reviewer acknowledgementpage 1 of 1 http://www.ojvr.org open access the editorial team of onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of onderstepoort journal of veterinary research. we appreciate the time taken to perform your review(s) successfully. in an effort to facilitate the selection of appropriate peer reviewers for the onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on https:// ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https://ojvr.org 2. in your ‘user home’ [https://ojvr.org/index. php/ojvr/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest(s). 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the onderstepoort journal of veterinary research. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 tel: 086 1000 381 abdalla a. latif adekunle b. ayinmode alaster samkange alfred maroyi alri pretorius ana m. tsotetsi-khambule andrea spickett anna-mari bosman annelize jonker annemarie labuscagne anthonius a. eze antoinette van schalkwyk awad shehata awoke k. gelaw ayana dinka ayesha hassim belinda blignaut brian j. willett celia hugo chantel j. de beer claude sabeta courtney a. cook david wallace delille wessels elibariki mwakapeje filomena iannino giulia simonato hassan y.a.h. mahmoud helena steyn heloise heyne imna malele jacqueline dabrowski johan esterhuizen junita liebenberg karama musafiri kerstin junker lia suzanne rotherham luís cardoso luke fiddes arnot lyndy j. mcgaw m a ghatee marco romito maryke m. henton mary-louise penrith matthew a. adeleke mirinda van kleef mohsen arbabi moritz jansen van vuuren motlatso t. hlokwe nabil qassem hailat nomakorinte gcebe oliver t. zishiri otto koekemoer paola debenedictis rhaksha bhoora seval bilge dagalp tanguy marcotty wilco botha wilna vosloo zuhair a. bani ismail 00 acknowledgement to reviewers http://www.ojvr.org https://ojvr.org https://ojvr.org https://ojvr.org https://ojvr.org/index.php/ojvr/user https://ojvr.org/index.php/ojvr/user mailto:publishing@aosis.co.za abstract introduction material and methods results and discussion conclusion acknowledgements references about the author(s) umberto molini department of pathobiology, school of veterinary medicine, faculty of agriculture and natural resources, university of namibia, neudamm campus, namibia gottlieb aikukutu central veterinary laboratory, windhoek, namibia juliet kabajani animal production and health laboratory, joint fao/iaea division of nuclear techniques in food and agriculture, international atomic energy agency, siebersdorf, austria siegfried khaiseb central veterinary laboratory, windhoek, namibia giovanni cattoli animal production and health laboratory, joint fao/iaea division of nuclear techniques in food and agriculture, international atomic energy agency, siebersdorf, austria william g. dundon animal production and health laboratory, joint fao/iaea division of nuclear techniques in food and agriculture, international atomic energy agency, siebersdorf, austria citation molini, u., aikukutu, g., kabajani, j., khaiseb, s., cattoli, g. & dundon, w.g., 2019, ‘molecular characterisation of infectious bursal disease virus in namibia, 2017’, onderstepoort journal of veterinary research 86(1), a1676. https://doi.org/10.4102/ojvr.v86i1.1676 research communication molecular characterisation of infectious bursal disease virus in namibia, 2017 umberto molini, gottlieb aikukutu, juliet kabajani, siegfried khaiseb, giovanni cattoli, william g. dundon received: 01 aug. 2018; accepted: 14 jan. 2019; published: 04 july 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract between july and september 2017, samples collected from six unvaccinated chickens in namibia were shown to be positive for infectious bursal disease virus (ibdv) by rt-pcr. partial sequence and phylogenetic analysis of the vp1 and vp2 genes from six viruses revealed that they all belong to the very virulent pathotype (genogroup 3) and are genetically very similar to ibdvs identified in neighbouring zambia. this is the first molecular characterisation of ibdv in namibia and has implications on the control and management of the disease in the country. keywords: poultry; infectious bursal disease virus; gumboro; namibia; phylogenetic analysis; vp1; vp2. introduction infectious bursal disease (ibd), also commonly known as gumboro disease, is a highly contagious immunosuppressive viral disease in poultry. the disease is caused by the ibd virus which is a member of the birnaviridae family of the avibirnavirus genus and infects lymphocytes in the bursa of fabricius of the host causing inflammation and subsequent atrophy of the organ. the resulting immunosuppression leaves the birds highly susceptible to secondary infections. the double-stranded rna genome of ibdv consists of two segments (a and b). segment a encodes four viral proteins (vp), namely, vp2, vp3, vp4 and vp5, while segment b encodes vp1. both vp1 and vp2 have been found to contribute to the virulence of ibdv (boot et al. 2005; gao et al. 2014; liu & vakharia 2004; yu et al. 2013) and segments of the vp1 and vp2 genes have been used to differentiate ibdvs by phylogenetic analysis (jackwood 2004). two serotypes of ibdv have been identified. serotype-1 is pathogenic to chickens, while serotype-2 is non-pathogenic (ismail et al. 1988). traditionally, serotype-1 viruses have been further classified into classical virulent, very virulent (vv), antigenic variant and attenuated strains. a more recent classification system proposed by michel and jackwood (2017) has grouped viruses into seven genotypes based on phylogenetic analysis of the hypervariable region of vp2 (michel & jackwood 2017). infectious bursal disease is regularly reported in africa, and circulating viruses have been well characterised in a number of countries, including egypt, ethiopia, nigeria, tanzania and tunisia (jenberie et al. 2014; kasanga et al. 2007; mardassi et al. 2004; nwagbo et al. 2016; shehata et al. 2017) however, little is known about the genetic makeup of ibdv in southern africa, with ibdvs having only been genetically characterised in zambia and south africa (kasanga et al. 2013; ndashe et al. 2016; vukea et al. 2014). in the past, incidences of ibd in namibia have been confined to rural areas. more recently, however, as the poultry industry and chicken populations have grown, the disease has begun to have more impact with significant mortalities being reported throughout the country. as yet, vaccination against ibd is not compulsory in namibia although live-attenuated d78 virus vaccines are available commercially. material and methods the samples analysed in this study were collected between july and september 2017 from rural villages in northern namibia located close to the border with angola (table 1). bursa of fabricius from dead chickens (3–6 weeks old) that had previously shown signs of illness, including depression, diarrhoea and prostration, were submitted to the central veterinary laboratory, windhoek. rna was extracted using the maxwell® 16 lev simplyrna tissue purification kit (promega) according to the manufacturer’s instructions with an elution volume of 50 µl. a fragment of the vp2 gene was amplified using the one-step reverse transcriptase – polymerase chain reaction kit (qiagen). the primer pair ibdv1 (5’ tcaggatttgggatcagc 3’) and ibdv2 (5’ tcaccgtcctcagcttac 3’), which produces an amplicon of 640 bp, was used as previously described (liu, giambrone & dormitorio 1994). the following thermal profile was used; reverse transcription at 50 °c for 30 minutes, initial denaturation at 95 °c for 15 min and then 40 cycles of denaturation at 95 °c for 30 seconds, annealing at 55 °c for 45 s and elongation at 72 °c for 45 s, followed by a final elongation at 72 °c for 5 min. for the vp1 gene, primer pair b-univ-f (5’ aat gag gag tat gag acc ga 3’) and b-univ-r (5’ cct tct cta ggt caa ttg agt acc 3’) was used to produce a 1050 bp amplicon (islam et al. 2012). the amplification conditions were as follows: reverse transcription at 50 °c for 30 min, initial denaturation at 95 °c for 15 min and then 35 cycles of denaturation at 95 °c for 10 s, annealing at 58 °c for 90 s and elongation at 68 °c for 30 s, followed by a final elongation at 68 °c for 7 min. amplified fragments were visualised on 1.0% – 1.5% agarose gels. positive rt-pcr amplicons were purified using a qiaquick pcr purification kit (qiagen) and were sent to lgc genomics (berlin, germany) for sequencing. all sequences generated were deposited in genbank under accession numbers mh237850 to mh237861. the staden package (http://staden.sourceforge.net/) was used to assemble the generated sequences. multiple sequence alignment was performed using muscle (http://www.ebi.ac.uk/tools/msa/muscle/) with default settings, incorporating all the sequences generated here combined with a selection of representative sequences available in genbank. phylogenetic trees were estimated using the neighbour-joining method available in mega 6 (tamura et al. 2013), employing the maximum composite likelihood model of nucleotide substitution and 1000 bootstrap replications. table 1: description of samples analysed in this study. ethical considerations this article followed all ethical standards for a research without direct contact with human or animal subjects. results and discussion from the phylogenetic analysis of the vp2, it can be seen that the viruses from namibia clustered in genogroup 3 (vvibdv) along with other viruses identified in africa, asia and europe (michel & jackwood 2017) (figure 1). it was also evident from the phylogenetic analysis that the viruses were genetically similar to viruses identified in zambia and south africa as opposed to those identified in other african countries (e.g. ethiopia, nigeria, egypt, tunisia and tanzania). interestingly, there was some genetic variation between the namibian viruses; samples g1 and g2 were identical and clustered together but were genetically distinct, with a 1.3% – 1.5% nucleotide divergence, from samples g5, g10, g12 and 5707. this implies that g1 and g2 do not share a recent common origin with samples g5, g10, g12 and 5707. samples g1 and g2 were collected in the ohangwena region of namibia which borders angola. there are several authorised live bird markets in this border region in which poultry originating from angola are both bought and sold. these markets may have been a source of viruses g1 and g2. however, namibia, also shares a border with zambia in the north-east of the country. given the similarity of the namibian samples with ibdv from zambia, as shown by the phylogenetic analysis, the two countries may also share a common origin or source of the virus. nonetheless, given that there is no published genetic data on ibdv available from angola, or for that matter from botswana or zimbabwe, despite disease occurrence being reported (kelly et al. 1994; mushi et al. 2006), no conclusion can yet be made on the origin of the ibdv investigated in this study. figure 1: neighbour-joining analysis using the mega6 software of a partial nucleotide sequence (403 bp) of the vp2 gene from the infectious bursal disease virus samples investigated (filled circles) and representative sequences from genbank. the numbers indicate the bootstrap values calculated from 1000 bootstrap replicates. the scale bar represents nucleotide substitutions per site. the different genogroups are indicated as described by michel and jackwood (2017). owing to the segmented nature of the ibdv genome, genome reassortment and recombination events, that have been shown to increase the virulence of the virus in addition to altering viral antigenicity, have been reported (boot et al. 2005; brandt et al. 2001; gao et al. 2014; jackwood & sommer-wagner 2011; liu & vakharia 2004; wei et al. 2008; yu et al. 2013). kasanga et al. (2013) identified a natural reassortant ibdv (kzc-104) in zambia in 2004 that consisted of a very virulent segment a and a classical attenuated segment b. the genome segments of the more recently identified viruses from zambia by ndashe et al. (2016) were both very virulent. therefore, to determine whether the namibian ibdvs were reassortants or not, a segment of the vp1 gene from the six samples was amplified by rt-pcr and sequenced. the results of the phylogenetic analysis of the vp1 segment shown in figure 2 clearly showed that the namibian viruses were not reassortants and were more similar to the genogroup 3 viruses described by ndashe et al. (2016) and not the genogroup 1 kzc-104 (genbank ab368969) reassortant described by kasanga et al. (2013). the tree also confirmed that samples g1 and g2 were genetically divergent (1.0% – 1.2 %) from samples g5, g10, g12 and 5707. figure 2: neighbour-joining analysis using the mega6 software of a partial nucleotide sequence (688 bp) of the vp1 gene from the infectious bursal disease virus samples investigated (filled circles) and representative sequences from genbank. the numbers indicate the bootstrap values calculated from 1000 bootstrap replicates. the scale bar represents nucleotide substitutions per site. the different genogroups are indicated as described by michel and jackwood (2017). the predicted amino acid sequence of the partial vp2 and vp1 was checked for characteristic aa residues. for vp2, all of the viruses possessed typical aa residues seen in other vvibdv (genogroup 3), namely, 222a, 242i, 253q, 256i, 294i and 299s (brandt et al. 2001; ndashe et al. 2016) (table 2). table 2: comparison of amino acid residues of the vp2 between the samples investigated and other infectious bursal disease viruses. likewise, the vp1 aa sequences possessed residues 145t, 146d, 147n, 242e and 287a. the tdn triplet has been shown by gao et al. (2014) to contribute to viral virulence, while 242e and 287a have been identified as possible virulent determinants (yu et al. 2010). at amino acid position 119 of the vp1, samples g5, g12 and 5707 possessed an aspartic acid (d) residue, while samples g1, g2, g10 and all of the other viruses included in the phylogenetic analysis possessed a glutamic acid (e) residue at this position. in addition, at amino acid position 252 of vp1, samples g5, g10, g12 and 5707 differed from all of the other viruses (including g1 and g2) by possessing valine (v) instead of an isoleucine (i) (table 3). the significance of these amino acid differences is unknown at present and requires further investigation. table 3: comparison of amino acid residues of the vp1 between the samples investigated and other infectious bursal disease viruses. conclusion this study has identified, for the first time in namibia, ibdvs that are genetically similar to viruses identified in neighbouring zambia. combined with data from further molecular epidemiological investigations throughout namibia and the region (including angola, botswana and zimbabwe), this study will allow for the design of targeted vaccination programmes and strategies by namibian veterinary authorities. it has also generated comparative data for those interested in the circulation of ibdv in southern africa. acknowledgements this work was supported through funding from the african renaissance funds and from the directorate of veterinary services, ministry of agriculture, water and forestry of namibia. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions u.m., s.k., g.c. and w.g.d. conceived and designed the study. u.m., g.a. and j.k. were involved in sample collection. u.m. and w.g.d. analysed the data and drafted the manuscript. all authors were involved in the revision and approval of the final 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genetics analysis version 6.0’, molecular biology and evolution 30, 2725–2729. https://doi.org/10.1093/molbev/mst197 vukea, p.r, willows-munro, s., horner, r.f. & coetzer, t.h., 2014, ‘phylogenetic analysis of the polyprotein coding region of an infectious south african bursal disease virus (ibdv) strain’, infection genetics and evolution 21, 279–286. https://doi.org/10.1016/j.meegid.2013.11.017 wei, y., yu, x., zheng, j., chu, w., xu, h., yu, x. et al., 2008, ‘reassortant infectious bursal disease virus isolated in china’, virus research 131, 279–282. https://doi.org/10.1016/j.virusres.2007.08.013 yu, f., qi, x., yuwen, y., wang, y., gao, h., gao, y., et al., 2010, ‘molecular characteristics of segment b of seven very virulent infectious bursal disease viruses isolated in china’, virus genes 41, 246–249. yu, f., ren, x., wang, y., qi, x., song, j., gao, y., et al., 2013, ‘a single amino acid v4i substitution in vp1 attenuates virulence of very virulent infectious bursal disease virus (vvibdv) in spf chickens and increases replication in cef cells’, virology 440, 204–209. https://doi.org/10.1016/j.virol.2013.02.026 abstract introduction material and methods results discussion acknowledgements references about the author(s) byron m. göpper department of anatomy and physiology, university of pretoria, south africa nina m. voogt department of anatomy and physiology, university of pretoria, south africa andre ganswindt department of anatomy and physiology, university of pretoria, south africa mammal research institute, department of zoology and entomology, university of pretoria, south africa citation göpper, b.m., voogt, n.m. & ganswindt, a., 2018, ‘first record of the marine turtle leech (ozobranchus margoi) on hawksbill turtles (eretmochelys imbricata) in the inner granitic seychelles’, onderstepoort journal of veterinary research 85(1), a1604. https://doi.org/10.4102/ojvr.v85i1.1604 research communication first record of the marine turtle leech (ozobranchus margoi) on hawksbill turtles (eretmochelys imbricata) in the inner granitic seychelles byron m. göpper, nina m. voogt, andre ganswindt received: 22 dec. 2017; accepted: 24 july 2018; published: 30 aug. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract ozobranchus spp. are leeches that feed solely on turtle blood. they are common ectoparasites found on a range of marine turtle species, with some species of the leech being implicated as vectors of fibropapilloma-associated turtle herpesvirus (fpthv). green (chelonia mydas) and hawksbill (eretmochelys imbricata) turtles are the two commonly occurring species in the inner granitic islands of the seychelles. routine monitoring of nesting turtles on cousine island, seychelles, allowed for opportunistic sightings of leeches on two hawksbill females. in both cases infestation was low, with three leeches collected off one female turtle and five off the other. no obvious signs of papillomas secondary to infection of fpthv were seen. all of the turtle leeches collected were determined to be ozobranchus margoi as they had five pairs of lateral digiform branchiae. the specimens were deposited in the seychelles natural history museum on mahé. to the best of our knowledge this is the first record of ozobranchus margoi recorded in the inner granitic seychelles on hawksbill turtles. introduction the genus ozobranchus is characterised, among other features, by their anterior abdominal somites that each possess a pair of gills that are divided distally into many branchiae (richardson 1969). they are the only non-piscicolid marine leeches to be permanent parasites of sea turtles and attach onto the turtle’s skin, particularly around the cloaca, head region and flippers (sawyer 1986). ozobranchs are stationary leeches, remaining on their host for their entire life cycle. numerous cocoons are deposited on the turtle’s plastron where they hatch. newly hatched leeches feed on the same host as their parents. this cycle is repeated, ultimately leading to large numbers of leeches on an individual (sawyer 1986). owing to the difficulties of studying their sea turtle hosts, very little is known about sea turtle leeches and whether they are able to survive partly without a host or utilise an alternate host (mcgowin et al. 2011). however, more knowledge about the life cycle of sea turtle leeches would be beneficial, as some species of ozobranchus have been implicated as mechanical vectors of fibropapilloma-associated turtle herpesvirus (fpthv), a neoplastic disease causing epithelial tumours in sea turtles (greenblatt et al. 2004). ozobranchus margoi parasitise several species of sea turtle, namely green turtles (chelonia mydas; richardson 1969), kemp’s ridley turtles (lepidochelys kempii; davies & chapman 1974), hawksbill turtles (eretmochelys imbricata; bunkley-williams et al. 2008), as well as loggerhead turtles (caretta caretta; insacco, violani & zava 2000). however, o. margoi show some degree of host preference and are most frequently associated with loggerhead turtles (bunkley-williams et al. 2008). geographically there have been reports of o. margoi from florida (davies 1978; davies & chapman 1974; mcgowin et al. 2011; sawyer, lawler & oversrteet 1975; truong 2014; truong & mcgowin 2011), north carolina (schwartz 1974), hawaii (balazs 1980), barbados (truong 2014), brazil (peralta et al. 2003; rodenbusch et al. 2012; truong 2014), puerto rico (bunkley-williams et al. 2008), uruguay (cordero 1929), adriatic sea (piccolo & manfredi 2003; scaravelli, affronte & costa 2003), italy (apathy 1890), mediterranean sea (insacco et al. 2000), tunisia (karaa et al. 2011), south africa (hughes, bass & mentis 1967), india (sanjeeva raj 1959), japan (oka 1927), taiwan (cheng-tsung & i-jiunn 2013; tseng, leu & cheng 2017) and australia (loop, miller & limpus 1995; richardson 1969). however, there are no published reports documenting the occurrence of o. margoi on turtles in the inner granitic seychelles. to the best of the authors’ knowledge, this is the first recorded report of o. margoi parasitising e. imbricata in the seychelles archipelago. material and methods observations were made on cousine island (-4.350577°s, 55.647527°e), a 25-hectare island with a 1-km stretch of beach. the island is situated in the inner granitic seychelles, seychelles archipelago, indian ocean. within the inner granitic islands, cousine island is an important nesting beach for hawksbill turtles (e. imbricata). the islands’ conservation and turtle monitoring programme has been running since 1991 (hitchins et al. 1999). while following cousine island’s standard routine monitoring protocols set out for nesting female hawksbill turtles, leeches were randomly spotted and opportunistically collected from two separate nesting female hawksbill turtles that came up to nest on cousine island’s beach during the 2015–2016 season. collection of specimens took place on 12 january 2016 and 22 january 2016. results all leeches were located attached to the soft tissue around the cloaca of both female turtles (figure 1). a total of eight leeches were collected, three off the female turtle with flipper tags sca 3814 (left) and sca 3815 (right), and five off the female turtle with flipper tags sca 7048 (left) and sca 7668 (right). five specimens were preserved in 70% ethanol and the remaining three in 10% formalin. subsequently, three of the specimens were deposited at the natural history museum, mahé, seychelles, for further studies and to make them available to other researchers. therefore, the specimens’ corresponding voucher numbers are: 1443/16 (collection date: 12 january 2016) preserved in 70% ethanol, 1444/16 (collection date: 22 january 2016) preserved in 70% ethanol and 1445/16 (collection date: 22 january 2016) preserved in 10% formalin. figure 1: ozobranchus margoi observed on nesting hawksbill turtle. photo taken of tail and cloaca while eggs were being laid. the bodies of all eight specimens showed a distinguishable trachelosome and urosome, with a posterior sucker clearly visible. furthermore, five pairs of gills were visible on the urosome (figure 2). based on these characteristics, the leeches were identified as ozobranchus margoi (davies 1978). figure 2: ozobranchus margoi specimen. discussion based on a literature search for published cases of o. margoi using various keyword combination searches in google scholar and the database on the sea turtle network (http://www.seaturtle.org/library/) no records of o. margoi parasitising e. imbricata in the seychelles could be found. samways et al. (2010), the main reference text for cousine island’s fauna, flora and ecology, also revealed no records; therefore, to the best of the authors’ knowledge, this is the first record of o. margoi parasitising on hawksbill turtles in the inner granitic seychelles. green and hawksbill turtles are the two commonly occurring species in the inner granitic islands of the seychelles. future standard operating procedures for monitoring protocols of nesting female turtles are encouraged to include the recording of any observations of leeches or fibropapillomas. no obvious signs of papillomas were seen on the turtles in this study and in both cases infestation of leeches was low. as leeches are common on green turtles in other regions of the world, it would be interesting to further investigate the occurrence of ozobranchus spp. in the seychelles sea turtle populations, especially to see if other species (e.g. o. branchiatus) are also present. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this paper. authors’ contributions b.m.g. and n.m.v. collected the samples, submitted them to the seychelles natural history museum and wrote the manuscript. a.g. made conceptual contributions and revisions to the manuscript. all authors contributed to editing the manuscript to its final form in preparation for publication. references apathy, s.t., 1890, ‘pseudobranchellion margoi (nova familia hirudinearum)’, orvos-termeszettudomanyi eresito 15, 122–127. balazs, g.h., 1980, ‘synopsis of biological data on the green turtle in the hawaii islands’, national oceanographic and atmospheric administration technical memorandum 7, 141. bunkley-williams, l., williams jr., e.h., horrocks, j.a., horta, h.c., mignucci-giannoni, a.a. & poponi, a.c., 2008, ‘new leeches and diseases for the hawksbill sea turtle and the west indies’, comparative parasitology 75, 263–270. 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samways, m.p., hitchins, p., bourquin, o. & henwood, j., 2010, tropical island recovery: cousine island, seychelles, wiley-blackwell, chichester. sanjeeva raj, p.s., 1959, ‘occurrence of ozobranchus margoi apathy (hirudinea: annelida) in the indian seas’, current science 28, 496–496. sawyer, r.t., 1986, leech biology and behaviour, clarendon press, oxford. sawyer, r.t., lawler, a.r. & overstreet, r.m., 1975, ‘marine leeches of the eastern united states and the gulf of mexico with a key to the species’, journal of natural history 9, 633–667. https://doi.org/10.1080/00222937500770531 scaravelli, d., affronte, m. & costa, f., 2003, ‘analysis of epibiont presence on caretta caretta from adriatic sea’, in d. margaritoulis & a. demetropoulos (eds.), proceedings of the first mediterranean conference on marine turtles, rome, october 24–28, 2001, pp. 211–225, barcelona convention – bern convention – bonn convention (cms), nicosia. schwartz, f.j., 1974, ‘the marine leech ozobranchus margoi (hirudinea)’, journal of parasitology 60, 889–890. https://doi.org/10.2307/3278927 truong, t.m., 2014, ‘investigating dna barcoding potentials and genetic structure in ozobranchus spp. from atlantic and pacific ocean sea turtles’, msc dissertation, wright state university, usa. truong, t.m. & mcgowin, a.e., 2011, ‘dna barcoding of sea turtle leeches (ozobranchus spp.) in florida coastal waters’, abstracts of the 75th anniversary meeting of the florida academy of sciences, florida institute of technology, melbourne, fl, p. 56. tseng, c.t., leu, j.h. & cheng, i.j., 2017, ‘on the genetic diversity of two species of the genus ozobranchus (hirudinida; ozobranchidae) from the atlantic and pacific oceans’, journal of the marine biological association of the united kingdom 98, 955–960. https://doi.org/10.1017/s0025315416001958 abstract introduction materials and methods results discussion recommendations acknowledgements references about the author(s) esmey b. moema department of biology, sefako makgatho health sciences university, pretoria, south africa pieter h. king department of biology, sefako makgatho health sciences university, pretoria, south africa johnny n. rakgole department of virology, sefako makgatho health sciences university, pretoria, south africa citation moema, e.b., king, p.h. & rakgole, j.n., 2019, ‘phylogenetic studies of larval digenean trematodes from freshwater snails and fish species in the proximity of tshwane metropolitan, south africa’, onderstepoort journal of veterinary research 86(1), a1726. https://doi.org/10.4102/ojvr.v86i1.1729 original research phylogenetic studies of larval digenean trematodes from freshwater snails and fish species in the proximity of tshwane metropolitan, south africa esmey b. moema, pieter h. king, johnny n. rakgole received: 09 jan. 2019; accepted: 18 apr. 2019; published: 17 sept. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the classification and description of digenean trematodes are commonly accomplished by using morphological features, especially in adult stages. the aim of this study was to provide an analysis of the genetic composition of larval digenean trematodes using polymerase chain reaction (pcr) and sequence analysis. deoxyribonucleic acid (dna) was extracted from clinostomatid metacercaria, 27-spined echinostomatid redia, avian schistosome cercaria and strigeid metacercaria from various dams in the proximity of tshwane metropolitan, south africa. polymerase chain reaction was performed using the extracted dna with primers targeting various regions within the larval digenean trematodes’ genomes. agarose gel electrophoresis technique was used to visualise the pcr products. the pcr products were sequenced on an applied bioinformatics (abi) genetic analyser platform. genetic information obtained from this study had a higher degree of discrimination than the morphological characteristics of seemingly similar organisms. keywords: digenean trematodes; classification; description; polymerase chain reaction; pcr; genetic composition; sequence analysis; nucleotide variations; molecular analysis. introduction the classification of digenean parasites, especially using only the larval stages, to determine the species level employing exclusively morphological characteristics is very challenging, because of the lack of genitalia that are regarded as the most important structures in the identification of these organisms (moema et al. 2013). it is also believed that digenean parasites might have experienced great morphological transformations forced by external adaptive forces (brooks, o’grady & glen 1985). gibson, jones & bray (2001) concurred with blair et al. (1998) who mentioned that phylogenies of the digenean trematodes based on their morphological characters and their life cycles have always been controversial. molecular characterisation in combination with morphological descriptions has been explored in a limited number of studies. examples include the study of blair et al. (1998) that presented the first molecular phylogenetic study for the superfamily hemiuroidea (whose members are parasitic in the stomach of marine and sometimes freshwater teleosts and elasmobranchs), which was first recognised under the name hemiurida by dollfus (1923). in dollfus’ classification, this superfamily was assigned three families, namely hemiruidae, accacoelidae and syncoeliidae. recent authors such as blair et al. (1998) tested for the resemblance between morphological and molecular data and explored the evolution of some morphological characters. in their concluding remarks, blair et al. (1998) said that morphological and molecular data were complementary, but there were some branches that were favoured by molecular data which were not supported by a single morphological synapomorphy (character state shared by two or more terminal groups including that inherited from their most recent common ancestor). the above-mentioned authors also concluded that molecular data provide more resolution in the phylogenetic relationships of digenean parasites. cribb, bray and littlewood (2001) presented the first phylogenetic analysis by providing explicit character matrices through the combination of a newly coded morphological matrix with new molecular data from the small subunit ribosomal rna gene deoxyribonucleic acid (ssrdna). the above-mentioned authors came up with a reasonably well-resolved tree by making use of complete ssrdna sequences from 75 digenean species representing 55 families in combination with 56 larval and adult morphological characters for those families. they also provided a historical review of previous classification keys and molecular phylogenetic studies of the digenean groups conducted previously. in gibson et al.’s (2001) study, the families schistosomatidae, sanguinicoilidae and spirorchiidae are grouped together as sister taxa of the superfamily schistosomatoidea. snyder (2004) did a study on the digenean parasites of the families schistosomatidae and spirorchiidae, generating deoxyribonucleic acid (dna) sequence data from the nuclear large subunit rdna (lsu) and nuclear small subunit (ssu) from the representatives of eight genera from both freshwater and marine turtles. the data generated were then incorporated into the pre-existing data from 10 genera of schistosomatidae and other selected members of the suborder diplostomata (sensu olson et al. 2003), including brachylaimidae, diplostomatidae, strigeidae and leucochloridiidae that constituted the outgroups. the ingroup had representatives from the following families: schistosomatidae, sanguinicolidae, spirorchiidae and clinostomatidae. the analyses performed by snyder (2004) of the combined lsu and ssu data using maximum prudence and bayesian deduction produced a single tree of identical topology. this author concluded that spirorchiidae is paraphyletic, thus making it taxonomically invalid. on the other hand, brooks et al. (1985) have always recognised this family as distinct but closely related to schistosomatidae and easily distinguishable by their reproductive organs and different definitive hosts. snyder’s (2004) recommendations were to either classify the two families, schistosomatidae and spirorchiidae as a single family, or that the latter family should be further broken down into smaller families. the study conducted by wilson et al. (2005) was to investigate features such as how many species occurred within ribeiroia, geographic distribution of this genus and the relationship of ribeiroia to other trematode species. they collected specimens belonging to this genus from africa (1 locality), the united states (8 localities) and the caribbean (2 localities), and pcr-based techniques were performed using tissue isolates from either metacercariae from amphibians or cercariae from infected molluscs, amplifying both the its1 and its2 regions. these authors reported the first internal transcribed spacer (its) sequence from either ribeiroia or cathaemasia spp. their study was the first to show that cercaria lileta was almost certainly a species of ribeiroia. the data obtained from the study recognised three closely related species of ribeiroia found in the caribbean (r. marini), africa (cathaemasia lileta) and some parts of america (cathaemasia ondatrae). the phylogenetic studies mentioned above are just a few reports from other parts of the world and just a handful from africa. the authors of this study are of the opinion that more studies on the phylogeny of trematodes need to be conducted so that we may have better insight into digenean parasite relationships in all ecosystems globally. thus, phylogenetic studies of larval digenean trematodes collected from various intermediate hosts were conducted around tshwane metropolitan. this research project was pursued from 2010 to 2015 in order to accurately place these parasites in their respective families and also to look at their relationships with other groups of digenean parasites. materials and methods sampling methods freshwater snail specimens of lymnaea natalensis were collected from various farm dams with scoop nets according to the method described by van eeden (1960). snails were squashed between two glass slides, the shell was removed and redial stages were sampled from the host’s tissue. cercariae were harvested from natural sheddings from freshwater snails. freshwater fish species, tilapia sparrmanii and pseudocrenilabrus philander, were sampled from the same waterbodies using hand nets. they were dissected; gills were removed and examined for cysts using a dissection microscope. metacercariae were excysted manually using fine forceps to expose the juvenile worms. deoxyribonucleic acid extraction and polymerase chain reaction specimens for pcr were suspended in 5% buffered saline solution (200 µl) and frozen in a -70 °c freezer until use. during the extraction process, specimens were thawed and excess buffered saline solution (bss: 800 ml distilled water, 8 g nacl, 0.2 g kcl, 1.44 g na2hpo4 and 0.24 g kh2po4; ph = 7.4) was removed. deoxyribonucleic acids were extracted using qiaamp dna mini kit (qiagen, valencia, ca) following the manufacturer’s instructions. briefly, specimens were transferred to a 220 µl lysis solution (i.e. 200 µl atl buffer and 20 µl proteinase k) in microcentrifuge tubes. samples were incubated at 56 °c for 4 hours followed by the addition of 200 µl of absolute ethanol. the dna in the lysate was allowed to bind to the spin columns and washed twice with the supplied buffers before being eluted in 200 µl of elution buffer. the extracted dna of parasites was stored at -20 °c until use. the genomic regions as presented in table 1 were amplified. the genomic regions of rdna that were targeted included its1, its2, dig and lsus. the universal primer pairs with their corresponding annealing temperatures (ta) were tested as shown in table 1. table 1: genomic regions and their corresponding primers used on various digenean parasites collected in the proximity of tshwane metropolitan. the pcr reaction was performed in a final volume of 25 µl. the reagents at a final concentration of 0.2 mm for dntps, 1.5 mm mgcl2, 0.2 µm each of primers and 1 u per reaction of biotec dna polymerase (bioline, london, united kingdom [uk]) or supertherm (jmr holdings, london, uk) were used. the pcr cycling condition was denatured at 92 °c – 94 °c with initial steps for 2 min. this step was to allow the splitting of double-stranded dna into single strands at the beginning of the reaction. following the initial steps was 30–40 three temperature cycles of 92 °c for 20 seconds with annealing temperature as indicated in table 1 and extension at 72 °c with steps of initial c for 60 s. the reaction was followed by a final extension of 72 °c for 5 min to allow for final steps of binding. amplicons were identified on agarose gel electrophoresis by matching the expected product size. the pcr amplification resulting from four parasitic samples at different developmental stages is presented in table 2. table 2: polymerase chain reaction amplification results from four parasitic samples at different developmental stages. sequencing of polymerase chain reaction amplicons all the positive pcr products generated were sequenced on an abi dna 3130xl analyser (applied biosystems, foster city, california, united states) using the same primer pairs as used for amplification. the sequences are generated on automated platforms and they do result in errors that need human interventions, for example, when there are ambiguous base-calls or groups, and hence, the sequences were edited using the chromaspro 1.49 (http://www.technelysium.com.au/chromaspro.html) software tool (technelysium pty ltd.). reference sequences were downloaded from genbank. study sequences and reference sequences were aligned with clusta1w (http://bips.ustrasbg.fr/fr/documentation/clusta1w/) (thompson, higgins & gibson 1994) as found in the bioedit 7.0.4 (http://www.mbio.ncsu.edu/bioedit/bioedit.html) software program (hall 1999). phylogenetic trees were constructed using neighbour-joining with a bootstrap method test of phylogeny with 1000 bootstrap replications; the amino acids substitution type and the poisson substitution model were used; and the pairwise distance homology was computed. the software program utilised was the mega 6 software program (tamura et al. 2013). ethical considerations this study fulfilled the requirements of the animal ethics committee (aec 02/05) and medunsa research ethics committee (bp 05/2005). freshwater fish species were kept in well-aerated glass aquaria and fed on fish flakes prior to dissections. upon dissection, the collected freshwater fish species were anaesthetised using clove oil. freshwater snails, on the other hand, were kept in well-aerated plastic containers and were also fed fish flakes prior to some of them being crushed for the collection of redial stages. results polymerase chain reaction amplification, sequencing and analysis polymerase chain reaction amplification amplification of dna yielded amplicons from only four specimens: clinostomatid metacercaria, 27-spined echinostomatid redia, avian schistosome cercaria and green strigeid metacercaria (excysted). the pcr amplification using primer pairs its1-19dg/its1-4s1dg (first round), its1-1dg/its1-2dg (second round), its2-f/its2-r, lsu/1500r and dig 125/1500r yielded bands ranging in size from 500 base pairs (bp) to 1500 bp, respectively (table 2). echinostomatid parasite amplification, sequencing and analysis the echinostomatid parasite was collected from lymnaea natalensis. it was described as 27-spined cercaria as petasiger variospinosus according to the morphological criteria (king & van as 2000). the redial stage was used for dna extraction. following dna extraction and pcr, a fragment of approximately 600 bp in length of dna was observed by electrophoresis. sequencing of the pcr product resulted in a 595 bp, spanning the 5.8s rrna, the its2 and the 28s rrna sequences. using the blast program, the parasite in this study with the accession number (kx034047) clustered with petasiger sp. with accession numbers (km972995 and km972992) (figure 1) and they share 95% homology. figure 1: phylogenetic tree of echinostomatid digenean parasites. note that the specimen in this study is highlighted with a diamond shape (♦). avian schistosome parasite amplification, sequencing and analysis an avian schistosomatid cercaria was identified as trichobilharzia sp. using morphological analysis (moema, king & baker 2008), collected from lymnaea natalensis. following dna extraction and pcr, a fragment of approximately 1500 bp in length of dna was observed by electrophoresis. sequencing of the pcr product resulted in a 659 bp of a 28s rrna partial sequence. using the blast program, the parasite in this study with the accession number (kx034046) is closely related to trichobilharzia regenti (figure 2) and they share 100% homology. figure 2: phylogenetic tree of avian schistosomatid parasites. note that the specimen in this study is highlighted with a diamond shape (♦). strigeid parasite amplification, sequencing and analysis the strigeid parasite was collected from tilapia sparrmanii and pseudocrenilabrus philander. it was described morphologically as the metacercaria of the family strigeidae because of its possession of pseudosuckers flanking the oral sucker (shoop 1989). following dna extraction and pcr, a fragment of approximately 1500 bp in length of dna was observed by electrophoresis. sequencing of the pcr product resulted in a 1255 bp, which is a partial sequence of the 28s rdna. the parasite in this study with the accession number (kx034049) was identified with the parasites belonging to the same superfamily diplostomoidea using the blast program (figure 3). the sequence in this study is closely related to cardiocephaloides longolis and strigeidae sp. and they share 94% homology. figure 3: phylogenetic tree of strigeid parasites. note that the specimen in this study is highlighted with a diamond shape (♦). clinostomatid parasite amplification, sequencing and analysis the clinostomatid metacercaria in this study was morphologically described as clinostomum tilapiae collected from tilapia sparrmanii. following dna extraction and pcr, a fragment of approximately 500 bp in length of dna was observed by electrophoresis. sequencing of the pcr product resulted in a 459 bp, which is a partial sequence of the 5.8 rrna, complete its2 and a partial 28s rrna sequences. using the blast program, the parasite in this study with the accession number (kx034048) clustered with clinostomum tilapiae (figure 4). recent african sequences with accession numbers (ky649349 up to kp649356) with the exception of ky649354 share 100% homology with the sequence in this study. figure 4: phylogenetic tree of clinostomatid parasites. note that the specimen in this study is highlighted with a diamond shape (♦). discussion the phylogenetic tree generated for the echinostomatid parasite in this study confirms the classification of this parasite as one of the digeneans belonging to the family echinostomatidae. this observation concurs with the results from the morphological descriptions made by king and van as (2000). the parasite formed a strong monophyletic group with other 27-spined echinostomatid isolates from the genbank under the genus petasiger. this discovery at the molecular level justifies the identification of this parasite at the morphological level. the family echinostomatidae also formed a sister clade to the family fasciolidae. this also confirms the observations made by olson et al. (2003) about the interrelationships of the parasites from the two families being more consistent than those in other families. the two families mentioned above are classified under the superfamily echinostomatoidea (jones, bray & gibson 2005). the morphological characterisation of the avian schistosome parasite in this study clearly shows that it belongs to the genus trichobilharzia (moema et al. 2008). this is also supported by the grouping of the avian schistosomatid cercaria in this study with other avian schistosome parasites’ sequences obtained from the genbank. the clades observed in this study concur with the findings made by snyder (2004), where he reported the existence of these clades from the three studies that he conducted. gibson et al. (2001) classified this group of parasites under the family schistosomatidae and subfamily bilharziellinae. these authors further mentioned that the parasites belonging to this family are blood flukes of birds, crocodiles and mammals. with regard to the strigeid parasite (green cyst), the molecular findings in this study show that the partial 28s rdna was covered. the newly constructed tree also demonstrates separate clusters formed by parasites belonging to the families diplostomatidae and strigeidae. the parasite in this study shows close links with the parasites of the genus cardiocephaloides longolis and strigeidae sp. the primer sets were designed to universally amplify genomes across digenean parasites. also because of the fact that there are no sequences similar to ours in the genbank database, it is difficult to analyse this parasite up to the species level. according to gibson et al. (2001), parasites of the family strigeidae are parasitic in fish and amphibians as second intermediate hosts. they further mentioned that adult forms of this family inhabit birds and rarely mammals as the definitive hosts. analysis of the clinostomum tilapiae sequence showed clustering with other clinostomum tilapiae sequences from other african countries (caffara et al. 2017). gibson et al. (2001) classified organisms of the genus clinostomum under the family clinostomatidae. these authors further mentioned that the metacercarial stages of these parasites are found in the muscles and abdominal cavity of freshwater fish, snails, snakes, frogs and salamanders. they also mentioned that adults of this family are found to inhabit mainly the buccal cavity or oesophagus of fish-eating birds, reptiles and mammals, including humans (rarely). in most cases, the phylogenetic studies were achieved using matured (adult) stages of digenean parasites. these include the studies conducted on digenean parasites at species level (barber, mkoji & loker 2000; barker et al. 1993; dzikowski et al. 2003; farjallah et al. 2009; huang et al. 2004; le, blair & mcmanus 2001; miller & cribb 2007; rinaldi et al. 2005; wilson et al. 2005). most of these phylogenetic studies were conducted on medically and veterinary important digeneans. other studies were performed at family level (chen et al. 2007; jousson et al. 1998), superfamily level (tkach pawlowski & mariaux 2003), suborder level (tkach et al. 2000) and on general digeneans (blair et al. 1996). the above--mentioned studies are just a few that are already documented. in this study, the larval stages of four families: (1) clinostomatidae, (2) schistosomatidae, (3) echinostomatidae and (4) strigeidae could be amplified and sequenced. from this study, it is evident that in future, specific primers that are able to differentiate parasites up to species level should be designed. the recent study also demonstrates that much more work needs to be done before we can understand parasite–host relationships in the localities studied. these parasites could have a profound negative impact on aquaculture. furthermore, these parasites could infect humans and cause minor medical conditions, such as trichobilharzia sp. causing swimmer’s itch. further experimental life-cycle studies using suitable hosts are therefore imperative in order to solve most of our cercaria, metacercaria and adult trematode questions of this study. recommendations the amount of sequence data of digenean parasites are lacking in the genbank database. we therefore recommend large-scale studies in the future based on molecular data. acknowledgements the authors thank the department of virology of the sefako makgatho health sciences university for their assistance in making this research project a success through their vast knowledge on phylogenetic studies and for allowing them to make use of their facilities. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.b.m. was responsible for the collection of samples, pcr work, analysis of results and manuscript write-up. p.h.k. was responsible for the collection of samples, morphological identification and manuscript write-up. j.n.r. was responsible for the pcr work 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loker, e.s., 2005, ‘a molecular phylogenetic study of the genus ribeiroia (digenea): trematodes known to cause limb malformations in amphibians’, journal of parasitology 91(5), 1040–1045. https://doi.org/10.1645/ge-465r.1 reviewer acknowledgement open accesshttp://www.ojvr.org page 1 of 1 the onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of the onderstepoort journal 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silva marco romito maria groot marinda oosthuizen mark c. williams martin van der leek maryke m. henton mary-louise penrith moritz jansen van vuuren muhammad k. khan neil duncan neil fourie nicola collins peter roeder peter thompson pierre dorny rudolph d. bigalke samson mukaratirwa shahn bisschop stina ekman tanguy marcotty tertius gous theo de waal tracy schmidt tshepo matjila vincent delespaux wanda markotter william wilson wilna vosloo 190 abstract introduction materials and methods results discussion conclusion acknowledgements references appendix 1 about the author(s) priscilla munzhelele nooitgedacht research station, department of agriculture, rural development, land and environmental affairs, animal research, non-ruminant sub-directorate, south africa department of agriculture and animal health, university of south africa, south africa james w. oguttu department of agriculture and animal health, university of south africa, south africa folorunso o. fasina department of production animal studies, university of pretoria, south africa citation munzhelele, p., oguttu, j.w. & fasina, f.o., 2016, ‘is a 10-sow unit economically sustainable? a profitability assessment of productivity amongst small-holder pig farmers, mpumalanga, south africa’, onderstepoort journal of veterinary research 83(1), a1011. http://dx.doi.org/10.4102/ojvr.v83i1.1011 original research is a 10-sow unit economically sustainable? a profitability assessment of productivity amongst small-holder pig farmers, mpumalanga, south africa priscilla munzhelele, james w. oguttu, folorunso o. fasina received: 10 july 2015; accepted: 21 oct. 2015; published: 12 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the majority of small-holder pig farmers in mpumalanga had between 1and 10-sow herds. the main aim of this study is to evaluate the current government agricultural intervention (supply of 10 sows and a boar) in terms of technical and economic feasibilities and ascertain whether the small-scale pig value chain system alleviates poverty. data were obtained from 220 randomly selected small-holder pig farmers using a semi-structured questionnaire. the results showed that 58% farrowed ≤ 10 piglets/born/sow/litter, 44.2% practiced no weaning method and many fed swill and leftovers alone (41.6%). pair-wise association revealed that the feeding of commercial feeds had a relationship with pigs in relatively good to very good body condition. pigs in poor body condition were positively correlated with the feeding of swill alone. the economic models for the 10-sow unit proved that pig farming is unprofitable if the current management and feeding systems that operate in the commercial industry are utilised. however, only through a combination of cooperative systems, benefits of economies of scale, reduction of preweaning mortalities and structured government inputs can pig production be profitable at this scale of production. introduction animal agriculture in general and pig production in particular are important economic activities globally (dietze 2011; mokoele et al. 2015; roelofse 2013). in south africa, pig production is distributed in all nine provinces, with higher concentrations in limpopo, north west, gauteng and kwazulu-natal, partially because of cultural and religious preferences and availability of feedstuffs. mpumalanga is placed sixth in terms of pig production, contributing approximately 8% of the national pig herd (daff 2012; mpg 2013). the province had a relatively high concentration of small-holder pig farmers otherwise known as emerging small-scale pig farmers. pig farming requires little space, yields a large number of offspring after a shorter gestation period than other small stock and can be combined with other forms of subsistence agriculture where land resources are scarce (daff 2012; makiwane et al. 2012). in addition, it plays a major role in poverty reduction and food security (fao 2004) and provides a form of investment, emergency cash and meat for home consumption (drucker & anderson 2004; mhlanga 2002). in mpumalanga, the commonly found breeds of pigs are the kolbroek, large white, landrace and their crosses. backyard pig farming and semi-intensive management systems in poorly designed pens are the most common small-holder pig farming practices in the rural and peri-urban areas of mpumalanga. in general, the farm families rely on family labour and the majority of products are meant for household consumption or converted to cash for the purpose of family maintenance. these contributions are very important for family incomes in mpumalanga, where poverty rates have ranged from 50.4% (1996), 59.1% (2004), 50.1% (2008) to 39.4% (2011) and the unemployment rate remains at 29.4% (mpg 2009, 2013). the mpumalanga department of agriculture, rural development, land and environmental affairs (dardlea) established the programme called masibuyele esibayeni, meaning back to the kraal (i.e. returning to the land), with similar programmes in gauteng and limpopo amongst others. the programme is aimed at helping the small-holder farmers to upgrade and boost productivity by improving the genetic pool of their livestock. for the pig component, dardlea provides farmers with 10 sows and 1 boar with improved genetics for breeding and provides supportive services to such farms. to evaluate the potential success of such a programme, we analysed profit determinants, the economic feasibility and viability of such small-holder projects and suggested options for improvement of the programme. materials and methods study area and data collection the study was approved by the ethical committee of the college of agriculture and environmental sciences, unisa (caes) with an ethical approval number: 2013/caes/140. a recent document targeting small-holder farmers indicated that at least 5889 small-holder farms exist in mpumalanga (daff 2013; figure 1). we used this number as the sample frame. the sample size was calculated for frequency using the formula: figure 1: map of mpumalanga with demarcation of agro-ecological zones. where, population size (for finite population correction factor or fpc) (n): 5889; hypothesised % frequency of outcome factor in the population (p): 50% ± 5; confidence limits as % of 100 (absolute ± %) (d): 5%; design effect (for cluster surveys-deff): 1. a total of 361 farms were needed. we continued to recruit small-holder pig farms randomly until we could identify no more farms of interest. a final list of 220 farmers was generated from the list provided by dardlea and the additions were made through consultations with farmers, extension officers, animal health technicians and community leaders. all identified farmers were visited and data were collected through the use of a semi-structured pretested questionnaire. direct observations were evaluated through a checklist, and photographic documentation was obtained, where necessary. the services of the extension officers and animal health technicians from dardlea, who were previously trained on questionnaire administration, were employed. the inclusion criteria were as follows: (1) ownership of ≥ 1 to ≤ 50 pigs and (2) resident within the province and active in the small-holder industry. the english questionnaire was translated and administered using local home languages (zulu, isindebele, shangaan and isiswati) for the understanding of the study participants. statistical analyses and management all responses were entered into microsoft excel 2007® spreadsheet and filtered. data were analysed using stata v9 (statacorp., texas, usa) and hypotheses were tested using appropriate analytical methods. to determine associations, all data were re-entered as 1 = yes and 0 = no and coded correctly for the stata programme. using pearson’s chi-square test, outputs were generated to associate certain variables and preferred methods, including markets, market determinants, treatment methods for sick pigs, feed preference, body conditions of the sows and age at weaning. to integrate economic analyses, a partial budgeting and return-on-investment (roi) model was developed in a microsoft excel 2007® spreadsheet. outcomes from the data obtained, including details from the field and published materials, were used to develop and validate the model. economic feasibility and viability of a 10-sow unit was tested for a 3-year farm operation. details of the inputs and outputs are available in the appendix. the sensitivity analyses were tested by varying some parameters, including the reduction in feed price, removal of farmer’s remuneration, transport cost and reduction or preweaning deaths. outputs were generated in tables and graphs, and the model is freely available in excel format for the use of small-holder farmers and development partners (see appendix). results descriptive statistics approximately 41% of the farms surveyed confirmed that the sows farrowed ≥ 11 piglets per litter and more than 58% farrowed ≤ 10/sow/litter. only about 19% weaned at 1 month (industry standard) and only 11% depended on commercial feed completely (table 1). the majority of the farmers mixed commercial feed with swill (41.6%) or fed swill and leftovers alone (47%), and about 69% resorted to home medication, allowed the animal to die or sent any sick animal for slaughter (table 1). only 27% sold their porkers at less than 6 months, whereas the majority (73%) marketed their pigs above 7 months. less than 10% of all sows were in adequate body condition (at least a score of 3). the prevailing local and market prices were the main determinant for marketing pigs and incomes arising from the sale were used mainly in the home or to maintain the remaining pigs (table 1). table 1: profit and market-related variables of small-holder farmers, mpumalanga. using the pearson’s chi–square test, the prevailing market price significantly influenced the preference for abattoirs (χ2 = 8.96, p < 0.005), auctions (χ2 = 135.51, p < 0.0001) and local slaughter slabs (χ2 = 72.71, p < 0.0001) as a means of disposal of final products (table 2). similarly, the prevailing local price of product had an influence on sales at auctions (χ2 = 39.74, p < 0.0001) and local slaughter slabs (χ2 = 114.39, p < 0.0001). age of pigs at sale slightly influenced the sale at auctions (χ2 = 6.11, p = 0.01), but significantly influenced sales at local slaughter slabs (χ2 = 28.97, p < 0.0001). there was a significant association between the use of ethno-veterinary preparations and sales at auctions (χ2 = 11.37, p = 0.001) or at local slaughter slabs (χ2 = 7.30, p < 0.01). furthermore, farmers who medicated their pigs themselves disposed off their products at the auctions (χ2 = 6.87, p < 0.01) or at the local slaughter slabs (χ2 = 11.35, p = 0.001) (table 2). finally, only the local slaughter slabs and the slaughter of sick animals were associated (χ2 = 6.58, p = 0.01). table 2: association between preferred methods of marketing, market price determinants and type of treatment for sick animals. there were associations between the feeding of commercial rations solely and poor body condition (χ2 = 9.75, p < 0.005) and poor–fairly good condition (χ2 = 5.46, p < 0.05). farmers who weaned their piglets at 1 month had their sows in very good body condition comparatively (χ2 = 8.55, p < 0.005), whilst those who weaned at about 3 months had good–very good body condition (χ2 = 6.46, p = 0.01) and those who did not wean at all had their sows in poor (χ2 = 8.80, p < 0.005) or good–very good condition (χ2 = 11.56, p = 0.001) (table 3). similarly, there were associations between weaning at 1 month and feeding of commercial ration (χ2 = 19.80, p < 0.0001), mixing of commercial ration and swill (χ2 = 11.47, p = 0.001) and mixing of swill and household remnants alone (χ2 = 10.62, p = 0.001) (table 4). table 3: association between body condition scores, types of feed used and age at weaning. table 4: association between types of feed used and weaning age. significant association existed between production of a larger number of piglets and feeding of commercial ration (χ2 = 11.57, p = 0.001). in contrast, the mixing of swill and commercial ration was associated with the production of low (χ2 = 17.25, p < 0.0001) to medium (χ2 = 23.11, p < 0.0001) numbers of piglets per litter (table 5). the feeding of swill only produced similar significant results (table 5). similarly, sows in poor body condition produced low (χ2 = 6.37, p = 0.01) to medium numbers of piglets per litter (χ2 = 5.44, p = 0.02) (table 5). only sows in very good body condition were associated with a large number of piglets per litter (χ2 = 7.77, p = 0.005). table 5: association between average number of piglets farrowed per sow per litter, types of feed used and body condition scores. economic models using partial budgeting and roi models, the 10-sow unit pig farm continues to utilise more cash (outflow) than the receipts that came into the farm account. feed (using commercial ration) accounted for at least 75% of the annual cash outflow for any 1 year (figure 2a; table 1-a1, table 2-a1, table 3-a1). with a 50% reduction in feed price through supplementation with swill and leftovers from the home, the model became economically viable towards the end of the third year of operation (figure 2b). however, a 100% reduction in feed price through complete replacement with swill would make the farm model break even at the beginning of the second year of operation with subsequent profits (figure 2c). with complete removal of remuneration for the farmer over a 3-year project cycle, the farm was still economically unsustainable (figure 2d) and similar results were obtained with a 60% reduction in transport cost (figure 2e) and improving the farm productivity through a 25% reduction in preweaning mortalities (figure 2f). figure 2: economic evaluation of a 10-sow unit using different scenarios. (a) economic evaluation of a 10-sow unit, south africa, (b) 50% reduction in feed price, (c) 100% removal of feed price, (d) no remuneration for the farmer, (e) transport cost reduction by 60% and (f) preweaning death reduced by 25%. discussion in mpumalanga, there are at least 15 auction facilities that are randomly dispersed. many of the small-holder farmers marketed their pigs at auctions or within the communities. because strong association existed between auctions and prevailing market price, it can be inferred that high pig populations at auctions are indications that the prevailing market prices are good. as such, market price is a driver for moving pigs to auctions. such pigs often evade anteand postmortem inspections and may inadvertently spread infectious diseases. it becomes necessary to identify each auction within mpumalanga and know the farms and road networks that support them so as to plan and apply intervention strategies where and when necessary, for example, in the case of a rapidly spreading animal disease or for surveillance purposes. secondly, market price had some degree of influence in moving pigs to the abattoirs but only a minority (8.3%) preferred this option for marketing. similar results have been reported from limpopo, where farmers travelled a long distance to obtain higher prices primarily at auctions and at abattoirs (mokoele et al. 2014). furthermore, because of the association that exists between local price and local markets (table 2), we inferred that the local market is highly influenced by prevailing local price, with minor influence from the age and sex of the pig. primarily, sick, unthrifty pigs, mature boars and late-maturing pigs are slaughtered locally and according to our results, only the local slaughter slabs were associated with the slaughter of sick animals. comparatively, prevailing market prices are higher than the local price range and better quality products are often sent to the auctions (51%) and commercial abattoirs (8.3%). it is known that pork from mature boars has ‘boar taint’, a pheromonal smell (androstenone and skatole), and generally attracts much lower prices compared with other pork. in addition, slow-growing, late-maturing pigs are characteristic of most small-holder pig producers, an indication that they will be presented for slaughter at later age. it is therefore not surprising that local market and age of pigs are associated. although a positive association exists between auctions and local slaughter slabs and home medication and ethno-veterinary usages, similar qualitative evaluations have revealed that small-holder pig farmers who sell at auction and locally tend to medicate the pigs themselves with ethno-veterinary preparations and also to use, sometimes incorrectly, long-acting oxytetracycline and ivermectin (fasina et al. 2012; mokoele et al. 2014). although dardlea has public veterinarians and animal health technicians in all the districts and municipalities within the province who provide free services to small-holder farmers, it appeared that small-holder pig farmers lacked information about veterinary services in the province, or intentionally refused to seek veterinary assistance; these farmers hardly consult veterinarians and para-veterinary professionals (table 2). reasons for this disconnect must be established and a drive to gain small-holder farmers’ trust and to make service accessible and affordable must be implemented. local slaughter for household consumption and sale within the community was prevalent in our study group (64%). this poses a high risk to animal and public health because preslaughter pig inspections are neglected, as highlighted above. whereas local slaughter for community sale is prohibited by law, slaughter for household consumptions is allowed under south african law (anonymous 2000 [meat safety act no. 40 of 2000]). there is an insignificant degree of association between auctions and sex of animals and the reason for this observation is evident. top quality breeding boars are priced beyond the reach of small-holder farmers and good commercial boars are equally expensive. as such, the small-holder farms often settle for lower quality boars sourced from auctions. this practice exposes the small-holder farms to risks of infectious animal diseases and the genetic value of the boars is doubtful. it will be important for agricultural authorities to revise their strategies, evaluate these gaps and devise means of addressing them. a good interim measure may be the creation of district and municipal pig-breeding centres to multiply quality genetics and distribute them to small-holder farmers at minimum costs. such an intervention would carry additional benefits of employment generation and provision of training in animal production, animal health and biosecurity. although it was established that the feeding of commercial rations alone is significantly associated only with poor to fairly good body condition, it was also established that such feeds are often rationed and pigs were underfed because of cost. however, results showed that well-conditioned animals tend to produce larger numbers of piglets per litter and early weaning was positively associated with good condition across the panel of feeding. the combination of good quality commercial ration supplemented with risk-free cooked swill may offer alternative feeding strategies in small-scale pig production. using partial budgeting and roi, the 10-sow unit pig farm is economically unsustainable with commercial feed. it appeared that the main driver of profitability in small-holder pig farms is the feed cost. whereas the profitability of a pig production unit increases with an increase in the number of live-born piglets per litter (kyriazakis & whittemore 2006), our results only partially agreed with this assertion. even when the production efficiency increased and preweaning mortality was reduced by 25%, a 10-sow pig production unit was still not able to break even in this analysis. feed is a pig farm input that is indisputably of utmost importance (kyriazakis & whittemore 2006); our analyses have confirmed this, as it was the most important determinant of profitability in small-holder farms. although using commercially compounded ration by farmers should yield better quality products and improve reproduction and overall production, at this scale of production it was not financially feasible and viable. previous studies have highlighted some of the reasons for the relatively high feed cost and suggested reasons why small-holder pig farmers rely on swill as an alternative form of feed (phengsavanh et al. 2010; roelofse 2013). we have similarly concluded that because of the infeasibility of feeding commercial ration, small-holder farmers will continue to feed swill and alternative feed sources for the unforeseeable future. we are aware that it is potentially possible for small-holder pig farmers to make profit (lapar & staal 2010; petrus et al. 2011; phengsavanh et al. 2011) and have demonstrated that in this economic analysis. however, the ≤ 10-sow unit, small-holder farmers in mpumalanga were able to achieve profitability mainly through the use of swill as a major source of feed. this practice is common to most small-holder pig farms elsewhere in south africa (gcumisa 2013; roelofse 2013). the feeding of swill comes with potential risk of spread of diseases to pigs (e.g. salmonellosis, campylobacteriosis, african swine fever, classical swine fever, porcine reproductive and respiratory syndrome and foot and mouth disease) and possible transmission of zoonotic diseases from pigs to humans (beltrán-alcrudo et al. 2008; daff 2005; haynes 2001). in addition, pig products originating from swill feeding may not reach the quality required by south african pig abattoirs. in view of the above risks and knowing that it is more realistic for small-holder farmers to make a profit with units of between 50 and 100 sows (roelofse 2013), we suggest that the agricultural authorities should assist farmers in the development of community self-help groups and farmers’ cooperatives. such calls have been made previously (munyai 2012) and the successes associated with such organisations by small-holder pig farmers have been documented in namibia (petrus et al. 2011), vietnam (lapar & staal 2010) and lao people’s democratic republic (phengsavanh et al. 2011). such cooperative organisations have the advantages of bulk purchase of feed with benefits of economies of scale and discounts (costales et al. 2007; lapar & staal 2010), reduced transport tariffs through bulk transport and better negotiating power. in addition, because an improvement in efficiency and reduction of preweaning loss by about 25% will improve profitability, it is necessary to implement the measures required to achieve these objectives. the government may also consider tax rebates on animal feed products that are directed to small-holder pig farmers. only through the combination of the above measures and interventions will small-holder farmers with ≤ 10-sow units be able to break even and use pig production as a means of poverty alleviation. conclusion small-holder pig production and health management will continue to be relevant in an emerging economy like south africa. however, for government agricultural interventions to provide the desired benefits, empirical evaluations for technical and economic feasibilities must be carried out. although a 10-sow unit is technically feasible, in mpumalanga and elsewhere in south africa, the current input systems negate the benefits that should come with such programmes. proposed models and revisions as suggested above may facilitate government interventions and make pig production more attractive to small-holder farmers. acknowledgements we thank the mpumalanga department of agriculture, rural development, land and environmental affairs (dardlea) for provision of access to the farmers. we thank the unisa m & d bursary for funding the work. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions p.m. was the project leader, f.o.f. supervised and organised the study and j.o. co-supervised the project. p.m. and f.o.f. prepared data, f.o.f. analysed the data and interpreted the data. p.m., f.o.f. and j.o. contributed to the drafting and submission of this article. references absa agri trend pork price, 19 june 2015, macro-economic outlook for commodity exports, viewed 02 july 2015, from http://www.absa.co.za/deployedfiles/absacoza/pdfs/commercial/sector%20focus/agribusiness%202015/06.june/agri%20trends%2019%20june%202015%20.pdf anonymous, 2000, meat safety act no 40 of 2000, viewed 10 july 2015, from http://www.acts.co.za/meat-safety-act-2000/ beltrán-alcrudo, d., lubroth, j., depner, k. & de la rocque, s., 2008, ‘african swine fever in the caucasus’, fao empres watch, pp. 1–8, viewed 12 june 2015, from http://www.fao.org/3/a-aj214e.pdf costales, a., delgado, c.l., catelo, m.a., lapar, m., tiongco, m., ehui, s.k. et al., 2007, scale and access issues affecting smallholder hog producers in an expanding peri-urban market, southern luzon, philippines, international food policy research institute, washington, dc. department of agriculture, forestry and fisheries (daff), 2005, the department of agriculture annual report 2004/5, national regulatory services (programme 7), viewed 06 july 2015, from http://www.nda.agric.za/docs/annual2005/natregser.pdf daff, 2012, a profile of the south african pork market value chain 2012, viewed 12 june 2015, from http://www.nda.agric.za/docs/amcp/pork2012.pdf daff, 2013, updated report on smallholder development working group meetings 2011/2012 to 2012/2013, viewed 17 october 2015, from http://www.nda.agric.za/doadev/sidemenu/smallholder/docs/small%20holder%20development%20meetings_print.pdf department of labour, 2015, minimum wage for farm worker sector with effect from 1 march 2014, viewed 02 july 2015, from http://www.labour.gov.za/dol/downloads/legislation/sectoral-determinations/basic-conditions-of-employment/2014farmworkerwages.pdf dietze, k., 2011, pigs for prosperity, diversification booklet number 15, food and agriculture organization of the united nations, rome, viewed 25 june 2015, from http://www.fao.org/docrep/015/i2471e/i2471e00.pdf drucker, a.g. & anderson, s., 2004, ‘economic analysis of animal genetic resources and the use of rural appraisal methods: lessons from southeast mexico’, international journal of agricultural sustainability 2(2), 77–97. fao, 2004, state of food insecurity in the world, monitoring progress towards the world food summit and millennium development goals, food and agriculture organization of the united nations, rome. fasina, f.o., lazarus, d.d., spencer, b.t., makinde, a.a. & bastos, a.d., 2012, ‘cost implications of african swine fever in smallholder farrow-to-finish units: economic benefits of disease prevention through biosecurity’, transboundary and emerging diseases 59(3), 244–255. gcumisa, s.t., 2013, ‘the untold story of the pig farming sector in rural kwazulu-natal: a case study of uthukela district’, master of agriculture dissertation, university of south africa. haynes, n.b. (ed.), 2001, keeping livestock healthy, 4th edn., storey publishing, north adams, ma. kyriazakis, i. & whittemore, c.t. (eds.), 2006, whittemore’s science and practice of pig production, 3rd edn., blackwell publishing, oxford. lapar, l. & staal, s., 2010, competitiveness of smallholder pig producers in vietnam. improving the competitiveness of pig producers in vietnam, project brief 5, international livestock research institute, nairobi. makiwane, m., makoae, m., botsis, h. & vawda, m., 2012, ‘a baseline study on families in mpumalanga’, human sciences research council, pretoria: human and social development, population health, health systems and innovation, cestii, viewed 02 july 2015, from file:///c:/users/user/downloads/mpumalanga%20family%20study%2030-07-2012.pdf mhlanga, f., 2002, community-based management of animal genetic resources: a participatory approaches framework, viewed 25 june 2015, from https://cgspace.cgiar.org/bitstream/handle/10568/3690/mhlanga.pdf?sequence=1 mokoele, j.m., janse van rensburg, l., van lochem, s., bodenstein, h., du plessis, j., carrington, c.a.p. et al., 2015, ‘overview of the perceived risk of transboundary pig diseases in south africa’, journal of the south african veterinary association 86(1), art. #1197, 9 pages. http://dx.doi.org/10.4102/jsava.v86i1.1197 mokoele, j.m., spencer, b.t., van leengoed, l.a.m.g. & fasina, f.o., 2014, ‘efficiency indices and indicators of poor performance among emerging small-scale pig farmers in the limpopo province, south africa’, onderstepoort journal of veterinary research 81(1), art. #774, 12 pages. http://dx.doi.org/10.4102/ojvr.v81i1.774 mpumalanga provincial government (mpg), 2009, mpumalanga economic profile, viewed 04 july 2015, from http://www.mpumalanga.gov.za/dedt/economic%20profile/mpu_econ_vol5_01_jul_2010.pdf mpg, 2013, socio-economic review and outlook of mpumalanga – june 2013, viewed 04 july 2015, from http://finance.mpu.gov.za/documents/ea.sero.june.2013.pdf munyai, f.r., 2012, ‘an evaluation of socio-economic and biophysical aspects of small-scale livestock systems based on a case study from limpopo province: muduluni village’, phd thesis, department of animal, wildlife and grassland science, university of the free state. petrus, n., mpofu, i., schneider, m. & nepembe, m., 2011, ‘the constraints and potentials of pig production among communal farmers in etayi constituency of namibia’, livestock research for rural development, 23 (7), viewed 15 june 2015, from http://www.lrrd.org/lrrd23/7/petr23159.htm phengsavanh, p., ogle, b., stür, w., frankow-lindberg, b.e. & lindberg, j.e., 2010, ‘feeding and performance of pigs in smallholder production systems in northern lao pdr’, tropical animal health and production 42(8), 1627–1633. phengsavanh, p., ogle, b., stür, w., frankow-lindberg, b. & lindberg, j., 2011, ‘smallholder pig rearing systems in northern lao pdr’, asian-australasian journal of animal sciences 24(6), 867–874. roelofse, j.j.h., 2013, ‘economic feasibility study of the establishment of smallholder pig farmers for the commercial market: empolweni case study’, master of industrial engineering dissertation, stellenbosch university. appendix 1 table 1-a1: project cash flow statement for a model 10-sow unit, mpumalanga, 2015 (year 1). table 2-a1: project cash flow statement for a model 10-sow unit, mpumalanga, 2016 (year 2). table 3-a1: project cash flow statement for a model 10-sow unit, mpumalanga, 2017 (year 3). abstract introduction materials and methods results discussion and conclusion acknowledgements references about the author(s) salah meradi institute of veterinary sciences and agronomic sciences, university of batna 1, algeria jacques cabaret national institute for agricultural research (inra), françois-rabelais university, france bourhane bentounsi institute of veterinary sciences, university of constantine 1, algeria citation meradi, s., cabaret, j., bentounsi, b., 2019, ‘sheep enteric cestodes and their influence on clinical indicators used in targeted selective treatments against gastrointestinal nematodes’, onderstepoort journal of veterinary research 86(1), a1648. https://doi.org/10.4102/ojvr.v86i1.1648 original research sheep enteric cestodes and their influence on clinical indicators used in targeted selective treatments against gastrointestinal nematodes salah meradi, jacques cabaret, bourhane bentounsi received: 29 apr. 2018; accepted: 19 dec. 2018; published: 28 feb. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract clinical indicators such as diarrhoea (disco) or anaemia (famacha©) are used as a measure for targeted selective treatments against gastrointestinal nematodes (gin). enteric cestodes such as moniezia may interfere directly with disco or indirectly with the famacha© score. we investigated 821 ouled djellal rams naturally infected in a steppe environment (gin alone, cestodes alone, gin and cestodes) or not. the rams were treated with ivermectin 2 months before being slaughtered to reduce the impact of nematodes on the clinical scores; however, persistent or newly acquired gins were not related to both scores. of the non-infected rams (n = 296), 26% identified as needing treatment against gin using the famacha score, and 34.5% using disco would have been thus selected. this implies that the clinical indicators used for the targeted selective treatment of gastrointestinal nematodes are not fully reliable when a low infection is recorded and may well be influenced by confounding factors. as expected, only disco was affected by cestode infection, and we suggest that the presence of moniezia should also be taken into consideration. introduction a wide diversity of opinions exists regarding pathogenicity of cestodes in sheep. cestodes from the genus moniezia are regarded as more pathogenic in young lambs, whereas other cestode genera also recorded in adult sheep are either considered less pathogenic (avitellina) or virtually non-pathogenic (stilesia) (soulsby 1982). studies by elliott (1984) did not consider moniezia as a cause of diarrhoea in sheep. conversely, cabaret et al. (2006) found a relationship between diarrhoea in lambs and the presence of moniezia. there is thus a real need to evaluate the influence of cestodes on sheep health and production, especially as they have marked their concomitant presence with gastrointestinal nematodes (gin) in steppe regions in eastern algeria (according to unpublished data from our laboratory). owing to the spread of anthelmintic resistance in gin, targeted selective treatments are being undertaken only for animals identified as sick to reduce the use of anthelmintics. this is based on the fact that in a flock, the populations of gastrointestinal parasites in small ruminants are highly aggregated and dispersed (gaba, ginot & cabaret 2005). performance indicators such as body weight (cottle 1991; stafford, morgan & coles 2009), milk production (hoste et al. 2002) or the use of clinical indicators, such as anaemia score using the famacha© system (van wyk & bath 2002), dag score (larsen et al. 1994) and the diarrhoea score disco (cabaret et al. 2006) were utilised for gin. disco was found to be the most relevant indicator in the steppe regions of eastern algeria and morocco (bentounsi, meradi & cabaret 2012; ouzir et al. 2011), where teladorsagia circumcincta, nematodirus and marshallagia marshalli were the most prevalent species, and haemonchus contortus was occasionally reported (bentounsi, meradi & cabaret 2012; meradi et al. 2011; ouzir et al. 2011). famacha© scores and body weight have not been reliable indicators for detecting lambs in need of treatment in our conditions (bentounsi, meradi & cabaret 2012). the clinical impact of enteric cestodes is poorly known and could bias the selection of sheep to anthelmintic treatment against gin. it was evaluated in this paper using clinical indicators of diarrhoea (disco) and anaemia (famacha©) obtained before slaughter of selected rams previously treated with ivermectin to also eliminate, in part, the bias owing to gin. it was expected that the diarrhoea score would be influenced and that the famacha© score would remain unchanged. the evaluation of these hypotheses was undertaken in rams in eastern algeria. materials and methods study site and experimental animals this study was carried out between november 2015 and february 2016 in sheep slaughtered at batna abattoir in eastern algeria. experimental animals were rams aged between 12 and 14 months, the accepted age for slaughter. the rams investigated in the present paper are of ouled djellal breed, grazed under an extensive system in the region of batna, which is characterised by a steppe climate. it is local practice to treat animals with subcutaneous injections of ivermectin 2 months before slaughter although they remain on their usual pastures. in our study, only treated rams were selected. a total of 821 rams were examined. famacha© and disco investigations the famacha© system is based on a semi-quantitative evaluation of the eye mucosal colour, which is classified into one of five categories on a colour chart, indicating a level of anaemia; 1 (red, non-anaemic) to 5 (white, severely anaemic) (van wyk & bath 2002). the disco indicator categorises a faecal sample according to the following consistency index; 1 (corresponds to normal sheep faeces in pellets), 2 (corresponds to ‘soft’ faeces [similar to cow pat]) and 3 (corresponds to diarrhoea [semi-liquid faeces]). these scores correlate to 40%, 25% and 15% dry matter in faeces, respectively (cabaret et al. 2006). these indicators (famacha© and disco) and faecal samples were taken from each animal in lairage at the slaughterhouse. animals under transport stress, with discomfort or that had conjunctivitis were excluded from scoring and faecal sampling. parasitological investigations faecal samples were collected per rectum to estimate gin. the gastrointestinal infection intensity was estimated by the number of nematode eggs per gram of faeces (epg) using a modified mcmaster method with a saline solution (1.18 specific gravity) sensitive to 15 epg (raynaud 1970). in addition, a flotation method was performed at a sensitivity of 7.5 epg (raynaud 1970) when the mcmaster result detected no eggs. the eggs were identified to genus level only, as marshallagia and nematodirus, and other gin. after slaughter, the small intestine was washed and the content was filtered through a sieve (250 µm mesh). cestodes were collected and transported to the parasitology laboratory, where the scoleces were counted and proglottids examined. the number of scoleces was used as an estimation of the number of parasites. samples from strobila of each cestode were taken and preserved in 10% formalin. cestodes were examined on a slide with cover glasses under a light microscope. the proglottids, after clearing and staining with acetic carmine, were identified on morphology (soulsby 1982). prevalence (% of ram infected) and abundance (average number of worms among rams) were calculated (bush et al. 1997). statistical analysis the data were analysed using the statistical package spss version 20. the univariate analyses of variance were performed to assess the significant differences in the famacha© score and disco in relation to infection groups. non-parametric spearman coefficient of correlation (rs) between clinical indicators and infection were also calculated. ethical considerations the experiment was approved by the local ethical committee for experimental animals in algeria and was conducted in compliance with the universal ethical standards. results the helminth fauna the overall prevalence of cestodes in rams studied in the region was 9.9%. the cestodes in decreasing order of prevalence were moniezia expansa (5.18%), avitellina centripunctata (3.35%), stilesia globipunctata (0.97%) and moniezia benedeni (0.36%). the prevalence of gin was 57.1%. the epg was low (see table 1). four parasitological groups could be established: no infection (296 rams), cestode infection only (32 rams), nematode infection only (444 rams) and cestode and nematode infection (49 rams) (see table 2). table 1: distribution of gastrointestinal nematode faecal egg counts in an abattoir (821 rams) in eastern algeria showing faecal egg count, prevalence (%p) and nematode categories. table 2: clinical indicators in relation to parasitological groups of rams indicating mean abundance ± standard deviation (ma ± sd). clinical indicators (famacha© and disco) the famacha© scores extended from 1 to 4 and were found in 42.9%, 37.8%, 18.4% and 0.9% of the rams, respectively. the disco values (from 1 to 3) were 62.7%, 28.5% and 8.8% of the rams, respectively. relationship between type of infection and the two clinical indicators disco was significantly (p = 0.00l) lower in the non-infected group and in the group with nematode infection only, and significantly (p = 0.0001) higher in the group with cestode infection only. in the group harbouring cestodes and nematodes, disco correlated with the number of cestodes (rs = 0.67; p = 0.0001) but not with nematode epg (rs = 0.25; p = 0.09). the group harbouring nematodes only had a significantly lower famacha© score (p = 0.003) (see table 2). clinical indicators could select the false-positive rams for treatment seventy-seven rams in the non-infected group (296 rams) had a famacha© score ≥ 3, which is often taken as an indication for treatment against gin; however, no infection could be detected. disco 2 or disco 3 values indicate necessity for treatment. disco 2 or disco 3 were found in 86 and 16 rams, respectively, from a total of 296 rams. by using clinical indicators as the only indication of a nematode infection, 77 and 102 rams would have been incorrectly treated as false positives. discussion and conclusion this low prevalence of enteric cestodes seems to be related to the steppe climate (cold semi-arid) of the region, which is unfavourable for the development of oribatid mites, intermediate hosts of cestodes (denegri 2001). it is also probably because of the age of the sheep slaughtered (soulsby 1982), as they were slightly over 1 year of age. similar prevalences within sheep of similar age groups have also been reported in turkey (4.43%), according to aydenizoz and yildiz (2003), and in several regions in eastern algeria such as el oued (13.2%), constantine (13.3%) and mila (12.1%) (bentounsi & meradi 2016). the very low infection rate of gin is related to the treatment given earlier: the rams are treated mainly with ivermectin 2 months before slaughter. nevertheless, they remained infected as some gin are resistant (bentounsi et al. 2007) or they become re-infected. use of these clinical indicators, designed to detect high infection levels, could leave low infections undetected. in the present study, disco was associated with cestode infection as previously shown for moniezia by cabaret et al. (2006). as expected, the clinical indicator, famacha© score, was not influenced by cestode infection and thus remains a good indicator for some gin (mainly haemonchus sp.) whatever the infection with cestodes. disco is possibly a more general clinical indicator, being susceptible to vary to different pathogens (coccidia, gin or moniezia among parasites), and thus requires parasitological diagnostics to be more effective in managing gastrointestinal tract infections. acknowledgements the authors are greatly indebted to the heads of the batna abattoir, idir kamel and mallem khalid, for their help in realising this work. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.m. conducted the research. b.b. conceived and directed the project and j.c. analysed the data. all authors have written and approved the final manuscript. references aydenizoz, m. & 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edn., baillière tindall, london. stafford, k.a., morgan, e.r. & coles, g.c., 2009, ‘weight-based targeted selective treatment of gastro-intestinal nematodes in a commercial sheep flock’, veterinary parasitology 164, 59–65. https://doi.org/10.1016/j.vetpar.2009.04.009 van wyk, j.a. & bath, g.f., 2002, ‘the famacha© system for managing haemonchosis in sheep and goats by clinically identifying individual animals for treatments’, veterinary research 33, 437–640. https://doi.org/10.1051/vetres:2002036 abstract introduction materials and methods results discussion acknowledgements references about the author(s) regina d. miambo department of para-clinics, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique school of life science, college of agriculture, engineering and science, university of kwazulu-natal, westville, south africa benigna laitela department of para-clinics, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique mokgadi p. malatji school of life science, college of agriculture, engineering and science, university of kwazulu-natal, westville, south africa sonia m. de santana afonso department of para-clinics, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique alberto p. junior department of para-clinics, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique johan lindh department of cell and molecular biology, uppsala university, uppsala, sweden samson mukaratirwa school of life science, college of agriculture, engineering and science, university of kwazulu-natal, westville, south africa citation miambo, r.d., laitela, b., malatji, m.p., de santana afonso, s.m., junior, a.p., lindh, j., et al., 2019, ‘prevalence of giardia and cryptosporidium in young livestock and dogs in magude district of maputo province, mozambique’, onderstepoort journal of veterinary research 86(1), a1709. https://doi.org/10.4102/ojvr.v86i1.1709 research communication prevalence of giardia and cryptosporidium in young livestock and dogs in magude district of maputo province, mozambique regina d. miambo, benigna laitela, mokgadi p. malatji, sonia m. de santana afonso, alberto p. junior, johan lindh, samson mukaratirwa received: 15 oct. 2018; accepted: 11 feb. 2019; published: 12 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: giardia and cryptosporidium species are significant zoonotic parasites of humans and domesticated animals. objectives: the study aimed to determine the prevalence of giardia and cryptosporidium in livestock and dogs of the magude district. method: the flotation technique (willis), modified ziehl-neelsen (mzn) and direct and indirect immunofluorescence (dif and iif) techniques were applied to determine the prevalence of giardia and cryptosporidium species in faecal samples of dog pups (156), goat kids (60) and calves (480) from the magude district of mozambique from february to september 2015. results: using willis, iif and dif, the prevalence of giardia in calves was 0%, 8.1%, and 6.0%; in dogs 0.6%, 8.3% and 5.7% and for goats 0% and 13.3% (iif was not performed), respectively. the prevalence of cryptosporidium in calves using willis, mzn, iif and dif was 0%, 3.8%, 4.7% and 0.4% and in dogs 0%, 0.6%, 6.4% and 0.6%, respectively. the parasite was not detected in goats. conclusion: results from the present study showed that iif performed better diagnosis of giardia and cryptosporidium, and that the mzn can be used as an alternative for cryptosporidium because of the high cost of iif. there is a need for identification of genotypes or subtypes of these parasites through application of molecular techniques in order to determine their zoonotic potential, and we advocate a ‘one health’ approach in the control and prevention of these parasites. keywords: zoonoses; giardia; cryptosporidium; dogs; calves; goats; mozambique. introduction protozoan species from the genus giardia and cryptosporidium are known to infect domestic and wild animals (taylor, coop & wall 2007) and are implicated as causative agents of diarrhoea in children, and as opportunistic infections in hiv-positive patients (fayer, morgan & upton 2000; irisarri-gutiérrez et al. 2017; morgan et al. 2000; pedersen et al. 2014; sow et al. 2016; wang et al. 2018). in domestic animals, the parasites are mainly prevalent in neonates and young animals (baroudi et al. 2018; de waal 2012; hamnes et al. 2006) with consequent economic loss because of different levels of morbidity and mortality (de graaf et al. 1999) particularly when they occur in concomitant infections with helminthic infections (taylor et al. 2007). there has been a description of two subtypes of cryptosporidium parvum (baroudi et al. 2018; fayer et al. 2000; santana et al. 2018; squire et al. 2017) and multiple genotypes within the species giardia duodenalis (ebner et al. 2015; feng & xiao 2011; itagaki et al. 2005; santín, trout & fayer 2007; sommer et al. 2018), and only a few are of zoonotic significance. depending on the purpose of the study, different techniques can be applied for the diagnosis of giardia and cryptosporidium. direct smears with or without staining and concentration techniques are mainly used routinely in the laboratory, and despite the relatively low cost, they have a disadvantage of low sensitivity (cheesbrough 1987; de waal 2012). in view of this limitation, immunological techniques based on the detection of antigens such as enzyme-linked immunosorbent assay (elisa), the immunofluorescence (if) staining method and the molecular test polymerase chain reaction (pcr) which detects the parasite deoxyribonucleic acid (dna) have been applied in epidemiological studies, and they have proved to be more sensitive and specific (geurden et al. 2008; gómez-couso, méndez-hermida & ares-mazás 2006; soares & tasca 2016). studies conducted in mozambique have reported prevalence of 8.1% for giardia intestinalis and 7.1% for cryptosporidium spp. in humans (irisarri-gutiérrez et al. 2017). mixed helminths infections of toxocara canis and ancylostoma spp. in dogs were reported by cruz and silva (1971) and santos, nhantumbo and alho (2013); however, there was no reference to giardia and cryptosporidium spp. the objective of this study was to determine the prevalence of giardia and cryptosporidium in dogs, cattle and goats in the magude district of maputo province, mozambique. materials and methods study area the study was conducted between february and september 2015 in the localities of the magude district (figure 1), maputo province, mozambique, namely magude sede, motaze, mapulanguene, panjane and mahele. the climate in the study area is dry sub-tropical, with an annual temperature average of 22 °c – 24 °c and the annual rainfall average of 630 mm (mae 2005). livestock production and agriculture associated with animal traction are the main livelihoods of the community (ine 2009). in the district, cattle are reared extensively, goats are housed at night and released in the morning to the communal grazing areas, and dogs are bred freely with many of them trained to shepherd cattle in grazing areas. figure 1: map of magude district and localities where dog and livestock samples were collected. sample collection and laboratory analysis a total of 696 faecal samples were collected from the rectum of calves (n = 480), goat kids (n = 60) and dog pups (n = 156) all less than 7 months of age using a latex glove. animals belonging to households pre-identified by the local veterinary technician were randomly selected at dip tanks during vaccination campaigns for calves, goats and dogs and a consent form was sought by each owner before sample collection. sample consistence was classified as normal (soft to hard) or diarrheic (watery) and then transferred into tubes with caps which were labelled with individual details of each animal (animal species, age if possible and identification number) and transported in a cooler box to the parasitology laboratory, faculty of veterinary medicine, eduardo mondlane university in maputo, mozambique, and kept at 4 °c until processed. a questionnaire was designed for dog owners and livestock farmers to collect information regarding animal husbandry, housing conditions, drinking water sources, feeding, treatment against parasitic infections and use of faeces in agricultural practices. copromicroscopic analysis faecal samples were processed for the detection of giardia and cryptosporidium (oo)cysts using the willis flotation technique as described by ueno and gonçalves (1998). identification of (oo)cysts was performed using morphological characteristics as described by taylor et al. (2007). to concentrate the (oo)cysts in faecal samples, the formol-ether technique was used as described by cheesbrough (1987). the pellet obtained from the concentration was used to prepare thin smears that were stained by the modified ziehl-neelsen (mzn) method as described by cheesbrough (1987) and observed under an optical microscope at 100× magnification for the presence or absence of cryptosporidium oocysts. the remainder of the pellet was transferred to eppendorf tubes and preserved at -20 °c for further processing for the detection of cryptosporidium and giardia by direct and indirect if tests (dif and iif). direct and indirect immunofluorescence the dif test was carried out using a kit (merifluor® cryptosporidium or giardia; meridian diagnostic, united states [us]) according to the manufacturer’s specification. all dog and goat samples were analysed; however, because of the lack of resources, only 237 calf samples were randomly selected and analysed by this technique. the iif test was conducted in all faecal samples except those from goats because the secondary antibody was derived from goats. for this technique, 25 µl of concentrated faeces by formol-ether method was transferred to an if slide. this was left to dry for approximately 5 minutes and fixed with absolute methanol. approximately 50 µl of primary antibody (anti-cryptosporidium parvum mab, abnova and anti-giardia lamblia pab, abnova, europe) diluted in (3%) bovine serum albumin (bsa) in phosphate-buffered saline (pbs) (1:500) was added to the smear, incubated for 1 h in a wet chamber and then washed three times in pbs tween-20 (0.05%). one drop of the secondary antibody coupled to fluorescein (goat pab to cryptosporidium parvum oocyst and giardia cysts or fitc, abcam) diluted in 3% bsa in pbs (1:1000) was added to the smear, incubated in the dark for 30 min and washed three times to remove the excess of fluorescein. to obtain the optimal dilution of the secondary antibody, serial dilutions were made starting from 1:10. a mounting reagent was added to the slide, covered with a coverslip and observed under a fluorescence microscope (100×). data analysis a sample was considered positive if at least one cyst or oocyst of giardia or cryptosporidium was identified in the slide. the prevalence (%) was calculated as the number of positive samples divided by the total number of samples analysed multiplied by 100 (thrusfield 1999). to analyse differences in the prevalence of giardia and cryptosporidium among the localities of the magude district, a general linear multivariate model was applied in statistical package for the social sciences (spss) version 20.0 and p < 0.05 was considered to be statistically significant. medicalc software was used to calculate the sensitivity and specificity of mzn and iif with the dif test used as a gold standard. ethical considerations this research was approved by the scientific board of the veterinary faculty, eduardo mondlane university, maputo, mozambique. results giardia cysts were detected in calves, young goats and pups and cryptosporidium oocysts in calves and dogs, as shown in table 1. prevalence values of giardia and cryptosporidium were high according to the iif test in pups (8.3%, ci: 8.0–8.5) (6.4%, ci: 6.1–6.6) and calves (8.1%, ci: 7.9–8.3) (4.7%, ci: 4.5–4.8), respectively. following this technique, the prevalence of giardia by the dif test was 5.7% (ci: 5.4–5.9) in pups, 6.0% (ci: 5.8–6.2) in calves and cryptosporidium by the mzn test in calves was 3.8% (ci: 3.6–3.9). in general, high prevalence values of giardia spp. and cryptosporidium spp. were recorded in the locality of magude sede for calves and dogs by iif and the lower in the locality of mahele for giardia, and this association was significant (p < 0.05) as represented in table 1. neither of these parasites was detected in dogs from motaze and mahele. the prevalence rate of giardia spp. in goat kids from motaze and magude sede was the same (6.66%) and in other localities, no positive was detected in this animal species. table 1: prevalence (%) of giardia spp. and cryptosporidium spp. in calves, goat kids and dog pups in different localities of the magude district, mozambique. all samples collected in calves and goat kids had normal consistency, whilst three of the 156 samples from pups were diarrhoeic (1.9%) and from these, only one was positive for giardia trophozoites (0.6%) by the willis method. the sensitivity and specificity of iif, willis and mzn are presented in table 2. the mzn method showed high sensitivity (100%) and specificity (96.20% and 100%) to detect cryptosporidium oocysts in calves and dogs, respectively. the iif method showed high sensitivity and specificity to both parasites, the sensitivity ranging between 88.89% and 100%, and the specificity between 95.38% and 98.51% for giardia spp.; with a sensitivity of 100% and specificity of 93.15% and 93.9% for cryptosporidium spp. table 2: sensitivity and specificity (95% confidence interval) of diagnostic tests used in the detection of cryptosporidium oocysts and giardia cysts in dogs and calves from the magude district of mozambique. other gastrointestinal helminths were observed in dog samples by the willis method, namely ancylostoma spp. with a prevalence of 60.3% (ci: 59.8–60.7) followed by toxocara spp. (5.8% [ci: 5.6–5.9]), trichuris vulpis (1.3% [ci: 1.2–1.4]), spirocerca lupi 0.6% ([ci: 0.5–0.7]) and taeniidae (1.9% [ci: 1.8–2.0]). in calves and goats, respectively, strongylid eggs were observed with prevalences of 50.8% (ci: 50.2–51.3) and 31.6% (ci: 30.5–32.6), eimeria spp. with 17.5% (ci: 17.1–17.8) and 41.6% (ci: 40.4–42.7) and moniezia spp. with 3.3% (ci: 3.1–3.4) and 11.6% (ci: 10.8–12.3). discussion the present study focussed on the diagnosis of giardia spp. and cryptosporidium spp. in livestock and dogs using copromicroscopical and immunological tests. this is the first study in mozambique reporting parasites of the genus giardia and cryptosporidium in a mixed farming (cropping and livestock) rural community set-up. the prevalence of 3.75% for cryptosporidium spp. in calves by mzn in the present study is slightly higher than the prevalence reported in calves from 3 to 8 months (1.4%) and lower than the prevalence found in calves less than 3 months (16.6%) in a study made by mtambo et al. (1997) in tanzania using the same diagnostic technique. the categorisation of animals into two groups compared to this study may have caused this discrepancy. on the other hand, on most of the farms sampled in this study, the animals were kept housed, thus increasing the chances of transmission between animals (taylor et al. 2007). the same factor may have contributed to the high prevalence of giardia spp. (49%) and cryptosporidium spp. (12%) found by hamnes et al. (2006) in calves between 3 and 183 days from norway using if tests. in addition, animals from this norwegian study were exposed to lower temperature conditions (between 3.6 °c and 14.0 °c) than from the animals of the magude district (between 18 °c and 35 °c). high temperatures may reduce the viability of oocysts in the environment, whilst at temperatures near to 4 °c, the parasites can remain viable for more than 1 month (adam 2014), hence increasing the risk of infection. in general, the prevalence rates were higher by iif compared to the dif test for both parasites. following the iif method by mzn, the prevalence of cryptosporidium in calves was also high and similar results were reported by mtambo et al. (1992). the high sensitivity of iif compared to mzn was also reported by ortega-mora et al. (1999) where same concentrated faecal samples of ewes were negative when analysed by mzn but positive by iif. diarrhoea is a common clinical sign in animals infected by giardia spp. and cryptosporidium spp. (dawson 2005; o’donoghue 1995). the low incidence of animals with diarrhoea may suggest a low pathogenic significance of these parasitic infections in dog pups and calves in the magude district. besides the low pathogenic significance, other factors associated with the absence of clinical signs in positive animals are: (1) the phase of excretion of (oo)cysts because the peak coincides with the peak of animals with diarrhoea which is between the ages of 8–14 days for cryptosporidium oocysts in cattle (causapé et al. 2002; olson et al. 2004) and between 2 and 4 weeks for giardia (geurden, vercruysse & claerebout 2010); (2) development of an immunological response with the advancing age of animals (huber, bomfim & gomes 2005) and (3) the virulence of the genotype involved (adam 2014). although it was not possible to confirm the species and genotypes of cryptosporidium and giardia based on the techniques used, the zoonotic potential of these parasites should be taken into consideration, especially for cryptosporidium spp. which is an opportunist in immune-compromised individuals such as those who are hiv-positive (morgan et al. 2000). in mozambique, clavero et al. (1999) isolated cryptosporidium spp. in hiv-infected humans. the evaluation of sensitivity and specificity for willis, mzn and iif techniques compared to the dif test showed a high sensitivity (100%) and specificity (96% – 100%) for the mzn test in the detection of cryptosporidium infections. results from our study indicate that the mzn technique is highly reliable in the diagnosis of cryptosporidium spp. in faecal samples. studies conducted by zaglool et al. (2013) and quílez et al. (1996) in the diagnosis of cryptosporidium spp. by mzn test indicated low sensitivities (73.3% and 79.3%, respectively) and a specificity approximating to the present study (95% and 100%, respectively). the high sensitivity of the mzn test to detect cryptosporidium spp. in this study can be attributed to the concentration of oocysts in faecal samples using the formalin-ether technique prior to analysis by subsequent tests. salleh et al. (2014) demonstrated that the sensitivity of mzn can be improved by the application of concentration techniques. in general, the sensitivity of iif and mzn tests in the detection of cryptosporidium spp. was similar (100%) in this study, these results were also similar to findings by rimhanen-finne et al. (2007). despite the similarity of results, the choice of diagnostic technique often depends on the availability of resources, time and the objective to be reached (diagnosis of specific parasite or multiple parasites) (chalmers 2014). in the iif technique specific antibodies against antigens produced by the parasite are used making it easy to read owing to the incidence of the fluorescent light in oo(cysts), indicated especially in cases of a low intensity of infections (robertson 2014). the disadvantage of mzn staining is that the oocysts may be easily confused with faecal debris that take up the stains (casemore, armstrong & sands 1985). the efficiency of iif compared to mzn was reported by ortega-mora et al. (1999) where the concentrated faecal samples of ewes were negative when analysed by mzn and positive by iif. ancylostoma spp. (60.25%) and toxocara spp. (5.76%) diagnosed in dogs of the magude district are of zoonotic potential and the epidemiology of this parasite is mainly associated with the high biotic potential of females and with transmammary infection by which larvae are transmitted to the offspring from the bitch (taylor et al. 2007; urquhart et al. 1998). the lower prevalence of toxocara spp. compared with ancylostoma spp. can be justified by the possible presence of animals with larvae in somatic tissues in which, instead of the larvae developing, maturing and producing eggs, they remain dormant in different tissues, and thus, there is a reduction or absence of eggs in faeces (taylor et al. 2007). there is a need for additional studies aimed at applying molecular techniques to identify the genotypes and subtypes of giardia and cryptosporidium involved in order to determine their zoonotic potential and to adopt effective control and prevention measures. acknowledgements the authors are grateful to the financier uem/asdi ‘impact of zoonotic diseases on public health and animal production in mozambique’. the authors are also thankful to all the collaborators from south africa (ms pulane malatji and dr oliver zishiri) and from mozambique (the agricultural technician from the magude district and the parasitology laboratory technician) involved in the sample collection. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions r.d.m., j.l., s.m.d.s.a. and s.m. conceived and designed the experiments r.d.m. and b.l. collected the samples from the field. r.d.m., s.m.d.s.a. and m.p.m. performed the experiments. r.d.m. and a.p.j. analysed the data. r.d.m. wrote the article. all authors read and approved the final manuscript. funding 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discussion conclusion acknowledgements references about the author(s) ivan g. horak department of veterinary tropical diseases, university of pretoria, south africa heloise heyne parasites, vectors and vector-borne diseases, agricultural research council-onderstepoort veterinary institute, south africa ali halajian department of biodiversity, university of limpopo, south africa shalaine booysen onderstepoort veterinary academic hospital, university of pretoria, south africa willem j. smit department of biodiversity, university of limpopo, south africa citation horak, i.g., heyne, h., halajian, a., booysen, s. & smit, w.j., 2017, ‘parasites of domestic and wild animals in south africa. l. ixodid ticks infesting horses and donkeys’, onderstepoort journal of veterinary research 84(1), a1302. https://doi.org/10.4102/ojvr.v84i1.1302 original research parasites of domestic and wild animals in south africa. l. ixodid ticks infesting horses and donkeys ivan g. horak, heloise heyne, ali halajian, shalaine booysen, willem j. smit received: 22 june 2016; accepted: 23 aug. 2016; published: 28 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the aim of the study was to determine the species spectrum of ixodid ticks that infest horses and donkeys in south africa and to identify those species that act as vectors of disease to domestic livestock. ticks were collected opportunistically from 391 horses countrywide by their owners or grooms, or by veterinary students and staff at the faculty of veterinary science, university of pretoria. ticks were also collected from 76 donkeys in limpopo province, 2 in gauteng province and 1 in north west province. all the ticks were identified by means of a stereoscopic microscope. horses were infested with 17 tick species, 72.1% with rhipicephalus evertsi evertsi, 19.4% with amblyomma hebraeum and 15.6% with rhipicephalus decoloratus. rhipicephalus evertsi evertsi was recovered from horses in all nine provinces of south africa and r. decoloratus in eight provinces. donkeys were infested with eight tick species, and 81.6% were infested with r. evertsi evertsi, 23.7% with a. hebraeum and 10.5% with r. decoloratus. several tick species collected from the horses and donkeys are the vectors of economically important diseases of livestock. rhipicephalus evertsi evertsi is the vector of theileria equi, the causative organism of equine piroplasmosis. it also transmits anaplasma marginale, the causative organism of anaplasmosis in cattle. amblyomma hebraeum is the vector of ehrlichia ruminantium, the causative organism of heartwater in cattle, sheep and goats, whereas r. decoloratus transmits babesia bigemina, the causative organism of babesiosis in cattle. introduction despite the long association between horses, donkeys and humans in south africa, it is strange that so little attention has been paid to the ixodid ticks with which they are infested. with the exception of surveys on the ticks that infest donkeys in botswana (mushi et al. 2003) and donkeys and horses in ethiopia (ferede et al. 2010; kumsa et al. 2012), there are no comprehensive studies on the ticks that infest these animals in sub-saharan africa. the data that are available are fragmented in that they generally have to be garnered from publications or surveys in which horses and donkeys as well as other animals were examined for ticks. in their extensive study of the zoogeography of the ixodid ticks of tanzania, yeoman and walker (1967) identified ticks in collections made from multiple host species including four horses and six donkeys. amblyommma variegatum, rhipicephalus appendiculatus, rhipicephalus decoloratus, rhipicephalus evertsi evertsi and a tick identified as rhipicephalus sanguineus were recovered from the horses and donkeys. in a similar study in kenya, collections were made from 19 horses and 3 donkeys (walker 1974). in addition to the aforementioned ticks, amblyomma gemma, dermacentor rhinocerinus, hyalomma rufipes, rhipicephalus jeanelli, rhipicephalus pulchellus, rhipicephalus simus (probably rhipicephalus preatextatus) and a tick belonging to the haemapysalis leachi group were identified. norval and his co-workers in zimbabwe examined collections from 39 horses and 11 donkeys and identified amblyomma hebraeum, h. rufipes, haemapysalis truncatum, r. appendiculatus, r. decoloratus, r. evertsi evertsi, rhipicephalus kochi, rhipicephalus sp. (near rhipicephalus punctatus), r. simus, rhipicephalus turanicus and rhipicephalus zambeziensis in the collections from horses, and a. hebraeum, h. rufipes, r. appendiculatus, r. decoloratus, r. evertsi evertsi and r. simus in those from donkeys (norval 1981, 1982, 1983; norval & mason 1981; norval, walker & colborne 1982). in their book on the rhipicephalus species of the world, walker, keirans and horak (2000) record a total of 25 rhipicephalus species and two subspecies on horses and 15 species and two subspecies on donkeys. only two of these species, r. appendiculatus and r. simus and the two subspecies, r. evertsi evertsi and r. evertsi mimeticus, are present in south africa, whereas the remainder occurs extralimitally. mushi et al. (2003) collected a. hebraeum, hyalomma sp. and r. evertsi evertsi from 12 donkeys examined at monthly intervals over a period of 7 months in their study devoted to the parasites of donkeys in the kgatleng district, botswana. ferede et al. (2010) collected a. variegatum, h. rufipes, r. evertsi evertsi, r. (boophilus) spp., rhipicephalus muhsamae and a tick they referred to as r. sanguineus from a total of 450 donkeys in a survey conducted in two districts in central oromia regional state, ethiopia. in the same region of oromia, kumsa et al. (2012) collected a. gemma, a. variegatum, h. rufipes, h. truncatum, r. decoloratus, r. evertsi evertsi and r. pulchellus from 1168 horses distributed in highland, midland and lowland localities. no serious effort was made to collect all the ticks present on the horses or donkeys in any of the abovementioned studies. in two southern african studies on the ticks of zebras, in which a few horses were included, horak, biggs and reinecke (1984b) collected h. rufipes, h. truncatum and a large number of larvae, nymphs and adults of rhipicephalus evertsi mimeticus from three horses in the khomas hochland of namibia, whereas horak, knight and de vos (1986) collected the larvae of amblyomma marmoreum and of margaropus winthemi, the adults of hyalomma glabrum, h. truncatum, rhipicephalus follis and rhipicephalus gertrudae and all stages of development of r. evertsi evertsi and of rhipicephalus glabroscutatus from two horses in the mountain zebra national park in the eastern cape province. it is interesting to note that tick collections have been made from more plains zebras (equus quagga), cape mountain zebras (equus zebra zebra) and hartmann’s mountain zebras (equus zebra hartmannae) than from domesticated horses or donkeys in south africa or namibia (horak et al. 1984b, 1986; horak, de vos & de klerk 1984a). the objectives of this study were to determine the species composition of ixodid ticks that infest horses and donkeys in south africa and to place in context the diseases they can potentially transmit to these animals and other domestic livestock. methods ticks were collected from horses in all nine provinces of south africa by their owners or grooms or by veterinary nurses and students from animals resident in the paddocks at the faculty of veterinary science, university of pretoria. in addition, s.b. (university of pretoria) collected ticks from horses presented for treatment at the university’s equine clinic. ticks previously collected from two horses in the mountain zebra national park (horak et al. 1986) have been included for completeness sake. ticks were collected by their owners or by a.h. and w.j.s. (university of limpopo) from 73 donkeys in limpopo province, 2 in gauteng province and 1 in north west province with the consent of the owners. although no attempt was made to make total collections, particular attention was paid to the head, shoulders, inner thighs, perianal region, tail brush and around the hooves. the ticks from each animal were placed in separate vials containing 70% ethyl alcohol or methylated spirits with a pencil-written label providing collection data. the ticks were identified and counted by the project leader i.g.h. (university of pretoria) and h.h. (arc-onderstepoort veterinary institute) using a stereoscopic microscope. the distribution by province of the various tick species was illustrated using a mosaic format. ethical considerations protocol v077/14 for the collection of ticks from horses and donkeys was submitted by i.g.h. and approved by the research committee and the animal ethics committee of the faculty of veterinary science, university of pretoria. results and discussion general the species and numbers of ticks collected from 391 horses and 76 donkeys are summarised in tables 1 and 2. the provincial distribution of the ticks collected from horses and donkeys is depicted in figure 1. a total of 5327 adult and immature ticks were collected from horses and a total of 487 from donkeys. the horses were infested with 17 tick species and donkeys with 8 tick species. a single donkey was the only animal from which rhipicephalus warburtoni was collected. the number of species and the total number of ticks collected from horses in the present survey were considerably higher than the 7 species and 917 ticks collected from horses in central oromia, ethiopia or those from donkeys in the kgatleng district, botswana, but similar to those of donkeys in central oromia. figure 1: the provincial distribution of ixodid ticks that infest horses and donkeys in south africa. table 1: ticks collected from 391 horses throughout south africa. table 2: ixodid ticks collected from 76 donkeys, mostly in limpopo province. with the exception of r. decoloratus and rhipicephalus microplus, of which the males are very small and engorged females large and rhipicephalus lunulatus, of which only one female tick was collected, more male than female ticks of all species were collected. twelve tick species were collected from horses in the eastern cape province, 10 from horses and donkeys in gauteng province and 9 from horses and donkeys in limpopo province (figure 1). rhipicephalus evertsi evertsi was present on horses in every province, and r. decoloratus on these animals in every province except the northern cape (figure 1). amblyomma spp. amblyomma hebraeum the bont tick, a. hebraeum, was the second most prevalent tick recovered from both horses and donkeys. this species was present on horses in each of the four northern provinces, as well as in kwazulu-natal and the eastern cape (table 1; figure 1). its presence in these provinces is in agreement with its overall geographic distribution as mapped by spickett (2013). the adults of a. hebraeum prefer large herbivores as hosts, whereas its immature stages and particularly the larvae infest the same hosts as the adults as well as smaller mammals, birds and tortoises (horak et al. 1987; horak, golezardy & uys 2007). the mean burden of adult ticks on 33 plains zebras examined in the kruger national park was 3 (horak et al. 1984a), compared to 5 on the 76 horses infested with adult ticks in the present study. horses on the same property as other livestock may thus serve as a reservoir of infestation and escape the sometimes rigorous acaricidal control regimens applied to cattle. amblyomma hebraeum is the vector of ehrlichia ruminantium, the causative organism of heartwater in cattle, sheep and goats and certain wildlife species (neitz 1937; norval & horak 2004). amblyomma marmoreum the south african tortoise tick, amblyomma marmoreum, is widespread in south africa, and whereas its adults and nymphs prefer leopard tortoises, stigmochyles pardalis, as hosts, its larvae infest a wide range of mammals as well as the larger ground-frequenting birds and reptiles (horak et al. 2006). the presence of larvae on the two horses and on four cape mountain zebras examined in the mountain zebra national park, where there are a large number of leopard tortoises, is thus not surprising (horak et al. 2006). hyalomma spp. hyalomma glabrum, hyalomma rufipes and hyalomma truncatum the three hyalomma species that are present in south africa are drought and heat-tolerant and are generally present in those regions where these climatic conditions prevail. previously, only two horses examined in the mountain zebra national park were infested with h. glabrum (horak et al. 1986). at that time, this tick was referred to as hyalomma marginatum turanicum, but it was subsequently reinstated as h. glabrum (apanaskevich & horak 2006). in the present study, more horses were infested with h. rufipes than with h. truncatum but the intensity of infestation with the latter tick was higher (table 1). in an earlier study carried out in zimbabwe, 12 out of 39 horses were found to be infested with h. rufipes and the same number with h. truncatum. there too the intensity of infestation with the latter species was higher (norval 1982). donkeys in the present study were also infested with both species (table 2). hyalomma rufipes was collected from horses in six provinces and h. truncatum in five (figure 1). hyalomma rufipes is the vector of babesia occultans, the causative organism of benign babesiosis in cattle, and h. truncatum is the vector of babesia caballi, the causative organism of equine piroplasmosis (de waal 1990; gray & de vos 1981). both species are two-host ticks and their immature stages feed on hares (horak & fourie 1991). consequently, infection with babesia spp. must pass transovarially from one generation of female ticks to the next generation of adult ticks. margaropus winthemi the one-host winter horse tick, m. winthemi, has a scattered distribution associated with the cooler, higher altitude regions of south africa (howell, walker & nevill 1978). during july 1984, three zebras in the mountain zebra national park were each found to be infested with more than 25 000 ticks in all stages of development (horak et al. 1986). penzhorn (1984) noted that of the 22 zebras in the park for which fairly accurate mortality dates were reported, 20 had died during the winter and that 19 of these deaths occurred between july and september. he regarded the late winter as a critical period for survival, probably because of the deteriorating condition of the forage and the cold weather. horak et al. (1986) reported that the large burdens of m. winthemi during winter probably exacerbated the effects of the already harsh conditions. although no m. winthemi were collected from horses in the free state, large numbers have been collected from gemsbok in the willem pretorius nature reserve in the centre of the province (fourie et al. 1991). rhipicephalus spp. rhipicephalus appendiculatus the provincial distribution of r. appendiculatus is similar to that of a. hebraeum (spickett 2013; figure 1). forty-one of the 391 horses examined were infested, and the mean intensity of infestation on these animals was 5 males and 4 female ticks. in zimbabwe 13 of 39 horses were infested and the mean intensity of infestation was 18 males and 13 female ticks (norval et al. 1982). ten of the 76 donkeys examined were infested. rhipicephalus appendiculatus is a three-host tick of which the adults prefer large domestic and wild ruminants as hosts (horak et al. 2007). it is the vector of theileria parva as well as buffalo-derived t. parva, the causative organisms of east coast fever and corridor disease, respectively, in cattle (norval & horak 2004). rhipicephalus decoloratus and rhipicephalus microplus excluding the western free state, the karoo and the northern cape province, r. decoloratus is widespread throughout the rest of south africa, whereas r. microplus is in the process of invading this region at the expense of r. decoloratus (horak et al. 2009; nyangiwe, harrison & horak 2013; spickett 2013; tønnesen et al. 2004). rhipicephalus decoloratus was present on horses in every province excepting the northern cape in the present study (figure 1). it prefers large ruminants as well as equids as hosts, whereas r. microplus is a cattle tick, but is in the process of adapting to other host species (horak et al. 2009). rhipicephalus decoloratus and r. microplus are one-host ticks and both transmit babesia bigemina, whereas r. microplus also transmits babesia bovis, the causative organisms of bovine babesiosis (de vos, de waal & jackson 2004). rhipicephalus evertsi evertsi with the exception of a large portion of the northern cape province, r. evertsi evertsi is present throughout south africa (spickett 2013). this species was collected from horses in all nine provinces in the present study (figure 1). its overall distribution includes much of east africa, eastern sudan and several sub-saharan countries in west africa (walker et al. 2000). more collections of r. evertsi evertsi have been made from horses, donkeys and plains zebras in the afrotropical region than any other rhipicephalus species (walker et al. 2000). it is the dominant species on horses in south africa and in oramia regional state in ethiopia, and on donkeys in south africa and botswana (kumsa et al. 2012; mushi et al. 2003; tables 1 and 2). the prevalence of r. evertsi evertsi on horses and donkeys in south africa and botswana was considerably higher than that on these animals in ethiopia. rhipicephalus evertsi evertsi is a two-host tick whose adults attach in the perianal region and on the inner thighs and inguinal regions of equids and the immature stages in the external ear canals of these animals. although the prevalence of infestation may be high, burdens of adult ticks are seldom large. infestations with immature ticks may be very large. for instance, a mean of more than 850 larvae and nymphs have been collected from the external ear canals of 33 plains zebras in the kruger national park (horak et al. 1984a). rhipicephalus evertsi evertsi transmits b. caballi and theileria equi, the causative organisms of equine piroplasmosis (de waal & potgieter 1987; norval & horak 2004), and theileria separata, the causative organism of ovine theileriosis (jansen & neitz 1956). it also transmits anaplasma marginale, the causative organism of anaplasmosis in cattle (potgieter 1981). engorging r. evertsi evertsi females secrete a paralysis-inducing toxin that affects lambs born during spring in the highveld regions of mpumalanga, the eastern free state and the north-eastern region of the eastern cape province (gothe 1981). rhipicephalus follis, rhipicephalus gertrudae and rhipicephalus simus with the exception of north west and mpumalanga provinces, one or more of these three species within the ‘r. simus’ group of ticks were present on horses in every province. rhipicephalus follis was found in the mountainous or higher altitude regions mainly in the eastern half of the country, r. gertrudae in the semi-arid or winter-rainfall regions with dry summers with r. simus in the warmer, moister lower altitude regions of the country (walker et al. 2000). rhipicephalus glabroscutatus the distribution of r. glabroscutatus stretches from the albany thicket biome in the eastern cape province through the fynbos biome to the west coast national park. it is a two-host tick, with all stages of development feeding around the hooves and below the fetlocks of its hosts. secondary bacterial infection of its attachment sites leads to foot abscesses and lameness, particularly in angora goats farmed in the albany thicket biome (macivor & horak 1987). species of which few were collected rhipicephalus lunulatus is a fairly rare species in south africa and is present in the warmer moist regions in the east of the country. its distribution is widespread in zimbabwe, east and west africa (walker et al. 2000). there is considerable controversy concerning the identity of the tick known as r. turanicus. the tick referred to by this name in south africa is not necessarily morphologically similar to ticks with the same name elsewhere, and further studies are required to establish its true identity. the distribution of r. warburtoni is more widespread than that plotted for it in the central and south-western free state by walker et al. (2000). its presence has been confirmed in the north of limpopo province (harrison, bown & horak 2011) as r. sp. (near warburtoni) and the collection of a male tick from a donkey is further proof of its presence there. r. zambeziensis is present in the limpopo valley and adjoining areas (norval et al. 1982), and its morphology, hosts and seasonal abundance are similar to those of r. appendiculatus. it is also a vector of east coast fever (lawrence, norval & uilenberg 1983). conclusion horses examined countrywide were found to be infested with a large variety of tick species of which r. evertsi evertsi was the most widespread. the prevalence of this tick is potentially important because it is the vector of the causative organisms of equine piroplasmosis. various other tick species collected from the horses are important vectors of diseases in domestic cattle. donkeys in limpopo province were infested with eight tick species of which r. evertsi evertsi was the most prevalent. acknowledgements the authors express their sincere thanks to the many owners, grooms, veterinary students and staff of the faculty of veterinary science who collected ticks for this project. part of this work is based on research supported by the south african research chairs initiative (sarchi – prof. wilmien j luus-powell) of the department of science and technology and national research foundation of south africa (grant no 101054). any opinion, finding and conclusion or recommendation expressed in this material is that of the authors, and the national research foundation (nrf) does not accept any liability in this regard. the participation of the senior author in the project was partially funded by a grant from the national research foundation and the university of pretoria. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors read and approved the manuscript. i.g.h. was the project leader. i.g.h. and h.h. identified the ticks that had been collected by s.b., 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distribution, commonwealth institute of entomology, london. walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. yeoman, g.h. & walker, j.b., 1967, the ixodid ticks of tanzania. a study of the zoogeoraphy of the ixodidae of an east african country, commonwealth institute of entomology, london. abstract introduction materials and methods results discussion acknowledgements references about the author(s) krzysztof w. romański centre for experimental diagnostics and biomedical innovations, wrocław university of environmental and life sciences, poland józef nicpoń centre for experimental diagnostics and biomedical innovations, wrocław university of environmental and life sciences, poland citation romański, k.w., nicpoń, j., 2018, ‘occurrence of the specific long spike burst pattern in the ovine proximal gallbladder as an indication of myoelectric regional variability’, onderstepoort journal of veterinary research 85(1), a1455. https://doi.org/10.4102/ojvr.v85i1.1455 original research occurrence of the specific long spike burst pattern in the ovine proximal gallbladder as an indication of myoelectric regional variability krzysztof w. romański, józef nicpoń received: 16 mar. 2017; accepted: 16 mar. 2018; published: 11 june 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the myoelectrical activity of the ovine gallbladder has not been fully recognised. five rams were fitted with six small intestinal and three gallbladder electrodes and a strain gauge force transducer was mounted near the gallbladder fundic electrode. in two series of successive experiments, the electromyographical and mechanical recordings were recorded over a period of 5–7 hours. the occurrence of the slow waves in the small bowel was regular, unlike those in the gallbladder. in the gallbladder infundibulum, the specific pattern, called the long spike burst pattern (lsbp), was observed. it comprised usually one or two parts of prolonged duration. the first part resembled the classical (short lasting) spike burst in the small bowel, and its amplitude was lower than that of the second part. the spike burst frequency of the second part was 2–3 times lower than that of the first part. during phase 1-like and phase 2a-like activities, the intensity of the gallbladder lsbp was reduced while enhanced after feeding. in fasted rams, the duration of a specific pattern, observed in the gallbladder infundibulum, was longer than in non-fasted animals and its amplitude was low. similar events were recorded in the gallbladder corpus, but the specific pattern was shorter and irregular. in the gallbladder fundus, mostly irregular short spike bursts were recorded. it is concluded that in sheep, specific types of the long-lasting groups of spikes occur in the upper gallbladder areas exhibiting myoelectrical regional variability. the character of an lsbp depends on feeding conditions. introduction sheep belong to the animal species in which the biliary tract contains well-developed gallbladders. its size is similar to that in man and dog; thus, it may store a substantial portion of the bile, and inflow and outflow of bile is almost continuous (aziz & khatra 1985). the gallbladder, similar to the small bowel, exhibits intense motor functions representing both myoelectrical and mechanical activities. the slow and rapid action potentials as the periodic deflections of the resting membrane potential can be distinguished (szurszewski 1987). slow waves are generated by at least some types of the interstitial cells of cajal (icc) (thuneberg 1999), the gastrointestinal pacemaker cells, because of slow transmembrane ion movements. interstitial cells of cajal along with the smooth muscle cells (smc) and with the cells expressing the platelet-derived growth factor receptors α+ (pdgfrα+ cells) form smc/icc/pdgfα+ syncytium (sip syncytium) responsible for pacemaker activity and provide propagation pathways for the slow waves (sanders, ward & koh 2014). usually, the slow waves (even at the peak of the upstroke phase) cannot reach the critical voltage threshold to evoke the rapid action potential (spike) responsible for mechanical activity. the spikes are usually organised in the small groups, namely the spike bursts, and they are restricted, in most cases, to the plateau phase of the slow waves. the spike bursts are omnipresent in the stomach and small bowel, and a similar situation exists in the gallbladder (buéno & praddaude 1979; szurszewski 1987). the spike bursts of longer durations, that is, which last longer than the plateau phase of the slow wave (over 3 sec in the stomach), can occur independently of the slow waves and induce longer contractions (sarna 2002). slow waves arrive in the stomach and small bowel regularly, also in sheep (roman´ski 2002a). they are also present in the ovine gallbladder, although not always detected there (roman´ski 2004a). thus, the ovine gallbladder exhibits intense motor activity and plays an important role in bile circulation (pass & heath 1977). the principal physiological roles of the gallbladder are to concentrate and excrete bile (lee & kuwer 2006; shaffer 2000). gallbladder emptying is more efficient after a meal, but during the fasting state periodic gallbladder contractions also evacuate bile into the duodenum (dodds, hogan & geenen 1989; ryan 1981; shaffer 2000). therefore, its motor function must be precise enough to ensure filling and emptying through the common bile duct as well as bile storage and mixing to prevent sedimentation of bile constituents. gallbladder motility also facilitates bile concentration. furthermore, the incessant but fluctuating motor activity of the gallbladder must be coordinated with that of the small bowel. in ruminants, there are no clear interdigestive periods, and ruckebusch (1989) suggested that periodic gallbladder contractions are also present, at least in sheep. therefore, the presence of specific motility patterns in the gallbladder can be expected, also in sheep (kaji, takamatsu & kojiya 2002). it was already reported that in sheep the migrating myoelectric complex (mmc)-like activity may occur in the gallbladder, as it was observed in the small bowel, and the ‘minute rhythm’ (mr) regularly arrived both in the small bowel and the gallbladder during its phase 2 activity (grivel & ruckebusch 1972; roman´ski 1996, 2002b, 2017; ruckebusch 1989). thus, the ovine gallbladder motility exhibits some similarities to the small intestinal motility. it seems likely that the regional differences in ovine gallbladder myoelectrical activity can also occur (buéno & praddaude 1979; roman´ski 2004a). therefore, the aim of this study was to characterise further the gallbladder motility in sheep under various feeding conditions with special emphasis directed towards the motility patterns and regional differences. materials and methods five healthy rams of polish merino breed, weighing between 38 kg and 43 kg, were used. before surgery, the animals were kept in large cages under normal day–night lighting conditions and fed good quality hay together with grain mixture. drinking water was not limited. animal preparation in the 24 h fasted rams, the general and local anaesthesia was applied just prior to surgery. a right-sided lateral laparotomy was performed. each ram was fitted with nine bipolar platinum wire electrodes for electromyographical recordings, and one strain gauge force transducer was attached for mechanical recordings. the teflon-covered electrodes (made by us) and the strain gauge force transducers embedded in rubber-teflon (silastic-type material) coat (rb products, madison, wi) were sutured to the serosal side of the gastrointestinal and gallbladder walls. electrode localisation: (1) the duodenal bulb, 6 cm distal to the middle of the pyloric ring; (2) the duodenum, 56 cm distal to the middle of the pyloric ring; (3) proximal jejunum, 200 cm distal to the duodenal electrode; (4) more distal jejunum, 100 cm distal to the first jejunal electrode; (5) subterminal ileum, 110 cm proximal to the middle of the ileocecal junction; (6) terminal ileum, 10 cm proximal to the middle of the ileocecal junction; (7) gallbladder infundibulum, about 1 cm distal to the cystic duct; (8) gallbladder corpus, 4 cm distal to the proximal gallbladder electrode; and (9) gallbladder fundus, 4 cm distal to the mid gallbladder electrode. the small strain gauge force transducer was attached next to the distal gallbladder electrode. it was used for additional confirmation of the correctness of myoelectrical recordings and for the assessment of types of gallbladder fundic contractions. the marked electrode and transducer wires were exteriorised over the skin, soldered to the plugs and fixed 2 cm from the skin incision. they were connected with the recording apparatus each time during the experimental periods and disconnected afterwards. after the surgery, rams were allowed at least two weeks to recover. experiments a total of 30 timed experiments, each lasting for 5–7 h, were conducted. four different conditions were considered. the first two were the experiments performed on 48 h fasted animals, with or without feeding during the experiment. fed animals received 250 g of a grain mixture which they consumed over 3–4 min. food was given during the late phase 2 of the duodenal mmc (and possibly the gallbladder), at about 70% of the mmc cycle. during further experiments performed in fasted and non-fasted animals, food was offered during the early phase 2 of the duodenal mmc (gallbladder mmc-like activity occurred concurrently with that in the duodenum), at about 30% of the mmc cycle. each experiment was conducted on each ram. the 10-channel electroencephalograph (reega duplex tr xvi), adapted for mechanical recordings, was used in the study. the myoelectrical and motor activities were recorded from nine electrodes and from the strain gauge force transducer. the small intestinal myoelectrical recordings serving for mmc and mr recognition in the small bowel were also helpful for interpretation of the gallbladder motility recordings. at least two full mmc cycles were recorded during each experiment. other details of the methodology are described elsewhere (roman´ski 2004a, 2016). data elaboration in the small-intestinal recording sites, the regular occurrence of the mmc cycles was observed. in the gallbladder, the presence of mmc-like activity was also detected, as suggested previously (roman´ski 1996). particular attention was given to phase 2 of the gallbladder mmc-like pattern. to characterise it, at least in part, several parameters were calculated and presented mostly in tables. they contained the spike bursts of different duration, especially the short spike bursts (classical, i.e., the myoelectrical correlates of shorter phasic contractions) and the long-lasting spike bursts. the classical spike burst frequency, amplitude, duration, number of spikes in one burst and number of spikes per second were calculated for the spike bursts noticed in all the gallbladder regions examined. similar parameters were calculated for the longer myoelectrical events. these episodes were observed in the upper gallbladder regions, namely in the gallbladder infundibulum and gallbladder corpus. they were called ‘the long spike burst patterns’ (lsbps), because the individual spikes were less frequent than those in the short spike bursts. the frequency, amplitude and duration of fundic longer and shorter contractions were calculated as well. statistics the mean values and standard deviations were calculated from the raw data. finally, the student’s t-test for paired values, preceded by the analysis of variance, was applied (snedecor & cochran 1971). the statistical significance was labelled when p < 0.05. ethical consideration the experimental model used in this article was approved by the ii local ethical committee for experimental animals in wrocław, poland. therefore, all the experiments were performed officially, according to polish and european laws. results in the small bowel, typical mmc cycles and mr episodes (both are not shown) were identified in the recordings. in the gallbladder, the pattern resembling the mmc in the small bowel occurred in a cyclical fashion. in both upper gallbladder regions, phase 1 of the mmc-like activity was less clear than that in the gallbladder fundus because it was interrupted by regularly arriving lsbps. phase 2 of the mmc arrived in the duodenum and the phase 2-like activity was seen in the gallbladder similar to that in the duodenum. the short spike bursts (the myoelectrical correlates of typical phasic contractions) were observed throughout this phase both in the small bowel and the gallbladder. the short spontaneous spike bursts arriving in the course of phase 2 of the gallbladder mmc-like activity (except the spike bursts forming the gallbladder mr, as seen in figure 2) are shown in table 1. the duration of the spike burst and the number of spikes in one spike burst were slightly greater in fed rams than in non-fed rams. the spike burst amplitude was significantly lower in fed animals than in non-fed animals and was slightly lower in the gallbladder fundus as compared with that in the gallbladder infundibulum. the frequency of the spike bursts was greater in fed rams than in fasted rams and especially higher in the gallbladder fundus and lowest in gallbladder infundibulum. the spike frequency measured within the spike burst was higher in fed rams than in non-fed rams, particularly in the experiments performed in non-fasted animals. in the gallbladder fundus, these values were slightly greater compared to other gallbladder regions examined (table 1). table 1: characteristics of the short spike bursts recorded during phase 2-like activity of the gallbladder migrating myoelectric complex as observed in the gallbladder infundibulum, corpus and fundus in various feeding conditions. the slow waves were regularly observed in the small bowel. they were observed occasionally in the gallbladder. both amplitude and frequency of the slow waves were lower than the spikes of the second part of the lsbp (figure 2). they can be seen in the gallbladder fundus (figure 1) and in the gallbladder corpus and fundus (figure 2a). figure 1: three fragments of the gallbladder myoelectrical recording in fasted rams. (a) recording during phase 1 of the migrating motility complex observed both in the duodenum (3 min after the onset of duodenal phase 1) and the gallbladder; (b) recording during phase 2a of the migrating motility complex observed both in the duodenum (14 min after the onset of duodenal phase 1) and the gallbladder; and (c) recording during phase 2b of the migrating motility complex observed both in the duodenum (25 after the onset of duodenal phase 1) and the gallbladder. figure 2: two fragments of gallbladder myoelectrical recordings in fasted rams after feeding. (a) the recording started 9 min after termination of feeding and (b) continued recording after feeding. in the gallbladder infundibulum of fasted and non-fasted rams, the short spike bursts occurred irregularly along with the lsbp pattern (table 2, figure 1). they appeared regularly, regardless of the mmc phase. long spike burst patterns arriving in the course of phase 2 of the gallbladder mmc-like activity usually contained two parts. the initial (shorter) part of higher spike frequency (table 2, figure 1) was observed in most cases at the beginning of lsbp. this part closely resembled typical prolonged (i.e. lasting longer than 2 sec) spike bursts. occasionally, the first part of the lsbp occurred without the second part. this part of the pattern (longer duration and usually higher amplitude) contained less frequent spikes resembling rather the long spike bursts described in the ovine colon (fioramonti & hubert 1980). the occurrence of the lsbp in the gallbladder infundibulum was regular and its absence in this region was very exceptional. in the gallbladder corpus, lsbps were shorter and absent more frequently than those in the gallbladder infundibulum (table 2). the lsbp present in the gallbladder corpus contained two parts in about 85% of episodes. in 10% of episodes, at the end of each pattern, more frequent spike burst again arrived. this was occasionally observed in the gallbladder infundibulum. the first part of the lsbp evolved into the second one abruptly or gradually, directly or through the transient short and irregular fragment distinct from both parts of the pattern. in about 5% of lsbps, their first part arrived again in the middle of the second part of the pattern. no lsbp was detected in the gallbladder fundus and in the small bowel. table 2: characteristics of the long spike burst pattern recorded in the gallbladder infundibulum and corpus in various feeding conditions. in the gallbladder infundibulum during phase 1 of mmc-like activity observed in fasted rams, the lsbp duration was 13 ± 6 sec with amplitude of 64 μv ± 18 µv in the gallbladder infundibulum. during phase 1 in non-fasted rams, the respective values were 9 ± 4 sec and 67 μv ± 20 μv. in the gallbladder corpus, the lsbps were less frequent and reduced in length. the lsbp occurred in both feeding regimes designed in this study. the lsbps observed during phase 2 of the gallbladder mmc-like activity were longer and developed further in the gallbladder infundibulum than in the gallbladder corpus regardless of feeding conditions (table 2). the duration of the whole pattern was the longest during the fasting period. as the amplitude was measured in the middle of the pattern, these values characterised exclusively its longer (second) part. both the amplitude and frequency of lsbp were the greatest in the experiments with feeding. in the gallbladder infundibulum and corpus, the number of spikes in the whole pattern was lower in the experiments performed on fasted rams than in the remaining experimental groups (table 2). in the gallbladder corpus, the duration of the first fragment of the lsbp was shorter than that in the gallbladder infundibulum (table 3). in both these gallbladder regions, the values were greater in fasted animals after feeding compared to non-fed animals. the amplitude of these spike bursts was lower in the gallbladder corpus than in gallbladder infundibulum and was the lowest after feeding. the frequency of spikes forming the first part of the lsbp was the lowest in fasted animals and feeding increased this value. the number of spikes in the shorter fragment of lsbp was not significantly smaller in the gallbladder corpus than in gallbladder infundibulum. similar differences of these values were observed between fed and non-fed animals (table 3, also see figure 2). table 3: characteristics of the shorter fragment of long spike burst pattern recorded in the gallbladder infundibulum and corpus in various feeding conditions. the duration of the second (longer) fragment of lsbp was only slightly shorter than the total duration of the pattern, and the changes related to the various feeding conditions were similar (table 1 and table 4). the frequency of the spikes forming this fragment was much lower than that of its first part, and it was higher in non-fasted animals than in fasted animals (table 3 and table 4). the number of spikes forming this part of the lsbp was smaller in fasted rams than in the remaining groups of the experiments (table 4). table 4: characteristics of the longer fragment of the long spike burst pattern recorded in the gallbladder infundibulum and corpus in various feeding conditions. the incidence of longer or shorter phasic contraction of the gallbladder fundus, recorded with the strain gauge method, followed in almost all cases the spike bursts. in fasted rams, the duration and frequency of longer contractions of the gallbladder infundibulum were significantly greater in fed animals than in non-fed animals (table 5). the amplitude was not significantly higher in fed rams as compared with non-fed rams. the duration of the shorter contractions was significantly reduced in fed rams, whereas both the amplitude and frequency were not significantly lower in fed animals than in non-fed animals. in non-fasted rams, the duration of longer contractions of the gallbladder fundus was significantly longer in fed animals than in non-fed animals and was also significantly prolonged as compared with the corresponding value obtained in fasted rams. the amplitude of longer contractions was slightly higher in fasted rams after feeding compared to non-fed rams. the frequency of longer contractions was slightly but significantly higher in non-fasted non-fed than in fasted non-fed rams. the duration of shorter contractions in non-fasted fed rams was significantly shorter than in non-fasted non-fed animals. the amplitude of shorter contractions in fasted rams was slightly lower after feeding compared to non-fasted animals. the frequency of shorter contractions in non-fasted non-fed rams was not significantly higher than in non-fasted fed animals (table 5). table 5: characteristics of the longer and shorter gallbladder fundic contractions recorded during phase 2-like activity of the putative gallbladder migrating myoelectric complex in various feeding conditions. discussion the results obtained in this study characterise in part the gallbladder myoelectrical activity with some regional differences. compared with the small-intestinal motility, it exhibits similarities and differences as it was initially reported earlier (roman´ski 1996, 2002b, 2004a). the mmc presented the principal pattern occurring in the ovine small bowel regardless of feeding conditions and a similar pattern appeared to occur also in the gallbladder. the duration and regularity of the spike bursts are similar in the small bowel and gallbladder, whereas the occurrence of the unique pattern in the upper gallbladder (lsbp) was specific for this organ. the shape, duration and spike frequency of the short-lasting spike bursts in the ovine gallbladder (representing the myoelectrical correlates of shorter phasic contractions) appeared not to be markedly different from those in the small bowel, but their amplitude was relatively low. this results from the thinner smooth muscle layer of the gallbladder wall as compared with that in the small bowel. not only the amplitude of the spike bursts but also the force of phasic contractions seemed to be smaller in the gallbladder than in the small bowel as was observed in the present and previous studies (grivel & ruckebusch 1972; roman´ski 2003). in the gastrointestinal tract, feeding evokes the overall increase of the myoelectric and contractile activity while suppressing the spike bursts and contraction amplitudes (heddle, miedema & kelly 1993; lester & bolton 1994; roman´ski 2003; wingate et al. 1979). a similar response to feeding was also observed in this study in the ovine gallbladder. long spike burst pattern is the novel event occurring in the upper ovine gallbladder. the first part was very similar to the typical but slightly longer spike burst, representing the myoelectrical correlate of phasic contraction. this part always occurred at the beginning of lsbp both in the gallbladder infundibulum and gallbladder corpus. as this part could appear twice within one lsbp and occurred sometimes in the various sites of the longer part of the pattern, it seems likely that the same typical spike bursts are superimposed on the lsbp arriving during this pattern. its second part was different because the spikes were less frequent than in the first part and their shape slightly differed from the typical spikes. during the second part of the lsbp, spikes occurred at a much lower frequency than during the first part, implying a different characteristic. similar events were also shown earlier (buéno & praddaude 1979; see also roman´ski 2004a). the slow waves were observed in the gallbladder infrequently. according to the suggestion of matsumoto, sarna and condon (1985) and of becker, duff and moody (1981), the slow waves can occur in canine and opossum gallbladder and are omnipresent there. in ovine gallbladder, they occurred at much lower frequency than the spikes observed within the second part of the lsbp (see also roman´ski 2004a). therefore, it is likely that lsbps contained only the spikes, not the slow waves. the question regarding the physiological role of lsbp is open. it is possible that it controls the episodes of gallbladder filling and emptying. relaxation and lowering of the pressure in the upper gallbladder region (pressure changes in ovine gallbladder are quite frequent, nejmark 1977) is necessary for gallbladder filling. soon after the pressure may rise because of the occurrence of a contraction induced at least by the first part of lsbp to evacuate a small portion of bile. the subsequent arrival of the second, longer part of lsbp increases tension in this region, which prevents bile inflow to the gallbladder from the common bile duct. it can occur especially when the amplitude of spikes forming the lsbp is high. this repeatable mechanism might be responsible for undisturbed bile inflow and outflow in cooperation with the sphincters located in the biliary tract, that is, mirizzi’s sphincter, lütkens’ sphincter and mainly the oddi’s sphincter (bagcivan et al. 2006; grivell et al. 2004; levina 1971; mirizzi 1940; see also nejmark 1977). the role of mirizzi’s and lütkens’ sphincters is difficult to assess because they represent the functional sphincters only. bile circulation between the liver, gallbladder and duodenum occurs during the interdigestive state and is more intense after a meal (roman´ski 2004b; ruckebusch 1989; scott & diamant 1988). it might be slightly increased because of the presence of the duct of luschka (spanos & syrakos 2006), but it is not known whether or not this duct occurs in sheep. the arrival of the longer lsbps, especially in fasted sheep, might help in reducing bile outflow during this period, facilitating bile storage and concentration. intensified lsbps after feeding might be responsible for bile evacuation, at least in part. different types of spike bursts, concerning their duration and amplitude, were found in the ovine gallbladder, and the question arises as to which types of contractions can be evoked by these spike bursts. this is also a question of terminology. according to the duration criterion, two types of contractions – phasic and tonic – can be distinguished. no myoelectric correlates of tonic contractions have been described. assuming that tonic contractions last several minutes or more (sarna 2002), it can be stated that the duration of lsbps and of other longer spike bursts arriving both in the gallbladder and small intestine, for example, the giant spike bursts, is too short to evoke tonic contractions. therefore, the contractions observed in the ovine gallbladder, lasting 5–10 sec, or even longer, should not be called tonic contractions. these contractions and their myoelectrical correlates, that is, giant-like contractions or giant-like spike bursts, respectively, can form specific patterns as lsbp in the gallbladder, but also similar events can be present in the small or large bowel (fioramonti & hubert 1980; sarna 2002). these longer spike bursts cannot probably be evoked by the slow waves because they last longer than their plateau phase. according to the definition of tonic contractions proposed by sarna (2002), any contractions that are shorter than several minutes could be classified as phasic contractions. however, this term has not yet been precisely defined. the applied electromyographical technique with inert teflon-platinum electrodes is apparently not harmful and yet sensitive enough to detect all the myoelectrical events in the normal gallbladder. however, not all the events, especially the slow waves, were regularly recorded here. therefore, it can be assumed that in the ovine gallbladder, the slow waves occur infrequently or their amplitude can be occasionally reduced to an undetectable level. some authors found neither slow waves nor spiking activity in the gallbladder of the dog or monkey. it may have been that techniques applied could explain this failure (becker et al. 1981; ludwick & bass 1967). thus, sheep may represent the suitable model for recording of the gallbladder myoelectrical activity. the spiking activity in the gallbladder was found also in the dog (itoh et al. 1982; traynor, dozois & dimagno 1984; ura, sarna & condon 1992). satisfactory recordings of the gallbladder electrical activity were obtained also in pigs (laplace 1976a, 1976b) and later in sheep (buéno & praddaude 1979). these scanty data suggest that application of the technique of recording of the gallbladder myoelectrical activity in sheep and in other 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https://doi.org/10.1007/bf01299823 abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) reza pasandideh department of animal science, khuzestan agricultural sciences and natural resources university, ahvaz, islamic republic of iran masoud reza seyfi abad shapouri department of pathobiology, shahid chamran university of ahvaz, islamic republic of iran mohammad taghi beigi nassiri department of animal science, khuzestan agricultural sciences and natural resources university, ahvaz, islamic republic of iran citation pasandideh, r., seyfi abad shapouri, m.r. & beigi nassiri, m.t., 2018, ‘immunogenicity of a plasmid dna vaccine encoding g1 epitope of bovine ephemeral fever virus g glycoprotein in mice’, onderstepoort journal of veterinary research 85(1), a1617. https://doi.org/10.4102/ojvr.v85i1.1617 original research immunogenicity of a plasmid dna vaccine encoding g1 epitope of bovine ephemeral fever virus g glycoprotein in mice reza pasandideh, masoud reza seyfi abad shapouri, mohammad taghi beigi nassiri received: 24 feb. 2018; accepted: 16 july 2018; published: 28 aug. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the aim of this study was to investigate the immunogenicity of a plasmid deoxyribonucleic acid (dna) vaccine encoding the g1 epitope of bovine ephemeral fever virus (befv) g glycoprotein in mice. a plasmid dna carrying the g1 gene was constructed and designated as pcdna3.1-g1. the expression of the target gene was confirmed in human embryonic kidney 293 (hek 293) cells transfected with pcdna3.1-g1 by indirect immunofluorescent staining. immunisation experiments were intramuscularly carried out by vaccinating 6-week-old female mice in four groups, including the pcdna3.1-g1 construct, pcdna3.1 (+) plasmid alone, bef-inactivated vaccine and phosphate-buffered saline (pbs) (1x) three times with 2-week intervals. fourteen days after the last immunisation, the animals were bled and the resulting sera were tested for anti-g1-specific antibodies by immunoblotting analysis, indirect enzyme-linked immunosorbent assay (elisa) and virus neutralisation (vn) test. serological assays showed that the pcdna3.1-g1 construct expressing g1 protein was able to elicit specific antibodies against this antigen. virus neutralisation test showed that pcdna3.1-g1 could induce anti-befv-neutralising antibodies in mice. our findings indicated that a new dimension can be added to vaccine studies for bovine ephemeral fever (bef) using eukaryotic expression plasmids encoding the g1 antigen in the future. introduction bovine ephemeral fever (bef) is a viral disease of cattle and water buffalos seen in africa, the middle east, australia and asia. infected cattle can show a wide spectrum of clinical signs, including a sudden onset of fever (41 °c – 42 °c) with loss of appetite, increased breathing and heart rate, stiffness, lameness, cessation of rumination and constipation (walker 2005; zheng et al. 2009). bovine ephemeral fever is an economically important disease, which can be spread rapidly and lead to considerable losses in the cattle industry, through reduced milk production in dairy herds, loss of condition in beef cattle and the immobilisation of draught animals (aziz-boaron et al. 2013; walker 2005). bovine ephemeral fever is caused by bef virus (befv) and transmitted through mosquitoes or biting midges. bovine ephemeral fever virus is classified as a member of the genus ephemerovirus in the family rhabdoviridae. bovine ephemeral fever virus has a bullet-shaped morphology, contains a 14.9 kb single-stranded, negative-sense ribonucleic acid (rna) genome, which encodes five structural proteins, including a nucleoprotein (n), a polymerase-associated protein (p), a matrix protein (m), a large rna-dependent rna polymerase (l) and a glycoprotein (g) spanning the viral envelope and a non-structural glycoprotein (gns). g protein is the main protective antigen of the virus and the target of anti-befv-neutralising antibodies and harbours five distinct antigenic sites – g1, g2, g3a, g3b and g4 – on its surface (cybinski et al. 1992; dhillon et al. 2000; kongsuwan et al. 1998). epitope-g1 is a linear site (y487–k503) in the c-terminal region of the ectodomain (trinidad et al. 2014) that only reacts with sera against befv, but other antigenic sites have cross-reactions with the sera against the related viruses besides befv (yin & liu 1997). the prevention and control of bef infection can be achieved through vaccination and treatment of affected cattle (aziz-boaron et al. 2013; wallace & viljoen 2005). various studies have been conducted to develop an efficient vaccine for bef, including live attenuated, inactivated, subunit g protein-based and recombinant vaccines (walker & klement 2015). an effective vaccination has been obtained using the befv g glycoprotein split from a semi-purified virus in cattle (bai et al. 1993). in addition, the befv g glycoprotein delivered in recombinant virus vectors has induced specific neutralising antibodies and cell-mediated immune responses in cattle (hertig et al. 1996; wallace & viljoen 2005). therefore, it appears that the recombinant expressed befv g protein may serve as a useful vaccine antigen (johal et al. 2008). deoxyribonucleic acid (dna) immunisation is a promising approach for vaccination by injection of an isolated eukaryotic expression plasmid encoding the antigen (watts & kennedy 1999). dna vaccination has been used successfully to immunise various animal species against many infectious agents and has several advantages over other vaccination approaches (corr et al. 1996; fynan et al. 1993; robinson, hunt & webster 1993; sakaguchi et al. 1996). however, no effort has been made so far regarding the evaluation of the efficacy of a dna vaccine based on befv g glycoprotein against bef. hence, the purpose of this study was to investigate the immunogenicity of a plasmid dna vaccine encoding the g1 epitope of bef virus g glycoprotein in mice. materials and methods virus, cell lines, bacterial strain and vector the strain of bef virus used in this study was procured from razi vaccine and serum research institute (hesarak, karaj, iran). basic local alignment search tool (blast) analysis based on g gene sequence showed that this strain had the highest identity with the yhl strain isolated in japan’s yamaguchi prefecture in 1966 (pasandideh et al. 2016). hamster lung (hmlu-1) cells were used to propagate the befv using roswell park memorial institute (rpmi) medium (bio idea, iran) supplemented with 5% fetal bovine serum (gibco, uk). human embryonic kidney 293 (hek 293) cells were used for plasmid transfection and expression experiments. hmlu-1 and hek 293 cell lines were received from the national cell bank of iran (ncbi) affiliated with the pasteur institute of iran. dh5α strain of escherichia coli (e. coli) (cinnagen, iran) and pcdna3.1 (+) eukaryotic expression vector (invitrogen, carlsbad, ca, united states of america [usa]) were used for the cloning and protein expression experiments. construction and preparation of expression vector the 420 base pairs (bp) fragment of befv g1 gene was previously cloned into the pcdna3.1 (+) vector under the control of the human cytomegalovirus (cmv) promoter using the g1 specific primers g1-fwd-kpni: 5’-gtgggtaccgccaccatggtgagagcttggtgtgaataca-3’ and g1-rev-bamhi: 5’-cattggatcctcaccaacctacaacagcagata-3’. then, the pcdna3.1-g1 construct containing the 420 bp fragment was transfected into hek 293 cell line to consider protein expression. finally, the expression efficiency was verified by indirect immunofluorescent staining (pasandideh et al. 2018). after verification of protein expression, the pcdna3.1-g1 construct was amplified in e. coli dh5α and purified with the endofree plasmid purification kit (qiagen, germany), according to the manufacturer’s instructions, and then used for immunisation of mice. mice and immunisation six-week-old female mice of n-mari strain were intramuscularly inoculated in four groups of five each. group a mice were immunised with 100 µg/animal of the endotoxin-free pcdna3.1-g1 construct in an appropriate rate of phosphate-buffered saline (pbs) (1x) in the anterior quadriceps muscle. group b mice were vaccinated with 200 µl/animal of bef-inactivated vaccine (kyoto biken laboratories, japan). control groups were only inoculated with 100 µl/animal of pbs (1x) or 100 µg/animal of empty pcdna3.1 (+) plasmid. immunisation was repeated two more times with 2-week intervals. fourteen days after the third immunisation, the animals were bled and the resulting sera were tested for anti-g1-specific antibodies by immunoblotting analysis, indirect enzyme-linked immunosorbent assay (elisa) and virus neutralisation (vn) test. a purified prokaryotic g1 protein with ~18 kda molecular weight, produced in our previous study, was used as a coating antigen to develop immunoblotting and indirect elisa in this study. detection of anti-g1-specific antibodies by immunoblotting immunoblotting analysis was used to investigate the immunogenicity of pcdna3.1-g1 and detect anti-g1-specific antibodies in the serum of mice. for this purpose, the 18-kda prokaryotic g1 protein was electrophoresed on 15% sodium dodecyl sulphate (sds)-polyacrylamide gel and then transferred to a nitrocellulose membrane by electroblotting at 60 volt (v) for 3 hours. the blotted membrane was blocked by pbs with 0.05% tween-20 (pbst) containing 5% skim milk overnight at 4 °c and then washed three times with pbst. the nitrocellulose membrane was cut into strips and incubated with the mouse sera diluted 1:20 in pbst containing 5% skim milk for 2 h at room temperature, individually. after washing with pbst, an anti-mouse igg (h+l)-hrp (bio-rad laboratories, usa) diluted 1:3000 in pbst containing 5% skim milk was added to the strips for 1 h at room temperature. after extensive washing, the strips were dipped in pbs containing h2o2, 4-chloro-l-naphthol and methanol to view the result of reaction. the colour developing reaction was stopped with distilled water. evaluation of anti-g1-specific antibody titers by indirect enzyme-linked immunosorbent assay anti-g1-specific antibody titers in the serum of each immunised mouse were determined by an indirect elisa. ninety-six-well immunoplates were coated with the prokaryotic g1 protein diluted 1:200 in carbonate coating buffer (0.5 m naho3/na2co3, ph 9.3) for 16 h at 4 °c. the final concentration of coating antigen was 0.25 µg/well by calculation. after washing three times with pbst to remove the unbound antigen, the plates were blocked with 300 µl of pbst containing 5% skim milk for 3 h at 37 °c. the plates were washed again with pbst and then incubated with the mouse sera (50 µl/well) diluted 1:100 in pbst containing 5% skim milk for 45 minutes at room temperature. after washing as previously mentioned, an anti-mouse igg (h+l)-hrp (bio-rad) diluted 1:3000 in pbst containing 5% skim milk was added for 45 min at room temperature. the plates were washed four times with pbst and finally 50 µl of the chromogen substrate (tetramethylbenzidine 1%, 0.1 m sodium acetate [ph 6] and h2o2 3%) was added to each well and incubated at room temperature in the dark for 10 min. the reaction was stopped by adding 50 µl of chloridric acid (0.1 m) and the absorbance was read immediately at 450 nanometers (nm) by an elisa spectrophotometer. detection of anti-bovine ephemeral fever virus neutralising antibodies mouse sera were tested for the presence of anti-befv-neutralising antibodies by vn assay. briefly, the sera were heat-inactivated at 56 °c for 30 min, and then 50 µl of two-fold serial dilutions of each serum was mixed with 50 µl of 100 tcid50 of befv in the 96-well tissue culture plate and then incubated for 1 h at 37 °c in 5% co2. after the incubation period, 50 µl of the vero cell suspension containing 15000 cells was added to each well and the plate was incubated for 4 days at 37 °c in a humidified incubator with an atmosphere of 5% co2. after the incubation, the cells were examined for befv-specific cytopathic effects (cpes) using an olympus ix71 inverted optical microscope (olympus australia, mt. waverley, australia). statistical analysis sas software (version 9.1; sas institute) was used for the analysis of elisa data. least significant difference (lsd) test was used to compare the mean of each group of mice, and a p-value of less than 0.01 was considered statistically significant. ethical consideration the authors declare that the project underwent ethical review and was given approval by an institutional animal care and use committee or by appropriately qualified scientific and lay colleagues. the care and use of experimental animals complied with local animal welfare laws, guidelines and policies. animal studies have been approved by the appropriate ethics committee of the khuzestan agricultural sciences and natural resources university (1/411/1189). results expression of g1 protein by the pcdna3.1-g1 construct the expression of g1 protein by pcdna3.1-g1 was confirmed using indirect immunofluorescence staining. the observation of intracytoplasmic fluorescence in the transfected cells with pcdna3.1-g1 in reaction to an anti-g1 monospecific polyclonal antibody, produced in our previous study (beygi nassiri, pasandideh & seyfi abad shapouri 2016), indicated that g1 protein was successfully expressed by pcdna3.1-g1 in the hek 293 cells (figure 1). figure 1: verification of g1 protein expression in immunofluorescence staining. (a) image of the transfected human embryonic kidney 293 (hek 293) cells with the pcdna3.1-g1 construct obtained by a fluorescence microscope and (b) transfected hek 293 cells with empty pcdna3.1 plasmid used as negative control. verification of anti-g1-specific antibodies using immunoblotting immunoblotting analysis was used to evaluate the immunogenicity of the pcdna3.1-g1 construct in mice. for this purpose, the 18-kda prokaryotic g1 protein was blotted onto a nitrocellulose membrane and exposed to serum of inoculated mice, separately. as shown in figure 2, the appearance of a distinct band with an approximate molecular weight of 18 kda for immunised mice by pcdna3.1-g1 and bef-inactivated vaccine indicated that specific antibodies against befv g1 protein were induced in these groups. figure 2: verification of anti-g1 antibodies against pcdna3.1-g1 and bovine ephemeral fever (bef)-inactivated vaccine in reaction to the 18-kda g1 protein using immunoblotting. lanes 1–4, respectively, show the membrane strips exposed to serum of inoculated mice with phosphate-buffered saline (1x), pcdna3.1, pcdna3.1-g1 and bef-inactivated vaccine; lane 5 shows the marker polypeptides. anti-g1-specific antibody titers after dna vaccination two weeks after the last immunisation, anti-g1-specific antibody titers in serum of inoculated mice were assayed using elisa. it was observed that anti-g1 antibody titers in mice immunised with pcdna3.1-g1 were significantly higher than those in control groups for plasmid and pbs 1x (p < 0.01). the most significant anti-g1-specific antibodies were elicited in mice vaccinated with bef-inactivated vaccine (p < 0.01) (table 1). table 1: anti-g1 antibodies produced in mice after intramuscular inoculation of plasmids and bovine ephemeral fever-inactivated vaccine measured by enzyme-linked immunosorbent assay. induction of neutralising antibodies against bovine ephemeral fever virus the presence of anti-befv-neutralising antibodies in the sera of immunised mice was investigated by vn assay. the sera collected from mice immunised with pcdna3.1-g1 and bef-inactivated vaccine neutralised all the virus activity up to 1:50 dilution and prevented befv-specific cpes from developing in vero cells (figure 3). therefore, our results indicated that immunisation with the pcdna3.1-g1 construct could elicit neutralising antibody responses against bef virus. as expected, the cpes caused by the virus proliferation were observed in the cells treated with the sera collected from the control groups for plasmid backbone and pbs (1x). figure 3: microscopic examination of the vero cells for evidence of viral cytopathic effects in virus neutralisation assay. (a and b) the cpes in the cells treated with the sera collected from control groups for pcdna3.1 and pbs, respectively; (c and d) the cells treated with the sera collected from the groups immunised with pcdna3.1-g1 and bovine ephemeral fever (bef)-inactivated vaccine, respectively. discussion natural bef infection leads to long-term immunity in affected animals (mackerras, mackerras & burnet 1940). hence, vaccination can be considered as an effective approach of prevention against the disease. so far, several studies have been developed to produce diverse vaccines for bef, including live attenuated, inactivated, subunit g protein-based and recombinant vaccines. live attenuated, inactivated and subunit vaccines are being used in the field (walker & klement 2015). however, according to our knowledge, no study has been performed to design a dna vaccine based on g1 gene for immunisation against bef and this was the first study in this area. in this study, a eukaryotic expression construct for g1 epitope of befv g glycoprotein gene was designed in order to evaluate its immunogenicity and efficacy in mice. serological assays showed that the pcdna3.1-g1 construct expressing g1 protein was able to induce specific immunity and produce antibodies against this antigen. however, the anti-g1-specific antibody titers against pcdna3.1-g1 were significantly lower than those against bef-inactivated vaccine. it may be for this reason that only an antigenic site of befv g glycoprotein gene was used in the pcdna3.1-g1 construct. virus neutralisation test showed that pcdna3.1-g1 could induce anti-befv-neutralising antibodies in immunised mice. in previous studies, it was found that befv g protein could induce virus-specific neutralising antibodies and confer passive protection against intracerebral infection of suckling mice (cybinski et al. 1990) and protect cattle against experimental intravenous befv challenge (hertig et al. 1996; johal et al. 2008; uren et al. 1994). the nucleotide and amino acid sequences of the g1 antigenic site of g glycoprotein have been highly conserved among all isolates, except for an amino acid substitution at position 499 for a few strains (kato et al. 2009; zheng & qiu 2012). however, amino acid variations detected in the main neutralisation sites (g1, g2 and g3) of the g protein did not affect the neutralisation properties of these epitopes (trinidad et al. 2014). high immunogenicity of g glycoprotein and the fact that g1 epitope has been genetically and antigenically conserved among various isolates of befv allow the use of g1 as a useful vaccine antigen. therefore, g1 antigen was chosen for application as a possible dna vaccine for immunisation of mice in this study. today, dna vaccines are widely considered because of many advantages, such as safety, stability, low costs and longer immunogenicity (porter & raviprakash 2017). the induction of anti-befv-neutralising antibodies in mice by the pcdna3.1-g1 construct expressing g1 protein in our research was consistent with the immunogenicity of subunit and recombinant vaccines based on g glycoprotein in previous studies. for example, vaccination using the befv g protein split from a semi-purified virus induced a neutralising antibody response and protected 50% of cattle in china (bai et al. 1993). in australia, administration of a g protein subunit vaccine with quil a adjuvant protected 100% of cattle against experimental challenge (uren et al. 1994). vaccination with recombinant new york board of health (nybh) strain of vaccinia virus expressing the befv g protein could elicit specific neutralising antibodies in cattle, but the protection experiment was inconclusive (hertig et al. 1996). in the same experiment, four doses of the neethling strain of lumpy skin disease virus expressing the befv g protein induced a specific neutralising antibody and cell-mediated immune responses in cattle but protection failed 10 weeks after the last dose (wallace & viljoen 2005). although various vaccines with different formulations have been developed for bef, there are few reports about the assessment of the vaccines under conditions in the field and their usage rates are often low. it appears that protective immunity for most of these vaccines continues for a limited period and their efficiency may be insignificant unless additional booster doses are administered at intervals of 6 months to 1 year (walker & klement 2015). therefore, other advanced technologies are needed to decrease the required number of doses and prolong the duration of protection. on the other hand, the cell-mediated responses may also be involved in protection against bef, especially for the longer-term sequelae that occur in some animals (della-porta & snowdon 1979; walker & klement 2015). regarding the ability of dna vaccines to induce protective humoral and significant cellular immune responses to the expressed antigens (khan 2013), it seems dna vaccination can be an appropriate approach against bef. however, as found in this study, the eukaryotic expression plasmids encoding the antigens usually induce fewer responses compared to live or inactivated pathogen immunisation (shah et al. 2011). we suggest the use of genetic adjuvants such as cytokine genes with the g1 antigen to improve the efficacy of the pcdna3.1-g1 construct in future studies. conclusion this study demonstrated that the pcdna3.1-g1 construct could induce immunity and protection against the befv in an animal model. our findings indicated that a new dimension can be added to vaccine studies for bef using eukaryotic expression plasmids encoding the g1 antigen in the future. obviously, further studies are needed to improve this type of vaccines and obtain more comprehensive information about their performance in the main hosts. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions r.p. performed the experiments and the data analysis and prepared the manuscript. m.r.s.a.s. conceived and designed the research and completed the manuscript revision. m.t.b.n. provided helpful suggestions regarding the study. all authors read and approved the final manuscript. references aziz-boaron, o., leibovitz, k., gelman, b., kedmi, m. & klement, e., 2013, ‘safety, immunogenicity and duration of immunity elicited by an inactivated bovine ephemeral fever vaccine’, plosone 8, e82217. https://doi.org/10.1371/journal.pone.0082217 bai, w., yan, j., zhang, z., jiang, c. & lin, x., 1993, ‘studies on a vaccine against ephemeral fever [bovine ephemeral fever virus; 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https://doi.org/10.1016/s0020-7519(99)00112-5 yin, z. & liu, j., 1997, animal virology, science press, beijing, china. zheng, f. & qiu, c., 2012, ‘phylogenetic relationships of the glycoprotein gene of bovine ephemeral fever virus isolated from mainland china, taiwan, japan, turkey, israel and australia’, virology journal 9, 1–8. https://doi.org/10.1186/1743-422x-9-268 zheng, f.y., lin, g.z., qiu, c.q., zhou, j.z., cao, x.a. & gong, x.w., 2009, ‘development and application of g 1-elisa for detection of antibodies against bovine ephemeral fever virus’, research in veterinary science 87, 211–212. https://doi.org/10.1016/j.rvsc.2009.03.010 abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) noluvuyo r. magadla department of agriculture and rural development, johannesburg, south africa department of production animal studies, university of pretoria, south africa wilna vosloo csiro-australian animal health laboratory, geelong, australia livio heath agricultural research council, onderstepoort veterinary institute, south africa bruce gummow department of production animal studies, university of pretoria, south africa discipline of veterinary science, james cook university, australia citation magadla, n.r., vosloo, w., heath, l. & gummow, b., 2016, ‘the african swine fever control zone in south africa and its current relevance’, onderstepoort journal of veterinary research 83(1), a1034. http://dx.doi.org/10.4102/ojvr.v83i1.1034 original research the african swine fever control zone in south africa and its current relevance noluvuyo r. magadla, wilna vosloo, livio heath, bruce gummow received: 15 aug. 2015; accepted: 03 dec. 2015; published: 23 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract african swine fever (asf) has been reported in south africa since the early 20th century. the disease has been controlled and confined to northern south africa over the past 80 years by means of a well-defined boundary line, with strict control measures and movement restrictions north of this line. in 2012, the first outbreak of asf outside the asf control zone since 1996 occurred. the objective of this study was to evaluate the current relevance of the asf control line as a demarcation line between endemic asf (north) areas and asf-free (south) area and to determine whether there was a need to realign its trajectory, given the recent outbreaks of asf, global climate changes and urban development since the line’s inception. a study of asf determinants was conducted in an area 20 km north and 20 km south of the asf control line, in limpopo, mpumalanga, north west and gauteng provinces between may 2008 and september 2012. the study confirmed that warthogs, warthog burrows and the soft tick reservoir, ornithodoros moubata, are present south of the asf control line, but no virus or viral dna was detected in these ticks. there appears to be an increasing trend in the diurnal maximum temperature and a decrease in humidity along the line, but the impact of these changes is uncertain. no discernible changes in minimum temperatures and average rainfall along the disease control line were observed between 1992 and 2014. even though the reservoirs were found south of the asf boundary line, the study concluded that there was no need to realign the trajectory of the asf disease control line, with the exception of limpopo province. however, the provincial surveillance programmes for the reservoir, vector and asf virus south of this line needs to be maintained and intensified as changing farming practices may favour the spread of asf virus beyond the control line. introduction african swine fever virus (asfv) is a highly contagious dna arbovirus belonging to the genus asfivirus of the family asfarviridae (fauquet et al. 2005) affecting domestic pigs. the virus replicates in both the mammalian host and ornithodoros moubata complex ticks (also called tampans), the arthropod host (dixon et al. 2004). the infection is characterised by high morbidity and mortalities of up to 100% in domestic pigs, but the presence of the disease can remain unnoticed in wild pigs, with neonatal common warthogs (phacochoerus africanus) developing viraemia high enough to infect ornithodoros ticks that feed on them (bastos et al. 2009; thomson 1985). the virus can also cause mortalities in ticks (kleiboeker & scoles 2001). the virus is maintained and transmitted through different cycles including the following: (1) the typical sylvatic cycle, where the asfv is maintained between warthogs and tampans with occasional spill-over to domestic pigs, (2) the endemic cycle (tampan or domestic pig cycle) which has been reported in east africa and (3) the domestic cycle, involving the domestic pig population where asfv can be transmitted by direct contact between infected and susceptible domestic pigs. the common warthog is the preferred vertebrate host for ornithodoros ticks that inhabit preexcavated burrows used for farrowing and shelter (arnot, du toit & bastos 2009). together the warthogs and ticks are the determinants of the sylvatic cycle for maintenance and transmission of african swine fever (asf) in the south african context (magadla 2015). in south africa, reports of asf date back to as early as 1926 when it was first recorded in the northern parts of the country, formerly known as transvaal (boshoff et al. 2007). in 1935, south africa instituted and gazetted a designated asf control area that mainly encompasses the limpopo province, the northern parts of north west and kwazulu-natal provinces and the north-eastern parts of mpumalanga province (figure 1). the designation of the area was based on the presence of epidemiologically significant factors (i.e. host, environmental and agent factors) and the presence of outbreaks (penrith, thomson & bastos 2004). the last reported outbreak in mpumalanga occurred in 1951. in 1996, an outbreak was reported just outside the control area in bela-bela, limpopo province (penrith et al. 2004). during this period, a number of cases or outbreaks occurred within the control zone in the limpopo province and were reported to the department of agriculture, forestry and fisheries (daff), south africa, and subsequently to the world organisation for animal health (oie). figure 1: spatial distribution of african swine fever outbreaks in south africa between 1993 and 2012. in january 2012, gauteng veterinary services reported to daff a suspected case of asf in a group of pigs that demonstrated clinical signs at a gauteng abattoir. this was the first outbreak of asf outside the control zone since 1996 (gauteng veterinary services 2012). within a period of 2 months from the confirmation of the index case, south africa reported an additional 16 outbreaks of asf to the oie, all diagnosed and confirmed outside the asf-controlled area in gauteng and mpumalanga provinces (oie 2014). the spatial distribution of the asf outbreaks that occurred in south africa between 1993 and 2012 is shown in figure 1. the article describes the first study to investigate the relevance of the asf control line as a demarcation between endemic asf (north) areas and asf-free (south) areas, since its institution. it also examines available climatic data along the control line to assess if there have been changes in climatic factors between 1993 and 2012 that could influence warthog or tampan distribution. materials and methods study area the asf control line, as determined by the animal diseases act of south africa (act 35 of 1984), was used as a reference for the study area. using the asf control line as a basis, a 20-km virtual boundary was built both north and south of the control line to traverse limpopo, mpumalanga, north west and gauteng provinces (figure 2). the asf control area in kwazulu-natal province was not included because of distance and limited resources. a sampling frame of farms in the study area was compiled using area maps obtained from the department of water affairs, pretoria, south africa, and the list of farms obtained from limpopo and gauteng provincial offices of daff. the total number of farms in the sampling frame was 1575. all samples were collected between may 2008 and september 2012. the aim of the survey was to establish the asfv distribution pattern along the control line. this would give an indication on whether the line served as a boundary for the disease in historic and recent outbreaks. figure 2: sampling area (between yellow lines) and spatial distribution of warthog burrows sampled for presence of tampans. survey design the survey was designed to sample 61 warthog burrows (i.e. the sampling unit was burrows and not farms). this number was based on the assumption that 20% of warthog burrows had infected tampans (pretorius et al. 2004) and the sample size was calculated using the formula: where n is the sample size, p is prevalence of warthog burrows with infected tampans and d = 10% is the margin of error at a 95% confidence interval (thrusfield 2005). the study was based on the assumption that 20 in every 100 farms had warthog burrows (pretorius et al. 2004) and using the formula n = n + negative binomial (n + 1, p), where n is the number of farms needed to be found and p is the proportion with warthog burrows with infected tampans (vose 2001). to be 95% confident of finding 61 burrows, 304 farms needed to be sampled. proportional weighting, based on the total number of farms in each province, was used to determine the number of farms to be sampled in each province. the selection of the 304 farms from the sample frame was carried out using survey toolbox, random village sampling (cameron 1999). warthog burrows were purposefully selected on each farm according to whether they were recently used by warthogs. warthog burrow sampling farms were visited between may and november during 2008–2012, and with the aid of farm staff warthog burrows were identified on each of the farms surveyed. each burrow was scraped 10 times using a spade specially modified for this purpose, spending a minimum of 30 min and a maximum of 45 min per burrow. scraping followed a set pattern of two scrapings each in the proximal (entrance) area, the deep areas, each of the sides and the bottom. a black plastic sheet was spread next to the burrow. the collected soil scrapings were spread on the black sheet under direct sunlight to facilitate detection of the tampans. all collected tampans were submitted to a central submission point, the onderstepoort veterinary institute, onderstepoort, gauteng, south africa, as part of their transboundary animal diseases programme. detection of african swine fever virus dna in tick samples at the onderstepoort veterinary institute, total dna was extracted from a pool of tampans crushed in a 1.5 ml eppendorf tube containing 1 ml of phosphate-buffered saline, supplemented with 1% foetal calf serum and 1% of a combination of antibiotics and an antimycotic. each pool of tampans was composed of tampans from the same warthog burrow. homogenates were centrifuged at 10 000 x g for 1 min and the supernatant frozen at -70 °c. dna was extracted from 200 µl of each tick homogenate and recovered in a final volume of 50 µl dna solution using the qiamp kit (qiagen gmbh, hilden), according to the manufacturer’s instructions. a nested polymerase chain reaction (pcr) that targets the c-terminal end of the p72 gene was used to screen soft tick samples for the presence of asfv dna (basto et al. 2006). all dna samples were tested for tick mitochondrial 16s recombinant dna according to published methodology (black & piesman 1994; vial et al. 2007) to exclude the occurrence of inhibitors present in the tick homogenates. questionnaire survey farm and warthog burrow data were collected from pig farm owners in the surveyed area by an interview-based questionnaire at the same time as the tick sampling was conducted and captured in microsoft excel 2010®. the questionnaire comprised 23 questions with subcomponents and the collected farm information included main farming activities, use of acaricides, presence of warthogs and other suid species on the farm, contact between warthogs and domestic pigs and estimated number of warthogs and warthog burrows on the farm. the warthog burrow information included individual burrow gps coordinates, habitat (classified into open veld, bushveld, riverine or wetlands, cultivated lands and others), the soil type (graded as sandy, rocky, muddy and clay), where the burrow was found, whether the burrow was active or inactive and the estimated number of tampans found (many [> 20], few [5–20] and very few [> 5]). spatial distribution the geographical distribution of the farms, warthog burrows and warthog burrows where tampans were found were mapped using the geographical information system software – arcgis 10.1 for desktop (esri 2012) and diva-gis 7.5.0.0 (hijmans et al. 2012). climate data the weather data, in monthly averages of minimum and maximum temperatures and millimetres of rainfall and humidity for the period 1993–2012 were obtained from the south african weather services in microsoft excel 2010®. the weather data were summarised into three seasonal averages: summer (december to february), autumn (march to may) and spring (september to november). winter (june to august) was omitted from the rainfall analysis as the study area is a summer rainfall area with very low potential rainfall in winter. the moving average of these three time periods was calculated using the formula: where mat is the moving average at time (t), n is the number of prior periods to include in the moving average and ‘at’ is the actual value at time (t). the centred moving average of the two time periods was calculated using the same formula. linear trend lines (y = mx + b, where m is the slope and b the intercept) were calculated and plotted using microsoft excel 2010 data analysis tools and charts. the linear regression analysis and time series graphs from microsoft excel 2010® data analysis tool were used to prove the statistical significance of values. results questionnaire survey results and presence of warthogs and burrows on farms a much higher proportion of farms had warthog burrows than anticipated when the study was designed. this resulted in the need to sample fewer farms than anticipated in order to meet the required sample size of 61 warthog burrows. table 1 shows that the required number of warthog burrows sampled exceeded the minimum number required in each province, thus ensuring that the power of the study was met. a total of 73 farms were surveyed in the study area, of which 86.3% had warthog burrows (table 1) and 72.6% had some sign of warthog activity, with warthogs seen on 66% of the farms during the visit (table 2). fifty-eight percent of the farms visited reported seeing warthogs on neighbouring farms. half the farms visited were wildlife farms, 30.1% farmed with livestock, 11% were crop farmers and the rest were in residential and mixed farming areas. only one property (a nature reserve) had obvious contact between domestic pigs and warthogs. eighteen farmers claimed to have seen an increase in the number of warthogs, and this was ascribed to conservation practices on their farms. although warthogs were not counted, 23.3% of farmers estimated they had between 1 and 20 warthogs on their farms, 17.8% had 21–40, 17.8% had 41–60 and 30.1% had more than 60 (table 2). table 1: number of farms and burrows sampled per province and prevalence of tampans. table 2: summary of farm information gathered through questionnaire survey. presence of tampans and their african swine fever virus infection status along the control line a total of 152 warthog burrows, three storm drains and two nests were sampled across 63 farms with burrows in the study area (table 1 and figure 2). the three storm drains and two nests (flattened grassy areas) were identified by farmers as areas where warthogs were residing on their property, which is why they were included in the sample. approximately 73.3% of warthog burrows were located in the bushveld area. the sampling teams found 61.8% of burrows were located in sandy soil, 17.2% in muddy soil, 12.7% in clay soil and 8.3% in rocky areas. out of the sampled warthog burrows, 92% had evidence of active use by animals. tampans were recovered from 20 (12.8%) of the sampled warthog burrows (95% confidence interval: 8.0% – 19.0%). table 1 shows the breakdown of recovered tampans by province. only 1 out of the 20 burrows from which tampans were recovered had no apparent signs of recent habitation. approximately 20% of burrows had < 5 tampans, 35% had between 5 and 20 tampans and 45% had more than 20 tampans. pools of tampans per burrow were tested for the presence of asfv dna. one of the farms, situated along the control line located in limpopo province in close proximity to the asf control line, had tampans in which asfv dna was found. however, no live virus was isolated from these pcr-positive tampan samples. climate changes along the control line the data for maximum and minimum average seasonal temperatures showed an increasing trend (figure 3). on regression analysis, there was a linear increase in maximum temperature between 1995 and 2012 (linear line; figure 3) that was statistically significant (p = 0.00018), but no significant change in minimum temperature (p = 0.6; r2 = 0.0017). the average humidity in the area along the asf control line showed a statistically significant decreasing trend (figure 4) (p = 0.003; r2 = 0.106) at a 95% confidence level, but the decrease observed in seasonal rainfall was not significant (p = 0.34; r2 = 0.015). figure 3: average seasonal maximum temperature along the african swine fever control line: 1993–2012. figure 4: mean seasonal humidity along the african swine fever control line: 1993–2012. discussion our study confirmed the presence of warthogs along the control line, but it is likely this has been the case throughout the existence of the control line. however, it was not anticipated that such a high proportion of farms would have warthog burrows on them and this impacted on the number of farms finally sampled. the original study design was based on pilot studies carried out in gauteng province where at the time approximately 20% of farms had warthog burrows (pretorius et al. 2004). despite the high prevalence of warthog burrows only 13% of them contained tampans, making the risk of transmission of asfv along the control line low. only 25% of the farmers in our study claimed to have observed an increase in warthog numbers over the past 5 years and this was thought by them to be mainly because of nature conservation practices on their farms. what has changed in recent times along the control line is an increase in game farming in south africa, including in the areas along the asf control line. a change from approximately 575 000 to 18.6 million wildlife animals has been documented between 1964 and 2007 (carruthers 2008), with a threefold increase in the number of wildlife farms between 1981 and 1992. the shift to wildlife-based production has been recognised as the most rapidly expanding agricultural activity (snijders 2012). the increase in the number of wildlife farms poses a potential risk for movement and increase in the number of warthogs. studies in the eastern cape have shown that common warthogs have the characteristics of an invasive species and have spread beyond the targeted introduction site (nyafu 2009). countering the potential effect of wildlife farms is increasing urban development and market crops along the control line, with farmers in the residential or communal farming areas, mixed farming and crop farming areas claiming that they had observed a decreasing number of warthogs over the years. the main reasons for this decrease were ascribed to hunting, changes in farming practices and changes in human population distribution, with signs of poaching traps identified on some farms. changes in farming practice towards crop farming could, however, influence the distribution of warthogs because they are known to forage crop fields (fao 2010) and are likely to gravitate towards cultivated areas, thus increasing the risk of virus being spread along the control line. expanding communal residential areas, where free-ranging domestic pigs are often kept, could increase the possibility of warthogs and tampans coming into contact with domestic pigs in these areas that could lead to outbreaks. adding to the complexity of factors playing a role along the control line is the effect of climate change (gummow 2010). the study showed that mean daily maximum temperatures have increased and humidity levels have decreased along the control line over the period of the study (1993–2012). if these trends continue, the area along the asf control line is likely to become dryer and this could also lead to warthogs moving southwards in search of better conditions for survival and thus lead to a wider distribution of tampans as well. tampans appear to be able to survive drier conditions than those that currently exist along the control line and the change in temperature and humidity is not likely to impact on the survivability of the tampans themselves (d’huart & grubb 2001). the overall infestation rate of the warthog burrows varied amongst provinces and was consistent with what other researchers have found (bastos et al. 2009; plowright, parker & pierce 1969). the study clearly showed that tampan-infested warthog burrows are widely spread throughout the study area both north and south of the asf control line. the greatest proportion of tampan-infested warthog burrows was in gauteng province (21.5% of burrows) and may partly be attributed to targeted sampling based on previous knowledge of where burrows were situated (pretorius et al. 2004). although asf dna was not detected in these tampans, the high proportion of infested burrows in this region supports the need for continued active surveillance along this part of the control line. in mpumalanga province, tampans were found in only 6.67% of warthog burrows on both sides of the control line and no viral dna was detected by pcr, suggesting that there is probably no need to shift the control line in these areas. tampans have previously been recorded in the mpumalanga province in the area along the kruger national park (penrith et al. 2004). our study mostly found tampans in areas bordering communal residential areas. the northern portion of the northwest province forms part of the asf-controlled area. the province borders south-western limpopo province, which is considered a high-risk area where tampans of o. moubata complex are found (penrith et al. 2004). in our study, an insignificant number of tampans (less than 5) was found in one warthog burrow situated north of the asf control line in northwest province and these tested negative for asfv dna. based on the small proportion of infested burrows found in this province, it seems therefore, that there is little need to shift the control line in northwest province. limpopo province forms the largest section of the asf control area where outbreaks of asf and tampans occur (penrith et al. 2004) and this province had the second highest proportion of tampan-infested burrows (17.5%). it is also the only province to have tampans, which tested positive for asfv during pcr screening. these were recovered from within a crop farming area situated south of but in close proximity to the asf control line, suggesting that the control line may need realigning in this area of the country. from the literature, it would seem that infection rates of tampans with asfv in the vicinity of the control line have always been low and our study confirms this. penrith et al. (2004) reported rates between 0.3% and 1.7%, whilst kleiboeker & scoles (2001) cited rates between 0% and 3.8%. one of the highest infection rates of asfv reported was in livingstone game park, zambia, where it was recorded to be 5.1% (wilkinson et al. 1988). a potential confounder in the study was the stage of ticks collected. wilkinson et al. (1988) emphasised that the overall infection rate of tampans depends on the relative proportions of different stages of ticks with a higher rate where the populations have a higher proportion of adults. the tampans collected for our study had approximately equal proportion of adults and nymphal stages, which may have decreased the sensitivity of detecting virus. future monitoring may benefit from looking only at adult ticks to detect virus. conclusion the study confirmed that warthogs, warthog burrows and tampans are found beyond the asf control line and that regular monitoring of the control line for asfv is recommended. only one farm had tampans infected with asfv, but no live virus was isolated. there is therefore limited evidence of asfv in tampans outside the asf control line at this time and the asf control line remains largely well positioned, with a possible exception in the limpopo province. changing farming practices to wildlife and crops and changing weather conditions along the control line may be creating environments that are suitable for wider spread of warthogs. this, coupled with increased informal settlement along the control line, could increase the risk of contact with domestic pigs where the virus could be amplified. regular monitoring of the control line is therefore recommended and the study serves as a basis for future monitoring. acknowledgements we acknowledge the gauteng, mpumalanga, limpopo and north west provincial veterinary services, daff and the agricultural research council for making the research possible and thank them for their support in carrying out this work. the authors are grateful for the funding received from the national research foundation, pretoria, south africa for this project. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions n.r.m. conducted the research as part of an msc at the university of pretoria under the supervision of b.g. and w.v. l.h. assisted with the molecular biology component of the project. n.m., b.g. and w.v. were all involved in the experimental design, acquiring funding, data analysis and writing up of the work. n.m. managed the field component of the project. references arnot, l.f., du toit, j. & bastos, a.d., 2009, ‘molecular monitoring of african swine fever virus using surveys targeted at adult ornithodoros ticks: a re-evaluation of mkuze game reserve, south africa’, onderstepoort journal of veterinary research 76, 385–392. basto, a.p., portugal, r.s., nix, r.j., cartaxeiro, c., boinas, f., dixon, l.k. et al., 2006, ‘development of a nested pcr and its internal control for the detection of african swine fever virus (asfv) in ornithodoros erraticus’, archives of virology 151, 819–826. bastos, a.d.s., arnot, l.f., jacquier, m.d. & maree, s., 2009, ‘a host species-informative internal control for molecular assessment of african swine fever 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‘wild property and its boundaries – on wildlife policy and rural consequences in south africa’, journal of peasant studies 39, 503–520. thomson, g.r., 1985, ‘the epidemiology of african swine fever: the role of free-living hosts in africa’, onderstepoort journal of veterinary research 52, 201–209. thrusfield, m., 2005, veterinary epidemiology, 3rd edn., blackwell, ames. vial, l., wieland, b., jori, f., etter, e., dixon, l. & roger, f., 2007, ‘african swine fever virus dna in soft ticks, senegal’, emerging infectious diseases 13(12), 1928–1931. vose, d., 2001, risk analysis: a quantitative guide, 2nd edn., john wiley & sons ltd, west sussex. wilkinson, p.j., pegram, r.g., perry, b.d., lemche, j. & schels, h.f., 1988, ‘the distribution of african swine fever virus isolated from ornithodoros moubata in zambia’, epidemiology and infection 101, 547–564. abstract introduction methods results discussion conclusion acknowledgements references about the author(s) gholamreza razmi department of pathobiology, ferdowsi university of mashhad, islamic republic of iran saeed yaghfoori department of pathobiology, ferdowsi university of mashhad, islamic republic of iran mehrdad mohri department of clinical sciences, ferdowsi university of mashhad, islamic republic of iran alirez haghparast department of pathobiology, ferdowsi university of mashhad, islamic republic of iran shahin tajeri department of pathobiology, ferdowsi university of mashhad, islamic republic of iran citation razmi, g., yaghfoori, s., mohri, m., haghparast, a. & tajeri, s., 2019, ‘the haematological, proinflammatory cytokines and igg changes during an ovine experimental theileriosis’, onderstepoort journal of veterinary research 86(1), a1629. https://doi.org/10.4102/ojvr.v86i1.1629 original research the haematological, proinflammatory cytokines and igg changes during an ovine experimental theileriosis gholamreza razmi, saeed yaghfoori, mehrdad mohri, alirez haghparast, shahin tajeri received: 11 mar. 2018; accepted: 12 oct. 2018; published: 04 feb. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract malignant ovine theileriosis is caused by theileria lestoquardi, which is highly pathogenic in sheep. theileriosis involves different organs in ruminants. little is known about the role of proinflammatory cytokines in the pathogenesis of t. lestoquardi infection. the aim of this study was to measure concentration changes of proinflammatory cytokines and immunoglobulin g (igg) during an ovine experimental theileriosis and correlate it with clinical and haematological parameters. during an experimental study, seven healthy baluchi sheep (four females and three males) about 6–8 months old were infected with t. lestoquardi by feeding of infected unfed ticks on the sheep’s ears. the infected sheep were clinically examined during the study and blood samples were collected on days 0, 2, 5, 7, 10, 12, 14, 17 and 21. the haematological parameters were analysed by an automatic veterinary haematology cell counter and the inflammatory cytokines interleukin-6 (il-6), tumour necrosis factor-α (tnf-α), interferon-γ (ifn-γ) and igg were measured by enzyme-linked immunosorbent assay. all infected sheep had temperatures above 40 °c on days 3–4 post infection (pi). the maximum temperature was noted on day 7, and it remained high until day 21. the parasitaemia of t. lestoquardi infection increased from 0.01% (day 7 pi) to 3.3% (day 21 pi). the mean white blood cell (wbc), red blood cell (rbc), lymphocyte, neutrophil and platelet values slightly increased on day 2 pi and decreased by day 17 and day 21 pi. the percentage parasitaemia and fever had a negative correlation with the numbers of wbcs, rbcs, lymphocytes, neutrophils and platelets. the serum concentration of il-6, tnf-α and ifn-γ cytokines increased and peaked on day 12 and thereafter decreased to levels lower than 0. out of all tested cytokines, the concentration of il-6 was significantly higher, as early as day 2 pi. no significant changes were observed for the igg levels during the course of disease. a significant and strong correlation was observed between il-6, tnf-α and ifn-γ values and a moderate correlation between il-6 and the numbers of lymphocytes in the present study. a strong correlation was determined between the percentage parasitaemia and haematological parameters in t. lestoquardi-infected sheep. in addition, preliminary results indicate that the measurement of the serum concentrations of il-6 in combination with haematological parameters could be considered a good marker to estimate the pathogenicity of t. lestoquardi strain. introduction ovine malignant theileriosis is an important tick-borne disease with high mortality rates. the disease is prevalent in the middle east, east and north africa, india, china, central asia and eastern and southern europe (ahmed et al. 2011; al-hamidhi et al. 2016; el imam & taha 2015). the agent of disease is t. lestoquardi, which is transmitted by hyalomma anatolicum ticks. the life cycle of t. lestoquardi is similar to that of t. annulata (ahmad et al. 2011; morrison 2015). briefly, when the ticks suck blood, the sporozoites are inoculated into blood and quickly enter monocytes and lymphocytes of associated lymph nodes near the tick bite. the sporozoites transform to trophozoites and develop to macroschizonts. the macroschizont development causes transformation and proliferation of the infected and non-infected lymphocytes and monocytes. later, the macroschizonts develop into microschizonts that produce many merozoites in infected lymphocytes or monocytes. the merozoites are released after lymphocyte disruption and enter the erythrocytes and transform to piroplasms with ring, dot and rod forms (ahmad et al. 2011; morrison 2015). the pathogenicity of theileria species is largely related to the ability of theileria schizonts to induce high proliferation of mononuclear leucocytes and the capacity to metastasise and multiply in non-lymphoid as well as lymphoid tissues (dobbelaere & küenz 2004; tretina et al. 2015). some studies have shown that the pathogenesis of acute theileriosis could be related to the high-level production of proinflammatory cytokines during the course of disease in t. annulata-infected cattle (glass et al. 2000; graham et al. 2001) and t. parava-infected cattle (yamada et al. 2009). so far, the role of proinflammatory cytokines in the immunopathogenesis of t. lestoquardi infection in sheep has been studied less than that of t. annulata and t. parva infection in cattle. the aim was to measure haematological parameters and proinflammatory cytokines (il-6, tnf-α, ifn-γ) and igg levels and to determine the correlation of the proinflammatory cytokine levels with haematological parameters during an ovine experimental theileriosis. methods experimental transmission in this study, seven baluchi sheep (4 females, 3 males) aged between 6 and 8 months were bought from a farm which had no history of theileriosis. the sheep were experimentally infected with t. lestoquardi as performed previously (yaghfoori et al. 2016, 2017). briefly, infected adult h. anatolicum with t. lestoquardi were prepared at the parasitology department, faculty of veterinary medicine of the ferdowsi university of mashhad, iran. for experimental transmission of t. lestoquardi, adult ticks of h. anatolicum (8 males, 22 females) were placed in cotton bags on the ears of each sheep. all sheep were clinically examined on days 0, 2, 5, 7, 10, 12, 14, 17 and 21, and the clinical signs recorded. the blood and lymph node smears were simultaneously prepared and thereafter blood samples (10 ml) were taken from the jugular vein into serum and ethylenediaminetetraacetic acid (edta) tubes. the blood and lymph node smears were stained using the giemsa method. the blood in serum tubes were centrifuged at ×1800 g rpm for 10 min and the serum samples were transferred to plain tubes and kept at -80 °c until the experiment was performed. the edta blood samples were kept at 4 °c until cell blood count (cbc) examination was performed. microscopic examination the blood and lymph node smears were stained using the giemsa method. the stained smears were examined for detection of trophozoites and schizonts of t. lestoqaurdi using a light microscope at ×1000 magnification. the parasitaemia of t. lestoquardi infection was determined by counting parasites in 100 microscopic fields in the blood smears (razmi et al. 2003). cell blood count determination total cell counts and differential wbc counts were measured on days 0, 2, 5, 7, 10, 12, 14, 17 and 21 in all sheep with an automatic veterinary haematology cell counter (nihon kohden, celltac α, nek–6450 k, tokyo, japan). sampling for cytokine measurement commercially available enzyme-linked immunosorbent assay (elisa) kits (biotechnology laboratory, china), including il-6, tnf-α, ifnγ and igg, were used to measure respective cytokine and antibody levels according to the manufacturer’s instructions. briefly, flat-bottomed 96-well plates were coated with cytokine-specific monoclonal antibodies labelled with biotin. a volume of 40 μl serum sample, 10 µl of anticytokine antibody and 50 μl of streptavidin-hrp were added to each well and incubated at 37 °c for 60 min. each well was washed with washing solution, then 50 μl chromogenic solution a and 50 μl chromogen solution b were added and incubated at 37 °c for 10 min. the reaction was stopped by adding 50 μl stop solution and optical density (od) was read at 450 nanometre (nm) in an elisa plate reader (elx800 absorbance reader, biotek, winooski, vt). cytokine concentrations (ng/l) were determined by using a standard curve. detection limits of il-6, tnf-α, ifnγ and igg were 2 ng/l – 600 ng/l, 5 ng/l – 400 ng/l, 5 ng/l – 300 ng/l and 0.02 mg/ml – 20 mg/ml, respectively. statistical analysis results were shown as mean ± sem. the wilcoxon test was used to compare the baseline and next values from the same group of animals. the correlations between different variables were analysed using the spearman’s test (spss software version 22). the strength of the correlation between the paired variables is shown as rs value and is by design constrained as follows: -1 ≤ rs ≤ 1. the strength of the correlation is ‘very weak’ if rs values are between 0.00 and 0.19, ‘weak’ if rs values are between 0.20 and 0.39, ‘moderate’ if rs values are between 0.40 and 0.59, ‘strong’ if rs values are between 0.60 and 0.79 and ‘very strong’ if rs values are between 0.80 and 1.0. a ‘positive correlation’ is a relationship between two variables in which both variables move in tandem. a ‘negative correlation’ is a relationship between two variables that move in opposite directions. the statistical analysis and the tables and chats draws were carried out using spss software (version 21). a correlation p < 0.05 was considered statistically significant. ethical considerations the experiment was performed according to the internal regulations declared by the animals support association, ferdowsi university of mashhad, iran. results in this study, all sheep were infected with t. lestoquardi and showed clinical signs, including fever, emaciation, lymph node enlargement, pulmonary and cardiac dysfunction, anaemia, icterus and death. six infected animals developed acute theileriosis and were euthanised on day 21 pi. one infected animal suddenly died on day 14 pi before it could be euthanised. the body temperature increased above 40 °c on days 3–4 pi and reached a maximum on day 21 pi (figure 1). pre-peripheral lymph node enlargement was detected on day 4 pi. schizonts of t. lestoqaurdi were microscopically detected on day 3 pi and piroplasm formed on day 5 pi. the parasitaemia of t. lestoquardi infection was first detected (0.01%) on day 5 pi and reached its highest rate (3.3%) on day 21 pi in infected sheep (figure 1). the mean wbc, rbc, lymphocyte, neutrophil and platelet values slightly increased on day 2 pi and thereafter decreased until day 21 pi (p < 0.05) (table 1). the levels of il-6 were markedly elevated on days 2 pi (p < 0.01) and 12 pi, in comparison to the other cytokines, and decreased to levels lower than on day 0 on days 17 and 21 pi (p < 0.05) (table 1). similar but lower peak levels of tnf-α and inf-γ were also observed on day 12 pi which decreased to the lowest level on day 17 and day 21 pi (p < 0.05) (table 1). the mean level of antibody igg changes was not significant during the course of the study (table 1). the serum concentrations of il-6 correlated strongly and significantly with the level of tnf-α and ifn-γ in serum samples (p < 0.01). the correlation between il-6 and the numbers of lymphocytes was moderate (p < 0.05) (table 2). the percentage parasitaemia and fever had a strong and significant negative correlation with the numbers of neutrophils, wbc, rbc and platelets (table 2). figure 1: change in body temperature (bar) and percentage of parasitaemia (line) during experimental t. lestoquardi infection in sheep. table 1: the mean levels of cell blood count, cytokine and antibody concentrations in sheep infected with t. lestoquardi during an experimental study. table 2: correlation between the mean levels of proinflammatory cytokines, immunoglobulin g, cell blood count, percentage parasitaemia and fever. discussion there is little information on the mechanism involved in the immunopathogenesis of t. lestoquardi infection despite its pathogenic nature (ahmad et al. 2011). in the present study, the haematological, proinflammatory cytokines and igg parameters were analysed during disease. during the course of infection, all animals showed a slight leukocytosis at the onset of t. lestoquardi infection, and then the rbc, wbc, neutrophil, lymphocyte and platelet numbers regularly decreased with increased percentage parasitaemia. these results agreed with the results of other authors (col & uslu 2006; elsadig et al. 2013; fry et al. 2016; hasanpour et al. 2008; leemans et al. 1999; nazifi et al. 2011; omer et al. 2002; sandhu et al. 1998) who reported anaemia, leukopenia, lymphopenia and thrombocytopenia in bovine and ovine theileriosis. the mechanism of anaemia is related to rbc lysis owing to activation of a complement system, erythrophagocytosis and increase in proinflammatory cytokine levels (graham et al. 2001; forsyth et al. 1999). in addition, rbc lysis may be the consequence of the damages caused to the rbc membrane because of oxidative stress (asri rezaei & dalir-naghadeh 2006; nazifi et al. 2011, 2013; razavi et al. 2011). a slight leukocytosis at the onset of infection could be attributed to proliferation of lymphocytes in the lymphoid organs as a defensive response to the theileria infection. leukopenia in the terminal stage could have resulted from large-scale destruction of lymphocytes by schizogony in lymphoid organs and infiltration of these cells into various organs (sandhu et al. 1998). the thrombocytopenia with decreased rbcs and wbcs could also be related to increased tnf-α production (forsyth et al. 1999) or suppression of the bone marrow by the parasite and its products (abd ellah 2015; mbassa et al. 1994). in this study, the levels of il-6, tnf-α and ifn-γ were increased from the onset to the middle course of disease and decreased in the terminal stage of disease in infected sheep. in vitro studies have reported high mrna expression levels of proinflammatory cytokines (il-1β, il-6 and tnf-α) in leucocytes infected with t. annulata or t. parva schizonts (brown et al. 1995; collins et al. 1996; mcguire et al. 2004; mckeever, nyanjui & ballingall 1997). some studies have shown a remarkable elevation in the levels of il-1β, il-6, ifn-γ and tnf-α cytokines in t. annulata-infected cattle (el-sebaei, el-ashker & el-boshy 2014; razavi et al. 2010; nazifi et al. 2010) and t. lestoquard-infected sheep (razavi et al. 2015) in farm conditions. despite the high correlation observed between il-6 levels with tnf-α and inf-γ levels during the course of disease, only il-6 was significantly increased. the sources of il-6 are monocytes and macrophages. the crucial role of il-6 is band t-cell activation and the induction of the acute phase response (hunter & jones 2015; tanaka & kishimoto 2012). during acute inflammation, il-6 is the first responding cell factor which will induce the generation of c-reactive protein (crp), serum amyloid a, fibrinogen and hepcidin in hepatocytes (reinhart et al. 2012; tanaka, narazaki & kishimoto 2014). il-6 has a longer plasma half-life than tnf-α or il-1β and is more reliably measurable in plasma than the other two cytokines. it also has other potential clinical uses, such as diagnosis and management of autoimmune rheumatic disorders. its major role as a biomarker of sepsis appears to be prognostic, and not diagnostic (faix 2013; reinhart et al. 2012). a correlation between high levels of il-6 with mortality rate has been reported in systemic bacterial infection in humans and dogs (nijsten et al. 1978; oda et al. 2005; rau et al. 2007), in malaria (day et al. 1999; mbengue et al. 2015) and canine babesiosis (goddard et al. 2016). the role of il-6 in immunopathogenesis of theileriosis is unclear. some studies have shown that high il-6 expression was related to virulence of theileria strains and host susceptibility (mckeever, nyanjui & ballingall 1997; yamada et al. 2009), and non-detectable il-6 expression correlated with resistance of sheep to t. annulata (schnittger et al. 2000) and african buffaloes to t. parva (okagawa et al. 2012). conclusion based on the results, the wbc, rbc, lymphocyte, neutrophil and platelet values decreased with increased percentage parasitaemia and temperature during the course of t. lestoquardi infection in sheep. in addition, a high correlation was observed between serum levels of il-6 with levels of tnf-α and inf-γ. preliminary results indicate that the measurement of the serum concentrations of il-6 cytokine in combination with haematological parameters could be considered a good marker to estimate the pathogenicity of the t. lestoquardi strain. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions g.r. was the leader of project. he did microscopic examination, analysed the data and wrote the manuscript. s.y. clinically examined the sheep, collected blood samples and performed elisa examination. m.m. performed haematological examination. a.h. analysed the immunology results. s.t. helped s.y. to do the experimental transmission of theileria lestoquardi by ixodid ticks. all the 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‘experimental theileria lestoquardi infection in sheep: biochemical and hematological changes’, acta tropica 173, 55–61. https://doi.org/10.1016/j.actatropica.2017.05.029 yaghfoori, s., razmi, g.r., mohri, m., razavizadeh, a.r. & movassaghi, a.r., 2016, ‘an experimental ovine theileriosis: the effect of theileria lestoquardi infection on cardiovascular system in sheep’, acta tropica 161, 55–61. https://doi.org/10.1016/j.actatropica.2016.05.014 abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) lillian mukandiwa department of paraclinical sciences, university of pretoria, south africa jacobus n. eloff department of paraclinical sciences, university of pretoria, south africa donald r. sibanda department of paraclinical sciences, university of pretoria, south africa vinny naidoo department of paraclinical sciences, university of pretoria, south africa citation mukandiwa, l., eloff, j.n., sibanda, d.r. & naidoo, v., 2016, ‘an acetone extract of clausena anisata may be a potential control agent for flies encountered in cutaneous myiasis’, onderstepoort journal of veterinary research 83(1), a1045. http://dx.doi.org/10.4102/ojvr.v83i1.1045 original research an acetone extract of clausena anisata may be a potential control agent for flies encountered in cutaneous myiasis lillian mukandiwa, jacobus n. eloff, donald r. sibanda, vinny naidoo received: 28 aug. 2015; accepted: 10 dec. 2015; published: 24 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract clausena anisata is a medicinal plant used traditionally to treat myiasis and as an insect repellent by various communities. we have previously demonstrated the effects of c. anisata extracts on blowfly feeding and development in our laboratory. the impact of c. anisata leaf extracts on populations of different fly species on farms in mpumalanga, south africa was investigated in this study under field conditions. flies were exposed to liver baits treated with acetone leaf extracts of c. anisata (150 mg/ml). fly numbers and composition on two farms, with and without c. anisata treated liver, were compared during a period of 12 weeks when fly populations were expected to be high. observations were made on fly behaviour and development, adult sizes and numbers. the flies exposed to liver treated with the leaf extract of c. anisata had a decreased rate of development, prolonged larval period, smaller body sizes and more sluggish behaviour compared to those subjected to the control treatment. no significant differences were, however, found between the numbers and sizes of flies on the treated and on the control farm, which was most likely due to the limited nature of the baiting programme we followed. the effects of c. anisata extracts on blowfly behaviour and development observed in previous laboratory studies were confirmed in this field evaluation. although the extracts did not have a significant effect on the overall population size in this experiment, we believe that the c. anisata leaf extract could be useful in integrated pest management based on its effect on larval development. in addition, species such as lucilia cuprina and chrysomya marginalis seemed to have been repelled by the c. anisata treated liver; as a result, further work should explore this aspect and how it can be used for the protection of animals. introduction myiasis, the infestation of live vertebrate animals with fly larvae, causes discomfort, loss in production, reproduction problems, blindness, lameness and even death in production animals (sotiraki & hall 2012). although myiasis has been recognised as a major disease from ancient times (sherman, hall & thomas 2000), in modern times the disease is still poorly controlled in the animal production industry of many countries. this leads to severe economic losses and animal deaths (sotiraki & hall 2012; wall 2012). economic losses occur through abortions, decreased milk production, lower production, lower fertility, poor hide quality, muscle damage and even death from toxicity or secondary infections. there are 18 different genera of flies that may be involved in cutaneous myiasis. they belong to seven families, namely calliphoridae, sarcophagidae, muscidae, phoridae, cuterebridae, gasterophilidae and oestridae (hall 1991). species of flies in the calliphoridae, sarcophagidae and muscidae families are the main cause of cutaneous myiasis (hall 1991). control of myiasis has largely relied on an integrated approach incorporating insecticides, husbandry practices (such as shearing, crutching, tail docking) (phillips 2009), fly trapping (urech et al. 2009; wall 2012) and, in australia, mulesing (tellam & bowles 1997). despite the integrated approach, a sustained reliance on the use of insecticides over many years has led to the inevitable development of blowfly populations resistant to insecticides (heath & levot 2015). other concerns with chemical compounds include the potential to cause human or animal toxicity. residues left in wool released in effluent during scoring may also lead to environmental contamination. consequently, there are increasing calls for the removal of certain blowfly control products from the international market, providing the impetus for non-insecticidal control methods (bates 2012). the fly population control method has been advocated as an alternative means of controlling myiasis (knipling 1979). the advantage of this system is that the control measure decreases the number of adult flies in the environment without having to rely on the exposure of the animal and wool to high concentrations of ectoparasiticides. an example of an effective system is the lucitrap system (bioglobal ltd, australia), which has been effectively used to reduce fly densities and strike incidence in australia (ward & farrell 2003) and south africa (scholtz et al. 2000). the lucitrap system consists of a translucent bucket made from tough ultraviolet-stabilised plastic and a removable lid with a flat surface, entrance cones that allow the sheep blowfly to enter but not leave the trap, and a bottle with the chemical attractant (lucilure) (levot 2009). the attractant consists of chemicals designed to mimic the odours of primary food sources of the sheep blowfly: fleece rot, animal carcasses, urine and faeces. clausena anisata (willd.) hook. f. ex. benth., belonging to the rutaceae family, is used for the treatment of myiasis in some communities in zimbabwe (chavunduka 1976) as a maggot-expelling agent. in ethiopia, the leaves of this plant are used to repel houseflies (karunamoorthi & husen 2012). it is also used as a repellent against various pests in different countries (boeke et al. 2004; mavundza et al. 2011; ndomo et al. 2008). our previous in vitro studies on the effect of the extracts of c. anisata on the blowfly larvae established that extracts of c. anisata deterred the second and third instar larvae of blowflies lucilia cuprina and chrysomya marginalis from feeding, resulting in lower pupae weights and smaller emerging adult flies (mukandiwa, eloff & naidoo 2012a; mukandiwa et al. 2012b). furthermore, a pyranocoumarin, seselin, isolated from the plant was identified as one of the compounds that deter blowfly larval feeding (mukandiwa et al. 2013). in this study we explored the possibility of suppressing populations by attraction and inhibition of the life-cycle through the use of traps baited with ox-liver mixed with a plant insecticidal extract. we evaluated the efficacy of liver treated with c. anisata extracts against myiasis-causing flies in field trials on farms in the mpumalanga province, south africa. materials and methods study sites fly populations were monitored at two farms in kwamhlanga, 90 km north-east of pretoria, south africa. this area was selected based on the high prevalence of myiasis in the summer months, with in the order of 60 cases occurring per month (area veterinarian, personal communication). farm 1 served as a control baited with liver treated with the solvent only, whereas farm two was baited with liver treated with c. anisata. the farms were more than 10 km apart (treated farm: gps coordinates 28°39’51”e, 25°21’17” and control farm gps coordinates 28°44’48’’e, 25°33’5”) and were matched with regard to grazing environment; sheep breed, stocking density, flock management and climate. both farms consisted of about 850 ha of predominantly natural pasture on which 100 sheep, 300 cattle and 80 goats are grazed. the migration of flies between the farms was eliminated as a factor because adult flies will not normally travel more than 3 km during their lifespan (gleeson & heath 1997). fly monitoring fly abundance was monitored between december 2011 and april 2012 using redtop flycatchers®. two traps were hung 100 m away from the sheep night-camps, 300 m apart, at a height of 1.2 m above the ground on trees as per the manufacturer’s instructions. the initial trapping (which included three trappings on separate occasions) was done at the beginning of the study to establish the fly numbers on the farms. the counts from the three trappings were averaged and the average counts served as the starting fly numbers in the study areas; thereafter, fly trapping was done every 4 weeks. each trapping lasted 48 hours and made use of rotten ox-liver as the bait. ox-liver is a good attractant of flies encountered in myiasis (blackwell et al. 1997; parker & welch 1991). following each trapping, all adult diptera were counted, measured in size and identified according to the descriptions given by howell, walker and nevill (1978). collection and preparation of plant material the leaves of c. anisata (willd.) hook. f. ex. benth. were collected in autumn from the national botanical garden, pretoria, south africa and dried at room temperature in a well-ventilated room. the plant species was identified by tree name tags and authenticated by the guide at the national botanical garden. the voucher specimen of the plant species, numbered pmdn317, is kept at the medicinal plant collection herbarium of the department of paraclinical sciences, university of pretoria, south africa. collection, drying and storage guidelines of the plant material followed were as outlined by mcgaw and eloff (2010). dried and powdered leaves of c. anisata (437 g) were extracted with acetone (5 l) at room temperature by continuous agitation on an orbital shaker (labotec®, model 202, south africa) for 6 h. the mixture was filtered and the solvent removed under reduced pressure at low temperature (40 °c – 50 °c) with a rotary evaporator. the extraction process was repeated twice and extracts combined to give 37.9 g of dry acetone extract. the dried extract was reconstituted in acetone to make a 150 mg/ml stock of extract, which was used for the assays. the whole process was repeated when more extract was needed. exposing flies to plant extract after the initial trapping, farm 2 was exposed for 4 weeks to ox-liver treated with a 150 mg/ml acetone extract of c. anisata, placed in the insectivorous bird feeders® (stride distributors cc, south africa) (figure 1). the concentration of extract was selected based on previous laboratory studies in which it was the most effective against blowfly larvae (mukandiwa et al. 2012b). the feeder consists of a bait bucket with a perforated bottom inside a migration bucket, also with a perforated bottom, over a tray. ideally, the feeding larvae migrate and drop into the feeding tray, where they can be eaten by birds. for this study the feeders were modified so that the bottom of the migration bucket was sealed to prevent the larvae from crawling out into the feeding tray of the feeder. the larvae fed on the bait in the smaller bucket until they crawled out, of their own accord, into the migration bucket, which contained wood shavings to allow for growth and development into subsequent stages. figure 1: the insectivorous bird feeder®. a mixture of 100 g of crushed ox-liver and 5 ml of plant extract (150 mg/ml) was placed in the bait bucket of the feeder (n = 10) around the sheep night-camps, 1.2 m above the ground. the feeders were left open for an hour in open space to enable the acetone to evaporate and then the lids were placed on top of the feeders to keep the rain out. two holes were made on either side of the feeders to allow gravid female flies to enter and lay eggs on the liver extract mixture so that the emerging larvae would become exposed to the plant extract. the feeders were inspected every 2 days and the insecticide mixture replaced every 6 days. after the 4 weeks of baiting, trapping was done with the redtop flycatcher® for 48 hours for fly number quantification (as described above) prior to another 5 weeks of exposing flies to the baited ox-liver. farm 1, which served as the control, was exposed to the same system as above with the exception that pure acetone was used in the bait (solvent control). at each inspection of the feeders (every 2 days) we checked for the presence of fly eggs on the baits, and larval growth, movement and feeding activity were visually evaluated. for each group of pupae, 50 normal-looking pupae were taken to the laboratory for the emerging flies to be assessed. statistical analysis the fly size data were evaluated by a non-parametric t-test for difference from the control group in spss 20 (ibm) as the counts were not normally distributed. sensory evaluation of the extract a sensory evaluation of the extract was conducted to determine the acceptability of the extract for use. the participants in this evaluation included farmers, animal health personnel (veterinarians, veterinary nurses and animal health technicians) and researchers in ethnoveterinary studies. the respondents were blinded as to what the sample was and were asked to comment on the smell. the respondents were asked to rate the extract on a scale of: very bad, bad, neutral, nice and very nice. a total of 50 respondents were used. the reason for undertaking this analysis was to ensure that the extract would be aesthetically acceptable to people who would have to work with it. results fly populations the flies caught in the redtop flycatchers® were composed of blowflies lucilia cuprina, chrysomya albiceps and c. marginalis, the fleshfly, sarcophaga haemorrhoidalis, and the housefly, musca domestica. the changes in the populations of flies by species over time are presented in table 1, whereas the changes in fly size are presented in table 2. the sizes of s. haemorrhoidalis on the treated farm and the control farm were not significantly different (p = 0.362) at the beginning of the study. however, as the study progressed, the flies on the treated farm were significantly smaller (p = 0.02 week 4; p < 0.00, week 8; p = 0.01 week 12) than those on the control farm at any given point in time. there was no particular pattern of change in size with the other fly species (table 2). table 1: number of flies over time on both the treated farm and the control farms. table 2: sizes (mean ± s.d.) (mm) of the different fly species captured from the treated farm during a 12-week period. observations flies were attracted to the baits of both farms and laid eggs. from the second day, according to inspections and by judging the stages of larvae observed in the baits after exposure, the eggs hatched within the mean standard period of 8–24 hours. however, by day 4 after hatching, all larvae, irrespective of species, that fed on the liver treated with c. anisata extract remained small, both in length and thickness, compared to the larvae that fed on liver treated with acetone only (figure 2). by day 4, fly larvae on the control farm (farm 1) were very active and began migrating into the migration bucket containing wood shavings (figure 1); pupation was observed on day 6. by day 14, 90% of the pupae had hatched. however, on the treated farm 2, the larvae were less active than the control group and more than 95% of them failed to crawl into the migration bucket, pupating instead in the bait bucket. figure 2: illustration of the different size of 4-day-old larvae from the control farm and the treated farm, from left to right. the larval stage was also prolonged for all the fly species that were on the treated liver, with the total period from hatching to pupation lasting 17–21 days. the larval stages lasted 4–6 days in the life-cycles of all the fly species encountered in this study, under the weather conditions in which this study was undertaken. a relatively small number, approximately 20%, of dead larvae were observed on the liver treated with the c. anisata extract. however, the dead larvae could not be counted as this could have disturbed the rest of the larvae. the majority of the emerging pupae (90%) from the treated groups appeared normal, and 75% of these pupae hatched after 2 weeks of pupation (days 35–42). in the control group, all the pupae hatched within 6–7 days of pupation (days 13–14). abnormal pupae were observed on the treated farm (figure 3). figure 3: (a, b) different forms of abnormal pupae emerging from the test farm (b–d) compared to the normal pupae from the control farm (a). of the 200 ± 5 pupae collected for laboratory assessment, 75% from the treated farm eclosed, yielding only the fleshfly, s. haemorrhoidalis; the rest of the pupae did not eclose. all the pupae from the control farm hatched, giving a mixed population of flies consisting of blowflies (77%), fleshflies (4%) and houseflies (19%). when the pupae from farm 2 that failed to hatch were cracked open, by holding the pupae between the forefinger and thumb and gently applying pressure, half-developed flies were found that had all their appendages (head, abdomen and legs) except the wings (figure 4). figure 4: half-developed flies from the pupae that failed to hatch naturally. to ascertain whether the absence of blowflies in the samples taken for laboratory assessment from the treated farm was due to the extract and not an artefact, untreated liver was hung around the sheep camps. the eggs laid were taken to the lab to develop into subsequent stages and 93% of the emerging flies were blowflies, lucilia and chrysomya spp. sensory evaluation of extract the majority of the respondents (64%) found the smell of the extract to be neutral (neither bad nor nice), with none of the respondents rating it very bad or very nice (figure 5). figure 5: summary of results on the sensory evaluation of the clausena anisata extract. discussion one of the major findings of this study was the prolonged time of 17–21 days taken for the baited larvae to pupate in comparison to 4–6 days for the control larvae. this is an important result, as the prolongation of the life-cycle increases the chances of death in the environment. ultimately this would reduce the total population, as the total number of life-cycles in one breeding season would be reduced. the data from the monitoring trappings indicated the presence of a mixed population of lucilia, chrysomya, sarcophaga and musca species, but surprisingly only s. haemorrhoidalis emerged from the pupae collected from the treated farm. when untreated liver was hung around the sheep camps on the treated farm, the flies that emerged from the collected eggs were largely lucilia cuprina, chrysomya albiceps and c. marginalis. this suggests that either the lucilia, chrysomya and musca species were more susceptible to the extract and did not survive to the adult stage, or they avoided the baits. the last option is most plausible, as the fly numbers and the size of the adult lucilia, chrysomya and muscidae species did not significantly change as the study progressed, as was observed with s. haemorrhoidalis. in previous studies under laboratory conditions the lucilia and chrysomya species laid eggs on the treated baits (mukandiwa et al. 2012b). the contradictory results may be because the flies had no other options as they were kept in a cage with only this site for oviposition. in a different setup where there are alternatives, the flies will avoid the c. anisata treated baits, suggesting the possibility that this extract may have potential benefits as a repellent that can be used on the animal as a wound dressing to keep blowflies away. this would also validate the traditional use in which the leaves are packed onto wounds to expel maggots (chavunduka 1976) and probably to keep the flies away. we have previously established the safety of c. anisata leaf extracts in a cytotoxicity study (mukandiwa et al. 2012b). however, further studies will need to be conducted in vivo to establish the efficacy and safety of the c. anisata extract as a wound dressing. if the argument above holds that the blowflies were repelled, it does not agree with earlier studies that evaluated the extract against the blowflies l. cuprina and c. marginalis (mukandiwa et al. 2012a, 2012b) under laboratory conditions. however, some conclusions can be drawn. the extract had a feeding deterrence effect on different fly species as the larvae on treated meat were smaller than on the control group both in the laboratory and in current studies. the extract also prolonged the pre-puparium stage in different fly species. therefore the effects of the acetone extract of c. anisata with regard to reduced larval sizes and the prolonged pre-puparium stage, although for different fly species, were the same under both laboratory and field conditions. at present, the mechanism of action of the extracts is unknown. however, based on the effects seen, 2 explanations are plausible, that is, the presence of feeding deterrents or the presence of juvenile hormone mimics. plant-derived compounds have been shown to affect the feeding and diet-selection behaviour of the larvae of blowflies (green, simmonds & blaney 2002; mukandiwa et al. 2012a, 2012b). some are toxic whereas others act as feeding deterrents; for example, phormia regina larvae avoided diets containing 10 ppm and 100 ppm azadirachtin and 10 ppm pyrethrum extract (green et al. 2004). the alkaloids arecoline, caffeine, quinine and nicotine, among others, reduce food consumption in blowfly larvae, resulting in reduced weights of the larvae (green et al. 2002). clausena anisata contains various alkaloids: atanisatin, clausanitin clausenol, clausenine girinimbine, heptaphylline, 3-methylcarbazole, 1-methyl-3,4-dimethoxy-2-quinolone, 3-formyl-1-hydroxycarbazole, murrayamine-a and ekeberginine furanoclausamine a and b, clausamine a-h, mukonal, glycosinine, mukonidine and clausine f clausanitine and mupamine (ojewole 2002). whereas the effects of each alkaloid listed here have not been established in laboratory studies, this class of compounds is known to act as feeding deterrents to insects and herbivores (war et al. 2012) in addition, c. anisata contains the coumarins imperatorin and xanthoxyletin, which have an anti-feeding effect on insects (gebreyesus & chapya 1983). we also isolated the pyranocoumarin, seselin (2h,8h-benzo[1,2-b:3,4-b’]dipyran-2-one,8,8-dimethyl), from the c. anisata extract as the feeding deterrent (anorexogenic agent) against blowfly larvae (mukandiwa et al. 2013). the other plausible and more likely reason for the prolonged life-cycle, 21 days in comparison to the 4–6 days of the control, may be more physiologically based, as plants are known to produce insect juvenile hormones (bede & tobe 2000). the major function of juvenile hormone is the maintenance of the larval status or the so-called juvenilising effect (dhadialla, retnakaran & smagghe 2005). the plant extract had effects similar to other insect growth regulators (igrs), which include reduced development and a prolonged larval period, a smaller body size at a given time compared to the control, and sluggish behaviour, delayed pupation and a reduced eclosion rate of pupae and adults. the igrs include the juvenile hormone analogues, ecdysone agonists and inhibitors and chitin synthesis inhibitors. ultimately, igrs control insect populations (mondal & parween 2000), albeit over a longer period of time. previous studies showed that igrs caused a decline in populations of the german cockroach (blattella germanica l.) in 3–4 months after the start of baiting or spraying treatment, with complete eradication after 12 months (mosson et al. 1995). our study period may therefore have been too short for the extract to begin to have effects on the total fly populations. this is supported by the lack of a clear pattern in the fly populations over our study. the other aim of this study was to ascertain the feasibility of using this plant extract in the field. the two major concerns were the possible unpleasant smell of the extract and chemical instability in the environment. for the first characteristic, the smell of the plant extract was scored by different individuals as being acceptable. this was an important finding, because the leaves of c. anisata, from which the extract was derived, are known to be densely dotted with glands and have a strong scent when crushed. the scent is highly unpleasant, characteristic of horse urine as suggested by the common afrikaans name, perdepis (horse urine) (schmidt, lotter & mccleland 2002). however, for this study the acetone leaf extract was rated by 64% as having a neutral smell, which implies either that the compounds that give c. anisata leaves their characteristic smell are not present in a high enough concentration in the acetone extract or that they were masked by other compounds. regarding our concerns about its stability, the extract proved to remain constantly effective despite being changed every 5 days. in the laboratory studies the larvae were exposed to the extract for 48 hours only. successful fly population control requires an integrated approach that would include removal of other food sources in the environment. this was clearly evident in this study, as the unscheduled death of a cow led to a massive increase in the number of flies trapped 2 weeks later, despite baits still having their desired effect. conclusion the effect on the larval growth and pupal shape make this extract a candidate for further evaluation in the search for new fly-control products. a longer study period is necessary to establish conclusively the effect of the extract on the total population of flies in the environment. furthermore, the extract seemed to repel the blowflies from the baits, suggesting that it may be used as a topical product on animals to repel blowflies. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions l.m. designed and conducted the experiments and drafted the manuscript. j.n.e. was involved in originating the research and writing up the manuscript. d.r.s. conducted the experiments and was involved in data collection and in writing up the manuscript. v.n. 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of pretoria, south africa alri pretorius new generation vaccines programme, agricultural research council-onderstepoort veterinary institute, south africa department of veterinary tropical diseases, university of pretoria, south africa selaelo i. tshilwane new generation vaccines programme, agricultural research council-onderstepoort veterinary institute, south africa junita liebenberg new generation vaccines programme, agricultural research council-onderstepoort veterinary institute, south africa helena steyn new generation vaccines programme, agricultural research council-onderstepoort veterinary institute, south africa mirinda van kleef new generation vaccines programme, agricultural research council-onderstepoort veterinary institute, south africa department of veterinary tropical diseases, university of pretoria, south africa citation thema, n., pretorius, a., tshilwane, s.i., liebenberg, j., steyn, h. & van kleef, m., 2016, ‘cellular immune responses induced in vitro by ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides’, onderstepoort journal of veterinary research 83(1), a1170. http://dx.doi.org/10.4102/ojvr.v83i1.1170 research project no.: 9/27/c243 and 30/01/v011 original research cellular immune responses induced in vitro by ehrlichia ruminantium secreted proteins and identification of vaccine candidate peptides nontobeko thema, alri pretorius, selaelo i. tshilwane, junita liebenberg, helena steyn, mirinda van kleef received: 03 feb. 2016; accepted: 14 apr. 2016; published: 30 aug. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract secreted proteins are reported to induce cell-mediated immunity characterised by the production of interferon-gamma (ifn)-γ. in this study three open reading frames (orfs) (erum8060, erum7760, erum5000) encoding secreted proteins were selected from the ehrlichia ruminantium (welgevonden) genome sequence using bioinformatics tools to determine whether they induce a cellular immune response in vitro with mononuclear cells from needle and tick infected animals. the whole recombinant protein of the three orfs as well as four adjacent fragments of the erum5000 protein (erum5000a, erum5000b, erum5000c, erum5000d) were successfully expressed in a bacterial expression system which was confirmed by immunoblots using anti-his antibodies and sheep sera. these recombinant proteins were assayed with immune sheep and cattle peripheral blood mononuclear cells (pbmcs), spleen and lymph node (ln) cells to determine whether they induce recall cellular immune responses in vitro. significant proliferative responses and ifn-γ production were evident for all recombinant proteins, especially erum5000a, in both ruminant species tested. thus overlapping peptides spanning erum5000a were synthesised and peptides that induce proliferation of memory cd4+ and cd8+ t cells and production of ifn-γ were identified. these results illustrate that a th1 type immune response was elicited and these recombinant proteins and peptides may therefore be promising candidates for development of a heartwater vaccine. introduction heartwater, a disease of cattle, sheep, goats and wild ruminants is caused by an intracellular rickettsiales, ehrlichia (cowdria) ruminantium (dumler et al. 2001; moshkovski 1947). heartwater affects sub-saharan africa and the caribbean where it is transmitted by ticks of the genus amblyomma. the only commercial vaccine against heartwater is a live blood vaccine that requires antibiotic treatment to prevent a serious course of the disease (van der merwe 1987). it is, however, risky to use in non-endemic areas where the vector resides because it contains viable organisms. it also requires a cold chain for storage and distribution and does not protect against all isolates (collins et al. 2003). attempts to develop an alternative to the blood vaccine have involved inactivated (martinez et al. 1994), attenuated (zweygarth et al. 2005) and recombinant dna vaccines that included genes coding for the major antigenic protein 1 (map1) (nyika et al. 2002), low molecular weight proteins of e. ruminantium (sebatjane et al. 2010), or a cocktail of four 1h12 e. ruminantium open reading frames (orfs) (pretorius et al. 2007, 2008). when a cocktail of the four 1h12 e. ruminantium orfs was tested in sheep as a dna vaccine as well as a dna vaccine prime or recombinant protein boost, it elicited complete protection after needle challenge. however, only limited protection was achieved with these vaccines when the animals were challenged by tick infestation. therefore, it is necessary that additional orfs need to be identified in order to improve the efficacy of a recombinant heartwater vaccine. approaches used to identify vaccine candidates for recombinant vaccine development are, in general, guided by the type of immune responses that are likely to mediate protection. it is known that a cellular t helper 1 (th1) immune response is fundamental in destruction of intracellular pathogens like e. ruminantium (totté et al. 1999). th1 responses are mediated by the cytokine interferon-gamma (ifn-γ), which is expressed primarily by cd4+ helper t cells and cytotoxic cd8+ t cells. thus, identification of antigens that induce similar responses is needed for evaluation as immunogenic agents. several e. ruminantium proteins have already been investigated for their ability to induce cellular immune responses in vitro. five orfs were selected from the e. ruminantium genome and their ability to induce proliferative responses and ifn-γ production was evaluated in vitro (sebatjane et al. 2010). all five recombinant proteins induced proliferation of immune peripheral blood mononuclear cells (pbmcs) and ifn-γ production. the corresponding five genes were each individually incorporated into pcmviubs, mammalian expression vector and tested as a potential vaccine in sheep using a dna prime-protein boost immunisation regimen. a cocktail of these dna constructs protected one out of five sheep against a virulent e. ruminantium (welgevonden) needle challenge. similarly, using a reverse vaccinology strategy, recombinant proteins that induced a cellular immune response in immune bovine and ovine pbmcs characterised by the induction of th1 cytokines that includes, ifn-γ, inos, gm-csf and tnf-α were identified (liebenberg et al. 2012). these studies all used needle infected animals to screen for protective antigens. however, indications are that antigens that induce protection against natural and not artificial challenge should be identified for inclusion in a dna vaccine. a successful dna vaccine may therefore need to contain a combination of recombinant proteins which induce cell-mediated immunity to ensure protection against heartwater. one of the complicating factors is that whole proteins from pathogens may contain epitopes, discrete sites recognised by lymphocytes presented in combination with major histocompatibility complex (mhc), which inhibit protective immune responses or induce immunopathology (wang et al. 2005). hence, these negative effects can be avoided if t cell epitopes that specifically stimulate immune responses are identified. including only the protective epitopes from multiple antigens and discarding the unnecessary sequence will also allow efficient use of limited space needed to package numerous antigens in a subunit vaccine. research directed at elucidating the epitopes of selected e. ruminantium proteins will provide a better understanding of which fragment of the protein is immunogenic for incorporation into a multivalent vaccine. furthermore, to ensure that the vaccine protects under field conditions, several immunogenic epitopes that are associated with cell-mediated immunity need to be identified and characterised using mononuclear cells from tick immune animals. in addition to several e. ruminantium promising vaccine targets which function extremely well as dna vaccines under experimental conditions, secreted proteins have been shown to be of particular relevance as protective antigens against several pathogens including chlamydia muridarum (murthy et al. 2007), ehrlichia canis and ehrlichia chaffeensis (doyle et al. 2006), mycobacterium tuberculosis (langermans et al. 2005) and anaplasma marginale (leal et al. 2000). these antigens have been reported to induce cell-mediated and humoral immunity characterised by the production of ifn-γ and antibodies. this indicates that secreted proteins may be potential vaccine candidates and warrants further investigation. in this study we therefore investigated whether secreted e. ruminantium recombinant proteins and peptides would induce similar immune responses in vitro by pbmc from needle infected and challenged (ni) and tick infected and challenged (ti) animals. materials and methods expression of recombinant proteins ehrlichia ruminantium genes of putatively secreted proteins were identified in silico using bioinformatics algorithms as described previously (liebenberg et al. 2012). protein expression was performed using the pet102/topo® expression system (invitrogen) according to the instructions of the manufacturer. briefly, the whole e. ruminantium orfs erum8060 (0.6 kb), erum7760 (0.75 kb) and erum5000 (1.47 kb) as well as the equally divided four 348 bp adjacent fragments (erum5000a, erum5000b, erum5000c, erum5000d) were pcr amplified using specifically designed primers (table 1-a1). plasmids of the expected size were sequenced to confirm the presence of inserts and that the orfs were in-frame. recombinant (his6-tagged) proteins were purified from soluble supernatant or the inclusion bodies using the protino® ni 150 prepacked columns kit (macherey-nagel) according to the instructions of the manufacturer. the purified proteins were assayed using sds-page analysis, western blot analysis using anti-his6 antibodies (roche) and heartwater immune sheep sera (figure 1-a1, supporting information). the recombinant proteins were acetone precipitated for use in immune assays as previously described (van kleef et al. 2002). the concentration of the recombinant proteins was determined using the pierce™ bca protein assay kit (thermo scientific). synthesis of peptides sixteen mer peptides overlapping by eight amino acids spanning the n-terminal fragment of erum5000 (erum5000a) (~348bp) (table 2-a1) were synthesised by genscript (usa) and the purity of the peptides were > 98% as analysed by high-performance liquid chromatography. the peptides were dissolved in water or 100% dimethyl sulfoxide to 1 mg/ml and stored at -20 °c. peptides were further diluted to 100 µg/ml in complete medium prior to use in immunological assays which include lymphocyte proliferation, ifn-γ enzyme-linked immunospot (elispot) assay and flow cytometry. immunological assays inoculation of animals as source of immune mononuclear cells sixto eight-month-old merino sheep (s147, s6010) and three heartwater naïve nguni cattle (b8460, b8347 and b8404) were immunised by the needle infection and treatment method and needle challenged as described previously (liebenberg et al. 2012). in addition to this, to mimic field or natural immunisation, four sheep (s6355, s6821, s6822, s6823) were tick infected and treated with ticks infected with the welgevonden strain in the laboratory as described previously (mahan et al. 1998) with some modifications. briefly, uninfected amblyomma hebraeum nymph ticks were infected by feeding on a sheep that had been infected intravenously with e. ruminantium welgevonden stock. engorged nymphs were allowed to moult to adults in the laboratory. a sheep was then infected by feeding 10 adults (5 males and 5 females) heartwater infected ticks on it. the sheep was monitored daily for the onset of clinical signs and treated on the third day of febrile reaction with terramycin®100 (pfizer). the sheep were tick challenged with the welgevonden infected ticks. heartwater infection of ticks and sheep were confirmed by pcs20 real-time pcr (steyn et al. 2008). all animal research was performed in accordance with the stipulations of the animal ethics committee at the arc onderstepoort veterinary institute and the university of pretoria animal use and care committee and section 20 approval from department of agriculture, forestry and fisheries. purification of peripheral blood mononuclear cells pbmcs were purified from whole blood under sterile conditions. briefly, blood was collected in bd (becton, dickinson) vacutainer®ethylenediaminetetraacetic acid (edta) tubes (becton, dickinson) and pbmcs were isolated by density gradient centrifugation (histopaque®-1077; sigma–aldrich®) as described by liebenberg et al. (2012). the cells were washed three times and counted using tc10™ automated cell counter (biorad) and the cells resuspended (4 × 106 cells/ml) in complete medium, rpmi-1640 (gibco® rpmi + glutamaxtm-i) (invitrogen) supplemented with 10% foetal bovine serum (fbs), 55 mm 2-mercaptoethanol and 1% gibco® pen strep (invitrogen). lymphocyte proliferation assay lymphocyte proliferation assays (lpa) were carried out in triplicate wells as described by van kleef et al. (2000). responder cells at a final concentration of 2 x 106 pbmc/ml were added to respective test wells together with one of the following: e. ruminantium crude antigen (1 µg/ml, positive control), cona (positive control), e. ruminantium recombinant proteins (10 µg/ml), synthetic peptides (10 µg/ml), erum5000a (positive control for peptides), e. ruminantium recombinant protein that tested negative previously (rerum4930, rnegative, negative control) or medium only (unstimulated pbmcs). proliferation was determined by measuring the incorporation of 0.5 µci of [methyl-3h] thymidine added during the final 18 h of the assay using a scintillation counter. results are presented as a stimulation index (si) ± standard deviation (s.d.), where si is the mean counts per min (cpm) of cells stimulated with antigen divided by mean cpm of unstimulated cells. only si two times higher than the negative recombinant protein and p ≤ 0.05 was considered to be significant antigen-specific proliferation. ifn-γ elispot assay the elispot assay was performed as described by (sebatjane et al. 2010). briefly, pbmcs (2 x 106 pbmc/ml) were seeded in elispot 96 well plates (millipore maips 4510) precoated with mouse anti-bovine ifn-γ mab cc302 coating antibody (1 μg/ml). pbmcs were stimulated with recombinant protein, peptides and controls as described for lpa. the plates were developed after 48 h incubation at 37 ºc in a humidified 5% co2 incubator. the number of spots per million cells (spmc) of the antigen was compared to the number of spmc of the corresponding negative control. only the number of spmc that were two times higher than the negative control and with a significant p value (p ≤ 0.05) as determined by student’s t-test were regarded as positive. additionally, for peptide responses only samples with at least 10 spmc were considered positive. cell surface staining immune pbmcs (2 x 106 cells/ml) were stimulated with antigens for 48 h at 37 °c. the cells were stained with the following commercial monoclonal antibodies: cd4 (igm, cell line gc50a), cd8 (igg1, cell line cact80c) and cd45ro (igg3, cell line ila116a) (washington state university monoclonal antibody centre, pullman, wa) at a 1:100 dilution in pn buffer (pbs, 0.5% fbs containing 0.2% sodium azide). following washing secondary antibodies goat anti-mouse igm-apc (invitrogen), goat anti-mouse igg1-pe (serotec) and goat anti-mouse igg3-fitc (serotec) were added at dilutions of 1:10, 1:40 and 1:10 respectively. all incubations were for 15 min at room temperature and washing was done twice with pn buffer. cells were fixed with 0.2% formaldehyde in pbs. samples were assayed on a fc 500 beckman coulter flow cytometer and data analysed using the kaluza software version 1.2 (beckman coulter). intracellular ifn-γ staining immune pbmcs were purified from s6821 and s6823 for intracellular ifn-γ staining using the bd cytofix/cytoperm™ kit (bd biosciences) and protocol. briefly cells (2 x 106 cells/ml) were incubated for 72 h at 37 °c in the presence or absence of 10 µg/ml peptide or recombinant protein. golgi stop solution was added 4 h prior to harvesting. cells were first surface stained as described above and subsequently, intracellular ifn-γ staining was performed with fluorochrome-conjugated anti-cytokine antibody (alexa fluor®488, serotec) at a dilution of 1:20. the cells were incubated at 4 °c for 30 min in the dark, followed by washing with the supplied buffers. the cells were analysed with an fc 500 beckman coulter flow cytometer and data analysed using the kaluza software version 1.2 (beckman coulter). major histocompatibility complex typing of experimental animals samples of genomic dna of animals used in this study were obtained from whole blood collected in bd vacutainer® k2e tubes containing edta. genomic dna purification was done using the generation®capture column kit (gentra systems) according to the instructions of the manufacturer. typing for ovine mhc ovar-drb1 and bola-drb3 was performed using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) as described by konnai et al. (2003) and van eijk, steward-haynes and lewin (1992), respectively. briefly, the second exon of ovar-dbr1 and bola-drb3 was amplified by nested pcr using different primers. the resulting dna (nested pcr product) was digested overnight at 37 °c with 5 u of either rsai, haeiii, psui, saci, sacii, ddei, ncii, hin1i or ecori restriction enzymes (roche), or at 60 °c with 5 u of bstni. however, drb rflp patterns could not be distinguished and thus the nested pcr products were cloned to pgem®-t easy vector (promega) and sequenced. the restriction patterns obtained were compared with published restriction maps (konnai et al. 2003; van eijk et al. 1992). statistical analysis the significance of differences between immunological assay results was determined by means of the student’s t-test. differences with p ≤ 0.05 were considered significant. results expression and purification of recombinant proteins three orfs (rerum5000, rerum7760, rerum8060) were chosen randomly out of a total of 24 exported proteins previously predicted by bioinformatics (table 1, liebenberg et al. 2012). they were successfully expressed in an escherichia coli bl21star™ (de3) host strain in conjunction with the pet102/d-topo® vector. recombinant rerum8060 and erum7760 were purified from both the soluble (s) and insoluble (i) fractions while rerum5000 was found to be insoluble and showed distinct bands at ~36 kda, ~42 kda and ~70 kda respectively in sds-page and in western blot assays that corresponded to their predicted molecular weights. in addition, heartwater immune sheep sera could detect rerum7760 and -8060 in both the soluble and insoluble fractions while rerum5000 could only be recognised in the insoluble fraction (figure 1-a1, supporting information). table 1: summary of results obtained from the selection of vaccine candidates using bioinformatics tools. proliferation induced by recombinant proteins no proliferation was induced in pbmc from the naïve s5408. each recombinant protein induced significant proliferation in pbmc from a different sheep (erum8060, ti s6355; erum7760, ni s6010; erum5000, ni s147; table 2). varying but significant proliferation was induced by all rproteins with pbmc from ni bovine (table 3). when spleen cells from ni and ti sheep and ni bovine were used, erum5000 performed the best out of the three rproteins with a si of 21 with lymph node cells from ti s6355. all three rproteins induced the highest significant proliferation with spleen and lymph node cells from ti s6355 compared to ni s6010. similarly, ovine spleen and lymph node cells responded with higher si than pbmc from the same animal. table 2: proliferative responses of peripheral blood mononuclear cells, spleen cells and lymph node cells from heartwater immune sheep stimulated with recombinant proteins. immune cells were obtained from a naïve sheep (s5408), needle infected and challenged sheep (s147, s6010) and tick infected and challenged sheep (s6355). ifn-γ responses induced by recombinant proteins no ifn-γ was induced in pbmc from the naïve s5408 (table 4). the highest significant response was induced by erum5000 with pbmc from ti s6822 (200 spmc) and lymph node cells from ti s6355 (272 spmc) (table 4). this was followed by erum8060 with pbmc from ni s147 (101 spmc) and spleen cells from ti s6355 (88 spmc). ovine spleen and lymph node cells responded with higher spmc than pbmc from the same animal. varying but significant proliferation was induced by erum5000 rprotein with pbmc from ni bovine (table 4). erum8060 and erum7760 only induced ifn-γ by pbmc from two bovine. table 3: proliferative responses and ifn-γ production of peripheral blood mononuclear cells from infected & treated immune cattle (b8347, b8404 and b8460) stimulated with rproteins. recombinant proteins were tested at a concentration of 10 μg/ml. table 4: ifn-γ by peripheral blood mononuclear cells, spleen cells and lymph node cells from heartwater immune sheep stimulated with recombinant proteins. immune cells were obtained from a naïve sheep (s5408), needle infected sheep (s147, s6010) and tick infected sheep (s6821, s6822, s6823, s6355). expression and purification of four recombinant protein fragments of erum5000 because rerum5000 induced significant and reproducible results, it was divided into four to provide a better understanding of which fragment of the protein is immunogenic for epitope mapping. ehrlichia ruminantium orf erum5000 (~1.39 kb) was pcr amplified into four consecutive ~348 bp fragments (erum5000a, erum5000b, erum5000c, erum5000d) using specifically design primers (table 1-a1). the corresponding recombinant proteins were produced in an e. coli topo tools expression system resulting in thioredoxin or his-tag fusion proteins with distinct bands at 34 kda (figure 1-a1). proliferation and ifn-γ secretion of peripheral blood mononuclear cells s in response to four fragments of rerum5000 pbmcs from ni bovine (b8374, b8404, b8460), sheep (s147, s6010), ti sheep (s6355, s6821, s6822, s6823) and a naïve sheep (s5408) were stimulated with the four fragment proteins, erum5000a, erum5000b, erum5000c and erum5000d. all four rproteins induced immune bovine pbmcs to proliferate with si ranging from ~2 to 18 (table 3). in correlation with the proliferation results, pbmcs from the naïve sheep (s5408) and one ni sheep (s6010) did not produce ifn-γ after they had been incubated with proteins (table 4). all proteins tested with immune bovine pbmcs induced significant production of ifn-γ (table 3). the highest magnitude of ifn-γ responses was obtained when tested with ti sheep pbmcs (s6821, s6822 and s6823). erum5000a induced the highest ifn-γ responses in all animals tested. erum5000d and erum5000c induced highest ifn-γ production in sheep as compared to erum5000b. however, erum5000b induced reproducible immune responses in both cattle and sheep. hence erum5000a and erum5000b fragments were further tested for their ability to induce immune responses in spleen and lymph nodes of ti and ni sheep. both fragments specifically induced immune cells from spleen and lymph node to secrete ifn-γ. once again ovine spleen and lymph node cells responded with higher spmc than pbmc from the same animal. peptide-specific lymphocyte proliferation and ifn-γ production significant proliferation and ifn-γ secretion after erum5000a stimulation provided the rationale for mapping erum5000a peptides for vaccine development. thus fourteen 16-mer peptides (p1–p14) overlapping by eight amino acids were synthesised to determine the exact minimal epitope sequence that can induce recall cellular immune responses. all peptides induced significant ifn-γ with spmc ranging from ~20 to 140 when tested with pbmc from ti sheep (figure 1) and ranged from 2 spmc to 160 spmc for cattle (table 5). the peptides that induced the best recall responses were p9 and p14 in ti sheep and p7 and p10 in cattle. figure 1: ifn-γ production by peripheral blood mononuclear cells stimulated with 16 mer overlapping peptides of erum5000a. table 5: proliferative responses and ifn-γ production of peripheral blood mononuclear cells from immune bovine (b8347; b8404 and b8406) stimulated with rprotein and peptides. cells were stimulated with peptides at a concentration of 10 mg/ml. determination of t cell subsets responsive to peptides of erum5000a in an attempt to assess phenotype of pbmcs responsive to 14 erum5000a peptides pbmcs from three sheep (s6821 s6822 and s6823) were used. cd4+ and cd8+ t cells expressing memory markers (cd45ro+) were measured by flow cytometry after 48 h stimulation. the percentages of cd4+ t cells expressing cd45ro+ was significantly induced by all peptides and varied between s6821 and s6822 with p2, p6, p8 and p14 showing best results (figure 2). whereas for pbmcs from s6823, only peptides p9 and p13 induced significant cd4+ t cells expressing cd45ro+. similarly, the percentage of cd8+ t cells expressing memory markers (cd45ro+) was remarkably high and varied between sheep. peptide p8 (aa 57–72) induced a high percentage of cd8+ t cells expressing memory markers (cd45ro+) in all three sheep followed by p14 (aa 105–116). figure 2: percentage of cells expressing cd4+cd45ro+ (a) and cd8+cd45ro+ (b) surface markers after peripheral blood mononuclear cells stimulation with erum5000a peptides for 48 h in vitro. intracellular ifn-γ staining analyses revealed that both cd4+ and cd8+ t cells from sheep 6821 produced high amounts of ifn-γ in response to p6, p8 and p11. similarly, cd4+ and cd8+ t cells from sheep 6823 produced high amounts of ifn-γ in response to p8 (table 6). in the case of other peptides lower ifn-γ production by both subsets was evident. these results also indicate that cd8+ t cells are the main producer of ifn-γ in the presence of p8. table 6: percentage of cd4+ and cd8+ t cells producing ifn-γ after in vitro stimulation of peripheral blood mononuclear cells from immune sheep (6821 and 6823) at a concentration of 10 μg/ml. the % increase was compared to medium. pcr-rflp typing of the ovar-drb1 and bola-drb3 genes from sheep the polymorphism of the ovine mhc class ii drb1 second exon (ovar-drb1, konnai et al. 2003) was studied in four sheep, (s147, s6821 s6822 and s6823) by pcr-rflp and bola-drb3 for three bovine (b8347, b8460 and b8404). when each amplified dna product was cleaved by the restriction enzymes, different patterns were observed. patterns, ovar-drb1 and bola-drb3 alleles based on restriction endonuclease enzyme digestion are listed (tables 7 and 8). three different alleles (*0332 and *0323, *0333) were obtained for sheep s147. sheep s6821 and s6823 has two different sets of alleles (*0109 and *0201) and (*0201 and *0801) respectively. whereas s6822 has homologous alleles (*0201, *0201). the alleles identified for s6821, s6822 and s6823 were also reported by konnai et al. (2003). for bovine, only one allele was similar to alleles reported by van eijk et al. (1992) and the remaining alleles were not present in any of the published work. therefore, new and unknown alleles were found in this study. discussion t cell responses characterised by the expression of ifn-γ are essential in protection against e. ruminantium infection (totté et al. 1999). a dna vaccine with four 1h12 orfs induced ifn-γ and protection in sheep against a needle challenge with e. ruminantium welgevonden but not against a tick challenge (pretorius et al. 2008). subsequently genome-based high through put technologies were then exploited to study e. ruminantium immunogenic antigens in an effort to improve this vaccine. many e. ruminantium proteins of unknown function, and some of the membrane-associated proteins, secreted, transporters, particularly the abc transport system and proteases were identified and immune characterised. expressed recombinant proteins were assayed using ni immune sheep or bovine pbmc and they induced recall t cell responses characterised by elevation of ifn-γ in vitro and in vivo (sebatjane et al. 2010), ifn-γ, tnf-α, gm-csf, inos and tlr4 expression (liebenberg et al. 2012). these studies all used ni animals to screen for protective antigens. however, indications are that antigens that induce protection against natural and not artificial challenge should be identified for inclusion in a dna vaccine. in this study we therefore investigated whether secreted e. ruminantium recombinant proteins would induce similar immune responses in vitro by pbmc from ni and ti animals. from a total of 27 secreted proteins (liebenberg et al. 2012) three were selected for this study. secreted proteins are regarded as promising vaccine candidates based on their ability to induce cell-mediated immunity characterised by production of ifn-γ (carlisle et al. 2007). these proteins were successfully expressed in e. coli. they induced varied but significant proliferative responses and production of ifn-γ in pmbc from most animals tested. recombinant proteins erum5000 and erum7760 induced the best response in pbmc from ti sheep whereas erum8060 induced the best results in pbmc from ni sheep. the responses induced by all three proteins were also greater in cells from spleen and lymph nodes than pbmc from ti sheep. this is expected because these compartments have larger numbers of lymphocytes than pbmc improving the chance for antigen encounter with specific t cells (blum & pabst 2007). the difference in immune responses obtained also suggests that the lymph node and spleen t cells may have distinct subsets of t lymphocytes (langeveld, gamadia & ten berge 2006). in addition, the spleen and ln cells of the tick infected animal produced the highest levels of ifn-γ protein, whereas the ln cells isolated from the needle infected animal remained unresponsive. this may be linked to the two different routes of infection that led to different homing patterns for the respective memory cells. this highlights the necessity to use ti animals to search for vaccine candidate antigens. in addition to the cellular immune response, serum of ni immune animal detected the recombinant proteins (figure 1-a1, supporting information) confirming that all proteins were secreted and exposed to the humoral immune system of the host during infection. however, when measuring il-4 (associated with th2 type responses) il-4 production was not detected (results not shown) thus the antibodies detected could be opsonising antibodies (igg2) associated with a th1 response. alternatively, the proteins may contain cross-reacting epitopes of a protein that induces a humoral immune response. it has been indicated that antibody-mediated immunity for intracellular parasites is not unusual (doyle et al. 2006). because the recombinant protein erum5000 induced the best overall response it was equally divided and four recombinant fragment proteins erum5000a, erum5000b, erum5000c and erum5000d were expressed. similar proliferative responses and production of ifn-γ were induced by the fragments in pbmcs isolated from the animals. however, the n-terminal fragment of erum5000 rprotein erum5000a induced the highest recall responses and was selected for epitope mapping. epitope mapping for this protein was done to identify vaccine candidates for incorporation into a multivalent vaccine. epitope mapping of erum5000 was determined using 14 overlapping peptides (p1–p14) spanning the fragment and pbmcs from ni cattle and ti sheep. the peptides that induced the best response in both cattle and sheep were p3, p5, p8 and p14. these peptides induced memory cd4+ and in particular cd8+ to proliferate and secrete ifn-γ. table 7: the polymerase chain reaction-restriction fragment length polymorphism patterns and ovar-drb1 alleles obtained using nine restriction enzymes. table 8: the polymerase chain reaction-restriction fragment length polymorphism patterns and bola-drb3 alleles obtained using three restriction enzymes. each recombinant protein induced immune responses and it varied between animals. similarly, peptides were recognised to various extents by immune animals. this can be expected because out bred animals were used for these assays. it has been indicated previously that mhc class ii molecules are highly polymorphic and different alleles vary in their peptide binding specificity (groothuis et al. 2005; sommer 2006). the results obtained from mhc typing using ovine mhc ii drb1 and bovine bola ii-drb3 and pcr-rflp highlighted the diversity between the animals because different alleles were found for each animal. this may also explain the variation of responses between sheep and bovine pbmcs. nevertheless, the data confirm that heartwater immune sheep and cattle were exposed to the proteins and peptides during infection and that recall immune responses developed in the hosts. both cd4+ and cd8+ t cells are ifn-γ-producing lymphocytes that are required in the development of protective immunity against heartwater. the cd4+ t cells play a major role to both cell-mediated and humoral immunity and act through the production of ifn-γ, as helper cells for immunoglobulin secretion and as effector cells to activate macrophages. cytotoxic cd8+ t cells limit bacteria replication by killing infected cells or by releasing cytokines that mediate intracellular killing of the pathogen through the production of inos (yewdell & haeryfar 2005). previously, t cell growth factors (mahan, smith & byrom 1994) and ifn-γ (totté et al. 1996) have been shown to inhibit e. ruminantium growth in vitro. the effect of ifn-γ may be because of upregulation of mhc class i and ii expression on monocytes leading to increased antigen presentation to immune cells, or by increased phagocytosis, reactive oxygen intermediates, nitric oxide and lysosomal enzyme production. hence the role of both cd4+ and cd8+ t cells, and ifn-γ production in the development of protective immunity against heartwater should be a high priority. in conclusion, we have demonstrated that predicted secreted e. ruminantium proteins induced a more robust th1 type response by ti than ni animals, as indicated by proliferation of mononuclear cells (blood, spleen and lymph node) and higher production of ifn-γ. furthermore, epitope mapping of erum5000a identified four peptides that were recognised by immune pbmcs from both ni cattle and ti sheep. these epitopes require further investigation as components for inclusion in the multiepitope dna vaccine against heartwater. acknowledgements we are very grateful to dr erich zweygarth and antoinette josemans for providing culture material for the purification of e. ruminantium elementary bodies and extraction of dna and ms e. faber for dna sequencing. this study was supported by joy liebenberg trust fund and south african department of science and technology. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions n.t. planned the study and did most of the experimental lab work, the data analyses as well as the preparation of manuscript. a.p. was the study supervisor, contributed to the planning of the study, the data analyses and the editing of the manuscript. s.i.t. contributed to the experimental lab work and the editing of manuscript; j.l. was responsible for the selection of vaccine candidates using bioinformatics tools and contributed to the editing of the manuscript; h.s. was responsible for the immunisation of the animals and contributed to the editing of the manuscript; m.v.k. is a programme manager and contributed to the editing of the manuscript. references blum, k.s. & pabst, r., 2007, ‘lymphocyte numbers and subsets in the human blood. do they mirror the situation in all organs?’, immunology letters 108(1), 45–51. http://dx.doi.org/10.1016/j.imlet.2006.10.009 carlisle, j., evans, w., hajizadeh, r., nadaf, m., shepherd, 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protein rerum5000i and four fragments, erum5000a, erum5000b, erum5000c and erum5000d, erum7760 and erum8060. the purified recombinant proteins are expressed and purified using championtm pet directional topo® expression system. recombinant proteins were detected by using (a) immune sheep sera and (b and c) anti-his antibodies. abstract introduction material and methods results discussion conclusion acknowledgements references about the author(s) juan scheun national zoological garden, south african national biodiversity institute, pretoria, south africa department of zoology and entomology, endocrine research laboratory, mammal research institute, faculty of natural and agricultural science, university of pretoria, pretoria, south africa adrian s.w. tordiffe department of paraclinical sciences, faculty of veterinary science, university of pretoria, onderstepoort, pretoria, south africa kirsten wimberger the cape parrot project, the wild bird trust, parktown, johannesburg, south africa andre ganswindt national zoological garden, south african national biodiversity institute, pretoria, south africa department of zoology and entomology, endocrine research laboratory, mammal research institute, faculty of natural and agricultural science, university of pretoria, pretoria, south africa citation scheun, j., tordiffe, a.s.w., wimberger, k. & ganswindt, a., 2020, ‘validating a non-invasive technique for monitoring physiological stress in the samango monkey’, onderstepoort journal of veterinary research 87(1), a1720. https://doi.org/10.4102/ojvr.v87i1.1720 original research validating a non-invasive technique for monitoring physiological stress in the samango monkey juan scheun, adrian s.w. tordiffe, kirsten wimberger, andre ganswindt received: 10 dec. 2018; accepted: 10 may 2019; published: 27 feb. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the non-invasive monitoring of physiological stress can provide conservation and wildlife managers with an invaluable tool for assessing animal welfare and psychological health of captive and free-ranging populations. a significant decrease in free-ranging primate populations globally and an increase in captive-housed primates have led to a need to monitor the stress and general welfare of these animals. we examined the suitability of three enzyme immunoassays (eias) for monitoring stress-related physiological responses in the samango monkey, cercopithecus albogularis erythrarchus. we conducted an adrenocorticotropic hormone (acth) challenge on a male and female at the national zoological garden, pretoria, south africa. individual faecal samples were collected 8 days preand post-acth administration and subsequently analysed for faecal glucocorticoid metabolite (fgcm) concentrations. during the study, biological stressors occurred for both the male and female. two of the three eias tested (11-oxoetiocholanolone i and ii) were able to reliably monitor fgcm alterations throughout the study period in both sexes. the 11-oxoetiocholanolone i eia, however, had the lowest mean deviation from the calculated baseline value and was thus chosen as the preferred assay. both the physiological activation of the stress response and the biological response to a stressor could be monitored with the chosen assay. the successful establishment of a reliable, non-invasive method for monitoring adrenocortical activity in c. albogularis erythrarchus will now allow conservationists, scientific researchers and wildlife managers to evaluate the level of stress experienced, and general welfare, by animals in captivity as well as free-ranging populations. keywords: acth challenge; animal welfare; samango monkey; non-invasive hormone monitoring; glucocorticoids; biological validation. introduction stress can be defined as a stimulus that may threaten, or appear to threaten, homeostasis (selye 1936). here, the perceived stressor triggers the so-called stress response, an adaptive mechanism aimed at restoring homeostasis of an individual. the initial step of this response includes the activation of the hypothalamic-pituitary-adrenal (hpa) axis, resulting in the production and secretion of glucocorticoids (gcs), such as cortisol and corticosterone, into the bloodstream (see sapolsky, romero & munck 2000). the temporal increase in plasma gc concentrations is responsible for the necessary adjustment of individual metabolism, increasing energy availability, enhancing cardiovascular activity and behavioural alterations, in order to restore homeostasis (reeder & kramer 2005). although these adaptation processes can be beneficial in the short term, a prolonged exposure to elevated gc concentrations may lead to numerous deleterious effects, most notably the suppression of the immune system, memory impairment and reproductive suppression (konstantinos & sheridan 2001; mcewen et al. 2008; sheriff et al. 2009; suter & schwartz 1985). because of their key role in the course of the stress response, gcs are often used as a physiological marker for the level of stress perceived by an animal (palme 2019; sheriff et al. 2011). although blood offers a robust matrix for measuring gc concentrations, the challenges associated with blood collection in the form of animal capture and restraint, along with the increase in gc concentrations as a result of the stressful procedure (feedback effect), render this approach impractical in many captive and free-ranging settings (heistermann 2010). consequently, monitoring adrenocortical activity using faeces as a hormone matrix has become an accepted non-invasive method for assessing the stress response of an animal. faecal samples are comparatively easy to collect with little to no disturbance to the animal and as a result, sampling is feedback-free (kersey & dehnhard 2014). further, faecal hormone values are less affected by episodic fluctuations of hormone secretions, as circulating hormone levels within the bloodstream accumulate within the faeces over an extended period of time (ganswindt et al. 2012; mcewen & wingfield 2003). however, as hormone metabolism and excretion may differ distinctively between species and sex (goymann 2012; rettenbacher et al. 2004), assays for non-invasive hormone monitoring need to be carefully validated in terms of its applicability for the intended hormone matrix to ensure a reliable quantification of respective hormone metabolites (touma & palme 2005). in this regard, the activation of the hpa axis, as a key part of the validation process, can be performed through biological and physiological means. in terms of biological stressors, factors such as animal handling, restraint, transportation and injury have all been shown to activate the hpa axis (bosson, palme & boonstra 2013; dehnhard et al. 2001; ganswindt et al. 2010; goymann et al. 1999; hämäläinen et al. 2014). however, individual variation, in terms of the neuroendocrine response to the presence of a stressor, can vary considerably and should be taken into account when using a biological stressor for assay validation (see gott et al. 2018; koolhaas et al. 2010). the physiological validation is conducted through the artificial activation of the hpa axis; this is achieved by injecting an individual with synthetic adrenocorticotropic hormone (acth) to increase gc production (acth challenge; palme 2019). primates are the mammal order most threatened by extinction (schipper et al. 2008), with hunting and deforestation presenting the most common threats (rovero et al. 2012). the forest dwelling samango monkey (cercopithecus albogularis) in south africa is one such species threatened by anthropogenic activities (linden et al. 2016; skinner & chimimba 2005; wimberger, nowak & hill 2017). changes in faecal glucocorticoid metabolite (fgcm) concentrations, according to environmental and biological stressors, can be used to provide insight into how this species has adapted to anthropogenic threats. analyses of gc in a related species, cercopithecus mitis stuhlmanni, showed that fgcm concentrations increased when individuals ate less preferred items (fallback foods) and decreased when they ate preferred items (e.g. insects, fruits, young leaves; foerster, cords & monfort 2012). furthermore, this change was more substantial in those female monkeys in the energetically demanding stages of late pregnancy and early lactation. similarly, the validation of an appropriate enzyme immunoassay (eia) for monitoring fgcm concentrations in the barbary macaque (macaca sylvanus) showed that individual fgcm concentrations increased during interactions with tourists (maréchal et al. 2011). thus, the validation of an appropriate eia for monitoring alterations in fgcm concentrations can offer an ideal tool for assessing the physiological reaction of primates to anthropogenic activities and changes in their natural environment. the aim of this study was to establish a suitable non-invasive tool for a reliable monitoring of fgcm concentrations, as a measure of stress, in c. albogularis erythrarchus. such a technique would be an ideal tool for assessing animal welfare of captive and free-ranging populations. material and methods adrenocorticotropic hormone challenge test an acth challenge was conducted on an adult male (6 years) and adult female (5 years) samango monkey (c. albogularis erythrarchus) housed at the national zoological garden (nzg), pretoria, south africa, from 17 october to 02 november 2012. both individuals were housed in separate, adjoining cages (6.5 m × 7.0 m × 3.5 m) since 2009, which allowed for direct visual, olfactory and vocal communication. each cage contained a sleeping room, suspended walkways, climbing poles, ‘monkey bars’, large trees as well as a number of resting platforms. the study was conducted outside of the defined reproductive period of the species, negating the possible effect that reproductive hormones, and the resulting behaviours, may have on the adrenal activity of the study animals. both animals were fed a mixed vegetable and fruit diet, while water was available ad libitum. regular veterinarian assessments confirmed that both individuals were healthy at the beginning of the study. to determine individual baseline fgcm concentrations, both cages were checked for faeces regularly and fresh faecal samples were collected from both individuals for a period of 7 days. collected material was homogenised and a 5 g – 10 g portion frozen at -20 °c. on day 8 of the experiment, both individuals were net-caught and hand-injected with zolazepam or tiletamine (3.5 mg/kg – 5 mg/kg body weight, zoletil®, virbac, south africa), before transferring them to the veterinary hospital at the nzg as part of an annual health assessment. once at the veterinary hospital, both individuals were intubated and maintained under anaesthesia on 1% – 2% isoflurane in oxygen. while under anaesthesia, individual spo2, etco2, blood pressure, electrocardiography as well as heart and respiratory rate were monitored. a physical examination, to account for any abnormalities or injuries, was conducted on both individuals. during the examination, the nzg veterinary staff confirmed a tail fracture in the male, which was presumably sustained during the net-capture event. the fracture was treated with a splinted bandage by the veterinarian and the animal received oral carprofen (rimadyl®, zoetis, south africa) at 2.5mg/kg once a day for 5 days for pain management. carprofen is a pain suppressor which has no direct effect on adrenal activity. the nzg staff monitored the tail recovery throughout the entire healing period; the need to change the bandage did not arise during the study period. throughout the intubation period, balanced intravenous fluids (ringer’s lactate) were administered to each individual at a rate of 10 ml/kg/h. a blood sample (10 ml) was collected from the caudal saphenous vein to obtain serum cortisol values. following this, each individual was injected with 10 iu (1.1 iu/kg – 1.5 iu/kg) synthetic acth (synacthen®, novartis, australia) intramuscularly. forty minutes after the synacthen injection, a second blood sample was taken to capture the induced rise in serum cortisol concentrations. both individuals were allowed to recover in a darkened cage for 3 hours before being released back into their respective individual enclosures. the male was released into a cordoned off section of its cage to allow the nzg staff to monitor his response to the tail bandage. this cordoned off section limited the direct contact (visual, olfactory) between study animals for the rest of the study. both the tail injury and the limited visual contact between individuals were regarded as possible biological stressors. all individual faecal droppings were collected for 48 h post-acth administration. following this, cages were checked for faeces regularly (5–10 times a day) for a further 5 days. in all cases, material from the middle of the dropping was collected to avoid any cross-contamination that may have occurred through contact with urine or any other environmental contaminants. faecal sample extraction and analysis all faecal sample extractions and analyses were conducted at the endocrine research laboratory, university of pretoria, south africa. faecal samples were lyophilised, pulverised and sieved through a fine mesh to remove any fibrous or undigested material (fieß, heistermann & hodges 1999). subsequently, 0.050 g – 0.055 g of the faecal powder was extracted by vortexing for 15 minutes with 1.5 ml 80% ethanol. following centrifugation for 10 min at 1500 × g, the supernatants were transferred into new microcentrifuge tubes and stored at -20 °c until analyses. the resulting steroid extracts were measured for fgcm concentrations using three eias: (1) an 11-oxoetiocholanolone i (detecting 11, 17-dioxoandrostanes), (2) an 11--oxoetiocholanolone ii (detecting fgcms with a 5β-3α-ol-11-one structure) and (3) a cortisol eia. initial assay selection was based on available eias already used for monitoring fgcm alterations in other non-human primates (e.g. hämäläinen et al. 2014; heistermann, palme & ganswindt 2006) including the vervet monkey (chlorocebus pygerythrus) (young et al. 2017). all assays were performed on microtiter plates as described by scheun et al. (2016). details for the three eias are described by palme and möstl (1997) for 11-oxoetiocholanolone i and cortisol, as well as möstl et al. (2002) for 11-oxoetiocholanolone ii assay. sensitivities for all three eias used were 0.6 ng/g dry weight. parallelism tests were performed for all three eias, with difference in slope being < 5% for the 11-oxoetiocholanolone i and cortisol, and < 3% for the 11-oxoetiocholanolone iieia. intraand inter-assay coefficients of variation, determined by repeated measurements of highand low-value quality controls, ranged between 1.9% – 11.7% (11-oxoetiocholanolone i), 6.1% – 11.7% (11-oxoetiocholanolone ii) and 9.5% – 11.4% (cortisol), respectively. serum analysis the two blood samples were put on ice for 30–60 min until clotted and subsequently centrifuged at 1500 × g for 15 min. the serum was then transferred into polystyrene tubes and stored at -20 °c until analysis at the veterinary hormone laboratory, faculty of veterinary science, university of pretoria. serum cortisol concentrations were determined using a coat-a-count© cortisol radio-immunoassay (siemens medial solutions diagnostics, tarrytown, new york, united states [us]). in brief, 25 µl standards, controls and samples were transferred in duplicates into coated tubes, respectively. one millilitre 125 i cortisol solution was added, and the tubes incubated for 45 min at 37 °c. subsequently, all tubes were thoroughly decanted, patted dry and counted for 1 min in a gamma counter (wallac wizzard2, perkin elmer) using multicalc software. sensitivity of the assay was 5.5 nmol/l. cross-reactivities of the antibody used are provided in the assay instruction manual. concentrations are given in mmol/l. data analysis individual baseline fgcm concentrations were determined for the respective data sets resulting from the three eias employed using an iterative process (brown et al. 1994; scheun et al. 2016). here, the mean and standard deviation (sd) value for each individual eia-specific data set were calculated. subsequently, all data points higher than the mean + 1.5 sd were removed and the mean and sd recalculated. this process was repeated until no value exceeds the mean + 1.5 sd, thus yielding the individual baseline value. periods of elevated fgcm concentrations were defined as the occurrence of two or more consecutive samples that exceed the calculated individual baseline level. to calculate the baseline stability of each eia, the mean absolute deviation (mad) from the calculated baseline value was determined. here, the calculated baseline fgcm value was subtracted from all pre-injection fgcm values for each eia-specific data set. the differences were noted as absolute values and the mean of the values was calculated, which subsequently gave the mad value for each eia. the mad values were converted to a percentage deviation value (mad/baseline value*100) to allow for the comparison between eias. gut passage time in similar sized primates has been reported as 20–36 h (bahr et al. 2000; rimbach et al. 2013; young et al. 2017). thus, endocrine response to the induced physiological stressor has been evaluated by focusing on alterations in fgcm concentrations up to 48 h post-induction. similarly, the endocrine response to the presence of the biological stressors focused on the alteration in fgcm concentrations from 48 to 191.5 h (male) and 48 to 199 h (female) post-injection. to determine whether an eia could successfully monitor changes in fgcm concentration we compared the (1) highest signal following both stressors and (2) the lowest mad value of the tested assays. ethical considerations the study was performed with the approval of the national zoological gardens ethics committee (reference p10/27). results adrenocorticotropic hormone challenge test serum gc concentrations increased considerably following the acth injection, with a 65.5% (male, pre-injection: 1.06 mmol/l, post-injection: 1.78 nmol/l) and 35.7% (female, pre-injection: 1.70 mmol/l, post-injection: 2.30 mmol/l) increase observed after 40 min. in total, 23 faecal samples from the male (13 preand 10 post-injection) and 19 faecal samples from the female (10 preand 9 post-injection) were collected during the monitoring period. for the male, the 11-oxoetiocholanolone i (53% ± 21.65% sd) had the lowest percentage mad value compared to the 11-oxoetiocholanolone ii (71.14% ± 25.52% sd) and cortisol eia (54.21% ± 39.41% sd). the cortisol eia (0.09% ± 0.24% sd) had the lowest percentage mad value in the female, followed by the 11-oxoetiocholanolone ii (0.30% ± 0.41% sd) and 11-oxoetiocholanolone i (0.51% ± 0.91% sd). a considerable increase in fgcm concentrations occurred after 23 h post-injection for both study individuals (figure 1). for the male, the most distinct increase in fgcm concentrations was detected using the cortisol eia (129.2% increase), whereas the 11-oxoetiocholanolone i and ii eias showed an increase of 21.3% and 71.1%, respectively (table 1). for the female, the 11-oxoetiocholanolone i eia showed the most distinct increase in fgcm concentrations (145.5%), followed by the 11-oxoetiocholanolone ii eia (75.5%), whereas the cortisol eia showed a decrease in fgcm concentration (-5.0%) post-acth administration (table 2). figure 1: the longitudinal faecal glucocorticoid metabolite response for both male (a) and female (b) study animals following adrenocorticotropic hormone administration (time: 0) and separation or injury. table 1: male baseline faecal glucocorticoid metabolite concentrations, as well as the mean absolute deviation from baseline, for all three enzyme immunoassays employed during the study. table 2: female baseline faecal glucocorticoid metabolite concentrations, as well as the mean absolute deviation from baseline, for all three enzyme immunoassays employed during the study. biological stressors for the male, the tail fracture and separation event co-occurred with a considerable increase in fgcm levels shown by two of the three eias tested (figure 1). the 11-oxoetiocholanolone i eia demonstrated elevated fgcm concentrations 71.5–103 h post-injection, whereafter fgcm levels decreased to the calculated baseline value; a peak fgcm concentration (peak response: 269.46%) was found at 96 h post-injection (table 1). similarly, the 11-oxoetiocholanolone ii eia showed elevated fgcm concentrations 71–191.5 h post-injection, before returning to the calculated baseline fgcm levels. a peak fgcm concentration (peak response: 198.30%) was found at 96 h post-injection (table 1). although the cortisol eia also showed a peak in fgcm concentrations at 103 h post-injection (peak response: 70.90%, table 1), no two consecutive samples, between 48 and 191.5 h were found to be above fgcm baseline concentrations. as such, no period of elevated fgcm concentrations could be defined for this assay for the period the biological stressors occurred. following the new separation setting with limited visual, olfactory contact, the 11-oxoetiocholanolone i eia showed the highest peak response in fgcm concentrations (979.62%) at 103 h post-injection for the female (figure 1, table 2). similarly, both the 11-oxoetiocholanolone ii and cortisol eias showed a peak response in fgcm concentration exceeding 400% of the respective calculated baseline fgcm values at 151 h and 122.5 h, respectively (table 2). determined fgcm concentrations did not return to baseline levels for any of the three eias during the post-injection observation period. discussion the ability of the three eias to detect a considerable increase in fgcm concentrations in response to the acth challenge, as well as the biological stressors monitored by default, confirms that monitoring alterations in fgcm concentrations can be used as a reliable measure of adrenal activity in c. albogularis erythrarchus. to obtain baseline serum cortisol concentrations, blood collection should occur within 3 min of the original stressor (martínez-mota et al. 2008; romero & reed 2005). as a result of the prolonged handling process of the animals in this study, we did not collect our first blood sample within the suggested time period. as such, the first male and female blood sample collected prior to the acth administration of the study may not be indicative of baseline cortisol levels. despite this, the comparison between the first and second blood samples can still be used as evidence that serum cortisol concentrations increase in c. albogularis erythrarchus following the stimulation of the adrenal cortex. the peak in fgcm concentration, as a result of the acth administration, occurred 23 h post-injection for the male and female study animal. the 23 h time delay, from injection to excretion, corresponds with the gut passage time of similar sized primates, such as the sykes’ monkey (c. mitis albogularis, 26.5 h, foerster & monfort 2010), the vervet monkey (chlorocebus pygerythrus, 29 h, young et al. 2017), the long-tailed macaque (macacafascicularis, 22 h, bahr et al. 2000) and brown spider monkeys (ateles hybridus, 24 h, rimbach et al. 2013). for the male, all three eias were indicative of an increase in hpa activity. however, only the 11-oxoetiocholanolone i and 11-oxoetiocholanolone ii eias were able to monitor the induced increase in fgcm concentrations in the female, with the cortisol eia displaying baseline fgcm values at 23 h post-injection. the physical injury to the tail of the male individual during the net-capture event may well explain the prolonged elevation of fgcm concentrations post-injection for two of the three eias used. here, the 11-oxoetiocholanolone i eia displayed the highest percentage fgcm response, as well as a prolonged elevation in fgcm concentration, throughout the presumed biological stressor period. similarly, the 11-oxoetiocholanolone ii eia also showed a considerable increase in fgcm concentrations, and the related period of elevated fgcm concentrations was even considerably longer than the period determined via the 11-oxoetiocholanolone i eia. the decrease in fgcm concentration throughout the biological validation period across assays may well be as a result of the tail fracture healing, leading to the decrease in the severity of the stress response and gc production. although numerous articles on the activation of the hpa axis in response to extrinsic and intrinsic factors, such as translocation, capture and handling, toxins and seasonality, exist (baker, gobush & vynne 2013; girard-buttoz et al. 2009; hämäläinen et al. 2014), only a few case studies describe the effect of physical injuries on adrenal activity. for example, ganswindt et al. (2010) showed that physical injury in african elephants (loxodonta africana) increased fgcm concentrations, which returned to baseline levels following recovery. likewise, kumar et al. (2014) found that asian elephants (elephas maximus) display elevated fgcm concentrations in response to physical injury. to our knowledge, no data exist on the effect of physical injury on adrenal activity for new or old-world primates; therefore, this may be the first, albeit brief, look into this topic. similar to the male in the study, the female exhibited a prolonged elevation of fgcm concentrations throughout the post-injection period, presumably because of the limited visual, olfactory contact with the male following acth injection. the inability of the female to interact with the male may explain why the elevated fgcm concentrations did not return to baseline levels. social instability and pair separation has been shown to increase the stress response and gc production in female squirrel monkeys (saimiri sciureus, lyons, ha & levine 1995) as well as common marmoset (callithrix geoffroyi, smith, birnie & french 2011). the disruption of social bonds, through the separation of individuals, is known to evoke a significant bio-behavioural stress response, while the maintenance of established pair bonds can act as a ‘social buffer’ to both intrinsic and extrinsic stressors (hennessy 1997). thus, social buffering may assist in moderating the hpa response, decreasing gc production and stress-related behaviour (hennessy, kaiser & sachser 2009). however, it is worth noting that the physiological response to social stressors can vary significantly between individuals and species; in this regard foerster, cords and monfort (2011) demonstrated that individual rates of agonism in c. mitis showed no measureable fgcm response. it should be noted that the prolonged elevation of fgcm concentrations from the initial increase post-injection, following the biological stressors perceived in both study animals, aggravates the interpretation of gut passage time within the study animals and additional research will have to be conducted to confirm the length found here. a clear difference in signal strength to the biological and physiological stressors was observed when comparing the respective results from the different eias between sexes. the most prominent of which was the lack of a response observed in the female, at 23 h post-injection, compared to the male when using the cortisol eia. similarly, all three eias demonstrated a drastic adrenal response, in terms of fgcm secretion, during the biological stressor perceived by the female, whereas the respective response was considerably lower across all three eias in the male. in trying to explain these sex-related differences, a potential sex-related difference in gc metabolism and excretion should be considered (see review: goymann 2012). for example, touma et al. (2003) demonstrated that corticosterone metabolism in mice (mus musculus) differ substantially between male and female individuals. furthermore, the perception of a stressor can vary significantly between individuals and sexes (goymann 2012; koolhaas et al. 2010; see stroud, salovey & epel 2002). for example, suomi (1991) showed that the activation of the stress response, and the degree of gc secretion, differs substantially between individual rhesus monkeys (macaca mulatta) faced with a similar social context. another factor that may explain the observed difference in response between sexes is the nature of the biological stressor experienced by each sex. the instability in social structure, such as the separation of individuals from conspecifics, has been shown to be a major stressor in female individuals and may explain the considerable fgcm increase seen in this regard (haller et al. 1999). with regard to the male, pain and discomfort have been shown to activate the stress response as discussed previously; however, the nature of the injury and the medical attention received by the male individual may have resulted in a lower stress response than that observed in the separation event of the female. thus, the differences observed between the sexes may well be explained by the stressor type and not as a result of the specific steroid metabolism of each sex. to pinpoint respective responses, additional research into the specific factors that control stress physiology of each sex would be required. from a technical perspective, both the 11-oxoetiocholanolone i and 11-oxoetiocholanolone ii eia were able to reliably monitor male and female alterations in fgcm concentrations across both the physiological and biological stressor periods; thus, both eias seem suitable to monitor adrenocortical activity in c. albogularis erythrarchus. conclusion two of the three assays used in the study were able to monitor changes in fgcm concentrations of c. albogularis erythrarchus male and female study animals. taking the mad values and percentage response into consideration, the 11-oxoetiocholanolone i eia was chosen as the most appropriate eia for monitoring physiological stress in the species. the non-invasive technique validated for c. albogularis erythrarchus now offers conservationists, managers and academic researchers a robust and feedback-free tool for monitoring the physiological stress experience in both captive and free-ranging environments. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors were responsible for experimental and project design. a.s.w.t. performed the experiments and provided veterinarian care. k.w. and a.g. collected and prepared samples for analysis. a.g. conducted sample analyses. j.s. performed data calculations. all authors co-wrote the manuscript. funding information all 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south africa chantel de beer agricultural research council-onderstepoort veterinary research, pretoria, south africa citation latif, a.a., ntantiso, l. & de beer, c., 2019, ‘african animal trypanosomosis (nagana) in northern kwazulu-natal, south africa: strategic treatment of cattle on a farm in endemic area’, onderstepoort journal of veterinary research 86(1), a1639. https://doi.org/10.4102/ojvr.v86i1.1639 original research african animal trypanosomosis (nagana) in northern kwazulu-natal, south africa: strategic treatment of cattle on a farm in endemic area abdalla a. latif, lundi ntantiso, chantel de beer received: 05 apr. 2018; accepted: 09 jan. 2019; published: 30 may 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract african animal trypanosomosis (aat) is caused by several species of the genus trypanosoma, a parasitic protozoan infecting domestic and wild animals. one of the major effects of infection with pathogenic trypanosome is anaemia. currently, the control policies for tsetse and trypanosomosis are less effective in south africa. the only response was to block treat all infected herds and change the dip chemical to one which controls tsetse flies during severe outbreaks. this policy proved to be less effective as demonstrated by the current high level of trypanosome infections in cattle. our objective was to study the impacts of aat (nagana) on animal productivity by monitoring the health of cattle herds kept in tsetse and trypanosomosis endemic areas before and after an intervention that reduces the incidence of the disease. the study was conducted on a farm in northern kwazulu-natal which kept a commercial cattle herd. there was no history of any cattle treatment for trypanosome. all cattle were generally in poor health condition at the start of the study though the herd received regular anthelminthic treatment. a treatment strategy using two drugs, homidium bromide (ethidium) and homidium chloride (novidium), was implemented. cattle were monitored regularly for 13 months for herd trypanosomosis prevalence (hp), herd average packed cell volume (h-pcv) and the percentage of the herd that was anaemic (ha). a total of six odour-baited h-traps were deployed where cattle grazed from january 2006 to august 2007 to monitor the tsetse population. glossina brevipalpis newstead and glossina austeni newstead were collected continuously for the entire study period. high trypanosomes hp (44%), low average h-pcv (29.5) and ha (24%) were rerecorded in the baseline survey. all cattle in the herd received their first treatment with ethidium bromide. regular monthly sampling of cattle for the next 142 days showed a decline in hp of 2.2% – 2.8%. however, an hp of 20% was recorded by day 220 and the herd received the second treatment using novidium chloride. the hp dropped to 0.0% and ha to 0.0% by day 116 after the second treatment. the cow group was treated again by day 160 when the hp and ha were 27.3% and 11%, respectively. the same strategy was applied to the other two groups of weaners and the calves at the time when their hp reached 20%. ethidium and novidium treatment protected cattle, that were under continuous tsetse and trypanosomosis challenge, for up to 6 months. two to three treatments per year may be sufficient for extended protection. however, this strategy would need to be included into an integrated pest management approach combining vector control for it to be sustainable. keywords: tsetse flies; trypanosomes; nagana; trypanocides treatment; kwazulu-natal. introduction african animal trypanosomosis (aat) is caused by several species of the genus trypanosoma, a parasitic protozoan infecting blood and tissues of the host animal (leak 1998). trypanosomes are transmitted by several vector species of blood sucking flies of the genus glossina, commonly known as tsetse flies. the tsetse belt in south africa is limited to north-eastern kwazulu-natal occupying 12,000 km² and is conserved mainly in nature reserves, national and private game parks and resorts (de beer et al. 2016; kappmeier green, potgieter & vreysen 2007). other man-made habitats such as exotic timber forests and patches of evergreen indigenous forests found around river beds are also suitable habitat for the tsetse flies. the habitat has been extended when most commercial cattle farming areas have moved into game farming areas and resorts, thus allowing bushes, trees and vegetation to grow, creating more suitable tsetse breeding sites. de beer et al. (2016) reported a greater extension of glossina brevipalpis than was previously known. currently, two species of tsetse species coexist, glossina brevipalpis newstead and glossina austeni newstead. the two species have different behavioural activities and g. austeni has been proven to be a more competent vector of trypanosome parasites (motloang et al. 2012). dispersal and re-invasion of the flies is continuous between different habitats (esterhuizen et al. 2005). there is a wealth of entomological information on the ecology of the two tsetse species covering geographical distribution, development of odour-baited traps and control methods since 1990 (de beer et al. 2016; hendrickx 2002, 2007; hendrickx et al. 2003; kappmeier 2000; kappmeier & nevill 1999a, 1999b; kappmeier, nevill & bagnall 1998; kappmeier green 2003). the limited grazing, small areas and degraded communal land force cattle to move to such tsetse habitat, exposing them to nagana challenge. one of the major effects of infection with pathogenic trypanosome is anaemia (leak 1998). the disease varies from acute to chronic forms. the acute form occurs soon after the infection, characterised by high parasitaemia and rapid fall of packed cell volume (pcv). the extent of the acute and chronic forms of the disease is determined by a number of factors: complete tolerance (no-illness) in the case of game animals; the virulence of the trypanosoma species, for example, trypanosoma congolense; and its level of parasitaemia (connor & van den bossche 2004; murray & gray 1984). the chronic stage that is typical in indigenous breeds can persist for an extended period in which case the affected animals lose condition and become increasingly anaemic and lethargic (itty 1996). marcotty et al. (2008) evaluated pcv values as an ‘indicator of trypanosomosis infections in cattle’ in tsetse infested areas. three different approaches are to be considered in studies of the impacts of aat on animal productivity (swallow 1999). the first approach involves longitudinal monitoring of trypanosome prevalence in cattle, their health and productivity in areas of known trypanosomosis risk. the second approach is to monitor the health and productivity of cattle herds kept in nearby areas of lower and higher levels of trypanosomosis risk during the same period of time. these two approaches have advantages in that the productivity indicators (herd average pcv and herd percentage anaemia) are measured for entire herds rather than for individual animals. these approaches have been adopted in aat studies conducted in kwazulu-natal. trypanosome infections in cattle were recorded at 21 of the 25 communal dip tanks which clearly showed that the disease was still abundant and highly prevalent in north-eastern kwazulu-natal (de beer et al. 2016; ntantiso et al. 2014). the third approach is to monitor the health and productivity of cattle herds before and after an intervention that reduces the incidence of trypanosomosis. this approach was one of the objectives of this study by following the parasitaemia and associated anaemia in a cattle herd. secondly, the study also aimed at investigating and appraising a control strategy based on herd treatment against trypanosomosis over time. currently, the control and management policies for tsetse and nagana are less effective involving the change of tick dipping chemicals to one which controls both ticks and tsetse such as pyrethroids formulations. the last mass cattle treatment with the trypanocide drug was reported in 1990 after a serious nagana outbreak. during this outbreak, about 10 000 cattle died of nagana and over 100 000 were treated using ethidium bromide (kappmeier et al. 1998). that once-off intervention was shown to be unsustainable over the years, and high levels of trypanosome infections in cattle is still documented (ntantiso et al. 2014; van den bossche et al. 2006). materials and methods boomerang farm boomerang farm is mainly a sugar cane plantation; however, a commercial cattle herd is kept there. it is situated next to the nhlozi gate in the western shores section of the isimangaliso wetland park. it has 180 head of cattle with an output of about 30 cattle sold per year. cattle were grazed into 300 hectares of indigenous forest during dry seasons. they were also supplemented by the cane sugar residue after the harvest. there was no history of any cattle treatment for trypanosome in recent years. all cattle were generally in poor health condition at the start of the study though the herd received regular anthelminthic treatment. tsetse population monitoring a total of six odour-baited h-traps (kappmeier 2000; kappmeier & nevill 1999b) were deployed on different sites where cattle grazed from january 2006 to august 2007. the traps were baited with odours to enhance trapping of g. brevipalpis (kappmeier & nevill 1999a). these baits consisted of 1-octen-3-ol and 4-methylphenol at a ratio of 1:8 that were released at 4.4 mg/hour and 7.6 mg/hour, respectively. the chemicals were dispensed from seven heat-sealed sachets (7 cm × 9 cm) made of low-density polyethylene sleeves (wall thickness 150 microns) placed near the entrance of the trap. a 300-ml brown glass bottle that dispensed acetone through a 6-mm hole in the lid at a rate of 350mg/hour was placed next to the h trap (kappmeier green 2002). flies were collected in a 20% ethanol solution to which an antiseptic, savlon® (johnson & johnson, pharmedica laboratories (pty) ltd. rattray road, east london, south africa) (0.4 ml/l) and formalin (0.4 ml/l) had been added to preserve the sampled flies as well as to combat ant and spider predation. traps were emptied and serviced every 14 days. the number of each species collected over this period was counted and results expressed as apparent density (ad), that is, the number of flies per trap per day. strategic use of trypanocides a treatment strategy using two drugs, homidium bromide (phenanthridinium bromide salt, ethidium – camco animal health, united kingdom) at a dose rate of 1.0 mg/kg and homidium chloride (phenanthridinium chloride salt, novidium – rhone-merieux, france) at a dose rate of 1.0 mg/kg, was attempted which would allow the herd of cattle to thrive in the high tsetse and trypanosomiasis challenge area. both drugs were administered intramuscularly, and both have therapeutic and prophylactic actions. ethidium was only used in the first cow treatment as an emergency, while treatment using novidium followed throughout the study period. novidium was not registered in south africa but a special import permit was granted by the department of agriculture, forestry and fisheries (daff). the adult cattle were treated and monitored for 13 months, while the calves born in 2005 and 2006 were treated and monitored for 8 months and 3 months, respectively. the herd average packed cell volume (h-pcv), the percentage of cattle with trypanosome infection referred to as the herd prevalence (hp) and the percentage of cattle in a herd with pcv of 24% or less referred to as herd anaemia (ha) (ntantiso et al. 2014; van den bossche & rowlands 2001) were all calculated. the hp, h-pcv and ha, which give a good indication of the health status of the herd (trail et al. 1991), were obtained from the three groups of cattle, that is, cows, weaners 2005 and calves 2006. the threshold for the treatment was decided arbitrarily to be hp of 20% which was noticed to produce ha of around 25%. sample processing and examination blood was collected from the tail or jugular veins using 10 ml vacutainer tubes coated with ethylenediamine tetraacetic acid as anticoagulant. sample processing was done on the site. blood from each sample was decanted into plain microhaematocrit capillary tubes that were sealed with cristseal and centrifuged for 5 min at 9000 revolution per minute (rpm). after centrifugation, the pcv was determined. animals with a pcv of 24% or less were considered anaemic (murray & dexter 1988; ntantiso et al. 2014; van den bossche & rowlands 2001). the buffy coat of each sample was extruded onto a microscope slide, covered with a cover slip and examined for motile trypanosomes under a compound microscope using ×40 magnification. trypanosoma congolense is the dominant species infecting cattle and buffalo in northern kwazulu-natal including the study farm (mamabolo et al. 2009; motloang et al. 2012; van den bossche et al. 2006). trypanosoma brucei was never detected in these reports, while trypanosoma vivax was not detected in cattle samples obtained from the study farm (boomerang farm) using molecular technique (mamabolo et al. 2009). therefore, trypanosoma infection in cattle, that is, the hp, in this study refers to infections with t. congolense. ethical considerations ethical approval for the study was obtained from the 3agricultural research council – animal ethics committee of the onderstepoort veterinary institute (ref. 07/20/c174). the research project was funded and approved by the department of agriculture, forestry and fisheries (daff) (project number: ov21/13/c142: epidemiology of animal trypanosomosis in kwazulu-natal, south africa). results tsetse population a total number of 3892 g. austeni and 4107 g. brevipalpis were collected over the 16-month period with the six odour-baited h-traps, and there was no tsetse free period (figure 1). the male to female ratio collected for g. austeni was 1:3.8 and for g. brevipalpis was 1:1.2. the monthly ad for g. austeni ranged from 3.9 in may 2006 at the end of the summer season to 0.4 in july 2007 in the middle of the winter season. the monthly ad for g. brevipalpis was the highest at 3.0 in march 2007, the middle of the summer season, and similar to g. austeni the lowest at 0.4 in july 2007. the average apparent densities for the collection period were similar for g. austeni (1.4 ± 0.9) and g. brevipalpis (1.4 ± 0.7). three peaks in fly numbers were observed within the collection period for g. austeni (april 2006, september 2006 and april 2007) as well as for g. brevipalpis (october 2006, march 2007 and august 2007) (figure 1). figure 1: monthly apparent density for glossina austeni and glossina brevipalpis at boomerang farm from january 2006 to august 2007. strategic treatment of adult cattle and weaned calves at boomerang farm using trypanocidal drugs table 1 and figure 2 show the results of hp, ha, h-pcv and timing of strategic treatment with ethidium bromide and novidium chloride. the primary survey conducted in june 2006 on the farm revealed very high trypanosomes hp (44%), h-pcv (29.5%) and ha (24%). subsequently, all cattle in the cow herd received treatment with ethidium bromide (first treatment). thereafter, the regular monthly sampling for the next 142 days showed a decline in hp of 2.2% – 2.8%. however, the hp of 16%, that is, five times the previous month, was recorded by day 185. this high level of infection was maintained for the following 2 months before the herd was treated by day 220 when the hp was 20% using novidium chloride. the hp and ha dropped to 0.0 and 0.0, respectively, by day 116 after the previous treatment. the cow group was treated again by day 160 after the previous treatment when the hp and ha were 27.3% and 11%, respectively. figure 2: trypanosome prevalence (%) and herd anaemia (%) for cows, and weaners at boomerang farm from 23 june 2006 to 05 august 2007. dark arrow indicates ethidium treatment and striped arrow indicates novidium treatment of cattle. table 1: herd average packed cell volume, trypanosome prevalence, herd anaemia and treatment strategy in cattle from june 2006 to august 2007 at boomerang farm. in december 2006, at a time when the hp of the adult cows was very low (2.8%), the weaned calves (2005 group) experienced a very high hp of 71.8% while 25.6% of them were anaemic. this demonstrated the high trypanosomes challenge during this period. forty-four days later, the hp of the group increased to 76% and the ha to 38.1%. all of the 2005 calves received treatment with novidium chloride (first treatment). the calves continued to be negative for trypanosomes infections with 0.0% ha for the following 80 days. this was followed by a second re-infection, and a high hp of 16.7% and 26.2% was recorded by day 116 and day 145, respectively, for the group treated with novidium chloride. this group remained with a very low hp (2.8%), with few cases being anaemic, ha was 5.6% for the rest of the observation period (43 days after the last treatment). the 2006 calf group was again examined in may and june 2007 where the hp was 26% and 20%, respectively. the calves were treated using novidium and remained uninfected for the following 43 days when the observations on the farm cattle were terminated. the 2006 calf group did not show signs of anaemia during the investigation period. figure 2 shows the correlation between hp and ha; high and low hp before and after treatment correlated well with high and low ha. discussion both g. austeni and g. brevipalpis had relatively high abundance throughout the study period. there were no tsetse free periods, and the cattle at boomerang farm were under constant vector pressure, which was higher in the hot or summer season. there was a positive association between these high tsetse apparent densities and infection prevalence in animals not treated with trypanocides. after treatment, this relationship did not exist, and it became apparent that these animals were protected against infection for up to 6 months. previous studies indicated that the vector competence of g. austeni for t. congolense, the most abundant trypanosoma species in north-eastern kwazulu-natal, was significantly higher than that of g. brevipalpis (motloang et al. 2012). however, cattle at dip tanks neighbouring a game park showed a similar high trypanosome prevalence in cattle where g. austeni was not recorded in the tsetse traps. the glossina brevipalpis population was high through the years (ntantiso et al. 2014) indicating that this species was most likely responsible for nagana transmission in cattle at these dip tanks. de beer et al. (2016) proposed that the high trypanosome infection prevalence in cattle recorded in certain areas might be the result of the greater densities of g. brevipalpis relative to their lower vector competence. the pathogenic t. congolense infecting cattle are responsible for the disease in kwazulu-natal (mamabolo et al. 2009; motloang et al. 2014; van den bossche et al. 2006). different strains of t. congolense with great variation in their virulence have been reported (bengaly et al. 2002; masumu et al. 2006; van den bossche et al. 2011). recently, two genetically distinct types of t. congolense, savannah and kilifi, have been isolated from cattle and tsetse flies in kwazulu-natal (mamabolo et al. 2009; motloang et al. 2014). of the two genetically distinct types of t. congolense isolated from cattle and tsetse flies in kwazulu-natal, the savannah sub-type is more prevalent and thought to be responsible for aat outbreaks in cattle. motloang et al. (2014) reported the first attempt to determine the geographical distribution of virulent t. congolense strains in kwazulu-natal. their results confirmed the higher virulence of the t. congolense savannah type compared to the kilifi type and indicated the prevalence of highly virulent strains to be higher in wildlife parks and in cattle near the parks than on farms further away. ntantiso et al. (2014) carried out intensive and systematic studies on the epidemiology of cattle trypanosomosis from 2005 to 2008 (ntantiso et al. 2014) in cattle neighbouring a game park. over their study period comprising 1318 observations, they found that 62% of the trypanosome-infected cattle were anaemic, compared to 20.0% anaemia in the uninfected group. these results demonstrated the virulence of trypanosomes in cattle near the game parks. the h-pcv recorded in boomerang cattle were higher in infected cattle compared to infected cattle grazed near the game parks during the same observation period as judged by the smaller percentage of ha in the boomerang cows group. other than the less virulent t. congolense challenge in boomerang cattle with reference to the findings by motloang et al. (2014), cattle also received better grazing supplemented with sugar cane residues. the first integrated tsetse and tick control was introduced in 1990, when a severe nagana outbreak occurred in the tsetse infested areas of north-eastern kwazulu-natal, about 10 000 cattle died of aat and 116, 000 were treated using ethidium bromide during this outbreak (kappmeier et al. 1998). the control measures included the use of pyrethroid dip, a chemical which has an insecticidal and acaricidal activity, on a 4-year interval to control the outbreak and challenge by the vector tsetse flies. this proposed management regime has not been followed, and 16 years following the 1990 outbreak, 76 cattle suspected to be infected with trypanosome were bled at one communal diptank at the edge of the hluhluwe-umfolozi park and the results were reported in a research communication by van den bossche et al. (2006). thirty-four per cent of cattle were found to be infected with t. congolense and 83% were anaemic. this once-off survey demonstrated that nagana was still prevalent and recommended further research to develop appropriate control methods. the intention of this study was to treat adult cows and calves at an arbitrary hp threshold of 20% before the disease produces significant production losses. the pcv of individual animals and the h-pcv are useful indicators of anaemia, and in trypanosome, endemic areas are the most typical signs of nagana in domestic animals (marcotty et al. 2008; murray & dexter 1988; trail et al. 1991). ethidium and novidium strategic treatment produced attractive results whereby cattle were protected for an extended period of up to 6 months. therefore, two to three treatments per year may be sufficient to keep cattle productivity on the farm under all year tsetse and trypanosomosis challenge. it is noted that the trypanosomes hp and the consequent ha reached very high levels in 2005 born calves (76% and 37%, respectively). if this group had not been treated, the weaned calves could have experienced a state of ‘stunted growth’ and become unfit for sale. additionally, calves, with reference to the group born in 2006, seemed to resist hp of up to 20% without showing recognised signs of anaemia (none of the calves were found with pcv equal or less than 24%). this observation proved the ‘arbitrary threshold for treatment’ adopted in this study. age-related resistance to trypanosomes is recognised where anaemia in infected calves was moderate (maclennan 1974; murray, morrison & whitelaw 1982; valli, forsberg & mcsherry 1978; wellde et al. 1981) as well as young animals which are less attractive to tsetse flies. ethidium and novidium were reported to give protection for a period of up to 4 months (brander & pugh 1977). there were some successes reported of farming in tsetse and trypanosomes challenge areas (holmes & scott 1982; logan et al. 1984; moloo et al. 1988; trail et al. 1985) and in zimbabwe and mozambique (boyt 1979; takken, taylor-lewis & woodford 1988). the strategic use of trypanocides requires close monitoring of the hp through veterinary supervision, surveillance and strict administration of the drugs (connor & van den bossche 2004; holmes & scott 1982) to avoid under-dosing or overdosing of a drug which may shorten the time for the trypanosomes to build resistance against the drug. the problem of development of resistance in trypanosomes is the threat to the sustainability of the strategy. it is noteworthy that an investigation into drug-resistant strains in kwazulu-natal was carried out and the results did not reveal the presence of any resistant strains (justin masumu, pers. comm., september 2010). in the absence of a tsetse eradication policy, integrated approaches can be applied for the control of trypanosomosis (holmes 1997; murray & black 1985) including animal treatment and tsetse fly suppression by using deltamethrin-treated cattle, targets and screens (bauer et al. 2011; hargrove, torr & kindness 2003; torr, maudlin & vale 2007). conclusion the tsetse and trypanosomosis high challenge had been continuous over the years (figure 1, table 1). the strategic treatment using ethidium bromide and novidium chloride produced promising results whereby cattle were protected for extended period of up to 6 months. therefore, two to three treatments per year may be sufficient to keep cattle productivity on the farm under tsetse and trypanosomosis continuous challenge. however, this strategy can be sustainable if an integrated management of tsetse and aat is implemented by suppression of the tsetse fly challenge. acknowledgements the project on tsetse and trypanosomosis ecology and epidemiology was funded by the department of agriculture, forestry & fisheries and the agricultural research council, south africa. the authors are grateful for the collaboration of the personnel of boomerang farm. 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author(s) mamohale e. chaisi department of veterinary tropical diseases, university of pretoria, south africa janine r. baxter department of veterinary tropical diseases, university of pretoria, south africa department of genetics, university of pretoria, south africa paidashe hove department of veterinary tropical diseases, university of pretoria, south africa biotechnology platform, agricultural research council, south africa chimvwele n. choopa department of veterinary tropical diseases, university of pretoria, south africa department of veterinary services, ministry of agriculture and livestock, zambia marinda c. oosthuizen department of veterinary tropical diseases, university of pretoria, south africa kelly a. brayton department of veterinary tropical diseases, university of pretoria, south africa department of veterinary microbiology and pathology, washington state university, united states zamantungwa t.h. khumalo department of veterinary tropical diseases, university of pretoria, south africa awelani m. mutshembele national zoological gardens, pretoria, south africa moses s. mtshali national zoological gardens, pretoria, south africa nicola e. collins department of veterinary tropical diseases, university of pretoria, south africa citation chaisi, m.e., baxter, j.r., hove, p., choopa, c.n., oosthuizen, m.c., brayton, k.a. et al., 2017, ‘comparison of three nucleic acid-based tests for detecting anaplasma marginale and anaplasma centrale in cattle’, onderstepoort journal of veterinary research 84(1), a1262. https://doi.org/10.4102/ojvr.v84i1.1262 research project no.: v067/13 original research comparison of three nucleic acid-based tests for detecting anaplasma marginale and anaplasma centrale in cattle mamohale e. chaisi, janine r. baxter, paidashe hove, chimvwele n. choopa, marinda c. oosthuizen, kelly a. brayton, zamantungwa t.h. khumalo, awelani m. mutshembele, moses s. mtshali, nicola e. collins received: 23 may 2016; accepted: 12 july 2016; published: 23 jan. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract several nucleic acid-based assays have been developed for detecting anaplasma marginale and anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. we evaluated the ability of the reverse line blot (rlb) hybridisation assay, two nested polymerase chain reaction (npcr) assays and a duplex real-time quantitative polymerase chain reaction (qpcr) assay to detect a. marginale and a. centrale infections in cattle (n = 66) in south africa. the lowest detection limits for a. marginale plasmid dna were 2500 copies by the rlb assay, 250 copies by the npcr and qpcr assays and 2500, 250 and 25 copies of a. centrale plasmid dna by the rlb, npcr and qpcr assays respectively. the qpcr assay detected more a. marginaleand a. centrale-positive samples than the other assays, either as single or mixed infections. although the results of the qpcr and npcr tests were in agreement for the majority (38) of a. marginale-positive samples, 13 samples tested negative for a. marginale using npcr but positive using qpcr. to explain this discrepancy, the target sequence region of the npcr assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. the results indicated sequence variation in the internal forward primer (am100) area amongst the south african a. marginale msp1β sequences, resulting in false negatives. we propose the use of the duplex qpcr assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both anaplasma spp. introduction bovine anaplasmosis is a tick-borne disease of cattle caused by the intra-erythrocytic rickettsia, anaplasma marginale (theiler 1910). the clinical manifestations of anaplasmosis include fever, progressive anaemia and icterus, and the disease has a case fatality rate of up to 36% (losos 1986). anaplasmosis is widely distributed around the world, and in south africa, it is endemic in most of the cattle-farming areas (de waal 2000; marufu et al. 2010; mtshali et al. 2007; potgieter 1979; stevens et al. 2007). five tick species have been implicated in the transmission of a. marginale in south africa: rhipicephalus decoloratus, rhipicephalus microplus, rhipicephalus evertsi evertsi, rhipicephalus simus and hyalomma marginatum rufipes (potgieter 1979; potgieter & van rensburg 1980). anaplasma marginale subsp. centrale, commonly referred to as anaplasma centrale, was first isolated in south africa by sir arnold theiler (theiler 1911) who originally classified it as ‘a. marginale variety centrale’. it causes a milder form of anaplasmosis, and a live blood vaccine containing a. centrale is used to immunise cattle against a. marginale in many countries, including south africa (de waal 2000; melendez et al. 2003; potgieter & van rensburg 1983). however, this vaccine causes variable protection against a. marginale and might not be effective against antigenically diverse, highly virulent stocks of a. marginale (bock & de vos 2001). the vaccine strain can cause reactions in adult cattle of susceptible breeds (bigalke 1980; pipano 1976) and it has been reported to cause severe anaplasmosis in splenectomised adult cattle (kuttler 1966; pipano, mayer & frank 1985). more recently, a strain of a. centrale that is closely related to the vaccine strain was associated with a case of clinical disease in a bovine in europe (carelli et al. 2008). the seroprevalence of a. marginale in south africa is known to be high (mtshali et al. 2007; stevens et al. 2007) and a number of novel a. marginale strains have been identified by the analysis of msp1α genotypes (de la fuente et al. 2007; mtshali et al. 2007; mutshembele et al. 2014). however, little work has been performed on the molecular detection of a. marginale and a. centrale in the field in south africa. infection by these organisms in endemic regions is usually low, asymptomatic and contribute to transmission by vectors. as these low infections can only be effectively detected using molecular methods (hofmann et al. 2015; schotthoefer et al. 2013; strik et al. 2007), various assays have been developed to detect a. marginale and a. centrale dna in vectors and hosts in different parts of the world. these include the reverse line blot (rlb) hybridisation assay (bekker et al. 2002), restriction fragment length polymorphism (rflp) assays (noaman & shayan 2010), nested polymerase chain reaction (npcr) assays (decaro et al. 2008; molad et al. 2006) and quantitative real-time polymerase chain reaction (qpcr) assays (carelli et al. 2007; decaro et al. 2008; futse et al. 2003; picoloto et al. 2010; reinbold et al. 2010; ueti et al. 2007). most of these assays have only been used to follow the organisms in experimentally infected cattle; however, the npcr assay designed by molad et al. (2006) and the qpcr tests developed by carelli et al. (2007) and decaro et al. (2008) have been used to detect a. marginale and a. centrale in field samples in israel and italy. the availability of all these molecular diagnostic assays of different sensitivities and cost makes the choice of an appropriate test for epidemiological studies difficult (bacanelli, ramos & araujo 2014). it is also important to assess the suitability of these assays in the detection of local a. marginale and a. centrale strains, as many different strains of a. marginale have been reported in south africa (mtshali et al. 2007; mutshembele et al. 2014) and elsewhere around the world (almazan et al. 2008; cabezas-cruz et al. 2013; de la fuente et al. 2001, 2007; pohl et al. 2013). we evaluated the ability of three different techniques, the rlb hybridisation assay (bekker et al. 2002), npcr assays (decaro et al. 2008; molad et al. 2006) and a duplex qpcr assay (decaro et al. 2008), in detecting a. marginale and a. centrale infections in cattle in south africa. to explain discrepancies between the npcr and qpcr assay results in the detection of a. marginale, the target sequence region of the npcr assay was evaluated by cloning and sequencing the msp1β gene from selected a. marginale-positive field samples. methods sample collection and dna extraction a total of 66 blood samples originating from cattle in mpumalanga (n = 42), western cape (n = 13) and kwazulu-natal (n = 11) provinces in south africa were included in the study. the samples were either obtained as frozen blood samples (obtained from the national zoological gardens, pretoria) or collected as fresh blood samples from cattle in the mnisi community area, bushbuckridge, mpumalanga province, south africa. fresh blood samples were collected in 9-ml vacutainer® edta tubes from the caudal vein of cattle that were at least 1-year old in accordance with the animal ethics code of the university of pretoria. genomic dna was extracted from the blood samples using a qiaamp dna blood mini kit (qiagen, usa), according to the manufacturer’s instructions. detection of anaplasma marginale and anaplasma centrale the samples were analysed for the presence of a. marginale and a. centrale using three pcr-based methods. reverse line blot hybridisation assay primers ehr-f and ehr-r (table 1) were used to amplify the v1 hypervariable region of the 16s rrna gene of anaplasma and ehrlichia species present in the samples. the pcr was performed in a 25-µl reaction mixture containing 1x platinum quantitative pcr supermix udg (invitrogen), 3 mm mgcl2, 200 µm dntps, 0.2 µm of each primer and 2.5 µl of template dna (approximately 200 ng). a touchdown thermal cycling programme was used as previously described (nijhof et al. 2005). pcr products were subjected to rlb hybridisation as described by nijhof et al. (2005) using the genusand species-specific oligonucleotide probes reported in bekker et al. (2002). table 1: oligonucleotide primers and probes used in this study for the detection of anaplasma marginale and anaplasma centrale. duplex real-time quantitative polymerase chain reaction the samples were analysed using the duplex qpcr assay reported by decaro et al. (2008) for simultaneous detection of a. marginale (detecting the msp1β gene) and a. centrale (detecting the groel gene), with minor modifications of the a. centrale probe to adapt it for use in the lightcycler real-time pcr system. the 20 µl reaction mixture contained 4 µl of faststart taqman mix (roche diagnostics), 0.5 µl udg, 0.6 µm of a. marginale-specific primers am-for and am-rev (table 1), 0.9 µm of a. centrale-specific primers ac-for and ac-rev (table 1), 0.2 µm of probes am-pb and ac-pb (table 1) and 2.5 µl of template dna (approximately 200 ng). dna extracted from the a. centrale vaccine strain purchased from onderstepoort biological products (obp) and sample 9410 obtained from dr helena steyn, onderstepoort veterinary institute (ovi), pretoria, south africa, were used as positive controls for a. centrale. sample 9410 was confirmed to have a. centrale infection by amplification and sequence analysis of the groel, msp2 and 16s rrna genes. samples c14 and f48 (originating from bovines in the mnisi community area) were used as positive controls for a. marginale. these samples were confirmed to contain a. marginale infections by amplification and sequence analysis of the msp1b gene. nuclease-free water was used as a negative control. thermal cycling was performed in a lightcycler v2 (roche diagnostics, mannheim, germany). thermal cycling conditions were udg activation at 40 °c for 10 min, pre-incubation at 95 °c for 10 min, 40 cycles of denaturation at 95 °c for 1 min, annealing–extension at 60 °c for 1 min and a final cooling step at 40 °c for 30 s. the results were analysed using the lightcycler software version 4.0 (roche diagnostics, mannheim, germany). a positive result was indicated by a cq value (quantification cycle, synonymous with the cp, crossing point, value given by the lightcycler instrument), the cycle at which fluorescence from amplification exceeds the background fluorescence. a lower cq correlates with a higher starting concentration of target dna in a sample. fam fluorescence (530 nm) was generated in a. marginale-positive samples, and lc-610 signals (610 nm) were generated in a. centrale-positive samples. nested polymerase chain reaction two npcrs were used to detect a. marginale and a. centrale in the samples as previously described (decaro et al. 2008; molad et al. 2006). external primers am456 and am1164 and internal primers am100 and am101 (table 1), specific for the msp1β gene of a. marginale, were used in the a. marginale-specific npcr. external primers ac1826 and ac2367 and internal primers cis1925 and cis2157 (table 1) were used to detect the msp2 gene of a. centrale. the optimised pcrs were performed in a final volume of 25 μl, containing 1x dreamtaq green pcr master mix (thermofisher scientific, south africa), yielding final concentrations of 2 mm mgcl2, 0.2 mm dntps, 1x dreamtaq™ reaction buffer and a proprietary amount of dreamtaq™ dna polymerase. for both primary pcrs, approximately 200 ng of genomic dna was used as template, and each external primer was added to a final concentration of 0.5 μm. the primary pcr thermal cycling conditions were 95 °c for 3 min, 35 cycles of 95 °c for 10 s, 62 °c for 30 s and 72 °c for 30 s, followed by a final extension at 72 °c for 7 min. the secondary pcr reaction mixes were prepared in the same way, except that each internal primer was added to a final concentration of 1 μm, and 1 μl of a 1:100 dilution of the primary pcr product was added as template. the secondary pcr thermal cycling conditions were the same as the primary pcr cycling protocol, except that the annealing temperature was at 66 °c (a. marginale) and 68 °c (a. centrale). the secondary pcr products were analysed by electrophoresis through a 2% agarose gel and stained with ethidium bromide. specificity and sensitivity of the reverse line blot, nested polymerase chain reaction and quantitative polymerase chain reaction assays the specificity of the rlb, npcr and qpcr assays in detecting closely related species has previously been assessed (bekker et al. 2002; carelli et al. 2007; decaro et al. 2008; molad et al. 2006). we analysed dna extracted from anaplasma sp. omatjenne, anaplasma phagocytophilum, babesia bovis and theileria parva using the rlb, npcr and qpcr assays. in order to determine the sensitivities of the assays, the msp1β and 16s rrna genes of a. marginale from sample f48, and the groel, msp2 and 16s rrna genes of a. centrale from sample 9410 were amplified with gene-specific primers (table 1) and cloned in the pjet vector (thermofisher scientific, south africa). clones with the correct insert were sequenced at inqaba biotechnologies (south africa) using vector primers. the sequences were assembled and aligned using the clc main workbench 7 (http://www.clcbio.com). plasmid dna was extracted from clones f48a (a. marginale msp1β gene), f48d (a. marginale 16s rrna gene), 9410c (a. centrale groel gene), 9410g (a. centrale 16s rrna gene) and 9410i (a. centrale msp2 gene) using the high pure plasmid isolation kit (roche diagnostics, mannheim, germany). the concentrations of the plasmids were determined using the powerwave xs2 microplate spectrophotometer (biotek, usa), and the copy number (copies/μl) was calculated using the formula below (ke et al. 2006): the linear ranges of detection of the assays were evaluated by analysing 10-fold serial dilutions of plasmid dna using the rlb, qpcr and npcr with an input of 2.5 μl of each dilution of dna. for the qpcr duplex assay, the dilutions were analysed in triplicate, and the means of the cq values were plotted against the log concentrations to generate standard curves for absolute quantification of a. marginale and a. centrale. pcr efficiency (e) was calculated from the slope of the curve using the formula below (bustin et al. 2009): amplification, cloning and sequencing of the msp1β gene to explain discrepancies between the npcr and qpcr assay results in the detection of a. marginale, the target sequence region of the npcr assay was evaluated by cloning and sequencing of the msp1β gene from selected a. marginale-positive field samples (c1, c14, c57, f48). primers am.f and am.r or am456 and am1164 (table 1) were used for the pcr. the 25 µl reaction mixture contained 1x dreamtaq green pcr master mix (thermofisher scientific, south africa), 0.5 µm of each primer (table 1) and 2.5 µl of template dna. purified pcr products were cloned into pgem®-t (promega, usa), and recombinant plasmids were sequenced at inqaba biotechnologies (south africa). the sequences were assembled and analysed using the clc main workbench 7 (http://www.clcbio.com). the identity of sequences obtained was determined by blast analysis (altschul et al. 1990), using the blastn function. genbank accession numbers sequences were submitted to genbank under the following accession numbers: ku647713–ku647720 (a. marginale msp1β gene), ku598853 (a. marginale 16s rrna gene), ku598854 (a. centrale 16s rrna gene), ku647711 (a. centrale groel gene) and ku647712 (a. centrale msp2 gene). statistical analysis the data were analysed using the statistical package for the social sciences (spss) version 23.0 (ibm spss, 2014). the fisher’s exact test was used to determine if the results of rlb, npcr and qpcr assays in detecting a. marginale or a. centrale infections in cattle were significantly different. the level of agreement between the results of the three assays was evaluated using the kappa score, at a 95% confidence interval (viera & garrett 2005). results specificity and sensitivity of the assays amplicons of approximately 500 bp were obtained from a. marginale clone f48d and a. centrale clone 9410g using the rlb pcr primers. amplicons of 246 bp (from a. marginale clone f48a) and 252 bp (from a. centrale clone 9410i) were obtained using npcr. as expected, qpcr products of approximately 95 bp and 77 bp were obtained from a. marginale clone f48a and a. centrale clone 9410c, respectively (results not shown). fam fluorescence (530 nm) was generated from a. marginale clone f48a, and lc-610 (610 nm) signals were generated from a. centrale clone 9410c. the efficiency of the duplex qpcr was 104% and 101% (figure 3) for a. marginale and a. centrale, respectively. no amplification was detected from the dna of anaplasma sp. omatjenne, a. phagocytophilum, b. bovis and t. parva or from the negative (water) control by the rlb, npcr or duplex qpcr assay. serial dilutions of each plasmid clone were prepared and tested using the rlb hybridisation assay (a. marginale clone f48d and a. centrale clone 9410g), npcr (a. marginale clone f48a and a. centrale clone 9410i) and duplex qpcr (a. marginale clone f48a and a. centrale clone 9410c). the smallest amounts of a. marginale plasmid dna that could be detected were 2500 copies per reaction for the rlb hybridisation assay (figure 1) and 250 copies per reaction for the npcr and qpcr assays (figures 2a and 3a). anaplasma centrale detection limits by the rlb, npcr and qpcr assays were 2500, 250 and 25 copies per reaction respectively (figures 1, 2b and 3b). figure 1: detection of serial dilutions (2.5x107 – 2.5x100 copies) of plasmid dna by the reverse line blot hybridisation assay. figure 2: detection of serial dilutions of plasmid dna by nested polymerase chain reaction. (a) lanes 1–8: 10-fold serial dilutions (2.5x107 – 2.5x100 copies) of anaplasma marginale plasmid dna (clone f48a; msp1β gene); lane 9: water negative control. (b) lanes 1–9: 10-fold serial dilutions (2.5x108 – 2.5x100 copies) of anaplasma centrale plasmid dna (clone 9410i; msp2 gene); lane 10: water negative control. m: 100 base pair marker; numbers on the left and right indicate molecular sizes in base pairs. figure 3: detection of 10-fold serial dilutions of plasmid dna by the duplex quantitative polymerase chain reaction assay. (a) anaplasma marginale plasmid dna (clone f48a; msp1β gene) 2.5x107 – 2.5x102 copies. (b) anaplasma centrale plasmid dna (clone 9410c; groel gene) 2.5 x 107 – 2.5 x 101 copies. detection of anaplasma marginale and anaplasma centrale in field samples by the reverse line blot, nested polymerase chain reaction and quantitative polymerase chain reaction assays the qpcr assays detected more a. marginaleand a. centrale-positive samples than either the rlb or npcr assays (figure 4a), either as single or mixed infections (figure 4b), although this difference was not statistically significant for a. centrale infections detected by the qpcr and npcr (figure 4a). the number of a. marginale-positive samples detected by qpcr was significantly different from the number of a. marginale-positive samples detected by rlb or npcr (p ≤ 0.05). both npcr and qpcr detected significantly more a. centrale-positive samples than the rlb (p ≤ 0.05; figure 4a). there was no significant difference between the number of a. marginale infections detected by the rlb and npcr assays (figure 4b). figure 4: (a) detection of anaplasma marginale and anaplasma centrale in south african cattle samples (n = 66) by the reverse line blot hybridisation assay (black), nested polymerase chain reaction (dark grey) and quantitative polymerase chain reaction (light grey). (b) proportion of single and mixed infections in south african cattle samples as detected by the three assays. single anaplasma marginale infection (grey), single anaplasma centrale infection (black), mixed anaplasma marginale and anaplasma centrale infections (hatched), no infection detected (white). the level of agreement between the results of the three assays was determined using kappa scores (table 2). for a. marginale, the agreement between the rlb and npcr assays and between the rlb and the qpcr assay was fair, whereas the agreement between the npcr and qpcr was moderate. for a. centrale, there was slight agreement between the results of the rlb and the npcr assays, and between the rlb and qpcr assays. the agreement between the npcr and the qpcr results was substantial (table 2). table 2: comparison of reverse line blot, nested polymerase chain reaction and quantitative polymerase chain reaction assays in the detection of anaplasma marginale and anaplasma centrale in cattle samples in south africa. the npcr and the qpcr assays had equivalent sensitivities in detecting a. marginale plasmid dilutions, and therefore, a substantial agreement between the tests was expected. however, the agreement was only moderate. although the two tests were in agreement for the majority (38) of a. marginale-positive samples, 13 samples that tested positive by the qpcr assay tested negative by npcr (table 2). dna smears were obtained in many of the a. marginale msp1β secondary pcr products from field samples, compared with clear bands obtained for a. centrale groel secondary pcr products (results not shown). to investigate the discrepancy in the detection of a. marginale by the npcr and qpcr assays, the msp1β gene was amplified, cloned and sequenced from selected field samples that yielded both sharp and ‘smeary’ pcr products to determine whether the target sites of the a. marginale npcr primers were conserved in the field samples examined. the sequence alignment indicated that the target sites of the external primers, am456 and am1164, and the internal reverse primer, am101, were identical in all of the samples. however, the target site of the internal forward primer am100 was not well conserved amongst the different a. marginale msp1β gene sequences from south africa (figure 5). figure 5: alignment of south african anaplasma marginale msp1β sequences generated in this study (ku647713–ku64747420) with published anaplasma marginale msp1β sequences m59845 (florida), af111196 and af111197 (south idaho) and af112479 (havana). the npcr and the qpcr tests were in agreement for 16 a. centrale-positive samples, but 11 samples tested positive for a. centrale using qpcr and negative using npcr (table 2). an attempt was made to amplify the msp2 gene from these samples, but it was not possible to obtain visible pcr products because of very low rickettsemias, and therefore sequence data could not be obtained. discussion in epidemiological studies, data on the prevalence of parasitic infections is highly dependent on the sensitivity of the diagnostic assay used (hofmann et al. 2015). we evaluated the ability of three published molecular assays in detecting a. marginale and a. centrale infections in blood samples from cattle in south africa. the rlb (bekker et al. 2002), npcr (decaro et al. 2008; molad et al. 2006) and qpcr (carelli et al. 2007; decaro et al. 2008) assays have previously been shown to be specific for detecting these infections in tick vectors and hosts. in addition, our results indicated that the tests do not detect dna from anaplasma sp. omatjenne, a species frequently encountered in south african field samples. our results indicate that the rlb assay is less sensitive than the npcr and qpcr assays in detecting a. marginale and a. centrale infections in cattle under the conditions prevailing when the tests were performed in south africa. the rlb assay is nevertheless a valuable screening tool for simultaneously detecting infections using species and genus specific (catchall) probes (bekker et al. 2002; gubbels et al. 1999). it has therefore been used extensively to reveal anaplasma/erhlichia and/or babesia/theileria infections in different hosts and vectors and in identifying novel species and variants of species in these genera (bhoora et al. 2009; bosman et al. 2010; ceci et al. 2014; chaisi et al. 2011; mans et al. 2011; nijhof et al. 2005; oosthuizen et al. 2009). in samples that contain mixed infections, however, the use of a single primer pair to amplify all infections decreases the sensitivity of the assay because of competition for primers between the different templates. organisms present at low infection levels could be masked by those with higher infection levels and could therefore be missed. misdiagnosis of carrier animals has important implications for disease control as outbreaks may occur when such animals are introduced to naïve animals in the presence of tick vectors (bilgic et al. 2013). in our study, the ‘catchall’ probe signal was very strong at the lowest detection limit of a. marginale, but the species-specific signal was very weak (figure 1). such low infections could easily be missed or regarded as ‘catchall’ signals only. optimisation of the concentration of the a. marginale probe used in the rlb assay might help in overcoming this problem. the a. centrale species-signal was strong and remained so throughout the detection range of the assay (figure 1). the npcr and duplex qpcr assays, which both detect the msp1β gene of a. marginale (carelli et al. 2008; molad et al. 2006), had the same detection limit (250 copies/reaction) in detecting a. marginale plasmid clones. however, the qpcr assay detected significantly more infections from field samples than the npcr assay. in other studies, the npcr assay was reported to be equally sensitive to the qpcr in detecting a. marginale infections (carelli et al. 2007; molad et al. 2006). in our study, dna smears were obtained in many of the a. marginale msp1β secondary npcr products from field samples. smearing in pcr products can indicate the addition of too much template dna (http://www.bio-rad.com/en-za/applications-technologies/pcr-troubleshooting); however, the amount of primary pcr product added was optimised, and many positive samples gave discrete pcr products, indicating that this was probably not the cause of the smears. smearing can also result if the sequence of one of the primers does not correspond with the sequence of the template. cloning and sequencing of the msp1β gene of a. marginale from selected field samples revealed a 12-bp deletion in the target region of the secondary pcr forward primer (am100). this primer would almost certainly fail to anneal to a. marginale strains containing the deletion, therefore yielding false negative results. the smears obtained in many of the a. marginale msp1β secondary pcr products from field samples were therefore likely to be due to the presence of the deletion in the msp1β gene in these samples. the use of a forward primer targeting a more conserved region of the gene would overcome this problem. although a. centrale is considered to be less pathogenic than a. marginale, the vaccine strain has been reported to cause severe anaplasmosis in adult cattle of susceptible breeds and in splenectomised adult cattle (bigalke 1980; kuttler 1966; pipano et al. 1976, 1985). more recently, a clinical case of bovine anaplasmosis attributed to a pathogenic strain of a. centrale that is closely related to the vaccine strain was reported in italy (carelli et al. 2008). it is therefore important to use assays that are specific and sensitive in detecting both a. marginale and a. centrale. our results indicate that the qpcr assay is ten times more sensitive than the npcr assay in detecting a. centrale infections. these results are corroborated by the higher prevalence of a. centrale detected in field samples by the qpcr assay than the npcr. however, decaro et al. (2008) found the npcr assay to be 1 log more sensitive than the qpcr assay in detecting a. centrale infections in cattle. the npcr targets the multi-copy msp2 gene and would be expected to be more sensitive than assays that target single-copy genes (hofmann et al. 2015; reinbold et al. 2010). however, msp2 is also a highly variable gene, and assays utilising such genes should target conserved regions of the gene so that the assay detects infections from a wide variety of hosts and geographical regions. although we were not able to sequence the msp2 gene of samples with conflicting a. centrale npcr and qpcr results, it is possible that this discrepancy is because of sequence differences in one or more of the primer target regions of south african a. centrale strains, as we observed with a. marginale msp1β sequences from south africa. variation of the msp2 gene of anaplasma spp. has previously been shown to occur between and within species, and amongst geographically different isolates (rymaszewska 2011). although probe-based qpcr is more expensive than npcr, it offers more advantages in that it is usually more sensitive, it is quantitative and has a short turn-around time, and the risk of carry-over contamination is much less than npcr (carelli et al. 2007). additionally, the duplex qpcr assay developed by decaro et al. (2008) offers a multiplex assay for simultaneous detection of low infections of both a. marginale and a. centrale using species-specific primers and probes in a single assay. it is therefore an invaluable tool for specific detection of these organisms in endemic regions. conclusion our results indicate that the duplex qpcr is more sensitive than the npcr and rlb assays in detecting carriers of bovine anaplasmosis in south africa. the rlb is the least sensitive method and detected fewer field samples than could be detected by the other methods. we found that there is variability in the msp1β gene target region of one of the internal primers of the npcr assay. this highlights the importance of testing the suitability of these assays in a new geographical region prior to deployment and also the difficulty of designing tests for these variable pathogens. surface proteins are often attractive targets as they provide good species specificity; however, these molecules are under tremendous selection pressure and are therefore frequently variable. acknowledgements this work is based on a research supported by the national research foundation (nrf) of south africa (grant number 81840 awarded to dr nicola collins) and technology innovation agency (tia), tshwane animal health cluster (grant tahc12-00037 awarded to professor marinda oosthuizen). any opinion, finding and conclusion or recommendation expressed in this material are that of the authors, and the funders do not accept any liability in this regard. the authors acknowledge dr erich zweygarth (freie universitat, berlin, germany) for providing anaplasma sp. omatjenne dna and dr charles byaruhanga (university of pretoria, south africa) for statistical assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.e.c. performed most of the experiments, analysed the data and wrote the 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tanzania ester adamson department of microbiology, parasitology and biotechnology, sokoine university of agriculture, tanzania jessica rowland department of microbiology, parasitology and biotechnology, sokoine university of agriculture, tanzania pedro m. palermo department of biological sciences, university of texas at el paso, united states mirende matiko department of microbiology, parasitology and biotechnology, sokoine university of agriculture, tanzania george e. bettinger department of biological sciences, university of texas at el paso, united states philemon wambura department of microbiology, parasitology and biotechnology, sokoine university of agriculture, tanzania john c. morrill orion research and management services, texas, united states douglas watts department of biological sciences, university of texas at el paso, united states citation nyundo, s., adamson, e., rowland, j., palermo, p.m., matiko, m., bettinger, g.e. et al., 2019, ‘safety and immunogenicity of rift valley fever mp-12 and armp-12δnsm21/384 vaccine candidates in goats (capra aegagrus hircus) from tanzania’, onderstepoort journal of veterinary research 86(1), a1683. https://doi.org/10.4102/ojvr.v86i1.1683 original research safety and immunogenicity of rift valley fever mp-12 and armp-12δnsm21/384 vaccine candidates in goats (capra aegagrus hircus) from tanzania salama nyundo, ester adamson, jessica rowland, pedro m. palermo, mirende matiko, george e. bettinger, philemon wambura, john c. morrill, douglas watts received: 17 aug. 2018; accepted: 16 nov. 2018; published: 31 jan. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract vaccination of domestic ruminants is considered to be an effective strategy for protecting these animals against rift valley fever (rvf), but available vaccines have limitations. therefore, the aim of this study was to determine the safety and immunogenicity of rvf virus (rvfv) mutagenesis passage 12 (mp-12) and armp-12δnsm21/384 vaccine candidates in goats (capra aegagrus hircus) in tanzania. goats were vaccinated intramuscularly with rvfv mp-12 or armp-12δnsm21/384, and then on day 87 post-vaccination (pv) all animals were revaccinated using the rvfv mp-12 vaccine candidate. serum samples were collected from the animals before and after vaccination at various intervals to test for rvfv using a vero cell culture assay and reverse transcription polymerase chain reaction and for rvfv-neutralising antibody using a plaque reduction neutralisation assay. serum samples collected before vaccination on days -14 and 0, and on days 3, 4 and 5 pv were negative for rvfv and neutralising antibody. all animals remained healthy, and viremia was not detected in any of the animals. rift valley fever virus antibody was first detected on day 5 pv at a 1:10 dilution in five of five animals vaccinated with the mp-12 vaccine and in five of eight animals vaccinated with armp-12δnsm21/384. titres then increased and were sustained at 1:40 to 1:640 through to day 87 pv. all animals that were revaccinated on day 87 pv with mp-12 developed antibody titres ranging from 1:160 to as high as 1:10 240 on days 14 and 21 pv. although the antibody titres for goats vaccinated with rvf mp-12 were slightly higher than titres elicited by the armp-12δnsm21/384 vaccine, these findings demonstrated that both vaccines are promising candidates for the prevention of rvf among tansanian goats. introduction rift valley fever (rvf) is an important zoonotic disease in africa and the arabian peninsula affecting both humans and animals, especially domestic ruminants (balkhy & memish 2003; pepin et al. 2010). the disease is caused by the rvf virus (rvfv), a negative single-stranded rna virus that belongs to the order bunyavirales, family phenuiviridae, genus phlebovirus (rima et al. 2017). the disease in animals is characterised by fever, ocular and nasal discharge, bloody diarrhoea, abortion storms in gestating ewes and 90% – 100% mortality in newborn lambs. in humans, the disease causes self-limiting febrile illness, but in about 1% – 2% of cases clinical symptoms progress to neurological disorder, vision loss, haemorrhagic fever and even death (madani et al. 2003). the disease was first identified during an epizootic and epidemic among sheep and humans on a farm in 1931 in the rift valley of kenya (munyua et al. 2010). subsequent outbreaks have been reported from numerous countries throughout africa and the arabian peninsula. outbreaks in east african countries usually occur following heavy rainfall that results in an increase in the abundance of mosquito vectors. in tanzania, outbreaks occur every 5–15 years with low-level transmission of rvfv between outbreaks (sumaye et al. 2013; woods et al. 2002). the first outbreak in tanzania was documented in 1977 and the most recent one occurred during 2006–2007 (anyamba et al. 2010; jost et al. 2010). in contrast to the most recent outbreak that affected humans and livestock in 52.4% of the regions in tanzania, previous outbreaks only affected livestock primarily in the northern parts of the country (faburay et al. 2017). as a result of the devastating impact of rvf on human and animal health in tanzania and other rvfv-enzootic countries, several vaccines have been developed, and some are currently being used in an attempt to prevent this disease among livestock in africa (faburay et al. 2017). vaccines offer the most promising control and prevention strategy for rvf because they can afford protection by inducing humoral and cell-mediated immune responses, as well as by enabling vaccinated animals to transfer colostrum that contains maternally acquired antibody to their offspring. (dar et al. 2013; labeaud, kazura & king 2010; morrill et al. 1987; morrill, mebus & peters 1997a; niklasson, meadows & peters 1984; pepin et al. 2010). therefore, a safe and efficacious vaccine that produces a rapid humoral response and long-term protective immunity could prevent human and animal disease and save economic resources in an outbreak situation (morrill et al. 2013b). however, the currently used rvf vaccines have not had a significant impact on the prevention of rvf in livestock, and approved vaccines are not available for human use (morrill & peters 2003). some of the promising rvf vaccine candidates being evaluated include the mutagenesis passage 12 (mp-12) vaccine and a recombinant candidate vaccine derived from mp-12, referred to as armp-12δnsm21/384 (caplen, peters & bishop 1985; saluzzo & smith 1990; won et al. 2007). rift valley fever mp-12 is a live attenuated mutagenised vaccine that was developed from a virulent egyptian rvfv strain, zh548, by 12 serial passages in human foetal lung fibroblast (mrc-5) cells in the presence of 5-flourouracil. as a result, mutations were induced in the large, medium and small rna segments resulting in attenuation of the virus through amino acid changes (vialat et al. 1997). although the mp-12 vaccine candidate was found to be safe and immunogenic in human volunteers, efforts to develop rvf mp-12 vaccine for human use were suspended because of other priorities (ikegami & makino 2009; pittman et al. 2016a, 2016b). moreover, extensive testing of the mp-12 vaccine found it to be safe and immunogenic in small laboratory animals, non-human primates, as well as in sheep and cattle (bird et al. 2009; morrill et al. 1987, 1991, 1997b, morrill & peters 2011). as a potential veterinary vaccine, mp-12 was not considered to be a promising candidate because it does not have biomarkers to distinguish naturally infected animals from vaccinated animals (diva). therefore, reverse genetic technology was used to develop a recombinant vaccine (armp-12δnsm21/384) that has nucleotides 21–384 deleted from the non-structural regions of the m segment to serve as a potential diva vaccine (ikegami et al. 2006; kalveram et al. 2011; won et al. 2007). safety and immunogenicity studies conducted in the usa demonstrated that the armp-12δnsm21/384 candidate vaccine was safe and immunogenic in sheep and calves using doses ranging from 1 × 103 through 1 × 105 plaque forming units (pfu) and was non-abortigenic and non-teratogenic in pregnant ewes vaccinated during the early gestation period (morrill et al. 2013a, 2013b). moreover, sheep vaccinated with this vaccine and then challenged with a virulent strain of rvfv were protected during experimental studies in canada (weingartl et al. 2014). although mp-12 and armp-12δnsm21/384 vaccine candidates have been shown to be safe and efficacious in sheep and calves in the united states (us), and the armp-12δnsm21/384 vaccine in sheep in canada, studies have not been conducted to assess the safety and immunogenicity of these vaccines in these target species or in goats in an rvfv-enzootic african country such as tanzania. therefore, the aim of this study was to assess safety and immunogenicity of mp-12 and armp-12δnsm21/384 vaccine candidates in goats (capra aegagru hircus) in tanzania. materials and methods study area animal experiments were conducted in an insect-proof animal biosafety level 2 (absl-2) facility and laboratory testing of blood samples from the animals was performed in a biosafety level 2 (bsl-2) virology laboratory located at sokoine university of agriculture (sua), morogoro, tanzania. the morogoro district is located at latitude 6°49’s and 37°39’e with an elevation peak at 1200 m above sea level. it is bordered by seven regions: tanga and manyara to the north; ruvuma, iringa and njombe to the south; the coastal region to the east; and dodoma to the west. it has a total of eight districts, namely, kilosa, mvomero, ulanga, gairo, kilombero, morogoro rural and morogoro district. experimental animals healthy c. aegagrus hircus goats 6–9 months old were used in this study. a total of 15 animals were purchased from local vendors in the mvomero district of the morogoro region of tanzania and housed in the sua absl-2 facility. prior to entering the facility, all animals were sprayed with steladone® 300 emulsifiable concentrate (ec) acaricide to remove and prevent introduction of ectoparasites. in addition, all animals were treated orally with 4 ml of 2.5% albendazole for possible parasites. the animals were individually identified using numbered ear tags and acclimatised in the facility for 2 weeks before use in the experiments. all 15 animals were housed in the same room of the facility. throughout the experiment, all animals were given fresh grass three times a day, supplemented with maize bran, a mineral block and water ad libitum, and were observed daily for elevated body temperature as a possible indication of illness. vero e6 cells and vaccine viruses the vero e6 cells used in this study were kindly provided by the university of texas at el paso (utep), texas, us. aliquots of 1.0 ml in freeze-dried form of the armp-12δnsm21/384 vaccine (lot no. 15/3/2017) were provided by the multi-chemical industry (mci) santé animale biopharmaceutical company in mohammedia, morocco. the identity of armp-12δnsm21/384 virus was confirmed at mci using a qualitative real-time polymerase chain reaction assay (nfon et al. 2012) targeting the l and m viral rna segments (morrill & peters 2011; njenga et al. 2015) followed by sequencing at the genewiz laboratories (genewiz global headquarters; us) using next generation sequencing technology (illumina method: 1 × 50 bp single read hiseq2500, high output, per lane [v4 chemistry]). the infectivity titre of the armp-12δnsm21/384 vaccine virus was 105.5 tissue culture infectious dose 50% (tcid50/ml in vero e6 cells. the mp-12 virus was originally obtained by utep from the world reference centre for emerging viruses and arboviruses, department of microbiology and immunology, university of texas medical branch, galveston, texas, usa. at utep, the identity of the mp-12 vaccine virus was confirmed using the plaque reduction neutralisation test (prnt) and a rvfv mp-12-specific monoclonal antibody (mab). the mab neutralised the infectivity titre of the mp-12 virus from 106.0 pfu/ml to 102.0 pfu/ml but did not neutralise the infectivity titre of sindbis and/or west nile viruses. a virus stock of rvf mp-12 was prepared at utep with an infectivity titre of 1.4 × 107.0 pfu/ml in vero e6 cells and was stored in 0.5 ml aliquots at −80°c. of this stock, 10 aliquots were provided to the sua virology laboratory to prepare working virus stocks to support this study. at sua, a working stock of the mp-12 virus was prepared in vero e6 cells with an infectivity titre of 1 × 107.0 pfu/ml. experimental design and vaccination the goats used in this study were divided into three groups: five animals for vaccination with mp-12, eight for armp-12δnsm21/384 and two animals for negative controls. each freeze-dried vial of armp-12δnsm21/384 was reconstituted in 2 ml of eagle’s minimum essential medium (emem) containing 4% foetal bovine serum (fbs) (thermo fisher scientific, carlsbad, ca, usa). each reconstituted vial contained 1 × 105.0 pfu/ml of the armp-12δnsm21/384 virus. the mp-12 vaccine virus was diluted in emem to yield a concentration of 1 × 105.0 pfu/ml from the initial concentration of 1.4 × 107.0 pfu/ml. one millilitre of each virus was loaded into separate 5 ml syringes in a class iia2 biosafety cabinet (nuaire, plymouth, mn, usa) and transported in a cool box on ice to the absl-2 animal facility. an 18-gauge needle was attached to each of the 5 ml syringes and the animals were vaccinated intramuscularly (im) in the neck area with 1 ml per animal. the two control animals were vaccinated likewise with 1 ml of emem containing 4% fbs. specimen collection and preparation blood samples (4 ml) were collected from the jugular vein of each manually restrained goat using a 6 ml vacutainer tube. serum (2 ml – 3 ml) was obtained from each of the animal blood samples after leaving the samples overnight at 4 °c followed by centrifugation at 1200 g for 10 minutes. aliquots of 0.5 ml – 1.0 ml of each serum sample were transferred to sterile prelabelled vials and stored at −80 °c in an ultra-low temperature freezer until tested for rvfv and/or rvfv-neutralising antibody. serum samples were collected 14 days before vaccination, as well as on day 0 immediately before vaccination, and were tested for rvfv using a vero e6 cell culture assay and for rvfv antibody using the prnt. samples obtained on days 3, 4 and 5 were also tested for rvfv using the same cell culture assay; thereafter, samples obtained on days 7, 14, 21, 28, 35, 70, 84 and 87 post-vaccination (pv) were tested to determine the neutralising antibody response using the prnt. on day 87 pv, all goats including the two emem control animals were revaccinated with 1 ml of 1 × 104.0 pfu/ml of the mp-12 vaccine. all animals were observed for signs of illness and each week rectal temperatures were recorded. blood samples were obtained on days 7, 14 and 21 following revaccination to determine the neutralising antibody response using the prnt, as described below. rift valley fever reverse transcription polymerase chain reaction prior to performing the rvf reverse transcription polymerase chain reaction (rt-pcr) assay, rna was extracted from serum samples collected from goats on day 14 before vaccination, on day 0 of vaccination and on days 3, 4, and 5 pv following the manufacturer’s instructions using the siam® viral rna mini kit (qiagen, hilden, germany). sera samples were pooled in groups of two, and mp-12 virus positive and negative control samples were included during rna extraction. after extraction, rna was stored at −80 °c. the qiagen one-step rt-pcr kit was used to test rna samples for rvfv rna. primers targeting the m segment (551 bp) – rvf forward 5’tgt gaa caa tag gca ttg g’3 and rvf reverse 3’gac tac cag tca gct cat tac 5’ (ibrahim et al. 1997) – were used at a concentration of 0.1 µm. mutagenesis passage 12 viral rna was used as a positive control, and master mix (buffer) was used as a negative control in the rt-pcr assay. thermocycler conditions were as follows: initial cdna synthesis at 50 °c for 30 min, pcr activation at 95 °c for 30 min, followed by 40 cycles at 95 °c for 30 seconds, 58 °c for 1 min and 72 °c for 2 min, then final extension at 72 °c for 10 min. the pcr amplicons, together with hi-lo™ dna marker (bionexus, inc. oakland, ca, usa), were loaded and separated on a 1.5% agarose gel (stained with 10 µl of gel red) using electrophoresis at 120 volts/20 cm for 45 min and visualised using a uv-transilluminators. virus isolation the sera samples obtained from goats on day 14 before vaccination and on day 0 of vaccination and samples obtained on days 3, 4 and 5 pv were diluted 1:2 in emem supplemented with 4% fbs. confluent monolayers of vero e6 cells were propagated in 24-well plates, and each culture was inoculated in duplicate with 50 µl of each serum sample. the cultures and inoculums were incubated for 1 h at 37 °c and agitated every 15 min to facilitate virus absorption. after absorption, 0.5 ml of emem supplemented with 4% fbs was added to each culture and incubation was continued at 37 °c with 5% co2. cultures were observed once daily for 10 days using an inverted microscope for cytopathic effect (cpe). after 10 days, all cpe-negative cultures were frozen, thawed and then passaged blindly in vero e6 cells using the same procedure; they were again observed once daily for 10 days for cpe. any cultures that developed cpe were harvested and stored in aliquots of 1.0 ml for further study using rt-pcr to determine if the cpe was caused by rvfv. if there was evidence of rvfv, all aliquots and any remaining cultures were destroyed by heating in an autoclave at 44.4 °c because of biosafety concern requirements that rvfv as a select agent must be kept in a bsl-3-plus laboratory. all animals used in the vaccine trials were kept isolated and quarantined in a holding facility separate from the absl-2 facility, and if confirmed to be infected with rvfv they were not used any further in this study. plaque reduction neutralisation test-80 serum samples collected from the goats on days 5, 7, 14, 21, 28, 35, 70, 84 and 87 pv and on days 7, 14 and 21 pv following revaccination were tested for rvfv-neutralising antibody. each serum sample was diluted 1:5 initially, followed by fourfold dilutions through 1:5120 in hanks’ balanced salt solution supplemented with 1% hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), penicillin and streptomycin and heat-inactivated fbs in a 96-well plate (thermo fisher scientific). each diluted test sera (75 µl) was mixed with an equal volume of virus suspension containing approximately 60–80 rvfv pfus. as a result, the final sera dilutions were 1:10, 1:40, 1:160, 1:640, 1:2560 and 1:10 240, containing virus ranging from 30 pfu to 40 pfu. the controls consisted of a mixture of an equal volume of 60–80 rvfv pfu with a 1:10 dilution of rvfv-positive antibody and a rvfv-negative antibody goat serum. the virus–serum dilution mixtures were incubated at 37 °c in the absence of co2 for 1 hour. next, vero e6 cells were seeded in 24-well tissue culture plates and incubated for 4–5 days at 37 °c and 5% co2 to provide 90% confluent monolayers. the growth media was then discarded from the vero e6 cell monolayers and 50 µl of each virus–serum dilution mixture was inoculated onto each of two wells of cell monolayers per sample. the virus positive antibody control serum mixtures were inoculated onto each of 20 culture wells and the virus-negative antibody control serum mixture was inoculated onto each of culture wells. cultures and inocula were incubated for 1 h at 37 °c and 5% co2 with agitation every 15 min. seakem agarose (1%) with an equal volume of 2x eagle’s basal medium with earle’s salt (ebme), hepes, sodium bicarbonate, 8% fbs and 1% penicillin, streptomycin and l-glutamine (thermo fisher scientific) was then prepared, and 0.5 ml was overlaid onto each cell culture. the agarose overlay was allowed to solidify and then the cultures were incubated for 2 days at 37 °c and 5% co2. each culture was then overlaid with 0.5 ml 1% agarose mixed with an equal volume of 2x ebme supplemented with 5% neutral red (thermo fisher scientific) and incubated overnight at 37 °c. the plaque forming units were counted and the dilution of serum that reduced the rvf mp-12 virus dose by 80% was considered as the neutralising antibody titre. clinical assessment of animals rectal body temperatures were recorded for each animal at the time of blood collection up to day 35 pv. in addition, their general health status was assessed by veterinary personnel once a day and recorded. animals that developed any sign of illness during the study were given a clinical examination by a veterinarian and samples were collected for analysis and diagnosis. statistical analysis data analysis was performed using r statistical analysis software version 3.4.1. analysis of the difference in antibody responses between goats vaccinated with the mp-12 or armp-12δnsm21/384 vaccines during the first vaccination and after mp-12 boosting were performed using the welch two-sample t-test with a significance level of p ≤ 0.05. ethical considerations the animal experiment was performed according to an experimental protocol reviewed and approved by the utep, el paso, texas, and the sua iacuc (institutional animal care and use committee) (ref # 559105-08 and sua/cmvbs/r.1, respectively). results clinical assessment of the animals the rectal body temperatures of all animals before vaccination with mp-12 or armp-12δnsm21/384 ranged from 38.2 °c to 38.5 °c. on day 1 after vaccination, the temperatures of all vaccinated animals had increased to 39.0 °c, and the control animals had a temperature of 40.0 °c. on day 2 pv and thereafter throughout the study, the temperatures of the animals ranged from 37.0 °c to 38.5 °c, including the control animals, and all animals remained healthy throughout the study (figure 1). figure 1: mean rectal body temperatures of goats (capra aegagrus hircus) vaccinated with rift valley fever mp-12 and armp-12δnsm21/384 vaccines. viremia serum samples obtained from all goats 14 days before vaccination and on day 0 immediately prior to vaccination with the mp-12 or armp-12δnsm21/384 vaccine were negative for rvfv rna as indicated using rt-pcr and rvfv isolation attempts in vero e6 cells. also, rvfv was not detected in any of the sera samples obtained on days 0, 3, 4 and 5 pv, nor from blind passage in vero e6 cells. therefore, there was no detectable viremia in the goats as a result of im vaccination with mp-12 or mp-12-nsm-del vaccines. immunogenicity all goats vaccinated with mp-12 or armp-12δnsm21/384 developed neutralising antibodies; however, the two control animals inoculated with only emem supplemented with 4% fbs did not produce neutralising antibodies (table 1). on day 5 pv, all five animals vaccinated with mp-12 had neutralising antibody titres of 1:10. on day 14 pv, three animals had neutralising titres of 1:40 and two had titres of 1:160. the antibody titres increased until day 28 and were either sustained or decreased through to day 87 pv, when all animals were revaccinated with 1 ml each of 1 × 104 pfu/ml of the mp-12 vaccine virus. the humoral immune response in these revaccinated animals was characterised by a rapid increase in neutralising antibody titres to peak titres of 1:640 on day 7 pv in all animals; on day 14 titres ranged from 1:640 to 1:10 240 and on day 21 pv from 1:2560 to 1:10 240 (table 1). table 1: rift valley fever neutralising antibody titres in goats (capra aegagrus hircus) vaccinated with 1 × 105.0 plaque forming unit (pfu)/ml of rift valley fever mp-12 and armp-12δnsm21/384 vaccine candidates and revaccinated with 1 × 104.0 pfu/ml of mp-12 on day 87 post-vaccination. in armp-12δnsm21/384 vaccinated goats, five of eight animals had neutralising antibody with titres of 1:10 on day 5 pv, and by day 7 pv all animals had antibody titres ranging from 1:10 to 1:160. antibody titres remained relatively constant until day 28, and by day 35 a slight increase was observed in titres that were as high as 1:640 in two animals. antibody titres then ranged from 1:40 to 1:160 until day 87 pv. after revaccination of all animals with the mp-12 vaccine on day 87 pv, antibody titres increased, ranging from 1:160 to 1:640 on days 7 and 14 pv, and from 1:160 to 1:2560 on day 21 pv. the antibody titres for the two emem control animals vaccinated with mp-12 and armp-12δnsm21/384 were 1:10 and 1:40 on day 7 pv, increasing to 1:160 for both animals by day 21 pv, thus in line with the titres observed for the animals initially vaccinated with mp-12 or armp-12δnsm21/384 vaccines (table 1). statistical analysis there was no significant difference in the antibody responses between goats vaccinated with mp-12 and those vaccinated with the armp-12δnsm21/384 vaccine (p = 0.10) during the first vaccination. however, the antibody titres for the goats that were revaccinated was significantly higher for the animals that received the mp-12 vaccine than for those that received the armp-12δnsm21/384 vaccine (p = 0.03). discussion the results of this study indicated that the rvf mp-12 and armp-12δnsm21/384 vaccine candidates elicited neutralising antibody in goats following vaccination using the im route. except for slightly elevated temperature of 39 °c to 40 °c on day 1 pv, all animals maintained normal body parameters such as appetite, well-being and normal rectal temperatures ranging between 37 °c and 38 °c. the transient, slightly elevated temperatures on day 1 pv in all animals, including the negative control animals, suggested that this observation was not related to the vaccines. the most likely reason was stress caused by manual handling of the animals during vaccination. other virulent rvfv infection-related symptoms such as haemorrhage, diarrhoea, nasal and ocular discharge were not observed during the entire pv period. there was no evidence of virus shedding as the control animals remained negative, while being confined in the same pens with the vaccinated animals. however, further studies are needed to exclude the possibility of shedding and/or spread of the vaccine virus, including experiments designed to evaluate viral shedding in excreta, such as nasal and ocular swabs, or testing for the potential spread to highly susceptible species, such as younger or immunocompromised animals. the rvf smithburn and clone 13 vaccines, which are the more commonly used vaccines in africa, especially the smithburn vaccine, warrant concern because of a link to foetal malformations, stillbirths and abortions during the first trimester of gestation (botros et al. 2006). moreover, experimental studies showed that clone 13 had a potential teratogenic effect among pregnant sheep (makoschey et al. 2016). although this study did not assess the safety of the vaccines in pregnant goats, our preliminary results showed that both the mp-12 and armp-12δnsm21/384 vaccines were safe and the antibody titres induced were considered to be high enough to protect african goats against rvfv infection. the potential protective efficacy based on antibody titres is supported by the results of a study that showed antibody titres in sheep of approximately 1:100 following vaccination with armp-12δnsm21/384 vaccine were protective against challenge with a virulent strain of rvfv (weingartl et al. 2014). moreover, studies involving the parent mp-12 vaccine revealed that antibody titres ranging from 1:10 to 1:20 in hamsters and 1:20 in rhesus macaques afforded protection against challenge with a virulent strain of rvfv (morrill & peters 2011; niklasson et al. 1984 1984). all five goats vaccinated with mp-12 and five of eight vaccinated with armp-12δnsm21/384 developed detectable neutralising antibodies by day 5 pv, demonstrating that the vaccines elicited a rapid humoral immune response comparable to results reported for sheep inoculated with a similar dose of armp-12δnsm21/384 vaccine (morrill et al. 2013a). moreover, the results were similar to those observed for pregnant sheep vaccinated with rvf mp-12 vaccine that developed detectable neutralising antibody from days 5 to 7 pv (morrill et al. 1991). goats vaccinated with the mp-12 vaccine developed neutralising antibodies with peak titres between 1:160 and 1:640 by day 35 pv, which were either sustained or decreased through day 87 pv prior to being revaccinated with the same vaccine. the rapid antibody immune response inducement and overall in increasing pattern of antibody titres suggested that the vaccine may possibly protect animals, even if administered after the onset of a rvf outbreak, as reported previously (bird et al. 2008). in our study, a robust antibody response was observed in all goats starting from day 7 after revaccination with the mp-12 vaccine. the antibody titres increased from 1:640 to 1:10 240 by day 21 post-revaccination, thus suggesting that the vaccine may afford protection to animals exposed to virulent rvfv in the field. a steady increase in neutralising antibody titres was observed in goats following vaccination with armp-12δnsm21/384, with peak titres measured on day 35 pv ranging from 1:40 to 1:640. these results demonstrated that the deletion of the non-structural region of the medium viral rna segment (nsm) did not affect immunogenicity and that the vaccine activated b-cells and dendritic cells for initiation of antibody development. following revaccination with the mp-12 vaccine, all goats elicited a rapid humoral immune response, and antibody titres were significantly higher than when the animals were first vaccinated, thus further demonstrating the potential of the vaccine to elicit strong immune responses in the field, if the vaccinated animals were exposed to virulent rvfv. the antibody responses of goats following single vaccination with mp-12 or armp-12δnsm21/384 did not differ significantly (p = 0.10), and therefore the armp-12δnsm21/384, with its potential for use as a diva marker vaccine, could have an advantage over the mp-12 vaccine. the results were comparable to those reported for studies conducted in sheep and calves in the usa following vaccination with mp-12 and armp-12δnsm21/348 (morrill et al. 1987, 1991, 1997b, 2013a, 2013b), in which animals developed detectable neutralising antibody by day 7 pv with a titre of 1:20. in this study, neutralising antibody were detected in most goats vaccinated with either vaccine on day 5 pv with titres of 1:10 and in all goats on day 7 with titres ranging from 1:10 to 1:160, slightly higher than titres reported for sheep in the usa study. the observation that sheep vaccinated with armp-12δnsm21/384 developed antibody titres that were comparable to those observed for goats in this study are an indication that these animals should also be protected following challenge with virulent rvfv (weingartl et al. 2014). overall, the antibody titres for goats in this study, following vaccinations with mp-12 or the armp-12δnsm21/384 vaccine candidate, were slightly lower than titres observed for sheep during a study in canada and sheep and cattle inoculated with these vaccines in the usa (morrill et al. 1987, 1991, 1997b, 2013a, 2013b; weingartl et al. 2014). however, the titres were comparable to those reported for goats, sheep and cattle vaccinated with rvf clone 13, despite the difference in laboratory testing procedures (daouam et al. 2015; dungu et al. 2010). comparison of antibody titres among different animal species and involving different laboratories must consider possible differences in genetics, age, nutritional and health status, environment and vaccination, as well as laboratory testing procedures. susceptibility differences may also contribute to variations among animal species in their ability to elicit immune responses to rvfv infection. for example, goats were reported to be more resistant to developing rvf disease than sheep, attributed in part to a lower and shorter viremia (nfon et al. 2012). therefore, the reduced amount of antigen produced in goats following vaccination, as opposed to sheep, may have resulted in a lesser amount of the vaccine virus being available to stimulate b cell secretion of antibody and may therefore have elicited a lower immune response in goats. while differences were observed in antibody titres elicited in goats vaccinated with either of the vaccines, the more critical criteria and promising feature regarding the assessment of the potential value of the mp-12 and armp-12δnsm21/384 vaccines was the fact that the antibody responses were consistent with moderate and predictive protective titres. the importance of this observation is that numerous studies in the usa and africa have demonstrated that antibodies are crucial for protection of animals against infection with rvfv (dungu et al. 2010; niklasson et al. 1984; morrill & peters 2003; njenga et al. 2015; pepin et al. 2010). conclusion the results of this study revealed that both the mp-12 and armp-12δnsm21/384 candidate vaccines elicited the production of antibody titres to levels that could possibly afford protection to goats without inducing adverse post-vaccinal reactions. thus, both vaccines are safe and should prove efficacious towards affording protection to this target species (goats) against virulent wild-type rvfv infection. other studies in progress to further evaluate the safety and immunogenicity of mp-12 and armp-12δnsm21/384 in goats and sheep, as well as evaluating other routes of vaccination, such as the intradermal and intranasal routes, will provide a better understanding of the overall safety and efficacy of the candidate vaccines for use in target domestic ruminant species of rvfv in africa. acknowledgements the authors would like to express their sincere appreciation to the research team under the feed the future innovation laboratory for rift valley fever control in agriculture from sokoine university of agriculture (sua) and the university of texas at el paso (utep) for their high level of cooperation throughout the study. the authors thank dr mhando anthony, peter marwa and shida mkuya for their technical assistance, including handling and maintaining the animals throughout the study and a special thanks to the multi-chemical industry (mci) santé animale biopharmaceutical company in mohammedia, morocco for providing the rvfv armp-12δnsm21/384 vaccine. the authors also thank ms linda salekwa for her technical advice and support throughout the study and the sua institutional animal care and use committee (iacuc) for their oversight during this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions the authors, d.m.w., g.e.b., p.w., m.m., and s.b. conceived and designed the experiments. the experiments were performed by s.b., e.a., j.m., e.a., l.m., and m.m., and the same authors participated in the acquisition, analysis, and interpretation of the data of the work. the authors, s.b., e.a., p.p., d.m.w., p.w., g.e.b., m.m., and j.m. prepared the manuscript. all authors participated in the revision of the manuscript, and agreed to be accountable for all aspects of the work and approved the final version this manuscript. funding information this study was funded under a subcontract from the university of texas at el paso (utep), texas who was awarded funding by united stated agency for international 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protein of rift valley fever virus suppresses virus-induced apoptosis’, journal of virology 81, 13335–13345. https://doi.org/10.1128/jvi.01238-07 woods, c.w., karpati, a.m., grein, t., mccarthy, n., gaturuku, p., muchiri, e. et al., 2002, ‘an outbreak of rift valley fever in northeastern kenya, 1997-98’, emerging infectious diseases 8, 138–144. https://doi.org/10.3201/eid0802.010023 about the author(s) susan d. kerfua nelson mandela african institute of science and technology, arusha, tanzania national livestock resources research institute, tororo, uganda gabriel shirima nelson mandela african institute of science and technology, arusha, tanzania lughano kusiluka department of global health and biomedical sciences, mzumbe university, tanzania chrisostom ayebazibwe national animal disease diagnostics and epidemiology centre, entebbe, uganda robert mwebe national animal disease diagnostics and epidemiology centre, entebbe, uganda sarah cleaveland institute of biodiversity, animal health and comparative medicine, university of glasgow, united kingdom daniel haydon institute of biodiversity, animal health and comparative medicine, university of glasgow, united kingdom citation kerfua, s.d., shirima, g., kusiluka, l., ayebazibwe, c., mwebe, r., cleaveland, s., et al., 2018, ‘corrigendum: spatial and temporal distribution of foot-and-mouth disease in four districts situated along the uganda–tanzania border: implications for cross-border efforts in disease control’, onderstepoort journal of veterinary research 85(1), a1716. https://doi.org/10.4102/ojvr.v85i1.1716 note: doi of original article: https://doi.org/10.4102/ojvr.v85i1.1528 corrigendum corrigendum: spatial and temporal distribution of foot-and-mouth disease in four districts situated along the uganda–tanzania border: implications for cross-border efforts in disease control susan d. kerfua, gabriel shirima, lughano kusiluka, chrisostom ayebazibwe, robert mwebe, sarah cleaveland, daniel haydon published: 11 dec. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the author list of this article published earlier, chrisostom ayebazibwe’s name was unintentionally misprinted as ‘chrisostome’. the correct name is ‘chrisostom’. the author sincerely regrets this error and apologises for any inconvenience caused. golezardy_81-85.indd in addition to the kruger national park, which is approximately 2 million ha in size, there are numerous smaller national and provincial nature reserves in south africa. besides the mammals, birds, reptiles, fish, arthropods and vegetation that are protected within these reserves the parasites of the various biota are coincidentally conserved with their hosts. the larger reserves are not only important from the conservation aspect, but also because of the opportunity they afford for studies in an environment in which there has been minimal human disturbance or pesticide usage. during the past 80 years several inventories of the arthropod and helminth parasites infesting wildlife in south africa have been published. bedford (1932, 1936) and haeselbarth, segerman & zumpt (1966) have listed the arthropods infesting domestic and wild animals, zumpt (1961) the mites, theiler (1962), walker (1991) and walker, keirans & horak (2000) the ticks, zumpt (1965) the myiasis-producing flies, round (1968) the helminths, ledger (1980) the lice, and segerman (1995) the fleas. in recent times, particular host species and nature reserves have been targeted for the collection of para sites. a number of these studies have been conducted in the cape province (now subdivided into the western cape province, the eastern cape province, and the northern cape province). during these surveys animals in the mountain zebra national park, the bontebok national park and the addo ele81 onderstepoort journal of veterinary research, 74:81–85 (2007) research communication ticks (acari: ixodidae) collected from animals in three western, semi-arid nature reserves in south africa h. golezardy1 and i.g. horak2* abstract golezardy, h. & horak, i.g. 2007. ticks (acari: ixodidae) collected from animals in three western, semi-arid nature reserves in south africa. onderstepoort journal of veterinary research, 74:81–85 the objective of this study was to make an inventory of the ixodid tick species infesting wild animals in three western, semi-arid nature reserves in south africa. to this end 22 animals in the kgalagadi transfrontier park, 10 in the west coast national park and 16 in the karoo national park were examined. fourteen tick species were recovered, of which hyalomma truncatum, rhipicephalus exoph thalmos and rhipicephalus glabroscutatum were each present in two reserves and the remainder only in one. the distributions of two of the 14 tick species recovered, namely rhipicephalus capensis and rhipicephalus neumanni, are virtually confined to the western semi-arid regions of southern africa. hyalomma truncatum, r. capensis and r. glabroscutatum were the most numerous of the ticks recovered, and eland, taurotragus oryx, were the most heavily infested with the former two species and gemsbok, oryx gazella, and mountain reedbuck, redunca fulvorufula, with r. glabroscutatum. keywords: geographic distribution, hosts, ixodid ticks, semi-arid nature reserves, wildlife * author to whom correspondence is to be directed. e-mail: ivan.horak@up.ac.za 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa, and division of parasitology, arc-onderstepoort veterinary institute, onderstepoort, 0110 south africa accepted for publication 15 september 2006—editor 82 ticks (acari: ixodidae) collected from animals in nature reserves in south africa phant national park were examined (horak, sheppey, knight & beuthin 1986; horak, fourie, novellie & williams 1991a; horak, boomker, spickett & de vos 1992; knapp, krecek, horak & penzhorn 1997), as well as animals in the andries vosloo kudu reserve (knight & rechav 1978; horak, spickett, braack & williams 1991b; horak & fourie 1991; horak et al. 1992), and the thomas baines nature reserve (horak, potgieter, walker, de vos & boomker 1983; petney, horak, howell & meyer 2004). the present paper provides an inventory of the ixodid ticks collected from wildlife in three semi-arid nature reserves in the erstwhile cape province, two in the region now known as the western cape province and one in the now named northern cape province. the helminths recovered from antelopes in two of these reserves have been recorded in a separate publication (boomker, horak, watermeyer & booyse 2000). the kgalagadi transfrontier park, that now incorporates the former kalahari gemsbok national park (24°45’–26°28’ s, 20°00’–20°50’ e), which lay within the borders of south africa, is located in a semiarid region in the northern part of northern cape prov ince and extends into the neighbouring country of botswana. the vegetation consists of a mosaic of lightly wooded grassland on the dune crests, pure grassland in shallow depressions between the dunes, and rhigozum trichotomum shrubby grassland in deeper hollows where the underlying calcrete is close to the surface (white 1983; acocks 1988). during october 1984 22 animals were examined for ticks in the central region of the former kalahari gemsbok national park. the west coast national park (33°06’–33°20’ s; 17°58’–18°11’ e), which incorporates the former langebaan nature reserve is situated in a semi-arid region on the western coast of the western cape province and comprises an area of 24 779 ha. the vegetation is classified as strandveld and isolated patches of coastal fynbos (white 1983; acocks 1988). the park lies within the winter rainfall region of south africa in which summers are hot and dry, and winters cold and wet. ten animals were examined during february 1990 in this park. the karoo national park (32°12’–32°20’ s; 22°18’– 22°39’ e), comprises an area of 17 706 ha near the town of beaufort west in the north-eastern part of the western cape province. it is a semi-arid region with hot summers and cold winters, and occasional snow on the higher mountain peaks. the vegetation consists of karroid broken veld (acocks 1988). sixteen animals were examined for ticks in this park during february 1991. the species and numbers of animals examined are summarized in table 1. the animals were either shot or chemically immobilized. the larger species that were shot were processed for ectoparasite recovery as described by horak et al. (1992) for greater kudus and the smaller animals as described by horak et al. (1986) for scrub hares. the animals that were immobilized were carefully scrutinized for ticks. the ticks collected from the processed material, or directly from the immobilized animals, were stored in 70 % alcohol for later identification and counting under a stereoscopic microscope. a total of 14 ixodid tick species were recovered in this way. only four tick species were recovered from the 22 animals examined in the south african portion of the kgalagadi transfrontier park (table 2), and individual burdens were small. no ticks were collected from the single red hartebeest and the two springbok examined. the species collected, namely hyalomma marginatum rufipes, hyalomma truncatum, rhipicephalus exophthalmos and rhipicephalus theileri are all adapted to harsh climatic conditions (howell, walker & nevill 1978; walker et al. 2000). the preference of r. exophthalmos for scrub hares and r. theileri for cape ground squirrels is evident from the collections made from these animals. the latter tick is also common on yellow mongooses, cyn ictis penicillata, and meercats, suricata suricatta, which share warrens with ground squirrels (horak, chap parro, beaucournu & louw 1999; walker et al. 2000). the species and numbers of ticks collected from animals in the west coast national park are summarized in table 3. six ixodid tick species were collected from the ten animals examined, and large numbers of h. truncatum, rhipicephalus capensis and rhipicephalus glabroscutatum were recovered. the distribution of r. capensis is virtually confined to the western winter rainfall region of the western cape province, while r. glabroscutatum occurs not only here and in the southern karoo, but also in the non-seasonal rainfall regions of the western and eastern cape provinces and in the valley bushveld of the latter province (walker et al. 2000). the eland, probably because of their larger size, harboured considerably more adult h. truncatum and r. capensis than the gemsbok (gallivan & horak 1997). conversely the gemsbok carried larger burdens of all stages of development of the two-host ticks rhipicephalus evertsi evertsi and r. glabroscutatum. all stages of the latter tick attach around the feet 83 h. golezardy & i.g. horak table 1 mammals examined for ticks in three western, semi-arid nature reserves in south africa host species scientific name number examined red hartebeest black wildebeest blue wildebeest bontebok springbok steenbok eland gemsbok grey rhebok mountain reedbuck rock hyrax cape ground squirrel scrub hare smith’s red rock rabbit alcelaphus buselaphus caama connochaetes gnou connochaetes taurinus damaliscus pygargus dorcas antidorcas marsupialis raphicerus campestris taurotragus oryx oryx gazella pelea capreolus redunca fulvorufula procavia capensis xerus inauris lepus saxatilis pronolagus rupestris 1 2 3 2 8 1 4 10 2 2 4 3 4 2 table 2 ixodid ticks collected from mammals in the south african part of the kgalagadi transfrontier park tick species tick life stage host species (total number of ticks collected) hyalomma marginatum rufipes hyalomma truncatum rhipicephalus exophthalmos rhipicephalus theileri adult adult adult all eland (5) blue wildebeest (3), eland (39), gemsbok (62) steenbok (2), gemsbok (2), scrub hares (28) cape ground squirrels (11) table 3 ixodid ticks collected from mammals in the west coast national park, south africa tick species tick life stage host species (total number of ticks collected) hyalomma truncatum larva adult rock hyrax (1) eland (609), gemsbok (131) ixodes pilosus group adult all eland (16) gemsbok (12) rhipicephalus capensis adult bontebok (4), eland (1 898), gemsbok (234) rhipicephalus evertsi evertsi immature all bontebok (16), springbok (2) eland (68), gemsbok (246) rhipicephalus gertrudae adult eland (6), gemsbok (2) rhipicephalus glabroscutatum immature all springbok (4) eland (25), gemsbok (7 960) table 4 ixodid ticks collected from mammals in the karoo national park, south africa tick species tick life stage host species (total number of ticks collected) amblyomma marmoreum larvae springbok (4), grey rhebok (58) hyalomma glabrum adult black wildebeest (21) rhipicephalus arnoldi immature rock hyrax (29), scrub hare (3), red rock rabbit (9) rhipicephalus distinctus all rock hyrax (129) rhipicephalus exophthalmos adult springbok (22), grey rhebok (2), mountain reedbuck (13) rhipicephalus glabroscutatum immature all rock hyrax (7) grey rhebok (274), mountain reedbuck (4 916) rhipicephalus neumanni adult black wildebeest (2), springbok (2), grey rhebok (2), mountain reedbuck (2) 84 ticks (acari: ixodidae) collected from animals in nature reserves in south africa and on the lower legs of their hosts (macivor & horak 1987). one of the bontebok harboured four adult r. capensis, and one of the springbok was infested with a small number of immature r. evertsi evertsi and the other with a similar number of immature r. glabroscutatum. seven ixodid tick species were recovered from the 16 animals examined in the karoo national park (table 4). the dominant species was r. glabro scuta tum and mountain reedbuck were the most heavily infested. five fairly rarely collected tick species were recovered, namely hyalomma glabrum, rhipicephalus arnoldi, rhipicephalus distinctus, r. exophthalmos and rhipicephalus neumanni. the adults of r. arnoldi infest smith’s red rock rabbits and the immature stages infest these animals and other sympatric small mammals (walker et al. 2000). all stages of development of r. distinctus infest rock hyraxes (horak & fourie 1986; horak et al. 1991a), and although the tick does not occur throughout the range of these small mammals its distribution is dependent on the presence of its hyrax hosts (walker et al. 2000). rhipicephalus neumanni is a tick of the semiarid central and western regions of south afri ca and southern regions of namibia (walker et al. 2000). it and r. capensis are the only ticks of the 14 species collected that have distributions virtually con fined to the south-western regions of the subcontinent. acknowledgements we are most grateful to south african national parks for placing the animals in the three reserves at our disposal, and for providing assistance and facilities to process the animals for tick recovery. the assistance of mr m.m. knight and dr j.p. louw with processing the carcasses or the immobilized animals for tick recovery is greatly appreciated. the national research foundation provided funds for the conduct of this project. the publication of this work has been facilitated through the integrated consortium on ticks and tick-borne diseases (icttd-3), financed by the international cooperation program of the european union through coordination action project no. 510561. references acocks, j.p.h. 1988. veld types of south africa with accompanying veld type map, 3rd ed. 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[johannesburg]: south african institute for medical research (publication no. 50). zumpt, f. 1965. myiasis in man and animals in the old world. london: butterworths. abstract introduction materials and methods results discussion acknowledgements references about the author(s) agnes wanyana college of veterinary medicine, makerere university, uganda kizito k. mugimba college of veterinary medicine, makerere university, uganda omony j. bosco college of veterinary medicine, makerere university, uganda halid kirunda national livestock resources research institute, tororo, uganda jessica l. nakavuma college of veterinary medicine, makerere university, uganda angélique teillaud interactions hôtes-agents pathogènes, université de toulouse, france école nationale vétérinaire de toulouse, toulouse, france mariette f. ducatez interactions hôtes-agents pathogènes, université de toulouse, france école nationale vétérinaire de toulouse, toulouse, france denis k. byarugaba college of veterinary medicine, makerere university, uganda citation wanyana, a., mugimba, k.k., bosco, o.j., kirunda, h., nakavuma, j.l., teillaud, a. et al., 2018, ‘genotypic characterisation of avian paramyxovirus type-1 viruses isolated from aquatic birds in uganda’, onderstepoort journal of veterinary research 85(1), a1510. https://doi.org/10.4102/ojvr.v85i1.1510 original research genotypic characterisation of avian paramyxovirus type-1 viruses isolated from aquatic birds in uganda agnes wanyana, kizito k. mugimba, omony j. bosco, halid kirunda, jessica l. nakavuma, angélique teillaud, mariette f. ducatez, denis k. byarugaba received: 14 july 2017; accepted: 16 may 2018; published: 25 june 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract avian paramyxovirus type-1 (apmv-1) viruses of the lentogenic pathotypes are often isolated from wild aquatic birds and may mutate to high pathogenicity when they cross into poultry and cause debilitating newcastle disease. this study characterised ampv-1 isolated from fresh faecal droppings from wild aquatic birds roosting sites in uganda. fresh faecal samples from wild aquatic birds at several waterbodies in uganda were collected and inoculated into 9–10-day-old embryonated chicken eggs. after isolation, the viruses were confirmed as apmv-1 by apmv-1-specific polymerase chain reaction (pcr). the cleavage site of the fusion protein gene for 24 representative isolates was sequenced and phylogenetically analysed and compared with representative isolates of the different apmv-1 genotypes in the genbank database. in total, 711 samples were collected from different regions in the country from which 72 isolates were recovered, giving a prevalence of 10.1%. sequence analysis of 24 isolates revealed that the isolates were all lentogenic, with the typical 111ggrqgr’l117 avirulent motif. twenty-two isolates had similar amino acid sequences at the cleavage site, which were different from the lasota vaccine strain by a silent nucleotide substitution t357c. two isolates, ndv/waterfowl/uganda/mu150/2011 and ndv/waterfowl/uganda/mu186/2011, were different from the rest of the isolates in a single amino acid, with aspartate and alanine at positions 124 and 129, respectively. the results of this study revealed that ugandan aquatic birds indeed harbour apmv-1 that clustered with class ii genotype ii strains and had limited genetic diversity. introduction avian paramyxovirus type 1 (apmv-1) belongs to the genus avulavirus, in the family paramyxoviridae, and is responsible for causing newcastle disease (nd), a highly infectious disease for poultry (alexander 2003). these viruses are usually grouped according to the pathotype based on the severity of the disease they may cause: velogenic (high virulence), mesogenic (mild virulence) and lentogenic (low virulence) viruses. the genetic basis of virulence is defined by the amino acid residues at the cleavage site of the fusion protein precursor (fo) (aldous & alexander 2001). the more virulent pathotypes that cause nd in poultry have multiple basic amino acids (at least three arginine or lysine residues between residues 113 and 116) with a phenylalanine at position 117 of the c-terminus of the f2 protein (also the n-terminus of the f1 protein) that make them cleavable by many proteases within the host tissues (aldous & alexander 2001; aldous et al. 2003). the avirulent strains have fewer basic amino acid residues, with a leucine residue at position 117 of the f-protein. this portion of the cleavage site of the f-protein has been used not only for pathotyping apmv-1 viruses but also for genotyping the different strains (aldous et al. 2003) among other typing methods that classify these strains into genotypes and lineages (kim et al. 2007; snoeck et al. 2009). most of the genotyping methods classify these viruses into two classes, i and ii, and further subdivide them into different genotypes and clades. the most recent classification has utilised the full f gene to classify these viruses further (diel et al. 2012; snoeck et al. 2013). the class ii viruses are the most commonly reported and studied viruses and are associated with disease in poultry, pet and wild birds. the role of apmv-1 recovered from wild birds has been alluded to in the epidemiology of nd in domestic poultry and aquatic birds in particular are thought to be the natural reservoirs (jorgensen et al. 2004). avirulent strains have particularly been consistently recovered from aquatic birds and their potential to mutate to virulent form upon passage in poultry has been confirmed (shengqing et al. 1997; takakuwa et al. 1998). the migration of wild bird populations along various migration pathways across the world constitutes a serious threat to possible spread of these viruses and pause a risk of transmission to domestic poultry (zarkov et al. 2005). studies have already shown similarities between strains recovered from aquatic birds and shorebirds with those isolated from live-bird markets in some parts of the world (kim et al. 2007), further confirming this threat. we have previously demonstrated that virulent apmv-1 strains circulate in live-bird markets in uganda in apparently healthy birds (byarugaba et al. 2014). however, despite the massive number of migratory and resident birds that rest along several waterbodies in uganda, no studies have been undertaken to understand whether these birds harbour apmv-1. with the biggest proportion of the poultry production sector in uganda being backyard, there is a high risk of transmission of the apmv-1 from aquatic birds into the poultry population that may result in economic losses to both the small-scale poultry farmers and the country. this study sought to establish if apmv-1 strains circulate in aquatic birds in uganda and how they compare genetically to others elsewhere. materials and methods sample collection fresh faecal samples were collected from aquatic bird (which includes all aquatic birds and waterfowl) roosting sites along various waterbodies across the country using sterile dacron swabs into cryovials containing virus transport medium supplemented with antibiotics (isotonic phosphate buffered saline, 2000 u/ml penicillin, 2 mg/ml streptomycin, 50 μg/ml gentamycin, 50 u/ml nystatin and 0.5% bovine serum albumin). the samples were stored and transported in dry shippers until delivered to the laboratory where they were stored at -80 oc until further use. a total of 711 samples were collected from various sites including musambwa island, makanaga bay, lutembe bay, mabamba bay, nakiwogo landing site, samuka island, macdonald bay, doho rice scheme, lake bisina island, murchison falls national park, queen elizabeth national park, and kibimba dam rice scheme. virus isolation the samples were inoculated (in triplicate) by the allantoic route into 9–10-day embryonated chicken eggs for virus isolation according to the world organisation for animal health (oie) manual of standards for diagnostic tests and vaccines (oie 2008). allantoic fluid was harvested 3 days post-inoculation and subsequently tested for haemagglutination (ha) using 1% chicken erythrocytes and haemagglutination inhibition (hi) with in-house–generated polyclonal anti-apmv-1 sera as described (oie 2008). the hi-positive samples were subsequently confirmed by polymerase chain reaction (pcr). confirmation by reverse transcription-polymerase chain reaction viral ribonucleic acid (rna) was extracted from all the hi-positive samples using the qiaamp viral rna mini kit (qiagen, germantown, md, usa) according to the manufacturer’s instructions. polymerase chain reaction was performed with a qiagen one-step reverse transcription-polymerase chain reaction (rt-pcr) kit (qiagen, usa) according to the manufacturer’s instructions, with the following apmv-1 primers fop1: 5’ tacacctcatcccagacagggtc 3’ (nucleotide position, 158–177) and fop2: 5’ aggcaggggaagtgatttgtggc 3’ (nucleotide position, 493–513) according to kho et al. (2000). the primers were used for the amplification of a 356 bp region corresponding to the cleavage activation site of f gene of apmv-1. the rt-pcr was performed in a 25 µl reaction volume containing 5 µl of 5x rt-pcr buffer, 11 µl of rnase-free h2o, 1 µl of 10 mmol/l dntps, 1.5 µl of 10 nmol/l of each primer, 2 µl of 50 mm mgcl2, 1 µl of enzyme mix (taq dna polymerase and reverse transcriptase) and 2 µl of viral rna extract. amplification was carried out in an applied biosystems veriti 96-well thermocycler with a single reverse transcription (rt) step of 50 ºc for 30 min, a denaturation step of the rt (95 ºc) for 15 min, followed by 40 cycles with 30 s denaturation at 95 ºc, 30 s of primer annealing at 58 ºc, 1 min of extension at 72 ºc and a final extension for 10 min at 72 ºc. the samples (including a known positive control) were then separated on a 1% agarose gel with a 100-bp marker. sequencing a total of 24 representative isolates were selected for sequencing of the partial cleavage site of the fusion gene. out of the 72 isolates, we selected 24 isolates chosen proportionally from each site including 7/23 from musambwa, 8/22 from lutembe, 5/8 from makanaga, 1/6 from samuka, 1/7 from nakiwogo and 1/6 from queen elizabeth national park. the fragments were run on a 1% agarose gel, excised from the gel and purified with qiaquick pcr purification kits (qiagen, usa) according to the manufacturer’s recommendations. sanger sequencing was carried out on the purified pcr products using the same primers that were used for the pcr. sequencing was performed on a 3130xl applied biosystems capillary sequencer at the plateau de génomique get-purpan, udear umr 5165 cnrs/ups, chu purpan, toulouse, france. phylogenetic analysis the basic local alignment search tool (blast) was used to find similar f gene sequences for apmv-1 in the genbank. sequences covering the cleavage site of the fusion gene representing all 18 genotypes of apmv-1 recently described by diel et al. (2012), including all the ugandan and east african sequences, were retrieved. the sequences were aligned together with the ugandan sequences generated in this study using clustal w and edited using bioedit software version 5.0.9 (hall 1999). phylogenetic analysis was performed using the mega version 5.05 program (tamura et al. 2011) with the neighbour-joining (nj) kimura 2-parameter method and 1000 bootstrap replicates. the amino acids around the fusion protein cleavage site were compared to representative sequences from each of the genotypes. vaccine strains – lasota, accession number: jf950510; hitchner bi, accession number: jn872151 and i-2, accession number ay935499 – were also included in the analysis. availability of data and materials the sequences of the cleavage site of the isolates analysed in this study were deposited in genbank with accession numbers lt549451, lt549452 and lt549453. these include ndv138/aquatic birds/uganda/2011 representing the 22 isolates with identical sequences and ndv150/aquatic birds/uganda/2011 and ndv186/aquatic birds/uganda/2011 as indicated in the phylogenetic tree legend in figure 2. ethics this study was approved by the college of veterinary medicine animal resources and biosecurity higher degrees research committee and uganda national council of science and technology (approval # hs 776). results occurrence of avian paramyxovirus type-1 from the 711 samples collected, 72 isolates were recovered. the prevalence at each site ranged from 0% to 36% and the average was estimated as 10.1% by hi (table 1). no isolates were recovered from doho rice scheme, lake bisina, mabamba, murchision falls and kibimba dam rice scheme. six isolates were obtained from queen elizabeth national park, 22 from lutembe, 8 from makanaga, 23 from musambwa, 7 from nakiwogo and 6 from samuka (table 1). musambwa island and lutembe bay provided the highest number of isolates, with queen elizabeth and nakiwogo providing the lowest number of isolates. prevalence was highest in musambwa, lutembe and makanaga. at the sites, the most common bird species were the grey-headed gull, white-winged tern, gull-billed tern, long-tailed cormorants, great cormorant, egyptian geese and others as indicated in table 1. figure 1: prevalence distribution of avian paramyxovirus-1 in aquatic birds roosting sites in uganda. table 1: occurrence of avian paramyxovirus type-1 by site among migratory aquatic birds. pathotypes of avian paramyxovirus type-1 in ugandan aquatic birds the deduced amino acid sequences of the f gene cleavage site were used to determine the pathotypes and are shown in table 2. all 24 isolates sequenced had a lentogenic motif of 111ggrqgr’l117 characteristic of the avirulent strains. however, isolates ndv150/waterfowl/uganda/2011 and ndv186/waterfowl/uganda/2011 were different from the rest of the 22 isolates in a single amino acid; aspartate and alanine at positions 124 and 129, respectively. the rest of the 22 isolates were identical at all positions. table 2: f-protein cleavage site motif of the newcastle disease viruses sequenced in this study. phylogenetic analysis: to determine the phylogenetic relationships between ugandan aquatic birds’ isolates, other ugandan strains from poultry and the rest of the world, the sequences of the 201-bp hypervariable region of the f gene were compared to the corresponding region of viruses available in genbank. results from the phylogenetic analysis clustered our isolates with genotype ii strains which had also been historically described as genotype ii or lineage 2. they were different from the recently isolated strains from poultry in uganda which belonged to genotype v as shown in figure 2. figure 2: phylogenetic analysis of partial f nucleic acid sequences of avian paramyxovirus type-1. the tree was generated by a neighbour-joining algorithm, and alignments were bootstrapped 1000 times (only bootstraps > 50 are shown). the strain names were edited to include origin of isolates where needed. genotypes are marked on the right. representative isolates from the 18 recently described genotypes (according to the new proposed classification by diel et al. (2012) are included and collapsed together, except for the genotype ii where the aquatic birds isolates from this study belong. discussion this study is the first to isolate and characterise apmv-1 from aquatic birds in uganda. most work on apmv-1 in many parts of the world has focused on poultry, where occurrence of both virulent strains and lentogenic strains of class ii has been reported with limited studies in wild birds (de almeida et al. 2013; miller, decanini & afonso 2010). virulent apmv-1 is a common cause of infections in birds and more than 230 bird species have been reported to be susceptible in experimental infections (usda/aphis/ws 2016) including those we found at the sites where we collected our samples, such as the cormorants. the economic impact of nd on the poultry industry in uganda and elsewhere is significant both in the backyard and commercial flocks. little is known about the apmv-1 strains circulating in wild birds, their evolution and their role in the epidemiology of the disease. czeglédi et al. (2006) speculated that class i and class ii genotype i are ancestral representatives of apmv-1 maintained by their natural hosts, the wild waterfowl. in the present study, we demonstrated the occurrence of apmv-1 among aquatic birds in uganda with a 10.1% prevalence, which was higher than that reported (2.1%) in other studies in africa (de almeida et al. 2013). a few studies in uganda have shown the occurrence of virulent apmv-1 strains (genotype v) that circulate among ugandan poultry and live-bird markets (byarugaba et al. 2014; otim et al. 2004). unlike in our study where the 24 sequenced isolates were clustered with genotype ii, other studies of apmv-1 in wild birds in africa and elsewhere have demonstrated the presence of both genotype i and ii strains in wild birds (miguel et al. 2013; snoeck et al. 2009), with a possible involvement of interor intracontinental bird migration. others have reported lentogenic apmv-1 in wild birds outside africa (banura et al. 2013; krapez et al. 2010; lindh et al. 2012; stanislawek et al. 2000; takakuwa et al. 1998). some of these studies indicate that wild birds may play a role as a potential source of virulent apmv-1 for poultry. moreover, it is suggested that velogenic apmv-1 might arise from lentogenic apmv-1 in nature through point mutations in the f-protein cleavage site, making them virulent for poultry (de leeuw et al. 2003; takakuwa et al. 1998). evolution of apmv-1 has continuously posed threats for emergence of new virulent strains and challenges for diagnosis of nd (cattoli et al. 2010; miller et al. 2010). toyoda et al. (1989) suggested that different strains of apmv-1 evolve through various degrees of accumulation of point mutations rather than gene exchange by recombination. such emerging virulent strains related to lentogenic strains (antigenically and genetically) have been suspected to have caused outbreaks in ireland in 1990 (toyoda et al. 1989; alexander et al. 1992) and in australia in 1998–2000 (gould et al. 2001; westbury 2001). phylogenetic analysis of apmv-1 isolates isolated in the current study showed that the partial f gene sequences clustered with those of genotype ii viruses. this is consistent with previous reports of a predominance of genotype ii viruses in wild birds (hoque et al. 2012). the earlier revelation that the highly virulent strains could evolve from viruses of low virulence by mutation (gould et al. 2001; westbury 2001) underscores the significance of more detailed genomic studies to ascertain possible epidemiological linkages of strains circulating in wild birds and poultry. although velogenic strains of apmv-1 have been isolated from wild birds suggesting an epidemiological link with strains in poultry (huovilainen et al. 2001; jorgensen et al. 2004; liu et al. 2008; snoeck et al. 2013; zarkov et al. 2005; zhu et al. 2010), the current study did not recover any virulent pathotype. our recent studies on apmv-1 in domestic poultry revealed a separate genotype (v) (byarugaba et al. 2014). this does not mean virulent strains may not occur in aquatic birds in the country and therefore more extensive molecular epidemiological and routine monitoring for apmv-1 in aquatic birds is important for early detection to prevent any possible spillover into domestic poultry. such detailed genomic studies will elucidate the exact role of wild birds in the ecology and epidemiology of apmv-1 in poultry and inform control strategies. acknowledgements the sample collection, virus isolation and sequencing of the isolates were supported by the world bank millennium science initiative project (grant # msi/03/32/2010) to denis k. byarugaba through the uganda national council of science and technology. we thank mathilde paul and agnès waret-szkuta (ihap, université de toulouse, inra, envt, toulouse, france) for their help with the cartography. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.k.b., a.w., h.k. and j.l.n. designed the study. a.w., d.k.b. and m.f.d. analysed the data and drafted the manuscript. a.w., j.b.o. and k.k.m. collected the samples, isolated apmv-1 strains and identified the viruses. a.t. and m.f.d. carried out the molecular characterisation and sequence analysis of the isolates. all authors read and approved the final manuscript. references aldous, e.w. & alexander, d.j., 2001, ‘detection and differentiation of newcastle disease virus (avian paramyxovirus type 1)’, avian pathology 30(2), 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r., 2005, ‘isolation of avian paramyxovirus type 1 (apmv-1) from free-living mallards (anasplatyrhynchos) (preliminary communication)’, bulgarian journal of veterinary medicine 8, 173–181. zhu, w., dong, j., xie, z., liu, q. & khan, m.i., 2010, ‘phylogenetic and pathogenic analysis of newcastle disease virus isolated from house sparrow (passer domesticus) living around poultry farm in southern china’, virus genes 40(2), 231–235. https://doi.org/10.1007/s11262-009-0436-0 madekurozwa_199-205.indd introduction the ultrastructure of the wall of avian ovarian follicles has been extensively studied in the domestic fowl (bellairs 1965; wyburn, aitken & johnson 1965a; wyburn, johnson & aitken 1965b; dahl 1971; rothwell & solomon 1977; perry, gilbert & evans 1978a, b; yoshimura & koga 1982 ), the domestic goose (kovacs, forgo & peczely 1992) and the japanese quail (ito, kihara, nakamura, yonezawa & yoshizaki 2003). these studies have highlighted marked developmental changes in the ultrastructure of the zona radiata, perivitelline, granulosa and thecal components of the follicular wall. the precocious development of the ovary and oviduct in the sexually immature ostrich has been described (madekurozwa 2002a, b, 2004, 2005). a more recent histological and immunohistochemical study demonstrated the presence of primordial, previtellogenic and vitellogenic developing follicles in the active ovary of the sexually immature ostrich (madekurozwa & kimaro in press). primordial follicles are composed of an oocyte surrounded by a layer of flat granulosa cells. the granulosa cells are enclosed in a single layer of squamous thecal cells. 199 onderstepoort journal of veterinary research, 73:199–205 (2006) ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (struthio camelus) m-c. madekurozwa1 and w.h. kimaro2 abstract madekurozwa, m-c. & kimaro, w.h. 2006. ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (struthio camelus). onderstepoort journal of veterinary research, 73:199–195 the ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. the follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. the granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. transosomes were observed along the apical and lateral plasma membranes of granulosa cells. the thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. the fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. the study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. in addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells. keywords: electron microscopy, follicles, ostrich, ovary, ultrastructure 1 department of anatomy and physiology, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: madex@op.up.ac.za 2 department of veterinary anatomy and cell biology, faculty of veterinary medicine, sokoine university of agriculture, p.o. box 3016, morogoro, tanzania accepted for publication 6 april 2006—editor 200 follicular wall in developing follicles of sexually immature ostrich (struthio camelus) a simple columnar or pseudostratified columnar gran ulosa cell layer surrounds the oocyte in previtellogenic follicles. the thecal layer in previtellogenic fol licles is undifferentiated. yolk accumulation is evident in the oocytes of vitellogenic follicles. a simple cuboidal cell layer surrounds the oocyte of these follicles. the thecal layer of vitellogenic follicles is clearly differentiated into theca interna and theca externa. there is currently a paucity of information on the morphology of ovarian follicles in the sexually immature ostrich. thus, the present study was undertaken to investigate the ultrastructure of ovarian follicles in this species. the results of this study complement the findings of the histological and immunohistochemical study carried out recently (madekurozwa & kimaro in press). materials and methods a total of 26 sexually immature female ostriches were used in the present study. the birds, which originated from commercial farms, were aged between 12 and 14 months, and weighed 90–100 kg. at a commercial abattoir the ostriches were electrically stunned and exsanguinated. ovarian tissue was immersion-fixed in 2.5 % glutaraldehyde in 0.1 m cacodylate buffer. thereafter, the tissue samples were post-fixed in osmium tetroxide, dehydrated and embedded in epoxy resin. ultrathin sections were stained with lead citrate and uranyl acetate. the samples were viewed with a philips cmio transmission electron microscope. results the ovary contained primordial, previtellogenic and vitellogenic follicles. the follicular wall enclosing the ovocyte was composed of four layers: the zona radiata; granulosa cell layer; basal lamina and thecal layer (theca interna and theca externa). zona radiata the zona radiata is the region in which cytoplasmic processes from the granulosa cells and the ovocyte interdigitate. in primordial follicles the zona radiata was poorly developed (fig. 1). in previtellogenic and vitellogenic follicles the zona radiata consisted of long, interdigitating cytoplasmic processes (fig. 2). interestingly, not all the granulosa cells exhibited cytoplasmic processes. in addition, in some cells only the peripheral regions of the apical membrane displayed cytoplasmic processes (fig. 3). perivitelline layer the perivitelline layer is an amorphous substance located between the cytoplasmic processes forming fig. 1 a simple cuboidal granulosa cell in a primordial follicle. the relatively smooth apical plasma membrane displays a few transosomes (arrows). note the presence of a vesicle (arrowhead) below the evaginating transosome. n: nucleus. o: ovocyte inset: a multivesicular body (arrowhead) in the granulosa cell of a primordial follicle fig. 2 part of the follicular wall and ovocyte (o) of a previtellogen ic follicle. cytoplasmic processes from granulosa cells and the ovocyte form the zona radiata (z). the granulosa cell layer (g) is composed of a simple columnar epithelium. b: basal lamina 201 m-c. madekurozwa & w.h. kimaro the zona radiata. none of the follicles studied in the present investigation exhibited a perivitelline layer. granulosa cell layer a simple cuboidal granulosa cell layer surrounded the ovocyte in primordial follicles (fig. 1). in previtellogenic and vitellogenic follicles, the granulosa cell layer was composed of either a simple columnar or a pseudostratified columnar epithelium (fig. 2). granulosa cells in primordial follicles were characterized by the presence of an ovoid, heterochromatic nucleus, with a prominent nucleolus (fig. 1). the spherical nucleus observed in previtellogenic and vitellogenic cells was either centrally or apically-located (fig. 2). the cytoplasm in primordial follicles contained relatively few organelles, which included mitochondria, rough endoplasmic reticulum (rer) cisterns, electron dense bodies, transosomes and multivesicular bodies (fig. 1). in addition, a dense network of microfilaments was observed in the cytoplasm. the subapical cytoplasm in previtellogenic and vitellogenic follicles contained numerous mitochondria, electron dense bodies and vesicles (fig. 3 and 4). some of the electron dense bodies contained membranes, which appeared to be remnants of transosomes (fig. 4). large amounts of rer and numerous vacuoles were also a typical feature of the granulosa cells in previtellogenic and vitellogenic follicles. in addition, occasional lipid droplets were observed in these cells. in all follicles desmosomes attached the apical plasma membrane of the granulosa cells to the ovocyte plasmalemma (fig. 3). transosomes were observed along the apical and lateral plasma membranes of the granulosa cells. transosomes along the apical fig. 3 apical portion of a granulosa cell. note the peripheral localization of the zona radiata (z). the sub-apical cytoplasm contains numerous vesicles (v) and electron dense bodies (arrowhead). desmosomes (arrows) attach the gran ulosa cell plasma membrane to the oolemma. o: ovocyte. g: granulosa cells fig. 4 supranuclear cytoplasm of a granulosa cell in a previtellogenic follicle. the apical plasma membrane exhibits trans osomes (arrow). electron dense bodies (arrowheads) within the granulosa cell contain transosomes. o: ovo cyte fig. 5 the transosomes formed from the apical plasma membrane are composed of inner (a), middle (b) and outer (c) membranes. granules (g) line the inner membrane. electron lucent (l) areas occur between the inner and middle membranes, as well as between the middle and outer membranes 202 follicular wall in developing follicles of sexually immature ostrich (struthio camelus) plasma membrane were most numerous in areas devoid of cytoplasmic processes. in the early stages of transosome formation a focal, electron dense area of the granulosa cell plasma membrane evaginated. directly below the evaginating apical membranes were vesicles with electron dense membranes (fig. fig. 6 transosomes formed along the lateral plasma membranes of the granulosa cells are composed of a single electron dense membrane lined with granules (arrowhead). arrows: desmosomes. n: nucleus fig. 7 a homogeneous basal lamina (b) separates the granulosa cells (g) from the thecal fibrocytes (t) fig. 8 portion of a vitellogenic follicle. fibrocytes (arrows) with elongated, heterochromatic nuclei form the theca interna. theca externa contains fibroblasts (arrowheads) and interstitial endocrine cells (t) separated by dense accumulations of collagen fibres (asterisks). b: blood vessel fig. 9 photomicrograph of the theca externa showing fibroblasts with elongated nuclei (n). the cytoplasm contains long cisterns of rer (arrows) and microfilaments (m). c: collagen fibres 203 m-c. madekurozwa & w.h. kimaro 1). upon release into the ovoplasm, the transosomes consisted of three electron dense layers, with the inner and middle, as well as the middle and outer layers separated by electron lucent areas. electron dense granules lined the inner layer of the transosome (fig. 5). transosomes, which formed along the lateral plasma membranes, were composed of a single electron dense membrane lined with granules (fig. 6). basal lamina the granulosa cell layer and thecal layer were separated by a homogeneous basal lamina, which was approximately 1 μm thick. the boundary between the basal lamina and the cells of the thecal layer was not well defined (fig. 7). thecal layer the thecal layer in primordial follicles consisted of two to three layers of fibrocytes. in previtellogenic and vitellogenic follicles the thecal interna was composed of fibrocytes with elongated, heterochromatic nuclei (fig. 8). the cytoplasm contained mitochondria, rer profiles and a few lipid droplets. the fibroblasts in the theca externa of previtellogenic and vitellogenic follicles contained elongated, euchromatic nuclei. these fibroblasts were characterized by the presence of long, parallel profiles of rer. in addition, the peripheral regions of the cytoplasm con tained numerous microfilaments (fig. 9). collagen fibres separated the layers of fibroblasts within the theca externa. fibroblasts within the same layer were commonly connected by desmosomes (fig. 10). sev eral blood vessels, nerves and interstitial endocrine cells were observed in the thecal layer. discussion based on the structure of the follicular wall it is evident that its primary function is the transportation of yolk precursors to the ovocyte. the transfer of yolk precursors from the granulosa cell layer to the ovocyte is aided by the presence of the zona radiata and transosomes. the zona radiata is composed of long interdigitating cytoplasmic processes, which effectively increase the surface area available for nutrient transfer. this is supported by research carried out on the domestic fowl in which it was shown that the zona radiata was most prominent during the period of rapid yolk deposition (rothwell & solomon 1977). in the current study, the zona radiata in the primordial follicles was poorly developed, implying that at this stage of development there is a limited movement of yolk precursors into the ovoplasm. conversely, the long cytoplasmic processes forming the zona radiata in previtellogenic and vitellogenic follicles suggested that the transfer and formation of yolk material was active in these follicles. the observation in the current study of an unevenly distributed zona radiata in previtellogenic and vitellogenic follicles is unusual, as research conducted in the domestic fowl (wyburn et al. 1965a; wyburn, johnson & aitken 1966; rothwell & solomon 1977; perry et al. 1978a), domestic goose (kovacs et al. 1992) and dove (zarnescu 2004) has demonstrated the presence of an evenly distributed zona radiata in previtellogenic and vitellogenic follicles. ultrastructural studies will need to be conducted on the follicles in sexually mature ostriches to determine whether the uneven distribution of the zona radiata is peculiar to this species. avian ovarian follicles contain membranous organelles known as transosomes which are involved in the transportation of the yolk precursors vitellogenin into the ovocyte (ito et al. 2003). transosomes are also known as unique organelles (schjeide, munn, mccandless & edwards 1966), terminal membranes (wyburn et al. 1965b), coated vesicles (perry et al. 1978a) and lining bodies (bellairs 1965; 1967; greenfield 1966; paulson & rosenberg 1974; ito et al. 2003; zarnescu 2004). based on the observation that transosomes also form between adjacent granulosa cells it is probable that these organelles are responsible for the transfer of nutrients between cells. as is the case in the domestic fowl (bellairs 1965; wyburn et al. 1965b; schjeide, hanzely, holshouser & briles 1974; rothwell & solomon 1977) and the domestic goose (kovacs et al. 1992), transosomes in the sexually immature ostrich were present in all fig. 10 desmosomes (arrow) connected some of the fibroblasts in the thecal layer 204 follicular wall in developing follicles of sexually immature ostrich (struthio camelus) follicular sizes. in the present study, transosomes formed in areas of the apical granulosa cell, which were devoid of cytoplasmic processes. in contrast, transosomes in the domestic goose (kovacs et al. 1992) and the japanese quail (ito et al. 2003) form at the tips of granulosa cell cytoplasmic processes. the morphology of transosomes in the ovarian follicles of the sexually immature ostrich was similar to that described in the domestic fowl (wyburn et al. 1965b; schjeide et al. 1966, 1974; schjeide, kancheva, hanzely & briles 1975), the domestic goose (kovacs et al. 1992) and the dove (zarnescu 2004). the inner membrane of the transosome was lined by granules, which have been described as being “ribosome-like” (kovacs et al. 1992; paulson & rosenberg 1974; zarnescu 2004). however, bellairs (1965) has stated that the granules are much larger than ribosomes. thus, the exact composition of the granules lining the inner membrane of transosomes is still unknown. the outermost layer of the transosome is derived from the ovocyte plasmalemma. it is unclear whether the middle layer of the transosome originates from the ovocyte plasmalemma or from the plasma membrane of the granulosa cell. it is known that the basal lamina plays an important role in the movement of yolk precursors from capillaries in the thecal layer to the granulosa cell layer. callebaut (1990) has reported the presence of yolk precursors and lipid droplets in the basal lamina of preovulatory follicles in the japanese quail. it was thought that the yolk precursors were en route from the thecal capillaries to the granulosa cell layer. although a close association between the fibrocytes in the thecal layer and the basal lamina was noted, no lipid droplets were observed in the basal lamina of the sexually immature ostrich. the thecal layer contains numerous capillaries, which act as a final conduit of yolk precursors from the liver. in addition to its role in nutrient transportation, the externa component of the thecal layer probably plays a role in the ovulation process of the mature follicle. controversy surrounds the cells forming the theca externa. researchers have described the cells in the theca externa as being “fibroblast-like” (perry et al. 1978a; zarnescu 2004) or “smooth muscle-like” (van nassauw, callebaut, harrisson & scheuermann 1992). contrary to the observations made in the japanese quail (van nassauw et al. 1992), the theca externa cells in the sexually immature ostrich did not display any ultrastructural features typical of smooth muscle cells. the findings of our previous study have shown that the cells in the theca externa are immunohistochemically distinct from the cells in the theca interna and stroma (madekurozwa & kimaro, in press). the theca externa showed strong smooth muscle actin immunoreactivity, whereas the fibroblasts in the stroma were immunonegative for smooth muscle actin. very few cells in the theca interna showed smooth muscle actin immunoreactivity. based on these findings it is probable that the dense meshwork of microfilaments observed in the present study were actin microfilaments. perry et al. (1978a) have suggested that the fibroblasts in the thecal layer have contractile abilities, which enable them to play a role in the rupture of the follicular wall during ovulation. in conclusion, the results of the present study have shown that the ultrastructure of developing follicles in the sexually immature ostrich is similar, but not identical to that of other avian species. the absence of the perivitelline layer in the ovarian follicles of the sexually immature ostrich is a notable difference. the amorphous material forming the perivitelline layer has been shown to be vitellogenin (ito et al. 2003). thus, the absence of the perivitelline layer in the sexually immature ostrich suggests that the transfer of vitellogenin from the granulosa cells into the ovocyte is very low at this stage of development. acknowledgements the authors thank the staff in the electron microscope unit for their technical assistance. the uni versity of pretoria and the national research foundation funded this study. the deutscher akademischer austauschdienst (daad) is gratefully acknowledged for providing dr w.h. kimaro with a scholarship, which enabled him to pursue a master’s degree at the university of pretoria. references bellairs, r. 1965. the relationship between oocyte and follicle in the hen’s ovary as shown by electron microscopy. journal of embryology and experimental morphology, 13:215– 233. bellairs, r. 1967. aspects of the development of yolk spheres in the hen’s oocyte, studied by electron microscopy. journal of embryology and experimental morphology, 17:267–281. callebaut, m. 1990. improved visualization of ultrastructural components in the avian ovarian granulosa basement membrane. cell biology international reports, 14:653–658. dahl, e. 1971. the fine structure of the granulosa cells in the domestic fowl and rat. zeitschrift für zellforschung, 119:58– 67. greenfield, m.l. 1966. the oocyte of the domestic chicken shortly after hatching, studied by electron microscopy. journal of embryology and experimental morphology, 15:297– 316. 205 m-c. madekurozwa & w.h. kimaro ito, y., kihara, m., nakamura, e., yonezawa, s. & yoshizaki, n. 2003. vitellogenin transport and yolk formation in the quail ovary. zoological science, 20:717–726. kovacs, j., forgo, v. & peczely, p. 1992. the fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose. cell and tissue research, 267: 561–569. madekurozwa, m-c. 2002a. progesterone and oestrogen receptor immunoreactivity in the vagina of the immature ostrich, struthio camelus. british poultry science, 43:450–456. madekurozwa, m-c. 2002b. a study of the immunohistochemical localization of the progesterone and oestrogen receptors in the magnum of the immature ostrich, struthio camelus. anatomia histologia embryologia, 31:317–320. madekurozwa, m-c. 2004. immunohistochemical localization of the progesterone and oestrogen receptors in the shell gland of sexually immature ostriches (struthio camelus) with active and inactive ovaries. research in veterinary science, 76:63–68. madekurozwa, m-c. 2005. morphological features of the luminal surface of the magnum in the sexually immature ostrich (struthio camelus). anatomia histologia embryologia, 34:350–353. madekurozwa, m-c. & kimaro, w.h. 2006. a morphological and immunohistochemical study of healthy and atretic follicles in the ovary of the sexually immature ostrich (struthio camelus). anatomia histologia embryologia (in press). paulson, j.l. & rosenberg, m.d. 1974. formation of lining bodies and oocyte bodies during avian oogenesis. devel opmental biology, 40:366–371. perry, m.m., gilbert, a.b. & evans, a.j. 1978a. electron microscope observations on the ovarian follicle of the domestic fowl during the rapid growth phase. journal of anat omy, 125:481–497. perry, m.m., gilbert, a.b. & evans, a.j. 1978b. the structure of the germinal disc region of the hen’s ovarian follicle during the rapid growth phase. journal of anatomy, 127:379– 392. rothwell, b. & solomon, s.e. 1977. the ultrastructure of the follicle wall of the domestic fowl during the phase of rapid growth. british poultry science, 18:605–610. schjeide, o.a., munn, r.j., mccandless, r.g. & ed wards, r. 1966. unique organelles of avian oocytes. growth, 30: 471–489. schjeide, o.a., hanzely, l., holshouser, s.j. & briles, w.e. 1974. production and fates of unique organelles (transosomes) in ovarian follicles of gallus domesticus under various conditions. cell and tissue research, 156:47–59. schjeide, o.a., kancheva, l., hanzely, l. & briles, w.e. 1975. production and fates of unique organelles (transosomes) in ovarian follicles of gallus domesticus under various conditions. ii. cell and tissue research, 163:63–79. van nassauw, l., callebaut, m., harrisson, f. & scheue rmann, d.w. 1992. smooth muscle cells in the walls of ovarian follicles in the japanese quail. cell and tissue research, 269:49–56. wyburn, g.m., aitken, r.n.c. & johnson, h.s. 1965a. the ultrastructure of the zona radiata of the ovarian follicle of the domestic fowl. journal of anatomy, 99:469–484. wyburn, g.m., johnson, h.s. & aitken, r.n.c. 1965b. specialised plasma membranes in the preovulatory follicle of the fowl. zeitschrift für zellforschung, 68:70–79. wyburn, g.m., johnson, h.s. & aitken, r.n.c. 1966. fate of the granulosa cells in the hen’s follicle. zeitschrift für zellforschung, 72:53–65. yoshimura, y. & koga, o. 1982. ultrastructural changes of the stigma of the follicle during the process of ovulation in the hen. cell and tissue research, 224:349–359. zarnescu, o. 2004. ultrastructural observations of previtellogenic ovarian follicles of dove. zygote, 12:285–292. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [878.740 1133.858] >> setpagedevice adedapo_31-36.indd introduction abrus precatorius is a popular medicinal plant. many medicinal uses are ascribed to this plant. the leaves, stem and roots are sweet-tasting due to the presence of glycyrrhizin, of which about 9–10 % is in the leaf (burkill 1997). it has been used in the treatment of coughs and vomiting in different animal species and therapeutic dose is said to be about 0.01 mg/kg body mass, above which toxicity results (gunsolus 1995). children are attracted by its brightly-coloured seeds and in some countries they play with the seeds and use them in school in their handiwork and to count. necklaces and other ornaments made from the seeds are worn by both children and adults. the seeds are also used for the treatment of diabetes mellitus and chronic nephritis (burkill 1997; cheeke 1998). the plant is also used in traditional medicine for the treatment of scratches, sores, and wounds caused by dogs, cats, and mice. it is also used with other ingredients to treat leucoderma. the leaf is commonly chewed or sucked to obtain its sweet taste. it is boiled with food e.g. cereal pulp, as a sweetener and even as a vegetable. fresh leaves may be pressed on the gum for sores in the mouth. the leaf is even used in many countries in preparations for skin cancer. the leaf has anodynal 31 onderstepoort journal of veterinary research, 74:31–36 (2007) studies on the toxicity of an aqueous extract of the leaves of abrus precatorius in rats a.a. adedapo1, o.a. omoloye1 and o.g. ohore2 abstract adedapo, a.a., omoloye, o.a. & ohore, o.g. 2007. studies on the toxicity of an aqueous extract of the leaves of abrus precatorius in rats. onderstepoort journal of veterinary research, 74:31– 36 the toxic effects of an aqueous extract of abrus precatorius were studied in 20 male white rats over a period of 18 days. the rats were divided into four groups of five rats per group. those in group a served as controls while the rats in groups b, c and d were dosed per os with 400 mg/kg, 800 mg/kg and 1 600 mg/kg of the extract, respectively. blood samples were collected for haematological and biochemical analysis and specimens of the liver, kidney and testes were taken for histopathological studies. the study showed that the extract of a. precatorius caused decreased levels of packed cell volume, haemoglobin concentration, red blood cell count, white blood cell count, mean corpuscular volume and mean corpuscular haemoglobin. the extract also resulted in increased levels of total serum protein, albumin, alanine amino transaminase, aspartate amino transferase, alkaline phosphatase and total bilirubin. histologically, testicular degeneration characterized by decreased numbers of lining cells of the epithelium as well as reduction in sperm cells with presence of scattered sertoli cells were noted. the study thus showed that aqueous extract of abrus precatorius is toxic and caution should be exercised in its use for medicinal purpose. keywords: abrus precatorius, haematology, histopathology, rats, serum biochemistry 1 department of veterinary physiology, biochemistry and pharm acology, university of ibadan, ibadan, nigeria 2 department of veterinary pathology, university of ibadan, ibadan, nigeria accepted for publication 27 september 2006—editor 32 toxicity of an aqueous extract of leaves of abrus precatorius in rats properties and a calming effect on the central nervous system (burkill 1995). the leaves are used for their anti-suppurative property. they are ground with lime and applied to acne sores, boils and abscesses. the plant is also traditionally used to treat tetanus and to prevent rabies (watt & breyer-brandwijk 1962). various african tribes use powdered seeds as oral contraceptives (watt & breyer-brandwijk 1962) while boiled seeds are eaten in certain parts of india (rajaram & janardhanan 1992). abrus precatorius contains ricin and abrin, which are among the most potent toxins described. seeds and foliage are all poisonous. in early stages of ricin toxicosis, animals were dull, later they showed signs of incoordination and in severe cases, there was profuse swelling, tetanic spasms of the muscles and con vulsion. clinical toxicosis reflects primary damage to the gastrointestinal tract and includes violent gastroenteritis followed by weakness and death (cheeke & shull 1985; galey 1996; adedapo 2002). abrin, which consists of abrus agglutinin (a haem aglutinin), and toxic lectins abrins a to d are the five toxic glycoproteins found in the seeds (budavari 1989; windholz 1989). abrin-a, one of four isoabrins from the plant, has the highest inhibitory effect on protein synthesis and consists of an a chain of 250 amino acids and a b chain of 267 amino acids (tah irov, lu & liaw 1994). the ld50 of abrin injected in mice is less than 0.1 μg/kg, making abrin one of the most toxic substances known (klaassen 2001). abrin is present in the leaf and is known to have action on hyperactivity of the system (burkill 1995). because of the hard and relatively impermeable coat of the mature seeds, they are considerably less toxic if swallowed whole. in fact it is claimed that all parts of this plant is toxic (cheeke 1998). however, they are more dangerous when the seeds are chewed or sucked because the toxic elements in the seeds are extracted and mixed with enzymes. clarke & clarke (1975) reported that the black and red seeds of a. precatorius, a perennial vine found throughout the tropics, contain the very poisonous phytotoxin, abrin, a substance very similar to ricin of the castor-oil seed. cheeke (1998) also showed that the plant is native to the tropics and that it grows by the seashore among the undergrowth and in hedges. this study seeks to establish the possible toxic effects of the leaves of this plant on animals using haematology, serum biochemistry and histopathology as indices of toxicosis especially that most works were done on the seeds rather than the leaves. materials and methods animals and experimental designs the animals used in this study were 20 adult male wistar rats of between 180 and 250 g body mass. the rats were maintained at the experimental animal house of the faculty of veterinary medicine, uni versity of ibadan. they were kept in rat cages and fed commercial rat pellets (ladokun and sons livestock feeds, nigeria ltd.) and allowed free access to clean fresh water. the twenty animals were allotted into four groups (a to d) of five animals per group. while the group a rats served as control experiment, groups b, c and d animals were administered with 400 mg/kg, 800 mg/kg and 1 600 mg/kg, doses of the extract, respectively. preparation of the aqueous crude extract of abrus precatorius freshly harvested leaves of the plants were used for the preparation of the extract. the plants were authent icated at the herbarium of department of botany and microbiology, university of ibadan, nigeria. the leaves were weighed and macerated using mortar and pestle. a specific quantity of water was added to ensure proper maceration and to also obtain an extract of 500 mg/ml concentration. thereafter, the solution was filtered through whatman filter paper, and the filtrate was then administered to the rats per os at predetermined dosages daily using stomach canula for 18 days. the control group received distilled water instead of extract. technique for obtaining blood and serum samples paired blood samples were collected by cardiac puncture from rats anaesthetized with diethyl ether into heparinised and non-heparinised bottles for haematological and serum biochemical studies, respectively. blood samples collected into clean nonheparinised bottles were allowed to clot and serum was separated from the clot and centrifuged according to groups into clean bottles for the biochemical analyses. determination of haematological parameters determination of haemoglobin concentration was as described by schalm, jain & carroll (1975) using the cyanomethaemoglobin method. packed cell volume (pcv) was determined by conventional method of filling the capillary tubes with blood as described 33 a.a. adedapo, o.a. omoloye & o.g. ohore by schalm et al. (1975). erythrocyte count was determined by the haemocytometer method as described by coles (1986). total leucocytes and differential leucocyte counts were also determined. erythrocytes indices were determined from values obtained for rbc count, haemoglobin concentration and pcv values. determination of serum biochemical parameters total protein was measured using the biuret reaction while albumin was measured by colorimetric estimation using the sigma diagnostics albumin re agent (sigma diagnostic®, uk), which contained bromocresol green (bcg). globulin was estimated as the difference between total protein and albumin. aspartate aminotransferase (ast), alkaline phosphatase (alp) and alanine aminotransferase (alt) were determined on a photoelectric colorimeter (gallenkamp and sons ltd; england) as described by toro & ackermann (1975) and duncan, prasse & mahaffey (1994). serum urea and creatinine levels were also determined on a photoelectric colorimeter (gallenkamp and sons ltd, england) as described by toro & ackermann (1975) and coles (1986). histopathology the liver, kidney and testes of all the animals were fixed in 10 % buffered formalin in labeled bottles, and processed routinely for histological examination. tissues embedded in paraffin wax were sectioned 5 μm thick, stained with haematoxylin and eosin, mounted on glass slides and then examined under a standard light microscope. statistical analysis the data were subjected to the student’s t-test and were considered significant at p < 0.05 (essex-sorlie 1995). results haematological changes the haematological changes produced in rats given different doses of a. precatorius are presented in table 1. rats given 400 mg/kg, 800 mg/kg and 1 600 mg/kg doses of the extract showed a significant decrease (p < 0.05) in the mean pcv level relative to the control, although these values did not indicate anaemia. the effect of the extract on the haemoglobin concentration also followed a similar decrease in rats treated with 400 and 1 600 mg/kg doses but not with the 800 mg/kg. all the dose levels caused a significant (p < 0.05) decrease in red blood cell counts as well as the mcv when compared to the control values. the leucogram showed that there was a significant decrease (p < 0.05) in white blood cell count in rats given 400 mg/kg relative to the controls, while a significant decrease (p < 0.05) was obtained for lymphocyte counts in rats dosed with 400 and 800 mg/kg of extract. there was a relative decrease in the neutrophil counts in rats dosed with 400 mg/kg of a. precatorius extract. serum biochemical changes the serum biochemical changes observed in rats given different doses of the extracts are shown in table 1 effects of the graded doses of the aqueous extracts of a. precatorius on haematological parameters of rats (n = 5) parameters control (a) 400 mg/kg (b) 800 mg/kg (c) 1 600 mg/kg (d) pcv (%) hb (g/l) rbc (x1012g/l) mcv (fl) mchc (g/dl) mch (pg) wbc (x109/l) lymphocytes (x109/l) neutrophils (x109/l) monocytes (x109/l) eosinophils (x109/l) 54.5 + 4.5 13.8 + 4.5 8.6 + 0.2 65.4 + 2.9 32.7 + 0.2 21.3 + 0.9 12.0 + 1.4 7.7 + 1.2 4.1 + 1.1 0.2 + 0.1 0 36.3 + 3.3a 11.9 + 1.5 6.9 + 0.2c 52.7 + 4.2e 32.9 + 0.6 17.2 + 1.9e 7.1 + 2.1f 5.0 + 1.1g 2.0 + 0.6h 0.1 + 0.1 0.1 + 0.1 40.6 + 2.5a 13.1 + 0.8 6.8 + 0.5c 59.6 + 2.7e 32.5 + 0.5 19.2 + 0.8e 12.5 + 0.1 6.4 + 0.6g 6.0 + 0.3h 0 0.1 + 0.1 34.0 + 0.5a 11.3 + 0.2b 6.2 + 0.2c 55.5 + 2.4 e 32.5 + 0.1 18.0 + 0.6e 12.2 + 0.1 7.3 + 2.1 4.6 + 0.6 0.1 + 0.1 0.1 + 0.1 superscripted items indicate significant values note: mean + s.d. 34 toxicity of an aqueous extract of leaves of abrus precatorius in rats table 2. it was only group d animals (1 600 mg/kg dose) that showed a significant increase (p < 0.05) in the levels of total protein, albumin and globulin. enzyme assay showed that there was a significant increase (p < 0.05) in the level of alp in rats administered with 1 600 mg/kg extract when compared to that of control. rats dosed with 400 mg/kg however, showed a significant decrease (p < 0.05) in alp when compared with the control group. the levels of ast and alt were also significantly elevated in rats dosed with 1 600 mg/kg extract. animals in group b (800 mg/kg) showed significant reduction in the levels of total bilirubin and conjugated bilirubin. group d (1 600 mg/kg) animals on the other hand, showed a significant increase (p < 0.05) in the levels of total bilirubin, conjugated bilirubin and unconjugated bilirubin relative to the control (table 2). histopathological studies histological examination revealed foci of lymphocytic infiltration at the portal areas of the liver. rats given the extract of the plant had scanty spermatozoa in the epididymis. there was severe degeneration of the epithelium of the seminiferous tubules characterized by disappearance of lining epithelium and presence of scattered sertoli cells. a few spermatogonia and a few intratubular multinucleated giant cells were also observed. discussion and conclusions there was a relative decrease in pcv, rbc and mcv in rats that were administered 400 mg/kg, 800 mg/kg and 1 600 mg/kg doses of a. precatorius extract. it thus showed that animals that browse the leaves of this plant may be subjected to depression in erythopoiesis and possibly anaemia over prolonged exposure. this might be as a result of the abrin content of the plant, which consists of abrus agglutinin (a haemaglutinin), and toxic lectins abrins a–d, which are the five toxic glycoproteins found in the seeds (budavari 1989; windholz 1989). the effects of anaemia are greatly influenced by its severity, duration and rate of development (taiwo & anosa 1995; macfarlane, reid & callander 2001). it may thus be safe to conclude that the aqueous extract of the leaves of a. precatorius can cause toxic effects on the red blood cells of rats. the study showed that there was a significant decrease in total white blood cell count in the group dosed with 400 mg/kg. toxic plants do not produce a direct effect on the white blood cells, such as neutrophils, lymphocytes, eosinophils and monocytes (swenson & reece 1993). however, in contrast, excessive ingestion of a wide variety of plants or their products has been found to cause hypoproliferative or non-regenerative anaemia. this is a stem cell disorder, characterized by reduced production of all blood components in the absence of a primary disease process infiltrating the bone marrow or suppressing haemopoiesis (olsen, keller & gerken 1984). the cause of the decrease in the low dose group in this study could not be ascertained. group b showed a significant decrease in the level of lymphocytes, which implies that lymphocytes that participate in immune responses have been suppressed. it therefore means that the body is vulnerable to infections (macfarlane et al. 2001). there was relative decrease in the level of neutrophils in the group b rats while the groups c and d rats showed an apparent increase, which might have been due to stress leading to demargination of neutrophils into circulation (bush 1991). table 2 effects of the graded doses of a. precatorius on the serum biochemical parameters of rats (n = 5) parameters control (a) 400 mg/kg (b) 800 mg/kg (c) 1 600 mg/kg (d) total protein (g/l) albumin (g/l) globulin (g/l) alt (u/l) ast (u/l) alp (u/l) total bilirubin (μmol) conj. bilirubin (μmol) unconj. bilirubin (μmol) 6.5 + 0.1 2.5 + 0.1 4.0 + 0.3 112.0 + 1.4 520.0 + 1.4 220.0 + 1.4 0.3 + 0.1 0.1 + 0.1 0.2 + 0.01 6.6 + 0.1a 3.4 + 0.1b 3.2 + 0.1c 109.0 + 1.4d 442 + 1.4e 62.0 + 1.4f 0.1 + 0.1f 0.02 + 0.01g 0.13 + 0.05 7.8 + 0.1a 3.2 + 0.1b 4.6 + 0.1 118.0 + 1.6d 660.0 + 1.6e 268.3 + 2.5f 0.3 + 0.01 0.1 + 0.02 0.2 + 0.01 26.5 + 4.4a 10.0 + 3.2b 16.5 + 4.2c 368.8 + 2.6 1904.8 + 8.6e 750.0 + 5.6f 0.9 + 0.4f 0.4 + 0.2f 0.5 + 0.04f superscripted items indicate significant values (p < 0.05) note: mean + s.d. 35 a.a. adedapo, o.a. omoloye & o.g. ohore there is increase in the level of alp in the groups c and d animals but a decrease in group b. this shows that at 800 mg/kg and 1 600 mg/kg, the plant extract can cause enzyme induction. an increased alp may be due to hepatic insufficiency, cholestasis or obstruction of the bile ducts (carlson 1996). there was also an increase in ast level following administration of the extract at 800 mg/kg and 1 600 mg/kg doses. the increase in ast may be due to hepatic insufficiency or muscle infection (abatan, arowolo & olorunsogo 1996). there was also an increase in alt following administration of the extract at 800 mg/kg and 1 600 mg/kg, which may be as a result of liver involvement (duncan et al. 1994; nelson & cox 2000). there is a significant increase in the total protein level in groups c and d. there was an increase in the albumin level in all three groups, but since a rise in albumin rarely occurs (bush 1991), the increase may be associated with dehydration and shock resulting in the elevation of the values of this parameter. there is significant increase in globulin in group d but a significant decrease in group b. the total bilirubin was increased in group d compared to the value of the control group. conjugated bilirubin to unconjugated ratio is less than 50 %. conjugated bilirubin is low, probably because of hepatic necrosis whereby ability of the liver to conjugate bilirubin is reduced particularly in rats administered with 800 mg/kg. increase unconjugated bili rubin level may be a pointer to hepatic injury (knoll 1998). histological examination revealed foci of lymphocyte infiltration in the portal areas of the liver while the testes showed marked testicular degeneration and severe disorganization of seminiferous tubules, which were devoid of spermatic cells. testicular degeneration involves a retrogressive change in the germinal epithelium of the seminiferous tubules (macfarlane et al. 2001). the pattern of testicular damage observed in this study is consistent with the effects of phoxim (atef, noussef, ramadam, nawito, el-sayed & el-rahman 1995), oestradiol valerate (kohler-samouilidis, papioannou, kotsaki-kovatsi & vadarakis 1998) and curcuma comosa extract (piyacchaturawati, timinkul & suksamran 1998). as the interstitium was devoid of leydig cells, the histological changes observed may also be due to decreased production of testosterone known to be responsible for normal testicular architecture (akinloye, igharha, olaniyi, alaka & oke 2000). in the case of the liver, there were cases of lymphocytic infiltration at the periportal area indicating a toxic effect, and probably a tissue reaction to the presence of the extract. the presence of glycosides in this plant may have been responsible for this observation (al-robai, abo-khatwa & jamal 1997; ade dapo, abatan, akinloye, idowu & olorunsogo 2003). this study thus showed that the extract of the leaves of abrus precatorius has potential toxic effects as shown by the effects it caused on the serum chemistry as well as 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southern and eastern africa, 2nd ed. london: livingstone publishers. windholz, m. 1989. an encyclopedia of chemicals drugs and biologicals, in the merck index, 3rd ed. rahway: merck & co. inc. abstract introduction materials and methods cultural isolation results discussion acknowledgements references about the author(s) olusola l. ajayi department of veterinary pathology, federal university of agriculture, nigeria richard e. antia department of veterinary pathology, university of ibadan, nigeria olufemi e. ojo department of veterinary microbiology and parasitology, federal university of agriculture, nigeria olajoju j. awoyomi department of veterinary public health and reproduction, federal university of agriculture, nigeria latifa a. oyinlola department of food science and technology, federal university of agriculture, nigeria oluwabusola g. ojebiyi department of veterinary pathology, federal university of agriculture, nigeria citation ajayi, o.l., antia, r.e., ojo, o.e., awoyomi, o.j., oyinlola, l.a. & ojebiyi, o.g., 2017, ‘prevalence and renal pathology of pathogenic leptospira spp. in wildlife in abeokuta, ogun state, nigeria’, onderstepoort journal of veterinary research 84(1), a1210. https://doi.org/10.4102/ojvr.v84i1.1210 original research prevalence and renal pathology of pathogenic leptospira spp. in wildlife in abeokuta, ogun state, nigeria olusola l. ajayi, richard e. antia, olufemi e. ojo, olajoju j. awoyomi, latifa a. oyinlola, oluwabusola g. ojebiyi received: 29 mar. 2016; accepted: 12 aug. 2016; published: 24 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract there is paucity of information on the prevalence of leptospirosis in wildlife in nigeria. this study investigated the prevalence and renal pathology of leptospirosis in wild animals in southwest nigeria. one hundred and five kidney samples were examined from 10 different wildlife species (antelope) greater cane rat (gcr), hare, african giant rat (agr), tree hyrax, civet cat, monitor lizard, python, bushbuck and partridge) using a combination of ellinghausen mccullough johnson harris (emjh) medium, microscopic agglutination test (mat), warthin–starry silver stain (wsss) and immunohistochemistry. chi-square test was used with confidence level set at 0.05 to ascertain associations between positive cases and sex and species. eighty-two (78.1%) samples were culturally positive, while 67.7% (63/93), 57.0% (16/28) and 66.7% (8/12) were wsss, mat and immunohistochemically positive, respectively. interstitial nephritis (41.0%) and tubular nephrosis (81.0%) were the most prominent histopathological changes. pathogenic leptospira organisms were highest in gcr (32.1%) and antelope (14.3%). serovars hardjo (11.54%), bratislava (3.9%), canicola (3.9%), icterohaemorrhagiae (15.4%), pomona (7.14%) gripptotyphosa (19.2%) and undetermined isolates were also detected in other animals. the result showed high prevalence of leptospira infection in the wild and the possibility of domestic animals and humans contracting the disease. this study is the first documentation of evidence of pathogenic leptospira species in wildlife in nigeria. introduction leptospirosis has been adjudged the most common and widespread zoonotic disease in the world (world health organization 1999). over the years, wildlife has been increasingly recognised as the reservoir host and environmental disseminator of different pathogenic leptospires (chin 2000; cirone et al. 1978; cox, smythe & leung 2005; hamir et al. 2001). increase in disease incidence in domestic animals (especially dogs) and change in serovars involved have been attributed to the endemicity of the disease in wildlife and increase in number of urban wildlife (okewole & ayoola 2009; prescott et al. 2002). in nigeria, leptospirosis has been demonstrated serologically in cattle, sheep and goats (agunloye 2002; diallo & dennis 1982; ezeh et al. 1990) with only one case report of a butcher in which serovar hardjoprajitno was isolated from his urine (ezeh et al. 1991). in dogs appropriately vaccinated with vaccine containing canicola and icterohaemorrhagiae, serological evidence indicated that there is emergence of new serovars of leptospirosis in southwest zone of nigeria (okewole & ayoola 2009). despite the zoonotic implication of leptospirosis, little is known of the epidemiology and the health risks of the disease in developing countries, especially in nigeria where game are regarded as a source of protein. this might be because of lack of awareness and the difficulty associated with the disease recognition and diagnosis. current diagnostic methods for leptospirosis in wildlife mostly depend upon demonstration of serum antibodies (boqvist, bergstrom & magnusson 2012; montagnaro et al. 2010) and a few instances of cultural isolation (ci) (felt et al. 2011). in the usa, canada, trinidad and tobago, serology, ci and silver impregnation of renal tissues have been used to determine the prevalence of the disease in the wild (adesiyun et al. 2006; alton et al. 2009; everard et al. 1983; twigg & cox 1976). the most commonly used serological test is the microscopic agglutination test (mat). despite the sensitivity of mat, it is difficult, labour intensive and sometimes the interpretation of the results is confusing and the test does not indicate active infection (wild et al. 2002). immunohistochemistry (ih) has been used in recent times to detect leptospira antigens in the tissues of infected dogs and pinniped populations (cameron et al. 2008; ross et al. 2011; wild et al. 2002). and more recently, ih and polymerase chain reaction were employed as leptospira diagnostic tools in wildlife in ontario, canada (shearer et al. 2014). presently, studies on the prevalence and renal pathology of leptospirosis in wildlife using the combination of ci, mat, warthin–starry silver stain (wsss) and ih are few in the literature. therefore, the paucity of information on leptospirosis in wildlife in nigeria and the possibility of people contracting the disease through improperly roasted game inspired us to investigate the prevalence of leptospira organism in wildlife, isolate and characterise the prevalent serovars, and examine the renal pathological changes associated with the disease using the combination of ci, mat, wsss and ih. materials and methods study location the study was carried out in abeokuta (the state capital of ogun state, nigeria) and its environs (figure 1). the city of abeokuta is located in the southwestern part of nigeria. its geographical coordinates are latitude 7° 15’ n and longitude 3° 35‘ e. the average daytime temperature is relatively high, generally above 28 °c, with an annual rainfall of 750 mm and average relative humidity of 74%. the city is about 81 km southwest of ibadan, the capital of oyo state, and 106 km north of lagos, the former capital city of nigeria. the state shares a boundary on the western part with the republic of benin. abeokuta lies at an altitude of about 157 m a.s.l. (adekunle & agbaje 2011). figure 1: a map of nigeria showing abeokuta, ogun state and the local villages and towns where games are sourced with its neighbouring states and the republic of benin. data source data were obtained from the wildlife butchers at the brewery area of lafenwa, abeokuta, ogun state, where hunters from different parts of the state gather to dress and sell their game. the samples were collected between october 2012 and january 2013. people from different part of the state, neighbouring states (especially lagos, oyo, ondo and osun states) and different parts of the world usually come to buy fresh game for pepper soup, roasted meat (locally called ‘suya’) and smoked meat. sample identification and collection ten different species of wildlife were examined and identified by the department of wildlife medicine, federal university of agriculture abeokuta. one hundred and five kidney samples (105) were collected from the 10 different species of wildlife with unknown history, which had been trapped and brought for sale by the wildlife hunters. twenty-two samples were collected from antelope (philantomba walteri), 54 from greater cane rat (gcr) (thyronomys swinderianus), 11 from hare (lepus microtis), 5 from african giant rat (agr) (cricetomys gambianus), 3 from tree hyrax (dendrohyrax dorsalis), 3 from civet cat (civettictis civetta), 2 from monitor lizard (varanus niloticus), 3 from python (python regius), and 1 from both bushbuck (tragelaphus scriptus) and partridge (perdix perdix). the sexes of these animals were identified and documented. visual estimation of their sizes was used to determine their ages, using small, medium and large sizes. the kidneys were collected into ice packs and taken to the department of veterinary pathology, federal university of agriculture abeokuta for subsequent bacteriological and pathological investigations. cultural isolation culture medium ellinghausen mccullough johnson harris (emjh) medium (ellinghausen & mccullough 1965; johnson & harris 1967) was used for isolation of the leptospira organisms, modified with the addition of 10% filtered rabbit’s serum (0.2 µm filter). antibiotics such as chloramphenicol (2 mg/400 ml), nalixidic acid (20 mg/400 ml) and neomycin (4 mg/400 ml) were added and enriched with calcium chloride (1 ml) and magnesium chloride (1 ml) as well as the addition of 5-fluorouracil (400 mg/l) to prevent the growth of other bacteria. leptospira isolation for each kidney, the renal capsule was removed and a tissue sample was taken using a sterile scalpel blade. tissues were macerated in sterile phosphate buffer solutions (pbs, ph 7.4) using a sterile toothed forceps and then allowed to stay for 10 min. about 5 drops of the tissue extract were inoculated into the medium and incubated at room temperature (28 °c – 30 °c) in the dark. the culture medium was examined after 24 hours for the growth of leptospira organism and later weekly under dark field microscopy. characterisation of leptospira isolates using microscopic agglutination test twenty-eight uncontaminated isolates were characterised as previously described (obregón et al. 2007) using six monoclonal antibodies (mabs) (table 1). the serogroups of mabs used include canicola, icterohaemorrhagiae, bratislava, grippotyphosa, pomona and hardjo. a 50% reduction in the number of free leptospires in the test sample was considered positive with or without agglutination and was recorded as the respective titre (senthil, ramadass & nachimutu 2001). table 1: reference rabbit leptospiral antisera used for characterisation of leptospiral isolates from wildlife. pathological changes samples of the kidney were grossly and histopathologically examined. they were collected into neutral buffered 10% formalin and processed via standard paraffin-embedding techniques. sections of the 105 kidney samples were cut at 5 µm and stained with haematoxylin and eosin stain, while 93 sections were stained with wsss. gross and histopathological grading of the observed lesions was performed. the severity of the gross lesions was graded as absent (-), mild (+), moderate (++) and marked (+++). for interstitial nephritis (in), scores were assigned as follows: + = 1–2 foci on section examined, ++ = 3–4 foci on section examined, +++ = > 5 foci on section examined. the severity or density of leptospira organism using wsss was determined according to the method of twigg and cox (1976) with slight modification using mild (+) for 1–2 foci with low density of the organism forming a thin layer on the apical part of tubular epithelial cells, moderate (++) for 3–4 foci with denser mass in the form of a thick rope around the lumen with a clear centre and marked (+++) = > 5 foci with entire lumen occluded by a tangled mass of leptospira organisms. the severity of tubular colonisation and zonal localisation (cortex, medulla and corticomedullary junction) of leptospira infection was also determined. immunohistochemistry three samples each were selected from four different species, namely antelope, hare, agr and gcr for ih. the rabbit immune sera-cocktail (table 1) used as primary antibodies against leptospira antigens in the kidney sections were graciously provided by prof. r.a. hartskeerl (who/fao/oie and national leptospirosis reference centre, kit biomedical research, amsterdam, the netherlands). ih was performed as previously described (ross et al. 2011). all steps were performed at room temperature. the sections were first incubated in 3% hydrogen peroxide for 15 min to quench endogenous peroxide. after a brief wash of tpbs, histomark (biotin streptavidin-hrp system, goat anti-rabbit igg [h+l] klp, gaitherburg, usa) detection system was used. non-specific binding was done by bathing in normal goat serum for 10 min. the tissues were then incubated with specific mab (1:800 dilutions in pbs) for 30 min. after a last wash in tpbs, the slides were incubated with streptavidin–biotin–horseradish peroxidase for 15 min. slides were then rinsed with distilled water and incubated with 3-amino-9-ethylcarbazole-peroxidase chromogen for 10 min, rinsed with distilled water and counterstained with mayer haematoxylin for 90 s, rinsed and mounted with glycerol for microscopic examination. statistical analysis descriptive statistics were used for both positive and negative cases for different levels of age, sex and species. chi-square test of association was used with confidence level set at 0.05 to ascertain associations between positive cases and sex and species of animals involved. results the prevalence of leptospira organism according to sexes and sizes in the wildlife using emjh medium is depicted in table 2. eighty-two (78.10%) samples were culturally positive for leptospira infection out of the 105 kidney samples examined. out of the 22 antelopes, 18 (81.80%) were positive and 4 (18.20%) were negative. twenty-seven (50.00%) of the 54 gcrs were positive, while 27 (50.00%) were negative. of the 11 hare samples examined, 7 (63.64%) were positive, while 4 (36.36%) were negative. out of the 5 and 3 samples examined from agr and tree hyrax, 4 (80.00%) and 2 (67.00%) were positive, respectively, and only 1 sample was negative in both species. two and 3 samples were examined from monitor lizard and python, respectively, with 100% positivity in each case. one kidney sample each was examined in the bushbuck and partridge and both were positive. table 2: the prevalence of leptospira organisms in 105 kidneys of wildlife in ellinghausen mccullough johnson harris medium according to sex and size. of the 105 animals, 52 (49.5%) were males and 53 (50.5%) were females. of the 52 male samples, 40 (76.9%) were positive, while 42 (79.3%) were positive of the 53 female samples. gcr had the highest infection prevalence of 57.5% compared with 17.5% and 15% in antelopes and hare, respectively. of the 42 positive females, gcr also showed the highest prevalence of 42.9%, while antelope and agr had 26.2% and 7.1%, respectively. other animals such as tree hyrax, monitor lizard, python and civet cat had 4.8% prevalence each. the hare and partridge showed the least prevalence of 2.4% each among the positive female animals. there were no significant (p > 0.05) sex and species differences in leptospira prevalence. of the 105 kidney samples, 30 animals (28.5%) were of small size and 40 (38.1%) medium size, while 35 (33.4%) were of large adult size. of the juvenile small-sized animals, 21 (70.0%) were positive; gcr and hare had the highest prevalence of 8 (38.0%) and 6 (28.5%), respectively. among the medium-sized animals, 33 (82.5%) were positive. antelope and gcr had the highest prevalence of 4 (12.0%) and 23 (69.7%), respectively. within the 35 large-sized adults, 28 (80.0%) were culturally positive, with antelope and gcr having the highest prevalence of 46.4% and 35.7%, respectively. table 3 depicts the characterisation and distribution of the leptospira isolates from different wild animals. of the 82 cultured isolates, 28 uncontaminated samples were used for mat. leptospira grippotyphosa had the highest isolates of 5 (19.23%) with agglutination titre of 1:3200. two of the 5 l. gripptotyphosa isolates were isolated from gcr, while 1 each was isolated from python, antelope and agr. this was closely followed by leptospira hardjo with 4 (15.38%) isolates; 3 (10.70%) from gcr and 1 from antelope with agglutination titre of 1:1600. there were three (10.70%) leptospira icterohaemorrhagiae isolates from 2 gcrs and 1 partridge with agglutination titre of 1:1600. leptospira pomona was 2 (7.00%) isolates, isolated from 2 antelopes with agglutination titre of 1:3200. one each of (4.00%) leptospira bratislava and leptospira canicola was isolated and characterised from two gcrs and showed agglutination titre of 1:1600 and 1:800, respectively. table 3: prevalence and characterisation of 28 leptospira isolates of wild animals using monoclonal antibodies in the microscopic agglutination test. of the 105 kidney samples collected, only 24 (22.90%) showed visible gross lesions. the type, severity and the distribution of the lesions are depicted in table 4. the gross changes revealed 2 kidneys with multiple foci of cortical haemorrhages from 1 antelope and 1 monitor lizard, while 8 (6.86%) animals (2 antelopes, 4 gcrs and 2 agr) had moderate-to-severe rough and pitted cortical surfaces with adherence of the renal capsule. mild-to-moderate cortical necrosis in 9 (5.88%) animals (5 antelopes, 2 gcrs and 1 each from agr and bushbuck) and 2 (1.96%) moderate multiple foci of pale nodules from antelope and gcr were observed. a moderate locally extensive red infarct was observed in the kidney of 1 tree hyrax and 2 renal hypoplasia from 1 gcr and 1 hare. of the 24 samples with gross lesions, 18 (75.00%) were culturally positive, while 64 (70.00%) from the remaining 81 samples without macroscopic lesions were also positive. of the 18 culturally positive samples with gross lesions, 7 each were from antelopes and gcrs, while 2 were from agrs and 1 each from bushbuck and monitor lizard. table 4: gross morphologic lesions of the kidney in relation to cultural isolation of leptospira organisms. results of histopathological changes, warthin–starry silver impregnation and ih of all the kidney sections collected are shown in the tables 5 and 6. in all cases (both leptospira positive and negative tissues), there were histopathological alterations in the kidneys. tubular nephrosis (81.0%), in (41.0%), interstitial fibrosis (20.0%) and protein cast (25.7%) were the most prominent histopathological changes observed in the kidney samples of both infected and non-infected animals, but these lesions were more marked in the infected animals. in was mostly cortical. the in was characterised by peritubular, perivascular and periglomerular lymphoplasmacytic inflammatory foci, and these coalesced in some sections (figure 2). the severity of in was mild in 35 (81.4%) animals, moderate in 7 (16.3%) and marked in only 1 (2.3%). gcr showed the highest prevalence of in with 24 (55.8%) of the 43 wild animals that demonstrated the lesion. figure 2: kidney section showing tubular degeneration and necrosis, tubular atrophy with moderate diffuse interstitial lymphoplasmacytic cellular infiltration in antelope positive with leptospira infection. table 5: renal localisation and tubular colonisation of leptospira organism in wildlife using warthin–starry silver stain. table 6: summary of the positive samples, histopathological changes, serovars identified and the methodologies used. of the 93 samples examined using wsss, 63 (67.7%) showed the presence of the organism. the morphological appearance of the organism was either intact or granular and in some cases intertwined, having a light brown to black appearance (figure 3). tubular colonisation showed that proximal convoluted tubules (pct) demonstrated the highest prevalence compared with distal convoluted tubules (dct) and collecting tubules. despite the fact that pct demonstrated the highest tubular colonisation, the corticomedullary junction showed the highest zonal localisation compared with the cortical and medullary zones. the severity of the tubular colonisation showed that the infection was mild in 43 (68.0%) kidneys, while it was moderate in 17 (27.0%) samples and severe in only 3 (5.0%) kidney samples. figure 3: photomicrograph of severely colonised proximal convoluted tubules and absence of interstitial mononuclear cells infiltration in the interstitium. ih of leptospira antigens in the three kidney samples of hare were all positive with three different serovars detected (l. icterohaemorrhagiae, l. canicola, l. bratislava) (figure 4). two of the three samples in antelope were positive for l. hardjo (figure 5), while l. bratislava and l. gripptotyphosa were detected in agr. only one of the three samples was positive in gcr for l. gripptotyphosa. figure 4: kidney section of antelope showing immunoreactivity of leptospira interrogans serovar hardjo antigen in the tubular lumen (arrows). figure 5: kidney section of hare showing strong immunoreactivity to leptospira interrogans serovar bratislava antigen in the lumen of distal convoluted tubule (arrow). in summary, five different leptospira serovars were isolated, characterised and detected from gcr using ci, mat and ih. three serovars each from antelope (l. hardjo, l. pomona and l. gripptotyphosa) and hare (l. canicola, l. icterohaemorrhagiae, l. bratislava) were detected, while two were detected from agr and one each from python and partridge using either mat or ih and both in some instances (table 6). the isolates from tree hyrax, monitor lizard and civet cat were not characterised because of contamination of their samples. discussion this study revealed a high prevalence of leptospirosis in wild animals in the study area (78.1%) compared with other studies elsewhere (millan et al. 2009; shearer et al. 2014). it also showed the endemicity of the infection in wild animals in abeokuta, nigeria, and possibly in the southwest zone of the country. factors responsible for this high prevalence are unknown, but it is possible to speculate that climatic conditions such as extended periods of rainfall and flooding, elevated temperatures throughout the year and high humidity favoured the prevalence of leptospira in the study area. during the last decade in nigeria, undocumented cases of canine leptospirosis in dogs have been observed in various clinics and post-mortem examinations. recently, serovars such as icterohaemorrhagiae, pomona, canicola, bratislava and gripptotyphosa have been serologically detected in dogs by okewole and ayoola, (2009) in southwest nigeria. in this study, these serovars were isolated and identified and possibly showed that wildlife might have been a source of infection to domestic animals. there seemed to be no significant difference in the prevalence of infection with respect to sex in this study. this agrees with the studies of hathaway, blackmore and marshall (1981) who observed no significant difference between male and female wildlife in their study. age-related occurrence of leptospirosis in the animals under investigation appears to be significant. the medium and large animals showed more prevalence than the small size. this might have been because of the fact that as the animal advances in age, the more the chances of exposure to the leptospira infection. this is in agreement with the works of hathaway et al. (1981) who observed marked differences in the age-specific prevalence of infection in adult animals in the wild. serology has been extensively used in the diagnosis of leptospirosis in the wild (khan et al. 1991). however, there have been few studies on isolation, characterisation and pathology (durfee et al. 1977). in this study, isolation and characterisation of leptospira isolates were performed to ascertain the prevalent serovars. of the 28 isolates for mat, 16 (57%) isolates were characterised, while 12 (43%) were not. serovars such as gripptotyphosa (n = 5), hardjo (n = 4), icterohaemorrhagiae (n = 3), pomona (n = 2), bratislava (n = 1) and canicola (n = 1) were identified in this study. this is in agreement with the earlier study of okewole and ayoola (2009) in which these serovars were identified serologically. the 12 unidentified isolates show that there are other serovars in wildlife in nigeria apart from those recognised. molecular characterisation of these isolates is underway and might likely show other unknown serotypes that were not previously documented. the higher number of gcr in this study is in agreement with the works of okarfor et al. (2013) who affirmed that gcr constitutes about 40.0% of mammalian species in the study area and their hunting rate is higher during the dry season compared with the rainy season. of the 16 characterised isolates, gcr had the highest prevalence (31.3%) of isolates with five different serovars. the source and high rate of infection in gcr might be attributed to the nature of their ecosystem, since they live in an environment that enhanced the epidemiology of the leptospira organism, such as marshy areas, river and lake banks (adeyeye, olaofe & ogunjana 2012; oboegbulem & okoronkwo 1990). moreover, gcr might possibly be a potential source of infection to other wild and domestic animals (especially cattle and dogs in peri-urban areas) and ultimately, a potential public health hazard to humans that consume smoked and inadequately cooked infected gcr. reports on isolation, characterisation and immunohistochemical detection of pathogenic leptospires in antelope are very rare in the literature, although serological evidence of l. hardjo has been demonstrated in 3.6% of 544 antelopes in colorado, usa (collins et al. 1981). in this report, of the 22 samples from antelope examined culturally and 6 isolates characterised with mat, 18 (81.8%) and 4 (66.7%), respectively, were positive, while 7 (36.8%) and 2 (66.7%) were positive of 19 and 3 samples examined with wsss and ih, respectively. in this study, serovars hardjo, pomona and gripptotyphosa were the most prevalent serovars in this specie. studies on isolation and characterisation of pathogenic leptospira from hare are also rare in the literature. in the work of hathaway et al. (1981), five hares were examined serologically and culturally, but none of them were positive. in this study, 11 kidneys of hares were examined culturally and 7 (71%) were positive and 3 different serovars were detected immunohistochemically. although the numbers of civet cat, pythons, partridge, monitor lizard and bushbuck examined in this study were few, the 100% positivity shows that larger population of these animals might have been infected with leptospirosis. this might pose a serious concern for the conservation of these animal species in the wild. the gross and histopathological changes observed in this study were consistent with those observed in domestic animals with renal leptospira infection (prescott et al. 2002). previous studies have associated in with leptospirosis in the literature (rossetti et al. 2004; scanziani, sironi & mandelli 1989; sterling & thiermann 1981; yener & keles 2001). in this study, of the 105 tissues examined histopathologically, in was observed in 41.0% and the likely aetiology (leptospires) was present in 78.0% with ci and 67.7% using wsss. this possibly showed a positive correlation between in and the presence of the organism in the renal tissues. the absence of in in the kidney tissues that were positive to leptospira organism might be because of the fact that the animals had overcome the infection and the inflammatory response had faded away with time, or that the little portion of the kidney examined might be devoid of such lesions as suggested by ross et al. (2011). interstitial fibrosis might have been because of previous subsiding inflammatory lesions induced by leptospira organism and subsequent scarification. on tubular colonisation, this study is in agreement with the report by twigg and cox (1976) in which pct, dct and collecting duct were consecutively colonised, but the number of animals involved in their study and the zonal localisation of leptospira colonies in the renal parenchyma were not documented. the use of diagnostic methods such as characterisation of leptospiral isolates using mabs, wsss and ih is rare in the literature (wild et al. 2002). to the best of our knowledge, the use of ci, characterisation, wsss, and ih have not been used to diagnose leptospirosis in wild animals in africa and very few studies have been reported in other parts of the world (cameron et al. 2008). the high prevalence of leptospirosis (78.1%) in this study showed the endemicity of the infection in the wild. this prevalence might increase in the nearest future because of genetic mutation of the present serovars and possibly as a result of change in climatic conditions such as increase in daily temperature and longer period of rainy season. thus, contact of wildlife with domestic animals should be prevented, especially in gcr domestication. vaccination of domestic animals should be encouraged as part of preventive measures with a polyvalent vaccine that contain all identified serovars to provide full coverage. future studies on the molecular characterisation of all the isolates in this study are warranted. this might detect and elucidate previously unrecognised pathogenic leptospira organisms as well as the unidentified isolates in this study. in conclusion, the high prevalence of leptospirosis in this study showed the endemicity of the disease in the wild and might be a potential source of infection to domestic animals and humans. both gross and histopathological changes showed that wild animals are susceptible to leptospirosis and might be a potential source of infection to both domestic animals and humans. the study also illustrates the necessity of combining of different diagnostic methods in the confirmation of leptospira infection. serovars gripptotyphosa and hardjo had the highest prevalence in gcr and antelope, respectively, while serovars bratislava, canicola, icterohaemorrhagiae, pomona and gripptotyphosa were also detected using mat and ih. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions o.l.a. and r.e.a. were project leaders and project designers; o.e.o., a microbiologist, contributed to leptospira isolation; o.g.o. was responsible for sample collection; l.a.o. provided geographical information and contributed to manuscript write up; and o.j.a. performed statistical analysis and contributed to manuscript write up. references adekunle, m.f. & agbaje, b.m., 2011, ‘public willingness to pay for ecosystem service functions of a peri-urban forest in abeokuta, ogun state, nigeria’, proceedings of the environmental management conference, federal university of agriculture, abeokuta, nigeria, 2012, viewed n.d., from http://www.unaab.edu.ng adesiyun, a.a., hull-jackson, c., mootoo, n., halsall, s., bennett, r., clarke, n.r. et al., 2006, ‘sero-epidemiology of canine leptospirosis in trinidad: serovars, implications for vaccination and public health’, journal of veterinary medicine b 53, 91–99. https://doi.org/10.1111/j.1439-0450.2006.00922.x adeyeye, e.i., olaofe, o. & ogunjana, k.e., 2012, ‘lipid profiles of the skin, muscle and liver of greater cane rat (thryonomys swinderianus): dietary implication’, elixir food science 53, 11749–11756. agunloye, c.a., 2002, ‘leptospiral agglutinating antibodies in sheep and goats in southwest nigeria’, phd thesis, university of ibadan, ibadan, nigeria. alton, g.d., berke, o., reid-smith, r., ojkic, d. & prescott, j.f., 2009, ‘increase in seroprevalence of canine leptospirosis and its risk factors, ontario 1998–2006’, canadian journal of veterinary research 73(3), 167–175. boqvist, s., bergstrom, k. & magnusson, u., 2012, ‘prevalence of antibody to six leptospira servovars in swedish wild boars’, journal of wildlife diseases 48(2), 492–496. https://doi.org/10.7589/0090-3558-48.2.492 cameron, c.e., zuerner, r.l., raverty, s., colegrove, k.m., norman, s.a., lambourn, d.m. et al., 2008, ‘detection of pathogenic leptospira of bacteria in pinniped populations via pcr and identification of a source of transmission for zoonotic leptospirosis in the marine environment’, journal of clinical microbiology 46(5), 1728–1733. https://doi.org/10.1128/jcm.02022-07 chin, j. 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epidemiology record 74, 237–242. abstract introduction the classification and types of stress the hypothalamo-pituitary-adrenocortical axis interactions between the genetic and physical state of cattle on the hypothalamo-pituitary-adrenocortical axis activity and stress perception stress and immune function stress and reproductive success the hypothalamo-pituitary-adrenocortical axis – significance in assessment of animal welfare in cattle conclusion acknowledgements references about the author(s) emma j. brown school of life sciences, university of kwazulu-natal, westville campus, south africa andre vosloo school of life sciences, university of kwazulu-natal, westville campus, south africa citation brown, e.j. & vosloo, a., 2017, ‘the involvement of the hypothalamo-pituitary-adrenocortical axis in stress physiology and its significance in the assessment of animal welfare in cattle’, onderstepoort journal of veterinary research 84(1), a1398. https://doi.org/10.4102/ojvr.v84i1.1398 review article the involvement of the hypothalamo-pituitary-adrenocortical axis in stress physiology and its significance in the assessment of animal welfare in cattle emma j. brown, andre vosloo received: 07 nov. 2016; accepted: 04 mar. 2017; published: 28 apr. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the intensification of cattle production has raised concern for animal welfare due to the stress that is associated with farming practices. the welfare of an animal is determined by the animal’s ability to cope with or adapt to its continuously changing environment and the biological cost that is associated with this adaptation and maintenance. stressors arise from various psychological, physiological and physical aspects of farming practices due to management and human–cattle interactions. measuring the activity of the hypothalamo-pituitary-adrenocortical (hpa) axis with plasma cortisol levels is a useful method for determining the effects of stress on animals as it is stimulated at the onset of a perceived stress. the activation of the hpa axis affects various target tissues or systems and can result in suppression of the immune system, increased susceptibility to disease and adverse effects on reproductive success in prenatal and neonatal calves. although some levels of stress associated with farming practices are unavoidable, improvements in farming methods need to be implemented in order to maintain or increase the efficiency of cattle production in a way that does not compromise the welfare of the animal. introduction cattle are considered the predominant farm animals that are required for the production of meat and milk for human consumption (phillips 2010). despite the obvious value of cattle for providing 18% of protein intake and 9% of energy intake in the human diet (phillips 2010), the recent intensification of cattle production has led to an increase in concern for animal welfare regarding management and human–cattle interaction (lynch 2010). members of the public and animal rights groups have placed cattle farming practices under great scrutiny, declaring that animal experimentation and intensive animal agriculture have led to insufficient attention to animal welfare (dohms & metz 1991). when formulating a definition for the term animal welfare, lynch (2010) states that this term cannot simply be explained by one definition. the u.k. farm animal welfare council formulated the five ‘freedoms’, which serve as a guideline when constructing a definition for the term animal welfare (lynch 2010). the five ‘freedoms’ are: (1) freedom from thirst, hunger and malnutrition, (2) freedom from discomfort, (3) freedom from pain, injury and disease, (4) freedom to express normal behaviour and finally (5) freedom from fear and distress. despite these guidelines, there is still great ambiguity that surrounds the term ‘welfare’. however, universal definitions that do exist have various determinants that encompass three main classes: (1) the production capacity of the animal, (2) the physiological function of the animal and (3) the feelings of the animal. an example of a universally accepted definition of the term welfare is the state of the animal in relation to its immediate environment and that the state of health, prosperity and well-being is attained by the ability of the animal to respond to external stimuli. the term ‘state’ refers to the feelings of the animal and the diverse physiological and behavioural responses as well as their general health (lynch 2010). in the livestock industry, it is economically essential to maintain high reproductive efficiency. to ensure optimal production is maintained, a balance needs to exist between increased production and the elimination of undesirable impacts of environmental stressors. in order to establish this balance, knowledge of how stress affects animals is essential (dreiling, carman & brown 1991). the perception of stress from either an internal or external stimulus (burdick et al. 2011) by an animal results in an abnormal or severe adjustment in its physiology (lynch 2010). the stressor, therefore, poses a threat to, and disrupts homeostasis (burdick et al. 2011) which is defined as the coordination of physiological processes that sustain a dependable state in an organism (chen et al. 2015). due to the fact that an animal’s immediate environment is not static and is subject to continuous unpredictable changes (möstl & palme 2002), all life forms must respond to these environmental changes (dohms & metz 1991) by stimulating a behavioural, autonomic, neuroendocrine or immunological response (von borell, dobson & prunier 2007). these responses are evoked due to the fact that the perception of stress directly stimulates the hypothalamo-pituitary-adrenocortical (hpa) axis (lay et al. 1996), which results in the release of stress-related hormones (burdick et al. 2011) in an effort to regulate homeostasis (manteca, mainau & temple 2013). activation of the hpa axis in response to stress (lay et al. 1996) enables the animal to cope with external or internal stress stimuli (lynch 2010). stress can be defined as either ‘good’ stress known as eustress or ‘bad’ stress known as distress (le fevre, matheny & kolt 2003). eustress is described as a positive stress and may have a beneficial effect on the animal (antoniou & cooper 2005). distress occurs when the demands that are placed on the body (both physiological and psychological) surpass the body’s capacity to expend energy in order to maintain homeostasis (le fevre et al. 2003). eustress is proposed not to be inherently bad for the animal (antoniou & cooper 2005; moberg 2000); however, overstimulation or under stimulation of the coping mechanism due to prolonged distress (le fevre et al. 2003; moberg 2000) can ultimately render the animal vulnerable to disease and failure to reproduce and develop properly (moberg 2000). increased disease susceptibility and failure to reproduce therefore indicate that the animal has difficulty coping (broom 1991), which poses a threat to the well-being of the animal (moberg 2000) and in turn, the welfare of the animal (broom 1991). it is therefore essential that one is able to differentiate between non-threatening eustress and distress that alters the biological state of the animal, resulting in adverse consequences for its welfare (moberg 2000). this review, therefore, serves to evaluate the physiology of stress, the effects that stress has on immune function and reproductive success and how this relates to the welfare and productivity of the animal. firstly, the evaluation must take into account the types of stress that cattle are subjected to and how these stressors may be classified. the involvement and role of the hpa axis must also be considered and mention must be given to the hormones that are secreted and the components that are activated. the review will then address the factors that contribute to how an animal perceives and reacts to a stress and the consequences that stress has on immune function and reproductive success. finally, a brief evaluation of the significance of the hpa axis in determining the biological functioning, and hence the welfare of the animal, will be given. the classification and types of stress origin cattle experience various stressors throughout the production cycle (carroll & forsberg 2007) which may arise from endogenous and exogenous sources (table 1). table 1: the exogenous and endogenous stressors that affect cattle. perception whether endoor exogenous in origin, stressors are perceived by the animal as either psychological, physical or physiological stress (carroll & forsberg 2007). as cattle usually experience a combination of stressor groups (lynch 2010), the cumulative stress response may have detrimental effects on the welfare of the animal (manteca et al. 2013). duration stress can be classified as chronic or acute, depending on the duration for which the animal is subjected to a particular stress. acute stress arises when an animal experiences a stressor for a short period of time and can be associated with the fight or flight response (hughes et al. 2013). acute stress is involved in preparing the immune system to stimulate adaptation for a short period of time (hughes et al. 2013). chronic stress is a result of long-term exposure to a stressor resulting in a prolonged disruption to the homeostatic state. the stress response shifts from preparing the immune system to suppressing the immune system. the transition between acute and chronic stress is dependent on the intensity of the psychological perception of the animal to a particular stressor. the duration for which the stressor triggers a stress response and the ability of the animal to overcome a stressful event are influenced by previous exposure, genetics, sex and the temperament of the animal (hughes et al. 2013). over the last century, our understanding of the physiology and behaviour of cattle has improved. a better understanding of the intricate regulatory processes, complex social structure and the highly developed learning ability of cattle has prompted the re-evaluation of the effect that farming practices and conditions have on both the effectiveness of production and the welfare of the animal (lynch 2010). lynch (2010) highlighted six areas of animal production that bring about stress to the animal and therefore may affect animal welfare: (1) the ill treatment and physical abuse of an animal, (2) neglect, by accident or ignorance, (3) inadequate design in accommodation and housing, leading to insufficient space, unsuitable floor types and poor feed and water access, (4) inadequate management and poor husbandry practices, (5) poorly executed mutilations, such as tail dockings, dehorning and castration and (6) poor condition of procedures such as transport and practices at the market and slaughter house. direct indications of harm to the welfare of cattle are reduced productivity and increased mortality (lynch 2010). there are, however, early warning approaches to measure and investigate the effects of stress (de kloet et al. 2005) due to the central control mechanisms of the hpa axis (lay et al. 1996). these early, direct and unbiased measurements of the biological state of the animal are vital for attempts to assess animal welfare and bring about improvements in housing and management of cattle in the production industry (broom 1991). the hypothalamo-pituitary-adrenocortical axis the hpa axis can be regarded as a crucial neuroendocrine system that is involved in the control of diverse physiological processes and adaptations to stress (mormède et al. 2007). when the environmental pressure of a perceived stress exceeds that to which an animal’s adaptive mechanisms can accomodate (kumar, manuja & aich 2012), this system produces energetic metabolites that arise either from energy storage tissues or from the transformation of proteins into energetic metabolites (mormède et al. 2007). the energy provided by this process is then used to fuel a behavioural, autonomic, neuroendocrine or immunological response (von borell et al. 2007) to help the animal cope with the stressor (mormède et al. 2007). the body systems that are paramount to this process of adaption to a perceived stress (kumar et al. 2012) are found within the central nervous system. the sympathetic-adrenomedullary (sam) axis and the hpa axis aid in the process of linking the initial perception of the stress to an adequate response (lynch 2010). for the purpose of this review, emphasis will be placed on the mechanisms of the hpa axis, due to the fact that it is involved in the regulation of bodily processes in response to long-term, chronic stress (kumar et al. 2012). there are many steps involved in the initial perception of a stress and the stimulation of an adequate response that will bring about the regulation of homeostasis (chen et al. 2015). moberg and mench (2000) outlined the biological model known as the general adaptation syndrome that animals have developed in order to cope with a perceived stress. the perception of stress requires a change in the biological function of the animal in order to cope with and reduce the negative effects associated with a stress response (figure 1b). the achievement of regaining homeostasis results in the normal biological function of the animal being restored (figure 1a) (lynch 2010). figure 1: the stages involved in the biological general adaptation syndrome in response to stress in animals. due to the fact that the sam is activated in response to a short-term or acute stress, its inability to rectify a stressful event results in the activation of the hpa axis (lynch 2010), which, as mentioned earlier, is involved in resolving long-term, chronic stress (kumar et al. 2012). figure 2 illustrates the hpa axis pathway, associated hormones and target systems. figure 2: the response of the hypothalamo-pituitary-adrenocortical axis to stress. cortisol, a primary glucocorticoid in cattle, is released from the adrenal cortex and distributed via the circulatory system to various target tissues or organs or systems in the body (figure 2) (burdick et al. 2011). in order for glucocorticoids to be transported via blood in the circulatory system, carrier proteins must be present (burdick et al. 2011). albumin is considered to be the main cortisol binding globulin (burdick et al. 2011). the severity of the effect that glucocorticoids exert on the target organs or tissues or systems is dependent upon six factors: (1) the amount of hormone that is secreted, (2) the duration of hormone secretion, (3) the peripheral blood concentration and cortisol binding globulins, (4) the abundance of glucocorticoid receptors in target tissues, (5) the tissue on which they exert an effect and (6) the extent of the breakdown of glucocorticoid metabolites (burdick et al. 2011). due to the fact that glucocorticoids are the final effectors of the hpa axis, they play a vital role in the control of homeostasis and the basal cortisol concentrations (lynch 2010). glucocorticoids also play a role in the mechanism of negative feedback (burdick et al. 2011). when the hypothalamus and anterior pituitary detect high concentrations of cortisol, the release of vasopressin (vp) and corticotrophin-releasing hormone (crh) from the hypothalamus and adrenocorticotrophic hormone (acth) from the anterior pituitary is inhibited, resulting in inhibition of the synthesis of cortisol from the adrenal cortex (burdick et al. 2011) and termination of the stress response (lynch 2010). although the hpa axis can be seen as advantageous in the restoration of the homeostasis to its normal state, failure to terminate the stress response can result in the overstimulation and dysregulation of the homeostatic system, resulting in a phenomenon known as allostatic load or overload (beerde 1997; lynch 2010). termination failure may be a result of stimulation and activation of an inadequate response to the perceived stressor or continuous habituation to the stimulus is not attained (lynch 2010). ultimately, the consequences of prolonged overor under-activity of the allostatic system are detrimental to immune function (beerde 1997) and the reproductive success of the animal (kumar et al. 2012), which in turn raises questions regarding its welfare (broom 1991). interactions between the genetic and physical state of cattle on the hypothalamo-pituitary-adrenocortical axis activity and stress perception the activity of the endocrine system, incorporating the hpa axis, is commonly used as an indicator of stress in animals (lynch 2010). although this method provides an accurate evaluation of animal welfare, one should be aware that the function of the hpa axis is highly heritable (mormede et al. 2011) and therefore results in differences both between different breeds of cattle and within various breeds of cattle (grandin 1997). this individual variation gives rise to differences in hpa axis activity (mormede et al. 2011) and generalised stress responses. individual variation is seen to be a result of the genetic and physical state of cattle and includes the breed, sex, age, temperament and behaviour of the species (lynch 2010). temperament can be defined as the reactivity of cattle to humans and their immediate environment. there are many factors that contribute to whether an animal may perceive a situation as being stressful, which include developmental history, prior experience and genetic factors. these factors contribute to whether the induced stress response is beneficial or harmful to the animal (burdick et al. 2011). temperament is influenced by genetics and is a hereditable trait which can affect an animal’s response to handling (grandin 1997). highly domesticated animals that are accustomed to routine handling have shown to have subtle responses to changes in the environment and human interaction, whereas wilder species have shown an increasingly elevated response to environmental changes and handling procedures (grandin 1997). as a result of more temperamental animals causing greater injury to themselves and to cattle handlers (burdick et al. 2011), cattle used in production industry are selected based on docility in order to improve animal welfare, performance and human safety (norris et al. 2014). wilder species of cattle are also seen to have higher basal cortisol concentrations which impact growth rates, reproduction and weakens immune responses to pathogens (grandin 1997). stress and immune function when an environmental or internal stress stimulus is perceived, it is paramount that the stress response is prompt, efficient and regulated in order to alleviate increased susceptibility to pathogens. stress affects the mechanisms of innate and adaptive immunity (lynch 2010), and although these systems are not mutually exclusive (salak-johnson & mcglone 2007), there is a complex interaction of communication between the two (lynch 2010). innate immunity refers to the ancient evolutionary mechanism that is evoked immediately or several hours (0–4 h) after the perception of an antigen. innate immunity includes the body’s physical barriers such as the skin and mucous membranes as well as complement and antigen non-specific cellular components. innate immunity is non-specific and the body’s first-line defence to a perceived pathogen (carroll & forsberg 2007). when functioning optimally, pathogens that are encountered on a daily basis are prevented from causing disease as their invasion is blocked by the body’s physical barriers (carroll & forsberg 2007). effector cells of the innate immune system such as macrophages, dendritic cells and b-cells, also known as professional antigen presenting cells, possess pattern recognition receptors that subsequently recognise the pathogen-associated molecular pattern and trigger the effector cells to perform their required function (medzhitov & janeway 2000). the pattern recognition receptors aid in detecting and eliminating the pathogens from the body (carroll & forsberg 2007) and account for the prompt kinetics of the innate immune response (medzhitov & janeway 2000). innate immunity also allows time for the acquired immune system to develop an antibody response to the detected pathogen, which may take several days or weeks (carroll & forsberg 2007). the cellular components of innate immunity are phagocytic cells such as neutrophils, monocytes and macrophages, which release anti-inflammatory mediators (carroll & forsberg 2007). natural killer cells are also components of innate immunity and serve as the link between innate and acquired immunity (lynch 2010). acquired immunity serves to adapt and build a specific immune response for each antigen that is encountered in the body. this type of immunity is characterised by its production of antibodies that are directed against specific antigens and also acquire the ability of immunologic memory that results in a faster and stronger immune response on subsequent detection of the same pathogen (carroll & forsberg 2007). dendritic and macrophage cells are specialised cells called antigen presenting cells (apcs) and present the detected antigen to a naïve lymphocyte (lynch 2010) (specialised white blood cells [carroll & forsberg 2007]), which evokes a humoral and cellular immune response (lynch 2010). the adaptive immune system is comprised of humoral and cellular immunity. humoral immunity is a part of the adaptive immune system that is evoked by the innate immune system and is known as the antibody-mediated immune response that is responsible for triggering specific b-cells to develop into plasma cells. a large number of antibodies are then secreted by these plasma cells and circulated in the blood and the lymph. antibodies are a group of proteins called immunoglobulins, whose functions differ (nauta 2010). immunoglobulin g (igg), immunoglobulin m (igm) and immunoglobulin a (iga) provide defence against viruses, bacteria and toxins, immunoglobulin e (ige) offers protection against parasites and allergens and immunoglobulin d (igd) has no evident role in defence. the antibodies of the humoral immune response act by attacking and invading the perceived pathogen, binding to it and subsequently marking the pathogen for destruction by cells called phagocytes. antibodies can be further categorised by those that activate complement serum proteins or those that bind to antigens. complement serum proteins that are activated by specific antibodies are then able to destroy the pathogen. antibodies that bind to the antigens are known as neutralising antibodies, and once bound the antigen is no longer able to recognise the host cell, therefore inhibiting the further infection of cells (nauta 2010; parham 2014). cellular immunity is also known as cell-mediated immunity (cmi) and is mediated primarily by small lymphocytes derived by the thymus – t cells. two types of t cells exist, the t helper cells and the t killer cells (nauta 2010). t helper cells play a crucial role in maximising the capabilities of the immune system by activating and directing other immune cells to destroy infected cells or pathogens. a second function of the t helper cells is to stimulate b-cells to secrete antibodies that activate phagocytes which subsequently activate the killer t cells (nauta 2010). the major function of killer t cells is its ability to recognise the cytotoxicity of cells infected with a virus and destroy these cells, as well as defending the organism against intracellular bacteria. intracellular bacteria are not detected by the antibodies and macrophages, and therefore, the clearance of infection depends on cytotoxic lymphocytes to eliminate the infected cells. the fact that killer t cells are highly specific with respect to the antigens that they recognise contributes to the uniqueness and effectiveness of the acquired immune response (nauta 2010; parham 2014). glucocorticoids directly influence the activity of the immune system (carroll & forsberg 2007). as previously mentioned, a stressor can be categorised as being acute or chronic. the degree of the perceived stress on the immune system and function may, therefore, be bi-directional. acute stressors may evoke an immuno-enhancing effect, resulting in the proliferation and differentiation of immune cells, whereas chronic stressors have an opposite effect by evoking an immunosuppressive response (carroll & forsberg 2007). the suppression of the immune system is firstly noticed at a cellular level, and as the stress persists, its effects can be examined across the entire immune system (carroll & forsberg 2007). the predominant stressors that result in immunosuppression are transport (chen et al. 2015) and handling (trunkfield & broom 1990). these stressors are seen to involve a complex mixture of unfavourable stimuli that act on the animal and, depending on the nature of methods used, result in lesser or greater effects in stress response. the transport procedure involves handling while loading and unloading, the removal from a familiar to an unfamiliar environment and disruption of social structure due to mixing with unfamiliar animals (trunkfield & broom 1990). studies conducted to measure cortisol concentrations during the transport procedures have shown an increase in blood cortisol concentrations, resulting in an increased neutrophil to lymphocyte ratio and ultimately causing increased disease susceptibility to bovine respiratory disease (chen et al. 2015). the suppression of the immune system may also result in a multifaceted disease complex (blecha 2000) that arises from viral–bacterial synergy (aich, potter & griebel 2009). when the immune system is impaired due to a chronic stress, the onset of a primary viral infection may increase the animal’s susceptibility to a bacterial infection (aich et al. 2009). an example of this phenomenon is bovine respiratory disease (blecha 2000). cattle whose immune system is already compromised by a viral infection and stress become more susceptible to bacterial pathogens that subsequently invade the bovine respiratory tract resulting in full-blown bovine respiratory disease (blecha 2000). temperament and genetics also influence the animal’s susceptibility to disease (burdick et al. 2011; hughes et al. 2013). studies conducted on steers found that there was an increased negative impact on the acquired immune function in steers that were more temperamental (burdick et al. 2011). components of the acquired immune system were affected, resulting in lower lymphocyte proliferation in vitro and decreased in vivo vaccine-specific immunoglobulin concentrations (burdick et al. 2011). other studies that were conducted on calves showed that more temperamental calves had a decreased response to vaccinations, which is an important part of acquired immunity. the unresponsiveness to vaccines was a result of higher concentrations of plasma glucocorticoids, which have an inhibitory effect on the immune system. genetics contributes to immune function due to its important role in determining the physical and physiological characteristics of an animal. animal production, therefore, favours the selection of animals that are genetically less susceptible to disease in order to improve their overall health, performance and productivity (hughes et al. 2013). the assessment of disease in animals is of considerable importance when evaluating animal welfare. animals that are kept in a way in which their immune systems are compromised and are ineffective in combating disease are indicative of management and housing systems that are inadequate and ultimately indicates that the welfare of the animal is at risk (broom 1991). stress and reproductive success the failure of an animal to reproduce not only results in the loss of genetic potential but also jeopardises the survival of the entire breed. an animal will make important physiological sacrifices to ensure that it maintains reproductive success, with only the most adverse threats preventing the animal from reproducing (moberg 1985). there are numerous ways in which stress influences reproduction and involves a number of paracrine, endocrine and neural systems (von borell et al. 2007). reproductive consequences in dairy cattle are easily examined due to their unique experience of repeated cycles of pregnancy during their lifetime (mallard et al. 1998). during the transition from pregnancy to motherhood, many physical, metabolic and physiological adjustments have to be made in order to accommodate pregnancy, parturition, as well as the onset of lactation. the exposure of the animal to environmental and management-related changes, including dietary changes, social regrouping, pen moves, encountering the milking parlour (sepúlveda-varas et al. 2013) and weaning (lynch 2010), induces stress in both mother and foetus during gestation (lay et al. 1997) and in the mother and calf after pregnancy (mallard et al. 1998). foetal development is a critical stage for developmental programming, which is a term that describes the association between environmental challenges that a mother is subjected to during pregnancy and the effect that this has on the developing foetus (harris & seckl 2011). as explained previously, the perception of a stress leads to the release of glucocorticoids due to the activation of the hpa axis (lay et al. 1997). foetal development is affected by glucocorticoids due to its interaction with gene expression (harris & seckl 2011). the activity of glucocorticoids associated with stress and non-stress activities is regulated by two receptors that differ in affinity for glucocorticoids. mineralocorticoid receptors have a high affinity for glucocorticoids, whereas glucocorticoid receptors have a low affinity for glucocorticoids. these receptors are present in various target cells that are widely distributed in tissues encountered in circulation (lynch 2010). glucocorticoids exert their effect on the developing foetus by binding to these receptors that then subsequently act as transcriptions factors, resulting in the alteration of gene expression. glucocorticoid receptors are abundantly present in the majority of foetal tissues, including the placenta, in early embryonic development. during early foetal development glucocorticoids aid in the normal development of the maturation of the lungs, correct brain development, remodeling of axons and dendrites as well as affecting cell survival. the expression of mineralocorticoid receptors only appears in later stages of development. glucocorticoids may have a range of effects on the developing foetus; however, the severity depends on the concentrations that the target tissues or organs or systems are subjected to (harris & seckl 2011). although chronic stress has an immunosuppressive effect on cattle (salak-johnson & mcglone 2007), the exposure of a pregnant cow to mild or acute stressors may be advantageous to the calf (lay et al. 1997). these advantages arise from the study of plasma cortisol concentrations, which are seen to be elevated in the calf (lay et al. 1997). when pregnant cattle were exposed to intermittent transport stress during gestation from 60 to 140 days, cortisol concentrations in the calf remained elevated for a longer period of time, resulting in prolonged activation of the hpa axis, and could further result in permanent activation (lay et al. 1997). the advantage of maintaining high blood cortisol concentrations in the calf may enable it to better cope with mild stress after birth; however, chronic stress may be seen as harmful due to the damaging effects of cortisol on immune function (lay et al. 1997). a calf is not only subjected to stress during pregnancy, as the requirement for the calf to respond to homeostatic changes continues after birth due to subjection to environmental and management associated stressors. calves are born with a functional immune system, which allows for the capability of the calf to respond to certain antigenic stimuli. due to the fact that maternal antibodies and proteins are not transferred to the calf via the placenta during pregnancy, at birth, the calf experiences an immunological naiveté. to ensure that the calf does not succumb to the infection of pathogens that could increase the risk of survival, it is paramount that the calf receives antibody-rich colostrum, which is produced by the mother (mallard et al. 1998). the consumption of colostrum is the mechanism by which the passive transfer of maternal antibodies to the neonatal calf is accomplished (donovan et al. 1986). the colostrum enables the calf to support its own defence mechanisms until they have become fully matured (mallard et al. 1998). the first 24 hours after calving are critical for the survival of the calf (stott et al. 1976). even though the uptake of colostrum provides the calf with antibodies to respond and survive the effects of pathogens, the calf is hugely at risk to environmental stressors, for instance, heat (stott et al. 1976). neonatal calves that are exposed to higher ambient temperatures have a marginally higher body temperatures which in turn increases the concentration of cortisol in the body. elevated cortisol concentrations in the blood influence the permeability of cells in the small intestine rendering the calf incapable of absorbing macromolecules such as immunoglobulins from colostrum. as a consequence, the transfer of passive immunity from the mother to calf is affected which in turn impairs the calf’s immunity, making it susceptible to disease and increasing the chances of mortality (stott et al. 1976). calves are subjected to further stress at the onset of weaning, which occurs due to the gradual decline in the availability of milk from the mother. the transition of the calf from nutritional and social dependence on the mother to complete independence gives rise to a number of stressors that the calf is subjected to. in cattle production, weaning occurs much earlier than natural weaning, with dairy calves being weaned several hours after birth (lynch 2010). the weaning of young calves brings about behaviour and nutritional stress, as young calves possess an immature ruminant digestive system that results in the alteration of metabolic and stress-related hormone levels, ultimately affecting immune responsiveness (pollock et al. 1992). in light of the above-mentioned factors, life expectancy and reproductive success of the mother and calf is often reduced, which is indicative of the animal battling to cope with its changing environment. the inability of the animal to cope with adverse stimuli suggests that the welfare of the animal may be compromised and ultimately questions reproduction procedures in the cattle production (broom 1991). the hypothalamo-pituitary-adrenocortical axis – significance in assessment of animal welfare in cattle the term ‘animal welfare’ has arisen in society to convey the ethical concerns about the quality of life that animals experience, particularly those animals that are involved in agriculture (duncan 2005). although the term ‘animal welfare’ is not expressed as a scientific concept, scientific methods are employed to identify, interpret and implement the societal concerns surrounding an animal’s quality of life, and therefore, animal welfare has become accepted as a scientific field (duncan 2005). without taking for granted that science plays a crucial role in solving animal welfare problems (duncan 2005), animal welfare must also be considered as a multifaceted issue that is not only comprised of scientific dimensions but also has ethical, economic and political considerations (carenzi & verga 2009). animal welfare must, therefore, be considered on a multi-disciplinary approach that combines researchers from different disciplines within the biological sciences (carenzi & verga 2009). various groups evaluating the welfare of an animal have different viewpoints depending on their profession (duncan 2005). for example, a veterinarian’s and a farmer’s biggest concern is the biological functioning of the animal regarding disease, injury, poor growth rates and reproductive problems (rushen et al. 2007). however, the public are more concerned about the emotions or the affective state of the animal, which entails suffering from unpleasant experiences, pain, fear and hunger (rushen et al. 2007). these varying viewpoints emphasise the multifaceted dimensions of the evaluation of animal welfare and that when conducting welfare assessments it is important that one keeps all considerations in mind (duncan 2005). although the driving force for the evaluation of animal welfare is the societal concerns of an animal’s feelings, one must remember that feeling is subjective. for example, humans are able to communicate about a certain experience and how that made them feel. it is therefore understood that when humans have an unpleasant experience they usually associate the same feelings with that experience. because animals communicate in a language that we are not able to understand, it is difficult to assess what each animal feels when it is subjected to a certain experience. the feelings of the animal are poorly defined therefore making them impossible to measure directly. alternatively, the use of good and poor biological functioning of the animal as an indicator of animal welfare is seen to be more advantageous than the assessment of the animal’s feelings because the variables that are involved are substantive and fairly easy to measure (duncan 2005). the evaluation of the hpa axis highlighted in this article provides a scientific approach to the evaluation of the biological functioning of the animal. the hpa axis is activated in response to an endogenous or exogenous stressor in order to help the animal cope with the perceived stress (lynch 2010). cortisol is the main product of the activation of the hpa axis and evokes a response from specific target tissues or systems, eventually restoring homeostasis (mormède et al. 2007). the cortisol levels also fluctuate with different types of stimuli that the animal is subjected to and can, therefore, be directly measured (mormède et al. 2007). prolonged stimulation of the hpa axis results in consequences for immune function and reproduction, which are indicators of the biological functioning of the animal (rushen et al. 2007). due to the fact that the hpa axis can provide measurable results of the quality of the biological functioning of the animal, its application to the evaluation of animal welfare ensures objectivity (duncan 2005). in light of the above, it is imperative that the evaluation of animal welfare incorporates both the biological functioning and the societal concerns of an animal’s feelings because society constantly poses questions regarding the ethicality of an animal’s quality of life in agriculture and science provides the evidence (duncan 2005). conclusion it is apparent that numerous stressors arising from both endogenous and exogenous aspects (lynch 2010) of the cattle production cycle have potentially inhibiting effects on overall productivity and well-being of an animal (carroll & forsberg 2007). the hpa axis functions as a coping mechanism to stress and re-adjusts an animal’s homeostatic state by increasing secretion of stress-related hormones (burdick et al. 2011) and bringing about a behavioural, autonomic, endocrine or immune response (kumar et al. 2012) to enable the animal to cope with the perceived stress (lynch 2010). immune and reproductive function are directly regulated by glucocorticoids (chen et al. 2015), the final product resulting from the activation of the hpa axis (lynch 2010). the effects that stress has on the immune function of an animal can be observed by the increase in the occurrence of diseases such as bovine respiratory disease that arise due to stressors associated with transport (chen et al. 2015). evaluation of reproductive success showed that stress negatively affects calves throughout their developmental stage, beginning with foetal development and continuing through to adulthood. even under the highest quality of management and handling, animals can still be subjected to unfavourable stress (lay et al. 1997). emphasis needs to be placed on the fact that good welfare is not achieved by the absence of negative experiences but rather by the higher occurrence of gratifying positive procedures (lynch 2010). helping an animal to attain optimum production potential can be desirable as long as there are no indications of poor welfare (broom 1991). emphasis should be placed on the fact there is nothing inherently bad about stress unless detrimental effects are observed (moberg 2000). it is, therefore, imperative that conclusions from scientific studies regarding animal welfare should be made on factual evidence rather than emotive grounds (broom 1991). in order to bring about improvements to animal welfare in the cattle production industry, studies should evaluate the preferences of these animals as one needs to know what an animal prefers in order for them to be treated in a humane way (broom 1991). the impact of stress on animals is too important to be avoided (moberg 2000) since animals are also sentient beings with the capability of feeling and suffering (lynch 2010). acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately 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ibadan, nigeria akwoba j. ogugua department of veterinary public health and preventive medicine, university of ibadan, nigeria folashade j. adedoyin department of veterinary public health and preventive medicine, university of ibadan, nigeria patricia i. otu department of veterinary public health and preventive medicine, university of ibadan, nigeria ayi v. kwaghe department of veterinary medicine, university of maiduguri, nigeria noah o. kolawole department of veterinary public health and preventive medicine, university of ibadan, nigeria oyinye j. okoro department of veterinary public health and preventive medicine, university of nigeria, nigeria charity a. agada department of veterinary public health and preventive medicine, university of agriculture, nigeria adeniyi o. tade department of veterinary public health and reproduction, federal university of agriculture, nigeria olufemi o. faleke department of veterinary public health and preventive medicine, uthman dan fodiyo university, nigeria anyanwu l. okeke national veterinary research institute vom, plateau state, nigeria ibikunle m. akanbi department of veterinary services, ministry of agriculture and rural development, oyo state, nigeria mofoluwake m. ibitoye department of veterinary services, ministry of agriculture and rural development, oyo state, nigeria morenike o. dipeolu department of veterinary public health and reproduction, federal university of agriculture, nigeria emma j. dale department of bacteriology and tb, animal & plant health agency, united kingdom perrett lorraine department of bacteriology and tb, animal & plant health agency, united kingdom andrew v. taylor department of bacteriology and tb, animal & plant health agency, united kingdom emmanuel a. awosanya department of veterinary public health and preventive medicine, university of ibadan, nigeria eniola o. cadmus department of preventive medicine and primary care, university of ibadan, nigeria judy a. stack department of bacteriology and tb, animal & plant health agency, united kingdom simeon i. cadmus department of veterinary public health and preventive medicine, university of ibadan, nigeria citation akinseye, v.o., adesokan, h.k., ogugua, a.j., adedoyin, f.j., otu, p.i., kwaghe, a.v. et al., 2016, ‘sero-epidemiological survey and risk factors associated with bovine brucellosis among slaughtered cattle in nigeria’, onderstepoort journal of veterinary research 83(1), a1002. http://dx.doi.org/10.4102/ojvr.v83i1.1002 original research sero-epidemiological survey and risk factors associated with bovine brucellosis among slaughtered cattle in nigeria victor o. akinseye, hezekiah k. adesokan, akwoba j. ogugua, folashade j. adedoyin, patricia i. otu, ayi v. kwaghe, noah o. kolawole, oyinye j. okoro, charity a. agada, adeniyi o. tade, olufemi o. faleke, anyanwu l. okeke, ibikunle m. akanbi, mofoluwake m. ibitoye, morenike o. dipeolu, emma j. dale, perrett lorraine, andrew v. taylor, emmanuel a. awosanya, eniola o. cadmus, judy a. stack, simeon i. cadmus received: 16 june 2015; accepted: 04 nov. 2015; published: 12 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract bovine brucellosis is endemic in nigeria; however, limited data exist on nationwide studies and risk factors associated with the disease. using a cross-sectional sero-epidemiological survey, we determined the prevalence of and risk factors for brucellosis in slaughtered cattle in three geographical regions of nigeria. serum samples from randomly selected unvaccinated cattle slaughtered over a period of 3 years (between december 2010 and september 2013) from northern, southern and south-western nigeria were tested for antibodies to brucella abortus using the rose bengal test. data associated with risk factors of brucellosis were analysed by stata version 12. in all, 8105 cattle were screened. an overall seroprevalence of 3.9% (315/8105) was recorded by the rose bengal test, with 3.8%, 3.4% and 4.0% from the northern, southern and south-western regions, respectively. bivariate analysis showed that cattle screened in northern nigeria were less likely to be seropositive for antibodies to brucella spp. than those from south-western nigeria (odds ratio = 0.94; 95% confidence interval: 0.73–1.22). however, logistic regression analysis revealed that breed ( p = 0.04) and sex ( p £ 0.0001) of cattle were statistically significant for seropositivity to brucella spp. the study found that brucellosis was endemic at a low prevalence among slaughtered cattle in nigeria, with sex and breed of cattle being significant risk factors. considering the public health implications of brucellosis, we advocate coordinated surveillance for the disease among diverse cattle populations in nigeria, as is carried out in most developed countries. introduction brucellosis is a disease responsible for serious economic losses in the livestock industry. it is a zoonotic disease causing morbidity in humans and thus constitutes an important public health problem globally (dean et al. 2012). besides the economic impact brucellosis has on livestock production, it is estimated that about 500 000 persons are also infected annually (pappas et al. 2006). the disease has been eradicated in most developed countries through the implementation of several extensive control programmes. on the other hand, developing countries have continued to experience an increasing trend of the disease because of lack of resources and coordinated control programmes. other major factors contributing to the disease in sub-saharan africa include increased pastoralism and transhumance and intensification of commercial livestock farms (ducrotoy et al. 2014). bovine brucellosis caused mainly by brucella abortus (and in some instances, where mixed farming is practiced, by brucella melitensis) is widespread in africa, where it remains one of the most important zoonotic diseases (ducrotoy et al. 2014; gameel et al. 1993; marcotty et al. 2009), with prevalence ranging from 6.6% to 41.0% among countries in west and central africa (akakpo 1987; bayemi et al. 2009; kubuafor, awumbila & akanmori 2000; schelling et al. 2003). although the prevalence of bovine brucellosis is high and variable in many african countries, surveillance across the continent is generally poor (marcotty et al. 2009; pappas et al. 2006). previous reports attributed the persistence and varying prevalence of the disease to factors that included purchase of infected cattle from the market for replacement or upgrading, nature of the animal production system, demographic factors, regulatory issues, climate, deforestation and wildlife interaction (avong 2000; muma et al. 2007; musa, jahans & fadalla 1990; oie 2011). most importantly, brucellosis is a contagious disease that is spread via direct contact with aborted foetuses, vaginal fluids, placentae and placental fluid. therefore, veterinarians, abattoir workers and other livestock keepers are at risk of infection. transmission can also be through the consumption of unpasteurised milk and milk products from infected animals (ibironke et al. 2008). sadly, however, adequate health and safety measures are rarely observed in most developing countries, hence increasing the chances of zoonotic transmission (swai & schoonman 2009). however, the fact that most animals, irrespective of where they originate, end up at the slaughter slabs or abattoirs is very important, because, apart from screening live animals at the herd level, screening slaughtered cattle at the abattoirs is also invaluable for the epidemiological investigation of bovine brucellosis. in nigeria, available seroprevalence studies have shown that bovine brucellosis is endemic in the country (ate et al. 2007; cadmus et al. 2006; ishola & ogundipe 2000; ocholi 1990). in addition, evidence abounds that the practice of transhumance (seasonal movement of people and livestock between summer and winter pastures) among the fulani pastoralists (a tribal group that holds the majority of the cattle population in nigeria) has facilitated the spread of the disease across nigeria (bale & kumi-diaka 1981; gameel et al. 1993). despite these, few broad-based epidemiological studies exist that provide empirical data on the burden and distribution of brucellosis among diverse cattle populations simultaneously across different geographical regions of nigeria. therefore, to fill this important epidemiological gap, we conducted a large survey over a period of 3 years to determine (1) the seroprevalence of brucellosis among slaughtered cattle across different regions of the country and (2) the risk factors associated with the disease. materials and methods study sites nigeria has a population of over 170 million people and about 13 million cattle according to the national population census published in 2006 and the draft report of the national agriculture sampling survey in 2011. the country is divided into six geographical regions of which three, namely northern, southern and south-western, were purposively selected for this study based on the slaughter cattle population in the areas. nigeria is located in the west african subregion and bordered in the north by niger republic and chad, in the south-west by benin republic and south-east by cameroon (figure 1). there is active transboundary movement of animals between nigeria and neighbouring countries. traditionally, agriculture including livestock farming is the mainstay of the people in nigeria, and this is characterised mostly by close interactions between humans and animals. figure 1: map of nigeria showing states in the regions where the study was conducted. northern region five states including benue, borno, niger, plateau and sokoto states (figure 1) were chosen from the northern region. borno and sokoto states are among the highest cattle-producing states in nigeria and also serve as a source of cattle to other parts of the country. southern region three states with major livestock activities, ebonyi, edo and enugu states (figure 1), were purposively selected in this region. cattle slaughtered in these states originate from the northern region as well as neighbouring african countries. south-western region in the south-western region, lagos, ogun and oyo states (figure 1) were selected because they account for the highest volume of cattle slaughtered in nigeria. in addition, cattle slaughtered in these states are sourced from northern nigeria, neighbouring african countries and a few locally bred animals within the region. duration of study, animal sampling, sample collection and handling the study was carried out over a period of 3 years between december 2010 and september 2013. the major abattoir in each chosen state was purposively selected for animal sampling. furthermore, all the abattoirs chosen receive their animal supply from diverse sources, which include: (1) local herds that are extensively managed, (2) trade cattle sourced from different markets within the state/region, (3) trade cattle sourced from different markets within northern nigeria and (4) markets from neighbouring african countries. on average in each abattoir, blood samples were collected randomly from at least 5% – 10% (based on the volume of slaughter, which ranged from between 20 and 1000 cattle per day) of cattle slaughtered during the study period. at sampling, the breed, sex and age of all animals were recorded. the age of the animals were estimated into ≥ 2 years (adults) and < 2 years (young adults) using the teething method (pace & wakeman 2003). these age groups were chosen because the puberty period of two common breeds in nigeria was estimated to be 19.0–23.5 months in rahaji (oyedipe et al. 1982) and 40.2 months in sokoto gudali (knudsen & sohael 1970). in addition, average body condition score of cattle slaughtered in each abattoir/state was documented as good, fair or poor (nicholson & butterworth 1986). when ≥ 70% of the animals slaughtered in a state were apparently healthy and well-fed, the overall body condition of animals slaughtered in the state was graded as good, when between 50% and 69% as fair and < 50% as poor (table 1). overall, all animals slaughtered were assumed to be unvaccinated against brucellosis because no programme for this exists in nigeria nor within the neighbouring african countries where the animals were sourced (ducrotoy et al. 2014). table 1: distribution of cattle screened according to sex, age, breed and geographical regions in nigeria. in all, approximately 10 ml of blood was collected per animal in a sterile vacutainer tube during slaughter by a trained technician. serum samples obtained from animals in each state for rose bengal test (rbt) were stored at -20 °c until they were transported under cool conditions and later assayed at the tuberculosis and brucellosis laboratories of the department of veterinary public health and preventive medicine, university of ibadan, nigeria. ethical consideration the protocols for this study were approved by the university of ibadan/university college hospital ethics committee (nhrfc/05/01/2008a). assay of samples the serum samples were tested with rbt as described by alton et al. (1988). the rbt antigen consisting of standardised b. abortus antigen (controls) was sourced from the animal and plant health agency, surrey, uk. briefly, equal volumes (30 μl) of antigen and test serum were mixed thoroughly on a plate using a stick applicator and the plate was rocked for 4 min. the appearance of agglutination within 1 minute was scored 2+ (++), whilst any agglutination between 1 and 4 min was scored 1+ (+). the absence of agglutination within 4 minutes of rocking was regarded as negative (−). statistical analysis data analysis was carried out using stata version 12. group differences were tested using chi-square statistics for categorical variables. a multivariable logistic regression was carried out using all the variables that were statistically significant at the 10% level with the main outcome measure (rbt) in bivariate analysis. all tests were two-tailed and statistical significance was set at p < 0.05. results in all, 8105 cattle were sampled from 11 states in the three geographical regions of nigeria. about two-thirds (59.3%) of the animals were of the bunaji breed, over half were male animals (54.1%) and the majority were adult (93.0%) (table 1). overall, the general body condition and physical health status of cattle slaughtered in the south-western, southern and northern regions were observed to be poor, fair and good, respectively (table 2). a seroprevalence of 3.9% was obtained by rbt (table 3). overall, the highest (4.0%) seroprevalence was recorded in the south-western region, followed by the northern (3.8%) and the lowest (3.4%) from the southern region (table 3). generally, the breed-specific result showed that the highest seroprevalence occurred amongst the mixed-breed cattle (6.1%), followed by rahaji (4.6%), other breeds (4.6%), bunaji (3.5%) and sokoto gudali (1.8%) breeds of cattle, respectively (table 3). higher age-specific seroprevalence was recorded in the adults (≥ 2 years) (3.9%) when compared to the young adults (< 2 years) (3.2%). in addition, the sex–specific result showed higher seroprevalence among the female (4.7%) than the male (3.2%) cattle (table 3). table 2: proportion of healthy cattle in good condition slaughtered based on states and geographical regions in nigeria. table 3: brucellosis seroprevalence in cattle slaughtered according to sex, age, breed and geographical region in nigeria using the rose bengal test. the results of the bivariate analysis showed that the breed ( p = 0.04) and sex ( p = 0.002) were significant factors associated with seropositivity of cattle for antibodies to brucella spp. in addition, cattle from the northern region showed less likelihood of being seropositive for antibodies to brucella spp. when compared to those from the south-western region [odds ratio (or) = 0.9; 95% confidence interval (ci): 0.73–1.22; table 3]. furthermore, our results revealed that female cattle were more likely to be seropositive in comparison to the male cattle (or = 1.5; 95% ci: 1.16–1.88), whilst the mixed-breed cattle were more likely to be seropositive than the bunaji breed (or = 1.8; 95% ci: 1.17–2.81; table 4). table 4: results of logistic regression analysis of variables significant at 10% level with the main outcome measure (rbt) in bivariate analysis. discussion a large sero-epidemiological survey of brucellosis was conducted among slaughtered cattle in nigeria, revealing an overall seroprevalence of 3.9%. it was observed that cattle screened in the northern and southern regions of nigeria were about 0.9 and 0.8 times less likely to be seropositive to brucella infection than those from the south-western region. despite the varying seroprevalence of bovine brucellosis found across the country, the findings indicated that the disease had spread among cattle that were slaughtered, hence reiterating its endemicity (although low) in nigeria. this has far-reaching public health implications considering the consumption of unpasteurised milk, lack of personal protective equipment by butchers and other risk practices among livestock workers in nigeria (ibironke et al. 2008). findings from this study are corroborated by an earlier study carried out in central oromiya, ethiopia, where variations were observed in the seroprevalence of bovine brucellosis among cattle in the agro-ecological areas studied (jergefa et al. 2009), in different countries in west africa (unger et al. 2003), and in small ruminants in different regions in ethiopia (teshale et al. 2006). however, the overall seroprevalence of 3.9% obtained in this study is lower than the 8.6% (cadmus et al. 2013) and 5.8% (cadmus et al. 2006) previously reported in south-western nigeria. although it is lower than the 12% from slaughtered cattle in tanzania reported by swai and schoonman (2009), it is comparable to the findings of investigators from other developing countries: 4.9% in slaughtered cattle in cameroon (shey-njila et al. 2005), 3.2% (berhe, belihu & asfaw 2007) and 3.5% (megersa et al. 2011) in ethiopia, 3.3% in central africa (nakouné et al. 2004) and 4.2% in eritrea (omer et al. 2000). the lower prevalence obtained in this study could be attributed to the fact that earlier studies in nigeria were localised to specific areas, in contrast to the widespread coverage in this study. therefore, the wider coverage and longer duration of this study might have had a diluting effect on the overall seroprevalence obtained. again, our findings show that cattle screened in northern and southern nigeria are less likely to be seropositive to antibodies to brucella species than compared to those from the south-western region (or = 0.9; 95% ci: 0.73–1.22) (table 3). this finding may be attributed to the fact that cattle herds in northern nigeria are less likely to be infected with brucella because of the effect of the persistent scorching sun in the area because the organism is less likely to survive in the environment under high temperatures (ducrotoy et al. 2014). the body condition of cattle screened could also be a contributing factor to the differences in seroprevalence observed across the geographical regions studied. fewer than 50% of the animals slaughtered in the south-west were apparently physically healthy and well-fed compared with ≥ 70% from the northern region. because body score provides an indication of the total health status of an animal (nicholson & butterworth 1986), it is plausible to infer that cattle slaughtered in south-western nigeria are more likely to be infected with brucellosis. our finding, though contrary to the assertion made by wadood et al. (2009), is supported by the report of kebede, ejeta and ameni (2008), who observed an increasing seroprevalence in animals with good, medium and poor body condition in a similar study conducted in ethiopia. furthermore, the fulanis do not often sell off animals, particularly the female cattle, unless they are sick and unproductive or when they are in serious need of funds. this practice of the fulanis was also reported in togo (dean et al. 2013). considering the results, one can infer that most of the unhealthy and emaciated animals that are sold cheaply end up in markets and abattoirs where traders have lower purchasing power. in south-western nigeria, more of the rich traders are concentrated in lagos, hence the slaughter of more healthy cattle, as opposed to ogun and oyo states that slaughter a larger population of sick animals (table 2). according to this scenario, one can safely infer that the epidemiology of brucellosis in slaughtered cattle in nigeria is also informed by the purchasing power of stakeholders in the industry. the difference in the breed-specific prevalence is in line with the findings of other researchers who have shown that breed was associated with brucellosis in cattle in nigeria (cadmus, adesokan & stack 2008; cadmus et al. 2013; junaidu, oboegbulem & salihu 2011; mai, irons & thompson 2012). this is also consistent with the reports by kubuafor et al. (2000) in ghana, karimuribo et al. (2007) in tanzania and matope et al. (2011), who associated the proportion of seropositive animals to breeds. our findings show that mixed breed (mostly crosses between rahaji and bunaji) had the highest seroprevalence. as reported earlier, genetic variation is an important factor in conferring resistance or tolerance of cattle breeds to diseases, whilst the antibody response of animals resistant to infection by b. abortus differed significantly from those of susceptible ones (martínez et al. 2010). the findings of this study also show that female cattle were more likely to be seropositive to antibodies to brucella spp. than male cattle (or = 1.5; 95% ci: 1.16–1.88). these results, though contrary to the reports by cadmus et al. (2013), are consistent with other reports that showed significantly higher seroprevalence in the female than male cattle (bekele et al. 2000; dinka & chala 2009; junaidu et al. 2011; kebede et al. 2008; kubuafor et al. 2000; megersa et al. 2011; tolosa et al., 2008). generally, female animals are kept for an extended period of time providing a longer time of exposure to the pathogen, which could in turn serve as a source of infection for other animals. moreover, female cattle are only culled when there is reduced reproductive performance or as a result of old age, at which time their risk of exposure would be high. in addition, the stress associated with pregnancy as well as calving, which tends to reduce immunity of female animals, may also explain the higher seroprevalence among female animals in this study. although this factor was not considered at the inception of the study, it is an important consideration for future studies because some of the female animals slaughtered during this study were pregnant. age did not play any significant role in seropositivity of cattle for antibodies to brucella spp. in this study. this is contrary to the normal pattern of brucellosis spread in a cattle population reported previously (berhe et al. 2007; mai et al. 2012; matope et al. 2011). however, it has been reported that young animals infected in utero could be latently infected, only to show evidence of infection in later years (hinic et al. 2009; nielsen 2000; robinson 2003). this finding may also be understandable because the cattle population screened were not from conventional herds but diverse cattle populations and mostly trade cattle. nonetheless, our finding agrees with that of jergefa et al. (2009), who showed no significant association between the age of cattle and seropositivity to brucella antibodies. the current findings could be attributed to the varying proportions of adult to young cattle that were screened (more than ten times), which is similar to more adult animals sampled by jergefa et al. (2009). again, we suspect that a sizeable proportion of the young cattle in our study could be from infected dams (a likely reason why they were sold), thus increasing the overall prevalence of brucellosis in the population against what is generally observed (bayemi et al. 2009). this study had some limitations. firstly, the use of purposive sampling resulted in sampling of more animals in south-western nigeria compared to other regions. the reason is that more animals destined for slaughter in nigeria are found in south-western nigeria, particularly lagos state (with the highest human population compared to other states in the country). this also necessitated more active routine screening of slaughtered cattle for brucellosis in south-western nigeria. the authors, however, believe that this difference did not significantly affect the results of this study, given the use of proportions in determining the relative prevalence of the disease. secondly, rbt was used as the screening tool in this study, as with many other brucellosis seroprevalence studies in africa (matope et al. 2011). its simplicity and relatively low cost (mcgiven 2013) are important considerations in low-resource settings like nigeria, where vaccination of cattle is seldom carried out. however, it is accepted that false-positive results because of cross-reactions may be obtained and that confirmation of positive results is recommended (oie 2011). lastly, bacteriological isolation of brucella spp. was not performed; this could have helped to confirm the species of brucella circulating among the cattle population screened and provided a better insight into the epidemiology of the disease. however, earlier studies in nigeria, other developing and developed countries, have found serological investigation useful for large-scale studies similar to this one. conclusion it was verified that bovine brucellosis is endemic in nigeria. higher seroprevalence was observed in the south-western compared to southern and northern regions, with the odds of brucellosis seropositivity lower in other regions compared to those in south-western nigeria. furthermore, our findings show that breed and sex of cattle play significant roles in the epidemiology of brucellosis in cattle population in nigeria. based on these findings, we suggest that more diverse epidemiological contexts (e.g. management systems, trade and transhumance systems, agro-ecological zones and climatic conditions) be studied across the country in order to provide a better platform for informed control measures to mitigate challenges peculiar to each region and the country at large. finally, we advocate for coordinated research to determine various social drivers responsible for the epidemiology of brucellosis in nigeria. acknowledgements partial funding support received by dr. cadmus from the john d. and catherine t. macarthur foundation, usa, under the higher education initiative in africa (grant no. 97944-0-800/406/99) is 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unger, f., munstermann, s., goumou, a., apia, c.n., konte, m. & hempen, m., 2003, ‘risk associated with bovine brucellosis in selected study herds and market places in four countries of west africa’, animal health working paper 2, international trypanotolerance centre, banjul. wadood, f., ahmad, m., khan, a., gul, s.t. & rehman, n., 2009, ‘seroprevalence of brucellosis in horses in and around faisalabad’, pakistan veterinary journal 29, 196–198. abstract introduction materials and methods results and discussions conclusion acknowledgements references about the author(s) inge-marié petzer department of production animal studies, university of pretoria, south africa joanne karzis department of production animal studies, university of pretoria, south africa edward f. donkin department of animal and wildlife sciences, university of pretoria, south africa edward c. webb department of animal and wildlife sciences, university of pretoria, south africa citation petzer, i-m., karzis, j., donkin, e.f. & webb, e.c., 2016, ‘a pathogen-specific approach towards udder health management in dairy herds: using culture and somatic cell counts from routine herd investigations’, onderstepoort journal of veterinary research 83(1), a1146. http://dx.doi.org/10.4102/ojvr.v83i1.1146 original research a pathogen-specific approach towards udder health management in dairy herds: using culture and somatic cell counts from routine herd investigations inge-marié petzer, joanne karzis, edward f. donkin, edward c. webb received: 31 dec. 2015; accepted: 17 apr. 2016; published: 30 aug. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a dedicated udder health diagnostic programme was developed and used over a 15-year period in south africa to analyse milk samples based on microbiological and cytological patterns within various groups and for individual cows and udder quarters in dairy herds. these pathogen-specific analyses are utilised for pro-active improvement and management of udder health in south african commercial dairy herds. the programme acts as a monitoring tool and identifies management areas at risk and individual cows with udder disease and uses both quarter and composite milk samples. intra-mammary infection (imi) is a dynamic situation and depending on the time a milk sample is taken, false-negative results may be obtained. a new imi and an infection that is curing may both have low somatic cell counts (sccs), masking the true bacterial status. scc in individual infected udder quarters may differ greatly depending on the causative bacterial species, its pathogenicity, the host immune status and the environmental factors involved. a pathogen-specific udder health approach was followed with repeated herd tests to take account of these udder health dynamics. the results of the herd imi investigation are applied in practice to assist veterinarians, udder health consultants and managers to make informed and specific detailed decisions at both a herd and on an individual cow basis regarding udder health. introduction mastitis is an endemic disease and is considered the most frequent and costly disease in the dairy industry responsible for the highest financial losses, which affects both the animal and the quality of the product (halasa et al. 2007; hogeveen et al. 2010). mastitis is complex and multifactorial in nature and generally results from an interaction between a variety of microbial infections, host factors and environmental and management factors and with generally poor treatment success. it is defined by the national mastitis council (nmc) as an inflammation of the mammary gland mainly caused by bacterial infection (nmc guidelines 2001). dealing with clinical mastitis cases remains important but damage to the udder parenchyma may lower a cow’s lifetime production potential and increases the risk of infecting fellow cows by the shedding of pathogens (degraves & fetrow 1993; white 2010). optimal management practices are considered to be the most effective way to control the disease. work done during the 1960s set the stage for our current understanding of mastitis and established standards for contagious mastitis control (davidson 1961; dodd et al. 1969; neave, dodd & kingwill 1966; neave et al. 1969). from this work, a five-point mastitis control programme was developed and was later upgraded to the nmc 10-point mastitis control plan (smith & hogan 2001). utilisation of this programme has led to a reduction in the prevalence and elimination of contagious mastitis from many south african farms. however, staphylococcus aureus and streptococcus agalactiae remain a challenge in individual farms in south africa and abroad (petzer et al. 2009). in a paper published by middleton (2013), he questioned what we have learned regarding s. aureus mastitis in the last 50 years. his answer summarised the global udder health dilemma. we have gained much knowledge but the basic fact that milking time hygiene is the main critical control point has not changed. decisions regarding the control of mastitis in a given herd will depend on the contagiousness, persistence and inflammatory nature of the main infecting species and strains. the use of historical data to evaluate true new intra-mammary infection (imi) rates, the extent of chronic carriers within the herd and the bacterial cure rates in combination with somatic cell count (scc) levels are all valuable measures to identify mastitis and stress-related causes of high scc. knowledge of the bacterial species present in the udders of cows in a herd and the ability to identify cows with and without imi can be used as a tool for in-depth management decisions. in order to control the transmission rate of contagious pathogens by limiting new imi, cows infected with contagious udder pathogens can be milked last, thereby limiting the risk of spreading this infection to the healthy cows. parlour supervision of the cows infected with contagious pathogens should then be intensified. it has been well established that the probability of imi cure depends on the cow, pathogen and treatment factors (barkema, schukken & zadoks 2006). although treatment success is influenced by choice and use of an appropriate product and also the lactation stage of the cow, the main factor influencing treatment success has been found to be treatment duration (barkema et al. 2006). in addition to the bacterial species or strains involved, their pathogenicity and possible antimicrobial resistance need to be considered, as well as the immune response of the cow. cure rates for s. aureus mastitis have been found to range from 3 to 74%. cure is lower in older cows, and in those with high scc, it increased chronic imi and increased the numbers of mammary quarters infected (barkema et al. 2006). spontaneous cure of new s. aureus imi may be as high as 21%, while in chronic cases, this figure can be as low as 3% (swinkels, schukken & cox 2012). only a few udder pathogens such as s. agalactiae are known for a high treatment success during lactation (keefe 1997). staphylococcus aureus and s. agalactiae imi are still present in dairy herds in south africa (petzer et al. 2013) and warrant a more efficient testing system to enable producers to eliminate both these pathogens from their herds. the scc measure has been used worldwide for several decades as a primary indicator of udder health in dairy herds (heeschen 2010; hillerton 1999; reneau 2001). more than 95% of the cells in milk are leucocytes consisting of variable proportions of macrophages that recruit the neutrophils and lymphocytes in the event of invading pathogens by releasing chemo-attractants (burvenich et al. 2000; sharma, singhand & bhadwa 2011; zeconni & smith 2000). the lymphocytes act mostly as memory cells for the immune system while neutrophils phagocytise and destroy the pathogens. less than 5% of scc consists of epithelial cells originating from the mammary gland (harmon 2001; lee, wooding & kemp 1980). the level of scc in milk depends on various factors of which imi is the most important. factors include parity, stage of lactation, milking frequency, stress (environmental, nutritional, systemic disease and day-to-day changes in management) and non-specific disturbance (nsd) where an increase in scc is not caused by pathogens (laevens et al. 1997; sandrucci et al. 2014; wegner et al. 1976; yagi et al. 2004). traditionally, bulk milk tank somatic cell count (bmscc) is used as a primary index when analysing herd udder health and is used as one of the quality criteria for payment by the secondary milk industry (ruegg & pantoja 2013). high bmscc could indicate udder health problems, whereas a too low level (below 100 000 cells/ml) should act as a warning that mastitis caused by coliform bacteria may increase (shuster, lee & kehrli 1996; suriyasathaporn et al. 2000). however, the bmscc provides only an estimation of the prevalence of infection and irritation of the udders of the cows. this is not a true reflection of the herd udder health status as milk from problem cows should have been excluded (cows with clinical mastitis, fresh in milk cows, cows under treatment and sometimes those with high scc). the bmscc can also be influenced by herd dynamics such as the average parity of cows, and the distribution of lactation stage seen in seasonality calving herds compared to herd with an all-year-round calving pattern (laevens et al. 1997). the impact of any individual cow on bmscc will further depend on its scc and the milk yield of the cow and the dilution factor within the tank. it has been estimated that there is likely to be a 10% increase in imi prevalence in a herd for every 100 000 cells/ml increase in bmscc (bradley 2007). in the year 2014, an estimated 145 million dairy farms operated globally and the largest average dairy herds were found in saudi arabia (8125 cows) followed by new zealand (393 cows) and south africa (238 cows) (lactodata 2014). the larger the herd, the more critical the interpretation of the bmscc needs to be and the less valuable it becomes as a pro-active udder health monitoring tool because of the dilution effect of the milk masking high scc of an individual cow. to evaluate changes in udder health status, the sccs of individual cows are often analysed together with herd clinical mastitis statistics (bradley & green 2001). an scc threshold is used as a guideline to estimate new imi, chronic imi and cure rates and those cows or udder quarters that remain without imi. there has never been absolute consensus regarding the correct threshold level to indicate imi. some consensus was reached regarding an scc standard of ≥ 200 000 cells/ml quarters milk as the indicator of imi at two world dairy summits (athens 1999; new zealand 2001). however, it was agreed at these summits that a tolerance range of up to 400 000 cells/ml was necessary for practical reasons (heeschen 2010). regardless of the international guidelines, countries still set their own scc thresholds, indicating that there is still no consensus at the operational level. the reliability of records of clinical mastitis cases depends on accurate detection of mastitis on a day-to-day basis on farms, and these records are often questionable. the aim of this article is to outline a fresh approach to herd udder health monitoring that facilitates an in-depth analysis of clinical and subclinical mastitis using software that has been developed and tested over the past 15 years at the faculty of veterinary science, university of pretoria. currently, more than 930 commercial dairy herds from south africa and neighbouring countries are benefiting from its application. sample analysis is paid for by the dairy producers, but it is partially subsidised by the university of pretoria to keep costs for whole herd analysis reasonable. in return, the university gains current udder health data that can be utilised for preand postgraduate student training and research. this data set provides current and historical data for research and contains at present results of over 1.6 million milk samples. the milk sample diagnostic (msd) programme that was developed provides an overview of the current and historical herd udder health situation and allows for analysis related to parity, days in milk and other herd groupings and facilitates decision making on an individual cow level based on current and historic species-specific and udder pathology information. it is used as a practical tool to identify challenges at the herd, cow and udder quarter level and guides decision making at an operational level. only by addressing the particular cause can the problem be eliminated. this udder health approach differs from the conventional approaches that use primarily scc and data of clinical mastitis cases as an aid to decision making. materials and methods milk samples and data udder quarter and composite milk samples (one milk sample taken from all four quarters) are taken in most cases from all lactating cows in a herd to establish the presence and prevalence of udder pathogens. professional milk samplers assist milk producers in south africa to take samples in an aseptic manner, ensuring that the sample quality is good and that the cold chain is maintained until the samples reach the milk laboratory. most milk samples reach the laboratory within 24 hours. on arrival at the milk laboratory (department of production animal studies, faculty of veterinary science, university of pretoria, south africa), the batch temperature of the milk samples is recorded. milk is then plated out on bovine tryptose agar, and cultures are read after 18–24 and 48 h (petzer et al. 2012). sccs were performed using a fossomatic 5000 (petzer et al. 2012). identification of mastitis pathogens is generally completed within 48–72 h after the samples have been received at the laboratory. in addition to scc, culture results, clinical appearance of milk and the general information about the cow are added to the msd programme. this includes information regarding calving dates, parity, milk yield, stage of pregnancy, status of the udder parenchyma (assessed by palpation) and teat canal scores. results of quarter milk and composite milk samples are analysed separately and are summarised in individual cow reports and various group reports. some of the various reports that will be discussed: the serial herd udder health report used for evaluation of composite cow milk samples provides an overview of the current and historical udder health status of the herd based on microbiological and scc results. the report also provides a perspective on the level of new, persistent and cured cases for each bacterial species isolated from the herds and indicates the scc distribution within the herd. a current herd udder health report for analysis of quarter milk samples summarises the scc and culture results of the current test of the herd as percentage of quarters with mastitis, or with nsd, imi with low scc, and the percentage of normal quarters. group reports focus on species identification, early post-partum reports (for the first 30 days in lactation) and a lactation stage report (5–90, 91–180 and ≥ 180-day groups) also differentiating between parities. the economic report provides financial information on probable loss in revenue as a result of milk not being produced because of an udder that is not completely healthy, indicated by the scc level. criteria used for diagnosis microbiology (culture) results and sccs are available for each quarter milk sample. many countries set their own operational scc threshold that is in accordance with recommendations of the world dairy summits of less than 400 000 cells/ml. an scc threshold of 300 000 was there for chosen for this programme, being midway between the 200 000 cells/ml recommended by the international dairy federation (idf) and the practical threshold of 400 000 cells/ml recommended at the world dairy summit. in this programme, quarters that tested bacteria negative with an scc of below 300 000 cells/ml milk were regarded as being normal (n); quarters that tested bacteria positive with an scc of equal or above 300 000 cells/ml milk were regarded as having mastitis (m); quarters that tested bacteria positive with an scc of below 300 000 cells/ml were identified as having a ‘teat canal infection’ (tci); and quarters that tested bacteria negative with an scc of equal or above 300 000 cells/ml were identified as having an nsd. the desired herd values aimed for are less than 5% of quarters with mastitis, less than 5% quarters with tci, less than 3% quarters with nsd and more than 50% normal quarters (giesecke, du preez & petzer 1994). results and discussions serial herd health report (composite milk samples) the serial herd udder health report is presented in two parts. the first part provides a summary overview of one to four consecutive herd examinations based on scc and culture results of composite milk samples, while the second part deals with the scc. the current herd udder health status is analysed and compared to results of previous examinations. positive progress or negative developments in the herd in terms of imi and scc trends is measured and evaluated. the various bacterial species isolated from individual cows are indicated as numbers and percentages of cows sampled (see table 1) while the scc are summarised according to six threshold levels (table 2). table 1: serial herd udder health report. case study part 1: bacteriology history report for four consecutive herd examinations of the same herd using composite cow milk samples. table 2: serial herd udder health report. part 2: somatic cell count history report for four consecutive herd examinations of the same herd based on the results of composite cow milk samples. part 1: serial herd microbiological report each bacterial species isolated from individual composite milk samples in the herd is indicated as a number and as a percentage of the total samples examined. of each bacterial species isolated, the number of new, repeat or persistent cases and cases cured are indicated. in the first examination of a herd, all cases are indicated as new infections. in consecutive herd examinations, new infections are indicated when a specific bacterial species has not been isolated from the same cow at the previous examination; persistent cases are those with identical bacterial isolations as shown previously; and ‘cases cured’ (those that are culture negative) in the current examination for the specific bacterial species were those that were isolated during the previous examination (table 1). prevalence of species-specific herd intra-mammary infections depending on the principal bacteria present and its prevalence in the herd, management strategies should be planned in collaboration with the herd manager. a policy of zero tolerance is followed in most cases where s. agalactiae and s. aureus are isolated, aiming at the eradication of these bacteria from all udders in these herds. this approach has proved to be practical and successful in south african herds over the past 15 years. the prevalence of s. aureus imi in more than 930 south african commercial dairy herds decreased from 14.08% in 2008 to 7.77% in april 2012 (petzer & karzis 2012) and to 5.14% in december 2014 (petzer, unpublished data). bacteriological results as shown in table 1 provide the veterinarian, udder health consultant and dairy manager and owner with detailed results to assist informed decision making regarding udder health management at the herd level. new intra-mammary infections depending on the species of bacteria and the level of new imis in the herd, deductions can be made regarding parlour hygiene and bedding management (table 1). management may prove to be inadequate and protocols may need to be revised and upgraded. the level of new imi can be a valuable measurement of effective parlour hygiene and of milker education, dedication and health, especially in the case of contagious mastitis bacteria such as s. aureus and s. agalactiae. it can also indicate ineffective separation of s. aureus– and s. agalactiae–positive cows or an incorrect milking order. the level of new infections may increase if there is a lack of biosecurity when new cows or heifers are introduced into a dairy herd without determining the status of their imi prior to allowing them on the farm, in the parlour and mixing them with the local herd. bacteria such as s. aureus and streptococcus pyogenes are known to be able to cause reverse zoonosis (messenger, barnes & gray 2014). when reverse zoonosis is suspected in a herd, milkers and people in close contact with the cows should be tested for the presence of these bacteria by requesting throat swabs. when most new imi are predominantly environmental bacteria such as the coliforms (escherichia coli, klebsiella spp. and serratia spp.) or streptococci other than s. agalactiae, then udder, feet and flank hygiene scores (cook & reinemann 2007) can be performed to quantify the challenge and identify areas of risk. sources may include inadequate management of bedding, which can cause increased levels of loose faeces on bedding surfaces (reneau et al. 2003), water pollution (mineral or microbial) or overcrowding. repeat (persistent) cases and cases cured when the same bacterial species is isolated from the same udder (composite milk samples) or the same quarter (quarter milk samples) on two consecutive examinations within a reasonable time period, it is regarded as a repeat or persistent imi. the percentage of cases that repeat and those cured (bacterial cure) indicate the level of chronicity and the problematic bacterial species or strains present in the herd. it may be an indication of ineffective mastitis treatment. udders of cows that repeat are palpated to identify possible parenchyma pathology because unsuccessful treatment may be as a result of fibrosis, nodules or atrophy of the udder parenchyma and not necessarily because of bacteria that are resistant to antimicrobials. ‘fibrosis’ (hardening udder quarter) is used as an indication of a more recent chronic case compared to ‘atrophy’ (shrinking udder quarter). application of results in a streptococcus agalactiae–positive herd the herd indicated in table 1 had 3.47% of cows infected with s. agalactiae at the examination dated 02 july 2015. although this percentage decreased from the previous examination on 04 june 2015 (from 5.34% to 3.47%), too many (40 of 45) of these infections were new s. agalactiae imi possibly indicating a relaxed parlour hygiene. five cases of s. agalactiae imi persisted and 33 had been cured since the june examination. the producer was asked to provide information on the whereabouts of 35 cows that were infected with s. agalactiae in june 2015 and which were not tested in july. these cows might have been dried off, removed from the herd or were merely not sampled. the target for new imi and persistent cases caused by s. agalactiae should both be < 5%. a revised management plan should include better prevention and follow-up of cases that did not cure. in this case where s. agalactiae imi has been isolated from a herd a partial ‘blitz therapy’ can be implemented because in this case positive cows have been identified. they should immediately, after conformation of their infections status, be separated from the rest of the cows for the treatment period and until they have been resampled and found to be cured (bacteria free). retesting of both the lactating herd and treated cows is essential for the successful elimination of s. agalactiae from the herd in a relative short period of time. a percentage of s. agalactiae–infected cows may have been missed during the laboratory examination because of the small volume of milk plated out; or because of sub-minimal concentrations of bacteria present in milk samples; or because of the presence of coagulase-negative staphylococci (cns) in udders with high scc initially masking the presence s. agalactiae (personal experience). a short laboratory turnover time in the case of s. agalactiae imi is crucial to the success of eliminating these bacteria from the herd (keefe 1997). case study: application of results in a staphylococcus aureus–positive herd in herds where a low prevalence of s. aureus is identified, the few positive animals should be culled as soon as possible, and management should focus on sound parlour and milking procedure hygiene. the lactating herd should be retested and quarter udder secretion samples of dry cows in late gestation should be included. when a herd is identified with a medium to high prevalence of s. aureus, a longer term strategy is adopted rather than that of culling all positive animals, although culling will form part of the action taken. an important action will be to upgrade the protocol, application of parlour management, the milking routine and the hygiene and monitoring strategies. other factors such as the within-herd prevalence, the contagiousness of the bacteria, the milk price, the current percentage of cows culled because of mastitis and the number of replacement heifers available must also be considered (bradley 2007). the herd indicated in table 1 was diagnosed with 78 (6.05%) s. aureus cows in october 2014 and was regarded as herd with a moderate level of infection prevalence. the producer would be advised to separate the s. aureus–positive cows, if possible for life, and to keep them in a s. aureus group. the results from november 2014 (table 1) showed an increased in the number of s. aureus cases to 214 (16.41%). of these, 159 cows (12.19%) had new s. aureus infections indicating that the preventative measures were inadequate, 44 cows (3.37%) showed persistent infection from the previous test, indicating in these cases a high possibility of chronic cases and only 11 cows (0.84%) were apparently ‘cured’. though the choice and duration of treatment should be discussed with the farmer, the probability of cure could be calculated for each s. aureus–positive cow using the system developed by sol et al. (1997). this formula incorporates parity (first lactation and higher), stage of lactation (early, mid or late), level of scc (above or below a linear score of 6.9 or approximately 800 000 cells/ml), the number of quarters per udder positive for s. aureus (less or more than 3) and quarter position (front or hind) of individual cows, as well as the treatment duration. it can be used to calculate the probability of s. aureus cure (swinkels et al. 2012) (figure 1). information for individual cows on parity, lactation stage, pregnancy status, milk yield, mastitis and scc history is available in the msd programme to aid in the decision making. udders of s. aureus cows should be palpated to identify gross parenchyma damage such as fibrosis, nodules and atrophy, which usually would make treatment ineffective. an informed decision could then be made regarding actions to be taken in case of each individual s. aureus cow, and a detailed action list for individual cows can be formulated for the manager. this might include intra-mammary therapy with or without an extended duration, early drying-off with therapy, inactivation of a quarter or culling of the cow. figure 1: a flow chart indicating events during the management of a streptococcus agalactiae intra-mammary infection outbreak in a dairy herd. in this particular herd (table 1) at the next examination date on 04 june 2015, the number of new cases had decreased to 22 cows (1.61%) of samples, while persistent infections remained relatively low at 32 (2.34%), as does the numbers with bacterial cure (1.32%). however, staphylococcus ‘cure cases’ need to be retested because of the nature of s. aureus to shed intermittently. this is the reason why these cows should remain separate from the rest of the herd for life. south african dairy managers and veterinarians are aware of the existence of reverse zoonosis where infections carried by people may pose a threat to udder health of cows. a noteworthy number of the work force (statistics south africa 2013) has immune systems compromised because of hiv with a consequence of an increased risk for disease. milkers with upper respiratory tract infections caused by s. aureus may pose a risk to the udder health of dairy cows if hygienic principles are not adhered to in the parlour (petzer et al. 2009). precautions such as the wearing of facial masks and placing milkers that have tested positive for s. aureus infections into strategic milking positions where they do not need to touch udders (like teat dipping) have been helpful (personal experience). application of results in herds with mainly gram-negative intra-mammary infection when imi with bacteria of environmental origin is predominant in a herd, the management focus should shift to camps, pasture, bedding and the parlour as sources and areas of risk. the specific bacterial species may provide information on the probable source. pseudomonas spp. for instance is often found in a water source, and listeria spp. is mostly present in silage (hogan & smith 2003). in south african pasture–based dairy herds, the prevalence of streptococcus uberis is increasing (petzer et al. 2009). part 2: serial herd somatic cell count report in the second part of the serial herd udder health report, six scc thresholds are calculated from the herd test (percentages and cumulative percentages). the scc increments are 125 000 cells/ml up to 500 000 cells/ml and become larger for higher scc levels. the herd target is to have more than 80% of lactating cows with scc of less than 250 000 cells/ml, while less than 5% should have an scc in excess of 750 000 cells/ml. herds with a high percentage (> 90%) of cows with low scc may be at a higher risk of contracting e. coli mastitis. in such a situation, management practices such as teatdip prior to milking and feeding immediately after milking to prevent cows from lying down would be advised to allow enough time for the teat canal to close. this is especially true of high-yielding herds where the immune system of cows is more likely to be weakened during the first trimester of their lactation (hogan & smith 2003). scc dynamics of four consecutive herd investigations from 26 october 2014 to 02 july 2015 are compared in table 2. the percentage of cows with scc below 250 000 cells/ml is still low in july 2015 (68.13%), although it has improved from 40.62% in november 2014. similarly, the percentage of cows with scc above 750 000 cells/ml decreased from 31.15% to 16.73% for the same period but is not yet on target. in herds with a high proportion of cows with high scc, a distinction is made between those with and without imi. in cases where most samples that showed high scc also had imi, the sources of these infections should be identified and eliminated or managed. when samples from which no bacteria were isolated form a significant portion of samples with high scc, possible stressors and causes of udder irritation need to be investigated. composite milk samples from cows identified with high scc can be tested on farm with the california milk cell test to gain insight into the inter-quarter scc relation. heat stress, mud stress, nutritional stress and social or handling stress are examples of stressors that can be responsible for increased scc (du preez 2000). differences in scc between quarters of the same udder are used. physiological changes and stressors to the cow will be more prone to cause an elevated scc in three or four quarters, while udder irritation is more often seen in only one or two quarters of an udder. the latter can be caused by incorrect milking techniques, incorrect milking machine settings or lack of adequate machine maintenance. cows that have recently been treated with intra-mammary antimicrobials could also test culture negative with a high scc. the probability of incorrect milking machine settings and incorrect use causing irritation can be investigated by using teat canal scoring (neijenhuis et al. 2001) on first lactating cows, 1–3 months into lactation. the teat canal is the first line of udder defence and a very important barrier preventing imi when damaged. pulsator function should be tested by performing the static test on all milking units followed by dynamic testing to check vacuum stability and level at the teat end during milking. milking machines with high milk lines as well as swing-over parlour systems are still used in south african dairies. teat canal damage is more likely occur in high–milk lines systems than in low milk lines because when over-milking occurs, the risk of a high–teat-end vacuum is greater in high-line systems that function at a higher system vacuum. too high–teat-end vacuum is known to be responsible for teat-end damage (reinemann et al. 2008). swing-over systems with automatic cluster removers are now installed in south african dairies. flow metres that measure the milk flow per unit and initiate cluster take-off are installed in these systems far above the level of the udder, and the milk lines transporting milk from the cluster to the flow metres are often more than 1.5 m – 2.0 m long. therefore, it is too long a delay in the time from when the take-off flow rate is reached until the cluster is actually removed, increasing the risk of over-milking. milking routine should be monitored on site, and a system lactocorder (wmb ag, balgach) can provide measurements of the milk let down time, rate of milk flow and the timing of cluster removal. automated inline monitoring systems such as afimilk programme (afimilk ltd, kibbutz afikim, israel) are available to identify cows that are in incorrect groups and to identify current and past trends in the milking routine. these can include the time from touch to attachment of clusters, milking speed, cluster fall-off, re-attachment and early detachments. reasons for insufficient stimulation or delay in take-off can be identified and rectified, whether by training or notifying the milkers or by correcting a milking machine fault. nutritional stress can contribute to high scc and practical methods such as bunker score and space (bolsen & pollard 2004), rumen filling (burfeind et al. 2010), faecal score and percentage of cows ruminating may be used as a starting point for the investigation in total mixed-ration herds, followed by an in-depth analysis of the feed when indicated. cows on pastures and in paddocks may suffer from stress because of mud during the rainy season. in south africa, the high temperature humidity index (thi) may often have a negative effect on the scc levels, milk yield and reproduction efficiency during the hot summer months (du preez, giesecke & hattingh 1990; giesecke et al. 1988). current herd udder health report (quarter milk samples) a summary of species-specific imi and sccs for herds is shown in table 3. the report is divided into two sections with the first part indicating results from lactating cows and the second part from non-lactating cows. the msd programme currently uses an scc level of 300 000 cells/ml in quarter milk samples as threshold to diagnose mastitis when imi is present and nsd in the absence of imi. quarters with an scc level below 300 000 cells/ml without imi are diagnosed as ‘normal’ and those with imi as ‘tcis’. dry cow secretions are only examined for the presence of bacteria and no scc is done. table 3: current herd udder health report based on somatic cell count and culture results of quarter milk samples from lactating and dry cows. two major concerns can be identified regarding the udder health status in the lactating cows in the herd indicated in table 3, namely the large percentage of quarters with high scc and the presence of s. aureus imi in the herd. staphylococcus aureus was isolated from 10.6% of quarters, cns from 12.4% and s. uberis and s. agalactiae each from 1.1%. of the 35.9% quarters with scc of 300 000 cells/ml and above (mastitis and nsd cases), only 6.6% had imi, indicating that something besides imi was prime reason causing high scc in the herd, even though 25.3% of all quarters had imi. more hind than front quarters were diagnosed with nsd and might indicate incorrect removal of clusters. of the dry cows sampled 4 weeks after intra-mammary treatment, 12% were infected with s. aureus, 34% with cns and 9% with s. uberis. this could indicate a poor cure rate for s. aureus and a possible high new imi occurring during the dry period for both cns and s. uberis when compared to the results of the lactating cows (table 3). an action list could be compiled for the producer which may include: s. aureus cows to be followed up by evaluating their mastitis history, performing udder palpation and evaluating criteria of each cow for her probability of cure. the milking machine should be checked, milk routine evaluated and other possible stressors investigated. group reports calving dates, pregnancy status, level of milk yield and status of the udder parenchyma can be entered into the msd programme as information additional to the laboratory results for individual cows. pathogen-specific group report reports of cows currently infected with specific udder pathogens are generated to use as on-farm action lists. as explained above, cows should be selected and separated, to deal with contagious imi such as s. aureus or s. agalactiae imi in a herd. cows that have had s. agalactiae imi and were cured (bacterial cure) may be returned to their previous groups, but s. aureus cases should remain in a separate group for life. reports of consecutive examinations provide information on the dynamics of imi in the individual cow and identify cows with persistent or chronic imi. application in herds during a streptococcus agalactiae mastitis outbreak during an s. agalactiae imi outbreak in a dairy herd, composite milk samples are taken from all lactating cows and quarter secretion samples are obtained from the dry cows not currently under antibiotic treatment. when the heifers presently in late gestation had been reared on fresh milk as calves, they are also sampled because of a risk of them having s. agalactiae imi (petzer & karzis 2012). the initial list of cows that tested positive for s. agalactiae is emailed to the producer within 24 hours after receiving the samples at the laboratory. managers should then immediately separate cows that tested positive for s. agalactiae, treat all quarters with of those cows with intra-mammary antimicrobials, and milk these cows last under strict hygiene conditions. udders of s. agalactiae cows should be palpated to identify gross pathology, in which case the cure rate may be low. any dry cow or heifers positive for s. agalactiae imi are also treated. after 10–14 days after the last intra-mammary treatment, the s. agalactiae–positive cow group is resampled (quarter milk samples) for micro-cytological analysis and composite samples are collected from the rest of the lactating herd. the percentages of s. agalactiae cases that are cured, those that persist and the number of new imi are analysed, and a management plan is formulated based on this information. cows with s. agalactiae imi are only regarded as being cured when no s. agalactiae can be isolated from any of their quarters, when the scc in all quarters is below 200 000 cells/ml and when there is only a small variation in the scc between quarters. the idf-recommended scc threshold of 200 000 cells/ml is used in this case to be extra cautious not to introduce a cow with persistent s. agalactiae imi again into the negative herd. depending on parlour hygiene and management, it is possible for one s. agalactiae–positive cow to initiate a new outbreak as was experienced in south african herds. monitoring of the herd should be continued on a regular basis, and intervals may be monthly or longer until all lactating cows have tested negative for s. agalactiae at least two or more consecutive tests. this may take 3–6 months depending on the initial s. agalactiae prevalence as well as the motivation and dedication of the producer and milkers (figure 1). for most major udder pathogens, different protocols are required. in the case of a s. uberis imi, different strains were identified (zadoks 2007). some strains were found to be more likely to cause chronic imi while others cured spontaneously after a period of only days. strain typing of s. uberis from individual cow samples on a herd basis has not been shown to be cost effective. when s. uberis is isolated repeatedly from the same cows, the herd manager is advised to increase the duration of intra-mammary treatment only in the event of clinical mastitis in those cows and to use intra-mammary dry cow therapy at drying-off in those specific cows. stage of lactation and parity the immune system of the lactating cow is known to be weakened during the peripartum period because of hormonal changes and many stressors. these can include calving and onset of lactation stress, social stress and stress because of the adaption to a new diet and the onset of a negative energy balance in the cow that can last up to day 100 of lactation (collard et al. 2000). because of vulnerability of the cow during this period, this stage of the production cycle is critical for monitoring udder health. udder health up to 30 days post-partum (multiparous and primiparous cows) the number of new imi and cases with elevated scc that occur from 5 to 30 days post-partum provides insight into the udder health during the dry period and calving hygiene. it is also an indication of bacterial cure rate during the dry period. however, there is also a risk of new imi post-partum when milking commences. knowledge about the prominent bacterial species causing imi in a herd enables advisors and managers to identify and deal with the sources and causes of infection timeously and effectively. the report that summarises the species-specific imi information including total, cured, persistent and new imi for cows between 5 and 30 days can be used to evaluate udder health during the dry period. the current species-specific imi and scc results of individual multiparous cows should be compared to their udder health in their previous late lactation. results for primiparous cows are indicated in a separate report as total numbers (and percentages) of species-specific imi that have been isolated. managers should aim to have less than 10% of cows with imi and scc in excess of 200 000 cells/ml during this period and an incidence of less than 5% of clinical mastitis (bradley 2007; green & bradley 2012). an scc threshold of 200 000 cells/ml is used to be more strict in management decisions of the post-partum group for they may be more susceptible to new imi because of calving stress, the onset of lactation, social stress and the possibility of developing a negative energy balance (barkema et al. 1999; fenwick et al. 2008). heifers are more prone than cows to develop severe udder oedema prior to calving and recently calved primiparous cows are said to have a greater prevalence of mastitis than older cows, despite having less mastitis later in lactation (barkema et al. 1998). heifers that are close to calving and primiparous cows should receive a diet with an adequate energy balance and without excessive sodium and potassium (nestor, hemken & harmon 1988). stress around calving should be limited by stimulating heifers to exercise and by avoiding overcrowding in order to reduce negative social interactions (hutjens & aalseth 2005). the imi profile of first lactation cows shortly post-partum is an indication of udder health challenges that have occurred mostly during late pregnancy (environmental bacteria) but can in some cases be traced back to the way they were reared as calves when fed infected milk and kept in groups (petzer & karzis 2012). when bacteria isolated from milk samples shortly post-partum are predominantly contagious, this may indicate treatment failure, the presence of chronic udder damage or new infections contracted during early lactation. when few cases are cured, treatment protocol needs to be revisited to indicate whether the correct antimicrobial product was used for the correct duration of time. udders of treated cows should be palpated to determine if there is chronic udder parenchyma damage (fibrosis, nodules or atrophy). a high rate of new imi caused by contagious bacteria could be an indication of poor milking hygiene of newly calved cows. trends of total, cured, new and persistent imi in a herd can be followed over time to improve management decisions. in the event where most imi are caused by environmental bacteria shortly post-partum, causes can be chronic infections or treatment failure but new environmental imi are more likely to have occurred during the dry period or at calving. most new imi caused by environmental bacteria are known to occur just after drying-off and shortly before and after calving (oliver & sordillo 1988). in these situations, daily pre-calving teat dipping during the high-risk periods in the dry period should be added to the management routine. no teat seal is currently registered on the south african market to assist in the prevention of new imi during the dry period of cows. udder health in early, mid and late lactation (90 days, 180 days and later in lactation) bacteriological and scc results of milk samples during early, mid and late lactation are compared to evaluate progress or failure of udder health management during lactation (table 4). important events occur during the first 90 days of lactation, which includes peak milk production and re-breeding. table 4: herd udder health status correlated with different stages of lactation using quarter milk samples. early lactation is also a high-risk period for metabolic diseases and multifactorial stress together with other diseases such as metritis and mastitis (bell 1995; pulfer 1991). the status of imi in cows at calving will determine udder health in that whole lactation. if recently calved cows have a high incidence of imi, little progress will be made in lowering the bmscc, as each animal cured during lactation would be replaced by another infected cow that has recently calved. the management aim should be to have less than 15% cows with scc in excess of 200 000 cells/ml up to 90 days in lactation (green & bradley 2012). mid-lactation is generally a lower risk period and a less eventful time for dairy cows than early lactation. during late lactation, the optimum body condition for cows should to be achieved, foetus growth accelerates and cows are prepared for drying-off. the timing of drying-off usually depends on the expected calving date and level of milk yield to allow for a sufficient dry period. concentrates may need to be reduced when milk yields are still high close to the date of drying-off to prevent excess udder oedema. while there may be a small rise in a cow’s scc in late lactation, sharp increases that are seen at this stage may be as a result of udder infection or irritation. general udder health of the herd in the herd used as an example (table 4), 71.38% of quarters were diagnosed as being normal; 11.6% with nsd, 7.95% with mastitis and 9.06% with tci. although the percentage of normal quarters is satisfactory, the 19.55% of quarter with an scc above 300 000 cells/ml milk is unacceptably high and so are the 17.01% cases of imi. the quarters with high scc in the herd originated from 7.95% mastitis and 11.6% nsd cases (table 4). the main reason for increased scc is imi. when no imi are found in many milk samples with high scc, the milking machine and stress factors (high thi, overcrowding, mud, nutritional shortcomings and inadequate management) may be indicated as causes (burvenich et al. 2000; du preez et al. 1990; sandrucci et al. 2014). the bacteria responsible for imi in this herd are cns (13.04%), s. uberis (2.14%), s. aureus (1.08%) and streptococcus dysgalactiae (0.72%). it was noted that 72.24% of imis in early lactation were because of mastitic quarters, while only 27.65% of imi identified in quarters in late lactation were mastitic (table 4). lactation stages: intra-mammary infections the portion of cows in early (34.78%), mid (27.53%) and late lactation (37.67%) differ in this herd. the percentages of imi and high scc quarters were calculated per lactation period (table 4). there were imi in 18.75% of quarters taken from cows in early, 14.46% in mid and 17.28% in late lactation (table 4). the percentage of imi detected in early lactation cows was high. this would warrant further investigation into the dry period and calving management, persistence of chronic cases, stress during early lactation and suppressed immunity of cows early in lactation. when the imi increases with days in milk, a distinction should be made between failure to cure of existing imi (that increased persistent cases) and an increase of new imi. persistent cases may be because of udder parenchyma damage, treatment failure or virulent or resistant pathogen strains and a suppressed host immune system. depending on the bacterial species involved, new infections may originate from the environment (bedding, pastures, water contamination and inadequate milking machine hygiene), the parlour hygiene during the milking routine or when biosecurity is lacking. lactation stages: somatic cell counts in the example in table 4 cows in early lactation had the highest percentage (28.10%) quarters with increased scc compared to mid (11.80%) and late lactation (17.28%) cows, although cows less than 5 days in milk (colostrum) are excluded. quarters identified with mastitis (13.54%) and nsd (14.58%) contributed almost equally to the high percentage of scc found in early lactation (table 4). therefore, in this herd, suspected causes for both imi and nsd in early lactating cows needed to be investigated. economic report estimations are made on the whole herd and do not take into account variations between cows. the msd programme is used to estimate milk production losses based on quarter milk sample with elevated sccs (giesecke et al. 1994; hortet & seegers 1998; sharma et al. 2011). the herd used as an example in table 5 had a daily milk production of 2650 litres. the daily milk loss in this herd because of elevated scc was estimated to be 126.55 litres. this loss represents an estimated loss of 3796 litres per month and 46 190 litres annually. this represented an estimated loss of 4.77% in potential milk production for this herd. table 5: estimated milk production losses in a herd associated with elevated quarter milk somatic cell counts of all lactating cows in the herd. when making a management decision regarding a s. agalactiae imi in a herd, it is helpful to have an indication of the current production loss because of s. agalactiae. in table 6, the daily milk production loss in the s. agalactiae–infected quarters was estimated to be 37.64 litres amounting to an estimated 1.4% production loss. although this case may not warrant blitz therapy to eradicate s. agalactiae, the focus should be on improving the milking routine. this calculation is only based on prevalence at the time of sampling and does not incorporate risks of new infection, shedding, cure rate and the number of persistent cases. table 6: estimated milk production losses in a herd associated with quarters infected with streptococcus agalactiae. conclusion there are many advantages of having species-specific imi information about udder health in the current msd system. it allows early detection of imi, rapid follow-up on information from tests; there is a short turnaround time after the receipting of milk samples and prompt communication of results to herd managers and owners. the programme firstly allows evaluation of the herd udder health situation enabling the consultant to identify the main causes of udder health problems in detail. this will assist in identifying and eliminating the sources of the problems timeously. at the same time, it provides information on each cow on parity, lactation stage, pregnancy status, production level and mastitis and scc history to enable informed decisions for individual cows and even individual udder quarters. management decisions can be based on sound information and cows that are cured or have persistent imi and new imi can be identified, based on actual bacterial identification. this improves the accuracy of the decisions made. this approach has proved to be practical and to build the confidence of dairy farm managers (personal experience). acknowledgements we acknowledge abaci systems for developing the msd computer programme, dr w.h. giesecke for the original concept of the diagnostic laboratory programme and the milk producers of south africa for their continuous use and support. thanks are also due to the staff of the milk laboratory of the department of production animal studies, faculty of veterinary science, onderstepoort for their continued quality work: mrs j.c. watermeyer, f. konaite, n. labuschagne, r. ludike, miss r. badenhorst, mr l.l. mohapi and k. malekane for performing the technical work and data logging. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions i-m.p. developed the concept for the msd computer programme and used and upgraded it, wrote the article and oversaw technical work and data capturing for the manuscript. j.k. assisted with literature search, assisted with the overall structure, verified data and edited the manuscript. e.f.d. assisted with the overall structure, interpretation of data and edited the manuscript. e.c.w. assisted with the overall structure and motivation and edited the manuscript. references barkema, h.w., deluyker, h.a., schukken, y.h. & lam, t.j., 1999, ‘quarter-milk 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versus long duration of treatment of clinical staphylococcus aureus mastitis’, xxvii world buiatrics congress, proceedings, lisbon, portugal, june 3–8, 2012, p. 150. wegner, t.n., schuh, j.d., nelson, f.e. & stott, g.h., 1976, ‘effect of stress on blood leucocyte and milk somatic cell counts in dairy cows’, journal of dairy science 59(5), 949–956. http://dx.doi.org/10.3168/jds.s0022-0302(76)84303-2 white, a., 2010, ‘mastitis, a practice approach’, journal of veterinary surgeon in general practice, livestock 15(7), 36–40. http://dx.doi.org/10.1111/j.2044-3870.2010.tb00325.x yagi, y., shiono, h., chikayama, y., ohnuma, a., nakamura, i. & yayou, k., 2004, ‘transport stress increases somatic cell counts in milk, and enhances the migration capacity of peripheral blood neutrophils of dairy cows’, journal of veterinary medicine 66(4), 381–387. http://dx.doi.org/10.1292/jvms.66.381 zadoks, r.n., 2007, ‘sources and epidemiology of streptococcus uberis, with special emphasis on mastitis in dairy cattle’, cab reviews: perspectives in agriculture, veterinary science, nutrition and natural resources 2, 15. http://dx.doi.org/10.1079/pavsnnr20072030 zeconni, a. & smith, k.l., 2000, ‘idf position paper on ruminant mammary gland immunity’, symposium on immunology of ruminant mammary gland, 11–14 june 2000, stresa, italy, pp. 1–120. apanaskevich_1-12.indd introduction it was originally assumed that only two species of the genus hyalomma koch, 1844, namely hyalomma (euhyalomma) truncatum koch 1844 and hyalomma (euhyalomma) rufipes koch 1844, occurred in south africa. in 1949 delpy described a new subspecies of h. (e.) rufipes, naming it hyalomma rufipes glabrum delpy, 1949. this name persisted until theiler (1956) raised it to species level as hyalomma glabrum, but in the same year hoogstraal (1956) synonymized it with hyalomma marginatum turanicum pomerantzev, 1946. he based this decision on a study of reared specimens of adult h. r. glabrum that were sent to him by gertrud theiler, and on some adults of h. (e.) m. turanicum originating from iran. no immature stages were studied. fur thermore, hoogstraal (1956) assumed that h. (e.) m. turanicum had been introduced into south africa on per sian sheep apparently imported from the medi ter ra nean region. since then the name h. (e.) m. turanicum has been used for this tick in all publications devoted to the hyalomma ticks of south africa. four years later, the subspecific status of h. (e.) rufipes was demonstrated by hoogstraal & kaiser (1960), and it 1 onderstepoort journal of veterinary research, 73:1–12 (2006) the genus hyalomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum delpy, 1949 (acari, ixodidae) as a valid species with a redescription of the adults, the first description of its immature stages and notes on its biology d.a. apanaskevich & i.g. horak department of veterinary tropical diseases, faculty of veterinary science, university of pretoria onderstepoort, 0110 south africa abstract apanaskevich, d.a. & horak, i.g. 2006. the genus hyalomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum delpy, 1949 (acari, ixodidae) as a valid species with a redescription of the adults, the first description of its immature stages and notes on its biology. onderstepoort journal of veterinary research, 73:1–12 for nearly 50 years the ixodid tick hyalomma marginatum turanicum, reputedly introduced into south africa on imported persian sheep, has been considered identical to the asian hyalomma (euhy alomma) marginatum turanicum pomerantzev, 1946. comparisons of this tick with the asian h. (e.) m. turanicum and other subspecies of hyalomma (euhyalomma) marginatum, however, reveal that it is an old taxon, namely hyalomma rufipes glabrum delpy, 1949. it is hereby reinstated as hy alomma (euhyalomma) glabrum, and its adults are redescribed and its immature stages described for the first time. the preferred hosts of its adults are large herbivores such as zebras, gems bok and eland, on which it occurs during summer. the preferred hosts of its immature stages are scrub hares and ground-frequenting birds, on which it is present during autumn and winter. data on its distribution and possible disease relationships are also provided. keywords: description, distribution, hosts, hyalomma (euhyalomma) glabrum, immature stages, seasonality accepted for publication 9 november 2005—editor 2 hyalomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum, 1949 became h. (e.) marginatum rufipes. there are thus cur rently two species and two subspecies of hyalomma recognized in south africa, namely h. (e.) truncatum, h. (e.) m. rufipes and h. (e.) m. tura nicum. the systematics of species within the h. (e.) mar ginatum group is one of the most complex in the subgenus euhyalomma filippova, 1984, and within the genus hyalomma as a whole. this group of ticks con sists of one extremely polymorphic species, namely hyalomma (euhyalomma) marginatum, which contains the four subspecies, hyalomma (euhy alomma) marginatum marginatum koch, 1844, h. (e.) m. rufipes, hyalomma (euhyalomma) margi natum isaaci sharif, 1928 and h. (e.) m. turanicum. apanaskevich (2003, 2004) has published a preliminary differentiation of these subspecies based on all their life stages. however, after large numbers of south african h. (e.) m. turanicum had been examined and compared with asian h. (e.) m. turanicum it was obvious that these taxa are entirely different. as a consequence we have now compared the morphological characters of males, females, nymphs and larvae of south african h. (e.) m. turanicum with those of h. (e.) m. marginatum, h. (e.) m. rufipes and h. (e.) m. isaaci, and those of h. (e.) m. turanicum from asia. the presence of a number of distinctive diagnostic characters on all developmental stages of south afri can h. (e.) m. turanicum has persuaded us to designate this taxon as a separate species within the h. (e.) marginatum group, namely hyalomma (euhya lomma) glabrum. hyalomma (euhyalomma) glabrum delpy, 1949 type specimens: the original, extremely brief description in the form of an identification key was based on adult specimens from the karoo, south africa (“karro”, delpy 1949). the deposition of the type spe cimens is unknown. gertrud theiler’s laboratoryreared specimens, from amongst which delpy described h. r. glabrum, are deposited in the tick museum at the onderstepoort veterinary institute (ovi), south africa, and could be considered as para types or syntypes. the male and female are illustrated under the name hyalomma turanicum in hoogstraal (1956). synonym: hyalomma rufipes glabrum delpy, 1949. material examined: 876 males, 111 females, 100 nymphs and 100 larvae from four localities in south africa (mountain zebra national park (32°15’ s, 25°27’ e), eastern cape province; karoo national park (32°16’ s, 22°32’ e), western cape province; and the farms “outuin” (30°10’ s, 18°02’ e), northern cape province; and “klipfontein” (33°20’ s, 23°19’ e), south-western eastern cape province). in addition to the abovementioned field collected specimens, we have also examined the laboratory-reared specimens in the gertrud theiler collection (de aar, 23.xi.1942; no.: 2850, 2851, 2852, 2853, 2854, 2855; 46 males, 88 females, nymphs and exuviae of nymphs, larvae and exuviae of larvae) deposited in the tick museum at the ovi. description the measurements are given as follows: minimum – maximum (average ± standard error, n = number of specimens examined). all measurements of adults are given in millimetres and those of immature stages in micrometres. male (fig. 1, 2a–i) conscutum (fig. 1): length 4.03–5.48 (4.72 ± 0.02, n = 100), width 2.77–3.63 (3.27 ± 0.02, n = 100), fig. 1 hyalomma glabrum, male, conscutum. bar = 1 mm 3 d.a. apanaskevich & i.g. horak fig. 2 hyalomma glabrum, male, a, genital structures: apron, postgenital sclerite, pregenital arch. bar = 200 μm; b, anal plates. bar = 500 μm; c, spiracular plate and circumspiracular setae (a—anterior; d—dorsal). bar = 400 μm; d, gnathosoma dorsally. bar = 500 μm; e, gnathosoma ventrally. bar = 500 μm; f, palp ventrally. bar = 400 μm; g, hypostome. bar = 400 μm; h, coxae. bar = 500 μm; i, genu iv: (i), dorsal view, (ii), medial view, (iii), lateral view. bar = 1 mm a b c a d e d f g h i (i) (ii) (iii) reviewer acknowledgement open accesshttp://www.ojvr.org page 1 of 1 the onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, 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reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the onderstepoort journal of veterinary research. please do not hesitate to contact me if you require assistance in performing this task. jana venter submissions@ojvr.org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 journal of veterinary research onderstepoort we appreciate the time taken to perform your review successfully. david paton donald king felicity burt guy palmer julie fitzpatrick martyn jeggo mathias borchert richard kock robert noad wilna vosloo 109 abstract introduction materials and methods results discussion conclusion acknowledgements references appendix 1 about the author(s) evans m. mathebula new generation vaccines programme, agricultural research council onderstepoort veterinary institute, south africa department of veterinary tropical diseases, university of pretoria, south africa frederika e. faber new generation vaccines programme, agricultural research council onderstepoort veterinary institute, south africa wouter van wyngaardt new generation vaccines programme, agricultural research council onderstepoort veterinary institute, south africa antoinette van schalkwyk molecular epidemiology and diagnostics, agricultural research council onderstepoort veterinary institute, south africa alri pretorius new generation vaccines programme, agricultural research council onderstepoort veterinary institute, south africa department of veterinary tropical diseases, university of pretoria, south africa jeanni fehrsen new generation vaccines programme, agricultural research council onderstepoort veterinary institute, south africa department of veterinary tropical diseases, university of pretoria, south africa citation mathebula, e.m., faber, f.e., van wyngaardt, w., van schalkwyk, a., pretorius, a. & fehrsen, j., 2017, ‘b-cell epitopes of african horse sickness virus serotype 4 recognised by immune horse sera’, onderstepoort journal of veterinary research 84(1), a1313. https://doi.org/10.4102/ojvr.v84i1.1313 original research b-cell epitopes of african horse sickness virus serotype 4 recognised by immune horse sera evans m. mathebula, frederika e. faber, wouter van wyngaardt, antoinette van schalkwyk, alri pretorius, jeanni fehrsen received: 13 july 2016; accepted: 29 sept. 2016; published: 24 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract identifying antigenic proteins and mapping their epitopes is important for the development of diagnostic reagents and recombinant vaccines. b-cell epitopes of african horse sickness virus (ahsv) have previously been mapped on vp2, vp5, vp7 and ns1, using mouse, rabbit and chicken monoclonal antibodies. a comprehensive study of the humoral immune response of five vaccinated horses to ahsv-4 antigenic peptides was undertaken. a fragmented-genome phage display library expressing a repertoire of ahsv-4 peptides spanning the entire genome was constructed. the library was affinity selected for binders on immobilised polyclonal immunoglobulin g (igg) isolated from horse sera collected preand post-immunisation with an attenuated ahsv-4 monovalent vaccine. the dna inserts of binding phages were sequenced with illumina high-throughput sequencing. the data were normalised using pre-immune igg-selected sequences. more sequences mapped to the genes coding for ns3, vp6 and vp5 than to the other genes. however, vp2 and vp5 each had more antigenic regions than each of the other proteins. this study identified a number of epitopes to which the horse’s humoral immune system responds during immunisation with ahsv-4. introduction african horse sickness virus (ahsv) is a member of the genus orbivirus in the family reoviridae (calisher & mertens 1998). it contains 10 linear double-stranded rna segments which code for seven structural (vp1–7) and at least five non-structural proteins (ns1–4, s10-orf2; belhouchet et al. 2011; roy, mertens & casal 1994; stewart et al. 2015; zwart et al. 2015). the gene segment coding for vp6 (segment 9) also codes for ns4, while segment 10 encodes ns3/3a and s10-orf2. the structural proteins are arranged in three concentric layers, with vp2 and vp5 forming the outer capsid and are associated with cell attachment and inducing apoptosis (manole et al. 2012; vermaak & theron 2015). the inner capsid consist of vp7 and vp3. it encloses the minor structural proteins vp1, vp4 and vp6 (replication complex) and the genome itself (maree & paweska 2005; mellor & hamblin 2004). there is currently no cure for ahs and the disease is controlled by vaccination. although the vaccine is effective, there is the potential that the attenuated vaccine may revert to virulence. in addition, infected animals cannot be differentiated from vaccinated animals, a factor which is important in non-endemic areas (kanai et al. 2014). there is thus a need for alternative ahs vaccines. although inactivated and subunit vaccines have also been developed, they are not yet commercially available (lelli et al. 2013; roy et al. 1996; scanlen et al. 2002). vaccination results in a predominantly humoral response with neutralising antibodies conferring protection (burrage et al. 1993). recombinant ahsv vp2 and vp5 alone or in combination induce protection in horses that is enhanced by the addition of vp7 (alberca et al. 2014; calvo-pinilla et al. 2014, 2015; kanai et al. 2014; martinez-torrecuadrada et al. 1996). understanding the immunogenicity of ahsv proteins and mapping their critical epitopes can contribute to developing an alternative recombinant vaccine. some b-cell antigenic regions on vp2, vp5 and ns1 of ahsv-4 have already been mapped using a variety of methods including phage display and pepscan (bentley et al. 2000; de la poza et al. 2015; martinez-torrecuadrada et al. 1999, 2001). this study focusses on b-cell epitopes mapped by phage display technology using sera from horses vaccinated with attenuated ahsv-4. phage display of peptides involves constructing a library of fragments of a gene or genome of interest fused to a gene encoding a phage surface protein (smith & petrenko 1997). the library of phage-displayed fusion peptides is then iteratively amplified and screened for target binders. traditionally, after a few rounds of selection, about 10–100 clones are picked and insert dna sequenced to identify the enriched peptides. this approach was used to map antigenic regions on vp7 (du plessis et al. 1994), vp5 (wang et al. 1995) and ns1 (du plessis, romito & jordaan 1995) of bluetongue virus (btv) and ahsv vp2 (bentley et al. 2000). owing to the small sample, there is a possibility that not all binding clones are identified when picking a subset. depending on the diversity of the clones after panning, clones that were enriched may not be among those picked for sequencing or tested for binding (domina et al. 2014; ngubane et al. 2013). using high-throughput sequencing, the entire pool of phages released after panning identifies all the selected peptides. during panning, peptides that do not bind the target of interest are often present. these are termed target-unrelated peptides (tups) and should be considered when analysing results (vodnik et al. 2011). using appropriate controls during panning, these sequences are used to normalise the experimental samples and the tups are removed. in conjunction with phage display, the large amount of data obtained from high-throughput sequencing has allowed rapid profiling of antigenic regions on the meningococcal virulence factor (neisseria adhesin a) by antibodies from vaccinated people (domina et al. 2014). this technique has been termed profiler (phage-based representation of immuno-ligand epitope). in other similar studies, using random peptide libraries in combination with high-throughput sequencing, peptides that bind to mycobacterium tuberculosis (ngubane et al. 2013) and patient-specific epitope motifs in serum from patients with peanut allergies (christiansen et al. 2015) have been identified. in the present study, phage display and high-throughput sequencing were used to dissect the immunogenicity of a live attenuated ahsv-4 vaccine in horses. the b-cell epitopes encoded by all 10 ahsv-4 segments as recognised by polyclonal antibodies from immunised horses were analysed. the epitopes identified here could be useful in diagnostic reagent development and, in conjunction with t-cell epitopes identified in a parallel study, have the potential to be included in an envisaged multi-epitope vaccine. materials and methods horse serum samples horse sera were obtained from a vaccine trial where five horses (horse 1–5) were each vaccinated twice subcutaneously with the live-virus–attenuated ahsv-4 vaccine strain (pretorius, faber & van kleef 2016). the research protocol was approved by both the animal ethic committees of the agricultural research council onderstepoort veterinary institute (arc-ovi) and onderstepoort biological products (obp), ltd. approval was also obtained from the south african department of agriculture, forestry and fisheries. blood was collected for serum 3 days before vaccination (day 0), 7 days after the booster inoculation on day 21 (day 28) and 31 days later (day 52). immunoglobulin g (igg) was purified from the sera using the protein g hp spin trap kit (ge healthcare life sciences) following manufacturer’s protocol. enzyme-linked immunosorbent assay of horse sera samples to check whether the vaccine induced a humoral response, the horse antisera were tested for the presence of anti–ahsv-4 antibodies. a 96-well microtiter plate (nunc polysorp) was coated with 50 µl of 10 µg/ml sucrose-purified ahsv-4 particles (huismans et al. 1987) diluted in pbs (ph 7.4). the plate was incubated at 4 °c overnight, emptied and blocked with 2% milk powder in pbs (mpbs). wells coated with mpbs only served as negative control. the plate was incubated for an hour at room temperature. three washes with pbs containing 0.05% tween-20 (pbst) were performed after every incubation. after washing, 50 µl of horse serum diluted 200x in mpbs was added to duplicate wells. this was incubated at 37 °c for an hour. after washing, 50 µl of protein a/g peroxidase (thermo scientific) diluted 1:10 000 in mpbst was added to each well and incubated at 37 °c for 45 min followed by washing. fifty microliters of substrate consisting of one o-phenylenediamine dihychloride tablet (sigma) dissolved in 5 ml of 0.1 m citrate buffer (ph 4.5) and 2.5 µl of 30% (v/v) hydrogen peroxide (saarchem) was added. the reaction was stopped after 10 min at room temperature by adding 50 µl of 2 n h2so4. the absorbance values were recorded at 492 nm. fragmented-genome phage display library construction this library was constructed essentially as previously described (du plessis & jordaan 1996; fehrsen et al. 2005; gupta et al. 1999) by using existing cdna copies of each ahsv-4 genome segment cloned into either pet102d-topo or pgemt (faber et al. 2016). large segments encoding vp1, vp2 and vp3 were cloned as two fragments. the segments were amplified from these plasmids using takara ex taq enzyme with pcr primer pairs t7a: tagttattgctcagcggtgg / trxfus: ttcctcgacgctaacctg (for pet102/d-topo) and t7b: taatacgactcactataggg / m13rev: caggaaacagctatgac (for pgem-t). annealing temperatures were 56 °c and 54 °c, respectively. dnase 1 digestion of four pools of similar sized amplicons yielded dna fragments of 50 bp – 600 bp that were gel purified using a qiaquick gel extraction kit (qiagen) before cloning in the pme1 linearised m13 phagemid vector pcvep1585042 (fehrsen et al. 2005). after electroporation of the phagemids into escherichia coli tg1 electroporation-competent cells (agilent technologies), the presence of inserts was confirmed by using pcr primers m13ff: gtaaaacgacgcgcag and m13rev: caggaaacagctatgac, while high-throughput sequencing was used to confirm full-genome representation in the library. phages were rescued for panning from bacterial cells containing the phagemids as described previously (van wyngaardt & du plessis 1998). affinity screening the library was screened for binders with purified iggs from horse sera collected on days 0, 28 and 52. the panning process was carried out as previously described (fehrsen et al. 2005) with minor modifications. wells of a microtiter plate (polysorp, nunc) were coated, in duplicate, with 100 µl of 20 µg/ml of igg diluted in pbs, for each time point of the five horses separately. the plate was incubated overnight at 4 °c. the next day, unbound iggs were discarded and the wells blocked with mpbs at room temperature for an hour. the wells were then washed three times with pbs. during this time, 1.12 × 1012 fusion phages from the library were pre-incubated for 20 min in 1 ml of mpbs containing 0.1% tween-20. after washing with pbs, 100 µl of the pre-incubated phages were added to each well and incubated at 37 °c for 1 hour. unbound phages where removed by washing the wells 10 times with 0.1% pbst and another 10 times with pbs. the bound phages were eluted with 100 µl of 0.1 m glycine–hcl (ph 2.2) elution buffer at room temperature for 10 minutes. the released phages were transferred to a new tube containing 50 µl of 1 m tris–hcl (ph 9.0) neutralising buffer. the phages were used to infect exponentially growing tg1 cells with an od600 of 0.5–0.6. this was incubated at 37 °c for 30 min and another 30 min shaking at 100 rpm. ten-fold dilutions of the infected cells were made and 100 µl of each dilution plated on tye containing 100 µg/ml ampicillin and 2% glucose plates. the remaining cells were centrifuged at 2000 g for 15 min, the pellet resuspended in pbs and 200 µl plated on similar 150-mm plates. the plates were incubated at 30 °c overnight and the number of output colonies recorded. colonies were scraped from the plates and phages rescued as above. after each round, half the rescued phages were used as input for the subsequent round and the remainder stored at -70 °c. these polyclonal pools of phages were tested in enzyme-linked immunosorbent assay (elisa) (van wyngaardt et al. 2004) for binding to the igg coated as for screening. four rounds of panning were performed. phagemid dna was isolated from the bacteria after each round and inserts were amplified by pcr for sequencing. high-throughput sequencing dna inserts were amplified from phagemids using takara ex taq enzyme at a tm of 56 °c and primers ff: cgt cggcagcgtcagatgtgtataagagacaggag gctagcaacgcgtcg and rev: gtctcgtgggctcg gagatgtgtataagagacagccaggcgcgccg. they bind very close to the insert and contain the illumina miseq sequencing platform adapters (underlined). high-throughput sequencing on gel-purified amplicons was done by inqaba biotech (south africa) using the illumina miseq v3 (150 cycles) platform. bioinformatic analysis of selected sequences the high-throughput paired-end sequencing data were analysed using the clc genomics workbench v7.5 (http://www.clcbio.com/products/clc-genomicsworkbench/) and microsoft® excel 2013. some fragments in the pool were represented by a single read where the second read did not meet the quality standard. after primer sequences were trimmed, low quality and sequences less than 50 bp were discarded. the sequences were mapped to a list containing the open reading frames (orfs) of all 10 ahsv-4 genome segments. the mapping profile of each segment was analysed individually for each horse. sequence distribution the sequences selected by immune igg were normalised by subtracting the pre-immune igg-selected sequences. to get an overall view of the reads mapping to each genome segment, data were converted to percentages: (number of reads mapping to a segment / total number of reads mapping the entire genome) × 100. the sequences were then further analysed by showing the position of each mapped sequence on the genome segment. for this purpose, the total number of mapped sequences for each segment was exported from clc to microsoft® excel without gaps. this was converted to proportions: total number of base matches per position / total number of base matches on the segment and illustrated by a graph. the data for all the horses were plotted on the same graph. the peaks were numbered with the most important criteria that all horses recognise the region. nucleic acid sequences were translated to amino acid sequences to confirm the in-frame display of peptides with the vector-encoded pviii, which is necessary to produce a functional pviii and natural ahsv peptides, and also to establish if the amino acid sequence mapping results in any notable change when compared to that using nucleotide sequences. after converting the unpaired and untrimmed nucleotides, the amino acid sequences that produced functional peptides were filtered from the pool by searching for the vector-encoded sequence on each peptide end (starting with snasf … or ending with … gap). the resultant peptides bearing the vector sequences were grouped to form a new sequence list, which was used to create a basic local alignment search tool (blast) database. the amino acid sequences of the 12 ahsv-4 proteins were used as reference and aligned against the new peptide sequence list using blast. each peptide that matched was illustrated on a graph by adding the number of times an amino acid aligned at a position on the respective proteins. for this, the alignment table in clc which shows where each peptide matches on the reference protein was used. a ‘per amino acid’ alignment graph was drawn in microsoft® excel. the position and number of peptide matches were illustrated as peaks and compared to the dna mapping. results enzyme-linked immunosorbent assay of horse antisera to confirm the presence of ahsv-specific antibodies in the sera of the vaccinated horses, they were tested against purified ahsv-4 in elisa. sera from day 28 after administration of the vaccine gave the highest signals that had all decreased by between 23% and 70% by 52 (figure 1). the day 0 signals indicated the sero-negative state of the horses prior to vaccination. igg was purified from all the samples and used to select binders from the phage library (below). figure 1: enzyme-linked immunosorbent assay showing the antisera of the five horses (h1–5) collected pre-vaccination (day 0) and post-vaccination (day 28 and 52) reacting with purified african horse sickness virus-4. each plotted value is an average of duplicate wells. milk powder–negative control background signals were subtracted from the experimental data. fragmented-genome library construction to map the epitopes on ahsv-4 which were recognised by immune horse sera, a phage-displayed peptide library was constructed. it comprised 5.63 × 105 clones with insert sizes ranging from 50 bp to 600 bp. this exceeded the minimum required size of 1.7 × 104 clones to cover the ±19-kb ahsv genome with a 99% probability (clarke & carbon 1976). this took into account the fact that a fragment can be cloned in one of two orientations, with three reading frames on either the nor c-terminus. this means that only 1/18 clones encoded a functional native ahsv-4 peptide (jacobsson et al. 2003). to confirm coverage, an aliquot of the library was subjected to high-throughput sequencing and mapped to the entire ahsv-4 genome. even though the depth of coverage varied across regions, there were no gaps. these results validated the theoretical calculations (figure 1-a1a). nevertheless, comparing the expected representation as a proportion of segment size and the actual numbers found by mapped sequences showed that not all the segments were represented equally in the library (figure 2). although the complete segment was represented, ns1 was the most underrepresented (figure 1-a1a). figure 2: proportional representation of the african horse sickness virus-4 genome segments according to (a) size and (b) actual number of clones in the primary unpanned fragmented-genome phage display library. segments are colour coded and named according to their encoded protein. affinity selection of binding peptides the fragmented-genome library was individually affinity selected for binders using antibodies from each horse and at each indicated time point. specific binding of the selected pools of phages to the respective iggs was demonstrated with elisa to show that the enrichment of phage pools was actually linked to their recognition by antibodies in the sera. there was an enrichment of phages with day 28 igg especially for horses 2 and 5 (figure 3) but no enrichment with immunoglobulins from day 0 sera and very little with day 52 igg. only horse 1 day 52 igg-selected phages showed similar enrichment to those selected with horse 1 day 28 igg. phage selected with horse 2 igg of both day 0 and day 52 time points showed elisa signals of at least twice that of the other horses. however, the unpanned library resulted in similar signals, which may suggest the presence of tups, non-specific binding or anti-phage antibodies in horse 2 serum. figure 3: enzyme-linked immunosorbent assay showing the pool of fusion phages in the unpanned library (r0) and phages selected during the four rounds of panning (r1 – r4) with day 0, 28 and 52 igg of all five horses, reacting with their respective igg. each plotted value is an average of duplicate wells. milk powder–negative control data have been subtracted from the experimental data. characterisation of selected phages it has been shown that comparing sequences from a selection round where there is no further enrichment with sequences from the unpanned library should give a clear change in binding pattern (christiansen et al. 2015). thus, pools of phages from panning round three for each time point were selected for further in silico characterisation. the dna inserts were amplified by pcr, gel purified and sequenced. the data were received as 2 × 75-bp paired-end reads for each dna fragment. the sequences were mapped to the orfs of each of the ahsv-4 genome segments. approximately 70% of the sequences mapped to the genome. the other 30% was either low quality or from untargeted sources and not taken into account. theoretically, affinity selection of a display library using a population of antibodies creates a sequence distribution pattern biased towards the selected genome regions. when the sequences were mapped to the orfs of ahsv-4 and the data for each time point pooled, a change in sequence distribution was observed (figure 4). the figure only reflects the proportional sequence match per gene and not their position on the gene. the sequence distribution was in agreement with the phage-igg elisa signals since day 28 selected sequences showed the most notable change in proportional hits to each gene when compared to day 0 and day 52 selected sequences. sequences mapping to the orf encoding for ns3, vp5 and vp6/ns4 increased by the largest amount on day 28 and had decreased to nearly day 0 levels by day 52. figure 4: proportional representation of paired-end sequence reads mapping to each segment after the third round of panning with anti-african horse sickness virus-4 igg. (a) day 0, (b) day 28 and (c) day 52. data represent the average of the five horses for each time point. segments are colour coded and named according to their encoded protein. the sequences identified with day 28 igg were further analysed to determine the regions on the individual genome segments with the highest number of overlapping sequences and thus potential antigenicity. in contrast to the complete coverage of a genomic segment prior to panning, sequences mapped only to specific positions on each segment (figure 1-a1b). the number of hits at a specific position was illustrated as a proportion of total hits for that genomic segment. for easy comparison, the results were combined on a single graph for all horses. for example, vp2 data of each horse were plotted on one chart (figure 5a) and the same for vp5 (figure 5b). the regions that were recognised by the antibodies from all or most of the horses were focussed on in this study. very few sequences mapped to the segment encoding ns1, which was therefore excluded from subsequent characterisation. figure 5: day 28 igg-selected vp2 (a) and vp5 (b) sequences, of all five horses, converted to matches per nucleotide position as a proportion of the total nucleotide matches per gene segment. numbers represent the identified potentially antigenic regions. data were normalised by subtraction of day 0 matches. usually with phage-displayed peptides, the inserts of single clones are sequenced, translated and then mapped to the amino acid sequence of the target protein. the in silico analysis described here was done with nucleic acid sequences. for phage-displayed peptides derived from randomly fragmented dna, there is a possibility that the dna insert is expressed as a functional fusion-phage protein but as a non-native peptide. to determine whether the nucleic acid matches described above were from phages expressing native ahsv-4 peptides, the sequences were also translated and aligned to the amino acid sequences of each protein. the translated sequences contained the primer/vector sequences and were selected based on the vector-encoded pviii amino acids at the cloning site. sequencing errors such as insertions and deletions could affect the correct translation of reads and result in discarding of reads/matches when the amino acid sequences are filtered for correct reading frame and thus the matching pattern. the translated sequences were aligned to the amino acid sequence of the ahsv proteins in a similar pattern as the nucleotide sequence mapping, for example, vp3 of horse 2 (figure 6). because the patterns were similar, the majority of sequences after panning were expressed on the phage as native ahsv peptides. this validates the less cumbersome route of using nucleotide sequences to map to the targets. however, there are exceptions; the gene segment coding for vp6 contains a second open reading frame in both btv and ahsv (belhouchet et al. 2011; zwart et al. 2015). this is a small, non-structural protein, named ns4. in this case, it is essential to look at the amino acid mapping to distinguish vp6 and ns4 matches (figure 7). for the ahsv-4 used in this study, ns4 is coded in the +2 reading frame from nucleic acid position 197 bp to 631 bp. the dna region coding for both proteins contains a major antigenic region recognised by the antibodies from all five horses, which is in fact an ns4 epitope (ns4-1). the regions flanking ns4-1 are vp6 epitopes but only vp6-1 is common between all the horses and not recognised by day 0 antibodies. in addition, segment 10 that encodes ns3 also contains a second reading frame (s10-orf2; stewart et al. 2015). in this case, less than 0.1% of the hits are in the s10-orf2. thus, the ns3 antigenic region remains the most recognised by the antibodies. figure 6: a comparison of the pattern of nucleic sequences mapping to the gene and the deduced amino acid alignments of the selected peptides using the day 28 vp3 data of horse 2 as an example. (a) nucleic acid mapping and (b) amino acid mapping. figure 7: a comparison of the pattern of nucleic acid sequences mapping to the gene and the deduced amino acid alignments of day 28 vp6 data of horse 2. the translated sequences are matched to either the vp6 or the ns4 amino acid sequence. ns4 is in the +2 reading frame from bp 197 to 631 but positioned with vp6 for ease of comparison. (a) nucleic acid mapping and (b) amino acid mapping. seventeen antigenic regions recognised by day 28 igg from most horses were identified (table 1). they were between 18 and 60 amino acids (aa) in length and mostly on the outer capsid proteins vp2 and vp5 (three each). two antigenic regions were identified on each of the inner core vp7, the sub-core vp3 and non-structural protein ns3. one region was identified by igg from all horses on each of the minor structural proteins vp1, vp4 and vp6 and the non-structural proteins ns2 and ns4. most of these regions were also identified by day 52 igg with the exception of vp2-2. table 1: potential antigenic regions on african horse sickness virus-4 proteins. discussion identifying antigenic regions on viruses and other pathogens remains important in vaccine and immunodiagnostic development. in this study, b-cell antigenic regions of ahsv-4 recognised by polyclonal sera from immunised horses were identified using phage display in conjunction with high-throughput sequencing of the selected sequences, in an approach similar to the profiler method (domina et al. 2014). this polyclonal pool of antibodies induced by the vaccination represents the complete antibody response that may contain neutralising antibodies. analyses were focussed on the phage-displayed peptides selected using antibodies from day 28 when the immunoglobulin levels were at their peak. the panning with day 52 igg yielded less enrichment of binders than day 28 igg (figure 3) even though there were anti–ahsv-4 antibodies in the day 52 serum (figure 1). the overlapping nucleotide sequences mapped predominantly to segments encoding ns3, vp6/ns4 and vp5. anti-ahsv ns3 antibodies have been found to occur at low concentrations in infected horses using immunoblotting (laviada et al. 1993), while high concentrations of anti-vp6 and vp5 antibodies were similarly detected using horse serum from early-stage infection (martinez-torrecuadrada et al. 1997). not all studies on ahsv antigenicity concur. the apparent differences may, among other reasons, be because of the time the serum was collected and methods used to determine antigenicity. a possible factor may be that the long fragments displayed in this study may adopt secondary structures and thus mimic discontinuous epitopes (fehrsen et al. 2005). this of course would not be the case with a linear or denatured peptide. it was not determined whether the epitopes on ns3, vp6/ns4 and vp5 induce neutralising antibodies. however, in a related orbivirus, candidate vaccines without ns3 or with non-functional vp6 protected sheep after challenge with a virulent btv strain (feenstra et al. 2014; matsuo, celma & roy 2010; matsuo & roy 2009; matsuo et al. 2011). this may suggest that ns3 and vp6 of ahsv do not induce neutralising antibodies. to account for the immuno-dominance of ns3 and vp6/ns4, it is possible that the virus uses these proteins to deceive the immune system during infection, as described for hiv1 using gp120, termed ‘deceptive imprinting’ (nara & garrity 1998). the virus uses the proteins to divert the immune system’s attention from the immunopathogenic hot spots on proteins or replication machinery, thus evading neutralisation. on the other hand, ahsv vp5 expressed in insect cells induced antibodies that neutralised the virus in a plaque reduction assay (martinez-torrecuadrada et al. 1999) and in combination with vp2 and vp7 protected horses against ahs (martinez-torrecuadrada et al. 1996). thus, the vp5 epitopes identified here might be able to induce neutralising antibodies. although multiple regions were selected with the different horse iggs, only those that are selected by igg from all horses are likely to be of interest. these regions are not unique to a particular animal and thus may be useful in developing a universal recombinant vaccine or diagnostic reagent. the regions with the most peptide sequences selected by phage display were ns3-1 (49 aa), ns3-2 (27 aa) and ns4-1 (30 aa). ns3-1 contains a proline-rich area (huismans et al. 2004). a proline-rich region was essential for plasma membrane targeting and viral release in the ebola and marburg virus matrix protein vp40 (reynard et al. 2011). ns3-1 could be involved in membrane binding and directing viral egress, thus being exposed to the immune system during virus replication. the other dominant region identified is contained on the vp6 encoding segment. this area maps to the ns4 amino acid sequences. because these ns3 and vp6 (and in effect ns4) proteins have previously been found to be non-neutralising and not critical for virus propagation, removing them from a vaccine construct may increase vaccine efficacy (stalhammar-carlemalm et al. 2007). three potentially antigenic regions were mapped on vp5. both vp5-1 and vp5-3 form part of regions found to react with serum from an ahsv-4–infected horse in immunoblots. vp5-3 contains a neutralising epitope (position 85–92) as recognised by a mouse monoclonal antibody (martinez-torrecuadrada et al. 1999). vp5-2 was not recognised by horse antibodies in an immunoblot; thus, it is possible that this region, identified by phage display, represents part of a discontinuous epitope. three antigenic regions were identified on vp2 (33-, 27and 18-aa long), with the first two near the n-terminal half and one at the c-terminus. these are not the same as previously identified epitopes mapped by others using horse antibodies by both phage display and pepscan. it is possible that the longer phage-displayed peptides in this study (up to 200 aa compared to 100 aa) allow formation of secondary structures, which can reveal additional antigenic regions. the region between amino acid positions 200 and 432, identified by only two horses in this study, was described as being dominant using mouse monoclonal antibodies, rabbit and horse polyclonal antibodies and pepscan (bentley et al. 2000; martinez-torrecuadrada et al. 2001). for vp2, the advantage of the large amount of data yielded by high-throughput sequencing of the released pool of phages instead of the traditional clone picking is clearly evident. two potential antigenic regions were mapped on vp7, one towards the centre (28 aa) and the other at the n-terminus (19 aa). these regions may have some relevance to neutralisation because the addition of vp7 in a mixture with vp2 and vp5 increased immunological protection of horses (martinez-torrecuadrada et al. 1996). furthermore, ahsv vp7 purified from bhk-infected cells was able to protect balb/c mice against lethal challenge, although it induced low antibody titres as detected with elisa, suggesting that such protection could conceivably also involve cell-mediated responses (wade-evans et al. 1997). this suggestion was supported recently where vp7 induced equine cytotoxic t-cell responses in vivo (faber et al. 2016). one region was mapped at the centre of ns2 (27 aa). in another study, recombinant ns2 and vp7 of ahsv serotype 3 reacted with hyperimmune guinea-pig sera to all nine serotypes and horse antisera to seven attenuated ahsv serotypes (bremer et al. 1994) which indicates both ns2 and vp7 contain conserved epitopes. the antigenicity of vp1, 3 and 4 remain largely unexplored. peptides representing these proteins were selected at a low frequency in this study. the size of an epitope varies between 5 and 15 binding residues (kringelum et al. 2013). the regions identified in this study are larger, each of which could possibly contain shorter minimal binding areas or are able to adopt a secondary structure and represent discontinuous epitopes as in the protein’s native form. phage display in conjunction with high-throughput sequencing methods have shown to be valuable tools to get a comprehensive antigenic profile of proteins. it was possible to confirm some of the antigenic regions identified by other methods, for example, by pepscan. as an alternative and to avoid potential bias from the display system used in this study, a bacteriophage t7 system can be used where the phages are released by lysis and not secreted through the cell membrane. this system was used to clone the complete human virome, made synthetically and codon optimised for e. coli expression. immuno-precipitation was used for selection thus avoiding solid-phase selection used in this study. although it is more expensive to set up than the fragmented-genome approach, together with high-throughput sequencing this method can also yield a wealth of data (xu et al. 2015). conclusion in conclusion, 17 horse-specific potential antigenic regions on both structural and non-structural proteins of ahsv-4 were identified using phage display combined with high-throughput sequencing. the antibodies used to identify the regions were from horses vaccinated with the serotype 4, which is included in the commercial ahsv vaccine, which is known to induce protection against african horse sickness. it needs to be confirmed whether these regions are in themselves involved in immunological protection. once confirmed as being potentially protective b-cell epitopes, they could conceivably be combined with suitable t-cell epitopes (faber et al. 2016) to form a vaccine construct capable of evoking both humoral and cellular immunity. apart from vaccine development, b-cell epitopes on ns2, vp6, ns3 and vp7 may also assist in the development of diagnostic tests. in addition to identifying antigenic regions elicited by a vaccine administered to the actual host animal, this study has assisted in developing approaches that can facilitate analysis of the large volume of phage display data. this approach is likely to be useful in monitoring the global antibody response in animals during vaccine trials. acknowledgements the work was funded by the economic competitiveness support programme, arc-ovi, south africa. competing interests the authors declare that they have no financial or personal relationships that may have 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random epitope library to map an antigenic determinant on the bluetongue virus outer capsid protein vp5’, journal of immunological methods 178, 1–12. https://doi.org/10.1016/0022-1759(94)00235-o xu, g.j., kula, t., xu, q., li, m.z., vernon, s.d., ndung’u, t. et al., 2015, ‘comprehensive serological profiling of human populations using a synthetic human virome’, science 348, aaa0698-1–aaa0698-9. https://doi.org/10.1126/science.aaa0698 zwart, l., potgieter, c.a., clift, s.j. & van staden, v., 2015, ‘characterising non-structural protein ns4 of african horse sickness virus’, plos one 10(4), e0124281. https://doi.org/10.1371/journal.pone,0124281 appendix 1 figure 1-a1: an example of a clc genomics workbench read mapping representation of sequenced inserts mapping to the african horse sickness virus-4 open reading frames segment encoding ns1. the sequences from the unpanned library (a) cover the entire gene without any gaps. the sequences from round three phages selected with day 28 igg (b) has gaps, indicating that only regions of interest have been selected. zweygarth_121-126.indd introduction heartwater or cowdriosis is an infectious, non-contagious, tick-borne disease caused by the intra cel lu lar rickettsial agent ehrlichia ruminantium. the dis ease affects cattle, sheep, goats and also some wild ruminants. it is transmitted by ticks of the genus amblyomma and has been reported from almost all african countries south of the sahara, from the adjacent islands in the indian and atlantic oceans (uilen berg 1983) and from some caribbean islands (perreau, morel, barré & durand 1980; birnie, burridge, camus & barré 1984). heartwater is usually an acute disease and may be fatal within hours or days after the onset of clinical signs. the mammalian cell culture system first described by bezuidenhout, paterson & barnard (1985) is the method of choice for in vitro isolation and propagation of e. ruminantium, whereas the successful prop agation of e. ruminantium in tick cells was only reported recently (bell-sakyi, paxton, munderloh & sumption 2000; bekker, bell-sakyi, paxton, martinez, bensaid & jongejan 2002; bell-sakyi 2004). initiation of infection in tick cell cultures was achieved using elementary bodies derived from mammalian cell cultures (bell-sakyi et al. 2000) or from other tick cell lines previously infected with mammalian cell culture forms (bell-sakyi 2004) but not directly from infected animals. attempts to infect tick cells with blood from sheep undergoing clinical responses following experimental e. ruminantium infection were unsuccessful (bell-sakyi 2004). the present experiments describe the first successful establishment of infection in ixodes scapularis (ide8) tick cell cultures directly from the blood of four sheep, each infected with a different south african stock of e. ruminantium, and the subsequent infection of endothelial cells from infected ide8 cells. 121 onderstepoort journal of veterinary research, 75:121–126 (2008) in vitro isolation of ehrlichia ruminantium from ovine blood into ixodes scapularis (ide8) cell cultures e. zweygarth*, a.i. josemans and h.c. steyn onderstepoort veterinary institute, private bag x5, onderstepoort, 0110 south africa abstract zweygarth, e., josemans, a.i. & steyn, h. 2008. in vitro isolation of ehrlichia ruminantium from ovine blood into ixodes scapularis (ide8) cell cultures. onderstepoort journal of veterinary research, 75:121–126 four stocks of ehrlichia ruminantium (welgevonden, ball3, nonile and blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into ixodes scapularis (ide8) tick cells using the leukocyte fraction of the blood of infected sheep. organisms of two of the e. ruminantium stocks (welgevonden and blaauwkrans) propagated in ide8 cells were also successfully used to infect bovine endothelial cells. all stocks were successfully propagated in ide8 cells using dulbecco’s modified eagle’s medium nutrient mixture ham f-12 containing 10 % foetal bovine serum (fbs). the technique should be included in any attempt to isolate uncharacterized e. ruminantium stocks. keywords: ehrlichia ruminantium, heartwater, in vitro isolation, tick cell line * author to whom correspondence is to be directed. e-mail: zweygarthe@arc.agric.za accepted for publication 14 april 2008—editor 122 in vitro isolation of ehrlichia ruminantium from ovine blood into ixodes scapularis (ide8) cell cultures materials and methods stocks of e. ruminantium four stocks of e. ruminantium isolated in south africa were used: the welgevonden stock was originally isolated by injecting a tick homogenate into a mouse; the tick had been collected on the farm welgevonden in the northern transvaal (du plessis 1985); the ball3 stock was isolated from a nonspecified host in the northern province (haig 1952); the blaauwkrans stock was isolated from an eland near port elizabeth in 1996 (zweygarth & josemans 2001); and the nonile stock from a sheep in kwazulu-natal (mackenzie & mchardy 1984). culture media uninfected and some infected tick cell cultures were propagated in l-15b medium (munderloh & kurtti 1989), which was supplemented with 5 % heat-inactivated foetal bovine serum (fbs), 10 % tryptose phosphate broth (tpb), 0.1 % bovine lipoprotein con centrate (icn, irvine, ca, usa), 100 iu/mℓ penicillin and 100 μg/mℓ streptomycin. the ph was adjusted to 7.2. infected tick cell cultures were also maintained in dulbecco’s modified eagle’s medium nutrient mixture ham f-12 (dme/f-12, sigma, st. louis, mo, usa; d 0547) containing 15 mm hepes and 1.2 g/ℓ sodium bicarbonate. this medium was further supplemented with 10 % (v/v) heat-inactivated fbs, 2 mm l-glutamine, 100 iu/mℓ penicillin and 100 μg/mℓ streptomycin and is referred to as df-12. df-12 medium was also used for infected and uninfected endothelial cell cultures. cell cultures the tick cell line ide8, derived from i. scapularis embryos (munderloh, liu, wang, chen & kurtti 1994) was used throughout these experiments. ide8 cultures, infected and uninfected, were maintained at 32 °c in complete l-15b medium, unless otherwise stated. ide8 cell cultures were used at passage levels between 54 and 62. three fifths of the medium was replaced weekly. propagation of infected and un infected bovine aorta endothelial (ba 886) cell cultures was carried out as described previously (zweygarth, vogel, josemans & horn 1997). sheep-derived infective culture inoculum each of the four e. ruminantium stocks—ball3, blaauwkrans, nonile or welgevonden—was used to infect a merino sheep by intravenous injection of a 5 mℓ blood stabilate. the body temperature of each sheep was monitored daily and a blood sample was drawn when it had risen to more than 41.5 °c, unless stated otherwise. blood was collected by venipuncture into sterile vac-u-test® tubes containing heparin (lithium heparin, 14.3 usp per mℓ blood) as anticoagulant and put on ice. the cooled blood was centrifuged (800 x g; 10 min; 4 °c) and the buffy coat was collected and washed with cold phosphate-buffered saline (pbs). the buffy coat was again collected, and the red blood cells were lysed for approximately 30 s in 20 mℓ sterile distilled water followed by the addition of 5 mℓ of a 5 x concentrated physiological nacl solution (4.5 % w/v). the leukocytes were centrifuged and the resulting cell pellet was re-suspended in 5 mℓ of df-12 or complete l-15b medium. the leukocyte suspensions were then inoculated into 25 cm² culture flasks containing ide8 cells. the cultures were incubated at 32 °c. attempts to infect tick cell cultures with the wel gevonden stock of e. ruminantium were also carried out according to the method described by byrom, yunker, donovan & smith (1991) with minor modifications. briefly, heparinized blood (lithium heparin, 14.3 usp per mℓ blood) was centrifuged (800 x g; 10 min; room temperature) and 3 mℓ of plasma were inoculated into each of two 25 cm² culture flasks containing a layer of ide8 cells. the cultures were put on a rocking platform for 3 h at three cycles per min at 25 °c, after which the plasma was decanted and the cell monolayer was rinsed three times with 5 mℓ pbs. finally df-12 medium was added. in vitro infection of bovine endothelial cells by e. ruminantium organisms derived from ide8 cell cultures ide8 cells infected with the welgevonden and blaauwkrans stocks of e. ruminantium were used to demonstrate infectivity of ide8-derived organisms for ba 886 cells. aliquots of 2.5 mℓ of infected ide8 cell suspension were distributed into culture flasks containing ba 886 cells. fresh df-12 medium was added to give a final volume of 5 mℓ. the cultures were then incubated at 37 °c and the medium was replaced every 3 days. ide8 and ba 886 cultures were monitored for infection by microscopic examinations. small samples from the cell layer were removed and smears were prepared. cytospin smears were made from cultures where some of the cells were in suspension. smears were allowed to dry before being fixed with methanol and stained with eosin-methylene blue. 123 e. zweygarth, a.i. josemans & h.c. steyn molecular characterization extraction and amplification of dna dna was extracted from the four stocks of e. ruminantium with the qiaamp dna kit extraction kit (qiagen). the primers for the pcs20 pcr diagnostic test (van heerden, steyn, allsopp, zweygarth, josemans & allsopp 2004) specific for e. ruminantium was used to sequence of e. ruminantium in the blood and in tick cell cultures. briefly, the pcrs were performed with 2 μℓ of genomic dna extracted from cell culture as template in a 50 μℓ reaction with 0.5 mm of each of the primers hh1f and hh2r, 2.5 mm dntp; 25 mm mgcl2, 10 x pcr reaction buffer and 0.5u of takara ex taq enzyme (takara shuzo co., ltd japan). pcr conditions were: incubation of 25 s at 94 °c, 35 cycles of denaturation at 94 °c for 30 s, annealing at 62 °c for 45 s, elongation at 72 °c for 30 s, final elongation at 72 °c for 10 min and hold at 4 °c (gene amp pcr system 9700, applied biosystems). each set of pcrs included a positive control containing 1 μℓ purified genomic dna of e. ruminantium (welgevonden) and a negative control containing 5 μℓ distilled water. ten microliter of each sample was separated on a 1 % agarose gel with molecular mass marker phix74. sequencing the pcr amplicons from samples showing bands of the expected size were purified with a high pure pcr product purification kit (roche) and sequenced using an abi prism 3100 automatic dna sequencer (bigdye terminator cycler sequencing kit, perkin elmer applied biosystems) with primers hh1f, hh2r (van heerden et al. 2004), and the data was assembled in gap 4 (staden, beal & bonfield 2000) and analysed using clustalx (thompson, gibson, plewniak, jeanmougin & higgins 1997). results sheep-derived infective culture inoculum leukocytes isolated from the blood of infected sheep were used as infective inoculum. all four south african e. ruminantium stocks were established successfully in ide8 cell cultures by this method. the welgevonden stock was detected in stained smears 9 days after initiation, when leukocytes harvested 14 days after infection were used. in contrast, all attempts to initiate the welgevonden stock in the “conventional” way (byrom et al. 1991), i.e. by incubating plasma from the infected animal together with host cells (ide8), failed (data not shown). during prolonged incubation of the latter ide8 cultures some cytotoxic effects induced by the ovine plasma became manifest as the ph of the medium failed to fall in the same way as it did in untreated controls. df-12 medium was used for these experiments. infection with the blaauwkrans and ball3 stocks of e. ruminantium was detected in stained smears in ide8 cell cultures after 21–29 and 18–25 days, respectively. both stocks were initiated using df-12 medium. leukocytes contained morula-like inclusions, presumably e. ruminantium. these were only demonstrated with the blaauwkrans stock before culture initiation. the results of successful culture initiations are shown in table 1. attempts to infect ide8 cell cultures using the nonile stock were carried out on two successive occasions. the first attempt was carried out on day 13 post infection of the donor sheep when its body temperature was 41.3 °c. a giemsa-stained cytocentrifuge smear prepared from the leukocyte inoculum revealed that more than 99 % of the cells were mononuclear. cultures initiated on this occasion remained negative throughout an observation period of 60 days. in contrast, the initiation experiment carried out the next day revealed a switch to a granulocyte cell distribution pattern, with mononuclear cells being a minor contaminating population only. thirteen days after initiation, infected tick cells were detected in giemsa-stained cytocentrifuge smears prepared from culture supernatant. complete l-15b medium was used for the initiation, but was replaced by df-12 medium 39 days after culture initiation due to unsatisfactory growth of the e. ruminantium organisms. the first subculture was carried out 100 days after culture initiation. these results are summarized in table 2. in vitro infection of ba 886 cells by e. ruminantium organisms derived from ide8 tick cell cultures ide8 cultures infected with the welgevonden stock for 149 days and the blaauwkrans stock for 165 days were used successfully to infect ba 886 cell cultures. when the welgevonden stock was used, both ba 886 cultures were positive 3 days after inoculation as determined by stained smears, whereas the blaauwkrans stock was detected in the two infected cultures on days 9 and 15, respectively. ba 886 cultures infected with both stocks were then subcultured after a further 15 days. the results are summarized in table 3. 124 in vitro isolation of ehrlichia ruminantium from ovine blood into ixodes scapularis (ide8) cell cultures molecular characterization sequence analysis using the pcs20 specific primers confirmed that the e. ruminantium stocks recovered from the tick cell cultures were the same as those which were injected into the donor sheep, showing that the organisms used to infect the sheep were also of the stocks isolated in the respective cultures. discussion the first continuous propagation of e. ruminantium in a tick cell line was achieved by bell-sakyi et al. (2000), who cultivated the gardel stock in the i. scapularis-derived cell line ide8 (munderloh et al. 1994). elementary bodies derived from bovine endothelial cell cultures of several stocks of e. ruminantium were used to establish continuous, infected tick cell cultures (bell-sakyi 2004). however, attempts to infect three different tick cell lines, avl/ctvm13, ide8 and ran/ctvm3, with blood from sheep undergoing clinical responses following experimental e. ruminantium infection were unsuccessful (bellsakyi 2004). similar unsuccessful results were obtained by us when we attempted to infect ide8 cultures using heparinized plasma from infected sheep. however, infective organisms are not only found free-floating in the blood or plasma of an infected animal but also in circulating leukocytes, in which the organisms are able to proliferate. logan, why ard, quintero & mebus (1987) observed e. ruminantium colonies in up to 35 % of neutrophils maintained in vitro for between 18 h and 5 days. further more, leukocytes from infected animals were able to transmit e. ruminantium to naive animals (ilemobade & blotkamp 1978). the experiments in the present study show that leukocytes may also be used to initiate in vitro cultures. in fact, ide8 tick cell cultures can be infected with e. ruminantium directly from the blood of infected sheep, provided leukocytes are used as the inoculum. cultures of all four stocks used in the experiments—ball3, blaauwkrans, nonile and welge vonden—were successfully initiated in ide8 cells using this technique. similar approaches were carried out with ehrlichia canis (ewing, munderloh, blouin, kocan, kurtti 1995) and with the other former ehrlichia species: ehrlichia equi (munderloh, madigan, dumler, goodman, hayes, barlough, nelson & kurtti 1996), the human granulocytic ehrlichiosis (hge) agent (munderloh, jauron, fingerle, leitritz, hayes, hautman, nelson, huberty, kurtti, ahlstrand, greig, mellencamp & goodman 1999), and ehrlichia phagocytophila (woldehiwet, horrocks, scaifer, table 1 infection of ide8 cell cultures using leukocytes isolated from sheep infected with three e. ruminantium stocks e. ruminantium stock number of flasks time to detection of e. ruminantium in ide8 cultures (days) time to first subculture (days) ball3 blaauwkrans welgevonden 2 3 2 18, 25 21, 21, 29 9, 9 63 60 37 table 2 infection of ide8 cell cultures using leukocytes isolated from sheep infected with the nonile stock of e. ruminantium inoculum number of flasks time to first detection of e. ruminantium in ide8 cultures (days) time to first subculture (days) mainly mononuclear cells mainly granulocytes 3 3 – (1) 13 (3) – (2) 100 (1) remained negative for 60 days (2) not applicable (3) detected in a cytocentrifuge smear of pooled samples from the three culture flasks table 3 in vitro infection of ba 886 cells by e. ruminantium organisms derived from ide8 tick cell cultures e. ruminantium stock time in ide8 culture (days) number of flasks time to detection of e. ruminantium in ba 886 cultures (days) time to first subculture (days) welgevonden blaauwkrans 149 165 2 3 3, 3 9, 15,15 15 15 125 e. zweygarth, a.i. josemans & h.c. steyn ross, munderloh, brown, edwards & hart 2002) (all now reclassified as anaplasma phagocytophilum [dumler, barbet, bekker, dasch, palmer, ray, rikihisa & rurangirwa 2001]) which have been successfully isolated from infected blood into i. scapularis cell cultures using leukocytes as infective inoculum. besides neutrophils, in vitro cultured macrophages also revealed the presence of inclusion bodies of e. ruminantium (sahu 1986). interestingly, infection of ide8 cells with the nonile stock using a leukocyte fraction containing almost exclusively mononuclear cells failed, although monocytes/macrophages have been described as containing the organism. in contrast, the nonile stock could be established in ide8 cells using granulocytes from the same infected sheep. ehrlichia ruminantium is regarded as an obligatory parasite of endothelial cells (martinez, sheikboudou, couraud & bensaid 1993) but it has been shown recently that not all e. ruminantium isolates can be initiated in endothelial cells. different types of host cell were required to isolate these organisms which have previously resisted all attempts at isolation in the conventional way (zweygarth, josemans, van strijp, van heerden, allsopp & allsopp 2002). consequently, isolating e. ruminantium using only endothelial cells limits the probability of success. here we show that ide8 cells can be used as an alternative to conventional methods. tick cell cultures may thus increase the chances of isolating organisms which do not generally grow in endothelial cells as the primary culture. both the welgevonden and blaauwkrans stocks in ide8 cells, which were used to inoculate ba 886 cells, not only infected them but also gave rise to continuous infected mammalian cell culture lines. similar results were reported by bell-sakyi et al. (2000). however, their ide8 cultures were not consistently infective for bovine pulmonary artery endothelial cells. the fact that endothelial cells can be infected with ide8-derived organisms makes the system very valuable, especially when cultures cannot be directly initiated in endothelial cells. infected ide8 cells can thus be used as a stable source of inoculum to identify suitable mammalian cell lines that support growth of the organisms. it has been reported recently that the kümm isolate, which resisted all attempts at conventional in vitro culture, was isolated in culture in non-endothelial cells (zweygarth et al. 2002). unlike others (bell-sakyi et al. 2000; bekker et al. 2002; bell-sakyi 2004), who only used a l-15b-based medium for the propagation of e. ruminantium in ide8 cultures, we also used df-12 medium, which was originally devised for the propagation of e. ruminantium in mammalian cell cultures at 37 °c (zweygarth et al. 1997). df-12 medium was a suitable alternative 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [595.276 841.890] >> setpagedevice oluwayelo_353-357.indd introduction chicken anaemia virus (cav) causes an economically important clinical and subclinical disease that is characterized by aplastic anaemia, thymus atrophy, intramuscular and subcutaneous haemorrhages, and immunosuppression in young chickens (mcnulty 1991). the virus is found in the organs (yuasa, taniguchi, imada & hihara 1983) and peripheral blood cells (imai, mase, tsukamoto, hihara & yuasa 1999) of infected chickens. its genome consists of circular single-stranded dna of 2.3 kb that has three partially overlapping open reading frames coding for proteins of 52 (vp1), 24 (vp2) and 14 (vp3) kda (noteborn, de boer, van roozelaar, karreman, kranenburg, vos, jeurissen, hoeben, zan tema, koch, van ormondt & van der eb 1991). it is possible to differentiate naturally occurring cav isolates using monoclonal antibody (mab) reactivity (mcnulty, mackie, pollock, mcnair, todd, mawhinney, connor & mcneilly 1990) and dna sequence determination (renshaw, soine, weinkle, o’connell, ohashi, watson, lucio, harrington & schat 1996). since the genome sequence of cav was first published (noteborn et al. 1991), several low and highpassage cav isolates have been sequenced (meehan, todd, creelan, earle, hoey & mcnulty 1992; renshaw et al. 1996; islam, johne, raue, todd & mul ler 2002). backyard chickens (gallus gallus domesticus), which constitute about 80 % of the estimated 150 million 353 onderstepoort journal of veterinary research, 75:353–357 (2008) research communication sequence and phylogenetic analysis of chicken anaemia virus obtained from backyard and commercial chickens in nigeria d.o. oluwayelu1, d. todd2 and o.d. olaleye3 abstract oluwayelu, d.o., todd, d. & olaleye, o.d. 2008. sequence and phylogenetic analysis of chicken anaemia virus obtained from backyard and commercial chickens in nigeria. onderstepoort journal of veterinary research, 75:353–357 this work reports the first molecular analysis study of chicken anaemia virus (cav) in backyard chickens in africa using molecular cloning and sequence analysis to characterize cav strains obtained from commercial chickens and nigerian backyard chickens. partial vp1 gene sequences were determined for three cavs from commercial chickens and for six cav variants present in samples from a backyard chicken. multiple alignment analysis revealed that the 6 % and 4 % nucleotide diversity obtained respectively for the commercial and backyard chicken strains translated to only 2 % amino acid diversity for each breed. overall, the amino acid composition of nigerian cavs was found to be highly conserved. since the partial vp1 gene sequence of two backyard chicken cloned cav strains (ngr/cl-8 and ngr/cl-9) were almost identical and evolutionarily closely related to the commercial chicken strains ngr-1, and ngr-4 and ngr-5, respectively, we concluded that cav infections had crossed the farm boundary. keywords: chicken anaemia virus, cloning, sequence analysis 1 department of veterinary microbiology and parasitology, university of ibadan, ibadan, nigeria 2 veterinary sciences division, department of agriculture and rural development for northern ireland, stormont, belfast bt4 3sd, united kingdom 3 department of virology, college of medicine, university of ibadan, ibadan, nigeria accepted for publication 14 august 2008—editor 354 chicken anaemia virus obtained from chickens in nigeria chicken population in nigeria, have been implicated in the epidemiology of some viral diseases of poultry in the country (abdu 1988; ohore, ozegbe, emik pe & okojie 2002) and concerns have been express ed about their ability to harbour cav. recent serologic studies on commercial and backyard chick ens in nigeria revealed a high prevalence of cav in fection (owoade, oluwayelu, fagbohun, ammer laan, mulders & muller 2004; emikpe, oluwayelu, ohore, oladele & oladokun 2005; oluwayelu 2006). while ducatez, owoade, abiola & muller (2006) reported sequence diversity of nigerian commercial chicken cavs, they did not investigate backyard chicken flocks. in the present study, we report the partial sequencing of the vp1 gene of three nigerian commercial cav strains, and the cloning and sequencing of cav dna obtained from nigerian backyard chickens. the nucleotide and amino acid sequences of the commercial cav strains were com pared with those of the backyard chicken cloned cav strains to determine the extent of genetic diversity between cavs circulating in the two types of chickens. materials and methods dna was extracted from pooled liver and thymus tissues of commercial chickens as previously described (oluwayelu, todd, ball, scott, oladele, emikpe, fagbohun, owoade & olaleye 2005). since tissue samples from infected backyard chickens were unavailable, sera of backyard chickens that were positive for cav antibodies by a modified blocking enzymelinked immunosorbent assay (elisa) were selected and dna extracted from them. poly merase chain reaction (pcr) was performed to amp lify a fragment in the vp1 gene of cav using primers and protocol as already described (olu wa yelu et al. 2005). amplified cav dna fragments from one of the cav antibody-positive backyard chicken sera were cloned into the plasmid vector, pcr® 2.1 topo® and trans formed into top 10 e. coli using topo ta cloning® kit (invitrogen). plasmid dnas of 12 selected colonies were purified using the wizard® plus minipreps dna purification kit (promega) and subsequently digested with ecori enzyme. the pcr products of plasmids containing the inserts of interest were then sequenced. the three cav strains from commercial chickens, designated ngr-1, ngr-4 and ngr-5 were sequenced bidirectionally using the bigdye® terminator v3.1 cycle sequencing kit (applied biosystems). amplification was done using the pcr primers as sequencing primers and the following thermal cycling conditions: 96 °c for 1 min, and then 25 cycles, each with 96 °c for 10 s, 50 °c for 5 s and 60 °c for 4 min. sequencing was carried out in an automated abi 3100 dna sequencer (applied biosystems). purified dnas of the six backyard chicken clones that were positive for the cav insert following ecori digestion were similarly sequenced. they were designated ngr/cl-1, ngr/cl-2, ngr/cl-5, ngr/cl-7, ngr/cl-8 and ngr/cl-9. sequence data obtained were analysed using the vector nti advance 9 software (invitrogen). phylogenetic and molecular evolutionary analyses were conducted using mega version 4 (tamura, dudley, nei & kumar 2007). the genbank accession numbers of the nigerian commercial chicken and backyard chicken cloned cav strains, as well as those of the 11 reference cav isolates are shown in table 1. results and discussion amplification of dna extracted from the commercial chicken tissues and backyard chicken sera yielded 733 bp cav-specific bands. alignment of the nucleotide sequence of the nigerian cav strains with those of reference isolates demonstrated 92–100 % sequence identity, with ngr/cl-1 and ngr/cl-7 strains having 100 % identity. with a maximum diversity of 6 %, the nigerian commercial chicken cav strains were more diverse than the backyard chicken cloned strains that had a maximum diversity of 4 %. while ngr-4 had the highest percentage identities with the backyard chicken cloned strains, there were high levels (98 %) of nucleotide identity between ngr-1 and a malaysian strain (smsc-1) and between ngr-4 and the bangladeshi virus (bd-3). alignment of the deduced amino acid sequences of all nine nigerian strains showed an overall sequence identity of 97–100 % while comparison of the sequences of the nigerian and 11 reference cav strains revealed that this fragment of the vp1 gene appeared to be highly conserved as only seven of the 20 strains compared had single amino acid substitutions not shared by others (table 2). phylogenetic analysis of the nucleotide sequences of the 20 cav strains showed four major clusters (fig. 1), with the backyard chicken cloned strains forming a unique cluster with cux-1m, cux-1n and cloned isolate 10. also, phylogenetic analysis of the amino acid sequences of the 20 cav strains showed four major clusters (data not shown). to date, there is no information about the characteristics of the cav strains that infect avian species 355 d.o. oluwayelu, d. todd & o.d. olaleye fig. 1 phylogenetic relationship among nigerian and reference cav strains based on nucleotide sequence of a fragment of the vp1 gene table 1 cav sequences used for multiple alignment analysis strain geographical origin genbank accession no. ngr-1 ngr-4 ngr-5 ngr/cl-1 ngr/cl-2 ngr/cl-5 ngr/cl-7 ngr/cl-8 ngr/cl-9 cux-1m cux-1n bd-3 smsc-1 a2 82-2 lf4 l-028 26p4 pallister cloned isolate 10 (cli 10) nigeria nigeria nigeria nigeria nigeria nigeria nigeria nigeria nigeria germany germany bangladesh malaysia japan japan china usa usa australia uk aj893509 aj893510 aj893511 am279653 am279654 am279655 am279656 am279657 am279658 m81223 m55918 af395114 af285882 ab031296 d31965 ay839944 u69549 d10068 s71488 u66304 ngr/ci-5 cux-1m/germany ngr/ci-8 cux-1n/germany ngr/ci-2 ngr/ci-7 ngr/ci-1 cii 10 ngr/ci-9 bd-3/bangladesh smsc-1/malaysia ngr-5 ngr-4 ngr-1 26p4/usa lf4/china 82-2/japan a2/japan pallister/australia l-028/usa 31 79 55 56 41 29 27 65 56 48 46 40 57 61 0.002 356 chicken anaemia virus obtained from chickens in nigeria other than commercial chickens. serologic surveys have shown that cav infection is prevalent in backyard chickens in nigeria (emikpe et al. 2005; oluwayelu 2006). the cav strains that infect backyard chickens may thus provide a basis for better understanding of the epidemiology of chicken infectious anaemia (cia). this study, which is the first report on cavs from backyard chickens in africa, is based on the genetic diversity of a fragment of the vp1 gene of three commercial and six backyard chicken cloned cav strains. the detection of 733 bp cav dnas by the pcr in these chickens confirmed the presence of cav in the chickens. the 6 % and 4 % nucleotide diversity obtained for the nigerian commercial and backyard chicken strains respectively translated to only 2 % diversity for both types of chickens at the amino acid level. since ngr/cl-1 and ngr/cl-7, which were 100 % identical at the nucleotide level were also 100 % identical at the amino acid level, it is likely that they are the same cav strain. ngr-4 and ngr-5 that did not grow in mdcc-msb1 cells (oluwayelu et al. 2005), formed a cluster with bd-3, which also did not grow in mdcc-msb1 cells in a previous study (islam et al. 2002) but the molecular basis for this has not yet been investigated. renshaw et al. (1996) suggested that 139q and/or 144q affected the rate of replication or spread of infection in mdcc-msb1 cells. it is likely that ngr-4 and bd-3 on one hand, and ngr-1 and smsc-1 on the other, share common evolutionary origins. since vaccination against cav is not done in nigeria at present, it is possible table 2 amino acid differences between nigerian and reference cav strains (numbering according to meehan et al. 1992.) amino acid position strain 251 254 265 275 287 290 294 299 321 322 357 364 370 consensus q e n y a a q n a r v i s ngr-1 r · t · t · · · · q · · · ngr-4 r · t · · · · · · q · · t ngr-5 r · t c · · · · · q · · · ngr/cl-1 · g t · · · · · · q · · · ngr/cl-2 · · · · · · · · · q · t · ngr/cl-5 · · · · · · · · · q m · · ngr/cl-7 · g t · · · · · · q · · · ngr/cl-8 · · · · · · · · · · · · · ngr/cl-9 · · · · · · · · · q · · · cux-1m · · · · · · · · r q · · · cux-1n · g t · · · · · · q · · · bd-3 r · t · · · · · · q · · t smsc-1 r · t · t p p · · q · · · a2 r · t · s · · · · q · · g 82-2 r g t · s · · · · q · · g lf4 r · t · s · · · · q · · · l-028 r · t · · · · · · q · · · 26p4 r g t · t · · · · q · · · pallister r · t · s · · y · q · · g cli 10 · q t · · · · · · q · · · amino acid residues similar to that of the consensus are indicated as dots (·) 357 d.o. oluwayelu, d. todd & o.d. olaleye that these cav strains were introduced into the country through importation of infected poultry, poultry vaccines or other biologicals. the close association of the nigerian backyard chicken cav strains with the commercial chicken strains in terms of genetic relatedness is an indication that cav infection is not restricted to the farm premises. the virus may have spread from the farm to the backyard chickens, or vice versa. the practice of culling and selling spent layers, some of which may be harbouring the virus and ultimately end up as backyard chickens, may contribute to dissemination of this virus in the field. the detection of six distinct cav variants from backyard chickens in this study, coupled with the fact that nigerian backyard chickens contain a mixed population of different breeds, suggest a need for further investigation of cav sequence diversity in these chickens and an evaluation of the specific economic losses caused by backyard chicken cav strains to the nigerian poultry industry. acknowledgements the authors gratefully acknowledge the john d. and catherine t. macarthur foundation for supporting this work with a staff development grant awarded to d.o. oluwayelu. we also thank mr c.a. latunji and dr a. ayinmode of the departments of zoology and veterinary microbiology and parasitology, university of ibadan, respectively, for their assistance in the phylogenetic analysis. we thank drs o.a. ola dele and b.o. emikpe of the faculty of veterinary medicine, university of ibadan, who assisted with the collection of samples. references abdu, p.a. 1988. infectious bursal disease in a flock of broilers and local nigerian chickens. bulletin of animal health and production in africa, 36:269–271. ducatez, m.f., owoade, a.a., abiola, j.o. & muller, c.p. 2006. molecular epidemiology of chicken anemia virus in nigeria. archives of virology, 151:97–111. emikpe, b.o., oluwayelu, d.o., ohore, o.g., oladele, o.a. & oladokun, a.t. 2005. serological evidence of chicken anaemia virus infection in nigerian indigenous chickens. onderstepoort journal of veterinary research, 72:101– 103. imai, k., mase, m., tsukamoto, k., hihara, h. & yuasa, n. 1999. persistent infection with chicken anaemia virus and some effects of highly virulent infectious bursal disease virus infection on its persistency. research in veterinary science, 67:233–238. islam, m.r., johne, r., raue, r., todd, d. & muller, h. 2002. sequence analysis of the full-length cloned dna of a chicken anemia virus (cav) strain from bangladesh: evidence for genetic grouping of cav strains based on the deduced vp1 amino acid sequences. journal of veterinary medicine (b), 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ozegbe, p.c., emikpe, b.o. & okojie, v.e. 2002. a survey of antibodies to newcastle disease virus in apparently healthy nigerian indigenous chickens (gallus gallus domesticus) in ibadan using elisa. african journal of clinical & experimental microbiology, 3:38–40. oluwayelu, d.o., todd, d., ball, n.w., scott, a.n.j., ola dele, o.a., emikpe, b.o., fagbohun, o.a., owoade, a.a. & olaleye, o.d. 2005. isolation and preliminary characterization of chicken anemia virus from chickens in nigeria. avian diseases, 49:446–450. oluwayelu, d.o. 2006. isolation and characterization of chick en infectious anemia virus in southwestern nigeria. ph.d. thesis, university of ibadan. owoade, a.a., oluwayelu, d.o., fagbohun, o.a., ammer laan, w., mulders, m.n. & muller, c.p. 2004. serologic evidence of chicken infectious anemia in commercial chicken flocks in southwest nigeria. avian diseases, 48: 202–205. renshaw, r.w., soine, c., weinkle, t., o’connell, p.h., ohashi, k., watson, s., lucio, b., harrington, s. & schat, k.a. 1996. a hypervariable region in vp1 of chicken anemia virus mediates rate of spread and cell tropism in tissue culture. journal of virology, 70:8872–8878. tamura, k., dudley, j., nei, m. & kumar, s. 2007. mega4: molecular evolutionary genetics analysis (mega) software version 4.0. molecular biology and evolution, 24:1596–1599. yuasa, n., taniguchi, t., imada, t. & hihara, h. 1983. distribution of chicken anemia agent (caa) and detection of neutralizing antibody in chicks experimentally inoculated with caa. national institute of animal health quarterly (japan), 23:78–81. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice luseba.indd introduction traditional medicine is part of the indigenous knowledge systems of people all over the world. traditional practices, used to keep their animals healthy and productive, and to treat and control diseases, constitute ethnoveterinary medicine (evm) (mathiasmundy & mccorkle 1989). traditional medicine is, in many rural areas of the republic of south africa (rsa), sometimes the only available alternative to expensive or unavailable modern orthodox health care for the management of both human and animal health. the high cost of pharmaceutical products and lack of access to veterinary services are significant reasons for farmers to use non-conventional medicines. small-scale farmers use plants extensive ly for the treatment of livestock diseases (cun ningham & zondi 1991; masika, sonandi & van averbeke 1997a, b; dold & cocks 1999, 2001; masika, van averbeke & sonandi 2000; van der merwe, swan & botha 2001). it is a common thread running through most traditional medicinal systems in africa (iwu 1994). in a previous ethnobotanic survey in this same study area, liengme (1981) recorded 32 medicinal plant species but only two of these plants, i.e. pterocarpus angolensis (fabaceae) and sarcostemma vim inale (asclepiadaceae) were reported as evm remedies in that study. other authors (mashabane, madzibane & potgieter 2000; kloppers 2001) also did not report any evm implications. this study is part of a regional project, the southern african development community (sadc) evm pro115 onderstepoort journal of veterinary research, 73:115–122 (2006) ethnoveterinary medicine practices among tsonga speaking people of south africa d. luseba1 and d. van der merwe2 abstract luseba, d. & van der merwe, d. 2006. ethnoveterinary medicine practices among tsonga speaking people of south africa. onderstepoort journal of veterinary research, 73:115–122 rapid rural appraisal methods were used to collate and code the indigenous knowledge on animal healthcare of tsonga speaking people of south africa. there was a rapport between local disease names as described by their clinical signs by the farmers and the local veterinary services important disease list. the perceived causes of diseases were physico-biological elements and no reference to ancestral guidance was recorded. males and old people were more knowledgeable but females and young people did show a certain degree of confidence during general discussions. plants were more frequently used than other non-conventional remedies with cattle being the most treated animals. farmers reported using 19 plant species belonging to 12 families. plants were collected from the wild when needed and no specific storage system was used. they were administered as decoctions or infusions of single plants. these remedies were used not only as alternatives to expensive pharmaceutical products but also because in certain diseases or chronic cases, they were thought to be more efficacious. keywords: ethnoveterinary medicine, medicinal plants, tsonga, south africa 1 agricultural research council, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110, south africa e-mail: lusebad@arc.agric.za 2 college of veterinary medicine, north carolina state university, 4700 hillsborough street, raleigh, nc, 27606, usa accepted for publication 13 march 2006—editor 116 ethnoveterinary medicine practices among tsonga people of south africa ject. it was perceived that no specific study had been done on the existence, purpose and effectiveness of indigenous veterinary remedies of the tsonga eth nic group in south africa. the present study was aimed at collecting, collating, validating and coding information pertaining to the practices of traditional veterinary medicine among the tsonga people. the tsongas are a minority ethnic group in the rsa. they are primarily rural farmers in the limpopo province bordering mozambique, who grow crops and keep cattle and goats (liengme 1981). materials and methods study area the study was conducted in the greater giyani municipality, which forms the biggest part of the former gazankulu homeland in the eastern part of the limpopo province of south africa (fig 1). the vegetation of the area includes two vegetation types in the savanna biome i.e. mixed lowveld bushveld and mopane bushveld (low & rebelo 1998). the area has a wet and hot summer with a mean temperature fig. 1 maps of south africa and the greater gi yani muni ci pal ity, lim po po prov ince �������������������� � �� � �� 117 d. luseba & d. van der merwe of 30 °c, and a dry and cool winter with a mean temperature of 18 °c. methodology rapid rural appraisal (rra) methods were used (beebe 1995). meetings with the traditional leader and the state veterinary service officers were conducted at the onset to explain the purpose of the research. oral interviews were thereafter conducted with farmers (approximately 200) between march 2002 and august 2003 at different communal dipping tanks in groups or individually. two traditional healers were interviewed individually. the interviews were conducted through a translator who in most of the cases was one of the local veterinary technicians. a general feedback session attended by 73 people was organized in march 2004 in order to correct, harmonize and share the information among farmers. key areas of investigation and discussions were the farmer’s socio-economic profile, animal husbandry, and local knowledge in animal healthcare (ethno-aetiology, ethno-diagnostics, treatments and disease control). detailed information on plants used was recorded including local name, indications, preparation, administration and dosage where applicable. this information was captured in an ms access based database called ovi evm mobile survey© developed at the onderstepoort veterinary institute (ovi). plant collection plants were collected under the guidance of the respondents. botanical data were collected using a collection form prescribed by the south african national biodiversity institute (sanbi). notes were also taken from discussions with respondents. pictures of the plants were taken with a digital camera and precise coordinates of the locations were taken with a gps instrument (etrex garmini™). three specimens of each species were collected, labelled and pressed according to the methods of fish (1999). one specimen was sent to the sanbi for identification, and two specimens were mounted and preserved in the herbarium at ovi. approximately 2 kg of fresh plant materials were collected, dried in the shade and stored in darkened glass jars for subsequent laboratory investigations. results and discussion farmers’ profile most of the respondents (70 %) were males above the age of 40. however, a considerable number of females and young males participated in the group discussions during interviews at the dipping tanks and the workshop. women were generally accepted at the dipping tanks contrary to practice among other ethnical groups such as the vhavenda (mabogo, personal communication 2004). the farmers in giyani and malamulele areas keep several domestic animal species including, in decreasing order of importance, cattle, goats, chickens, sheep, dogs and donkeys. traditional healers were generally not consulted for animal healthcare. they were rarely consulted to find lost animals or for protecting animals against witchcraft. most of the farmers (87 %) learnt about traditional treatments from relatives and other farmers. there was very little reference to ancestral guidance as occurs in traditional human medicine. farmers were interested in knowing the medicines used by others and they were all willing to share the information. this is contrary to traditional healers, who tend to keep their knowledge to themselves, as it is the source of their livelihood. the results of this research and our laboratory findings on the validation were keenly awaited by the respondents and other farmers. ethnoveterinary indications the major disease problems in the investigated region, as reported by the state veterinary service, were high calf mortality, retained placenta, worm infection, lumpy skin disease, sweating sickness in calves, blackquarter, footrot and abscesses caused by ticks, heartwater (cowdriosis) and eye infection (c. mabaso, personal communication 2001). cattle were the most commonly treated, followed by goats, chickens and sheep. van der merwe et al. (2001) reported similar results for tswana speaking people. there was almost no mention of treating dogs, cats and donkeys. this is probably because rural africans do not generally keep animals as pets and because non-production animals are perceived as being more resistant than humans to different kind of ailments. production animals are also more important because of their socio-economic importance in the african’s life. this has far reaching implications on training of animal healthcare officers in the country, and service rendering to african communities needs to be focused on production animals. perceived causes and diagnoses from the description of disease signs by farmers and known disease patterns, major livestock diseases could be coded (table 1). however, it was difficult to accurately match the indigenous names of the dis118 ethnoveterinary medicine practices among tsonga people of south africa eases with the conventional ones as noted previously by mathias-mundy & mccorkle (1989). the causes of disease and patterns were not always clearly defined but physico-biological elements were considered as major causes of diseases as demonstrated elsewhere (van der merwe et al. 2001). the farmers considered drinking dirty water as a cause of redwater (bovine babesiosis). according to farmers, heartwater is suspected when an over-excited animal runs and suddenly falls dead with froth from the mouth. it was thought to be caused by excessive accumulation of blood in the head. this is why this condition was also treated traditionally by cutting a small edge of the animal’s ear to let the blood flow. it was also thought that tick borne diseases such as heartwater and redwater are propagated through tick saliva. tick bites were recognised by farmers as the biggest causes of wounds. wounds were well defined and treated with irritant plants. blackquarter was suspected when the animal failed to wake up and was limping and, sometimes, showed swellings on the legs. these clinical signs were reported to occur in spring or after frost in winter. internal parasite infection was strongly linked to diarrhoea and lack of appetite but differentiating nematode infections from tapeworms or flukes were often non-specific and difficult to categorize. newcastle disease in chickens was matched with signs of greenish diarrhoea and paralysis occurring when seasons change. there was no mention of supernatural causes of animal diseases and no rituals were observed when diagnosing or treating livestock. plant use and veterinary consistency plants were predominantly used in the treatment of livestock diseases compared to other traditional remedies (table 2). data on plant usage were analysed according to the criteria of veterinary consistency as defined by kansonia & ansay (1997). there is consistency when the same plant genus or family is mentioned at least twice for treating the same illness. nineteen species belonging to 12 plant families were reported, the most common families being euphorbiaceae and fabaceae with four plants each. plants mentioned by traditional healers (balanites mau ghamii (balanitaceae), combretum paniculatum (combretaceae), diospyros mespiliformis (ebeneceae), dombeya rotundifolia (sterculiaceae), and ele phantorrhiza elephantina (fabaceae) were unknown to farmers individually, but they were able to identify the plants from digital pictures during the feedback session. plants were not processed or mixed with other materials in most cases. they were used as single plant decoctions or infusions for dosing animals or crushed and used topically for wound treatment. this is in contrast to traditional healers who frequently use complex mixtures. this agrees with findings in madik we, northwest province (van der merwe et al. 2001) but contradicts those of masika et al. (2000) in the eastern cape province. farmers gave many reasons why ethnoveterinary medicines are still in use including the claim of the absence of side effects or being more efficacious than pharmaceuticals. the farmers also indicated that traditional medicines are preferred because there are no restrictions or withdrawal periods for consumption of meat from treated animals. in general, traditional medicines were used as second choice after pharmaceutical medicines, for chronic cases and when pharmaceutical medicines were ineffective. tsonga people make lesser use of phytotherapy as shown by the shorter list of 19 plants recorded in this study when compared to that of other ethnic groups i.e. zulu who use more than 40 species (cunningham & zondi 1991), the xhosa who use 53 species (dold & cocks 2001) and the batswana who use approximately 50 species (van der merwe et al. 2001). ethnically, tsongas are a minority group in south afri ca and it is assumed that loss of tradition in a minority group is greater than in bigger groups. young people are not keen to use evm, probably due to lack of information and interest or to rural exodus as suggested by van der merwe et al. (2001). furthermore, the study area is a buffer zone for the kruger national park. it is a national policy to effectively prevent the transfer of controlled diseases, such as foot–and-mouth disease, from certain game carrier species to livestock. for many decades, dipping to control ticks and other veterinary services table 1 list of tsonga important animal disease names and corresponding conventional names local name conventional name xihlakahla/ nyongwa shimeme foloja jenejene dzohana matsumba mbuphye gugumagala gallsickness (anaplasmosis) heartwater abortion newcastle disease (ncd) blackquarter wounds, lumpy skin disease warts bottle jaw 119 d. luseba & d. van der merwe have been provided free of charge which have contributed to lesser use of ethnoveterinary medicines. the availability of a plant per se does not seem to influence its uses more than traditional and sociocultural characteristics and inheritance. some common plants occurring in the region (liengme 1981) that were frequently used by many other ethnic groups were not reported. to name a few of these, plants such as acacia tortilis (fabaceae), albizia versicolor (fabaceae), brachylaena discolor (asteraceae), combretum erythrophylum (combretaceae), dicrostachys cinerea (fabaceae), grewia occidentalis (tiliaceae), leonotis spp. (lamiaceae), lippia ja vanica (verbenaceae), ozoroa reticulata (anacardi aceae), sclerocarya birrhea (pedaliaceae) and zizi phus mucronata (rhamnaceae) were not reported. there were three similar usages of evm plants among tsongas and other ethnic groups. dicerocary um eriocarpum (pedaliaceae) is used by both tsongas and batswana (van der merwe et al. 2001) for dystocia and retained placenta. aloe zebrina (asphodelaceae) is used for wounds or burns and e. elephantina for heartwater. more plants were used in common with vhavenda who are living in a much closer region (ovi evm mobile survey©, unpublished data, 2003). these are aloe marlothi, cassia abbreviata (fabaceae), cissus quandrangularis (vitaceae), d. eriocarpum, d. rotundifolia , e. elephantina, jatropha curcas (euphorbiaceae), p. angolensis, s. viminale, senna italica (fabaceae), solanum lichtensteinii (solanaceae), synadenium cupulare (euphorbiaceae) and terminalia sericea (combretacae). mode of action, preparation and storage the mode of action of plants was not always well explained by farmers. in case of general illness, a plant supposedly stimulates blood circulation and would act as a cleansing factor. the doctrine of signature (i.e. plant that produces a red decoction is given for a disease that in its signalment shows red, such as haemoglobinuria with babesiosis) was also present. for instance, d. eriocarpum is used both topically as a lubricant during dystocia and per os in the treatment of retained placenta because of the soapy character of the infusion. water was exclusively used as a solvent, but there was no precise amount of plant material per volume of water. qualitative estimates such as colour change (red colour for p. angolensis and e. elephantina) of the liquid after a certain period when plant material is soaked or the formation of a slippery infusion (d. eriocarpum) are used to determine adequate concentration of preparations. it is, therefore, difficult to standardize such remedies and make objective recommendations. however, a 1 l bottleful and a big horn are often used for dosing adult cattle, while amounts of a quarter to a half bottleful are used for calves. plants were collected when needed in the wild but medicines that were perceived to be essential, such as roots of jatropha zeyheri (euphorbiaceae) and ground bark of p. angolensis, were often stored. farmers were aware that medicinal plants are getting scarce and would indiscriminately use fresh materials from nurseries—contrary to general practice in human traditional medicine (van der merwe et al. 2001). they also indicated that fresh material was more effective than dried material. traditional healers store large numbers of medicinal plants and recommend that plant materials be ground and stored in preferably dark bottles. they acknowledged that ground plant material might lose efficacy after a year. bundled, intact medicinal plants were stored in shade for longer periods. some medicines were stored in a refrigerator, but were not allowed to freeze. table 2 list of plant family and botanical names followed by tsonga names, plant part (s) used, indication(s), preparation and dosage where applicable family and botanical name tsonga name indication plant part preparation asclepiadaceae sarcostemma viminale neta wound aerial part grind and apply directly on the wound. very effective against maggots asphodelaceae aloe marlothii mhangani newcastle disease(ncd) leaves leaves are crushed and the juice is mixed with drinking water for treating chickens against ncd. it is also planted in the yard to protect the household against lightning strike asphodelaceae aloe zebrinae chovoloti wound leaves succulent fresh leaves are crushed and applied on wound; it is good against maggots 120 ethnoveterinary medicine practices among tsonga people of south africa family and botanical name tsonga name indication plant part preparation balanitaceae balanites maughamaii nulu diarrhoea leaves grind the leaves and mix with cold water. dose with 1 l bottle for adults and a 340 ml bottle for calves. after treatment, animals are housed only after they have eaten combretaceae combretum paniculatum mpfunta for fertility problems root bark grind root bark, make a decoction, sieve, dose approximately 500 ml. use only when animal is not fertile, give twice at 2 days, interval combretaceae terminalia sericea konono wound leaves grind leaves, mix with water and apply on the wound; cover with cattle dung ebeneceae diospyros mespiliformis ntoma for milk production bark grind bark and mix with hippopotamus fat; dose and also rub into vagina euphorbiaceae euphorbia cooperi mokhoto/nkonde blackquarter aerial part crush succulent stems, mix with water and drench the animal. it is also planted in the yard to prevent lightning strike euphorbiaceae jatropha curcas nhlampfura constipation seeds crush one or two seeds, mix with water and drench the animal for treatment of constipation in cattle and goats euphorbiaceae jatropha zeyheri xidomeja/ mudomeja general ailments, illthrift, and diarrhoea roots together with p. angolensis, they are the most used plants. fresh or dry roots are ground, soaked in water and dosed to animals for all sorts of ailments, unthriftiness, and diarrhoea euphorbiaceae synadenium cupulare mdleve eye infection, blackquarter milky latex apply milky latex on the third eyelid for eye infections or on the skin of the limping leg for treatment of blackquarter. protect the eye since the latex can damage it fabaceae cassia abbreviata lumanyama worm infestation bark grind the bark, soak in water overnight or boil, cool, sieve and drench the animals fabaceae elephantorrhiza elephantina xixuvari heartwater, blackquarter and as appetite stimulant or tonic aerial parts and bulb grind and soak plant material in cold water for 6 h. when the water becomes reddish, dose 1 l; give every morning until the animal recovers fabaceae pterocarpus angolensis vhangazi/ murhotso general illness, unthriftiness, gallsickness, intestinal worms, blackquarter bark chop the bark, soak in cold water, dose once only with bottle or horn (approximately 750 ml) after the water has changed to reddish or boil for 30–60 min. to prevent “blood diseases” (blackquarter/anthrax?), the potion is given twice annually, when the seasons change fabaceae senna italica ximbangambangana diarrhoea and gallsickness bark grind the bark, soak in water and dose cattle for diarrhoea and gallsickness pedaliaceae dicerocaryum eriocarpum dinda/dindza dystocia, retained placenta aerial part crush the aerial parts and mix with water. it is used as lubricant during difficult calving and drenched (5 l) for retained placenta solanaceae solanum lichtensteinii ndhulwani respiratory problems aerial part crush aerial part and mix with water, instill the fluid in the nostril to clear up the airways in case of upper respiratory problems table 2 (continues) 121 d. luseba & d. van der merwe family and botanical name tsonga name indication plant part preparation sterculiaceae dombeya rotundifolia shilubare/xiluvarhi newcastle disease leaves and flowers grind leaves/flowers and mix with chicken feed during the outbreaks. it is used generally when seasons change to prevent the disease vitaceae cissus quandrangularis nyangala wound treatment, tick repellent, lumpy skin disease aerial parts crush aerial parts, mix with water and use as poultice. it is used to control maggots and ticks. can be used to prevent secondary wound infection due to tick bites. in case of lumpy skin disease, crush the stem, mix with water, red soil (tsumani), and pork fat; smear the whole body table 2 (continues) other evm remedies other indications included the treatment of gallsickness with an infusion of a portion of ground abomasum of a kudu (tragelaphus strepsiceros). hippopot amus fat was smeared onto the vulva of cows to attract bulls in cases of female infertility. a pinch of powder of ground shell from the large terrestrial snails named wumba, the tsonga name for achatina spp., was applied to the eyeball, usually two times every second day for treating chronic eye infections. mixtures of clay and water or salt and water were used for treating intestinal conditions like diarrhoea. a mixture of salt, potassium permanganate and coca cola™ was used for the same condition. conclusion the tsonga people of south africa practice evm, although to a lesser extent than the vhavenda, batswana and xhosa. however, there are some similar usages of medicinal plants showing a consistency across ethnic groups in the country. women and young people do have a knowledge of evm, and it seems that they participate and learn during open discussions. organizing surveys and workshops during school holidays and when women are not involved in other activities can encourage this. it was also shown that organizing a feedback session at the end of the research period was important. not only was it possible to have a consensus on the results, but farmers also learnt from others. it was also an opportunity to make recommendations such as the appropriate use of medicinal materials, harvesting methods and methods for growing medicinal plants. there is evidence to suggest that farmers have a unique knowledge of disease causes and patterns (masika et al. 1997a), which is not always comparable to scholastic understanding. however, it is often related to the type of treatment used. this should be taken into account in any disease control campaign since people relate easily to what is already embedded in their traditions. it was also shown that production animals are important in rural communities and efforts to improve animal productivity through effective health management are of utmost importance. further studies to explain the rationale behind the use of these remedies should be conducted based on biological and chemical analyses. these should form the bases of recommendations made to communities. acknowledgements the authors are grateful for funding from the department of science and technology, south africa and dr jenny turton for obtaining funding. the co-operation and patience of the communities, especially chief ngobe, mr m. mabunda and the state veterinary services of greater giyani, limpopo province, south africa are gratefully acknowledged. dr c. mabaso is thanked for chairing and helping in the general organization of the feedback session with the communities. references beebe, j. 1995. basic concepts and techniques of rapid appraisal. human organization, 54:42–51. cunningham, a.b. & zondi, a.s. 1991. cattle owners and traditional medicines used for livestock. institute of natural resources, university of natal, pietermaritzburg (investigational report, no. 69). 122 ethnoveterinary medicine practices among tsonga people of south africa dold, a.p. & cocks, m.l. 1999. preliminary list of xhosa plant names from the eastern cape province, south africa. bothalia, 29:267–292. dold, a.p. & cocks, m.l. 2001. traditional veterinary medicine in the alice district of the eastern cape province, south africa. south african journal of science, 97:375–379. fish, l. 1999. preparing herbarium specimens. pretoria: national botanical institute. iwu, m.m. 1994. african medicinal plants in the search for new drugs based on ethnobotanical leads, in ethnobotany and the search for new drugs. chichester: wiley. kansonia, k. & ansay, m. 1997. a recognition of rural knowledge: medicinal plants and traditional veterinary medicine of central africa (testing the traditional veterinary pharmacopoeia), in ethnoveterinary medicine: alternatives for livestock development, proceedings of an international conference held in pune, india: 14–18. kloppers, r.j. 2001. a comparison of plants utilised by tsonga people in south africa and mozambique. southern african ethnobotany: the official newsletter of the indigenous plant use forum, 1:32–41. liengme, c.a. 1981. plants used by the tsonga people of gazankulu. bothalia, 13:501–518. low, a.b. & rebelo, a.g. 1998. vegetation of south africa, lesotho and swaziland, 2nd ed. pretoria: department of environ mental affairs and tourism. mashabane, l.g., madzibane, j. & potgieter, m.j. 2000. a comparative analysis of mopane’s uses between the vatsonga and vhavenda. southern african ethnobotany: the official newsletter of the indigenous plant use forum, 1:11–12. masika, p.j., sonandi, a. & van averbeke, w. 1997a. perceived causes, diagnosis and treatment of babesiosis and anaplasmosis in cattle by livestock farmers in communal areas of the central eastern cape province, south africa. journal of south 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice barson_nematodes.indd introduction the study of parasitic diseases of fish and other aquatic organisms in south africa only started to gain attention over the past few decades as scientists began to realise their significance in fisheries and aquaculture (safriel & bruton 1984; hoffman & prinsloo 1996). only a few researchers have published their work on nematode parasites in the country, and these include prudhoe & hussey (1977), mashego (1977, 1982), mashego & saayman (1981), boomker (1982, 1994a, b), saayman, mashego & mokgalong (1991) and mokgalong (1996). the results of this study will contribute to this existing body of knowledge. the sharptooth catfish, clarias gariepinus (burchell, 1822), the host species investigated in this study, is widely distributed in africa (safriel & bruton 1984; skelton 2001) and is an excellent species for aquaculture and biological research (hoffman & prinsloo 1996). the study was carried out as a reconnaissance survey of the internal parasites that are found in c. gariepinus from the rietvlei dam, which can be used in the fish health assessment index that has been developed for south african fish by avenant-oldewage (2001). the objectives of this paper were to specifically identify and classify the nematode parasites collected from c. gariepinus based on their morphological features, and to note their prevalence and mean intensity in the rietvlei dam. materials and methods study area the rietvlei nature reserve (25°41’22” s; 26°37’48” e) lies between pretoria and johannesburg in gauteng province (fig. 1). developed out of the rietvlei 87 onderstepoort journal of veterinary research, 73:87–94 (2006) nematode parasites of clarias gariepinus (burchell, 1822) from the rietvlei dam, south africa m. barson1, 2 and a. avenant-oldewage1 abstract barson, m. & avenant-oldewage, a. 2006. nematode parasites of clarias gariepinus (burchell, 1822) from the rietvlei dam, south africa. onderstepoort journal of veterinary research, 73:87–94 catfish, clarias gariepinus, from the rietvlei dam near pretoria, south africa were examined for nem atode parasites. two species, procamallanus laeviconchus in the stomach and contracaecum spp. larvae in the abdominal cavity, were found. the morphology of these species, based on light and scanning electron microscopy, and how they compare with previously described specimens are discussed. infection rates were mild compared to previous surveys although contracaecum spp. had a high prevalence of 86 %. keywords: clarias gariepinus, contracaecum, nematode, parasite, procamallanus, rietvlei dam 1 department of zoology, university of johannesburg, kingsway campus, p.o. box 524, auckland park, johannesburg 2006, south africa 2 author to whom correspondence is to be directed. e-mail: barson@science.uz.ac.za. present address: department of biological sciences, university of zimbabwe, p.o. box mp 167, mt pleasant, harare, zimbabwe accepted for publication 10 january2006—editor 88 nematode parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa water scheme, it is solely responsible for conservation of the sesmylspruit catchment area, and the rietvlei dam (25°32’30” s; 28°16’46” e) currently supplies 27 % of pretoria’s water requirements (wessels 1998). a smaller dam, the marais dam, lies approximately 4 km upstream, and the two are separated by a wetland (fig. 1). further upstream, just before the sesmylspruit enters the reserve, the stream receives effluent from a number of industries and a wastewater treatment plant. field collection of fish and parasites and identification fish were collected in may 2003 from the rietvlei dam using large mesh gill nets. the fish were dissected and the mesenteric cavity examined for parasites. the gastrointestinal tract was then dissected from the rectum to the oesophagus and all nematodes encountered were carefully detached from the stomach or intestinal mucosa. the internal organs of each fish were also examined for parasites or cysts. the nematodes were fixed in glacial acetic acid and preserved in 70 % ethyl alcohol. some larval nematodes were stained with horen’s trichome stain according to the method of khalil (1991). specimens for scanning electron microscopy (sem) examination were preserved in absolute alcohol after which they were processed for sem as detailed by barson & marshall (2004). the parasites were identified based on their observed morphology as well as from drawings. light micrographs were taken with a zeiss axioplan microfig. 1 the location of the rietvlei dam inside the rietvlei nature reserve. x indicates point of sampling pretoria rietvlei nature reserve johannesburg rietvlei dam marais dam sesmylspruit south africa 0 0 ,1 0 ,5 1 km 2 km 89 m. barson & a. avenant-oldewage scope, and scanning electron micrographs with a jeol 6100 scanning microscope. drawings and measurements were done with a zeiss 25 standard light microscope equipped with a drawing tube. the descriptions by yamaguti (1961), chabaud (1974), hartwich (1974) and moravec (1975) were used to aid in the identification of the parasites. parasite prevalence and mean intensities were measured and calculated as defined by mar golis, esch, holmes, kuris & schad (1982). voucher specimens were deposited in the zoological collection of the university of johannesburg (formerly rand afrikaans university), south africa. results and discussion two nematode genera were found in c. gariepinus from the rietvlei dam, namely procamallanus laeviconchus (wedl, 1862) and larvae of contracaecum spp. procamallanus laeviconchus (wedl, 1862) (fig. 2 and 3) this is a small ovoviviparous nematode that is prevalent in most african freshwater fishes, notably in sil uroids (khalil 1970; moravec 1975; mashego 1977; mashego & saayman 1981; boomker 1982, 1994a, fig. 2 procamallanus laevionchus female drawings. (a) whole worm. scale bar = 400 μm; (b) anterior end. scale bar = 100 μm; (c) mid-body. scale bar = 200 μm; (d) posterior end. scale bar = 20 μm. a = anus, ap = anal (tail) processes, bc = buccal capsule, ep = excretory pore, go = glandular oesophagus, int = intestine, nr = nerve ring, ov = ovary, ut = uterus, v = vulva, vg = vagina go ov int v ut vg bc ep nr ov int ut ap a a b c d 90 nematode parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa b; chishawa 1991; saayman et al. 1991; douëllou 1992; khalil & polling 1997). one out of seven hosts was infected (prevalence 14 %) with an intensity of 13 nematodes. only female specimens were obtained and described, most of the characteristics matching those described by moravec (1975) and boomker (1982). diagnostic measurements show that the rietvlei dam specimens have the same size range as those described by boomker (1982) although they were somewhat larger with respect to the distance of anus to tail and length of the glandular portion of oesophagus (table 1). this study additionally showed the three-dimension al aspect of the head of p. laeviconchus with the sem (fig. 3f), the buccal capsule of which still fits the fig. 3 procamallanus laevionchus photomicro graphs of female specimen. (a) head region showing buccal capsule; (b) mid-body; (c) motile larvae in region of the vulva (arrow); (d) posterior end with uterus extension full of larvae and eggs; (e) eggs (arrow) ejected from vulva; (f) scanning electron micrograph of head showing three-dimensional structure of buccal capsule (arrow); (g) scanning electron micrograph of anterior end showing annulations and lateral papillae. scale bars: (a)–(d) = 200 μm; (e) = 100 μm; (f)–(g) = 10 μm a b c d e f g 91 m. barson & a. avenant-oldewage description by moravec (1975). microscopical observation showed three terminal tail processes (fig 2d), but these were, however, not observed on the sem photograph of the posterior end of the worm (fig. 3g); the specific specimen was probably still in its fourth larval stage. eggs and motile larvae at various stages of development were observed, some eggs having been squeezed out through the genital opening (fig. 3b and c). measurements of l3 larvae are reflected in table 2 procamallanus laeviconchus is always found deeply attached to the mucosa of pyloric region of the host’s stomach wall and has been shown to cause severe pathological effects (paperna 1996). apart from the finding of mashego & saayman (1981) who recorded a total of 23 worms in one fish, the intensity of 13 worms in one fish from the rietvlei dam is considerably high when compared to the low numbers recorded by boomker (1982, 1994a) from c. gariepinus. moravec (1975) states that p. laeviconchus infection is widespread in many african fish families. in neighbouring zimbabwe, chishawa (1991) and douëllou (1992) recorded it from the clariidae and schilbeidae from lake kariba. in nigeria, opara & okon (2002) reported it from oreochromis niloticus (cichlidae) and yakubu, omoregie, wade & faringoro (2002) from c. gariepinus (clariidae) and ti lapia zilli (cichlidae). khalil (1970) recovered p. laeviconchus from seven fish species from ghana and belonging to the mormyridae, schilbeidae and mochokidae. the list of african helminths by canaris & gardner (1967) includes procamallanus mazabukae yeh, 1957 infecting homa fish from zambia and pro camal lanus spiralis infecting heterobranchus anguilaris (clariidae) from northern africa. the former does not, however, appear in khalil & polling’s (1997) updated checklists, while the latter has probably been renamed as spirocamallanus (santos, cárdenas & lent 1999). however, other procamallanus species in africa are known to infect amphibians (canaris & gardner 1967; anderson 1992). many species of protable 1 comparative diagnostic measurements of procamallanus laevionchus infecting c. gariepinus from the rietvlei dam (this study) and hartbeespoort dam (boomker 1982), and in clarias sp. from the nile river, egypt (moravec 1975) measurement* rietvlei dam (2003, this study) n = 7 nile river (moravec 1975) n = 8 hartbeespoort dam (boomker 1982) n = 5 length (mm) maximum width buccal capsule length buccal capsule width length of muscular part of oesophagus length of glandular part of oesophagus distance of nerve ring from anterior end distance of vulva to anus (mm) distance of anus to tail distance of vulva to tail (mm) 6.2–8.9 (7.6)** 145–280 (222) 60–90 (78) 58–70 (62) 380–510 (441) 960–1020 (983) 105–213 (173) 2.0–3.3 (2.7) 140–340 (227) 2.4–3.4 (2.9) 3.7–7.4 136–204 (69) 57–63 360–516 666–990 189–207 – – 1.8–3.0 7.0–8.9 181–216 83–96 60–70 411–491 775–927 208–226 3.0–3.5 117–138 3.1–3.6 * all measurements in μm unless otherwise stated ** mean values in parentheses n number of specimens measured table 2 diagnostic measurements of contracaecum sp. l3 larvae infecting c. gariepinus from the rietvlei dam measurement range n = 9 mean body length (mm) body width head diameter ventricular appendix length ventricular appendix width intestinal caecum length (mm) intestinal caecum width length of tail from anus to tip 22.0–35.0 680–780 120–160 510–1 040 100–160 1.24–2.2 120–180 130–250 27.6 710 140 790 120 1.72 160 200 * all measurements in μm unless otherwise stated n number of specimens measured 92 nematode parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa fig. 4 contracaecum sp. larvae. (a) head. scale bar = 200 μm; (b) anterior end showing ventricular region. scale bar = 500 μm; (c) posterior end. scale bar = 200 μm. a = anus, ag = anal glands, ic = intestinal caecum, int = intestine, l = lip (labium), nr = nerve ring, o = oesophagus, va = ventricular appendix, ve = ventriculus camallanus infecting freshwater fishes have also been recorded in europe (moravec 1994) and in the neotropical region (santos et al. 1999). contracaecum spp. larvae (fig. 4) third-stage larvae (l3) with two blind caeca branching off from the intestinal tract at the junction of the oesophagus and midgut (fig 4b). ventricular appendix shorter and pointing posteriorly; intestinal caecum longer and pointing anteriorly. tail curved with terminal spine (fig. 4c). reproductive system not fully developed. contracaecum larvae have been recorded from catfish and other fish species from many water bodies nr o ic ic ve va int a ag l o a b c 93 m. barson & a. avenant-oldewage in south africa (whitfield & heeg 1977; mashego & saayman 1981; boomker 1982, 1994a, b; saayman et al. 1991), zimbabwe (chishawa 1991; douëllou 1992; barson 2004), and east africa (malvestuto & ogambo-ongoma 1978; aloo 2001). it is a cosmopolitan parasite of fish-eating birds and mammals (hartwich 1974; anderson 1992) and can reach alarming intensities without affecting the condition of the host (mashego & saayman 1981; boomker 1982; paperna 1996), an adaptation that probably ensures that the larvae survive to reach the final host without killing the intermediate host. contracaecum larvae are difficult to differentiate into species except when using molecular analysis or alternatively infecting experimental hosts to obtain adult worms. adult contracaecum species from fish-eating birds have only been studied and recorded by ortlepp (1938, cited by mokgalong 1996), saayman et al. (1991) and mokgalong (1996) in south africa. canaris & gardner (1967) listed nine adult contracaecum species from african waterbirds, while barson & marshall (2004) recorded four species from zimbabwean birds. while a high prevalence (86 %) and a mean intensity of 16.3 (intensity range 3–44) were recorded from rietvlei dam, 100 % infection levels are very common, with intensities as high as 700–2 000 worms per fish (mashego & saayman 1981; boom ker 1982, 1994a). this makes contracaecum one of the most prevalent fish parasites in south africa and the fact that its life cycle involves migratory bird species (e.g. cormorants) can justify this observation. pa perna (1996) urges aquaculturists to control aquatic birds on fishponds as an effective means of reducing contracaecum infection. acknowledgements we thank the university of johannesburg and the water research fund for southern africa (warfsa) for funding this study, university of johannesburg zo ology technical staff and student assistants for field and laboratory assistance, and spectrau unit and graphics staff for technical support. references aloo, p.a. 2001. occurrence of larval contra cae cum (nematoda: heterocheilidae) in three teleostean species from lake naivasha, kenya. east african journal of science, 3:1–12. anderson, r.c. 1992. nematode parasites of ver te brates: their de velopment and transmission. wal ling ford: cab inter national. avenant-oldewage, a. 2001. protocol for the assessment of fish health based on the health index. report and manual for training of field workers to the rand water board. ver een iging: rand water (report no. 2001/03/31). barson, m. 2004. the occurrence of contracaecum sp. larvae (nematoda: anisakidae) in the catfish clarias gariepinus (burchell) from lake chivero, zimbabwe. onderstepoort jour nal of veterinary research, 71:35–39. barson, m. & marshall, b.e. 2004. first record of contracaecum spp. (nematoda; anisakidae) in fish-eating birds from zimbabwe. journal of the south african veterinary association, 75:74–78. boomker, j. 1982. parasites of south african freshwater fish. i. nematodes of the catfish [clarias gariepinus (burchell, 1822)] from the hartbeespoort dam. onderstepoort journal of veterinary research, 49:41–51. boomker, j. 1994a. parasites of south african freshwater fish. vi. nematode parasites of some fish species in the kruger national park. onderstepoort journal of veterinary research, 61:35–43. boomker, j. 1994b. parasites of south african freshwater fish. vii. nematode parasites of some scaled fishes from the hartbeespoort dam, transvaal. onderstepoort journal of veterinary research, 61:197–199. canaris, a.g. & gardner, s.l. 1967. a guide to helminth species described from african vertebrates. morgantown: west virginia university library. chabaud, a.g. 1974. keys to subclasses, orders and superfamilies, in cih keys to the nematode parasites of vertebrates, edited by r.c. anderson, a.g. chabaud & s. wilmott. slough: commonwealth agricultural bureaux, 1:6–17. chishawa, a.m.m. 1991. a survey of the parasites of three siluriformes [sic] fish species in lake kariba. kariba: uni versity of zimbabwe (university lake kariba research station bulletin, 1/91). douëllou, l. 1992. a survey of fish parasites in lake kariba. kariba: university of zimbabwe (university lake kariba research station bulletin, 1/92). hartwich, g. 1974. keys to genera of the ascaridoidea, in cih keys to the nematode parasites of vertebrates, edited by r.c. anderson, a.g. chabaud & s. wilmott. slough: com monwealth agricultural bureaux, 2:1–15. hoffman, l.c. & prinsloo, j.f. 1996. the potential of freshwater fish in south africa. food industries of south africa, 30: 1–2. khalil, l.f. 1970. on some nematodes from the freshwater fishes of ghana with the description of a new species, spironoura petrei n. sp. journal of helminthology, 46:63–68. khalil, l.f. 1991. techniques for identification and investigative helminthology: techniques for processing platyhelminths and acanthocephalans, in helminthology manual, edited by l.f. khalil. london: international institute of parasitology. khalil l.f. & polling, l. 1997. check list of the helminth parasites of african freshwater fishes. department of zoology/ biology. sovenga: university of the north. malvestuto, s.p. & ogambo-ongoma, a. 1978. observations of the infection of tilapia leucosticta (pisces: cichlidae) with contracaecum (nematoda: heterocheilidae) in lake naivasha, kenya. journal of parasitology, 64:383–384. margolis, l., esch, g.w., holmes, j.c., kuris, a.m. & schad, g.a. 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accepted: 01 dec. 2015; published: 13 june 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a retrospective study covering a period of ten years (2001–2010) was conducted using postmortem meat inspection records of the department of veterinary services in gaborone to determine the prevalence of bovine fasciolosis in botswana. meat inspection records of monthly and annual returns from the two main export abattoirs in the country were examined, as well as the data collected on the total number of cattle slaughtered and the number of livers condemned due to fasciola gigantica infection. only 1250 of the approximately 1.4 million cattle slaughtered were infected with f. gigantica (0.09%, 95% confidence intervals [ci] 0.0% – 0.3%). no distinct seasonal pattern was observed in condemnation rates of livers. however, the pattern of distribution of fasciolosis was higher (but not significant) in cattle that originated from areas with high rainfall and more permanent water bodies than those from relatively low rainfall areas with a transitory water system. it is recommended that a longitudinal survey should be carried out at the abattoirs and farms to determine the prevalence of the disease in cattle of different ages, sex and breed as well as the place of origin in the country. the present study indicated that the prevalence of fasciolosis in cattle is low and the disease is therefore of less significance in botswana than other african countries for which information is available. introduction fasciolosis, also known as liver fluke disease, is a hepatic parasitic infection caused by trematodes of the genus fasciola which affects numerous mammalian species, mainly ruminants (gajewska, smaga-kozlowska & wisniewski 2005) in most countries of the world. the two most important species are fasciola hepatica and fasciola gigantica. these liver flukes are significant pathogens of domestic animals, in particular cattle and sheep (jones, hunt & king 2006). as fasciola are haematophagous, their infection usually results in anaemia (phiri, phiri & harrison 2007) and can cause a high proportion of mortalities, especially in small ruminants and calves (mungube et al. 2006). lymnaeid snails, the intermediate hosts of fasciola spp., play a vital role in the epidemiology and distribution of fasciolosis (coelho & lima 2003; pfukenyi & mukaratirwa 2004). therefore, fasciolosis is prevalent in areas where climatic conditions are favourable for the survival of the snail intermediate host, such as marshland pastures (mcgavin & zachary 2007). in most african countries, the prevalence of fasciolosis in ruminants has been determined through slaughterhouse surveys (mungube et al. 2006; mwabonimana et al. 2009; phiri et al. 2005b). information gathered on animals slaughtered at an abattoir can be a convenient and relatively inexpensive source of information (roberts & suhardono 1996), with the condemnation rates providing a useful guide to the prevalence of the subacute, mild or chronic forms of the disease in the regions served by the abattoirs (pfukenyi & mukaratirwa 2004). the data can be used to determine trends in prevalence and significance of the disease, especially where the reporting system is reliable (roberts & suhardono 1996). in botswana, both the traditional (cattle post) grazing areas and commercial ranches (large fenced farms) send their animals for slaughter to various abattoirs throughout the country. the cattle are usually brought to these abattoirs on foot or by road or rail transport. meat inspection in the abattoirs is carried out independent of the owners. the prevalence of a number of diseases, notably bovine cysticercosis, has been reported from data collected at abattoirs in the country. however, no recorded studies have been carried out to determine the prevalence of fasciolosis in cattle using abattoir records. the objective of the present study was to determine the prevalence of fasciola gigantica infections in slaughtered cattle based on the records from the two main export abattoirs in botswana for the period 2001–2010. materials and methods sampling method the study involved the retrieval and analysis of meat inspection data from two major botswana meat commission (bmc) export abattoirs at lobatse and francistown (figure 1). these are the largest abattoirs in botswana, with wide catchment areas, and are located in the southern and northern parts of the country, respectively. figure 1: map showing location of the two sampled export abattoirs in botswana. the bmc abattoirs are parastatal organisations jointly owned by the government of botswana and private companies, whereas smaller municipal abattoirs are owned by the local governments in the districts. the selected abattoirs covered all regions of the country, and consequently a range of climatic conditions, and serviced both the communal and commercial beef sector farmers, with concurrent different systems of cattle management. data for the ten-year period, from 2001 to 2010, were collected to estimate the prevalence of fasciolosis in cattle in botswana. meat inspection is performed by certified meat inspectors in accordance with the standards of the livestock and meat industries act of 2007 of the republic of botswana, under the supervision of the chief veterinary officer in the department of veterinary services of the ministry of agriculture. data collection and analysis the data were obtained from the two main export abattoirs for the period from january 2001 to december 2010. records of monthly and annual returns from the abattoirs were scrutinised with regard to the number of cattle slaughtered and the corresponding number of livers condemned as a result of infection with f. gigantica. the prevalence of fasciolosis was calculated as the number of cattle infected with fasciola expressed as a percentage of the total number of cattle slaughtered, and was calculated annually for each abattoir. the overall prevalence for the 10-year period (2001–2010) was also determined for each abattoir, together with their 95% ci. data obtained for the prevalence of bovine fasciolosis were entered, validated and calculated in microsoft excel 2007 spreadsheet, and later transferred into ibm statistics programme for social sciences (ibm spss) version 21.0 for windows (ibm corporation, new york, usa) for statistical analysis. the mann–whitney u test was used for the analysis. descriptive statistics were used to calculate the mean and the 95% ci for the different subgroups. results the results of the number of cattle slaughtered and condemned livers in each of the two abattoirs over the 10-year period are displayed in table 1. out of the 929 937 and 464 784 cattle slaughtered at lobatse and francistown abattoirs, 17 and 1232 livers were condemned, respectively. in total, only 1250 livers (0.09%; 95% ci 0.08% – 0.09%) out of almost 1.4 million livers examined were condemned because of f. gigantica infestation. table 1: the number of cattle slaughtered and livers condemned due to fasciola gigantica infections at two export abattoirs in botswana. a 10-year summary to compare the trend in prevalence of bovine fasciolosis between the two export abattoirs is presented in figure 2. the annual prevalence at the francistown abattoir varied between 0% and 1.35%, and at the lobatse abattoir between 0% and 0.01% (figure 2 and table 1). the overall 10-year prevalence of fasciolosis in cattle between the two abattoirs varied between 0.002% at lobatse and 0.265% at francistown. the prevalence at francistown abattoir (0.27%; 95% ci 0.25–0.28) was significantly higher than that at lobatse abattoir (0.002%; 95% ci 0.00–0.00) (p [0.003] < 0.05). figure 2: ten-year (2001–2010) annual trend of the prevalence of fasciolosis in cattle at two export abattoirs in southern and northern botswana. the supply of cattle for lobatse abattoir, at which prevalence is lower, originates from the southern half of the country, mainly from the two major cattle-producing areas of gantsi and kgalagadi districts, in the south-west and western parts of the country, but also from southern, south-east, kgatleng and kweneng districts. in contrast, the francistown abattoir is supplied by the central and north-east districts. the southern and western parts of the country receive less rainfall compared to the central and north-eastern areas of the country. there are also more and larger rivers in the central and north-eastern parts of botswana. discussion the findings from this study demonstrate that although fasciolosis was present in cattle slaughtered at the two main export abattoirs in botswana, the prevalence was low. this is the first systematic record of the prevalence of bovine fasciolosis in the country, and it provides evidence for the need for more extensive epidemiological investigations to be undertaken in different regions of the country. the mean overall prevalence (0.1%) of fasciolosis in cattle slaughtered at the two main export abattoirs in botswana was much lower than that reported from other countries in sub-saharan africa. similar abattoir studies in zimbabwe (pfukenyi & mukaratirwa 2004), kenya (kithuka et al. 2002; mungube et al. 2006) and tanzania (mellau, nonga & karimuribo 2010; nonga et al. 2009) reported a higher prevalence of 37.1%, 8%, 26%, 16.5% and 8.6%, respectively. the present findings rank among the lowest in africa, and suggest that liver fluke infection in slaughtered cattle in botswana is not of clinical economic importance. the abnormal surge in condemnations of fasciola-infected livers in 2006 (n = 269), and even more striking in 2007 (n = 773), even though the latter was a drought year in botswana, could be attributed to the high rainfall received by the country in 2005 and the above average rainfall in 2006, which led to higher infections due to increased contact between cattle and contaminated pastures. the most plausible explanation, however, could be that the actual drought that prevailed in 2007 could have led to increased supply (sale) of cattle to the francistown abattoir as a management measure by farmers, to mitigate the high costs associated with the purchase of supplementary feeds during drought. this highlights the potential bias of extrapolating results from abattoirs to the general population. the limitations associated with abattoir studies include the bias of age and low prevalence of clinical disease (robertson & blackmore 1985), as most often the relatively younger and healthier animals are sent for slaughter. the difference in prevalence of fasciolosis might be attributable to ecological and climatic variations in different areas as well as animal husbandry practices, which differ between countries. this variation could also be due to different internal parasite control between countries. in botswana, the commonly used veterinary drugs are sold to farmers at subsidised prices from the livestock advisory centres (lacs) to improve livestock production. therefore, farmers regularly use anthelmintics, and although they use blanket treatment or indiscriminate dosing of their animals, they indirectly could be treating for fasciolosis, as well. one commonly used anthelmintic is albendazole (valbazen®), which is a broad-spectrum drug with flukicidal activity, as well as killing adult trematodes, nematodes and cestodes. triclabendazole, which is a highly effective flukicide against both adult and juvenile flukes, and the recommended drug in most countries, is not available in botswana, most probably because of its high cost. the low prevalence might also be attributed to the provision of better veterinary services to the farming community by private veterinary practitioners, whose numbers have increased in recent years, and who also provide advice to farmers on livestock management, which could reduce the prevalence of fasciolosis. in addition, owing to the large size of the bovine liver, it is also possible that the prevalence of fasciolosis was underestimated in the present study since some livers with partial infection could have been passed as fit for human consumption after trimming of the affected parts. another possibility might be that farmers chose to send their healthiest animals to the main abattoirs and the less healthy or poor conditioned cattle to the local council abattoirs and meat inspection slabs situated around the country, where they expect less scrutiny during meat inspection of their animals. the pattern of distribution of f. gigantica infections from this study showed that areas that receive higher rainfall had a higher prevalence than did cattle from the drier areas. a comparison of the findings between the two abattoirs showed that the francistown abattoir, in the northern region, recorded a higher prevalence of the disease than the lobatse abattoir, in the southern part of the country. the result is not surprising as the francistown abattoir receives cattle from high rainfall areas in the north-east and central districts. the north-east district borders matebeleland province in zimbabwe, where a prevalence of 36.1%, based on an abattoir study, has been reported (pfukenyi & mukaratirwa 2004). this area receives an annual rainfall of more than 1000 mm, which provides good conditions for the survival of the intermediate host snail, lymnaea natalensis. the lowest prevalence of fasciolosis recorded at lobatse abattoir in the southern part of the country could be attributed to the fact that the abattoir is largely supplied of cattle from kgalagadi and gantsi districts, where relatively dry conditions exist, which are unfavourable for the survival of the intermediate host snail. the mean annual rainfall for lobatse is 550 mm, and it is even lower (250 mm – 300 mm) in gantsi and kgalagadi districts. the observation from this study is, to some extent, in agreement with studies from other parts of africa, where higher prevalence of fasciolosis in cattle was reported following periods of high rainfall and from areas with high rainfall than during drought periods and from areas with lower rainfall (kithuka et al. 2002; mungube et al. 2006; pfukenyi & mukaratirwa 2004). this is further evidence that rainfall has a direct influence on the occurrence of liver flukes (kithuka et al. 2002), and the origin of cattle examined at a particular abattoir would be expected to have a strong influence on the prevalence of the disease (phiri et al. 2005b). fasciolosis is enzootic in areas with a mean annual rainfall of over 1000 mm where l. natalensis is widely distributed (pfukenyi & mukaratirwa 2004) (table 2). table 2: annual rainfall and temperature for the catchment areas of two export abattoirs in botswana. the cool and humid climate in the central and north-east districts therefore probably provides the optimal conditions for the survival of the intermediate host snail and the liver fluke. lymnaea natalensis, a freshwater snail, is common and widely distributed in tropical and subtropical africa, including botswana (brown & kristensen 1989; seddon et al. 2010), and can tolerate a wide range of conditions, including changes affecting regional wetlands (seddon et al. 2010). the snail is found in a great variety of habitats, including natural permanent water bodies, man-made dams, reservoirs, ponds and even cattle drinking troughs (pfukenyi & mukaratirwa 2004; seddon et al. 2010). these water bodies increase the risk of acquisition of infection (ogunrinade & ogunrinade 1980). therefore, the higher prevalence of the disease in cattle slaughtered at the francistown abattoir was probably related to the presence of numerous streams and rivers in the north-east catchment area, as opposed to the ephemeral water system that is prevalent in most of the southern region catchment areas where the lobatse abattoir is located. a similar study in zambia by phiri et al. (2005b) found a higher prevalence in areas prone to flooding. there was no distinct seasonal pattern in liver condemnation rates and therefore the prevalence of fasciolosis. in contrast, elsewhere in africa seasonal differences have been observed, with a high prevalence of the disease reported during the rainy or post-rainy season (asanji & williams 1984; mzembe & chaudhry 1981; nonga et al. 2009; pfukenyi & mukaratirwa 2004; phiri et al. 2005a). a study in zimbabwe found that a snail population builds during the beginning of the dry season and then drops during the cold, dry months in winter, but again increases during the rainy season, with a concomitant peak in liver condemnations at the abattoirs (pfukenyi & mukaratirwa 2004). this is also a likely scenario in north-eastern botswana. conclusion in conclusion, the findings of the present abattoir study have provided preliminary baseline data on the prevalence of bovine fasciolosis in botswana. these results suggest that f. gigantica is not a major cause of liver condemnation in abattoirs in botswana, and thus only low annual financial losses would have been incurred as a consequence of condemnation of f. gigantica infected livers during the 10-year period. there is a need, however, for a cross-sectional study of fasciolosis in cattle of all ages to determine the real situation in botswana and to have a better understanding of the epidemiology of this important parasitic disease of ruminants to subsequently allow the design and implementation of appropriate control measures. acknowledgements the authors would like to thank the staff of the two abattoirs and the director of veterinary services, botswana, for allowing access to the meat inspection records. the college of agriculture in botswana and murdoch university in australia are also acknowledged for their financial support. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions m.e.m. conceptualised the research and was responsible for data collection, samples analysis, performing calculations and writing the manuscript. i.d.r. was the lead researcher and supervisor, made contributions to modify the research concept, advised on statistics and edited the article. references asanji, m.f. & williams, m.o., 1984, ‘the effect of sex on seasonal variation in single and double infection of cattle in sierra leone by dicrocoelium hospes and faciola gigantica’, veterinary parasitology 15, 247–255. http://dx.doi.org/10.1016/0304-4017(84)90076-1 brown, d.s. & kristensen, t.k., 1989, a field guide to african freshwater snails, southern african species, danish bilharziasis laboratory, chatollenlund. coelho, l.h.l. & lima, w.s., 2003, ‘population dynamics of lymnaea columella and its natural infection by fasciola hepatica in the state of minas gerais, brazil’, journal of helminthology 77, 7–10. http://dx.doi.org/10.1079/joh2002138 gajewska, a., smaga-kozlowska, k. & wisniewski, w., 2005, ‘pathological changes of liver in infection of fasciola hepatica’, wiadomosci parazytologiczne 51, 115–123. jones, t.c., hunt, r.d. & king, n.w., 2006, veterinary pathology, wiley blackwell, ames. kithuka, j.m., maingi, n., njeruh, f.m. & ombui, j.n., 2002, ‘the prevalence and economic importance of bovine fasciolosis in kenya: an analysis of abattoir data’, onderstepoort journal of veterinary research 69, 255–262. mcgavin, m.d. & zachary, j.f., 2007, pathologic basis of veterinary disease, mosby elsevier, st louis, mo. mellau, l.s.b., nonga, h.e. & karimuribo, e.d., 2010, ‘a slaughterhouse survey of liver lesions in slaughtered cattle, sheep and goats at arusha, tanzania’, research journal of veterinary sciences 3, 179–188. http://dx.doi.org/10.3923/rjvs.2010.179.188 mungube, e., bauni, s., tenhagen, b.a., wamae, l., nginyi, j. & mugambi, j., 2006, ‘the prevalence and economic significance of fasciola gigantica and stilesia hepatica in slaughtered animals in the semi-arid coastal kenya’, tropical animal health and production 38, 475–483. http://dx.doi.org/10.1007/s11250-006-4394-4 mwabonimana, m.f., kassuku, a.a., ngowi, h.a., mellau, l.s.b., nonga, h.e. & karimuribo, e.d., 2009, ‘prevalence and economic significance of bovine fasciolosis in slaughtered cattle at arusha abattoir, tanzania’, tanzania veterinary journal 26, 68–74. mzembe, s. & chaudhry, m., 1981, ‘the epidemiology of fascioliasis in malawi, part ii: epidemiology in the definitive host’, tropical animal health and production 13, 27–33. http://dx.doi.org/10.1007/bf02237882 nonga, h., mwabonimana, m., ngowi, h., mellau, l. & karimuribo, e., 2009, ‘a retrospective survey of liver fasciolosis and stilesiosis in livestock based on abattoir data in arusha, tanzania’, tropical animal health and production 41, 1377–1380. http://dx.doi.org/10.1007/s11250-009-9325-8 ogunrinade, a. & ogunrinade, b., 1980, ‘economic importance of bovine fascioliasis in nigeria’, tropical animal health and production 12, 155–160. http://dx.doi.org/10.1007/bf02242647 pfukenyi, d.s. & mukaratirwa, s., 2004, ‘a retrospective study of the prevalence and seasonal variation of fasciola gigantica in cattle slaughtered in the major abattoirs of zimbabwe between 1990 and 1999’, onderstepoort journal of veterinary research 71, 181–187. http://dx.doi.org/10.4102/ojvr.v71i3.258 phiri, a.m., phiri, i.k., sikasunge, c.s. & monrad, j., 2005a, ‘prevalence of fasciolosis in zambian cattle observed at selected abattoirs with emphasis on age, sex and origin’, veterinary medicine 52, 414–416. http://dx.doi.org/10.1111/j.1439-0450.2005.00872.x phiri, a.m., phiri, i.k., siziya, s., sikasunge, c.s., chembensofu, m. & monrad, j., 2005b, ‘seasonal pattern of bovine fasciolosis in the kafue and zambezi catchment areas of zambia’, veterinary parasitology 134, 87–92. http://dx.doi.org/10.1016/j.vetpar.2005.06.010 phiri, i.k., phiri, a.m. & harrison, l.j.s., 2007, ‘the serum glucose and [beta]-hydroxybutyrate levels in sheep with experimental fasciola hepatica and fasciola gigantica infection’, veterinary parasitology 143, 287–293. http://dx.doi.org/10.1016/j.vetpar.2006.09.001 roberts, j.a. & suhardono, 1996, ‘approaches to the control of fasciolosis in ruminants’, international journal for parasitology 26, 971–981. http://dx.doi.org/10.1016/s0020-7519(96)80074-9 robertson, i.d. & blackmore, d.k., 1985, ‘abattoir data as a source of epidemiological information: a neglected resource’, proceedings of the 4th international symposium on veterinary epidemiology and economics, singapore, november 18–22, 1985, pp. 377–381. seddon, m., van damme, d., graf, d.l., appleton, c. & bennett, l., 2010, ‘lymnaea natalensis’, in iucn red list of threatened species, international union for conservation of nature, cambridge. mbao_9-15.indd introduction east coast fever, an often fatal disease of cattle in the east ern, central and southern parts of africa, is caused by theileria parva, an obligate intracellular protozoan parasite. it is mostly transmitted by the three-host ixodid ticks rhipicephalus appendiculatus and rhipicephalus zambeziensis. the disease is a major constraint to livestock development in the affected regions (young, groocock & kariuki 1988). control methods include immunization by the infection-and-treatment method (i&t) (radley, brown, burridge, cunningham, kirimi, purnell & young 1975) in which doses of t. parva cryopreserved stabilates (cunningham, brown, burridge & purnell 1973) are inoculated simultaneously with a long acting tetracycline. the method is widely applied in the field (uilen berg 1999; marcotty, billiouw, chaka, berkvens, losson & brandt 2001; fandamu, thys, duchateau & berkvens 2006). currently, stabilates are stored in liquid nitrogen. the maintenance of the cold chain up to the farm level is complicated which makes the method less 9 onderstepoort journal of veterinary research, 74:9–15 (2007) comparison of the survival on ice of thawed theileria parva sporozoites of different stocks cryoprotected by glycerol or sucrose v. mbao1, d. berkvens2, p. dorny2,3, p. van den bossche2,4 and t. marcotty2* abstract mbao, v., berkvens, d., dorny, p., van den bossche, p. & marcotty, t. 2007. com parison of the survival on ice of thawed theileria parva sporozoites of different stocks cryoprotected by glycerol or sucrose. onderstepoort journal of veterinary research, 74:9–15 stabilates of theileria parva sporozoites are mostly delivered in liquid nitrogen tanks to the east coast fever immunization points. using an in vitro titration model, we assessed the loss of infectivity of several stabilates when they are stored in ice baths for up to 24 h. comparisons, with respect to rates of loss of infectivity, were made between t. parva stocks (chitongo and katete), cryoprotectants (sucrose and glycerol) and method of assessment (in vivo and in vitro techniques). chitongo and katete stabilates showed similar loss dynamics. the losses were 1–4 % (depending on parasite stock) and 3 % per hour of storage for glycerol and sucrose stabilates respectively, and the loss rates were not significantly different. the results suggest that chitongo stabilates and sucrose cryoprotected suspensions can be delivered on ice as is done for katete. a graphical relationship of in vitro effective dose at 50 % infectivity (ed50) and in vivo protection rate was made. the relationship showed a 35 % loss of protection for a relatively low corresponding increase of ed50 from 0.006 to 0.007 tick equivalent. keywords: cold-chain, immunization, in vitro, sporozoites, theileria parva, zambia * author to whom correspondence is to be directed. e-mail: tmarcotty@itg.be 1 department of veterinary and livestock development, ministry of agriculture and cooperatives, p.o. box 670050, mazabuka, zambia. present address: department of animal health, institute of tropical medicine, nationalestraat 155, b-2000 antwerp, belgium 2 department of animal health, institute of tropical medicine, nationalestraat 155, b-2000 antwerp, belgium 3 department of parasitology, faculty of veterinary medicine, ghent university, salisburylaan 133, b9820 merelbeke, bel gium 4 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa accepted for publication 15 september 2006—editor 10 survival of theileria parva sporozoite stocks cryoprotected by glycerol or sucrose appropriate for rural circumstances and is expensive for livestock keepers. this problem was partly alleviated in the eastern province of zambia by stor ing the stabilate on ice for a few hours between thaw ing and inoculation. this method allows the distribution of the service using bicycles instead of motor cars. studies conducted in this region using a glycerated t. parva katete stock stabilate (geysen, bishop, skil ton, dolan & morzaria 1999) showed that after 6 h of storage on ice stabilates could still protect 90 % of the immunized animals (marcotty et al. 2001). in the southern province of zambia, the i&t immunization against ecf makes use of the milder chitongo strain (geysen et al. 1999). there is no information on how long stabilates of this t. parva strain can be stored on ice without appreciable loss in their potency. in this study, the loss of infectivity of stabilates due to storage on ice was assessed by in vitro titration (marcotty, speybroeck, berkvens, chaka, besa, madder, dolan, losson & brandt 2004). the performance of the chitongo stock was compared with that of the katete stock with the aim of determining the suitability of the deferred immunization technique using the chitongo stock. various studies indicated that sucrose is a promising t. parva stabilate cryoprotectant. it is cheap and more user friendly than glycerol. ongoing work seems to indicate that sucrose stabilates have higher infectivity, and therefore better infectivity, on recovery from cryopreservation compared to glycerol counterparts (mbao, unpublished data 2005). since it is not known whether stabilates cryoprotected by sucrose show similar survival when stored on ice as glycerol-cryoprotected stabilates, the infectivities during ice storage of sucrose and glycerol katete stabilates were compared. a considerable amount of work on in vitro assessment of t. parva stabilates has been done (wilkie, kirvar & brown 2002; marcotty et al. 2004; mbao, speybroeck, berkvens, dolan, dorny, madder, mulumba, duchateau, brandt & marcotty 2005). this has shown the potential of the technique as an economical and ethical way of assessing stabilate infectivity for determining immunizing doses, comparison of stocks and evaluating effects of various pro cesses during stabilate production and cryopres ervation. however, a relationship between in vitro and in vivo assays to predict actual immunization potential of stabilates is yet to be made. in the present work, we evaluated this link by establishing a graphical relationship between effective doses that give 50 % infectivity (ed50) (in vitro) to proportions of animals successfully immunized (in vivo). when a stabilate is stored on ice, its effective dose for respective levels of infectivity increases with time. these doses can easily be assessed in vitro and compared to the proportions of animals successfully immunized with similar stabilates stored in similar conditions. materials and methods stabilate preparations animals two friesian heifers kept in a tick-proof stall were inoculated with 1 ml of t. parva katete subcutaneously below the right parotidian lymph nodes. they were checked daily for rectal temperature and once the parotidian nodes were palpably swollen, biopsies were aspirated and smears prepared for calculating percentages of schizont-infected lymphoblasts. thin jugular blood smears were made to check for parasitaemia. nymphal rhipicephalus appendiculatus ticks were applied on day 10 post inoculation. the moulted ticks (adults) were pre-fed for 4 days on new zealand white rabbits kept at the institute of tropical medicine animal quarters. these ticks were used to produce stabilates k2g and k2s as described below. another friesian heifer was used in a similar way to infect a batch of ticks with t. parva chitongo for the production of stabilate c1g. stabilates used one chitongo and three katete stabilates were used. katete stabilates were all produced from batches of adult r. appendiculatus ticks, infected as nymphs with the same t. parva seed stabilate. the first glycerated katete stabilate (k1g) that was used in this study had been produced and tested in vivo by marcotty et al. (2001). the two other katete stabilates, k2g and k2s, were produced for the present work from a single batch of infected ticks but were cryoprotected using 7.5 % (w/v) glycerol and 0.3 m (10 % w/v) sucrose, respectively. the chitongo stab ilate (c1g) was cryoprotected with 7.5 % glycerol. production for all stabilate productions, infected nymphs were allowed to moult in an incubator at 22 °c and 85 % relative humidity. eight to 12 weeks after engorgement, the resulting adult ticks were fed on rabbits for 4 days to induce sporogony of the parasite. har vested ticks were manually ground in a mortar using a pestle for 15 min in minimum essential medium supplemented with bovine serum albumin (mem/bsa) 11 v. mbao et al. (purnell, brown, cunningham, burridge, kirimi & ledger 1973) at a concentration of 20 ticks per 1 ml. for glycerol stabilates, an equal amount of chilled mem/bsa with glycerol at 15 % (w/v) was added drop-wise (oie 2005). for the sucrose stabilate, an equal amount of 0.6 m sucrose/mem/bsa solution was added to give final concentration of 0.3 m. the 0.6 m sucrose medium had been prepared by dissolving 30.8 g of sucrose grains (sigma #s1888) in 150 ml of mem/bsa solution. the extracts were stirred continuously in an ice bath. the k2g and k2s stabilates were prepared in a single session and from a single batch of ticks, the only difference being that, at the stage where the cryoprotectant was added, half the extract was mixed with glycerol and the other with sucrose as described above. some portions of the stabilates were further diluted to a final concentration of one tick per 1 ml. all suspensions were aliquoted into 1.5 ml nalgene® cryogenic vials (1 ml per vial). vials were then placed in polystyrene boxes and transferred to a –80 °c freezer for 24 h before being plunged into liquid nitrogen for storage. ice bath storage storage on ice meant keeping stabilate vials in a polystyrene box filled with water and pieces of melting ice (about 3 °c). groups of six vials of each stabilate were thawed at 37 °c for 5 min and stored on ice for different periods. the k1g stabilate was stored on ice for 3, 8 or 24 h. the c1g stabilate storage times were 1, 3, 6, 12 or 24 h. finally, the thawed k2g and k2s stabilates were kept on ice for 6, 12 or 24 h before the titration. stabilate for in vivo titration had been stored on ice for 8, 12, 16, 24 or 32 h as reported by marcotty et al. (2001). in vitro titrations peripheral blood mononuclear cells (pbmc) were isolated from one friesian heifer throughout the study. the protocol for in vitro titration of t. parva tick-derived stabilates was described by marcotty et al. (2004) and modified by mbao et al. (2005). the k2g and k2s stabilates stored on ice, including a control group (thawed at titration), were transferred to separate falcon tubes and centrifuged. the supernatants, being arbitrarily allocated to separate rows in a 96-well microtitration plate (12 rows by eight columns), were then serially diluted eight times (two-fold dilution). all stabilate groups were titrated in parallel in three sessions. a session was taken as a titration at a given time, sharing a batch of pbmc, culture media and stabilate diluents, thereby forming a cluster for the purpose of statistical analyses (marcotty et al. 2004). for controls, stabilate freshly thawed from liquid nitrogen storage was used. the c1g and k1g stabilates were titrated in single sessions. the stabilate was diluted 12 times (1.5 fold dilution). table 1 presents the various sessions and microtitration plate setup. after 10 days of incubation, 100 μl of each microtitration well were transferred to a separate slide as a cytospin smear. to avoid cross contaminations between various stabilates, each row (corresponding to a different stabilate) was assigned to a particular cyto-centrifuge block and sampling was done from the lowest to the highest stabilate concentration. experimental animals were maintained and treated humanely according to the guidelines laid down by the ethics commission of the institute of tropical medicine of antwerp, belgium (dg003-mm-k-rip). details of animals used for production of k1g by marcotty et al. (2001) are fully described in the given reference. in vivo titrations the in vivo titrations data used to compare with in vitro was obtained from marcotty et al. (2001). the relationship between storage on ice and proportions of successfully immunized animals are illustrated in fig. 1. table 1 sessions and number of microtitration plate wells read for stabilates stored on ice k1g k2g k2s c1g number of sessions plates/session wells read 1 2 188 3* 4* 569 3 4 575 1 2 120 * same sessions and plates as for k2s k = katete c = chitongo g = glycerol s = sucrose 12 survival of theileria parva sporozoite stocks cryoprotected by glycerol or sucrose statistical analysis infectivity losses of katete and chitongo (k1g and c1g) in function of time of storage on ice data were analysed by logistic regression in stata® (stata corporation, texas). the proportion of positive wells was the response variable and explanatory variables were the dose (natural log of tick equivalents [ln t.e.]) and storage time on ice. storage time was considered both as a discrete variable and, in a simplified model, as a continuous variable. the two models were compared by means of a likelihood ratio test. the level of significance was set at 5 %. estimation of residual infectivity was conducted by using ratios of effective doses [edcontrol/edx] where x = time of storage on ice. this was calculated using a non-linear combination of estimators which also calculates the ratios’ respective confidence intervals (mbao et al. 2005). infectivity losses of glycerol and sucrose stabilates (k2g and k2s) in function of time of storage on ice the comparison of the infectivity of stabilates cryoprotected with glycerol or sucrose was conducted by a logistic regression using the gllamm command (generalised linear latent and mixed models) in stata®, with vial and session as random effects. the explanatory variables were stabilate cryoprotectant (sucrose or glycerol), stabilate dose (ln t.e.) and time of storage on ice. the response variable was the proportion of positive wells. interactions between time and stabilate as well as dose and stabilate were tested. initially, all variables except the dose were entered as discrete variables. this was the saturated model. the non-significant interactions were dropped and the analysis redone with time as continuous variable. this was the simpler model. when the two models were not statistically different (p > 0.05), the simpler model was adopted. comparison of the viability estimations in in vivo and in vitro experiments (k1g in vivo and in vitro) results from the in vivo evaluation of the k1g stabilate were obtained from previous experimental work (marcotty et al. 2001). the best fit curve on observed proportions of protected animals was calculated in a logistic model. taking the time of storage as a common axis for the in vivo and in vitro infectivity loss evaluations, the in vitro ed50 estimates were plotted against predicted proportions of protected animals (in vivo) that had been inoculated with k1g. �������� ��� ����� ����� ������������ � �� � � � �� � �� �� � � �� � � ���� ��� ���� ���� � �� �� �� �� �� � �� � �� �� �� � � � � � � � � ������������������������� ���������������������������� ������������ ���������������������!���"�#� ����$��%��� �&� ����'�( ��������������������� ��������!���"�#� ����$��%�����&� ����'�( ) ��*�) � �����) �������������������� ) ��*�) � �����) ���������������������������������� � fig. 1 effect of storing theileria parva stabilates on ice on the ability to induce immunity in cattle (marcotty et al. 2001) 13 v. mbao et al. results for all stabilates, the time of storage on ice was taken as a continuous variable as the likelihood ratio tests comparing these models to the models using the time as a discrete variable were not significant (p > 0.05). infectivity losses of katete and chitongo (k1g and c1g) in function of time of storage on ice the two stabilates were kept in separate models as they were tested separately. for k1g, the residual infectivity after each hour of storage on ice was estimated to be 0.99 of the infectivity in the preceding hour (95 % ci: 0.96–1.02). the effect of storage time was not significant whether time was a discrete variable (p = 0.99, p = 0.24 and p = 0.55 for times 3, 8 and 24 h, respectively) or continuous variable (p = 0.45). the residual infectivity of c1g after storage on ice was 0.96 of the infectivity in the preceding hour (95 % ci: 0.93–1.00). similarly, storage time was not significant for discrete time (p = 1, p = 1, p = 0.17 and p = 0.17 for times 3, 6, 12 and 24 h, respectively) or continuous time (p = 0.07) (fig. 2). infectivity losses of glycerol and sucrose stabilates (k2g and k2s) in function of time of storage on ice since the stabilates were tested in parallel, infectivity losses of the two stabilates were analysed in one �� �� �� �� � �� � �� �� �� &� & &� & &� &� &� �� ������ ���� � �� � �� � � �� �� � � � �� � �� �� � � � �� � �� � �� + �� �� �� �� � �� � �� �� �� &� & &� &� &� &� �� ������ ���� � �� � �� � � �� �� � � � �� � �� �� � � � �� � �� � �� , fig. 2 (a) titration curves of k1g (katete) stored on ice for 0, 3, 8 and 24 h (from left to right). [ln t.e.] is the natural log of the stabilate dose expressed in tick equivalents; and (b) c1g (chitongo) stored on ice for 1, 3, 6, 12, 24 h (from left to right) �� �� �� �� � �� � �� �� �� &� & &� & &� &� �� ������ ���� � �� � �� � � �� �� � � � �� � �� �� � � � �� � �� � �� �� �� �� �� � �� � �� �� �� ������ ����� !������ � �� � �� � � �� �� � � � �� � �� �� � � � �� � �� � �� � � � � � � � �� � �� � �� � �� � � � � � fig. 3 titration curves of katete-k2g (——) and k2s (-----) stabilates stored on ice for 0 (control), 6, 12 and 24 h (from left to right). (ln t.e.) is the natural log of the stabilate dose expressed in tick equivalents fig. 4 correlation between proportions of protected animals against in vitro ed50 14 survival of theileria parva sporozoite stocks cryoprotected by glycerol or sucrose model. the k2g had a residual infectivity of 0.99 of that in the preceding hour of storage. the effect of storage time was not significant (p = 0.35) for the studied time periods. the k2s had 0.97 residual infectivity per hour of storage. here, effect of storage time was significant (p = 0.04). the interaction between stabilate and storage time was not significant (p = 0.45). base infectivity, i.e. infectivity after production and cryo-storage (before storage on ice) for sucrose was 10 times higher that of glycerol (95 % ci: 6.2–16.7) (fig. 3). comparison of the viability estimations in in vivo and in vitro experiments (k1g in vivo and in vitro) the graphical representation shows that an increase of ed50 from 0.006 to 0.007 tick equivalents (in vitro) results in the protection proportion dropping from 92 % to 57 % (in vivo) (fig. 4). discussion results showed that the loss of infectivity after shortterm storage on ice of katete and chitongo strain t. parva stabilates with glycerol or sucrose as cryoprotectant was minimal. this observation is in line with the one of musisi, quiroga, njuguna, kamwendo & chamambala (1996) who found that animals were protected with a trivalent stabilate stored on ice for 15 h. similar results were obtained by marcotty et al. (2001) who observed successful immunization in 90 % of cattle inoculated with stabilates stored on ice for up to 6 h. except for the sucrose-stabilate (k2s), the effect of storage on ice for up to 24 h was not statistically significant. it is assumed that an effect of storage time on infectivity (reduced infectivity) will be observed for longer storage periods than investigated in this work. however, it was observed that keeping stabilate on ice for longer periods resulted in contamination of the cultures on several occasions. this was most likely due to fungal proliferation (from stabilate tick material) at this temperature. the observed dynamics of infectivity loss for the katete and chitongo strains were similar. this indicates that the deferred immunization technique that has been applied in the eastern province of zambia since 1996 could be used in the southern province where the chitongo stock is used. this would greatly simplify the delivery of stabilate to the remote areas and make delivery much cheaper. regarding the two different strains used here, the infectivity loss dynamics observed may, therefore, be true for other t. parva stocks in other regions of africa. how ever, it may be necessary to similarly test such stocks to confirm this assumption. since the interaction between stabilate and time was not significant when comparing katete glycerol and sucrose stabilates, it is assumed that the stabilate cryoprotected with sucrose did not lose infectivity faster or slower than the stabilate cryprotected with glycerol. in terms of base infectivity (infectivity at time 0), sucrose stabilate appeared to have higher titres despite having been prepared from the same batch of ticks and during the same production session. this was also seen in comparisons of other glyc erol/sucrose stabilates prepared with a similar protocol (mbao, unpublished data 2005) and may be due to lower toxicity of sucrose for sporozoites. however, the observed difference in titre may also be due to an effect on the host cells, for instance, glycerol may be more toxic to lymphocytes. it would be necessary to conduct a more direct comparison (without the use of lympho cytes) of the effects of these two cryoprotectants for a good conclusion, e.g. by comparing the proportion of live sporozoites on recovery from cryopreservation using reverse transcription-polymerase chain reaction. sucrose therefore remains a potentially cheaper and better candidate for field stabilates. a loss of protection in vivo by 35 % (92–57 %) in going from in vitro ed50 of 0.006 to 0.007 seems to be a drastic loss. in terms of stabilate dilutions, this would be similar to diluting a stabilate by a factor of 1.2 times. practically, this dilution is too small to account for the corresponding large loss in protection. cunningham, brown, burridge, musoke purnell, radley & sempebwa (1974) observed a decrease in protection of only about 10 % on diluting stabilate from 1/150 to 1/450 (3 times dilution). therefore, it is assumed that the observed anomaly is rather due to the small number of animals that had been used in the in vivo trial (about four animals per storage period). the in vivo trial also lacked repetitions. in the assumption that in vitro result are more accurate, these two factors could have resulted in an underestimation of residual infectivity of the stabilate. on the other hand, the in vitro model assumes that sporozoites are live or dead i.e. infective or not. some sporozoites might, however, be weakened by storage on ice but remain infective in vitro. the same sporozoites may no longer be infective in vivo because of more hostile conditions like non-specific immune reactions. this would result in an overestimation of residual infectivity if we assume the in vivo result to be more accurate. in short, this lack of 15 v. mbao et al. agreement between in vivo and in vitro observations could be explained by either the lack of accuracy of the in vivo trial or, more likely, by the different conditions to which weakened sporozoites are exposed in the two techniques. such biases and limitations should be considered carefully when using the in vitro model. notwithstanding the considerations outlined in effecting a relationship between the techniques, the in vitro technique remains a valuable alternative to in vivo testing as it remains more ethical and cheaper. further, in vitro titration/evaluation has the advantage that sessions are easily repeated to offset random variation seen in in vivo titrations. ideally, parallel in vitro and in vivo trials using the same thawed stabilate and a larger number of animals should be set up, preferably with several repetitions to confirm the relation between in vivo and in vitro t. parva infections. in conclusion, sucrose protected stabilates lose infectivity at the same rate as glycerol protected ones. chitongo and katete strains have similar infectivity losses when stored on ice. chitongo stabilates used in the southern province of zambia can therefore be delivered to the immunization points in this way (up to 6 h). this would reduce the costs and complications associated with stabilate delivery in liquid nitrogen thus making the i & t immunization option available and affordable to more cattle keepers. further, this finding could be valid for other strains of t. parva in other regions affected by east coast fever. acknowledgements this work was funded by a belgian technical cooperation phd scholarship. the authors thank the laboratory staff of the institute for tropical medicine, antwerp, where the work was done. we are particularly grateful to anke van hul for the help in the preparation and reading of microscopic slides. references cunningham, m.p., brown, c.g.d., burridge, m.j. & pur nell, r.e. 1973. cryopreservation of infective particles of theileria parva. international journal for parasitology, 3: 583–587. cunningham, m.p., brown, c.g.d., burridge, m.j., musoke, a.j., purnell, r.e., radley, d.e. & sempebwa, c. 1974. east coast fever: titration in cattle of suspensions of theileria parva derived from ticks. british veterinary journal, 130:336–345. fandamu, p., thys, e., duchateau, l. & berkvens, d. 2006. perception of cattle farmers on east coast fever immunizations in southern zambia. tropical animal health and production, 38:9–16. geysen, d., bishop, r., skilton, r., dolan, t.t. & morzaria, s. 1999. molecular epidemiology of theileria parva in the field. tropical medicine and international health, 4:a21–a27. marcotty, t., billiouw, m., chaka, g., berkvens, d., los son, b. & brandt, j. 2001. immunization against east coast fever by the infection and treatment method: evaluation of the use of ice baths for field delivery and appraisal of an acid formulation of long-acting tetracycline. veterinary parasitology, 99:175–187. marcotty, t., speybroeck, n., berkvens, d., chaka, g., besa, r.k., madder, m., dolan, t.t., losson, b. & brandt, j. 2004. in vitro titration of theileria parva tick derived stabilates. parasitology, 128:131–137. mbao, v., speybroeck, n., berkvens, d., dolan t., dorny, p., madder, m., mulumba, m., duchateau, l., brandt, j. & marcotty, t. 2005. comparison of manu al and homogenizer methods for preparation of tick-derived stabilates of theileria parva: equivalence testing using an in vitro titration model. parasitology, 131:45–49. musisi, f.l., quiroga, j.c., njuguna, l.m., kamwendo, s.p. & chamambala, k.e. 1996. infectivity of theileria parva trivalent stabilate after thawing and maintenance at 4 °c for up to 15 hours. international journal for parasitology, 26:175–179. oie 2004. manual of diagnostic tests and vaccines for terrestrial animals, 5th ed. www.oie.int/fr/normes/mmanual/a_0062.htm (consulted 20/02/06). purnell, r.e., brown, c.g.d., cunningham, m.p., burridge, m.j., kirimi, i.m. & ledger, m.a. 1973. east coast fever: correlation between the morphology and infectivity of theileria parva developing in its tick vector. parasitology, 66: 539–544. radley, d.e., brown, c.g.d., burridge, m.j., cunningham, m.p., kirimi, i.m., purnell, r.e. & young, a.s. 1975. east coast fever: 1. chemoprophylactic immunization of cattle against theileria parva (muguga) and five theilerial strains. veterinary parasitology, 1:35–41. uilenberg, g. 1999. immunization against diseases caused by theileria parva: a review. tropical medicine and international health, 4:a12–a20. wilkie, g.m., kirvar, e. & brown, c.g.d. 2002. validation of an in vitro method to determine infectivity of cryopreserved sporozoites in stabilates of theileria spp. veterinary parasitology, 104:199–209. young, a.s., groocock, c.m. & kariuki, d.p. 1988. integrated control of ticks and tick-borne diseases of cattle in africa. parasitology, 96:403–432. barson_cestodes.indd introduction fish helminthology in southern africa is not as widely studied as other aspects of aquatic parasitology and fish biology. this is probably because helminths mainly infect the internal organs, predominantly the gastrointestinal tract which, for humans, does not com prise the edible portion of the fish. although fishermen and anglers regularly encounter encysted “grubs” (metacercariae) in the skin and muscles of fish (b. marshall, personal communication 2002), they regard them as just a nuisance, notwithstanding the biological and economic impact they may have on the fish species. only a few studies on cestode and trematode parasites of fish in south africa have been documented (whitfield & heeg 1977; boomker, huchzermayer & naude 1980; mashego 1977, 1981, 1982, 2001; mashe go & saayman 1989; brandt, van as, schoonbee & hamilton-attwell 1981; van as, schoonbee & brandt 1981; britz, van as & saayman 1985; saayman, mashego & mokgalong 1991, luus-powell & mashego 2003). in 1984, van as and basson compiled a checklist of south african freshwater fish parasites, but many new species have been discovered since then (khalil & polling 1997). paperna (1996) published a concise update of the parasitic diseases of fish in africa, in which the occurrence and geographical distribution, life cycles, pathology, epizootiology and control of the parasites is described. the tapeworms (cestoda) and flukes (digenea) infect internal organs of fish (roberts & janovy 2000) and their life cycles involve more than one intermediate host, including planktonic copepods, molluscs and fish. piscivorous birds, in which some helminths 101 onderstepoort journal of veterinary research, 73:101–110 (2006) on cestode and digenean parasites of clarias gariepinus (burchell, 1822) from the rietvlei dam, south africa m. barson1, 2 and a. avenant-oldewage1 abstract barson, m. & avenant-oldewage, a. 2006. on cestode and digenean parasites of clarias gariepinus (burchell, 1822) from the rietvlei dam, south africa. onderstepoort journal of veterinary research, 73:101–110 sharptooth catfish, clarias gariepinus, from the rietvlei dam near pretoria, south africa were examined for internal platyhelminth parasites. two adult cestodes, polyonchobothrium clarias (stomach) (prev alence 71 %, mean intensity = 5, n = 7) and proteocephalus glanduliger (anterior intestine) (prevalence 14 %, mean intensity = 2, n = 7), were found in the gut while metacercariae of one larval digenean, ornithodiplostomum sp. (prevalence 14 %, mean intensity = 140, n = 7), were found encysted in the muscles. the morphology of these species, based on light and scanning electron microscopy as well as histological analysis, and how they differ from previously described specimens, are discussed. ornithodiplostomum is a new record in southern africa. infection levels of the host fish were mild compared to records from previous surveys. keywords: cestode, clarias gariepinus, digenean, platyhelminth, rietvlei dam, south africa 1 department of zoology, university of johannesburg, kingsway campus, p.o. box 524, auckland park, johannesburg 2006, south africa 2 author to whom correspondence is to be directed. e-mail: barson@science.uz.ac.za. present address: department of biological sciences, university of zimbabwe, p.o. box mp 167, mt pleasant, harare, zimbabwe accepted for publication 23 january2006—editor 102 cestode and digenean parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa develop into adult stages, are important in that they can disseminate parasite eggs over long distances, making it difficult to control the spread of infections between water bodies in different catchments (saayman et al. 1991). in wetland systems such as the rietvlei dam locality, there is a high diversity of aquatic birds, both resident species (e.g. ducks) and migratory species (e.g. cormorants) (m. barson, personal observation 2003). while there is much need to understand the interactions between fish and birds in the transmission of helminth infections, only a few parasitological studies on piscivorous birds in africa are documented (beverly-burton 1963; ukoli 1968; saayman et al. 1991; mokgalong 1996; barson 2004; barson & marshall 2004). the sharptooth catfish, clarias gariepinus, (burchell, 1822) investigated in this study, is a widely distributed food fish in africa (safriel & bruton 1984; skelton 2001) and is one of the best species being targeted for aquaculture and biological research. this study was carried out as a survey of the internal parasites that are found in c. gariepinus from the rietvlei dam, which can be used in the fish health assessment index that has been developed for south african fish by avenant-oldewage (2001). the objectives of this paper were to specifically identify and classify the internal platyhelminth parasites collected from the host fish (clarias gariepinus) based on their morphological features, and to note their prevalence and mean intensity in the rietvlei dam. fig. 1 the location of the rietvlei dam inside the rietvlei nature reserve. x indicates point of sampling. bar = 2 km pretoria rietvlei nature reserve johannesburg rietvlei dam marais dam sesmylspruit south africa 0 0 ,1 0 ,5 1 km 2 km 103 m. barson & a. avenant-oldewage materials and methods study area the rietvlei nature reserve (25°41’22” s, 26°37’48” e) lies between pretoria and johannesburg in the gauteng province, the economic hub of south africa (fig. 1). it is solely responsible for conservation of the sesmylspruit catchment area, and the rietvlei dam (25°32’30” s, 28°16’46” e) currently supplies 27 % of pretoria’s water requirements (wessels 1998). the dam is supported by the smaller marais dam, which lies approximately 4 km upstream, and the two are separated by a wetland (fig. 1). further upstream, just before the sesmylspruit enters the reserve, effluent from a number of industries and a wastewater treatment plant is discharged into the stream, directly affecting the two dams. collection of fish and parasites fish were collected from the rietvlei dam using large mesh gill nets in may 2003. the gastrointestinal tract was dissected from the rectum to the oesophagus and parasites encountered were carefully detached from the stomach or intestinal mucosa. portions of the skin between the lateral line and dorsal fin of each fish were peeled off with forceps and fillets of muscle tissue were cut and examined for encysted parasitic forms. the liver, spleen, gall bladder and kid neys of each fish were also examined for parasites or cysts. trematode cysts from the muscle were teased manually to release metacercariae, which were fixed in hot alcohol-formal-acetate (afa) and preserved in 70 % ethyl alcohol. cestodes from the intestinal tract were swirled in 0.1 % sodium chloride (saline) to relax them, fixed in hot afa and preserved in 70 % ethyl alcohol. cestodes were stained with aqueous acetocarmine solution as described by khalil (1991). digenean trematode metacercariae were stained in delafield’s haematoxylin and counterstained in eosin. standard microtechnique procedures were used to prepare transverse serial sections of cestodes, which were embedded in synthetic resin (transmit lm) with a curing point of 70 °c. the specimens were sectioned with a rotary resin microtome (anglio scientific) at 5 μm thickness, and the sections were stained with azan, a trichrome stain. the parasites were identified based on their morphology, and using drawings and light micrographs taken with a zeiss axioplan microscope. scanning electron micrographs were taken with a jeol 6100 scanning electron microscope (sem). drawings and measurements were done with a zeiss standard 25 microscope equipped with a drawing tube. the following keys were consulted for identification: ya maguti (1958) and gibson, jones & bray (2002) for the trematodes, and yamaguti (1959), freze (1965), schmidt (1986), khalil, jones & bray (1994) and rego (1994) for the cestodes. parasite prevalence and mean intensities were calculated as defined by margolis, esch, holmes, kuris & schad 1982. voucher specimens were deposited in the zoological collection of the university of johannesburg (formerly rand afrikaans university), south africa. results and discussion the platyhelminth endoparasites found in c. gariepinus include two adult cestode species, poly onchobothrium clarias and proteocephalus glan duliger, and digenean metacercariae of the genus ornithodip lostomum (table 1). cestoda polyonchobothrium clarias (woodland, 1925), (fig. 2 and 5) scolex rectangular with a flat to slightly raised rostellum armed with a crown of 26–30 hooks (mean 28; n = 6). rostellum divided into two semicircles each bearing 13–15 hooks. hooks at the end of each semicircle smaller than the others. two longitudinally elongated bothria in line with the gaps between the crowns of hooks. immature proglottids of strobila not completely segmented. some mature segments apparently fused as shown by sem (fig. 5e). testes medullary; uterus anterior to ovary, highly folded and occupying the greater portion of gravid proglottids. vitellaria cortical. eggs unoperculate and embryonated. measurements of structures are given in table 2. polyonchobothrium clarias is widely distributed in siluroid fishes from african freshwater fishes, having been recorded from nigeria in the north african catfish clarias lazera (= c. gariepinus) cuvier & valenciennes, 1840 (aderounmu & adeniyi 1972). it was also reported in the bagrid catfish chrysichthys thonneri steindachner, 1912 from gabon, the mudfish clarias anguillaris (linnaeus, 1758) and heterobranchus bidorsalis geoffroy saint-hilaire, 1809 from senegal (khalil 1973), and in c. anguillaris from egypt (amin 1978). in southern africa, p. clarias was first observed and recorded by mashego (1977) from c. gariepinus in seven dams in the le bowa region, limpopo province, south africa. the only 104 cestode and digenean parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa other record of the parasite was from the middle letaba dam (saayman et al. 1991), also in the limpopo province. its high prevalence in the rietvlei system, as well as in the vaal dam (m. barson & a. avenant-oldewage, unpublished data 2003), seems to suggest that the cestode is widely distributed in c. gariepinus in the country. in zimbabwe, chishawa (1992) and douëllou (1992) recorded an intestinal cestode from the brown squeaker, synodontis zambezenis peters, 1852, and c. gariepinus from lake kariba. although they mistook it for larval p. clarias, it was apparently an adult with 38 hooks on its apical crown. larvae only occur in copepod intermediate hosts. the low intensity of p. clarias in hosts from the rietvlei dam (up to 11 worms per fish) would be expected to inflict minimal damage on the host tissue (paperna 1996), whereas high parasitic loads in the gall bladder have been shown to cause granulomatous nodules and fibrosis (wabuke-bunoti 1980). mashego (1977) recorded intensities of up to 200 in c. gariepinus from lebowa. in nigeria, aderounmu & adeniyi (1972) reported a heavy infection of 123 worms per host in c. gariepinus, causing nodules at the point of attachment. barson & avenant-olde wage (unpublished data 2003) recorded more than 100 individuals infecting one specimen of c. gariepinus in the vaal dam. the fact that the tapeworms physically fig. 2 polyonchobothrium clarias (a) mature proglottid, (b) scolex, (c), (d) gravid proglottids (scale bar = 200 μm), (e) eggs (scale bar = 50 μm), b = bothrium, ec = excretory canal, gp = genital pore, hk = hook, ov = ovary, r = rostellum, t = testis, ut = uterus, vt = vitel laria gp ut ec t ov vt ut eggs a b c d e r hk b ov 105 m. barson & a. avenant-oldewage resisted detachment from the gut mucosa suggests that the suction created by the bothria and the clasp of the apical hooks could cause severe pathological effects in heavy infections. proteocephalus glanduliger (janicki, 1928) fuhrmann, 1933 (fig. 3 and 5f) scolex unarmed, with four cup-shaped suckers arranged symmetrically around a protrusible rostellum; neck region not differentiated into well-formed proglottids. glandular organ present in one specimen but not apparent in the other, approximately similar in size to suckers. all proglottids broader than long. genital pores lateral and alternating. testes medullary as shown in resin sections (fig. 3e and 5f). specimens much larger than those described by freze (1965) and mashego (2001) (table 3). despite the abundance and diversity of proteocephalid cestodes in african freshwater fish (khalil & polling 1997), only one species, proteocephalus glanduliger has been recorded in south africa from c. gariepinus (mashego 1977, 2001; van as & basson 1984; saayman et al. 1991). only two specimens of this cestode were recovered from one of the seven catfish from the rietvlei dam. mashego table 1 prevalence and intensity of endohelminths of c. gariepinus from rietvlei dam parasite species location in host n* prevalence (%) intensity mean intensity polyonchobothrium clarias stomach mucosa, anterior intestine 7 71.4 1–11 5.0 proteocephalus glanduliger anterior and midintestine 7 14.0 1–2 2.0 ornithodiplostomum sp. metacercariae mid-dorsal muscles between dorsal fin and lateral line 7 14.0 0–140 140.0 *n = sample size table 2 polyonchobothrium clarias measurements (in μm, unless otherwise stated) n = 6 range mean length (mm) maximum width number of proglottids scolex size (l*w)1 number of hooks in apical rostellum mean size of hooks mean size of immature proglottids (l*w) mean size of mature proglottids (l*w) mean size of gravid proglottids (l*w) mean egg size 23–43 280–701 133–230 420–680*160–230 26–30 35–57 80–180*146–170 122–179*129–222 240–378*280–710 28–42.5*21–38 29.8 506 183.0 527*198 28 44 150*151 151*186 330*506 32.8*27.0 1 l*w : l = length, w = width table 3 proteocephalus glanduliger measurements (in mm, unless otherwise stated) measurement 1 2 mean strobila length strobila maximum width no. of proglottids scolex (l*w) 1 size of apical organ (l*w) size of suckers (mean l*w) size of mature proglottid (l*w) size of gravid proglottid (l*w) size of cirrus sac (l*w) size of testes 7.04 0.52 58 1.08*0.82 0.36*0.28 0.36*0.32 0.13*0.45 0.14*0.03 – 13.0 1.08 45 1.28*0.92 – 0.48*0.40 – – – 10.02 0.80 51.5 1.20*0.88 0.36*0.28 0.44*0.36 – – – 1 l*w : l = length, w = widths 106 cestode and digenean parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa fig. 3 proteocephalus glanduliger (a, d) scolex (scale bar = 400 μm), (b) gravid proglottid, (c) mature proglottid, (e) transverse section (scale bars = 200 μm), ec = excretory canal, go = glandular organ, cs = cirrus sac, lm = longitudinal muscles, s = sucker, sg = shell gland, t = testis, tg = tegument, ut = uterus, vt = vitellaria a b c d e go ut ec t vt vt ut ov s sg cs ec cs ec t vt lm tg 107 m. barson & a. avenant-oldewage (1977) recorded it in 11 of 337 hosts and mashego (2001) in 11 of 115 hosts, with a mean intensity of seven parasites per fish. while the present specimens were much larger (table 3) than those described by janicki (freze 1965) and mashego (2001) from c. anguillaris and c. gariepinus, respectively, the glandular organ at the apex of the scolex was much smaller and almost equal in size to the suckers (fig 3a). serial sectioning of the present material confirmed the medullary positioning of the testis and vitellaria (fig. 3e and 5f), a characteristic of the genus proteocephalus (freze 1965; schmidt 1986; rego 1994). the histology of the worm as observed from the sections resembles the description of p. glanduliger by mashego (2001) in c. gariepinus from four south african dams. proteocephalid cestodes have been found in c. gariepinus from the neighbouring zim babwe (barson 2004; chishawa 1991; douëllou 1992) but as they were not specifically identified, comparifig. 4 ornithodiplostomum sp. metacercariae (a) encysted (scale bar = 400 μm), (b) drawing showing main features (scale bar = 350 μm), (c) whole worm (scale bar = 250 μm), (d) anterior end showing oral sucker and pharynx (left arrow) and ventral sucker (right arrow), (e) posterior end showing rudimentary genital/excretory opening (scale bars = 150 μm), eo = excretory opening, ic = intestinal caecum, os = oral sucker, ph = pharynx, t = testes, to = tribocytic organ, vs = ventral sucker a b c ed os ph ic vs to t eo to t 108 cestode and digenean parasites of clarias gariepinus (burchell, 1822) from rietvlei dam, south africa fig. 5 polyonchobothrium clarias (photographs) (a) scolex (sem) showing the arrangement of hooks around the rostellum (scale bar = 200 μm); (b) mature proglottid (lm); (c) gravid proglottid (lm) (scale bars = 400 μm); (d) embryonated egg (lm) (scale bar = 50 μm); (e) strobila (sem) showing genital openings (go) and fused segment (fs) on the ventral surface; (f) light micrographs of azan stained transverse section through early mature proglottid (scale bar = 60 μm), t = testes, vt = vitellaria a b c ed f son with their south african counterparts cannot be made. in african fish species other than the clariidae, many proteocephalid species have been described from the sudan (khalil 1963, 1973; jones 1980), the democratic republic of congo, egypt, liberia and senegal (khalil 1973; khalil & polling 1997). trematoda ornithodiplostomum sp. metacercariae (fig. 4) metacercariae coiled and encysted in tough white cysts formed by the host in the dorso-lateral muscles and a thin transparent wall, secreted by the par asite (fig. 4a), thus difficult to identify. the few successfully excysted metacercariae were identified as belonging to the family diplostomidae and closely resemble ornithodiplostomum sp. body indistinctly bipartite with no pseudosuckers (as in other diplostomes), but with a large circular holdfast organ (tribo cytic organ). developing, immature testes apparent; subterminal eversible genital apparatus also clear from photomicrographs (fig. 4c–e). the present specimens match the description of adult ornithodiplostomum sp. that occurs in the podicipedidae (grebes) and anatidae (ducks) from the table 4 ornithodiplostomum sp. measurements (μm) n = 3 range mean length maximum width size of oral sucker (l*w)1 size of pharynx (l*w) size of ventral sucker (l*w) size of tribocytic organ (l*w) size of anterior testes size of posterior testes 544–656 320–356 97–115*60–75 48–55*45–52 75–82*64–70 138–150*115–124 44–60*31.5–42.5 62–73.3*50–58 602 336 108*66.5 50*49.5 80–68 142.3*119.5 53.3*40 67*52.5 1 l*w : l = length, w = width 109 m. barson & a. avenant-oldewage holarctic region and africa (niewiadomska 2002). this is the first record of this genus in south africa, neither appearing in the latest checklist (khalil & polling 1997) nor in canaris & gardner’s (1967) checklist of helminths of african vertebrates. how ever, related genera such as diplostomum, neodiplo stomum and postodiplostomum are quite common in southern africa (mashego 1977; prudhoe & hus sey 1977; khalil & polling 1997). the prevalence of this parasite was low (14 %) but the intensity of infestation was very high (table 1). only three metacercariae were successfully recovered and measured (table 4) from the cysts. some were indistinct and difficult to identify (e.g. fig 4a), thus it cannot be concluded that all the metacercariae found were ornithodiplostomum sp., or even exclusively diplostomid. experimental infection of suitable bird hosts with these metacercariae is the only way to obtain adult parasites which can then identified to species level. acknowledgements we acknowledge the assistance of the university of johannesburg (uj) and the water research fund for southern africa (warfsa) in funding this study; dr r. greenfield, g. o’brien, prof. v. wepener, drr m.a. tsotetsi, m. mathonsi & t. muteveri (zoology, uj) for field and laboratory assistance; e. lutsch (zoology, uj), dr w. oldewage (spectrau) and mr e. karim (uj graphics) for technical support. references aderounmu, e.a. & adeniyi, f. 1972. cestodes in fish from a pond at ile-ife, nigeria. the african journal of tropical hydro biology and fisheries, 2:151–156. amin, o.m. 1978. intestinal helminths of some nile fishes near cairo, egypt, with redescriptions of camallanus kirandensis baylis, 1928 (nematoda) and bothriocephalus aegyptiacus rysavy and moravec, 1975 (cestoda). journal of parasitology, 64:93–101. avenant-oldewage, a. 2001. protocol for the assessment of fish health based on the health index. report and manual for training of field workers to the rand water board. ver een iging: rand water (report no. 2001/03/31). barson, m. 2004. a study of the helminth parasites of fish and fish-eating birds in the manyame catchment, zimbabwe. m.phil. thesis, university of zimbabwe. barson, m. & marshall, b.e. 2004. first record of contracaecum spp. (nematoda; 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fight for wetlands. http:// www.boma.wildnetafrica.com. whitfield, a.k. & heeg, j. 1977. on the life cycles of the cestode ptychobothrium belones and nematodes of the genus contracaecum from lake st. lucia, zululand. south african journal of science, 73:121–122. yamaguti, s. 1958. systema helminthum. vol. i. digenetic trem atodes of vertebrates. part i & ii. new york & london: interscience publishers, inc. yamaguti, s. 1959. systema helminthum. vol. ii. the cestodes of vertebrates. new york & london: interscience publishers, inc. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice madekurozwa_209-216.indd introduction although a low latitude inhabitant, the mature ostrich (struthio camelus) is a seasonal breeder, with reproductive activity being influenced by photoperiod and probably feed availability (sauer 1972; degen, weil, rosenstrauch, kam & dawson 1994; hicks-all dredge 1998). likewise, studies on the immature ostrich have revealed a seasonality in the differentiation of the reproductive systems of both male and female birds (madekurozwa 2002a, b, 2004, 2005; made kurozwa, chabvepi, matema & teerds 2002). in one particular study changes in the morphology of the uterine region of the oviduct were found in immature ostriches having either active or inactive ovaries (madekurozwa 2004). however, despite the information gained from this study, our knowledge of the seasonal morphological changes in the uterus is incomplete as there do not appear to have been any ultrastructural studies carried out on this oviducal region in the immature ostrich. in addition, it would appear from the literature that ultrastructural investigations of the uterus in avian species have concentrated on mature birds during the breeding season (breen & de bruyn 1969; wyburn, johnston, draper & davidson 1973; bakst & howarth 1974; bakst 1978). 209 onderstepoort journal of veterinary research, 74:209–216 (2007) ultrastructural features of the uterus in the sexually immature ostrich (struthio camelus) during periods of ovarian inactivity and activity m-c. madekurozwa* department of anatomy and physiology, faculty of veterinary science, university of pretoria onderstepoort, 0110 south africa abstract madekurozwa, m-c. 2007. ultrastructural features of the uterus in the sexually immature ostrich (struthio camelus) during periods of ovarian inactivity and activity. onderstepoort journal of veterinary research, 74:209–216 the ultrastructure of the surface epithelium and tubular glands of the uterus in the immature ostrich is described. in ostriches with inactive ovaries the uterus is lined by a non-ciliated simple columnar epithelium, with basally located heterochromatic nuclei. scanning electron microscopy revealed that these non-ciliated cells have a dense microvillous cover. a simple columnar to pseudostratified columnar epithelium, comprised of non-ciliated and ciliated cells, lines the uterus in birds with active ovaries. the ciliated cells possess a wide luminal region, which contains a nucleus and various organelles. an accumulation of secretory granules was observed in the apical regions of the non-ciliated cells, as well as in a few ciliated cells. in addition to non-ciliated and ciliated cells, a cell type with rarefied cytoplasm was also identified. these cells appear to correspond to calcium secreting cells identified in other avian species. the results of this study indicate that, although uterine differentiation is present in immature ostriches with active ovaries, the production of secretory product appears to occur mainly in non-ciliated epithelial cells. keywords: electron microscopy, ostrich, uterus, ultrastructure * e-mail: madex@op.up.ac.za accepted for publication 13 march 2007—editor 210 features of uterus in sexually immature ostrich (struthio camelus) during ovarian inactivity and activity in light of the paucity of ultrastructural information on the uterus of immature birds, and of the immature ostrich in particular, the present study was undertaken. the ultrastructural changes occurring in the surface epithelium and tubular glands of the uterus in the immature ostrich during ovarian inactivity and activity are described. materials and methods forty sexually immature female ostriches aged between 12 and 14 months and weighing 90–100 kg, were used. twenty-five of the birds had active ovaries, which contained 10–25 small, predominantly yellow-yolk follicles, with the diameters of the largest follicles ranging from 11 to 19 mm. these birds were sampled between september and december, a period of increasing daylength in the southern hemisphere. fifteen ostriches with inactive ovaries were collected from march to june, a period of decreasing daylength. the birds were killed at a commercial abattoir, employing a standard slaughter pro tocol. oviducts were collected 10–15 min after the birds were killed. for transmission and scanning electron microscopy tissue samples from the major part of the uterus were fixed by immersion in 3 % glutaral dehyde buffered in 0.067 m sodium cacodylate buffer (ph 7.2). after fixation the tissue samples for trans mis sion electron microscopy were dehydrated through a graded series of ethanol and embedded in an epoxy resin. semi-thin sections were stained with tolui dine blue, while ultra-thin sections were stained with uranyl acetate and lead citrate. the samples were viewed with a philips cmio transmission electron microscope (fei, eindhoven, the netherlands). tissue samples for scanning electron microscopy were dehydrated through a graded series of acetone and thereafter dried in a critical point dryer. the tissues were then mounted on aluminium stubs and coated with gold palladium before they were viewed in a philips xl20 scanning electron microscope (fei, eindhoven, the netherlands). additional samples for light microscopy were immersion-fixed in bouin’s fluid for 12 h. after fixation, tissues were processed routinely for histology and embedded in paraffin wax. the presence of acid and neutral mucopolysaccharides was demonstrated using a combined alcian blue-periodic acid schiff (pas) technique (mowry 1956). results immature ostriches with inactive ovaries scanning electron microscopy (sem) revealed the presence of a series of uniform undulating primary mucosal folds, which were separated by deep crypts. non-ciliated, microvillous cells lined the surface of the uterus in these birds (fig. 1). the microvillous cover was generally dense, although occasional cells with only a few microvilli were also observed. transmission electron microscopy showed that the non-ciliated cells formed a simple columnar epithelium. the cells contained basally located, large heterochromatic nuclei. most of the organelles, which included mitochondria, electron dense bodies, rough endoplasmic reticulum (rer) and ribosomes, were located in the supranuclear area of the cell (fig. 2). the electron dense bodies varied in shape and heterogenicity. a series of junctional complexes linked the apical regions of adjacent cells. in addition, the lateral plasma membranes displayed simple folding, both apically and basally. in birds with inactive ovaries the uterine epithelium did not stain with alcian blue or pas. immature ostriches with active ovaries the mucosa of the uterus was thrown into thick lamellar primary folds, which still retained the arrangement observed in birds with inactive ovaries. deep crypts indented the primary folds, resulting in the formation of numerous secondary folds (fig. 3). cil iated and non-ciliated cells, with the former being pre dominant, lined the mucosal folds. tufts of cilia completely covered adjacent non-ciliated cells in most areas. however, in areas lined by only a few ciliated cells, the non-ciliated cells and the openings of tubular glands were clearly visible (fig. 4). the glandular openings were circumscribed by a raised area, which possessed a few microvilli (fig. 4). transmission electron microscopy revealed that in birds with active ovaries a simple columnar to pseudostratified columnar epithelium lined the uterus. non-ciliated cells formed the simple columnar epithelium, while the pseudostratified epithelium consisted of both non-ciliated and ciliated cells. the ciliated cells exhibited well-developed, regularly arranged cilia, which were intermingled with microvilli. the elongated or irregular shaped, euchromatic nuclei, were located in the wide luminal regions of the cells (fig. 5). several mitochondria, rer profiles, electron dense bodies and bundles of fibrils 211 m-c. madekurozwa fig. 4 scanning electron micrograph showing non-ciliated (nc) and ciliated (cc) cells in the uterus of an immature ostrich with an active ovary. the mucosal surface is indented with crypts (arrows) resulting in the formation of secondary folds. in addition, occasional tubular gland openings are observed (arrowhead). inset: scanning electron micrograph of a tubular gland opening (t) in the uterus of an immature ostrich with an active ovary. the raised rim of the tubular gland opening possesses a few microvilli (arrows). c: tuft of cilia fig. 1 scanning electron micrograph of non-ciliated microvillous cells in the uterus of an immature ostrich with an inactive ovary. some cells (asterisk) have a sparse covering of microvilli fig. 2 transmission electron micrograph of the non-ciliated simple columnar epithelium in an immature ostrich with an inactive ovary. most of the organelles which include electron dense bodies (e) are concentrated in the supranuclear cytoplasm. junctional complexes (arrows) and folding (arrowhead) of the lateral plasma membranes unite adjacent cells fig. 3 scanning electron micrograph of undulating mucosal folds in the uterus of an immature ostrich with an active ovary. the primary mucosal folds are further divided into secondary folds (s) by crypts (arrows) 212 features of uterus in sexually immature ostrich (struthio camelus) during ovarian inactivity and activity were identified in the supranuclear cytoplasm. secretory granules occurred in the supranuclear regions of a few cells (fig. 6). in some areas slender non-ciliated cells were observed between the ciliated cells. the nuclei of the non-ciliated cells were elongated, heterochromatic fig. 5 survey transmission electron micrograph of the pseudostratified columnar epithelium lining the uterus in an immature ostrich with an active ovary. ciliated cells typically contain elongated or irregular shaped nuclei (n). the apical plasma membrane exhibits cilia (arrows) and microvilli (arrowheads) fig. 6 transmission electron micrograph showing the occurrence of secretory granules (arrows) in ciliated cells in the uterus of an immature ostrich with an active ovary fig. 7 survey transmission electron micrograph of the pseudostratified columnar epithelium lining the uterus in an immature ostrich with an active ovary. slender non-ciliated cells (nc) alternate with ciliated cells (cc). the non-ciliated cells contain secretory granules (arrows). vacuoles (arrowheads) occur either in the middle or apical regions of the non-ciliated cells fig. 8 transmission electron micrograph of the pseudostratified columnar epithelium lining the uterus in an immature ostrich with an active ovary. a few cells with electron lucent cytoplasm (c) and a basal accumulation of mitochondria (m) occur in the surface epithelium 213 m-c. madekurozwa and basally placed. secretory granules were concentrated mainly in the apical regions of the cells (fig. 7). other organelles and inclusions present in the supranuclear cytoplasm included mitochondria, profiles of rer and vesicles. vacuoles containing membranous profiles were observed in either the middle or apical regions of the non-ciliated cells. in both non-ciliated and ciliated cells folding of the lateral plasma membrane, in the apical parts of the cells, was present although not extensive. wellformed junctional complexes were observed apically, as well as distally along the lateral plasma membrane. occasional cells with rarefied cytoplasm were observed in the epithelium. the location of these electron lucent cells varied, with cells being observed in both the apical and basal regions of the epithelium. the apical electron lucent cells had a wide luminal region, which contained a round nucleus with peripheral clumps of heterochromatin. an accumulation of mitochondria was observed in the narrow basal region of the cell (fig. 8). a second type of electron lucent cell was observed close to the basal lamina. these cells contained irregular shaped heterochromatic nuclei. profiles of rer, mitochondria and vacuoles were observed in the cytoplasm, which contained relatively few organelles (fig. 9). in addition to the electron lucent cells, a few cells with condensed cytoplasm and heterochromatic, elongated nuclei were also observed in the epithelium. apically placed electron dense bodies were the only recognizable organelles in these cells. groups of pyramidal shaped cells, which displayed numerous microvilli on their luminal surfaces, formed the tubular glands (fig. 10). the lumina of the glands were devoid of secretory material. heterochromatic fig. 9 transmission electron micrograph of an electron lucent cell located in the basal region of the epithelium. rer profiles (arrows) are a prominent feature of these cells. arrowheads: basal lamina fig. 10 survey transmission electron micrograph of a tubular gland in the uterus of an immature ostrich with an active ovary. g: gland cells. l: lumen. inset: the luminal regions of the gland cells contain several electron dense bodies (arrows) and accumulations of vesicles (v). intricate folding (i) of the plasma membrane and junctional complexes (arrowheads) unite adjacent gland cells. l: lumen 214 features of uterus in sexually immature ostrich (struthio camelus) during ovarian inactivity and activity nuclei, which were either round or elongated in shape, were placed centrally or basally in the cells. distributed in the supranuclear cytoplasm were mitochondria, vesicles, microfilaments and profiles of rer. the presence of a few lipid droplets and electron dense bodies of various sizes was noted in some cells. well-developed junctional complexes and folding of the lateral plasma membranes characterized the apical regions of the gland cells (fig. 10). the majority of non-ciliated surface epithelium cells stained strongly with alcian blue and pas, indicating the presence of both acidic and neutral mucopolysaccharides. tubular gland cells and the majority of ciliated cells were negative for both alcian blue and pas. occasional ciliated cells displayed supranuclear diastase-resistant pas staining, which indicated the presence of neutral mucopolysaccharides. discussion the present study has established that the uterus in the immature ostrich undergoes ciliogenesis and gland formation, which appear to be associated with the presence of an active ovary. although hormonal studies on the immature ostrich have not been carried out, based on the results of the present and previous studies (madekurozwa 2002a, b, 2004, 2005), it would appear that the ovarian follicles in birds with active ovaries produce hormones in sufficient quantities to stimulate oviducal differentiation. these oviducal changes are most likely stimulated by oestrogen, as it is known that in birds small yellow-yolk follicles produce oestrogen whilst preovulatory follicles secrete progesterone (tilly, kowalski & johnson 1991). furthermore, research in both birds (wrenn 1971; anderson & hein 1976; perche, laine, pageaux, laugier & sandoz 1989) and mammals (sawyer, olson & gorell 1984; verhage, mavrogianis, boice, li & fazleabas 1990; abe & oikawa 1993) has indicated that the stimulation of ciliogenesis and tubular gland development is primarily a function of oestrogen. as revealed by sem, ciliogenesis in the uterus of immature ostriches with active ovaries was patchy. similar observations were also made in the magnum of immature ostriches (madekurozwa 2005). to date there is no published information on the distribution of ciliated cells in the uterus of the mature ostrich. as a result it is impossible to determine whether the uneven distribution of ciliated cells observed in the current study is a common observation in the ostrich or if it is peculiar to immature ostriches. transmission electron microscopic observations of both non-ciliated and ciliated cells in the uterus of the immature ostrich with an active ovary showed that the morphology of these cells is similar to cells in the uterus of the domestic fowl (johnston, aitken & wyburn 1963), pekin duck (balachandran, bhatnagar & geissinger 1985), japanese quail (yamamoto, ozawa & nagai 1985) and mature ostrich (muwazi, baranga, kayanja & schliemann 1982). an interesting observation of the present study was the occurrence of secretory granules in the majority of non-ciliated cells, but only in a few ciliated cells. based on the alcian blue-pas staining reaction, the non-ciliated cells contained a mixture of acid and neutral mucopolysaccharides, while ciliated cells only demonstrated the latter mucopolysaccharide. although the exact nature of these mucopolysaccharides in the immature ostrich is unknown, it is known that non-ciliated cells in the uterus of the domestic fowl produce the proteoglycan, ovoglycan, which is a component of the eggshell matrix (fernandez, moya, lopez & arias 2001). in addition, the non-ciliated cells in the domestic fowl also produce osteopontin, a matrix protein involved in the mineralization of the eggshell (fernandez, escobar, lavelin, pines & arias 2003). the cuticle and shell pigment are thought to be produced by both ciliated and non-ciliated cells (breen & de bruyn 1969; baird, solomon & tedstone 1975). in addition to typical non-ciliated and ciliated epithelial cells, cells with either rarefied or condensed cytoplasm were also observed. cells with rarefied cytoplasm have been identified in the uterine region of the mature ostrich (muwazi et al. 1982), as well as in the domestic fowl (ljungkvist 1967). ljungkvist (1967) identified the electron lucent cells as unicellular calcium secreting glands. further research by solomon, fryer & baird (1975) identified the surface epithelium of the uterus as the primary site of calcium secretion, although the exact cells involved were not demonstrated. in addition to the electron lucent cells, a few cells with condensed cytoplasm were observed in the uterine epithelium of the immature ostrich. based on the morphological features displayed it is likely that these cells were degenerating. to date no detailed ultrastructural studies have been conducted on the regression of the oviducal epithelium in birds. light microscopic studies conducted on the japanese quail (eroschenko & wilson 1974) and the 215 m-c. madekurozwa pied myna (gupta & maiti 1987) indicate that large sections of the oviducal epithelium slough off during regression. further ultrastructural studies need to be carried out on immature ostriches with regressive oviducts to ascertain the mode and stages of involution in this species. in addition to the surface epithelium, tubular glands located in the lamina propria also make a significant contribution to the secretions of the uterus. in this study sem revealed the presence of several tubular gland openings in the uterus of birds with active ovaries. in the magnum of the immature ostrich the glandular openings took the form of pores surrounded by non-ciliated cells (madekurozwa 2005). the raised rim, which surrounded the glandular openings in the uterus of the immature ostrich, was not observed in the magnal region. although bakst & howarth (1974) observed glandular openings in the magnum of the domestic hen, no mention of these structures was made in the description on the uterus. this might have been due to the presence of numerous cilia, which effectively obscured the gland openings. the ultrastructural morphology of the tubular gland cells in the uterus of the immature ostrich is similar to that of the japanese quail (yamamoto et al. 1985) and the domestic fowl (breen & de bruyn 1969). as in the japanese quail (yamamoto et al. 1985) and domestic fowl (breen & de bruyn 1969) the gland cells in the immature ostrich were devoid of secretory granules. research conducted by jande, tolnai & lawson (1981) and yamamoto et al. (1985) has shown that gland cells are involved in the transportation of calcium, which is utilized in the calcification of the eggshell. furthermore, wasserman, smith, smith, brindak, fullmer, krook, penniston & kumar (1991) have localized a calcium pump in the microvillar plasma membrane of gland cells and the calcium binding protein, calbindin-d28k, in the cytoplasm of the gland cells. these findings suggest that the tubular glands are the primary sites of calcium secretion in the uterus. however, as mentioned previously, cells in the surface epithelium are also thought to play a role in calcium secretion (ljungkvist 1967; solomon et al. 1975). in conclusion, the present ultrastructural study has shown cellular differentiation in the form of ciliated cells and tubular glands in the uterus of immature ostriches with active ovaries. a significant finding is the presence of secretory granules in most non-ciliated cells. however, the presence of secretory granules in only a few of the ciliated cells indicates that the secretory cells in the uterus of the immature ostrich are not fully functional. acknowledgements the author thanks technical staff in the university of pretoria’s department of pathology, electron microscope unit and department of education innovation (creative studios) for their assistance. the university of pretoria and the national research foundation (thuthuka programme) funded this study. references abe, h. & oikawa, t. 1993. observations by scanning electron microscopy of oviductal epithelial cells from cows at follicular and luteal phases. anatomical record, 235:399–410. anderson, r.g.w. & hein, c.e. 1976. estrogen dependent ciliogenesis in the chick oviduct. cell and tissue research, 171:459–466. baird, t., solomon, s.e. & tedstone, d.r. 1975. localisation and characterization of egg shell porphyrins in several avian species. british poultry science, 16:201–208. bakst, m. 1978. scanning electron microscopy of the oviducal mucosa apposing the 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research | vol 85, no 1 | a1583 | 27 june 2018 original research spatial and temporal distribution of foot-and-mouth disease in four districts situated along the uganda–tanzania border: implications for cross-border efforts in disease control susan d. kerfua, gabriel shirima, lughano kusiluka, chrisostome ayebazibwe, robert mwebe, sarah cleaveland, daniel haydon onderstepoort journal of veterinary research | vol 85, no 1 | a1528 | 27 august 2018 original research immunogenicity of a plasmid dna vaccine encoding g1 epitope of bovine ephemeral fever virus g glycoprotein in mice reza pasandideh, masoud reza seyfi abad shapouri, mohammad taghi beigi nassiri onderstepoort journal of veterinary research | vol 85, no 1 | a1617 | 28 august 2018 original research lay perceptions, beliefs and practices linked to the persistence of anthrax outbreaks in cattle in the western province of zambia doreen c. sitali, mwamba c. twambo, mumba chisoni, muma j. bwalya, musso munyeme onderstepoort journal of veterinary research | vol 85, no 1 | a1615 | 29 august 2018 original research prevalence and aetiology of coccidiosis in broiler chickens in bejaia province, algeria nedjima debbou-iouknane, hama benbarek, abdelhanine ayad onderstepoort journal of veterinary research | vol 85, no 1 | a1590 | 18 september 2018 original research comparative evaluation of dry and liquid rime lamp in detecting trypanosomes in dead tsetse flies peter nambala, janelisa musaya, kyoko hayashida, emmanuel maganga, edward senga, kelita kamoto, john chisi, chihiro sugimoto onderstepoort journal of veterinary research | vol 85, no 1 | a1543 | 03 october 2018 original research detection of virulence factors of south african lactococcus garvieae isolated from rainbow trout, oncorhynchus mykiss (walbaum) cornelia m. meyburgh, robert r. bragg, charlotte e. boucher onderstepoort journal of veterinary research | vol 85, no 1 | a1568 | 04 october 2018 original research prevalence of canine babesia and ehrlichia co-infection and the predictive value of haematology yolandi rautenbach, johan schoeman, amelia goddard onderstepoort journal of veterinary research | vol 85, no 1 | a1626 | 09 october 2018 57 65 73 79 87 93 99 108 page i of ii table of contents i research communication genetic characterisation of african swine fever virus from 2017 outbreaks in zambia: identification of p72 genotype ii variants in domestic pigs edgar simulundu, yona sinkala, herman m. chambaro, andrew chinyemba, frank banda, lynnfield e. mooya, joseph ndebe, simbarashe chitanga, chitwambi makungu, gift munthali, paul fandamu, ayato takada, aaron s. mweene onderstepoort journal of veterinary research | vol 85, no 1 | a1562 | 26 june 2018 research communication prevalence of methicillin-resistant staphylococcus aureus among large commercial pig herds in south africa shani van lochem, peter n. thompson, cornelius h. annandale onderstepoort journal of veterinary research | vol 85, no 1 | a1561 | 17 july 2018 research communication first record of the marine turtle leech (ozobranchus margoi) on hawksbill turtles (eretmochelys imbricata) in the inner granitic seychelles byron m. göpper, nina m. voogt, andre ganswindt onderstepoort journal of veterinary research | vol 85, no 1 | a1604 | 30 august 2018 review article amphistome infections in domestic and wild ruminants in east and southern africa: a review davies m. pfukenyi, samson mukaratirwa onderstepoort journal of veterinary research | vol 85, no 1 | a1584 | 18 october 2018 original research genetic characterisation of virulence genes associated with adherence, invasion and cytotoxicity in campylobacter spp. isolated from commercial chickens and human clinical cases samantha reddy, oliver t. zishiri onderstepoort journal of veterinary research | vol 85, no 1 | a1507 | 15 february 2018 original research prevalence of mastitis pathogens in south african pasture-based and total mixed ration-based dairies during 2008 and 2013 david blignaut, peter thompson, inge-marié petzer onderstepoort journal of veterinary research | vol 85, no 1 | a1482 | 31 may 2018 original research occurrence of the specific long spike burst pattern in the ovine proximal gallbladder as an indication of myoelectric regional variability krzysztof w. romański, józef nicpoń onderstepoort journal of veterinary research | vol 85, no 1 | a1455 | 11 june 2018 original research genotypic characterisation of avian paramyxovirus type-1 viruses isolated from aquatic birds in uganda agnes wanyana, kizito k. mugimba, omony j. bosco, halid kirunda, jessica l. nakavuma, angélique teillaud, mariette f. ducatez, denis k. byarugaba onderstepoort journal of veterinary research | vol 85, no 1 | a1510 | 25 june 2018 1 6 10 13 26 35 42 vol 85, no 1 (2018) issn: 0030-2465 (print) | issn: 2219-0635 (online)onderstepoort journal of veterinary research 50 http://www.ojvr.org open access table of contents ii original research peste des petits ruminants virus infection of black bengal goats showed altered haematological and serum biochemical profiles shahana begum, mohammed nooruzzaman, murshida parvin, nijaya mohanto, rokshana parvin, mohammad r. islam, emdadul h. chowdhury onderstepoort journal of veterinary research | vol 85, no 1 | a1595 | 15 october 2018 original research molecular analysis of shiga toxin-producing escherichia coli o157:h7 and non-o157 strains isolated from calves maryam kohansal, ali ghanbari asad onderstepoort journal of veterinary research | vol 85, no 1 | a1621 | 17 october 2018 113 123 correction corrigendum: spatial and temporal distribution of foot-and-mouth disease in four districts situated along the uganda–tanzania border: implications for cross-border efforts in disease control susan d. kerfua, gabriel shirima, lughano kusiluka, chrisostom ayebazibwe, robert mwebe, sarah cleaveland, daniel haydon onderstepoort journal of veterinary research | vol 85, no 1 | a1716 | 11 december 2018 reviewer acknowledgement onderstepoort journal of veterinary research | vol 85, no 1 | a1713 | 03 december 2018 130 131 page ii of ii madekurozwa.indd introduction the motility of the oviduct in birds is regulated by hormonal and neural factors (wechsung & houvenaghel 1988). previous studies on the oviduct of the domestic fowl (gilbert & lake 1963; costagliola, mayer, vittoria, carrese, lamanna & cecio 1997), the japanese quail (arjamaa & talo 1983) and the pigeon (atoji, mizutani, yamamoto & suzuki 2000) have demonstrated the presence of an extensive neural network in both the intermuscular and muscular regions of the organ. these neural networks are known to play a significant role in the transportation of the egg in the oviduct by co-ordinating muscle contraction (talo & kekalainen 1976; wechsung & houvenaghel 1988). a dysfunction in oviducal motility results in a condition known as egg-binding in which the formed egg is retained within the oviduct (rosskopf & woerpel 1984; krautwald-junghanns, kostka & hofbauer 1998). ruptured oviduct and peritonitis are possible consequences of this condition (rosskopf & woerpel 1984). although the exact cause of egg-binding is unknown, it is possible that a disturbance in the innervation to the oviduct might be involved. however, before the possible role of the nerves of the oviduct 131 onderstepoort journal of veterinary research, 73:131–137 (2006) immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in nerves in the oviduct of the sexually immature ostrich, struthio camelus m.-c. madekurozwa* department of anatomy and physiology, faculty of veterinary science, university of pretoria onderstepoort, 0110 south africa abstract madekurozwa, m.-c. 2006. immunoreactivities to protein gene product 9.5, neurofilament protein and neuron specific enolase in nerves in the oviduct of the sexually immature ostrich, struthio camelus. onderstepoort journal of veterinary research, 73:131–137 the present study investigated the distribution of nerves in the oviduct of the sexually immature ostrich. the presence of protein gene product 9.5, neurofilament protein and neuron specific enolase nerve fibres were demonstrated in the infundibulum, magnum, isthmus, shell gland and vagina. nerve fibres containing protein gene product 9.5, neuron specific enolase and neurofilament protein were particularly numerous in the tunica muscularis and intermuscular connective tissue areas of the shell gland and vagina. the presence of a large number of nerves in these oviductal regions is probably important in the coordination of muscle contraction. an interesting finding of the study was the presence of protein gene product 9.5 and neuron specific enolase immunopositive nerve fibres in the walls of blood vessels. it is likely that these nerves are autonomicin nature and play a role in the regulation of blood flow. this study has shown the presence of an extensive neural network in the oviduct of the ostrich. in addition, the results of the investigation have shown that the neuronal markers protein gene product 9.5, neurofilament protein and neuron specific enolase can be used to demonstate nerve fibres in the ostrich keywords: immunohistochemistry, nerves, neurofilament protein, neuron specific enolase, ostrich, oviduct, protein gene product 9.5 * e-mail: madex@op.up.ac.za accepted for publication 30 march 2006—editor 132 immunoreactivities to protein gene product 9.5, neurofi lament protein and neuron specifi c enolase in ostrich in this condition can be investigated it is necessary to study their normal distribution within the oviduct. in mammals plasma levels of oestrogen influence the density of nerves in the female reproductive tract (haase, buchman, tietz & schramm 1997; brauer, llodra, scorza, chavez, burnstock, thrasivoulu & cowen 1999; zoubina & smith 2001; zoubina, mize, alper & smith 2001). although the effect of oestrogen on the innervation of the avian oviduct is unknown it was decided to use prepubertal ostriches in the present study to avoid any possible effects of high oestrogen levels on oviductal nerve density. the distribution of nerves within the oviduct of these birds was studied using antibodies against protein gene product 9.5 (pgp), neurofilament protein (np), and neuron specific enolase (nse). a marker for afferent nerves, np, has been identified in the uterus of the human (khong, tee & kelly 1997), the bovine (wrobel & kujat 1993) and the guinea pig (alm, lund berg, wharton & polak 1988a). the general neuronal markers, pgp and nse, have been utilized to demonstrate nerve fibres in the uterus of the horse (bae, corcoran & watson 2001) and the guinea pig (alm, lundberg, wharton & polak 1988b; lundberg, alm, wharton & polak 1988). materials and methods fifteen prepubertal female ostriches, with quiescent ovaries, were used in the study. the birds were aged between 12 and 14 months, with body mass of 90– 100 kg. at a commercial ostrich abattoir the ostriches were electrically stunned and exsanguinated. tissue samples were collected 10–15 min after slaughter from the infundibulum, magnum, isthmus, shell gland pouch and cranial vagina. five samples were taken from each region. the tissue samples were immersion-fixed in either 4 % paraformaldehyde (ph 7.2) or bouin’s fluid for 14 h. some of the samples were fixed in bouin’s fluid because the antibody against protein gene product 9.5 (dakocytomation, den mark) is only suitable for use on paraffin sections. the tissue samples fixed in paraformaldehyde were placed, for 24 h at 4 °c, in a 30 % sucrose solution made up in 0.01m phosphate buffered saline solution 1–1 (pbs, ph 7.4). thereafter, they were snap frozen in oct compound (sakura, ca, usa) in an isopentane/dry ice slurry. the samples were then stored at –80 °c. the tissue samples fixed in bouin’s fluid were processed routinely for histology and embedded in paraffin wax. the immunostaining technique was performed on 10 μm thick cryostat sections and 5 μm thick paraffin sections, using a lsab-plus kit (dakocytomation, denmark). the cryostat sections were air dried for 60 min at room temperature after which they were rinsed in pbs. endogenous peroxidase activity in both cryostat and paraffin sections was blocked using a 3 % (v/v) hydrogen peroxidase solution in water for 5 min. the slides were then rinsed in pbs for 5 min. thereafter, the paraffin sections were microwaved at 750 w for three cycles of 5 min each. after being allowed to cool for 20 min the sections were rinsed with pbs. the paraffin sections were then incu bated for 30 min at room temperature with a polyclonal antibody against pgp (dakocytomation, denmark), at a dilution of 1:50. the cryostat sections were incubated for 30 min with monoclonal antibodies against np (dakocytomation, denmark), and nse (dakocytomation, denmark) at dilutions of 1:75 and 1:50, respectively. after this incubation all the slides were rinsed with pbs and then incubated for 15 min with a biotinylated secondary antibody (lsabplus kit, dakocytomation, denmark). thereafter, the slides were rinsed in pbs and subsequently incubated for 15 min with the streptavidin peroxidase component of the lsab-plus kit. slides were then rinsed in pbs and bound antibody was visualized after the addition of a 3,3’-diaminobenzidine tetrachloride solution (lsab-plus kit, dakocytomation, denmark). slides were counterstained with mayer’s haematoxylin for 20 s before being dehydrated in graded concentrations of ethanol. in the negative controls, the primary antibodies were replaced with either normal mouse or normal rabbit serum. a histological section of nerve was used as a positive control. no background staining was detected in the negative control sections, whilst nerve fibres immunoreactive for pgp, np and nse were observed in the positive control sections. a subjective assessment of the density and distribution of immunoreactive nerves (bae et al. 2001) was performed under the x 40 objective of a light microscope and was graded subjectively as: – = no nerve fibres observed; + = very few nerve fibres observed; + = a small number of nerve fibres observed; ++ = a moderate number of nerve fibres observed and +++ = a large number of nerve fibres observed. the relative density of immunoreactive nerves is summarized in table 1. 133 m.-c. madekurozwa results nerve fibres immunoreactive for pgp, np and nse were observed in all the oviductal regions studied. furthermore, immunoreactivity was demonstrated in nerve fibres in all the tissue layers of the oviduct with the exception of the epithelium. in the ostrich, the two layers of the tunica muscularis were separated by a wide connective tissue area, which is referred to as the intermuscular connective tissue area in this study. distribution of pgp immunoreactive nerve fibres few pgp-immunoreactive nerve fibres were found in the lamina propria-submucosa of the proximal regions of the oviduct (fig. 1a). in the tunica muscularis the density of nerve fibres containing pgp appeared to be similar in the shell gland and vagina, but lower in the infundibulum, magnum and isthmus (table 1). nerve fibres within the muscle bundles coursed parallel to the smooth muscle cells, whilst nerve fibres between the muscle bundles were orientated obliquely (fig. 1b). within the intermuscular connective tissue area, nerve bundles containing pgp were seen close to blood vessels. in addition, a few pgp-immunoreactive nerve fibres were occasionally present in the walls of the blood vessels. pgp-immunopositive nerve bundles in the tunica serosa did not appear to be accompanied by blood vessels. table 1 relative frequency and distribution of protein gene product 9.5 (pgp)-, neurofilament protein (np)and neuron specific enolase-immunoreactive nerves in the oviduct of the ostrich region of oviduct protein gene product neurofilament protein neuron specific enolase infundibulum epithelium lamina propria-submucosa tunica muscularis intermuscular region tunica serosa – – + + + – + + + + – + + + – magnum epithelium lamina propria-submucosa tunica muscularis intermuscular region tunica serosa – + ++ + + – ++ ++ ++ + – + + + + isthmus epithelium lamina propria-submucosa tunica muscularis intermuscular region tunica serosa – + + + + – + ++ ++ ++ – ++ ++ ++ ++ shell gland epithelium lamina propria-submucosa tunica muscularis intermuscular region tunica serosa – ++ +++ ++ ++ – ++ +++ +++ ++ – + +++ +++ ++ vagina epithelium lamina propria-submucosa tunica muscularis intermuscular region tunica serosa – +++ +++ ++ + – + +++ +++ + – + +++ +++ + the density of immunoreactive nerves was graded semiquantitatively as follows: – = none observed; + = very few observed; + = a small number observed; ++ = a moderate number observed; +++ = a large number observed 134 immunoreactivities to protein gene product 9.5, neurofi lament protein and neuron specifi c enolase in ostrich fig. 1 (a–b) protein gene product 9.5 (pgp)-immunoreactive nerve fibres in the oviduct of the ostrich (a) pgp-immu no reactive nerve fibre (arrow) in the lamina propria-sub mucosa of the magnum. (b) pgp-immunoreactive nerve fibres (arrow) in the tunica muscularis of the isthmus are positioned parallel to the longitudinal axes of the smooth muscle cells (m). note the blood vessel (v) associated with the nerve scale bar = 50 μm a b fig. 2 np-immunoreactive nerve fibres (arrow) in the intermuscular connective tissue area of the vagina. v: blood vessel. m: smooth muscle cells scale bar = 50 μm a b c d fig. 3 (a–d) neuron specific enolase (nse)-immunoreactive nerve fibres in the oviduct of the ostrich (a) nse-immunoreactive nerve fibres (arrow) in the lamina propria-submucosa of the isthmus. (b) nse-immunoreactive nerve fibres (arrows) distributed in the wall of a blood vessel (v) in the intermuscular connective tissue area of the shell gland. (c) a nse-immunoreactive nerve cell body (arrow) is observed in a nerve bundle (arrowheads). m: smooth muscle cells. (d) nerve cell bodies (arrow) with nse-immunoreactive cytoplasm are observed in the intermuscular connective tissue area of the vagina. m: smooth muscle scale bar = 50 μm 135 m.-c. madekurozwa distribution of np immunoreactive nerve fibres in the shell gland and the vagina the density of np immunoreactive nerve fibres was higher in the tunica muscularis than in the lamina propria-submucosa (table 1). in all the oviductal regions, the np immunoreactive nerve fibres within the tunica muscularis ran parallel to the longitudinal axis of the smooth muscle cells (fig. 2a). the intermuscular connective tissue area, located between the muscle layers of the tunica muscularis, contained several np immunoreactive nerve bundles (fig. 2b). these nerve bundles were in most cases associated with blood vessels. no np immunoreactive nerve fibres were demonstrated with in the blood vessel walls in any of the oviductal regions studied. a few large nerve bundles containing np immunoreactivity were observed in the tunica ser osa. these nerve bundles appeared to course independently of blood vessels. distribution of nse immunoreactive nerve fibres in general nerve fibres in the lamina propria-submucosa coursed parallel to the epithelium (fig. 3a). in the shell gland and vagina the density of nse immunoreactive nerve fibres appeared to be greater in the tunica muscularis than in the lamina propriasubmucosa (table 1). the tunica muscularis in all the regions studied contained nse-immunoreactive nerve fibres which were orientated parallel to the longitudinal axis of the smooth muscle cells. nerve bundles containing nse immunoreactivity were observed in the intermuscular connective tissue area of the oviduct. in addition to coursing along side blood vessels, nse-immunopositive nerve fibres were seen within the walls of blood vessels (fig. 3b). in all the oviducal regions studied, a few nerve bundles within the tunica muscularis and intermuscular connective tissue area contained nse-immunoreactive nerve cell bodies (fig. 3c). the nerve cell bodies occurred either singly or in small groups, and were characterized by the presence of abundant nse-immunoreactive cytoplasm and a large vesicular nucleus with a prominent nucleolus. occasionally, the nerve cell bodies appeared to be independent of nerve bundles (fig. 3d). the frequency of occurrence of the nerve cell bodies did not appear to differ between the various oviductal regions. a moderate number of nse-immunoreactive nerve bundles was observed in the tunica serosa of the isthmus and shell gland, whilst few nerve bundles or nerve fibres were seen in the serosal layers of the other regions of the oviduct. in all cases nerve bundles within the tunica serosa did not appear to be accompanied by blood vessels. discussion the present study is the first documentation of nerve distribution in different regions of the ostrich oviduct. the neuronal markers used showed that the tunica serosa and the intermuscular connective tissue area contained predominantly nerve bundles, whilst the tunica muscularis and lamina propria-submucosa con tained mainly isolated nerve fibres. furthermore, the markers used indicated that nerve density is greater in the tunica muscularis and intermuscular connective tissue area, and less in the lamina propria-submucosa. these findings are in general agreement with previous studies in the domestic fowl (gilbert & lake 1963; costagliola et al. 1997) and the pigeon (atoji et al. 2000). in addition, as noted by gilbert & lake (1963), the nerve density in the shell gland and vagina is greater than in the more proximal regions of the oviduct. the large number of nerve fibres in the tunica muscularis of the shell gland and the vagina is undoubtedly important in the coordination of muscle contraction during oviposition. interestingly, the results of the present study are contrary to the results of an electron microscopic study carried out in the quail oviduct in which it was demonstrated that very few nerve fibres were present in the tunica muscularis (arjamaa & talo 1983). these findings are surprising as it is assumed that the tunica muscularis in the avian oviduct would have an extensive neural network to coordinate the transportation of the ovum. however, as explained by arjamaa & talo (1983) the muscular contractions of the quail oviduct occur as a result of electrical activity induced as the ovum stretches the smooth muscle cells. in the present study, pgp and nse-immunoreactive nerve fibres were seen in the walls of blood vessels. since both pgp and nse are general neuronal markers, demonstrating both efferent and afferent nerves, it is possible that the nerves observed in the walls of the blood vessels were autonomic. admit tedly, the antibodies used precluded the identification of sympathetic and parasympathetic nerve fibres. however, research carried out by sorger, pittman & soderwell (1983) has shown the presence of a sympathetic innervation to blood vessels in the uterus of the hamster. furthermore, in the pigeon nicotinamide adenine dinucleotide phosphate reduced diaphorase (nadph-d)-reactive nerve fibres, which are known to be parasympathetic in nature, have been observed around blood vessels in the oviduct (atoji et al. 2000). 136 immunoreactivities to protein gene product 9.5, neurofi lament protein and neuron specifi c enolase in ostrich likewise, a study on the innervation of the oviduct in the domestic fowl demonstrated a perivascular autonomic nerve network, which was thought to regulate blood flow (costagliola et al. 1997). in addition to having an influence on blood flow it is known that autonomic nerve fibres play a role in the regulation of glandular secretion. indeed, a study of the uterine innervation in the mare indicated the presence of nerve fibres around endometrial glands (bae et al. 2001). as the ostriches used in the present study were sexually immature with quiescent ovaries, developed tubular glands were not observed in the oviduct. a study using oviductal material from sexually mature ostriches is necessary to determine the innervation of oviductal tubular glands in this species. the structure and location of nerve cell bodies in the ostrich oviduct correlated well with descriptions of similar cells in the domestic fowl (gilbert & lake 1963; costagliola et al. 1997). gilbert & lake (1963) reported that in the domestic fowl single and paired nerve cell bodies were found in large nerve bundles in the intermuscular connective tissue area. furthermore, by employing nadph-d histochemistry cos tag li ola et al. (1997) were also able to demonstrate a few nerve cell bodies in the circular layer of the tunica muscularis. in the present study nse immunohistochemistry demonstated the presence of nerve cell bodies in the intermuscular connective tissue area, as well as in the tunica muscularis. furthermore, as in the domestic fowl (gilbert & lake 1963), some nerve cell bodies appeared to occur independently of nerves. interestingly, the nerve cell bodies identified in the present study were immunopositive for nse, but not for pgp or np. in conclusion, the results of the study indicate that the oviduct of the ostrich has an extensive neural network, which was demonstrated by the neuronal markers pgp, np and nse. acknowledgements the author thanks ms m. smit for technical advice. prof. t.a. aire’s help in reviewing the manu script is gratefully appreciated. the national re search foundation of south africa (thuthuka programme) and the university of pretoria funded the study. references alm, p., 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice ojvr 83_1 book file.indb open access reviewer acknowledgement http://www.ojvr.org open access page 1 of 1 the onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. therefore, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of the onderstepoort journal of veterinary research. we appreciate the time taken to perform your review successfully. in an effort to facilitate the selection of appropriate peer reviewers for the onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on http://www. ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please contact us at submissions@ojvr. org so that we can submit your details to the editor-in-chief for consideration. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ojvr.org 2. in your ‘user home’ [http://www.ojvr.org/ index.php/ojvr/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the onderstepoort journal of veterinary research. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 fax: +27 21 975 4635 journal of veterinary research onderstepoort luus-powell_323-329.indd introduction the pentastomida represent an ancient taxon, comprising some 131 species in seven families (almeida & christoffersen 1999). adults of most species inhabit the nasal passageways and lungs of snakes, lizards and crocodilians, while others are found in the air sacs of gulls and terns, and in the nasopharynx and sinuses of canids and felids (bakke 1972; banaja, james & riley 1975; riley 1986). two species, raillietiella bufonis and raillietiella indica, use amphibians as final hosts (ali, riley & self 1982). the pentastomid life cycle usually includes a vertebrate intermediate host in which larvae undergo several moults to reach the infective stage (riley 1986; winch & riley 1986a; riley & huchzermeyer 2000), but insects have been reported as intermediate hosts of some raillietiellid parasites, while reighardia sternae, a parasite of gulls, has a direct lifecycle (banaja et al. 1975; riley 1986). two families of pentastomids, sebekidae and subtriquetridae, use various freshwater fish species as intermediate hosts (fain 1961; overstreet, self & vliet 1985; winch & riley 1986a, b; junker, boomker & booyse 1998). the sebekiid genera, agema, alofia, leiperia and selfia, have to date only been recorded from crocodilian final hosts, but a single sebekia species is also thought to mature in a che323 onderstepoort journal of veterinary research, 75:323–329 (2008) pentastomid parasites in fish in the olifants and incomati river systems, south africa wilmien j. luus-powell1*, antoinette jooste1 and kerstin junker2 abstract luus-powell, wilmien j., jooste, antoinette & junker, kerstin. 2008. pentastomid parasites in fish in the olifants and incomati river systems, south africa. onderstepoort journal of veterinary research, 75:323–329 during parasitological field surveys of freshwater fish, sebekiid and subtriquetrid pentastome larvae were recovered from the body cavity or swim bladder of several fish species from various localities in limpopo and mpumalanga provinces, south africa. sebekia wedli was recovered from the body cavity of marcusenius macrolepidotus (mormyridae) from flag boshielo dam, limpopo province, and alofia sp. and subtriquetra rileyi were found in the swim bladder of oreochromis mossambicus (cichlidae) from the phalaborwa barrage, limpopo province. the latter species was also collected from the swim bladder of o. mossambicus in dams in the phalaborwa region and the ga-selati river, limpopo province. a single specimen of sebekia okavangoensis was present in the body cavity of clarias gariepinus (clariidae) in a dam on a sugarcane farm in the komatipoort region, mpumalanga province. pentastomid infections in the mormyridae and clariidae represent new host records. keywords: alofia sp., clarias gariepinus, marcusenius macrolepidotus, oreochromis mossambicus, pentastomida, sebekia okavangoensis, sebekia wedli, south africa, subtriquetra rileyi * author to whom correspondence is to be directed. e-mail: powellw@ul.ac.za 1 department of biodiversity, university of limpopo (turfloop cam pus), private bag x1106, sovenga, 0727 south africa 2 department of veterinary tropical diseases, university of pre toria, private bag x04, onderstepoort, 0110 south africa accepted for publication 22 august 2008—editor 324 pentastomid parasites in fi sh in olifants and incomati river systems, south africa lonian host (dukes, shealy & rogers 1971; riley, spratt & winch 1990). the sebekiids diesingia and pelonia, on the other hand, have hitherto been reported exclusively from chelonian final hosts (sambon 1922; overstreet et al. 1985; junker & boomker 2002). adults of the subtriquetridae are known from crocodilians only (riley et al. 1990). in south africa infective larvae of sebekia wedli, leiperia cincinnalis and subtriquetra rileyi have been reported from the cichlids tilapia rendalli (redbreast tilapia) and oreochromis mossambicus (mozambique tilapia) from the phabeni dam in the kruger national park (knp) (junker et al. 1998). junker, boomker & bolton (1999) recorded the nile crocodile, croco dylus niloticus, as the definitive host of s. wedli and l. cincinnalis, as well as of alofia nilotici, alofia sim psoni, sebekia cesarisi and sebekia okavango ensis in south africa. the final host of s. rileyi is as yet unknown. the pentastomid parasites included in this publication are products of a number of parasitological studies on freshwater fish from sev eral local ities in two provinces of south africa. materials and methods during fish health surveys, a total of 1 047 fish, belonging to 14 species were collected at nine localities in south africa and examined for parasites (tables 1 and 2). fish were captured using gill nets of stretched mesh sizes, ranging from 30–120 mm, and transported live to the field laboratory where they were kept in con tainers with well aerated water. immediately before dissection, fish were killed by decapitation. encysted larvae were removed from their cysts and placed in water to unfold. in order to prevent them from contracting, they were fixed by adding small quantities of 70 % ethanol to the water over a period of approximately 1 h, after which they were transferred to 70 % ethanol. pentastomids were mounted and cleared in hoyer’s medium for identification. measurements were taken from whole mounted specimens according to the schematic layout proposed by riley et al. (1990). hook and oral cadre morphology, combined with the number of annuli, were used as identification criteria. the ecological terms prevalence, mean intensity and abundance are used in accordance with bush, lafferty, lotz & shostak (1997). standard deviation was not calculated, since too few specimens were collected which renders standard deviation values meaningless (rózsa, reiczigel & majoros 2000). results pentastomid larvae were encountered at four localities in the olifants river drainage system in limpopo province (table 2). eight encysted infective s. wedli larvae were collected from the body cavity of five of 29 marcusenius macrolepidotus (mormyridae) (bulldog) in flag boshielo dam, constituting a new intermediate host record for this parasite. while a single cyst was located on the swim bladder, the remainder were found between fat deposits on the mesenteries. cysts had a yellowish colour and closely resembled those of clinostomum metacercariae, although slightly smaller in size. no free-living larvae were detected in the swim bladder. the prevalence of infection was 17.2 %, with a mean intensity of 1.6 (range 1–3) and an abundance of 0.3. subtriquetra rileyi was free-living in the swim bladder of o. mossambicus from dams in the pic region. the prevalence of infection was 6.3 %, the mean intensity 1.9 (range 1–6) and the abundance 0.1. no pentastomid larvae were recovered from sharptooth catfish, clarias gariepinus (clariidae), the only other host examined at these sites at the time (table 2). at the phalaborwa barrage, s. rileyi, and a total of three infective larvae of an alofia sp. were recovered from o. mossambicus. concurrent infections of s. rileyi and alofia sp. occurred in two hosts, with the former moving freely in the swim bladder while the alofia sp. was encapsulated. combined, the two species had a prevalence of 30 %, a mean intensity of 2.4 (range 1–5) and an abundance of 0.7. clarias gariepinus and labeo rosae (cyprinidae) examined at this site harboured no pentastomid larvae (table 2). this is the first published record of an alofia species from fish intermediate hosts in south africa. in the ga-selati river, three s. rileyi larvae were recovered from one o. mossambicus of 36 examined, resulting in a prevalence of 2.7 %, and an abundance of 0.08. larvae were moving freely in the swim bladder. no pentastomid larvae were found in c. gariepinus at this site (table 2). with respect to the incomati river drainage system, mpumalanga province, a single infective larva of s. okavangoensis was encysted on the mesenteries of c. gariepinus, from a dam on a sugarcane farm in the komatipoort region. this is the first report of c. gariepinus as intermediate host of a pentastomid. 325 w.j. luus-powell, a. jooste & k. junker t a b l e 1 l o ca lit ie s a t w h ic h f is h w e re c o lle ct e d in l im p o p o a n d m p u m a la n g a p ro vi n ce s, s o u th a fr ic a l o c a li ty c o -o rd in a te s s a m p li n g d a te s c o m m e n ts l im p o p o p ro v in c e : o li fa n ts r iv e r s y s te m f la g b o sh ie lo d a m 2 4 °4 9 ′ s , 2 9 °2 4 ′ e d e c 1 9 9 8 ; n o v 1 9 9 9 ; f e b , s e p t a n d d e c 2 0 0 0 f o rm e rl y kn o w n a s a ra b ie d a m ; p re vi o u sl y p a rt o f m p u m a la n g a p ro vi n ce g a -s e la ti r iv e r 2 3 °9 2 ′ s , 2 3 °9 2 ′ e a p ri l, ju ly , o ct 2 0 0 2 ; ja n , a p ri l, ju l a n d o ct 2 0 0 3 ; ja n 2 0 0 4 – p ic : p h o sp h a te m in e 2 4 °0 1 ′ s , 3 1 °0 5 ′ e a p ri l, ju ly a n d o ct 2 0 0 2 ; ja n , a p ri l, ju ly a n d o ct 2 0 0 3 ; ja n 2 0 0 4 ; ju n e , o ct 2 0 0 5 ; ja n a n d a p ri l 2 0 0 6 p a rt o f th e p h a la b o rw a i n d u st ri a l c o m p le x (p ic ). p e n ta st o m id d a ta f ro m t h e p ic w e re c o m b in e d f o r th e ca lc u la tio n o f in fe ct io n s ta tis tic s p ic : fe rt ili ze r p la n t 2 3 °5 9 ′ s , 3 1 °0 5 ′ e ju n e a n d o ct 2 0 0 5 ; ja n a n d a p ri l 2 0 0 6 p ic : co p p e r m in e 2 4 °0 0 ′ s , 3 1 °0 5 ′ e ju n e a n d o ct 2 0 0 5 ; ja n a n d a p ri l 2 0 0 6 p h a la b o rw a b a rr a g e 2 4 °0 4 ′ s , 3 1 °0 8 ′ e ju n e a n d o ct 2 0 0 5 ; ja n a n d a p ri l 2 0 0 6 w a te r so u rc e f o r th e p ic n w a n e d il u p h e p h e d a m 2 2 °3 9 ′ s , 3 0 °2 5 ′ e a p ri l a n d o ct 1 9 9 6 ; m a rc h 1 9 9 7 ; m a rc h , m a y a n d o ct 1 9 9 8 ; m a rc h , m a y, j u ly a n d o ct 1 9 9 9 ; ju n e 2 0 0 1 – t za n e e n d a m 2 3 °4 8 ′ s , 3 0 °1 0 ′ e ju n e 1 9 9 7 ; n o v 1 9 9 8 ; ja n , m a rc h , m a y, a u g , n o v a n d d e c 1 9 9 9 ; ja n , a p ri l, ju ly a n d n o v 2 0 0 0 ; f e b , m a y a n d o ct 2 0 0 1 – m p u m a la n g a p ro v in c e : in c o m a ti r iv e r s y s te m k o m a tip o o rt 2 4 °3 0 ′ s , 3 1 °3 0 ′ e ju ly 2 0 0 6 s a m p lin g s ite w a s a s u g a r ca n e f a rm d a m l o sk o p d a m 2 5 °2 2 ′ s , 2 9 °1 0 ′ e m a y 2 0 0 1 – b ly d e c a n yo n d a m 2 4 °3 2 ′ s , 3 0 °4 8 ′ e ju ly a n d n o v 1 9 9 9 – 326 pentastomid parasites in fi sh in olifants and incomati river systems, south africa t a b l e 2 t h e p re va le n ce o f p e n ta st o m id in fe ct io n s in f is h c o lle ct e d a t va ri o u s lo ca lit ie s in s o u th a fr ic a d ra in a g e s y s te m n o . o f in fe c te d h o s ts /h o s ts e x a m in e d l im p o p o p ro v in c e m p u m a la n g a p ro v in c e o li fa n ts r iv e r in c o m a ti r iv e r f is h s p e c ie s f la g b o s h ie lo d a m g a -s e la ti r iv e r p h a la b o rw a in d u s tr ia l c o m p le x p h a la b o rw a b a rr a g e n w a n e d il u p h e p h e d a m t za n e e n d a m k o m a ti p o o rt l o s k o p d a m b ly d e c a n y o n d a m a le s ti d a e h yd ro cy n u s vi tt a tu s m ic ra le st e s a cu tid e n s n c n c n c n c n c n c n c n c n c 0 /3 3 n c n c 0 /1 2 0 /2 n c n c n c n c c ic h li d a e o re o ch ro m is m o ss a m b ic u s t ila p ia r e n d a lli 0 /1 2 0 /3 1 /3 6 n c 1 3 /2 0 7 n c 1 2 /4 0 n c 0 /4 5 n c 0 /1 8 n c n c n c 0 /3 n c n c n c c la ri id a e c la ri a s g a ri e p in u s 0 /5 0 /1 9 0 /4 5 0 /2 0 0 /1 5 0 /3 1 /4 0 /3 0 /2 c y p ri n id a e b a rb u s m a to zz i c yp ri n u s ca rp io l a b e o b a rb u s m a re q u e n si s l a b e o b a rb u s p o ly le p is l a b e o r o sa e 0 /2 n c n c n c 0 /2 n c n c n c n c n c n c n c n c n c n c n c n c n c n c 0 /1 5 n c n c 0 /2 1 n c n c n c n c n c n c n c n c n c n c n c n c n c 0 /1 0 /3 n c 0 /3 n c n c n c 0 /3 n c m o c h o k id a e s yn o d o n tis z a m b e ze n si s 0 /5 n c n c n c n c n c n c n c n c m o rm y ri d a e m a rc u se n iu s m a cr o le p id o tu s p e tr o ce p h a lu s w e ss e ls i 5 /2 9 n c n c n c n c n c n c n c 0 /1 6 3 0 /1 3 5 0 /7 4 0 /2 7 n c n c 0 /4 n c 0 /8 n c s c h il b e id a e s ch ilb e in te rm e d iu s 0 /4 n c n c n c 0 /1 7 0 /4 n c n c n c n c n o t co lle ct e d 327 w.j. luus-powell, a. jooste & k. junker discussion the fact that s. wedli, s. okavangoensis and the alofia sp. had reached the final larval stage, as evidenced by double hooks and rows of annular spines typical for infective sebekiid larvae (winch & riley 1986a), confirms that m. macrolepidotus, c. gariepinus and o. mossambicus are indeed the respective true intermediate hosts for these pentastomids. similarly, winch & riley (1986a) found only the infective stage of sebekia to be encysted. morphological characteristics of infective larvae of s. wedli from m. macrolepidotus correspond well with those recorded from o. mossambicus and t. rendalli (junker et al. 1998). ranging from 73–77, the number of annuli in infective larvae from m. macrolepidotus was similar to that observed in the latter hosts, namely 71–76 (junker et al. 1998). other characteristics that support the identification of s. wedli were the single row of chloride cell pore caps on the anterior border of the annuli and the overall appearance of the oral cadre. the latter is readily distinguished from congeneric african species by appearing open anteriorly and having a less pronounced ovoid profile (riley & huchzermeyer 1995). the arrangement of the chloride cell pore caps in an irregular field at the anterior border of the annuli, about 2–3 cells deep, and the mitre-shaped appearance of the oral cadre, observed in the specimen from c. gariepinus confirm its identification as s. oka vangoensis as described by riley & huch zermeyer (1995). all infective sebekia and alofia larvae found during this study were encysted, the cysts being attached to either the swim bladder or the mesenteries. conversely, junker et al. (1998) recorded encysted as well as free-living infective s. wedli larvae from t. rendalli and o. mossambicus. the majority were, however, encysted and the authors speculated that free-living infective larvae had only recently moulted into this developmental stage. junker et al. (1998) recovered s. wedli from the swim bladder only, and not from the mesenteries. they did, however, collect encysted infective larvae of another sebekiid, l. cincinnalis from this site in t. rendalli and o. mossambicus. the literature suggests that sebekiids may occupy a number of sites in their intermediate hosts, and overstreet et al. (1985) reported infective larvae of sebekia mississippiensis under the connective tissues lining muscle, kidney, liver and swim bladder of a variety of fish intermediate hosts. sebekia wedli larvae appear to be host specific in flag boshielo dam, as none of the other seven fish species examined at the time, including 12 o. mossambicus and three t. rendalli, harboured pentastomid larvae (table 2). sebekia wedli had a relatively high prevalence of 40.5 % in t. rendalli in the knp, but only three of 119 o. mossambicus harboured this pentastome (junker et al. 1998). hence, it is possible that the sample size with respect to the other hosts in flag boshielo dam was too small to detect infection. although mormyrids were studied intensively at nwanedi-luphephe and tzaneen dams, limpopo prov ince, and loskop and blyde canyon dams, mpu malanga province (table 2), pentastomid larvae were only recovered from m. macrolepidotus from flag boshielo dam. flag boshielo dam has a large population of nile crocodiles. these crocodilians have long been established as a final host of s. wedli (sambon 1922), and junker et al. (1999) reported this pentastomid from nile crocodiles in south africa. mormyrids are bottom feeders, favouring the muddy bottomed margins of rivers and floodplains (skelton 2001), and are thus likely to ingest pentastome eggs, which are shed in crocodile faeces, and settle onto the bottom substrate. it remains a matter of speculation as to why s. wedli was not observed in intermediate hosts at any of the other dams, since both loskop and bly de canyon dams, as well as tzaneen dam also support crocodile populations. possibly sample sizes at all these dams were too small to detect pentastome infections. prevalence data on pentastomids in nile corocodiles in any of the above dams, including flag boshielo, are lacking. clarias gariepinus constitutes an important component of the diet of nile crocodiles (guggisberg 1972; whitfield & blaber 1979), and its own feeding habits would readily expose it to ingesting pentastome eggs. riley & huchzermeyer (2000) noted that clari as spp. were common in swamp forest pools in the northern congo republic and postulated that it might serve as intermediate host for the pentastomids, agema silvaepalustris, alofia parva and s. oka vangoensis, that were recovered from swamp forest dwarf crocodiles, osteolaemus tetraspis, in that region. however, no proof of the role of clarias in the transmission of pentastomids has so far been presented. the prevalence of infection in c. gariepinus is low and only one of four hosts at the komatipoort locality was infected, while a total of 112 collected at eight additional localities did not habour the parasite (table 2). ten c. gariepinus from the knp also har328 pentastomid parasites in fi sh in olifants and incomati river systems, south africa boured no pentastomids (junker, unpublished data 1996). winch & riley (1986b) concluded that the absence of subtriquetra subtriquetra in bottomfeed ing tilapia spp. in trinidad, despite its presence in aequidens pulcher, another bottom-feeder in the same reservoir, was probably due to an immune response. this could also be an explanation for the low prevalence of pentastomids in c. gariepinus. to date, several fish species belonging to a number of families world-wide have been recorded as intermediate hosts of sebekiids, namely sebekia oxycephala in a. pulcher (cichlidae) (blue acra) and tilapia sp. (cichlidae) in trinidad and gambusia affi nis (poeciliidae) (mosquitofish) in florida (boyce, cardeilhac, lane, buergelt & king 1984; winch & riley 1986a). fundulus grandis (fundulidae) (gulf killifish), lepomis macrochirus (centrarchidae) (bluegill), micropogonias undulatus (sciaenidae) (atlantic croaker), micropterus salmoides (centrarchidae) (largemouth bass) and xiphophorus helleri (poeciliidae) (swordtail) are confirmed intermediate hosts for sebekia mississippiensis (overstreet et al. 1985). fain (1961) lists alestes macrophthalmus (alestidae) (torpedo robber), bathybates ferox (cichlidae), chry sichthys brachynema (claroteidae) (salmontail catfish), chrysichthys mabusi (claroteidae), lates mi crolepis (latidae) (forktail lates), lates niloticus (latidae) (nile perch), mastacembelus sp. (mas tacembelidae) and oreochromis niloticus (= tilapia nilotica) (cichlidae) (nile tilapia) as intermediate hosts of l. cincinnalis in central africa. the latter was also present in serranochromis meridianus (cichlidae) (lowveld largemouth) in south africa (junker 2002). few data exist on the intermediate hosts of subtriquetra, which has sofar been recovered from a. pulcher, o. mossambicus and t. rendalli (winch & riley 1986b; junker et al. 1998). fain (1961) reports s. subtriquetra from brazilian fish in general. while s. rileyi has now been reported from intermediate fish hosts from several localities in south africa, its final host has not been identified. according to riley et al. (1990) adult subtriquetrids are exclusive to crocodilians, but adult s. rileyi were absent in crocodiles harbouring other pentastome infections in the knp, suggesting the involvement of a different final host, perhaps piscivorous terrapins or birds (junker 2002). acknowledgements the division for research development and administration, university of limpopo, is thanked for financial assistance. the research at phalaborwa was supported by a grant from the national research foundation of south africa (thuthuka programme; ttk2004081000029). we are grateful to the department of biodiversity, university of limpopo, for infrastructure and messrs h.e. hattingh and j. theron for technical assistance during some of the field surveys. references ali, j.h., riley, j. & self, j.t. 1982. amphibians as definitive hosts for pentastomids: railietiella bufonis n. sp. from bufo lemur in puerto rico and a reassessment of railietiella indica gedoelst, 1921. systematic parasitology, 4:279–284. almeida, de oliveira, w. & christoffersen, m.l. 1999. a cladistic approach to relationships in pentastomida. journal of parasitology, 85:695–704. bakke, t.a. 1972. reighardia sternae (diesing, 1864) ward, 1899 (pentastomida: cephalobaenida) from the common gull (larus canus l.) in a norwegian locality. norwegian journal of zoology, 20:273–277. banaja, a.a., james, j.l. & riley, j. 1975. an experimental investigation of a direct life-cycle in reighardia sternae (diesing, 1864), a pentastomid parasite of the herring gull (larus argentatus). parasitology, 71:493–503. boyce, w., cardeilhac, p., lane, t., buergelt, c. & king, m. 1984. sebekiosis in captive alligator hatchlings. journal of the american veterinary medical association, 185:1419–1420. bush, a.o., lafferty, k.d., lotz, j.m. & shostak, a.w. 1997. parasitology meets ecology on its own terms: margolis et al. revisited. journal of parasitology, 83:575–583. dukes, g.h., shealy, r.m. & rogers, w.a. 1971. sebekia oxycephala (pentastomida) in largemouth bass from lake st. john, concordia parish, louisiana. journal of para sitology, 57:1028. fain, a. 1961. les pentastomides de l’ afrique centrale. annales du musée royale de l’afrique centrale, série 8, 92:1–115. guggisberg, c.a. 1972. crocodiles. cape town: purnell. junker, k., boomker, j. & booyse, d.g. 1998. pentastomid infections in cichlid fishes in the kruger natioanl park, and description of the infective larva of subtriquetra rileyi n. sp. onderstepoort journal of veterinary research, 65:159–167. junker, k., boomker, j. & bolton, l. 1999. pentastomid infections in the nile crocodile (crocodylus niloticus) in the kruger national park, south africa, with a description of the males of alofia simpsoni. onderstepoort journal of veterinary research, 66:65–71. junker, k. 2002. a study on the pentastomida parasitising crocodilian and chelonian final hosts, with special emphasis on the south african pentastome fauna. ph.d. thesis. university of karlsruhe. junker, k. & boomker, j. 2002. description of pelonia africana n. g., n. sp. (pentastomida: sebekidae) from the lungs of pelomedusa subrufa and pelusios sinuatus (chelonia) in south africa. onderstepoort journal of veterinary research, 69:53–59. overstreet, r.m., self, j.t. & vliet, k.a. 1985. the pentastomid sebekia mississippiensis sp. n. in the american alligator and other hosts. proceedings of the helminthological society of washington, 52:266–277. 329 w.j. luus-powell, a. jooste & k. junker riley, j. 1986. the biology of pentastomids. advances in parasitology, 25:45–128. riley, j., spratt, d.m. & winch, j.m. 1990. a revision of the genus sebekia sambon, 1922 (pentastomida) from crocodilians with description of five new species. systematic parasitology, 16:1–25. riley, j. & huchzermeyer, f.w. 1995. pentastomid parasites of the family sebekidae fain 1961 in west african dwarf crocodiles osteolaemus tetraspis cope, 1851 from the congo, with a description of alofia parva n. sp. onderstepoort journal of veterinary research, 62:151–162. riley, j. & huchzermeyer, f.w. 2000. diet and lung parasites of swamp forest dwarf crocodiles (osteolaemus tetraspis osborni) in the northern congo republic. copeia, 2:582–586. rózsa, l., reiczigel, j. & majoros, g. 2000. quantifying parasites in samples of hosts. journal of parasitology, 86: 228–232. sambon, l.w. 1922. a synopsis of the family linguatulidae. journal of tropical medicine and hygiene, 25:188–206; 391–428. skelton, p.h. 2001. a complete guide to the freshwater fishes of southern africa. halfway house: southern book publishers (pty) ltd. whitfield, a.k. & blaber, s.j.m. 1979. predation on striped mullet (mugil cephalus) by crocodylus niloticus at st. lucia, south africa. coopeia, 2:266–269. winch, j.m. & riley, j. 1986a. morphogenesis of larval sebekia oxycephala (pentastomida) from a south american crocodilian (caiman sclerops) in experimentally infected fish. zeitschrift für parasitenkunde, 72:251–264. winch, j.m. & riley, j. 1986b. studies on the behaviour, and development in fish, of 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice elias_289-298.indd introduction the vascular system of the male reproductive tract in birds has, in general, been poorly studied. the only comprehensive report of both the arterial supply and venous drainage is that of nishida (1964) in the domestic fowl. a general description of the arterial supply to the male reproductive organs of the pigeon has also been provided (bhaduri, biswas & das 1957) and a more recent study (elias, aire & soley 2007) has described the macroscopic features and variations in the arterial supply to the reproductive tract of the male ostrich. the venous system has received even less attention, with most of the information being supplied by nishida (1964) and some incidental data being provided from a study on the renal portal and venous systems of the fowl kidney by kurihara & yasuda (1975). descrip289 onderstepoort journal of veterinary research, 75:289–298 (2008) macroscopic features of the venous drainage of the reproductive system of the male ostrich (struthio camelus) m.z.j. elias1, t.a. aire and j.t. soley* department of anatomy and physiology, faculty of veterinary science, university of pretoria onderstepoort, 0110 south africa abstract elias, m.z.j., aire, t.a. & soley, j.t. 2008. macroscopic features of the venous drainage of the reproductive system of the male ostrich (struthio camelus). onderstepoort journal of veterinary research, 75:289–298 the macroscopic features of the venous drainage of the reproductive system of the male ostrich were studied in six pre-pubertal and three sexually mature and active birds. each testis was drained by one to four testicular veins. the right testicular veins drained the right testis and epididymis and its appendix to the caudal vena cava and to the right common iliac vein, whereas the left testicular veins drained the left testis and epididymis and its appendix exclusively to the left common iliac vein. a number of variations in the drainage pattern based on the point of entry and number of testicular veins were observed. the cranial aspect of the testis was also linked to the caudal vena cava or common iliac vein via the adrenal veins. the cranial, middle and caudal segments of the ductus deferens (and ureter) were drained by the cranial, middle and caudal ureterodeferential veins respectively, to the caudal testicular veins, the caudal renal veins and pudendal/caudal part of the internal iliac veins. in some specimens, the caudal ureterodeferential veins also drained into the caudal mesenteric vein. the surface of the phallus was drained by tributaries of the pudendal vein. the basic pattern of venous drainage of the reproductive organs of the male ostrich was generally similar to that described for the domestic fowl. however, important differences, including the partial fusion of the caudal renal veins, drainage of the cranial aspect of the testes via the adrenal veins, drainage of the caudal ureterodeferential veins into the caudal mesenteric vein and the presence of veins draining the surface of the phallus, were observed. although significant, these differences may simply reflect variations in the normal pattern of venous drainage of the reproductive tract of birds which could be verified by studying more specimens and more species. keywords: macroscopic features, male reproductive system, ostrich, struthio camelus, veins * author to whom correspondence is to be directed. e-mail: john.soley@up.ac.za 1 present address: veterinary faculty, department of preclinics, eduardo mondlane university, maputo, mozambique accepted for publication 10 september 2008—editor 290 macroscopic features of venous drainage of reproductive system of male ostrich (struthio camelus) tions of the venous system involving the male reproductive tract of birds presented in textbooks [e.g. baumel (1975), lake (1981) and baumel (1993)] simply reflect the work of nishida (1964) and kurihara & yasuda (1975). the only reference to drainage of the reproductive organs in the male ostrich is by bezuidenhout (1999) who notes that “as the caudal vena cava passes cranially and ventrally it receives veins from the gonads, adrenals and surrounding tissues”. in view of the lack of comparative data on the vascularization of the male reproductive tract of birds, and considering the need for a better understanding of the reproductive biology of the ostrich as a farmed animal, this paper presents a description of the gross pattern of venous drainage in the male ostrich. variations in the basic pattern are also described. the terminology used is that of nomina anatomica avium (baumel, king, breazile, evans & berge 1993). materials and methods the torsos of nine male ostriches with viscera intact, but which had been skinned and from which the limbs had been removed, were obtained from the oryx abattoir in krugersdorp, gauteng province and from the klein karoo abattoir in oudtshoorn, western cape province, south africa. the specimens comprised six semi-adult birds (12–14 months old) and three sexually mature and active birds. the torsos were prepared as follows for latex injection and the formation of resin casts, respectively. the venous system of five of the birds (four semiadults and one sexually mature bird) was flushed free of blood by rinsing with physiological saline injected through the caudal vena cava (5–10 cm above the vessel’s origin between the cranial poles of the testes), after which blue “latex”, (revertex chemicals company®, pretoria) was subsequently injected into the veins via the same route using a 50 mℓ syringe. the torsos were trimmed of excess tissue and immersion fixed in a 10 % formalin bath for a number of days. the fixed specimens were rinsed in running water for 2 days after which they were carefully dissected to expose the latex-filled veins. the pattern of venous drainage was described and digitally recorded with a nikon 4500 coolpix digital camera. for the preparation of resin casts of the veins, four torsos (two semi-adults and two sexually active birds) were injected with a coloured resin (pigment preparation pty. ltd., pretoria) via the caudal vena cava using the same technique employed for the latex injections. each specimen was subsequently kept for a number of days in a cold room before tissue maceration in a bath containing a 20–30 % sodium hydroxide solution. the corrosion casts obtained were carefully washed clean and the pattern of veins described and digitally recorded. results venous drainage of the testes the testes were drained via testicular veins (venae testiculares) that were variable in number (ranging from one to four for each testis) and point of termination (fig. 1, 2a–c and 3). these veins were observed to arise from a well-developed venous network located in the testicular capsule (fig. 3). based on the relationship of the testicular veins to the caudal vena cava and the common iliac veins, the following variations were observed: type a in this variation, the testicular veins draining the right testis, the epididymis and its appendix, and the cranial aspect of the ductus deferens and ureter, joined both the caudal vena cava (just cranial to its origin) and the right common iliac vein. the vessels draining the left testis and associated structures emptied exclusively into the left common iliac vein, caudal to the confluence of the two common iliac veins (fig. 1, 2b and 3) this was the most common pattern noted and occurred in five (55.5%) of the specimens examined. type b this variation was observed in four (44.5 %) of the specimens and was characterized by the right testicular veins draining only into the caudal vena cava, while the left testicular veins again emptied into the left common iliac vein (fig. 2a and c). as noted above, the testes displayed a well-developed venous network situated in the testicular capsule, particularly in the sexually mature birds, and from which the testicular veins emanated. due to the complexity of this network and the formation of numerous tributaries, it was difficult in some specimens to accurately determine the exact number of testicular veins present. however, based on obvious connections between the testicular veins and the caudal vena cava and common iliac veins, four basic numerical patterns were observed. type i in this variation at least four testicular veins were observed to drain the right testis while a similar number drained the left testis (fig. 291 m.z.j. elias, t.a. aire & j.t. soley fig. 1 a diagramatic sketch showing, in ventral view, the main veins draining the reproductive tract of the male ostrich. the testes are shown reflected laterally to illustrate the testicular veins and associated vessels v. ureterodeferentialis cranialis v. adrenalis testis vv. testiculares v. femoralis v. portalis renalis caudalis ren, lubus medius fused portion of caudal renal veins ureter anastomosis interiliaca v. iliaca interna v. ureterodeferentiales caudales phallus v. renalis caudalis ductus deferens appendix epididymidis v. iliaca communis tributaries to v. pudenda cloaca v. mesenterica caudalis v. lateralis caudae v. pudenda v. ischiadica vv. ureterodeferentiales mediae v. cava caudalis 292 macroscopic features of venous drainage of reproductive system of male ostrich (struthio camelus) fig. 2a–c diagramatic sketches showing, in ventral view, the main variations of the testicular veins in respect of their number and location appendix epididymidis v. ureterodeferentialis cranialis v. adrenalisv. cava caudalis v. adrenalis ductus deferens testis vv. testiculares v. iliaca communis v. cava caudalis v. adrenalis ductus deferens testis vv. testiculares appendix epididymidis v. ureterodeferentialis cranialis v. adrenalis v. iliaca communis appendix epididymidis v. ureterodeferentialis cranialis v. adrenalis v. cava caudalis v. adrenalis ductus deferens testis vv. testiculares v. iliaca communis 293 m.z.j. elias, t.a. aire & j.t. soley 1 and 3). this was the most common pattern noted and occurred in three (33.4 %) of the specimens examined. type ii in this instance three testicular veins (essentially cranial, middle and caudal vessels) drained the right testis and two testicular veins (cranial and caudal) the left testis (fig. 2a). this variation occurred in two (22.2 %) of the specimens. type iii this type was characterized by the presence of two testicular veins draining the right testis and a single, large testicular vein draining the left testis. the latter was formed by a large number of branched tributaries (fig. 2b). this variation was present in two (22.2 %) of the specimens examined. type iv this variation was also observed in two (22.2 %) of the specimens and consisted of a single testicular vein draining the right testis and two veins draining the left testis (fig. 2c). each of these vessels was composed of numerous tributaries. it was observed in all specimens that a small vessel (effectively an additional testicular vein?) emanated from the venous network at the cranial aspect of each gonad and emptied into the adrenal vein. (fig. 1, 2a–c, 3 and 4). in the ostrich this vessel was prominent and well-developed on the left side of the body where it drained into the left common iliac vein. the adrenal vein was smaller on the right side where it emptied into the caudal vena cava. in addition to draining the cranial aspect of the testis, the adrenal vein drained part of the appendix epididymidis, the fig. 3 ventral view of the reproductive tract of the ostrich illustrating the testes (t) and associated veins. the testes have been reflected laterally to expose more clearly the relevant veins. note the extensive venous network within the testicular capsule. v. cava caudalis (1), vv. iliacae communes dextra et sinistra (2, 2’), vv. renales caudales dextra et sinistra (3, 3’), vv. ureterodeferentiales craniales dextra et sinistra (4, 4’), vv. testiculares dextra et sinistra (5, 5’), appendix epididymidis (6), branch draining the cranial pole of the testis to the adrenal vein (7). v. adrenalis sinistra (8). arrow at top left indicates caudo-cranial direction. latex injection fig. 4 ventral view of the thoraco-abdominal region of a pre-pubertal bird illustrating the testes (t) and associated veins. v. cava caudalis (1), vv. iliacae communes dextra et sinistra (2, 2’), v. renalis caudalis dextra (3), vv. testiculares dextra et sinistra (5, 5’), v. adrenalis sinistra (6). note the branches from the latero-dorsal body wall (4) draining into the adrenal vein (6) as well as a testicular vein (7) that drains the left testis into the left common iliac vein via the adrenal vein. arrow top left indicates caudo-cranial direction. latex injection 294 macroscopic features of venous drainage of reproductive system of male ostrich (struthio camelus) adrenal gland and the cranio-lateral body wall of the thoraco-abdominal region. very fine branches of the adrenal vein also drained the cranial division of the kidney. the position and course of the caudal vena cava of the ostrich were similar to that described for the fowl (gallus domesticus) (nishida 1964). it was formed by the confluence of the right and left common iliac veins in the vicinity of the cranial poles of the testes, the adrenal glands and cranial divisions of the kidneys, where it lay almost directly below the aorta. in the material studied, the caudal vena cava varied between 2–4 cm in width, and 20–28 cm in length. it lay slightly to the right of the median plane and coursed obliquely cranioventrally (fig. 1 and 3) towards the right atrium of the heart. drainage of the ductus deferens the cranial aspect of each deferent duct was drained by the most caudal testicular vein through the cranial ureterodeferential vein (vena ureterodeferentialis cranialis) (fig. 1, 2a–c and 3). fig. 5 a ventral view of the middle seg ment of the reproductive tract showing the region of fusion between the left and right caudal renal veins (1), vv. ureterodeferentiales mediae dextra et sinistra (2, 2’), vv. renales caudales dextra et sinistra (3, 3’). arrow top left indicates caudo-cranial direction. latex injection fig. 6 a corrosion cast (dorsal view) showing the main components of the venous system involved in drainage of the male reproductive tract: v. cava caudalis (1), vv. iliacae communes (2), vv. renales caudales (3). vv. ischiadicae (4), fused portion of caudal renal veins (5), vv. portales renales caudales (6), anastomosis interiliaca (7), vv. iliacae internae (8) and v. caudae medianae (9). arrow top left indicates caudo-cranial direction. latex injection 295 m.z.j. elias, t.a. aire & j.t. soley fig. 7 ventro-lateral view of the pelvic region displaying the vv. iliacae internae dextra et sinistra (1, 1’), vv. pudendae dextra et sinistra (2, 2’), v. mediana caudae (3), vv. ureterodeferentiales caudae (4), ductus deferentes et ureteres dextrae et sinistrae (5, 5’), v. mesenterica caudalis (6). note the caudal ureterodeferential vein (4—single white arrow) draining the ductus deferens (5) to the caudal mesenteric vein (6), arrow top left indicates caudocranial direction. latex injection fig. 8 ventro-lateral view of the pelvic region showing the v. iliaca interna sinistra (1), v. pudenda sinistra (2), v. caudae medianae (3), v. ureterodeferentialis caudalis (4), ductus deferentes et ureteres sinistrae (5). in this view the exact positioning of the veins in the pelvic region has been obscured to illustrate the anastomosis between the v. mesenterica caudalis (7) and the left internal iliac vein (1) via a large unnamed vein (6). arrow top left indicates caudo-cranial direction. latex injection the middle portions of the deferent duct and ureter were drained by the middle ureterodeferential veins (venae ureterodeferentiales mediae). these vessels were variable in number, size and point of entry along the caudal renal veins into which they emptied. the caudal renal veins also drained the caudal part of the cranial division and all of the middle and caudal divisions of the kidney as well as the ureter in those regions. the caudal renal veins were observed to fuse with each other at a level between the caudal and middle renal lobes (fig. 1, 5 and 6) to form a single vessel approximately 3–5 cm long, before again dividing cranially into two separate vessels. in all specimens studied, the middle ureterodeferential veins emptied into both the fused and separated parts of the caudal renal veins. the cranial continuation of the caudal renal veins drained ventro-caudally into the common iliac veins (fig. 1 and 3), while the femoral veins (vena femoralis) entered the common iliac veins dorso-laterally. each common iliac vein (vena illiaca communis) emptied into the caudal vena cava in the vicinity of the cranial aspect of the testes. the caudal part of the deferent duct and the cloaca were drained by the caudal ureterodeferential veins (venae ureterodeferentiales caudales) to the pudendal veins and to the caudal portion of the internal iliac veins. each pudendal vein (vena pudenda), after receiving the caudal lateral vein (from the wall 296 macroscopic features of venous drainage of reproductive system of male ostrich (struthio camelus) of the pelvic region) formed the internal iliac vein (fig. 7 and 8). the two internal iliac veins proceeded cranially and were connected by a transverse vessel, the interiliac anastomosis, 2–5 cm caudal to the caudal pole of the kidney. the anastomosis was linked caudally to the caudal median vein (fig. 6 and 7). in three specimens (33.3 %), tributaries from the caudal deferent duct, together with branches from the rectum and cloaca, were observed to drain into the caudal mesenteric vein (vena mesenterica caudalis) in addition to the branches draining into the pudendal vein. the caudal mesenteric vein emptied into the caudal median vein (fig. 7). in one of these specimens the caudal mesenteric vein, in addition to its connection to the caudal median vein, was linked to the left internal iliac vein by a large unnamed vessel draining the wall of the rectum (fig. 8). drainage of the phallus a number of superficial tributaries displaying no particular pattern drained the mucosa, but not the substance of the phallus, (fig. 1 and 9) to the pudendal vein. these vessels were relatively large (fig. 9). a venous network of fine vessels was situated at the root of the phallus which also drained into the pudendal vein (fig. 1). discussion the gross pattern of venous drainage of the male reproductive tract in the ostrich is basically similar to that described for the fowl (nishida 1964). however, certain noteworthy variations were observed in the material studied which may reflect unreported differences between avian species. in both the ostrich and fowl (nishida 1964) the testicular veins arise from a well-developed network located in the testicular capsule. in the fowl both sets (left and right) of testicular veins are reported to empty into the corresponding common iliac vein and the caudal vena cava as illustrated in the sketch by nishida (1964). in other studies on the fowl, however, the testicular veins are shown to empty only into the caudal vena cava (nickel, schummer & seiferle 1977). a similar situation is also illustrated for the veins draining the single ovary of gallus (baumel 1993). in five of the ostriches studied, the drainage pattern of the right testis resembled that described by nishida (1964) (testicular veins emptied into the right common iliac vein and the caudal vena cava) whereas the testicular veins from the left testis emptied exclusively into the left common iliac vein. in four birds, however, the testicular veins emanating from the right testis drained only into the caudal vena cava, with those from the left testis again emptying into the left common iliac vein. in the ostrich, therefore, the route of drainage of the right testicular veins reflects both patterns reported in the fowl. however, the exclusive drainage of the left testicular veins into the corresponding common iliac vein in all the ostrich specimens examined appears to be a unique feature and contrasts sharply with the observation that in the ostrich the veins from the gonads empty into the caudal vena cava fig. 9 ventro-lateral view of the phallus showing the v. pudenda dextra (1) receiving large, superficial tributaries (2) from the root and body of the phallus (3). arrow top left indicates caudo-cranial direction. latex injection 297 m.z.j. elias, t.a. aire & j.t. soley (bezuidenhout 1999). the slightly more cranial positioning of the right testis reported in the ostrich (soley 1992; soley & groenewald 1999) may provide an explanation for that drainage into both the right common iliac vein and the caudal vena cava or only into the caudal vena cava, and to the exclusive drainage of the more caudally positioned left testis into the left common iliac vein. little information is available in the literature regarding the numerical patterns of the testicular veins. the illustration of nishida (1964) shows one large vein draining the right testis and three vessels draining the left testis, similar to the type iv variation reported in the present study. as the authors did not have a full english translation of the text available, it is possible that a range of variations were described by nishida (1964). the illustration by nickel et al. (1977) demonstrates at least three veins draining both the left and right testes respectively which corresponds closely with the most common variation (type i) seen in the ostrich involving four testicular veins draining each testis. these observations would suggest that a range of numerical variations is typical for the testicular veins of birds. an interesting observation in the ostrich was the presence of a small vessel emanating from the venous network at the cranial aspect of each gonad and which drained into the adrenal vein. this drainage route for the testes has not been reported in the fowl although baumel (1993) illustrates some vv. ovaricae reaching the caudal vena cava via the adrenal vein. in the ostrich, the adrenal vein on the left side of the body drained into the left common iliac vein whereas that on the right side emptied into the caudal vena cava. in the fowl both the left and right adrenal veins reportedly drain into the caudal vena cava (goodchild 1969; baumel 1993). again, the unequal positioning of the ostrich testes may account for this phenomenon. in the ostrich, drainage of the cranial and middle segments of the ductus deference parallels that described in the fowl (nishida 1964).the cranial aspect of the ductus deferens and ureter are drained by the cranial ureterodeferential veins to the most caudal testicular veins and from there to the common iliac veins or caudal vena cava. however, it is not clear whether the cranial ureterodeferential veins in the fowl (vv. ureto-deferentiales anteriores – nishida 1964) reach the common iliac veins independently or via the caudal testicular veins. in both the ostrich and fowl, middle ureterodeferential veins (vv. ureto-deferentiales mediae – nishida 1964) drain the middle segments of the ductus deferens and ureter to the caudal renal veins (v. renalis efferens – nishida 1964). a marked difference in the ostrich is that some of the middle ureterodeferential veins enter the fused portion of the left and right caudal renal veins, a situation lacking in the fowl as fusion of the caudal renal veins does not occur (akester 1964; kurihara & yasuda 1975; baumel 1993). fusion of the caudal renal veins has only previously been reported in a single 5-day old chicken embryo (miller 1903). why this phenomenon should be a consistent feature in the ostrich, or what its functional significance is, remains unknown. in six of the nine specimens, the caudal ureterodeferential veins in the ostrich were observed to drain into the pudendal vein and the caudal aspect of the internal iliac vein which formed the continuation of the former vessel. in three specimens, however, the caudal ureterodeferential veins were seen to drain into the caudal mesenteric vein in addition to the branches draining into the pudendal vein. in the fowl the caudal ureterodeferential veins (vv. ureto-deferentiales posteriors – nishida 1964) are shown draining only into the pudendal vein (v. pudenda interna – nishida 1964). as a caudal mesenteric vein (v. coccygomesenterica – nickel et al. 1977) has been described in the fowl (nickel et al. 1977; baumel 1993) it is possible that in this species branches of the caudal ureterodeferential veins do in fact empty into this vessel but have not yet been described. it was also noted that in the ostrich the caudal mesenteric vein drained into the caudal median vein which in turn was linked to the interiliac anastomosis. in the fowl, both the caudal median vein and the caudal mesenteric vein appear to drain into the interiliac anastomosis (baumel 1993, page 467), although alternative drainage patterns have also been described (kurihara & yasuda 1975; baumel 1993). the large tributaries draining the mucosa of the phallus to the pudendal vein in the ostrich have not been described in the fowl. this omission may be due to the relatively small size of the phallus and the attendant difficulties in revealing the blood vessels in this species. in conclusion, the pattern of venous drainage of the reproductive organs of the male ostrich follows the basic pattern described in the domestic fowl by nishida (1964). although the differences observed between the ostrich and fowl are significant, they may simply reflect variations in the normal pattern of venous drainage of the reproductive tract of birds which could be verified by studying more specimens and more species. 298 macroscopic features of venous drainage of reproductive system of male ostrich (struthio camelus) acknowledgements we thank the swedish international development agency, project uem iniveveter sida-sarec 2001–2003, for funding this study, mrs w. oliver for preparing the original art work and mr l. de villiers for assistance in obtaining the specimens. references akester, a.r. 1964. radiographic studies of the renal portal system in the domestic fowl (gallus domesticus). journal of anatomy, 98:365–376. baumel, j.j. 1975. heart and blood vessels, in the anatomy of domestic animals. vol. 2, edited by r. getty. philadelphia: saunders company. baumel, j.j. 1993. systema cardiovascular, in handbook of avian anatomy. nomina anatomica avium, 2nd ed., edited by a.s. king, j.e. breazile, h.e. evans & j.c.v. berge. cambridge: academic press. baumel, j.j., king, a.s., breazile, j.e., evans, h.e. & berge, j.c.v. (eds) 1993. handbook of avian anatomy. nomina anatomica avium, 2nd ed. cambridge: academic press. bhaduri, j.l., biswas, b. & das, s.k. 1957. the arterial system of the domestic pigeon (columba livia gmelin). ana tomischer anzeiger, 104:1–14. bezuidenhout, a.j. 1999. anatomy, in the ostrich, biology, production and health, edited by d.c. deeming. cambridge: cabi publishing. elias, m.z.j., aire, t.a. & soley, j.t. 2007. macroscopic features of the arterial supply to the reproductive system of the male ostrich (struthio camelus). anatomia, histologia & embryolgia: journal of veterinary medicine series c, 36: 255–262. goodchild, w.m. 1969. the venous system of the adrenal glands of gallus domesticus. british poultry science, 10:183– 185. kurihara, s. & yasuda, m. 1975. morphological study of the kidney in the fowl. ii. renal portal and venous systems. japanese journal of veterinary science, 37:363–377. lake, p.e. 1981. male genital organs, in form and function in birds, edited by a.s. king & j. mclelland. london: academic press. miller, a.m. 1903. the development of postcaval vein in birds. american journal of anatomy, 2:283–298. nickel, r., schummer, a. & seiferle, e. 1977. circulatory system, in anatomy of the domestic birds. berlin: verlag parey. nishida, t. 1964. comparative and topographical anatomy of the fowl. xlii. blood vascular system of the male reproductive organs. japanese journal of veterinary science, 26:211– 221. soley, j.t. 1992. a histological study of spermatogenesis in the ostrich (struthio camelus). ph.d. thesis. university of pretoria. soley, j.t. & groenewald, h.b. 1999. reproduction, in the ostrich, biology, production and health, edited by d.c. deeming. london: cabi publishing. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice horak_13-25.indd introduction the majority of ticks that have been collected from tortoises in south africa belong to the genus amblyomma and of these amblyomma marmoreum, colloquially known as the south african tortoise tick, is probably the most prevalent (theiler & salisbury 1959). although records of ticks on tortoises in south africa date back to the 18th and 19th centuries (theiler 1943; walker & schulz 1984), it was not until theiler & salisbury (1959) and norval (1975) published their descriptions of ticks in the “amblyomma marmoreum group” and on the ecology of a. marmoreum, respectively, that the taxonomy and biology of the latter tick was comprehensively addressed. in their paper, theiler & salisbury (1959) illustrated and described all stages of development of a. marmoreum 13 onderstepoort journal of veterinary research, 74:13–25 (2006) hosts, seasonality and geographic distribution of the south african tortoise tick, amblyomma marmoreum i.g. horak1, i.j. mckay2,* heloise heyne2 and a.m. spickett2 abstract horak, i.g., mckay, i.j., heyne, heloise & spickett, a.m. 2006. hosts, seasonality and geographic distribution of the south african tortoise tick, amblyomma marmoreum. onderstepoort journal of veterinary research, 74:13–25 the tortoise tick amblyomma marmoreum was collected from large numbers of reptiles and other animals during the course of numerous surveys conducted in south africa. a total of 1 229 ticks, of which 550 were adults, were recovered from 309 reptiles belonging to 13 species, with leopard tortoises, geochelone pardalis being the most heavily infested. the 269 birds sampled harboured 4 901 larvae, 217 nymphs and no adult ticks, and the prevalence of infestation was greatest on hel meted guinea fowls, numida meleagris. only two larvae were recovered from 610 rodents, including 31 spring hares, pedetes capensis, whereas 1 144 other small mammals yielded 1 835 immature ticks, of which 1 655 were collected from 623 scrub hares, lepus saxatilis. the 213 carnivores examined harboured 2 459 ticks of which none were adult. a single adult tick and 6 684 larvae and 62 nymphs were recovered from 656 large herbivores, and a total of 4 081 immature ticks and three adults were collected from 1 543 domestic animals and 194 humans. adult male and female a. marmoreum were most numerous on reptiles during january and february, and larvae during march. the largest numbers of larvae were present on domestic cattle and helmeted guineafowls in the eastern cape province during march or april respectively, whereas larvae were most numerous on helmeted guineafowls, scrub hares and the vegetation in north-eastern mpu malanga province during may. in both provinces nymphs were most numerous between october and december. amblyomma marmoreum appears to be most prevalent in the western regions of the western and eastern cape and free state provinces, and the north-eastern regions of the northern cape, kwazulunatal, mpumulanga and limpopo provinces. keywords: amblyomma marmoreum, birds, carnivores, domestic animals, geographic distribution, large herbivores, reptiles, seasonality, small mammals, vegetation 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa, and department of zoology and entomology, university of the free state, bloemfontein, 9301 south africa 2 department of parasitology, arc-onderstepoort veterinary institute, onderstepoort, 0110 south africa * present address: school of geosciences, university of witwatersrand, private bag x3, wits, 2050 south africa accepted for publication 22 june 2005—editor 14 south african tortoise tick, amblyomma marmoreum and mapped its distribution and supplied a brief host list, while norval (1975) described its ecology, life cycle and seasonality in the eastern cape province and provided a more comprehensive list of hosts. some years later walker & schulz (1984) recorded the burdens of a. marmoreum on tortoises in the addo elephant national park in the eastern cape province, while norval (1983) listed its hosts and mapped its distribution in zimbabwe. interest in a. marmoreum gained momentum with the unpublished discovery by bezuidenhout & olivier in 1985 and bezuidenhout in 1986 (cited by bezuidenhout 1987, and oberem & bezuidenhout 1987) that it could both acquire and transmit ehrlichia (cow dria) ruminantium, the causative organism of heart water in domestic and wild ruminants. in addition, bezuidenhout (1988) demonstrated that the leopard tortoise, a preferred host of all stages of development of the tick, could be infected with e. (cowdria) ruminantium and that the tick could acquire infection from an infected tortoise. he also demonstrated that two hosts of the immature stages, namely hel meted guineafowls and scrub hares could act as sub clinical reservoirs of e. (cowdria) ruminantium. walker & olwage (1987) have published colour illustrations of a male and female tick and have mapped the distribution of a. marmoreum in africa. horak, macivor, petney & de vos (1987a) have provided host lists as well as the mean intensity and prevalence of infestation on these hosts, while dower, petney & horak (1988) determined the tick burdens, and the detachment periods and weights of the various life stages of a. marmoreum on naturally infested tortoises in the eastern cape province. the pres ence, and occasionally also the seasonality, of the immature stages of a. marmoreum on a large number of host species have been recorded by horak and his co-workers in several published and unpublished surveys of parasites of domestic and wild animals conducted in south africa (horak & fourie 1986; horak, jacot guillarmod, moolman & de vos 1987b; dower et al. 1988; fourie & horak 1990; horak, williams & van schalkwyk 1991a; horak, fourie, novellie & williams 1991b; horak, spickett, braack & williams 1991c; horak, knight & williams 1991d; horak & fourie 1991; horak, boomker, spickett & de vos 1992; horak, spickett, braack & penzhorn 1993; boomker, horak & ram say 1994; horak & boomker 1998; horak 1999; horak, braack, fourie & walker 2000; horak, macivor & greeff 2001; horak, gallivan, braack, boomker & de vos 2003; horak & matthee 2003; uys & horak 2005). fielden, magano & rechav (1992) have compared the length of the life cycle of a. marmoreum on tortoises and on guinea pigs in the laboratory, and fielden & rechav (1994) and rechav & fielden (1995) have determined its attachment sites and sea sonal abundance on leopard tortoises. burridge (2001) and burridge & simmons (2003) have described its introduction as well as that of other ticks into the united states of america on imported reptiles and allan, simmons & burridge (1998) have reported its establishment on a reptile-breeding facil ity in florida. peter, burridge & mahan (2000) have demonstrated the competence of a. marmoreum as a potential effective vector of e. (cow dria) ruminantium in that country. the present paper is based on the findings pertinent to a. marmoreum in the numerous surveys conducted in south africa as well as on a large number of tick collections made from reptiles, particularly tortoises that have not previously been published. its purpose is to record the presence and the seasonality of adult and immature a. marmoreum on tortoises, and that of its larvae and nymphs on mammals and birds as well as of its free-living larvae on vegetation. furthermore the coordinates of the localities at which the abovementioned collections were made have been used to up-date theiler & salisbury’s (1959) map of the tick’s distribution in south africa. materials and methods ticks were collected manually from several live reptiles, chiefly tortoises, and preserved in 70 % ethyl alcohol for subsequent identification and counting. ticks were also collected from domestic and wild animals in various regions of the country, as well as from the vegetation within the kruger national park in north-eastern mpumalanga province. the techniques used to collect ticks in the various surveys have been described by horak et al. (1987b; 1991c; 1992), dower et al. (1988) and spickett, horak, van niekerk & braack (1992), and are not repeated here. the findings presented pertain only to a. marmoreum and not to the other ticks collected during the various surveys. the animal species sampled have been divided into six groupings, namely reptiles, birds, small mammals, carnivores, large herbivores, and domestic animals and humans, and the numbers of a. marmoreum collected from each host species within each of the groupings have been summarized in tabular format. the scientific names of the animals 15 i.g. horak et al. examined are included in the tables and are not repeated in the text. the monthly mean adult and immature tick burdens of all tortoises examined over a period 6 years, irrespective of species and the year in which they were sampled, have been used to construct a pattern of annual seasonality for a. marmoreum. a similar process has been followed with the immature tick burdens of helmeted guineafowls and of cattle examined at monthly intervals over a period of 2 years in the eastern cape province, and of helmeted guineafowls and scrub hares examined at monthly intervals over periods of 2 and 6 years, respectively in north-eastern mpumalanga province. the seasonality of free-living larvae on the vegetation was determined from the means of consecutive monthly collections made over a period of 13 years and 8 months in a landscape zone within the southern kruger national park. the geographic coordinates of the localities at which the various collections were made have been added to those of theiler & salisbury (1959), and have been used to redefine the distribution of a. marmoreum within the borders of south africa. results and discussion hosts a total of 309 reptiles belonging to 13 species were examined and with the exception of the karoo padloper, of which only two specimens were sampled, all tortoise species were infested with adult a. marmoreum (table 1). the largest numbers of adult and immature ticks were recovered from leopard tortoises. comparisons between reptile species are, however, not possible because of differences in the sampling techniques. most collections were done manually and focused on the more visible and hence detachable ticks, but 14 leopard tortoises were kept in cages over water until all the ticks present on most of them had detached (dower et al. 1988). the prevalence of infestation on the various species of reptiles can also not be deduced from the available data as the majority of collections were made from live animals and no records were kept of the number of reptiles on which no ticks were seen. whereas other animals harbour only the immature stages of a. marmoreum, reptiles, and more particularly tortoises, harbour all stages of development. this pattern of host preference dictates that the tick’s life cycle can be completed only in those localities where tortoises and certain other large reptile species are present. in the present study the ratio of larvae to nymphs to adults on tortoises and other reptiles was 2.7:1.0:3.0, indicating that the number of immature ticks would have been unable to maintain the adult population. however, as most of the collections made from these animals were not design ed to be total recoveries, and focused mainly on the more visible adult ticks, these ratios are not surprising. in a more detailed study on the seasonal occurrence of a. marmoreum, leopard tortoises in a large, fenced enclosure containing natural savannatype vegetation in the national zoolog ical gardens, pretoria, were examined for ticks at monthly intervals (rechav & fielden 1995). over a period of a year the ratio of larvae to nymphs to adults on these animals was 5.7:2.7:1.0, indicating an adequate number of immature ticks to maintain the adult burdens on the tortoises. five bird species, comprising 269 individuals, were examined for ticks (table 2). these examinations were thorough as they were conducted on dead birds and hence both the prevalence and intensity of infestation recorded are more reliable than they would have been had they been based on manual collections of ticks from live birds. a large proportion of helmeted guineafowls and francolins were infested and many larvae were collected from these birds. the largest number of ticks recovered from a single guineafowl consisted of 585 larvae and one nymph collected from a bird examined during february 1984 in the mountain zebra national park in the eastern cape province. helmeted guineafowls and francolins are not only large birds, but also spend most of their lives on the ground, factors that both probably contribute to their success as hosts of the immature stages of a. marmoreum. ticks were collected from a total of 1 754 small mammals belonging to 11 species, the majority of which were killed for survey purposes (table 3). amongst these there were 579 murid rodents and 31 springhares, a very large rodent, all of which were sampled in localities in which tortoises, or other animals harboured a. marmoreum, and yet only two bush karoo rats were infested, each with a single larva. this supports the contention of petney, horak, howell & meyer (2004) that rodents are not good hosts of amblyomma spp. they arrived at this conclusion after recovering only five unhealthy-looking larvae and a nymph of amblyomma hebraeum from just six of 169 collections made from striped grass mice in a habitat heavily contaminated with 16 south african tortoise tick, amblyomma marmoreum table 2 amblyomma marmoreum collected from birds host species number examined number infested number of ticks collected larvae nymphs males females total helmeted guineafowl, numida meleagris 231 177 4 175 206 0 0 4 381 greywing francolin, francolinus africanus 7 4 129 0 0 0 129 cape francolin, francolinus capensis 7 3 15 1 0 0 16 crested francolin, francolinus sephaena 23 13 582 10 0 0 592 swainson’s francolin, francolinus swainsonii 1 0 0 0 0 0 0 total 269 197 4 901 217 0 0 5 118 table 1 amblyomma marmoreum collected from reptiles host species number examined number infested number of ticks collected larvae nymphs males females total leopard tortoise, geochelone pardalis 59 56 336 92 301 83 812 geometric tortoise, psammobates geometricus 64 54 9 7 52 12 80 karoo tortoise, psammobates oculifer 11 11 0 1 9 4 14 tent tortoise, psammobates tentorius tentorius 24 11 8 8 14 2 32 tent tortoise, psammobates tentorius trimeni 5 1 0 0 1 0 1 angulate tortoise, chersina angulata 52 6 0 5 12 1 18 areolate padloper, homopus areolatus 73 68 58 63 32 20 173 speckled padloper, homopus signatus 11 2 81 0 1 0 82 greater padloper, homopus femoralis 4 4 0 1 4 0 5 karoo padloper, homopus boulengeri 2 2 2 5 0 0 7 white-throated monitor, varanus exanthematicus 1 1 0 1 1 0 2 puff adder, bitis arietans 2 1 0 0 1 0 1 gaboon adder, bitis gabonica 1 0 2 0 0 0 2 total 309 217 496 183 428 122 1 229 17 i.g. horak et al. larvae of this tick. with the exception of scrub hares, of which 43.6 % were infested with a. marmoreum, and which collectively also harboured surprisingly large numbers of nymphs, other small mammals also appeared not to be suitable hosts of this parasite. fifteen carnivore species, comprising 213 individuals that were killed for survey and other purposes, were examined. the prevalence of infestation was highest on black-backed jackals (table 4). the average prevalence of infestation on the 656 herbivores, all of which had been killed for survey purposes, was 28.2 % and varied between 2.4 % on 41 warthogs to 72.7 % on 11 elands (table 5). the only black wildebeest infested was a very old animal, which harboured a burden of 146 larvae and two nymphs, while 34 infested greater kudus had mean burdens of 92 larvae. with the exception of a single male tick on a bontebok ram examined during december 1979 in the bontebok national park in the western cape province (horak, brown, boomker, de vos & van zyl 1982), no large herbiv orous animal was infested with adult ticks. the ticks from domestic cats and dogs were collected either by the staff at veterinary clinics when the animals were brought in for treatment, or by their owners and hence cannot be regarded as complete collections or as a true indication of the prevalence of infestation on these animals. those collected from horses, cattle, sheep and goats were recovered from animals that had been slaughtered for survey purposes and had thus been thoroughly processed for the recovery of external and internal parasites. the ticks recorded on humans were collected as part of an on-going survey to identify those species that bite people in south africa (horak, fourie, heyne, walker & needham 2002). the incomplete nature of the collections made from dogs and cats is endorsed by the fact that larger numbers of nymphs than larvae were recovered, whereas on the other domestic animals, for which table 3 amblyomma marmoreum collected from small mammals host species number examined number infested number of ticks collected larvae nymphs males females total pouched mouse, saccostomys campestris 14 0 0 0 0 0 0 namaqua rock mouse, aethomys namaquensis 425 0 0 0 0 0 0 striped grass mouse, rhabdomys pumilio 91 0 0 0 0 0 0 swamp rat, otomys irroratus 2 0 0 0 0 0 0 bush karoo rat, otomys unisulcatus 47 2 2 0 0 0 2 spring hare, pedetes capensis 31 0 0 0 0 0 0 rock dassie, procavia capensis 102 21 108 3 0 0 111 rock elephant shrew, elephantulus myurus 296 7 10 1 0 0 11 cape hare, lepus capensis 67 8 15 4 0 0 19 scrub hare, lepus saxatilis 623 272 1 260 395 0 0 1 655 smith’s red rock rabbit, pronolagus rupestris 56 17 36 3 0 0 39 total 1 754 327 1 431 406 0 0 1 837 18 south african tortoise tick, amblyomma marmoreum the collection procedures had been more thorough, the converse was true (table 6). the two horses that were examined had both been used as trails horses in the mountain zebra national park, where four of 14 cape mountain zebras and eight of 11 elands examined during the same period were infested. the cattle were examined on farms in valley bushveld in the eastern cape province (horak 1999) and in false upper karoo in south-western free state province (fourie & horak 1990), the sheep on farms in valley bushveld and eastern province thorn veld (horak et al. 1991a), and the goats in valley bushveld and in noorsveld in the eastern cape province (horak et al. 1991d; 2001) and in mixed bushveld in limpopo province (boom ker et al. 1994). the prevalence and intensity of infestation was greater on cattle and sheep than on goats, while the intensity of infestation was greater on cattle than on sheep (table 6). horak et al. (2002) noted that the comparatively small total number of only 194 collections of ticks taken from humans over four decades reflects the tendency in south africa for individuals and healthcare personnel to discard attached specimens, and more particularly larvae, once they have been re moved. in addition, a large proportion of persons living or working in rural environments in the eastern, northeastern and northern regions of the country regard table 4 amblyomma marmoreum collected from carnivores host species number examined number infested number of ticks collected larvae nymphs males females total black-backed jackal, canis mesomelas 8 8 243 27 0 0 270 hunting dog, lycaon pictus 8 1 0 16 0 0 16 bat-eared fox, otocyon megalotis 2 1 1 0 0 0 1 cheetah, acinonyx jubatus 3 2 26 18 0 0 44 caracal, caracal caracal 51 35 1 360 15 0 0 1 375 african wild cat, felis lybica 1 1 1 0 0 0 1 lion, panthera leo 24 17 159 203 0 0 362 leopard, panthera pardus 6 5 43 202 0 0 245 yellow mongoose, cynictis penicillata 80 3 11 0 0 0 11 white-tailed mongoose, ichneumia albicauda 2 2 7 0 0 0 7 banded mongoose, mungos mungo 2 1 7 0 0 0 7 spotted hyaena, crocuta crocuta 10 2 0 15 0 0 15 aardwolf, proteles cristatus 1 1 27 0 0 0 27 civet cat, civettictis civetta 7 6 16 46 0 0 62 large-spotted genet, genetta tigrina 8 6 13 3 0 0 16 total 213 91 1 914 545 0 0 2 459 19 i.g. horak et al. tick-bite as a normal occurrence and would seldom consider retaining specimens. thus, the eight instances of bites by a. marmoreum are likely to represent only a small proportion of bites on humans due to this species. the large numbers of a. marmoreum larvae collected from the vegetation by drag-sampling with flannel strips implies that they quest for hosts from this vantage point. this would seem to be an unnecessary strategy if tortoises and other land-bound reptiles are their only hosts. by questing from the vegetation the larvae are unlikely to be particularly host-specific and will attach to a variety of animals, an observation supported by the present findings. norval (1975) stated that adult ticks attach in greater numbers around the bases of the hind-legs of tortoises, and nymphs around those of the forelegs as well as on the head and neck, while larvae are evenly distributed between the two sites. fielden & rechav (1994) and burridge, simmons & allan (2000) report most adults on the upper soft-skinned parts of the hind-legs and in the hollows in front of these legs, as well as around the base and on the ventral surface of the tail. larvae and nymphs attach mainly in soft skinned localities that are protected by the carapace, particularly the neck and upper legs (fielden & rechav 1994). uys & horak (2005) collected equal numbers of a. marmoreum larvae from the bodies and from the wings of cresttable 5 amblyomma marmoreum collected from large herbivores host species number examined number infested number of ticks collected larvae nymphs males females total cape mountain zebra, equus zebra zebra 14 4 180 1 0 0 181 warthog, phacochoerus africanus 41 1 2 0 0 0 2 giraffe, giraffa camelopardalis 6 1 0 2 0 0 2 impala, aepyceros melampus 229 67 2 227 23 0 0 2 250 black wildebeest, connochaetes gnou 13 1 146 2 0 0 148 bontebok, damaliscus pygargus dorcas 47 14 90 4 1 0 95 springbok, antidorcas marsupialis 22 2 4 0 0 0 4 african buffalo, syncerus caffer 1 1 8 1 0 0 9 eland, taurotragus oryx 11 8 167 0 0 0 167 greater kudu, tragelaphus strepsiceros 120 34 3 131 2 0 0 3 133 red forest duiker, cephalophus natalensis 23 12 40 8 0 0 48 gemsbok, oryx gazella 26 15 457 13 0 0 470 grey rhebok, pelea capreolus 62 20 210 4 0 0 214 reedbuck, redunca arundinum 21 2 12 0 0 0 12 mountain reedbuck, redunca fulvorufula 20 3 10 2 0 0 12 total 656 185 6 684 62 1 0 6 747 20 south african tortoise tick, amblyomma marmoreum ed francolins, fewer from their tails and the least from their heads and upper necks. they suggest that this attachment pattern might be a strategy to avoid competition with the larvae of a. hebraeum and hyalomma marginatum rufipes, of which more than 84 % attach to the heads and upper necks of the birds. seasonality the countrywide seasonality of all stages of development of a. marmoreum on tortoises is graphically illustrated in fig. 1. male and female ticks were most numerous on tortoises during january and february. thereafter, with the exception of july, the only month during which female ticks outnumbered males, the numbers of female ticks remained low even when the numbers of males started to increase on tortoises from october to december. norval (1975) recorded the largest numbers of adult ticks on leopard tortoises on a farm in the eastern cape province from january to march, whereas rechav & fielden (1995) recorded the largest numbers of adults on these animals in the national zoological gardens, pretoria from october to january, with males most numerous from october to may and females from september to december. female ticks can take up to 60 days (norval 1975) to 91 days (fielden et al. 1992) to complete feeding on artificially infested tortoises, and dower et al. (1988) recorded a period as long as 73 days before all females and 111 days before all males had detached from tortoises that they had collected in the field. the january and february peak in adult tick numbers may therefore reflect an accumulation of ticks that had attached a month or two earlier. the longer period of attachment of male ticks may in part explain the preponderance of male compared to female ticks on tortoises. larvae were most numerous on tortoises, irrespective of the localities in which they had been sampled, during march (fig. 1), and on helmeted guineafowls and cattle in inland valley bushveld in the eastern cape province from february to april with an april peak on the guineafowls and a march peak on the cattle (fig. 2a and b). larvae were most numerous on helmeted guineafowls, scrub hares and the vegetation in the lowveld of north-eastern table 6 amblyomma marmoreum collected from domestic animals and from humans host species number examined number infested number of ticks collected larvae nymphs males females total dogs 915 8 2 13 0 0 15 cats 20 1 0 2 0 0 2 horses 2 2 14 0 0 0 14 cattle 58 32 1 942 17 0 0 1 959 sheep 175 81 1 132 30 0 0 1 162 goats 373 82 851 73 0 0 924 humans 194 7 2 3 3 0 8 total 1 737 213 3 943 138 3 0 4 084 fig. i the seasonal occurrence of amblyomma marmoreum on tortoises in south africa ��������� �� �� ������������� � � � �� � � � � � � �� �� �� � � � ��� �� ��� � �� � �� ��� � � � � � � � � � � � � � � � � � ! � �� 21 i.g. horak et al. ��������� �� �� ��� "#�$� %�&��'() ����$(* �� � � � � �� � � � � � � �� �� � � � � � � �� � � � � � � � � ! � � � �� � � � � � � �� �� �� �� � � �� �� �� �� �� �� � �� �� � � � � � � �� � �� ��� � �� �� � �� � � �� � �� �� �� �� �� � � �� � � � ��������� �� �� �����+�#,(� ���'(-�. ! �� �� �� �� �� �� �� � � � �� � � � � � � � � ! � �� � �� ��� ��������� �� �� ��� ��"�� ���$'(-�. ) � �� � � � � � � � � ! � �� � � � � �� � � � � � � �� �� �� �� � � �� �� �� �� �� �� �� � ��������� �� �� ����+ ����'() ����$(* �� / � � � �� � � � � � � �� �� � � � � � � � � �� � � � � � � �� �� �� �� � � ��� ��� ��� ��� ��� �� � �� �� �� �� �� � � � �� � � � � � � � � ! � �� � �� ��� � � � �� � � � � � � �� �� � � � � � � � � �� � � � � � � �� �� �� �� � � ��������� �� �� ����"#�$� %�&��'(-�. * ��� �� �� �� �� � �� � � � � � � �� � � � � � � � � ! � �� � �� ��� fig. 2 the seasonal occurrence of the larvae and nymphs of amblyomma marmoreum on (a) helmeted guinea fowls and (b) domestic cattle in valley bushveld in the eastern cape province, and (c) helmeted guineafowls, and (d) scrub hares, and of larvae on (e) vegetation in the lowveld of north-eastern mpumalanga province. knp = kruger national park 22 south african tortoise tick, amblyomma marmoreum mpumalanga province from march or april to june or july, with peaks in may (fig. 2c–e). very few if any larvae were present from october to december or january in either of the provinces. norval (1975) collected most larvae from a naturally infested leopard tortoise during april and from the vegetation on a farm in the eastern cape province from february to june with a peak in may. rechav & fielden (1995) recorded the largest numbers of larvae on tortoises in the national zoological gardens, pretoria from january to may with a peak in february and march. nymphs were most numerous on tortoises, irrespective of the localities in which they had been examined, during april (late summer) and during sep tember (spring) (fig. 1). nymphs were most numerous on helmeted guineafowls and on cattle in inland valley bushveld from september to december (fig. 2a and b), and on scrub hares in the lowveld from october to december (fig. 2d). however, nymphs were most numerous on guineafowls in the latter habitat from december to august (fig. 2c). the largest numbers of nymphs were present on tortoises in the national zoological gardens, pretoria from may to october with a peak in june and july (rechav & fielden 1995). compared to 1 604 larvae, only six nymphs were collected during nearly 14 years of monthly dragsampling the vegetation in the lowveld. these nymphs have not been incorporated in fig. 2e. on the other hand, norval (1975) collected 449 larvae and 27 nymphs from the vegetation by this sampling method and the latter were most numerous from october to january. the present results indicate that the seasonal pattern of occurrence of the immature stages of a. marmoreum in the north-east of the country is similar to that in the south-east. norval (1975) thought that hosts other than tortoises did not play a significant role in the life cycle or seasonality of a. marmoreum because of the small number of ticks he encountered on these hosts. however, the substantial numbers presently recorded on some host species, other than tortoises, as well as the proportion of these animals that are infested, imply that they may well play an important role in the tick’s life cycle. in his experiments on the life cycle of a. marmoreum norval (1975) found that larvae required up to 30 days to complete feeding on tortoises and nymphs up to 51 days, whereas larvae he fed on the ears of sheep engorged in 6–12 days and nymphs in 8–20 days. dower et al. (1988) recorded a mean of 35 days and a range of 8–104 days for larvae, and a mean of 21 days and range of 4–47 days for nymphs to detach from tortoises collected in the field. as there was no way of knowing exactly how many days before capture these ticks attached to the latter tortoises, all these periods are probably considerably longer. fielden et al. (1992) compared the length of the life cycle of a. marmoreum on tortoises and on guinea pigs in the laboratory. on tortoises the period between larval attachment and the emergence of adults varied between 69 and 172 days, whereas on guinea pigs it varied between 63 and 95 days. domestic chickens, and hence presumably guineafowls, have higher body temperatures than those of mammals, and larvae of a. hebraeum fed on chickens engorged and detached in 4–8 days compared to 5–10 days on rabbits infested at the same time (holley & petney 1988). if a similar phenomenon occurs when the immature stages of a. marmoreum feed on guineafowls as opposed to sheep, the length of the life cycle could be reduced even further. according to norval (1975) the life cycle of a. marmoreum in the eastern cape province can be completed in either 1 or 2 years. he proposed that for it to be completed in 1 year, it was necessary for larvae to feed on tortoises in late summer and the resultant nymphs and subsequent adults to feed on tortoises in spring and in early summer respectively. fielden et al. (1992) suggest that host selection by the immature stages could be important in determining whether the life cycle is completed in 1 or in 2 years, an opinion with which we concur. provided the larvae and the nymphs feed on mammals or birds in late summer and in spring respectively and the adults on tortoises in early summer the life cycle can be completed in 1 year. geographic distribution theiler & salisbury (1959) mapped the distribution of a. marmoreum in south africa from the geographic coordinates of 49 localities at which collections were made. we have now added more than 100 localities to their map (fig. 3). most collections have been made in the western regions of the western and eastern cape and free state provinces, and in the north-eastern regions of the northern cape, kwazulu-natal, mpumalanga and limpopo provinces. the seemingly large number of localities at which a. marmoreum was present in north-eastern mpumalanga and limpopo provinces are a reflection of the numerous surveys conducted in these regions by horak and his co-workers over a period of 25 years. the several collections made in other regions imply that these are also suitable hab23 i.g. horak et al. itats for the tick. very few collections of a. marmoreum have been made in the eastern highveld regions of the eastern cape, free state and mpuma langa provinces and inland mountainous regions of kwazulu-natal. we are, however, convinced that future collections will indicate an even wider distribution for a. marmoreum in south africa than that now proposed. acknowledgements we are indebted to mr p.d. burdett who was responsible for many of the tick collections from reptiles, and thank the south african national parks for placing the animals as well as their staff and facilities in the various national parks at our disposal. we gratefully acknowledge the assistance of messrs c. cheney, j. sithole, m.m. knight, e.j. williams and the late b.d. de klerk with processing the carcasses of the survey animals for the recovery of ectoparasites, and that of mrs e.l. visser, miss m. l. horak, mr a.c. uys and mr e.j. williams who assisted with the recovery of ticks from the processed material. dr r. williams of the onderstepoort veterinary institute constructed the distribution map. references allan, s.a., simmons, l.-a. & burridge, m.j. 1998. establishment of the tortoise tick amblyomma marmoreum (acari: ixodidae) on a reptile-breeding facility in florida. journal of medical entomology, 35:621–624. bezuidenhout, j.d. 1987. natural transmission of heartwater. onderstepoort journal of veterinary research, 54:349–351. bezuidenhout, j.d. 1988. sekere aspekte van hartwater oordraging, voorkoms van die organisme in bosluise en in vitro kweking. d.v.sc. thesis, university of pretoria. boomker, j., horak, i.g. & ramsay, k.a. 1994. helminth and arthropod parasites of indigenous goats in the northern transvaal. onderstepoort journal of veterinary research, 61:13–20. burridge, m.j., simmons, l.-a. & allan, s.a. 2000. introduction of potential heartwater vectors and other exotic ticks into florida on imported reptiles. journal of parasitology, 86: 700–704. burridge, m.j. 2001. ticks (acari:ixodidae) spread by the inter national trade in reptiles and their potential roles in dissemination of diseases. bulletin of entomological research, 91:3–23. burridge, m.j. & simmons, l.a. 2003. exotic ticks introduced into the united states on imported reptiles from 1962 to 2001 and their potential roles in international dissemination of diseases. veterinary parasitology, 113:289–320. dower, kathy m., petney, t.n. & horak, i.g. 1988. the developmental success of amblyomma hebraeum and amblyomma marmoreum on the leopard tortoise, geochelone fig. 3 the geographic distribution of amblyomma marmoreum within the borders of south africa 24 south african tortoise tick, amblyomma marmoreum pardalis. onderstepoort journal of veterinary research, 55: 11–13. fielden, l.j., magano, s. & rechav, y. 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animals in south africa. xxvi. the mosaic of ixodid tick infestations on birds and mammals in the mountain zebra national park. onderstepoort journal of veterinary research, 58:125–136. horak, i.g., spickett, a.m., braack, l.e.o. & williams, e.j. 1991c. parasites of domestic and wild animals in south africa. xxvii. ticks on helmeted guineafowls in the eastern cape province and eastern transvaal lowveld. onderstepoort journal of veterinary research, 58:137–143. horak, i.g., knight, m.m. & williams, e.j. 1991d. parasites of domestic and wild animals in south africa. xxviii. helminth and arthropod parasites of angora goats and kids in valley bushveld. onderstepoort journal of veterinary research, 58:253–260. horak, i.g. & fourie, l.j. 1991. parasites of domestic and wild animals in south africa. xxix. ixodid ticks on hares in the cape province and on hares and red rock rabbits in the orange free state. onderstepoort journal of veterinary research, 58:261–270. horak, i.g., boomker, j., 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matthee, sonja 2003. parasites of domestic and wild animals in south africa. xliii. ixodid ticks of domestic dogs and cats in the western cape province. onderstepoort journal of veterinary research, 70:187–195. norval, r.a.i. 1975. studies on the ecology of amblyomma marmoreum koch 1844 (acarina: ixodidae). journal of parasitology, 61:737–742. norval, r.a.i. 1983. the ticks of zimbabwe. vii. the genus am blyomma. zimbabwe veterinary journal, 14:292–305. oberem, p.t. & bezuidenhout, j.d. 1987. heartwater in hosts other than domestic ruminants. onderstepoort journal of veterinary research, 54:271–275. peter, t.f., burridge, m.j. & mahan, s.m. 2000. competence of the african tortoise tick amblyomma marmoreum (acari: ixodidae), as a vector of the agent of heartwater (cowdria ruminantium). journal of parasitology, 86:438– 441. petney, t.n., horak, i.g., howell, d.j. & meyer, s. 2004. striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks. onderstepoort journal of veterinary research, 71:313–318. rechav, y. & fielden, l.j. 1995. seasonal abundance of the tortoise tick amblyomma marmoreum (acari: ixodidae) on the leopard tortoise. journal of medical entomology, 32:161– 165. spickett, a.m., horak, i.g., van niekerk, andrea & braack, l.e.o. 1992. the effect of veld-burning on the seasonal abundance of free-living ixodid ticks as determined 25 i.g. horak et al. by drag-sampling. onderstepoort journal of veterinary research, 59:285–292. theiler, gertrud 1943. ticks in the south african zoological survey collection. part ii. onderstepoort journal of veterinary science and animal industry, 18:85–89. theiler, gertrud & salisbury, lois e. 1959. ticks in the south african zoological survey collection—part ix—“the amblyomma marmoreum group”. onderstepoort journal of veterinary research, 28:47–124. uys, a.c. & horak, i.g. 2005. ticks on crested francolins, francolinus sephaena and on the vegetation on a farm in limpopo province, south africa. onderstepoort journal of veterinary research, 72:339–343. walker, jane b. & schulz, k.c.a. 1984. records of the bont tick, amblyomma hebraeum, from the angulate tortoise, chersina angulata, and the leopard tortoise, geochelone pardalis. onderstepoort journal of veterinary research, 51: 171–173. walker, jane b. & olwage, a. 1987. the tick vectors of cowdria ruminantium (ixodoidea, ixodidae, genus amblyomma) and their distribution. onderstepoort journal of veterinary research, 54:353–379. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice stevens_87-95.indd introduction south africa has been accorded a foot-and-mouth (fmd) disease-free without vaccination status by the world organisation for animal health (oie) with the exception of the wildlife reserve, the kruger national park (knp), and adjacent private wildlife parks which are considered as the only endemically infected areas in the country due to the presence of persistently infected african buffaloes, syncerus caffer. surrounding the knp and adjacent parks is a ‘buffer zone with vaccination’, a narrow strip of farmland immediately adjacent to the knp where all cattle are regularly vaccinated against fmd and weekly surveillance and strict movement control programmes are in place (brückner, vosloo, kloeck & weaver 2003; thomson, vosloo & bastos 2003). a ‘buffer zone where vaccination is not allowed’, surrounds the ‘buffer zone with vaccination’, and separates the endemically infected area from the fmd-free zone. two of the country’s provinces are affected by this control effort, viz. mpumalanga and limpopo. as an 87 onderstepoort journal of veterinary research, 74:87–95 (2007) influence of dipping practices on the seroprevalence of babesiosis and anaplasmosis in the foot-and-mouth disease buffer zone adjoining the kruger national park in south africa k.b. stevens1*, a.m. spickett2, w. vosloo2, 5, d.u. pfeiffer1, e. dyason3 and b. du plessis4 abstract stevens, k.b., spickett, a.m., vosloo, w., pfeiffer, d.u., dyason, e. & du plessis, b. 2007. influence of dipping practices on the seroprevalence of babesiosis and anaplasmosis in the foot-and-mouth disease buffer zone adjoining the kruger national park in south africa. onderstepoort journal of veterinary research, 74:87–95 a serological survey of bovine babesiosis and anaplasmosis was conducted in the foot-and-mouth disease buffer zone surrounding the kruger national park in south africa between 2001 and 2003 to determine whether the withdrawal of government-subsidized dipping in certain regions had affected the seroprevalence of these tick-borne diseases. seroprevalence of anaplasma marginale and babesia bovis increased during the study period. this increase was greater in limpopo province where farmers had to supply their own acaricide than in mpumalanga province where dipping materials were provided by the local veterinary services. the number of animals testing positive for b. bigemina decreased in both provinces during the study period, which was attributed to possible vector displacement rather than more effective tick control measures. responses to a questionnaire on ticks and tick-borne diseases revealed local knowledge on the subject to be highly variable and sometimes incorrect. keywords: bovine anaplasmosis, bovine babesiosis, dipping practices, fmd buffer zone, kruger national park, seroprevalence, south africa * author to whom correspondence is to be directed. e-mail: kstevens@rvc.ac.uk 1 royal veterinary college, hawkshead lane, north mymms, hatfield, al9 7ta, united kingdom 2 onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 3 department of agriculture, private bag x 9487, polokwane, 0700 south africa 4 csir, private bag x 11309, nelspruit, mbombela, mpumalanga, 1200 south africa 5 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, 0002 south africa accepted for publication 11 october 2006—editor 88 seroprevalence of babesiosis and anaplasmosis in foot-and-mouth disease in south africa incentive for farmers to present their animals for weekly inspection and disease interventions, the south african government has traditionally provided the means and infrastructure for tick control in communal farming areas such as these. babesia bovis, the cause of asiatic babesiosis or redwater, babesia bigemina, the cause of african babesiosis or redwater and anaplasma marginale, the cause of gallsickness or anaplasmosis, are all tick-borne parasites of major economic importance in southern africa (norval 1994). confirmed vectors of b. bigemina are rhipicephalus (boophilus) decoloratus and rhipicephalus (boophilus) microplus (nomenclature according to horak, camicas & keirans 2002), while b. bovis is transmitted only by the latter tick species. the parasites are widespread in south africa, except in very low rainfall areas, coinciding with the distribution of their tick vectors; b. bigemina being more widespread than b. bovis which occurs mainly in high rainfall areas. young animals commonly harbour a clinically inapparent form of the redwater while acute, nonfatal infections in adult cattle become latent. euro pean cattle breeds are more susceptible to the disease than indigenous breeds but a high mortality rate may occur in sanga and zebu breeds (de vos & potgieter 1994). all cattle breeds develop latent infections after recovery from the disease. however, european breeds may retain b. bovis infections for life, remaining infective for ticks for up to 2 years while zebu types lose this infection within 2 years. babesia bigemina infections rarely persist for more than 1 year in any cattle breed and only remain infective for ticks for 4–7 weeks (de vos & potgieter 1994). anaplasmosis in cattle is arthropod-borne and caused by anaplasma marginale, an obligate intra-erythrocytic rickettsia. haematophagous insects and iatrogenic means may mechanically transmit the parasites and biological transmission is by ixodid ticks. rhipicephalus (b.) decoloratus has been considered the most important vector because disease occurrence mainly coincides with the distribution of this tick species. four other tick species, r. (b.) microplus, rhipicephalus simus, rhipicephalus evertsi evertsi and hyalomma marginatum rufipes have been shown to be capable of experimental transmission of anaplasmosis, their distribution overlapping that of r. (b.) decoloratus (de vos & potgieter 1994; potgieter & stoltsz 1994). clinically, anaplasmosis ranges from inapparent infection to severe disease and high mortality, being characterized by fever, progressive anaemia and icterus. animals that are subclinically infected or that recover from the disease usually remain carriers of the parasite for life (potgieter & stoltsz 1994). owing to budgetary constraints and political reforms government subsidization of cattle dipping was suspended in certain provinces, including limpopo province, between 1994 and 2002 resulting in farmers having to supply their own acaricides instead of relying on the local veterinary services for the provision of free-dipping materials as they had done in the past. by investigating the spatio-temporal patterns of the prevalence of b. bovis, b. bigemina and a. mar ginale between 2001 and 2003 in the ‘buffer zone with vaccination’, this study aimed to determine whether these regional changes in tick control measures had affected the seroprevalence of these three diseases, and to investigate by means of a questionnaire the perceptions of local farmers regarding ticks and tick-borne diseases. materials and methods study area a longitudinal study was designed to investigate spatio-temporal variation in the seroprevalence of b. bovis, b. bigemina and a. marginale antibodies in cattle in the fmd buffer zone adjoining south africa’s knp. the park is situated in the north-east part of the country and spans two provinces—limpopo to the north and mpumalanga to the south. at the time of the study, the veterinary services in mpumalanga were responsible for providing all dipping materials and maintaining dipping facilities, while in limpopo, farmers in ‘the buffer zone without vaccination’ were expected to supply their own acaricides. however, following changes in local departmental policy and availability of funds, farmers in both provinces are at present being supplied with free acaricides. recruitment of study population and sample collection twenty-five of the 121 diptanks in the whole buffer zone (the areas with and without fmd vaccination) were selected by stratified random sampling to participate in the study (14 in limpopo and 11 in mpumalanga). participating herd owners were chosen randomly at each diptank, and between 30 and 40 animals of varying ages were randomly selected per owner at each diptank. the original intention was to sample 30 animals older and ten younger than 2 years of age at each diptank. however, this proved impossible to do as the younger age-group was 89 k.b. stevens et al. either absent from, or poorly represented at the diptanks, resulting in insufficient data to allow for any meaningful comparison between age-groups. therefore, approximately 40 animals, of any age, were sam pled from each diptank. cattle were sampled once a year between april and august, from 2001 to 2003. blood was collected in 10 ml sterile, plain vacuum tubes, using a 20-gauge needle from the caudal vein in adult cattle and the jugular in calves while the animals were restrained in a crush. at the laboratory, the tubes were centrifuged, the serum decanted and the sera were frozen and stored for later analysis. each sampled animal received an ear-tag with a unique identification number. un fortunately, due to loss of ear-tags, sale of animals, death or absenteeism it was not always possible to sample the same animals each year, particularly by the third year of the study, and new animals had to be recruited from each owner to ensure that 40 animals, or as close to the number as was practical, were sampled. questionnaire completion each owner of a sampled herd completed a questionnaire providing data on their herd, dipping practices, their general perception of ticks and tick-borne diseases and the number of animals that had died from a tick-borne disease, giving a total of 72 questionnaires. only those animals where cause of death was confirmed by blood smear were accepted as having died of a tick-borne disease. certain questions were only addressed to the mpumalanga herdsmen on request of the directorate of veterinary services who required the information for a separate study. laboratory analyses the standard indirect fluorescent antibody test (ifa) as described by joyner, donnely, payner & brocklesby (1972) was used to test all blood samples for the presence of antibodies to b. bovis and b. bigemina while the competition inhibition elisa for anaplasmosis (visser, mcguire, palmer, davis, shkap, pipa no & knowles 1992; ndung’u, aguirre, rurangirwa, mcelwain, mcguire, knowles & palmer 1995) was used to test for antibodies to a. margi nale. statistical analysis to account for clustering at the provincial and diptank levels, both serological and questionnaire data were analysed using stata’s survey command series (intercooled stata 7.0 for windows (stata corporation, college station, tx)), with province as the stratification variable and diptank (or diptank group) as the cluster (primary sampling unit) variable. diptanks were grouped according to proximity resulting in seven diptank groups (five in limpopo and two in mpumalanga; fig. 1). questionnaire data were evaluated using contingency tables to identify significant differences in dipping frequency between provinces and in the number of deaths due to anaplasmosis or babesiosis between provinces. serological data were analysed using contingency tables to identify significant differences in seroprevalence within years and between provinces. logistic regression with province, diptank (or diptank group) and year as independent variables, was used to identify risk factors ������� ��� � �� ������ �� ��� � �� � � � � � � � ����� �� ������ ������ �� ��� ���� fig. 1 location of the 25 diptanks and the seven diptank groups in the foot-and-mouth disease buffer zones surrounding the kruger national park 90 seroprevalence of babesiosis and anaplasmosis in foot-and-mouth disease in south africa associated with the prevalence of each disease. statistical analyses were conducted using spss 14.0 for windows (spss inc., chicago, il) and intercooled stata 7.0 for windows (stata corporation, college station, tx). arcview gis 3.3 (esri, redlands, ca, usa) was used for spatial data management and presentation. results questionnaire study of the 72 herdsmen who completed the questionnaire, the majority owned herds comprised of nguni crossbreeds (41, 57 %), nguni cattle (14, 19 %), or a mixture of the two (4, 6 %), while five herds (7 %) comprised brahmin crossbreeds. herd size varied from 3 to 198 animals with a mean herd size of 43 animals (sd 42.9). seven of the 72 herdsmen (10 %) farmed only cattle. all other respondents kept chickens either alone (29 of the herdsmen, 40 %), or in combination with goats (24, 33.3 %), sheep (2, 3 %) or goats and sheep (12, 17 %). sixty-three of the 72 herdsmen (88 %) were aware that they were required by law to dip their cattle. however, all respondents agreed that dipping was good for their animals, although opinions varied as to why it was advantageous with most being of the opinion that it had more than one benefit. sixty-five (90 %) felt that it protected against diseases transmitted by ticks, 58 (81 %) that it prevented damage to cow’s teats, and 54 (75 %) that it prevented damage to hides, while 43 (60 %) said that the animals looked clean after dipping and 13 (18 %) admitted to dipping in order to avoid getting into trouble with the government. fifty-six of the herdsmen (78 %) felt that those who failed to bring their cattle for regular dipping should be fined. there was a significant difference between provinces in summer dipping frequency (chi = 4.3, df = 1, p = 0.04) with just over 80 % of mpumalanga farmers dipping once a week in summer, although this figure rose to almost 100 % in limpopo province (table 1). sixty-four (89 %) of the farmers said they dipped all their cattle, while one (1 %) admitted to dipping only part of his herd. however, 65 (88 %) of the respondents felt that their animals would get sick if not dipped, yet only 53 (82 %) of these said that if for some reason the diptank was closed they would buy acaricide in order to treat their animals. the remainder were content to wait until the diptank reopened, even if this took a few months. mpumalanga and limpopo herdsmen appeared equally aware of alternative methods of tick control. the most well-known alternative form of treatment was hand-spraying (48, 67 %), followed by pour-on remedies (32, 44 %), spray-races (12, 17 %), handdeticking (10, 14 %), used motor oil (5, 7 %), disinfectant (1, 1 %) and traditional medication (1, 1 %). twenty-three (32 %) of the herdsmen questioned preferred one or more of these alternative methods of tick control to dipping due to ease of administration (10, 43 %), efficacy (5, 22 %) or cost (3, 13 %). general consensus as to how long it would take an animal to get sick after being bitten by a tick varied. a quarter of respondents (18 people) thought that it would take less than 15 days, 14 (19 %) did not know, 11 (15 %) felt it would take between 16 and 28 days, 9 (13 %) that it would take 1–2 months, and 2 (3 %) thought it would take more than 2 months. mpumalanga herdsmen were asked whether it was the presence of ticks on an animal that caused it to become sick or whether it was due to diseases carried by the ticks. four (11 %) felt that it was the presence of the tick while eight (23 %) answered that it was due to diseases carried by the parasites. however, 17 (49 %) felt that it was due to a combination of the two, and two (6 %) did not know. twelve of the mpumalanga herdsmen (34 %) agreed that only one tick was necessary to make an animal sick, while the rest either did not know (8, 23 %) or felt that it re quired a number of ticks, although the actual amount varied. five (14 %) thought 2–5 ticks were needed, four (11 %) thought that 6–15 were necessary, while one (3 %) herdsman thought it required 16–30 parasites and another (3 %), that it required more than 30. table 1 dipping frequency of 72 cattle herds in summer and winter in the foot-and-mouth disease buffer zones in mpumalanga and limpopo provinces season limpopo province mpumalanga province weekly (n [%]) fortnightly (n [%]) weekly (n [%]) fortnightly (n [%]) summer winter 35 (97) 26 (72) 1 (3) 10 (28) 28 (82) 27 (79) 6 (18) 7 (21) 91 k.b. stevens et al. only nine (26 %) of the mpumalanga herdsmen felt that they were solely responsible for the health of their cattle. five (14 %) thought the government was responsible while 17 (49 %) felt the responsibility fell jointly on them and the government. at the time of the study dipping was free of charge in mpumalanga. the herdsmen were therefore asked what they would do if required to pay r0.50 (approximately us$0.08) per animal to cover the cost of each dipping. only 12 (34 %) said that they would pay the r0.50 and continue to bring all their animals, five (14 %) responded that they would stop bringing their animals, four (11 %) would dip only if their cattle had ticks, two (6 %) said they would only bring some cattle for dipping and one person (3 %) said they would dip their cattle less frequently. thirty-two (91 %) of the mpumalanga herdsmen were satisfied with the current dipping system, yet only 18 (49 %) of the limpopo herdsmen felt the same way, citing poor efficacy of the dipping chemical (11, 61 %), lack of dipping chemical (4, 22 %) and inability to afford their own acaricide (4, 22 %) as reasons for dissatisfaction. there was no significant difference in the number of herds that experienced deaths due to either babesiosis (six herds) or anaplasmosis (seven herds) at the province level. seroprevalence study serology data was analysed at both the provincial and diptank group level. there was no significant difference in seroprevalence of b. bovis between the two provinces for any of the 3 years. in mpu malanga, the proportion of animals testing seropositive for b. bovis decreased slightly over the 3 years (51.4–48.1 %), but increased in limpopo from 44.2 to 59.7 % (table 2). there was a significant difference in the seroprevalence of b. bigemina between the two provinces in 2003; 33.1 % of animals tested positive in limpopo while only 17.1 % tested positive in mpumalanga (chi = 28.8, df = 1, p = 0.01; table 2). in mpumalanga, seroprevalence of b. bigemina halved during the study period (35.6–17.6 %) and decreased in limpopo from 46.6 to 33.1 % (table 2). there was a significant difference in seroprevalence of a. marginale between the two provinces in 2001; 75.1 % of animals tested positive in mpumalanga while 58.6 % tested positive in limpopo (chi = 26.9, df = 1, p = 0.05; table 2). in mpumalanga, mean seroprevalence of a. marginale fluctuated but was over 75 % for 2 of the 3 years, while in limpopo the proportion of seropositive animals increased during the trial period from 58.6 to 78.4 % (table 2). the proportion of animals seropositive for b. bovis differed significantly between diptank groups for each of the 3 years, although no diptank group exhibited a consistently high or low seroprevalence during the study period (table 3). between 2001 and 2003, the proportion of animals seropositive to b. bovis decreased slightly in four of the diptank groups. the proportion of animals seropositive for b. bigemina also differed significantly between diptank groups for each of the 3 years, with diptank groups 5 and 6 displaying consistently low seroprevalences for the disease (table 3). between 2001 and 2003 the proportion of animals testing positive for b. bigemina table 2 seroprevalence of babesia bovis, babesia bigemina and aanaplasma marginale in cattle in the foot-and-mouth disease buffer zones in mpumalanga and limpopo provinces between 2001 and 2003 province year cattle sampled (n) seropositive cattle (n [%, 95 % ci]) b. bovis b. bigemina a. marginale mpumalanga 2001 385 198 (51.4, 46.3–56.5) 137 (35.6, 30.8–40.6) 289 (75.1, 70.4–79.3) 2002 397 199 (50.1, 45.1–55.2) 145 (36.6, 31.8–41.5) 230 (57.9, 52.9–62.8) 2003 432 208 (48.1, 43.3–53.0) 76 (17.6, 14.1–21.5) 354 (81.9, 78.0–85.5) limpopo 2001 536 237 (44.2, 40.0–48.5) 250 (46.6, 42.4–51.0) 314 (58.6, 54.3–62.8) 2002 490 247 (50.4, 45.9–54.9) 154 (31.4, 27.3–35.7) 345 (70.4, 66.2–74.4) 2003 486 290 (59.7, 55.2–64.1) 161 (33.1, 29.0–37.5) 381 (78.4, 74.5–82.0) 9 2 s e ro p re va le n ce o f b a b e sio sis a n d a n a p la sm o sis in fo o t-a n d -m o u th d ise a se in s o u th a frica table 3 seroprevalence of babesia. bovis, babesia. bigemina and anaplasma marginale between 2001 and 2003 for the seven diptank groups in the foot-and-mouth disease buffer zones in mpumalanga and limpopo provinces province diptank group seropositive cattle [n (%, 95% ci)] b. bovis b. bigemina a. marginale 2001 2002 2003 2001 2002 2003 2001 2002 2003 limpopo province 1 48 (40.0, 31.1–49.3) 14 (21.9, 12.5–34.0) 27 (36.0, 25.2–47.9) 53 (44.2, 35.1–53.5) 28 (43.8, 31.4–56.7) 26 (34.7, 24.0–46.5) 52 (43.3, 34.3–52.7) 23 (35.9, 24.3–48.9) 42 (56.0, 44.1–67.5) 2 92 (80.7, 72.2–87.5) 84 (69.4, 60.4–77.5) 89 (74.2, 65.4–81.7) 74 (64.9, 55.4–73.6) 38 (31.4, 23.3–40.5) 43 (35.8, 27.3–45.1) 79 (69.3, 59.9–77.6) 86 (71.1, 62.1–79.0) 102 (85.0, 77.3–90.0) 3 79 (69.9, 60.6–78.2) 73 (64.0, 54.5–72.8) 83 (68.0, 59.0–76.1) 80 (70.8, 61.5–79.0) 50 (43.9, 34.6–53.5) 51 (41.8, 32.9–51.1) 72 (63.7, 54.1–72.6) 73 (64.0, 54.5–72.8) 96 (78.7, 70.4–85.6) 4 2 (5.7, 0.7–19.2) 36 (85.7, 71.5–94.6) 24 (58.5, 42.1–73.7) 17 (48.6, 31.4–66.0) 15 (35.7, 21.5–52.0) 22 (53.7, 37.4–69.3) 21 (60.0, 42.1–76.1) 32 (76.2, 60.5–87.9) 40 (97.6, 87.1–100.0) 5 16 (10.4, 6.1–16.3) 40 (26.8, 19.9–34.7) 67 (52.3, 43.4–61.2) 26 (16.9, 11.3–23.8) 23 (15.4, 10.0–22.3) 19 (14.8, 9.2–22.2) 90 (58.4, 50.2–66.3) 131 (87.9, 81.6–92.7) 101 (78.9, 70.8–85.6) mpumalanga province 6 105 (55.6, 48.2–62.8) 104 (50.2, 43.2–57.2) 95 (39.4, 33.2–45.9) 63 (33.3, 26.7–40.5) 50 (24.2, 18.5–30.6) (36 (14.9, 10.7–20.1) 144 (76.2, 69.5–82.1) 128 (61.8, 54.8–68.5) 206 (85.5, 80.4–89.7) 7 93 (47.4, 40.3–54.7) 95 (50.0, 42.7–57.3) 113 (59.2, 51.8–66.2) 74 (37.8, 30.9–44.9) 95 (50.0, 42.7–57.3) 40 (20.9, 15.4–27.4) 145 (74.0, 67.2–79.9) 102 (53.7, 46.3–60.9) 148 (77.5, 70.9–83.2) p-value < 0.001 < 0.001 0.04 < 0.001 < 0.001 < 0.001 0.09 < 0.001 0.14 χ2 (df 6) 190.4 100.85 62.3 111.0 61.51 67.5 46.6 73.9 42.4 93 k.b. stevens et al. decreased in six of the seven diptank groups. the proportion of animals seropositive for a. mar ginale only differed significantly between diptank groups in 2002. the number of diptank groups where more than 70 % of the animals tested positive for a. marginale increased over the 3 years from two to six. diptank group 1 consistently recorded the lowest a. marginale seroprevalence. logistic regression revealed that none of the risk factors, year, province, diptank (or diptank group), were significantly associated with the prevalence of b. bovis. however, year was significantly associated with both a. marginale (p = 0.006) and b. bigemina (p = 0.001), while diptank group was a significant risk factor for b. bigemina (p = 0.018) (table 4). discussion anaplasma marginale was the most prevalent of the three potentially disease-causing blood parasites studied and the number of animals testing positive for it increased in both provinces between 2001 and 2003, although this increase was almost three times greater in limpopo than in mpumalanga (19.8 versus 6.8 %, respectively). between 2001 and 2003 the seroprevalence of b. bovis decreased very slightly in mpumalanga from 51.4 to 48.1 % but increased in limpopo from 44.2 to 59.7 %. babesia bigemina was the least prevalent of the three parasites in both provinces and was the only one for which the proportion of seropositive animals decreased markedly over the 3 years, achieving a mean seroprevalence of 17.6 % in mpumalanga by 2003 and 33.1 % in limpopo. although seroprevalence of a. marginale and b. bovis increased in both provinces during the study period, the greater increase observed in limpopo, where farmers were expected to supply their own acaricides, suggests that this was due to the removal of government-subsidised dipping, although the antibody seroprevalence of anaplasma may have been additionally influenced by increased mechanical transmission by biting flies. of the 72 farmers who completed the questionnaire, 71 claimed to dip all cattle every week in summer. however, as replies to the questionnaire revealed local knowledge of ticks and tick-borne diseases to be highly variable and sometimes incorrect, farmers might not have been fully aware of the need for regular dipping and therefore, once expected to provide their own acaricides, might not have been dipping as often as they said they were. it is interesting to note that over 60 % of the questionnaire respondents felt that either the government was responsible for the health of their cattle, or that they and the government were jointly responsible. although this question was only addressed to the mpumalanga herdsmen, it is likely that the farmers in limpopo were of the same opinion. thus, if the herdsmen felt that it was the province’s duty to provide free acaricides, they may have resisted supplying their own. although the veterinary service in limpopo has retable 4 final logistic regression model for factors associated with the seroprevalence of babesia bovis, babesia bigemina and anaplasma marginale in cattle in the foot-and-mouth disease buffer zones in mpumalanga and limpopo provinces (year, province and diptank group included as independent variables) disease risk factor ß std error p-value 95 % ci b. bovis year 0.146 0.13 0.278 –0.13 to 0.42 province 0.627 0.53 0.246 –0.46 to 1.72 diptank group –0.203 0.16 0.207 –0.53 to 0.12 constant –291.67 262.67 0.278 –835.0 to 251.69 b. bigemina year –0.362 0.10 0.001 –0.57 to 0.15 province 0.447 0.43 0.314 –0.45 to –1.343 diptank group –0.226 0.09 0.018 –0.41 to –0.04 constant 723.98 200.71 0.001 308.79 to 1139.18 a. marginale year 0.350 0.11 0.006 0.11 to 0.59 province –0.541 0.33 0.119 –1.23 to 0.15 diptank group 0.191 0.11 0.088 –0.03 to 0.42 constant –700.11 229.65 0.006 –1175.18 to –225.04 94 seroprevalence of babesiosis and anaplasmosis in foot-and-mouth disease in south africa sumed responsibility for the provision of dipping ma terials, it is doubtful whether this on its own will be sufficient to decrease the seroprevalence of b. bovis and a. marginale in the region. the questionnaire responses identified a very real need to educate the local farmers on the importance of ticks and tick-borne diseases before the success of any dipping policy, whether government subsidized or otherwise, is guaranteed. of the three parasites studied, b. bigemina was the only one to show a marked decrease in seroprevalence between 2001 and 2003. it is not possible to attribute this to good tick control strategies and, in contrast the increased seroprevalence of b. bovis and a. marginale to poor tick control strategies, as the tick burden would be the same for all three diseases. rhipicephalus (b.) microplus is the sole vector of b. bovis in south africa, whereas b. bigemina can be spread by both r. (b.) microplus and r. (b.) decoloratus (de vos & potgieter 1994). furthermore, tønnesen, penzhorn, bryson, stoltsz & masibigiri (2004) found that when r. (b.) microplus and r. (b.) decoloratus occurred in the same region, r. (b.) micro plus tended to displace r. (b.) decoloratus over time and that this displacement was rapid and complete at communal diptanks. thus, the overall decrease in seroprevalence of b. bigemina might indicate a gradual displacement of r. (b.) decoloratus by r. (b.) microplus in the region and, in fact, a recent study in limpopo found r. (b.) microplus to be more plentiful than r. (b.) decoloratus at the four communal grazing areas investigated (rikhotso, stoltsz, bryson & sommerville 2005). future research into the temporal abundance and distribution of r. (b.) microplus and r. (b.) decoloratus in the region would provide an indication as to whether one vector is in fact displacing the other, or if the contrasting trends in seroprevalence of b. bovis and b. bigemina can be attributed to some other cause. based on a study of bovine babesiosis in zimbabwe, norval, fivaz, lawrence & daillecourt (1983) defined the following five epidemiological situations on the basis of the frequency of occurrence of serological positives and disease history: (i) enzootically (endemically) stable situations (81– 100 % positive sera) (ii) situations approaching endemic stability (61– 80 % positive sera) (iii) endemically unstable situations (21–60 % positive sera) (iv) minimal disease situation (1–10 % positive sera) (v) disease-free situations (0 % positive sera). they further indicated that clinical babesiosis was most possible at localities where the percentage of serological positives for either b. bovis or b. bigemina was in the range of 21–60 %, while the disease was not a cause of significant cattle mortality at localities where no babesia antibodies were detected or where the percentage of serological positives was over 80 %. in the current study, despite the implementation of what could be considered an intensive dipping strategy in both provinces (table 1), which should ostensibly minimize tick infestation and thus babesia infection challenge, a minimal disease situation was only found for b. bigemina in mpumalanga during 2003 (table 2). the remainder displayed an endemically unstable situation. as regards the seven diptank groups during 2003 (table 3), only diptank groups 5 (limpopo) and 6 (mpumalanga), established a minimal disease situation for b. bigemina. diptank groups 2 and 3 (limpopo) were in a situation approaching endemic stability for b. bovis, while all other groups were endemically unstable for both babesia species, the latter situation being indicative of high risk of clinical disease (norval et al. 1983) and a probable cause for concern should dipping be disrupted in any way. conclusion seroprevalence of a. marginale and b. bovis increased during the study period and for both parasites this increase was greater in limpopo where farmers had to supply their own acaricide than in mpumalanga where dipping materials were provided by the local provincial veterinary services, suggesting that regional differences in dipping policy may have affected the seroprevalence of these two diseases. conversely, the number of animals testing positive for b. bigemina decreased during the study period, particularly in mpumalanga. the contradictory behaviour of this parasite may be the result of vector displacement rather than more effective tick control measures. responses to a ques tion naire on ticks and tick-borne diseases revealed local knowledge on the subject to be highly variable and sometimes incorrect. acknowledgements we thank brenda botha and heloise heyne for their considerable assistance with the collection of samples in the field, as well as the many officers of the provincial veterinary services involved, especially the animal health inspectors at the various diptanks. 95 k.b. stevens et al. references brűckner, g.k., vosloo, w., kloeck, p.e.l.g. & weaver, d.b. 2003. eradication of foot-and-mouth disease by using stamping-out and vaccination: the examples of the outbreaks due to serotype o and sat-1 in south africa in foot-and-mouth disease: control strategies, edited by b. dodet & m. vicari. symposium proceedings, lyon, france 2–5 june 2002: 135–143. de vos, a.j. & potgieter, f.t. 1994. bovine babesiosis, in infectious diseases of livestock with special reference to southern africa, edited by j.a.w. coetzer, g.r. thomson & r.c. tustin. cape town: oxford university press. horak, i.g., camicas, j.-l. & keirans, j.e. 2002. the ixodidae, amblyommidae and nuttalliellidae (acari: ixodida): a world list of valid tick names. experimental and applied acarology, 28:7–54. joyner, l.p., donnely, j., payner, r. & brocklesby, d.w. 1972. the indirect fluorescent antibody test for the differentiation of infections with babesia divergens or babesia major. research in veterinary science, 13:515–518. ndung’u, l.w., aguirre, c., rurangirwa, f.r., mcelwain, t.f., mcguire, t.c., knowles, d.p. & palmer, g.h. 1995. detection of anaplasma ovis infection in goats by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay. journal of clinical microbiology, 33: 675–679. norval, r.a.i. 1994. vectors: ticks, in infectious diseases of livestock with special reference to southern africa, edited by j.a.w. coetzer, g.r. thomson & r.c. tustin. cape town: oxford university press. norval, r.a.i., fivaz, b.h., lawrence, j.a. & daillecourt, t. 1983. epidemiology of tick-borne diseases of cattle in zimbabwe. i. babesiosis. tropial animal health and production, 15:87–94. potgieter, f.t. & stoltsz, w.h. 1994. bovine anaplasmosis, in infectious diseases of livestock with special reference to southern africa, edited by j.a.w. coetzer, g.r. thomson & r.c. tustin. cape town: oxford university press. rikhotso, b.o., stoltsz, w.h., bryson, n.r. & som merville, j.e. 2005. the impact of 2 dipping systems on endemic stability to bovine babesiosis and anaplasmosis in cattle in 4 communally grazed areas in limpopo province, south africa. journal of the south african veterinary asso ciation, 76:217–223. thomson, g.r., vosloo, w. & bastos, a.d.s. 2003. the epidemiology and control of foot-and-mouth disease in subsaharan africa foot-and-mouth disease: control strategies, edited by b. dodet & m. vicari. symposium proceedings, lyon, france, 2–5 june 2002: 125–134. tønnesen, m.h., penzhorn, b.l., bryson, n.r., stoltsz, w.h. & masibigiri, t. 2004. displacement of boophilus decoloratus by boophilus microplus in the soutpansberg region, limpopo province, south africa. experimental and applied acarology, 32:199–208. visser, e.s., mcguire, t.c., palmer, g.h., davis, w.c., shkap, v., pipano, e. & knowles, d.p. jr. 1992. the anaplasma marginale msp5 gene encodes a 19 kilodalton protein conserved in all recognized anaplasma species. infection and immunity, 60:5139–5144. howell_153-161.indd introduction equine encephalosis virus (eev) is classified in the genus orbivirus of the family reoviridae (verwoerd, huismans & erasmus 1979). in southern africa three orbiviruses transmitted by culicoides spp. have been identified as real or potential pathogens of domestic animals, namely bluetongue virus (btv) in ruminants and african horse sickness virus (ahsv) and eev in equids. the genome of each of these viruses con sists of ten dsrna segments that encode seven structural and three non-structural proteins (verwoerd & huismans 1972; huismans 1979). genome segment 2 encodes vp2 which is one of two outer capsid proteins and determines serotype-specificity and also serves as the dominant antigen inducing protective immunity (huismans, van der walt, cloete & erasmus 1987; scanlen, paweska, verschoor & van dijk 2002). gorman, tay lor & walker (1983) reviewed the genetic relationships of the orbiviruses and drew attention to the complexity of antigenic variation within each sero logical group. studies on the natural occurrence of ahsv (mcintosh 1958; howell 1962), btv (howell 1969) and more recently of eev (erasmus, boshoff & pieterse 1996; howell, groenewald, visage, bos man, coetzer & guthrie 153 onderstepoort journal of veterinary research, 75:153–161 (2008) prevalence of serotype specific antibody to equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004) p.g. howell†, jane p. nurton, daleen nel, carina w. lourens and a.j. guthrie* equine research centre, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa abstract howell, p.g., nurton, jane p., nel, daleen, lourens, carina w. & guthrie, a.j. 2008. prevalence of serotype specific antibody to equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004). onderstepoort journal of veterinary research, 75:153–161 cohorts of yearlings were sampled over a period of 6 years in a retrospective serological survey to establish the annual prevalence of serotype specific antibody to equine encephalosis virus on thoroughbred stud farms distributed within defined geographical regions of south africa. seasonal seroprevalence varied between 3.6 % and 34.7 %, revealing both single and multiple serotype infections in an individual yearling. during the course of this study serotypes 1 and 6 were most frequently and extensively identified while the remaining serotypes 2, 3, 4, 5 and 7 were all identified as sporadic and localized infections affecting only individual horses. this study of the seasonal prevalence of equine encephalosis virus has a corollary and serves as a useful model in the seasonal incidence of the serotypes of african horse sickness and bluetongue in regions where the respective diseases are endemic. keywords: equine encephalosis virus, horses, neutralizing antibody, orbivirus, prevalence, seroepidemiology, serotypes * author to whom correspondence is to be directed. e-mail: alan.guthrie@up.ac.za † present address: 384 amberglen, p.o. box x004, howick, 3290, south africa accepted for publication 21 april 2008—editor 154 equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004) 2002) have identified by serum-virus neutralization (svn), the existence of multiple serotypes within each serogroup in southern africa. this multiplicity of serotypes amongst these three viruses, within the endemic regions of southern africa has a profound influence on the epi demiology of these infections. the persistence of the individual serotypes of these viruses, when vector populations are inactive or appear to be absent on account of low winter temperatures or dry climatic periods is an important determinant in their regional distribution. meiswinkel, nevill & venter (1994) noted that there was as yet no evidence that these viruses were transmitted transovarially in culicoides spp., while du toit (1962) and nevill (1971) were of the opinion that in the case of btv, vertebrate reservoir hosts such as cattle, which exhibit inapparent infections, were necessary to bridge the winter months. where ahsv and eev are concerned, donkeys and zebras in warm, lowlying areas where culicoides are active throughout the year may fulfil a role similar to that of cattle for btv (nevill 1971; barnard 1993). the onset of summer with more favourable environmental conditions, in particular higher temperatures and precipitation, will promote the emergence of greater numbers of culicoides, higher infection rates and older individuals in the population, all of which will increase the level of transmission. the further spread of the individual serotypes during the summer months occurs by either dispersal of infected competent species of culicoides or the translocation of viraemic vertebrate hosts. donkeys and free-living zebras have been monitored for the presence of group specific antibody to ahsv and eev (paweska, gerdes, woods & williams 1999; venter, paweska, williams & nevill 1999). these surveys confirmed the high prevalence of positive sera from unvaccinated donkeys for both ahsv and eev and established a distribution pattern for these infections. in 1993 barnard & paweska reported the identification of antibody against individual serotypes of eev in a cohort of zebras confined to the kruger national park in which neutralising antibody to seven serotypes of eev was identified. in all these studies, however, the susceptible status of the donors and the time of infection were not established. a similar shortcoming prevailed in a retrospective survey conducted amongst groups of thoroughbred mares whose ages were not established at the time of sampling, but whose sera showed a high prevalence of virus neutralizing antibody to one or more of the seven validated serotypes of eev. in this study (howell et al. 2002), the locality or time at which the infections took place were not established. the substantial adverse economic consequences of bluetongue (bt) and african horse sickness (ahs) require annual polyvalent prophylactic immunization. in terms of the response to individual serotypes included in the polyvalent vaccines the resulting immunity in individual animals is variable. serotype specific antibody derived from immunization, natural challenge or the transfer of colostrum cannot be distinguished by conventional svn assays, thus sero-epidemiological surveys are inconclusive in establishing seroconversion to natural challenge and obscure the regional prevalence of the serotypes of btv and ahsv in their hosts. within the endemic regions, unpredictable seasonal epidemics of ahs may occur, during the course of which, numerous different serotypes may be recovered from blood or tissue samples taken from clinically affected horses. these isolates, however, represent only a small sample and do not reflect the actual prevalence of the individual serotypes. a more reliable approach to resolve this shortcoming is the demonstration of seroconversion in unvaccinated animals to natural challenge. the epidemiology of eev would appear in many respects to be similar to that of ahsv and btv, thus this usually benign infection in equids may be considered as a model for the study of the distribution and seasonal appearance of the multiple serotypes of all three orbiviruses. equine encephalosis generally has minor economic implications and no vaccine has been developed, thus the high seroprevalence is uncomplicated by antibody derived from prophylactic immunization. on account of the generally mild clinical signs and ephemeral character of the disease the majority of infections pass unnoticed and the collection of samples for serological surveys to detect serotype specific seroconversion is unbiased. the objective of this retrospective serological investigation was to establish the annual occurrence of the individual serotypes of eev and their distribution and prevalence within cluster groups of horses on stud farms within defined geographical regions. the monitoring of the successful transmission of individual serotypes would provide some indication of the origin and character of the endemic cycles of infection exhibited by eev during the midto late summers included in the study period. 155 p.g. howell et al. materials and methods approximately 3 200 (range 3111–3329) thoroughbred foals were registered with the national horseracing authority of southern africa from each of the foaling seasons (august to december) from 1997 to 2002 in south africa. of these registered foals, approximately 500 (15 %), were subsequently consigned to the 1999 to 2004 national yearling sale (nys) when they were about 18 months old (table 1). the respective groups of yearlings evaluated in this study are referred to as the 1999–2004 yearling crops. mares typically foaled in foaling boxes and were moved to pastures within 48 h of foaling. weanlings and yearlings were reared on pasture. yearlings were stabled for up to 2 months prior to the nys. midge control was not practised on the farms. most mares and foals, however, were treated weekly with pyrethroids to prevent tick infestations. foals generally remained on the farm where they were born until sold at the nys, except when their dams were sent for breeding (september to december). blood collection blood was collected by jugular venipuncture into vacuum tubes without anticoagulant from each of the yearlings consigned to the nys from 1999 to 2004. the samples were held overnight at 10 °c. after separation of the clot the serum was dispensed into vials, indexed and stored at –20 °c until required for serological tests. enzyme-linked immunosorbent assay of antibody a 1:5 stock dilution of all the serum samples was assayed for group specific antibody to eev. a competitive elisa (celisa) technique, using an extracted serotype 1 eev antigen as described and validated by crafford (2001) was used. the percentage inhibition (pi) based on the inclusion of appropriate controls were obtained after the addition of an orthophenylene diamine/h2o2 substrate. the reaction was terminated by the addition of 1 m h2so4 and the intensity of the colour reaction recorded using a 492 nm filter. samples exhibiting inhibition of < 40 % were set aside and excluded from further examination. assay of serum neutralising antibody assays of serotype specific svn antibody in the stored serum samples which had a pi > 40 % were performed in a microtitre system, as described previously (howell et al. 2002). twofold dilutions (initial dilution of 1:10) of each serum were incubated with approximately 100 tcid50/100 μℓ of each reference antigen which was added in equal volume to the serum dilutions. after incubation at 37.5 °c in a 5 % co2 gassed incubator, a vero cell suspension of 480 000 cells per mℓ was added in a volume of 80 μℓ. the progress of the cytopathic effect of the virus was recorded daily. the serum dilution end-points were determined on the development of 50 % destruction of the monolayers inoculated with the dilution of the back titration of the virus representing 100 tcid50. the concentration of virus used for the assay of each batch of sera was monitored and where the concentration of virus fell within the range of 30–300 tcid50, the assay of antibody was accepted. whilst a serum neutralization titre of ≥ 10 was considered positive, a titre of > 80 was interpreted as confirmation of an unambiguous response to infection with the specific serotype of eev. statistical analysis relative risks (rr) were used to quantify the relationships between risk factors and seroprevalence, respectively, between four age groups of the yearlings included in this study. results interpretation of serological tests at a serum dilution of 1:5 the celisa identified 680 positive sera with 95 % of the samples exceeding a pi of 60 %. table 1 summary of size of foal crop, number and percentage of yearlings sampled at the 1999–2004 national yearling sales year of sampling size of crop yearlings sampled percentage of crop sampled 1999 2000 2001 2002 2003 2004 3189 3207 3329 3228 3178 3111 516 513 483 481 500 499 16.2 16.0 14.5 14.9 15.7 16.0 156 equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004) table 2 influence of age on susceptibility to natural eev challenge month of birth foals seropositive percent relative risk (rr) august september october november/december 442 1 022 1 037 491 118 245 205 112 0.27 0.24 0.20 0.23 1.35 1.21 1.00 1.15 table 3 prevalence of primary and multiple serotype infection of yearlings yearling crop yearlings sampled seropositive yearlings percentage seropositive seropositive yearlings single serotype infections multiple infections individuals immune profile 1999 516 148 28.7 148 0 – 2000 513 90 17.5 87 1 1 1 eev1, eev5 eev1, eev6 eev1, eev7 2001 483 146 30.2 142 4 eev1, eev7 2002 481 167 34.7 165 2 eev1, eev6 2003 500 111 22.2 94 9 1 6 1 eev1, eev6 eev1, eev7 eev6, eev7 eev4, eev6 2004 499 18 3.6 14 1 1 1 1 eev1, eev6 eev6, eev4, eev6, eev7 eev1, eev3, eev7 year 1 year 2 year 3 challenge challenge j a s o n d j f m a m j j a s o n d j f m a m j j birth stabled loss of colostral protection sampling at nys fig. 1 seasonal exposure of the horse population to the transmission of equine encephalosis virus in south africa prior to the national yearling sales 157 p.g. howell et al. cape coast serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 81 7.4 6 2000 86 18.6 16 2001 81 32.1 26 2002 82 63.4 52 2003 77 1.3 1 2004 116 0.9 1 robertson serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 129 83.7 108 2000 115 3.5 1 3 2001 96 9.4 6 3 2002 122 60.0 71 2 2003 131 6.1 7 2 2004 127 0.0 highveld serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 13 0.0 2000 23 21.7 1 2 2 2001 28 25.0 3 2 2 2002 15 20.0 1 1 1 2003 30 20.0 1 2 3 2004 26 15.4 1 3 2 karoo serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 27 7.4 1 1 2000 24 12.5 2 1 2001 27 48.2 4 4 1 2 2 2002 22 0.0 2003 29 55.2 11 10 2004 25 0.0 ceres serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 61 8.2 5 2000 61 4.9 2 1 2001 48 4.1 2 2002 52 46.2 24 2003 40 15.0 6 2004 38 0.0 eastern cape serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 67 1.5 1 2000 74 33.8 25 1 2001 79 65.8 50 1 1 2002 53 5.6 3 2003 52 23.1 11 2 1 2004 52 17.3 6 3 kwazulu-natal serotype yearling crop number percentage seroconverted 1 2 3 4 5 6 7 1999 138 18.8 26 2000 130 26.2 28 2 5 1 2001 124 29.0 32 9 2002 135 9.0 7 1 6 2003 141 44.0 54 2 13 1 2004 115 3.5 1 1 2 fig. 2 distribution of stud farms included in the survey with numbers of yearlings sampled per year, percentage of yearlings seropositive for equine encephalosis virus and numbers of yearlings positive for type specific antibody for each of the seven recognised serotypes of equine encephalosis virus for each of the years investigated (1999–2004) 158 equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004) yearlings which appeared to have been exposed to a single serotype, invariably had svn antibody titres of 1:240 to > 1:320 to the homologous antigen. eighteen horses (0.6 %) were identified in the latter years of the survey which exhibited high pi (80 %) by celisa but low svn end-points. these horses were considered to have been recently infected in the period immediately preceding their shipment to the nys and were excluded from the distribution map, as they did not fulfil the required criteria of an unambiguous response to natural infection. as a retrospective study, a second serum sample to confirm a rising antibody titre in these horses was not available for examination. influence of age on susceptibility to infection an analysis of the percentage of seropositive yearlings, classed according to the month in which they were born (august to november/december over the 6 years of the survey) is given in table 2. the relative risk of infection (rr) during the following summer showed no significant difference between foals born in august in which colostral protection would have waned and those born in november/ december, when maternal antibody may have been expected to provide some measure of protection against natural infection (see fig. 1). seasonal serconversion to eev a summary of the primary and multiple infections that took place in each cohort of yearlings is given in table 3. these data confirm the variable annual seroprevalence of eev. in the 1999, 2001 and 2002 yearling crops the seroprevalence varied from 28.7 % to 34.7 % of the population sampled, whereas in 2004 the seroprevalence was only 3.6 %. notwithstanding the relatively short period of exposure, 30 yearlings were infected with more than one serotype. the profile of these multiple infections invariably included the predominant serotype in the region and a random assortment of one or other additional heterologous serotype. distribution of serotypes of eev a distribution map with an analysis of the serotypes identified in each cluster during the 6 years covered by this survey is presented in fig. 2. the insert for each cluster indicates the year of sampling, bearing in mind that infection took place during the previous summer, the number of yearlings sampled, those that seroconverted and the percentage prevalence of eev. the number of positive sera is occasionally at variance with the prevalence of individual serotypes due to the detection of antibody to more than one serotype in individual horses. although all of south africa’s stud farms were not represented at the nys, the sample is, however, representative of the main thoroughbred breeding regions of the country. although each cluster was located in a region of common topography and climate, no attempt was made to associate these environmental conditions and their influence on the vector population with the seasonal variations in the prevalence of the serotypes within the region or on individual farms. apart from recognizing the primary role of the vector in the transmission of each serotype, the dynamics of the culicoides populations within the terrain of each cluster requires further study. during the 6-year duration of this survey, serotypes 1 and 6 were predominant and account for 65 % and 30 %, respectively, of the recorded seroconversions. serotype 7 represented by the isolate 21/20, the most recently identified serotype recovered in 2000, was detected in only 4.5 % of the samples. antibody homologous to the remaining serotypes namely 2, 3, 4 and 5 was only sporadically identified, except within the kwazulu-natal cluster where serotypes 2, 4 and 5 were identified during different seasons in the 6-year period of sampling. serotype 2 was identified on two separate stud farms 70 km apart on the highveld during the 2000 seasonal infection and during the same season in the sera of four yearlings included in the karoo cluster. the two stud farms on which these yearlings were infected were separated by approximately 400 km from the highveld cluster, and were situated in a distinct ecological environment, on the banks of a major river system. an evaluation of the seasonal prevalence of the two dominant serotypes suggests a possible pattern. it would appear that a season or two of high prevalence is followed, in the same cluster, by a dramatic reduction of homologous seroconversion in the following year. this was evident with serotype 1 in the 2001 season in kwazulu-natal and eastern cape and in the 2002 season in the robertson and ceres clusters. a similar seasonal reduction of infection was observed with serotype 6 in kwazulu-natal and robertson in 1999. of note in relation to the high prevalence of serotype 6 in the southern clusters, during the 1998 and 2001 seasons, is the absence of any seroconversion to serotype 1 in the yearlings in the remaining clusters. 159 p.g. howell et al. discussion the data obtained from this study have provided some insight into the complexity of the epidemiology of eev infections in an endemic area, where a multiplicity of serotypes have circulated. the unique susceptibility of the yearling population has provided an opportunity to follow successive annual cycles of transmission of eev between vector and host, in the absence of any complicating previous immunological stimulation. over a period of 6 years the monitoring of successive groups of yearlings has confirmed the ongoing transmission of the seven serotypes of eev currently identified in south africa. a schematic representation of the complex interrelationship between the age of the horse, colostral protection and the onset and termination of the transmission of the serotypes of eev is illustrated in fig. 1. the climatological and ecological factors which determine the magnitude of the vector population are operative well before the onset of transmission in midsummer. within the transmission cycle the highest prevalence of infection in the yearlings is most likely to occur between february and may during their second year, while the stabling of the yearlings in the months preceding the sales in march of the following year would reduce the risk of exposure. it would therefore appear that the antibody profile detected in individual yearlings sampled in march is a reflection of the prevalence of a particular serotype transmitted during the preceding season. the decline in the concentration of serotype specific, passively transferred colostral antibody after birth, will determine the susceptibility of the foal to the seasonal transmission of the homologous virus during midto late summer. unpublished data have shown that antibody to specific serotypes of eev, homologous with the variable profile identified in the serum of the mare at parturition, may be detected in the serum of the foal, for 3–5 months after birth and only occasionally in individuals for a period not exceeding 6 months. no data are, however, available to establish the protective value of the diminishing low concentrations of antibody to homologous challenge, during the midto late summer period. in a separate study conducted on the 1997 cohort of yearlings (guthrie, howell, gardner, swanepoel, nur ton, harper, pardini, groenewald, visage, hedges, balasuriya, cornel & maclachlan 2003) it was shown that seroconversion to west nile virus (wnv) was more frequent in foals born in august (rr 4.7) when compared with those born in november and december (rr 1.0). maternally transferred antibody to the monotypic virus appeared to provide protection to challenge during early summer. in contrast, the highly variable immune status of mares to eev (howell et al. 2002) fails to provide the foal with an effective polyvalent immunity to natural challenge from the unpredictable array of serotypes identified in this study. the risk of infection therefore appears to be unaffected by the transfer of colostral antibody. two distinct cycles of infection have been identified in this survey. a high prevalence of a single serotype during a particular season as recorded in the kwazulu-natal and the robertson clusters where serotype 6 was involved in yearlings sampled in 1999, or of serotype 1, which was dominant in the yearling samples of 2000 from the robertson and cape coast clusters. unpublished records of the recovery of virus from horses during the 1999 outbreak of ahs in the western cape, showed a similar pattern, with ahs serotype 7 and eev serotype 1 were transmitted concurrently over a period of 8 weeks. this is in marked contrast to the low and sporadic appearance of all seven serotypes of eev identified in clusters that were separated one from another by spatial distance or physical topographical barriers. at most, only a few yearlings in any one season showed seroconversion to these serotypes, occasionally in successive seasons, but more frequently as isolated infections. outer capsid protein vp2 determines eev serotype. more significant in the epidemiology of the orbiviruses is the role of other viral proteins, which could independently determine the efficacy whereby the serotype replicates, is transmitted and maintained over time in an isolated niche. the seroconversion detected in the serum of a yearling reflects the successful transmission of a specific serotype by a competent vector. it was not possible to establish whether an index case early in the season provided the viraemia to initiate an ongoing cycle of transmission between the vectors and other horses, which would account for the high prevalence of serotype 6 in the robertson cluster for foals born in the 1999 season. the two distinctive epidemiological patterns, however, raise the question as to whether transmission is determined by the composition and dynamics of the vector population in a given area, or the genetic characteristics of the particular virus strain circulating at that time. the isolated seroconversions are of great significance and raise many unanswered questions with respect to the distribution of the serotypes and their persistence in a given 160 equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004) locality during the inter-epidemic periods which may encompass one or more years. it was reported that following the first isolation of eev serotype 2 (cascara prototype) in 1967, the examination of a number of serum samples from horses in various parts of south africa showed a high percentage of the sera with homologous antibody, indicating a widespread prevalence of this serotype during the preceding summer (erasmus, adelaar, smit, lecatsas & toms 1970). during the course of this survey only five yearlings (0.7 %), from two separate clusters were shown to have been infected after an interval of 37 years with serotype 2. similarly, in a recent survey amongst aged brood mares on stud farms it was found that only 1.5 % had been previously exposed to this serotype (howell et al. 2002). the immune profile of these mares clearly showed that the persistently low prevalence of certain serotypes was not the outcome of immunological pressure within the equine population which limited the transmission of the specific serotype. the mechanism whereby these sporadic and isolated seroconversions to a particular serotype occur is yet to be established. previous experience has shown that equids in the incubation period of infection with ahsv may be transported and subsequently provide an index case for further transmission by hitherto uninfected vectors. this translocation of the virus with other natural movements of equids over time might explain the redistribution of individual serotypes as independent members of the serological group. the persistence of individual serotypes of eev that are only sporadically transmitted does not appear to lead to dissemination after infection of individual horses. this is apparent from the low prevalence of seroconversion in the study population and unlike the current strains of eev serotype 6 and 1, are not capable of initiating large scale transmission. the prevalence of eev infection has a parallel in the seasonal occurrence of ahsv and btv, where recovery of individual serotypes follows a similar pattern and suggests that the role of the immune status of the mammalian hosts does not affect the prevalence of the individual serotypes in the vector population. future investigations into the interaction between vector and virus may characterize the persistence of the serotypes in an isolated locus as a unique manifestation of the complex epidemiology of the orbiviruses in southern africa. acknowledgements the authors thank the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria for providing laboratory facilities and support without which this study would not have been possible and dr melvyn quan for assistance with preparation of figures used in this paper. this study was supported in part by funds donated by the thoroughbred horseracing trust and the south african horse import and export council. references barnard, b.j.h. 1993. circulation of african horsesickness virus in zebra (equus burchelli) in the kruger national park, south africa, as measured by the prevalence of type specific antibodies. onderstepoort journal of veterinary research, 60:111–117. barnard, b.j.h. & paweska, j.t. 1993. prevalence of antibodies against some equine viruses in zebra (zebra burchelli) in the kruger national park, 1991–1992. onderstepoort journal of veterinary research, 60:175–179. crafford, j.e. 2001. development and validation of enzymelinked immunosorbent assays for detection of equine encephalosis virus 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2002. the protective efficacy of a recombinant vp2-based african horsesickness subunit vaccine candidate is determined by adjuvant. vaccine, 20:1079–1088. venter, g.j., paweska, j.t., williams, r. & nevill, e.m. 1999. prevalence of antibodies against african horse sickness (ahs) and equine encephalosis (ee) virus in donkeys in southern africa, in equine infectious diseases viii, edited by u. wernery, j.f. wade, j.a. mumford & o.r. kaaden. newmarket: r & w publications (newmarket) ltd. verwoerd, d.w. & huismans, h. 1972. studies on the in vitro and the in vivo transcription of the bluetongue virus genome. onderstepoort journal of veterinary research, 39: 185–191. verwoerd, d.w., huismans, h. & erasmus, b.j. 1979. orbiviruses, in comprehensive virology, edited by h. fraenkel-conrat & r.k. wagner. new york: plenum press. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods results & discussion conclusion and recommendations acknowledgements references about the author(s) abdalla a. latif school of life sciences, university of kwazulu-natal, durban, south africa bonginkosi nkabinde epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa brian peba epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa olivier matthee epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa ronel pienaar epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa antoinette josemans epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa daniel marumo epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa karien labuschagne epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa nada a. abdelatif medical research council, durban, south africa andreas krüger department of tropical medicine, bundeswehr hospital, hamburg, germany ben j. mans epidemiology, parasites and vectors, agricultural research council-onderstepoort veterinary research, pretoria, south africa department of life and consumer sciences, college of agriculture and environmental sciences, university of south africa, johannesburg, south africa department of tropical veterinary diseases, faculty of veterinary sciences, university of pretoria, pretoria, south africa citation latif, a.a., nkabinde, b., peba, b., matthee, o., pienaar, r., josemans, a. et al., 2019, ‘risk of establishment of canine leishmaniasis infection through the import of dogs into south africa’, onderstepoort journal of veterinary research 86(1), a1634. https://doi.org/10.4102/ojvr.v86i1.1634 original research risk of establishment of canine leishmaniasis infection through the import of dogs into south africa abdalla a. latif, bonginkosi nkabinde, brian peba, olivier matthee, ronel pienaar, antoinette josemans, daniel marumo, karien labuschagne, nada a. abdelatif, andreas krüger, ben j. mans received: 16 mar. 2018; accepted: 05 dec. 2018; published: 28 may 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract canine leishmaniasis is a vector-borne disease caused by protozoa of the genus leishmania that affect dogs, humans and wildlife. sandflies of the genera phlebotomus and lutzomyia are the primary vectors. canine leishmaniasis is an exotic and controlled disease in south africa. the main purpose of our risk assessment study was to evaluate the likelihood that this exotic disease could enter and be established in south africa through importation of live dogs. risk analysis to the spread of the disease follows the world organization for animal health (oie) formal method of quantitative risk assessment documented as a step-by-step process. we have identified and discussed 11 possible risk factors involved in three steps for final assessment. the annual average number of diagnostic tests performed on imported dogs from 44 countries for 2011–2015 was 1158. leishmania is reported to occur in 21/44 (47.7%) exporting countries. a total of 71.1% of leishmania positive dogs were imported from these endemic countries. the yearly percentage of leishmania positive dogs ranged from 0.2% to 2%. three confirmed clinical and fatal cases of leishmaniasis in dogs of unidentified origin have been reported by our laboratory and the state veterinarians. the disease has been reported in neighbouring countries as well as the putative sandfly vectors. this study concluded that the risk for the introduction and degree of uncertainty of leishmania in imported dogs in south africa are moderate. risk mitigation and recommendations such as investigations into possible occurrence of autochthonous leishmaniasis in the country, surveillance in its wildlife reservoirs and systematic surveillance of sandfly populations are discussed. keywords: canine leishmaniasis; risk assessment; phlebotominae; ticks; south africa. introduction canine leishmaniasis (canl) is a vector-borne disease caused by protozoa of the genus leishmania (kinetoplastida: trypanosomatidae) that affect dogs in tropical and subtropical regions of the old world (africa, asia, and southern europe) and the new world (south america and north america) (dantas-torres 2009; otranto & dantas-torres 2009). the most important species of leishmania for dogs is leishmania infantum (syn. leishmania chagasi), which causes visceral and cutaneous diseases in humans (baneth et al. 2008; duprey et al. 2006). can l is expanding its geographical range from northern argentina to the northern united states covering 18 states and southern canada (duprey et al. 2006). in europe, the extension of the range has been reported in italy, where there is evidence that canl is currently expanding into continental climate areas of northwestern italy, far from the recognised disease-endemic areas along the mediterranean coasts (ferroglio et al. 2005). leishmaniasis is one of the major infectious diseases afflicting the world’s poorest populations living mainly in rural and suburban areas (alvar, yactayo & bern 2006). sandflies of the genera phlebotomus are the vectors in the old world and lutzomyia in the new world, that is, insects responsible for parasite transmission. progress in phlebotomine sandfly research that included taxonomy and systematics, vector competence, eco-epidemiology and vector control has recently been reviewed (bates et al. 2015). the prevalence of canine leishmania infection in endemic areas is considerably higher than that of apparent clinical illness (baneth et al. 2008), indicating that not all infected dogs develop the disease. this has also been demonstrated by an experimental model of infection, which showed that dogs developed variable immune responses, and although some became sick, other infected dogs remained clinically healthy over 5 years of observation (pinelli et al. 1994). the immunofluorescence antibody test (ifat), which uses whole promastigote antigen, is highly specific and sensitive for the detection of exposure to leishmania, but may lack sensitivity to detect infected but clinically healthy dogs (mettler et al. 2005). although 88% – 100% of the dogs showing clinical signs were found serologically positive, only 30% – 66% of the subclinically infected ones were positive (porrozzi et al. 2007; solano-gallego et al. 2001). dogs with symptomatic as well as asymptomatic infections are infectious to sandflies. however, infectiousness was shown to be higher in dogs with clinical disease (baneth et al. 2008). infected dogs in non-endemic areas may also contribute to the maintenance of the leishmania parasites within the canine population through possible non-sandfly vector transmission modes of infection as documented in hematophagous ectoparasites, such as ticks (solano-gallego et al. 2009). recently, three species of the sandfly genus sergentomyia were demonstrated to have a high rate of l. infantum-positive females under natural conditions. this finding indicated that these species were the natural vectors of canl in the mont-rolland area of senegal and contradicts the notion that species of the phlebotomus genus in the old world are the only vectors of leishmaniasis (senghor et al. 2016). leishmania vertical infection was demonstrated experimentally in puppies born to infected female and male beagles, and transmission was assumed to be transplacental (boggiatto et al. 2011; rosypal et al. 2005). transplacental infection of litters in dog breeding may be the most important mechanism of transmission (duprey et al. 2006). the occurrence of l. infantum infection in dogs in 18 states in the united states and two provinces in canada where the vector sandflies have not been identified is now well established (duprey et al. 2006). research into the sandfly populations of south africa has never been carried out systematically and the species records came out from only ad hoc and limited surveys. there are about 17 species of phlebotomines known in south africa and the last record dates back to 1987 (braack et al. 1981; davidson 1979, 1980, 1987; lewis 1967, 1971; zielke 1971); however, not much is known about their distribution and their ecology, biology and disease relationships. a case of cutaneous leishmaniasis in a sheep from the eastern transvaal and human cases of cutaneous leishmaniasis and canl were reported from the country (grové 1970; van der lugt, carlyon & waal 1992; van der lugt & stewart 2004). there has been an increase in the number of imported dogs into the country from countries where canl is endemic or has reported its occurrence. the main objective of this study was to evaluate the likelihood that canl could enter and be established in south africa through importation of live dogs. materials and methods leishmania tests the leishmania test is an ifat using l. infantum antigens (ag) antigen slides are prepared from culture suspension of promastigote of l. infantum (itmap 263 – clone 10) according to the protocol adopted and modified from the oie manual (oie 2016), which mentions the sensitivity as 96% and specificity as 98%. a titre of ≥ 1:50 is considered positive for south africa. molecular tests and dna (deoxyribonucleic acid) sequence analysis were performed as confirmatory tests. a real-time polymerase chain reaction (pcr) capable of detecting and differentiating the l. donovani complex (l. donovani, l. infantum and l. chagasi), the l. brasiliensis complex as well as other leishmania spp. (such as l. major, l. mexicana and l. tropica) was used for diagnostic screening (schulz et al. 2003). this test is based on the 18s rrna gene and amplicons were sequenced directly from both directions to obtain consensus sequences. as a positive control, we used the l. infantum ifat ag. amplification of the 18s gene was performed using primers 609f and 706r previously published (maia da silva et al. 2004) that yield a fragment of ~900 bp (base pair) spanning the v7–v8 hypervariable region. sequencing was performed at inqaba biotech (south africa), and sequences were analysed using blastn analysis (altschul et al. 1990) and assigned to a species complex based on 100% identity. phlebotomine sandfly species one ad hoc collection of sandflies from kuleni, kwazulu-natal, was made. flies were collected using light traps and samples were preserved in 70% alcohol until identification. sandflies were processed and identified morphologically and genetically as described by krüger 2017. assessment of risk factors our import risk assessment regarding the spread of the disease follows the world organization for animal health formal method of quantitative risk assessment (oie 2016). the risk assessment is a formal method to deal with hazards and associated risks and has been documented as a step-by-step process. the hazard identification (infectious pathogens, i.e. leishmania parasites) is the first step and considered separately from the risk assessment. the risk assessment process follows three steps: (1) entry (release) assessment that describes the pathways necessary for the introduction of the hazard, (2) exposure assessment (description of pathways necessary for the hazard to occur following entry) and (3) consequence assessment (identification of the consequences of disease entry and establishment such as the effect on human health and animal health). hazard identification canine leishmaniasis is an exotic and controlled disease in south africa. the main purpose of our risk assessment study was to evaluate the likelihood that this exotic canl could be established in south africa through importation of live infected dogs. risk factors identified in release assessment (likelihood of entry) risk factors identified in release assessment included confirmed diagnosis at port of entry, missed diagnosis at port of entry, imported parasitic infected or uninfected vector ticks, diseases reported in exporting countries, report of the disease in neighbouring and regional countries and infected dogs of unknown origin or illegal entry into the country. risk factors identified in exposure assessment (likelihood of target population to be exposed) risk factors identified in exposure assessment included presence of sandfly vectors and other means of transmission such as mechanical or direct transmission (vertical transmission, venereal and dog biting wounds). consequences assessment (likelihood of occurrence and magnitude) the consequences assessment included disease transmission to canine/wildlife animals and human health (zoonosis). risk assessment risk assessment, risk management and risk communication are components of risk analysis. the oie provides an import risk analysis standard (oie 2016). the four components of the risk analysis involve hazards identification, risk assessment, risk management and risk communication. in the present study, we have identified the hazards and assessed risks step-by-step but have not covered the risk management and risk communication as adopted by the oie (2016). however, some aspects of risk management were highlighted to provide guidelines to reduce the risk of introduction of canl in the country and assist in planning future management policies based on scientific data, published evidence and expert opinion. a qualitative risk assessment was conducted, and so the likelihood of release and exposure, as well as the magnitude of the consequences, was expressed as negligible, low, moderate and high. a scenario tree was used, detailing the release (entry), exposure and consequence pathways of leishmania introduction into south africa. the likelihood is a product of all the steps in the scenario tree (peeler, reese & thrush 2015). this assessment is summarised using a risk matrix, which combines the likelihood of the hazard and the consequences of that hazard to produce an overall risk estimate (desvaux et al. 2016; dufour et al. 2011). the final risk estimate considers the degree of uncertainty in the available data and scientific evidence (costard 2008). ethical considerations the agricultural research council-onderstepoort veterinary research parasitology diagnostics laboratory is accredited by south african national accreditation system and approved by the department of agriculture, forestry and fisheries. results & discussion risk factors identified in release assessment (likelihood of entry) probability of a screening test (immunofluorescence antibody test) missing an infected animal at a port of entry the number of dogs imported into south africa during 2011–2015 and tested for the controlled leishmaniasisis is shown in figure 1. the average number of tests performed per year was 1158. table 1 shows the number of diagnostic tests performed for the disease (1574 tests) on dogs imported from 44 countries into south africa. leishmania is reported to be endemic or to occur in 21/44 (47.7%) exporting countries (table 1). a high percentage of dogs (71.1%) were imported from canl endemic countries or where the disease has been reported. the yearly percentage of seropositive ranged from 0.2% to 2.0% (figure 2). figure 1: number of diagnostic ifat for canine leishmania per year (2011–2015); dogs intended for importation into south africa. figure 2: percentage of leishmania positive ifat from 2011 – 2015, dogs intended for importation into south africa. table 1: canine leishmania serological tests (ifat) performed for dogs for importation into south africa (1574). missed diagnoses at port of entry serology employing the ifat, which uses whole promastigotes antigen, is highly specific and sensitive for the detection of canl and is the recommended diagnostic method (oie 2016). in canl, high ab titres are associated with high parasitaemia and disease. some of the dogs remain seronegative for extended periods of time after infection. this long incubation period results in false negative results, which has an implication in the diagnosis of the disease in imported asymptomatic dogs. thus, the test may lack sensitivity (about 4%) to detect clinically healthy but infected dogs (mettler et al. 2005; oliva et al. 2006). a missed diagnosed case in durban north, ethekwini (2012) a 4-year-old golden spaniel male dog was imported into south africa from italy in october 2010. it was released from quarantine and moved to durban. in march 2011 (6 months later), the dog was tested for leishmania antibodies using the ifat and the results were positive at a 1/50 dilution. a re-test was carried out in june 2012 (15 months after the first test) and the result was positive at 1/800 dilution. the dog was euthanised on 30 august 2012 (22 months after importation) and the post-mortem confirmed infection with leishmania (anon 2012). a missed diagnosed case in cape town (2014): two dogs, a 4-year-old male and a 6-year-old female bull terrier, were imported from angola into south africa on 5th of february 2014 and landed at cape town. both tested ifat negative for leishmania 76 days before importation and were not tested again within the prescribed 30-day period before movement. the dogs remained negative on testing at destination and were released. the female dog was presented 6 days later at the veterinary clinic with a generalised nodular skin condition. the biopsy samples and giemsa’s stained smears showed leishmania amastigotes intracellularly in macrophages and cutaneous fibroblasts which confirmed the diagnosis (grewar & brady 2014). autochthonous leishmaniasis cases leishmania case study in durban (2015) a3-year-old rottweiler male dog, with an uncertain history, was presented at a private veterinary clinic in durban in february 2015. the patient showed general poor body condition, loss of body weight, scruffy coat, bilateral ocular discharge, hair loss on tips of tail and ears without pruritis being evident at the time. two months later, the skin showed exfoliative dermatitis, multiple areas of localised alopecia, skin thickening and ulceration particularly on pressure points, pin bones and elbows. other signs included generalised lymphadenopathy. isolation and identification of parasite from lymph node, bone marrow and spleen aspirate were successful in cell culture. the bone marrow cell culture demonstrated extensive leishmania growth, with typical leishmania cells in giemsa stained smears. a sample of the organism tested positive by pcr. sequencing of the cell cultures obtained from the dog and the standard cell culture used for ifat indicated that both were 100% identical to each other and to the leishmania donovani/chagasi/infantum complex. leishmania case reports in dogs in durban (1964) and free state (1987) leishmaniasis was diagnosed twice in dogs: in 1964 in a dog from durban whose life history was uncertain and in 1987 in a dog from the free state which had never left the country (van der lugt & stewart 2004). leishmaniasis reported in sheep (1992) a case of cutaneous leishmaniasis was reported in a sheep from the eastern transvaal (van der lugt et al. 1992). the skin lesions were described and the amastigote stage of leishmania species was identified. imported parasitic infected/uninfected vector ticks tick-associated transmission of leishmania has been reported (mckenzie 1984) (see risk factors identified in ‘exposure’ assessment). tick inspection is the responsibility of the veterinary authority at entry ports. leishmaniasis reported in exporting countries the dogs came from 8/15 african countries with history of the disease, 5/8 countries in the middle east, 3/12 countries in asia, 1/4 of european countries, from the two south american countries, brazil and argentina, as well as from the united states and canada (2/3) (table 1). report of the disease in neighbouring countries or countries in the region leishmaniasis has been reported in several countries neighbouring or in the southern region; angola, botswana, malawi, mozambique, namibia and zambia (alvar et al. 2012; world health organization 2010). autochthonous cutaneous leishmaniasis identified as belonging to the l. tropica group has been reported in 34 cases in namibia since the 1970s. leishmania parasites have also been isolated from hyraxes (procavia capensis) and from naturally infected phlebotomus rossi sandflies, which are possible vectors of the human disease (campbell, gordon & emms 1979; grové 1970, 1989; grové & van dyk 1974; noden & soni 2015; rutherfoord & uys 1978). if autochthonous leishmaniasis is not monitored, it can suddenly become an epidemic should ecological and environmental conditions change (noden & van der colf 2013). a nurse was evaluated for cutaneous leishmaniasis, after showing skin lesions 3 weeks after travel to botswana and visiting a game reserve. the biopsy revealed promastigotes of l. tropica (schwartz et al. 2012). a 26-year-old man from angola with no history of travel outside the country had visceral leishmaniasis. the parasite was isolated and biochemically characterised using molecular tools and identified as l. infantum, a parasite not endemic to this region (jimenez et al. 1994). recently, a dog that had never left angola nor had its parents was found seropositive with dat titres of 800 and ≥6400 and was also found to be pcr-positive and confirmed to be infected with l. infantum by dna sequence analysis. this case is strongly suggestive of an autochthonous infection (vilhena et al. 2014). two autochthonous cases of cutaneous leishmaniasis in zambia were described in humans, both of whom also had tuberculosis. amastigotes were cultured from blood and identified in skin, bone marrow, liver and spleen (naik et al. 1976). two cases of cutaneous leishmaniasis in malawi were diagnosed by histopathology in 1 year. the authors suggested that the infection may be more prevalent in this region than what was previously thought (pharoah et al. 1993). risk factors identified in exposure assessment (likelihood of the target population to be exposed) presence of sandfly vectors (biological transmission) female phlebotomine sandflies are the only vectors of leishmania spp. and also the main mode of parasite transmission (dantas-torres et al. 2012; killick-kendrick 1990). sandfly situation in south africa identification of sandflies in kuleni, kwazulu-natal: out of 13 sandflies (11 females and 2 males), 10 were dissected for micromorphological identification. of these, nine were grassomyia (formerly sergentomyia) squamipleuris, one male could only be specified as sergentomyia spec., but not g. squamipleuris. g. squamipleuris is supposedly a reptile-feeder and not known as a mammalian leishmania vector. it has been reported before from transvaal and zululand (de meillon 1955). on the other hand, very little is known about the host preferences for most sandfly species, but it was suggested that sergentomyia schwetzi may bite dogs (senghor et al. 2016). records of phlebotomus sp.: research into the sandfly populations of south africa and of the southern african neighbouring countries has never been carried out systematically and species records came only from ad hoc and limited surveys. the last known record of phlebotomines in south africa dates back to 1987 (braack et al. 1981; davidson 1980, 1981, 1983, 1987; lewis 1967, 1971; zielke 1971). the first phlebotomine sandfly collection from botswana, southern africa, was carried out in 2014/2015 (krüger 2015). during a pilot survey in the north of the country, 41 specimens were collected, of which 37 were morphologically and genetically identified to be species of the genera sergentomyia and phlebotomus (krüger 2017). the sandfly fauna of southern africa accounts for about 49 species (krüger 2015) and 17 different species are known so far from south africa and of south west africa/namibia (zielke 1971). only a few specimens of the genus phlebotomus were caught whereas the majority belongs to the genus sergentomyia. vectorial capacity of phlebotomine sandflies: phlebotomus rodhaini, a putative vector of zoonotic leishmaniasis, was recorded recently from botswana (krüger 2017) and this species has also been reported to occur in namibia and south africa (davidson 1981). phlebotomus rossi was reported to be infected with leishmania parasites in namibian loci of cutaneous leishmaniasis and its distribution covers namibia, south africa and zimbabwe (davidson 1987; killick-kendrick 1990). the status of p. rossi, the suspected vector of cutaneous leishmaniasis in south west africa, was revised by lewis and legder (1976). most sergentomyia spp. feed preferentially on cold-blooded vertebrates, being proven vectors of reptile leishmania species. their possible role in the circulation of mammalian leishmaniasis in the old world has been considered where leishmania dna and the parasites have been identified in several species, while some species were reported to feed on mammals, including man (ayari et al. 2016; berdjane-brouk et al. 2012; maia et al. 2015; senghor et al. 2016). recently, three species of the genus sergentomyia were demonstrated to have a high rate of l. infantum-positive females under natural conditions. this finding indicated that these species were the natural vectors of canl in the mont-rolland area of senegal and contradicts the notion that species of the phlebotomus genus in the old world are the only vectors of leishmaniasis (senghor et al. 2016). three records of sandflies biting a man in south west africa show that further observations on these nocturnal insects in south africa are necessary (lewis 1971). however, several aspects of the fly–parasite relationships must be elucidated to confirm the fly species as a vector of leishmaniasis, such as obtaining data on the species richness, abundance, biting and infection rates, taking of blood meals from reservoir hosts (e.g. rodents) as well as from humans to confirm using xenodiagnostic attempts (maia & depaquit 2016; pech-may et al. 2010). ticks, mechanical, and direct mode of transmissions tick transmission sandflies are the accepted biological vectors of leishmania parasites. currently, other modes of disease transmission have been reported in the literature. reports of ticks as vectors of leishmania were studied by scientists in different disease-endemic areas. rhipicephalus sanguineus has long been suspected to transmit leishmania infantum in studies carried out in laboratory and natural conditions. the role of the brown dog ticks, r. sanguineus, as vectors of leishmania is highlighted herein. conclusive evidence of xenodiagnosis was reported by mckenzie (1984). in that study, a tick pick-up and transmission using r. sanguineus nymphs fed on two naturally infected dogs and the subsequent adult stage were fed on two uninfected dogs. transmission attempt was performed on one recipient infected dog concluded that r. sanguineus was able to transmit l. infantum to a susceptible dog through normal feeding. infections were monitored in dogs using serology, xenodiagnosis with ticks and direct culture of bone marrow and lymph node aspirates. based on this study, dantas-torres (2011) concluded that r. sanguineus was able to transmit l. infantum to a susceptible dog through normal feeding, and as such demonstrated that the tick-vector theory deserves attention. the study by solano-gallego et al. (2012) has shown high prevalence of l. infantum dna in r. sanguineus (males and females) removed from l. infantum seropositive and seronegative dogs. this study was also supported by the results obtained by colombo et al. (2011). these authors showed that live parasites were detected in newly moulted r. sanguineus adult ticks obtained as nymphs, which engorged on dogs in the endemic area, and thus could confirm transstadial transmission. dantas-torres et al. (2010a) reported for the first time the retrieval of l. infantum kdna in salivary glands of r. sanguineus ticks and recommended further studies to assess the competence of ticks as vectors of leishmania parasites. this was followed by studies of medeiros-silva et al. (2015) who reported the presence of the parasite dna in the intestines and salivary glands of r. sanguineus and viable l. infantum could be successfully isolated. in an experimental injection of l. infantum promastigotes into the haemocoel through the coxa of engorged females, the subsequent eggs and larvae were found positive for l. infantum kdna (dantas-torres et al. 2010b; dantas-torres, latrofa & otranto 2011). these authors concluded that their results have shown, for the first time, the transovarial passage of l. infantum kdna in r. sanguineus. moreover, the potential transovarial and transstadial passage of kdna through ticks was confirmed by pcr (dabaghmanesha et al. 2016). r. sanguineus removed from leishmania-infected dogs was found to be infective to hamsters (coutinho et al. 2005). all these results highlight the potential of r. sanguineus as a vector of l. infantum. however, in his review article entitled ‘ticks as vectors of leishmania parasites’, dantas-torres (2011) emphasised the need for further research to better understand the participation of r. sanguineus in the epidemiology of leishmaniasis. presence of the brown dog tick r. sanguineus in the country: in south africa, all life stages of r. sanguineus feed on domestic dogs (walker, keirans & horak 2000). the tick was collected from dogs in the northern cape, eastern cape, north west province and the high veld (bryson et al. 2000; horak 1995; matthee et al. 2010; nyangiwe, horak & bryson 2006). it is a known vector of ehrlichia canis and babesia vogeli in south africa. other modes of transmission other modes of transmission included transplacental transmission (boggiatto et al. 2011; rosypal et al. 2005). boggiatto et al. (2011) described the first and novel report of disseminated l. infantum parasites as identified by qpcr in 8-day-old pups born to a naturally infected seropositive dog with no travel history. this is considered the first report of vertical transmission of l. infantum in naturally infected dogs in north america, emphasising that this novel means of transmission could possibly sustain infection within populations. other modes of direct transmission were through dog bite wounds (duprey et al. 2006; naucke, amelung & lorentz 2016), venereal transmission (silva et al. 2009), blood transfusions (freitas et al. 2006; owens et al. 2001) and haematophagous insects and fleas (colombo et al. 2011; coutinho & linardi 2007; dantas-torres 2006; daval et al. 2016; de morais et al. 2013; ferreira, fattori & lima 2009; gustavo et al. 2010; otranto & dantas-torres 2009; seblova et al. 2014; slama et al. 2014). the authors concluded that the occurrence and persistence of limited canl foci (e.g. in households or in kennels) is a threat for further spread of the disease in non-endemic areas should competent vectors be introduced. consequences assessment (likelihood of occurrence and magnitude) disease transmission to dogs/wildlife animals leishmania parasites are zoonotic multi-host parasites, which may be maintained in several mammalian species in nature. roque and jansen (2014) reviewed the mammalian species known to be infected with leishmania spp. in the americas, highlighting those that can maintain and act as a source of the parasite in nature. these host reservoirs were presented separately in each of seven mammal orders; marsupialia, cingulata, pilosa, rodentia, primata, carnivora and chiroptera responsible for maintaining leishmania species in the wild (more than 80 species of animals). ashford et al. (1973) proved that the hyraxes procavia habessinica and heterohyrax brucei are the natural reservoirs of cutaneous leishmaniasis in highland ethiopia. further cases of cutaneous leishmaniasis were diagnosed in namibia by finding amastigotes in sections of excised lesions from hyraxes. a culture of tissue from the tip of the nose of one animal in diphasic blood-agar medium showed active promastigotes 14 days after inoculation. iberian hares (lepus granatensis) were recently deemed responsible for an outbreak of human leishmaniasis in spain (carillo, moreno & cruz 2013; ruiz-fons, ferroglio & gortázar 2013). jackals and foxes may play a role in the spread of zoonotic l. tropica (talmi-frank et al. 2010). faiman et al. (2013) reported that two species of rodents, the levant voles (microtus guentheri) and tristram’s jirds (m. tristrami) as reservoirs of l. major in israel. human health (zoonosis) in south africa and neighbouring countries the classification of visceral and cutaneous forms of leishmaniasis as observed in human disease cannot be applied to the infection in other mammals. dogs infected with l. infantum present viscero-dermal disease, where parasite isolation is common even from intact skin. moreover, leishmania species associated with human cutaneous infection have been observed in rodent viscera (roque et al. 2010). four cases of cutaneous leishmaniasis were reported from the republic of south africa and from south west africa/namibia in 1970 (grové 1970). in this report, it was stated that two of patients had never been out of south africa or south west africa; and the other two probably contracted the infection in south or south west africa. the lesions were typical nodules or ulcers and the diagnosis was proven by histology which also showed typical intracellular amastigotes of leishmania. this was the first report of cutaneous leishmaniasis in southern africa. again, cutaneous leishmaniasis from south west africa/namibia was reported in 1978 and the authors made the following comment ‘this adds a further dimension to the characterisation of this disease in southern africa’ (grové 1989; rutherfoord & uys 1978). in the years 1974 and 1976, autochthonous cases of human cutaneous leishmaniasis were reported in zambia (campbell, gordon & emms 1976; grové & van dyk 1974; naik et al. 1976). two cases of cutaneous leishmaniasis in malawi were diagnosed by histopathology in northern malawi in 1 year (pharoah et al. 1993). these authors also made the following comment ‘the leishmania species responsible could not be identified, but the infection may be more prevalent in this region than previously thought’. a 26-year-old man from angola with no history of travel outside the country was diagnosed with typical symptoms of visceral leishmaniasis. the parasite was isolated and characterised using both kinetoplast dna and nuclear dna probes and showed a strong homology with l. infantum (jimenez et al. 1994). a patient contracted cutaneous leishmaniasis 3 weeks after travel to botswana and a visit to a game reserve (schwartz et al. 2012). risk assessment risk assessment, risk management and risk communication are components of risk analysis. a qualitative risk assessment was conducted, and so the likelihood of release and exposure as well as the magnitude of the consequences were expressed in terms of negligible, low, moderate and high. a scenario tree was used, detailing the release (entry), exposure and consequence pathways of leishmania introduction into south africa. the likelihood is a product of all the steps in the scenario tree (peeler et al. 2015). this assessment is summarised using a risk matrix, which combines the likelihood of the hazard and the consequences of that hazard to produce an overall risk estimate (desvaux et al. 2016; dufour et al. 2011). the final risk estimate considers the degree of uncertainty in the available data and scientific evidence (costard 2008). figure 3 depicts the ‘risk pathway’ scenario tree for the introduction of leishmania. for entry, canl is reported in exporting countries, in neighbouring and regional countries and as such, the risk is considered high. after diagnosis, if the test result is negative, there is a probability that this is because of a missed case and not necessarily because the test is true negative. if the sandfly vector is present (two putative vector species present), then this leads to a high chance of transmission to dogs, wildlife and zoonotic cases arising. if transmission was to occur through mechanical means (venereal, vertical or through blood transfusion) then, transmission to dogs and other wildlife would be low and confined in a dog population however, negligible in humans. if autochthonous and cases of infected dogs, assumed to be of unknown origins or illegal importation, transmission by sandflies and therefore to dogs/wildlife and humans would be high. in a transmission scenario is to occur in dogs, wildlife and humans, there are no effective control or treatment measures currently in place to effectively maintain the spread. considering these different risk factors, a moderate likelihood of the introduction of leishmania is expected. this is further summarised in table 2, which shows the likelihood of the leishmania entry and the consequence of this hazard, which in our case are both moderate and this results in a moderate risk estimate. for a risk assessment to be complete, uncertainty about the available data and scientific evidence should be given. table 3 shows the degrees of uncertainty in available sources. reliable and complete data needed for the quantification of the risk of introduction of leishmania is lacking but there is strong scientific evidence available in multiple references, although some conclusions reported by review authors vary. a moderate degree of uncertainty is concluded from our risk assessment and therefore the final risk estimate is given as moderate. figure 3: risk pathway and probability (negligible to high) for entry (release), exposure and consequence assessment for introduction of canine leishmaniasis. table 2: combination risk matrix of the likelihood of exposure and entry of leishmania and the consequence assessment. table 3: degree of uncertainty in the quality and availability of data and scientific evidence in relation to qualitative risk assessment. conclusion and recommendations not much attention has been given to the reported and published autochthonous leishmaniasis cases involving rural dogs and humans since 1970 in south africa and southern african countries in general. it seems as if the risk has either not been considered or has been underestimated or neglected. autochthonous leishmaniasis ecology and epidemiology should be studied under current conditions of climatic and environmental change. considering that only few reports of leishmaniasis infection in wildlife host reservoirs are available in south africa, long-term investigations must be undertaken in this field. attention should be given to surveillance of the reservoir infection in rodents and hyraxes in view of a higher incidence of infection in these animal species encountered in africa. the main route of transmission of canl parasite to mammals is via the bite of the female phlebotomine sandfly (killick-kendrick 1990). two putative and suspected vector species have been reported to occur in south africa, namibia and botswana: p. rodhaini and p. rossi (davidson 1981, 1987; killick-kendrick 1990; krüger 2017; lewis & legder 1976). the abundance and vector capacity of the two species have not been demonstrated. the occurrence and persistence of limited canl foci (direct transmission, vertical, venereal and blood transfusion) is a threat for further spread of the disease in non-endemic areas should the competence of these putative vectors be established. where sandfly populations are likely to have a lower vectorial capacity than in endemic areas because of lower vector densities, the probability of establishment following introduction of an infected dog remains high, according to the model (efsa panel animal health and welfare 2015). only few authors in south africa and southern africa have made contributions to the studies on sandflies. this resulted in very few collections being made, and currently there is not much information about the distribution of phlebotomine sandflies and their ecology, biology and disease relationships. this emphasises the need for improving our knowledge of the vector competence of these two sandfly species and of the distribution and abundance of other known vectors present in africa. acknowledgements the collaboration and assistance of the private veterinarian and the state veterinary office, durban, and the allerton veterinary laboratory is acknowledged. the authors are grateful to annett michel (hamburg) for technical assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.a.l. and n.a.a. were responsible for data analysis and manuscript writing. b.n., b.p., o.m., r.p., d.m. and a.j. were responsible for samples diagnostics and data collection. b.j.m. was responsible for data revision and manuscript writing. k.l. was responsible for field sampling and data collection. a.k. was responsible for insect identification and manuscript writing. funding information the parasitology diagnostic project, number 15/08/1p01, was funded by the agricultural research council-onderstepoort veterinary research (arc-ovr) and the department of agriculture, forestry and fisheries, south 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animals. this is perhaps not surprising considering the amount of assistance required to collect ticks from dead or immobilized animals of this size, and the time that has to be spent identifying and counting immature ticks in representative samples of these collections and often all the several thousand adult ticks that a single large herbivore may harbour. amongst those who have attempted to do so are horak, potgieter, walker, de vos & boomker (1983), rechav, zeederberg & zeller (1987), horak, fourie, novellie & williams (1991a), horak, anthonissen, krecek & boomker (1992a) and zieger, horak, cauldwell & uys (1998). prior as well as subsequent to these published studies the opportunity to sam231 onderstepoort journal of veterinary research, 74:231–242 (2007) ticks associated with the three largest wild ruminant species in southern africa i.g. horak1*, h. golezardy2 and a.c. uys2 abstract horak, i.g., golezardy, h. & uys, a.c. 2007. ticks associated with the three largest wild ruminant species in southern africa. onderstepoort journal of veterinary research, 74:231–242 the objective of this study was to assess the host status of the three largest southern african wild ruminants, namely giraffes, giraffa camelopardalis, african buffaloes, syncerus caffer, and eland, taurotragus oryx for ixodid ticks. to this end recently acquired unpublished data are added here to already published findings on the tick burdens of these animals, and the total numbers and species of ticks recorded on 12 giraffes, 18 buffaloes and 36 eland are summarized and discussed. twenty-eight ixodid tick species were recovered. all stages of development of ten species, namely amblyomma hebraeum, rhipicephalus (boophilus) decoloratus, haemaphysalis silacea, ixodes pilosus group, margaropus winthemi, rhipicephalus appendiculatus, rhipicephalus evertsi evertsi, rhipicephalus glabroscutatum, rhipicephalus maculatus and rhipicephalus muehlensi were collected. the adults of 13 species, of which the immature stages use small mammals as hosts, namely haemaphysalis aciculifer, hyalomma glabrum, hyalomma marginatum rufipes, hyalomma truncatum, ixodes rubicundus, rhipicephalus capensis, rhipicephalus exophthalmos, rhipicephalus follis, rhipicephalus gertrudae, rhipicephalus lounsburyi, rhipicephalus lunulatus, rhipicephalus pravus group and rhipicephalus simus, were also collected. keywords: african buffaloes, eland, giraffa camelopardalis, giraffes, ixodid ticks, syncerus caffer, taurotragus oryx, tick burdens * author to whom correspondence is to be directed. e-mail: ivan.horak@up.ac.za 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa; division of parasitology, onderstepoort veterinary institute, onderstepoort, 0110 south africa; and department of zoology and entomology, university of the free state, p.o. box 339, bloemfontein, 9300 south africa 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa. a.c. uys’ present address: p.o. box 652, newlands, 0049 south africa accepted for publication 23 march 2007—editor 232 ticks associated with largest wild ruminant species in southern africa ple more of these large herbivores has arisen and the present paper reports the results of the latter collections. however, since we thought it unlikely that, in the near future, similar exhaustive tick collections would be made from such large animals in southern africa, we decided to combine the earlier published data with the more recent findings in order to give a more comprehensive overview of the numbers and species of ticks that may infest giraffes, african buffaloes and eland in a variety of habitats. giraffes occur in several associations of dry savanna, varying from scrub to woodland. they do not occur in forest or desert and generally are not found in open plains. they are present in north-eastern limpopo and mpumalanga provinces, south africa, and in the north-eastern parts of namibia (skinner & smithers 1990), but in recent times have been introduced into a number of regions in south africa in which they previously did not occur. african buffaloes prefer savanna-type habitats and need a plentiful supply of grass, shade and water. they occur in herds, which increase in size in the dry season, but become more fragmented during the wet season because of the usual plentiful supply of water and grazing (skinner & smithers 1990). the current distribution of african buffaloes in south africa is mostly patchy. large numbers are present in the kruger national park (knp) and the hluhluweimfolozi park in the north-eastern regions of the limpopo, mpumalanga and kwazulu-natal prov inces, with smaller populations in national, provincial and privately owned reserves in these and nearly all other provinces of south africa. eland are gregarious animals usually occurring in small herds, and are as at home in arid semi-desert scrub associations as they are in montane grassland (skinner & smithers 1990). historically they were present virtually throughout south africa, but overexploitation reduced their numbers. however, because they are a sought after species, reintroductions have resulted in their now occupying virtually the same regions in which they originally occurred. one of the serious shortcomings of the past and current studies is that they are biased towards localities and seasons at which animals were, or have been, made available for survey purposes and are thus not necessarily representative of the entire ixodid tick fauna present within the geographic distribution of the host species being examined. nevertheless a remarkably large number of tick species were recovered. moreover, because of the large size of the hosts, large numbers of adult ticks were collected (gallivan & horak 1997), and the greater certainty with which the adult ticks can be identified has augmented the accuracy of our identification of the immature stages, and hence our results. materials and methods the localities at which animals were examined, includ ing those of the earlier studies of horak et al. (1983, 1991a, 1992a), rechav et al. (1987) and zieger et al. (1998), are summarized in table 1. with the exception of free state and gauteng provinces at least one of the three large wild ruminant species was sampled in each province of the republic of south africa. in addition, six of the giraffes were exam ined in the etosha national park in northern namibia (horak et al. 1992a), and two of the eland on a game ranch in central province, zambia (zieger et al. 1998). ticks were recovered from the animals in the earlier surveys as described by the authors of those studies. the carcasses of giraffes, african buffaloes and eland in the present surveys were processed for tick recovery and the ticks identified and counted as described by horak, boomker, spickett & de vos (1992b). when animals had been immobilized, and not killed, an attempt was made to collect as many adult ticks as possible, with particular attention being paid to the preferred sites of attachment of the various species. a total of 12 giraffes, 18 african buffaloes and 36 eland were examined, but in the tables devoted to each host species only the results for those animals sampled within a particular tick species’ distribution range are given for that particular tick. results and discussion the species and numbers of ticks collected from the giraffes, buffaloes and eland are summarized in tables 2, 3 and 4, and the total numbers of male and female ticks recovered and, where it was possible to calculate this, the ratio of males to females for each species are summarized in table 5. a calculated total of 450 709 ticks, belonging to 28 species and three subspecies, were collected from the 66 animals examined. of these 57 688 were adults, of which at least half were identified and counted. although this may seem like a large number of ticks, probably three times as many would 233 i.g. horak, h. golezardy & a.c. uys have been recovered had the digestion technique of van dyk & mckenzie (1992) been used. however, the fine structures of tick larvae and nymphs are often damaged during the period of digestion required in the latter technique, and thus, although tick recovery may be more complete, it is achieved at the expense of accurate species determination. two of the 28 species recovered are one-host ticks, namely margaropus winthemi and rhipicephalus (boophilus) decoloratus. five are two-host ticks, these being hyalomma glabrum, hyalomma marginatum rufipes, hyalomma truncatum, rhipicephalus glabroscutatum and rhipicephalus evertsi of which both subspecies of the latter, namely evertsi and mimeticus were collected. the remaining 21 species are all three-host ticks. all stages of development of amblyomma hebraeum, r. (boophilus) decoloratus, haemaphysalis silacea, ixodes pilosus group, m. winthemi, rhipicephalus appendiculatus, r. evertsi evertsi, r. glabroscutatum, rhipicephalus maculatus and rhipicephalus muehlensi were collected. only the adults of 13 species, namely haemaphysalis aciculifer, h. glabrum, h. marginatum rufipes, h. truncatum, ixodes rubicundus, rhipicephalus capensis, rhipicephalus exophthalmos, rhipicephalus follis, rhipicephalus gertrudae, rhipicephalus lounsburyi, rhipicephalus lunulatus, rhipicephalus pravus group and rhipicephalus simus, of which the immature stages use small mammals as hosts, were recovered. in addition the larvae and nymphs of the south african tortoise tick, amblyomma marmoreum, nymphs and adults of the tropical bont tick, amblyomma variegatum, a single male rhipicephalus longiceps, 30 rhipicephalus supertritus males and 16 rhipicephalus zambeziensis nymphs were retable 1 localities at which large wild ruminants were examined for ixodid ticks country and province locality co-ordinates vegetation type (van der merwe 1983; white 1983; acocks 1988) south africa western cape west coast national park 33°06’ s, 17°59’ e strandveld and patches of coastal fynbos eastern cape thomas baines nature reserve 33°23’ s, 26°28’ e false macchia, eastern province thornveld and valley bushveld andries vosloo kudu reserve 33°07’ s, 26°40’ e valley bushveld mountain zebra national park 32°15’ s, 25°27’ e karroid merxmeullera mountain veld, replaced by karoo on the higher slopes and karroid broken veld in the north northern cape kgalagadi transfrontier park 24°45’–26°28’ s, 20°00’–20°50’ e lightly wooded grassland on dune crests and grassland in depressions between dunes kwazulu-natal eastern shores park 28°08’ s, 32°30’ e zululand palm veld, subdivision of coastal thornveld and coastal communities imfolozi nature reserve 28°15’ s, 31°57’ e zululand thornveld and lowveld north west sa lombaard reserve 27°35’ s, 25°29’ e dry cymbopogon-themeda veld mpumalanga pretoriuskop (knp) 25°10’ s, 31°16’ e lowveld sour bushveld lower sabie (knp) 25°07’ s, 31°55’ e lowveld mtethomusha nature reserve 25°29’ s, 31°17’ e lowveld namibia okaukuejo, etosha national park 19°11’ s, 15°55’ e mopane savanna zambia central mtendere game ranch 15°05’ s, 28°16’ e miombo woodland knp = kruger national park 234 ticks associated with largest wild ruminant species in southern africa covered. ten of the 28 tick species recovered occur only in south africa. they are h. silacea, h. glabrum, i. pilosus, i. rubicundus, m. winthemi, r. capensis, r. follis, r. glabroscutatum and r. lounsburyi. with the exception of ticks of the i. pilosus group and i. rubicundus, of which more females than males were recovered, considerably more males than females of all other species, of which both sexes were present, were collected. mating in ixodes species may occur in the preparasitic phase of the life cycle (fourie & horak 1994), and many males thus never attach. this behaviour is probably largely responsible for the female-biased parasitic populations of the two ticks in this genus in the present study. the male-biased populations of the other tick species is due to the propensity of parasitic male ixodid ticks of several species to spend extended periods on their hosts (londt 1976; jordaan & baker 1981), whereas the females may require only a few days to two weeks to mate and engorge before detaching. furthermore the larger size of engorging females is likely to make them more susceptible to removal by grooming or predation than the considerably smaller males. amblyomma species the larvae of amblyomma hebraeum infest a wide variety of hosts, including ungulates, carnivores, lago morphs and birds, and all stages of development, but particularly the adults, infest very large hosts (horak, macivor, petney & de vos 1987a). the males may remain attached for several months (jordaan & baker 1981), thus augmenting the already male-biased population of adult ticks. amblytable 2 giraffes examined and the number infested within the distribution range of each tick tick species hosts examined (infested) life stage total no. of ticks locality amblyomma hebraeum 6 (6) larvae nymphs males females 773 554 2 406 512 knp hyalomma marginatum rufipes 12 (8) males females 374 77 knp, etosha hyalomma truncatum 12 (12) males females 1 649 619 knp, etosha rhipicephalus (boophilus) decoloratus 6 (6) larvae nymphs males females 2 472 2 538 1 503 821 knp rhipicephalus appendiculatus 6 (6) larvae nymphs males females 168 682 49 4 knp rhipicephalus evertsi evertsi 6 (6) larvae nymphs males females 452 190 106 20 knp rhipicephalus evertsi mimeticus 6 (5) males females 14 5 etosha rhipicephalus longiceps 6 (1) males 1 etosha rhipicephalus pravus group 6 (3) males females 32 20 knp rhipicephalus simus 6 (4) males females 22 6 knp knp = kruger national park 235 i.g. horak, h. golezardy & a.c. uys omma hebraeum is present in the bushveld and low veld regions of northern, eastern and southeastern south africa (walker & olwage 1987). amblyomma marmoreum is known as the south african tortoise tick, and all stages of development may be found on a variety of these reptiles, with leopard tortoises, geochelone pardalis, generally the most heavily infested (horak, mckay, heyne & spickett 2006). the larvae infest a wide variety of mammals and birds, and are often found on mammals and birds of several species that are present within the tick’s distribution range (horak et al. 2006). amblyomma variegatum does not occur in south africa, but is present in most sub-saharan countries table 3 african buffaloes examined and the number infested within the distribution range of each tick tick species hosts examined (infested) life stage total no. of ticks locality amblyomma hebraeum 18 (18) larvae nymphs males females 47 815 6 437 6 154 1 764 knp, eastern shores, imfolozi, thomas baines, mtethomusha amblyomma marmoreum 18 (4) larvae nymphs 8 26 knp, eastern shores, imfolozi, thomas baines, mtethomusha haemaphysalis silacea 15 (7) larvae nymphs males females 81 357 135 78 eastern shores, imfolozi, thomas baines hyalomma truncatum 17 (2) males females 6 0 knp, imfolozi, mtethomusha rhipicepalus (boophilus) decoloratus 18 (11) larvae nymphs males females 1 640 290 248 160 eastern shores, imfolozi, thomas baines, mtethomusha rhipicephalus appendiculatus 18 (17) larvae nymphs males females 139 389 32 715 1 360 774 knp, eastern shores, imfolozi, thomas baines, mtethomusha rhipicephalus evertsi evertsi 18 (15) larvae nymphs males females 721 35 96 45 knp, eastern shores, imfolozi, thomas baines, mtethomusha rhipicephalus follis 1 (1) males females 40 32 thomas baines rhipicephalus maculatus 14 (14) larvae nymphs males females 15 379 16 772 946 398 eastern shores, imfolozi rhipicephalus muehlensi 16 (15) larvae nymphs males females 11 775 495 193 141 eastern shores, imfolozi, mtethomusha rhipicephalus simus 18 (9) males females 121 64 knp, eastern shores, imfolozi, mtethomusha knp = kruger national park 236 ticks associated with largest wild ruminant species in southern africa table 4 eland examined and the number infested within the distribution range of each tick tick species hosts examined (infested) life stage total no. of ticks locality amblyomma hebraeum 4 (4) larvae nymphs males females 32 394 2 405 3 488 530 knp, andries vosloo kudu reserve, thomas baines nr amblyomma marmoreum 15 (8) larvae 167 knp, mountain zebra np, av kudu reserve, thomas baines nr amblyomma variegatum 2 (2) nymphs males females 4 60 26 mtendere game ranch haemaphysalis aciculifer 2 (1) males 2 knp haemaphysalis silacea 4 (4) larvae nymphs males females 7 686 933 1 409 386 andries vosloo kudu reserve, thomas baines nr hyalomma glabrum 11 (11) males females 955 191 mountain zebra np hyalomma marginatum rufipes 16 (16) males females 907 470 kgalagadi transfrontier park, thomas baines nr, sa lombaard nr hyalomma truncatum 32 (27) males females 1 702 650 mountain zebra np, kgalagadi transfrontier park, west coast np, sa lombaard nr ixodes pilosus group 4 (4) larvae nymphs males females 2 817 177 22 64 andries vosloo kudu reserve, thomas baines nr ixodes rubicundus 11 (7) males females 34 81 mountain zebra np margaropus winthemi 11 (9) larvae nymphs males females 12 792 6 874 1 915 893 mountain zebra np rhipicephalus (boophilus) decoloratus 8 (5) larvae nymphs males females 3 106 3 988 3 581 848 knp, av kudu reserve, thomas baines nr, mtendere game ranch rhipicephalus appendiculatus 8 (8) larvae nymphs males females 18 025 1 489 8 106 3 345 knp, av kudu reserve, thomas baines nr, mtendere game ranch rhipicephalus capensis 2 (2) males females 1 506 408 west coast np rhipicephalus evertsi evertsi 34 (31) larvae nymphs males females 6 900 2 128 1 574 392 knp, mountain zebra np, av kudu reserve, thomas baines nr, west coast np, sa lombaard nr, mtendere game ranch 237 i.g. horak, h. golezardy & a.c. uys to the north (walker & olwage 1987). its host spectrum is similar to that of a. hebraeum and its presence on the two eland examined in zambia is thus to be expected. haemaphysalis species the two male haemaphysalis aciculifer recovered from an eland in the knp during september 1979 (horak et al. 1983; table 4), were the only specimens of this species reported in the park until 2000, when horak, braack, fourie & walker (2000) recorded 13 males and two females from a honey badger, mellivora capensis. this tick is seldom recovered in large numbers but appears to be present in north-eastern mpumalanga province and thence along the eastern and southern coastal and adjacent inland regions to the south-western region of the cape province (theiler 1962; horak, keep, spickett & boomker 1989; horak & boomker 1998). haemaphysalis silacea is present in north-eastern kwazulu-natal and in the eastern cape provinces (walker 1991), and is associated with vegetation classified as valley bushveld (acocks 1988) and with greater kudus, tragelaphus strepsiceros, common duikers, sylvicapra grimmia, cape grysbok, rhaphicerus melanotis, and helmeted guineafowls, numida meleagris (horak & knight 1986; macivor & horak 2003). seven of the 18 buffaloes and all four eland examined in valley bushveld-type vegetation in the north-eastern kwazulu-natal and the eastern cape provinces were infested. hyalomma species delpy (1949) described the tick now known as hyalomma glabrum as hyalomma rufipes glabrum, and the latter name persisted until theiler (1956) raised it to species level as hyalomma glabrum. in the same year hoogstraal (1956) synonymized it with asian hyalomma marginatum turanicum, and since then it has been considered identical to the latter tick. recent studies have, however, revealed that it is a separate taxon, and apanaskevich & horak (2006) have subsequently reinstated it as hyalomma glabrum. howell, walker & nevill (1978) have illustrated its distribution (as h. m. turanicum) in the central and western regions of south africa. the adults prefer large animals such as eland and cape mountain zebras, equus zebra zebra, and its immature stages infest scrub hares, lepus saxatilis, and ground-frequenting birds (apanaskevich & horak 2006). tick species hosts examined (infested) life stage total no. of ticks locality rhipicephalus exophthalmos 11 (1) males females 0 2 mountain zebra np rhipicephalus follis 11 (11) males females 983 447 mountain zebra np, thomas baines nr rhipicephalus gertrudae 2 (1) males females 4 2 west coast np rhipicephalus glabroscutatum 14 (13) larvae nymphs males females 6 510 2 846 868 499 mountain zebra np, andries vosloo kudu reserve, west coast np rhipicephalus lounsburyi 11 (2) males 3 mountain zebra np rhipicephalus lunulatus 2 (2) males females 218 48 mtendere game ranch rhipicephalus simus 6 (4) males females 39 5 knp, av kudu reserve, thomas baines nr rhipicephalus supertritus 2 (2) males 30 mtendere game ranch rhipicephalus zambeziensis 2 (1) nymphs 16 knp knp = kruger national park np = national park nr = nature reserve table 4 cont. 238 ticks associated with largest wild ruminant species in southern africa the distribution of hyalomma marginatum rufipes is more extensive than that of h. glabrum, which it almost entirely overlaps (howell et al. 1978). its host preferences are the same as those of the latter tick (walker 1991), but include giraffes and buffaloes, which are generally not present within the distribution range of h. glabrum (tables 2 and 3). with the exception of the eastern coastal regions and some adjacent inland areas and the southern coastal regions, hyalomma truncatum is present virtually throughout the country (howell et al. 1978). the adults are found on the same large hosts as the former two ticks, and its immature stages infest scrub hares and rodents (walker 1991). ixodes species ticks of the ixodes pilosus group are present in the southern coastal and adjacent regions of the western and eastern cape provinces and in southeastern kwazulu-natal province, where all stages of development may be encountered on antelopes, caracals, caracal caracal and domestic dogs (horak, jacot guillarmod, moolman & de vos 1987b; horak et al. 1989; horak & boomker 1998; horak & matthee 2003). the colloquial name given to ixodes rubicundus is karoo paralysis tick, because of the paralysis associated with infestation of small domestic livestock in the karoo regions of the country (howell et al. 1978; fourie & horak 1994). the hosts of the adults are wild and domestic ruminants, caracals and domestic dogs (horak et al. 1987b, 1991a; fourie & horak 1993; horak & matthee 2003). the immature stages infest smith’s red rock rabbits, pronolagus ru pestris, caracals and rock elephant shrews, elephan tulus myurus (horak et al. 1991a; fourie, horak & woodall 2005). margaropus winthemi this tick is commonly known as the winter horse tick, and its distribution in the cooler higher-lying regions of the country suggests that its original hosts table 5 male to female ratios of tick species collected from giraffes, african buffaloes and eland in southern africa tick species total no. of adult ticks male:female ratio male female male female amblyomma hebraeum amblyomma variegatum haemaphysalis aciculifer haemaphysalis silacea hyalomma glabrum hyalomma marginatum rufipes hyalomma truncatum ixodes pilosus group ixodes rubicundus margaropus winthemi rhipicephalus (boophilus) decoloratus rhipicephalus appendiculatus rhipicephalus capensis rhipicephalus evertsi evertsi rhipicephalus evertsi mimeticus rhipicephalus exophthalmos rhipicephalus follis rhipicephalus gertrudae rhipicephalus glabroscutatum rhipicephalus longiceps rhipicephalus lounsburyi rhipicephalus lunulatus rhipicephalus maculatus rhipicephalus muehlensi rhipicephalus pravus group rhipicephalus simus rhipicephalus supertritus 12 048 60 2 1 544 955 1 281 3 357 22 34 1 915 5 332 9 515 1 506 1 776 14 0 1 023 4 868 1 3 218 946 193 32 182 30 2 806 26 0 464 191 547 1 269 64 81 893 1 829 4 123 408 457 5 2 479 2 499 0 0 48 398 141 20 75 0 4.29 2.31 – 3.33 5.00 2.34 2.65 0.34 0.42 2.14 2.92 2.31 3.69 3.89 2.80 – 2.14 2.00 1.74 – – 4.54 2.38 1.37 1.60 2.43 – 1.0 1.0 – 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 – 1.0 1.0 1.0 – – 1.0 1.0 1.0 1.0 1.0 – total (excluding ixodes spp.) 42 805 14 682 2.92 1.0 239 i.g. horak, h. golezardy & a.c. uys were cape mountain zebras, on which it still occurs in very large numbers during the winter months (horak et al. 1991a). the burdens of the eland (table 4), examined at the same time as zebras in the mountain zebra national park, although large, did not nearly approach those of the latter animals. rhipicephalus species rhipicephalus (boophilus) decoloratus. using molecular analyses supported by an analysis of morphological characters murrell, campbell & barker (2000) and beati & keirans (2001) decided that the genus rhipicephalus is paraphyletic with respect to the genus boophilus and consequently murrell & barker (2003) proposed the use of the above nomenclature. we have chosen to follow their proposal, although many people prefer to retain boophilus as a generic name. giraffes and eland can generally be considered as good hosts of this tick, whereas afri can buffaloes appear to be resistant to infestation, with more than half of the adult ticks reported here coming from a single, approximately 6-month-old calf. norval (1984) and horak, golezardy & uys (2006) have discussed the apparent resistance of african buffaloes to artificial and natural infestations with this tick, and it would seem that this resistance is acquired, rather than innate. adult rhipicephalus appendiculatus are common parasites of african buffaloes, eland, greater kudus, male nyalas, tragelaphus angasii, and domestic cattle in wooded savannas from south-eastern south africa in the south to kenya and uganda in the north (walker, keirans & horak 2000). the immature stages are found on the same hosts as the adults, but also infest smaller antelopes and scrub hares (walker et al. 2000). the eland included in the present study and more particularly those from zambia, were heavily infested with adult ticks (table 4). the buffaloes, which were examined mainly during the early winter months, harboured very large numbers of larvae (table 3). rhipicephalus capensis, rhipicephalus follis, rhipicephalus gertrudae and rhipicephalus simus have several morphological characteristics in common and also have similar life cycles during which the immature stages infest murid rodents and the adults the larger species of antelopes (walker et al. 2000). adult r. gertrudae also infest domestic dogs and primates, including humans (brain & bohrmann 1992; horak, fourie, heyne, walker & needham 2002; horak & matthee 2003), while those of r. simus infest domestic and wild equids, suids and carnivores (horak et al. 1987b, 2000; walker et al. 2000). rhipicephalus capensis occurs almost exclusively in the western coastal regions of the west ern cape province, south africa; r. gertrudae is present in the same western regions, but its distribu tion extends east to the southern and southern-central regions of the country and north into namibia; r. follis is only found in south africa and is present to the east and north-east of r. gertrudae’s distribution range and generally in mountainous terrain; while, with the exception of the more arid regions of central and western south africa and southern namibia, the distribution range of r. simus effectively overlays those of the other three species and extends north into africa to a latitude of approximately 9° south. rhipicephalus evertsi evertsi and rhipicephalus evertsi mimeticus are two-host ticks and all stages of development may infest the same host species. excluding the deserts and regions of high rainfall, the former tick is found throughout sub-saharan africa, whereas the latter is confined to the arid and semi-arid regions of namibia, western botswana and parts of angola (walker et al. 2000). the adults of rhipicephalus exophthalmos parasitize antelopes, domestic ruminants and scrub hares in the south-eastern and north-western regions of south africa and in a broad, central band from the south to the north of namibia (walker et al. 2000). its immature stages prefer elephant shrews, el ephantulus spp., as hosts (fourie et al. 2005). the two-host tick, rhipicephalus glabroscutatum, is present in the fynbos and karoo regions of the western cape province, and also in the karoo and the valley bushveld regions of the eastern cape province (walker et al. 2000). it is a common parasite of the feet and lower legs of small and large antelope in these regions (horak & boomker 1998; macivor & horak 2003). infestation with its adults is a contributory cause of foot abscess in domestic goats in the valley bushveld regions of the eastern cape province (macivor & horak 1987). the distribution in south africa of rhipicephalus maculatus and rhipicephalus muehlensi is confined to the coastal bush and adjacent inland regions of north-eastern kwazulu-natal (walker et al. 2000). the recovery of an r. maculatus nymph from the vegetation and adults from an elephant, loxodonta africana, in the southern regions of the knp (braack, maggs, zeller & horak 1995), and of r. muehlensi adults from one of the buffaloes examined in the mtethomusha nature reserve, just south of the knp, suggest that they have been introduced into these 240 ticks associated with largest wild ruminant species in southern africa reserves on animals translocated from north-eastern kwazulu-natal. all stages of development of both ticks may infest the same host species, but the adults of r. maculatus prefer thick-skinned animals such as buffaloes and bush pigs, potamochoerus larvatus, and those of r. muehlensi nyalas (horak, boomker & flamand 1991b; 1995). rhipicephalus longiceps is a rarely encountered tick and apparently is present only in certain regions of namibia and angola (walker et al. 2000). rhipicephalus lounsburyi prefers the higher mountainous regions of the eastern cape province (walker et al. 2000) and two eland in the mountain zebra national park were infested. the adults attach around the feet of their antelope and sheep hosts, while the only known hosts of its immature stages are four-striped grass mice, rhabdomys pu milio, from which a larva and two nymphs have been collected (walker et al. 2000; horak, fourie & braack 2005). although rhipicephalus lunulatus is fairly widespread in sub-saharan africa its distribution in south africa is limited to the eastern regions (walker et al. 2000). the adults have a wide host range and would seem to attach around the feet and lower legs of their hosts (walker et al. 2000). the eland examined in zambia had fairly large burdens of adult ticks (table 4), while a multimammate mouse, mastomys sp., and a scrub hare examined on the same ranch as the eland were infested with nymphs (zieger et al. 1998). ticks belonging to the rhipicephalus pravus group have been collected from scrub hares in the knp (horak, spickett, braack & penzhorn 1993), and were assigned to rhipicephalus sp. near pravus by walker et al. (2000). however, the ticks collected in the same park from three of the giraffes, appear to us to be very similar, if not identical to the true r. pravus of east africa. the immature stages of rhipicephalus supertritus are unknown, but are assumed to be similar in appearance to those of r. appendiculatus. it is apparently commonest in central africa, including northern zimbabwe and mozambique, parts of zambia and southern tanzania (walker et al. 2000), and both eland examined in zambia were infested. acknowledgements we are most grateful to ezemvelo kzn wildlife, sanparks, and the provincial division of nature conservation of the eastern cape province for placing the animals included in the recent surveys at our disposal, and for providing assistance and facilities to process them for tick recovery. we are particularly indebted to messrs johan sithole and eddie williams for their assistance in the latter respect. the university of pretoria and the national research foundation provided funds for the conduct of this project. the publication of this work has been facilitated through the integrated consortium on ticks and tick-borne diseases (icttd-3), financed by the international cooperation program of the europe an union through coordination action project no. 510561. references acocks, j.p.h. 1988. veld types of south africa with accompanying veld type map, 3rd ed. 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brown ticks of the world. cambridge: academic press. white, f. 1983. the vegetation of africa. a descriptive memoir to accompany the unesco/aetfat/unso vegetation map of africa. paris: unesco. yeoman, g.h. & walker, jane b. 1967. the ixodid ticks of tanzania. a study of the zoogeoraphy of the ixodidae of an east african country. london and reading: commonwealth institute of entomology. zieger, u., horak, i.g., cauldwell, a.e. & uys, a.c. 1998. ixodid tick infestations of wild birds and mammals on a game ranch in central province, zambia. onderstepoort jour nal of veterinary research, 65:113–124. hyera.indd introduction several free-living game animal species, particularly the african buffalo, syncerus caffer, are important reservoirs of foot-and-mouth disease virus (fmdv) for livestock in southern africa (bengis, thomson, hedger, de vos & pini 1986). some of these species occur in large numbers in botswana, especially along the chobe river, the okavango delta and, as such, the two areas are recognized as the main foot-andmouth disease high-risk zones in the country. goats and sheep can be infected experimentally with fmdv (dellers & hyde 1964; geering 1967; burrows 1968; mcvicar & sutmoller 1968; anderson, doughty & anderson 1976) and natural infection with fmdv has been reported both in sheep (zaikin 1959; littlejohn 1970; hedjazi, ansari & nadalian 1972) and in goats (hedjazi et al. 1972). carrier states have been demonstrated both in sheep (bur rows 1968; mcvicar & sutmoller 1968, 1972) and in goats (mcvic ar & sutmoller 1968, 1972; anderson et al. 1976). it is estimated that there are about 2.2 million goats and 0.4 million sheep in botswana (anon. 1996). the role of goats and sheep in the epidemiology of fmd in botswana is not known. the study reported here was carried out in order to determine the level of exposure of tswana goats and sheep to the south african territories (sat) serotypes of fmdv in northwestern botswana with the ultimate goal of highlighting the epidemiological roles that these animals might play in the transmission of the disease to cattle. 143 onderstepoort journal of veterinary research, 73:143–147 (2006) a serological survey for antibodies to foot-and-mouth disease virus in indigenous tswana goats and sheep in kasane, maun and shakawe districts in northwestern botswana j.m.k. hyera1, m. letshwenyo2, k.b. monyame1, g. thobokwe1, a.r. pilane2, n. mapitse2 and e.k. baipoledi1 abstract hyera, j.m.k., letshwenyo, m., monyame, k.b., thobokwe, g., pilane, a.r., mapitse, n. & baipoledi, e.k. 2006. a serological survey for antibodies to foot-and-mouth disease virus in indigenous tswana goats and sheep in kasane, maun and shakawe districts in northwestern bo tswana. onderstepoort journal of veterinary research, 73:143–147 a serological survey was conducted in apparently healthy, unvaccinated indigenous tswana goats and sheep in kasane, maun and shakawe districts in northwestern botswana in order to determine in these animals, the levels of exposure to the south african territories (sat) serotypes: sat 1, sat 2 and sat 3 of foot-and-mouth disease virus (fmdv). a total of 250, 142 and 134 goat sera originating respectively from kasane, maun and shakawe districts were tested for fmdv antibodies against the three sat serotypes by the liquid phase blocking enzyme-linked immunosorbent assay and 26 of 250 (10.4 %), 5 of 142 (3.5 %) and 18 of 134 (13.4 %) were positive either to sat 1 or sat 3, or to both serotypes.none of the goats’ sera was positive to sat 2 serotype.all sheep sera (n = 9) tested negative against all three serotypes of the virus.the findings are discussed in relation to results of other serological surveys carried out elsewhere. keywords: botswana, fmd antibodies, fmd virus, indigenous goats, indigenous sheep 1 botswana national veterinary laboratory, private bag 0035, gaborone 2 department of animal health and production, private bag 0032, gaborone, botswana accepted for publication 16 march 2006—editor 144 foot-and-mouth disease virus in indigenous tswana goats and sheep in bo tswana materials and methods serum samples individual healthy, unvaccinated tswana goats and sheep were bled aseptically from the jugular vein using plain vacutainer tubes. the blood samples were held on ice in a box to prevent haemolysis and after the blood had coagulated serum was harvested into sterile tubes which, after identification, were properly packed in a cool box and thereafter transported to the national veterinary laboratory (nvl) in gaborone by the quickest available means. a total of 526 goat sera (250 from kasane, 142 from maun and 134 from shakawe) and nine sheep sera (from kasane) were submitted to the nvl where they were stored at –20 °c until used. the age of the animals from which blood samples were taken was also recorded. serum antibody assay sera were tested for fmd antibodies against sat 1, sat 2 and sat 3 serotypes of fmdv in microtitre enzyme-linked immunosorbent assay (elisa) plates fig. 1 map of northwestern botswana illustrating the geographical distribution of seropositive goats ��������� ������� �� � � ��� �� �� � � ��� �� �� �� �� �� �� ������� �� ������ ���������� �������� ����!� ������� ������ ��"�� #� ����� ��$���$� �� %�������& ��!� ���� '� ��� ������ ��"����� ( � � � � � ) �� *� �� ��"� �$���+� "� ,���$� �$���� �$�"� �++����!� � ������� �� � � ��� -���� �� .�/��+�"� � *���� �( ( �( �((��� 0�1 0�) 0�( 0�� ��$� �� 2���� �� � 3����� 4���� 5$�� �� 6���� ���$��� ������ ���$��� 4�7��� table 1 prevalence of fmd antibodies in indigenous goats in northwestern botswana district number of sera tested number of sera positive prevalence (%) ± se1 kasane maun shakawe 250 142 134 26 5 18 10.40 ± 3.78 3.52 ± 3.03 13.43 ± 5.77 1 standard error at 95 % confidence 145 j.m.k. hyera et al. (nunc immunoplates, maxisorp surface) by the liquid phase blocking elisa (lpbe) using methods described elsewhere (hamblin, barnett & hedger 1986; hamblin, barnett & crowther 1986; hamblin; kitching, donaldson & crowther 1987). the sera were tested in duplicate wells; firstly, the tests were performed on a screening basis at the fixed dilution of 1/16 (1/32 after antigen addition) and thereafter all positive and doubtful sera were semi-titrated from dilutions of 1/32–1/256 (after addition of antigen). antigen and known positive and negative sera were included in each elisa plate as controls. optical density (od) values were determined at a wavelength of 492 nm using a microplate elisa reader (titer-tek multiskan; labsystems) linked to a compatible computer using elisa data interchange (edi) version 2.16 software (genesi; windows for microplate based assays). the antibody titres were calculated by the method of kaerber (1931) and were expressed in logarithms to base 10 (log10). statistical analysis prevalences or seropositivity rates were determined by dividing the total number of positive serum samples by the total number of samples tested (thrusfield 1995) and were expressed as a percentage. relative frequency was also calculated according to thrusfield (1995) and was expressed as a percentage.the standard error (se) of percentages at 95 % confidences was calculated according to the method described by swisnscow (1980). the statistical table of armitage (1971) was used to assess the statistical significance between prevalence rates. probability (p) values of < 0.05, < 0.01 and < 0.001 were considered respectively, as significant, very significant and highly significant. results a total of 49 of the 526 (9.30 ± 2.50 %) goat sera were seropositive to fmdv. table 1 shows the prevalence of antibodies to fmdv in northwestern botswana by district. the fmd antibody prevalences in kasane and shakawe were similar (∆ % = 3.03, se = 3.52; p > 0.05) but differed very significantly both between kasane and maun (∆ % = 6.08, se = 2.47; p < 0.01) and between maun and shakawe (∆ % = 9.90, se = 3.33; p < 0.01). table 2 presents the prevalences within various locations of the three districts; the highest prevalence was recorded in kazungula, kasane district followed by jao island in sha kawe district. the geographical distribution of the seropositive goats is illustrated in fig. 1. table 3 presents the distribution of positive goat sera by age group.the frequency of positive test results was comparatively higher in the adult than in the young goats (∆ % = 42.8, se = 9.13; p < 0.001). serotype-wise 45 of 526 (8.50 ± 2.38 %), 0 of 526 (0 %) and 9 of 526 (1.70 ± 0.62 %) goat sera exhibited antibodies to sat 1, sat 2 and sat 3, respectively.the seropositivity rate to sat 1 was higher than that to sat 3 and the difference between the table 2 prevalence of fmd antibodies in indigenous goats within several locations of three districts in northwestern botswana district location number of positive sera total sera tested prevalence (%) kasane kazungula kachikau lesoma kilo 256 mabele muchenje mabozo satau kavimba parakurungu kataba 5/12 0/23 0/11 0/12 9/53 0/12 1/16 4/39 3/53 2/21 2/8 41.67 0.00 0.00 0.00 16.98 0.00 16.67 10.26 5.66 9.52 25.00 maun senkoyo mababe xaxaba 3/101 1/30 1/11 2.97 3.33 9.09 shakawe xhau 1 xhau 2 gumbo jao island 3/40 1/48 2/8 12/38 7.50 2.08 25.00 31.58 146 foot-and-mouth disease virus in indigenous tswana goats and sheep in bo tswana two rates is highly significant (∆ % = 6.80, se = 1.34; p < 0.001). table 4 shows the distribution of antibody titres against the three sat serotypes in goats. none of the goats examined had positive test results to sat 2 and none of the nine sheep sera tested had antibodies to all three sat serotypes. discussion the occurrence of buffaloes in the northwestern districts of botswana poses a constant threat of infection with fmdv to cattle, goats and sheep. consequently, cattle in kasane and shakawe districts are vaccinated annually against the three sat serotypes of the virus using inactivated trivalent (sat 1, sat 2 and sat 3) vaccine. goats and sheep on the other hand are not vaccinated against any of the fmdv serotypes, although during fmd outbreaks all three livestock species (cattle, goats and sheep) are subject to the same quarantine procedures normally put in place. therefore demonstration of fmd antibodies either in goats or in sheep would indicate exposure to fmd field virus. the significantly high frequency of fmd antibodies occurring in adult goats is probably a reflection of this type of exposure, the african buffalo possibly being the primary source of virus. the fmd antibodies demonstrated in the small number of young goats are, perhaps, residues of de clining maternally-derived antibodies. a similar serosurvey in a fmd enzootic area in kenya showed that both goats and sheep were frequently exposed to infection with fmdv as evidenced by a high proportion of seropositive animals (anderson et al. 1976). no seropositive sheep was found in the present study. however, the number of sheep samples tested in this study is too small to justify making meaningful conclusions. natural infection of sheep with fmdv has been reported by zaikin (1959), littlejohn (1970) and, more recently, by knowles, samuel, davies, kitching & donaldson (2001). the disease in sheep and goats has been reported as occurring frequently in certain provinces of iran (hedjazi et al. 1972) and the frequency of occurrence was associated with the emergence of exotic strains of the virus particularly those strains belonging to sat 1 such as a22 (hedjazi et al. 1972). in the serosurvey reported here, the prevalence of fmd antibodies against sat 1 in goats was significantly higher than that against the other sat serotypes; this observation concurs with the findings of hedjazi et al. (1972). it is on record that goats and sheep become carriers after exposure to fmd virus (burrows 1968; mcvicar & sutmoller 1968, 1972). when investigating the occurrence of carrier state in indigenous goats and sheep in kenya, anderson et al. (1976) found a very low incidence of fmd carriers in goats but none in sheep. no records are available on the occurrence of fmdv carrier states in goats and sheep in botswana. there are also no reports on the occurrence of clinical fmd in these animals in the country. more studies are therefore needed to determine the exact role, if any, of tswana goats and sheep in the epidemiology of fmd in botswana. table 3 distribution of positive goat sera by age group age group number of positive sera relative frequency (%) adult1 35 71.40 young2 14 28.60 total 49 100.00 1 more than one year 2 less than one year table 4 distribution of fmd antibody titres by sat serotypes of fmd virus in indigenous tswana goats in northwestern botswana titre1 number of sera per sat serotype sat 1 sat 2 sat 3 < 1.60 ≥1.60, < 2.00 > 2.00 481 36 9 526 0 0 517 9 0 1 expressed in log10 147 j.m.k. hyera et al. acknowledgements this work was funded by the government of botswana. we thank the veterinary staff from the three districts and those at the nvl for their cooperation and technical assistance. we are grateful to dr j.f.c. nyange (nvl, pathology section) for critically reading the manuscript. the figure was made by mr calistus bodilenyane and mr anthony dingalo of the veterinary epidemiology and economics section of the department of animal health and production. this paper is published with permission of the director of animal health and production, ministry of agri culture, republic of botswana. references anderson, e.c., doughty, w.j. & anderson, j. 1976. the role of goats in the epizootiology of foot-and-mouth disease in kenya. journal of hygiene, 76:395–402. anon. 1996. agricultural statistics. ministry of agriculture, botswa na, gaborone. armitage, p. 1971. statistical methods in medical research, ox ford: blackwell scientific publication. bengis, r.g., thomson, g.r., hedger, r.s., de vos, v & pini, a. 1986. foot-and-mouth disease and the african buffalo (syncerus caffer). carriers as a 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p. 1972. experimental footand-mouth disease in sheep and goats: an epizootiological model. archiv für die gesamte virusforschung, 38:85–96. swinscow, t.d.v. 1980. statistics at square one. london: british medical association. thrusfield, m. 1995. veterinary epidemiology, 2nd ed., london: blackwell scientific publication. zaikin, d.g. 1959. clinical picture of foot-and-mouth disease in sheep under pasture maintenance conditions. veterinariya, 36:32–34. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) maryam kohansal department of medical biotechnology, fasa university of medical science, iran department of biology, payame noor university (pnu), iran ali ghanbari asad department of medical biotechnology, fasa university of medical science, iran citation kohansal, m. & ghanbari asad, a., 2018, ‘molecular analysis of shiga toxin-producing escherichia coli o157:h7 and non-o157 strains isolated from calves’, onderstepoort journal of veterinary research 85(1), a1621. https://doi.org/10.4102/ojvr.v85i1.1621 original research molecular analysis of shiga toxin-producing escherichia coli o157:h7 and non-o157 strains isolated from calves maryam kohansal, ali ghanbari asad received: 27 feb. 2018; accepted: 15 aug. 2018; published: 17 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract shiga toxin-producing escherichia coli (stec) o157 and non-o157 are food-borne pathogens and contaminants of foods of animal origin. this study was conducted to investigate the presence of virulence and integrase genes in stec isolates from diarrhoeic calves in fars province, iran. five hundred and forty diarrheic neonatal calves were randomly selected for sampling. rectal swabs were collected and cultured for isolation and identification of e. coli following standard methods. the isolates were analysed for the presence of class 1 integrons and bacterial virulence factors using polymerase chain reaction (pcr). antimicrobial susceptibility testing was performed using the kirby–bauer disc diffusion method. out of 540 diarrhoeic faecal samples, 312 (57.7%) harboured e. coli and 71 (22.7%) of them were identified as stec: 41(69.5%) carried the stx2 gene, 21 (35.6%) carried the stx1 gene and 3 (5%) carried both. twenty-six (44%) of the isolates showed the eae gene. among the stec isolates examined for susceptibility to eight antimicrobial agents, erythromycin and penicillin (96.8%) resistance were most commonly observed, followed by resistances to ampicillin (71.8%), tetracycline (62.5%) and trimethoprim/sulfamethoxazole (39%). integrons were detected by pcr in 36% of the stec tested isolates, 57 (89%) of which showed resistance to at least three antimicrobial agents. our findings should raise awareness about antibiotic resistance in diarrhoeic calves in fars province, iran. class 1 integrons facilitate the emergence and dissemination of multidrug-resistance (mdr) among stec strains recovered from food animals. introduction new research provides the strongest evidence that shiga toxin-producing escherichia coli (stec) non-o157:h7 and in particular serogroup o157 are linked to severe gastrointestinal diseases (dehkordi et al. 2014). the clinical manifestations of stec infection can vary, from asymptomatic carriage to very serious illnesses such as haemolytic uremic syndrome (hus), thrombocytopenic purpura (ttp) and haemorrhagic colitis (hc) (thomas et al. 2012). estimates vary, but experts suggest that gastrointestinal infections are responsible for approximately 1.5 million deaths per year, over 90% of which are in developing countries (montenegro et al. 2011). for instance, non-o157:h7 serogroups are found in more than 36 000 cases of infections annually and at least 73 000 are infected with o157:h7 serogroup in the united states (us) (zhao et al. 2001). studies have revealed that o157 and non-o157 strains of cattle origin can cause the disease in humans via consumption of raw milk and undercooked meat. in fact, cattle, especially young animals, are known to be the primary reservoirs of both non-o157 and o157 stec (moura et al. 2012). the pathogenicity of stec is associated with shiga toxin (stx) encoded by shiga toxinogenic (stx) genes 1, 2 (stx1 and stx2) and an outer membrane protein which is encoded by the chromosomal eae gene (pradel et al. 2008). the problems with some new stec strains isolated from neonatal calf diarrhoea (ncd) (rigobelo et al. 2008), which is recognised as a disease complex characterised by acute, undifferentiated diarrhoea in newborn calves, are that antibiotic multiresistance (de verdier et. al. 2012) and stec strains can be transmitted to humans by contact occupational exposure and the food chain (schroeder et al. 2002). epidemiological observations show high levels of antimicrobial resistance in bacterial pathogens from veterinary and human medicine (zhao et al. 2001). this has led to the discovery that these bacteria are able to acquire antibiotic resistance by resistance-conferring genes, many of which are carried on transposons, plasmids or integrons (bakhshi, najibi & sepehri-seresht 2014). an integron is mainly composed of an integrase gene that encodes a site-specific recombinase, by which an insertion site of integron is recognised. moreover, an integron contains a variable region which is the place for gene cassettes to be inserted (white et al. 2001). depending on the sequence of the encoded integrases (inti) catalysing excision and integration of deoxyribonucleic acid (dna) units, eight distinct integron classes have been identified up to now, and class 1 integrons have shown to be the major contributors to multidrug-resistant (mdr) infections in the enterobacteriaceae family (singh et al. 2005). many studies in various countries including iran have shown that the distribution of integrons among enteric bacteria has increased over time (eftekhari et al. 2013; gonzalez et al. 1998; hamada, oshima & tsuji 2003; martinez-freijo et al. 1998, 1999; najibi et al. 2012). in iran, only a few studies have reported antimicrobial resistance properties and virulence genes in the pathogenic e. coli (bakhshi et al. 2014; shahrani et al. 2014). unfortunately, there is no conclusive data on the distribution of virulence genes and the antimicrobial resistance properties of stec strains isolated from iran, particularly from fars, which is one of the major agricultural and animal husbandry areas in iran, with nearly 400 000 cattle and 8 000 000 sheep and goats (shams et al. 2012). materials and methods study design and study areas sampling and escherichia coli identification a total of 540 recto-anal mucosal swabs from diarrhoeic calves (< 30 days of age) were collected over 1 year from november 2015 to november 2016. these calves were raised on 33 farms from eight geographic areas in fars province, including industrial, semi-industrial and traditional farms, with a herd size of 25–500 cows. these farms had a recognised scouring problem in neonatal calves. sick calves which showed abnormal faecal consistency and/or signs of dehydration and weakness were selected. none of them had been vaccinated. all samples were immediately placed in cooled boxes and transported to the laboratory. the swab samples were incubated overnight at 37 °c in trypticase soy broth (tsb) (merck kgaa, darmstadt, germany). each sample was then streaked onto macconkey’s agar (mc, merck, germany) (24 hours at 37 °c). lactose positive colonies were cultured on eosin methylene blue agars (emb, merck, germany) (24 h at 37 °c). green colonies with a metallic lustre were considered typical e. coli colonies. such colonies were confirmed as e. coli using standard biochemical tests (citrate utilisation, indole production, glucose, lactose fermentation, urease negative and hydrogen sulphate production). the biochemically confirmed e. coli colonies were subjected to dna analysis. antimicrobial susceptibility and multidrug resistance antimicrobial susceptibility testing against eight antimicrobials was performed on 52 o157 and 12 non-o157 stec isolates using the disk diffusion method on mueller hinton agar plates (merck, germany) based on the clinical and laboratory standards institute (clsi) guidelines (wayne 2012a). the following antibiotics (padtanteb, iran) were applied: chloramphenicol (c: 30 µg), erythromycin (e: 25 µg), ampicillin (am: 10 µg), trimethoprim/sulfamethoxazole (sxt: 30 µg), penicillin (p: 10 µg), enrofloxacin (enr: 10 µg), cefixime (cfm: 5 µg) and tetracycline (tet: 30 µg). the zone diameters were measured (to the nearest millimetre) and interpreted as intermediate (i), susceptible (s) or resistant (r) according to clsi protocol (wayne 2012a); intermediate strains were considered susceptible. based on the definition proposed by an international expert, the mdr phenotype was resistant to three or more antimicrobial classes (magiorakos et al. 2012). e. coli, atcc 25922 (sensitive to all these drugs), recommended by clsi, was used as a quality control. the specified range of quality control result was published in m100-s22 (wayne 2012b). dna extraction a single colony of overnight tsb culture was suspended in 100 µl of distilled water and exposed to boiling for 10 min at 100 °c. after a 13 min freeze, the frozen cell pellets were centrifuged at 14 000 rpm for 10 min (dehkordi et al. 2014) and the supernatant, containing bacterial dna, was subjected to pcr analysis. polymerase chain reaction detection of virulence factors and class 1 integron in shiga toxin-producing escherichia coli strains polymerase chain reaction assays were used to detect the presence of the following virulence genes coding regions including stx1, stx2 and eae. to detect class 1 integron in confirmed stec isolates, a pcr protocol was employed. preparation of the dna samples was done as described in previously published paper (dehkordi et al. 2014). primer sequences, sizes of pcr products and pcr conditions are shown in table 1. dna from e. coli o157:h7 edl933 strain and atcc 25922 strains were used as positive and negative controls, respectively. the amplified dna products were separated by 1.5% agarose gel electrophoresis (sigma-aldrich, st. louis, mo, united states). the gels were stained with ethidium bromide (merek, germany). visualisation of amplified products was done by ultraviolet (uv) illumination and photographed using a kodak camera system (gel logic 200). table 1: primers and polymerase chain reaction conditions used in this study. statistical analysis the chi-square (χ2) test and fisher’s exact test were used to assess whether integron-positive strains were significantly more resistant than integron-negative strains for each of the tested antibiotics. a p value < 0.05 was considered statistically significant. statistical calculations were made using graphpad prism for windows version 5 (graphpad software, san diego, ca). results isolation and characterisation of shiga toxin-producing escherichia coli in calves from 540 diarrhoeic calves, 312 samples (57.7%) were positive for e. coli. shiga toxin–producing escherichia coli strains were isolated from 71 (22.7%) out of the 312 samples, which possess stx1 and/or stx2. twelve (3.57%) isolates were classified as e. coli o157:h7 and 59 (31.19%) as non-o157. characterisation of virulence genes of 312 e. coli strains, 71 isolates (22.7%) were identified as stec. the virulence genes stx2, stx1 and eae were detected at 76%, 46.4% and 53.5% in stec isolates, respectively. of isolates that were not characterised as stec, 101 (32.3%) were positive for eae gene (figures 1 and 2). these findings are summarised in table 2. out of 59 non-o157 strains (pnu6, pnu11, pnu12 and pnu16) in the diarrhoeic calves, four were positive for both the stx1 and stx2 genes and three non-o157 harboured all of the stx1, stx2 and eae genes. table 2: distribution of virulence genes in shiga toxin-producing escherichia coli strains. figure 1: agarose gels electerophoresis of shiga toxin-producing escherichia coli isolates. (a) polymerase chain reaction amplification of the stx1 (551 bp) and stx2 (118 bp) genes. lanes 1–3, stx1; lanes 1–3 and 5, stx2 and (b) polymerase chain reaction amplification of the eae gene (840 bp) (lanes 1–4, 6 and 7). lane m, 100 bp molecular size markers; lanes cand c+, negative and positive control. figure 2: frequency of occurrence of tested virulence genes in 71 stec strains. antibiotic susceptibility the antimicrobial susceptibility of 52 non-o157 and 12 o157 stec isolates was determined by the disk diffusion method. the resistance patterns of the e. coli o157 strains were to penicillin and ampicillin (91% – 8%), followed by tetracycline, erythromycin and cefixime (66% – 25%). nine (75%) of the 12 o157 strains exhibited multidrug resistance (mdr, resistant to ≥ 3 antimicrobial classes). the most common mdr phenotypes were am-e-p-tet, which accounted for 15% of the 12 o157 strains. all of the examined non-o157 strains showed resistance to trimethoprim/sulfamethoxazole. the resistance patterns of all non-o157 strains to tested antibiotics were as follows: erythromycin (98%), penicillin (91%), ampicillin (73%), tetracycline (65%), chloramphenicol (40%), cefixime (25%) and enrofloxacin (21%). forty-eight (92%) of the 52 non-o157 strains displayed multidrug resistance. the most frequently observed mdr profiles am-c-e-p-tet-enr–sxt (33% of the 52 non-o157 strains) were associated with 10 of these isolates (pnu4, pnu29, pnu31, pnu33, pnu38, pnu41, pnu49, pnu50, pnu51, pnu52). the resistance patterns of 64 stec isolates are shown in table 3 and figure 3. figure 3: antimicrobial susceptibility patterns in 71 shiga toxin-producing escherichia coli strains. table 3: antibiotic resistance pattern in shiga toxin–producing escherichia coli strains. integrons class 1 integrons were detected among 23 (36%) of the stec isolates (figure 4 and table 4). integron-positive strains were significantly more resistant to enrofloxacin, trimethoprim/sulfamethoxazole and tetracycline than integron-negative strains (p < 0.05). nevertheless, resistance to ampicillin, erythromycin, penicillin, cefixime and chloramphenicol could not be directly related to the presence of integrons (table 5). all of the integron-positive strains displayed multidrug resistance. the most prevalent mdr phenotypes in integron-positive strains were am-cfm-e-p-tet (26% of the 52 non-o157 strains). figure 4: polymerase chain reaction amplicons of shiga toxin-producing escherichia coli integrons. polymerase chain reaction amplification of the class 1 integron, integrase. bp, base pair; int1, integrase gene. table 4: overview of the integron-positive shiga toxin–producing escherichia coli strains. table 5: comparison of the resistances between integron-positive and integron-negative strains was done using the p-values listed in the table. discussion antibiotic resistance developed in stec isolates from humans and animals (van meervenne et al. 2013). integrons, which are known to be associated with many antimicrobial resistance genes, were suspected to serve as pools of antimicrobial resistance genes worldwide (el-sokkary & abdelmegeed 2015). class 1 integrons are commonly found in gram-negative pathogens (maguire et al. 2001). in this study, the presence of major virulence factors and resistance to antimicrobials belonging to classes generally utilised in iran was investigated in zoonotic stec isolates from calves with diarrhoea. owing to the close contact of humans with animals, the presence of virulence and antimicrobial resistance genes in e. coli strains harboured by animals leads to public health concerns (torkan et al. 2016). escherichia coli, which has been implicated as an aetiological factor of calf diarrhoea, harbours many virulence genes that enable it to cause disease in a particular host (nagarjuna et al. 2015). in the present study, among 312 e. coli strains from diarrhoeic calves, 71 (22.7%) were stec. the results are in agreement with those of dastmalchi et al. (2012), who screened 51 e. coli isolates from diarrhoeic calves in the urmia region, which is located in west azerbaijan province, iran, and illustrated that 19.6% of isolates were stx positive. most epidemiological studies in diarrhoeic calves in iran have disclosed that the prevalence of stec infection ranges between 6.4% and 34.5% (pourtaghi, dahpahlavan & momtaz 2013; shahrani et al. 2014). these discrepancies can be attributed to the small sample size and geographical differences. in other words, stec prevalence in calves may be influenced by environmental factors (dastmalchi et al. 2012). higher prevalence of the stx2 gene (54 isolates) compared to the stx1 gene (33 isolates) in this study corroborates the findings of previous reports in iran (dastmalchi et al. 2012; tahamtan, hayati & namavari 2010). however, these results contrast with other reports that have shown that most stec from diarrhoeic calves only produce stx1, whereas stx2-positive strains are the dominant types in healthy calves (nguyen, vo & vu-khac 2011). the differences in these findings suggest that stx2 may be associated with a majority of e. coli isolates from diarrhoeic calves in iran. shiga toxin producing e. coli infection, which is associated with diarrhoea in calves, may result in severe diseases in humans such as hus and hc (bastos et al. 2006). the diarrhoeal phase of diseases associated with stec is usually self-limiting, and the role of early antimicrobial treatment in the prevention of hus is still regarded as controversial (shahrani et al. 2014). current recommendations and the available data suggest that not only do antibiotic exposure increase the risk of hus in children via inducing expression of stx through replication of temperate bacteriophages carrying stx-encoding genes (ochoa et al. 2007), it turns out to have another perilous effect on the frequency of stec antimicrobial resistance (shahrani et al. 2014), which could result in an increase of frequency of stec and perhaps greater shedding. resistance could contribute to greater contamination of animal food products with stec (torkan et al. 2016). several reports have documented that a significant increase of antimicrobial resistance in stec strains isolated from animals and humans has acquired antibiotic resistance genes almost 20 years ago (zhao et al. 2001). in stec strains, class 1 integrons are strongly associated with multidrug resistance (colello et al. 2015). previous studies have reported the occurrence and prevalence of class 1 integrons to be ranging from 2.7% to 41.0% among stec isolates in germany (askar et al. 2011), argentina (colello et al. 2015), belgium (van meervenne et al. 2013), north america (nagachinta & chen 2009), brazil (cergole-novella et al. 2011) and us (singh et al. 2005; zhao et al. 2001). class 1 integrons appear to be common in the endemic stec strains. in the present study, class 1 integron was identified in 23 (36%) out of 71 stec isolates. our data revealed low distribution of class 1 integrons among stec isolates from calves with diarrhoea in the south of iran compared with a similar study in northern iran in 2014 for which the authors found a higher percentage (53%) of the strains containing integron class 1 (bakhshi et al. 2014). the various percentages of class 1 integrons in different parts of the world could be attributed to the characteristics of the analysed collection and differences in the prevalence of antibiotic consumption in each country (kargar et al. 2014). in general, exposure to antibiotics, heavy metals or biocides and a high multiplicity of other different environmental factors are among the main reasons for an increase of cells containing integrons (baquero, martínez & cantón 2008). all integron-positive strains examined in this study were resistant to at least three different antibiotics (mdr). similarly, high percentages of mdr phenotypes among integron-positive stec strains have been reported in argentina (nagachinta & chen 2009) and iran (bakhshi, najibi & sepehri-seresht 2014). however, other authors (colello et al. 2015; van meervenne et al. 2013) have reported a lower rate (less than 90%) of stec in diarrhoeic calves. the highest resistances among the integron-positive strains were found to enrofloxacin (17%), trimethoprim/sulfamethoxazole (39%) and tetracycline (62%). the integron-positive strains were significantly more resistant to these antibiotics than the integron-negative strains. the resistance to enrofloxacin (enr), trimethoprim/sulfamethoxazole (sxt) and tetracycline (tet) is related to the presence of the integron. the significant association between resistance to fluoroquinolones, tetracycline, trimethoprim and sulfonamides (enr, tet, sxt) tested and integron existence could be explained because of the fact that many fluoroquinolone, tetracycline, trimethoprim and sulfonamide resistant genes have been reported within integron structures, including gyra, gyrb, qnr, teta, tetb, tetc, tetd, sul1, sul2, sul3 and dfra1 (kaplan et al. 2013; wang et al. 2010). conclusion we report the presence of class 1 integrons in the most familiar stec strains from diarrhoeic calves. results imply that stx2, stx1 and eae putative virulence gene, the inti integrase gene and resistance to erythromycin, penicillin, ampicillin, tetracycline and trimethoprim/sulfamethoxazole were the most commonly detected characteristics of the stec strains isolated from diarrhoeic calves in southern iran. our investigation demonstrated that calves are possible reservoirs of stec strains and developed resistance to multiple classes of antimicrobials. emerging data suggest an association between mdr and integrons which may play a significant role in the dissemination of resistance genes. therefore, it is advised to stop routine antimicrobial treatment and conduct further molecular studies to detect other antimicrobial resistance and virulent genes in stec isolates obtained in this study. acknowledgements 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entomology, rhodes university, south africa citation horak, i.g., pearcy, a. & lloyd, k.j., 2017, ‘parasites of domestic and wild animals in south africa. li. ticks infesting leopard tortoises stigmochelys pardalis, hingeback tortoises kinixys zombensis and angulate tortoises chersina angulata’, onderstepoort journal of veterinary research 84(1), a1303. https://doi.org/10.4102/ojvr.v84i1.1303 original research parasites of domestic and wild animals in south africa. li. ticks infesting leopard tortoises stigmochelys pardalis, hingeback tortoises kinixys zombensis and angulate tortoises chersina angulata ivan g. horak, ashley pearcy, kyle j. lloyd received: 22 june 2016; accepted: 30 aug. 2016; published: 28 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the objective of the study was to record the tick species collected from three species of tortoise, each in a different province of south africa. ticks were collected from leopard tortoises, stigmochyles pardalis, in the southern region of the kruger national park, mpumalanga province; from hingeback tortoises, kinixys zombensis, in the enseleni nature reserve, kwazulu-natal province and from angulate tortoises, chersina angulata, in the west coast national park, western cape province. of the 63 leopard tortoises examined, 58 were infested with amblyomma marmoreum and 49 with amblyomma hebraeum, and all stages of development of both species were recovered. amblyomma nuttalli was collected from 25 hingeback tortoises, and all stages of development were present. all 24 angulate tortoises examined were infested with amblyomma sylvaticum, and large numbers of larvae, nymphs and adults were collected. three snake species and a sand lizard were also infested with a. sylvaticum. the adults of a. marmoreum, a. nuttalli and a. sylvaticum were identified as specific parasites of the family testudinidae, whereas all stages of development of a. hebraeum were classified as generalists. introduction the history of ticks on tortoises in south africa dates back to the 18th century. the first tick with a strictly south african distribution to be described was collected from a tortoise by sparrman during his travels in the cape (theiler 1943). this new species was described as acarus sylvaticus (now known as ablyomma sylvaticum) by de geer in 1778. ticks on tortoises were again reported by arbousset and daumas in the 1846 account of their exploratory tour of the cape of good hope (walker & schulz 1984). the majority of ticks that have been documented on tortoises in south africa belong to four species of amblyomma and all are ornate. theiler (1943) published illustrations and a description of amblyomma sylvaticum, and theiler and salisbury (1959) illustrated and described ticks of ‘the amblyomma marmoreum group’, including amblyomma nuttalli and a. marmoreum, whereas walker and olwage (1987) have produced colour illustrations of amblyomma hebraeum and a. marmoreum. the sexes of the four amblyomma species are dimorphic and the males are the most brightly coloured. within south africa, a. marmoreum is the most widespread of the four species and is present in all nine provinces, although only patchily in some (horak et al. 2006a). it is most prevalent in the fynbos, albany thicket and savanna biomes and the eastern region of the nama karoo biome. relatively few collections have been made in the large grassland biome of south africa. although a. hebraeum is not regarded as a tick of tortoises, it is regularly found on leopard tortoises within its own distribution range from the albany thicket biome in the south of the eastern cape province through the savanna biome of the east and northern regions of the country to the eastern region of north west province (spickett 2013). the third species, a. nuttalli, is widespread in africa, but has a distribution virtually restricted to the indian ocean coastal belt biome in north-eastern kwazulu-natal (horak et al. 2006b). the core distribution of the fourth species, a. sylvaticum, lies within the fynbos biome of the western and northern cape provinces (horak et al. 2006b). extensive host records for a. marmoreum have been provided by horak et al. (2006a). all stages of development infest leopard tortoises, stigmochyles pardalis, as well as various other tortoise species, whereas the larvae, and to a lesser extent nymphs, infest carnivores, equids, bovids, leporids and ground-frequenting birds. the usual hosts for all stages of development of a. hebraeum are large herbivores, whereas the larvae and nymphs also infest small herbivores, carnivores, hares and ground-frequenting birds (horak et al. 1987). walker and schulz (1984) collected a. hebraeum nymphs from 13 of 29 leopard tortoises and from 4 of 7 angulate tortoises, chersina angulata examined in the southern region of the albany thicket biome, whereas dower, petney and horak (1988) collected a. hebraeum nymphs and a few adults from 14 leopard tortoises in the same biome. walker and schulz (1984) also collected the adults of a. hebraeum from a leopard tortoise in the savanna biome in north-eastern kwazulu-natal. horak et al. (2006b) recorded nine males of a. nuttalli on five of seven hingeback tortoises, kinixys zombensis, examined in the indian ocean coastal belt biome. they also recorded fairly large numbers of a. sylvaticum in all stages of development on 124 of 138 angulate tortoises and a few on other tortoise species examined in the fynbos biome (horak et al. 2006b). norval (1975) studied the life cycle of a. marmoreum in the laboratory, feeding all stages of development on tortoises. including the time the ticks spent off-host while moulting and laying eggs, and using the maximum period of feeding for each life stage, the life cycle took 242 days to complete. however, when the larvae and nymphs were fed on sheep, the life cycle could be completed in 193 days. amblyomma marmoreum larvae detached from naturally infested tortoises between 8 and 104 days after capture, nymphs between 4 and 47 days, males between 1 and 111 days and females between 1 and 73 days (dower et al. 1988). norval (1974) also studied the life cycle of a. hebraeum in the laboratory, feeding the larvae on rabbits, the nymphs on sheep and the adults on calves. on these hosts, the life cycle was completed between 114 and 165 days, with the larvae feeding for 4–15 days before detaching, the nymphs for 6–9 days and the females for 6–12 days. a single female tick laid 18 765 eggs (norval 1974). however, on naturally infested tortoises, a. hebraeum nymphs spent 1–125 days (mean 40 days) before detaching, males 14–92 days (mean 56 days) and females 15–77 days (mean 49 days) (dower et al. 1988). aeschlimann (1967) fed all stages of development of a. nuttalli on tortoises, and the life cycle took between 130 and 196 days to complete. according to him, santos dias in mozambique counted 22 891 eggs laid by a single female tick. no studies have been conducted on the life cycle of a. sylvaticum. fielden and rechav (1994) recorded most a. marmoreum larvae (49.6%) on the head and neck of leopard tortoises, followed by the anterior legs and the anterior and posterior armpits. most nymphs attached to the head and neck (53.6%), followed by the anterior legs. most males attached to the posterior armpit (35.3%), followed by the posterior legs and around the base of the tail and on the tail, and most females attached around the base of the tail and on the tail (47.8%), followed by the posterior legs and the posterior armpit. pearcy and beyer (2013) report that the larvae and nymphs of a. sylvaticum attach to the soft tissue around the base of the neck and the sockets of the front and hind limbs of angulate tortoises. most males attach on the outer shell between the scutes and the remainder on the body of the tortoise, whereas approximately equal numbers of females attach to the shell and to the body. there are no attachment data for a. nuttalli. the aim of this study was to record collections of ticks from tortoises subsequent to the publications of horak et al. (2006a, 2006b). here we report on collections of a. marmoreum and a. hebraeum from leopard tortoises, of a. nuttalli from hingeback tortoises and of a. sylvaticum from angulate tortoises and explore the possibility of host specificity amongst the four ticks. the likelihood of transmission of diseases to domestic livestock is also discussed. methods ticks were collected by a.p. (university of witwatersrand) from a single speke’s hingeback tortoise, kinixys spekii, and 63 leopard tortoises between skukuza, lower sabie and pretoriuskop in the southern kruger national park between september 2011 and february 2013. ticks were also collected by a.p. from 24 angulate tortoises at duinepos in the west coast national park over a 5-day period between 27 and 31 october 2012 (pearcy & beyer 2013). the sites of attachment of all stages of development on the angulate tortoises were recorded. ticks were collected by k.j.l. (rhodes university) from 25 hingeback tortoises in the enseleni nature reserve in north-eastern kwazulu-natal between 03 december 2012 and 28 january 2013. the ticks collected from each animal were placed in separate vials containing 70% ethyl alcohol with a pencil-written label providing collection data. the ticks were identified and counted by i.g.h. (university of pretoria) using a stereoscopic microscope. the tortoise species sampled, and the species and numbers of ticks recovered have been summarised in table 1. table 1: ixodid ticks collected from three tortoise species in three regions of south africa. cumming (1998) considered that an afrotropical ixodid tick had to infest at least 50 host species to be considered a generalist. for a tick to be considered a specialist at least 10 collections had to be made and at least 90% of all collections had to come from a single taxon. using these criteria, cumming (1998) classified a. hebraeum as a generalist. we, however, decided to decrease the number of host species infested to 20 for a tick to be considered as a generalist, instead of the 50 species cumming suggested for the whole of africa. we only consider ticks as specialists if at least 20 collections had been made and at least 90% of all collections were made from a single taxon. furthermore, animals of at least four other species unrelated to the taxon of specificity were examined at the same locality or localities. ethical considerations the south african national parks research committee approved the projects for the capture of tortoises and removal of ectoparasites. fieldwork in the enseleni nature reserve was conducted under a permit issued by ezemvelo kzn wildlife (scrp151) and rhodes university ethical standards committee (zool-16-2012). results fifty-eight out of 63 leopard tortoises examined in the southern kruger national park were infested with a. marmoreum and 49 with a. hebraeum (table 1). one tortoise was not infested with either species, whereas 49 were infested with both, 13 with a. marmoreum only and four with a. hebraeum only. fifty-six tortoises were infested with the adults of a. marmoreum and eight with the adults of a. hebraeum. amblyomma marmoreum males outnumbered females by 4.4 to 1. all 25 of the hingeback tortoises examined in the enseleni nature reserve were infested with a. nuttalli. only one larva was collected, and male ticks outnumbered females by 4.8 to 1 (table 1). a single a. nuttalli male was collected from a speke’s hingeback tortoise in the southern kruger national park. the 24 angulate tortoises examined in the west coast national park were all infested with a. sylvaticum, and substantial numbers of larvae, nymphs and male ticks were collected. males outnumbered females by 4.6 to 1 (table 1). three species of snake and a sand lizard were also infested with a. sylvaticum. according to our adaptation of cumming’s approach, and making use of all the data available to us from horak and his co-worker’s past surveys, the adults of a. marmoreum, a. nuttalli and a. sylvaticum are specialists on the family testudinidae, whereas the immature stages of a. marmoreum and all stages of development of a. hebraeum are generalists. more than 95% of collections of adult a. marmoreum were made from testudinidae, whereas several species within six other families, unrelated to testudinidae, were examined for ticks in the same localities. besides testudinidae, the immature stages of a. marmoreum were collected from more than 60 other species unrelated to testudinidae. more than 95% of collections of adult a. nuttalli were made from testudinidae, whereas nyalas (tragelaphus angasii), bushbuck (tragelaphus scriptus), red duikers (cephalophus natalensis) and bushpigs (potamochoerus larvatus) were examined within the same region (horak, boomker & flamand 1991, 1995). more than 95% of collections of adult a. sylvaticum were made from testudinidae, whereas eland (tragelaphus oryx), gemsbok (oryx gazella), bontebok (damaliscus pygargus pygargus), springbok (antidorcas marsupialis) and hyraxes (procavia capensis) were examined in the same national park as the angulate tortoises from which a. sylvaticum was collected (golezardy & horak 2007). the adults of a. hebraeum were collected from more than 25 species of mammals and the immature stages from more than 40 species of mammals as well as from five species of birds. discussion amblyomma marmoreum horak et al. (2006b) recorded 80 collections of a. marmoreum from 82 leopard tortoises, and we now add a further 58 collections from 63 leopard tortoises. the comparatively small number of larvae and nymphs recovered is not unusual. even with careful scrutiny, the numbers of larvae and nymphs on leopard tortoises were underestimated by rechav and fielden (1995). these stages, and especially the larvae, infest leopard tortoises, other tortoise species, several reptile species and a variety of other hosts (horak et al. 2006a). very few adult ticks have been recovered from any hosts other than tortoises, and using a modified version of cumming’s (1998) approach, we have classified the adults as specialists on testudinidae, whereas the immature stages are generalists. amongst the testudinidae, more collections of a. marmoreum adults have been made from leopard tortoises than from any other species of tortoise (horak et al. 2006b; table 1). rechav and fielden (1995) reported that the male to female ratio of a. marmoreum adults differed depending on the season during which leopard tortoises were examined in the national zoological gardens, pretoria. they found that although there was an increase in numbers of both sexes between september and october (spring), the male to female ratio was constant at approximately 2:1, whereas in april (autumn) females were virtually absent and the ratio was approximately 20:1. the ratio of males to females on leopard tortoises examined in the albany thicket biome in the addo elephant national park during september and october was 1.8:1 (walker & schulz 1984), 2.8:1 on tortoises captured between february and may in the same biome close to grahamstown (dower et al. 1988), 4.1:1 on tortoises examined during february in the bontebok national park in the fynbos biome in the western cape province (horak & boomker 1998) and 4.4:1 on the tortoises examined in this study. these differences could be because of seasonal fluctuations in the abundance of male and female ticks, differences in the length of time that males or female ticks remained attached to their tortoise hosts, or because of the diligence with which ticks were collected. bezuidenhout (1988) demonstrated experimentally that leopard tortoises could be infected with ehrlichia ruminatium, the causative organism of heartwater in domestic and wild ruminants, and that a. marmoreum could acquire infection from an infected tortoise. later peter, burridge and mahan (2000) showed that a. marmoreum larvae could acquire infection from goats with clinical heartwater and that after moulting the nymphs could transmit infection to susceptible sheep. the larvae of a. marmoreum are generalists and naturally feed on a large variety of animals (horak et al. 2006a). it is thus possible that they could acquire infection while feeding on an infected ruminant, and the ensuing nymphs transmit infection to a susceptible animal. it would, however, appear that few nymphs infest domestic or wild ruminants (horak et al. 1987). consequently, the role that a. marmoreum might play in the epidemiology of heartwater in the field has yet to be determined. amblyomma hebraeum the adults of a. hebraeum are generalists, and besides tortoises, they infest equids, suids and large bovids (horak et al. 1987). the immature stages are also generalists and infest the same hosts as the adults, but in addition feed on leporids and ground-frequenting birds (horak et al. 1987). however, few larvae or adult ticks are found on tortoises (horak et al. 2006b; table 1). cumming (1998) also classified a. hebraeum as a generalist but without discriminating between the immature stages and the adults. the larvae, whose mouthparts are long and relatively slender, possibly do not infest tortoises because their skins are too impenetrable (the mouthparts of a. marmoreum larvae are shorter and more robust than those of a. hebraeum). adult ticks possibly do not infest tortoises because they prefer warm-blooded hosts. too few adult ticks were collected to determine a meaningful sex ratio. amblyomma hebraeum is the most effective vector of e. ruminantium in south africa. infection can be acquired by larvae feeding on an infected host and transmitted by the ensuing nymphs, or acquired by nymphs and transmitted by adult ticks. if indeed tortoises are naturally infected with e. ruminantium, infection could be acquired from them by nymphs of a. hebraeum and transmitted to susceptible livestock by the ensuing adults. whether this occurs in nature has yet to be proved. amblyomma nuttalli despite extensive surveys conducted on a variety of host species in many of the national and provincial nature reserves, including north-eastern kwazulu-natal, horak and his co-workers had never collected a. nuttalli (horak et al. 1991, 1995), whose adults we regard as specialists on testudinidae. amblyomma nuttalli is widespread in the afrotropical region (burridge 2001), whereas its core distribution in south africa is north-eastern kwazulu-natal, where theiler (1962) records 10 collections. aeschlimann (1967) reports that the distribution of a. nuttalli is associated with forests in côte d’ivoire. with the exception of the knysna and alexandria forests biomes in the western and eastern cape provinces, the indian ocean coastal belt biome in north-eastern kwazulu-natal is possibly the closest to a forest habitat in south africa. extralimittally adult and immature ticks have been recovered from tortoises, varanid lizards, a python and from puffadders and gaboon adders, whereas the immature stages have been collected from small forest dwelling antelopes, reptiles and ground-frequenting birds (aeschlimann 1967; burridge 2001; theiler 1962). amblyomma sylvaticum of the four amblyomma species, only a. sylvaticum has a strictly south african distribution, and even in south africa, its distribution is confined to the coastal and adjacent inland regions of the northern, western and western region of the eastern cape provinces. although angulate tortoises appear to be the hosts of choice for all stages of development, a. sylvaticum is known to infest other tortoise species, and the immature stages infest lizards and snakes (horak et al. 2006b; walker 1991; table 1). amongst the testudinidae, more collections of the adults of a. sylvaticum have been made from angulate tortoises than from any other species of tortoise (horak et al. 2006b). walker (1991) reports a collection of nymphs and adult ticks from a mole snake, pseudaspis cana and here we record a collection of all stages of development from this snake. mole snakes are large and sturdy and live underground in abandoned animal burrows and are prone to long fasts (branch 1998). it is perhaps this behaviour that renders them more prone to infestation with adult ticks than other snakes. general with the exception of a. hebraeum, of which too few adult ticks were collected, the male to female ratios presently recorded for the three other amblyomma spp. were remarkably similar. the ratio for a. marmoreum was 4.4:1, for a. nuttalli 4.8:1 and for a. sylvaticum 4.6:1. the similarity might be fortuitous but generally reflects the fact that male ticks remain attached for longer than females (dower et al. 1988; norval 1975), leading to a preponderance of males. the gender ratios also affirm that the collections were thorough, in that female ticks are generally larger than males, and hence, they should theoretically be easier to see and collect. however, the high ratio of males to females reflects careful collection of the smaller males. conclusion four of the nine amblyomma species that are present in south africa infest tortoises. leopard tortoises are one of the preferred hosts of a. marmoreum adults, hingeback tortoises are one the preferred hosts of a. nuttalli adults, and angulate tortoises are one the preferred hosts of a. sylvaticum adults. the adults of these three species are specialist parasites of the family testudinidae. the fourth species, a. hebraeum, which usually infests warm-blooded animals, frequently infests leopard tortoises within its own distribution range, and its adults and immature stages are generalists. acknowledgements the authors express their sincere thanks to sanparks for permission to conduct studies on tortoises in the kruger national park and the west coast national park, and to ezemvelo kzn wildlife for permission to study the ecology of hingeback tortoises in the enseleni nature reserve and to collect ticks from them. the participation of the senior author in the project was partially funded by a grant from the national research foundation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors read and approved the manuscript. a.p. collected ticks from leopard tortoises and from angulate tortoises and ascertained the sites of attachment of ticks on angulate tortoises. k.j.l. collected ticks from hingeback tortoises, and i.g.h. identified all the ticks and compiled the first draft of the manuscript. references aeschlimann, a., 1967, ‘biologie et écologie des tiques (ixodoidea) de côte d’ivoire’, acta tropica 24, 281–405. bezuidenhout, j.d., 1988, ‘sekere aspekte van hartwateroordraging, voorkoms van die organisme in bosluise en in vitro kweking’, dvsc thesis, university of pretoria. branch, w.r., 1998, field guide to snakes and other reptiles of southern africa, struik publishers, cape town. burridge, m.j., 2001, ‘ticks (acari: ixodidae) spread by the international trade in reptiles and their potential roles in dissemination of diseases’, bulletin of entomological research 91, 3–23. cumming, g.s., 1998, ‘host preference in african ticks (acari: ixodida): a quantitative data set’, bulletin of entomological research 88, 379–406. https://doi.org/10.1017/s0007485300042139 dower, k.m., petney, t.n. & 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entomology 32, 161–165. https://doi.org/10.1093/jmedent/32.2.161 spickett, a.m., 2013, ixodid ticks of major economic importance and their distribution in south africa, agri connect, pretoria. theiler, g., 1943, ‘ticks in the south african zoological survey collection. part ii’, onderstepoort journal of veterinary science and animal industry 18, 85–89. theiler, g., 1962, the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region), project s 9958, report to the director of veterinary services, onderstepoort, mimeographed. theiler, g. & salisbury, l.e., 1959, ‘ticks in the south african zoological survey collection. part ix. “the amblyomma marmoreum group”’, onderstepoort journal of veterinary research 28, 47–124. walker, j.b., 1991, ‘a review of the ixodid ticks (acari: ixodidae) occurring in southern africa’, onderstepoort journal of veterinary research 58, 81–105. walker, j.b. & olwage, a., 1987, ‘the tick vectors of cowdria ruminantium (ixodoidea, ixodidae, genus amblyomma) and their distribution’, onderstepoort journal of veterinary research 54, 353–379. walker, j.b. & schulz, k.c.a., 1984, ‘records of the bont tick, amblyomma hebraeum, from the angulate tortoise, chersina angulata, and the leopard tortoise, geochelone pardalis’, onderstepoort journal of veterinary research 51, 171–173. abstract introduction materials and methods results discussion acknowledgements references appendix 1 about the author(s) timothy y. woma department of veterinary tropical diseases, university of pretoria, south africa morbilliviruses research laboratory, national veterinary research institute, nigeria pius s. ekong morbilliviruses research laboratory, national veterinary research institute, nigeria dauda g. bwala morbilliviruses research laboratory, national veterinary research institute, nigeria john o. ibu morbilliviruses research laboratory, national veterinary research institute, nigeria louisa ta’ama morbilliviruses research laboratory, national veterinary research institute, nigeria dyek y. dyek morbilliviruses research laboratory, national veterinary research institute, nigeria ladi saleh morbilliviruses research laboratory, national veterinary research institute, nigeria david shamaki morbilliviruses research laboratory, national veterinary research institute, nigeria demo j.u. kalla animal production programme, abubakar tafawa balewa university, nigeria dalan bailey school of immunity and infection, university of birmingham, united kingdom haruna m. kazeem department of veterinary microbiology, ahmadu bello university zaria, nigeria melvyn quan department of veterinary tropical diseases, university of pretoria, south africa citation woma, t.y., ekong, p.s., bwala, d.g., ibu, j.o., ta’ama, l., dyek, d.y. et al., 2016, ‘serosurvey of peste des petits ruminants virus in small ruminants from different agro-ecological zones of nigeria’, onderstepoort journal of veterinary research 83(1), art. #1035, 9 pages. http://dx.doi.org/10.4102/ojvr.v83i1.1035 original research serosurvey of peste des petits ruminants virus in small ruminants from different agro-ecological zones of nigeria timothy y. woma, pius s. ekong, dauda g. bwala, john o. ibu, louisa ta’ama, dyek y. dyek, ladi saleh, david shamaki, demo j.u. kalla, dalan bailey, haruna m. kazeem, melvyn quan received: 05 aug. 2015; accepted: 14 oct. 2015; published: 11 mar. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract peste des petits ruminants, caused by the peste des petits ruminants virus (pprv), is a highly contagious and economically important transboundary viral disease of domestic and wild small ruminants and a major hindrance to small-ruminant production in nigeria. the seroprevalence and distribution of pprv antibodies in small ruminants in rural households, farms, live animal markets and slaughter slabs across the six different agro-ecological zones of nigeria were determined. a total of 4548 serum samples from 3489 goats and 1059 sheep were collected in 12 states. a pprv competitive enzyme-linked immunosorbent assay was used to test the samples and the data analysed with r statistical software version 3.0.1. the study animals included all ages and both sexes. the overall prevalence estimate of sera positive for pprv antibodies was 23.16% (n = 1018 positive samples per 4548 total samples, 95% confidence interval: 21.79% – 24.57%). there were significant differences in the seroprevalence between the states (p = 0.001). taraba state had the highest seroprevalence of 29.51%, whilst the lowest seroprevalence of 14.52% was observed in cross river state. there were no significant differences in the pprv seroprevalence between male and female animals (p = 0.571), age (p = 0.323) and between species (p = 0.639). these data indicate the current seroprevalence to pprv in the small-ruminant population in nigeria. introduction peste des petits ruminants (ppr) is a highly contagious and economically important transboundary viral disease of domestic and wild small ruminants (balamurugan et al. 2010). outbreaks of ppr occur regularly in small ruminants throughout nigeria and are characterised by pyrexia, depression, anorexia, diarrhoea, respiratory distress, mucopurulent oculo-nasal discharge with matting of the eyelids, necrotic oral lesions that produce a foetid smell and sometimes abortion in pregnant animals. the disease is caused by the peste des petits ruminants virus (pprv), which is classified in the genus morbillivirus within the family paramyxoviridae (king et al. 2012). there are many documented reports of the endemic nature of pprv in nigeria (el-yuguda et al. 2010; emikpe & akpavie 2010; ezeibe et al. 2008; ibu et al. 2008; isoun & mann 1972; obi et al. 1983; obidike et al. 2006; odo 2003; ogunsanmi et al. 2003; okoli 2003; shamaki 2002; taylor & abegunde 1979; ularamu et al. 2012). outbreaks have continued to occur in the country during the last 40 years, despite the introduction of the tissue culture rinderpest vaccine against pprv in the 1980s and the use of the homologous pprv vaccine (diallo 2003; diallo et al. 1989) since 1998. the national veterinary research institute vom, nigeria, produces a 50-dose pprv vaccine vial using the nigeria 75/1 strain, with the recommendations that small ruminants must be vaccinated from 3 months of age and thereafter every 12 months. small ruminants play an important role in agricultural food production and in sustainable employment in nigeria and the control and eradication of ppr is therefore a priority, in order to ease poverty and improve the health and husbandry of animals kept by resource-poor people in this developing country. this study was designed to determine the seroprevalence and distribution of pprv antibodies in small ruminants in rural households, farms, live animal markets and slaughter slabs across different states in all the agro-ecological zones of nigeria. this information will be helpful to develop a progressive control programme with the aim to eradicate ppr from nigeria. materials and methods study area each agro-ecological zone consists of administrative structures called states, subdivided into local governments. two states were selected at random from each agro-ecological zone: the north-eastern agro-ecological zone is montane savannah and samples were collected from adamawa and taraba states; the south-eastern agro-ecological zone is tropical rain forest and samples were collected from anambra and imo states; the south-southern agro-ecological zone is mangrove or swamp and samples were collected from akwa ibom and cross river states; the north-central agro-ecological zone is guinea or derived savannah and samples were collected from kwara and plateau states; the north-western agro-ecological zone is sudan and sahel savannah and samples were collected from kano and sokoto states; the south-western agro-ecological zone is tropical rain forest and samples were collected from ogun and ondo states (iloeje 2001). sample size and sample collection the openepi v2 open-source calculator, sspropor (kevin, andrew & minn 2009) was used to calculate the total number of samples to be collected per state using the equation: sample size (n) = deff × np(1 − p)/[d2/z21 − α/2 × (n − 1) + p(1 − p)]    [eqn 1] where deff = design effect for cluster surveys, n = population size, p = hypothesised % frequency of outcome factor in a population and d = confidence limits as ± %. the calculated sample size per state was 379 (table 1). table 1: apparent and true prevalence of peste des petits ruminants virus antibodies in small ruminants in nigeria, 2010–2013. the combined population of domestic small ruminants in nigeria in 2007 was estimated at 90 million (faostat 2007). nigeria has a 36-state structure, and a figure of 2 000 000 small ruminants per state was estimated for sample size calculations (in effect an infinite population). a percentage frequency of outcome factor in the population was hypothesised to be 44% ± 5% based on a study by shamaki (2002). a confidence limit of 5% and design effect of one was used. multistage sampling was performed with four hierarchical stages. the first level of selection was the state. two states were selected randomly from each agro-ecological zone of the country. within each state, local governments, districts and villages were selected randomly. within each selected village, animals were sampled from different homes, from the live animal market and from the slaughter slabs. these were selected purposely because of logistical constraints. about 5 ml of blood was collected by venipuncture. sera were separated within 24 h after centrifugation at a relative centrifugal force of 1000 for 10 min. all samples were then stored at -20 °c until used. competitive enzyme-linked immunosorbent assay the commercially available competitive enzyme-linked immunosorbent assay (c-elisa) kit (idvet, france) for pprv was used for this study. this diagnostic kit detects antibodies directed against the nucleoprotein of pprv and was developed by a fao reference laboratory (cirad-emvt, montpellier, france). the microplates were supplied in strips, already precoated with pprv recombinant nucleoprotein. the kit contained anti-nucleoprotein horseradish peroxidase (np-hrp) concentrated conjugate (10×), positive and negative controls, dilution buffers, wash concentrate (20×), substrate solution and stop solution (0.5 m h2so4). the test was performed according to the manufacturer’s instructions (libeau et al. 1995). the elisa microplates were read with an immunoscan reader (flow laboratories, uk) with a filter of 450 nm. the optical density (od) was recorded and the test was validated when the mean value of the negative control od (odnc) was greater than 0.7 and the mean value of the positive control od was less than 0.3 of the odnc. the od values were converted to competition percentage (cp) using the following formula: cp = od sample/odnc × 100. the samples with cp 35% (cut-off) were considered positive for pprv infection. statistical analysis the overall and group apparent prevalence (total positive/total sample analysed) and the exact binomial 95% confidence intervals (ci) were computed. pearson’s chi-squared test was used to determine if there were significant differences in the number of positive and negative test results within each variable. a p < 0.05 was considered significant for all tests. the true prevalence (tp) together with 95% ci for all categories was calculated using the rogan–gladen adjusted estimator of ‘true’ prevalence (rogan & gladen 1978). tp was calculated using the following equation: tp = (ap + sp − 1) / (se + sp − 1)    [eqn 2] where ap = apparent prevalence, sp = specificity and se = sensitivity. the sensitivity of the c-elisa test has been reported to be 90% and specificity 98% (saliki et al. 1993). all analyses were performed in r statistical software version 3.0.1 (r 2015). results at a 95% confidence level, a sample size of 379 animals per state was calculated. a total of 4548 field serum samples were collected from 12 states across the agro-ecological zones of the country. no animal was known to have been vaccinated against pprv before or at the time of sampling. some animals appeared to have clinical signs compatible with ppr during sampling (figure 1a–e). small ruminants were transported in vehicles that were often overcrowded, over long distances from the north of the country to the live animal markets in the south (figure 2a). on arrival at the markets, the animals were tied together as they awaited buyers (figure 2b). figure 1: clinical signs (compatible with peste des petits ruminants) observed during sampling (2010–2013) in various locations in nigeria: (a) frothy salivation and muco-purulent nasal discharge; (b) soiled anal region because of profuse diarrhoea; (c) matted eyelids, salivation and nasal discharges; (d) raised hair coat, depression and increased rectal temperature and (e) frothy salivation and death. figure 2: (a) small ruminants were transported in an overcrowded vehicle from the north-eastern part of the country to the south. note the exposure to weather elements (sun, rain) in addition to the stress of transportation over long distances without feed or water. (b and c) live animal market in a south-western nigerian city. the overall apparent prevalence estimate of sera positive for pprv antibodies was 22.38% (n = 1018/4548, 95% ci: 21.79–24.57), with a range of 14.5% – 30.1%. the overall tp estimate was 23.16% (table 1). there were significant differences in the distribution of pprv antibodies in small ruminants between states (p = 0.001) (table 1, figure 3). the highest seroprevalence was in taraba state (29.51%), whilst the lowest seroprevalence was observed in cross river state (14.52%). the highest seroprevalence of pprv in sheep where n > 6 was in kano state (33.12%, n = 157) and in goats it was in taraba state (27.97%, n = 236) (figure 4). the seroprevalence varied between localities, with a range of 12.64% (ikot-omin, cross river state) to 33.33% (kassa, taraba state) (figure 4). no spatial pattern was evident in the distribution of pprv antibodies in small ruminants – the seroprevalence did not appear to be higher in states neighbouring other countries, nor higher in one region of the country. figure 3: prevalence of peste des petits ruminants virus antibodies in each locality indicated by coloured circles. the colour of each state indicates the overall prevalence in that state. from the 4548 small ruminants that were sampled, 42.3% were male animals and 57.7% were female animals (table 1). true prevalence was 22.58% for male animals and 23.59% for female animals. there was no significant difference in the pprv seroprevalence between male and female small ruminants (p = 0.571). the animals that were sampled ranged in age from 3 months to 3 years. the tp of pprv in animals above 1 year of age was 22.55% and 24.40% in animals between 3 and 12 months of age. there was no significant difference between the seroprevalence of pprv in small ruminants over 1 year of age and those between 3 and 12 months of age (p = 0.323) (table 1). a total of 3489 serum samples were collected form goats and 1059 serum samples from sheep. the tp in goats was 22.93% and it was 23.91% in sheep. there was no significant difference between the seroprevalence of pprv in sheep and goats (p = 0.639) (table 1). the sample size in each locality varied from 43 (ikot eneobong, cross river state) to 111 (kassa, taraba state) with a median of 95 (figure 4). in most localities in the south of nigeria, sheep were seronegative for pprv. in this region, the sample sizes in sheep were either small (akwa ibom, anambra and imo states) or no sheep were available for sampling (cross river state), as most farmers breed goats and sheep are scarce. however, in idanre and fajaw, both in ondo state, all 14 and 17 sheep, respectively, were seronegative for pprv. the highest seroprevalence of pprv in sheep in a locality where the sample size was greater than two was in gaya, kano state (38.24%, n = 34). the largest number of sheep sampled in a locality (n = 44) was in bafarawa (sokoto state), where the seroprevalence of antibodies to pprv in sheep was 22.73%. figure 4: the proportion of sheep and goats seropositive to peste des petits ruminants virus in nigeria. the size of the pie symbol is relative to the number of samples collected. goats were present in all localities sampled, with a range of 43 (ikot eneobong, cross river state) to 104 (brangaville, akwa ibom state). the prevalence in goats ranged from 12.36% (ijebu-ode, ogun state) to 34.00% (maihula, taraba state) (figure 4). the seroprevalence of pprv antibodies in each animal category was very similar (20.44% in male adult sheep, 22.29% in female adult sheep, 21.64% in young male sheep, 23.78% in young female sheep, 23.89% in adult male goats, 22.09% in adult female goats, 26.21% in young male goats and 24.02% in young female goats). the highest seroprevalence was 66.67% in young female sheep from kwara state (n = 18). discussion analysis of 4548 samples from sheep and goats confirmed the endemic nature and wide distribution of pprv throughout nigeria. this study found an overall current seroprevalence of pprv antibodies in small ruminants in nigeria to be 23.16%. taylor and abegunde (1979) in the 1970s reported a seroprevalence of 57% and 44% in sheep and goats respectively. obi et al. (1983) reported 52.2% and 53.7% in sheep and goats in the 1980s, whilst shamaki (2002) reported a seroprevalence of 44% from samples analysed between 1995 and 1999. these differences may be because of the sampling locations, sample frames, seasons of the year, different sampling techniques and laboratory tests used. it is also possible that the higher seroprevalence obtained previously was because of sampling during outbreaks. the worldwide eradication of rinderpest virus, which cross-reacts with pprv in serological tests, may also have been responsible for the lower seroprevalence we found in this study. these results are similar to a study in ethiopia, which reported a seroprevalence of 33% in sheep and 67% in goats in the 1990s, but another study a decade later found the seroprevalence to be 9% in goats and 13% in sheep (abraham et al. 2005; roeder et al. 1994). the difference was less in sudan (51.9% compared to 67.2%) in studies conducted 10 years apart (haroun et al. 2002; saeed et al. 2010). taraba state in the north-east agro-ecological zone had the highest seroprevalence rate of 29.51%, whilst cross river state in the south-southern agro-ecological zone had the lowest seroprevalence rate of 14.52%. this observation may be because of the prevailing management practices in the various states and rainfall, which affects the growth of grasses. most of the small ruminants in taraba state are on a free range (agro-pastoralism) system because the farmlands for crops are at a distance from most villages and the zone is a savannah with a longer dry season period. most animals in cross river state are tied at home and grass is provided because crops are planted in the backyard. cross river state is a rain forest zone with abundant rain that enables grass to grow almost all the year round. the sheep breeds observed in taraba state were of the uda, balami and yankassa breeds, whilst those seen in cross river state were predominantly of the west african dwarf (wad) stock. the wad breeds of goats were also seen in cross river state, while those in tarabastate were a mix of both the wad and the sokoto red. it is not clear whether these breed differences in small ruminants had any effect on the difference in seroprevalence rates observed in the two states. transportation, management practices and marketing play major roles in the epidemiology of pprv. we observed that animals from different households and localities are usually transported together in close proximity (figure 2a) to the market. at the market, the animals are tied together (figure 2b) in stalls as they await buyers. any animal not sold is returned to the flock and new animals bought also end up in new flocks. these practices are conducive to outbreaks of diseases. new infections leading to outbreaks normally occur when animals are transported together or when they are tied together (transportation stress, overcrowding and malnutrition) in the live animal markets or kept for safety reasons with a relative from another locality. in this study, there was an over-representation of female animals (2623 female animals and 1925 male animals). this may be because of the practice of sacrificing male animals when the need to sell or slaughter animals arises. the c-elisa is an ideal test to carry out a serological survey of pprv antibodies in small ruminants. the c-elisa was used in this study because of the merits it has over other serological techniques, which are more laborious and time consuming. the results of this study may have been biased by the sampling technique employed. because of the logistical constraints and the complexity of the pastoral husbandry practices, it was not possible to systematically select the different villages, farms, households and the animals to include in this study. live sheep and goat markets and slaughter slabs were also sampled randomly in each locality visited. a more accurate seroprevalence may emerge if randomised sampling is carried out where the actual small-ruminant population in each state (data such as these are currently not available in nigeria) is taken into account. an important observation during the course of sampling is the apparent lack of awareness amongst the small-ruminant owners of the vaccination status of their flocks. the control and eradication of ppr is a priority in order to ease poverty and improve the health and husbandry of animals kept by people in developing countries. it is encouraging to note that between 18 and 23 november 2013, heads of veterinary laboratories and directors of veterinary services from the ecowas subregion gathered in lagos to develop a framework for the progressive control of ppr in the region. this type of subregional approach also took place in southern africa and a panafrican strategy for the progressive control of pprv has also been developed (elsawalhy et al. 2010). this study provides an overview of the current antibody seroprevalence to pprv in the small-ruminant population in nigeria and also confirms a low level of awareness about vaccination amongst small-holder farmers in rural settings. acknowledgements this work was supported by a grant (rfa 2 no.48) from the agricultural research council of nigeria through the competitive agricultural research grant scheme. we sincerely thank all the field staff who helped during sample collection and other logistics. d. shamaki is a recipient of an iaea project on ppr (crp d32026). t.y. woma is a recipient of an iaea intern fellowship. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions t.y.w. was responsible for experimental and project design and wrote the draft manuscript. p.s.e. performed the statistical analysis d.g.b., j.o.i., l.t., d.y.d. and l.s. performed most of the experiments. h.m.k., d.j.u.k., d.b. and d.s. made conceptual contributions. m.q. was the project leader and contributed in writing the manuscript. references abraham, g., sintayehu, a., libeau, g., albina, e., roger, f., laekemariam, y. et al., 2005, ‘antibody seroprevalences against peste des petits ruminants (ppr) virus in camels, cattle, goats and sheep in ethiopia’, preventive veterinary medicine 70, 51–57. balamurugan, v., sen, a., venkatesan, g., yadav, v., bhanot, v., riyesh, t. et al., 2010, ‘sequence and phylogenetic analysis of the structural genes of virulent isolates and vaccine strains of peste des petits ruminants virus from india’, transboundary and emerging diseases 57, 352–364. diallo, a., 2003, ‘control of ppr: classical and new generation of vaccines’, devbiol (basel karger) 114, 85–91. diallo, a., taylor, w.p., lefevre, p.c. & provost, a., 1989, ‘atténuationd’unesouche de virus de la peste des petits ruminants: candidat pour un vaccine homologue vivant’, revue d’élevage et de médecine vétérinaire des pays tropicaux 42, 311–319. elsawalhy, 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(eds.), 2012, virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses (ictv), elsevier academic press, san diego, ca. libeau, g., préhaud, c., lancelot, r., colas, f., guerre, l., bishop, d.h. et al., 1995, ‘development of a competitive elisa for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein’, research in veterinary science 58(1), 50–55. obi, t.u., ojo, m.o., durojaiye, o.a., kasali, o.b., akpavie, s.o. & opasina, b.a. 1983, ‘ppr in goats in nigeria: clinical, microbiological and pathological features’, zentralbl veterinarmed b. 30, 751–761. obidike, r., ezeibe, m., omeje, j. & ugwuomarima, k., 2006, ‘incidence of peste des petits ruminants haemagglutinins in farm and market goats in nsukka, enugu state, nigeria’, bulletin of animal health and production in africa 54, 148–150. odo, b.i. 2003, ‘comparative study of some prevalent diseases of ecotype goats reared in south-eastern nigeria’, small ruminant research 50, 203–207. ogunsanmi, a., awe, e., obi, t. & taiwo, v., 2003, ‘peste des petits ruminants virus (ppr) antibodies in african gray duiker (sylvicapragrimmia)’, african journal of biomedical research 6, 59–61. okoli, i.c., 2003, ‘incidence and modulating effects of environmental factors on trpanosomosis, peste des petits ruminants (ppr) and bronchopneumonia of west african dwarf goats in imo state, nigeria’, livestockresearch for rural development 15, 9–11. roeder, p.l., abraham, g., kenfe, g. & barrett, t., 1994, ‘ppr in ethiopian goats’, tropical animal health and production 26(2), 69–70. rogan, w.j. & gladen, b., 1978, ‘estimating prevalence from the results of a screening test’, american journal of epidemiology 107, 71–76. saeed, i.k., ali, y.h., khalafalla, a.i. & rahman-mahasin, e.a., 2010, ‘current situation of peste des petits ruminants (ppr) in the sudan’, tropical animal health and production 42, 89–93. saliki, j., libeau, g., house, j.a., mebus, a. & dubov, e.j., 1993, ‘monoclonal antibody based blocking elisa for specific detection and titration of ppr virus antibody in caprine and ovine sera’, journal of clinical microbiology 31, 1075–1082. shamaki, d., 2002, ‘some aspects of serological and molecular epidemiology of peste des petits ruminants (ppr) in nigeria’, phd thesis, university of ibadan, nigeria. taylor, w.p. & abegunde, a., 1979, ‘the isolation of peste des petits ruminants virus from nigerian sheep and goats’, research in veterinary science 26, 94–96. ularamu, h.g., owolodun, o.a., woma, t.y., audu, b.j., aaron, g.b., chollom, s.c. et al., 2012, ‘molecular diagnosis of recent suspected outbreaks of peste des petits ruminants (ppr) in yola, adamawa state, nigeria’, african journal of biotechnology 11, 1158–1162. appendix 1 table 1-a1: small-ruminant peste des petits ruminants virus seroprevalence survey from different localities in nigeria, 2010–2013. table 2-a1: nigerian individual state peste des petits ruminants virus seroprevalence data based on species of small ruminants, 2010–2013. table 3-a1: peste des petits ruminants virus seroprevalence data based on category of small ruminants in nigeria, 2010–2013. abstract introduction materials and methods ethical consideration results discussion conclusion acknowledgements references about the author(s) gabriel m. shirima department of health and biomedical sciences, the nelson mandela african institution of science and technology, tanzania john s. kunda national institute of medical research, ministry of health, tanzania citation shirima, g.m. & kunda, j.s., 2016, ‘prevalence of brucellosis in the human, livestock and wildlife interface areas of serengeti national park, tanzania’, onderstepoort journal of veterinary research 83(1), a1032. http://dx.doi.org/10.4102/ojvr.v83i1.1032 note: this article is partially based on the author’s thesis of the degree of doctor of philosophy at the department of veterinary clinical studies, university of glasgow, united kingdom, available here: http://theses.gla.ac.uk/4826/1/2005shirimaphd.pdf research communication prevalence of brucellosis in the human, livestock and wildlife interface areas of serengeti national park, tanzania gabriel m. shirima, john s. kunda received: 04 aug. 2015; accepted: 17 dec. 2015; published: 24 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract between 2005 and 2006, a cross-sectional survey was carried out in domestic ruminants in agropastoral communities of serengeti district, tanzania to determine the seroprevalence of brucellosis in domestic–wildlife interface villages. both the rose bengal plate test (rbpt) and competitive enzyme linked-immunosorbent assay (c-elisa) were used to analyse 82 human and 413 livestock sera from four randomly selected villages located along game reserve areas of serengeti national park. although both cattle (288) and small ruminants (125) were screened, seropositivity was detected only in cattle. the overall seroprevalence based on c-elisa as a confirmatory test was 5.6%. in cattle both age and sex were not statistically associated with brucellosis seropositivity (p = 0.63; 95% ci = 0.03, 0.8 and 0.33; 95% ci = 0.6, 3.7, respectively). overall herd level seropositivity was 46.7% (n = 7), ranging from 25% to 66.7% (n = 4–10). each village had at least one brucellosis seropositive herd. none of the 82 humans tested with both rbpt and c-elisa were seropositive. detecting brucella infection in cattle in such areas warrants further investigation to establish the circulating strains for eventual appropriate control interventions in domestic animals. introduction brucellosis is caused by a variety of brucella species and is a disease of major socio-economic importance in domestic animals worldwide, especially in developing countries where disease control programmes are either non-existent or inadequate. the disease also occurs in wild animals, thus posing a danger of transmission between domestic and wild animals in wildlife–livestock interface areas (assenga et al. 2015; shirima 2005; shirima et al. 2007). brucella species that cause disease in terrestrial animals include brucella abortus, brucella melitensis, brucella suis, brucella ovis and brucella canis (corbel 1988). b. abortus, b. melitensis, b. suis and b. canis can all cause human brucellosis, with different degrees of severity. serengeti is one of the districts housing national parks in tanzania. serengeti national park occupies more than 50% of the area of serengeti district. livestock keepers in serengeti district who entirely depend on livestock production and practise limited agriculture observed domestic animals commingling with wildlife throughout the year in search of pastures and water. this is also common in villages located at interface areas of serengeti national park (fyumagwa 2012). the presence of zoonotic diseases in domestic animals around the serengeti ecosystem could be a source of cross-transmission to wildlife because commingling between the two populations has been reported elsewhere (assenga et al. 2015; fyumagwa et al. 2009; shirima et al. 2007). brucella infection has been reported in wildebeests (connochaetes taurinus), buffaloes (syncerus caffer) (fyumagwa et al. 2009), and cattle (bugwesa et al. 2009) in the serengeti ecosystem, but has not been reported from small ruminants and humans from the same interface areas. assenga et al. (2015) and fyumagwa et al. (2009) suggested further studies in livestock and human populations adjacent to wildlife areas and characterisation of the circulating pathogens for future interventions. therefore, this seroprevalence study was conducted to elucidate the disease extent and distribution in livestock and humans in villages located in the wildlife–livestock interface area in serengeti district so as to devise control and preventive strategies. materials and methods study location the study was conducted in serengeti district, tanzania. the district has an area of 10 373 km2 with a human population of about 249 420 (nbs 2012). the serengeti national park occupies about 7000 km2 whereas the game reserve occupies 258 km2 of its area. the human population occupies only 659 km2, including farming and livestock practices. study villages four villages, namely bisarara, ketembere, masinki, and nyichoka, bordering game reserves around serengeti national park were selected randomly for the study between november 2005 and march 2006. four herds were selected randomly from each village for sampling with the exception of one village where three herds were selected. in these villages, cattle and small ruminants were kept together and thus one household was considered to constitute one herd. sample size the livestock sample size was estimated to provide 80% power of getting a seropositive animal with 95% confidence. based on an estimate of 50% brucellosis prevalence and 0.05 error margin, the sample size was calculated as described by martin, meek and willeberg (1987) to obtain the total number of domestic ruminants to be screened from the district: where: n = the required sample size; p = estimated prevalence = 0.5; z = level of confidence as 1.96 and d = desired precision level = 0.05. blood sampling and processing livestock sampling it was estimated to sample at least 100 animals from four herds in each village with 25–30 animals per herd. the animals were sampled randomly from herds with more than 30 animals whereas in herds with 1–30 animals, all were tested. blood sample and data collection from animals whole blood (10 ml) was collected in plain vacutainers from each animal. individual bio-data such as sex, age, species, and abortion history in the previous year were collected. blood samples were processed on the day of collection. samples were centrifuged for 5 min using a mobile spin centrifuge (vulcan technologies, usa) at the serengeti veterinary office. serum was aliquoted into eppendorf tubes (eppendorf-netheler-hinz gmbh, hamburg, germany) in duplicate and kept at -20 °c. all serum samples were subjected to the rose bengal plate test (rbpt) as screening test at sokoine university of agriculture, tanzania. rbpt-positive samples were confirmed by competitive enzyme-linked immunosorbent assay (c-elisa). the kits and antigen used for screening and confirmation were kindly donated by the animal and plant health agency (apha), weybridge, uk (batch numbers sg269 and sg276). the agglutination test and c-elisa were performed and interpreted as described by apha protocol (perret et al. 2001). the elisa test results were measured by elisa reader multiscan rc version 6.0 (labsystems, helsinki, finland) at 450 nm. the plate results were considered invalid if any of the following applied: the binding ratio was less than 10. the optical density (od) of the mean of the 6 negative ods was less than 0.70. the optimal mean negative od was 1.0. the od of the mean of the 6 positive wells was greater than 0.10. the mean od of the four conjugate control wells was less than 0.70 (shirima 2005). the cut-off value for c-elisa positivity was based on the conjugate control where the cut-off was taken as 60% of the mean of the od of the four conjugate control wells. any test sample giving an od equal to, or below this value, was considered positive. all results were expressed as a percentage of the conjugate control and referred to as percentage positive values (shirima 2005). blood collection from humans in each livestock-keeping household, family members were approached to identify volunteers for blood samples following discussions about the purpose of the project and the potential of the brucellosis problem in the district. blood was aseptically collected from the brachial vein using a disposable 5 ml-syringe (ha young corporation, korea). the blood was immediately transferred into a plain vacutainer and serum separation and testing was processed as described in the previous section (shirima 2005). statistical analysis the data collected were stored and analysed using version 7.1.1.14 of the epi-info software package (centres for disease control and prevention, atlanta, ga, usa). seroprevalence with 95% confidence interval was calculated for individual and herd level estimates. the relationship between dependent variables (herd and animal level) and outcome (serostatus) was explored using the mantel–haenzel test (epi-info software 2015). ethical consideration blood collection from humans was carried out after obtaining ethical clearance from the ministry of health and social welfare, tanzania (ethical clearance number nimr/hq/vol. iv/b575). sampling of livestock was done with verbal consent from the herd owners who were informed about the purpose of the project. results herd structure both cattle and small ruminants (goats and sheep) constituted a herd, as they stayed together. the herd size ranged from 9 to 150 domestic ruminants. the majority of herds (80%) had a herd size between 9 and 40 animals, with one herd having more than 100 animals. in the study area; 296 cattle, 75 goats and 42 sheep were screened for brucella antibodies. livestock seropositivity the prevalence of brucellosis in domestic ruminants based on c-elisa was 5.6% in livestock–wildlife interface areas of serengeti district. although cattle and small ruminants were kept together, seropositivity was only detected in cattle. there was no significant difference (p = 0.63) in seropositivity between female (3.2%) and male (3.8%) animals from this study. in addition, seropositivity was not statistically influenced by age between young animals (≤ 2.5 years, n = 159) and adults (> 2.5 years, n = 254) or sex (p = 0.33; p = 0.63). the overall brucella seropositivity at herd level was 46.7%, although it ranged from 25% to 66.7%. village level seropositivity ranged from 1.9% to 11% with highest level observed in nyichoka village (11%), followed by bisarara (8.6%), and the lowest seroprevalence was observed in ketembere village (1.9%) (table 1). table 1: herd and individual level brucella seropositivity in selected villages of serengeti district. between the years 2005 and 2006, 20 cases of abortions were reported from the study herds with one seropositive herd at masinki village experiencing seven cases of abortion. however, there was no statistically significant association between animal level c-elisa seropositivity and abortion (p = 0.66, ci = 0.02, 7.2). human brucella seropositivity in total, 82 humans (47 males and 35 females) volunteered for brucellosis screening. none tested positive with either rbpt or c-elisa. discussion the serological survey conducted revealed that only cattle from the four villages surveyed were exposed to brucella infection. the individual and herd seroprevalence of 5.6% and 46.6% noted from this study were comparable to other studies in agropastoral and pastoral communities in tanzania (assenga et al. 2015; bugwesa et al. 2009; shirima et al. 2007). the infection in cattle suggests exposure to the bacteria because vaccination using s19 is not practised in tanzania, including serengeti district. infection in cattle residing close to wildlife areas could perpetuate transmission between cattle and wildlife animals and thus maintain the disease in the two populations. brucella seropositivity was not detected in sheep and goats in the study area despite the fact that all the animals were kept together. this could be attributed to the extent of environmental contamination and duration of the infection in the study herds. these findings were comparable to those of assenga et al. (2015), where seropositivity was significantly low in small ruminants compared to cattle. however, several studies have indicated brucella infection in both cattle and small ruminants that cohabited (gamal et al. 2014; shirima 2005). although cattle and small ruminants from the same herd could be exposed to brucella infection, no study has been conducted to elucidate the circulating brucella species. encountering brucella seropositivity in all villages could indicate the spatial distribution of the disease and the potential to perpetuate the disease within and between villages, depending on how densities and contact rates among animals increases. similar observations were reported by jiwa et al. (1996), kadohira et al. (1997), and shirima (2005), who suggested that commingling of herds at grazing and watering points were risk factors for disease transmission. conclusion in conclusion, anti-brucella antibodies were present in cattle in the serengeti ecosystem, suggesting that the infection might circulate in the ecosystem. therefore, further studies are required to establish spatial and temporal distribution and characterise brucella species for eventual control interventions in livestock. acknowledgements we are grateful to the both ministry of livestock and fisheries development and ministry of health social welfare for granting permission to undertake this project. we thank the dfid-uk for funding this work. we also owed thanks to vla weighbridge for supplying testing reagents. we extend our sincere thanks to technicians from sokoine university for assisting with analysing the samples. the generous cooperation of herd owners and extension personnel is highly appreciated. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions g.m.s. was the team leader and responsible for rbpt and c-elisa testing, interpreting the results, and writing the manuscript. j.s.k. was responsible for field blood collection, serum separation, carrying out rbpt screening and working on the manuscript. references assenga, j.a., matemba, l.e., muller, s.k., malakalinga, j.j. & kazwala, r.r., 2015, ‘epidemiology of brucella infection in the human, livestock and wildlife interface in the katavi-rukwa ecosystem, tanzania’, bmc veterinary research 11, 189. http://dx.doi.org/10.1186/s12917-015-0504-8 bugwesa, z.k., fyumagwa, r.d., mdaki, m.r., kuya, s. & hoare, r., 2009, ‘seroprevalence of brucella abortus in livestock-wildlife interface in serengeti ecosystem’, proceedings of the 7th tawiri scientific conference, arusha, tanzania, december 02–04, 2009. corbel, m.j., 1988, ‘brucellosis’, in j.a. laing, w.j. brinley morgan & w.c. wagner (eds.), fertility and infertility in veterinary practice, 4th edn., pp. 190–221, baillière tindall, london. epi info 7.1.5.2 software, 2015, centres for disease control and prevention, atlanta, ga. fyumagwa, r.d., 2012, ‘anthropogenic and natural influence on disease prevalence at the human-livestock-wildlife interface in the serengeti ecosystem’, phd thesis, norwegian university of science and technology, trondheim, norway. fyumagwa, r.d., wambura, p.n., mellau, l.s.b. & hoare, r., 2009, ‘seroprevalence of brucella abortus in buffaloes and wildbeests in the serengeti ecosystem, a threat to humans and domestic ruminants’, tanzania veterinary journal 26(2), 62–67. gamal, w., falk, m., mandy, c.e., heinrich, n. & uwe, r., 2014, ‘detection of brucella melitensis in bovine milk and milk products from apparently healthy animals in egypt by real-time pcr’, journal of infection in developing countries 8(10), 1339–1343. http://dx.doi.org/10.3855/jidc.4887 jiwa, s.f.h., kazwla, r.r., tungaraza, r., kimera, s.i. & kalaye, w.j., 1996, ‘bovine brucellosis serum agglutination test prevalence and breed predisposition according to prevalent management systems in the lake zone of tanzania’, preventive veterinary medicine 26, 341–346. kadohira, m., mcdermott, j.j., shoukri, m.m. & kyule, m.n., 1997, ‘variation in the prevalence of antibody to brucella infection in cattle by farm, area and district in kenya’, epidemiology and infection 118, 35–41. martin, s.w., meek, a.h. & willeberg, p., 1987, veterinary epidemiology: principles and methods, iowa state university press, ames, ia. national bureau of statistics (nbs), 2012, population and housing census, viewed n.d., from http://nbs.go.tz/nbs/takwimu/census2012/basic_dem/ perret, l., brew, s., stack, j., tucker, j. & macmillan, a.p., 2001, guide to the eia techniques used in the diagnosis of brucellosis at the vla, weybridge, veterinary laboratory agency, new haw, addlestone, surrey. shirima, g.m., 2005, ‘the epidemiology of brucellosis in animals and humans in arusha and manyara regions of tanzania’, phd thesis, university of glasgow. shirima, g.m., cleaveland, s., kazwala, r.r., kambarage, d.m., nigel, f., mcmillan, a. et al., 2007, ‘sero-prevalence of brucellosis in smallholder dairy, agropastoral, pastoral, beef ranch and wildlife animals in tanzania’, bulletin of animal health and production in africa 55, 13–21. mirzaiedehaghi.indd introduction malignant theileriosis of sheep is a highly fatal acute or subacute disease caused by the tick-borne protozoan parasite, theileria lestoquardi which is transmitted by ticks of the family ixodidae, particularly hyalomma anatolicum anatolicum (hooshmand-rad & hawa 1973; morel & uilenberg 1981). the chemotherapeutic efficacy of a number of compounds includ ing parvaquone (clexon) and buparvaquone (butalex) has been tested for the treatment of the disease. tests have shown that these two compounds are effective (hooshmand-rad 1989) but they are not easily and quickly eliminated from the body of animals (mchardy, wekesa, hudson & randall 1985) which can constitute a public health hazard if milk and meat of treated animals are consumed by humans. the therapeutic effect of the alka loids of the plant peganum harmala has been investigated for the treatment of tropical theileriosis in cattle which is caused by theileria annulata and it was shown that they are effective (fan, liang, men, gao, li, zhao, hu, dang, zhang, preston & yin 1997; hu, fan, liang, zhao, dang, gao, dong, preston & yin 1997) in addition, it has been shown that these alkaloids, when inoculated intramuscularly do not infiltrate the muscular tissue surrounding the inoculation site but are also rapidly eliminated from the body (puzii & serov 1983). the objective of this study was to study the effect of total alkaloids of p. harmala when used for the treatment of naturally infected cases of ovine malignant theileriosis. materials and methods one hundred sheep suffering from natural infections of malignant theileriosis and belonging to farmers were selected. they were of different ages but all were in the primary phase of the disease as determined by microscopic examination of giemsa-stained prescapular lymph node biopsy smears in which 153 onderstepoort journal of veterinary research, 73:153–155 (2006) research communication treatment of natural ovine malignant theileriosis with a chloroform extract of the plant peganum harmala m. mirzaiedehaghi* pathobiology department, school of veterinary medicine, shahid bahonar university of kerman kerman, iran abstract mirzaiedehaghi, m. 2006. treatment of natural ovine malignant theileriosis with a chloroform extract of the plant peganum harmala. onderstepoort journal of veterinary research, 73:153–155 one hundred sheep naturally infected with theileria lestoquardi were treated with a chloroform extract of the plant peganum harmala. the treatment was continued for 5 days, the dose of extract being 5 mg/kg per day. sixty-five of the sheep responded to treatment and recovered but 35 did not and died. the cure rate was 65 %. keywords: malignant ovine theileriosis, peganum harmala extract, sheep, theileria lestoquardi * e-mail: dr_mirzaie_mo@mail.uk.ac.ir accepted for publication 19 january 2006—editor 154 treatment of ovine malignant theileriosis with chloroform extract of peganum harmala fairly numerous schizonts were present. similar examination of blood smears revealed that piroplasm parasitaemia was rare. in addition, only one or both prescapular lymph node(s) were slightly to moderately enlarged and the rectal temperature was between 40.5 and 41.9 °c. a chloroform extract of p. harmala was prepared from the stems and leaves of the plant according to the method described by mirzaie & shaddel (2004). this was administered intramuscularly to the sheep at a dosage rate of 5 mg/kg body mass once a day for 5 days (fan et al. 1997). after treatment the rectal temperature of the sheep was measured daily in the morning until it dropped to normal values. their vigor, appetite, visible mucous membranes and other signs were observed clin ically every day. in addition, giemsa-stained smears prepared every three days from biopsy material of a prescapular lymph node and blood smears were examined for 20 days after commencement of the treatment. results before treatment lymph node biopsy smears from enlarged prescapular nodes and blood smears showed schizonts or piroplasms, respectively, of t. lestoquardi in all the sheep. however, 12–20 days after the commencement of the treatment schizonts and piroplasms could not be detected in 65 of the sheep. in addition other clinical signs and fever disappeared in these sheep and they all recovered. the clinical signs and parasitemia of the other 35 sheep became progressively more severe and all of them died. discussion and conclusion in this study 65 % of the sheep recovered after treatment but in another experiment reported by mirzaie & shaddel (2004) five sheep which had been experimentally infected with t. lestoquardi and then treated with an extract containing the alkaloids of p. harmala, all recovered. the reasons for this apparent discrepancy in the success rate of the treatment in our experiment and that of mirzaie & shaddel (2004) could be the different environmental conditions in which their sheep were maintained, the small numbers of sheep they used and the fact that the infection was experimentally induced. our results are in agreement with that of other experiments in which the alkaloids of p. harmala were used for the treatment of tropical theileriosis in cattle (agaev, mirzabekov, gumbatov & mirzabekov 1977; vecherkin, puzii, romakhov & tribunskii 1977; charyev & khudaiberdyev 1978; levchenko 1978, 1979; puzii, vetchorkin, romakhov & vecherkin 1979; puzii, vecherkin, toptaev, tsyganova & duisheev 1982; fan et al. 1997; hu et al. 1997). references agaev, a.a., mirzabekov, d.a., gumbatov, m.g. & mirzabekov, k.d. 1977. the protection of imported breeding cattle from blood parasitic diseases in azerbaidzhan. ussr, azerbaidzhanskii nauchno-issledovatel’skii institut infor matsii i tekhniko-ekonomicheskikh issledovanii gosplana azerbaidzhanskoi ssr: abstracts of papers of the scientific con ference dedicated to the 75th anniversary of the foundation of the azerbaidzhani veterinary research institute: 95–96. charyev, o.c. & khudaiberdyev, d. 1978. the therapeutic efficacy of “garmala” against theileriasis.trudy-turkmenskogo-sel’skokhozyaistvennogo-instituta, 20:107–108. fan, b., liang, j., men, j., gao, f., li, g., zhao, s., hu, t., dang, p., zhang, l., preston, p.m. & yin, h. 1997. effect of total alkaloid of peganum harmala l. in the treatment of experimental haemosporidian infections in cattle. pro ceedings of the european union international symposium on ticks and tickborne diseases,tropical animal health and production, 29:4 (supplement), 77s–83s. hooshmand-rad, p. 1989. chemotherapy in ovine malignant theileriosis. archive institute razi, 40:1–8. hooshmand-rad, p. & hawa, n.j. 1973. transmission of theil eria lestoquardi in sheep by hyalomma anatolicum anatolicum.tropical animal health and production, 5:103–109. hu, t.j., fan, b.t., liang, j., zhao, s., dang, p., gao, f., dong, m.x., preston, p.m. & yin, h. 1997. observations on the treatment of natural haemosporidian infections by total alkaloid of peganum harmala l. in cattle. proceedings of the european union international symposium on ticks and tickborne diseases. tropical animal health and produc tion, 29:4 (supplement), 72s–76s. levchenko, f.f. 1978. comparative study of 3 chemotherapeutic preparations against natural theileriasis in cattle. trudy nauchno issledovatel’skogo veterinarnogo instituta tadzhikskoi ssr, 8:117–119. levchenko, f.f. 1979. the efficacy of peganum harmala alkaloids in the treatment of theileriasis in cattle. trudy nauch no issledovatel’skogo veterinarnogo instituta tadzhikskoi ssr, 9:89–92. mchardy, n., wekesa, l.s., hudson, a.t. & randall, a.w. 1985. antitheilerial activity of bw720c (buparvaquone): a comparison with parvaquone. research in veterinary science, 39:29–33. mirzaie, m. & shaddel, f. 2004. effect of total alkaloids of peganum harmala l. on experimentally ovine malignant theileriosis caused by theileria lestoquardi. spring meeting and malaria meeting, 4–7 april, 2004, chester college, chester, united kingdom, sp11. morel, c. & uilenberg, g. 1981. the nomenelature of some theileria species (sporozoa,babesioidea) of domestic ruminants. revue d elevage et de medecine veterinaire des pays tropicaux, 34:139–143. puzii, a.d. & serov, v.m. 1983. persistence of pegarmin (a preparation of the alkaloids of peganum harmala) in the body (of cattle). veterinariya, moscow, ussr, 5:62–64. puzii, a.d., vecherkin, s.s., toptaev, v.i., tsyganova, g.a. & duisheev, n.a. 1982. tests for teratogenic and em155 m. mirzaiedehaghi bryotoxic properties of pegarmin (alkaloid of the plant peganum) and its therapeutic use in cattle infected with theileria annulata.trudy vsesoyuznogo instituta eksperimental’noi veterinarii, 56:31–38. puzii, a.d., vetchorkin, s.s., romakhov, v.g. & vecherkin, s.s. 1979. application of alkaloids of peganum harmala for treatment of theileriasis in cattle. xxi world veterinary congress, 1–7 july 1979, moscow. summaries. vol. 2, sec. iii: parasitology: 22. vecherkin, s.s., puzii, a.d., romakhov, v.g. & tribunskii, m.p. 1977. the alkaloids of harmel in the treatment of theileriasis.veterinariya moscow, 10:77–78. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice horak_193-198.indd introduction african buffaloes, syncerus caffer, are large bovids that prefer savanna-type habitats and require a plentiful supply of grass, shade and water for optimal survival. they occur in herds, which increase in size in the dry season, but decrease during the wet season because of the usual abundance of both food and water (skinner & smithers 1990). large numbers of these animals are present in the kruger national park (knp) in north-eastern mpumalanga and limpopo provinces, and in the umfolozi and hluhluwe nature reserves (recently combined to form the hluhluwe-imfolozi park) in the north-eastern regions of kwazulu-natal province (kzn), with smaller populations in national, provincial and privately owned reserves or in buffalo breeding projects in these and nearly all other provinces of south africa. 193 onderstepoort journal of veterinary research, 73:193–198 (2006) the host status of african buffaloes, syncerus caffer, for rhipicephalus (boophilus) decoloratus i.g. horak1*, h. golezardy2 and a.c. uys2 abstract horak, i.g., golezardy, h. & uys, a.c. 2006. the host status of african buffaloes, syncerus caf fer, for rhipicephalus (boophilus) decoloratus. onderstepoort journal of veterinary research, 73: 193–198 the objective of this study was to assess the host status of african buffaloes, syncerus caffer, for the one-host tick rhipicephalus (boophilus) decoloratus. to this end the r. (b.) decoloratus burdens of ten buffaloes examined in three north-eastern kwazulu-natal province (kzn) nature reserves were compared with those of medium-sized to large antelope species in these reserves and in the southern kruger national park (knp), mpumalanga province. the r. (b.) decoloratus burdens of the buffaloes were considerably smaller than those of the antelopes in the knp, but not those in the kzn reserves. the life-stage structure of the r. (b.) decoloratus populations on the buffaloes, in which larvae predominated, was closer to that of this tick on blue wildebeest, connochaetes taurinus, a tick-resistant animal, than to that on other antelopes. a single buffalo examined in the knp was not infested with r. (b.) decoloratus, whereas a giraffe, giraffa camelopardalis, examined at the same locality and time, harboured a small number of ticks. in a nature reserve in mpumalanga province adjacent to the knp, two immobilized buffaloes, from which only adult ticks were collected, were not infested with r. (b.) decoloratus, whereas greater kudus, tragelaphus strepsiceros, examined during the same time of year in the knp harboured large numbers of adult ticks of this species. african buffaloes would thus appear to be resistant to infestation with r. (b.) decoloratus, and this resistance is expressed as the prevention of the majority of tick larvae from developing to nymphs. keywords: african buffaloes, ixodid tick, rhipicephalus (boophilus) decoloratus, syncerus caffer, tick resistance * author to whom correspondence is to be directed: e-mail: ivan.horak@up.ac.za 1 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, and division of parasitology, arc-onderstepoort vet erinary institute, onderstepoort, 0110 south africa 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. a.c. uys’ present address: p.o. box 652, new lands, 0049 south africa accepted for publication 5 april 2006—editor 194 host status of african buffaloes, syncerus caffer, for rhipicephalus (boophilus) decoloratus african buffaloes may be infested with exceptionally large numbers and species of ixodid ticks (yeoman & walker 1967; walker 1974; horak, potgieter, walker, de vos & boomker 1983a), and because of their own large size a large percentage of these ticks are usually adults (horak, macivor, petney & de vos 1987; gallivan & horak 1997). there is, however, some doubt as to their suitability as hosts for the one-host tick rhipicephalus (boophilus) decoloratus. in tanzania no r. (b.) decoloratus were present in 25 collections from buffaloes examined by yeoman & walker (1967), while in kenya and zimbabwe only one of 119, and one of 135 collections, respectively, were positive (walker 1974; mason & norval 1980). in north-eastern kzn, south africa, only one of four fairly exhaustive collections made from buffaloes was positive, and it contained only larvae of this species (horak et al. 1983a). in an attempt to confirm this observation norval (1984) infested a tame, hand-reared, tick-free, yearold african buffalo with 10 000 larvae of r. (b.) decoloratus on each of three occasions over a period of 3 months. the first infestation yielded 201 engorged females, the second 421, and the third, which was accompanied by considerable irritation and grooming, only 63 engorged females. according to norval (1984) the latter result is similar to what one would expect on brahman cattle resistant to rhipicephalus (boophilus) micro plus. an opportunity to do tick counts on african buffaloes arose when one of these animals was slaughtered in the knp for survey purposes and ten were slaughtered during a survey to determine the prevalence of bovine tuberculosis in buffaloes in the north-eastern kzn nature reserves. an additional two animals were immobilized for other purposes in a north-eastern mpumalanga province nature reserve and were also examined for ticks. the main objective of the present communication is to compare the r. (b.) decol or a tus burdens of these buffaloes with those of medium-sized and large antelopes in the kzn nature reserves and in the knp. materials and methods during september 1985 a single buffalo was shot near satara tourist rest camp in the southern knp, mpumalanga province. during june and july 1994 four buffaloes were darted and then shot in the umfolozi nature reserve, four in the hluhluwe nature reserve, and two in the eastern shores nature reserve, all in north-eastern kzn. the animals were skinned and their skins were processed for tick recovery as described by horak, boomker, spickett & de vos (1992). two buffaloes were immobilized and carefully examined for adult ticks in the mtethomusha nature reserve, mpumalanga province, adjacent to the south-western boundary of the knp. the ticks recovered from all the buffaloes were identified and counted. their burdens of r. (b.) decoloratus are com pared with those of medium-sized and large antelopes processed in the same way, namely impalas, aepyceros melampus, greater kudus, tragelaphus strepsiceros, and blue wildebeest, connochaetes taurinus, in the knp, and nyalas, tragelaphus angasii, in the north-eastern kzn nature reserves (horak, de vos & brown 1983b; horak et al. 1992; horak, boomker & flamand 1995; horak, gallivan, braack, boomker & de vos 2003). the tick burden of the single buffalo examined in the knp is also compared to that of a giraffe, giraffa camelopardalis, shot at the same time and place. the tick burdens of the kzn buffaloes are compared with those of nyalas and impalas examined in north-eastern kzn reserves during the same time of year, but not in the same year as the buffaloes. while the adult tick burdens of the buffaloes examined in the mtethomusha nature reserve during july 1999 are compared with those of greater kudus examined in the south of the knp during july 1981 and 1982. with the exception of some of the nyalas, the length of the idiosoma of engorging female r. (b.) decoloratus collected from the animals was measured. those ticks of which the idiosoma measured 4.0 mm or more, were regarded as standard females, i.e. female ticks that will engorge and detach within the next 24 h. results the r. (b.) decoloratus burdens of individual buffaloes are summarized in table 1. the largest number of ticks of this species were collected from the only buffalo calf examined, an animal approximately 6 months of age. the overall ratio of larvae to nymphs to males and females on all the buffaloes was 10.5: 1.8: 1.6: 1.0. if, however, the tick burden of the calf is excluded from the comparison, the ratios are 36.1: 2.8: 1.6: 1.0. the numbers of r. (b.) decoloratus on the slaughtered buffaloes are compared with those on impalas and greater kudus in the knp, and on nyalas in the north-eastern kzn reserves in table 2. the overall ratio of the various life stages on the 305 antelopes examined is 10.7: 5.7: 2.0: 1.0. in table 3 the r. (b.) decoloratus burdens of the buffaloes are compared with those of nyalas, im195 i.g. horak, h. golerzady & a.c. uys palas and kudus examined during the same time of year (but not in the same year), at the same localities, or in a locality adjacent to that in which the buffaloes were examined. the mean tick burdens of the buffaloes examined in kzn exceeded those of nyalas and impalas examined at the same localities, but the life-stage ratios on the buffaloes were markedly skewed in favour of larvae. the buffalo examined in the knp was not infested, but a giraffe examined at the same time and locality harboured a small burden of r. (b.) decoloratus. the immobilized buffaloes examined for adult ticks in the mthetomusha nature reserve, adjacent to the knp, were not infested, whereas all eight kudus examined in the south of the knp were infested with adult ticks. the life-stage structure of the r. (b.) decoloratus pop ulation on buffaloes in north-eastern kzn is compared in table 4 to that of this tick on blue wildebeest in the knp. the tick life-stage ratios on the wildebeest, which are tick-resistant animals, were 21.0 larvae: 4.0 nymphs: 1.4 males: 1.0 females. table 1 numbers of rhipicephalus (boophilus) decoloratus collected from african buffaloes in five nature reserves in south africa host date locality number of rhipicephalus (boophilus) decoloratus larvae nymphs males females total buffalo 1* buffalo 2* buffalo 3 buffalo 4 buffalo 5** buffalo 6 buffalo 7 buffalo 8 buffalo 9 buffalo 10 buffalo 11 buffalo 12 buffalo 13 july 1999 july 1999 sept 1985 june 1994 june 1994 june 1994 june 1994 june 1994 june 1994 june 1994 june 1994 july 1994 july 1994 mtethomusha reserve mtethomusha reserve kruger national park umfolozi reserve umfolozi reserve umfolozi reserve umfolozi reserve hluhluwe reserve hluhluwe reserve hluhluwe reserve hluhluwe reserve eastern shores eastern shores – – 0 590 234 180 200 288 6 10 116 8 44 – – 0 28 178 0 0 82 0 2 0 0 0 0 0 0 8 186 2 0 48 0 2 2 0 0 0 0 0 4 (0) 120 (8) 0 0 28 (2) 6 (0) 2 (0) 0 0 0 0 0 0 630 718 182 200 446 12 16 118 8 44 total ratio 1 676 10.5 290 1.8 248 1.6 160 (10) 1.0 2 374 total excluding buffalo calf no. 5 ratio 1 442 36.1 112 2.8 62 1.6 40 (2) 1.0 1 656 * = immobilized and examined only for adult ticks ** = calf, approximately 6 months old ( ) = number of standard female ticks, i.e. with idiosoma > 4.0 mm in length table 2 total numbers of rhipicephalus (boophilus) decoloratus collected from african buffaloes and antelopes at various localities in south africa host (number examined) locality number of rhipicephalus (boophilus) decoloratus larvae nymphs males females total buffalo (1) buffaloes (10) kruger national park kwazulu-natal 0 1 676 0 290 0 248 0 160 (10) 0 2 374 ratio 10.5 1.8 1.6 1.0 impalas (135) kudus (95) nyalas (75) kruger national park kruger national park kwazulu-natal 226 612 161 815 11 905 101 981 107 711 5 355 37 724 35 959 2 100 18 024 (355) 18 140 (360) 1 313* 384 341 323 625 20 673 total (305) ratio 400 332 10.7 215 047 5.7 75 783 2.0 37 477 1.0 728 639 ( ) = number of standard female ticks, i.e. with idiosoma > 4.0 mm in length * = length of idiosoma of maturing females not measured 196 host status of african buffaloes, syncerus caffer, for rhipicephalus (boophilus) decoloratus discussion it is arguable as to whether r. (b.) decoloratus was brought to southern africa on the cattle of the early herdsmen that came to this country from further north in africa or whether it is actually a parasite of wild herbivores in this country and has adapted to cattle. whatever the tick’s origin, wherever its distribution overlaps that of cattle, impalas, bushbuck, trage laphus scriptus, greater kudus, nyalas and burchell’s zebras, equus burchelli, in south africa these animals have all proved to be excellent hosts (baker & ducasse 1967; horak et al. 1983a, 1992, 1995, 2003; horak, de vos & de klerk 1984). rhipicephalus (boophilus) decoloratus is a one-host tick and where exhaustive tick collections have been made the approx imate life-stage structure of parasitic populations is larvae 11: nymphs 6: males 2: females 1 (table 2). this stepped-down population structure probably results from the loss of larvae and newly-moulted nymphs, followed by the loss of nymphs and newly-moulted adults at the time of the larval and nymphal moults respectively. the 2: 1 ratio of males to females is most likely a consequence of two factors, namely the greater loss of engorging females, because of their larger size, than of males during grooming, and the persistence of males on cattle, and probably other hosts, after females originating from the same infestation have detached (londt 1976). in systems in which tick predation by red-billed oxpeckers, buphagus erythrorhynchus, occurs naturally, or is encouraged, engorged female r. (b.) decoloratus would be a preferred food item of these birds (bezuidenhout & stutterheim 1980), thus further affecting the male: female ratio. not all large wild herbivores are susceptible to infestation with r. (b.) decoloratus, and blue wildebeest seem to have an innate resistance to infestation with this and other ticks (horak et al. 1983b). although larval burdens may be fairly large on blue wildebeest, there is a sharp decline in numbers between the larval and the nymphal stages, resulting in a lifetable 3 total numbers of rhipicephalus (boophilus) decoloratus collected from african buffaloes and antelopes examined during the same time of year at the same or adjacent localities host (number examined) locality number of rhipicephalus (boophilus) decoloratus larvae nymphs males females total buffaloes (10) nyalas (13) impalas (2) buffalo (1) giraffe (1) buffaloes (2)* kudus (8)** kwazulu-natal reserves kwazulu-natal reserves kwazulu-natal reserves kruger national park kruger national park mtethomusha reserve kruger national park 1 676 452 52 0 0 – – 290 200 34 0 4 – – 248 26 30 0 37 0 2 448 160 (10) 36 (10) 36 (0) 0 8 (2) 0 1 245 (38) 2 374 714 152 0 47 0 3 693 * = immobized and only adult ticks collected ** = only adult ticks taken into consideration ( ) = number of standard female ticks, i.e. with idiosoma > 4.0 mm in length table 4 numbers of rhipicephalus (boophilus) decoloratus collected from african buffaloes and blue wildebeest host (number examined) locality number of rhipicephalus (boophilus) decoloratus larvae nymphs males females total african buffaloes (10) kwazulu-natal reserves 1 676 290 248 160 (10) 2 374 mean ratio ratio excluding buffalo calf no. 5 in table 1 167.6 10.5 36.1 29.0 1.8 2.8 24.8 1.6 1.6 16.0 (1.0) 1.0 1.0 237.4 blue wildebeest (47) kruger national park 19 722 3 805 1 271 940 (88) 25 738 mean ratio 419.6 21.0 81 4.0 27 1.4 20 (1.9) 1 547.6 ( ) = number of standard female ticks, i.e. with idiosoma > 4.0 mm in length 197 i.g. horak, h. golerzady & a.c. uys stage structure of larvae 21: nymphs 4 (table 4). if one were to exclude the tick burden of the kzn buffalo calf, which harboured fairly substantial numbers of nymphs and adult ticks, the life-stage structure on buffaloes would be 36.1 larvae to only 2.8 nymphs. this skewed life-stage distribution is underscored by the fact that only one of four buffaloes exhaustively examined for ticks in the hluhluwe nature reserve during september 1978 was infested with r. (b.) de coloratus and then harboured only 64 larvae (horak et al. 1983a). the appa rent resistance of african buffaloes to infestation with r. (b.) decol oratus probably stems from an anci ent association between these animals and the tick, and seems to be similar to that in indigenous nguni cattle in south africa (spickett, de klerk, ens lin & scholtz 1989). the resistance of hosts to ticks can be expressed in several ways. in the case of r. (b.) decoloratus on afri can buffaloes and blue wildebeest, resistance interferes with the development of larvae to nymphs, but apparently not with the development of nymphs to adults, nor with the engorgement of female ticks (table 4). in blue wildebeest this resistance appears to be innate and is present in very young calves (horak et al. 1983b). in african buffaloes resistance would seem to be acquired in that young calves and tick-naïve animals are susceptible to infestation with r. (b.) decoloratus and only become resistant after re peated infestations (norval 1984, table 1). although a large proportion of r. (b.) decoloratus larvae may develop to adults on apparently susceptible hosts such as impalas and greater kudus (horak et al. 1992, 2003), only a small percentage of the resultant females engorge (table 2), seemingly because of a degree of resistance in these animals. of the total of 36 164 female ticks on the impalas and kudus ex amined in the knp only 715 (2 %) were of standard size (table 2). furthermore females that do mature on resistant animals may fail to reach full engorgement before detaching, and these smaller ticks will produce fewer eggs than fully engorged females. in contrast to blue wildebeest, which are resistant to infestation with a large variety of tick species (horak et al. 1983b), african buffaloes seem to be resistant only to infestation with r. (b.) decoloratus. in support of this contention the ten buffaloes examined in the north-eastern kzn nature reserves during the current surveys were infested with a total of ten ixodid tick species and their total burdens varied from 5 911 to 58 498 ticks. rhipicephalus (boophilus) decoloratus accounted for only 2 374 out of the total of 236 845 ticks collected from these animals, of which 229 920 of the latter were immature and 6 925 adults. this is still a large number of adult ticks considering that it was mid winter and that most adult ticks are encountered on wild life and on domestic cattle in south africa during summer (baker & ducasse 1967; rechav 1982; horak et al. 1984, 1992, 1995, 2003). however, this large number of adult ticks on buffaloes even during winter is not surprising considering that the larger the host species the more adult ticks it is likely to harbour (horak et al. 1987; gallivan & horak 1997). african buffaloes and eland, taurotragus oryx, are the largest wild bovids in south africa and consequently, irrespective of the season, are usually infested with more adult ticks than other host species (horak et al. 1983a, 1987). the absence, or virtual absence, of adult r. (b.) decoloratus on the buffaloes is thus the more conspicuous. judging by the small tick burdens of impalas and nyalas examined at the same localities in northeastern kzn as the buffaloes (table 3), this region is marginal for r. (b.) decoloratus. because of their large size and hence the amount of meat that can be obtained from them, buffaloes that are to be culled are usually slaughtered in winter to avoid the carcass being exposed to both the heat of the sun and to exploitation by blowflies. the same applies in respect of heat stress in buffaloes that are to be chemically immobilized for any length of time. winter is a season during which the numbers of r. (b.) decoloratus on wildlife may be at their lowest (horak et al. 1984, 1992), and hence it is not an ideal time during which to compare the tick burdens of various host species, particularly if the region is marginal for r. (b.) decoloratus. conclusion african buffaloes appear to acquire resistance to infestation with the one-host tick r. (b.) decoloratus, and this resistance is expressed as the prevention of the majority of tick larvae from developing to nymphs. the correctness of this conclusion can, however, only be verified when buffaloes are exhaustively examined for ticks at a locality in which susceptible hosts belonging to other species are heavily infested with r. (b.) decoloratus. acknowledgements we are most grateful to ezemvelo kzn wildlife, sanparks and the provincial division of nature con servation of mpu malanga province for placing the 198 host status of african buffaloes, syncerus caffer, for rhipicephalus (boophilus) decoloratus animals in their reserves at our disposal, and for providing assistance and facilities to process the animals for tick recovery. we are particularly indebted to dr pete rogers who facilitated the arrangements for the studies in the kzn nature reserves. the assistance of ms m. cohen and dr j.p. louw with processing the immobilized animals or carcasses for tick recovery is greatly appreciated. funds for the conduct of this project were provided by the university of pretoria and the national research foundation. this work has been facilitated through the integrated con sortium on ticks and tick-borne diseases (icttd3), financed by the international cooperation program of the european union through coordination action project no. 510561. references baker, maureen k. & ducasse, f.b.w. 1967. tick infestation of livestock in natal. i. the predilection sites and seasonal variations of cattle ticks. journal of the south african veterinary medical association, 38:447–453. bezuidenhout, j.d. & stutterheim, c.j. 1980. a critical evaluation of the role played by the red-billed oxpecker buphagus erythrorhynchus in the biological control of ticks. onderstepoort journal of veterinary research, 47:51–75. gallivan, g.j. & horak, i.g. 1997. body size and habitat as determinants of tick infestations of wild ungulates in south africa. south african journal of wildlife research, 27:63–70. horak, i.g., potgieter, f.t., walker, jane b., de vos, v. & boomker, j. 1983a. the ixodid tick burdens of various large ruminant species in south african nature reserves. onderstepoort journal of veterinary research, 50:221–228. horak, i.g., de vos, v. & brown, moira r. 1983b. parasites of domestic and wild animals in south 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19:679–700. skinner, j.d. & smithers, r.h.n. 1990. the mammals of the southern african subregion. pretoria: university of pretoria. spickett, a.m., de klerk, d., enslin, c.b. & scholtz, m.m. 1989. resistance of nguni, bonsmara and hereford cattle to ticks in a bushveld region of south africa. onder stepoort journal of veterinary research, 56:245–250. walker, j.b. 1974. the ixodid ticks of kenya. a review of present knowledge of their hosts and distribution. london and reading: commonwealth institute of entomology. yeoman, g.h. & walker, j.b. 1967. the ixodid ticks of tanzania. a study of the zoogeoraphy of the ixodidae of an east african country. london and reading: commonwealth institute of entomology. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [878.740 1133.858] >> setpagedevice abstract introduction materials and methods results discussion acknowledgements references about the author(s) imna i. malele vector & vector borne disease institute, tanga, tanzania johnson o. ouma biotechnology research institute, kenya agricultural and livestock research organization, muguga, kenya africa technical research centre, vector health international, arusha, tanzania hamisi s. nyingilili vector & vector borne disease institute, tanga, tanzania winston a. kitwika vector & vector-borne diseases centre, kigoma, tanzania deusdedit j. malulu vector & vector borne disease institute, tanga, tanzania henry b. magwisha tanzania veterinary laboratory agency, dar es salaam, tanzania eliningaya j. kweka division of livestock and human diseases vector control, tropical pesticides research institute, arusha, tanzania department of medical parasitology and entomology, catholic university of health and allied sciences, mwanza, tanzania citation malele, i.i., ouma, j.o., nyingilili, h.s., kitwika, w.a., malulu, d.j., magwisha, h.b. et al., 2016, ‘comparative performance of traps in catching tsetse flies (diptera: glossinidae) in tanzania’, onderstepoort journal of veterinary research 83(1), a1057. http://dx.doi.org/10.4102/ojvr.v83i1.1057 research project no.: becanet grant no. 2/2007 and who/tdr no. a80132 original research comparative performance of traps in catching tsetse flies (diptera: glossinidae) in tanzania imna i. malele, johnson o. ouma, hamisi s. nyingilili, winston a. kitwika, deusdedit j. malulu, henry b. magwisha, eliningaya j. kweka received: 08 sept. 2015; accepted: 17 dec. 2015; published: 23 june 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study was conducted to determine the efficiency of different tsetse traps in 28 sites across tanzania. the traps used were biconical, h, ngu, nzi, pyramidal, s3, mobile, and sticky panels. stationary traps were deployed at a distance of 200 m apart and examined 72 h after deployment. the results showed that 117 (52.2%) out of the 224 traps deployed captured at least one glossina species. a total of five glossina species were captured, namely glossina brevipalpis, glossina pallidipes, glossina swynnertoni, glossina morsitans, and glossina fuscipes martinii. biconical traps caught tsetse flies in 27 sites, pyramidal in 26, sticky panel in 20, mobile in 19, s3 in 15, ngu in 7, h in 2 and nzi in 1. a total of 21 107 tsetse flies were trapped, with the most abundant species being g. swynnertoni (55.9%), followed by g. pallidipes (31.1%), g. fuscipes martinii (6.9%) and g. morsitans (6.0%). the least caught was g. brevipalpis (0.2%). the highest number of flies were caught by ngu traps (32.5%), followed by sticky panel (16%), mobile (15.4%), pyramidal (13.0%), biconical (11.3%) and s3 (10.2%). nzi traps managed to catch 0.9% of the total flies and h traps 0.7%. from this study, it can be concluded that the most efficient trap was ngu, followed by sticky panel and mobile, in that order. therefore, for tsetse fly control programmes, ngu traps could be the better choice. conversely, of the stationary traps, pyramidal and biconical traps captured tsetse flies in the majority of sites, covering all three ecosystems better than any other traps; therefore, they would be suitable for scouting for tsetse infestation in any given area, thus sparing the costs of making traps for each specific glossina species. introduction there are 33 species and subspecies of tsetse flies (diptera: glossinidae) in africa, infesting 36 countries south of the sahara (gooding & krafsur 2005). tsetse flies transmit trypanosomes that cause african trypanosomiasis (at) in humans and livestock. the disease has negative effects on the hosts as it causes low productivity, leading to mortality when untreated. in livestock, some of the direct losses caused by the disease include abortion, reduced milk yield and poor calf crop. given its role in transmitting at in livestock and humans, the tsetse fly deserves special attention, particularly with regard to its response to different sampling methods in a variety of ecological settings. studies on tsetse ecology and control have mainly relied on the availability of an efficient sampling method, a number of which have been described. the main sampling methods include the use of fly nets and fly rounds (pollock 1982), sticky materials (vreysen, khamis & van der vloedt 1996), and fabric traps (challier & laveissiere 1973; dransfield & brightwell 1997; ndegwa & mihok 1999; vale 1982; vale, flint & hall 1986). most of the traps used in the foregoing studies were developed in west and southern africa for riverine and savannah tsetse species, respectively. interestingly, the sampling studies carried out thus far have revealed variable efficiency of the traps in capturing tsetse flies. in view of this, the efficiency of traps to capture certain tsetse species has been enhanced through modification of various designs of traps for use against particular target species in relation to the environment (ndegwa & mihok 1999; ndegwa, mihok & oyieke 2001). traps basically function through visual stimuli. in the field, however, the visual stimuli can be greatly obstructed by vegetation, particularly for forest species. in such cases, attraction of flies to traps is enhanced through the use of odour attractants. three groups of natural odours have been determined so far from the host animals: those found in urine (e.g. phenols), breath (e.g. acetone) and skin secretions (e.g. octenols (vale 1993). the response of tsetse flies to particular odours varies amongst species (green 1986; kuzoe & schofield 2004). principally, tsetse traps are made up of blue and black textile materials and white netting. the blue colour has been found to be a visual stimulus or attractant to flies. field observations on tsetse trapping show that all tsetse traps attract tsetse flies to land on them. however, the difference in their relative trapping efficiency is based on their designs, especially the entrance for flies into the retaining cage. this has been the basic reason for modifying conventional traps and designing new ones to suit the target species in different types of habitats. considerable advances have been made in the development of efficient traps for tsetse flies, as trapping is increasingly being used for population suppression and control of tsetse flies. currently, the common traps used for sampling and monitoring economically important tsetse species include the biconical trap (challier et al. 1977), developed for sampling glossina morsitans; f3 and epsilon (flint 1985; green & flint 1986) for glossina pallidipes; ngu (dransfield & brightwell 1997) for g. pallidipes; nzi (mihok 2002) for tsetse flies, horse flies, deer flies and stable flies; s3 (ndegwa & mihok 1999) for glossina swynnertoni and the pyramidal (goutex & lancien 1986) for glossina tachinoides. however, it has been found that the difference in trap efficiency is related to the behavioural differences between the species and varies between different populations of the same species. in some cases, minor modifications are required to improve the efficiency of a trap. for example, the s3 trap underwent a series of modifications before it was perfected (ndegwa & mihok 1999). knowledge of the response of particular tsetse species to specific traps with or without odours is important for enhancing the efficiency of tsetse fly suppression operations and the formation of barrier systems used in tsetse control or eradication campaigns. in tanzania, despite the vast area that is infested by tsetse flies, tsetse trapping has mainly depended on traps developed outside the country, targeting different vegetation types and tsetse species, except for the mobile, scoop (kuzoe & schofield 2004) and sticky panel (vreysen et al. 1996). the dominant tsetse species are the savannah tsetse species, which include g. pallidipes, g. swynnertoni and g. morsitans morsitans. other species that are not widely distributed include glossina austeni, glossina brevipalpis, glossina longipennis, glossina fuscipes martinii and g. fuscipes fuscipes. unpublished results (vvbd – tanga) show that the response of tsetse flies to traps in tanzania varies from one area to another even within the same species. it is likely that such variation could affect the results obtained from tsetse surveys, particularly data on fly density and distribution. this study was aimed at investigating the efficiency of different traps for different tsetse species so that, if need be, a single trap could be used for sampling different tsetse species if it proved to be efficient against several tsetse species. for example to trap g. swynnertoni, several traps have been used, including pyramidal traps (malele et al. 2007; mramba et al. 2013), epsilon traps (auty et al. 2012), rectangular cloth targets and small leg panels (mramba et al. 2013). however, the comparative performance of several traps against the species has not been documented. variations in response in relation to traps and fly species have a negative impact, especially when planning for tsetse and trypanosomiasis control. furthermore, such variations could lead to underestimation of the production losses and public health harm caused by tsetse flies and tsetse-borne diseases. on the other hand, if one trap is found to be efficient against several tsetse species, then the cost of making several traps to suit several species present in an area could be avoided. apart from sampling, efficient tsetse sampling traps have been used elsewhere as cheap control devices against the vector (madubunyi 1988). we report the results of studies carried out to determine the relative efficiency of different tsetse traps in trapping various species of tsetse flies in different ecological settings in the serengeti ecosystem (mara region), the western ecosystem (kigoma and tabora regions) and the southern ecosystem (selous game reserve, which covers lindi and the south-eastern part of the morogoro region), and we recommend the most efficient trap for each species according to the ecological zonation in the three ecosystems in tanzania. the data presented were collected from 2008 to 2012. materials and methods study areas serengeti ecosystem the serengeti ecosystem is situated in the mara region, northern tanzania, and consists of serengeti national park (senapa), ngorongoro conservation area and maswa game reserve. the site comprises a savannah habitat with a wide range of wild game, including wildebeests, elephants, antelopes, lions, wild pigs, buffaloes and giraffes, most of which serve as a source of blood meals for tsetse. tsetse fly species found in the serengeti include g. morsitans, g. pallidipes, g. brevipalpis and g. swynnertoni, with the predominant species being g. swynnertoni. western ecosystem uvinza (kigoma): the uvinza site is found in western tanzania and borders the moyowosi game reserve to the west and mpanda/uvinza game reserve and ilunde and chakulu forest reserves to the east. the habitat is mainly miombo woodland. the wild animals found at uvinza include buffaloes, antelopes and wild pigs migrating from the neighbouring game reserves. however, part of the area is used for cattle ranching. the common tsetse flies in the uvinza area are g. morsitans, g. pallidipes, g. f. martinii and g. brevipalpis. ugalla (urambo): the ugalla game reserve is the key component of the study in the ugalla area. the climate is defined by a distinct wet season from december to june and a dry season from july to november. the vegetation consists of dry zambezian miombo woodland; wooded grassland with reduced tree cover is the most widespread vegetation type in the area. wild animals are common in the ecosystem. the herbaceous layer is dominated by hyperrhenia species, with a shrub layer of saplings of the canopy trees. the livelihoods of the local people around ugalla game reserve consist of a mixture of activities such as livestock keeping, agriculture, fishing, hunting, beekeeping and the harvesting of forest products (lutabingwa 2006). tsetse sampling was conducted at kangeme, lumbe, ukumbi-siganga and usinga. common tsetse species in the area are g. morsitans and g. pallidipes. southern ecosystem selous game reserve: the park varies from rolling grassy woodlands and plains to rocky outcrops cut by the rufiji river – the lifeblood of the park, with tributaries that form a network of lakes, lagoons and channels. this is one of the areas in tanzania with a high density of wild animals that include, naming but a few, antelopes, crocodiles, hippos, and black-and-white colobus monkeys in the riverine forests. during the dry season from june to october, animals tend to concentrate along the river linked to the rufiji in lake tagalala, where waterbuck, reedbuck and bushbuck are abundant. in the dry season, there is a notable migration of elephants between the selous game reserve and mozambique’s niassa game reserve. tsetse sampling was conducted along game-viewing and camping sites owned by mivumoni river lodge and selous luxury camp of serena hotels. common tsetse species include g. morsitans and g. pallidipes (malele et al. 2013). traps eight traps, namely the biconical, pyramidal, ngu, mobile, sticky panel, s3, nzi and h, were compared for relative efficiency in trapping different tsetse fly species in a total of 28 sites in the three ecosystems (serengeti, western and southern tanzania). deployment of the traps in the field was as described by vale (1982) and fao (1992). the traps were deployed at an interval of about 200 m apart in a latin square study design (days × treatments × sites) and remained at one site for 72 h before being transferred to a new site. tsetse flies caught in each trap at each site were identified to sex and species levels, pooled and recorded. tsetse catches by the sticky panel and mobile traps were included in the analysis just for comparison purposes because any fly that lands on sticky panels is retained and those trapped by a mobile trap are scooped (sucked into a retaining cage), whereas tsetse flies may visit and leave other stationary traps without entering the retention cage. data analysis data on the tsetse catches from the 28 sites of the three ecosystems were collected and recorded on sheets, entered in microsoft excel and then transferred to epi info (2014) analytical software for analysis. one-way analysis of variance was used to analyse the efficiency of each trap for the five species trapped. tsetse fly counts were used as independent variables and trap type, species and ecosystem as grouping variables. the overall comparison of the traps’ sampling efficiency regardless of the tsetse fly species was done using generalised linear model univariate analysis. traps and sexes were considered for a full factorial model for main-effect analysis. separation of means was done at the 95% confidence interval and a significance level of 5% in all the statistical tests. results overall counts of glossina species from various areas overall, a total of 21 107 tsetse flies were caught from the 28 sites (table 1). six sites with the highest counts contributed 12 358 tsetse flies (58.5%); these were death valley, 5928; uvinza, 1661; hippo area, 1298; hembe, 1289; retima pool, 1101 and mareo, 1081. the remaining 8749 flies were caught at the remaining 22 sites. table 1: descriptive statistics for sites. the trapping performance of stationary traps for different glossina species demonstrated that although the ngu traps caught tsetse in only 7 sites compared with the biconical and pyramidal traps, which caught flies in over 25 sites, ngu trapped more tsetse flies than any other traps used in the study. by ranking the means, ngu traps were found to be significantly more efficient (p < 0.05) than biconical and pyramidal traps (table 2). table 2: overall counts of positive traps for glossina species. five glossina species were caught at the following proportional percentages in decreasing order: g. swynnertoni (55.9%), g. pallidipes (31.1%), g. f. martini (6.9%), g. morsitans (6.0%) and g. brevipalpis (0.2%). the average species count of g. swynnertoni was significantly different (p = 0.05) from that of all other species; g. pallidipes was significantly different from g. morsitans, g. f. martinii and g. brevipalpis, but the last three species were not significantly different from one another. the performance of traps for each ecosystem is shown in table 3. nearly all the traps were able to catch flies in the serengeti ecosystem, except for the h trap. table 4 shows the occurrence of different tsetse species per ecosystem. table 3: comparison of means of trap performance per ecosystem. table 4: mean rank of tsetse species per ecosystem. out of the 224 traps deployed, only 117 (52.2%) captured flies. table 5 shows that 73 (62.4%) out of these 117 traps caught a single glossina species, whereas 42 (35.9%) traps caught two species each, and only 2 (1.7%) traps had three species each. the following traps demonstrated consistent efficiency, in decreasing order, for trapping g. swynnertoni: mobile, sticky panel, pyramidal, and biconical. biconical traps caught tsetse flies at 27 sites, pyramidal at 26, sticky panel at 20, mobile at 19, s3 at 15, ngu at 7, h at 2 and nzi at 1. table 5: single, double or triple species trapped by different traps. glossina swynnertoni was captured by 90 traps, g. pallidipes by 52, g. morsitans by 10 and g. f. martinii by 8; the least trapped tsetse fly was g. brevipalpis, caught by 3 traps. glossina pallidipes and g. swynnertoni were sympatric species recorded in 29 (24.8%) occurrences (table 5). out of the 21 107 tsetse flies trapped, 1449 were trapped at uvinza and kagerankanda in kigoma. g. f. martinii was only found in the western part of the country, along the lake shores of lake tanganyika and along the rivers draining into lake tanganyika. most of the g. f. martinii flies were not sorted into their respective sexes. of the sorted flies (19 658), females were significantly more numerous than males (one male to two females) (p < 0.05). also, more females were trapped by sticky panels than by mobile traps, and more males were trapped by mobile traps than by sticky panels. in total, however, the mobile traps caught more flies than the sticky panels (table 6). table 6: mean tsetse sexes per mobile trap versus sticky panel. discussion five species of glossina were recorded in this study. proportionally, the species ranged in decreasing order from g. swynnertoni (55.9%) to g. pallidipes (31.1%), g. f. martinii (6.9%), g. morsitans (6.0%) and g. brevipalpis (0.2%). g. swynnertoni was the dominant species in the serengeti ecosystem, whereas g. morsitans was the dominant species in the western ecosystem. species dominance in specific regions is likely due to the ready and continuous availability of preferred hosts in those regions. confirmation of preferred hosts is only achievable through blood meal analysis, which was outside the scope of this study. combining an analysis of tsetse-trapping efficiency with an analysis of the origin of the blood meals for some of the most commonly used tsetse traps would add value and is a subject for further investigation. on trap performance, the ngu traps caught tsetse flies at only 7 sites compared with the biconical and pyramidal traps, which caught flies in over 25 sites; however, ngu trapped more tsetse flies than the rest of the traps used in the study. although this demonstrates the superior efficiency of the ngu trap, it is unclear whether the failure to catch flies at most of the sites was a result of interand intra-species differences in behaviour and response to ngu. all the traps used in this study, with the exception of h and nzi, which trapped very few flies, could be used for sampling tsetse flies in the serengeti ecosystem, although their efficiency varies. in the western zone, all traps except nzi and s3 could be used for sampling or trapping flies. in the southern zone, the mobile, biconical, pyramidal and sticky panel traps could be used for sampling or trapping purposes. again, g. swynnertoni could be trapped by all the traps used in the study except the h trap. the performances of the sticky panel and mobile traps showed no significant difference in the proportion of flies caught. the catches were 16% by sticky panel and 15.4% by mobile trap. it was noted, however, that the mobile trap can be used to catch g. swynnertoni, g. brevipalpis and g. morsitans, whereas the sticky panel is more suitable for catching g. swynnertoni. the suitability of the sticky panel for catching g. swynnertoni is consistent with the observations of mramba et al. (2013), although they used a different type of sticky panel, that is, all blue-legged panels. on the other hand, ngu performed better in catching g. pallidipes, g. morsitans, g. swynnertoni and g. f. martinii. in this study, ngu performed best for g. pallidipes, and this concurs with earlier findings for this trap, which was developed for savannah species (dransfield et al. 1986). the pyramidal trap has been the trap of choice in many studies in tanzania because of its simplicity in deployment; however, its trapping ability was not as superior as ngu, although it still was able to trap about 12.4% of the total flies in this study, second to ngu (amongst stationary traps) and slightly better than the biconical trap, which is usually used as a sampling device (takken 1984) (table 6). the suitability of biconical as a sampling trap (unbiased towards any one species) was demonstrated by its being one of the traps that trapped nearly all tsetse species in the study sites (table 5). it has been documented that the h trap is good for trapping g. brevipalpis (kappmeier 2000), which is typically a forest species. however, in this study the performance of the trap against g. brevipaplis was not good; perhaps vegetation cover could have influenced the results in the present study. trapping was carried out mostly in savannah wooded areas and not in forested areas, where the species is mostly found. for g. m. morsitans, the mobile trap performed better, followed by ngu (figure 1). when evaluating the overall performance of traps regardless of species, and by analysing the data considering the density of flies sampled regardless of species, significant statistical difference was found among the traps, with ngu having the highest sampling efficiency (figure 2). this affirms the superior performance of ngu, as already discussed earlier. only ngu trapped more flies as a stationary trap than the mobile trap, which sucks in any fly in the vicinity. figure 1: trapping performance of different traps for different species of tsetse flies. figure 2: overall tsetse fly species mean number per trap. from the foregoing observations, it can be concluded that the different tsetse traps being used in tanzania have varying trapping efficiencies, which in some cases seems to be dependent on the tsetse fly species being sampled and the ecological setting. however, pyramidal and biconical traps can be used for sampling or trapping tsetse flies, hence saving the cost of having different traps developed for each specific glossina species. acknowledgements this work was funded by becanet grant no. 2/2007 and who/tdr no a80132. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions i.i.m., j.o.o., h.s.n., w.a.k. performed the study, i.i.m., j.o.o., w.a.k. wrote the manuscript, h.b.m., e.j.k., d.j.m. performed the statistical analysis of the data. all 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entomological research 86, 289–296. abstract introduction research method and design results discussion acknowledgements references about the author(s) david ndeereh department of veterinary services, kenya wildlife service, kenya andrew thaiyah department of clinical studies, university of nairobi, kenya gerald muchemi department of public health, pharmacology and toxicology, university of nairobi, kenya antoinette a. miyunga forensics and genetics laboratory, kenya wildlife service, kenya citation ndeereh, d., thaiyah, a., muchemi, g. & miyunga, a.a., 2017, ‘molecular surveillance of spotted fever group rickettsioses in wildlife and detection of rickettsia sibirica in a topi (damaliscus lunatus ssp. jimela) in kenya’, onderstepoort journal of veterinary research 84(1), a1265. https://doi.org/10.4102/ojvr.v84i1.1265 original research molecular surveillance of spotted fever group rickettsioses in wildlife and detection of rickettsia sibirica in a topi (damaliscus lunatus ssp. jimela) in kenya david ndeereh, andrew thaiyah, gerald muchemi, antoinette a. miyunga received: 20 may 2016; accepted: 17 aug. 2016; published: 30 jan. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract spotted fever group rickettsioses are a group of tick-borne zoonotic diseases caused by intracellular bacteria of the genus rickettsia. the diseases are widely reported amongst international travellers returning from most sub-saharan africa with fever, yet their importance in local populations largely remains unknown. although this has started to change and recently there have been increasing reports of the diseases in livestock, ticks and humans in kenya, they have not been investigated in wildlife. we examined the presence, prevalence and species of rickettsia present in wildlife in two regions of kenya with a unique human–wildlife–livestock interface. for this purpose, 79 wild animals in laikipia county and 73 in maasai mara national reserve were sampled. dna extracted from blood was tested using the polymerase chain reaction (pcr) to amplify the intergenic spacer rpme-trnafmet and the citrate synthase-encoding gene glta. rickettsial dna was detected in 2 of the 79 (2.5%) animals in laikipia and 4 of the 73 (5.5%) in maasai mara. the pcr-positive amplicons of the glta gene were sequenced to determine the detected rickettsia species. this revealed rickettsia sibirica in a topi (damaliscus lunatus ssp. jimela). this is the first report of spotted fever group rickettsioses in wildlife and the first to report r. sibirica in kenya. the finding demonstrates the potential role of wild animals in the circulation of the diseases. introduction spotted fever group (sfg) rickettsioses are a group of tick-borne zoonotic diseases caused by over 20 species of the intracellular bacterium rickettsia (azad & beard 1998; parola, paddock & raoult 2005; raoult & roux 1997; todar 2012). they occur worldwide and are associated with diseases in animals and humans (azad & beard 1998; parola et al. 2005) and are diagnosed in some international travellers returning from sub-saharan africa (freedman et al. 2006; jensenius, parola & raoult 2006) including kenya (rutherford et al. 2004; yoshikawa et al. 2005). the clinical manifestations in humans are non-specific and mimic those of other diseases such as malaria and flu-like illnesses (krauss et al. 2003; roch et al. 2008). they are transmitted by different species of ixodidae ticks and reservoirs include various species of domestic and wild animals (cowan 2003; todar 2012). because wild animals are hosts of many species of ticks, the diseases can be of particular concern where wildlife shares habitats and other resources with humans and domestic animals (grootenhuis & olubayo 1993). the importance of sfg rickettsioses as causes of illnesses in kenya is underreported and underappreciated despite being reported amongst foreign travellers who visit game reserves (richards et al. 2010; rutherford et al. 2004; yoshikawa et al. 2005). this may be attributed to lack of awareness and the challenges of making diagnoses in febrile patients in africa (ari et al. 2011; brah et al. 2015). the diseases may therefore be amongst the ‘fevers of unknown origin’ whose aetiologies are often not the focus of health providers or are difficult to diagnose because of lack of resources (brah et al. 2015). nevertheless, there have been increasing reports in kenya of sfg rickettsioses in humans, domestic animals and ticks (macaluso et al. 2003; maina 2012; mutai et al. 2013; richards et al. 2010; rutherford et al. 2004; yoshikawa et al. 2005) but information about their presence in wildlife is lacking. in a situation where emerging and re-emerging zoonotic infections are on the increase with over 70% originating from wildlife (jones et al. 2008), it is important that studies extend to include all aspects of disease epidemiology. wildlife plays an important role of disease epidemiology but is often neglected in surveillance and detection of diseases. wildlife can be important reservoir hosts of many tick-borne pathogens including sfg rickettsiae, which can be transmitted to domestic animals and humans in areas where wildlife and domestic animals share habitats and other resources. this study investigates sfg rickettsioses at the wildlife–livestock interfaces in laikipia county and maasai mara national reserve. it evaluates their presence, prevalence and identifies the species of sfg rickettsiae circulating in wildlife. research method and design study areas and sampling procedure blood was collected from immobilised wild animals in laikipia county and maasai mara national reserve between february 2014 and october 2015. laikipia county is about 9500 km2 and is located in the central region of kenya to the northwest of mt. kenya between 0.88n 36.18e and 0.2667s 37.38e. the maasai mara national reserve is approximately 1510 km2 between 1.22s 34.75e and 1.75s 35.42e within narok county in southwestern kenya along the border with tanzania. the main human populations in both areas are pastoralists whose livelihoods are dependent on livestock. other forms of land use include agriculture, commercial ranching, wildlife conservation and ecotourism. the two areas comprise some of the most important areas for biodiversity in kenya, and they have large populations of free-ranging wildlife, which share habitats and other resources with humans and domestic animals providing a likely interface for disease transmission. the sampling sites in each study area were selected based on high interaction of livestock and wildlife and accessibility to enable darting of the animals. the coordinates of each sampling site were recorded using a global positioning system (gps) (garmin gps 12 xl, garmin olathe, ks, usa) and entered into a geographical information system (gis) database. convenience sampling of the animals was employed because of the difficulties of constructing a sampling frame in wildlife to allow for random sampling. this method allowed for readily available animals of the target species to be sampled. the target species were those most common in the study areas with the high tendency to interact with livestock. these included buffalo (syncerus caffer), zebra (equus burchellii), grant’s gazelle (nanger granti), common waterbuck (kobus ellipsiprymnus ssp. ellipsiprymnus), impala (aepyceros melampus), topi (damaliscus lunatus ssp. jimela), coke’s hartebeest (alcelaphus buselaphus) and wildebeest (connochaetes taurinus). to facilitate sample collection, the animals were immobilised following the protocols recommended by mckenzie (1993) by experienced personnel to ensure a humane exercise as much as possible. at least 30 ml of blood was collected from each animal by jugular venipuncture into edta-coated tubes and split into four aliquots. each aliquot was labelled with information identifying the sample number, date, location and animal species and stored frozen in liquid nitrogen (-196 °c) until required for processing. dna extraction genomic dna was extracted from preserved edta blood samples using the manufacturer’s instructions for the dneasy® blood and tissue kits (qiagen gmbh, hilden, germany). however, these instructions were modified slightly in order to optimise the amount of dna extracted by increasing the amount of blood from 50 µl to 100 µl as recommended by the manufacturer to 200 µl and reducing the amount of ae buffer from 200 µl to 150 µl. extracted dna quality was evaluated using the agarose gel electrophoresis protocol in which an aliquot of the extracted dna was run on 1.2% agarose gel. screening for rickettsia host dna was screened for evidence of rickettsia using pcr assay to amplify the intergenic spacer rpme-trnafmet and the citrate synthase-encoding gene glta using previously described primer sets shown in table 1. the amplifications were carried out in a total volume of 25 µl reaction mix containing 1 µl deoxyribonucleotide triphosphates solution (dntps), 2.5 µl standard taq buffer (biolabs®, new england, uk), 1 µl each of reverse and forward primers, 17.25 µl of dnase/rnase-free® pcr grade water (qiagen, hilden, germany), 0.25 µl of taq dna polymerase (biolabs®, new england, uk) and 2 µl of template dna. the amplifications were performed in applied biosystems veriti® 96-well thermocycler (applied biosystems, california, usa). the pcr conditions were as follows: 3 min initial denaturation at 95 °c, 35 cycles of 30 s denaturation at 95 °c, 30 s primer annealing at temperatures specific for each of the primers (table 1), one minute extension at 72 °c and a final 10 minute extension at 72 °c. the mixture was then maintained at 4 °c. a negative control using dnase/rnase-free® pcr grade water (qiagen, hilden, germany) was included for quality control. the positive control used was dna extracted from rickettsia africae isolated from a tick in kenya. the pcr products were visualised using agarose gel electrophoresis to check for the presence of band and size of amplicon. table 1: primers used for polymerase chain reaction amplification and sequencing of spotted fever group rickettsiae in wildlife. dna sequencing and analysis the glta-positive pcr amplicons of, or close to, the expected product sizes were purified using qiaquick® purification kit (qiagen, hilden, germany) following the manufacturer’s instructions. sequencing was done by direct cycle sequencing using the abi prism bigdye terminator v3.1 cycle sequencing kit and the sequences analysed in an abi310 dna analyser (applied biosystems, california, usa). traces were assembled and primer regions trimmed using geneious v 8.1.6 software. consensus nucleotide sequences were used to query the genbank database, and the highest similarity was identified by basic local alignment search tool (blastn) available from the national center for biotechnology information (bethesda, md). this was used to assign identity to the recovered species. the study sequences along with those with closest match in genbank were aligned using muscle (edgar 2004). the evolutionary history was inferred by using the maximum likelihood method based on the tamura 3-parameter model (tamura 1992). the phylogeny tree with the highest log likelihood (-1233.9127) was developed. the percentage of trees in which the associated taxa clustered together was shown next to the branches. initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and bionj algorithms to a matrix of pair-wise distances estimated using the maximum composite likelihood (mcl) approach and then selecting the topology with superior log likelihood value. the tree was drawn to scale with branch lengths measured in the number of substitutions per site. the analysis involved 15 nucleotide sequences. codon positions included were 1st+2nd+3rd+noncoding. there were a total of 774 positions in the final dataset. evolutionary analyses were conducted in mega6 (tamura et al. 2013). results rickettsial infection in animals animals were sampled in 17 locations in laikipia county and 19 locations in maasai mara national reserve and the neighbouring areas during six separate expeditions between february 2014 and october 2015 (table 2). the sampled areas in laikipia county included nine sites in ol pejeta conservancy, five sites in adc mutara ranch and two sites in mpala ranch all of which incorporate cattle ranching and wildlife conservation as well as one site in kiamariga sub-location, which is a community land where free-ranging wildlife interacts with livestock (figure 1). in maasai mara, sampled areas included seven sites inside the national reserve and 12 sites in neighbouring community group ranches where wildlife interacts freely with livestock (figure 1). table 2: animals sampled in each site in both study areas and corresponding dates. figure 1: sampled sites in laikipia and maasai mara. in total, 152 animals comprising of eight different species were sampled in both areas. these comprised of 79 in laikipia and 73 in maasai mara. all the animals responded well to the immobilisation drugs and induction times ranged between 8 and 12 min. no complications were encountered during immobilisation and handling except for a few animals, which had slightly elevated body temperatures that were attributed to physical exertion during darting as well as psychological stress and fear. these animals were cooled by applying copious amounts of water on the whole body. two of the 79 (2.5%) animals in laikipia were found infected with sfg rickettsioses. infections were found in a zebra (equus burchellii) and a buffalo (syncerus caffer) representing a prevalence of 2.6% and 3.2% in these species, respectively. four of the 73 (5.5%) animals in maasai mara were infected with sfg rickettsioses. infections were found in 1 of the 35 wildebeests (connochaetes taurinus) and 3 of the 30 topi (damaliscus lunatus ssp. jimela) representing a prevalence of 2.9% and 30%, respectively. infections were detected by amplification of the intergenic spacer rpme-trnafmet and glta gene. these findings are summarised in table 3. a representative gel image of pcr amplification of glta gene is shown in figure 2. table 3: animals species sampled and prevalence of spotted fever group rickettsioses. figure 2: gel image of polymerase chain reaction amplifications of the glta gene. identification of rickettsia spp. rickettsia sibirica was identified in one sample obtained from a topi (damaliscus lunatus ssp. jimela) in maasai mara national reserve. the isolate yielded a partial glta sequence of 779 bp that had 99% identity to r. sibirica isolated from china accession number km288711 in the genbank. the study sequence was submitted to the genbank and provided accession number kx244606. the maximum likelihood phylogeny tree drawn using the study sequence and those similar to it in genbank is shown in figure 3. from the tree, the study sequence has a close relationship with other sequences from the same species in genbank, such as r. sibirica from senegal, while having a more distant relationship with the same species from other continents. figure 3: a phylogenetic relationship between rickettsia sibirica isolated from topi in kenya and other rickettsia sequences in the genbank. discussion sfg rickettsioses can potentially be a public health concern in areas such as laikipia and maasai mara which have unique human–livestock–wildlife interfaces that can potentially facilitate transmission of zoonotic infectious pathogens across different species. sfg rickettsioses were detected in 2.5% and 5.5% of wildlife sampled in laikipia and maasai mara, respectively. this is the first report of the presence of the diseases in wildlife in kenya, which demonstrates that wildlife may play a role in their spread. the finding of the presence of sfg rickettsioses in wildlife is consistent with a study by zhang, fan and bi (1995) who, using pcr reported a prevalence of 7.4% in wild mice in china. inokuma et al. (2008) and ortuno et al. (2007) also reported the presence of sfg rickettsioses using pcr in a deer in japan and a wild boar in spain, respectively. other studies by boretti et al. (2009) and barandika et al. (2007) using similar methods reported no detection in wild foxes in switzerland and wild small mammals in spain. the finding of low prevalence in wildlife is also comparable to a study in domestic animals by maina (2012) who reported a prevalence of 3.7% in dogs and 7.7% in cats in western kenya and no detection in cattle, sheep and goats. it is also comparable to a study by kleinerman et al. (2013) who reported a prevalence of 2.0% in camels but no detection in horses in israel. the finding, however, contrasts several other studies that have reported higher prevalence in domestic animals in kenya. using pcr, mutai et al. (2013) reported a higher prevalence of 16.3% in cattle and 15.1% in sheep but a lower prevalence of 7.1% in goats from various parts of kenya. likewise, kamani et al. (2015) reported a higher prevalence of 18.8% in camels in nigeria. the presence of r. sibirica has not been reported before in kenya. the pathogen is widely distributed in north asia (jensenius, fournier & raoult 2004) with no reports available about its detection in africa. it is the causative agent of north asian tick typhus also called siberian tick typhus (jensenius, fournier & raoult 2004). the illness is characterised by fever, malaise, headache, myalgias and regional lymphadenopathy (jensenius, fournier & raoult 2004; ramos et al. 2013), which may be confused with those of other febrile infections leading to misdiagnosis. it is therefore of interest to understand how local populations in laikipia and maasai mara cope with infections by r. sibirica. the study documents the presence of sfg rickettsioses in wildlife, which suggests that wildlife can play a role in the epidemiology of the diseases. the finding underscores the risks for zoonotic transmission of sfg rickettsioses to humans and domestic animals at the wildlife–livestock interfaces in laikipia and maasai mara. it is recommended that serological and molecular studies be initiated to determine sfg rickettsioses prevalence in local residents. acknowledgements this study was made possible through the facilitation of the head of the veterinary services department at kenya wildlife service. the authors acknowledge the assistance provided by moses yongo, the molecular biologist at the forensic and genetics laboratory at the kenya wildlife service. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions the authors contributed equally in the study conception and design, data collection and analysis and preparation of the manuscript. references ari, m.d., guracha, a., fadeel, m.a., njuguna, c., njenga, m.k., kalani, r. et al., 2011, ‘challenges of establishing the correct 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‘estimation of the number of nucleotide substitutions when there are strong transition-transversion and g + c-content biases’, molecular biology and evolution 9, 678–687. tamura, k., stecher, g., peterson, d., filipski, a. & kumar, s., 2013, ‘mega6: molecular evolutionary genetics analysis version 6.0’, molecular biology and evolution30(12), 2725–2729. todar, k., 2012, ‘rickettsial diseases including typhus and rocky mountain spotted fever’, todar’s online textbook of bacteriology, university of wisconsin, department of bacteriology, madison, wisconsin, viewed n.d., from http://www.textbookofbacteriology.net yoshikawa, h., kimura, m., ogawa, m., rolain, j. & raoult, d., 2005, ‘laboratory-confirmed mediterranean spotted fever in a japanese traveller to kenya’, american journal of tropical medicine and hygiene73(6), 1086–1089. zhang, j.z., fan, m.y. & bi, d.z., 1995, ‘detection of spotted fever group rickettsiae in ticks and rodents by polymerase chain reaction technique in people’s republic of china’, acta virologica 39(5–6), 263–267. skinner_237-239.indd introduction birth records from mammals bred in captivity are an important source of information (skinner, moss & skinner 2002), especially where the species are endangered, vulnerable or valuable for other reasons. the white rhinoceros, ceratotherium simum, and cape buffalo, syncerus caffer, are members of the so-called “big five” which play an important role in ecotourism and in the game ranching industry. both species command extremely high prices at game sales for aesthetic reasons, their importance as trophy animals and the scarcity of “disease-free” (e.g. foot-and-mouth disease, corridor disease and bovine tuberculosis) buffaloes. it is therefore useful to garner as much information as possible from breeding records of captive animals to improve basic knowledge and husbandry. in this paper are analysed the breeding records of white rhinoceros and cape buffaloes from a number of years’ data collected routinely at the lichtenburg game breeding centre and the solole private nature reserve, and compared with published data, where available. 237 onderstepoort journal of veterinary research, 73:237–239 (2006) research communication captive breeding of the white rhinoceros, ceratotherium simum, and the cape buffalo, syncerus caffer j.d. skinner1, h.m. dott1, a. matthee2 and l. hunt3 abstract skinner, j.d., dott, h.m., matthee, a. & hunt, l. 2006. captive breeding of the white rhinoceros, cera totherium simum, and the cape buffalo, syncerus caffer. onderstepoort journal of veterinary re search, 73:237–239 breeding records of 40 white rhinoceros and 155 cape buffalo were analysed. three rhinoceros cows bred in captivity, themselves conceived for the first time at 84, 87 and 95 months of age, respectively. rhinoceros cows breed throughout the year. there is no evidence of a relationship between calving interval and month of birth. calving intervals were normally distributed about the mean of 34 months and there were no significant differences between bulls, cows or sex of calf. there was no difference in the sex ratio of calves born to young cows nor older cows. the male:female ratio of the calves was 1:1. younger cows did not have shorter birth intervals. although captive cape buffaloes breed throughout the year, there is a preponderance of births in midsummer. there was some evidence that larger cows produce heavier calves and that season of birth may influence birth weight. male calves weighed 41.20 ± 0.68 kg vs 39.00 ± 0.73 kg (range 24–60 kg) for female calves but this difference was not significant. calving intervals were normally distributed about the mean of 395 days and the male:female ratio of the calves was 1:1.2. keywords: birth weights, breeding season, buffalo, calving interval, rhinoceros, sex ratio 1 veterinary wildlife unit, faculty of veterinary science, uni versity of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: john.skinner@up.ac.za 2 national zoological gardens, p.o. box 754, pretoria, 0001 south africa 3 solole private nature reserve, p.o. box 824, sun valley, 7975 south africa accepted for publication 24 april 2006—editor 238 captive breeding of white rhinoceros, cera totherium simum and cape buffalo, syncerus caffer methods rhinoceroses data were routinely collected from 1980–2003 at the lichtenburg game breeding centre in the north west province (26°15’ s; 26°17’ e). the rhinoceroses are free ranging and maintained in a 2 500 ha enclosure on natural veld (mixed sweet veld type, acocks 1988) supplemented with lucerne hay in winter when the nutritional level of natural forage declines markedly. the adult sex ratio is 1 male:5 females. data on births are routinely collected and recorded immediately after calves are born, as they are precocial and accompany their dams from the outset. date of conception is calculated using a gestation length of 16 months (owen-smith 1988). buffaloes data were routinely collected from 1998 to 2001 at the solole private nature reserve, phalaborwa, mpu malanga province (23°56’ s; 31°09’ e). the buffaloes are retained in paddocks and fed with teff and/or lucerne hay. cows are mated with wild captured bulls and the calves are removed and weighed at birth before being isolated from their dams and reared artificially on foster mothers in order to keep them disease free. cows are placed in three categories according to size: extra large, large and medium. statistical analysis the means are presented ± the standard error. means were compared using a student t-test or a one-way analysis of variance. results white rhinoceroses age at first conception for three cows bred and born at lichtenburg was 84, 87 and 95 months. a total of 40 calves were born, but only 33 calving intervals are available. the calving intervals were normally distributed about the mean (34 ± 1.8 months; range 21–61 months). there was no significant difference in calving interval between dams (anova f = 1.745; df 5.24; p = 0.16) (table 1). the calving interval after a male calf (35 ± 3.0 months) was not significantly different from that after a female calf (33 ± 2.0 months; t = 0.39; df 31; p = 0.69). there is not a clear-cut calving season but 12 (30 %) of the calves were born in march and april. there is no evidence of a relationship between calving interval and month of birth, e.g. in january three calves were born and there was an annual interval of c 32 months until the next calf was born. the sex ratio was 1:1. buffaloes the monthly distribution of buffalo calf births over a period of 3 years is illustrated in fig. 1. the mass of the 155 buffalo calves were normally distributed about the mean of 40.06 ± 0.51 kg (range 24–60). females (39.0 ± 0.73) were lighter than �� �� �� �� � � �� �� �� �� � � �� � �� � �� � � ��� �� � � �� � � �� � � �� � � ���� � �� � � ���� � � � � � � � � � � fig. 1 distribution of buffalo births at solole private nature reserve table 1 calving intervals in days for white rhinoceros dams dams number (n) mean variance dam 296 dam 406 dam 407 dam 585 dam 932 dam 933 10 10 5 4 2 2 910.8 923.8 1 272.4 1 254.5 869.5 1 088.5 92 161.9 39 932.1 182 524.8 81 867.0 2 244.5 324 012.5 table 2 variation in buffalo calf masses (kg mean ± s.e.) according to years year 1 year 2 year 3 females males all 33.45 ± 2.19 38.00 ± 1.18 37.73 ± 1.31 37.35 ± 1.01 39.75 ± 0.93 38.44 ± 0.71 41.72 ± 0.92 43.30 ± 1.07 42.44 ± 0.7 239 j.d. skinner et al. males (41.2 ± 0.68; t = –2.207; df 153; p = 0.029). this was particularly evident in year 1. the calves born in year 3 (42.44 ± 0.7; n = 76) were heavier than those born in years 1 (37.73 ± 1.31; n = 22) and 2 (38.44 ± 0.71; n = 57; f = 14.59; df 2 152; p < .001). in year 1 the calves born to cows designated large and extra large (39.12 ± 1.5; n = 8) were heavier than those designated medium (31.45 ± 1.28; n = 11; t = 3.88; p = 0012; df 17). there were 62 confirmed calving intervals (395.8 ± 4.82 days), which were normally distributed about the mean; the calving interval was unaffected by the sex of the preceding calf (t = –0.52; df 60; p = –0.516). the sex ratio was 1 male:1.2 females. discussion very few data are available for comparison with those presented here. age at first conception was 88.2 months (n = 3) for white rhinoceroses, whereas at matobo national park, zimbabwe, this varied from 72–138 months (rachlow & berger 1998). owensmith (1988) states that young white rhinoceros fe males produced a higher proportion of male calves and exhibited shorter birth intervals but qualifies this by stating that, at that time, there was a predominance of young rhinoceroses in captivity. our data do not support this view and the overall sex ratio is 17 males:19 females. the calving interval of 34 ± 1.8 months is slightly longer than those cited by owensmith (1988) for wild rhinoceroses in hluhluwe-imfolozi park in natal (2.63 years; n = 53) and those held in an enclosure in the kruger national park (2.70 years; n = 19). these are of the same order as rhinoceroses in matobo national park, zimbabwe (2.85 years; n = 23, owen-smith 1988; 2.9 years; n = 21, rachlow & berger 1998) but less than for rhinoceroses in kyle national park, zimbabwe (3.45 years; n = 23). rachlow & berger (1998) suggest that rate of calving may be inversely related to population density. the differences may be related to the most beneficial habitats, as the nutritive value of grasses in the veld at lichtenburg shows a marked decline in winter. on the other hand, the differences are not great and are more likely to be spurious, particularly as one bull is adequate to cover five cows, and there is no competition between bulls. the peak in births around march to april corresponds with a similar pattern in hluhluwe-imfolozi park. there is no evidence of a relationship between calving interval and month of birth or significant difference in calving interval between dams. although the buffaloes breed throughout the year (fig. 1) husbandry may, in part, be responsible for apparent peaks, as there were no peaks in records from the national zoological gardens in pretoria (skinner et al. 2002). the fact that calves are removed from their dams at birth in the solole private nature reserve may also influence breeding patterns. for example, in the wild, the calving interval is at least 60 days longer, depending on locality (bertschinger 1996). in addition, bulls were not permanently present in the cow paddocks. moreover, the level of nutrition is possibly the most important factor determining reconception in buffalo cows. those at solole receive supplementary feeding to maintain their condition. calves born in the first year were lighter than in years 2 and 3. it was in this year that the difference between male and female calves was most marked and the large cows had significantly heavier calves than medium-sized cows possibly due to greater uterine capacity. conclusions because both species are aseasonal or opportunistic breeders and, despite apparent seasonal peaks in calving, they do lend themselves to manipulative breeding and the possibility of increasing calving rates. the key to this would appear to lie in reducing the intercalving interval, either by removal of offspring, in the case of rhinoceroses, or hormonal induction of ovulation in the case of buffalos. both approaches could be tested experimentally. references acocks, j.p.h. 1988. veld types of south africa, with accompanying veld type map. pretoria: government printer (memoirs of the botanical survey of south africa, no. 57). bertschinger, h. 1996. reproduction in the african buffalo: a review, in proceedings of a symposium on the african buffalo as a game ranch animal, edited by b.l. penzhorn. onderstepoort 1996. owen-smith, r.n. 1988. megaherbivores. cambridge: cambridge university press. rachlow, j.l. & berger, j. 1998. reproduction and population density: trade-offs for the conservation of rhinos in situ. animal conservation, 1:101–106. skinner, j.d., moss, d.g. & skinner, d.c. 2002. inherent seasonality in the breeding seasons of african mammals: evidence from captive breeding. transactions of the royal society of south africa, 57:25–34. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [878.740 1133.858] >> setpagedevice abolnik_147-152.indd introduction pigeon paramyxovirus type 1 (ppmv-1) is an antigenic variant of avian paramyxovirus type 1 (apmv-1; newcastle disease virus) that was first identified by monoclonal antibody binding studies (alexander, wil son, thain & lister 1984; alexander, russell, par sons, abu elzein, ballouh, cernik, engstrom, feve reiro, fleury, guittet, kaleta, kihm, kosters, lomniczi, meister, meulemans, nerome, petek, pokomunski, polten, prip, richter, sághy, samberg, spanoghe & tumova 1985; king 1996; lana, snyder, kink & marquart 1988). ppmv-1 strains caused outbreaks among racing and show pigeons in europe in 1981 and re-emerged in 1985 causing a panzootic that continues today (biancifiori & fioroni 1983; alexander et al. 1985; wilson 1986; kaleta 1992a, b; ujvári, wehman, kaleta, werner, savić, nagy, czifra & lomniczi 2003; aldous, fuller, mynn & alexander 2004). in pigeons and doves clinical signs of infection include neurological signs such as torticollis and paralysis, and the excretion of large volumes of green, watery diarrhea (alexander et al. 1984; 1985; lemahieu, de vriese & bijnens 1985). in chickens, intracerebral pathogenicity (icpi) values for ppmv-1 are typical of mesogenic newcastle 147 onderstepoort journal of veterinary research, 75:147–152 (2008) characterization of pigeon paramyxoviruses (newcastle disease virus) isolated in south africa from 2001 to 2006 c. abolnik1*, g.h. gerdes1, j. kitching2, s. swanepoel3, m. romito1 and s.p.r. bisschop4 abstract c. abolnik, g.h. gerdes, j. kitching, s. swanepoel, m. romito & s.p.r. bisschop. 2008. characterization of pigeon paramyxoviruses (newcastle disease virus) isolated in south africa from 2001 to 2006. onderstepoort journal of veterinary research, 75:147–152 pigeon paramyxovirus type 1 (ppmv-1), a variant of newcastle disease virus that primarily affects doves and pigeons has been isolated in south africa since the mid-1980s. phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into south africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in europe and the far east since the early 1990s. during 2006, a ppmv-1 virus was isolated from an african ground hornbill (bucorvus leadbeateri) which became acutely infected with ppmv-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related ppmv-1 strain was also isolated from doves collected nearby. the hornbill isolate had icpi and mdt values characteristic of ppmv-1 strains. the threat of ppmv-1 to poultry production and biodiversity in southern africa highlights the importance of monitoring the spread of this strain. keywords: african ground hornbill, newcastle disease virus, paramyxovirus, phylogenetic characterization, pigeon * author to whom correspondence is to be directed. email: abolnikc@arc.agric.za 1 arc-onderstepoort veterinary institute, private bag x5, onder stepoort, 0110, south africa 2 stellenbosch provincial veterinary laboratory, private bag x1, elsenburg, 7607, south africa 3 deltammune laboratories, p.o. box 14167, lyttleton, centurion, 0140, south africa 4 poultry reference laboratory, university of pretoria, private bag x04, onderstepoort, 0110 south africa accepted for publication 14 april 2008—editor 148 pigeon paramyxoviruses (newcastle disease virus) isolated in south africa disease viruses but in most cases, ppmv-1 isolates have increased their virulence for chickens after passage, and therefore represent a threat to poultry production (alexander & parsons 1986; king 1996; kommers, king, seal & brown 2001). besides pigeons, doves and chickens, ppmv-1 viruses have also been isolated from kestrels, falcons, cockatoos, budgerigars, pheasants, swans and a robin (alexander et al. 1985; lister, alexander & hogg 1986; kaleta 1992b; werner, römer-oberdörfer, köllner, manvell & alexander 1999; monne, beato, capua & mandola 2006; aldous et al. 2004). phylogenetic analysis has classified ppmv-1 strains into a discrete lineage, vib (lomniczi, wehman, herczeg, bal lagi-pordany, kaleta, werner, meulemans, jorgen sen, manté, gielkens, capua & damoser 1998), recently re-classified as lineage 4b. lineage 4b was further split into subgroups 4bi and 4bii (aldous et al. 2004). large die-offs in doves and pigeons have occasionally been reported in various parts of south africa since the 1980s after the first isolation of ppmv-1 from doves during an outbreak in september 1986 (pienaar & cilliers 1987). in the present study, the phylogenetic relationships between 21 south african ppmv-1 viruses isolated from doves, pigeons, chickens, a duck and an african ground hornbill (bucorvus leadbeateri) were investigated, and the pathogenicity of the african ground hornbill isolate for chickens was determined. materials and methods isolates virus isolation was performed at the onderstepoort veterinary institute, stellenbosch provincial veterinary laboratory, the university of pretoria’s poultry reference laboratory and deltammune laboratory by inoculation into the alantoic cavities of 9 to 10-day-old embryonated specific antibody-negative fowl eggs. isolates are indicated in table 1. table 1 south african pigeon paramyxovirus isolates isolate year host town, provincea accession number za469/ppmv1/02 piza04n230 doza05n240 doza05n247 piza05n277 doza05am68313 doza05n417 doza05n539 doza06n549 doza06n589 doza06n591 ckza06n606 doza06up470 piza06n642 doza06n621 -za06n690 piza06n699 piza06n635 doza06n757 blza06n779 dkza06n828 2002 2004 2005 2005 2005 2005 2005 2005 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 28-week-old layers racing pigeons doves laughing dove pigeon laughing dove doves doves doves doves doves 4-week-old broilers dove pigeon doves unknown pigeon pigeon laughing doves african ground hornbill muscovy duck mooirivier, kzn oudtshoorn, wc kimberly, nc kuilsrivier, wc pretoria, gp marikana, nwp montagu, wc belville, wc cape town, wc darling, wc kimberly, nc sibasa, lp polokwane, lp cape town, wc cape town, wc germiston, gp middleburg, mpu stellenbosch, wc dwaalboom, nwp dwaalboom, nwp worchester,wc ay445669 ef030962 ef030953 ef030954 ef030963 ef030952 ef030955 ef030956 ef030957 ef030958 ef030959 ef030951 ef030961 ef030950 ef030960 ef030964 a kzn kwazulu-natal wc western cape nc northern cape lmp limpopo nwp north west gp gauteng mpu mpumahlanga 149 c. abolnik et al. pathogenicity tests icpi and mdt tests were performed according to standard procedures (oie manual of standards: diagnostic tests and vaccines 2000). reverse transcription-polymerase chain reaction and nucleotide sequencing viral rna was extracted from alantoic fluid using trizol® reagent (gibco, invitrogen). the fusion (f) protein gene was targeted in a one-step rt-pcr using the oligonucleotide primer pair and thermal cycling parameters described elsewhere (abolnik, horner, maharaj & viljoen 2004). cycle sequencing was performed using the abi prism® big dye™ terminator cycle sequencing ready reaction kit (applied biosystems) and the reverse primer from the rt-pcr to span the region containing the f0 peptide cleavage site. reactions were analysed with an abi3130™ genetic analyser (applied biosystems). sequence analysis nucleotide and amino acid sequence analyses and alignment were carried out using bioedit (hall 1999) and clustalw software (http://www.ebi.ac.uk/clustalw/index.html). phylogenies were reconstructed with mega 3.1 software (kumar, tamura & nei 2004) using the neighbour-joining tree inference method with the kimura 2-parameter substitution model and 1 000 bootstrap replicates to assign confidence levels to branches. results and discussion phylogenetic analysis of partial f protein genes indicated that the south african ppmv-1 isolates do not cluster together as a single geographical entity, but instead are split between the two lineages 4bi and 4bii (fig. 1). one of the south africa isolates, piza05n277, isolated from a pigeon in pretoria (gau teng province) in may 2005 shared 99 % nucleotide sequence identity with the other international lineage 4bi viruses, but only 98–99 % and 94–95 % sequence identities with other south african lineage 4bi and lineages 4bii viruses, respectively. therefore, we suspect that this strain was recently introduced into the country. the muscovy duck (cairina moschata) (dkza06n828) was a suspected organophosphate poisoning case and probably contracted ppmv-1 through contact with faeces from infected doves. ckza06n606 was isolated from 4-week-old broilers and is only the second reported case of ppmv-1 infection of chickens in south africa. the african ground hornbill isolate, blza06n779 shared 99.4 % sequence identities in the partial fusion protein gene with doza06n757 isolated from doves on the same farm during the outbreak. an mdt value of 89.4 h was obtained for blza06n779, which falls within the 90h time limit for mesogenic viruses. the icpi value obtained was 0.33, which indicates that it is not velogenic but does not differentiate between mesogenic and lentogenic viruses. the south african lineage 4bii strains formed a single clade and these viruses were mainly obtained from the western cape province, apart from single isolates from gauteng (-za06n690) and kwazulunatal provinces (za469/ppmv1/02). nucleotide sequence homology within the south african clade varied from 96.7 % to 99.1 %. relatively longer branch lengths suggest that the lineage 4bii strains may have been circulating in south africa for an extended period. the sequences of 112rrqkrf117 and 112rrkkrf117 at f0 for lineages 4bi and 4bii, respectively (fig. 2), are in agreement with other reports (collins, strong & alexander 1994; mase, imai, sanada, sanada, yuasa, imada, tsukamoto & yama guchi 2002; meulemans, van den berg, de caesstecker & boschmanns 2002; terregino, cattoli, gros sele, bertoli, tisato & capua 2003). although the bootstrap values supporting the phylogenetic relationships within lineages 4bi and 4bii are low, amino acid sequences (fig. 2) support the phylogenetic relationships: lineage 4bi clade (c) is distinguished from clades (a) and (b) (fig. 1) by t3, l10 and r27 substitutions, and lineage 4bii clade (d) differs from clade (e) by a p36→s substitution. we have demonstrated that exotic strains of pigeon paramyxoviruses were introduced into south africa on multiple occasions. routes of introduction into south africa are speculative, although they are most likely via the importation of infected pigeons for racing and ornamental purposes as reported in other countries (aldous et al. 2004). a second case of ppmv-1 infection of chickens in south africa is reported here, highlighting the importance of the threat of ppmv-1 to poultry, but ppmv-1 also potentially threatens biodiversity of wild birds, particularly rare indigenous dove and pigeon species. we report the first isolation of ppmv-1 from an african ground hornbill (bucorvus leadbeateri), an increasingly rare species, which became acutely infected with ppmv-1 and died, although the virus did not display increased pathogenicity for chickens. it is likely that the ground hornbill became infected by scavenging off the dead doves in the area. our findings suggest that ppmv-1 150 pigeon paramyxoviruses (newcastle disease virus) isolated in south africa fig.1 phylogenetic tree of a 374-bp region of the fusion protein gene of ppmv-1 viruses. south african strains are framed (bold type) pukpi99065 pukpi99128 pebpi98328 pdepi99063 paepi00377 pbepi98327 99299 piepi00246 piepi00242 -iepi00242 pukpi00248 pcapi91374 paepi00318 pukpi02440 pukpi02440 pukpi99131 piepi01387 piepi00339 pukpi02422 pukpi02423 pukpi99064 piza05n277 pukpi01421 se-8/00 paepi00366 de-1266/01 de-690/00 se-9/00 se-7/00 pdkpi00316 pdkpi01322 pitdo01321 pukpi01426 pukpi01383 piepi01369 pukpi02435 pukpi02434 pukpi02431 pukpi02429 blza06n779 doza06n757 piza06n699 doza06up470 doza05am68313 dkza06n828 doza05n240 doza06n591 ckza06n606 99106 pukpi02428 pigeon/italy/1166/00 yu vo-595/01 ptrbu95211 de-2663/98 patp97205 doza06n549 piza06n635 piza06n642 doza05n539 doza06n621 doza05n417 piza04n230 doza06n589 doza05n247 za469/ppmv1/02 -za06n690 jp/gunma-pg/2000 pdeck95331 pittd00177 pitpi99232 dove/italy/2736/00 pfrpi98372 pukpi98355 jp/shiga-pg/96 piepi96349 de-1383/99 pdepi94217 pdkpi3202 de-51/93 jp-utsunomiya-pg/95 jp/tochigi-pg/95 de-60/93 de-61/93 pdepi94216 de-48/92 es-3/92 de-55/94 patpi95295 pdepi9593 pukpi90213 88 67 64 1 2 55 27 41 7 5 55 8 14 31 36 2 71 53 41 36 46 38 34 57 60 41 9 72 41 62 93 61 5174 72 54 54 66 42 72 58 64 38 41 23 79 35 57 21 17 45 97 8 7 8 12 15 0 0 6 9 14 14 gb western cape, kwazulu-natal and gauteng provinces (e) western cape province (d) lineage 4bii europe, south africa and japan northern cape, western cape, north-west and limpopo province (c) north-west, limpopo and mpumalanga provinces (b) gauteng province (a) lineage 4bi europe, united arab emirates and south africa 0.005 40 61 46 49 151 c. abolnik et al. 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . p i z a 0 5 n 2 7 7 m g s k p y t r i p a p l m l i t r i t l v l s c i c l t s s l d g r p l a a a g i v v t g d k a i n i y t s s q t g s i i v k l l p n m p k d k e a c a k a p l e a y n r t l t t l l t p l g d s i r r i q g s v s t s g g r r q k r f i g a i i g s b l z a 0 6 n 7 7 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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. t . f . . . l . . . . . . . . . . . i . . . . . . . . . . . . . . . . . . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s . . . . . . . . . . . . . . . . . . . . v . . k . . . . . . . . . . f ig . 2 m u lti p le a m in o a ci d s e q u e n ce a lig n m e n t o f p a rt ia l s o u th a fr ic a n p ig e o n p a ra m yx o vi ru s fu si o n p ro te in s e q u e n ce s. t h e f u si o n p ro te in c le a va g e s ite ( f 0 ) is in d ic a te d in b o ld 152 pigeon paramyxoviruses (newcastle disease virus) isolated in south africa infection may be lethal to species other than doves and pigeons. identification of molecular virulence determinants of paramyxoviruses other than the f0 cleavage site, and identification of host-specific factors that affect virulence are research areas that require more attention. acknowledgements we thank elsa cornelius, piekie combrink, hope kekana and magdeline dreyer for performing virus isolations, and emily 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods results ethical considerations discussion conclusion acknowledgements references about the author(s) chantel j. de beer agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa department of zoology and entomology, university of the free state, south africa gert j. venter agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa department of veterinary and tropical diseases, university of pretoria, south africa karin kappmeier green agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa johan esterhuizen agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa department of vector biology, liverpool school of tropical medicine, united kingdom daniel g. de klerk agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa jerome ntshangase agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa marc j.b. vreysen joint food and agriculture organization/international atomic energy agency division of nuclear techniques in food and agriculture, insect pest control laboratory, austria ronel pienaar agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa makhosazana motloang agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa lundi ntantiso makhathini research station, jozini, south africa abdalla a. latif agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa agricultural research council – onderstepoort veterinary institute, parasites, vectors & vector-borne diseases, south africa citation de beer, c.j., venter, g.j., kappmeier green, k., esterhuizen, j., de klerk, d.g., ntshangase, j. et al., 2016, ‘an update of the tsetse fly (diptera: glossinidae) distribution and african animal trypanosomosis prevalence in north-eastern kwazulu-natal, south africa’, onderstepoort journal of veterinary research 83(1), a1172. http://dx.doi.org/10.4102/ojvr.v83i1.1172 research project no: 000773 original research an update of the tsetse fly (diptera: glossinidae) distribution and african animal trypanosomosis prevalence in north-eastern kwazulu-natal, south africa chantel j. de beer, gert j. venter, karin kappmeier green, johan esterhuizen, daniel g. de klerk, jerome ntshangase, marc j.b. vreysen, ronel pienaar, makhosazana motloang, lundi ntantiso, abdalla a. latif received: 09 feb. 2016; accepted: 11 mar. 2016; published: 09 june 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract an unpredicted outbreak of african animal trypanosomosis or nagana in 1990 in north-eastern kwazulu-natal necessitated an emergency control programme, utilising the extensive cattle-dipping system in the area, as well as a reassessment of the tsetse and trypanosomosis problem in the province. since 1990, sporadic blood sampling of cattle at the dip tanks in the nagana-infested areas were undertaken to identify trypanosome species involved and to determine the infection prevalence in cattle. the distribution and species composition of the tsetse populations in the area were also investigated. from november 2005 to november 2007 selected dip tanks were surveyed for trypanosome infection prevalence. during april 2005 to august 2009 the distribution and abundance of tsetse populations were assessed with odour-baited h traps. the tsetse and trypanosome distribution maps were updated and potential correlations between tsetse apparent densities (ads) and the prevalence of trypanosomosis were assessed. glossina brevipalpis newstead and glossina austeni newstead were recorded in locations where they have not previously been collected. no significant correlation between tsetse relative abundance and nagana prevalence was found, which indicated complex interactions between tsetse fly presence and disease prevalence. this was epitomised by data that indicated that despite large differences in the ads of g. austeni and g. brevipalpis, trypanosome infection prevalence was similar in all three districts in the area. this study clearly indicated that both tsetse species play significant roles in trypanosome transmission and that it will be essential that any control strategy, which aims at sustainable management of the disease, should target both species. introduction the discovery that trypanosoma brucei plimmer & bradford was the cause of african animal trypanosomosis (aat), also known as nagana, can be dated back to the 1880s when it was recorded for the first time in north-eastern kwazulu-natal (formerly known as zululand), south africa (bagnall 1993; bruce 1895; steverding 2008). in 1895, sir david bruce stated that game animals were the reservoir hosts of the causative trypanosome species and that these protozoan parasites were transmitted between their mammalian hosts by tsetse flies (diptera: glossinidae) (bruce 1895). four species of tsetse, that is, glossina morsitans morsitans westwood, glossina pallidipes austen, glossina brevipalpis newstead and glossina austeni newstead have been recorded in south africa. g. m. morsitans was the only species that was encountered in the most northern parts of the country (this included the kruger national park and parts of the limpopo and mpumalanga provinces), bordering onto zimbabwe in the north and mozambique in the east (fuller 1923). in 1897 after a large reduction of cattle and wildlife during the rinderpest epizootic of 1896–1897, g. m. morsitans completely disappeared from south africa (fuller 1923). the other three species of tsetse, that is, g. pallidipes, g. brevipalpis and g. austeni, remained present in the former zululand in the north-eastern part of kwazulu-natal province (fuller 1923). glossina pallidipes was the predominant species in north-eastern kwazulu-natal, and based on its abundance it was considered the most important vector of aat at that time. g. brevipalpis and g. austeni, which were restricted to areas with mostly dense vegetation, were not considered as important vectors of aat. the sheer abundance of g. pallidipes was illustrated by large numbers of flies being trapped on certain occasions, that is, in 1932, 2 million flies were collected within a month after deployment of 1000 harris traps and nearly 8 million flies over that entire year (harris 1932). the presence of these large numbers of tsetse flies in north-eastern kwazulu-natal resulted in severe outbreaks of nagana between 1942 and 1946 (du toit 1954). a campaign that was largely reliant on newly developed synthetic insecticides such as ddt (1,1,1-trichloro-2,2-di (4-chlorophenyl)ethane) and hch (hexachlorobenzene) was launched between 1945 and 1952 to eradicate g. pallidipes. these chemicals were extensively used as part of residual aerial spraying campaigns that were aimed at creating a zone free of g. pallidipes in zululand (du toit 1954). surveys in 1953 failed to detect any g. pallidipes and it was concluded that this species was eradicated from zululand (du toit 1954). the programme was very successful because it integrated several control tactics (aerial spraying, trapping and bush clearing). furthermore, it was sustainable as the entire g. pallidipes population was targeted and kwazulu-natal is today still free of this species. this was one of the first tsetse fly control programmes that was implemented following area-wide integrated pest management (aw-ipm) principles (klassen 2005; vreysen, robinson & hendrichs 2007). an additional benefit from this campaign was the apparent removal of g. brevipalpis from the hluhluwe–imfolozi park (du toit 1954). in areas where g. brevipalpis and g. austeni were found in the absence of g. pallidipes, no control efforts were implemented against these species, and consequently, these two species remained present in zululand (kappmeier, nevill & bagnall 1998). from 1955 onwards, only sporadic cases of nagana were recorded in zululand (bagnall 1993, 1994). however, in 1990, a severe outbreak of nagana once again occurred in north-eastern kwazulu-natal. cattle mortalities during this outbreak were exacerbated by the co-occurrence of a severe drought (emslie 2005; kappmeier et al. 1998). emergency control measures were implemented that relied on the extensive dipping network that was mainly used for tick control in kwazulu-natal (kappmeier et al. 1998). as part of the control measures, the active ingredient of the acaricide dipping product was changed from amitraz to the pyrethroid cyhalothrin for a period of 2 years (bagnall 1993). whereas the pyrethroid cyhalothrin is very effective for controlling both ticks and tsetse flies (bagnall 1993), the efficacy of amitraz is limited to ticks and is considered less effective against dipteran flies (hall & fischer 1984). the adopted cattle-dipping regime in combination with animal treatments using trypanocidal drugs brought the disease outbreak under control (kappmeier et al. 1998). the 1990 outbreak illustrated that nagana had remained a serious debilitating problem in kwazulu-natal, with mixed infections of trypanosoma congolense broden and trypanosoma vivax ziemann (bagnall 1993). a survey to determine the prevalence of trypanosoma species in 1994 indicated the highest trypanosomosis prevalence in the ubombo district of kwazulu-natal, while the lowest prevalence was found to be in the hlabisa district, which surrounds the hluhlwe–imfolozi park (de waal et al. 1998). periodic screening of cattle in the tsetse-infested area indicated that t. congolense was the dominant species (mamabolo et al. 2009; motloang et al. 2012; van den bossche et al. 2006), t. vivax being present to a lesser extent (mamabolo et al. 2009; motloang et al. 2014). recently trypanosoma theileri (laveran) and t. brucei were detected based on the alignment of 18s rrna gene sequences which were comparable with trypanosome sequences available at the national centre for biotechnology information (ncbi) (taioe 2014). two sub-genotypes of trypanosoma congolense, the savannah and kilifi were identified with pcr (mamabolo et al. 2009). mixed infections of these two sub-genotypes were also observed (gillingwater, mamabolo & majiwa 2010). these surveys showed that trypanosome infections in cattle at dip tanks close to the hluhluwe–imfolozi park were higher than at dip tanks further away from this game reserve (motloang et al. 2014; ntantiso et al. 2014; van den bossche et al. 2006). long-term management of nagana by dipping and/or curative treatment of infected cattle with trypanocidal drugs is neither cost effective nor sustainable (bagnall 1994; shaw 2009), and it became evident that a long-term solution to the nagana problem in south africa could only be attained if vector control was to be included. initial surveys showed that g. brevipalpis and g. austeni were still the only species present in kwazulu-natal (kappmeier green 2002). between 1993 and 1999, extensive tsetse fly surveys were conducted in an area of approximately 12 000 km2 using odour-bated sticky xt traps (cross-shaped targets) to assess the distribution of each species (figure 1a, b) (kappmeier & nevill 1999a, kappmeier green & venter 2007). the survey indicated that g. brevipalpis was present in southern and northern bands, commonly associated with game reserves and other protected areas. the distribution of g. austeni was continuous from south to north and did not extend as far west as that of g. brevipalpis. g. austeni also was very common in communal farming areas (kappmeier green 2002). the 1993–1999 survey did not establish the southernmost distribution limit of g. brevipalpis and g. austeni, as the sampling frame was only designed to determine broad distribution limits. it was, however, suggested that the southernmost limit of the tsetse fly distribution in south africa, and therefore africa, could roughly be regarded as the southernmost extent of the umfolozi river (kappmeier green 2002). the 1993–1999 survey also showed that g. brevipalpis and to a lesser extent g. austeni were present close to the coastal areas apparently not sampled in the 1950s (du toit 1954). figure 1: apparent density of glossina brevipalpis (a), glossina austeni (b) and trypanosome prevalence in cattle at dip tanks (c) in north-eastern kwazulu-natal. the entomological sampling data obtained from 1993 to 1999 (kappmeier green 2002) as well as environmental and climatic variables were used to develop a probability of presence model for these two species (hendrickx 2002; hendrickx et al. 2003). the model predicted a broader geographical distribution range for both g. brevipalpis and g. austeni than what was indicated by the survey data; however, hendrickx et al. (2003) suggested that the model overestimated tsetse fly distribution. knowledge on the accurate distribution of the target insect, as well as the interaction of the insect with the causative agent it transmits, is vital for the successful implementation of any proposed control campaign and will directly affect its outcome, sustainability and cost (shaw 2009; vreysen et al. 2007). additional ongoing efforts to generate continental as well as national atlas of tsetse and aat (cecchi et al. 2014, 2015) will be benefited from continuously updated data sets. the development of an improved trap for g. brevipalpis and g. austeni (kappmeier 2000) and enhanced artificial odour system for g. brevipalpis (kappmeier & nevill 1999a) allowed a more effective sampling of both species in south africa. subsequently, as a starting point, the prediction model of hendrickx (2002) was validated through selective trapping in the area. from 2005 to 2009, researchers at the agricultural research council-onderstepoort veterinary institute (arc-ovi) carried out various studies. these included a transect study, based on the results of the distribution survey and prediction model, to determine the distribution of g. austeni to the west of the hluhluwe dam (esterhuizen, kappmeier & nevill unpublished data) and to assess fly presence and densities at dip tanks (gillingwater et al. 2010; mamabolo et al. 2009; motloang et al. 2012; ntantiso et al. 2014). in addition, tsetse population genetics (koekemoer et al, unpublished data) and morphometric studies (de beer et al. unpublished data) were conducted to assess the level of gene flow between the tsetse populations in kwazulu-natal and those in southern mozambique and swaziland. the present paper collates the data of these different studies to update the existing maps on the distribution of tsetse flies and trypanosomosis in north-eastern kwazulu-natal. correlation between tsetse apparent densities (ads) and infection prevalence of the disease in livestock were also investigated. materials and methods study area the tsetse-infested area (± 16 000 km2) in south africa is confined to the north-eastern part of kwazulu-natal province. the area stretches roughly from the umfolozi river (-28.5204, 32.3123) in the south to the border of mozambique (-26.8692, 32.8342) in the north, and from the indian ocean coast in the east up to the west of the hluhluwe–imfolozi park (-28.33416, 31.691222) (kappmeier green 2002). although some commercial cattle farms are present, it is predominantly a subsistence cattle-farming area with numerous communal farms that are interspersed with a number of protected areas. the protected areas consist of provincial and private game parks and reserves. the area surrounding the extensive fresh water lake system, isimangaliso wetland park, is a proclaimed world heritage site. these areas not only contain a wide variety of game animals, such as birds, rodents and smaller primates, but also large numbers of bigger mammals that are potential hosts for tsetse. the target area contains a number of state forests, mostly pine and eucalyptus plantations, and commercial sugarcane farms. the climate is subtropical with average minimum temperatures ranging from 10.52 °c ± 1.26 °c in july (winter) to 21.71 °c ± 0.67 °c in january (summer). the average maximum temperature ranges from 24.65 °c ± 0.77 °c in july to 30.86 °c ± 1.38 °c in january. the average minimum relative humidity ranges from 39.92% ± 8.23% in july to 62.04% ± 6.39% in october, whereas the average maximum relative humidity ranges from 92.23% ± 3.73% in june to 94.94% ± 3.13% in february. the area receives an average maximum of 132.92 mm ± 92.95 mm of rain in january, and most of the precipitation is received in the hot season from october to march. however, it can also rain in the cold dry season from april to september with a minimum average 9.27 mm ± 9.68 mm of rain in july. on average, more rain is received in the coastal area as compared with the interior. tsetse fly sampling tsetse fly collections were made at 18 sites located in four magisterial districts: ingwavuma, ubombo, hlabisa and the northern part of enseleni (table 1; figure 1a, b). a total of 77 odour-baited h traps (kappmeier 2000; kappmeier & nevill 1999b) were deployed at these 18 sites from april 2005 to april 2009. the number of trap days ranged from 723 at ocilwane (northern enseleni) to 1661 at false bay park (eastern hlabisa district). the traps were baited with artificial odour baits to enhance trapping of g. brevipalpis (kappmeier & nevill 1999a). these consisted of 1-octen-3-ol and 4-methylphenol at a ratio of 1:8 that were released at 4.4 mg/h and 7.6 mg/h, respectively. the chemicals were dispensed from seven heat-sealed sachets (7 cm ´ 9 cm) made from low-density polyethylene sleeves (wall thickness 150 µm) placed near the entrance of the trap. a 300 ml glass bottle that dispensed acetone through a 6-mm hole in the lid at a rate of ca. 350 mg/h was placed next to the trap (esterhuizen 2007; kappmeier green 2002). flies were collected in a 20% ethanol solution to which savlon® (johnson & johnson, pharmedica laboratories [pty] ltd. rattray road, east london, south africa) (0.4 ml/l) and formalin (0.4 ml/l) had been added to preserve the sampled flies as well as to protect them from ant and spider predation. traps were emptied and serviced every 14 days. the number of each species of tsetse fly collected over this period was counted, and results for each species expressed as ad, that is, the number of flies per trap per day. table 1: tsetse h trap catches from april 2005 to august 2009 in the north-eastern kwazulu-natal. trypanosomosis survey a large number of communal dip tanks, constructed, maintained and operated by the provincial department of veterinary services, are routinely used for tick control and are homogeneously distributed throughout the tsetse-infested area. the tick control policy, as dictated by the department of veterinary services, consists of weekly dipping of cattle in the summer and fortnightly dipping during the winter. cattle in the communal farming areas as well as on the commercial farm boomerang roam around freely. the trypanosome infection prevalence in cattle was determined at 27 dip tanks from november 2005 to november 2007 (figure 1c). all animals that congregated at a particular dip tank were considered as one herd as they grazed together and were managed using the same animal husbandry practice (emslie 2005; ntantiso et al. 2014). herd size was very variable and ranged from 13 to 6411 animals, and the number of cattle owners at each dip dank was likewise very variable and ranged between 5 and 296. cattle were screened on the day they were scheduled for routine dipping. these herds were divided into groups of 30 to 40 cattle, and 2 to 3 animals in each group were randomly sampled. table 2 lists the number of cattle sampled at each dip tank. table 2: trypanosome infection rates as determined by buffy coat examination of live trypanosomes collected from cattle at dip tanks from november 2005 to november 2007 in the north-eastern kwazulu-natal. blood was collected from the tail or jugular vein of adult animals using 10-ml vacutainer tubes containing the anticoagulant edta (bd vacutainer®; bd, plymouth, united kingdom) (ntantiso et al. 2014). the blood was transferred to micro-haematocrit centrifuge capillary tubes (marienfeld-superior, lauda-königshofen, germany) that were sealed with cristaseal (hawksley) and centrifuged in a haematocrit centrifuge for 5 min at 9000 rpm. the buffy coat of each specimen was extruded onto a microscope slide, covered with a cover slip and examined for motile trypanosomes under a compound microscope using a 40-times magnification (paris, murray & mcodimba 1982). the trypanosomosis infection prevalence at each dip tank was expressed as the number of cattle that had a positive identification of trypanosomes in their buffy coat as a proportion of the number of cattle screened. data analysis all data were analysed using the statistical software graphpad instat (version 3.00, 2003). tsetse relative abundance was expressed as the ad of each species, that is, the number of flies collected per trap per day. for comparison of the relative abundance of g. brevipalpis and g. austeni populations, a paired test was used to differentiate between mean tsetse fly ad. the data were not normally distributed and the non-parametric wilcoxon matched-pairs test was used. to evaluate the ad of each tsetse species per district, a one-way analysis of variance (anova) was used to differentiate between the mean tsetse fly ad. the data were not normally distributed, thus a non-parametric method (kruskal–wallis test) was used. additionally, dunn’s multiple comparison tests were used if the p value < 0.05. proportional differences in trypanosome infection prevalence were determined with chi-square (χ2) analysis with the yate’s continuity correction. linear regression analysis was carried out on trypanosome infection prevalence and tsetse fly ad, as well as infection prevalence and distances from game reserves. all statistical tests were done at the 5% significance level. maps were developed in arcgis desktop 10.1 (esri, 2011). for the trypanosome infection prevalence, an inverse distance weighted interpolation method was used with a power 2 function and a variable search radius setting at 10 points. results tsetse distribution and abundance a total of 77 h traps were deployed at 18 sites located in four magisterial districts, that is, ingwavuma, ubombo, hlabisa and enseleni (table 1; figure 1a, b). these traps collected a total of 216 449 g. brevipalpis (ad = 2.64 flies/trap/day) and 17 097 g. austeni (ad = 0.21 flies/trap/day) between 01 april 2005 and 31 august 2009 (table 1; figure 1a, b). while both tsetse species were collected in the three northerly districts, only g. brevipalpis was trapped (with six h traps) at ocilwane in the northern part of the enseleni district in the south (table 1; figure 1a). the overall ad of g. brevipalpis (2.64 flies/trap/day) was significantly higher (p < 0.001) than that of g. austeni (0.21 flies/trap/day). this might, however, be an artefact resulting from the intrinsic biases of the trapping system. comparison of the ads in the three northerly districts indicated that the ad of g. brevipalpis was significantly higher than that of g. austeni in the ingwavuma (p = 0.008) and hlabisa (p < 0.001) districts (table 1; figure 1a, b). the ad of g. brevipalpis (0.03 flies/trap/day) in the ubombo district was significantly lower than that of g. austeni (0.33 flies/trap/day) (p < 0.002) (table 1). glossina brevipalpis was most abundant in the hlabisa district (3.76 flies/trap/day), and the ad was significantly higher (p < 0.001) than that in the ubombo district (0.03 flies/trap/day); an area where previously no g. brevipalpis were collected (kappmeier green 2002) (table 1; figure 1a). significantly higher numbers (p = 0.025) of g. austeni (ad = 0.33 flies/trap/day) were collected in the ubombo district as compared with the hlabisa (0.02 flies/trap/day) and the ingwavuma districts (0.04 flies/trap/day) (table 1). no g. austeni was trapped at ocilwane in the enseleni district (table 1; figure 1b). these trapping data indicated that the monzi forest in the hlabisa district maintained the most southerly distribution of g. austeni in kwazulu-natal (and africa). the greatest variations in ads among sites within the same district (table 1) were observed in the hlabisa district for both g. brevipalpis (p < 0.001) and g. austeni (p < 0.001). the ad of g. brevipalpis in the hluhluwe–imfolozi park (10.74 flies/trap/day) was significantly higher than that observed at kuleni (0.17 flies/trap/day). there were also significant differences in ad of the g. brevipalpis populations of st lucia (7.25 flies/trap/day) and kuleni (0.17 flies/trap/day). g. austeni was most abundant at boomerang (0.78 flies/trap/day), and this ad was significantly higher than that obtained in the hluhluwe–imfolozi park (0.02 flies/trap/day). in the ubombo district, the ad of g. brevipalpis at false bay park (1.55 flies/trap/day) was significantly higher (p = 0 .044) than that at phinda (0.06 flies/trap/day) and lower mkhuze (0.06 flies/trap/day). the ad of g. austeni was significantly higher at false bay park (0.60 flies/trap/day) (p = 0.002) in the ubombo district as compared with the ad in mkhuze (0.01 flies/trap/day). finally, in the ingwavuma district there were no significant differences in relative abundance between individual sites for both g. brevipalpis (p = 0.114) and g. austeni (p = 0.113). trypanosome infection at dip tanks a total of 1034 blood samples were collected from cattle at 25 dip tanks in the tsetse-infested area and were examined microscopically for the presence of trypanosomes (table 2). the number of cattle screened per dip tank ranged from 27 at ekuphindisweni to 54 at boomerang (table 2). data of trypanosome prevalence (table 2) were, as was done for the tsetse data, grouped per magisterial districts (table 1). the results showed that trypanosome infection was widespread in north-eastern kwazulu-natal with positive infected cattle found at 21 (84.0%) of the 25 dip tanks surveyed (table 2; figure 1c). trypanosome prevalence ranged from 20% in the ubombo district to 3% in the enseleni district and was not significantly different (p = 0.05, χ2 = 7.91, df = 3) between the four magisterial districts. notwithstanding the fact that there were no significant differences between the four districts, the proportional representation of positive animals at the 25 individual dip tanks differed significantly (p < 0.001, χ2 = 173.30, df = 24). significant differences in the proportion of positive animals per dip tank were also found within the districts of hlabisa (p < 0.001, χ2 = 51.462, df = 6), ingwavuma (p < 0.001, χ2 = 59.078, df = 9) and ubombo (p < 0.001, χ2 = 51.211, df = 6). these proportional differences in infection prevalence found between individual dip tanks seemed to be linked to the distance between the dip tank and a protected area or game reserve. the highest trypanosome prevalence (48%) was recorded at the mseleni dip tank which was situated next to the northern parts of isimangaliso wetland park in the ubombo district (table 2). similarly, trypanosome prevalence in cattle was high at dip tanks that were close to protected areas and/or game parks (figure 1c). especially in the hlabisa district, a significant positive linear regression (r2 = 0.62, p = 0.04) was evident between trypanosome prevalence and the distance between the dip tank and the protected areas or game parks. the same trend was also observed for the other two districts, but it was not significant (ingwavuma: r2 = 0.22, p = 0.17 and ubombo: r2 = 0.19, p = 0.41). the correlation between infection prevalence and distance from the game parks was further epitomised by the absence of infected animals at the two dip tanks located more than 20 km further from a game park (table 2). tsetse relative abundance and trypanosome infection prevalence the trypanosome prevalence in cattle, as determined with the buffy coat technique, at the 18 dip tanks was correlated with the tsetse relative abundance, as determined with odour-baited h traps. only dip tanks that had h traps deployed in a 12 km radius were selected for the regression analyses (table 2). no linear correlation could be found between trypanosome prevalence and the relative abundance of either g. brevipalpis (r2 = 0.01, p = 0.68), g. austeni (r2 = 0.05, p = 0.40) or the total tsetse catches (r2 = 0.04, p = 0.43). however, at some of the dip tanks with high trypanosome prevalence in the cattle, for example, mseleni (48%) and mkhumbikazana (37%) in the ubombo district, the ad for g. austeni was relatively high in the absence of g. brevipalpis (figure 1a, b). in contrast, at the ekuphindisweni dip tank in hlabisa district where the screened cattle showed a similar high infection prevalence (37%), the relative abundance of g. brevipalpis was high, in the absence of g. austeni (figure 1a, b). the relative abundance of both g. brevipalpis and g. austeni was relatively high at the dip tank on the commercial farm boomerang that is located to the west of and next to the southern extent of the isimangaliso wetland park and where the highest trypanosome prevalence (44%) was recorded in the hlabisa district. these apparent discrepancies in ad of tsetse flies at the dip tanks with high trypanosome infection prevalence indicate that a number of factors, such as the tsetse and trypanosomosis monitoring regimes used (a trap placement radius of 12 km could be too large), may influence the outcome of such a regression. ethical considerations materials used in the study posed no health risk to researchers, and no vertebrate animals were harmed. the study was done as part of a project on national assets (000773) at the arc-ovi in collaboration with the food and agriculture organization (fao)/international atomic energy agency (iaea) division of nuclear techniques in food and agriculture and the department of technical cooperation of the iaea under project raf 5069. discussion baseline data on tsetse distribution, relative abundance and species composition, as well as the extent of the trypanosomosis problem, are essential for the development of an appropriate cost effective, sustainable area-wide control strategy (leak, ejigu & vreysen 2008; shaw 2009; vreysen 2005). previous surveys, using xt sticky traps, indicated that the g. brevipalpis population was restricted to two distinct bands in the north and south of the tsetse fly infested area in kwazulu-natal (kappmeier green 2002) (figure 1a). these two bands were also more or less defined in the tsetse distribution prediction model for this species (hendrickx et al. 2003; kappmeier green, potgieter & vreysen 2007), except for a few patches predicted centrally. during the recent surveys, the h trap sampled low numbers of g. brevipalpis in at least five sites in a central area in the tsetse-infested belt and located east of the mkuzi game reserve and north of the southern previously defined distribution band (figure 1a). therefore, these positive trap catches could in part be used to update the prediction model of hendrickx (hendrickx et al. 2003) and indicate that g. brevipalpis is also present in the central and southern parts of ubombo district, where it was previously not sampled. whether this distribution is patchy or continuous still needs to be verified. during the 1993–1999 surveys (kappmeier green 2002), g. austeni was sampled at the hluhluwe dam situated east of hluhluwe–imfolozi park (figure 1b). a transect study was therefore carried out along the hluhluwe river, starting at the hluhluwe dam and progressing to the west into hluhluwe–imfolozi park, with the aim to determine if g. austeni could be found in the park as predicted by the distribution model of hendrickx et al. (2003). all traps along the transect sampled g. austeni, but at very low ads (0.02 flies/trap/day) (table 1). the presence of g. austeni along this transect also confirms the accuracy of the g. austeni distribution prediction model (hendrickx et al. 2003). glossina brevipalpis was likewise trapped in the transect traps, but this was expected considering that it had previously been collected inside and outside the hluhluwe–imfolozi park (kappmeier green 2002). taken into account that g. austeni is primarily restricted to and does not disperse far from dense vegetation (esterhuizen 2007; esterhuizen et al. 2005), and in view that bush clearing in the communal farming areas has increased considerably (kappmeier et al. 1998), it is likely that game reserves and protected areas will become more important in sustaining g. austeni populations in the future. the survey data of 1993–1999 indicate the southernmost extent of the umfolozi river (-28.5204, 32.3123) to be the most southerly distribution of g. brevipalpis (kappmeier green 2002). glossina austeni was not trapped south of the st lucia estuary, where the umfolozi river enters the lake (kappmeier green 2002). the tsetse prediction model of hendrickx et al. (2003) suggested a very low probability of occurrence for g. austeni in the south of the umfolozi river, but a much higher probability of occurrence for g. brevipalpis. during the 2005–2009 surveys, g. brevipalpis was collected at the ocilwane dip tank which is 5 km south of the umfolozi river – an area which had not previously been surveyed (figure 1a). the presence of g. brevipalpis at this site also corresponds to what was predicted by hendrickx et al. (2003), so that the predictions were confirmed at this site. the results of the recent surveys also indicate that the most southern limit of g. brevipalpis, and the factors that will determine this in the area, remain undefined. available data indicate that the southern limit for g. austeni seems to be defined as the umfolozi river, as was previously assumed (kappmeier green 2002). similarly, the most western extent of the distribution of each species needs to be verified. available trap catches indicate that the most western distribution limit of g. austeni was -28.10357, 32.08377 (in hluhluwe–imfolozi park), whereas g. brevipalpis was collected much further west at -28.04500, 32.03900, that is, west of ocilwane (figure 1a, b). the trap catches have consequently been used to update the tsetse distribution prediction models for both g. brevipalpis and g. austeni (hendrickx 2007). since only data up to 2009 are presented, ongoing and future surveys will have to be processed and incorporated into these distribution maps and models. the available trap data have partly validated the prediction model, emphasising the fact that such models can be valuable tools for the planning of tsetse and trypanosomosis intervention practices and their value should not be underestimated. furthermore, the new distribution data will need to be taken into consideration in the suggested tsetse control strategy proposed by kappmeier green et al. (2007), which needs to be modified accordingly. trypanosome infections in cattle were recorded at 21 of the 25 dip tanks which clearly showed that the disease was still abundant and highly prevalent in north-eastern kwazulu-natal at the time of the trypanosomosis survey (figure 1c). a potential shortcoming of the trypanosome survey may have been that infections were not identified to species level. it was, however, previously indicated that t. congolense is the dominant species in the area (mamabolo et al. 2009; motloang et al. 2012; van den bossche et al. 2006). the data on the spatial distribution of the trypanosome infection in north-eastern kwazulu-natal indicated that the disease was widespread in cattle, that is, from areas close to the mozambique border in the north to a dip tank close to the umfolozi river in the south, covering the entire length of the tsetse-infested area of north-eastern kwazulu-natal (figure 1c). in general, higher infection rates were found in dip tanks located near game reserves and protected natural areas (figure 1c), an observation also made by van den bossche et al. (2006) and ntantiso et al. (2014) for dip tanks near the hluhluwe–imfolozi park. this game–livestock–tsetse interface poses a high risk for cattle getting infected not only for the hluhluwe–imfolozi park (ntantiso et al. 2014), but possibly throughout north-eastern kwazulu-natal. in 1994, the highest prevalence of trypanosomosis was recorded in the ubombo district (kappmeier et al. 1998). although in the present study the dip tank with most of the cattle infected (mseleni 48%) was found in this district, the trypanosome prevalence was not significantly different from that obtained in the ingwavuma and hlabisa districts confirming that trypanosomosis remains a problem throughout the whole area. the absence of a significant linear correlation between trypanosome prevalence and the relative abundance of tsetse flies in north-eastern kwazulu-natal may partly be attributed to the coexistence of the two tsetse species, each with a different vectorial capacity and/or competence. previous studies indicated that the vector competence of g. austeni for t. congolense, the most abundant trypanosoma species in north-eastern kwazulu-natal, was significantly higher than that of g. brevipalpis (motloang et al. 2012). our data seem to corroborate this greater vector competence of g. austeni in view of the relative high trypanosome prevalence recorded in cattle at the mseleni dip tank where only g. austeni flies were sampled (mbazwana ad = 0.15 flies/trap/day). however, cattle at the ekuphindisweni dip tank showed a similar high trypanosome prevalence, but g. austeni was not sampled in the traps; only g. brevipalpis was trapped in relatively high numbers (ekuphindisweni ad = 1.59 flies/trap/day) indicating that g. brevipalpis was most likely responsible for transmission in this area. in general, high ads of g. brevipalpis in the vicinity of dip tanks resulted in trypanosome infection rates that were as high as those found at dip tanks where g. austeni was present but at low ads. hendrickx et al. (2003) also noted that low g. austeni densities coincide with areas of higher trypanosomosis prevalence in the northern communal areas. however, the very high ads of g. brevipalpis (on average 12.6 times higher than that of g. austeni) and the much lower trap catches of g. austeni might be an artefact resulting from the intrinsic biases of the trapping system. it is known that g. austeni responds poorly to traps (kappmeier 2000), and it is not attracted to any of the artificial odours (kappmeier & nevill 1999a; kappmeier green 2002) used to bait traps. essentially, mark–release–recapture studies will be needed to confirm the low efficacy of the sampling tool used for g. austeni. if the relative abundance of g. breviplapis is indeed a reflection of actual population densities, it can be hypothesised that the high trypanosome infection prevalence in certain areas might be the result of the greater densities of g. brevipalpis despite their lower vector competence. the relative low vector competence of g. brevipalpis previously found in south africa has probably resulted in an underestimation of the importance of g. brevipalpis in the epidemiology of nagana in north-eastern kwazulu-natal. vector competence studies in the laboratory have shown that the susceptibility of g. brevipalpis for t. congolensis can be as high as 12.3% (moloo, okumu & kuria 1998). in uganda, it was shown that the infection rates for t. congolensis in g. brevipalpis collected in field could be 2.6% (harley 1967a, 1967b; moloo, kutuza & boreham 1980). in addition, trypanosome infection rates in tsetse flies are not constant and can change over time and between populations. in uganda, infection rates varied considerably with the different seasons and with the age of the g. brevipalpis population (harley 1966, 1967a, 1967b). both g. brevipalpis and g. austeni will readily feed on cattle (clausen et al. 1998; moloo 1993) and can therefore play a role in the transmission of trypanosomosis in kwazulu-natal. similar average trypanosome infection rates in the three northerly districts seem to support this hypothesis even though the relative abundance and distribution of g. austeni and g. brevipalpis were clearly different. conclusion the dynamics of tsetse populations can fluctuate in space and time and need to be monitored regularly for the development and implementation of an effective control strategy. differences in fly density, distribution and ecology imply that different control strategies may be needed for the control of both these species in the nagana-infected area. the role that game parks and other protected areas may play in sustaining tsetse populations, as well as the circulation of trypanosomes in game animals, and the potential dispersal of both the vector and pathogen from these areas need further investigation. furthermore, it will be essential that any strategy that aims at finding a sustainable solution to the nagana problem in north-eastern kwazulu-natal needs to target both tsetse fly species. acknowledgements the authors thank the department of science and technology and the joint fao/iaea division of nuclear techniques in food and agriculture for financial support. also, the staff of the parasites vectors and vector-borne diseases programme of arc-ovi and the staff of the kwazulu-natal tsetse research station (ztrs) are thanked for their assistance. we thank dr ben mans for constructive comments on earlier drafts of this manuscript. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions a.a.l., k.k.g, j.e. and l.n. conceptualised and designed the study. the acquisition of data was done by c.j.d.b., d.g.d.k., j.n., r.p., k.k.g, m.j.b.v. and j.e. c.j.d.b., g.j.v., m.j.b.v., and a.a.l contributed to the analysis, interpretation of data and writing of the manuscript. all authors read and approved the 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http://dx.doi.org/10.1007/978-1-4020-6059-5 vreysen, m.j.b., 2005, ‘monitoring sterile and wild insects in area-wide integrated pest management programmes’, in v.a. dyck, j. hendrichs & a.s. robinson (eds.), sterile insect technique: principles and practice in area-wide integrated pest management, 1st edn., pp. 325–361, springer, dordrecht, the netherlands. http://dx.doi.org/10.1007/1-4020-4051-2_12 abstract introduction material and methods results discussion acknowledgements references about the author(s) pelin f. polat department of internal medicine, faculty of veterinary medicine, harran university, sanliurfa, turkey adem şahan department of internal medicine, faculty of veterinary medicine, harran university, sanliurfa, turkey gürbüz aksoy department of internal medicine, faculty of veterinary medicine, harran university, sanliurfa, turkey mehmet o. timurkan department of virology, faculty of veterinary medicine, atatürk university, erzurum, turkey ender dinçer advanced technology education, research and application center, mersin university, mersin, turkey citation polat, p.f., şahan, a., aksoy, g., timurkan, m.o. & dinçer, e., 2019, ‘molecular and restriction fragment length polymorphism analysis of canine parvovirus 2 (cpv-2) in dogs in southeast anatolia, turkey’, onderstepoort journal of veterinary research 86(1), a1734. https://doi.org/10.4102/ojvr.v86i1.1734 original research molecular and restriction fragment length polymorphism analysis of canine parvovirus 2 (cpv-2) in dogs in southeast anatolia, turkey pelin f. polat, adem şahan, gürbüz aksoy, mehmet o. timurkan, ender dinçer received: 29 jan. 2019; accepted: 19 mar. 2019; published: 27 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract canine parvovirus-2 (cpv-2) is the aetiological agent of an infectious viral disease of dogs, characterised by diarrhoea and vomiting. mutations of the cpv-2 genome have generated new variants circulating worldwide. this article reports the molecular analysis of cpv-2 variants collected in the dog population in southeast anatolia, turkey. twenty blood samples previously taken for the laboratory diagnosis of dogs with suspected parvovirus were screened for cpv-2 by polymerase chain reaction (pcr). of the 20 samples, 18 tested positive for cpv-2. partial vp2 gene sequencing and restriction fragment length polymorphism (rflp) analysis revealed cpv-2a (n = 1), cpv-2b (n = 16) and cpv-2c (n = 1) variants. phylogenetic analysis based on the partial length vp2 gene showed that cpv-2b (n = 15) variants showed sequences clustering separately in the phylogenetic tree. the cpv-2c sample was phylogenetically related to chinese strains and indonesia strain, whereas the cpv-2a sample was phylogenetically related to the portuguese strain. these results, which are the first to demonstrate the presence of cpv-2c in the dog population of southeast anatolia, turkey, indicate that cpv-2a/2b/2c variants co-exist in turkey’s dog population. keywords: canine parvovirus variants; molecular characterisation; turkey; rflp; restriction fragment length polymorphism. introduction canine parvovirus type 2 (cpv-2) causes an infectious viral disease of dogs, characterised by diarrhoea, vomiting and heart failure in pups. although its origin is still unknown, cpv-2 is probably derived from feline panleukopenia virus (fpv) or fpv-like carnivore parvovirus, which is widespread worldwide with different frequencies (calderon et al. 2009; hayes et al. 1979; mittal et al. 2014; nandi & kumar 2010) cpv-2 (now included in the species carnivore protoparvovirus 1) is a non-enveloped, single-stranded deoxyribonucleic acid (dna) virus of genus protoparvovirus, subfamily parvovirinae, and family parvoviridae (catmore et al. 2019). the virus has a 5.2 kb genome with two major open reading frames (orfs). one orf encodes for the vp1 and vp2 capsid proteins, while the other orf expresses non-structural proteins (ns1 and ns2). the vp2 capsid protein is the most important viral determinant for host range. amino acid mutations of this protein have important biological consequences, such as canine–feline host range and antigenic properties (calderon et al. 2009; dei giudici et al. 2017; mira et al. 2017; muz et al. 2012). in the late 1970s, cpv-2 emerged to become widespread in dog populations worldwide. after an adaptation period, cpv-2 caused a pandemic in dogs in 1978–1980. new antigenic variants, named cpv-2a and cpv-2b, resulted from mutations in cpv-2. in 2001, a new antigenic variant, cpv-2c, with amino acid substitution 426 (asp→glu) in the vp2 capsid protein, was found in italy (battilani et al. 2001; decaro & buonavoglia 2012). these three variants can be distinguished by only one amino acid change at residue 426 (asn in cpv-2a, asp in cpv-2b and glu in cpv-2c) (pérez et al. 2012), using polymerase chain reaction (pcr)-based genotyping assay and sequencing (chou et al. 2011). these three variants are separated from the original cpv-2 by five to six amino acid residues (battilani et al. 2001). during the 2000s, a change to residue 297 (ser→ala) created two new variants, reported as a new cpv-2a/2b (battilani et al. 2001). the distribution of cpv-2 variants varies between countries, and the cpv-2a/2b/2c variants currently circulate at different rates. for example, recent epidemiological investigations have revealed that cpv-2a is the main variant circulating in australia (woolford et al. 2017), india (nandi & kumar 2010), korea (geng et al. 2015) and greece (ntafis et al. 2010), while cpv-2b has been reported in taiwan (chou et al. 2011), italy (dei giudici et al. 2017; mira et al. 2018; tucciarone et al. 2018), south africa, north america, greece (ntafis et al. 2010) and iraq (sheikh et al. 2017). the cpv-2c variant has also been identified in spain, the united states (us), portugal, germany (decaro &buonavoglia 2012; pérez et al. 2012), argentina (calderon et al. 2009), uruguay (pérez et al. 2012) and sweden (sutton et al. 2013). finally, all three variants have been found co-circulating in tunisia and greece (decaro & buonavoglia 2012). vaccination is the only protection from the disease, and inactivated and modified live virus vaccines have recently been used to immunise dogs (zhou et al. 2017). new mutations (y324i and t440a) have recently emerged on the vp2 protein because of antigenic drift in the global dog population. the presence of antigenic variants has raised concerns about the efficacy of existing vaccines because of the risk of vaccine failure (pratelli et al. 2001; zhou et al. 2017). in turkey, cpv vaccines (nobivac – intervet; vanguard – pfizer; parvodog – merial; and quantum – schering) with 2a and 2b variants have been used to immunise the dog population. there have been few molecular characterisation studies of cpv-2 variants in turkey (table 1 and figure 1). some studies have reported that cpv-2a/2b is present in dogs (karapınar, dincer & ozkan 2018; timurkan & oguzoğlu 2015; yesilbag et al. 2007; yılmaz, pratelli & torun 2005), while cpv-2c has been detected in the blood samples of cat collected from ankara province (muz et al. 2012). however, there are no reports yet of cpv-2c in turkey’s dog population. figure 1: illustrative map of canine parvovirus-2 variant (2a/2b/2c) locations in the studies. table 1: studies of canine parvovirus -2 genotyping of dogs in turkey. the aim of this study was to characterise cpv-2 variants, using pcr and restriction fragment length polymorphism (rflp) methods, from clinically ill dog samples in southeast anatolia, turkey (sanliurfa province). material and methods samples blood samples (n = 20) from dogs with gastroenteritis were investigated. these dogs were submitted to the department of internal medicine (faculty of veterinary medicine, harran university, turkey) by citizens from the centre and other districts of sanliurfa province in 2017. only blood (taken for blood gas analysis and leucocyte level) samples were taken for diagnosis of the disease. no stool samples were taken. the clinical samples were stored at –20 °c until used in the study. because these blood samples were collection material, no ethical approval was required. clinical symptoms in the sampled dogs included fever, anorexia, lethargy, vomiting, severe bloody diarrhoea, severe weight loss and leucopenia. all the sampled dogs were strays without any vaccination history. information about each dog is given in table 2. table 2: sample no, age, accession numbers and clinical sings of dogs infected with canine parvovirus. deoxyribonucleic acid extraction, polymerase chain reaction and restriction fragment length polymorphism analysis cpv-2 genomic dna was extracted from clinical specimens using a high pure viral nucleic acid kit (roche diagnostics, mannheim, germany) following the manufacturer’s recommendations. purified viral genomic dna was eluted in 50 µl of elution buffer and then stored at –20 °c until pcr was performed. the pcr was performed using hfor and hrev primers for detecting the partial vp2 gene (630 base pairs [bp]) of cpv-2, according to the protocol reported elsewhere (battilani et al. 2001). the amplified pcr products were visualised using 1% agarose gel electrophoresis stained with ethidium bromide. the size of the pcr amplicons was determined using the 100 bp marker (dna ladder, thermo fisher scientific, us). for rflp analysis, viral genomic dna was amplified using 555for-5’-(caggaagatatccagaagga)-3’ (from 4003 to 4022)/555rev-5’-(ggtgctagttgatatgtaataaaca)-3’ (from 4585 to 4561) primers to obtain the partial vp2 gene, region (583nt) (buonavoglia et al. 2001). the amplified pcr amplicons were then digested using the enzyme mboii (fastdigest, thermo fisher scientific, us) for rflp characterisation, which distinguished cpv-2a/2b variants from cpv-2c (buonavoglia et al. 2001). the 583-bp pcr amplicons were digested with 2 µl (5u/1 µl) of the restriction enzyme mboii, 1 µl of 10x mboii buffer and 8 µl of molecular grade water for 1 hour at 37 °c on a heat block. subsequently, digested and non-digested amplicons were assessed on 1.5% agarose gel electrophoresis stained with ethidium bromide (figure 2). figure 2: restriction fragment length polymorphism analyses of the partial vp2 gene polymerase chain reaction amplicon (583 base pairs) of canine parvovirus-2a/2b/2c variants. line m: 100 base pairs deoxyribonucleic acid ladder (thermoscientific, united states); line 1: undigested canine parvovirus2a; line 2: undigested canine parvovirus-2b; line 3: undigested canine parvovirus-2c; line 4; canine parvovirus-2a undigested with mboii; line 5: canine parvovirus-2b undigested with mboii; line 6: canine parvovirus-2c digested with mboii. deoxyribonucleic acid sequencing and comparative analysis sequence analysis was performed on all positive samples. the pcr amplicons were cleaned with a genejet pcr purification kit (thermo fisher scientific, us) before sequencing in an abi prism 310 genetic analyzer (applied biosystem, ca, us) with hfor and hrev primers for pcr. the sequence data were submitted to the ddbj/embl/genbank databases under the following accession numbers: mg780275–mg780292 (table 2). the acquired dna sequences were compared with other cpv reference strains available from genbank database (http://www.ncbi.nlm.nih.gov). phylogenetic analysis included three canine (nobivac, intervet; vanguard, pfizer; quantum, schering) and two feline (felocell, pfizer; purevax, merial) vaccine strains and one fpv (accession no. m38246). the bioedit program was used for nucleotide and amino acid alignment comparisons (http://www.mbio.ncsu.edu/bioedit/bioedit.html) (hall 1999). phylogenetic analyses were conducted with the neighbour-joining method, using mega6 (tamura et al. 2013). ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results twenty blood specimens from dogs showing signs of gastroenteritis (bloody diarrhoea, vomiting, etc.) were tested for cpv-2 using pcr and rflp. of these samples, 18 were identified by pcr as positive for cpv-2, whereas 2 samples were negative. restriction fragment length polymorphism and sequence analysis of the pcr products revealed that only one sample was positive for cpv-2c; the other samples were positive for cpv-2a and cpv-2b (figures 2 and 3). figure 3: phylogenetic tree based on the partial vp2 gene of canine parvovirus-2 shows canine parvovirus variants circulating in sanliurfa, turkey, and reference sequences. the phylogeny was performed neighbour-joining method based on 1000 replicates by mega 6 software. each sequence is indicated with accession number, country and canine parvovirus-2 variants. in this study, canine parvovirus-2a sequence is indicated with ‘square’, canine parvovirus-2b sequence is indicated with ‘triangle’ and canine parvovirus-2c sequence is indicated with ‘circle’. the dogs were aged between 1.5 and 5 months; 40% (8/20) were female and 60% (12/20) were male. according to the clinical data, the dogs had not been known previously vaccinated. all dogs were of mixed breed. the partial vp2 gene segment of cpv-2 was obtained using hfor and hrev oligonucleotides from the buffy coat (n = 18) of the dog samples. obtained pcr amplicons were sequenced using the same primer set. the sequence analysis revealed amino acid divergences between the sequences analysed in this study and related samples from genbank at residues 297, 300, 305, 316, 324, 375 and 440 (table 3). sequence analysis results showed that 16 samples were characterised as cpv-2b, 1 was characterised as cpv-2a and 1 was characterised as cpv-2c, according to the 426 residue of the vp2 protein amino acid sequence (table 3). the nucleotide similarity (homology) of our variants was 99.0% – 100.0% for the turkish strains and 97.8% – 99.0% for the other world strains. no co-infections by multiple cpv-2 variants were detected in the investigated samples. the phylogenetic tree (figure 3) revealed that (1) 15 of the tr-urf/cpv-2b variants formed a separate group within the reference cpv-2b sequences; (2) the tr-urf/cpv-2a variant was more similar to the portuguese strain (accession no. kr559896); and (3) the tr-urf/cpv-2c variant was more similar to chinese strains (accession no.gu380303, mf467242, mf467229, ky386853 and ky626000) and the indonesia strain (accession no. lc216910). finally, comparison of the 18 turkish cpv sequences to the 3 vaccine strains revealed that the vaccine strains were from a separate branch. table 3a: amino acid changes in vp2 partial gene of canine parvovirus-2a, canine parvovirus-2b and canine parvovirus-2c. table 3b: amino acid changes in vp2 partial gene of canine parvovirus-2a, canine parvovirus-2b and canine parvovirus-2c. in this study, some amino acid changes were observed at residues 297, 300, 305, 324, 375, 426 and 440 compared to the reference genes (accession number: m38245) (table 3). we detected 297 (ser→ala), 300 (ala→gly), 305 (asp→tyr), 324 (tyr→ile) and 375 (asn→asp) substitutions in all cpv-2a/2b/2c variants compared to the reference strain. we also found substitutions at 426 (asn→asp) as cpv-2a, (asn→asn) as cpv-2b and (asn→glu) as cpv-2c. discussion canine parvovirus type 2, an important viral agent of domestic and wild canids, causes haemorrhagic gastroenteritis and myocardial disease, especially in the young. although cpv-2 has a dna genome, it displays high rates of nucleotide changes leading to the emergence of new variants (2a/2b/2c) (shackelton et al. 2005; pérez et al. 2012). canine parvovirus-2a/2b variants are prevalent in both asian countries, such as china, korea, india and japan (geng et al. 2015; zhao et al. 2016), and european countries, such as italy (dei giudici et al. 2017), greece (ntafis et al. 2010), portugal (miranda & thompson 2016) and switzerland (nandi & kumar 2010). the cpv-2c variant has been reported on several continents, including europe (portugal, greece, ltaly, spain, france, belgium, sweden and the united kingdom) (decaro & buonavoglia 2012; decora et al. 2006; mira et al. 2017; ntafis et al. 2010; sutton et al. 2013), africa (tunisia), south america (uruguay, brazil and argentina) (calderon et al. 2009), australia (woolford et al. 2017) and asia (geng et al. 2015; nandi & kumar 2010; sheikh et al. 2017). this study provides the first molecular and sequence analysis of cpv-2 strains from the dog population in southeast anatolia, turkey. according to demeter et al. (2010), mboii-based rflp analysis is misleading for detection of cpv-2c because cpv-2a can show the same rflp results as cpv 2c. however, figueiredo et al. (2017) showed that cpv-positive pcr amplicons were not digested with mboii enzyme and identified as cpv-2/2a/2b. this suggests that mboii-based rflp analysis is unreliable if used alone to identify cpv-2 variants in samples. instead, rflp analysis can be used in conjunction with sequence analysis for the characterisation of cpv-2 variants. nucleic acid-based techniques (conventional and real-time pcr) have been developed to differentiate the old variant strains used in vaccines from new variants. in addition, pcr–rflp assay has recently provided a time-saving and low-cost method for detecting and characterising cpv-2 variants in dog samples (chou et al. 2011). however, molecular techniques, rflp and pcr–rflp may not enable molecular typing of clinical samples (parthiban et al. 2010), so sequence analysis is required to accurately distinguish the cpv variant from indicator amino acid residues (castro et al. 2011). therefore, recent studies have used sequencing and rflp analysis together to provide greater precision in the molecular typing of variants. in turkey, the cpv-2c variant has only been demonstrated in cats in ankara province (muz et al. 2012), while cpv-2 variants in turkey have only been characterised for a few provinces (dincer 2017; karapınar et al. 2018; timurkan & oguzoğlu 2015; yesilbag et al. 2007). one study of mersin province found that cpv-2b was the major variant in cpv-infected dogs (dincer 2017), which is in line with our results (16/18 [88.8%] of our samples were cpv-2b). on the other hand, other studies have detected more cpv-2a than cpv-2b samples in infected dogs. muz et al. (2012) found that cpv-2c obtained from one cat had the amino acid residues 297s, 300a, 305d, 311n, 323d and 324y. these changes have also been found in the corresponding reference sequence for fplv (genebank no: m36246). however, we detected amino acid residues 297a, 300g, 305y, 311d, 323n and 324i, which indicate that these two variants may be quite different from each other. to better understand this, a larger gene region will need to be examined in turkey to determine the origin viruses precisely. the vp2 protein, which has amino acid residues at 87, 101, 297, 300, 305, 323, 324, 375 and 440, is a major antigenic determinant that plays a pivotal role in the host distribution of the virus and an important role in modulating host response. additionally, changes in the vp2 protein amino acid residues may increase pathogenicity (dei giudici et al. 2017; muz et al. 2012). in this study, we detected amino acid residues substitutions on the vp2 protein at 297, 300, 305, 323, 324, 426, 375 and 440 (table 3). we also showed differences in the amino acid residues between the three canine vaccines and the sanliurfa province field sequences, including 297, 300, 305, 316, 324, 375 and 440. in iraq, cpv-2b isolates obtained from dogs have demonstrated similar differences in the vp2 protein amino acid residues (sheikh et al. 2017). these changes may both reduce vaccine efficacy and increase the pathogenicity of cpv-2 variants. in particular, residue 324i shows strong positive selection in all carnivore parvoviruses (lin et al. 2014). residue 323, which lies adjacent to y324i, plays a role in host range as previously reported (dei giudici et al. 2017; yılmaz et al. 2005). although the function of residue y324i has not been fully determined, lin et al. (2014) reported that its mutation was responsible of viral shedding for up to 2 months in ill dogs. recently, amino acid changes have been reported in field isolates of cpv-2 in turkey, excluding amino acid residue y324i, in dogs (dincer 2017; timurkan & oguzoğlu 2015) and cats (muz et al. 2012). in this study, we detected the 324i mutation in all our cpv-2a/2b/2c variants, and to the best of our knowledge, only dincer (2017) has previously reported such a change in mersin province, turkey. in addition, the 324i mutation, which is common in asian cpv-2 variants, has been found in the isolates from taiwan (lin et al. 2014), india and japan (geng et al. 2015). in europe, the 324i mutation has been reported in cpv-2a isolates from hungary (cságola et al. 2014). residue 440a, which is important antigenically and varies within the cpv-2 variants, is located in the vp2 protein gh loop (muz et al. 2012). the t440a mutation has been reported in italian cpv-2a (dei giudici et al. 2017), korean cpv-2a (yoon et al. 2009), taiwanese cpv-2a (chiang et al. 2016), italian cpv-2b (dei giudici et al. 2017) and argentine and italian cpv-2c (dei giudici et al. 2017; calderon et al. 2009). recent studies have reported 440a mutation in the dog population in turkey (muz et al. 2012; timurkan & oguzoğlu 2015). we also detected 440a mutation in cpv-2a and cpv-2c variants in our dog samples. this indicates that further studies of 440a and 324i are needed to better understand the relationship between this mutation and the severity of clinical symptoms. previous studies have reported that cpv-2 variants (cpv-2a/2b/2c) can infect and cause disease in cats through transfer from dogs, although cat-to-cat transfer is also possible (battilani et al. 2006; ikeda et al. 2000). the dog trade and transport between countries play important roles in expanding the geographical distribution of cpv-2 variants, facilitated by free movement of people and their companion animals between the european union and other countries worldwide. a similar situation occurs in other continents, such as asia and south america. as a result, closely related cpv-2 viral sequences have been detected across continents (tucciarone et al. 2018). mira et al. (2017), for example, identified cpv-2c with an asian-origin variant in a dog transported from thailand to italy. their genome analysis of amino acid changes showed that cpv-2c is more closely related to asian than european variants. awad et al. (2018) reported that egyptian fpv isolates were closely related to portuguese isolates. as a result, new variants can arise in fields where they have not been previously reported via the international transportation of animals. vaccines play a pivotal role in protecting dog populations against cpv infection. updating currently available vaccines has become important because of the emergence of new variants of cpv. some studies (decaro & buonavoglia 2012) have reported that cpv-2-based vaccines protect against all cpv-2 variants, while others have shown that cpv-2c was detected in dogs regularly vaccinated with vaccines containing the original cpv-2 (ntafis et al. 2010; pratelli et al. 2001). geng et al. (2015) reported that dogs became infected with cpv-2 despite having been vaccinated for that virus. thus, current vaccines may not protect sufficiently against different cpv variants. calderon et al. (2009) showed that vaccinated dogs have become infected with cpv-2c in argentina. more recently, sutton et al. (2013) reported that vaccinated dogs were confirmed with severe haemorrhagic gastroenteritis caused by cpv-2c. although the pathological and epidemiological conditions are not completely known, cpv-2c causes a more severe disease in adult dogs than cvp-2a/2b variants (chiang et al. 2016). in australia, cpv-2c has been detected in vaccinated dogs even though the vaccination schedule was followed completely using commercial vaccines against cpv-2b. these dogs presented with nonspecific clinical symptoms (without vomiting or with mild diarrhoea) (woolford et al. 2017; wilson et al. 2014). there may be a variety of reasons for vaccine failure, including improper administration techniques, inappropriate vaccine handling, maternal antibodies, the virus variant used and the degree of attenuation of the vaccine virus (hernandez–blanco & catala–lopez 2015). monitoring both old and new cpv variants is important to understand virus evolution and develop preventive measures such as vaccines. the vaccines used and the immune status of puppies determine the vaccination efficacy (chiang et al. 2016; woolford et al. 2017). phylogenetic analysis based on partial vp2-region variants in turkey demonstrated that, in this study, the sequences were located among viruses from other countries. the vaccine and field viruses formed distinct genetic branches. specifically, amino acid substitutions are pivotal to genetic complexity and may result in vaccine failure and other disadvantages. new vaccines that include currently circulating strains should therefore be used to ensure appropriate and effective immunisation in turkey. consequently, this study provided the first molecular identification of cpv-2c and demonstrated the circulation of cpv-2a/2b variants in the dog population of southeast anatolia, turkey. moreover, this study offered the first use of rflp and pcr/sequence analysis for the detection and characterisation of cpv-2 variants from clinical samples obtained from dogs with gastroenteritis in turkey. although cpv-2 is a dna virus, the detected amino acid substitutions indicated that cpv-2 has evolved continuously. these mutations on the cpv-2 vp2 protein may cause currently used vaccines to fail. regular epidemiological surveys and molecular studies can identify new cpv-2 genetic variants and changes. future epidemiological and molecular surveys will help to better trace the distribution of cpv-2c and other variants in dogs in turkey. moreover, the use of appropriate test systems, such as serological and/or molecular tests, will reveal the real incidence of cpv-2c in field samples in turkey. finally, vaccination programmes and the vaccines used in dogs should be revised considering the cpv-2 variants in the field. all the obtained data will enable more effective control of cpv-2 infections in the country. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.f.p. and a.s. were responsible for the specimen collection and recording of information about each dog. e.d. was responsible for the project planning, laboratory assays and general overview. g.a. was responsible for the data interpretation. e.d. and m.o.t. were responsible for the manuscript preparation. all authors read and approved the final manuscript. funding information this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability statement additional data not included in this article will be made available on request. disclaimer the views and opinions expressed in this article are those of the author(s) and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references awad, r.a., khalil, w.k.b. & attallah, a.g., 2018, ‘epidemiology and diagnosis of feline panleukopenia virus in egypt: clinical and 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digestive cells. then a kinete develops from each zygote and this migrates from the gut to the salivary glands (schein, warnecke & kirmse 1977; mehlhorn, schein & warnecke 1978; fawcett, büscher & doxsey 1982). the kinete develops in e cells of the salivary gland by integration with the metabolism of the e cells. few sporoblasts are produced relative to the number of piroplasms ingested and it is thought that theileria is susceptible to digestion by the tick or tick innate immunity (pur nell & joyner 1968). when the tick next feeds the sporoblast grows and divides to produce thousands of sporozoites. tick salivation enables sporozoites to enter cattle and infect lymphocytes. there the schizont stage develops and undergoes further nuclear division (shaw & young 1995). the infection of the lymphocytes induces them to divide, each daughter cell taking dividing schizonts with it. the final stage in the lymphocytes is the merozoite which develops within the schizont. merozoites are released into the blood plasma and infect erythrocytes 157 onderstepoort journal of veterinary research, 73:157–162 (2006) research communication cloned theileria parva produces lesser infections in ticks compared to uncloned t. parva despite similar infections in cattle a.r. walker*, f. katzer, d. ngugi and d. mckeever centre for tropical veterinary medicine, royal (dick) school of veterinary studies university of edinburgh, roslin, eh25 9rg, scotland, uk abstract walker, a.r., katzer, f., ngugi, d. & mckeever, d. 2006. cloned theileria parva produces lesser infections in ticks compared to uncloned t. parva despite similar infections in cattle. onder stepoort journal of veterinary research, 73:157–162 experimental transmissions of cloned theileria parva in cattle with rhipicephalus appendiculatus ticks were compared to transmissions with uncloned t. parva during studies on the potential for genetic recombination during syngamy of theileria to produce antigenic diversity for evasion of bovine immunity. prevalence and abundance of t. parva infection in adult ticks, which resulted from the feeding of nymphs on the calves, were significantly higher in the uncloned compared to the cloned t. parva. development of sporoblasts of t. parva in the ticks to produce infective sporozoites was similar. there was no statistically significant difference in the clinical course of infection in cattle between cloned and uncloned t. parva. it was concluded that cloned t. parva has characteristics that reduce its viability during the tick stages of its life cycle. keywords: clone, east coast fever, infection, rhipicephalus appendiculatus, theileria parva * author to whom correspondence is to be directed. e-mail: alanw@staffmail.ed.ac.uk accepted for publication 29 march 2006—editor 158 cloned theileria parva infections in ticks compared to uncloned t. parva in which they are known as piroplasms. successful immunity, mediated mainly by cytotoxic lympho cytes, may confine infections with potentially fatal doses of sporozoites to ones that produce few clinical signs and very low parasitaemias of piroplasms in which form the theileria may be transmitted to more ticks (mckeever, taracha, innes, machugh, awina, goddeeris, & morrison 1994). existing vaccination procedures using the infection and treatment method manipulate this cycle to produce sporozoites in cryopreserved form which are inoculated to mimic natural infection. to develop syn thetic antigen vaccines it is necessary to understand how theileria evades cattle immunity (morzaria, nene, bishop & musoke 2000). this evasion is thought to be mediated by continual generation of antigenically diverse strains during recombination in the tick (mckeever 2001). cloning theileria is a technique for studying the genetics of strain diversity. this communication is from a project which used transmissions with cloned t. parva to produce recombined stocks with hypothetical potential for immune evasion. we found abnormally low levels of infection in ticks with cloned theileria. therefore results of previous transmission experiments with uncloned theileria of the same strains were compared with the performances of cloned theileria in both cattle and tick infections in order to determine what levels of transmissibility can be expected when planning experiments. theileria parva of two strains which had been isolated from two sites in kenya (muguga and mar ikebuni) were used. both strains have been maintained as cryopreserved stabilates used for cattle to tick transmissions. the uncloned muguga strain consists of a fairly homogeneous parasite population which can be distinguished by four genetic satellite markers (ms7, ms14, ms16 and ms25) developed by oura, odongo, lugega, spooner, tait & bishop (2003). the uncloned marikebuni strain, however, is very diverse, and at least 48 out of the 60 markers described by oura et al. (2003) reveal polymorphism between its constituents. clones of these two strains were produced by infecting 1 x 107 peripheral blood mononuclear cells (pbmc) in vitro with a dose of t. parva stabilate equivalent to that carried by one infected tick. at 48 h after infection the cells were plated out by limiting dilution with non-infected pbmc in u bottom 96-well plates (morzaria, dolan, norval, bishop & spooner 1995). the wells were scored for growth after 14 days and cells were harvested for dna extraction and typing by satellite markers. male calves were friesian at the centre for tropical veterinary medicine (ctvm) and boran at the international livestock research institute (ilri) in kenya. all were without previous exposure to t. parva. they were kept in isolation pens (20 °c at ctvm) and monitored daily during infections for temperature, schizonts in the lymph node draining the site of infection and piroplasms in venous erythrocytes. the theileria used as inocula were either stabilated spor ozoites from infected adult ticks that had been partially fed for 4 days, or cell culture containing schiz onts in pbmc, or in infected ticks. the doses of theileria were calculated from previous in vivo and in vitro titra tions to give patent piroplasm para sita emias (0.1 % or more of erythrocytes infected). infec tions were controlled when necessary with tetra cyc line (short acting) injected intramuscularly at 10 mg/kg for up to 5 days starting from first pyrexia and appearance of schizonts. experience has shown that this has no effect on infections in the ticks. the ticks used were r. appendiculatus of a stock originating in the 1960s from muguga in kenya which have been maintained at ctvm since then. in addition, one experiment at ilri used a stock desig nated as muguga, of the same origin as above but maintained at ilri since the 1970s. transmissions were done by application of uninfected nymphal ticks to calves at a time corresponding with expected peak of piroplasm parasitaemia in red blood cells. engorged nymphs detaching from the cattle were maintained at 28 °c, 85 % relative humidity for 28 days until completion of post moult development. adults of both sexes in equal numbers (minimum sample of 32 ticks) that had detached on the day of peak piroplasm parasitaemia were assessed for infection with t. parva by incubation for 5 days at 37 °c and 100 % relative humidity, followed by dissection and staining of their salivary glands with methyl green and pyronin (walker, mckellar, bell & brown 1979). counts were made by microscopy of sporoblasts per pair of salivary glands. data were not normally distributed thus analysis used chi square, mann whitney u test and kendall’s rank correlation. percentages were converted by arcsin and abundances of theileria spor oblasts in ticks were transformed to log10(n +1) to normalize the overdispersion of this variable (büscher & otim 1986). two batches of uncloned and cloned theileria in equal infection levels in ticks were compared for degree infectivity of the sporozoites for bovine cells after the theileria were matured by partially feeding the ticks. batches of ten ticks were surface sterilized and then ground in commercial medium using tissue grinders to release sporozoites. the suspensions of sporozoites were used to infect 159 a.r. walker et al. cultures of bovine peripheral blood mononuclear cells in an infectivity titration (wilkie, kirvar & brown 2002). historical data on transmissions using uncloned thei leria could not be standardized for use in the comparisons with cloned theileria of this study. three of the ten transmissions reported here representing cloned theileria were of mixed infections using two different clones of theileria sufficiently distinguishable by genetic markers for analysis of genetic recombinations. the outcome of these mixed infections was examined by polymerase chain reaction (pcr) using a sub-set of published satellite markers (oura et al. 2003) which distinguish between the parent theileria using their specified pcr conditions. the dna used for this was extracted from lymph node and blood sampled repeatedly during the infections. from lymph node cells dna was extracted using the promega wizard sv genomic dna purification system and for blood samples the qiagen qiaamp dna blood mini kit was used. the theileria types in the ticks were also determined by pcr using the same satellite markers on dna that was extracted either from dissected salivary glands or from whole ground ticks, with the same method as for lymph node cells. the analysis of the mixed infections in fig. 1 shows that the infections of animals bz114 and bz115 were dominated by the t. parva marikebuni clone and that the t. parva muguga clone was barely detectable during the infection (lanes 11 and 12). this was also seen in the infections in the ticks, where ticks fed on animal bz114 did not pick up t. parva muguga and for bz115 muguga alleles were only barely detectable (data not shown). we considered these mixed infections were more characteristic of infections from single sources and did not exclude them from further comparisons with single source infections. table 1 shows data from ten transmissions using cloned t. parva that met the criteria of producing patent infection with pyrexia at 40.0 °c and piroplasms detectable by microscopy. these are compared with seven transmissions using uncloned t. parva meeting the same criteria. sufficient data for a statistical comparison were obtained by combining results from infections with both strains of t. parva. experience at ctvm and ilri have shown that although these strains are different in immunological cross reactivity, they produce similar infections in cattle and ticks. moreover, the five clinical variables measured in days were used to compare the muguga and marikebuni strains by chi square tests between different pairs of calves. no significant differences were found between these pairs. the grouping of this data was considered justified for further comparison using mann whitney u test. the threshold for recording pyrexia was strict, at lane 1 negative control lane 2 stabilate 64 (marikebuni) lane 3 stabilate 70 (marikebuni) lane 4 stabilate 81 (muguga) lane 5 stabilate 72 (marikebuni) lane 6 calf 211 infected with stabilate 72 lane 7 clone a3 lane 8 clone 13 lane 9 muguga clone 1, ilri stabilate 3308 lane 10 marikebuni clone 1, ilri stabilate 4210 lane 11 calf bz114, day 21 of infection with stabilates 3308 + 4210 lane 12 calf bz115, day 21 of infection with stabilates 3308 + 4210 1 650 bp 1 000 bp 850 bp 650 bp 500 bp 400 bp 1 2 3 4 765 12111098 fig. 1 polymorphism in pcr size detected within the pim gene of t. parva 1 6 0 c lo n e d t h e ile ria p a rva in fe ctio n s in ticks co m p a re d to u n clo n e d t . p a rva table 1 infection characteristics of cloned compared to uncloned t. parva in cattle and ticks. ten transmissions with cloned t. parva and seven transmissions with uncloned t. parva, all meeting the criteria of patent disease with pyrexia and production of piroplasms. analysis by mann-whitney u test strain of t. parva clone number infection method day 1st pyrexia day 1st schizont day 1st piroplasm day max. piroplasm days pyrexic max. % piroplasms prevalence of sporoblasts per tick (%) abundance of sporoblasts per tick muguga muguga marikebuni marikebuni marikebuni marikebuni marikebuni marikebuni muguga + marikebuni muguga + marikebuni 3308 3308 3262 3262 13 + 17 4210 4210 a3 3308 + 4210 3308 + 4210 stabilate ticks ticks ticks cells stabilate stabilate cells stabilates stabilates 8 12 9 6 8 12 7 9 10 10 10 10 11 6 6 11 8 6 7 6 12 17 15 14 10 14 12 10 12 11 17 18 17 16 12 18 15 14 18 19 11 3 9 9 4 5 9 4 9 9 5.6 0.1 1.2 0.1 0.01 1.2 2.0 0.01 5.8 2.6 60 1.7 35 0 0.9 52 38 14 19 6 17.9 0.3 4.5 0 0.3 13.0 4.2 0.7 0.9 0.01 medians 9.0 7.5 12.0 17.0 9.0 1.2 16.5 0.8 muguga muguga muguga muguga muguga marikebuni marikebuni not not not not not not not stabilate stabilate stabilate stabilate stabilate stabilate stabilate 12 6 8 8 6 12 9 11 5 5 6 6 10 6 13 10 10 12 12 14 12 14 16 16 15 16 16 17 6 11 9 7 11 6 10 0.1 7.9 5.5 3.2 7.3 0.2 6.0 95 51 88 59 58 95 100 121 17.9 63.7 29.7 7.9 78 44 medians 8.0 6.0 12.0 16.0 9.0 5.5 88.0 58.0 probability 0.65 0.20 0.56 0.25 0.27 0.15 0.003* 0.002* * significantly different at p < 0.01 161 a.r. walker et al. 40.0 °c, and the number of days pyrexic was recorded up to day 18. when ticks were used to infect, day 0 of cattle infection was counted as day 4 of tick feeding. the course of infection of the calves using cloned theileria was similar to that using uncloned theileria. the median values of the characteristics measured in days were the same or representing milder disease in the cloned theileria infections. the number of days pyrexic is the best single measure of these infections; it was 9.0 in both the infections with cloned t. parva and with uncloned t. parva. none of the comparisons of these characters between cloned and uncloned t. parva were significantly different. although the level of piroplasm parasitaemia was more than twofold higher in the transmissions of uncloned theileria, this difference was not significant. in contrast, the differences of infection with theileria of the ticks from the transmission with cloned compared to uncloned theileria were large and highly significant for both characteristics of prevalence of infection and abundance of infection. the relation between piroplasm parasitaemia and tick infection was examined using data from the uncloned t. parva transmissions. kendall’s rank correlations showed an inverse relationship, with negative and nearly significant correlations (piroplasms against prevalence –0.5, p = 0.06; piroplasms against abundance –0.49; p = 0.06). the results obtained at ctvm contrasted greatly with those from ilri where, in totals of 29 transmissions with uncloned t. parva muguga and separately t. parva marikebuni, compared to 17 similar transmissions with cloned t. parva of both strains, the uncloned transmissions resulted in median tick infections having prevalence of 60.0 % ticks infected and abundance of 20.8 sporoblasts per tick compared to cloned transmissions with prevalence of 72.0 % and abundance of 22.0 (paul spooner, personal communication 2005). table 2 shows that similar high levels of infectivity of sporozoites for bovine cells were found with the batches of cloned and uncloned t. parva marikebuni strain. there was no significant difference when compared by chi square test. it was important that only simple conclusions were drawn from the analysis because of the highly variable clinical course of infections with theileria. we con sider that we have found a characteristic of cloned t. parva that may have an influence on studies using clones and that this needs to be reported so that it can be incorporated into experimental procedures. cloned t. parva produced a course of disease in calves with a clinical pattern without significant differences from that produced by uncloned t. parva. the cloned t. parva produced an acute infection that was potentially fatal and with sufficient piroplasms in the peripheral blood to expose feeding ticks to the typical massive numbers of the microgamete and macrogamete stage of theileria in the tick’s gut. cloned t. parva infections in ticks were characterized by prevalences and abundances of infection that were both significantly lower than obtained with uncloned t. parva. it can be considered that this difference was due to the cloned theileria infections producing a median of maximum piro plasm parasitaemias of 1.2 % compared to 5.5 % for the un cloned theileria. however, these data are not significantly different and it is clear from table 1 that low piroplasm parasitaemias can give rise to high prevalence and abundance of infection in some cases. young, dolan, morzaria, mwakima, norval, scott, sherriff & gettinby (1996) demonstrated, with transmissions in 113 calves of uncloned t. parva muguga to r. appendiculatus, that there is a significant and positive linear regression of piroplasm parasitaemia with both prevalence and abundance of infection in ticks. however, this relationship was not simple; a large increase in piroplasm parasitaemia related to only a small increase in infections in the ticks. the data we present do not support a proposition that the difference in tick infections between cloned and uncloned t. parva was due directly to the difference in piroplasm parasitaemias. the infections of the ticks with cloned t. parva in the tick’s salivary glands table 2 in vitro assessment of infectivity of sporozoites of t. parva marikebuni uncloned and clone 13. data are mean numbers of wells with growth out of a total of 96 available wells for each concentration of cells per well 9 000 cells per well 3 000 cells per well 1 000 cells per well 300 cells per well total 4 day fed ticks uncloned cloned cloned 92 96 96 67 91 88 37 61 56 14 28 22 210 262 262 5 day fed ticks uncloned cloned 96 93 91 71 80 29 38 14 305 207 162 cloned theileria parva infections in ticks compared to uncloned t. parva matured normally to produce sporozoites of infectivity to cattle cells that were similar to uncloned t. parva. these results indicate that there was a characteristic of cloned t. parva that either reduced the rate of syngamy or viability of the zygotes and kinetes in the tick. it may be important that the meiotic reduction division of t. parva is thought to occur in the kinete stage (gauer et al. 1995). the tick gut and haemolymph between the gut and salivary glands are potentially hostile from the likely effects of an innate immune system of this arthropod. theileria parva in high levels of infection in r. appendiculatus can harm the salivary glands and thus it is likely that this species of tick has evolved immune defences against it (watt & walker 2000). genetic experiments using cloned theileria should be adjusted to this anomaly. acknowledgements the work at the centre for tropical veterinary medicine was supported by the wellcome trust. we thank the royal (dick) school of veterinary studies, and the international livestock research institute for provision of facilities and encouragement for such clinical investigations. we are specially grateful for the considerable experimental contributions to this study of paul spooner at ilri. references büscher, g. & otim, b. 1986. quantitative studies on theileria parva in the salivary glands of rhipicephalus appendiculatus adults: quantitation and prediction of infection. international journal of parasitology, 16:93–100. fawcett, d.w., büscher, g. & doxsey, s. 1982. salivary gland of the tick vector of east coast fever. 3: the ultrastructure of sporogony in theileria parva. tissue and cell, 14:83– 206. gauer, m., mackenstedt, u., mehlhorn, h., schein, e., zapf, f., njenga, e., young, a. & morzaria, s. 1995. dna 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) natascha v. meunier department of pathology and pathogen biology, royal veterinary college, united kingdom faculty of epidemiology and population health, london school of hygiene and tropical medicine, united kingdom peregrine sebulime department of wildlife and aquatic animal resources, makerere university, uganda richard g. white faculty of epidemiology and population health, london school of hygiene and tropical medicine, united kingdom richard kock department of pathology and pathogen biology, royal veterinary college, united kingdom citation meunier, n.v., sebulime, p., white, r.g. & kock, r., 2017, ‘wildlife-livestock interactions and risk areas for cross-species spread of bovine tuberculosis’, onderstepoort journal of veterinary research 84(1), a1221. https://doi.org/10.4102/ojvr.v84i1.1221 note: richard g. white and richard kock are joint senior authors. research note wildlife-livestock interactions and risk areas for cross-species spread of bovine tuberculosis natascha v. meunier, peregrine sebulime, richard g. white, richard kock received: 14 apr. 2016; accepted: 21 july 2016; published: 23 jan. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the transmission of diseases between livestock and wildlife can be a hindrance to effective disease control. maintenance hosts and contact rates should be explored to further understand the transmission dynamics at the wildlife-livestock interface. bovine tuberculosis (btb) has been shown to have wildlife maintenance hosts and has been confirmed as present in the african buffalo (syncerus caffer) in the queen elizabeth national park (qenp) in uganda since the 1960s. the first aim of this study was to explore the spatio-temporal spread of cattle illegally grazing within the qenp recorded by the uganda wildlife authority (uwa) rangers in a wildlife crime database. secondly, we aimed to quantify wildlife-livestock interactions and cattle movements, on the border of qenp, using a longitudinal questionnaire completed by 30 livestock owners. from this database, 426 cattle sightings were recorded within qenp in 8 years. thirteen (3.1%) of these came within a 300 m–4 week space-time window of a buffalo herd, using the recorded gps data. livestock owners reported an average of 1.04 (95% ci 0.97–1.11) sightings of uganda kob, waterbuck, buffalo or warthog per day over a 3-month period, with a rate of 0.22 (95% ci 0.20–0.25) sightings of buffalo per farmer per day. reports placed 85.3% of the ungulate sightings and 88.0% of the buffalo sightings as further than 50 m away. ungulate sightings were more likely to be closer to cattle at the homestead (or 2.0, 95% ci 1.1–3.6) compared with the grazing area. each cattle herd mixed with an average of five other cattle herds at both the communal grazing and watering points on a daily basis. although wildlife and cattle regularly shared grazing and watering areas, they seldom came into contact close enough for aerosol transmission. between species infection transmission is therefore likely to be by indirect or non-respiratory routes, which is suspected to be an infrequent mechanism of transmission of btb. occasional cross-species spillover of infection is possible, and the interaction of multiple wildlife species needs further investigation. controlling the interface between wildlife and cattle in a situation where eradication is not being considered may have little impact on btb disease control in cattle. introduction circumstantial evidence suggests that transmission of a range of diseases between wildlife and livestock occasionally occurs either directly or through a vector (bengis, kock & fischer 2002; miller, farnsworth & malmberg 2013; siembieda et al. 2011). transmission between species is dependent on the population distribution and timing of contacts (renwick, white & bengis 2007), and where weak inter-species transmission occurs, spread within species needs to be high for the disease to persist in the ecosystem. the wildlife-livestock interface has been well studied for bovine tuberculosis (btb) in countries with intensive control programmes, and wildlife have been implicated as a reservoir of infection to cattle (palmer 2013). molecular studies indicate that isolates of btb have been found in both domestic and wild animals supporting cross-species transmission, although the direction of transmission is not proven (de garine-wichatitsky et al. 2013; musoke et al. 2015). mathematical models are useful to estimate the contact rates and effective transmission between species, which can assist with the management of intervention strategies (barron, nugent & cross 2013; brooks-pollock & wood 2015; hardstaff et al. 2013; zanella et al. 2012). gps collaring studies have also assisted with estimating these contact rates and probabilities of wildlife-livestock interactions over landscapes and time (brook et al. 2013; caron et al. 2016; miguel et al. 2013). in uganda, african buffalo (syncerus caffer) are maintenance hosts for btb in the queen elizabeth national park (qenp), with reports of the disease dating back to the 1960s (woodford 1982a). infection levels have not notably increased in buffalo since this time, with kalema-zikusoka et al. (2005) proposing that the drastic population decreases because of poaching in the 1970s and 1980s limited the spread of the infection. alternately, the lack of fencing may allow buffalo to expand to other reserves according to natural resource fluctuations, maintaining lower density populations and limiting disease spread. both cattle and buffalo numbers are increasing which may bring animals into closer contact because of grazing pressures and impact on potential cross-species interactions (plumptre et al. 2010). communities that keep livestock, within qenp and in neighbouring villages, are not fenced out of the park, and there is much anecdotal evidence of wildlife coming into contact with livestock. because of limited grazing land, some farmers are known to drive their livestock illegally into qenp for extra forage, potentially coming into closer contact with wildlife (g. kalule, pers. comm., november 2014). the first aim of this study was to explore the spatio-temporal spread of cattle illegally grazing within the qenp, uganda, recorded by the uganda wildlife authority (uwa) rangers in a wildlife crime database. secondly, we aimed to quantify wildlife-livestock interactions at the northern border of the qenp demarcated by the nyamugasani river, using a longitudinal questionnaire. materials and methods ethics statement all research activities in uganda were reviewed and approved by uwa, the uganda national council for science and technology (uncst) and the royal veterinary college research ethics committee (urn201041290/ ppb01253). study area our study site was qenp, a 1978 km2 wildlife protected area situated in western uganda (00°12’s, 30°00’e) adjacent to the democratic republic of congo and forms part of the greater virunga landscape, which is comprised of multiple wildlife reserves (uganda wildlife authority 2012). large herbivores present in qenp include african buffalo, uganda kob (kobus kob thomasi), defassa waterbuck (kobus ellipsiprymnus), warthog (phacochoerus africanus) and african elephant (loxodonta africana). predators include lion (panthera leo), spotted hyena (crocuta crocuta) and leopard (panthera pardus). livestock owners were from the region on the north-western border of the park and were predominantly basongora pastoralists with a majority of ankole cattle, an indigenous breed. there are no physical barriers preventing animal movement across the qenp borders except in the far south of the park. wildlife crime database the wildlife crime database was part of a uwa project that was undertaken in partnership with the wildlife conservation society of new york. rangers on daily patrols recorded illegal activities and animal sightings within the qenp, including grazing of domestic animals on park land. a data set was obtained in march 2015 from the uwa department of research and monitoring containing gps coordinates for buffalo and cattle sightings within the protected area. the sightings were recorded from january 2006 until the end of march 2014 with variable quality of reporting. uganda kob were not recorded in this database as they were numerous and the task became burdensome to the field staff recording the data. spatio-temporal analysis was conducted to identify overlapping areas of buffalo and cattle sightings. each sighting was representative of one herd on a specific day, and sightings were considered repeats if recorded within immediate proximity and time. temporal limits were 2, 4 and 6 weeks based on the survival time of mycobacteria in the environment (tanner & michel 1999). spatial limits were 150 m, 300 m and 500 m to take into account the variability of the gps equipment and recording methods, and movement of animals around the recorded site. any buffalo and cattle recorded sharing the same site on the same day were additionally highlighted. analysis and mapping were done in r (r core team 2013) and quantum gis (qgis development team 2015) with downloaded base maps (http://www.gadm.org). interaction surveys farmers or employed herdsmen accompany their cattle herds to grazing and watering sites and return to enclose their animals near the homestead overnight. twenty-five livestock owners from nyakatonzi (northern border of qenp) and five livestock owners from katwe kabatoro (community within qenp) were identified to take part in a longitudinal survey recording daily wildlife sightings (figure 1). these farmers, all members of the muhumuza nyakatonzi cattle keepers association, were selected from within each parish in the study area. one member of the household was required to be able to read english. verbal consent was obtained from the livestock owner after a prepared statement was read in the local language; participants could withdraw from the study at any time and were compensated for their participation. the livestock owners were asked to give monthly demographic information such as herd size, sales and losses of cattle. daily information was requested on wildlife sightings by the livestock owners, noting the location of the sighting, the species involved and distance from domestic cattle, as well as grazing with other cattle herds, that is, how many cattle herds interact with his herd. this information was collected over 3 months from december 2014 to march 2015. an animal health technician conducted weekly visits to ensure questionnaires were completed. the survey period was preceded by a pilot survey for 1 month in november 2014 to test the questionnaires and the logistics and adjusted after consultation with the animal health technician involved. figure 1: study site in western uganda showing location of study farms, communal grazing areas and watering sites. inset: queen elizabeth national park. weather information was obtained from uwa for the weather station at mweya outpost for 2002–2013 (figure 1). local regression smoothing was applied to the time-series data of the wildlife sightings to observe trends. the number and incidence of sightings (average sightings per farmer) were calculated and analysed in r statistical software. mixed models were used to examine the relationship between the outcomes of sightings and distance to wildlife with the following variables: location type, herd size, location, distance to park, month and species where appropriate. farmer id was added as a random effect for all models. herd size was divided into three categories (5–24, 25–75 and > 75). the distance between wildlife and livestock was condensed from five into two categories: ‘near’ (from: almost touched, 10 m) and ‘far’ (from: 50 m, 100 m and > 100 m). this was done to compensate for observer bias of distance, and the true ‘near’ distance is more likely to range from 5 m to 50 m. bukangara was the reference location in the statistical models. the analysis focused on ungulates, which were comprised of african buffalo, uganda kob, waterbuck and warthog, with additional individual focus on buffalo. results wildlife crime database the wildlife crime database contained 5989 buffalo sightings and 426 cattle sightings for the years january 2006 – march 2014. the cattle were distributed along the northern borders of the park, whereas buffalo concentrations were higher to the south-west of lake george and in the southern region of qenp, ishasha sector. the spatial distribution of animals was similar by year and season; however, the total number of cattle sightings per year was variable. the number of cattle and buffalo sightings within qenp per year is summarised in table 1, highlighting the number of interactions within 4 weeks and 300 m. the locations and numbers of the wildlife-cattle interactions are shown in figure 2. the areas where interactions occur follow the high density spatial distribution of the cattle along the northern border of qenp. there were 13 interactions at the 300 m–4 week limit (3.1% of cattle sightings) with 57 interactions at our maximum limits. nine interactions were seen within 1 day at any distance < 500 m. the closest distance between species within 6 weeks was 101 m. figure 2: queen elizabeth national park showing points of spatio-temporal overlap (n) between buffalo and cattle within (a) 2 weeks and 150 m, (b) 4 weeks and 150 m, (c) 6 weeks and 150 m, (d) 2 weeks and 300 m, (e) 4 weeks and 300 m, (f) 6 weeks and 300 m, (g) 2 weeks and 500 m, (h) 4 weeks and 500 m and (i) 6 weeks and 500 m. table 1: number of sightings per year of cattle and buffalo in the queen elizabeth national park in the wildlife crimes database from uganda wildlife authority. farmer interaction surveys thirty farmers participated in the longitudinal survey reporting wildlife-cattle interactions over a 3-month period from december 2014 until march 2015. the total cattle study population was reported to be 1869 adults and 381 calves on average per month. the average male:female ratio was 1:3, and the calf:adult ratio was 1:5. the median herd size was 38 animals (mean 62, range 5–237). farmers sent herds out to graze for an average of 10.9 hours (min–max 4.9–18.1 hours; s.d. 1.6) per day. the number of cattle herds seen mixing with other herds at the grazing points was on average five herds per day (median 4, s.d. 4.7). at the watering points, five herds on average were also seen per day (median 4, s.d. 4.1). over the 3-month period, an average of six animals left a herd per month because of death, sale, theft or being lost during grazing. on average five animals entered a herd per month by buying in, births, gifts or being found whilst grazing. this resulted in an average loss of one animal per month per herd. more details of these gains and losses are shown in tables 2 and 3. table 2: average losses of cattle reported per herd per month. table 3: average gains of cattle reported per herd per month. in total, there were 2744 daily reports from farmers covering the 3-month period from 24 december 2014 until 25 march 2015. farmers reported wildlife sightings on 73.1% of farmer days. ungulates, including buffalo, were seen by farmers on 52.7% of reported days (2901 total sightings), with a mean incidence of 1.04 (95% ci 0.97–1.11) animal sightings per farmer per day. buffalo were seen on 18.8% of reported days at a rate of 0.22 (95% ci 0.20–0.25) sightings per farmer per day. in addition to ungulates, elephants were seen on 57.6% of days, predators (lion, leopard, hyena) on 13.2% of days (figure 3) and other wildlife were mentioned in 9.2% of the reports. these included reports of hippopotamus, nile crocodile, rabbit (most likely scrub hare) and unspecified snake and bird species. ungulates were seen more frequently at grazing areas compared with watering points and homesteads. the total number of sightings decreased over the 3 months studied (figure 4). uganda kob (n = 1257) were the most commonly seen ungulate, followed by buffalo (n = 635), warthog (n = 525) and waterbuck (n = 484). the beginning of the rainy season usually falls within this time period (months 1–3 in figure 5) based on data from the mweya weather station. figure 3: number of daily sightings per farmer of elephant, predators and ungulates for the period december 2014–march 2015. (a) grazing area, (b) watering point and (c) homestead. figure 4: number of sightings per farmer per ungulate type, plotted per day for the period december 2014–march 2015. (a) grazing area, (b) watering point and (c) homestead. figure 5: (a) monthly rainfall and (b) mean monthly min–max temperature at uganda wildlife authority mweya weather station in queen elizabeth national park for 2002–2013. nyamugasani grazing and watering points were the areas most frequently used by farmers. nyamugasani had the highest absolute number of ungulate sightings (52.1%) and buffalo sightings (51.7%). relatively, rwehingo grazing area (or 2.1; 95% ci 1.5–3.1; p < 0.001), nyamugasani grazing area (or 1.4; 95% ci 1.0–1.9; p = 0.05) and grazing on multiple sites (or 4.1; 95% ci 1.2–13.8; p = 0.02) were associated with greater odds of ungulate sightings compared with bukangara, in the mixed univariate model (table 4). when examining buffalo sightings, rwehingo (or = 1.8, 95% ci 1.1–2.9; p = 0.02) and multiple sites (or 5.2, 95% ci 1.9–14.4; p = 0.002) had increased odds; however, there was no association with specific watering locations. ungulates (n = 72, 2.6%) and buffalo (n = 10, 0.4%) were seldom reported close to the homesteads. table 4: grazing and watering locations showing the area visited and the percentage of ungulate and buffalo sightings at each location. there were greater odds of an ungulate sighting, within the first 2 months of the study or from farmers with larger herd sizes (or 2.7), using the univariate analysis (table 5). the evidence of a herd size effect was lost in multivariate models as there was irregular distribution of herd sizes amongst parishes. muruti parish tended to have larger herds, and kyakitale had smaller herds than average, although homesteads from these locations were closest to the park borders. homesteads closer to the park were more likely to see ungulates with a 0.97 decrease in odds (95% ci 0.95–0.99) of a sighting per 100 m increase in straight-line distance to the qenp border (p < 0.01). the average distance of homesteads, where sightings took place, was 1100 m from the border of qenp, and where no sightings took place, the average distance was 2400 m. table 5: risk factors associated with sightings of ungulates and buffalo reporting odds ratios for univariate mixed models with farmer as a random effect. analysing the subset of sightings, during the 3-month period, 14.7% of the ungulate sightings and 12.0% of the buffalo sightings were reported as ‘near’ distance to the cattle herd. of these, buffalo were never reported as ‘almost touching’, and only n = 6/1612 (0.4%) ungulates (two uganda kob, four waterbuck) were reportedly close enough to touch the livestock. an additional 31.1% of buffalo sightings were considered to be roughly 50 m away compared with 40.8% of the ungulate sightings. with the univariate mixed model, ungulates had greater odds of a ‘near’ distance sighting at the homestead (or 2.0) (table 6). those with larger herd sizes were more likely to report a ‘near’ sighting with buffalo and ungulates. there was an inverse relationship between homestead distance from the park boundary and a ‘near’ sighting (or 0.95, 0.93–0.97 per 100 m). table 6: risk factors associated with distance (near vs. far) of cattle from wildlife reported for ungulates and buffalo including odds ratios for univariate mixed models with farmer as a random effect. discussion this study described reports of wildlife proximity to livestock around qenp using an existing wildlife sightings database as well as daily sighting reports from livestock owners. the value of wildlife sighting reports is limited in that absence of a reported sighting is not necessarily absence of an animal. a benefit of the longitudinal livestock owners study is that herdsmen remain with the cattle for most of the day, which would increase the likelihood of a reported sighting. we reported a larger number of sightings compared with similar research on the border of the kruger national park in south africa (brahmbhatt et al. 2012). however, only fenced areas of the park were included in that study implying that contacts were reported when wildlife or livestock crossed this barrier, whereas free movement is possible at qenp. the daily reporting of sightings by livestock owners minimised recall bias, although there was a risk of reporting fatigue as the study progressed. employment of a field technician performing weekly visits encouraged completion of the questionnaires by livestock owners. the tapering of wildlife sightings over time may still be a consequence of fatigue, but the levels of uganda kob sightings did not differ significantly by month on pearson’s chi-squared test (p = 0.1). this supports the outcome that the decrease of total sightings is a real finding. this may be related to the onset of the rainy season and would need to be confirmed with an extended study across seasons. ryan, knechtel and getz (2006) showed a seasonal change in the home range of buffalo related to water availability and quality of pastures, as well as changes in herd size, which may account for the decreased number of sightings over time. although water availability may not be a factor, a perennial river is the main water source on the qenp boundary. additionally, in late january, large areas of qenp were subject to wildfires, which probably influenced the grazing patterns of the wildlife, including areas adjacent to our study site. animals may have moved deeper into the park in search of grazing until there was sufficient regrowth in the burnt areas. although elephant and predator sightings were not the focus of this study from a disease point of view, their sightings, especially the high number close to the homesteads, were associated with destruction of property and loss of livestock. these species are a source of conflict between the wildlife authority and community farmers, who do not feel appropriately compensated for their losses (b. sunday, pers. comm., november 2014). the role of ungulates, besides buffalo, in the transmission and maintenance of btb is not known in the wildlife of qenp. btb was confirmed in uganda kob for the first time in qenp in 2011, after a cluster of mortalities was investigated (predict consortium 2014; b. ssebide, pers. comm., march 2015). in warthog, m. bovis was isolated from a necropsy in 1997 (kalema-zikusoka et al. 2005) confirming the ongoing infection in this species since previously reported by woodford (1982b). considering the close contact these species have with buffalo and cattle, this association should be further explored. warthog may contribute to disease in a similar manner as wild boar in europe (hardstaff et al. 2013; palmer 2013) or uganda kob likewise to the kafue lechwe antelope in zambia (munyeme & munang’andu 2011). nevertheless, environmental conditions differ and the wetlands favoured by kafue lechwe may aid survival and spread of m. bovis in its habitat. close contact does not necessarily equate to an effective transmission event, and the potential for disease transmission across species is likely influenced by a number of factors. firstly, the variability in infectiousness of the animals because of disease progression and species will impact transmission. in an experimental study in south africa, it was suggested that african buffalo do not commonly shed high quantities of m. bovis in nasal and oral secretions and are unlikely to transmit infection through water in free range conditions (michel et al. 2007). this, however, did not preclude the possibility of the spread of other mycobacterial species. conversely, in cattle, nasal excretion of m. bovis was seen more commonly (menzies & neill 2000). the tendency to graze cattle illegally within qenp would therefore allow for contamination of the wildlife grazing areas as well as the communal pastures and watering points. a second factor influencing effective transmission is survival of the pathogens dependant on environmental conditions. we used up to 6 weeks as an indicator of survival time of m. bovis as shown by tanner and michel (1999), but this may be an overestimate. jackson, de lisle and morris (1995) showed an inverse relationship of bacterial survival with high temperature implying that under the hot conditions found at our study site (20 °c – 30 °c), m. bovis is unlikely to survive beyond 4 weeks unless in the shade (duffield & young 1984). it should also be considered that for oral transmission to take place, a susceptible animal must graze the specific location where an infectious animal had been within this time frame. under extensive grazing conditions, this is likely to be an uncommon occurrence. the possibility of infection transmission will also be influenced by the movement and distribution of animals regulating direct and indirect contact rates. there were limitations to the distances reported in this study, as they were an approximation. animals may have moved around the recorded point and contacts between species may have gone unnoticed. no buffalo were reported to be in direct contact with cattle in the livestock owner reports and no buffalo came within 100 m of cattle in the ranger reports. in a study by miguel et al. (2013) using gps collared animals, direct contact between buffalo and livestock was also limited. for aerosol transmission, few bacilli carried in droplets can transmit btb infection (neill, bryson & pollock 2001). close contact between cattle herds occurs with congestion at the entrance to watering points and kraaling at night which would encourage droplet transmission between cattle (gannon, hayes & roe 2007), whereas this close contact was rare between wildlife and cattle. additionally, pollock et al. (2006) reviewed evidence indicating that in outdoor conditions, transmission rates for btb were low. the prevalence of btb in individual cattle around qenp is estimated at 2.8% (95% ci 1.5–5.4) (meunier, unpublished data), whereas in buffalo btb is reported at 21.6% (kalema-zikusoka et al. 2005). considering the lack of control measures in cattle for btb, it is not unreasonable to expect higher levels of btb infection in local cattle if a high frequency of transmission from buffalo was taking place. alternately, ankole cattle, the widely kept local breed, may be less susceptible than expected to btb (ameni et al. 2007). in south africa, there is evidence of the same spoligotypes of btb present in both wildlife and livestock (hlokwe et al. 2014). identification of the strain types involved in infection of wildlife and livestock bordering qenp could give a better indication if inter-species spread is occurring. conclusion this study quantified reports of wildlife seen near to livestock around qenp showing that inter-species transmission of btb infection is possible and showed feasible high-risk areas for interactions on the border of qenp. sharing of grazing and water resources occurred frequently, which would favour environmental transmission. in contrast, infrequent close contact between species occurred which could possibly facilitate aerosol transmission. although considering the environmental conditions, host determinants and pathogen factors, transmission of btb between species is likely a rare spillover event. other diseases, particularly those with tick vectors, could be easily transmitted within the time frames and distances reported in our study (caron et al. 2013). regarding the high number of sales in cattle herds, disease introductions in cattle from other livestock are likely to be a high-risk event, even though farmers may first implicate wildlife as a source of infection. disease control at the point of movement and sale of cattle will presumably have a greater impact on the spread of btb in livestock than controlling the interface between wildlife and cattle in a situation where eradication is not being considered. however, this intervention does not address the spread of the disease within wildlife. it is unknown what role other wildlife such as the uganda kob, waterbuck and warthog play in infection transmission within this system, although they were regularly sighted near cattle and homesteads. acknowledgements the authors acknowledge mr. yonah kajunwa, for his work as our field technician and translator; dr kalule and the ministry of agriculture animal industry and fisheries for their support; the uganda wildlife authority for providing the wildlife and weather data sets; ruby chang for statistical advice; and the muhumuza nyakatonzi cattle keepers association and the livestock owners for their participation. this work was funded by the royal veterinary college. n.v.m. is funded by a bloomsbury colleges phd scholarship. r.g.w. 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tropical animal health and production 14, 155–160. http://dx.doi.org/10.1007/bf02242146 zanella, g., bar-hen, a., boschiroli, m.l., hars, j., moutou, f., garin-bastuji, b. et al., 2012, ‘modelling transmission of bovine tuberculosis in red deer and wild boar in normandy, france’, zoonoses and public health 59(suppl.), 170–178. http://dx.doi.org/10.1111/j.1863-2378.2011.01453.x horak_163-174.indd introduction the kruger national park, located in the lowveld of north-eastern south africa, is approximately 1.9 million ha in size and has been managed as a wildlife conservation area since its proclamation in 1926. in 1954, during a period of fire suppression within the park, a burn plot experiment was launched. the history and a description of the experimental burn plots have been recorded by trollope, potgieter, biggs & trollope (1998) and are briefly repeated here. the experiment was initially designed to obtain an understanding of the effects of season and frequency of burning on vegetation, and was conducted in four major veld types of the park, in each of which four separate replicates were laid out. each replicate is approximately 100 ha in extent and comprises 11 to 13 treatment sub-plots and an un-burned control, each approximately 7 ha in size, in a contiguous rib163 onderstepoort journal of veterinary research, 73:163–174 (2006) effect of burning on the numbers of questing ticks collected by dragging i.g. horak1, g.j. gallivan2, a.m. spickett3 and a.l.f. potgieter4 abstract horak, i.g., gallivan, g.j., spickett, a.m. & potgieter, a.l.f. 2006. effect of burning on the numbers of questing ticks collected by dragging. onderstepoort journal of veterinary research, 73:163–174 sixteen experimental burn plot replicates, in groups of four, in four landscape zones of the kruger national park, south africa, and from which wildlife are not excluded, have been subjected to fixed, regular burning regimens since 1954. in 1999, a study to determine the effect of burning on ixodid ticks questing for hosts from the vegetation of the plots was initiated, and six sub-plots, with identical histories, within each of two of the burn plot replicates in combretum collinum/combretum zeyheyri woodland on granite, were selected. with few exceptions these 12 sub-plots, as well as unburned vegetation adjacent to each of the replicates, were sampled for ticks at monthly intervals for a period of 39 months by dragging with flannel strips. the existing regimen of burning during august or during october on individual sub-plots was continued during this time. a total of 14 tick species was recovered from the plots of which nine could be considered major species. sufficient numbers for statistical analysis of only eight species were, however, collected. burning appeared to have little short-term effect on the number of ticks recovered. in the longer term, the response varied from no change, an increase, or a decrease in the numbers of ticks collected each year after burning. tick species, life cycle, seasonality, questing strategy, host preference and host utilization of the habitat were important determinants of the effect of burning. keywords: experimental burn plots, fire, amblyomma hebraeum, amblyomma marmoreum, haemaphysalis leachi, rhipicephalus appendiculatus, rhipi cephalus (booph ilus) decoloratus, rhipicephalus evertsi evertsi, rhipicephalus simus, rhipicephalus zambeziensis 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa; e-mail: ivan.horak@up.ac.za 2 187 cluny street, ottawa, ontario, canada. k1g oj9 3 division of parasitology, arc-onderstepoort veterinary institute, onderstepoort, 0110 south africa 4 scientific services, kruger national park, skukuza, 1350 south africa. present address: p.o. box 578, hartenbos, 6520 south africa accepted for publication 30 march 2006—editor 164 effect of burning on numbers of questing ticks collected by dragging bon-like spatial design. essentially unaltered burning regimens have been applied and monitored on the various treatment plots until the present. recently, however, projects to determine the effect of burning on a wider range of elements than just the vegetation were initiated, and small mammals, reptiles, birds, insects and ixodid ticks were included. surveys on the seasonality of ticks infesting animals in the park were conducted intermittently between 1978 and 1994 by examining various species at monthly intervals for periods ranging from 13 to 72 consecutive months (horak 1998). a survey on the seasonality and species diversity of ticks questing for hosts from the vegetation was initiated in 1988 (horak, spickett & braack 2000b). in the latter study ticks were collected at approximately monthly intervals for 164 consecutive months, by dragging flannel strips over the vegetation of two landscape zones in the south of the park. in september 1988 part of the vegetation in one of the zones was subjected to a controlled total burn and the effect of this fire on the numbers and seasonality of questing ticks was determined (spickett, horak, van niekerk & braack 1992). tick numbers were reduced after the burn, but increased after varying periods thereafter depending on tick species, patterns of seasonal occurrence, and host preference. as part of the initiatives to make greater use of the experimental burn plots and in view of our ongoing involvement in tick research in the park we were invited to determine the effect of burning on the numbers of ticks questing from the vegetation in the experimental burn plots. this report records the month ly tick counts in sub-plots within two replicates of the experimental burn plots before and after burns as well as on unburned vegetation adjacent to the replicates over a period of 39 consecutive months. materials and methods three of four experimental burn plot replicates are located just north and one just south of the road between the tourist rest camps of skukuza and pre torius kop, and two of the northerly replicates were selected for drag-sampling (fig. 1). the sub-plots in the more westerly replicate have for convenience been identified as the nwaswitshaka burn plots (25°08’ s; 31°38’ e), while those in the more easterly replicate are referred to as the skukuza burn plots (25°10’ s; 31°44’ e). both sets of replicates are in a landscape zone described as combretum collinum/combretum zeyheyri woodland on granite (zone 3a: gertenbach 1983). each replicate is surrounded by an approximately 4 m wide fire-break, which is kept reasonably free of vegetation by means of a road-grader, and is subdivided by similar fire-breaks into 12 sub-plots, each approximately 7 ha in size. wildlife has free access to all the experimental burn plot replicates. the subplots within the nwaswitskaka and skukuza experimental burn plot replicates are paired and at the commencement of the survey the sub-plots of each pair had been subjected to the same burning regimen for the previous 45 years. six sub-plots that had received the same treatment were selected at each locality. one sub-plot was not burned (control), a second was burned the first week of august each year, a third sub-plot was burned the first week of august every second year, a fourth was burned the first week of august every third year, a fifth sub-plot was burned the first week of october every second year, and a sixth was burned the first week of october every third year. unburned vegetation adjacent to the fire-break surrounding the 12 sub-plots comprising the burn plot replicate was selected as a second control. as chance would have it the experiskukuza burn plots nwaswitshaka burn plots nwaswitshaka river 400 400 tourist road fig. 1 configuration of the experimental burn plot replicates located north and south of the road between the tourist rest camps of skukuza and pretoriuskop. the sub-plots in the westerly replicate are referred to as the nwa switshaka burn plots and those in the easterly replicate as the skukuza burn plots 165 i.g. horak et al. mental burning regimen required that all five subplots at both localities were burned in 2000, three during august and two during october. ticks were collected at approximately monthly intervals from january 1999 to march 2002 by dragsampling the vegetation of the selected sub-plots. this was done by an operator dragging a wooden spar, with 10 x 10 cm x 100 cm weighted flannel strips attached to it, for 200 m over the vegetation of each sub-plot. after each drag all instars of all ticks on the flannel strips were collected by means of forceps and stored in 70 % ethanol in labelled glass vials. at every occasion the drags were repeated six times at approximately 50 m intervals in each subplot, and a total of 42 x 200 m long drags were thus made within each of the two experimental burn plot replicates and their outside control localities every month. with few exceptions sampling was performed during the third week of each month so that it did not coincide with the experimental burning. there were no drags during february 1999 because of rain, and none in february and march 2000 because of flooding in the southern regions of the park following more than 400 mm rain during february (fig. 2). rain during september 2000 again prevented sampling, while one of the sub-plots and both outside control areas could not be sampled during september 2001 because they were still smouldering after a huge accidental fire in the south-east of the park had jumped the fire-breaks surrounding the burn plots. data analysis the tick counts were log10 transformed prior to analysis to reduce the effects of overdispersion (petney, van ark & spickett 1990). the seasonality of each species and stage was then tested for both locations using autocorrelation (spss inc. 1994). amblyomma marmoreum, rhipicephalus appendiculatus and rhip i cephalus zambeziensis exhibited a pronounced periodicity with peaks from the summer to early winter and a marked decline in numbers in the late winter and early spring. the analyses for these species were restricted to the period from january to july, the months with the highest numbers. this reduced the number of drags with zero counts, and because burning occurred in the late winter or early spring, col lections from the preceding burn year were eliminated. to analyse the effect of burning, the year after burning was classed as the first, second or third, or unburned (control). however, the decline in tick numbers in 2001 following the burning of both sets of five sub-plots in 2000 was also confounded by the marked decrease in rainfall from 1999/2000 to 2000/2001 (fig. 2). to allow for temporal variation among calendar and climatic years, a second variable was created with four periods corresponding to the calendar year after burning: period 1 [prior to burn month (august or october) in 1999], period 2 (from burn month in 1999 to burn month in 2000), period 3 (from burn month in 2000 to burn month in 2001), and period 4 (october 2001 to march 2002). the total tick data and data for each species were then analysed using a three factor analysis of variance with location, period and year after burning. because year after burning was not balanced across periods, and there were often significant period effects, a multiple classification analysis (spss inc. 1994) was used to adjust the means for the effect of the other variables. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� �� �� � � � � � � ������������������������ �������������������������������������������������������������������������� ����������������������� � ����������������������������������������������������� fig. 2 monthly rainfall (mm) measured at the skukuza tourist rest camp during the collection period. the numbers in the figure represent total annual rainfall (june to may) 166 effect of burning on numbers of questing ticks collected by dragging results fourteen tick species were collected (table 1). ambly omma hebraeum and rhipicephalus (boophilus) decoloratus were the most common species at both sites. amblyomma marmoreum, haemaphysalis leachi, r. appendiculatus, rhipicephalus evertsi evertsi, rhipicephalus simus and r. zambeziensis were also commonly collected. with the exception of h. leachi and r. simus, whose adults were most routinely recovered, larvae were the most frequently collected stage. total ticks more ticks were collected from the nwaswitshaka burn plots than from the skukuza burn plots (p ≤ 0.001). there was no evidence of seasonality at either location. there was a significant difference among periods (p = 0.002) with lower numbers in table 1 total numbers (proportions %) of ticks collected from two sets of experimental burn plots in the kruger national park, south africa tick species stage of development locality skukuza nwaswitshaka amblyomma hebraeum ll nn aa 39 980 25 1 (84.21) (0.05) 35 704 23 0 (65.66) (0.04) amblyomma marmoreum ll nn 102 1 (0.21) 1 690 2 (3.11) rhipicephalus (boophilus) decoloratus ll aa 3 928 1 (8.27) 10 895 0 (20.04) dermacentor rhinocerinus ll 5 (0.01) 24 (0.04) haemaphysalis leachi ll nn aa 2 3 921 (0.01) (1.94) 5 4 960 (0.01) (0.01) (1.77) haemaphysalis spinulosa aa 0 1 haemaphysalis zumpti aa 0 2 hyalomma truncatum ll aa 1 0 1 1 rhipicephalus appendiculatus ll nn aa 418 10 5 (0.88) (0.02) (0.01) 939 56 6 (1.73) (0.10) (0.01) rhipicephalus evertsi evertsi ll aa 535 0 (1.13) 1 816 1 (3.34) rhipicephalus follis aa 1 0 rhipicephalus simus ll nn aa 12 2 455 (0.03) (0.96) 14 1 340 (0.03) (0.63) rhipicephalus turanicus aa 0 1 rhipicephalus zambeziensis ll nn aa 1 023 37 9 (2.15) (0.08) (0.02) 1 847 39 3 (3.40) (0.07) (0.01) total 47 477 (100.00) 54 375 (100.00) ll = larvae nn = nymphs aa = adults 167 i.g. horak et al. period 3 (2000/2001) than in the other periods (fig. 3). comparing between years after burning, the second year after burning and the unburned sub-plots had significantly higher tick numbers than the first and third years after burning (table 2). burning per se appeared to have little effect on the number of ticks collected, as there was no consistent pattern in the number of ticks recovered 1–2 weeks after a burn and the number recovered in the preceding month or in the subsequent 1–2 months. in 2000/2001 there was a marked drop in tick numbers relative to the preceding years in the burned sub-plots, but little change from the other years in the unburned sub-plots. amblyomma hebraeum amblyomma hebraeum larvae were the most commonly collected ticks, accounting for 84.2 % of those collected at skukuza and 65.7 % of those at nwa swit shaka. the numbers did not differ significantly between the two locations (p > 0.15), and there was no evidence of seasonality. there was a significant difference among periods (p = 0.01) with periods 2 (1999/2000) and 4 (2001/2002) having higher numbers than periods 1 (1999) and 3 (2000/2001) (fig. 4a). there was also a significant difference among years after burning (p = 0.001) with the second year after burning and unburned sub-plots having higher numbers than the first and third years after burning (table 2). the difference among years after burning was more pronounced at nwaswitshaka than at skuku za. at nwaswitshaka the decrease in the numbers of a. hebraeum larvae in period 3 occurred on both the unburned and burned sub-plots suggesting that it was related to the drier conditions in 2000/2001, but there was no effect at skukuza. burning per se appeared to have little effect on the number of a. he braeum larvae recovered, as there was no consistent pattern in the number collected 1–2 weeks after a burn and the number collected in the preceding month or in the subsequent 1–2 months. amblyomma marmoreum amblyomma marmoreum larvae were collected from january to october (fig. 4b), with the highest numbers from march to august. they were most common at nwaswitshaka (p < 0.001). the numbers were highest in 2000, and none were collected in 2002 (p < 0.001). there was no significant difference (p = 0.12) among the years after burning. rhipicephalus (boophilus) decoloratus rhipicephalus (boophilus) decoloratus larvae were the second most commonly collected tick (table 1). the numbers were significantly higher (p < 0.001) at nwaswitsha ka, where they accounted for 20.0 % of the ticks collected, than at skukuza where they accounted for 8.3 % of the ticks collected. there was no evidence of seasonality. the numbers were significantly lower in period 3 than in the other periods (p < 0.001), and significantly lower in period 4 than in period 2 (p < 0.05) (fig. 4c). the numbers were higher in the second year after burning than in the first and third years and in the unburned sub-plots (p = 0.009). burning per se did not appear to affect the numbers of r. (b.) de coloratus larvae. ����������������������������������������������������������������������������������������� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �! !� ! �!� �! �!� �! !� ! "#��$%� ��������������������"#��$%����������������������"#��$%�����������������������"#��$%� fig. 3 total number of ticks collected each month from the vegetation of all plots combined at skukuza and at nwa switsha ka. period 1 [prior to burn month (august or october) in 1999]; period 2 (from burn month in 1999 to burn month in 2000); period 3 (from burn month in 2000 to burn month in 2001); and period 4 (october 2001 to march 2002). indicates month of burning 1 6 8 e ffe ct o f b u rn in g o n n u m b e rs o f q u e stin g ticks co lle cte d b y d ra g g in g ��������� �� �� ��� &��'�# � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ������������ ����������� ������ ���� &��'�# ( � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������� ��� ���� &��'�# � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �! !� ! �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%����������������������"#��$%�����������������������"#��$%� ��������������� �������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� ��������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � !� ! �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� ������������� ������ �%�&�� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� fig. 4 total number of (a) amblyomma hebraeum larvae, (b) amblyomma marmoreum larvae, (c) rhipicephalus (boophilus) decoloratus larvae, and (d) haemaphysalis leachi adults collected each month from the vegetation of all plots combined at skukuza and at nwaswitshaka. period 1 [prior to burn month (august or october) in 1999]; period 2 (from burn month in 1999 to burn month in 2000); period 3 (from burn month in 2000 to burn month in 2001); and period 4 (october 2001 to march 2002). indicates month of burning 1 6 9 i.g . h o r a k e t a l. ������������ �������������� &��'�# � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� ������������ ��� ��� ��� ��� &��'�# � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� ������������ ����� �%�&�� ( � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������������� �������������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� ������������ ������������ &��'�# � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������������� �������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������� �� � � � � � �� �� �� �� � � � ! �!� �! �!� �! !� ! !� ! "#��$%� ��������������������"#��$%�����������������������"#��$%����������������������"#��$%� fig. 5 total number of (a) rhipicephalus appendiculatus larvae, (b) rhipicephalus evertsi evertsi larvae, (c) rhipicephalus simus adults, and (d) rhipicephalus zambeziensis larvae collected each month from the vegetation of all plots combined at skukuza and at nwaswitshaka. period 1 [prior to burn month (august or october) in 1999]; period 2 (from burn month in 1999 to burn month in 2000); period 3 (from burn month in 2000 to burn month in 2001); and period 4 (october 2001 to march 2002). indicates month of burning 170 effect of burning on numbers of questing ticks collected by dragging haemaphysalis leachi most of the h. leachi collected were adults (table 1). the numbers did not differ between the two locations (p > 0.5), and there was no evidence of seasonality. the numbers of h. leachi adults changed markedly over time (p < 0.001), with few in the first year, an increase over the next 6 months, then a decrease to august 2001 and reappearance in november 2001 (fig. 4d). the numbers were lower the first year after burning than in the other years (p < 0.001). the effect of burning per se was difficult to determine because adult tick numbers usually decline on the vegetation in the park during the late winter (horak, emslie & spickett 2001). rhipicephalus appendiculatus rhipicephalus appendiculatus exhibited a pronounced seasonal periodicity. larvae were present from february to october with peak occurrence from may to august, nymphs were present from june to february with a peak in august to october, and the adults were collected from february to april (fig. 5a). rhipicephalus appendiculatus larvae and nymphs were most common at nwaswitshaka (p = 0.035 and p < 0.001, respectively). there was a marked increase in number of larvae from 1999 to 2000 in both areas and a decline in 2001 (p < 0.001). the number of nymphs also appeared to increase from 1999/2000 to 2000/ 2001, but did not appear to decline in 2001/2002. the numbers of larvae were higher in the first year after burning and lowest in the third year after burning (p = 0.004). the immediate effect of burning on lar vae could not be assessed because the seasonal decline in numbers coincided with the timing of the fires. nymphs were collected 1–2 weeks after burning, but the numbers were too low to assess the immediate impact of the fire. rhipicephalus evertsi evertsi rhipicephalus evertsi evertsi larvae were present throughout the year, with no evidence of seasonality (fig. 5b). they were more common at nwaswitshaka than at skukuza (p < 0.001). there was a significant difference among periods (p < 0.001) as the numbers declined from period 1 to period 3, then increased in period 4. the numbers were significantly higher on the burned sub-plots than on the unburned sub-plots (p = 0.004). rhipicephalus simus most of the r. simus collected were adults (table 1). they were more common at sku ku za than at nwaswitshaka (p = 0.028). the numbers increased from 1999 to 2001/2002, and there was a marked seasonality with the highest numbers from january to march and the lowest numbers from august to october (fig. 5c). the numbers increased with year after burning (p < 0.001). rhipicephalus zambeziensis rhipicephalus zambeziensis was more common than r. appendiculatus in both areas. there was a pronounced seasonality for all stages (fig. 5d). lar vae were collected from april to november with a peak in july, nymphs were collected from may to january with a peak in august, and adults were collected from february to april. rhipicephalus zambe zi ensis larvae were more common at nwaswitshaka than at sku kuza (p = 0.001), but the number of nymphs did not differ between the two areas. the numbers of larvae decreased from the first to the third year after burning, but the change was not statistically significant (p = 0.16). the immediate effect of burning on larvae could not be assessed because the seasonal decline in numbers coincided with the timing of the table 2 adjusted means (log10 transformed) for the number of ticks collected each month in the burn plots by year after burning. values with the same superscript are not significantly different (p > 0.05) tick species and stage of development year after burning 1 2 3 unburned total ticks amblyomma hebraeum larvae amblyomma marmoreum larvae (january to july) rhipicephalus (boophilus) decoloratus larvae haemaphysalis leachi adults (december 1999 to march 2002) rhipicephalus appendiculatus larvae (may to july) rhipicephalus evertsi evertsi larvae rhipicephalus simus adults rhipicephalus zambeziensis larvae (may to july) 1.81a 1.47a 0.15a 0.75a 0.37 0.67a 0.40a 0.15 0.75a 2.12b 1.71b 0.16a 1.02 0.75a 0.32b,c 0.36a 0.24a 0.67a 1.86a 1.28a 0.01a 0.75a 0.77a 0.12c 0.40a 0.30a 0.45a 2.10b 1.74b 0.19a 0.86a 0.65a 0.48a,b 0.21 0.33a 0.90a 171 i.g. horak et al. fires. as with r. appendiculatus, r. zam be ziensis nymphs were collected on burned sub-plots 1–2 weeks after burning, but the numbers were too low to determine the immediate impact of the burn. discussion dragging is only effective for sampling those ticks and developmental stages that quest for hosts from the vegetation and that are inclined also to attach to flannel strips. hence dragging may limit the tick species and life stage spectrum of any investigation that relies on it as the only sampling method. amongst the 14 tick species collected from the burnplots the larvae of a. hebraeum, a. marmoreum. r. (b.) decoloratus, r. appendiculatus, r. e. evertsi and r. zambe ziensis quest from vegetation and are inclined to attach to flannel. although the nymphs and adults of r. appendiculatus and r. zambeziensis also quest from vegetation, they are less inclined to attach to flannel than the larvae. on the other hand, the adults of h. leachi, that prefer carnivores as hosts (horak, braack, fourie & walker 2000a), and of r. simus, that prefer monogastric animals such as carnivores, equids and suids as hosts (walker, keirans & horak 2000), also quest from the vegetation and readily attach to flannel. they are probably enticed to do so by chemicals exuded by the omnivorous, monogastric persons dragging the flannel strips, and they are also amongst the tick species that are most frequent ly recorded as attaching to humans (horak, fourie, heyne, walker & needham 2002). the adults of the rhinoceros tick d. rhinocerinus also quest from vegetation, but prefer grass species with long, thick stems, which, although present, are sparse on the burn-plots. the nymphs and adults of a. hebraeum and a. marmoreum, the larvae and nymphs of d. rhinocerinus, h. leachi and r. simus, the larvae and adults of h. truncatum and the adults of r. e. evertsi (the latter two ticks both have two-host life cycles) all quest for hosts from the soil surface or its immediate vicinity and are seldom collected by dragging. with the exception of h. truncatum, all the major tick species that occur in the park were collected from the experimental burn-plots in sufficient numbers to make analysis possible. without any doubt h. truncatum was also present on the plots as adults and larvae on the soil surface and as adults on large animals such as giraffes and zebras and as larvae and nymphs on scrub hares and gerbils (horak, potgieter, walker, de vos & boomker 1983; horak, de vos & de klerk 1984; horak, spickett, braack & penzhorn 1993; horak 1998; braack, horak, jordaan, segerman & louw 1996). in our experience, however, very few, if any, h. truncatum can be collected by drag-sampling and consequently its favoured host animals have to be examined in order to determine its presence at a particular locality. the effects of burning on tick populations are complex because fires may affect both ticks and their host species (spickett et al. 1992). fires alter the physical environment and the nature and quatity of food and cover available to animals (bigalke & willan 1984). questing ticks may be destroyed in a fire (bigalke & willan 1984), but some species may also exhibit avoidance strategies that enhance survival (frost 1984). the direct impact on other free-living stages, and on small mammal hosts, will depend on the intensity of the burn. increased temperatures and a decrease in soil moisture on burned areas (cass, savage & wallis 1984) may increase the mortality of free-living ticks, although the increased temperatures may also accelerate temperature-dependent development (spickett et al. 1992). burning may also affect tick populations by altering the spatial distribution of preferred and other hosts. many herbivores select the new growth in the burned areas (gureja & owen-smith 2002; tomor & owen-smith 2002; wron ski 2003; zavala & holdo 2005), but the populations of small mammals and birds requiring ground cover may decline (frost 1984). all stages of development of a. hebraeum, the most commonly collected species at both locations, can be found on large herbivorous animals, the size of kudu bulls and larger, as well as on warthogs (horak, macivor, petney & de vos 1987; horak, boomker, de vos & potgieter 1988; horak, boomker, spickett & de vos 1992). with the exception of rodents, on which they do not occur, the nymphs and particularly the larvae can be found on most smaller animals, including antelopes, zebras, large and small carnivores, hares, guineafowls and leopard tortoises (horak et al. 1984; 1993; 2000a; horak, spickett, braack & williams 1991; braack et al. 1996; horak 1998). this, coupled with the fact that all stages of development are present throughout the year in the park (horak et al. 1992), and that the females lay exceptionally large numbers of eggs (norval 1974), ensure that the larvae of a. hebraeum are more numer ous on the vegetation in most habitats in the park than those of other ticks (spickett, horak, braack & van ark 1991). fire did not appear to have an immediate effect on the numbers of questing larvae, suggesting that many survived the fire. while there were statistically significant differences among the years after burning, there was not a consistent 172 effect of burning on numbers of questing ticks collected by dragging trend, indicating that fire has no long-term effect probably because engorged female a. hebraeum are con tinuously brought on to the burn plots by their various large herbivorous hosts. all stages of development of a. marmoreum prefer leopard tortoises (horak, mckay, heyne & spickett 2006). fire may kill many tortoises because of their limited ability to escape (branch 1998), but larger bird species such as helmeted guineafowls and pheasants, as well as carnivores, large herbivores and scrub hares can harbour considerable numbers of larvae and some nymphs of this tick (horak et al. 1991; 1993; 2000a; 2006; uys & horak 2005). burning did not have a significant effect on the numbers of questing a. marmoreum larvae. there was a seasonal decline in the number of larvae at the time of the burns, and the 4–6 month delay between the burns and reappearance of questing larvae was probably adequate for re-colonization of the burned plots by the preferred hosts. the one-host tick, r. (b.) decoloratus infests especially kudus, bushbuck, impalas and zebras in the park (horak et al. 1983; 1984; 1992; horak, gallivan, braack, boomker & de vos 2003). it is present throughout the year, but is most numerous on its hosts during spring and mid-summer, and probably completes two if not three life cycles annually. consequently animals browsing or grazing on the burn plots, or just walking through shortly after the spring burns, will carry in large numbers of engorged female ticks. the interval between the detachment of these females and the hatching of larvae from the eggs they have laid can be as short 40 days during the summer (spickett & heyne 1990). burning did not appear to have an immediate effect on the number of larvae, suggesting that substantial numbers survived the fire, or that the changes in microclimate after the burn accelerated development (spickett et al. 1992). the increase in numbers in the second year after the burn may be the consequence of increased use of the burned area by preferred hosts the first year after burning. the occurrence of two other major tick species, namely r. appendiculatus and r. zambeziensis in nearly equal numbers on the burn-plots is fortuitous. both ticks are present in the park, but often separately in habitats only a few km apart (horak 1998). judging by the small numbers collected from the burn plots these were not located in an ideal habitat for either species and probably lie in a transitional zone. minshull & norval (1982) recorded an increase in the number of r. appendiculatus larvae collected the year after burning in hyperrhaenia grassland on clay, but not on sandy soil. this is consistent with increases in r. appendiculatus and r. zambeziensis larvae the year after burning in the present study. this is probably caused by the influx of “burn grazers” after the fire. the larvae and nymphs of d. rhinocerinus, h. leachi, r. simus and r. turanicus use small rodents as hosts (norval 1984; walker et al. 2000; horak & cohen 2001; petney, horak, howell & meyer 2004), as do the larvae of h. truncatum (braack et al. 1996). while many small mammals survive fire, there are often marked declines in the populations of some species after a burn because of the habitat changes (frost 1984). a reduction in the population of the hosts of their immature stages would explain the significant reduction in the numbers of adult h. leachi and r. simus collected the first year after the burn. a pack of wild dogs, with a den in the vicinity of the plots, probably played a role in seeding the area with engorged female h. leachi and particularly r. simus (norval 1984; horak et al. 2000a). the immature stages and adults of r. e. evertsi utilize zebras as hosts, and can be found on these animals throughout the year (horak et al. 1984). gureja & owen-smith (2002) and tomor & owen-smith (2002) list zebras as one of the species preferentially using burnt areas. these animals were often seen on the plots, both before and after burns, and probably seeded them with engorged nymphs and females as well as with the females of r. simus, which also feed on them. surveys on the seasonality of ticks on eight host species, ranging in size from gerbils to kudus, and including helmeted guineafowls, as well as on the vegetation of two landscape zones in the park, have been conducted intermittently since 1978 (horak 1998). these have shown that the only stages of development present on the vegetation in substantial numbers at the time of the burns in august or october are larvae of r. (b.) decoloratus and nymphs of r. appendiculatus and r. zambeziensis. thus, a burn in august or october is only likely to have a direct effect on peak numbers of single life stages of the abovementioned three major tick species. the larvae of a. hebraeum and r. e. evertsi may also be pres ent at this time, but are non-seasonal without defined peaks in numbers. depending on the heat generated by the fire, various life stages of other ticks questing for hosts on the soil surface, or moulting or ovipositing in sheltered spots, may also be adversely affected. one of the reasons for the apparent lack of effect of fire on the numbers of ticks questing for hosts from 173 i.g. horak et al. the vegetation in the present investigation could be the small size of the individual sub-plots. although 7 ha may seem large, in the context of a large wildlife reserve and free access by animals, the plots should probably have been 10 times larger. the apparent ease with which the 7 ha sub-plots can be re-colonised by small mammals or accessed by large animals either for foraging, loafing or simply as walkways, add innumerable variables to the already com plex analysis of the effects of fire on questing ticks. finally, the free-living stages of ticks of sub-saharan africa have been exposed to intermittent fires for millions of years and have probably been selected for survival strategies as yet unknown to us. consequently they have, and probably will, successfully survive the effects of fire for centuries to come. conclusion unless animals are excluded from recently burned localities the effects of fire on free-living tick numbers are likely to be minimal and of short duration. acknowledgements we are indebted to south african national parks for placing their staff and facilities in the park at our disposal. we are also most grateful to those staff members of the kruger national park and the several nature conservation students who assisted us with the many drags that were made. this 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [878.740 1133.858] >> setpagedevice abstract introduction materials and methods results discussion conclusion acknowledgments references about the author(s) fatemeh bakhshi department of biology, islamic azad university, urmia branch, urmia, iran reza pilehchian langroudi razi vaccine and serum research institute, agricultural research, education and extension organization (areeo), alborz, karaj, iran bahram golestani eimani department of biology, islamic azad university, urmia branch, urmia, iran citation bakhshi, f., pilehchian langroudi, r. & golestani eimani, b., 2016, ‘enhanced expression of recombinant beta toxin of clostridium perfringens type b using a commercially available escherichia coli strain’, onderstepoort journal of veterinary research 83(1), a1136. http://dx.doi.org/10.4102/ojvr.v83i1.1136 research note enhanced expression of recombinant beta toxin of clostridium perfringens type b using a commercially available escherichia coli strain fatemeh bakhshi, reza pilehchian langroudi, bahram golestani eimani received: 18 dec. 2015; accepted: 31 mar. 2016; published: 30 june 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract clostridium perfringens beta toxin is only produced by types b and c and plays an important role in many human and animal diseases, causing fatal conditions that originate in the intestines. we compared the expression of c. perfringens type b vaccine strain recombinant beta toxin gene in the escherichia coli strains rosettatm(de3) and bl21(de3). the beta toxin gene was extracted from pjetβ and ligated with pet22b(+). pet22β was transformed into e. coli strains bl21(de3) and rosettatm(de3). recombinant protein was expressed as a soluble protein after isopropyl β-d-1-thiogalactopyranoside (iptg) induction in strain rosettatm(de3) but not in bl21(de3). expression was optimised by growing recombinant cells at 37 °c and at an induction of 0.5 mm, 1 mm, 1.5 mm iptg. expression was evaluated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). the recombinant protein was purified via ni-nta and was analysed using western blot. we concluded that e. coli strain rosettatm(de3) can enhance the expression of c. perfringens recombinant beta toxin. introduction clostridium perfringens, an anaerobic gram-positive, rod-shaped bacterium, is able to form environmentally resistant spores. clostridium perfringens produces seventeen toxins. four of them – iota, alpha, beta and epsilon – are major toxins and are used for classifying c. perfringens into five types – a, b, c, d and e (nilo 1980). beta toxin is only produced by types b and c and plays an important role in many human and animal diseases via pore formation in the endothelial cell membrane (michlard et al. 2009; nagahama et al. 2015). clostridium perfringens beta toxin (cpb) forms a multimeric transmembrane pore in the endothelial cell membrane and is the cause of cell lysis (steinthorsdottir et al. 2000). clostridium perfringens has a circular chromosome of approximately 3.6 mega-base pairs with a low content of g+c (about 25%) (canard et al. 1992; casjens 1998). the genome c. perfringens includes extra chromosomal genetic elements in the form of plasmid and phage-encoded mobile genes that can vary in size and composition (bruggemann 2005; cavalcanti et al. 2004). clostridium perfringens beta toxin gene (cpb), which encodes a protein made up of 309 amino acids, is located on the different large plasmids that are carried by types b and c (rokos, rood & duncan 1978). expression of cpb in escherichia coli has been shown and a molecular analysis revealed that it has sequence homology with alpha-toxin, gamma-toxin and leukocidin of staphylococcus aureus (hunter et al. 1993). in the present study, the beta toxin gene of the c. perfringens vaccine strain (cwb cn228) was expressed in e. coli and purified proteins were analysed. this vaccine strain was isolated in iran (brooks & entessar 1957), and its beta toxin gene was sequenced, described and deposited in genbank hq179547.1. materials and methods the expressions of c. perfringens type b vaccine strain recombinant beta toxin gene in the e. coli strains rosettatm(de3) and bl21(de3) were compared. the methods used have been described elsewhere (pilehchian langroudi et al. 2011). plasmid pjet1.2/blunt, pfu dna polymerase, dntps, t4 dna ligase, ndei and xhoi res and generuler™, 100 bp plus dna size markers, pageruler™, and extraction kit, sds, proteinase k, lysozyme and plasmid extraction kit were purchased from fermentas (thermo scientific™ germany). high pure pcr product purification kit for dna fragments recovery was purchased from vivantis technologies sdn bhd (selangor, malaysia). sheep primary antibody and conjugate anti-sheep horseradish_peroxidase (hrp) were purchased from dako company (glostrup, denmark). clostridium perfringens type b vaccine strain (cwb cn228), e. coli strain top10, which was applied as a cloning host, and e. coli strains bl21(de3) and rosetta™(de3) (novagen, merck millipore, germany) as expression hosts were prepared at the razi institute. clostridium perfringens was cultured anaerobically in the liver extraction media at 37 °c for 5 h after the centrifugation supernatant was removed and discarded; the whole genomic dna was extracted using the phenol–chloroform method. the gene cpb was amplified via pcr using one pair of specific primers consisting of ndei at the 5, end of the forward and xhoi at the 3, end of the reverse primers. (pilehchian langroudi et al., 2011). pfu dna polymerase was used for amplification of the blunt-end pcr product. after ligation of the cpb gene into the pjet1.2/blunt and producing pjetβ (pjet1.2/blunt beta) recombinant vector, the e. coli strain top10 competent cell was transformed using pjetβ, and the screening of recombinant clones was done via antibiotic resistance and colony pcr. nucleotide sequencing was carried out via source bioscience co. (berlin, germany). the pjetβ recombinant vector was purified from e. coli/top10/pjetβ cells and was digested using ndei and xhoi. after digestion, the cpb gene was extracted from the gel and was purified. pet22b(+) was digested using the same enzymes, was purified from the electrophoresis gel and was ligated using cpb gene. the ligation product was applied on the 1% electrophoresis agarose gel to show the formation of recombinant plasmid. escherichia coli strains bl21(de3) and rosetta™(de3) were selected as expression hosts. after preparation of the competent cells, they were transformed using pet22β [pet22b(+) beta] expression vector so that e. coli/bl21/pet22β and e. coli/rosetta™/pet22β were produced. to confirm the presence of the recombinant pet22β expression vector, recombinant cell screening was done, as previously described (pilehchian langroudi et al., 2011). colony pcr was done using recombinant individual e. coli colonies. after purification, the recombinant pet22β expression vector was sequenced. expression of clostridium perfringens beta toxin protein recombinant cells were cultured in lb/amp media and were incubated at 37 °c to od600 = 0.6–0.7. to induce protein expression, 0.5 mm isopropyl β-d-1-thiogalactopyranoside (iptg) was added and growth was continued for 18 h; in additional trials, different concentrations of iptg (0.5 mm, 1 mm and 1.5 mm) were used. effects of a temperature gradient (25 °c, 31 °c and 37 °c) and time variation (3, 6 and 18 h) were considered. the expressed protein analysis was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). negative controls including of the e. coli/bl21/pet22 and e. coli/rosettatm/pet22 were used for each analysis. the recombinant beta protein, which contains a 6-his tag at the c-terminal, was purified with ni-nta resin. the bacterial pellet was suspended in the lysis buffer and the cells were lysed with sonication repeated six times on ice for a duration of 5 min. the cell lysate was centrifuged at 13 680 g, and the clarified supernatant was loaded on ni-nta resin at the flow rate of 1 ml/min. the column was washed with five volumes of wash buffer, and finally, the protein was eluted by adding elution buffer, as previously described (pilehchian langroudi et al. 2013). the purified protein was analysed using sds-page and western blot. the protein concentration was determined using a standard procedure, as previously described (bradford 1976). results the electrophoresis result showed that genomic dna was extracted and the pcr analysis revealed that the cpb gene was amplified. after successful production of the recombinant pjetβ cloning vector and the e. coli/top10/pjetβ cells and also successful subcloning of the pet22β, e. coli strains bl21(de3) and rosettatm(de3) cells were transformed using this expression vector. the colony pcr result showed the presence of the recombinant pet22β plasmids in both recombinant e. coli colonies. sequencing revealed that the inserted gene was consistent with the cbp gene in genbank (hq179547.1). after expression, the sds-page analysis showed that the recombinant protein was well expressed between 2 and 4 h after induction via 0.5 mm iptg in recombinant e. coli/rosettatm(de3) but not in e. coli/bl21 (figure 1). the use of different concentrations of iptg had no significant effect on protein expression, but the effects of temperature and time on expression levels were significant, and in the case of time the effect continued up to 18 h (figure 2). recombinant protein was purified using ni-nta resin, and the result showed a very sharp band of the purified protein as an approximately 35-kda using sds-page and western blot analysis (figure 3). figure 1: comparison of beta toxin expression in (a) escherichia co-li/rosettatm/pet22β and (b) escherichia co-li/bl21/pet22β. figure 2: (a) effects of iptg concentration on expression. (b) effects of different temperature on expression. (c) effects of time on recombinant clostridium perfringens beta toxin expression. figure 3: sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis of purified recombinant beta toxin. (a) sodium dodecyl sulfate polyacrylamide gel electrophoresis. (b) western blot using sheep primary antibody and conjugate anti-sheep horseradish_peroxidase. discussion iranian variant type b was isolated in 1954 from intestinal contents of sheep and goats. the three strains of clostridium welchii type b isolated were different from the classical type b strains in their production of kappa and non-production of lambda and hyaluronidase toxins. two of the strains were isolated from young goats and the other from an adult sheep (brooks & entessar 1957). by now, some vaccines have been manufactured using the secreted cpb protein of c. perfringens types b and c. it has been about five decades since the vaccine against c. perfringens types b and c, cpb protein was produced in iran (pilehchian langroudi 2015) genetic engineering, helping to produce recombinant protein that as a good alternative to native toxins belonging to the c. perfringens species (nijland et al. 2007). in 1998, cpb of c. perfringens was expressed and secreted in bacillus subtilis in the form of mutant protein (beta toxoid). the mutation of two amino acids that were located in the membrane binding region affected the lethal dose in mice (steinthorsdottir et al. 1998). previously, a genetic construct containing c. perfringens epsilon toxin protein (etx) and cpb genes was produced in iran. the fusion gene was expressed as a soluble protein in the e. coli strain rosetta™ and its immunogenicity was studied in mice. (pilehchian langroudi et al. 2013). in the present study, recombinant plasmid pet22bβ was transformed into e. coli strains bl21(de3) and rosettatm(de3). the sds-psage analysis showed no cpb band from e. coli/bl21/ pet22β but showed a cpb band from e. coli/rosettatm/pet22β. the sequencing analysis of purified pet22β revealed that the inserted gene had 987 bp, demonstrating a 99% – 100% identity with cpb genes that were previously deposited in genbank. the recombinant toxin was expressed 30 min after induction with iptg and continued for 18 h. it has been reported that the expression of recombinant beta toxin in e. coli started 30 min after induction with 0.5 mm iptg and continued up to 6 h (steinthorsdottir et al. 1995). it was revealed that different concentrations of iptg had no obvious effect on the protein expression level. a previous report showed that there was no more expression after induction beyond 1 mm iptg (goswami et al. 1996). the optimal thermal condition for protein expression is 37 °c. at present, accessing recombinant bacterium and manufacturing a recombinant vaccine is possible. (michlard et al. 2009) the strain used in this work was c. perfringens type b vaccine strain, which is the iran variant of this species (brooks & entessar 1957). although the beta toxin gene is significantly similar to other genes, it is unique for iran. this strain is one of the active ingredients in the tetravalent enterotoxaemia vaccine in iran, so it is very important that we use its beta toxin protein in the future recombinant vaccine. conclusion we concluded that pet22b(+) and e. coli strain rosettatm(de3) are suitable expression vectors and hosts that can enhance the expression and production of c. perfringens recombinant beta toxin. therefore, the e. coli/rosettatm/pet22β clone could be used for further research on recombinant vaccine production. acknowledgments the present study was conducted by the research fund from razi vaccine and serum research institute. the authors would like to thank yalda pilehchian (m.sc.) for the assistance of final proof reading. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions f.b., was the student, r.p.l. the supervisor, and b.g.i. the consultant supervisor. references bradford, m., 1976, ‘a rapid and sensitive for the quantitation of microgram quan-tities of protein utilizing the principle of protein-dye binding’, analytical biochemistry 27(1–2), 248–254. http://dx.doi.org/10.1016/0003-2697(76)90527-3 brooks, m. & entessar, f., 1957, ‘anomalous clostridium welchii type b strains isolated in iran’, british veterinary journal 113, 506–509. bruggemann, h., 2005, ‘genomics of clostridia pathogens: implication of extra chromosomal elements in pathogenicity’, current opinions in microbiology 8(5), 601–605. http://dx.doi.org/10.1016/j.mib.2005.08.006 canard, b., saint-joanis, b. & cole, s., 1992, ‘genomic diversity and organization of virulence genes in the pathogenic anaerobe 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perfringens beta-toxin: expression of wild type and mutant toxins in bacillus subtilis’, fems microbialogy letters 158(1), 17–23. http://dx.doi.org/10.1111/j.1574-6968.1998.tb12794.x steinthorsdottir, v., fridriksdottir, v., gunnarsson, e. & andresson, o., 1995, ‘expression and purification of clostridium perfringens beta toxin glutathione s-transferase fusion protein’, fems microbialogy letters 1309(2–3), 273. http://dx.doi.org/10.1016/0378-1097(95)00218-t steinthorsdottir, v., halldorsson, h. & andresson, o., 2000, ‘clostridium perfringens beta toxin forms multimeric transmembrane pores in human endothelial cells’, microbial pathogenesis 28, 45–50. http://dx.doi.org/10.1006/mpat.1999.0323 abstract introduction materials and methods results ethical consideration discussion conclusion acknowledgements references about the author(s) felistas ndhlovu division of veterinary services, department of livestock and veterinary services, zimbabwe daud n. ndhlovu department of clinical veterinary studies, university of zimbabwe, zimbabwe sylvester m. chikerema department of clinical veterinary studies, university of zimbabwe, zimbabwe mhosisi masocha department of geography and environmental science, university of zimbabwe, zimbabwe mudavanhu nyagura department of pre-clinical veterinary studies, university of zimbabwe, zimbabwe davies m. pfukenyi department of clinical veterinary studies, university of zimbabwe, zimbabwe citation ndhlovu, f., ndhlovu, d.n., chikerema, s.m., masocha, m., nyagura, m. & pfukenyi, d.m., 2017, ‘spatiotemporal patterns of clinical bovine dermatophilosis in zimbabwe 1995–2014’, onderstepoort journal of veterinary research 84(1), a1386. https://doi.org/10.4102/ojvr.v84i1.1386 original research spatiotemporal patterns of clinical bovine dermatophilosis in zimbabwe 1995–2014 felistas ndhlovu, daud n. ndhlovu, sylvester m. chikerema, mhosisi masocha, mudavanhu nyagura, davies m. pfukenyi received: 13 oct. 2016; accepted: 16 may 2017; published: 27 june 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a retrospective study of clinical bovine dermatophilosis outbreaks and cases for the period 1995–2014 was conducted, using data obtained from the division of veterinary services (dvs). a total of 3856 outbreaks and 26 659 cases of dermatophilosis were reported countrywide during this period. the post rainy season accounted for 37.9% of the outbreaks followed by the rainy season (26.7%), cold dry season (22.1%) and the hot dry season (13.2%). a retrospective space–time scan statistic in satscan™ was used to detect clusters. from this study, it was evident that dermatophilosis was spreading from the north-west of zimbabwe through the central to the north-east during the period 2010–2014. five clusters were identified mainly in the central and north-western regions of zimbabwe. the primary cluster was centred at ungwe, gokwe district in midlands; the second, third, fourth and fifth likely clusters were centred at bonga (mashonaland central), arda (mashonaland west), nsenga (matabeleland north) and zanda in gokwe, respectively. the findings of this study suggest the continued spread of dermatophilosis across the country; as such the department of livestock and veterinary services are advised to develop measures aimed at managing this spread such as dipping, quarantine, movement control and raising farmer awareness. introduction dermatophilosis is an economically important disease of livestock caused by dermatophilus congolensis, a gram-positive actinomycete that produces motile zoospores which invade the skin (hadrill & walker 1996; samson et al. 2010). although it has a worldwide distribution, the disease is reported mainly in african countries (awad, nadra-elwgoud & el-sayed 2008). the disease is of particular importance in the tropics and sub-tropics where it causes substantial losses (woldemeskel & taye 2002). dermatophilosis has been reported in cattle, camels, buffaloes, donkeys, cats, dogs, wildlife and man (awad et al. 2008; amor et al. 2011) but is more severe in ruminants (dalis et al. 2009; woldemeskel & mersha 2010). although tick-free animals are susceptible to infection and clinical disease, the disease is more severe in animals infested with amblyomma variegatum ticks (ambrose 1996). as such, occurrence and spread of dermatophilosis in cattle has been associated with a. variegatum (ahoussou et al. 2010). walker (1996) postulated that an immunosuppressive agent secreted in the tick’s saliva or waste metabolites led to the development of dermatophilosis. clinically, bovine dermatophilosis presents as an exudative dermatitis with lesions that can be localised or generalised (koney et al. 1996). the lesions are distributed mainly in the inguinal area, scrotum and front legs in males; in females, they are mainly around the udder and external genitalia and on the back in both sexes (chatikobo et al. 2001; dalis et al. 2009). this distribution has been reported to adversely affect mating and fertility. a carrier state has been reported in cattle by stewart (1972), such carrier cattle do not present with observable clinical signs and were postulated to be the chief means of survival for d. congolensis. economically, bovine dermatophilosis is important due to morbidity and mortality, damage to hides and its effect on draught animal power (samui & hugh-jones 1990). hide condemnations are an important cause of economic loss for the leather industry and in countries where cattle hides are processed for human consumption (cadmus & adesikan 2009). in addition, introduction of exotic breeds to improve meat and milk production has been frustrated in other parts of africa (koney 1996) by this disease. the disease is also of public health significance as it can be transmitted to humans (awad et al. 2008; amor et al. 2011). in zimbabwe, bovine dermatophilosis is a notifiable disease with two statutory instruments (si) developed in 2010 for its management and control. statutory instrument 166 of 2010 (animal health [dermatophilosis areas] order 2010a) defines certain districts in the country as dermatophilosis prescribed areas. si 167 of 2010 (animal health [dermatophilosis] regulations 2010b) regulates what should be done by farmers and authorised persons for the control of dermatophilosis. the si place a legal obligation on the farmer or anyone to report occurrences of suspected bovine dermatophilosis to the nearest veterinary offices. persistent droughts and the land resettlement programme have been postulated as the reasons for the spread (chatikobo et al. 2009), while ndhlovu and masika (2013) cited the decrease in government support to the smallholder farmer dipping programme and the economic challenges faced by the country at the time as further contributing factors. studies on bovine dermatophilosis in zimbabwe have focused on tick control, prevalence and distribution of the disease in certain areas of the country (chatikobo et al. 2001, 2004, 2009; ndhlovu & masika 2013, 2015). however, little has been done on the spatiotemporal characteristics of the disease. various spatial statistical analysis techniques and software are available. these techniques use spatially referenced data for the development and analysis of spatial models (li, guo & elkan 2011). satscan™ (2016) is a free downloadable software that can be used to analyse spatial, temporal and space–time data using the spatial, temporal and space–time scan statistics within the software satscan™ version 9.4.4 (software for the spatial and space-time scan statistics 2016). the scan tests detect the location and test the significance of clusters using a search or scanning window (heres, brus & hagenaars 2008). the scanning window moves across space and/or time; for each location and size of the window, the number of observed and expected cases are counted (kulldorf et al. 2005). scan statistics can be used to detect and test the presence of purely spatial, purely temporal and space–time clusters (szonyi, wade & mohammed 2010). the detection and scanning is performed, briefly, as follows: for purely spatial analysis a circular or oval scanning window is used and in space–time analysis a cylindrical window is utilised, the base of this cylinder represents the geographic search area while the height represents the time frame (szonyi et al. 2010; wolff et al. 2014). the observed and expected number of cases inside the search window is compared with those outside the window, and a likelihood ratio test is performed to confirm if the risk of disease inside the window is the same as in the outside (szonyi et al. 2010). the window with the highest likelihood ratio is considered the most likely cluster and a p-value is assigned to it. the current study was undertaken to determine the spatial and temporal trends, associated with the occurrence of dermatophilosis in zimbabwe, as well as to identify possible clustering of the disease. this study used passively collected data on clinical dermatophilosis. findings from this study will assist in the formulation of targeted control strategies aimed at preventing further spread of the disease with the ultimate goal of improving smallholder livestock production and productivity. materials and methods study population and data collection a retrospective study of clinical bovine dermatophilosis outbreaks and cases for the period 1995–2014 was conducted. dermatophilosis outbreaks and cases were identified through the division of veterinary services (dvs) disease reporting system. this system is made up of a computerised database created using disease and epidemiology reports submitted from 60 districts and 8 provincial veterinary offices of the country. data in the system are collected through an animal disease surveillance system that combines both passive and active elements. the passive component is based on regular disease reporting by farmers with subsequent follow-up by veterinary officers (vo), this system can suffer from under reporting. dermatophilosis is notifiable in zimbabwe (statutory instrument 166 of 2010 [animal health {dermatophilosis areas} order 2010a]; si 167 of 2010 [animal health {dermatophilosis} regulations 2010b]), as such under reporting is mitigated as reporting the occurrence of the disease is mandatory. this system is augmented through monthly visits conducted by veterinarians, animal health inspector and veterinary extension assistants (veas) to dip tanks and farms. while these visits are not specifically for the detection of bovine dermatophilosis, they increase the likelihood that the disease will be detected and appropriate follow-ups made. the data on disease occurrences that are reported and investigated are initially captured in a disease investigation form (dif). the dif has a number of sections that capture biographical data of the farmer reporting such as, the geographical location, number of cases, herd size, clinical signs and whether it is an initial or a follow-up report. the section for diagnosis is divided into two parts, the first is completed by an officer other than a veterinarian and in this section the tentative diagnosis is captured. the final section is for the provisional/definitive diagnosis, this part is filled in by the veterinarian upon referral of the report by a non-veterinarian and also in the event that the disease occurrence is reported directly to the veterinary officer. disease reports were generated when vo, animal health inspectors (ahis) and/or veas investigated disease occurrences that were reported by livestock owners. diagnoses were made by vos, ahis and veas with reports from ahis and veas further verified by the vos for accuracy. diagnoses were based on the presenting clinical signs and the provisional diagnosis was verified based on adherence to a prescribed case definition for bovine dermatophilosis, geographical area, and season. cases presenting with a case definition that satisfied any one of the following clinical signs (lesions): small paint brush-like clumping of hair, multiple circumscribed scabs over 1 cm in diameter and confluent progressive lesions as described by hadrill and walker (1996), were considered clinically to be bovine dermatophilosis cases. the officers making the diagnoses were trained in animal health and disease recognition and possessed qualifications ranging from diploma in animal health to veterinary science degrees. data on new cases were used to avoid cases that were reported as follow-ups and chronic cases; this was done by filtering the data set to include only records that were flagged as new and excluding all records that were flagged as follow-ups in the database. data manipulation: geo-referencing and epidemiological units disease data were captured in microsoft excel® (2013); these data were geo-referenced using the coordinate system (latitude and longitude). in the event that a farm or dip tank (epi-unit) had different geo-references (latitudes and longitudes) for the number of times reports were made from that location, the coordinates were corrected for each by using the list of dip tanks and farms with global position system (gps)-derived coordinates that had been collected by the dvs since 2010. data were checked for multiple entries; these occurred when the same case and the related information was entered more than once, such additional entries were deleted. records that had zero cases and deaths were deleted. outbreaks and cases were identified to an epidemiological unit (epiunit) which was either a dip tank or a farm. an outbreak was defined as the occurrence of a disease event at a point in time and place that complied with the case definition of bovine dermatophilosis, and these data were used for temporal analysis. cases were defined as the total number of cattle at a point in time and place, presenting with clinical signs that complied with the case definition of bovine dermatophilosis. the individual clinical cases data were used in the spatial analysis using satscan™ version 9.4.4 (software for the spatial and space–time scan statistics 2016) and in temporal analysis. temporal analysis data were analysed using the software package for social scientists (spss) (international business machines® spss® version 21 2012). clinical dermatophilosis cases and outbreaks were tested for normality using the kolmogorov–smirnov test, a p-value of < 0.05 indicated non-normality of the distribution. cases and outbreaks were further pooled according to seasons of the year. seasons of the year were defined according to chikerema et al. (2012) as follows: rainy (december–february), post rainy (march–may), cold dry (june–august) and hot dry (september–november). this definition of seasons is a variation to that described in the climate handbook of zimbabwe meteorological services of zimbabwe (1981). the handbook defines the seasons as follows: main rainy season; mid-november to mid-march, post rainy season; mid-march to mid-may, cool season; mid-may to august and hot season; september to mid-november. according to the climate handbook of zimbabwe (meteorological services of zimbabwe 1981), the mean temperature was reported to be highest, at 32 °c, in october the mid-point of the hot dry season, 12.8 °c – 18.6 °c in july at the mid-point of the cold dry season and a low of 9 °c in january the mid-point of the rainy season. average annual rainfall for zimbabwe was reported to be 675 mm, of this; the least rainfall, that is, ≤ 0.2%, was received in october and april the mid-points of the hot dry and post rainy seasons, respectively, while the most average rainfall of ≤ 100 mm was received during rainy season, and ≤ 10 mm of rainfall received in the cool dry season (meteorological services of zimbabwe 1981). descriptive statistics, that is, proportions of pooled seasonal cases and outbreaks, were computed. the relationship between the number of clinical cases and outbreaks, as dependent variables, and the categories – month, season and year – as independent variables, were evaluated using the kruskal–wallis one-way anova for independent samples, and the median test both non-parametric tests. post-hoc multiple pairwise comparison tests were performed using the same test in spss® (international business machines® spss® version 21 2012). a p-value of < 0.05 was considered as the significance level. separate box-plots for the pooled dermatophilosis clinical outbreaks and cases, according to month, season and year, were constructed. space–time analysis to detect and test clusters, the scan test in satscan™ version 9.4.4 (software for the spatial and space–time scan statistics 2016), a statistical software, was used, and the p < 0.05 was considered as significant. the null hypothesis was that the number of cases were randomly distributed throughout the dip tanks and farms and that the time frames of occurrence of cases were similar, the alternative hypothesis was that cases were clustered. the variables used for the analysis were the location, coordinates, dates and number of cases. retrospective space–time analysis was used, using the space–time permutation probability model, scanning for areas with high rates. the study period was from 01 january 1995 to 31 december 2014 and time precision was a month. the maximum spatial cluster size was set at 25% of the population at risk while the maximum temporal cluster size was at 20% of the study period and time aggregation was set at 3 months. these parameters which were lower than those recommended (kulldorf 2015) were selected to make the scan more specific. the scan test detects and tests for clusters in the following manner, the observed and expected number of cases inside the search window are compared with those outside the window and a likelihood ratio test is computed to test if the risk of disease inside the window is the same as outside (szonyi et al. 2010). the window with the highest likelihood ratio is considered the most likely cluster and a p-value is assigned to it. the likelihood ratio test is computed by comparing the likelihood or probability of occurrence of an event or parameter under the alternate and null hypothesis (dohoo, martin & stryhn 2003). the likelihood ratio test was run with 999 monte carlo replications. monte carlo is a type of simulation that relies on repeated random sampling and statistical analysis to compute the results (raychaudhuri 2008); these statistical analyses are performed using a computer (munfrom et al. 2011). the output of the analysis was projected onto quantum geographical information system (qgis) version 2.14.5-las palmas de g.c. (2017) an open source geographic information management system software. results temporal distribution of dermatophilosis outbreaks a total of 3856 outbreaks with 26 659 cases of clinical bovine dermatophilosis were reported countrywide during the period 1995–2014. the temporal distribution of outbreaks and cases reported for the period are indicated as box-plots in figures 1, 2 and 3. the post rainy season accounted for 37.91% of the outbreaks followed by; the rainy season (26.73%), cold dry season (22.14%) and the hot dry season (13.22%). dermatophilosis was reported throughout the year with most outbreaks occurring in the first third of year (figure 1) with the minimum and maximum monthly median outbreaks for the period being 4 and 15 during october and the period february–april respectively. the study period 1995–2008 was characterised by fewer outbreaks (minimum and maximum median outbreaks of 1 and 11.5 in 1997 and 2006 respectively) than the period 2009–2014 which had comparably more outbreaks (minimum and maximum median outbreaks of 19.5 and 54.5 during 2009 and 2014 respectively) (figure 2). the number of cases across the years were characterised by a notable spike in 1999 (median 644) and more cases for the period 2010–2014. the post rainy season accounted for most of the outbreaks and cases followed by the rainy season (figure 3). the distribution of median outbreaks across seasons was significantly different (p = 0.013). multiple pairwise comparisons showed that outbreaks during the hot dry season were significantly different (p = 0.022) from those during the post rainy season, a significant difference (p = 0.030) was also observed between the rainy and hot dry season. the median number of cases differed significantly across seasons (p = 0.0001). the median number of cases during the hot dry season differed significantly from the number during the rainy season (p = 0.001), the post rainy season (p = 0.0001) and cold dry season (p = 0.044). figure 1: monthly distribution of dermatophilosis (a) outbreaks and (b) cases for the period 1995–2014. figure 2: annual distribution of dermatophilosis (a) outbreaks and (b) cases for the period 1995–2014. figure 3: seasonal distribution of dermatophilosis (a) outbreaks and (b) cases for the period 1995–2014. clusters detected five likely clusters of varying sizes were detected using satscan™ (2016); the locations of the clusters are shown in figure 4. details of the clusters are summarised in table 1. the clusters were located mainly in the north-west parts of zimbabwe and were of varying spatial and temporal sizes. figure 4: likely clusters of clinical dermatophilosis cases in zimbabwe for the period 1995–2014. table 1: likely clusters from a space–time scan statistic (space–time permutation model) of clinical dermatophilosis. ethical consideration the division of veterinary services, zimbabwe, consented to the use of the data. farmers handle the animals and treat them with due care taken for their welfare. discussion findings of this study were limited by the type of data used, which were collected passively. such data make it difficult to identify instances of missing data because of either underreporting or failure by staff to capture the data electronically. however, these findings can be used as baseline data, by animal health authorities and researchers to develop targeted disease control strategies and research on putative risk factors for the occurrence of dermatophilosis. the disease was reported for all the years from 1995 to 2014 although at different levels. the findings of this study suggest an increase in the number of outbreaks and cases starting from 1999 onwards as shown by the increase in median outbreaks and cases. the first increase in 1999–2000 could have been due to the effect that the start of the land reform programme had on dipping and other animal health related activities. during this period as stockowners moved their cattle from one area to another, dipping schedules could have been missed or the dipping services not yet well established at the new properties, leading to increased outbreaks of tick-borne and tick associated diseases. previously, lawrence, foggin and norval (1980) reported that the disruptions in dipping and other animal health activities during the war of independence led to increased livestock deaths due to diseases. the second peak around the period 2006–2007 could have been associated with the start of the economic challenges that peaked in 2008. this also could have led to disruptions in animal health control activities. the general increase in outbreaks throughout the years and particularly around 2014 could be due to the fact that the disease had attained pandemic proportions as was previously predicted by chatikobo et al. (2009). increased reporting of the disease by officers may also have contributed to gradual increase in outbreaks reported. the data used in the study was based on a passive surveillance system, as such, there is a possibility of underor over-reporting due to misdiagnosis; not all farmers report disease occurrences to veterinary authorities and they usually treat their livestock without such consultation, the disease is notifiable in zimbabwe, it is mandatory for farmers to report it to national authorities. notable were the low number of outbreaks and cases characterising the period 1995–1998. the computerised information management system for capturing disease data was established in 1995, as such it is possible that that reports on dermatophilosis and other diseases were not captured in the system as officers were not yet used to the system resulting in underreporting during the early years of the system being established. the current study suggested that dermatophilosis outbreaks occurred throughout the year and most outbreaks occurred during the rainy and post rainy seasons. due to the established association between a. variegatum and dermatophilosis (ahoussou et al. 2010; stachurski, zoungrana & konkobo 2010), the seasonality of the disease could be explained by the seasonality of the tick infestation burdens. unganai (1996) reported that the main precipitation months are december to february although precipitation can span the months of october to april depending on the region of the country. norval (1983) and walker et al. (2003) reported that a. variegatum was present throughout the year although heavier infestations occurred during the warmer months (september–may) than in the cooler months (june–september). this temporal distribution implies that control of the disease should not only be limited to the rainy and post rainy season but throughout the year. given the chronic nature of the disease, it is likely that some of the cases reported during other seasons were not new cases/infections; in the data collection, follow-up cases were excluded so this proportion might be minimal. it is postulated in this study, that farmers who report a dermatophilosis occurrence and are given advice do not usually return to give the same report later. disease clusters occur when cases of the disease occur in closer succession and in a smaller area than would be expected by chance alone. such clusters occur in space and time (wolff et al. 2014). five clusters were identified mainly in the central and north-western regions of zimbabwe. the primary cluster (cluster 1) which occurred between 01 january 1999 and 31 december 1999 was located in ungwe, gokwe district in the midlands province, bordering with kadoma in mashonaland west province. the space–time cluster corroborates reports by chatikobo et al. (2009) who reported that outbreaks of dermatophilosis occurred in this area around 1999. the explanation given by the farmers who were interviewed was that cattle in the area had not been dipped for the past five-six months resulting in outbreaks of the disease (chatikobo et al 2009). the second cluster (cluster 2) was centred at bonga dip tank, mbire district in mashonaland central province. cluster 2 had a radius of 239.7 km and included the mashonaland west, mashonaland central and mashonaland east provinces with a cluster period from 01 january 2011 to 31 december 2014. the bonga cluster had the largest radius and longest period spanning 4 years. this cluster could have resulted from inter district and inter provincial movement of a. variegatum tick infested, infected or carrier cattle from the south-western provinces going eastwards. most of the cattle markets are lucrative in the north-east parts of zimbabwe. the third cluster was centred at arda gokwe north district in the midlands province, this cluster had a radius of 13.3 km and was clustered within the period 01 january 1999 to 30 march 1999. location of cluster 3 was consistent with reports that dermatophilosis has initially and consistently been reported from the north-west of zimbabwe, this as a result of its association with a. variegatum that is prevalent in this part of zimbabwe (chatikobo et al. 2001; norval 1983; walker et al. 2003). nsenga farm in binga district, matabeleland north province was the centre for cluster 4, spanning a period from 01 january 2010 to 31 march 2010. according to chatikobo et al. (2009) dermatophilosis spread from this north-west area going north-eastwards. the fifth cluster was centred at zanda in gokwe district. clusters 1, 3 and 5 were located in the part of the country where dermatophilosis has been consistently reported from (chatikobo et al. 2009; ndhlovu & masika 2015). an examination of the cluster periods suggests that dermatophilosis, according to the current study period, might have started from clusters 1 and 3 (01 january 1999), then spread to clusters 5 and 4 and finally cluster 2 (31 december 2014). the spatial and temporal proximities of clusters 1, 3 and 5 suggest that it could be possible that movement of disease from one area to the other was due to an increase in cattle movements within and between districts; such movements could be for sale purposes or for socially related activities. furthermore, these three clusters were associated with the period when the land reform was at its peak; this programme was associated with movements of livestock from one district to another district where people had been allocated land for resettlement. the space–time analysis is in agreement with findings by chatikobo et al. (2009) who reported that dermatophilosis spread from matabeleland north, midlands, and mashonaland west in that order. during the study by chatikobo et al. (2009), dermatophilosis had been reported from three provinces, the current study suggests that the disease and its associated tick a. variegatum had spread further north-eastwards to include two more provinces, that is, mashonaland central and mashonaland east provinces. unlimited livestock movement both legal and illegal might have contributed to this spread. according to estrada-peña et al. (2008), the southern limit for a. variegatum was between latitudes 17°s and 18.5°s. these two facts imply that dermatophilosis and a. variegatum have the potential of spreading north-eastwards to new areas in mashonaland east and manicaland provinces. findings of this study suggest that dermatophilosis occurs throughout the year and that the disease and associated tick a. variegatum were spreading from the north-west to the north-eastern parts of zimbabwe. conclusion findings from this study suggest that bovine dermatophilosis was spreading from north-west to the north-east of zimbabwe; this can be used by the dvs and farmers to plan for the control and management of dermatophilosis through a knowledge of where the disease is most likely to occur next. interventions that can be instituted may include pre-movement inspection of cattle to ensure that they are not tick infested and that they are clinically disease-free; movement bans should be imposed on clinically affected animals, and these should be treated promptly. dipping of cattle prior to the movement can aid in reducing the spread of the disease and its associated tick to new areas. a policy of culling previously infected animals that are repeatedly infected should be considered. at destination point, cattle from a dermatophilosis area must be quarantined for at least a month to detect any carriers. the occurrence of space–time clusters indicates areas where more rigorous dermatophilosis control measures should be implemented together with raising stock-owner awareness about the disease. targeted research should be undertaken to identify the drivers responsible for the spread of dermatophilosis and to determine the socio-economic implications of the disease to the state and livestock keepers. acknowledgements the authors acknowledge the assistance provided by the directorate of veterinary services who allowed the use of the disease data sets from the division of veterinary services. competing interests the 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accepted: 03 oct. 2016; published: 31 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a total of 118 sera were collected during 2016 from two groups of dromedaries from kebili and medenine governorates in the south of tunisia. the aim of this study was to provide the first serological investigation of four emerging vector-borne diseases in two groups of dromedaries in tunisia. sera were tested by elisa and serum neutralisation test to identify west nile virus (wnv), bluetongue virus (btv), epizootic haemorrhagic disease virus (ehdv) and rift valley fever virus (rvfv). in the first group, the seroprevalence for btv was 4.6%, while in the second group, it was 25.8% for wnv and 9.7% for btv. only serotype 1 was detected for btv in the two groups. no evidence for circulation of rvf and ehd viruses was revealed. results indicated that dromedaries can be infected with btv and wnv, suggesting that this species might play a significant role in the epizootiology of these viral diseases in tunisia and neighbouring countries. introduction the economic value of the dromedary as a source of meat and transportation with the renewed interest around the middle east respiratory syndrome coronavirus (mers-cov) in the arabian peninsula raises the question of the impact of the dromedary on new and emergent diseases in north africa. the effective control of west nile (wn), bluetongue (bt), epizootic haemorrhagic disease (ehd) and rift valley fever (rvf) depends on a thorough understanding of the possible implications of dromedaries in the replication or the dissemination of the west nile virus (wnv), bluetongue virus (btv), epizootic haemorrhagic disease virus (ehdv) and rift valley fever virus (rvfv). tunisia is located at the interface between the sub-saharan region and europe. it has encountered several episodes of emerging vector-borne diseases such as wn, bt and ehd. tunisia has experienced three major wn epidemics that have particularly affected humans in 1997, 2003 and 2012 (ben hassine et al. 2015). in 2015, an equine with clinical signs was reported for the first time in the south of tunisia in the oases of tozeur (oie 2015). since the first occurrence of bt in 1999, outbreaks have been reported in tunisia and three serotypes, namely btv2, btv1 and btv4 were identified in 2000, 2006 and 2009, respectively (hammami 2004; sghaier et al. 2014). more recently, other bt outbreaks because of serotype 1 in 2011 and serotype 4 in 2013 have been reported (lorusso et al. 2013; sghaier et al. 2014). in 2015, a study suggested an active circulation of rvfv and evidence of human exposure in the population of tunisia was reported (bosworth et al. 2016). ehdv was detected for the first time in tunisia in 2006. recently, clinical cases in cattle were reported in countries surrounding the mediterranean basin, including morocco, algeria and tunisia, with the emergence of ehdv serotype 6 (ben dhaou et al. 2016). the aim of the present study was to provide the first serological investigation of four emerging vector-borne diseases in two groups of dromedaries in tunisia with a focus on wnv, btv, ehdv and rvfv. materials and methods this study was carried out in 2016 in the governorates of kebili and medenine (figure 1). these two governorates were selected because of their relatively high dromedary density and cross-border animal movement within the region (ayari-fakhfakh et al. 2011). these factors represent high-risk zones for disease transmission in tunisia. figure 1: geographical location of sampled dromedaries in the south of tunisia (kebili and medenine governorates). the first lot of 87 sera was collected from dromedaries intercepted at the country border posts by the tunisian veterinary services in the region of ben guerden (governorate of medenine) where the dromedaries usually circulate in the arid desert. the second lot of 31 sera was collected from individual dromedaries in contact with sheep herds in the oases of kebili, which are characterised by traditional irrigation where the main practice is flooding (mekki et al. 2013). sera were tested by enzyme-linked immunosorbent assay (elisa) for wnv, btv, rvfv and ehdv. igg antibodies to rvfv were detected using id screen® rvf competition multi-species elisa kits (id vet; innovative diagnostics, montpellier, france). for ehd, all the sera were tested by a blocking competitive elisa (lsi vet ehdv blocking, lsi, france) to detect the specific anti-ehdv vp7 antibodies. sera were screened for the presence of group-specific btv antibodies by using the c-elisa idexx bluetongue competition® assay (idexx bt, hoofddorp, the netherlands). the usutu virus (usuv)and wnv-specific antibodies were detected using a commercially available competition elisa, (id screen® west nile competition, id vet, montpellier, france). serum neutralisation (sn) test for 26 btv serotypes and for wnv–usuv was used on positive samples (di gennaro et al. 2014). the test was conducted at the istituto zooprofalitico sperimentale dell’abruzzo e del molise ‘g. caporale’ (izsam) and oie reference laboratory for btv and wnd, under biosafety level 3 conditions as previously described (oie 2016). results and discussion seropositivity of btv was detected in the two groups (4.6% in the group of medenine vs. 9.7% in the group of kebili). this study confirms the circulation of serotype 1 in tunisia that was detected for the last time in 2011 (sghaier et al. 2014). for btv, tunisia has adopted a vaccination strategy in sheep using a bivalent vaccine (btv1 and btv4), but dromedaries are not vaccinated. three species of culicoïdes were predominantly detected in the south of tunisia (gabes governorate): culicoïdes jumineri, culicoïdes sahariensis and culicoïdes submarimitimus although culicoïdes imicola is considered the main vector of btv (mellor 1996; sghaier et al. 2009). according to batten et al. 2011, dromedaries seem to act as reservoirs, possibly playing a role in the spread of the disease by helping the virus to get through the geographic barrier which is represented by the sahara desert. this desert stands between the tropics and subtropics, where btv is endemic, and north africa, where it periodically causes epizootics. however, there is scarce information about the clinical manifestations of bt in dromedaries. south american camels are susceptible to btv infection, but they develop only a mild form of the disease (schulz et al. 2012). for wnv, this study revealed that 25.8% of the dromedaries, which live in oases, were seropositive. this result is consistent with the findings reported by ben hassine et al. (2015), where a high seropositivity in horses was found in the oases of kebili that were identified as high-risk areas for wnv circulation in tunisia. moreover, the presence of culex pipiens in the south of tunisia and isolation of wn virus from c. pipiens in central tunisia (lineage 1) confirms this finding (wasfi et al. 2016). recent studies in north africa reported wnv seropositivity rates in dromedaries ranging from 13% (touil et al. 2012) to 29% (el-harrak et al. 2011). similar to human and horse populations, in which less than 1% of wnv-infected individuals become severely ill, it appears that the majority of camels infected with wnv are asymptomatic and recover uneventfully. no evidence for the circulation of rvf and ehd viruses are revealed in this study. serologic evidence of rvf in dromedaries is frequently reported (swai & sindato 2015), yet the description of clinical signs is rare. subclinical, mild forms and healthy carriers of the virus (paweska 2015; swanepoel & coetzer 2004) have been reported. the presence of the rvf competent vector c. pipiens and aedes caspius in oases make this ecosystem favourable for rvf transmission in tunisia where aedes could be responsible for the initiation of an outbreak and culex maintains the virus activity (soti et al. 2012). for ehdv, dromedaries seem not to be involved in the disease transmission. the study conducted by wernery et al. (2013) using elisa test showed that 29% of dromedaries from the united arab emirates have antibodies to ehdv. conclusion this study, for the first time, reports infection of dromedaries with wnv and btv in tunisia, confirming the reported infections of dromedaries with btv and wnv in north africa and the middle east. a larger serosurvey with an entomological monitoring and a syndromic surveillance in a one health concept is needed to understand the implications of dromedaries in the transmission of emerging arthropod-borne viruses which can be very useful for the purposes of control and planning surveillance activities not only in tunisia but also for all the regions of north africa. acknowledgements this study was partially funded by iaea (raf50/68 regional project), italian ministry of health izs am 01/10 rc and eu grant fp74613996 vmerge, and is catalogued by the vmerge steering committee as vmerge0011. the authors would like to thank spana-tunisia for logistical support. competing 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dromedaries’, journal of camel practice and research 20(2), 135–137. abstract introduction materials and methods results and discussion conclusion acknowledgements references about the author(s) edgar simulundu department of disease control, university of zambia, zambia yona sinkala department of veterinary services, ministry of fisheries and livestock, zambia herman m. chambaro department of veterinary services, ministry of fisheries and livestock, zambia andrew chinyemba department of disease control, university of zambia, zambia frank banda department of veterinary services, ministry of fisheries and livestock, zambia lynnfield e. mooya department of veterinary services, ministry of fisheries and livestock, zambia joseph ndebe department of veterinary services, ministry of fisheries and livestock, zambia simbarashe chitanga department of biomedical sciences, university of zambia, zambia chitwambi makungu department of veterinary services, ministry of fisheries and livestock, zambia gift munthali department of veterinary services, ministry of fisheries and livestock, zambia paul fandamu department of veterinary services, ministry of fisheries and livestock, zambia ayato takada division of global epidemiology, hokkaido university research center for zoonosis control, japan aaron s mweene department of disease control, university of zambia, zambia citation simulundu, e., sinkala, y., chambaro, h.m., chinyemba, a., banda, f., mooya, l.e. et al., 2018, ‘genetic characterisation of african swine fever virus from 2017 outbreaks in zambia: identification of p72 genotype ii variants in domestic pigs’, onderstepoort journal of veterinary research 85(1), a1562. https://doi.org/10.4102/ojvr.v85i1.1562 research communication genetic characterisation of african swine fever virus from 2017 outbreaks in zambia: identification of p72 genotype ii variants in domestic pigs edgar simulundu, yona sinkala, herman m. chambaro, andrew chinyemba, frank banda, lynnfield e. mooya, joseph ndebe, simbarashe chitanga, chitwambi makungu, gift munthali, paul fandamu, ayato takada, aaron s mweene received: 06 oct. 2017; accepted: 16 may 2018; published: 26 june 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract african swine fever (asf) is a contagious haemorrhagic disease associated with causing heavy economic losses to the swine industry in many african countries. in 2017, zambia experienced asf outbreaks in mbala district (northern province) and for the first time in isoka and chinsali districts (muchinga province). meanwhile, another outbreak was observed in chipata district (eastern province). genetic analysis of part of the b646l gene, e183l gene, cp204l gene and the central variable region of the b602l gene of asf virus (asfv) associated with the outbreaks in mbala and chipata districts was conducted. the results revealed that the asfv detected in mbala district was highly similar to that of the georgia 2007/1 isolate across all the genome regions analysed. in contrast, while showing close relationship with the georgia 2007/1 virus in the b646l gene, the asfv detected in chipata district showed remarkable genetic variation in the rest of the genes analysed. these results suggest that the georgia 2007/1-like virus could be more diverse than what was previously thought, underscoring the need of continued surveillance and monitoring of asfvs within the south-eastern african region to better understand their epidemiology and the relationships between outbreaks and their possible origin. introduction african swine fever virus (asfv) causes african swine fever (asf), an acute, contagious and devastating haemorrhagic disease that affects swine. because of its high mortality rates (up to ≈100%), serious socio-economic impact, high capacity for transboundary dissemination and lack of an effective vaccine or treatment, asf is considered to be one of the most challenging animal diseases to contend with (costard et al. 2009). historically, asf was first described in kenya in 1921 and is considered to be endemic in many african countries (penrith et al. 2013). indeed, reports of outbreaks in at least 26 african countries during 2009–2011 testify to the disease’s continued eminent presence and the threat it poses to the pig industry in sub-saharan africa (penrith et al. 2013). based on sequence analysis of the variable c-terminus of the b646l gene encoding the p72 capsid protein, asfv field strains are genetically categorised into 24 genotypes (achenbach et al. 2016; bastos et al. 2003; quembo et al. 2018). traditionally, while all the genotypes have been circulating within south-eastern africa, only genotype i has been reported from and predominates in west africa (brown et al. 2017). moreover, genotype i asfv was introduced into europe, south america and the caribbean, but has only remained endemic in sardinia (italy) (costard et al. 2009). however, the global asf situation changed dramatically following the introduction of a highly virulent genotype ii asfv, which was circulating in south-eastern africa, into georgia in 2007 (costard et al. 2009; rowlands et al. 2008). the virus spread rapidly and eventually advanced into eastern europe, a situation that has raised serious concerns over the global pig industry (vergne et al. 2017). in recent years, zambia has suffered frequent sporadic asf outbreaks in almost all its provinces, which have been associated with multiple genotypes (simulundu et al. 2017, 2018; thoromo et al. 2015). in 2017, asf outbreaks occurred in mbala (northern province), isoka and chinsali (muchinga province) districts. furthermore, an outbreak was also observed in may 2017 in chipata district (eastern province). with the aim of increasing knowledge about the epidemiology of asf, findings on genetic analysis of distinct genome regions of asfvs associated with outbreaks in mbala and chipata districts are reported. materials and methods on 26 april 2017, an asf outbreak in zambia, which occurred in mbala district in free-range domestic pigs, was reported (world organisation for animal health [oie] 2017a). the affected village had 48 pigs in which 15 cases and 11 deaths (73.3% case fatality rate) were recorded. the disease was observed in chinsali and isoka districts for the first time (oie 2017b). in these two districts, a total of 1895 cases with 1891 deaths (99.8% case fatality rate) were reported. the asf outbreak in chipata district involved a farmer whose 28 indigenous pigs were affected and 20 had died (71.4% case fatality rate). for laboratory diagnosis, dna was extracted from tissue homogenates on samples (kidneys, lymph nodes, tonsils and spleens) collected from pigs that died from the disease. molecular diagnosis was conducted by polymerase chain reaction (pcr) using the asf diagnostic primer set ppa1/ppa2 (agüero et al. 2003; yabe et al. 2015). for genetic characterisation, international standardised procedures for studying the molecular epidemiology of asf (oie 2013) were applied on pcr-positive samples. specifically, the c-terminal end of the p72 (b646l) gene, p54 (e183l) gene, p30 (cp204l) gene and the central variable region (cvr) within the b602l gene were amplified using the primer pairs p72u/p72d, ppa722/ppa89, p30-f/p30-r and cvr1/cvr2 or orf9l-f/orf9l-r, respectively (simulundu et al. 2017, 2018). purified pcr products were sequenced directly via the sanger technology using a 3500 genetic analyzer (applied biosystems, foster city, ca, usa). the sequences were deposited in genbank (accession no. lc322009-lc322016). two asfvs, designated zam/2017/mbala/1 and zam/2017/chipata/1, were genetically characterised. evolutionary relationships were inferred using the neighbour-joining method, while evolutionary distances were computed using the p-distance method. the genetic trees were generated using mega6 software, version 6.06 (tamura et al. 2013). results and discussion nucleotide sequence comparisons using the basic local alignment search tool (blast) (http://blast.ncbi.nlm.nih.gov/blast.cgi) revealed that the p72, p54 and p30 sequences of zam/2017/mbala/1 were 100% identical to those of georgia 2007/1 (genbank accession no. am999764-am999766). in contrast, blast showed that the p72 and p30 nucleotide sequences of zam/2017/chipata/1 were 99% – 100% similar to those of zam/14/chipata, which was detected in domestic pigs in the same district (simulundu et al. 2018). the p54 nucleotide sequence of zam/2017/chipata/1 showed 100% similarity to that of kli/88/2, which was found in petauke district, eastern province, in 1988 (simulundu et al. 2017). phylogenetic analysis showed that both zam/2017/mbala/1 and zam/2017/chipata belonged to p72 genotype ii (figure 1). in contrast, the p54 phylogeny revealed that zam/2017/mbala/1 belonged to genotype iia while zam/2017/chipata/1 grouped under genotype viiia (figure 2a). thus, the p54 genetic tree showed that p72 genotype ii viruses were separated into distantly related genotypes iia, iib (solely comprising zam/14/chipata) and iic (composed of asfvs associated with outbreaks in tanzania and malawi in 2011) and the zambian virus that fell into genotype viiia (figure 2a). consistent with the p72 and p54 evolutionary relationships, zam/2017/mbala/1 was closely related to zam/13/mbala and georgia 2007/1 viruses in the p30 tree (figure 2b). in contrast, zam/2017/chipata/1 and zam/14/chipata formed a distinct group that was distantly related to the georgia 2007/1-like viruses (figure 2b). figure 1: phylogenetic relationships of the p72 gene of african swine fever viruses detected in domestic pigs during 2017 in zambia. the evolutionary relationships were conducted by mega6 software using the neighbour-joining method, with evolutionary distances being computed by the p-distance method. numbers at branch nodes indicate bootstrap values ≥ 50%. the genbank accession numbers of strains included in the analyses are indicated in parenthesis and the p72 genotypes are shown after the country of origin of the strains or the right bracket. virus strains characterised in this study are in bold. bar, number of substitutions per site. figure 2: phylogenetic relationships of the p54 (a) and p30 (b) genes of african swine fever viruses detected in domestic pigs during 2017 in zambia. the evolutionary relationships were conducted by mega6 software using the neighbour-joining method, with evolutionary distances being computed by the p-distance method. numbers at branch nodes indicate bootstrap values ≥ 50%. the genbank accession numbers of strains included in the analyses are indicated in parenthesis and the p54 genotypes are shown after the country of origin of the strains or the right bracket. virus strains characterised in this study are in bold. bar, number of substitutions per site. while amino acid sequence analysis of the cvr revealed that the sequence of zam/2017/mbala/1 possessed ten copies (bndbndbnaa) of tetramer repeats that were 100% similar to those circulating in eastern europe since 2007, the sequence of zam/2017/chipata/1 was 100% identical to that of zam/14/chipata, with 14 copies (bnabndbtdbnaag) (simulundu et al. 2017, 2018). notably, while the case fatality rate in mbala district was lower (73.3%) than that in isoka and chinsali (99.8%), it was comparable to that observed in chipata (71.4%) where asf is endemic. perhaps this could be explained by the fact that since 2013 mbala district has had prior experience with asf associated with genotype ii virus (simulundu et al. 2017, 2018) and thus some pigs may have developed some level of immunity. moreover, antibodies to asfv were detected in the serum of pigs in the district by elisa (oie 2017a), a finding which suggests possible persistent circulation of the virus in the area. meanwhile, isoka and chinsali districts were affected by asf for the first time and possibly the naïve pig population was highly susceptible to asfv infection. in zambia, p72 genotype ii asfv similar to georgia 2007/1 isolate was first detected in 1993 in lusaka province (simulundu et al. 2017). about two decades later, this viral genotype was associated with outbreaks in mbala district, northern province, probably as a result of introduction from neighbouring tanzania and not from eastern province (simulundu et al. 2018). furthermore, in 2014, a genotype ii variant was associated with an outbreak in chipata district (simulundu et al. 2017, 2018). along with the identification of yet another genotype ii variant, zam/2017/chipata/1, and the observation that asfvs associated with outbreaks in 2011 in tanzania and malawi belonged to p54 genotype iic, which is different from that of georgia 2007/1 (genotype iia) (figure 2a), these findings suggest that in the south-eastern african region, these viruses could be diverse, probably because of the presence of a sylvatic cycle and long-term endemicity. the p30 phylogeny provided further evidence on the genetic diversity of p72 genotype ii viruses as zam/2017/chipata/1 and zam/14/chipata belonged to a group that was phylogenetically distinct from that of georgia 2007/1-like viruses (figure 2b). meanwhile, reminiscent of the eastern europe situation, the geographical extent of genotype ii asfv in south-eastern africa appears to be expanding as this virus has been detected in madagascar, malawi, mauritius, mozambique, tanzania, zambia and zimbabwe (simulundu et al. 2017; van heerden et al. 2017). historically, genotype ii asfv has been circulating in mozambique and zambia for about 20–25 years (bastos et al. 2004; boshoff et al. 2007; penrith et al. 2007; simulundu et al. 2017). this genotype was introduced in madagascar in 1997, most likely from mozambique. tanzania had its first experience with asf outbreaks associated with genotype ii virus during 2010–2012, possibly as a result of spread from malawi (misinzo et al. 2012). the virus appears to have become endemic in domestic pigs in tanzania. this virus spread to mauritius in 2007, probably from madagascar, and was eradicated in 2008 (lubisi et al. 2009). the identification of genotype ii asfv in domestic pigs in zimbabwe in 2015 was traced to outbreaks in an endemic region in mozambique (van heerden et al. 2017). more recently, p72 genotype ii asfvs were detected in soft tick reservoir hosts in mozambique, providing evidence for the first time of a possible sylvatic origin of these viruses in south-eastern africa (quembo et al. 2018). these observations underscore the need of continued surveillance and monitoring of asfvs within the south-eastern african region to better understand their epidemiology and the relationships between outbreaks and their possible origin. conclusion the present study investigated the molecular epidemiology of asf outbreaks that occurred in 2017 in northern, muchinga and eastern provinces of zambia. genetic analysis showed that the outbreaks in the northern region were caused by genotype ii asfv, which was highly similar to the georgia 2007/1 isolate. although belonging to genotype ii, the asfv associated with the outbreak in eastern province was genetically diverse, suggesting that these outbreaks were not related. overall, this study supports the idea that genotype ii asfv circulating in south-eastern africa is genetically diverse, probably because of the long-term endemicity in domestic pigs and a possible sylvatic origin. acknowledgements this study was supported in part by the world bank livestock development and animal health project (project id no. p122123), the japan initiative for global research network on infectious diseases and the japan agency for medical research and development/japan international cooperation agency (jica) within the framework of the science and technology research partnership for sustainable development. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions e.s., y.s., h.m.c. and a.c. conceived and designed the experiments. e.s., y.s., h.m.c., a.c., f.b., l.e.m., j.n., s.c. and c.m. were involved in sample collection and processing. e.s., y.s., h.m.c. and a.c. performed the experiments. g.m., p.f., a.t. and a.s.m. made conceptual contributions. all authors were involved in data analysis, writing, revising and approving the final version of the manuscript. references achenbach, j.e., gallardo, c., nieto-pelegrín, e., rivera-arroyo, b., degefa-negi, t., arias, m. et al., 2017, ‘identification of a new genotype of african swine fever virus in domestic pigs from ethiopia’, transboundary and emerging diseases 64, 1393–1404. 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1974) or institutions where they were held captive. parasites are known to influence fitness and, more recently, have also been shown to influence behaviour (zimmer 2000). the endoparasitic taxa carried by each individual in the pride are influenced not only by how pride members associate, but also by the general health of the individual, the size of the parasite suprapopulation as well as ecological factors. here we report on the endo parasites of a small pride of lions in the southwestern kalahari, namibia, as determined by opportunistic collection of faeces during field ob servations. materials and methods research subjects a small pride of lions on the western fringe of the kalahari desert in southeastern namibia was studied. the pride was held in a 500 ha enclosure on a private reserve, intu afrika kalahari game reserve (24°33’ s, 18°31’ e), situated approximately 60 km 71 onderstepoort journal of veterinary research, 73:71–75 (2006) research communication faecal helminth egg and oocyst counts of a small population of african lions (panthera leo) in the southwestern kalahari, namibia y. smith* and o.b. kok department of zoology and entomology, university of the free state, p.o. box 339 bloemfontein, 9300 south africa abstract smith, y. & kok, o.b. 2006. faecal helminth egg and oocyst counts of a small population of african lions (panthera leo) in the southwestern kalahari, namibia. onderstepoort journal of veterinary research, 73:71–75 an endoparasite survey of a small pride of african lions (panthera leo) was conducted at intu afrika kalahari game reserve, southwestern namibia, during winter and summer of 2003 and 2004, respectively. overall, 23 fresh lion scats were collected opportunistically during fieldwork trials. a flotation technique was employed for the diagnosis of parasites. three nematodes, ancylostoma braziliense, gnathostoma spinigerum and uncinaria stenocephala and two coccidians, toxoplasma gondii and isospora felis were recorded. by using the mcmaster method for quantification, a maximum number of 14 866 oocysts per gram of faeces was obtained for i. felis during winter 2003. endoparasite taxa carried by the different individuals in the pride were found to be related to their levels of association. rates of infection were relatively low as a result of the habitat, semi-captive conditions and earlier sporadic deworming. keywords: coccidians, endoparasites, kalahari desert, nematodes, panthera leo ∗ author to whom correspondence is to be directed. e-mail: liongirl@mweb.com.na accepted for publication 1 august 2005—editor 72 faecal helminth egg and oocyst counts of african lions (panthera leo) in namibia north-east of mariental. the pride consists of two adult males, an adult female and two cubs. the younger male and female are siblings and are closely bonded. the pride was being rehabilitated and was provided with a live animal once a week. fieldwork trials on aspects of their behaviour were performed during winter (may to july 2003) and again during summer (february and march 2004). the cubs were born during november 2003 and were included in the summer trials. faecal collection and preservation intestinal parasite surveys were conducted by opportunistic collection of scats during field observations. a total of 23 fresh samples were collected from the adult lions during both summer and winter. during the summer, six samples of scat from the cubs were also obtained, four from the male and two from the female. nematode eggs, cestode eggs and proglottids, and oocysts in the faeces were preserved in 10 % formalin in 750 ml glass bottles within 12 h after collection. a standard operating procedure (sop cvi/07/02/014 faecal examination: diagnostic and quantitative) obtained from clinvet international (pty) limited was employed. eggs of taenia spp. could be identified by using a flotation technique and expelled proglottids were located by washing over a sieve with 0.5 mm apertures. the resulting residues were suspended in a small amount of water and examined under a stereoscopic microscope for the presence of proglottids. a quantitative parasite egg and coccidian oocyst count was done according to the modified mcmaster method described by reinecke (1983). results the eggs of three nematodes, ancylostoma braziliense, gnathostoma spinigerum and uncinaria stenocephala, and the oocysts of two coccidians, toxoplasma gondii and isospora felis, were recovered and identified following soulsby (1982) and the experience of the laboratory staff of clinvet international. all these species, excluding uncinaria, are listed by boomker, penzhorn & horak (1997) as typical helminth and coccidian parasites of african lions. table 1 indicates the seasonal occurrence of the parasite species recovered. intact and fragmented nematodes were also found in the scat during the summer trial. they were present in each flo ta tion performed. a total of six samples, three from each season, were negative for any eggs, oocycts or worms when quantitative counts were done with the mcmaster method. all of these were collected from the adult lions. as shown in table 2, the intensity of eggs and oocysts of the endoparasites decreased during the subsequent summer trial except for the hookworm, a. braziliense, which increased. results differed during the flotation and the mcmaster slide investigations. two species of parasite, a. braziliense and t. gondii, were present in both the male lions. flotation results for the older male indicated a. braziliense and the mcmaster slide results showed only the presence of t. gondii. the younger male’s flotation results indicated only t. gondii, while the mcmaster slide demonstrated the presence of a. braziliense. the female had the highest egg counts in both the flotation and the mcmaster slide counts. flotation showed that the female harboured the oocysts of i. felis, but they were not present in the mcmaster slide. results for the female cub’s flotation included a. braziliense and i. felis, whereas the former and t. gondii were present in the mcmaster slide. in the case of the male cub, flotation indicated the presence of a. braziliense, t. gondii and g. spinigerum, while the mcmaster slide showed only t. gondii. the female cub had a higher egg and oocyst count than the male cub. uncinaria stenocephala was not present during the summer trials. discussion egg and oocyst counts during both the winter and summer trials indicate low rates of infection. research in the serengeti and ngorongoro crater (müller-graf 1995) indicated that median values per gram faeces may reach up to 5 700 for coelozoic helminths with no apparent ill-effects to the host. with the exception of a. braziliense, all egg counts decreased from the winter to the summer trials (table 2). the scope of the increase of the former is reduced by the increase in sample size since two cubs were included in the summer trials. the results, however, may be attributable to the high egg production of a. braziliense which permits a higher fecundity (hinz 1988). schmidt & roberts (1985) state that some 25 000– 30 000 eggs may be produced per day by a female in the gastrointestinal tract. in respect of the histozoic coccidians and specifically i. felis, oocyst counts subside after initial infection which explains the substantial decrease in oocyst count from winter to summer. cysts persist in the tissue, egg production all but ceases and immunity responses after the first infection is generally permanent (schmidt & roberts 73 y. smith & o.b. kok 1985). the high oocyst count for i. felis during the winter trials indicates a new infection for the female. further to the former, the pride was dewormed one year prior to the inception of the fieldwork and the animals are fed, albeit live animals reducing exposure to some of the nematodes. the specific habitat experienced drought conditions for two years before the research and this would have assisted in reduction of viable parasite eggs in the environment. the discrepancies between endoparasite presence in the flotation and mcmaster slide can be explained in terms of the opportunistic nature of the scat collection and the random dispersion of endoparasite eggs and oocysts in faecal matter. when calculated in total, the female had the highest egg and oocyst counts using both the flotation and mcmaster slide methods. schmidt & roberts (1985) call this phenomenon overdispersion and state that parasite infrapopulations are not randomly dispersed, but that a minority of the hosts will harbour a majority of the parasites. a relative large number of intact and fragmented nematodes were found in the scat samples. in the case of both species of hookworms present, the eggs, after voiding, require warm and moist conditions to hatch into l1 or rhabditiform larvae (schmidt & roberts 1985). the majority of scats collected were in the form of diarrhoeic faeces. the lions were fed sporadically during the summer trials and engorged themselves when large ungulates were killed. the diarrhoeic faeces had to be left for a day or two to permit drying prior to being collected. during this time, some of the nematode eggs could have hatched. alternatively, nematode eggs also hatch in warm, moist soil and as a result of the rains some of the hatched larvae in the scat may also have been present in the environment (schmidt & roberts 1985). table 1 endoparasite suprapopulation in african lions during two contrasting periods of study at intu afrika kalahari game reserve a. braziliense g. spinigerum u. stenocephala t. gondii i. felis winter 2003 older male female younger male summer 2003 older male female younger male male cub female cub table 2 egg and oocyst counts of endoparasites in the pride of african lions at intu afrika kalahari game reserve sample size infected samples infected hosts mean intensity maximum intensity winter 2003 a. braziliense g. spinigerum t. gondii i. felis 11 11 11 11 4 1 4 7 3 1 2 3 133 666 233 3 320 200 666 400 14 866 summer 2004 a. braziliense g. spinigerum t. gondii i. felis 17 17 17 17 4 1 7 1 3 1 4 1 173 66 173 66 600 66 200 66 74 faecal helminth egg and oocyst counts of african lions (panthera leo) in namibia all of the helminth endoparasites found are transmitted either via the environment or by transplacental transmission. according to noble & noble (1988) congenital transmission of hookworms has been demonstrated in domestic dogs, but not in domestic cats. however, this has not been investigated in lions and remains to be proved or disproved. the infection of the female cub with i. felis was most likely due to contact with the female. curio (1988) reported that faecal egg counts matter correlate positively with dominance rank in the yellow baboon (papio cynocephalus). the data collected on the kalahari lion in this investigation does not support this. although feeding habits of the older male should support greater exposure to parasites, especially t. gondii which has the herbivore as an intermediate host, behaviour may play a more prominent role in terms of the parasite burden. the older male spent most of his time alone and did not allogroom often and, as a result, his exposure to endoparasite eggs and oocysts from other members of the pride was reduced. the younger male had the lowest egg and oocyst counts, which is contradictory to published data. the circumstances in which the lions were held were, how ever, unnatural and under more natural conditions the younger male would have left the pride for a nomadic lifestyle. all the behaviour of the younger male indicated that he was under considerable social pressure. dominance from the older male was constant and turned violent as the younger male grew older and began to resist. when an animal is under stress, adrenal glands become hyperactive and corticosteroids are released (h. bertschinger, personal communication 2004). this impacts negatively on the animal in question and reduces its resistance to parasites (esch, whitfield gibbons & bourque 1975). wilson (1976) makes mention of what hans seyle called the general adaptation syn drome in 1956, in which there is a sequence of three stages, namely the alarm stage when adrenal corticotropical hormones are released, a stage of resistance in which the adrenal glands enlarge due to a continuous increased demand for corticosteroids, and finally, a stage of exhaustion is reached when the body is not able to withstand the increased corticosteroid levels which increases the chances of infection. aggressive interactions are named as the most potent of stressors. exposure to infection in the younger male was also increased by the protracted periods spent with the female and later, the cubs. the positive response determined in the younger male in terms of resistance to the parasites could be as a result of his response to the pressure. in all our observations of the animals’ feeding, the younger male consumed the largest amounts of meat (on one occasion he fed for 4 h and 22 min) with an average consumption of 50 kg, and also drank water for 8.39 min, the longest time recorded. although he was not weighed, he was obviously the most overweight of the lions and, as a result, he may have had the physiological resources to resist parasite infections. the relatively low egg and oocyst counts and corresponding infection levels of the lion pride are as a result of a variety of factors. firstly, the habitat was clean and parasites present in the environment would have been reduced by the two years of drought and the sporadic deworming prior to the inception of this study. isolation of the lions from the greater reserve may also have been responsible for the fewer taxa present. finally, the relative safety and lack of natural pressure on the animals would have permitted a well-functioning immune system allowing for a strong defense response to endoparasites. acknowledgements we thank mr howard hebbard for permission to conduct research at intu afrika kalahari game reserve, the national research foundation of south africa and the business community in namibia, notably siemens, engen, proforce, sirkel motors and the windhoek observer, for generous financial contributions towards the project, and the staff of clinvet international for assistance with the laboratory work and the identification of parasites. references bjork, k.e., averbeck, g.a. & stromberg, b.e. 2000. parasite and parasite stages of free-ranging wild lions (panthera leo) of northern tanzania. journal of zoo and wild life medicine, 31:56–61. boomker, j., penzhorn, b.l. & horak, i.g. 1997. parasites of lions (panthera leo) and leopards (panthera pardus): a doc umentation. proceedings of a symposium on lions and leopards as game ranch animals, onderstepoort, pre toria: 131–142. curio, e. 1988. behaviour and parasitism, in parasitology in focus, edited by h. melhorn. berlin: springer-verlag. esch, g.w., whitfield gibbons, j. & bourque, j.e. 1975. an analysis of the relationship between stress and parasitism. the american midland naturalist, 93:339–353. hinz, e. 1988. geomedical aspects of parasitology, in para sitology in focus, edited by h. melhorn. berlin: springer-verlag. 75 y. smith & o.b. kok le roux, p.l. 1958. pharyngostomum cordatum (dies, 1850), galoncus perniciosus (v. linstow, 1885) and gnathostomum spinigerum (owen, 1836) infections in a lion in northern rhodesia. transactions of the royal society of tropical med icine and hygiene, 52:14. müller-graf, c.d.m. 1995. a coprological survey of intestinal parasites of wild lions (panthera leo) in the serengeti and ngorongoro crater, tanzania, east africa. journal of parasitology, 81:812–814. müller-graf, c.d.m., woolhouse, m.e.j. & packer, c. 1999. epidemiology of an intestinal parasite (spirometra spp.) in two populations of african lions (panthera leo). parasitology, 118:407–415. noble, e.r. & noble, g.a. 1988. parasitology: the biology of animal parasites. santa barbara: febiger. reinecke, r.k. 1983. veterinary heminthology. durban: butterworths. rodgers, w.a. 1974. weights, measurements and parasitic infestation of six lions in southern tanzania. east african wildlife journal, 12:157–158. schmidt, g.d. & roberts, l.s. 1985. foundations of parasitology. st. louis: mosby college publishing. soulsby, e.j.l. 1982. helminth, arthropods and protozoa of domestic animals. philadelphia: lea & febiger. wilson, e.o. 1976. sociobiology: the new synthesis. cambridge: harvard university press. zimmer, c. 2000. parasite rex. london: arrow books. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice hay_249-253.indd introduction six species of the plant family rubiaceae, viz. pachystigma latifolium, pachystigma pygmaeum, pachystigma thamnus, pavetta harborii, pavetta schumanniana and fagodia homblei cause gou siekte, a syndrome characterized by heart failure and sudden death, when ingested by ruminants (theiler, du toit & mitchell 1923; pretorius & terblanche 1967; kellerman, coetzer, naudé & botha 2005). a number of cardiodynamic changes (pretorius & terblanche 1967) as well as myocardial lesions that consist of a loss of myofilaments, replacement of myocytes with collagenous tissue, lengthening of sarcomeres and cardiac dilatation (newsholme & coetzer 1984) have been reported. apart from the economic impact on farming with domestic ruminants the plant has attracted the attention of researchers for its possible use as a model for studying heart failure. one model that was successfully used in sheep was that produced by addition of dried pachystigma pygmaeum leaves to a normal diet (schutte & du plooy 1990). a later study also succeeded in inducing heart failure in rats by injecting crude extracts of pavetta harborii intraperitoneally (hay, pipedi, schutte, turner & smith 2001). administration of dried leaves or crude extracts to induce heart failure can, however, have limitations because of seasonal variations in plant toxicity resulting in variations in the degree of heart failure that develops. theiler et al. (1923) found that factors such as soil type and seasonal climate changes caused variations in the toxicity of pachystigma pygmaeum (= vangueria pygmaea) 249 onderstepoort journal of veterinary research, 75:249–253 (2008) cardiotoxic effects of pavetamine extracted from pavetta harborii in the rat l. hay1*, r.a. schultz2 and p.j. schutte1 abstract hay, l., schultz, r.a. & schutte p.j. 2008. cardiotoxic effects of pavetamine extracted from pavetta harborii in the rat. onderstepoort journal of veterinary research, 75:249–253 previous studies have shown that crude extracts from pavetta harborii as well as dried plant material have cardiotoxic effects on rats and sheep that can lead to heart failure. the active component has since been isolated and identified. this substance has been named pavetamine. the aim of this study was to determine whether pavetamine has cardiotoxic effects similar to those seen in previous reports, when administered to rats intraperitoneally. sprague dawley rats received two doses, initially 4 mg/kg and then 3 mg/kg pavetamine respectively and were monitored for 35 days before cardiodynamic parameters were measured by inserting a fluid-filled catheter into the left ventricle via the right carotid artery. these values were compared to those of control rats that had received only saline. pavetamine significantly reduced systolic function and body mass in the treated rats, which indicates that it has the potential to induce heart failure in this animal model. keywords: cardiodynamics, cardiotoxic, pavetamine, pavetta harborii, rat * author to whom correspondence is to be directed. e-mail: lhay@ul.ac.za 1 department of physiology, university of limpopo, medunsa campus, p.o. box 130, medunsa, 0204 south africa 2 section of toxicology, private bag x05, arc-onderstepoort veterinary institute, onderste poort, 0110 south africa accepted for publication 17 june 2008—editor 250 cardiotoxic effects of pavetamine extracted from pavetta harborii in the rat and it should therefore be considered that this might also be the case for other species of this family. fourie, erasmus, schultz & prozesky (1995) succeeded in isolating and describing the active compound which has become known as pavetamine. it be came available in small quantities for further investigation. subsequent investigations have shown that pavetamine inhibits protein synthesis in the cardiac muscle of rats but that it has no effects on other tissues (schultz, fourie, basson, labuschagne & prozesky 2001). the aim of this study was to investigate in rats whether pavetamine has cardiodynamic effects typical to those described previously for the dried material of pachystigma pygmaeum and crude extracts of pavetta harborii given to sheep and rats respectively. methods experimental animals the investigation conforms to the guide for the care and use of laboratory animals (nih publication no. 85-23, revised 1996). ethics approval was obtained from the animal ethics committee of the arconder stepoort veterinary institute. healthy, young, male sprague dawley rats (n = 20) of the same age were used and equally divided into a control and a treated group. they were kept individually in separate cages and had free access to water and nutritionally balanced rat cubes (epol pty. ltd. sa). pavetamine from dried p. harborii plant material was extracted by the method described by fourie et al. (1995). the rats in the treated group each received an intraperitoneal injection of 4 mg/ kg pavetamine in 0.5 mℓ saline on day 1 and a second injection of 3 mg/kg in 0.5 mℓ saline on day 20. the control rats only received similar quantities of normal saline administered by the same route. cardiodynamic data were recorded for the individual experimental animals on 1 day from days 35–39. these data were recorded for the individual rats over a period of 5 days as time constraints on the procedure limited the number of animals that could be done in 1 day. the animals were anaesthetized by administering an intramuscular injection of 0.5 mℓ of a mixture of ketamine (100 mg/mℓ) (kyron laboratories pty ltd. johannesburg, sa) and xylazine (2 %) (premier pharmaceuticals co ltd. johannesburg, sa) at a ratio of 1:3. the anaesthesia was maintained through out the procedure by further 0.1 mℓ injections at 20 min intervals or as needed. a constant anaesthetic plane was continually assessed by means of the tarsal pinch reflex. the following surgical procedure and cardiodynamic monitoring on each rat were performed in a sterile environment on an operation table prewarmed to maintain the body temperature of the animals. each rat was placed on its back and its right carotid artery surgically exposed and separated from the accompanying vagus nerve. care was taken not to damage the nerve. a catheter (cordis 2.5 f) was inserted into the left ventricle of the heart via the exposed artery. during this procedure care was taken to minimize blood loss. the catheter was connected to a fluidfilled hewlett-packard quartz transducer which was connected to a hewlett-packard 8 channel recording system (hp7758) and the data obtained were stored on a magnetic tape for later analysis. the pressure recordings were monitored on an oscilloscope to confirm that the catheter was in the left ventricle. the following indices were monitored and computed: peak left ventricular pressure (lvsp), left ventricular end-diastolic pressure (lvedp), heart rate (hr), the maximum rate of increase of left ventricular pressure (+lvdp/dtmax) indicating left ventricular (lv) contractility, the time constant t calculated from the lv pressure curve as the negative inverse of the slope of the regression line of the lv pressure versus lv -dp/dt with a variable pressure asymptote, which gives an indication of the rate of the left ventricular isovolumic relaxation (weisfeldt, weis, frederiksen & yin 1980), and the cardiac work (cw) that was calculated by multiplying hr and sbp. directly after the last recordings the rats received a lethal dose (500 mg/kg) of sodium pentobarbitone (kyron laboratories (pty) ltd). statistical analysis was done with gen stat (2000) and the student t-test was used to compare the treated to the non-treated groups taking p < 0.05 as significant. results fig. 1 shows that the rats receiving pavetamine had slower mass gains than those of control rats. after the second administration of pavetamine the treated rats showed no further mass gains up to the termination of the experiment. table 1 summarizes the effects of pavetamine on cardiac function. both t and lvedp were not signif251 l. hay, r.a. schultz & p.j. schutte icantly affected by the pavetamine intervention. the other parameters that were measured were all significantly reduced. the reduction in dp/dtmax and cw were identical at 30.9 % but since these two parameters are affected by different factors, this similarity is probably purely coincidental. discussion it is not clear how the pavetamine caused a decrease in the mass gain of the treated group. previous studies did not reveal possible effects of these plant toxins on the mass of treated animals. in a study investigating the affect of pavetamine on protein synthesis in the different tissues of rats it was shown to have a detrimental effect on cardiac tissue but other tissues were not affected despite a loss in their mass (2 % within the first 24 h) (schultz et al. 2001). in another study where pavetta harborii extracts were administered to rats, protein synthesis in cardiac tissue was compromised to the extent that it lead to a degeneration in the myofilaments (hay et al. 2001). a similar degeneration of myocardial fibres was also seen in sheep to which dried pachystigma pygmaeum leaves had been administered (schutte, els, booyens & pienaar 1984). these pathological changes in the cardiac tissue could be the underlying cause of the reduced cardiodynamic function (systolic and diastolic) associated with the disease as described by hay et al. (2001) and by schutte & du plooy (1990). when a minimum critical dosage of the plant material is administered to animals and if enough time is allowed for the disease to progress fully, it will eventually reach a stage where typical clinical signs of congestive heart failure will manifest. these signs were described in detail by pre torius & terblanche (1967). our results show that the pavetamine significantly reduced the systolic component of the cardiodynamic function of the rats but did not affect the diastolic table 1 the cardiodynamic parameters of control rats and rats receiving pavetamine control pavetamine %δ p≤ t(ms) lvedp (mmhg) dp/dtmax (mmhg.s –1) cw (mm hg.beats.min–1) lvsp (mmhg) hr (beats.min–1) 52.11 ± 1.9 6.9 ± 1.3 2 746 ± 105 40 570 ± 1 799 158.4 ± 7.8 258 ± 11 52.10 ± 1.6 6.4 ± 1.4 1897 ± 107 28 010 ± 2 386 119.5 ± 6.8 231 ± 7 0 –7.2 –30.9 –30.9 –24.5 –10.5 ns ns 0.001 0.001 0.002 0.05 see text for meanings of abbreviations 0 50 100 150 200 250 300 350 400 450 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 time (days) m a s s ( g ra m ) control rats: a = 0.5 mℓ saline; b = 0.5 mℓ saline a b pavetamine rats: a = 4 mg/kg; b = 3 mg/kg fig. 1 the effects of pavetamine administration on rat body mass over a period of 39 days 252 cardiotoxic effects of pavetamine extracted from pavetta harborii in the rat component measured from t, nor did it influence the lvedp. this is in contrast to the significant length ening in t that was found in rats to which crude extracts of pavetta harborii had been administered (hay et al. 2001). a possible explanation for this could be that the concentration of the pavetamine used in this study was too low or that the time period over which the toxin was administered was too short, or both, to cause these additional changes associated with heart failure, namely a high lvedp and a reduced diastolic function. all the indicators of systolic function in our study were significantly reduced but the heart rate did not increase significantly, suggesting that the clinical signs usually associated with advanced congestive heart failure (pretorius & terblance 1967) had not yet fully developed. both cw and dp/dtmax were significantly decreased and the substantial reduction in lvsp implies that the reduction in dp/dtmax was sufficient to alter cardiac effectiveness. it is known that altered loading conditions of the heart can affect an index such as dp/dtmax (broughton & korner 1980). in our study only the afterload was significantly altered and this would therefore suggest that a slight overestimation of dp/dtmax would have occurred in the treated group. this fact combined with the lower lvsp would therefore suggest that this factor should not detract from our observations on dp/dtmax. the hr was significantly reduced in the treated rats and this was unexpected in the light of a diminished contractile force. similar observations were made in rats receiving crude extracts (hay et al. 2001). however, in goats administered pavetamine resulted in tachycardia and ecg changes (fourie et al. 1995). likewise, in the sheep studies heart rate was initially not affected but later arrhythmias appeared and in the final stages of congestive heart failure tachycardia occurred (pretorius & terblanche 1967). various changes in the ecg were described by pretorius & terblanche (1967). this would suggest that the toxin contained in the dried plant material affected the electrophysiology of the pacemaker cells. the authors of that paper suggested that ischaemia could be responsible for the observed ecg changes (pretorius & terblanche 1967). t-wave inversion was a common occurrence in sheep that received the dried plant material of pachystigma pygmaeum (pretorius & terblanche 1967; pretorius et al. 1973). t-wave inversion is also a well known phenomenon associated with hypokalemia (ganong 2001) and since electrophysiological affects could also involve potassium ions this aspect should be further investigated. therefore a possible effect of the toxin on the ions and the associated affect it might have on the membranes of the pacemaker cells should not be disregarded when the hr observations are considered. as mentioned before pretorius et al. (1973) confirmed ca2+ abnormalities in myocardial cells exposed to this toxin. ca2+ abnormalities can technically also impact on the cell membranes of damaged myocardial cells via complex interactions in the ca2+/na exchange system. this has been confirmed to take place in cases where myocardial cells were damaged in other ways than by this toxin (pieske 1998). in conclusion, the results suggest that pavetamine administered to rats diminishes their systolic function, but the effects on diastolic function and hr suggest that the rats were not in the advanced stages of congestive heart failure associated with a high lvedp and compensatory tachycardia. further dose response studies need to be done to determine the dose of pavetamine required to induce advanced congestive heart failure similar to the clinical picture described in sheep (pretorius & terblanche 1967; kellerman et al. 2005). acknowledgements our special thanks to mr o.h.i. majane (ul), ms l. la buschagne, ms k.m. basson (arc-ovi) and ms m.f. smith (arc-biometry unit) for their valuable contributions during this project. funding was provided by the gauteng province (department of agriculture, conservation and environment) and the north-west province (department of agriculture, conservation, environment and tourism). references broughton, a. & korner, p.i. 1980. steady-state effects of preload and afterload on isovolumic indices of contractility in autonomically blocked dogs. cardiovascular research, 14: 245–253. fourie, n., erasmus, g.l., schultz, r.a. & prozesky, l. 1995. isolation of toxin responsible for gousiekte, a plantinduced cardiomyopathy of ruminants in southern africa. onderstepoort journal of veterinary research, 62:77–87. ganong, w.f. 2001. review of medical physiology, 20th ed. new york: lange medical books/mc graw-hill. hay, l., pipedi, m., schutte, p.j, turner, m.l. & smith, k.a. 2001. the effect of pavetta harborii extracts on cardiac function in rats. south african journal of science, 97:481– 484. 253 l. hay, r.a. schultz & p.j. schutte kellerman, t.s., coetzer, j.a.w., naudé, t.w. & botha, c.j. 2005. plant poisonings and mycotoxicoses of livestock in southern africa, 2nd ed. cape town: oxford university press. newsholme, s.j. & coetzer, j.a.w. 1984. myocardial pathology of ruminants in southern africa. journal of the south african veterinary association, 55:89–96. pieske, b. 1998. new aspects of the pathophysiology of heart failure. wiener medizinische wochenschrift, 148:108–20. pretorius, p.j. & terblanche, m. 1967. a preliminary study on the symptomatology and cardiodynamics of gousiekte in sheep and goats. journal of the south african veterinary med ical association, 38:29–53. pretorius, p.j., terblanche, m., van der walt, j.d. & van ryssen, j.c.j. 1973. cardiac failure in ruminants caused by gousiekte, in cardiomyopathies. vol. 2, edited by e. bajusz & g. rona. baltimore: university park press. schultz, r.a., fourie, n., basson, k.m., labuschagne, l., prozesky, l. 2001. effect of pavetamine on protein synthesis in rat tissue. onderstepoort journal of veterinary research, 68:325–330. schutte, p.j., els, h.j., booyens, j. & pienaar, j.g. 1984. ultrastructure of myocardial cells in sheep with congestive heart failure induced by pachystigma pygmaeum. south african journal of science, 80:378–380. schutte, p.j. & du plooy, w.j. 1990. cardiodynamic improvement following pge1 administration in sheep with congestive heart failure. prostaglandins leukotrienes and essential fatty acids, 41:45–49. theiler, a., du toit, p.j. & mitchell, d.t. 1923. gousiekte in sheep. reports of the director of veterinary education and research, union of south africa, 9 and 10. weisfeldt, m.l., weis, j.l., frederiksen, j.t. & yin, f.c.p. 1980. quantification of incomplete left ventricular relaxation: relationship to the time constant for isovolumic pressure fall. european heart journal, 1:119–129. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) gerda fouche chemistry department, faculty of natural and agricultural sciences, university of pretoria, pretoria, south africa olubukola t. adenubi phytomedicine programme, department of paraclinical sciences, university of pretoria, onderstepoort, south africa tlabo leboho council for scientific and industrial research (csir) biosciences, pretoria, south africa lyndy j. mcgaw phytomedicine programme, department of paraclinical sciences, university of pretoria, onderstepoort, south africa vinny naidoo biomedical research centre, faculty veterinary science, university of pretoria, onderstepoort, south africa kevin w. wellington council for scientific and industrial research (csir) biosciences, pretoria, south africa jacobus n. eloff phytomedicine programme, department of paraclinical sciences, university of pretoria, onderstepoort, south africa citation fouche, g., adenubi, o.t., leboho, t., mcgaw, l.j., naidoo, v., wellington, k.w. et al., 2019, ‘acaricidal activity of the aqueous and hydroethanolic extracts of 15 south african plants against rhipicephalus turanicus and their toxicity on human liver and kidney cells’, onderstepoort journal of veterinary research 86(1), a1665. https://doi.org/10.4102/ojvr.v86i1.1665 original research acaricidal activity of the aqueous and hydroethanolic extracts of 15 south african plants against rhipicephalus turanicus and their toxicity on human liver and kidney cells gerda fouche, olubukola t. adenubi, tlabo leboho, lyndy j. mcgaw, vinny naidoo, kevin w. wellington, jacobus n. eloff received: 25 june 2018; accepted: 20 feb. 2019; published: 22 july 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract hot water and hydroethanolic (70:30) extracts were prepared from 15 plant species, which were investigated to discover eco-friendly and less expensive tick control methods as an alternative to synthetic acaricides. a contact bioassay was used to determine the acaricidal activity of these extracts against the cattle tick, rhipicephalus turanicus (acari: ixodidae) at a concentration of 20% (200 mg/ml). the hydroethanolic extracts had better activity than the hot water extracts against r. turanicus. the hydroethanolic extract from tabernaemontana elegans (leaves) had the best mortality (87.0%). this was followed by calpurnia aurea (stems) with a mortality of 75.0%, schkuhria pinnata (whole plant) with a mortality of 67.0% and aloe rupestris (leaves) with a mortality of 66.6%. the toxicity of the plant extracts was also investigated and it was found that most of the hydroethanolic and hot water extracts were either safe or very safe on human vero kidney and liver hepg2 cells. from this study, it was evident that botanicals have the potential to be developed as environmentally benign natural acaricides against r. turanicus. keywords: rhipicephalus turanicus; contact bioassay; acaricidal activity; vero cells; hepg2 cells; toxicity; water; ethanol. introduction rhipicephalus turanicus is a tick species found in several mediterranean countries (mumcuoglu et al. 1993) as well as in africa, asia and europe (li et al. 2017). ticks are vectors of various pathogens such as bacteria, viruses and protozoa (boldbaatar et al. 2006; kocan, blouin & de la fuente 2011). r. turanicus is known to be a vector of rickettsial diseases such as q-fever and north asian tick typhus that is caused by rickettsia sibirica (berdyev 1980). this tick species parasitises a wide range of hosts including humans and dogs. when heavy tick infestations occur in livestock, huge economic losses result through anorexia, blood loss, decrease in productivity, depression of immune function, damage to hides, general stress and irritation, toxicosis, transmission of pathogens, treatment costs and also death (elango & rahuman 2011; ghosh, azhahianambi & yadav 2007; tian et al. 2011). the global annual economic loss due to ticks and tick-borne diseases equates to around $7 billion (bagavan et al. 2009; zahir et al. 2010). current tick control strategies aim to reduce tick numbers to acceptable levels, reduce chemical residue risks, prevent production loss and reduce the dependence on chemical acaricides by exploiting different control treatments for different herd groups (ghosh et al. 2007). tick control has been accomplished by the use of chemical acaricides, which are effective when properly applied. the use of chemical acaricides has, however, resulted in environmental contamination, contamination of milk and meat products with insecticide residues and the development of acaricide-resistant ticks (graf et al. 2004). the high cost of developing new chemical acaricides and the lack of suitable immunising agents have led to a renewed interest in the use of botanicals as an alternative for the control of cattle ticks (madzimure et al. 2011; zaman et al. 2012). botanicals have been perceived to be reasonably safe with fewer risks to the environment and also marginal impact on both human and animal health (isman 2008; panella et al. 2005; su & mulla 1998). when compared with chemical acaricides, herbal extracts have several advantages, for example, they are biodegradable, do not accumulate and pollute the environment, they are less toxic to the environment and to non-targeted species, and are also less likely to have resistance developed against them (liang et al. 2003). there have been several reports by researchers obtaining promising results in controlling ticks by using botanicals (cetin et al. 2009; coskun et al. 2008; koc et al. 2013). after many years of displaying promise, botanicals have acquired a foothold in several markets and are finally attaining a standing with the agrochemical industry (isman 2015). in 2011, the environmental protection agency (epa) approved requiem®, an insecticide based on a terpenoid-rich extract of dysphania (= chenopodium) ambrosioides (l.) mosyakin and clemants (isman 2015). captiva®, an insecticide based on an extract of capsicum oleoresin and garlic oil, was approved by the epa in 2014 (isman 2015). in light of the above, a large number of plant extracts from several south african plants were investigated to identify novel botanicals that could be used as an alternative to synthetic acaricides against ticks. these plant species were based on a comprehensive literature survey and ethno-veterinary use by traditional communities to control ticks in livestock. in previous work, we reported on the acaricidal activity of organic extracts of south african plants against the larvae of both rhipicephalus decoloratus (koch 1844) (acari:ixodidae) (fouche et al. 2016a) and rhipicephalus (boophilus) microplus (wellington et al. 2016), and also on anthelmintic activity against haemonchus contortus (fouche et al. 2016b). furthermore, we also reported on the acaricidal activity against rhipicephalus turanicus (fouche et al. 2017). herein we report on the results of screening 36 extracts that were investigated for acaricidal activity against the tick species, r. turanicus and on their toxicity on human liver and kidney cells. materials and methods plant material collection fifteen plants (aloe rupestris baker, calpurnia aurea ssp. aurea (aiton) benth., cissus quadrangularis l., cleome gynandra l., clematis brachiata thumb., ficus sycomorus l., monsonia angustifolia e. mey. ex a. rich., pelargonium luridium (andrews) sweet, schkuhria pinnata (lam.) kuntze ex thell., sclerocarya birrea (a.rich.) hochst., senna italica subsp arachoides (burch.) lock and tabernaemontana elegans stapf.) were selected on the basis of available literature and ethno-veterinary usage in south africa. production of dried, ground plant material and preparation of the hot water and hydroethanolic extracts plant material was dried in an oven at 30 °c – 60 °c and ground to fine particles using a hammer mill. hot water and ethanol/water (30:70) extracts were prepared from the plants (table 1). the hydroethanolic extract was prepared at room temperature by adding the solvent mixture (350 ml) to the ground plant material (30 g), which was followed by stirring for 1 hour. after stirring, the contents were allowed to settle and then the solvent was removed. the residue was re-extracted by adding the same volume of solvent and stirring for 1 h again. for the third extraction the same volume of solvent was used but this time the mixture was stirred overnight (24 h). after the third extraction, all the extracts were combined and the ethanol was evaporated using a rotary evaporator. a freeze drier or genevac personal evaporator was used to remove water, and the extracts were then stored in a cold room at −20 °c. table 1: plant species, plant part used for the solvent extraction, plant family, the mass and percentage of extract obtained. hot water extracts were prepared by combining ground plant material (30 g) and hot water (350 ml). the mixture was stirred while boiling at 100 °c on a hot plate for 1 h and filtered with the aid of a hydraulic pump. the concentrated extract was lyophilised to remove traces of the solvent. ticks adult stages (both sexes) of r. turanicus (acari: ixodidae) were obtained from clinvet international, bloemfontein, south africa and kept at the phytomedicine laboratory, paraclinical sciences, faculty of veterinary sciences, university of pretoria. the ticks were housed in glass humidity chambers, closed by a removable cover at an average temperature of 25 °c ± 1 °c and relative humidity of 75% ± 10%, which was maintained by placing saturated sodium chloride solution in the glass chamber. the vials, in which the ticks were stored, were covered with cotton mesh (to allow normal air exchange) and set on a square glass plate placed at the base of the chamber on four small bearings so that the edges of the plate were at a distance of 1.5 cm from the walls. the saturated saline solution on the floor of the glass chamber also prevented the ticks from reaching the walls (zorloni, penzhorn & eloff 2010). determination of the acaricidal activity the contact bioassay described by zorloni et al. (2010) was employed. one microliter of a 20% (200 mg/ml) concentration of each extract was dropped on the dorsum of each tick (n = 10) for 1 minute before storing them in a vial covered with a perforated stopper. the same procedure was followed for the negative control (acetone or distilled water) and positive control (cypermethrin, 5 mg/ml). each treatment was performed in triplicate on each of three different occasions to yield nine replicates. the percentage mortality was determined 24 h after exposure by observing under a stereomicroscope (american optical corporation). the ticks were recorded as alive and active if they exhibited normal behaviour on exposure to carbon dioxide (co2) from human breath (host-associated stimulus) or after being physically stimulated with plastic tweezers, whereas they were confirmed dead based on signs of cuticle darkness, halted malpighian tubules movement and haemorrhagic skin lesions. those showing some difficulty in movement or maintaining a normal posture were termed weak or very weak if there was no leg coordination or a loss of a righting reflex (a reflex that corrects the orientation of the body when it is taken out of its normal upright position). the percentage mortality in all the experimental groups was corrected by applying abbott’s formula (abbott 1987): determination of the toxicity of the plant extracts viable cell growth after incubation of vero african green monkey kidney cells (atcc® ccl-81tm) and hepg2 human liver cancer cells (atcc® hb-8065tm) with test samples was determined using the tetrazolium-based (mtt) colorimetric assay to determine the cell viability (mcgaw, steenkamp & eloff 2007; mosmann 1983). for vero cells, minimum essential medium (mem) was used as a growth medium and hepg2 cells were cultured in dulbecco’s minimum essential medium (dmem). doxorubicin hydrochloride (pfizer) was used as a positive control. each plant extract was tested in quadruplicate and the assays were repeated three times (mcgaw et al. 2007; mosmann 1983). statistical analysis data on tick mortality and cytotoxicity were presented as the arithmetic mean values ± standard error of the mean (mean ± sem). significance was analysed using one-way analysis of variance followed by tukey’s multiple comparison test on graphpad prism 7.02 (graphpad software, san diego, ca). values were considered to differ statistically when p ≤ 0.05. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results extraction and yield the assayed plants represent 13 plant families with the capparidaceae and geraniaceae families being represented by two plant species. the leaves were the most common plant part used followed by the whole plant, stem, bark, root and fruit (table 1). the mass that was obtained for each of the extracts is shown in table 1. from 30 g ground plant material 3% – 4% yields were obtained. determination of the acaricidal activity the method used to determine the acaricidal activity of the plant extracts was the contact bioassay described by zorloni (2008). the plant extracts were exposed in vitro to the adults of r. turanicus for efficacy testing. the pyrethroid insecticide, cypermethrin, was used as a positive control. the hydroethanolic extracts had higher activity than the hot water extracts. t. elegans (leaves) had the best mortality (87.0%). this was followed by c. aurea (stems) with a mortality of 75.0%, s. pinnata (whole plant) with a mortality of 67% and a. rupestris (leaves) with a mortality of 66.6%, as shown in table 2. table 2: the mortality, corrected mortality and toxicity of the fifteen indigenous south african plant species screened against r. turanicus. the extract of c. aurea (leaves, flowers) had the best acaricidal activity (60%), which was followed by s. pinnata (whole plant) and g. deserticola (whole plant) each with an activity of 53%. poor activity (10% – 47%) was observed for the remainder of the hot water extracts as shown in table 2. determination of the toxicity of the plant extracts on vero cells a cut-off of 100 µg/ml was used for toxicity (nondo et al. 2015) and, therefore, many of the extracts of the plants had low toxicity on vero cells as the lc50 values were greater than 100 µg/ml. the lc50 values were compared with that of the positive control, doxorubicin (lc50 = 1.52 µg/ml), as shown in table 2. the hydroethanolic (lc50 = 49 µg/ml) and hot water (lc50 = 69 µg/ml) extracts of t. elegans were toxic to vero cells. toxicity on hepg2 cells only the hydroethanolic extract of m. angustifolia and t. elegans with lc50 values of 3.0 µg/ml and 71.0 µg/ml compared with the positive control, doxorubicin (lc50 = 0.34 µg/ml), which was toxic as shown in table 2. overall, for most of the plants, the lc50 values were greater than 1000 µg/ml and were therefore classified to be safe. comparative toxicity of hydroethanolic and hot water extracts on vero and hepg2 cells it was apparent that for many of the plants the hydroethanolic and hot water extracts were more toxic to the hepg2 cells than the vero cells (table 2). for h. rigidula, m. angustifolia, p. luridum, s. pinnata and s. italica, there was a significant statistical difference. discussion it is evident from the results in table 2 that the hydroethanolic extracts had better mortality against ticks than the hot water extracts. furthermore, most of the hydroethanolic and hot water extracts were either safe or very safe on vero and hepg2 cells. the best mortality (87%) was observed for the hydroethanolic extract of t. elegans (leaves), which was also toxic (lc50 < 100 µg/ml) to vero (lc50 = 49 ± 5 µg/ml) and hepg2 (lc50 = 71 ± 7 µg/ml) cells. phytochemical research has shown that t. elegans is rich in monoterpenoid indole alkaloids such as coronaridine, voacangine, hydroxycoronaridine, isovoacangine, 11-hydroxycoronaridine, voacristine, 19-epi-voacristine, isovoacristine, ibogamine, 10-methoxyibogamine, 11-methoxyibogamine and 19-epi-heyneanine (bombardelli et al. 1976; danieli et al. 1980; gabetta, martinelli & mustich 1975; kam & sim 2002; prakash chaturvedula et al. 2003; van der heijden, brouwer & verpoorte 1986). a range of biological activities have been reported for these indole alkaloids such as anticholinesterase, antihypertensive, antimicrobial and antimalarial properties, central nervous system-stimulating activity and particularly antitumor activity (andrade et al. 2005; de almeida et al. 2004; delorenzi et al. 2001; kam et al. 2004; medeiros et al. 2011; perera et al. 1985; prakash chaturvedula et al. 2003; van beek et al. 1984; vieira et al. 2008). β-carboline indole alkaloids have been isolated from the methanol leaf extract of t. elegans (mansoor et al. 2009). it is likely that alkaloids may also have been extracted by the hydroethanolic extract which may, therefore, be responsible for the acaricidal activity against r. turanicus. acaricidal activity was also noted for the hydroethanolic extract of calpurnia aurea (stems) and the hot water extract of c. aurea (leaves, flowers), which had mortality values of 75% and 60%, respectively. the latter was found to be very safe (lc50 > 100 µg/ml) against both vero and hepg2 cells. our results were in agreement with those of zorloni et al. (2010) who also reported acaricidal activity for this plant species but for acetone leaf extracts. in their report, 20% and 10% acetone leaf extracts of c. aurea either killed or severely compromised the mobility of unfed adults of rhipicephalus pulchellus ticks. the main chemical constituents of c. aurea are phenolic compounds (adedapo et al. 2008), which are said to be accountable for the attraction behaviour of over 12 species of ixodid ticks (mcdowell & wallade 1986; wood et al. 1975; yoder & stevens 2000). the efficacy of the extract of c. aurea (leaves) may be ascribed to its capacity to lure and also both kill or ruthlessly compromise the mobility of ticks. zorloni (2008) reported that c. aurea used to control ticks on animals in southern ethiopia had comparable acaricidal activities to the plant in south africa despite growing under widely diverse environmental conditions. the hydroethanolic extract of aloe rupestris (leaves) had the second best mortality (66.6%) but was found to be slightly toxic (lc50 = 100–500 µg/ml) against vero (lc50 = 153 ± 5 µg/ml) and hepg2 cells (lc50 = 496 ± 11 µg/ml). this acaricidal activity correlates with a report on that of another aloe species, aloe ferox, which has been used as a tick control remedy in some south african villages (moyo & masika 2009). aloe species have also been used in ethiopia to control ectoparasites (gemeda et al. 2014). it has been shown by phytochemical analyses that numerous aloe species contain diverse carbohydrate polymers (notably glucomannans) and an array of other low molecular weight phenolic compounds comprising alkaloids, anthraquinones, anthrones, benzene and furan derivatives, chromones, coumarins, flavonoids, phytosterols, pyrans and pyrones (cock 2015). 7-o-methylaloesin was isolated from the leaf exudate of a. rupestris (bisrat et al. 2000). the hydroethanolic extract of s. pinnata (whole plant) had a mortality of 67% against r. turanicus and was found to be slightly toxic (lc50 = 100–500 µg/ml) against both vero (lc50 = 460 ± 7 µg/ml) and hepg2 cells (lc50 = 116 ± 40 µg/ml). this plant and its varieties have been used as either insect repellents or insecticides mostly against fleas (heiser 1945). phytochemical investigations have afforded polyacetylenes (bohlmann & zdero 1977), diterpenes (bohlmann et al. 1980), sesquiterpene lactones (delgado, hernández & romo de vivar 1984; herz & govindan 1980; pérez et al. 1984; pettei et al. 1978) and phenylpropanoids (delgado & tejeda 1998). some of these compounds have biological activities (delgado et al. 1998). these compounds may, therefore, be responsible for the observed acaricidal activity against r. turanicus. both the hot water and hydroethanolic extract of g. deserticola (whole plant) had poor activity of 53% and 16.7%, respectively, against r. turanicus. the hot water extract was found to be very safe (lc50 > 1000 µg/ml) against both vero and hepg2 cells. it has been found in phytochemical investigations that the genus gnidia is abundant in chromones, coumarins, diterpene esters, flavonoids, lignans and neolignans (bhandurge et al. 2013). none of the extracts of senna italica subsp arachoides (roots, leaves, fruits) had efficacy against r. turanicus. acaricidal activity was reported for the root extract of s. italica subsp. arachoides against adults of hyalomma rufipes with the ethyl acetate extract being the most potent out of several extracts (hexane, chloroform, dichloromethane, ethyl acetate and methanol) tested (magano et al. 2008). for c. quadrangularis (stem), the hot water and hydroethanolic extracts had poor activity (22.2% and 13.8%, respectively) against r. turanicus. this is in contrast to what was reported by santhoshkumar et al. (2012) who found that the aqueous extract of c. quadrangularis (stem) had acaricidal activity against r. (b.) microplus. furthermore, the aqueous stem extract attached to silver nanoparticles were found to be more active against r. (b.) microplus than the free aqueous stem extract. similarly, the hot water and hydroethanolic extracts of c. gynandra (leaves) had poor activity (27.8% and 30.5%, respectively). malonza et al. (1992), however, reported on the acaricidal activity of the leaf extract of this plant but against nymphal and adult amblyomma variegatum and r. appendiculatus tick species. from the results of screening the extracts against vero and hepg2 cells (table 2) it is evident that none were as toxic as the positive control, doxorubicin (vero cells: lc50 = 1.52 µg/ml ± 0.32 µg/ml; hepg2: lc50 = 0.34 µg/ml ± 0.14 µg/ml). the use of ethno-veterinary leads for investigating plants for acaricidal activity against ticks has compounded the probability of discovering novel, natural acaricides capable of combatting r. turanius and also other tick species. botanicals with validated bioactivity and little to no toxicity, may have commercial value to the livestock industry. conclusion the hydroethanolic extracts of t. elegans (leaves), a. rupestris (leaves), c. aurea (stems) and s. pinnata have good to very good acaricidal activity against r. turanicus but are slightly toxic or toxic to vero and hepg2 cells. the hot water extracts of c. aurea (leaves and flowers), g. deserticola (whole plant), and s. pinnata (whole plant) have poor acaricidal activity (53% – 60%) but are safe or very safe on vero and hepg2 cells. from these results, it is evident that these plant extracts have potential as acaricidal agents against r. turanicus. compound isolation from these plant extracts could result in the discovery of novel natural acaricidal agents less toxic to non-targeted species and the environment. acknowledgements the authors thank the technology innovation agency (tia) of south africa for financial support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions g.f. conceptualised the study. g.f., k.w.w. and t.l. did the literature search and plant selection. t.l. prepared the plant extracts. j.n.e. conceptualised the study in a joint application and supervised the students and postdoctoral fellow. v.n. supervised determination of acaricidal activity. l.j.m.g. supervised the determination of cytotoxicity. o.t.a. determined the acaricidal activity against adult ticks of r. turanicus. k.w.w. wrote the first draft of the manuscript. funding information the technology innovation agency (tia) of south africa provided financial support to conduct this study. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references abbott, w., 1987, ‘a method of computing the effectiveness of an insecticide’, journal of the american mosquito control association 3, 302–303. adedapo, a.a., jimoh, f.o., koduru, s., afolayan, j.a. & masika, p., 2008, ‘antibacterial and antioxidant properties 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https://doi.org/10.1007/bf00988590 yoder, j.a. & stevens, w.b., 2000, ‘attraction of immature stages of the american dog tick (dermacentor variabilis) to 2,6-dichlorophenol’, experimental and applied acarology 24, 159–164. zahir, a.a., rahuman, a.a., bagavan, a., santhoshkumar, t., mohamed, r.r., kamaraj, c. et al., 2010, ‘evaluation of botanical extracts against haemaphysalis bispinosa neumann and hippobosca maculata leach’, parasitology research 107, 585–592. zaman, m.a., iqbal, z., abbas, r.z., khan, m.n., muhammad, g., younus, m. et al., 2012, ‘in vitro and in vivo acaricidal activity of a herbal extract’, veterinary parasitology 186, 431–436. zorloni, a., 2008, ‘evaluation of plants used for the control of animal ectoparasitoses insouthern ethiopia (oromiya and somali regions)’, m.sc. thesis, phytomedicine programme, university of pretoria. zorloni, a., penzhorn, b.l. & eloff, j.n., 2010, ‘extracts of calpurnia aurea leaves from southern ethiopia attract and immobilise or kill ticks’, veterinary parasitology 168, 160–164. vanniekerk.indd introduction despite the small size of most birds their capacity for rapid and maintained population growth can contribute significantly to the demography of those tick spe cies that use them as hosts. certain species daily fly considerable distances, and may alight in diverse habitats during such flights, thus enhancing their potential for the acquisition, transport and dissemination of ticks. furthermore, the seasonal migratory species cover considerable distances in a relatively short period of time and can therefore transport ticks from one country or even continent to another (hoogstraal, kaiser, traylor, gaber & guindy 1961). few of the migratory birds examined in egypt by hoogstraal et al. (1961) were infested, and those that were, harboured only small numbers of ticks. in con trast, it would appear as if a large proportion of migratory and non-migratory ground-frequenting birds in rural and wildlife regions of south africa are infested. the individual burdens of those species commonly referred to as game birds, namely guineafowls, spur fowls and francolins, may be large, often exceeding 100 and sometimes 1 000 immature ticks (horak, spickett, braack & williams 1991b; uys & horak 2005). in southern africa, the adults of four tick species, haemaphysalis hoodi, ixodes pterodromae, ixodes theilerae and ixodes uriae prefer birds as hosts (walker 1991). the adults of two other species, hyalomma marginatum rufipes and rhipicephalus 123 onderstepoort journal of veterinary research, 73:123–130 (2006) birds as hosts of immature ixodid ticks in free state province, south africa d.j. van niekerk, l.j. fourie and i.g. horak department of zoology and entomology, university of the free state p.o. box 339, bloemfontein, 9300 south africa abstract van niekerk, d.j., fourie, l.j. & horak, i.g. 2006. birds as hosts of immature ixodid ticks in free state province, south africa. onderstepoort journal of veterinary research, 73:123–130 the objective of this study was to determine the species spectrum of ixodid ticks infesting birds in free state province, south africa. to this end a large number of birds belonging to several species were examined for ticks and a total of 180 birds belonging to 39 species at 17 localities were infested, and ticks belonging to eight species were recovered. the immature stages of only two, namely amblyomma marmoreum and hyalomma marginatum rufipes, were sufficiently prevalent and numerous to safely assume that they regularly use birds as hosts. helmeted guineafowls, numida meleagris, were the most heavily infested and one harboured a total of 319 larvae and four nymphs. amongst the other species an eastern clapper lark, mirafra fasciolata, was infested with 69 larvae and a nymph, but no other bird harboured more than 40 ticks. the larvae and nymphs of h. m. rufipes were most numerous on birds from april to august keywords: amblyomma marmoreum, birds, hyalomma marginatum rufipes, ixodid ticks, seasonal occurrence accepted for publication 28 march 2006—editor 124 birds as hosts of immature ixodid ticks in free state province, south africa turan icus, although they usually infest mammals, may infest ostriches, and those of the latter tick, also the larger raptors (theiler 1962; norval 1982; walker, keirans & horak 2000). birds, however, serve as hosts of the immature stages of a number of tick species (theiler 1962). helmeted guineafowls, numida meleagris, are good hosts of the immature stages of amblyomma hebraeum, amblyomma marmoreum, haemaphysalis silacea, h. m. rufipes and hyalomma glabrum (at the time referred to as hyalomma marginatum turanicum) (horak & williams 1986; rechav, zeederberg & zeller 1987; horak et al. 1991b). crested francolins, dendroperdix sephaena, are good hosts of the immature stages of a. hebraeum, a. marmoreum and h. m. rufipes (uys & horak 2005), and cape francolins, pternistis capensis, are hosts of the immature stages of haemaphysalis aciculifer (horak & boomker 1998). birds of several other species may be infested with the larvae of a. marmoreum and the immature stages of h. m. rufipes and h. glabrum (h. m. turanicum) (norval 1975; norval 1982; horak & boomker 1998; horak, fourie, novellie & williams 1991a). because the adults of hyalomma spp. are often the cause of lameness in small domestic stock (kok & fourie 1995), and those of ixodes rubicundus and rhipicephalus warburtoni of toxin-induced paralysis in these animals (fourie, horak & marais 1988; fourie, petney, horak & de jager 1989), a number of surveys to determine the host spectrum of these ticks in free state province, south africa were conducted. several birds were examined during these surveys, while other birds were examined for ticks when ringed by one of us (djvn). this paper pre sents the findings of these investigations and discusses the role played by birds as hosts of the immature stages of ixodid ticks in south africa. materials and methods ticks were collected from birds at various localities in free state province, south africa (table1). the names of the birds and the sequence in which they are listed follow maclean (1993), and their common names are those recorded by hockey, dean & ryan (2005). larger birds were processed for tick recovery as described by horak & williams (1986) and smaller birds as described by horak et al. (1991a). when live birds were ringed the head, neck, body, wings and tail were carefully examined with the naked eye and all visible ticks collected. excluding the ticks from one of the helmeted guineafowls, which was particularly heavily infested, the mean monthly burdens of immature h. m. rufipes were calculated irrespective of the year in which the birds were examined. results and discussion the species and numbers of birds examined and the species and numbers of ticks they harboured are summarized in table 2. the immature stages of eight ixodid tick species were recovered from 180 birds belonging to 39 species. larvae and nymphs of h. m. rufipes were the most numerous and prevalent, followed by the larvae of a. marmoreum. a single helmeted guineafowl in the willem pretorius nature reserve harboured the largest infestation of both species, with 209 larvae and a nymph of a. marmoreum and 106 larvae and three nymphs of h. m. rufipes. in addition it was also infested with three larvae of rhipicephalus evertsi evertsi and a single larva of rhipicephalus gertrudae. with the exception of an eastern clapper lark, mirafra fasciolata, at glen agricultural college, which harboured 69 larvae and a nymph of h. m. rufipes, and four other birds that each harboured 20 or more larvae and nymphs, no bird was infested with more than 20 ticks. amblyomma marmoreum infestation with this tick was most prevalent on helmeted guineafowls in the willem pretorius nature reserve, and four of the six birds examined were infested. fifteen smaller birds belonging to 11 species were also examined in this reserve, but not one was infested. of the 170 birds, other than helmeted guineafowls, examined elsewhere in the province, 35 were infested and their individual burdens never exceeded five ticks. adult a. marmoreum, colloquially known as the south african tortoise tick, infests tortoises, and more particularly the leopard tortoise, geochelone pardalis as hosts (horak, mckay, heyne & spickett 2006). the immature stages infest tortoises, some of the larger reptiles, a large number of bird species, hares, antelopes, carnivores and domestic livestock (norval 1975; horak & fourie 1991; horak et al. 1991a; 2006; horak, braack, fourie & walker 2000). helmeted guineafowls appear to be good hosts of the larvae of this tick. in previous surveys several of them harboured more than 50 larvae with 585 being the largest number recovered. however, the number of nymphs rarely exceeded five (horak & williams 1986; horak et al. 1991b). thirteen of 16 helmeted guineafowls examined in an earlier study in the mountain zebra national park, eastern cape prov125 d.j. van niekerk, l.j. fourie & i.g. horak ince were infested, but not one of 27 smaller birds belonging to five other species (horak et al. 1991a). other game birds that are regularly infested, and of which individuals may harbour large burdens, are francolins and spurfowls (horak & boomker 1998; uys & horak 2005). smaller birds are either less exposed to the larvae of a. marmoreum because of their size, or because they spend less time on the ground, or they may be less susceptible. the larvae of this tick quest for hosts from the vegetation (horak et al. 2006), whereas the activity of many of the smaller birds is confined to the soil surface and hence they may not even come into contact with the larvae. few nymphs are collected from the vegetation by sampling with flannel strips (spickett, horak, van niekerk & braack 1992; horak et al. 2006), indicating that they probably quest for hosts from the soil surface. this may explain the larger number of nymphs collected from small birds than from helmeted guineafowls (table 2). larvae are generally most numerous on the vegetation from late summer to midwinter, and nymphs from spring to midsummer (norval 1975; rechav et al. 1987; spickett et al. 1992; horak et al. 2006). the presence of this tick on birds could thus also be expected to be seasonal, but because too few ticks were collected from too few birds this could not be verified. hyalomma species three tick species belonging to the genus hyalomma, namely h. truncatum, h. m. rufipes and h. glabrum (h. m. turanicum) are present in south africa (walker 1991). both h. truncatum and h. m. rufipes are widespread and there is considerable overlap in their distributions (howell, walker & nevill 1978). hyalomma glabrum, which until recently was considered to be identical to asian hyalomma marginatum turanicum (apanaskevich & horak 2006), has a more restricted distribution (howell et al. 1978), and the only region of free state province in which there is substantial table 1 localities in free state province at which ticks were collected from birds tick species locality coordinates amblyomma marmoreum bishop’s glen* salopia* slangfontein* wolwespruit* 29°00’ s; 26°21’ e 29°12’ s; 25°58’ e 30°08’ s; 25°24’ e 28°51’ s; 25°32’ e amblyomma marmoreum and hyalomma marginatum rufipes willem pretorius nr glen agricultural college preezfontein* doornhoek* 28°18’ s; 27°15’ e 28°54’ s; 26°21’ e 29°50’ s; 25°23’ e 28°53’ s; 25°43’ e hyalomma marginatum rufipes bloemfontein morning star* floradale* sandveld nr wolwekop* nooitgedacht* ventersvlei* soetdoring nr 29°08’ s; 26°10’ e 28°48’ s; 27°14’ e 28°57’ s; 26°13’ e 27°38’ s; 25°42’ e 29°27’ s; 26°40’ e 29°13’ s; 25°54’ e 29°14’ s; 25°55’ e 28°50’ s; 26°04’ e hyalomma glabrum tussen-die-riviere nr 30°29’ s; 26°15’ e haemaphysalis leachi tussen-die-riviere nr 30°29’ s; 26°15’ e ixodes rubicundus bishop’s glen* tussen-die-riviere nr slangfontein* 29°00’ s; 26°21’ e 30°29’ s; 26°15’ e 30°08’ s; 25°24’ e rhipicephalus evertsi evertsi willem pretorius nr 28°18’ s; 27°15’ e rhipicephalus gertrudae tussen-die-riviere nr willem pretorius nr 30°29’ s; 26°15’ e 28°18’ s; 27°15’ e rhipicephalus warburtoni slangfontein* 30°08’ s; 25°24’ e * = farm nr = nature reserve 1 2 6 b ird s a s h o sts o f im m a tu re ixo d id ticks in f re e s ta te p ro vin ce , s o u th a frica table 2 ixodid ticks collected from birds in free state province, south africa. bird names and sequence according to maclean (1993) and hockey et al. (2005) bird number infested number of ticks collected other tick species common name scientific name amblyomma marmoreum hyalomma marginatum rufipes larvae nymphs larvae nymphs cattle egret bubulcus ibis 1 h. m. rufipes 1 male orange river francolin scleroptila levaillantoides 1 8 swainson’s spurfowl pternistis swainsonii 1 1 1 helmeted guineafowl numida meleagris 10 233 3 139 6 h. glabrum 12 ll, 4 nn; r. e. evertsi 4 ll; r. gertrudae 2 ll northern black korhaan afrotis afraoides 3 2 5 caspian plover charadrius asiaticus 1 1 2 crowned lapwing vanellus coronatus 3 15 14 double-banded courser rhinoptilus africanus 1 1 namaqua dove oena capensis 1 1 spotted eagle owl bubo africanus 1 14 10 rufous-cheeked nightjar caprimulgus rufigena 5 2 1 4 melodious lark mirafra cheniana 6 1 1 8 13 eastern clapper lark mirafra fasciolata 22 2 8 108 8 spike-heeled lark chersomanes albofasciata 34 7 2 93 7 i. rubicundus 3 ll; r. warburtoni 5 ll, 15 nn red-capped lark calandrella cinerea 1 1 pink-billed lark spizocorys conirostris 1 2 2 large-billed lark galerida magnirostris 5 12 1 1 ashy tit parus cinerascens 1 6 african red-eyed bulbul pycnonotus nigricans 2 1 1 r. gertrudae 1 ll ant-eating chat myrmecocichla formicivora 5 6 2 h. leachi 1 n cape robin-chat cossypha caffra 4 2 5 12 karoo scrub-robin cercotrichas coryphoeus 7 2 15 32 r. gertrudae 1 ll kalahari scrub-robin cercotrichas paena 1 1 1 2 7 d .j. v a n n ie k e r k , l .j. f o u r ie & i.g . h o r a k bird number infested number of ticks collected other tick species common name scientific name amblyomma marmoreum hyalomma marginatum rufipes larvae nymphs larvae nymphs chestnut-vented tit-babbler parisoma subcaeruleum 2 1 1 desert cisticola cisticola aridulus 1 2 cloud cisticola cisticola textrix 5 1 6 1 blackchested prinia prinia flavicans 2 2 1 marico flycatcher bradornis mariquensis 1 2 fiscal flycatcher sigelus silens 3 2 7 pririt batis batis pririt 1 2 cape wagtail motacilla capensis 1 1 african pipit anthus cinnamomeus 2 3 pipit anthus sp. 2 1 6 4 bokmakierie telophorus zeylonus 1 3 1 pied starling spreo bicolor 4 8 2 wattled starling creatophora cinerea 31 59 29 cape weaver ploceus capensis 1 i. rubicundus 2 ll red bishop euplectes orix 5 3 5 i. rubicundus 1 female bunting emberiza sp. 1 1 totals 180 263 20 515 181 ll = larvae nn = nymphs table 2 cont. 128 birds as hosts of immature ixodid ticks in free state province, south africa overlap in the geographic distribution of this tick and that of h. m. rufipes is the south-west (howell et al. 1978). these are all two-host ticks and the immature stages of h. truncatum and h. m. rufipes can readily be differentiated from each other (arthur 1975a; b), whereas those of the latter tick and h. glabrum are somewhat similar in appearance (apanaskevich & horak 2006). the only birds examined in the south-west region of the free state province were those on the farms “slangfontein” and “preezfontein” and in the tussen-die-riviere nature reserve. helmeted guinea fowls in the reserve were infested with larvae and nymphs of h. glabrum (table 2). hyalomma m. rufipes was the most numerous of the ticks recovered and the majority of bird species examined in free state province were infested. it was also the most numerous tick collected by hoogstraal et al. (1961) from birds migrating from east africa to europe and to asia via egypt. all but seven of the 1 025 immature ticks they collected from 340 birds belonged to this species. in the present survey seven of ten helmeted guineafowls examined were infested and one of these birds harboured the highest individual total for a bird of any species, namely 106 larvae and three nymphs. helmeted guineafowls are not only the largest birds examined in the present survey, but they also spend most of the day on the ground, making them readily accessible to the larvae of h. m. rufipes. the bare heads and upper necks of these birds are a favoured attachment site for larvae of several tick species (horak & williams 1986), and probably also for those of h. m. rufipes. although the heads and upper necks of crested francolins, d. sephanea are not bare, more than 90 % of h. m. rufi pes larvae have been recovered from here on these birds (uys & horak 2005). the overall ratio of 515 h. m. rufipes larvae to 181 nymphs on birds implies a satisfactory translation of one developmental stage to the next and that birds are thus suitable hosts of the tick. the adults of h. m. rufipes attach to large animals such as african buffaloes, syncerus caffer, eland, taurotragus oryx, giraffes, giraffa camelopardalis and domestic cattle (norval 1982; rechav et al. 1987; horak, swanepoel & gummow 2002). they are most numerous on these animals during the summer months (londt, horak & de villiers 1979; rechav et al. 1987; fourie, kok & heyne 1996; dreyer, fourie & kok 1998). the immature stages use birds, cape hares, lepus capensis and scrub hares, lepus saxatilis as hosts (norval 1982; rechav et al. 1987; horak & fourie 1991), and are most numerous on helmeted guineafowls and hares from late summer to late winter (rechav et al. 1987; horak & fourie 1991). in the present survey most immature ticks were collected from birds between april and august with a peak in may (fig. 1). the discrete separate periods of seasonal occurrence of the adults and the immature stages imply that a single life cycle is completed annually. hyalomma m. rufipes is probably the principal vector in south africa of the virus causing crimean congo haemorrhagic fever (horak et al. 2002; swanepoel & burt 2004). although certain bird species would seem to be susceptible to experimental infection with the virus and can then transmit it to h. m. rufipes (zeller, cornet & camicas 1994), according to swanepoel & burt (2004) it seems unlikely that this would occur naturally. the larvae and nymphs require 22 days to complete feeding on experimentally infested rabbits (knight, norval & rechav 1978), and if they require the same length of time to feed on birds, their long-distance translocation is a distinct possibility, particularly on migratory species. the hosts and seasonal occurrence of adult and immature h. glabrum [at the time considered to be h. m. turanicum by horak et al. (1991a)] are similar to those of h. m. rufipes. its host spectrum is, however, curtailed by the limits of its own distribution in south africa. the immature stages of h. truncatum infest scrub hares and rodents (horak et al. 1991a). if they are found on birds it can safely be assumed that they ����������� ��� ��� ������ � � � � �� � � � � � �� � � � �� � �� � � � � � � � � � � �� ���� ���� ��� ������ � ���� ���� �������� �� �!�� ����� "�� ���� #� �$�%& fig. 1 seasonal occurrence of the immature stages of hyalomma marginatum rufipes on birds in free state province, south africa 129 d.j. van niekerk, l.j. fourie & i.g. horak are ‘stragglers’ from a heavily contaminated environment. other ticks the remaining five tick species must be considered ‘stragglers’ and their presence on the birds reflecting local or seasonal abundance rather than host preference. the immature stages of haemaphysalis leachi and r. gertrudae parasitize murid rodents (norval 1984; walker et al. 2000), and those of i. ru bicundus and r. warburtoni parasitize rock elephant shrews, elephantulus myurus (fourie, horak & van den heever 1992; walker et al. 2000). however, an engorged female i. rubicundus was recovered from a red bishop, euplectes orix. the immature stages of r. e. evertsi prefer domestic and wild equids as hosts, but may also be found on a large variety of other animals, and occasionally also on birds (walker et al. 2000). acknowledgements we are most grateful to the farmers, landowners and nature reserve managers and the management of glen agricultural college for permission to collect birds and ticks on the properties under their authority. mr e. j. williams was responsible for the collection of ticks from many of the birds and dr g. kopij for those from the starlings at bloemfontein and excelsior. references apanaskevich, d.a. & horak, i.g. 2006. the genus hyalomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum delpy, 1949 (acari, ixodidae) as a valid species with a re-description of the adults, first description of the immature stages and notes on its biology. onderstepoort journal of veterinary research, 73:1–12. arthur, d.r. 1975a. the larvae of some ixodid ticks (acarina) from the eastern cape province of south africa. bulletin of entomological research, 65:405–421. arthur, d.r. 1975b. the nymphs of some ixodid ticks (acarina) from the eastern cape province of south africa. bulletin of entomological research, 65: 423–431. dreyer, karin, fourie, l.j. & kok, d.j. 1998. tick diversity, abundance and seasonal dynamics in a resource-poor urban environment in the free state province. onderstepoort journal of veterinary research, 65:305–316. fourie, l.j., horak, i.g. & marais, l. 1988. an undescribed rhipicephalus species associated with field paralysis of angora goats. journal of the south african veterinary association, 59:47–49. fourie, l.j., petney, t.n., horak, i.g. & de jager, c. 1989. seasonal incidence of karoo paralysis in relation to the infestation density of female ixodes rubicundus. veterinary parasitology, 33:319–328. fourie, l.j., horak, i.g. & van den heever, j.j. 1992. the relative host status of rock elephant shrews elephantulus myurus and namaqua rock mice aethomys namaquensis for economically important ticks. south african journal of zoology, 27:108–114. fourie, l.j., kok, d.j. & heyne, h. 1996. adult ixodid ticks on two cattle breeds in the south-western free state, and their seasonal dynamics. onderstepoort journal of veterinary research, 63:19–23. hockey, p.a.r., dean, w.j.r. & ryan, p.g. 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african ground-feeding birds to hyalomma marginatum rufipes ticks. american journal of tropical med icine and hygiene, 50:676–681. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice abstract introduction material and methods results discussion acknowledgements references about the author(s) ahmad oryan department of pathobiology, shiraz university, iran mahboobe mosadeghhesari department of pathobiology, shiraz university, iran saeed zibaee razi vaccine and serum research institute, mashhad, iran ali mohammadi department of pathobiology, shiraz university, iran citation oryan, a., mosadeghhesari, m., zibaee, s. & mohammadi, a., 2017, ‘identification and phylogenetic analysis of contagious ecthyma virus from camels (camelus dromedarius) in iran’, onderstepoort journal of veterinary research 84(1), a1257. https://doi.org/10.4102/ojvr.v84i1.1257 research communication identification and phylogenetic analysis of contagious ecthyma virus from camels (camelus dromedarius) in iran ahmad oryan, mahboobe mosadeghhesari, saeed zibaee, ali mohammadi received: 13 may 2016; accepted: 25 jan. 2017; published: 24 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract contagious ecthyma is a highly contagious disease affecting domestic and wild ruminants such as sheep, goats and camels. the identification and characterisation of a parapoxvirus (ppv) infecting camels is described here. the virus was detected in dromedary camels (camelus dromedarius) from kerman and shiraz in iran. ppv-specific amplification by polymerase chain reaction (pcr) further confirmed that the disease was associated with ppv infection. phylogenetic analysis of orf011 (b2l) gene sequences showed 99.79% and 82.13% similarity of the ppv identified in this study with the jodhpur isolate and the bovine papular stomatitis virus (bpsv) isolates (ce41), respectively. moreover, phylogenetic analysis of the orf045 gene indicated that the shiraz sample was in all probability closely related to vr634 and to f00.120r and pcpv776. in conclusion, the results suggest that camel ppv (cppv) is a likely cause of contagious ecthyma in dromedary camels in iran. introduction camel contagious ecthyma (cce), also known as orf, auzdik or contagious pustular dermatitis, is a worldwide skin disease in camelids characterised by self-limiting proliferative lesions (nagarajan et al. 2010). these nodular lesions followed by papules and vesicles mostly appear around the mouth, nares, lips and eyes or in some instances, other sites (abubakr et al. 2007; venkatesan et al. 2010). they then develop into scabs and healing may take up to 20 days – 30 days or even more (nagarajan et al. 2010). this disease is caused by pseudocowpox virus (pcpv) in cattle, whereas its main causative agent in sheep, goats, camels and humans is orf virus (orfv) (davari et al. 2013; nagarajan et al. 2010). however, pcpv has been reported as the etiologic agent of the disease in camels (abubakr et al. 2007; nagarajan et al. 2010). the orfv or cce virus belongs to the genus parapoxvirus (ppv), the subfamily chordopoxvirinae of the poxviridae family (fleming, wise & mercer 2015; harvey et al. 2015; klein & tryland 2005; venkatesan et al. 2010). morbidity is high, whereas its mortality and fatality rates are low to moderate. nevertheless, this debilitating disease can lead to high mortality in younger animals due to secondary infections and trouble in suckling and eating (hosamani et al. 2006). the genome of orfv contains a linear double-stranded dna molecule of approximately 134 kbp – 139 kbp in length (hautaniemi et al. 2010; li et al. 2013; rovozze & burke 1973). the genes at the terminal ends are mostly responsible for viral replication, whereas those located in the central sites are essential for virus virulence and pathogenesis (li et al. 2013). for instance, the well-conserved orf045 gene encodes the late transcription factor vltf-1, whereas the envelope or orf011 (b2l) gene encodes a highly immunogenic envelope protein stimulating a strong antibody response (kottaridi et al. 2006; zhang et al. 2014). orfv is sometimes transmitted to humans via direct contact with the infected animal (torfason & gunadottir 2002). clinical signs of pox, contagious ecthyma and papillomatosis are similar and difficult to distinguish (khalafalla, al-busada & el-sabagh 2015a; khalafalla et al. 2015b; mosadeghhesari et al. 2014; nagarajan et al. 2010). polymerase chain reaction (pcr) and sequencing methods may be useful for genetic characterisation and classification of ppvs (inoshima et al. 2001; khalafalla et al. 2015a). the ppvs in iranian dromedaries (camelus dromedarius) have not yet been characterised at the genetic level, whereas their relationship to ppvs in domestic animals also remains unclear. also, the orf011 and orf045 genes of camel ppv (cppv) were used for viral detection, examining the outbreak of cppv infection in camels by pcr and for phylogenetic analysis. material and methods sampling sixty skin samples were collected from two outbreaks of orfv which occurred in 2013 in dromedary camels of shiraz and kerman cities, southern and eastern iran, respectively. biopsies of the skin and the lip of the affected camels were sent to razi research vaccine and serum institute. the samples collected from the diseased camels were analysed for pcr assay. dna extraction and primer design dna of the samples from the skin and lip lesions was extracted by high pure® extraction kit (roche diagnostics gmbh, mannheim, germany). briefly, 50 mg of the tissue samples was homogenised in 200 μl of phosphate buffer saline (pbs) and mixed with 200 μl of binding buffer and 50 μl of proteinase k then incubated at 72 °c for 10 min. the process was continued according to the manufacturer’s instructions. according to inoshima, morooka and sentsui (2000), a set of primers including pan-parapoxvirus primer 1 (ppp-1) and ppp-4 were applied based on the sequence of the orf011 gene. the sequences of ppp-1 and ppp-4 were 5’-gtc gtc cac gat gag cag ct-3’ and 5’-tac gtg gga agc gcc tcg ct-3’, respectively (inoshima et al. 2000). in addition, the primers 045f (5’-cct act tct cgg agt tca gc-3’) and 045r (5’-gca gca ctt ctc ctc gta g-3’) were applied based on the sequence of 045 gene of orfv, isolate ov-sa00 (accession no. ay186732) (delhon et al. 2004). polymerase chain reaction for amplification of the orf011 gene, the pcr assay was carried out as follows: 1 μg of dna isolated from the infected or uninfected cells was added to 50 μl of the pcr mixture containing 0.2 mm primers (ppp-1 and ppp-4), 0.2 mm datp, dctp, dgtp and dttp, 10 mm tris–hcl (ph 8.3), 50 mm kcl, 1.5 mm mgcl2 and 1 u of amplitaq gold dna polymerase. dna was amplified with a dna thermal cycler by a two-step reaction. the mixture was denatured at 95 °c for 9 min, five cycles of 94 °c for 1 min, 50 °c for 1 min, 72 °c for 1 min, 25 repeated cycles at 94 °c for 1 min, 55 °c for 1 min and finally 72 °c for 1 min (inoshima et al. 2000). for amplification of the orf045 gene, the pcr assay was carried out in a reaction mixture of 50 μl containing 1 μg (10 μl) of extracted dna, 5 μl of dntp (10 mm), 5 μl of 10x pcr buffer, 2 μl of mgcl2 (50 mm), 2 u/tube of taq polymerase, 50 pmol of each of the primers and 33 μl of distilled water. forty cycles of denaturation (at 95 °c, for 10 s), annealing (at 47 °c for 10 s) and extension (at 74 °c for 10 s) were followed by a 15-min incubation at 78 °c to final extension of the primers. the amplification products (10 μl) were analysed by 2% agarose gel in tris–boric acid–edta (tbe) buffer and stained with sybr green (1 μg/ml) (kottaridi et al. 2006). the amplicons were visualised using a uv transilluminator ‘gel doc’ system. sequencing of the polymerase chain reaction products containing orf011 and orf045 genes the pcr products with the expected sizes (594 bp and 393 bp) were purified from the gel using the bioneer (daejeon, south korea) gel extraction kit, according to the manufacturer’s procedures, and then were sequenced by the use of neighbour-joining method (bioneer). phylogenetic analysis comparison among the isolates of this study and those of the orfv available in genbank, ncbi database, was made using the online blast program. sequence identities of the nucleotides were analysed by clc program version 5.5. the sequences were assembled into multiple sequence alignment. a phylogenetic tree derived from the nucleotide sequences was constructed for the orfv, using neighbour-joining method of clc program version 5.5. the sequences of the orf011 and orf045 genes of orfv used in phylogenetic analysis are presented in tables 1 and 2. table 1: nucleotide sequences of orf011 gene that were aligned in the samples of the present study. table 2: nucleotide sequences of orf045 gene that were aligned in the samples of the present study. results polymerase chain reaction and dna sequencing the pcr method was performed to amplify the orf011 gene. this method successfully detected the dna from members of the ppv genus and was able to diagnose the orfv infection. to confirm the causative agent, a partial region of the orf011 gene was amplified. the primers ppp-1 and ppp-4 were described as the universal primers to amplify most species of orfvs that have been used in the present study. a 594-bp product representing the region (157 nt – 750 nt) of the orf011 gene was amplified from shiraz samples. the 594-bp dna fragment was sequenced and the nucleotide sequence data confirmed orfv. another pcr test was performed on dna purified from kerman samples as template and the pair of 045f/045r primers to amplify the orf045 gene. pcr resulted in the amplification of the gene orf045 with an expected dna fragment (392 bp). phylogenetic analysis the samples used in this study included kerman (msoke) and shiraz (msosh) isolates. the sequences aligned with msoke and msosh were based on the orf011 gene (table 1) and the gene orf045 (table 2), respectively. as stated previously, the samples of this study and the previous isolates were aligned and the phylogenetic tree was drawn by clc program version 5.5. results of the phylogenetic tree related to the orf011 gene have been shown in figure 1a that there is a main clade or lineage containing sv252/11 and other isolates. a branch of the main clade contains three clades including msoke and cce virus isolate from jodhpur, msoke/jodhpur isolate sister to bovine papular stomatitis virus (bpsv) isolate ce41, and clade msoke/jodhpur and ce41 sisters to brazil isolates. msoke had 99.77% and 82.13% similarity with jodhpur and ce41 isolates, respectively. in addition, msoke had minimum distance (0.20) to jodhpur and ce41. the results of phylogenetic tree from gene orf045 with two principal clades analysis are shown in figure 1b including clade orfb and other sequences, clade gq329670/msosh and gq329669/hm589037 while another branch contains other sequences. indeed, msosh was closely related to pcpv from finland (vr634), another pcpv from finland (f00.120r) and then also to pcpv from austria (pcpv776). figure 1: phylogenetic analysis of the orf virus based on the (a) orf011 and the (b) orf045 gene. the phylogenetic trees were constructed using the neighbour-joining algorithm in clc main workbench 5.5 with 1000 bootstrap replicates. discussion several procedures have been developed to detect orfv, while most of them are time-consuming, costly and sometimes associated with low specificity, reduced sensitivity and with cross reactions (inoshima et al. 2001; khalafalla et al. 2015a; kottaridi et al. 2006). assays based on pcr are considered as rapid, sensitive and specific tests for identifying the causative agents of diseases (andrade et al. 1983; inoshima et al. 2001, 2002). several pcr protocols have been described for detecting the dna of ppv (inoshima et al. 2000, 2001, 2002; torfason & gunadottir 2002). two target genes to generate pcr amplicons from cce dna for sequence analysis are the open reading frame orf011 and orf045 which encode vltf-1 (khalafalla et al. 2015b; kottaridi et al. 2006). sequencing of the suspected samples showed that msosh had close relation to pcpv from finland (vr634) followed by another pcpv from finland (f00.120r) and pcpv from austria (pcpv776). additionally, msoke had 99.77% and 82.13% similarity to jodhpur and ce41 isolates, respectively. khalafalla et al. (2015b) considered that camel orfv isolates can be divided into two genetic clades including the asian lineage of isolates from saudi arabia, bahrain and india and the african lineage of isolates from sudan, based on the sequence of the orf011 gene among camels in sudan. they showed that the camel orfv is diverse from viruses close to pcpv and orfv. nagarajan et al. (2010) analysed and compared the sequence of indian pcpv with several sequences related to orfv, pcpv and bpsv available in the database. they showed that orfv from different regions of the world had 92.3% – 92.7% and 96.2% – 96.5% sequence similarity with camel pcpv at the nucleotide and amino acid level, respectively. orfv has previously been cultivated on cell cultures such as vero, lamb testis (lt) and ovine testis (oa3.ts), whereas the cell culture was done on chicken embryo fibroblast (cef) in this study. it is because cpe includes cell detachment, rounded cells and aggregation of cells. meanwhile, many researchers have studied the effects of orfv on various cell cultures. housawi et al. (2012) compared the immunogenicity and protective efficacy of three locally developed live orfv vaccines in vero cell culture. for production of a live attenuated vaccine against orfv, a wild virus strain has been attenuated through serial passages on the primary cef tissue cultures (mercante et al. 2008). the isolation and attenuation of the virus in iran would therefore be necessary for protection of camels. a recent study performed by mombeni et al. (2013) in iran reported severe cases of cce that caused a morbidity and mortality rate of 70.6% and 6.0%, respectively. this points to the potential importance of this disease in iran. this study has indicated that orfv with an apparent close similarity to pcpv can be a major cause of contagious ecthyma in iranian camels. acknowledgements the authors are thankful to mrs majidi from the razi vaccine and serum research institute for her valuable contribution to this study. the authors are also indebted to dr shahriari and dr varshovi for their assistance in providing the samples. the authors would like to thank the authorities of shiraz university for partial financial support of the project. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors contributed equally in all stages of the manuscript preparation and approved its final version. 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subtropical countries in africa, asia, australia and some european countries (horak 1971; kelly & henderson 1973). in zimbabwe and the rest of southern africa, calicophoron microbothrium, with bulinus tropicus as its intermediate host (swart & reinecke 1962a; over 1982; chingwena, mukaratirwa, kristensen & chimbari 2002), has a wide distribution and has been incriminated in the majority of outbreaks of amphistomosis in ruminants (dinnik & dinnik 1962; dinnik 1965; horak 1971). the epidemiology of amphistomosis has been reported mostly in india and other tropical countries (okafor, mbata & anosike 1988; rolfe, boray, nichols & collins 1991; prasad & varma 1999; bedarkar, narladkar & deshpande 2000), and knowledge on 95 onderstepoort journal of veterinary research, 73:95–100 (2006) observations on mass production of calicophoron microbothrium metacercariae from experimentally and naturally infected bulinus tropicus m. mavenyengwa1, s. mukaratirwa1, m. obwolo1 and j. monrad2 abstract mavenyengwa, m., mukaratirwa, s., obwolo, m. & monrad, j. 2006. observations on mass production of calicophoron microbothrium metacercariae from experimentally and naturally infected bulinus tropicus. onderstepoort journal of veterinary research, 73:95–100 in an attempt to establish an ideal method for mass production of calicophoron microbothrium metacercariae, a study was carried out to compare the shedding capacities of bulinus tropicus naturally and experimentally infected with c. microbothrium. a total of 906 f1 b. tropicus between 4 and 5 weeks old were each experimentally infected with two c. microbothrium miracidia and monitored for 12 weeks. the infected snails were fed on dried lettuce and fish flakes and were kept in 1 l plastic aquaria housed in a snail room where temperature, light and humidity were controlled. seventy-four percent of the experimentally infected snails died during the prepatent period and of the remaining, only 13.2 % developed patent infection, while 12.5 % were refractory. snail growth rate was poor and the average shedding rate was 20 cercariae per snail per day. compared to the experimentally infected snails, 2 200 adult b. tropicus, collected from the field and naturally infected with c. microbothrium, yielded high numbers of metacercariae. eighty-four percent of the snails died within 7 weeks of the study with peak mortality occurring from the 2nd to the 4th week of infection and coinciding with an overall decrease in the number of cercariae shed. keywords: bulinus tropicus, calicophoron microbothrium, mass production, metacercariae, snails 1 faculty of veterinary science, paraclinical veterinary studies, university of zimbabwe, harare, zimbabwe 2 danish centre for experimental parasitology, frederiksberg, copenhagen, denmark accepted for publication 16 november 2005—editor 96 mass production of calicophoron microbothrium metacercariae from infected bulinus tropicus other aspects of the disease such as its pathogenesis, perceived mechanisms of resistance (butler & yeoman 1962; horak 1967, 1971) and clinical pathology (horak 1967) is limited, available related literature having been assembled from field observations. in order to acquire full knowledge of the pathogenesis of the disease, the mechanisms of resistance and the clinical pathology, systematic experimental infections using susceptible hosts are required, followed by time series observations and sampling strat egies. for such studies to succeed large stocks of viable amphistome metacercariae are required (swart & reinecke 1962a) and the first step is to develop a routine technique for establishing and maintaining infected snails for harvesting metacercariae. swart & reinecke (1962b) successfully infected large numbers of first generation (f1) b. tro p icus under laboratory conditions, while rolfe, boray & collins (1994) used naturally infected b. tropicus for obtaining infective metacercariae for experimental infection in sheep. the objective of this study was to establish a method of harvesting amphistome metacercariae in large quantities for experimental studies. materials and methods experimental infection of b. tropicus with c. microbothrium miracidia collection and culture of c. microbothrium eggs live, sexually matured c. microbothrium collected from the rumens of cattle slaughtered at a local abattoir, were preserved in physiological saline until they were confirmed to be c. microbothrium according to eduardo (1982a, b, c). to obtain the eggs, the flukes were crushed with a pestle in a mortar before mixing with distilled water. the contents were then serially sieved through sieves of 425 and 150 μm and finally a 63 μm sieve from which the eggs were collected. the eggs were then washed into a conical flask and left to stand for 10 min before the excess water was decanted and the sediment transferred into petri dishes for incubation. the eggs were incubated at 25 °c until day 12 when hatching occurred upon exposure to a 40 watt light bulb. propagation and maintenance of b. tropicus juveniles first generation b. tropicus juveniles used in this study were bred from adult b. tropicus collected from natural habitats and maintained in the laboratory. after hatching, the juvenile snails were kept in 1 l plastic aquaria containing de-chlorinated water from an artificial pond located outside the snail room and fed on dried lettuce and commercial fish flakes. the same source of water was used for the maintenance of the naturally infected snails in the laboratory. experimental infection of b. tropicus with c. microbothrium miracidia a total of 906 f1 b. tropicus, between 3 and 4 weeks old, were individually exposed for 4 h to 2 newly hatched miracidia of c. microbothrium in 10 ml plastic cups, each containing 3 ml of de-chlorinated pond water, before being pooled into 1 l plastic aquaria holding a maximum of 10 snails each. at the time of the transfer, stereoscopic examination failed to reveal any miracidia remaining in the plastic cups. the infected snails were housed in a snail room at 30 °c and observed once per day for 12 weeks to record the mortality pattern, the growth rate and any abnormalities that could occur during the study period. florescent bulbs supplied light during the day. de-chlorinated pond water (ph 6.9) was used in the aquaria and the water was replaced every 2 days. the snails were fed on dried lettuce and commercial fish flakes. at 4 weeks post-infection each snail was exposed to light for 1 h every 2 days to stimulate cercariae shedding. snails shedding cercariae were placed in separate aquaria and kept in the dark for mass production of cercariae. mass production of c. microbothrium metacercariae from experimentally infected b. tropicus durie’s (1955) technique for harvesting large numbers of amphistome metacercariae was used with some modifications. inside a 500 ml glass beaker painted black on the outside, and midway to the capacity graduation mark, a cellulose acetate strip 10 mm wide was pasted right around the wall. dechlorinated water from an artificial pond outside the snail room was added into the beaker to midway the width of the cellulose acetate strip. bulinus tropicus shedding cercariae were then placed in nylon gauze bags, dropped into the beaker and placed in a covered wooden box fitted with a light source, making sure the light source was directly above the beaker and high enough to avoid overheating the water. these snails were exposed in this way every 2 days for 3 h before they were returned to the dark aquaria. meta cercariae harvested at each exposure were counted and kept moist at 25 °c. 97 m. mavenyengwa et al. mass production of c. microbothrium metacercariae from naturally infected b. tropicus a total of 2 200 adult b. tropicus, naturally infected and shedding c. microbothrium metacercariae, were collected from a single natural habitat and maintained in the laboratory in dark 5 l plastic aquaria filled with de-chlorinated pond water, with each aquarium holding approximately 80 snails. immediately after collection, the snails were individually screened and each one that either failed to shed cercariae or shed other than amphistome cercariae was discarded after several daily exposures to direct sunlight. the identity of the cercariae shed by the b. tropicus was confirmed by feeding 15 000 metacercariae to a sheep, which was sacrificed at day 56 post-infection. the mature amphistomes recovered from the rumen of the sheep were identified as c. microbothrium, according to eduardo (1983). the management regimen and observation of the naturally infected b. tropicus was similar to that described for the experimentally infected group excepting that lettuce and fish flakes formed the basal diet and that mud collected from the natural habitat was added into the aquaria in an attempt to emulate the natural habitat conditions. for mass production of cercariae, nylon gauze bags (2 mm) each holding up to 20 snails were used to confine snails during shedding to minimize ingestion of metacercariae by the snails. the open-end of each snail bag was stapled, individually immersed in a plas tic container holding 500 ml of de-chlorinated pond water and exposed to sunlight for 3 h before being returned to the dark aquaria. the harvested metacercariae were then stored under moist conditions at 25 °c until required for use. the harvesting procedure was repeated on alternate days until few or no cercariae were released from the snails. the growth and mortality rates and any abnormalities observed during the study period were recorded. results experimental infection of b. tropicus with c. microbothrium eggs and embryo development a large number of eggs were obtained by crushing mature c. microbothrium flukes. the eggs were oval in shape, colourless and filled with vitelline cells. the anterior end was tapered and operculated while the posterior end was broad. on the 8th day of incubation there was a decrease in the number of vitelline cells and an increase in the size of the embryo, which in many eggs had assumed a concave position. by day 12 of incubation the embryo had taken up most of the shell space and some embryos could be seen wriggling inside the shell, signifying maturity and readiness to hatch. exposure of eggs to an artificial light source on this day stimulated hatching spontaneously. experimental infection of b. tropicus and mass production of metacercariae confinement of individual snails in small plastic cups with small volumes of de-chlorinated pond water facilitated the infection as it promoted close contact between the snail and the miracidia. upon exposure to the snails, the miracidia became very active and made numerous oscillatory movements around the snail and/or the snail faecal material. the majority of the snails retreated from the miracidia by migrating out of the water. after vigorous movement during the first 30 min of contact the snails became relatively inactive apparently indicating successful miracidial penetrations as was supported by failing to observe miracidia microscopically in the cups after the snails had been removed. the attempt at mass production of c. microbothrium using experimentally infected b. tropicus was largely unsuccessful as low numbers of cercariae were recovered and snail mortality was high. of the 906 b. tropicus infected 673 (74.3 %) died during the 13 weeks of the study, 120 (13.2 %) shed cercariae and 113 (12.5 %) survived the infection but remained refractory. the first cercariae were released on day 42 post-infection, at an average rate of 20 cercariae per snail per day. as illustrated in fig. 1, snail losses occurred throughout the study period. after a high proportion (34 %) of snails had been lost in the first 4 weeks of infection, a steady loss occurred from weeks 5–12, when only 25.7 % of the infected snails remained. the growth rate of the snails was poor and the majority remained stunted. only 8 % of the infected snails had marginal increases in shell length, ranging from about 2 to 3 mm over the study period. one percent of snails survived for 20 weeks outside the study period before they were discarded. mass production of c. microbothrium metacercariae from naturally infected b. tropicus the shedding rate of naturally infected snails could not be determined because of the large numbers of 98 mass production of calicophoron microbothrium metacercariae from infected bulinus tropicus cercariae that were released per exposure. cer cariae emergence commenced soon after exposure to direct sunlight, initially in small numbers but peaking within an hour by which time they were so many swimming cercariae. the cercariae encysted approximately 10 min after release, at the level of the water meniscus. however, perhaps because of a shortage of space for cercariae released, some cercariae encysted below the meniscus, some on the water surface and some on the bottoms of the containers. shedding had almost ceased by the end of 2 h, while cercariae encystment was completed after 3 h. after recovery of high numbers of metacercariae during the first 3 weeks, the numbers started to diminish gradually, in concert with the death of high yielding snails (fig. 1). only 15 % of the 2 200 b. tropicus naturally infected with c. microbothrium survived infection till the end of the study. snail mortalities occurred throughout the study period, with peak mortality from the 2nd to the 4th week of collection from the field (fig. 1). after a 95 % survival rate of the snails at the end of the first week post-collection, at the end of week 3 postcollection only 46 % remained. snail mortality decreased from the 5th week until the end of the study and during this period the shedding rate reduced drastically. discussion the crushing of adult flukes to harvest c. microbothrium eggs as done in this study, proved simpler, faster, cheaper and less labour intensive than where mature flukes are left to deposit the eggs after collection (tripathi & srivastava 1987; prasad, malviya, varma & dwivedi 1994). although detailed parasite systematics and biology studies were not done in this study, the observed development pattern and behavioural characteristics of c. microbothrium larval stages are similar to those described in other amphistome species (dutt 1980; sey 1991; gupta 1993). since the miracidia were observed to become less active within 2 h of hatching, exposure of miracidia to snails should be fairly rapid to achieve infection while the miracidia are still energetic. to counter this problem it is advisable to serially expose incubated eggs to a light source in batches 1 h apart, so that active and energetic miracidia are always available for each batch of snails to be infected. this study confirmed that, as reported previously (swart & reinecke 1962a; dinnik 1962; over 1982; chingwena et al. 2002) b. tropicus is the intermediate host of c. microbothrium in southern africa including zimbabwe. while c. microbothrium did infect b. tropicus in the laboratory, with a minimum prepatent period of 42 days, the experimentally infected snails did not yield large numbers of metacercariae due to high mortality, poor growth rate and low per capita shedding rate. most experimentally infected snails were lost during the first 4 weeks of the study, most likely from the stress of infection. the infection dose used in this study was adequate considering that in the field, the dose might be multi-miracidial unless susceptible fig. 1 weekly survival rates (%) of bulinus tropicus, either experimentally or naturally infected with calicophoron microbothrium miracidia ��������� ������ ������ �� ������� ���� ������ �� � � �� � � �� �� � �� � � ��������� �� �� � ��� �� ���������� �� �� � ��� �� ��������� ���� ������ ������ ���� ���� ������ �� ��� �� �� �� � 99 m. mavenyengwa et al. snails invoke retraction strategies as observed in this study to avoid multiple infections. swart & reinecke (1962b) massively infected a large number of b. tropicus with c. microbothrium under laboratory conditions. in this study, massive infection as described by swart & reinecke (1962b), was attempted as a sideline but caused 100 % mortality a few days after exposure and was consequently abandoned. apparently only 13.2 % of the experimentally infected snails became infected. no dissections were done to determine the actual number of snails that had become infected, but this result was similar to that obtained by prasad et al. (1994) who confirmed through dissection that only 15.2 % of 550 indo planorbis exustus experimentally infected with param phistomum epiclitum became infected. the individual snail infection method used in this study could be expected to succeed but it appears that the study failed to reproduce the critical natural parameters an infected snail would require to survive the infection in the field. swart & reinecke (1962a) reported that water ph, temperature, water maturity, aeration and light source were among the parameters that had to be adjusted to sustain the infected snails, but they did not report survival rates. unlike the experimentally infected b. tropicus, naturally infected b. tropicus produced large numbers of c. microbothrium cercariae and the production was sustained for 3 weeks after which it started to decline. however, snail mortality was comparatively greater than the experimentally infected group, peaking at 3 weeks and coinciding with a fall in total weekly cercariae production. the death of the snails was possibly related to the stress of stimulation and massive release of cercariae. cercariae are released through rupturing of snail tissues and considering the production capacity displayed by the naturally infected snails in the present study, both the damage to the snail tissues and the massive physiological/nutritional strain of the maturing cercariae are likely to have overwhelmed these intermediate hosts. the cercariae production rate decreased and coincided with the peak in snail mortality at 3 weeks. it is possible and logical to conclude that the peak in mortality at 3 weeks was due to the death of high yielding snails under severe shedding stress and therefore died quicker leaving less productive snails. this is contrary to the report by swart & reinecke (1962b) where heavily infected snails were sustained for up to 10 months despite regular exposures to a 40 watt yellow light. from this study, cercariae shedding peaked at 3 h after exposure to sunlight and confirms swart & reinecke’s (1962b) findings where cercariae emergence peaked in the first 2–3 h, and it would appear any exposure exceeding 3 h at a given time can lead to snail fatigue and high mortalities. in nature the infected snails may conceivably have many ways of avoiding excessive shedding, which include aestivation and residing in the shade of aquatic plants to avoid light exposure (mavenyengwa, personal observation 2003). under such conditions shedding can be expected to be regulated, thus prolonging snail survival. the possibility that addition of mud from the natural habitat to the aquaria may have favoured development and survival of the naturally infected snails, should be investigated. no growth retardation tendencies or signs of calcium deficiencies were found among the naturally infected snails. this is possibly related to the mineral content of the habitat soil and subsequent adjustments of the water ph to simulate field conditions. induction of experimental amphistomosis in the final host requires large numbers of viable metacercariae for infection (swart & reinecke 1962a; horak 1967) and observations made in this study highlight some of the difficulties that can be encountered in an attempt to raise such numbers of c. microbothriuminfected b. tropicus under laboratory conditions. swart & reinecke (1962b) using the mass infection technique succeeded in infecting a large number of young snails confined in a container although they neither mention the number of snails infected nor the quality of the water used. in the present study, raising such snails proved laborious, time consuming and produced insufficient quantities of metacercariae for experimental purposes. the use of naturally infected b. tropicus for mass production of cercariae proved relatively easy but had disadvantages including working with an unknown parasite species, which can only be determined at the end of the study. this constraint can, however, be overcome by conducting primary infections in a single ruminant host to obtain the adult amphistomes for confirmation of parasite identity before embarking on any major work on experimental amphistomosis. the other disadvantage is the probability of getting mixed infections from a single habitat. however, despite these disadvantages, it was found that the use of naturally infected snails for harvesting metacercariae of c. microbothrium was relatively easier, more efficient, and time conserving provided that stable habitats with naturally infected b. tropicus (common in zimbabwe soon after the rainy season) 100 mass production of calicophoron microbothrium metacercariae from infected bulinus tropicus are identified and the seasonal occurrence of amphistomosis is considered. acknowledgements danida through the enreca-livestock helminths research project is acknowledged for the provision of funds. our tribute also goes to all support staff in the depart ment of paraclinical veterinary studies, university of zimbabwe, for their assistance in the collection of samples, and to the staff at blair research labor atory for the provision of facilities for maintenance and shedding of the snails. references bedarkar, s.n., narladkar, b.w. & deshpande, p.d. 2000. seasonal prevalence of snail-borne fluke infections in ruminants of marathwada region. journal of veterinary parasitology, 14:51–54. boray, j.c. 1969. studies on intestinal paramphistomosis in sheep due to paramphistomum ichikawai fukui, 1922. vet erinary medical review, 4:290–308. butler, r.w. & yeoman, g.h. 1962. acute intestinal paramphistomiasis in zebu cattle in tanganyika. veterinary record, 74:227–231. chingwena, g., mukaratirwa, s., kristensen, t.k & chimbari, m. 2002. larval trematode infections in freshwater snails from the highveld and lowveld areas of zimbabwe. journal of helminthology, 76:283-293. dinnik, j.a. & dinnik, n.n. 1962. the growth of param phistomum microbothrium fischoeder to maturity and its longevity in cattle. bulletin of epizootic diseases in africa, 10: 27–31. dinnik, j.a. 1965. the snail hosts of certain paramphistomatidae and gastrothylacidae (trematoda) discovered by the late dr p.l. le roux in africa. journal of helminthology, 39:141– 150. durie, p.h. 1955. a technique for the collection of large numbers of paramphistome (trematoda) metacercariae. australian journal of agricultural research, 6:200–202. dutt, s.c. 1980. paramphistomes and paramphistomiasis in domestic ruminants in india, edited by a. singh. ludhiana, india: punjab agricultural university press. eduardo, s.l. 1982a. the taxonomy of the family param phistomidae fischoeder, 1901 with special reference to the morphology of the species occurring in ruminants. i. general considerations. systematic parasitology, 4:7–57. eduardo, s.l. 1982b. the taxonomy of the family paramphistomidae fischoeder, 1901 with special reference to the morphology of the species occurring in ruminants. ii. revision of the genus paramphistomum fischoeder, 1901. systematic parasitology, 4:189–238. eduardo, s.l. 1982c. techniques for examining paramphistomes. journal of helminthology, 56:117–119. eduardo, s.l. 1983. the taxonomy of the family paramphistomidae fischoeder, 1901, with special reference to the morphology of the species occurring in ruminants. iii. revision of the genus calicophoron nasmark, 1937. systematic parasitology, 5:25–79. eduardo, s.l. 1988. hosts and geographical distribution of paramphistomid species occurring in ruminants. philippine journal of veterinary animal science, 14:25–70. gupta, n.k. 1993. amphistomes, systematics and biology. oxford and ibh publishing co. pvt, new dehli, india. horak, i.g. 1967. host-parasite relationships of paramphistomum microbothrium fischoeder, 1901, in experimentally infested ruminants, with particular reference to sheep. onderstepoort journal of veterinary research, 34:451–540. horak, i.g. 1971. paramphistomiasis of domestic ruminants. advances in parasitology, 9:33-72. kelly, d.j. & henderson, w.k. 1973. calicophoron calicophorum (trematoda: paramphistomidae) and paramphistomiasis in domestic cattle in the east kimberley district of western australia. tropical animal health and production, 5: 192–195. mukaratirwa, s., kristensen, t.k., siegismund, h.r. & chandiwana, s.k. 1998. genetic and morphological variation of populations belonging to the bulinus tropicus/ trancatus complex (gastropoda: planorbidae) in southwestern zimbabwe. journal of molluscan studies, 64:435–436. okafor, f.c., mbata, g. & anosike, j. 1988. studies on paramphistomum cervi (schrank, 1790) infections of ruminants in imo state, nigeria, with special reference to the role played by bulinus b. forskalii (ehrenberg) in their transmission. bulletin of animal health and production in africa, 36: 142–146. over, h.j. 1982. ecological basis for parasite control: trematodes with special reference to fascioliasis. veterinary parasitology, 11:85–97. prasad, a., malviya, h.c., varma, t.k. & dwivedi, p. 1994. development of paramphistomum epiclitum fischoeder 1904 in the snail host. indian journal of animal sciences, 64: 568–573. prasad, a. & varma, t.k. 1999. on the prevalence and community dominance among paramphistomes infecting domestic ruminants. veterinary parasitology, 13:129–133. rolfe, p.f., boray, j.c., nichols, p. & collins, g.h. 1991. epidemiology of paramphistomosis in cattle. inter national journal for parasitology, 21:813–819. rolfe, p.f., boray, j.c. & collins, g.h. 1994. pathology of infection with paramphistomum ichikawai in sheep. international journal for parasitology, 24:995–1004. sey, o. 1991. handbook of the zoology of amphistomes. boca raton: crc press. swart, p.j. & reinecke, r.k. 1962a. studies on param phistomiasis. i. the propagation of bulinus tropicus krauss 1848. onderstepoort journal of veterinary research, 29: 183–187. swart, p.j. & reinecke, r.k. 1962b. studies on param phistomiasis. ii. the mass production of metacercariae of param phistomum microbothrium fischoeder 1901. onderstepoort journal of veterinary research, 29:189–195. tripathi, h.n. & srivastava, h.d. 1987. amphistomes of ruminants. 2. life history of two species paramphistomum srivastava and tripathi (1980) of sheep, goats and buffaloes. indian journal of animal sciences, 57:1043–1056. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjdffile false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice segwagwe_61-70.indd introduction induction of ovulation with gnrh is used in the ovsynch protocol (pursley, wiltbank, stevenson, otto bre, garverick & anderson 1997); it has also been used at the end of a protocol where the initial treatment used was a combination of intra-vaginal progesterone releasing device containing 1.9 g of progesterone (cidr device) and 5 mg oestradiol ben zo ate (rivera, goni, chaves, ferrero & bo 1998). the proportion of cows ovulating within 24–48 h of using gnrh, tended to be higher in the group of cows where the initial treatment was a combination of progesterone and oestradiol benzoate, compared to where either a cidr devise or calf removal was used (rivera et al. 1998). emergence of a new follicular wave can be synchronized by the injection of oestradiol benzoate at the time of cidr insertion (adams 1998). this explains the synchronous induction of ovulation with gnrh at the end of a treatment regime that involved the cidr device and oestradiol benzoate (rivera et al. 1998). interestingly, the function of the corpus luteum that formed as a sequel to this ovulation depended on the stage of development of the ovulatory follicle at the time of the induction of the ovulation; a corpus luteum formed after the ovulation of the dominant follicle that were 61 onderstepoort journal of veterinary research, 73:61–70 (2006) the effect of gnrh or oestradiol injected at pro-oestrus on luteal function and follicular dynamics of the subsequent oestrous cycle in non-lactating cycling holstein cows b.v.e. segwagwe1*, k.l. macmillan2 and p.d. mansell2 abstract segwagwe, b.v.e., macmillan, k.l. & mansell, p.d. 2006. the effect of gnrh or oestradiol injected at pro-oestrus on luteal function and follicular dynamics of the subsequent oestrous cycle in non-lactating cycling holstein cows. onderstepoort journal of veterinary research, 73:61–70 oestrous synchronization involves synchronization of ovarian follicular turnover, new wave emergence, and finally induction of ovulation. the final step can be synchronized by the parenteral administration of either gnrh or oestradiol benzoate. this study investigated corpus luteum and follicular emergence after ovulation had been induced by the administration of either gnrh or oestradiol benzoate. the injection of oestradiol benzoate may have delayed the emergence of the first follicular wave subsequent to the induced ovulation; administration of oestradiol benzoate or gnrh lowered the progesterone rise so that the maximum dioestrous concentration of progesterone on day 9 was lower when cows were treated during pro-oestrus compared to the spontaneously ovulating controls. one implication of findings from the present study is that induction of ovulation with either oestradiol benzoate or gnrh, administered 24 or 36 h after withdrawal of the cidr device, respectively, may lower fertility. future studies must identify the timing of administration relative to the time of cidr device withdrawal and the optimum concentration of oestradiol benzoate or gnrh that would not have untoward effects on the development of the corpus lutea, particularly within the first week of dioestrus. keywords: corpus luteum, gnrh, oestradiol benzoate, ovarian follicles, ovulation, progesterone * author to whom correspondence is to be directed 1 applied neurobiology group, faculty of veterinary medicine, uni versity of glasgow, 464 bearsden road, glasgow, g61 1qh, uk. e-mail: besegwagwe@yahoo.co.uk 2 the university of melbourne, veterinary clinical centre, 250 princess highway, werribee, 3030, vic australia accepted for publication 9 november 2005—editor 62 gnrh or oestradiol on luteal function and follicular dynamics of oestrous cycle in holstein cows at the growing or early static phase at the time of gnrh administration maintained the highest concentration of plasma progesterone compared to cows ovulating spontaneously (rivera et al. 1998). oestradiol benzoate is frequently used to induce ovulation and reduce the interval and variation of onset of oestrus (day, burke, taufa, day & macmillan 2000). more than 90 % of cows came into oestrus within 3 days of induction of ovulation with oestradiol benzoate (day et al. 2000). this synchronized occurrence of oestrus allows fixed-time insemination with resulting conception rates in heifers so inseminated within 48 h approaching 50 % (hanlon, william son, wichtel, steffert, craigie & pfieffer 1996). a typically normal corpus luteum was formed after treatment of anovulatory anoestrous beef cows with progesterone and induction of ovulation with oestradiol benzoate (fike, day, inskeep, kinder, lewis, short & hafs 1997). recruitment of a new wave of follicles occurred earlier after induction of ovulation with gnrh given 24 h after prostaglandin f2α (pgf2α), compared to untreated controls (taponen, katila & rodriguezmartinez 1999), although induction of ovulation with gnrh may also be associated with shortened luteal lifespan (taponen et al. 1999) and reduced pregnancy rates (schmitt, diaz, drozt & thatcher 1996). while an injection of gnrh during metoestrus did not affect the development of the corpus luteum when compared to that in untreated controls (taponen, rodriguez-martinez & katila 2000) it did cause a predictable release of leutinizing hormone (lh) and a significant increase in serum progesterone due to an increase in the proportion of large luteal cells on day 10 (mee, stevenson, alexander & sasser 1993). luteal plasma progesterone concentration may be decreased after induction of ovulation with a gnrh agonist compared to that which occurs after spontaneous oestrus (lucy & stevenson 1986). peters, ward, warren, gordon, mann & webb (1999) compared the effect of gnrh and spontaneous ovulation (control) in an ovsynch protocol on post-ovulation luteal function. plasma progesterone concentrations increased more rapidly in the gnrh group than in the control group and were significantly higher on days 3 and 4 after ovulation. the size of the pre-ovulatory follicle has been positively correlated with the plasma concentrations of progesterone post-ovulation (moreira, de la sota, diaz & thatcher 2000). there is no other information in the literature where a comparison of the post-ovulation corpus luteum and follicular development after treatment with oestra diol benzoate or gnrh at pro-oestrus was made in the same trial. it was therefore hypothesized that the injection of oestradiol benzoate at pro-oestrus would cause delayed emergence of the first postovulation follicular wave and that post-ovulatory corpus luteum development and plasma progesterone concentrations would be similar in cows induced to ovulate with either oestradiol benzoate or gnrh. materials and methods experimental design and treatment of animals each cow in a group of 14 non-lactating cycling holsteins was injected intramuscularly with 25 mg of pgf2-alpha (lutalyse®, pharamacia & upjohn) on day –17 (where day 0 was the day of ovulation which was defined as the day on which an ovulatory follicle was last seen). a cidr® device (genetics australia, bacchus marsh, victoria), was inserted into the vagina of every cow on day –10, and left in situ for 8 days. every cow was injected intramuscularly with 2 mg oestradiol benzoate (cidirol®, gen etics australia) on that day and then with a further 25 mg of pgf2a on day –2. this was where the cidr devices were withdrawn. the cows were randomly allocated to three subgroups (n = 5 sub-group a; n = 5 sub-group b; n = 4 sub-group c). in round 1, cows in sub-group a were each injected intramuscularly with 1 mg oestradiol benzoate, 24 h after cidr device removal, (day –1), while every cow in the sub-group b was injected intramuscularly with 250 μg gnrh analogue (fert agyl®, intervet australia) 36 h after cidr device removal. cows in sub-group c received no further pro-oestrous treatment. the entire treatment procedure was repeated three times in a cross over design with each of the 14 cows receiving one of the three pro-oestrous treatments at a given round. a new cidr device was inserted into the vagina of every cow on day 11 of each round, and an intramuscular injection of 2 mg oestradiol benzoate on the same day so that day – 10 of one round was day 11 of the preceding round. each sub-group received the three pro-oestrous treatments following the same direction of order so that there were three sequences of the pro-oestrous treatments for the entire duration of the experiment, as shown in table 1. oestrus was observed using heatwatch® (ddx incorporated, denver, usa, www.heatwatch.com), 63 b.v.e. segwagwe, k.l. macmillan & p.d. mansell over a 72-h period beginning on the day of cidr removal. however, the heatwatch® was left on and recorded for the entire duration of the experiment. ultrasonography sequential examination of structures on the ovary of every cow was completed using an 7.5 mhz real time b-mode transrectal transducer connected to an ultrasound (aloka ssd-500®, aloka, tokyo, japan). this daily examination commenced on day –5 and ended on day 10 of each round. measurements of ovarian structures included the location and diameter of the follicles (mm), particularly those with a diameter of 4 mm or more, the location and the diameter of the corpus luteum. the growth rate of the first dominant follicle was defined as the change in diameter during the time interval from emergence until day 10, which was calculated by taking the difference in the diameter of the dominant follicle between day of emergence and day 10 divided by the number of days from emergence to day 10. ovulation was assumed to have occurred for an individual cow, after the disappearance of a follicle measuring over 8 mm in diameter, followed by the appearance of a corpus luteum at this site, as well as an increase in plasma progesterone concentration. blood sampling whole blood samples (10 ml) were collected from the coccygeal vein of every cow every alternated day from day –5 until day 9 (except for the catheterised cows on the days of intensive jugular bleeds (days –1 and 0). this sample was collected into a heparinised tube using a 22-gauge sterile venipuncture needle. plasma was separated using a centrifuge (clements sg 400®, clements, sydney, nsw, australia) at 2 000 rpm for at least 15 min. the plasma was pipetted into 5-ml plasma tubes in duplicates and stored immediately at –20 °c until assayed. just prior to assaying, the plasma samples were thawed in a cold room, at 2.5 °c overnight in addition, an intravenous catheter was surgically inserted into the jugular vein of each of the six cows on day –2, comprising of two cows per treatment sub-group. each of these selected six cows was placed in an individual pen. the whole blood (10 ml) was collected from the catheterised jugular vein at 0, 4, 8, 12, 14, 16, 20, 24 and 36 h relative to the time (t) of oestradiol benzoate treatment (t0, t4, t8, t12, t14, t16, t20, t24 and t36, respectively). plasma was separated, transferred into plasma tubes and stored until further assaying. the catheters were withdrawn from the jugular vein of each of the six cows after the last jugular bleed (36 h); at this time these cows were removed from the individual pens to join the rest of the cows in the trial. hormonal assays progesterone a commercial radioimmunoassay kit specific for progesterone was used (spectria® progesterone 125-i coated tube radioimmunoassay, orion diag nostica, finland). the principle of the test is that progesterone in the “unknown” samples competes with 125-i labelled progesterone tracer, for binding sites on tubes coated with polyclonal rabbit progesterone antibodies. a gamma counter, packard auto-gamma 5780® (united technologies, packard, australia) was used to measure 125-i progesterone bound to the polyclonal rabbit progesterone antibodies in the test tube. the progesterone concentrations (standards in bovine steer plasma and unknown samples) and quality control data were determined by the meth ods and computer program previously described (bur ger, lee & rennie 1972). the intraassay coefficients of variation (cv) for high, medium and low standard controls 11.3 %, 8.8 % and 8.2 %, respectively. the inter-assay cv for high, medium and low standard controls was 12.1 %, 18.1 % and 14.9 %, respectively. the minimum detectable concentration of progesterone was 0.12 ng/ml. oestradiol oestradiol concentrations were measured using methods previously described (prendiville, enright, crowe, finnerty, hynes & roche, 1995; burke, day, table 1 the sequence of treatments received by cows in the three sub-groups through the three rounds of the experiment sub-group round sequence 1 2 3 a b c oestradiol benzoate gnrh none gnrh none oestradiol benzoate none oestradiol benzoate gnrh o-g-n g-n-o n-o-g 64 gnrh or oestradiol on luteal function and follicular dynamics of oestrous cycle in holstein cows bunt & macmillan 2000). specifically, 200 μl aliquots of plasma and standards were extracted in 16 mm x 100 mm borosilicate glass tubes, using 2 ml diethyl ether by mixing in a multiple vortexer for a minimum of 15 min. the tubes were transferred into a freezing methanol bath, the solvent layer was decanted into 12 mm x 75 mm borosilicate glass tubes, and the solvent was evaporated in a fume hood. standards and samples were reconstituted in 300 μl of assay buffer (0.1 m pbs, ph 7.0, with 0.1% gelatine and 0.1% sodium azide). the first antibody (50 μl: kit antibody diluted 1:10 in assay buffer) was added to each tube. tubes were vortexed and incubated for 1 h at room temperature. lyophilised 125-oestradiol-17β was reconstituted in 20 ml assay buffer, and 50 μl was added to each tube; tubes were vortexed and incubated for an additional 2 h at room temperature. second antibody (300 μl of kit second antibody, covalently bound to magnetic particles) was added to each tube, vortexed, and incubated at room temperature for 20 min. placing the tubes in magnetic racks separated bound and free fractions. the supernatant was decanted and counts per minute in pellets were determined with a gamma counter. the intra-assay coefficients of variation (cv) for high, medium and low standard controls 3.5 %, 7.9 % and 7.1 %, respectively. the inter-assay cv for high, medium and low standard controls was 19.0 %, 20.8 % and 12.1 %, respectively. the minimum detectable con centration of oestradiol was 0.25 pg/ ml. statistical analysis one-way analyses of variance and fisher’s least significant difference were used to compare means from each round. a cross-over design was used to analyse all rounds combined, the effects being sequence vs animal within sequence, round vs residual and treatment vs residual. the pkcross command of stata was used (statacorp 2001). the effect of diameter of the ovulatory follicle at the time of treatment on the diameter of the corpus luteum on day 10, and the progesterone concentration on day 9 were analysed using the analysis of covariance. the interaction of treatment and the size of the ovulatory follicle, as well as cow were omitted from the statistical model, as they were not significant. results animals excluded from analysis the progesterone concentration of cow 9898 declined from 1.14 ng/ml to below 1 ng/ml on day –3 of round 2. after injection of 1 mg oestradiol benzoate on day –1, the progesterone concentration of cow 9898 remained below 0.1 ng/ml. this cow did not show behavioural signs of oestrus, did not ovulate, and did not develop a corpus luteum. this cow was excluded from the round 2 analyses. oestrus and follicular development none of the cows in the gnrh group (0/14) exhibited behavioural signs of oestrus while every cow in the control (14/14) and oestradiol benzoate groups (13/13) exhibited behavioural signs of oestrus (p < 0.01). all cows treated with gnrh (14/14), oestradiol benzoate (13/13) or left untreated (14/14) ovulated (p > 0.05). the interval from cidr device removal to onset of oestrus did not differ between oestradiol benzoate cows and control (44.7 ± 5.6 vs 46.5 ± 7.4 h for oestradiol benzoate and control cows, respectively. the dominant follicle of the first follicular wave emerged earlier in the control and gnrh treated cows than in those treated with oestradiol benzoate (p < 0.05, table 2, fig. 1). the growth rates of the first dominant follicle of cows in all the treatment groups were similar (averaged 1.24 ± 0.05 mm/day, p = 0.35, table 2, fig. 1). the dominant follicle ceased to grow on day 7.3 ± 0.2 (p = 0.55). there was early divergence in growth rate of the two largest follicles of the gnrh treated cows when compared to control (p < 0.05, table 2), but not to oestradiol benzoate treated cows (p > 0.05). however, the day of divergence of the growth rates of the two largest follicles in the first follicular wave did not differ (p > 0.12). on day 10, diameter of the dominant follicle tended to be smaller in the oestradiol benzoate treated cows (p = 0.08, table 2, fig. 1). concentration of plasma oestradiol the interval from emergence to peak oestradiol concentration was similar among all treatment groups (p > 0.24, tables 2). the peak oestradiol concentration was only different between the gnrh and control cows (p < 0.05, table 2) corpus luteum and progesterone profiles the diameter of the corpus luteum on day 10 and the growth rate of the follicles did not differ between the three treatment groups (p > 0.12, table 2, fig. 2). the corpus luteum was detectable earlier, by ultra sonography, in the control cows compared to the either the oestradiol benzoate or gnrh groups (p < 0.05, table 2). 65 b.v.e. segwagwe, k.l. macmillan & p.d. mansell the progesterone rise was earlier in control cows than in oestradiol benzoate or gnrh treated cows (p < 0.05, table 2). the plasma progesterone on day 9 was higher in the control than in oestradiol benzoate or gnrh treated cows (p > 0.05, table 2) discussion treating non-lactating holstein cows with either 1 mg oestradiol benzoate or 250 μg of gnrh during pro-oestrus influenced the development of the subsequent corpus luteum. cows treated with either table 2 follicular and corpus luteum development for all rounds combined parameter (mean ± sem) group p*control (n = 14) oestradiol benzoate (n = 13) gnrh (n = 14) total size of the ovulatory follicle just before ovulation (mm) 16.0 ± 0.7b (12–20) 14.8 ± 0.8b (11–20) 12.6 ± 0.8a (10–19) 14.4 ± 0.5 (10–20) 0.004 size of the ovulatory follicle at treatment (mm) 12.9 ± 0.9a (10–20) 12.1 ± 0.8a (9–18) 10.9 ± 0.7a (7–18) 11.9 ± 0.5 (7–20) 0.21 emergence day of first wave dominant follicle (day) 0.8 ± 0.2a (0–2) 1.7 ± 0.9b (0–3) 0.8 ± 0.9a (0–3) 1.1 ± 0.9 (0–3) 0.02 divergence day of first wave (day) 2.2 ± 0.2a (1–3) 2.8 ± 0.3ab (1–6) 3.0 ± 0.3b (2–5) 2.7 ± 0.2 (1–6) 0.10 day of reduction of growth of the first dominant follicle (day) 7.6 ± 0.3a (6–9) 7.2 ± 0.2a (6–8) 7.1 ± 0.3a (6–10) 7.3 ± 0.2 (6–10) 0.55 diameter of the first dominant follicle on day 10 (mm) 15.6 ± 0.8ab (10–20) 14.3 ± 0.5a (12–18) 16.3 ± 0.7b (10–20) 15.4 ± 0.4 (10–20) 0.08 growth rate of the first dominant follicle (mm/day) 1.18 ± 0.08a (0.63–1.67) 1.23 ± 0.07a (0.90–1.75) 1.31 ± 0.09a (0.60–2.14) 1.24 ± 0.05 (0.60–2.14) 0.35 first day corpus luteum detected, days (day) 2.6 ± 0.3a (1–4) 3.1 ± 0.2b (2–4) 3.3 ± 0.2b (2–4) 3.0 ± 0.1 (1–4) 0.02 diameter of corpus luteum on day 10 (mm) 25.5 ± 1.1a (20–30) 23.1 ± 0.7a (19–28) 22.4 ± 1.2a (17–34) 23.7 ± 0.6 (17–34) 0.12 growth rate of the corpus luteum (mm/day) 1.9 ± 0.2a (0.3–2.6) 1.8 ± 0.5a (0.5–2.3) 1.8 ± 1.0a (0.3–4.3) 1.8 ± 0.7 (0.3–4.3 0.99 maximum plasma pro gesterone concentration on day 9 (ng/ml) 5.0 ± 0.3b (2.7–8.3) 3.6 ± 0.2a (2.6–5.2) 3.6 ± 0.4a (1.4–6.6) 4.0 ± 0.2 (1.4–8.3) 0.02 progesterone rise (ng/ml/day) 1.22 ± 0.12b (0.68–2.07) 0.88 ± 0.06a (0.64–1.30) 0.87 ± 0.10a (0.31–1.62) 0.99 ± 0.06 (0.31–2.07) 0.02 maximum plasma oestradiol concentration (pg/ml) 1.4 ± 0.1a (0.9–2.5) 1.7 ± 0.1ab (1.1–2.6) 2.0 ± 0.3b (1.0–4.0) 1.7 ± 0.1 (0.9–4.0) 0.10 interval to peak oestradiol plasma concentration post-ovulation (days) 5.7 ± 0.3a (5–7) 5.5 ± 0.2a (5–7) 5.3 ± 0.2a (5–7) 5.5 ± 0.1 (5–7) 0.43 *p value derived from the crossover analysis a, b means with common superscripts did not differ at a significance level of 0.05 66 gnrh or oestradiol on luteal function and follicular dynamics of oestrous cycle in holstein cows 1 mg oestradiol benzoate or 250 μg gnrh had a significantly smaller corpus luteum on day 10; this corpus luteum secreted less progesterone than the corpus luteum formed after cows ovulated spontaneously. the consistent association of the treatment groups with the smaller corpus luteum strongly suggested an effect of induction of ovulation 24 or 36 h after cidr device removal with oestradiol benzoate and gnrh, respectively. these findings support the hypothesis that the development of corpus luteum in cows treated with gnrh and oestradiol benzoate at pro-oestrus was similar. however, the findings that control cows had a larger corpus luteum on day 10 than that of cows treated with gnrh or oestradiol benzoate was unexpected. the reasons for this outcome remain unclear. reduced plasma concentrations of progesterone in cattle during the luteal phase following ovulation with a gnrh agonist compared to spontaneous oestrus has previously been reported (schmitt et al. 1996); in those studies, cows that had been treated with gnrh at pro-oestrus and subsequently �� �� �� �� �� � � � � ��������� ��� � � �� � � � � � �� �� � �� � � �� �� �� � �� �� � � � � � �� �� � �� ��� � �� �� � � � � � � � ��������� ��� � � �� �� � � � � � �� � �� � � �� � � � � �� �� � � � � � � � � � �� � � �� �� � ���������������������������� ����������������� fig. 1 mean diameter of the first dominant follicle (a) and plasma oestradiol 17β (b) ± sem during the combined rounds � � �� � �� ��������� ��� � � �� � � � � � �� �� � �� � � � �� � �� � � � � �� � �� � � � � �� � � � � � � � � � ��������� ��� � � �� �� � � � � � �� � �� � �� � �� � � � �� � �� � � � � � � �� � �� � � �� �� � ���������������������������� ����������������� fig. 2 mean diameter of the corpus luteum (a) and plasma progesterone concentration (b) ± sem of the three treatment groups during days 1–10 for all rounds combined 67 b.v.e. segwagwe, k.l. macmillan & p.d. mansell had lower progesterone concentration also experienced short oestrous cycles. in the current study, the progesterone concentrations with corpus luteum diameter differed only on days 9 or 10 for progesterone concentration and corpus luteum diameter, respectively. transrectal ovarian ultrasonography and blood sampling were discontinued on days 9 and 10 of each round, respectively; therefore, the ovarian function after day 10 could not be described. future studies should perform similar trials and sequentially examine ovarian structures and steroids for the entire oestrous cycle in order to account for the effect of induction of a dominant follicle on ovarian function after day 10. the experiment was not designed to produce ovulatory follicles of different sizes at the time of administration of the pro-oestrous treatments. it was therefore not surprising that these follicles were similar at the time of the pro-oestrous treatments. interestingly, the final measured diameter of the ovulatory follicles was smaller in gnrh treated cows than when cows were either not treated or treated with oestradiol benzoate, when all rounds were combined (table 2). the ovulatory follicle diameter just prior to ovulation was left as an observation, and could not be included in the analysis as a covariate, or in the linear regression that analysed the relationship of the ovulatory follicle with the either the diameter of the corpus luteum on day 10 or progesterone concentration on day 9. this was due to the likelihood of a treatment effect that had been given 2 days prior to ovulation in the majority of cows. however, when the size of the ovulatory follicle at the time of treatment was added to the analysis, there was a relationship with the plasma progesterone concentration on day 10, but not with the diameter of the corpus luteum on day 9. these results must be interpreted with caution due to the low power of the performed tests. other studies have found an association between the maturity of the ovulatory follicle and the subsequent corpus luteum function has been reported previously (burke, mussard, grum & day 2001). the physiological importance of higher concentration of progesterone within the first days after ovulation is equivocal. while some workers reported that lower progesterone concentrations before, but not after insemination, were associated with lower fertility (vasconcelos, silcox, rosa, pursley & wiltbank 1999), it has also been shown that the early rise in plasma progesterone concentration within 5 days after ai was positively correlated with the mechanism of maternal recognition of pregnancy (mann & lam ming 1999). supporting the latter study is the recent report that pregnancy rates were lower in cows treated with oestradiol benzoate when the new wave had just emerged compared to when the newly emerged follicle ovulated spontaneously; the ovulatory follicle was smaller when it had just emerged, compared to oestradiol benzoate administration at dominance (lane, austin, roche & crowe 2001). perhaps this lowered fertility was due to the corpus luteum development post-ovulation, as seen in this study. synchronization protocols are usually initiated when the cows are at different stages in terms of ovarian follicular development. the relevance of the present study is that the subsequent corpus luteum may be partly compromised in cows where treatments for induction of ovulation are done when the follicle has only recently emerged. the concept of synchronization of emergence of a new wave as described previously (burke et al. 2000), could become most important so that the fertility of the ovulatory follicle was not compromised by the induction of ovulation with oestradiol benzoate or gnrh. there was a tendency for the corpus to be detected earlier when cows were in the control group. no reason was evident why these corpus luteuma were detected earlier in this group. on average, the corpus luteuma were first detected on day 3.0 ± 0.1 (range 1.0–4.0; table 2). this is in agreement with other studies (singh, pierson & adams 1997; kot & ginther 1999). although the corpus luteuma may start to form soon after ovulation (reynolds & redmond 1999), the most intensive growth in luteal tissue will occur between days 3 and 4 (kot & ginther 1999); changes in echogencity and histomorphology occur by day 3 (singh et al. 1997), corresponding with the increase in plasma concentrations that occurs at this time (wiltbank, shiao, bergflet & ginther 1995). treating non-lactating cycling holstein cows with 1 mg oestradiol benzoate at pro-oestrus delayed wave emergence of the subsequent cycle by 0.9 days. this delay was less than that reported in other studies (burke et al. 2000). in addition, the first dominant follicle reportedly emerges on average between days 0.2 to 4 of ovulation (knopf, kastelic, schal lenberger & ginther 1989). the emergence of the new follicle of cows in the control group in the current study was within this normal range limiting any phys iological significance of the delay. perhaps the magnitude of the delay when oestradiol benzoate was injected at pro-oestrus could be due to the minor role of oestradiol in the control of fsh during the periovulatory period (findlay, clarke & robertson 1990). studies using ovariectomised cow models (kesner 68 gnrh or oestradiol on luteal function and follicular dynamics of oestrous cycle in holstein cows & convey 1982) or immuno-neutralisation against either oestrogen or inhibin (kaneko, nakanashi, akagi, arai, taya, watanabe, sasamoto & hase gawa 1995) support the role of inhibin, but not oestradiol, in the control of fsh secretion during the preovulatory period. also, suppression of the fsh surge with charcoal-extracted bovine follicular fluid delayed emergence of a new wave by 2 days (turzillo & fortune 1990). collectively, these series of studies showed that oestradiol had a positive feedback e ffect on the secretions of fsh during the follicular phase, but exert a minor effect in suppressing fsh secretion in the absence of inhibin during the 24-h period following the preovulatory lh surge. it is therefore proposed that the slight delay in the suppression of emergence of the next wave in cows treated with 1 mg oestradiol benzoate at pro-oestrus was due to the decline in inhibin around ovulation; hence, the secondary fsh surge, which is thought to play a role in recruitment of a new cohort of ovarian follicles (kaneko, watanabe, taya & sasamoto 1992), was minimally suppressed, in every cow treated with oestradiol benzoate. there were no differences in the time from ovulation to the plasma maximum oestradiol concentration between the three treatment groups. these similarities cast further doubts on the significance of the delayed follicular emergence of oestradiol benzo atetreated cows in relation to subsequent follicular development. it would be expected that delayed follicular emergence would have delayed the peak oes tra diol concentration. when follicular emergence was delayed by 2 days in cows treated with charcoal extracted bovine follicular fluid, there was an associated delay in the interval from ovulation to peak plasma oestradiol concentrations (turzillo & fortune 1990). interestingly, the peak plasma oestradiol concentration occurred on day 5 and began declining by day 7. this decline in plasma oestradiol concentration coincided with the time when the dominant follicle reduced in its growth rate in each of the treatment groups. this is in agreement with a one study that showed that the morphological appearance of a dominant follicle was positively correlated with its functionality (ali, lange, gilles & glatzel 2001). this decline in plasma oestradiol also co incided with the time when a new follicular wave usually emerged in three-wave cycles (knopf et al. 1989). in this study, ultrasonography was not continued for sufficient time to retrospectively identify the emergence of a new follicular wave between days 6 and 10. however, since by definition, dominance of a follicle is lost when a new wave emerges (ireland, mihm, austin, diskin & roche 2000), and the decrease in plasma oestradiol on day 7 indicated this loss of dominance was similar to that reported previously (rhodes, fitzpatrick, entwistle & kinder 1995). none of the gnrh treated cows exhibited behavioural signs of oestrus, while all of the controls and oestradiol benzoate treated cows did show behavioural signs of oestrus. this effect of gnrh has been reported in other studies (mcdougall, cullum, anniss & rhodes 2001). the preovulatory lh surge released by gnrh decreased the concentration of serum and intrafollicular oestradiol within 1 h of treatment (kobayashi, coe & stevenson 1995), consequently inhibiting the expression of oestrus (stevenson, hoffman, nichols, mckee & krehbiel 1997). expression of oestrus in cows can be affected by whether or not the minimum threshold concentration of oestradiol in plasma required to induce oestrous behaviour has been achieved (glencross, esslemont, bryant & pope 1981). it was not surprising that none of the gnrh-treated cows in the present study exhibited behavioural signs of oestrus. other authors have found that with the ovsynch protocol, 30% of cows may show some behavioural signs of oestrus before the second injection of gnrh (stevenson, kobayashi & thompson 1999). this will occur in a population of cows where the first gnrh in the ovsynch protocol has been administered randomly to cows at various stages of the follicular development. in summary, induction of ovulation with either oestradiol benzoate or gnrh resulted in synchronized ovulation. cows that were induced to ovulate with oestradiol benzoate had delayed emergence of the new follicular wave (by 0.9 days) compared to gnrh-treated or control cows. dominant follicle that ovulated spontaneously were larger than those induced to ovulate with either oestradiol benzoate or gnrh. consequently, the corpus luteum of cows that ovulated spontaneously had a greater growth rate, resulting in a significantly larger corpus lutea on day 10. this corpus lutea secreted more progesterone than those formed when cows had been in duced to ovulate with either gnrh or oestradiol benzoate. these induced ovulations were followed by different patterns of corpus lutea development. one implication of findings from the present study is that induction of ovulation with either oestradiol benzoate or gnrh, administered 24 (oestradiol benzoate) or 36 h (gnrh) after withdrawal of the cidr device, respectively may lower fertility. the effect of this induction on the corpus lutea development after day 10 needs to be studied. in addition, future stud69 b.v.e. segwagwe, k.l. macmillan & p.d. mansell ies must identify the timing of administration relative to the time of cidr device withdrawal and the optimum concentration of oestradiol benzoate or gnrh that would not have untoward effects on the development of the corpus lutea, particularly within the first week of dioestrus. references adams, g.p. 1998. control of ovarian follicular wave dynamics in mature and prepubertal cattle for synchronization & superstimulation. proceedings of the xxth world buiatrics conference, sydney 6–10 july 1998 2:595–605. ali, a., lange, a., gilles, m. & glatzel, p.s. 2001. morpholo gical and functional 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods ethical consideration results discussion conclusion acknowledgements references about the author(s) evelyn madoroba bacteriology section, agricultural research council-onderstepoort veterinary institute, south africa college of agriculture and environmental sciences, university of south africa, florida campus, south africa daniel kapeta college of agriculture and environmental sciences, university of south africa, florida campus, south africa awoke k. gelaw bacteriology section, agricultural research council-onderstepoort veterinary institute, south africa citation madoroba, e., kapeta, d. & gelaw, a.k., 2016, ‘salmonella contamination, serovars and antimicrobial resistance profiles of cattle slaughtered in south africa’, onderstepoort journal of veterinary research 83(1), a1109. http://dx.doi.org/10.4102/ojvr.v83i1.1109 research project no.: 000791-y5 original research salmonella contamination, serovars and antimicrobial resistance profiles of cattle slaughtered in south africa evelyn madoroba, daniel kapeta, awoke k. gelaw received: 02 nov. 2015; accepted: 25 jan. 2016; published: 26 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract antimicrobial resistant salmonella are among the leading causes of foodborne infections. our aim was to determine salmonella contamination during cattle slaughter in south african rural abattoirs (n = 23) and environmental samples. furthermore, antimicrobial resistance patterns of the salmonella isolates were determined. samples of cattle faeces (n = 400), carcass sponges (n = 100), intestinal contents (n = 62), hides (n = 67), and water from the abattoirs (n = 75) were investigated for salmonella species using microbiological techniques and species-specific polymerase chain reaction targeting the inva gene. in total 92 salmonella species isolates were recovered. the salmonella mean frequency of occurrence on hides, carcasses, and intestinal contents was 35.37% (n = 81). eleven faecal samples (2.75%) tested positive for salmonella. the predominant serovar was salmonella enteritidis. diverse serovars that were identified on carcasses were not necessarily found on the hides and intestinal contents. the inconsistent occurrence of the diverse salmonella serovars on hides, carcasses, and intestinal contents implies that in addition to carriage on hides and in intestinal contents, other external factors also play an important role regarding carcass contamination. the 92 salmonella were serotyped and tested for susceptibility towards the following antimicrobials: ampicillin, cefotaxime, enrofloxacin, kanamycin, and oxytetracycline using the disk diffusion method. most salmonella (n = 66; 71.7%) isolates were resistant to at least one antimicrobial with highest resistance observed towards oxytetracycline (51.90%), which highlights the need for strict hygiene during slaughter and prudent antimicrobial use during animal production. in conclusion, cattle slaughtered in south african rural abattoirs harbour diverse salmonella serovars that are resistant to antimicrobials, which could be a public health risk. the findings should assist policymakers with improving implementation of hygienic slaughter of cattle in rural abattoirs, which is paramount from socioeconomic, public health, and epidemiological standpoints. introduction bacteria are among the leading causes of foodborne illnesses (buzby & roberts 2009). foodborne outbreaks result in socioeconomic challenges as a result of hospitalisations and associated medications, particularly among the vulnerable groups such as the elderly and immunocompromised individuals (gragg et al. 2013). among the bacteria, salmonella has been frequently linked to gastroenteritis worldwide (skov et al. 2007). salmonella causes salmonellosis, which is characterised by nausea, abdominal pain, diarrhoea, and sometimes fever that results in morbidity and in some instances mortality in both animals and human beings (nørrung & buncic 2008; velge, cloeckaert & barrow 2005). a study by majowicz et al. (2010) found that, globally, salmonella infection is responsible for an estimated 93.8 million cases of human gastroenteritis and 155 000 fatalities annually. salmonella has been associated with a number of food-producing animals, which makes animals and their products important sources of human infections (acha & szyfres 2001; davies, dalziel & gibbens 2004). the risk of salmonella contamination may be present at any stage of food animal production ranging from the live animal to environmental factors (alexander, warnick & wiedmann 2009; troutt & osburn, 1997). at the farm level, cattle hides may become exposed to salmonella through contact with contaminated faeces, feed, or the environment, which poses a risk to food safety if these organisms are transferred on the carcass during slaughter (arthur et al. 2007; brichta-harhay et al. 2008). cattle may also be contaminated with salmonella during transportation through contact with faeces of other animals. during slaughter, some procedures such as evisceration and splitting may contribute to carcass contamination (hui 2012). this is exacerbated by the asymptomatic carrier status of some cattle, which may pose a risk along the food chain (fegan et al. 2005; tadesse & tessema 2014). therefore, some of the sources of salmonella contamination are already present well before the animals are presented for slaughter. in this regard, strict hygienic processes during slaughter procedures are paramount in order to reduce the chances of meat contamination. in south africa, the meat safety act (no. 40 of 2000) provides for specific requirements on how red meat abattoirs should slaughter cattle. however, in some rural abattoirs meat inspection may not be routinely carried out, which often compromises safe meat handling. this potentially exposes rural communities to various foodborne pathogens including salmonella spp. there is paucity of information regarding prevalence of diseases and hygiene measures taken during cattle slaughter among communal cattle producers. therefore, it is imperative to obtain such information for better understanding of the potential risk of spreading salmonella from cattle slaughtered in rural abattoirs through the food chain and to institute proper and relevant situation-specific management strategies for foodborne diseases. the prevalence of antimicrobial resistance among foodborne pathogens has increased during recent decades (economou & gousia 2015). the increase in antimicrobial resistance among gram-negative bacteria such as salmonella may be attributed to the overuse of antimicrobials in food-producing animals for growth promotion, treatment of infection, or for prophylaxis (economou & gousia 2015). in south africa, the fertilizers, farm feeds, agricultural remedies, and stock remedies act (act 36 of 1947) makes provision for antimicrobial administration in animals (henton et al. 2011). this allows individuals such as farmers to access stock remedies over the counter (henton et al. 2011), which may contribute to antimicrobial overuse. however, the medicines and related substances control act (act 101) also controls veterinary medicines whereby antimicrobials for animal use are only prescribed by a veterinarian (henton et al. 2011). there is limited information regarding salmonella contamination of carcasses by hides and intestinal contents during cattle slaughter in south african rural abattoirs. information about antimicrobial resistance of salmonella isolates recovered from cattle in south african rural communities is scant. this study was aimed at contributing to knowledge about the extent of contamination of cattle carcasses by hides and intestinal contents during slaughter and antimicrobial resistance of the salmonella isolates. the objectives of this study were therefore to determine the presence and serovar diversity of salmonella on cattle hides, carcasses, and intestinal contents of cattle slaughtered in rural abattoirs (n = 23) of vhembe district in limpopo province of south africa. in addition, the occurrence of salmonella spp in water samples (used in abattoirs) and fresh cattle faeces from communities that supply rural abattoirs with animals for slaughter was determined in a parallel study. furthermore, antimicrobial resistance patterns of the salmonella isolates recovered from slaughtered cattle and environmental samples were determined. materials and methods study area and design vhembe district municipality is located in the northern part of limpopo province of south africa and it comprises four local municipalities; namely mutale, musina, makhado, and thulamela. in this study, samples were collected from cattle slaughtered in rural abattoirs of vhembe district from all the four local municipalities. a cross-sectional study involving 23 rural abattoirs that slaughter cattle was conducted between march 2011 and april 2012. to our knowledge, these represent all the rural abattoirs that had permission to slaughter cattle in vhembe district during the study period. the names of the abattoirs have been withheld for confidentiality. cattle for slaughter at the rural abattoirs were obtained from farms in any of the four local municipalities of vhembe district. in addition, some of the cattle were brought for slaughter by community members in preparation for events such as weddings, funerals, or other important family functions. the study targeted collection of matched 100 hide samples, 100 carcass swabs, and 100 rectal swabs. the sample size was determined using the following formula described in the european food safety authority (efsa) report on ‘development of harmonised survey methods for foodborne pathogens in foodstuffs in the european union’ (käsbohrer et al. 2010): where n = sample size; zα = desired confidence level at 95% (equivalent to zα value of 1.96); and l = accuracy, which was set at 0.05 in this study; p = annual expected prevalence. the annual expected salmonella prevalence in south african cattle is not formally documented, hence an approximate prevalence of 18.75%, which was based on a retrospective study by kidanemariam, engelbrecht and picard (2010) was used in this study. assuming a prevalence of 18.75%, the estimated sample size is 234, hence this study targeted 300 samples from cattle hides, carcasses, and intestinal contents. a parallel study involving collection of freshly voided cattle faeces (n = 400) was carried out in order to ascertain the possible risk of salmonella shed by cattle in vhembe district communities and to obtain information about asymptomatic animals that had potential of contaminating carcasses during slaughter. sample size was also based on the assumptions that were made for slaughtered cattle. water samples used in the abattoirs (n = 75) were also analysed for the presence of salmonella. sample collection carcasses samples were collected from carcasses (n = 100) using premoistened commercial beef carcass sampling polywipe kits (thermofischer scientific, waltham, ma, usa) as described in the united kingdom meat industry guide. using a sweeping motion, the sponge was rubbed firmly across the carcass from the hind quarter covering an area of 1000 cm2 per carcass. the polywipe sponge was placed in a sample bag, labelled appropriately and kept cool (but not frozen) by immediately placing in insulated cooler boxes containing frozen freezer blocks and transported to the feed and food analysis laboratory, bacteriology section of the agricultural research council-onderstepoort veterinary institute (arc-ovi). hides samples were collected from external hide surfaces (n = 67) by rubbing using a premoistened sterile sponge covering 500 cm2. briefly, sterile square metal templates covering 100 cm2 areas were placed onto hides and two sponges were used to swab five consecutive areas (antic et al. 2010). each sponge was placed in a separate stomacher bag and transported to the arc-ovi feed and food analysis laboratory within 24 h. intestinal contents intestinal contents (n = 62) were collected from the slaughtered animals and the samples were placed in sterile containers. the containers were immediately placed in cooler boxes containing frozen freezer packs. water water samples used in the abattoirs (n = 75) were collected according to instructions in the water research commission no tt117/99 (2000). briefly, the tap was opened and water was allowed to run for 3 min, followed by filling sterile bottles to about three quarters full. the bottles containing water were immediately placed in cooler boxes containing frozen freezer packs. faeces freshly voided faeces (n = 400) from cattle in the local communities were collected in sterile containers as a parallel study to determine the potential risk of salmonella during slaughter and among inhabitants of vhembe district. microbiological analysis salmonella isolation and identification for carcass and hide sponges, the samples were mixed thoroughly with maximum recovery diluents. the fluid mixture from the hide and carcass sponges (25 ml) was inoculated into 225 ml of non-selective pre-enrichment liquid medium containing buffered peptone water (bpw) supplemented with 1% tween 80 (1:10 v/v). for cattle faeces and intestinal contents, 10-g samples were inoculated into 90 ml of bpw supplemented with 1% tween 80. the inoculated bpw was incubated aerobically at 37 ºc for 18 ± 2 h. after incubation, 1-ml aliquots of the samples from bpw were inoculated into rappaport–vassiliadis soy broth (rvs) and tetrathionate broth (mktt) (rv; merck, darmstadt, germany), followed by incubation at 42 ºc for 24 h. loopfuls of the broth were plated onto xylose lysine desoxycholate agar (xld), rapidsal, and brilliant green agar (bga), followed by incubation at 37 ºc for 24 h. the plates were examined for the presence of typical salmonella colonies and further identified on the basis of biochemical tests. isolates that were positive for lysine decarboxylase, were motile, did not produce urease, produced hydrogen sulfide on triple sugar iron agar, fermented dulcitol, and had variable reactions to mannitol were identified as salmonella spp. isolation of salmonella from water samples was adopted from the standing committee of analysts (2002; hammarstrom & ljutov 1954). briefly, the bacteria in water were concentrated by passage through membrane filters. the membrane filters were transferred into bpw, followed by incubation at 37 ºc for 18 ± 2 h. a portion of broth (10 ml) was transferred into enrichment selective media (90 ml), followed by subculture onto xld, rapidsal, and bga. the plates were incubated at 37 ºc for 24 h and examined for colonies that are typical of salmonella species. for all samples, internal quality control was performed in parallel with the test samples and involved the use of salmonella typhimurium atcc14028 as positive control and escherichia coli atcc 25922 as a negative control. in addition, for water samples, blank control filters containing sterile distilled water (100 ml) and ringer’s lactate solution were included. this was done after normal sterilisation of the filtration unit. colonies that were pink and black centred on xld, pink on bga, and purple on rapidsal agar (merck, darmstadt, germany) were considered typical of salmonella spp and were subcultured onto blood tryptose agar (bta), followed by incubation at 37 ºc for 24 h. the pure cultures were confirmed using the following biochemical tests: triple sugar iron agar, urea agar, malonate broth, phenol red dulcitol broth, lysine decarboxylase broth, decarboxylase broth control, and thio-gelatine semisolid agar to test for motility. escherichia coli atcc 25922 and salmonella typhimurium atcc 14028 were included as negative and positive controls respectively. isolates that were confirmed as salmonella were preserved in nutrient broth supplemented with 35% glycerol and stored at -20 °c for subsequent species-specific polymerase chain reaction (pcr), antimicrobial susceptibility testing, and serotyping. salmonella serotyping salmonella serotyping was done as prescribed in the kauffman–white scheme. briefly, the salmonella isolates were tested against polyvalent and monovalent antisera for the presence of agglutination. the bacteria were tested for the presence of somatic (o) antigens, flagellar (h), and vi antigens. antimicrobial susceptibility test salmonella isolates (n = 92) were tested for antimicrobial susceptibility towards the following antimicrobial agents: ampicillin (amp), cefotaxime (cxt), enrofloxacin (enr), kanamycin (k), and oxytetracycline (ot) using the kirby–bauer disk diffusion method. briefly, the salmonella spp were suspended in physiological saline until the turbidity was equivalent to 0.5 mcfarland standard. the bacterial suspensions were inoculated onto mueller hinton agar, and disks were placed on the inoculated agar. the inoculated plates were incubated at 37 ºc for 24 h. the inhibition zones were measured using calipers and results were interpreted as sensitive, intermediate, or resistant according to clinical laboratory standards. the reference strains of e. coli atcc 25922 and salmonella typhimurium atcc 14028 were included alongside the field isolates. molecular identification the pcr was used to confirm identification made by phenotypic tests. dna extraction the salmonella isolates were resuscitated by inoculation into nutrient broth, followed by incubation at 37 ºc for 2 h. loopfuls from the nutrient broth were streaked onto nutrient agar, followed by incubation at 37 ºc for 24 h. the dna was extracted from salmonella colonies using the cell lysis method. briefly, salmonella cells were suspended in 1 ml of sterile distilled water. the bacterial suspensions were boiled at 99 ºc for 10 min, followed by cooling at room temperature and centrifugation at 13 000 rpm for 5 min. the supernatants were transferred into clean sterile eppendorf tubes and debris was discarded. the crude supernatant was used as dna template in the pcr reactions. polymerase chain reaction the 25-µl pcr reactions contained 12.5 µl dreamtaq master mix (fermenats; ontario, canada), 10 µm of each primer targeting the inva gene (invaf-5’-gtgaaattatcgccac gttcgggcaa-3’; invar–5’-tcatcgcaccgtcaaagg aacc-3’ (marlony et al. 2003), crude dna extract (5 µl), and molecular grade water (5.5 µl). part of the inva gene has been shown to be specific for salmonella, and if detected, it may be used to confirm the genus (nucera et al. 2006; rahn et al. 1992). the pcr mixture was amplified in a thermocycler (eppendorf, hamburg, germany) using the following conditions: denaturation at 95 ºc for 5 min, 35 cycles of denaturation at 95 ºc for 1 min, annealing at 56 ºc for 30 sec, and elongation at 72 ºc for 1 min. final extension was done at 72 ºc for 7 min. e. coli atcc 25922 and salmonella typhimurium atcc 14028 were included as negative and positive controls, respectively. in the negative pcr control, molecular grade water was used instead of dna. agarose gel electrophoresis the pcr amplicons were subjected to electrophoresis through 1.5% ethidium bromide stained agarose gel at 3 v/cm for 1 h. a 100 bp molecular weight marker was used for determining the size of amplicons. gels were visualised under ultraviolet light and the results were recorded using a gel documentation system (bio-rad; hercules, ca, usa). ethical consideration this study was approved by the agricultural research council. dr loock and dr mampane of limpopo provincial veterinary services offered permission to collect samples from rural abattoirs with the assistance of mr muthapuli. results salmonella species isolated table 1 shows a summary of the frequency of isolation of salmonella on hides, carcasses, and intestinal contents and the associated serovars. on average, the frequency of salmonella isolation on hides, carcasses, and intestinal contents was 35.37% (n = 81). most of the salmonella were isolated from hides (59.70%; 40/67), followed by carcasses (30%; n = 30). the frequency of occurrence of salmonella in intestinal contents was 17.74% (11/62). all the salmonella isolates had the inva gene successfully amplified. table 1: distribution of salmonella serovars according to samples types. no salmonella was isolated from the 75 water samples. out of the 400 freshly voided cattle faeces that were tested, 2.75% (n = 11) were positive for salmonella. salmonella serovars overall, out of the 92 salmonella spp isolated from hides, carcasses, intestinal contents, and freshly voided faeces, the predominant serovar was s. enteritidis (n = 8; 8.7%). other serovars that were identified include, s. heidelberg (n = 2; 2.2%), s. aberdeen (n = 1; 1.1%), s. hayindongo (n = 1; 1.1%), s. mbandaka (n = 2; 2.2%), s. anatum (n = 2; 2.2%), s. othmarschen (n = 1; 1.1%), s. nigeria (n = 2; 2.2%), s. tennessee (n = 1; 1.1%), s. cardoner (n = 1; 1.1%), s. senftenberg (n = 2; 2.2%), and s. pretoria (n = 1; 2.2%). the remainder of the isolates could not be serotyped to serovar level. these salmonella isolates belonged to omd and ome serogroups because they reacted to these anti-salmonella polyvalent somatic antisera. there were no monovalent antisera to further confirm the serovars of salmonella isolates that belonged to these groups. the distribution of salmonella serovars is summarised in table 1. antimicrobial resistance patterns overall, 66 (71.7%) of the salmonella isolates were resistant to at least one of the tested antimicrobial agents resulting in 14 resistance patterns (table 2). the serovars that were associated with the various resistance patterns are included in table 2. most salmonella were resistant to oxytetracycline (51.90%), followed by ampicillin (39.24%), kanamycin (29.11%), cefotaxime (26.58%), and enrofloxacin (11.39%). multidrug resistance (resistance to ≥ 3 antimicrobials) was observed in 25.32% of the salmonella isolates. table 2: summary of antimicrobial resistance patterns of salmonella isolates from this study. discussion the aim of this study was to determine the contamination of hides, carcasses, and intestinal contents of cattle slaughtered in 23 rural abattoirs of vhembe district, south africa by salmonella serovars, with a view to determining potential sources of contamination during cattle slaughter. all the sample types, with the exception of water were contaminated with salmonella to varying degrees. the serovars were diverse, and most of the salmonella belonged to group omd and ome and they were not typed to serovar level. numerous salmonella serovars belong to omd and ome groups; hence it is challenging to establish the public health significance of these isolates. some salmonella serovars that were observed on the cattle carcasses were not necessarily observed on the hides or intestinal contents, suggesting other potential contamination sources that were not analysed in this study. despite extensive cattle production in rural areas, most of the salmonella isolates were resistant to at least one of the tested antimicrobials and multidrug resistance was also observed. the hides, carcasses, and intestinal contents of cattle in this study showed notable levels of salmonella contamination. salmonella isolates on the hides are usually transferred from the environment or from faecal contamination during transportation of the animals to the abattoir (antic et al. 2010). the contamination of hides by salmonella that was observed in this study is not unusual because it is often not practical to control salmonella at the farm level; therefor, contaminated cattle are usually presented for slaughter (antic et al. 2010; bacon et al. 2000; small et al. 2002; vivas & buncic 2004). studies involving control measures for decontamination of hides have been done, but the practicalities are uncertain (mies et al. 2004). however, it is plausible to present relatively clean animals for slaughter to minimise the risk of carcass contamination during slaughter. the presence of salmonella in intestinal contents could be related to the asymptomatic carrier status of some cattle that continue to shed salmonella without showing any clinical signs (cummings et al. 2010). this could result in presentation of contaminated animals for slaughter, which poses a risk of transfer on carcasses. information on the prevalence of salmonella carrier status in african cattle is limited. in ethiopia, salmonella carrier status was observed to be 7.07% and 43.81% among cattle and pigs respectively (tadesse & tessema 2014). these asymptomatic animals may become a source of spreading salmonella in the herd and transmission of foodborne salmonellosis in humans. the proportion of carcass contamination in this study was relatively high and the potential sources of contamination were diverse. the carcasses could have been contaminated during hide removal or during evisceration. although molecular epidemiology tools such as pulsed-field gel electrophoresis and whole genome sequence analysis were not used to prove the unequivocal similarity of strains, it is highly likely that salmonella mbandaka and salmonella nigeria that were isolated from carcasses could be a result of cross-contamination from hides. likewise, the s. enteritidis on carcasses could have been transmitted from intestinal contents and faecal matter. however, s. enteritidis is not regularly isolated in cattle. nevertheless, 6 out of 232 s. enteritidis from cattle, poultry, sheep, goats, and pigs were isolated from cattle in a study that was undertaken in south africa from 1999 to 2006 (kidanemariam et al. 2010). use of the same equipment for slaughtering different animals and inadequate sterilisation of the utensils, which was observed during sampling, could have contributed to high chances of salmonella contamination. in some of the abattoirs, workers sharpened knives that were used during slaughter on unconventional objects such as stones despite the potential risk of contamination. it would be paramount to study the role of such practises in salmonella contamination of cattle carcasses. this was outside the scope of this study and constitutes a limitation of the study. some unconventional slaughter practises in a small proportion of the rural abattoirs could have also exacerbated the frequency of carcass contamination. for instance, despite the provisions of the procedure for cattle slaughter that are elaborated in the meat safety act (no. 40 of 2000), some workers in rural abattoirs still used the head of the animal as floor-support during hide removal, which increases the chances of contamination. such unhygienic practises lead to cross-contamination by foodborne pathogens including salmonella. this could have contributed to the inconsistent diversity of salmonella serovars that were observed on hides, carcasses, and intestinal contents. the separation of ‘clean’ and ‘dirty’ areas is usually proposed as one of the control measures to curb cross-contamination. however, separating ‘clean’ and ‘dirty’ areas may be extremely challenging in rural abattoirs from this study because animal slaughter took place in one room without compartments. this set-up highlights the need for strict physical and chemical decontamination programmes and regular inspection of the slaughter process as well as monitoring of the effectiveness of refrigeration of final carcass to minimise proliferation of salmonella (sofos & geornaras 2010). the use of chemical decontamination is not always acceptable in different geographical areas, and in some cases, the use of chemicals needs to be followed by rinsing of the chemicals with water (hugas & tsigarida 2008). little attention has been focused on the use of biological control such as competitive microbial cultures and bacteriophages. despite the potential effectiveness of physical, chemical, or biological control measures, the starting-point for producing safe food should be based on good hygienic practice and good manufacturing practice that are underpinned by hazard analysis critical control points management principles (nørrung & buncic 2008). in addition, training of both food handlers and consumers plays an important role in overall food safety. our findings are in contrast with a recent study that was conducted in abattoirs that slaughter cattle and pigs in vhembe district, south africa where salmonella were not isolated (tanih et al. 2015). the different findings could be because of the variation in number of abattoirs, source of animals, and types of samples. in addition, different isolation approaches were used. compared to tanih et al. (2015), our protocol used a relatively larger sample volume (25 ml) for pre-enrichment in buffered peptone water. in addition, this study used both mktt and rvs for selective enrichment and xld, bga, and rapidsal agar for culture that increases the likelihood for the recovery and detection of salmonella species. in the current study, it was demonstrated that salmonella isolates were shed in faeces of cattle from communities in areas that supply rural abattoirs in vhembe district, which poses a potential environmental health risk. this is particularly important because it was observed that cattle manure was used for boosting growth of vegetables such as cabbage, which may be consumed raw as salads. in addition, salmonella-infected cattle may pose a risk as they may cause cross-contamination during slaughter. in this study, diverse salmonella serovars were isolated from cattle hides, carcasses, intestinal contents, and faeces. s. anatum, s. tennessee, and s. pretoria were isolated from hides, but were not isolated from the carcass and intestinal contents. this indicates possible environmental contamination. in a similar study, s. anatum was isolated from hides, faeces, and subiliac lymph nodes (gragg et al. 2013). salmonella anatum was the predominant serovar found in a study in ethiopia that was aimed at determining antimicrobial resistance profiles and salmonella serovars in slaughterhouse personnel, the environment in the slaughterhouse, and beef cattle (sibhat et al. 2011). although s. anatum, s. tennessee, and s. pretoria have been rarely linked to clinical cases, s. anatum was implicated in the infection of a patient who consumed contaminated unpasteurised orange juice (krause, terzagian & hammond 2001). salmonella tennessee was deemed the causative agent of a nationwide outbreak of salmonellosis in the usa, which was likely linked to environmental contamination of a peanut butter plant (sheth et al. 2011). salmonella enteritidis was found on one of the carcasses, four intestinal contents, and three cattle faecal samples. it is likely that the s. enteritidis was transmitted from the intestinal contents, but this finding needs to be confirmed by strain typing techniques such as pulsed-field gel electrophoresis and whole genome sequence analysis. globally, salmonellosis in humans has been associated with s. enteritidis and s. typhimurium (hendriksen et al. 2011). the presence of s. cardoner and s. senftenberg on two carcasses from this study, but not on the hides or intestinal contents, highlights the presence of other sources of contamination that were not analysed in this study. although the information about clinical cases related to s. cardoner and s. senftenberg is scant, it is paramount to be vigilant about hygiene in order to reduce the risk of infection, particularly among individuals who may be immunocompromised. most salmonella in this study were resistant to ot. tetracycline resistance among food production animals has been attributed to selection pressure exerted from diverse sources such as prophylaxis, veterinary therapy, and use of antibiotics for animal growth promotion (chopra & roberts 2004; khachatourian 1998). the mechanisms of antimicrobial resistance may be broadly divided into genetic and phenotypic. genetic resistance may be because of chromosomal mutation, or acquired genes that are harboured on transposons or plasmids (khachatourian 1998). tetracycline resistance may occur through tetracycline modification, ribosome protection, and tetracycline efflux (chopra & roberts 2004). multidrug resistant salmonella were predominant in this study. this finding is significant because antimicrobial resistance carrying plasmids could be co-localised with virulence genes, which enhances invasiveness of the bacteria (brichta-harhay et al. 2011; helms, simonsen & molbak 2004). this could further complicate management of infection associated with multidrug resistant salmonella. multidrug resistant foodborne pathogens from carcasses highlight the risk associated with the challenge of treating both human and animal infections. the situation may be exacerbated in immunocompromised individuals who are usually highly susceptible. our findings are in harmony with observations of multidrug resistant salmonella isolated from meat and environmental sources in other studies (poppe et al. 2005). the presence of multidrug resistant salmonella pathogens warrants further investigation into general cattle farming practices and handling of veterinary drugs that might contribute to selection pressure in animals that are raised using extensive farming. the results further warrant a holistic and multidisciplinary approach to biosecurity and safety. it would be interesting to establish if there is any association between the antimicrobial resistance and virulence genes in the salmonella isolates from this study in future. it is important to determine the serovars of all omd, ome salmonella groups for epidemiological purposes. in addition, it is paramount to establish the salmonella pulsotypes and compare the patterns to those found in humans and other geographical areas. further studies are required to study the contamination of cattle carcasses by salmonella and the different serovars that are involved in different regions of south africa. it would be important to determine the link between similar serovars using technologies with higher resolution such as whole genome sequence analysis. conclusion the hides of cattle presented for slaughter in rural abattoirs of vhembe district are highly contaminated with salmonella and this may pose a risk of carcass contamination during slaughter. some asymptomatic cattle presented for slaughter contribute to carcass contamination because of salmonella in the intestines that has a high chance of being transferred onto the carcass. this could be exacerbated by not following proper slaughter procedure in some of the abattoirs. the differences among salmonella serovars on hides, carcasses, and intestinal contents illustrates that there are other sources of contamination during slaughter. antimicrobial resistance among salmonella from cattle and environmental samples of vhembe district was high. this poses a risk to consumers because the salmonella may proliferate along the food value chain. together, there is a need for regular assessment and inspection during animal slaughter in vhembe district rural abattoirs. acknowledgements this work was supported by the red meat research and development trust under cost centre 11/04/c246 and national research foundation-technology and human resources for industry programme under grant tp2011071300017. the work was done at the agricultural research council-onderstepoort veterinary institute. we are grateful to dr loock and dr mampane for giving permission to access rural abattoirs in vhembe district and for allocating mr muthaphuli to assist with abattoir identification and sample 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vivas, a.l. & buncic, s., 2004, ‘potential for use of hide-carcass microbial counts relationship as an indicator of process hygiene performance of cattle abattoirs’, food protect trends 24, 814–820. water research commision no tt117/99., 2000, quality of domestic water supplies: sampling guide, pretoria, south africa. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) alsagher o. ali division of infectious diseases, animal medicine department, south valley university, egypt hassan y.a.h. mahmoud division of infectious diseases, animal medicine department, south valley university, egypt citation ali, a.o. & mahmoud, h.y.a.h., 2017, ‘epidemiological studies based on multi-locus sequence typing genotype of methicillin susceptible staphylococcus aureus isolated from camel’s milk’, onderstepoort journal of veterinary research 84(1), a1425. https://doi.org/10.4102/ojvr.v84i1.1425 original research epidemiological studies based on multi-locus sequence typing genotype of methicillin susceptible staphylococcus aureus isolated from camel’s milk alsagher o. ali, hassan y.a.h. mahmoud received: 27 jan. 2017; accepted: 10 aug. 2017; published: 22 sept. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract one hundred milk samples were collected from camel’s milk for the isolation of staphylococcus aureus. thirty-one isolates were s. aureus, 45 were other forms of staphylococci and 24 represented other bacteria. five isolates from s. aureus were methicillin resistant s. aureus (mrsa) and 26 samples were methicillin susceptible s. aureus (mssa). the whole genome sequence of s. aureus was annotated and visualised by rapid annotation using subsystem technology (rast) which is a fully-automated service for annotating complete or nearly complete bacterial genomes. four isolates from mssa strains were subjected to multi-locus sequence typing (mlst). three multi-locus sequences types or sequence types (mlst/st) were found, namely st15, st1153 and st130. the phylogenetic analysis of the concatenated sequences of the seven genes forming the mlst profile of s. aureus classification revealed a high degree of similarity and close relationship between the st15 and st1153 while the third st (st130) was located in a different cluster. introduction staphylococcus aureus is a prominent pathogen causing a wide array of diseases in different animal species as well as in human beings. it has been subjected to numerous studies from different countries all over the world, from egypt (aly & abo-al-yazeed 2003), sudan (shuiep et al. 2009), saudi arabia (zahran & al-saleh 1977), ethiopia (semereab & molla 2001), morocco (benkerroum et al. 2003; khedid, faid & soulaimani 2003) and the usa (solyman et al. 2013). several molecular procedures have been established and used to identify and evaluate s. aureus isolates. coagulase gene (coa gene) typing is considered a simple and effective method for typing s. aureus isolates from human patients and bovine mastitic milk (aarestrup, dangler & sordillo 1994; da silva & da silva 2005; talebi-satlou, malahat & habib 2012; weese & van duijkeren 2010). epidemiological studies based on analysis of this gene have shown that s. aureus isolates could be divided into a number of subtypes (aslantaş et al. 2007). the multi-locus-sequence typing (mlst) has been used widely to outline the population genetic structure of s. aureus as well as other bacterial species. the mlst genotyping could identify different strains, track the spread of methicillin resistance and identify epidemic clones (holden et al. 2013; kurt et al. 2013; nübel et al. 2010). whole-genome sequencing provides the best discriminatory power between closely related bacterial isolates; hence, because of the rapidly decreasing cost and time required for this technology, it could conceivably become a viable tool in diagnostic laboratories in the near future to reconstruct intercontinental and local transmission of s. aureus lineages (harris, feil & holden 2010; köser, holden & ellington 2012). this present study aimed to investigate the effectiveness of mlst as a method of typing s. aureus isolates from camel origin as well as the implementation of multiple sequence alignment and phylogenetic analysis to clarify the molecular characterisation of mlst genes. materials and methods a total of 100 raw milk samples were collected from dairy she-camels in red sea governorate (al-shalateen area), egypt. animals were examined for body temperature, pulse rate and respiratory rate. animals were apparently healthy with no local or systemic infection. the milk samples were collected and stored in sterile plastic tubes. isolation and culturing of staphylococcus aureus staphylococcus aureus was isolated on baired-parker media. three to four typical colonies were harvested and picked up by a sterile metal bacteriological loop then immersed in glycerol and kept at -70 °c – -80 °c for further studies. biochemical tests the coagulase test was performed by two different methods, namely the slide coagulase test and the tube coagulase test (field & smith 1995). detection of meca gene the meca gene was utilised to affirm the classification of s. aureus into methicillin resistant s. aureus (mrsa) or methicillin susceptible s. aureus (mssa). the meca gene was analysed by multiplex pcr in addition to the mecc gene which was identified recently as a homologue for meca. the femb gene was used as a corroborative gene for s. aureus (alsagher 2016; nakagawa et al. 2005; paterson et al. 2014). whole genome sequencing and analysis staphylococcus aureus dna was used to obtain the genomic sequence of s. aureus by shotgun sequencing (sanger institute, uk). artemis was used to manipulate the genome sequence of s. aureus. this is a free genome browser and annotation tool that permits visualisation of sequence features, next generation information and the results of investigations inside the context of the sequence and furthermore its six-frame translation (alsagher 2016; rutherford et al. 2000). sequence alignments, translations and comparisons were done utilizing bioedit (version 7.0.9.0) (hall 1999). the blast algorithm was used to search the ncbi gen bank (http://www.ncbi.nlm.hih.gov/) databases for homologous sequences. neighbour-joining trees (saitou & nei 1987) were constructed on the basis of genetic distances and estimated by two-parameter method (kimura 1980) using mega6 (http://www.megasoftware.net) (tamura et al. 2013). the reliability of the trees was estimated by 500 bootstrap confidence values. to construct the phylogenetic tree, we used 15 sts retrieved from the mlst websites (http://www.mlst.net/) (7, 8, 9, 11, 17, 19,126, 127, 128, 132, 1146, 1147, 1148, 1149 & 1150). in addition, 3 sts (15, 130 & 1153) were extracted from the bacterial chromosome of local isolates. these sts were used to construct the phylogenetic tree. results standard culturing methods and the application of coagulase tests for 100 milk samples of she-camels revealed that 31 isolates were s. aureus, 45 isolates were other forms of staphylococci, and 24 isolates were other bacteria. according to the presence or absence of the meca gene in s. aureus isolates, five isolates were mrsa and 26 were mssa. the whole genome sequence of s. aureus was annotated by using subsystem technology (rast) which is a fully-automated service for annotating complete or nearly complete bacterial genomes (http://rast.nmpdr.org/) and artemis (rutherford et al. 2000). four isolates from s. aureus were sequenced by the shotgun sequencing technique and the whole genome sequence was obtained. the mlst genes were annotated, extracted and subjected to further analysis to predict the sequence typing by using the mlst websites (http://www.mlst.net/). one local isolated (a11) gave st 15 (cc 5), local isolates (a12 & a13) gave st 130 (cc 130) and the local isolate (a15) gave st1153 (cc 1153) (table 1). table 1: the sequence similarity and length of multi-locus sequence typing genes of staphylococcus aureus local isolate (a11) and their reference alleles of st15. the seven genes used in s. aureus mlst genotyping (yqil, aroe, glpf, gmk, pta, tpi & arcc) were highly polymorphic and they were divided into numerous numbers of different alleles. the allelic composition of each sequence type (st), similarity and length were shown for st15, st130 and st1153 in (tables 1, 2 and 3) respectively. table 2: the sequence similarity and length of multi-locus sequence typing genes of staphylococcus aureus local isolate (a12 and a13) and their reference alleles of st130. table 3: the sequence similarity and length of multi-locus sequence typing genes of staphylococcus aureus local isolate (a15) and their reference alleles of st1153. the multiple sequence alignment of the translated amino acid sequences of the concatenated mlst genes were shown and aligned with selected other sequence types of s. aureus (figure 2). the evolutionary history was inferred by using the maximum likelihood method based on the tamura-neimodel (tamura & nei 1993). the analysis involved 18 nucleotide sequences. all positions containing gaps and missing data were eliminated. there were a total of 3186 positions in the final data set. the phylogenetic analysis revealed that st15, st130, st1153 and other sequence types used to construct the phylogenetic tree were divided into two main clusters. the st15 and st1153 were closely related to each other and gathered in one cluster away from the third st130 (figure 1). figure 1: molecular phylogenetic analysis by maximum likelihood method of multi-locus sequence typing concatenated sequences of staphylococcus aureus. discussion classification and characterisation of s. aureus isolates is an essential and preliminary step to understanding the epidemiological status of this contagious bacterium. nowadays, mlst genotyping is widely used to investigate the dynamic nature of s. aureus. the implementation of the new techniques such as the whole genome sequence can potentially facilitate the mining of bacterial chromosomes for all the s. aureus genes. in our study, traditional culturing methods and the coagulase test revealed that 31% of the isolates were s. aureus, 45% isolates were other forms of staphylococci and 4% were other pathogens. these results were higher than the 14% reported by abdel hameid et al. in 2004. using the meca gene to classify s. aureus isolates into 16.12% mrsa and 83.87% mssa, our results contradict those obtained by monecke et al. (2011b) which showed that absence of meca gene and the rarity of antibiotic resistance related genes in s. aureus isolates from camel’s milk. the mlst analysis of four isolates of mssa revealed that three sequence types were recorded (st15, st130 and st1153) in (a11, a12, a13 and a15) local isolates respectively (table 4, figures 1 and 2). the presence of st15 in mssa isolates was in agreement with another study in livestock and humans (sanz 2013). st130 was found in two local isolates (a12 and a13), both of which were mssa strains. this finding was in agreement with the findings of shore et al. (2011) and le maréchal et al. (2011) in sheep and human isolates respectively. the st1153 found in a15 local isolate was in agreement with a study of enany et al. (2010) and moneck et al. (2011a) which indicated that the pvl+mssa had hallmark characteristics from pvl+mrsa strains in that it possessed a novel mlst (st1009) with a single variant (st1153) as well as double locus variants (st1216) (table 4, figure 2). figure 2: multiple sequence alignment of amino acids of concatenated sequences of multi-locus sequence typing genes. table 4: the sequence type and the colonal complex of local isolates of staphylococcus aureus isolates. conclusion mlst genotyping is a useful way to classify s. aureus which can also provide a highly efficient molecular epidemiological investigation. three sequence types (st15, st130 and st1153) were obtained in this study from mssa isolated from she-camel’s milk. phylogenetic analysis revealed close similarity between st15 and st1153, while the other st130 is located in a separate cluster of other sequence types. further investigations using this approach are needed to make a clear molecular epidemiological survey on s. aureus from camel’s 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introduction the inoculation of theileria parva sporozoites into cattle usually causes east coast fever (ecf), an acute and often fatal lymphoproliferative disease of major economic importance in eastern, central and southern africa (young, groocock & kariuki 1988). this obligate intracellular parasite is transmitted mainly by the three-host tick, rhipicephalus appendiculatus, from which sporozoites can be extracted. these sporozoites extracts are then cryopreserved as stabilates and used for immunization by the infec tion and treatment (i&t) method, challenge of immune or vaccinated animals, in vitro testing and re search investigations. the i&t method of immuni zation (radley, brown, burridge, cunningham, kir imi, purnell & young 1975) is the only means currently available 207 onderstepoort journal of veterinary research, 73:207–213 (2006) infectivity of theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing: evaluation by in vitro titration v. mbao1, d. berkvens2, t. dolan3, n. speybroeck2, j. brandt2, p. dorny2, 4, p. van den bossche2, 5 and t. marcotty2* abstract mbao, v., berkvens, d., dolan, t., speybroeck, n., brandt, j., dorny, p., van den bossche, p. & marcotty, t. 2006. infectivity of theileria parva sporozoites following cryopreservation in four suspension media and multiple refreezing: evaluation by in vitro titration. onderstepoort journal of veterinary research,73:207–213 theileria parva sporozoite stabilates are used for immunizing cattle against east coast fever and in in vitro sporozoite neutralization assays. in this study, we attempted to identify a cheaper freezing medium and quantified the infectivity loss of sporozoites due to refreezing of stabilates, using an in vitro technique. pools of stabilates prepared using minimum essential medium (mem), roswell park memorial institute (rpmi 1640), foetal calf serum (fcs) and phosphate-buffered saline (pbs) were compared. all were supplemented with bovine serum albumin except the fcs. rpmi 1640 was as effective as mem in maintaining sporozoite infectivity while the infectivity in pbs and fcs reached only 59 % and 67 %, respectively. in a second experiment, a stabilate based on mem was subjected to several freeze-thaw cycles including various holding times on ice between thawing and refreezing. refrozen stabilate gave an average sporozoite infectivity loss of 35 % per cycle. the results indicate that rpmi can be used as a cheaper freezing medium for t. parva stabilates and that refrozen stabilate doses need to be adjusted for the 35 % loss of infectivity. keywords: cryopreservation, culture media, in vitro, refreezing, sporozoites, theileria parva, ticks * author to whom correspondence is to be directed: e-mail: tmarcotty@itg.be 1 department of veterinary and livestock development, ministry of agriculture and cooperatives, p.o. box 670050, mazabuka, zambia. present address: department of animal health, institute of tropical medicine, nationalestraat 155, b-2000 antwerp, belgium 2 department of animal health, institute of tropical medicine, nationalestraat 155, b-2000 antwerp, belgium 3 livestock services limited, p.o. box 24437, 00502 karen, nairobi, kenya 4 department of parasitology, faculty of veterinary medicine, ghent university, salisburylaan 133, b9820 merelbeke, bel gium 5 department of veterinary tropical diseases, faculty of vet erinary sciences, private bag x04, onderstepoort, 0110 south africa accepted for publication 11 april 2006—editor 208 infectivity of theileria parva sporozoites: evaluation by in vitro titration for immunizing cattle against ho m ol ogous challenge (uilenberg 1999). the technique requires simultaneous inoculation of t. parva sporozoites and a long acting tetracycline. it is widely used in several provinces of zambia and other countries in the region (ui len berg 1999; marcotty, billiouw, chaka, berkvens, losson & brandt 2001; fandamu, thys, duchateau & berkvens 2006). univalent stabilates are used in zambia but stabilates containing several t. parva stocks are required elsewhere, e.g. in tanzania (morzaria, nene, bishop & musoke 2000). the extraction of t. parva sporozoites into different media has been described for both experimental and field-use stabilates (cunningham, brown, burridge, joyner & purnell 1973a; cunningham, brown, burridge & purnell 1973b; purnell, brown, cunningham, burridge, kirimi & ledger 1973; kimbita, silayo & dolan 2004). these media include bovine serum and eagle’s minimum essential medium (mem) supplemented with bovine serum albumin (bsa). the stabilates for field immunization are routinely produced using mem supplemented with bsa. well-characterized and homogeneous stabilates need to be available for immunizing cattle against east coast fever, use in in vitro sporozoite neutralization assays and research in general. for developing countries, they should be cheap and easy to produce. the powdered formulation of mem is much cheaper to import into africa than the liquid form but is not always readily available. therefore, other media for their efficiency in maintaining sporozoite infectivity were evaluated. two media to test and compare with mem were selected, namely the powder formulation of roswell park memorial institute (rpmi 1640) and phosphate-buffered saline (pbs). rpmi 1640 is the most common medium used for cell culture while pbs, which does not contain nutrients, is a very basic buffer solution found in most laboratories. bsa, on the other hand, is expensive. hence, the possibility of using fcs instead of mem with bsa was investigated. the objective of the second study was to quantify the loss of infectivity for stabilates undergoing a refreezing step after production. this technique is used in the production of polyvalent ecf vaccines to allow titration of individual components before mixing them. it is also envisaged that, with the onset of veterinary services privatization in zambia and several other countries in the region, stabilate refreezing may be considered by some animal health service providers in an attempt to salvage left-over doses after an immunization campaign. refreezing may also be useful in cases where homogeneous stabilate needs to be used at different time periods for research work. ensuring homogeneity of the stabilates by pooling and refreezing aliquots for use in particular sets of experiments is one practical method of removing variability in infectivity seen in stabilate taken from different storage vials (v. mbao, unpublished observations 2005). from immunization protocol using re frozen stabilates (njuguna & musisi 1996), it is known that t. parva sporozoites do survive refreezing cycles and it is expected that they lose some viability at each cycle. it is, however, not known to what extent. there is therefore need to have empirical data on the effect of such a process for quality assurance. in this study, sporozoite infectivity was evaluated after single and multiple refreezing cycles. multiple cycles were included to amplify any effects there might be, and to aid in calculating the average loss per cycle. titration of stabilates was done in vitro. equivalence testing (mbao, speybroeck, berkvens, dolan, dorny, madder, mulumba, duchateau, brandt & marcotty 2005) was used to calculate the effect of alternative media on sporozoite viability compared with the standard medium (mem/bsa) and quantify loss of sporozoite infectivity due to refreezing. a random effect model was applied in view of the levels of confounders at tick batches, grinding pools and storage vials. materials and methods media all media and additives were obtained from invitrogen (carlsbad, california), unless otherwise stated. the following media were used for preparation of the sporozoite suspensions: mem (with earle’s salts), rpmi-1640 (powder formulation), pbs and heat inactivated fcs. the mem, rpmi and pbs solutions were supplemented with bsa (acros organics, belgium) at 35 g/l and all of them, including fcs, with l-asparagine (bdh biochemical, uk) at 100 mg/l hepes (25 mm/l), penicillin-streptomycin at 100 iu/ ml and kanamycin at 100 μg/ml. the ph of media was adjusted to 7.0–7.2 using sodium bicarbonate. stabilate preparations three batches of nymphal r. appendiculatus ticks were infected at different times and locations with the t. parva katete stock and allowed to moult to adults in an incubator at 22 °c and 85 % relative humidity. six to 8 weeks after engorgement as nymphs, the adult ticks were fed on rabbits for 4 days to in209 v. mbao et al. duce sporogony of t. parva (fao 1984) and removed. the ticks in batches 1 and 2 were divided randomly into groups as shown in table 1 and ground separately in each of the four media in the volumes shown. for batch 1, eight groups were ground (table 1) using an omni-mixer homogeniser® (omni inter national, usa, model 17106) following the standardized international protocol (oie 2005) with some modifications (mbao et al. 2005). the batch 2 ticks were ground using an ultraturax® tissue homogeniser (jan ke & kunkel kg, staufen, germany, model tp18/2). batch 3 ticks were ground manually using a mortar and pestle for 15 min (cunningham et al. 1973b). the different methods of grinding were used due to different laboratory set-ups in the three places where the stabilates were produced. an equal volume of chilled medium with 15 % (w/v) glycerol was added drop-wise to the ground up tick supernatant (fao 1984). the extracts were stirred continuously in an ice bath. the 13 suspensions containing the numbers of ticks per ml (tick equivalents [t.e.]) are shown in table 1. all suspensions were aliquoted into 1.5 ml cryogenic vials (nalgene) (1 ml per vial), cooled in an ultra freezer at –80 °c for 24 h and then stored in liquid nitrogen. refreezing cycles the stabilate from batch 3 was used for the stabilate refreezing experiment. a ‘cycle’ was defined as a refreeze and subsequent thaw process. vials were thawed by placing them in a water bath at 37 °c for 5 min. before the first refreeze cycle, thawed vials were pooled to ensure homogeneity, centrifuged (400 g for 10 min) to remove fungi and yeasts (marcotty et al. 2004) and supernatants re-aliquoted. in a first step, two titration sessions were set up to compare control and single-refreeze stabilates. aliquots of the thawed, pooled and centrifuged stabilate material were kept on ice (control material, not for refreezing) while the rest was refrozen. refreezing was for 1.5 h in a –80 °c freezer. in a second step, five groups of vials from the same homogeneous pool were subjected to several cycles. all groups were refrozen once. after 1.5 h, four of these groups were thawed and refrozen. after a further 1.5 h, three groups from the previous four were refrozen and this continued until the last group had undergone a fifth cycle. each group had been subdivided into three subgroups and each of these subgroups was held on ice for 5, 30 and 60 min before a subsequent refreezing. in vitro titrations the protocol for in vitro titration of t. parva tickderived stabilates that was used is that described by marcotty, speybroeck, berkvens, chaka, besa, madder, dolan, losson & brandt (2004) and modified by mbao et al. (2005). briefly, test stabilate is serially diluted in 96-well microtitration plates and then bovine peripheral blood mononuclear cells are added to the wells. the plates are incubated for 10 days at 37 °c in 5 % co2 after which cyto-centrifuged samples are taken, giemsa stained and microscopically examined for t. parva macroschizonts. titrations for media comparison were conducted in two stages. the first stage comprised six sessions in which all four media were titrated in parallel. there were four sessions for tick batch 1 stabilates and two for tick batch 2 stabilates. in the second stage, only mem and rpmi 1640 stabilates were compared in six sessions: four sessions for tick batch 1 stabilates and two for tick batch 2 stabilates. the total number of microtitration wells read for the first and second stages were 855 and 574, respectively. stabilates from batch 1 were diluted serially six times (1.5 times per dilution step) and those from the second (batch 2) 12 times (1.5 times per dilution step). this was necessitated because the first batch had lower infectivity, as determined by a preliminary in vitro titration. table 1 number of ticks ground per batch per medium medium batch 1 batch 2 batch 3 vol* (ml) final conc. (t.e./ml) mem pbs rpmi fcs 20 5 200 200 200 200 20 5 200 200 200 200 25 10 500 500 500 500 50 10 1 000 * volume of medium per group 210 infectivity of theileria parva sporozoites: evaluation by in vitro titration the multiple frozen and thawed stabilates (cycles 1–5) were titrated in parallel a day after the refreezing cycles. six and two titration sessions for the multiple freezing and control experiments, respectively were conducted. a total number of 1 115 and 335 microtitration wells were read for multiple freezing and control experiments, respectively. statistical analysis data from the two experiment stages of media comparison (i.e. all four media and mem vs rpmi) were pooled. wells either scored positive or negative. the binary results were analysed by logistic regression using generalized linear latent and mixed models (gllamm) in stata8/se® (statacorp, 2003). the proportion of positive wells was the response variable. the three explanatory variables were: the natural log of the stabilate concentration in tick equivalents (ln t.e.), the experiment stage and the stabilate medium. several random effects that could affect the model were identified, namely the tick batch, grinding pools and stabilate storage vials. random effects are factors or hidden variables that do not interest us but still have an effect on the variability of the data and as such should not be ignored during the analysis. comparison of respective infectivities of test stabilate media to those of mem stabilates were conducted by calculating the ratios of effective doses edmem/edx  where ed = the effective dose, in tick equivalent, of stabilate that results in a given proportion of wells to be positive and x = test media. the ratios were calculated by use of non-linear combination of estimators (nlcom) in stata8/se® (mbao et al. 2005) which also fits 95 % confidence intervals around the estimates (ratios). the level of significance was set at 5 %. the same model was used for the assessment of the effect of multiple freezing. the natural log of the stabilate concentration (ln t.e.), the cycle number and the holding time were continuous explanatory variables. the cycle was regarded as a continuous variable so as to enable estimation of a loss of infectivity per cycle. the response variable was proportion of pos itive wells and ‘session’ of titration was a random effect. ratios of effective doses, edx/edx + 1  where x = cycle number or holding time, were calculated to compare sporozoite infectivities at each cycle and holding time. to express this as infectivity losses between subsequent cycles, the calculated ratios were subtracted from unit (1-ratio) e.g. a ratio of 0.99 per cycle is actually 1 % loss of infectivity per cycle. results differences in the infectivity of stabilates prepared and stored in pbs (n = 215), rpmi (n = 502) and fcs (n = 212) were not statistically significant (p > 0.05) when compared to mem (n = 500). estimates of ed ratios were 0.59, 1.03 and 0.67 for pbs, rpmi and fcs, respectively (table 2). the regression curves of predicted values, comparing infectivities, were table 2 ratios of effective doses (edmem/edx) with lower and upper 95 % confidence limits medium estimate* lower limit upper limit pbs rpmi fcs 0.59 1.03 0.67 0.31 0.63 0.35 1.14 1.67 1.29 * this ratio is an indicator of the relative infectivity of stabilates in comparison to the reference (mem) mem = minimum essential medium x = test stabilate medium (pbs, rpmi or fcs) table 3 percentage loss of sporozoite infectivity (1– ed ratio) after several cycles of refreezing with lower and upper 95 % confidence limits cycle estimate lower limit upper limit single multi 32 % 35 % 5 % 28 % 52 % 40 % ed (effective dose) ratio = edcycle/edcycle +1 211 v. mbao et al. very close, with mem and rpmi showing a superimposition of their curves (fig. 1). while the dose (ln t.e.) was significant (p < 0.001), the stage did not significantly influence the outcome (p = 0.10). a single re-freezing cycle resulted in a significantly lower infectivity (p = 0.03) than the control kept on ice for 1.5 h (fig. 2). the ed ratio was 0.68 (95 % ci: 0.48–0.95) and the estimated loss in infectivity was 32 %. the loss during multiple freeze cycles was also significant (p < 0.001). on average, the ed ratio was 0.65 (95 % ci: 0.60–0.72) giving an estimated loss of 35 % per cycle (fig. 3). in both single and multiple refreezing cycles, dose effect was significant (p < 0.001). these results are shown in table 3. holding times were not significant (p = 0.88) nor was the random effect of session (p = 0.34). a stabilate kept on ice for 1 h did not lose more than 30 % of its infectivity. discussion sporozoite infectivities for stabilates prepared in pbs, rpmi and fcs when compared to mem were not statistically significantly different. however, the rpmi stabilates were closest in infectivity to mem stabilates as shown by the equivalence test estimate (minimum 0.63 times). this observation agrees with the findings of kimbita et al. (2004) when they compared different media including leibovitz-15, optimem and iscove’s mem. in their study, rpmi and mem stabilates showed similar infectivity when compared to l-15. gray & brown (1981) showed that neat serum could have inhibitory effects on sporozoite infectivity and this could explain why the ed of fcs was lower than that of mem. however, the effect may not be very pronounced as the confidence interval is wide. fcs is expensive (about €107/l) due to the stringent standards required for international distribution. if it could be produced locally in existing fig. 2 titration curves of stabilate that underwent a single refreeze/thaw cycle (broken line) and control stabilates (1.5 h storage on ice) (solid line) � �� � �� �� � �� � �� �� � � � � �� �� � � � �� � ��� ��� ��� ��� ��� �� �� ��� ��� ��� � ���� ��� ����� �� ����� �������� � ������ ������� � ������ ������� �� ����� ��� � �� � �� �� � �� � �� �� � � � � �� �� � � � �� � ��� ��� ��� ��� ��� �� �� ��� ��� ��� � ���� ��� ����� �� ��������� � ����������� � ����������� �� ���������� �� ���������� �� fig. 1 titration curves of rpmi (thin continuous line), mem (thick broken line), fcs (thick continuous line) and pbs (thin broken line) based sporozoite stabilates � �� � �� �� � �� � �� �� � � � � �� �� � � � �� � ��� ��� ��� ��� �� �� ��� ��� ��� � ���� ��� ����� �� ����� �������� � ����� ������ � ������ ������ �� ���� ��� � � � fig. 3 titration curves of multiply frozen stabilates (cycles 1–5) 212 infectivity of theileria parva sporozoites: evaluation by in vitro titration regional laboratories with nationally or regionally accepted quality control standards from known naive dams, it could be very much cheaper than international grade fcs. newborn calf serum might also be used (€67.4/l) and would be much cheaper from local sources. the cost of using locally produced reagents would then be lower than commercial media supplemented with bsa (1 l of rpmi with bsa at 3.5 % costs €72). the pbs stabilates were also less infective than mem stabilates but still showed a reasonably high infectivity (59 % of that of mem). it could be that the nutritive value of the defined medium components may not be as critical as their buffering and cryoprotective qualities during storage. if this is the case, pbs supplemented with bsa has the potential to be the cheapest alternative medium (€62/l). although pbs and fcs were found to maintain a high proportion of the infectivity of the sporozoite dose, their suitability needs further investigation. one refreezing cycle significantly decreased the infectivity of sporozoites in comparison to storage for 1.5 h on ice. the latter should not affect the infectivity as marcotty et al. (2001) found that keeping a stabilate on ice for up to 6 h did not reduce sporozoite infectivity significantly. although the actual freezing temperature was not measured in these experiments, 1.5 h was found to be sufficient to freeze the vial contents to –60 °c (njuguna & musisi 1996). this is below the critical temperature range (–20 to –50 °c) at which ice crystals form and raise extra-cellular solute concentrations, a process that is detrimental to cell integrity in cryopreservation (farrant 1970). subsequent refreezing cycles caused similar losses in infectivity. refreezing of stabilates seems to induce considerable loss in infectivity. attempts to refreeze stabilates left over from field immunization would result in low quality stabilates that may not be protective upon inoculation. it would be helpful to have this information in the extension packages for stabilate delivery especially for the private sector involved in ecf immunizations. a re-titration of such stabilate may be necessary to determine appropriate immunizing doses. this would be beyond the expertise of the service provider. moreover, they may not have appropriate equipment to undertake a standard refreezing process. in unavoidable situations, e.g. polyvalent ecf vaccine production, it would be advisable that the infectivity loss is considered and immunizing doses adjusted accordingly. the process of refreezing may be useful in research work for reducing variability arising from different storage vials. this involves thawing the stabilate, pooling the contents of vials and refreezing for later use. holding the stabilates for up to 1 h on ice did not reduce the quality or infectivity of stabilates significantly. this is important as the process of preparing stabilates, particularly centrifuging and aliquoting, takes time before the stabilate is ready for freezing. in addition, including “batch” as a random effect not only takes into account the tick batches but the different methods of grinding (omni-mixer homogen iser®, ultraturax® and manual) used in these exper iments. in conclusion, the study confirms the findings of kimbita et al. (2004) that rpmi 1640 is as effective as mem in supporting sporozoite infectivity. therefore, it is recommended that rpmi 1640 that is properly supplemented can be used as an alternatively cheaper freezing medium in t. parva stabilate production where mem is either too costly or not available. we also showed that there is an estimated loss in sporozoite infectivity of 35 % when stabilates are refrozen. this loss needs to be adjusted for in both research and field use stabilates. acknowledgements the study was supported financially by the belgian government through the “assistance to the vet erinary services of zambia” (asveza) project in collaboration with the government of the republic of zambia. we are grateful to the technical staff at the centre for ticks and tick borne diseases, lilongwe, malawi, where some of the stabilates were produced, and at the institute for tropical medicine, antwerp, where the titrations were conducted. we thank anke van hul for her invaluable help in microscopic slide processing. references cunningham, m.p., brown, c.g.d., burridge, m.j., joyner, l.p. & purnell, r.e. 1973a. east coast fever: the infectivity for cattle of infective particles of theileria parva harvested in various substrates. international journal for para sitology, 3:335–338. cunningham, m.p., brown, c.g.d., burridge, m.j. & pur nell, r.e. 1973b. cryopreservation of infective particles of theileria parva. international journal for parasitology, 3:583–587. fandamu, p., thys, e., duchateau, l., berkvens, d. 2005. perception of cattle farmers on east coast fever immunisations in southern zambia. tropical animal health and production, 38:9–16. fao 1984. tick and tick-borne disease control, a practical field manual. vol. ii. rome: food and agriculture organization. 213 v. mbao et al. farrant, j. 1970. mechanisms of injury and protection in living cells and tissues at low temperatures, in current trends in cryobiology, edited by a.u. smith. new york: plenum press. gray, m.a. & brown, c.g.d. 1981. in vitro neutralisation of thei leria sporozoite infectivity with immune serum, in advances in the control of theileriosis, edited by a.d. irvin, m.p. cunningham & a.s. young. the hague: martinus nij hoff. kimbita, e.n., silayo, r.s. & dolan, t.t. 2004. theileria parva: in vitro studies on the effects of holding temperature, ph and medium on sporozoite infectivity. tropical animal health and production, 36:341–351. marcotty, t., billiouw, m., chaka, g., berkvens, d., losson, b. & brandt, j. 2001. immunisation against east coast fever by the infection and treatment method: evaluation of the use of ice baths for field delivery and appraisal of an acid formulation of long-acting tetracycline. veterinary parasitology, 99:175–187. marcotty, t., speybroeck, n., berkvens, d., chaka, g., besa, r.k., madder, m., dolan, t.t., losson, b. & brandt, j. 2004. in vitro titration of theileria parva tick derived stabilates. parasitology, 128:131–137. mbao, v., speybroeck, n., berkvens, d., dolan t., dorny, p., madder, m., mulumba, m., duchateau, l., brandt, j. & marcotty, t. 2005. comparison of manual and homogenizer methods for preparation of tick-derived stabilates of theileria parva: equivalence testing using an in vitro titration model. parasitology, 131:45–49. morzaria, s., nene, v., bishop, r., & musoke, a. 2000. vaccines against theileria parva. annals of the new york academy of sciences, 916:464–473 njuguna, l.m. & musisi, f.l. 1996. bulk-freezing of theileria parva stabilates for vaccine production. international journal for parasitology, 26:67–70. oie 2005. manual of diagnostic tests and vaccines for terrestrial animals, 5th ed. http://www.oie.int/fr/normes/mmanual/a_00062.htm (20 febru ary 2006). purnell, r.e., brown, c.g.d., cunningham, m.p., burridge, m.j., kirimi, i.m. & ledger, m.a. 1973. east coast fever: correlation between the morphology and infectivity of theileria parva developing in its tick vector. parasitology, 66: 539–544. radley, d.e., brown, c.g.d., burridge, m.j., cunningham, m.p., kirimi, i.m., purnell, r.e. & young, a.s. 1975. east coast fever: 1. chemoprophylactic immunization of cattle against theileria parva (muguga) and five theilerial strains. veterinary parasitology, 1:35–41. statacorp 2003. stata/se 8.0 for windows statistical software. texas: stata corporation. uilenberg, g. 1999. immunisation against diseases caused by theileria parva: a review. tropical medicine and international health, 4:a12–a20. young, a.s., groocock, c.m. & kariuki, d.p. 1988. integrated control of ticks and tick-borne diseases of cattle in africa. parasitology, 96:403–432. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [878.740 1133.858] >> setpagedevice abstract introduction materials and methods results ethical considerations discussion conclusion acknowledgements references about the author(s) georgina c. cole department of companion animal clinical studies, university of pretoria, south africa adrian s.w. tordiffe department of paraclinical sciences, university of pretoria, south africa gerhard steenkamp department of companion animal clinical studies, university of pretoria, south africa citation cole, g.c., tordiffe, a.s.w. & steenkamp, g., 2017, ‘assessment of a portable lactate meter for field use in the white rhinoceros (ceratotherium simum)’, onderstepoort journal of veterinary research 84(1), a1399. https://doi.org/10.4102/ojvr.v84i1.1399 original research assessment of a portable lactate meter for field use in the white rhinoceros (ceratotherium simum) georgina c. cole, adrian s.w. tordiffe, gerhard steenkamp received: 17 nov. 2016; accepted: 25 july 2017; published: 10 nov. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract blood lactate is a predictor of mortality in critically ill humans and animals. handheld lactate meters have the potential to be used in the field to evaluate the condition of severely injured rhinoceroses but have not been compared with laboratory-based methods. agreement between a handheld lactate meter and a laboratory method was assessed, as was the stability of rhino blood lactate in the anticoagulant sodium fluoride/potassium oxalate (fluoride/oxalate). blood samples were obtained from 53 white rhinos that had been immobilised for management reasons. lactate was measured by means of a handheld meter using whole blood in heparin (wbhep), whole blood in fluoride/oxalate (wbfo) and fluoride/oxalate plasma (pfo). results were recorded in both blood (bl) and plasma (pl) modes and compared to an established laboratory method for measuring plasma lactate. to assess the stability of lactate over time, blood lactate in fluoride/oxalate was measured on the handheld meter at intervals for up to 91 h. agreement was best using wbfo in pl mode, with small bias (−0.16), tight 95% limits of agreement (loa) (−1.46, 1.14) and a pc (95% ci) of 0.97 (0.92, 0.99). the agreement was improved for all sample types when using the pl mode compared to the blood lactate (bl) mode. blood lactate was stable in fluoride/oxalate for 91 h, with a mean change from baseline of 0.15 (−0.178, 0.478) mmol/l (mean, 95% ci). the handheld meter was found to be suitable for field use in white rhinos but provided more reliable results with the device in pl mode. furthermore, rhino blood lactate was found to be stable in fluoride/oxalate for as long as 3 days. introduction lactate is produced during anaerobic glycolysis where the conversion of pyruvate to lactate facilitates continued adenosine triphosphate (atp) production in the absence of oxygen (lagutchik et al. 1996). increased blood lactate levels may occur as a result of decreased oxygen delivery in circulatory or septic shock, increased oxygen demands such as exercise or shivering, or decreased oxygen utilisation in conditions such as systemic inflammatory response syndrome (sirs) or renal failure (cohen, woods & krebs 1976; fall & szerlip 2005; huckabee 1961a, 1961b). blood lactate measurement in human patients has been extensively studied and demonstrated to be an effective predictor of mortality and useful in monitoring response to therapy in numerous critically ill patient groups (aslar et al. 2004; cerovic et al. 2003; fall & szerlip 2005; lee d cady et al. 1973; mcnelis et al. 2001; nguyen et al. 2004). lactate measurement in horses is predictive of mortality in sick neonates (borchers et al. 2012; corley, donaldson & furr 2005; henderson et al. 2008) and adult emergencies (delesalle et al. 2007; furr, lessard & white 1995; johnston, holcombe & hauptman 2007; schulman, nurton & guthrie 2012; tennent-brown et al. 2010). in bovine medicine, lactate can be useful as a prognostic indicator in cattle with respiratory disease (coghe et al. 2000; helena et al. 2015) and abomasal disorders (buczinski, boulay & francoz 2015; figueiredo et al. 2008). in dogs, lactate measurement has prognostic value in systemically ill animals (lagutchik et al. 1998; stevenson et al. 2007b), dogs with hypotension (ateca, dombrowski & silverstein 2015), septic peritonitis (cortellini, seth & kellett-gregory 2014) and babesiosis (nel et al. 2004). lactate is also a predictor of gastric necrosis and outcome in dogs with gastric dilatation-volvulus (de papp, drobatz & hughes 1999). rhino poaching has increased dramatically in south africa in recent years, with the number of rhinos killed rising from 13 in 2007 to 1215 in 2014 (department of environmental affairs 2015). rhinos that survive a poaching event may have serious injuries such as gunshot wounds to the head, abdomen, thorax and limbs, or systemic complications because of prolonged immobilisation with synthetic opioids. these animals may also have severe facial injuries with open sinuses and exposed bone resulting from traumatic horn removal. other injuries such as fractures of the axial or appendicular skeleton and muscle trauma have been reported. little is known about the assessment of critically injured rhinos or how to treat them. the practical difficulties associated with treating wild animals for severe injuries make major interventions, intensive care and long-term analgesia challenging. identifying prognostic indicators in critically injured rhinos that have initially survived a poaching event may help to focus treatments in injured animals as well as monitor response to therapy. it may improve the welfare of injured rhinos in those cases where long-term survival is unlikely, and euthanasia may be more appropriate. it could also potentially enable more effective use of resources so that time and money can be directed towards cases with a higher probability of success and may help identify areas for future research. the use of blood lactate as a prognostic indicator in rhino species has not been investigated. laboratory measurement of blood lactate requires special sample handling. blood samples must be kept at 4 °c and plasma separated almost immediately to prevent falsely elevated lactate levels occurring from continued cellular glycolysis (sacks 2008). rhinos in south africa are mostly free ranging and are kept in game reserves or on private property that is often remote, making this type of sample handling impractical. handheld lactate meters designed for human use have been validated in a number of animal species making animal-side lactate measurement feasible and practical in emergency settings (buczinski et al. 2014; burgdorf-moisuk et al. 2012; delesalle et al. 2007; evans & golland 1996; stevenson et al. 2007a; tennent-brown et al. 2007; thorneloe, bédard & boysen 2007; tynan et al. 2015). results are available in minutes allowing treatment decisions based on results to be made at the animal’s side. this is especially pertinent in a free-ranging species where a diagnosis must be made and treatments administered within the short period that the animal is immobilised. the roche accutrend plus system (roche diagnostics ltd. ch-6343 rotkreuz, switzerland) is a portable handheld device that measures blood lactate and has been designed for use in human healthcare. it has been evaluated and found to be accurate for use in horses (tennent-brown et al. 2007), dogs (acierno & mitchell 2007; karagiannis et al. 2013) and cats (acierno et al. 2008). its use in the rhino has not been evaluated. accordingly, the aim of this study was to evaluate a handheld lactate meter for field use in the white rhino by comparing it with a laboratory bench-top analyser. the effects of sample type (blood versus plasma) and anticoagulant (heparin versus sodium fluoride/potassium oxalate) on the agreement of the handheld device with the laboratory method were also investigated. the accutrend measures l-lactate in a drop of blood applied to a disposable test strip. the test strips are made up of several layers. the top layer is a mesh, and below that is glass fibre where leucocytes and erythrocytes are filtered out. plasma enters the bottom layer of the strip where lactate oxidase converts lactate to pyruvate using the electron carrier bis-[2-hydroxyethyl]-4-hydroxyiminocyclohexa-2, 5-dienylidene ammonium chloride (pennell & tracy 1999): a further chemical reaction then occurs where the reduced electron carrier reacts with phosphomolybdic acid to form the dye molybdenum blue. the colour intensity of the dye is measured by reflectance photometry, and the l-lactate concentration is calculated (stevenson et al. 2007a; tennent-brown et al. 2007). a result appears in the digital display in 60 s. the accutrend can be set to read either blood lactate (bl mode) or plasma lactate (pl mode). the machine measures plasma lactate concentration, and an algorithm is used to calculate whole blood lactate from this value. the algorithm is specific for human blood and may not give accurate results when used in other species. the cobas integra 400 plus bench-top analyser (roche, f. hoffmann-la roche ltd grenzacherstrasse 1244070 basle, switzerland) is a laboratory-based analyser that measures lactate in 192 µl of plasma that is automatically pipetted from a sample by the machine. the manufacturer’s instructions state that either heparinised plasma or plasma in sodium fluoride/potassium oxalate (fluoride/oxalate) may be used. the test principle is an enzymatic colorimetric method. it is an established method of laboratory-based lactate detection in humans (de backer 2003; toffaletti et al. 1992) and is currently used to measure lactate in a variety of mammalian species at the onderstepoort veterinary academic hospital. lactate levels will increase in blood samples after collection unless plasma is immediately separated or antiglycolytic agents are used. a further aim of this study was therefore to assess the stability of rhino blood lactate in the antiglycolytic agent fluoride/oxalate over time. materials and methods study population and capture study subjects were 53 white rhinos (ceratotherium simum) from three locations in south africa: 48 animals were from a game farm with a large breeding population of white rhinos (reserve 1) and three from a private game reserve with a small population (less than 10) of rhinos (reserve 2). furthermore, two white rhinos from another private game reserve (reserve 3) were included in the lactate stability study only. animals were immobilised for management reasons such as translocation, horn removal or both. on reserves 1 and 3, rhinos were darted from a vehicle. supplementary feeding from vehicles was routinely carried out, meaning that the animals were habituated and that darting was carried out with minimal pre-capture stress or activity. the three rhinos in reserve 2 were darted from a helicopter. rhinos were darted with a combination of etorphine (captivon 9.8 mg/ml, wildlife pharmaceuticals south africa, karino) and azaperone (stresnil, 40 mg/ml, schering plough animal health, kenilworth, new jersey, usa). butorphanol (20 mg/ml, kyron laboratories, johannesburg, south africa) was given intravenously once rhinos were recumbent to improve blood oxygenation. drug dosages used were standard for rhino capture (burroughs et al. 2012) and calculated according to the size of the animal which was estimated by the veterinarian in charge of immobilisation. rhinos were put into life-stage categories based on age (if known) or size and breeding activity (if actual age unknown): calf (< 1 year), yearling (1–2 years), sub-adult (2–7 years) and adult (> 7 years). ambient temperatures ranged from 22 °c to > 35 °c, and immobilisation took place during daylight hours. blood sampling and lactate measurement blood samples were taken from the auricular vein, or occasionally the radial vein in the case of calves. blood was collected in evacuated tubes (vacuette® and vaccutainer®, beckton, dickinson & co, new jersey, usa) containing lithium heparin and also tubes containing fluoride/oxalate using an 18g double-ended vacuum tube needle. lactate was measured on the accutrend within 5 min of blood collection using either heparinised whole blood (wbhep) or whole blood in fluoride/oxalate (wbfo) by placing one drop of fresh blood on to the lactate test strip according to the manufacturer’s instructions. to ascertain which type of sample (blood or plasma) and which mode on the accutrend (bl or pl) would give results in closest agreement to those of the bench-top analyser, both whole blood (in the field) and plasma (in the laboratory) were used, and results were recorded in both bl and pl modes. this is similar to the method used by tennent-brown et al, assessing the accuracy of accutrend in horses (tennent-brown et al. 2007). the manufacturer’s instructions state that only fresh blood or heparinised whole blood should be used on the accutrend. in this study, wbhep, wbfo and fluoride/oxalate plasma (pfo) were used. regular control checks were performed throughout the study using lactate control solutions according to the manufacturer’s instructions to ensure proper functioning of the instrument. a glass pasteur pipette with a rubber dropper was used to apply blood or plasma to the test strip throughout the study. blood tubes were placed in an upright position in a cooler bag containing ice blocks and left to stand. to test the stability of rhino blood lactate in fluoride/oxalate, lactate from eight blood samples collected into fluoride/oxalate tubes as described above was measured on the accutrend at various time intervals. blood tubes were not inverted each time, allowing separation of the plasma and cellular component of blood to occur with gravity, and plasma was aspirated from the top of the tube for each lactate measurement. therefore, whole blood was used for the first measurement (t = 0) and plasma for all subsequent measurements. for the remaining samples, plasma from the fluoride/oxalate tubes was separated from the cellular fraction by centrifugation within 8 h of collection and frozen at −20 °c. samples were then transferred on ice to the onderstepoort veterinary academic hospital and stored at −80 °c (within 48 h of collection) for analysis at a later date. at the time of analysis, pfo from all study subjects was removed from the freezer and defrosted at room temperature. plasma lactate for each rhino was then measured concurrently with the accutrend and with the cobas bench-top analyser. daily quality controls and calibrations were run as part of the laboratory’s standard protocols. statistics comparisons made between lactate measured on the accutrend and the cobas bench-top analyser were analysed using bland altman statistical analysis (bland & altman 1986), passing and bablok regression analysis (passing & bablok 1983) and concordance correlation (lawrence & lin 1989). all calculations were performed using medcalc® version 12.2.0.0. results for comparison of lactate measurement techniques, blood samples were taken from 51 rhinos (48 from reserve 1 and 3 from reserve 2) consisting of 37 females and 14 males. sampling of some individuals on consecutive days allowed for greater than 51 comparisons between groups (n = 57). of the female rhinos 65% were adults, 11% sub-adult, 13% juvenile and 11% calves. of the male rhinos, 50% were adult, 14% sub-adult, 7% juvenile and 29% calves. the cobas bench-top analyser has a measuring range of 0.2 mmol/l to 15.5 mmol/l. lactate measured on the cobas bench analyser (n = 57) ranged from 0.9 to 14.3, mean 4.6 (mmol/l). the accutrend reads lactate values from 0.8 mmol/l to 21.7 mmol/l in bl mode and 0.7 mmol/l to 26 mmol/l in pl mode. lactate measured with the accutrend for all sample types combined ranged from 0.9 mmol/l to 13.8 mmol/l in bl mode and 0.8 mmol/l to 16.5 mmol/l in pl mode. plasma lactate measured with the cobas bench-top analyser (cobas) was compared with lactate measured with the accutrend for each of the following sample types and meter settings: heparinised whole blood with the accutrend in bl mode (wbhep bl) and in pl mode (wbhep pl), fluoride/oxalate whole blood with the accutrend in bl and pl mode (wbfo bl, wbfo pl), and plasma in fluoride/oxalate with the accutrend in bl and pl mode (pfo pl, pfo bl). bland–altman analysis (bland & altman 1986) assesses agreement between two methods by plotting the differences between paired measurements against the average of those measurements. the magnitude of the bias (average of differences) describes the level of agreement, (0 = perfect agreement). the limits of agreement (95% loa) are bias plus or minus 1.96 x standard deviation (+ 1.96 s.d.) and describe where 95% of the differences between the measurement techniques are likely to lie. loa refer to actual measurements of the technique in question (lactate in mmol/l) and if they encompass differences that are not clinically significant then the two methods may be considered to agree. bland–altman analysis indicated good agreement between all group comparisons with a mean difference close to zero for all comparisons over the whole range of lactate values (figure 1). when the accutrend was compared to the cobas bench-top analyser the smallest mean differences were seen when wbhep was used in the bl mode and when wbfo was used in the pl mode (0.11 and −0.16 respectively). the 95% loa were narrowest when wbfo was used in the pl mode, but widest when wbhep was used in the bl mode. figure 1: bland – altman plots of rhino blood lactate concentration. the plots describe agreement between plasma lactate measured with a laboratory method (cobas) and the accutrend handheld meter. lactate is measured on the accutrend with different sample types using either the blood or plasma mode. (a) plasma in fluoride/oxalate, plasma mode, (b) plasma in fluoride/oxalate, blood mode, (c) whole blood in fluoride/oxalate, plasma mode, (d) whole blood in fluoride/oxalate, blood mode, (e) whole blood in heparin, plasma mode and (f) whole blood in heparin, blood mode. concordance analysis results are displayed in table 1 as concordance correlation coefficient pc and 95% ci. highest level of agreement (pc, 95% ci) was seen between the comparisons cobas – wbfo pl and cobas – pfo pl with values of 0.97 (0.92, 0.99) and 0.96 (0.94, 0.98) respectively. poorest agreement, with pc, 95% ci values of 0.89 (0.82, 0.94) and 0.85 (0.78, 0.90) was seen when lactate measured on the cobas bench-top analyser was compared with lactate measured on the accutrend in pl and bl mode using wbhep. table 1: bland – altman, passing and bablock and concordance analysis of plasma lactate measured with a laboratory method compared with the accutrend handheld meter. lactate is measured on the accutrend with different sample types using either the blood or plasma mode. when passing and bablok regression was performed, the 95% confidence intervals for the intercept a did not include the value zero for all, apart from the comparison cobas – pfo bl indicating that systematic differences occur between measurements for all other comparisons (table 1). for slope b the 95% ci did not include the value one for the following comparisons: cobas – pfo bl, cobas – wbfo bl and cobas – wbhep bl; indicating that proportional differences exist between the measurements. there were no comparisons where both a and b were not significantly different from zero and one, and the cusum linearity test yielded p < 0.01 for all comparisons indicating deviation from linearity. comparisons between methods were split according to lactate concentration to see if agreement was improved at lower lactate concentrations. this was not done for comparisons cobas – wbfo bl and cobas – wbfo pl because of the small sample size. results are shown in table 2. bland–altman agreement was improved with both a smaller mean difference and narrower 95% loa when lactate was < 5 mmol/l for the comparison cobas – pfo bl. smaller 95% loa but slightly larger mean differences were seen for comparisons cobas – wbhep bl, cobas – wbhep pl and cobas – pfo pl when lactate concentration was < 5 mmol/l. values of pc were lower, and 95% ci wider, for all groups when concordance analysis was performed on comparisons split according to lactate concentration < 5 mmol/l and ≥ 5 mmol/l. improved agreement was seen with passing and bablok regression when lactate < 5 mmol/l for the following comparisons: cobas – pfo bl, cobas – pfo pl and cobas – wbhep pl, where intercept a and slope b were not significantly different from zero and one, indicating no systematic or proportional differences, whereas for lactate ≥ 5 mmol/l the 95% ci for intercept a and slope b did not include zero and one. however, p was still less than 0.01 for all groups, indicating poor fit of a linear model. table 2: bland – altman, passing and bablock and concordance analysis of plasma lactate measured with a laboratory method compared with the accutrend handheld meter with analysis split according to laboratory method lactate concentration < or ≥ 5 mmol/l. lactate is measured on the accutrend with different sample types using either the blood or plasma mode. the stability of lactate in fluoride/oxalate over time is shown in figure 2. six rhinos were included in the study (2 from reserve 3, 3 from reserve 2 and one from reserve 1). the two rhinos in reserve 3 were each sampled twice (3 months apart), so 8 samples were available in total. lactate was measured on the accutrend for each sample between four and seven times over a minimum of 14.3 to a maximum of 91.3 h. lactate remained stable with a mean change (mean, 95% ci) from baseline to last measurement of 0.15 (−0.18, 0.48) mmol/l and 0.14 (−0.13, 0.4) mmol/l with the accutrend in pl mode and bl mode, respectively (table 3). figure 2: lactate stability in sodium fluoride/potassium oxalate over time. the graph illustrates the measured concentration of lactate in 8 rhino blood samples (each sample designated by a shape marker) with sodium fluoride/potassium oxalate over time using the accutrend meter set in either blood (bl, dashed line) or plasma (pl, solid line) mode. table 3: stability of rhino blood lactate in sodium fluoride/potassium oxalate over time. ethical considerations this research (project number v059-12) was approved by the university of pretoria animal ethics committee. the ethical standards required in the university of pretoria’s code of ethics for researchers and the policy guidelines for responsible research have been upheld throughout the research process. oral consent was given by the owners or caregivers of the rhinos for their inclusion in this study. only rhinos being immobilised for management purposes were used in the study; so no animals were anesthetised solely for the purposes of research. discussion bland–altman analysis indicated good agreement between the accutrend and the cobas bench-top analyser when wbfo and pfo were used in both the pl and the bl mode. moderate agreement was seen when wbhep was used in both the bl and pl modes. the highest level of agreement with a small bias, narrowest loa and pc closest to one was seen when wbfo was used on the accutrend in the pl mode. when pfo was used on the accutrend in pl mode, agreement with the cobas bench-top analyser was also good with small bias, narrow loa and pc value close to one. for all sample types in this study (blood and plasma), the pl setting had better agreement with the cobas bench-top analyser than the bl setting. a study evaluating the accutrend in dogs also found that when whole blood was used the plasma setting provided better agreement with the reference method than the blood setting (karagiannis et al. 2013). an equine study assessed the accusport (which uses the same technology as the accutrend) and found the highest level of agreement to a laboratory method when plasma was used in the plasma mode (evans & golland 1996). it has been suggested that the algorithm used by the accusport and accutrend analysers to convert measured plasma lactate values to blood values is accurate only for humans and may not be suitable for other animals because of differences in lactate distribution between blood cells and plasma (evans & golland 1996; tennent-brown et al. 2007). the results of the current study suggest that when measuring lactate in rhino blood or plasma on the accutrend, the plasma mode may yield more accurate results. the poorest agreement with the cobas bench analyser was seen when wbhep was used on the accutrend, with wide loa and pc < 0.90 for both pl and bl modes. the accutrend manufacturer’s instructions recommend the use of heparinised whole blood or fresh whole blood; and therefore anticoagulant choice is unlikely to affect results here. the reason for the relatively poor agreement seen with wbhep in the current study may be related to sampling methods. at the time of sampling, blood was placed into two of each of plain, edta, heparin and fluoride/oxalate tubes. extra blood was stored for future studies. in the time taken to fill all the tubes it is possible that the blood lactate concentration in the rhino may have changed. studies indicate that lactate levels change over time during chemical immobilisation in rhinos, often decreasing in the initial period post immobilisation (buss et al. 2015; miller et al. 2013; morkel et al. 2010) presumably as the metabolic acidosis caused by pre-capture exertion begins to normalise and because of the effects of butorphanol administration. the lactate concentration in the first tube filled may therefore be different from that in the last tube. as the order of tubes filled was random and not recorded, we were unable to test this hypothesis. as pfo was the sample type used for analysing lactate on the cobas bench-top analyser, it may be that agreement with heparin samples are poor because of differences in lactate concentration between the heparin and fluoride/oxalate samples and not because of inaccuracy of the accutrend. lactate analysis on the accutrend (in both bl and pl modes) using pfo was performed on the same day as the laboratory method analysis in a laboratory under controlled conditions, whereas analysis of wbhep on the accutrend was performed on several different days, under variable field conditions. it is possible that the effects of climate and inter-assay variation may have had a negative effect on agreement between methods. however, the wbfo analysis on the accutrend was also performed in field conditions over several days, and when wbfo was used in the pl mode, agreement with the cobas bench-top analyser was greater than for all other sample types. the sample size for the wbfo group was smaller and with a narrower range of lactate values than the other groups. with a larger sample size and a greater range of lactate values the differences between heparin and fluoride/oxalate samples may have been less marked. owing to the instability of blood lactate in heparin and the impracticalities of separating plasma in the field, only heparinised whole blood was measured on the accutrend, and it was not possible to assess agreement between the accutrend and the cobas bench-top analyser using heparinised plasma in this study. studies in horses have demonstrated improved accuracy of the accusport and accutrend when measuring lactate levels less than 10 mmol/l (evans & golland 1996; schulman, nurton & guthrie 2012), or less than 5 mmol/l (tennent-brown et al. 2007) respectively. in the current study, tighter loa were observed, with reasonable preservation of bias, when lactate was less than 5 mmol/l for all groups examined. additionally, passing and bablok regression results indicated a lack of proportional and systematic differences for pfo pl, pfo bl, and wbhep pl with lactate < 5 mmol/l. however, when concordance analysis was performed there was poorer agreement for all groups when split according to low or higher lactate concentration than with results combined. the majority of blood lactate values measured in this study were < 10 mmol/l and larger sample size and a greater range of lactate values would be needed to properly assess the performance of the accutrend at higher lactate values. although the results of this study indicate that the accutrend may have better accuracy when measuring lower lactate levels, the accutrend results will likely be sufficient to follow trends when measuring lactate in rhinos in the field. when assessing a new method of measuring a clinical variable, an important point to consider is if the new method is interchangeable with the old method over the clinically relevant range of measurements (altman 2009). the clinically relevant range in the rhino has yet to be determined; however, the accutrend had a good level of agreement with the cobas bench-top analyser over the range of lactate values tested here (0.8 mmol/l – 16.5 mmol/l in pl mode). for this device to be of use to veterinarians treating rhinos, it must be suitable for use in field conditions. the instruction manual for the accutrend states that for lactate testing the instrument must be operated between 15 °c and 35 °c, on a level surface. on one occasion during the study, ambient temperatures exceeded 35 °c. the device indicated a temperature error. this was an advantage as it meant that it was not possible to record an erroneous result when the machine was outside of its temperature range. the field conditions in this study were variable and the device was at times operated on the back of a vehicle moving over rough terrain. lactate in whole blood was measured in the field in this study (plasma was tested in the laboratory), and when the device was in pl mode there was good agreement with the cobas bench-top analyser. overall the device performed well in the field in this study and is therefore likely to be suitable for field measurement of lactate in the white rhino providing the manufacturer’s limitations are observed. continuation of cellular glycolysis after venepuncture results in a rise in blood lactate levels over time if blood is kept in tubes containing heparin or edta (astles, williams & sedor 1994). the addition of sodium fluoride/potassium oxalate (an antiglycolytic agent) maintains stable lactate levels in human blood at room temperature for 8 h (astles et al. 1994) or in equine blood refrigerated for up to 6 h (tennent-brown et al. 2010; williamson et al. 1996). the rhino blood samples in this study were stored for 14 h to 91 h under highly variable conditions. field temperatures ranged from 22 °c to > 35 °c, and although blood tubes were stored in cooler bags with ice, it was often not possible to replenish ice regularly meaning blood storage temperatures for the first 4–10 h in the field would have been variable. the maximum change in blood lactate over time in this study was 1.5 mmol/l for the pl setting with a mean (95% ci) change of 0.53 (−0.08, 1.13) mmol/l (table 3). the mean change is small and is unlikely to be clinically significant. a maximum change of 1.5 mmol/l could be clinically significant at lower lactate levels; however, this maximum difference of greatest magnitude was seen in a sample with a high initial lactate level, and although not fully assessed here it can be seen from the graph (figure 2) that when lactate levels were low the changes over time were minimal (< 0.6 mmol/l for all lactate values < 4 mmol/l) suggesting that this would not interfere with diagnosing a clinically important hyperlactaemia at lower lactate levels. the overall mean (95% ci) change for all samples from baseline to final measurement was 0.15 (−0.178, 0.478) mmol/l. these results are similar to those in a study that reported a mean increase of 0.15 (0–0.3) mmol/l in controlled laboratory conditions with human samples kept in fluoride/oxalate for 24 h at room temperature (astles et al. 1994). the apparent stability of rhino blood lactate in fluoride/oxalate tubes over a long period and in variable temperature conditions has important practical implications for clinicians wishing to measure blood lactate in the field where a portable handheld lactate meter is not available. normal resting values for blood lactate in white rhinos (and other rhino species) without the effects of pre-capture exertion or respiratory depressant immobilising drugs are currently not available in the literature. there were variable levels of pre-capture exertion in this study, and the effects of butorphanol and time from darting which are factors that have been shown to effect lactate levels in rhinos (buss et al. 2015; miller et al. 2013) have not been examined here. in addition, a range of lactate values was desirable in this study to assess the accuracy of the accutrend at low and high lactate values. the utility of lactate testing in the field and the ability of blood lactate levels to predict outcome in critically injured rhinos still need to be evaluated. lactate levels greater than 2 mmol/l would be considered elevated in humans (levy 2006), 1.5 mmol/l is the upper limit of normal in horses (tennent-brown 2011), whereas dogs have normal values of 0.3 mmol/l – 2.5 mmol/l (sharkey & wellman 2013). these are fairly narrow ranges of normal lactate, which seem to be conserved across mammalian species, and rhinos may well have a similar range. as lactate in free-ranging rhinos is only measured when rhinos are immobilised it will be difficult to differentiate elevated lactate levels caused by disease, from transient elevations caused by exertion or immobilising drugs. studies in humans (husain et al. 2003; mcnelis et al. 2001; nguyen et al. 2004) and horses (johnston et al. 2007; tennent-brown et al. 2010; wotman et al. 2009) have found that serial lactate levels may be more useful in predicting outcome and monitoring response to treatment than single measurements. critically injured rhinos are now being immobilised repeatedly for treatment and re-assessment of injuries. some are being boma restricted or confined to smaller areas to make management easier. in these circumstances, serial lactate measurements may be possible. a handheld meter such as the accutrend evaluated in this study could be a suitable field tool for this sort of investigation. as results are available in a short time period and test strips are inexpensive, it may also be a useful tool for physiological monitoring during immobilisation; in field or captive situations in conjunction with other observed clinical parameters such as heart or respiratory rate and monitoring tools such as pulse oximetry. conclusion the accutrend system provided results in close agreement to those measured on a laboratory based analyser when whole blood or plasma in fluoride/oxalate was used with the device set in plasma mode. it is light, portable, easy to operate in field conditions and is suitable for field use in white rhinos. rhino blood lactate is stable in sodium fluoride/potassium oxalate for as long as 3 days, this has implications for veterinarians working in remote locations who wish to measure lactate. acknowledgements jackie clark of roche diagnostics ltd. donated accutrend meters for the study. thanks go to dr cobus raath of wildlife vets for his assistance, support and facilitating data collection. this work was funded by the zebra foundation of the british veterinary zoological society. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions g.c.c. was responsible for the study design, data collection, statistical analysis and manuscript writing. this research was undertaken as part of postgraduate studies for a master of science (veterinary research) at the university of pretoria. g.s. 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particularly for the resource-poor, at-risk populations and farmers in africa. so far, resistance to one or more of the trypanocides used in livestock has been reported in at least 13 africa countries (ndung’u, murilla, mdachi, mbwambo, sinyangwe, machila, delespaux, geerts, brandt, peregrine, mcdermott, holmes & eisler 1999; anene, onah & nawa 2001). the main factors associated with the emergence of resistance include the prolonged and improper use of the trypanocidal drugs in both humans and livestock. human sleeping sickness is caused by trypanosoma brucei rhodesiense and t. brucei gambiense, both of which also infect some domestic and wild animals. results of studies on the role of the livestock reservoir in the epidemiology of t. b. rhodesiense sleeping sickness have indicated that an aggressive chemotherapy policy should be pursued in livestock 17 onderstepoort journal of veterinary research, 74:17–22 (2007) occurrence of multiple drug resistance in trypanosoma brucei rhodesiense isolated from sleeping sickness patients j.m. kagira1* and n. maina2 abstract kagira, j.m. & maina, n. 2007. occurrence of multiple drug resistance in trypanosoma brucei rhodesiense isolated from sleeping sickness patients. onderstepoort journal of veterinary research, 74:17–22 the occurrence of cross-resistance among melarsoprol-resistant trypanosoma brucei rhodesiense isolates was investigated in this study. the isolates, t. b. rhodesiense ketri 237, 2538, 1992, 2709, 2694 and 3530, had been obtained from sleeping sickness patients in kenya and uganda between 1960 and 1985. five groups consisting of six mice each were inoculated intraperitoneally with 105 parasites of each isolate, and 24 h later treated with either melarsoprol, homidium chloride, diminazene aceturate or isometamidium chloride. the control group comprised infected but untreated mice. the mice were monitored for cure for a period of 60 days post-treatment. the mean prepatent period in the control mice was 5 days while the mean survival period was 22 days. five of the stabilates, ketri 237, 2538, 2709, 2694, and 3530, were confirmed to be melarsoprol resistant. cross-resistance was observed, with the majority of the isolates being resistant to homidium chloride (5/6) and diminazene aceturate (5/6), but all were sensitive to isometamidium chloride (6/6). however t. b. rhodesiense ketri 1992, which was previously considered as melarsoprol resistant, was sensitive to all the drugs tested. in conclusion, our study has revealed the existence of cross-resistance among the melarsoprol resistant isolates which could only be cured by isometamidium. keywords: cross-resistance, kenya, melarsoprol, trypanocidals, trypanosoma brucei rhodesiense, uganda * author to whom correspondence is to be directed. e-mail: jkagira@yahoo.com 1 kenya agricultural research institute-trypanosomiasis research centre (kari-trc), p.o. box 362, kikuyu, kenya 2 department of biochemistry, jomo kenyatta university of agriculture and technology, p.o. box 62000 nairobi, kenya accepted for publication 15 september 2006—editor 18 multiple drug resistance in trypanosoma brucei rhodesiense isolated from sleeping sickness patients in endemic areas (angus 1996; fervre, coleman, odiit, magona, welburn & woolhouse 2001). this is due to an incidence of up to 20 % of t. b. rhodesiense in cattle in endemic areas (hide, angus, holmes, maudlin & welburn 1998). the widespread use of trypanocides, especially diminazene aceturate (diminazene) and isometamidium chloride (isometamidium), acts as a selection pressure for the de velopment of resistant trypanosomes (angus 1996). it is hypothesized that trypanosomes infective for humans can be selected for drug resistance during treatment of livestock and that they might subsequently be introduced to humans by tsetse flies. indeed, a study carried out during the 1988– 1990 sleeping sickness epidemic in the busoga district in uganda showed that the disease is up to five times more likely to be transmitted by the cattle-flyhuman transmission cycle than the human-fly-human cycle (hide, tait, maudlin & welburn 1996). reduced sensitivity of t. b. rhodesiense isolates to trypanocides used in cattle has been reported in kenya and uganda (van hoeve & grainge 1965; matovu, iten, enyaru, schmid, lubega, brun & kaminsky 1997). mice infected with t. b. rhodesi ense isolated from a human patient were refractory to diminazene administered at the normal dosage levels (matovu et al. 1997). in another study, a t. b. rhodesiense stock isolated from cattle was found to be clearly resistant to diminazene and isometamidium (enyaru, matovu, lubega & kaminsky 1998). in the latter study, diminazene at 14 mg/kg was not sufficient to cure all mice, while 33 % of mice treated with isometamidium at 2 mg/kg were not cured. these studies showed that the control of sleeping sickness by treatment of the animal reservoir could face serious problems since the drug-resistant parasites would most likely not be eliminated by diminazene and isometamidium administered at the recommended dosage levels (matovu et al. 1997; who 2001). a reduced virulence of resistant isolates has been observed in many pathogenic organisms. in trypanosomes, the existence of such a scenario has been inconsistent, although most studies have shown that the resistant strains are less virulent than the parental clone (kaminsky & zweygarth 1989; egbe-nwiyi, igbokwe & onyeyili 2005). due to the presence of mixed infections of trypanosome strains with different drug sensitivities in a single host, it is postulated that competition between the strains will arise (mu tugi 1993). the sensitive trypanosomes will grow faster and develop an infection earlier than the slower growing resistant strains, a situation that may result in selecting out of the latter. indeed, it has been shown that although suramin resistance in t. evansi is highly stable, its spread is quite limited (mutugi 1993). it has been suggested that lack of spread of suramin-resistant t. evansi is occasioned by poor survival of the resistant isolates as compared to the sensitive and more pathogenic ones (mutugi, boid & luckins 1996). the possible existence of such a scenario in t. b. rhodesiense isolates has not yet been investigated to date. the objective of this study was to determine the pathogenicity and possible occurrence of cross-resistance among melarsoprol-resistant t. b. rhodesiense cryopreserved at the trypanosomosis research centre (trc) in kenya. standardized tests and methods of interpretation of results (eisler, brandt, bauer, clausen, delespaux, holmes, ilemobade, machila, mbwambo, mcdermott, mehlitz, murilla, ndung’u, peregrine, sidibe, sinyangwe & geerts 2001) were used in this study. materials and methods ethical review all the procedures used in this study were reviewed and approved by the institutional animal care and use committee (iacuc) of the trc in nairobi, kenya. trypanosomes six t. b. rhodesiense isolates, which had been determined to be resistant to melarsoprol, were used. the isolates included t. b. rhodesiense numbers ketri 237, 2538, 1992, 2709, 2694 and 3530 (hereafter referred by their specific numbers). these isolates were characterized by other workers and found to be melarsoprol-resistant (baachi, nathan, livingston, valladares, mccann, bitonti, sjoersma, saric, sayer, njogu & clarkson 1990; brun, schumacher, schomidiumid, kunz & burri 2001). in this study, the trypanosomes were multiplied in gamma-irradiated swiss white mice. at the rising wave of parasitaemia, the mice were euthanased by placing them in an atmosphere of carbon dioxide and trypanosomes harvested through cardiac puncture. mice swiss white mice weighing 20–30 g were obtained from the laboratory-animal breeding colony at the trc. the mice were kept in cages where they were fed on commercial pellets and water ad libitum. 19 j.m. kagira & n. maina drugs isometamidium chloride (samorin®, merial, france), diminazene aceturate (berenil®, hoechst, ireland) and homidium chloride (novidium®, may & baker, uk) were prepared by dissolving in sterile distilled water. melarsoprol (arsobal®, aventis) was prepared using propylene glycol as the diluent. experimental design pathogenicity groups of six mice were inoculated intraperitoneally with 105 parasites of the isolates studied. the mice, which also served as the control groups for the sensitivity studies (see below), were monitored for the pre-patent period, parasitaemia pattern and survival period. the parasitaemia levels were estimated daily using the rapid matching method (herbert & lumsden 1976). mice in extremis were euthanased and necropsied. sensitivity study groups of six mice were inoculated intraperitoneally with 105 parasites of the isolates listed above. twenty-four hours after inoculation, trypanocidal drugs were administered, also intraperitoneally. isometamidium, diminazene, homidium and melarsoprol were given at dosage rates of 1.0 mg, 20 mg, 1.0 mg and 10 mg/kg body mass, respectively. after treatment, the parasitaemia was monitored daily during the first week, three times a week during the second week and twice a week thereafter in wet smears of tail blood. the treated groups were monitored until relapse occurred or until 60 days posttreatment, when the mice were euthanased. interpretation the results of the study were interpreted as described by eisler et al. (2001). a trypanosome isolate was considered as drug sensitive if at least five out of six treated mice were cured. if fewer than five mice were cured, the isolate was considered resistant. results pathogenicity study (table 1) the mean pre-patent period in the infected nontreated mice was 5 days (range 4–6 days). the parasitaemia pattern varied between the isolates, with most isolates producing several peaks of parasitaemia before killing the mice. the mean survival period was 22 days (range 17–29 days). mice infected with ketri 237 had the shortest survival period table 1 pathogenicity of the different t.b. rhodesiense isolates isolate number mean prepatent period days (range) mean survival period in days (range) ketri 2538 ketri 1992 ketri 237 ketri 2694 ketri 3530 ketri 2709 6 (4–6) 6 (4–12 5 (4–7) 4 (4–5) 4 (3–6) 4 (3–5) 18 (16–20) 24 (22–31) 17 (16–18) 29 (24–35) 22 (17–29) 22 (12–32) table 2 sensitivity of t.b. rhodesiense isolates to different trypanocidal drugs isolate number number of mice cured* time to relapse (days)* ismm hm da mel b ismm hm dm mel b ketri 2538 ketri 1992 ketri 237 ketri 2694 ketri 3530 ketri 2709 5/6 6/6 6/6 6/6 6/6 6/6 0/6 6/6 0/6 4/6 1/6 0/6 2/6 6/6 0/6 4/6 4/6 4/6 0/6 6/6 0/6 0/6 5/6 1/6 53 0 0 0 0 0 8 0 9 29 11 9 13 0 12 32 22 11 7 0 4 4 15 7 *ismm = isometamidium chloride da = diminazene aceturate hm = homidium chloride mel b = melarsoprol resistant isolates = 0/6–4/6 sensitive isolates = 5/6–6/6 20 multiple drug resistance in trypanosoma brucei rhodesiense isolated from sleeping sickness patients while those infected with ketri 2694 had the longest. the most noticeable lesions at post mortem examination of all the mice that died were enlarged spleens and congestion of most organs. sensitivity study (table 2) five of the isolates investigated were confirmed to be melarsoprol-resistant. cross-resistance was observed in these isolates, with the majority being resistant to homidium and diminazene, and only sensitive to isometamidium. however, different levels of sensitivities to specific drugs were observed among the isolates. ketri 2694, 3530, and 2694 were found to have a higher sensitivity to diminazene when compared to that of other isolates. ketri 2694 was also relatively sensitive to homidium when compared to that of the other isolates. all mice infected with ketri 1992 were cured with all the drugs used. this is despite the fact that the parasite was previously recorded as being resistant to melarsoprol. the time taken before a relapse occurred varied between isolates and the drugs used, with the shortest time being observed mainly in melarsoprol-treated groups. for the homidium-treated groups, ketri 2538 had the least number of days to relapse while ketri 2694 had the most. however, for diminazene-treated groups, ketri 2709 had the least number of days to relapse. for melarsoprol, ketri 237 and 2694 had the least number of days to relapse while ketri 3530 had the most. discussion previous studies have shown that t. b. rhodesiense infections in livestock can be effectively cured (angus 1996; matovu et al. 1997). thus, mass chemotherapy of animals acting as reservoirs of t. b. rhodesiense has been advocated as an effective strategy for control of the spread of sleeping sickness (fevre et al. 2001). this approach can be hindered by the emergence of resistant trypanosomes, which can also be spread from humans to cattle and vice versa (matovu et al. 1997). it is also evident that resistance of t. b. brucei to diminazene is a significant problem in trypanosomosis-endemic areas (ndung’u et al. 1999; anene et al. 2001; anene, ezeokonkwo, mmesirionye, tettey, brock, barrett & de koning 2006). from the stocks analysed in this study, melarsoprolresistant isolates were also shown to be cross-resistant to both diminazene and homidium. the latter are curative livestock trypanocides and are used at a dosage lower than the one used in this study. the cross-resistance between arsenicals and diamidines has been widely investigated and is partially associated with loss of adenosine p2 transporters in the resistant trypanosomes (carter & fairlamb 1993; barrett & fairlamb 1999; de koning, anderson, stew art, burchmore, wallace & barrett 2004). since diminazene is the most commonly used drug in the field, the occurrence of resistance in 5/6 of the tested isolates is particularly important in controlling the spread of resistant isolates. fortunately, only a few cases of melarsoprol resistance have been reported in t. b. rhodesiense patients in recent years (matovu et al. 1997; kibona, matemba, kaboya & lubega 2006). the lack of spread of these isolates could be due several factors, which include: low trans mis sibility of resistant isolates, low transmission capacity from humans to livestock and reduction in the incidence rates of the rhodesian type of sleeping sickness (brun et al. 2001). on the contrary, the emergence of numerous cases of patients with t. b. gam biense resistant to melarsoprol is of great concern (brun et al. 2001; matovu, seebeck, enyaru & kaminsky 2001). in endemic countries, this has necessitated the change of the treatment regimen for late-stage disease from melarsoprol to eflornithine (brun et al. 2001; legros, ollivier, gas tellu-etchegorry, paquet, burri, jannin & büscher 2002). the large proportion of homidium-resistant isolates in the current study was unexpected, especially because a related phenanthridinium compound, isometamidium, cured all the infected mice. the biochemical mechanism of homidium resistance is not clear, although there is evidence that homidium-resistant t. brucei clones accumulate smaller amounts of the drug than their sensitive counterparts (frommel & balber 1987). homidium was extensively used in the 1960s and 1970s when most of the parasites used in this study were isolated. however, its usefulness was curtailed by widespread emergence of resistance (scott & pegram 1974). thus, one cannot rule out the possible spread of the homidiumresistant isolates from livestock to humans during the two decades. it can be hypothesized that this phenomenon could have led to the subsequent emergence of resistance to both melarsoprol and diminazene, as observed in this study. the existence in cattle of t. b. rhodesiense resistant to homidium and other drugs used for routine treatment was also reported in kenya in 1960s (van hoeve & grainge 1965). cross-resistance between homidium and diminazene was reported in cattle (schonfield, rottcher & moloo 1987; codja, mulatu, majiwa, leak, rowlands, authie, d’ieteren & peregrine 1993). 21 j.m. kagira & n. maina isometamidium is as regarded a hybrid molecule which contains homidium and an additional moiety of m-amidinophenyl-azo-amine that is part of the diminazene molecule (wragg, washbourn, brown & hill 1958). these combined properties of diminazene and homidium could be responsible for curing 100 % of the isolates used in this study. the prophylactic use of this drug in the field could thus be an effective tool in controlling the spread of melarsoprol-resistant trypanosomes should they emerge. cross-resistance between isometamidium and diminazene rarely occurs in the field and, as such, they are used as a sanative combination to curtail the development of resistance to either drug (whitesand 1960). the pathogenicity of trypanosomes used in these studies was not significantly different from that of the corresponding sensitive stabilates. in most cases, the prepatent period ranges between 3–6 days (kagira, unpublished data 2005). however, there was a wide range in the survival period of mice infected with the different isolates. the relationship between the virulence of the trypanosomes and sensitivity to different drugs could not be accurately assessed, as we did not have the original stabilates before resistance emerged. however, mice infected with more sensitive isolates (ketri 1992 and 2694) survived longer when compared to those that were highly resistant. other studies have documented reduced virulence amongst resistant t. brucei and t. evansi strains (mutugi 1993; egbe-nwiyi et al. 2005). thus, it is hypothesized that sensitive trypanosomes will grow faster and hinder the spread of the resistant ones (mutugi et al. 1993). in conclusion, our study has revealed the existence of multiple resistance among the melarsoprol-resistant isolates. the results suggest that isometamidium can be used to curtail the spread of such resistance. however, the results should also be considered with caution since drug pharmacokinetics and immune response differ between animal species, and responses in mice might not be able to be directly extrapolated to the situation in cattle. acknowledgements the study was funded by government of kenya. we thank dr john thuita of the primate division, trypanosomiasis research centre, who participated in the development of the protocols. we are also grateful for the technical assistance provided by 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medicine and para sitology, 38:177–180. scott, j.m. & pegram, r.g. 1974. a high incidence of trypanosoma congolense strains resistant to homidium bromide in ethiopia. tropical animal health and production, 6: 215–221. van hoeve, k. & grainge, e.b. 1965. drug sensitivity of trypanosoma brucei subgroup trypanosomes isolated from cattle in alego. east africa trypanosomiasis research organisa tion report for 1965. who 2001. recommendations of a scientific working group on sleeping sickness. world health organisation (who) report. whitesand, e.f. 1960. recent work in kenya on control of drug resistant cattle trypanosomiasis. proceedings of the 8th isctrc meeting, jos, nigeria: 141–154. wragg, w.r., washbourn, k., brown, k.n. & hill, j. 1958. metamidium: a new trypanocidal drug. nature, 182: 1005–1006. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) christo j. botha department of paraclinical sciences, university of pretoria, south africa sarah j. clift department of paraclinical sciences, university of pretoria, south africa gezina c.h. ferreira department of paraclinical sciences, university of pretoria, south africa mxolisi g. masango food, feed and veterinary public health, agricultural research council-onderstepoort veterinary institute, south africa citation botha, c.j., clift, s.j., ferreira, g.c.h. & masango, m.g., 2017, ‘geigerin-induced cytotoxicity in a murine myoblast cell line (c2c12)’, onderstepoort journal of veterinary research 84(1), a1465. https://doi.org/10.4102/ojvr.v84i1.1465 original research geigerin-induced cytotoxicity in a murine myoblast cell line (c2c12) christo j. botha, sarah j. clift, gezina c.h. ferreira, mxolisi g. masango received: 05 may 2017; accepted: 22 aug. 2017; published: 31 oct. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern africa. the toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. the objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (c2c12) using methyl-thiazol-tetrazolium (mtt) and lactate dehydrogenase (ldh) assays, annexin v and propidium iodide (pi) flow cytometry and transmission electron microscopy (tem). mouse myoblasts were exposed to 2.0 mm, 2.5 mm and 5.0 mm geigerin for 24, 48 and 72 h. a concentration-dependent cytotoxic response was observed. apoptosis was detected by means of annexin v flow cytometry during the first 24 h and apoptotic bodies were also visible on tem. according to the ldh and pi flow cytometry results, myoblast cell membranes were not injured. we concluded that the murine myoblast cell line (c2c12) is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts. introduction geigeria poisoning, locally referred to as ‘vermeersiekte’, is an important plant poisoning in southern africa and in certain seasons, large numbers of sheep have been affected (grosskopf 1964; kellerman, naudé & fourie 1996). all plants of the genus geigeria may cause intoxication, but there are two species which are of particular economic importance in southern africa, namely, geigeria ornativa and geigeria aspera (kellerman et al. 2005). the toxicity induced by these geigeria species is attributed to various sesquiterpene lactones and several have been isolated; for example, vermeeric acid (rimington, roets & steyn, 1936), geigerin (rimington & roets 1936), geigerinin (de villiers 1959) and vermeerin (grosskopf 1964). rimington et al. (1936) proposed that vermeeric acid is the cause of vermeersiekte, but inexplicably the compound could not be re-isolated by various workers (grosskopf 1964; kellerman et al. 2005). in a preliminary trial, rimington and roets (1936) drenched geigerin to only one sheep, without ill effect; however, subcutaneous injection and oral dosing to cats induced toxicity. thus, it is generally believed that a combination of different sesquiterpene lactones induces poisoning (kellerman et al. 2005). the oral administration of an extract containing geigerin, ivalin and dihydrogriesenin to sheep induced ‘vermeersiekte’ (kellerman et al. 2005). the plant toxins are cumulative, as under natural conditions sheep have to graze geigeria ornativa for approximately 2–3 weeks before clinical signs are noticed (grosskopf 1964; kellerman et al. 1996). furthermore, in experimental studies to reproduce the syndrome, sheep received plant material over lengthy periods before exhibiting clinical signs (botha et al. 1997; pienaar et al. 1973; van heerden, van der lugt & durante 1993). however, progress to elucidate the specific or combination of toxic sesquiterpene lactones that will induce ‘vermeersiekte’ has been hampered because of the lack of a suitable small laboratory animal model. such a model would be required to test the isolated fractions because it would simply not be feasible to prepare sufficient quantities of the fractions for administration to sheep over extended periods (grosskopf 1964). the overarching aim was to establish an in vitro tissue culture model to replace the use of sentient animals in toxicity testing, which can then be used in future studies to screen other and/or combinations of sesquiterpene lactones implicated in ‘vermeersiekte’ for cellular toxicity. the specific objective of this study was to investigate the cytotoxicity induced by geigerin in a mouse myoblast cell line (c2c12). cytotoxicity of geigerin was determined using the methyl-thiazol-tetrazolium (mtt) and lactate dehydrogenase (ldh) assays as well as annexin v and propidium iodide (pi) flow cytometry. transmission electron microscopy (tem) was used to investigate subcellular changes induced by geigerin in vitro. materials and methods chemicals and reagents all the chemicals, reagents and cell culture media were purchased from sigma aldrich (south africa) unless otherwise stated. cell culture flasks and plates were purchased from aec amersham (south africa). geigerin geigerin, isolated from g. aspera in the 1980s, has been preserved in dried form in a locked safe deposit as part of the natural toxin collection of the department of paraclinical sciences, faculty of veterinary science, onderstepoort. before commencement of the study, a sub-sample of the pure compound was sent to the department of chemistry, university of pretoria, for structural verification by nuclear magnetic resonance (nmr). it was confirmed that the geigerin had not degraded (figure 1). geigerin stock solutions were prepared in acetone. the final acetone concentration did not exceed 0.5%. the working solution was prepared using the corresponding cell culture medium. figure 1: geigerin, a sesquiterpene lactone, isolated from geigeria aspera. cell cultures the murine skeletal muscle c2c12 cell line (crl-1772) was obtained from the american type culture collection (atcc, manassas, va). the myoblasts were grown in dulbecco’s modified eagle’s medium (dmem), supplemented with 10% foetal calf serum (fcs) and 4 mm glutamine, in a humidified atmosphere of 5% co2 at 37 °c. the cells were cultured in 75 cm2 cell culture flasks. cytotoxicity assays exposure of c2c12 cell cultures to geigerin the cell cultures were seeded at a density of 1 × 106 cells/well in 6-well microtitre plates for flow cytometry and at 2 × 103 cells/well in 96-well plates for the mtt and lactate dehydrogenase (ldh) assays. after 24 h of seeding the cells, geigerin at concentrations of 2.0 mm, 2.5 mm and 5.0 mm were added to each experimental well. the plates were then incubated for 24, 48 and 72 h in a humidified atmosphere of 5% co2 at 37 °c. control wells contained cells and the corresponding culture medium only. cells exposed to triton-x (0.01%) and staurosporine (1 µm) were used as internal quality controls (positive controls) for necrosis (ldh and pi flow cytometry) and apoptosis (annexin v flow cytometry) assays, respectively. methyl-thiazol-tetrazolium assay the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (mtt) assay was based on the modified method described by mosmann (1983). at the end of the exposure period, the supernatants in each well were discarded and the wells were washed with 200 µl phosphate buffered saline (pbs). then dmem (200 µl), containing 5% fcs and 30 μl of mtt (5 mg/ml in pbs), was added to each well. the plates were gently shaken and incubated for 2 h at 37 °c in 5% co2 atmosphere. the supernatants were discarded and 100 μl dimethyl sulphoxide (dmso) was added and the plates were gently shaken for 5 min to solubilise the formed formazan. absorbance (570 nm) was measured using an enzyme-linked immunosorbent assay (elisa) reader (synergy ht, bio-tek instruments, winooski, vt, usa). cell survival was calculated as a percentage of the geigerin tested relative to the negative controls (cells exposed to media only). three independent experiments were carried out with three replicate wells for each geigerin concentration. lactate dehydrogenase assay damaged plasma membranes, indicated by ldh release, were evaluated by measuring ldh activity using the cytotox-one homogeneous membrane integrity assay kit (promega, madison, wi). at the end of the exposure period, 100 µl of the cell culture medium was removed from each experimental well and transferred into a 96-well opaque-walled tissue culture plate. an equal amount (100 µl) of the cytotox-one reagent was added to the wells and the plate was incubated overnight at room temperature. at the end of the incubation, 50 µl of the stop solution was added to each well and absorbance was measured using an elisa reader (synergy ht, bio-tek instruments, winooski, vt, usa) at 540 nm excitation and 590 nm emission wavelengths. ldh release was calculated as the percentage absorbance of each sample relative to the negative controls (cells exposed to media only). two independent experiments were carried out with two replicate wells for each geigerin concentration. annexin v flow cytometry assay an annexin v-fluorescein isothiocyanate (fitc) apoptosis detection kit (life technologies, south africa) was used to detect apoptotic cells. at the end of the exposure period, cells were trypsinised with trypsin-versene ethylenediaminetetra-acetic acid (edta), washed with dulbecco’s pbs (dpbs) and centrifuged at 950 g for 3 min. the supernatant fluid was carefully discarded and pellets were resuspended in 1 ml dpbs and centrifuged as before. the pelleted cells were then resuspended in 300 µl of the annexin binding buffer. a cell suspension of 100 µl was transferred to sterile 2 ml eppendorf tubes. an amount of 5 µl annexin v solution was added to each cell suspension and the tubes were incubated for 15 min at room temperature. after the incubation period, 400 µl of binding buffer was added to each tube. samples were mixed gently and kept on ice. fluorescence was measured immediately using a flow cytometer (cytomics fc 500, beckman coulter, miami, fl) and analysed using the kaluza 1.1 software package. the induction of apoptosis was recorded as annexin v binding activity (%). two experiments were carried out for each geigerin concentration. propidium iodide flow cytometry assay pi was used to stain cells with damaged plasma membranes. at the end of the exposure period, cells were trypsinised, washed with dpbs and centrifuged as described for the annexin v assay. the supernatant was carefully discarded and pellets were resuspended in 1 ml dpbs and centrifuged as before. the pelleted cells were then resuspended in 300 µl of the binding buffer. a cell suspension of 100 µl was transferred to sterile 2 ml eppendorf tubes. an amount of 1 µl pi solution was added to each cell suspension and the tubes were incubated for 15 min at room temperature. after the incubation period, 400 µl of binding buffer was added to each tube. samples were mixed gently and kept on ice. fluorescence was measured immediately using a flow cytometer (cytomics fc 500, beckman coulter, usa) and analysed using the kaluza 1.1 software package. the pi uptake was estimated as the percentage fluorescence of each sample relative to the controls. two experiments were carried out for each geigerin concentration. transmission electron microscopy the cell cultures were seeded at a density of 2.5 × 105 cells/well in 6-well microtitre plates and after 24 h incubation, 2.0 mm, 2.5 mm and 5.0 mm geigerin were added to each experimental well. the plates were then incubated for a further 24, 48 and 72 h at 37 °c. control wells contained cells and the corresponding culture medium only. subcellular changes in the myoblasts were investigated following the modified method of glauert (1980). at the end of the exposure period, cells were fixed in 2.5% glutaraldehyde in sodium phosphate buffer for 15 min before detachment using a cell scraper. loose cells were transferred to 2 ml eppendorf tubes, fixed for another 1 h and pelleted using centrifugation at 830 g for 3 min. the pelleted cells were post-fixed in 1% aqueous osmium tetroxide for 1 h, followed by washing and dehydration in buffer and graded alcohols. cells were subsequently embedded in absolute resin at 60 °c. following overnight curing, ultra-thin sections were prepared and stained with lead citrate and uranyl acetate. the prepared sections were viewed using a transmission electron microscope (philips cm10) operated at 80 kv. statistical analysis basic descriptive statistics were performed (mean ± sem) and data were expressed as percentages compared to the negative controls. linear mixed model analysis, also known as restricted maximum likelihood (reml) analysis, was applied to the mtt activity values to model the correlation over 72 h in an analysis of repeated measurements. a combined analysis over 3 weeks was performed to test for differences between the three time periods, the three different concentrations and the period by concentration interaction effects. an antedependence model of order 1 was found to best model the correlation over time, allowing for variances to change over time. means were compared using tukey’s test at 5% level. all data were analysed using the statistical programme genstat® (payne 2015). results methyl-thiazol-tetrazolium assay a concentration-dependent cytotoxic response was recorded following the exposure of c2c12 myoblasts to geigerin at 2.0 mm, 2.5 mm and 5.0 mm concentrations (figure 2). comparison between the concentration means revealed a significant difference (p < 0.05), but no statistical difference occurred when the three incubation periods were compared. the greatest reduction in cell survival (%) was observed when the c2c12 cells were exposed to 5.0 mm geigerin after 24 h (49.6 ± 1.8%); 48 h (43.3 ± 1.2%) and 72 h (31.2 ± 1.2%). figure 2: assessment of cell survival using methyl-thiazol-tetrazolium assay following exposure of c2c12 myoblasts to geigerin (2.0 mm, 2.5 mm and 5.0 mm) for 24, 48 and 72 h. apoptosis analysis myoblasts undergoing apoptosis after exposure to geigerin for 24, 48 and 72 h were detected using the annexin v flow cytometry assay. a concentration-dependent increase in annexin v binding activity was recorded only at the 24 h exposure period (figure 3) for the different geigerin concentrations (i.e. 67.66% for 2.0 mm; 81.15% for 2.5 mm; 89.48% for 5.0 mm) when compared to the negative controls (37.81%). these results indicate that apoptosis was induced in the myoblasts exposed to geigerin for 24 h. figure 3: annexin v activity (flow cytometry) following exposure of c2c12 myoblasts to geigerin (2.0 mm, 2.5 mm and 5.0 mm), media only (control) and staurosporine (positive control) for 24 h. (a) control, (b) geigerin 2.0 mm, (c) geigerin 2.5 mm, (d) geigerin 5.0 mm and (e) straurosporine 1 µm. necrosis analysis the induction of necrosis following exposure of the c2c12 myoblasts to geigerin at 2.0 mm, 2.5 mm and 5.0 mm concentrations for 24, 48 and 72 h was investigated using the ldh and pi flow cytometry assays. insignificant ldh leakage (less than 20%) was recorded after 24 and 48 h exposure to geigerin, but no ldh leakage was measured after 72 h (figure 4a). uptake of the pi dye by the myoblasts was also not substantial when compared to that of the control cells (figure 4b). collectively, these results indicate that there was very little damage to the cell membranes of the c2c12 cells following exposure to geigerin at the different concentrations and exposure periods. figure 4: (a) lactate dehydrogenase enzyme leakage and (b) propidium iodide uptake (flow cytometry), following exposure of c2c12 myoblasts to geigerin (2.0 mm, 2.5 mm and 5.0 mm), media only (control) and triton-x (positive control) for 24, 48 and 72 h. transmission electron microscopy ultrastructural examination of the cultured myoblasts revealed regular apoptotic changes, namely, blebbing of the cell membrane with formation of apoptotic bodies as well as nuclear invagination and chromatin condensation towards the nuclear periphery following exposure to geigerin for 24 h when compared to the control cells (figure 5). in general, mitochondrial membranes remained intact throughout the exposure period. the integrity of the cell and nuclear membranes remained intact throughout the exposure period similar to the control cells (figure 5). the golgi bodies and endoplasmic reticula (er) appeared normal and evenly distributed in the cytoplasm of the cells. after 72 h exposure to geigerin, the er vesicles became enlarged and the ribosomes appeared disorganised and detached when compared to control cells (figure 5). with prolonged exposure to 5.0 mm geigerin, the mitochondrial matrix (including the cristae) became less electron-dense compared to the control cells. rare necrotic cells (i.e. swollen or enlarged because of hydropic change, nuclear membrane indentation and loss of cell membrane contiguity) were noticed following exposure of the cells to the highest concentration of geigerin (5.0 mm) for 72 h (figure 5). figure 5: electron micrographs showing c2c12 myoblasts exposed to 5.0 mm geigerin for 24, 48 and 72 h. (a) control, (b) geigerin 5.0 mm (24 h), (c) geigerin 5.0 mm (48 h) and (d) geigerin 5.0 mm (72 h). discussion the cytotoxicity results obtained using the mtt assay indicated that geigerin affected the activity of the mitochondrial dehydrogenase enzyme (figure 2). the inhibition of mitochondrial oxidative phosphorylation by sesquiterpene lactones isolated from g. aspera has been demonstrated previously (narasimhan, kim & safe 1989; van aswegen, vermeulen & potgieter 1979). decreased enzyme activities (such as mitochondrial succinate dehydrogenase involved in mitochondrial electron transfer chain reactions) as a result of mitochondrial damage have been linked to reduced atp synthesis, which is crucial for metabolic functions and growth of cells (schulze-osthoff et al. 1992). apoptosis is an important, highly-conserved and well-controlled process of cell death that is induced by a variety of physiological and pathological conditions (zhang et al. 1998). it has been demonstrated that specific death receptors (e.g. fas, tumour necrosis factor) and mitochondria both play a critical role in the initiation of apoptosis via their activation of caspases (cysteine-requiring proteases) (sawai & domae 2011). phosphatidylserine, an amino-phospholipid located on the inner surface of the plasma membrane, has been used as an early marker of cells undergoing apoptosis (krysko, d’herde & vandenabeele 2006). during apoptotic cell death, phosphatidylserine is actively translocated to the outer surface of the plasma membrane, where it can be detected by using annexin v (masango, ellis & botha 2015). in the current study, apoptosis was induced in the c2c12 myoblasts only at the 24 h period following exposure to geigerin (figure 3). previous studies have reported induction of apoptosis by sesquiterpene lactones in cultured cells (dirsch, stuppner & vollmar 2001; zhang, ong & shen 2004; zhang et al. 2005, 2013). the presence of apoptotic cells was furthermore confirmed by tem after exposure to geigerin (figure 5). necrosis was not observed when utilising in vitro assays following exposure of c2c12 myoblasts to geigerin (figure 4), but with tem a few necrotic cells were observed at the highest exposure level after 72 h (figure 5). moderately high geigerin concentrations were required to induce cytotoxicity in this study. it could be because of the cumulative effect of sesquiterpene lactones associated with this disease. sheep have to ingest large quantities of the plant material over prolonged periods of time before they develop clinical signs (botha et al. 1997; grosskopf 1964; kellerman et al. 1996). the established murine myoblast cell line could also be used to compare the relative toxicities of other more toxic αβ-unsaturated-γ-lactones, such as geigerinin, ivalin and vermeerin, also implicated as a cause of the disease (grosskopf 1964; kellerman et al. 2005; rodriguez, towers & mitchell 1976). in addition, metabolic activation with s9-mix should also be done in order to establish if the biotransformed sesquiterpene lactones are more toxic (hostanska et al. 2014). conclusion in summary, exposure of the c2c12 myoblasts to increasing geigerin concentrations resulted in concentration-dependent cytotoxicity. apoptosis was the main mechanism through which geigerin-induced the observed cell death. some ultrastructural lesions typical for necrosis were also observed at the highest geigerin exposure levels after 72 h. based on the cytotoxicity observed, supported by tem findings, it is concluded that the murine myoblast cell line (c2c12) could be used as a suitable in vitro model to evaluate cytotoxicity induced by other and/or combinations of sesquiterpene lactones implicated in ‘vermeersiekte’ in sheep, with and without metabolic activation. future studies should also investigate subcellular effects of these myotoxins on the differentiated myotubes and/or other cell lines. acknowledgements this work was supported by the national research foundation (nrf) of south africa. the authors would like to thank dr mirinda van kleef for support with the flow cytometry and mr chris van der merwe for the tem. they would also like to express their gratitude to marie smith, stats4science, who performed the statistical analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.j.b. was the principal investigator. m.g.m. and g.c.h.f. performed the assays. s.j.c. made conceptual contributions. all authors read and approved the final manuscript. references botha, c.j., gous, t.a., penrith, m.l., naudé, t.w. labuschagne, l. & retief, e., 1997, ‘vermeersiekte caused by geigeria burkei harv. subsp. burkei var. hirtella merxm. in the northern province of south africa’, journal of the south african veterinary association 68, 97–101. https://doi.org/10.4102/jsava.v68i3.884 de villiers, j.p., 1959, ‘the 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5, 239–249. zhang, g., yan, g., gurtu, v., spencer, c. & kain, s.r., 1998, ‘caspase inhibition prevents staurosporine-induced apoptosis in cho-k1 cells’, apoptosis 3, 27–33. https://doi.org/10.1023/a:1009655018726 abstract introduction materials and methods results discussion acknowledgements references about the author(s) jonas thoromo department of veterinary microbiology and parasitology, sokoine university of agriculture, tanzania edgar simulundu department of disease control, the university of zambia, zambia herman m. chambaro ministry of fisheries and livestock, lusaka, zambia liywalii mataa ministry of fisheries and livestock, lusaka, zambia caesar h. lubaba ministry of fisheries and livestock, lusaka, zambia girja s. pandey department of disease control, the university of zambia, zambia ayato takada division of global epidemiology, hokkaido university research center for zoonosis control, japan gerald misinzo department of veterinary microbiology and parasitology, sokoine university of agriculture, tanzania aaron s. mweene department of disease control, the university of zambia, zambia citation thoromo, j., simulundu, e., chambaro, h.m., mataa, l., lubaba, c.h., pandey, g.s. et al., 2016, ‘diagnosis and genotyping of african swine fever viruses from 2015 outbreaks in zambia’, onderstepoort journal of veterinary research 83(1), a1095. http://dx.doi.org/10.4102/ojvr.v83i1.1095 original research diagnosis and genotyping of african swine fever viruses from 2015 outbreaks in zambia jonas thoromo, edgar simulundu, herman m. chambaro, liywalii mataa, caesar h. lubaba, girja s. pandey, ayato takada, gerald misinzo, aaron s. mweene received: 15 oct. 2015; accepted: 18 dec. 2015; published: 29 apr. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in early 2015, a highly fatal haemorrhagic disease of domestic pigs resembling african swine fever (asf) occurred in north western, copperbelt, and lusaka provinces of zambia. molecular diagnosis by polymerase chain reaction targeting specific amplification of p72 (b646l) gene of asf virus (asfv) was conducted. fourteen out of 16 domestic pigs from the affected provinces were found to be positive for asfv. phylogenetic analyses based on part of the p72 and the complete p54 (e183l) genes revealed that all the asfvs detected belonged to genotypes i and id, respectively. additionally, epidemiological data suggest that the same asfv spread from lusaka to other provinces possibly through uncontrolled and/or illegal pig movements. although the origin of the asfv that caused outbreaks in domestic pigs in zambia could not be ascertained, it appears likely that the virus may have emerged from within the country or region, probably from a sylvatic cycle. it is recommended that surveillance of asf, strict biosecurity, and quarantine measures be imposed in order to prevent further spread and emergence of new asf outbreaks in zambia. introduction african swine fever (asf) is a highly contagious and fatal haemorrhagic disease of domestic pigs caused by a complex dna virus of the genus asfivirus, family asfarviridae. most isolates of asf virus (asfv) cause an acute haemorrhagic fever with mortality rates that can reach 100% in susceptible pigs. asfv circulates in an ancient sylvatic cycle involving soft ticks of the ornithodoros species and warthogs (phacochoerus aethiopicus) as well as in domestic pig populations with or without involvement of ornithodoros ticks; other wild suids, in particular bushpigs (potamochoerus larvatus, potamochoerus porcus) can act as asymptomatic reservoirs of the virus but are not associated with ticks and their role, if any, in the epidemiology of the disease is unknown (muhangi 2014). although asf is known to be endemic in sub-saharan africa (penrith et al. 2013), it is occasionally introduced into new regions or continents. for example, in june 2007, asf was introduced into the republic of georgia and subsequently spread into neighbouring countries including armenia, azerbajan, the russian federation, ukraine, and belarus (beltrán-alcrudo et al. 2008; chapman et al. 2011). recently, asf has spread into some of the member states of the european union including poland, latvia, lithuania, and estonia (pejsak et al. 2014). these events stress the need for concerted international efforts in the control of asf. in zambia, asf was first reported in 1912 in the vicinity of fort jameson, now called chipata, in eastern province (wilkinson et al. 1988). subsequently, there have been repeated reports of its occurrence in the same area, where it is now considered to be endemic. laboratory confirmation was done on only five occasions between 1974 and 1983 (wilkinson et al. 1988). however, a recent study confirmed an outbreak of asf in lusaka in october 2013 (yabe et al. 2015). although the zambian government has endeavoured to control the disease, outbreaks continue to be reported across the country in non-endemic regions (yabe et al. 2015). in early 2015, outbreaks of a haemorrhagic disease resembling asf that killed a number of domestic pigs were reported in north western, copperbelt, and lusaka provinces of zambia. the present study was aimed at confirming and conducting genotypic characterisation of asfvs involved in these outbreaks. materials and methods study area, sample collection, and processing tissue samples were collected from domestic pigs in districts with suspected asf cases based on clinical signs, post-mortem lesions, and reports from local veterinary officials and farmers during january to april 2015. samples were obtained from kitwe district in the copperbelt province; chongwe, chilanga (linda area), and lusaka (chamba valley area) in lusaka province; and solwezi district in north western province (figure 1). eight samples were collected from domestic pigs in lusaka province, four samples came from north western, and four from the copperbelt provinces, making a total of 16. tissues collected included mesenteric lymph nodes, spleen, kidney, and liver. samples were collected in sterile tubes and transported on ice to the laboratory where they were stored at -80 °c until use. tissues were homogenised in phosphate-buffered saline at a ratio of 1:10 (w/v). tissue homogenates from each animal were pooled and used for laboratory diagnosis and molecular analyses. figure 1: map showing sampling locations for african swine fever in domestic pigs. dna extraction one µl of proteinase k (20 mg/ml) was added to 100 µl of 10% tissue homogenate followed by overnight incubation of the tissue-enzyme mixture at 55 °c. then the tissue-enzyme mixture was heated at 95 °c for 10 min followed by centrifugation at 6000 g for 5 min. the supernatant containing dna was collected and the pellet discarded. diagnosis of african swine fever virus using polymerase chain reaction asfv dna was detected by polymerase chain reaction (pcr) using ppa1/2 primers, as previously reported by agüero et al. (2003). pcr was carried out in gene amp pcr system (applied bio-systems, foster city, ca usa) using taq polymerase (thermoscientific, waltham, ma usa), according to the manufacturer’s instructions. pcr products were separated by electrophoresis on a 1.5% agarose gel stained with gelred nucleic acid stain (phenix research products, candler, usa). genotyping of african swine fever virus genotyping of asfv was performed by partial amplification of the p72 (b646l) and complete p54 (e183l) gene amplification using pcr. primers including p72u/d and ppa89/722 were used in the amplification of p72 and p54 genes, respectively (bastos et al. 2003; nix et al. 2006). the amplification conditions previously described by gallardo et al. (2009) were used in the present study. pcr products were purified from agarose gels using a nucleospin gel and pcr clean-up kit (macherey-nagel, düren, germany) and subjected to dideoxynucleotide cycle sequencing using big dye terminator cycle sequencing kit version 3.1 (applied biosystems, foster city, ca usa). products from dideoxynucleotide cycle sequencing reaction were purified by ethanol precipitation and separated on a 3500 genetic analyzer (applied biosystems, foster city, ca usa). chromatograms for both the forward and the reverse primer reactions were read using sequence scanner v1.0 software (applied biosystems, foster city, ca usa). the obtained sequences were deposited in genbank/embl/ddbj databases under accession numbers lc088171-lc088176. data analysis asfv nucleotide sequences representing the 22 p72 genotypes (boshoff et al. 2007) were obtained from genbank database and analysed in mega 6.06 (tamura et al. 2013) along with those determined in the present study. the p72 and p54 nucleotide sequences were aligned in mega 6.06 using clustal w. phylogenetic reconstruction was conducted using the neighbour-joining method. phylogenetic tree reliability was supported using the bootstrap method with 1000 replications. results outbreak history in solwezi district in north western province, an outbreak of a pig disease similar to asf was observed in january–february 2015 at a farm with a total of 66 pigs. twenty pigs died as a result of the disease whereas 46 were depopulated by government. at another farm in the same district 7 pigs died from a similar disease and the rest (23 pigs) were depopulated by government. epidemiological investigations revealed that all the pigs at the primary farm were brought from the copperbelt province. during this time period, 4 pigs died from a herd of 68 at a farm in kitwe district of the copperbelt. the rest of the pigs (64) were depopulated by government officials. the origin or source of the disease was not established. in chongwe district (lusaka province), the disease came about after a boar was bought from kaunda square in lusaka from a farmer whose pigs had started dying and was selling them off. the farmer in question had initially bought the pigs from chilanga district. this was during a time when it was not mandatory to test animals meant for breeding for asf. the case was reported in april 2015. during the sampling period, more than 600 pigs died in lusaka province including chilanga (linda area), chongwe, and lusaka (chamba valley area) either through depopulation or from disease. clinical signs and post-mortem findings clinical signs and gross pathological findings of the screened animals in this study were similar to those of asf, and hence this disease was highly suspected. the animals presented with incoordination, inappetence, cyanosis of the ears, petechial haemorrhages on leg extremities, tachypnoea, and recumbence. on post-mortem examination pigs had oedematous lungs, fluids in body cavities, and haemorrhages and congestion in internal organs such as the spleen, lymph nodes, intestine, and kidneys. detection of african swine fever virus genome in total, 16 clinical samples from four different districts in zambia were analysed using pcr. out of the 16 samples, asfv was detected in 14 samples using three different pairs of primers that specifically amplify different parts of asfv genome including b646l and e183l. nucleotide sequence and phylogenetic analysis the p72 and p54 nucleotide sequences of asfv detected in domestic pigs in kitwe, solwezi, chongwe, lusaka, and chilanga were found to be 100% similar to each other. phylogenetic analysis of the p72 gene sequences from this study clustered into genotype i (figure 2a). similarly, phylogenetic analysis of the e183l (p54) nucleotide sequences showed that the 2015 zambian asfv belonged to genotype id (figure 2b). moreover, they were different from other genotype i asfvs such as those from west africa (genotype ib) and america and europe (genotype ia and ic) (figure 2b). figure 2: phylogenetic relationships based on nucleotide sequences of part of p72 (a) and p54 (b) genes of african swine fever virus detected in tissues collected from diseased pigs in zambia. discussion although zambia has experienced many outbreaks of asf, genetic characterisation of asfvs involved in outbreaks has been conducted only for some viruses. available genotyping studies were performed before the year 2008 (lubisi et al. 2005, 2007; nix et al. 2006). however, asf outbreaks in domestic pigs have continued to occur in zambia, including the one reported in 2013 (yabe et al. 2015) and those reported in this study. in this study, clinical and post-mortem findings consistent with those of asf were observed suggesting that outbreaks of a highly fatal haemorrhagic disease afflicting domestic pigs in north western, copperbelt, and lusaka provinces of zambia could be asf. asfv infection of diseased pigs was confirmed through detection of different parts of the asfv genome by pcr. the finding that nucleotide sequences of the p72 and p54 genes of asfv under study were 100% identical suggests that these outbreaks were caused by the same virus. phylogenetic analyses based on the p72 nucleotide sequences confirmed that the 2015 asfv from zambia belonged to genotype i (figure 2a). genotype i asfvs have a wide geographical distribution and have been previously reported in europe, south america, the caribbean islands, west africa, east africa, and southern africa (lubisi et al. 2005). based on the finding that the p54 nucleotide sequences of 2015 zambian asfv belonged to the southern african genotype 1d, it seems likely that this virus may have originated from within the country (or possibly from the southern african region) and may not have been imported from other geographical locations. epidemiological data gathered from farmers and veterinarians among the different provinces of zambia during the 2015 asf outbreaks suggest that asf spread within zambia was facilitated by movement of infected pigs and/or pig products from affected areas to disease free areas. for instance, in chongwe district (lusaka province), the disease came about after a boar was bought from kaunda square (lusaka district) from a farmer who was selling off pigs because his pigs had started dying. similarly, asf broke out in solwezi after a farmer purchased pigs from the copperbelt province. it is therefore recommended that future control and prevention strategies of asf in zambia should focus on early detection through screening of the animals and timely confirmation of the disease, strict quarantine measures, biosecurity, stock movement restrictions including culling and proper disposal of infected and in-contact animals, and decontamination of affected premises. acknowledgements we thank participating farmers and veterinarians in zambia. this work was supported in part by the japan initiative for global research network on infectious diseases and japan science and technology agency/japan international cooperation agency within the framework of the science and technology research partnership for sustainable development. j.t. was supported by a scholarship from the wellcome trust (grant wt087546ma) to southern african centre for infectious diseases surveillance, sokoine university of agriculture, morogoro, tanzania. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions j.t. and e.s. conducted most of the experiments. e.s. and g.m. were responsible for experimental and project design. h.m.c. and g.s.p. participated in sample collection and preparation. l.m. and c.h.l. were involved in epidemiological investigations and data analysis. a.t., g.m. and a.s.m. were project leaders and made conceptual contributions. all authors participated in writing of the manuscript. 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health and production 47(2), 459–463. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) yolandi rautenbach department of companion animal clinical studies, university of pretoria, south africa johan schoeman department of companion animal clinical studies, university of pretoria, south africa amelia goddard department of companion animal clinical studies, university of pretoria, south africa citation rautenbach, r., schoeman, j., goddard, a., 2018, ‘prevalence of canine babesia and ehrlichia co-infection and the predictive value of haematology’, onderstepoort journal of veterinary research 85(1), a1626. https://doi.org/10.4102/ojvr.v85i1.1626 original research prevalence of canine babesia and ehrlichia co-infection and the predictive value of haematology yolandi rautenbach, johan schoeman, amelia goddard received: 09 mar. 2018; accepted: 20 aug. 2018; published: 09 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract canine babesiosis and ehrlichiosis are important tick-borne infections in south africa. many south african general veterinary practitioners perceive co-infection with ehrlichia spp. as a common occurrence in dogs with babesiosis. studies about the prevalence of co-infection in south african dogs are lacking. this retrospective study aimed to determine the prevalence of ehrlichia co-infection in dogs with babesiosis. additionally, the predicative value of specific haematological variables for co-infection was evaluated. the study population consisted of 205 dogs diagnosed with canine babesiosis presented to the onderstepoort veterinary academic hospital (ovah) in 2006 and between 2011 and 2013. the babesia-infected dogs were grouped based on presence or absence of an ehrlichia spp. co-infection. ehrlichia spp. co-infection was confirmed using polymerase chain reaction. positive and negative predictive values (ppvs and npvs) of leukopenia or thrombocytopenia for co-infection were also calculated. the prevalence of babesia spp. and ehrlichia spp. co-infection in this cohort of dogs was 2%. in the babesiosis dogs, the ppv of leukopenia for co-infection with ehrlichia spp. was 1.3%, and the npv 97.4%. similarly, the ppv and npvs of thrombocytopenia for co-infection were 2.1% and 100%, respectively. co-infection with ehrlichia spp. was a rare occurrence in dogs with babesiosis presented to the ovah. normal leukocyte or platelet counts confidently ruled out the presence of concurrent ehrlichiosis in this cohort of dogs. however, the diagnosis of ehrlichia co-infection based on the presence of thrombocytopenia or leukopenia would have been associated with false positive results in more than 97.4% of cases. introduction canine babesiosis and ehrlichiosis are important tick-borne infections in south africa, resulting in severe clinical disease (collett 2000; rautenbach, boomker & de villiers 1991; van heerden 1982). in south africa, canine babesiosis is predominantly caused by the virulent babesia rossi sp. (matjila et al. 2008). although several ehrlichia spp. are able to cause natural disease in dogs, only e. canis and e. ruminantium occur in southern africa (kelly 2000; neer et al. 2002). severe thrombocytopenia is a common finding in canine babesiosis (kettner, reyers & miller 2003; scheepers et al. 2011), as is leukopenia (scheepers et al. 2011). similarly in canine ehrlichiosis, thrombocytopenia and leukopenia are common haematological abnormalities in both the acute and chronic phase of the infection (kelly 2000; shipov et al. 2008). despite the presence of severe thrombocytopenia, clinical bleeding is uncommon in canine babesiosis because of marked platelet activation and marked hyperfibrinogenaemia (goddard et al. 2015b; liebenberg et al. 2013); however, excessive bleeding is seen in cases with concomitant ehrlichia spp. infections (van heerden, reyers & stewart 1983), most likely secondary to the thrombocytopenia and thrombocytopathia reported in this disease (neer 1998). in endemic areas, infection with multiple tick-borne pathogens is possible in individual animals, especially secondary to a heavy tick infestation (shaw et al. 2001). a single tick species can act as a vector for multiple pathogens and simultaneous infection with different organisms is possible (schouls et al. 1999; shaw et al. 2001). in south africa, a 19% infection rate of e. canis was noted in rhipicephalus sanguineus ticks (mtshali et al. 2017), but to the authors’ knowledge no reports are available on the infection rate of b. rossi in haemaphysalis elliptica ticks. studies about the prevalence of babesia and ehrlichia co-infections in dogs in south african are lacking. the majority of veterinary practitioners in south africa have reported diagnosing canine ehrlichiosis in their practices and more than half consider it an occasional to common co-infection in dogs with babesiosis (collett 2000). approximately one-quarter of these practitioners based their diagnosis of concurrent ehrlichia spp. infection on the presence of normal white cell count or leukopenia, while half of the clinicians based their diagnosis on the presence of a thrombocytopenia (collett 2000). in addition, a considerable proportion of practitioners are inclined to treat for ehrlichia spp. co-infection in dogs with babesiosis, without the former being definitively confirmed (collett 2000). tetracycline, chloramphenicol, imidocarb dipropionate and amicarbalide are effective in the treatment of ehrlichiosis (neer 1998). however, doxycycline is considered the drug of choice and a 4-week oral course is recommended based on the current consensus statement of the infectious disease study group of the american college of veterinary internal medicine (eddlestone et al. 2007; neer et al. 2002). currently, there are conflicting results about the efficacy of doxycycline in clearing e. canis from the blood and tissues of infected dogs (eddlestone et al. 2007; harrus et al. 1998; iqbal & rikihisa 1994; mcclure et al. 2010), raising questions about the dosage and duration of doxycycline treatment and the development of potential drug resistance. the first objective of this study was to determine the prevalence of ehrlichia spp. co-infection in dogs with babesiosis presented to the onderstepoort veterinary academic hospital (ovah). the second objective was to determine the predictive value of thrombocytopenia or leukopenia for co-infection with ehrlichia spp. in dogs with babesiosis. we hypothesised that the prevalence of co-infection would be low in dogs presented with babesiosis and, additionally, that the presence of leukopenia or thrombocytopenia would not be predictive of concurrent ehrlichia infection. materials and methods this retrospective study included the review of medical records of client-owned dogs naturally infected with canine babesia spp., from two previously performed prospective clinical research studies. the dogs were presented for veterinary care to the ovah, gauteng, south africa, between january and march 2006 (study 1) and from october 2011 to april 2013 (study 2). the research protocols were approved by the university of pretoria’s animal ethics committee (v070-05 and v055-11, respectively). several studies originating from these two cohorts of dogs have been published (goddard et al. 2015a, 2015b, 2016; rees & schoeman 2008; schoeman & herrtage 2007, 2008). infection with babesia parasites was initially diagnosed by demonstration of intra-erythrocytic trophozoites on stained thin blood smears, and the species was confirmed as b. rossi by polymerase chain reaction (pcr) and reverse line blot (rlb) (matjila et al. 2004, 2008). all samples were also screened for b. vogeli and e. canis. owner consent was obtained for enrolment of all the dogs in this study. animals suitable dogs sampled during the second study were of any breed and either sex, > 12 weeks of age and weighed > 3 kg, while dogs from the first study did not have weight or age restrictions. both groups of dogs had a demonstrable parasitaemia on a stained thin peripheral blood smear and clinical signs consistent with clinical babesiosis. dogs were excluded if any signs of concurrent chronic or inflammatory disease conditions, any obvious infections or wounds, or any signs of trauma were present. vaccination, drug therapy (drugs affecting platelet function or serum glucose concentrations, calcium-containing preparations, catecholamines or other adrenergic drugs, insulin therapy) or any unrelated metabolic illness four weeks prior to presentation were also reasons for exclusion. sample collection and analyses peripheral venous blood was collected at presentation prior to any treatment. blood samples were collected from the jugular vein from each dog with a 21-gauge needle by careful venepuncture with minimal stasis. the blood samples were collected into serum and ethylenediaminetetraacetic acid (edta) vacutainer plastic tubes (becton dickinson [bd] biosciences, new jersey). the edta sample was used to perform a complete blood count (cbc), pcr and rlb assays. because of the prolonged period between the two study populations, cbc data from two different automated cell counters were available: advia 2120 (siemens, munich, germany) and cell-dyn 3700 (abbott diagnostics, santa clara, ca, united states). deoxyribonucleic acid extraction and polymerase chain reaction deoxyribonucleic acid (dna) was extracted from 200 μl edta-anticoagulated whole blood using a blood and tissue extraction kit (qiamp blood and tissue extraction kit, qiagen, venlo, netherlands) according to the manufacturer’s instructions. molecular diagnosis of b. rossi and exclusion of other babesia, theileria, ehrlichia and anaplasma spp. were performed using pcr and rlb. polymerase chain reaction was conducted with a set of primers that amplified a 460–540 base pair fragment of the 18s ssu rrna spanning the v4 region, a region conserved for babesia and theileria. the ehrlichia pcr amplified the v1 hypervariable region of the 16s ssu rrna. the membrane used for rlb included probes for b. vogeli, b. rossi, b. canis and e. canis (matjila et al. 2008). statistical analysis the babesia-infected dogs were divided into groups, based on presence or absence of ehrlichia spp. co-infection. descriptive statistics were performed using a commercial software package (spss statistics 23.0® software; spss inc, statacorp, college station, tx, united states). the prevalence of ehrlichia spp. co-infection in babesiosis dogs was defined as the percentage of babesiosis dogs that tested positive on pcr for ehrlichia spp. the respective positive predictive values (ppvs) of leukopenia and thrombocytopenia for co-infection with ehrlichia spp. were calculated. ppv = tp / (tp + fp) × 100, where tp represents the true positives (babesia-infected dogs with leukopenia or thrombocytopenia co-infected with ehrlichia spp.) and fp represents the false positives (babesia-infected dogs with leukopenia or thrombocytopenia without ehrlichia spp. co-infection). the respective negative predictive values (npvs) of a normal leukocyte count and platelet count for the exclusion of co-infection with ehrlichia spp. were also determined. npv = tn / (tn + fn) × 100, where tn represents the true negatives (babesia-infected dogs with normal leukocyte counts or platelet counts without ehrlichia spp. co-infection) and fn represents false negatives (babesia-infected dogs with normal leukocyte counts or platelet counts with ehrlichia spp. co-infection). thrombocytopenia was defined as a platelet count < 200 × 109/l (population-based reference interval [ri]: 200–500 × 109/l) and a leukopenia was defined as a leukocyte count < 6 × 109/l (ri: 6–15 × 109/l). results study population characteristics of the 205 dogs sampled that were naturally infected with babesia spp., only 198 dogs were included in the study. two dogs were excluded because of negative pcr results for any infectious agents and six dogs did not have any pcr results available for analysis. the median age (range) and weight (range) of all the infected dogs were 18 months (9–36) and 18.2 kg (8.5–28.5) (table 1), respectively, and included 127 male and 71 female dogs. the most prevalent breeds were mixed-breed dogs (29%), followed by boerboels (12%) and jack russell terriers (8%). the remaining breeds each represented less than 5% of the population. the median age (range) and weight (range) of the babesia-infected dogs were 18 months (9.0–36.5) and 18.3 kg (8.6–28.5), respectively. the median age (range) and weight (range) of the co-infected dogs were 12 months (6–18) and 6.7 kg (4.2–23.9), respectively (table 1). table 1: population characteristics, leukocyte count and platelet count for dogs with babesia spp. and ehrlichia spp. infection. co-infection prevalence based on pcr results, 191 of the dogs were solely infected with b. rossi (96%), four dogs were infected with b. vogeli (2%), of which two were co-infected with e. canis (1%), one dog was co-infected with both b. rossi and b. vogeli (0.5%) and two dogs infected with b. rossi were co-infected with e. canis and e. ruminantium (1%). the prevalence of babesia spp. and ehrlichia spp. co-infection in this cohort of dogs was 2%. predictive value of haematology results the median leukocyte count (range) and platelet count (range) of all the infected dogs were 6.7 × 109/l (4.8–11.90) and 17 × 109/l (4–49), respectively. in the babesia-infected dogs, the median leukocyte count (range) was 6.67 × 109/l (4.8–11.90) and the median platelet count (range) was 16 × 109/l (4–48). the median leukocyte count (range) in co-infected dogs was 8.86 × 109/l (3.88–11.21), and the platelet count (range) was 58 × 109/l (31–100) (table 1). the ppv of leukopenia for co-infection with ehrlichia spp. in babesiosis dogs was 1.3%, while the npv was 97.4% (table 2). the ppv and npv of thrombocytopenia for co-infection with ehrlichia spp. in babesiosis dogs was 2.1% and 100%, respectively (table 3). table 2: contingency table illustrating the relationship between infection status and leukocyte count. table 3: contingency table illustrating the relationship between infection status and platelet count. discussion published data on the prevalence of co-infection with multiple tick-borne pathogens in south african dogs are very limited. our study showed that the prevalence of concurrent infections with babesia and ehrlichia spp. in south african dogs is very low. our results concur with those of a previous molecular survey conducted on 1138 blood specimens collected from domestic dogs in south africa between 2000 and 2006, which reported a 2% prevalence of b. rossi and e. canis co-infection (matjila et al. 2008). interestingly, the survey reported that b. rossi and e. canis co-infections were found in all of the sampled areas except for the free state and eastern cape provinces (matjila et al. 2008). similar to our findings, 1.5% of dogs were concurrently infected with b. rossi and e. canis, 1.3% of the dogs had a b. vogeli and e. canis co-infection and only one dog had a concurrent b. rossi and b. vogeli infection in the subset of survey samples collected from the ovah (matjila et al. 2008). our results confirmed that b. rossi is the most common cause of babesiosis in dogs presented to the ovah (matjila et al. 2008). this correlates with the high percentage of babesia-infected dogs presented to the ovah that are infested with h. elliptica, the tick vector for b. rossi (horak 1995; matjila et al. 2008). the b. vogeli and e. canis co-infection can be explained by the fact that r. sanguineus is the vector for both of these pathogens (groves et al. 1975; uilenberg et al. 1989). the mixed infections with b. rossi and e. canis or b. vogeli is most likely because of the overlapping distribution pattern of r. sanguineus and h. elliptica and the fact that these vectors commonly infest the same host (horak 1995; matjila et al. 2008). two of the dogs in our study were co-infected with both e. canis and e. ruminantium. ehrlichia ruminantium causes heartwater or cowdriosis in all domestic and some wild ruminants and is transmitted by ticks of the genus amblyomma (allsopp 2010). the main vector of heartwater in southern africa is a. hebraeum (allsopp 2010). currently, eight different srrna genotypes of e. ruminantium are known, of which only the pretoria north genotype has been identified in dogs (allsopp 2010). this genotype was isolated in south african dogs that presented with clinical symptoms of ehrlichiosis but that tested negative using a pcr assay specific for north american e. canis (allsopp & allsopp 2001). the e. ruminantium organism infecting dogs has not yet been isolated in culture (allsopp 2010; allsopp & allsopp 2001), thus prohibiting further investigation into its pathogenicity, host specificity and the identification of its vector. recently, a low number of a. hebraeum ticks were found in a south african cohort of dogs and cats (mtshali et al. 2017). these ticks were negative for the presence of e. canis dna (mtshali et al. 2017), but the samples were not screened for e. ruminantium dna. positive predictive value and npv are utilised for assessing the probability of an accurate diagnosis (petrie & watson 2013). there is an interdependence between prevalence, sensitivity, specificity and predictive values (hernaez & thrift 2017). prevalence can also be interpreted as the probability that the disease is present before the test is performed (pretest probability) and affects predictive values (petrie & watson 2013). when disease prevalence is low, the npv is higher and consequently the ppv lower (hernaez & thrift 2017; petrie & watson 2013). thus, in a low prevalence setting, a negative result confidently excludes the presence of disease (hernaez & thrift 2017; petrie & watson 2013). the opposite is true when the prevalence of the disease increases (petrie & watson 2013). the predictive value of the presence of leukopenia or thrombocytopenia for concurrent ehrlichiosis in our population of dogs with babesiosis was very low, 1.3% and 2.1%, respectively. in contrast, the predictive value of normal leukocyte count or platelet count for the absence of concurrent ehrlichiosis was very high, 97.4% and 100%, respectively. therefore, in light of the low prevalence of co-infection in this population of dogs with babesiosis, a negative test result (i.e. normal leukocyte count or normal platelet count) confidently rules out the presence of concurrent ehrlichiosis. furthermore, a positive test result (i.e. leukopenia or thrombocytopenia) may result in incorrect diagnosis of co-infection, leading to an over-diagnosis of concurrent ehrlichiosis in babesia-infected dogs. the median age of the population of infected dogs in this study was 18 months, which is consistent with previous reports that the majority of dogs with babesiosis are young (jacobson 2006; mellanby et al. 2011). more male than female dogs were presented with babesiosis, 64% compared to 36%, mirroring previous reports (jacobson 2006). the most common breeds in this study were mixed-breed, followed by boerboel and jack russell terrier. these findings are similar to the reported literature, with mixed-breed dogs most commonly presented for babesiosis while toy breed dogs appear to be less likely to be presented for babesiosis (jacobson 2006; mellanby et al. 2011). a limitation of this study is the fact that the haematology was performed on two different haematology analysers and therefore the results are not comparable. however, the population-based ris remained unchanged over the study period; therefore, the classification of a leukopenia or thrombocytopenia remained consistent. a further limitation regarding the reported prevalence of ehrlichia co-infection in south african dogs with babesiosis is that the findings of this study are only applicable to the geographical area of this cohort of dogs. the prevalence might be higher in areas where regular tick prevention is not practised or in areas with a higher concentration of tick vectors. in light of the retrospective nature of this study, the inference was made that the analysed data were unbiased; however, some babesiosis cases with concurrent ehrlichiosis could have inadvertently been excluded. therefore, the results reported here should be confirmed with a prospective study. conclusion it can thus be concluded from these results that concurrent ehrlichiosis is uncommon in dogs with babesiosis presented to the ovah, situated north of pretoria in gauteng. although thrombocytopenia is a common finding in dogs with babesiosis, its ppv for co-infection is very low, similar to the presence of leukopenia. therefore, the diagnosis of ehrlichia co-infection based on thrombocytopenia or leukopenia is associated with many fp results. acknowledgements competing interests the authors declare that they have no financial or 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accepted: 22 oct. 2018; published: 30 jan. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract caprine pediculosis is an ectoparasitic disease of great concern among goat farmers in india. it may be caused by either sucking lice or chewing lice; the latter one results in severe skin lesions, leading to production loss. this study evaluated the effectiveness of the macrocytic lactone drug, ivermectin, administered via subcutaneous injection, against chewing lice bovicola (damalinia) caprae infestation in naturally infested goats. the study was conducted on 20 goats with severe b. caprae infestation. animals of group a (n = 10) were treated using a single dose of ivermectin (200 µg/kg body weight) subcutaneously and animals of group b (n = 10) underwent placebo therapy using normal saline. the animals were examined on days 0, 3, 7, 14, 21, 28, 42 and 56 for lice counts. there was 100% elimination of lice in all animals of group a and effective protection from re-infection remained at least for 21 days. considerable improvement in haematological parameters was also observed by day 21. based on this study, ivermectin injected via a subcutaneous route can be used effectively for the therapeutic and prophylactic management of chewing lice infestation in goats maintained under an extensive grazing system. introduction caprine pediculosis is considered as a major ectoparasitic condition commonly seen among goats reared under an extensive grazing system, mainly during the winter season (iqbal et al. 2014). it is reported from almost all parts of the world, especially from the dehradun valleys of the subtropical himalayas, tropical asia and parts of africa (adesh et al. 1994; ajith et al. 2017a). based on their feeding habits, aetiological agents of caprine pediculosis can be classified into two subgroups: sucking lice (suborder: anoplura), which includes blood feeders such as linognathus stenopsis and l. africanus, and chewing or biting lice (suborder: mallophaga), which includes non-blood feeders such as bovicola (damalinia) caprae and b. limbata (taylor, coop & wall 2007). the common chewing lice found among goats in india were b. caprae (ajith et al. 2017a; giri, kashyap & dewangan 2013). these are small (approximate size: 1 mm – 2 mm) permanently parasitic insects of the phthiraptera family, seen close to the skin, and complete their whole life cycle on the host body surface and transmission occurs only by direct physical contact (smith & sherman 2009; taylor et al. 2007). agroclimatic region, breed, immune status, system of rearing and hygiene are the major factors affecting the prevalence and distribution of lice among goats (ajith et al. 2017a). alopecia, skin irritation, papulo-crustous dermatitis, self-excoriation, anaemia and stress-associated production loss and growth reduction were often found to be associated with caprine pediculosis (taylor et al. 2007). chewing lice infestation induced t-helper cells-2 (th2)-dominant immune response and conferred pro-oxidative haemato-biochemical damages in goats (ajith et al. 2017b). successful control of chewing lice in goat herds depends on the method of application and efficacy of insecticide, which, in turn, depends on the distribution of insecticide on the body surface and redistribution to untreated parts. failure in treatment may occur because of incorrect application, inability to reach effective licicidal concentration on skin surface and development of insecticidal resistance (keys, toothey & arul 1993). conventionally, topical insecticides such as amidines, organophosphates, carbamates and pyrethroids were used as dips or spray to control chewing lice infestation in animals. but low safety (potential for being toxic to the domestic animals), public health concerns (topical preparations can spread to animal handlers), practical difficulties (the distribution of insecticide depends on hair length and external application is not suitable because caprine pediculosis is more prevalent during winter season) and risk of environmental contamination (goats are mainly reared under extensive rearing system, especially in india, so topically applied insecticide may get spread into the pasture) limited their therapeutic use in food animals, especially those reared under an extensive grazing system. later, the pour-on formulation of synthetic pyrethroids became more popular and acceptable because of high efficiency, ease of application and rapid distribution on the body (mcewan jenkinson et al. 1986). flumethrin 1% pour-on (bayticol® pour-on, 1% weight/volume [w/v]; bayer, germany) was reported to be 100% effective in the treatment of chewing lice (b. caprae) infestation in goats, preventing re-infestation for up to 42 days under experimental conditions (garg, katoch & bhushan 1998). the possible demerit of that study design was the lack of possible routes of transmission, because chewing lice essentially need close contact or manual transfer of lice for re-infection. pour-on treatments were reported to be less effective in goats because of the variability in hair fibre characteristics and lice occur more close towards skin surface (taylor et al. 2007). also, rapid development of resistance, high toxicity and concerns of residues in milk as well as meat and chance of environmental contamination discourage the use of topical pyrethroids in food animals, especially maintained under extensive system of rearing (lakshmanan et al. 2013). macrocyclic lactones (avermectins and milbemycins) are a group of endo-ectoparasiticidal agents with a wide margin of safety and broad spectrum of activity. injectable forms of agents such as ivermectin, doramectin and moxidectin were used against various ectoparasites and endoparasites, especially against haematophagous ectoparasites such as ticks, fleas and sucking lice, but not used clinically against chewing lice in goats because of lack of scientific evidence (smith & sherman 2009; taylor et al. 2007). ivermectin is an antiparasitic drug with a broad spectrum of activity, high efficacy and wide margin of safety. the pharmacokinetic study of ivermectin in goats showed that it persists in significant concentrations in skin and hair for a minimum of 17 days post-treatment after subcutaneous route of administration (lespine et al. 2005). even though topical preparations of macrocyclic lactones such as selamectin, eprinomectin, ivermectin and doramectin were found to be effective and giving persistent activity against biting lice infestation in dogs, cats, cattle and sheep, efficacy of subcutaneous injection of avermectins against chewing lice infestation is not yet investigated in goats (holste et al. 1997; lloyd et al. 2001; rugg et al. 1995; shanks et al. 2003). in view of this, a field study was designed to evaluate the effects of chewing lice on haematological parameters and the effectiveness of subcutaneous ivermectin in the management of chewing lice infestation among goats reared under extensive grazing system under natural conditions. materials and methods a herd of 24 black bengal male goats kept for meat purpose, coming under the age group of 10–12 months with severe b. caprae infestation (cumulative count above 100 in 10 cm × 10 cm areas at six sites on the neck, shoulder, withers, inguinal, flank and rump), were included in the study. no animals used in the study population previously received any type of ectoparasiticidal therapy. animals without any systemic signs of disease only were included. all animals of the herd were weighed and dewormed using oral albendazole suspension at the rate of 10 mg/kg body weight 3 weeks prior to the field study, and 20 animals showing no parasitic ova in faecal smear examination on day 0 of the clinical trial were randomly grouped into two groups: group a and group b. animals of group a (treatment group [n = 10]) were treated using ivermectin (hitek injection, 1% w/v; virbac india, mumbai, india) on day 0 at the dose rate of 200 µg/kg body weight subcutaneously. animals of group b (n = 10) kept as control and were given a placebo therapy using 0.6 ml normal saline subcutaneously on day zero. animals were kept under the same environmental and management conditions throughout the study and thus facilitated natural transmission of lice to the treatment group by constant exposure to infested untreated animals. all animals were allowed to graze for 8 h a day in the same grazing area, where green grass and fresh water were available. animals were examined on days 0, 3, 7, 14, 21, 28, 42 and 56 for lice counts and severity of infestation was assessed by summation of total motile lice, counted using the standard counting technique (lice number was counted in 10 cm × 10 cm areas at each of the six sites on the neck, shoulder, withers, inguinal, flank and rump) (higgs, love & morcombe 1994; holdsworth et al. 2006). percentage reduction was calculated by garg’s formula and reduction efficacy was calculated using abbott’s formula (abbott 1925; garg et al. 1998). garg’s formula: where, ta = infestation on treated animals after treatment tb = infestation on treated animals before treatment ca = infestation on control animal after treatment cb = infestation on control animals before treatment. abbott’s formula: where, c = parasite count of control group t = parasite count of treatment group. blood samples were collected from the jugular vein of all animals of both groups on day 0 (before treatment) and day 21 to evaluate the alterations in haematological parameters such as haemoglobin (hb), total erythrocytic count (tec), total leukocytic count (tlc) and differential leukocytic count (dlc) by standard methods (jain 1986). ethical considerations the study was conducted as per the world association for the advancement of veterinary parasitology (waavp) guidelines for evaluating the efficacy of ectoparasiticides against biting lice (holdsworth et al. 2006). this work was approved under the project department of science and technology – sustainable agriculture and rural transformation holistic initiative (dst-sarthi) (seed/sarthi/hp/19/2012), funded by the department of science and technology, ministry of science and technology, government of india, and was conducted in compliance with the universal ethical standards. results the mean number of b. caprae observed on each of the six sites during the study period was recorded (table 1). animals of group a showed a significant reduction in the number of lice by day 3 and complete elimination of lice population was found on day 14. even though ivermectin-treated animals were in constant exposure to untreated lice-infested animals, they remained free from lice and lice eggs from day 14 to day 21 (figure 1), which suggests that ivermectin via subcutaneous route could produce high licicidal concentration on skin surface at least for 21 days. in group a, two animals showed mild levels of lice infestation (cumulative count < 10 lice) on day 28 and all animals showed mild infection by day 56. there was a significant (p < 0.05) increase in lice count on animals of group b during the course of the study. anaemia, leucocytosis, neutrophilia and eosinophilia were observed in chewing lice-infested animals before treatment and significant (p < 0.05) improvement towards normal values was noted in group a animals after treatment (table 2 and figure 2). no animals in the treatment group showed any adverse reactions. figure 1: effect of ivermectin (200 µg/kg body weight via subcutaneous route) on mean number of lice (bovicola caprae). figure 2: alterations in haematological profile of lice (bovicola caprae) infested goats before and after treatment (mean). table 1: mean lice (bovicola caprae) count and intact lice egg (nits) in different body regions of goats. table 2: alterations in haematological profile of lice-infested goats before and after treatment (mean ± standard error). discussion and conclusion in this study, the use of ivermectin via subcutaneous route was found effective in controlling chewing lice infestation on goats under field conditions. treatment of caprine pediculosis had not been studied extensively, and the studies conducted on sheep and cattle were usually extrapolated for deciding therapeutic strategy in goats (constable et al. 2016). the previous literature on the efficacy of macrocyclic lactones, especially ivermectin via subcutaneous route, in biting or chewing lice infestation in animals remained variable. some earlier studies reported that injectable avermectins were not effective, even when topical pour-on formulations showed high efficacy in the same animal herd (chick et al. 1993; cleale et al. 2004; titchener, parry & grimshaw 1994). recently, several studies reported the beneficial and persistent effect of injectable avermectins in chewing lice infestation in ruminants. morsy, habib and haridy (2001) observed complete cure from b. caprae infection in all goats parenterally treated with a combination of ivermectin and clorsulon by 1–2 weeks. colwell (2002) reported that injectable moxidectin prevented re-infestation with b. bovis for up to 35 days. these variations in results may be attributed to the altered resistance pattern of the parasite and improved distribution, stability, persistent activity and pharmacokinetic properties of the injectable preparations of avermectins. constable et al. (2016) appreciated these recent studies showing excellent efficacy and persistent effect by recommending both topical and injectable macrocyclic lactone-based products against both sucking and chewing lice in cattle. the reports of resistance and tolerance towards conventional insecticides like pyrethroids in cattle and horse with chewing lice infestation are alarming (ellse, burden & wall 2012; sands et al. 2015). also, these topical preparations are highly prone to environmental contamination and have a severe public health impact. ivermectin is a highly lipophilic compound that dissolves in most of the organic solvents but is practically insoluble in water, which provides long duration of action and makes it least capable of environmental contamination. it has exceptional potency against endoparasites as well as ectoparasites at extremely low doses, which accounts for its wide margin of safety; hence, toxicity to ivermectin is very rare. the greatest bioavailability, longer duration of activity and better efficacy is achieved by subcutaneous route of administration when compared with the topical application (canga et al. 2009). recent reports on the better therapeutic efficacy of newer generation insecticides such as fipronil (phenylpyrazoles) and imidacloprid (neonicotinoids) in controlling chewing lice infestation of dogs and horses are worth outstanding (kužner et al. 2013; mencke et al. 2004). some essential oils and oil-based formulations are also shown to have in vitro licicidal, as well as ovicidal, effect and are suggested as environment-friendly toxicologically safe alternative in controlling chewing lice infestation in donkey (sands, ellse & wall 2016; talbert & wall 2012). as with other parasitic diseases, antigens from the lice saliva might evoke strong humoral immune response, resulting in anaemia, mild neutrophilia and severe eosinophilia. in this study, significant level of anaemia, leucocytosis, neutrophilia and eosinophilia was observed in chewing lice-infested animals. such alterations in haematological parameters were previously reported in calves and goats infested with chewing or biting lice or sucking lice (ajith et al. 2017b; devaney et al. 1992; otter et al. 2003). even though chewing or biting lice feed on epidermal exudates and debris from upper skin layers, the increased infestation of these parasites may lead to severe inflammatory reactions on skin surfaces. the resultant tissue scaling and inflammatory changes on skin surface cause oxidative stress and alter haemato-biochemical values in caprine pediculosis (ajith et al. 2017b). several vasodilators, anticoagulants, histamine binding proteins and immunosuppressive compounds are present in the saliva of human lice (jones 1998). the possible role of these chemicals present in lice saliva in haematological changes needs to be explored in the future studies. the significant improvement in the haematological parameters on day 21 of ivermectin therapy can be attributed to the recovery from lice-induced damages. in lice-infested goats, the significant decrease in serum mineral, calcium, phosphorus, copper and zinc, levels was also reported earlier (dede, deger & deger 2003). the current study showed complete clearance of lice infestation in all animals by day 14 after subcutaneous ivermectin therapy and the treated animals remained uninfected from day 14 to day 21, even when the maximum chance for natural transmission from infested untreated animals was provided. even after day 21, the lice counts remained significantly low till day 56 of therapy, which makes clear that ivermectin has persistent licicidal activity and long half-life after subcutaneous administration in goats and is efficient in controlling chewing lice. hence, it can be concluded that injectable ivermectin can maintain licicidal concentrations on the skin surface and is 100% effective in controlling chewing lice (b. caprae) infestations for a minimum of 21 days. acknowledgements 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discussion conclusion acknowledgements references appendix 1 about the author(s) nola j. parsons southern african foundation for the conservation of coastal birds, bloubergrant, south africa bayworld centre for research and education, port elizabeth, south africa tertius a. gous veterinary pathologist, helderberg, south africa adam m. schaefer harbor branch oceanographic institution, florida atlantic university, united states ralph e.t. vanstreels laboratory of wildlife comparative pathology, university of são paulo, brazil citation parsons, n.j., gous, t.a., schaefer, a.m. & vanstreels, r.e.t., 2016, ‘health evaluation of african penguins (spheniscus demersus) in southern africa’, onderstepoort journal of veterinary research 83(1), a1147. http://dx.doi.org/10.4102/ojvr.v83i1.1147 original research health evaluation of african penguins (spheniscus demersus) in southern africa nola j. parsons, tertius a. gous, adam m. schaefer, ralph e.t. vanstreels received: 31 dec. 2015; accepted: 23 mar. 2016; published: 20 sept. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the african penguin (spheniscus demersus) is an endangered seabird that breeds along the coast of namibia and south africa, and disease surveillance was identified as a priority for its conservation. aiming for the establishment of baseline data on the presence of potential pathogens in this species, a comprehensive health assessment (blood smear examination, haematology, biochemistry and serology) was conducted on samples obtained from 578 african penguins at 11 breeding colonies and a rehabilitation centre. there were 68 penguins that were seropositive for at least one of seven pathogens tested: avian encephalomyelitis virus, avian infectious bronchitis virus, avian reovirus, infectious bursal disease virus, newcastle disease virus, mycoplasma gallisepticum and mycoplasma synoviae. all samples were seronegative for avian influenza virus subtypes h5 and h7 and infectious laryngotracheitis virus. the apparent prevalence of babesia sp. and borrelia sp. in blood smears was consistent with previous studies. babesia-infected individuals had a regenerative response of the erythrocytic lineage, an active inflammatory response and hepatic function impairment. these findings indicate that african penguins may be exposed to conservation-significant pathogens in the wild and encourage further studies aiming for the direct detection and/or isolation of these microorganisms. introduction the african penguin (spheniscus demersus) is considered an endangered species (birdlife international 2015) that breeds from central namibia to south africa’s eastern cape province (hockey, dean & ryan 2005) (figure 1). there has been more than a 60% decrease in the population between 2001 and 2009, mainly attributable to changes in overall abundance and local availability of prey (crawford et al. 2006, 2011; sherley et al. 2013). the levels of breeding success were deemed inadequate to sustain the african penguin population, and among other conservation efforts, limiting mortality through controlling the spread of disease was suggested to try to maintain an equilibrium situation (crawford et al. 2006). figure 1: the natural distribution of the african penguin (light grey area) showing the sample collection sites (black dots). disease is a major ecological force that has the potential to cause significant effects especially in threatened populations (friend, mclean & dein 2001) and heard et al. (2013) showed that the threat of disease increases with the level of extinction risk in all species. however, there is limited knowledge on the effects of disease on population dynamics of seabirds (lewison et al. 2012) or even for the role of disease as a major threat to species at risk of extinction (heard et al. 2013). while a single disease outbreak could decimate a population, the true cost of disease may be associated with chronic attrition of the population (friend et al. 2001) and thereby influence metabolic rate, life history traits and social status (barbosa & palacios 2009). comprehensive health assessments of free-ranging avian species have rarely been reported in the literature (smith et al. 2008). modern conservation efforts can be enhanced by the availability of comprehensive health assessment data at a population level (karesh & cook 1995). disease is often listed as a predicted threat to threatened species but this is generally a precautionary approach because there is a lack of surveillance data necessary to fully evaluate the threat (heard et al. 2013). therefore, health assessments and the compilation of baseline data on the presence of parasites and potential pathogens fill a critical data gap, particularly for endangered species. if a species is negatively affected by a major threat other than disease, that species is more likely to be simultaneously threatened by disease (heard et al. 2013). several parasites have been recorded from the african penguin: seven trematode species, two nematode species, one argasidae tick species and two louse species (brandão, moreira & luque 2014). only the trematode cardiocephaloides physalis has caused mortality in the african penguin (randall & bray 1983; horne, bray & bousfield 2011); however, all parasite species may affect the fitness of the host, predispose the individual to disease, cause poor breeding productivity and nest desertion (brandão et al. 2014; duffy 1983; kanarek, horne & zaleśny 2013). a large-scale health assessment was conducted on the african penguin following the methods reported by karesh et al. (1999), smith et al. (2008) and travis et al. (2006) on other penguin species, using blood smear examination, haematology, biochemistry and serology. adult penguins in the breeding season on the colonies in south africa as well as penguins admitted for rehabilitation were sampled. additional samples included banked serum samples from penguins previously admitted to the southern african foundation for the conservation of coastal birds (sanccob) and previous colony samples. methods sampling procedures a total of 578 samples from the breeding range were analysed in this study. these samples were obtained from african penguins bled at western cape and eastern cape breeding colonies as well as from african penguins bled on admission for rehabilitation to sanccob (cape town, western cape); collected from various namibian colonies in 2009 and from areas in the western cape from 2010 to 2013 (figure 1). african penguins are admitted for rehabilitation because of oiling, debilitation, injuries, arrested moult and eggs and chicks admitted for hand-rearing (parsons & underhill 2005). table 1 summarises the distribution of the sampling effort in relation to the sample collection site, month and year, clinical history and laboratory examinations. table 1: summary of the sampling effort and tests evaluated during this health assessment of african penguins. colony samples were collected from penguins that were visually healthy on examination by a veterinarian. all penguins sampled at breeding colonies were adults, with the exception of 11 chicks and 1 juvenile sampled in the western cape 2007–2008 group. birds selected were either resting in the colony or sitting on nests with medium to large chicks. handling time was 5–10 min per bird, and the birds were released near their nest sites. samples collected from penguins in the namibia 2009 group were obtained on the second day of admission to the centre, and all were adults. samples collected from penguins in the rehabilitation 2010–2013 group were obtained within the first 3 days of admission to the centre and comprised 53 chicks, 25 juveniles and 87 adults. for 365 of the total samples, sex was determined through routine dna analysis by molecular diagnostic services (pty) ltd (durban, south africa): 186 male penguins (51%) and 179 female penguins (49%). haematology blood (5 ml – 20 ml) was collected through veni-puncture of the jugular vein using a 21-g needle (25 mm × 0.8 mm), immediately transferred into ethylenediaminetetraaceticacid and serum clot activator tubes (vacuette®; greiner bio-one, austria) and stored at 4 °c for up to 60 h until being analysed. serum clot activator tubes were centrifuged and serum transferred into separate eppendorf tubes and immediately frozen at -20 °c. blood smears were prepared, air-dried, fixed in methanol and stained with modified wright–giemsa stain (kyro-quick®; kyron laboratories [pty] ltd, benrose, south africa). all slides were examined for blood parasites for 10 min using a 50× oil immersion lens with a 10× eyepiece. haematology and biochemistry analyses were performed following routine laboratory procedures at idexx laboratories (pty) ltd (cape town, south africa) (see parsons et al. 2015b for details). serology the frozen serum samples were submitted to the western cape provincial veterinary laboratory (stellenbosch, south africa) for haemagglutination inhibition assay (hia) for avian influenza virus subtypes h5 and h7 (aiv h5, aiv h7) and newcastle disease virus (ndv) and for serum plate agglutination (spa) testing for mycoplasma gallisepticum (mg) and mycoplasma synoviae (ms). the hia testing for avian influenza virus was done according to the protocol for non-chicken species (world organisation for animal health 2014). additionally, samples were submitted to idexx laboratories (pty) ltd (johannesburg, south africa) for indirect enzyme-linked immunosorbent assay (elisa) testing for avian infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev), avian reovirus (arv), infectious bursal disease virus (ibdv), mg and ms (table 2). elisa testing used secondary antibodies targeting chicken igy. in the case of mycoplasma spp., spa and elisa were used to test different subsets of samples. because of the occurrence of herpesvirus respiratory infections at the same facility (parsons et al. 2015a), a limited number of samples were submitted to agrilabs (pioneerfoods [pty] ltd, malmesbury, south africa) to be tested for infectious laryngotracheitis virus (iltv, also referred to as gallid herpesvirus 1) through indirect elisa. table 2: diagnostic results for pathogens tested during this health assessment of african penguins. data analysis statistical significance was set at 0.05 and tests were two-tailed using spss 21 for windows (ibm corp., 2011, armonk, ny, usa). fisher’s exact test was used to evaluate if the seroprevalence (number of positive samples/number of samples tested) for mycoplasma spp. was different in relation to the serological test (spa or elisa). the data set presented by parsons et al. (2015b) was used as haematological reference values for comparison with seropositive individuals; this data set comprises the seronegative and blood parasite-negative, apparently healthy adult african penguins sampled at colonies in this study. mann-whitney tests were used to compare haematological results between reference values and individuals that were seropositive for aev, mg (spa test) or two or more pathogens. haematological results of individuals seropositive for other pathogens were not included in this analysis because of insufficient sample size (less than five samples). because c. 60% of the blood parasite-positive individuals were chicks, a different data set had to be used as reference values to evaluate the haematological results of these individuals; seronegative and blood parasite-negative apparently healthy african penguin chicks admitted to sanccob were used as a reference data set. mann–whitney tests were used to compare haematological results between these reference values and individuals positive for babesia sp. on the other hand, borrelia sp.–positive and mixed infection–positive individuals were not included in this analysis because of insufficient sample size. results a total of 578 individuals were screened; of those, 68 penguins were seropositive for at least one of the nine pathogens tested (table 2); of these, 12 individuals were seropositive for more than one of the diseases tested: aev + ibdv (2 samples), aev + ibv (1), aev + ibdv + ibv (1), aev + mg (1), arv + ibv (1), ibdv + ibv (2), ibdv + ms (1), mg + ms (2), and mg + ndv (1). all samples were seronegative for aiv h5, aiv h7 and iltv. samples tested for antibodies against mycoplasma spp. using spa were more frequently positive (4.2% for mg and 8.1% for ms) than those tested using elisa (0.5% for both mg and ms); this occurred for both mg (p < 0.01) and ms (p = 0.03). haematological results for seropositive individuals are presented in table 3. table 3: morphometry, haematology and serum chemistry results of seropositive individuals, in relation to reference values for healthy adult wild african penguins. blood smears revealed 33 samples were positive for babesia sp., 2 individuals were positive for borrelia sp. and 1 individual was positive for both babesia sp. and borrelia sp. (table 2); no other blood parasites were observed. these blood parasites were morphologically consistent with those documented by earlé et al. (1993) and yabsley et al. (2012). only two blood smear–positive individuals (babesia-positive) were also found to be seropositive: one was seropositive to mg (spa test) and the other was seropositive to both aev and ibdv. haematological results for blood smear–positive individuals are presented in table 4. table 4: morphometry, haematology and serum chemistry results of blood smear–positive individuals, in relation to reference values for healthy african penguin chicks admitted to the southern african foundation for the conservation of coastal birds (this study) and healthy adult wild african penguins. of the positive individuals, 66 (97%) were adults compared to two (3%) chicks. there was no difference across genders. of the positive adults, there were 49 (74%) that were sampled as healthy individuals in wild colonies (90% unknown breeding status, 10% sitting with chicks) and 17 (26%) sampled when admitted for rehabilitation (94% oiled, 6% injured). there was a significant difference in the prevalence of seropositive individuals between the three geographical areas: namibia, western cape and eastern cape. complete details on the sampling effort and serological and blood smear results in relation to age group and sex are provided in table 1-a1, and in relation to breeding colony in table 2-a1. ethical considerations research permits to conduct this work were obtained by the department of environmental affairs (dea) (res2012/61 ext, res2011/19, and res2010/58), capenature (aaa007-00047-0056, aaa004-0508-0035, aaa004-000120-0035 and aaa007-00040-0035) and south african national parks (parsn1027). procedures were approved by the animal ethics committee of the dea, and all blood samples were collected by veterinarians (n.j.p., t.a.g.) registered with the south african veterinary council. where applicable, arrive guidelines for reporting in vivo animal experiments (kilkenny et al. 2010) have been adhered to. discussion our results should be interpreted taking into account the characteristics and inherent limitations of the serological tests used in this study. because serological tests specifically designed for african penguins are not currently available, we used commercial tests designed for poultry. the indirect elisa tests used in this study rely on the basic assumption that antibodies against chicken igy can also reliably recognise penguin igy. while these specific commercial tests have not undergone thorough validation to estimate their sensitivity and specificity when applied to samples from african penguins, other studies on the antigenic properties of penguin immunoglobulins corroborate the validity of their basic methodological assumption (bizelli et al. 2015; graczyk et al. 1994, 1995). unfortunately, the lack of serological tests specifically designed or validated for penguins is a recurrent methodological limitation of serological inquiries in these species (karesh et al. 1999; nunes et al. 2012; smith et al. 2008; travis et al. 2006; uhart et al. 2004), which hopefully will be overcome through ongoing research aiming at the production of secondary antibodies specifically targeting penguin igy (bizelli et al. 2015). on the other hand, the hia used to test for ndv, aiv h5 and aiv h7 is not subject to this limitation because it does not rely on the recognition by secondary antibodies. it is also worth noting that this is not a comprehensive study into all pathogens and parasites that can affect the health of african penguins on an individual or population level. further studies looking at epidemiology as well as interaction between parasites, pathogens and fitness of individuals are encouraged. avian encephalomyelitis virus (picornaviridae) seropositivity to aev was identified in the namibian and the western cape samples and in penguins admitted for rehabilitation at sanccob; overall seroprevalence was relatively low (2.9%). aev has been documented in domestic birds in south africa (odend’hal 1983), but it has never been demonstrated to infect penguins by direct diagnostic methods. serological surveys examining penguins in peru and at the falkland and galapagos islands have only found negative results (smith et al. 2008; travis et al. 2006; uhart et al. 2004), whereas karesh et al. (1999) found antibodies against aev in southern rockhopper penguins (eudyptes chrysocome) in argentina, with seroprevalence (3%) similar to that observed in this study. aev infections seldom cause clinical disease in adult chickens, but can lead to significant decreases in egg production and hatchability; however, in young chickens, aev can produce paralysis, ataxia and muscular dystrophy (tannock & shafren 1994). in this study, aev seropositive penguins had slightly lower serum sodium and chloride concentrations; this cannot be explained by the pathogenesis of aev infection and is therefore interpreted as an incidental finding. avian infectious bronchitis virus (coronaviridae) seropositivity to ibv was identified in the namibian and the western cape samples and in penguins admitted for rehabilitation at sanccob; overall seroprevalence was relatively low (3.6%). few studies have tested penguins for antibodies against ibv. karesh et al. (1999) found a seroprevalence between 23% and 47% (depending on the titre cutpoint) in southern rockhopper penguins in argentina, whereas smith et al. (2008) did not detect antibodies against this pathogen in humboldt penguins (spheniscus humboldti) in peru. dna from coronaviruses has been detected in the tissues of beachcast carcasses of magellanic penguins (spheniscus magellanicus) in brazil; however, it is unclear whether these viruses were associated with disease (niemeyer et al. 2012). coronaviruses such as ibv are known to cause respiratory, intestinal and reproductive diseases in both domestic and wild birds (gerlach 1994). however, the significance of this infection in penguins is unclear. individuals that were seropositive for ibv had significantly lower body mass but not head length than otherwise healthy adults, suggesting poorer body condition compared to those that were seronegative. however, this result should be interpreted with caution considering the low sample size. avian influenza virus (orthomyxoviridae) we found no serological evidence of highly pathogenic influenza a virus (subtypes h5 and h7), despite past evidence of their circulation in wild birds in south africa (abolnik et al. 2012; cumming et al. 2011). penguin seropositivity to aiv has been demonstrated by studies in the antarctic (abad et al. 2013; morgan & westbury 1981; wallensten et al. 2006) and subantarctic (abad et al. 2013), and hurt et al. (2014) have demonstrated that the aiv h11n2 present in penguins on the antarctic peninsula is an evolutionarily distinct lineage, not closely related to aiv strains from migratory flying birds. on the other hand, the few serological studies on penguins at lower latitudes conducted to date have failed to demonstrate exposure to aiv (karesh et al. 1999; smith et al. 2008; travis et al. 2006). however, this is unlikely to result from an absence of circulation of these viruses, as their worldwide distribution has been extensively documented (olsen et al. 2006). it is likely that these negative results reflect the fact that aiv occurrence is highly variable and species and location dependent (hanson et al. 2008). it must also be considered that antibodies against aiv subtypes other than h5 and h7 would have gone undetected by the tests used in this study. avian reovirus (reoviridae) antibodies against arv were detected in wild african penguins sampled in namibia and the western cape, with a low overall seroprevalence (0.9%). reovirus-like agents with some similarity to reference chicken reovirus strain were isolated in african penguins that died at a zoo in the united kingdom (gough et al. 2002). however, in that case, the birds were seronegative to the one-way neutralisation test, and it was unclear what role the virus played in their deaths (gough et al. 2002). surveys in peru and on the falkland and galapagos islands have found only seronegative penguins (smith et al. 2008; travis et al. 2006; uhart et al. 2004). on the other hand, karesh et al. (1999) detected antibodies against arv in 23% of southern rockhopper penguins sampled in argentina. arv has been documented in domestic birds worldwide, including south africa, and may lead to a broad variety of clinical presentations (gerlach 1994; van loon et al. 2001). infectious bursal disease virus (birnaviridae) antibodies against ibdv were detected in wild african penguins sampled in namibia and the western cape and in penguins admitted for rehabilitation at sanccob; overall seroprevalence was relatively low (2.7%). antibodies against ibdv have been demonstrated in penguins by studies using elisa in brazil (nunes et al. 2012) and virus neutralisation tests in crozet archipelago and at various locations in antarctica (gardner, kerry & riddle 1997; gauthier-clerc et al. 2002; watts, miller & shellam 2009), whereas studies using agar-gel diffusion tests have failed to obtain positive results in south america (karesh et al. 1999; smith et al. 2008; travis et al. 2006). watts et al. (2009) argue that ibdv serotype 1 is endemic and widespread in antarctic birds, with emperor penguins (aptenodytes forsteri) playing a key role in the virus’ persistence in antarctica. ibdv is known to cause disease in young chickens, in which it can produce bursal lymphoid depletion and high mortality (world organisation for animal health 2008). no clinical signs of disease have been observed in any of the seropositive penguin species in the wild (gardner et al. 1997; gauthier-clerc et al. 2002; nunes et al. 2012; watts et al. 2009). gough et al. (2002) reported the isolation of ibdv serotype 2 from the tissues of african and macaroni penguins (eudyptes chrysolophus) deceased at a zoo in the united kingdom and considered that although the infection was not primarily responsible for the deaths, it may have exacerbated concurrent disease conditions. unfortunately, in this study, we did not have a sufficient number of seropositive penguins to evaluate the potential health effects of exposure to ibdv. infectious laryngotracheitis virus (herpesviridae) there were no positive samples in serology testing for iltv (also known as gallid herpesvirus 1) despite previous evidence that african penguins are susceptible to herpesvirus-like infections (kincaid, bunton & cranfield 1988; parsons et al. 2015a). previous studies on other penguin species have also failed to identify antibodies against this virus (karesh et al. 1999; smith et al. 2008). wild african penguin chicks have presented herpesvirus-like respiratory infections, which were not detected by molecular or serological tests targeting iltv, suggesting that a different herpesvirus was involved (parsons et al. 2015a). newcastle disease virus (paramyxoviridae) five individuals were seropositive to ndv (also known as avian paramyxovirus type 1), all of which were sampled in the western cape. penguins that were seropositive for ndv have been demonstrated in the antarctic (morgan & westbury 1981), argentina (karesh et al. 1999), macquarie island (morgan et al. 1981) and south shetland islands (thomazelli et al. 2010). thomazelli et al. (2010) determined that the strains detected in penguins at the south shetlands islands had low pathogenicity. ndv infection has also been demonstrated in captive penguins in the united states (pierson & pfow 1975), where a velogenic neurotropic strain was identified, and in israel (haddas et al. 2014), where the pathogenicity of the strain could not be determined. it is clear that penguins are susceptible to this virus and that some ndv strains, presumably those with low pathogenicity, circulate in wild penguin populations. ndv has also been demonstrated in great white pelicans (pelecanus onocrotalus) in the western cape (assunção et al. 2007). it is interesting to note that one of the individuals identified as seropositive was a penguin that had been rehabilitated at sanccob 7 years earlier and, at that time, received vaccination for ndv. the vaccination consisted of an initial ocular spray vaccination on admission to the centre with live lasota strain (nobilis® nd lasota, kempton park, south africa) followed by an intramuscular injection of inactivated lasota strain (lomovac, tad, germany) (n.j. parsons, unpublished data). there is no literature, to our knowledge that determines how long the vaccination antibodies remain detectable in a penguin following vaccination. although it is unlikely that antibodies are still circulating 7 years after vaccination, it is possible that vaccination may have interfered with the results. sanccob stopped routinely marking all penguins before release into the wild in august 2005, but routinely vaccinated for ndv up until august 2008. mycoplasma spp. serological tests for mg and ms have not been routinely used in wild penguin species. there was inconsistency between the serological tests, with a higher frequency of positives when samples were tested with spa compared to elisa testing. while different subsets of samples were tested with each test, this discrepancy suggests an inherent difference in the sensitivity and specificity of the two tests. it is also important to consider that cross-reactivity with other mycoplasma spp. from african penguins in this study is possible. multiple mycoplasma spp. (excluding mg and ms) have been demonstrated to occur in penguins (banks, cary & hogg 2009; buckle et al. 2013; dewar et al. 2013; frasca et al. 2005). furthermore, frasca et al. (2005) found cross-reactivity of antibodies against mycoplasma sphenisci to antibodies against mg and ms in agglutination tests. therefore, caution should be used when interpreting these results. mg and ms potentially cause respiratory disease, sinusitis, conjunctivitis and synovitis in domestic and wild birds (jordan 1975). mycoplasma sphenisci was described in an african penguin showing signs of upper respiratory tract disease in a north american aquarium (frasca et al. 2005) and m. lipofaciens was identified from the lungs of a fiordland penguin (eudyptes pachyrhynchus) after post-mortem examination showed bronchopneumonia (buckle et al. 2013). on the other hand, m. sphenisci and other mycoplasma spp. have been detected in the faeces of apparently healthy penguins in antarctica and subantarctic islands (banks et al. 2009; dewar et al. 2013). in this study, african penguins seropositive to mg in the spa test had considerably lower serum concentrations of sodium, chloride and creatinine and higher concentrations of potassium, suggesting impairment of kidney function. although mg and ms are known to produce renal lesions, these tend to be less prominent than respiratory and articular lesions (jordan 1975; lockaby et al. 1998). future studies will be necessary to identify which species of mycoplasma occurs in african penguins and to confirm if it produces significant renal disease. it is worth noting that great white pelicans have been shown to have high prevalence (98%) of mycoplasma spp. in south africa (assunção et al. 2007). this species breeds sympatrically with and often predates on african penguins (mwema, de ponte machado & ryan 2010). furthermore, because great white pelicans are known to feed on avian offal in agricultural areas (crawford, cooper & dyer 1995), they could play a key role in spreading pathogens such as mycoplasma spp. from domestic animals to seabirds (assunção et al. 2007). babesia sp. and borrelia sp. although we did not fully characterise the blood parasites, their morphology was consistent with babesia peircei and relapsing fever borrelia as previously described in african penguins in the same region (earlé et al. 1993; yabsley et al. 2012). the apparent prevalence of babesia sp. in wild african penguins in this study (1.5% – 3.0%) is similar to that observed in previous studies, as is the higher frequency of babesia sp. and borrelia sp. among chicks and individuals undergoing rehabilitation (brossy et al. 1999; earlé et al. 1993; yabsley et al. 2012). the pathological significance of babesia sp. to penguins is not clear, and so far, this parasite has only been associated with only mild regenerative anaemia (brossy et al. 1999; cunningham et al. 1993; vanstreels et al. 2015). in this study, african penguin chicks with babesia sp. had significantly different haematological and serum chemistry values compared to healthy chicks. babesia-infected penguins had abnormalities in erythrocyte size and lower haemoglobin concentration, suggesting a regenerative response of the erythrocytic lineage, presumably to the haemolysis caused by the parasite. higher white blood cell counts in babesia-infected penguins indicate an active inflammatory response to the parasite and/or a stress response. finally, higher serum levels of creatinine kinase and lower serum levels of uric acids and albumin indicate impairment of hepatic function and may also be partly related to haemolysis (see harrison & lightfoot 2006). conclusion considering the decreasing trend of the african penguin population (crawford et al. 2011), disease is yet another significant threat to the species in addition to poor nutrition, environmental degradation and anthropogenic impacts (woods et al. 2009). serological surveillance can be a powerful tool to track the prevalence of pathogens that are otherwise difficult to detect in wildlife populations (gilbert et al. 2013). the reported seroprevalence in this study is consistent with previously reported studies on wild penguins, suggesting that these are endemic pathogens or natural, apathogenic flora. it must also be borne in mind that the presence of antibodies indicates past exposure to a pathogen and does not necessarily indicate presence of the organism or active infection. in addition, cross-reaction of tests with other antigens and microorganisms may interfere with specificity of the results (barbosa & palacios 2009). studies addressing the direct detection and isolation of pathogenic organisms in penguins are encouraged and, in combination with serological investigations, should provide deeper insight on their epidemiology in these birds. acknowledgements sanparks and capenature assisted tremendously in the transport to and from the island colonies as well as allowing access to their colonies. sampling was assisted by sanparks and capenature staff as well as sanccob staff, volunteers and interns and mystic aquarium staff. south african marine rehabilitation and education centre generously allowed use of their premises for laboratory work in the eastern cape. sanccob is supported by a wide range of donors, including the international fund for animal welfare, hans hoheisen charitable trust and the national lottery distribution trust fund. this research is supported by the sea research foundation (mystic aquarium), the georgia aquarium and the leiden conservation foundation. additionally, n.j.p. is supported by a professional development programme bursary granted by the national research foundation and r.e.t.v. is supported by coordenação de aperfeiçoamento de pessoal de nível superior. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions n.j.p. and t.a.g. designed and coordinated the study, collected samples and compiled results. a.m.s. and r.e.t.v. conducted epidemiological and statistical analyses. 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27 february 2015, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.03.04_ai.pdf yabsley, m.j., parsons, n.j., horne, e.c., shock, b.c. & purdee, m., 2012, ‘novel relapsing fever borrelia detected in african penguins (spheniscus demersus) admitted to two rehabilitation centers in south africa’, parasitology research 110, 1125–1130. appendix 1 table 1-a1: details of sampling effort and serological results in relation to age group and sex. table 2-a1: details of sampling effort and serological results in relation to sampling location and/or clinical history. abstract introduction bovine tuberculosis in cattle and current control measures mycobacterium bovis infection in wildlife and current control approaches challenges in the control of bovine tuberculosis in cattle challenges in the control of mycobacterium bovis infection in african buffalo the way forward? can vaccination contribute to bovine tuberculosis control in south africa? conclusion acknowledgements references footnote about the author(s) luke f. arnot † department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa bovine tuberculosis and brucellosis research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa anita michel bovine tuberculosis and brucellosis research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation arnot, l.f. & michel, a., 2020, ‘challenges for controlling bovine tuberculosis in south africa’, onderstepoort journal of veterinary research 87(1), a1690. https://doi.org/10.4102/ojvr.v87i1.1690 note: †, 1970–2018. review article challenges for controlling bovine tuberculosis in south africa luke f. arnot, anita michel received: 03 sept. 2018; accepted: 18 oct. 2019; published: 27 feb. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract all effects taken together, bovine tuberculosis (btb) has a long-term detrimental effect on bovine herds and many wildlife species in south africa. the disease is not only found in domestic cattle but also in african buffaloes and has to date been diagnosed in 21 wildlife species, including several rare and endangered species, thus having a potentially serious effect on conservation and biodiversity. in cattle, btb is mostly characterised by sporadic outbreaks, but bovine herds chronically infected with the clinical disease are not uncommon. presently, the recognised btb control strategy in south africa is based on ‘test and slaughter’, using the intradermal tuberculin test, followed by the slaughter of animals that have tested positive. affected herds are placed under veterinary quarantine with movement restrictions until the outbreak is eradicated; this can take several years or last indefinitely if the outbreak cannot be eradicated. the same measures apply to infected buffalo populations, often with no prospect of ever being eradicated. this strategy is neither practical nor viable in the context of a communal farming system and becomes unethical when dealing with valuable wildlife reservoir hosts. transmission of btb between wildlife and cattle has been demonstrated and emphasises the need for an effective, affordable and culturally acceptable control strategy to curb the spread of btb in south africa. in countries with similar challenges, vaccination has been used and found to be promising for treating wild and domestic reservoir species and may hence be of value as a complementary tool for btb control in south africa. keywords: african buffalo; bovine tuberculosis control; cattle; conservation; game farming; mycobacterium bovis. introduction bovine tuberculosis (btb) is an economically important and widespread disease in cattle caused by mycobacterium bovis (m. bovis) with the potential to establish to certain wild animal species. the organism belongs to the so-called mycobacterium tuberculosis complex, which is characterised by the ability of its members to cause tuberculosis in mammalian species, including humans. mycobacterium bovis has been circulating within european mediterranean bovine herds from biblical times (myers & steele 1969), spreading to western europe with the movement of cattle across europe (muwonge et al. 2019). apart from being an impediment to livestock production and human livelihoods, btb is a threat to the health of wildlife species and people, particularly in developing countries (ayele et al. 2004; hlokwe et al. 2016; michel et al. 2015; miller et al. 2017a; renwick, white & bengis 2007). in south africa, the zoonotic significance of m. bovis is poorly understood, but in light of its soaring hiv statistics btb can potentially contribute significantly to the human tuberculosis burden (hsrc 2018; olea-popelka et al. 2017; park et al. 2010). following the spillover of btb from cattle to wildlife, african buffalo (syncerus caffer) emerged as the maintenance host, with far-reaching implications for multispecies ecosystems because infected buffaloes act as a source of m. bovis to any mammalian species sharing the habitat with them (de vos et al. 2001). as a consequence, m. bovis has infected and been diagnosed in 21 wildlife species (hlokwe et al. 2019; michel et al. 2015), including near-threatened, rare and endangered wildlife species such as white and black rhinoceros and wild dog (higgitt et al. 2019; miller et al. 2016, 2017b, 2018). in addition to buffalo, greater kudu (tragelaphus strepsiceros) has shown the potential to act as a maintenance host and most probably responsible for introducing m. bovis into a conservation area (michel et al. 2009). warthogs (phacochoerus africanus), a social species that show predominantly lung and intestinal tuberculous lesions, are considered potential btb reservoir hosts in ecosystems supporting high population densities (michel et al. 2015); however, conclusive evidence is difficult to obtain in large reserves where other reservoir species are present – an assessment which is also true for lions (panthera leo) (michel & van helden 2019). a future negative impact on wildlife conservation efforts is considered inevitable unless the spread of btb can be addressed urgently and effectively. the aim of this article was to review the current challenges and opportunities in controlling btb in the domestic and wild reservoir species in south africa. bovine tuberculosis in cattle and current control measures bovine tuberculosis spread to south africa and other colonies with the importation of cattle from europe in the early 1800s and was first diagnosed in a bovine in south africa in 1880 (henning 1956; myers & steele 1969). importation of large numbers of cattle mostly from europe and also australia and south america before the existence of btb screening or control measures led to the increase in the number of cattle diagnosed with tuberculous lesions at major abattoirs in south africa (cousins et al. 2004). for this reason, btb received attention as a major livestock disease as far back as 1911 under the diseases of stock act and later when the btb control and eradication scheme was launched in 1969. during the 1970s, there was a clear emphasis on establishing btb-free commercial cattle herds, and a herd accreditation scheme was strongly promoted (michel, sibanda & de klerk-lorist 2019). the control interventions were highly successful and led to a drastic decrease in btb prevalence in the commercial cattle farming sector where the herd prevalence decreased from 11.8% in 1971 to 0.39% in 1995 (michel et al. 2008). the decentralisation of state veterinary services and the re-prioritisation of disease control efforts coupled with budgetary constraints severely hampered btb control in subsequent years, with the number of cattle tested annually plummeting in all provinces from the late 1990s onwards (cloete 2015). as a consequence of these and other challenges in controlling this disease, btb has slowly been re-emerging in commercial cattle production throughout south africa (michel et al. 2008, 2019). in the communal cattle farming sector, the btb prevalence had remained unknown until recently where studies have revealed a high variability in animal prevalences ranging from very low (< 0.5%) to high (>15%) (musoke et al. 2015; sichewo, etter & michel 2019). surveillance of cattle herds for btb is the responsibility of the provincial veterinary services, whereby budget allocations and availability of human resources determine surveillance activities. cattle owners who want their animals tested can do so at their own expense. however, once an outbreak of btb is suspected or confirmed through meat inspection at abattoirs or ante mortem testing, the control measures laid down in the bovine tuberculosis manual of the bovine tuberculosis scheme, issued by the department of agriculture, forestry and fisheries, are to be applied (daff 2016). state veterinarians assume complete control of all diagnoses and control measures. this means infected farms are placed under quarantine, and cattle are tested using the comparative or single intradermal tuberculin test, also referred to as tuberculin skin test (tst). test-positive cattle are branded and slaughtered, and the remaining herd is subjected to a repetition of these procedures at 3-month intervals until the herd is cleared of any suspect or positive reactor animals during which time the quarantine can be lifted. compensation may be paid for condemnations only. mycobacterium bovis infection in wildlife and current control approaches in south africa, m. bovis has affected wildlife species for many decades and was first identified in a greater kudu in the eastern cape in 1928, followed by identification in a duiker (sylvicapra grimmia) in 1929 (paine & martinaglia 1928). these cases were presumed to be isolated incidents. the first confirmed case in a buffalo within a recognised wildlife reserve in south africa occurred when a buffalo was found to be infected within the hluhluwe–imfolozi park (hip) in 1986, followed by a lion in 1992 and kudus, bushpigs (potamochoerus larvatus) and baboons (papio ursinus) in the same reserve in 1998 (buss 2015). the hip reserve was only fenced in the 1950s, and it is presumed that btb was introduced by spillover from adjacent communal cattle to buffalo during co-mingling prior to hip being fully fenced (hlokwe et al. 2011; wadge 2007). the first suspected case of m. bovis infection within the kruger national park (knp) was an impala that died in 1977, but the diagnosis was never confirmed by culture (buss 2015). this unconfirmed case was thought to be an isolated incident, until the first index case was confirmed in a young buffalo bull in the southern section of the park in july 1990 (bengis et al. 1996). a btb-positive dairy herd on a farm located south of the crocodile river was reported in the 1950s, and other outbreaks on cattle farms in the same area were reported in 1982, 1983 and 1984 (cloete 2015). the crocodile river forms the southern boundary of the knp and was not fenced at the time. buffaloes were known to cross the river at night to graze on the opposite bank (bengis et al. 1996), thus coming into contact with cattle on infected farms adjoining the knp. soon after this index case in 1990, it was found that only buffalo herds in the southern-most part of the knp were infected with btb. there was a drive to contain the spread of btb from infecting herds located further north within the reserve (caron, cross & du toit 2003). several proposals were put forward to prevent btb from spreading, including creating a buffalo-free zone using a fenced off cordon and intensive culling of buffalo within the cordon to protect unaffected herds, implementing a form of metaphylaxis vaccination, or a combination of these options, as opposed to letting the btb take its natural course (caron et al. 2003; de vos et al. 2001). one such proposal was to create a buffalo-free zone by culling all buffaloes starting at the olifants river in the south, then extending for a 20-km zone in a northerly direction. any buffalo within the 20-km zone would be culled, and then the culling zone would be extended southwards to eliminate all buffaloes to the south of the river, thus eliminating the spread to herds north of the buffalo-free zone (bartlett 1997). however, during the planning phase, it was found that btb had already spread to buffalo herds to the north of this zone (bartlett 1997), and the plan was abandoned. by 2006, buffalo herds in the far northern areas of the knp were found to be infected (buss 2015). in 2008, buffaloes within the gonarezhou national park in zimbabwe tested positive, and the same strain circulating within the kruger buffalo was isolated, implying that btb from the knp had spread across south africa’s northern border into zimbabwe (de garine-wichatitsky et al. 2010). it was ascertained by molecular characterisation of btb strains that the knp had been infected by a single m. bovis parent strain (sb0121), suggesting a single introduction into the knp (hlokwe, van helden & michel 2013, 2014; michel et al. 2009), which was most likely from the infected dairy farm adjacent to the southern border of the knp during the 1950s. the madikwe game reserve (mgr), located in the far northern regions of the north west province, was home to a very valuable disease-free1 buffalo herd comprising outstanding genetic stock. this herd originated from 53 buffaloes imported back into south africa from european and american zoos (de klerk-lorist 2015). in 2012, prior to an auction, three of the 51 buffaloes tested positive for the disease and were confirmed to be infected with m. bovis (hlokwe et al. 2016). initial epidemiological investigations failed to reveal how btb had been introduced into mgr, but genetic characterisation by spoligotyping and variable number of tandem repeat (vntr) typing made it possible to trace back the m. bovis isolate to wildlife in kwazulu-natal (kzn). it is strongly suspected that amongst the estimated 8000 antelopes translocated into mgr, the m. bovis strain was introduced by another bovid species from kzn. as can be seen from the case above, even under strict biosecurity with regard to cattle and buffalo movements, introduction of m. bovis into an unaffected area by other bovid species is a reality. in 2014, a btb prevalence survey amongst communal cattle within the mnisi area, located adjacent to the western border of the knp, revealed that 0.39% of these communal cattle were infected with the knp strain of m. bovis (musoke et al. 2015). this finding proved for the first time that spillback from wildlife reservoir hosts in knp back into adjacent communal cattle was indeed happening. this finding has far-reaching consequences for the national bovine herd in that wildlife reservoir hosts will continue to spread m. bovis amongst themselves in an uncontrolled manner and will constitute a persistent risk to cattle herds that are in close proximity. in a most recent study in northern kzn close to but not adjoining the hip, communal cattle were found to be co-infected with different m. bovis strains all of which were shared with buffaloes in hip (sichewo et al. 2019). with the increasing human population pressure at the wildlife or livestock interface of conservation areas, such as the knp and hip, spillover and spillback incidents will continue to pose a significant risk to cattle unless btb can be controlled within the wildlife reservoir host. vice versa, in the absence of btb control in cattle, exposure of wildlife hosts to m. bovis continues to exist making spillover inevitable. the application of control measures for m. bovis infection in buffalo presents a dilemma for state veterinary services because the bovine tuberculosis manual has been written exclusively with a view on controlling btb in cattle and thereby ignoring the need to manage infected buffalo herds (and potentially other wildlife species) on commercial ranches (michel et al. 2015, 2019). while the presence of m. bovis prohibits the open trade in wildlife species in general, pre-movement testing for btb is only mandatory for buffaloes. in essence, movement of buffaloes from disease-free populations is subject to a negative comparative tst. should the tst yield inconclusive results, the test must be repeated after 3 months. particulars for test interpretation and management of infected herds are similar to those for cattle herds and are set out in the veterinary procedural notice (vpn) (daff 2017). for all practical purposes, infected buffalo ranches are placed under quarantine indefinitely, unless the owner can provide satisfactory evidence that m. bovis has been successfully eradicated from the population. challenges in the control of bovine tuberculosis in cattle the introduction of m. bovis strains into south africa with infected cattle from europe during colonial times promoted a high genetic diversity of strains, representing the european (eu)-1 and possibly the eu-2 clonal complexes (michel et al. 2008). eu-1 is primarily circulating within cattle in the republic of ireland and the united kingdom, and former colonies of britain but is absent from the rest of africa (smith et al. 2011). eu-2 strains are dominant in the iberian peninsula from where they were possibly disseminated by international trade (rodriguez-campos et al. 2012). in south africa, two major interventions could contribute to the decline of the presence of btb in cattle, namely, the rinderpest epidemic at the end of the 19th century as well as the implementation of the tuberculosis scheme in 1969 specifically aimed at eradication of btb. in spite of the drastic reductions in the number of infected cattle, the diversity of the prevailing btb strains remained high, with spoligotype sb0130 being most prevalent as shown in a study in 2014 (hlokwe et al. 2014). over and above, compared to a previous study using m. bovis isolates dating to the end of the 20th century (michel et al. 2008), an increased interspecies spread of btb has been recorded indicating the transmission of m. bovis between cattle and wildlife (hlokwe et al. 2014; sichewo et al. 2019). the persistence of highly diverse m. bovis strains in the national cattle herd is not only testimony to an impaired efficacy of the prevailing control strategy but may also point towards a potential selection for highly successful m. bovis outbreak strains that are able to evade conventional control measures. the ‘test and slaughter’ approach forms the foundation of btb eradication in developed countries. looking at the reasons behind the success of this control strategy, we find five essential requirements that must be in place: a uniform (commercial) cattle farming system, stringent test interpretation (accepting an ‘overkill’ of cattle), financial compensation for cattle slaughtered, adequate government resources guaranteeing completion of the eradication campaign and the absence of an m. bovis wildlife reservoir (amanfu 2006; smith et al. 2011). the significance of the latter is demonstrated by the inability of countries including, but not limited to, the united kingdom, ireland, spain and new zealand to eradicate btb from the cattle population in spite of large financial commitments (reviriego gordejo & vermeersch 2006). in south africa, buffaloes are highly effective maintenance hosts of btb, which facilitate the persistence and continued horizontal spread not only to other wildlife species but also to cattle beyond the border of game and veterinary control fences, thus jeopardising btb control in cattle (musoke et al. 2015; sichewo et al. 2019). it is practically impossible to eradicate btb where there is a possibility of contact between buffalo and cattle herds (phepa, chirove & govinder 2016). in 2015, the number of cattle in south africa was estimated to be 13.9 million. of these, 58% reside in the commercial sector while about 42% of the national herd are kept in communal farming systems (daff 2015, 2017). cattle belonging to different small-scale communal farmers mix together over common grazing lands. these cattle congregate periodically in large numbers at communal dip tanks for dipping and other basic veterinary procedures, and at communal watering points. the aggregation of large numbers of communal cattle at these points provides an ideal opportunity for the spread of btb amongst these cattle. communal cattle, in contrast to commercially farmed cattle, constitute more than a commercial value to their owners; the number of cattle owned by a communal cattle farmer is a measure of wealth and social status within the community. apart from being a source of animal protein, cattle also serve as a source of cash for emergency expenditure, draught power, fertiliser, hides and are highly important for cultural practices (meltzer 1995; scholtz et al. 2008). the control of btb or any other controlled disease that requires the compulsory removal of animals amongst communal cattle herds therefore imposes unique and difficult to accept challenges on the owners and their families which are currently not dealt with in a culturally sensitive manner. from a technical point of view, the tst has severe shortcomings in the communal farming sector: impractical: testing of cattle is conducted at the crush pen of the relevant dip tank facility and disrupts the regular dipping activity on the day of tuberculin injection. in addition, cattle owners have to return their cattle to the dip tank after 72 h for the reading of the skin reactions. as a consequence, high rates of farmer non-compliance and a resultant poor testing coverage are observed. unethical: in contrast to a developed country setting where a certain ‘overkill’ or wastage of healthy animals because of the limited specificity of the tst is acceptable or affordable, this approach is to be rejected on ethical grounds in a developing country where food security is not guaranteed. uncertain quality: for the ‘test and slaughter’ strategy to be a successful control method, the tst must be performed by competent personnel with functioning, calibrated testing equipment. in reality, financial constraints and lack of trained personnel can render the tst a non-reliable method generating test results of questionable quality without checks and balances. unsustainable: quarantine measures instituted in a communal farming area mean that all farmers, whether their herds are infected or not, are collectively denied market access for their animals, rendering the control strategy unsustainable and unacceptable to farmers. for all practical purposes, the quarantine remains in place indefinitely because eradication of btb is difficult or impossible to achieve in the absence of earmarked financial resources and adequate compensation for slaughtered animals. the control of btb in many developing countries, including south africa, is severely hindered because of general government fatigue of chronic, neglected, difficult-to-control livestock diseases affecting primarily marginalised farming communities. as a result, very few communally owned cattle are routinely tested for btb. the frequently poorly regulated movement of animals and lack of systematic and verifiable livestock identification also complicate the issue (amanfu 2006). challenges in the control of mycobacterium bovis infection in african buffalo south africa is blessed with an amazing array of wildlife species, which is one of the greatest attractions for tourism in south africa, contributing billions of rands into the south african economy each year, with tourism contributing 2.9% of the total gross domestic product (gdp) of south africa in 2016 (statistics sa 2018). the wildlife industry, together with ecotourism, hunting and game sales grew by 20.3% per year in the past 15 years and created over 140 000 permanent jobs (oberem 2016). however, there is a possibility that m. bovis infection can pose a severe threat to the wildlife diversity, on the one hand, and the income from tourism, on the other hand. african buffaloes play an important ecological role as bulk grazers and key prey species. their commercial use is based on their iconic status for ecotourism, game farming and hunting safaris. the wide utilisation explains their popularity, resulting in a widespread distribution throughout conservation areas and privately owned game operations (michel & bengis 2012; prins 1996). whether they are free-ranging or are actively translocated, through their cardinal role as a maintenance host of btb, buffaloes also serve as a disease amplifier and effective disseminator of m. bovis, and thus ongoing surveillance and efficacious control measures are needed for buffaloes and possibly other species sharing the same habitat. a case of mycobacterium orygis infection in a buffalo was diagnosed in 2012 (gey van pittius et al. 2012) and again in a different herd in 2017 (verrynne, unpublished data), indicating the presence of this mycobacterium tuberculosis complex (mtbc) organism in south africa and its ability to cause positive tst reactions. in spite of extensive follow-up testing in the second case over a period of 2 years, no indication for the spread of the organism within the herd was found. for the time being, the btb control measures, including the tst prescribed for cattle, have been adopted for buffaloes, which bear several inherent impediments to ensuring the quality of the diagnosis made and the health and welfare of the buffalo during testing: immobilisation causes severe stress and potential fatalities direct and severe consequences of the ‘test and removal’ control approach: unethical culling of buffalo as a result of the limited test specificity undue financial losses to buffalo owners boma confinement may predispose buffaloes to shedding and transmission of m. bovis quality of test results may be compromised by: the current lack of sufficient validation data required to set adequate interpretation criteria the absence of trained state veterinary officials competent to conduct and interpret the tst performing the tst in buffaloes is a difficult and expensive process, as the animals need to be confined in a boma where they are chemically immobilised twice within 72 h to perform the tst. the interferon-gamma assay (bovigam®), an alternative btb test that can be used as an ancillary test at the discretion of the state veterinarian, mitigates the need to confine the buffalo in bomas for 3 days but still necessitates immobilising buffaloes in order to obtain blood samples (michel et al. 2011). in addition, the bovigam® assay requires the whole blood sample to reach a laboratory for stimulation within 8 h, which is not always possible in remote locations. the way forward? the future success of controlling btb in cattle and wildlife populations in south africa will depend on an effective, affordable, practical, sustainable, and culturally and ethically acceptable control programme in cattle and buffalo that is potentially expandable to other wildlife species. to achieve this goal, it will be important for the south african government as the custodian of livestock animal health, conservation agencies as well as producers’ organisations to enter into consultations and private–public partnerships to address strategies for the control of btb in a stakeholder-sensitive manner. the diverse spectrum of economic, cultural and species settings in which btb control must be effectively applied renders a ‘one-size-fits-all’ control strategy ineffective and unrealistic. in specific commercial settings, ‘test and slaughter’ with compensation through industry-driven initiatives can be a viable control strategy, while vigilant surveillance at population and individual animal level (the latter at slaughter and pre-movement), community-based animal health care, increased education and involvement of communal cattle farmers with regard to suitable and locally relevant btb control could be implemented in communal small-scale herds. in addition to all of the above, a vaccination strategy for domestic and wildlife reservoir species could afford the opportunity to significantly reduce the prevalence of diseased animals and limit the onward spread of the disease (buddle et al. 2018). can vaccination contribute to bovine tuberculosis control in south africa? in developed countries with an effective btb control programme in cattle and a btb wildlife reservoir host, it has been shown that eradication in livestock depends on concurrent and successful control in the wildlife reservoir (de lisle et al. 2002). in south africa, the situation is more complex as btb in cattle is poorly controlled or uncontrolled in some areas and is a potential source of infection to valuable healthy wildlife populations in conservation areas and also in commercial game operations. therefore, vaccination of both cattle and wildlife reservoir species should form part of a comprehensive strategy to combat btb in south africa. the aim of vaccinating domestic or wild m. bovis reservoir species is to induce an immune response that ideally protects the animals against infection. however, incomplete protection, meaning infection not being prevented in the vaccinate, is still highly beneficial if the infection cannot progress to a disease, resulting in significantly reduced disease severity (palmer & thacker 2018). several tb vaccine approaches have been explored in humans and in animals; compared to subunit and dna vaccine candidates, bacillus calmette-guerin (bcg) appears to be the most successful candidate in animals. the bcg vaccine is presently the only vaccine registered for the control of tuberculosis in humans and the most common vaccine used experimentally in the control of btb in cattle. after a series of controlled vaccination experiments, a recent vaccination trial in free-ranging cattle in new zealand showed that low-dose bcg protected cattle from either becoming infected or from developing gross lesions with a trial efficacy of 86% (nugent et al. 2018). if applied systematically, vaccination is expected to reduce m. bovis prevalence and can contribute significantly to an integrated btb control in cattle using lethal and non-lethal approaches (buddle et al. 2011, 2013). however, one drawback of bcg vaccination is that it causes a positive tst reaction for several months, which appears to drop to a vaccine reactor rate of 10% after about 9 months post vaccination (chambers et al. 2014). the development of diva (differentiating infected from vaccinated animals) tests has been explored and may mitigate this problem in future (buddle et al. 2013). in wild reservoir hosts, vaccination holds the potential to slow down interspecies transmission to vulnerable wildlife species and also to cattle, which is a major benefit where other control measures are not feasible or not desirable. bacillus calmette-guerin has also been researched in affected wildlife, including european badgers (chambers et al. 2014), white-tailed deer (palmer, thacker & waters 2007), european wild boar (gortazar et al. 2014) and brush tailed possums (buddle, wedlock & denis 2006; tompkins et al. 2009) with beneficial but variable effects. de klerk et al. (2010) found that bcg was able to reduce the number of lesions in experimentally infected yearling african buffalo although not statistically significant. the use of the live attenuated bcg vaccine also seems attractive because it requires only a single dose, which makes its application in wildlife more practical. on the other hand, the use of a live vaccine in animals destined to enter the human food chain could raise concerns of potentially negative implications for immunocompromised patients (norouzi et al. 2012) as bcg organisms may persist in the edible tissues (palmer et al. 2012). the use of inactivated vaccines against btb would therefore be preferred because of being environmentally safer and more stable under field conditions, thereby causing no disease in non-target wildlife species where safety challenges have not been conducted when compared to the live bcg vaccine (garrido et al. 2011). recent studies using an inactivated m. bovis vaccine have demonstrated protective responses comparable or superior to bcg in a number of wild and domestic animal species, indicating promising prospects for the future use of these vaccines. these inactivated m. bovis vaccines provided good protection in european wild boar (garrido et al. 2011), red deer (lopez et al. 2016), cattle (jones et al. 2016; van der heijden et al. 2017) and sheep (balseiro et al. 2017). when used for vaccination of cattle, the same inactivated m. bovis vaccine induced strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination. these cattle were challenged with live bcg vaccine, and although not statistically significant, recovery of bcg after challenge was lowest in the group vaccinated with inactivated m. bovis as compared to bcg and unvaccinated control animals (van der heijden et al. 2017). in general, more stringent requirements for vaccine use have been postulated for cattle than for wild life. it should be emphasised that this strongly depends on the context in which the vaccine is to be applied. for a vaccine to be considered successful in cattle in epidemiological settings in which conventional control measures are either absent or not implemented because they are deemed impractical, unethical, unaffordable, wasteful or culturally unacceptable, the hallmark of full protection against infection is not justified, similar to the requirements of a wildlife vaccine (buddle et al. 2006). under those circumstances, vaccination can be regarded as the best tool available to perform the task (buddle et al. 2018). the same principle of local context also applies to the significance of interference of a vaccine with future diagnostic testing. while it is not a concern for wildlife without commercial use, in countries where a high number of wild animals are traded, determining the btb status by tb testing is relevant. for this reason, it is of utmost importance to define the expectations and evaluate the limitations of a vaccine in the local context of farming systems and environments. in summary, btb control in south africa faces unique challenges within the livestock sector, the wildlife industry and in conservation areas alike, which cannot be successfully addressed using conventional resource-intensive control measures based on culling alone. vaccination offers a valuable adjunct control tool that has been demonstrated to effectively protect animals from tb disease and can slow down its transmission in multiple species. prior work conducted in south africa has established infection models for both cattle and african buffalo, which form a suitable foundation for future vaccine efficacy trials under controlled and field conditions (buddle et al. 2013; de klerk et al. 2006, 2010; van der heijden et al. 2017). conclusion it is essential that btb be well-controlled within south africa because of the risk it poses to livestock production, commercial game operations, wildlife conservation and human health and livelihoods. however, current approaches to btb control are not or less effective than required to fulfil the mandate of the tuberculosis scheme. the underlying reasons for this inefficacy are manifold and economic, political, technical, social and ethical in nature. to improve the overall btb control, a new strategy is urgently needed, which takes into account affordability, feasibility and cultural acceptability of the measures to be implemented. vaccination offers a promising strategy as an integral part of the btb control in south africa. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. funding information this research received no specific grant from any funding agency in the public, commercial, or 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276, 2987–2995. https://doi.org/10.1098/rspb.2009.0414 van der heijden, e.m.d.l., chileshe, j., vernooij, j.c.m., gortazar, c., juste, r.a., sevilla, i. et al., 2017, ‘host immune response profiles of calves following vaccination with live bcg and inactivated mycobacterium bovis vaccine candidates’, plos one 12(11), e0188448. http://doi.org/10.137/1journal.pone0.188448 wadge, m.j.h., 2007, ‘a vulnerability analysis of hluhluwei mfolozi park for the period 1980–2000’, msc thesis, university of witwatersrand. footnote 1. free from foot-and-mouth disease, corridor disease, brucellosis and bovine tuberculosis. abstract introduction materials and method results discussion acknowledgements references about the author(s) marcia sanhokwe department of livestock and pasture science, university of fort hare alice, south africa johnfisher mupangwa department of livestock and pasture science, university of fort hare alice, south africa patrick j. masika department of livestock and pasture science, university of fort hare alice, south africa fort cox college of agriculture and forestry, middledrift, south africa viola maphosa department of livestock and pasture science, university of fort hare alice, south africa voster muchenje department of livestock and pasture science, university of fort hare alice, south africa citation sanhokwe, m., mupangwa, j., masika, p.j., maphosa, v. & muchenje, v., 2016, ‘medicinal plants used to control internal and external parasites in goats’, onderstepoort journal of veterinary research 83(1), a1016. http://dx.doi.org/10.4102/ojvr.v83i1.1016 note: †, 1966–2015 original research medicinal plants used to control internal and external parasites in goats marcia sanhokwe, johnfisher mupangwa, patrick j. masika, viola maphosa, voster muchenje received: 17 july 2015; accepted: 21 oct. 2015; published: 29 apr. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the use of medicinal plants plays a major role in the primary health care of animals in south africa. a survey was conducted to document medicinal plants used to control parasites in goats in kwezi and ntambethemba villages in the eastern cape province, south africa. information from 50 farmers and 3 herbalists was obtained through the use of a structured questionnaire, and a snowball sampling technique was used to identify key informants. the obtained data were analysed using proc freq of sas (2003), and fidelity level values were determined to estimate the healing potential of the mentioned plants. the survey revealed nine plant species belonging to eight families that were used to control parasites in goats. asphodelaceae (22.22%) was the most frequently used plant family. leaves were the most used plant parts, constituting 60.38%. they were prepared either as infusions or decoctions of single plants or in mixtures. aloe ferox, acokanthera oppositifolia and elephantorrhiza elephantina were the plants having the highest fidelity level for their use to control parasites, each scoring 100%, followed by albuca setosa (83.33%). the study revealed low knowledge about ethno-veterinary medicine in the study area. it also revealed that information on ethno-veterinary medicine in this area is mostly confined to older people and there is danger that this knowledge can be lost before being passed on to other generations. therefore, there is an urgent need to document information on these plant species so that the future generation can benefit. further investigation should be carried out to validate the efficacy and safety of the above-mentioned plants so as to provide cheap alternative ways of controlling parasites. introduction goats play an important role in the socio-economic activities of people, especially in developing countries, by providing food and income (peacock 2005). however, parasites limit goat productivity as they reduce fertility, cause skin irritation and suck blood, ultimately leading to death (molefe et al. 2012). gastrointestinal parasites such as haemonchus contortus and fasciola hepatica are a major health problem in small ruminants (vatta & lindberg 2006). external parasites such as ticks, lice and mites have also been reported in goats. most of these parasites are more prevalent in developing countries because of inappropriate housing and lack of adequate veterinary services (mungube et al. 2006). commercial drugs are mostly used to control parasites; however, these are expensive and are out of reach for many resource-poor farmers. some of the parasites have also developed resistance against these drugs (clark, stephen & cawley 1996), and the drugs can pollute the environment (wall 2007). this has led farmers to resort to alternative measures that include the use of medicinal plants to treat and control livestock parasites. there is also a belief that natural products are safe to use and harmonious with the biological system (erasto 2003). knowledge on the use of ethno-veterinary medicine is passed on orally, and there is a danger that this information might disappear because of technical and socio-economic changes. therefore, this study was conducted to document the medicinal plants used to control internal and external parasites in goats in the chris hani district municipality, south africa. this will help in the pharmacological study of these plants and in the development of therapeutic drugs that have fewer side effects than synthetic chemicals. materials and method study site the survey was conducted in chris hani district municipality in two local municipalities, namely emalahleni and tsolwana, where one village each was randomly selected, namely kwezi and ntambethemba, respectively. the area lies within latitude 31°70’63–32°31’34s and longitude 27°23’41–27°51’17e. it receives an average annual rainfall of 483 mm, with most rain occurring in summer. the study area has an average minimum and maximum temperature of 7 °c and 22 °c, respectively (institute for soil, climate and water 2008). the area is covered by eastern mixed nama karoo, sub-arid thorn bushveld, south-eastern mountain grassland and moist bushland veld types (acocks 1975). sampling procedure villages were randomly selected, and farmers who keep goats were identified using the snowball sampling procedure. this sampling technique, which involved approaching goat farmers and herbalists with more knowledge on the plants used in treating internal and external parasites, in turn directed us to other potential respondents (patton 1990). interviews were conducted amongst 50 farmers and 3 herbalists. data collection structured questionnaires were used to collect data. the data collected included demographic information such as gender, age, source of information and employment status. information gathered included local name of plant used, condition of plant used (dry or fresh), plant parts used, method of preparation, adverse effects, source of knowledge, parasites affecting livestock, other methods used to control parasites, dosage and their source of knowledge. this survey was carried out in accordance with the university of fort hare research and ethics policy (ethical certificate number map011ssan01). plants were collected with the help of herbalists and were authenticated by a botanist, professor grierson at the university of fort. data analysis descriptive statistics were obtained using proc freq of sas (2003). fidelity level (fl) values were determined to report the most used plants in the different communities, as this can be an indication of their possible efficacy (table 1). this was calculated for plant species that had been reported more than three times. fl is the percentage of respondents who use a certain plant for the same main function (sofowara 1982) and was calculated as: where na is the number of respondents who claim the use of a plant species to treat a particular ailment and n is the number of informants who use the plant as medicine for any ailment (alexiades 1996). table 1: fidelity level indices of plant species used to control parasites in the study area. results demographic information the demographic data of respondents are shown in table 2. the majority of the households were male headed (73.58%). the most dominant age group within these heads was above 51 years (84.91%), followed by those aged 31–50 years (13.21%). most of the respondents (43.40%) never went to school. it was found that most of the respondents were unemployed (71.70%) and depended on government grants (39.62%) and selling livestock (35.85%) as their source of income. table 2: demographic data on distribution of respondents. livestock inventory all respondents owned livestock, which included cattle (92.45%), sheep (71.70%), goats (100.00%) and chickens (75.47%). farmers’ reasons for keeping goats were for multiple purposes such as meat, milk, income and for cultural or religious reasons. most of the farmers kept goats for cultural or religious reasons (58.49%). most of the farmers had more than 40 sheep, whilst cattle, goats and chickens were kept in smaller numbers ranging from 1 to 10. prevalence of diseases and parasites gallsickness, heartwater, redwater, diarrhoea and bloating are some of the diseases that were reported to affect goats in the area. the most prevalent tick-borne diseases in the area were heartwater (10.44%), redwater (11.54%) and gallsickness (60.02%). these diseases were more prevalent in summer. government veterinary officers helped the farmers to identify the diseases, and this helped in providing the right treatment. most of the farmers (88.68%) treated their goats when suffering from these diseases. all respondents acknowledged both internal and external parasites to cause huge problems. diseases, parasites, stock theft and poor rangelands were reported as the main challenges faced by the farmers in livestock rearing. parasites (47.17%) were reported as the most problematic challenge that the farmers are facing. common parasites in the study area were fleas (30.77%), lice (65.38), mites (75.38%), ticks (84.62%) and helminths (100%), all of which were reported to be more prevalent in summer than any other season of the year. figure 1 shows the prevalence of parasites in the study area. farmers were able to tell that a goat had been infested with parasites through loss in body condition (32.08%), loss of appetite (33.96%) and rubbing against poles (33.96%). figure 1: perceived prevalence of parasites in chris hani district in eastern cape province, south africa. parasite control the study revealed that most of the farmers (96.23%) dipped their goats once a month. the government provided commercial drugs for use in a spray race or dip tanks. dazzle dip (diazinon 30%) was one of the commercial drugs that they used to control external parasites in the area. dipping was carried out once a month. those farmers who did not dip (3.77%) felt there was no need to do that as they believed that goats are resistant to parasites. the majority of the respondents (69.23%) used medicinal plants, a few used commercial drugs (11.54%), and a proportion (19.23%) used both medicinal plants and commercial drugs to control parasites in their herd. farmers used medicinal plants for various reasons, that is, they are effective (69.81%), cheap (1.89%), easily accessible (13.21%) and easy to use (15.09%), whilst others (11.54%) indicated that they did not have knowledge about the plants. overall, nine plants belonging to eight families used to control parasites in goats were reported as shown in table 3. asphodelaceae was the most frequently mentioned plant family (22.22%). aloe ferox was the most used plant in the area (43.40%). different plant parts such as leaves, roots, tubers and bark were used in preparing the remedies. most of the farmers used leaves (60.38%) in preparing the medicine. for their preparation method, (60.38%) respondents used decoctions and (39.62%) infusions. no side effects were reported by the respondents. some of the respondents combined more than one plant in the preparation of medicines. others also mixed plant extracts with nonplant materials such as epsom salts, flour, butter, potassium permanganate, rock salt and oil cakes. fl values were determined so as to estimate the medicinal use values and the relative preference of species by the local communities in this study area. accordingly, a. ferox, e. elephantina and acokanthera oppositifolia were the plants having the highest fl values for their use to control parasites, each scoring 100.00%, followed by a. setosa (83.33%). most of the respondents (75.47%) reported that they acquired their knowledge orally from their parents and from other farmers. about 88.68% of respondents did not put any conservation methods in place to prevent plants from becoming extinct. the reasons were that there is not enough land to cultivate the plants (50.94%), and 49.06% of the respondents believed the plants are abundant in the wild and there is no need to conserve them. those who practised conservation cultivated the plants in their gardens and used plant parts such as leaves, which does not destroy the whole plant. table 3: plants used to control parasites in goats. discussion in this study the demographic characteristics of the farmers were similar to those reported by mwale and masika (2009) in the eastern cape and limpopo provinces of south africa (luseba & van der merwe 2006). the majority of the households were male headed, whilst the most dominant age group consisted of older generations. most of the respondents who were using medicinal plants and had essential knowledge were generally older than 51 years. the findings of this study agree with those of wanzala et al. (2005), who mentioned that information on medicinal plants is mostly stored in the memory of a few older people entrusted with it within communities. most of these older people are unemployed and rely on grants for their survival. this concurs with findings by masika, van averbeke and sonandi (2000) who reported that most of the farmers rely on grants. the reason could be that most of them are old and poor, so grants are their major source of income. the reasons for keeping goats were similar to those cited by bester, ramsay and scholtz (2009) and masika and mafu (2004). most of the farmers were keeping goats for cultural reasons and for cash income. the use of goats for milk was unpopular as reported previously by mahajana and cronje, (2000) and masika and mafu (2004). furthermore, most of the farmers mentioned parasites as the most problematic challenge that they were facing and this concurs with studies by mwale and masika (2009). parasites might be a problem in this area because the animals are raised on poorly managed pastures where parasites are abundant. infestation with internal and external parasites was reported to be more prevalent in the summer season, which could be because of poor management of pastures (masika & mafu 2004), inappropriate housing (mungube et al. 2006) and climatic conditions in the tropics (webb & mamabolo 2004; phiri et al. 2007), which are favourable for parasite development. findings from this study revealed that farmers usually dipped their goats to control parasites. however, this contradicts the findings by kunene and fossey (2010), who observed that most goats are hardly dipped in rural communities. the farmers questioned in this study are not provided with commercial drugs to control internal parasites. the majority of the respondents (69.23%) used medicinal plants, whilst others bought their own commercial drugs (11.54%) and a proportion (19.23%) used both medicinal plants and commercial drugs to control parasites in their herd. farmers gave reasons why medicinal plants are still in use, which included the efficacy of the plants, no side effects, and cheap and easy accessibility. moreki, dikeme and poroga (2010) attributed the wide use of ethno-veterinary medicine in villages to lack of knowledge about the use of commercial drugs and their high price in most areas. however, gueye (1999) argued that the use of ethno-veterinary medicine is the only option for most resource-limited farmers in africa because of lack of veterinarians in the rural areas. farmers reported that they were able to define diseases using clinical signs, but it should be kept in mind that some diseases exhibit similar signs (differential diagnosis) and that may affect the accuracy of the diagnosis. wrong diagnosis of disease will result in incorrect dosage, and this can affect the efficacy of the herbal remedy. the most frequently mentioned plant family was asphodelaceae. maphosa and masika (2010) reported similar findings. this could be as a result of its vast natural distribution, with 13 genera (treutlein et al. 2003). aloe ferox and bulbine latifolia were the plant species reported that belong to the family asphodelaceae. in this study, leaves were the most used plant parts mentioned, which concurs with previous studies (gebrezgabiher et al. 2013; maphosa & masika 2010; masika & afolayan 2003; mohammed & seyoum 2013; setlalekgomo & setlalekgomo 2013), who reported the highest percentage use of leaves for ethno-veterinary purposes. the use of leaves in preparation of herbal remedies reduces loss of plants from the natural habitats as it does not destroy the whole plant. on the other hand, this study contradicts findings by cheikhyoussef et al. (2011), who reported that roots were the most commonly used plant part. the use of roots in preparation of herbal remedies is not advised, as this results in loss of medicinal plants from their habitats. decoction was the most commonly used preparation method in this study. this is in agreement with maphosa and masika (2010) and djoueche, azebaze and dongmo (2011), who reported decoction as the most used method for preparing medicine. decoction involves boiling part of the plant in water for a few minutes. this process extracts water-soluble polar compounds and the high temperature is responsible for reducing the toxicity of thermolabile compounds that may be poisonous to the animals (djoueche et al. 2011). however, some thermolabile compounds may also be lost in the process, which can affect the efficacy of the plant. some of the respondents combined more than one plant in preparation of medicines. the use of combined plant materials was also reported by masika et al. (2000) and maphosa and masika (2010) but contradicts findings by van der merwe, swan and botha (2001), who reported use of a single plant. other respondents mixed plant materials with nonplant substances such as epsom salts, rock salt, butter, oil cake, flour and potassium permanganate. these findings were similar to previous reports of mixing with nonplant materials (djoueche et al. 2011; maphosa & masika 2010; mohammed & berhanu 2011; mohammed & seyoum 2013). mixing of plant materials with nonplant substances influences the absorption of compounds contained in the plants (djoueche et al. 2011). epsom salts are known to have a laxative effect. oil cakes increase bile secretion, which promotes the solubilisation of non–water-soluble compounds (djoueche et al. 2011). rock salt has emulsifying properties assisting formation of stable emulsions in the gastrointestinal tract and therefore increasing the solubilisation of alkaline compounds in plant extracts, thus increasing their absorption (djoueche et al. 2011). addition of butter is known to improve the flavour and reduce the chances of animals vomiting (mohammed & seyoum 2013). farmers in the eastern cape province are aware of the toxicity of some plants such as a. oppositifolia and therefore add more water to the herbal preparations and boil the plant material before administering it to the animals. they also mixed a. oppositifolia with other plant materials such as a. ferox. akocanthera oppositifolia is toxic because of the cardiac glycosides it contains. this is in consonance with studies by maphosa and masika (2010), who reported the awareness of poisonous plants by farmers in the eastern cape province. adding large quantities of water before boiling results in the extract becoming more dilute and therefore the toxicity of the plant will be reduced. boiling the plant extract also reduces toxicity by evaporating aromatic poisonous compounds. some of the plants that were being used by farmers to control parasites in this study area have been reported to possess pharmacologically active substances. of the nine plants species used to control parasites in goats in chris hani district, a. ferox was the most frequently used plant as previously reported (maphosa & masika 2010; moyo & masika 2009; setlalekgomo & setlalekgomo 2013). the plant a. ferox has a laxative effect because of the presence of glycoside aloin (steenkamp & stewart 2007; eloff & mcgaw 2014). it is also known as an insect repellent; is used to treat heartwater and gallsickness (van wyk, van oudtshoorn & gericke 2002), poultry diseases, sheep scab, and to control ticks in cattle (moyo & masika 2009). elephantorrhiza elephantina is used to treat heartwater (eloff & mcgaw 2014; luseba & van der merwe 2006; van der merwe et al. 2001) and used in goats to control helminths (maphosa & masika 2010; okoli, tamboura & hounranghe-adote 2010) and in humans for high blood pressure (mathias-mundy & mccorkle 1989). elephantorrhiza elephantina possesses antibiotic properties (van wyk & wink 2004; van wyk et al. 2002). it relieves inflammation in animals and is also used as a purgative (cocks 2006). centella coriacea contains triterpenoids that have antibiotic and purgative effects. agapanthus praecox has been reported to contain saponins that have antibiotic, analgesic, laxative, anti-oedema, anti-inflammatory and immunoregulatory effects (van wyk et al. 2002). albuca setosa is used in the management of diabetes mellitus (oyedemi et al. 2011). acokanthera oppositifolia has been reported to treat anthrax and tapeworms (dold & cocks 2001). fl values of a. ferox, a. oppositifolia, a. setosa and e. elephantina were high, showing that most people in the area prefer these plants and constantly use them in controlling parasites. in this study, the most cited plants had the highest fl, contrary to findings by njoroge (2012). trotter and logan (1986) reported that plants that are constantly used by people in a certain area are more likely to contain bioactive substances. validation of these plants is important so as to isolate the active compounds and produce drugs from these plants. some plant species had low fl values because some of the respondents did not know the preparation methods and dosages. most of the informants acquired their knowledge from their elders, as reported by mwale et al. (2005) and mwale and masika (2009). ethno-veterinary knowledge is not written and is accepted orally from elders, analogous with earlier studies (farooq et al. 2008; giday, asfaw & woldu 2009). it is important to document ethno-veterinary knowledge so that it will not be lost and to increase the use of common plants. the bulk of plant matter is collected from the natural vegetation, which poses a major threat to its existence. farmers should use plant parts such as leaves rather than the whole plant to prevent plants from becoming extinct (maroyi 2012). other respondents cultivate the plants in their gardens, and this is not recommended as it is believed that monoculture conditions do not trigger the production of secondary metabolites (schippmann, leaman & cunningham 2003). plants that are cultivated are believed not to possess power compared with wild plants. therefore, it is advisable to use wild plants that grow under stress conditions and competition, as they possess secondary metabolites. conclusion and recommendation the study revealed nine plant species that are used to control parasites in goats in chris hani district municipality. it also revealed that information about ethno-veterinary medicine in this area is mostly confined to older people and there is a danger that this knowledge can be lost before being passed on to other generations. therefore, there is an urgent need to document these medicinal plants before the death of knowledgeable people in the study area. further research should be carried out to assess the efficacy and safety of the plants mentioned, especially those with the highest fl. acknowledgements the authors wish to acknowledge the national research foundation (grant number t219) for funding the research. we would also like to thank professor grierson for identification of plant species. cooperation of farmers, herbalists and extension officers of chris hani district municipality is greatly acknowledged. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions m.s. designed the study, conducted the questionnaire survey, analysed data and prepared the manuscript. p.j.m. was the supervisor, participated in the project design, interpreted the data and performed a critical evaluation of the manuscript. j.m. was the supervisor, assisted with data interpretation and edited the manuscript. v.m. was the co-supervisor and edited the manuscript. v.m. 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splenomegally and, in some cases, death (friedhoff & soule 1996; knowles 1996). most clinical cases of equine piroplasmosis in southern africa are caused by t. equi, which usually shows a high prevalence, while b. caballi infection is usually not clinically apparent and usually has a lower prevalence (de waal 1995, 2000; zweygarth, lopezrebollar, nurton & guthrie 2002). horses infected with t. equi and/or b. caballi may remain carriers for long periods and act as sources of infection for ticks, which in turn act as vectors of the disease (de waal 2000; zweygarth et al. 2002). in south africa, the 141 onderstepoort journal of veterinary research, 75:141–146 (2008) prevalence of theileria equi and babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern free state province, south africa m.y. motloang1, 2*, o.m.m. thekisoe3, a. alhassan3, m. bakheit1, m.p. motheo1, f.e.s. masangane1, m.l. thibedi1, n. inoue3, i. igarashi3, c. sugimoto4 and p.a. mbati1 abstract motloang, m.y., thekisoe, m.m.o., alhassan, a., bakheit, m., motheo, m.p., masangane, f.e.s., thibedi, m.l., inoue, n., igarashi, i., sugimoto, c. & mbati, p.a. 2008. prevalence of theileria equi and babesia caballi infections in horses belonging to resource-poor farmers in the north-eastern free state, south africa. onderstepoort journal of veterinary research, 75:141– 146 the prevalence of theileria equi and babesia caballi infections in the north-eastern free state province of south africa was determined by examination of thin and thick giemsa-stained blood smears, ifat and pcr. no parasites were detected by microscopy from any blood samples collected at five study sites, qwaqwa, kestell, harrismith, vrede and warden. of the tested serum samples, 28/29 (96.5 %), 20/21 (95.2 %) and 42/42 (100 %) were positive by ifat for t. equi infections in harrismith, kestell and qwaqwa, respectively, and 5/29 (17.2 %), 13/21 (61.9 %) and 30/42 (71.4 %) were sero-positive for b. caballi infections in harrismith, kestell and qwaqwa, respectively. all dna samples from the study sites were negative for b. caballi infections by pcr, but five samples, two from each of kestell and warden and one from vrede, were pcr positive for t. equi infections. the high prevalence of antibodies against t. equi and b. caballi in the sampled horses indicates that the animals had been exposed to t. equi and b. caballi infections but the absence of parasitaemia and very low number of positive pcr samples, however, imply that t. equi and b. caballi are endemically stable in the north-eastern free state province. keywords: babesia caballi, free state, theileria equi * author to whom correspondence is to be directed. e-mail: motloangm@arc.agric.za 1 parasitology research programme, university of the free state, qwaqwa campus, phuthaditjhaba, 9866 south africa 2 arc-onderstepoort veterinary institute, parasitology division, onderstepoort, 0110 south africa 3 national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, hokkaido, 080-8555, japan 4 center for zoonosis control, hokkaido university, sapporo, hokkaido, 060-0818 japan accepted for publication 14 april 2008—editor 142 theileria equi and babesia caballi infections in horses in north-eastern free state province, south africa ixodid tick, rhipicephalus evertsi evertsi is the main vector of t. equi and b. caballi (de waal & potgieter 1987). theileria equi and b. caballi infections are usually diagnosed by microscopic examination of giemsa or wright-stained blood films (saal 1964), but these methods have poor sensitivity during very low parasitaemia. serological assays, such as enzyme-linked immunosorbent assay (elisa) and immunofluorescent antibody test (ifat) are also commonly used for detection of t. equi and b. caballi infections (bose, jorgensen, dalgleish, friedhoff & de vos 1995; bruning 1996; weiland 1986) but cannot distinguish between current and past infections due to persistence of antibodies even when parasites are cleared from the animal. pcr is the commonly used molecular technique for the diagnosis of t. equi and b. caballi and has been reported to be highly specific and sensitive (bashiruddin, camma & rebelo 1999; rampersad, cesar, campbell, samlal & ammons 2003). the prevalence of some of the tick-borne diseases, poultry diseases and helminth infections in domestic livestock of resource-poor farmers in the northeastern free state province has recently been reported, as well as the ticks occurring in the region (hlatshwayo, mbati & dipeolu 2002; mbati, hlatshwayo, mtshali, mogaswane, de waal & dipeolu 2002; nyaile, thekisoe, bisschop & mbati 2003; thekisoe, mbati & bisschop 2003; tsotetsi & mbati 2003; mtshali, de waal & mbati 2004). the current study was aimed at documenting t. equi and b. caballi infections in horses in the north-eastern free state province by conventional parasitological methods, ifat and dna amplification by pcr. a questionnaire survey in order to assess the perceptions, attitudes and expectations of farmers in the study area with regard to t. equi and b. caballi infections was also conducted. materials and methods description of the study area the north-eastern free state province includes towns situated in the thabo mofutsanyane district municipality which borders lesotho on the south, motheo, lejweleputswa and northern free state district municipalities on the south-west, west and north, respectively, while mpumalanga and kwazulunatal provinces are on the north-east and eastern borders, respectively. blood samples were collected between august 2003 and october 2005 from horses of 38 resource-poor farmers located in five study sites within the north-eastern free state: harrismith (four farms), kestell (three farms), qwa qwa (16 villages), vrede (11 farms) and warden (four farms). fig. 1 shows the exact locations of the study sites within thabo mofutsanyane district. collection of blood samples a total of 122 blood samples were collected from horses at the five localities, viz. 29 from harrismith, 21 from kestell, 42 from qwaqwa, 19 from vrede •vrede •qwaqwa •harrismith•kestell •warden lesotho free state province thabo mofutsanyane district nn nn fig. 1 maps of the free state province and the thabo mofutsanyane district municipality showing the location of the five study sites: harrismith, kestell, qwaqwa, vrede and warden. the maps were obtained from the website of the municipality demarcation board of the republic of south africa (www.demarcation.org.za) 143 m.y. motloang et al. and 11 from warden. ten milliliters of blood were collected in both silicone and edta-coated vacutainers by venipuncture of the jugular vein, and were kept in a cooler-box before transport to the laboratory. blood samples were also collected from seven horses known to be infected with t. equi parasites from kaalplaas farm of the onderstepoort veterinary institute (ovi), pretoria, and were used as controls for t. equi. preparation of thin and thick blood smears thin and thick blood smears were prepared from the unclotted blood in the edta-coated vacutainers and stained with giemsa as described by de waal (1999). the parasitaemia was estimated by counting parasitized red blood cells (rbc) in eight different fields consisting of an average of 800 red blood cells per field using a formula in which the number of infected rbc is divided by the total number of uninfected rbc x 100. a smear was considered negative based on the criterion described by de waal & potgieter (1987), i.e. smears that reveal no parasites after 5 min examination are judged to be negative. serum preparation and dna extraction the blood in silicone-coated vacutainers was left for 24 h at room temperature and then centrifuged at 200 x g for 5 min (de waal & potgieter 1987). the resulting sera were then placed in cryogenic vials and stored at –35 °c until used. genomic dna was extracted from horse blood by the phenol-chloroform method (sambrook & russel 2001) with minor modifications. briefly, 400 μℓ of blood was treated with five units of te (10 mm tris-hcl [ph 7.4], 1 mm edta) buffer and then centrifuged at 14 000 x g for 2 min. the resulting pellet was washed five times in te, removing the erythrocyte ghost layer with every wash, and re-suspended in 400 μℓ of distilled water. after incubation at 37 °c for 1 h with 100 μg/mℓ of proteinase k, 1 % sds and 1 % sarcosine, dna was extracted with phenol-chloroform and precipitated with ethanol at –34 °c, and was finally dissolved in 20 μℓ distilled water. indirect immunofluorescent test (ifat) antigen slides, conjugate and control sera were obtained from the ovi. batches of antigen slides were removed from storage at –20 °c, incubated at 37 °c for 10 min and then fixed in cold acetone at –20 °c. two-fold dilutions, of the test and control sera, starting at 1/40, 1/80 and 1/160, were prepared using pbs (tenter & friedhoff 1986). a drop (~ 4 μℓ) of each diluted serum was pipetted into each respective circle of the antigen slide, incubated at 37 °c for 30 min and then washed off with pbs. a second wash was done using distilled water and the slides were allowed to air dry, after which 2 μℓ of conjugate dilution was added and incubated at 37 °c for 30 min. the conjugate was washed in pbs for 10 min and after air-drying, mounting fluid consisting of 50 % glycerin and 50 % pbs was placed on each slide and covered with 24 x 50 mm cover slip. the slides were observed under a fluorescent microscope using a 50x water objective (tenter & fried hoff 1986). test readings were ranked as negative for slides with no specific fluorescence when observed under the fluorescent microscope, positive for those that presented strong fluorescence at a serum dilution of 1/80 and positive/negative for those that showed a weak fluorescence (ovi 1999). polymerase chain reaction (pcr) a primer pair targeting the ema1 gene (accession no. ay058899) of t. equi with a product of 743 bp was used for amplification of t. equi dna, and a primer pair designed from the bc48 gene (accession no. ab017700) with a product of 179 bp were used for amplification of b. caballi dna (table 1). the pcr was conducted in a total volume of 50 μℓ containing pcr buffer (10 mm tris-hcl (ph 8.3), 50 mm kcl, 1.5 mm mgcl2), 2 mm each of the 4 dntps, 5 pmol of each primer and five units of amplitaq gold dna polymerase (applied biosystems japan ltd., tokyo, japan). the reaction mixture was heated at 94 °c for 10 min (denaturation step) and subjected to 35 cycles each at 94 °c for 45 s, 60 °c for table 1 pcr primers used for amplification of t. equi and b. caballi dna parasite target gene primer sequence theileria equi ema1 f: 5’gcatccattgccatttcgag3’ r: 5’tgcgccatagacggagaagc3’ babesia caballi bc48 f: 5’cggctgctatggttattcag3’ r: 5’agagtgcaaccgagcaatgc3’ 144 theileria equi and babesia caballi infections in horses in north-eastern free state province, south africa 1 min (annealing), and 2 min at 72 °c with a final extension at 72 °c for 7 min. the pcr products were electrophoresed on a 1 % agarose gel, stained with ethidium bromide and visualized under uv light. the positive pcr products were purified using the qiaquick gel extraction kit (qiagen, usa). the nucleic acid sequence was determined with the bigdye terminator cycle sequencing kit (applied biosystems, japan). questionnaire survey questionnaires were completed by a total of 23 farmers in harrismith, kestell and qwaqwa. the aim of the survey was to determine the level of knowledge of resource poor farmers in the north-eastern free state province concerning equine piroplasmosis, ranging from knowledge of its cause and transmission, whether they could recognize the clinical signs and whether they had knowledge on control measures for the disease. results microscopy and ifat neither t. equi nor b. caballi was detected in the thick and thin blood smears made from the blood on samples from harrismith, kestell, qwaqwa, vrede and warden (table 2). however, antibodies against t. equi and b. caballi were detected by ifat in the blood of some of the horses in harrismith, kestell and qwaqwa. out of a total of 99 samples from these three study sites, 98 % were t. equi positive and 48 % were b. caballi positive (table 2), while 58 % had mixed infections. theileria equi was detected in three of the seven horses from kaalplaas in both thick and thin smears, which were used as a positive control group for t. equi, while serology showed the presence of antibodies against t. equi in all of them (table 2). gene amplification by pcr all the blood samples from kaalplaas were pcr pos itive for t. equi, while only five samples (two from kestell, two from warden and one from vrede) were pcr positive for this parasite (fig. 2). these pcrpositive products were sequenced and the sequence fragments were identical ranging from 83–100 % nu cleic acid sequence identities, with other ema1 genes in the ncbi genbank database determined by blastn search (data not shown) (www.ncbi. nlm.nih.gov/blast). all the north-eastern free state province and kaalplaas dna samples were pcr neg ative for b. caballi using primers targeting the bc48 gene. questionnaire survey none of the farmers in the study sites who completed questionnaires had any scientific knowledge on equine piroplasmosis. forty-eight percent of them admitted observing clinical signs such as nasal discharge where affected animals appeared to suffocate from accumulation of mucus with occasional table 2 microscopic, ifat and pcr results for detection of t. equi and b. caballi infections study site no. of samples microscopy (%) ifat (%) pcr (%) t. equi b. caballi t. equi b. caballi t. equi b. caballi harrismith kestell qwaqwa vrede warden kaalplaas 29 21 42 19 11 7 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 3 (43) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 28 (96.5) 20 (95.2) 42 (100) nd* nd 7 (100) 5 (17.2) 13 (61.9) 30 (71.4) nd nd 3 (43) 0 (0) 2 (9.5) 0 (0) 1 (5.2) 2 (18.1) 7 (100) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) nd* – not done 2 3 4 5 + –1 700 bp m 500 bp 100 bp fig. 2 five dna samples positive by pcr for theileria equi using ema1 primers. lane m: 100 bp marker; lanes 1–2: kestell samples; lane 3: vrede sample; lanes 4–5: warden samples; lane +: positive control (t. equi—usda strain); and lane –: negative control (b. caballi—usda strain) 145 m.y. motloang et al. sneezing, but these were signs which could not necessarily be linked to equine piroplasmosis. all of them indicated that they believed ticks did have an effect on their livestock’s health although they could not specifically single out the disease(s) they transmitted. a common inexpensive method of tick control used by those who owned a relatively small number of livestock included burning the grass of the grazing area in winter while dipping in an acaricide was done by those who owned a larger numbers of animals. discussion in this study, neither t. equi nor b. caballi was detected by microscopy in the samples from qwaqwa, harrismith, kestell, vrede and warden. beaver, jung & cupp (1984) stated that blood smears are useful for studying the morphological changes of blood cells and blood parasites. however, the main disadvantage of this method is that the volume of blood used for preparing the smears is small, making the detection of a low parasitaemia and of carrier animals difficult (ambrosio & de waal 1990). demon stration of parasites by microscopy was only successful in three of the seven control horses from kaalplaas which had a known history of t. equi infections. indirect immunofluorescent test is the most widely used serological diagnostic test. as shown in table 2, the prevalence of antibodies against t. equi in the samples from kestell, harrismith and qwaqwa was higher than that of b. caballi. results of the current study correspond to those of a previous seroepidemiological survey of t. equi and b. caballi in the northern and eastern cape provinces in which it was found that the prevalence of t. equi infections was relatively higher than that of b. caballi (gummow, de wet & de waal 1996). moreover, t. equi parasites are reported to propagate faster than b. caballi (holman, frerichs, chieves & wagner 1993), which also explains why the prevalence and pathogenicity of t. equi is higher than those of b. caballi in endemic areas (schein 1988; de waal 2000; alhas san, pumidonming, okamura, hirata, battse tseg, fujisaki, yokoyama & igarashi 2005). all the dna samples from the study areas were negative for b. caballi infections by pcr. babesia caballi generally produces a low parasitaemia (potgieter, de waal & posnett 1992; holman et al. 1993) which makes it extremely difficult to detect in blood smears and even dna probes have been reported to detect b. caballi-specific dna at irregular intervals during the clinical course of an infection (posnett & ambrosio 1991; holman et al. 1993). the respective sequences of t. equi were identical to other ema1 genes of t. equi in the genbank database, thereby confirming that our results are not falsely positive. the two pcr-positive animals from kestell also showed the presence of antibodies by ifat but parasitaemia was not within detectable levels by microscopic examination. the current study has demonstrated a high prevalence of antibodies against t. equi and b. caballi in the horses by ifat. although the presence of antibodies did not distinguish current from previous infections, these results do, however, indicate that the horses in the study region are exposed to t. equi and b. caballi infections. however, it can be concluded that five t. equi pcr-positive animals were still harbouring the parasites, although at a very low parasitaemia level, while the remainder might have recovered from the infections before the sampling period. furthermore, the questionnaire survey revealed that these animals had never been treated for equine piroplasmosis, which suggests that they are possibly randomly re-exposed to t. equi and b. caballi infections several times during their lifetime as the vector tick (r. evertsi evertsi) is ever present in the region (hlatshwayo et al. 2002; mbati et al. 2002). the pcr method, with its high specificity, can be used in confirmatory diagnosis while ifat is useful in large-scale epidemiological surveys. in conclusion, this study has demonstrated the existence of t. equi and b. caballi in the north-eastern free state province which confirms previous reports that this tick-borne disease is widespread in south africa (de waal 1995; gummow et al. 1996). however, the negative giemsa-stained blood smears, high incidence of positive serological assay, presence of the tick vector (hlatshwayo et al. 2002; mbati et al. 2002) and absence of clinical signs from the horses imply that the study region is endemically stable for t. equi and b. caballi infections. data from this study can serve as a basis for future largescale epidemiological studies on equine piroplasmosis in this region. there is a need for scientific education for resource-poor farmers on diseases of economic and veterinary importance as this can improve their overall control. acknowledgements the authors are grateful to the horse owners in the study sites for their co-operation. we thank mr j. komape; ms m. sentle; mr a. spickett and mrs a. spickett from the ovi and dr b. visser, university of 146 theileria equi and babesia caballi infections in horses in north-eastern free state province, south africa the free state, for their technical support and training in diagnostic assays. this study received financial support from two grants made available to prof. p.a. mbati (university of the free state, qwaqwa campus [senate research grant – 250135073] and the national research foundation [gun 2050679]) as well as one grant, the japanese society for the promotion of science—asia-africa platform grant made available 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [595.276 841.890] >> setpagedevice sahle_289-299.indd introduction there are seven immunologically distinct serotypes of foot-and-mouth disease (fmd) virus with different geographical distributions. the south african territories (sat) serotypes of fmd virus are prevalent in sub-saharan africa where outbreaks attributed to them have been recorded in many countries in east, west and southern africa (vosloo, bastos, sangare, hargreaves & thomson 2002a). the sat serotypes have been shown to be endemic to most african buffalo (syncerus caffer) populations in southern africa and, although not studied in such detail elsewhere, evidence suggests that buffaloes in east africa are also persistently infected with sat-1, sat-2 and sat-3 (hedger, forman & woodford 1973; thomson & bastos 2004; vosloo & thomson 2004). although these animals generally do not show clinical disease, they excrete virus throughout the acute phase of the disease (gainaru, thomson, bengis, esterhuysen, bruce & pini 1986) during which time they can infect other susceptible species. this is followed by a persistent infection where virus can only be found in the oro-pharyngeal region and, for buffaloes in particular, this period has been shown to be up to 5 years in a single animal (condy, hedger, hamblin & barnett 1985). circumstantial as well as experimental evidence have pointed to persistently infected buffaloes precipitating disease 289 onderstepoort journal of veterinary research, 74:289–299 (2007) study of the genetic heterogeneity of sat-2 foot-and-mouth disease virus in sub-saharan africa with specific focus on east africa m. sahle1, 2#, r.m. dwarka1, e.h. venter2 and w. vosloo1, 2* abstract sahle, m., dwarka, r.m., venter, e.h. & vosloo, w. 2007. study of the genetic heterogeneity of sat-2 foot-and-mouth disease virus in sub-saharan africa with specific focus on east africa. onderstepoort journal of veterinary research, 74:289–299 the epidemiology of serotype sat-2 foot-and-mouth disease was investigated in sub-saharan africa by phylogenetic analysis using the 1d gene encoding the major antigenic determinant. fourteen genotypes were identified of which three are novel and belong to east africa, bringing the total number of genotypes for that region to eight. the genotypes clustered into three lineages that demonstrated surprising links between east, southern and south-western africa. one lineage was unique to west africa. these results established numerous incursions across country borders in east africa and long term conservation of sequences for periods up to 41 years. ethiopia, kenya and uganda have all experienced outbreaks from more than one unrelated strain, demonstrating the potential for new introductions. the amount of variation observed within this serotype nearly equalled that which was found between serotypes; this has severe implications for disease control using vaccination. keywords: 1d gene, east africa, foot-and-mouth disease, phylogenetic study, sat-2, sub-saharan africa * author to whom correspondence is to be directed. e-mail: vosloow@arc.agric.za 1 exotic diseases division, arc-onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110, south africa # present address: national animal health research centre, ethiopian agricultural research organization, p.o. box 04, sebeta, ethiopia accepted for publication 7 may 2007—editor 290 genetic heterogeneity of sat-2 fmd virus in sub-saharan africa when in close contact with other susceptible species (dawe, flanagan, madekurozwa, sorensen, anderson, foggin, ferris & knowles 1994a; dawe, sorenson, ferris, barnett, armstrong & knowles 1994b; vosloo, bastos, kirkbride, ester huysen, janse van rensburg, bengis, keet & thom son 1996; bastos, boshoff, keet, bengis & thomson 2000; vosloo, bastos & boshoff 2006) and new vari ants of virus being generated that could have severe implications on disease control reliant on the use of vaccines (vosloo et al. 1996). previous studies focusing mainly on buffalo isolates have indeed demonstrated large numbers of fmd virus variants pres ent within specific geographic regions and in only a few cases could the transmission of virus from carrier buffaloes to susceptible livestock and wildlife be verified by phylogenetic analysis (dawe et al. 1994a; bastos et al. 2000; vosloo, boshoff, dwarka & bas tos 2002b; vosloo et al. 2006). other wild ungulates do not become long term carriers of fmd virus, but can spread the disease during acute infection (hed ger, condy & golding 1972; hedger 1981; thomson, bengis & brown 2001; thomson, vosloo & bastos 2003). the causative virus, fmd virus, is one of two members of the aphthovirus genus belonging to the picornaviridae family. the single stranded rna genome is 8 500 nucleotides in length and encodes a single open reading frame. the viral rna dependant rna polymerase lacks proof reading ability which leads to significant nucleotide changes during each round of viral replication (sobrino, dávila, ortin & domingo 1983). the rate of change for rna viruses ranges between 10–1 and 10–4 substitutions per nucleotide per year (reviewed in domingo, bar anowski, escarmis & sobrino 2002) while rates in excess of 10–2 substitutions per nucleotide per year within the vp1-vp3 coding region have been found during an outbreak of fmd virus (sobrino, palma, beck, dávila, de la torre, negro, villanueva, ortin & domingo 1986; villaverde, martinez-salas & domingo 1988) with the estimation that clones from a single isolate differ in approximately 0.6–2 genomic positions, contemporary isolates in 2–20 positions and different isolates from a single outbreak differ in 50–100 genomic positions (reviewed in domingo, escarmis, martinez, martinez-salas & mateu 1992; sobrino, saiz, jimenez-clavero, nunez, rosas, bara nowski & ley 2001). the best method to date to differentiate between fmd virus isolates has been the determination of the rna sequence encoding the vp1 protein which contains the major antigenic determinants of the virus (beck & strohmaier 1987; samuel, knowles & kitching 1988). despite the success in elucidating the epidemiology of the disease, sequence data do not predict with accuracy the influence on the antigenicity of the virus and therefore cannot at present be used in isolation to recommend vaccine strains. this is of particular interest in regions where vaccination is used to control and eradicate the disease, as it is imperative that vaccines be used that are antigenically closely related to viruses circulating in the field (hunter 1998). of the sat serotypes most outbreaks in domestic animals have been recorded for sat-2 (thomson & bastos 2004) giving credence to the fact that sat-2 may be most efficient in crossing species barriers (bastos 2001). this serotype has also spread into the middle east on at least two occasions (ferris & donaldson 1992; bastos, haydon, sangare, boshoff, edrich & thomson 2003b). in contrast, most buffaloes first become infected with sat-1, followed by sat-2 and lastly by sat-3 as determined by serological responses in young buffaloes in the kruger national park (knp), south africa (thomson & bastos 2004). the genetic diversity of sat-2 fmd isolates has been published previously for african countries with specific focus on southern africa (vosloo, knowles & thomson 1992; vosloo, kirkbride, bengis, keet & thomson 1995; bastos et al. 2003b) with limited data on isolates from east africa. the present study was carried out to determine the genetic variability of the sat-2 fmd isolates in east africa and to elucidate their epidemiology on a subcontinental basis. materials and methods viruses included in this study a total of 41 sat-2 fmd virus isolates from ethiopia, sudan, kenya, uganda, tanzania and eritrea isolated between 1975 and 2000 were supplied by the world reference laboratory (wrl) for fmd at the institute for animal health, pirbright (united kingdom). these isolates were propagated once on ibrs-2 cells before further processing. nucleic acid isolation and rt-pcr amplification total rna was extracted from cell culture supernatant using a guanidium thiocyanate-silica method (boom, sol, salimans, jansen, wertheim-van dillen & van der noordaa 1990). the rna viral template was reverse transcribed using amv reverse transcrip291 m. sahle et al. tase (promega) with antisense primer (p1) of beck & strohmaier (1987) and dna amplification has been described previously (bastos 1998). the p1 primer complementary to the conserved 2a/b junction site and the forward primer binding within 1c (vp3) termed vp3ab (5’-cactgctaccactcrg agtg-3’) (bastos 1998), were used to amplify an approximately 880 bp fragment. dna purification and cycle sequencing the pcr amplicon was excised from a 1.5 % agarose gel and purified using the qia quick gel extraction kit (qiagen). purified pcr products were sequenced using the big dye® version 3.0 cycle sequencing kit and the abi prism 310 genetic analyzer (applied biosystems). two sequencing reactions were performed per isolate using identical upstream and downstream primers as in the pcr. phylogenetic analysis the dapsa program (harley 2001) was used to align generated nucleotide sequences to data previously published for 26 isolates bringing the total number of isolates to 67 from 20 countries (table 1). a homologous region of 648 bp corresponding to the complete vp1 encoding gene and 6 nucleotides of the 2a region was used for phylogenetic analysis. phylogenetic reconstructions [neighbour joining (nj) and minimum evolution (me)] were carried out using methods of analysis included in mega version 2.0 (kumar, tamura, jakobsen & nei 2001), with p-distance, pair-wise deletions of gaps and confidence levels assessed by 1 000 bootstrap replications. a gamma shape parameter of 0.9059 as determined in model test (posada & crandall 1998) was used to construct the minimum evolution tree. parsimony and upgma analysis were performed using mega version 2.0. average pair-wise comparisons were conducted to estimate divergence within and between lineages and genotypes. a variability plot of sequences of the 1d gene of all virus isolates included in this study was determined using mega version 1.02 (kumar, tamura & nei 1993) with numbers of variable sites in overlapping windows of 10 and > 70 % variation taken as an indication of hypervariability. results phylogenetic analysis complete 1d gene sequences (648 bp) were used to determine phylogenetic relationships between the 67 sat-2 isolates from sub-saharan africa and one isolate that had caused outbreaks in saudi arabia during 2000 (table 1). nj, upgma, me and parsimony methods produced trees with similar topology (results not shown) indicating that the recovered phylogeny is a good estimate and reliable. only the me tree is shown (fig. 1). the me tree revealed three lineages which are summarized below and were broadly geographically linked, with lineage i con sisting of isolates from east africa, angola, the democratic republic of congo (drc; zaire) and saudi arabia, lineage ii containing isolates from west africa, while lineage iii encompassed southern and east africa. lineage i genotype g (eritrea 1998 and saudi arabia 2000), genotype h (rwanda 2000), genotype i (kenya and uganda 1957–1998), genotype j (uganda 1998 and drc 1982), genotype k (angola 1974), genotype l (uganda 1975–1976), genotype m (sudan 1977), and genotype n (ethiopia 1991) lineage ii genotype e (ghana, nigeria and senegal 1975– 1991) and genotype f (gambia and senegal 1979– 1983) lineage iii genotype a (south africa 1983–1995), genotype b (ethiopia, burundi, kenya, tanzania, malawi 1975– 1999), genotype c (zambia and botswana 1996– 1998), genotype d (zimbabwe and botswana 1983– 2000) previously, 11 genotypes were described for subsaharan africa based on genetic distance and bootstrap support (bastos et al. 2003b) and in this study the genotypes are assigned the same alphabetical letters to facilitate comparison. the phylogeny corresponded well to what was previously described for sat-2 in sub-saharan africa (bastos et al. 2003b, sangare, bastos, venter & vosloo 2004). three new genotypes were demonstrated in east africa, viz. genotype l that contained isolates from uganda (1975–1976), genotype m with two isolates obtained in sudan during 1977 and genotype n with a single isolate from ethiopia made during 1991. in this study, the number of isolates from east africa was increased compared to earlier studies and the previously described genotype g was shown to contain two more isolates obtained in eritrea during 1998, genotype h remained the same with a single isolate 292 genetic heterogeneity of sat-2 fmd virus in sub-saharan africa table 1 summary of sat-2 fmd viruses included in this study virus designations country of origin year of sampling reference genbank accession no. ken/3/57 kenya 1957 unpublished aj251473 ang/4/74# angola 1974 bastos et al. (2003b) af479417 mal/3/75# malawi 1975 bastos et al. (2003b) af367099 nig/2/75 nigeria 1975 sangare et al. (2004) af367139 sen/7/83 sénégal 1983 sangare et al. (2004) af479414 sen/5/75 sénégal 1975 bastos et al. (2003b) af367099 tan/1/75 tanzania 1975 this study ay343970 uga/51/75 uganda 1975 this study ay343963 ken/2/76 kenya 1976 this study ay343940 uga/3/76 uganda 1976 this study ay343964 uga/8/76 uganda 1976 this study ay343965 sud/6/77 sudan 1977 this study ay343939 sud/9/77 sudan 1977 this study ay442014 gam/8/79 gambia 1979 sangare et al. (2004) af426093 gam/9/79 gambia 1979 sangare et al. (2004) af426078 zai/1/82 zaire 1982 bastos et al. (2003b) af367100 pal/5/83 south africa 1983 bastos et al. (2003b) af367102 zim/7/83 zimbabwe 1983 van rensburg & nel (1999) af136607 ken/1/84 kenya 1984 this study ay344505 ken/2/84 kenya 1984 this study ay343941 ken/1/85 kenya 1985 this study ay343942 ken/1/86 kenya 1986 this study ay343943 tan/1/86 tanzania 1986 this study ay343971 ken/1/87 kenya 1987 this study ay343944 ken/2/87 kenya 1987 this study ay343945 ken/2/88 kenya 1988 this study ay343946 ken/1/89 kenya 1989 this study ay343947 eth/1/90 ethiopia 1990 this study ay343935 eth/2/90 ethiopia 1990 this study ay343936 gha/2/90 ghana 1990 sangare et al. (2004) af426081 bun/1/91 burundi 1991 bastos et al. (2003b) af367111 eth/1/91 ethiopia 1991 this study ay343937 eth/2/91 ethiopia 1991 this study ay343938 gha/8/91 ghana 1991 sangare et al. (2004) af426083 ken/8/91 kenya 1991 this study ay343949 ken/28/91 kenya 1991 this study ay343948 ken/33/91 kenya 1991 this study ay343950 uga/3/91 uganda 1991 this study ay343966 ken/1/92 kenya 1992 this study ay343953 ken/3/92 kenya 1992 this study ay343951 ken/6/92 kenya 1992 this study ay343952 ken/1/94 kenya 1994 this study ay343954 ken/2/94 kenya 1994 this study ay343955 ken/3/95 kenya 1995 this study ay343957 ken/7/95 kenya 1995 this study ay343956 knp/31/95* south africa 1995 bastos et al. (2003b) af367119 uga/9/95 uganda 1995 this study ay343967 ken/1/96 kenya 1996 this study ay343960 ken/7/96 kenya 1996 this study ay343959 ken/11/96 kenya 1996 this study ay343958 zam/10/96* zambia 1996 bastos et al. (2003b) af367121 bot/1/98* botswana 1998 bastos et al. (2003b) af367122 bot/31/98* botswana 1998 bastos et al. (2003b) af367125 eri/1/98 eritrea 1998 this study ay343933 eri/4/98 eritrea 1998 this study ay343934 eri/12/98 eritrea 1998 bastos et al. (2003b) af367126 ken/7a/98 kenya 1998 this study ay343961 ken/16/98 kenya 1998 this study ay343962 uga/19/98 uganda 1998 this study ay343969 uga/28/98 uganda 1998 this study ay343968 zim/267/98* zimbabwe 1998 bastos et al. (2003b) af367130 ken/5/99 kenya 1999 bastos et al. (2003b) af367131 ken/7/99 kenya 1999 bastos et al. (2003b) af367132 ken/9/99 kenya 1999 bastos et al. (2003b) af367135 rwa/1/00 rwanda 2000 bastos et al. (2003b) af367134 sau/6/00 saudi arabia 2000 bastos et al. (2003b) af367132 zim/1/00* zimbabwe 2000 bastos et al. (2003b) af367136 all unmarked isolates were obtained from cattle * buffalo isolate # species of origin not known 293 m. sahle et al. i iii ii b a c d e g m n h j i 100 100 100 99 100 100 100 100 100 71 100 100 100 100 100 99 72 100 94 100 99 88 99 77 95 95 100 100 5 k l f ken/11/96 ken/6/92 ken/8/91 uga/3/91 ken/7/95 ken/28/91 ken/1/89 ken/2/84 ken/1/96 ken/7/96 ken/1/85 ken/1/92 ken/1/94 ken/3/92 ken/2/94 uga/9/95 ken/7a/98 ken/2/88 ken/3/57 ang/4/74 uga/3/76 uga/8/76 uga/51/75 zai/1/82 uga/28/98 uga/19/98 rwa/1/00 sud/6/77 sud/9/77 eth/2/91 sau/6/00 eri/12/98 eri/1/98 eri/4/98 gam/9/79 gam/8/79 sen/7/83 gha/8/91 gha/2/90 nig/2/75 sen/5/75 zim/7/83 bot/1/98 zim/267/98 zim/1/00 zam/10/96 bot/31/98 knp/31/95 pal/5/83 eth/2/90 eth/1/91 eth/1/90 bun/1/91 ken/7/99 ken/5/99 ken/9/99 ken/16/98 ken/33/91 tan/1/86 mal/3/75 ken/3/95 ken/2/76 tan/1/75 ken/1/86 ken/1/84 ken/1/87 ken/2/87 100 fig. 1 minimum evolution tree based on the 1d gene depicting genetic relationships of sat-2 fmd isolates from sub-saharan africa. bootstrap values were estimated based on 1 000 replications. i–iii depict the major lineages, while a–n indicate genotypes 294 genetic heterogeneity of sat-2 fmd virus in sub-saharan africa 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 k e n / 3 / 5 7 t t s a g e g a e v v t t d p t t h g g k v t t p r r v h t d v a f l l d r s t h v h t n t t a f v v d l m d t k e k a l v g a i l r s a t y y f c d l e v a c t a n / 1 / 7 5 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . s . . . . . . . q q . . . . . l . . a s . . . . . . . . i . . u g a / 5 1 / 7 5 . . . . . . . . . . . . . . . . . . . . s . g a . . . . . . . . . . . . . . . . . . . . q k . s . a . . . l . . . . . . . . . . . . . . . . . . . . . m d i t . k e n / 2 / 7 6 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . n . . . . . . . q q . . . . . l . . a s . . . . . . . . i . . u g a / 3 / 7 6 . . . . . . . . . . . . . . . . . . . . s . g a . . . . . . . . . . . . . . . . . . . . q k . s . a . . . l . . . . . . . . . . . . . . . . . . . . . m . i t . u g a / 8 / 7 6 . . . . . . . . . . . . . . . . . . . . s . g . . . . . . . . . . . . . . . . . . . . . q k . s . a . . . l . . . . . . . . . . . . . . . . . . . . . m . i t . s u d / 6 / 7 7 . . . . s . . . d . i . . g . a . . . . t e g . a . . i . . . . . . . . . . . . . . . . . k . . . a . . . . . . . r . . . . . . . . . . . . . . . . . . . . . . s u d / 9 / 7 7 . . . . s . . . d . i . . g . a . . . . t e g . a . . i . . . . . . . . . . . . . . . . . k . . . a . . . . . . . r . . . . . . . . . . . . . . . . . . . . . . k e n / 1 / 8 4 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . n . . . . . . . q q . . . . . l . . a s . . . . . . . . i . . k e n / 2 / 8 4 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 / 8 5 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 / 8 6 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . n . . . . . . . q q . . . . . l . . a s . . . . . . . . i . . t a n / 1 / 8 6 . . . . . . . . d . . . . . . s . . . . s . e e k . . m . . . . . . v . . . f . . . . . . k a t . n . . . . . . . q q t . . . . l . . a s . . . . . . . . i . . k e n / 1 / 8 7 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . n . . . . . . . q q . . . . . l . . a s . . . . . . . . i . . k e n / 2 / 8 7 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . n . . . . . . . q q . . . . . l . . a s . . . . . . . . i . . k e n / 2 / 8 8 . . . . . . . . d . . . . . . s . . . . t . m a a . . . . . . . . . . . . . f . . . . . . k . t . a . . . . . . n . q . . . . . l . . . t . . . . . . . . i . . k e n / 1 / 8 9 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . e t h / 1 / 9 0 . . . . . . . . d . . . . . . s . . . . n . l e k . . m . . . . . . v . . . f . . . . . s k . t . n . . . . . . . q h . . . . . l . . a s . . . . . . . . i t . e t h / 2 / 9 0 . . . . . . . . d . . . . . . s . . . . n . l e k . ? m . . . . . . v . . . f . . . . . s k . t . n . . . . . . . q h . . . . . l . . a s . . . . . . . . i . . e t h / 1 / 9 1 . . . . . . . . d . . . . . . s . . . . n . l e k . . m . . . . . . v . . . f . . . . . s k . t . n . . . . . . . q h . . . . . l . . a s . . . . . . . . i . . e t h / 2 / 9 1 . . . . . . . . d . . . i . . . . . . . s . . p a . . i . . . . . . . . . . . . . . . . . k . t . n i . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 8 / 9 1 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . r . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 2 8 / 9 1 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 3 3 / 9 1 . . . . . . . . d . . . . . . s s . . . s . v e k . . m . . . . . . v . . . f . . . . . s k . t . n . . . l . . . q h . . . . . l . . a s . . . . . . . . i . . u g a / 3 / 9 1 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . r . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 / 9 2 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 3 / 9 2 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 6 / 9 2 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 / 9 4 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 2 / 9 4 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 3 / 9 5 . . . . . . . . d . . . . . . s . . . . s . v e k . . m . . . . . . v . . . f . . . . . . k . t . k . . . . n . . q q . . . . . l . . a s . . . . . . . . i . . k e n / 7 / 9 5 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . u g a / 9 / 9 5 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 / 9 6 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 7 / 9 6 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 1 / 9 6 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . n l . d . k g k . . . n . . . . . . . . . . . . . . . . . . . . i . . e r i / 1 / 9 8 . . . . . . . . d . . . . . . s . . . . n . q e g . . k . . e . . . . . . . . . . . . . . k . s . . . . . . . . . k . . . . . . . . . a s . . . . . . . . i . . e r i / 4 / 9 8 . . . . . . . . d . . . . . . s . . . . n . q e g . . k . . e . . . . . . . . . . . . . . k . s . . . . . . . . . k . . . . . . . . . a s . . . . . . . . i . . k e n / 7 a / 9 8 . . . . . . . . d . . . . . . . . . . . t . . a a . . . . . . . . . . . . . . . . . . . . k . t . a . . . . . . . . . . . . . . . . . . . . . . . . . . . i . . k e n / 1 6 / 9 8 . . . . . . . . d . . . . . . s s . . . s . v e k . . m . . . . . . v . . . f . . . . . s k . t . n . . . l . . . q h . . . . . l . . a s . . . . . . . . i . . u g a / 1 9 / 9 8 . . . . . . . . d . . . . . . . . . . . s . r n . . . i . . . . t . . . . . . . . . . . . k . s . a . . . . . . . . . . . . . . . . . . . . h . . . . . . i . . u g a / 2 8 / 9 8 . . . . . . . . d . . . . . . . . . . . s . r n . . . i . . . . t . . . . . . . . . . . . k . s . a . . . . . . . . . . . . . . . . . . . . h . . . . . . i . . 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 v g k h k h v f w q p n g a p r t t q l g d n p m v l s r n n v t r f a i p f t a p h r l l s t v y n g e c e y t k t v t a i r g d r e v l a q k y s s a k h s . . t . t r . y . . . . . . . . . . p . . . . . . . f a h . g . . . . . . . y . . . . . . . a . m . . . . . k . . d r . s . . . . . . a . . . a . . a d s r . t . . e . a r . . . . . . . . . . . n e . . e . . . . f . h . k . . . . . . . y . . . . . . . . . . . . . . . a . s . p . s . . . . . . q a . . a . . a . g r . t . . t . t r . y . . . . . . . . a . a . . . . . . . f a h . g . . . . . v . y . . . . . . . a . r . . . . . k . . d r . . . . . . . . a . . . a . . a . s r . a . . e . a r . . . . . . . . . . . n e . . . . . . i f . h . k . . . . . . . y . . . . . . . . . . . . . k . a . s . p . s . . . . . . q a . . a . . a . g g . t . . e . a r . . . . . . . . . . . n e . . . . . . . f . h . k . . . . . v . y . . . . . . . . . . . . . . . a . s . p . s . . . . . . q a . . a . . a . g r . t . . e . . r . . . . . . . . . . . . v . . . . . . . f a h . g . . . . . . . y . . . . . . . . . . . . . . . . . n . . s n p . . . . . a . . . a . h k d v a . . . . e . . r . . . . . . . . . . . . v . . . . . . . f a h . g . . . . . . . y . . . . . . . . . . . . . . . . . n . . s n p . . . . . a . . . a . . k d v a . . . . . . t r . . . . . . . . . . . . t . . . . . . . f a h . r . . . . . . . y . . . . . . . a . r . . . . . k . . d r . s . . . . . . a . . . a . . a d s r . t . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . . . t r . . . . . . . . . . . . t . . . . . . . f a h . r . . . . . . . y . . . . . . . a . r . . . . . k . . d r . s . . . . . . a . . . a . . a d s r . t . . t . . r . y . . . . . . . . . . v . . . . . . . f a h . g . . . . . . . y . . . . . . . a . . . . . . . r . . d k . . . . . . . . a . . . a . . a d s r . a . . . . t r . . . . . . . . . . . . t . . . . . . . f a h . r . . . . . . . y . . . . . . . a . r . . . . . k . . d r . s . . . . . . a . . . a . . a d s r . t . . . . t r . . . . . . . . . . . . t . . . . . . . f a h . r . . . . . . . y . . . . . . . a . r . . . . . k . . d r . s . . . . . . a . . . t . . a d s r . t . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . s r . y . . . . . . . . . . . . . . . . . . f a h . g . . . . . v . y . . . . . v . a . r . . . . . k . . d r . s p . . . . . a . . . a . . a d s r . . . . e . t r . y . . . . . . . . . . . . . . . . . . f a h . g . . . . . v . y . . . . . v . a . r . . . . . k . . d r . s p . . . . . a . . . a . . a d s r . . . . e . t r . y . . . . . . . . . . . . . . . . . . f a h . g . . . . . v . y . . . . . v . a . r . . . . . k . . d r . s p . . . . . a . . . a . . a d s r . . . . e . . r . . . . . . . . . . . . t . . . . . . . y . h . g . . . . . . . y . . . . . . . . . . . . . . . d . k . a s . . . . . . . a . . . a . . a n t . . t . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . t . . r . y . . . . . . . . . m t . . . . . . . f a h . g . . . . . . . y . . . . . . . a . . . . . . . k . . d r . s . . . . . . a . . . a . . a e s r . t . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . s . s . . . . v . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . t . t r . y . . . . . . . . a . a . . . . . . . f a h . g . . . . . v . y . . . . . . . a . r . . . . . n . k d r . s . . . . . . a . . . a . . a . s r . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . e . . r . . . . . . . . . . . . . . . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . d . t r . . . . . . . . . . . . . . . . . . . . y a k g g . . . . . . . . . . . . . . . . . . . . . . . t . . . . a . . . . . . . a a . . a . . a d n v . t . . d . t r . . . . . . . . . . . . . . . . . . . . y a k g g . . . . . . . . . . . . . . . . . . . . . . . t . . . . a . . . . . . . a a . . a . . a d n v y t . . e . . r . . . . . . . . . . . p k f . . . . . . f . h k k . . . . . . . . . . . . . . . . . . . . . . . k . . e k t i . . . . . . a . . . . . . a . t . . a . . t . . r . y . h . . . . . . . . t . . . . . . . f a h . g . . . . . . . y . . . . . . . a . . . . . . . k . . d r . s . . . . . . a . . . a . . a e s r . t . . d . . c . . . . . . . . . . . v . . . . . . . . f . h . g . . . . . . . . . . . . . . . . . . . . . . . d . n s k . . . . . . . . q . . q . . . a . t r . a . . d . . r . . . . . . . . . . . v . . . . . . . . f . h . g . p . . . . . . . . . . . . . . . . . . . . . d . n s k . . . . . . . . q . . q . . . a . t r . a 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 a l p s t f n f g f v t a d k p v d v y y r m k r a e l y c p r a l l p a y t h a g g d r f d a p i g v a k q l l . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . d . s d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d . . n r . . . . . . . . . e . . . h . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . n d r . . . . . . . . . e . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d . . n r . . . . . . . . . e . . . h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . d . . n r . . . . . . . . . e . . . h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . s . n . r . . . . . . . . . e . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . s . n . r . . . . . . . . . e . . . c . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . g n r . . . . . . . . . e . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . h . . . . n . . . . . . . . . . . . . . . . . p . . . . . q . g n r . . . . . . . . . e . . . c . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . g d r . . . . . . . . . e . . . c . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . g n r . . . . . . . . . e . . . c . . . r . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . g n r . . . . . . . . . e . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . h . . . . r . . . . . . . . . . . . . . . . . p . . . . . q . n n r . . . . . . . . . e . . . c . . . . . . . . h . . . . r . . . . . . . . . . . . . . . . . p . . . . . q . n n r . . . . . . . . . e . . . c . . . . . . . . h . . . . r . . . . . . . . . . . . . . . . . p . . . . . q . n n r . . . . . . . . . e . . . . . . . . . . . . h . . . . a . . . . . . . . . . . . . . . . . . . . . . . d . v . r . . . . . . . . . e r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . g i r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . y . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . n n r . . . . . . . . . k . . v c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . q . . . . . . . . v . . . . . . . . . . . . . . . . . . . p . . . . . d . . s r . . . . . . . . . e r . t . . . q . . . . . . . . v . . . . . . . . . . . . . . . . . . . p . . . . . d . . s r . . . . . . . . . e r . t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . q d r . . . . . . . . . e . . . . . . . . . . . . h . . . . q . . . . . . . . . . . . . . . . . p . . . . . q . g i r . . . . . . . . . e . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . d . k n r . . . . . . . . . e r . . y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . d . k n r . . . . . . . . . e r . . s f ig . 2 d e d u ce d a m in o a ci d a lig n m e n t o f v p 1 o f th e n e w ly s e q u e n ce d is o la te s p e rf o rm e d in t h is s tu d y (t a b le 1 ) 295 m. sahle et al. from rwanda made during 2000 (rwa/1/00), while genotype i expanded with several more isolates from kenya and uganda in addition to demonstrating that this genotype had circulated for 41 years (1957–1998). genotype j, that previously contained only one isolate from the drc made during 1982 (zai/1/82), had two more isolates from uganda 1998 (uga/19/98 and uga/28/98) and although these differed by 22 % from the former, the cluster was supported by a bootstrap value of 95 %. the within genotype variation based on pair-wise comparisons was up to 26 % while between genotype differences varied from approximately 30–44 %. closely related viruses, with sequence homology of > 94 %, caused a number of outbreaks between 1984 and 1998 in kenya and uganda, suggesting a direct epidemiological association between these outbreaks and possible long-term conservation (geno type i, lineage i; fig. 1). a historical isolate made in 1957 in kenya (ken/3/57) clustered within this genotype with a high bootstrap support, but differed by 24 % from the former cluster. in addition, three outbreaks caused by isolates from lineage iii within genotype b had occurred in kenya between 1976 and 1995 (ken/2/76 and ken/3/95), 1984–1987 (ken/1/84, ken1/86, ken1/87, ken2/87) and 1991–1999 (ken/33/91, ken/16/98, ken/4/99, ken/5/99, ken/7/99, ken/9/99). while the isolates obtained within an outbreak differed by less than 2 % from each other, the distances between these outbreak clusters were 9–13 % and each cluster was supported by bootstrap of 100 %. outbreaks from tanzania during 1975 (tan/1/75) and 1986 (tan/1/86) as well as from malawi during 1975 (mal/3/75) clustered within genotype b and seemed to be related to the ongoing outbreaks in the eastern african region based on a bootstrap support of 99 % (fig. 1). uganda suffered from unrelated outbreaks between 1975–1976 (genotype l; uga/51/75, uga/3/76, uga/8/76) and 1998 (genotype j) bringing the total number of unrelated outbreaks to three, all from different genotypes. the introduction into rwanda during 2000 was unrelated to any of these previous outbreaks in neighbouring countries. during 1990– 1991 ethiopia had at least two separate introductions, with one isolate clustering within genotype n (eth/2/91; lineage i) and eth1/90, eth/2/90 and eth/1/91 clustering within genotype b, lineage iii. a single isolate from burundi, made in 1991 also grouped in genotype b, indicated that this genotype had been circulating in east and southern africa for 24 years. genetic variability and distribution of mutations the invariable sites over the 1d gene for all the isolates included in the study were 236/638 (37 %), with 56 % (358/638) parsimoniously informative sites and 54 singletons. for the deduced amino acid sequences, the invariable sites were 42 % (91/216), 43 % (93/216) parsimoniously informative sites and 32 singletons occurred. amino acid variability was plotted to determine whether mutations were randomly distributed or localized to specific regions of the vp1 gene. the result of the amino acid hypervariability plots of 67 isolates from africa indicated the hyper-variable regions were located at amino acid positions 45–50, 107–111, 135–141 and 148– 160 (within the g-h loop) as well as 198–202, the c-terminal part of the protein. a putative hyper-variable site was also identified at positions 21–28, which corresponds with a t-cell epitope identified on o kaufbeuren (collen, dimarchi & doel 1991) and was also recognised as hyper-variable for sat-1 (sahle, dwarka, venter & vosloo 2007). when comparing only the newly generated deduced amino acid sequences of the 48 east african isolates, the rgd cell attachment site of the virus at amino acid positions 144–146 within the g-h loop was completely conserved across all isolates (fig. 2). the c at the base of the 1d loop (position 134) was maintained as well as the r at position +1, the i at position –1 and the l at position +4. of the previously described neutralisation sites identified by monoclonal antibodies at positions +2, +3 and +10 and +12 (crowther, rowe & butcher 1993; bastos et al. 2003b), only +3 was moderately conserved with three (v/a/l) options, while at positions +2 and +10, five different amino acids occurred and at position +12 seven differences were found (fig. 2). previously bastos et al. (2003b) found that for representative isolates from sub-saharan africa, but with few of them being from east africa, the +2 and +3 sites showed moderate levels of variation, while the +10 and +12 sites showed high levels. for the east african isolates, only the +3 site showed moderate variation. the vp1/2a cleavage site contained predominantly amino acid sequences vp1(k/r)q/ 2a(l/t/v)(l/c/h/s/y) with the q at the cleavage site conserved over all isolates. discussion phylogenetic analysis has been of great benefit in determining possible origins of fmd outbreaks, interspecies transmission, tracing spread of virus over vast distances and ultimately to better understand 296 genetic heterogeneity of sat-2 fmd virus in sub-saharan africa the epidemiology of the disease in sub-saharan africa (vosloo et al. 1992; dawe et al. 1994a; vosloo et al. 1995; bastos et al. 2000; bastos 2001; bastos, haydon, forsberg, knowles, anderson, bengis, nel & thomson, 2001; sangare, bastos, marquardt, venter, vosloo & thomson 2001; bastos, anderson, bengis, keet, winterbach & thomson 2003a; bastos et al. 2003b; sangare, bastos, venter & vosloo 2003; sahle, venter, dwarka & vosloo 2004; sangare et al. 2004; vosloo & thomson 2004; vosloo et al. 2006). sat-2 isolates from east africa have not been studied in detail and compared to those obtained from other regions to better understand and assess the molecular epidemiology of sat-2 in sub-saharan africa. the phylogeny has expanded with three new genotypes identified in east africa, bringing the total number to eight belonging to two different lineages. the previously identified lineages (bastos et al. 2003b; sangare et al. 2004) could not be followed in this study, as the inclusion of more isolates has altered the structure of the phylogeny at that level, albeit not on genotype level. only three lineages were assigned in this study that covered east africa and south-western africa, one consisting solely of west african isolates and the third from east and southern africa. these linkages between different geographical regions of the subcontinent demonstrate clearly the potential for fmd virus to disperse over considerable distances and emphasize the need to investigate the main factors which play a role in exchange of subtypes of the virus between countries and its spread within and between regions. transboundary transmission of the disease due to animal movement is possible as a number of countries share common boundaries and animal trading across borders is common practice (ndiritu 1984). added to this, the population of susceptible hosts for fmd in east african countries is high [the cattle and sheep population were estimated to be 57.6 and 98.9 million, respectively (mcdermott & arimi 2002)], and can easily maintain cycles of fmd epizootics. the livestock and the livestock production systems, illegal trading of animal and animal products as well as the presence of cloven-hoofed wild animals in the region favour the transmission of disease between neighbouring countries and could lead to endemic cycles. a study performed in west africa indicated clearly that the role of sheep and goats in the epidemiology of fmd is not fully understood either due to a real low prevalence of disease or, more likely, because clinical disease is not apparent and the importance of these species is overlooked (bronsvoort, tanya, kitching, nfon, haman & morgan 2003). in sudan it was shown that sheep and goats play an important role in the epidemiology based on serological studies following natural infection (abu elzein, newman, crowther, barnett & mcgrane 1987). cross-border transmission was aptly demonstrated where an outbreak in saudi arabia was shown to cluster with three isolates previously obtained from eritrea which was possibly due to spread of virus to saudi arabia arising from trade in livestock between the two countries (bastos et al. 2003b). similarly rare incursions of sat-1 into the middle east have been recorded (knowles & samuel 2003). within a geographical region such as east africa, cross border movement most probably leads to dissemination of disease between various countries sharing borders. more surprising was the demonstration that isolates from angola and drc clustered with the east african lineage i which is supported by a high bootstrap value. however, due to the low numbers of isolates available from central and south-western africa, it is not possible to determine whether these were accidental introductions over large distances, or whether there are indeed similar isolates circulating within this geographical region. a total of 14 genotypes were identified in sub-saharan africa. of these, six may be extinct (e, f, k, l, m and n) as no isolates similar to those included in this study have been found since 1996. however, in endemic areas the disease is often not reported nor material submitted for further investigation, implying that the exact distribution and occurrence of serotypes is not known. bronsvoort et al. (2003) found by using questionnaires that the prevalence may be up to 58 % in specific regions of mali but outbreaks are not reported to veterinary services. investigations into more recent isolates may prove that these and new genotypes are currently circulating within subsaharan africa. east africa demonstrates the most variation of all regions in sub-saharan africa with at least eight genotypes in two lineages consisting almost exclusively of cattle isolates, compared to southern africa where three genotypes have been described (bastos et al. 2003b), the latter consisting mostly of buffalo isolates. previously, bastos et al. (2003b) found that the highest rates of nucleotide substitution for sat-2 groups were those that were recovered from cattle, while the lowest rates were recovered from wildlife. they speculated that these different rates could have been due to a higher number of cases during cattle outbreaks resulting in more virus replication and more opportunity for divergence although 297 m. sahle et al. their study could not support this assumption statistically. from the data included in this study, it is clear that within east africa at least, more variation is observed, giving credence to the fact that the disease is most probably maintained by livestock. other factors cannot be excluded in generating this diversity. the role of buffaloes in these regions is largely unknown, and more studies into the presence of sat serotype viruses in buffaloes in the area could provide an explanation. in addition, the role of other wildlife species is also not clear. in southern africa and in the knp in particular, it has been shown that impalas can play an important role in transmitting disease to other species (vosloo et al. 2006). in addition to transboundary movement of livestock which could spread disease, introduction of strains due to vaccine cannot be excluded. the latter could explain the long term conservation of genotypes observed over extended time periods. a number of east african countries have had separate incursions of disease belonging to different lineages and genotypes such as kenya, ethiopia and uganda. these genetic differences lead to antigenic differences (vosloo, dwarka, bastos, esterhuysen, sahle & sangare 2004) and have an important bearing on the use of vaccination to control the disease. cross neutralisation studies have shown that even within a genotype of sat-2, the antigenic relationships are sufficiently poor to warrant specific vaccines strains and there will probably be no protection between genotypes (vosloo et al. 2004). countries will have to consider the strains and genotypes included in vaccines to ensure that vaccination will be effective. these differences could also have a negative impact on diagnostic tests relying on antigenic relationships between viruses and should be considered when diagnoses are required. in contrast tanzania, rwanda and eritrea had outbreaks caused by single genotypes, but this could be due to under representation, rather than a true reflection of the current status. the seven serotypes of fmd virus cluster into lineages that differ by approximately 30–50 % over the 1d gene (knowles & samuel 2003). in this study it was observed that lineages differ by up to 44 % from each other, nearly as much as was found for serotypes, indicating the high level of mutation found in sat-2. a similar level of variation was observed for sat-1 when isolates from over sub-saharan africa were investigated (sahle et al. 2007). knowles & samuel (2003) suggested that variation of up to 20 % could be used for inclusion into a sat topotype. however, it was found in this study that certain genotypes that corresponded to geographical locations (ie topotypes) had up to 26 % within group variation (genotype d) when pairwise comparisons were performed and it seems plausible that these cut-off levels should be redefined, especially for the sat types. the hyper-variable regions of the east african isolates compared to those identified for sat-1 and sat-2 with 135–141 and 148–160 corresponding to the βg-βh loop (bastos et al. 2001; bastos et al. 2003b; vosloo et al. 2006; sahle et al. 2007). sites 107–111 correspond to the βf-βg loop shown to be hyper-variable for sat-1 and sat-3 (bastos 2001; bastos et al. 2003a, b), while 45–50 correspond to the βb-βc loop identified on o1 bfs (acha raya, fry, stuart, fox, rowlands & brown 1989) and shown to be hyper-variable for sat-1 (vosloo et al. 2006; sahle et al. 2007). this high level of variation around the immunologically important gh loop which also plays a role in cell receptor recognition (logan, abu-ghazaleh, blakemore, cur ry, jack son, king, lea, lewis, newman & parry 1993) could have a severe impact on the efficacy of vaccines. as was demonstrated in previous studies investigating sat-2, the arginine at position 148 was conserved in all isolates investigated. the role of this change from leucine (as in most other serotypes) to arginine is not clear as the leucine has been shown to stabilize alpha helix formation (france, piatti, newman, toth, gibbons & brown 1994). this study adds to our understanding of the molecular epidemiology of sat-2 fmd isolates in subsaharan africa and demonstrates clearly that control of this disease should be seen as a regional priority due to the virus’ ability to spread over vast distances. it also indicates that our understanding of the factors that lead to the generation of variants, disappearance and re-emergence of strains and regional patterns is inadequate and that more research is needed to ensure better prediction of disease emergence and effective control policies. acknowledgements we thank n. ferris of the institute for animal health, pirbright, uk for supplying the majority of virus isolates for this study. we are also very grateful to all technical staff of the exotic diseases division of the onderstepoort veterinary institute for their technical assistance. particular thanks must be expressed to a.d.s. bastos, k. boshoff, h.g. o’neill and f.f. maree for valuable discussions. the work was supported by intervet international. 298 genetic heterogeneity of sat-2 fmd virus in sub-saharan africa references abu elzein, e.m.e., newman, b.j., crowther, j.r., barnett, i.t. & mcgrane, j.j. 1987. the prevalence of antibodies against foot-and-mouth disease in various species of sudanese livestock following natural infection. revue d’élevage et de médecine vétérinaire des pays tropicaux, 10:7– 12. acharya, r., fry, e., stuart, d., fox, g., rowlands, d. & brown, f. 1989. the 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great britain, horizon bioscience. vosloo, w., bastos, a.d.s. & boshoff, c.i. 2006. retrospective genetic analysis of sat-1 type foot-and-mouth disease outbreaks in southern africa. archives of virology, 151: 285–289. palmer_299-314.indd introduction outbreaks of pest blackflies (diptera: simuliidae) have been one of the most serious problems affecting agriculture and tourism along the middle and lower orange river following the completion of the gariep and vanderkloof dams in the late 1970s (palmer 1997; myburgh & nevill 2003). the blackfly problem is attributed mainly to high winter flows, which provide suitable habitat for over-wintering blackfly larvae. the main pest species is simulium chutteri, although there are times when simulium damnosum and simulium impukane are problematic. adult female blackflies usually need a blood meal to complete the development of eggs, and their large numbers and tendency to crawl into ears, noses and eyes, make them problematic to livestock and people. all outdoor activities are seriously affected, particularly stock farming, irrigation farming, river rafting and other tourist activities. chemical control of pest blackfly in south africa began in the 1960s with the use of ddt, a wide-spectrum larvicide (howell & holmes 1969). in 1991 the 299 onderstepoort journal of veterinary research, 75:299–314 (2008) evaluation of larvicides in developing management guidelines for long-term control of pest blackflies (diptera: simuliidae) along the orange river, south africa r.w. palmer1* and n.a. rivers-moore2 abstract palmer, r.w. & rivers-moore, n.a. 2008. evaluation of larvicides in developing management guidelines for long-term control of pest blackflies (diptera: simuliidae) along the orange river. onderstepoort journal of veterinary research, 75:299–314 in 2000 and 2001 orange river levels were higher than normal: associated serious outbreaks of blackfly had a substantial detrimental impact on the local economy. the poor control was attributed to the suspected development of larval resistance to temephos. a long-term solution to blackfly control, through the identification of a suitable replacement to temephos for use during high flow conditions, was proposed. this study, however, failed to identify or register a suitable larvicide for use during high flow conditions. although permethrin was highly effective against blackfly larvae, it was rejected because of its detrimental impacts on non-target fauna. various formulations of locally produced dry bacillus thuringiensis var. israelensis (b.t.i.) were tested, but these were ineffective against blackflies. the study also confirmed that resistance to temephos has developed among simulium chutteri in the middle and lower orange river. the feasibility of “reversing” the resistance to temephos through the use of the synergist piperonyl butoxide (pbo) was investigated, but the results were not favourable. furthermore, pbo was highly toxic to blackflies and non-target organisms, and was not recommended for further testing. this means that b.t.i. currently remains the only symptomatic measure of treatment currently applied. although resistance to b.t.i. has not been reported for blackflies elsewhere in south africa, there is a need to remain vigilant and to implement an operational strategy that minimizes the risks of resistance developing. keywords: larvicide trials, orange river, pest blackflies, resistance, simuliidae ∗ author to whom correspondence is to be directed. e-mail: rob@nepid.co.za 1 nepid consultants, p.o. box 4349, white river, 1240 south africa 2 institute for water research, p.o. box 94, rhodes university, grahamstown, 6140 south africa accepted for publication 14 august 2008—editor 300 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa department of agriculture initiated a programme to control these pests along the middle and lower orange river (palmer 1997). the programme was initially based on aerial applications of two larvicides, namely temephos, an organophosphate, with abate® 200ec being the only product registered for use in south africa, and the bacterium bacillus thuringiensis var. israelensis (b.t.i.), with teknar® hp-d and vectobac® 12as being two products registered for use in south africa. the bacterial larvicides, which contain protein toxins produced by the naturally occurring soil bacterium b.t.i. are generally the preferred method of blackfly control because of their high target specificity and low impact on the environment. however, these products are relatively bulky and are suitable for use when flows in the orange river are moderate to low (< 100 m3/s). at river levels exceeding 300 m3/s it becomes difficult to airlift the volumes needed to treat the orange river effectively. under these conditions it is preferable to use abate® 200ec, an organophosphate that is more concentrated and easier to airlift than b.t.i. between 1991 and 1999 the control programme was supported by research conducted by the onderstepoort veterinary institute with funding from the water research commission and the red meat producers organization. the research aimed to ensure that the control programme was both effective and environmentally safe (palmer 1997; myburgh 1999). the con trol programme was highly effective during this period, but research was discontinued in 1999. serious outbreaks of blackflies were experienced in 2000 and 2001 as a result of river levels being higher than normal, and this led to appeals from organized agriculture for the problem to be rectified, and for a long-term solution be found. the aim of this study was to find a suitable larvicide for use against blackflies in the orange river during high flow conditions. the findings of the dispersal properties of various products and their toxicity to blackfly larvae and common non-target organisms, using flowthrough gutters and field trials are described. materials and methods study area the main study area for this project was the orange river downstream of vanderkloof dam (fig. 1). the great fish river near grahamstown was also used to conduct trails to investigate larval resistance to temephos, as the river contains populations of the main pest species, s. chutteri, which had not previously been exposed to temephos. alternative larvicides were tested in the orange river at upington, and in various rivers near grahamstown, in the eastern cape province. the latter were chosen because of the availability of mean daily flow data, and their small size which reduced the volume of larvicide needed. criteria for evaluation the criteria used to evaluate the larvicides were based on a hypothetical ideal larvicide which should have the following characteristics: • effective against blackflies and target specific • easy to airlift and apply • disperses rapidly and evenly in water fig. 1 map of the middle and lower orange river downstream of van der kloof dam (formerly pk le roux dam), with the main blackfly problem area highlighted in grey 301 r.w. palmer & n.a. rivers-moore • carries for long distances downstream • good shelf life • cheap • safe to handle • without problems of cross resistance with temephos. larvicides selected for evaluation this study was limited to investigating currently available commercial products for blackfly control, as it was well beyond its budget and scope to investigate new products for development. the study relied largely on the experience of the world health organ ization’s onchocerciasis control programme in west africa (ocp). the ocp has tested over 50 larvicides and hundreds of formulations for use against pest blackflies since it inception in 1974 (kur tak, back, chalifour, doannio, dossou-yove, duval, guillet, meyer, ocran & wahle 1989). most of the tests were conducted during the early phases of the ocp. the ocp was discontinued in 2002 (lévêque et al. 2003), and since then very few investigations of new products for blackfly control have been undertaken. the larvicides that were most commonly used by the ocp are listed in table 1. this provided a good starting point for identifying a suitable larvicide for use in the orange river. the use of alternative organophosphates was not considered because of the possible cross-resistance with temephos, while the use of carbamates was not considered because of the high impacts on non-target fauna. the most suitable candidate was therefore one of the synthetic pyrethroids, a group of powerful broadspectrum insecticides (mueller-beilschmidt 1990) which act as neurotoxins. the two pyrethroid compounds that were commonly used in west africa were permethrin and etofenprox. although permethrin is more toxic to non-target aquatic fauna than etofenprox, it has a lower mammalian toxicity. permethrin is particularly suited for large rivers and was considered to be the most suitable candidate larvicide for testing in south africa. while synthetic pyrethroids generally degrade to varying degree by exposure to light, permethrin is a newer, light-stable pyrethroid (mueller-beilschmidt 1990). several agrochemical companies were contacted for samples of permethrin, or similar products to replace temephos, for trial purposes. the only company to express an interest in supplying alternative products to temephos was wefco marketing. the other companies approached indicated that they were not prepared to take the risk, mainly because of legal implications and public concerns about environmental safety. wefco marketing suggested using piperonyl butoxide (pbo) in combination with temephos, as piperonyl butoxide combined with temephos or permethrin has the ability to “reverse” the table 1 main characteristics of the blackfly larvicides used by onchocerciasis control programme in west africa. [data from dr jean-marc hougard, institut de recherche pour le développement, france] family organophosphates pyrethroids carbamate bacteria common name temephos phoxim1 pyraclofos permethrin etofenprox carbosulfan b.t. h-14 formulation ec2 ec ec ec ec ec wd3 % active ingredient 20 50 50 20 30 25 < 2 toxicity class4 iii ii ii ii iii ii iii toxicity against ntf5 low medium medium high medium high low dose6 1507 150 120 45 60 120 500 average carry at (km) 10 m3/s 12 3 unused unused unused unused 1.5 100 m3/s 16 5 18 7 6 9 5 300 m3/s 20 unused 23 8 8 unused unused 1 chlorphoxim until 1991 2 emulsion concentrate 3 water dispersible 4 according to the who (1988) classification of active ingredient: ii, moderately hazardous; iii, slightly hazardous 5 toxicity against non-target fauna according to the criteria of the ecological group 6 in mℓ of formulation per m3/s 7 300 mℓ in clear water 302 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa development of resistance (jones 1998). larvicide synergy is a useful approach since the combined exposure of two or more larvicides causes more adverse effects than the sum of their individual effects (cox 1998). this opened the possibility of continuing with the operational use of temephos, but trials were needed on blackflies from non-resistant populations, such as the great fish river, as well as on presumed resistant populations in the orange river. wefco marketing supplied small quantities of four formulations of permethrin and three formulations of pbo for initial trials, while basf supplied small quantity of abate® 200ec. another possible option was the use of a powdered formulation of b.t.i., which would be lighter and therefore more easily air lifted than the standard liquid concentrate, particularly during high flow conditions. although the operational application of powdered formulations of b.t.i. could be a problem because of wind and equipment available, the powder could be mixed with water prior to application. plant health products supplied small quantities of two locally pro duced powdered and granular b.t.i. formulations for initial testing, which were toxic to mosquitoes (m. mor ris, personal communication 2005). the products and formulations tested during this study are listed in table 2. dispersal properties the dispersal properties of the various products were observed by mixing the product with various volumes of water and applying this mixture to a jar of standing clear water. this simple test provided a rapid visual indication of how the product is likely to behave when applied in a river and was used as the first step in screening potential larvicides. for m u lations that were buoyant or sank to the bottom were immediately rejected. viscosity viscosity of b.t.i. products can be a serious problem, so the relative viscosity of the b.t.i. was measured by filling a small (125 mℓ) cup containing a small hole (approx. 3 mm in diameter), and timing the cup to empty. this was performed four times for each of two b.t.i. concentrations (8 and 24 g/ℓ) plus a control of 0 g/ℓ. gutter trials gutter trials were conducted in the great fish river at the pigott’s bridge gauging weir (q9h012), 40 km from grahamstown (33°05’53.5” s; 26°26’42.2” e). this site was chosen because of accessibility, the weir provided sufficient head that allowed water to be gravitated into the gutters, and there were high populations of s. chutteri within close proximity to the trial site. the trials were conducted using a flowthrough four-gutter system in which river water gravitated from the gauging weir. the gutters were 3.45 m long and 0.15 m wide and were made of a galvanized tin alloy (fig. 2). blackfly larvae were obtained from the main river (5 min walk away) by cutting lengths of cyperus reeds which were trailing in fast current. twenty reeds were placed in each gutter and wedged in position using 0.20 m lengths of dowels. larvae were given at least 1.5 h to settle table 2 products and formulations tested during this study for potential use for blackfly control active ingredient class formulation suppliers b.t.i. bacterial toxin dry powder 500:5001 dry powder 500:2502 slow-release granules plant health products permethrin pyrethroid larvex™ 0.5 % w/v rd 95/a 200 g/ℓ rd 95/b 200 g/ℓ permethrin (unlabelled) wefco marketing piperonyl butoxide synergist rd 96a rd 96/b pbo (unlabelled) wefco marketing temephos organophosphate abate® 200ec sa cyanamid basf wefco marketing 1 formulation consists of 500 g b.t.i. fermentation broth plus 500 g carrier, mixed and dried down 2 formulation is twice as concentrated as the 500:500 dry powder, and consists of 500 g b.t.i. fermentation broth plus 250 g carrier, mixed and dried down 303 r.w. palmer & n.a. rivers-moore before exposure to chemical larvicides, whereas for b.t.i. trials larvae were given an 1.5 h to settle. the only species of blackfly that was present was s. chutteri. flow in each gutter was determined prior to each application by holding a container (11.8 ℓ) at the exit of each channel and timing it to fill. larvicide was applied over 10 min in the header chamber at the top end of each gutter to ensure homogenous mixing, using a 60 mℓ syringe. water temperature at the time of each application was recorded. larval mortality was assessed 1 h after application for chemical products and 24 h for b.t.i. products. the assessment was based on the abundance of live larvae in an untreated control gutter and compared with the abundance of live larvae in treated gutters. abundance of larvae was based on a 10-point visual ranking method described by palmer (1994). the abundance ratings were converted to population densities to determine efficacy, although this method is unlikely to detect mortalities less than 50 %. at least 15 counts were made per gutter. field trials belmont valley field trials were undertaken at two sites in the belmont valley near grahamstown (site 1: 33°19’25.2” s; 26°36’00.8” e; site 2: 33°19’21.4” s; 26°36’49.7” e). the stream was chosen because of its small size, close proximity to grahamstown and high populations of blackflies. a road bridge crosses the stream and culverts were used to measure the stream flow using equations 1a–c (gordon et al. 1992). q = 1000va [1a] a = 0.5r2(θ–sinθ) [1b] θ = 2 cos–1(r – d) [1c] r where q = discharge in ℓ/s; v = average current speed in m/s; a = cross-sectional area in m2; θ is in radians. the only species of blackfly that was present at site 1 was the pest species simulium nigritarse, but simulium adersi was also present further downstream at site 2. larvicide was applied with a 1 ℓ hand-held garden sprayer over a period of 8 min. water temperature at the time of the application was recorded. larval mortality was assessed 3 h after application for chemical trials and 24 h after application for b.t.i. trials. the assessment of efficacy was based on the abundance of live larvae on the stones-in-current in an untreated stretch of stream compared to the abundance of live larvae in the treated section, before and after application. abundance of larvae and assessment of efficacy was based on the same method as used for the gutter trials, and sample size was also at least 15 stones or reeds. buffalo river a field trial was undertaken in eastern cape province in the buffalo river at a gauging weir near king willi am’s town (r2h010 – 32°56’26.5” s; 27°27’41.3” e). the site was chosen because of the high populations of blackflies and close proximity to an accurate gauging weir. the most common species of blackfly that was present was simulium hargreavesi, but other species present were simulium vorax, s. nigritarse and s. damnosum. larvicide was again applied with a 1 ℓ hand-held garden sprayer over a period of 8 min. water temperature at the time of the application was recorded. larval mortality was assessed 3 h after application. the assessment of efficacy was based on the abundance of live larvae in the stonesin-current in before and after application. abundance of larvae was based on the same method as used in the gutter trials. at least 15 counts were made. orange river field trials were conducted in the orange river at upington and kanoneiland in august 2005 and october 2006. for the earlier trials, the same methods as those described above were used. the most common species present was s. chutteri, although s. damnosum was also present. in the latter field trials, two formulations of abate® 200ec were applied by boat, one to each of two channels on either side of the orange river at kanoneiland. fig. 2 flow-through gutter system used for larvicide trials on s. chutteri larvae 304 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa impacts on non-target organisms impact of permethrin on non-target organisms was evaluated during selected field trials. population densities of aquatic invertebrates were estimated before and after application using the visual method of assessment developed for estimating blackfly pop ulations (palmer 1994). results dispersal properties initial screening of the physical behaviour of the products in water showed that permethrin and piperonyl butoxide formulations dispersed well. of the formulations tested, permethrin rd95b dispersed table 3 viscosity (mℓ/s) for tap water and two concentrations of b.t.i. (500: 250) sample flow time (s): 0 g/ℓ (125 mℓ tap water) flow time (s): 8 g/ℓ (1 g in 125 mℓ) flow time (s): 24 g/ℓ (3 g in 125 mℓ) 1 2 3 4 37.9 38.5 36.8 38.5 39.4 37.3 37.5 36.8 38.1 37.9 37.6 37.4 mean 37.9 37.8 37.8 flow rate (mℓ/s) 3.3 3.3 3.3 table 4 conditions of application and results of flow through gutter trials to test the toxicity of permethrin rd 95/b to blackfly larvae at pigott’s bridge, great fish river trial no. date water temp. (°c) gutter channel flow (ℓ/s) dosage (mg/ℓ) efficacy (%) 1 05.06.05 11.5 1 2 3 4 1.23 1.07 1.28 1.01 0.01626 0.03738 0.04688 0.09901 98 99 99 100 2 05.06.05 11.5 1 2 3 4 1.76 1.64 1.60 1.54 0.00201 0.00366 0.00833 0.01732 62 74 94 99 table 5 conditions of application and results of field trials to test the toxicity of permethrin rd95b to blackfly larvae trial no. date site water temp. (°c) flow (ℓ/s) dosage (mg/ℓ) efficacy (%) 1 03.05.05 belmont valley site 1 11.5 69 0* 0.024 0.048 0 0 63 2 03.06.05 belmont valley site 2 12.0 69 0* 0.024 0.048 0.072 0 25 88 88 3 06.06.05 buffalo river 12.0 279 0.050 99 4 07.06.05 belmont valley site 1 11.0 91 0 0.02 0 82 5 08.06.05 belmont valley site 1 10.0 70 0 0.040 0 83 6 08.06.05 belmont valley site 2 10.2 70 0 0.030 0 56 * control 305 r.w. palmer & n.a. rivers-moore the best by far. the abate® 200ec dispersed well. the density of larvex™ 0.5 % w/v was very low and floated on the surface, so this product was excluded from further testing. the standard formulation of powdered b.t.i. mixed well in water, but the more concentrated formulation was dense and settled rapidly when applied to water. viscosity the powdered formulation of b.t.i. mixed well with small quantities of water, but the concentrated formulation formed sticky lumps when mixed in larger volumes. the viscosity of the supernatant was low and not significantly different from that of tap water (student’s t-test; p < 0.05; d.f. = 3) at concentrations of 8 and 24 g/ℓ (table 3). by contrast, the viscosity of the lumps of the concentrated formulation was exceedingly high. to undertake the viscosity trials the formulation was dissolved in a separate beaker prior to draining through a cup so as to prevent the drainage hole from being blocked with lumps. larvicide trials permethrin gutter trials gutter trials conducted at pigott’s bridge confirmed that permethrin rd95b is highly effective against s. chutteri at dosages as low as 0.016 mg/ℓ (table 4). river trials river trials conducted at two sites in the belmont valley indicated that permethrin achieved between 63 % and 88 % mortality of s. nigritarse at a dosage of 0.0483 mg/ℓ (table 5). the trials assumed that flows at the two sites were the same, but the results suggest that flows at the downstream site were slightly lower, and therefore dosages higher, than upstream. a subsequent trial in the buffalo river achieved 99 % mortality at a slightly higher dosage of 0.0502 mg/ℓ (table 5). mortality curves for simulium using permethrin rd95b were constructed for both gutter trial and river trial experiments. the mortality curve for the gutter trials was significant (p < 0.05; d.f. = 8; intercept = 9.80 ± 0.75; slope = 1.50 ± 0.35), while the mortality curve for the river trials was not significant (p < 0.05; d.f. = 5; intercept = 21.83 ± 3.64; slope = 11.38 ± 2.47). the lc50 values for gutter trials (0.001 mg/ℓ) were, however, 30 times lower than for the river trials (0.033 mg/ℓ), suggesting that the effects of higher flow rates may have an impact on effective concentrations of permethrin (fig. 3). similar findings were made in west africa, and the researcher recommended the use of a closed-circuit trough system for screening conventional larvicides for blackfly control (ocran 1989). impacts on non-target organisms a series of trials were conducted to assess the target specificity of permethrin in local rivers. initial trials were conducted in the bloukrans river near grahamstown, and the buffalo river near king williams town, because of their small size, ease of logistics and ability to measure stream flows. invertebrate diversity in the bloukrans river was low prior to a trial application of permethrin in june 2005. individuals of the caddisfly macrostemum capense and gyrinidae beetles were absent from these aquatic invertebrate communities, while chironomids were present in low numbers only. the aquatic invertebrate communities were largely unaffected by applications of permethrin at concentrations of 0.02, 0.03 and 0.04 mg/ℓ (tables 6a and b). invertebrates which were affected negatively by the application of permethrin were limpets (ancylidae) and flatworms (turbellaria). the diversity of invertebrates in the buffalo river, near king williams town, was very low prior to appli cation because the site is highly polluted from domestic and industrial wastes. this meant that the fauna that were present were highly tolerant species. despite this, the application of permethrin rd95b at a concentration if 0.05 mg/ℓ had major impacts on non-target organisms, particularly water boatmen and mayflies, followed by caddisflies and fig. 3 mortality curves for simulium spp. based on applications of permethrin rd95b at different concentrations, for gutter and river trials 306 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa flatworms (table 6c). the only taxon that appeared to be unaffected was non-biting midges. the numbers of non-biting midges appeared to increase after application, but this was probably because they were small and must have been largely overlooked prior to application, whereas after application they were about the only fauna left alive, so they were more visible. the intention was to use permethrin for blackfly control in the orange river, so a trial was conducted to investigate its impacts on non-target fauna in the orange river. permethrin was applied at a concentration of 0.1 ppm from a road bridge to the left channel of the river at the kanoneiland. the composition of aquatic invertebrates was assessed before and after application at blaauwskop, 5.4 km downstream of the bridge. the application had a devastating impact on non-target fauna: 100 % mortality was recorded for various families of mayflies, damselflies, water boatmen and hydroptilid caddisflies (table 6d). prior to application the population of blackfly larvae was very high, but very few (3 %) survived the application. other taxa that were detrimentally affected included non-biting midges, limpets and two species of caddisfly that are important table 6a relative abundance of invertebrate taxa in the bloukrans river (belmont valley site 2) before and after application of permethrin at 0.03 mg/ℓ taxa application rating efficacy (%) turbellaria (flatworm) before after 221412121432211 111111112132124 99 ancylidae (limpets) before after 221212212212121 111212111111112 99 cheumatopsyche afra (caddisfly) before after 322333131341122 214122323211233 0 chironomidae (non-biting midge) before after 111111111111111 111111111111111 0 macrostemum capense (caddisfly) before after 111111111111111 111111111111111 0 baetidae (mayfly) before after 232212422322223 422234322422223 0 gyrinidae (water boatmen) before after 111111111111111 111111111111111 0 table 6b relative abundance of invertebrate taxa in the bloukrans river (belmont valley site 1) before and after application of permethrin at 0.04 mg/ℓ taxa application rating efficacy (%) chironomidae (non-biting midge) before after 111111111111111 211111111111111 n/a ancylidae (limpets) before after 133322221222232 123111322122222 0 cheumatopsyche afra (caddisfly) before after 121211112221232 121211112121212 0 turbellaria (flatworm) before after 221232222232222 222211112112221 0 macrostemum capense (caddisfly) before after 111111111111111 111111111111111 0 baetidae (mayfly) before after 112324222224232 212232222222332 0 gyrinidae (water boatmen) before after 111111111111111 111111111111111 0 307 r.w. palmer & n.a. rivers-moore table 6c relative abundance of invertebrate taxa in the buffalo river before and after application of permethrin at 0.05 mg/ℓ taxa application rating efficacy (%) gyrinidae (water boatmen) before after 211212222222222 111111111111111 100 baetidae (mayfly) before after 322323434433353 111211111111111 99 macrostemum capense (caddisfly) before after 321221211411112 111211111112111 89 turbellaria (flatworm) before after 221231212462132 111213211212124 74 cheumatopsyche afra (caddisfly) before after 333433334443443 212321131223232 69 ancylidae (limpets) before after 324112222222323 212214122122122 37 chironomidae (non-biting midge) before after 221121221222213 222232232223222 0 table 6d relative abundance of invertebrate taxa in the orange river river before and after application of permethrin at 0.10 mg/ℓ at kanoleiland, left channel, 5.4 km upstream, at discharge of 12.92 m3/s, on 14/08/2005 taxa application rating efficacy (%) baetidae (mayfly) before after 111224123231112 111111111111111 100 heptageniidae (mayfly) before after 112331112134111 111111111111111 100 leptophlebiidae (mayfly) before after 113411113114121 111111111111111 100 gyrinidae (water boatmen) before after 211112221111121 111111111111111 100 coenagrionidae (damselfly) before after 112121111111111 111111111111111 100 hydroptilidae (caddisfly) before after 112211111121111 111111111111111 100 simuliidae (blackfly) before after 81567767716677(10) 502513143331121 97 chironomidae (non-biting midge) before after 89688818877188(10) 664555555177777 82 ancylidae (limpets) before after 111241323231122 112111311111111 80 amphipsyche scottae (caddisfly) before after 331132241531312 221113211112122 63 cheumatopsyche thomasseti (caddisfly) before after 231121233331322 321311213221221 35 turbellaria (flatworm) before after 113111235311225 203411113451111 18 ecnomidae (caddisfly) before after 111111111121211 121112111111111 n/a elmidae (beetle) before after 211111111111121 111111121111111 n/a 308 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa predators of blackflies, cheumatopsyche thomasseti and amphipsyche scottae. the results were conclusive evidence that permethrin is unsuitable for use in the orange river because of its detrimental impact on non-target fauna. temephos and piperonyl butoxide gutter trials gutter trials using the abate® 200ec formulation of temephos showed that the product was effective against s. chutteri in the great fish river, but ineffective in the orange river (table 7). gutter trials conducted at pigott’s bridge, great fish river, indicated that piperonyl butoxide rd95b is highly effective against s. chutteri at dosages as low as 0.01 mg/ℓ (table 8). however, temephos (abate® 200ec) combined with pbo in various combinations showed no mortality (table 8). field trials two field trials using abate® 200ec (obtained from wefco marketing) were conducted in the great fish river, on 13 december 2005, one at coniston and one at carlisle bridge. these trials were undertaken table 7 conditions of application and results of flow through gutter trials to test the toxicity of various formulations of temephos to blackfly larvae trial no. date water temp. (°c) gutter channel flow (ℓ/s) dosage (mg/ℓ) efficacy (%) abate® 200ec (great fish river) 2* 19.07.05 12.5 1 2 3 4 0.97 1.23 1.31 1.29 0 0.010 0.050 0.100 0 0 0 50 3*** 12.12.05 22.0 1 2 3 4 0.53 0.68 0.81 0.81 0 0.050 0.100 0.300 0 40 77 90 4*** 13.12.05 25.0 1 2 3 – 1.14 0.97 0 0.080 0.150 0 22 68 5*** 14.12.05 27.0 1 2 3 1.08 1.11 1.04 0 0.05 0.50 0 65 92 abate® 200ec (orange river) 6** 02.10.06 20 1 2 3 4 1.89 1.74 1.82 1.56 0.000 0.050 0.100 0.500 0 0 0 0 7*** 03.10.06 17 1 2 3 4 1.73 dry 1.72 1.67 0.050 0 0.100 0.500 0 – 0 0 8** 03.10.06 18 1 2 3 4 1.67 1.78 dry 1.71 1.000 5.000 0 12.000 0 0 0 0 9*** 04.10.06 19 1 2 3 4 1.68 1.85 1.85 1.91 5.000 10.00 20.00 30.00 0 0 0 0 * old product supplied by sacyanamid ** new product supplied by basf *** new product supplied by wefco marketing 309 r.w. palmer & n.a. rivers-moore to confirm the results of the gutter trials, that temephos is effective in the great fish river. larval numbers prior to application were assessed at 0.2, 0.5, 1.0 and 4 km downstream of carlisle bridge, and 5.5, 6.8 and 10.8 km downstream of coniston. larval numbers were consistently high at all sites (rating between 9 and 10). water temperature at the time of application was moderate (20 °c), and the water was turbid (secchi depth 7 cm). the flow at the pigott’s bridge gauge was estimated at 5.2 m3/s, whereas the flow at carlisle bridge was assessed at 4.5 m3/s. larvicide was applied at a concentration of 0.1 ppm at coniston and 0.05 ppm at carlisle bridge. larval counts conducted the following day indicated highly successful control at both concentrations. the 0.05 ppm application from carlisle bridge achieved 97 % blackfly mortality at 4 km downstream. the 0.1 ppm application achieved 99 % mortality at cranford, 10.8 km downstream of the point of application, and larval counts at the farm mowbray, 17.7 km downstream, indicated mortality of 45 %. the results of these trials confirmed that the abate® 200ec formulation was highly effective against blackflies in the great fish river. the next step was to conduct a similar test in the orange river. a field trial using abate® 200ec was conducted in the orange river at the top end of kanoneiland on 4 october 2006. flows in the two channels on either side of kanoneiland were estimated at 62 and 26 m3/s for the right and left channels, respectively. larval numbers prior to application were very high (rating of 10). two formulations of abate® 200ec were applied by boat, both at 0.1 ppm. the left channel received 8 ℓ of abate® 200ec, obtained from wefco marketing, and the right channel received 18.5 ℓ of abate® 200ec from basf. larval counts conducted the following day at the road bridge, 7 km downstream of the point of application, showed no indication of mortality. the results of these trials confirmed that larval resistance to temephos has developed in the orange river. powdered and granular b.t.i. preliminary results from the orbital shaking table indicated that the powdered formulation of b.t.i. (500:500) is toxic to s. chutteri, but results were intable 8 conditions of application and results of flow through gutter trials to test the toxicity of piperonyl butoxide combined with temephos (abate® 200ec) trial no. date water temp. (°c) gutter channel flow (ℓ/s) dosage (mg/ℓ) efficacy (%) pbo only (great fish river) 1 09.06.05 10.0 1 2 3 4 1.83 1.72 1.52 1.72 0 0.01 0.05 0.10 0 82 97 99 pbo plus temephos (great fish river) 20.07.05 13.2 1 2 3 4 1.60 1.61 1.34 1.66 0 0.01 ppm (80 % temephos; 20 % pbo) 0.05 ppm (80 % temephos; 20 % pbo) 0.1 ppm (80 % temephos; 20 % pbo) 0 0 0 0 pbo plus temephos (orange river) 1 15.08.05 14.6 1 2 3 4 1.84 1.76 1.91 2.08 100 % pbo (0.5 ppm pbo only) 80 % pbo; 20 % temephos (0.5 ppm) 20 % pbo; 80 % temephos (0.5 ppm) 0 % pbo (0.5 ppm temephos only) 0 0 2 5 2 15.08.05 14.6 1 2 3 4 1.84 1.75 1.91 2.08 100 % pbo (0.1 ppm pbo only) 80 % pbo; 20 % temephos (0.1 ppm) 20 % pbo; 80 % temephos (0.1 ppm) 0 % pbo (0.1 ppm temephos only) 0 0 0 0 2* 04.10.06 18.0 1 2 3 4 1.68 1.85 1.85 1.91 10 % pbo (0.019 pbo + 0.2 temephos) 20 % pbo (0.038 pbo + 0.2 temephos) 50 % pbo (0.095 pbo + 0.2 temephos) 50 % pbo (0.095 pbo only) 0 0 0 0 * pbo was applied first and temephos was applied 2 h later 310 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa conclusive. a mortality of 98 % was obtained for larvae exposed for 1 h at a dosage of 2 mg/ℓ, compared to 78 % mortality at half the dosage (table 9). however, it took a long time to record the results and by the time the control group was counted, 3 h had passed and mortality was 86 %. clearly, the closedsystem shaking table is unsuitable for trials exceeding 1 h in duration. gutter trials gutter trials conducted at pigott’s bridge, great fish river, indicated that the powdered formulation of b.t.i. (500:250) is ineffective against s. chutteri, even at dosages of 19.38 mg/ℓ (table 10). a possible source of error was that walking past the gutters sometimes cast a shadow on the gutters and this would temporarily stop larvae from feeding. if this occurred during larvicide application, mortality would be reduced. however, this did not occur and larvae were feeding during the time of application, so the poor results could not have been caused by a lack of larvicide ingestion. flow volumes in the gutter trials ranged between 0.75 and 0.91 ℓ/s and this created average current speeds of between 0.5 and 0.8 m/s, which is within the flow preference for s. chutteri. the poor results are therefore considered to be unrelated to inadequate experimental design, and were probably caused by inadequate toxicity. the formation of sticky lumps which remained behind in the syringe, could partly explain the poor results at lower concentrations, but this formulation problem is unlikely to have affected the results at higher concentrations. gutter trials conducted at upington on the orange river using granular b.t.i. also showed that this formulation was ineffective against s. chutteri (table 10). river trials a field trial conducted in belmont valley, grahamstown, on 4 june 2005 indicated that the powdered formulation of b.t.i. (500:250) is ineffective against s. nigritarse at a dosage of 4.57 mg/ℓ (water temperature 11.5 °c; flow 62 ℓ/s; 170 g applied; and 0 % mortality). higher dosages were not undertaken because such concentrations would be impractical for aerial application, even if the product is applied as a dry powder. a possible reason for the poor efficacy was that the active ingredient could have settled in the sticky mass at the base of the sprayer. however, it is unlikely that all active ingredient remained in the sprayer. table 9 conditions of application and results of orbital shaking table to test the toxicity of bacillus thuringiensis var. israelensis (500:500) to blackfly larvae at upington date water temp. (°c) container no. dosage (mg/ℓ) no. dead no. alive % dead 07.09.04 not recorded 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0* 0.000000 0.000001 0.000002 0.000004 0.000008 0.000015 0.000031 0.000061 0.000122 0.000244 0.000488 0.000977 0.001953 0.003906 0.007813 0.015625 0.031250 0.062500 0.125000 0.250000 0.500000 1.000000 2.000000 38 26 23 14 30 23 28 29 8 24 15 21 12 30 23 18 47 34 39 30 48 26 41 56 6 10 5 8 12 15 14 10 40 4 22 37 13 36 34 26 29 26 31 11 11 4 11 1 86 72 82 64 72 61 67 74 17 86 40 36 48 45 40 41 62 57 56 73 81 87 78 98 * control 311 r.w. palmer & n.a. rivers-moore discussion permethrin the results of this study indicate that the permethrin formulation rd95b is highly effective against blackflies at a concentration of 0.043 mg/ℓ, and was by far the most promising larvicide tested. however, the chemical has two problems, namely potential for resistance and impacts on the environment. resistance to permethrin has been reported for a wide variety of insects and cross-resistance to a range of synthetic pyrethroids has been reported (cox 1998). the use of permethrin, or any additional replacement larvicide for temephos, in the orange river blackfly control programme would have to be used within a careful management framework to avoid resistance developing again. permethrin decomposes rapidly in water and, while it is known to be highly toxic to aquatic organisms, including fish, it is relatively safe to people. muirheadthomson (1977) noted that permethrin was 40 times more toxic than abate®, but raised concerns on its effects on non-target organisms. wide spectrum larvicides may result in the undesirable eradication of most aquatic invertebrates and a change in ecosystem equilibrium and functioning. kreutzweiser & kings bury (1987) noted that river systems took 1–18 months to recover from applications of permethrin. impacts on non-target organisms may often be detected through increases in the density of drifting invertebrates. kreutzweiser & kingsbury (1987) reported a major increase in downstream drift of nontarget organisms following an application of permethrin in forest streams in canada. such impacts would be exacerbated through further exposure to aquatic invertebrates as they drift downstream with the “slug” of larvicide (muirhead-thomson 1977). while synthetic pyrethroids have been shown to have moderate acute toxicity to birds (ld50 = 1 000 mg/kg) (spiop 1986), the us environmental protection agency has classified permethrin as a carcinogen, since it causes cancerous tumours in lung and liver tissue of mice (cox 1998). permethrin has been widely reported as being highly toxic to aquatic invertebrates (mueller-beilschmidt 1990; cox 1998), with an lc50 of less than 1.0 ppb, which is similar to that used for pest blackfly control (smith & stratton 1986). the aquatic invertebrate groups most sensitive to permethrin are mayfly, damselflies and zooplankton (anderson 1982; smith & stratton 1986; spiop 1986). such impacts could potentially table 10 conditions of application and results of flow through gutter trials to test the toxicity of various dry formulations of bacillus thuringiensis var. israelensis to blackfly larvae trial no. date water temp. (°c) gutter channel flow (ℓ/s) dosage (mg/ℓ) efficacy (%) standard powder formulation (500:250) (great fish river) 03.06.05 11.5 1 2 3 4 0.88 0.86 0.75 0.83 0* 0.9 2.2 6.0 0 0 0 0 double strength powder formulation (500:500) (great fish river) 04.06.05 11.2 1 2 3 4 0.85 0.91 0.82 0.86 0* 3.7 8.1 19.4 0 0 0 0 04.06.05 11.2 1 2 3 granular formulation (orange river) 03.10.06 18 1 2 3 4 1.62 1.79 1.79 1.85 0.5 0.9 1.9 9.0 0 0 0 0 * control 312 larvicides in developing management guidelines for control of pest blackfl ies (diptera: simuliidae), south africa have a significant indirect effect on fish, through diminished food supplies (spiop 1986). permethrin is highly toxic to fish (cox 1998), particularly at lower water temperatures, and to smaller fish (hill 1985), since fish lack the enzymes to break permethrin down (haya 1989). the lc50 values of many fish species tested is less than 1.0 ppm (world health organization 1990), the same concentration recommended for pest blackfly control. permethrin shows intermediate toxicity to fish within the spectrum of pyrethroid toxicities, although it is also noted that pyrethroids in general are highly toxic to fish, with about 40 % of lc50 values for fish being less than 1 ppb (smith & stratton 1986). differences in mortalities between gutter trials and river trials at the same concentrations recorded in this study may be attributed to differences in exposure time and/or interactions between permethrin and suspended organic matter. muirhead-thomson (1987) reported that pyrethroids were more toxic to fish in the laboratory than in natural water because they adhere to suspended organic matter in the water and sediment. temephos formulations of temephos (abate® 200ec) were highly effective against blackfly in the great fish river, but the same products were ineffective in the orange river. the results indicate clearly that larval resistance to temephos has developed in the orange river, but the mechanisms of resistance were not investigated in this study. temephos has a number of chemical bonds that are available for metabolic attack by oxidases or esterases. laboratory studies in west africa have shown that resistance to temephos among the s. damnosum complex is associated mainly with detoxication by esterases, but oxidase enzyme systems are also involved in some members of the complex (kurtak 1990). cross-resistance tests showed no cross-resistance to carbamate insecticides with these mechanisms, but negative correlations with most pyrethroids (kurtak 1990). the feasibility of “reversing” the resistance through the use of piperonyl butoxide was investigated. the results showed that piperonyl butoxide on its own is highly toxic to blackfly larvae. this contradicts the generally held view that piperonyl butoxide is nontoxic on its own. various combinations of piperonyl butoxide and temephos showed no enhanced toxicity as predicted. b.t.i. formulations the physical properties of the standard dry powder concentrated formulation of b.t.i. (500:250) were unsuitable for blackfly control, firstly because of poor vertical dispersion and rapid settling in water. the implication of this is that downstream carry is likely to be limited. secondly, the product forms sticky lumps when mixed with water and this is likely to cause clogging problems with application equipment, should the product be applied as a wet formulation. a possible solution to this problem would be to apply the product as a dry powder, but this is likely to restrict applications to periods when wind is not blowing. the slow-release granular formulation, by contrast, showed excellent dispersal properties. a more serious problem was that gutter and field trials found that all formulations tested were ineffective against blackflies. this was unlikely to have been due to low water temperatures, since water temperatures always exceeded the 10 °c temperature threshold of efficacy found by de moor & car (1986). concentrations of b.t.i. applied in these trials were also within the range used by parkes & kalff (2004), who applied b.t.i. to larval simuliids in rivers in southern quebec at concentrations of 1 g/ℓ/s and achieved in excess of 80 % mortality. similarly, de moor & car (1986) achieved equivalent mortalities in s. chut teri in the orange river, at concentrations of 1.6 ppm per 10 min. conclusions various potential larvicides for use during high flow conditions were investigated in laboratory, gutter and river trials, but none was found to be suitable. this study therefore failed to identify or register a suitable larvicide for use during high flow conditions. permethrin was highly effective against blackfly larvae, but was rejected because of its detrimental impacts on non-target fauna. various formulations of locally produced dry b.t.i. were tested, but these were ineffective against blackflies. trials with 20 % ec formulations of temephos in the great fish river found that the product is effective, but gutter and river trials in the orange river showed no efficacy, even at dosages that were 300 times the recommended dose. the results confirmed that larval resistance to temephos has developed in the orange river. the feasibility of “reversing” the resistance to temephos through the use of piperonyl butoxide was in313 r.w. palmer & n.a. rivers-moore vestigated. various combinations of piperonyl butoxide and temephos were tested, but none showed enhanced toxicity, as predicted. the results showed that piperonyl butoxide alone is highly toxic to blackfly larvae and non-target organisms, and is therefore not recommended for use in blackfly control. the natural recovery from resistance will depend on the rate at which the population mixes with non-resistant populations. the middle and lower orange river is geographically isolated, therefore the resistant population of blackflies is likely to remain so for some time. how long reversal to resistance will take, is unknown, but findings elsewhere have shown that the development of resistance is much more rapid than its reversal. the risks of larval resistance to temephos were well known when the programme started in 1991, and nothing was done to monitor resistance or test the development of resistance when it was first suspected in 2000. this underscores the need for radical improvements in the management of the control programme. resistance to b.t. toxins has for many years been considered remote because of the complexity of the toxin, containing multiple toxins with multiple target sites (mcgaughey & whalon 1992; mcgaughey 1994). however, resistance to b.t.i. has been documented in the laboratory for at least eight species of pest, and the diamond backed moth (plutella xylostella) has developed widespread resistance in the field (tabashnik 1994). possible physiological mechanisms of resistance include changes in the gut ph or enzymes that would deactivate the toxic protein (mcgaughey & whalon 1992). in some moths, resistance is due to changes in the binding sites in the insect mid gut (mcgaughey & whalon 1992; tabashnik 1994). although resistance to b.t.i. has not been reported for blackflies (kurtak et al. 1989), there is a need to remain vigilant and to implement an operational strategy that minimizes the risk of resistance developing. acknowledgements we thank the water research commission, agrinoord kaap, the orange river producers alliance and plant health products for financial support. the wrc steering committee, and in particular steve mitchell (water research commission) and dirk steenkamp (national department of agriculture), are thanked for their input comments over the course of this research. nkosinathi mtwa and vivian mcpher son are thanked for field assistance. keith craig (kwan dwe private game reserve, eastern cape prov ince) and mike palmer (strowan farm, grahamstown) are thanked for facilitation in the great fish river trials. two anonymous reviewers are thanked for their comments and suggestions. references 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) shahana begum department of pathology, bangladesh agricultural university, bangladesh mohammed nooruzzaman department of pathology, bangladesh agricultural university, bangladesh murshida parvin department of pathology, bangladesh agricultural university, bangladesh nijaya mohanto department of pathology, bangladesh agricultural university, bangladesh rokshana parvin department of pathology, bangladesh agricultural university, bangladesh mohammad r. islam department of pathology, bangladesh agricultural university, bangladesh emdadul h. chowdhury department of pathology, bangladesh agricultural university, bangladesh citation begum, s., nooruzzaman, m., parvin, m., mohanto, n., parvin, r., islam, m.r. et al., 2018, ‘peste des petits ruminants virus infection of black bengal goats showed altered haematological and serum biochemical profiles’, onderstepoort journal of veterinary research 85(1), a1595. https://doi.org/10.4102/ojvr.v85i1.1595 original research peste des petits ruminants virus infection of black bengal goats showed altered haematological and serum biochemical profiles shahana begum, mohammed nooruzzaman, murshida parvin, nijaya mohanto, rokshana parvin, mohammad r. islam, emdadul h. chowdhury received: 07 dec. 2017; accepted: 28 aug. 2018; published: 15 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in bangladesh, veterinarians often claim to reduce the mortality of natural peste des petits ruminants (ppr) outbreaks with the help of supportive fluid and electrolyte therapy. information on haematological and biochemical parameters of ppr-infected goats, which is often altered because of associated tissue damages, is necessary to formulate the appropriate supportive therapy. this study determined the haematological and serum biochemical parameters of black bengal goats naturally infected with ppr virus. blood and serum samples from 13 ppr-affected black bengal goats from 13 field outbreaks and 5 healthy goats were collected and analysed by routine haematological and biochemical examination. haematological analysis of prr-affected goats showed severe anaemia characterised by significant decrease in the values of haemoglobin, total erythrocyte counts (tecs) and packed cell volume (pcv). on the contrary, ppr-affected goats showed marked leucocytosis with absolute increase in lymphocytes and neutrophils counts compared to the healthy goats. biochemical analysis revealed significant decrease in total protein and albumin level and increased creatine kinase, aspartate transaminase and alanine transaminase that mirrored the gross and histopathological changes in the ppr-affected goats. significant increase in the values of sodium and chloride ions was found in the sera of ppr-infected goats. peste des petits ruminants virus altered the haematological and serum biochemical parameters of the infected goats. antidiarrheal agents with aqua solution together with other drugs to support liver and kidney function could help improve therapy of ppr-infected goats. introduction peste des petits ruminants (ppr) is an acute, highly contagious viral disease of goats, sheep and other related species. peste des petits ruminants is one of the main transboundary animal diseases and presents a major threat to animal production in ppr endemic areas because of high case fatality reaching 90% in native animals and also because of trade restriction (diallo 2003). the disease is caused by the ppr virus (pprv) which is a single-stranded negative sense ribonucleic acid (rna) virus under the genus morbillivirus, family paramyxoviridae and antigenically related to the rinderpest, canine distemper and measles virus (gibbs et al. 1979). the virus is circulating in africa, the middle east and central to south-east asia in four genetically distinct lineages, three of which (i, ii, iii) were first described in africa, including guinea, ivory coast, senegal, mali, burkina faso, ghana, nigeria, uganda and tanzania, and the fourth one (iv) in asia (banyard et al. 2010; chowdhury et al. 2014; rahman et al. 2016). however, the asian lineage was recently introduced in some countries of africa and europe, indicating continuing spread of the virus across the continents (maganga et al. 2013; parida et al. 2016). the first outbreak of ppr in bangladesh was recorded in 1993 (islam et al. 2001). since then the outbreaks of ppr are being recorded regularly across the country (chowdhury et al. 2014; bhuiyan et al. 2014; rahman et al. 2011, 2016). clinical ppr outbreaks are often marked by high fever, mucopurulent discharges from nose, eyes and mouth; severe diarrhoea; enteritis; bronchopneumonia and erosive stomatitis with high morbidity and mortality rates (bhuiyan et al. 2014). in the majority of ppr outbreaks, average flock morbidity and mortality of 75% and 59%, respectively, and a case fatality rate of 74% were recorded (chowdhury et al. 2014). in 2010, there were approximately 84 000 hospital cases of ppr in bangladesh, with an estimated direct annual loss of tk1842 million (us$24.56) (siddiky 2013). lack of quality vaccines together with poor presumptive diagnosis hindered the success of ppr control programmes (haider et al. 2017). veterinarians often claim to reduce the mortality of clinical ppr outbreaks to some extent with the help of supportive therapy. moreover, a recent study shows that supportive therapy in the form of fluid and antibiotics combined with ppr-specific hyperimmune serum can reduce the mortality in ppr-infected goats (yousuf et al. 2015). however, the knowledge of haematological, serum biochemical and electrolyte profiles of clinically ppr-infected goats, which is often altered because of diarrhoea and mucopurulent discharge, is needed to better describe the disease status and formulate appropriate supportive therapy. therefore, this study was designed to assess the serum biochemical and haematological profiles of ppr-infected black bengal goats in bangladesh. materials and methods animals a total of 13 flocks of black bengal goats with suspected ppr outbreaks were selected from two villages in the mymensingh district of bangladesh during the period july 2015 to june 2016. the detailed descriptions of the flocks are shown in table 1. blood and nasal swab were collected from one ppr-infected goat of each flock that showed clinical signs of ppr. in addition, blood samples were collected from five apparently healthy goats that served as controls. the five healthy goats were obtained from ppr seronegative flocks other than the 13 infected flocks. all animal handling, necropsy and sample collection was performed by trained veterinarians. the study was carried out in accordance with the recommendation of the animal experimentation ethics committee of bangladesh agricultural university, mymensingh (ref. no.: aeec/01/2015). the protocol was approved by the animal experimentation ethics committee. prior approval was obtained from the animal owners regarding further use of the initially collected diagnostic samples. table 1: morbidity and mortality of black bengal goats naturally infected with peste des petits ruminants virus. necropsy and histopathology a total of seven dead goats were collected from seven ppr-suspected flocks and routine necropsy was performed in the department of pathology. gross pathological changes were recorded. the tissue samples of lips, trachea, lungs, spleen, lymph node, intestine, kidney, heart, spleen and liver were collected in 10% neutral buffered formalin and processed for histopathological study following standard procedures (luna 1988). in addition, bronchial and mesenteric lymph nodes were collected aseptically and stored at -80 oc for molecular detection of the virus. molecular detection of the peste des petits ruminants virus blood and nasal swabs from ppr-suspected sick goats were soaked onto small pieces of filter paper as described elsewhere (bhuiyan et al. 2014; forsyth & barrett 1995). in brief, a few drops of blood were poured on the filter papers, from the base towards the tip, until it was completely soaked. nasal swab were collected with sterilised filter paper which was directly applied on the nostrils of ppr-suspected goats. the filter papers were air-dried avoiding direct sunlight and stored at -70 oc in screw-capped tubes. the smeared filter papers were directly used in polymerase chain reaction (pcr) tubes as a source of rna template as described previously (bhuiyan et al. 2014). in addition, rna was extracted from 20% tissue homogenate of bronchial and mesenteric lymph nodes of ppr-suspected dead goats using rneasy mini kit (qiagen, germany). the presence of ppr virus was detected by an established reverse transcription-pcr (rt-pcr) technique (forsyth & barret 1995) to amplify a 448 bp fragment of the f gene. the rt-pcr was performed with qiagen one-step rt-pcr kit (qiagen, germany) as per the manufacturer’s instruction. competitive enzyme-linked immunosorbent assay approximately 7 ml of blood per goat was collected aseptically from the jugular vein of 13 pprv rt-pcr positive goats after 10–14 days of onset of clinical signs. in addition, blood samples from five healthy goats were collected as control. after collection, 2 ml of blood was transferred to a sterile vial containing ethylenediaminetetraacetic acid (edta) at the rate of 1 mg/ml blood for routine examination of blood. approximately 5 ml of blood was transferred to another sterile tube without edta for serum separation. after clotting of the blood, serum samples were obtained by centrifugation at 900× g for 10 min and stored at -20 °c for serological and biochemical analysis. the amount of pprv nucleoprotein-specific antibody in serum samples was quantified by a competitive enzyme-linked immunosorbent assay (elisa) kit (id screen ppr competition, id-vet, montpellier, france). haematological analysis routine haematological examination of whole blood samples was performed by standard method to determine the values of haemoglobin (hb), erythrocyte sedimentation rate (esr), packed cell volume (pcv), total erythrocyte count (tec), total leucocyte count (tlc) and differential leucocyte count (dlc) (lamberg & rothstein 1978). biochemical analysis for determination of serum electrolytes (sodium, potassium and chloride ions), samples were analysed from a commercial lab (pure diagnostic center, mymensingh, bangladesh) with an automated electrolyte analyzer genlyte 3000a (ivd) as per kit instruction (electrolyte solution, biogen, gmbh, germany). other serum biochemical constituents such as inorganic phosphorus (p), calcium (ca), total protein (tp), albumin, glucose, bilirubin and blood urea nitrogen (bun), creatine kinase (ck), alkaline phosphatase (alp), alanine transaminase (alt) and aspartate transaminase (ast) were determined with the help of an automated t80 ultraviolet–visible spectroscopy (uv/vis) spectrophotometer (pg instruments, uk) at bangladesh agricultural university central laboratory, mymensingh, bangladesh. statistical analysis data were statistically analysed using the software package graphpad prism version 5.0. differences in haematological and biochemical profiles between pprv-infected and healthy goats were evaluated statistically by one-tailed non-parametric mann–whitney u test. a p value of ≤ 0.05 was considered significant. results outbreak investigation a total of 13 ppr-suspected flocks of black bengal goats were investigated in the present study. detailed description of the affected flocks is given in table 1. out of 125 goats examined, 99 showed clinical signs of ppr such as high fever (105 °f), profuse nasal and ocular discharge and severe diarrhoea; 70 goats died within 7–12 days after onset of clinical signs. therefore, the average morbidity and mortality of the ppr-suspected flocks were 80.44% and 65.67%, respectively. the case fatality varied from 25% to 100%, with an average of 56.36%. it is noteworthy that none of the examined animals received any supportive therapy. molecular detection of the virus the blood and nasal swabs from 13 ppr-suspected goats were collected on small pieces of filter paper and analysed by rt-pcr to amplify a 488 bp fragment of f gene of ppr virus. the filter paper rt-pcr method successfully detected pprv in all of the tested nasal swabs. however, blood samples from only five ppr-suspected goats produced a positive result in the rt-pcr. it is noteworthy that goats that were positive with nasal swabs, but negative with blood were sampled at much later stages of fever. in addition, the rt-pcr method successfully amplified pprv in lymph node samples collected during necropsy from ppr-suspected goats. pathological investigation erosive and necrotic spots on the lip and tongue with severely congested trachea were observed in the dehydrated carcasses. significant lesions were found in the lungs, which included congestion and consolidation, mostly in the apical lobes, together with white necrotic foci on the surface (figure 1a). congestion and fragility of the liver (figure 1b) and atrophy of the spleen (figure 1c) were found. the kidney (figure 1d) and the mesenteric lymph nodes (figure 1e) were enlarged two to three times and showed congestion. haemorrhagic streaks were seen in the duodenum and the terminal ileum and severe congestion was also observed in the testicles of two male goats (figure 1f). figure 1: post-mortem examinations of peste des petits ruminants–infected black bengal goats showed severe haemorrhage and congestion of visceral organs. (a) lungs showed severe congestion with white necrotic foci on the surface. (b) congested and fragile liver and (c) atrophy of the spleen. (d) kidneys showed congestion and (e) mesenteric lymph nodes were enlarged and congested. (f) congestion and swelling of testis were found in peste des petits ruminants–infected male goat. representative images of one out of seven dead goats are shown. the histopathological changes in the lungs, kidney and lymph nodes of ppr-infected goats were marked by severe haemorrhage and congestion. the affected lungs showed syncytial cell formation in the alveoli and clumps of mononuclear cells infiltration in the inter-alveolar space and bronchial wall (figure 2a). massive haemorrhage in the tubular areas of kidneys was found in some goats (figure 2b). severe haemorrhage and congestion with brown colour haemosiderin deposition were found in kidneys (figures 2c & 2d). the spleen was congested with haemosiderin deposition (figure 2e). severe congestion and haemorrhage with stagnation of bile pigments were found in the liver parenchyma (figure 2f). taken together, natural ppr infections of black bengal goats showed severe haemorrhage and congestion in different visceral organs. figure 2: histopathological findings of peste des petits ruminants–infected black bengal goats showed haemorrhage and mononuclear infiltration. (a) peste des petits ruminants–infected lungs showed clumps of large mononuclear and multinucleated cells within the alveoli (h&e stain, 250×). (b) kidney showed haemorrhage in the tubular area (h&e stain; 62.5×). (c) and (d) kidneys showed severe haemorrhages and congestion with haemosiderosis (h&e stain, 250×). (e) haemosiderosis with severe congestion in the spleen (h&e stain, 250×). (f) congestion with stagnant of bile pigments in the liver (h&e stain, 250×). haematological profiles of naturally peste des petits ruminants–infected goats to investigate the haematological profiles of goats with natural ppr outbreaks, serum samples were collected from 13 infected and 5 healthy black bengal goats. the serum samples were first screened for the presence of pprv-specific antibodies by a competitive elisa. all serum samples from ppr-suspected goats showed a high level of pprv-specific antibody when goats survived 10 days after onset of clinical signs (figure 3a). the control goats did not show any pprv-specific antibody response. then, we performed routine examination of whole blood samples from ppr-infected as well as healthy goats. there was a significant decrease in the values of hb concentration (figure 3b), pcv (figure 3c) and tec (figure 3e) in the ppr-infected goats. on the other hand, tlc (figure 3f) was significantly increased in ppr-infected goats as compared to healthy animals. further analysis of the dlc showed a significant increase in the absolute number of lymphocytes and neutrophils (figure 3g). however, the other blood parameters such as esr (figure 3d) and absolute monocytes, eosinophils and basophils counts (figure 3g) remained comparable between ppr-infected and healthy goats. collectively, natural ppr infection in goats induced severe anaemia and leucocytosis. figure 3: haematological profile of peste des petits ruminants virus–infected black bengal goats showed marked anaemia and leucocytosis. blood samples were collected from healthy and peste des petits ruminants–affected goats and the amount of (a) peste des petits ruminants virus–specific antibody, (b) haemoglobin, (c) packed cell volume, (d) erythrocyte sedimentation rate, (e) total erythrocyte counts, (f) leucocyte count and (g) differential leucocyte count were quantified. data indicate mean ± sem from 5 healthy and 13 peste des petits ruminants virus–infected goats. one-tailed mann–whitney test. p ≤ 0.05 was considered statistically significant. serum biochemical analysis of natural peste des petits ruminants virus–infected goats different biochemical parameters of serum samples obtained from ppr-infected goats were quantified and compared with healthy goats. peste des petits ruminants–infected goats showed significantly lower level of total protein (figure 4b) and albumin (figure 4c) in their sera compared to healthy goats. no significant difference was found in the amount of glucose (figure 4a), bilirubin (figure 4d) and bun (figure 4e) between ppr-infected and healthy goats. further analysis of the serum enzymes showed a significant increase in the level of ck (figure 5b), ast (figure 5c) and alt (figure 5d) in the ppr-infected goats compared to the healthy goats. however, the amount of alp remained constant between the infected and healthy goats. figure 4: peste des petits ruminants virus–infected black bengal goats showed altered serum biochemical profiles. serum samples were collected from healthy and peste des petits ruminants–affected goats and the level of (a) glucose, (b) total protein, (c) albumin, (d) bilirubin and (e) blood urea nitrogen was quantified. data indicate mean ± sem from 5 healthy and 13 peste des petits ruminants virus–infected goats. one-tailed mann–whitney test. p ≤ 0.05 was considered statistically significant. figure 5: natural peste des petits ruminants virus–infected black bengal goats showed elevated serum enzymes. serum samples were collected from healthy and peste des petits ruminants–affected goats and the amount of (a) alkaline phosphatase, (b) creatine kinase, (c) aspartate transaminase and (d) alanine transaminase was measured. data indicate mean ± sem from 5 healthy and 13 peste des petits ruminants virus–infected goats. one-tailed mann–whitney test. p ≤ 0.05 was considered statistically significant. furthermore, we determined the concentration of some important serum electrolytes in ppr-infected and healthy goats. we found significantly higher levels of sodium (figure 6a) and chloride (figure 6e) ions in sera of ppr-infected goats than healthy goats. however, other electrolytes such as potassium, calcium and phosphorus level remained normal in both animal groups (figures 6b, 6c & 6d). of note, among infected animals, five goats with a very high level of sodium and chloride ions were suffering from severe diarrhoea and showed early mortality. collectively, goats with natural pprv infection showed an abnormal serum biochemical profile marked by hypoproteinaemia, increased kidney and liver enzymes and higher sodium and chloride ions. figure 6: serum electrolytes analysis of natural peste des petits ruminants virus–infected black bengal goats showed elevated level of sodium and chloride ions. serum samples were collected from healthy and ppr-affected goats and the values of (a) sodium, (b) potassium, (c) calcium, (d) phosphorus and (e) chloride were quantified. data indicate mean ± sem from 5 healthy and 10–13 peste des petits ruminants virus–infected goats. one-tailed mann–whitney test. p ≤ 0.05 was considered statistically significant. discussion peste des petits ruminants virus infection induced marked anaemia, leucocytosis and altered serum enzyme and electrolyte profiles in black bengal goats. outbreak investigation showed that all 13 goats included in the study were naturally infected with pprv. pathological investigation of all ppr-suspected goats showed marked bronchopneumonia and haemorrhagic lesions in different visceral organs that were typical of natural pprv outbreaks (chowdhury et al. 2014; rahman et al. 2011, 2016). however, the haemorrhagic change in kidney was not reported earlier. in bangladesh, veterinarians generally treat ppr-infected goats with antibiotic, antihistaminic, dextrose saline and astringent mixture (chowdhury et al. 2014). veterinarians often claim to reduce mortality in infected flocks by controlling secondary bacterial infections and counteracting shock. however, information on the haemato-biochemical profiles of ppr-infected goats, which are often altered because of viremia and associated tissue destruction, is necessary to formulate an appropriate supporting therapy against devastating clinical ppr outbreaks. all serum samples from the confirmed ppr outbreaks showed pprv-specific antibody titres during the later stage of the disease, particularly if the goat survived more than 10 days after onset of clinical signs. thus, supportive therapy could provide a critical window for the development of adaptive immunity in infected goats during the first 10–12 days of infection. the routine haematological examination of the ppr-infected goats revealed a significant reduction in the values of hb, pcv and tecs compared to healthy goats which was because of widespread haemorrhages throughout the body. a significant decrease in the values of hb and erythrocyte count was also reported earlier (das et al. 2015; sharma et al. 2012) and expected in ppr-infected goats because of the massive haemorrhage in different visceral organs which we also observed during pathological investigations. we found an increase in the absolute number of lymphocytes and neutrophils in the ppr-infected goats. similar findings of the leucocytosis in the ppr-infected goats were shown by some other studies indicating acute inflammation in the studied animals (das et al. 2015; ezeasor, emikpe & anosa 2015; maina, gitao & gathumbi 2015). although others reported some degree of leucopoenia in the ppr-infected goats (sharma et al. 2012), such differences between research groups could possibly be explained by a variation in the disease status as well as nutritional condition of the infected animals. a decrease in total protein and albumin indicates liver and kidney dysfunction, which was mirrored by the gross and histopathological and biochemical findings of the infected goats. on the other hand, the level of important enzymes such as ck, ast and alt increased significantly in the ppr-infected goats that correlated with the damage in liver and kidney by the virus (al-dubaib 2009; chowdhury et al. 2014; kul et al. 2007). diarrhoea together with an increased nasal, oral and ocular discharge was shown to produce severe dehydration and death in the ppr-infected animals (chowdhury et al. 2014). in addition, pathological changes in the liver and kidney might lead to the fluid and electrolyte imbalance in the infected goats. we found a significantly higher concentration of sodium and chloride ions in the blood of ppr-infected goats than that of the healthy goats. other electrolytes such as potassium, calcium and phosphorus remained unaltered. increased sodium and chloride ions in ppr-infected goats were also found in similar other studies (das et al. 2015; kataria, kataria & gahlot 2007). our study also found an association of the increased sodium and chloride level with the mortality in five severely diarrheic goats. in addition, the comparable level of glucose in the infected as well as healthy goats denied the necessity of applying dextrose saline as supportive therapy which is being used in the field to treat ppr-infected goats. conclusion in conclusion, haematological analysis of ppr-affected goats showed severe damage in the liver and kidneys, with marked anaemia, leucocytosis and altered serum biochemical profiles. the significant kidney lesions and increased level of ck highlight an increased tropism of the virus towards kidneys. therefore, veterinarians should suggest antidiarrheic therapy in the form of oral or intravenous application of isotonic aqua solution together with other supportive drugs to support liver and kidney function for the treatment of natural ppr outbreaks. a clinical scoring system can be devised and applied in the field with a treatment plan informed by the haematological and serum biochemical profiles. however, it requires a further comprehensive study. acknowledgements we would like to acknowledge the bangladesh agricultural university research system (baures) for providing administrative support to conduct the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.b. performed experiments, analysed and interpreted the data, and drafted the manuscript. m.m.p. and n.m. performed experiments. m.n., r.p. and m.r.i. analysed and interpreted the data and drafted the manuscript. e.h.c. was responsible for conception and design, analysis and interpretation of data, drafting of the manuscript and supervision of the study. funding information this study was funded by research grants from the bangladesh academy of science–united states department of agriculture (bas-usda), dhaka, bangladesh (grant no. pals ls04) and the international atomic energy agency (iaea) (grant no. ras/5/060). 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(ed.), 2013, economic impact of transboundary animal diseases in saarc countries saarc agriculture centre, dhaka. yousuf, m.a., giasuddin, m., islam, s.s. & islam, m.r., 2015, ‘management of an outbreak of peste des petits ruminants with antibiotic combined hyperimmune serum therapy’, asian journal of medical and biological research 1, 220–234. https://doi.org/10.3329/ajmbr.v1i2.25616 botha_307-314.indd introduction poisoning of livestock by cardiac glycoside-containing plants has the greatest economic impact of all plant-associated poisonings in the republic of south africa (kellerman, naudé & fourie 1996). collectively they are held responsible for 33 % of all mortalities from plant poisonings of cattle and 10 % of those in small stock. the majority of the cardiac glycoside poisonings in small stock is ascribed to krimpsiekte, arguably the most economically important plant poisoning of small stock in the little karoo and southern great karoo (botha 2003; kellerman, coetzer, naudé & botha 2005). it is estimated that more than 26 000 sheep and goats succumb annually (kel lerman et al. 1996). chemically, two major groups of cardiac glycosides, namely the cardenolides and bufadienolides, are recognized. poisoning by bufadienolide-containing plants surpasses cardenolide-induced poisonings in importance and may be either acute or chronic. tulp poisoning (induced by various moraea species) and slangkop poisoning (caused by various drimia 307 onderstepoort journal of veterinary research, 74:307–314 (2007) a potential krimpsiekte vaccine c.j. botha1*, j.e. crafford2, v.p. butler jr3, m.n. stojanovic3 and l. labuschagne4 abstract botha, c.j., crafford, j.e., butler, v.p. jr, stojanovic, m.n. & labuschagne, l. 2007. a potential krimpsiekte vaccine. onderstepoort journal of veterinary research, 74:307–314 krimpsiekte, a chronic form of cardiac glycoside poisoning, is an important plant-induced intoxication of small stock in south africa. it is caused by cumulative, neurotoxic bufadienolides, such as cotyledoside. a cotyledoside-bovine serum albumin conjugate was synthesized to immunize animals. the efficacy of the cotyledoside-conjugate in inducing an immunological response was ascertained in rabbits (n = 4) and sheep (n = 4) by determining cotyledoside antibody titres with an elisa using cotyledoside-hen ovalbumin as antigen. the formation of anticotyledoside antibodies was induced in both rabbits and sheep following immunization with the cotyledoside-protein conjugate. protection provided by the vaccine was demonstrated by challenging sheep (n = 4) with repeated, daily doses of cotyledoside (0.015 mg/kg) administered intravenously, commencing 45 days after the initial vaccination. one control animal died on day 3 of the challenge period and the other was severely affected after administration of the third cotyledoside dose. the immunized ewes (n = 2) remained clinically unaffected and the challenge was suspended following six daily injections. vaccination as a means of preventing krimpsiekte seems to be quite feasible and deserves further investigation. keywords: cardiac glycoside, cotyledoside, krimpsiekte, immunization, rabbits, sheep, vaccine * author to whom correspondence is to be directed: e-mail: christo.botha@up.ac.za 1 department of paraclinical sciences, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 3 department of medicine, college of physicians and surgeons, columbia university, 630 west 168th street, new york ny 10032 united states of america 4 toxicology division, arc-onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa accepted for publication 26 april 2007—editor 308 potential krimpsiekte vaccine species) induce only acute intoxication as these species contain non-cumulative bufadienolides (kellerman et al. 1996, 2005). members of three genera of the crassulaceae (cotyl edon, tylecodon and kalanchoe), colloquially referred to as plakkies, on the other hand, may cause either acute or chronic poisoning. krimpsiekte, the chronic form of the poisoning, is a neuromuscular affliction of small stock following ingestion of plants that contain atypical cardiac glycosides (such as cotyledoside and tyledoside d), generally referred to as cumulative, neurotoxic bufadienolides. tyleco don wallichii and tylecodon ventricosus are probably the most important species of the group of plants causing krimpsiekte (botha, van der lugt, erasmus, kellerman, schultz & vleggaar 1997; botha, kellerman, schultz, erasmus, vleggaar & retief 1998). in this syndrome the cardiac, respiratory and gastrointestinal signs, typical of acute poisoning are diminished and the neuromuscular signs increase. small stock tire easily, lag behind the flock and frequently lie down. often they assume a characteristic stance, with the back arched, limbs tucked in under the body and head down, sometimes trembling. the animals may be recumbent for long periods. of particular concern is evidence that secondary poisoning of humans can occur if meat or edible tissues obtained from carcasses of animals that have died of krimpsiekte are consumed (kellerman et al. 2005). this is a real danger, as rural people with meagre means and financial constraints, commonly consume animals that die in the veld. immunoprophylaxis against plantand fungal poisonings is currently receiving some attention; for instance, australian researchers are evaluating immunization as a means of preventing the mycotoxicosis lupinosis in sheep (than, anderton, cockrum, payne, stewart & edgar 1994). most phytotoxins are not immunogenic due to their small molecular size and, in order to induce immunity following parenteral administration to an animal, must first be coupled to carrier proteins (silbart, rasmussen & oliver 1997). various attempts at vaccinating stock against plant toxins have been unsuccessful for a number of reasons. one of these is that the immunological response is overwhelmed due to the high toxin concentrations, which occur in acute toxic exposures (edgar 1994). krimpsiekte, on the other hand, is a chronic manifestation that can be induced by repeated exposures to small doses of the cumulative bufadienolide; a vaccine, therefore, could conceivably prevent this intoxication. if a prophylactic vaccine could be produced the incidence of, and mortalities caused by, krimpsiekte could be curtailed. technically the development of a vaccine to prevent krimpsiekte is quite feasible (butler & chen 1967; schmidt & butler 1971). antibodies have been raised against digoxin, a cardenolide cardiac glycoside. sheep and rabbits immunized by repetitive administration of digoxin-protein conjugates developed high serum titres of antidigoxin antibodies of high specificity and affinity (schmidt & butler 1971; butler, smith, schmidt & haber 1977b). immunized rabbits were protected from the toxic effects of a lethal dose of digoxin (butler et al. 1977b). however, antibody binding of digoxin interferes with the renal excretion of digoxin, thus greatly prolonging the half-life of this compound (schmidt, kaufman & butler 1974; butler, schmidt, smith, haber, raynor & demartini 1977a; butler et al. 1977b). the first objective of this project was to synthesize a cotyledoside-protein conjugate with which to immunize animals. the second aim was to evaluate the efficacy of the conjugate in inducing an immunological response in rabbits and sheep by determining cotyledoside antibody titres in an elisa. the third objective was to demonstrate the efficacy of the vaccine (conjugate) by challenging sheep with the neurotoxic, cumulative bufadienolide, cotyledoside. materials and methods preparation of cotyledoside-protein conjugates cotyledoside has previously been extracted and pur ified from t. wallichii (botha et al. 1997). coty l edo side-protein conjugates were prepared by a mixed anhydride procedure (erlanger, borek, beiser & lieberman 1957). cotyledoside (1 mg) was heated in a closed container (oil bath) at 100 °c in pyridine (200 μl) in the presence of succinic anhydride (1 mg) and a catalytic amount of dimethylaminopyridine for 8 h. during this time, the reaction mixture turned distinctly brown. the reaction was followed by removal of aliquots (2 μl) with a micropipette, quenching them in methanol (100 μl) and confirming by mass spectra (shimadzu lcms 2010a) the disappearance of the starting material, with the concomitant production of succinylated cotyledoside. no further characterization was performed on this material. the reaction was stopped by the removal of heat and addition of ethyl acetate. solvent was removed by a stream of nitrogen and the residue was partitioned between 0.01 m hcl solution and ethyl acetate (1 ml each). the organic layer was 309 c.j. botha et al. dried over magnesium sulphate. after evaporation of solvent under vacuum, the residue was dissolved in tetrahydrofuran (thf, 100 μl). the acid was transformed into the n-hydroxysuccinimide (nhs) ester by the addition of dicyclohexyl carbodiimide, (dcc, 1 mg) and nhs (1 mg). within 1 h, the precipitate was filtered through a wool pad and added directly to a protein solution (5 mg of either bovine serum albumin (bsa) or hen ovalbumin (ova) in 2 ml water) and the ph was adjusted to 8 by adding nahco3. the reaction mixture was stirred overnight and then filtered, thf was evaporated under vacuum and excess small molecules were removed by gel filtration (pd10 column, pharmacia). accumulat ed protein fractions had characteristic cotyledoside uv peaks (~300 nm), indicating attachment between 4–8 cotyledosides per protein (fig. 1). cotyledoside elisa maxisorb 96-well plates (nunc) were coated with 50 μl antigen (ova-cotyledoside conjugate) at a dilution of 10 μg/ml in coating buffer (0.1 m carbonatebicarbonate buffer, ph 9.6) in alternate rows. the re maining rows were coated with ova instead of antigen at the same concentration. the plates were sealed and incubated overnight at 4 °c. plates were washed five times with pbs supplemented with 0.05 % tween 20 and blotted dry (washing), then blocked with 100 μl pbs supplemented with 5 % skimmed milk powder per well and incubated for 1 h at room temperature followed by washing. sera were loaded in duplicate rows (e.g. row a coated with antigen and row b coated with ova) and a two-fold dilution series (50 μl per well) (using blocking buffer and starting at 1:500 for rabbit sera and 1:200 for sheep sera) was made across the plate followed by incubation and washing. when testing rabbit sera 50 μl biotinylated goat anti-rabbit serum at a dilution of 1:1 000 was added to all wells and incubated. after washing, 50 μl strepavidin horseradish-peroxidase (hrpo) conjugate at a dilution of 1:1 000 was added to all wells. for testing sheep sera rabbit antisheep hrpo conjugate was used at 1:1 000. fol lowing further incubation and washing, 50 μl substrate (ortho-phenylenediamine at a concentration of 0.04 mg/ml and containing 0.05 % h2o2 30 % v/v) was added to all wells and the plates incubated for 10 min in the dark after which 50 μl stop solution (1n h2so4) was added to all wells. the plates were read in a biotek el808 plate reader at 450 nm and results recorded. the net optical density was calculated by subtracting the value obtained for the ovacoated well from the corresponding antigen-coated well. rabbit immunization new zealand white rabbits (n = 4) were housed at the university of pretoria biomedical research centre. they were kept individually in rabbit cages, had free access to water and were fed a commercial ration. for immunization, an emulsion was prepared by adding the antigenic hapten-carrier conjugate (2 mg/ml) or pure bsa (sigma) dissolved in normal saline (2 mg/ml) to an adjuvant, at first to complete and later to incomplete freund’s (sigma) (table 1). two rabbits (a male and a female) were immunized with bsa-cotyledoside conjugate and the other two control rabbits (a male and a female) were immunized with similar volumes of bsa on days 0, 21 and 49 (table 1). a large area on the back of each rabbit was shaved and they were each immunized by intradermal administration of the infig. 2 rabbit exhibiting granulomatous skin lesions at the injection sites fig. 1 the cotyledoside-protein conjugate. note that the conjugation could also possibly occur at position c-4 , or at both positions c-2 and c-4 310 potential krimpsiekte vaccine t a b l e 1 im m u n iz a tio n r e g im e n f o r th e r a b b its a n d s h e e p r a b b it s e x p e ri m e n ta l (n = 2 ) c o n tr o ls ( n = 2 ) d a y c o m p o s it io n d o s e ( m l) s it e s r o u te c o m p o s it io n d o s e ( m l) s it e s r o u te 0 0 .5 m l c o ty le d o si d e c o n ju g a te + 0 .5 m l c o m p le te f re u n d ’s 0 .1 4 in tr a d e rm a lly , o n b a ck 0 .5 m l b s a + 0 .5 m l c o m p le te f re u n d ’s 0 .1 4 in tr a d e rm a lly , o n b a ck 2 1 0 .5 m l c o ty le d o si d e c o n ju g a te + 0 .5 m l in co m p le te f re u n d ’s 0 .1 4 in tr a d e rm a lly , o n b a ck 0 .5 m l b s a + 0 .5 m l in co m p le te f re u n d ’s 0 .1 4 in tr a d e rm a lly , o n b a ck 4 9 0 .2 5 m l c o ty le d o si d e c o n ju g a te + 0 .2 5 m l in co m p le te f re u n d ’s 0 .1 2 in tr a d e rm a lly , o n b a ck 0 .2 5 m l b s a + 0 .2 5 m l in co m p le te f re u n d ’s 0 .1 2 in tr a d e rm a lly , o n b a ck s h e e p e x p e ri m e n ta l (n = 2 ) c o n tr o ls ( n = 2 ) d a y c o m p o s it io n d o s e ( m l) s it e s r o u te c o m p o s it io n d o s e ( m l) s it e s r o u te 0 1 m l c o ty le d o si d e c o n ju g a te + 1 m l c o m p le te f re u n d ’s 0 .4 2 s u b cu ta n e o u s, in n e r th ig h 1 m l b s a + 1 m l c o m p le te f re u n d ’s 0 .4 2 s u b cu ta n e o u s, in n e r th ig h 2 1 0 .2 5 m l c o ty le d o si d e c o n ju g a te + 1 m l in co m p le te f re u n d ’s 0 .2 5 2 s u b cu ta n e o u s, b ri sk e t 1 m l b s a + 1 m l in co m p le te f re u n d ’s 0 .2 5 2 s u b cu ta n e o u s, b ri sk e t 311 c.j. botha et al. oculum at four sites on the back (fig. 2). on days 21 and 49 the rabbits were weighed and bled from an ear vein (2 ml) to determine antibody titres using the ova-cotyledoside in an elisa. on day 61 the rabbits were anaesthetized (with a xylazine/ketamine combination [xylavet 2 %, intervet sa (pty) ltd and anaket, bayer animal health division]) and exsanguinated by inserting a hypodermic needle into the heart. all the blood was collected for serology. sheep immunization four mutton merino ewes, weighing 38.5–44.5 kg, were housed in a pen with a concrete floor at the toxicology biolab, onderstepoort veterinary institute (ovi). they had free access to water and were fed the ovi standard concentrate and eragrostis hay. the sheep were randomly allocated to two groups. two of the sheep were initially immunized by subcutaneous inoculation of an emulsion containing equal volumes of bsa-cotyledoside plus complete freund’s adjuvant. the other two animals served as controls and were vaccinated with the same volumes of bsa dissolved in saline and complete freund’s adjuvant (table 1). a booster vaccination containing cotyledoside conjugate plus incomplete freund’s adjuvant was prepared and inoc ulated 3 weeks later (table 1). the serum antibody titres of all four sheep were determined by elisa before vaccination, 3 weeks after the initial vaccination and 3 weeks after the booster vaccination (day 42). cotyledoside challenge in sheep commencing 45 days after the initial vaccination, all four sheep were challenged with daily injections of cotyledoside. cotyledoside (20 mg), previously extracted and purified from t. wallichii (botha et al. 1997), was dissolved in 2 ml ethanol and 78 ml normal saline (0.025 % m/v). each animal received daily intravenous injections of 0.015 mg/kg cotyledoside (botha et al. 1997). during the challenge study clinical examinations were performed daily. in order to avoid any bias the senior author (cjb), who interpreted the clinical presentations, was blinded to the immunization status of the sheep. results rabbit immunization all the rabbits developed an injection site reaction, noticeable as erythema and oedema, followed by granulomatous lesions, superficial skin necrosis and scab formation (fig. 2). high anticotyledoside antibody titres were detected on termination of the experiment (on day 61) in the two rabbits that were immunized with the bsa-cotyledoside conjugate (fig. 3). as antibodies were raised in rabbits following immunization, it was decided to continue with the project in sheep, two being subsequently vaccinated. sheep immunization all four sheep developed slight to severe injectionsite reactions, but this did not hamper their habitus. anticotyledoside antibodies were raised in the two sheep following immunization with the cotyledosideprotein conjugate (fig. 4). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1 :5 0 0 1 :1 0 0 0 1 :2 0 0 0 1 :4 0 0 0 1 :8 0 0 0 1 :1 6 0 0 0 1 :3 2 0 0 0 1 :6 4 0 0 0 1 :1 2 8 0 0 0 1 :2 5 6 0 0 0 1 :5 1 2 0 0 0 1 :1 0 2 4 0 0 0 dilutions o d v a lu e s rabbit 1 rabbit 2 rabbit 3 rabbit 4 fig. 3 optical density values (490 nm) obtained in a cotyledoside elisa for the rabbit sera on day 61. rabbits 3 and 4 were controls 312 potential krimpsiekte vaccine cotyledoside challenge in sheep the cotyledoside challenge was suspended when clinical signs reminiscent of krimpsiekte were elicited. one of the sheep developed jerky, forced abdominal respiration and made grunting noises shortly after the third injection administered in the morning. she became paretic during the afternoon and tended to assume the krimpsiekte posture the next day (fig. 5). due to ethical considerations no further cotyledoside was administered to this ewe. it transpired that this was a control animal. another ewe had rumen stasis on the 4th day, but did not exhibit signs of paresis and was standing. this animal was not judged to be severely affected and it was decided to administer another dose of cotyledoside to this animal. however, shortly thereafter, her condition rapidly deteriorated and she developed severe respiratory distress and died during the afternoon. it was then revealed that this was also a control sheep. as no sign of toxicity was observed in the other two immunized ewes the daily cotyledoside administrations were continued for a further 2 days (in total six daily injections). these sheep remained clinically unaffected and the challenge was stopped as they had received cotyledoside for a longer period (1.5– 2.0 times) than the control animals. these two sheep remained clinically unaffected for 6 months. discussion this preliminary trial verified that the cotyledosideprotein conjugate used did indeed induce the formation of high anticotyledoside antibody titres in rabbits and sheep. in addition, the prophylactic potential of this “monovalent vaccine” was demonstrated in the limited challenge study in the sheep. the cotyledoside conjugate induced an immunological response, which prevented cotyledoside-induced krimp siekte in the two sheep immunized with it. the death of one of the control animals was unexpected as none of the sheep in previous experiments died after receiving similar amounts of cotyledoside intravenously, and although affected, following cessation of the administration of the toxin they all recovered spontaneously or after administration of strained ruminal content (botha et al. 1997; botha, rundberget, swan, mülders, flåøyen 2003). 0 0.5 1 1.5 2 2.5 1: 20 0 1: 40 0 1: 80 0 1: 1 60 0 1: 3 20 0 1: 6 40 0 1: 12 8 00 1: 25 6 00 dilutions o d v al u es sheep 10 sheep 12 sheep 14 sheep 16 fig. 4 optical density values (490 nm) obtained in a cotyledoside elisa for the sheep sera on day 42. sheep 10 and 16 were controls fig. 5 affected sheep (sheep 16) assuming the krimpsiekte posture 313 c.j. botha et al. as the duration of challenge of the two cotyledoside-conjugate immunized sheep was 1.5–2.0 times longer than that of the other two sheep, the daily cotyledoside administration to them was stopped to prevent saturation of the induced antibodies with cotyledoside. this could eventually have resulted in excess circulating cotyledoside and, as consequence, death. it can be concluded that immunization of sheep and goats can most probably be utilized as a means of preventing krimpsiekte. one possible drawback is that animals with high anti body titres might be able to accumulate toxic amounts of cotyledoside, which could be released when the antibody-toxin complex is degraded (butler et al. 1977a and b). however, the cotyledosideimmunized sheep remained asymptomatic for 6 months after the challenge period, which provides some evidence that release of cotyledoside from degrading antibodies will not result in recrudescence of toxicity. another disadvantage of krimpsiekte immunoprophylaxis is the perceived interference in the elimination of cotyledoside and its subsequent retention, which would greatly increase the total body burden of cotyledoside. thus, carcasses of animals protected by immunization might not be suitable for human consumption (edgar 1994; silbart et al. 1997; botha 2003). however, secondary poisoning from consumption of immunized animals that have consumed t. wallichii may or may not constitute a problem. conceivably, such protected animals could dispose of antibody-bound glycoside by metabolic degradation or conjugation, e.g. to sulphates and glucuronides, which could be excreted in bile. another question that needs to be addressed is the degree of cross-reactivity between different krimpsiekte-inducing cumulative bufadienolides. in preliminary trials, similar vaccines produced from cardenolides (digoxin, ouabain) and non-cumulative bufadienolides (proscillaridin) were unable to protect small stock from acute poisoning induced by dosing 4–5 g/kg milled, fresh t. wallichii leaves, ostensibly through lack of cross-immunity (j.p.j. joubert, unpublished data 1985). this lack of crossimmunity can possibly be circumvented by preparing a polyvalent vaccine consisting of the major bufadienolides. the above drawbacks, however, should not place any constraints on continuing this preliminary investigation. in addition, the antibodies produced could conceivably be used diagnostically in an immunoassay to detect exposure of sheep (live or slaughtered) to cotyledoside and related bufadienolides. acknowledgements authors m.n. stojanovic and v.p. butler were supported by the national science foundation, usa, grants ccf-0621600 and che-0533096. m.n. stojanovic is a leukemia and lymphoma so ciety scholar. references botha, c.j., van der lugt, j.j., erasmus, g.l., kellerman, t.s., schultz, r.a. & vleggaar, r. 1997. krimpsiekte, associated with thalamic lesions, induced by the neurotoxic cardiac glycoside, cotyledoside, isolated from tylecodon wallichii (harv.) toelken subsp. wallichii. onderstepoort journal of veterinary research, 64:123–128. botha, c.j., kellerman, t.s., schultz, r.a., erasmus, g.l., vleggaar, r. & retief, e. 1998. krimpsiekte in a sheep following a single dose of tylecodon ventricosus (burm.f.) toelken and the isolation of tyledoside d from this plant species. onderstepoort journal of veterinary research, 65:17–23. botha, c.j., rundberget, t., swan, g.e., mülders, m.s.g. & flåøyen, a. 2003. toxicokinetics of cotyledoside following intravenous administered to sheep. journal of the south african veterinary association, 74:7–10. botha, c.j. 2003. krimpsiekte, a paretic/paralytic syndrome of small stock in south africa. ph.d. thesis, norwegian school of veterinary science, oslo. butler, v.p. & chen, j.p. 1967. digoxin specific antibodies. proceedings of the national academy of sciences, 57:71–78. butler, v.p., schmidt, d.h., smith, t.w., haber, e., raynor, b.d. & demartini, p. 1977a. the effects of sheep digoxin-specific antibodies and their fab fragments on digoxin pharmacokinetics in dogs. the journal of clinical investigation, 59:345–359. butler, v.p., smith, t.w., schmidt, d.h. & haber, e. 1977b. immunological reversal of the effects of digoxin. federation proceedings, 36:2235–2241. edgar, j.a. 1994. vaccination against poisoning diseases, in plant-associated toxins, agricultural, phytochemical and ecological aspects, edited by s.m. colegate & p.r. dorling. wallingford: cab international. erlanger, b.f., borek, f., beiser, s.m. & lieberman, s. 1957. steroid-protein conjugates. i. preparation and characterization of conjugates of bovine serum albumin with testosterone and with cortisone. journal of biological chemistry, 228:713–727. kellerman, t.s., naudé, t.w. & fourie, n. 1996. the distribution, diagnosis and estimated economic impact of plant poisonings and mycotoxicoses in south africa. onderstepoort journal of veterinary research, 63:65–90. kellerman, t.s., coetzer, j.a.w., naudé, t.w. & botha, c.j. 2005. plant poisonings and mycotoxicoses of livestock in southern africa, 2nd ed. cape town: oxford university press. schmidt, d.h. & butler, v.p. 1971 immunological protection against digoxin toxicity. journal of clinical investigation, 50: 866–871. schmidt, d.h., kaufman, b.m. & butler, v.p. 1974 persistence of hapten-antibody complexes in the circulation of immunized animals after a single intravenous injection of hapten. journal of experimental medicine, 139:278–294. 314 potential krimpsiekte vaccine silbart, l.k., rasmussen, m.v. & oliver, a.r. 1997. immunoprophylactic intervention in chemical toxicity and carcinogenicity. veterinary and human toxicology, 39:37–43. than, k.a., anderton, n., cockrum, p.a., payne, a.l. stewart, p.l. & edgar, j.a. 1994. lupinosis vaccine: positive relationship between anti-phomopsin igg concentration and protection in victorian field trials, in plant-associated toxins, agricultural, phytochemical and ecological aspects, edited by s.m. colegate & p.r. dorling, wallingford: cab international. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) stuart j.g. gordon institute of veterinary, animal and biomedical sciences, massey university, new zealand charlotte bolwell institute of veterinary, animal and biomedical sciences, massey university, new zealand chris w. rogers institute of veterinary, animal and biomedical sciences, massey university, new zealand godfrey musuka mailman school of public health, columbia university, zimbabwe patrick kelly ross university school of veterinary medicine, st. kitts, west indies alan guthrie equine research centre, university of pretoria, south africa philip s. mellor the pirbright institute, pirbright, united kingdom chris hamblin the pirbright institute, pirbright, united kingdom citation gordon, s.j.g., bolwell, c., rogers, c.w., musuka, g., kelly, p., guthrie, a. et al., 2017, ‘a serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in zimbabwe’, onderstepoort journal of veterinary research 84(1), a1505. https://doi.org/10.4102/ojvr.v84i1.1505 research communication a serosurvey of bluetongue and epizootic haemorrhagic disease in a convenience sample of sheep and cattle herds in zimbabwe stuart j.g. gordon, charlotte bolwell, chris w. rogers, godfrey musuka, patrick kelly, alan guthrie, philip s. mellor, chris hamblin received: 07 july 2017; accepted: 19 sept. 2017; published: 14 nov. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a convenience sample of sheep and cattle herds around the cities of harare, kwekwe and bulawayo, located in the highveld region of zimbabwe, was used to estimate the sero-prevalence and sero-incidence of bluetongue virus (btv) and epizootic haemorrhagic disease virus (ehdv) antibodies. a competitive enzyme-linked immunosorbent assay was used to identify serum antibodies against btv and ehdv across three rainy seasons. the median sero-prevalence of btv and ehdv antibodies in cattle was 62% (interquartile range [iqr]: 30–89) and 56% (iqr: 5–77), respectively. in sheep, the median sero-prevalence of btv and ehdv was 41% (iqr: 19–63) and 0% (iqr: 0–21), respectively. median sero-incidences of btv and ehdv antibodies in cattle of 43% (iqr: 22–67) and 27% (iqr: 9–57) respectively were recorded. the median sero-incidence of btv in sheep was 14% (iqr: 6–23). based on these preliminary findings, animal health workers in zimbabwe should continue to monitor the exposure rates of cattle and sheep to btv and consider the possibility of strains emerging with increased pathogenicity. there are no previous published reports of antibodies against ehdv in zimbabwe so the possibility of epizootic haemorrhagic disease existing in domestic livestock should now be considered by zimbabwean animal health officials. seroconversions to btv and ehdv occurred predominantly at the end of each rainy season (march and april), which generally corresponds to high numbers of the culicoides vectors. btv isolations were made from three individual cows in two of the sentinel herds and all three were identified as serotype 3. this is the first time btv serotype 3 has been recorded in zimbabwe, although its presence in neighbouring south africa is well documented. introduction bluetongue virus (btv) and epizootic haemorrhagic disease virus (ehdv) are members of the orbivirus genus within the reoviridae family and are transmitted between ruminants by biting midges of the genus culicoides (diptera: ceratopogonidae). twenty six distinct btv serotypes have been recognised and seven serotypes for ehdv have been identified (cêtre-sossah et al. 2014; maclachlan et al. 2015). while bluetongue (bt) remains endemic in many tropical and subtropical areas, where the vector population is abundant, this disease was previously considered to be exotic in europe, with only a few sporadic outbreaks in cyprus, greece and the iberian peninsula prior to 1998 (musuka & kelly 2000). however, several different btv serotypes have since been recorded in a number of mediterranean regions and in 2006 the first btv cases were identified in north-western europe in domestic ruminants (orłowska et al. 2016). epizootic haemorrhagic disease (ehd) has been endemic in north america since 1955 affecting mainly white-tailed deer and, more rarely, cattle and sheep. ehdv has been isolated from epidemics in australia, south-east asia and africa, and more recently on reunion island and in countries surrounding the mediterranean basin (cêtre-sossah et al. 2014; maclachlan et al. 2015). higher mortalities from bt and ehd tend to occur in areas where the diseases are not endemic. cyprus and spain have recorded mortality rates up to 70% and 75%, respectively, in bt outbreaks in sheep (gambles 1949; manso-ribeiro et al. 1957). during an ehd outbreak in japan in 1959, 10% of the cattle infected with the ibaraki strain of ehdv serotype 2 died (thevasagayam 1998). bt is an important endemic disease of ruminants in zimbabwe with the occurrence of the btv being previously documented (blackburn, searle & phelps 1985; jorgensen, halliwell & honhold 1989; mushi et al. 1990; musuka & kelly 2000). mortality rates from bt have reached over 33% in sheep in zimbabwe (musuka 1999). no clinical cases of ehd have been reported in zimbabwe to date and there are no published data on the sero-prevalence or sero-incidence of antibodies against ehdv in ruminants in the country (gordon 2010; musuka 1999). although one study has isolated btv from culicoides imicola and culicoides magnus (gordon et al. 2015), the vector competence of other culicoides species in zimbabwe has not been documented and needs further investigation. the aim of this study was, therefore, to contribute further information on the epidemiology of these two orbiviruses in zimbabwe by determining the sero-prevalence and sero-incidence of btv and ehdv in cattle and sheep in select ruminant and sentinel herds established around three zimbabwean highveld towns. some of the viruses, isolated from blood samples from the animals in the sentinel herds that had seroconverted, were also identified. materials and methods identification of sampling herds blood samples collected from cattle and sheep were used to determine the sero-prevalence and sero-incidence of btv and ehdv on six selected farms located in the central highveld plateau of zimbabwe, adjacent to the cities of harare, kwekwe and bulawayo (figure 1). the climate in these regions favours culicoides vectors. farms were selected on the basis of age (> 1 year), herd size (> 8) and owners’ cooperation and were typical of the zimbabwean highveld, having similar soil, vegetation and agricultural practices (gordon 2010; gordon et al. 2015; musuka 1999). the cattle and sheep on these premises had no history of prophylactic immunisation against btv or ehdv, which meant that subsequently detected antibodies against these viruses would have been derived from natural challenge. figure 1: the location of the three highveld cities in zimbabwe around which the sampling herds were established. detection of antibodies against bluetongue virus and epizootic haemorrhagic disease virus to determine sero-prevalence venous blood samples (5 ml) were collected in ethylenediaminetetraacetic acid (edta) vacutainers from a total of 137 cattle and 72 sheep on the participating farms at the start of the three rainy seasons (1999/2000; 2000/2001; 2001/2002). plasma, separated by centrifugation from these blood samples, was assayed by competitive enzyme-linked immunosorbent assays (c-elisa) for btv antibodies (afshar et al. 1987, 1997; anderson 1984) and ehdv antibodies (thevasagayam et al. 1995). detection of antibodies against bluetongue virus and epizootic haemorrhagic disease virus to determine sero-incidence animals that were negative for antibodies against btv and/or ehdv by c-elisa at the beginning of each rainy season were used to establish the sentinel herds. sentinel herds consisted of a total of 44 sheep and 65 cattle. four sentinel herds, two of cattle and two of sheep, were established on farms near harare. two sentinel herds, one of cattle and one of sheep, were established outside kwekwe and two similar sentinel herds were established in the bulawayo area. whole blood was collected in edta vacutainers every 2 weeks throughout each rainy season (november–april). plasma was separated by centrifugation and assayed for antibodies against btv and ehdv, using the c-elisa methods described above. no further samples were collected from animals once they had seroconverted. virus isolation and serotyping the centrifuged red blood cells from the edta blood samples collected 2 weeks before sentinel animals seroconverted to either btv or ehdv were washed and lysed following methods described by hamblin et al. (1992) and musuka and kelly (2000). the chance of isolating free virus was improved by assaying the blood samples collected before animals seroconverted. virus isolations were made by intravenous inoculation of 11-day-old embryonated hens’ eggs (iah, uk) and by intracerebral inoculation of 1to 2-day-old suckling mice (cd-1, uk) using techniques described by gordon et al. (2017). the viruses isolated were typed using virus type-specific antisera and bhk-21 cells in microtitre plates using neutralisation test methods modified from hazrati and ozawa (1965). statistical analysis data were described using simple descriptive statistics. the non-parametric data have been presented as medians with interquartile ranges (iqrs). the shapiro–wilk test was used to test the data for normality, while a kruskal–wallis test was used to test the effect of season on the median prevalence of btv and ehdv. stata ic v12 (college station, tx, usa) was used to perform all analyses, with levels of significance set at p < 0.05. results bluetongue virus and epizootic haemorrhagic disease virus sero-prevalence and sero-incidence the results generated by c-elisa for the 137 cattle samples showed that the small per-farm sample size (12; iqr: 10–21) resulted in a large between-farm variation in the btv and ehdv sero-prevalence (table 1). the highest btv and ehdv sero-prevalence was recorded for samples collected during the 2000/2001 season. however, there was only a significant difference recorded between the sero-prevalence of antibodies to ehdv in cattle over the three seasons (p = 0.043). the median sero-prevalence of btv and ehdv in the cattle sampled across the three rainy seasons studied was 62% (iqr: 30–89) and 56% (iqr: 5–77), respectively. table 1: the sero-prevalence of antibodies against bluetongue virus and epizootic haemorrhagic disease virus in cattle in selected zimbabwean highveld herds across three rainy seasons. the results from the 72 sheep also showed that the small per-farm sample size (12; iqr: 11–13) resulted in a large between-farm variation in the btv and ehdv sero-prevalence (table 2). the sero-prevalence of btv antibodies in sheep was highest in the third season (2001/2002), but the difference between years was not significant (p = 0.513). the median sero-prevalence of btv and ehdv over the three seasons was 41% (iqr: 19–63) and 0% (iqr: 0–21), respectively. table 2: the sero-prevalence of antibodies against bluetongue virus and epizootic haemorrhagic disease virus in sheep in selected zimbabwean highveld herds across three rainy seasons. the nine sentinel cattle herds, established over the three rainy seasons, comprised a total of 65 cattle with a median of 9 (iqr: 5–14) cattle per farm (table 3). the small sentinel herd sample size resulted in a large between-sentinel herd variation in the sero-incidence of btv and ehdv. median sero-incidences for btv and ehdv in cattle of 43% (iqr: 22–67) and 27% (iqr: 9–57), respectively, were recorded. table 3: the sero-incidence of bluetongue virus and epizootic haemorrhagic disease virus in cattle in selected zimbabwean highveld herds across three rainy seasons. the six sentinel sheep herds, established over the three rainy seasons, comprised a total of 44 sheep, with a median of 9 (iqr: 7–11) sheep per farm. only one sheep seroconverted against ehdv and seven sheep converted against btv across all three seasons. the median sero-incidence of btv over the three seasons was 14% (iqr: 6–23). viral serotyping virus isolations were made from three individual cows from two of the farms where the sentinel cattle were located during the 2001/2002 season. the virus type, determined by neutralisation assays for each isolate, was btv serotype 3. no ehdv isolations were made. discussion sheep and cattle in the selected herds in this study were commonly exposed to btv, concurring with previous studies in zimbabwe. musuka and kelly (2000) recorded sero-prevalences of antibodies against btv in sheep and goats of up to 100% in the north-east of zimbabwe while jorgensen et al. (1989) reported an overall sero-prevalence of 71% in indigenous goats at 25 farms across zimbabwe. prevalence rates of btv antibodies, ranging from 64% to 84%, have been reported in sheep and goat populations in south africa (gerdes 2004). in north africa, btv sero-prevalences in algeria were found to be only 14% in sheep and 29% in cattle (madani et al. 2011). in 455 calves sampled in kenya, btv antibodies were found in 94% of these animals (toye et al. 2013). cattle rarely show clinical signs of bt, however, and are, therefore, not vaccinated in zimbabwe (musuka & kelly 2000). it should be noted, however, that the strain of btv serotype 8, which recently emerged in europe, appears more pathogenic in cattle than is usually the case for btv (maclachlan & guthrie 2010) so the situation in zimbabwe may change. consequently, animal health officials in zimbabwe should continue to monitor the exposure rates of cattle to btv and consider the possibility of emerging btv strains with increased pathogenicity. cattle sentinel herds recorded high sero-prevalences and sero-incidences of antibodies against ehdv in the selected highveld populations in zimbabwe. antibodies against ehdv were detected in 64% of calves tested in kenya (toye et al. 2013) and in 38% of cattle and sheep sampled on reunion island (desvars et al. 2015). the low sero-prevalence and sero-incidence rates against this disease recorded in sheep suggest that cattle, which are likely to be the preferred hosts of culicoides vector species, as in other countries, may be a more natural host of this virus in zimbabwe, compared to sheep. whereas ehdv can cause a fulminant haemorrhagic disease syndrome in white-tailed deer, similar infections of ruminant livestock are usually subclinical or clinically inapparent. nonetheless, it is now widely accepted that ehdv infections in some circumstances have resulted in disease in cattle (maclachlan et al. 2015). maclachlan et al. (2015) reported strains of ehdv serotypes 2, 6 and 7 as the apparent recent cause of a bt-like disease syndrome of cattle in northern and southern africa, reunion island, north america and the mediterranean basin, including algeria, israel, jordan, morocco, tunisia and turkey. affected cattle in north africa and israel exhibited bt-like disease signs when infected with ehdv serotype 7 (maclachlan & guthrie 2010). ibaraki disease, an acute febrile disease in cattle caused by a variant of ehdv serotype 2, was first reported in the central and western parts of japan in 1959 (inaba 1975). the host and virus factors that may lead to the expression of ehd in cattle remain, however, undetermined (maclachlan et al. 2015). in zimbabwe, the sero-prevalence of antibodies against ehdv was, however, lower than the sero-prevalence of btv antibodies in both cattle and sheep, suggesting that domestic ruminants in this country are less susceptible to ehdv than btv, or that the vector species of culicoides, which transmit ehdv, are different to those that transmit btv in zimbabwe. venter (2015) classified c. imicola as the most important vector of btv to livestock in south africa. in the sudan, mellor, osborne and jennings (1984) isolated btv only from c. imicola while ehdv isolates were made from culicoides schultzei group midges. lee (1979) also reported the isolation of ehd group viruses from c. schultzei collected in nigeria. gordon et al. (2015) recorded c. imicola as the most abundant species trapped in zimbabwe and only recorded very low numbers of schultzei group midges. the highest prevalence of both btv and ehdv in cattle was detected during the second rainy season (2000/2001). this was a season which recorded above-average rainfall (department of meteorological services, belvedere, harare, zimbabwe). during each rainy season, most seroconversions to the btv and ehdv occurred towards the end of the season. mellor (1990) states that this delay reflects the increase in vector numbers and the viraemia, which develops in the infected ruminants, providing a source of infection for further vectors. large populations of culicoides vectors have been reported previously in zimbabwe in areas with high average annual rainfall and summer temperatures (gordon et al. 2015; musuka et al. 2001). large catches of culicoides have also been recorded in the warm, summer rainfall areas of south africa (venter 2015; venter, nevill & van der linde 1997). coetzee et al. (2012) state that bt is commonly reported in south africa in late summer in areas with high rainfall and after good rains. rainfall data would need to be utilised in future studies to demonstrate the association between rainfall, culicoides numbers and the sero-incidence of btv and ehdv in zimbabwe. in this study, the only btv serotype identified was serotype 3. while this serotype has not been previously reported in zimbabwe, coetzee et al. (2012) reported that serotypes 1–6 are commonly encountered in neighbouring south africa. sghaier et al. (2017) recently identified serotype 3 in tunisia while desvars et al. (2015) attributed btv-3 as the cause for a clinical outbreak in merino sheep on reunion island. previous studies have identified btv serotypes 1, 2, 8, 10, 11, 12, 15, 16 and 18 from culicoides vectors in zimbabwe, although these studies were also restricted to samples from the highveld region (blackburn et al. 1985; gordon et al. 2015). as yet, no data exist on the distribution of btv or ehdv serotypes in the middleveld or lowveld plateaus of zimbabwe. conclusion these results report on the sero-prevalence of btv and ehdv in selected ruminant herds on the highveld of zimbabwe. most seroconversions to btv and ehdv occurred in march and april towards the end of each rainy season, which coincides with the peak abundance of culicoides vectors. the btv serotype isolated and identified in the blood samples from sentinel herd animals, prior to seroconversion, was serotype 3 which, while not reported previously in zimbabwe, is commonly encountered in south africa. furthermore, btv serotyping in all zimbabwean studies to date has only been with samples collected in highveld locations making it difficult to evaluate their national distribution. while the findings in this study are somewhat dated, no subsequent work has been conducted in zimbabwe on these orbiviruses, highlighting the need for further research on the distribution of btv and ehdv in zimbabwe. there have been no previous published reports of ehd in shown so these findings have shown the presence of antibodies against ehdv in domestic livestock in another african country. acknowledgements the authors acknowledge the cooperation provided by the cattle and sheep establishments used in this study. this study was made possible by the generous support of the wellcome trust and the biomedical research and training institute. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions the 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‘seasonal abundance and parity of stock-associated culicoides species (diptera: ceratopogonidae) in different climatic regions in southern africa in relation to their viral vector potential’, onderstepoort journal of veterinary research 64, 259–271. abstract introduction material and methods results discussion conclusion acknowledgements references about the author(s) eran dvir department of companion animal clinical studies, faculty of veterinary science, university of pretoria, pretoria, south africa tel hai academic college, upper galilee, israel chantal rosa department of companion animal clinical studies, faculty of veterinary science, university of pretoria, pretoria, south africa northwest veterinary specialists, runcorn, united kingdom ian handel royal (dick) school of veterinary studies and the roslin institute, university of edinburgh, scotland, united kingdom richard j. mellanby royal (dick) school of veterinary studies and the roslin institute, university of edinburgh, scotland, united kingdom johan p. schoeman department of companion animal clinical studies, faculty of veterinary science, university of pretoria, pretoria, south africa citation dvir, e., rosa, c., handel, i., mellanby r.j. & schoeman, j.p., 2019, ‘vitamin d status in dogs with babesiosis’, onderstepoort journal of veterinary research 86(1), a1644. https://doi.org/10.4102/ojvr.v86i1.1644 original research vitamin d status in dogs with babesiosis eran dvir, chantal rosa, ian handel, richard j. mellanby, johan p. schoeman received: 20 apr. 2018; accepted: 03 dec. 2018; published: 28 mar. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract canine babesiosis is a virulent infection of dogs in south africa caused principally by babesia rossi. hypovitaminosis d has been reported in a wide range of infectious diseases in humans and dogs, and low vitamin d status has been associated with poor clinical outcomes. however, the relationship between vitamin d status and canine babesiosis has not been investigated. the objective of this study was to examine the relationship between the presence and severity of b. rossi infection and vitamin d status of infected dogs. owners with dogs with a confirmed diagnosis of b. rossi infection and of healthy control dogs were invited to enrol onto the study. vitamin d status was assessed by measurement of serum concentrations of the major circulating vitamin d metabolite, 25-hydroxyvitamin d (25[oh]d). dogs with babesiosis (n = 34) had significantly lower mean serum 25(oh)d concentrations than healthy dogs (n = 24) (37.76 ± 21.25 vs. 74.2 ± 20.28 nmol/l). the effect of babesiosis on serum 25(oh)d concentrations was still significant after adjusting for any effect of age, body weight and sex. there was a negative relationship between serum 25(oh)d concentrations and disease severity in dogs with babesiosis. serum concentrations of creatinine and alanine aminotransferase and time to last meal were not associated with serum 25(oh)d concentrations in dogs with babesiosis. in conclusion, dogs with babesia rossi infections had lower serum 25(oh)d concentrations than healthy dogs. the inverse correlation between 25(oh)d concentrations and the clinical severity score indicate that hypovitaminosis d might be a helpful additional indicator of disease severity. keywords: vitamin d; 25-hydroxyvitamin d; babesiosis; dog; babesia rossi. introduction babesiosis is a disease of considerable global significance that causes morbidity and mortality in humans and many domestic and wild animals, including dogs (mellanby et al. 2011; schoeman 2009). canine babesiosis is characterised by a wide range of clinical signs including pallor related to the anaemic state and often icterus (schoeman 2009). the clinical course can vary from chronic or subclinical through to peracute and fatal, depending on the virulence of the species and the susceptibility of the host (bohm et al. 2006). even in referral centres with access to advanced veterinary care, mortality rates range from 7% to 10% (bohm et al. 2006; schoeman et al. 2007). consequently, there is a clear need to understand more about the factors involved in modulating the risk of babesia infections in dogs and to develop therapeutic approaches that improve patient outcomes. the importance of vitamin d in maintaining skeletal health has been known for nearly a century (elder & bishop 2014). however, there is increasing interest in exploring the importance of vitamin d in regulating non-skeletal tissue following the discovery that a wide range of tissues, notably immune cells, can express the vitamin d receptor (mellanby 2016; provvedini et al. 1983). these observations resulted in numerous studies that have examined the relationship between vitamin d status, typically assessed by serum concentrations of the major vitamin d metabolite 25-hydroxyvitamin d (25[oh]d), and the development and outcome of a wide range of infectious diseases (youssef et al. 2011). for example, a link has been made between vitamin d status and the development of mycobacteria, hiv and respiratory infections (kearns et al. 2015). studies in dogs and cats have similarly found a relationship between hypovitaminosis d and the presence of an infectious disease. such observations include a study of spirocerca lupi infections in dogs that found that infected dogs had a lower vitamin d status than healthy dogs (rosa et al. 2013). in addition, cats with either mycobacteria or feline immunodeficiency virus (fiv) infections were also found to have lower 25(oh)d concentrations than healthy cats (lalor et al. 2012; titmarsh et al. 2015b). although there have been very few studies that have examined the relationship between vitamin d status and babesiosis in any species (kules et al. 2014; mert et al. 2009), numerous studies have linked low vitamin d status to both the development and outcome of infections in human and experimental models of malaria, a disease that has a similar pathophysiology to canine babesiosis (luong & nguyen 2015; reyers et al. 1998). for example, plasma 25(oh)d concentrations were lower in children with severe malaria compared to healthy children (cusick et al. 2014). furthermore, supplementation with vitamin d ameliorated experimental cerebral malaria in mice (he et al. 2014). lastly, the administration of vitamin d alongside arteether improved survival in mice with cerebral malaria compared to either agent used alone (dwivedi et al. 2016). a proteomic approach revealed that vitamin d binding protein concentrations were lower in dogs with babesiosis, indicating that vitamin d metabolism may be altered in infected dogs (kules et al. 2014). the hypothesis of this study was that dogs with babesiosis would have a lower vitamin d status than healthy dogs. the aim of the study was to measure serum concentrations of 25(oh)d in healthy dogs and in dogs with confirmed babesiosis. material and methods selection of cases this prospective study was performed on dogs with canine babesiosis presented to the onderstepoort veterinary academic hospital (ovah) of the university of pretoria in south africa. an initial diagnosis was made upon admission and included detection of large babesia spp. parasites on a stained thin capillary blood smear. dogs were excluded from the study if they were less than 1 year old and had a history of previous exogenous corticosteroid therapy, known concurrent disease or ehrlichia canis morulae detected on the blood smear. after admission the dogs were further excluded if concurrent disease was identified during their hospital stay or if their blood samples were positive for babesia vogeli or e. canis by polymerase chain reaction (pcr) and reverse line blot (rlb) (matjila et al. 2004). the pcr was conducted with a set of primers that amplified a 460–540 base pair fragment of the 18s small subunit (ssu) rrna spanning the v4 region, a region conserved for babesia and theileria. the ehrlichia pcr amplified the v1 hypervariable region of the 16s ssu rrna (bekker et al. 2002; schouls et al. 1999). the membrane used for rlb included probes for b. vogeli, babesia rossi, b. canis and e. canis. twenty-four healthy dogs were used as the control group. these dogs were admitted to the ovah for blood donation as part of the ovah blood bank. they were considered healthy on the basis of a physical examination and routine laboratory testing (i.e. haematology and serum biochemical analysis). study design a physical examination was conducted, and history, including patient age, sex and duration of illness, was determined. blood was collected upon admission and all dogs with babesiosis had a haematology and serum biochemistry analysis performed. blood was taken from the jugular vein by needle venepuncture in all cases. serum samples were allowed to clot and the tubes were spun down within 1 h. the serum was used for general diagnostic serum biochemistry and an additional tube was placed in dedicated plastic storage tubes and stored at -80 °c until 25(oh)d analysis. the severity of the babesiosis cases was scored using the following parameters, with each criteria receiving a score of 1: basal serum cortisol > 275 nmol/l, basal plasma acth > 20 pg/ml, basal serum t4 < 2.8 nmol/l, serum albumin < 20 g/dl, serum potassium < 3.6 mmol/l, serum phosphate > 1.94 mmol/l, serum creatinine > 133 µmol/l, 2, serum bile acids ≥ 55 µmol/l, serum glucose < 3.3 mmol/l, presence of icterus, per cent of band cells of the total white cell count > 10% and body temperature < 37.8 °c (bohm et al. 2006; keller et al. 2004; nel et al. 2004; schoeman et al. 2007). each case received an accumulated score (maximum 12). in the healthy dogs, anamnesis, haematology, serum biochemistry, urinalysis and faecal analysis, including a modified centrifugal faecal flotation, were performed to rule out canine babesiosis or any other systemic disease. exclusion criteria for all dogs of all groups included the following: less than 1 year of age, concurrent diseases, treated with medications that could influence vitamin d concentrations (corticosteroids, anticonvulsants, calcium channel blockers, diuretics). the appetite of all dogs diagnosed with babesiosis was recorded as time (in hours) since the last meal, based on information obtained from the owners. serum samples for 25(oh)d analysis were frozen within 1 hour of collection and stored at -80 °c prior to being sent to the laboratory for analysis on dry ice. serum 25(oh)d concentrations were measured and validated as described in detail elsewhere (gow et al. 2011; mawer et al. 1990). samples were extracted using acetonitrile and applied to c18 silica sep-paks and metabolites separated by straight phase high-performance liquid chromatography. a hewlett-packard zorbax-sil column eluted with hexane:propan-2-ol:methanol (92:4:4) was used. then serum 25(oh)d2 and 25(oh)d3 were measured separately by application to a second zorbax-sil, the column eluted with hexane:propan-2-ol (98:2) and quantified by uv absorbance at 265 nm (radioimmunoassay) and corrected for recovery (sensitivity 5 nmol/l, intraand inter-assay coefficients of variation 3.0% and 4.2%, respectively). results were expressed as total serum 25(oh)d concentrations. the specialist assay laboratory csb3 participates successfully in the vitamin d quality assurance scheme and is accredited to the international organization for standardization (iso) 9001:2008 and iso 13485:2003. statistical analyses general linear models were used to assess the relationship between variables of interest and 25(oh)d concentrations. residual models were inspected for normality, heteroscedasticity and trend with other variables to assess model assumptions. models were fitted using the r statistical system. a p-value of < 0.05 was considered statistically significant in all statistical tests performed. ethical considerations the dogs were only included in the study with owner consent. the study was reviewed and approved by the animal use and care committee of the university of pretoria (protocol v070-05). results thirty-four dogs with babesiosis and 24 healthy control dogs were included in the study. dogs included in both groups were of different breeds and more than 1 year of age. the common breeds in the babesiosis group were cross breed (10), boerboels (4), german shepherd dogs (4), fox terriers (3) and other breeds with ≤ 2 dogs. their age ranged from 12 to 156 months (median 24 months). the common breeds in the control group were beagles (5), boerboels (4), greyhounds (3), fox terriers (3) and other breeds with ≤2 dogs. their age ranged from 12 to 120 months (median = 30 months). the babesiosis group had significantly lower mean serum 25(oh)d concentrations than the control group (37.8 ± 21.3 vs. 74.2 ± 20.3 nmol/l; figure 1). the effect of babesiosis on serum 25(oh)d concentrations was still significant after adjusting for any effect of age, body weight and sex (table 1). figure 1: serum 25-hydroxyvitamin d concentrations in dogs with babesia and healthy dogs. the black line indicates the mean concentration in each group. table 1: estimates from linear models of 25-hydroxyvitamin d concentrations exploring the relationship between vitamin d status and the presence of babesiosis, adjusting for other co-variables. the severity score of the babesiosis cases ranged from 0 to 10 with a median of 2. the negative relationship between serum 25(oh)d concentrations and disease severity was still significant after adjusting for any effect of age, body weight and sex (table 2). the relationship between 25(oh)d concentrations in canine babesiosis and alanine aminotransferase (alt), creatinine and time since last meal was explored in a linear model including additional co-variables of age, weight and sex. none of the clinical or biochemical parameters were associated with 25(oh)d concentrations in dogs with babesiosis. table 2: estimates from linear models of 25-hydroxyvitamin d concentrations in dogs with babesiosis, exploring the relationship between vitamin d status and disease severity and adjusting for other co-variables. discussion this study revealed that hypovitaminosis d is associated with canine babesiosis and that 25(oh)d concentrations are lower in patients with more severe disease. reduced serum 25(oh)d concentrations could be attributed to anorexia or alterations in hepatic function. however, our analysis revealed that the decrease in 25(oh)d concentrations in dogs with babesiosis was independent of serum creatinine and alt, thereby suggesting that the effect of renal insufficiency or liver damage was unlikely to be responsible for development of hypovitaminosis d. moreover, our analysis revealed that hypovitaminosis d was not significantly correlated with the time from the last meal. our findings are consistent with previous studies, which have identified an association between hypovitaminosis d and the presence of an infectious disease and inflammation (holowaychuk et al. 2012). examples of recent studies showing this relationship are in dogs with leishmaniasis (rodriguez-cortes et al. 2017) and s. lupi infections (rosa et al. 2013) and in cats with mycobacteriosis (lalor et al. 2012) and fiv infection (titmarsh et al. 2015b). the association between disease severity and vitamin d status identified in this study, with lower vitamin d concentrations associated with more severe disease, is also consistent with several previous studies in companion animals. serum 25(oh)d concentrations were found to be negatively correlated to disease severity in studies of canine chronic enteropathies (titmarsh et al. 2015a) and canine congestive heart failure (osuga et al. 2015). the findings of our study differed from a smaller analysis of babesiosis in sheep, which found no difference in vitamin d status in infected and control animals (mert et al. 2009). the relationship between low vitamin d status and human malaria has also been extensively reported, with the pathophysiology of human malaria sharing many similarities with canine babesiosis (luong & nguyen 2015). children with severe malaria were found to have significantly lower 25(oh)d concentrations than community children (cusick et al. 2014). despite the growing evidence that infectious diseases in companion animals and humans are associated with low vitamin d status, it still remains unclear whether the low serum 25(oh)d concentrations are the cause or consequence of the infectious state. a longitudinal study of 25(oh)d concentrations in humans with malaria found that vitamin d status did not change during the course of the infection, suggesting that 25(oh)d concentrations are unaffected during the evolution of an infectious process (newens et al. 2006). the same could apply in canine babesiosis; however, additional research is required to more stringently examine whether low vitamin d status influences the risk of infectious disease development in both humans and companion animals. moreover, the hypovitaminosis d seen in canine babesiosis could be because of an excess of vitamin d consumption during the inflammatory process resulting from a rapid conversion of 25(oh)d to the bioactive form 1,25-dihydroxyvitamin d by inflammatory cytokines (autier et al. 2014). alternatively, hypovitaminosis d could be present prior to babesiosis infections and predispose these dogs to disease development and increased disease severity. the time of the meal did not appear to influence the vitamin d results, making this hypothesis less likely in this study, but other factors involved in vitamin d storage and turnover affecting the serum 25(oh)d concentrations prior to disease could not be excluded. ideally, the dietary vitamin d content should have also been evaluated in this study. hypovitaminosis d might have immunological consequences during the course of canine babesiosis infections, especially because vitamin d has a direct effect on the innate and adaptive immune response. the relationship between vitamin d status and immune function deserves further study in canine babesiosis, because it has been recently shown that dogs with canine babesiosis have alterations in their lymphocyte populations. a lower percentage of cd3+ and cd4+ lymphocytes were found in complicated babesiosis cases, and a lower percentage of cd8+ t lymphocytes was identified in babesiosis compared to healthy dogs, suggesting the presence of functional immune suppression (rautenbach et al. 2017). we have previously reported that the active vitamin d metabolite, 1.25-dihydroxyvitamin d, can profoundly influence the phenotype and function of innate immune cells ex vivo (besusso et al. 2015). whether vitamin d status alters disease susceptibility and the evolution of the infection in vivo remains unclear. in order to examine the relationship between vitamin d and infectious diseases, studies have investigated whether vitamin d supplementation can influence the outcome. in an experimental murine model of cerebral malaria, the co-administration of vitamin d alongside arteether was found to significantly improve survival. the improvement in outcome was associated with partially restored blood–brain barrier integrity and reduced serum pro-inflammatory cytokine concentrations (dwivedi et al. 2016). another study also reported that the oral administration of vitamin d, both before and after experimental malaria infection, protected mice from the development of cerebral malaria. there was reduced accumulation of pathogenic t cells in the brain and substantial improvement of the blood–brain barriers of malaria-infected mice in vitamin d–treated mice (he et al. 2014). based on these observations, further studies exploring the role of vitamin d supplementation in canine babesiosis would be informative. conclusion we have shown that dogs with b. rossi infections have lower serum concentrations of 25(oh)d. further studies are required to investigate whether vitamin d status plays a causative role in the development of babesiosis and whether improvement of vitamin d status can result in enhanced treatment outcomes. the inverse correlation between 25(oh)d concentrations and the clinical severity score indicate that hypovitaminosis d might be a helpful additional indicator of disease severity. acknowledgements the authors thank all the owners, vets and nurses involved in the care of the patients included in the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.d., r.j.m. and j.s. were responsible for experimental and project 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research, south africa michael crampton biosciences unit, council for scientific and industrial research, south africa boet weyers onderstepoort biological products, onderstepoort, south africa malefa tselanyane biosciences unit, council for scientific and industrial research, south africa tsepo tsekoa biosciences unit, council for scientific and industrial research, south africa rachel chikwamba biosciences unit, council for scientific and industrial research, south africa citation mokoena, t., chakauya, e., crampton, m., weyers, b., tselanyane, m., tsekoa, t. et al., 2017, ‘evaluation of plant-produced clostridium perfringens type d epsilon toxoid in a vaccine against enterotoxaemia in sheep’, onderstepoort journal of veterinary research 84(1), a1271. https://doi.org/10.4102/ojvr.v84i1.1271 original research evaluation of plant-produced clostridium perfringens type d epsilon toxoid in a vaccine against enterotoxaemia in sheep tinyiko mokoena, ereck chakauya, michael crampton, boet weyers, malefa tselanyane, tsepo tsekoa, rachel chikwamba received: 31 may 2016; accepted: 27 nov. 2016; published: 25 apr. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract enterotoxaemia (pulpy kidney) is a common bacterial disease of sheep caused by clostridium perfringens type d epsilon toxin. it has mortality rates of up to 30% in non-vaccinated animals. current vaccines from whole cell cultures are expensive to manufacture and can induce local inflammatory responses in sheep. they usually have reduced immunogenicity because of the difficulty of standardising the inactivation step in vaccine manufacturing. in the current study, we evaluated the safety and potency of a recombinant plant-made epsilon toxoid protein (r-etox) as an affordable and safer alternative vaccine for developing countries. results of injection site reactions, rectal temperature and toxin neutralisation test in single and prime–boost inoculations of mice, guinea pigs and sheep suggest that the product is not toxic to animals and could protect sheep against enterotoxaemia. introduction enterotoxaemia (commonly known as pulpy kidney disease) is a disease of economic importance in sheep, with a mortality rate of between 10% and 30% in non-vaccinated lambs. although it also affects calves and goats, the symptoms are less severe, causing a non-fatal subacute disease (morris et al. 2012). the disease is precipitated when sheep are fed a rich ration, such as green pastures, or if the diet is changed abruptly. the protein-rich material stimulates the growth of clostridium perfringens type d, an anaerobic bacterium which occurs in the intestinal tract, to multiply rapidly and to produce highly active epsilon toxin (etx) that has been shown to be the major cause of the disease. etx is produced as an inactive protoxin which is activated through a complex process involving trypsinisation where 13 and 29 amino acid residues are removed at the n-terminus and c-terminus, respectively (bokori-brown et al. 2011; garcia et al. 2013). the toxin causes an efflux of intracellular k+ ions resulting in cell apoptosis and sudden death of the animal (chandran et al. 2010; fennessey et al. 2012). farmers usually encounter dead animals in the morning and post-mortem examination shows oedema in many organs including the brain, heart, lungs and kidneys. because of the rapid progression of the disease, antibiotics are of no use and vaccination is the only solution (titball 2009). currently available vaccines in south africa are formalin-treated alum or oil emulsion of whole cell culture or bacterial filtrates of c. perfringens (c. welchii) type d. these vaccines usually have reduced immunogenicity because of the difficulty of standardising the inactivation step (mathur et al. 2010), and some components of the toxoids induce local inflammatory responses or allergic reactions (jiang et al. 2014). moreover, the media used for anaerobic fermentation of the bacteria are not readily available in many developing countries, including south africa, where it has to be imported, thereby making the manufacturing process expensive. plants have proven to be useful vehicles for producing high volume but low-cost biologics for different applications and could provide a solution to potential production challenges for enterotoxaemia. the world’s first regulatory opportunity for a plant-made vaccine for veterinary purposes occurred in early 2006 when a newcastle disease virus vaccine for chickens demonstrated technical and industrial feasible (sparrow et al. 2007). this provided a regulatory opportunity for other plant-made vaccines to be approved. since then, several viral antigens have been expressed in plants, including rabies, norwalk virus, anthrax, avian reovirus and avian influenza virus (gunn et al. 2012). the advantages of using plants as expression systems include vaccine antigen production that is safe, potentially more cost-effective and scalable than current methods (chen & lai 2013; rybicki et al. 2012; šmídková et al. 2010). moreover, plant tissues may act as adjuvants and increase immunogenicity of antigens in animals or, alternatively, plant materials can be freeze-dried to increase the antigen dose on a per-gram basis (wang & coppel 2008). moreover, a plant-made veterinary product in powder form can potentially be easily stored and transported under limited or no refrigeration without degradation (gunn et al. 2012). despite all these advantages, poor expression levels of some antigens and the complex regulatory problems surrounding plant-produced vaccines remain a barrier to commercialisation. in a recent study, rybicki et al. (2012) highlighted some of the hurdles in commercialising products in south africa including lack of financial support, lack of manufacturing infrastructure, and an uncertain regulatory and entrepreneurial environment for commercial development of novel plant-made products (rybicki et al. 2012). in the current study, we explore plant-based expression to demonstrate the feasibility of producing affordable vaccines for animals in developing countries. materials and methods to demonstrate expression, we used transient expression by agroinfiltration of nicotiana benthamiana plant leaves. for purposes of this study, the nomenclature etx refers to the c. perfringens type d epsilon toxin gene, r-etx is the recombinant epsilon protein produced from the plant matrix before activation by trypsinisation and r-etox refers to the plant-produced recombinant epsilon toxin protein after trypsinisation and formalin inactivation. to get to the stage of evaluating the toxicity and potency of the r-etox in animals, the experiments were conducted in three sequential steps viz: transient expression of recombinant c. perfringens epsilon toxin (r-etx) in n. benthamiana leaves, purification and toxin inactivation. transient expression of epsilon toxin open reading frame in nicotiana benthamiana the r-etx was produced by expressing a chemically synthesised (geneart, gmbh) n. benthamiana plant-codon optimised etx open reading frame (orf) sequence (ncbi: ay858558). the 864-bp sequence is a truncated version of the native gene with 13 n-terminus and 29 c-terminus amino acids removed. the orf sequences were cloned into the magnicon vector pich11599 (icon genetics, halle, germany) with plch17388 (apoplast protein targeting) or pich17620 (cytosolic targeting) together with the plch14011 (integrase) for transfection into plant leaves (figure 1a) using agrobacterium-mediated transfer using vacuum infiltration (giritch et al. 2006). once in planta the integrase assembles the 5′ and 3′ modules into a replication-competent tobacco mosaic virus. the assembled dna construct is then transcribed and spliced to generate a functional infective replicon that produces the r-etx protein. figure 1: expression of epsilon toxin open reading frame in nicotiana benthamiana. (a) schematic representation of the plasmid constructs, (b) sodium dodecyl sulphate–polyacrylamide gel analysis of the agroinfiltrated recombinant recombinant toxoid and (c) western blot analysis of recombinant toxoid protein. protein extraction leaf samples (10 g) from transfected n. benthamiana plants were harvested between 3, 6 and 9 days post-infection (dpi) and homogenised in extraction buffer (100 mm glycine, 40 mm ascorbic acid, 1 mm edta [ph 9.5]) using a 1:1 (w/v) ratio of extraction buffer to harvested plant material (g). protein purification the resulting green juice was clarified by filtration through four layers of cheese cloth then loaded onto an equilibrated capto q column (ge healthcare, piscataway, nj) with three column volumes of buffer a (50 mm diethanolamine, ph 9.5) and the flow-through/wash fraction collected. the unbound proteins were washed and the bound proteins were eluted. the r-etx-containing fraction was finally ‘polished’ by superdex 220-pg size-exclusion chromatograph (bio-rad, hercules, ca) in 50 mm diethanolamine buffer, ph 9.5. the r-etx was then trypsinated and subsequently formalin inactivated as described (chandran et al. 2010) resulting in a protein code named r-etox, which was then used for subsequent analysis. residual toxicity tests and formulation methods were also according to chandran et al. (2010) with minor modifications. the formalin treatment was conducted to ensure that there was complete processing of the r-etx into r-etox as per standard protocols. immunoblot analysis purified r-etx was resolved on 12% reducing sodium dodecyl sulphate–polyacrylamide gel (sds–page) followed by coomassie blue staining. the amount of protein loaded was approximately 4 μg – 8 μg of sample. pageruler™ plus prestained protein ladder (#sm1811; fermentas, hanover, md) with high-range molecular weight markers was loaded for comparison of the molecular weights. immunoblot analysis was carried out according to standard protocols where proteins from sds–page were transferred onto a polyvinylidene difluoride membrane (bio-rad, hercules, ca) then, treated with polyclonal guinea pig anti-etx antibody (obp) and rabbit anti-guinea pig igg-hrp monoclonal antibody (st. louis, mo). bound antibodies were detected using ecl detection kit (aec-amersham, midrand, south africa). enzyme-linked immunosorbent assay analysis to quantify the accumulation of the r-etx in the leaves using enzyme-linked immunosorbent assay (elisa), either six leaf discs from each of three transfected plants or aliquots of the purified samples were used. leaf discs were ground up in 300 µl of extraction buffer (4 m urea and 100 mm dithiothreitol [dtt]) (voinnet et al. 2003) in liquid nitrogen and then analysed. elisa analysis was carried out using crude r-etx samples and native etx as protein standards using a protocol derived from chandran et al. (2010). the r-etx protein was detected using polyclonal guinea pig etx antibody (sera) as primary antibody, followed by hrp-conjugated anti-guinea pig polyclonal antibody (abcam, cambridge, uk) and detected using calorimetry using an ecltm-plus kit (amersham pharmacia, amersham, uk) according to the manufacturer’s instructions. for comparison, calculated antigen concentrations in the samples were normalised to the fresh leaf weight to obtain the yield of r-etx per kg fresh weight (fw). protein identification using mass spectrometry protein bands of interest were trypsin digested as per the protocol described by shevchenko et al. (2007). briefly, gel bands were destained using 50 mm nh4hco3/50% meoh followed by in-gel protein reduction (50 mm dtt in 25 mm nh4hco3) and alkylation (55 mm iodoacetamide in 25 mm nh4hco3). proteins were digested overnight at 37 °c using 50 ng – 500 ng tryspin depending on the gel piece size. digests were resuspended in 35 µl 2% acetonitrile/0.2% formic acid and analysed using a dionex ultimate 3000 rslc system coupled to a qstar elite mass spectrometer. peptides were first de-salted on an acclaim pepmap c18 trap column (75 μm × 2 cm) for 8 min at 5 μl/min using 2% acetonitrile/0.2% formic acid, and then separated on acclaim pepmap c18 rslc column (75 μm × 15 cm, 2 µm particle size). peptide elution was achieved at a flow rate of 500 nl/min with a gradient of 4% – 60% b in 30 min (a: 0.1% formic acid; b: 80% acetonitrile/0.1% formic acid). nanospray was performed using a microionspray head assembled with a new objective, picotip emitter. an electrospray voltage of 2.0 kv – 2.8 kv was applied to the emitter. the qstar elite mass spectrometer was operated in information dependent acquisition using an exit factor of 7.0 and maximum accumulation time of 2.5 seconds. mass spectrometry (ms) scans were acquired from m/z 400 to 1500 and the three most intense ions were automatically fragmented in q2 collision cells using nitrogen as the collision gas. collision energies were chosen automatically as function of m/z and charge. protein pilot v4.0.8085 using paragon search engine (ab sciex, foster city, ca) was used for comparison of the obtained ms/ms spectra with protein sequences in a uniswiss 2011 database. proteins with a threshold above ≥ 99.9% confidence were reported. in vivo toxicity in mice the formalin-inactivated protein (r-etox) was formulated into alum, montanide gel and isa 70 (seppic, paris, france) as 50 µg and 150 µg doses in 0.2 ml volumes. the toxicity of the formulations was evaluated using 5 groups of 10 female cd-1 mice (8–10 weeks old; weighing approximately 37 g each) and eight guinea pigs immunised subcutaneously. some animals were inoculated with single doses, whereas others in a prime–boost regimen at a 3-week interval between the vaccinations. vaccination and challenge experimental animals the obp commercial vaccine (enterotoxaemia alum-precipitated vaccine for sheep and goats) and phosphate-buffered saline were used as the positive and negative controls. all animals were observed for mortalities and bled at weekly intervals until the 35th day. safety in sheep was determined by subcutaneously injecting groups of two sheep in the inner thigh with 1 ml of formulated candidate vaccines as described above. subsequently, the animals were observed for an allergic reaction at the injection site and rectal temperatures were measured. we therefore monitored the animals 5 days before and 14 days after vaccination and recorded the size of lesions and the time taken before healing. this was used as an indication of the safety of the vaccine. the antitoxin units (iu) were determined by mixing the native epsilon toxin protein (etx) with varied dilutions of serum from vaccinated guinea pigs. the residual toxicity in mice was determined by inoculating 0.2 ml of the mixture (dilution range 33 iu/ml – 2.5 iu/ml) intravenously into 10 mice. lack of mortality in the mice 14 days post-infection indicated neutralisation of etx. based on the dilution factor, the number of iu of the test serum was determined and was directly correlated to the potency of the vaccine. a value of 5 iu per ml of serum was considered to be protective according to the european pharmacopoeia (edqm 2008). results to assess expression of the plant-codon optimised etx orf (figure 1a), plant extracts from agroinfiltrated plant leaves were analysed by sds–page (figure 1b) and western blot analysis (figure 1c). there is no commercially available pure ext protein to use as a positive control; hence, a non-formulated vaccine etx protein (obp positive control) was used in the analysis. the plant expression results show that the plant-codon optimised etx orf sequences could be expressed in n. benthamiana leaves at levels detectable by sds–page (figure 1b). the r-etx was detectable at a molecular weight of approximately 35 kda on sds–page (figure 1b). this is higher than the predicted 32 kda for the truncated recombinant protein but migrating at similar molecular weight was the positive control. in the different plant extracts harvested at 3 dpi, 6 dpi and 9 dpi, the band corresponding to the predicted r-etx (approximately 35 kda) was clearly visible. we then used immunoblotting to confirm the identity of the proteins using a polyclonal primary anti-etx antibody (figure 1c). a 35-kda protein was detected in plant extracts at the same molecular weight position as the positive control. in addition, a smaller band corresponding to the 28-kda molecular weight size band was detectable in the positive control. surprisingly, a similar band size was detectable in infiltrated plant extracts as detected by both sds–page and western blot analysis. this suggested a second orf or possible degradation product. to further corroborate the results of the sds–page and western blot analysis, we excised the bands indicated as band a1 to band a3 (figure 1b) and sequenced them by ms. table 1 shows the sequencing results including the coverage for the three bands. plant extracts contain complex mixtures of proteins with similar isoelectric points and co-migrating at the same molecular weight on sds–page. ms can therefore show the predominant proteins in each of the bands. protein band a1 (~35 kda) excised from lane 1 (see figure 1b) was confirmed to be predominantly epsilon toxin (r-etx) from c. perfringens with 65% protein coverage. the next abundant protein from the band a1 was trypsin, which is used in the peptide digestion during sequencing. the sequencing results confirmed that the protein sample being formulated for the vaccine was predominantly r-etx. we then sequenced the corresponding protein band in the plant extracts, migrating at the same molecular weight (b and a2), and confirmed that it was also epsilon toxin from c. perfringens at 68% sequence coverage, followed by two stress-related proteins from the tobacco extract. band a3 was shown to be glucan-endo-1,3-beta-d-glucanase, which is one of the stress-related proteins. in summary, the sequencing results confirmed that the epsilon toxin was expressed in plant leaves at levels detectable by sds–page. the sequencing results also corroborated the sds–page and western blot analyses. table 1: sequencing data for the putative r-etx protein bands from sds–page by mass spectrometry peptide finger printing. quantification of the protein using elisa showed that the best protein yield of r-etx was 380 mg kg-1 fw in the apoplast when harvested at 9 dpi. the yield varied with the time of harvesting and subcellular targeting of the protein (figure 2). with a focus on maximising the protein yield, the apoplast vector combination at 9 dpi was chosen to produce bulk plant material. the protein was purified, trypsinated, inactivated, formulated and injected into animals for safety and potency. figure 2: effect of subcellular targeting of recombinant toxoid protein on accumulation. transient expression of the recombinant toxoid protein in agroinfiltrated nicotiana benthamiana leaves as measured by an anti-epsilon toxin enzyme-linked immunosorbent assay on crude extracts at 3, 6 and 9 days post-infiltration. the safety of the candidate vaccines was investigated using a mouse and guinea pig lethality test (table 2) and also by examining injection site reaction as indicated by the diameter and duration of lesions on the injector site reaction. to assess the allergic reactions to the vaccine or adjuvant, the rectal temperatures of the vaccinated sheep was monitored. at 2 weeks post-inoculation, all mice survived the 50 µg or 150 µg r-etox doses, while all but one group of guinea pigs had 100% survival (table 2). none of the inoculated mice and guinea pigs showed clinical signs of disease at the time of observation irrespective of the vaccine formulation. furthermore, we examined the safety of the candidate vaccines in sheep (table 3). the rectal body temperature of vaccinated sheep remained within the same range as the positive control. all groups except the 150 µg isa 70 and the control group had no injection site reaction to the vaccination (table 4). the lesions in the 150 µg isa 70 group started at 3 days post-inoculation and lasted until 12 days. the commercial vaccine group (alum) had a similar pattern with injection site reaction observed 8 days post-vaccination and lasting up to 5 days. despite these variations, the vaccine’s overall inference was that the vaccine formulations were safe in animals. table 2: safety of plant-based enterotoxaemia vaccine candidates in mice and guinea pigs. table 3: safety of enterotoxaemia candidate vaccines in sheep. table 4: safety of enterotoxaemia candidate vaccines in sheep. we then investigated the potency of the candidate subunit vaccines using the toxin neutralisation assay as per the european pharmacopoeia and as described by lobato et al. (2010). the toxin neutralisation assay result was acceptable for regulatory approval of candidate enterotoxaemia vaccines. results of the toxin neutralisation test showed that vaccination with the r-etox led to a steady rise in the serum potency to the acceptable level of 5 iu in most regimens (figure 3). alum was generally the least potent adjuvant with single vaccinations not reaching the potency level at 3 weeks after vaccination (figure 3). figure 3: efficacy of recombinant toxoid vaccine candidates using serum of vaccinated guinea pigs. the alum formulations did reach the acceptable potency threshold in a prime–boost vaccination regimen. all formulations with the isa 70 and montanide adjuvants were potent with values 10 iu – 33 iu. we also observed that the best serum potency was with 50 µg of antigen of montanide formulation in a booster vaccination. increasing the amount of antigen to 150 µg did not result in an increase in serum potency (figure 3). overall, the results suggest that all the vaccine formulations tested were potent enough to translate to a protection level similar to or better than the commercial vaccines in the field. in all cases, the negative controls showed no serum potency and remained negative throughout the experiment. ethical considerations all experiments were approved by the necessary ethics committees at the council of science and industrial research (csir) and obp. as per the advice, if the prime vaccination was safe, no boost vaccine was administered to spare the animals from unnecessary pain. to reduce the number of experimental animals, the guinea pigs used for the safety study were also used in the toxin neutralisation assay. discussion native epsilon toxin from c. perfringens type d is a highly toxic protein that requires enzymatic activation and detoxification by chemical inactivation with formaldehyde as part of vaccine-manufacturing process. this makes it expensive and relatively unsafe to manufacture. despite considerable efforts in producing a second-generation subunit vaccine (langroudi, shamsara & aghaiypour 2013; lobato et al. 2010; souza et al. 2010; walmsy & kirky 2008), a plant-based subunit vaccine for enterotoxaemia disease remains unavailable on the market. however, a number of strategies for production of recombinant etx protein have been reported. the epsilon type d toxin has been recombinantly expressed in escherichia coli with the ultimate purpose of producing a vaccine from a relatively less toxic insoluble form of the toxoid (souza et al. 2010). to date, no reports of production of this important veterinary molecule in plants have been made. in this article, we have successfully expressed the truncated r-etx in a highly immunogenic form and at potentially viable levels for commercial implementation using a transient deconstructed virus strategy in n. benthamiana. identity of the plant-made r-etx was confirmed by peptide mass fingerprinting ms. electrophoresed r-etx cross-reacted with antibodies raised against the native toxin in a western blot experiment, showing a positive signal at around 35 kda size (figure 1b), which is about 3 kda higher than the predicted mass. this is not very surprising as bands ranging from 26 kda to 32 kda have been detected before even with the native c. perfringens etx extracts possibly because of post-transcriptional modifications (chandran et al. 2010). our results also show 28 kda etx protein band in the positive control as reported in the literature. interestingly, we detected the 28 kda in plant extracts by both sds–page and western blot analysis, but results from sequencing showed undetectable levels of r-etx. considering that we used polyclonal antibodies raised in vegetable or grass eating animals, it is conceivable that there is non-specific binding to some plant proteins such as the stress-related glucan-endo-1,3-beta-d-glucanase. however, it is not clear why the plant-expressed r-ext migrated at a higher molecular weight than predicted. we speculate that the higher molecular weight could be due to glycosylation as reported with other plant-produced proteins (van dolleweerd et al. 2014). the r-etx is predicted to have four n-glycoslyation sites at positions 31, 96, 260 and 369. having said that, the sds–page, western blot analysis and ms all confirmed the identity of the plant-produced r-etx elisa also confirmed the conservation of that the protein to cross-react with etx-specific antibodies. to confirm the efficacy and safety of r-etox, guinea pigs were vaccinated. this study convincingly demonstrated the potency of the plant-made molecule in the animal model using different adjuvants. interestingly, the use of isa 70 adjuvant resulted in the most potent formulation where even a single 50-µg dose vaccination led to efficient protection of the animals (table 2). increasing the quantity of antigen did not lead to increased potency. dose titration experiments will be done in future work to properly determine the minimum effective dose for possible commercial implementation. furthermore, duration of immunity (based on target species serum neutralising antibodies) will be determined during the envisaged field testing. based on our results, the potential for commercial production of an enterotoxaemia vaccine in plants is promising. plant-based expression will present several advantages for production of this subunit vaccine in resource-limited markets. the system has potential for minimising the cost and complexity of production of the bulk antigen for formulation. the typical current production technology for pulpy kidney disease (enterotoxaemia) vaccine relies on clostridium fermentation under anaerobic conditions. the conditions required for optimal production of toxin must be precisely controlled by process engineers (robertson et al. 2011). in contrast, the technology reported here will use greenhouse cultivation and transfection by vacuum infiltration of n. benthamiana plants, processes that require simple invariable conditions and semi-skilled technical staff. conclusion we have reported time production of enterotoxaemia vaccine based on the c. perfringens type d epsilon toxoid and empirically demonstrated the safety and antigenicity of the product in animal models from which can be inferred that the product can protect sheep against the disease. acknowledgements this study was funded by onderstepoort biological products (obp), the csir parliamentary grant and the technology innovation agency (tia) of the republic of south africa. the authors would like to acknowledge stella manganye at csir, lorraine zondi and calvin molepo at onderstepoort biological products (obp) for technical support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.c. conceived and designed the experiments. t.m., t.t., m.t. and b.w. performed the 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reports 21, 1020–1026. https://doi.org/10.1007/s00299-003-0619-4 wang, l. & coppel, r.l., 2008, ‘oral vaccine delivery: can it protect against non-mucosal pathogens?’, expert review of vaccines 7, 729–738. https://doi.org/10.1586/14760584.7.6.729 abstract introduction conclusion acknowledgements references about the author(s) celia abolnik department of production animal studies, university of pretoria, south africa citation abolnik, c., 2017, ‘history of newcastle disease in south africa’, onderstepoort journal of veterinary research 84(1), a1306. https://doi.org/10.4102/ojvr.v84i1.1306 review article history of newcastle disease in south africa celia abolnik received: 04 july 2016; accepted: 05 aug. 2016; published: 24 feb. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract poultry production in south africa, a so-called developing country, may be seen as a gradient between two extremes with highly integrated commercial enterprises with world-class facilities on one hand and unimproved rural chickens kept by households and subsistence farmers on the other. although vaccination against newcastle disease is widely applied to control this devastating infection, epizootics continue to occur. since the first official diagnosis in 1945, through the sporadic outbreaks of the 1950s and early 1960s, to serious epizootics caused by genotype viii (late 1960s–2000), genotype viib (1993–1999), genotype viid (2003–2012) and most recently genotype viih (2013 to present), south africa’s encounters with exotic newcastle disease follow global trends. importation – probably illegal – of infected poultry, poultry products or exotic birds and illegal swill dumping are likely routes of entry. once the commercial sector is affected, the disease spreads rapidly within the region via transportation routes. each outbreak genotype persisted for about a decade and displaced its predecessor. introduction newcastle disease (nd) is a highly contagious and lethal avian disease caused by certain strains of avian paramyxovirus type 1 (apmv-1) that is transmitted by the faecal–oral route in avian species. apmv-1 is a member of the avulavirus genus, viruses that have a membrane-encapsulated single-stranded rna genome of negative polarity (alexander 1997; mayo 2002). apmv-1 is split into two classes: class i consists of apmv-1 viruses commonly isolated from wild birds, whereas the stains infecting poultry fall within class ii (diel et al. 2012). each class is further divided into genotypes; the current classification is summarised in table 1 along with pertinent characteristics of each genotype. these genotypes have emerged over the course of several decades, due in part to the inherent propensity of rna viruses to evolve rapidly, as well as immune pressure that drives strain selection within the host (miller et al. 2009). table 1: current classification of avian paramyxovirus type 1 (newcastle disease virus). many avian species have been diagnosed with apmv-1 infection, but clinical signs tend to vary with the pathogenicity of the isolate and the species of the bird. chickens are highly susceptible to apmv-1 infection; strains are therefore further classified based on the symptoms they cause in this species. lentogenic strains typically cause subclinical infections or mild respiratory disease, with coughing, gasping, sneezing and rales. mesogenic strains potentially cause acute respiratory disease and neurologic signs in some chickens, but the mortality rate is usually low. velogenic strains cause severe, often fatal disease. most poultry infected with velogenic apmv-1 (synonymous with ‘nd’) appear lethargic and inappetant with conjunctivitis and ruffled feathers. additional symptoms often include watery, greenish or white diarrhoea, respiratory signs, cyanosis and swollen heads. neurologic signs include tremors, clonic spasms, paresis or paralysis of the wings and/or legs, torticollis and circling. egg production often declines dramatically, and eggs may be misshapen, abnormally coloured and rough or thin-shelled, with watery albumin. sudden death, with no or few symptoms, is also common with velogenic apmv-1 strains (alexander 1997). the intracerebral pathogenicity index (icpi) provides a relative measure of a virus’ pathogenicity. the most virulent viruses will give indices that approach the maximum score of 2.0, whereas lentogenic and asymptomatic enteric strains will give values close to 0.0 (oie 2012). the presence of multiple basic amino acids at the fusion (f) protein cleavage site (f0) is another important indicator of viral virulence (alexander 1997). nd is a severe global problem affecting poultry production. in africa its effect is particularly severe on backyard poultry flocks. poultry production in south africa, a so-called developing country, may be seen as a gradient between two extremes, with highly integrated commercial enterprises with world-class facilities on one hand and unimproved rural chickens kept by households and subsistence farmers on the other. although vaccination is widely applied to control the disease, epizootics continue to occur. a description of south africa’s experience with this devastating disease in the context of global outbreaks follows. early global and south african outbreaks nd may have been prevalent in scotland as early as 1898, when a disease decimated domestic fowl in the scottish northwest region (maminiaina et al. 2010). the first confirmed outbreaks of nd occurred in 1926 in java, indonesia, and in 1927, in newcastle-upon-tyne, england, but the disease could also have been present in korea in 1924 (konno, ochi & hashimoto 1929). the following letter in 1892 to agricultural journal (south africa) may, however, be the earliest account yet: can any of your readers give me some information as to the causes, prevention, and treatment of a disease that from time to time attacks my fowls, and seems to resist all known treatment. the disease apparently begins with a cough, and gasping breathing, the fowl trembles all over, and when it sits down one leg is stretched in front the other behind the body. there seems to be a gradual paralysis of both legs. the patient has all along an excellent appetite, which is maintained to the very end of the disease-invariably death. some of the cases make gurgling noise when breathing, and they generally live in this dying state for months. aloes, rusty water, lime, and paraffin-remedies recommended by “fowl” authorities have all been tried-as well as homoeopathic doses of aconite, without benefit. i have two english imported canaries ill, and the symptoms are very similar. as the canaries are valuable birds i am anxious to know of a cure. (mr t. duncan greenlees, fort england, grahamstown, query no. 221fowl-sickness 1892) another example of possible nd citing extracts from a letter to the transvaal agricultural journal in 1903: i have a considerable number of mixed breed fowls running on my place here. they suffer from various diseases known to us locally as ‘lame-sickness’, ‘eye-sickness’, ‘sore throat,’ ‘sore nostrils,’ &c., the right names of which we do not know. ‘lame-sickness’, the fowl becomes unable to walk, and appears to have paralysis of the legs, the wings droop, the foeces [sic] are loose, weakness sets in and the bird dies … sore throat,’ ‘sore nostrils,’ &c.-the fowl eventually chokes, suffocates and dies. it resembles a child’s croup’ … there is another disease, which is difficult to describe. the fowl appears to get into a fit, dances about, and suddenly falls down and dies. (ada rosenbloom, hamans kraal 1903) although many of the symptoms described in these two cases are not pathognomonic for nd, and indeed, the description of unilateral paresis or paralysis is suggestive of a marek’s disease infection, the presence of neurological signs makes nd a strong possibility. nd may therefore have been present in south africa since at least the late 1800s. newcastle disease outbreaks in south africa, 1945–1970s nd was officially diagnosed for the first time in south africa in 1945, after a severe disease with respiratory, nervous and intestinal symptoms, and high mortalities swept through poultry in the natal (kwazulu-natal) province. the diagnosis was made by serum neutralisation tests performed at weybridge in england. kashscula believed that nd was already present in the port of durban in september 1944 and because the symptoms and post-mortem findings in natal so closely resembled those described by hudson in mombassa, kenya in 1935, it was ‘almost certain’ that the disease had been brought by ship from some harbour on the east coast of africa. hudson considered that the infection had spread south to lindi and there were persistent rumours suggesting that the entire african east coast had been affected (kaschula et al. 1945). in retrospect, this outbreak appears to have been part of a panzootic that swept through italy, palestine and the whole of central europe during the second world war (kluge 1964). the 1944/1945 outbreak in south africa was confined almost entirely to the sugarcane belt of natal. the free-ranging flocks of the indian population suffered the heaviest losses. the disease was eventually stamped out, but 100 000 fowl are estimated to have perished. it was strongly suspected that the mombassa outbreak in 1935 was not the first african case of nd (kaschula et al. 1945). confirmed nd appeared in cape town’s windermere township in july, 1949. by september it had spread to port elizabeth and de aar in the cape province. by january 1950 nd had spread to natal and was also confirmed in johannesburg. more outbreaks flared up in durban during january of 1951 and 1953. with the application of strict control measures, the disease was suppressed until early october, 1954 when nd reappeared in johannesburg. by the end of december 1954 the disease had been stamped out, but it was estimated that more than 300 000 fowls perished in these outbreaks of the early 1950s. vaccines had recently been developed and more than a million birds were vaccinated with an attenuated live virus (de kock 1954). in july 1960, the mild lentogenic type of nd, termed the ‘american type’, was diagnosed in south africa for the first time. the strain of virus was so mild that symptoms of infection were apparent only to the careful observer (kluge 1964). velogenic nd re-emerged in the northern outskirts of pretoria in august 1961 and reached evaton in november, where it re-emerged in june 1962. thereafter, an intensive control campaign was successfully concluded and no further outbreaks occurred for several years (kluge 1964). in 1967, a serious nd outbreak occurred on a farm in the cape flats near cape town, despite control measures that included quarantine and vaccination. by july 1968, the bellville and stellenbosch regions were also affected, and the disease spread throughout the district. this strain was referred to as the ‘asiatic type’, whereas the milder ‘american type’ was simultaneously circulating in the transvaal (gauteng), natal and eastern cape provinces (oosthuizen 1979). the outbreaks caused serious losses while they lasted, but the control measures instituted by the state (quarantine and other zoo-sanitary measures, in conjunction with immunisation of all birds in the surrounding and proclaimed areas) seemed to effectively control the disease and eliminated the infection in each instance. the live mesogenic komarov strain was used as a vaccine and no immunisation against nd was practised or allowed at any time except during outbreaks of the disease and then only under state supervision. it was believed that the disease was not enzootic in south africa and the localised nature of the outbreaks indicated that the infections were probably introduced from some unknown external sources. therefore, nd was not considered to be of major importance for south africa (de almeida et al. 2013; kluge 1964). south africa experienced one of its most severe nd epidemics from 1970 to 1972. the rapid expansion of the industry since 1960 had created large concentrations of susceptible poultry. farms accommodating hundreds of thousands of chickens were established in close proximity to one another and near the urban markets. the method of eradication previously employed to control nd had become impractical because of the rapid dissemination of the infection in certain regions (coetzee 1980). the first outbreak occurred in the town of potgietersrus (mokopane), and later, in september 1970 on the outskirts of pretoria in backyard poultry. by december, one of the major commercial broiler operations in johannesburg was affected. the failure of the campaign to eradicate nd was attributed to two reasons. firstly, there was a lack of adequate funds for slaughtering out with compensation – as had previously been applied – and secondly, as a result of the development of huge broiler operations. the industry suffered great economic losses and by 1971 nd was believed to be enzootic in the country. by 1974 outbreaks of nd had subsided and occurred only in unvaccinated birds on small poultry farms and among unvaccinated poultry kept in backyards. outbreaks were reported fairly frequently in consignments of imported birds at the quarantine station at the international airport in johannesburg. these consignments were evidently infected while they were handled by dealers prior to shipment, since all birds, virtually without exception, developed nd from 3 to 7 days after their arrival in south africa. vaccination against nd became a common practise at the airport quarantine station (anonymous, sapv-pluimveebulletin, february 1974). subsequent to the severe outbreak of the 1970s, only small sporadic outbreaks occurred over the next two decades. genetic characterisation of newcastle disease strains in south africa begins the sources of sporadic nd outbreaks since the 1950s were never resolved and it was presumed that the periodic outbreaks were spill-overs from an unknown reservoir, which some suggested to be village chickens, but wild birds were also suspected (verwoerd 1995b). the national veterinary laboratories at allerton in kwazulu-natal, stellenbosch in the western cape and the university of pretoria routinely isolated apmv-1s from the national flock during outbreaks. to resolve the molecular epidemiology of nd in south africa, a project was started at the onderstepoort veterinary institute in pretoria, whereby all the available apmv-1 isolates were subjected to reverse-transcription polymerase chain reaction (pcr) amplification of a portion of the f gene, followed by dna sequencing and phylogenetic analysis (abolnik 2007). in the course of these molecular investigations, virulent genotypes were identified (described below) as well as genotype i and ii vaccine derivatives. genotype vi was, and still is, occasionally isolated from pigeons (and in some cases, chickens), but will not be discussed in this review. thus the strains responsible for causing sporadic epidemics in south african poultry since the 1960s were identified as follows. genotype viii outbreak (1960s–1994; 2000) genotype viii is one of the smallest exotic nd groups in the world and is likely to have originated in southeast asia. these strains are velogenic and were isolated in poultry in singapore and western china between 1979 and 1985, argentina in 1970, japan in 1991, taiwan in 1995 and italy in 1994 (aldous et al. 2003; liang et al. 2002; mase et al. 2002; rui et al. 2010; tsai et al. 2004). in south africa, genotype viii was isolated in 1968, 1974 and in the early 1990s up to 1994. herczeg and co-workers suggested that this genotype was enzootic in south africa since the 1960s (herczeg et al. 1999). most isolations of genotype viii were made in the 1990s in the kwazulu-natal province, one of the largest and most intensive poultry-producing areas in south africa, but two more genotype viii strains were isolated in the latter part of the decade. these late isolates did not appear to be as virulent as those prior to 1994 since the only clinical signs reported were a slight drop in egg production and nasal excretions. whether this was due to improved and widespread vaccination in the 1990s or attenuation is unknown. the reservoir of genotype viii in south africa between 1994 and 2000 is also unknown since despite intensive active surveillance and the ongoing molecular epidemiological study, no genotype viii viruses were isolated by the national laboratories during this period. no genotype viii strains have been detected in south africa since june 2000 (abolnik 2007; unpublished laboratory data). genotype viib (1993–1999) in june 1993, a neuro/respirotropic ndv was isolated from an outbreak at a large commercial poultry producer near hartebeespoort, gauteng province. within 6 months nd had spread throughout southern africa causing devastating losses in all types of poultry. an estimated one million mortalities per week were recorded at the peak of the outbreak. the major outbreak of 1993/1994 was eventually brought under control by improved vaccination procedures and biosecurity measures, but sporadic outbreaks continued to occur for the remainder of the decade (huchzermeyer & gerdes 1993; verwoerd 1995a, 1995b). in retrospect, the incurrence of genotype viib into south africa in the early 1990s was concurrent with the rise of this pandemic strain in many parts of east asia and europe in the 1990s. extensive phylogenetic analysis demonstrated that the genotype viib strain responsible for the outbreaks in south africa originated in southern europe (abolnik 2007) and not the opposite, as was originally reported (herczeg et al. 1999). genotype viib may even have been introduced from abroad into south africa on multiple occasions since a genotype viib virus had been isolated from an ostrich in south africa in 1991, 2 years prior to the outbreak index case at hartebeespoort, yet the ostrich virus shared only 97% nucleotide sequence identities with the early south african genotype viib. the south african genotype viib outbreak strains from 1993, however, shared 100% sequence identity with spanish (1990) and portuguese (1991) isolates (abolnik 2007). it is possible that day-old chicks, eggs or unprocessed poultry products were imported into south africa from europe, or via a neighbouring country trading with europe, before the infection was reported there. genotype viib was also present in mozambique in 1993 (herczeg et al. 1999) and reached botswana. genotype viib was not detected in south africa after 1999 (abolnik 2007). genotype viid (2003–2012) generally, apmv-1 strains infect waterfowl such as geese and ducks without causing overt clinical symptoms. these species therefore usually act only as carriers of the virus. however, nd outbreaks have occurred frequently in chinese geese since 1997 (alexander 1997; takakuwa et al. 1998; yin & liu 1997). in 1999, an outbreak of nd in shanghai goose flocks caused moderate morality rates of 10% – 20% in adult geese but up to 100% mortalities were observed in geese under 2 weeks of age. a novel virus isolate, sf02, was determined to be the etiological agent of these outbreaks. it was named ‘goose paramyxovirus’ and was subsequently classified as genotype viid. genotype viid is highly pathogenic to chickens, pigeons, partridges and ducks. icpis of 1.80–1.94 have been recorded, but chickens inoculated with either live or inactivated lasota vaccines were fully protected against challenge with genotype viid (jinding et al. 2005; liu et al. 2003; zou et al. 2002; zou & gong 2003). from 1999 to 2000, an outbreak of velogenic viscerotropic ndv occurred in a single commercial flock as well as village chickens in a relatively small geographic area of the kwazulu-natal province. the pathogen was identified as a genotype viid virus. for almost 3 years afterwards there were no reports of nd outbreaks, nor were any virulent viruses isolated by national laboratories. then, towards the end of september 2003, a velogenic nd virus was isolated from village chickens near pietermaritzburg. this was close to the locations of the outbreaks of 1999 and 2000, but the infection did not seem to spread. the 2003 genotype viid strain was phylogenetically linked to the 1999/2000 isolates (abolnik et al. 2004). almost exactly 1 year later, in october 2004, an outbreak of genotype viid started in commercial flocks of the camperdown/richmond districts. although the index case was a commercial farm, workers reported that a disease with similar symptoms had been killing fowls in their villages for several weeks prior. a sample was obtained for testing. it was shown to be a genotype viid strain, almost identical to the isolate from the commercial farm on which the village workers were employed. the spill-over was attributed to poor on-farm biosecurity practices. interestingly, around this time a separate sub-genotype viid was introduced into the outbreak region in kwazulu-natal (kzn); four isolates shared a very recent common ancestor with two chinese goose viruses isolated in 2001. this genotype viid subgroup circulated concurrently with the epidemic genotype viid strain that had arisen in 1999/2000 but appeared to be displaced within months and did not spread beyond the kzn province. after december 2004, genotype viid spread northwards along the major poultry-producing corridor between kzn and gauteng/north west provinces, affecting both commercial and backyard flocks. outbreaks were usually associated with poor vaccination practices although sometimes mortalities were reported in well-vaccinated flocks. the index case in the northern region appears to have been contaminated hatching eggs sourced from a supplier in kwazulu-natal. over the next few months, the disease spread westwards to the northern cape, southwards to the cape provinces and eastwards into mpumalanga province. the epidemic peaked in september 2005, and by 2006, it had begun to subside in most regions, except for the western and eastern capes where cases were still increasing. the western and eastern cape outbreaks were characterised by at least three separate introduction events from northern regions as established by mapping specific genetic markers. during the epidemic in commercial and backyard chickens, mortalities were recorded in peafowl, hadeda ibis chicks, geese, ostriches, pheasants and doves (abolnik 2007). no cases of genotype viid were detected in south africa after 2012 (unpublished laboratory data). genotype viih (2013 to present) genotype viih has caused outbreaks in the poultry of bali, indonesia and malaysia since 2007, and an isolate was also recovered from a wild egret in china (dimitrov et al. 2016). genotype viih has since reached southern africa. in december 2011 it caused an outbreak in vaccinated broilers in mozambique, where it still persists (mapaco et al. 2016). in late august 2013, genotype viih was diagnosed in a flock of commercial layers in lephalale, limpopo province and in a flock of free-range broilers near potchefstroom, north west province. by october 2013, the strain had spread to the free state province and had been confirmed in zimbabwe. by november 2013, the outbreaks had spread to gauteng and reached kwazulu-natal by july 2014 and mpumalanga in october 2014. the south african genotype viih viruses share a very recent common ancestor with strain sukorejo/019/10, with 98% nucleotide sequence identities across the complete genome (data will be published elsewhere). the sukorejo virus was isolated during an outbreak in commercial poultry that circulated in indonesia in 2009 and 2010 (xiao et al. 2012). imported poultry, poultry products or exotic birds from south east asia are the likely source of genotype viih in the southern african region and while it is likely that genotype viih spread to south africa across a neighbouring international border, the exact epidemiological source remains unclear. conclusion small-scale poultry production has enormous potential to stimulate the socio-economic growth of resource-poor households. chickens provide a good source of protein and ready cash for households, help to sustain the rural economy and contribute to the prevention of urban migration, yet nd is the single largest threat to poultry production in africa. the commercial sector also bears a huge economic burden as a result of the disease. in addition to production losses incurred during outbreaks and the chronic costs of control through vaccination, with a constant nd-endemic status valuable trade and export markets remain inaccessible. from genotype viii in the 1960s and 1970s, to genotype viib in the 1990s, genotype viid in the 2000s and now genotype viih, each genotype displaced its predecessor and circulated for approximately a decade (figure 1). this is an unusual trend, since in other nd-endemic regions (e.g. east asia) numerous genotypes are in co-circulation at any time. each of south africa’s outbreaks was caused by exotic strains of nd introduced at periodic intervals. the illegal importation of poultry, poultry products or exotic birds was the likely source of infection. genotypes viib and viih index cases were found inland, but genotype viid was likely introduced through the port of durban, by illegal harbour swill dumping (abolnik 2007). infected swill has indeed been implicated in the introduction of infectious diseases into south africa before. in 2000, foot and mouth disease was introduced into kwazulu-natal by contaminated swill (brückner et al. 2002). classical swine fever was probably also introduced from asia into the western cape in 2005 by illegal swill feeding of pigs (penrith 2013). any presence of the disease in neighbouring counties is likely to spread throughout the region, as people, vehicles and poultry move across the borders. once nd is established in the south african commercial sector, it spreads rapidly along major transportation routes via movement of infected poultry and/or fomites. this is facilitated firstly by the large commercial producers themselves who transport hatching eggs and day-old chicks between their operations, buy in supplemental eggs at pack stations on commercial layer farms, frequently transport manure between sites in uncovered trucks, and secondly by the cull buyers, that is vendors of spent layer hens. figure 1: timeline of newcastle disease virus outbreaks in south africa since the late 1960s, which were caused by successive outbreaks of genotypes viii, viib, viid and viih, respectively. in developed countries, commercial chickens have almost no resale value after their usefulness has expired. in south africa, however a spent layer hen with a replacement value of about r50 (±$4.40) can be sold for about r35 (±$3.10) allowing the farmer to recoup most of the expense. cull buyers pick up batches of thousands of birds with trucks and transport them to central depots. some large commercial operations even have their own cull depots and in this way birds from different farms are brought together. from the depot, fowls (perhaps several hundred or less) are sold to smaller buyers, who then supply birds to informal markets including townships and rural areas. where the market is good, large cull buyers will often travel over long distances, for example, between gauteng and the western cape or kwazulu-natal. therefore culls that originate in the cape can very easily, and are commonly, be sold in kwazulu-natal. interestingly, cull chickens are not significantly cheaper than frozen meat, but these older chickens are coveted because consumers consider them to be tastier. therefore, in south africa, a layer hen still has a value at the end of her production life. this makes the layer market very competitive as about 25 million layer hens are in production in south africa at any given time (southern african poultry association 2012). the cull-buyer industry is legal, albeit largely unregulated and uncontrolled. a positive consequence of a cull-buyer system is that it appears to have limited the need for large live bird markets in south africa. live bird markets are key epidemiologic points for the spread of nd and other dangerous diseases like highly pathogenic avian influenza in other developing countries in africa and asia. commercial live vaccines such as lasota, vg/ga, clone 30 and b1 (genotype ii) are applied for prevention and control of nd in south africa, yet outbreaks continue to occur. these classical vaccines have been experimentally demonstrated in vivo to provide complete protection against clinical disease and mortality induced with virulent strains (including genotype vii) that are antigenically mismatched to the vaccine strain, and still either significantly reduce or completely prevent shedding. even though some of these experiments were conducted with ‘sub-optimal’ vaccine doses (dortmans, peeters & koch 2012), titres of 104 eid50/ml live vaccine applied under ideal laboratory conditions still equates to a significant number of live replicating viruses. current mass-immunisation methods involving vaccine application by spraying or drinking water are possibly insufficient to guarantee that each bird in the house receives an adequate vaccine dose (dortmans et al. 2012). concurrent infection with immunosuppressive pathogens may also contribute to poor immunity levels so facilitating nd viral spread (jeon et al. 2008). nd may be spread in supposedly well-vaccinated flocks and maintained over a period of years (dortmans et al. 2012). the efficacy of transmission from birds shedding low nd virus titres under field conditions remains to be investigated. clearly what is urgently needed in south africa is stricter implementation of good biosecurity measures as well as enforcement of the existing control policies to prevent newly-introduced exotic strains from becoming epidemics. the ultimate goal should be to attain ‘nd-free’ status. acknowledgements competing interests the author declares that she has no financial or personal relationships that may have inappropriately influenced her in writing this article. references abolnik, c., 2007, ‘molecular epidemiology of newcastle disease and avian influenza in south africa’, phd thesis, dept. of zoology, university of pretoria, viewed 01 july 2016 from http://upetd.up.ac.za/thesis/available/etd-06202007-123059/ abolnik, c., horner, r.f., bisschop, 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sheng wu hua xue yu sheng wu wu li xue bao (shanghai) 35(9), 801–805. zou, j., shan, s.h., yao, l.t. & gong, z.x., 2002, ‘sequence analysis of f gene of sf02 isolate of goose paramyxovirus and its identification by multiplex rt-pcr’, sheng wu hua xue yu sheng wu wu li xue bao (shanghai) 34(4), 439–444. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) doreen c. sitali department of disease control, university of zambia, zambia mwamba c. twambo department of veterinary services, ministry of fisheries and livestock, zambia mumba chisoni department of disease control, university of zambia, zambia muma j. bwalya department of disease control, university of zambia, zambia musso munyeme department of disease control, university of zambia, zambia citation sitali, d.c., twambo, m.c., chisoni, m., bwalya, m.j. & munyeme, m., 2018, ‘lay perceptions, beliefs and practices linked to the persistence of anthrax outbreaks in cattle in the western province of zambia’, onderstepoort journal of veterinary research 85(1), a1615. https://doi.org/10.4102/ojvr.v85i1.1615 original research lay perceptions, beliefs and practices linked to the persistence of anthrax outbreaks in cattle in the western province of zambia doreen c. sitali, mwamba c. twambo, mumba chisoni, muma j. bwalya, musso munyeme received: 20 feb. 2018; accepted: 16 july 2018; published: 29 aug. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract anthrax, a neglected zoonotic disease that is transmitted by a spore-forming, rod-shaped bacterium, bacillus anthracis, has reached endemic proportions in the western province of zambia. transmission of anthrax from the environment as well as between cattle has been observed to be partly because of entrenched beliefs, perceptions and traditional practices among cattle farmers in the known outbreak areas. this study was aimed at exploring lay perceptions, beliefs and practices that influence anthrax transmission in cattle of the western province. a mixed-methods study was conducted from august to december 2015. quantitative data were collected using a cross-sectional survey. qualitative data were generated by interviewing professional staff and community members. five focus group discussions and five key informant interviews were conducted. thematic analysis of interview data was performed using nvivo software. the findings suggested that cattle anthrax was biologically as well as culturally maintained. cattle farmers were reluctant to have their livestock vaccinated against anthrax because of perceived low efficacy of the vaccine. also, the cattle farmers did not trust professional staff and their technical interventions. popular cultural practices that involved exchange of animals between herds contributed to uncontrolled cattle movements between herds and subsequent transmission of anthrax. these findings imply the need for professional staff to be culturally competent in handling socio-cultural issues that are known to be barriers for disease control in animals. there is a need to develop a policy framework that will foster integrated control of anthrax across disciplines. introduction anthrax is a severe infectious disease caused by a bacterium known as bacillus anthracis. anthrax can be found naturally in soil and commonly affects domesticated and wild herbivores. although anthrax is perceived to be a weapon of bioterrorism in most countries in the world (siamudaala et al. 2006), it has ecologically emerged to be a significant public health threat in the western province of zambia. in this region, the disease has reached endemic proportions with interminable outbreaks in both cattle and humans (siziya 2017). between 1989 and 1995, the western province recorded 1626 suspected cases of cattle anthrax of which 51 were confirmed cases (siamudaala et al. 2006), and 1216 cases were recorded between 1999 and 2007 (munang’andu et al. 2012). furthermore, zambia is categorised under the countries where the problem of anthrax in africa has reached a hyper-endemic state given the incessant and the long-drawn-out outbreak periods that seem unceasing in the recent years (world anthrax data site 2007). the disease has had a modifying effect across families, public health institutions and ecotourism in the affected areas of the western province as well as other parts of zambia, such as the luangwa valley (kamboyi 2015). despite the western province being the most sparsely populated place in zambia, with a human population density of less than five people per square kilometre (iucn 2003), outbreaks and occurrence of anthrax in cattle are markedly more noticeable in this area than any other area in zambia. the western province has recorded six outbreaks of anthrax in cattle and humans between 2011 and 2016 compared to southern province that has not recorded any outbreaks since 2011 (office of the auditor-general 2015). the cattle population in the western province is estimated to be over 760 000 but is continuously under serious threat not only to anthrax but also to contagious bovine pleuralpneumonia among other diseases (national livestock epidemiology and information centre 2015). the place has the highest incidence and prevalence of anthrax in zambia, with cultural practices and beliefs of the local people being identified as significant risk factors coupled with the ecological set-up (kamboyi 2015; munang’andu et al. 2012; siamudaala et al. 2006). the disease endemicity is a result of appropriate mix of environmental and epidemiological factors. ecological factors include the cyclical rainfall pattern, flooding, evaporation potential, temperature and the geology of the floodplains. epidemiological factors include the increase in cattle and human populations on the floodplains during the dry season, leading to anthropogenic pressure, transhumance grazing system (munang’andu et al. 2012) and human behavioural factors (sitali et al. 2017). notwithstanding that ecological and epidemiological factors have been researched to some extent, human practices and behavioural factors have not been scrutinised and are still poorly understood (mumba et al. 2018). outbreak investigation reports and reviews of anthrax outbreaks in the western province have indicated that the disease has persisted in the province because of entrenched cultural beliefs and practices of local communities (mwambi et al. 2017). however, this phenomenon has been poorly explored thus far. it is crucial for programme implementers to consider local beliefs, practices and perceptions surrounding the disease if control measures are to yield the most significant results. modifying factors such as demographics and socio-economic status, among others, are vital in predicting how a community responds to information leading to health behaviour. in this study, the overall objective was to assess and determine the perceptions, beliefs and cultural practices, and other anthropogenic factors, related to the contraction of anthrax by cattle in the western province. these factors have been partly discussed in a previous paper (sitali et al. 2017); in this article, we describe the observational and situational analysis data of anthrax in the western province as it relates to the endemicity in cattle, zoonotic implication and public health impact of the disease. materials and methods study area three districts that are endemic to anthrax in the western province were conveniently selected for the study. these were mongu, nalolo and limulunga (figure 1). the western province lies in the upper zambezi basin also called the barotse floodplain located at coordinates 14°19’–16°32’s and 23°15’–23°33’e and covering about 5500 km2 in extent (iucn 2003). the maximum flooded area is estimated at 10 750 km2 (welcomme 1975) when floods of all tributaries of the zambezi river are taken into account. the floodplain stretches from the confluence of the lungwebungu river with the zambezi river in the north extending southwards for a distance of 250 km until ngonye falls. soils are composed of the kalahari sands stretching several metres deep underlain by calcareous rocks (munang’andu et al. 2012). elsewhere, calcareous soils have been associated with prolonged survival of anthrax spores (hugh-jones & hussaini 1975; new et al. 2002). the main human activities in the study area are traditional cattle farming, fishing and rice farming, and to a lesser extent, maize and cassava farming in the upper forest lands. the cattle population in the study area is estimated at over 760 000 (national livestock epidemiology and information centre 2015). the zambezi floodplain alone contains a population of over 225 000 people in an estimated 28 000 sparsely spaced households (central statistical office zambia 2014). given the landmass of the floodplain to be 10 750 km2 at its maximum (welcomme 1975), this gives a human population density of 20.6 people/km2 in the floodplain during the dry season. however, it was estimated that another 200 000 people live on the plain margin (turpie 2004) and their livelihood is also dependent on the floodplain, thereby exerting more pressure on the natural resource utilisation of the floodplain. figure 1: map of zambia showing four districts of the western province. study design the study was a mixed-methods design employing both quantitative and qualitative methods. a cross-sectional survey was concurrently conducted with five focus group discussions and five key informant interviews from august to december 2015. quantitative data were reported in a paper by sitali et al. (2017). data collection and analysis respondents for the focus group discussions were purposively sampled from the survey participants based on whether they had lost cattle because of anthrax, had a family member who suffered from anthrax or died from the same. five key informant interviews with professional staff were held as follows: one with a senior veterinary officer, three with veterinary assistants and one officer working for the ministry of health. an unstructured topic guide with open-ended questions was used to guide the interviews. the interview guide focused on the experiences of key informants with communities in controlling for anthrax, and the challenges faced. all the interviews took place in the respondents’ environments, either their office or home. the interviews lasted for a minimum of 30 min to 1 h. also, five focus group discussions were held with community members as follows: one in limulunga, two in mongu and two in nalolo district. each focus group had an average of 11 participants. two of the focus group discussions were held with female participants only; one was held with men only to identify gender differences in perceptions and practices. two of the focus group discussions were held with both sexes. field observations were supplemented with informal discussions with community members and professional staff from the health and livestock departments. a topic guide was used to guide the discussions, focusing on the beliefs, and perceptions, of respondents towards professional staff and control measures. common cattle-rearing practices and cultural practices surrounding anthrax were also explored. piloting of the tools was not performed as is the standard practice in qualitative research. however, at the end of each field day, recorded narratives were audio played to identify areas that needed further probing or clarification. the questions in the interview guide were then adjusted accordingly. this was repeated until saturation of data (no new themes emerge from data) was reached. interviews were audio-recorded. all the interviews were moderated by the principal investigator and a research assistant took field notes. focus group discussions were either held at a village or crush-pen. focus group discussions were held in the local language (lozi) to facilitate understanding. the narratives were audio-taped and later translated into english and transcribed into computer files. nvivo 12 for windows was used to help manage the data. broad coding followed by fine coding was performed in nvivo software to facilitate the identification of themes. framework matrices were formulated to help cross-check information from community members with that of professional staff to identify similar and contradicting views between professional and community members. illustrative quotations that represented the themes were used to present results. information was summarised through a logical risk chart of pathogen transmission and contamination based on the lay practices, beliefs and cultural understanding of the disease. ethical consideration the study received ethical approval from the university of zambia biomedical research ethics committee (unzabrec), protocol number 013-08-15. permission to collect data was obtained from the provincial veterinary officer of western province. informed verbal consent was obtained from all the participants before interviewing them. results the results presented in this article were from the focus group discussions, key informant interviews, field observations as well as review of annual veterinary records. four major themes and three sub-themes were generated from the focus group discussions and interviews. these were: popular beliefs of community members perceptions of community members common practices of community members cattle-rearing practices carcass disposal practices cattle vaccination practices factors influencing beliefs and practices. popular beliefs of community members farmers believed that the vaccine made their animals sick instead of protecting them from anthrax. according to them, cattle continued to die even after being vaccinated. therefore, community members did not consider the vaccine to be effective in protecting their animals. one female respondent had this to say about cattle vaccination: ‘we have observed that the vaccines are injected in our cattle but what we have seen is that the disease continue to escalate. because just after vaccination the cattle would die and again a week after, another.’ (fgd 2, male farmer, aged 52) a senior veterinary staff explained that this belief was as a result of the manner in which government conducted anthrax vaccinations. he explained that anthrax was classified as a management disease which meant that the farmer bore the cost of vaccination as opposed to diseases of national economic importance (dneic) such as contagious bovine pleuropneumonia (cbpp) for which the government sponsors annual vaccination. however, most cattle farmers preferred to be reactive rather than proactive because they were only willing to have their cattle vaccinated against livestock diseases once there were cattle mortalities. because there were usually no clinical signs observed before the cattle died, most animals at risk may already have been infected such that once vaccinated, some of those already infected succumbed to the disease as the vaccine itself is a live attenuated strain. also, community members believed that veterinarians introduced anthrax into their animals when they went to vaccinate the animals. one key informant explained how this belief came about in the area where he was working. he said: ‘…they have various reasons to give; their belief is that, when veterinary people come to their area to vaccinate animals, they bring diseases. our animals usually die after they have vaccinated them. moreover, this belief, it is like it emanated from the early years of 1970s when the first outbreak of cbpp occurred in western province. because the vaccine that was being used then, the route of injection was the tail swish, so animals reacted by losing their tails, then because of that they believed that veterinary staff brings diseases to their animals.’ (key informant 5, male, veterinary officer, aged 38) because of these beliefs, most farmers avoided taking their cattle for vaccinations because of fear of losing them from anthrax. some key informants explained that the situation in some veterinary camps was so severe that the veterinary officers had to work with police officials during vaccination campaigns. the police officers had to handcuff some of the resisting farmers to get their animals vaccinated. sometimes, police officers were required to protect veterinary officers against hostile farmers (figure 2). figure 2: cattle owner handcuffed by police officer to allow for cattle vaccination. perceptions concerning veterinary staff from the focus group discussions, it was also evident that community members had certain perceptions concerning veterinary staff. respondents suspected that veterinary staff worked with a reputed meat-processing company to wipe out their cattle population from their province. veterinary staff were also suspected to be practicing satanism (satanism is a group of ideological and philosophical beliefs based on satan). this suspicion came as a result of veterinary staff collecting blood samples from animals for sero-surveillance for cbpp. one key informant explained what commonly happened in his veterinary camp, he said: ‘the other part again is where you do sero-surveillance, when you are doing sero-surveillance; you have to collect blood samples…… so those when you collect blood from the animals they say these vets are practicing satanism, where do they take the blood of our animals? so again even then, we had to work with the police to convince them to take blood samples. usually, after you have handcuffed someone, usually they give in.’ (key informant 5, male, veterinary officer, aged 38) common practices among community members cattle-rearing practices cattle are the primary source of livelihood for most families in the western province. cattle are sold to pay for school and medical fees, household necessities and dowry (lobola), and are used for draught power and manuring crop fields. because of this, cattle loaning and exchange is common. most farmers in the province also sell their cattle to a meat-processing company that is based in the provincial capital. it is therefore common for most farmers to trek their cattle on foot to the central abattoir. during trekking, farmers commonly use bush tracks that lead to interchange of diseases among cattle herds they directly encounter on the way or share grazing areas with. also, when lobola is paid in the form of cattle, these have to be trekked to the woman’s family. sometimes, cattle are loaned to another herder to take care of under the mafisa agreement (see below) and therefore had to be taken to the plains for manuring crop fields and to plough the fields. these scenarios lead to uncontrolled animal movements that result in the spread of animal diseases including anthrax. in the period before 1991, government had workers who manned and controlled animal movements and had infrastructure to regulate movement of cattle. these were laid off during the structural adjustment programme that was undertaken in 1991 (muuka et al. 2012). this led to vandalism of the infrastructure and breakdown in animal movement controls. one participant explained the scenario in his village and said: ‘the problem of anthrax we do not have a law on the movement of cattle. so the cattle that bring the diseases are those from shang’ombo (a neighbouring district). in the past there used to be buffer zones from shang’ombo there, there used to be camps, so those people used to control the spread of diseases, but today the fences (cordon lines) have been damaged.’ (fgd 3; male participant; farmer; aged 62) in the western province of zambia, cattle farmers commonly practice a traditional cattle risk-management system called mafisa. in this system, farmers apportion out their cattle to other people to herd as a way of reducing the risk of losing animals from diseases and cattle raiders (sumbwa 2000). if the person herding the cattle under the mafisa agreement takes good care of the animals, and they multiply, they are rewarded with an animal at the end of the year. therefore, the system also served as a form of social support. however, if a cattle has died while under the care of a herder, he was obliged to produce evidence that it was not sold but died on its own. the herder is expected to dry the meat and or preserve the skin (figure 4 left) and horns for the owner’s inspection to prove that he did not sell the animal. this practice has led to persistence of anthrax spores in the environment as the spores have been found to survive in these animal products for years. one respondent in a focus group discussion explained what happened in the mafisa agreement in bulozi. he said: ‘it can die (meaning cattle) at the mafisa the one keeping it will not do anything about it until the owner comes. i can say that sometimes the owner of the cattle for mafisa is not around, went somewhere, because of fear of getting into trouble what will he do, just skin that cattle and dry it to wait for the owner to come and get it when he is back.’ (fgd 3 participant, male, farmer, aged 56) in some instances, calves that are born within the mafisa contracts are taken back to where the owner lived. in this way, infected calves transmitted anthrax from one herd to the other. it is also a common practice for some farmers to send their animals to the plains for manuring crop fields and therefore cattle diseases were also transmitted between herds. furthermore, sometimes, farmers distribute their animals to other relatives or areas when they heard rumours of a disease. if the cattle were already infected, they transmitted the disease to other herds. this study also observed that uncontrolled cattle movement was a common practice in the province. cattle were commonly used to pay lobola. therefore, once a woman was married off, cattle were moved to the woman’s family. moreover, cattle were also commonly used as a form of payment to traditional healers when one fell ill. because of these trade-offs, cattle were commonly moved between herds, leading to possible transmission of animal diseases including anthrax. carcass disposal practices farmers also explained that because veterinary officers rarely responded to reports of animal mortalities, they attempted to diagnose the cause of death in cattle themselves. therefore, it was common for farmers to open a carcass and examine for an enlarged spleen in the animal. this is usually performed in the plains where animals were being grazed. this practice therefore leads to exposure of the vegetative cells into open air (oxygen) and forms spores which contaminate the grazing areas and water points ready for infecting another animal. one respondent had this to say about carcass disposal: ‘what i want to say is that us that have cattle the problem we face is caused by the vet, we do not have a vet officer here nearby to diagnose the cause of death of the cattle and what disease it was suffering from so that we know what to do. so when it dies, we just get it and eat because we do not know what sickness it had.’ (fgd 1 participant, female, aged 47) commonly, most farmers eat contaminated meat and dispose of cattle’s remains by burying (chanda, mulubwe & mwale 2017; chavwanga 2013). disinfection of burial sites or carcasses is not performed, facilitating disease transmission. this was because, most of the times, veterinary staff are not available to supervise carcass disposal because of logistical challenges of transport and lack of fuel for incineration. villagers butcher the animals and share the meat among themselves because of poor access to veterinary services. this practice leads to the dissemination of anthrax bacterium and contaminates the environment where other cattle are grazing (figure 3). in instances where veterinary staff supervised disposal of the carcasses, the common method of disposal is by burying without decontamination of sites. this is because of lack of fuel for incineration and scarcity of firewood in the floodplains, and lack of disinfectants such as formaldehyde. often, community members exhume cattle remains (figure 4 right) and consume them because of high poverty levels. incineration is the recommended method of choice disposal of anthrax carcasses (food and agriculture organisation of the united nations 2003). figure 3: community members burying anthrax carcass in grazing field. figure 4: preserved hide from anthrax carcass (left); exhumed remains of incinerated anthrax carcass (right). cultural practices affecting cattle-rearing in the western province, the local inhabitants depend on a transhumance grazing system that is tied to the flooding regime. annually, herdsmen along with their families take cattle into the wetlands during the dry season (june–november). therefore, at the peak of the dry season, cattle herds are concentrated around the lagoons and oxbow lakes. as the grass becomes overgrazed, cattle inhale the spores from the grass. they also ingest spores by drinking water from the lagoons where spores have been concentrated by incubator conditions (munang’andu et al. 2012). this migration sometimes transmitted anthrax between the floodplains and the highland areas. cattle vaccination practices the study also established that most farmers in the province did not vaccinate their cattle against anthrax. when provincial veterinary records for animal vaccinations were checked, it was found that only about 10% of the farmers vaccinated their cattle. most key informants explained that most farmers avoided having their cattle vaccinated because of misconceptions and beliefs concerning the vaccine and veterinary staff. one veterinary camp officer explained how he experienced resistance from farmers in his district. he said: ‘what we realize in … [name withheld] is that people are very difficult there … there is a lot of non-compliance by the farmers to have their animals vaccinated. even despite this veterinary assistant acquiring some vaccine, others were still resisting to pay for the animals. they have various reasons to give; their belief is that, when vets come to our area to vaccinate the animals, they bring diseases. our animals usually die after they have vaccinated them …’ (key informant 1, male, veterinary officer, aged 62) apart from misconceptions and beliefs, most veterinary staff indicated that several logistical barriers affected farmers’ ability to have their cattle vaccinated. among the most notable ones were the lack of cold chain equipment in the veterinary camps and low socio-economic status of farmers. one farmer said: … the issue that affects us is that money is difficult to find with the poverty we have in our homes even just to manage to pay for one cattle to be vaccinated is a challenge … (fgd 2 participant, farmer, aged 77) other barriers were reduced access to the vaccine and lack of funding for logistical support for vaccinations and geographical remoteness of most areas. these barriers were further compounded by lack of government funding for anthrax control as a result of the disease being classified as a management disease. also, communities had inadequate access to social services such as schools, health facilities and markets and therefore felt neglected. they lacked information on how anthrax affected their animals and how they got affected themselves. because of this, most of them had poor health promotion–seeking behaviours such as not having cattle vaccinated. in the western province, it was common for most cattle herds to be owned by several members of a kinship, some of whom lived away from the village or even overseas. therefore, decisions to have cattle vaccinated were often delayed because they needed to be made collectively. also, some veterinary staff explained that most farmers were of low educational status and did not understand the vaccination schedules. therefore, because of poor understanding of the schedules, most farmers thought that their cattle were protected against anthrax once they received vaccinations for other diseases. table 1 summarises the findings in a logical risk chart. table 1: barriers identified to affect anthrax outbreaks in the western province of zambia. discussion this article set out to explore the lay beliefs, perceptions and practices linked to the persistent transmission of anthrax in cattle in the western province. lay perceptions and beliefs the study observed that community members had certain beliefs and perceptions that influenced their response to technical interventions. firstly, community members had a negative attitude towards the anthrax vaccine because they believed that the vaccine was not effective in protecting their cattle against anthrax. therefore, they were not willing to have their cattle vaccinated. this observation resonates with the postulation of the health belief model. the health belief model postulates that an individual is more likely to seek help or treatment when they see the efficacy of taking such action to reduce susceptibility or reduce the risk of disease (perceived benefits) (burke 1950). the model has been applied to predict health-related behaviours such as vaccinations. therefore, there is a need to sensitise farmers on the importance of cattle vaccinations. vaccinating cattle during outbreaks should be avoided to address misconceptions concerning the efficacy of the vaccine. equally so, the community members did not seem to trust professional veterinary staff as they suspected them of practicing satanism. the lack of trust by community members could be attributed to reduced access to information and lack of community engagement as expressed by community members. some studies in other zoonotic diseases like ebola have demonstrated similar trends (dingwall 2015). dingwall indicated that during the ebola outbreak in west africa, health professionals failed to collaborate with traditional healers, who were often more accessible and more trusted than government health systems. public health communications were poorly designed and may well have promoted rather than reduced risk behaviour. research evidence has shown that community involvement in control programmes should be fostered to enhance trust and cooperation (dingwall 2015). this finding confirms the importance of involving local communities in programmes to enhance cooperation and trust. dingwall (2015) therefore concluded that ‘community engagement is the one factor that underlies the success of all other control measures’. therefore, the importance of community engagement cannot be overemphasised. lay practices affecting anthrax transmission the study also established that the communities were engaged in specific cultural practices that facilitated transmission of anthrax between animals. firstly, the communal ownership of cattle made it difficult for herders to make timely decisions to vaccinate their animals. other studies have indicated that most pastoral communities tend to have a communal ownership of cattle, which leads to difficulties in making decisions. people working in urban areas yet owning cattle back home leave their animals under the husbandry of caretakers on the floodplains (munang’andu et al. 2012). when an animal dies in the absence of the owner, the caretaker would keep the head, hide or other suitable body parts as evidence that the animal was not sold but died of a disease when the owner returned. such traditional practices predisposed both cattle and human beings to the risk of anthrax infection. further, communities in the western province practiced the transhumance grazing system. the annual migration of cattle between the upland and the floodplains put anthropogenic pressures on the limited pastures and water points (munang’andu et al. 2012). this can be attributed to the fact that during the dry season (may–october), most watering points tend to be dry, with only muddy water remaining in the oxbow lakes and lagoons apart from the main river channel. the grass is depleted from most of the floodplains except the dambos around the oxbow lakes and lagoons. animals graze close to the ground and inhale the spores. it is also likely that they ingest spores by drinking muddy water that has been contaminated by spores persisting in the micro-environments around the oxbow lakes and lagoons that serve as the only sources of water. this situation could have facilitated the transmission of anthrax to animals. although the farmers in the western province are not nomadic, the annual migration system promotes the transmission of diseases into new areas as is the case with the masai of tanzania (mangesho et al. 2017). carcass disposal practices when an animal died also contributed to the transmission of animal anthrax. in the western province, it was common for community members to slaughter and eat meat from infected carcasses. therefore, when cattle died, it was butchered and shared right from where it died. this practice promoted the dissemination of anthrax bacterium into the environment. incineration was rarely practiced as professional staff did not commonly respond to calls for animal mortalities. processing of animal products disseminated the spores even further to the immediate environment and beyond. a study conducted in bangladesh also found that community members disposed of their carcasses in water bodies where humans and animals drank water (hassan et al. 2015), leading to ingestion or inhalation of spores by other cattle. these findings underscore the importance of professional staff to supervise and educate community members on the safe disposal practices of the carcass. tuchili et al. (1997) were able to isolate b. anthracis from preserved smoked dry meat in the western province, indicating that the processing and preservation methods employed do not inactivate the anthrax spores. further studies carried out by tuchili et al. (1997) showed the presence of anthrax spores from cattle hides that had been preserved and stored for a long time. this poses a significant public health threat, considering that large quantities of these products are sold to the general public outside the western province without any quality assurance tests and public health inspections, thereby increasing the risk of anthrax distribution from endemic to non-endemic urban areas every year. consistent with other studies (chakraborty et al. 2012; chirundu et al. 2009; hassan et al. 2015), this study established that few farmers vaccinated their animals against anthrax. participants in the focus group discussions cited various reasons for failure to have their animals vaccinated. the most significant ones were poverty, difficulties in accessing the vaccine, inadequate access to veterinary services and lack of cold chain facilities. key informants mentioned logistical challenges such as lack of cold chain facilities, funding for anthrax control, reduced access to the vaccine and poor attitude of the farmers as some of the factors that perpetuated anthrax in the western province. these findings were consistent with other literature that postulates that anthrax affects poor communities with poor veterinary infrastructure and occurs among marginalised populations (food and agriculture organisation of the united nations 2003). anthrax has also persisted in countries where public health infrastructure is weak. there is therefore need to improve public health infrastructure. conclusion the study established that anthrax infection in cattle was not only biologically determined but also culturally maintained. the study found that cattle owners were reluctant to have their cattle vaccinated because they believed that the vaccine made their animals sick. furthermore, community members did not trust professional staff and their technical interventions. popular cultural practices that involved exchange of animals between herds and uncontrolled animal movements contributed to the transmission of anthrax between cattle. we therefore argue that current technical approaches to control anthrax must be backed by the social, and cultural, framework. also, there must be strategic annual vaccinations of cattle coupled with improved public health awareness campaigns aimed at promoting active participation by the general public in the control of the disease. lastly, there is a need to improve veterinary extension services and restore veterinary infrastructure to control animal movements in the province. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions d.c.s. and m.m. participated in the conception of the project, data collection and writing of the manuscript. m.c.t. participated in the identification of data collection sites, data collection and critical review of 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sensing and geophysical information systems for public health, anxieties over anthrax, viewed 16 february 2016 from http://www.vetmed.1su.edu/whocc/mp_world.htm abstract introduction materials and methods ethical approval results discussion conclusion acknowledgements references about the author(s) oliver t. zishiri department of genetics, university of kwazulu-natal, south africa nelisiwe mkhize department of genetics, university of kwazulu-natal, south africa samson mukaratirwa department of genetics, university of kwazulu-natal, south africa citation zishiri, o.t., mkhize, n. & mukaratirwa, s., 2016, ‘prevalence of virulence and antimicrobial resistance genes in salmonella spp. isolated from commercial chickens and human clinical isolates from south africa and brazil’, onderstepoort journal of veterinary research 83(1), a1067. http://dx.doi.org/10.4102/ojvr.v83i1.1067 original research prevalence of virulence and antimicrobial resistance genes in salmonella spp. isolated from commercial chickens and human clinical isolates from south africa and brazil oliver t. zishiri, nelisiwe mkhize, samson mukaratirwa received: 30 sept. 2015; accepted: 11 dec. 2015; published: 26 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract salmonellosis is a significant public health concern around the world. the injudicious use of antimicrobial agents in poultry production for treatment, growth promotion and prophylaxis has resulted in the emergence of drug-resistant strains of salmonella. the current study was conducted to investigate the prevalence of virulence and antimicrobial resistance genes from salmonella isolated from south african and brazilian broiler chickens as well as human clinical isolates. out of a total of 200 chicken samples that were collected from south africa 102 (51%) tested positive for salmonella using the inva gene. of the overall 146 salmonella-positive samples that were screened for the irob gene, most of them were confirmed to be salmonella enterica with high prevalence rates. all the salmonella isolates obtained were subjected to antimicrobial susceptibility testing with ten antibiotics. salmonella isolates from south african chickens exhibited resistance to almost all antimicrobial agents used. all the samples were further subjected to the polymerase chain reaction in order to screen some common antimicrobial and virulence genes of interest, namely spic, pipd, misl, orfl, pse-1, tet a, tet b, ant (3”)-la, sul 1 and sul. all the salmonella-positive isolates exhibited resistance to at least one antimicrobial agent; however, antimicrobial resistance patterns demonstrated that multiple drug resistance was prevalent. the findings provide evidence that broiler chickens are colonised by pathogenic salmonella harbouring antimicrobial resistance genes. therefore, it is evident that there is a need for prudent use of antimicrobial agents in poultry production systems in order to mitigate the proliferation of multiple drug resistance across species. introduction the increasing human population around the world places a huge demand on food in order to ensure the survival of mankind. this exerts pressure on a number of food industries such as poultry production systems, where growth promotion agents are used in an effort to satisfy the increasing food demand. the presence of salmonella in chicken meat and its related products has often caused them to be unsafe for human consumption (centers for disease control [cdc] 2013). salmonella is classified as one of the most common zoonotic foodborne pathogens that cause outbreaks and sporadic cases of gastroenteritis in humans throughout the world (humphrey 2000). in the united states of america a total of 19 531 infections, 4563 hospitalisations and 68 deaths associated with foodborne diseases were reported in 2012 (cdc 2013). epidemiological studies have reported numerous times that foods of animal origin, particularly poultry, are major vehicles associated with illnesses caused by salmonella (dallal et al. 2010). salmonella can grow as surface-associated aggregates on food surfaces and equipment (chia et al. 2009), commonly described as biofilms. the cells that develop as biofilms are potential sources of contamination on food products, which can result in infection in human hosts (chia et al. 2009). the reservoir of salmonella is the gastro-intestinal tract (git) of a wide range of domestic and wild animals, and a variety of foodstuffs of both animal and plant origins are potential sources of infection (thong et al. 2002; thorns 2000). according to the centers for disease control and prevention (cdc) the genus salmonella is divided into two species, salmonella enterica and salmonella bongori (cdc 2013). the species s. enterica is further subdivided into six subspecies that are designated by taxonomic names such as s. enterica subsp. enterica, s. enterica subsp. salamae, s. enterica subsp. arizonae, s. enterica subsp. diarizonae, s. enterica subsp. houtenae and s. enterica subsp. indica (cdc 2013). salmonella is associated with approximately 2500 serovars. these serovars are separated based on differences in their lipopolysaccharide layer with regard to their somatic (o) and flagellar (h) antigens (amagliani, brandi & schiavano 2012). with regard to the o antigen, salmonella is divided into 50 serogroups and then further divided into more than 2500 serovars based on the h antigens present (amagliani et al. 2012). the majority of the serovars of salmonella belong to s. enterica and the most common serovar associated with zoonotic infection is salmonella enteriditis, followed by salmonella typhimurium (amagliani et al. 2012). serovars that are generally found in food products of animal origin include s. enteriditis, s. typhimurium, salmonella gallinarum, salmonella weltevreden and salmonella infantis (foley et al. 2011). salmonellosis in both humans and animals results from various vehicles of salmonella serovars such as s. enteritidis, s. infantis, salmonella kentucky, and salmonella heidelberg. these vehicles that cause infection appear to be more prevalent in poultry than in any other food animal (foley et al. 2011). the ability of bacteria to infect the host relies on the genetic determinants called virulence genes, located in the salmonella pathogenicity islands (spi). according to groisman and ochman (1997), spis are portions of dna that have been acquired from other microorganisms by horizontal gene transfer and they are absent in non-pathogenic strains. at least 60 virulence genes associated with spis (groisman & ochman 1997) have been mapped so far and they all serve different functions. some facilitate colonisation for the pathogen to survive host defence mechanisms and some are responsible for multiplication inside the host. however, during contamination the host infection outcomes depend on various factors such as age, environment and genetics to influence the host status (van asten & dijk 2005). a well-known virulence gene in salmonella is the salmonella invasion gene a (inva), which is responsible for host invasion (galan, ginocchio & costeas 1992). this gene is vital because it is conserved in all salmonella and is found in spi-1. hence it is used by researchers as a marker to detect salmonella isolated from different sources. various studies from both developed and developing countries have focused on investigating the prevalence of genes encoding for virulence in salmonella. these countries include the united states of america (zou et al. 2011), senegal and gambia (dione et al. 2011), brazil (borges et al. 2013; dias de olivieira et al. 2003) and nigeria (smith et al. 2015). in south africa information on the prevalence of virulence genes in salmonella of animal and human origin is limited. therefore, these among other facts motivated us to embark on the current study in which we evaluated the prevalence of virulence genes in salmonella such as inva, spic, pipd, misl and orfl. the sip proteins, which include sipa, sipb, sipc and sipd, were some of the first virulence genes to be characterised in salmonella based on their involvement in the invasion of cultured epithelial cells (kaniga, trollinger & galan 1995). sipc is directly involved in the translocation process and is delivered to the cytosol of the host cell, where it may also have an effector function (hersh et al. 1999). the spic gene was investigated in this study because it is involved in the interaction with the intercellular membrane and trafficking, thereby hindering correct cellular function (uchiya & nikai 2008). the misl is an spi-3 encoded protein that is involved in other aspects of pathogenesis such as chronic infection and host specificity (blanc-potard et al. 1999). the orfl virulence gene is found in spi-4 and is involved in adhesion, autotransportation and colonisation (hughes et al. 2008). another important virulence gene is the pipd, which is found in spi-5 and is a type iii secreted effector associated with the spi-1 system (hughes et al. 2008). the use of antimicrobial agents in poultry production for treatment purposes, growth promotion and prophylaxis raises a major concern with regard to antimicrobial resistance and multidrug resistance, which are frequently observed among many salmonella serovars (duong et al. 2006). increasing evidence demonstrates that antimicrobial usage in animals promotes the emergence of a wide range of resistant zoonotic pathogens such as salmonella, which compromises the effectiveness of antibiotic treatments used in humans when an infection occurs (gyles 2008). the variety of antibiotics that are administered in veterinary practice therapeutically has caused selective pressure, resulting in an increase in genetic sequences that confer resistance on microorganisms. antimicrobial-resistant salmonella has been recognised as a public health concern for decades in developed and developing countries and the evolving resistance in this pathogen limits the therapeutic options available to physicians for the treatment of human salmonellosis (foley et al. 2011). extensive use of antibiotics in chicken production systems for non-therapeutic purposes such as growth promotion results in the resistance of bacteria to these antimicrobial agents. bacteria use both natural and acquired resistance mechanisms to protect themselves against agents that could harm them. acquired resistance arises from mutations, gene transfer by conjugation or transformation, transposons, integrons and bacteriophages (cogliani, goossens & greko 2011). it is therefore necessary to determine bacterial resistance to antibiotics of all classes, the phenotypes they exhibit and the mutations responsible for resistance using molecular genetic analysis methods. the impact of antimicrobial resistance on human health is of great concern for the treatment of various infections that arise from food of animal origin (glenn et al. 2011), where combinations of broad-spectrum antibiotics need to be administered in order to control infection. therefore, understanding the mechanisms of antibiotic resistance, the location of genes on a chromosome or plasmid and their expression will assist in developing screening and control strategies that are urgently needed in order to reduce the spread and evolution of resistant bacteria. against this background, this study aimed to investigate the prevalence of virulence and antimicrobial resistance genes in chicken samples from south africa as well as imports from brazil and human clinical isolates. genetic characterisation of the antimicrobial resistance and virulence genes present in salmonella is essential in understanding the pathogenicity and prevalence of resistance that exists in this zoonotic foodborne pathogen. materials and methods sample collection broiler chicken caecum samples were collected on the day of slaughter from poultry slaughterhouses within the durban metropolitan area in kwazulu-natal province of south africa between march and october 2014. samples were collected in batches of 25 per month. in total 200 samples were randomly collected over the eight-month period. all the samples were aseptically collected in plastic screw-top tubes containing 45 ml of 0.1% w/v peptone-water and stored on ice until transported back to the university of kwa-zulu natal (westville campus), where enrichment of the samples was done immediately on arrival. south africa imports more than 50% of chicken products from brazil because domestic production cannot meet the current demand. whenever chicken products are imported in batches from other countries such as brazil, quality assurance, routine disease surveillance, screening and testing are conducted before the products are conveyed to the food chain. it was therefore crucial to include some samples that originated from brazil in our study. accordingly, salmonella isolates from brazilian broiler chickens (24) and human clinical samples (20) from patients emanating from the coastal region of kwazulu-natal province in south africa were provided by the national health laboratory service of south africa. enrichment enrichment was carried on south african (sabc) chicken samples only. ten millilitres of rinse peptone-water from the samples were incubated at 37 °c for 24 h. after incubation 0.1 ml aliquots from the peptone-water samples were inoculated into tubes containing 10 ml of rappaport vassiliadis (rv) broth medium and incubated at 42 °c for 48 h (ahmed & shimamoto 2012). microbiological analysis after enrichment, a loopful of the broth culture was streaked onto xylose-lysine-deoxycholate (xld) agar plates and incubated at 37 °c for 24 h. black colonies with typical phenotypic characteristics were regarded as suspect salmonella. suspected salmonella colonies were picked and inoculated on tsb broth and incubated while shaking at 37 °c for 24 h. the resulting culture was used for dna extraction and some was used for susceptibility tests. the remaining culture was used for 60% glycerol stocks that were then stored at -80 °c for future purposes. dna extraction genomic dna of all the salmonella isolates was extracted from the culture using the zymoresearch fungal and bacterial genomic dna minipreptm kit following the manufacturer’s instructions. a positive salmonella control was prepared by isolating genomic dna from a reference strain of known salmonella broth culture. after dna extraction, a nanodrop spectrophotometer was used to check the concentration and quality of the isolated dna and extracted dna was then stored at -20 °c until use in polymerase chain reaction (pcr). confirmation of salmonella using polymerase chain reaction pcr was performed on the dna extracted from all the detected and obtained samples. the inva gene was used to confirm the presence of salmonella. a 25 µl pcr reaction was used for amplification of the inva gene. the primers used for detection of the inva gene are presented in table 1. the pcr reaction was carried out in a total volume of 25 µl containing 12.5 µl dreamtaq green pcr master mix, 1 µl inva primer (forward), 1 µl inva primer (reverse), 4 µl of template dna and 6.5 µl dh2o. amplification was carried out in a thermo-cycler using 34 cycles consisting of denaturation for 30 s at 95 °c, annealing for 30 s at 58 °c, extension for 1 min at 72 °c and final extension for 5 min at 72 °c. pcr products were run on a 1.5% agarose gel using electrophoresis at 70 volts for 60 min to detect a 284 base pair product size of the inva target gene. furthermore, the irob gene that is unique to s. enterica species was used to confirm the identity of the species. the primers used are presented in table 1 and everything was done following the same procedure used for inva gene amplification, except for the annealing temperature, which in this case was 55 °c for 40 s. table 1: primers used to confirm salmonella spp. detection of virulence genes the primers used to detect virulence genes are depicted in table 2. the pcr reactions was carried out in a total volume of 25 µl and under the following conditions: spic gene (initial denaturation at 94 °c for 12 min, 1 min of denaturation at 94 °c, 30 s of annealing at 54 °c and 5 min of extension at 72 °c for a total of 34 cycles; 5 s were added to the extension time each cycle); misl and orfl genes (3 min at 94 °c, 35 cycles of 1 min at 94 °c, 1 min at 58 °c and 1 min at 72 °c and finally 5 min at 72 °c) and for pipd gene (94 °c for 5 min, 34 cycles of 25 s of denaturation at 94 °c, 30 s of annealing at 56 °c and 50 s of extension at 72 °c and a final cycle at 5 min at 72 °c). gel electrophoresis of amplified products was then carried out in 1.5% agarose in a 1x tbe buffer containing gelred. after the gels were run, pcr products were visualised using the chemidoctm imaging system. table 2: primers used to detect virulence genes in salmonella spp. antimicrobial susceptibility testing antimicrobial resistance of the 146 salmonella isolates were tested against ten antimicrobial agents using the kirby-bauer disc diffusion method on mueller hinton agar following the guidelines of the clinical and laboratory standards institute (clsi) (clsi 2008). the antimicrobials selected were those commonly used in the poultry industry, namely gentamicin (10 µg), amoxicillin (10 µg), erythromycin (10 µg), chloramphenicol (30 µg), tetracycline (10 µg), trimethoprim (1.25 µg), ampicillin (10 µg), streptomycin (25 µg), trimethoprim-sulfamthoxazole (25 µg) and kanamycin (30 µg). the oxoid antibiotic discs were impregnated with the concentrations of each antibiotic. first, mueller hinton agar was inoculated with 0.1 ml of nutrient broth samples, which had been inoculated with a loopful of glycerol stocks of positive samples and then incubated at 37 °c for 24 h. with the use of a glass hockey stick, the culture was spread onto the agar for even distribution of the organisms that demonstrated the presence of salmonella after pcr, thereafter; discs impregnated with antibiotics were evenly placed on plates and then incubated at 37 °c for 24 h. the inhibition zones were measured and scored as sensitive (s), intermediate susceptibility (i) and resistant (r) according to the clsi recommendations. escherichia coli atcc 25922 was used as a reference strain for antibiotic disc control (bacci et al. 2012). detection of antimicrobial resistance genes genomic dna of salmonella extracted was used to detect antimicrobial resistance genes. the primer sets used to detect antimicrobial resistance genes are presented in table 3. the pcr reactions were carried out in total volumes of 25 µl each using the following conditions: pse-1 gene (initial denaturation at 94 °c for 12 min, 1 min of denaturation at 94 °c, 30 s of annealing at 57 °c and 5 min of extension at 72 °c for a total of 34 cycles; 5 s was added to the extension time each cycle); ant (3”)-la gene (3 min at 94 °c, 35 cycles of 1 min at 94 °c, 1 min at 58 °c and 1 min at 72 °c and finally 5 min at 72 °c); tet a and tet b gene (94 °c for 5 min, 34 cycles of 25 s of denaturation at 94 °c, 30 s of annealing at 55 °c and 50 s of extension at 72 °c and a final cycle at 5 min at 72 °c). sul1 and sul2 detection was carried in the same way as for tetracycline genes, but with annealing temperatures of 65 °c and 52 °c respectively. gel electrophoresis of the amplified products was then carried out in 1.5% agarose in a 1x tbe buffer containing gelred. after the gels were run, the pcr products were visualised using the chemidoctm imaging system. table 3: primers used to screen antimicrobial resistance genes in salmonella spp. ethical approval the animal studies were approved by the appropriate ethics committee of the university of kwazulu-natal (reference: 012/15/animal); therefore, they have been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments. results culture identification out of the 200 samples collected from sabc slaughterhouses, only 102 (51%) were confirmed positive for salmonella. the 102 salmonella isolates together with the 24 salmonella isolates from brazilian chickens (bbc) and 20 human clinical isolates (sahc) obtained from national health laboratory service (nhls) made the total of samples used in this study 146. out of the 146 samples screened for the irob gene, most were confirmed to be salmonella enterica with the following prevalence rates: 17 (85%) of human clinical samples, 70 (68.6%) of south african chicken samples and 17 (70.8%) of brazilian chicken samples. figures 1 and 2 illustrate the amplification pcr products for inva and irob gene respectively. figure 1: detection of the 284 bp inva gene amplicon from eight representative salmonella isolates by agarose gel electrophoresis. figure 2: detection of the 606 bp irob gene amplicon from nine representative salmonella isolates by agarose gel electrophoresis. detection of virulence genes in salmonella spp. pcr was used to screen for all four virulence genes and all the genes screened were depicted in figures 3, 4, 5 and 6. all the amplicon sizes were consistent with the sizes that were expected. table 4 shows that spic (47%), pipd (35%), misl (2%) and orfl (20.6%) genes were harboured in south african broiler chicken isolates. it was also demonstrated that brazilian broiler salmonella isolates harboured all the genes, with the following prevalence rates: spic (83%), pipd (87.5%), misl (29%) and orfl (25%). the same table presents the results for south african human clinical isolates that were harbouring 85% of spic gene, followed by pipd (80%), then misl (75%), and lastly 20% of orfl. figure 3: detection of the 309 bp spic gene amplicon from eight representative salmonella isolates by agarose gel electrophoresis. figure 4: detection of the 550 bp misl gene amplicon from seven representative salmonella isolates by agarose gel electrophoresis. figure 5: detection of the 350 bp orfl gene amplicon from nine representative salmonella isolates by agarose gel electrophoresis. figure 6: detection of the 400 bp pipd gene amplicon from nine representative salmonella isolates by agarose gel electrophoresis. table 4: prevalence of virulence genes in salmonella isolates from three different origins. antimicrobial susceptibility testing a total of 146 salmonella isolates from different origins were tested for resistance to ten antimicrobial agents using the disc diffusion method. the incidences of resistance for all the isolates tested are presented in table 5. salmonella isolated from sabc exhibited resistance to all ten antimicrobials and the highest rates of resistance observed were: tetracycline (93%), trimethoprim-sulfamthoxazole (84%), trimethoprim (78.4%), kanamycin (74%), gentamicin (48%), ampicillin (47%), amoxicillin and chloramphenicol (31%), erythromycin (18%) and streptomycin (12%). isolates from brazilian broiler chickens also demonstrated resistance to all antimicrobials tested and 100% of the isolates exhibited complete resistance to ampicillin, amoxicillin and tetracycline (83%), trimethoprim (66.7%), erythromycin (62.5%), trimethoprim-sulfamthoxazole (50%), kanamycin (16.7%), gentamicin and streptomycin (12.5%) and chloramphenicol (4.2%). salmonella isolates from south african human clinical isolates exhibited resistance to eight antimicrobial agents. the highest resistance rates were observed for erythromycin and amoxicillin, both at 30%. multidrug resistance was also observed across all isolates tested and table 6 summarises the resistance patterns of salmonella isolates in the current study. the resistance patterns provide evidence for multidrug resistance and from table 6 it can be seen that the salmonella isolates used have the potential to confer resistance to more than two antimicrobial agents. table 5: antimicrobial susceptibility tests on salmonella isolates of different origins. table 6: antibiotic resistance patterns of salmonella isolates illustrating multiple drug resistance. molecular detection of antimicrobial resistance genes in salmonella antimicrobial resistance genes were detected in all 146 salmonella isolates, regardless of antimicrobial susceptibility, using the disc diffusion method since this method is presumptive and needs to be supplemented with other tests. observed pcr results indicated that detected salmonella contained antimicrobial resistance genes known to confer resistance. six antimicrobial resistance genes were screened in total: pse-1, ant (3”)-la, tet a, tet b, sul 1 and sul 2; the phenotypes of the genes are illustrated in figures 7, 8, 9, 10, 11 and 12 respectively. the prevalence rates of the genes detected are presented in table 7. in the sabc isolates the most prevalent antimicrobial resistance gene that was detected was pse-1 gene (56%), known to confer resistance to ampicillin. this gene was followed by tet a (44%), ant (3”)-la (32%) and tet b (28%), known to confer resistance to tetracycline, gentamicin and tetracycline respectively as depicted in table 7. in the brazilian broiler chicken isolates tet a and sul 1 genes (83%) were the most prevalent genes, followed by sul 2 (79%), ant (3”)-la (75%), pse-1 (63%) and tet b (33%). finally, in the south african human isolates the gene that showed the highest prevalence was ant (3”)-la (80%), followed by tet a (70%). the tet b, sul 1 and sul 2 genes all exhibited 60% prevalence and lastly, the pse-1 gene reported a (50%) prevalence. these results are partially consistent with the antimicrobial susceptibility testing because most of the genes were detected in isolates that exhibited resistance. figure 7: detection of the 412 bp pse-1 gene amplicon from eight representative salmonella isolates by agarose gel electrophoresis. figure 8: detection of the 526 bp ant (3”)-la gene amplicon from eight representative salmonella isolates by agarose gel electrophoresis. figure 9: detection of the 210 bp tet a gene amplicon from seven representative salmonella isolates by agarose gel electrophoresis. figure 10: detection of the 659 bp tet b gene amplicon from four representative salmonella isolates by agarose gel electrophoresis. figure 11: detection of the 350 bp sul 1 gene amplicon from seven representative salmonella isolates by agarose gel electrophoresis. figure 12: detection of the 720 bp sul 2 gene amplicon from eight representative salmonella isolates by agarose gel electrophoresis. table 7: prevalence of antimicrobial resistance genes screened from 146 salmonella isolates. discussion the injudicious use of antimicrobials leads to resistance in various bacteria such as salmonella, and therefore antibiotic resistance in foodborne bacterial enteric pathogens is an almost inevitable consequence of the irrational use of antimicrobial drugs in animal production systems (newell et al. 2010). findings from the current study demonstrated the presence of salmonella in broiler chicken isolates at farm level. out of the 200 samples tested for salmonella only 102 (51%) samples tested positive using pcr, amplifying the inva gene. these results were almost in agreement with results presented in previous studies (antunes et al. 2003; capita, alonso-calleja & prieto 2007; chuanchuen & padungtod 2009; hao van et al. 2007), which reported salmonella prevalence as 57%, 49%, 60% and 53.3% respectively. cortez et al. (2006) conducted a study on the identification of salmonella isolates from chicken abattoirs and reported that out of 288 samples collected 52 (18%) samples were tested positive. these results are relatively low and contrary to results obtained from the current study. moreover, similar previous studies (dogru et al. 2010; kaushik et al. 2014; van nierop et al. 2005; zewdu & cornelius 2009) also reported very low salmonella prevalence – 8%, 23.7%, 19.2% and 8% respectively – compared to the current study. information from the literature provides proof that broiler chickens are potential carriers of salmonella. however, various factors contributing to the high prevalence of salmonella isolates were detected in the present study. feed, housing and the hygiene status of the farms where the chickens were reared may be some of the factors. the presence of salmonella species at farm level is a serious concern because it shows that the potential exists for the pathogens to disseminate from the farms to communities. this is of great concern because the majority of south africans depend on food sold in informal retail outlets where hygienic conditions are questionable and promote the accumulation and proliferation of pathogens, posing a danger to consumers. the presence of salmonella in chicken meat is quite serious because south africa imports a significant amount of poultry products from other countries such as brazil, china and the usa. consumers are therefore at risk of contracting salmonellosis from either local or imported poultry products. it is worth noting that the greatest attribute salmonella uses to survive in a host cell is pathogenicity. various studies have probed the significance of pathogenicity in a pathogen–host interaction and most concluded that pathogenicity is indeed significant for the pathogen to survive and proliferate. pathogenicity helps salmonella to invade and destroy epithelial cells in the host intestines (uchiya & nikai 2008) and then to colonise other cell lines. all this happens as a result of the presence of the genetic determinants responsible for virulence in salmonella spp. in the current study the prevalence of four virulence genes was established. the virulence genes were selected on the basis of their functions and danger to chickens and humans. all the genes detected belonged to different salmonella pathogenicity islands (sp1). it was not part of the scope of the study to detect these islands, but the information was sourced from the literature. salmonella pathogenicity islands sp1-2, sp1-5, sp1-3 and sp1-4 encode for spic, pipd, misl and orfl genes respectively. the function of the spic gene is to interact with intercellular membrane trafficking in such a manner as to alter it, hindering the correct cellular functioning (uchiya & nikai 2008). the results obtained from south african broiler chickens present spic gene as the most prevalent virulent gene that was detected among the four genes that were screened. the prevalence rates reported were 47%, 35%, 2%, and 20.6%, all of them corresponding to the following genes respectively: spic, pipd, misl and orfl. the detection of these genes in isolates obtained at a farm level on the carcasses of healthy chickens emphasises the fact that healthy chickens with no sign of illness can be carriers of pathogenic salmonella (skyberg, logue & nolan 2006). this demonstrates the risk of chicken meat to consumers and implies the possible dissemination of virulence genes. the presence of the same virulence genes in human clinical samples demonstrates the dissemination and distribution of virulence genes, although the origin of the salmonella that infected the patients was not known because no background information had been obtained from patients about possible sources of infection with salmonella. salmonella isolates detected from brazilian imported chicken meat also demonstrated the presence of all the virulence genes screened during the study, with spic and pipd genes as the most prevalent, having a prevalence rate of 83% and 87% respectively. the genes with the lowest prevalence rates were orfl (25%) and misl (29%); both have been reported to be responsible for the survival of salmonella in host macrophages. virulence genes have also been detected in previous studies from brazil (borges et al. 2013; castilla et al. 2006; dias de olivieira et al. 2003), west africa (dione et al. 2011), colombia (sánchez-jiménez et al. 2010) and england (hughes et al. 2008). therefore, the detection of virulence genes from chicken meat imported from other countries and then sold in south africa indicates the possibility of a transfer of salmonella pathogenic strains from other countries to south africa. although virulence genes are common in local salmonella strains, receiving foreign strains can worsen the situation by increasing the prevalence of genes encoding for pathogenicity and increasing genetic diversity of salmonella strains in south africa. antimicrobial resistance is a global public health problem. the increasing antimicrobial resistance in salmonella is a forward irreversible reaction, but it can be reduced if certain precautions are followed worldwide. tetracycline is a commonly used antimicrobial agent in human and animal medicine because it is cheap and easily accessible. tetracycline resistance has been reported worldwide, and it comprises three types of resistance mechanism, namely tetracycline efflux, tetracycline modification and ribosomal protection (roberts 2005). during the current study salmonella isolates from brazilian chicken isolates, south african chicken isolates and south african human isolates exhibited resistance to tetracycline, with prevalence rates of 83%, 93% and 10% respectively. both genes known to confer resistance to tetracycline, namely tet a and tet b, were detected in some isolates that exhibited resistance. the prevalence of tet a was higher than tet b in all the groups of isolates screened. this was quite similar to the findings in previous studies that reported the same pattern (chuanchuen & padungtod 2009; miko et al. 2005). not all of the confirmed salmonella isolates were harbouring tet a and tet b genes, leading to the prevalence of the genes being low compared to the prevalence of salmonella resistance to tetracycline. these results imply that there might be other antimicrobial genes conferring resistance that were not detected in the study, as there are other determinants that confer resistance to tetracycline, namely tet c, tet d, tet r, tet m and several others. however, in the human isolates the prevalence of resistance was very low, but the two genes were detected even in the isolates that were susceptible to tetracycline. ampicillins and amoxicillin are among the drugs of choice for treating salmonellosis (de toro et al. 2011). in the current study, salmonella isolates from brazilian chickens demonstrated high resistance to these two drugs compared to other isolates. a gene conferring resistance to β-lactamase, namely pse-1, was detected in most of the salmonella isolates found to be resistant to ampicillin and amoxicillin. llanes, kirchgesner and plesiat (1999) reported that resistance to β-lactam is due to the production of the pse-1 enzyme. according to glenn et al. (2011), salmonella spp. isolated from food-producing animals have been reported to carry the pse-1 gene and it is one of the most prevalent β-lactamases. the current study demonstrated high prevalence of salmonella isolates with the pse-1 gene compared to similar studies (bacci et al. 2012; chuanchuen & padungtod 2009) that reported very low prevalence rates of 0% and 5% respectively. south african studies have detected the presence of pse-1 gene in aquatic systems and in livestock production (igbinosa 2015; igbinosa & okoh 2012). although studies related to the mapping pse-1 gene have been conducted, there is still a paucity of information on the prevalence of this gene in south africa. more information on such genes can contribute to the solution of developing new drugs. since high prevalence rates of pse-1 gene were observed, this implies that the presence of β-lactamase in foodborne pathogens is increasing. batchelor, hopkins and threlfall (2005) speculated that the increasing presence of β-lactamase in pathogenic bacteria limits the therapeutic use of antimicrobial agents. the ant (3”)-la gene is one of the aminoglycoside resistance determinants. it has been detected in a number of bacterial pathogens, but information about this gene’s prevalence in salmonella is limited, especially in africa, where little research has been done. in the current study it was detected in some of the gentamicin-resistant salmonella isolates. moreover, genes conferring resistance to sulfamethoxazole (sul 1 and sul 2) were also detected in most salmonella isolates that exhibited resistance to trimethoprim-sulfamethoxazole. the results showed that some isolates were even harbouring both genes. multidrug resistance is an increasing problem that has been reported in both animal and human medicine. salmonella isolates used in the present study illustrated a high rate of multidrug resistance. the results presented in table 6 show patterns illustrating multidrug resistance, which can be confirmed if an isolate is resistant to more than two antibiotics. there may be several possible explanations for such outcomes but the main one could be a lack of compliance with legislation governing the amount and type of antimicrobial agents used in the south african poultry industry and also in human medicine. multidrug resistance has a negative impact on therapy in both animal and human medicine. various studies have proven that infections caused by multidrug-resistant salmonella strains are more dangerous than the infections caused by susceptible strains, since they extensively delay therapy, placing patients’ lives at risk (martin et al. 2004; varma et al. 2005). the presence of multidrug-resistant salmonella strains in chickens and also in humans has serious implications for public health systems and for the economy. overall, the results obtained from the study demonstrate that the detected salmonella strains harboured both virulence and antimicrobial resistance genes. the potential exists for the random dissemination of these genes, with negative implications for the health and welfare of chickens and humans and could result in increased antimicrobial resistance. since some developed and developing countries have prohibited the use of some antimicrobial agents as feed additives in animal husbandry, especially poultry, south africa should take note of what has been happening in other countries with regard to the regulation of antimicrobial use and attempt to prevent the escalating antibiotic resistance problem. conclusion in conclusion, all of the 12 genes examined in this study were successfully amplified in the salmonella isolated from different origins. these findings indicate that the selective pressure caused by the variety of antibiotics administered therapeutically in veterinary practice and poultry production systems for growth promotion and prophylaxis has resulted in an increase in genes conferring resistance to salmonella. it is difficult to make comparisons between the salmonella surveillance surveys conducted in different countries as the prevalence of salmonella varies regionally and isolation rates depend upon the country, sample plan and methodology used. the data from this study indicate the dissemination of antimicrobial resistance genes in salmonella isolated from broiler chickens at the abattoir level. the emergence and dissemination of antimicrobial-resistant salmonella in food animals has major public health implications, especially for large-scale suppliers who export their products both regionally and internationally. therefore, foodborne salmonellosis should constantly be monitored. future work in the area of this study should include organism specificity by serotyping positive salmonella samples in order to determine the serovars of salmonella which are most prevalent in broiler chickens. furthermore, phylogenetic analyses should also provide interesting insights into determining how closely related the positive salmonella isolates are to each other. acknowledgements the authors would like to acknowledge the college of agriculture, engineering and science as well as the school of life sciences at the university of kwazulu-natal (westville campus) for financial support; nhls of south africa for providing the project with isolates from brazilian broiler chickens and from human clinical isolates. lastly, we would like to express our most sincere gratitude to the various poultry slaughterhouses within kwazulu-natal province in south africa, who for reasons of confidentiality remain anonymous, for providing the chicken samples for this study. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. there is no conflict of interest from all the parties involved in this publication and all parties gave their consent to publish this work. authors’ contributions o.t.z. was the project 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94–105. abstract introduction materials and methods discussion acknowledgements references appendix 1 about the author(s) mohsen arbabi department of parasitology, kashan university of medical sciences, iran amir abdoli department of parasitology, kashan university of medical sciences, iran abdolhossein dalimi department of parasitology, tarbiat modares university, iran majid pirestani department of parasitology, tarbiat modares university, iran citation arbabi, m., abdoli, a., dalimi, a. & pirestani, m., 2016, ‘identification of latent neosporosis in sheep in tehran, iran by polymerase chain reaction using primers specific for the nc-5 gene’, onderstepoort journal of veterinary research 83(1), a1058. http://dx.doi.org/10.4102/ojvr.v83i1.1058 research project no: 92019029 original research identification of latent neosporosis in sheep in tehran, iran by polymerase chain reaction using primers specific for the nc-5 gene mohsen arbabi, amir abdoli, abdolhossein dalimi, majid pirestani received: 10 sept. 2015; accepted: 24 sept. 2015; published: 11 aug. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract little is known about latent infection and molecular characterisation of neospora caninum in sheep (ovis aries). in this study, 330 sheep samples (180 hearts and 150 brains) were analysed for n. caninum dna by nested polymerase chain reaction (pcr) targeting the nc-5 gene. neospora caninum dna was detected in 3.9% (13/330) of sheep samples. the parasite’s dna was detected in 6.7% of heart samples (12/180) and 0.7% (1/150) of brain samples. no clinical signs were recorded from infected or uninfected animals. sequencing of the genomic dna revealed 96% – 99% similarity with each other and 95.15% – 100% similarity with n. caninum sequences deposited in genbank. to our knowledge, this is the first report on the use of pcr to identify latent neosporosis in sheep in iran. the results of this study have the potential to contribute to our understanding of the role of n. caninum-infected sheep in the epidemiology of neosporosis. introduction neospora caninum is a worldwide protozoan having a variety of animal hosts (dubey & schares 2011; dubey, schares & ortega-mora 2007). domestic and wild canids are definitive, whereas different bird and mammalian species (such as cattle, water buffalo, and sheep) serve as intermediate hosts (dubey & schares 2011). abortion, especially in dairy cattle, is one of the major consequences of neosporosis in animal husbandry (almeria & lópez-gatius 2013) that lead to significant economic losses (reichel et al. 2013). moreover, ovine abortion and reproductive failure due to neosporosis have been reported in several studies (dubey & lindsay 1990; howe et al. 2008, 2012; jolley et al. 1999; moreno et al. 2012; pena et al. 2007). in different studies, antibodies to n. caninum have been detected in 1.1% – 8.3% of sheep in the west of iran (ezatpour et al. 2015; gharekhani & heidari 2014), 27.7% in pakistan (nasir et al. 2012), 2.1% in turkey (gökçe et al. 2015), 10.3% in china (liu et al. 2015), 16.8% in greece (anastasia et al. 2013), 3% in argentina (hecker et al. 2013), and 13.1% in south-eastern brazil (da silva andrade et al. 2012). however, there is little information describing the detection of nucleic acids resulting from latent neosporosis in sheep. until now, different genes such as internal transcribed spacer sequences, 18s-like ribosomal dna (small-subunit rdna), and nc-5 genes have been used for molecular diagnosis of neosporosis (reviewed by al-qassab, reichel & ellis [2010]; goodswen, kennedy & ellis [2013]). however, studies have indicated that the nc-5 gene is one of the most highly sensitive and specific for the detection of neosporosis (almerıa et al. 2002; dubey et al. 2014; hughes et al. 2006; kaufmann et al. 1996; paula et al. 2004; yamage, flechtner & gottstein 1996) because it is repeated in the n. caninum sequence (al-qassab et al. 2010). hence, the main objective of this study was to investigate detection and molecular characterisation of latent neosporosis in sheep (ovis aries) in tehran, iran, by polymerase chain reaction (pcr) using primers specific for the nc-5 gene. materials and methods animals and study area a total of 330 samples from healthy slaughtered sheep (180 hearts and 150 brains) were purchased from an abattoir in vavan (located in the vicinity of tehran) from april to september 2014. the animals tested originated from different counties (eslamshahr, shahriar, robatkarim), all of which are located between 50 km and 200 km from tehran. these locations have hot summers and moderate winters. no clinical signs such as fever, lymphadenitis, nasal and ocular discharges, or jaundice were recorded in any of the animals before slaughter. dna extraction the whole brain and heart of each sheep were individually rinsed with distilled water, packaged, and refrigerated. approximately 200 g – 250 g of different segments of brain and heart were homogenised with a pestle and mortar in liquid nitrogen, and dna was extracted using a phenol–chloroform extraction method as described in our recent report (abdoli et al. 2015). to prevent dna cross-contamination, all materials that were used between different tissue samples were decontaminated with sodium hypochlorite solution (2.5%) and rinsed with distilled water. the concentration of dna was determined by nanodrop spectrophotometer (thermo fisher scientific, wilmington, de, usa) for each sample. overall, the dna concentration ranged between 150 ng/μl and 200 ng/μl. nested polymerase chain reaction nested pcr was conducted using specific primers for the nc-5 gene. the first round of pcr was conducted using a pair of n. caninum–specific primers, np21plus (5’-cccagtgcgtccaatcctgtaac-3’) and np6plus (5’-ctcgccagtccaacctacgtcttct-3’) (muller et al. 1996). nested pcr was performed with the primers np6 (5’-cagtcaacctacgtcttct-3’) and np7 (5’-gggtgaaccgagggagttg-3’) (hughes et al. 2006). each amplification was performed in 20-µl reaction mixtures containing 10 µl of 2x master mixes (dfs master mix, bioron gmbh, ludwigshafen, germany), each of the respective primers (10 pmol for the first round reaction and 25 pmol for nested pcr), 7 µl of distilled water, and 1 µl of template dna. one microlitre of the first round product was used as the template for nested pcr. for each reaction, a negative control (double distilled water) and a positive control (dna extracted from the nc-5 strain of n. caninum) were included. amplification was performed with initial denaturation for 5 minutes at 94 °c, followed by 40 cycles at 94 °c for 40 seconds (denaturation), annealing at 62 °c in the first round, and 56 °c in nested pcr for 40 seconds, extension at 72 °c for 40 seconds, and final extension at 72 °c for 10 minutes. pcr products were electrophoresed on a 1.5% agarose gel stained with safe stain (sinaclon, tehran, iran) and visualised under ultraviolet trans-illumination. nucleotide sequence analysis four positive pcr products (from the second reaction) were amplified with a master mix containing pfu dna polymerase (thermo fisher scientific, waltham, usa, cat. no. ep0501), extracted from the gel (vivantis gel purification kit, selangor darul ehsan, malaysia) according to the manufacturer’s protocols. then the products were sequenced in the forward and reverse directions by sequetech (mountain view, ca, usa) (abdoli et al. 2015). the sequences were edited with bioedit sequence alignment editor (hall 1999), aligned with nc-5 partial sequences from other hosts by clustalx2.12 (larkin et al. 2007) and compared with sequences of n. caninum available in genbank. phylogenetic trees were inferred and evolutionary analyses were conducted using the tamura three-parameter option of the neighbour-joining model with mega6 software (http://www.megasoftware.net/) (tamura et al. 2013). the bootstrap scores were calculated for 1000 replicates (tamura et al. 2013). results neospora caninum dna was detected in 13 out of 330 sheep samples (3.9%). the infection rates in the heart and brain samples were 6.7% (12/180) and 0.7% (1/150), respectively. four nucleotide sequences of the nc-5 gene with a length of 227 bp (figure 1) were submitted to the genbank database (genbank accession numbers kr106181, kr106182, kr106183, kr106184). the results demonstrated our sequences shared 96% – 99% similarity with each other (figures 2 and 3) and 95% – 100% similarity with n. caninum deposited in genbank (appendix figure 1). phylogenetic trees showed intraspecific variations between our isolates and other n. caninum specimens deposited in genbank (figure 2). analysis of our sequences showed 96.9% – 97.8% similarity with n. caninum isolated from sheep (dq077661) in the uk and 96.9% – 99.1% similarity with n. caninum isolated from sparrows (passer domesticus) in iran. interestingly, one of our samples (kr106181) showed 100% similarity with n. caninum isolated from wolves (canis lupus) (kf649846) in the united states. figure 1: polymerase chain reaction products of four neospora caninum positive samples. figure 2: phylogenetic relationships among neospora caninum specimens based on a fragment of the nc-5 sequence. figure 3: partial sequences of the nc-5 gene from four isolates of neospora caninum from sheep samples. discussion although an association between ovine abortion and neosporosis has been reported in different studies (dubey & lindsay 1990; howe et al. 2008, 2012; jolley et al. 1999; moreno et al. 2012; pena et al. 2007), there is little information describing molecular detection of latent neosporosis in sheep. here, we found a total infection rate of 3.9% (13/330) in our sheep samples. interestingly, 12 out of 13 positive samples were detected in the hearts and one positive sample was diagnosed in the brain. in previous studies, the seroprevalence of n. caninum has been reported in a range of 1.1% – 8.3% of sheep from the west of iran (ezatpour et al. 2015; gharekhani & heidari 2014). moreover, n. caninum dna was detected in 8.5% (asadpour et al. 2013) and 0.9% of aborted ovine fetuses in iran (sasani et al. 2013). şuteu et al. detected n. caninum dna in the diaphragm tissues of 2 out of 181 (1.1%) slaughtered goat kids in romania (şuteu et al. 2013). in the majority of studies, n. caninum was detected in brain samples from aborted or naturally infected sheep (asadpour et al. 2013; bishop et al. 2010; dubey & lindsay 1990; sasani et al. 2013; silva et al. 2009). in this regard, silva and colleagues detected n. caninum dna in 2 out of 102 slaughtered goats (1.92%) in brazil. interestingly, both positive samples were isolated from brain samples, whereas all heart and tongue samples were negative (silva et al. 2009). santos et al. (2010) detected n. caninum dna in 5 out of 100 brain samples of beef cattle in brazil, whereas none of the heart samples were positive (santos et al. 2010). these results are dissimilar to our report, in which most of the positive samples were detected in the heart samples rather than in the brain samples (6.7% versus 0.7%). our results also indicated that in sheep the heart is more susceptible to n. caninum infection than the brain. latent neosporosis can reactivate in conditions such as immunosuppression and pregnancy (andrianarivo et al. 2005; hemphill, vonlaufen & naguleswaran 2006; magaña et al. 2015; mazuz et al. 2016; pabón et al. 2007; rettigner et al. 2004). latently infected animals are also a source of n. caninum infection for canine definitive hosts. in the current study, we used the nc-5 gene for detection and phylogenetic analysis of n. caninum. this gene is repeated in the n. caninum sequence (al-qassab et al. 2010); hence, it is presented as a highly sensitive and specific gene for detection of neosporosis (kaufmann et al. 1996; yamage et al. 1996). the earlier study in this regard was conducted by yamage et al. (1996), who compared the sensitivity and specificity of different primers for diagnosis of n. caninum. in this study, five forward (np1, np3, np5, np7, np21) and four reverse (np2, np4, np6, np8) oligonucleotide primers that derived from the nc-5 genes were compared for the detection of neosporosis in experimentally infected mice. among 19 combinations of forward and reverse primers, the np21/np6, np7/np6, and np2l/np4 primer pairs were able to detect at least 10 pg genomic dna with a specific single band (yamage et al. 1996). the nc-5 gene can also discriminate n. caninum from other related apicomplexan parasites (toxoplasma gondii and sarcocystis species) (kaufmann et al. 1996). thus, the nc-5 gene has been used as a highly sensitive and specific gene for detection of neosporosis (almerıa et al. 2002; dubey et al. 2014; hughes et al. 2006; paula et al. 2004; yamage et al. 1996). hence, we selected the nc-5 gene for sensitive and specific detection of neosporosis in the current study. we also sequenced four positive samples for phylogenetic analysis. we found that our sequences displayed similarity levels of 96% – 99% with each other (figure 3) and 95% – 100% with n. caninum sequences deposited in genbank (appendix figure 1). in comparison with molecular diagnosis, few studies have been conducted on the phylogenetic analysis of n. caninum with the nc-5 gene (auriemma et al. 2014; čobádiová et al. 2013; hughes et al. 2006). blast analyses indicated greater than 94% (čobádiová et al. 2013), 96% (auriemma et al. 2014), and 97% (hughes et al. 2006) similarities between their sequences and other n. caninum sequences deposited in genbank. it therefore seems that the nc-5 gene is not a suitable biomarker for phylogenetic analysis and discrimination of genetic diversity for n. caninum. instead, this gene is rather a highly sensitive and specific biomarker for the diagnosis of neosporosis. the use of ribosomal dna, its-1, and recently microsatellites have been recommended for discriminating between n. caninum isolates (al-qassab et al. 2010). taken together, the results of this study provide molecular and epidemiological information about latent n. caninum infection in sheep in iran. it can be expected that in future these results will contribute to revealing the role of latent n. caninum infection in the epidemiology of neosporosis in sheep. acknowledgements this work was supported by the iran national science foundation (grant no. 92019029). the authors would like to thank mr. mahdi rezapour, ali karaji, and ali shafiei for their help in preparing the samples. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.a. conceived the study design, analysed and interpreted the data and reviewed the manuscript; a.a. collected the data, prepared the samples, performed molecular assays, designed the tables and figures and 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evolutionary genetics analysis version 6.0’, molecular biology and evolution 30, 2725–2729. http://dx.doi.org/10.1093/molbev/mst197 yamage, m., flechtner, o. & gottstein, b., 1996, ‘neospora caninum: specific oligonucleotide primers for the detection of brain “cyst” dna of experimentally infected nude mice by the polymerase chain reaction (pcr)’, journal parasitology 82, 272–279. http://dx.doi.org/10.2307/3284160 appendix 1 figure 1-a1: sequence alignment of nc-5 gene of neospora caninum from mammalian and bird hosts. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) susan d. kerfua nelson mandela african institute of science and technology, arusha, tanzania national livestock resources research institute, tororo, uganda gabriel shirima nelson mandela african institute of science and technology, arusha, tanzania lughano kusiluka department of global health and biomedical sciences, mzumbe university, tanzania chrisostome ayebazibwe national animal disease diagnostics and epidemiology centre, entebbe, uganda robert mwebe national animal disease diagnostics and epidemiology centre, entebbe, uganda sarah cleaveland institute of biodiversity, animal health and comparative medicine, university of glasgow, united kingdom daniel haydon institute of biodiversity, animal health and comparative medicine, university of glasgow, united kingdom citation kerfua, s.d., shirima, g., kusiluka, l., ayebazibwe, c., mwebe, r., cleaveland, s., et al., 2018, ‘spatial and temporal distribution of foot-and-mouth disease in four districts situated along the uganda–tanzania border: implications for cross-border efforts in disease control’, onderstepoort journal of veterinary research 85(1), a1528. https://doi.org/10.4102/ojvr.v85i1.1528 original research spatial and temporal distribution of foot-and-mouth disease in four districts situated along the uganda–tanzania border: implications for cross-border efforts in disease control susan d. kerfua, gabriel shirima, lughano kusiluka, chrisostome ayebazibwe, robert mwebe, sarah cleaveland, daniel haydon received: 22 aug. 2017; accepted: 16 july 2018; published: 27 aug. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract foot-and-mouth disease (fmd) is one of the major trans-boundary animal diseases in east africa causing economic loss to farmers and other stakeholders in the livestock industry. foot-and-mouth disease occurs widely in both uganda and tanzania with annual outbreaks recorded. with the recent introduction of the progressive control pathway for fmd control (pcp-fmd) in eastern africa, knowledge of the spatial and temporal distribution of fmd at the border area between uganda and tanzania is helpful in framing engagement with the initial stages of the pcp. retrospective data collected between 2011 and 2016 from four districts located along the border areas of uganda and tanzania, recorded 23 and 59 fmd outbreaks, respectively, for the entire study period. analysis showed that 46% of the 82 recorded outbreaks occurred in 20% of sub-counties and wards immediately neighbouring the uganda–tanzania border and 69.5% of the outbreaks occurred during the dry months. while the serotypes of the fmd virus responsible for most outbreaks reported in this region were not known, previous research reported south african territory (sat) 1, sat 2 and o to be the serotypes in circulation. the results from this study provide evidence of the endemic status of fmd on the uganda–tanzania border and emphasise that the border area should be given due consideration during fmd control drives and that cross-border coordination should be prioritised. with the limited data on circulating serotypes in this area, there is a need for more vigilance on fmd case detection, laboratory diagnostic confirmation and provision of more complete documentation of outbreaks. this work further recommends more studies on cross-border livestock movement coupled with phylogenetics in order to understand the spread of the fmd in the border area. introduction foot-and-mouth disease (fmd) is an endemic livestock disease in east africa (food and agriculture organization of the united nations [fao 2007]) caused by the foot-and-mouth disease virus (fmdv). the disease affects cattle, goats, sheep, pigs and other cloven hooved animals including wildlife. the virus is transmitted when susceptible animals are exposed to animals that have the virus. although close proximity between animals may be necessary, some information suggests that the virus can be carried by the wind. typically, the virus causes livestock to exhibit fevers; blisters in the mouth, tongue, hooves and udder; loss of appetite; anorexia; and excessive salivation. while the virus affects livestock of all ages, the young are most likely to die due to the disease, while older animals become less productive. once the virus is active, it must run its course, which is why prevention of fmd is most effective. foot-and-mouth disease virus exists as seven serotypes: a, o, c, asia 1, sat 1, sat 2 and sat 3. six of these serotypes (except for asia 1) are known to have circulated in east africa (vosloo et al. 2002). serotype o is the most predominant serotype and serotype a is widely distributed across east africa. among the sats, sat 2 is the most predominant, while sat 3 is the least common. serotype c has never been reported in tanzania but was last reported in uganda in 1971 and in kenya in 2004. since then, it has not been reported in east africa (sangula 2010). outbreaks of fmd occur widely throughout east africa with cases recorded every year in parts of uganda and tanzania. challenges remain with the implementation of control measures, such as vaccination and movement restriction, and thus fmd remains poorly controlled in both countries (ministry of agriculture animal industry and fisheries [maaif] 2012; vosloo et al. 2002). as a result, farmers, traders and national governments continue to incur direct and indirect costs associated with the disease annually (baluka, ocaido & mugisha 2014; kivaria 2003). locally, fmd outbreaks affect the sale and purchase of livestock and livestock products, resulting in loss of income. internationally, endemic countries are restricted from exporting their livestock and livestock products (james & rushton 2002), resulting in loss of revenue. in addition, governments of affected countries may impose quarantine and vaccination campaigns, which are costly and may not be effective (james & rushton 2002). in recent years, growing attention has been given to understanding the epidemiology of fmd in uganda and tanzania (ayebazibwe et al. 2010; balinda et al. 2010; genchwere & kasanga 2014; kasanga et al. 2011; kivaria 2003; namatovu et al. 2015). research momentum has also been generated as a result of the introduction of the progressive control pathway for foot-and-mouth disease (pcp-fmd), a step-wise framework developed by the fao and the world organization for animal health (oie) to assist fmd-endemic countries in reducing the impact of the disease (fao 2011). the pathway in its initial stages requires the countries to have information on fmd outbreaks, including what serotypes are circulating, to understand the epidemiology of the disease and design strategic controls. uganda and tanzania are currently in the initial stages of the pcp-fmd, and both countries require accurate outbreak information to strategise the implementation of control measures (fao 2011). however, information is still limited on the epidemiology of fmd, particularly at the border region between uganda and tanzania. border regions remain central in the epidemiology of trans-boundary animal diseases and these regions often suffer the burden of trans-boundary livestock diseases (di nardo, knowles & paton 2011). the uncontrolled movement of people and animals along borders has been documented as one of the major factors for the introduction and continued circulation of animal diseases, particularly fmd (fèvre et al. 2006; otte, nugent & mcleod 2004). between 2001 and 2008, sub-counties in uganda that were adjacent to the ugandan–tanzanian border registered more outbreaks compared to the other ugandan sub-counties situated along the uganda–democratic republic of congo border, uganda–kenya border, uganda–rwanda border and the uganda–south sudan border (ayebazibwe et al. 2010). in addition, a study in tanzania reported on the increased number of fmd outbreaks around kagera (picado et al. 2010), a tanzanian region, which includes the missenyi and kyerwa districts and borders uganda and rwanda. thus, the border area between tanzania and uganda remains an important area where more information on fmd outbreaks is helpful to inform strategies on regional control of the disease, which would be of great relevance and benefit to east african community (eac). therefore, the objective of this study was to generate more information on spatial and temporal fmd outbreak patterns in the districts located along the border of uganda and tanzania. materials and methods study area the study comprised of four districts located along the uganda–tanzania border. the study included two districts, isingiro and rakai, in uganda located north of the uganda–tanzanian border, and the two districts of missenyi and kyerwa, in tanzania located south of the uganda–tanzania border (figure 1). these districts were purposively chosen because they share an international border. isingiro and rakai districts are composed of 10 and 20 sub-counties, respectively. in uganda, a sub-county is the third lowest administrative level that is made up of several parishes, and within each parish is a number of villages. in tanzania, the missenyi district comprises of 20 wards, whereas the kyerwa district has 18 wards. a ward in tanzania serves as an administrative structure and is composed of several villages. the sub-counties and wards in uganda and tanzania are shown in figure 1. in this study, sub-counties and wards that share an international border with either country will be addressed as adjacent or close to the international border. figure 1: study areas isingiro and rakai districts in uganda and missenyi and kyerwa districts in tanzania. data collection and management ugandan fmd outbreak records for the districts of rakai and isingiro were retrieved from the national animal disease diagnostics and epidemiology centre (naddec) that carries out routine national surveillance in response to disease outbreaks. in the maaif (uganda), the fmd reporting system works in such a way that fmd is considered as one of the notifiable diseases of public concern and has to be reported within 24 h to the commissioner of animal health (cah). a suspected fmd case is reported by the affected farmer to the animal health staff (animal husbandry officer [aho] or veterinary officer [vo]) in the sub-county who reports to the district veterinary officer [dvo]). the dvo establishes an interim quarantine and reports to the cah by either an official letter or e-mail. the cah dispatches a team to investigate the outbreak and confirm its presence or absence. the team collects appropriate samples which are analysed in the regional laboratories using mostly antibody and antigen enzyme-linked immunosorbent assay (elisa) tests and occasionally polymerase chain reaction (pcr). after the confirmation of fmd, control measures such as quarantine and/or vaccination are instituted. in tanzania as well, fmd is considered a notifiable disease and must be reported within 24 hours. for the tanzanian border districts of missenyi and kyerwa, retrospective data were retrieved from the records of the dvos. in tanzania, the system for reporting fmd is such that, a farmer reports to the livestock field officer (lfo) who goes to the affected farm to confirm the presence of fmd based on clinical signs. the lfo reports to the dvo. ideally, appropriate samples are collected from the infected animals by the dvo and transported to the national laboratory (tanzania veterinary laboratory agency) for confirmation of fmd through antibody and antigen elisa tests. however, in this study, an fmd outbreak was defined as the presence of fmd clinical signs in at least one herd of cattle in a village within 1 month of the report of an outbreak. most of the outbreak diagnoses were performed by the vos in the districts, who based their diagnoses mainly on clinical signs exhibited by the affected animals. cattle were considered as fmd-positive cases if they exhibited two or more of the following clinical signs – lesions in the mouth, on the gum, on the tongue, on the hooves and lesions on the mammary glands (in the case of females) accompanied by excessive salivation, fever, anorexia and lack of appetite. for this study, data on serotypes that were in circulation during the study period were retrieved from the dvos’ records as well as from naddec records. other sources of this information were research works that had previously been carried out in this region (genchwere & kasanga 2014; namatovu et al. 2015). in the areas of this study, the seasons comprised wet and dry periods which are annually bimodal. the wet period consists of rainy days which occur in march and april and from september to november. the long dry season is characterised by periods of high temperatures averaging 27.5 °c – 30 °c and extends from december to february and from may to august (isingiro district local government [idlg] 2011; rakai district local government 2013). the dry season in the kagera region extends from december to february, and then from may to september, while the wet season is in march and april and then from october to december (fao n.d.). constraints of the study most of the data in this study consisted of reported cases based on clinical signs of fmd. a few samples were taken to the respective reference laboratories in the two countries for analysis to confirm the presence or absence of the disease by tests such as real-time or conventional pcr and antigen/antibody elisas, among others. owing to lack of tests carried out for most of the reported cases, one cannot rule out that the symptoms could have been caused by other diseases that exhibit clinical signs similar to those of fmd such as bovine papular stomatitis, vesicular stomatitis, malignant catarrhal fever and bluetongue (holliman 2005). other details were not included during data collection. over 60% of the data lacked elements such as geographic positioning system (gps) locations, number of cases registered during the outbreaks and number of animals at risk. cases in this study comprised only cattle because data were only collected for cattle. this study was based on retrospective data retrieved from monthly reports by the dvos of the study districts and may be subject to underreporting by farmers owing to political, social and other reasons (dhikusooka et al. 2015; sutmoller et al. 2003). data analysis the data that were analysed comprised of the number of outbreaks in each sub-county and/or ward, the month in which the outbreak occurred, the district in which the sub-county/ward was located and whether the sub-county and/or ward was directly adjacent to the international border between tanzania and uganda. data analysis was performed in r software, version 3.3.2 (r core team 2013), using generalised linear mixed effect models (package lme4) to describe the relationship between the response variable (number of outbreaks modelled as poisson distribution) and the fixed (season, wet-dry, border adjacency [yes, no] and year of outbreak) and random (district, sub-county and/or ward, year) effects. model selection was undertaken, and the significance of fixed effects was determined using likelihood ratio tests (lrts). maps were prepared using qgis 2.16.0 (open source geospatial foundation 2016) reflecting the sub-counties/wards where fmd outbreaks had occurred over the last 6 years. because no gps data on the exact location of affected villages were collected, analysis was conducted at the scale of sub-counties/wards where the affected villages were located (figure 2). figure 2: spatial distribution of foot-and-mouth disease in sub-counties and/or wards in border districts of uganda and tanzania between 2011 and 2016. results spatial distribution between 2011 and 2016, a total of 23 outbreaks were reported to have occurred in the two ugandan districts and 59 in the two tanzanian districts located along the uganda–tanzania border. throughout the entire period, the district of missenyi in tanzania had the highest number of outbreaks (36) followed by kyerwa (23), isingiro (15) and rakai (8) (refer to figure 2 on annual frequency trends). the latter two districts are in uganda. of the 82 outbreaks, 38 outbreaks (46%) occurred in sub-counties and/or wards that shared an international border with either country. overall, the analysis showed that sub-counties/wards adjacent to the border reported three times more fmd outbreaks than the sub-counties/wards that were not adjacent to the border (x2 = 5.643, df = 1, p < 0.001). temporal distribution of foot-and-mouth disease analysis of temporal data showed that the months with no outbreaks over the 6-year period in the two ugandan border districts were january, march, july and october–december. isingiro district reported the highest number of outbreaks to have occurred in august (6), whereas rakai registered the most outbreaks in february (3) (figure 3). in missenyi district of tanzania, most outbreaks occurred in june (10) and july (10), whereas in kyerwa the highest number of outbreaks was reported in april (7). september was the only month with no outbreaks in both tanzanian districts. the analysis showed that outbreaks were 2.7 times more frequent in the dry season than in the wet season (x2 = 18.311, df = 1, p < 0.001). overall, the distribution of outbreaks was such that on average, outbreaks in the four districts occurred within an interval of three months apart; however, it is not clear if a pattern exists in the distribution of the reported outbreaks within the districts. figure 3: temporal distribution of the number of outbreaks in selected border districts of uganda and tanzania. serotype distribution overall, from the data available on serotypes, three serotypes (o, sat 1 and sat 2) were found to be in circulation in the border region between 2011 and 2016 as shown in table 1. table 1: foot-and-mouth disease serotypes responsible for some of the outbreaks between 2011 and 2016 in the districts at the border of uganda and tanzania. discussion the results showed that fmd outbreaks occurred in all four districts at the uganda–tanzania border over the period (2011–2016), reflecting the endemic nature of fmd in this border region. overall, the study revealed that sub-counties and/or wards that were adjacent to the international border between uganda and tanzania were significantly more affected by fmd and most outbreaks occurred during dry season (figures 2 and 3). the high occurrence of outbreaks in ugandan sub-counties adjacent to the border is consistent with findings by di nardo et al. (2011), who reported that border regions usually suffer the burden of trans-boundary livestock diseases fuelled by trade (both legal and illegal) between neighbouring countries. the increased number of outbreaks in this area is consistent with findings by picado et al. (2010). in addition, di nardo et al. (2011) identified the trans-boundary area between uganda and tanzania as one of the main risk areas for fmd circulation in eastern africa (di nardo et al. 2011). according to the isingiro district local government, isingiro district shares its border with tanzania, which creates an opportunity for cross-border trade but also predisposes the district to an influx of pastoral immigrants from tanzania who come with potentially infected animals, creating a constant potential source of diseases (idlg 2011). so, while trade benefits border communities, it also creates or facilitates the trans-border spread of livestock diseases (domenech et al. 2016). this produces a continual introduction or re-introduction of the disease from either side owing to uncontrolled human and animal migration (di nardo et al. 2011). in the missenyi district (kakunyu ward), the same village of bubale was consistently affected for 4 years, perhaps attributable to the location of a major livestock market in bubale. also in uganda, the kakuuto sub-county reported the most number of outbreaks in the rakai district, while the sub-county of endinzi reported the most in the isingiro district. both districts had recurrent outbreaks probably because of their major cattle markets located near the border. these finding are consistent with those that have shown that livestock markets are one of the major factors for fmd circulation and distribution in sub-saharan africa (di nardo et al. 2011). compared to the ugandan districts, the tanzanian districts had higher numbers of outbreaks. the two countries have different livestock disease policies for disease control. in uganda, control of fmd is deemed a public good, with vaccines and vaccinations offered at a subsidised cost to farmers whenever available (muleme et al. 2012). the situation in tanzania is different. the national livestock policy by the ministry of livestock development (mld, 2006) does not mention governmental subsidisation of fmd vaccination costs. therefore, farmers may be reluctant to vaccinate for fmd because of the full cost they must incur, which may contribute to the spread of the disease in the country. however, despite the imposed quarantines and regular vaccinations in the ugandan district of isingiro, fmd outbreaks are still frequent. this may be the result of resistance by some residents and leaders to imposed quarantines for economic and political reasons (kabasongora 2014; new vision 3 november 2016). occasionally, in the rakai district, some farmers take their animals into tanzania to elude vaccination, citing vaccine ineffectiveness. they later return with their animals that may have been infected from tanzania, complicating efforts in controlling fmd in rakai (j. kabagenyi [animal husbandry officer, rakai district] pers. comm., 15 february 2016). consequently, the continuous presence of fmd pockets on either side of the uganda–tanzania border increases the risk of fmd outbreaks in both countries, given that the outbreaks may spread beyond the border region. in addition, wildlife is responsible for circulating sat strains of the fmd virus, as has been studied in some parts of africa (jori & etter 2016; vosloo et al. 2005). however, it is not clear if the presence of wildlife is the reason for fmd persistence in the kibale ward of the kyerwa district. the rumanyika orugundu game reserve stretches into some parts of the kamuli and the kibale wards (figure 2). the kamuli ward reported only one outbreak during the entire 6-year period. some studies have shown that areas located at wildlife–livestock interfaces have more fmd outbreaks than areas that do not harbour any wildlife (ayebazibwe et al. 2010; sinkala et al. 2014). nevertheless, wards in kyerwa, such as murongo and kibingo, that accommodate the ibanda national game reserve did not register any outbreaks during the entire study period. in the isingiro district, all the sub-counties located near lake mburo game reserve, save for kabingo (in 2013), and did not report any outbreaks as well. it may appear that the presence of wild life may not be a major factor in fmd persistence in this border area. further studies on risk factors for fmd persistence or new introductions in these areas are needed to determine the reasons behind persistent outbreaks. although genchwere and kasanga (2014) showed that most farmers in tanzania were aware of fmd signs and farmers’ descriptions of fmd had a high correlation with positive elisa results obtained from fmd outbreaks during their study, more reported cases should be confirmed by laboratory analysis. this would also address the dearth of information on fmd serotypes presented in outbreak report in both countries, mainly due to the limited laboratory and field capacity (dhikusooka et al. 2015; namatovu et al. 2013). muleme et al. (2012) reported on poor outbreak investigations and revealed that out of 121 outbreaks reported in uganda between 2001 and 2010, the causal serotype was determined for only nine outbreaks. this further complicates any efforts for disease control given that circulating serotypes are unknown (kitching et al. 2007). the presence of serotype o has recently been confirmed by other studies carried out in the missenyi and rakai districts (genchwere & kasanga 2014; kerfua et al. 2013; namatovu et al. 2015). serotypes sat 1 and sat 2 have been previously detected by studies carried out in the lake zone region where both the missenyi and kyerwa districts are located (swai, mrosso & masambu 2009). also, the 2013 fmdv sat 2 isolate from the isingiro district in uganda was closely related (97% similarity) to the vp 1 nucleotide coding sequences found in the isolate from tanzania (tan43/2009). this raises the question of whether the virus is spread between the two countries through trans-boundary movement of people and animals (namatovu et al. 2015). most of the outbreaks occurred in the dry months of the year, consistent with previous findings (ayebazibwe et al. 2010; kivaria 2003). during the dry months, farmers move their animals for long distances in search of pasture and water and the interaction between animals at the water and pasture sources may facilitate easy spread of infectious diseases (vanderwaal et al. 2017). the patterns of outbreaks in all four districts also showed that the outbreaks occurred on average 3 months apart (data not shown), implying probable circulation of the virus between the districts. this argument may be inconclusive given that sequence and serotype data for this study were very limited. conclusion areas adjacent to the borders and livestock markets may play a major role in fmd spread and thus fmd control can be strategised based on emphasis on such hot spots. wildlife may not be as important in fmd spread in some areas and so further studies on risk factors have to be carried out for those areas that are persistently affected. this study underscores the need for better outbreak data documentation and the need for laboratory analysis and confirmation of fmdv serotypes during outbreaks reported in either country. it also recommends the need to determine genetic relatedness of fmdvs responsible for outbreaks in this region in order to improve our understanding of fmdv circulation and assist with its control. it is also imperative that neighbouring countries coordinate regional control strategies and enact and enforce laws regulating movement of livestock across borders for better management of fmd at border districts. acknowledgements this study was financed by the program for enhancing health and productivity in livestock (pehpl) based at the nelson mandela african institute of science and technology, tanzania, and supported by the bill & melinda gates foundation. the authors thank the national animal disease diagnostics and epidemiology centre which is under the ministry of agriculture animal industry and fisheries (maaif) of uganda, the district veterinary officers and animal health officers and livestock field officers in both countries. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this paper. authors’ contributions s.d.k. is a phd student and was responsible for data collection, analysis and also preparation of the draft manuscript. g.s. is an academic supervisor and is one of the current project leaders and helped to edit the manuscript. l.k. was one of the project leaders, is an academic supervisor and aided in editing the manuscript. s.c. and c.a. are also academic supervisors and were helpful in conceptual contributions. r.m. was responsible for providing critical information on fmd outbreaks in uganda and d.h. was helpful in concept building, data analysis and editing of the manuscript. 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w.b. karesh, m.d. kock, p.j. nyhus & a. yang (eds.), conservation and development interventions at the wildlifelivestock interface: implications for wildlife, livestock and human health, pp. 67–73, international union for conservation of nature, gland, switzerland. vosloo, w., bastos, a.d.s., sangare, o., hargreaves, s.k. & thomson, g.r., 2002, ‘review of the status and control of foot-and-mouth disease in sub-saharan africa’, revue scientifique et technique (international office of epizootics) 21, 437–449. https://doi.org/10.20506/rst.21.3.1349 abstract introduction materials and methods results and discussion conclusion acknowledgements references about the author(s) georges m. tshilenge department of preclinical medicine, faculty of veterinary medicine, university of kinshasa, kinshasa xi, the democratic republic of the congo mfumu l.k mulumba department of clinical medicine, faculty of veterinary medicine, university of kinshasa, kinshasa xi, the democratic republic of the congo gerald misinzo department of microbiology and parasitology, sokoine university of agriculture, morogoro, united republic of tanzania rob noad department of pathobiology and population sciences, london school of hygiene and tropical medicine, royal veterinary college, hertfordshire, united kingdom william g. dundon animal production and health laboratory, joint food and agriculture orginazation/international atomic energy agency, vienna, austria department of nuclear sciences and applications, international atomic energy agency, vienna, austria citation tshilenge, g.m., mulumba, m.l.k., misinzo, g., noad, r. & dundon, w.g., 2019, ‘rift valley fever virus in small ruminants in the democratic republic of the congo’, onderstepoort journal of veterinary research 86(1), a1737. https://doi.org/10.4102/ojvr.v86i1.1737 original research rift valley fever virus in small ruminants in the democratic republic of the congo georges m. tshilenge, mfumu l.k mulumba, gerald misinzo, rob noad, william g. dundon received: 07 feb. 2019; accepted: 07 may 2019; published: 10 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract rift valley fever (rvf) is a zoonotic viral disease caused by the rvf phlebovirus (rvfv) that infects a variety of animal species including sheep and goats. sera (n = 893) collected between 2013 and 2015 from randomly selected indigenous sheep and goats in seven provinces of the democratic republic of the congo (drc) were tested for the presence of specific immunoglobulin g (igg) and m (igm) against rvfv, using two commercially available enzyme-linked immunosorbent assays. the reverse transcription polymerase chain reaction (rt-pcr) was also used to detect rvfv nucleic acid. there was significant variation in true seroprevalence of rvfv for both sheep and goats between the seven provinces investigated. values ranged from 0.0 (95% confidence interval [ci] 0.0–6.55) to 23.81 (95% ci 12.03–41.76) for goat and 0.0 (95% ci 0.0–7.56) to 37.11 (95% ci 15.48–65.94) for sheep, respectively. one serum (1.85%) out of 54 that tested positive for igg was found to be igm-positive. this same sample was also positive by rt-pcr indicating an active or recent infection. these findings report the presence of rvfv in small ruminants in the drc for the first time and indicate variations in exposure to the virus in different parts of the country. keywords: rift valley fever; ruminants; serology; democratic republic of the congo; prevalence. introduction rift valley fever (rvf) is a zoonotic viral haemorrhagic fever caused by rvf phlebovirus (rvfv) which is a negative single-stranded ribonucleic acid (rna) virus that belongs to the order bunyavirales, family phenuiviridae (previously named bunyaviridae) and genus phlebovirus (plyusnin et al. 2011). the disease infects both large and small ruminants and, as such, has a significant socio-economic impact on livestock in many african countries (martin et al. 2008). pregnant ruminants infected with rvfv are subject to high rates of abortion, while high mortality is seen among young animals, characterised by hepatic necrosis and pantropic haemorrhage (coetzer 1988). the disease was first described in 1931 as an enzootic hepatitis among sheep in kenya (daubney, hudson & garnham 1931). the spread and dissemination of the disease can be facilitated by a number of different factors including vectors such as mosquitoes, ticks and flies (linthicum et al. 1985; mweya et al. 2013; turell et al. 2007), climate (anyamba, linthicum & tucker 2001; walsh, willem de smalen & mor 2017) and infected livestock trade and movement (kenawy, abdel-hamid & beier 2018). rift valley fever phlebovirus is also a significant threat to human health. the virus can be transmitted by mosquito bites, contact with infected tissues, blood or amniotic fluid (especially in aborted material) in slaughterhouses, and handling of contaminated meat during food preparation (mroz et al. 2017). prevention and control of rvf in humans therefore rely on preventing the disease in domestic animals in the peridomestic environment. currently, the best and most efficient way to do this is to vaccinate all susceptible animals (dungu, lubisi & ikegami 2018; kenawy et al. 2018). several countries in africa, the arabian peninsula and islands in the indian ocean have reported rvf outbreaks of different severities (king et al. 2010; madani et al. 2003; maganga et al. 2017; nanyingi et al. 2017; nguku et al. 2010; oyas et al. 2018; shoemaker et al. 2002; sindato et al. 2015). in the democratic republic of the congo (drc), although no rvf outbreaks have been reported to date, seropositivity of cattle to rvfv was first described in 2009 (mulumba et al. 2009) and has recently been confirmed (tshilenge et al. 2018). however, no information is available on the seroprevalence of rvfv in small ruminants (goat and sheep) in the country. this study was therefore undertaken to fill this knowledge gap and to provide a clearer picture of the presence of rvfv in the drc. materials and methods samples were collected in 7 of the 26 provinces of drc (figure 1 and table 1), namely mongala (2°09’n, 21°31’e), south ubangi (3°15’n, 19°46’e), north ubangi (4°1’n, 21°01’e), kwilu (5°02’s, 18°50’), lomami (6°08’s, 24°29’e), south kivu (2°42’s, -27°2’e) and tanganyika (7°04’s, 29°43’e). figure 1: map of democratic republic of the congo showing locations of sample collection (adapted with permission from www.d-maps.com). territories with seroprevalences > 10% are indicated with black triangles. table 1: serological status by location to anti-rift valley fever immunoglobulin g antibodies. mongala, north ubangi, south ubangi and kwilu are low-altitude regions (100 m – 700 m) characterised by tropical and equatorial climates with annual rainfalls of 1600 mm – 2000 mm, 90% relative humidity and an average annual temperature of 31 °c. these four provinces are prone to flooding during seasonal rainfall. lomami is situated at an altitude of 700 m – 1200 m with a rainfall of 1200 mm/year – 1800 mm/year, an annual average temperature of 22 °c and 78% relative humidity. south kivu is a high-altitude, mountainous region characterised by dense rainforests and an equatorial climate with an average annual rainfall of 1800 mm, an average temperature of 18 °c and 80% relative humidity. tanganyika is located on the shores of lake tanganyika on the western branch of the great rift valley in the humid tropical climate zone. the average annual temperature is 22 °c with an average annual precipitation of 700 mm and 85 °c of relative humidity. the samples were collected from 2013 to 2015 from randomly selected sheep and goats following owners’ consent and under the supervision of the animal health and production department (dpsa) and the central veterinary laboratory, kinshasa. the location, age and sex were recorded for each animal. samples from animals less than 4 months of age were discarded to avoid maternal antibodies. all farms visited had variable herd size ranging from 2 to 50. blood samples were transported to the laboratory (central veterinary laboratory [cvl], kinshasa) on ice packs (approximately 4 °c). twenty-four hours following whole blood collection, sera were harvested and kept at -20 °c until further use. sera were screened for the presence of specific immunoglobulin g (igg) antibodies to rvfv using the id screen® rvf competition multi-species enzyme-linked immunosorbent assay (elisa) kits (id-vet; innovative diagnostics, grabels, france). the reported sensitivity (se) and specificity (sp) values for this kit are 98% and 100%, respectively (kortekaas et al. 2013). all igg-positive sera were screened for specific anti-rvfv igm antibodies using the id screen® rvf igm capture (id-vet; innovative diagnostics, grabels, france), per manufacturers’ instructions. viral rna was extracted from goat sera using a purelink® rna mini kit (invitrogen, waltham, ma, united states [us]) and following the manufacturer’s instructions. for the reverse transcription polymerase chain reaction (rt-pcr), primers rvf1 and rvf2 were used to amplify a target segment of 551 bp (base pairs) which encodes a portion of the g2 glycoprotein gene as described previously (tshilenge et al. 2018). a descriptive statistical analysis was performed. true seroprevalence and exact 95% confidence interval (95% ci) were computed using an epidemiological calculator available at http://epitools.ausvet.com.au/content.php?page=trueprevalence. this method calculates the true prevalence from survey testing results using a test of known sensitivity and specificity (rogan & gladen 1978). comparisons of true seroprevalence between groups as well as associations between seropositivity were made using a nonparametric fisher test. the threshold of significance was set at a p value of 0.05. data analyses were performed using epi info 7 (cdc, atlanta, ga, us). ethical considerations the study protocol was implemented with approval from the direction of agriculture, central veterinary laboratory of kinshasa/animal health and production department. consent for blood sampling of herds was obtained from owners. animals were bled using conventional protocols. results and discussion a total of 893 sera were tested for anti-rvfv-nucleoprotein igg antibodies by competitive elisa. the true seroprevalence in goats ranged from 0.0% (95% ci 0.0–6.55) to 23.81% (95% ci 12.03–41.76), while in sheep, the true seroprevalence ranged from 0.0% (95% ci 0.0–7.56) to 37.11% (95% ci 15.48–65.94) (table 1). the seroprevalence varied significantly between the provinces from which samples originated, indicating that environmental and/or geographical factors play a role in rvf epidemiology in the drc. the highest seroprevalences [37.11% (95% ci 15.48–65.94) for sheep and 23.81% (95% ci 12.03–41.76) for goats] were recorded in the low-altitude region of businga, nord-ubangi province, where average rainfall ranges from 1600 mm to 1800 mm with a relative humidity of around 90%. this humid environment would explain higher numbers of potential insect vectors for rvfv compared to the less humid climates of tanganyika and south kivu where a seroprevalence of < 10% in both sheep and goats was recorded. it is known that during heavy rains, the numbers of fresh water species mosquitoes increase and play an important role in amplifying rvf (ikegami & makino 2011). therefore, the monitoring and/or control of vectors for rvfv in regions of the drc with high humidity would be opportune. low true seroprevalence values of < 10% were detected in the low-lying humid regions in the provinces of sud-ubangi, mongala and kwilu, suggesting that climate and the presence of insect vectors may not be the only factors involved in rvfv transmission in the drc. the territories sampled in nord-ubangi province are located close to the border with the central african republic which has reported the presence of rvfv in the past (gonzalez et al. 1989; nakounne, selekon & morvan 2000), and so the transboundary movement of previously infected animals should also be taken into account when considering the results of this study. the seroprevalences of > 10% recorded in nord-ubangi and lomani are in agreement with seroprevalence values reported in other african countries, for example 24.7% in madagascar (jeanmaire et al. 2011), 29.7% and 22.0% in tanzania (sindato et al. 2015), 34% and 22% in sudan (eisa 1984), 85% and 12.2% in mauritania and cameroon (zeller et al. 1995) and 19.3% in senegal (chevalier et al. 2005). in contrast, the seroprevalence values of < 10% recorded in mongala, kwilu, sud kivu and tanganyika are similar to studies from togo (i.e. 6.1%) (zeller et al. 1995), côte d’ivoire (i.e. 9.71%) (formenty, domenech & zeller 1992), burkina faso (i.e. 7.67 %) (boussini et al. 2014) and gabon (i.e. 6.47%) (maganga et al. 2017). a single sample from a goat was positive for igm antibodies and the animal was confirmed by rt-pcr to be at the acute viraemic phase of rvf infection at the time of sampling. amplification of rvfv nucleic acid from this serum by rt-pcr produced an expected dna fragment of 551 bp. this confirms the utility of rt-pcr in the detection of early to acute viraemia which occurs between 4 and 8 days post-infection. the goat in question was identified in north ubangi province which borders with the central african republic, once again highlighting the influence transboundary movement of previously infected animals can potentially have on the outcome of seroprevalence studies such as this one. conclusion despite the lack of reports of rvf outbreaks in the drc, it is evident that small ruminant populations in some parts of the country are exposed to the virus. more in-depth serological, molecular epidemiological and virological surveys of livestock need to be conducted in order to more fully understand the dynamics of rvfv circulation in the drc. vaccination in at-risk areas and the monitoring of vectors and animal movement should limit the spread of disease in susceptible animal populations and prevent transmission to humans. acknowledgements competing interests the authors declare that they have no conflict of interests regarding the publication of this study. authors’ contributions g.m.t., m.l.k.m. and r.n. conceived and designed the study. g.m.t. collected the samples. g.m.t., m.l.k.m., g.m. and w.g.d. analysed the data. g.m.t., w.g.d., g.m. and r.n. drafted the manuscript. all authors read and approved the final manuscript funding information this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). data availability statement data 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domestic ungulates in cameroon and several west african countries (1989–1992) following the 1987 mauritanian outbreak’, research in virology 146(1), 81–85. https://doi.org/10.1016/0923-2516(96)80593-0 abstract introduction materials and methods results and discussion conclusion acknowledgements references appendix 1 about the author(s) helena c. steyn vaccine development and diagnostics, onderstepoort veterinary research, pretoria, south africa alri pretorius vaccine development and diagnostics, onderstepoort veterinary research, pretoria, south africa citation steyn, h.c. & pretorius, a., 2020, ‘genetic diversity of ehrlichia ruminantium field strains from selected farms in south africa’, onderstepoort journal of veterinary research 87(1), a1741. https://doi.org/10.4102/ojvr.v87i1.1741 original research genetic diversity of ehrlichia ruminantium field strains from selected farms in south africa helena c. steyn, alri pretorius received: 12 feb. 2019; accepted: 01 nov. 2019; published: 25 june 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract heartwater is a tick-borne disease caused by the intracellular rickettsial parasite ehrlichia ruminantium and transmitted by amblyomma hebraeum ticks. heartwater is problematic in endemic areas because it causes high mortality in ruminants and leads to economic losses that threaten productivity and food security. this may indicate that there is augmented genetic diversity in the field, which may result in isolates that are more virulent than the ball3 and welgevonden isolates. the genetic diversity of e. ruminantium was investigated in this study, focussing on the pcs20 gene region and four polymorphic open reading frames (orfs) identified by subtractive hybridisation. the 16s ribosomal ribonucleic acid gene confirmed e. ruminantium in brain, blood and tick genomic deoxyribonucleic acid samples (n = 3792) collected from 122 farms that were randomly selected from seven provinces of south africa where heartwater is endemic. the conserved e. ruminantium pcs20 quantitative polymerase chain reaction (qpcr) assay was used to scan all collected field samples. a total of 433 samples tested positive with the qpcr using the pcs20 gene region, of which 167 were sequenced. the known stocks and field samples were analysed, and phylogenetic trees were generated from consensus sequences. a total of 25 new clades were identified; of these, nine isolates from infected blood could be propagated in cell cultures. these clades were not geographically confined to a certain area but were distributed amongst heartwater-endemic areas in south africa. thus, the knowledge of strain diversity of e. ruminantium is essential for control of heartwater and provides a basis for further vaccine development. keywords: ehrlichia ruminantium; molecular epidemiology; phylogenetic analyses; qpcs20 real-time pcr; subtractive hybridisation polymorphic orfs. introduction heartwater is a tick-borne disease caused by the obligate intracellular rickettsial parasite, ehrlichia ruminantium. amblyomma hebraeum tick species transmit the e. ruminantium pathogens to livestock and wild ruminants. heartwater is a major problem in the endemic areas of south africa (sa) because it causes mortality in cattle, sheep and goats, especially the angora goats that supply the mohair industry, and is responsible for high economic losses in both communal farming and the commercial sectors (allsopp 2010). molecular detection of e. ruminantium in animals and ticks focussed on the following genes or regions: the pcs20 region (steyn et al. 2008; van heerden et al. 2004), the 16s ribonucleic acid (rna) gene sequence (allsopp et al. 1997) and the map1 gene family (allsopp et al. 2001; barbet, byrom & mahan 2009; faburay et al. 2008). other methods of detection include loop-mediated isothermal amplification (lamp) (nakao et al. 2010), multiple-locus variable tandem repeats (mlva) (pilet et al. 2012) and multi-locus sequence typing (mlst) (adakal et al. 2009; nakao et al. 2011). however, there is not a single method/gene that provides the full picture of genetic diversity of e. ruminantium. genetic diversity amongst e. ruminantium isolates has been confirmed in previous epidemiological studies using the map1 gene family with known strains and field isolates in infected sheep and cattle from different ecological heartwater-endemic origins in africa. some of these isolates were from zimbabwe (kwekwe, crystal springs, plumtree and palm river), botswana (sunnyside), sa (welgevonden), nigeria (nigeria d225), senegal (senegal), sudan (um-banein) and caribbean (antiqua, gardel) (allsopp et al. 2001; faburay et al. 2008; reddy et al. 1996). the map1-2 paralog showed the greatest diversity (79%) in the map1 gene family while the rest were more conserved (98% – 99%) (barbet et al. 2009). the zimbabwe stains, highway, crystal springs and nyatsanga, were closely related while the senegal and antiqua strains were identical to each other. currently, we have 17 e. ruminantium reference strains from different geographical origins in sa, sub-saharan africa and the caribbean islands propagated in cell culture (allsopp, bezuidenhout & prozesky 2005). in spite of identifying additional isolates, including the crystal springs stock (zimbabwe), isolated from a dog (allsopp & allsopp 2001), the breed strain isolated from an angora goat (du plessis et al. 1983), rietgat (mahan et al. 1998), skukuza (peter et al. 1999) and warmbath isolates (mahan et al. 1998), there is little knowledge of the current diversity within e. ruminantium in sa. this highlights the need for an epidemiological survey in sa to determine the genetic diversity and occurrence of the pathogens in the field. genomic deoxyribonucleic acid (dna) from these 17 reference strains were used for sequence analysis (pcs20 region, 16s ribosomal rna [16s rrna], glta, groel, ftsz, sodb, nuob and map1) to demonstrate the extensive recombination that occurs between strains and to distinguish one strain from another by phylogenetic analysis (allsopp et al. 1997, 2005; allsopp & allsopp 2001, 2007). recombination is likely to occur when either the mammalian or the vertebrate host is infected with more than one isolate, or two infected ticks with two different isolates feed on the same animal. double infections have been demonstrated before in the ruminant host, for example, the kümm isolate, where two distinct genotypes were isolated from one blood sample, as described previously by zweygarth et al. (2002). however, it is also likely that recombination occurs in the tick during feeding where the tick is already infected with a strain from a previous feeding on a different infected animal (allsopp et al. 2007). recombination was also observed in several field samples. nakao et al. (2011) found recombination between known and unknown strains in uganda similar to the recombination between known strains that were described in sa (allsopp et al. 2007; aguiar et al. 2013; frutos et al. 2007). this study reports on a molecular epidemiological study of e. ruminantium in field samples from sa that were collected from different heartwater-endemic areas. this was done to characterise field isolates and confirm genetic diversity within e. ruminantium in sa. this study focussed on comparison of the conserved pcs20 gene region in different isolates. the pcs20 gene region is conserved and useful for the isolation of e. ruminantium in an epidemiological study but is not polymorphic enough to distinguish between new isolates that clustered together in the pcs20 phylogenetic tree. for example, the pcs20 region could not differentiate between the welgevonden stock and the ball3 strain that was used to develop the blood vaccine. therefore, four polymorphic open reading frames (orfs) (erum0250, erum1290, erum6300 and erum8340) were chosen for in-depth genotype characterisation. these orfs were previously identified by subtraction hybridisation using e. ruminantium dna from welgevonden and senegal stocks (unpublished). they are highly polymorphic and have an identity that varies between 66% and 92%. the orfs showed homology to unknown proteins or had no significant similarity to known sequences in the public databases. eight of these stocks were further characterised using the small-subunit 16s rrna gene to confirm that all the samples detected and used as reference strains belonged to e. ruminantium species. materials and methods study area this study was conducted in heartwater-endemic areas of sa where the vector tick (a. hebraeum) occurs. a total of 3792 samples (blood, n = 2385; brain, n = 4; and a. hebraeum ticks, n = 1403) were collected from randomly selected cattle, sheep and goats from 122 selected farms and rural areas including communal dip tanks (figure 1). samples were collected using the random geographic coordinate method (cameron 1999). a geographic area where heartwater occurs was first divided into districts. farms were randomly selected in districts with a high mortality rate because of heartwater or where continuous problems with the disease were reported. this included the eastern cape (ec) (grahamstown, bathurst, port alfred, alexandria and east london districts), kwazulu-natal (vryheid, pongola, paulpietersburg, umzinto, mzinga, hluhluwe, ladysmith, bulwer and ulundi districts), mpumalanga (mp) (kruger national park, nelspruit, witrivier, barberton and lydenburg districts), limpopo (lm) (potgietersrus district, and on a farm springbokfontein [sbf] in the tolwe district where a heartwater vaccine trial was also conducted), north west (nw) province (rustenburg district) and gauteng (gp) (roodeplaat and pretoria-north districts) (figure 1). samples (n = 40) were collected as negative controls in the free state (warden district) where the tick vector does not occur. in all of these cases, the disease status of the animals was unknown. figure 1: a map of south africa indicating the commercial farms and dip tanks in rural areas used to collect blood and tick (amblyomma hebraeum) samples for characterisation of ehrlichia ruminantium in the endemic regions of south africa. the heartwater sample collection sites are indicated with green dots. samples and extraction of deoxyribonucleic acid blood from cattle was collected from the coccygeal vein, while in sheep and goats it was collected from the jugular vein, into sterile heparin vacutainer® tubes. the local veterinarian who did the post-mortem collected brain tissue samples from four animals (two bovines, one sheep and one goat), and they were kept at -20 °c until used. ticks were collected from the same animals that were bled in the field or by drag sampling as described previously (horak et al. 2006). each live tick was washed with 70% ethanol, rinsed in distilled water, dried with tissue paper and cut in two and homogenised in 1.5 ml sucrose potassium glutamate (spg), and the homogenate was stored at -20 °c until used. genomic dna was extracted from whole blood (200 µl) or homogenised tick extract (200 µl) using the qiaamp® dna mini kit (qiagen) according to the manufacturers’ instructions and was stored at -20 °c until further use. phylogenetic screening and analyses using deoxyribonucleic acid sequence of the pcs20 region, 16s ribosomal ribonucleic acid gene and four polymorphic genes the standard pcs20 polymerase chain reaction (pcr) diagnostic assays, pcr/probe and quantitative polymerase chain reaction (qpcr) (taqman) probe, were performed as described (steyn et al. 2008; van heerden et al. 2004) and were used to screen the collected blood and tick samples for the presence of e. ruminantium. samples that tested positive were further analysed by amplifying the pcs20 region using a pcr method previously published (van heerden et al. 2004) with the hh1f and hh2r primers (table 1-a1) that gave a 720 bp amplicon. the small-subunit 16s rrna gene method described by allsopp et al. (1997) was used to confirm that all the samples detected and used as reference strains belonged to e. ruminantium. four polymorphic orfs, erum0250, erum1290, erum6300 and erum8340, previously identified using subtractive hybridisation assays (unpublished) were selected for analysis. primer sets for these orfs were designed for pcr and sequencing (idt whitehead sci.) (table 1-a1) and were used to amplify all pcs20-positive samples and all known strains. positive (welgevonden genomic dna) and negative (no genomic dna) controls were included in each run. four 25 µl pcr reactions of each sample amplified with each orf primer set were pooled after amplification. the pooled amplicons were purified with msb® spin pcrapace kit (stratec molecular, berlin, germany) and sequenced with the primers from each amplicon. sequencing was done using the bigdye terminator version 3.1 cycle sequencing kit (applied biosystems) on an abi 3100 sequencer (applied biosystems), and kits were used as recommended by the manufacturer. each amplicon was sequenced three times to obtain a consensus sequence. internal primers were required for the sequencing of the two largest orfs: erum1290 and erum8340. sequences of each sample in an orf were cut to the same length. sequence alignment as well as phylogenetic trees were inferred and visualised using the qiagen clc genomics workbench version 9 (http://www.clcbio.com/products/clc-genomics-workbench/). all the trees were unrooted. culturing and characterisation of field isolates blood stabilates were prepared in 10 ml aliquots of blood collected from animals in the field as described previously (brayton et al. 2003). briefly, infected blood (5 ml) was mixed with spg (5 ml) (sucrose, 0.218 m; kh2po4, 0.0038 m; k2hpo4, 0.0072 m; and glutamate, 0.0049 m; bovarnick, miller & snyder 1950). these stabilates were stored in liquid nitrogen until used. the blood stabilate was thawed at 25 °c in water diluted 1:300 in spg and 10 ml were used to infect a sheep within 20 minutes by the intravenous (iv) route (brayton et al. 2003). a total of nine naïve 8-month-old merino sheep obtained from heartwater-free areas were used in this trial. two blood stabilate samples used were collected from the current study: (1) blood stabilate from a sick angora goat (2 ml) from the farm riverside (ec province); and (2) blood stabilate from a sick sheep (10 ml) from the farm grootvallei in the potgietersrus district (lm province). in addition to these, six naïve sheep were each infected with six different blood stabilates from sheep that died of heartwater during a vaccine trial at the sbf farm (named as sbf1, sbf2, sbf4, sbf5, sbf6 and sbf7) (collins et al. 2003; pretorius et al. 2008). one sheep was infected with the omatjenne isolate (10 ml) collected from namibia (du plessis 1990). the omatjenne isolate was not previously propagated in cell cultures but was included in this study to serve as a reference strain. the temperatures of the infected sheep were observed daily after inoculation, and the animals were monitored for clinical signs of heartwater, which include heavy breathing and loss of appetite, depression, hanging head, stiff gait, excessive chewing movement and nervous signs. blood was collected from the jugular vein into sterile heparin vacutainer® tubes 3 days after an increase in temperature above 41.5 °c was observed for in vitro culturing, and the sheep were treated thereafter with terramycin®100. blood collected at the peak infection was used to initiate cell cultures for further characterisation as described previously (zweygarth & josemans 2001). once the cultures were established, the isolates were characterised using the pcs20 region (method described above), and these isolates were named reference strains. the small-subunit 16s rrna gene (allsopp et al. 1997) was used to confirm that all the samples detected and used as reference strains belonged to e. ruminantium. the 16s rrna gene was amplified and sequenced with vector-specified primers, t7 forward and sp6 reverse (table 1-a1), and these amplicons were cloned in pgem-t easy vector (promega, usa) and transformed into top10 (invitogen®) electrocompetent cells. plasmids with the correct-sized inserts were sequenced three times as described above and the consensus sequences of each were analysed. genbank accession numbers the genbank nucleotide accession numbers of new strains and uncultured isolates within the pcs20 gene region, erum0250, erum1290, erum6300 and erum8340 orfs, and the 16s rrna gene were submitted to genbank (table 2-a1). ethical considerations all animal work was performed in accordance with the stipulation of the animal ethics committee at the agricultural research council – onderstepoort veterinary institute animal ethics committee (ethical clearance number: 9/15/p001). the animal ethics was approved 2006/2007 on project numbers: department of agriculture ov9/23/c167 grant, the fp6 eu inev epigenevac fp6-003713 grant and the red meat research and development trust of south africa. results and discussion screening of field samples with the pcs20 quantitative polymerase chain reaction (taqman) probe assay the field samples collected in this study included blood, brain samples and a. hebraeum ticks obtained from cattle, sheep and goats (table 1). from 3788 field samples, 25% tick (344/1403) and 3.5% (85/2381) blood samples tested positive with the pcs20 assay. the observed low numbers of blood samples that tested positive for e. ruminantium could be attributed to the disease state of the animals. previous experiments have shown that the pcs20 test can only detect high levels of e. ruminantium in blood that develop one day before the onset of and during the febrile reaction (steyn et al. 2008). most of the ticks collected from the animals (data not shown) were positive while the blood sample collected from the same animal tested negative. it is possible that the ticks were collected prior to the febrile reaction and the pathogen could not yet be detected in the blood. other possibilities to consider are that the animals were immune to heartwater because of endemic stability (repeated exposure). in addition, the farmers regularly treat their animals with antibiotics, especially the angora goat farmers, if they appeared sick or depressed. in spite of this, two sick animals were found in the field, one angora goat from the riverside farm in the ec and one sheep from the farm grootvallei in the lm province that died from heartwater. two bovine brain samples were sent in from the nw province to confirm heartwater. these all tested positive with the pcs20 taqman probe assay. table 1: the combined results for pcs20-positive samples collected from cattle, sheep, goats and ticks in the six heartwater-endemic regions of south africa. characterisation of field strains using pcs20 from the field samples (blood and ticks, n = 3792) that originated from different geographical sites (122 farms) in sa (figure 1), only 433 blood and tick samples tested positive with the pcs20 assay. of these, only 167 field samples could be successfully sequenced and used to infer a pcs20 gene region maximum likelihood unrooted tree (figure 1-a1). in order to simplify this tree, identical sequences from the same farm were represented by a single sequence, and all new sequences were compared to the 17 known reference strains (figure 2). a detailed description of all the sites these samples were collected from is listed in table 3-a1. even though the pcs20 region is conserved (collins et al. 2003) and is used for heartwater diagnosis (steyn et al. 2008), it was still possible to distinguish 25 novel clusters in our study. figure 2: an unrooted phylogenetic tree of collected field isolates using the pcs20 gene region. the tree was constructed using the reference (blue) cultured strains and representatives of new isolates (red) in south africa, with a neighbour-joining maximum composite likelihood approach. propagated new stocks are indicated in green. the strains and field isolates are consistently separated into two major clades: the welgevonden (sa) and the east and west africa clades. the overall southand west african clustering is similar to other studies albeit using different genes including map1 and other housekeeping genes (adakal et al. 2009; allsopp et al. 2001, 2007, 2009; barbet et al. 2009; faburay et al. 2008; martinez et al. 2004; nakao et al. 2011). similar to pcs20, the map1 gene family is also separated into these two major clades (allsopp et al. 2001). the map1 family is conserved between strains (98% – 99%) except for map1-2 paralog with a diversity of 79% (barbet et al. 2009). in most studies on map1, chrystal-springs and mara87/7 cluster together, but were not identical (allsopp et al. 2001). in the current pcs20 analysis, we could not detect any sequence difference between these two strains. although not identical in sequence, welgevonden and the nonile strains clustered together in the map1 gene analysis. in the current pcs20 analysis, nonile and um-banein were identical and clustered together with gardel. this was also the case using the16s rrna gene. senegal and kümm1 clustered together in both genes tested in our study. furthermore, this study could define heterogeneity of strains in different locations in sa, as more than one isolate was found in an area and even on a farm. for example, nine field isolates that clustered together with the welgevonden, ball3 and vosloo strains were geographically distributed in four provinces (three lm, two mp, three ec and one nw) on seven different farms (figure 2). this is similar to previous findings using e. ruminantium and other ehrlichia spp. different genotyping methods of field samples collected in sa, africa and caribbean islands, using the map1 gene family, housekeeping genes or the pcs20 gene region, indicated that there is no geographical pattern on the distribution of e. ruminantium (allsopp et al. 2001, 2007; pilet et al. 2012). ehrlichia chaffeensis isolates from different geographic areas in the united states showed no diversity between them (yu, mcbride & walker 2007), while in some cases, isolates from different geographic areas belonged to the same group (i.e. from tennessee, florida and arkansas). comparison amongst various isolates using the outer membrane protein of e. chaffeensis p28-19 gene revealed that heterogeneity exists between isolates. this p28 gene belongs to the pfam01617 protein family and is present in e. chaffeensis, ehrlichia canis, ehrlichia ewingii, e. ruminantium and ehrlichia muris (yu et al. 2007). this gene sequence showed more diversity, and it is present throughout the coding sequences. culturing of new field isolates naïve sheep were infected with the omatjenne, 758-gvl-lm, 1166-rve-ec and the sbf 1–7 isolates for propagation in cell cultures. the animals showed clinical signs of heartwater of heavy breathing and loss of appetite, depression, hanging head, stiff gait, excessive chewing movement and nervous symptoms. all the sheep had a temperature of 42 °c for 3 days and were bled on the third day of febrile reaction and treatment was administered for 3 days. interestingly, the sheep infected with the omatjenne isolate showed identical heartwater signs. the new isolate names, origin and type of cell cultures used for propagation are listed in table 2. table 2: the nine new isolates established in cell culture. six blood isolates propagated in cell cultures from the lm province originated from the sbf farm. these blood isolates from the sbf farm were positive for e. ruminantium with the pcs20 assay before and after they were propagated in cell cultures confirming that these strains were the same as the original sequences. the sbf farm was used for a dna vaccine experimental field trial (pretorius et al. 2008), and six novel strains were found on this farm as well as 38 other isolates (figure 1-a1). in total, nine new strains were propagated in cell culture and were sequenced to confirm that the sequence was the same as the original blood sample. three new strains sbf6, sbf7 and grootvallei and 59 field genotypes (figure 1-a1) from other areas clustered together in this new cluster that was closely related to welgevonden strain but differ with two base pairs. two sbf isolates, four new sbf strains and 10 field isolates were identical to welgevonden. two new strains sbf2 and sbf5 and 34 other field isolates clustered with mara87/7. a new cluster formed close to welgevonden and originated from the 758-glv-lm (grootvallei farm, potgietersrus) and sbf farm (sbf6 and sbf7) and both were in the lm province. the welgevonden, ball3 and vosloo strains cluster together with two new strains (sbf1 and sbf4) from the sbf farm. the um-banein (sudan) and nonile (kwazulu-natal [kzn]) cluster together. however, kümm2, omatjenne and 1166-rve-ec (riverside farm) clustered on their own and had an identity similarity between 81% with the rest of the e. ruminantium sequences. the riverside strain named after the farm was isolated from an angora goat kid from the ec. the original blood sample (1166-rve-ec) used for sequencing and phylogenetic analysis was identical to the welgevonden isolate. a merino sheep was subsequently infected with the goat blood stabilate at the arc-ovi to establish this isolate in cell culture (ide8 cells). however, after propagation in cell culture, the sequence was identical to the kümm2 strain and the original isolate was lost. it could be that there was more than one e. ruminantium isolate in the original goat blood sample. similar results were obtained by zweygarth et al. (2002). they could distinguish between the two isolates, kümm1 and kümm2, by culturing the kümm strain in different cell lines using ba cells and the ide8 tick cell line. one of the isolates preferred growth in the ide8 cells, while the other was suppressed. the opposite was also confirmed where it was shown that the welgevonden strain grows in the ba cells and kümm2 does not (zweygarth et al. 2002). therefore, it was possible that the original riverside sample was infected with two isolates and at least one of these preferred growth in the ide8 tick cell line. the known kümm1 strain is the only south african strain that clustered together with the west african group but is not identical to them. interestingly, two field isolates from the ec and two from two farms in kzn grouped closely together with the western african cluster. it is the first time that we have found isolates that are close to the west africa clade. 16s ribosomal ribonucleic acid gene the 16s rrna gene sequence was used to confirm that the new isolates were that of e. ruminantium. results from the phylogenetic tree confirmed that the new strains belonged to the e. ruminantium species (figure 3). three of the new sbf (4, 6 and 7) strains clustered with mara87/7 and grootvallei, and the three sbf (1, 2 and 5) strains clustered with welgevonden. none of the new strains clustered with the western african clade except for the old kümm1 strain. the um-banein and the gardel strains were closely related within the pcs20 gene regions with only five nucleotide difference; we found that these two strains also grouped together using the 16s rrna v1 loop gene region. the kümm2 and omatjenne strains clustered on their own in the 16s rrna gene but closer to the west africa strains, and in the pcs20 gene region they cluster on their own but closer to the west africa and mara/blaauwkrans group. the kümm2 and omatjenne strains were previously described by allsopp and allsopp (2007) as non-pathogenic strains that cluster closely to e. ruminantium that was isolated from namibia (du plessis & kümm 1971). interestingly, the omatjenne was isolated from a hyalomma truncatum adult tick (du plessis 1990) and that could explain why it differs from e. ruminantium. although, it fits in the e. ruminantium group and potentially has the same clinical symptoms as heartwater, further in-depth characterisation is required. this may be done by genome sequencing of the kümm and omatjenne strains determining their host tick species and evaluating their pathogenicity in ruminants in order to reclassify them. figure 3: an unrooted phylogenetic tree was constructed using the 16s ribosomal ribonucleic acid sequence comparing the newly cultured strains (diamonds) with the reference strains in south africa using a neighbour-joining maximum composite likelihood approach. phylogenetic analyses using deoxyribonucleic acid sequence of four polymorphic genes the four polymorphic orfs (erum6300, 8340, 0250 and 1290) had different polymorphism profiles (figures 4 and 5). erum6300 divided the known strains into clusters that were similar to the pcs20 clusters, and only one field strain could be sequenced. however, with the erum8340, more field isolates could be distinguished from each other while known strains (grootvallei and sbf4) could not be sequenced. with the erum0250 and erum1290, more field isolates and known strains could be distinguished from each other. this indicates that even if the strains had identical pcs20 sequences, they however do differ from each other, confirming the huge genetic diversity in the field. figure 4: phylogenetic tree constructed (a) from erum0250 deoxyribonucleic acid sequence data of 11 reference strains and 25 field isolates and (b) from erum1290 deoxyribonucleic acid sequence data of 16 ehrlichia ruminantium reference strains, seven new strains and 33 field isolates from blood and ticks in the heartwater-endemic regions of south africa. figure 5: phylogenetic tree constructed (a) from erum6300 data of 16 ehrlichia ruminantium reference strains, seven new strains and one field isolate and (b) from erum8340 nucleic acid sequence of 16 e. ruminantium reference strains, seven new strains and 13 field isolates collected from blood and ticks in the heartwater-endemic regions of south africa. conclusion the current study identified 25 new e. ruminantium clusters based on pcs20 data. in addition, nine new isolates were propagated in cell culture. this study showed that there is major genetic diversity in sa, and this knowledge is essential for control of heartwater and for vaccine development. the impact of moving animals from one endemic area to another could increase recombination of strains to more virulent ones. thus, continuous epidemiological studies are needed to identify and grow isolates in vitro for further characterisation. as recombination has been shown to occur in many genes, future epidemiological studies should focus on complete genome sequencing of all cultured isolates. in addition, the cross-protection status of newly cultured isolates should be determined and compared with the highly virulent welgevonden strain, as we currently base all heartwater vaccine studies on this strain. acknowledgements the authors thank dr e. zweygarth and mrs a. josemans for culturing the new strains and dr selaelo ivy tshilwane for editing the manuscript. competing interests the authors have declared that no competing interest exists. authors’ contributions h.c.s. did the planning, execution and analysis of all experimental work and preparation of the manuscript. a.p. assisted with sequencing analysis and manuscript editing. funding information this study was funded by the south african department of agriculture ov9/23/c167 grant, the fp6 eu inev epigenevac fp6-003713 grant and red meat research and development trust of south africa. data availability statement the sequence data is available in genbank (see table 2-a1). disclaimer the 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strijp, m.f., van heerden, h., allsopp, m.t. & allsopp, b.a., 2002, ‘the kümm isolate of ehrlichia ruminantium: in vitro isolation, propagation and characterization’, the onderstepoort journal of veterinary research 69(2), 147–53. appendix 1 table 1-a1: primer sequences used to amplify and sequence the, erum0250, erum1290, erum6300, erum8340 orfs, hh1f, hh2r, 16sf and 16sr, cowf, cowr and cowtm probe. table 2-a1: genbank accession numbers of the 16sr rna and pcs20 gene region. table 3-a1: a summary of the samples collected in south africa. figure 1-a1: an unrooted phylogenetic tree of all collected field isolates and reference strains using the pcs20 gene region. the tree was constructed with a neighbor-joining maximum composite likelihood (mcl) approach. the data is summarised in figure 2. lidetu_73-79.indd introduction echinococcosis/hydatidosis is a zoonotic disease caused by tapeworms of the genus echinococcus that occurs throughout the world and causes considerable economic losses and public health problems in many countries. taxonomically only four species are accepted and regarded as valid. these are echinococcus granulosus, echinococcus multilocularis, echinococcus oligartrus and echinococcus vogeli (thompson 1986). the disease which is caused by e. granulosus has a broad geographical distribution, with highly endemic areas in countries of africa, latin america, australia and asia (soulsby 1986). it is considered endemic in the entire mediterranean zone including egypt and all countries in the middle east (haridy, ibrahim & morsy 2000; anderson, ouhelli & kashani 1997 cited by dalimi, motamedi, hosseini, mohammadian, malaki, ghamari & ghaffari far 2002). the domestic dog as a definitive host of the adult e. granulosus plays the most important role in the spread of infection via contamination of 73 onderstepoort journal of veterinary research, 74:73–79 (2007) the prevalence, organ distribution and fertility of cystic echinoccosis in feral pigs in tropical north queensland, australia d. lidetu1 and g.w. hutchinson2 abstract lidetu, d. & hutchinson, g.w. 2007. the prevalence, organ distribution and fertility of cystic echinococcosis in feral pigs in tropical north queensland, australia. onderstepoort journal of veterinary research, 74:73–79 an investigation was carried out to study the prevalence of echinococcus granulosus hydatidosis in feral pigs (sus domesticus) in the charters towers region of tropical north queensland. data were collected from a total of 238 carcasses, which were hunted and shot in the burdekin river catchment area. organs of the abdominal, thoracic, and pelvic cavities were examined for the presence of hydatid cysts. in the laboratory, cysts and hydatid cyst fluids were examined under a stereoscopic binoc ular microscope and a compound microscope. an overall prevalence of e. granulosus hydatid cysts in feral pigs was found to be 31.1 %. there was no significant difference in either sex or age between infected and non-infected feral pigs. the predilection sites of cysts were livers (23 %) and lungs (62 %), with more cysts in lungs (252) than livers (48). the ratio of livers to lungs infected with fertile cysts was 1:4 compared to 1:8 sterile cysts. the overall fertility of cysts was 70.1 %. the percentage of fertile cysts in liver and lung was 79.2 % and 68.7 %, respectively. the diameter of fertile cysts ranged from 15 to over 60 mm. there was no significant difference in size between fertile and non-fertile cysts in lungs. the high prevalence rate and fertility of cysts in feral pigs confirm that feral pigs can take part in the sylvatic cycle of the parasite in the region. the public health significance of this observation is potentially very important. keywords: australia, echinococcus granulosus, feral pigs, north queensland, organ distribution, prevalence, sylvatic hydatidosis 1 ethiopian institute of agricultural research, national animal health research center, p.o. box 04, sebeta, ethiopia. email: deslidetu@yahoo.com 2 australian institute of tropical veterinary and animal sciences, james cook university, townsville, qld. 4811, australia. present address: nsw agriculture, elizabeth macarthur agricultural institute, pmb 8, camden, nsw 2570, australia accepted for publication 9 october 2006—editor 74 cystic echinoccosis in feral pigs in tropical north queensland, australia environment creating the domestic life cycle. the maintenance of e. granulosus in wild or feral animals without the involvement of humans or domestic ungulates demonstrates the existence of sylvatic cycles. there is evidence of the sylvatic cycle in australia, north america, and in east and south africa (rausch 1986). based on morphological, developmental and biochemical criteria thompson & kumaratilake (1982), thompson (1987), lymbery & thompson (1989) and thompson & lymbery (1990) classified e. granulosus in australia into three intraspecific variants, namely the tasmanian sheep strain, the australian mainland strain and the australian sylvatic strain. the sylvatic cycle exists in north queensland between dingoes and macropods (banks 1985). kumaratilake & thompson (1982) found two infected feral pigs in western australia, but none of the five cysts were fertile. subsequently, thompson (1987) re ported that 12 out of 25 feral pigs trapped were infected with the parasites. sakamoto, tani, hutchin son, copeman, thompson & sakamoto (1989) and hutchinson & copeman (1988) conducted epidem i ological and histopathological studies in north queens land and suggested that feral pigs could play an important role in the sylvatic cycle of the parasite. the sample size taken during these studies was small. the objective of this study was to further investigate the prevalence, organ predilection and cyst fertility levels of hydatid disease in feral pigs in tropical north queensland. materials and methods study area this study was conducted in the charters towers region of dalrymple shire in sub coastal north queensland, approximately 120 km inland from townsville (19.35° s, 146.78° e). the total area was estimated to be up to 30 000 km2 in extent and is in the seasonally dry tropics with one annual wet season, which is generally between december and april. the start of the wet season may vary depending on the monsoon. the mean annual rainfall in charters towers is 570 mm. the annual average daily maximum and minimum temperatures are 27.4 °c and 12.7 °c, respectively. study design and sampling procedure studies to establish the prevalence of hydatid disease in animals depend mainly on collection of data obtained from animals slaughtered in abattoirs. in wildlife reliable and accurate records to study this disease depend on the number of animals which can be trapped or killed for survey purposes. limit ations of surveillance data for cystic hydatidosis have been reviewed recently (schantz 1997). therefore, this study was designed on the use of a chiller depot which has been assigned permanently in the study area to store the carcasses of feral pigs shot in the field. feral pigs are hunted for sport and for export as game meat to europe especially to germany. fieldshot animals were identified with ear tags with code numbers and deposited the same day in the chiller depot after their intestinal tracts were removed. the weight of each carcass was measured and it and the hunter’s registration number were recorded on tags which were fastened to each carcass. data, such as mass, sex, age group and physical condition of the carcasses were collected on a monthly basis. for subsequent meat inspection prior to export certification hearts, the lungs, livers, kidneys and spleens were not removed from carcasses. age estimations were based on dentition. the animals were assigned to one of two broad age groups, i.e. 9–15 months as young, and above 16 months as adult. data on suckling or weaner pigs were not available. organs of the abdominal, thoracic and pel vic cavities were examined for the presence of hydatid cysts by observation and palpation, with special emphasis being placed on the livers and lungs. hydatid lesions were differentiated from other cystic parasitic diseases based on fao/unep/who guide lines (1981), and were removed intact from the infected organs, stored on ice and transported to a laboratory in townsville within 4 h. in the laboratory, the size of the cysts was measured. small lesions < 5 mm diameter and those that could not be confidently differentiated as hydatids, were excluded. the presence and number of cysts were recorded according to organ distribution. the contents of each cyst were then aspirated with a hypodermic syringe, transferred into vials or petri dishes and examined under a stereoscopic binocular microscope for the presence of brood capsules. drops of hydatid cyst fluid (hcf) were also mounted on slides with cover slips and observed under a compound microscope for the presence (fertile cysts) or absence (sterile cysts) of protoscolices. a fertile cyst was recorded as viable if it contained protoscolices showing flame-cell activity. cysts which contained no protoscolices or were calcified, were considered as infertile. the cysts were then incised with 75 d. lidetu & g.w. hutchinson a scalpel to determine the stages of degeneration. all were examined for the presence of an identifiable laminated membrane indicating that the cyst was of echinococcus origin. this structure could be detected in hydatid cysts which were either live or partially necrotic, and it appeared to persist for some time after death of the parasite (banks 1985). age and sex, predilection sites, fertility and sterility of cysts were factors which were considered for statistical analysis. the chi-square method of analysis was employed to determine statistical differences in the fertility of cysts in both sexes and age groups of the pigs. numbers and sizes of the cysts were calculated and differences tested for statistical significance using a one-way analysis of variance using statistix version 3.5 (analytical software, st. paul, mn, usa). results all feral pigs were of the same black type (fig. 1). a total of 238 feral pigs was examined, 137 being male and 101 female. of these 103 were classified as young and 135 as adult. the overall prevalence of e. granulosus was 31.1 % (74/238) with the age group prevalence of the disease being 31.1 % in both young and adult feral pigs alike. the sex-specific prevalence was 32.1 % and 29.7 % for male and female feral pigs, respectively (tables 1 and 2). in all 74 of the infected feral pigs 56 lungs, 17 livers and one kidney were found harbouring one or more cysts. the prevalence of e. granulosus metacestodes in the principal sites of predilection was calculated to be 56/74 (75.7 %) in the lung and 17/74 (22.93 %) in the liver. mean numbers of cysts per organ were 2.8 in livers and 5.5 in lungs (table 3). a single sterile cyst was found in a kidney. no cysts were found in the hearts or other organs. the frequency distribution of the number of cysts found in infected pigs shows the level of infection. a total of 301 confirmed hydatid cysts was found in the livers and lungs of all the infected feral pigs (table 3). the percentage of fertile cysts in the livers and lungs was 79.2 % and 68.7 %, respectively (table 3). the overall fertility rate of cysts in livers and lungs was 70.1 %. live protoscolices were found in 19/38 (50 %) of all fertile liver cysts and 109/173 (63.2 %) of all fertile cysts from lungs. the ratio of livers to lungs infected with fertile cysts was about 1:4 compared with 1:8 for sterile cysts. the diameter of fertile cysts ranged from 15–60 mm (table 4). of all cysts in either organ only a very few cysts showed necrosis and calcification. the mean size of sterile cysts from the livers (33.7 mm) was significantly larger than that of the fertile cysts (21.0 mm) (table 4) but in lungs there was no difference between the two types (mean 31 mm). there was no significant difference in the fertility of cysts in pigs of either sex (p = 0.803). discussion in this study, importance was given to feral pigs as they have the ability to transmit diseases and parasites important to livestock and humans (kanameda � �� �� �� �� ��� ��� ��� ��� ��� ��� � � � � � �� �� �� �� �� �� ������� ��� � � � � � � �� � � �� �� � �� fig. 1 frequency distribution of hydatid cysts in feral pigs table 1 frequency distribution of hydatid cysts in infected feral pigs by sex sex no. examined no. infected no. not infected % infected male female 137 101 44 30 93 71 32.1 29.7 total 238 74 164 31.1 chi-square 2.587 degrees of freedom 1 significance 0.108 thus sex and infection are independent 76 cystic echinoccosis in feral pigs in tropical north queensland, australia 1990). the use of dogs for pig hunting and the way these dogs are fed during the hunting season may contribute to the maintenance of hydatid disease in feral pigs. this situation has stimulated number of stud ies in different parts of australia (banks 1985; baldock et al. 1985; thompson & kumaratilake 1982; hutchinson & copeman 1988; sakamoto et al. 1989). the present study showed an overall prevalence of 31.1 % of e. granulosus hydatid cysts in feral pigs, which is comparable to, but slightly higher than that reported in a preliminary study in the same area which was 8/29 (27 %); the number of samples were, however, small (hutchinson & copeman 1988). there were no statistical differences due to age or sex (tables 1 and 2). gemmell & brydon (1960) found no differences in sex-specific prevalence in beef and dairy cattle in new south wales but showed an increase in prevalence with age. similarly, baldock, arthur & lawrence (1985b) found no differences in sex-specific prevalence in queensland cattle, but the prevalence increased linearly with age from 17 months to 4 years. several factors may affect the variation in prevalence with origin of hosts. these include climate, stocking rate and the abundance of infected definitive and other intermediate hosts. similar factors may be responsible for the variation in prevalence in feral pigs between queensland and western australia. as very young (i.e. suckling pigs) were not sampled, as such animals are not accepted by the game processors, the true age prevalence could not be determined, but would be expected a priori to be similar to that in cattle. table 2 frequency distribution of hydatid cysts in infected feral pigs by age age group no. examined no. infected no. not infected % infected young adult 134 104 43 31 93 71 32.1 29.8 total 238 74 164 31.1 chi-square 2.587 degrees of freedom 1 significance 0.108 thus sex and infection are independent table 3 distribution and percentage of fertile hydatid cysts per organ in infected feral pigs organ infected no. of feral pigs no. of cysts % fertile mean no. of cysts fertile sterile total liver lung kidney 17 56 1 38 173 0 10 79 1 48 252 1 79.2 68.7 0 2.8 5.5 0 total 74 211 90 301 70.1 4.1 degrees of freedom 1 significance p = 0.108 table 4 differences in the mean size (± standard error) of fertile liver and lung hydatid cysts in feral pigs cyst type mean size (mm) ± s.e no. of feral pigs fertile liver lungs 21.6 ± 2.02 31.2 ± 0.58 22 40 sterile liver lungs 33.7 ± 3.66 31.1 ± 2.87 6 15 for cysts in the liver there was a significant difference in size due to fertility/sterility f (1, 26) = 7.84, p = 0.0095 for cysts in the lung there was no significant difference in size due to fertility/sterility f (1, 53) = 0.0, p = 0.9680 77 d. lidetu & g.w. hutchinson the lungs and livers were favoured sites for hydatid cyst development in feral pigs as has been shown to be the case with other studies in pigs or other host species. in this study cysts were found in lungs five times more often than in livers. about 13.5 percent of the total feral pigs sampled had cysts in both organs, but no cysts were found in any other organ except for a single cyst in a kidney. banks (1985), however, found three infected feral pigs of which two harboured single hepatic cysts, one of which was fertile, and the third had disseminated infection with some fertile cysts in the liver, lungs, spleen and kidneys. in other susceptible host species such as ungulates, hydatid cysts also occur predominantly in the lungs and livers. in cervids (cervinus spp.) cysts are rarely found in organs other than the lungs (rausch 1975). in macropods cysts are often found in the thoracic cavity, mainly in the lungs, although occasional cysts occur in the pleural cavity attached to the lungs by a thin peduncle (banks 1985). in pigs the liver is often involved alone or in combination with the spleen and kidneys (reviewed in rausch 1997). unicystic forms of infection seem to be more common than multicystic forms. very few multicystic forms were found in this study. at present there are no means by which to estimate the ages of hydatid cysts in feral pigs without conducting an experimental infection study to determine growth rates of the cysts. without this information it is not possible to determine when feral pigs became infected. while the majority of cysts observed in this study were between 21 and 30 mm in diameter there was no significant differences (p > 0.05) between the mean size of cysts in livers and lungs. the diameter of metacestodes of the sylvatic strain of e. granulosus in experimentally infected cattle increased linearly with time. hepatic cysts increased at a rate of 0.3 mm per month, so that after 1 year the predicted mean diameter was only 3.9 mm (banks 1985). according to banks’ (1985) estimate, a cyst with a diameter of 5 mm could be between 1 and 2 years of age. growth rates in sheep are believed to be similar (sweatman & williams 1963). dew (1925) claimed that hydatid cysts in domestic pigs reached 40 mm in only 3 months. unfortunately no information has been found of similar studies in feral animals. as there are host species differences and other factors, such as the immune response, environmental and climatic factors, it cannot be established if the natural growth rate of hydatid cysts in feral pigs is similar to this, or closer to that in herbivores. the high percentage (69–80 %) of fertile hydatid cysts in feral pigs was surprising in view of the limited information on the fertility rate that was obtained in previous studies, and the very low (< 1 %) rate in cattle cysts in the same region. although a high proportion of cysts were fertile, the numbers of protoscoleces observed in each cyst were relatively low, usually less than 50 and they were frequently nonviable. this precluded the possibility of having the protoscolices typed to strain. in contrast, from a single fertile cyst of sheep origin several thousand protoscolices can be found. in the present study, there was no statistically significant difference in the fertility of cysts in pigs of either sex (p = 0.843). fertile liver cysts were, with one exception, smaller than 30 mm diameter, whereas some lung cysts up to 60 mm contained protoscolices. the reasons for the high fertility level of e. granulosus in feral pigs and whether feral pigs harbour the sylvatic strain of the parasite are not yet known. the low proportion of fertile cysts in cattle (baldock et al. 1985b; banks 1985) suggests that cattle play little part in continuing the life cycle of the parasite. our result supports the view that feral pigs might play an important role in maintaining the sylvatic life cycle of e. granulosus. banks (1985) noted that in those parts of northern queensland hundreds of kilometres from the nearest sheep, no evidence was found for the existence of domestic animal cycle spill-over into wild and feral animals which share the same habitat. since, in northern queensland the mainland domestic strain has been identified (baldock et al. 1985a) and propagation of the mainland domestic strain between dingoes and feral pigs has been suggested (banks 1985) further study is clearly necessary to better understand the relationships between strains and their host assemblages. banks (1985) described the two most significant intermediate hosts as the black-striped wallaby (macro pus dorsalis) and the feral pig (sus domesticus) with a prevalence of 21.8 % and 9.4 %, respectively. in the present study, the 31 % prevalence of the disease in feral pigs was higher than that obtained previously. this demonstrates that feral pigs have a role in the epidemiology of the disease in this region. there is no direct evidence that feral pigs are a substantial diet of dingoes, in distinction to wallabies, which have been identified in stomach contents (banks 1985). whether feral pigs are infected from dingo faeces, and if dingoes are capable of being infected from pig-derived cysts is yet to be clarified. 78 cystic echinoccosis in feral pigs in tropical north queensland, australia using a chiller depot for disease surveys in wild or feral animals could be of use for the storage of animals shot in the field and it could become an economical way for collecting information. however, such surveys must be planned carefully as killed feral animals are not true random samples from the population at risk, especially where size of the population of the stock is difficult to estimate or it is impossible to apply techniques of sample size estimation. the use of tables of binomial confidence limits or capture and release techniques (cannon & roe 1982) would be applicable to estimate the sample size for this type of disease survey. this survey was conducted under such conditions for number of reasons. firstly, a simple, economical and satisfactory method of surveying feral pig hydatid disease was not found. secondly, the chiller depot serves not only as a convenient source of data and specimens, which would otherwise be logistically difficult to obtain in sufficient numbers, but it is also a source which enables the tracing of information on the habitat of the feral animals and behaviour which can be obtained by personal communication with the professional hunters. the public health significance of this investigation is important. hunters’ dogs which may be fed offal from killed or scavenged dead feral pigs, could become infected, and if patent infections develop, they may shed e. granulosus eggs within and around urban centres. after being voided in the faeces of a dog, eggs can contaminate extensive areas through transportation by blowflies. thompson (1987) has suggested such a cycle already exists in perth. no data are available on human hydatidosis in north queensland and no pig hunting dogs were found to be infected with e. granulosus (hutchinson & copeman 1988), but this leaves no grounds for complacency. baldock et al. (1985a), in determining the origin of human infections, suggested the domestic strain of the parasite was most commonly encountered in human cases. in their study campos-bueno, lopezabente & andres-cercadillo (2000) described the risk factors for hydatidosis traceable to the family to be of greater relative importance than those attributable to working directly with livestock. from the results of this study, we speculate that the sylvatic pig form may be a potential source of human cases of hydatidosis for humans. acknowledgements the australian international development bureau (aidb) is acknowledged for the opportunity to undertake this postgraduate study at the then graduate school of tropical veterinary science, james cook university, north queensland, townsville. mr glen de’ath is thanked for his advice and assistance with the statistical analysis. references baldock, f.c., thompson, r.c.a. & kumaratilake, l.m. 1985a. strain identification of e. granulosus in determining origin of infection in case of human hydatid disease in australia. transactions of royal society of tropical medicine and hygiene, 79:238–241. baldock, f.c., arthur, r.j. & lawrence, a.r. 1985b. a meatworks survey of bovine hydatidosis in southern queensland. australian veterinary journal, 62:335–339. banks, d.j.d. 1985. the epidemiology of echinococcosis in trop ical queensland, ph.d. dissertation, james cook univer sity. campos-bueno, a., lopez-abente, g. & andres-cercadillo, a.m. 2000. risk factors for echinococcus granulosus infection: a case control study. the american journal of tropical medicine and hygiene, 62:329–334. cannon, r.m. & roe, r.t. 1982. livestock disease surveys: a field manual for veterinarians. australian bureau of animal health, department of primary industry. canberra: australian government publishing service. dalimi, a., motamedi, g.h., hosseini, m., mohammadian, b., malaki, h., ghamari, z. & ghaffari, far, f. 2002. echinococcosis/ hydatidosis in western iran. veterinary parasitology, 105:161–171. dew, h.r. 1925. the histogenesis of the hydatid parasite (taenia echinococcus) in the pig. medical journal of australia, 1: 101–130. fao/undp/who 1981. guidelines for surveillance and control of echinococcosis/hydatidosis. geneva: switzerland gemmel, m.a. & brydon, p. 1960. hydatid disease in australia. v. observation on hydatidosis in cattle and pigs in new south wales and the economic loss caused by the larval stage of e. granulosus (batsch, 1786; rudolphi, 1801) in food animals in australia. australian veterinary journal, 36: 73–78. haridy, f.m., ibrahim, b.b. & morsy, t.a. 2000. sheepdog-man. the risk zoonotic cycle in hydatidosis. journal of the egyptian society of parasitology, 30:423–429. hutchinson, g.w. & copeman, d.b. 1988. research report. graduate school of tropical veterinary science, james cook university. kanameda, m. 1990. studies of selected diseases of feral pigs in townsville area. m.sc. thesis, james cook university. kumaratilake, l.m. & thompson, r.c.a. 1982. intraspecific variation in e. granulosus. australian situation and perspectives for the future. transactions of royal society of tropical medicine and hygiene, 76:13–16. lymbery, a.j. & thompson, r.c.a. 1989. genetic differences between cysts of e. granulosus in the same host. international journal for parasitology, 19:961–964. rausch, r.l. 1975. taeniidae, in diseases transmitted from animals to man, edited by w.d. hubbert, w.f. mcculloch & p.r. schnurrenberger. springfield: c. thomas. rausch, r.l. 1985. life-cycle patterns and geographic distribution of echinococcus species, in the biology of echino79 d. lidetu & g.w. hutchinson coccus and hydatid disease, edited by r.c.a. thompson. boston: allen & unwin. rausch, r.l. 1997 echinococcus granulosus: biology and ecology, in compendium on cystic echinococcosis in africa and in middle eastern countries with special reference to morocco, edited by f.l. andersen, h. ouhelli & m. kachani. provo: brigham young university. sakamoto, t., tani, s., hutchinson, g.w., copeman, d.b., thompson, r.c.a. & sakamoto, h. 1989. studies on echinococcosis in australia. i. histopathological observations on echinococcosis of cattle in australia. journal of the faculty of agriculture, iwate university, 18:323–337. schantz, p.m. 1997 sources and uses of surveillance data for cystic echinococcosis, in compendium on cystic echinococcosis in africa and in middle eastern countries with special reference to morocco, edited by f.l. andersen, h. ouhelli & m. kachani. provo: brigham young university. soulsby, e.j.l. 1986. helminths, arthropods and protozoa of domesticated animals. london: bailliere tindall. sweatman, g.k. & williams, j.r. 1963. comparative studies on the biology and morphology of echinococcus granulosus from domestic livestock, moose and reindeer. para sitology, 53:339–390. thompson, r.c.a. 1986. the biology and systematics of echinococcus. london: academic press. thompson, r.c.a. 1987. outbreak of hydatid disease in western australia. parasitology today, 3:261–262. thompson, r.c.a. & kumaratilake, l.m. 1982. intra-specific variation in e. granulosus. australian situation and perspectives for the future. transaction of royal society of tropical medicine and hygiene, 76:13–16. thompson, r.c.a. & lymbery, a.j. 1990. echinococcus: biology and strain variation. international journal for parasitology, 20:457–470. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) stuart j.g. gordon institute of veterinary, animal and biomedical sciences, massey university, new zealand charlotte bolwell institute of veterinary, animal and biomedical sciences, massey university, new zealand chris w. rogers institute of veterinary, animal and biomedical sciences, massey university, new zealand godfrey musuka international center for aids care and treatment programs, mailman school of public health, columbia university, united states patrick kelly ross university school of veterinary medicine, st. kitts, west indies alan guthrie equine research centre, faculty of veterinary science, university of pretoria, south africa philip s. mellor international research centre, the pirbright institute, united kingdom christopher hamblin international research centre, the pirbright institute, united kingdom citation gordon, s.j.g., bolwell, c., rogers, c.w., musuka, g., kelly, p., guthrie, a. et al., 2017, ‘the sero-prevalence and sero-incidence of african horse sickness and equine encephalosis in selected horse and donkey populations in zimbabwe’, onderstepoort journal of veterinary research 84(1), a1445. https://doi.org/10.4102/ojvr.v84i1.1445 research communication the sero-prevalence and sero-incidence of african horse sickness and equine encephalosis in selected horse and donkey populations in zimbabwe stuart j.g. gordon, charlotte bolwell, chris w. rogers, godfrey musuka, patrick kelly, alan guthrie, philip s. mellor, christopher hamblin received: 02 mar. 2017; accepted: 17 mar. 2017; published: 10 may 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract sentinel herds and samples submitted by private equine practitioners were used to determine the sero-prevalence and sero-incidence of african horse sickness virus (ahsv) and equine encephalosis virus (eev) in horse and donkey populations in the highveld region of zimbabwe. the sero-prevalence and sero-incidence of antibodies against these viruses were determined using the competitive enzyme-linked immunosorbent assay (elisa) for the detection of serum antibodies. in donkeys, the median sero-prevalence of ahsv antibodies, across the three rainy seasons under study, was 75% (inter quartile range [iqr] 67–83), with a seasonal median sero-incidence of 45% (iqr 40–63). in horses, the median sero-prevalence of eev antibodies was 63% (iqr 21–73), with a median seasonal sero-incidence of 10.5% (iqr 10–14), while in donkeys the median sero-prevalence of eev antibodies was 80% (iqr 67–90), with a median seasonal sero-incidence of 50% (iqr 40–60). this study highlighted the significant levels of exposure of donkeys to ahsv and horses and donkeys to eev in zimbabwe despite equine encephalosis remaining unreported by zimbabwean veterinarians to date. most seroconversions in sentinel herd animals to ahsv and eev occurred towards the end of the rainy season in march, april and may corresponding to the time of the year when the culicoides vectors are in high abundance. in order to determine the clinical significance of these infections, blood and spleen samples, submitted by private equine veterinary practitioners over a 5-year period, from horses showing characteristic clinical signs of african horse sickness were tested for the presence of viral antigen using the antigen capture elisa. the median sero-prevalence of ahsv antigen in horses recorded from these samples was 38% (iqr 33–88). the predominant ahsv antigen from these samples was serotype 7 (33%) followed by serotype 2 (26%) and serotypes 4 and 8 (16% each). african horse sickness virus serotypes 3 and 9, identified in this study, had not been previously reported in zimbabwe. introduction african horse sickness (ahs) and equine encephalosis (ee) are significant horse diseases in zimbabwe caused by arthropod-borne viruses belonging to the reoviridae family, genus orbivirus. mortality rates of up to 95% because of ahs have been recorded in unvaccinated horses in south africa (coetzer & guthrie 2004). namibia lost an estimated 10% of its horse population to ahs in 2011 (liebenberg et al. 2016). the donkey is resistant to both ahs and ee but is considered to be an ideal sentinel to determine the prevalence and distribution of ahs virus (ahsv) and ee virus (eev) through the detection of specific antibodies resulting from natural infection (howell et al. 2002). in contrast to ahs, ee is perceived as an emerging infectious disease and was first identified in 1967 by erasmus et al. (1970) in horses in south africa. ee has also been reported in botswana, kenya, namibia, zimbabwe (coetzer & guthrie 2004; grewar et al. 2015; paweska et al. 1999), ethiopia, ghana, the gambia (oura et al. 2012) and israel (wescott et al. 2013). infections of horses with the eev manifest typically as subclinical or mild disease (crafford et al. 2011). crafford (2002) described ee morbidity rates in horses in south africa exceeding 74% with mortality rates up to 5%. the distribution of ahs and ee in southern africa seems to be governed by a number of factors including the prevalence and seasonal incidence of the major arthropod vector(s), which are certain species of culicoides biting midges (diptera: ceratopogonidae) (liebenberg et al. 2016; mellor & hamblin 2004). this suggests that the incidence of these diseases will have the same seasonality as the vector species. gordon et al. (2015) found the greatest diversity and highest median counts of culicoides to be in the central highveld region of zimbabwe where climatic and geophysical conditions favoured optimal breeding and larval development. although the vector competence of most culicoides species in zimbabwe has not been studied in detail, gordon et al. (2015) isolated orbiviruses from culicoides imicola which included eev. in zimbabwe, blackburn and swanepoel (1988) and musuka and kelly (2000) reported that ahs was diagnosed in late summer and autumn, which corresponds to high recorded numbers of the culicoides vector (gordon 2010). the objectives of this study were to determine the sero-prevalence and sero-incidence of ahsv and eev in horses and donkeys from sentinel herds established around harare and bulawayo in zimbabwe. the serotypes of ahsv in animals from the sentinel herds and from the samples of suspected clinical cases in horses were also determined. materials and methods identification of sampling herds donkeys and horses on selected farms around the cities of bulawayo and harare, in zimbabwe, were sampled to determine the sero-prevalence and sero-incidence of ahsv and eev antibodies. both locations lie in the central highveld of zimbabwe and experience climatic conditions that favour the abundance of the culicoides vector(s). farms were chosen based on a minimum number (> 6) of adult (> 18-month-old) donkeys or horses present and on-farm owner compliance. the farms included in the study were all of similar size and contained soil types, vegetation types and husbandry practices typical of farms in the highveld of zimbabwe. determining the sero-prevalence of african horse sickness virus and equine encephalosis virus antibodies in sentinel herds venous blood samples (5 ml) were taken from all horses on participating farms using ethylene diamine tetra-acetic acid (edta) vacutainers and tested for eev antibodies to determine the sero-prevalence of eev in these herds. blood samples were also taken from all donkeys on participating farms and tested for antibodies against ahsv and eev to determine the sero-prevalence of ahsv and eev in these donkey herds. due to the widespread use of the live, attenuated, ahs vaccine (onderstepoort biological products®) in horses in zimbabwe, the sero-prevalence of ahsv in horses could not be determined as ahsv-specific antibodies may have been derived from vaccine viruses. the detection of antibodies against ahsv was determined using a competitive enzyme-linked immunosorbent assay (elisa) following the methodology described by hamblin et al. (1992). the detection of antibodies against eev was determined using an elisa following the methodology described by crafford (2002) and subsequently published by crafford et al. (2011). determining the sero-incidence of african horse sickness virus and equine encephalosis virus antibodies in sentinel herds sentinel herds of donkeys and horses were established, at the same farms used to determine the sero-prevalence of antibodies to these two viruses during three rainy seasons (1999–2002). these sentinel herds were used to determine the sero-incidence of ahsv antibodies in donkeys and eev antibodies in horses and donkeys by determining the rates at which sentinel animals seroconverted to ahsv or eev. the sentinel herds were established using healthy adult horses and donkeys of both sexes that tested as seronegative against ahsv antibodies (donkeys) and eev antibodies (horses and donkeys). a total of three horse sentinel herds were established around harare and six around bulawayo. two donkey sentinel herds were established on farms around harare and one around bulawayo. venous blood samples from the animals in the sentinel herds were taken using edta vacutainers and tested for antibodies against ahsv and eev every 2 weeks, during each of the three rainy seasons (november–april), until seroconversion was detected. private equine veterinary practitioners’ samples private equine veterinary practitioners, working within the highveld region of zimbabwe, were asked to monitor for suspected clinical cases and deaths from ahs in horses located within their veterinary practice catchment area over a 5-year period from 1998 to 2003. during this period, spleen samples, blood samples and combined spleen and blood samples were submitted by private equine veterinary practitioners from horses suspected to have died from or suffering from clinical ahs. no clinical cases of ee in horses or donkeys had been reported by practising veterinarians in zimbabwe to that time although all submitted samples were tested for both ahsv and eev. the blood samples submitted were collected in edta from live patients and the spleen tissue from dead patients following the recommendations outlined by hamblin et al. (1992). some practitioners submitted both ante-mortem blood samples and post-mortem spleen samples from fatal clinical cases. all submitted blood and spleen samples were washed and lysed using techniques recommended by hamblin et al. (1992) and musuka and kelly (2000). a serogroup specific, indirect, sandwich, antigen capture elisa developed by hamblin et al. (1991) and crafford et al. (2003) was used to detect the presence of ahsv and eev antigens respectively in these samples. virus isolations were made by intracerebral inoculation of 2-day-old suckling mice (cd-1, uk) and intravenous inoculation of 11-day-old embryonated hens’ eggs (iah, uk). in each case, 100 μl of a 1 in 10 dilution of lysed blood or homogenated spleen was inoculated. brains from dead mice and hearts from dead embryos were homogenised as a 1 in 10 suspension and serially blind passaged up to three times in baby hamster kidney (bhk) tissue culture cells to amplify any virus present and to adapt them to growth in cell culture. viral serotyping the ahs viruses were typed by virus neutralisation, using microtitre plates and bhk cells, following methods similar to those described by howell (1962). virus neutralisation tests were performed on viruses isolated from edta blood and spleen samples submitted by private practitioners as well as those isolated from edta blood samples from sentinel herd animals 2 weeks before they seroconverted. assaying the blood samples collected before animals seroconverted maximised the chance of isolating free virus. ahsv positive sera, from sentinel animals that had seroconverted but were negative for virus isolation, were serotyped in neutralisation tests using defined serotype-specific ahs viruses, using microtiter plates and bhk cells, following methods modified from blackburn and swanepoel (1988). statistical analysis data were examined using simple descriptive statistics. data were tested for normality using the shapiro–wilk test. non-parametric data are presented as median and inter quartile range. the effect of season on prevalence was tested using a kruskal–wallis test. all analyses were performed using stata ic v12 (college station, tx, usa). the level of significance was set at p < 0.05. results sero-prevalence and sero-incidence of african horse sickness virus and equine encephalosis virus antibodies serum decanted from blood samples collected from 279 horses was tested for eev antibodies over the three rainy seasons to determine the sero-prevalence of eev in these herds. the small per farm sample size (22; inter quartile range [iqr] 14–28) provided a large between-farm variation in the sero-prevalence of eev (table 1). the highest prevalence was recorded during the second rainy season (2000/2001) although no significant difference between years in sero-prevalence was found (p = 0.059). the median sero-prevalence of eev across the three rainy seasons under study in the horses sampled was 63% (iqr 21–73). table 1: the sero-prevalence of antibodies against equine encephalosis virus in horses in zimbabwe, during three rainy seasons for the period 1999–2002. serum decanted from blood samples collected from 155 donkeys was tested for antibodies against ahsv and eev to determine the sero-prevalence of ahsv and eev (table 2). the median sero-prevalence of ahsv and eev across the three rainy seasons under study was 75% (iqr 67–83) and 80% (iqr 67–90), respectively (table 2). table 2: the sero-prevalence of antibodies against african horse sickness virus and equine encephalosis virus in donkeys in zimbabwe during three rainy seasons for the period 1999–2002. a total of six horse sentinel herds were established over three rainy seasons to determine the sero-incidence of eev antibodies in horses. the sentinel herds consisted of a total of 76 horses and a median sero-incidence of eev of 10.5% (iqr 10–14) was recorded. one to two donkey sentinel herds were established during each rainy season under study to determine the sero-incidence of ahsv and eev antibodies in donkeys. the sentinel herds consisted of a total of 18 donkeys, with each sentinel herd in each rainy season comprising 4–5 donkeys. a median sero-incidence of 45% (iqr 40–63) and 50% (iqr 40–60) was recorded for ahsv and eev, respectively. private equine veterinary practitioners’ samples during a 5-year period from 1998 to 2003, 22 spleen samples, 92 edta blood samples and 11 combined spleen and edta blood samples were submitted by private equine veterinary practitioners from 125 horses suspected to have died from or suffering from clinical ahs (table 3). based on antigen detection from samples submitted by private practitioners, there was an overall median sero-prevalence of ahsv antigens in horses of 38% (iqr 33–88) (table 3). three of the 125 samples tested positive for eev antigens. table 3: horse samples submitted by private equine veterinary practitioners for the detection of african horse sickness virus antigens. viral serotyping the predominant ahsv serotype identified from private equine veterinary practitioners’ horse spleen and blood samples and blood samples from donkey sentinel herd animals showing seroconversion was serotype 7 (19/57, 33%) followed by serotype 2 (15/57, 26%), serotypes 4 and 8 (9/57, 16% each) and serotype 6 (3/57, 5%). serotypes 3 and 9 (1/57, 2%) were identified for the first time in zimbabwe. attempts to serotype eev were unsuccessful. discussion this study recorded a high median sero-prevalence of antibodies against ahsv in selected donkey herds and a high median sero-prevalence of antibodies against eev in selected horse and donkey herds in the highveld region of zimbabwe. a sero-prevalence for antibodies against eev of 56% – 87% in horses and donkeys and a sero-prevalence of ahsv of 44% – 64% in horses were previously reported in zimbabwe (blackburn 1982; blackburn, searle & phelps 1985; blackburn & swanepoel 1988; musuka 1999; musuka & kelly 2000; paweska et al. 1999). howell et al. (2008) found the seasonal sero-prevalence of antibodies against eev in south africa to vary between 3.6% and 34.7%. because of the annual polyvalent prophylactic immunisation against ahsv practised in horses in zimbabwe at the time of this study, serotype-specific antibody derived from immunisation, natural challenge or the transfer of colostrum could not have been distinguished by conventional serum viral neutralisation assays (howell et al. 2008). since sero-epidemiological surveys would, therefore, have been inconclusive in establishing seroconversion to natural ahsv challenge, horse sera were not tested for antibodies against ahsv in this study. no donkeys in this study had received prophylactic immunisation against ahsv meaning that antibodies against ahsv were most likely derived from natural challenge. the median seasonal sero-incidence of eev antibodies was found to be higher in donkey sentinel herds than in horse sentinel herds. this could be because of the ahs vaccine used in horses conferring some form of protection against eev in this species but may also reflect the fact that donkeys are rarely stabled at night in zimbabwe and are thus more at risk of exposure to the culicoides vector(s) than are most horses. furthermore, the high incidence of ahsv and eev antibody detection in donkeys should be interpreted with caution as the sentinel herd sample size of donkeys in this study was small. the prevalence and incidence rates, however, show that horses and donkeys face a significant rate of exposure to both ahsv and eev in zimbabwe. previously, ee in southern africa was thought to be mainly distributed in south africa and botswana (paweska et al. 1999), and so these results have emphasised the importance of this disease in horses and donkeys in zimbabwe. the sentinel herds were situated in the highveld of zimbabwe, which is characterised by high rainfall and temperature that favour the abundance of the culicoides vector(s). the sero-incidence of ahsv and eev antibodies in sentinel herds at some of the farms closely matched the number of culicoides vectors trapped per night at these farms, as reported in previous studies by gordon (2010) and gordon et al. (2015). most sero-conversions to ahsv and eev in this study occurred from january to april, 3–4 months after the beginning of the annual rainy season. this delay reflects the time taken for vector numbers to increase after the beginning of the annual rains and the time for a viraemia to develop in animals, which then act as sources of infection for the vectors (mellor 1990). the clinical cases and samples submitted by practitioners also displayed this apparent seasonal latency. this temporal pattern of a delay in sero-conversions has been previously reported by blackburn and swanepoel (1988) and musuka and kelly (2000) where most cases of ahs were diagnosed in late summer and autumn coinciding with the end of the zimbabwean rainy season in march to may. however, further studies utilising rainfall data would be required in order to demonstrate an association between rainfall and the sero-incidence of ahsv and eev. the predominant ahsv serotype recorded in this study was serotype 7, which was identical to the findings reported in the 1980 outbreak of ahs in the north west of harare (blackburn & swanepoel 1988). in the current study, ahsv serotypes 3 and 9 were identified for the first time in zimbabwe. despite the detection of eev antigen in three of these samples submitted by private practitioners, no serotypes for eev could be identified. in a study on ee in thoroughbred yearlings in south africa, serotypes 1 and 6 were most frequently identified, while the remaining serotypes 2, 3, 4, 5 and 7 were only identified sporadically in localised infections of ee affecting individual horses (howell et al. 2008). conclusion this study highlighted the significant levels of exposure of horses and donkeys to ahsv and eev in zimbabwe. the sero-incidence of ahsv and eev infection and the clinical cases of ahs were highest at the end of the rainy season in march and april coinciding with peak abundance of the culicoides vector(s). the main ahsv serotype responsible for causing clinical ahs in the blood and spleen samples submitted by private equine veterinary practitioners and blood samples from sentinel herd animals showing seroconversion was serotype 7, corroborating previous ahsv identification research findings in zimbabwe. this study identified the presence of ahsv serotypes 3 and 9 in zimbabwe for the first time. african horse sickness continues to be a serious problem in zimbabwe despite the extensive vaccination programme conducted in horses across the country at the time of this study. as ee is largely undiagnosed by equine veterinary practitioners in zimbabwe, the high reported sero-prevalence and sero-incidence of this disease will hopefully alert zimbabwean veterinarians to the possibility of its occurrence in both horses and donkeys. acknowledgements the authors would like to acknowledge the support and cooperation provided by the horse and donkey establishments in zimbabwe which permitted sentinel herds to be established and monitored over all the rainy seasons under study. the authors are also grateful for the assistance provided by the private equine veterinary practitioners who provided the blood and spleen samples from horses with suspected ahs clinical cases. the authors would also like to acknowledge the support provided by the biomedical research and training institute and the wellcome trust. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions s.j.g.g. wrote the manuscript and acted as the project leader. g.m., p.k., p.s.m. and c.h. helped design the project, establish and monitor the sentinel herds, test for serum antibodies and antigens and assist with the virus isolation and identification. c.b., c.w.r. and a.g. made conceptual contributions and assisted with the data analysis, statistical work and the editing of the final manuscript. references blackburn, n.k., 1982, 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and infection 140, 1982–1986. https://doi.org/10.1017/s0950268811002688 paweska, j.t., gerdes, g.h., woods, p.s.a. & williams, r. 1999, ‘equine encephalosis in southern africa: current situation’, in u. wernery, j.f. wade, j.a. mumford & o.r. kaaden (eds.), proceedings of the eighth international conference on equine infectious diseases, dubai, uae, march 23–26, 1998, pp. 303–305. wescott, d.g., mildenberg, z., bellaiche, m., mcgowan, s.l., grierson, s.s., choudhury, b. et al., 2013, ‘evidence for the circulation of equine encephalosis virus in israel since 2001’, plos one 8, e70532. https://doi.org/10.1371/journal.pone.0070532 phologane_267-277.indd introduction foot-and-mouth disease (fmd) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. although mortality in adult animals is generally low, the production losses when intensively farmed livestock are infected can be significant. the aetiological agent of this disease, the foot-and-mouth disease virus (fmdv), belongs to the aphthovirus genus of the picornaviridae family. the virion consists of an icosahedral shell composed of 60 copies each of four structural proteins, vp1, vp2, vp3 and vp4 surrounding a singlestranded positive-sense rna genome of approxi267 onderstepoort journal of veterinary research, 75:267–277 (2008) molecular characterization of sat-2 foot-and-mouth disease virus isolates obtained from cattle during a four-month period in 2001 in limpopo province, south africa b.s. phologane1, 2†, r.m. dwarka1, d.t. haydon3, l.j. gerber2 and w. vosloo1, 4* abstract phologane, b.s, dwarka, r.m, haydon, d.t., gerber, l.j. & vosloo, w. 2008. molecular characterization of sat-2 foot-and-mouth disease virus isolates obtained from cattle during a fourmonth period in 2001 in limpopo province, south africa. onderstepoort journal of veter inary research, 75:267–277 foot-and-mouth disease (fmd) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. the virus is a single-stranded rna virus that has a high rate of nucleotide mutation and amino acid substitution. in southern africa the south african territories (sat) 1-3 serotypes of fmd virus are maintained by large numbers of african buffaloes (syncerus caffer), which provide a potential source of infection for domestic livestock and wild animals. during february 2001, an outbreak of sat-2 was recorded in cattle in the fmd control zone of south africa, adjacent to the kruger national park (knp). they had not been vaccinated against the disease since they form the buffer between the vaccination and free zones but in the face of the outbreak, they were vaccinated as part of the control measures to contain the disease. the virus was, however, isolated from some of them on several occasions up to may 2001. these isolates were characterized to determine the rate of genetic change in the main antigenic determinant, the 1d/2a gene. nucleotide substitutions at 12 different sites were identified of which five led to amino acid changes. three of these occurred in known antigenic sites, viz. the gh-loop and c-terminal part of the protein, and two of these have previously been shown to be subject to positive selection. likelihood models indicated that the ratio of non-synonymous to synonymous changes among the outbreak sequences recovered from cattle was four times higher than among comparable sequences isolated from wildlife, suggesting that the virus may be under greater selective pressure during rapid transmission events. keywords: 1d (vp1) gene, cattle, foot-and-mouth disease, mutation rate, sat-2 * author to whom correspondence is to be directed. e-mail: vosloow@arc.agric.za 1 agricultural research council, onderstepoort veterinary insti tute, exotic diseases division, private bag x05, onder stepoort, 0110 south africa 2 department of biomedical science, tshwane university of tech nology, private bag x680, pretoria, 0001 south africa 3 division of environmental and evolutionary biology, university of glasgow, glasgow, g12 8qq, uk 4 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa † deceased accepted for publication 2 july 2008—editor 268 molecular characterization of sat-2 foot-and-mouth disease virus isolates obtained from cattle in south africa mately 8 500 nucleotides. seven immunologically distinct serotypes (a, o, c, sat-1, sat-2, sat-3 and asia 1) have been identified, of which the south african territories (sat) types 1–3, are endemic in sub-saharan africa (brooksby 1982). historically, the sat-2 serotype has been responsible for almost half of fmd outbreaks in domestic animals in southern africa (thomson & bastos 2004). eradication of the disease in sub-saharan africa is not possible owing to the presence of large numbers of free-living african buffaloes (syncerus caffer), which are efficient maintenance hosts for the sat types of fmdv and provide a potential source of infection for domestic livestock and wild animals (thom son & bastos 2004; vosloo & thomson 2004). these viruses can persist in an individual buffalo and in an isolated herd for at least 5 and 24 years, respectively (condy, hedger, hamblin & barnett 1985). the use of fencing to separate buffaloes and livestock as well as vaccination of cattle with viruses that are antigenically closely related to those carried by nearby buffalo form an integral part of fmd control in southern african countries (hunter 1998; brück ner, vosloo, du plessis, kloeck, connoway, ek ron, weaver, dickason, schreuder, marais & moga jane 2002). despite these control measures, outbreaks of fmd do occur if some of the control measures are compromised, one of which, the sat2 outbreak reported here, occurred in 2001 on farms in the limpopo province adjacent to the kruger national park (knp), home to free-ranging, infected african buffaloes. the disease remains a major economic concern for the livestock industry in many developing countries and a continued threat to countries that are disease free due to its potential negative impact on trade in agricultural products. thus, rapid identification of the serotypes of fmdv that are responsible for an outbreak is essential for selection of an appropriate emergency vaccine and to trace the origin and spread of an outbreak (callens & de clercq 1997). genetic characterization of outbreak strains is also important to determine if variants exist in the field, especially for the sat types in which it has been shown that more significant genetic variation occurs compared to other serotypes (bastos, haydon, forsberg, knowles, anderson, bengis, nel & thomson 2001; bastos, anderson, bengis, keet, winterbach & thomson 2003a; bastos, haydon, sangare, boshoff, edrich & thomson 2003b; sahle, dwarka, venter & vosloo 2007; sahle, dwarka, ven ter & vosloo 2007a, b). foot-and-mouth disease viral rna consists of a single open reading frame flanked by two non-coding regions both predicted to exhibit complex secondary structures (mason, grubman & baxt 2003). because rna viruses lack proof-reading capacity, their genomes undergo high mutation rates, shown to be between 10–5 and 10–4 base mis-incorporations per nucleotide site per genome replication (holland, spind ler, horodyski, grabeu, nichol & vande pol 1982; drake 1993; drake & holland 1999). this, together with a very short generation time, can result in as much as 0.5–1.5 % of the genome changing each year (haydon, samuel & knowles 2001b). the analysis of rna sequences from field isolates indicated that fixation of mutations occurs along the entire fmdv genome, but mutations are preferentially accumulated in structural regions, which are the targets of selective pressures (martin, nunez, sobrino & dopazo 1998; fares, moya, escarmis, baranowski, domingo & barrio 2001; haydon, bastos, samuel & knowles 2001a). the high antigenic diversity of fmdv stems from its extreme genetic heterogeneity (mateu, martinez, rocha, andreu, parejo, giralt, sobrino & domingo 1989). antigenic variants can be selected for in immune or partially immune hosts (gebauer, de la torre, gomes, maten, barahona, tiraboshi, bergman, de mello & domingo 1988), although diez, mateu & domingo (1989) reported that antigenic variants may accumulate over time even in the absence of antibody selection. studies have shown that cattle and buffalo carriers (subclinically infected animals) may be a significant source of genetic variation (salt, samuel & kitching 1996; vosloo, bastos, kirkbride, esterhuysen, janse van rensburg, bengis, keet & thomson 1996; vosloo, de klerk, boshoff, botha, dwarka, keet & haydon 2007). in particular, capsid protein vp1, encoded by the 1d gene, greatly contributes to the antigenicity of fmdv (mateu et al. 1989; mateu, camarero, giralt, andreu & domingo 1995) due to the existence of an immuno-dominant site, located between beta-strands g and h (the g-h loop) of vp1 which also acts as the cell receptor recognition site (kitson, mccahon & belsham 1990). other sites on the capsid involving vp2 and vp3 also play a role in the antigenic characteristics of the virus (stave, card, morgan & vakharia 1988; kitson et al. 1990). during february 2001 an fmd outbreak was detected in cattle in the buffer zone in limpopo province, republic of south africa affecting various dip tanks in the communal farming district. the outbreak followed severe flooding in the area that damaged 269 r.m. phologane et al. the game fence surrounding the knp and buffaloes were observed on the communal farms. this study concerned the investigation of the genetic variation accumulating in the 1d gene among 11 sat-2 isolates obtained from a single outbreak over 111 days. sequencing of the full-length 1d gene of these sat-2 outbreak strains enabled us to determine the position of substitutions in the main antigenic determinant. in response to the outbreak, immediate widespread vaccination of cattle herds was conducted. in order to examine whether vaccination resulted in a change in selective pressures on the virus the ratio of non-synonymous to synonymous changes among the outbreak sequences were compared with those from closely related strains isolated from wildlife in the adjoining knp. materials and methods viruses included in this study eleven viruses were isolated from bovine epithelial tissue samples taken from clinically affected animals on various farms involved in the sat-2 outbreak over 111 days (table 1). these samples were first propagated on primary pig kidney (pk) cells and, if necessary, passaged on ib-rs-2 (instituto biologico rim suino) cells. once positive cultures were obtained, the viruses were stored in glycerol at –70 °c for further characterization. twelve sat-2 1d sequences were selected from previous publications for phylogenetic and other analysis. table 1 summarizes all relevant information regarding the viruses used in this study. rna extraction, cdna synthesis and genomic amplification genomic rna was extracted from cultured viruses by a modified silica/guanidinium thiocyanate nucleic acid extraction method (boom, sol, salimans, jansen, wert heim-van dillen & van der noordaa 1990) and reverse transcribed using amv-rt (pro mega) and the 2a/b junction primer, p1, of beck & strohmaier (1987) as described by bastos (1998). genomic amplification of the 1d gene was performed using a forward primer binding within the 1c gene (vp3) termed vp3-ab (5′ cac tgc tac cac tcr gag tg 3′) (bastos et al. 2003b) and a reverse primer, p1 (5′ gaa ggg ccc agg gtt gga ctc 3′) which is complimentary to the highly conserved 2a/b junction site (beck & strohmaier 1987), resulting in a product of approximately 880 bp. pcr was performed in a 50 μℓ volume in the presence of 3 μℓ cdna, 200 μm dntps, 25 pmol of each primer, 1 x taq buffer (roche) and 2.5 u of taq polymerase (roche). thermal cycling profile included an initial denaturation step at 96 °c for 1 min, followed by 30 cycles of denaturation at 96 °c for 20 s, annealing at 56 °c for 30 s and extension at 70 °c for 30 s. pcr product purification and nucleotide sequence determination the pcr products of 880 bp were excised from the 1.5 % agarose gel and purified using the qiaquick gel extraction kit (qiagen). sequencing of the full length 1d gene (648 bp) was performed using the big dye® version 3.1 cycle sequencing ready reaction kit and the abi prism 310 genetic analyser (applied biosystems). two independent cycle sequencing reactions were performed per sample using the sense and anti-sense primers utilized in the pcr. phylogenetic analysis nucleotide sequences were trimmed to correspond to the full length 1d gene (648 bp) and six nucleotides of the 2a gene of all sat-2 outbreak strains and aligned with published sequences (obtained from genbank, table 1) using the dapsa program (harley 2001). phylogenetic trees were constructed using maximum likelihood methods (swofford 2001) assuming an hky85 model (hasegawa, kishino & yano 1985) of nucleotide substitution with rate heterogeneity. support for the phylogenetic groups within the tree was estimated from 1 000 bootstrap re-samples using the neighbour-joining method (swofford 2001) with the same model of nucleotide substitution. statistical parsimony (implemented in the program tcs [clement, posada & crandell 2000]) was utilized to reconstruct the sequence of mutations throughout the course of the outbreak. this parsimony-based algorithm outputs a rooted network indicating likely haplotype transitions. the underlying population genetic algorithm on which the statistical basis of the output is determined is complex and described in detail elsewhere (templeton, crandall & sing 1992) and is particularly suited for application to very closely related sequences. the ratio of non-synonymous to synonymous substitutions (dn/ds) for the sequences detailed in table 1 was calculated using the program codeml (yang 1997) which approximates the dn/ds ratio over the estimated phylogeny. different dn/ds ratios over dif270 molecular characterization of sat-2 foot-and-mouth disease virus isolates obtained from cattle in south africa ferent parts of the tree were estimated. because the viruses in this study were replicating in vaccinated hosts, the hypothesis that they might be under more intense selective pressure than viruses in the rest of the phylogeny was tested and therefore two dn/ds ratios were fitted: one to non-outbreak strains, and the other to the monophyletic group constituting the outbreak strains (indicated in fig. 1). since the simpler model is nested within the more complex model, a likelihood-ratio rest (lrt) could be used to determine the level of support for the more complex model. results phylogenetic analysis the maximum-likelihood tree showed that the 11 outbreak sequences (sar/1/01 to sar/11/01) were all closely related to each other and grouped in a table 1 summary of sat-2 serotype isolates included in this study virus designation country of origin date of sampling place of origin grid reference species genbank accession no. days post outbreak sar/1/01♠ rsa 01/02/2001 orinoco dt, lp 31°05’ e–24°45’ s bov ay442903 1 sar/2/01♦ rsa 01/02/2001 orinoco dt, lp 31°05’ e–24°45’ s bov ay442904 1 sar/3/01♦ rsa 01/02/2001 orinoco dt, lp 31°05’ e–24°45’ s bov ay442905 1 sar/4/01♦ rsa 01/02/2001 orinoco dt, lp 31°05’ e–24°45’ s bov ay442906 1 sar/5/01♦ rsa 06/02/2001 orinoco dt, lp 31°05’ e–24°45’ s bov ay442907 6 sar/6/01♦ rsa 04/02/2001 newington, lp 31°18’ e–24°48’ s bov ay442908 4 sar/7/01♠ rsa 05/02/2001 dwarsloop, lp 31°06’ e–24°47’ s bov ay442909 5 sar/8/01♦ rsa 05/02/2001 dwarsloop, lp 31°06’ e–24°47’ s bov ay442910 5 sar/9/01♦ rsa 05/02/2001 dwarsloop, lp 31°06’ e–24°47’ s bov ay442911 5 sar/10/01♠ rsa 22/05/2001 craigieburn, lp 30°58’ e–24°40’ s bov ay442912 111 sar/11/01◊ rsa 22/05/2001 craigieburn, lp 30°58’ e–24°40’ s bov ay442913 111 knp/18/95 rsa 1995 mondzweni 31°38’ e–24°34’ s buf af367118* – knp/31/95 rsa 1995 mondzweni 31°38’ e–24°34’ s buf af367119* – knp/19/89 rsa 1989 ripape 31°37’ e–24°44’ s buf af367110* – knp/183/91 rsa 1991 water affairs weir 31°56’ e–25°08’ s buf af367112* – knp/32/92 rsa 1992 boyela vlakteplaas 31°17’ e–22°54’ s buf af367115* – knp/18/88 rsa 1988 orpen gate 31°24’ e–24°28’ s imp af367138* – knp/19/88 rsa 1988 rabelais dam 31°30’ e–24°27’ s imp af367106* – knp/20/88 rsa 1988 timbavati river 31°28’ e–24°26’ s imp af367107* – zim/267/98 zim 1998 chizarira 28°00’ e–17°47’ s buf af367130* – zam/7/96 zam 1996 mulanga 25°30’ e–17°10’ s buf af367120* – zam/10/96 zam 1996 mulanga 25°30’ e–17°10’ s buf af367121* – bot/18/98 bot 1998 nxaraga 23°15’ e–19°40’ s buf af367123* – rsa = republic of south africa; lp = limpopo province; zim = zimbabwe; zam = zambia; bot = botswana; bov = bovine; buf = buffalo; imp = impala; – = not applicable; passage history of viruses (pk = primary pig kidney cells, rs = ibrs-2 cell line): ♠pk1, rs1; ♦pk1; ◊pk1,rs2 * bastos et al. 2003b 271 r.m. phologane et al. knp/19/89/buffalo knp/18/95/buffalo knp/31/95/buffalo knp/18/88/impala knp/20/88/impala knp/19/88/impala knp/183/91/buffalo knp/32/92/buffalo zam/7/96/buffalo zam/10/96/buffalo bot/18/98/buffalo zim/267/98/buffalo sar/8/01/cattle sar/9/01/cattle sar/3/01/cattle sar/4/01/cattle sar/5/01/cattle sar/6/01/cattle sar/7/01/cattle sar/1/01/cattle sar/10/01/cattle sar/11/01/cattle sar/2/01/cattle 0.1 100 100 96 65 99 80 100 96 99 a 100 fig. 1 the maximum likelihood phylogeny of the outbreak sequences and a number of closely related sequences from wildlife obtained in the knp, zimbabwe, botswana and zambia. levels of bootstrap support (estimated using the neighbor-joining method) are indicated on the branches. the ‘a’ indicates the position downstream from which a different dn/ds ratio was fitted to the rest of the tree 272 molecular characterization of sat-2 foot-and-mouth disease virus isolates obtained from cattle in south africa single cluster with 100 % bootstrap support (fig. 1). the outbreak isolates grouped with viruses previously obtained from the knp during an outbreak in impalas in 1988 (knp/18-20/88) and isolates obtained from buffaloes in the park between 1989 and 1995 (knp/19/89, knp/18/95 and knp/31/95) (12– 13 % nucleotide differences). this cluster was supported by 99 % bootstrap. the buffalo isolates from zambia, zimbabwe and botswana grouped according to topotype patterns previously observed for sat-2 (fig. 1; bastos et al. 2003b). the estimated transition:transversion ratio for this set of sequences was 2.51:1, and the shape parameter for the ϒ-distribution governing rate heterogeneity was estimated to be 0.27. mutation analysis the coding region included in the analysis contained 648 nucleotides from the 1d/2a gene, translated into 216 amino acid residues (214 aa 1d and 2 aa of 2a, fig. 2). three of the initial isolates (sar/24/01) had 100 % identical nucleotide sequences and were assumed to represent the ancestral state. on the same day and the same farm, orinoco, sar/1/01 was isolated that differed by 0.309 % from the other isolates. on day 4, sar/6/01, isolated on the farm newington, differed by 0.154 %, while the three dwarsloop isolates (sar/7-9/01) obtained at day 5 differed by 0.154 %, 0.463 % and 0.309 %, respectively, from the original viruses. sar/5/01 was isofig. 2 sequence alignments of 216 amino acids corresponding to the 1d/2a gene of the 11 sat-2 outbreak strains obtained during a single outbreak that affected various farms during 2001. cell attachment site of the viruses (rgd), in the g-h loop is highlighted and underlined. ‘ .’ indicates amino acids identical to sar/2/01 10 20 30 40 50 60 70 80 sar/2/01 ttsagegadv vttdpsthgg qvkekrrmht dvafvldrft hvhtnkttfn vdlmdtnskt lvgallrast yyfcdleiac sar/1/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/3/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/4/o1 .......... .......... .......... .......... .......... .......... .......... .......... sar/5/01 .......... .......... ......l... .......... .......... .......... .......... .......... sar/6/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/7/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/8/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/9/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/10/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/11/01 .......... .......... .......... .......... .......... .......... .......... .......... 90 100 110 120 130 140 150 160 sar/2/01 vgehkrvywq pngaprttql gdnpmvfsnk gvtrfavpyt aphrllstvy ngeckytasv tairgdravl aakytntkht sar/1/01 .......... .......... .......... .......... .......... ....e..... .......... .......... sar/3/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/4/o1 .......... .......... .......... .......... .......... .......... .......... .......... sar/5/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/6/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/7/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/8/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/9/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/10/01 .......... .......... .......... .......... .......... .......... .......... .......... sar/11/01 ..k....... .......... .......... .......... .......... .......... .......... .......... 2a 170 180 190 200 210 215 sar/2/01 lpstfnfghv tadasvdvyy rmkraelycp rpllpaydha nrdrfdapig vekq lc sar/1/01 .......... .......... .......... .......... .......... .... .. sar/3/01 .......... .......... .......... .......... .......... .... .. sar/4/o1 .......... .......... .......... .......... .......... .... .. sar/5/01 .......... .......... .......... .......... .......... .... .. sar/6/01 .......... .......... .......... .......... s......... .... .. sar/7/01 .......... .......... .......... .......... .......... a... .. sar/8/01 .......... .......... .......... .......... .......... .... .. sar/9/01 .......... .......... .......... .......... .......... .... .. sar/10/01 .......... .......... .......... .......... .......... .... .. sar/11/01 .......... .......... .......... .......... .......... .... .. 273 r.m. phologane et al. lated at day 6 from orinoco and similarly differed by 0.154 %. on day 111 two isolates were obtained from craigieburn and differed by 0.309 % and 0.463 % from the ancestral state. the tcs analysis of the nucleotide sequences (fig. 3) indicated that this divergence probably accumulated as a result of 12 independent mutations (11 in vp1 and 1 in the 2a gene). five of these mutations resulted in non-synonymous amino acid changes: position 27: g-t, arginine to leucine (sar/5/01); 83: g-a, glutamic acid to lysine (sar/11/01); 135: a-g, lysine to glutamic acid (sar/1/01); 201: a-g, asparagine to serine (sar/6/01); and 211: t-c, valine to alanine (sar/7/01). the latter three of these changes arose in known antigenic sites, the g-h loop (135) and the c-terminus region (201 to 211) (kitson et al. 1990). al these amino acid substitutions were due to nucleotide differences in the second position of the codon, except the change on amino acid position 135, which occurred in the fist position of the codon. none of the changes became fixed in isolates over time. the tcs analysis (fig. 3) shows that one variant (sar/1/01) was present within the first 3 days of the outbreak together with a theoretical genotype not sampled. this latter variant was not isolated again and may have been a dead-end for this particular virus. the ancestral virus seemed to have spread to other farms. between days 4–110 five more variants were sampled of which three (sar/5-7/01) resulted from three separate direct changes from the ancestral state, and two viruses (sar/8-9/01) represented viruses arising from genotypes that were not sampled (fig. 3). at day 111 two viruses were isolated, one resulted from an absent genotype (sar/10/01), while the last isolate (sar/11/01) was fig. 3 the rooted cladogram supported by statistical parsimony in the program tcs. the square indicates the identified root of the genealogy. small circles indicate genotypes that were not sampled. nucleotide substitutions are labelled on the arrows (those in bold are non-synonymous). where a genotype is removed from its putative ancestor by more than one mutational change, the order of these changes is unknown 93 403 602 632 80 336 c-t 465 247 489 648 291135 d a y s 1 – 3 d a y 1 1 1 sar/8/01/cattle sar/9/01/cattle sar/5/01/cattle sar/6/01/cattle sar/7/01/cattle sar/1/01/cattle sar/10/01/cattlesar/11/01/cattle g-t d a y s 4 – 1 1 0 sar/2-04/01/cattle g-a c-t c-t c-t t-c t-c t-c t-c a-g a-g 274 molecular characterization of sat-2 foot-and-mouth disease virus isolates obtained from cattle in south africa related to sar/10/01 and originated from a single difference between the two isolates. the overall average dn/ds ratio calculated in codeml over the phylogeny shown in fig. 1 was 0.070, indicating strong purifying selection. when two dn/ds ratios were fitted, one over the outbreak clade, and another for the rest of the tree, the dn/ds ratio for the main tree declined to 0.066, but increased to 0.264 in the outbreak clade. the lrt indicated that this more complex model was a significant improvement over the simpler model (χ2 = 4.1, p < 0.05) and suggested that the four-fold increase in the dn/ds ratio in the outbreak clade should be regarded as significant. discussion in the case of an fmd outbreak, rapid identification of the serotypes of fmdv that are responsible for the outbreak is essential for selection of an appropriate emergency vaccine and helps to trace the origin and spread of the outbreak (callens & de clercq 1997; vosloo, bastos & boshoff 2006) as well as wildlife movements (vosloo, bastos, michel & thomson 2001). south africa experienced an outbreak caused by the pan-asian serotype o in 2000 (sangare, bastos, marquardt, venter, vosloo & thomson 2001) that was introduced by the illegal use of swill as pig feed (brückner et al. 2002). this serotype does not occur in the buffalo populations in southern africa and is therefore exotic to the region and had to be eradicated with urgency. the outbreak was finally controlled by stamping out, vaccination using an imported vaccine of known efficacy to the outbreak strain and strict quarantine measures, resulting in severe financial losses to both commercial and small scale farmers in the area. implementation of control measures depends on accurate genetic characterization of the virus strain responsible for an outbreak. phylogenetic analysis of the sat-2 outbreak in 2001 revealed that all 11 virus samples collected from the infected premises clustered in one strongly supported clade, which was related to viruses isolated from buffaloes and impalas living in adjacent wildlife reserves, strongly suggesting that wildlife was the origin of the outbreak (fig. 1). this clade has previously been shown to belong to a topotype that covers the geographical regions of north eastern south africa (knp), south eastern zimbabwe and mozambique (bastos et al. 2003b). the same geographic areas form a topotype for sat-1 (bastos et al. 2001) and sat-3 (bastos et al. 2003b), respectively. the isolates from zambia (zam/7/96 and zam/10/96) clustered together as did viruses from botswana (bot/18/98) and zimbabwe (zim/267/98), in accordance with their previous description into two separate topotypes, albeit with overlapping distribution ranges (bastos et al. 2003b). the knp/ south zimbabwe/mozambique topotype differs significantly from other topotypes in the region and the clustering profile of the outbreak isolates within this southern topotype, together with the outbreak’s proximity to the knp, is a strong indication that the virus originated in the knp. this hypothesis is supported by the observation that fencing, constructed to prevent the co-migration of wildlife and cattle in the areas adjacent to the national park, was washed away, and had not been repaired at the time of the outbreak and that buffaloes were observed in the farming areas. the outbreak occurred in the buffer zone without vaccination and animals were therefore fully susceptible to fmd infection. although the first cases were found on 1 february 2001, the age of lesions on certain farms suggested that the outbreak had been ongoing for some time prior to that (e. dyason, personal communication 2001). this could explain why a variant (sar/1/01) was found on the farm ori noco at day 1. after diagnosing the outbreak, cat tle were vaccinated during the weeks of 5 february and 5 march 2001 with a trivalent vaccine containing sat-1, sat-2 and sat-3 strains. therefore, all the isolates acquired between days 1–6 were most probably from unvaccinated animals and no immune pressure was present. the vaccination cover at craigieburn was limited since the area is mountainous and it was difficult to find the animals during the vaccination campaigns. it was therefore not surprising when fresh lesions were found at day 111 (22 may 2001), most probably in unvaccinated animals (information kindly supplied by e. dyason). although no samples were characterized between days 6 and 111, it is clear that the disease had been spreading between farms in the intervening period. the antigenic relationship of the outbreak virus with the vaccine was acceptable with r1-values of 0.4 and 0.35 with the two vaccine strains, respectively (j.j. esterhuysen, personal communication 2002). it was previously shown that a sat-1 vaccine with similar r1-values to the outbreak strain protected pigs from needle infection (cloete, dungu, van staden, ismail-cassim & vosloo 2008). there is therefore no reason to believe that the vaccine was not protective and the outbreak was controlled using vaccination and movement restrictions. the last clinical cases were observed on 30 may 2001. 275 r.m. phologane et al. the 11 cattle outbreak isolates in this study accumulated 12 independent mutations (or changes at 1.85 % of sequenced sites) (fig. 3) over 4 months, which is rapid compared to two previous studies of genetic change in buffaloes. vosloo et al. (1996) showed a substitution rate of 1.64 % per year over a similar genetic region for a sat-2 virus in carrier buffaloes and vosloo et al. (2007) estimated a rate of 1.1 % per year in an outbreak of sat-1 in buffalo that also led to the establishment of a carrier state. it is not known whether fmdv might evolve faster in cattle than buffaloes, or whether the faster rate of change observed in this study might have arisen as a result of vaccine induced selection pressure. it may also indicate that rates increase during transmission events compared to sequential isolates from carrier animals. the proportion of these substitutions that were non-synonymous is comparable between this study, 5/12, versus 15/25 in vosloo et al. (1996). interestingly, two of the observed non-synonymous changes (pos 135 in sar/1/01 and 201 in sar/6/01) are at sites previously identified to be subject to positive selection (haydon et al. 2001a). when a separate dn:ds ratio is estimated for branches of the phylogeny associated with the recent outbreak in cattle, our analysis revealed it to be significantly higher (by a factor of 4) compared to that arising over the rest of the phylogeny. such an increase may have occurred as a result of the change of host (from buffalo to cattle), or the presence of vaccine selection, but it may also have arisen as a result of the different time-scales over which viral isolates were collected (111 days versus 13 years). all the isolates in this study were first propagated on cell culture to obtain sufficient material for amplification and sequencing (table 1). this could have led to selection of variants that are better adapted to cell culture growth and could have an impact on the results. however, all isolates were subjected to the same regime of being isolated initially on one passage of primary pig kidney cells, while sar/1/01, sar/7/01 and sar/10/01 were also passaged once on ibrs-2 cells and sar/11/01 was passaged twice on these cells. no nucleotide changes specific to the viruses passaged on ibrs-2 were observed (results not shown) and the passage history should therefore not influence the results of this study. three of the amino acid changes are in known antigenic regions, sar/1/01 at position 135 within the gh loop and sar/6/01 (position 201) and sar/8/01 (position 211) within the c-terminal region. we have not investigated antigenic differences using these cell passaged viruses to determine whether any of the changes lead to new antigenic variants. it was previously shown that genetic and antigenic changes could occur after cell passage but that the virus could also be remarkably stable over many cell passages (domingo, dávila & ortín 1980; gonzalez, saiz, laor & moore 1991; meyer, pacciarini, hilyard, ferrari, vakharia, donini, brocchi & molitor 1994). this study has demonstrated that nucleotide substitutions can arise sufficiently quickly in the 1d gene of fmdv and that genetic data can provide high resolution information on the disease transmission between farms. information of this sort has high potential value in identifying elusive transmission routes of disease. acknowledgements we honour billy phologane who sadly passed away before the paper could be published. we want to express our sincere gratitude to dr edwin dyason for providing the field information for this paper and for critically reading the manuscript. mr jan esterhuysen provided the r1-values of the outbreak isolate. references bastos, a.d.s. 1998. detection and characterisation of footand-mouth disease virus in sub-saharan africa. onderstepoort journal of veterinary research, 65:37–47. bastos, a.d., anderson, e.c., bengis, r.g., keet, d.f., winterbach, h.k. & thomson, g.r. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice abstract introduction research method and design results discussion conclusion acknowledgements references about the author(s) alicia cloete south african field epidemiology training programme, national institute for communicable diseases, university of pretoria, south africa the school for health systems and public health, university of pretoria, south africa department of agriculture, forestry and fisheries, animal health, pretoria, south africa cornelia gerstenberg department of agriculture, forestry and fisheries, animal health, pretoria, south africa natalie mayet south africa regional global disease detection centre, national institute for communicable diseases, south africa stefano tempia center for respiratory diseases and meningitis, national institute for communicable diseases, south africa influenza division, centers for disease control and prevention, united states of america influenza program, centers for disease control and prevention, pretoria, south africa citation cloete, a., gerstenberg, c., mayet, n. & tempia, s., 2019, ‘brucellosis knowledge, attitudes and practices of a south african communal cattle keeper group’, onderstepoort journal of veterinary research 86(1), a1671. https://doi.org/10.4102/ojvr.v86i1.1671 original research brucellosis knowledge, attitudes and practices of a south african communal cattle keeper group alicia cloete, cornelia gerstenberg, natalie mayet, stefano tempia received: 23 july 2018; accepted: 19 dec. 2018; published: 18 feb. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract brucellosis remains an animal and public health concern in south africa, given the intensity and widespread distribution of outbreaks in cattle. we conducted a cross-sectional survey among cattle keepers in the whittlesea community of the eastern cape province of south africa, which utilises communal grazing. individual cattle keepers (n = 227) who attended prearranged meetings in selected villages were interviewed using a structured questionnaire to assess their knowledge, attitude and practices (kap) regarding bovine brucellosis. we compared kap scores between previous brucellosis-affected villages and unaffected villages. we compared attitude and practices scores between those who had heard of brucellosis and those who had not and between those above the 75th percentile knowledge score and those below. the kap for the study population were described using frequency tables. scores of different groups were compared using the welch t-test or the wilcoxon rank-sum test. knowledge scores of those who had heard of brucellosis (60%) showed a bimodal distribution with a 0/18 primary peak and 5–6/18 secondary peak. attitude scores showed a median of 7/14 (interquartile range [iqr] 6–9), with 98% requesting more information on brucellosis. practices scores showed a median of 6/18 (iqr 3–8), with high-risk practices identified that could facilitate brucellosis transmission. there were significant differences in attitude and practices scores between the groups above and below the 75th percentile knowledge score. the community showed poor knowledge, poor to average practices and average to good attitude. identified high-risk practices highlight the risk of potential introduction and transmission of brucellosis between cattle and zoonotic transmission to humans. introduction brucellosis has been identified as a neglected tropical disease by the world health organization (who 2006). globally, it is regarded as the most common zoonotic infection among humans and remains endemic in many countries (franco et al. 2007; pappas et al. 2006). in resource-limited countries, communities and individuals who rely on livestock keeping for their livelihood are more at risk of zoonotic exposure because of close contact with livestock, and they may be less likely to be diagnosed and treated correctly (marcotty et al. 2013; who 2006). knowledge on brucellosis and the impact thereof is thought to be lacking among livestock farmers across all levels of production in south africa (department of agriculture, forestry & fisheries [daff] 2017; national department of agriculture [nda] 1997). literature review brucellosis background brucella abortus mainly affects cattle, which clinically may present with abortion, reduced fertility, decreased milk production, orchitis and joint problems (olsen & tatum 2010; who 2006). brucella abortus may be transmitted to humans mainly through the ingestion of non-pasteurised dairy products and through direct contact with birth material from infected cattle (olsen & tatum 2010; zinsstag et al. 2007). awareness of the disease, vaccination and testing of cattle, quarantine of infected herds and slaughter of positive reactors are crucial aspects of brucellosis control in cattle (godfroid et al. 2004; olsen & tatum 2010). zoonotic transmission should be mitigated mainly through disease control in livestock (olsen & tatum 2010) and also through the pasteurisation of milk (who 2006; zinsstag et al. 2007). brucellosis in south africa a recent publication by the south african daff stated that there is currently great concern for brucellosis transmission given the intensity and widespread distribution of outbreaks in cattle throughout the country (daff 2017). established brucellosis control measures in south africa are currently undergoing discussion and revision to optimise collaboration between government, industry and farming communities (daff 2017). south african legislation states that all heifers between the ages of 4 and 8 months should be vaccinated with an efficient remedy (national department of agricultural economics and marketing 1986), of which the b. abortus strain 19 and rb51 vaccines are currently registered (daff 2016). livestock farmers are responsible for ensuring that their cattle herds are vaccinated and government veterinary services assist farmers where resources allow (daff 2017). at the moment, most cattle farmers are at risk of acquiring brucellosis-positive cattle in their herds, as there is generally poor compliance with brucellosis vaccination requirements, and testing is not currently compulsory for all cattle (daff 2017). furthermore, a lack of proper fencing, mixing of animal groups and communal grazing practices create potential risks of spread among livestock in areas where communal grazing is practised (alusi 2014; nda 1997). regarding zoonotic transmission, human cases of brucellosis are considered under-diagnosed and under-reported in the country, as is the case in many resourced-limited countries where brucellosis is endemic in cattle (wojno et al. 2016). brucellosis knowledge, attitude and practices several brucellosis knowledge, attitude and practices (kap) studies have been conducted and published in other resource-limited countries including tajikistan, jordan, egypt, uganda, nigeria and kenya. knowledge, attitude and practices surveys provide critical and relevant information that help to explore potential risk factors, as well as potential intervention and prevention strategies for disease. studies conducted in uganda (kansiime et al. 2014; nabirye et al. 2017), kenya (obonyo & gufu 2015), jordan (musallam, abo-shehada & guitian 2015), nigeria (buhari et al. 2015) and tajikistan (lindahl et al. 2015) highlighted the need for brucellosis education for improved prevention, management and control of the disease. each study showed particular kap trends and highlighted the importance of such studies to understand country-specific circumstances in order to address specific shortcomings. statement of the problem there is currently a paucity of information on brucellosis in humans and livestock in south africa, as well as on the brucellosis kap in different farming systems, including communal grazing settings. the current risk of community members acquiring the disease from communal livestock is unknown and may only be extrapolated from the information available from other countries. a better understanding of the kap regarding brucellosis could increase the understanding of brucellosis risk factors for cattle and humans, influence local awareness programmes and guide policy on brucellosis control interventions (lindahl et al. 2015). objectives we aimed to determine the current kap of cattle keepers regarding bovine brucellosis, in the whittlesea community of the eastern cape province of south africa where communal grazing of cattle is practiced. we also sought to compare the kap of cattle keepers between previously brucellosis-affected and unaffected villages; to establish if there was any significant difference between attitude and practices scores between those who had heard and not heard of brucellosis previously, as well as between those who scored above the 75th percentile knowledge score and those who scored below. contribution to field this study was conducted to gather critical information that is important for the understanding of potential zoonotic transmission of bovine brucellosis in communal farming settings in south africa. the information obtained may be utilised to guide local awareness programmes and policy on disease control interventions to promote both cattle and human health. research method and design setting the whittlesea community consists of 48 villages that are located in the lukhanji local municipality of the chris hani district in the eastern cape province of south africa (figure 1), with an estimated human population of 56 500 in 2011 (statistics south africa). communal land lies in between the scattered villages where sheep, goats and cattle from adjacent villages are grazed together. the whittlesea community is serviced by the local queenstown state veterinary office, which is part of the eastern cape province government veterinary services. during 2008–2009, eastern cape province government veterinary services succeeded in eradicating b. abortus from a communal cattle population after an outbreak occurred in the lukhanji municipal area (fisher 2010). nine out of the forty-eight villages of the whittlesea community had laboratory-confirmed brucellosis-positive cattle in the 2008–2009 outbreak, and extensive awareness campaigns were conducted throughout the area (fisher 2010). because of an intense and timeous disease control response, further spillover to cattle that originated from other villages was prevented. brucellosis eradication from the affected villages was achieved through a combination of testing, slaughtering, vaccination, retesting and movement control measures. figure 1: location of the lukhanji municipal area of the chris hani district in the eastern cape province of south africa. design we conducted a cross-sectional survey using a structured questionnaire to investigate kap regarding brucellosis and associated risk factors among cattle keepers (representing their households) in the whittlesea community. this community was selected for the study given the communal grazing setup, its history of bovine brucellosis infection and the existing regular interaction between livestock keepers and local government veterinary services. study population and sampling strategy the cattle keeper population of the whittlesea community was extrapolated from official vaccination records. in 2016, the queenstown state veterinary office vaccinated 20 419 cattle against anthrax, and we estimated an average of 10 cattle per owner, leading to a calculated population size estimate of 2042 cattle keepers. these cattle and cattle keepers originated from the 48 villages of whittlesea community but were considered as a single epidemiological unit because of free movement and intervillage contact. the required study sample size was calculated as 323 cattle keepers, assuming a 50% proportion of any investigated kap attributes at 95% confidence intervals and 5% precision, using epi info™ 7 (centers for disease control and prevention, ga, united states of america [us]). the whittlesea community was approached as a unit through a meeting with the village and community leaders. through queenstown state veterinary office we established that the most effective and efficient means of sampling would be to conduct cattle keeper meetings, using the villages as conjugation points. we selected a total of 18 of the 48 villages (37.5%) of the whittlesea community: all villages that had laboratory-confirmed brucellosis-positive cattle during the 2008–2009 outbreak were purposefully selected (nine in total), and nine previously negative villages were selected through simple random sampling without replacement, using microsoft® excel 2010 (microsoft corporation, redmond, wa, us). the presumed negative villages tested negative for brucellosis during the 2008–2009 outbreak and during subsequent years. the meeting venues were selected for convenience to enable maximum contact with cattle keepers from the whittlesea community with the available resources. for each of the 18 participating villages, all eligible cattle keepers who attended prearranged cattle keeper meetings were interviewed. an estimation of a good attendance of approximately 20 cattle keepers per meeting was envisioned to meet the calculated sample size. procedure a pretested kap survey questionnaire consisting of 49 questions (some with additional subquestions) was used as the data collection tool. it was divided into five sections: (1) demographics; (2) knowledge on brucellosis as a disease; (3) attitudes towards animal health, human health and brucellosis; (4) self-reported practices relating to meat and dairy product consumption; and (5) self-reported practices relating to cattle husbandry. the questionnaire contained binary, multiple selection and open-ended questions. we adapted the questionnaire from a brucellosis kap survey conducted in tajikistan (lindahl et al. 2015) to fit a rural south african context. we also added questions to capture necessary information on human health-seeking behaviour, communal grazing practices and information transfer routes. (the full questionnaire is available from the author or publisher). the team of interviewers consisted of five animal health technicians (ahts) from the queenstown state veterinary office who were trained by the principal investigator to successfully interview participants using the questionnaire. the ahts also pretested the draft kap survey questionnaire on five random cattle keepers during the initial site visit in 2016 before the questionnaire was further adapted and finalised for use. the questionnaires were translated verbally into xhosa by the interviewers as required. two teams consisting of two to three interviewers conducted two meetings per day to cover the 18 villages during a specified 5-day study period in 2017. cattle keepers originating from the selected villages were interviewed. all interviewees were aged 18 years and older and able to communicate verbally in either english or xhosa. a cattle keeper was referred to as a person who owns cattle or a person responsible for the cattle on the owner’s behalf. cattle keeper meetings were organised in the selected villages on prearranged dates and times. participants were consecutively enrolled in the study and interviewed as they arrived at the central meeting point. afterwards an informative talk on brucellosis was conducted with a question-and-answer session. analyses questionnaire responses were captured and coded using stata version 14 (statacorp, college station, tx, usa). missing data were excluded for the kap analyses. a denominator was thus defined for each individual question to illustrate the total number of responses received and recorded for the kap questionnaires. descriptive statistics were used for demographic characteristics and brucellosis kap of the respondents. we allocated scores to specific questions in the kap survey questionnaire following a scoring sheet developed by brucellosis experts of daff. (the scoring sheet is available from the author or publisher.) for the knowledge section, we distinguished between participants who stated they had heard of brucellosis previously and those who had not. only the participants who indicated they had heard of brucellosis were further assessed in the knowledge section, and a maximum score of 18 could be achieved from 9 questions. a participant could achieve a maximum score of 14 from 9 questions in the attitude section and a maximum score of 18 from 11 questions in the practices section. a higher score would imply better attitude and practices, respectively. no previous benchmark scores were available for comparison. in addition, we categorised the respondents’ level of knowledge, attitude and practices, respectively, as high or moderate to low using the 75th percentile of the individual scores for further analysis. the welch t-test or the wilcoxon rank-sum test was used to assess differences between knowledge, attitude and practices scores, respectively, between brucellosis-affected and unaffected village groups. the welch t-test or the wilcoxon rank-sum test was also used to assess differences between attitude and practices scores between those who had heard and not heard of brucellosis previously, as well as between those who scored above and those below the 75th percentile knowledge score. stata version 14 was used to perform statistical analyses. potential benefits and hazards no harm was associated with collecting information on kap regarding brucellosis and associated risk factors. the potential gains included using the opportunity to also educate the community regarding brucellosis during the contact sessions. recruitment procedures participants were recruited on a voluntary basis as they arrived to attend prearranged cattle keeper meetings. informed consent an information sheet and informed consent document was used by the interviewers to explain the study process and purpose and to obtain consent. the study was voluntary and anonymous. data protection questionnaires were administered once off, and the participants’ names and contact details were not collected. a unique identifier was assigned to each participant and used as a study number to aid data collection and capturing. the completed questionnaires and the consent forms have been stored securely by the principal investigator. the electronic database has been securely stored by the principal investigator, and only co-authors have access to the database. results socio-demographic characteristics of respondents a total of 227 cattle keepers were interviewed during the study period, 73% of whom were male (table 1). the median age of cattle keepers was 62 years (interquartile range [iqr] 51–71 and range of 21–93). the median number of household members was 4 (iqr 3–5 and range of 1–11). in terms of household education level, 41.6% (94/226) of respondents indicated some level of primary school, 46.0% (104/226) some level of secondary school and 12.0% had completed tertiary education. the predominant language in the households was xhosa (99.1%). the median travel distance to a healthcare facility was 1 km (iqr 1–4 and range of 0–40). the median number of cattle kept by each household was 10 (iqr 5–19 and range of 1–313). many of the cattle keeper households also owned goats (63%) and/or sheep (55%). table 1: selected socio-demographic characteristics of cattle keeper households in the whittlesea community, south africa, 2017. knowledge on brucellosis as a disease in cattle and in humans out of 227 participants, 136 (59.9%) indicated they had heard of brucellosis previously. table 2 shows the responses to some of the knowledge questions of these 136 participants. the main sources of information on brucellosis were veterinary services (53.7%; 73/136), community gatherings or talks (17.6%; 24/136), friends or family members (13.2%; 18/136) and radio or television (11.0%; 15/136). the majority of respondents reported having heard of brucellosis a year ago, with a noticeable peak detected for a response of 8–9 years ago as well. table 2: knowledge of brucellosis among participants that reported having heard about the disease, whittlesea community, south africa, 2017 (reflecting the outcome of some of the knowledge questions). when asked which animals could become infected with b. abortus, 50.0% (65/130) of respondents did not know, 36.9% (48/130) stated cattle, 23.8% (31/130) sheep and goats and 17.7% (23/130) all animals. when asked about the transmission of brucellosis in cattle, 68.8% (88/128) of respondents did not know and 19.5% (25/128) stated abortion. participants reported the following as signs of brucellosis in cattle: 19.2% (24/125) abortion and 12% (15/125) weak calves, whereas 78.4% (98/125) of respondents did not know. only 16.8% (22/131) of respondents knew that humans could become infected with brucellosis. among these respondents, drinking raw milk (66.7%; 12/18), assisting with calving or handling placenta (22.2%; 4/18), slaughtering an infected animal (16.7%; 3/18) and handling an abortion (11.1%; 2/18) were reported as possible means of zoonotic transmission. when asked about the symptoms of brucellosis in humans, 87.3% (103/118) of participants did not know, and 8.5% (10/118) cited fever. the fact that brucellosis is a government-controlled disease was acknowledged by 55.6% (74/133) of respondents, and 50.0% (67/134) of respondents were aware of a brucellosis vaccine for cattle. the chronic nature of brucellosis in cattle was familiar to 30.1% (40/133) of respondents, whilst 9.0% (12/134) of respondents were aware of the availability of treatment for brucellosis in humans. attitudes towards animal health, human health and brucellosis when asked if they ensured that new cattle were healthy before buying or receiving them, 33.7% (71/211) responded positively (table 3). to ensure cattle health, 37.5% (30/80) of respondents stated that they sought veterinary advice, 28.8% (23/80) bought from people they knew or trusted and 22.5% (18/80) relied on their own experience. when asked what action would be taken if a cow aborted, 46.2% (102/221) of respondents indicated that they would contact veterinary services for help, 21.7% (48/221) did not know what to do and 19.0% (42/221) would do nothing. abortion in cattle was considered a serious condition by 57.8% (130/225) of respondents. when asked if they would get rid of cattle that had a chronic disease, only 20.4% (46/225) of respondents concurred. table 3: responses of participants to attitude questions towards animal health, human health and brucellosis, whittlesea community, south africa, 2017 (reflecting the outcome of some of the attitude questions). the statement ‘raw milk is better and healthier than boiled (pasteurised) milk’ was agreed to by 46.9% (105/224) of respondents, whilst 62.4% (138/221) of respondents agreed to the statement ‘humans can get certain diseases from slaughtering animals’. when asked if participants considered it a serious situation if they could get sick from cattle that have brucellosis, 51.8% (115/222) of respondents did not concur. when asked if a participant would be willing to wear gloves if it helped to prevent disease transmission, 77.4% (123/159) concurred (this question was relocated from the practices section). participants were asked what action they would take if they experienced fever or influenza-like symptoms: 93.2% (204/219) of respondents replied that they would go to a health facility or doctor. the majority of respondents (98.7%; 220/223) stated that they would like to receive more information on brucellosis. the preferred reported methods of communication were as follows: farmers’ day meetings with veterinary services (58.3%; 126/216) and community meetings with veterinary services (54.6%; 118/216). these two options were commonly chosen as participants indicated that they were then able to ask questions as well. only 4.2% (9/216) stated they preferred an information pamphlet, and only 3.2% (7/216) preferred information via radio or television. practices relating to meat and dairy consumption and cattle husbandry milk was consumed by 89.0% (202/227) of respondents and 36.8% (74/201) of respondents stated that they consumed boiled or pasteurised milk exclusively (table 4). milk sources were indicated by respondents as follows: 80.0% (148/185) obtained milk from their own cows, 21.1% (39/185) from commercial stores and 20.5% (38/185) from informal stores. a majority of 84.8% (184/217) respondents indicated that it was practical to boil milk. regarding meat practices, 83.1% (187/225) of respondents indicated that they practised home slaughter of cattle in their household. table 4: responses of participants regarding practices relating to dairy consumption and cattle husbandry, whittlesea community, south africa, 2017 (reflecting the outcome of some of the questions on practices). when asked about cattle origin, 49.1% (109/222) indicated that they had bought their cattle from people in the community, 26.6% (59/222) had obtained their cattle as an inheritance, 16.2% (36/222) had bought their cattle from other communities or areas and 7.2% (16/222) had bought their cattle from commercial farmers. only 7.6% (17/225) of respondents indicated that they had purchased or received new cattle during the last year. in contrast, 60.9% (109/179) of respondents stated that they sold cattle on a regular basis. of these, 75.0% (114/152) of respondents indicated that they sold to local markets or auctions, 42.8% (65/152) sold to people in the community, and only one participant indicated sale directly to an abattoir. moreover, 98.2% (221/225) of respondents indicated that their cattle shared grazing and/or water with other livestock, and 91.5% (205/224) of respondents claimed that they could not keep their cattle separate from other livestock. only 6.9% (15/218) of respondents indicated that they had inquired if cattle had been tested for brucellosis before they bought or received them. regarding assistance during parturition, 21.7% (49/226) stated that a household member had helped to deliver a calf and 22.9% (48/210) of respondents stated that a household member had handled placental membranes. in terms of action that would be taken if an aborted foetus was found, 35.0% (79/226) of respondents stated that they would bury the foetus, and 25.7% (58/226) would give it to dogs. similarly, in terms of action that would be taken if placenta was found, 36.8% (81/220) of respondents stated that they would bury the placenta, and 30.9% (68/220) would give it to dogs. when asked if their cattle were vaccinated against brucellosis, 44.7% (101/226) of respondents stated yes, and 25.2% (57/226) stated that they did not know. when asked if their cattle had been tested for brucellosis before, 47.8% (107/224) of respondents stated yes, and 16.5% (37/224) stated that they did not know. from respondents who indicated their cattle had been tested, 86.8% (72/83) said that their cattle had tested negative, whilst 12.1% (10/83) did not know the testing results. the number of years ago that cattle were last said to be tested followed a bimodal pattern, with 49% (49/100) stating 1–2 years ago and 33% (33/100) stating 8 years ago. knowledge, attitude and practices scores for the knowledge section, 91 out of 227 (40%) participants indicated that they had not heard of brucellosis and were awarded a knowledge score of zero. the overall knowledge score of those who indicated that they had heard of brucellosis showed a bimodal distribution, with a main peak of zero and a second of 5–6 (out of a possible maximum of 18). for all participants, the overall median attitude score (out of a possible maximum of 14) was 7 (iqr 6–9), and the overall median practices score (out of a possible maximum of 18) was 6 (iqr 3–8). knowledge, attitude and practices scores for all participants are illustrated in figure 2, which shows comparative scoring between the three sections. attitude scores were the highest, followed by practices scores and then knowledge scores. figure 2: boxplot showing knowledge, attitude and practices score distribution for communal cattle keepers of the whittlesea community, south africa, 2017. comparison of scoring between different groups no significant difference in the knowledge (wilcoxon rank-sum test: p-value = 0.786), practices (wilcoxon rank-sum test: p-value = 0.405) and attitude (welch t-test: p-value = 0.553) scores was observed between previously brucellosis-positive and -negative villages. there was no significant difference in the attitude (welch t-test: p-value = 0.072) and practice (wilcoxon rank-sum: p-value = 0.137) scores between respondents who had heard about brucellosis previously and those who had not. there were statistically significant differences in the attitude (welch t-test: p-value = 0.041) and practices (wilcoxon rank-sum: p-value = 0.007) scores between respondents above and those below the 75th percentile of knowledge scores. trustworthiness reliability the structured kap survey questionnaire was piloted prior to use during a previsit to the whittlesea community. after the ahts received training on questionnaire administration and data collection, a copy of the questionnaire was given to each to interview a random cattle keeper in the community. after completion of this exercise, the questionnaire was edited based on the findings and additional inputs provided by the ahts. prior to official interviews and data collection, the ahts received repeat training by the principle investigator to promote uniformity of the interview and recording process. validity all eligible cattle keepers who attended the prearranged meetings were interviewed. the overall turnout to the meetings was rated satisfactory by the ahts who normally service the villages. overall, only 227 cattle keepers were successfully interviewed, compared to the calculated sample size of 323. the smaller sample size may impact our power to detect ‘real’ differences. discussion approximately 60% of respondents stated that they had heard of brucellosis previously. our overall findings on the cattle keepers of the whittlesea community showed that the knowledge levels were poor, resulting in poor to average practice scores regarding brucellosis, whilst the attitude score tended to be average to good. there were significant differences between the attitude (p-value = 0.041) and practices (p-value = 0.007) scores of the groups above and those below the 75th percentile knowledge score. this finding may suggest that poor practices were a result of poor knowledge rather than poor attitude in the study population. the kap scores did not differ significantly between previously brucellosis-affected and unaffected villages, confirming the initial assumption that there were no significance differences between the villages (sampling sites). furthermore, the attitude and practices scores did not differ significantly between the groups who had heard of brucellosis previously and those who had not, suggesting that, to a certain extent, attitude and practices may be a result of community or cultural traits that are not influenced merely by individual awareness of brucellosis. a relatively high number of participants stated they had heard of brucellosis previously (60%). similar results were seen in brucellosis kap studies conducted in northern uganda (nabirye et al. 2017) and kenya (obonyo & gufu 2015), where 63% and 79% of community participants had heard of brucellosis, respectively. brucellosis kap studies conducted in egypt (holt et al. 2011), nigeria (buhari et al. 2015), uganda (kansiime et al. 2014) and jordan (musallam et al. 2015) showed that 83%, 93%, 99.3% and 100% had heard of brucellosis, respectively. contrasting results were found in a brucellosis kap study in tajikistan, where only 15% had heard of brucellosis (lindahl et al. 2015). the main source of brucellosis information was stated as unspecified media in the jordan study (musallam et al. 2015), community health workers in the kenya study (obonyo & gufu 2015), parents in the nigeria study (buhari et al. 2015) and friends or family members in the tajikistan study (lindahl et al. 2015). of particular interest in the whittlesea community was that most had heard of brucellosis through veterinary services, indicating the importance of the role of government veterinary services in this regard. the bimodal distribution of when the participants had last heard of brucellosis probably reflected yearly interaction with government veterinary services on brucellosis and the dedicated awareness campaign during the last outbreak in 2008–2009. the low overall knowledge scores of participants pointed toward a lack of detailed information and/or ineffective information transfer. participants generally knew what government was doing regarding brucellosis, namely control and vaccination, but they lacked more in-depth knowledge with particular reference to zoonotic implications and disease prevention. poor knowledge of brucellosis in humans specifically needs to be addressed in future awareness and education campaigns. poor overall knowledge scoring was also reported in the tajikistan (lindahl et al. 2015), northern uganda (nabirye et al. 2017) and nigeria (buhari et al. 2015) brucellosis kap studies, with poor to average knowledge reported by the kenya study (obonyo & gufu 2015) and good knowledge scoring reported by the jordan (musallam et al. 2015) and egypt studies (holt et al. 2011). the tajikistan (lindahl et al. 2015), kenya (obonyo & gufu 2015), egypt (holt et al. 2011) and jordan (musallam et al. 2015) studies showed good knowledge on brucellosis as a zoonosis, whilst the nigeria study (buhari et al. 2015) showed poor knowledge in this area. we established that there were opportunities for improvement regarding attitude toward zoonotic disease transmission through slaughter practices and benefits of pasteurised or boiled milk consumption. correct information on these practices is necessary to equip community members to safeguard themselves. attitude toward the importance of abortion and especially chronic disease in cows also showed avenues for improvement, which would facilitate the identification of disease in these animals. the findings of human health-seeking behaviour patterns and access to human healthcare facilities showcased the importance of healthcare professionals, who should be equipped to enable identification and treatment of brucellosis in humans. respondents showed a very positive attitude toward receiving more information on brucellosis, and it is critical for the relevant authorities to take note of preferred means of communication. the overall attitude scores of participants were average to good in the whittlesea community. similarly, the brucellosis kap study conducted in northern uganda (nabirye et al. 2017) showed a very positive attitude among community participants. in contrast, the kenya study (obonyo & gufu 2015) found an unfavourable attitude among community participants. in the tajikistan brucellosis kap study (lindahl et al. 2015), 63% of respondents requested more information on brucellosis, preferring an educational booklet; in contrast, the kenya study (obonyo & gufu 2015) found that 97% requested more information and preferred the local fm radio stations for information transfer. these different information-channel preferences, as well as the different sources of current information, highlight the importance of establishing how a target community wants to be reached prior to conducting awareness or education campaigns. several high-risk practices were identified. even though a greater sales trend was observed compared to new acquisitions of cattle, there was a lack of proper health investigation in purchased and received cattle, adding to the risk of brucellosis entry into the communal cattle population. this risk was further exacerbated by the overall disease transmission risk of the communal nature of grazing cattle. this highlights the importance of treating communal cattle as a single epidemiological unit and aiming disease prevention measures at keeping the communal herd protected against disease introductions. this would subsequently serve to prevent spillover to the human population. similar high-risk practices of untested introductions and mixing with potentially infected cattle on communal grazing lands have been identified in kenya (obonyo & gufu 2015), nigeria (buhari et al. 2015) and uganda (kansiime et al. 2014). potential zoonotic transmission risks were identified that could have led to brucellosis in humans if present in the cattle population. raw milk was still being consumed, which could potentially be decreased through educating people on the benefits of boiling milk. most households reported home slaughter of livestock. slaughtering of brucellosis-infected cattle is considered a high-risk activity, as the persons conducting slaughter and handling contaminated meat may be exposed if precautions are not taken (galinska & zagórski 2013; sadler 1960). education on the proper handling and disposal of placenta and aborted foetuses is required to decrease potential human exposure to and environmental contamination with brucellosis. the positive attitude shown towards the use of gloves for protection should be noted by the relevant authorities and cattle keepers should take responsibility for using gloves as a measure of protection. the practices results reflected a lack of effective communication during contact sessions between cattle keepers and government veterinary services in terms of what cattle were vaccinated and tested for. the ongoing brucellosis vaccination and testing of cattle creates an ideal opportunity to promote brucellosis awareness and to convey important information. the impact of the intense 2008–2009 brucellosis awareness and testing campaigns was reflected in the bimodal pattern of testing history, as stated by respondents, as well as the feedback on when respondents had last heard of brucellosis. the overall practice scores of respondents were poor to average, with several high-risk behaviours identified in this community. brucellosis kap studies conducted in tajikistan (lindahl et al. 2015), kenya (obonyo & gufu 2015), northern uganda (nabirye et al. 2017), jordan (musallam et al. 2015), egypt (holt et al. 2011) and nigeria (buhari et al. 2015) also revealed high-risk activities, including the handling of cattle birth material without protection. tajikistan (lindahl et al. 2015), kenya (obonyo & gufu 2015) and northern uganda (nabirye et al. 2017) also reported the consumption of unpasteurised dairy products as a high-risk activity, whilst in the jordan (musallam et al. 2015) and egypt (holt et al. 2011) studies the majority of participants boiled milk before consumption but not always before making cheese. limitations only 227 interviews out of the 323 (70%) calculated sample size were achieved. the lack of accurate recorded information on cattle and cattle keepers limited the validity of the sample size calculation. however, according to the local ahts, the turnouts to the meetings were good compared to what is normally experienced. cattle keepers who attended our meetings may have been more exposed to information compared to cattle keepers who do not routinely attend meetings. recall bias may have occurred if participants had difficulties to recall information shared during previous information campaigns. whereas the scoring system was developed and agreed upon by brucellosis experts of daff, the assigned questions’ scores may have been impacted by the perceived importance of kap in the local context. recommendations the findings of this study contain useful information to understand the communal farming setting in the whittlesea community that could be used to influence the approach to local brucellosis control and prevention strategies. targeted, consistent brucellosis awareness and information campaigns could address the community’s high-risk practices by increasing their knowledge through utilising their positive attitudes and heeding the request for brucellosis information. the number of similarities and differences described between this study and other brucellosis kap studies from different countries further highlights the importance of establishing kap in local settings. existing contact sessions between government veterinary services and cattle keepers could be utilised optimally to create awareness of brucellosis and to provide relevant information on animal and human health and disease prevention. information transfer would probably be more efficient if provided through methods that are acceptable to the community, which in this study was indicated as community or farmer’s meetings with government veterinary services. it would be of benefit if particular attention could be paid to improving knowledge of the community on brucellosis as a zoonotic disease and to ensuring that healthcare practitioners are equipped to identify and treat the disease. it is also useful to note that relative findings of this study could also be applicable to other zoonoses present in the country that have similar risk factors, for example mycobacterium bovis. similar kap studies could be used for the whole country to determine baseline kap for different communities prior to embarking on awareness and education campaigns in order to adapt the approach and content thereof to optimise efficient and effective information transfer. knowledge, attitude and practices studies could then subsequently be repeated as an evaluation tool to determine the effectiveness of conducted awareness and education campaigns. conclusion in conclusion, the cattle keepers of the whittlesea community showed poor knowledge, poor to average practices and average to good attitude pertaining to brucellosis and related factors. bovine brucellosis is currently prevalent throughout south africa, leaving this community at risk of introducing brucellosis into the cattle population, as new cattle brought in from other locations are not always tested, and test results are generally not requested. if brucellosis-positive cattle enter the communal cattle population, there is a risk of spread between cattle as a result of communal grazing practices and a lack of fencing. this highlights the importance of treating communal cattle as a single epidemiological unit regarding disease prevention, detection and control. community members are at risk of acquiring brucellosis if the disease is present in the cattle population through several high-risk practices, including consumption of raw milk and the handling of cattle birth material. this kap study provides necessary information to address shortcomings in knowledge and practices within the study area to improve both animal and human health. acknowledgements the authors acknowledge the valuable assistance of the queenstown state veterinary office in the organisation and application of the kap interviews. they also acknowledge the manuscript reviews performed by prof. lucille blumberg (national institute for communicable diseases) and dr bernice harris (university of pretoria). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.c. was the primary investigator. c.g. and n.m. supervised the project design and write-up. s.t. provided statistical support for the analysis. disclaimer the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the relevant institutions or funder. references alusi, p.m., 2014, 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https://doi.org/10.7196/samj.2016.v106i9.11020 world health organization (who), 2006, the control of neglected zoonotic diseases: a route to poverty alleviation, who, geneva. zinsstag, j., schelling, e., roth, f., bonfoh, b., de savigny, d. & tanner, m., 2007, ‘human benefits of animal interventions for zoonosis control’, emerging infectious diseases 13(4), 527–531. https://doi.org/10.3201/eid1304.060381 abstract introduction history of peste des petits ruminants research in africa global recognition of peste des petits ruminants peste des petits ruminants in africa research on peste des petits ruminants in africa recommendations conclusion acknowledgements references about the author(s) samuel e. mantip department of animal health and production, university of abomey-calavi, abomey calavi, benin viral research division, national veterinary research institute, vom, nigeria david shamaki viral research division, national veterinary research institute, vom, nigeria souabou farougou department of animal health and production, university of abomey-calavi, abomey calavi, benin citation mantip, s.e., shamaki, d. & farougou, s., 2019, ‘peste des petits ruminants in africa: meta-analysis of the virus isolation in molecular epidemiology studies’, onderstepoort journal of veterinary research 86(1), a1677. https://doi.org/10.4102/ojvr.v86i1.1677 review article peste des petits ruminants in africa: meta-analysis of the virus isolation in molecular epidemiology studies samuel e. mantip, david shamaki, souabou farougou received: 01 aug. 2018; accepted: 22 oct. 2018; published: 26 mar. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract peste des petits ruminant (ppr) is a highly contagious, infectious viral disease of small ruminant species which is caused by the peste des petits ruminants virus (pprv), the prototype member of the morbillivirus genus in the paramyxoviridae family. peste des petits ruminant was first described in west africa, where it has probably been endemic in sheep and goats since the emergence of the rinderpest pandemic and was always misdiagnosed with rinderpest in sheep and goats. since its discovery ppr has had a major impact on sheep and goat breeders in africa and has therefore been a key focus of research at the veterinary research institutes and university faculties of veterinary medicine in africa. several key discoveries were made at these institutions, including the isolation and propagation of african ppr virus isolates, notable amongst which was the nigerian pprv 75/1 that was used in the scientific study to understand the taxonomy, molecular dynamics, lineage differentiation of pprv and the development of vaccine seeds for immunisation against ppr. african sheep and goat breeds including camels and wild ruminants are frequently infected, manifesting clinical signs of the disease, whereas cattle and pigs are asymptomatic but can seroconvert for ppr. the immunisation of susceptible sheep and goats remains the most effective and practical control measure against ppr. to carry out ppr vaccination in tropical african countries with a very high temperature, a thermostable vaccine using the rinderpest lyophilisation method to the attenuated nigeria 75/1 ppr vaccine strain has been developed, which will greatly facilitate the delivery of vaccination in the control, prevention and global eradication of ppr. apart from vaccination, other important questions that will contribute towards the control and prevention of ppr need to be answered, for example, to identify the period when a susceptible naïve animal becomes infectious when in contact with an infected animal and when an infectious animal becomes contagious. keywords: peste des petits ruminants virus; lineages; isolates; molecular; epidemiology; vaccine; sheep; goat. introduction peste des petits ruminant (ppr) is a highly contagious, infectious viral disease of small ruminants species which is caused by the peste des petits ruminants virus (pprv), the prototype member of the morbillivirus genus in the family paramyxoviridae and the order mononegavirales (gibbs et al. 1979). currently there are seven known members of the genus morbillivirus, which include the measles virus (mv), rinderpest virus (rpv), pprv, canine distemper virus (cdv), phocine distemper virus (pdv), cetacean morbillivirus (cemv) and feline morbillivirus (fmv) (woo et al. 2012). cemv is further grouped into three genetically distinct viruses: the porpoise morbillivirus (pmv), pilot whale morbillivirus (pwmv) and dolphin morbillivirus (dmv). peste des petits ruminants virus has a linear negative-stranded rna genome that consists of 15 948 nucleotides and the pprv genome that carries six transcriptional units or genes; each encodes for a contiguous and non-overlapping protein except the p gene, which also expresses c and v non-structural proteins (mahapatra et al. 2003). the six pprv genes encode six structural proteins, namely, the nucleocapsid protein (n), phosphoprotein (p), fusion protein (f), matrix protein (m), hemagglutinin-neuraminidase protein (hn), large protein (l) and two non-structural proteins, namely c and v, in the order of 3’-n-p/c/v-m-f-hn-l-5’ (bailey et al. 2005; mahapatra et al. 2006; (rahaman et al. 2003). the molecular epidemiology of the ppr virus, based on the sequence comparison of a small region of the f gene (322 nt [nucleotideprotein or basepair]; forsyth & barrett 1995), the n gene (255 nt; couacy-hymann et al. 2002) or the h gene (298 nt; kumar et al. 2014), has defined the existence of four distinct lineages, that is, lineages i–iv of the virus (banyard et al. 2010; dhar et al. 2002; kwiatek et al. 2011; libeau, diallo & parida 2014; shaila et al. 1996). the nomenclature of lineages i and ii is slightly different for analyses depending on whether the n or f gene is being considered (lineage ii of the f gene data is considered to be lineage i based on n gene data and vice versa). however, recently a comparison has been made between genetic data derived from the f, n and h genes and the partial n gene sequence revealed a greater variability than the f and h genes; therefore, lineage classification according to the n gene is considered the most accurate way to type novel isolates, as this region is more divergent between lineages and between isolates of the same lineages (kumar et al. 2014). all pprv strains belong to a single serotype, but the different strains have been grouped into four distinct lineages. historically, lineages i–iii have been found in africa and numbered according to the apparent spread of the virus from west africa (lineages i and ii) to east africa (lineage iii). lineage iv was mainly restricted to the middle east and asia, with a few exceptions of lineage iii being reported in yemen and oman, and mixed lineages of lineages iii and iv in the united arab emirates and qatar. however, lineage iv has now established its presence all across the ppr-endemic areas, with frequent outbreaks in africa, by replacing other lineages (kwiatek et al. 2011; libeau et al. 2014; mantip et al. 2016; muniraju et al. 2014; woma et al. 2015) (mantip et al. 2016). recently, the evolution of pprv and its relationship with its prototype rpv has been suggested using molecular phylogenetic techniques with virus full genome sequence data. bayesian phylogenetic studies found that rrpv is more closely related to measles virus than to pprv (furuse, suzuki & oshitani 2010; pomeroy, bjornstad & holmes 2008). the origin of ancestral pprv and its relationship with other morbilliviruses have recently been estimated using bayesian analysis of the complete virus genome sequences (kumar et al. 2014). a bayesian phylogenetic analysis of all four viral lineages mapped the time to the most recent common ancestor at the beginning of the 20th century, a few decades before the first recorded description of the virus in 1942. lineage iii pprv is proposed to have been the first to have diverged (parida et al. 2015). the estimated probability for the root location of an ancestral virus and individual lineages was determined as being nigeria for pprv, senegal for lineage i, nigeria/ghana for lineage ii, sudan for lineage iii and india for lineage iv (muniraju et al. 2014). the life cycle of pprv is 6 hours – 8 hours in cultured cells (kumar et al. 2014). the initial step of infection of pprv starts with the binding of the virus to the host cell and delivery of the nucleocapsid into the host cell cytoplasm; this plays an important role in the pathogenesis of the virus and susceptibility to the host (kumar et al. 2014). the first interaction of the host and pathogen (attachment) is mediated by the binding of the virus to the cell receptors through its h protein (kumar et al. 2014). similar to mv and cdv, pprv has two natural cellular receptors: the signaling lymphocyte activation molecule (slam) or cd150 protein and nectin-4. the signaling lymphocyte activation molecule is exclusively expressed on immune cells (lymphocyte, macrophages and dendritic cell surface but not on epithelial cells), while nectin-4 is the epithelial cell receptor, but is not expressed in lymphocytes and dendritic cells (adombi et al. 2011; munir, munir & zohari 2013; pawar et al. 2008). the existence of these two different receptors may explain why morbilliviruses are both lymphotropic and epitheliotropic (kumar et al. 2014). following the release of nucleocapsid from the viral envelope, viral transcription starts in the cytoplasm. the rdrp present in the infecting virion initiates synthesis of the mrna as well as complementary rna (crna). the rdrp of all paramyxoviruses is believed to attach at the genomic promoter (gp) on genomic rna, from where the transcription begins (baron et al. 2017; barrett, banyard & diallo 2005). the process of assembly and release of the morbillivirus including pprv is not well understood. like other enveloped viruses, paramyxoviruses also form virus particles when all the structural components of the virus, including viral glycoproteins and viral rnps, have assembled at selected sites on the membranes where virions bud, then pinch off to achieve particle release (kumar et al. 2014), allowing the transmission of infections to new cells and hosts (harrison, sakaguchi & schmitt 2010). apart from the endemic presence of ppr in sub-saharan africa from the past decades, in recent years, field data and laboratory findings have confirmed the dramatic spread of ppr towards the south of africa, affecting gabon, the democratic republic of congo, somalia, kenya and tanzania (swai et al. 2009). an outbreak of ppr was reported for the first time in october 2012 in angola and in july 2015 in zambia (baron et al. 2016). the risk of ppr introduction is now high for neighbouring countries with major sheep/goat populations, such as the republic of south africa and mozambique. in addition to the spread in africa, many asian countries are now infected, including china. after an initial identification in tibet in 2007 (wang et al. 2009), this country experienced a major ppr epizootic in 2013–2014 (wang et al. 2009). peste des petits ruminant affects sheep and goats, although goats are often more severely affected than sheep (lefevre & diallo 1990). however, variable seroprevalence has been observed in sheep and goats after an outbreak (abraham et al. 2005; ayari-fakhfakh et al. 2011; ozkul et al. 2002; swai et al. 2009). various factors may explain these differences: livestock management practices, host density, strain virulence (couacy-hymann et al. 2007) as well as host species and breeds (diop, sarr & libeau 2005). for instance, sahelian goats are considered more resistant than guinean dwarf goats, while alpine goats are very sensitive after experimental infections (hammouchi et al. 2012). peste des petits ruminant is not considered as pathogenic in cattle, domestic and wild african buffaloes (syncerus caffer) although 10% or more of these species may seroconvert when exposed to pprv in enzootic regions (abraham et al. 2005; couacy-hymann et al. 2007; ozkul et al. 2002). in a nationwide serological survey recently implemented in senegal (2015), seroprevalence rates as high as 80% were observed in regions where both cattle and small ruminants were abundant, without any reported clinical sign in cattle (baron et al. 2016). conversely, high case fatality rates (96%) were reported in india in domestic buffaloes (bubalus bubalis) and the disease was experimentally reproduced in these animals (govindarajan et al. 1997). in addition, ppr has been suggested to occur as a disease in camelids; a respiratory syndrome was the main sign in ethiopia and sudan (khalafalla et al. 2010; roger et al. 2000). other wild ruminants, including representatives of the gazellinae, tragelaphinae caprinae subfamilies, may show serious illness and mortality when infected with pprv from neighbouring small ruminant populations. in specific conditions, wildlife may play an important role in ppr epidemiology, as was seen in the arabian peninsula (kinne et al. 2010). the incubation period of the disease is typically 4–6 days, although it may range between 3 and 14 days. during the acute stage of the disease, animals show pyrexia (up to 41 °c) that may last for 3–5 days and that can be accompanied by depression, anorexia and dryness of the muzzle. watery nasal and lachrymal discharges gradually become mucopurulent with excessive salivation. erosive lesions formed in the oral cavity may become necrotic. in severe cases of the disease, these necrotic lesions progress with the appearance of a deposit of fibrin (caseous deposit) on the tongue. in later stages of the disease, animals develop diarrhoea and coughing with laboured, abdominal breathing. finally, the animal may become dyspnoeic, suffering from progressive weight loss and emaciation, ultimately leading to its death. in some cases, particularly in mild infections, animals may convalesce, returning to a pre-infection health status within 10–15 days of infection. the morbidity rate can reach 100% with a high case fatality rate in the acute form of the disease (pope et al. 2013). at an early stage of infection, virus excretion is very high in the exhaled air. by analogy with rpv, this probably allows non-contact transmission over at least a few metres (idnani 1944). nasal and ocular discharges, saliva and faeces also contain large amounts of viral antigen (abubakar et al. 2012). in goats, pprv–rna or antigen is excreted in the faeces for at least 2 months after a natural infection (abubakar et al. 2012; ezeibe et al. 2008), although it is not known if this is an infectious virus. because pprv is quickly inactivated in the environment, its transmission most often occurs by direct contact between infected and healthy animals. however, indirect transmission through recently (within hours) contaminated material cannot be excluded and should be considered in epidemiological models and risk-based control measures (baron et al. 2016). because of its rapid spread in immunologically naïve flocks, a common belief is that pprv can only persist in large populations and only if new susceptible hosts (newborn, migrating or purchased animals) are available (wamwayi et al. 1995). the economic losses that are associated with ppr can be substantial. peste des petits ruminant has severe implications on the livelihoods of pastoralists and farmers across the world. the world’s populations of 2.1 billion small ruminants are critical assets for poor rural households in developing countries, providing quality protein, milk, nutrition, fertiliser, wool and fibre, as well as income opportunities and financial flexibility. around 80% of those animals are found in regions where ppr, is present (fao 2013). the virus is now present or expected to exist in more than 70 countries and it keeps spreading, and appearing in areas or countries where it did not exist before, almost every year, and causes an estimated $1.5 to $2 billion financial loss each year. the control, prevention and eradication of ppr are linked to other global challenges such as poverty alleviation, food security and nutrition, and resilience building, in particular for smallholder farmers and women. history of peste des petits ruminants research in africa in africa, ppr has often been confused with the closely related rpv, the history of which dates back to as far as the late 4th century ad (curasson 1932; henning 1956). rinderpest virus is predominantly a disease of large ruminants and is linked historically with devastating epidemics, often with very high mortality rates. in contrast to the extensive historical data available on rpv, the first recorded description of pprv was in 1942 when gargadennec and lalanne identified a disease closely related to rpv in the ivory coast in west africa. early observations suggested that the disease was not transmissible from small ruminants to in-contact cattle and this led them to assume it was a novel virus distinct from rpv (gargadennec & lalanne 1942). because of the similarity between rpv and ppr, they have common names such as ovine rinderpest, goats’ plaque, pseudo-rinderpest, pneumoenteritis complex, stomatitis-pneumoenteritis and plague of small ruminants or kata; however, because of the official discovery of ppr in the ivory coast which is a french-speaking colony, pest des petits ruminants (ppr), which means plague of small ruminants, is commonly used worldwide. in 1956, it was shown by mornet and colleagues that pprv and rpv are closely related antigenically (pastoret, 2006). in 1979, pprv was classified as a morbillivirus under the family paramyxoviridae and the order mononegavirales (gibbs et al. 1979). peste des petits ruminant was first isolated in sheep cell culture in 1962 (gilbert, y. & monnier, j., 1962) and was observed for the first time under electron microscope in 1967 (bourdin & laurent-vautier 1968). peste des petits ruminant has been an important disease of sheep and goats in africa ever since its discovery and has therefore been a major focus of research in virtually all the veterinary research institutes and university faculties of veterinary medicine in africa. several key discoveries with regard to the nature of pprv and its epidemiology, through serological and genetic methods, were carried out in african institutions that contributed towards the understanding and control of ppr. in 1962, gilbert and monnier in their effort to develop a homologous ppr vaccine carried out the first adaptation of pprv in tissue culture when a cytopathic effect (cpe) of the virus was observed in primary sheep liver cells, manifested by the appearance of syncytia (gilbert & monnier 1962). years later, pprv was adapted in vero cells and a homologous live attenuated ppr vaccine was developed. this was the first ppr vaccine that was developed in africa for worldwide ppr vaccination by continuous passage of nigeria 75/1 strain in vero cells and is now commonly used in african countries. based on circumstantial evidence, such as strong seasonal incidence and the fact that sheep/goats could be protected by vaccination and by avoiding low lying pastures, it was stipulated that outbreaks of ppr are always attributed to changes in weather, that is, either during the onset of the rainy season or during the dusty harmattan season. once animals are infected by ppr, sooner or later they begin to manifest the clinical signs and symptoms of the disease and can easily transmit the virus to susceptible naïve populations, thereby leading to the spread of the disease in a particular population. consequently, as the transmission of pprv from virus-excreting infected to naïve animals mainly occurs through close contact, the most important sanitary preventive measure consists of restricting the importation of susceptible animals from infected areas to disease-free areas. outbreaks can be controlled by stamping out infected specimens followed by disinfection of premises and compensation of affected farmers. however, as most of the ppr-endemic regions are in africa, such drastic measures are difficult to implement. thus, vaccination is the main means available for the effective prevention and control of ppr in those countries (baron et al. 2016). the main characteristic of the pathogenesis of pprv infection, as for other morbilliviruses, is the profound but transient virus-induced immunosuppression, with the consequence of increased susceptibility to opportunistic infections that impact the final outcome of the infection (baron et al. 2014; pope et al. 2013). this immunosuppression is a consequence not only of direct effects of virus multiplication in lymphoid cells but also of the different strategies developed by morbilliviruses to overcome the host protective response, such as interference with both the innate and induced immune responses. however, this immunosuppressive effect is transient and recovery from the disease is usually accompanied by the establishment of a strong, specific and long-term protective immune response (servet-delprat et al. 2003). attenuated morbillivirus vaccines seem to have less immunosuppression capacity compared to wild-type viruses, but still induce a strong protective immunity (sen et al. 2010). with that characteristic, the live attenuated rinderpest vaccine (plowright & ferris 1962) was one of the key factors in the success of the global rinderpest eradication programme. after plowright and ferris’ success, attempts were also made to develop a live attenuated ppr vaccine which, however, was unsuccessful (benazet 1973; gilbert & monnier 1962). following those failures, and considering the close antigenic relationship between rpv and pprv, the live attenuated rinderpest vaccine was tested in small ruminants for protection against ppr. despite the lack of a detectable pprvneutralising antibody, these animals resisted the pprv challenge and the rpv vaccine was successfully used to protect small ruminants against ppr (rowland & bourdin 1970; taylor & abegunde 1979; taylor, al busaidy & barrett 1990). continued research on the development of a homologous ppr live attenuated vaccine was finally successful in 1989, with the attenuation of the pprv strain nigeria 75/1 by means of serial passage in vero cells (diallo et al. 1989b). several field trials of this vaccine conducted between 1989 and 1996 demonstrated its high efficacy in protecting sheep and goats against ppr. anti-pprv antibodies generated by vaccinated animals last for at least 3 years, equal to the effective economic life of the animals. during the development of this vaccine, different wild-type pprv strains were used as challenge viruses and all failed to induce disease in vaccinated animals, demonstrating the efficacy of the vaccine against virulent pprv strains of any lineage. the availability of a homologous vaccine for ppr was fortunate as the use of rinderpest vaccine in any animal species was forbidden once a country was declared free of rinderpest. following this first success, other pprv strains were successfully attenuated by serial passage in cell culture (sen et al. 2010). amongst them, the pprv/nigeria 75/1 (lineage ii) and the pprv/sungri/96 (lineage iv) are currently the most used vaccines and have been extensively tested and validated, including the determination of full genome sequences (diallo 2003, 2004; diallo et al. 2007; riyesh et al. 2011; singh & bandyopadhyay 2015; singh et al. 2009). like all paramyxoviruses, pprv is heat sensitive. field distribution of the live attenuated pprv vaccines therefore needs a cold chain from the manufacturer to the point of delivery to the animal. unfortunately, most of the countries where ppr is endemic are in regions of hot climate and have poor infrastructure with inconsistent electricity supply. this issue has been addressed by attempting to improve the freeze-drying procedures for the attenuated pprv vaccines using various stabilisers (riyesh et al. 2011; saravanan et al. 2010; sarkar et al. 2003; silva, carrondo & alves 2011; silva et al. 2014; worrall et al. 2000). with such improvements, it has been possible to keep the vaccine at 45 °c for at least 14 days with minimal loss of potency (worrall et al. 2000). one significant problem with the live attenuated pprv vaccines is that animals that have received this vaccine cannot be distinguished serologically from animals that have been infected and recovered. this makes epidemiological serosurveillance of the disease impossible in areas where a vaccination programme has been or is being implemented. a way to combine vaccination and disease serosurveillance activities for better management of the disease would be the use of vaccines, enabling differentiation of infected and vaccinated animals (diva). with the advent of recombinant dna technology, different approaches are being followed to develop vaccines that enable this differentiation, to allow countries to implement both vaccination and disease surveillance programmes at the same time (diallo et al. 2007). the development of in vitro systems for isolation and large scale propagation of pprv in the 1960s paved the way for serological, antigenic and genetic studies of the virus as well as the development of diagnostic assays, thereby improving the understanding of the virus. global recognition of peste des petits ruminants currently, ppr is the fastest expanding and potentially the most economically important disease of sheep and goats in many regions of the developing countries where these domestic animals play an integral and important role in sustainable agriculture and development (baron et al. 2016). peste des petits ruminant has spread so alarmingly during the last two decades that it has become a matter of concern for the food and agriculture organization of the united nations (fao) and the international organization epizootic (oie), which have now initiated efforts for its control and eradication (oie 2015). peste des petits ruminants was first identified in nigeria (west africa) in 1942. it is currently believed to be endemic across much of west africa (banyard et al. 2010). west africa includes 16 countries distributed over an area of approximately 5 million square kilometres. almost all of these countries have experienced significant outbreaks of pprv. in recent years, material submitted to regional reference laboratories (rrls) has confirmed the presence of either antibodies to the virus or the detection of viral nucleic acid in samples from burkina faso (2008), ghana (2010), nigeria (2007, 2013, 2014) and senegal (2010) (banyard et al. 2010). peste des petits ruminants virus strains from both lineages i and ii are currently circulating across west africa although undoubtedly many outbreaks are not characterised at the molecular level. characterisation of pprv in nigeria from a recent outbreak indicated that pprv lineage iv is also circulating alongside pprv lineage ii in nigeria (mantip et al. 2016). east africa is generally used to specifically refer to the area now comprising the countries of kenya, tanzania and uganda but often includes somalia, djibouti, ethiopia and eritrea. peste des petits ruminants virus is endemic across the majority of these countries, with genetic typing of the virus in 1996 determining a virus circulating in ethiopia as belonging to lineage iii (banyard et al. 2010). previous isolation of viruses that group in lineage iii includes two isolates from wildlife in oman (1983) and the united arab emirates (1986), an isolate from sudan (1972) and an unexpected virus isolate from sheep in southern india (1992) (banyard et al. 2010). more recently, the confirmation of endemicity of pprv across east africa has been shown through the detection of antibodies to pprv in kenya (1999 and 2009) and uganda (2005 and 2007). molecular tools have characterised some viruses as belonging to lineage iii, with isolates being characterised in sudan (2000), uganda (2007) and most recently in tanzania (2008 and 2010) (banyard et al. 2010). somalia was also affected by pprv in 2006, with the central regions being most seriously affected (nyamweya et al. 2009). peste des petits ruminants virus was also seen in ethiopia in 2008 and 2009 (nyamweya et al. 2009). despite the absence of molecular typing for the recent kenyan and somalian outbreaks, it is likely that the virus circulating in these areas is lineage iii. central africa includes a core region of the african continent, namely, burundi, the central african republic (car), angola, cameroon, chad, gabon, the democratic republic of the congo (drc) and rwanda. phylogenetic analysis has shown that lineage iv viruses are circulating across central africa (banyard et al. 2010). geopolitically, northern africa includes seven countries or territories: algeria, egypt, libya, morocco, sudan, tunisia and the western sahara. algeria, morocco, tunisia, mauritania and libya together are sometimes referred to as the maghreb (or maghrib), while egypt is a transcontinental country by virtue of the sinai peninsula, which is in asia. historically, it is hypothesised that pprv spread into north and east africa from west africa, moving up through trade routes through sudan and egypt and into the middle east. historically, pprv had been detected in 1987 and 1990 in egypt (ismail & house 1990). molecular typing has characterised a recent discovery of pprv in egypt as lineage iv (banyard et al. 2010). with the transcontinental status of egypt it is suggested that the remainder of north africa was totally free from pprv until recently when an extensive outbreak occurred in morocco. in 2008, local veterinary services reported 257 outbreaks across 36 of morocco’s 61 provinces. despite low mortality and morbidity rates, this outbreak was of great significance because of commercial trade between morocco and both algeria and spain. genetic characterisation of the moroccan virus classified it as a lineage iv virus (fao 2009; khalafalla et al. 2010). in the arabian peninsula and the middle east, the presence of pprv was analysed in saudi arabia and it was concluded that the disease was not circulating in the country (al-naeem, elzein & al-afaleq 2000). however, by april 2002 an outbreak with a case mortality rate of 100% was reported in sheep and goats (housawi et al. 2004) and since then further serological surveys and outbreaks have been reported (al-dubaib 2009; el-rahim et al. 2005; housawi et al. 2004). seroprevalence of both sheep and goats has also been reported in north jordan (al-majali, hussain & amarin 2008) and in lebanon with a seroprevalence of up to 48.6% being reported (attieh, 2007). in the remaining areas of the arabian peninsula, lineage iv virus has been detected in a game reserve in the united arab emirates (kinne et al. 2010) as well as in qatar (2010). interestingly, in qatar, both lineages iii and iv are circulating, with both lineages being isolated from goats in 2010 (banyard et al. 2010). the situation in qatar is further complicated by the recent detection of pprv in wild deer populations. however, the role of sylvatic pprv and potential transmission to domestic species remains unknown (de nardi et al. 2012). within yemen, the most southern region of the peninsula, lineage iii virus continues to circulate, with no introduction of lineage iv having been reported in yemen or oman (banyard et al. 2010). peste des petits ruminants virus has been reported in pakistan since 1991, with initial epidemics in the punjab region being characterised by using pcr in 1994 (amjad et al. 1996). since then both the spread of the virus and an increase in reporting meant that pprv has been documented on several occasions in pakistan. outbreaks that affected afghan sheep (bulkhi) occurred in the punjab (durrani et al. 2010), lahore (rashid, asim & hussain 2008) and islamabad (zahur et al. 2009). currently, only the lineage iv virus has been identified in pakistan (banyard et al. 2010). peste des petits ruminants virus is endemic across much of india, and an improvement in veterinary services, reporting networks and diagnostic capabilities across india has led to an increase in awareness of the disease (banyard et al. 2010). the virus was first reported in southern india in 1987 (shaila et al. 1989), where it seemed to remain for several years before spreading across the entire country and surrounding regions. molecular characterisation of virus isolates from india shows that virtually all isolates analysed belong to lineage iv. more recent epidemiological studies of pprv in india have characterised a number of closely related lineage iv viruses being present (dhar et al. 2002). the virus continues to be reported periodically across india, with recent reports documenting the presence of pprv in rajasthan (kataria, kataria & gahlot 2007) in the north, the kolkata region in the east (saha, lodh & chakraborty 2005), karnataka and maharashtra in the south-west (chavran, digraskar & bedarkar 2009; santhosh et al. 2009) and across the southern peninsula (raghavendra et al. 2008). these reports reflect the endemicity of the disease across the entire country. the presence of pprv is poorly characterised across great stretches of the middle east, with outbreaks in iraq most frequently being reported. detection of pprv in iraq dates back to 2000 where a virus causing high morbidity and low mortality rates was characterised (barhoom, hassan & mohammed 2000). however, retrospective sero-analysis has shown that the virus was previously circulating in 1994 (shubber et al. 2004). iran has also had pprv circulating for many years, with initial detection back in 1995, leading to the extensive spread of the virus across the country at a huge economic cost (bazarghani et al. 2006). more recently in 2009, the virus from iran has been characterised at the genetic level, being grouped with lineage iv viruses (banyard et al. 2010). the recent detection of pprv in areas of the near east has highlighted the potential for spread of pprv into areas that have never previously been documented. tajikistan borders afghanistan where pprv is believed to be present and was previously reported in association with rpv outbreaks (kwiatek et al. 2007). peste des petits ruminants virus is also believed to be present in kazakhstan, although only very few seropositive animals have been identified (lundervold et al. 2004). the recent detection of pprv in china was officially reported in the ngari region of western tibet in july 2007 (wang et al. 2009). previously, pprv infection had been recognised in several countries bordering the south-western region of china, including india, nepal (2009), bangladesh (2000 and 2009), pakistan (2004 and 2009) and afghanistan (banyard et al. 2010). all the recent virus strains detected in south-west asia and the middle east belong to lineage iv and the tibetan isolate is of the same lineage and is closely related to isolates from both india and tajikistan. the terrain of western and south-western ngari permits uncontrolled animal movement, and a small ruminant trade exists between tibet and the bordering nations such as india and nepal. these factors suggest that animals from a neighbouring country in south-west asia are the most likely source of pprv in tibet (wang et al. 2009). serological detection of antibodies to pprv has also been detected in samples from vietnam (maillard et al. 2008) and samples submitted to rrls from bhutan have recently been typed as lineage iv virus (banyard et al. 2010). these findings suggest that the virus may be present across a greater area than what is currently thought. it is possible that pprv has spread into many other bordering countries, but the unfamiliarity of local human populations with the disease means that it may remain either unnoticed or be misdiagnosed as a different disease with similar clinical manifestations (banyard et al. 2010). recent reports of pprv in areas close to european borders have increased its profile both scientifically and in the media. while this devastating disease of small ruminants has continued to plague agriculture across africa and asia for many years, the threat of its spread into developed countries has greatly renewed research interest in the virus. the detection of pprv in turkey in 1996 raised initial awareness of the virus and questioned the potential for pprv to spread across the rest of europe (ozkul et al. 2002). indeed, there have been numerous reports of pprv in turkey, having now also been reported in western turkey, bursa province (yesilbag et al 2005) and mugla and aydin provinces (toplu 2004) in the aegean district. throughout 2005, 78 separate outbreaks of pprv were recorded across turkey, with quarantine and vaccination being used to prevent further spread of the disease (tufan 2006). in 2007, the first outbreak of fatal pprv was reported in kirikkale province, central anatolia (kul et al. 2007), suggesting the spread of the virus into the central belt of the country, and in 2009 infection of sheep in the middle and eastern black sea region of turkey was reported (albayrak & alkan 2009). these reports suggest that pprv is present across much of turkey and diagnostic samples received by rrls have confirmed turkish pprv isolates as belonging to lineage iv (banyard et al. 2010). consequently, the moroccan outbreak and the potential existence of as-yet-unidentified foci of pprv infection across other territories in northern africa also increase the threat of movement of infected animals into southern europe. historical exchanges exist between morocco and spain where both ovine and caprine populations are important. an increase in human population and in turn the small ruminant population, across such areas undoubtedly increases the risk of further emergence of pprv across north africa. it is therefore essential that europe maintains surveillance of the disease in order to successfully contain the disease should it be imported (minet et al. 2009). peste des petits ruminants in africa generally in africa, ppr occurs throughout the year, but it has been documented in various reports that the prevalence of the disease varies according to climatic conditions and seasons. obi et al. (1983b) in a study carried out in western nigeria showed that ppr may be encountered almost throughout the year, with peaks in the wet months of june–august. they also observed that at the university of ibadan teaching and research farm, 192 (74%) cases recorded in goats between 1973 and 1980 were in april–july, while out of the five natural outbreaks that were investigated, three occurred in the wet season (june–july) and the remaining two were recorded in november–december. wosu et al. (1990) in a study in the nsuka area reported that ppr incidence was higher during the dry harmattan season (december–january) than in the rainy season, with a peak in april. they attributed this to the dry, cold and dusty harmattan with the accompanying poor nutrition at that time of the year. seasonal occurrence of the disease was also observed by whitney, scott and hill (1967) and nduka and ihemelandu (1973). other factors that have been reported to affect the prevalence of ppr in africa include the acquisition of animals from local markets, mass transportation of animals during major festivals (bonniewell 1980), local husbandry and customary practices of renting males and all year-round kidding and constant introduction of new stock, leading to an increased number of susceptible animals (obi et al. 1983b). during the observation of the first cases of ppr in nigeria, it was thought that the disease was a problem of dwarf goats in the wet and humid southern parts of nigeria. however, serological surveys in southern nigeria by obi et al. (1983a) and in northern nigeria by gibbs et al. (1979) showed widespread infection in the country. bourdin (1983) reported that the highly acute form of the disease most frequently seen in goats was rare in sahelian sheep. sheep and goats of all ages and sexes can be affected during an outbreak (obi et al. 1983a). bourdin reported that in goat populations, the highest incidence occurs in young animals between 1 and 24 months, with losses of up to 50%. consequently, it had been thought that pprv is restricted in its host range, being confined to its natural infections to sheep and goats, with the latter species being more often affected than the former (losos 1986). the isolation of pprv from dorcas gazelle, which died of a natural infection in the united arab emirates (furley, taylor & obi 1987), has broadened our understanding of the host range of the virus. in cattle, ppr was not shown to be pathogenic although there was seroconversion (gibbs et al. 1979). the investigations on a new pathology of camels in ethiopia and sudan (roger et al. 1997) have implicated pprv in a natural outbreak of an acute febrile infection characterised by respiratory signs. results of experimental transmission indicate that cattle and pigs do not suffer from clinical disease (nawathe & taylor 1979). shamaki (2002) reported the probable role of wildlife (warthog) and camel in the epidemiology of ppr in nigeria. elaborately, lineage differentiation of ppr is determined by the sequence comparison of a small region of the f gene (forsyth & barrett 1995) or n gene (couacy-hymann et al. 2002), depending on the reaction components used by the testing laboratory. historically, african isolates of pprv were numbered lineages i–iii according to the proposed spread of the virus from west africa to east africa. following this nomenclature, the n gene primer sets (couacy-hymann et al. 2002) typed west african viruses from senegal, guinea, guinea-bissau, ivory coast and burkina faso as belonging to lineage i. the isolates derived from ghana, mali and nigeria then formed lineage ii and those detected in ethiopia and sudan were from lineage iii. data derived from the f gene material reversed the classification of lineages i and ii isolates, and historically this difference has been maintained (shaila et al. 1996). the current molecular characterisation of pprv virus isolates divides them into four genetically distinct lineages: lineage i being represented mainly by western african isolates from the 1970s and recent isolates from central africa; lineage ii by west african isolates from the ivory coast, guinea and burkina faso; lineage iii by isolates from eastern africa, sudan, yemen and oman; and lineage iv includes all viruses isolated from recent outbreaks across the arabian peninsula, the middle east, southern asia and recently across several african territories. consequently, data generated by pcr and sequencing are routinely used to construct phylogenetic trees for pprv and ascribe different isolates to the different lineages in order to understand the spread and epidemiological movement of the virus (dhar et al. 2002; ozkul et al. 2002; shaila et al. 1996). research on peste des petits ruminants in africa the continuous incursion of pprv into previously unaffected regions of africa, the middle east and asia stimulated research interest in pprv in africa, and right from the identification of pprv in nigeria, different techniques have been employed to contribute towards the control and prevention of pprv. the techniques and approaches used to improve pprv isolation and molecular research and diagnosis right from inception include virus isolation, detection of viral antigen and nucleic acid sequencing as outlined below. virus isolation during the inception of pprv research, the commonly used cell culture systems employed in pprv isolation were primary lamb kidney and ovine skin tissues (bourdin, p. & laurent-vautier, 1968) (gilbert, y. & monnier, j., 1962)(taylor, w. p. & abegunde, 1979). the use of vero cells was later employed (hamdy et al. 1976). the sensitivity of pprv isolation was then increased by growing the virus in lamb’s or goat’s kidney cells (ali & taylor 1984). peste des petits ruminants virus has also been adapted to grow in other continuous cell lines including madin-darby bovine kidney epithelial cell line and baby hamster kidney-21 fibroblast cell line (mdbk and bhk-21) (diallo et al. 1989b). however, vero cells derived from african green monkey kidney were the most widely used cell line for pprv before the discovery of the goat or dog slam. following the discovery of a protein receptor on the cell surface of morbillivirus susceptible hosts, the slam, also known as cd150, is used preferentially by wild-type morbilliviruses to bind to the host (banyard, baron & barrett 2005; baron 2005; cocks et al. 1995; cosby et al. 2002; kruse et al. 2001; mcquaid & cosby 2002; ono et al. 2001; sidorenko & clark 1993; tatsuo et al. 2000; tatsuo, ono & yanagi 2001; yanagi, takeda & ohno 2006). however, although lymphoid tissues are major sites of morbillivirus replication, it is also observed that they can infect and replicate in epithelial cells of other organs, such as the alimentary track epithelial cells, and lung and kidney cells by utilising other types of cell receptors (hashimoto et al. 2002; leonard et al. 2008; plowright 1964; takeuchi et al. 2003; wohlsein et al. 1993; wohlsein et al. 1995). the infection efficacy for those cells is up to 100–1000 times less than that of the lymphoid cells (hashimoto et al. 2002; takeda et al. 2007), but because they are easy to maintain in culture in vitro, they have been favoured preferentially for morbillivirus isolation. in the case of pprv, primary cultures of bovine kidney, goat kidney, sheep kidney and lung cells have all been utilised for isolation and maintenance (abu elzein et al. 1990; ali & taylor 1984; lefevre & diallo 1990; taylor & abegunde 1979; taylor, al busaidy & barrett 1990). however, not only is the availability of primary cells becoming more problematic, their quality is also not guaranteed and there is considerable variation from batch to batch. these drawbacks in the use of primary cell cultures have stimulated the use of cell lines for pprv isolation, in particular, the african green monkey kidney (vero) cell line (lefevre & diallo 1990). unfortunately, as with other morbilliviruses, pprv isolation using these cells is inefficient: the likelihood of isolating the virus is very low, and even if it is successful, it often requires multiple, sequential blind passages and many weeks in culture before the development of any cpe can be observed (abu elzein et al. 1990; albayrak & alkan 2009; gulyaz & ozkul 2005; lefevre & diallo 1990). following the identification of the slam as the main receptor used by mv, cdv and rpv wild-type strains (banyard et al. 2005; hsu et al. 2001; tatsuo et al. 2000, 2001), vero or cho cells expressing human, dog or bovine slam proteins have been used as a highly efficient means of isolation and propagation of these morbilliviruses (tatsuo et al. 2001; seki et al. 2003; tatsuo et al. 2001). based on the discovery of slam as a possible protein for the isolation and propagation of pprv, a cv1 cell line stably expressing the goat slam protein was developed for the isolation of pprv (adombi et al. 2011). this cell line, designated as chs-20, is used to isolate pprv from clinical specimens of sick sheep and goats with suspected ppr specimens collected in 2008 and 2009 from different locations in nigeria and cote d’ivoire, respectively (adombi et al. 2011). preliminary tests carried out demonstrated a high sensitivity for isolating pprv from pathological samples, indicating the recent use of slam in pprv isolation and propagation in place of the previous methods. agar gel immunodiffusion test agar gel immunodiffusion (agid) was widely used to identify pprv and it can detect 42.6% of ante-mortem and necropsy specimens (abraham & berhan 2001; obi 1984). agar gel immunodiffusion can be used to test the presence of both antigen and antibodies and can give results within 2 h – 4 h when rinderpest (rp) hyperimmune serum is used, while it needs 4 h – 6 h with ppr hyperimmune serum (obi 1984). one of the important advantages of this test is that it is highly specific (92%), though it cannot differentiate between ppr and rp. hyperimmune serum standard ppr anti-serum was made by immunising sheep with 5 ml of pprv with a titre of 104 tcid50 (50% tissue culture infective dose) per millilitre administered at weekly intervals for 4 weeks. the animals were bled 5–7 days after the last injection. standard rp hyperimmune anti-serum is also effective in detecting ppr antigen (abraham et al. 2005). counter immuno-electrophoresis counter immuno-electrophoresis (ciep) used the same principle as the agid except that the gel is electrically charged to improve the sensitivity of the test (abraham et al. 2005). enzyme-linked immunosorbent assay for antigen detection a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (elisa) was found to be highly effective in the detection of antigen in tissues and secretions of infected goats (saiki et al. 1988). another format of antigen elisa which was more widely used is immunocapture elisa (abraham et al. 2005). it gives a reliable result within 2 h in pre-coated plates and samples maintained at room temperature for a period of 7 days with no more than 50% reduction in response (libeau et al. 1994). the immunocapture elisa allows for a rapid differential diagnosis of ppr and rp viruses, and this is of great importance as the two diseases have similar geographical distributions and may affect the same animal species. the main advantages of this assay include rapidity, specificity, robustness and simplicity. it is suitable for routine diagnosis of ppr and rp from field samples such as ocular and nasal swabs. complementary dna probes for the differentiation between ppr and rp, the use of [p32]-labelled complementary dna (cdna) probes derived from the n-protein gene of the two viruses had been described (diallo et al. 1989b). it could differentiate between the two viruses without the need of virus isolation. cdna directed against the matrix protein, fusion protein and phosphoprotein gene was found to cross-hybridise to a much greater extent and was not suitable for use as discriminating probes (diallo et al. 1989a). unfortunately, this hybridisation cannot be used widely because it requires fresh specimens and in addition to the short half-life of [p32], there is a constraint with the handling of isotopes. therefore, probes using non-radioactive labels such as biotin (pandey, baron & barrett 1992) or dioxin (libeau et al. 1995) were developed. the biotin-labelled cdna was found to be as specific as the one using the radioactive label and more rapid in differentiation between ppr and rp (pandey et al. 1992). however, it was reported elsewhere that the expected sensitivity responsiveness had never been obtained using non-radioactive labels (diallo et al. 1995). reverse transcription polymerase chain reaction conventional serological techniques and virus isolation are normally used to diagnose morbillivirus infection in samples submitted for laboratory diagnosis. however, such techniques are not suitable for use on decomposed tissue samples; the polymerase chain reaction (pcr) has been proved invaluable for analysis of such poorly preserved field samples. saiki et al. (1988) first demonstrated the efficiency of amplifying in vitro a selected sequence flanked by two oligonucleotide primers of opposite orientation. the method consists of repetitive cycles of dna denaturation, primer annealing and extension by a dna polymerase effectively doubling the target with each cycle, leading, theoretically, to an exponential rise in dna product. when the pcr starting material is dna, high purification of the nucleic acid is not necessary; therefore, the procedure is greatly simplified. these qualities have made the pcr one of the essential techniques in molecular biology today and have made it the most widely used laboratory technique in disease diagnosis. since the genome of all morbilliviruses consists of a single strand of rna, it must be first copied into dna, using reverse transcriptase, in a two-step reaction known as reverse transcription/polymerase chain reaction (rt-pcr). reverse transcription/polymerase chain reaction has been shown to be useful for the rapid detection of morbillivirus-specific rna in samples submitted for laboratory diagnosis (shaila et al. 1996). it has proved especially useful in identifying the new morbilliviruses found in marine mammals (barrett et al. 1993). both genus-specific and universal morbillivirus primer sets have been produced, which can be used to distinguish all known morbilliviruses (forsyth & barrett 1995). two sets of primers have been made based on sequences in the 3’ end of n genes (messenger sense), which are the least conserved regions between the two viruses. they enable specific amplification of 300 base pair (bp) fragments for rpv and pprv (couacy-hymann et al. 2002). reverse transcription/polymerase chain reaction, using phosphoprotein (p) universal primer and fusion (f) protein gene-specific primer sets to detect and differentiate between ppr and rp, was described by some researchers (barrett et al. 1993; couacy-hymann et al. 2002; forsyth & barrett 1995). advancement in modern technology, awareness and availability of molecular tools in africa led to an increased interest in pprv following rpv eradication; outbreaks of pprv have been genetically typed, enabling molecular epidemiological assessment of lineage distribution across endemic areas of africa. this in turn has led to speculation surrounding the emergence of a dominant lineage of pprv in new areas (kwiatek et al. 2011). certainly, the recognition of the importance of ppr as an obstacle to the development of sustainable agriculture across africa has led to the laboratory confirmation of ppr disease, with subsequent reporting of cases to the scientific press (banyard 2016). the advent of molecular technologies over the last 20 years has led to studies into the genetic composition of different isolates of pprv, which has fostered a greater understanding of the molecular epidemiology of ppr in africa. from a genetic point of view, four distinct lineages of pprv have been described based on genetic data derived from the nucleoprotein gene (couacy-hymann et al. 2002). furthermore, the rapid development of sequencing technologies has revolutionised sequence generation from samples with the result that full genome data can be derived from samples with relative ease, enabling phylogeneticists to assess isolates based on any part of the genome (banyard 2016). most recently, the first bayesian full genome assessment of pprv has concluded that from an evolutionary perspective, pprv emerged at the beginning of the 20th century, a few decades before the first recorded description of pprv in 1942 (muniraju et al. 2014). a bayesian phylogenetic analysis of all pprv lineages mapped lineage iii pprv as the first to have diverged from an ancestral virus. the estimated probability for the root location of an ancestral pprv and individual lineages was determined as being nigeria for pprv as a whole, senegal for lineage i, nigeria/ghana for lineage ii, sudan for lineage iii and india for lineage iv. in recent years, pprv has extended its boundaries southwards in africa, as far as southern tanzania (2008) and zambia (2015) and the democratic republic of congo and angola (2012) (banyard et al. 2010; fao 2013). peste des petits ruminant outbreaks have also been reported across north africa, including tunisia (2006), morocco (2008) and algeria (2011). the hypothesis that dominant lineages may enter areas and overwhelm existing lineages of the virus is of great interest. certainly, the extensive detection of lineage iv virus in africa in recent years has led some to hypothesise that lineage iv is a dominant lineage that is replacing other lineages across different areas (banyard 2016). historically, the detection of lineage iv pprv in central africa was the first detection of this lineage on the african continent (banyard 2016). while reports of pprv infection are frequently reported in the scientific literature and to the oie, genetic characterisation is required to understand the epidemiology of the virus (banyard 2016; banyard, baron & barrett 2005). the expansion of lineage iv virus across africa has been a slow process when considering reports of genetic characterisation of this lineage in different areas. whether local extinction of other lineages occurs in africa is unclear as reports where genetic data exist for local outbreaks are few (banyard 2016). the reported expansion of lineage iv virus across central and east africa and the explosion of lineage iv virus between 2008 and 2012 across north africa have further fuelled ideas surrounding the enhanced pathogenicity of lineage iv strains of the virus (kwiatek et al. 2011). a recent study in nigeria has demonstrated an extensive distribution of lineage iv virus alongside a fairly limited distribution of the lineage ii virus that has been documented historically (mantip et al. 2016; woma et al. 2015). whether this increased representation of lineage iv virus in different regions indicates a dominance of viruses from this lineage is unclear. certainly, as with other morbilliviruses (baron 2005; barrett, banyard & diallo 2005), the determinants of virulence for pprv remain almost completely undefined and as such potential lineage-driven dominance remains speculative, especially as many regions are unable to genetically type circulating viruses. in addition, recent reports of lineages i, ii and iv in uganda (luka et al. 2012) and tanzania (parida et al. 2015), lineages ii and iv in nigeria (mantip et al. 2016; woma et al. 2015) and lineage iii in sudan suggest that other lineages of the virus remain present in the region despite the emergence of lineage iv (banyard et al. 2010). the apparent expansion of asian lineage iv across africa is supported by a constant increase in the incidence of disease, suggesting an increase in virulence (libeau, diallo & parida 2014). virus lineages circulating in africa is shown in table 1. table 1: lineages of peste des petits ruminants virus circulating in different countries of africa, based on partial n/f gene sequence analysis. diagnosis and control of peste des petits ruminants in africa peste des petits ruminants is recognised by farmers in africa through the exhibition of profuse nasal discharge and diarrhoea by infected sheep and goats, mostly manifested during the onset of the rainy season and harmattan period. a presumptive diagnosis is usually based on clinical signs. several diseases and conditions may however be confused with ppr in sheep and goats because of the similarity in clinical signs. these include rinderpest, bluetongue and contagious caprine pleuropneumonia. diagnosis of the disease may also be complicated as a result of secondary bacterial infections specifically caused by mannheimia haemolytica. therefore, in addition to clinical observations, a differential diagnosis must be confirmed by laboratory diagnostic techniques. the laboratory tests currently available for diagnosis of the disease can be grouped into three categories: (1) those detecting virus or viral antigen (e.g. virus isolation, antigen capture elisa and lateral flow devices), (2) those detecting genetic material from the virus (e.g. rt-pcr, real-time pcr and lamp [loop-mediated isothermal amplification] pcr) and (3) those detecting antibodies against the virus (e.g. virus neutralisation test, competitive elisa and indirect elisas). however, the efficiency of laboratory diagnosis can be greatly influenced by the integrity of the sample received, which is often affected by the conditions of the samples by collection and transportation. there is no specific treatment against the disease, although a broad spectrum of antibiotics can be administered against secondary bacteria that could aggravate the disease condition because of the animal’s compromised immune status. control of ppr in non-infected countries may be achieved using classical measures, such as the restriction of importation of sheep and goats from affected areas, isolation, quarantine, slaughter and proper disposal of carcasses and contact fomites and decontamination of affected premises in case of infection introduction. control of ppr outbreaks can also rely on movement control (quarantine) combined with the use of focused ‘ring’ vaccination and prophylactic immunisation in high-risk populations. homologous ppr vaccine attenuated after 63 passages in vero cells (adu et al. 1990; diallo et al. 1989b) is presently used and can produce a solid immunity for 3 years (libeau et al. 1995). the pprv homologous vaccine was found to be safe under field conditions even for pregnant animals and it induced immunity in 98% of the vaccinated animals (libeau et al. 1995). the ppr vaccine seed is available through the pan african veterinary vaccine centre (panvac) at debre-zeit, ethiopia, for african countries. recently, mariner et al. (2017) developed a ppr thermostable vaccine using the rinderpest lyophilisation method to the attenuated nigeria 75/1 ppr vaccine strain; the thermostability of the vaccine is sufficient enough to be used without a cold chain for up to 30 days, which will greatly facilitate the delivery of vaccination in the control, prevention and global eradication of ppr. recommendations a regional strategic vaccination campaign should be adopted across african countries. this can be achieved by the use of a ppr vaccine that is highly efficacious in protecting animals against all pprv strains. an epidemiological participatory technique, to assess livestock owners’ perceptions of vaccination success and serological surveys at a defined time period after vaccination to determine vaccinated animals’ immune response, should be adopted. a periodic surveillance to understand the epidemiological situation to help define the status of ppr in africa should be adopted. the purpose of this is to determine the early detection of the appearance of the disease or virus incursion; to demonstrate the absence of clinical disease or infection with pprv; and to subsequently determine and monitor the prevalence, distribution and occurrence of the disease or infection across africa. peste des petits ruminants causes an acute fatal disease in its natural host (sheep and goats), but a subclinical infection in cattle, buffaloes, camel and antelope. there is indeed a threat of the emergence of a new virulent morbillivirus of bovine and camelid, similar in manner to the way pprv has emerged in sheep and goats. host susceptibility, resistance and transmissibility to pprv are not well understood. a systems biology approach through the mapping of the ‘infectious window’, that is, the period when an infected animal is able to infect other animals, which will be useful in unravelling the host factors associated with susceptibility, resistance and transmissibility to pprv, should be carried out. this will contribute immensely in providing an instantaneous protection solution strategy and will equally contribute in avoiding unnecessary pre-emptive culling during epidemics. conclusion because of the virulent nature of ppr in the majority of infected sheep and goats, most especially in naïve populations, even with the availability of an effective attenuated vaccine, ppr is still considered a disease of higher economic importance than other small ruminant diseases in ppr-endemic areas of africa. active surveillance for ppr is not carried out and the economic impact of the disease on the african livestock industry is thus difficult to precisely quantify. nevertheless, not all farmers choose to vaccinate their animals and outbreaks of ppr amongst unvaccinated sheep and goats continue to occur on an annual basis. clinical disease amongst unvaccinated sheep and goats in the field can be severe, and it is not uncommon for sheep and goat farmers in africa to suffer significant losses amongst their flocks as a result of seasonal outbreaks of ppr. the campaign for the total eradication of ppr by fao/oie has stimulated the involvement of all the stakeholders in small ruminant production in the control, prevention and eventual eradication of ppr. acknowledgements the authors are grateful to the national veterinary research institute, vom, nigeria, for providing funding support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.e.m. was responsible for drafting the document, proofreading and manuscript preparation. s.e.m., d.s. and s.f. assisted with proofreading and manuscript preparation. all authors read and approved the final version of the manuscript. references abraham, g. & berhan, a., 2001, ‘the use of antigen – capture enzyme linked immunosorbent assay (elisa) for the diagnosis of rinderpest and peste des petits ruminants in 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investigations of a peste des petits ruminants (ppr) outbreak in afghan sheep in pakistan’, pakistan veterinary journal, 29, 174–178. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) harry a. musonye department of biochemistry, microbiology and biotechnology, kenyatta university, nairobi, kenya ezekiel m. njeru department of biochemistry, microbiology and biotechnology, kenyatta university, nairobi, kenya ahmed hassanali department of chemistry, kenyatta university, nairobi, kenya lydia m. langata department of biochemistry, microbiology and biotechnology, kenyatta university, nairobi, kenya dominic mijele department of veterinary services, kenya wildlife service, nairobi, kenya titus kaitho department of veterinary services, kenya wildlife service, nairobi, kenya edward king’ori department of veterinary services, kenya wildlife service, nairobi, kenya james nonoh department of biochemistry, microbiology and biotechnology, kenyatta university, nairobi, kenya citation musonye, h.a., njeru, e.m., hassanali, a., langata, l.m., mijele, d. et al., 2019, ‘16s rrna gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine’, onderstepoort journal of veterinary research 86(1), a1724. https://doi.org/10.4102/ojvr.v86i1.1724 original research 16s rrna gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine harry a. musonye, ezekiel m. njeru, ahmed hassanali, lydia m. langata, dominic mijele, titus kaitho, edward king’ori, james nonoh received: 24 dec. 2018; accepted: 20 feb. 2019; published: 17 july 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract several types of odours are involved in the location of host animals by tsetse (diptera: glossinidae), a vector of animal african trypanosomiasis. host animals’ ageing urine has been shown to be the source of a phenolic blend attractive to the tsetse. nevertheless, limited research has been performed on the microbial communities’ role in the production of phenols. this study aimed at profiling bacterial communities mediating the production of tsetse attractive phenols in mammalian urine. urine samples were collected from african buffalo (syncerus caffer), cattle (bos taurus) and eland (taurotragus oryx) at kongoni game valley ranch and kenyatta university in kenya. urine samples, of each animal species, were pooled and left open to age in ambient conditions. bacteriological and phenols analyses were then carried out, at 4 days ageing intervals, for 24 days. phenols analysis revealed nine volatile phenols: 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. eight out of 19 bacterial isolates from the ageing urine revealed the potential to mediate production of phenols. 16s rrna gene characterisation of the isolates closely resembled enterococcus faecalis kub3006, psychrobacter alimentarius pamc 27887, streptococcus agalactiae 2603v, morganella morganii sub.sp. morganii kt, micrococcus luteus nctc2665, planococcus massiliensis strain es2, ochrobactrum pituitosum aa2 and enterococcus faecalis ogirf. this study established that some of the phenols emitted from mammalian urine, which influence the tsetse‘s host-seeking behaviour, are well characterised by certain bacteria. these results may allow the development of biotechnological models in vector control that combines the use of these bacteria in the controlled release of semiochemicals. keywords: trypanosomiasis; tsetse; phenols; bacteria; mammalian urine. introduction animal african trypanosomiasis (aat) has for years continued to suppress livestock health and productivity in sub-saharan africa. it is an endemic parasitic disease of domestic livestock with negative impact on people’s income on the african continent. the disease covers about 11 million square kilometers of sub-saharan africa occurring in 37 countries (cecchi et al. 2009). cattle, sheep, camels, goats and pigs are the main economically important domestic species in which the infection is most commonly diagnosed (auty et al. 2015). the direct and indirect cost caused by this disease is estimated in billions of dollars (chitanga et al. 2011). four biological factors have been described as variables that manipulate epidemiology of aat in tsetse infested areas of africa. these factors are parasite, vector, livestock and reservoir hosts (van den bossche et al. 2010). trypanosoma species are the causative parasites for aat. the species include trypanosoma vivax, trypanosomabrucei brucei and trypanosoma congolense (lai et al. 2008). these parasites cause moderately mild infections in wild animals as compared to domestic animals where they cause cruel, and often fatal disease. currently, there is little evidence of successful vaccine development for the trypanosomes as the parasite apparently has the capacity to evade mammalian immune defences (esterhuizen et al. 2011; cnops, magez & de trez 2015; scolari et al. 2016). control of aat as a measure to improve the production capacities of rural-based communities in regions infested by tsetse has been addressed by other methods. the methods primarily include use of insecticides, trypanocidal drugs, trapping vectors, pesticide treatments and sterile male release strategies (hargrove et al. 2012; holmes 2013; shaw et al. 2015). nevertheless, these methods have their own weaknesses. for instance, use of trypanocidal drugs has been limited by efficacy factors because of multiple drug resistance (moti et al. 2015). another key concern is fake and substandard drugs as well as drug safety, in terms of residues in food-producing animals (baker et al. 2013). spraying of insecticides in the environment is expensive and toxic and drives the evolution of resistance in target vectors (scolari et al. 2016; touré, ramirez & sommerfeld 2015). tsetse fly species (diptera: glossinidae) belonging to the genus glossina are the main vectors of trypanosomes. the species are subgrouped into morsitans, palpalis and fusca that inhabit savannah, riverine environments and forests in that order (rayaisse et al. 2011). of the three groups of glossina spp., the savannah and riverine groups inhabit areas suitable for grazing and watering. they live among livestock and wildlife where they feed on bloodstream meal from infected hosts and pick trypanosomes, the aat disease causing parasites. the tsetse flies’ ability to detect and locate suitable hosts from which to feed on is critical for their survival and reproduction (lord et al. 2017). they achieve this through short-range visual and long-range odour sensation (omolo et al. 2009). different odour sources that include, but are not limited to, urine, faeces, skin surface, manure and the breath of cattle have been shown to enhance host location by the tsetse vector. among these, mammalian urine and the breath of cattle have been researched. in their studies, alkhaldy, edwards and combet (2018), spiehs et al. (2018) and tangtrakulwanich et al. (2015) identified phenols in mammalian urine. according to hassanali et al. (1986) and baldacchino et al. (2014), mammalian urine is composed of phenolic compounds that are odorous and highly attractive to tsetse. among the several phenols present in the urine of mammals, for instance, cattle or buffaloes, 4-cresol and 3-n-propylphenol are the most attractive to glossina spp. (madubunyi et al. 1996; owaga et al. 1988; saini 1990). however, for riverine tsetse, these phenols are not very attractive (hall et al. 1990). tsetse attractant phenols in mammalian host urine are believed to form slowly from the pro-attractants (mihok & lange 2012). these attractants have been identified as a combination of sulphates, glucuronates and other unabsorbed precursors in urine. studies have suggested the involvement of microbial communities in the breakdown of these conjugates into tsetse attractive phenols (omolo et al. 2009). the attraction of tsetse species to livestock and wildlife is therefore attributable to phenolic compounds in urine. it is essential to, therefore, profile the bacterial communities associated with the production of tsetse attractive phenols in mammalian urine to help in the generation of intervention tools in aat control and elimination. materials and methods study area sample collection was carried out at the kongoni game valley ranch (0.7754° s, 36.3715° e) and the kenyatta university cattle farm (1.1767° s, 36.9365° e) in kenya. the kongoni game valley ranch is situated in an agro-pastoralist semi-arid ecosystem and consists of mostly acacia woodland with thickets along lake naivasha. the kenyatta university cattle farm is characterised by a semi-intensive farming system. urine sample sources disease-free male and female african buffaloes (syncerus caffer), domestic cattle (bos taurus) and eland (taurotragus oryx) were used in this study. the choice of these species was influenced by the reported presence of tsetse attractive phenols in cattle and buffalo urine (hassanali et al. 1986; madubunyi et al. 1996). eland have been shown to be resistant to trypanosomiasis (pappas 2002). however, it has not been known if the animal’s urine has the potential to attract tsetse. it was therefore thought to be of interest to determine if there are tsetse attractant cues and their bio-agents in the urine. urine sample collection fresh buffalo and eland urine samples were collected in collaboration with the kenya wildlife service veterinary department. collection was opportunistic, as the selected wildlife urinated naturally, during de-snaring and translocation activities. fresh cattle urine was also collected while the animals were urinating naturally. sterile 50 ml universal bottles were used in collecting the urine samples. samples were collected over a period of 2 weeks. the samples were immediately placed in a cool box at 4 °c and transferred to the kenyatta university microbiology research laboratory for bacteriological analysis and the preparation of phenols analysis. sample preparation once in the laboratory, the 50 ml bottle urine portions collected for the 2 weeks, for each animal species, were aseptically pooled together into sterile transparent plastic bottles measuring 1000 ml. the total pooled samples for each species measured about 800 ml. these pooled samples, for each species, were then divided into two, each measuring 400 ml. one was preserved at -20 °c to defer any chemical transformation, and the other was left open and allowed to age in ambient conditions, throughout the study period (24 days), in the laboratory. evaluation of phenols generation in mammalian urine samples at ageing intervals extraction and clean-up phase of mammalian ageing urine the liquid–liquid extraction (lle) method was used in the extraction. briefly, the hydrophobic organic phase of pooled urine samples for each animal was extracted using analytical grade dichloromethane (dcm) (loba chemie, mumbai india), three times (30 ml, 30 ml and 30 ml), at intervals of 4 days, for 24 days. during the extraction, 50 ml of urine was put in a separating funnel, mounted on a stand, with the stopcock at the bottom closed; 30 ml of dcm was then added to the urine sample, in the separating funnel, to form two layers. the contents in the separating funnel were then shaken thoroughly, four times, with thumb held firmly on the stopper. during the shaking, the stopcock was opened periodically to vent the vapour pressure build up. the separating funnel was then mounted back on the stand. the stopper was taken off, the stopcock opened and the lower organic layer was drained into a beaker. these steps were repeated with two more 30 ml dcm portions. the bottom organic layers for each fermenting interval, of each study animal, were pooled together and put in conical flasks. the top hydrophilic aqueous layers were also kept separate until extraction was complete and the product isolated. further purification of the organic layers was realised using anhydrous sodium sulphate (na2so4), a drying agent. the agent was added to the organic phase in a conical flask using a spatula and swirled gently. the sulphate was added until it was seen to form floating solids from an initial clump like appearance. the mixture was left to stand for about 5 minutes to ensure complete absorption of water. complete absorption of water was determined to have been achieved after the extracted contents turned into a clear liquid, from a cloudy like solution. the dried extracted organic layer was decanted and transferred to a beaker. the layer was then subjected to a rotary pump at 40 °c to concentrate the sample. all the dcm was evaporated until organic compounds, in forms of wax-like substances, were formed at the walls of the rotary flask. thereafter, 2 ml of dcm was added into the rotary flask and swirled round to ensure collection of the wax-like substance from the walls. the substances for each sample were passed through a 0.22 µm millipore filter and the contents transferred to bijou bottles, wrapped in aluminium foils, and kept at –20 °c for subsequent analysis. gas chromatography-mass spectrometry analysis for the gas chromatography-mass spectrometry (gc-ms) analysis, a stock solution was prepared. in this preparation, 1 mg of a sample was separately weighed and dissolved in 1 ml dcm to make a stock solution, 1 mg/ml, from which an experimental sample with a final concentration of 100 ng/µl was prepared. in addition, 1 ml of internal standard, a control solution, was aliquoted and analysed. the samples were analysed by gc-ms on an isq 7000 (thermo fisher scientific, massachusetts, united states [us]), using the following operating conditions: (1) transfer line temperature of 280 °c, inlet temperature of 270 °c and column oven temperature programmed from 35 °c to 280 °c with the initial temperature maintained for 5 min, then 10 °c/min to 280 °c for 10.5 min and the final one at 50 °c/min to 285 °c for 29.9 min. (2) a hp-5 ms low-bleed capillary column fitted gc (30 m × 0.25 mm internal diameter 0.25 μm) (restek, bellefonte, us). (3) carrier gas helium at a flow rate of 1.25 ml/min. a quadruple temperature of 180 °c and an ion source temperature of 250 °c maintained by the isq 7000 mass selective detector. the mass spectrometry ion source temperature was set at 230 °c. electron impact mass spectra were achieved at acceleration energy of 70 ev. (4) one microlitre aliquot of extract was automatically injected in the split/splitless mode via an auto sampler. fragment ions were analysed over 40–550 m/z mass range in the full scan. the filament delay of 5 min was used. (5) raw data (retention time, peak area, analyte name, % quality) files were analysed and exported to the access database, used for storage and retrieval, using gc-ms software (thermo scientific’s mass frontier). in addition, a wide range of de-convolution and analysis programmes were used, bundled with ms libraries that included a mass spectral deconvolution system (amdis) with national institute of standard technology (nist) ms library (version 2.0, 2011) and tag finder. bacterial isolation during urine ageing intervals one millilitre of urine portion was drawn from each of the ageing urine samples for bacteriological analysis. this was carried out at an ageing interval of 4 days, for 24 days. each urine sample was aseptically subjected to serial dilution. aliquots of 0.1 ml, making a 10-6 dilution factor, were aseptically pipetted and each inoculated by spread plate method on the cysteine–lactose–electrolyte-deficient (cled) medium (oxoid, basingstokes, uk medium) in threes. the plates were subsequently inverted and incubated for 24 hours at 37 °c. for purification, morphologically different colonies were sub-cultured on nutrient agar (na) (himedia, mumbai india) plates by streak plating. the isolated cultivable bacterial cultures were grouped into different groups based on their morphological appearance on cled and na. the groups were then preserved on glycerol stock at –20 °c in 20% (v v-1) for further analysis. screening bacterial isolates for their ability to mediate production of phenols in urine samples of the selected mammals to screen the bacterial isolates, collected, fresh urine under refrigeration was obtained and subjected to sterilisation using 0.22 nm millipore filters. aliquots of 50 ml sterilised urine were aseptically transferred into 500 ml erlenmeyer flasks. a consortium of isolated morphologically different bacterial communities was inoculated in sterilised urine portion and incubation was carried out for 3 days at room temperature. each of the isolated bacterium was also subjected to the sterilised urine separately. the involved bacteria were tested by inoculating aliquot portions of pooled fresh sterile urine with 10 μl of 24-h-old cultures of the isolated bacterial cell suspension, 1 × 104 colony forming unit (cfu)/ml, in a 0.9% nacl solution to obtain a final concentration of 1 × 1011 cfu/ml. these treatments were then incubated at room temperature, under aerobic conditions. the isolates, purity in each flask was established by inoculating a portion onto na on each extraction day and examining these following a period of growth. in this analysis, blanks were used for control studies. the blanks were obtained through aseptic incubation of replica sterile urine for 3 days at room temperature. for phenols assays, 50 ml of each sample was obtained from test flasks after incubation and extracted with dichloromethane. the extracts were examined by gc-ms and the peak matching the major phenolic components of the selected animals’ urine was checked for in the chromatograms. molecular characterisation of bacteria screened for phenols production genomic dna (gdna) was directly extracted from bacterial colonies obtained from a pure culture on a na plate. the extraction and purification was done using a dneasy tissue kit (qiagen, hilden german), following the manufacturer’s instructions. the dna samples were then stored at –20 °c for further use. a polymerase chain reaction (pcr) amplification of the small subunit (ssu) 16s rrna gene was carried out using 27-forward (5’-agagtttgatcmtggctcag-3’) and 1492-reverse (5’-cggttaccttgttacgactt-3’) primers. the reaction was performed in a 50 µl volume, containing 10×pcr buffer (5 µl), 2 µl each of primers, 2 mm dntp mixture (2.5 µl), taq dna polymerase (1 µl), dho (pcr water), up to 50 µl, and template dna (1 µl). the pcr conditions were pre-denaturation at 94 °c for 5 min, 36 cycles of denaturation at 94 °c for 1 min, annealing at 54 °c for 1 min and extension at 72 °c for 2 min, and finally extension at 72 °c for 10 min. genomic dna (gdna) and pcr products, 5 µl each, were inspected for quality by gel electrophoresis. the 100-bp dna ladder, for pcr products, and 1-kb dna ladder, for gdna (fischer scientific, united kingdom [uk]), were loaded along with samples on their respective gels as molecular weight markers. the products were then visualised under ultra violet (uv) trans-illuminator light. the gels were then photographed using a digital photograph. purification of the pcr products was carried out using exonuclease-shrimp alkaline phosphatase (exo-sap mix) reagent following the manufacturer’s instructions. after purification, the pcr products were then sequenced. the primers used for sequencing were 27f and 1492r for the 16s rrna gene. sequences were generated by sanger (capillary) sequencing, using the ab1 3730 dna sequencer (applied biosystems, us). data analysis data obtained from the bacterial isolates ability to mediate phenols production were subjected to analysis of variance (anova) using statistical analysis software (sas) (2010). means pair-wise comparison was carried out using tukey’s hsd (honestly significant difference) at 5% level. generated sequences were assembled and edited using finch tv (mishra et al. 2010). consensus (contig) building was carried out using dna baser software (zhang, fu & zhang 2012). for describing the isolates’ phylogenetic relationship, the 16s rrna gene sequences were aligned using clustal x version 2 software (larkin et al. 2007), together with related sequences obtained from the ncbi genbank database using the blast search program (altschul et al. 1997). the evolutionary history of the isolates was then presented in the form of a phylogenetic tree. the tree was inferred, based on the jukes–cantor model, by the maximum likelihood method (jukes & cantor 1969), at 1000 iterations, using mega 7 (kumar, stecher & tamura 2016). nucleotide sequence accession number: the 16s rrna sequences for the isolates in this study have been deposited in the ncbi genbank under accession numbers mk123487-mk123505. ethical considerations authority to conduct the research was approved by the national commission for science, technology and innovation (nacosti/p/17/73722/18052). results phenolic compounds in dichloromethane extracted urine demonstration of formation of phenols at urine ageing intervals showed several peaks in the chromatogram. the peaks indicated the presence of phenolic compounds and other non-phenolic compounds (peaks 10 and 11). through the study, a total of nine volatile phenolic compounds were detected in the urine headspace. the compounds were identified as 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. a representative chromatogram of the phenols identified with their peak identities is shown in figure 1. figure 1: gas chromatography-mass spectrometry chromatogram for phenolic extracts of day twenty buffalo, cattle and eland urine samples. (a) buffalo; (b) cattle; (c) eland. peaks identities: (1) 4-cresol, (2) phenol, (3) ortho-cresol, (4) 3-cresol, (5) 4-ethylphenol, (6) 3-ethylphenol, (7) 3-propylphenol, (8) 4-propylphenol and (9) 2-methyloxyphenol. bacterial isolates ability to mediate production of phenols in mammalian urine samples a total of 19 morphologically different bacteria colonising ageing mammalian urine were isolated. urine fermentation with mixed bacteria (m.b) revealed the presence of eight phenolic compounds (table 1). a comparison between individual isolates incubated in sterile urine samples and that of mixed culture samples showed some similarities in the type of phenolic compounds detected. the resultant phenolic concentrations varied, at p ≤ 0.05 when compared across test bacteria. of the 19 bacteria previously isolated, eight revealed a significant association with volatile phenols (table 1). these included b2, b22, e11, e49, b25, b34, b42 and b70. the volatile phenols of biological origin detected in urine samples incubated with these bacteria were also detected in naturally colonised mammalian urine samples (figure 1). table 1: quantitative and qualitative analysis of phenolic compounds using cattle, buffalo and elands’ sterile fresh urine incubated with various bacterial isolates. as shown in table 1, para-cresol was significantly present in urine samples incubated with e11 (425989.00 mg/l), e49 (170206.67 mg/l), b25 (333066.00 mg/l), b34 (104538.67 mg/l), b70 (432655.67 mg/l) and m.b (159542.33 mg/l), at (p ≤ 0.0001). o-cresol was only observed in urine samples incubated with b2 (98100.67 mg/l) and m.b (141549.33 mg/l), at p ≤ 0.0001. the m-cresol was revealed in the presence of b34 (35703.33 mg/l) and m.b (165767.33 mg/l), at p ≤ 0.0001. the 3-ethylphenol was recorded when urine was subjected to b22 (273865.33 mg/l) and m.b (163582.00 mg/l) at p ≤ 0.0001. 4-ethylphenol was revealed in urine incubated with b22 (216816.33 mg/l) and m.b (87753.67 mg/l), at p ≤ 0.0001. 3-propyphenol was significantly associated with e11 (543339.67 mg/l), b25 (97421.33 mg/l), b42 (27813.33 mg/l), b70 (546006.33 mg/l) and m.b (273865.33 mg/l), at p ≤ 0.0001. 4-propylphenol was significant in m.b (98887.67 mg/l), e11 (13797.3 mg/l) and b70 (12797.33 mg/l), at p ≤ 0.0001. phenol was significant at e11 (133811.3 mg/l), b70 (127144.67 mg/l), m.b (42106.67 mg/l), b25 (122922.33 mg/l) and e49 (14589.0 mg/l), at p ≤ 0.0001. molecular characterisation of bacterial isolates screened for potential to mediate production of phenols the molecular characterisation with 16s rrna gene confirmed the bacteria to belong to different species and strains. molecular analysis of the 16s rrna gene of bacteria associated with phenols production authenticated the existence of a varied population of bacteria in cattle, buffalo and eland urine at different ageing intervals. genomic dna extracted from pure bacterial cultures showed an intact band when loaded to an 0.8% agar rose gel run at 80 v for about 30 min. the 1500 bp amplified region of 16s rrna gene revealed different band intensities when visualised in 1.2% agar rose gel run at 80 v for 1 h. at approximately 1500 bp, all pcr products showed a definite and appropriately sized band in all lanes. on sequencing the 19 bacterial isolates, 14 different species belonging to different strains were revealed. the query sequence in the ncbi genebank database, using blast based on 16s rrna gene sequencing, showed the relatedness of the tested organism with the same identity within different genera (table 2). table 2: bacterial strains showing significant similarity with mammalian urine bacterial isolates tested for their ability to mediate production of phenols. as shown in table 2, the phylogenetic relationship of the isolates revealed the presence of the planococcus massiliensis strain es2 (99% 16s rrna gene sequence similarity with isolate b2); psychrobacter alimentarius pamc 27889 (99% 16s rrna gene sequence similarity with isolate b22); providencia rettgeri rb151 (99% 16s rrna gene sequence similarity with isolate b5); enterococcus faecalis og1rf (99% 16s rrna gene sequence similarity with isolate b70); enterococcus faecalis kub3006 (90% 16s rrna gene sequence similarity with isolate e11); morganella morganii subsp. morganii kt (99% 16s rrna gene sequence similarity with isolate b25); micrococcus luteus nctc 2665 (99% 16s rrna gene sequence similarity with isolate b34); ochrobactrum pituitosum aa2 (92% 16s rrna gene sequence similarity with b42); alcaligenes faecalis jq135 (99% 16s rrna gene sequence similarity with isolate b74), streptococcus agalactiae 2603v/r (99.9% 16s rrna gene sequence similarity with isolate e49), bacillus pumilus glb197 (99% 16s rrna gene sequence similarity with isolate b10); bacillus cereus mly1 (99% 16s rrna gene sequence similarity with isolate b12); bacillus cereus cmcc p0021 (99% 16s rrna gene sequence similarity with isolate b71); bacillus amyloliquefaciens subsp. plantarum ucmb5033 (99% 16s rrna gene sequence similarity with isolate b73); bacillus cereus atcc 4342 (99% 16s rrna gene sequence similarity with isolate e8); bacillus cereus issfr-3f (99% 16s rrna gene sequence similarity with isolate b7); bacillus cereus m3 (99% 16s rrna gene sequence similarity with isolate e51); bacillus megaterium yc4-r4 (99%16s rrna gene sequence similarity with isolate e48) and bacillus safensis u17-1 (99% 16s rrna gene sequence similarity with isolate e53). evolutionary relatedness of the 19 bacterial isolates screened for potential to mediate production of phenols in mammalian urine was evaluated based on their 16s rrna gene. a phylogenetic tree based on these isolates is shown in figure 2. the bacteria were grouped into seven clusters. the clusters were grouped based on isolates similarity in relation to mediation of urine volatile phenols. furthermore, some bacterial communities were clustered depending on when they were isolated from urine, whereas others were grouped along their genotypic relatedness. figure 2: molecular phylogenetic analysis of bacterial communities with and without the potential to mediate production of phenols in mammalian urine by the maximum likelihood method. clusters 1 and 2 exclusively contain bacterial communities that showed negative results for phenol mediation. also, worthy of note is that bacteria that fell under these clusters were isolated between days, 16 and 24 of ageing mammalian urine. the two clusters were supported by bootstrap values of 100% and 56%, respectively. cluster 1 comprised of isolates b7, e51, e8, b12 and b71, whereas cluster 2 was represented by isolates e48, b73, b10 and e53. the closed neighbours for all the isolates in these two clusters were dominated by different members of the bacillus spp. bacteria having ability to mediate formation of phenols in mammalian urine were grouped in closely related phylogenetic positions. they were grouped together in clusters 3, 4, 6 and 7. the clusters were supported by bootstrap values of 99%, 100%, 87% and 91%, respectively. cluster 3 was represented by isolates e11 and b70. these isolates were closely related to different strains of enterococcus faecalis. clusters 4 and 6 were represented by only one isolate each, b34 and b42, respectively. the close neighbour for isolate b34 was micrococcus luteus nctc 2665, while that for b42 was ochrobactrum pituitosum aa2. cluster 7 comprised of three isolates, b25, b2 and e49, whose close neighbours were morganella morganii subsp. morganii kt, planococcus massiliensis strain es2 and streptococcus agalactiae 2603v/r in that order. cluster 5, supported by a bootstrap value of 91%, comprised of isolates b22, b5 and b74 represented by closest neighbours, psychrobacter alimentarius pamc 27889, providencia rettgeri rb151 and alcaligenes faecalis jq135, respectively. nevertheless, a unique trend was noted with this cluster. not all the representative isolates were positive for mediation of phenols production. isolates b5 and b74 showed negative results when tested for potential to mediate production of phenols in mammalian urine (table 1). in terms of genotypic relatedness, these isolates were distantly related to their counterparts in clusters 1 and 2. the evolutionary history was inferred by using the maximum likelihood method based on the jukes–cantor model (jukes & cantor 1969). the bootstrap consensus tree inferred from 1000 replicates (felsenstein 1985) is taken to represent the evolutionary history of the taxa analysed (felsenstein 1985). the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (felsenstein 1985). initial tree(s) for the heuristic search were obtained automatically by applying neighbour-join and bionj algorithms to a matrix of pair-wise distances estimated using the maximum composite likelihood approach and then selecting the topology with a superior log likelihood value. the analysis involved 39 nucleotide sequences. there were a total of 1494 positions in the final dataset. evolutionary analyses were conducted in mega7 (kumar et al. 2016). one representative sequence from each urine isolate operational taxonomic units (otus) was included in the tree along with the sequence from the ncbi database. nd (not detected) is indicated against isolates that were negative for phenols production. bacterial isolates with potential to mediate phenols production are indicated against their associated phenols in brackets. the far left values, from 1 to 7, show the number of clusters. discussion phenolic compounds in dichloromethane extracted urine the presence of phenolic compounds in dcm urine extracts of this study confirmed that mammalian urine has a large range of phenolic compounds. these findings are in agreement with previous studies that identified phenols in animal urine (alkhaldy et al. 2018; spiehs et al. 2018; tangtrakulwanich et al. 2015). the presence of tsetse attractant cues in eland ageing urine provides more information on which animals to target when controlling tsetse. eland have been shown to be resistant to trypanosomiasis (pappas 2002). however, it has not been known if the animal’s urine has the potential to attract tsetse. some of the phenolic compounds detected in this study, for instance, phenol, 4-cresol and 3-propylphenols, have been previously linked to morsitans flies behavioural response (brightwell, dransfield & kyorku 1991; hassanali et al. 1986; madubunyi et al. 1996; owaga et al. 1988; vale, hall & gough 1988). bacterial isolation at urine ageing intervals this study was able to cultivate 19 morphologically different bacteria in ageing urine samples when compared to the study carried out by íñigo et al. (2016). in their research, they sought to characterise microbes found in human urine using culture independent methods. they reported 336 bacterial species in the urine samples. the lower number of bacteria identified in this study, as compared to íñigo et al.’s (2016) findings, can be attributed to the variation in the species under study or in the methods used to culture and identify the isolates. bacterial isolateʼs ability to mediate production of phenols in urine samples of the selected mammals when the sterilised fresh urine was fermented with a mixture of isolated bacteria, the amounts of detected phenols were varied. in a study involving mixed bacterial cultures and microbes in soil, kai et al. (2009) observed that the cultures had potential to produce diverse volatile organic compounds. the differences in the concentration of phenols, in this study, can be attributed to multivariate factors. for example, there must have been competition among the mixed bacteria, where some inhibited activities of their neighbours who might have been responsible for mediation of phenols production. andreev et al. (2017) documented a study related to this suggestion. some of the bacteria could also not be producing any volatiles but are essential for the existence of others that mediate phenols production in urine. variation in the amounts of detected phenols can also be attributed to the presence of microbes that naturally prevent the formation of phenols. this is likely achieved through enzymatic activities of the associated bacteria. the activities work against the production of phenols by other species. such a phenomenon has been reported by troccaz et al. (2013). urine inoculated with a consortium of bacteria may be comprising bacteria that mediate production of detected phenols as well as those that break the phenols down once they are generated. these findings suggest that different bacterial communities co-existing in urine are essential determinants for the release of volatile organic compounds’ complexes needed to elicit tsetse’s behavioural response. when the sterilised urine samples were inoculated with individual bacteria for 3 days, the results revealed a potential of eight isolates to mediate production of volatile phenols. these findings concur with previous studies (schulz & dickschat 2007; troccaz et al. 2013). troccaz et al. (2013) profiled different bacterial volatiles in human urine while in their study that sought to establish different volatiles emitted by bacteria, schulz and dickschat (2007) associated about 65 percent of bacteria investigated with production of the volatiles. in addition, tasin et al. (2018) associated volatiles that determine the behavioural role of insects to microorganisms. the presence of volatile phenols from specific bacteria inoculated in urine samples may be attributed to multiple factors. firstly, it might be because of a difference in nutritional activities of different bacterial species inoculated in urine samples as suggested by tasin, knudsen and pertot (2012) and teira et al. (2011). in a nutrients manipulation experiment, teira et al. (2011) demonstrated that bacterial phylogenetic groups react differently to an alteration in nutrients. secondly, the bacterial individual metabolism could be linked to a difference in the phenol’s profiles observed. by the use of metabolic pathways of glycolysis, or degradation of amino acids, bacteria are able to generate aromatic compounds (schulz & dickschat 2007). according to todar (2012), many volatile organic compounds are generated during primary and secondary metabolism in microorganisms. therefore, the production of secondary metabolic phenols in mammalian urine can be said to be limited to a certain phylogenetic group or are species-specific. characterisation of bacterial isolates with potential to mediate production of tsetse attractive phenols in mammalian urine the significant similarity of bacterial isolates that was positive for mediation of production of phenols, with enterococcus faecalis kub3006, psychrobacter alimentarius pamc 27887, streptococcus agalactiae 2603v, morganella morganii sub.sp. morganii kt, micrococcus luteus nctc2665, planococcus massiliensis es2, ochrobactrum pituitosum aa2 and enterococcus faecalis ogirf suggests the potential of these bacterial communities to mediate production of phenols in mammalian urine. these findings are supported by earlier studies by alkhaldy et al. (2018), schoefer et al. (2003), schneider and blaut (2000), schneider et al. (1999) and bokkenheuser, shackleton and winter (1987) who demonstrated the ability of different bacteria to metabolise phenols and other volatiles. in this study, bacteria having the ability to mediate the formation of phenols were clustered in closely related phylogenetic positions. this clustering suggests that bacterial communities with potential to mediate production of phenols are restricted to a limited phylogenetic group. therefore, the bacteria may have evolved to colonise mammalian urine where they mediate production of volatile phenols that influence the tsetse’s host-seeking behaviour. in addition, biochemical pathways that are responsible for the production of phenols by bacterial species may be conserved across taxonomic groups of these bacteria. isolate b2, which was closely related to psychrobacter alimentarius pamc 27887, may have retained critical genes that code for the mediation of production of phenols. unlike its counterparts in that cluster, this isolate showed potential for the mediation of production of phenols. enterococcus faecalis strains showed potential to produce 4-cresol, phenol and 3-propylphenol. these phenols have been previously reported to attract tsetse to their host animals (bursell et al. 1988; gikonyo et al. 2000; hassanali et al. 1986). these results closely relate to a study carried out by trocazz et al. (2013) who documented the role of enterobacteriaceae in the production of p-cresol, phenol and other volatiles in human urine. the origin of enterococcus faecalis varies from animals, environmental and human sources. however, the intestinal tract of animals and humans is the natural habitat of this bacterium (klein 2003). the bacterium is tolerant to a wide range of stress and can survive for a long time outside their natural intestinal hosts (arias & murray 2012). this long interaction with fermenting urine provides a platform for the generation of urine phenols. production of phenols suggests this bacterium’s ecological importance in the tsetse’s host-seeking behaviour and transmission of aat. morganella morganii sub.sp. morganii, just like enterococcus faecalis strains, showed the potential to generate 4-cresol, phenol and 3-propylphenol. these phenols have an ecological role in the attraction of tsetse vectors to the host (bursell et al. 1988). these findings are comparable to a study carried out by marshall et al. (2016), engl and kaltenpoth (2018). in their study, the researchers established that morganella morganii produces phenol as a sex pheromone of the new zealand grass grub (costelytra zealandica). morganella morganii is often found in the human gut and animal gut (jones-dias et al. 2016). the bacterium has also been found commonly in the livestock environment (fischer et al. 2016; rodrigues et al. 2017; wang et al. 2017). through its interaction with animals, it is likely that this bacterium gains access to mammalian urine where it mediates production of associated urine phenols. in this study, streptoccoccus agalactiae 2603v bacterium showed the potential to mediate production of phenol and 3-propylphenol which are known to attract tsetse. the incidences of this bacterium in the study samples compares well with other findings. for example, production of 3-methylbutanoic acid volatiles in sterilised milk strongly correlated with the growth of staphylococcus aureus when mixed with staphylococcus agalactiae (chen et al. 2018). the bacterium has been isolated in different bovine environments (holmøy et al. 2018; miranda et al. 2018; reyes et al. 2017; skjstrup et al. 2018; svennesen et al. 2019). there is a possibility that the prevalence of this bacterium in the bovine environment creates an opportunity for the bacterium to colonise mammalian urine and thus mediate phenols’ formation. micrococcus leteus, nctc 2665, in this study, showed the ability to generate 4-cresol and 3-cresol. previous studies, for example, field experiments involving tsetse behavioural responses by hassanali et al. (1986) profiled 4-cresol from the excretory products of buffalo as a fundamental component of the tsetse host location. mediation of the production of phenols, especially 4-cresol, by this bacterium in this study may explain earlier reports of the involvement of skin bacteria in the production of volatiles that determine the host-seeking behaviour of some insects (verhulst et al. 2011). the micrococcus spp. main natural habitat is mammalian skin; the secondary habitat is dairy products, meat, soil and water (grice et al. 2009; grice & segre 2011). in related studies that targeted the analysis of mosquito attractive cues in animals and human skin bacteria, busula et al. (2017) reported the ability of volatiles, generated by bacteria on cow skin, to attract mosquito species. it is therefore noteworthy to suggest that this bacterium plays a role in the production of phenols in wildlife and livestock when urine comes into contact with animal skin. this study established that psychrobacter alimentarius pamc 27889 constitutes a proportion of ageing mammalian urine flora. the bacterium showed the ability to generate 3and 4-ethylphenols. the 3-ethylphenol has been previously attributed to the attractiveness of cattle urine to g. pallidipes austen and glossinna morsitans morsitans westwood (bursell et al. 1988). a study carried out by broekaert et al. (2013) established sea food spoilage to volatile compounds associated with psychrobacter spp. the researchers established volatile compounds that were linked to psychrobacter spp. in a related study, trexler et al. (2003) reported that gravid aedes abopictus oviposited more often in water inoculated with a member of psychrobacter spp. members of psychrobacter spp. have been previously isolated in diverse environments (hamm et al. 2016; meziti et al. 2010; yang et al. 2017). the association of pyschrobacter spp. with bovine environment could be a favourable prospect for psychrobacter alimentarius pamc 27889 to colonise mammalian urine, which supports the formation of urine phenols. ochrobactrum pituitosum aa2 bacteria were found to have the potential of generating 3-propylphenol, which is a critical compound of tsetse attraction to the host. a study involving rhizospheres profiled this bacterium as having the ability to produce volatile organic compounds that promote plant growth (baysal & silme 2017). members of ochrobactrum spp. have been isolated from several ecological niches, for instance, water, soils, animals, plants and humans (dini-andreote et al. 2014; kulkarni et al. 2017). planococcus massiliensis strain es2, in this study, was found to have the potential to generate o-cresol when inoculated in sterilised urine. currently, it is not clear if the o-cresol has the potential to attract tsetse. brega et al. (1990) documented the presence of this particular phenolic compound in human urine even though the presence was not linked to this bacterium. this bacterium was previously isolated in the human gut, sea water, clamp prawns and the marine environment (seck et al. 2016; too et al. 2017). the ability to survive in different environments may be related to their presence in the fermenting urine of mammals where they are associated with the production of o-cresol as established in this study. conclusion this study sought to profile bacterial communities in mammalian urine that mediate the production of tsetse attractive phenols. the study revealed that some bacterial communities colonising mammalian urine seem to be well characterised by certain volatile phenolic compounds. the bacteria identified, that appeared to mediate production of tsetse attractive phenolic compounds in mammalian urine, include psychrobacter alimentarius pamc 27887, enterococcus faecalis kub3006, streptococcus agalactiae 2603v, morganella morganii sub.sp. morganii kt, micrococcus luteus nctc 2665, ochrobactrum pituitosum aa2 and enterococcus faecalis ogirf. the study opens potential pathways of reducing the attraction of savannah tsetse to host animals and enhancing ‘push–pull’ models in vector control that combines the use of repellents on host animals and attraction to traps. this study hypothesises that tsetse attraction to livestock and wildlife can be controlled significantly by achieving an optimum solution in the reduction of bacteria that mediate the production of phenols. this may enhance the controlled release of the semiochemicals in the grazing fields. however, more research needs to be carried out to substantially reduce the attraction of tsetse to their host animals. acknowledgements the authors acknowledge the kenya wildlife service veterinary department for their guidance during wildlife sample collection. the authors are indebted to the international centre of insect physiology and ecology (icipe) in nairobi for providing space for phenols analysis. they also thank the department of chemistry, kenyatta university, for allocating laboratory space for extraction. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions h.a.m., j.n., e.m.n. and a.h. conceived the idea for the study, designed the experiment, and prepared and reviewed the manuscript. h.a.m., l.m.l., d.m., t.k. and e.k. collected the data. h.a.m. performed the experiments and analysed the data. all authors read and approved the final manuscript. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement the data generated during the current study is not publicly available because it was part of the authors’ msc. thesis which is still under examination at 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asian-australasian journal of animal sciences 30(11), 1660. https://doi.org/10.5713/ajas.17.0553 zhang, y., fu, b. & zhang, x., 2012, ‘dna cryptography based on dna fragment assembly’, in 2012 8th international conference on information science and digital content technology (icidt 2012), ieee, jeju, south korea, june 26–28th, 2012, vol. 1, pp. 179–182. junker_315_337.indd introduction guineafowls (numididae) originated on the african continent, and with the exception of an isolated population of helmeted guineafowls in north-west moroc co, their natural distribution is restricted to subsaharan africa (del hoyo, elliott & sargatal 1994). in the wake of commercial game bird farming, but also as ornamental birds in aviculture, they have been introduced to many other parts of the world, such as france, hungary, italy, greece, the united kingdom, the usa, australia and different regions of the former ussr (haziev & khan 1991). according to belshaw (1985) guineafowls were imported into the southern mediterranean region several millennia before turkeys and hundreds of years before junglefowls from which today’s domestic chickens were derived. currently four genera of guineafowls are recognized, namely acryllium gray, 1840, agelastes bonaparte, 1850, guttera wagler, 1832 and numida linnaeus, 1766 (del hoyo et al. 1994). many publications on the helminth fauna of guineafowls originate from northern and western africa, where, second only to the introduced and native domestic fowls, they are farm-reared as a source of protein. the economic importance of guineafowls and domestic fowls within the poultry industry, as well as the fact that domestic fowls are kept by many private households to augment their income, necessitated a better understanding of factors, such as gastro-intestinal parasites, influencing the success315 onderstepoort journal of veterinary research, 74:315–337 (2007) a check list of the helminths of guineafowls (numididae) and a host list of these parasites k. junker and j. boomker* department of veterinary tropical diseases, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract junker, k. & boomker, j. 2007. a check list of the helminths of guineafowls (numididae) and a host list of these parasites. onderstepoort journal of veterinary research, 74:315–337 published and personal records have been compiled into a reference list of the helminth parasites of guineafowls. where data on other avian hosts was available these have been included for completeness’ sake and to give an indication of host range. the parasite list for the helmeted guineafowls, numida meleagris, includes five species of acanthocephalans, all belonging to a single genus, three trematodes belonging to three different genera, 34 cestodes representing 15 genera, and 35 nematodes belonging to 17 genera. the list for the crested guineafowls, guttera edouardi, contains a single acanthocephalan together with 10 cestode species belonging to seven genera, and three nematode species belonging to three different genera. records for two cestode species from genera and two nematode species belonging to a single genus have been found for the guineafowl genus acryllium. of the 70 helminths listed for n. meleagris, 29 have been recorded from domestic chickens. keywords: acanthocephalans, cestodes, check list, guineafowls, host list, nematodes, trematodes * author to whom correspondence is to be directed. e-mail: joop.boomker@up.ac.za accepted for publication 9 may 2007—editor 316 check list of helminths of guineafowls (numididae) and host list of parasites ful rearing of these birds. consequently studies have been conducted to assess the extent to which guineafowls and domestic fowls can serve as alternative hosts for their respective helminths and possibly be adversely affected by them (hodasi 1969, 1976; fabiyi 1972; fatunmbi & olufemi 1982; vercruysse, harris, bray, nagalo, pangui & gibson 1985). in southern africa ortlepp (1937, 1938a, b, 1963), saayman (1966), crowe (1977) and verster & pta sins ka-kloryga (1987) have published on the helminth fauna of guineafowls. no data on the helminths infecting species of the guineafowl genus agelastes could be found, and we are of the opinion that the comparatively short parasite lists for the genera acryllium and guttera reflect a lack of data rather than an absence of parasites. the check list herein is intended as a quick reference aid and is split into two sections. the first section con tains the parasites listed under their scientific names and authorities. synonyms are provided either as generic synonyms in the case where whole genera have been synonymized or specific synonyms. the second section lists the hosts and their synonyms alphabetically, together with their respective parasites, also in alphabetical order. the synonymy of the acanthocephalan genus med iorhynchus van cleave 1916 is as given by van cleave (1947) and schmidt & kuntz (1977) and specific synonymy is according to yamaguti (1963). for an in-depth review of the involved history of this genus’s nomenclature the reader is referred to van cleave (1947). the taxonomy of digenean trematodes follows yamaguti (1958), but since the application of molecular techniques to this group has recently led to many changes, the reader is encouraged to consult the latest literature. the classification of cestodes is based on the works of khalil, jones & bray (1994). information on generic synonyms and type species follows khalil et al. (1994), while that on other species as well as the hosts and geographic distribution has mainly been derived from yamaguti (1959), schmidt (1986) and additional published records. as regards nematode taxonomy, the authors have followed the cih keys to the nematode parasites of vertebrates (anderson, chabaud & willmott, 1974– 1983) and, where differences have occurred, have accepted the validity of genera and species as listed by gibson (2005). with regard to generic synonyms, only synonyms listed in the cih keys and by gibson (2005) have been included in the check list. specific synonyms, type species and other species, as well as much of the data on hosts and geographic distribution are according to yamaguti (1961) and gibson (2005). host and geographic data have been supplemented by including additional literature references. the families and subfamilies of cestodes and nematodes are listed according to the system of khalil et al. (1994) and the cih keys, respectively, but genera within these families are presented in alphabetical order. synonyms have been arranged chronologically. the hosts and geographic localities per author are listed alphabetically. if several authors made reference to the same host, the authors are listed in chronological order. the nomenclature and taxonomy of the avian hosts mainly follows peterson (1999) and has been supplemented by lepage (2007). avian orders and families, as well as the nomenclature of southern african hosts follow hockey, dean & ryan (2005). in order to avoid excessive duplication, helmeted guineafowls are listed below as n. meleagris only without regards to the subspecies. a total of nine subspecies of n. meleagris are currently recognized (del hoyo et al. 1994, peterson 1999). these are: n. m. coronatus gurney, 1868, n. m. galeatus pallas, 1767, n. m. marungensis schalow, 1884, n. m. meleagris (linnaeus, 1758), n. m. mitratus pallas, 1767, n. m. papillosus reichenow, 1894, n. m. rei chenowi ogilvie-grant, 1894, n. m. sabyi hartert, 1919 and n. m. somaliensis neumann, 1899. del hoyo et al. (1994) give a detailed list of the geographic range of the various subspecies of helmeted guineafowls. in the case of the crested guineafowls, guttera edouardi (hartlaub, 1867) we follow hockey et al. (2005) and lepage (2007). crowe (1978, cited in hockey et al. 2005) had synonymized g. edouardi with guttera pucherani (hartlaub, 1861), but this decision was reversed and g. edouardi reinstated (little & crowe 2000, cited in hockey et al. 2005). peterson (1999) still lists g. edouardi as a subspecies of g. pucherani. hosts listed in the literature as gallus domesticus or gallus gallus domesticus are referred to below as domestic chicken. lepage (2007) lists domestic chicken as unconfirmed subspecies, g. g. domesticus (no authority given), of the red junglefowl, gallus gallus (linnaeus, 1758). however, this subspecies is not included in the five subspecies listed by peterson (1999). 317 k. junker & j. boomker parasite/host check list phylum acanthocephala class archiacanthocephala order gigantorhynchidea family gigantorhynchidae hamann, 1892 genus mediorhynchus van cleave 1916 echinorhynchus zoega in müller, 1776, in part; gigantorhynchus hamann,1892, in part; heteroplus kostylev, 1914; empodius travassos, 1916; micracanthorhynchus travassos, 1917; lei pe racanthus bhalerao, 1937; disteganius lehmann, 1953, nomen nudum; empodisma yamaguti, 1963 type species: mediorhynchus papillosus van cleave, 1918 1. mediorhynchus empodius (skrjabin, 1913) meyer, 1933 ardea, ardeotis arabs, numida meleagris yamaguti (1963), belgium, russia 2. mediorhynchus gallinarum (bhalerao, 1937) van cleave, 1947 domestic chicken yamaguti (1963), india, philippines talbot (1971), papua and new guinea gallinaceaous birds schmidt & kuntz (1977), (east-) africa, india, papua and new guinea, philippines numida meleagris junker & boomker (2006), south africa 3. mediorhynchus numidae (baer, 1925) meyer, 1933 numida meleagris meyer (1932), namibia oosthuizen & markus (1967), south africa 4. mediorhynchus selengensis harris, 1973 numida meleagris vercryusse et al. (1985), burkina faso schmidt & kuntz (1977) synonymized this spe cies with m. gallinarum. 5. mediorhynchus taeniatus (von linstow, 1901) dollfus, 1936 empodius segmentatus de marval, 1902 ardeotis arabs dollfus (1951) in yamaguti (1963), mauritania chlamydotis undulata dollfus (1951) in yamaguti (1963), morocco guttera edouardi southwell & lake (1939), democratic republic of the congo numida meleagris von linstow (1901), kenya meyer (1932), africa, malawi southwell & lake (1939), democratic republic of the congo graber (1959), chad fabiyi (1972), nigeria hodasi (1976), ghana crowe (1977), south africa burhinus oedicnemus, chlamydotis macquenii, otis tarda meyer (1932), africa, malawi phylum plathyhelminthes class trematoda order digenea family brachylaemidae joyeaux & foley, 1930 genus postharmostomum witenberg, 1923 type species: postharmostomum gallinum (witenberg, 1923) 1. postharmostomum gallinum (witenberg, 1923) crested guineafowl khan, khan & rayaz (1984), pakistan domestic chicken yamaguti (1958), hawaii, japan, russian turkestan numida meleagris yamaguti (1958), north africa family dicrocoeliidae odhner, 1911 genus dicrocoelium dujardin, 1845 type species: dicrocoelium lanceatum stiles & hassal, 1898 1. dicrocoelium macrostomum odhner, 1911 coturnix coturnix yamaguti (1958), russia numida meleagris lesbouyries (1941), egypt junker & boomker (2007b), south africa genus lutztrema travassos, 1941 type species: lutztrema olliquum (travassos, 1917) numida meleagris hodasi (1976), ghana class cestoda subclass eucestoda order cyclophyllidea family davaineidae braun, 1900 subfamily davaineinae braun, 1900 genus abuladzugnia spasskii, 1973 type species: abuladzugnia gutterae (ortlepp, 1963) 1. abuladzugnia gutterae (ortlepp, 1963) cotugnia gutterae ortlepp, 1963 guttera edouardi ortlepp (1963), mozambique numida meleagris junker & boomker (2007b), south africa 318 check list of helminths of guineafowls (numididae) and host list of parasites 2. abuladzugnia transvaalensis (ortlepp, 1963) cotugnia transvaalensis ortlepp, 1963 numida meleagris ortlepp (1963), south africa genus cotugnia diamare, 1893 ershovitugnia, spasskii, 1973; pavugnia spasskii, 1984; ros telug nia spasskii, 1984 type species: cotugnia digonopora (pas quale, 1890) diamare, 1893 1. cotugnia crassa fuhrmann, 1909 guineafowl hudson (1934), east africa bwangamoi (1968), uganda numida meleagris fuhrmann (1909) in ortlepp (1963), the white nile baer (1925), namibia baer (1926), east africa, west africa ortlepp (1963), tanzania the white nile rises from lake victoria in uganda and enters the sudan where it joins the blue nile in karthoum to form the nile. white nile is one of the states of sudan. 2. cotugnia digonopora (pasquale, 1890) diamare, 1893 taenia digonopora pasquale, 1890 anser, columba livia, gallus gallus, numida mele agris schmidt (1986), africa, burma, india, indonesia, philippines guineafowl baylis (1934), uganda 3. cotugnia meleagridis joyeux, baer & martin, 1936 numida meleagris joyeux, baer & martin (1936), northern soma liland graber (1959), chad fabiyi (1972), nigeria hodasi (1976), ghana 4. cotugnia shohoi sawada, 1971 acryllium vulturinum schmidt (1986), somalia 5. cotugnia tuliensis mettrick, 1963 numida meleagris schmidt (1986), zimbabwe genus davainea blanchard, 1891 type species: davainea proglottina (davaine, 1860) blanchard, 1891 1. davainea nana fuhrmann, 1912 guttera edouardi ortlepp (1963), zambia numida meleagris fuhrmann (1912), northern africa junker & boomker (2007b), south africa vanellus cinereus schmidt (1986), africa, japan 2. davainea paucisegmentata fuhrmann, 1909 numida meleagris baer (1926), sudan, west africa schmidt (1986), africa, europe 3. davainea paucisegmentata var. dahomeensis joyeux & baer, 1928 numida meleagris schmidt (1986), france 4. davainea proglottina (davaine, 1860) blanchard, 1891 taenia proglottina davaine, 1860; davainea varians sweet, 1910; davainea dubius meggitt, 1916 alectoris graeca, bonasa umbellus, gallus gallus, perdix perdix schmidt (1986), cosmopolitan domestic chicken baer (1926), south africa magwisha, kassuku, kyvsgaard & permin (2002), tanzania numida meleagris nfor, ajanusi, agbede & esievo (1999), nigeria genus numidella spasskaya & spasskii, 1971 type species: numidella numida (fuhrmann, 1912) spasskaya & spasskii, 1971 1. numidella numida (fuhrmann, 1912) spasskaya & spasskii, 1971 davainea numida fuhrmann, 1912; raillietina (paroniella) numida (fuhrmann, 1912) fuhrmann, 1920; raillietina (pa roniella) magninumida jones, 1930 guineafowl baylis (1934), uganda guttera, numida meleagris schmidt (1986), africa, cuba, north america guttera baer (1933), zimbabwe baer (1933) lists ‘guttera eduardi elliot’ as host. none of the subspecies of guttera edouardi listed in lepage (2007) has been described by elliot, but lepage (2007) lists guttera pucherani verreauxi (elliot, 1870). meleagris gallopavo, numida meleagris jones (1930), north america numida meleagris baer (1925), namibia ortlepp (1963), south africa fabiyi (1972), nigeria genus porogynia railliet & henry, 1909 polycoelia fuhrmann, 1907, preoccupied type species: porogynia paronai (moniez, 1892) railliet & henry, 1909 1. porogynia paronai (moniez, 1892) railliet & henry, 1909 taenia paronai moniez, 1892; linstowia lata fuhrmann, 1901; polycoelia lata (fuhrmann, 1901) fuhrmann, 1907; ma lika numida woodland, 1929; raillietina (paroniella) wood landi baylis, 1934 319 k. junker & j. boomker guttera edouardi, numida meleagris, pternistis natalensis schmidt (1986), africa, europe guineafowl woodland (1928), sudan baylis (1934), uganda guttera edouardi ortlepp (1963), zambia numida meleagris baer (1925), namibia baer (1926), east africa, west africa woodland (1928), sudan ortlepp (1963), south africa, swaziland cruz e silva (1971), mozambique fabiyi (1972), nigeria genus raillietina fuhrmann, 1920 kotlania lópez-neyra, 1929; nonarmiella movsesyan, 1966; nonar mina movsesyan, 1966; kotlanotaurus spasskii, 1973; roytmania spasskii, 1973; skrjabinotaurus spasskii & yurpalova, 1973; oschmarinetta spasskii, 1984 type species: raillietina tetragona (molin, 1858) 1. raillietina angusta ortlepp, 1963 raillietina (raillietina) angusta ortlepp, 1963 numida meleagris ortlepp (1963), south africa 2. raillietina cohni (baczynska, 1914) fuhrmann, 1924 davainea cohni baczynska, 1914; raillietina (ransomia) cohni (baczynska, 1914) fuhrmann, 1920; raillietina (raill ietina) cohni (baczynska, 1914) fuhrmann, 1924 gallus gallus, numida meleagris, pterocles exustus, pterocles orientalis arenarius schmidt (1986), africa, nepal domestic chicken baer (1926), east africa 3. raillietina echinobothrida (megnin, 1880) fuhrmann, 1924 taenia echinobothrida megnin, 1880; taenia botrioplites piana, 1881; davainea parechinobothrida magalhães, 1898; davainea penetrans baczynska, 1914; raillietina (john stonia) echinobothrida (megnin, 1880) fuhrmann, 1920; raillietina (raillietina) echinobothrida (megnin, 1880) fuhrmann, 1924; raillietina (fuhrmannetta) echinobothrida (megnin, 1880) stiles & orleman, 1926 columba livia, gallus gallus, gallus gallus bankiva, meleagris gallopavo, numida meleagris, perdix perdix, phasianus colchicus schmidt (1986), cosmopolitan domestic chicken baer (1926), west africa le roux (1926), south africa joyeux et al. (1936), northern somalia poulsen, permin, hindsbo, yelifari, nansen & bloch (2000), ghana magwisha et al. (2002), tanzania permin, esmann, hoj, hove & mukaratirwa (2002), zimbabwe gallus gallus bankiva baer (1933), zimbabwe numida meleagris baer (1933), zimbabwe southwell & lake (1939), democratic republic of the congo cruz e silva (1971), mozambique ayeni, dipeolu & okaeme (1983), nigeria 4. raillietina pintneri (klaptocz, 1906) fuhrmann, 1924 davainea pintneri klaptocz, 1906; raillietina (ransomia) pintneri (klaptocz, 1906) fuhrmann, 1920; raillietina (raillietina) pintneri (klaptocz, 1906) fuhrmann 1924; kotlania pintneri (klaptocz, 1906) lópez-neyra, 1931 guttera baer (1933), zimbabwe baer (1933) lists ‘guttera eduardi elliot’ as host. none of the subspecies of guttera edouardi listed in lepage (2007) have been described by elliot, but lepage (2007) lists guttera pucherani verreauxi (elliot, 1870). guttera edouardi, numida meleagris schmidt (1986), africa guttera edouardi ortlepp (1963), mozambique, zambia numida meleagris baer (1925), namibia baer (1926), sudan, west africa graber (1959), chad ortlepp (1963) south africa, swaziland fabiyi (1972), nigeria 5. raillietina somalensis sawada, 1971 raillietina (raillietina) somalensis sawada, 1971 acryllium vulturinum schmidt (1986), somalia 6. raillietina steinhardti baer, 1925 raillietina (ransomia) steinhardti baer, 1925 guttera edouardi ortlepp (1963), mozambique, zambia numida meleagris yamaguti (1959), africa verster & ptasinska-kloryga (1987), south africa 7. raillietina tetragona (molin, 1858) fuhrmann, 1924 taenia tetragona molin, 1858; taenia longicollis molin, 1858; davainea tetragona (molin, 1858) blanchard, 1891; davainea bothrioplitis fillippi, 1892; raillietina (ransomia) tetragona (molin, 1858) fuhrmann, 1920; raillietina (raillietina) tetragona (molin, 1858) fuhrmann, 1924; kotlania tetragona (mo lin, 1858) lópez-neyra, 1931; raillietina (raillietina) galli (ya maguti, 1935) sawada, 1955 gallus gallus, guttera edouardi, lagopus lagopus, lagopus muta, meleagris gallopavo, numida meleagris, pavo cristatus, pavo muticus schmidt (1986), cosmopolitan domestic chicken baer (1926), east africa, west africa le roux (1926), south africa poulsen et al. (2000), ghana 320 check list of helminths of guineafowls (numididae) and host list of parasites magwisha et al. (2002), tanzania permin et al. (2002), zimbabwe numida meleagris baer (1926), east africa, west africa ayeni et al. (1983), nigeria haziev & khan (1991), republic of bashkortostan 8. raillietina tetragonoides (baer, 1925) fuhrmann, 1932 raillietina (ransomia) tetragonoides baer, 1925; raillietina (raillietina) tetragonoides (baer, 1925) fuhrmann, 1932; raillietina (raillietina) tetragona var. cohni (baczynska, 1914) lópez-neyra, 1944 numida meleagris baer (1925), namibia schmidt (1986), africa 9. raillietina toyohashiensis sawada & chikada, 1972 numida meleagris schmidt (1986), japan (zoo) genus skrjabinia fuhrmann, 1920 raillietina (skrjabinia) fuhrmann, 1920; brumptiella lópez-neyra, 1929; armacetabulum movsesyan, 1966; markewitchella spasskii & spasskaya, 1972; daovantienia spasskii & spasskaya, 1976 type species: skrjabinia cesticillus (molin, 1858) fuhr mann, 1920 1. skrjabinia cesticillus (molin, 1858) fuhrmann, 1920 taenia cesticillus molin, 1858; davainea cesticillus blan chard, 1891; raillietina (raillietina) mutabilis rüther, 1901; railli etina (skrjabinia) cesticillus (molin, 1858) fuhrmann, 1920 colinus virginianus, coturnix coturnix, gallus gallus, lagopus lagopus, lagopus lagopus scotica, lyrurus tetrix, meleagris gallopavo, numida meleagris, perdix perdix, phasianus colchicus, tetrao urogallus, tetrastes bonasia schmidt (1986), cosmopolitan domestic chicken baer (1926), west africa le roux (1926), south africa joyeux et al. (1936), northern somalia poulsen et al. (2000), ghana magwisha et al. (2002), tanzania permin et al. (2002), zimbabwe numida meleagris nfor et al. (1999), nigeria 2. skrjabinia deweti ortlepp, 1938 numida meleagris ortlepp (1938a), south africa subfamily idiogeninae fuhrmann, 1907 genus idiogenes krabbe, 1867 ersinogenes spasskaya, 1961; paraidiogenes movsesyan, 1971 type species: idiogenes otidis krabbe, 1867 1. idiogenes sp. numida meleagris hodasi (1976), ghana family dilepididae railliet & henry, 1909 genus choanotaenia railliet, 1896 type species: choanotaenia infundibulum (bloch, 1779) railliet, 1896 1. choanotaenia infundibulum (bloch, 1779) railliet, 1896 taenia infundibulum bloch, 1779 domestic chicken poulsen et al. (2000), ghana magwisha et al. (2002), tanzania numida meleagris haziev & khan (1991), republic of bashkortostan nfor et al. (1999), nigeria family paruterinidae fuhrmann, 1907 genus octopetalum baylis, 1914 type species: octopetalum gutterae baylis, 1914 1. octopetalum gutterae baylis, 1914 ascometra gutterae (baylis, 1914) baer,1955 guttera edouardi, numida meleagris baer (1926), east africa baer (1955), democratic republic of congo, malawi, south africa schmidt (1986) africa, france 2. octopetalum numida (fuhrmann, 1909) baylis, 1914 rhabdometra numida fuhrmann, 1909; octopetalum longi cirrosum baer, 1925; unciunia sudanea woodland, 1928; ascometra numida (fuhrmann, 1909) baer, 1955 guineafowl baylis (1934), uganda guttera edouardi baer (1955), sub-saharan africa ortlepp (1963), south africa, zambia numida meleagris baer (1925), namibia baer (1926), sudan, west africa woodland (1928), sudan baer (1955), sub-saharan africa ortlepp (1963), central africa, north africa, south africa, southern africa, swaziland fabiyi (1972), nigeria hodasi (1976), ghana genus metroliasthes ransom, 1900 hexaparuterina palacios & barroeta, 1967 type species: metroliasthes lucida ransom, 1900 1. metroliasthes lucida ransom, 1900 alectoris graeca, alectoris rufa, coturnix coturnix, gallus gallus, gallus gallus bankiva, guttera edouardi, meleagris gallopavo, numida meleagris, perdix perdix schmidt (1986), africa, australia, europe, india, north and south america, russia numida meleagris southwell & lake (1939), democratic republic of congo 321 k. junker & j. boomker family hymenolepididae ariola, 1899 subfamily hymenolepidinae perrier, 1897 genus echinolepis spasskii & spasskaya, 1954 type species: echinolepis carioca (maghalães, 1898) spasskii & spasskaya, 1954 1. echinolepis carioca (maghalães, 1898) spasskii & spasskaya, 1954 davainea carioca maghalães, 1898; taenia conardi zürn, 1898; hymenolepis carioca (maghalães, 1898) ransom, 1902; hymenolepis pullae cholodkovsky, 1913; weinlandia rustica meggitt, 1926; hymenolepis rustica fuhrmann, 1932; dicranotaenia carioca (maghalães, 1898) skrjabin & mathevossian, 1945; dicranotaenia rustica (meggitt, 1926) skrjabin & mathevossian, 1945 alectoris graeca, bonasa umbellus, colinus virgini anus, coturnix coturnix, gallus gallus, meleagris gallopavo schmidt (1986), cosmopolitan domestic chicken le roux (1926), south africa magwisha et al. (2002), tanzania numida meleagris baer (1926), west africa genus hymenolepis weinland, 1858 triorchis clerc, 1903 preoccupied; cloacotaenia wolffhügel, 1938; amphipetrovia spasskii & spasskaya, 1954; australiolepis spasskii & spasskaya, 1954; orlovilepis spasskii & spasskaya, 1954; staphylepis spasskii & oshmarin, 1954; arhynchotaenia saakova, 1958 nec pagenstecher, 1877; schmelzia yamaguti, 1959; woodlandia yamaguti, 1959; arhynchotaeniella schmidt, 1986; cloacotaeniella schmidt, bauerle & wertheim, 1988; amazilolepis schmidt & daily, 1992 type species: hymenolepis di m inuta (rudolphi, 1819) weinland, 1858 1. hymenolepis cantaniana (polonio, 1860) ransom, 1909 taenia cantaniana polonio, 1860; davainea oligophora maghalães, 1898; davainea cantaniana railliet & lucet, 1899; hymenolepis inermis (yoshida, 1910) fuhrmann, 1932 colinus virginianus, coturnix coturnix, gallus gallus, meleagris gallopavo, numida meleagris, pavo cristatus, perdix perdix, phasianus colchicus, tetrao parvirostris, tetrastes bonasia, turnix suscitator schmidt (1986), cosmopolitan domestic chicken le roux (1926), south africa magwisha et al. (2002), tanzania le roux (1926) chose to retain the name h. inermis for his unarmed specimens and not to accept the synonymy of h. inermis and h. cantaniana since the latter had been described as having an armed rostellum. numida meleagris hodasi (1976), ghana junker & boomker (2007b), south africa genus hispaniolepis lòpez-neyra, 1942 satyolepis spasskii, 1965 type species: hispaniolepis villosa (bloch, 1782) lópez-neyra, 1942 1. hispaniolepis falsata (meggitt, 1927) lópez-neyra, 1942 hymenolepis falsata meggitt, 1927. numida meleagris myers, wolfgang & kuntz (1960), sudan chlamydotis undulata schmidt (1986), egypt 2. hispaniolepis fedtschenkoi (solowiow, 1911) lópezneyra, 1942 hymenolepis fedtschenkoi solowiow, 1911; hymenolepis gwil etica dinnik, 1938 gallus gallus, lyrurus tetrix, numida meleagris, tetraogallus himalayensis, tetraogallus caucasicus, tetrastes bonasia schmidt (1986), russia, europe, asia, africa 3. hispaniolepis hilmyi (skrjabin & mathevossian, 1942) lópez-neyra, 1942 hymenolepis tetracis hilmy, 1936 numida meleagris schmidt (1986), liberia 4. hispaniolepis villosa (bloch, 1782) lópez-neyra, 1942 numida meleagris baer (1926), east africa genus ortleppolepis spasskii, 1965 type species: ortleppolepis multiuncinata (ortlepp, 1963) spasskii, 1965 1. ortleppolepis multiuncinata (ortlepp, 1963) spasskii, 1965 hispaniolepis multiuncinata ortlepp, 1963 guttera edouardi ortlepp (1963), zambia numida meleagris junker & boomker (2007b), south africa phylum nemathelminthes class nematoda subclass adenophorea order enoplida superfamily trichinelloidea hall, 1916 family trichuridae (ransom, 1911) railliet, 1915 subfamily capillariinae railliet, 1915 genus aonchotheca lópez-neyra, 1947 avesaonchotheca auct.; baruscapillaria auct.; capillaria auct.; ptero thomix auct.; skrjabinocapillaria skarbilovich, 1946 1. aonchotheca caudinflata (molin, 1858) 322 check list of helminths of guineafowls (numididae) and host list of parasites calodium caudinflata molin, 1858; capillaria blomei tra vassos, 1915; trichosoma longicollis rudolphi, 1819 chrysolophus, columba, coturnix, gallus, lagopus, lyrurus, otis, numida meleagris, passer, perdix, phasianus, sturnus, tetrao, turdus yamaguti (1961), europe, north america yamaguti (1961) lists aonchotheca caudinflata from otis without giving the host’s species name. it is therefore not clear whether otis refers to the current genus otis, ardeotis, neotis or chlamydotis. domestic chicken magwisha et al. (2002), tanzania numida meleagris ayeni et al. (1983), nigeria genus capillaria zeder, 1800 trichosoma rudolphi, 1819; trichosomum creplin, 1829; thominx dujardin, 1845; tridentocapillaria barus & sergeeva, 1990; aonchotheca auct; baruscapillaria auct.; ptherominx auct.; tri chocephalus auct. type species: capillaria anatis (schrank, 1790) travassos, 1915 1. capillaria anatis (schrank, 1790) trichocephalus capillaris rudolphi, 1809 anas, anser, clangula, lyrurus, melanitta, merganser, perdix, phasianus yamaguti (1961), europe, sakhalin, siberia domestic chicken magwisha et al. (2002), tanzania numida meleagris nfor et al. (1999), nigeria genus eucoleus dujardin, 1845 capillaria auct.; thominx auct.; trichocephalus auct. 1. eucoleus annulatus (molin, 1858) trichosoma annulatus molin, 1858 bonasa, chrysolophus, colinus, gallus, lyrurus, meleagris, numida meleagris, perdix, phasianus, syr ma ticus, tetrao yamaguti (1961), asia, europe, north and south america domestic chicken magwisha et al. (2002), tanzania numida meleagris fabiyi (1972), nigeria hodasi (1976), ghana vercruysse et al. (1985), burkina faso subclass secernentea order rhabditida superfamily rhabditoidea family strongyloididae chitwood & mcin tosh, 1934 genus strongyloides grassi, 1879 1. strongyloides avium cram, 1929 gallus yamaguti (1961), north america, puerto rico numida meleagris fabiyi (1972), nigeria order strongylida superfamily strongyloidea family syngamidae leiper, 1912 subfamily syngaminae baylis & daubney, 1926 genus syngamus siebold, 1836 cyathostoma auct.; ornithogamus ryjikov, 1948 type species: syngamus trachea (montagu, 1811) siebold, 1836 1. syngamus trachea (montagu, 1811) siebold, 1836 fasciola trachea montagu, 1811; syngamus trachealis siebold, 1836; strongylus trachealis nathusius, 1937 in ortlepp (1923); strongylus pictus creplin, 1849; sclerostomum syngamus diesing, 1951 in ortlepp (1923); syngamus furcatus theob., 1896; syngamus primitivus molin, 1861; syngamus sclerostomum molin, 1861 galliformes, passeriformes; rarely anseriformes, “arde iformes”, “pelicaniformes”, piciformes, otidiformes yamaguti (1961), africa, australia, europe, india, north and south america domestic chicken magwisha et al. (2002), tanzania numida meleagris hodasi (1976), ghana nfor et al. (1999), nigeria order ascaridida superfamily heterakoidea family heterakidae railliet & henry, 1912 subfamily heterakinae railliet & henry, 1912 genus heterakis dujardin, 1845 ganguleterakis lane, 1914; raillietakis freitas, 1956; inglisakis freitas, vicente & santos, 1969 type species: heterakis vesicularis (frölich, 1791) 1. heterakis vesicularis (frölich, 1791) ascaris vesicularis frölich, 1791; ascaris papillosa, bloch, 1782, in part anas, colinus, coturnix, cygnus, gallus, lagopus, meleagris, numida meleagris, oreortyx pictus, otis, pavo, perdix, phasianus colchicus, polyplectron, tetrao yamaguti (1961), africa, europe, north america lophophorus, lophura yamaguti (1961), nepal yamaguti (1961) lists heterakis vesicularis from otis with out giving the host’s species name. it is therefore not clear whether otis refers to the current genus otis, arde otis, neotis or chlamydotis. 2. heterakis brevispiculum gendre, 1911 domestic chicken, numida meleagris, pternistis bicalcaratus 323 k. junker & j. boomker yamaguti (1961), africa, puerto rico, south america numida meleagris fabiyi (1972), nigeria hodasi (1976), ghana 3. heterakis dispar (schrank, 1790) ascaris dispar schrank, 1790 alectoris, anas, anser, anser cygnoides, branta, berni cla, cairina, chloephaga, glaucidium, numida meleagris, strix, surnia, tadorna yamaguti (1961), cosmopolitan domestic chicken permin, magwisha, kassuku, nansen, bisgaard, frandsen & gibbons (1997), tanzania 4. heterakis gallinarum (schrank, 1788) ascaris gallinarum schrank, 1788; heterakis gallinae gmelin, 1790; heterakis longicaudata von linstow, 1879 acryllium, alectoris, anas, anser, bonasa, cairina, chrysolophus, colinus, corvus, coturnix, cupidonia, domestic chicken, francolinus, houbara, lagopus, lophophorus, lophura, lyrurus, meleagris, otis, pavo, pedioecetes, perdix, phasianus, pterocles, strix, syrmaticus, tetrao, tragopan, tympanuchus yamaguti (1961), cosmopolitan yamaguti (1961) lists heterakis gallinarum from otis without giving the host’s species name. it is therefore not clear whether otis refers to the current genus otis, ardeotis, neotis or chlamydotis. domestic chicken poulsen et al. (2000), ghana magwisha et al. (2002), tanzania permin et al. (2002), zimbabwe numida meleagris ayeni et al. (1983), nigeria verster & ptasinska-kloryga (1987), south africa haziev & khan (1991), republic of bashkortostan santa cruz, ortis de rott & resoagli (1998), argentina 5. heterakis tenuicauda von linstow, 1883 alectoris graeca, alectoris graeca saxatilis yamaguti (1961), turkestan acryllium vulturinum canavan (1929) in yamaguti (1961), east africa family ascaridiidae travassos, 1919 genus ascaridia dujardin, 1845 cotylascaris sprent, 1971 type species: ascaridia hermaphrodita (frölich, 1789) railliet & henry, 1914 1. ascaridia calcarata (gendre, 1909) numida meleagris yamaguti (1961), africa junior synonym of ascaridia numidae (leiper, 1908) according to sprehn (1932) in yamaguti (1961). 2. ascaridia compar (schrank, 1790) travassos, 1913 ascaris compar schrank, 1790 alectoris, coturnix, gallus, lyrurus, numida meleagris, oreortyx pictus, perdix, tetrao, tetrastes yamaguti (1961), america, europe, india, philippines 3. ascaridia galli (schrank, 1788) freeborn, 1932 ascaris galli schrank, 1788; fusaria inflexa zeder, 1800 (baylis 1932, cited in yamaguti 1961); fusaria reflexa zeder, 1800, in part; fusaria strumosa zeder, 1800, in part (lópez-neyra 1946, cited in yamaguti 1961); heterakis brasiliensis magalhães, 1892 (pinto & lins de almeida 1935, cited in yamaguti 1961); heterakis granulosa von linstow, 1906 (baylis 1932, cited in yamaguti 1961); ascaridia hamia lane, 1914 domestic chicken, guineafowl yamaguti (1961), europe, japan alectoris, bonasa, cairina, colinus, duck, ithaginis, ly rurus, meleagris, numida meleagris, perdix, pha sianus, streptopelia, tetrao, tympanuchus yamaguti (1961), cosmopolitan domestic chicken poulsen et al. (2000), ghana magwisha et al. (2002), tanzania permin et al. (2002), zimbabwe numida meleagris ayeni et al. (1983), nigeria verster & ptasinska-kloryga (1987), south africa haziev & khan (1991), republic of bashkortostan 4. ascaridia lineata (schrank, 1866) ascaris lineata schrank, 1866 alectoris, anas, anser, bonasa, duck, francolinus, gal l us, goose, meleagris, meleagris ocellata, numida, partridge, phasianus, pigeon, tympanuchus yamaguti (1961), africa, brazil, china, cuba, europe, formosa, india, malaya, north america, philippines, puerto rico, turkestan domestic chicken le roux (1926), south africa 5. ascaridia numidae (leiper, 1908) travassos, 1913 heterakis numidae leiper, 1908 alectoris, guttera yamaguti (1961), africa guineafowl yamaguti (1961), puerto rico bwangamoi (1968), uganda numida meleagris graber (1959), chad yamaguti (1961), africa, the white nile myers et al. (1960), sudan fabiyi (1972), nigeria hodasi (1976), ghana vercruysse et al. (1985), burkina faso verster & ptasinska-kloryga (1987), south africa the white nile rises from lake victoria in uganda and enters the sudan where it joins the blue nile in karthoum to form the nile. white nile is one of the states of sudan. 324 check list of helminths of guineafowls (numididae) and host list of parasites 6. ascaridia perspicillum (rudolphi, 1803) ascaris perspicillum rudolphi, 1803 anas acuta, domestic chicken, meleagris gallopavo, numida meleagris, pavo cristatus, tetrao urogallus, tetrastes bonasia rupestris, turdus viscivorus yamaguti (1961), europe, hawaii, india, indonesia, japan, malaya superfamily subuluroidea family subuluridae (travassos, 1914) yorke & maplestone, 1926 subfamily subulurinae travassos, 1914 genus subulura molin, 1860 allodapa auct. type species: subulura acutissima molin, 1860 1. subulura acuticauda (von linstow, 1901) railliet & henry, 1914 oxysoma acuticauda von linstow, 1901; heterakis acuticauda (von linstow, 1901) von linstow, 1909 numida meleagris von linstow (1901), kenya yamaguti (1961), africa 2. subulura brumpti (lopez-neyra, 1922) allodapa brumpti lopez-neyra, 1922 alectoris graeca, anas, colinus virginianus texanus, domestic chicken, meleagris gallopavo, numida, perdix perdix, streptopelia orientalis yamaguti (1961), europe, palestine, cyprus, cuba, puerto rico, panama, north america, africa, china domestic chicken hodasi (1969), ghana mukaratirwa, hove, esmann, hoj, permin & nansen (2001), zimbabwe numida meleagris graber (1959), chad hodasi (1976), ghana nfor et al. (1999), nigeria 3. subulura dentigera ortlepp, 1937 numida meleagris ortlepp (1937), south africa 4. subulura differens (sonsino, 1890) heterakis differens sonsino, 1890 alectoris graeca, centropus phasianus, domestic chicken, euplectes orix, numida meleagris, perdix perdix canescens, pternistis bicalcaratus yamaguti (1961), cosmopolitan 5. subulura suctoria (molin, 1860) heterakis suctoria molin, 1860; ascaris forcipata rudolphi, 1819, in part caprimulgus, podager, nyctibius yamaguti (1961), brazil burhinus, coturnix, numida, pternistis yamaguti (1961), south africa (transvaal) coturnix coturnix, lagopus lagopus, phasianus colchicus, phasianus colchicus mongolicus, phasianus colchicus principalis yamaguti (1961), russia, turkestan domestic chicken permin et al. (1997), tanzania permin et al. (2002), zimbabwe guttera edouardi junker & boomker (2007b), south africa numida meleagris ortlepp (1937), south africa fabiyi (1972), nigeria vercruysse et al. (1985), burkina faso 6. subulura strongylina (rudolphi, 1819) ascaris strongylina rudolphi, 1819; strongylus spiculatus cob bold, 1861 (boughton 1939, cited in yamaguti 1961) bonasa, bucco, callipepla, caprimulgus, chelidoptera, colinus, cuculus, gallus, malocoptila, monasa, nonnula, numida meleagris, odontophorus, perdix, podager, tetrao, tympanuchus yamaguti (1961), north america, puerto rico crypturellus, odontophorus capueira, tinamus yamaguti (1961), brazil domestic chicken permin et al. (1997), tanzania poulsen et al. (2000), ghana order spirurida diesing, 1861 superfamily thelazioidea family thelaziidae skrjabin, 1915 genus oxyspirura drasche in stossich, 1897 cramispirura skrjabin, 1931 type species: oxyspirura cephaloptera (molin, 1860) 1. oxyspirura mansoni (cobbold, 1879) filaria mansoni cobbold, 1879; spiroptera emmerezii emmerez & mégnin, 1901 (marotel & carougeau 1902, cited in yamaguti 1961) domestic chicken, gallus gallus, meleagris gallopavo, pavo cristatus yamaguti (1961), atlantic and pacific islands, australia, democratic republic of congo, formosa, india, japan, north america numida meleagris hodasi (1976), ghana superfamily spiruroidea family gongylonematidae (hall, 1916, subfam.) sobolev, 1949 genus gongylonema molin, 1857 type species: gongylonema musculi (rudolphi, 1819) neumann, 1894 1. gongylonema ingluvicola ransom, 1904 gongylonema sumani bahlerao, 1933 (baylis 1939, cited in yamaguti 1961) 325 k. junker & j. boomker gallus, meleagris, pheasants yamaguti (1961), cosmopolitan domestic chicken poulsen et al. (2000), ghana magwisha et al. (2002), tanzania permin et al. (2002), zimbabwe numida meleagris nfor et al. (1999), nigeria 2. gongylonema congolense fain, 1955 cairina moschata fain (1955), democratic republic of the congo gallus fain (1955), burundi, democratic republic of the congo, rwanda guttera edouardi junker & boomker (2007b), south africa numida meleagris fain (1955), burundi, democratic republic of the congo, rwanda fabiyi (1972), nigeria graber (1976), ethiopia hodasi (1976), ghana vercruysse et al. (1985), burkina faso junker & boomker (2007b), south africa scleroptila levaillantii fain (1955), rwanda 3. gongylonema sumani bhalerao, 1933 gallus gallus bhalerao (1933), india numida meleagris fain & thienpont (1958), burundi superfamily habronematoidea family habronematidae (chitwood & wehr, 1932) ivaschkin, 1961 subfamily habronematinae chitwood & wehr, 1932 genus cyrnea seurat, 1914 seurocyrnea strand, 1929; skrjabinochona guschkanskaja, 1931; chenspirura hsü, 1957 nec kou, 1958 type species: cyrnea eurycerca seurat, 1914 1. cyrnea eurycerca seurat, 1914 alectoris, coturnix, francolinus, phasianus, merops yamaguti (1961), africa, europe numida meleagris ortlepp (1938a), southern africa alectoris rufa (“perdix rouge” in yamaguti [1961]) yamaguti (1961), corsica 2. cyrnea parroti seurat, 1917 cyrnea seurati lopéz-neyra, 1918; habronema numidae ortlepp, 1938; cyrnea numidae (ortlepp, 1938) alectoris barbara yamaguti (1961), algeria alectoris rufa yamaguti (1961), spain numida meleagris ortlepp (1938b), malawi, south africa, swaziland fabiyi (1972), nigeria vercruysse et al. (1985), burkina faso chabaud (1958) divided the genus cyrnea into the two subgenera procyrnea chabaud, 1958 and cyrnea chabaud, 1958, subsequently raising them to genus level (chabaud, 1975). he also synonymized cyrnea (cyrnea) numidae ortlepp, 1938 and cyrnea (cyrnea) seurati lopéz-neyra, 1918 with cyrnea (cyrnea) parroti seurat, 1917. our specimens of cyrnea parroti collected from numida meleagris in south africa comply with ortlepp’s (1938b) description of c. numidae, but the arrangement of cephalic structures in apical view is that of c. parroti. not having examined otlepp’s (1938b) specimens we adopt the classification of chabaud (1958) and list ortlepp’s specimens as c. parroti. genus sicarius li, 1934 type species: sicarius dipterum (popova, 1927) li, 1934 1. sicarius caudatus quentin & wertheim, 1975 numida meleagris junker & boomker (2007b), south africa pycnonotus capensis quentin & wertheim (1975), israel quentin & wertheim (1975) described s. caudatus from p. capensis present in the collection of the “hel minth ological laboratory jerusalem” and list jerusalem as locality. it should be noted that p. capensis is endemic to south africa (lepage 2007). we therefore conclude that the authors were either looking at birds kept in captivity in israel, making it difficult to determine the geographic origin of the parasites or did not have any information on the original locality if the birds had been collected in south africa. 2. sicarius renatae cancrini, balbo & iori, 1991 acryllium vulturinum cancrini, balbo & iori (1991), somalia subfamily histiocephalinae gendre, 1922 genus hadjelia seurat, 1916 gilsonia gedoelst, 1919; stellobronema guschanskaja, 1937; sobolevicephalus parukhin, 1964 1. hadjelia truncata (creplin, 1825) spiroptera truncata creplin, 1825; hadjelia inermis (gedoelst, 1919) aceros corrugatus ortlepp (1964), malucca islands, indonesia columba livia tadros & iskander (1975), egypt guttera edouardi, numida meleagris junker & boomker (2007b), south africa tockus erythrorhynchus, tockus leucomelas ortlepp (1964), south africa tockus fasciatus semifasciatus cram (1927, cited in ortlepp 1964), africa 326 check list of helminths of guineafowls (numididae) and host list of parasites coracias benghalensis, halcyon smyrnensis, upupa epops singh (1949), india chabaud & campana (1950) synonymized h. inermis with h. truncata. ortlepp (1964) did not follow this and recorded his specimens as h. inermis. tadros & iskander (1975) synonymized h. inermis, h. parva and h. lhuillieri with h. truncata, designating h. truncata as the new type species of the genus. family tetrameridae travassos, 1914 subfamily tetramerinae railliet, 1915 genus tetrameres creplin, 1846 tropisurus diesing, 1835; tropidurus wiegmann, 1835, preoccupied; gynaecophila gubanov, 1950; petrowimeres tschertkova, 1953; microtetrameres auct. type species: tetrameres paradoxa (diesing, 1835) 1. tetrameres fissispina diesing, 1861 acanthophorus horridus von linstow, 1876; acanthophorus tenuis von linstow, 1876; filaria pulicis von linstow, 1894 alectoris, anas acuta, anas clypeata, anas platyrhynchos, anas querquedula, aythya ferina, bucephala clangula, columba livia, cygnus melanocoryphus, fulica atra, gallus, melanitta fusca, meleagris, meleagris gallopavo, mergus merganser, nycticorax nycticorax, perdix, somateria molissima, tachybaptus fluviatilis yamaguti (1961), africa, canton, europe, for mosa, guam, india, malaya, north and south america, philippines, russian turkestan, siberia, turkey domestic chicken le roux (1926), south africa poulsen et al. (2000), ghana magwisha et al. (2002), tanzania guineafowl le roux (1926), south africa numida meleagris fabiyi (1972), nigeria hodasi (1976), ghana vercruysse et al. (1985), burkina faso 2. tetrameres numida junker & boomker 2007 numida meleagris junker & boomker (2007a), south africa chabaud (1975) divided the genus tetrameres into the two subgenera tetrameres (tetrameres) creplin, 1846 and tetrameres (microtetrameres) travassos, 1915. we adopt the view of anderson (1992) and consider the two as valid genera. superfamily acuarioidea family acuariidae (railliet, henry & sisoff, 1912, subfam.) subfamily acuariinae railliet, henry & sisoff, 1912 genus acuaria bremser, 1811 cheilospirura auct. type species: acuaria anthuris (rudolphi, 1819) 1. acuaria hamulosa (diesing, 1851) spiroptera hamulosa diesing, 1851; cheilospirura hamulosa diesing, 1861; spiroptera perforans cento scudi, 1911 coturnix coturnix, gallus gallus, meleagris, pheasant yamaguti (1961), cosmopolitan domestic chicken le roux (1926), south africa poulsen et al. (2000), ghana magwisha et al. (2002), tanzania numida meleagris fabiyi (1972), nigeria hodasi (1976), ghana genus synhimantus railliet, henry & sisoff, 1912 type species: synhimantus laticeps (rudolphi, 1819) 1. synhimantus spiralis (linstow, 1883) dispharagus spiralis linstow, 1883 accipiter, alectoris, bonasa, ciconia, colinus, columba, coracias, corvus, gallus, meleagris, metopidius, numida meleagris, passer, perdix, phasianus, quiscalus, turdus, turdus migratorius yamaguti (1961), cosmopolitan numida meleagris fabiyi (1972), nigeria hodasi (1976), ghana vercruysse et al. (1985), burkina faso genus dispharynx railliet, henry & sisoff, 1912 type species: dispharynx nasuta (rudolphi, 1819) 1. dispharynx nasuta (rudolphi, 1819) spiroptera nasuta rudolphi, 1819 passer domesticus yamaguti (1961), europe gallus gallus yamaguti (1961), africa, america, australia, ceylon, cuba, formosa domestic chicken, turkeys gibbons, jones & khalil (1996), no geographic data given numida meleagris verster & ptasinska-kloryga (1987), south africa host/parasite check list order tinamiformes family tinamidae (tinamous) genus tinamus hermann, 1783 subulura strongylina genus crypturellus brabourne & chubb, 1914 crypturus subulura strongylina 327 k. junker & j. boomker order galliformes domestic chicken mediorhynchus gallinarum postharmostomum gallinum choanotaenia infundibulum davainea proglottina echinolepis carioca hymenolepis cantaniana raillietina cohni raillietina echinobothrida raillietina tetragona skrjabinia cesticillus acuaria hamulosa aonchotheca caudinflata ascaridia galli ascaridia lineata ascaridia perspicillum capillaria anatis dispharynx nasuta eucoleus annulatus gongylonema ingluvicola heterakis brevispiculum heterakis dispar heterakis gallinarum oxyspirura mansoni subulura brumpti subulura differens subululra strongylina subulura suctoria syngamus trachea tetrameres fissispina gallinaceaous birds, galliformes mediorhynchus gallinarum syngamus trachea family numididae (guineafowls) crested guineafowl postharmostomum gallinum guineafowl cotugnia crassa cotugnia digonopora numidella numida octopetalum numida porogynia paronai ascaridia galli ascaridia numidae tetrameres fissispina genus numida linnaeus, 1766 1. numida meleagris (linnaeus, 1758) (helmeted guinea fowl) phasianus meleagris numida meleagris galeatus pallas, 1767 numida galeata numida meleagris meleagris (linnaeus, 1758) numida ptilorhyncha numida meleagris mitratus pallas, 1767 numida mitrata numida meleagris marungensis schalow, 1884 numida frommi, numida marungensis, numida meleagris bodalyae, numida meleagris frommi, numida meleagris maxima, numida meleagris rikwae, numida mitrata frommi, nu mida mitrata maximia, numida mitrata rikwae, numida rikwae mediorhynchus empodius mediorhynchus gallinarum mediorhynchus numidae mediorhynchus selengensis mediorhynchus taeniatus dicrocoelium macrostomum lutztrema sp. postharmostomum gallinum abuladzugnia gutterae abuladzugnia transvaalensis choanotaenia infundibulum cotugnia crassa cotugnia digonopora cotugnia meleagridis cotugnia tuliensis davainea nana davainea paucisegmentata davainea paucisegmentata var. dahomeensis davainea proglottina echinolepis carioca hispaniolepis falsata hispaniolepis fedtschenkoi hispaniolepis hilmyi hispaniolepis villosa hymenolepis cantaniana idiogenes sp. metroliasthes lucida numidella numida octopetalum gutterae octopetalum numida ortleppolepis multiuncinata porogynia paronai raillietina angusta raillietina cohni raillietina echinobothrida raillietina pintneri raillietina steinhardti raillietina tetragona raillietina tetragonoides raillietina toyohashiensis skrjabinia cesticillus skrjabinia deweti acuaria hamulosa ascaridia calcarata ascaridia compar ascaridia galli ascaridia lineata ascaridia numidae ascaridia perspicillum aonchotheca caudinflata capillaria anatis cyrnea eurycerca 328 check list of helminths of guineafowls (numididae) and host list of parasites cyrnea parroti eucoleus annulatus gongylonema congolense gongylonema ingluvicola gongylonema sumani hadjelia truncata heterakis brevispiculum heterakis dispar heterakis gallinarum heterakis vesicularis oxyspirura mansoni sicarius caudatus sicarius renatae subulura acuticauda subulura brumpti subulura dentigera subulura differens subulura strongylina subulura suctoria strongyloides avium syngamus trachea dispharynx nasuta synhimantus spiralis tetrameres fissispina tetrameres numida genus: guttera wagler, 1832 numidella numida raillietina pintneri ascaridia numidae 1. guttera edouardi (hartlaub, 1867) (crested guineafowl) numida edouardi (hartlaub, 1867); guttera pucherani edouardi (hartlaub, 1867) mediorhynchus taeniatus abuladzugnia gutterae davainea nana metroliasthes lucida octopetalum gutterae octopetalum numida ortleppolepis multiuncinata porogynia paronai raillietina pintneri raillietina steinhardti raillietina tetragona gongylonema congolense hadjelia truncata subulura suctoria genus: acryllium gray, 1840 heterakis gallinarum 1. acryllium vulturinum gray, 1840 (vulturine guineafowl) cotugnia shohoi raillietina somalensis heterakis tenuicauda sicarius renatae family odontophoridae (new world quails) genus oreortyx baird, 1858 ortyx in yamaguti (1961) 1. oreortyx pictus (douglas, 1829) (mountain quail) ortyx picta ascaridia compar heterakis vesicularis genus callipepla wagler, 1832 lophortyx subulura strongylina genus colinus goldfuss, 1820 ascaridia galli eucoleus annulatus heterakis vesicularis heterakis gallinarum subulura strongylina synhimantus spiralis 1. colinus virginianus (linnaeus, 1758) (northern bobwhite) tetrao virginianus echinolepis carioca hymenolepis cantaniana skrjabinia cesticillus 1a. colinus virginianus texanus lawrence, 1853 subulura brumpti genus odontophorus vieillot, 1816 subulura strongylina 1. odontophorus capueira (spix, 1825) (spot-winged wood-quail) perdix capueira subulura strongylina family phasianidae (partridges, francolins, spur fowls, pheasants, etc.) partridge ascaridia lineata pheasant acuaria hamulosa gongylonema ingluvicola turkey dispharynx nasuta genus meleagris linnaeus, 1758 acuaria hamulosa ascaridia galli ascaridia lineata eucoleus annulatus gongylonema ingluvicola heterakis vesicularis heterakis gallinarum synhimantus spiralis 329 k. junker & j. boomker tetrameres fissispina 1. meleagris gallopavo linnaeus, 1758 (wild turkey, common turkey) echinolepis carioca hymenolepis cantaniana metroliasthes lucida numidella numida raillietina echinobothrida raillietina tetragona skrjabinia cesticillus ascaridia perspicillum oxyspirura mansoni subulura brumpti tetrameres fissispina 2. meleagris ocellata cuvier, 1820 (ocellated turkey) agriocharis ocellata ascaridia galli genus bonasa stephens, 1819 ascaridia galli ascaridia lineata eucoleus annulatus heterakis gallinarum subulura strongylina synhimantus spiralis 1. bonasa umbellus (linnaeus, 1766) (ruffed grouse) tetrao umbellus davainea proglottina echinolepis carioca genus tetrastes keyserling & blasius, 1840 ascaridia compar 1. tetrastes bonasia (linnaeus, 1758) (hazel grouse) bonasa bonasia, bonasia bonasia, tetrao bonasia hispaniolepis fedtschenkoi hymenolepis cantaniana skrjabinia cesticillus 1a. tetrastes bonasia rupestris (brehm, 1831) ascaridia perspicillum genus tetrao linnaeus, 1758 aonchotheca caudinflata ascaridia compar ascaridia galli eucoleus annulatus heterakis vesicularis heterakis gallinarum subulura strongylina 1. tetrao urogallus linnaeus, 1758 (western caper caillie) tetrao major skrjabinia cesticillus ascaridia perspicillum 2. tetrao parvirostris bonaparte, 1856 (black-billed ca per caillie) tetrao urogalloides hymenolepis cantaniana genus lyrurus swainson, 1832 aonchotheca caudinflata ascaridia compar ascaridia galli capillaria anatis eucoleus annulatus heterakis gallinarum 1. lyrurus tetrix (linnaeus, 1758) (black grouse) tetrao tetrix hispaniolepis fedtschenkoi skrjabinia cesticillus genus tympanuchus gloger, 1841 cupidonia ascaridia lineata heterakis gallinarum 1. tympanuchus phasianellus (linnaeus, 1758) (sharptailed grouse) pedioecetes phasianellus (linnaeus, 1758), tetrao phasianellus ascaridia galli ascaridia lineata heterakis gallinarum subulura strongylina genus lagopus brisson, 1760 aonchotheca caudinflata heterakis gallinarum heterakis vesicularis 1. lagopus lagopus (linnaeus, 1758) (willow ptarmi gan) tetrao lagopus raillietina tetragona skrjabinia cesticillus subulura suctoria 1a. lagopus lagopus scotica (latham, 1787) lagopus scotica skrjabinia cesticillus 2. lagopus muta (montin, 1781) (rock ptarmigan) raillietina tetragona genus tetragallus gray, 1832 1. tetraogallus caucasicus (pallas, 1811) (caucasian snowcock) tetrao caucasica hispaniolepis fedtschenkoi 2. tetraogallus himalayensis gray, 1843 (himalayan snowcock) megaloperdix nigelli hispaniolepis fedtschenkoi genus alectoris kaup, 1829 caccabis in yamaguti (1961) 330 check list of helminths of guineafowls (numididae) and host list of parasites ascaridia compar ascaridia galli ascaridia numidae cyrnea eurycerca heterakis dispar heterakis gallinarum synhimantus spiralis tetrameres fissispina 1. alectoris barbara (bonnaterre, 1792) (barbary partridge) caccabis petrosa, perdix barbara cyrnea parroti 2. alectoris graeca (meisner, 1804) (rock partridge) perdix graeca davainea proglottina echinolepis carioca metroliasthes lucida heterakis tenuicauda subulura brumpti subulura differens 2a. alectoris graeca saxatilis (bechstein, 1805) caccabis saxatilis chukar heterakis tenuicauda lepage (2007) states that the original alectoris graeca has been split into four species, namely alectoris graeca, alectoris chukar (gray, 1830), alectoris philbyi lowe, 1934 and alectoris magna (prjevalski, 1876). 3. alectoris rufa (linnaeus, 1758) (red-legged part ridge) caccabis rufa, coturnix rufa, tetrao rufus metroliasthes lucida cyrnea eurycerca cyrnea parroti genus francolinus stephens, 1819 ascaridia lineata cyrnea eurycerca heterakis gallinarum genus scleroptila blyth, 1852 1. scleroptila levaillantii (valenciennes, 1825) (redwinged francolin) francolinus levaillantii, perdix levaillantii gongylonema congolense genus pternistis wagler, 1832 subulura suctoria 1. pternistis natalensis (smith, 1834) (natal spurfowl) francolinus natalensis, pternistes natalensis porogynia paronai 2. pternistis bicalcaratus (linnaeus, 1766) (doublespurred spurfowl) francolinus bicalcaratus, tetrao bicalcaratus heterakis brevispiculum subulura differens genus perdix brisson, 1760 aonchotheca caudinflata ascaridia compar ascaridia galli capillaria anatis eucoleus annulatus heterakis vesicularis heterakis gallinarum subulura strongylina synhimantus spiralis tetrameres fissispina 1. perdix perdix (linnaeus, 1758) (grey partridge) tetrao perdix davainea proglottina hymenolepis cantaniana metroliasthes lucida raillietina echinobothrida skrjabinia cesticillus subulura brumpti 1a. perdix perdix canescens buturlin, 1906 subulura differens genus coturnix bonnaterre, 1791 aonchotheca caudinflata ascaridia compar cyrnea eurycerca heterakis gallinarum heterakis vesicularis subulura suctoria 1. coturnix coturnix (linnaeus, 1758) (common quail) tetrao coturnix dicrocoelium macrostomum echinolepis carioca hymenolepis cantaniana metroliasthes lucida skrjabinia cesticillus acuaria hamulosa subulura suctoria genus ithaginis wagler, 1832 ascaridia galli genus tragopan cuvier, 1829 ceriornis heterakis gallinarum genus lophophorus temminck, 1813 heterakis gallinarum heterakis vesicularis genus gallus brisson, 1760 aonchotheca caudinflata ascaridia compar ascaridia lineata eucoleus annulatus gongylonema congolense 331 k. junker & j. boomker gongylonema ingluvicola heterakis vesicularis strongyloides avium subulura strongylina synhimantus spiralis tetrameres fissispina 1. gallus gallus (linnaeus, 1758) (red junglefowl) gallus ferrugineus, phasianus gallus davainea proglottina echinolepis carioca hispaniolepis fedtschenkoi hymenolepis cantaniana metroliasthes lucida raillietina cohni raillietina echinobothrida raillietina tetragona skrjabinia cesticillus acuaria hamulosa dispharynx nasuta gongylonema sumani oxyspirura mansoni 1a. gallus gallus bankiva temminck, 1813 metroliasthes lucida raillietina echinobothrida genus lophura fleming, 1822 gennaeus heterakis gallinarum 1. lophura nycthemera (linnaeus, 1758) (silver pheasant) euplocamus nycthemerus, gennaeus nycthemerus, phasianus nycthemerus heterakis vesicularis genus syrmaticus wagler, 1832 graphophasianus eucoleus annulatus 1. syrmaticus soemmeringii (temminck, 1830) (copper pheasant) graphophasianus soemmeringii, phasianus soemmeringii heterakis gallinarum genus phasianus linnaeus, 1758 aonchotheca caudinflata ascaridia galli ascaridia lineata capillaria anatis cyrnea eurycerca eucoleus annulatus heterakis gallinarum synhimantus spiralis 1. phasianus colchicus linnaeus, 1758 (common pheasant, ring-necked pheasant) hymenolepis cantaniana raillietina echinobothrida skrjabinia cesticillus heterakis vesicularis subulura suctoria 1a. phasianus colchicus mongolicus brandt, 1844 phasianus mongolicus turkestanicus subulura suctoria 1b. phasianus colchicus principalis sclater, 1885 phasianus principalis subulura suctoria genus chrysolophus gray, 1834 thaumalea aonchotheca caudinflata eucoleus annulatus heterakis gallinarum genus polyplectron temminck, 1807 heterakis vesicularis genus pavo linnaeus, 1758 heterakis gallinarum heterakis vesicularis 1. pavo cristatus linnaeus, 1758 (indian peafowl) hymenolepis cantaniana raillietina tetragona ascaridia perspicillum oxyspirura mansoni 2. pavo muticus linnaeus, 1766 (green peafowl) raillietina tetragona order anseriformes syngamus trachea family anatidae (ducks, geese and swans) duck ascaridia galli ascaridia lineata goose ascaridia lineata genus anser brisson, 1760 cotugnia digonopora ascaridia lineata capillaria anatis heterakis dispar heterakis gallinarum 1. anser cygnoides (linnaeus, 1758) (swan goose) cygnopsis cygnoides heterakis dispar genus branta scopoli, 1769 heterakis dispar 1. branta bernicla (linnaeus, 1758) (white-bellied brant) 332 check list of helminths of guineafowls (numididae) and host list of parasites anas bernicla “bernicla” heterakis dispar genus cygnus bechstein, 1803 1. cygnus atratus (latham, 1790) (australian black swan) chenopsis atrata heterakis vesicularis 2. cygnus melanocoryphus (molina, 1782) tetrameres fissispina genus chloephaga eyton, 1838 heterakis dispar genus tadorna boie, 1822 todorna heterakis dispar genus cairina fleming, 1822 ascaridia galli heterakis dispar heterakis gallinarum 1. cairina moschata (linnaeus,1758) (muscovy duck) gongylonema congolense genus anas linnaeus, 1758 ascaridia lineata heterakis dispar heterakis gallinarum heterakis vesicularis subulura brumpti 1. anas platyrhynchos linnaeus, 1758 (mallard) anas boschas tetrameres fissispina 2. anas clypeata linnaeus, 1758 (northern shoveler) anas spathula, spatula clypeata tetrameres fissispina 3. anas acuta linnaeus, 1758 (northern pintail) dafila acuta ascaridia perspicillum tetrameres fissispina according to lepage (2007) a. acuta has been split into a. acuta and anas eatoni, but some authors consider a. eatoni a subspecies of a. acuta. peterson (1999) lists the two as separate species. 4. anas querquedula linnaeus, 1758 (garganey) querquedula querquedula capillaria anatis tetrameres fissispina genus aythya boie, 1822 1. aythya ferina (linnaeus, 1758) (common pochard) anas ferina, aristonetta ferina, nyroca ferina tetrameres fissispina genus somateria leach, 1819 1. somateria mollissima (linnaeus, 1758) (common eider) anas mollissima tetrameres fissispina genus melanitta boie, 1822 oedemia, oidemia capillaria anatis 1. melanitta fusca (linnaeus, 1758) (velvet scooter) anas fusca, oidemia fusca tetrameres fissispina genus clangula leach, 1819 1. clangula hyemalis (linnaeus, 1758) (oldsquaw, longtailed duck) anas hyemalis, harelda hyemalis, ereunetes occidentalis capillaria anatis genus bucephala baird, 1858 1. bucephala clangula (linnaeus, 1758) (common gol deneye) anas clangula, clangula clangula, glaucionetta clangula tetrameres fissispina genus mergus linnaeus, 1758 merganser capillaria anatis 1. mergus merganser linnaeus, 1758 (common merganser) tetrameres fissispina order turniciformes family turnicidae (buttonquail) genus turnix bonnaterre, 1791 1. turnix suscitator (gmelin, 1789) (barred buttonquail) tetrao suscitator hymenolepis cantaniana order piciformes syngamus trachea order galbuliformes family bucconidae (puffbirds) genus bucco brisson, 1760 subulura strongylina genus malocoptila gray, 1841 subulura strongylina 333 k. junker & j. boomker genus nonnula sclater, 1854 subulura strongylina genus monasa vieillot, 1816 subulura strongylina genus chelidoptera gould, 1837 subulura strongylina order bucerotiformes family bucerotidae (hornbills) genus aceros hodgson, 1844 1. aceros corrugatus (temminck, 1832) (wrinkled hornbill) buceros corrugatus, rhyniceros corrugatus hadjelia truncata genus tockus lesson, 1830 1. tockus erythrorhynchus (temminck, 1823) (red-billed hornbill) buceros erythrorhynchus hadjelia truncata 2. tockus fasciatus semifasciatus (hartlaub, 1855) (allied hornbill) lophoceros semifasciatus hadjelia truncata 3. tockus leucomelas (liechtenstein, 1842) (southern yellow-billed hornbill) buceros leucomelas hadjelia truncate some authors consider t. leucomelas a subspecies of tockus flavirostris (rüppell, 1853) (lepage 2007) order upupiformes family upupidae (hoopoes) genus upupa linnaeus, 1758 1. upupa epops linnaeus, 1758 (hoopoe) hadjelia truncata family coraciidae (rollers) genus coracias linnaeus, 1758 synhimantus spiralis 1. coracias benghalensis (linnaeus, 1758) (indian roller) corvus benghalensis hadjelia truncata genus halcyon swainson, 1821 1. halcyon smyrnensis (linnaeus, 1758) (white-throated kingfisher) hadjelia truncata family meropidae (bee-eaters) genus merops linnaeus, 1758 cyrnea eurycerca order cuculiformes family cuculidae (cuckoos, coucals, anis, road runners, couas, etc.) genus cuculus linnaeus, 1758 subulura strongylina genus centropus illiger, 1811 1. centropus phasianinus (latham, 1802) (pheasant coucal) cuculus phasianinus subulura differens order strigiformes family strigidae (typical owls) genus strix linnaeus, 1758 heterakis dispar heterakis gallinarum genus surnia dumeril, 1805 heterakis dispar genus glaucidium boie, 1826 heterakis dispar family nyctibiidae (potoos) genus nyctibius vieillot, 1816 subulura suctoria family caprimulgidae (nightjars) genus podager wagler, 1832 subulura strongylina subulura suctoria genus caprimulgus linnaeus, 1758 subulura strongylina subulura suctoria order columbiformes family columbidae (pigeons and doves) pigeon ascaridia lineata genus columba linnaeus, 1758 aonchotheca caudinflata synhimantus spiralis 1. columba livia gmelin, 1785 (rock pigeon) 334 check list of helminths of guineafowls (numididae) and host list of parasites cotugnia digonopora raillietina echinobothrida hadjelia truncata tetrameres fissispina genus streptopelia bonaparte, 1855 spilopelia ascaridia galli 1. streptopelia orientalis (latham, 1790) (oriental turtledove) columba orientalis, turtur orientalis subulura brumpti order gruiformes family rallidae (rails, crakes, moorhens and coots) genus fulica linnaeus, 1758 1. fulica atra linnaeus, 1758 (common coot) tetrameres fissispina family otididae (bustards and korhaans) yamaguti (1961) lists aonchotheca caudinflata, heterakis gallinarum and heterakis vesicularis from otis without giving the host’s species name. it is therefore not clear whether otis refers to the current genus otis, ardeotis, neotis or chlamy dotis. otidiformes in yamaguti (1961) syngamus trachea genus otis linnaeus, 1758 1. otis tarda linnaeus, 1758 (great bustard) mediorhynchus taeniatus genus ardeotis le maout, 1853 1. ardeotis arabs (linnaeus, 1758) (arabian bustard) choriotis arabs, otis arabs mediorhynchus empodius mediorhynchus taeniatus genus chlamydotis lesson, 1839 houbara hetrakis gallinarum according to lepage (2007) the common name houbara has been split into chlamydotis undulata and chlamydotis macqueenii. 1. chlamydotis undulata (jacquin, 1784) (houbara bustard) otis houbara, psophia undulata mediorhynchus taeniatus hispaniolepis falsata 2. chlamydotis macqueenii gray, 1832 (macqueen’s bustard) otis macqueenii mediorhynchus taeniatus order charadriiformes family pteroclidiae (sandgrouse) genus pterocles temminck, 1815 calopterocles heterakis gallinarum 1. pterocles exustus temminck, 1825 (chestnut-bellied sandgrouse) pteroclidurus exustus, pterocles senegalensis raillietina cohni lepage (2007) lists pterocles senegalensis as synonym for pterocles exustus. 2. pterocles orientalis arenarius (pallas, 1775) (eastern black-bellied sandgrouse) pterocles arenarius raillietina cohni lepage (2007) and peterson (1999) list pterocles arenarius as subspecies of pterocles orientalis (linnaeus, 1758). family jacanidae (jacanas) genus metopidius wagler, 1832 synhimantus spiralis family burhinidae (thick-knees) genus burhinus illiger, 1811 oedicnemus subulura suctoria 1. burhinus oedicnemus (linnaeus, 1758) (eurasian thick-knee) charadrius oedicnemus, oedicnemus crepitans mediorhynchus taeniatus family charadriidae (plovers, dotterels, lapwings) genus vanellus brisson, 1760 1. vanellus cinereus (blyth, 1842) (grey-headed lapwing) hoplopterus cinereus, microsarcops cinereus, pluvianus cinereus davainea nana order falconiformes family accipitridae (eagles, hawks, buzzards, kites, vulures) genus accipiter brisson, 1760 synhimantus spiralis order ciconiiformes family podicipedidae (grebes) genus tachybaptus reichenbach, 1853 1. tachybaptus novaehollandiae (stephens, 1826) (australian grebe) 335 k. junker & j. boomker podiceps fluviatilis, podiceps novaehollandiae tetrameres fissispina family ardeidae (herons, egrets and bitterns) “ardeiformes” syngamus trachea genus ardea linnaeus, 1758 mediorhynchus empodius genus nycticorax forster, 1817 1. nycticorax nycticorax (linnaeus, 1758) (black-crowned night-heron) ardea nycticorax tetrameres fissispina family pelecanidae (pelicans) “pelicaniformes” syngamus trachea family ciconiidae (storks) genus ciconia brisson, 1760 synhimantus spiralis order passeriformes syngamus trachea family corvidae (crows and ravens) genus corvus linnaeus, 1758 heterakis gallinarum synhimantus spiralis family pycnonotidae genus pycnonotus boie, 1826 1. pycnonotus capensis (linnaeus, 1766) (cape bulbul) sicarius caudatus family muscicapidae (old world flycatchers) genus turdus linnaeus, 1758 aonchotheca caudinflata synhimantus spiralis 1. turdus viscivorus linnaeus, 1758 (mistle thrush) ascaridia perspicillum 2. turdus migratorius linnaeus, 1766 (american robin) planesticus migratorius synhimantus spiralis family sturnidae (starlings) genus sturnus aonchotheca caudinflata family passeridae (old world sparrows, sowfinches and relatives) genus passer brisson, 1760 aonchotheca caudinflata synhimantus spiralis 1. passer domesticus (linnaeus, 1758) (house spar row) fringilla domestica dispharynx nasuta genus euplectes swainson, 1829 1. euplectes orix (linnaeus, 1758) (red bishop) pyromelana oryx subulura differens family fringillidae (canaries and buntings) genus quiscalus vieillot, 1816 synhimantus spiralis acknowledgements the first author was supported by a claude leon foundation postdoctoral fellowship grant. funds for this research were made available by 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systema helminthum. new york: interscience publishers. yamaguti, s. 1963. acanthocephala. vol. v, in systema helminthum. new york: interscience publishers. fandamu_37-43.indd introduction theileria parva is a tick-borne protozoan parasite of cattle that causes an acute, often fatal lymphoproliferative disease known as east coast fever (ecf). theileria parva multiplies in bovine lymphocytes by schizogony resulting in the production of merozoites that invade red blood cells (rbcs) and develop into piroplasms (conrad, denham & brown 1986). the major clinical signs in t. parva infections are enlargement of lymph nodes, especially those adjacent to sites of parasite inoculation, pyrexia, lymphocyte destruction resulting in leukopenia, interstitial pneumonia, pulmonary oedema and death (jura & losos 1980; irvin & mwamachi 1983; irvin & mor rison 1987). other signs include blood-tinged diarrhoea, lacrimation and corneal opacity, splenic enlargement and lymphomata in the kidneys. animals usually die within 30 days after infection (irvin & morrison 1987). some animals die within 24 days as a result of respiratory distress while others die later following secondary infections and wasting (irvin & mwamachi 1983; irvin & morrison 1987). anaemia has not been recognised as a major feature in classical t. parva infections (maxie, dolan, jura, tabel & flowers 1982) but it has been described in cattle infected with 37 onderstepoort journal of veterinary research, 74:37–43 (2007) red blood cell volume as a predictor of fatal reactions in cattle infected with theileria parva katete p. fandamu1, t. marcotty2, j.r.a. brandt2, l. duchateau3, n. speybroeck2, t.t. dolan4 and d. berkvens2* abstract fandamu, p., marcotty, t., brandt, j.r.a., duchateau, l., speybroeck, n., dolan, t.t. & berkvens, d. 2007. red blood cell volume as a predictor of fatal reactions in cattle infected with theileria parva katete. onderstepoort journal of veterinary research, 74:37–43 a comparison of mean corpuscular volume (mcv) and packed cell volume (pcv) was made between cattle undergoing lethal and non-lethal reactions following experimental infections with the apicomplexan protozoa, theileria parva katete. this work confirmed that anaemia occurs in infected animals. however, the fall in pcv was steeper in lethal reactions compared to non-lethal reactions. our results show that animals with initially lower mcv values are more prone to fatal reaction, despite having normal pcv profiles. the study also found that small red blood cells are more likely to be infected with t. parva. these findings suggest that animals with a higher proportion of small red blood cells in circulation will be more likely to succumb to t. parva infections. the potential for using mcv as a predictor of the outcome of infection challenge is discussed. keywords: anaemia, cattle, mean corpuscular volume, packed cell volume, prediction of outcome, theileria parva, zambia * author to whom correspondence is to be directed. e-mail: dberkvens@itg.be deceased 15 january 2007 1 department of veterinary and livestock development, mazabuka veterinary research station, p.o. box 670050, mazabuka, zambia 2 department of animal health, institute of tropical medicine, nationalestraat 155, b-2000 antwerpen, belgium 3 department of physiology, biochemistry and biometrics, facul ty of veterinary medicine, ghent university, salisburylaan 133, b-9820, belgium 4 livestock services limited, p.o. box 24437, 00502 karen, nairobi, kenya accepted for publication 28 september 2006—editor 38 red blood cell volume in cattle infected with theileria parva katete certain stocks of t. parva (norval, perry & young 1992; mbassa, balemba, maselle & mwaga 1994). the pathogenesis of the anaemia in t. parva infections is not well understood (mbassa et al. 1994). in infections with the related theileria annulata and theileria mutans parasites, anaemia is common and is attributed to the replication of the parasite in the erythrocytes (young, purnell, payne, brown & kanhai 1978; norval et al. 1992). the severity of t. parva infections has been linked to the quantity of the infective dose (radley, brown, burridge, cunningham, peirce & purnell 1974; dolan, young, losos, mcmillan, minder & soulsby 1984), to the t. parva stock (mbogo, kariuki, nguni & mchardy 1996) and to the cattle breed (paling & geysen 1981) but there are no reports of the role of anaemia in the pathogenicity or virulence of t. parva. the objective of this study was to confirm the decline of packed cell volume (pcv) in animals infected by t. parva katete and to associate the magnitude of this decline with the severity of the clinical reaction. in addition, we investigated the association between the size of rbcs and severity and lethality of ecf infection and compared the diameter of infected and non-infected rbcs to evaluate whether there is a preference of t. parva merozoites for larger or smaller cells. materials and methods animals a retrospective analysis was conducted using haematological data from 18 t. parva susceptible belgian black and white dairy cattle aged approximately 10 months that had been used in t. parva experiments between 1996 and 2000. the animals (16 heifers and two steers) were housed and fed on hay and concentrates and water ad libitum. these experiments were performed under the control of the ethi cal committee for experimental animals of the institute of tropical medicine of antwerp (belgian registration number la 1100120) which guarantees that animals do not suffer unnecessarily. the animals were inoculated with t. parva katete stabilates derived from the same reference stock v1. this stock was isolated from cattle in katete district of eastern province of zambia in 1983 (berkvens, geysen & lynen 1989). stabilates used and doses inoculated were administered randomly and independently of mean corpuscular volume (mcv) and pcv values. the stabilates were injected subcutaneously in front of the right parotid lymph node. blood samples were collected from the external jugular vein in ethylene diaminotetraacetic acid (edta) tubes for pcv and mcv measurements. all samples were processed on the day of collection. blood and lymph smears were prepared daily from day 7. theileria parva infections were confirmed by the presence of schizonts in the lymph node biopsies 7–20 days after inoculation. classification of reactions was according to morzaria, irvin, voigt & taracha (1987). non-lethal reactions were recorded when infected animals (confirmed by the presence of schizonts) survived for more than 24 days without chemotherapy. haematological examinations the pcv and the mean corpuscular volume (mcv) were measured directly using a technicon h1e haem atology analyzer (bayer, tarrytown, ny) together with other haematological parameters. the cor puscular size is determined by direct measurement and not derived from pcv and red blood cell counts (feldman, zinkl & jain 2000). additional examinations were carried out on blood smears to determine the mean diameters of infected and non-infected rbcs. red blood cells were measured prior to detection of piroplasms in blood (day 7), at 1 % parasitaemia and when maximum parasitaemia was recorded. measurements were done using a micro meter (mahr 46eh digital micrometer head, germany) fitted on a microscope at x1000 magnification. examinations were performed around the “tail” of the smears where the cells were evenly spread. twenty circular rbcs were measured from 3–4 fields for slides obtained before parasitaemia. following the detection of piroplasms, the sizes of all infected cells in a particular field were measured, provided that they contained a single piroplasm and were circular. thereafter, four unparasitized rbcs with normal shape and nearest to an infected cell were measured and an average size calculated. statistical analysis data analysis was done in stata se/8.0 (statacorp 2003). the pcv and mcv profiles were drawn using cubic splines (five cross-median knots). the pcv, mcv, parasitaemia and rbc diameters were analysed using mixed models with animals as random effect. to ensure normality of the response variables, pcv values were arcsine transformed (osborne 2002) while rbc diameters were transformed using natural logarithms. parasitaemia values were logarithm transformed. lethality group (lethal or nonlethal), period of infection (days 0–7 or days 14–21) and the period x lethality group interaction were used as categorical fixed effects for pcv and mcv 39 p. fandamu et al. regressions. separate analyses were done on rbc diameters before and during parasitaemia using the lethality group, the presence of piroplasms and the interaction between these two as categorical fixed effects. results the animals that suffered lethal reactions died on average 20 days after infection (18–23 days) following a period of high fever and parasitosis. the reactions of all animals undergoing non-lethal infections were also characterized by fever and parasitosis. however, parasitaemia was significantly higher in lethal than non-lethal reactions (p = 0.004) in the period 14–21 days. the mean parasitaemia (proportion of rbc infected by a piroplasm) in the lethal group was 3.88 (1.54–9.77) while that of the nonlethal group was 0.51 (0.18–1.41). packed cell volume packed cell volume profiles for animals with lethal and non-lethal reactions are shown in fig. 1. in the early stage of infection (days 0–7) there was no significant difference in pcv values between animals with lethal and those with non-lethal reactions (p = 0.915) (fig. 3a). in both groups, the pcv was significantly lower in the later stage compared to the early stage (p < 0.001). however, the pcv in the later stage of infection (days 14–21) decreased significantly more in lethal reactions (p < 0.001). mean corpuscular volume the mcv profiles following t. parva infection are shown in fig. 2. when the mcv values were compared in the early stage of infection, there was a statistically significant difference between animals that developed lethal reactions and those that did not (p < 0.001). animals that had lethal infections had lower initial mcv values (smaller cells) than those that suffered non-lethal infections. the boxand-whisker plot in fig. 3b further shows that animals that suffered lethal reactions had a lower median mcv value than those that had non-lethal reactions. late in the infection, mcv values remained lower in animals with lethal reactions (p < 0.001). ������� �� ��� � �� �� �� �� � � � �� � � ��� ������������� � � �� �� �� �� �������� �� ��� ��� ������������� � � � �� � � � � �� �� �� � �� �� �� �� fig. 1 packed cell volume (pcv) as a function of days post infection in animals with non-lethal and lethal reactions. open circles are individual observations and the solid line corresponds to a cubic spline function fitted through the observations ������� �� ��� ��� ������������� � � �� �� �� �� �������� �� ��� �� �� �� �� �� �� �� � � � �� �� � ��� ������������� � � �� �� �� �� �� �� �� �� �� �� � � � �� �� � fig. 2 mean corpuscular volume (mcv) as a function of days post infection in animals with non-lethal and lethal reactions. open circles are individual observations and the solid line corresponds to a cubic spline function fitted through the observations 40 red blood cell volume in cattle infected with theileria parva katete when plotting the initial frequency of rbc sizes in individual animals, the distribution of rbc sizes was wider in animals with non-lethal reactions (fig. 4). while the lower limit of sizes was the same in both groups, the upper limit was lower in lethal t. parva infection. sizes of parasitized and unparasitized erythrocytes in t. parva infections animals that suffered lethal reactions had smaller rbc diameters before parasitaemia was detected (p < 0.001) (table 1). in addition, for both lethal and non-lethal reactions, the diameter of parasitized �� ������� ������ �� ������� ������ �� �� �� �� �� � � � � � � �� � � �� �� � � � �� �� �� �� �� � � � � � � �� � � �� � � fig. 3 the box-and-whisker plots of the average packed cell volume (pcv) and mean corpuscular volume (mcv) values for cattle infected with t. parva katete in the first 7 days post infection. (a) pcv values and (b) mcv values. dots represent outlier values. boxes represent the 25th, 50th (median) and 75th percentiles (including outliers). whiskers include all observations except outliers table 1 mean diameter (95 % confidence interval) of red blood cells (μm) lethal non-lethal before parasitaemia 4.87 (4.81–4.93) 5.19 (5.12–5.26) during parasitaemia low parasitaemia infected rbc non-infected rbc 4.46 (4.41–4.51) 5.05 (5.01–5.09) 4.60 (4.55–4.65) 5.21 (5.17–5.25) maximum parasitaemia infected rbc non-infected rbc 4.45 (4.40–4.51) 5.04 (5.00–5.08) 4.59 (4.54–4.65) 5.20 (5.16–5.24) 41 p. fandamu et al. fig. 4 red blood cell volume (in fl) histograms from animals in the study taken in the first stage of infection (0–7 days) using a haematology analyzer. animals 1–10 had lethal reactions; 11–18 had non-lethal reactions � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � �� �� �� � � � �� �� � � � � � � �� �� �� �� � �� �� � �� �� �� � �� �� �� � �� �� �� �� �� ����� �� 42 red blood cell volume in cattle infected with theileria parva katete rbcs was significantly smaller than non-parasitized cells at low and maximum parasitaemia (p < 0.001). the average size of uninfected cells was significantly larger during the parasitaemia of lethal reactions than before. discussion this study confirmed that anaemia occurs in animals infected with the t. parva katete stock. in the early stage of infection, the pcv profiles were similar in both outcome groups but the decrease in pcv was steeper in animals undergoing lethal reactions as the infection progressed. before day 24 in both lethal and non-lethal infections, the mcv did not respond, presenting as a normocytic (non-regenerative) anaemia. this contrasts with our unpublished experimental observations with the t. parva katete infections in belgian black and white cattle that during recovery following chemotherapy the mcv values increased, showing a regenerative response. it is possible that chemotherapy destroys most of the schizonts, halts lymphodestruction and allows the homeostatic mechanism to switch focus from a lymphoid to an erythroid response. the other major finding was that animals that suffered lethal reactions had lower initial mcv values. before infection, these animals were not anaemic as their pcv values were within normal range. no effect of age on mcv could be observed in this study as the number of observations was limited and the range of ages narrow. birgel junior, d’angelino, ben esi & birgel (2001) reported that pcv and rbc counts were significantly higher in younger jersey cattle while mcv did not seem to be significantly affected by age. in contrast penny, scofield & cem browicz (1966) working with different breeds (fries ian, hereford, shorthorn, charolais, guernsey and ayrshire) with ages ranging from less that 2 years to over 10 years reported that pcv increased in bulls of ages 3–8 years compared other age groups while there was no significant difference in rbc and mcv values with age. one possible explanation for the deaths in animals with low mcvs could be that there is an increased affinity of t. parva for small rbcs which would contribute to increased destruction or clearance of infected cells and subsequently to anaemia. this hypothesis is supported by the fact that uninfected red blood cells were larger during parasitaemia than before in lethal reactions in our study. indeed, if the smallest cells of a pool become infected, the average size of the remaining cells will increase as observed. waugh, narla, jackson, mueller, suzuki & dale (1992) reported that aging rbcs lose both surface area and volume and are smaller than younger cells. older (smaller) cells might be more easily infected by merozoites than younger (larger) cells. shaw & tilney (1995) suggested that the deformability of the erythrocyte membrane may be a crucial factor in determining host cell susceptibility to invading t. parva merozoites. it may be that older cells lose some of the characteristics that prohibit the initial interaction with merozoites, making it easier for the parasite to enter the cell. therefore, as parasitized cells are lysed or damaged by the parasite, or parasitized and non parasitized cells are coated with parasite antigen and removed by the immune system, animals with a pool of old cells may be more likely to develop anaemia than animals with a population of younger cells. it has been reported that following t. parva infections, there are considerable losses in parasitized rbcs and anaemic changes in animals with severe parasitaemia (barnett 1957). additionally, shiono, yagi, kumar, yamanaka & chikayama (2004) reported an increasing number of igg bound red blood cells that were prone to removal by phagocytosis in animals infected with theileria sergenti when a rise in parasitaemia was recorded. otsuka, yamasaki, yamato & maede (2002) also reported immunological phagocytosis of igg bound red blood cells in dogs infected with babesia gibsoni which resulted in severe anaemia. therefore, animals with a higher proportion of smaller cells may experience a higher fatality rate as parasite invasion of these fragile cells would lead to more severe anaemia. the anaemia may exacerbate the respiratory distress caused by pneumonia and oedema induced by the infiltration of the lungs with infected and dividing lymphoblasts (irvin & morrison 1987) that is considered to be the cause of death in most t. parva infections (irvin & mwamachi 1983). the invasion of older rbcs observed in this study is in contrast to the findings of mbassa et al. (1994) who reported that t. parva merozoites infected immature stages of erythrocytes. a possible explanation for this difference could be that mbassa and colleagues were studying natural t. parva challenge in tanzania and were thus looking at a different parasite and cattle with a different genetic make up. these findings offer the possibility of predicting the possible outcome of t. parva infection based on an assessment of the initial mcv values and could 43 p. fandamu et al. contribute to improved diagnosis and control. it must be noted that mcv is only one additional parameter affecting the outcome of t. parva which is equally influenced by the parasite strain, the dose and cattle breed. our observations pertain only to t. parva katete in belgian black and white dairy animals and need to be extended to other breeds and other t. parva stocks. it might also have relevance for other blood parasites of animal health importance such as babesia spp. acknowledgements we express our sincere thanks to all members of staff of the laboratory for clinical biology and the department of animal health at the institute of tropical medicine, antwerp, belgium for their assistance in looking after the experimental animals and processing the samples. this work was conducted within the framework of the belgium government funded project “assistance to the veterinary services of zambia (asveza)”. references barnett, s.f. 1957. theileriasis control. bulletin of epizootic diseases of africa, 5:343–357. berkvens, d.l., geysen, d.m. & lynen, g.m. 1989. east coast fever immunisation in the eastern province of zambia, in theileriosis in eastern, central and southern africa, edited by t.t. dolan. proceedings of a workshop on east coast fever immunisation held in lilongwe, malawi, 20–22 september 1988: 83–86. birgel, e.h. junior, d’angelino, j.l., benesi, f.j. & birgel, e.h. 2001. reference values of the erythrogram of jersey breed, raised in são paulo state. arquivo brasileiro de 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haematological values for the clinically normal bull. british veterinary journal, 122:239–247. radley, d.e., brown, c.g.d., burridge, m.j., cunningham, m.p., peirce, m.a. & purnell, r.e. 1974. east coast fever: quantitative studies of theileria parva in cattle. experimental parasitology, 36:278–287. shaw, m.k. & tilney, l.g. 1995. the entry of theileria parva merozoites into bovine erythrocytes occurs by a process similar to sporozoite invasion of lymphocytes. parasitology, 111: 455–461. shiono, h., yagi, y., kumar, a., yamanaka, m. & chikayama, y. 2004. accelerated binding of autoantibody to red blood cells with increasing anaemia in cattle experimentally infected with theileria sergenti. journal of veterinary medicine, b51:39–42. waugh, r.e., narla, m., jackson, c.w., mueller, t.j., suzuki, t. & dale, g.l. 1992. rheologic properties of senescent erythrocytes: loss of surface area and volume with red blood cell age. blood, 79:1351–1358. young, a.s., purnell, r.e., payne, r.c., brown, c.g.d. & kanhai, g.k. 1978. studies on the transmission and course of infection of a kenyan strain of theileria mutans. parasitology, 67:99–115. open access reviewer acknowledgement http://www.ojvr.org open access page 1 of 1 the onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. therefore, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of the onderstepoort journal of veterinary research. we appreciate the time taken to perform your review successfully. in an effort to facilitate the selection of appropriate peer reviewers for the onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on http://www. ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please contact us at submissions@ojvr. org so that we can submit your details to the editor-in-chief for consideration. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ojvr.org 2. in your ‘user home’ [http://www.ojvr.org/ index.php/ojvr/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the onderstepoort journal of veterinary research. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 fax: +27 21 975 4635 acknowledgement to reviewers http://www.ojvr.org http://www.ojvr.org http://www.ojvr.org mailto:submissions@ojvr.org mailto:submissions@ojvr.org http://www.ojvr.org http://www.ojvr.org http://www.ojvr.org/index.php/ojvr/user http://www.ojvr.org/index.php/ojvr/user mailto:publishing@aosis.co.za abstract introduction materials and methods results discussion acknowledgements references about the author(s) lezaan roux department of veterinary tropical diseases, bovine tuberculosis and brucellosis research programme, faculty of veterinary science, university of pretoria, onderstepoort, south africa alicia j. mccall department of agriculture and rural development, veterinary services, kwazulu-natal province, hluhluwe, south africa anita l. michel department of veterinary tropical diseases, bovine tuberculosis and brucellosis research programme, faculty of veterinary science, university of pretoria, onderstepoort, south africa citation roux, l., mccall, a.j. & michel, a.l., 2019, ‘detection of native interferon-γ in nyala (tragelaphus angasii): towards diagnosing tuberculosis’, onderstepoort journal of veterinary research 86(1), a1796. https://doi.org/10.4102/ojvr.v86i1.1796 research communication detection of native interferon-γ in nyala (tragelaphus angasii): towards diagnosing tuberculosis lezaan roux, alicia j. mccall, anita l. michel received: 13 june 2019; accepted: 27 aug. 2019; published: 06 nov. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract mycobacterium bovis is the main cause of tuberculosis in wildlife. in south africa, african buffaloes (syncerus caffer) are a wildlife maintenance host while a number of other species are considered spillover hosts. nyala (tragelaphus angasii), a large antelope species from southern africa, is frequently traded and can be infected with m. bovis. interferon gamma (ifn-γ) release assays that detect cell-mediated immune (cmi) responses to m. bovis infection have shown promise in elephants, rhinoceroses and buffaloes. the bovigam® assay is a commercial ifn-γ release assay designed to detect tuberculosis in cattle and has been validated in buffaloes. we tested the suitability of the bovigam® assay to detect native ifn-γ release in nyala. blood samples collected from 17 nyalas were stimulated with different mitogens and ifn-γ release measured. we found that incubating whole blood with phorbol 12-myristate 13-acetate and calcium ionophore (pma/cai) resulted in the highest levels of ifn-y release. samples stimulated with tuberculin purified protein derivatives of m. bovis (ppdb) and m. avium (ppda) did not show significant ifn-γ production. an intradermal tuberculin test (idt) and culture of tissues from 15 of the 17 culled nyala were also performed, which supported the findings of the bovigam® assay, suggesting the potential value of this assay for the diagnosis of tuberculosis in nyala. keywords: interferon gamma; mycobacterium bovis; nyala; tragelaphus angasii; tuberculosis. introduction bovine tuberculosis (btb), caused by mycobacterium bovis (m. bovis), has been diagnosed in numerous wildlife species. some of these wildlife species act as maintenance hosts, including african buffalo (syncerus caffer) and greater kudu (tragelaphus strepsiceros), while others are considered potential maintenance hosts such as lion (panthera leo) and warthog (phacochoerus africanus), or spillover hosts including chacma baboon (papio ursinus), leopard (panthera pardus), impala (aepyceros melampus) and other bovidae (michel et al. 2015). nyala is a bovid native to southern africa that is regularly sold and translocated in large numbers across the country. currently, there is little knowledge on their role in the epidemiology of btb, and to the best of our knowledge, there is currently only one report on the occurrence and diagnosis of bovine tuberculosis in nyala in south africa (hlokwe, van helden & michel 2014). methods currently used to diagnose tuberculosis in wildlife include bacterial cultures and for some species cell-mediated immune (cmi) response assays such as the intradermal tuberculin test (idt) and interferon gamma (ifn-γ) assays (maas, michel & rutten 2013). bacterial culture is the gold standard in the diagnosis of tuberculosis and is most successfully performed on post-mortem tissue samples; but it can take up to 8 weeks to make an initial analysis and requires additional tests, such as polymerase chain reaction (pcr), to identify the pathogen present. the idt has been used in several wildlife species but requires the capture and immobilisation of animals twice over the period of 72 h and has not yet been validated for nyala (cousins & florisson 2005). the ifn-γ assay detects ifn-γ released from t-cells of an m. bovis infected host. the advantages of the ifn-γ assay are that only whole blood, not tissue, is needed for the analysis; the analysis can be carried out within days; and the animal is captured only once for blood collection. however, the ifn-γ assay is species or at least family specific and will have to be designed and/or optimised for wildlife species. mitogens known to cause optimal ifn-γ release independent of the host’s infection status are used as positive controls in the ifn-γ assay, but the mitogen’s efficacy might also be species specific (angkawanish et al. 2013; maas et al. 2012; morar et al. 2013; schiller et al. 2009). bovigam® is a commercial igra kit used to analyse ifn-γ release in cattle but has been successfully validated for buffalo plasma (michel et al. 2011). we tested whole blood collected from nyala and stimulated with a variety of mitogens and bovine and avian tuberculin in order to identify the conditions for native ifn-γ release in this species. culture and pcr results were used to verify the cmi responses. materials and methods animals and sample collection tissue and blood samples were collected from 15 and 17 nyalas, respectively, in the phinda private game reserve in northern kwazulu-natal for ifn-γ assay and skin test before being culled for population management purposes. blood, 2 ml × 9 ml, was collected in heparin vacutainer tubes and stimulated within 8 hours. upon completion of all testing procedures, the nyalas were humanely euthanised and tissue samples collected from lungs, head lymph nodes, thoracic lymph nodes and kept frozen at −20 °c. interferon gamma assay aliquots of 1 ml blood were incubated, respectively, with roswell park memorial institute (rpmi) 1640 medium cell culture media (negative control), purified protein derivative of bovine tuberculin (ppdb, 24 µ/ml), purified protein derivative of avian tuberculin (ppda, 48 µg/ml) and mitogens: pokeweed mitogen (pwm, 5 µg/ml and 20 µg/ml); phorbol 12-myristate 13-acetate and calcium ionophore (pma/cai, 100 ng/ml and 2 µg/ml); concanavalin a (cona, 10 µg/ml); phytohemagglutinin (pha, 10 µg/ml) and at 37 °c for 24 h. the supernatants were harvested and stored at –20 °c. the ifn-γ assay was carried out according to the manufacturer’s instructions (bovigam® 1g, asurequality australia pty ltd). optical density (od) values (od at 450 nm – 650 nm) values of mitogen and antigen-stimulated samples were blanked by subtracting the values of the animals’ specific negative controls. intradermal tuberculin test two sites on opposite sides of the neck were shaved and skin fold thickness measured with calipers followed by injection of 0.1 ml of ppdb and ppda, respectively. the skin reactions were read after 72 h by measuring the increase in skin fold thickness and clinically evaluated. bacterial culture and polymerase chain reaction tissue handling and culture were performed as described before (michel et al. 2017). acid fast bacterial colonies were subjected to a multiplex pcr method previously described by kim et al. (2013). polymerase chain reaction products were run on a 2% agarose gel at 100 v for minimum of 1 h. the dna fragments were viewed using chemidoc™ xrs system (bio-rad laboratories, south africa). ethical considerations ethical clearance to conduct this study was obtained from the animal ethics committee (aec) of the university of pretoria on 22 august 2016 (ethical clearance number: v078-16). results whole blood samples (n = 17) were incubated, respectively, with pma/cai, both concentrations of pwm, cona, ppdb and ppda. only two samples were incubated with pha. pma/cai stimulation yielded the highest release of ifn-γ with blanked od values ranging from −0.255 to 3.573 (figure 1). blanked od ranges for samples incubated with 5 µg/ml pwm, 20 µg/ml pwm, cona and pha were −0.097 to 1.83, −0.04 to 2.112, −0.282 to 0.068 and −0.239 to −0.011, respectively. negligible amounts of ifn-γ were detected in the antigen-stimulated samples, with od values ranging from −0.259 to 0.016 for ppdb and −0.241 to 0.195 for ppda. figure 1: optical densities of plasmas from nyala whole blood samples stimulated with different mitogens and antigens. average blanked optical density values (490 nm – 690 nm) of mitogen or antigen-stimulated samples. values were corrected by subtracting the antigen or mitogen values with the negative sample values of respective animals. error bars represent standard error of the mean. the skin test was evaluated in 14 adult and one subadult nyala. the skin fold thickness at the bovine injection site remained unchanged in 11 animals and increased by between 0.2 mm and 0.5 mm in four animals. the skin fold thickness at the avian injection site remained unchanged in 10 animals and increased by between −0.3 mm and 0.4 mm in five animals. all animals showing a reaction at the bovine injection site also showed a reaction at the avian injection site. subtraction of the avian increase from the bovine increase resulted in net increases of between −0.3 mm and 0.4 mm. with the exception of two animals which showed small hard swellings at the bovine injection site (net differential increases of 0.1 mm and 0.4 mm, respectively) none of the animals showed clinical signs at the injection sites. all skin test results were considered negative for bovine tuberculosis. bacterial culture of tissues from nyala 3, 5 and 17 showed growth on the löwenstein-jensen media after 10 weeks. the resulting bacterial colonies were all identified as non-tuberculous mycobacteria (table 1). table 1: results of bacterial culture (and polymerase chain reaction) of tissue samples from nyala. discussion the increasing spread of m. bovis infection in wildlife in south africa causes concern to game owners, conservation bodies and state veterinary services. the successful control relies on validated diagnostic assays for ante mortem testing of host species at risk of transmitting m. bovis. cmi assays showed promise in detecting m. bovis infection in cattle and buffalo and were considered for potential use in nyala. this study aimed at determining which mitogen will result in sufficient t-cell stimulation and, therefore, optimal ifn-γ production. not only did the pma/cai incubation result in the highest release of ifn-γ, but also the high od values achieved showed that the bovigam® kit can be successfully used with nyala samples. this was even more evident in the antigen-stimulated samples that showed significantly and consistently lower od values compared to the positive control. these results and conclusions indicated absence of exposure to m. bovis and were supported by the findings of the post-mortem examination, the idt, bacterial culture and pcr results. the findings further demonstrate that the presence of non-tuberculous mycobacteria in three nyala did not cause any cross-reactions potentially resulting in false positive test results upon stimulation with ppdb in the bovigam® assay. in conclusion, the findings of this study highlight the potential value of the commercial bovigam® assay in the diagnosis of m. bovis infection in nyala and justify a validation study in this species. acknowledgements the authors thank the management of the phinda game reserve for permission to collect the samples. they acknowledge ms pamela wambulawaye for conducting the bacterial culture and polymerase chain reaction (pcr). competing interests the authors have declared that no competing interest exists. authors’ contributions l.r. performed the interferon gamma assay and interpreted test data, part of the bacterial culture work and wrote the manuscript. a.j.m. performed and interpreted the skin test, blood sample collection and stimulation, as well as post-mortem examination and tissue sample collection. a.l.m. designed the project, added conceptual contributions and wrote the manuscript. funding information the national research foundation of south africa funded this research (grant number: 109337). data availability statement data are available for sharing. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references angkawanish, t., morar, d., van kooten, p., bontekoning, i., schreuder, j., maas, m. et al., 2013, ‘the elephant interferon gamma assay: a contribution of diagnosis of tuberculosis in elephants’, transboundary and emerging diseases 60(suppl 1), 53–59. https://doi.org/10.1111/tbed.12098 cousins, d.v. & florisson, n., 2005. ‘a review of tests available for the use in the diagnosis of tuberculosis in non-bovine species’, revue scientifique et technique / office international des epizooties 24(3), 1039–1059. https://doi.org/10.20506/rst.24.3.1635 hlokwe, t., van helden, p. & michel, a.l., 2014, ‘evidence of increasing intra and inter-species transmission of mycobacterium bovis in south africa: are we losing the battle?’, preventative veterinary medicine 115(1–2), 10–17. https://doi.org/10.1016/j.prevetmed.2014.03.011 kim, y., choi, y., jeon, b., jin, h., cho, s. & lee, h., 2013, ‘a simple and efficient multiplex pcr assay for the identification of mycobacterium genus and mycobacterium tuberculosis complex to the species level’, yonsei medical journal 54(5), 1220–1226. https://doi.org/10.3349/ymj.2013.54.5.1220 maas, m., michel, a.l. & rutten, v.p.m.g., 2013, ‘facts and dilemmas in diagnosis of tuberculosis in wildlife’, comparative immunology, microbiology and infectious diseases 36(3), 269–285. https://doi.org/10.1016/j.cimid.2012.10.010 maas, m., van kooten, p.j.s., schreuder, j., morar, d., tijhaar, e., michel, a.l. et al., 2012, ‘development of a lion-specific interferon-gamma assay’, veterinary immunology and immunopathology 149(3–4), 292–297. https://doi.org/10.1016/j.vetimm.2012.07.014 michel, a., cooper, d., jooste, j., de klerk, l.-m. & jolles, a., 2011, ‘approaches towards optimising the gamma interferon assay for diagnosing mycobacterium bovis infection in african buffalo (syncerus caffer)’, preventative veterinary medicine 98(2–3), 142–151. https://doi.org/10.1016/j.prevetmed.2010.10.016 michel, a.l., de klerk-lorist, l.-m., buss, p., hofmeyr, m., cooper, d. & bengis, r.g., 2015, ‘tuberculosis in south african wildlife: lions, african buffalo and other species’, in h. mukundan, m.a. chambers, w.r. waters & m.h. larsen (eds.), tuberculosis, leprosy and mycobacterial diseases of man and animals: the many hosts of mycobacteria, pp. 365–385, cab international, croydon. michel, a.l., lane, e.p., de klerk-lorist, l-m., hofmeyr, m., van der heijden, e.m.d.l., botha, l. et al., 2017, ‘experimental mycobacterium bovis infection in three white rhinoceroses (ceratotherium simum): susceptibility, clinical and anatomical pathology’, plos one 12(7), e0179943. https://doi.org/10.1371/journal.pone.0179943 morar, d., schreuder, j., mény, m., van kooten, p.j.s., tijhaar, e., michel, a.l. et al., 2013, ‘towards establishing a rhinoceros-specific interferon-gamma (ifn-γ) assay for diagnosis of tuberculosis’, transboundary and emerging diseases 60(s1), 53–59. https://doi.org/10.1111/tbed.12132 schiller, i., waters, w.r., vordermeier, h.m., nonnecke, b., welsh, m., keck, n.a. et al., 2009, ‘optimization of a whole-blood gamma interferon assay for detection of mycobacterium bovis-infected cattle’, clinical and vaccine immunology 16(8), 1196–1202. https://doi.org/10.1128/cvi.00150-09 abstract introduction materials and methods results discussion acknowledgements references about the author(s) peter nambala department of basic medical sciences, university of malawi, malawi janelisa musaya department of pathology, university of malawi, malawi kyoko hayashida research centre for zoonosis control, hokkaido university, japan emmanuel maganga mikolongwe veterinary college of agriculture and food security, limbe, malawi edward senga department of basic medical sciences, university of malawi, malawi kelita kamoto department of basic medical sciences, university of malawi, malawi john chisi department of basic medical sciences, university of malawi, malawi chihiro sugimoto research centre for zoonosis control, hokkaido university, japan citation nambala, p., musaya, j., hayashida, k., maganga, e., senga, e., kamoto, k. et al., 2018, ‘comparative evaluation of dry and liquid rime lamp in detecting trypanosomes in dead tsetse flies’, onderstepoort journal of veterinary research 85(1), a1543. https://doi.org/10.4102/ojvr.v85i1.1543 original research comparative evaluation of dry and liquid rime lamp in detecting trypanosomes in dead tsetse flies peter nambala, janelisa musaya, kyoko hayashida, emmanuel maganga, edward senga, kelita kamoto, john chisi, chihiro sugimoto received: 11 sept. 2017; accepted: 25 july 2018; published: 03 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract xenomonitoring is an important approach in assessing the progress of trypanosomiasis control as well as in estimating the endemicity of trypanosomes in affected areas. one of the major challenges in this approach is the unavailability of sensitive and easy to use xenomonitoring tools that can be used in the remote areas where the disease occurs. one tool that has been used successfully in detecting the parasites in tsetse flies is the repetitive insertion mobile element loop-mediated isothermal amplification (rime lamp). this tool has recently been modified from the liquid form to dry form for use in remote areas; however, uptake for use in the field has been slow. field-collected tsetse flies were used to evaluate the performance of dry rime lamp over the conventional liquid rime lamp. all the samples were also subjected to internal transcribed spacer 1 (its1) ribosomal deoxyribonucleic acid (dna) polymerase chain reaction (pcr) as a standard. its1-pcr-positive samples were further sequenced for confirmation of the species. a total of 86 wild tsetse flies were left to dry at room temperature for 3 months and dna was extracted subsequently. all 86 flies were glossina morsitans morsitans. from these, dry rime lamp detected 16.3% while liquid rime lamp detected 11.6% as infected with trypanosomes. ten positive samples on its1-pcr were sequenced and all were shown to be trypanosomes. the use of dry rime lamp in the field for xenomonitoring of trypanosomes in tsetse flies will greatly contribute towards control of this neglected tropical disease as it provides the cheapest, fastest and simplest way to estimate possible human infective trypanosome infection rates in the tsetse fly vectors. introduction human african trypanosomiasis (hat) is an important public health problem that affects rural populations of 36 sub-saharan african countries (franco et al. 2014). the disease is caused by trypanosomes belonging to the subgenus trypanozoon (bruce 1915). trypanosoma brucei rhodesiense causes an acute disease in eastern, central and southern parts of africa, while trypanosoma brucei gambiense causes chronic diseases and occurs in west and central africa (brun & blum 2012). other parasites of the subgenus trypanozoon are trypanosoma brucei brucei, trypanosoma evansi and trypanosoma equiperdum, which cause nagana, surra and dourine in livestock, respectively. the epidemiology of sleeping sickness follows the parasite–vector interactions within a particular environment (dale et al. 1995); therefore, the risk of host infection is through a tsetse fly bite. in malawi, it has been shown that tsetse flies are infected with the human infective t. b. rhodesiense (musaya et al. 2017). therefore, deploying control measures in areas infested with infected tsetse flies would be ideal in reducing the prevalence of the disease. in limited-resource settings, tools that are sensitive and easy to use will be very useful in the surveillance of trypanosome infection rate in tsetse flies. in any particular environment, success of trypanosomiasis control is dependent upon the use of appropriate diagnostic tools. detection of trypanosomes in vectors for surveillance has previously depended on dissection of tsetse flies for mid-guts and salivary parts for microscopy and polymerase chain reaction (pcr) for amplification of trypanosome deoxyribonucleic acid (dna) (basheer et al. 2013; malele et al. 2011). these techniques are labour intensive and not conducive for control programmes. not all tsetse flies positive for trypanosome dna are indicative of an established infection as some trypanosome dna may originate from the blood meal that the flies had taken prior to analysis (macleod et al. 2007). however, for control purposes any tsetse fly infection is important for deployment of control strategy instead of waiting until infection is established in humans or animals. despite technological advancement in the diagnosis of trypanosomes, the uptake of these techniques has been minimal and many diagnostic settings continue to rely on insensitive and unsatisfactory parasitological or serodiagnostic techniques (njiokou et al. 2004; ouma et al. 2000) a more sensitive diagnostic and surveillance technique called repetitive insertion mobile element loop-mediated isothermal amplification (rime lamp) has been developed and evaluated for detection of trypanosomes using tsetse fly mid-guts (alibu et al. 2015; njiru et al. 2008a). however, this test still poses a challenge in tropical and developing countries where the disease is endemic as the reagents and field samples still require expensive equipment, which are unfortunately not available in most places. a technique for use in surveillance and control programmes should be able to detect trypanosomes in a variety of specimens that can be easily collected and stored with very minimal logistical requirements. dry rime lamp for detection of hat has been developed through modification of the conventional liquid rime lamp (hayashida et al. 2015). the unique feature in the components of dry rime lamp is that all reaction reagents are air-dried on a lid of individual reaction tubes for easy storage, transportation and use at room temperatures unlike liquid rime lamp, which requires cold chain for storage of reagent components. dry rime lamp was demonstrated to have robust performance in detecting hat in artificially parasite mixed blood (hayashida et al. 2015). however, use of dry rime lamp for detection of trypanosomes in field-collected samples has not been demonstrated. detection of trypanosomes in tsetse flies may accelerate the critical advances in the control of both hat and animal african trypanosomiasis (aat). in this study, we demonstrate that dry rime lamp has the potential to be used as a surveillance tool to detect trypanosomes in field-caught tsetse flies. the study was carried out in the liwonde wildlife reserve, machinga district, malawi. materials and methods study area the fieldwork was conducted in the liwonde wildlife reserve, machinga district, malawi. this is located in the eastern part of malawi, and being a protected area, it is rich in wildlife. wildlife provides the main source of blood meals for tsetse flies. the main feature is the shire river, which flows along the wildlife reserve and forms a valley. on the highlands east of this area, land use is characterised by mixed-crop agriculture, livestock and higher human population densities. a game fence, which is irregularly maintained, physically marks the boundary between the protected and settlement areas. tsetse collection tsetse flies were collected using stationary epsilon traps that were placed near the main camp for 24 hours. fly round was also used to cover an area of about 5 kilometres in the same location where traps were set. fly rounds involved the use of a blue or black screen tent mounted in the body of a car that was baited with tsetse fly odour attractants (3-n propyl phenol, 1-octen-3-ol, 4-methyl phenol). collected dead and live tsetse flies were dried at room temperature and individually put in single eppendorf tubes containing desiccants. cotton wool was placed in the middle of the tubes to avoid contact of the desiccants with the tsetse fly. the flies were stored at room temperature for a period of 3 months before analysis. isolation of parasite deoxyribonucleic acid dry whole tsetse flies were transferred to new eppendorf tubes and crushed using a sterilised plastic pestle. parasite dna was extracted from the crushed tsetse flies using roche dna isolation kit (mannheim, germany) as per the manufacturer’s instructions. the dna was suspended in 100 µl of pcr water and stored at -20 °c. amplification of trypanosome deoxyribonucleic acid using repetitive insertion mobile element loop-mediated isothermal amplification dry rime lamp was used to amplify the parasite dna. liquid rime lamp was also used alongside the dry rime lamp. six lamp primers (table 1) were used to detect the trypanozoon group of the trypanosome parasite for both dry and liquid rime lamp (hayashida et al. 2015; njiru et al. 2008a; njiru et al. 2008b). for dry rime lamp, all lamp reagents were placed on the lid of each 0.2-ml microtube and air-dried as described by hayashida et al. (2015). the purified genomic dna from the dead tsetse fly was used as a reaction template, 2 μl of dna plus 1 μl of 25x lamp buffer (500 mm tris–hcl, 250 mm kcl and 100 mm mgso4), 1.5 μl of 100 mm mgso4, and 0.1% triton x-100 in nuclease-free water were added to make a 25-μl reaction mix. the tubes were turned upside down and mixed well so that the dried reagents were completely reconstituted. the tubes were incubated at 65 °c for 60 min and the amplification reactions were visualised using a battery-operated light-emitting diode (led) illuminator (figure 1). figure 1: visualisation of dry repetitive insertion mobile element loop-mediated isothermal amplification using battery-operated led light. positive and negative samples turn green and orange, respectively, under led light. table 1: nucleotide sequences of internal transcribed spacer 1-polymerase chain reaction and repetitive insertion mobile element loop-mediated isothermal amplification primers. for the liquid rime lamp, amplification was performed using 25 μl total volume for one reaction: 2 µl of template dna, 1 μl of 25x lamp buffer (500 mm tris–hcl, 250 mm kcl and 100 mm mgso4), 1.5 μl of 100 mm mgso4, 1.4 μl of 25 mm dntps, 1 μl of each primer, 1 μl of bst 2.0 dna polymerase (new england biolabs, japan), 2 μl of 2 m trehalose, 1 μl of colori-fluorometric indicator (cfi; 3 mm hydroxy-naphthol blue (mp biomedicals, aurora, oh, usa) and 0.35% v/v gelgreen (10,000x solution; biotium, hayward, ca, usa) and volume adjusted with nuclease-free water containing 0.1% triton x-100. the amplification was monitored on the fam channel using a real-time pcr machine (cfx96; bio rad, japan) at 62 °c for 60 min. amplification of trypanosome deoxyribonucleic acid using serum-resistant associated gene loop-mediated isothermal amplification all samples were also subjected to serum-resistant associated gene (sra) lamp that is specific for the detection of the sra responsible for infectivity of t. b. rhodesiense to humans. six primers used for sra lamp are also shown in table 1 (hayashida et al. 2015). amplification was performed using 25-μl total volume for one reaction: 2 μl of template dna, 1 μl of 25x lamp buffer (500 mm tris–hcl, 250 mm kcl and 100 mm mgso4), 1.5 μl of 100 mm mgso4, 1.4 μl of 25 mm dntps, 1 μl of each primer, 1 μl of bst 2.0 dna polymerase (new england biolabs), 2 μl of 2 m trehalose, 1 μl of cfi and volume adjusted with nuclease-free water containing 0.1% triton x-100. the amplification was monitored in a real-time pcr machine at 62 °c for 60 min. visualisation of dry repetitive insertion mobile element loop-mediated isothermal amplification reaction colori-fluorometric indicator dye that contains gelgreen was included in the dry tube to enable direct visualisation of amplified dna products on a battery-operated handheld led illuminator that emits 500 nm wavelength of light (figure 1). amplified dna was detected by direct visual inspection of colour change of cfi dye under led light. positive and negative samples turn green and orange, respectively, under led light at 500-nm wavelength. confirmatory tests: internal transcribed spacer 1–polymerase chain reaction and sequencing internal transcribed spacer 1–polymerase chain reaction: alongside lamp, the extracted dna samples were also analysed with internal transcribed spacer (its1)–pcr, which detects both the trypanozoon and other trypanosome parasites and distinguishes the species. the pcr assays were performed in a dna thermal cycler (applied biosystems, california, usa). the its1-pcr used two oligonucleotide primers (table 1) for amplification of its1 as described by njiru et al. (2005). each amplification reaction was made in a final volume of 20 μl containing 5 µl of 2x ampdirect plus (sh imadzu, japan), 0.5 µm of each oligonucleotide primer for its1-pcr, 0.025 u of biotaq hs dna polymerase (bioline, uk) and 1 μl of dna template. the thermocycling programme for the its1-pcr was 10 min at 95 °c, followed by 35 cycles of 30 sec at 94 °c, 1 min at 55 °c, 1 min at 72 °c and a final cycle of 7 min at 72 °c. after pcr, 5 μl of each sample was run on a 1.5% agarose gel and stained with gelred (biotium), a non-carcinogenic gel staining fluorescence dye. all samples were also subjected to sra pcr alongside sra lamp to detect t. b. rhodesiense. both forward and reverse primers for sra pcr were those described by welburn et al. (2001). a final volume of 20 μl was used for each reaction. the reaction conditions were the same as described above for its1-pcr, except the annealing temperature was set as 58 °c. sequencing of internal transcribed spacer 1–polymerase chain reaction products: ten samples positive on its1-pcr were randomly chosen for sequencing. the pcr products were purified using exosap-it reagents (affymetrix, usa) as per the manufacturer’s instruction or cloned into pgemt-easy vector (promega) if multiple traces were observed. the sequencing reactions were performed using bigdye 3.1 terminator cycle sequencing kit and sequenced with an automated 3500 series genetic analyser (applied biosystems). the resulting sequences were analysed with the target sequences using mega7 software (sudhir, stecher & tamura 2016) and blast (https://blast.ncbi.nlm.nih.gov/blast.cgi). statistical analysis data were double entered and verified using microsoft excel software. all statistical analyses were performed using r version 3.3.2 (r core team 2016). results a total of 86 mature tsetse flies were captured. all tsetse flies belonged to glossina morsitans morsitans species. of these tsetse flies, 31 (36%) were male tsetse flies while 55 (64%) were female tsetse flies. all mature tsetse flies were further analysed using dry and liquid rime lamp to compare the diagnostic performance of these two tools in detecting trypanozoon trypanosomes in field-caught samples. the results were further confirmed through its-pcr and sequencing. we also assessed using sra pcr and sra lamp whether trypanozoon-positive samples were t. b. rhodesiense parasites (the causative agent of hat) or not. detection of trypanozoon trypanosomes using dry and liquid repetitive insertion mobile element loop-mediated isothermal amplification to evaluate the diagnostic performance of dry rime lamp as a potential field surveillance tool, we first used dry rime lamp to analyse all 86 field-caught tsetse flies. dry rime lamp detected 14 of 86 (16.3%) samples as trypanozoon positive. total infected female and male tsetse flies were 12 (14%) and 2 (2.3%), respectively. to verify dry rime lamp results, all 86 samples were subjected to a well-established conventional liquid rime lamp (alibu et al. 2015; malele et al. 2013). liquid rime lamp detected 10 of the 86 (11.6%) samples as trypanozoon positive (table 2). furthermore, infected female and male tsetse flies detected using liquid rime lamp were 9 (10.5%) and 1 (1.2%), respectively. all liquid rime lamp–positive samples were also positive using dry rime lamp and the proportion of positive agreement between the two tests was 75% and that for negative agreement was 96%, suggesting that there is no significant difference in the diagnostic performance of dry and liquid rime lamp in detecting trypanozoon trypanosomes in field-collected samples. table 2: diagnostic performance of dry repetitive insertion mobile element loop-mediated isothermal amplification against liquid repetitive insertion mobile element loop-mediated isothermal amplification for the detection of trypanosomes. confirmation of repetitive insertion mobile element loop-mediated isothermal amplification results through internal transcribed spacer 1–polymerase chain reaction our group and others have previously described its1-pcr as the most specific test for the detection of trypanosomes in tsetse flies (alibu et al. 2015). therefore, all 86 tsetse fly samples were reanalysed using its1-pcr as a confirmatory test for both dry and liquid rime lamp. its1-pcr detected 22 of 86 (25.6%) samples as trypanosome positive with trypanosomes infection rate in female and male g. morsitans at 30.9% and 12.9%, respectively (p = 0.06201, chi-square test) (table 3). its1-pcr showed 5 of 86 (5.8%) of the samples to be trypanozoon positive giving the expected pcr product size of 480 bp (figure 2) and 17 of 86 (19.8%) samples to be positive for other animal trypanosome species. when a comparison of the results was made, four of five its1 trypanozoon-positive samples were also positive using dry and liquid rime lamp, hence confirming our lamp results as a true reflection of trypanozoon infection in field-caught tsetse flies. thus, in total its1-pcr detected 22 of the 86 tsetse flies as infective with trypanosomes. mixed infection was observed in one of the five trypanozoon-positive samples, indicating that each tsetse fly has the potential of infecting both human and domestic animals (table 3). figure 2: representative positive products amplified by internal transcribed spacer 1-pcr, polymerase chain reaction were electrophoresed. sample 14 (300 bp): trypanosoma godfleyi; sample 32, 40 and 02 (710 bp): trypanosoma congolense forest; sample 39 (400 bp): trypanosoma simiae; sample 50, 59, 62, 68 (250 bp): trypanosoma vivax; sample 83 (480 bp): trypanozoon. table 3: detection of trypanosome deoxyribonucleic acid by internal transcribed spacer 1-polymerase chain reaction, in glossina morsitans morsitans from liwonde. because rime lamp was developed as a field surveillance tool with high sensitivity (njiru et al. 2008a), the results of our dry and liquid rime lamp when used in field situations are in line with findings of others (malele et al. 2013). the diagnostic performance of dry rime lamp was higher than that of its1-pcr (table 4). concordance was also observed, and the sensitivity of dry lamp comparing its1-pcr was 80% (95% confidence interval [ci]: 28% – 99%) and specificity was 88% (95% ci: 78% – 94%). dry rime lamp detected 16.3% of the tsetse flies as trypanozoon positive compared to its1-pcr, which only detected 5.8% as trypanozoon parasites. table 4: diagnostic performance of dry repetitive insertion mobile element loop-mediated isothermal amplification against internal transcribed spacer 1-polymerase chain reaction for the detection of trypanosomes. confirmation of trypanosome species through sequencing because microscopy was not carried out on all field-caught tsetse flies, 10 samples were randomly picked from the 21 clear, single-band–positive its1-pcr products which were sequenced and confirmed as trypanosoma species. the species confirmed were trypanozoon (480 bp), trypanosoma vivax (250 bp), trypanosoma godfleyi (300 bp), trypanosoma simiae (400 bp) and trypanosoma congolense (7100 bp). randomly sequenced samples that were also positive on dry and liquid rime lamp were all (three samples) confirmed as trypanozoon species. this suggests that dry rime lamp can be an alternative surveillance tool because of its user-friendly simplicity compared to its1-pcr and liquid rime lamp. trypanosoma brucei rhodesiense status in tsetse flies using serum-resistant associated gene loop-mediated isothermal amplification and serum-resistant associated gene polymerase chain reaction because both dry and liquid rime lamp and its1-pcr detect all trypanozoon trypanosomes subspecies without distinguishing the subspecies, we next tested if trypanozoon-positive samples detected by rime lamp and its1-pcr are positive for t. b. rhodesiense, which is responsible for human infections in malawi (bruce et al. 1914; chisi et al. 2011; madanitsa et al. 2009). all trypanozoon-positive samples were subjected to sra lamp and sra pcr as described in the ‘materials and methods’ section to distinguish t. b. rhodesiense from other members of the trypanozoon group. both sra lamp and sra pcr were negative, suggesting that trypanozoon species detected in rime lamp and its1-pcr were t. b. brucei, a parasite responsible for aat. discussion the infection rate of trypanosomes in wild tsetse flies in malawi was previously shown to be higher compared to other countries in eastern and central africa where hat and aat is endemic (alibu et al. 2015). in malawi, the number of new cases of hat trypanosomiasis infection over the past 10 years have remained constant (franco et al. 2014) compared to other countries such as tanzania, uganda, zambia and kenya where the disease is almost under control. this indicates that the disease transmission is not well controlled and humans are still exposed to possibly highly infected vectors and if control is to be possible, surveillance tools should be able to use any available sample materials for the detection of the parasite. in this study, the infective rate in flies was 25.6% by its1-pcr and there was a difference between male (12.9%) and female (30.9%) tsetse flies with higher rates in female g. m. morsitans. unlike in other insect vectors such as sand flies, mosquitoes and black flies (aransay, scoulica & tselentis 2000; dyab et al. 2015), both male and female tsetse feed on blood and therefore both are vectors for trypanosomes. however, in natural field situations, female tsetse flies usually have higher infection rates than male tsetse flies and this is partly because female tsetse flies have a longer lifespan than male tsetse flies (leak 1999) and our results were in agreement with this observation. during this study, microscopy was not carried out; however, the ten positive samples on its1-pcr that were sequenced confirmed that the samples under study were indeed trypanosomes as they aligned with trypanosome species after blast analyses. comparative analysis of dry lamp and liquid lamp showed that dry lamp was in concordance with the conventional liquid lamp where it detected 9 out of 10 positives detected by liquid lamp. furthermore, dry lamp was able to detect five more samples, which were negative on liquid lamp. this finding is in line with other findings on using lamp for xenomonitoring of trypanosomes in tsetse flies (malele et al. 2013). the ability of its1-pcr in detecting trypanozoon subspecies was far less to that of dry rime lamp. this is partly because the its1 region is estimated to have only 100–200 copies per haploid genome compared to 500 copies in the rime region, hence making it less sensitive (bhattacharya, bakre & bhattacharya 2002). therefore, when using pcr for detection of trypanosomes, there is a high possibility of missing out many cases and obtaining misrepresentative data. as shown in table 3, the performance of dry and liquid rime lamp was comparable, detecting 16.3% and 11.6%, respectively. using dry lamp as a simple tool for xenomonitoring of trypanosomes in tsetse flies is thus encouraging. the present study has demonstrated that whole dry dead tsetse flies can be used as samples in both lamp and conventional pcr. several other studies have been conducted on the use of tsetse fly mid-guts for lamp and pcr (cunningham et al. 2016; enyaru et al. 2010). the use of whole dead tsetse flies is advantageous for xenomonitoring of trypanosomes in tsetse flies as different species of the trypanosome parasite are found in different parts of the tsetse fly. a good example is t. vivax, which only develops in the proboscis of the tsetse fly. this is in contrast to the t. brucei group, which develops in the mid-gut of the vector (leak 1999), hence increasing the possibility of detecting the parasite. there is no need for special storage conditions of the dead flies as they can be stored at room temperature, hence reducing surveillance costs compared to use of mid-guts, which quickly become degraded and require liquid nitrogen tanks to be carried to the field for storage. the dry flies used in this study were left for 3 months at room temperature. the study has demonstrated robust detection of the dry rime lamp method in dead whole tsetse flies. the sensitivity of the dry lamp was high enough to allow for the detection of a single infected tsetse fly after a long period of storage at room temperature. though the sample size used in this study was small (86 flies), it still has a potential application for large-scale xenomonitoring for generation of epidemiological data for decision-making. to minimise surveillance costs and save time, use of dry lamp and dead tsetse flies is recommended over the conventional liquid lamp and tsetse fly mid-guts. acknowledgements we would like to thank african parks for giving us access and permission to the liwonde wildlife reserve to do sample collection. this study was partially supported by a research grant from the international collaborative research program for tackling the neglected tropical diseases (ntds) challenges in african countries (jp18jm0510001), the japan agency for medical research and development. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this paper. authors’ contributions p.n. was responsible for sequencing samples and drafted the manuscript. j.m. analysed the data and wrote the article. k.h. was responsible for experimental design. e.s. and k.k. were responsible for performing the molecular experiments. e.m. collected the flies from the field. j.c and c.s. assisted in study design, provided resources and assisted in 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means of serum-resistance-associated (sra) gene’, lancet 358, 2017–2019. https://doi.org/10.1016/s0140-6736(01)07096-9 abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) aaron e. ringo national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan gabriel o. aboge department of public health, pharmacology and toxicology, faculty of veterinary medicine, university of nairobi, nairobi, kenya paul f. adjou moumouni national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan seung hun lee national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan charoonluk jirapattharasate department of pre-clinic and applied animal science, faculty of veterinary science, mahidol university, phutthamonthon nakhonpathom, thailand mingming liu national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan yang gao national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan huanping guo national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan weiqing zheng department of disinfection and vector control, nanchang center for disease control and prevention, nanchang, china artemis efstratiou national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan eloiza m. galon national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan jixu li national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan oriel thekisoe unit for environmental sciences and management, north-west university, potchefstroom, south africa noboru inoue obihiro university of agriculture and veterinary medicine, obihiro, japan hiroshi suzuki national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan xuenan xuan national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, japan citation ringo, a.e., aboge, g.o., adjou moumouni, p.f., hun lee, s., jirapattharasate, c., liu, m. et al., 2019, ‘molecular detection and genetic characterisation of pathogenic theileria, anaplasma and ehrlichia species among apparently healthy sheep in central and western kenya’, onderstepoort journal of veterinary research 86(1), a1630. https://doi.org/10.4102/ojvr.v86i1.1630 original research molecular detection and genetic characterisation of pathogenic theileria, anaplasma and ehrlichia species among apparently healthy sheep in central and western kenya aaron e. ringo, gabriel o. aboge, paul f. adjou moumouni, seung hun lee, charoonluk jirapattharasate, mingming liu, yang gao, huanping guo, weiqing zheng, artemis efstratiou, eloiza m. galon, jixu li, oriel thekisoe, noboru inoue, hiroshi suzuki, xuenan xuan received: 12 mar. 2018; accepted: 15 jan. 2019; published: 13 june 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract tick-borne diseases (tbds) caused by theileria, babesia, anaplasma and ehrlichia species are common in tropical and subtropical regions. in this study, we investigated the presence and genetic diversity of theileria spp., anaplasma ovis, b. ovis, e. ruminantium and anaplasma spp. in sheep from the machakos and homa bay counties of kenya. in order to improve the diagnosis and control of ovine tbds, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (pcr). the assays were conducted using primers based on theileria spp. 18s rrna, anaplasma ovis major surface protein-4 (aomsp4), b. ovis 18s rrna, e. ruminantium pcs20 and anaplasma spp. 16s rrna. the overall infection rates for theileria spp., a. ovis, e. ruminantium and anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. the overall co-infection was 47/76 (61.8%). all theileria spp. positive samples were confirmed to be of theileria ovis on sequencing. a phylogenetic analysis of the 18s rrna gene sequences of t. ovis revealed that all isolates of this study clustered with t. ovis sequences extracted from the genbank suggesting this gene is highly conserved. e. ruminantium pcs20 sequences were in the same clade on the phylogenetic tree. however, three aomsp4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. the 16s rrna sequencing revealed uncharacterised anaplasma spp. and a. ovis. the phylogenetic analyses of the uncharacterised anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the genbank. this study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in kenya, which is useful for the diagnosis and control of tbds. keywords: sheep; pcr; kenya; tick-borne pathogens; phylogenetic analysis. introduction tick-borne diseases (tbds) caused by theileria, babesia, anaplasma and ehrlichia are common in tropical and subtropical regions of the world where there is increased interaction of hosts, pathogens and vectors (bilgic et al. 2017). over the years, limited attention of tbd studies has been directed to sheep and goats compared to cattle (yin et al. 2007). however, small ruminants are becoming important in a number of countries as far as socio-economic importance is concerned. therefore, more attention is now being directed towards pathogens of sheep and goats (bilgic et al. 2017). the tick-borne pathogens of small ruminants include theileria ovis, t. separata, t. lestoquardi, t. sp. ot1, t. sp. ot3, t. luwenshuni and t. recondita which cause theileriosis. babesia ovis and b. motasi cause babesiosis. anaplasma ovis causes anaplasmosis, whereas a. phagocytophilum causes tick-borne fever and ehrlichia ruminantium causes ehrlichiosis (bilgic et al. 2017). t. lestoquardi, b. ovis, b. motasi, a. phagocytophilum and e. ruminantium are considered to be pathogenic, while the rest with the exception of the recently detected t. sp. ot1 and t. sp. ot3 are non-pathogenic in sheep and goats (razmi & yaghfoori 2003; schnittger et al. 2000; uilenberg 1995). the pathogenicity of t. sp. ot1 and t. sp. ot3 do not have conclusive evidence (yin et al. 2007). economic losses incurred from the tbds include mortality, production losses, veterinary costs and tick control (jonsson, bock & jorgensen 2008). in sheep, t. ovis and a. ovis manifest themselves as sub-clinical infections (bilgic et al. 2017). animals that survive the acute phase of infection develop a life-long carrier state, which is associated with significant production and economic losses (gharbi et al. 2006; uilenberg 1995). therefore, the pathogens regarded as less pathogenic should equally be considered important, as they continuously infect ticks when they are in the carrier state, resulting in new infection to uninfected animals (razmi & yaghfoori 2003). the eastern part of africa is one of the three regions of sub-saharan africa which are considered to be highly populated by sheep and other livestock (hanotte et al. 2000). regardless of the large population of sheep in the region, little information is available on the presence and genetic diversity of tick-borne pathogens. this study was conducted to fill in this information gap. blood samples were collected from sheep in machakos and homa bay counties in kenya and tested for the occurrence and genetic diversity of ovine tick-borne pathogens using pcr and sequencing. materials and methods study area blood samples were collected from sheep in machakos and homa bay counties (figure 1). machakos is located in central kenya, about 63 km south-east of nairobi. the local climate is semi-arid with hilly terrain and an altitude of 1000 m – 2100 m above sea level. the climate of this area is temperate and subtropical whereby temperatures are modified by altitude. the area has summer rainfall, while the vegetation is savannah, and rain starts at the end of march to may. figure 1: a map of kenya showing the two counties where samples were collected: (1) homa bay in western kenya and (2) machakos in central kenya. the red triangles are locations of sample collection farms. homa bay county is located in western kenya along lake victoria. the county is near mount homa and ruma national park, located 420 km south-west of nairobi. the climate is semi-arid with temperatures ranging from 26 °c in the coldest months (april and november) to 34 °c during the hottest months (january and march). the county receives an average rainfall of 1100 mm, long rains in march to may and short rains in september to november (climate-data.org 2018). the farmers in both counties are mainly pastoralists with large groups of livestock involving cattle, sheep and goats. the breeds of sheep are mainly red maasai. sample collection and dna extraction a total of 76 blood samples were collected in august 2011 (dry season) from clinically healthy sheep, using sterile needles and edta-coated vacutainer tubes (boenmed, jiangsu, china). an average of 3 ml – 5 ml of blood was drawn from the jugular vein of the animal and refrigerated until transported to the laboratory. fifty-two and 24 samples were collected in machakos and homa bay counties, respectively. samples were collected randomly in five locations and mostly from pastoralist farms. male and female sheep of around 2 years of age and above were targeted. dna was extracted from 200 µl of blood using the qiaamp dna blood mini kit (qiagen, hilden, germany), following manufacturer’s protocol, and stored at -30 °c until the time of use. molecular detection of tick-borne pathogens all samples were screened using pcr with primers obtained from previous studies for theileria spp. 18s rrna, b. ovis 18s rrna, a. ovis major surface protein 4 (aomsp4), anaplasma spp. 16s rrna and e. ruminantium pcs20 genes (table 1). the reaction mixture had a final volume of 10 µl, containing 0.5 mm of each primer, 1 µl of 10×standard ex taq buffer, 1 µl of dntp mix and 0.1 µl of ex taq polymerase (takara – shiga, japan), 1 µl of dna template and 5.9 µl of double distilled water. the double distilled water was used as a negative control, while positive controls were positive samples from a previous study (ringo et al. 2018). polymerase chain reactions were run in a thermal cycler (bio rad, hercules, ca, united states [us]). polymerase chain reaction conditions consisted of initial denaturation at 95°c for 5 minutes, followed by 35 cycles of denaturation for 1 min at 94 °c, 1 min annealing at differing temperatures which can be found in table 1 and 1.30 min extension at 72 °c. the final extension was set to 10 min at 72 °c. the same pcr conditions were used for anaplasma spp. pcr and nested pcr. as for e. ruminantium, the reaction mixture was performed in a semi-nested pcr consisting of initial denaturation for 3 min at 94 °c, followed by 25 cycles of denaturation for 0.30 seconds at 94 °c, annealing for 0.45 seconds at 61 °c and 1 min at 72 °c extension. the final extension was done at 72 °c for 10 min. the pcr products were electrophoresed on a 2% agarose gel and then stained with ethidium bromide and viewed under uv transilluminater. table 1: list of primers used for polymerase chain reaction assays. cloning and sequencing for sequencing, 3–5 positive samples per detected pathogen were randomly selected. amplicons were purified using a qiaquick gel extraction kit (qiagen, germany) per the manufacturer’s protocol. the concentration of the extracts was checked using the nano drop 2000 spectrophotometer. the template (6 µl) was ligated into a pgem-t easy vector (2 µl) (promega, us), with t4 dna ligase and restriction buffer (each 1 µl) added and incubated at 16 °c for 3 hours and then at 4 °c overnight. transformation of the plasmid into escherichia coli dh5α competent cells (prepared in-house) was performed. lysogeny broth (lb) was added and incubated at 37 °c in a shaker incubator for 1 h and then inoculated on lb agar plates and incubated at 37 °c overnight. colonies were picked and put in lb broth with an antibiotic (ampicillin) 50 µg/ml (wako, saitama, japan) incubated at 37 °c overnight in a shaker incubator. plasmid was extracted using the nucleospin® plasmid quickpure (macherey-nagel-germany) kit. the samples were sequenced using a bigdye terminator v3.1 cycle sequencing kit (applied biosystems, us) and a 3100 genetic analyzer (applied biosystems, foster city, calafornia, us). the alignment of the sequences was performed using lasergene v14.1 (dnastar, madison, wi, us). the nucleotide sequence identities and similarities were determined by using a genbank blastn analysis. phylogenetic analysis the sequences obtained in this study were compared with sequences deposited in the genbank by phylogenetic analysis using mega version 7.0 software (kumar, stecher & tamura 2016). the maximum likelihood method was used to construct phylogenetic trees for t. ovis, a. ovis, e. ruminantium and uncharacterised anaplasma spp. bootstrap analysis with 1000 replications was used to estimate the confidence of the nodes and branches of the trees. nucleotide sequence accession numbers sequences obtained in this study were deposited in the genbank database of the national center for biotechnology information using bankit. the sequences were assigned the following accession numbers: mf360021 to mf360025 for t. ovis 18s rrna; mf360026 to mf360029 for a. ovis msp4; mg637125 to mg637127 for uncharacterised anaplasma spp. 16s rrna; and mg544303 to mg544305 for e. ruminantium pcs20. ethical considerations the owners of the selected farms were informed about the study and provided their approval for sample collection from their sheep. all the procedures were approved and carried out according to ethical guidelines for the use of animal samples permitted by obihiro university of agriculture and veterinary medicine (permit for animal experiment: 280080, dna experiment 1219-2; pathogen: 2015727). results overall infection rates polymerase chain reaction results revealed that 59/76 (77.6%) sheep were infected with at least one pathogen. seventeen sheep (22.4%) were not infected with any of the screened pathogens. theileria spp. had an overall infection rate of 39/76 (51.3%), a. ovis 26/76 (34.2%), e. ruminantium 6/76 (7.9%) and uncharacterised anaplasma spp. 31/76 (40.8%). theileria ovis was the only theileria species identified following the sequencing of all pcr-positive theileria spp., while for anaplasma spp., the uncharacterised anaplasma spp. and a. ovis were identified. meanwhile, b. ovis and t. lestoquardi were not detected in this study. infection rates based on location in homa bay, 20/24 (83.3%) sheep were infected with at least one pathogen, while 4/24 (16.7%) were not infected with any of the pathogens screened. the infection rates for each pathogen in homa bay were 13/24 (54.2%), 8/24 (33.3%), 2/24 (8.3%) and 10/24 (41.7%) for t. ovis, a. ovis, e. ruminantium and uncharacterised anaplasma spp., respectively. in machakos, 39/52 (75%) sheep were infected with at least one pathogen. thirteen of the 52 (25%) sheep were not infected with any of the screened pathogens. the infection rates for each pathogen in machakos were 26/52 (50%), 18/52 (34.6%), 4/52 (7.7%) and 14/52 (26.9%) for t. ovis, a. ovis, e. ruminantium and uncharacterised anaplasma spp., respectively. mixed infections several sets of co-infections were revealed in this study (table 2). multiple infections were found in 47/76 (61.8%) samples. theileria ovis was the pathogen most frequently associated with multiple infections (35/76; 46.1%) followed closely by a. ovis (34/76; 44.7%) (table 2). theileria ovis + a. ovis co-infection had the highest overall prevalence. a triple infection (a. ovis + t. ovis + uncharacterised anaplasma spp.) with an overall prevalence of 6/76 (7.9%) was revealed in this study (table 2). table 2: mixed infections detected in sheep in this study. comparative sequence analysis the pcr products of all the t. ovis, a. ovis and e. ruminantium isolated in this study were of the expected sizes of 520 bp (base pair), 347 bp and 279 bp, respectively. the three uncharacterised anaplasma spp. amplified products were of different sizes (398 bp, 367 bp and 344 bp). the percentage of identity among the five t. ovis 18s rrna nucleotide sequences of this study (mf360021 – mf360025) ranged from 99.61% to 100.00%. these sequences shared 100% identity with previous sequences deposited in genbank from sudan (ay260171 and mg333457) and tanzania (ay260174). furthermore, the four a. ovis msp4 nucleotide sequences of this study (mf360026 – mf360029) shared 97.98% – 100.00% nucleotide identity and had 98% nucleotide identity with the other sequences from tunisia (ky659324, ky659320, km285220 and kc432643). meanwhile, the three e. ruminantium pcs20 sequences isolated in this study (mg544303 – mg544305) shared a nucleotide identity of 99.64% and had 100% sequence identity with other genbank sequences from ethiopia (gu797236), benin (kx356089), the ivory coast (kx356090) and cameroon (jq039914 and jq039939). as for uncharacterised anaplasma spp. 16s rrna, the three sequences from this study (mg637125–mg637127) shared 100% nucleotide identity and showed 100% nucleotide identity with sequences from japan (ay969011) and the united states (kj942183). phylogenetic analysis phylogenetic trees of t. ovis, a. ovis, uncharacterised anaplasma spp. and e. ruminantium were constructed based on 18s rrna, msp4, 16s rrna and pcs20 gene sequences, respectively, which have been generated in this study and those downloaded from genbank. the t. ovis sequences from this study clustered with the t. ovis sequences from the genbank, making a separate clade from other theileria species (figure 2). three a. ovis msp-4 sequences from this study were in the same clade, while one sequence formed a separate branch (figure 3). similarly, for e. ruminantium, all the three sequences from this study were located in the same clade (figure 4). meanwhile, the uncharacterised anaplasma spp. sequences of this study appeared in the same clade separated from other anaplasma sequences from the genbank (figure 5). figure 2: phylogenetic analysis of theileria ovis based on 18s rrna. the sequences generated in this study are shown in bold font. numbers at the nodes represent the percentage of occurrence of clades in 1000 bootstrap replication of the taxa. the 18s rrna gene sequence of babesia major (eu622907) was used as an outgroup. figure 3: phylogenetic analysis of anaplasma ovis based on aomsp4. the sequences determined in this study are shown in bold font. numbers at the nodes represent the percentage of occurrence of clades in 1000 bootstrap replication of the taxa. the msp4 gene sequence of anaplasma marginale (kx989519) was used as an outgroup. figure 4: phylogenetic analysis of ehrlichia ruminantium based on pcs20. the sequences in bold font are from this study. the numbers at the nodes represent the percentage of occurrence of clades in 1000 bootstrap replication of the taxa. the pcs20 gene sequence of ehrlichia chaffeensis (cp007477) was used as an outgroup. figure 5: phylogenetic analysis of uncharacterised anaplasma spp. based on 16s rrna. the sequences in bold font are from this study. the numbers at the nodes represent the percentage of occurrence of the clades in 1000 bootstrap replication of the taxa. the 16s rrna gene sequence of anaplasma ovis (kx579073) was used as an outgroup. discussion despite the wide distribution of ovine tbds in tropical and subtropical regions of the world (ros-garcía et al. 2013), and how important they are in livestock improvement (bilgic et al. 2017; jongejan & uilenberg 1994; jonsson et al. 2008), very little information is available regarding their presence and distribution (ros-garcía et al. 2013). in the present study, we performed molecular detection and analysis of tick-transmitted protozoan and rickettsial pathogens in blood samples of sheep collected from homa bay and machakos counties, kenya. the following pathogens were detected: t. ovis, a. ovis, e. ruminantium and uncharacterised anaplasma spp. theileriosis in sheep can be caused by several theileria spp. (berggoetz et al. 2014) – in this study, only t. ovis was detected (51.3%). the study revealed t. ovis as the most prevalent pathogen, which is in contrast to previous studies (mwamuye et al. 2017; omondi et al. 2017; wamuyu et al. 2015) that did not detect this pathogen in kenya. however, adjou moumouni et al. (2015) reported this pathogen in cattle reared in the town of ngong which is in kajiado, a county bordering machakos county in the south-west. the prevalence of t. ovis was compared to other studies from neighbouring countries. in ethiopia, gebrekidan et al. (2014) reported a prevalence of 92%, while el imam et al. (2016) reported a prevalence of 88.6% in the sudan. normally, t. ovis is known to cause a benign type of theileriosis and is less pathogenic to sheep (razmi et al. 2003; schnittger et al. 2000; uilenberg 1995). this protozoa is considered of low economic importance in sheep (mtshali et al. 2016). however, it cannot be neglected, as it can cause disease in sheep under stressful situations. the 18s rrna gene sequence of t. ovis from this study shared high identity (100%) with isolates from neighbouring countries including the sudan (ay260171 and mg333457) and tanzania (ay260174). this could be because of the movement of animals in huge numbers in this region by the pastoralists, which leads to an increased probability of animals from different areas crossing over in search of grazing pastures. this could lead to transmission of ticks from one herd to the other. moreover, the phylogenetic tree showed all 18s rrna sequences (figure 2) in this study in the same cluster with other t. ovis sequences extracted from the genbank, which suggest that similar genotypes are circulating in the field. anaplasmosis caused by a. ovis generally is considered to cause sub-clinical symptoms in sheep, although this disease has been reported to be prevalent in high severity in bighorn sheep (renneker et al. 2013; tibbitts et al. 1992). acute disease tends to be associated with stress factors such as co-infection, hot weather, deworming, vaccination, heavy tick burden, long distance transportation and animal movement (renneker et al. 2013). in this study, a prevalence of 34.2% was detected for this pathogen – this is higher than previously reported in kenya by omondi et al. (2017). however, this pathogen was not detected in the maasai mara and shimba hills national reserves (mwamuye et al. 2017; wamuyu et al. 2015) located in the south-west of kenya about 150 km from the study areas. data from other studies in africa showed a high prevalence of a. ovis in tunisia (belkahia et al. 2014), algeria (aouadi 2017) and south africa (ringo et al. 2018). these observations suggest that a. ovis has a wide distribution in different geographical areas of africa. however, in kenya and other parts of sub-saharan africa, large numbers of sheep are owned by the pastoralists, who travel long distances daily in sunny weather, searching for pastures and water (byaruhanga et al. 2016). this potentially leads to animal stress and eventually may result in severe disease. the low production of local breeds of sheep owned by pastoralists could be associated with this infection as they are easily exposed to stress and to pathogens that are endemic in sub-saharan africa (bilgic et al. 2017). phylogenetic analysis of a. ovis msp4 revealed that the three sequences isolated in this study appear in the same clade with other sequences extracted from the genbank (figure 3), while the remaining sequence from this study formed a separate branch (figure 3). this shows considerable genetic divergence of sequences isolated from this study, revealing that different genotypes of this parasite are circulating in the field. ehrlichia ruminantium, transmitted by ticks of the genus amblyomma, causes heartwater in domestic and wild ruminants in sub-saharan africa, madagascar and some caribbean islands (allsopp 2010). the disease is one of the major obstacles to improving livestock production in africa (cangi et al. 2017). in this study, e. ruminantium was detected in sheep from both machakos and homa bay counties, with an overall occurrence of 7.9% – this supports the previous studies in kenya (omondi et al. 2017) that reported a similar prevalence. the presence of several amblyomma tick species in kenya, including amblyomma variegatum, has been reported by omondi et al. (2017), which suggests that ovine ehrlichiosis is endemic in kenya. in the phylogenetic analysis, all the three sequences obtained in this study (figure 4) clustered together, which shows that the gene is highly conserved in the study areas. the uncharacterised anaplasma sp. was detected in this study. this study reports the pathogen for the first time in kenya. the overall prevalence is 31.6% from both sampled counties. phylogenetic analyses showed the two sequences of this bacterium clustered together (figure 5). the overall co-infection rate in this study was 61.8%, with t. ovis showing high association in co-infection. the co-infection (t. ovis + a. ovis) (27.3%) was the most common and this could be associated with a relatively higher infection rate of the two pathogens in this study. moreover, the two pathogens share the same vector, rhipicephalus evertsi evertsi, which has been reported in kenya by omondi et al. (2017). in the two counties, the co-infections were relatively similar, except for t. ovis + a. ovis co-infection which had a prevalence of 4% and 14% in homa bay and machakos, respectively. this could be attributed to the fact that the two pathogens had higher infection rates in both counties and machakos had a higher number of samples compared to homa bay. in this study, all pathogens were detected from apparently healthy animals, which suggest that sheep in kenya have established an enzootic stability status against these pathogens. it shows that animals were infected at the early stages of their lives and developed immunity against the detected pathogens. these animals carry the pathogens without displaying any clinical signs, but they continuously transmit the pathogens to ticks and eventually to new animals. babesia ovis and theileria lestoquardi were not detected in this study, which supports the previous study (omondi et al. 2017), although further studies with greater coverage may be needed to ascertain their absence. conclusion the results obtained in this study indicate the occurrence and diversity of t. ovis, a. ovis, e. ruminantium and uncharacterised anaplasma spp. in sheep from machakos and homa bay counties. mixed infections are common in the study area and, therefore, disease diagnosis can be complex. consequently, proper diagnostic tests are required for accurate diagnosis. further studies covering larger sample size and wider geographical coverage, including blood work, more epidemiological data and tick collection are required to estimate the risk factors associated with these diseases and their economic importance. acknowledgements this study was supported by a grant-in-aid for scientific research from the ministry of education, culture, sports, science and technology of japan and a grant from the japan society for the promotion of science core-to-core program japan (26304036 and 17f17105). the authors thank the state veterinarians and animal health technicians in kenya for collecting blood samples in the study areas. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions the collection of samples was supervised by g.o.a. the research was designed by a.e.r., p.f.a.m., x.x. and n.i. laboratory experiments were performed by a.e.r., s.h.l., m.l., y.g., h.g., a.e., e.m.g. and j.l. the manuscript was written by a.e.r., p.f.a.m., c.j., w.z., o.t., h.s. and x.x. references adjou moumouni, p.f., aboge, g.o., terkawi, m.a., masatani, t., cao, s., kamyingkird, k. et al., 2015, ‘molecular detection and characterization of babesia bovis, babesia bigemina, theileria species and anaplasma marginale isolated from cattle in kenya’, parasites and vectors 8(1), 496. https://doi.org/10.1186/s13017-015-1106-9 aktas, m., altay, k. & dumanli, n., 2007, ‘determination of prevalence and risk factors for infection 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https://doi.org/10.1007/s00436-007-0689-2 kubi_223-229.indd introduction the complex epidemiology of animal trypanosomosis is greatly affected by the number of infected tsetse transmitting the disease (lambrecht 1980). many ecological and physiological factors have been suggested to affect tsetse infection rates (lambrecht 1980). of particular epidemiological interest is the age prevalence of metacyclic trypanosomal infections in tsetse. this age-prevalence relationship has been described by various authors for different tsetse species (e.g. harley 1966). recently, a field study conducted in the zambezi valley (zimbabwe) described this relationship for glossina pallidipes using a mathematical model (woolhouse, hargrove & mcnamara 1993). at the same time, this model provided estimates of the developmental period of trypanosomes in tsetse and age-dependent susceptibility to infection. this paper describes a study conducted in the eastern province, zambia in which the age-prevalence relationship of trypanosomal infections in another tsetse species, glossina morsitans morsitans was investigated. whereas previous studies (woolhouse et al. 1993; woolhouse, bealby, mcnamara & silutongwe 1994) described this age-prevalence relationship for mature or metacyclic infections, this 223 onderstepoort journal of veterinary research, 74:223–229 (2007) age prevalence of trypanosomal infections in female glossina morsitans morsitans (diptera: glossinidae) on the plateau area of eastern zambia c. kubi1, m. billiouw2 and p. van den bossche1, 3* abstract kubi, c., van den bossche, p. & billiouw, m. 2007. age prevalence of trypanosomal infections in female glossina morsitans morsitans (diptera: glossinidae) on the plateau area of eastern zambia. onderstepoort journal of veter inary research, 74:223–229 trypanosomal infections in female glossina morsitans morsitans were investigated in an area in the eastern province of zambia between 1992 and 1994. a total of 4 416 flies were captured, aged using the ovarian ageing method and screened for trypanosomal infections in both the mouthparts, salivary glands and the midgut. congolense-type infections were identified in 4.8 % of the flies. vivax-type and immature infections were identified in 1.8 % and 6.8 % of the flies, respectively. the prevalence of con golense-type, vivax-type and immature infections increased with age. for vivax-type infections the age-prevalence relationship could be described by a model assuming a constant per capita rate of infection. for congolense-type and midgut infections, a polynomial term was added to the model significantly improving the fit. the per capita at which flies become infected was significantly higher for immature compared to mature infections. observations strongly suggest that tsetse acquire new midgut infections at any age and that maturation of these infections is not limited to those obtained during the first blood meal. keywords: age-prevalence, glossina morsitans morsitans, trypanosoma congolense, trypanosoma vivax, zambia * author to whom correspondence is to be directed: e-mail: pvdbossche@itg.be 1 department of animal health, institute of tropical medicine, nationalestraat 155, b-2000 antwerp, belgium 2 thorite road, 269 garneton, kitwe, zambia 3 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa accepted for publication 19 march 2007—editor 224 trypanosomal infections in female glossina morsitans morsitans (diptera: glossinidae) of zambia study gives an important additional dimension to the analysis by also investigating the age-prevalence relationship of immature or midgut infections. in an attempt to clarify the age-dependent maturation process of immature infections the fitted age-prevalence models of mature and immature infections are compared. material and methods study site the study was conducted between 1992 and 1994 in an area situated between 31°47’–31°55’ e and 13°55’–14°12’ s in katete district, eastern province, zambia. it is a highly cultivated area with a dense cattle population of about eight head of cattle per km2. bovine trypanosomosis, transmitted by g. morsitans morsitans, is one of the major constraints for agricultural development. tsetse sampling tsetse were captured using man-walked fly rounds (potts 1930) and f3-traps (flint 1985). the odour bait used was acetone at a release rate of 250 mg/h. tsetse were sampled in different vegetation types (brachystegia, riverine and combretum woodland) during rainy, cold-dry and hot-dry seasons. fly dissection flies were dissected within 4 h after collection. physio logical age determination of females was conducted as described by saunders (1960) and challier (1965). each fly was assigned to an ovarian age category (oc), 0–7, depending on its ovarian configuration. categories 0–1 correspond to ages 0–8 and 9–16 days, respectively and ocs 2–6 correspond to additional intervals of 9 days, from 17–25 to 53–61 days, respectively. depending on the content of the uterus, ocs 1–7 were subdivided into a (egg or first instar larva), b (second instar larva) or c (third instar larva). ovarian category 0 was subdivided into a (immature egg) and b (mature egg). the categories were transformed into days corresponding to the pivotal age of each category. mouthparts, salivary glands and midgut dissections were performed using the method described by lloyd & johnson (1924). infections in the tsetse were identified according to the site of trypanosomal infestation. infections in the proboscis alone were recorded as vivax-type, in the proboscis and the midgut as congolense-type and in the midgut alone as immature infections. the salivary glands were examined for mature brucei-type infections. throughout the analysis it was assumed that midgut or immature infections either mature into congolense-type infections or remain immature for the rest of the fly’s life. changes in the age-prevalence re lation ship of immature infections are thus due to either maturation of immature infections into congolensetype infections, acquisition of new midgut infections, or a combination of both. the age-prevalence relationship of the sum of immature and congolensetype infections, on the other hand, is only influenced by the development of new midgut infections. conse quently, the maturation of midgut infections or transmissibility was expressed as – congolensetype infec tions/immature infections + congolensetype infections (%) per oac. statistical data analysis the raw fly data consist of three dichotomous response variables indicating, for each fly, the presence or absence of congolense-type, vivax-type and immature infections and a set of three explanatory variable, i.e. the year and month of fly capture and the ovarian age of the fly. the data are expressed in the form of prevalence of infection. the first part of the analysis explored the effect of the three explanatory variables on the prevalence of the different infection types. it was assumed that the responses are from underlying binary distributions and consequently logistic regression was used to model the prevalences. hypothesis testing was done by means of χ2-tests. the significance level was set at 0.01. since there was no significant interaction between the effects of age and time (year, month) for any of the infection types, the analysis of the ageprevalence curves proceeded using the pooled data. the second part of the analysis estimated the per capita rate (λ) at which flies become infected and the developmental period (τ) of the trypanosomes in the tsetse fly. since logistic regression fits prevalences that are bounded between 0 and 1, it was not appropriate for this purpose. instead, we described the age-prevalence relationship for mature infections by the model used by woolhouse et al. (1993): y(a) = 1 – exp[–λ (a – τ)] or ln (1 – y(a)) = λτ – λa for a > τ; y(a) = 0 for a < τ the age-prevalence relationship for immature infections was described using the following model: 225 c. kubi, m. billiouw & p. van den bossche ln (1 – y(a) – z(a)) = λ ‘a where y(a) is the proportion of infected flies at age a (a is the pivotal age of each of the ocs) and z(a) is the proportion of tsetse with immature infections at age a. the log-linear model was fitted to the ageprevalence data using least squares, i.e. with the assumption of approximate normality of ln (1 – y) or ln (1 – y – z). polynomial terms were added when they significantly improved the model fit. the model’s assumptions are discussed by woolhouse (1989). the per capita rate at which tsetse become infected with immature infections (λ ‘) was compared with the per capita rate at which tsetse become infected with mature infections (λ) using a t-test. the glim statistical software package was used for all statistical analyses. results over the 2-year study period, 4 416 female g. morsitans morsitans were sampled, screened for the presence of trypanosomal infections and aged using the ovarian dissection method. the annual totals were 2 162 and 2 254 flies for 1992 and 1993, respectively. monthly sample sizes, pooled over both years, ranged from 175 in january to 599 in may. congolense-type infections, identified in 212 flies (4.8 %), were the dominant infection type. a total of 81 (1.8 %) flies had a vivax-type infection. the difference in prevalence is significant (χ2 = 61, p < 0.01). a total of 295 (6.8 %) flies had immature or midgut infections. one fly (0.02 %) had a brucei-type infection. brucei-type infections were not included in the analysis. temporal variation in 1992, the overall prevalence of trypanosomal infections was 277 flies out of a total of 2 162 dissected flies (12.8 %). in 1993, a total of 311 flies out of 2 254 dissected flies (13.8 %) were infected. the difference in prevalence between the 2 years was not significant (χ21 = 0.93, p < 0.01). average monthly prevalence of trypanosomal infections ranged from 8 % in june to 17.5 % in july. this variation was significant (χ211 = 30, p < 0.01). the shape of the monthly variation did not significantly differ between both years (month.year interaction: χ211 = 19, p < 0.01). the effects of year and month on the prevalences, allowing for the effect of the age structure of the flies, were explored for congolense-type, vivax-type and the sum of immature and congolense-type infections. results are presented in table 1. table 1 logistic regression analysis of deviance of terms affecting the prevalence of congolense-type, vivax-type and immature + congolense-type infections in female g. morsitans morsitans terms included terms added χ2-value d.f. p-value congolense-type infections – – – age age year month age year month 3.2 15.5 87.1 3.3 18.1 1 11 1 1 11 0.07 0.16 < 0.001a 0.07 0.07 vivax-type infections – – month – age age age + monthc year month month.yearb age year month age.monthb 3.5 25.7 13.9 14.2 3.5 25.0 11.9 1 11 12 1 1 11 11 0.06 0.007a 0.30 < 0.001a 0.06 0.009a 0.37 immature + congolense-type infections – – month – age age age + monthc year month month.yearb age year month age.monthb 1.6 39.9 18.3 19.5 31.7 36.4 6.9 1 11 12 1 1 11 11 0.20 < 0.001a 0.10 < 0.001a 0.19 < 0.001a 0.8 a significant at p = 0.01 b interaction term only c main effect + interaction term 226 trypanosomal infections in female glossina morsitans morsitans (diptera: glossinidae) of zambia there was no significant yearly variation in the prevalences of any of the infection types and no effect of year on the shape of the monthly variation of any of the infection types. for congolense-type infections, only age had a significant effect on prevalence. monthly variation in prevalences were not significant. for vivax-type infections both age and month, but not their interaction, were significant factors. the monthly prevalence of vivax-type infections varied from 0.7 % in october to 3.6 % in december. for the sum of immature and congolense-type infections both age and month, but not their interaction, were significant factors explaining the variation in the prevalence. average estimates of infection prevalences with age, for the whole of the study period, were obtained, ignoring the effects of month and year. age-prevalence relationship the observed age-prevalence relationship for congolense-type and vivax-type infections are shown in fig. 1a and b. the prevalence of congolense-type infections ranged from 0 % (ocs 0a and 0b) to 9.5 % (oc 6b). for vivax-type infections the prevalence varied between 0 % (oc 0a) to 6.1 % (oc 6b). the model ln (1 – y) = λτ – λa was fitted to the age prevalence data of the congolense-type and vivax-type infections to obtain estimates of the per capita infection rate (λ) and developmental period (τ). the fitted curves are listed in table 2 and plotted, together with the observed prevalences, in fig. 1a and b. for the congolense-type infections, the model included a cubic term of age which significantly improved the fit (f1,16 = 5.95, p < 0.01). the model table 2 parameter estimates of age-prevalence models for three trypanosomal infection-types in female g. morsitans morsitans estimate ± s.e. parameter congolense-type –0.02107 –0.002672 2.292 e-07 0.009337 0.0004328 9.391 e-08 1 age age3 vivax-type 0.007950 –0.0008496 0.005302 0.0001323 1 age immature + congolense-type –0.00448 0.00000032 0.0003929 0.00000012 age age3 �������� � �� �� � �� �� �� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � � �� � � � �� �� �� � � �� � � � � � � � � � �� �� � � � fig. 1 variations in prevalence of trypanosomal infections in female glossina morsitans morsitans with fly age (by ovarian category) a congolense-type infection. the best fit model (line) is ln (1 – y) = 0.02107 – 0.002672 age + 2.297 e-07 age3 b vivax-type infection. the best fit model (line) is ln (1 – y) = 0.007950 – 0.0008496 age c congolense-type infection ( ) + immature. the best fit model (line) is ln (1 – y) = –0.004448 age + 0.00000032 age3 and immature ( ) only 227 c. kubi, m. billiouw & p. van den bossche explained 87 % and 71 % of the variation in ln (1 – y) for congolense-type and vivax-type infections, respectively. the parameter estimates (+ standard e rror) for congolense-type infections were τ = 7.9 + 3.7 days and λ = 0.00261 + 0.00043 per fly per day at age = τ. for vivax-type infections, the parameter estimates (+ standard error) were τ = 9.4 + 6.5 days and λ = 0.00085 + 0.00013 per fly per day at age = τ. the prevalence of immature infections varied between 0 % (oc 0a) to a maximum of 12.2 % (oc 5c). the observed prevalences for immature only and the sum of immature and congolense-type infections are presented in fig. 1c. the model for immature + congolense-type infections was fitted without intercept (f1,16 = 0.01, p < 0.05). a cubic term significantly improved the fit (f1.17 = 6.1, p < 0.05). this model explains 91.5 % of the variation of the prevalence of immature + congolense-type infections. the fitted curve is plotted together with the observed prevalences in fig. 1c. the parameter estimates of the fitted model are given in table 2. the per capita rate (λ) at which flies become infected was estimated at 0.00447 + 0.00039 per fly per day at day 0. the estimate of λ‘ at age τ for mature infections is significantly lower (t33 = 2.958; p = 0.0028) than the estimate of λ for immature infections at age 0. the observed and fitted ratio of congolense-type infections/immature infections + congolense-type infections, for each of the age groups, is plotted in fig. 2. the ratio increases rapidly to a value of about 0.5 at the age of about 30 days after which it remains almost constant. discussion except for studies conducted by clarke (1969) and woolhouse et al. (1994) little information is available on the prevalence of trypanosomal infections in zambian tsetse species. contrary to clarke’s observations, congolense-type infections are the most prevalent in our study area. this is in accordance with the high bovine trypanosomosis prevalence, caused by t. congolense, in the plateau area of the eastern province. the per capita rate at which female g. morsitans morsitans acquire congolense-type infections, suggests that 0.26 % of the flies become infected per day. at a feeding interval of 4 days (rogers 1988) and a 75 % feeding preference for cattle (as is the case in the study area), this corresponds to a successful infection every 72 blood meals on cattle. the prevalence of t. congolense infections in cattle is high in the study area. at a prevalence of 40 %, 3.5 % of the feeds on infected cattle develop and mature in female g. morsitans morsitans. this figure is slightly higher than the 2.5 % obtained by rogers & boreham (1973) from an analysis of data on g. swynnertoni in tanzania. the estimated developmental period of congolensetype infections in g. morsitans morsitans corresponds well with the average 8–9 days observed by elce (1971). nantulya, doyle & jenni (1978), however, observed development periods up to 40 days. the estimated developmental period for vivax-type infections agrees with the 5–13 days reported by davies (1977). results from this study show that the prevalence of vivax-type infections rises approximately linearly with fly age. this is what could be expected and reinforces the observations that tsetse flies can readily infect themselves with t. vivax throughout their life. this is most likely related to the relatively simple developmental cycle of t. vivax in the tsetse fly. the age-prevalence curve of immature + congolense-type infections strongly suggests that, in accordance with laboratory observations (welburn & maudlin 1992), wild tsetse also acquire new trypanosomal midgut infections at any age. consequently, infected blood meals can give rise to immature infections even when these blood meals are taken by non-teneral flies. there is some conflicting evidence concerning the ease with which non-teneral tsetse flies can infect themselves with t. congolense. many authors, using both laboratory-reared tsetse and tsetse emerging � � � � �� � � � � � �� � � � � � � � � �� � � � �� �� �� � � � � �� � �� � �� �� �� �� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �������� � �� �� �� ��� ��� ��� �� �� ��� ��� ��� fig. 2 variations in the ratio of congolense-type infections/ congolense-type + immature infections ( ) of female glossina morsitans morsitans with fly age (by ovarian category) 228 trypanosomal infections in female glossina morsitans morsitans (diptera: glossinidae) of zambia from wild pupae, have described the significantly higher susceptibility of teneral compared to non-teneral flies to t. congolense infections (owaga 1981; distelmans, d’haeseleer, kaufman & rousseeuw 1982; mwangelwa, otieno & reid 1987; welburn & maudlin 1992; dale, welburn, maudlin & milligan 1995). on the other hand, harley (1967) found an increasing t. congolense-infection prevalence with increasing age in females of several tsetse species. it is nevertheless generally accepted that tsetse, once fed, are relatively but not completely refractory to trypanosomal infections. consequently, infections acquired by non-teneral wild flies are expected to play a minor role in the epidemiology of tsetsetransmitted trypanosomosis. according to our observations, the prevalence of congolense-type infections increases substantially, though not linearly, with increasing age. the fit of the model to the age-prevalence data was improved by an additional cubic term. this age-prevalence model can be explained by variations in incubation time of infections obtained in young flies, age-dependent decrease in susceptibility to infection or increased mortality of tsetse infected with a metacyclic t. congolense infection (dale et al. 1995; wool house et al. 1993). the developmental or incubation period of congolense-type infections can vary (dale et al. 1995). midgut or immature infections, however, must develop immediately after the ingestion of the infected blood meal. assuming that maturation is restricted to infections obtained during the first blood meal, the prevalence of midgut infections in the youngest age categories cannot be lower than the maximum prevalence of mature, congolense-type infections in the subsequent age categories. this could not be confirmed by our observations. the maximum congolense-type infection prevalence (8.6 % in oc 6) is higher than the maximum midgut-infection prevalence in the first three age categories (6 %). furthermore, our results show that between ocs 2 and 6 the congolense-type infection prevalence almost doubles from 4.5 % to 8.6 %. this increase in congolense-type infections cannot be due to retarded maturation of trypanosomal infections obtained during the first blood meal, since the prevalence of immature infections in the first age categories only varies between 0 % and 4 %. consequently, the increase in the congolense-type infection prevalence with increasing age is a result of maturation of newly acquired midgut infections. moreover, if few of the newly acquired midgut infections in non-teneral tsetse would mature, one would expect a decreasing ratio of congolense-type infections/immature + congolense-type infections once the developmental period of the infections acquired during the first blood meal is over. this is not the case. although our results indicate that maturation of trypanosomal congolense-type infections is not restricted to those obtained during the first blood meal, the shape of the ratio of congolense-type infections/ immature infections + congolense-type infections curve suggests that trypanosomal infections obtained early in life contribute more to the congolensetype prevalence than those acquired at a later age. moreover, there is a significant difference between the force-of-infection for immature infections (λ‘) and the force-of-infection for mature congolense-type infections (λ). this implies that immature infections do not all mature to become mature infections some weeks later. there are two obvious explanations: either there is an increased mortality of immature infected flies, or there is some recovery from infections. welburn, maudlin & ellis (1989) showed that, under laboratory conditions, it can take up to 7 days to remove an incoming infection from the midgut of a tsetse fly. consequently, some of these “non-permanent” midgut infections will be detected and classified as midgut infection for a particular age group but will be removed from the midgut in a higher age group. observations described in this paper confirm the age-prevalence relationship of trypanosomal infections in wild tsetse reported in other recent studies and another tsetse species (woolhouse et al. 1993; woolhouse et al. 1994). moreover, our results clearly indicate that the increase in the prevalence of congolense-type infections with increasing age is due to the maturation of midgut infection acquired by non-teneral tsetse flies. contrary to laboratory observations (welburn & maudlin 1992), these newly acquired infections substantially contribute to the overall congolense-type infection rate of the tsetse population. acknowledgements the authors are grateful to the staff of the chipopela research station where the work was conducted. drs m.e.j. woolhouse and r. de deken are kindly acknowledged for their comments on the manuscript. the work was funded by the belgian development co-operation through the assistance to the veterinary services of zambia project. 229 c. kubi, m. billiouw & p. van den bossche references challier, a. 1965. amélioration de la méthode de détermination de l’âge physiologique des glossines. etudes faites sur glossina palpalis gambiensis vanderplank, 1949. bulletin de la société pathology exotique, 58:250–259. clarke, j.e. 1969. trypanosome infection rates in the mouthparts of zambian tsetse flies. annals of tropical medicine and parasitology, 63:15–34. dale, c., welburn, s.c., maudlin, i. & milligan, p.j.m. 1995. the kinetics of maturation of trypanosome infections in tsetse. parasitology, 111:187–191. davies, h. 1977. tsetse flies in nigeria, 3rd ed. ibadan: oxford university press. distelmans, w., d’haeseleer, f., kaufman, l. & rousseeuw, p. 1982. the susceptibility of glossina palpalis palpalis at different ages to infection with trypanosoma congolense. annales de la société belge médecine tropicale, 62:41–47. elce, b.j. 1971. the transmission of trypanosoma congolense through glossina morsitans and the white mouse. transactions of the royal society of tropical medicine and hygiene, 65:239. flint, s. 1985. a comparison of various traps for glossina spp. (glossinidae) and other diptera. bulletin of entomologic research, 75:529–534. harley, j.m.b. 1966. studies on age and infection rate of glossina pallidipes austen, g. palpalis fuscipes and g. brevipalpis newst. in uganda. bulletin of entomologic research, 57:23–37. harley, j.m.b. 1967. further studies on age and trypanosome infection rate in glossina pallidipes aust., g. palpalis fuscipes and g. brevipalpis newst. in uganda. bulletin of ento mologic research, 5:459–477. lambrecht, f.l. 1980. ecological and physiological factors in the cyclic transmission of african trypanosomiasis. insect science and its application, 1:47–54. lloyd, l.l. & johnson, w.b. 1924. the trypanosome infections of tsetse flies in northern nigeria and a method of estimation. bulletin of entomologic research, 14:25–257. mwangelwa, m.i., otieno, l.h. & reid, g.d.f. 1987. some barriers to trypanosoma congolense development in glossina morsitans morsitans. insect science and its applica tion, 1:33–37. nantulya, v.m., doyle, j.j. & jenni, l. 1978. studies on trypansoma (nannomonas) congolense 2. observations on the cyclical transmission of three field isolates by glossina morsitans morsitans. acta tropica, 35:339–344. owaga, m.l.a. 1981. trypanosome infection rate in the tsetse species; glossina pallidipes austen, in a rural situation in kenya. insect science and its application, 1:411–416. potts, w.h. 1930. a contribution to the study of numbers of tsetse-fly (glossina morsitans westw.) by quantitative methods. south african journal of science, 27:491–497. rogers, d.j. 1988. a general model for the african trypanosomiasis. parasitology, 97:193–212. rogers, d.j. & boreham, p.f.l. 1973. sleeping sickness survey in the serengeti area (tanzania) 1971 ii. the vector role of glossina swynnertoni austen. acta tropica. 30:24– 35. saunders, d.s. 1960. the ovulation cycle in glossina morsitans westwood (diptera: muscidae) and a possible method of age determination for female tsetse flies by examination of their ovaries. transactions of the royal society of tropical medicine and hygiene, 112:221–238. welburn, s.c., maudlin, i. & ellis, d.s. 1989. rate of trypanosome killing by lectins in midguts of different species and strains of glossina. medical and veterinary entomology, 3:77–82. welburn, s.c. & maudlin, i. 1992. the nature of the teneral state in glossina and its role in the acquisition of trypanosome infection in tsetse. annals of tropical medicine and parasitology, 86:529–536. woolhouse, m.e.j. 1989. on the interpretation of age-prevalence curves for schistosome infections of host snails. parasitology, 99:47–56. woolhouse, m.e.j., hargrove, j.w. & mcnamara, j.j. 1993. epidemiology of trypanosome infections of the tsetse fly glossina pallidipes in the zambezi valley. parasitology, 106:479–485. woolhouse, m.e.j., bealby, k., mcnamara, j.j. & silutongwe, j. 1994. trypanosome infections of the tsetse fly glossina pallidipes in the luangwa valley, zambia. international journal of parasitology, 24:987–993. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) christo j. botha department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa louis g.j. ackerman † department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa mxolisi g. masango analytics and institutional research unit, university of the witwatersrand, johannesburg, south africa luke f. arnot ‡ department of production animal studies, faculty of veterinary science, university of pretoria, onderstepoort, south africa citation botha, c.j., ackerman, l.g.l., masango, m.g. & arnot, l.f., 2020, ‘failure of diplodiatoxin to induce diplodiosis in juvenile goats’, onderstepoort journal of veterinary research 87(1), a1712. https://doi.org/10.4102/ojvr.v87i1.1712 note: †, 1940–2017; ‡, 1970–2018. original research failure of diplodiatoxin to induce diplodiosis in juvenile goats christo j. botha, louis g.j. ackerman, mxolisi g. masango, luke f. arnot received: 09 nov. 2018; accepted: 08 july 2019; published: 05 mar. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract diplodiosis is an important neuromycotoxicosis of ruminants in south africa when grazing on harvested maize fields in winter. it is believed to be caused by mycotoxin(s) synthesised by stenocarpella (diplodia) maydis. although several metabolites have been isolated from s. maydis culture material, none of these have been administered to ruminants to reproduce the disease. the objectives of this study were to isolate diplodiatoxin and to administer it to juvenile goats. diplodiatoxin, considered as a major metabolite, was purified from s. maydis-infected maize cultures (coligny 2007 isolate). following intravenous administration of 2 mg and 4 mg diplodiatoxin/kg body weight for five consecutive days to two juvenile goats, no clinical signs reminiscent of diplodiosis were observed. based on previous experimental results and if diplodiatoxin was the causative compound, the dosage regimen employed was seemingly appropriate to induce diplodiosis. in addition, intraruminal administration of 2 mg/kg diplodiatoxin to one goat for three consecutive days also did not induce clinical signs. it appears as if diplodiatoxin alone is not the causative compound. other metabolites and/or mixtures of diplodiatoxin and other mycotoxins, when available in sufficient quantities, should also be evaluated. keywords: diplodiosis; diplodiatoxin; stenocarpella maydis; mycotoxin; neuromycotoxicosis. introduction stenocarpella (diplodia) maydis (berk.) sutton is one of the most prevalent cob and stalk rot pathogens of maize responsible for a decline in grain quality and yield (flett & mclaren 1994). stenocarpella maydis-infected maize is also associated with intoxication in ruminants, resulting in the neuromycotoxicosis known as diplodiosis (kellerman et al. 2005). diplodiosis is an important toxicosis in south africa because of the fact that many farmers utilise harvested maize fields, locally called stover, as an important winter food source (kellerman et al. 2005). the first record of diplodiosis in south africa is reported by van der bijl (1914) who cites government veterinary surgeon webb who submitted maize cobs in august 1912 from mooi river, kwazulu-natal province. mr webb wrote (van der bijl 1914): i am also sending you some specimens of mealies taken from fields in which cattle have become sick, showing symptoms of intoxication and paralysis due, i believe, to poisoning to fungi on the mealies. (p. 231) later, mitchell (1919) reproduced diplodiosis in cattle by feeding naturally infected maize cobs as well as a s. maydis culture grown on sterile maize kernels. furthermore, diplodiosis has also been recorded in argentina (odriozola et al. 2005), australia (darvall 1964) and brazil (riet-correa et al. 2013). in south africa, diplodiosis is prevalent in late winter where cattle and sheep graze on harvested s. maydis-infected maize fields (kellerman et al. 2005). under field conditions, diplodiosis becomes apparent from 6 days to 2 weeks after the animals were exposed to s. maydis-infected maize cobs and stalks (kellerman et al. 2005). during experimental reproduction of the disease, where ruminants were fed s. maydis-cultured maize kernels, clinical signs were noticeable after a short latent period of 2–8 days (kellerman et al. 1985). the clinical signs continued for 1–4 days. initially the animal’s back is slightly arched, there are mild tremors, particularly noticeable over the flank and shoulders, as well as lacrimation and salivation. the animal assumes a wide-based stance and is reluctant to move. locomotor abnormalities, such as a high-stepping gait, hypermetria and falling, are noticed. these signs are progressive and eventually the animal is unable to rise and becomes paralysed. however, livestock can recover completely if they receive symptomatic and supportive treatment and good nursing care (kellerman et al. 2005). since the first report of diplodiosis by van der bijl in 1914, several efforts have been made to isolate the principal toxins responsible for this mycotoxicosis. different metabolites, namely, diplodiatoxin (steyn et al. 1972), stenocarpin (marais 1990), carpellin (marais 1990), dipmatol (ackerman et al. 1995), diplonine (snyman et al. 2011) and chaetoglobosins k, l, m and o (rogers et al. 2014; wicklow et al. 2011), have been isolated from s. maydis-contaminated maize. a major shortcoming is that none of these s. maydis metabolites isolated thus far has been administered to ruminants to reproduce diplodiosis (masango et al. 2015b). therefore, the specific role, if any, that the different metabolites play in diplodiosis has not yet been established. diplodiatoxin (c18h28o4) (figure 1) has initially been isolated and characterised from s. maydis-infected maize cultures using bioassay-guided isolation from chloroform–methanol extracts (steyn et al. 1972). figure 1: the chemical structure of diplodiatoxin purified from stenocarpella maydis-infected maize culture material. furthermore, diplodiatoxin has also been detected in s. maydis-infected maize kernels and diseased stalk material as well as from ears collected during a natural field outbreak using nuclear magnetic resonance (nmr) and mass spectrometric data (rogers et al. 2014; wicklow et al. 2011). however, diplodiatoxin was reported to account for only 10% of the total toxicity of the maize culture material based on toxicity studies in chickens (steyn et al. 1972). diplodiatoxin was reported to induce liver degeneration in chickens (louw 1969). in acute and subacute toxicity studies in rats, a decrease in body weight and feed intake, dullness, irritability, tremors and convulsions as well as liver damage and inhibition of the brain acetylcholinesterase activity were reported (rahman et al. 2002; rao et al. 2003). in in vitro toxicity studies, diplodiatoxin induced a concentration-dependent cytotoxicity (masango et al. 2014) and the accompanying cell death was because of necrosis and caspase-dependent apoptosis (masango, ellis & botha 2015a). the cell death was characterised by mitochondrial damage, cytoplasmic vacuolation and nuclear fragmentation (masango et al. 2015a). as diplodiatoxin is considered a major metabolite synthesised by s. maydis (rogers et al. 2014; wicklow et al. 2011), the aim of this study was to isolate and purify diplodiatoxin from s. maydis-infected maize cultures and to administer it to juvenile goats in an attempt to reproduce diplodiosis in a target animal. materials and methods isolation and purification of diplodiatoxin stenocarpella maydis-infected maize cultures (coligny 2007 isolate) were prepared by the grain crops institute (arc-gci, potchefstroom, south africa). milled culture material (2.3 kg) was soaked in hexane (1.8 l) overnight. the solvent was separated from the culture material using a buchner funnel and the material was washed with portions of hexane (100 ml/filtration; 1 l in total). the defatted meal was placed in open trays in a fume hood to dry overnight. the dried, defatted maize culture was placed in a plastic bucket with a lid. an ammonia solution was prepared (250 ml of 32% ammonia in 5 l of water) and added to the maize and mixed well to form a slurry. the mixture was left at room temperature for over a weekend. methanol (2.5 l) was added to the hydrolysate slurry. the mixture was stirred well and left for 1 h with occasional stirring. the liquid was removed by pressing through a mutton cloth (4.2 l obtained). the solution was acidified to ph 2 with concentrated hydrochloric acid (hcl) (200 ml), extracted with chloroform and evaporated to dryness. the crude extract was purified using flash chromatography. a column was prepared using silica gel (80 ml, merck silica gel 60, 0.040 mm – 0.063 mm) packed in a glass column (30 mm × 150 mm). the crude extract was dissolved in chloroform (chcl3) and placed on the column and eluted with hexane:ethyl acetate:acetic acid (200:100:12.5). different fractions were collected and combined according to thin layer chromatography (tlc) analysis. the column was finally eluted with acetone (150 ml). the fraction containing the diplodiatoxin (tlc, rf 0.42; hexane:acetone:acetic acid [1:4:6 drops]) was re-chromatographed using hexane:acetone:acetic acid (400:100:12.5) to give diplodiatoxin. the compound was recrystallised using ethyl acetate to produce pure diplodiatoxin (pure on tlc). the structure was confirmed with deuterium (1h) and carbon-13 nuclear magnetic resonance (13c nmr) and compared with literature values (ichihara et al. 1986; steyn et al. 1972). animal trial three juvenile, weaned saanen-cross goats weighing between 8 kg and 11 kg were purchased. on arrival the goats were examined to ensure they were clinically healthy. the goats were housed in individual concrete pens at the onderstepoort veterinary animal research unit and the study was completed there. the goats had free access to water and were fed a standard ration. during a 3-week adaptation period the goats were dewormed (ivermectin; ivomec injection, merial, halfway house, gauteng, south africa), vaccinated against enterotoxaemia (enterotoxaemia alum precipitated vaccine, onderstepoort biological products, onderstepoort, gauteng, south africa) and received an anticoccidial agent (toltrazuril; baycox 5%, bayer animal health, isando, gauteng, south africa). clinical examinations were performed weekly. blood samples (5 ml serum and 5 ml collecting tube containing ethylenediaminetetraacetic acid (edta) as anticoagulant) were collected before and periodically during the trial from the vena jugularis. these were submitted to determine serum enzyme activities (aspartate transaminase [ast] and gamma-glutamyl transferase [ggt]), other chemical pathology parameters (total serum proteins, albumin, urea and creatinine) and complete blood counts. a single goat was dosed at a time and based on the results the subsequent dose level was decided upon before the next animal was used. the dose was estimated based on trials conducted at the onderstepoort veterinary institute (t.s. kellerman 1984, [arc-ovi] personal observation). diplodiatoxin was dissolved in dimethylsulphoxide (dmso) and administered intravenously (two goats) and intraruminally (one goat). the goats were observed for a minimum of 3 weeks after receiving the last dose. ethical considerations ethical clearance was provided by the animal ethics committee, university of pretoria (project number v123-16). results the chemical extraction and isolation procedure yielded 370.9 mg of pure diplodiatoxin (figure 1). following intravenous and oral administration of diplodiatoxin to juvenile goats, no clinical signs reminiscent of diplodiosis were observed. the clinical pathology parameters determined fluctuated within normal reference ranges. the dosing regimen and results are presented in table 1. table 1: toxicity of diplodiatoxin for juvenile saanen-cross goats. discussion the dosage regimen was estimated based on a dosing experiment conducted to determine the toxicity of local s. maydis cultures for boer goats (t.s. kellerman 1984, personal observation). in kellerman’s study, three goats each received 5 g of s. maydis-infected maize culture material/kg for three consecutive days. all three goats exhibited locomotor abnormalities 3–4 days after dosing commenced. in the current study, the yield of pure diplodiatoxin obtained from 2.3 kg culture material was 371 mg, equating to 0.161 mg diplodiatoxin/g. thus, 5 g of culture material contained approximately 0.81 mg of diplodiatoxin. the doses administered intravenously to two animals during the current study were 2.5 to 5 times higher and were administered for a longer period. thus, based on the previous experimental results, the dosage regimen employed in the current study was seemingly appropriate to induce clinical signs. although individual variation in susceptibility or breed differences might play a role, it seems unlikely that diplodiatoxin is the causative compound. snyman et al. (2011) isolated a neurotoxin, diplonine, which induced clinical signs in guinea pigs that were considered as similar to diplodiosis in livestock. however, excessively high oral doses were required to induce neurological signs. doses equivalent to 200 g culture material/kg and higher were administered. these doses are unphysiological and livestock will not be able to ingest these massive amounts of maize cobs and stover. in addition, masango et al. (2014) compared the in vitro cytotoxicity of diplonine, diplodiatoxin and dipmatol on neuro-2a, chinese hamster ovary (cho-k1) and madin-darby bovine kidney epithelial (mdbk) cell lines and concluded that diplodiatoxin was the most cytotoxic and diplonine was not cytotoxic. in the current study, diplodiatoxin was also administered intraruminally via a stomach tube, as it is possible that diplodiatoxin could be bioactivated by rumen microbes to a more toxic metabolite (kellerman et al. 2005). although diplodiatoxin was only dosed to one animal at 2 mg/kg for three consecutive days, no clinical signs were noticed. it is also conceivable that one or more of the other metabolites synthesised by s. maydis may induce clinical signs or a combination of mycotoxins might have a synergistic or additive effect. wicklow et al. (2011) proposed that a mixture of the different chaetoglobosins might be the causative agent. their supposition is based on disruption of actin polymerisation which may interfere with the myelination process. in experimental studies of s. maydis-induced perinatal mortality, spongiform degeneration of myelin is a common feature and it was concluded that the unknown toxin acts mainly on myelin (kellerman et al. 1991; prozesky et al. 1994). the hypothesis is also supported by evidence of demyelination of nerves when vervet monkeys (cercopithecus aethiops) were fed s. maydis-infected maize culture as part of their daily diet (fincham et al. 1991). conclusion it appears as if diplodiatoxin alone is not the causative compound. other metabolites and/or mixtures of diplodiatoxin with other mycotoxins, when available in sufficient quantities, should also be evaluated. acknowledgements this work was supported by the maize trust of south africa (project no. mtm 15-10). the authors would like to thank prof. bradley flett, grain crops institute (arc-gci), for the culture material, dr gerda fouché for summarising the isolation and purification of diplodiatoxin as performed by the late dr louis g.j. ackerman, and antonette van wyk for assistance during the animal trials. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.j.b. was the principal investigator. l.f.a. conducted the clinical trial. l.g.j.a. performed the extraction, isolation and purification of diplodiatoxin. m.g.m. assisted with planning of the study and made conceptual contributions. funding information this work was supported by the maize trust of south africa (project no. mtm 15-10). data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references ackerman, l.g.j., combrink, s., horak, r.m., kuhn, m., learmonth, r.l., lübben, a. et al., 1995, ‘a novel hydroxylated fatty acid isolated from cultures of diplodia maydis on maize’, in book of abstract – poster b1, 5th frank warren national organic chemistry conference, 4–7 april 1995, aventura aldam resort, ventersburg, free state, south africa. darvall, p.m., 1964, ‘mouldy corn cobs, a danger to cows’, queensland agricultural journal 90, 692–693. fincham, j.e., hewlett, r., de graaf, a.s., taljaard, j.j.f., steytler, j.g., rabie, c.j. et al., 1991, ‘mycotoxic peripheral myelinopathy, myopathy and hepatitis caused by diplodia maydis on vervet monkeys’, journal of medical primatology 20, 240–250. flett, b.c. & mclaren, n.w., 1994, ‘optimum disease potential for evaluating resistance to stenocarpella maydis ear rot in corn hybrids’, plant disease 78, 587–589. https://doi.org/10.1094/pd-78-0587 ichihara, a., kawagishi, h, tokugawa, n. & sakamura, s., 1986, ‘stereoselective total synthesis and stereochemistry of diplodiatoxin, a mycotoxin from diplodia maydis’, tetrahedron 27(12), 1347–1350. https://doi.org/10.1016/s0040-4039(00)84255-0 kellerman, t.s., coetzer, j.a.w., naudé, t.w. & botha, c.j., 2005, plant poisoning and mycotoxicoses of livestock in southern africa, 2nd edn., oxford university press, cape town. kellerman, t.s., prozesky, l., schultz, a.r., rabie, c.j., van ark, h., maartens, b.p. et al., 1991, ‘perinatal mortality in lambs of ewes exposed to cultures of diplodia maydis (stenocarpella maydis) during gestation’, onderstepoort journal of veterinary research 58, 297–308. kellerman, t.s., rabie, c.j., van der westhuizen, g.c.a., kriek, n.p.j. & prozesky, l., 1985, ‘induction of diplodiosis, a neuromycotoxicoses, in domestic ruminants with cultures of indigenous and exotic isolates of diplodia maydis’, onderstepoort journal of veterinary research 52, 35–42. louw, w.k.a., 1969, ‘toxic metabolites of diplodia maydis (berk.) sacc.’, m.sc. 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accepted: 26 mar. 2019; published: 26 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract trypanosoma evansi is enzootic in camels in egypt, and water buffaloes act as a reservoir for camel infection. molecular techniques have contributed towards understanding the epidemiology of t. evansi. trypanosoma evansi was detected in acute and chronic stages of the disease in male and female mice by polymerase chain reaction (pcr) using two primers. two experiments were conducted. in experiment i, two groups consisting of 26 female and 26 male mice received 104 trypanosome by i/p inoculation for each mouse. in experiment ii, 42 female and 42 male mice were inoculated i/p with 102 trypanosome/mouse. in addition, five mice were kept as uninfected control for each group. mice were monitored daily for parasitaemia level during the pre-patent period using the micro-haematocrit centrifugation technique (mhct) and conventional pcr. the primer pairs, (trypanosoma brucei) tbr1/2 and terotat1.2 (t. evansi rode trypanozoon antigen type [rotat] 1.2), detected the infection after 24 hours earlier than mhct in both experiments. the course of infection that was detected by mhct revealed three waves of parasitaemia in female mice and two waves in male mice in the chronic stage of infection. in addition, pcr was able to detect t. evansi in different organs in the chronic stage (i.e. disappearance of parasite from blood). application of the two primer sets on blood samples from camels showed that all samples were positive by tbr1/2 primers and only 32 of 44 were positive by terotat1.2 primers. acutely and chronically trypanosoma-infected mice were detected by pcr in blood and organs. tbr1/2 primers were more sensitive than terotat1.2 primers in detecting trypanosoma-infected mice, and more reliable in detecting field-infected camels and excluding carrier animals. keywords: trypanosoma evansi; acute; chronic; mice; pcr; polymerase chain reaction; organs. introduction trypanosoma evansi belongs to the genus trypanosoma and subgenus trypanozoon, and is the first pathogenic trypanosome to be discovered globally. trypanosoma evansi was hypothesised to originate from trypanosoma brucei (tbr), by lacking genes necessary for mitochondrial development with loss of ability to undergo growth and differentiation in the insect vector and adaptation to a non-cyclic mode of transmission for the parasite (carnes et al. 2015; luckins 1988). trypanosoma evansi has been able to spread rapidly by non-specific mechanical vectors, such as tabanids and stomoxes (herrera et al. 2004; otto et al. 2010). trypanosoma evansi is enzootic in camels in egypt, and water buffaloes act as a reservoir for camel infection (elhaig, youssef & el-gayar 2013; hilali et al. 2004). surra is an insect-borne parasitic disease caused by t. evansi with wide distribution globally. trypanosoma evansi infects a wide diversity of mammalian hosts, including animals and humans. however, camels, horses and dogs remain the most critical hosts for this parasite, and bovine hosts are very efficient reservoirs (desquesnes et al. 2013; fernandez et al. 2009; rjeibi et al. 2015). the syndromes associated with t. evansi infection are severe and fatal, especially in the late stage of the disease. the disease varies from chronic to lethal acute accompanied with progressive weakness, emaciation, depletion, recurrent fever, enlarged lymph nodes and death (omer, mousa & al-wabel 2007; saleh, bassam & sanousi 2009). the underestimation of the medical and economic impacts of t. evansi has contributed to its ability to spread silently via healthy carriers. although t. evansi is inapparent in most instances, the parasite affects livestock productivity causing mortality, reduced animal production and reproduction performance, low carcass quality, and decreased animal strength (desquesnes et al. 2013; reid 2002). diagnosis of t. evansi infection relies on the detection of the parasite in the blood or tissue fluids of infected animals. parasitological techniques cannot always detect ongoing infections as the level of parasitaemia is often low and fluctuating, particularly during the chronic stage of the disease, which exhibits very low parasitaemia (nantulya 1990). consequently, the sensitivity and specificity of parasitological diagnostic tests are unacceptable in situations where confirmation of presence or absence of t. evansi in livestock is necessary prior to the introduction of new animals or after implementation of control and eradication programmes (viljoen & luckins 2012). the nucleic acid-based assays are considered to be the most powerful tools for the detection of t. evansi in several animals and vectors (sukhumsirichart et al. 2000). molecular techniques enable researchers to identify and characterise the newly introduced t. evansi strains, detect mixed infections, study the disease epidemiology and understand the interaction between vectors and reservoirs (fernandez et al. 2009). therefore, the objective of this study was to compare and evaluate the sensitivity and specificity of the two primer sets in the detection of acutely and chronically trypanosoma-infected male and female mice. materials and methods all procedures for animal infection, euthanasia and sample collections were approved by the animal reproduction research institute, agricultural research center, egyptian committee. trypanosoma evansi strain trypanosoma evansi strain (isolated from naturally infected camels) was obtained from the department of parasitology, faculty of veterinary medicine, cairo university. the parasite was propagated in laboratory mice and preserved in liquid nitrogen, according to shumei et al. (1996), for further processing. mice swiss albino mice (73 females and 73 males) weighing 25 g – 30 g were purchased from the laboratory animal building, research institute of ophthalmology, giza. the mice were housed in a pathogen-free environment for 2 weeks prior to the initiation of experiments. the mice were subjected to feed and water ad libitum. experiment i two groups of mice, 26 females and 26 males, were kept separately. twenty-one mice from each group received 104 trypanosome/mouse by i/p inoculation (infective dose [id]), according to sharma et al. (2012). the other five mice from each group were served as uninfected control groups. three mice were sacrificed daily from each group after infection. for each mouse, blood samples were collected from a tail vein to detect the pre-patent period and estimate the peak of parasitaemia and molecular assays. in addition, liver, spleen, kidneys, brain and ovaries or testis were collected on 1, 3, 5, and 7 days post-infections (dpi). a section from each organ was preserved at -20 °c for molecular examination. experiment ii two groups of mice, 47 females and 47 males, were kept separately. forty-two mice from each group were inoculated i/p with 102 trypanosome/mouse (id), according to sharma et al. (2012). five mice from each group were served as an uninfected normal control group. three mice from each group were sacrificed from 1–7 dpi and were examined by micro-haematocrit centrifugation technique (mhct) to detect the pre-patent period and estimate the peak of parasitaemia. all remaining mice were subsequently followed up biweekly for the assessment of parasitaemic waves during the course of infection by mhct from peripheral blood. samples were collected, as mentioned before (see experiment i), from each mouse during parasitaemic and aparasitaemic waves. parasitological examination all blood samples were collected from caudal vein blood in heparinised tubes and then centrifuged for 5 minutes at 5000 revolutions per minute. the capillary tubes were examined under the microscope (×10) for the detection of trypanosomes using mhct (woo 1970). molecular detection of trypanosoma evansi in blood and tissue samples of experimentally infected mice in both experiments deoxyribonucleic acid extraction from blood and tissue samples dna was extracted from each blood sample according to the procedure used by sarataphan et al. (2007). briefly, 50 µl of each blood sample was lysed twice in 500 µl of 0.1 m ammonium chloride and then centrifuged. the sediment was re-suspended in 50 µl of 0.002% sodium dodecyle sulphate and 50 µl of 5% chelex-100® (sigma) suspension in te buffer (10 mm tris–hcl, 0.1 mm edta, ph 8.0). the mixture was heated at 70 °c for 8 min followed by heating to 100 °c for 10 min. finally, the mixture was centrifuged at 10 000 rpm for 5 min at room temperature, and the supernatant was stored at -80 °c for molecular detection of trypanosomes by polymerase chain reaction (pcr). for tissue samples, 0.5 g of each tissue was washed in 500 µl of phosphate-buffered saline and then grinded in liquid nitrogen. the obtained pellet was suspended in 300 µl of 10% chelex-100® suspension in te buffer. the tissue samples were subsequently processed as stated above. conventional polymerase chain reaction sensitivity for detection of trypanosoma evansi to determine the detection limit of the standard pcr assay and establish a standard dilution for detection, 10-fold serial dilutions of known aliquots of t. evansi (1.0 × 106 trypanosomes) were used for seeding the non-infected camel blood. dilutions were subjected to dna extraction as described above and then processed by conventional pcr as described below using tbr f-tbr r (masiga et al. 1992) and (t. evansi rode trypanozoon antigen type) terotat920 f-terotat1070 r (konnai et al. 2009) primer sets. deoxyribonucleic acid amplification by conventional polymerase chain reaction assays on blood and tissue samples from trypanosoma-infected mice in both experiments pcr analysis was done by using primer pairs tbr f-tbr r (masiga et al. 1992) and terotat920 f-terotat1070 r (konnai et al. 2009) in standard pcr procedures. briefly, the pcr mixture of 25 µl contained 2x taq master mix, 25 pmol of each primer, 2 µl of deoxyribonucleic acid (dna) template and up to 25 µl nuclease-free water were added. pcr using tbr f (5′-gaa tat taa aca atg cgc ag-3′) and tbr r (5′-cca ttt att agc ttt gtt gc-3′) was performed in a thermocycler (nexus gradient eppendorf, germany) as follows: an initial denaturation step at 95 °c for 4 min, 30 cycles of denaturation at 95 °c for 45 seconds, annealing at 52 °c for 45 s, and extension at 72 °c for 60 s and final extension at 72 °c for 10 min. pcr using terotat920 f (5′-ctg aag agg ttg gaa atg gag aag-3′) and terotat1070 r (5′-gtt tcg gtg gtt ctg ttg ttg tta-3′) was carried out in a thermocycler (eppendorf thermal cycler, germany) as follows: an initial denaturation step at 95 °c for 4 min, 35 cycles of denaturation at 95 °c for 60 s, annealing at 52 °c for 60 s, and extension at 72 °c for 60 s and final extension at 72 °c for 10 min. amplicons were resolved on a 1.5% agarose gel stained with ethidium bromide (sigma) and photographed under ultra violet (uv) light (sambrook and russell 2001). positive control (t. evansi dna) and negative control (reaction mixtures without dna) were included in each pcr run. application of polymerase chain reaction assay on field blood samples from camels forty-four camels’ blood samples were collected in sterile heparinised tubes from different locations in giza and nobariya provinces for detection and isolation of t. evansi. each sample was tested by mhct, and 0.5 ml of blood was inoculated i/p into a laboratory mouse to investigate parasitic infection. dna was extracted, and the pcr assay was conducted as mentioned before. ethical considerations ethical approval was provided by the committee at the animal reproduction research institute agricultural research center, egypt (23-2016). results following-up the course of infection in experimentally infected mice different parasitaemic patterns were appeared in the infected groups according to the id in mice. in experiment i, the pre-patent period (appearance of parasite in blood) was detected at 3 dpi and reached the peak of parasitaemia at 7 dpi (figure 1, experiment i). in experiment ii (figure 1, experiment ii), the pre-patent period was determined at 5 dpi and parasitaemia peaked at 9 dpi, and three infection patterns were observed as follows: six mice showed persistent parasitaemia and died within 12–34 dpi (g1). in contrast, other six mice showed the disappearance of parasitaemia at 15 dpi and remained stable at this level until the end of the experiment (g2). twelve mice of the group (g3) displayed three waves of parasitaemia at 9, 22 and 34 dpi, with the disappearance of parasitaemia at 15, 29 and 46 dpi (table 1). figure 1: infection in experimentally infected mice. the course of the parasitemia during the period of the experiment i (a) (104 infective dose) and experiment ii (b) (102 infective dose) by micro-haematocrit centrifugation technique. the days post infection is plotted against the approximate no. of parasite in 50 ul blood. the plotted curves represent the different disease patterns obtained in mice during both experiments. table 1: summary of experimental infection. detection limit of trypanosoma evansi using conventional polymerase chain reaction pcr sensitivity was estimated using two primer sets terotat1.2 and tbr1/2 with 10-fold serial dilution of 106 parasites/ml. using terotat1.2 primer set, the minimal detection limit was 10 parasites/ml blood. no extra bands were amplified except the 151 bp (base pairs) target dna band (figure 2a). employing tbr1/2 primer set, the lowest detection limit was 0.0001 parasites/ml blood despite the appearance of two extra non-specific bands in addition to the 164 bp, specifically amplified band (figure 2b). figure 2: polymerase chain reaction detection limit by conventional polymerase chain reaction. lane m: 100 base pairs molecular weight deoxyribonucleic acid marker. lanes 1–12: different dilutions and -ve: negative control. trypanosoma brucei 1/2 detects trypanosoma evansi at lower dilutions (10-4 parasite/ml) than t. evansi rode trypanozoon antigen type 1/2 (10 parasite/ml of blood). (a) terotat; (b) tbr. conventional polymerase chain reaction detection of trypanosoma evansi in blood in both experiments it was found that the assay was able to detect t. evansi dna at 1 dpi in both experiments. high detection sensitivity was obtained by using tbb1/2 primer sets (figures 3a and 4a) with convenient band intensity on the agarose gel throughout the course of infection. however, by using rotat1.2 primer sets, the intensity of the amplified bands was clearly observed and increased gradually from the first dpi until the peak of parasitaemia in both experiments (figures 3b and 4b). it was observed that there was no variation in the results between female and male mice. figure 3: direct polymerase chain reaction from mouse blood injected by 104 trypanosome. (a) polymerase chain reaction amplification using trypanosoma brucei 1/2 primer sets. the band 164 base pairs was high intensity and consistently visible throughout the course of infection. (b) polymerase chain reaction amplification using t. evansi rode trypanozoon antigen type 1.2 primer sets. the bands intensity was clearly observed and increased gradually from the first day post infection until the peak of parasitemia. m: 100 base pair molecular weight deoxyribonucleic acid marker, +ve: positive control, -ve: negative control, numbers: days post infection. letters: mice were sacrificed at day 1, 3, 5 and 7 post infection. figure 4: direct polymerase chain reaction from mouse injected with 102 trypanosome. (a) polymerase chain reaction amplification using trypanosoma brucei 1/2 primer sets. (b) polymerase chain reaction amplification using t. evansi rode trypanozoon antigen type 1.2 primer sets. m: 100 base pair molecular weight deoxyribonucleic acid marker, +ve: positive control, -ve: negative control, numbers: days post infection. letters: mice were sacrificed at day 1, 3, 5 and 7 post infection. detection of trypanosoma evansi in different female and male organs in both experiments the pcr technique could detect the infection at 24 hours post infection (pi) in both experiments in different examined organs, such as liver, spleen, kidney, testis and brain. the specific bands of the used primers appeared at 164 bp and 151 bp dna fragments for tbr and terotat primers, respectively. furthermore, tbr1/2 primers displayed clear bands at 1 dpi, and terotat1.2 primers showed a faint band at 1 dpi, with an increased band intensity as the infection progressed towards the parasitaemic stage (figures 5 and 6). in addition, in the chronic stage, no variation was detected between the used primers (figure 7). figure 5: polymerase chain reaction r amplification of deoxyribonucleic acid extracted from tissues of infected male mice (parasitaemic stage) using trypanosoma brucei primer set. m: 50 base pairs molecular weight deoxyribonucleic acid marker, +ve: positive control, -ve: negative control, 1, 3, 5, 7: days post infection and l (liver, ) s (spleen), k (kidneys), t (testes) and b (brain). figure 6: polymerase chain reaction amplification of deoxyribonucleic acid extracted from tissues of infected male mice (parasitaemic stage) using t. evansi rode trypanozoon antigen type primer set. m: 50 base pairs molecular weight deoxyribonucleic acid marker, +ve: positive control, -ve: negative control, 1, 3, 5, 7: days post infection and l (liver), s (spleen), k (kidneys), t (testes) and b (brain). figure 7: polymerase chain reaction amplification of deoxyribonucleic acid extracted from tissues of infected male mice (chronic) using (a) trypanosoma brucei primer set and (b) t. evansi rode trypanozoon antigen type primer set. m: 50 base pairs molecular weight deoxyribonucleic acid marker, +ve: positive control, -ve: negative control, 7: days post infection and l (liver), s (spleen), k (kidneys), t (testes) and b (brain). detection and isolation of trypanosoma evansi from clinical samples of camels all blood samples of camels were negative for t. evansi by mhct or mouse inoculation. meanwhile, pcr showed that all the tested samples were positive for the presence of the parasite using tbr1/2 primer sets. however, by using terotat1.2 primer, only 32 out of 44 animals were positive for t. evansi infection (figure 8). figure 8: polymerase chain reaction results of field blood samples with trypanosoma brucei 1/2 (164 base pairs) and t. evansi rode trypanozoon antigen type (rotat) 1.2. (151 base pairs) primers sets: m: 100 base pairs molecular weight deoxyribonucleic acid marker, +ve: positive control trypanosoma evansi deoxyribonucleic acid and -ve: negative control. (a) tbr and (b) terotat discussion molecular diagnostic techniques represent essential tools for the detection of t. evansi infection and are widely spread in the detection of surra globally because they are rapid, accurate and reliable (desquesnes & davila 2002; sengupta et al. 2010). monitoring parasitiaemic level in t. evansi is important for evaluating the health status of the host, determination of disease stage and risk of transmission of disease between animals in the field. experimental infection of t. evansi was done in mice with two different inoculation doses to follow up the disease and determine which technique is more suitable technique for the early detection of t. evansi. infection of mice with 104 and 102 trypanosomes in experiment i and experiment ii, respectively, showed variable disease patterns, which could be ascribed to the inoculum dose and sex of mice. microscopic examination by mhct showed no difference in parasitaemia in the pre-patent periods between male and female mouse groups in experiment i 3 dpi. however, the progress of infection was delayed in male mice compared to female mice as the peaks of parasitaemia were achieved earlier in female than male mice. differences in infection progress and parasitaemic peaks in t. evansi infection are sex dependant, and may be influenced by a stronger immune response against t. brucei in male mice compared to female ones carvalho et al. (2018). all remaining trypanosoma-infected mice were died by the 9 dpi because of acute parasitaemia. in experiment ii (102 id), the pre-patent period was delayed 2 days before compared to experiment i as a result of decreased inoculum dose. similar results were observed by sharma et al. (2012) after t. evansi experimental infection in mice, and the parasite was detected at 3 days and 4.5 days post-inoculation of 104 and 102 parasites, respectively. the different patterns of infection observed after inoculation of 102 parasites are a reflection of levels of immune response elicited among animals. female mice showed consistent parasitaemia until death (g1), which can be ascribed to a depressed immune status usually observed in the acute form of the disease. a persistent chronic undetectable infection in g2 is a reflection of adaptive immune resistance against infection, which represents a carrier state. fluctuating parasitaemia throughout the course of infection in g3 mirrored antigenic variation among the strains of t. evansi and evasion of host immune response (maudlin, holmes & miles 2004). the mechanism is a result of the parasite’s ability to periodically switch its major variant surface glycoprotein yielding a parasitaemic relapse (herrera et al. 2004). two parasitaemic waves were observed in male mice. most of males died from acute parasitaemia where only three mice showed one wave of parasitaemic disappearance and were dead by the second wave of parasitaemia. when the results of mhct (parasitological test) and pcr (molecular technique) were compared, conventional pcr with tbr1/2 and terotat1.2 primer sets was able to detect the parasite as early as 1 dpi in mice. however, mhct detected parasite at 3 dpi in experiment i and at 5 dpi in experiment ii. all tests were able to detect a peaking parasitaemia. these results are in line with those of previous studies (ashour et al. 2013; sengupta et al. 2010). however, fernandez et al. (2009) determined the pre-patency 12 hours post infection in mice by tbr1/2 and 1 dpi by its1 primers sets. striking differences in the intensity of the bands amplified by terotat1.2 primer sets from day 1 until the appearance of a consistent parasitaemia were ascribed to differences in the parasite count in the blood samples. gonzález et al. (2006) and fernandez et al. (2009) confirmed that the proportion of dna is usually a reflection of the number of the parasite in a blood sample. therefore, they recommended the use of at least 100 ng dna in the sample when parasitaemia is lower than 103 parasites/ml to ensure the detection of the parasite by pcr assay. in tbr1/2 pcr, the intensity of the amplified bands showed little or no difference through the course of infection. this corresponded to the high number of repeat copies of the target sequence, which overcomes low parasitic dna concentration in the sample. in the current study, sybr green real-time pcr assay was carried out using terotat1.2 primer sets, as described by konnai et al. (2009). conventional pcr results were able to detect the pre-patent periods (i.e. appearance of parasite in blood) at 1 dpi in both experiments i and ii in mice. these results were further confirmed by melting curve analysis, which was rapid and avoided the downstream procedure of the conventional pcr. taylor, boyle and bingham (2008) detected the parasite 6 dpi in rats injected with 104 trypanosomes using its-1 taqman real-time pcr; however, sharma et al. (2010) detected the parasite 1.5 dpi in mice infected with 104 parasites and 3 dpi with 102 parasites by the same assay. discrepancies between our results and those obtained in previous studies may be because of variations in real-time conditions and different target sequences used in the techniques. animals infected with t. evansi are characterised by fluctuating parasitaemia as a result of interactions between the host immune response and the ability of the parasite to evade immunity by antigenic variation. parasitaemia is usually accompanied by the rise and fall of body temperature of the host (dargantes et al. 2009). during these periods, the demonstration of t. evansi in the blood of animals is obvious; however, low-level parasitaemia results in the difficulty of parasite detection. the polymerase chain reaction was able to detect t. evansi during the chronic phase of the disease in mice, while mhct could not. the limited sensitivity and false-negative results of mhct may be attributed to the fluctuation of parasitaemia and the presence of parasite with low number (nantulya 1990). our results are in agreement with those obtained by ashour et al. (2013) and ramírez-lglesias et al. (2011) in mice and rabbits, respectively. differences between our results and those obtained in previous studies may also be because of diversity of trypanosome strains, different pcr conditions, different primer sets and dna extraction methods, as mentioned by fernandez et al. (2009). in this work, chelex resin was used for dna extraction from blood samples. the method provided high dna yield with convenient purity, and was less laborious and overcame the toxic effect and risk of using organic extraction methods (herrera et al. 2005). application of pcr for t. evansi field diagnosis in camels showed variable results among the primers sets. the mhct and mouse inoculation were not able to detect the parasite in the field samples. this might have been influenced by preservation methods and the duration transportation of samples before laboratory examination. in the same vein, holland et al. (2001) reported that trypanosomes in samples from animals with high (>104 trypanosomes/ml of blood) and low parasitaemia (250 trypanosomes/ml of blood) could not be detected beyond 8 and 3 h after storage at 4 °c and 27 °c, respectively. however, positive results were recorded for all samples using conventional pcr and tbr1/2 primer sets, where terotat1.2 pcr was able to detect 32 out of 44 samples. the low sensitivity of the rotat1.2 pcr compared with that obtained by tbr1/2 was attributed to several factors such as number of copies of the target dna sequence on the genome, parasitaemic level, parasitic dna concentration and non-rotat1.2 (variable surface glycoprotein) vsg t. evansi variant existence that was previously reported in kenya (sengupta et al. 2010). similar results were obtained by elhaig et al. (2013), who reported that tbr primers showed higher sensitivity and specificity than five other primer sets for the detection of t. evansi and were able to detect parasitaemia below one parasite per millilitre of blood. in conclusion, this study demonstrated that pcr was accurate, sensitive and reliable in the detection of the early t. evansi-infected mice, and also able to discriminate chronically infected carrier animals. in addition, pcr with tbr1/2 was more sensitive than terotat1.2. however, tbr1/2 could detect field infection even when samples were negative by conventional methods. therefore, this study will contribute towards understanding the course of the disease and finding suitable diagnostic tools for t. evansi. furthermore, the use of molecular pcr for screening of newly introduced animals will help in excluding the carrier animals and detecting the early infected animals for saving free herds. acknowledgements the authors would like to thank the faculty and staff members of the department of parasitology, faculty of veterinary medicine, cairo university, for providing them with tyrpanosoma evansi. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors contributed equally to this work. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references ashour, a., abou el-naga, t., barghash, s. & salama, m., 2013, ‘trypanosoma evansi: detection of trypanosoma evansi dna in naturally and experimentally infected animals 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ankara university of veterinary medicine, ankara, turkey feray alkan department of virology, faculty of veterinary medicine, ankara university of veterinary medicine, ankara, turkey citation dağalp, s.b., babaoglu, a.r., dogan, f., farzani, t.a. & alkan, f., 2020, ‘an assessment of bovine herpes virus 4 as a causative agent in abortions and neonatal death’, onderstepoort journal of veterinary research 87(1), a1761. https://doi.org/10.4102/ojvr.v87i1.1761 original research an assessment of bovine herpes virus 4 as a causative agent in abortions and neonatal death seval b. dağalp, ali r. babaoglu, firat doğan, touraj a. farzani, feray alkan received: 07 mar. 2019; accepted: 28 aug. 2019; published: 06 feb. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract numerous viruses, including bovine viral diarrhoea virus (bvdv), bovine herpes virus 1 (bohv-1) and bovine herpes virus 4 (bohv-4), and other pathogens are the most common causes of reproductive disorders and are responsible for huge economic losses in livestock production. this study investigates the aetiological role of bohv-4 in fertility problems such as abortions, stillbirth and birth with unviable calves. retrospective samples from 38 animals, including 17 aborting cows, 17 aborted foetuses, three stillborn calves and one unviable newborn calf were analysed. the bohv-4 genome was detected in 25 (65.7%) animals by polymerase chain reaction. in 14 of these infected animals, we detected co-infection with bvdv, while the co-presence of bohv-1 was also detected in one animal. in addition to the high prevalence of bohv-4 genome in materials related to fertility problems, isolation of bohv-4 from the brain of one stillborn calf indicated a causal link between bohv-4 and fertility problems, such as abortion, stillbirths or birth with unviable calves. keywords: abortion; bohv-4; cow; co-infection; viruses. introduction previous studies have identified several viruses associated with reproductive disorders in cattle, such as abortion, neonatal death and births with congenital abnormalities. in such cases, bovine viral diarrhoea virus (bvdv) and bovine herpes virus 1 (bohv-1) (czaplicki & thiry 1998; kirkbride 1992; mckercher & wada 1964; miller & van der maaten 1987; murray 1990; schiefer 1974; wellemans, van opdenbosch & mammerickx 1986) are often detected along with bovine herpes virus 4 (bohv-4). the detection of bohv-4 even in healthy cattle (czaplicki & thiry 1998; kirkbride 1992; monge et al. 2006) makes the investigation of the epidemiology and pathogenetic mechanisms of this infection an interesting subject for researchers. bovine herpesvirus 4 (bohv-4) is a member of the rhadinovirus genus of the gamma herpes virinae sub-family (ictv 2017). antigenically, closely related strains of bohv-4 with similar restriction patterns form two main groups, namely, genotype 1 and genotype 2, which possibly correspond to the movar-like and dn 599-like reference strains, respectively. genotype 3 has also been reported recently (verna et al. 2012). as with other herpes viruses, animals experimentally infected with bohv-4 develop a latent infection, followed by virus reactivation and re-excretion after dexamethasone treatment and stress factors such as calving (dubuisson et al. 1989). this virus naturally infects blood mononuclear cells, such as macrophages and monocytes, and displays a specific tropism towards vascular endothelia, mammary tissue, endometrium and foetal tissues. clinical manifestations associated with bohv-4 in cattle include postpartum metritis (monge et al. 2006), abortion (czaplicki & thiry 1998; kirkbride 1992; schiefer 1974; wellemans et al. 1986), mastitis (izumi et al. 2006; miyano et al. 2004; wellenberg et al. 2000), pneumonia, keratoconjunctivitis, encephalitis and diarrhoea in both natural and experimental infections (goyal & naeem 1992; thiry et al. 1990). although most bohv-4 isolates are considered mild pathogens or completely apathogenic for cattle, bohv-4 antibodies have higher prevalence in abortion cases than in clinically asymptomatic cattle (altamiranda et al. 2015; naeem, goyal & werdin 1989). in turkey, numerous studies have investigated bohv-1 and bvdv as aetiological agents of fertility problems, such as abortion, and have conducted serological screening of antibodies against these agents (alkan & burgu 1993; alkan et al. 2005, 2009, 2018; bilge-dagalp et al. 2008; oğuzoğlu et al. 2012; tan et al. 2006). there have also been virological and serological studies on bohv-4 infection. these studies show that bohv-4 infection is very common and is increasing significantly in cattle herds in turkey (aslan, azkur & gazyagc 2015; bilge-dagalp et al. 2007, 2008, 2011; dağalp et al. 2010; tuncer-göktuna et al. 2016; yildirim et al. 2011). however, there is no published data about the detection of bohv-4 in samples from aborted foetuses and stillborn or unviable calves. the present study investigates the presence of bohv-4 genome in samples from aborting cows, aborted foetuses, stillborn calves and unviable calves to investigate the interaction between bohv-4 and these fertility problems. it also investigated whether there is any significant association between co-infection of bohv-4 and two other abortifacient pathogens, bvdv and bohv-1. materials and methods sampled animals we used retrospective samples from 38 animals submitted to our laboratory for routine diagnosis, including 17 aborted foetuses, four stillbirths or unviable calves and 17 aborting cows from four different cattle herds (totalling 500–700 animals). in total, the 26 samples from 17 cows comprised leukocytes (n = 15), vaginal swabs (n = 9) and placental materials (n = 2). other samples comprised various tissues from foetuses aborted at seventh to eighth month of gestation and from stillborn or unviable calves (table 1). to increase the possibility of detecting viruses, each sample from the same animal was tested for bohv-4 by polymerase chain reaction (pcr) assay along with two other frequently detected pathogens in turkey, namely, bovine herpes virus 1 (bohv-1) and bvdv. table 1: distribution of materials and animals tested and polymerase chain reaction results. viral nucleic acid extraction and polymerase chain reaction extraction of deoxyribonucleic acid (dna) for all samples was carried out according to sambrook, fritsch and maniatis (1989). total rna was extracted using a high pure viral nucleic acid kit (roche, germany) according to the manufacturer’s recommendations. complementary dna (cdna) synthesis was performed using the first strand cdna synthesis kit (thermo scientific, united states) according to the manufacturer’s protocol. the primer sets specific for gb of bohv-4 f: (5’-cccttctttaccaccacctaca-3’) and r: (5’-tgccatagcagagaaacaatga-3’) (goltz et al. 2004), gc of bohv-1 f: (5’-ctgctgttcgtagcccacaacg-3’) and r: (5’-tgtgacttggtgcccatgtcgc-3’) (van engelenburg et al. 1993) and 5’-utr of bvdv f: (5’-atg ccc wta gta gga cta gca-3’) and r: (5’tca act cca tgt gcc atg tac -3’) (vilcek et al. 1997) were used in the pcr techniques that are described elsewhere with some minor modifications (wellenberg et al. 2001). specific pcr products (615 base pair (bp), 397 bp and 288 bp of bohv-4, bohv-1 and bvdv, respectively) were visualised in transilluminator after electrophoresis in 1% agarose gel containing 10-mg/ml ethidium bromide. ethical considerations this study was performed within the scientific research projects of ankara university (project no.: 10b3338005). results the pcr results are shown in table 1, including the description of animals and related materials. out of 38 animals, the bohv-4 genome was detected in 25 (65.7%) animals, with 14 (36.8%) and one (2.6%) co-infection rates for bvdv and bohv-1, respectively. moreover, out of 38 animals, 19 (50.9%) animals were found to be positive for bvdv and one (2.6%) animal was found to be positive for bohv-1 (table 1). the estimated rate of bohv-4 positivity was 76.1% (16/21) for aborted foetuses and stillborn or unviable calves and 82.5% (14/17) for cattle (table 2). in total, 78.9% (30/38) of the sampled animals were positive for at least one of the tested viruses. the data showed that almost all tissues from an aborted foetus with bohv-4 were found positive (table 1). table 2: results according to sampled herds and clinical cases. discussion bovine herpes virus 4 is a co-factor in cattle abortion cases along with other pathogens, including viruses, bacteria and protozoans. they also play a clear role in certain other reproductive disorders such as endometritis (chastant-maillard 2015; cvetojević et al. 2016; frazier et al. 2002; reed, langpap & bergeland 1979). researchers have, therefore, focussed on detecting bohv-4 in semen, uterine and blood samples from aborting cows in herds with postpartum metritis (cvetojević et al. 2016; dağalp et al. 2012; nikolin et al. 2007; nikolin, vesna & radosavljević 2008). however, the connection between bohv-4 and abortions remains unclear because the virus has been isolated from both diseased and healthy cattle (czaplicki & thiry 1998; deim, szeredi & egyed 2007; leboeuf 2013). our results (tables 1 and 2) show that bohv-4 infection has a very high prevalence in the samples obtained from herds i and ii. because very few animals were sampled from herds iii and iv, we could not analyse the prevalence of bohv-4 infection. however, our previous results and knowledge from veterinarians suggest that bohv-4 infection is also increasing significantly in these herds, resulting in an increase in economic losses (bilge-dagalp 2007, 2008, 2011; dağalp et al. 2012). the pathogenesis of bohv-4 infection has been questioned because of the detection of bohv-4 from both healthy individuals and cattle with a wide variety of clinical signs (monge et al. 2006; wellenberg et al. 2000). in our study, out of 38 animals tested by pcr targeting the gb gene region of bohv-4, 25 animals, including aborting cows (n = 14) and aborted, stillborn and unviable calves (n = 11), tested positive for the virus. because of the use of retrospective samples, different materials (blood, vaginal and placental samples) could be tested for aborting cows. however, it is not yet possible to say which of these materials should be used to diagnose bohv-4 infection, although vaginal specimens appear superior to blood samples (table 1). similarly, several tissue samples from an aborted foetus gave positive results for bohv-4, which indicate that bohv-4 is transferred to the foetus transplacentally. in addition, isolation of bohv-4 from a brain sample of a stillborn calf (animal no. 2 in table 1) in another study (unpublished data) is very important for showing transplacental transfer and the virus’ causative role in fertility problems. in short, our study has substantially determined a link between fertility problems such as abortion and stillbirths and bohv-4 because we detected the virus in various foetal tissue samples, while the other studied pathogens, especially bvdv, were absent in most of the aborted foetuses (tables 1 and 2). verna et al. (2012) likewise concluded that the detection of bohv-4 as a sole agent offers indirect evidence of the virus’ involvement in bovine abortion. several reports indicate that bohv-4 has an immunosuppressive effect (egyed 2000; egyed et al. 1996) and contributes to disease development by stimulating inflammatory reactions (donofrio et al. 2005). szenci et al. (2016) reported that local multiplication of histophilus somni may produce certain chemicals (pge2) that reactivate latent bohv-4 from some local macrophages, while multiplication of the reactivated virus decreases the efficiency of local macrophage functions (opsonisation, antigen presentation and killing activity), which further promotes local bacteria multiplication. for this reason, we also tested all our samples for bvdv and bohv-1, which are very common infections in turkey, as well as for bohv-4. the rate of detection of bvdv was as high as for bohv-4 in herd i, which had an acceptable sample size of aborting cows and foetuses for this assessment (table 2). one of the six aborted foetuses was also co-infected with bohv-4 and bvdv. however, it is not possible to conclude whether this signifies an interaction between bvdv and bohv-4 infection in these cases, although bohv-4 has been identified as an important factor affecting the immune system. this could explain the high rates of these infections in herds i and ii. based on our knowledge, few studies have investigated clinical cases, including abortions and the effects of bohv-4 along or together with these other viruses. yilmaz, coskun and sahin (2016) had reported that 66.66% of aborted calves (8/12) were positive for bvdv, although bohv-1 and bohv-4 were not detected in the sampled animals. similarly, another study (tuncer-göktuna et al. 2016) using pcr analysis for investigating the role of herpes viruses and pesti-viruses in cases of ruminant abortion between 2007 and 2015 in western turkey did not find bohv-4 in any of the tested samples from 60 aborted foetuses, while two and 31 calves aborted analysed using elisa were found positive for bohv-1 and bvdv antigens, respectively. however, some other studies have reported the presence of bohv-4 and its co-infection with other pathogens (cvetojević et al. 2016; frazier et al. 2002; reed et al. 1979). cvetojević et al. (2016) detected bohv-4 in 21% of examined samples (21/100), co-infection of bohv-4 with bvdv in two samples and one sample infected with neospora caninum. otherwise, several studies have proposed that bohv-4 is a secondary agent, often associated with secondary bacterial infections (chastant-maillard 2015; donofrio et al. 2008; klamminger et al. 2017; jacca et al. 2014; nak et al. 2011) and also fungi or other viruses (drolet, werdin & goyal 1986; fabian et al. 2008; frazier et al. 2002). gagnon et al. (2017) suggested that because bohv-4 is a frequent risk or a secondary factor in cattle infections, higher bohv-4 seroprevalence in cattle with respiratory or reproductive diseases could be expected. thus, our data detecting two viral pathogens in the same aborted foetus, stillborn calves and aborting cows (tables 1 and 2) are not surprising. unfortunately, we were unable to test both foetuses and their mothers. therefore, more detailed investigations are needed to establish a clear link between bohv-4 infection and abortion as well as the potential interaction of bohv-4 with other abortifacient pathogens in the same sample. in this study, there were two main limiting factors. firstly, it did not test for the presence of any additional microorganisms (bacteria, etc.). secondly, it did not include serological results to show the positivity rate for bohv-4 or the other studied viruses in aborting cows and/or healthy cattle in these herds. in conclusion, we consider that bohv-4 alone or in conjunction with other pathogens, especially bvdv, contributed to the development of the reproductive disorders reported in this study, and possibly to others as well. further studies using epidemiological data from bohv-4 infections and molecular characterisation of field bohv-4 in different clinical cases are needed to understand their pathogenesis and responsibility for economic losses affecting cattle husbandry in turkey. acknowledgements competing interests the authors have declared that no competing interests exist. author’s contributions all authors contributed equally to this work. funding information this study was supported by a grant from the scientific research projects (project no: 2004 0810 068 and 10b3338005), ankara university. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily 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introduction materials and methods results and discussion conclusion acknowledgements references about the author(s) shiva j. jyothi department of veterinary biotechnology, college of veterinary science, p.v. narsimha rao telangana veterinary university, hyderabad, india sunil r. patil department of veterinary biotechnology, college of veterinary science, p.v. narsimha rao telangana veterinary university, hyderabad, india narasimha y. reddy department of veterinary biotechnology, college of veterinary science, p.v. narsimha rao telangana veterinary university, hyderabad, india rao p. panduranga biovet pvt ltd., malur, karnataka, india uma madala ella foundation, turkapally, hyderabad, india gnana m. prakash department of animal genetics and breeding, college of veterinary science, p.v. narsimha rao telangana veterinary university, hyderabad, india kalyani putty department of veterinary biotechnology, college of veterinary science, p.v. narsimha rao telangana veterinary university, hyderabad, india citation jyothi, s.j., patil, s.r., reddy, n.y., panduranga, r.p., madala, u., prakash, g.m. et al., 2020, implications of a conserved region of bluetongue virus protein vp2 in cross-neutralisation of bluetongue virus serotypes, onderstepoort journal of veterinary research 87(1), a1816. https://doi.org/10.4102/ojvr.v87i1.1816 original research implications of a conserved region of bluetongue virus protein vp2 in cross-neutralisation of bluetongue virus serotypes shiva j. jyothi, sunil r. patil, narasimha y. reddy, rao p. panduranga, uma madala, gnana m. prakash, kalyani putty received: 13 sept. 2019; accepted: 25 june 2020; published: 08 oct. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract bluetongue (bt) is a vector-borne disease of ruminants caused by bluetongue virus (btv). twenty-nine different serotypes of btv are currently reported throughout the world. the main objective of this study is the development of a subunit vaccine model that could potentially be adapted to provide broad spectrum protection against multiple btv serotypes, which the conventional vaccines fail to address. to this end, three different btv proteins (conserved region of viral protein [vp]2, vp5 and ns1) were expressed and purified in an escherichia coli expression system. the immunogenicity of these proteins was tested in murine models using the montanidetm isa 201 vg adjuvant. balb/c mice were immunised thrice (with individual proteins and a mixture of three proteins) at two-week intervals and were monitored until day 40 post-infection/vaccination. protein-specific antibodies directed against the recombinant proteins were detected by indirect enzyme-linked immunosorbent assay. neutralising antibody (nab) titres and cross-neutralisation against a range of btv serotypes (btv-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24) were determined by serum neutralisation test. the recombinant proteins elicited higher nab titres compared with the inactivated vaccine group, except for btv-1, where the inactivated vaccine group elicited higher nab titres. additive effect of the three proteins was not observed as the nab titres generated with a combination of conserved vp2, vp5 and ns1 was similar to those of the individual protein groups. whilst btv-12 could only be neutralised by serum raised against the inactivated vaccine group, btv-5 and -24 could not be neutralised by any of the groups tested. our cumulative data suggest that the conserved regions of vp2 (cvp2), vp5 and ns1 could play an important part in the novel vaccine design against multiple btv serotypes. importantly, given that vp2 was already known to elicit a serotype-specific immune response against bt, we report, for the first time, that the conserved region of vp2 has the ability to induce cross-protective immune response. keywords: bluetongue; subunit vaccine; conserved vp2; cross-neutralisation; broad-spectrum protection. introduction bluetongue virus (btv) is a member of the family reoviridae and is the causative agent of bluetongue (bt) disease, a haemorrhagic disease of sheep and some species of wild ruminants (mertens & diprose 2004). so far, 29 serotypes (kartika lakshmi et al. 2018; maan et al. 2016) of btv have been identified worldwide and 24 serotypes have been reported to circulate in india (hemadri et al. 2017; krishnajyothi et al. 2016). as per a recent study conducted during 2014–2018 by our group, btv serotypes btv-1, -2, -4, -9, -12, -16, -21 and -24 seemed to be the circulating serotypes in india (personal observation, unpublished data). an inactivated pentavalent vaccine consisting of btv serotypes btv-1, -2, -10, -16 and -23 is currently used in some parts of india. however, circulation of multiple serotypes, often within the same animal, and little or no cross-protection between serotypes makes btv control and prevention a challenge (hemadri et al. 2017). bluetongue virus has a double-stranded rna (dsrna) genome composed of 10 linear segments encoding seven structural proteins (viral protein [vp]1–vp7) and five non-structural proteins (ns1, ns2, ns3a, ns3b and ns4) (rao et al. 2017). vp2, a major viral serotype determinant, is responsible for virus attachment and haemagglutination. vp5, coded by segment 6, induces membrane permeabilisation during onset of the infection and can cause syncytia formation, affect specificity of virus neutralisation and also show partial correlation with virus serotype. ns1 is a viral protein translation enhancer that co-localises with the centrosome and may play a role in disrupting and blocking cell division in mammalian cells, and it is highly conserved among different btv serotypes. it contains epitopes associated with both t-cell and humoral responses, and antibody responses against ns1 protein may be important contributors to immune protection (anderson, hagglund & breard; 2014; rao et al. 2017). protein-based vaccines for bt were based on the initial observation that vp2, in combination with vp5, elicits protective immunity in vaccinated sheep, following which several studies reported potential vaccines for other immunogenic proteins of btv including ns1 (anderson, hagglund & breard 2013, 2014; jones et al. 1996; marin-lopez et al. 2014; mohd jaffar et al. 2014; roy et al. 1990). vp2 is the most variable protein unique to each btv serotype; sequence data indicate that the most conserved region of vp2 (cvp2) between serotypes was evident from 338 to 383 amino acids predicted with two major histocompatibility complex class i (mhci) and two major histocompatibility complex class ii (mhcii) binding sites and three b-cell epitopes, suggesting cvp2 has a strong immunogenicity potential (shiva jyothi et al. 2018). conventional vaccines, though efficient, are not without disadvantages. incomplete attenuation or inactivation, ability of vaccine strains to reassort with field strains, adverse clinical reactions to vaccination and emergence of new serotypes limit the use of ‘serotype-specific protection conferring’ conventional vaccines. with this perspective, the main objective of this study was to develop a broad-spectrum subunit vaccine model for bt that can confer protection against a range of btv serotypes. here, cvp2, vp5 and ns1 proteins were expressed in escherichia coli. immunogenicity potential of these proteins was tested in balb/c murine models and compared with the conventional inactivated pentavalent vaccines. finally, the cross-neutralisation potential of the proposed vaccine model was evaluated and discussed. materials and methods virus and cell lines bluetongue virus serotypes btv-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24 isolated during bt outbreaks in 2011–2016 were plaque purified and their identities were confirmed by real-time polymerase chain reaction (maan et al. 2016). the virus was propagated in bhk-21, and vero cell lines were grown in minimal essential media (gibco, united states [us]) supplemented with 10% foetal bovine serum (gibco, us) at 37 °c in an atmosphere containing 5% co2 (world organisation for animal health 2012). generation of recombinant plasmids the conserved region of vp2 (234 bp; nucleotide positions: 1012–1246, genbank accession: kp339225) was amplified from btv-16 complementary dna using primers f (bamhi) 5’ggatccgatgcgtttctatgtgttgctaat3’ and r (ecori) 5’gaattctcagtcaaagaggttaacgcgcc3’ and cloned into expression vector prset-b (invitrogen, us). for vp5 and ns1, codon optimised artificially synthesised btv-16 full length genes (genbank accession numbers: kf664138 and kf387525 for vp5 and ns1, respectively) were cloned in pet-28b (+) (invitrogen, us). protein expression by autoinduction and purification the constructed plasmids were transformed into e. coli bl21 (de3) competent cells (sigma, us) according to the manufacturer’s protocol and previously described procedure (hanahan et al. 1991). protein expression by autoinduction, as described in an earlier study, was followed for all three proteins (studier 2005). the proteins were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and western blotting (shi & jackowski, 1998) to detect his-tagged cvp2 (9.35 kda), vp5 (59.8 kda) and ns1 (61.2 kda). protein purification was done using nickel (ni-nta) affinity chromatography (qiagen, us) as per the manufacturer’s instructions. refolding of proteins purified under denaturing conditions was done as mentioned in a previous study (anderson et al. 2014). final confirmation of the eluted proteins was done using sds-page along with western blotting. mice immunisation thirty-six (6to 8-week-old) female balb/c mice were housed at the animal house of the college of veterinary science, hyderabad. they were divided into six groups (groups 1–6) of six mice each; groups 1–4 were injected subcutaneously on days 0, 14 and 28 with 25 µg of each protein in montanidetm isa 201 vg emulsion (table 1). the commercially available inactivated pentavalent vaccine (raksha-blu, indian immunologicals, india) containing btv-1, -2, -10, -16 and -23 was used to immunise the group 5 mice. six non-immunised balb/c mice administered with montanidetm isa 201 vg in phosphate-buffered saline were used as the control group (group 6). all the animals in this study were examined daily for the presence of reactions, if any, until day 40 and sacrificed; blood was then collected by heart puncture for serological analyses. table 1: protocol of mice immunisation. antibody detection (indirect enzyme-linked immunosorbent assay and serum neutralisation assay) antibody response against specific proteins was monitored by indirect enzyme-linked immunosorbent assay (elisa) using 1/200 dilution of collected serum samples (anderson et al. 2014). neutralising antibodies were determined (savini et al. 2004). plaque-purified btv-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24 were used as antigens. serum samples obtained two weeks after the third immunisation were serially diluted (1/10, 1/20, 1/40, 1/80, 1/160, 1/320, 1/640 and 1/1280), incubated with an equal quantity of 100 tissue culture infective dose (tcid)50/50 µl and added to confluent monolayers of vero cells in 96-well plates. plates were observed under an inverted microscope at 12-h interval for cytopathic effect (cpe), and the final titre was taken on day 5. all dilutions were performed in quadruplicate, and the assays were repeated at least three times. the neutralisation titres were determined as the highest dilution of serum giving a 50% neutralisation endpoint and expressed as a log10 reciprocal of the highest positive serum dilution. statistical analysis data were presented as means and standard deviations, where applicable, and the statistical significance of differences between groups was determined as p-values using student’s t-test in excel. p-value < 0.05 was considered to be significant. ethical consideration all the animal experimentation protocols were approved by the host institute’s institutional animal ethics committee (iaec no. 16/6/iaec/2017/16.05.17). results and discussion the development of effective vaccines for viruses with multiple distinct serotypes is highly challenging. subunit vaccines address the drawbacks conventional vaccines pose and have an advantage of being differentiating infected from vaccinated animals (diva) compliant. humoral response is the main component of the immune response against btv; contribution of the cellular component, although it appears to be important, is not well understood (anderson et al. 2013; marin-lopez et al. 2014). the major aim of the subunit vaccine design in this study was to determine its (1) role in humoral immune response and (2) potential to induce cross-serotype reactive immune responses. recombinant bluetongue virus proteins’ expression and purification conserved region of vp2 was detected in the soluble fraction of the e. coli cell lysate; vp5 and ns1 were detected in inclusion bodies and purified under denaturing conditions followed by refolding of the eluted proteins. purified proteins were desalted by dialysis and their concentrations were found to be 1.8 mg/ml, 1.1 mg/ml and 0.45 mg/ml for cvp2, vp5 and ns1, respectively. sodium dodecyl sulfate polyacrylamide gel electrophoresis gel analysis detected proteins of desired size with up to 90% purity indicated by the absence of bands of higher and lower molecular weights than the desired sizes (figure 1a and b). figure 1: identification of purified btv recombinant proteins and detection of protein-specific antibodies by indirect elisa. (a) ni-nta purification of recombinant btv proteins, and sds-page analysis of purified cvp2, vp5 and ns1: arrowheads indicate recombinant btv proteins at the expected molecular weights. a (lane 1): cvp2, 9.35 kda; b (lane 1): vp5, 59.8 kda; c (lane 1): ns1, 61.2 kda. lane m: protein marker. (b) western blot confirmation of tagged proteins: his-tagged recombinant btv proteins were detected using anti-his antibodies. lane m: protein marker; a (lane 1): cvp2, 9.35 kda; b (lane 1): vp5, 59.8 kda; c (lane 1): ns1, 61.2 kda. (c, d, e) protein-specific antibodies against btv cvp2, vp5 and ns1 in immunised mice. mice were vaccinated thrice with a 2-week interval using btv recombinant proteins (table 1). the cod values were calculated as (cod = od sample – od background) and compared with preimmunised and control group. several host systems were reported earlier in the successful expression of recombinant btv proteins, including baculoviral, bacterial, yeast and mammalian cells (mohd jaffar et al. 2014). in this study, we have succeeded in making use of the e. coli expression system, suggesting that the btv proteins can be expressed in simpler bacterial expression systems without the use of complex eukaryotic expression systems. despite the difficulties in protein expression (particularly for the larger btv proteins) often observed with the e. coli expression systems, such as formation of inclusion bodies, potential misfolding of proteins and lack of post-translational modifications, this system is preferred for its ease of handling and scalability of production of potential vaccine candidates. autoinduction depends on glucose catabolite repression and lactose induction to tightly control protein expression (studier 2005). because of the reduced need for sample processing, higher yields of the target protein and ease of culture scale up, this system when compared with isopropyl β-d-1-thiogalactopyranoside (iptg) induction is a very attractive method for achieving high throughput protein expression. specific antibody responses to the recombinant proteins in mice indirect elisa was used to evaluate specific antibody responses to cvp2, vp5 and ns1 in mice on day 40 post-immunisation. all three proteins induced protein-specific immune responses when compared with preimmunisation and control animal data (figure 1c, d and e). results support previous findings that vp2, ns1 and ns2 induced specific antibody responses in cattle, although the role that ns1 and ns2 antibodies play against btv infection was not studied (anderson et al. 2014). in a previous study, specific responses for two domains of vp2 encompassing amino acids 63–471 and 555–956 and for vp5 lacking the first 100 amino acids was observed (mohd jaafar et al. 2014); findings in this study added novel information of strong specific immune response to cvp2 encompassing 78 (position 337–415) amino acids. one limitation of this study was that, since the main objective here was to determine if the recombinant proteins were immunogenic, single dilution (1/200) was used in i-elisa, and so the antibody levels were not titrated. also, monitoring of antibody responses to the individual antigens in group 4 mice injected with a mix of the three proteins would also have provided better insights into understanding the proteins’ dynamics when administered together. cross-reactive neutralising antibody responses in mice by day 40 post-immunisation, all the mice (except control mice) showed detectable neutralising antibody (nab) levels by serum neutralisation test (table 2). serum neutralisation test analysis revealed that anti-cvp2 (group 1) antibodies could neutralise btv-1, -2, -4, -9, -10, -16, -21 and -23. high nab titres of log 3.1 were produced for btv-4, -9, -10 and -16, followed by a titre of log 2.5 for btv-21, titre of log 1.9 for btv-2 and log 1.6 for btv-1 and -23. no neutralisation was observed for btv-5, -12 and -24. anti-vp5 (group 2) antibodies produced high neutralisation titres of log 3.1 for btv-4, -9, -10, -16, -21 and -23, followed by titre of log 2.5 for btv-2, log 1.3 for btv-1 and no neutralisation of btv-5, -12 and -24. anti-ns1 (group 3) had nab titres of log 3.1 for btv-4, -16, -21 and -23, followed by a titre of log 1.6 for btv-9 and -10. absence of neutralisation was seen for btv-1, -2, -5, -12 and -24. sera from group 4 (cvp2+vp5+ns1) mice produced high neutralising titres of log 3.1 for btv-4, -10 and -16, followed by a titre of log 2.5 for btv-9, -21 and -23, titre of log 1.9 for btv-2 and titre of log 1.3 for btv-1. no neutralisation was observed for btv-5, -12 and -24. sera from animals vaccinated with the commercial inactivated vaccine (group 5) were found to neutralise btv-23 with titres of log 2.8; titre of log 2.5 for btv-1 and -2; titre of log 2.2 for btv-10, -12 and -16 and titre of log 1.9 for btv-4 and -9. no neutralisation was observed for btv-5, -21 and -24. inhibition of virus-induced cpe was not observed in the sera of the control animals (immunised with montanidetm isa 201 vg emulsion alone). table 2: serum neutralisation test antibody titres expressed as log10 values. neutralising antibody titres have been shown to be an essential component of the protective immune response against btv (anderson et al. 2014; huismans et al. 1987; roy et al. 1990). the neutralising ability of cvp2 strengthens the hypothesis that the cvp2 is immunologically significant corroborating the in silico findings of b-cell epitopes in the conserved region (shiva jyothi et al. 2018), thereby making a way for an important line of further investigation. to the best of our knowledge, this study presents the first report of immunisation of the cvp2 segment. however, there are reports where individual or a combination of virus proteins have been evaluated as vaccines for bt. recombinant vp2 and vp5 have been shown to elicit neutralising antibodies (with nab titres of up to log 2.408) and protect sheep against homologous btv challenge, but not a cross-reactive antibody response (anderson et al. 2013, 2014; huismans et al. 1987; mohd jaffar et al. 2014; roy et al. 1990). in this study, the results indicate a cross-reactive potential of cvp2, but not against all the tested serotypes. a maximum nab titre of log 3.1 was observed in immunised mice. in general, the non-structural btv proteins have predominantly been associated with cross-serotype cellular immune responses (andrew et al. 1995). ns1, being one of the most conserved btv proteins, was included in this study with the aim of detecting neutralising antibodies against btv serotypes and to check for its role (if any) in eliciting cross-neutralisation between serotypes. although its role in humoral immunity is not clearly known, ns1 has been shown to be a strong inducer of cytotoxic t lymphocytes (ctl) in sheep, inducing both homotypic and heterotypic t-cell responses (anderson et al. 2013; andrew et al. 1995). this could explain the neutralisation of fewer serotypes when compared with other groups and consistent low nab titres in the group injected with ns1 in this study. the major intention in including a group of mice injected with all the three recombinant proteins was to assess if there exists any additive effect in nab production. our findings indicate either an equivalent or less (even though not significant), but not higher, nab production against any of the btv serotypes tested. however, it cannot be ignored that the doses of recombinant proteins and the inactivated vaccine are not ideally comparable. interestingly, in the group vaccinated with the commercial inactivated vaccine harbouring btv serotypes btv-1, -2, -10, -16 and -23, cross-neutralisation with btv-4, -9 and -12 was noticed, albeit with low nab titres. further studies in the natural host may shed more light into the role of ctl responses and additive effects of ctl and humoral immune responses. high nab titres observed in this study with the recombinant proteins indicate the added advantage of the designed subunit vaccine’s potential adaptability over the commercial inactivated vaccine. the subunit vaccine using cvp2, vp5 and ns1 seems promising for further development as a btv vaccine that is safe, broad spectrum and potentially diva compliant. the potentiality of these recombinant proteins should further be evaluated in natural host along with challenge studies for it to be ultimately used as a vaccine candidate. conclusion in this study, we present the stages in the development and evaluation of a novel designed recombinant subunit btv vaccine model that is effective against multiple btv serotypes. the central hypothesis of this study was that the conserved epitope of btv vp2 protein, as part of a subunit vaccine, should confer broad-spectrum immunity against most of the available serotypes of btv. because the developed subunit vaccine with all three proteins was shown to induce a specific immune response to the administered proteins and could also neutralise a range of btv serotypes, we believe that the recombinant btv proteins cvp2, vp5 and ns1 are of potential significance. additional investigations on the determinants of the cellular immune response of these proteins and immunogenicity studies in the natural host with subsequent btv challenge will give better insights into the immune response generated by these vaccines. acknowledgements we are thankful to the ella foundation, turkapally, for their generous offer to carry out the expression studies of the research. competing interests the authors have declared that no competing interest exists. authors’ contributions s.j.j. performed experiments, analysed data and co-wrote the manuscript; s.r.p. assisted in performing experiments; y.n.r. reviewed the study protocol; r.p.p. served as a scientific advisor; u.m. provided technical help for protein purification; n.r.h. served as a scientific advisor and k.p. designed the experiment, supervised the research and co-wrote the manuscript. funding information this study was funded by the department of biotechnology project, government of india (sanction number: bt/pr9711/adv/90/158/2013; date 01 sept. 2016). data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references anderson, j., 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gel electrophoresis’, in b. hames (ed.), gel electrophoresis of proteins: a practical approach, pp. 1–50, oxford university press, oxford. shiva jyothi, j., putty, k., patil, s.r. & reddy, y.n., 2019, ‘in-silico approach to determine the possibility of a recombinant broad-spectrum vaccine for bluetongue disease’, indian journal of animal research 53(9), 1229–1233. http://www.indianjournals.com/ijor.aspx?target=ijor:ijar1&volume=53&issue=9&article=019 studier, f.w., 2005, ‘protein production by auto-induction in high density shaking cultures’, protein expression and purification 41(1), 207–234. https://doi.org/10.1016/j.pep.2005.01.016 world organisation for animal health, 2012, terrestrial animal health code, oie, paris. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) inge-marié petzer department of production animal studies, university of pretoria, south africa joanne karzis department of production animal studies, university of pretoria, south africa edward f. donkin department of animal and wildlife sciences, university of pretoria, south africa edward c. webb department of animal and wildlife sciences, university of pretoria, south africa eric m.c. etter department of production animal studies, university of pretoria, south africa department environment and societies, french agricultural research centre for international development (cirad), france citation petzer, i-m., karzis, j., donkin, e.f., webb, e.c. & etter, e.m.c., 2017, ‘somatic cell count thresholds in composite and quarter milk samples as indicator of bovine intramammary infection status’, onderstepoort journal of veterinary research 84(1), a1269. https://doi.org/10.4102/ojvr.v84i1.1269 original research somatic cell count thresholds in composite and quarter milk samples as indicator of bovine intramammary infection status inge-marié petzer, joanne karzis, edward f. donkin, edward c. webb, eric m.c. etter received: 22 may 2016; accepted: 27 nov. 2016; published: 24 mar. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the objective of the study was to establish an operational somatic cell count (scc) threshold to predict the presence of intramammary infection (imi) in composite milk samples and compare findings with those in quarter milk samples. south african dairy producers now preferred composite milk samples for herd udder health analysis because of increasing cow numbers, convenience of sampling and lower cost. a retrospective study was conducted on 345 461 composite and 89 638 quarter milk samples from south african herds. variance estimates for the proportion of quarter samples testing positive were adjusted to account for the lack of their independence within individual cows. the imi at scc thresholds of 150 000 cells/ml and 200 000 cells/ml differed only by 3.26% in composite milk samples. youden’s index indicated the optimum scc thresholds for composite and quarter milk samples as 150 000 cells/ml and 200 000 cells/ml, respectively. at 150 000 cells/ml, sensitivity (95% confidence intervals [ci]) in composite milk samples was 65.3% (64.0%, 66.6%) and specificity was 66.8% (65.7%, 67.9%); and in quarter milk samples, sensitivity at 200 000 cells/ml was 70.8% (69.5%, 72.0%) and specificity was 63.6% (62.4%, 64.8%). the likelihood of infection for udders and quarters, respectively, was 1.034 and 1.327 at an scc threshold of 150 000 cells/ml and 0.864 cells/ml and 1.177 cells/ml at 200 000 cells/ml. the area under the curve of the receiver operating characteristics graph was 0.7084 and 0.7277 for composite and quarter samples, respectively, indicating that the scc test could be considered as a good indicator of imi in both sample types. introduction bovine mastitis remains a major challenge and the disease responsible for most economic losses in dairy cows in developed countries despite improvements in management of subclinical mastitis over the past decade (geary et al. 2012). more than 80% of financial losses because of mastitis have been estimated to occur as a result of the subclinical form of the disease indicating that this should be the key focus in proactive udder health management (giesecke et al. 1994). there is a correlation between the bulk milk somatic cell count (bmscc) and the estimated percentage of cows with intramammary infection (imi) in a herd (smith, hillerton & harmon 2001). bmscc is useful for indicating milk quality, safety and suitability for manufacturing of dairy products (miller et al. 1993; reneau & packard 1991; sheldrake, hoare & mcgregor 1983) but it does not provide information about udder health of the individual cow. no consensus exists worldwide regarding the legal limits of bmscc in milk for human consumption. in the european union, australia, new zealand, canada and switzerland, the legal bmscc limit is 400 000 cells/ml; in south africa, 500 000 cells/ml; in the usa, 750 000 cells/ml; and in brazil, 1 million cells/ml (ruegg & pantoja 2013). somatic cell counts (sccs) from quarter samples have been generally accepted and used as the operational measure of inflammation of the bovine lactating gland since 1960 (barkema et al. 1999; biggs 2009; harmon 2001; heeschen 1996, 2010; schukken et al. 2003), as an indicator of the severity of imi (harmon 1994) and as an indicator of economic losses (degraves & fetrow 1993). the scc threshold level used to describe normal milk, imi and subclinical mastitis, however, has often been and still is a controversial subject (heeschen 2010). in an international dairy federation (idf) bulletin (kästli 1967), an udder quarter was considered to be normal when no pathogens were isolated and the scc was < 500 000 cells/ml milk. however, more recently, quarter milk samples with an scc of ≤ 100 000 cells/ml from which no microorganisms have been isolated and without a history of recent infection are considered to be normal (harmon 2001; smith et al. 2001), whereas those with an scc of < 200 000 cells/ml have been regarded as an indication of an inflammatory response and that the quarter is likely to be infected (dohoo & leslie 1991; laevens et al. 1997; smith et al. 2001; schepers et al. 1997). in 2001, the national mastitis council defined subclinical mastitis as an infected quarter with an scc ≥ 200 000 cells/ml in the absence of clinical changes to milk (smith et al. 2001) based on findings by degraves and fetrow (1993), harmon (1994) and hillerton (1999). this decision was endorsed at an idf world dairy summit in new zealand (harmon 2001), but it was agreed that a tolerance range of up to 400 000 cells/ml was necessary for practical reasons. further controversy still exists in literature regarding mastitis cure, as the term ‘cure’ may be used when describing only clinical cure of mastitis, clinical cure with reduced scc, or when clinical symptoms disappear, scc has reduced and mastitis pathogens are absent (hiitiö et al. 2012; roy et al. 2012; swinkels, schukken & cox 2012). the current quarter milk scc threshold of 200 000 cells/ml does not distinguish between principal udder pathogens although pathogens are known to differ in their pathogenic effects (barkema et al. 1999; pantoja, hulland & ruegg 2009; petzer et al. 2009, 2012). current information on scc threshold levels for pathogen-specific imi is needed because scc is known to differ between udder pathogens and over time (petzer et al. 2009, 2012; zadoks & fitzpatrick 2009). the average number of lactating cows per herd in south africa increased from 100 in 1997 to 238 in january 2015, and in provinces, it ranges from 76 in the northern cape to 769 in the eastern cape (lactodata 2013, 2015). quarter milk samples for routine udder health herd examination have been replaced by composite milk samples because of practical and financial reasons. in composite samples, milk from the four udder quarters is combined, with a consequent dilution effect that has required different interpretation of the results from quarter milk samples. although an international scc threshold has been established for the indication of imi in quarter milk, this has not been agreed for composite milk samples. identification of pathogens by phenotypical classification, biochemical analysis and genotyping is costly and may well become impractical in both small and large south african herds in future. knowledge of udder pathogens in herds remains of immense importance for a correct focus of proactive strategies and monitoring of udder health in dairy herds (ruegg 2011; schukken et al. 2003). early detection of imi will assist management, lessen the severity and duration of mastitis, reduce parenchyma damage, improve bacterial cure and lessen the risk and duration of bacterial shedding and development of new imi (giesecke, du preez & petzer 1994; schukken et al. 2012). a screening test is required to enhance the likelihood of identifying individual cows at risk of having imi within herds (reneau & packard 1991). such a test can be considered successful when it is simple, inexpensive and reproducible, widely available and with sufficient discriminatory power. scc was chosen in this study for the evaluation of composite milk samples, to be used as a screening test that could identify individual cows with possible imi. in this article, the incidence of imi in composite milk samples at various scc thresholds with indications of probable accuracy (sensitivity and specificity) and an ‘optimum’ scc threshold were evaluated. results of composite and quarter samples were evaluated and compared to results obtained at similar scc thresholds. materials and methods study design and study population a retrospective observational study was conducted on milk samples of lactating cows from south african commercial dairy herds over a period of more than 4 years (january 2008 till april 2012). samples originated from approximately 830 commercial south african dairy herds that submitted samples as part of their routine udder health monitoring programme and also from herds with increased bmscc who had sought help. the lactating cow numbers in herds tested varied from approximately 30–1700 cows, whereas their intervals for herd examinations ranged from monthly and three-monthly, to annually and bi-annually, or in some cases only a once-off test. data selection and sampling in the majority of cases, all lactating cows within herds were sampled and not only selected individuals or groups. a total of 386 031 composite cow and 95 228 quarter milk samples were initially under consideration for use, of which 10.51% and 5.87%, respectively, were defined as being unsuitable and were excluded from the data set. samples were regarded as unsuitable when any visible abnormalities such as dirt, floccules, blood or watery milk were detected. sample results were excluded from this study when cultures indicated contaminated, mixed bacterial growth or when there were doubtful scc results or missing identification information. the final data set comprised of 345 461 composite and 89 638 quarter milk samples. results were exported to and captured in the milk sample diagnostic (msd) computer program and were identified by producer, cow number, quarter position, date of processing and sample type. in most cases, information regarding parity, milk yield and days in milk was not provided. the msd program was developed over several years by abaci south africa for the milk laboratory at the university of pretoria (faculty of veterinary science) to assist in the analysis of milk samples received from dairy herds (petzer et al. 2016). milk samples were taken by professional samplers or milkers trained according to a standard operating procedure (giesecke et al. 1994). prior to sampling, first milk was stripped from all quarters and teat ends were carefully cleaned and disinfected with methylated alcohol. approximately 10 ml of foremilk was collected aseptically into sterile marked sample tubes and kept refrigerated until shipment. in the case of composite milk samples, the same procedure was followed, only approximately equal volumes of milk from each of the four quarters were collected in one sample tube. samples were transported on ice to reach the milk laboratory at the university of pretoria (faculty of veterinary science) within 48 hours after sampling. temperatures and conditions such as sample tubes cleanliness and appearance were noted on arrival at the laboratory, and samples which were spoiled or of doubtful quality were not processed. samples were plated out at the laboratory on the day of their arrival. laboratory methods microbiology and sccs were performed by the milk laboratory at the university of pretoria (faculty of veterinary science), on all milk samples under consideration for this study. milk was plated on bovine blood tryptose agar plates (oxoid, supplied by quantum biotechnologies [pty] ltd, ferndale, south africa). inoculated agar plates were incubated aerobically at 37 °c ± 1 °c and examined after 18–24 hours and 48 hours. colonies were initially identified based on colony morphology, haemolysis and potassium hydroxide test (koh) results (idf document 132). the catalase reaction was used to differentiate between staphylococci and streptococci. staphylase, a coagulase test (oxoid, supplied by quantum biotechnologies [pty] ltd, ferndale, south africa), was used to distinguish between coagulase positive (staphylococcus aureus) and coagulase-negative staphylococci (cns). the staph api test (biomerieux south africa [pty] ltd, randburg, south africa) was used for the identification of staphylococci. streptococci species were differentiated into the various lancefield groups using a strepkit (oxoid, supplied by quantum biotechnologies [pty] ltd, ferndale, south africa). gram-negative organisms were diagnosed using dnase and macconkey agar (oxoid supplied by quantum biotechnologies [pty] ltd, ferndale, south africa) and api 20e (biomerieux south africa [pty] ltd, randburg, south africa). imi was defined as a pure culture when only one species grew on an inoculum. growth of two distinct species was regarded as mixed growth, and a sample was considered to be contaminated when more than two species were present. samples with growth of two and more colonies from the same bacterial species were recorded (smith et al. 2001) except in the case of s. aureus where one and more colonies were recorded. somatic cells were counted by fluoro-opto-electronic means using a fossomatic 5000 (rhine ruhr, p.o. box 76167, wendywood 2144, south africa). data analysis in this study, all pure bacterial cultures isolated from milk samples were considered as imis. a further study, using the same data set investigated and analysed, results from 16 different udder pathogens. scc was compared with the presence and absence of organisms at 11 threshold levels. results were analysed at scc thresholds with increments of 50 000 cells/ml up to 500 000 cells/ml, and thereafter, the increments escalated to 750 000 cells/ml and greater. two-by-two tables were generated. true-positive and false-positive cases were defined as those milk samples that had scc above the level investigated and where true-positive cases were culture positive and false-positive cases were not. true-negative and false-negative samples were described as having scc levels below the level of investigation but with either imi (false negative) or without imi (true negative). the results were analysed using a genstat program (payne et al. 2012). the likelihood ratios were determined using the category-oriented likelihood method (moosapour et al. 2011). in the case of composite milk samples, sensitivity, specificity and 95% confidence intervals (ci) of binomial proportions around these estimates were determined using the mid-p exact test from the openepi freeware program. quarter samples cannot be considered to be independent but rather clustered within cows (potentially multiple sample sets per cow) (ampe et al. 2012). sample dependency of scc values within each cow was modelled to take into account individual cow factors that may affect quarter samples. an estimator of cluster variance was determined as described by scheaffer, mendenhall and ott (1996). the 95% confidence intervals were adjusted for clustering by using the design effect (cluster variance or sample random sample variance) (scheaffer et al. 1996): where = the estimated proportion of unweighted quarters with imi, n = the number of cows, ai = the number of quarters with imi per cow and mi = the number of quarters tested per cow. the estimated variance of the portion [] of quarters with imi was calculated using the following formula (scheaffer et al. 1996): the area under the curve (auc) of the receiver operating characteristic (roc) curves was used to estimate the discriminative power of the scc measurement over the range of possible thresholds and was calculated with the trapezoid method (perkins & schisterman 2006). the best threshold point for balancing the sensitivity and specificity ratios of the scc, the one closest to the (0.1) point, was taken as the threshold point that best differentiated between those udders (composite milk) or quarters with imi and those without. youden’s index which indicates the maximum distance between the roc curve and the diagonal chance line was calculated by subtracting 1 from the sum of the maximum sensitivity and specificity. results composite milk samples the scc threshold levels of primary interest initially focused on in this study were 100 000 cells/ml, 200 000 cells/ml and 400 000 cells/ml for they were the levels in quarter milk samples that have been recommended by the international mastitis council (imc) and at a dairy world summit. because scc threshold levels used on the various farms may well differ from those recommended by the imc, other scc ranges were also investigated in an attempt to minimise diagnostic error under different circumstances. the percentage of imi in this data set of 345 461 composite milk samples was 43.37% (table 1). the percentage of samples with imi at scc threshold levels of 100 000 cells/ml, 200 000 cells/ml and 400 000 cells/ml was 10.89%, 18.31% and 26.26%, respectively. of the 149 817 culture-positive samples, 57.78% had scc in excess of 200 000 cells/ml milk compared with 26.67% of culture-negative samples. the ratio in composite samples of culture-negative to culture-positive samples was 2.960, 2.268 and 1.821 to 1 at scc thresholds of 100 000 cells/ml, 200 000 cells/ml and 400 000 cells/ml, respectively (table 1). table 1: the presence and absence of intramammary infection within each somatic cell count range in composite milk samples. sensitivity of scc thresholds as an indicator of imi in composite samples was 87.0% with 95% upper and lower confidence intervals of (85.5%, 88.5%) at the lowest scc range investigated (50 000 cells/ml) but dropped noticeably as scc increased to 74.9% (73.5%, 76.3%) at 100 000 cells/ml, 57.8% (56.6%, 59.0%) at 200 000 cells/ml and 39.2% (38.2%, 40.2%) at 400 000 cells/ml scc threshold ranges. at the 200 000 threshold, the specificity was 73.3% (72.2%, 74.5%) (table 2). the likelihood ratios for culture-negative samples ranged from 3.039 at 50 000 cells/ml to 0.468 at 750 000 cells/ml, whereas the likelihood ratio became larger than 1 at the 150 000 cells/ml scc range. the youden’s index that balances sensitivity and specificity to obtain a mathematical optimum scc threshold was indicated at 150 000 cells/ml in composite samples with a sensitivity of 65.3% (64.0%, 66.6%) and specificity of 66.8% (65.7%, 67.9%) (table 2). table 2: analysing intramammary infection in composite milk samples at different somatic cell count thresholds. quarter milk samples of the 89 638 quarter milk samples analysed, 43 746 (48.8%) had an scc of above 200 000 cells/ml. the percentage of quarter milk samples in this data set from which microorganisms were isolated was 33.99% representing 30 467 milk samples (table 3). the prevalence of imi at various scc ranges was determined with standard errors at 95% confidence intervals. at scc ranges of 100 000 cells/ml, 200 000 cells/ml and 400 000 cells/ml, the prevalence (upper and lower 95% ci) of imi was 20.4% (19.7%, 21.2%), 29.1% (28.3%, 30.9%) and 41.1% (38.9%, 43.3%). table 3: quarter milk samples indicating the presence and absence of intramammary infection for each somatic cell count range. the percentage of quarter milk samples with imi was 18.73% at 200 000 cells/ml and 22.14% at 400 000 cells/ml. (table 3). the ratio of quarter samples without imi to those with imi remained positive up to an scc level of 750 000 cells/ml. only at scc above 750 000 cells/ml, there was a clear negative ratio of uninfected to infected samples. although discriminative measures are mostly used to make policy decisions, predictive measures are most useful in predicting the probability of a disease in an individual. the likelihood ratio for identifying culture-negative samples at various scc thresholds ranged from 3.145 more likely at 50 000 cells/ml to 0.446 at 750 000 cells/ml. the youden’s index at 100 000 cells/ml was 0.311 and increased to the highest values of 0.344 and 0.345 at scc thresholds of 200 000 cells/ml and 250 000 cells/ml quarter milk (table 4). the youden’s index ranges from 0 for a poor to 1 for the perfect test indicating the best balance between the sensitivity and specificities of tests. table 4: sensitivity and predictability of quarter intramammary infections for various somatic cell count thresholds. the result of the auc of the roc that expresses the discriminative power of the ssc test to identify imi in both composite and quarter milk samples was 0.7084 in composite and 0.7277 in quarter milk samples (table 5). the closer the auc is to 1, the higher the diagnostic accuracy of the test. table 5: receiver operating characteristic curves and area under the curve for the efficacy of somatic cell count test to identify intramammary infection in quarter and composite milk samples. discussion the average number of cows in south african dairy herds has increased especially in the eastern cape and natal provinces (lactodata 2015) with some herds exceeding 1700 cows. in order to be able to make effective decisions at herd and individual cow level, all lactating cows in a dairy herd were tested both for microbiology and scc in the milk laboratory at the university of pretoria (faculty of veterinary science). this is, however, not the system used in other south african laboratories. owing to the practical difficulty of sampling all cows in the large herds and analytical costs, only cows previously identified with high scc counts or those found to have high scc in the present test would be cultured. in some laboratories, the chosen scc threshold for culturing may be as high as 500 000 cells/ml milk (personal communication). information of all four quarters, all with possible different infection status, pathogen strains and inflammatory responses, is amalgamated into a composite milk sample. these results can be expected to be less informative and the values less sensitive than those from quarter milk samples. the current study has indicated that at a 500 000 cells/ml scc threshold, 66.18% and 45.52% of imi may be missed in composite and quarter milk samples (tables 1 and 3). few test results in a laboratory are either simply positive or negative, and there is usually a gradual change between results from those considered to be normal to what may be abnormal. dohoo and leslie (1991) indicated that an scc threshold of 200 000 cells/ml of milk was not an absolute value but the scc threshold was associated with probability of imi. milk samples with scc below that threshold could not be considered to be culture negative, but they were only more likely to be associated with no imi. it was in this spirit that this large data set was analysed in order to quantify the likelihood of predicting the culture-negative samples as a whole at various scc thresholds. this data set provided an overview of the on-farm operational conditions experienced by south african commercial dairy herds at the time. it did not attempt to distinguish between farming systems, management variations and levels, dairy breeds, cow age, parity, days in milk, quarter position, yield or stress factors originating from environmental, milking machine use, maintenance or machine settings, possible nutrition short comings, social stress or systemic diseases that may challenge the immunity of dairy cows. this study was intended to serve as an initial study to identify the relation between imi in general and scc levels, whereas the second part of this study will report on scc finding regarding major and minor pathogen groups as well as for 16 specific udder pathogens. failure to isolate bacteria from quarter samples with high scc does not necessarily always indicate that the udder or quarter has no imi. bacteria may be missed because of very low concentrations present in the milk (ruegg & pantoja 2013). other studies have found that in 10% – 25% of cases of quarters with high scc where they failed to isolate bacteria, the possibility existed that bacterial numbers were too low to detect with the laboratory technique (volume of milk used) or bacteria were excreted intermittently by the udder, as is known to occur with s. aureus imi (dohoo & leslie 1991; pantoja et al. 2009; schepers et al. 1997). schepers et al. (1997) found that 50.2% of the variation in scc could be explained by the presence of imi. some other factors also known to increase scc are days in milk, month of sampling, udder quarter position, parity, interaction between stage of lactation and parity (schepers et al. 1997). scc is also known to increase in the absence of imi during stressful events such as extreme environmental temperatures. transportation stress has been shown to change the peripheral blood neutrophil function by enhancing their migration capacity across the blood udder barrier with a consequent increase in scc (yagi et al. 2004). wegner et al. (1976) reported an increase in both blood and milk leucocytes concentrations following heat stress. green et al. (2006) in two consecutive summers found increased proportions of cows with scc above 200 000 cells/ml without evidence of higher imi and considered that heat stress was presumably responsible for 70.8% of the overall scc increase. in such situations, it may not be beneficial to treat cows with intramammary antimicrobials. quarter milk samples the most accurate relationship between imi and scc exists where quarter milk samples are taken, and reduced milk yields have been observed and when scc has exceeded 100 000 cells/ml (schukken et al. 2003). sccs of milk from healthy quarters have been found to be consistently low, and a threshold of below 200 000 cells/ml has been proposed to be the most practical level for defining udder health (dohoo & leslie 1991; pantoja et al. 2009; schepers et al. 1997). in this study, the percentage of quarter milk samples with imi at 100 000 (15.59%) and at 200 000 cells/ml (18.73%) differed only slightly indicating a reduced risk of missing imi when using a threshold of 200 000 cells/ml, compared to a threshold of 100 000 cells/ml. furthermore, over 63.03% of all quarter samples tested were culture negative at the 200 000 cells/ml scc threshold, indicating the usefulness of scc as a screening test in quarter milk samples. compared with similar studies by eberhart et al. (1979) and malinowski et al. (2006), the percentage of uninfected quarters in this study at threshold levels of 100 000 and 200 000 cells/ml was noticeably higher than their findings of 50.0% and 59.6%, respectively. although the sensitivity ratio predicting accuracy in identifying imi at 50 000 cells/ml was relatively high at 88.9% with 95% upper and lower confidence intervals of (87.0%, 90.7%), it reduced rapidly to 81.9% (80.8%, 83.0%), 70.8% (69.5%, 72.0%) and 57.3% (56.1%, 58.1%) at scc levels of 100 000 cells/ml, 200 000 cells/ml and 400 000 cells/ml, respectively. these sensitivity ratios, although slightly lower, compared favourably with those found by schepers et al. (1997) of 83.2%, 74.5% and 60.8% at similar scc thresholds. the specificity that predicted accuracy of culture-negative results increased from a low of 49.2% (49.0%, 50.3%) at 100 000 cells/ml to 75.3% (74.1%, 76.5%) at 400 000 cells/ml (table 4). specificity values reported by this study for scc ranges of 100 000 cells/ml, 200 000 cells/ml and 400 000 cells/ml were, however, markedly lower than the 80.5%, 89.6% and 95.0% reported by schepers et al. (1997). hillerton (2000) proposed sensitivity of 80% and specificity of 99% as appropriate target values for screening tests, but these were clearly impractical in this study, even for quarter milk samples. the 95% upper and lower ci for sensitivity and specificity that was adapted for clustering varied little for all scc threshold levels, indicating that cow factors had little or no influence on the scc levels of the quarters in this study, but it should be taken into consideration that this could possibly have been masked by the large data set. clinicians are often more interested in the predictive values of a test indicating a disease than in the sensitivity or specificity ratios of the test. ruegg and pantoja (2013) found positive predictive values relatively poor for indicating recovery from imi when using the 200 000 scc thresholds. the likelihood ratio (table 4) obtained in this study confirmed that finding of ruegg and pantoja (2013). the likelihood ratio 1.177 at an scc level of 200 000 cells/ml increased to below 1 only at an scc threshold of 300 000 cells/ml. a likelihood ratio of < 1 indicates that the scc level is more commonly associated with lack of imi than with imi. although the highest youden’s index (0.345) was found to be at an scc threshold of 250 000 cells/ml in quarter milk, the threshold of 200 000 cells/ml with an almost similar value (0.344) was selected. this was done because of its higher sensitivity (70.8% vs. 66.7%) at an scc of 200 000 cells/ml with the aim of identifying cases with imi rather than those without (table 4). even the highest values obtained with the youden’s index were closer to 0 than 1 indicating only fair accuracy. the efficacy of the scc test for indicating imi in quarter milk was found to be good but not excellent when the auc in the roc curve was determined at 0.7277 (table 5). different populations, for example, younger cows or herds with a low or high bmscc, may justify the use of different scc thresholds for screening purposes. when the prevalence of imi in a herd is high, the scc threshold used may shift towards higher specificity levels, in order to identify the true-negative cows, whereas sensitivity might be of greater value in identifying true-positive cases in herds where imi prevalence is low. thus prior to analysing the data for practical operations, clarity should be obtained as to the requirement for that specific dairy herd. the operational threshold selected should depend on what the practitioner wants to achieve with this information and what is therefore the most important aspect to optimise. one of the first questions to answer is whether it is more important to identify samples with imi or samples without imi. because sensitivity and specificity ratios are inversely related in most cases, one of the two will be favoured when setting a threshold in a test. when the aim is to eradicate imi with streptococcus agalactiae from a herd through separation and treatment of cows with infected udders, for instance, it could be beneficial to test all cows in the herd, even if the prevalence is low, or to set a low threshold because of the low sensitivity at higher scc levels. when culling is the purpose for testing, as may be the case with s. aureus, the scc threshold may be increased to high levels such as 800 000 cells/ml (sol et al. 1997; swinkels et al. 2012). in the latter case, the scc test should rather be used as a screening and not a diagnostic test to select cows for further bacterial analysis. when a decision needs to be made whether subclinical imi with s. aureus should be treated with the scc level, the number and position of quarters infected, parity, days in milk and treatment regime known, the probable outcome of treatment success can be calculated (sol et al. 1997). knowledge of cows or quarters infected with cns may be of little to no benefit and the cost of further testing may not be justified. cows with cns are neither treated nor separated when infected. it may therefore be beneficial to use variable scc thresholds when evaluating dairy herds depending on the main udder pathogens isolated from that herd. composite milk samples few studies have been conducted on composite milk samples compared with the number of studies conducted investigating scc and imi in quarter milk samples. a composite sample is a combination of milk from the four udder quarters, and it is to be expected that the sensitivity and specificity of this sample type should differ at similar scc levels from that in quarter milk samples. in this study, composite milk samples of 52.91% and 59.84% had scc ≤ 150 000 cells/ml and ≤ 200 000 cells/ml, respectively, and 15.05% and 18.31% were culture positive at the same scc levels (table 1). this is a disturbingly high percentage if only viewed from a point of possible misdiagnosis of imi when choosing either of these scc thresholds. however, it needs to be considered that a substantial portion of these imi may be minor pathogens and cns. the proportion of major pathogens and contagious species isolated from low scc was not examined in this article, but will be reported in a subsequent article. sensitivities of the scc test for the composite milk samples were 65.3% with 95% ci of (64.0%, 66.6%) at 150 000 and 57.8% (56.6%, 59.0%) at 200 000 cells/ml and decreased to 25.5% (24.7%, 26.3%) at the highest scc range. these results indicated that scc for composite milk samples at 200 000 cells/ml was less able to detect imi than at scc for quarter milk samples at 200 000 cells/ml where the sensitivity was 70.8%. although still less precise, results of the test at scc of 150 000 cells/ml were more comparable with the results obtained in quarter milk samples (tables 2 and 4). specificity and the ability to detect culture-negative samples accurately using scc were higher in composite samples than quarter milk samples. it increased from 66.8% (65.7%, 67.9%) at the scc threshold of 150 000 cells/ml to 73.3% (72.2%, 74.5%) at 200 000 cells/ml compared to 63.6% (62.4%, 64.8%) for quarter milk sample at an scc threshold of 200 000 cells/ml (tables 2 and 4). dohoo and leslie (1991) reported in composite samples overall higher sensitivity (83.4%) and slightly lower specificity (58.9%) for major pathogens at 200 000 cells/ml. the scc threshold point in composite samples where sensitivity and specificity were evenly balanced using the youden’s index was indicated at 150 000 cells/ml (table 2). this was found to be the optimum mathematical scc threshold to identify imi when examining composite samples without including any further information such as cow and environmental information. the likelihood ratios were lower in composite samples than those at similar scc thresholds in quarter milk samples. at 150 000 cells/ml and 200 000 cells/ml, the likelihood ratios to detect culture-negative composite samples were 1.034 and 0.864 compared to 1.177 in quarter samples at 200 000 cells/ml (tables 2 and 4). comparing quarter and composite cow milk sample results the percentage of samples from which organisms were isolated at a threshold of 200 000 cells/ml was surprisingly similar in composite (18.31%) and quarter (18.73%) milk samples (tables 1 and 3). it was, however, possible that the dilution effect of the composite milk sample caused more imi to be at a subminimum level of detection for conventional laboratory microbiological detection systems (dohoo & leslie 1991; pantoja et al. 2009; schepers et al. 1997). for every one infected composite or quarter milk sample at an scc threshold of 50 000 cells/ml, 3.969 composite and 6.589 quarters were uninfected, respectively. at an scc threshold of 200 000 cells/ml, the ratio of infected to uninfected was 1 to 2.268 in composite milk samples compared to 1 to 4.340 in quarter milk samples (tables 1 and 3) making it more likely to correctly identify quarters with imi using scc. although specificity of the scc test was higher in composite milk samples at a threshold of 200 000 cells/ml than for quarter milk samples, this difference between the sample types remained fairly constant at all scc levels tested. sensitivities of scc for composite and quarter samples, however, started at a similar level at the 50 000 cells/ml scc threshold (87.0% and 88.9%) but differed increasingly as the scc level increased. sensitivity decreased more rapidly in composite samples to 25.5% and quarter samples to 44.9% at the high scc level (tables 2 and 4). an ‘optimum’ threshold was sought for the scc test that could indicate imi in milk samples at a level effective for screening of samples to be cultured. in composite milk samples, the best threshold level proved to be 150 000 cells/ml, whereas scc threshold levels from 200 000 cells/ml were indicated for quarter milk (tables 2 and 4). this result is in agreement with the 200 000 cells/ml threshold recommended by the national mastitis council when using the scc test to indicate the presence of imi in quarter milk samples (smith et al. 2001). the auc was found to be 0.7084 in composite and 0.7277 in quarter samples indicating that scc was a good but not an excellent test for indicating imi in both sample types (table 5). conclusion the large data set used in this study was a reflection of actual samples received by the laboratory for analysis and interpretation. samples were received from herds that differed in management levels, exposure to environmental conditions, microbial imi and cow factors (age, parity, days in milk and milk yield). the scc test was found to be good but not excellent as an indicator of imi in both quarter and composite sample types. the current threshold for scc of 200 000 cells/ml used to detect only imi in quarter milk samples was reconfirmed to be optimal. based on the findings of this study, an scc threshold of 150 000 cells/ml milk can be recommended for use in composite milk samples and could be used in practice as a selection criteria to select samples for culturing in large herds. this study indicated that the likelihood of identifying culture-negative samples at an scc threshold of 200 000 cells/ml in quarter milk samples was 1.177 compared with 1.034 in composite milk samples at an scc threshold of 150 000 cells/ml. although less true culture-negative milk samples can be expected to be identified for composite milk compared to quarter milk samples because of a lower sensitivity of the scc test in composite samples, it was indicated that scc could be used at the lower threshold level to indicate imi in composite milk samples. it should be noted, however, that these optimal statistically determined scc threshold levels need to be adapted depending on specific operational circumstances. even at these ‘optimal’ scc levels, using the scc as the only test to predict the presence of imi is helpful as screening test to select samples for microbiological determinations, but is not ideal as a stand-alone test to indicate imi. acknowledgements we gratefully acknowledge the milk producers of south africa for their continuous use and support of the milk laboratory and for allowing us to use the data to gain knowledge. many thanks to the staff of the milk laboratory of the department of production animal studies, faculty of veterinary science, onderstepoort, for their continued quality work: mrs jc watermeyer, f konaite, n labuschagne, r ludike, miss r badenhorst, p malokotsa, k madiba, mr ll mohapi and k malekane for performing all technical work and data logging; mrs m. smith for data management and prof. gf fosgate for his assistance with the model to determine estimators of cluster variance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions i.m.p. developed the concept of the article, performed data analysis, wrote the manuscript and oversaw all technical work and data capturing. j.k. assisted with literature search and with the overall structure, verified data and edited the manuscript. e.f.d. assisted with the overall structure, interpreted data and edited the manuscript. e.c.w. assisted with the overall motivation and edited the manuscript. e.m.c.e. assisted with the data analysis. references ampe, b., goethals, k., laevens, h. & duchateau, l., 2012, ‘investigating clustering in interval-censored udder quarter infection times in dairy cows using a gamma frailty model’, preventive veterinary medicine 106, 251–277. https://doi.org/10.1016/j.prevetmed.2012.04.004 barkema, h.w, van der ploeg, j.d., schukken, y.h., lam, t.j.g.m., benedictus, g. & brand, a., 1999, ‘management style and its association with bulk milk somatic cell count and rate of clinical mastitis’, journal of dairy science 82, 1655–1663. https://doi.org/10.3168/jds.s0022-0302(99)75394-4 biggs, a.m., 2009, ‘optimising the use of scc information’, in proceedings of the 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62(suppl.), 59–70. https://doi.org/10.1186/2046-0481-62-s4-s59 abstract introduction materials and methods phenotypic characterisation of bacteria antibiotic susceptibility test detection of extended spectrum β-lactamase production results discussion conclusion acknowledgements references about the author(s) samuel n. akpan department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of ibadan, ibadan, nigeria olubusola a. odeniyi department of microbiology, faculty of sciences, university of ibadan, ibadan, nigeria oluwawemimo o. adebowale department of veterinary public health and preventative medicine, college of veterinary medicine, federal university of agriculture, abeokuta, nigeria selim a. alarape department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of ibadan, ibadan, nigeria olanike k. adeyemo department of veterinary public health and preventive medicine, faculty of veterinary medicine, university of ibadan, ibadan, nigeria citation akpan, s.n., odeniyi, o.a., adebowale, o.o., alarape, s.a. & adeyemo, o.k., 2020, ‘antibiotic resistance profile of gram-negative bacteria isolated from lafenwa abattoir effluent and its receiving water (ogun river) in abeokuta, ogun state, nigeria’, onderstepoort journal of veterinary research 87(1), a1854. https://doi.org/10.4102/ojvr.v87i1.1854 original research antibiotic resistance profile of gram-negative bacteria isolated from lafenwa abattoir effluent and its receiving water (ogun river) in abeokuta, ogun state, nigeria samuel n. akpan, olubusola a. odeniyi, oluwawemimo o. adebowale, selim a. alarape, olanike k. adeyemo received: 18 feb. 2020; accepted: 25 june 2020; published: 15 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract untreated abattoir effluent constitutes potential reservoir for transmission of pathogenic strains of multiple antibiotic-resistant bacteria by pollution of surface and ground water sources. this study was carried out to determine the antibiotic resistance and extended spectrum β-lactamase (esbl) production profiles of gram-negative bacteria isolated from effluent collected from lafenwa municipal abattoir and its receiving surface water, ogun river, in abeokuta, ogun state, nigeria. twelve effluent and 18 water samples were collected for this study. total heterotrophic and coliform counts were estimated, bacterial identification was performed using standard culture-based procedures, whilst antibiotic resistance profiles of isolated bacteria against five antibiotics (ceftazidime, cefpodoxime, cefotaxime, ertapenem and amoxicillin-clavulanate) and detection of esbls were done using disk diffusion and double-disc synergy tests. a total of 54 gram-negative bacteria were isolated, including salmonella spp. (9), escherichia coli (15), klebsiella spp. (7), shigella spp. (5), pseudomonas spp. (12) and enterobacter spp. (6). both enterobacteriaceae and pseudomonas isolates (31% and 66.6%, respectively) were resistant to all selected antibiotics except ertapenem (98% susceptibility). overall, 77% isolates had multiple antibiotic resistance index (mari) values, but none of the antibiotic-resistant isolates showed evidence of esbl production. the presence of multiple antibiotic-resistant isolates in the effluent and receiving water of lafenwa abattoir suggests a major risk to public health and food safety. current methods of waste disposal at the abattoir are unacceptable and greatly reduce the qualities of the processed meat and contaminate the environment. there is a need for improved abattoir waste management and water treatment strategies. keywords: antibiotic resistance; multiple antibiotic resistance index; gram-negative bacteria; abattoir effluent; surface water. introduction the water ecosystem is often degraded when the inherent potential of a water body to purify itself is lowered; hence, a river or stream is said to be polluted (denise & john 2014). pollution is the presence of significant quantity of contaminants in the environment. waste management, hygiene and sanitation in nigeria pose a big challenge, especially with many complex issues intertwined with much larger sociocultural problems (adedeji & adetunji 2011). the adequate facilities to ensure safe disposal of abattoir wastes in a manner that will not constitute a potential hazard to public, animal and environmental health are considered very essential and still underdeveloped in many developing countries (nafarnda et al. 2012). moreover, abattoirs in nigeria have no facilities for waste treatment; wastes are either disposed on open dumps or are discharged into nearby streams, hence constituting an environmental menace (adeyemo 2002). in particular, the lafenwa municipal abattoir effluents and wastes are channelled into ogun river, which is used by indigenes of surrounding communities for drinking and domestic purposes, without prior treatment to remove bacteria and other noxious substances (adebowale et al. 2016). abattoir effluent contains several million colony forming units (cfu) of total aerobic bacteria count and faecal coliforms (omole & longe 2008). several previous studies have reported the isolation of pathogenic bacterial and fungal species, such as staphylococcus aureus, streptococcus, salmonella spp., escherichia coli, aspergillus, mucor, saccarhomyces spp. and penicillium spp., from abattoir wastewater (adesomoye, opera & makinde 2006; adebowale et al. 2016; coker, olugasa & adeyemi 2001). isolated pathogens threaten public and environmental health by introducing resistant strains or variants, which could migrate into ground or surface water, animals and other vectors. multiple antibiotic-resistant enterobacteriaceae from waste water and sludge of slaughterhouses have also been documented and intestinal contents/faeces of slaughtered animals at abattoirs, such as sheep, goats and cattle, are significant reservoirs (martins da costa, vaz-pires & bernardo 2006; um et al. 2016). multidrug resistance especially against antibiotics is becoming an emerging challenge to public, animal and environmental health, calling for its strict judicious and stewardship use in food animals. although few studies have reported the bacteriological assessment of lafenwa abattoir effluent and its receiving water, none have focused on the antibiotic-resistant potential of these pathogens. hence, this current study aimed at investigating this gap. materials and methods study area the study area is located in abeokuta north local government area, ogun state, nigeria, with a total land mass of 808 km2 and a population of 201 329 (nigeria population commission [npc] 2006). the lafenwa municipal abattoir is situated on geographical coordinates 7° 10′ 0″ n and 3° 3′ 0″ e. the abattoir is a major slaughtering facility in ogun state where a total number of 150–200 cattle are slaughtered daily for public consumption, and waste effluents are discharged directly into an adjoining water body (ogun river). the slaughtering activities commence from 06:00 to 12:00 every day, except on sundays (adebowale et al. 2016). sample collection effluent and water samples were collected between may and june 2019. at the abattoir, sampling points were divided into point a (channel draining the trippery and guttery section) and point b (main outflow channel discharging into the river) (adebowale et al. 2016). on the river, the sampling points were divided into upstream (75 m before the point of inflow of abattoir effluent into the river), midstream (at the point of abattoir effluent release into river) and downstream (75 m after the point of effluent inflow) (adebowale et al. 2016). in total, 12 effluent and 18 water samples were collected fortnightly over a 6-week period. collected samples were transported on ice to the laboratory for microbial analysis. bacterial isolation bacteria were isolated according to the process defined by harrigan and mccance (1976). firstly, 9 ml of sterile distilled water was dispensed into seven test tubes. then, 1 ml of the effluent was dispensed into the first test tube and serially diluted at a ratio of 1:10. water samples from the receiving surface water body were diluted serially at a ratio of 1:7. about 1 ml of each of the effluent and water samples was inoculated on aseptically prepared macconkey agar (ma) (himedia laboratories pvt. ltd, mumbai, india), eosine methylene blue (himedia india) and salmonella shigella agar (himedia india) using the pour plate technique. plates were incubated at 35 °c ± 2 °c for 18 –24 hours. the total heterotrophic and coliform counts were determined on nutrient agar (na) and ma plates. the maximum limit of heterotrophic count for drinking water is 1.0 × 10² cfu/ml (environmental protection agency [epa] 2002), whereas the maximum limit for wastewater is 400 cfu/ml. morphologically distinct colonies were sub-cultured twice on corresponding media. pure colonies obtained were stored both on na slants and nutrient broth containing 15% glycerol (volume/volume [v/v]), for further investigation. phenotypic characterisation of bacteria the pure isolates were characterised based on gram status using the potassium hydroxide water (koh) test. using a sterile loop, a colony of overnight bacterial culture was gently mixed with a drop of 3% koh (v/v) on a clean microscope slide. the loop was gently raised from the koh-bacterium mixture to a height of 1 cm. a viscous drawing reaction indicated positivity of the test (gram-negative reaction), and a non-viscous reaction showed that the isolate was gram-positive (powers 1995). furthermore, biochemical characterisation of bacteria isolates was conducted using the catalase, urease, methyl red, voges proskauer, citrate and sugar fermentation tests. antibiotic susceptibility test the disc diffusion technique according to bauer et al. (1996) was conducted to determine the antibiotic susceptibility pattern of isolates. firstly, one colony of overnight bacterial culture was inoculated into normal saline and standardised to 0.5 mcfarland. then, previously prepared sterile mueller hinton (mh) agar plates (biolab, wadeville, germiston, south africa) were inoculated, carefully spread over the entire surface of the agar using sterile swab and allowed for 5 min to dry. using a sterile forceps, following antibiotic discs – cefotaxime, cefpodoxime, amoxicillin-clavulanate, ceftazidime and ertapenem were placed onto the surface of the mh agar, and incubated at 35 °c ± 2 °c for 18–24 h. the zone of inhibition was read post-incubation and recorded following the clinical and laboratory standards institute guidelines (clinical and laboratory standards institute [cli] 2014). isolates showing resistance to any of the third-generation cephalosporins were further investigated for extended spectrum β-lactamase (esbl) production. detection of extended spectrum β-lactamase production the detection of esbls producing bacteria was carried out using the double-disc synergy test kit, following the clinical and laboratory standards institute (clsi) (2014) guidelines. bacteria isolates resistant to any of the third-generation cephalosporin were standardised to 0.5 mcfarland. subsequently, the standardised broth was gently spread over mh agar plates, and three antibiotics (ceftazidime, cefpodoxime and cefotaxime) were placed at a distance of 20 mm from amoxicillin-clavulanate (augmentin) disc, which was placed at the centre. the plates were incubated at 35 °c ± 2 °c for 18–24 h. at the end of the incubation period, the plates were observed for isolates showing enhancement towards the augmentin disc. calculation of multiple antibiotic resistance indices the multiple antibiotic resistance indices (mari) were calculated according to the formula described by krumperman (1983): where a = total number of antibiotics to which an isolate shows resistance b = total number of antibiotics to which the isolate was exposed. ethical considerations this article followed all ethical standards for a research without direct contact with human or animal subjects. results the mean total heterotrophic and coliform counts of both the effluent from the two points and water samples from ogun river were estimated and are presented in table 1. the mean total heterotrophic counts (thc) for the effluent from sampling point b were higher (30.0 × 106 cfu/ml) than that of sampling point a (13.5 × 106 cfu/ml), with both points exceeding the maximum limit of 400 cfu/ml for wastewater. also, thc for all the water samples collected from ogun river exceeded the maximum limit of 1.0 × 10² cfu/ml for drinking water. table 1: mean total heterotrophic and coliform counts of the abattoir effluents and its receiving water body (ogun river). pathogenic bacteria including salmonella, pseudomonas, enterobacter, klebsiella and shigella were isolated from water sources used for meat processing and effluents of the abattoir. the frequency of occurrence of different types of bacterial species isolated from the abattoir wastewater is presented in figure 1. figure 1: frequency of occurrence of isolated bacterial species in the abattoir wastewater. the antibiotic sensitivity and resistance patterns of isolated gram-negative bacteria were determined and are presented in figures 2–4. the enterobacteriaceae isolates showed resistance to amoxicillin-clavulanate (4.76%), cefpodoxime (7.14%), cefotaxime (11.90%), ceftazidime (4.76%) and ertapenem (2.38%). a total of 16.6% of pseudomonas isolates were resistant to amoxicillin-clavulanate, 25% to cefpodoxime, 16.6% to cefotaxime and 8.33% to ceftazidime. meanwhile, pseudomonas isolates were 100% sensitive to ertapenem, suggesting a high potency of action of that class of antibiotics (carbapenems) against pseudomonas organisms. figure 2: cultured agar plates of isolates with zones of bacterial inhibition (l21, r32) and escherichia coli resistance to antibiotic discs (r30). figure 3: antibiotic sensitivity and resistance pattern for enterobacteriaceae isolates. figure 4: antibiotic sensitivity and resistance pattern for pseudomonas isolates. the mari of isolates against the selected antibiotics are presented in table 2. the study results showed that 77% mari of the bacterial isolates were between values 0.2 and 1 (table 2). table 2: multiple antibiotic resistance indices of isolates to selected antibiotics. discussion this study revealed high heterotrophic and coliform counts in both the effluents from lafenwa abattoirs and the receiving water body (ogun river). the results of this study are similar to those of umunnakwe, akagba and aharanwa (2013) who assessed the bacterial contents of the aba river, where bacterial counts exceeded the permissible standards. a study by adebowale et al. (2016), which investigated potential bacterial zoonotic pathogens at lafenwa abattoir, also showed that mean total bacteria and coliform counts for the waste water and the receiving surface water exceeded the epa (2002) and world health organization (who) (2004) maximum recommended and permissible limits of 400 cfu/ml and 200 cfu/ml, respectively. the high heterotrophic counts in the effluent and water samples indicated the presence of high levels of dissolved salts and organic micronutrients, which support the growth of microbial populations. the primary sources of these microorganisms are the wastes from the slaughtered animals. also, the high counts of thc and total coliform counts (tcc) recorded in the midstream (13.6 × 106 cfu/ml and 24 × 106 cfu/ml) and downstream (5.2 × 106 cfu/ml and 15 × 106 cfu/ml) indicated a correlation between the bacteriological quality of the discharged effluents and the river water. total coliform counts for the water samples exceeded the maximum limits of 1.0 × 10² cfu/ml recommended by epa (2002) in drinking water. this is indicative of faecal contamination at different points of river sampling. all water samples were found to have coliform counts, which exceeded surface water limits of 200/100 ml, and wastewater limits of 400/100 ml, indicative of diffuse faecal pollution of ogun river. this is in agreement with the assertion of adeyemi and adeyemo (2007) that the presence of faecal coliforms attributed to poor sanitary hygiene standards and lack of efficient waste management control systems in rivers and streams is very common in developing countries. the results of isolated bacteria showed that e. coli had the highest percentage frequency of 28%. this result tallies with the findings of nwankwo, magaji and tijani (2015) with a reported frequency of 30.5%, which was also the highest amongst all the bacterial isolates. however, the total heterotrophic and coliform water samples obtained downstream showed lesser values, suggestive of dilution effect with respect to time, surface area and rainfall (adebowale et al. 2016). the occurrence of other pathogenic bacteria in abattoir water sources used for meat processing and effluents, such as salmonella, pseudomonas, enterobacter, klebsiella and shigella, which were also isolated in this study, has also been widely reported (galvin et al. 2010; nafarnda et al. 2012). these bacterial organisms have been implicated as the causes of typhoid fever, shigellosis, pneumonia, meningitis, urinary tract infections (uti), cellulitis and neonatal sepsis (neu 1985). as shown in figure 4, isolated e. coli from the receiving water showed resistance to all the five selected antibiotics, an indication of strong inactivation or hydrolysing action against the different classes of antibiotics (β-lactam/β-lactamase inhibitors, carbapenems and cephems). antibiotic resistance in e. coli is of particular concern because it is the most common gram-negative pathogen in humans, multidrug-resistant strains and is easily transferable to other strains (rasheed et al. 2014). when an organism becomes resistant to first-, secondand third-line antibiotic agents, it becomes a nightmare for the physician and the duration of an infection becomes prolonged (kim & aga 2007; schmidt 2002). the mari showed that 77% of the bacteria isolated in this study were between values 0.2 and 1 (table 2). according to chitanand et al. (2010), mari values less than 0.2 are considered to be an original strain resistance to an antibiotic on first exposure whilst values above 0.2 infer that the effluent and receiving water are high-risk point sources (points where the risk of entry, contact or infection with pollutant is the highest, which are basically the points at the river site where pollution is high) (mthembu 2008). therefore, the mari values ≥ 0.2 obtained infer that the slaughtered animals from which the wastes were obtained have been exposed to frequent antibiotic therapy over time, leading to bacterial resistance to selected antibiotics. this is a pointer to the indiscriminate use of antibiotics on livestock by farmers for growth promotion, prophylactics and therapeutic purposes. here, the major incriminated stakeholders are uneducated nomadic cattle rearers who serve dual functions of producers and health personnel to large livestock herds, some in the hinterlands, with no form of training or monitoring on the use of antibiotics. these animals are later sold to buyers and slaughtered in abattoirs, with attendant risks involved (alhaji 1976). none of the isolates tested in this study were positive to the ddst for esbl production, suggesting that other mechanisms of bacterial resistance could be responsible. furthermore, none of the gram-negative bacteria isolates (salmonella spp., e. coli, klebsiella spp., shigella spp., pseudomonas spp. and enterobacter spp.) from downstream showed resistance to the selected antibiotics, which may have been because of the possibility of river purification by dilution (as a result of distance from the point source) and rainfall. conclusion multidrug-resistant bacteria are difficult to treat, thereby increasing the spread of resistance and complicating efforts to combat them in animals and humans. this study exposed the increasing trend and prevalence of multidrug-resistant bacteria and a high risk of transfer of multiple resistant genes to other microbial populations in the aquatic ecosystem. thus, ogun river is a reservoir and foci for the spread of multidrug-resistant bacteria to aquatic life, animals and humans. the multiple antibiotic-resistant pathogens isolated from high mari reported in this study should alert government, public health and veterinary authorities about the dangers of irresponsible use of antibiotics in animals and the rationalisation of antibiotics use in nigeria. also, the role of animal wastes in the spread and maintenance of multiple antibiotic-resistant strains of pathogenic gram-negative bacteria through the water environment as shown in this study calls for the attention of relevant authorities for appropriate action in water sanitation and management, proper effluent treatment and disposal, policy formulation and enforcement, protection of public and ecosystem health, and curbing the menace of antimicrobial resistance in nigeria. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. funding information this research was partially funded by the african water association. data availability statement the data sets used and/or analysed in the current study are available from the corresponding author upon reasonable request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references adebowale, o.o., jayeola, a., adeyemo, o.k. & kperegbeyi, e., 2016, ‘potential bacterial zoonotic pathogens isolated from a major abattoir and its receiving surface water in abeokuta, nigeria’, alexandria journal of veterinary science 50(1), 94–98. https://doi.org/10.5455/ajvs.222912 adedeji, o.b. & adetunji, v.e., 2011, ‘aquatic pollution in nigeria: the way forward’, advances in environment biology 5(8), 2024–2032. adesemoye, a.o., opere, b.o. & makinde, 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about the author(s) gerda fouche chemistry department, faculty of natural and agricultural sciences, university of pretoria, pretoria, south africa olubukola t. adenubi phytomedicine programme, department of paraclinical sciences, university of pretoria, onderstepoort, south africa tlabo leboho council for scientific and industrial research (csir) biosciences, pretoria, south africa mbokota c. khosa agricultural research council – tropical and subtropical crops, nelspruit, south africa lyndy j. mcgaw phytomedicine programme, department of paraclinical sciences, university of pretoria, onderstepoort, south africa vinny naidoo biomedical research centre, faculty veterinary science, university of pretoria, onderstepoort, south africa kevin w. wellington council for scientific and industrial research (csir) biosciences, pretoria, south africa jacobus n. eloff phytomedicine programme, department of paraclinical sciences, university of pretoria, onderstepoort, south africa citation fouche, g., adenubi, o.t., leboho, t., khosa, m.c., mcgaw, l.j., naidoo, v. et al., 2021, ‘corrigendum: acaricidal activity of the aqueous and hydroethanolic extracts of 15 south african plants against rhipicephalus turanicus and their toxicity on human liver and kidney cells’, onderstepoort journal of veterinary research 88(1), a1951. https://doi.org/10.4102/ojvr.v88i1.1951 note: doi of original article published: https://doi.org/10.4102/ojvr.v86i1.1665 correction corrigendum: acaricidal activity of the aqueous and hydroethanolic extracts of 15 south african plants against rhipicephalus turanicus and their toxicity on human liver and kidney cells gerda fouche, olubukola t. adenubi, tlabo leboho, mbokota c. khosa, lyndy j. mcgaw, vinny naidoo, kevin w. wellington, jacobus n. eloff published: 27 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published version of this article, fouche, g., adenubi, o.t., leboho, t., mcgaw, l.j., naidoo, v., wellington, k.w. et al., 2019, ‘acaricidal activity of the aqueous and hydroethanolic extracts of 15 south african plants against rhipicephalus turanicus and their toxicity on human liver and kidney cells’, onderstepoort journal of veterinary research 86(1), a1665. https://doi.org/10.4102/ojvr.v86i1.1665, the fourth author, mbokota c. khosa, was omitted from the ‘authors’ and ‘affiliations’ sections. the indicated author should be added as the fourth author, and the following affiliation should be added as his affiliation: agricultural research council – tropical and subtropical crops, nelspruit, south africa. the authors’ contributions section is hereby update to: authors’ contributions g.f. conceptualised the study. m.c.k. was involved in the collection of some of the plant material and in the preparation of the extracts used in the biological screening assays. g.f., k.w.w. and t.l. performed the literature search and plant selection. t.l. prepared the plant extracts. m.c.k. was also involved in the fractionation and isolation process in the natural product chemistry laboratory. j.n.e. conceptualised the study in a joint application, and supervised the students and postdoctoral fellow. v.n. supervised determination of acaricidal activity. l.j.m.g. supervised the determination of cytotoxicity. o.t.a. determined the acaricidal activity against adult ticks of r. turanicus. k.w.w. wrote the first draft of the manuscript. abstract introduction materials and methods results discussion acknowledgements references appendix 1 about the author(s) junita liebenberg vaccines and diagnostics development programme, arc-onderstepoort veterinary research, pretoria, south africa helena c. steyn vaccines and diagnostics development programme, arc-onderstepoort veterinary research, pretoria, south africa antoinette i. josemans epidemiology, parasites and vectors programme, arc-onderstepoort veterinary research, pretoria, south africa erika faber vaccines and diagnostics development programme, arc-onderstepoort veterinary research, pretoria, south africa erich zweygarth department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation liebenberg, j., steyn, h.c., josemans, a.i., faber, i. & zweygarth, e., 2020, ‘in vitro propagation and genome sequencing of three ‘atypical’ ehrlichia ruminantium isolates’, onderstepoort journal of veterinary research 87(1), a1769. https://doi.org/10.4102/ojvr.v87i1.1769 original research in vitro propagation and genome sequencing of three ‘atypical’ ehrlichia ruminantium isolates junita liebenberg, helena c. steyn, antoinette i. josemans, erika faber, erich zweygarth received: 25 mar. 2019; accepted: 01 oct. 2019; published: 24 june 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract three isolates of ehrlichia ruminantium (kümm 2, omatjenne and riverside), the causative agent of heartwater in domestic ruminants, were isolated in ixodes scapularis (ide8) tick cell cultures using the leukocyte fraction of infected sheep blood. all stocks were successfully propagated in ide8 cells, whereas initiation attempts using endothelial cell cultures were unsuccessful. therefore, the new technique should be included in any attempt to isolate field strains of e. ruminantium to enhance the probability of getting e. ruminantium isolates which might not be initiated in endothelial cells. draft genome sequences of all three isolates were generated and compared with published genomes. the data confirmed previous phylogenetic studies that these three isolates are genetically very close to each other, but distinct from previously characterised e. ruminantium isolates. genome comparisons indicated that the gene content and genomic synteny were highly conserved, with the exception of the membrane protein families. these findings expand our understanding of the genetic diversity of e. ruminantium and confirm the distinct phenotypic and genetic characteristics shared by these three isolates. keywords: ehrlichia ruminantium; heartwater; in vitro isolation; tick cell line; comparative genomics. introduction the intracellular rickettsial agent ehrlichia ruminantium causes a disease commonly known as heartwater or cowdriosis. it is an infectious, non-contagious disease which affects mainly cattle, sheep, goats and some wild ruminants. it is transmitted by ticks of the genus amblyomma and has been reported from almost all african countries south of the sahara, from the adjacent islands of the indian ocean and atlantic ocean (uilenberg 1983) and from some caribbean islands (birnie et al. 1984; perreau et al. 1980). the method of choice for in vitro isolation of e. ruminantium is infection of endothelial cells (bezuidenhout, paterson & barnard 1985), the cell type in which organisms occur in infected animals (cowdry 1926). however, several isolates (allsopp et al. 2007; du plessis & kümm 1971; steyn 2009) have failed to establish in endothelial cell cultures (bezuidenhout & brett 1992; bezuidenhout et al. 1988). besides endothelial cells, tick cell lines have been used to initiate e. ruminantium in in vitro cultures and have even allowed the establishment of infection directly from the leukocytes of sheep blood (zweygarth, josemans & steyn 2008). therefore, attempts were made to isolate ‘atypical’ e. ruminantium stocks in tick cells, atypical in the sense that they could not be initiated by using the classical ways of infecting endothelial cells (bezuidenhout et al. 1985; byrom et al. 1991). since the early reports of the e. ruminantium omatjenne and kümm stocks, it has been clear that these stocks share many phenotypical and genetic characteristics, but they differ from all other isolates (allsopp et al. 1997, 2001; du plessis 1985, 1990). the kümm stock was prepared from a goat, which was diagnosed with heartwater, from the northern province of south africa, a heartwater endemic area (du plessis & kümm 1971). sheep injected with a lymph node suspension from goat developed heartwater symptoms. after more than 100 passages in mice it was still found to be pathogenic in mice, sheep and goats, but non-pathogenic to cattle (du plessis 1982). many attempts were made to culture this organism in endothelial cells; however, it was only established in culture in 2002 using different monocyte cell lines (zweygarth et al. 2002). it was observed that the kümm stock comprised two 16s rrna (16s ribosomal ribonucleic acid) genotypes, a 16s genotype typical of west african isolates (kümm1) isolated in a canine macrophage-monocyte cell line (dh82) and a 16s genotype identical to e. ruminantium (omatjenne) (kümm2) isolated in a sheep blood mononuclear cell line (e2). the omatjenne genotype originated from the farm omatjenne in the otjiwarongo district of namibia, a heartwaterand amblyomma-free area (du plessis 1990). healthy cattle on this farm reacted positively to e. ruminantium antigen using an indirect fluorescent antibody (ifa) test. subsequently, ticks were collected from cattle on the farm and homogenates of individual ticks injected into mice. the serum of a single mouse, inoculated with homogenate prepared from a hyalomma truncatum tick, tested positive in the ifa test. the original infective agent was non-pathogenic to mice, calves and sheep. only after passaging through three generations of a. hebraeum, it became fatal to sheep and mice (du plessis 1990). the organisms observed in brain smears of the sheep closely resembled those of the kümm stock; fewer colonies of small size compared with those typically observed in animals infected with other e. ruminantium isolates. both stocks were atypical in that they are highly pathogenic to mice, but apparently non-pathogenic to bovine and could not be cultured in endothelial cells. the kümm stock was described as atypical in that it infected mouse peritoneal macrophages (du plessis 1982). because of the differences in pathogenicity and anomalous behaviour in cell culture, it was questioned whether the kümm stock belonged to the species e. ruminantium (du plessis 1982). likewise allsopp et al. (1997) suggested that e. ruminantium (omatjenne) (then cowdria ruminantium [omatjenne]), not to be confused with ehrlichia sp. (omatjenne) later renamed anaplasma sp. (omatjenne), may belong to a different species because of its difference in vector specificity and virulence. phylogenetic studies revealed that all e. ruminantium stocks analysed routinely grouped into one of two major clades, a west african clade and a southern/eastern african clade, except for kümm2 and/or omatjenne that clustered either as a unique group or in one of the major clades (allsopp & allsopp 2007; allsopp et al. 1997, 2001; steyn 2009; van heerden et al. 2004). even isolates from several other geographical areas of africa, the indian ocean islands and the caribbean cluster with the southern and eastern african isolates can be included in a worldwide clade (cangi et al. 2016). all these studies, however, were conducted with a limited number of genes, which do not necessarily allow the identification of recombination events. furthermore, only small numbers of e. ruminantium isolates with different genotypes have been isolated in cell culture, which limits studies to link variation in dna sequence to phenotypic characteristics. therefore, there is a need to establish more isolates in cell culture that would enable us to conduct experiments to determine virulence, cross-protection between isolates, and to produce whole genome sequences. this study reports on the isolation of ‘atypical’ e. ruminantium in tick cell cultures. in addition, we generated draft genome sequences of all three ‘atypical’ isolates and determined the differences between them and previously characterised e. ruminantium genomes. materials and methods infective agents three stocks of e. ruminantium were used. the kümm2 genotype (zweygarth et al. 2002), which was derived from the kümm stock (du plessis 1982), was originally isolated from a naturally infected goat in rust de winter (limpopo province, south africa). the e. ruminantium omatjenne isolate was isolated from a single h. truncatum tick collected from a heartwaterand amblyomma-free area of namibia (du plessis 1990). its complete 16s rdna (16s ribosomal rna) sequence was determined and submitted to genbanktm (accession number c. ruminantium [omatjenne] u03776) (allsopp et al. 1997). the original inoculums from which the kümm and omatjenne stocks were isolated are no longer available and the complete history of the blood stabilates used in this study is unknown. the third isolate was derived from the blood obtained from a sick angora goat from the farm riverside (26.83°e, -33.45°s; grahamstown, eastern cape province, south africa) (steyn 2009). cell cultures the tick cell line ixodes scapularis (ide8), derived from ixodes scapularis embryos (munderloh et al. 1994) was used. it was propagated in l-15b medium (munderloh & kurtti 1989), which was supplemented with 5% heat-inactivated foetal bovine serum (fbs), 10% tryptose phosphate broth, 0.1% bovine lipoprotein concentrate (mp biomedicals, santa ana, ca, united states [us]), 100 iu/ml penicillin and 100 μg/ml streptomycin. the ph was adjusted at approximately 7.0. infected tick cell cultures were maintained in dulbecco’s modified eagle’s medium nutrient mixture ham f-12 (dme/f-12; sigma, st. louis, mo, us; d 0547) containing 15 mm hepes and 1.2 g/l sodium bicarbonate, further supplemented with 10% (volume per volume [v/v]) heat-inactivated fbs, 2 mm l-glutamine, 100 iu/ml penicillin and 100 μg/ml streptomycin, and are referred to as df-12. infection of ixodes scapularis (ide8) cell cultures each of the e. ruminantium stocks was used to infect merino sheep by intravenous injection of 5 ml of an infectious blood stabilate. the sheep subsequently developed symptoms associated with e. ruminantium infection. the body temperature of each sheep was monitored daily and a blood sample was collected when it exceeded 41.0 °c. blood collected at the peak febrile reaction was used to initiate cell cultures according to the method described by zweygarth et al. (2008). thereafter the sheep were treated with tetracycline. blood was collected by venipuncture into sterile vac-u-test® tubes containing heparin (lithium heparin, 14.3 united states pharmacopoeia (usp) per ml blood) as anticoagulant and stored in ice. the cooled blood was centrifuged (800 × g; 10 min; 4 °c) and the buffy coat collected and washed with cold physiological phosphate-buffered saline (pbs). the buffy coat was re-collected, and the red blood cells were lysed for approximately 30 seconds in 18 ml sterile distilled water followed by the addition of 2 ml of 10-fold concentrated hanks’ balanced salt solution to restore physiological tonicity. the lysate was centrifuged for 5 min at 290 × g at 18 °c. the resulting cell pellet was re-suspended in 5 ml df-12 and distributed into 25 cm² culture flasks containing ide8 cells. the cultures were incubated at 32 °c. dna preparation and sequencing the e. ruminantium elementary bodies were isolated from the cell culture material on discontinuous percoll density gradients (mahan et al. 1995) and the bacterial dna was extracted with the dneasy blood & tissue kit (qiagen, gmbh, hilden, germany). the genomes were sequenced using illumina nextera paired-end libraries on the illumina miseq and/or hiseq platforms (illumina, san diego, ca, us). de novo assembly sequencing reads were processed and assembled using clc genomics workbench version 7.0 (https://www.qiagenbioinformatics.com/). default parameters were used for quality trimming, and adapter sequences were removed. trimmed reads < 50 bp were discarded. several de novo assemblies using different combinations of parameters were performed for each data set. the following stringent parameters were used in all assemblies: mismatch cost, insertion cost and deletion cost of three, and length fraction and similarity fraction = 0.9. we varied the minimum contig length, also whether to use global alignment and whether to perform scaffolding or not. contigs <500 bp and <10-fold coverage were discarded; the remaining contigs were blasted with the national center for biotechnology information’s (ncbi) nucleotide blast (blastn, https://blast.ncbi.nlm.nih.gov/blast.cgi) to identify and remove contaminating data (phix, tick, and mycoplasma spp.). the sequences of the contigs from different clc assemblies for each data set were compiled and joined in gap4 (bonfield, smith & staden 1995). discrepancies between different assemblies of the same data sets were checked and incorrect contig sequences were removed. the resulting contigs were ordered to the welgevonden strain (cr767821) using progressive mauve (darling, mau & perna 2010) of mauve version 2.4.0 (darling et al. 2004). in silico multi-locus sequence typing multi-locus sequence typing (mlst) loci (glta, groel, lepa, lipa, lipb, secy, sodb and suca), as described by adakal et al. (2009), were selected for the analysis. the homologous sequences of the eight genes were identified in the complete genome sequences of welgevonden (nc_005295.2/cr767821.1) and gardel (nc_006831.1/cr925677.1) or the gap4 alignments of incomplete genome sequences. these included ball3, mara87/7, blaauwkrans and kwanyanga from south africa (unpublished genomes), and senegal (nz_mquj00000000.1) and sankat 430 (bddn01000001 to bddn01000183) from west africa. in addition, orthologous genes from other ehrlichia species, as well as the closely related anaplasma species, were included. these were e. canis strain jake (nc_007354), e. chaffeensis strain arkansas (nc_007799), e. muris as145 (nc_023063), a. marginale strain saint maries (nc_004842), a. phagocytophilum strain hz (nc_007797), a. centrale strain israel (nc_013532) and a. ovis strain haibei (nz_cp015994). the aligned concatenated sequences were analysed in clc genomics workbench 7.0. a maximum likelihood phylogenetic tree (unweighted pair group method using arithmetic averages [upgma], bootstrap analysis with 100 replicates) was created and pairwise comparisons were performed to illustrate relationship between the e. ruminantium isolates and the other ehrlichia and anaplasma species. whole genome comparisons the ordered contig sequences of each genome were concatenated and submitted to ncbi. protein-coding genes were annotated using ncbi prokaryotic genomes annotation pipeline (pgap) (tatusova et al. 2016) and the resulting genbank files were used in whole genome comparisons with the welgevonden and gardel sequences. whole-chromosome alignments were performed locally with blastall (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/latest/); the tabular view option (-m = 8) allowed visualisation of the alignments in the artemis comparison tool (act) programme (carver et al. 2005). in addition, blast ring image generator (brig) (alikhan et al. 2011) was used to compare whole genomes and subsets of genes. we used the locus tags for coding sequence (cds) from the original annotation and publication (collins et al. 2005) of the welgevonden strain of e. ruminantium. the corresponding reference sequence database (refseq) locus tags created subsequently by the ncbi prokaryotic genome annotation pipeline are listed in table 1-a1. ethical consideration experiments were performed in accordance with the stipulations of the animal ethics committee at arc-onderstepoort veterinary research and approved by the south african department of agriculture, forestry and fisheries under section 20 of the animal disease act of 1984. results infection of ide8 cell cultures leukocytes isolated from the blood of infected sheep were used as infective inoculum. all three e. ruminantium stocks were established successfully in ide8 cell cultures by this method (table 1). the kümm2 stock was detected in giemsa-stained smears 28 days after initiation. infection with the omatjenne stock was first demonstrated on day 47 post-infection. the riverside isolate was detected in stained smears in ide8 cell cultures after 25 days. it was not possible to initiate in vitro cultures of any of the three isolates using bovine endothelial cells as host cells (data not shown). table 1: infection of ide8 cell cultures using leukocytes from ovine blood isolated from sheep infected with three e. ruminantium stocks. genome assembly the average coverage of all assemblies was greater than 100-fold, and the draft genome sequences of kümm2, omatjenne and riverside isolates comprised 6, 7 and 9 contigs, respectively (table 2). all contigs of these three genomes were successfully mapped to the reference genome (figure 1-a1). the total length of the joined contig sequences ranged between 1448 megabases (mb) and 1455 mb. the remaining gaps were in repeat regions, including both tandem repeats and dispersed repeats with large repeat units. table 2: assembly information. in silico multi-locus sequence typing analysis multi-locus sequence typing was used to illustrate the relationship between different e. ruminantium isolates. the south african isolates (welgevonden, ball3, mara87/7, blaauwkrans and kwanyanga), as well as the isolate from the caribbean (gardel), shared more than 99% identity across the eight genes selected for multi-locus sequence typing (mlst), whereas the west african isolates shared 98% identity with the south african isolates (table 2-a1). the mlst sequences of the three ‘atypical’ isolates were identical, but had only 88% identity compared with other e. ruminantium isolates and were separated into a distinct clade in the phylogenetic tree (figure 1). the other three species of ehrlichia shared 88% – 89% identity (genetic distances 0.11–0.13), whereas different anaplasma species are more diverse with 68.5% – 92.5% identity (distance of 0.08–0.15). in fact, the genetic distance between a. marginale and a. centrale (0.08, 92.5% identity) is considerably closer than that between the ‘atypical’ and other e. ruminantium isolates (0.12, 88% identity). figure 1: phylogenetic comparison of the multi-locus sequence typing; unweighted pair group method using arithmetic averages tree constructed from the aligned concatenated sequences of the eight selected genes. general genome features all three genomes have a low guanine-cytosine (gc) content (28.94% – 28.95%) and a low protein-coding capacity (65%) (table 3), which is comparable with the 27.5% gc and 63% – 64% coding capacity of other e. ruminantium genomes (frutos et al. 2006; nakao, jongejan & sugimoto 2016). in each genome, one set of rrna genes, 36 transfer rna (trna) genes, transfer-messenger rna (tmrna) and two non-coding rna (ncrna) genes were identified. table 3: properties of the annotated genomes. the genomes generated in this study still have gaps because of dispersed repeats and large tandem repeats, and certain of the assembled tandem repeats are missing repeat units. these facts could contribute to the smaller genome size (1.45 mb) of the draft genomes compared with 1.5 mb reported for the welgevonden and gardel sequences (collins et al. 2005; frutos et al. 2006). it could also explain the slightly higher protein-coding capacity calculated for the assemblies because the repeat regions are mostly located in non-coding regions. the gene content and genomic synteny were highly conserved between the three genomes as well as compared with previously sequenced genomes (figure 2 and figure 2-a1). small inversions and rearrangements were found in the area around the shift in gc-skew value between 690 kilobases (kb) and 740 kb as well as other regions where duplications are found (at 630 kb, 1185 kb and 1248 kb) (figures 1-a1 – 3-a1). a high rate of dna reorganisation in the terminus region is often observed between closely related bacteria, which may be associated with the mechanism of chromosome separation after replication (hughes 2000). figure 2: blast ring image generator alignments of the three isolates against e. ruminantium welgevonden. (a) whole genome comparisons of the nucleotide sequence (blastn) and (b) the amino acid sequences comparisons of the coding sequence (blastp). single nucleotide polymorphism (snp) and insert or deletion (indel) analysis revealed that when the repeat regions were excluded, only four snps and four indels distinguished the kümm2 and omatjenne sequences. the riverside sequence differed from either kümm2 or omatjenne by 328 snps and 16 indels. comparison of membrane protein families although gene content and genomic synteny are highly conserved, variation was observed between the genomes generated in this study and those reported previously. the variation was mainly limited to genes predicted to encode hypothetical proteins or membrane proteins and specifically the members of the four membrane protein families described previously (collins et al. 2005). we identified the orthologous families in the kümm2, omatjenne and riverside genomes, and found that their arrangement and number were identical in all the three genomes sequenced in this study. there were, however, variations compared with other e. ruminantium isolates. the major antigenic protein 1 (map1) family has been described in various isolates. the nucleotide sequences of the members of map1 family in kümm2, omatjenne and riverside show a high degree of similarity to welgevonden (figure 3a), except for map1-5. in the syntenic locus of map1-5, two smaller open reading frames (orfs) were identified in the ‘atypical’ genomes (figure 3b). figure 3: genomic comparison of the map1 multigene family. (a) blast ring image generator alignments of the three isolates against selected e. ruminantium welgevonden gene sequences. (b) linear representation of the alignments indicating members of the family in black with adjacent genes in grey. the locus tag prefixes for kümm2 (edl79), riverside (edl81) and omatjenne (edl80) were omitted to simplify the drawing. two other families described in collins et al. (2005), here designated membrane family 1 (erum2240–erum2340; erum2400; erum2410) and membrane family 2 (erum2750–erum2800; erum3600–erum3630), were less conserved at nucleotide level (figure 4a) and both of these families have one member less compared with the welgevonden annotation (figure 4b). three of the genes in membrane family 2 are in opposite orientation in the ‘atypical’ strains as compared with welgevonden. figure 4: genomic comparison of membrane families (1) (erum2240–2410) and (2) (erum2750–3630). (a) brig alignments of the three isolates against selected e. ruminantium welgevonden gene sequences. (b) linear representation of the alignments indicating members of the families in black with adjacent genes in grey. the locus tag prefixes for kümm2 (edl79), riverside (edl81) and omatjenne (edl80) were omitted to simplify the image. orthologs of four predicted membrane proteins (erum7990, erum8000, erum8010 and erum8020; membrane family 3) were identified in the same relative location in the three newly sequenced genomes (figure 4-a1). in all three genomes at this location, however, membrane family 3 expanded to seven members and five more members were identified at 82 mb upstream (figure 4-a1, table 3-a1). discussion in vitro isolation of heartwater organisms in cell culture is undoubtedly the most valuable aid for research in heartwater. for years, e. ruminantium was isolated from the blood or plasma of reacting animals by cultivation in ruminant endothelial cells, although it was recognised quite early that it was not possible to initiate in vitro cultures of some strains using this approach (bezuidenhout & brett 1992; bezuidenhout et al. 1988). it was shown in previous experiments that leukocytes obtained from four sheep each of which was infected with a different stock of e. ruminantium were able to infect ide8 tick cell cultures and endothelial cell cultures simultaneously (zweygarth et al. 2008). we applied this technique to the three ‘atypical’ strains that could not be isolated in endothelial cells, the kümm (du plessis 1982), omatjenne (du plessis 1990) and riverside (steyn 2009) isolates. here we show that all three isolates could be established in ide8 tick cell cultures and continuously propagated for 169 days or more. these cultures provided excess material for additional molecular characterisations of the isolates. the most distinctive characteristic of the ‘atypical’ isolates is the fact that they cannot be initiated in ruminant endothelial cell cultures. however, some of the reported anomalies may be contributed to other experimental factors. the experiments were conducted using uncharacterised inoculums passaged many times in different animals during the pre-pcr era when it was difficult to detect pathogen-free animals. hence, the original kümm stock comprised two organisms or one of them was introduced over the years during passaging. there is also a possibility that the initial omatjenne agent is not the same as the organism we have cultured in this study. the pathogenicity and vector specificity of the cultured organisms need to be verified experimentally. the sequences of the concatenated mlst loci of the three ‘atypical’ isolates were identical; however, they formed a distinct clade in tree topology. these results were confirmed by genome sequences. although these three sequences only differed by a few snps and indels, as well as variation and small gaps in repeat regions that may be ascribed to assembly errors, they are markedly different from other e. ruminantium genomes. the 16s rrna and map1 gene sequences identified the omatjenne agent, and later kümm2, as e. ruminantium in the southern african clade (allsopp et al. 1997; van heerden et al. 2004). in contrast, tree topology and pairwise comparison of eight genes presented in this study may support an argument for the ‘atypical’ isolates to be classified as a separate species. the genetic distances and identity shared between the ‘atypical’ isolates and other e. ruminantium isolates are similar to the distances and percentage identity between the different species of ehrlichia and anaplasma. whether these three isolates indeed represent a unique species needs to be validated. all the e. ruminantium genomes sequenced thus far are syntenic (frutos et al. 2006; nakao jongejan & sugimoto 2016), and it is known that anaplasma spp. and ehrlichia spp. share conserved gene order (dunning hotopp et al. 2006; pierlé et al. 2012). the synteny is also observed for the three isolates sequenced in this study with a few exceptions in the membrane protein families. of the four families analysed, the map1 family was the most conserved one across all e. ruminantium genomes. the paralogs are maintained in the same order in all genomes, but in place of map1-5, two small orfs were detected in the genomes presented here. the map1-5 gene is truncated compared with the other paralogs in all isolates analysed thus far. it was also identified as one of the paralogs that undergoes balancing selection, a type of selection that is reported to maintain genetic variation in genes that are involved in evasion of host immune response (salim et al. 2019). most variation was detected in membrane family 1 and membrane family 2. the nucleotide sequences differed significantly between orthologous genes, and the number, order and, in some cases, orientation of the paralogs were different in new annotations. in membrane family 3, the nucleotide sequences between orthologs were conserved, but this family was expanded from four to 12 members in the ‘atypical’ genomes. at present, the function of these putative proteins is unknown, and it has not been shown that all members of these two protein families are expressed. therefore, it is not known whether these variations have an effect on the expression or function of the predicted proteins encoded by these orfs. it is known that anaplasma and ehrlichia spp. present a wide range of paralogous genes encoding various functions that ensure survival in diverse host and vector environments (dunning hotopp et al. 2006). several studies have reported a high level of genetic diversity among e. ruminantium isolates (allsopp & allsopp 2007; cangi et al. 2016; nakao et al. 2011). in contrast, here we found that the genome sequences of the three ‘atypical’ isolates from distant geographical areas and diverse habitats are almost identical. excluding repeat regions, kümm2, from the limpopo province of south africa, and omatjenne, from the much drier and heartwater-free otjiwarongo district of namibia, differ by four snps and four indels only. in fact, there were more substitutions and small deletions detected between the parental welgevonden strain and its daughter strain after 11–13 passages in a different cell culture environment (frutos et al. 2006). although the current results have not connected any genetic variation to the phenotypes that distinguish these isolates, variations in the membrane protein families may contribute to the ability of these organisms to infect different cells. a comprehensive snp analysis, including all genomes sequenced, may elucidate the determinants of diversity. the synteny conservation in e. ruminantium genomes suggests that at least some of the phenotypes are associated with small polymorphisms. in view of this, we are in the process of generating whole genome sequences of all the e. ruminantium isolates we have in cell culture. in addition, we need to establish more field isolates in culture to conduct phenotypic and genotypic analyses in the future work. to date, no standard cell line has been designated for isolation of e. ruminantium, and it is clear that the ‘atypical’ isolates cannot be easily isolated in bovine endothelial cells. we therefore recommend the use of both ruminant endothelial cells and tick cell cultures concurrently. the two methods complement each other and should be used when isolating field strains of ehrlichia spp. acknowledgements the authors thank prof. katherine m. kocan, oklahoma state university, stillwater, ok, us, for the provision of the ide8 cells and the gauteng department of agriculture and rural development for funding support. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. author’s contributions j.l. and e.z. conceptualised and wrote the article. e.z. performed the cell culture experiments and interpreted results. j.l. assembled and analysed the sequencing data. h.c.s. performed experimental work involving animals and contributed to the editing of the article. a.i.j. and e.f. contributed to experimental laboratory work and reviewed the article. all authors read and approved the final article. funding information prof. katherine m. kocan, oklahoma state university, stillwater, ok, us, provided the ide8 cells. this work was funded by the gauteng department of agriculture and rural development, south africa (project: identification of ehrlichia ruminantium proteins involved in host and vector cell invasion). data availability statement the nucleotide sequences and annotation of the three genomes are available from ncbi (https://www.ncbi.nlm.nih.gov/genome/microbes/). the accession numbers are listed in table 3. disclaimer the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors or funder. references adakal, h., meyer, d.f., carasco-lacombe, 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different ehrlichia ruminantium isolates’, veterinary microbiology 101(4), 279–291. https://doi.org/10.1016/j.vetmic.2004.02.015 zweygarth, e., josemans, a.i. & steyn, h.c., 2008, ‘in vitro isolation of ehrlichia ruminantium from ovine blood into ixodes scapularis (ide8) cell cultures’, onderstepoort journal of veterinary research 75(2), 121–126. https://doi.org/10.4102/ojvr.v75i2.10 zweygarth, e., josemans, a.i., van strijp, m.f., van heerden, h., allsopp, m.t. & allsopp, b.a., 2002, ‘the kümm isolate of ehrlichia ruminantium: in vitro isolation, propagation and characterization’, onderstepoort journal of veterinary research 69(2), 147–153. appendix 1 table 1-a1: the refseq locus tags of the members of the membrane protein families. table 2-a1: pairwise comparison table of the multi-locus sequence typing alignment. the upper comparison shows percentage of identical bases in alignment positions between two isolates and the lower comparison depicts the jukes-cantor distance between sequences. table 3-a1: membrane family 3 of the welgevonden isolate compared to kümm2, omatjenne and riverside. figure 1-a1: mauve alignment of the contigs of the three draft genome sequences against the linearised genome sequence of e. ruminantium welgevonden (top panel). the second, third and bottom panels represent the ordered contig sequences, separated with vertical red lines, of kümm2, omatjenne and riverside respectively. each coloured block represents a segment of the genome sequence that aligned to a homologous part of another genome without any genome rearrangements. blocks above the centre line of each panel is in the forward orientation relative to the reference genome sequence, while those below the centre line indicate regions that align in the inverse orientation. the height of the similarity profile inside a block corresponds to the average level of conservation in that region. figure 2-a1: act alignment of the three draft genome sequences against the whole genome sequence of e. ruminantium welgevonden. the coloured lines drawn between two adjacent linearised chromosomes show the location of homologous genes and indicate the same (red) or opposite (blue) orientation relative to the chromosome immediately above. figure 3-a1: circular representations of the genomes generated with dnaplotter. in each circle the tracks from outside to inside represent: (a) coding sequences on the forward (blue) and (b) reverse strands (red), % gc plot, and (c) gc skew [(gc/(g+c)]. figure 4-a1: act alignment of the e. ruminantium kümm2 sequence (bottom sequence in each panel) against e. ruminantium welgevonden (top sequence in each panel) to depict the expansion of membrane family 3 in the kümm2 isolate. the coloured lines drawn between the sequences show the location of homologous genes in the same (red) or opposite (blue) orientation. top (a) four e. ruminantium welgevonden open reading frames matching open reading frames in two locations (yellow lines) in the e. ruminantium kümm2 annotation. middle (b) the syntenic matches of the four welgevonden orfs to seven kümm2 open reading frames. bottom (c) the same four welgevonden open reading frames matching five kümm2 open reading frames upstream. golezardy_81-85.indd in addition to the kruger national park, which is approximately 2 million ha in size, there are numerous smaller national and provincial nature reserves in south africa. besides the mammals, birds, reptiles, fish, arthropods and vegetation that are protected within these reserves the parasites of the various biota are coincidentally conserved with their hosts. the larger reserves are not only important from the conservation aspect, but also because of the opportunity they afford for studies in an environment in which there has been minimal human disturbance or pesticide usage. during the past 80 years several inventories of the arthropod and helminth parasites infesting wildlife in south africa have been published. bedford (1932, 1936) and haeselbarth, segerman & zumpt (1966) have listed the arthropods infesting domestic and wild animals, zumpt (1961) the mites, theiler (1962), walker (1991) and walker, keirans & horak (2000) the ticks, zumpt (1965) the myiasis-producing flies, round (1968) the helminths, ledger (1980) the lice, and segerman (1995) the fleas. in recent times, particular host species and nature reserves have been targeted for the collection of para sites. a number of these studies have been conducted in the cape province (now subdivided into the western cape province, the eastern cape province, and the northern cape province). during these surveys animals in the mountain zebra national park, the bontebok national park and the addo ele81 onderstepoort journal of veterinary research, 74:81–85 (2007) research communication ticks (acari: ixodidae) collected from animals in three western, semi-arid nature reserves in south africa h. golezardy1 and i.g. horak2* abstract golezardy, h. & horak, i.g. 2007. ticks (acari: ixodidae) collected from animals in three western, semi-arid nature reserves in south africa. onderstepoort journal of veterinary research, 74:81–85 the objective of this study was to make an inventory of the ixodid tick species infesting wild animals in three western, semi-arid nature reserves in south africa. to this end 22 animals in the kgalagadi transfrontier park, 10 in the west coast national park and 16 in the karoo national park were examined. fourteen tick species were recovered, of which hyalomma truncatum, rhipicephalus exoph thalmos and rhipicephalus glabroscutatum were each present in two reserves and the remainder only in one. the distributions of two of the 14 tick species recovered, namely rhipicephalus capensis and rhipicephalus neumanni, are virtually confined to the western semi-arid regions of southern africa. hyalomma truncatum, r. capensis and r. glabroscutatum were the most numerous of the ticks recovered, and eland, taurotragus oryx, were the most heavily infested with the former two species and gemsbok, oryx gazella, and mountain reedbuck, redunca fulvorufula, with r. glabroscutatum. keywords: geographic distribution, hosts, ixodid ticks, semi-arid nature reserves, wildlife * author to whom correspondence is to be directed. e-mail: ivan.horak@up.ac.za 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa, and division of parasitology, arc-onderstepoort veterinary institute, onderstepoort, 0110 south africa accepted for publication 15 september 2006—editor 82 ticks (acari: ixodidae) collected from animals in nature reserves in south africa phant national park were examined (horak, sheppey, knight & beuthin 1986; horak, fourie, novellie & williams 1991a; horak, boomker, spickett & de vos 1992; knapp, krecek, horak & penzhorn 1997), as well as animals in the andries vosloo kudu reserve (knight & rechav 1978; horak, spickett, braack & williams 1991b; horak & fourie 1991; horak et al. 1992), and the thomas baines nature reserve (horak, potgieter, walker, de vos & boomker 1983; petney, horak, howell & meyer 2004). the present paper provides an inventory of the ixodid ticks collected from wildlife in three semi-arid nature reserves in the erstwhile cape province, two in the region now known as the western cape province and one in the now named northern cape province. the helminths recovered from antelopes in two of these reserves have been recorded in a separate publication (boomker, horak, watermeyer & booyse 2000). the kgalagadi transfrontier park, that now incorporates the former kalahari gemsbok national park (24°45’–26°28’ s, 20°00’–20°50’ e), which lay within the borders of south africa, is located in a semiarid region in the northern part of northern cape prov ince and extends into the neighbouring country of botswana. the vegetation consists of a mosaic of lightly wooded grassland on the dune crests, pure grassland in shallow depressions between the dunes, and rhigozum trichotomum shrubby grassland in deeper hollows where the underlying calcrete is close to the surface (white 1983; acocks 1988). during october 1984 22 animals were examined for ticks in the central region of the former kalahari gemsbok national park. the west coast national park (33°06’–33°20’ s; 17°58’–18°11’ e), which incorporates the former langebaan nature reserve is situated in a semi-arid region on the western coast of the western cape province and comprises an area of 24 779 ha. the vegetation is classified as strandveld and isolated patches of coastal fynbos (white 1983; acocks 1988). the park lies within the winter rainfall region of south africa in which summers are hot and dry, and winters cold and wet. ten animals were examined during february 1990 in this park. the karoo national park (32°12’–32°20’ s; 22°18’– 22°39’ e), comprises an area of 17 706 ha near the town of beaufort west in the north-eastern part of the western cape province. it is a semi-arid region with hot summers and cold winters, and occasional snow on the higher mountain peaks. the vegetation consists of karroid broken veld (acocks 1988). sixteen animals were examined for ticks in this park during february 1991. the species and numbers of animals examined are summarized in table 1. the animals were either shot or chemically immobilized. the larger species that were shot were processed for ectoparasite recovery as described by horak et al. (1992) for greater kudus and the smaller animals as described by horak et al. (1986) for scrub hares. the animals that were immobilized were carefully scrutinized for ticks. the ticks collected from the processed material, or directly from the immobilized animals, were stored in 70 % alcohol for later identification and counting under a stereoscopic microscope. a total of 14 ixodid tick species were recovered in this way. only four tick species were recovered from the 22 animals examined in the south african portion of the kgalagadi transfrontier park (table 2), and individual burdens were small. no ticks were collected from the single red hartebeest and the two springbok examined. the species collected, namely hyalomma marginatum rufipes, hyalomma truncatum, rhipicephalus exophthalmos and rhipicephalus theileri are all adapted to harsh climatic conditions (howell, walker & nevill 1978; walker et al. 2000). the preference of r. exophthalmos for scrub hares and r. theileri for cape ground squirrels is evident from the collections made from these animals. the latter tick is also common on yellow mongooses, cyn ictis penicillata, and meercats, suricata suricatta, which share warrens with ground squirrels (horak, chap parro, beaucournu & louw 1999; walker et al. 2000). the species and numbers of ticks collected from animals in the west coast national park are summarized in table 3. six ixodid tick species were collected from the ten animals examined, and large numbers of h. truncatum, rhipicephalus capensis and rhipicephalus glabroscutatum were recovered. the distribution of r. capensis is virtually confined to the western winter rainfall region of the western cape province, while r. glabroscutatum occurs not only here and in the southern karoo, but also in the non-seasonal rainfall regions of the western and eastern cape provinces and in the valley bushveld of the latter province (walker et al. 2000). the eland, probably because of their larger size, harboured considerably more adult h. truncatum and r. capensis than the gemsbok (gallivan & horak 1997). conversely the gemsbok carried larger burdens of all stages of development of the two-host ticks rhipicephalus evertsi evertsi and r. glabroscutatum. all stages of the latter tick attach around the feet 83 h. golezardy & i.g. horak table 1 mammals examined for ticks in three western, semi-arid nature reserves in south africa host species scientific name number examined red hartebeest black wildebeest blue wildebeest bontebok springbok steenbok eland gemsbok grey rhebok mountain reedbuck rock hyrax cape ground squirrel scrub hare smith’s red rock rabbit alcelaphus buselaphus caama connochaetes gnou connochaetes taurinus damaliscus pygargus dorcas antidorcas marsupialis raphicerus campestris taurotragus oryx oryx gazella pelea capreolus redunca fulvorufula procavia capensis xerus inauris lepus saxatilis pronolagus rupestris 1 2 3 2 8 1 4 10 2 2 4 3 4 2 table 2 ixodid ticks collected from mammals in the south african part of the kgalagadi transfrontier park tick species tick life stage host species (total number of ticks collected) hyalomma marginatum rufipes hyalomma truncatum rhipicephalus exophthalmos rhipicephalus theileri adult adult adult all eland (5) blue wildebeest (3), eland (39), gemsbok (62) steenbok (2), gemsbok (2), scrub hares (28) cape ground squirrels (11) table 3 ixodid ticks collected from mammals in the west coast national park, south africa tick species tick life stage host species (total number of ticks collected) hyalomma truncatum larva adult rock hyrax (1) eland (609), gemsbok (131) ixodes pilosus group adult all eland (16) gemsbok (12) rhipicephalus capensis adult bontebok (4), eland (1 898), gemsbok (234) rhipicephalus evertsi evertsi immature all bontebok (16), springbok (2) eland (68), gemsbok (246) rhipicephalus gertrudae adult eland (6), gemsbok (2) rhipicephalus glabroscutatum immature all springbok (4) eland (25), gemsbok (7 960) table 4 ixodid ticks collected from mammals in the karoo national park, south africa tick species tick life stage host species (total number of ticks collected) amblyomma marmoreum larvae springbok (4), grey rhebok (58) hyalomma glabrum adult black wildebeest (21) rhipicephalus arnoldi immature rock hyrax (29), scrub hare (3), red rock rabbit (9) rhipicephalus distinctus all rock hyrax (129) rhipicephalus exophthalmos adult springbok (22), grey rhebok (2), mountain reedbuck (13) rhipicephalus glabroscutatum immature all rock hyrax (7) grey rhebok (274), mountain reedbuck (4 916) rhipicephalus neumanni adult black wildebeest (2), springbok (2), grey rhebok (2), mountain reedbuck (2) 84 ticks (acari: ixodidae) collected from animals in nature reserves in south africa and on the lower legs of their hosts (macivor & horak 1987). one of the bontebok harboured four adult r. capensis, and one of the springbok was infested with a small number of immature r. evertsi evertsi and the other with a similar number of immature r. glabroscutatum. seven ixodid tick species were recovered from the 16 animals examined in the karoo national park (table 4). the dominant species was r. glabro scuta tum and mountain reedbuck were the most heavily infested. five fairly rarely collected tick species were recovered, namely hyalomma glabrum, rhipicephalus arnoldi, rhipicephalus distinctus, r. exophthalmos and rhipicephalus neumanni. the adults of r. arnoldi infest smith’s red rock rabbits and the immature stages infest these animals and other sympatric small mammals (walker et al. 2000). all stages of development of r. distinctus infest rock hyraxes (horak & fourie 1986; horak et al. 1991a), and although the tick does not occur throughout the range of these small mammals its distribution is dependent on the presence of its hyrax hosts (walker et al. 2000). rhipicephalus neumanni is a tick of the semiarid central and western regions of south afri ca and southern regions of namibia (walker et al. 2000). it and r. capensis are the only ticks of the 14 species collected that have distributions virtually con fined to the south-western regions of the subcontinent. acknowledgements we are most grateful to south african national parks for placing the animals in the three reserves at our disposal, and for providing assistance and facilities to process the animals for tick recovery. the assistance of mr m.m. knight and dr j.p. louw with processing the carcasses or the immobilized animals for tick recovery is greatly appreciated. the national research foundation provided funds for the conduct of this project. the publication of this work has been facilitated through the integrated consortium on ticks and tick-borne diseases (icttd-3), financed by the international cooperation program of the european union through coordination action project no. 510561. references acocks, j.p.h. 1988. veld types of south africa with accompanying veld type map, 3rd ed. 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[johannesburg]: south african institute for medical research (publication no. 50). zumpt, f. 1965. myiasis in man and animals in the old world. london: butterworths. abstract acknowledgements references appendix 1 appendix 2 about the author(s) william c. mcmahon centre for viral zoonoses, department of medical virology, faculty of health sciences, university of pretoria, pretoria, south africa jessica coertse centre for viral zoonoses, department of medical virology, faculty of health sciences, university of pretoria, pretoria, south africa centre for emerging zoonotic and parasitic diseases, national institute of communicable diseases, national health laboratory services, johannesburg, south africa teresa kearney ditsong national museum of natural history, pretoria, south africa mammal research institute, department of zoology and entomology, faculty of natural and agricultural sciences, university of pretoria, pretoria, south africa mark keith ditsong national museum of natural history, pretoria, south africa lourens h. swanepoel department of zoology, university of venda, thohoyandou, south africa wanda markotter centre for viral zoonoses, department of medical virology, faculty of health sciences, university of pretoria, pretoria, south africa citation mcmahon, w.c., coertse, j., kearney, t., keith, m., swanepoel, l.h. & markotter, w., 2021, ‘surveillance of the rabies-related lyssavirus, mokola in non-volant small mammals in south africa’, onderstepoort journal of veterinary research 88(1), a1911. https://doi.org/10.4102/ojvr.v88i1.1911 research communication surveillance of the rabies-related lyssavirus, mokola in non-volant small mammals in south africa william c. mcmahon, jessica coertse, teresa kearney, mark keith, lourens h. swanepoel, wanda markotter received: 22 oct. 2020; accepted: 03 june 2021; published: 03 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the reservoir host of mokola virus (mokv), a rabies-related lyssavirus species endemic to africa, remains unknown. only sporadic cases of mokv have been reported since its first discovery in the late 1960s, which subsequently gave rise to various reservoir host hypotheses. one particular hypothesis focusing on non-volant small mammals (e.g. shrews, sengis and rodents) is buttressed by previous mokv isolations from shrews (crocidura sp.) and a single rodent (lophuromys sikapusi). although these cases were only once-off detections, it provided evidence of the first known lyssavirus species has an association with non-volant small mammals. to investigate further, retrospective surveillance was conducted in 575 small mammals collected from south africa. nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr) assay of 329 brain samples did not detect any lyssavirus ribonucleic acid (rna). serological surveillance using a micro-neutralisation test of 246 serum samples identified 36 serum samples that were positive for the presence of mokv neutralising antibodies (vnas). these serum samples were all collected from gerbilliscus leucogaster (bushveld gerbils) rodents from meletse in limpopo province (south africa). mokola virus infections in limpopo province have never been reported before, and the high mokv seropositivity of 87.80% in these gerbils may indicate a potential rodent reservoir. keywords: bushveld gerbil; lyssavirus; mokola; non-volant small mammal; rabies-related; reservoir; rodent; surveillance. the mokola virus (mokv), a rabies-related lyssavirus, represents one of 17 recognised species within the lyssavirus genus, all capable of causing a fatal encephalitic disease (walker et al. 2018). the mokola virus is exclusively endemic in africa with only 30 sporadic cases reported since its discovery more than 50 years ago (figure 1; table 1) (coertse et al. 2017; kgaladi et al. 2013). the reservoir host of mokv is still unknown, with spillover dead-end hosts such as domestic cats (felis catus) and dogs (canis familiaris), most commonly reported to be infected with mokv. this has led to the hypothesis that the reservoir of mokv might be a prey species that interacts with domesticated animals via a prey-to-predator pathway (kgaladi et al. 2013). non-volant small mammals (i.e. shrews, sengis and rodents) have been suggested as possible reservoir hosts considering that previous mokv isolations were in shrews (crocidura spp.), four in nigeria and one in cameroon (causey et al. 1969; kemp et al. 1972; le gonidec et al. 1978), and a single reported case in a rodent (lophuromys sikapusi) in the central african republic (saluzzo et al. 1984). to investigate further, nucleic acid and serological surveillance were retrospectively conducted, targeting non-volant small mammals from specific locations in south africa. figure 1: geographical distribution of all reported mokola virus cases (n = 30) in the african continent. table 1: summary of all reported mokola virus cases in africa. non-volant small mammals were captured and sampled in accordance with the field procedure guidelines of sikes and gannon (2011) during the period of 2015–2017 from two different sites in south africa: meletse area in limpopo province (24.5914° s, 27.6258° e) and secunda area in mpumalanga province (26.5158° s, 29.1914° e). all the species investigated were designated as of least concern by the international union for conservation of nature red list of threatened species. morphological species identification followed classifications by meester et al. (1986), newbery (1999), as well as monadjem et al. (2015). following morphological identification, animals were anesthetised with isofor (safeline pharmaceuticals, south africa), after which blood was collected by cardiac puncture (1% – 3% volume/body mass) in 0.8 ml minicollect serum separator tubes (greiner bio-one, austria). serum was separated from whole blood by centrifugation (centrifuge 5418, eppendorf, germany) at 4300 g for 5 min and transferred to 2.0 ml sarstedt tubes (sarstedt inc.). animals that were not collected as voucher specimens were marked with a unique tattoo number near the base of their tail, and released back to their respective capture sites. voucher specimens were euthanised with an overdose of isofor, after which their organs were harvested (i.e. brain, tongue, salivary glands, heart, kidney, lungs, pectoral muscle, spleen, intestines, rectum and bladder) in 2.0 ml sarstedt tubes for a broader pathogen surveillance study and immediately stored in liquid nitrogen until storage at –80 °c. carcasses were placed in a 3 l pathopak (intelsius solutions, united kingdom [uk]) containing 80% ethanol and were submitted to ditsong national museum of natural history and the natural history collection for public health and economics for voucher-based morphological identification, and museum archiving. total ribonucleic acids (rnas) were extracted from brain samples (n = 329) (nine shrews, four sengis and 316 rodents) using trizol™ reagent (invitrogen, united states [us]), followed by nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qrt-pcr) assay as previously described (coertse et al. 2019). serum samples (n = 246) (three shrews, four sengis and 239 rodents) were subjected to serological surveillance using a micro-neutralisation test as previously described (smith & gilbert 2017), during which mokv 12/458 (2012, felis catus, durban, kwazulu-natal, south africa) (coertse et al. 2017) was used as challenge virus. if a reduction or absence of fluorescence was observed at the 1:25 serum dilution during initial screening, the serum sample was subjected to follow-up screening (in duplicate) at 1:10, 1:50, 1:250 and 1:1250 serum dilutions. the 50% end-point (ed) neutralisation titre was calculated by the reed and muench method (1938) and considered positive for mokola virus neutralising antibodies (mokv vnas) when they had a 50% ed neutralisation titre at a serum dilution of ≥ 25 (i.e. where ≤ 5 out of the 10 counted fields contain infected cells at the 1:25 serum dilution). if additional material was available, non-volant small mammals that tested positive for the presence of mokv vnas were subjected to genetic species identification with the cytochrome b (cytb) barcoding pcr assay as previously described (greenberg et al. 2012). template deoxyribonucleic acid (dna) required for the barcoding assay was extracted from various biological sample types (such as blood, kidney, heart and pectoral tissue) using the quick-dna™ miniprep plus kit (zymo research, us). all of the brain samples were negative for the presence of viral rna with the pan-lyssavirus qrt-pcr assay (appendix table 1-a1). negative results were expected as these animals were apparently healthy individuals and did not exhibit any visible signs of disease. an overall mokv seropositivity of 87.80% (36 out of 41) was observed for the gerbils (gerbilliscus leucogaster) tested from meletse at the cut-off 1:25 serum dilution (figure 2; appendix tables 1-a1, 2-a1, 3-a1 & 4-a1). the titre ranges for this rodent species were high when compared to another serological surveillance study conducted in zimbabwe (foggin 1988). foggin identified mokv vnas in 5.63% (18 out of 320) of all rodents that were tested. an overall mokv seropositivity of 17.57% (13 out of 74) was observed for gerbils which neutralised mokv infection at various serum dilutions that ranged from 1:8, 1:16 to 1:32. none of the other mokv serological surveillance studies have tested this rodent species for the presence of mokv vnas (aghomo et al. 1990; kemp et al. 1972; nottidge, omobowale & oladiran 2007; ogunkoya et al. 1990). even though mokv has been shown to cross-react in serological assays with other closely-related lyssaviruses (kuzmin et al. 2008), cross-reactivity with other phylogroup ii lyssaviruses was not investigated in this study. figure 2: graphical representation of the micro-neutralisation test results of the gerbilliscus leucogaster serum samples from meletse (n = 36). of the 36 gerbils showing mokv seropositivity, only 28 were genetically identified with the cytb barcoding pcr assay (table 2). the same identification was obtained from morphological examination of 24 voucher specimens (table 2). eight gerbils could not be identified to species level as they were released and no additional sample material was available. the highveld gerbil, gerbilliscus brantsii, is sympatric with g. leucogaster, however, based on known museum records, no g. brantsii has been caught at meletse before (rautenbach 1982) and these were, therefore, allocated to g. cf. leucogaster. the variability observed in the per cent identity (i.e. 83.78% – 100.00%) between the individual gerbils is expected since previous molecular characterisation assays performed on the gerbilliscus genus have recorded intra-species genetic variation that range from 1% to 20% (aghová et al. 2017; colangelo et al. 2007). table 2: genetic and morphological species identification and voucher information for all gerbilliscus leucogaster serum samples from meletse, limpopo province that were positive for the presence of mokola virus neutralising antibodies. members of the gerbilliscus genus are nocturnal and terrestrial, exhibit no sexual dimorphism (skinner & chimimba 2005) and occupy simple to complex, deep burrows (i.e. warrens) (de graaff 1981; granjon & dempster 2013). they are physiologically, morphologically and behaviourally adapted to live in arid climates (granjon & dempster 2013; monadjem et al. 2015). gerbilliscus leucogaster, however, is less arid adapted and can be found along rivers and drainage lines in open grasslands and wooded savannas (dempster 2013; monadjem et al. 2015). the breeding pattern and social organisation of g. leucogaster rodents are not well-understood, however, studies have reported a communal nature (de graaff 1981; smithers 1971) with burrows being occupied by a pair (skinner & chimimba 2005) and some warrens housing families or several adults (choate 1972). the ecological nature of bushveld gerbils may potentially be the reason why this specific rodent species are more likely to be mokv seropositive compared to solitary rodent species belonging to the steatomys and rhabdomys genera occurring at meletse. more nucleic acid and serological surveillance studies in non-volant small mammal populations are required to obtain a better understanding of mokv distribution, prevalence and its potential reservoir species. brain and serum samples in this study were collected from seemingly healthy small mammals in areas that do not coincide with areas where previous mokv cases have been reported in south africa. surveillance should be expanded to areas where mokv spillover infections in cats and dogs have previously been reported. furthermore, because lyssavirus distribution and dynamics might be influenced by seasonality, surveillance efforts should also include samples that were collected in different seasons and over multiple years. this expansion, together with representative sample sizes of certain non-volant small mammal species, will collectively increase the possibility of identifying more of these animals that are infected or that have previously been exposed to mokv. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions w.c.m. performed all experiments associated with this study which forms part of his m.sc. medical virology degree. j.c. and w.m. provided academic guidance and supervised the overall process and operations of this study. t.k., m.k. and l.h.s. assisted with non-volant small mammal sample collection and species identification in the field. t.k. provided museum information from the ditsong national museum of natural history. all authors contributed equally to the construction of this research communication. ethical considerations this study formed part of a larger surveillance programme of the bio-surveillance and ecology of emerging zoonoses research group in the centre for viral zoonoses that focuses on zoonotic pathogens in bats and non-volant small mammals. the overall research had animal ethical clearance from the university of pretoria’s animal ethics committee (aec) (principal investigator: w.m.; project reference number: ec071-15) and had permission to do research in terms of section 20 of the animal diseases act of 1984 (act no. 35 of 1984) from the department of agriculture, land reform and rural development (dalrrd) (project name: epidemiology of zoonotic pathogens in rodents, shrews and sengis in southern africa; project reference number: 12/11/1/1/8). sampling permits were obtained from limpopo’s department of economic development, environment and tourism (za/lp/73972 [2016–2017] and za/lp/83642 [2017–2018]) and mpumalanga’s tourism and parks agency (mpb.5583 [2017]). the m.sc. committee from the university of pretoria’s school of medicine, faculty of health sciences approved the protocol of this research project (project reference number: 13057368). individual animal ethical clearance (principal investigator: wm.; project reference number: h008-18), as well as research ethical clearance was obtained from the university of pretoria’s aec and research ethics committee (project reference number: 426/2018). funding information this study was funded by the south african research chair in infectious diseases of animal (zoonoses) from the national research foundation of the department of science and innovation, w.m. (uid98339), as well as additional grants awarded to w.m. by the nrf (uid92524, uid85756 and uid91496). the national research foundation for funding the equipment based at the dna sanger sequencing facility in the faculty of natural of agricultural sciences, university of pretoria (uid78566) and the poliomyelitis research foundation. data availability the authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. disclaimer the contents of this research communication are solely the responsibility of the authors. the opinions, findings and conclusions expressed do not necessarily reflect the official view of the national research foundation. references aghomo, h.o., tomori, o., oduye, o.o. & rupprecht, c.e., 1990, ‘detection of mokola virus neutralising antibodies in nigerian dogs’, research in veterinary science 48(2), 264. https://doi.org/10.1016/s0034-5288(18)31005-1 aghová, t., šumbera, r., piálek, l., mikula, 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mammals of botswana, trustees of the national museum of rhodesia, salisbury. smith, t.g. & gilbert, a.t., 2017, ‘comparison of a micro-neutralization test with the rapid fluorescent focus inhibition test for measuring rabies virus neutralizing antibodies’, tropical medicine and infectious disease 2(3), 24. https://doi.org/10.3390/tropicalmed2030024 walker, p.j., breyta, r., blasdell, k.r., calisher, c.h., dietzgen, r.g., fooks, a.r. et al., 2018, ‘rhabdoviridaee’, in j.h. kuhn & s.g. siddel (eds.), ictv report negative-sense rna viruses, journal of gen eral virology, 99, 447–448. viewed 24 may 2020, from https://talk.ictvonline.org/ictv-reports/ictv_online_report/negative-sense-rna-viruses/w/rhabdoviridae appendix 1 table 1-a1: non-volant small mammal species included in the surveillance of mokola virus in south africa. table 2-a1: sampling event details of collected gerbilliscus leucogaster serum samples from meletse, limpopo province and their seropositivity. table 3-a1: the 50% end-point neutralisation titres of all gerbilliscus leucogaster serum samples from meletse, limpopo province that were positive for the presence of mokola virus neutralising antibodies. table 4-a1: micro-neutralisation test results of all non-volant small mammal serum samples that tested negative for the presence of mokola virus neutralising antibodies. appendix 2 additional references bingham, j., javangwe, s., sabeta, c.t., wandeler, a.i. & nel, l.h., 2001, ‘report of isolations of unusual lyssaviruses (rabies and mokola virus) identified retrospectively from zimbabwe’, journal of the south african veterinary association 72(2), 92–94. https://doi.org/10.4102/jsava.v72i2.624 causey, o.r. & kemp, g.e., 1968, ‘surveillance and study of viral infections of vertebrates in nigeria’, nigerian journal of science 2, 131–135. familusi, j.b. & moore, d.l., 1972, ‘isolation of a rabies-related virus from the cerebrospinal fluid of a child with “aseptic meningitis”’, african journal of medical 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https://doi.org/10.1023/a:1008120511752 sabeta, c.t., blumberg, l., miyen, j., mohale, d., shumba, w. & wandeler, a., 2010, ‘mokola virus involved in a human contact (south africa)’, fems immunology & medical microbiology 58(1), 85–90. https://doi.org/10.1111/j.1574-695x.2009.00609.x sabeta, c.t., markotter, w., mohale, d.k., shumba, w., wandeler, a.i. & nel, l.h., 2007, ‘mokola virus in domestic mammals, south africa’, emerging infectious diseases 13(9), 1371. https://doi.org/10.3201/eid1309.070466 von teichman, b.f., de koker, w.c., bosch, s.j.e., bishop, g.c., meredith, c.d. & bingham, j., 1998, ‘mokola virus infection: description of recent south african cases and a review of the virus epidemiology: case report’, journal of the south african veterinary association 69(4), 169–171. https://doi.org/10.4102/jsava.v69i4.847 mukaratirwa_173-180.indd introduction trichinellosis is a parasitic zoonosis manifesting in humans as a syndrome with specific clinical signs and symptoms of variable intensity (kocieka 2000). the infection is contracted by eating raw or undercooked infected meat resulting in various symptoms that range from mild subclinical to classic forms depending on the extent of invasion, the parasite load, the species of trichinella involved and the immune response of the host (clausen, meyer, krantz, moser, gomme, kayser, albrectsen, cui, wang, wu & jin 1997; taratuto & venturiello 1997; kocieka 2000). 173 onderstepoort journal of veterinary research, 75:173–180 (2008) experimental infections of baboons (papio spp.) and vervet monkeys (cercopithecus aethiops) with trichinella zimbabwensis and successful treatment with ivermectin s. mukaratirwa1*, b.m. dzoma1, e. matenga1, s.d. ruziwa1, l. sacchi2 and e. pozio3 abstract mukaratirwa, s., dzoma, b.m., matenga, e., ruziwa, s.d., sacchi, l. & pozio, e. 2008. experimental infections of baboons (papio spp.) and vervet monkeys cercopithecus aethiops) with trichinella zimbabwensis and successful treatment with ivermectin. onderstepoort journal of veterinary research, 75:173–180 experimental trichinella zimbabwensis infections were established in three baboons (papio sp.) and four vervet monkeys (cercopithecus aethiops) and the clinical-pathological manifestations assessed. the infected animals showed clinical signs ranging from fever, diarrhoea, periorbital oedema and muscular pain in varying degrees. one baboon became blind due to the infection. levels of creatinine phosphokinase and lactate dehydrogenase increased to reach a peak on day 42 post-infection (pi) for both baboons and monkeys. blood parameters such as packed cell volume, levels of red blood cells and white blood cells did not change significantly from the normal ranges except for the levels of eosinophils which peaked above the normal ranges at day 28 and 56 pi in baboons and at day 56 pi in monkeys. two baboons and two monkeys died during the course of the experiment. they were emaciated and showed lesions such as ascites, hydropericardium, congested liver and enlarged gall bladder. histopathological findings of various muscles included a basophilic transformation of muscle cells, the dis appearance of sarcomere myofibrils and basophilic sarcoplasm with the presence of trichinella larvae in the sarcoplasm. these changes were mainly in the massetter and were of various intensities in the tail, gastrocnemius and biceps muscles. five consecutive treatments with an oxfendazole-levamisole combination on surviving animals failed to clear the infection whereas ivermectin cleared the infection after one treatment in two monkeys and after two treatments in a baboon. keywords: baboons, cercopithecus aethiops, ivermectin, levamisole, monkeys, oxfendazole, papio spp., pathology, trichinella zimbabwensis * author to whom correspondence is to be directed. e-mail: mukaratirwa@ukzn.ac.za 1 department of paraclinical veterinary studies, faculty of veterinary science, university of zimbabwe, p.o. box mp 167 mount pleasant harare, zimbabwe 2 department of animal biology, university of pavia, piazza botta 9, 27100 pavia, italy 3 department of infectious, parasitic and immunomediated diseases, istituto superiore di sanità, viale regina elena 299, 0061 rome, italy accepted for publication 21 april 2008—editor 174 experimental infections of baboons and vervet monkeys with trichinella zimbabwensis the description of a new non-encapsulated species, trichinella zimbabwensis, by mukaratirwa & foggin (1999), pozio foggin, marucci, la rosa, sacchi, corona, rossi & mukaratirwa (2002), which affects both mammals and reptiles is a new addition to the already existing five encapsulated species infecting only mammals (trichinella spiralis, trichinella nativa, trichinella britovi, trichinella murrelli and trichinella nelsoni) and the two non-encapsulated species trichinella pseudospiralis occurring in both mammals and birds and trichinella papuae occurring in both mammals and reptiles (pozio, owen, marucci & la rosa 2004a). the only report of a natural infection of t. zimbabwensis is in farmed crocodiles (crocodylus niloticus) in zimbabwe (foggin, vassilev & widdowson 1997), where animals are reared for their meat and skin. experimental infections have shown that t. zimbabwensis readily infects domestic pigs and laboratory mice and rats (mukaratirwa & foggin 1999; mu karatirwa, nkulungo, matenga & bhebhe 2003) confirming its chance to complete the entire life cycle irrespective of whether the host is homoiothermic or poikilothermic (pozio, marucci, casulli, sacchi, mukaratirwa, foggin & la rosa 2004b). however, the epidemiology and the public health significance of this parasite are still obscure. non-human primates by virtue of their close relationship to humans are good models for the study of the clinical-pathological manifestations of trichinellosis and its treatment. the objective of this study was to evaluate the clinical-pathological manifestations of baboons (papio spp.) and vervet monkeys (cercopithecus aethiops) experimentally infected with t. zimbabwensis and their response to treatment. materials and methods experimental animals two adult female (codes sfb and bfb) and one male (code smb) baboons and two adult female (codes bfm and sfm) and two male (codes smm and bmm) vervet monkeys were randomly selected for the study. these animals were born and bred at the animal house unit of the faculty of veterinary science, university of zimbabwe. they were reared on a commercial primate diet and water was available ad libitum. a week before the infection, biopsies were collected from the animals while they were under anaesthesia with a combination of xylazine (2 mg/kg) and ketamine (10 mg/kg) administered intramuscularly, from the massetter, biceps and gastrocnemius muscles to ensure that the animals were free from trichinella spp. infection. restraint and experimental infection of animals the t. zimbabwensis used was an isolate derived from a naturally infected crocodile. it was maintained in the laboratory by serial passages in rats. firststage larvae were collected from five skinned carcasses of infected rats following methods described by pozio et al. (2002). each experimental animal was anaesthetized with a combination of xylazine (2 mg/kg) and ketamine (10 mg/kg) intramuscularly. two of the baboons were infected with doses ranging from 9 000–16 600 larvae per kg, whereas 17 600–25 000 larvae per kg were used to infect three of the monkeys (table 1). one baboon and one monkey were kept as uninfected controls. sample collection and analysis muscle biopsies and blood samples were collected while the animals were under anaesthesia at weekly intervals after infection and at each time of collection the rectal temperatures were recorded. the muscle biopsies were processed by the hcl-pepsin digestion method (pozio et al. 2002) to detect the l-1 stage of t. zimbabwensis and the resulting larval counts were expressed as larvae per gram (lpg) of muscle. a portion of the muscle biopsy from each muscle was preserved in 3 % glutaraldehyde and processed for electron microscopy. the levels of creatinine kinase (ck) and lactate dehydrogenase (ldh) were determined in serum samples. the clinical signs were recorded daily until the end of the experiment. the animals were treated for 5 consecutive days with a combination of levamisole (40 mg/kg) and oxfendazole (50 mg/kg) and dexamethasone (5 mg/kg) on days 35 and 51 post-infection (pi) for the monkeys and baboons, respectively. an intramuscular injection of ivermectin (300 μg/kg) was administered after the levamisole-oxfendazole treatment. the efficacy of treatment was monitored through weekly collection of muscle biopsies from the masseter muscles to detect viable first stage larvae. histological and ultrastructure studies of the nurse cell-parasite complex muscle biopsies and muscles of animals which died during the study were cut into small sections and in 175 s. mukaratirwa et al. part fixed in 10 % buffered formalin and in part preserved in glutaraldehyde for ultrastructural studies. for histology, fixed specimens were dehydrated using different grades of alcohol, cleared in xylene embedded in paraffin wax at 58–60 °c and finally, 3 μm sections were stained by haematoxylin and eosin stain (anderson & gordon 1996). sections were examined by light microscopy using 5–40x magnification. small pieces of the biopsy from the masseter muscle of a baboon, collected 51 days pi, were fixed for 4 h at 4 °c in 0.1 m cacodylate buffer (ph 7.2) containing 2.5 % glutaraldehyde. the samples were then washed in the same buffer and post-fixed for 1.5 h at 4 °c with 1 % oso4 in cacodylate buffer. all samples were dehydrated in ethanol and embedded in epon 812 for sectioning. for light microscopy, semithin sections (0.5 μm) were stained with 0.5 % toluidine blue. thin sections (80 nm), stained with uranyl acetate and lead citrate, were examined under a zeiss em 900 transmission electron microscope. results parasitological aspects the pre-infection muscle biopsies from all experimental animals were negative for trichinella larvae. in the infected baboons that died, muscles with the highest lpg were the diaphragm, psoas, laryngeal, and tongue in this order (table 1). in monkeys, the diaphragm, tongue and masseter had the highest lpg compared to the other muscles (table 1). no relationship was observed between the infective dose, the severity of the disease and the lpg in both baboons and monkeys. clinical manifestations the clinical signs observed are summarized in table 2. the time between infection and onset of the first clinical signs ranged from 9 to 30 days pi. the rectal temperatures ranged between 38.4–40 °c in the baboons and 38.9–39.9 °c in the monkeys for a period of 8 weeks. table 1 number of larvae per gram (lpg) of muscle in baboons and monkeys that died due to trichinella zimbabwensis infection larvae per gram of muscle animal code infective dose/kg day of death pi diaphragm massetter tongue psoas laryngeal muscle digital flexus hamstring muscle eye sfb 16 600 50 621 260 820 965 545 552 910 37 smb 16 400 49 9 987 465 888 1 819 1 600 287 479 144 bmm 17 600 35 255 nd 215 nd 135 75 115 nd bfm 25 000 30 100 168 165 nd nd 86 nd 1 nd = not determined pi = post infection table 2 clinical manifestations of baboons and monkeys infected with trichinella zimbabwensis animal code sfb smb bfb bfm sfm smm bmm infective dose/kg 16 600 16 400 7 300 25 000 24 500 24 500 17 600 clinical signs and symptoms day of the first manifestation pi fever diarrhoea depression periorbital oedema muscular pain alopecia blindness death 9 16 12 30 30 26 38 49 dpi 11 16 12 30 30 26 no 50 dpi 16 16 12 30 30 26 no s 14 16 12 30 30 26 no 30 dpi 13 19 12 30 30 26 no s 18 21 12 30 30 26 no 36 dpi 10 21 12 30 30 26 no s pi = post infection, dpi = days post infection, no = not observed, s = survived 176 experimental infections of baboons and vervet monkeys with trichinella zimbabwensis diarrhoea was observed in all infected animals from day 16 pi (table 2). gradual depression and deterioration in body condition accompanied by alopecia, muscle pain on palpation and wasting were observed in all infected animals and these became marked from day 26 pi. blindness of both eyes was only observed in one baboon (sfb). clinical biochemistry and blood parameters levels of ck ranged from 197 to 3409 ui/ℓ in baboons and from 78 to 2679 ui/ℓ in the infected monkeys. the levels of cpk in the infected baboons gradually increased from day 0 to day 28 pi and then increased tenfold to reach a peak on day 42 pi. after the peak, there was a sharp drop to reach normal levels from day 56 pi (fig. 1a and b). in infected monkeys, the ck levels slightly decreased from day 0 pi to day 28 pi before starting to increase with a peak on day 56 pi. levels of ldh ranged from 118 to 1587 ui/ℓ in baboons and from 203 to 1469 ui/ℓ in monkeys. in both monkeys and baboons, ldh reached a peak at day 42 pi. packed cell volume (pcv) and white cell counts did not change significantly in the course of the infection. however, the level of eosinophils peaked at day 28 and 56 pi above the normal ranges in baboons and monkeys, respectively. treatment the 5-day-treatment with oxfendazole-levamisole combination failed to kill trichinella larvae; on the contrary, the single ivermectin treatment successfully destroyed the larvae in all the surviving infected monkeys (bmm and smm) but in the baboon (bfb) a low level of larvae was still detected in the biopsy (table 3). in this animal, no living larvae were detected in another muscle biopsy collected after a second treatment with ivermectin. histopathology and ultrastructure of muscle tissues all the animals which died due to the infection (sfb, smb, bfm and fmm) were emaciated and showed ascites, hydropericardium, congested livers and enlarged gall bladders. histopathological findings of various muscles included a basophilic transformation of muscle cells, the disappearance of sarcomere myofibrils and a sarcoplasm that was basophilic. the changes also included pale, swollen muscle fibres with the presence of trichinella larvae in the sarcoplasm, and the loss of myofibrils in affected muscles. these changes were mainly in the masseter, and were of various intensities in the tail, gastrocnemius and bicep muscles. biliary fibrosis, eosinophilic myositis, lymphocytic enteritis and dilatation of lymphatic vessels of the large and small intestines were also observed in sfb. in muscles infected with t. zimbabwensis, clear mod ifications of the muscle fibre architecture were observed. the myofilaments appeared modified in a nurse-cell-like structure (fig. 2a). the thick collagen capsule was absent, and no inflammatory reaction was observed around the nurse cell-larva complex. transmission electron micrographs revealed the ultrastructural changes in the muscle fibres. in the nurse-cell-like structure the contractile elements 3 500 3 000 2 500 2 000 1 500 1 000 500 0 iu /ℓ 0 14 28 42 56 60 days post-infection baboon (n = 2) a ldhck 3 500 3 000 2 500 2 000 1 500 1 000 500 0 iu /ℓ 0 14 28 42 56 60 days post-infection monkey (n = 2) b fig. 1 kinetics of creatinine phosphokinase (ck) and lactate dehydrogenase (ldh) in a baboons bfb and smb (a) and monkeys sfm and bmm (b) infected with trichinella zimbabwensis 177 s. mukaratirwa et al. fig. 2 larvae of trichinella zimbabwensis in muscles of a baboon 51 days post infection a longitudinal section of an intracellular larva (l), surrounded by a nurse-cell-like structure (nc). note the absence of the typical collagen capsule. no inflammatory cells were observed around the infected muscle fibres. mc = normal muscle cell. bar = 200 μm b tem micrograph showing a trichinella larva (l) surrounded by a nurse-cell-like structure (nc). note the loss of contractile elements. bar = 5 μm c details of the nurse-cell-like structure (nc) showing a hypertrophic nucleus with the nucleolus (nu) in the transcriptional phase. bar = 5 μm d details of the outer zone of the nurse-cell-like structure (nc) showing bands of collagen fibres (arrows). mc = normal muscle cell, n = fibroblast nucleus, nc = nurse-cell-like structure. bar = 5 μm a b c d 178 experimental infections of baboons and vervet monkeys with trichinella zimbabwensis were replaced by a sarcoplasmic reticulum (fig. 2b). at higher magnification, in the cytoplasm of this structure, characterized by the presence of a smooth and rough endoplasmic reticulum, it was possible to observe hypertrophic nuclei each of which contained a prominent nucleolus (fig. 2c). this detail was indicative of the presence of an intense transcriptional activity. at the peripheral zone of the nurse-cell larva complex, bundles formed by irregularly arranged collagen fibres were observed (fig. 2d). discussion in this study, t. zimbabwensis was able to establish in both baboons and monkeys and the clinical signs observed did not differ from those already reported in humans infected with the non-encapsulated species t. pseudospiralis (ranque, faugére, pozio, la rosa, tamburrin, pellissier & brouqui 2000). the normal rectal temperatures of baboons and monkeys are 36–39 °c and 36–40 °c, respectively (poole 1987). in the course of trichinellosis in humans, fever is one of the most common sign expected (clausen et al. 1997) and in this study it was recorded in both infected baboons and monkeys. the observation of diarrhoea in infected animals in this study around day 16 pi could be related to the early stages of intestinal invasion which manifests clinically as diarrhoea of several days’ duration and abdominal pains (kociecka 2000). the pathophysiology of trichinellosis in the small intestines includes restricted absorption, disturbed motility of the intestines, diarrhoea or constipation and the presence of cellular infiltrates in the lamina propia (ruitenberg, elgersma, kruizing & leenstra 1977; castro & bullick 1983; gustowska, ruitenberg, elgersma & kociecka 1983; ruitenberg & buys 1986). in humans infected with t. spiralis, pathomorphology of the intestinal mucosa includes lesions to the epithelium involving the brush border, lamina propia and smooth muscles of the jejunum, deformation of villi, stimulated enterocyte proliferation at villi margins, hyperplasia in the crypts of lieberkuhn and the presence of massive cellular infiltrates in the mucosal sublayer (castro & bullick 1983). lesions may persist until day 65 pi (kociecka 1981a; gustowska et al. 1983). in this study, similar intestinal lesions were noted from a baboon that died on day 49 pi. periorbital and facial oedema is a common clinical sign of t. spiralis and t. pseudospiralis trichinellosis (kociecka 1981a) and this agrees with the findings from this study where all affected animals manifested periorbital oedema. the observation that the intensity of clinical disease and the muscle larval yields were not proportional to the initial infective doses given to the animals might suggest host factors that determine disease resistance since the parasite and environment factors were similar for all the animals. the experimental infection of swiss cd1 mice reveals the existence of structural changes of the infected muscle cells 4 months pi. these structures were very similar to those observed in crocodiles 18 months pi (pozio et al. 2002). in the baboon infected with the same species of trichinella, the myofilaments of the infected muscle cell had lost their integrity and appear transformed in a nurse-cell-like structure surrounded by a bundle of collagen fibres irregularly arranged (a true collagen capsule being absent) 51 days pi. these results suggest that t. zimbabwensis is able to infect different mammalian species in which it reproduces the same morphological changes as have been described in reptiles (pozio et al. 2002, 2004b). the choice of treatment for trichinellosis in humans varies according to the clinical severity of the infection and the strain or species of trichinella involved (andrews, ainsworth & abernethy 1994). drugs administered to infected humans include anthelmintics, glucocorticosteroids, immuno-modulating drugs and preparations which compensate protein and electrolyte deficits (kociecka 2000). in this study, since table 3 larvae per gram (lpg) of masseter muscle following treatment with oxfendazole-levamisole combination and subsequent use of ivermectin in three monkeys and one baboon infected with trichinella zimbabwensis animal code lpg after oxfendazolelevamisole treatment lpg after one ivermectin treatment lpg after second ivermectin treatment bmm smm *sfm bfb 11.1 9.5 6.4 44.1 0 0 – 7.5 – – – 0 * died 36 days post infection 179 s. mukaratirwa et al. adult worms had long been expelled the treatment was targeted at the larvae in muscles. injectable ivermectin was able to clear the infection after 7 days following a single dose unlike the oxfendazolelevamisole combination which failed to clear the infection in both the monkeys and baboons when administered for 5 consecutive days. however, it was not determined whether the effects of ivermectin were potentiated by the earlier use of the combination of oxfendazole-levamisole or were due solely to the anthelmintic effects of the drug. disturbances in blood muscle enzyme activities are related primarily to activities of those enzymes that are muscle bound, and these include ck, ldh and occasionally aspartate aminotransferase (ast). this follows the damage of the muscle cell by the l-1 resulting in an increase in permeability of the muscle cell membranes (boczon, winiecka, kociecka, hadas & andrezejewska 1981). in 75–90 % of human cases of trichinellosis, an increase in the activity of ck was noted between weeks 2 and 5 pi and involved an increase of several fold and ldh levels fluctuated from week 1 to week 6 pi. in this study, ldh and ck peaked around day 42 pi in both baboons and monkeys although there were phases where the levels of both enzymes dropped. this is unsurprising since the increase in ck and ldh activity in blood is not correlated to the clinical severity of trichinellosis (boczon et al. 1981) and there could also be some host differences in reacting to different trichinella spp. the peak around day 42 pi of both enzymes could be related to the fact that almost all of the parasites are expected to be in the muscles by that period resulting in marked muscle membrane damage (boczon et al. 1981). the settling of trichinella larvae in the muscles results in the basophilic transformation of the muscle cells, encapsulation of the larvae and the development of a capillary network surrounding the affected cell (gabryel & blotna 1969; gabryel, gustowska & blotna-filipiak 1995). these changes appear early and persist for as long as the encapsulated larva remains viable. in this study the basophilic changes were observed together with an eosinophilic infiltration around the affected cell. the muscles that were preferred most by the parasite were the diaphragm, psoas, laryngeal, tongue and masseter muscles in that order. the masseter muscle, however, had the greatest histopathological changes attributable to the parasite. trichinella pseudospiralis and t. spiralis larvae have been shown to have the massetter muscle as their main predilection sites in monkeys (kociecka 1981b), and this probably explains the high histopathological changes in the massetter muscles from our animals. leukocytosis appears early and rapidly increases between weeks 2 and 5 of the disease and subsides in parallel to clinical signs while eosinophilia persists (kociecka 2000). however, an extremely severe course of trichinellosis may be accompanied by eosinopenia and/or lymphopenia which is a manifestation of immunosuppression (dupoy-camet, paugam, picard & ancelle 1994). in our study, eosinophilia was noted, albeit between weeks 4 and 8 pi. however, eosinophilia has been reported to regress slowly and may persist from several weeks to 3 months pi, and no relationship has been noted between the clinical course of disease and eosinophilia (dupoy-camet et al. 1994). the clinical, parasitological, pathological, biochemical and haematological picture of t. zimbabwensis infection in the baboons and monkeys in this study closely simulates those reported for t. pseudospiralis and t. spiralis infection in humans and other primates (clausen et al. 1997; taratuto & venturiello 1997; ranque et al. 2000). the fact that t. zimbabwensis could successfully infect non-human primates to give clinical features characteristic of other trichinella species in humans could have relevant implications in human infection as chances of humans getting infected with t. zimbabwensis are pres ent in zimbabwe where crocodile meat is consumed. acknowledgements we thank the university of zimbabwe for financial support and staff of the university animal house and parasitology section, department of paraclinical veterinary studies for their assistance in handling the animals and processing of the samples. references anderson, g. & gordon, c.k. 1996. 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [595.276 841.890] >> setpagedevice article information authors: nma b. alhaji1 ismail a. odetokun2 aminu shittu3 joshua onyango4 umar m. chafe5 muhammed s. abubakar6 issa a. muraina7,8 folorunso o. fasina9 hu suk lee10 affiliations: 1epidemiology unit, ministry of livestock and fisheries development, nigeria 2department of public health and preventive medicine, university of ilorin, nigeria 3department of theriogenology and animal production, usmanu danfodiyo university, nigeria 4animal welfare and veterinary health, university of northampton, united kingdom 5department of medicine and surgery, usmanu danfodiyo university, nigeria 6department of pathology and microbiology, usmanu danfodiyo university, nigeria 7diabetes and nutritional science division, king’s college, united kingdom 8national veterinary research institute, akure, nigeria 9department of production animal studies, university of pretoria, south africa 10international livestock research institute, regional office for east and southeast asia, hanoi, vietnam correspondence to: hu suk lee email: h.s.lee@cgiar.org postal address: international livestock research institute, regional office for east and southeast asia, 17a nguyen khang street, cau giay district, hanoi, vietnam dates: received: 30 june 2015 accepted: 06 oct. 2015 published: 15 dec. 2015 how to cite this article: alhaji, n.b., odetokun, i.a., shittu, a., onyango, j., chafe, u.m., abubakar, m.s. et al., 2015, ‘time-series analysis of ruminant foetal wastage at a slaughterhouse in north central nigeria between 2001 and 2012’, onderstepoort journal of veterinary research 82(1), art. #1010, 13 pages. http://dx.doi.org/10.4102/ojvr.v82i1.1010 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. time-series analysis of ruminant foetal wastage at a slaughterhouse in north central nigeria between 2001 and 2012 in this original research... open access • abstract • introduction • materials and methods    • study area    • data sources    • seasonal-trend decomposition and statistical analyses • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references • appendix 1 • appendix 2 abstract top ↑ in developing countries, foetal wastage from slaughtered ruminants and the associated economic losses appear to be substantial. however, only a limited number of studies have comprehensively evaluated these trends. in the current study, secondary (retrospective) and primary data were collected and evaluated to estimate the prevalence of foetal wastage from cattle, sheep and goats slaughtered at an abattoir in minna, nigeria, over a 12-year period (january 2001 – december 2012). time-series modelling revealed substantial differences in the rate of foetal wastage amongst the slaughtered species, with more lambs having been wasted than calves or kids. seasonal effects seem to influence rates of foetal wastage and certain months in the year appear to be associated with higher odds of foetal wastage. improved management systems are suggested to reduce the risk of foetal losses. introduction top ↑ ruminant livestock (cattle, sheep and goats) are a source of food (meat and milk) and are also used to produce animal feed, clothing, shelter, weapons, utensils and industrial items (church 1993). food animals also play an important role in providing financial security for rural populations and in improving a nation’s economy, especially in developing countries (alhaji 2011; alhaji & odetokun 2013). in nigeria, the annual consumption of animal protein (meat, milk, eggs and fish) amounts to 8.8 kg/person compared to the global average of 41.9 kg/person (food and agriculture organization [fao] 2013). countries such as australia and the usa consume approximately 111.5 kg/person/year and 120.2 kg/person/year, respectively (fao 2013). the comparison suggests that nigeria still rates as a developing country, with low per capita production and consumption of animal protein (nwakpu & ugwu 2004). nigeria’s cattle, sheep and goat populations were recently estimated at 19.2 million, 38.5 million and 57.4 million, respectively (fao 2014), with annual growth rates that are too low to satisfy the requirements of the increasing human population. the nigerian population was estimated at over 174 million in 2013 (population reference bureau 2013) and a relative increase in wealth will mean that the demand for meat as a source of protein will continue to grow. the high demand for sources of animal protein has led to practices such as indiscriminate slaughter of young and pregnant female animals, which is seen routinely in some nigerian abattoirs (taiwo, aluko & olufowobi 2006). this practice results in wastage of scarce protein available to consumers and a decrease in the livestock growth capacity of the country owing to low herd replacement rates (cadmus & adesokan 2010). the indiscriminate slaughter of pregnant females and the consequent wastage of embryos and foetuses are regarded as major destructive mechanisms that counteract food production efforts (abassa 1995). the magnitude of foetal losses due to the slaughter of reproductively active dams has been reported amongst both large (cows) and small (ewes and does) ruminants from several abattoirs in the south west (cadmus & adesokan 2010; oyekunle, olubanjo & fasina 1992), south east (wosu 1988; wosu & dibua 1992), north central (alhaji 2011; alhaji & odetokun 2013), north west (garba et al. 2010; muhammad, ashiru & abdullahi 2007) and north east (bokko 2011; chaudhari & bokko 2000) zones of nigeria. the practice of slaughtering pregnant ruminants has had a negative effect on the national herd size and total meat production, restricting availability of animal protein. the associated economic losses constrain the contribution of livestock to the gross domestic product in the country (alhaji 2011; alhaji & odetokun 2013; ngbede et al. 2012). substantial foetal calf wastage has been recorded at an abattoir in northern nigeria (alhaji 2011), which translated to an average annual financial loss of close to us$290 000 (ngbede et al. 2012). reasons for the reported continuous slaughter of pregnant ruminants should be investigated to predict associated trends and facilitate sound strategic planning and decision-making in combating future foetal livestock wastage in nigeria. none of the aforementioned reports have adequately addressed an empirical trend analysis of ruminant foetal wastage. specifically, seasonal slaughter trends should be studied to determine slaughter patterns to allow future predictions. previous limitations in this regard may be due to a lack of detailed abattoir slaughter records, only short periods covered by available information and incomplete data. we present an empirically sound trend analysis to evaluate and compare seasonal patterns and variations of ruminant foetal wastage in nigeria over a 12-year period. materials and methods top ↑ study area the study was conducted at the minna metropolitan abattoir (mma), a major slaughterhouse in the capital of niger state, north central nigeria (6°1′n; 9°5′e) (see figure 1). in 2007, the human population in the city was estimated at just more than 304 000. figure 1: map of nigeria, with the location of minna in the northern central region of nigeria indicated. data sources secondary data on animals presented at the abattoir between 2001 and 2010 were obtained retrospectively from the records of the zonal veterinary office, niger state ministry of livestock and fisheries development, minna. in addition, primary data were obtained for 2011 and 2012 from direct anteand post-mortem inspections in the lairage and slaughterhouse of the mma. the retrospective data were considered valid based on the detailed pregnancy statuses and foetal wastages recorded. all the female animals presented for slaughter between 2011 and 2012 were assessed for pregnancy status and foetal wastage at the slaughterhouse and the data recorded. the data were entered into a spreadsheet and exported to statistical software applications (stata version 11.2 [statacorp, college station] and r version 3.2.2 [https://cran.r-project.org/bin/windows/base/]). abattoir foetal wastage was defined as the removal and subsequent discard of one or more foetuses from the gravid uterus of a female animal. seasonal-trend decomposition and statistical analyses monthly and total foetal wastage rates were expressed as the total number of pregnant females (npreg) divided by the total number of female animals slaughtered (npreg + not-preg). boxplots were generated to display the upper and lower quartiles of groups of numerical data, with the whiskers indicating variability outside these parameters. foetal wastage, throughputs and slaughter rates of pregnant animals were analysed by month and by season across all animals combined and for individual species. a seasonal-trend decomposition procedure based on a locally weighted regression (stl) (cleveland et al. 1990) was used for the time-series analyses. months were used as the data time units for seasonality and trend. stl is a useful technique to visualise time-series data sets in which periods can be decomposed into three separate parts: trend, seasonal and remainder components. the stl procedure involves a sequence of applications with locally weighted regression (loess), which identify data patterns that do not conform to the mathematical polynomial equations. annual patterns were determined as seasonal components over the 12 years, followed by smoothing to determine trends. the remaining components were the monthly residuals from the seasonal and trend fits. monthly variations in foetal wastage were illustrated using a seasonal cycle subseries plot that assumed yearly periodicity. the seasonal cycle subseries plots display horizontal lines for the mean rate of foetal wastage for each month over the entire study period, whereas the end of vertical lines emanating from the horizontal lines indicate the specific rates of foetal wastage for that month in each year of the study. based on the seasonal cycle subseries plot, patterns between and within groups can be visualised for the total study period. the plot basically shows the average foetal wastage for each month (january–december) of the study period plotted as a horizontal line. the vertical lines drawn from each horizontal line display the individual patterns for the relevant month in each year. however, the seasonal cycle subseries plots were interpreted with caution, as the horizontal lines (average for vertical lines) were substantially affected by large values. as the stl cycle subseries plots do not provide any statistical comparisons, we used logistic regression analysis for additional analyses of the monthly or seasonal differences, as applied elsewhere (lee et al. 2014). the univariable logistic regression model was employed for each animal with an odds ratio and 95% confidence interval (ci) for each month to compare average monthly foetal wastage statistically. the month with the lowest values of foetal wastage (november) was used as a reference month. differences were considered statistically significant at p < 0.05. results top ↑ in the period under review, a total of 536 405 animals were slaughtered at the mma, of which 260 849 were cattle, 8051 sheep and 267 505 goats (table 1). a total of 9948 pregnant animals were slaughtered, representing 4.02% (95% ci: 3.94–4.10) of the total number of female animals slaughtered during 2001–2012. however, proportionately more pregnant sheep (15.89%; 95% ci: 14.80–17.03) were slaughtered than cattle (4.53%; 95% ci: 4.42–4.64) or goats (2.99%; 95% ci: 2.89–3.09). also, more male (53.83%; 95% ci: 53.69–53.96) than female animals (46.17%; 95% ci: 46.04–46.31) were slaughtered (table 1). in total, foetuses of 5929 calves, 755 lambs and 4740 kids were wasted (table 1). table 1: summary of the number of ruminants slaughtered at minna municipal abattoir, nigeria, 2001–2012. figure 2 shows the mean monthly and seasonal throughputs across all animal species slaughtered. the monthly average throughput was 1242 (figure 2a), with the lowest throughput obtained in february (995), whereas august produced the highest throughput (1489). daily throughput was highest (1366) in the late rainy season, with the lowest throughput (1079) reported for the late dry season (figure 2b). the evaluation of monthly slaughter patterns showed that more pregnant animals were slaughtered in august and early dry to early rainy seasons (figure 2c, d). total monthly and seasonal foetal wastage patterns across all species were similar (figure 2e, f). figure 2: monthly and seasonal rates of throughput, pregnant animals slaughtered and foetal wastages observed for the period 2001–2012 at the minna metropolitan abattoir, nigeria. (a) monthly throughput (all species), (b) seasonal throughput (all species), (c) monthly slaughter (all pregnant animals), (d) seasonal slaughter (all pregnant animals), (e) monthly rate of foetal wastages (all species) and (f) seasonal rate of foetal wastages (all species). the stl plots (figure 3) indicate that seasonality appears to be the strongest component in determining slaughter patterns during the study period. for cattle, the stl plot of the trend component (figure 3a, top row) appears fairly consistent, except for a sharp increase between 2002 and 2003. the seasonal component (figure 3a, middle row) shows a single peak (≤ 7%) per year, with the highest foetal wastage rates observed from the middle to later months of the year (may–october). the remainder component appears to have random variations (figure 3a, bottom row), although a large positive residual was noted in october 2002. in addition, the seasonal cycle subseries plot shows that the mean monthly foetal wastage rates were greatest in october (χ2 = 167.75; p < 0.0001) (figure 4a; appendix 1 and 2). the cycle subseries plot also shows monthly variations across different years, as indicated by the vertical peaks (figure 4a). compared with november as standard, the odds of calf wastage were slightly higher in july, august and october, but lower in february, march and december (table 2). figure 3: seasonal-trend decomposition of monthly rates of slaughter and foetal wastage at the minna metropolitan abattoir, nigeria, for the period 2001–2012. (a) cattle, (b) sheep and (c) goats. figure 4: cycle subseries plot from data of (a) cattle, (b) sheep and (c) goats slaughtered at minna metropolitan abattoir, nigeria, during the period 2001–2012. table 2: univariable logistic regression results for foetal wastage rate, expressed as odds ratios, for the period 2001–2012. data are organised according to month for each animal species. for sheep data, the stl plot of the trend component (figure 3b, top row) shows major fluctuations during the 12-year study period. the seasonal component (figure 3b, middle row) shows two peaks (> 10%) per year, with the highest foetal wastage rates between december and february, and march and june, respectively. other months were quiet with regard to foetal wastages. the remainder component (figure 3b, bottom row) shows random variations, although large positive residuals were noted intermittently throughout the study period. the seasonal cycle subseries plot shows that mean monthly foetal wastage rates were greatest in may and least between september and november (χ2 = 36.78; p = 0.0001) (figure 4b; appendix 1 and 2). the odds of lamb wastage were significantly higher between february and september every year (table 2). for goats, the stl plot of the trend component shows consistent minor fluctuations over the study period, with a significant peak in 2006 and a lesser one in 2008 (figure 3c, top row). the seasonal component (figure 3c, middle row) indicates two peaks (≤ 6%) per year, with the highest foetal wastage rates between august and december, january and march, and may and july. the remaining components also have random variations (figure 3c, bottom row), although large positive residuals were noted in march 2006 and october 2007. the seasonal cycle subseries plot shows that mean monthly foetal wastage rates were greatest in october, closely followed by a peak in march (χ2 = 88.17; p < 0.0001) (figure 4c; appendix 1 and 2). the odds of kid wastage were slightly elevated in february, march, may and june, but lower in april (table 2). the odds for foetal wastages varied for individual species and across months, but were generally higher for sheep than for goats or cattle (table 2). discussion top ↑ this study used the stl method to evaluate the trends and seasonal patterns of abattoir slaughter and foetal wastage at a nigerian abattoir over a 12-year period. based on significant data sets from slaughtered cattle, sheep and goats, variations with regard to trend, seasonality and remainder components were observed. seasonality (particularly late rains) affected slaughter patterns and foetal wastage at the mma between 2001 and 2012 most notably. the results showed that annual trends alone, without considering the effect of other parameters, could not predict the rates of ruminant foetal losses at the studied abattoir. there was no consistent trend observed for cattle, sheep or goats with regard to slaughter and foetal wastage. slaughter of pregnant ruminants has also been reported from cameroon (ndi, tambi & agharih 1993), ethiopia (mekibib, desta & tesfaye 2013), ghana (atawalna et al. 2013), mali (wilson & traore 1988), tanzania (assey et al. 1998), gambia (goossens et al. 1998), zambia (zulu et al. 2013), australia (ladds, summers & humphrey 1975) and new zealand (lawton, mead & baldwin 2000). in nigeria, the agricultural year is divided into two major seasons, namely the rainy season (april–october) and the dry season (november–march). foetal wastage peaked in the dry seasons or immediately afterwards, which coincides roughly with periods when most of the animals lose significant body condition owing to drought-related food scarcity. the sale of extensively raised cattle appears to surge during this period to recover some funds given the sparse feed supply. in addition, as no controlled breeding is observed for these livestock, pregnant animals may be sold off to an abattoir, which subsequently results in foetal wastages. although previous studies have concluded that annual foetal losses amongst cattle occur more often in the dry seasons in slaughterhouses and abattoirs across nigeria (cadmus & adesokan 2010; chaudhari & bokko 2000; ngbede et al. 2012; nwakpu & osakwe 2007; tizhe et al. 2010) and cameroon (ndi et al. 1993), our study shows a similar result also for goats. however, a different pattern was observed for foetal wastage of sheep, as foetal wastage rates were relatively lower between november and march. it is unclear whether this pattern was due to the relative paucity of data for sheep compared to other species; however, we are aware that festivities notably affect patterns of sheep slaughter in nigeria. the adoption of feed reservation in stores for use in periods of feed scarcity and the practice of zero grazing may alleviate these practices and reduce associated foetal losses. the highest overall foetal wastage, specifically for cattle and goats, was observed during the dry season of 2010. an earlier report on foetal calf wastage at the mma showed a similar trend in 2009 (alhaji 2011). it should be kept in mind that seasonal slaughter patterns and trends may be affected to varying extents by circumstances such as occasions and festivities with no fixed annual dates (e.g. eid), salary increases and improved living standards, as well as uncertainties and risks such as drought. the model used to analyse small-ruminant foetal wastage at the minna abattoir revealed multiple peaks per year across the seasons, as well as inconsistent trend influences. these fluctuations are not unexpected. analysis of the dates of eid el kabir from 2001 to 2012 showed that foetal wastage peaked in the months leading up to the festival. other studies have similarly associated higher sheep and goat slaughter, together with higher levels of lamb and kid foetal wastage, around festive periods (alade, sadisu & gambo 2011; bokko 2011; garba et al. 2010; tizhe et al. 2010). observant nigerians prepare for such festivals effectively through earlier purchase of small ruminants, predominantly sheep but also goats. during the festivities, these animals (primarily sheep) are slaughtered mostly in homesteads, and therefore the available data about sheep slaughter were relatively scant. efforts should be directed at encouraging abattoir slaughter even during such festivities and special occasions. our results show that more goats were slaughtered than cattle or sheep at the minna abattoir, although more foetal sheep losses were observed than for the other ruminants. this is similar to observations in other studies (alade et al. 2011; khan & khan 1989; mukasa-mugerwa & tekelye 1988). it is possible that the relatively small size and associated easier handling, ready availability, preference for taste and relatively lower costs may drive the preferential slaughter of small ruminants over large ruminants. this may apply to the slaughter of goats, but not necessarily for sheep. seasons and festivities affect the slaughter of sheep and many home slaughters of this species occur around eid. our study highlighted slaughter trends across ruminant livestock and confirmed seasonal patterns of foetal losses amongst pregnant ruminant species presented for slaughter at the mma. the trend and seasonality will likely continue for the foreseeable future unless significant changes occur in animal management practices in the industry. although legislation currently exists to prevent foetal wastage, adherence to such legislation is difficult to enforce. although we have offered possible reasons for the observed patterns, future studies should use empirical evaluations to explain the current patterns and predict the associated economic cost to the nation. increased purchasing power and urbanisation, together with a growing population, will likely result in a continuing rise in the demand for meat. the current trends and seasonality of foetal wastage at abattoirs are incompatible with meeting future challenges. the mainly pastoralist and transhumant systems of ruminant management influence the marketing structure for livestock in nigeria. herds are moved regularly in response to seasonal changes, the quality of grazing and the challenge posed by tsetse flies (southward in search of pastures and returned northward during tsetse fly infestation in the southern areas), and marketing systems closely follow this pattern. during these movements, sick and healthy animals are sold in open livestock markets along the routes, with live goats and sheep bought more regularly than cattle owing to the substantial price differences between small and large ruminants (lawal-adebowale 2012). whether this pattern influenced the pattern of animal slaughters at the abattoir could not be confirmed in this study. it may be possible that peak slaughter occurs when more animals transit through the minna area and an independent evaluation elsewhere may present a different result. our evaluation is subject to some limitations. first, the data were inconsistent for some years, with many missing values. this may have introduced bias and shifted the observed trends and seasonality. future planning at the abattoir should consider targeted and detailed data sets to minimise such error. second, the dynamics of domestic ruminant supply observed for the studied abattoir may not be representative of all nigerian abattoirs and a similar study elsewhere in nigeria might reveal a different outcome. finally, we cannot confirm whether censorship (by butchers who want to use or sell foetal products and avoid confiscation) affected the data used for this study. because the abattoir staff change often and there is no standardised format for reporting the slaughters, the possible effect of variation in data entry could not be determined. a standardised format for reporting is encouraged for future use at the abattoir. conclusion top ↑ this empirical trend analysis of ruminant foetal wastage at the mma demonstrated that seasonality is a major factor in determining slaughter patterns of pregnant ruminants. this slaughter pattern is likely to continue. we recommend a change in the current animal management practices to and enforcement of laws guiding against the indiscriminate slaughter of pregnant ruminants at nigerian abattoirs. future studies need to explore the use of empirical evaluations in analysing the current pattern and quantify the economic losses associated with abattoir foetal wastages. acknowledgements top ↑ we thank the staff of the niger state ministry of livestock and fisheries development, minna, for providing materials used in this research. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions n.b.a. (ministry of livestock and fisheries development), i.a.o. (university of ilorin) and a.s. (usmanu danfodiyo university) collected the data. f.o.f. (university of pretoria), h.s.l. (international livestock research institute) and a.s. contributed to data analyses and statistics. j.o. (university of northampton), u.m.c. (usmanu danfodiyo university), m.s.a. 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31st annual conference of nigerian society of animal production, pp. 140–142, kano, march 12–16, 2006. tizhe, m.a., kubkomawa, h.i., waba, y.e. & addass, p.a., 2010, ‘foetal wastage in ruminants and sustainable livestock industry in nigeria’, journal of agriculture and veterinary sciences 2, 72–80. wilson, r. & traore, a., 1988, ‘livestock production in central mali: reproductive performance and reproductive wastage in ruminants in the agro-pastoral system’, theriogenology 29(4), 931–944. pmid: 16726414, http://dx.doi.org/10.1016/0093-691x(88)90230-0 wosu, l., 1988, ‘calf wastage through slaughtering of pregnant cows in enugu abattoir, nigeria’, revue d’élevage et de médécine véterinaire des pays tropicaux 41, 97–98. wosu, l. & dibua, e., 1992, ‘lamb and kid wastage through slaughtering of pregnant ewes and goats at enugu and nsukka abattoirs in anambra state, nigeria’, small ruminant research and development in africa. proceedings of the first biennial conference of the african small ruminant research network, pp. 10–14, nairobi, december 10–14, 1990. zulu, v., mwanza, a.m., banda, f.c., yasuda, j. & yoshida, m., 2013, ‘cattle reproductive wastage in zambia: a case of mongu abattoir’, bulletin of the faculty of agriculture, kagoshima university 63, 49–54. appendix 1 top ↑ table 1-a1: case–control evaluation of monthly calf wastage using logistic regression analysis. table 2-a1: case–control evaluation of monthly lamb wastage using logistic regression analysis. table 3-a1: case–control evaluation of monthly kid wastage using logistic regression analysis. appendix 2 top ↑ figure 1-a2: monthly and seasonal rate of throughput, slaughter of pregnant animals and foetal wastage for cattle, minna metropolitan abattoir, nigeria, during 2001–2012. (a) monthly throughput for cattle, (b) seasonal throughput for cattle, (c) monthly slaughter of pregnant cattle, (d) seasonal slaughter of pregnant cattle, (e) monthly rate of foetal wastages in cattle and (f) seasonal rate of foetal wastages in cattle. figure 2-a2: monthly and seasonal rate of throughput, slaughter of pregnant animals and foetal wastage for sheep, minna metropolitan abattoir, nigeria, during 2001–2012. (a) monthly throughput for sheep, (b) seasonal throughput for sheep, (c) monthly slaughter of pregnant sheep, (d) seasonal slaughter of pregnant sheep, (e) monthly rate of foetal wastages in sheep and (f) seasonal rate of foetal wastages in sheep. figure 3-a2: monthly and seasonal rate of throughput, slaughter of pregnant animals and foetal wastage for goats, minna metropolitan abattoir, nigeria, during 2001–2012. (a) monthly throughput for goat, (b) seasonal throughput for goat, (c) monthly slaughter of pregnant goat, (d) seasonal slaughter of pregnant goat, (e) monthly rate of foetal wastages in goat and (f) seasonal rate of foetal wastages in goat. karzis_281-288.indd introduction the aim of this study was to determine withdrawal periods and tissue tolerance of three different intramammary antibiotics in goats with clinical mastitis, and to evaluate the effects of related factors. one of the most threatening diseases to dairy goat production is mastitis (smith & sherman 1994). mastitis in goats is treated with the same preparations 281 onderstepoort journal of veterinary research, 74:281–288 (2007) withdrawal periods and tissue tolerance after intramammary antibiotic treatment of dairy goats with clinical mastitis j. karzis1*, e.f. donkin2 and i.m. petzer1 abstract karzis, j., donkin, e.f. & petzer, i.m. 2007. withdrawal periods and tissue tolerance after intramammary antibiotic treatment of dairy goats with clinical mastitis. onderstepoort journal of veterinary research, 74:281–288 the aim of this study was to determine withdrawal periods (wp) and tissue irritation after administration of three intramammary antibiotics [curaclox lc (norbrook (ark ah)], spectrazol milking cow (schering-plough ah) and rilexine 200 lc [logos agvet (virbac)] in goats with clinical mastitis. withdrawal periods in goats with clinical mastitis treated with curaclox lc, were not significantly different from those recommended for use in cows (72 h) with (67 h) or without (48 h) the 24 h mandatory safety margin while spectrazol caused a significantly longer withdrawal period (122 h) than that recommended for use in cattle with (60 h) and without (36 h) the 24h safety margin. the withdrawal period of clinical mastitis cases treated with rilexine 200 lc was 48 h compared to the 96 h recommended for use in cows. a linear model of regression with factors influencing the wp in goats with clinical mastitis was as follows: wp = 30.21 + 4.692 (sampling time) + 22.11 (udder pathology) – 13.6 (floccules) – 0.00649 (milk yield). somatic cell counts (scc) of milk from udder halves with clinical mastitis ranged from 7 053 x 103 to 7 948 x 103 cells per ml without isolations of bacteria and between 6 476 x 103 and 8 479 x 103 cells per ml with isolations of bacteria. most of the variation in scc could not be explained and the california milk cell test (cmct) and scc on their own were not reliable methods for mastitis diagnosis. however, cmct and scc were indicators of udder irritation. in goats without clinical mastitis, spectrazol milking cow caused the least tissue irritation followed by rilexine 200 lc and curaclox lc. for goats with clinical mastitis, rilexine 200 lc caused the least irritation, followed by curaclox lc while spectrazol milking cow caused the most irritation. keywords: antibiotic withdrawal periods, california milk cell test, clinical mastitis, dairy goats, somatic cell count, tissue tolerance * author to whom correspondence is to be directed e-mail: jkarzis@mweb.co.za 1 department of production animal studies, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of animal and wildlife sciences, faculty of natural and agricultural sciences, university of pretoria, 0001 south africa accepted for publication 27 march 2007—editor 282 withdrawal periods and tissue tolerance after antibiotic treatment of dairy goats with mastitis containing antibiotics used for treating bovine mastitis. limited research has shown that residues persist for a longer period in goat milk than in milk from cows (bangen, skjerve, grave & soli 1992). antibiotic residues may pose a serious health hazard for humans consuming goat milk, as anaphylactic and allergic reactions may occur due to these residues, as well as the danger of development of resistant strains of bacteria. the production of cheese may also be seriously affected if there are antibiotic residues in the milk. therefore, the consequences of mastitis and antibiotic treatment lead to economic losses in the goat dairy farming industry (smith & sherman 1994). the main causes of mastitis are poor hygiene during milking as well as inefficient use of milking machines (joy, gallego, perez-sempere & molina 1989). in the past somatic cell counts (scc), california milk cell test (cmct) and conductivity measurements were used in the diagnosis of mastitis in goats. residue tests and screening tests for the presence of antibiotic residues in the milk after treatment for mastitis have also been used (paape 2000). mastitis in goats, as in cattle, may be subclinical or clinical, and may be peracute to chronic. in goats the diagnosis of subclinical mastitis is not well defined. the predisposing factors and pathogenesis of udder infection and inflammation in goats are similar to those in cattle and sheep in which the galactogenous route of infection is the most important. injuries to teats will usually result in severe mastitis. clinical signs of mastitis in goats resemble those in cattle and sheep (smith & roguinsky 1977). early indications of clinical mastitis include a decrease in milk production in the affected udder half and, in some, lameness on the affected side as the goat attempts to avoid contact of the hindlimb with tender half of the udder. visual inspection of the udder may reveal asymmetry and the affected gland may be swollen (acute) or atrophied (long term inflammation) (smith & sherman 1994). palpation may disclose the presence of heat, tenderness and swelling or multiple abscesses (acute mastitis) or atrophy, nodules and fibrosis (chronic mastitis). particular care is needed in the clinical examination of the goat’s milk because of its apparent normality even though there may be severe inflammatory changes in the udder (radostits, gay, blood & hinchcliffe 2000). if the teat is cold and oedematous or the secretion red and watery, gangrenous mastitis should be expected (smith & sherman 1994). the goat may be systemically ill in some cases of mastitis, showing signs of fever, anorexia and depression. materials and methods model system herds used in trials three experimental trials were conducted. trial 1 and 2 were conducted at the faculty of veterinary science, university of pretoria (herd a), while trial 3 was conducted on a commercial goat dairy in the limpopo province of south africa (herd b). trial 4 was conducted on a small-scale goat dairy near onderstepoort (herd c). trial 1 herd a consisted of 15 lactating multiparous saanen goats 14 of which were in early lactation. trial 1 commenced in winter when ambient temperatures were low (± 10 °c), with no rainfall. goats in trials 1 and 2 were milked with a six-point milking machine. it had a low milk line, with a system vacuum of 36 kpa and a pulsation rate of 74 pulses per min. trial 2 trial 2 was conducted in herd a using 14 lactating multiparous saanen goats 13 of which were in mid lactation, and one in late lactation. trial 2 commenced in spring with moderate (± 24 °c) ambient temperatures and light rainfall in the evenings. trial 3 trial 3 was conducted in a large commercial herd (350 lactating goats) (herd b), using 64 saanen, saanen/indigenous crossbreeds and toggenburg goats. all of the goats were in early and mid lactation and 57 of the 64 were in their first and the rest in their second lactation. trial 3 commenced in summer when ambient temperatures were very high (± 30 °c), with high humidity and light rainfall. goats in trial 3 were milked using a ten point quick exit milking system, with a low milk line and a system vacuum of 36 kpa. the pulsation rate ranged between units from 78.6 to 103.1 pulses per min. trial 4 herd c consisted of 13 lactating saanen and saanen/toggenburg crossbred dairy goats and were milked by hand. only four goats with clinical mastitis from this herd were selected for study. herds a, b and c were on total mixed rations (tmr). in trials 1 to 3 results of clinical udder examinations, milk yield, age and stage of lactation were evaluated 283 j. karzis, e.f. donkin & i.m. petzer and selection of experimental animals was done by the principle of pairing. daily milk yield was categorized as low (less than 1.3 l), medium (1.3–1.5 l) and high (more than 1.5 l). (in trial 4 only goats with clinical mastitis were selected.) goats with clinical mastitis were identified by the presence of floccules in the milk and high scc with or without detectable udder damage. clinical examination clinical procedure clinical examination of the mammary parenchyma was performed post milking in the lactating goats according to the method described by giesecke & van den heever (1974) and giesecke, du preez & petzer (1994). body temperature the rectal temperature of the goats was taken at every milking in trial 1. in trials 2, 3 and 4 temperatures were taken only of those animals when they were suspected to be sick. sampling in all trials goats were milked at 12-hourly intervals and foremilk samples were taken aseptically from udder halves of all goats according to the method described by giesecke et al. (1994). udder halves were milked separately and udder half milk yields were recorded. in trials 1 to 3 sampling continued after treatment until scc returned to baseline values and there were at least two consecutive negative thermo resistant inhibitory substances (tris) tests for each goat (karzis 2005). tests performed on goat milk at each 12-hourly milking an aseptic foremilk sample was taken for testing followed by the cmct, which was done in the dairy according to standard procedures (karzis 2005). the milk laboratory (department of production animal studies, faculty of veterinary science, uni versity of pretoria) performed microbiology and sccs on all milk samples. a fossomatic 5000 (rhine rühr) was used for scc. withdrawal periods (wps) for antibiotic residues in milk were determined using the tris test and the colour dye (for curaclox lc) (karzis 2005). antibiotic treatment products investigated all the products used are available commercially as intramammary infusions for the treatment of bovine mastitis. • in trial 1, six untreated goats acted as controls, while eight goats were treated with curaclox lc [nor brook (ark ah)], containing 75 mg sodium ampicillin, 200 mg sodium cloxacillin and a blue dye. • in trial 2, seven of the goats were untreated controls and the other seven were treated with spectra zol milking cow (schering-plough ah), containing 250 mg cefuroxime. • two products were investigated in trial 3. twenty untreated goats acted as controls and 20 goats were treated with rilexine (s4) 200 lc [logos agvet (virbac)], containing 100 mg ceph a lexin, 100 mg neomycin sulphate and 10 mg predni solone, while 12 goats served as controls and 12 were treated with curaclox lc. • in trial 4, one goat was treated with spectrazol milking cow and three were treated with curaclox lc. administration of antibiotics the entire content of the intramammary infusion syringe containing antibiotics, formulated for the treatment of bovine mastitis was injected into each udder half of the goat. intramammary antibiotics were administered to both udder halves of goats in the treatment groups at three consecutive milkings of 12 hourly intervals in trials 1 to 3. in trial 4 the udder half with clinical mastitis was treated. data management all data were entered and stored in microsoft excel and were analysed using the statistical programme genstat (2003). the correlation coefficient (pearson’s coefficient of correlation) is a measure of the linear relationship between two random variates (–1 < r < 1) (draper & smith 1981). the tabled critical values for determining significance of a correlation coefficient are dependent on the pairs of data in the sample. generally, a coefficient of + 0.9 is an indication of a very strong correlation, + 0.7 indicates a strong correlation, + 0.5 a moderate and – 0.3 to + 0.3 a weak correlation (rayner 1969). 284 withdrawal periods and tissue tolerance after antibiotic treatment of dairy goats with mastitis graphs were drawn of the means of each variable over time using sigma plot 9.0 (www.rockware.com/catalog/pages/sigmaplot2.html) results withdrawal periods after intramammary treatment of goats with clinical mastitis see table 1. regression of data of clinical mastitis cases see table 2. linear regression model withdrawal period (wp) as measured by tris = 30.21 + 4.692 (sampling time) + 22.11 (udder pathology) – 13.6 (floccules) – 0.00649 (milk yield). analysis of variance of scc of data of clinical mastitis cases tables of scc see table 3. table 1 goats with clinical mastitis: withdrawal periods (wp) of intramammary antibiotics compared to wp recommended for use in cattle with or without the 24 h safety margin (one sample t-tests) trials and products used mean ± sd f probability: recommended for use in cattle f probability: wp recommended for use in cattle – 24 h trial 1 (curaclox lc) trial 2 (spectrazol milking cow) 67 + 41.33 122 + 37.37 p = 0.619 p = 0.020 p = 0.090 p = 0.007 p is significant at the 5 % level (p < 0.05) table 2 clinical mastitis in goats: regression model term in model adjusted r2 standard error of regression f probability sampling times (06:00 and 18:00) udder parenchyma (damaged = 1, normal = 0) floccules (present = 1, absent = 0) 68.10 % 90.50 % 94.10 % 9.30 5.06 4.00 p < 0.001 p < 0.001 p = 0.035 p is significant at the 5 % level (p < 0.05) table 3 goats with clinical mastitis: differences in log scc of treatment and control groups, infected and non-infected udder halves, stages of lactation and parity for both treatment times trials and products used time treated and non-treated infected and non-infected stage of lactation parity goats with clinical mastitis (cura clox lc, spectrazol and rilexine) 06:00 p < 0.001 p = 0.173 p < 0.001 p < 0.001 18:00 p < 0.001 p < 0.001 p < 0.001 p < 0.001 trial 1 (curaclox lc), goats without clinical mastitis 07:00 p = 0.227 p < 0.001 19:00 p = 0.078 p < 0.001 trial 2 (spectrazol), goats without clinical mastitis 06:00 p < 0.001 18:00 p < 0.001 p is significant at the 5 % level (p < 0.05) table 4 goats with clinical mastitis: correlation matrix (curaclox lc, spectrazol and rilexine) excluding withdrawal period as measured by tris (degrees of freedom = 332) log scc 1 1 milk yield 2 r2 = –0.283 1 cmct 3 r2 = 0.408 r2 = –0.002 1 parity 4 r2 = 0.225 r2 = –0.617 r2 = –0.021 1 1 2 3 4 285 j. karzis, e.f. donkin & i.m. petzer graphs of scc see fig. 1. tables of statistical linear correlation coefficients see tables 4 and 5. discussion a negative tris test on cow’s milk is an indication that the milk is safe for human consumption, i.e. that it is below the maximum residue limits (mrls) and within the safe tolerance levels for antibiotic residues in milk (μg/kg) fixed by codex alimetarius, ecregulations and food and drug administration (usfda). the mrls (ec) for ampicillin, cloxacillin, cephalosporins and neomycin were 4, 30, 100 and 500, respectively, and mrls codex for ampicillin, cloxacillin, cephalosporins and neomycin were 4, 4, 100 and 500, respectively (honkanen-buzalski & reybroeck 1997). in this investigation the contents of a whole tube of the respective intramammary antibiotic was inserted into the udder half of each goat as is recommended for the use in cattle. it can therefore be expected that this leads to an antibiotic concentration in the goats’ udders, that’s treated, greater than would be the case in cows’ udders since the goat udder is smaller than that of a cow udder and goats produce a smaller volume of milk than cows. withdrawal periods the wp of a particular commercially available preparation recommended for use in cattle has a 24 h safety margin added to the longest wp obtained in the trial for the preparation. in this study this 24 h safety margin has not been added and, in practice, a 24 h safety margin should be added to all the wps determined in this investigation. there is no significant difference at the 5 % level of significance in wp determined and the wp recommended for use in cattle (72 h) for curaclox lc with (p = 0.619) or without (p = 0.090) the 24 h safety. table 5 goats with clinical mastitis: correlation matrix (curaclox lc, spectrazol & rilexine) including withdrawal period as measured by tris (degrees of freedom = 16) logscc 1 1 milk yield 2 r2 = –0.812 1 cmct 3 r2 = 0.839 r2 = –0.833 1 parity 4 r2 = 0.167 r2 = –0.438 r2 = 0.228 1 withdrawal period (tris) 5 r2 = 0.563 r2 = –0.270 r2 = 0.379 r2 = –0.362 1 1 2 3 4 5 ������� ��� ��� ��� ��� � � � �� �� �� �� �� �� �� � ��� ��� ��� ��� ��� ��� � ����� ������ ������ ������ ������ ������ ������� �� �� �� � � � � � � �� � �� �� � � fig. 1 goats with clinical mastitis. the mean scc of udder halves with clinical mastitis in treatment groups (t1 = curaclox lc, t2 = spectrazol milking cow, t3 = rilex ine 200 lc) versus control group (rx = treatment) 286 withdrawal periods and tissue tolerance after antibiotic treatment of dairy goats with mastitis however, for spectrazol milking cow the wp determined (122 h) is significantly longer that the wp recommended for use in cattle (60 h) with (p = 0.020) and without (p = 0.007) the 24 h safety margin (table 1). the sample size for clinical mastitis cases treated with rilexine 200 lc was insufficient for similar statistical analysis. there is no significant difference between wp as measured by tris and persistence of the dye in the milk nor is there a significant difference between left and right udder halves with clinical mastitis. this is an indication that the presence of colour dye in the milk had, in this case, a reliable application for the goat producer. no significant effect on wp of goats with clinical mastitis is shown between udder halves from which bacteria were isolated or not, as measured by tris (p = 0.324) and persistence of the dye in the milk (p = 0.938). a moderate positive correlation (r2 = 0.563) is present between wp and log scc and a weak negative correlation (r2 = –0.362) between wp and parity (table 5). the wp is excluded in table 4 and there are 332 ° of freedom as opposed to 16 ° of freedom when the wp is included as seen in table 5 (karzis 2005). regression model of all clinical data the linear regression model from data in table 3 is valid because the final r2 is 95.7 %, standard error of regression = 3.41. although the regression model improves with the addition of terms, the f probability decreases from a 1 % significance level (p < 0.001) to a 5 % (p = 0.035) and a 10 % level (p = 0.085) (see table 2). this is a biologically meaningful model that was used to determine which are the most important factors affecting wp as measured by tris. this model is therefore still meaningful even though the end significance is at the 10 % level. according to the linear model of regression the wp, as measured by tris, increased at the evening milking by 4.7 h. additional stress of goats in trials 2 and 3 from the change of regular milking times to 12 hourly intervals and milking in the dark may have influenced the scc. when chronic udder parenchyma damage was present the wp increased by 22 h. a possible explanation could be that the antibiotic residues took longer to be excreted from udders with chronic damage due to the anatomical changes. the linear model of regression shows that the presence of floccules in the milk decreased the withdrawal time by 13.6 h. therefore, this implies that goats with clinical mastitis without udder damage have a shorter wp as measured by tris. the regression model indicates that when milk yield decreased, the wp decreased by an insignificant 0.00649 h. analysis of variance of somatic cell count (scc) there is a highly significant difference (p < 0.001) in mean log scc in the goats with clinical mastitis between the treatment groups t1 (curaclox lc), t2 (spectrazol milking cow) and t3 (rilexine 200 lc) and the control group (table 3). the scc was the highest in t1 followed by t2, c and t3 at 06:00 and from highest to lowest at 18:00 (t2, t1, c to t3). in goats without clinical mastitis curaclox lc caused the most tissue irritation (karzis 2005) while spectrazol milking cow (fig. 1) caused the most tissue irritation in goats with clinical mastitis compared to those treated with curaclox lc and rilexine 200 lc (table 3). fig. 1 shows that mean scc was the highest for goats treated with spectrazol milking cow, lower for those treated with curaclox lc and lowest for those treated with rilexine 200 lc. the mean scc of goats in the control group was higher than that of goats in the t3 group at all times except at treatment time, and at 12 h after the treatment commenced. a decrease in mean scc was evident during treatment in trial 3. this may have been due to the presence of prednisolone, a corticosteroid in rilexine 200 lc, which may help in reducing inflammation in udders with clinical mastitis. the presence of bacteria did not affect the mean log scc of clinical udder halves significantly at 06:00, (p = 0.173). however, the presence of bacteria affected the mean log scc significantly (p < 0.001) at 18:00 (table 3). this could be explained by the increase in the wp in evening milking as shown in the regression model (table 2). mean log scc in udder halves with clinical mastitis differed highly significantly (p < 0.001) between stages of lactation (table 3). the scc was high in early lactation, decreased in mid lactation and increased in late lactation. the increase in scc in late lactation was in agreement with that reported in the literature on both goats and cattle. the increase in scc in late lactation was only detected in goats with clinical mastitis. in the latter the scc decreased in late lactation for goats in trials 1 and 2 (karzis 2005). it can therefore be deduced that the mean scc in goats with clinical mastitis was influenced by stage of lactation. 287 j. karzis, e.f. donkin & i.m. petzer there is a highly significant difference (p < 0.001) between mean log scc and parity (table 3). mean scc was high in the first lactation, decreased in the second lactation, increased in the third to fifth lactations and decreased again in the seventh lactation at 06:00 and 18:00. this pattern differed from that in trial 1 and 2 in goats without clinical mastitis, where scc increased at the seventh lactation (karzis 2005). california milk cell test (cmct) the sample size is inadequate to perform chisquared tests on the data from goats with clinical mastitis. mean cmct scores of milk from goats in the control group (c) and of those in t2 (spectrazol milking cow) were unstable throughout the trial. however, mean cmct score of milk from goats in t1 (curaclox lc) and in t3 (rilexine 200lc) increased with antibiotic treatment (karzis 2005). there is a strong positive correlation between cmct and log scc (table 5) (including wp tris) and a moderate positive correlation between cmct and log scc (table 4) (excluding wp tris). table 4 excluding wp tris is a stronger correlation model (332 ° of freedom) than table 5 including wp tris (16 ° of freedom). this positive correlation did not explain why scc increased the most after treatment with spectrazol milking cow, while cmct increased the most after treatment with rilexine 200 lc. cmct and scc were both unreliable methods for determining intramammary infection in goats and should be used in conjunction with udder palpation and microbiological tests for effective mastitis diagnosis. correlations table 4 shows a correlation matrix with 332 pairs of data excluding wp as measured by tris (karzis 2005). the only correlation that shows any linear relationship in table 4 is a moderate negative correlation between parity and milk yield (r2 = –0.617). the correlation matrix in table 5 shows a fairly strong negative correlation between log scc and milk yield (r2 = –0.812) and between cmct and milk yield (r2 = –0.833), a fairly strong positive correlation between log scc and cmct (r2 = 0.839) and a positive correlation between log scc and wp. there were low, mid and high yielding goats in the clinical group. a weak negative correlation (r2 = –0.270) was present between wp and milk yield and a moderate negative correlation (r2 = –0.438) between milk yield and parity. both (tables 4 and 5) show moderate negative correlations between milk yield and parity, indicating that milk yield decreased with increasing parity in saanen goats with clinical mastitis. conclusion the withdrawal periods of antibiotic from udder halves with clinical mastitis treated with curaclox lc, were not significantly different from those recommended for use in cattle with or without the 24 h safety margin. however, withdrawal periods of antibiotic from udder halves with clinical mastitis treated with spectrazol lactating cow were significantly longer (122 h) than those recommended for use in cattle with (60 h) or without the 24 h safety margin. the wp as measured by tris and the persistence of the dye colour in milk did not differ significantly in goats treated with curaclox lc. the variability in scc was largely unexplained, and an increased scc did not necessarily indicate an intramammary inflammation in goats, as it does in cows. milk from goats with clinical mastitis had higher scc than goats without clinical mastitis. the mean log scc was significantly different in goats with clinical mastitis between treatment groups and the control group, between stages of lactation and for different parities. the presence of bacteria did not influence the mean log scc of milk from udder halves with clinical mastitis significantly at 06;00 but it did at 18:00. according to the linear model of regression the wp increased at evening milking by 4.7 h, with chronic udder damage by 22 h, and when floccules were present in the milk it decreased by 13.6 h. in goats without clinical mastitis curaclox lc caused the most tissue irritation while spectrazol milking cow caused the most tissue irritation in goats with clinical mastitis and the least in goats without clinical mastitis. further studies are necessary on scc of goat milk as an indicator of tissue irritation in goats with and without clinical mastitis. the mean cmct score of milk from goats in trial 1 (curaclox lc) and trial 3 (rilexine 200 lc) increased with antibiotic treatment. there is a strong positive correlation between cmct and log scc (table 5) (including wp tris, 16 degrees of freedom) and a moderate positive correla288 withdrawal periods and tissue tolerance after antibiotic treatment of dairy goats with mastitis tion between cmct and log scc (table 4) (excluding wp tris, 332 degrees of freedom). the correlation matrix shows a strong negative correlation between log scc and milk yield and cmct and milk yield, a negative correlation between milk yield and parity, a strong positive correlation between log scc and cmct and a positive correlation between log scc and wp. further investigations are necessary to determine withdrawal periods in goats with clinical mastitis and those without. acknowledgements we thank the national research foundation for the funding of this research, mr tobie fourie and the staff of the limpopo melkery, mr daan delport, the staff of the milk laboratory, faculty of veterinary science, the staff of the pharmacy, onderstepoort veterinary academic hospital, the staff of the onderstepoort teaching animal unit for all their help, and marie smith of the agricultural research council silverton for the statistical analysis. references bangen, m., skjerve, e., grave, k. & soli, n.e. 1992. prescribing of drugs for food producing animals in norway. information about withdrawal times. journal of veterinary pharmacology and therapeutics, 15:180–187. draper, n. & smith, h. 1981. applied regression analysis, 2nd ed. new york: wiley & sons. genstat® for windows® 2003. introduction 7th ed., edited by r. w. payne. vsn international. giesecke, w.h., du preez, j.h. & petzer, i.m. 1994. practical mastitis control in dairy herds. south africa: butterworths. giesecke, w.h. & van den heever, l.w. 1974. a guide to the testing of stock remedies (act 36/1974) for the treatment and control of septic mastitis of cows (mastitis remedies). pretoria: department of agricultural technical services (technical communication, no. 123). honkanen-buzalski, t. & reybroeck, w. 1997. residues and contaminants milk and milk products: antimicrobials. international dairy federation special issue, 9701:26-28. joy, s., gallego, l., perez-sempere, j.i. & molina, a. 1989. the exploitation of dairy sheep of the menchegan breed with mechanical milking, ii.technical and economic rates. proceedings of 4th international symposium on milking of small ruminants, kibbutz shefayim, israel, 1989: 368. karzis, j. 2005. intramammary antibiotics in dairy goats: withdrawal periods and tissue tolerance. m.sc. thesis, uni versity of pretoria. paape, m.j. 2000. situation regarding the legal limit for somatic cell counts for goats in the united states. proceedings of the 7th international conference on goats, tours, france, 2000: 755–756. radostits, o.m., gay, c.c., blood, d.c. & hinchcliffe, w. 2000. veterinary medicine. textbook of the diseases of cattle, sheep, pigs, goats and horses, 9th ed. london: w.b. saunders ltd. rayner, a.a. 1969. a first course in biometry for agriculture students. pietermaritzburg: university of natal press. smith, m.c. & roguinsky, m. 1977. mastitis and other diseases of the goat’s udder. journal of the american veterinary medical association, 171:1241–1248. smith, m.c. & sherman, d.m. 1994. goat medicine. baltimore & maryland: lippincott williams & wilkins. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) cornelia m. meyburgh department of microbial, biochemical and food biotechnology, university of the free state, south africa robert r. bragg department of microbial, biochemical and food biotechnology, university of the free state, south africa charlotte e. boucher department of microbial, biochemical and food biotechnology, university of the free state, south africa citation meyburgh, c.m., bragg, r.r. & boucher, c.e., 2018, ‘detection of virulence factors of south african lactococcus garvieae isolated from rainbow trout, oncorhynchus mykiss (walbaum)’, onderstepoort journal of veterinary research 85(1), a1568. https://doi.org/10.4102/ojvr.v85i1.1568 original research detection of virulence factors of south african lactococcus garvieae isolated from rainbow trout, oncorhynchus mykiss (walbaum) cornelia m. meyburgh, robert r. bragg, charlotte e. boucher received: 20 oct. 2017; accepted: 05 july 2018; published: 04 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract lactococcus garvieae is a gram-positive bacterium that causes mortalities in freshwater and marine fish worldwide and therefore results in severe economic losses in the aquaculture industry. apart from the apparent integral role of the exopolysaccharide (eps) capsule in pathogenesis, factors associated with virulence of this bacterium are poorly understood. however, recent studies have indicated that the ability of l. garvieae to cause disease does not depend on the presence of the eps capsule. lack of knowledge of virulence factors, pathogenesis and serology of l. garvieae is an impediment to the development of effective typing methods and control measures. this study, therefore, aimed to detect the presence of eps capsules and other putative virulence factors in south african l. garvieae fish pathogenic isolates and a non-virulent isolate, and to identify possible candidates for subunit vaccine development. no indication of the presence of the eps capsule was detected by negative staining or amplification of the eps biosynthesis gene cluster in the virulent isolates or the avirulent strain, discrediting the notion that the eps capsule is the sole determinant of virulence. however, a set of putative virulence factor genes was detected in all isolates, and candidates for subunit vaccine development (enolase, lactate dehydrogenase phosphoenolpyruvate-protein phosphotransferase) were identified by identification of extracellular proteins of virulent strains. introduction infectious disease caused by bacteria causes severe fiscal loss in aquaculture (austin & austin 2012). lactococcus garvieae, a pathogen of importance in the aquaculture of freshwater and marine fish (bragg & broere 1986; collins et al. 1983; eldar et al. 1996; kusuda et al. 1991), is described as a gram-positive, facultative anaerobic, non-motile bacterium that does not produce endospores. it is generally described as an α-haemolytic bacterium (ravelo et al. 2001), but has been noted as β-haemolytic (teixeira et al. 1996). a south african gram-positive cocci isolate, initially described as streptococcus spp. (bragg & broere 1986), were reclassified as enterococcus spp. and l. garvieae based on 16s ribosomal deoxyribonucleic acid dna (rdna) sequencing (bekker et al. 2011). the bacterium is considered an emerging zoonotic agent, placing immunocompromised individuals at risk (gibello et al. 2016). antimicrobial agents show strong in vitro activity against l. garvieae, but perform poorly under field conditions because of anorexia of infected fish (bercovier et al. 1997) and possibly the ineffective metabolism of antibiotics in fish (romero, feijoó & navarrete 2012). dissemination of antibiotic resistance in bacteria has grown into a global public health concern, accelerated by the unregulated and injudicious administration of antibiotics in humans and animals (heuer et al. 2009). in aquaculture, chemotherapeutic treatment has led to the emergence of multiple resistance in streptococcal fish pathogens (aoki et al. 1990; austin & austin 2012). the spread of antibiotic resistance is aided by various mechanisms of horizontal gene transfer, of which plasmid-mediated transfer is the most widely documented in streptococcal fish pathogens. vaccination is considered the best option to control lactococcosis because of the poor efficiency of chemotherapeutic agents under field conditions and the risks associated with the spread of antibiotic resistance determinants. bacteria employ a wide repertoire of virulence factors to promote survival within the host, some of which cause imminent damage to the host. early studies on toxins of a non-lancefield streptococcus sp. (presumably l. garvieae) isolated from yellowtail showed the presence of a haemolytic toxin in culture supernatant (kusuda & hamaguchi 1988). a study by aguado-urda et al. (2012) characterised five plasmids in a clinical isolate of l. garvieae strain 21881. the largest of these plasmids (68 798 bp [base pair]), pgl5, was shown to encode putative virulence factors, including a protein that posesses the enzymatic domain corresponding to the family of actin-adp-ribosyltransferases (aguado-urda et al. 2012). full genome sequencing of l. garvieae strains atcc 49156® and lg2 by morita et al. (2011) identified several genes encoding putative virulence factors showing significant similarity to virulence factors of related species. a comparative genome analysis of a virulent strain lg2 and a non-virulent strain atcc® 49156 of l. garvieae identified a 16.5 kilobase (kb) capsule gene cluster, which is present in lg2 but absent in atcc® 49156 (morita et al. 2011). the capsular gene cluster consists of 15 genes, of which 8 (eps-r, x, a, b, c, d and cps-l, w) are conserved in the exopolysaccharide (eps) biosynthesis gene cluster of four lactococcus lactis strains isolated from human faecal samples (morita et al. 2011). analyses indicate that the capsular gene cluster is a genomic island, because of the presence of insertion sequences (iss) on both ends of the capsular gene cluster. even though the polysaccharide capsule is widely regarded as a major virulence factor of l. garvieae, it has been shown that non-capsulated strains lgper and atcc® 49156 are pathogenic to rainbow trout, causing 89% and 98% mortality, respectively (türe et al. 2014). these results suggest that the presence of the polysaccharide capsule cannot be directly correlated with pathogenicity in fish. despite the integral role of the polysaccharide capsule in pathogenesis, none has been structurally characterised. understanding of the underlying genetic basis of variability in eps structure and its likely interrelationship with serological variability in l. garvieae is lacking presently. lack of knowledge of virulence factors, pathogenesis and serology of l. garvieae is an impediment to the development of effective typing methods and control measures. therefore, this study aims to investigate the prevalence of various putative virulence factors in south african l. garvieae isolates and to speculate on their role in the process of infection in order to further elucidate the pathogenesis of lactococcosis in rainbow trout and identify possible targets for recombinant vaccine development. materials and methods isolates used in this study cultures used in this study were obtained from a study by bragg and broere (1986) during which these bacteria were isolated from diseased rainbow trout, oncorhynchus mykiss (walbaum), in the former eastern transvaal (currently mpumalanga) area and johannesburg, gauteng. symptoms observed in diseased rainbow trout included extreme exophthalmos and rupture of one or both eyes, haemorrhaging of the ocular chamber, melanosis, enlargement of the spleen and haemorrhaging of intestines (bragg & broere 1986). geographic origins and isolate numbers (a1–a12), which will hereafter be used to refer to isolates, are presented in table 1. freeze-dried isolates were revived in brain heart infusion (bhi) broth (merck, 1.10493) and incubated under aerobic conditions at 30 °c (a1–a3, a5, a6, a11 and a12) for 48 hours or anaerobic conditions at 37 °c (a4, a7–a10) for 48 h using an anaerobic jar with a gas-generating kit (oxoid™ br0038, basingstoke, united kingdom). culture purity was confirmed by gram staining. revived isolates were stored in commercially available cryogenic vials (microbank™, pro-lab diagnostics, toronto, canada) at -20 °c. table 1: isolate numbers and geographic origins of isolates used in the current study. identification of isolates by 16s ribosomal dna sequencing isolates were cultivated in tryptic soy broth (tsb) (merck, 1.05459) under aerobic conditions at either 30 °c or 37 °c for 18 h with no agitation. dna was extracted according to the method described by thanh (2006). universal sequence primers 8f (5’-agagtttgatcctggctcag-3’) and 1525r (5’-aaggaggtgatccagcc-3’) were used to amplify a 1517 base pair (bp) sequence of the 16s rdna gene by polymerase chain reaction (pcr) (beumer & robinson 2005). amplicons were visualised by gel electrophoresis on a 1% agarose gel (sambrook & russell 2001). for purposes of purification of the pcr product, the pcr products were run on a 2% weight per volume (w/v) low-melt agarose (nusieve® gtg® agarose). bands were excised under ultraviolet illumination. the pcr products were purified from gel slices using a wizard® sv gel and pcr clean-up system (promega, madison, wi). sequencing reactions were prepared using applied biosystems™ bigdye® terminator v.3.1 sequencing kit. samples were submitted for sanger sequencing at an in-house facility at the university of the free state. sequence data were viewed using geneious version 9 (http://www.geneious.com, kearse et al. 2012) and sequences were used to query the national centre for biotechnology information (ncbi) database using the nucleotide–nucleotide blast (blastn) algorithm (altschul et al. 1990). phenotypic characterisation of exopolysaccharides negative staining with nigrosine was performed on isolates a1–a12 to determine the presence of eps capsules. pseudomonas aeruginosa and an avirulent reference strain of l. garvieae (ncfb657) were included as positive and negative controls, respectively. genotypic characterisation of exopolysaccharides the l. garvieae eps synthesis gene cluster, as described in l. garvieae lg2 (miyauchi et al. 2012), was amplified in isolates a1–a3, a5, a6, a11 and a12 using the takara la pcr kit (takara bio inc. #rr002a, kyoto, japan). amplicons were visualised on a 1% agarose gel. detection of putative virulence factor genes by polymerase chain reaction the presence of putative virulence factor genes, as identified by morita et al. (2011), in south african isolates were investigated by pcr using primers designed by türe and altinok (2016). the presence of the following genes was investigated: haemolysins 1, 2 and 3 (hly1, -2, -3), nadh oxidase (nox), superoxide dismutase (sod), pneumococcal adherence and virulence factor a (pava) and pneumococcal surface adhesin a (psaa) (table 2). amplicons were visualised on a 1% agarose gel. products were purified using a wizard® sv gel and pcr clean-up system (promega, usa). sequencing was performed using the applied biosystems™ bigdye® terminator v.3.1 sequencing kit. obtained sequences were viewed and edited with geneious 9 (kearse et al. 2012) and used to query the ncbi database using the blastn algorithm (altschul et al. 1990). table 2: genetic loci and predicted gene products of candidate virulence genes, previously identified in lactococcus garvieae lg2 (morita et al. 2011), under investigation in this study. detection of extracellular virulence factors lactococcus garvieae strains a1–a3, a5, a6, a11, a12 and ncfb657 were cultivated in tsb media for 24 h at room temperature. a negative control, consisting of sterile tsb, was included. cells were pelleted by centrifugation at 20 000 x g for 2 minutes, and the supernatants filtered with a 0.2 μm syringe filter unit to remove residual cellular components. amicon® ultra-4 10k centrifugal filter units (merck) with a molecular weight cut-off (mwco) of 10 kda were used to concentrate extracellular proteins. a volume of 4 ml filtered supernatant was applied to the centrifugal filter and the unit was centrifuged at 7197 x g for 20 min. the eluate was discarded, the centrifugal filter was inverted inside a 50 ml falcon tube and again centrifuged at 7197 x g for 20 min to collect the concentrated protein fraction. extracellular proteins, concentrated by amicon® ultra-4 10k centrifugal filter units (merck), were visualised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the polyacrylamide gel was stained and destained using the rapid fairbanks coomassie blue protein staining and destaining method (wong et al. 2000). protein identification prepared samples were submitted to the facility for genomics and proteomics at the university of the free state for identification by nanoscale liquid chromatography coupled to tandem ms (nano lc-ms/ms) and by querying the swiss-prot annotated protein sequence database (bateman et al. 2015) using the mascot search engine that uses a probability-based scoring algorithm based on the molecular weight search (mowse) algorithm (perkins et al. 1999). results identification of isolates amplification of the 16s rdna gene fragment by pcr yielded the expected ± 1500 bp product, visualised by gel electrophoresis in figure 1. the nucleotide blast search results, tabulated in table 3, identified isolates a1–a3, a6, a11 and a12 as l. garvieae, a4 as enterococcus faecalis, a7 as e. durans and a8–a10 as e. faecium. these results are consistent with the results of bekker et al. (2011), who also identified this set of 12 isolates as 5 enterococcus spp. and 7 l. garvieae isolates using 16s rdna sequencing (bekker et al. 2011). no reports of fish-pathogenic enterococcus spp. have been published; therefore, isolates a4 and a7–a10 were excluded from further experiments in this study. figure 1: visualisation of 16s polymerase chain reaction products of isolates a1–a12 on a 1% weight per volume (w/v) agarose gel. bands in the expected size range (± 1500 bp) were observed. table 3: top basic local alignment search tool search hits generated for 16s ribosomal dna genes amplified from isolates a1–a12. phenotypic characterisation of exopolysaccharides negative staining as shown in figure 2, no halos were observed in any of the isolates including the negative control ncfb657, in comparison with the positive control p. aeruginosa, where a clear halo was observed, indicating the presence of a polysaccharide capsule. figure 2: negative staining of lactococcus garvieae a1–a12 using nigrosin, visualised using 100x magnification. l. garvieae ncfb657 and pseudomonas aeruginosa were included as negative and positive controls, respectively. (a), a1; (b), a2; (c), a3, (d), a4; (e), a5; (f), a6; (g), a7; (h), a8; (i), a9; (j), a10; (k), a11; (l), a12; (m), l. garvieae ncfb657; (n), p. aeruginosa. genotypic characterisation of exopolysaccharides the amplification of the eps biosynthesis gene cluster by long-range pcr was attempted as described by miyauchi et al. (2012). figure 3 shows a product of ~ 750 bp for all isolates tested, consistent with the product sizes observed in the kg+ (non-capsulated) strains tested in the study by miyauchi et al. (2012). the presence of insertion sequences (iss) flanking the eps biosynthesis gene cluster in the kg− strain lg2 used in the study by miyauchi et al. (2012) may suggest that the eps biosynthesis gene cluster could be inserted at different genomic loci in strains other than lg2. if this should happen, the primers used by miyauchi et al. (2012) will fail to amplify this gene cluster in other strains as these primers flank the gene cluster including the iss. therefore, primers that amplify the first gene in the cluster, a transcriptional regulator (epsr), were designed. however, pcr amplification of this gene yielded no product in any isolates tested. figure 3: visualisation of lr pcr products on a 1% agarose gel. a band of ~ 750 bp was observed in all isolates tested, indicating the absence of an exopolysaccharide gene cluster in the genomes of these isolates. detection of extracellular proteins extracellular proteins of eight l. garvieae strains, purified and concentrated by amicon® ultra-4 10k centrifugal filter units (merck), were visualised on a 12% sds-page gel. more protein bands were observed in strain a3 in comparison to other strains, as can be seen in figure 4. no clear bands were observed in the negative control, which consisted of sterile tsb medium, indicating that the growth medium did not contribute any of the protein bands detected and identified in supernatants (table 4). figure 4: extracellular proteins of eight lactococcus garvieae strains, including the reference strain ncfb657, visualised on a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. a negative control (sterile tryptic soy broth (tsb)) was included. table 4: top protein scores generated by query of the swiss-prot protein sequence database with the search engine mascot, following nano liquid chromatography–tandem mass spectrometry analysis of extracellular proteins from lactococcus garvieae strain a3. detection of putative virulence factor genes by polymerase chain reaction visualisation of pcr products by gel electrophoresis, as presented in figure 5, followed by sequencing and nucleotide database search confirmed the presence of seven candidate virulence genes in genomes of seven south african isolates as well as an avirulent reference strain, ncfb657. figure 5: putative virulence factor genes were detected by polymerase chain reaction assays. products are shown visualised on 1% agarose gels. the first lanes on all gels are size markers, and the subsequent lanes represent virulence genes in the following order: hly1; hly2; hly3; nox; sod; pava; psaa. (a), a1; (b), a2; (c), a3, (d), a5; (e), a6; (f), a11; (g), a12; (h), ncfb657. discussion all putative virulence genes under investigation were present in the genomes of all south african isolates used in this study, as well as the reference strain ncfb657. these results are in accordance with the findings of türe and altinok (2016), who reported the presence of this set of putative virulence factor genes in the genomes of a total of 34 l. garvieae strains, including the avirulent reference strains atcc® 49156 and atcc® 4392 and isolates from diseased rainbow trout in turkey, france, iran, spain and italy. no phenotypic proof of the presence of a polysaccharide capsule was collected in this study using light microscopy. these observations alone, however, should not be considered definitive proof of the absence of capsules, as yoshida et al. (1997) reported that neither negative staining using muir’s method or indian ink, nor the quellung reaction was successful in visualising l. garvieae kg− capsules. the eps capsule influences a variety of host interactions, including adhesion, invasion and sensitivity to opsonophagocytosis, and is a well-recognised virulence factor of l. garvieae. the eps locus of l. garvieae lg2 was reported to be a genomic island, attributed to the presence of iss is982 on both ends of the eps biosynthesis cluster, differences in guanine-cytosine (gc) content between the lg2 chromosomal average and cluster (39% and 31%, respectively) and demonstration that this locus was probably inserted into the locus syntenic to four sequenced l. lactis strains (morita et al. 2011). it was postulated that this gene cluster might have been present in the avirulent non-capsulated reference strain atcc® 49156, originally isolated from diseased yellowtail, and was lost during repeated subculturing (morita et al. 2011). the negative results obtained using the pcr amplification of the eps biosynthesis cluster in this study may reflect a similar phenomenon as the attenuation of atcc® 49156 by repeated subculturing. the strains used in this study were isolated from diseased rainbow trout in 1986 and has been repeatedly subcultured under laboratory conditions, which may be selective for the non-capsulated phenotype as no selective pressure in the form of host immune mechanisms is applied. presence of the capsule confers fitness only when growth occurs under in vivo conditions. therefore, deletion of the eps biosynthesis cluster, and subsequently the loss capsulated kg− phenotype, is a possible explanation for the results obtained. however, in light of recent findings by türe and altinok (2016), the isolates investigated in this study may not have been capsulated at the time of isolation. previous immunoproteomic studies on l. garvieae did not identify enolase, 60 kda chaperonin or phosphoenolpyruvate-protein phosphotransferase as antigens recognised by rabbit antisera (shin et al. 2007) or olive flounder (paralichthys olivaceus) antisera (shin et al. 2009). however, l-lactate dehydrogenase was reported as an antigen common to both kg+ and kg− serotypes (shin et al. 2009). although enolase, 60 kda chaperonin and phosphoenolpyruvate-protein phosphotransferase have not been identified as antigens in l. garvieae, the identification of these extracellular proteins is of interest as they have been described as moonlighting proteins with involvement in virulence and regulation of virulence genes in gram-positive bacteria. enolase (ec 4.2.1.11) is a glycolytic enzyme responsible for catalysing the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the second last step of glycolysis, and the reverse reaction in gluconeogenesis (lee et al. 2006). additionally, this enzyme plays a role in virulence of gram-positive and gram-negative bacteria (bergmann et al. 2001; nogueira et al. 2013; pancholi & fischetti 1998). numerous studies on the moonlighting activities of enolase in prokaryotes have highlighted its involvement in binding of host plasmin(ogen), a process of critical importance to invasion of host tissues (bergmann et al. 2001; henderson & martin 2011; pancholi & fischetti 1998). binding of host plasmin(ogen) to its surface endows the bacterial cell with protease activity, which facilitates invasion and dissemination in the host (winram & lottenberg 1996). surface-associated enolase with plasmin(ogen) binding activity has been reported in human streptococcal pathogens streptococcus pyogenes (pancholi & fischetti 1998), streptococcus pneumoniae (bergmann et al. 2001) and piscine pathogen streptococcus iniae (kim et al. 2007). a study by wang et al. (2015) confirmed the immunogenicity of s. iniae recombinant α-enolase in mice, showing that immunisation with α-enolase elicited a significant increase in specific immunoglobulin g in comparison with the control group and ultimately protected mice against systemic s. iniae infection (wang et al. 2015). the ability of s. iniae α-enolase to confer protection in nile tilapia (lafrentz, shoemaker & klesius 2011) and turbot (scophthalmus maximus) (zhang, zhang & sun 2014) has been previously suggested. the prokaryotic 60 kda chaperonins (cpn60) is a highly conserved group of proteins that mediate intracellular protein folding to ensure correct functioning (henderson, fares & lund 2013). cpn60 is found on the cell surface of a variety of bacteria where it functions mainly as an adhesin. among the members of the order lactobacillales reported to produce extracellular cpn60 are lactobacillus johnsonii (bergonzelli et al. 2006), l. lactis (katakura et al. 2010), streptococcus agalactiae (hughes et al. 2002) and streptococcus suis (wu, zhang & lu 2008). diverse ligands of bacterial cell-surface cpn60 have been identified, ranging from mucin, lactoferrin, invertase and glycosphingolipids to integrin receptors cd11/cd18, αvβ3 and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (henderson et al. 2013). proteomic analysis of s. iniae atcc® 29178 and l. garvieae kg9408 (capsulated) by 2-dimensional gel electrophoresis identified cpn60 in both fish pathogens (shin et al. 2006), but subsequent immunoproteomic analyses by immunoblotting with rabbit and olive flounder antiserum did not identify this protein as an antigen of either capsulated or non-capsulated l. garvieae (shin et al. 2007, 2009). phosphoenolpyruvate (pep)-protein phosphotransferase (ec 2.7.3.9) is a key enzyme in the bacterial phosphotransferase system (pts) (deutscher et al. 2014). the multifunctionality of s. pneumoniae pep-protein phosphotransferase (ptsa) has been investigated by demonstrating its presence on the cell surface of s. pneumoniae using immunofluorescence techniques and using recombinantly expressed ptsa (rptsa) to screen a filamentous phage display library to identify peptides capable of inhibiting adhesion of s. pneumoniae to human lung adenocarcinoma cells (mizrachi nebenzahl et al. 2016). these peptides showed homology to various human ecm proteins, including multimerin i, protocadherin 19 and collagen type viiα1, suggesting that ptsa acts as an adhesin of s. pneumoniae (mizrachi nebenzahl et al. 2016). furthermore, rptsa was identified as a candidate target for vaccine development by showing that immunisation of mice with rptsa offered protection against pneumococcal challenge via different routes (mizrachi nebenzahl et al. 2016). pore-forming toxins are secreted by a variety of gram-negative and gram-positive pathogenic bacteria to disrupt lipid bilayers of host cells (iacovache, van der goot & pernot 2008). the putative virulence gene hly1 was reported to display 56% amino acid sequence homology to a protein in e. faecalis containing motif pf03006 conserved in proteins with haemolytic activity (morita et al. 2011). proteins containing this motif are classified in the haemolysin-iii related family (http://pfam.xfam.org/; finn et al. 2014). based on the structural features of hly1, it is classified as a member of the α-pore-forming toxins, which include colicins of escherichia coli (iacovache et al. 2008). another putative haemolysin under investigation in this study, encoded by hly2, was reported to display 72% amino acid homology to with a haemolysin in s. suis (morita et al. 2011). the gene product of hly3 was reported to display 59% amino acid sequence similarity to a haemolysin in s. pyogenes (morita et al. 2011), an organism associated with β-haemolytic activity. reactive oxygen species (ros) have various noxious effects on cells (yesilkaya et al. 2000). protection against oxidative stress is afforded by the enzymatic removal of ros by catalase and sod. the lab are catalase negative (collins et al. 1983); therefore, sod (ec 1.15.1.1) represents a major defence mechanism against oxidative stress in l. garvieae. the detoxifying properties of sod can also enhance intraphagocytic survival of bacteria upon generation of bactericidal ros as a result of respiratory burst (fang et al. 2015). the enzyme nox reduces molecular oxygen to h2o or h2o2 and was proposed to perform an important role in defence against o2 toxicity and o2 sensing, which are essential functions during aerobic growth of lab (muchnik et al. 2013). the importance of nox in the virulence of s. pneumoniae has been established by yu et al. (2001) by demonstrating significant attenuation of a nox-deficient mutant, leading to the conclusion that nox is required for in vivo proliferation in o2-rich environments. random peptide library phage display was used to identify several extracellular matrix proteins as ligands to recombinant nox, suggesting that the enzyme may play a multifunctional role in virulence by additionally acting as an adhesin (muchnik et al. 2013). adherence to host cells is a prerequisite step in the colonisation processes of pathogenic and commensal microbes (kline et al. 2009). morita et al. (2011) reported that protein lcgl_1330 of l. garvieae lg2 shared 62% amino acid identity with s. pneumoniae tigr4 pava and contained a fibronectin-binding motif (pf05833). pneumococcal adherence and virulence factor a (pava) is a cell surface-localised fibronectin-binding protein that lacks both typical signal sequences for secretion as well as the lpxtg anchorage motif characteristic of cell surface proteins (holmes et al. 2001). another protein with amino acid sequence homology (49%) to a s. pneumoniae cell surface-exposed virulence determinant, pneumococcal surface antigen a (psaa), was identified in lg2 by morita et al. (2011). this protein was originally identified as a putative adhesin (berry & paton 1996); however, its structure was not found to be consistent with the function of adhesion (johnston et al. 2004). its function was revealed to be a divalent metal ion-binding lipoprotein component of an atp-binding cassette (abc) transporter for mn2+ (johnston et al. 2004). conclusion the major virulence factor of l. garvieae defined thus far is the antiphagocytic polysaccharide capsule. with the aid of next-generation sequencing of full genomes, several other putative virulence factors have been identified. despite rapid advances in the areas of molecular biology and bioinformatics, its pathogenic processes are inadequately understood. no polysaccharide capsule was detected in any of the south african strains studied. putative virulence factors with roles in adhesion, cytolytic activity, oxidative stress tolerance and metal homeostasis were identified in a set of seven south african fish pathogenic l. garvieae isolates, as well as an avirulent isolate. detection of extracellular proteins of south african fish pathogenic l. garvieae isolates in this study identified candidates that may be investigated as subunit vaccines in future. specific virulence factors responsible for the pathogenicity of l. garvieae could not be identified, as putative virulence factor genes were present in both the fish pathogenic isolates and the avirulent isolate. all virulence factors discussed in this study, with the exception of haemolysins, are virulence lifestyle factors that indirectly contribute to host damage. these virulence lifestyle factors aid in the infection process by evasion of the host’s innate immune system, cofactor homeostasis, systemic invasion and dissemination in the host and adhesion to host tissues. future studies should focus on investigating differential expression of virulence lifestyle and true virulence genes during growth in the host environment versus laboratory conditions. 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pretoria, pretoria, south africa vinny naidoo biomedical research centre, faculty of veterinary science, university of pretoria, pretoria, south africa department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa citation chipangura, j.k., ganswindt, a. & naidoo, v., 2020, ‘a report on the housing vervet monkeys adjacent to domestic cats as a means of environmental enrichment’, onderstepoort journal of veterinary research 87(1), a1870. https://doi.org/10.4102/ojvr.v87i1.1870 original research a report on the housing vervet monkeys adjacent to domestic cats as a means of environmental enrichment john k. chipangura, andre ganswindt, vinny naidoo received: 01 apr. 2020; accepted: 07 sept. 2020; published: 26 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in current research guidelines, much focus is placed on ethical management of animals and the application of principles of reduction, refinement and replacement. of these refinements through environmental enrichment is an important aspect when housing primate to prevent behavioural problems. in this study, we investigated the co-housing of domestic cats and vervet monkeys as a novel method of enrichment based on the cohabitation and stress alleviation effect of horses housed with goats and from seeing cats cohabitating with vervet monkeys in an animal sanctuary. the study used a habituation method whereby the cats were stepwise introduced to the monkeys by sight and smell but with physical separation. assessment included changes in behaviour, weight and faecal glucocorticoid metabolite (fgcm) concentrations over time. on the first day of housing, the vervets whilst inquisitive kept their distance. the vervets housed in cages that were closest to the cats were the most active and during the first minute of introduction made more alarm calls, which stopped a few days later. the fgcms were non-significantly different. the results of this study provide evidence that vervet monkeys and domestic cats could potentially be housed together without overt aggression. we thus suggest further observations to ascertain if the co-housing could have long-term benefits for vervet monkeys, from the companionship that would be offered by the cats. keywords: vervet monkeys; domestic cats; stress; faecal glucocorticoid metabolites. introduction experimental animals play an important role in biomedical research. whilst the use of animal models is meant to generate reliable results that could allow for extrapolation to humans or veterinary medicine, stress in the research animals can at times become a confounder. these stressful conditions tend to confound research data by altering the physiological status of the animal (hubrecht & kirkwood 2010). for laboratory animals held in captivity, housing and environmental conditions can become the stressors. stress can impact the well-being of animals by resulting in effects such weight loss, immune system dysregulation and even aggression (honess & marin 2006). to improve these circumstances, good welfare practice advocates for the full implementation of the 3rs (reduction, replacement, refinement) in research (hubrecht & kirkwood 2010; russell & burch 1959). of the 3rs, refinement is aimed at methods used to minimise the severity of handling of animals in research and thus the stress experienced by animals (newberry 1995; olsson & dahlborn 2002). examples of refinement techniques include gentle handling and providing animals with appropriate housing that allows for the expression of species-specific behaviours (newberry 1995; olsson & dahlborn 2002), with enrichment of the home environment also being an essential component. whilst the housing conditions for laboratory animals may be improved through methods such as the provision of toys, logic puzzles and offering different types of bedding, the most important enrichment tool is to provide an environment where animals would have an opportunity to express species-specific behaviours, which for primates is social housing – an important aspect to consider (coleman & novak 2017; hubrecht & kirkwood 2010). for proper social housing, one requires adequate space that not only takes into account floor space by height because of the arboreal nature of primate natural environments. unfortunately, the latter is not always possible in research centres, as adequate space is a limiting factor for economic reasons and when taking into account disease mitigation strategies, which would require wholly indoor housing. vervet monkeys (chlorocebus pygerythrus) are one common non-human primate (nhp) species used in biomedical research (jorgensen et al. 2017). despite their widespread use, there is limited information available describing their optimal environmental enrichment. with vervets in the wild living in large social groups, it is not surprising that these animals’ social needs require attention when in captivity to prevent behavioural abnormalities, whilst at the same time maintaining the integrity and quality of research data (seelig 2007). in contrast, the rhesus macaque (macaca mulatta) represents the most commonly used nhp species in research and numerous published studies exist on their optimal enrichment in captivity (e.g. coleman & novak 2017; novak et al. 1998; reinhardt 1999; weed et al. 2003; wooddell et al. 2019). as a result, enrichment methods developed for macaques are often adopted for vervet monkeys. whilst these interventions may be successful, generalisations on husbandry of nhps can lead to further problems because the animals’ psychological response to interventions may be very species-specific. the university of pretoria biomedical research centre (upbrc) housed adult vervet monkeys over a period of 10 years, for which numerous environmental enrichment techniques have been tried on the colony. the specific facility had five primate rooms of which four were in use. each room had an indoor and outdoor area. whilst the indoor area is one large space per primate room, the indoor area can if needed be subdivided into smaller cage spaces. both the indoor and outdoor areas are sufficiently high for the placement of branches to stimulate arboreal behaviour. whilst the indoor areas are physically separated by brick walls, the outdoor pens are separated by a double fence system. as a result of the permanent housing of these primates, our laboratory is constantly looking into ways to better enrich the environment of these permanently housed primates. a potential opportunity presented itself when a university graduate student wanted to undertake research on disease-free cats. in a laboratory animal research environment, the optimal housing conditions for cats should allow for sufficient space, allow for group housing and provide for opportunities for behaviours such as climbing (geret et al. 2011). based on the similarity in housing requirements between cats and nhps, we asked the question as to whether the animals could be housed in the same environment and whether both species would benefit from their interaction. we based this supposition on a previous study that showed the cohabitation and stress alleviation effect of housing horses with goats (winter 1996). in our consideration, we felt that the cats would be minimally intimidating to the nhps because of their size. we also undertook an extensive literature review, where we found no information on co-housing of cats and primates. at present the only information we were able to find on interspecies co-housing was for different nhp species in the same cage, which were for rhesus and cynomolgus macaques (rehrig & wyatt 2012). as a result, a general internet search was undertaken, where numerous reports of primates and cats forming strong bonds could be found, together with one specific mention of a vervet baby bonding with two cats at a sanctuary (carlson 2017). furthermore, we had the opportunity to visit a local vervet sanctuary in south africa, where we were able to observe a group of feral cats making their home in the enclosure with no adverse interactions between the two groups. no information was also available on the potential for disease transmission between the two species. for this study, we describe our experience with the housing and the results from stress-monitoring testing that was also undertaken, as a pilot study to look at the potential of co-housing of a primate and non-primate species. materials and methods animals used in the study vervet monkeys the vervets used in this study were resident at the upbrc and were not part of any other research study at the time of the experiment. the colony is unique in that the individual history of the animals is unknown, including their age and relationship with each other, as the animals were all wild caught at an unknown date before being moved to their current home. the animals were resident at the upbrc since 2003 and were estimated to have an age of 9 years when they arrived at the facility. the study animals were thus likely very close to their lifespan of typically 25 years in captivity. the colony comprised six females and four males. at the time of their arrival, the animals were housed in individual commercial primate cages. since then their habitat has been modified to a design that includes both indoor and outdoor areas (figure 1). the colony is currently kept in heterosexual pairs or groups of three in ceiling height (3 m height) cages and surface area of 5.7 m2, with an indoor and outdoor component since 2011, with both enclosures having artificial trees therein to the current period of 2017 when the study was undertaken. the vervets had free choice of selecting being indoors or outdoors at any time of the day. the cages were cleaned at least once a day during the observational period and the vervets fed twice daily, in the morning with fruits and primate biscuits (wheat bran, maize meal, protein, vitamin, mineral (pvm) primate supplement (pvm nutritional sciences, south africa) and vitamin c (junglevites chewey c, pharmanatura, south africa) and in the afternoon with fruits and vegetables. potable municipal water was provided ad libitum. all male vervets were castrated in 2011 prior to heterosexual pair housing. room temperature for the indoor cages was maintained at 20 ºc – 25 ºc, with a relative air humidity of approximately 50% and a 12-h artificial light/dark cycle. food enrichment was provided in the form of raisins, rusks, sunflower seeds and nuts and environmental enrichment by providing hard plastic toys, balls, foraging containers, plastic crates, climbing wooden logs, puzzle feeders, swing ropes and tyres. figure 1: cage layout for the cats and vervet monkeys. (a) inside cage area; (b) outside pens; (c) double fence separating the outer pens; (d) inside observation area; (e) outside observation area; thick arrow, room entry point; thin arrow, animal outer access hatches; large solid areas, bricked walls; thin line, expanded metal mesh. domestic cats eight neutered domestic cats that were assigned to form part of another long-term study, which has been postponed, were used. the cats were 6 weeks old at the start of housing, and were not exposed to monkeys before. the colony comprised four females and four males. before the experiment, the cats were group housed in the same unit as the primate, albeit in a separate room (room 5). the room as for the primates was equipped with an indoor and outdoor section, water bowl, food bowl, litter tray, cat beds and soft blankets. the enclosure was cleaned at least once a day. the cats were fed twice a day with a commercial cat diet and water was provided ad libitum. environmental enrichment included scratch poles, tree stumps, hiding places and commercial cat toys. the inner room was separated from the primates by brick wall, whilst for safety reasons the outdoor areas of the primates and cats were separated by a movable double expanded metal grids, with a 30 cm separation between the two grid layers. experimental procedures six days prior to housing the animal species next to each other separated by a barrier, the vervets and cats were monitored in their accustomed enclosures with behavioural observations and faecal sample collection taking place three times per day. further animals were weighed a day before the housing experiment began to establish baseline weights. the vervets were well accustomed to being weighed as this was a common procedure during their housing at the facility and they participated in the procedure without any aggression or overt signs of stress. for the weighing the animals entered into a smaller cage of known weight on wheels, which is subsequently weighed on a calibrated platform scale. the vervet monkeys were kept in their usual social groups, during the introduction of the domestic cats as next-door neighbours. no changes were made to the animals’ diets and water was provided ad libitum. the cats were allowed outdoor access to the enclosure next to the vervets during the day (8:00–14:00), whilst they were returned to their indoor housing at night for the first month. after 1 month, the cats were left permanently in the enclosure next to the vervets, with a free choice of inside or outside access. to prevent injuries, the cats and vervets were never in direct contact, with a double expanded-metal mesh separating them always, but they could see and smell each other via the cage fencing. to minimise confounding variables, only personnel whom the primates were accustomed to could work in the primate and cat unit. staff members, responsible for monitoring the animals, had at least 3 years of experience working with the vervets and were the same staff who took care of the kittens. during the first week of introduction, we monitored the domestic cat and vervet behaviour on an hourly basis, which we decreased to twice daily during the second week to ensure that the animals did not injure themselves. to prevent bias in behavioural observations, behaviours of the animals were monitored by closed-circuit video. behavioural observations the cats and vervets were monitored for the full duration of the experiment by closed-circuit video (hikvision dvr – model:ds-9016hfi-s [hikvision corporate solutions, alberton, south africa] and samsung camera – model: shc-721ap [samsung electronics, johannesburg, south africa]). as we had no idea about what behaviour the animals would elicit on the introduction, we decided to monitor them against predator avoidance behaviour, which was previously described. it was believed that if the vervets were to be stressed by the presence of the cats, they would elicit a typical predator avoidance behaviour. we also made the assumption that the vervet monkeys may associate the domestic cats with servals (felis serval), which are their natural predators, although the two species are different in size (guy & curnoe 2013).the following behaviours were chosen for analysis, as described by seyfarth, cheney and marler (1980) as relevant for primates facing a potential predator as looking in direction of predator: alarm calls defined as short tonal calls produced in a series of inhalations and exhalations or climbing high up tree branches provided in the enclosure. for alarm calls the total number of calls was recorded, whilst for the other behaviours the number of animals showing the behaviour was recorded. faecal sample collection faecal samples from both species were collected for 6 days prior to co-housing to determine species-specific baseline values. after introducing the domestic cats, faeces were collected for both species for another 6 days. the enclosures were inspected three times a day, in the morning (7:00–10:00), at noon and afternoon (14:00–15:00) and all faeces were collected into individually labelled plastic bottles. samples were homogenised using a wooden spatula before placing into plastic bottles. all available samples, including samples produced overnight, were collected. all samples were stored at −20 °c within 1 h of collection until analysis. for the vervets, individual sampling was possible because individuals were fed biscuits mixed with different food colorants (robertsons, durban, south africa). the domestic cats used communal litter trays and the faecal sample could only be identified to a particular cat when the cat was observed defecating. in total 311 samples were collected for the vervets and 79 for the cats. faecal steroid extraction and analysis at the endocrine research laboratory, faculty of veterinary science, university of pretoria, south africa, the frozen faecal samples were lyophilised, pulverised and sifted through a wire-mesh strainer to separate faecal powder from undigested material. about 0.10 g – 0.11 g faecal powder from each sample was then extracted by adding 3 ml 80% (volume/volume) ethanol. the suspension was vortexed for 15 min and subsequently centrifuged at 1500 × g for 10 min. the supernatant (1.5 ml) was then transferred into labelled eppendorf safe-lock micro test tubes and stored at -20 °c until further analysis. faecal steroid extracts were measured for faecal glucocorticoid metabolite (fgcms) concentrations, using established enzyme immunoassays (eia) for fgcm monitoring in cats (schatz & palme 2001) and vervet monkeys (young et al. 2017). respective eias used antibodies against 11-oxoetiocholanolone (detecting 11, 17-dioxoandrostanes, for the cats) and cortisol (for the vervet monkeys). detailed assay characteristics, including a full description of the assay components and cross-reactivities, are provided for both eias by palme and möstl (1997). assay procedures followed the protocols published by ganswindt et al. (2002). sensitivity of the 11-oxoetiocholanolone eia was 2.4 ng/g dry weight (dw)and for the cortisol eia it was 0.6 ng/g dw. intra-assay coefficients of variation determined by repeated measurements of high and low value quality controls were 4.8% and 5.8% for 11-oxoetiocholanolone and 4.0% and 4.8% for cortisol measurements. inter-assay coefficients of variation were 8.1% and 12.6% for 11-oxoetiocholanolone and 12.7% and 14.7% for the cortisol eia. statistical analysis the behaviour of the vervets was monitored on the closed-circuit video recording and then analysed using descriptive statistics prior to cat introduction, immediately post-introduction, and after 3 days of housing. to check for significant changes in behaviour before and during co-housing, the data were checked for normality using the shapiro–wilk test and analysed using the kruskal–wallis (kw) test. one-way analysis of variance (anova) on ranks and multiple comparisons using tukey’s post hoc test were used to isolate the group or groups that differ from others. differences in animal weight before and after 6 days of co-housing were examined for cats and vervets using either a wilcoxon signed rank or paired t-test. differences in fgcm concentrations for the day prior to introduction, of introduction and days 1 and 2 post-introduction for cats and vervets were analysed using kw one-way anova on ranks (for the cats as no individual sampling could be conducted) or repeated measured anova (for the vervets). the individual median fgcm concentration was calculated in cases where more than one sample per day was collected from an individual. normality of available data sets was analysed using shapiro–wilk’s test. the statistical analyses were performed using the software programme sigma plot 12.5. the significance level was set at 0.05. ethical consideration the university of pretoria animal ethics committee (up-aec) approved the use of vervet monkeys and domestic cats that were resident at the upbrc for this study (protocol number v118/15). the up-aec is registered with the south african national health research ethics council (nhrec), and follows the south african national standard for the care of research animals (sans10386). results behavioural observations on the first day of housing next to cats, it was observed that the vervets, in general whilst interested with the presence of the cats (looking in direction of cats, pacing up and down), still maintained their distance from the cage boundary. the four vervets housed in two cages that were closest to the domestic cats, with only 30 cm (group 1) and 3 m (group 2) between the enclosures, respectively, were the most active. during the first 1 min of introduction, the vervets looked more often at the cats, climbed up to the top of the cage and made more alarm calls (table 1). the calls were made whilst looking in the direction of the cats, with the other vervets in the cages furthest from the cats responding by looking at the direction of the caller. the calls were significantly reduced by day 3 and no calls were made by day 14. none of the vervets showed redirected aggression to cage mates and no aggression was evident for any other point of the study. the cats demonstrated inquisitive behaviour as they explored the new environment although without urine marking. we attributed the absence of urine markers rather to the habituation of the cats to using a litter tray. table 1: frequency of recorded responses of the vervets. when comparing the number of alarm calls made 1 min before the cats were introduced, at the time when the cats were introduced and 3 days after the cats were introduced, the difference in the median values (2, 31 and 15, respectively) amongst the groups was found to be greater than that would be expected by chance (p = 0.001), and the pairwise multiple comparison test showed evidence of a statistically significant difference only for 1 min before the cats were introduced and at the time the cats were introduced. for the number of vervets looking in the direction of the cats, the difference in the median values amongst the groups was greater than that would be expected by chance (p = 0.029), and the pairwise multiple comparison test showed evidence of a statistically significant difference only for 1 min before the cats were introduced and at the time the cats were introduced. weight alterations related to changes in housing conditions there was a no significant (t = 0.218, df = 9, p = 0.832) increase in average weight for the vervets from 5.5 kg ± 0.82 kg (mean ± standard deviation [sd]) before housing to 5.6 kg ± 0.81 kg during housing (table 2). in contrast, there was a significant increase (w = 36, n = 8, p < 0.001) in weight gain for the cats during housing, with the overall average weight of 3.7 kg ± 0.72 kg prior compared with 3.9 kg ± 0.73 kg during co-housing (table 3). the latter was as expected for young growing cats. table 2: vervet monkeys’ weight (kg) before and during housing next to domestic cats. table 3: domestic cats’ weight (kg) before and during housing next to vervet monkeys. glucocorticoid alterations related to changes in housing conditions there was no significant (f = 0.59, n = 10, p = 0.627) difference in fgcm levels for the vervets before, during and on days 1 and 2 post-introduction, with the overall mean fgcm concentrations of 95.22 ± 43.04 ng/g dw (mean ± sd) prior and 116.49 ± 62.07 ng/g dw during co-housing. similarly, there was no significant (h = 2.04, df = 3, p = 0.564) difference in fgcm levels for the cats before, during and on days 1 and 2 post-introduction), with the overall mean fgcm concentrations of 0.33 ± 0.17 µg/g dw (mean ± sd) prior and 0.38 ± 0.28 µg/g dw during co-housing. discussion this study was aimed at evaluating the potential of housing vervet monkeys (c. pygerythrus) next to domestic cats (felis silvestris catus), which we believed would be appropriate based on the similar housing requirements between these species, the size of the cats and an observed natural instance at a local vervet sanctuary where the two species tolerated each other well. despite the potential benefits of companionship to both the species related to reduction in stress, because of the possibility of the animals responding adversely to the interaction including aggression or self-injury, we ran the experiment as a pilot study, with a fence being maintained between the two groups, and used faecal glucocorticoids as one of the main indicator parameters. on the first day of housing, both vervets and domestic cats were aware of the other species although they kept their distance. no aggression was evident in any of the cage groupings. the monkeys (two males and two females) housed in the cages that were closest to the domestic cats were the most vocal. our findings of no aggression are similar to those of jorgensen et al. (2017), who noted that vervets rarely show overt aggression on the day of introduction to new housing system when pair housed with another vervet. the vervets however responded in a similar way reported for animals in the wild when they encounter leopards (seyfarth et al. 1980) by showing an increase in directed looking and climbing behaviour and making alarm calls. the alarm calls were clearly directed towards the cats as the caller looked directly at the cats, with the animals in the furthest enclosure looking at the direction of the caller. this was an unexpected finding as the vervets have been in captivity for over 15 years and it indicates that alarm calls were likely a learnt behaviour during infancy to avoid predators (seyfarth et al. 1980). with the animals becoming accustomed to the cats in what can be considered no longer than a routine acclimatisation period, this would indicate that cats could be companions to primates. however, the next step would be to evaluate the direct interactions of the two species to ascertain if companionship bond is formed as seen in the report of a vervet baby raised with cats. another important parameter in the monitoring of animal welfare is their change in body weight, which may increase or decrease. animals can lose weight (poole 1997) because of inappetence or a reduction in food and water intake. even when the animal maintains a normal appetite during stress, the underlying causes of stress may increase energy expenditure, contributing to a net loss of energy that will subsequently lead to weight loss (hubrecht & kirkwood 2010; poole 1997). in contrast, the animals may have an increase in weight because of cortisol-mediated deposition of fat in the abdominal region together with increased liver weights. with none of the animals inducted into the study having lost weight, it can thus be concluded that housing vervet monkeys and domestic cats separated by a physical barrier was not a stressful event to either species. with three pivotal parameters of stress (weight loss, faecal glucocorticoid concentrations and behavioural parameters) all being relatively stable, the results of this study provide the first step in demonstrating that vervets and domestic cats could be co-housed and this can be used as a way of utilising limited laboratory animal space. however, as the study relied on physical separation, the next step would be to explore the possibilities of co-housing vervets and cats in the same enclosure. acknowledgements the authors would like to thank the university of pretoria biomedical research centre (upbrc) staff for looking after the animals, endocrine research laboratory staff for analysing the samples and everyone else who assisted in the completion of this research. competing interests the authors have declared that no competing interests exist. authors’ contributions j.k.c. and v.n. conceived the study and participated in the data analysis and write-up of the manuscript. a.g. 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https://doi.org/10.1002/ajp.23073 young, c., ganswindt, a., mcfarland, r., de villiers, c., van heerden, j., ganswindt, s. et al., 2017, ‘faecal glucocorticoid metabolite monitoring as a measure of physiological stress in captive and wild vervet monkeys’, general and comparative endocrinology 253, 53–59. https://doi.org/10.1016/j.ygcen.2017.08.025 abstract introduction materials and methods results discussion acknowledgements references about the author(s) oday a. aljumaili department of veterinary clinical studies, faculty of veterinary medicine, universiti putra malaysia, serdang, malaysia laboratory of vaccines and immunotherapeutics, institute of bioscience, universiti putra malaysia, serdang, malaysia muhammad b. bello laboratory of vaccines and immunotherapeutics, institute of bioscience, universiti putra malaysia, serdang, malaysia department of veterinary microbiology, faculty of veterinary medicine, usmanu danfodiyo university, sokoto, nigeria swee k. yeap laboratory of vaccines and immunotherapeutics, institute of bioscience, universiti putra malaysia, serdang, malaysia abdul r. omar laboratory of vaccines and immunotherapeutics, institute of bioscience, universiti putra malaysia, serdang, malaysia department of pathology and microbiology, faculty of veterinary medicine, universiti putra malaysia, serdang, malaysia aini ideris department of veterinary clinical studies, faculty of veterinary medicine, universiti putra malaysia, serdang, malaysia laboratory of vaccines and immunotherapeutics, institute of bioscience, universiti putra malaysia, serdang, malaysia citation aljumaili, o.a., bello, m.b., yeap, s.k., omar, a.r. & ideris, a., 2020, ‘protective efficacy of inactivated newcastle disease virus vaccines prepared in two different oil-based adjuvants’, onderstepoort journal of veterinary research 87(1), a1865. https://doi.org/10.4102/ojvr.v87i1.1865 original research protective efficacy of inactivated newcastle disease virus vaccines prepared in two different oil-based adjuvants oday a. aljumaili, muhammad b. bello, swee k. yeap, abdul r. omar, aini ideris received: 28 mar. 2020; accepted: 25 june 2020; published: 28 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract despite the availability of newcastle disease (nd) vaccines for more than six decades, disease outbreaks continue to occur with huge economic consequences to the global poultry industry. the aim of this study is to develop a safe and effective inactivated vaccine based on a recently isolated newcastle disease virus (ndv) strain ibs025/13 and evaluate its protective efficacy in chicken following challenge with a highly virulent genotype vii isolate. firstly, high titre of ibs025/13 was exposed to various concentrations of binary ethylenimine (bei) to determine the optimal conditions for complete inactivation of the virus. the inactivated virus was then prepared in form of a stable water-in-oil emulsion of black seed oil (bso) or freund’s incomplete adjuvant (fia) and used as vaccines in specific pathogen-free chicken. efficacy of various vaccine preparations was also evaluated based on the ability of the vaccine to protect against clinical disease, mortality and virus shedding following challenge with highly virulent genotype\vii ndv isolate. the results indicate that exposure of ndv ibs025/13 to 10 mm of bei for 21 h at 37 °c could completely inactivate the virus without tempering with the structural integrity of the viral hemagglutin-neuraminidase protein. more so, the inactivated vaccines adjuvanted with either bsoor fia-induced high hemagglutination inhibition antibody titre that protected the vaccinated birds against clinical disease and in some cases virus shedding, especially when used together with live attenuated vaccines. thus, genotype vii-based ndv-inactivated vaccines formulated in bso could substantially improve poultry disease control particularly when combined with live attenuated vaccines. keywords: newcastle disease virus; genotype vii; inactivated vaccine; water-in-oil emulsion; virus shedding. introduction newcastle disease (nd) is one of the most important avian diseases that significantly militate against poultry production all over the world (alexander & senne 2008). the disease is particularly important in chicken where it inflicts huge economic losses because of high mortality, reduced egg production as well as restrictions in international trade of poultry and poultry products (oie 2012). the aetiology of the disease is newcastle disease virus (ndv), a highly pleomorphic enveloped virus that belongs to the genus avulavirus in the family paramyxoviridae (nagai, hamaguchi & toyoda 1989). the virus encloses a 15.2 kb negative-stranded non-segmented ribonucleic acid (rna) genome that houses six genes encoding the following structural proteins: nucleocapsid protein (np), phosphoprotein (p), matrix protein (m), fusion protein (f), hemagglutinin-neuraminidase protein (hn) and large protein (l) (murulitharan et al. 2013; yusoff & tan 2001). among these proteins, f is considered to be the major molecular determinant of ndv pathogenicity in chickens (de leeuw et al. 2005). as a rule, virulent ndv strains are known to possess multiple basic amino acid residues (arginine and lysine) between amino acid positions 112–116 and a phenylalanine residue at position 117 in the f protein. on the contrary, isolates of low virulence are considered to be those with monobasic f cleavage site and a leucine residue at position 117 (oie 2012). in addition, isolates of ndv can be pathotypically classified into lentogenic (mildly virulent), mesogenic (moderately virulent) and velogenic (highly virulent) strains (bello et al. 2018a; vijayarani et al. 2010) based on the mean death time (mdt) performed in 9–10-days-old embryonated chicken eggs and the intracerebral pathogenicity index in 1-day-old chicks. furthermore, ndv is classified into more than 18 phylogenetically distinct genotypes (bello et al. 2018b; diel et al. 2012; snoeck et al. 2013). interestingly, despite this high genetic diversity, all ndv isolates are serologically grouped into a single serotype because they share a fairly similar immunodominant epitopes (gogoi, ganar & kumar 2017). thus, any strain of ndv could theoretically elicit immune response capable of cross-protecting against any other strain of the virus. over the years, lasota, b1 and other genotype ii-based live attenuated vaccines have been used to curtail the menace of ndv in the global poultry industry (kapczynski, afonso & miller 2013). however, the vaccines have frequently been associated with the severe post-vaccinal respiratory reactions that may predispose the birds to secondary bacterial infections. furthermore, they can only protect against clinical disease but are unable to block the replication and shedding of phylogenetically divergent virulent isolates especially those belonging to genotype vii taxon (hu et al. 2011). currently, members of genotype vii taxon are the most rapidly evolving group of ndv directly linked with the ongoing fourth pandemic of the disease (miller et al. 2015). they are also widely distributed and have been the most predominantly circulating ndv strains in malaysia (roohani et al. 2015) and many other countries over the past two decades (esmaelizad et al. 2016; zhang et al. 2012). effective control of these viruses is therefore of paramount importance in improving the productivity of the global poultry industry. the current trend in the control of virulent ndv infection is the development of genotype-matched vaccines based on the currently circulating ndv isolates (bello et al. 2018a). these vaccines have been shown to be more effective not only in disease prevention, but also in blocking the replication and shedding of the virulent virus post-challenge (bello et al. 2020; hu et al. 2011; xiao et al. 2012). while efforts have been put in place to develop live attenuated vaccines based on genotype vii ndv, very few studies exist on genotype-matched inactivated ndv vaccines. therefore, in the present study, we developed a safe and effective inactivated vaccine based on a recently circulating ndv isolate, formulated it in different adjuvants and evaluated its protective efficacy against the challenge with another virulent genotype vii ndv isolate. materials and methods virus preparations newcastle disease virus strain ibs 025/13 is a velogenic isolate with intracerebral pathogenicity index (icpi) of 1.86. the virus was initially isolated from a well-vaccinated broiler farm in malaysia and was shown to be a naturally recombinant ndv with np and p genes derived from genotype ii (vaccine strain), while the m, f, hn and l genes being more closely related to genotype vii isolates (satharasinghe et al. 2016). the virus was propagated in 9-days-old specific pathogen-free (spf) embryonated chicken eggs and titrated using the methods described by reed and muench (1938). virus inactivation the 0.1 m binary ethylenimine (bei) was prepared according to the methods of king (1991). briefly, 0.041 g of 2-bromo-ethylamine hbr (bea) sigma (st. louis, missouri, united states) was dissolved in 2 ml of 0.175 n naoh merck (darmstadt, germany) and incubated for 60 min at 37 °c. the bei solution was then mixed with 109.2 eid50 of ndv strain ibs025/13 to make its final concentration of 4 mm and 10 mm in the bei–virus mixture. the mixture was afterwards incubated in a shaker at 37 °c for 21, 48, 72 and 96 h. finally, residual bei was hydrolysed from the samples by sodium thiosulfate (merck) treatment at the concentration of 10 times of the final bei concentration. viability test on the inactivated virus to test the viability of the inactivated virus, two blind passages were performed in 9-days-old spf embryonated eggs via allantoic cavity inoculation. during each passage, 108.6 eid50 of the inactivated virus (ndv ibs025/13) was inoculated into five eggs and embryonic mortality was monitored for 6 days before harvesting allantoic fluid for the next passage or storage (pharmacopoeia 2008). hemagglutination assay hemagglutination assay (ha) titres for live and the inactivated viruses were determined using 1% chicken erythrocytes suspension as described previously (beard, hopkins & hammond 1975). briefly, 50 µl of allantoic fluids containing either live or inactivated ndv ibs025/13 was added in triplicates to the first wells of 96-well microtitre plate. the allantoic fluid was then diluted in a twofold fashion up to well 11, while the 12th well was left as phospate buffered saline (pbs) control. next, 25 µl of 1% chicken red blood cells suspension was added to each well followed by 30 min incubation at room temperature. hemagglutination assay titre was determined as the highest dilution of the virus that agglutinated 1% chicken erythrocyte suspension was considered the ha titre. gas chromatography mass spectroscopy nigella sativa seeds (black seed) were purchased from a commercial dealer in malaysia. cold pressed method was used in extracting black seed oil (bso) using manual press machine. the gas chromatography (gc) profile of bso in this work was obtained using shimadzu gcms-qp2010 system. a bp × 5 (5% diphenyl + 95% dimethylpolysiloxane) 30 m (length) × 0.25 mm i.d. with 0.25 μm film thickness was utilised with a flow rate of 0.80 ml/min. a microliter injection was made in split less mode at the temperature of 250 °c. the gc oven was firstly held at 50 °c for a minute and later increased at 3 °c/min to 300 °c; it was again increased at the rate of 10 °c/min to 340 °c. the mass spectrometer was operated in electron power mode at 70 ev. the data were collected from m/z 40 to m/z 700 (khalid & shedeed 2016). preparation of inactivated newcastle disease virus in oil adjuvant the inactivated virus was prepared as water-in-oil (w/o) emulsion according to the method of mady et al. (2013) with slight modification. the oil phase consisted of 28% of oil soluble surfactant span 80 mp biomedicals inc. (santa ana, california, united states) mixed with 72% of freund’s incomplete adjuvant (fia) sigma-aldrich (schnelldorf, germany) or bso, while the water phase was made up of 12% aqueous soluble surfactant tween 80 (sigma-aldrich) mixed with 88% inactivated virus suspension. to prepare the homogenised w/o emulsion, the aqueous phase was added to the oil phase at a ratio of 3:4 in a drop-wise fashion with continuous mixing for 30 min. the hydrophilic lipophilic balance (hlb) was later adjusted to 3 as previously described by stone et al. (1978). stability test stability testing of emulsion involves determination of stability at long-term storage at 4 °c and 25 °c. the test was performed as described by cessi and nardelli (1974) and el-bagoury et al. (2015). vaccination and challenge study embryonated spf eggs were obtained from veterinary research institute (vri), ipoh, malaysia, and hatched in sterile hatchery facility in our laboratory. after hatching, the birds were transferred to the experimental animal facility where they were fed with commercial feeds and allowed to drink water ad libitum. a total of 81 birds were randomly divided into seven groups each comprising 11 chickens (with the exception of the first group that had 15 birds that served as negative control). three groups (groups 2–4) were vaccinated twice subcutaneously at 10 and 24 days with different inactivated ndv vaccine formulations (table 1). the remaining three groups (groups 5–7) were vaccinated at 10 days old with different inactivated vaccine formulations via subcutaneous route and live ndv vaccine via eye drop (table 1). at days 7, 14, 21 and 28, serum samples were collected and analysed using hemagglutination inhibition (hi) test as described by bello et al. (2020). the chickens were later challenged with 105 eld50 of velogenic genotype vii strain ibs002/11 (roohani et al. 2015) at 38 days old via eye drop. sera samples were collected from all groups to evaluate the hi titre. table 1: vaccination of specific pathogen-free chickens with different newcastle disease virus vaccines. pathogenicity scoring system chickens were clinically examined and scored on daily basis for 14 days following challenge with the virulent ndv isolate as recommended by allan and borland (allan & borland 1979). briefly, chickens were scored 0 if they showed no any clinical symptom, 1 if they had some respiratory symptoms or greenish diarrhoea, 2 when they were moribund and 3 when they were dead. the average scores from each group were measured as group’s total score. virus shedding measurement cloacal swab samples were collected from six birds in each group on days 5, 7 and 10 post-challenge (dpc). the samples were transferred into 1 ml sterile pbs, mixed thoroughly and kept at -70 °c until use. total rna was extracted from the swab samples using rneasy® mini kit plus qiagen (hilden, germany) following manufacturer’s recommendations. measurement of viral load was performed based on one-step taqman-based real-time polymersase chain reaction (pcr) as described by rasoli et al. (2014). the assay was carried in a final volume of 10 µl consisting of 5 µl iscript™ one-step rt-pcr kit for probes (2× reaction buffer containing 0.25 mm of each deoxynucleotide (dntp), magnesium ions, itaq dna polymerase, stabilisers) (bio-rad, united states of america), 1 µl of iscript reverse transcriptase (bio-rad), 0.5 µl of 20 mm of forward and reverse primers and 0.5 µl of 5 mm of probe with 3 µl of rna template, and sterile nuclease-free distilled water was used to top-up to the required volume. the thermocycling programme for the assay was 95 °c for 10 min for initial denaturation followed by 40 cycles of denaturation at 95 °c for 2 min, annealing at 58 °c for 30 s, extension at 72 °c for 15 s and plate read (rasoli et al. 2014). potency test of newcastle disease inactivated vaccine inactivated nd vaccine formulations that gave the best results were analysed further based on potency tests b as established by the european pharmacopoeia monograph 870 (pharmacopoeia 2008). a total of 120 chickens were randomly divided into two groups: bei-fia (n = 60) and bei-bso (n = 60). each group was further subdivided into three sub-groups (20 birds per sub-group) and immunised with 1/25, 1/50 and 1/100 dilution of the vaccines via intramuscular injection. the birds were later challenged with 105 eld50 of ndv strain ibs002/11 via intraocular inoculation. the birds that survived the challenge and showed no any clinical signs were considered protected. the vaccine was considered acceptable if the actual protection was up to 50% based on european pharmacopoeia monograph (monograph 0870 2007). ethical considerations all animal experiments were conducted in accordance with guidelines of the laboratory animal care as approved by the institutional animal care and use committee at the faculty of veterinary medicine, universiti putra malaysia (reference no. upm/iacuc/aup-r096/2015) results chemical inactivation of newcastle disease virus ibs025/13 two different concentrations (4 mm and 10 mm) of bei were used for virus inactivation. our results showed that exposure of ndv ibs025/13 to 4 mm of bei for 21 h could not inactivate the virus, as embryonic mortality was recorded in the inoculated spf eggs from the first passage. however, when the exposure was extended to 48, 72 and 96 h, no embryonic mortality was recorded at first passage in the inoculated eggs. however, at the second passage, dead embryos were observed 120 h post-inoculation, indicating incomplete inactivation of the virus. on the contrary, incubation of the virus with 10 mm bei for 21, 48, 72 or 96 h completely inactivated the virus as evidenced by the lack of embryonic mortality at 144-h post-inoculation in both the first and second passages. effect of binary ethylenimine on hemagglutination titre to test whether exposure to bei would affect the titre of inactivated virus, the ha test was performed before and after the chemical treatment. our result showed that bei treatment only had a mild effect on the ha titre of the inactivated virus which changed from 10 log2 to 9 log2 (table 2). table 2: effect of binary ethylenimine inactivation on the viral hemagglutination assay titre. gas chromatography mass spectroscopy analysis of black seed oil based on gc–ms analysis, the following constituents were found in bso: thujene <alpha-> (3.4%), pinene <alpha-> (0.7%), pinene <beta-> (0.7%), cymene <para-> (7.1%), trans-4-methoxy thujane (0.9%), thymoquinone (7.5%), 2,4-decadienal, (e,z)-(cas) trans,cis-2,4-decadienal (1.2%), trans,trans-2,4-decadienal (1.9%), n-hexadecanoic acid (6.7%), 5-isopropenyl-2-methyl-7-oxabicyclo[4.1.0]heptan-2-ol (1.8%), 9,12-octadecadienoic acid (z,z)-,methyl ester (cas) methyl linoleate (30%), 9-octadecenoic acid (e)-(26.6%), hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester (0.5%), e,e,z-1,3,12-nonadecatriene-5,14-diol (9.3%) and stigmast-5-en-3-ol, (3.beta.)-(cas) 24.beta.-ethyl-5.delta.-,cholesten-3.beta.-ol (0.8%). stability tests based on the tests performed in this study, our results showed that the optimised w/o vaccine preparation formed an emulsion that remained intact for more than 3 months at 25 °c or 6 months at 4 °c. this indicates the stability of the inactivated vaccine emulsified in oil phase (data not shown). hemagglutination inhibition test post-vaccination antibody titre in the chickens’ sera was determined using hi test with homologous antigens. all birds had no detectable ndv antibody titre just before vaccination. similarly, the control group showed no hi antibody reactions throughout the study. on the contrary, chickens vaccinated with various nd vaccines showed increasing antibody titre throughout the experimental period. generally, higher antibody titres were recorded in groups vaccinated with live vaccines compared to those vaccinated with inactivated vaccines. in addition, inactivated vaccines formulated in fia elicited higher hi titre than those formulated in bso adjuvant (table 3). table 3: geometric mean hemagglutination inhibition titre (log2 ± sd) in specific pathogen-free chickens vaccinated with different newcastle disease vaccines. scoring of mortality, morbidity and pathogenicity challenged chickens from all the groups were observed on a daily basis and accordingly scored based on disease severity and mortality. in the control group, all the birds displayed signs of nd, recorded 100% (15/15) mortality and accordingly had the highest average score for nd clinical signs (1.86 ± 0.55) compared to the other groups (data not shown). chickens in group 2 (those vaccinated with 109.47 eid50 at days 10 and 24) recorded average score of 0.09 ± 0.09. all other groups recorded no mortality or any obvious clinical signs. cloacal virus shedding virus shedding from cloaca was determined from all the groups at different time points post-challenge. our results indicate that virus shedding was observed in all the groups immunised with only the adjuvanted inactivated vaccines (groups 2–4), although differences exist in both the load and duration of the virus shedding among the groups. interestingly, no virus shedding was observed in groups 5–7 which consisted of chicken vaccinated with various combinations of live and inactivated vaccines. at all the time points, group i had a significantly higher virus shedding than the remaining groups (p < 0.05). in fact, at 5 dpc, only group i demonstrated virus shedding (table 4). at 7 dpc and 10 dpc, groups 2–4 were still showing virus shedding even though the load had begun to decline in comparison to group i (unvaccinated control). table 4: cloacal virus shedding of specific pathogen-free chickens following challenged with genotype vii newcastle disease virus. vaccine potency potency outcomes were calculated based on the protection percentage according to reed and muench method (tables 5 and 6) and the results were expressed as protective dose 60 (pd60). the vaccination of chickens with diluted bei–bso vaccine showed that the protection percentages of chickens inoculated with 0.02 ml (1/25 of the field dose), 0.01 ml (1/50 of the field) and 0.005 ml (1/100 of the field dose) were 95%, 81% and 42%, respectively, and the pd60 was 10−7.612. in contrast, the vaccination of chickens with diluted bei–ifa vaccine showed that the protection percentage of chickens inoculated with 0.02 ml (1/25 of the field dose), 0.01 ml (1/50 of the field) and 0.005 ml (1/100 of the field dose) were 98%, 90% and 52%, respectively, and the pd60 was 10−7.532. table 5: potency test of binary ethylenimine-inactivated newcastle disease virus vaccine formulated in black seed oil as adjuvant. table 6: potency test of binary ethylenimine–inactivated newcastle disease virus vaccine formulated in freund’s incomplete adjuvant. discussion despite the intensive use of lasota and hitchner b1 vaccines in poultry, outbreaks of nd are still a common occurrence throughout southeastern asia where the most predominantly circulating ndv isolates are taxonomically classified as genotype vii (roohani et al. 2015). these genotype vii isolates are currently considered to be the most economically important ndv strains in malaysia because of their huge contribution to the ongoing fourth nd pandemic (miller et al. 2015). furthermore, they are also frequently isolated among the farms that have vaccinated using the conventional genotype ii-based nd vaccines. as genotype mismatch between the genotype ii-based nd vaccines and the circulating genotype vii ndv is considerably believed to be responsible for the sub-optimal protective efficacy of the current vaccines (miller et al. 2009), development of new vaccines based on the currently prevalent genotype vii ndv stands to improve the effectiveness of nd control in the global poultry industry. hence, this study was carried out to characterise the efficacy of a chemically inactivated genotype-matched vaccine against genotype vii ndv isolates in malaysia. newcastle disease virus strain ibs025/13 is a highly virulent (mdt 58.5, icpi 1.68) genotype vii isolate that grows to high titre in chicken-embryonated eggs. in the present study, the virus was successfully inactivated using a chemical compound called bei. this compound has been shown to inactivate viruses by alkylating their genomic rna, thereby interfering with the replication process (delrue et al. 2012). however, when used in excessive quantities, the compound may destroy the structural and immunogenic integrity of the viral surface glycoproteins. thus, it is always necessary to optimise the conditions for the development of inactivated vaccines using bei. based on the results obtained in this study, exposure of ndv strain ibs025/13 to 10 mm of bei for 21 h at 37 °c completely inactivated the virus. furthermore, the ha titre of the virus before and after bei treatment remained fairly similar, signifying that the chemical can effectively inactivate the virus without destroying the viral hn protein. thus, exposure of virulent ndv to 10 mm of bei for 21 h at 37 °c could be regarded as the optimal condition for the development of inactivated nd vaccines. a cardinal requirement for all inactivated vaccines is the need to completely inactivate the entire virus particles in the vaccine preparations. in effect, the inactivation process should render the virus incapable of undergoing any replication and at the same time spare the outer structural proteins of the virus so that neutralising immune response can be elicited in the vaccinated host. this is, however, highly challenging, as some viruses have evolved the strategy of avoiding inactivation by forming virus aggregates, leading to vaccine failure. thus, all inactivated vaccines are required to be checked for evidence of virus growth after inactivation to avoid the risk of their reversion to virulence. interestingly, when inactivated ibs025/13 was inoculated into spf chicken-embryonated eggs in two consecutive passages, no embryonic mortality was recorded even at 6 days post-inoculation. this signifies the complete and stable inactivation of the virus. conventionally, inactivated nd vaccines are prepared in commercially available adjuvants such as mineral oil, which are known for their potent immunomodulatory activity. however, in spite of their track record of enhancing the magnitude of immune response to different pathogens, these adjuvants have many drawbacks that limit their prospects in vaccine delivery. for instance, some mineral oils are known to cause severe tissue reactions when used as adjuvants in birds. they may also contain carcinogenic substances that could constitute the risk of developing cancer among the poultry meat consumers. thus, alternative immunomodulatory agents with enhanced efficacy and minimum toxicity are urgently needed in poultry vaccine industry. interestingly, recent evidences indicate that n. sativa oil, a potent anticancer, anti-inflammatory and antioxidant agent (sharma et al. 2009) could serve as an excellent adjuvant for a variety of viral pathogens. it is a non-specific immunomodulatory agent that profoundly enhances cytokine gene expression as well as cell-mediated and humoral immune responses (umar et al. 2015). to investigate its efficacy as an adjuvant for ndv vaccine, we prepared a w/o emulsion of our inactivated ndv in nigella sativa oil and used in it in spf chicken vaccination. our findings reveal a steady rise in hi antibody titres from the point of vaccination up to 4 weeks post-vaccination in all the vaccinated groups, with higher antibody titres generally recorded in groups vaccinated with a combination of live and inactivated vaccines. this is not unexpected as live vaccines are replicating vaccines whose immunogenicity is generally more robust than that the inactivated vaccines (senne, king & kapczynski 2004). however, even in the birds vaccinated with inactivated ndv adjuvanted in bso, we observed a mean hi antibody titre of log2 6.19 ± 0.95, which is known to be above the protective threshold of 5 log2 (kapczynski et al. 2013). indeed, when combined with live vaccine, inactivated ndv emulsified in bso induced the mean hi titre of log2 8.73 ± 0.50 which is comparable to the titre in birds vaccinated with commercial ndv vaccine. thus, given its low toxicity and profound immunomodulatory effects, as established by previous researchers (salem 2005; umar et al. 2015; zaoui et al. 2002), bso can be efficiently used as an adjuvant for inactivated ndv vaccines especially in combination with the live vaccine. while all vaccinated birds were protected from overt clinical signs and mortality, virus shedding was noted in all the groups vaccinated with the inactivated vaccines alone. this indicates that these vaccines could only protect against the clinical disease but not against virulent virus infection and replication. nevertheless, the magnitude and duration of virulent virus shedding in those groups was generally lower than those of the control group whose magnitude of the virus shedding was high from day 5 post-challenge. similar findings were previously reported (fentie et al. 2014; kapczynski & king 2005). interestingly, virus shedding was not recorded in any of the groups vaccinated with a combination of live and killed vaccines (groups 5–7) throughout the observation period. it is possible that the combination of the two different vaccine preparations robustly stimulates the immune system to produce a strong mucosal immunity that prevents the replication and shedding of the challenged virulent virus. in conclusion, we have shown that the optimal conditions for chemical inactivation of ndv ibs025/13 are by exposure to bei at 37 °c for 21 h. these conditions completely inactivate the virus without compromising the structural integrity of the surface glycoproteins of the virus. we have also successfully prepared the inactivated vaccines as stable w/o emulsions (for both bso and fia) that elicited strong immune responses against clinical disease and mortality because of virulent genotype vii ndv challenge in chickens. finally, bso proved to be an efficient adjuvant as it elicited a comparable immune response with the freunds incomplete adjuvant and yet reduces vaccine residues and minimises tissue reactions in the animal tissue. this makes it a potential substitute of the widely used mineral oil in adjuvanting inactivated nd vaccines. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. funding information this research was funded by grants from the trgs-moe, grant number 5 535 402, and hicoe-moe grant number 6 369 101, government of malaysia. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not 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medicine, sudan university of science and technology (sust), khartoum-north, sudan sana i. mohammed virology department, central veterinary research laboratory (cvrl), soba, khartoum, sudan mihad hakeem epidemiology department, central veterinary research laboratory (cvrl), soba, khartoum, sudan ihsan h. ahmed viral vaccine production department, central veterinary research laboratory (cvrl), soba, khartoum, sudan nussieba a. osman department of pathology, parasitology and microbiology, college of veterinary medicine, sudan university of science and technology (sust), khartoum-north, sudan citation alhussain, a.e.m., abdalla, n.a.s., mohammed, s.i., hakeem, m., ahmed, i.h. & osman, n.a., 2020, ‘detection of peste des petits ruminants virus in pneumonic lungs from apparently healthy sheep and goats slaughtered at al-hasaheisa slaughterhouse, gezira state, central sudan’, onderstepoort journal of veterinary research 87(1), a1892. https://doi.org/10.4102/ojvr.v87i1.1892 original research detection of peste des petits ruminants virus in pneumonic lungs from apparently healthy sheep and goats slaughtered at al-hasaheisa slaughterhouse, gezira state, central sudan alaa e.m. alhussain, nahid a.s. abdalla, sana i. mohammed, mihad hakeem, ihsan h. ahmed, nussieba a. osman received: 08 july 2020; accepted: 14 oct. 2020; published: 17 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the study aimed to investigate the presence of peste des petits ruminants (ppr) in pneumonic lung tissues from clinically apparently healthy sheep and goats and further demonstrating its prevalence in gezira state, central sudan. during march 2019, 99 pneumonic lung samples were collected from apparently healthy sheep (80) and goats (19) from al-hasaheisa slaughterhouse located in al-hasaheisa locality, gezira state. using the haemagglutination (ha) test for the detection of peste des petits ruminants virus (pprv) antigen, an overall antigenic prevalence of 86.9% was demonstrated in sheep and goats lung tissue homogenate. of note, the prevalence of pprv is higher in goats (100%) compared to sheep (83.7%). in this study, the reported increasing prevalence of ppr in central sudan might be because of insufficient vaccination of animals. the findings of the present study indicated the widespread of ppr amongst sheep and goats in al-hasaheisa, gezira state. detection of pprv antigen in the pneumonic lung samples is an indication of exposure of these animals to pprv or presence of ppr viral infection and demonstrates the role of ppr as the cause of pneumonia in small ruminants. in fact, the circulation of the virus in clinically apparently healthy animals poses a threat for other in-contact susceptible animals and could play a significant role in the spread of the disease. keywords: peste des petits ruminants; pprv; sudan; sheep; goats; pneumonic lungs; haemagglutination test. introduction peste des petits ruminants (ppr) is a highly contagious and fatal viral disease affecting domestic and wild small ruminants in developing countries (edo, deneke & abdela 2017; oie 2019). peste des petits ruminants virus (pprv) causes high rates of deaths with morbidity and mortality rates ranging from 90% to 100% (dhar et al. 2002; singh & prasad 2008) amongst infected small animals, hence has a major economic effect in livestock, particularly in endemic areas, including the inter-tropical regions of africa, the arabian peninsula, the middle east and asia (oie 2019). peste des petits ruminants virus also causes high rates of abortion in goats (abubakar, ali & khan 2008). peste des petits ruminants is caused by pprv, classified in the small ruminant morbillivirus (srmv) species, in the morbillivirus genus, in the paramyxoviridae family (gibbs et al. 1979; maes et al. 2019). like other morbillivirus members, pprv is highly pathogenic for its natural hosts ‘small ruminants’ (gibbs et al. 1979). peste des petits ruminants has been existing in sudan since the first reported outbreak in 1971 in sheep and goats in three areas in elgedarif (elhag ali 1973), which was at first diagnosed as rinderpest but later confirmed as ppr (elhag ali & taylor 1984). peste des petits ruminants outbreaks were then reported in goats in sinnar, central sudan, during 1971–1972 and in mieliq in 1972 (elhag ali & taylor 1984). afterwards, ppr outbreaks were reported in gezira state during 1990s (el hassan et al. 1994). after the 2000s, outbreaks of ppr were reported in many parts of sudan in sheep and goats (osman 2005; osman et al. 2018; saeed et al. 2004, 2010, 2017), camels (khalafalla et al. 2010; kwiatek et al. 2011) and dorcas gazelles (asil et al. 2019), affecting the economy of the country. in recent years, ppr was demonstrated in lung samples collected from clinically healthy animals that showed lesions on post-mortem (pm) in slaughterhouses in sudan (saeed et al. 2017). sheep, goats and cattle are the main sources of meat production in sudan. knowing that ppr is endemic causing significant losses amongst small ruminants, it is important to investigate its presence in the slaughtered animals. the present study aimed to investigate the presence of ppr and demonstrate the prevalence of pprv antigen in pneumonic lung tissues from clinically apparently healthy sheep and goats slaughtered at al-hasaheisa slaughter house, gezira state, central sudan. materials and methods study area the study was conducted in al-hasaheisa slaughterhouse located in al-hasaheisa locality, gezira state (central sudan) (figure 1). gezira state has an area of 27 549 square kilometres (km2) and is located between the coordinates 14°30’ north and 33°30’ east in central sudan. gezira state is composed of seven localities; however, the study was conducted only in al-hasaheisa locality in the western area of the state. lung tissue samples were collected from al-hasaheisa abattoir, which is located at the margins of the town. sheep, goats and cattle were slaughtered daily, except on monday, in al-hasaheisa abattoir. figure 1: map of sudan showing the study area ‘al-hasaheisa area in gezira state, central sudan’. reference virus the live attenuated pprv nigeria 75/1 vaccine strain was obtained as a freeze-dried preparation, dissolved into 0.5 millitres (ml) of sterile distilled water, stored at -20°c till used. the ppr vaccine was the positive control pprv antigen used in the ha test and kindly provided by the viral vaccine department, central veterinary research laboratory (cvrl), soba, khartoum. sample collection and preparation during march to april 2019, a total of 99 lung samples with pneumonic lesions were collected from clinically apparently healthy sheep and goats, without considering its different sex, ages and breeds, from al-hasaheisa abattoir. these tissues were collected from sheep (80 samples) and goat (19 samples), preserved into sealed plastic bags, frozen at −20 °c. a 20% (w/v) tissue homogenate containing viral antigen was prepared from sheep and goats lung tissues in phosphate-buffer saline (pbs) (ph 7.2–7.4) supplemented with antibiotics [1 ml of penicillin (200 international units [iu]/ml), 1 ml of streptomycin (100 micrograms [µg]/ml), 1 ml of gentamycin (10 000 µg/ml)] and antifungal [0.5 ml of fungizone (50 000 iu/ml)] following the standard procedure. the homogenate was distributed into eppendorf tubes and stored at −20 °c and then used as antigen source for pprv in the haemagglutination (ha) test. preparation of red blood cells suspension whole blood was collected, from healthy non-immunised chickens, in vacutainers containing anticoagulant ‘alsever’s solution’, red blood cells (rbcs) were washed three times and 0.8% of rbcs suspension was prepared in pbs (ph 6.8) following the standard procedure, the rbcs suspension was used as an indicator in the ha test. haemagglutination test the ha test was used for identifying and quantifying pprv antigen in the 20% tissue suspension of sheep and goats lung tissues following the procedure described previously by ezeibe, wosu and erumaka (2004) and osman (2005) with some modifications for the test conditions. to achieve highest ha titres, the ha test was performed in 96-well v-shaped bottom microtiter ha plates using pbs (ph 6.8) as diluent, chicken rbcs suspension at 0.8% concentration, and the test plates were incubated in a refrigerator at 4 °c for 16–17 min. positive results were indicated by the formation of the ha sheet in the wells containing pprv and negative results were indicated by the formation of the sharp red button of sediment rbcs at the bottom of the well. red blood cells control appeared as a sharp red button (figure 2). the end-point dilution for the haemagglutinating virus, which is equal to one ha unit (1 hau), was determined as the last well that showed complete ha of rbcs. the haemagglutinationtitre (ha titre) of the virus in the sample was determined as the reciprocal of the end-point dilution and expressed as hau. figure 2: haemagglutination test plate for detection of peste des petits ruminants virus antigen showing positive haemagglutination result as a diffused sheet or film and negative ha result as a sharp red button of sediment red blood cells. peste des petits ruminants virus control (column 1), antigen samples (columns 2–11), red blood cells control (column 12). (samples are in vertical position). ethical consideration this article followed all ethical standards for research without direct contact with human or live animal. results to investigate the presence of ppr in sheep and goats pneumonic lungs from al hasaheisa slaughterhouse, the ha test for the detection of pprv antigen was performed on 20% lung tissue homogenates prepared from the lung samples. the ha results took 16–17 min to appear, and the elution phenomenon happens rapidly at 18 min post-incubation, where all positive ha results turned negative. with these optimised conditions (pbs with ph 6.8, using v-shaped bottom ha plates and incubation at 4 °c), the ha test could detect pprv antigen. results of the ha test revealed that 86/99 lung samples were found positive, with an overall higher antigenic prevalence of 86.9%, whilst 13 (13.1%) samples were found negative. moreover, the ha titres achieved ranged from 2 to 256 hau, with a mean titre of 17. moreover, 2 (2.0%) samples achieved the highest ha titre of 256 hau and 2 (2.0%) samples achieved the highest ha titre of 128 hau. apparently, most of the samples (29/99, 29.3%) achieved a titre of 8 hau (figure 3). figure 3: detection of pprv in pneumonic lungs of both sheep and goat collected from al-hasaheisa slaughterhouse, sudan. distribution of haemagglutination titers. considering the animals’ species under study, in goats, all samples tested (100%, 19/19) were found positive, whereas in sheep, of the 80 tested lung samples, 67 (83.7%) proved positive, whereas 13 (16.3%) lung samples proved negative (figure 4). amongst goats, ha results revealed that ha titres ranged from 2 to 256 hau with a mean titre of 22.7, one sample only (5.3%) achieved the highest ha titre of 256 hau, whereas most of the samples (7/19, 36.8%) achieved a titre of 4 hau (figure 4). amongst sheep, ha results similarly revealed that ha titres ranged from 2 to 256 hau with a mean titre of 15.6, one sample only (1.2%) achieved the highest ha titre of 256 hau and 2 (2.5%) samples achieved the highest ha titre of 128 hau, whereas most of the samples (25/67, 31.3%) achieved a titre of 8 hau (figure 4). figure 4: detection of pprv in pneumonic lungs of sheep or goat collected from al-hasaheisa slaughterhouse, sudan. distribution of haemagglutination titers. discussion this study was performed to investigate the presence of ppr and further demonstrate its prevalence in pneumonic lung samples from clinically apparently healthy sheep and goats slaughtered at al-hasaheisa slaughterhouse, gezira state, central sudan. ninety-nine pneumonic lung samples from sheep (80) and goats (19) were screened using the ha test for detection of pprv antigen. in this study, the ha test was used because of the demonstrated ha properties of pprv as stated earlier (ezeibe et al. 2004; ramachandran et al. 1993; wosu 1985, 1991). in a recent study by rahman et al. (2020), the ha test was proved to be as sensitive as an immuno-capture enzyme-linked immunosorbent assay (ic-elisa) for detection of pprv in nasal swabs and faecal samples from clinically ppr-suspected camels in pakistan. a 20% lung tissue homogenates were prepared from sheep and goats pneumonic lung samples and used as an antigen in the ha test as performed earlier by ezeibe et al. (2004) and osman et al. (2008). chicken rbcs were used in the ha test because it is the most sensitive rbcs for detection of pprv antigen and it causes highest ha titers in a relatively short time as documented by ezeibe et al. (2004) and osman et al. (2008). red blood cells suspension with 0.8% concentration in pbs (ph 6.8) was used for obtaining the optimal result of the ha test for pprv, according to ezeibe et al. (2004). it is known that virus members of paramyxoviridae and orthomyxoviridae cause ha of rbcs and the results were obtained after using the normal conditions of the ha test (pbs with ph 7.4, u-shaped bottom ha plates and incubation at room temperature 20 °c – 25 °c). in this study, using these optimised conditions (pbs with ph 6.8, using v-shaped bottom ha plates and incubation at 4 °c), the ha test could detect only pprv but not other viruses. results of the ha test indicated 86/99 positive samples with an overall higher antigenic prevalence of 86.9%. this indicated the wide distribution of ppr in the central part of the country, which might be affected by the animal movement in these areas. another study conducted during 2002–2004 in sudan, using the ha test, revealed higher prevalence (92.5%) amongst sheep and goats than that obtained in this study (osman 2005; osman et al. 2008). the prevalence of pprv antigen obtained from gezira state in this study is much higher compared to 54.3% prevalence in sheep and goats lung tissue samples, using ic-elisa, reported recently in the central states of sudan (saeed et al. 2017). these results appear much higher compared to 15% antigenic prevalence demonstrated in pneumonic lung samples from sheep and goats, using reveres passive haemagglutination test (rpha), in white nile state in sudan during 2008–2009 (ishag, saeed & ali 2015). also it is much higher compared to 42.6% prevalence demonstrated in sheep tissue samples from ppr-suspected outbreaks in sudan during 2008 using ic-elisa (saeed et al. 2010). in a recent study, lower prevalence (18.3%) of pprv antigen was demonstrated in lung samples from different animal species using ic-elisa (saeed et al. 2017). in this study, the reported increasing prevalence of ppr is an indication of exposure of these animals to the virus and might be because of insufficient vaccination of animals. considering the species basis, all the goat samples (19/19 samples) were found positive with an overall prevalence of 100%. the prevalence of pprv antigen obtained from gezira state in this study is much higher compared to 66.8% prevalence reported recently amongst goats in the central states of sudan (saeed et al. 2017). these results appear much higher to that described recently by saeed et al. (2017), who reported a prevalence of 21.1% in lung samples from clinically apparently healthy goats in sudan using ic-elisa. in another study, a lower overall prevalence (63%) of pprv was demonstrated amongst goats in eritrea by using an immunofluorescence test (sumption et al. 1988). another study performed on pneumonic lungs of goats in bitlis and van slaughterhouses, turkey, revealed 40% (17/42) prevalence of pprv antigen amongst goats by using an immunohistochemical test (ihc) (yener et al. 2004), which is much lower than the prevalence achieved in this study. considering the species basis, 67/80 of the sheep samples were positive with an overall antigenic prevalence of 83.7%. the prevalence of pprv antigen obtained from gezira state in this study is much higher compared to 70.7% prevalence reported recently amongst sheep in the central states of sudan (saeed et al. 2017). these results appear much higher to that described recently by saeed et al. (2017), who reported a prevalence of 15.4% in lung samples from clinically apparently healthy sheep in sudan using ic-elisa. a much lower antigenic prevalence of 42.6% was demonstrated in sheep, using ic-elisa, during 2008 in sudan (saeed et al. 2010). in the same study conducted in 2008, a lower overall prevalence of 27.3% was demonstrated amongst sheep in gezira state using ic-elisa (saeed et al. 2010). the findings indicated the presence and the higher prevalence of ppr amongst sheep and goat populations in al-hasaheisa gezira state, sudan, considering that these lung samples were collected from clinically apparently healthy animals. detection of pprv antigen in the pneumonic lung samples is an indication of exposure of these animals to pprv or ppr viral infection and demonstrates the role of ppr as the cause of pneumonia in small ruminants. in fact, the circulation of the virus in clinically apparently healthy animals, that might excrete the virus in the environment, poses a threat for other in-contact susceptible animals and could play a significant role in the spread of the disease. results of the study demonstrated the widespread of the disease not only in al-hasaheisa town but also in its neighbouring villages because most of these animals found in al-hasaheisa were brought from the neighbouring villages. the higher prevalence achieved indicated the potential exposure of these animals to pprv and the wide distribution of the disease amongst small ruminants in central sudan, which is considered as ppr endemic areas. for control and rapid elimination of the disease, local authorities should increase the awareness of small ruminant owners, regarding the economic problem and losses caused by the disease and the importance of animal vaccination against the disease. acknowledgements the authors would like to thank dr mohammed hayati, al-hasaheisa slaughterhouse, gezira state, for his help in sample collection to the staff of the departments of virology, molecular biology and the viral vaccine production, the central veterinary research laboratory, soba and sudan university of science and technology (sust), for assistance with the laboratory experiment. competing interests the authors have declared that no competing interests exist. authors’ contributions a.e.m.a. performed sample collection and preparation, laboratory work, data analysis and results interpretation, and prepared the drafted manuscript. n.a.s.a., s.i.m., m.h. and i.h.a. helped with the laboratory work. n.a.o. designed and supervised the research work, performed laboratory work, data analysis and results interpretation, prepared and finalised the manuscript. funding information the research in this study was funded by a research project for nussieba a. osman from the republic of the sudan, sudanese ministry of higher education and scientific research, commission of scientific research and innovation (grant no. 2017/972). data availability statement data sharing is not applicable 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is human-specific and maintained in human-to-human transmission cycles, while cryptosporidium parvum (formerly cryptosporidium parvum genotype 2) is maintained by a number of different animal reservoir host species including bovines (xiao, fayer, ryan & upton 2004). cryptosporidium parvum also causes disease in humans and is, therefore, a zoonosis that is trans23 onderstepoort journal of veterinary research, 74:23–29 (2007) prevalence and determinants of cryptosporidium spp. infection in smallholder dairy cattle in iringa and tanga regions of tanzania e.s. swai1*, n.p. french2, e.d. karimuribo3, j.l. fitzpatrick4, m.j. bryant5, d.m. kambarage3 and n.h. ogden6 abstract swai, e.s., french, n.p., karimuribo, e.d., fitzpatrick, j.l., bryant, m.j., kambarage, d.m. & ogden, n.h. 2007. prevalence and determinants of cryptosporidium spp. infection in smallholder dairy cattle in iringa and tanga regions of tanzania. onderstepoort journal of veterinary research, 74:23–29 the prevalence of cryptosporidium spp. infection in a cross-sectional study of dairy cattle, from two contrasting dairying regions in tanzania, were determined by staining smears of faecal samples with the modified ziehl-neelsen technique. of the 1 126 faecal samples screened, 19.7 % were positive for cryptosporidium spp. the prevalence was lower in tanga region than in iringa region. the prevalence of affected farms was 20 % in tanga and 21 % in iringa. in both regions, the probability of detecting cryptosporidium oocysts in faeces varied with animal class, but these were not consistent in both regions. in tanga region, cryptosporidium oocysts were significantly more likely to be found in the faeces of milking cows. in iringa region, the likelihood that cattle had cryptosporidium-positive faeces declined with age, and milking cattle were significantly less likely to have cryptosporidiumpositive faeces. in this region, 7 % of cattle were housed within the family house at night, and this was marginally associated with a higher likelihood that animals had cryptosporidium-positive faeces. our study suggests that even though herd sizes are small, cryptosporidium spp. are endemic on many tanzanian smallholder dairy farms. these protozoa may impact on animal health and production, but also on human health, given the close associations between the cattle and their keepers. further studies are required to assess these risks in more detail, and understand the epidemiology of cryptosporidium spp. in this management system. keywords: cryptosporidium, dairy cattle, epidemiology, iringa, prevalence, tanga, tanzania * author to whom correspondence is to be directed. e-mail: emasw@yahoo.co.uk 1 veterinary investigation centre, box 1068, arusha, tanzania 2 epicentre, institute of veterinary, animal and biomedical sciences, college of sciences, massey university, palmerston north, new zealand 3 department of veterinary medicine and public health, faculty of veterinary medicine, sokoine university of agriculture, morogoro, tanzania 4 moredun research institute, pentlands science park, bush loan, penicuik, uk 5 department of agriculture, university of reading, uk 6 groupe de recherche en épidémiologie des zoonoses et santé publique, faculté de médecine vétérinaire, université de montréal, canada accepted for publication 15 september 2006—editor 24 cryptosporidium spp. infection in smallholder dairy cattle in tanzania mitted from cattle to humans. for some bovine-associated species, such as c. andersoni, there is no evidence to date for associations with infections or disease in humans (xiao et al. 2004). cryptosporidium infections are transmitted by the faeco-oral route, and infections in livestock can be transmitted to humans by direct contact and by faecal contamination of drinking water (ramirez et al. 2004). the disease is readily transmissible, as oocysts persist for long periods in a suitable environ ment (castro-hermida, gonzalez-losada & ares-mazas 2002), and low numbers of oocysts may produce infection in susceptible hosts (ramirez et al. 2004). enteric disease caused by cryptosporidium spp. is usually self-limiting in immunocompetent individuals but debilitating and persistent in those immunocompromised (ramirez et al. 2004). crypto spor idium infection has been termed as an aids-defining illness, and its prevalence amongst aids sufferers in africa is sometimes high (gatei, green sill, ashford, cuevas, parry, cunliffe, beeching & hart 2003). human infections in africa have been associated with c. parvum (tumwine, kekitiinwa, nabukeera, akiyoshi, rich, widmer, feng & tzipori 2003), which may be of livestock origin because cryptosporidium spp. infections in livestock appear to be common (matovelo, landsverk & posoda 1984; esrony, kambarage, mtambo, muhairwa & kusiluka 1996; mtambo, sebatwale, kambarage, muhairwa, maeda, kusiluka & kazwala 1997). smallholder dairy production is now a widespread practice in east africa, having received support for agricultural development and improved standards of living, as well as being a system that may improve human nutrition (mdoe 1993; thorpe, chabari, maloo, muinga, mukhebi, mullins, muriethi, mussukuya, nyambaka, ole-maki, otieno, perry, rugema & wekesa 1993). in this study we have investigated the prevalence of cryptosporidium spp. infections amongst smallholder dairy cattle in two regions of tanzania. we have also attempted to investigate potential risk factors for infection in these cattle that may point to methods by which infections can be controlled and/or their transmission to humans limited. materials and methods the study area the study sites are described in detail in ogden, swai, beauchamp, karimuribo, fitzpatrick, bryant, kambarage & french (2005). briefly, the study was carried out in two regions of tanzania, namely the coastal tanga region, that lies between 38–39° e and 4–6° s, and the inland highland iringa region, lying between 35–36° e and 7–8° s. the study took place in two of the six administrative districts in iringa region (iringa urban and iringa rural, now kilolo), and five of the six administrative districts in tanga region (korogwe, lushoto, muheza, pangani and tanga). study farm selection farms in each study region were randomly selected by means of the random number generator in epiinfo version 6 (cdc, atlanta, usa), from a sampling frame of 3 001 in tanga and 500 in iringa using the databases of the tanga and iringa dairy devel opment projects. farms at both study sites were estimated to have an average of three to four dairy animals of any age and both sexes, therefore a farm sample size of 200 in each study region was considered necessary to provide between 600 and 800 animals. the sample size in each region was calculated to provide 80 % ability to detect an odds ratio of 2.0, assuming a critical probability of 0.05, and exposure that occurs in 50 % of the population, a prevalence of 20 % in the unexposed group and a design effect of 2.0 (french & tyrer 1997). farms recorded to have more than ten animals were excluded from the selection process because farms of this size are not considered as ‘smallholder’ farms (tanga dairy development programme 1999), although a small number of selected farms had more than ten cattle by the time sampling began. study animals the population of interest consisted of all smallholder mixed dairy farmers and dairy cows from the five administrative districts of tanga and two of the iringa study regions. a ‘dairy animal’ referred to crosses of bos taurus cattle (mainly ayrshire, friesian and jersey) with the bos indicus breed, the indigenous tanzania short horn zebu (tshz) or boran. the level of exotic blood varied from first to third filial generation (f1, f2 and f3). the animal husbandry practices included both zero and open grazing systems. over 60 % of the cattle were stall-fed throughout the year. data collection data were collected from farms by two separate teams of researchers, one in each region. faecal sampling and data collection were carried out between january and april 1999. one person in each region collected farmand animal-level data on a 25 e.s. swai et al. structured questionnaire, which was completed on all selected farms on a single visit. the information collected concerned farm and animal events that occurred during 1998, including the numbers of animals that had died or suffered ill health on each farm (not reported here), parasite control, feeding methods and feed types, cattle movements on and off the farm, and grazing and housing practices. the response to many of these questions were investigated as explanatory variables in analysis of positivity/negativity for cryptosporidium spp. infection. these included variables that could be considered as farm-level variables including ‘farm class’ (whether the farm was located in an urban, rural or periurban area), frequency of contact with extension officers (rare, moderate or intensive) and farmer attendance at a dairy development project training course. animal housing details, including building, flooring and bedding types, were recorded and used as explanatory variables in statistical analyses. the administrative district in which the farm occurred was also considered as an explanatory variable in the analysis. animal-level variables included age (centre), sex, breed, filial generation, source (homebred or broughtin), source of brought-in animals (charity gift, dairy development project credit agreement, or cash purchase), whether or not the animal had been zerograzed or allowed to graze at pasture in the three months prior to the onset of the sampling period (october to december 1998), and anthelmintic treatments during this period. factorized ‘classes’ of adult female animals were also created as follows: pregnant cows (all females that have had one calf and were pregnant at the time of the visit), milking cows (all females that were in milk at the time of the visit), and ‘dry cows’ (cows that were not in milk, nor pregnant at the time of the visit). laboratory analysis of samples during the visit to each farm, a fresh rectal faecal sample was collected from each animal into a sterile, airtight, 10 ml plastic tube. collected faecal samples were labelled and transported in a cool-box to local laboratories prior to despatch in refrigerated containers to sokoine university of agriculture in morogoro. here, the presence of cryptosporidium spp. oocysts in faeces samples was detected using the modified ziehl-neelsen staining technique as described by henriksen & pohlenz (1981). briefly, faecal smears were prepared on a microscope slide, air dried and fixed with methanol for 5 min. fixed smears were stained with dilute carbol fuchsin (1:10) for 3–5 min and washed with tap water. smears were decolourized using acid alcohol, then counterstained with 0.5 % malachite green solution for 1 min. smear slides were air dried and then examined under the microscope at 400x magnification. cryptosporidium spp. oocysts appear as pink to red, spherical to ovoid bodies against a green to purple background. samples were considered positive if at least one morphologically distinct cryptosporidium spp. oocyst was observed. statistical analysis data files of questionnaires and laboratory results were prepared in epi-info version 6.04b (cdc, usa). the presence or absence of oocysts in faeces samples was the outcome variable in mixed effects logistic regression analyses performed in stata version 6 for windows (stata corporation, college station, texas, usa). the animaland farm-level variables described above served as explanatory variables. associations between explanatory variables and the outcome were investigated singly and in multivariable models. in multivariable models, backwards and forwards substitution and elimination were performed to find the most parsimonious model from which no explanatory variables could be removed without significantly affecting model deviance. cross tabulations and correlations were performed on explanatory variables to identify highly associated variables that could not be incorporated in the same multivariable models. in all models, the farm identification (id) number was considered a random effect to account for clustering of animals by farm, and the level of significance was p < 0.05 throughout. separate statistical analyses were performed for the data from the two regions because previous studies have indicated that parasite ecology and epidemiology may be very different in the two regions (ogden et al. 2005; swai, french, karimuribo, fitzpatrick, bryant, brown & ogden 2005). results farm response rate all selected 200 farms from each of tanga and iringa regions were visited during the period of january to april 1999. in tanga, a total of 697 animals kept on 185 farms (92.5 % of the sample) were examined and sampled. fifteen farms (7.5 %) had no animals during the actual survey period. in iringa, a total of 698 animals from 195 farms (97.5 % of the sample) were examined and sampled. three farms (1.5 %) had no animals and animals on two farms (1 %) could 26 cryptosporidium spp. infection in smallholder dairy cattle in tanzania table 1 the proportions of cattle in each category of each variable investigated during the study variable categories no. of animals (%) iringa tanga animal-level variables sex male female 182 (26) 516 (74) 146 (21) 551 (79) source of animal homebred brought-in 406 (58) 292 (42) 436 (63) 261 (37) filial generation f1 f2 f3 350 (50) 347 (49.9) 1 (0.1) 217 (31) 459 (66) 21 (3) adult cow ‘class’ milking cows dry cows pregnant cows 158 (22.6) 60 (8.6) 65 (9.3) 137 (20) 50 (7) 86 (12.3) breed ayrshire cross friesian cross jersey cross simmental cross sahiwal cross tshz cross boran cross 403 (58) 305 (44) 1 (0.1) 0 0 150 (22) 549 (78) 169 (24) 604 (86) 12 (2) 5 (1) 12 (2) 541 (77) 121 (17) age < 3 years 3 to < 6 years > 6 years 440 (63) 165 (24) 93 (13) 396 (57) 214 (31) 87 (12) grazing history in last 3 months of 1998 zero grazing semi/free grazing 423 (61) 275 (39) 626 (90) 71 (10) anthelmintic treatment in 1998 yes no 614 (88) 84 (12) 313 (45) 384 (55) farm-level variables farm classification peri-urban urban rural 109 (16) 391 (56) 198 (28) 117 (17) 318 (46) 262 (37) housing details cowshed with roof cowshed no roof traditional kraal or boma 531 (76.1) 80 (11.4) 87 (12.5) 644 (92.4) 39 (5.6) 14 (2) sleeping area cubicle in cow shed cow shed floor family house 398 (57) 247 (35.3) 53 (7.6) 374 (53.6) 315 (45.2) 8 (1.14) floor/bedding type concrete left over forage dried grass hardcore soil wood 335 (47.9) 0 (0) 23 (3.3) 67 (9.5) 201 (28.7) 72 (10.3) 402 (57.6) 69 (9.9) 16 (2.3) 38 (5.4) 168 (24.1) 4 (0.6) frequency of extension officer contact rare moderate intensive 15 (2) 596 (85) 87 (13) 6 (0.8) 659 (94.5) 32 (4.7) district (iringa) iringa urban iringa rural (kilolo) 461 (66) 237 (34) na district (tanga) tanga muheza pangani korogwe lushoto na 341 (48.8) 185 (26.5) 26 (4) 64 (9) 81 (12) na = not applicable 27 e.s. swai et al. not sampled because the owner could not be traced. the number of animals examined per herd ranged from 1–13 animals. the age range of animals examined varied from 1 day to 13 years. the characteristics of the sample of cattle in each region are detailed in table 1. prevalence of cryptosporidium spp. oocysts in faeces samples results were available from 444 of the 697 animals sampled in tanga, and 682 of the 698 animals sampled in iringa. the missing results arose due to loss of labels during transport to laboratories. of the 1 126 faecal samples screened in both tanga and iringa, 222 (19.7 %) were positive for cyptosporidium spp oocysts. the prevalence amongst study animals investigated was higher in iringa than in tanga (table 2). in tanga region, the highest prevalence was observed in cattle in tanga, korogwe, muheza and least in lushoto and none in pangani districts (table 2), although these differences were not significant (likelihood ratio statistic [lhr] = 4.89, df = 4, p > 0.1). in tanga region, on 37 of the 185 farms sampled (20 %, 95 % confidence interval [ci] = 14.5– 26.5) at least one animal tested positive for cryptosporidium infection. in iringa region, 41 of 195 farms (21 %, ci = 15.5–27.4) had at least one animal positive. in iringa region, two factors remained significant in the multivariable model. first, the likelihood that faecal samples were positive decreased significantly with animal age (coefficient = –0.009, se = 0.004, p = 0.017). second, faecal samples from milking cows were significantly less likely to be positive than those of other classes of cattle (odds ratio [or] = 0.56, ci = 0.32–0.97, p = 0.038). faecal samples from animals that were kept in the family house at night were positive more often than samples from animals housed in other buildings but the difference was not significant (or = 1.75, ci = 0.92–3.34, p = 0.09). in tanga, milking cows were significantly more likely to have positive faeces samples than other animal classes (or = 2.19, ci = 1.08–4.39, p = 0.028). the random effect of farm id number was negligible in tanga region (coefficient = 0.001, se = 0.735), but strong in iringa region (coefficient = 0.472, se = 0.237). discussion in this study, there was evidence that cryptosporidium spp. are endemic on smallholder dairy farms in two different regions of tanzania. the detected prevalence of infection in the cattle was intermediate in comparison with that observed in other studies in tan zania, from 5.3 % (mtambo et al. 1997) up to 62 % (lema 1990; esrony et al. 1996). it was generally lower than observed in studies in europe for instance up to 36 % in dairy cattle in germany (joachim, krull, schwarzkopf & daugschies 2003) and 80 % in calves britain (scott, smith, mtambo & gibbs 1995). while the method of detection used in the present study is a rapid screening method, there was no attempt to concentrate faecal oocysts and the method has low sensitivity of detection compared to other methods (gobet, buisson, vagner, naciri, grap pin, comparot, harly, aubert, varga, camerlynck & bonnin 1997). furthermore, our cross-sectional study would not capture the potential for seasonal variations in prevalence that, in studies in europe, may be considerable (huetink, van der giessen, noordhuizen & ploeger 2001). table 2 the prevalence of cryptosporidium spp. in dairy cattle by regions and districts in iringa and tanga. 95 % ci = confidence intervals adjusted for the farm effect district number of animals positive % positive (95% ci) iringa region iringa rural iringa urban all 43/201 138/481 181/682 21.3 (18.4–25.0) 28.6 (26.5–30.9) 26.5 (24.7–28.4) tanga region tanga muheza korogwe lushoto pangani all 24/228 12/134 4/38 1/33 0/11 41/444 10.5 (8.6–13.0) 8.9 (5.6–12.2) 10.5 (8.6–13.0) 3.03 (0.07–7.7) 0 (0–28.5) 9.3 (7.7–11.0) 28 cryptosporidium spp. infection in smallholder dairy cattle in tanzania our prevalence estimates are likely, therefore, to be lower than the true prevalence in the cattle, but it could be expected that features of cattle demography and management on smallholder dairy farms are not conducive to the maintenance of endemic cycles of cryptosporidium spp. by the cattle alone. these include the low number of cattle on individual farms, often consisting of just one or two animals, low rates of reproduction amongst the cattle, and a low rate of production of cryptosporidium-naïve and susceptible calves (french, tyrer & hirst 2001). the mostly zero-grazed management may reduce the potential for farm-to-farm transmission. despite these features, which could all result in fade-out of infectious diseases (anderson & may 1992), crypto sporidium spp. were detected in faeces of the cattle in nearly all the administrative districts in both regions studied. there was evidence of geographic variations in the prevalence, and possibly the dynamics of infection. first, the detected prevalence of infection was higher in cattle in iringa region than in tanga region. this is consistent with unpublished observations for prevalence of infection in other species in tanzania (dogs, pigs and cattle in other management systems) in which prevalence is generally higher in regions that have a cooler climate, suggesting that the high ambient temperature and humidity in coastal tanga may be sub-optimal for survival of cryptosporidium spp. outside the host. second, the risk factors for infection varied between the two regions. in iringa region, younger animals were more likely to be positive, and milking adult cows were particularly unlikely to be detectably infected. in tanga region, there was no association of detected infection with age, but milking adult cows were particularly likely to be positive. in addition, the farm effect in iringa region was high suggesting that unmeasured management characteristics of individual farms had a considerable effect on cryptosporidium spp. epidemiology, which was not the case in tanga region. this may also suggest that important features of the epidemiology of these infections may differ in the two regions, which could also be associated with differences in climate between the regions. in a study of cryptosporidium spp. dynamics in cattle of farms in the usa, however, infections in younger animals were associated with the zoonotic c. parvum, while infections in older animals were associated with non-zoonotic species or genotypes (santin, trout, xiao, zhou, greiner & fayer 2004). thus our observed regional differences in the epidemiology of cryptosporidium spp. could possibly signify differences in the risk of human infections. our study indicated that smallholder dairy farmers and their families often live in close proximity to their cattle, even within the family home. the probability of transmission of zoonotic cryptosporidium spp. from cattle to humans on these farms may, therefore, be high. farmers keep their cattle within the family home at night to prevent the theft of these valuable commodities (karimuribo, personal communication 2002). it is not clear, however, why the detected prevalence of infection in cattle that are brought into the home at night was particularly high; this warrants further investigation. in this study we have demonstrated the presence of cryptosprodium spp. infection in smallholder dairy cattle in two regions of tanzania. the epidemiology of infection may differ in different regions with potential consequences for human health. some cattlekeeping practices are likely to enhance direct bovineto-human transmission. further studies are needed to understand the dynamics of transmission cycles and the genetic diversity of cryptosporidium spp. on the farms, and to identify and if possible alter management practices that are risk factors for human infections. acknowledgements this study was funded by the uk department for international development. we 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akiyoshi, d.e., rich, s.m., widmer, g., feng, x. & tzipori, s. 2003. cryptosporidium parvum in children with diarrhea in mulago hospital, kampala, uganda. american journal of tropical medicine hygiene, 68:710–715. xiao, l., fayer, r., ryan, u. & upton, s.j. 2004. cryptosporidium taxonomy: recent advances and implications for public health. clinical microbiology review, 17:72–97. http://www.ojvr.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on https:// ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these 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somayeh namroodi stephan zientara tlabo leboho william dundon http://www.ojvr.org� https://ojvr.org� https://ojvr.org� https://ojvr.org� https://ojvr.org/index.php/ojvr/user https://ojvr.org/index.php/ojvr/user mailto:publishing@aosis.co.za abstract introduction materials and methods results discussion acknowledgements references appendix 1 about the author(s) abiodun j. fatoba discipline of genetics, school of life sciences, college of agriculture, engineering and sciences, university of kwazulu-natal, westville, south africa oliver t. zishiri discipline of genetics, school of life sciences, college of agriculture, engineering and sciences, university of kwazulu-natal, westville, south africa damer p. blake department of pathobiology and population sciences, the royal veterinary college, hawkshead lane, hertfordshire, united kingdom sunday o. peters department of animal science, berry college, mount berry, georgia, united states jeffrey lebepe department of biodiversity and evolutionary biology, school of life sciences, college of agriculture, engineering and sciences, university of kwazulu-natal, durban, south africa samson mukaratirwa department of biological sciences, school of life sciences, college of agriculture, engineering and sciences, university of kwazulu-natal, durban, south africa matthew a. adeleke discipline of genetics, school of life sciences, college of agriculture, engineering and sciences, university of kwazulu-natal, westville, south africa citation fatoba, a.j., zishiri, o.t., blake, d.p., peters, s.o., lebepe, j., mukaratirwa, s., et al. 2020, ‘study on the prevalence and genetic diversity of eimeria species from broilers and free-range chickens in kwazulu-natal province, south africa’, onderstepoort journal of veterinary research 87(1), a1837. https://doi.org/10.4102/ojvr.v87i1.1837 original research study on the prevalence and genetic diversity of eimeria species from broilers and free-range chickens in kwazulu-natal province, south africa abiodun j. fatoba, oliver t. zishiri, damer p. blake, sunday o. peters, jeffrey lebepe, samson mukaratirwa, matthew a. adeleke received: 27 nov. 2019; accepted: 25 june 2020; published: 17 sept. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study was conducted from january to october 2018 with the objective to determine the prevalence and genetic diversity of eimeria species in broiler and free-range chickens in kwazulu-natal province, south africa. a total of 342 faecal samples were collected from 12 randomly selected healthy broiler chicken farms and 40 free-range chickens from 10 different locations. faecal samples were screened for the presence of eimeria oocysts using a standard flotation method. the species of eimeria isolates were confirmed by amplification of the internal transcribed spacer 1 (its-1) partial region and sequences analysis. among broiler and free-ranging chickens, 19 out of 41 pens (46.3%) and 25 out of 42 faecal samples (59.5%) were positive for eimeria infection. molecular detection revealed the following species: eimeria maxima, eimeria tenella, eimeria acervulina, eimeria brunetti and eimeria mitis in all the samples screened. similarly, polymerase chain reaction assays specific for three cryptic eimeria operational taxonomic units were negative for all the samples. phylogenetic analysis of the its-1 sequences supported species identity with the greatest variation detected for e. mitis. this study provides information on the range and identity of eimeria species, and their genetic relatedness, circulating in commercially reared broilers and free-ranging chickens from different locations in kwazulu-natal province. keywords: chickens; coccidiosis; eimeria; genetic diversity; molecular diagnosis; prevalence. introduction poultry production has become a major driving force in the economy of many developing countries, which are countries characterised by low income and gross domestic product per capita (alders & pym 2009). south africa produced 129.3 million chickens throughout the nine provinces in 2017, of which 7% were from kwazulu-natal (south african poultry association 2017). the vulnerability of chickens under commercial production to parasitic diseases such as coccidiosis is a major threat to the productivity and viability of the south african poultry industry. coccidiosis is an enteric disease that reduces performance and affects the welfare of chickens, leading to high morbidity and mortality in the absence of effective control (blake & tomley 2014). globally, the annual burden of preventing/controlling coccidiosis has been estimated to exceed $3 billion (blake & tomley 2014). eimeria, a parasite of the phylum apicomplexa, is the causative agent of this disease, and its species such as eimeria necatrix, eimeria maxima, eimeria acervulina, eimeria praecox, eimeria mitis, eimeria brunetti and eimeria tenella are also known to infect chickens (nematollahi, moghaddam & niyazpour 2008). mixed infections are common (haug et al. 2008; jenkins et al. 2008), thereby complicating diagnosis and effective control. the emergence of three cryptic eimeria genotypes, referred to as operational taxonomic units (otus) x, y and z, has added further complexity. these were first detected circulating among commercial chickens reared in australia (cantacessi et al. 2008). the three otu genotypes have since been reported in several african countries, such as nigeria, tanzania, ghana, uganda and zambia (clark et al. 2016; jatau et al. 2016). the widespread occurrence of these cryptic genotypes could pose a significant risk to vaccine development and application (clark et al. 2016). effective control of coccidiosis in chickens relies on strict management practices, supplemented by timely application of anticoccidial drugs and/or vaccines (godwin & morgan 2015) underpinned by proper diagnosis and identification. traditional diagnostic methods include evaluation of the location and the characteristics of gross pathology (lesion scoring) and microscopic analysis of oocyst morphology (kumar et al. 2014). however, the relative complexity and requirement of expertise for these methods necessitated the development of molecular alternatives, including genusand species-specific polymerase chain reaction (pcr) assays (lew et al. 2003). the use of nuclear and mitochondrial genetic markers (e.g. internal transcribed spacer [its] sequences, 18s ribosomal rna, cytochrome oxidase subunit i [coi]) has proven effective in the identification and taxonomic classification of protozoan parasites, including eimeria (kumar et al. 2015a; ogedengbe et al. 2018; tan et al. 2017). thus, its-1 sequences have served as genetic markers to identify eimeria species (cook et al. 2010; oliveira et al. 2011). based on the observed diversity, its-based species-specific primers have been developed for use in the identification of eimeria species (lew et al. 2003). however, studies from various countries have reported nucleotide variations in the its-1 region within eimeria species isolates (bhaskaran et al. 2010; kumar et al. 2015a; lew et al. 2003). genetic diversity among species and strains of eimeria could pose a major risk to the control of coccidiosis in the future. as such, knowledge defining naturally occurring genetic diversity becomes imperative to understand the pathogenicity and epidemiology of eimeria that infect chickens (morris & gasser 2006). there is a dearth of information on eimeria occurrence and diversity in south africa. as such, reports on circulating eimeria species in kwazulu-natal province together with information on their occurrence in commercial chickens are not available. this study, therefore, aimed to determine prevalence and genetic diversity of eimeria species in both broiler and free-range chickens in kwazulu-natal province. materials and methods study area kwazulu-natal is the second most populous province among the nine provinces in south africa. it has a population of approximately 10 million people and land size of 94 000 km2 located between latitude 28°99’s and longitude 30°97’e. the capital city of pietermaritzburg has a warm and subtropical climate throughout the year, especially around the coastline, but gets colder in the inland areas. the poultry industry in kwazulu-natal province is one of the producers of broiler birds in south africa with a total of 6.7 million broiler birds in 2017, contributing 6.4% to the national broiler production (south african poultry association 2017). sample collection a total of 342 chicken faecal samples were collected from 12 broiler farms consisting of 41 pens (1–5 pens per farm) and free-range chickens. the age of broiler chickens at the time of sampling ranged from 3 to 10 weeks, with the exception of a single farm consisting of 12-week-old chickens. in addition, 42 faecal samples of 40 free-ranging 3-week-old village chickens were randomly collected from four localities. the 342 samples were collected randomly once from the following locations: pietermaritzburg, phoenix, scottburg, stanger, chatsworth, westville, maphumulo, umvoti, port sherpstone and shongweni of kwazulu-natal province from january to october 2018. detailed information on the number of pens per farm, number of samples per pen, number of farms per location and number of chickens per location is shown in appendix 1 tables 1-a1 and 2-a1. there were no clinical signs of coccidiosis among the chickens on any of the farms sampled. samples were collected following the procedure described by kumar et al. (2014). briefly, in the broiler farms, faecal samples were collected following a pre-determined ‘w’ pathway in each pen to allow random sampling. fifty-millilitre conical tubes containing 10 ml of 2% potassium dichromate were used to collect faeces up to 20 ml of the tube and stored at 4 °c until further use. depending on the size of the pen, four to eight 50-ml conical tubes of faecal samples were collected per pen and the content was mixed together vigorously. sample processing and microscopic oocyst identification samples were processed based on the procedures described by kumar et al. (2014), with minor modifications. two grams of faecal samples were weighed into a beaker and mixed with 100 ml of distilled water. this was stirred with a glass rod and later filtered through a gauze. the filtrate was transferred into a new 50-ml conical tube and filled to the brim with saturated salt solution. this was then centrifuged at 800 × g for 10 minutes. the supernatant was decanted and the sediment was transferred into a new 50-ml tube and then later pelleted at 14 000 × g for 3 min. oocysts per gram (opg) were counted using a mcmaster counting chamber following a standard protocol (haug, williams & larsen 2006). samples with opg greater or equal to 250 opg were selected for deoxyribonucleic acid (dna) extraction. photomicrograph images of unsporulated oocysts were taken randomly from each farm sampled using an omax compound microscope containing a 5 mp camera at 400×. dna extraction total genomic dna was extracted using a quick-dnatm fecal/soil microbe miniprep kit (zymo research, united states [us]) based on the manufacturer’s protocol with minor modifications. faecal samples in the bashing beadstm lysis tube (0.1 mm and 0.5 mm) were processed on a vortex genie at maximum speed for 25 min, instead of 20 min as recommended by the manufacturer’s protocol. dna quality and concentration were checked on an agarose gel (1.5%) and nanodroptm 1000 spectrophotometer (thermo scientific, us) at 260 nm absorbance. polymerase chain reaction amplification a nested pcr protocol targeting the genomic its-1 region was used to detect each eimeria species. genusand species-specific primers were used as described by lew et al. (2003). each 25 µl pcr contained 12.5 µl 2x dreamtaq green pcr master mix (thermo scientific, us), 1 µl of each forward and reverse primer (10 µm of stock solution; table 1), 5.5 µl nuclease free water and 5 µl dna template. thermal cycling was done as follows: initial denaturation at 94 °c for 3 min, 30 cycles of 94 °c for 30 s, 56 °c for 30 seconds and 72 °c for 90 s and a final extension at 72 °c for 15 min. the primary pcr product (1 µl of the 25 µl) was used as template for the nested pcr containing species-specific primers in each tube. the same thermal cycling conditions were used for the species with varying annealing temperature as follows: 55 °c for e. mitis, 56.7 °c for e. tenella, 61 °c for e. acervulina, 62 °c for e. maxima, 61 °c for e. necatrix, 61 °c for e. praecox and 61 °c for e. brunetti. nuclease-free water replaced the dna template for the negative control. amplification of nested pcr products was checked on 1.5% (w/v) agarose gel at 100 v for 30 min and visualised under ultraviolet light using a bio-rad chemidoctm mp system (bio-rad, us). similarly, the samples were also screened for the presence of three cryptic eimeria otus by targeting the its-2 genomic region using the primers and thermal cycling procedure described by fornace et al. (2013), as shown in table 2. the pcr products were sent for sequencing at inqaba biotech (south africa). sequencing was done with both forward and reverse primers using big dye chemistries in an abi 3500xl genetic analyzer, pop-7tm (thermo scientific, us). table 1: genusand species-specific internal transcribed spacer-1 primers used in the study. table 2: primers used for the detection of three cryptic eimeria operational taxonomic units. sequence analysis a total of 28 its-1 sequences were viewed, edited and trimmed. consensus sequences were generated from both forward and reverse sequences using bioedit version 7.0.5.3 software (hall 1999). the sequences were submitted to national center biotechnology information and assigned accession numbers (appendix 1 table 3-a1). also, the sequences were compared with selected published sequences from the genbank. sequence alignment was performed using the clustalw programme. pairwise percentage identity (appendix 1 figure 1-a1) was carried using sequence demarcation tool (sdt) version 1.2 software (muhire, varsani & martin 2014). genetic distance within eimeria species isolates from this study was calculated with mega version 6.0 (tamura et al. 2013) using the tamura 3-parameter model. phylogenetic analysis of internal transcribed spacer-1 sequences the genetic diversity that exists between the its-1 sequences generated in this study (n = 28) and those of american, chinese, indian, australian, egypt, sudan and swedish eimeria species isolates published in genbank (appendix 1 table 4-a1) were analysed. phylogenetic analyses using the maximum likelihood (ml) method were carried out with mega version 6.0 (tamura et al. 2013). the nucleotide substitution model that best fitted the data set was identified using model-test in mega6. based on the akaike information criterion, the jukes–cantor model was identified as the best model. gaps in the alignment were treated as missing characters. bootstrap iteration was based on 1000 replicates and the percentage value was indicated at each node. neospora caninum (genbank accession number: af038860.1) and toxoplasma gondii (eu025025.1) were used as out-group species to root the tree. statistical analysis data generated were analysed using the statistical package for the social sciences (spss) software version 25.0. descriptive statistics were used to determine the prevalence of detected eimeria species. ethical consideration the protocol for this study was approved by the university of kwazulu-natal animal research ethics committee and assigned the reference number arec/058/017d. results polymerase chain reaction amplification and microscopic unsporulated oocyst detection among broiler and free-ranging chickens, 19 out of 41 pens (46.3%) and 25 out of 42 samples (59.5%) were positive for eimeria infection (figure 1). the highest level of eimeria infection was observed in the following locations in both broiler and free-ranging chickens as shown in figure 2: phoenix (7/41; 17.1%), scottburg (4/41; 9.8%), shongweni (8/42; 19%), port sherpstone (7/42; 16.7%) and maphumulo (7/42; 16.7%). figure 1: images of unsporulated eimeria oocysts detected in faecal samples from infected farms. figure 2: occurrence of eimeria infection in different locations in kwazulu-natal. using the species-specific nested pcr assay, five eimeria species were identified (e. tenella, e. maxima, e. acervulina, e. brunetti and e. mitis) in all screened samples (figure 3). in broiler farms, e. tenella had the highest prevalence (13/19; 68.4%), followed by e. maxima (9/19; 47.4%) based on pens which were positive. however, in free-ranging chickens, e. mitis (24/25; 96%) and e. maxima (23/25; 92%) had the highest prevalence. the lowest prevalence was observed for e. acervulina (5/19; 26.3%) and e. brunetti (3/25; 12%) in broiler and free-ranging chickens, respectively (figure 4). figure 3: amplification of eimeria species by polymerase chain reaction. m: 100 bp dna marker; l2: negative control; l3–7: samples. (a) eimeria mitis 328 bp; (b) eimeria tenella 278 bp; (c) eimeria maxima 145 bp; (d) eimeria acervulina 321 bp; (e) eimeria brunetti 311 bp. figure 4: prevalence of eimeria species in both broilers and free-range chickens in kwazulu-natal. dna amplification of eimeria species the most common mixed species combinations detected in broiler and free-ranging chicken faecal samples were e. tenella + e. maxima (4/19; 21.1%) and e. mitis + e. maxima + e. acervulina (11/25; 44%), respectively. other combinations were e. mitis + e. maxima (2/19; 10.5%), e. tenella + e. mitis (1/25; 4%), e. acervulina + e. maxima (1/25; 4%), e. acervulina + e. tenella (2/19; 10.5%), e. mitis + e. tenella (3/19; 15.8%), e. acervulina + e. tenella + e. maxima (1/19; 5.3%), e. acervulina + e. mitis + e. tenella (2/19; 10.5%), e. tenella + e. acervulina + e. mitis + e. maxima (7/25; 28%), e. tenella + e. mitis + e. brunetti + e. maxima (1/25; 4%), e. acervulina + e. mitis + e. tenella + e. maxima (3/19; 15.8%) and e. tenella + e. acervulina + e. mitis + e. maxima + e. brunetti (2/25; 8%). overall, among the broiler farms, scottburg farm had the highest prevalence level of mixed species (e. acervulina + e. mitis + e. tenella + e. maxima; 75%), whilst mixed species (e. acervulina + e. mitis + e. maxima) with a prevalence of 44% was the highest among all locations with the free-range chickens. cryptic eimeria otus were not detected in all the samples screened. internal transcribed spacer-1 sequence analysis internal transcribed spacer-1 sequences of e. mitis, e. maxima, e. tenella, e. acervulina and e. brunetti from this study showed high homology with sequences from eimeria species present in the genbank as follow: 90% – 93% identity for e. mitis, 99.31% for e. maxima, 99% – 100% for e. tenella, 99.38% for e. acervulina and 100% for e. brunetti. the overall mean genetic distance within eimeria species isolates from kwazulu-natal in south africa calculated by ml (tamura 3-parameter model) with 1000 bootstrap replicates was 1.14 ± 0.08. mean genetic distance per species was as follows: e. mitis (0.13 ± 0.014), e. maxima (0.09 ± 0.020), e. tenella (0.09 ± 0.012), e. acervulina (0.02 ± 0.005) and e. brunetti (0.02 ± 0.006). phylogenetic analysis of internal transcribed spacer-1 sequences maximum likelihood with the jukes–cantor model was used to create the phylogenetic tree (figure 5) of the 28 its-1 sequences generated in this study, together with reference eimeria its-1 sequences of american, chinese, indian, australian and swedish isolates. irrespective of their geographical locations, the its-1 sequences of all five species clustered in distinct clades. among the e. tenella clade, all the seven e. tenella sequences from this study clustered with e. tenella sequences from china, egypt and india with a very strong support. similarly, all the five and eight sequences of e. acervulina and e. mitis from this study, respectively, clustered with e. acervulina and e. mitis sequences of america, india, sudan and sweden with a very strong support. all the e. maxima sequences from this study clustered with e. maxima sequences from america and india with low support. within the e. brunetti clade, all the three sequences from this study clustered with e. brunetti sequences from india and australia with a very strong support. genetic distances between its-1 sequences of eimeria isolates in this study and those of a public database were as follow: e. mitis (0.12 ± 0.013), e. acervulina (0.02 ± 0.005), e. maxima (0.07 ± 0.016), e. tenella (0.07 ± 0.010) and e. brunetti (0.01 ± 0.004). figure 5: maximum likelihood tree (tamura-3 model) of internal transcribed spacer-1 sequences of eimeria species. percentage of bootstrap (1000 replicate) values is indicated in each node. the scale bar indicates sequence substitution per site. sequences in this study are in different colours and shapes. discussion coccidiosis is an enteric disease that poses a threat to efficient poultry production (ogedengbe, hanner & barta 2011), compromising economic productivity and chicken welfare. for effective diagnosis, control and epidemiology of the disease, the identification of specific species of eimeria is essential. understanding the occurrence of genetic diversity and regional population structure are important (hamza, al-massodi & jeddoa 2015; morris & gasser 2006). in this study, eimeria infection had an overall prevalence of 46.3% (19 out of 41 pens) and 59.5% (25 out of 42 samples) across different farms and locations, which was higher than the 29.4% found among eimeria parasites from kwazulu-natal and limpopo (malatji et al. 2016). however, it was lower than previous reports from other regions including ethiopia (56%; luu et al. 2013), romania (91%; gyorke et al. 2013), anhui province, china (87.75%; huang et al. 2017) and two north indian states (81.3%; kumar et al. 2015b). molecular diagnosis using nested species-specific its-1 primers was used to identify five species of eimeria (e. tenella, e.maxima, e. acervulina, e. brunetti and e. mitis) circulating in both commercial broiler and free-range chickens in kwazulu-natal province. this is similar to the study of debbou-iouknane, benbarek and ayad (2018), who reported the same five species of eimeria among broilers farms in bejaia region of algeria. the prevalence of one or more species of eimeria in broiler farms in this study could be influenced by the different anticoccidial used in various farms (carvalho et al. 2011), although our study did not document anticoccidial use in the farms. the most prevalent species among broiler farms in this study was e. tenella (68.4%), which is in agreement with other studies that have reported a high prevalence that ranges from 80.67% to 100% in anhui province, china, trinadad and indonesia (brown et al. 2018; hamid et al. 2018; huang et al. 2017). the high prevalence of e. tenella poses a major concern to the health status of chickens because it is associated with caecal lesions causing haemorrhage, oedema and anaemia (iacob & duma 2009). however, e. mitis (96%) had the highest prevalence among free-ranging village chickens in this study. the reason for this is unclear as it is contrary to reports of most studies where e. acervulina and e. tenella are known to be highly prevalent in most farms because of their high reproductive potentials (williams 2001). co-infection with multiple eimeria species is a common finding in many poultry farms (aarthi et al. 2010; haug et al. 2008). we also found multiple infections (57.9% and 100%) to be common in both chicken types, with two or more species among the samples examined. eimeria tenella + e. maxima (21.1%) and e. mitis + e. maxima + e. acervulina (44%) were the most common co-infections. this is in line with different studies which reported the frequency of e. maxima in most mixed species infection (kaboudi, umar & munir, 2016). mixed infections among eimeria species poses a challenge to the control of coccidiosis in chickens as it can increase pathogenicity of the disease among birds (jekins et al. 2008). it could also serve as a potential threat to the effectiveness of anticoccidial vaccine, and this has warranted the combination of different eimeria strains in some species, such as e. maxima, in the design of anticoccidial vaccines. the efficacy of anticoccidial vaccines is under threat, especially with the recent upsurge of new eimeria variants (otus), which was first detected circulating among commercial birds in australia (cantacessi et al. 2008). the presence of these otus (otux, otuy and otuz) has also been reported across much of the southern hemisphere (clark et al. 2016; fornace et al. 2013; jatau et al. 2016). in this study, none of the samples was positive for any of the three otus. this could be because of the geographical location of our study sample, which is on latitude 28°s. although a study has reported the distribution of these cryptic species (otus) in the northern hemisphere (jatau et al. 2016), a more elaborate study by clark et al. (2016) in 20 different countries from five continents has opined that these otus are distributed towards the south of the 30°n latitude. the study reported eight different countries to be populated with otus with the following distribution: otuz was found in all the eight countries south of the 30°n latitude and otux was detected south of 30°n in six out of the eight countries, whilst otux, otuy and otuz were only detected in nigeria among all the african countries at the same geographical location (clark et al. 2016). similarly, its-1 sequences belonging to five different eimeria species were generated in this study. the similarity of the sequences generated in this study when compared with published eimeria species sequences ranged from 90% to 93% in e. mitis, 99.31% in e. maxima, 99% to 100% in e. tenella, 100% in e. brunetti and 99.38% in e. acervulina. although the ml tree, as shown in figure 5, grouped all five species of eimeria into five distinct clades, some level of variation existed within species of eimeria in this study and that of the public database, as indicated by their mean genetic distances. the lowest genetic distance of 0.01 was observed among e. brunetti isolates. similar its-1 sequence variations among e. mitis, e. tenella and e. maxima have also been reported by different authors (bhaskaran et al. 2010; kumar et al. 2015a; lew et al. 2003; thenmozhi, veerakumari & raman 2014). in conclusion, this study characterised eimeria species in broiler and free-range chickens based on molecular diagnostic techniques and determined their diversity in kwazulu-natal province. the study reports the presence of five eimeria species (e. tenella, e. maxima, e. acervulina, e. brunetti and e. mitis), all of which are regarded as pathogenic. although none of the chickens showed clinical signs of coccidiosis during sampling, the high prevalence of these pathogenic parasites in the study area suggests that subclinical infection is common in all infected chickens. thus, effective control strategies remain imperative to curtail coccidial infection in poultry farms in the study areas. a survey on the types of anticoccidial used among commercial farms and their efficacy should be conducted to understand the impact of this disease. this will also help in the implementation of policies for the control of this disease in kwazulu-natal province. acknowledgements financial support by the national research foundation of south africa (grant numbers: 112886 and 112768) is gratefully acknowledged. competing interests the authors have declared that no competing interest exists. authors’ contributions a.j.f. was involved in design, collection of samples, laboratory work, result analysis and manuscript writing. o.tz. co-supervised the research and corrected the manuscripts. d.p.b. and s.o.p. were involved in grant writing for the research and correcting the manuscript. j.l. provided technical support and corrected the manuscript. s.m. provided guide for sample 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biology and evolution 30(12), 2725–2729. https://doi.org/10.1093/molbev/mst197 tan, l., li y., yang, x., ke, q., lei, w., mughal, m.n. et al. 2017, ‘genetic diversity and drug sensitivity studies on eimeria tenella field isolates from hubei province of china’, parasites & vectors 10(1), 137. https://doi.org/10.1186/s13071-017-2067-y thenmozhi, v., veerakumari, l. & raman, m., 2014, ‘preliminary genetic diversity study on different isolates of eimeria tenella from south india’, international journal of advanced veterinary science and technology 3(1), 114–118. https://doi.org/10.23953/cloud.ijavst.194 williams, r.b., 2001, ‘quantification of the crowding effect during infections with the seven eimeria species of the domesticated fowl: its importance for experimental designs and the production of oocyst stocks’, international journal for parasitology 31(10), 1056–1069. https://doi.org/10.1016/s0020-7519(01)00235-1 appendix 1 table 1–a1: summary of samples collected in broiler farms and the outcome of eimeria detection. table 2–a1: summary of samples collected in free-range chickens and the outcome of eimeria infection. table 3–a1: summary of samples collected in free-range chickens and the outcome of eimeria infection. table 4–a1: its-1 sequences of eimeria species downloaded from genbank. figure 1–a1: pairwise percentage identity of its-1 sequences of different eimeria species. abstract introduction materials and methods results and discussion conclusion acknowledgements references about the author(s) louis j. la grange department of agriculture, rural development, land and environmental affairs, chief directorate veterinary services, veterinary public health, mbombela, south africa school of life sciences, college of agriculture, science and engineering, university of kwazulu-natal, durban, south africa samson mukaratirwa school of life sciences, college of agriculture, science and engineering, university of kwazulu-natal, durban, south africa one health center for zoonoses and tropical veterinary medicine, school of veterinary medicine, ross university, basseterre, saint kitts and nevis citation la grange, l.j. & mukaratirwa, s., 2020, ‘experimental infection of tigerfish (hydrocynus vittatus) and african sharp tooth catfish (clarias gariepinus) with trichinella zimbabwensis’, onderstepoort journal of veterinary research 87(1), a1876. https://doi.org/10.4102/ojvr.v87i1.1876 research communication experimental infection of tigerfish (hydrocynus vittatus) and african sharp tooth catfish (clarias gariepinus) with trichinella zimbabwensis louis j. la grange, samson mukaratirwa received: 20 apr. 2020; accepted: 17 aug. 2020; published: 05 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract trichinella zimbabwensis naturally infects a variety of reptilian and wild mammalian hosts in south africa. attempts have been made to experimentally infect piranha fish with t. zimbabwensis and t. papuae without success. tigerfish (hydrocynus vittatus) and african sharp tooth catfish (clarias gariepinus) are accomplished predators cohabiting with nile crocodiles (crocodylus niloticus) and nile monitor lizards (varanus niloticus) in southern africa and are natural hosts of t. zimbabwensis. to assess the infectivity of t. zimbabwensis to these two hosts, 24 african sharp tooth catfish (mean live weight 581.75 ± 249.71 g) randomly divided into 5 groups were experimentally infected with 1.0 ± 0.34 t. zimbabwensis larvae per gram (lpg) of fish. forty-one tigerfish (mean live weight 298.6 ± 99.3 g) were randomly divided for three separate trials. an additional 7 tigerfish were assessed for the presence of natural infection as controls. results showed no adult worms or larvae of t. zimbabwensis in the gastrointestinal tract and body cavities of catfish sacrificed at day 1, 2 and 7 post-infection (p.i.). two tigerfish from one experimental group yielded 0.1 lpg and 0.02 lpg of muscle tissue at day 26 p.i. and 28 p.i., respectively. no adult worms or larvae were detected in the fish from the remaining groups sacrificed at day 7, 21, 28, 33 and 35 p.i. and from the control group. results from this study suggest that tigerfish could sustain t. zimbabwensis under specific yet unknown circumstances. keywords: clarias gariepinus; hydrocynus vittatus; tiger fish; infectivity; trichinella zimbabwensis. introduction the genus trichinella belongs to the family trichinellidae and the order trichuridae, class nematode (pozio et al. 2009). trichinella establishes in the host when larvae in raw or undercooked meat are consumed (dupoy-camet 2000). parasites in the genus complete both intermediate and definitive life cycle stages in a single host. larvae are intracellular parasites of striated muscle tissue and adult nematodes parasitise the intestinal epithelium (gottstein, pozio & nöckler 2009). trichinella zimbabwensis has the widest host range and is the most prevalent of the three known trichinella spp. (i.e. t. nelsoni, t. zimbabwensis and trichinella t8) in the greater kruger national park (gknp). it naturally infects various reptilian and wild mammalian hosts in south africa (la grange & mukaratirwa 2020; mukaratirwa et al. 2019). amongst the confirmed natural hosts, the highest prevalence has been recorded in nile crocodile (crocodylus niloticus) (la grange, govender & mukaratirwa 2013). in the last two decades, significant strides were made in identifying natural hosts of trichinella spp. in the gknp (mukaratirwa et al. 2019) and continued surveillance to identify additional hosts for t. zimbabwensis is still required (la grange & mukaratirwa 2020; la grange et al. 2013). previous experimental studies involving fish elsewhere showed some trichinella species are able to survive for short periods in the gut (moretti et al. 1997; tomašovičová 1981), suggesting some tropical fish to act as definitive or paratenic hosts for the parasite. pienaar (1968) reported the presence of 46 fish species, including predatory species such as african sharp tooth catfish (clarias gariepinus), tigerfish (hydrocynus vittatus), madagascar mottled eel (anguilla marmorata) and common long fin eel (a. mossambicus) in the knp. catfish are accomplished scavengers and predators, and consume a variety of food ranging from plankton to small mammals (bruton 1979; skelton 2001). tigerfish are apex predators that occupy the same trophic level as nile crocodiles where they cohabit (roux et al. 2018). the feeding behaviour of these fish makes them potential hosts of t. zimbabwensis. this study aimed at assessing the infectivity of t. zimbabwensis to catfish and tigerfish which cohabit natural environments with nile crocodile, a known natural host of the parasite. materials and methods twenty-four african sharp tooth catfish from a research breeding and holding facility of the university of limpopo, south africa were housed outdoors in moveable ponds at a research facility at tipperary farm, nelspruit, south africa. they were fed commercial koi pellets daily (aqua plus, midfeeds, south africa). tigerfish were collected from two sites along the komati river (31˚55’07.1’’e, 25˚29’41.4’’s and 31˚58’02.3’’e, 25˚29’12.8’’s) and housed indoors on a private farm near one of the collection sites. ponds were equipped with an inline filtration system and submersible pump (6500 l/h). a water heater (ultrazap, 3 kw) was additionally installed during two of the trials to maintain average water temperature within the fish’s natural range. water temperature was recorded for 4 h intervals in the ponds and komati river using temperature loggers (hobo water temp pro v2, onsetcomp). trichinella zimbabwensis larvae, previously derived from a nile crocodile, were maintained in a colony of rats housed at the biological resource unit (bru) at the westville campus of the university of kwazulu-natal. infected rats were euthanised and larvae isolated using artificial muscle digestion (nöckler & kapel 2007). additional trichinella-infected muscle tissue was collected and mixed into a homogenous, pooled sample containing 50 larvae per gram (lpg) to infect catfish via natural feeding. ten catfish were infected using an orogastric tube (table 1). fish in group 1 (n = 5) were sedated with 0.3 ml/l of 2-phenoxyethanol in water. once stage 2 anaesthesia was reached (gardner 2017), the fish were individually infected with approximately 500 larvae administered via a canine urinary catheter (sovereign®, french gauge 8, sherwood medical, st. louis, united states of america). the procedure was repeated for group 2 (n = 5) but without anaesthetic, to rule out possible negative effects of anaesthetic on the establishment and/or development of t. zimbabwbensis larvae. table 1: results from experimental infection of african sharp tooth catfish (clarias gariepinus) with trichinella zimbabwensis. an additional 10 fish were infected using blank gelatin capsules (size 0, medisurge, south africa). capsules containing approximately 500 larvae were administered orally. group 3 (n = 5) was infected with, and group 4 (n = 5), without anaesthesia (table 1). four fish (group 5) were infected through natural feeding with approximately 1000 larvae/fish (table 1). ten grams of infective tissue (50 lpg) was used to prepare infective boluses (approximately 500 larvae). two boluses were offered to each fish on the day of infection and were readily consumed by the fish. fresh chicken hearts (mikon farming abattoir, south africa) were ‘loaded’ with a 2 ml solution of infective material (approximately 300 larvae/ml) using a 3 ml syringe and 25 gauge needle. each fish received a single infected chicken heart. three separate trials were conducted (table 2). table 2: results from experimental infection of tigerfish (hydrocynus vittatus) with trichinella zimbabwensis. tigerfish intended for trial 1 (t1) (n =1), trial 2 (t2) (n = 3) and trial 3 (t3) (n = 3) died during the respective first weeks of acclimation because of stress, preserved (frozen) and retained as a control group. fish were euthanised on day 1, 2 and 7 p.i. (catfish) and 7, 21, 28, 33 and 35 p.i. (tigerfish) using 0.6 ml/l of 2-phenoxyethanol. fish were allowed to reach stage 4 anaesthesia (gardner 2017) before being decapitated. each fish was dissected ventrally and opened lengthwise from the cloaca to the head. the body cavity was rinsed with 0.9% saline solution and the wash collected in a glass petri dish. the small intestines and stomach were separated into specimen dishes containing 0.9% saline solution (justine, briand & bray 2012), cut open and the mucosa carefully scraped. the contents and the wash liquid were examined for the presence of t. zimbabwensis larvae and/or adults. additionally, a representative sample of the whole body comprising 100 g of muscle tissue was collected, artificially digested (nöckler & kapel 2007) and examined for the presence of t. zimbabwensis larvae. the average weight ± standard deviation (sd) was expressed per group. water temperature was expressed as average daily water temperature ± sd for each group. mean temperature of the ponds during each trial was compared with the mean temperature of the river during the corresponding time frame using student t-tests. ethical consideration the study protocol was approved by the animal research ethics committee of the university of kwazulu-natal (ref: arec/029/016m and 030/019d). results and discussion results from the experimental trials are shown in tables 1 and 2. no larvae or adults of t. zimbabwensis were observed in the gastrointestinal tract, body cavities and muscle tissue of catfish infected using different modes at day 1, 2 or 7 p.i. (table 1). because of prolonged national power failures during the last 2 weeks of t1g1, four tigerfish died at day 23 p.i and two at day 26 p.i. from oxygen depletion of the water. the two surviving fish were euthanised at day 28 p.i. analysis of the two tigerfish that died on day 26 p.i. yielded a total of 10 dead larvae (1 lpg of muscle) from one fish. of the two that were euthanised one fish yielded a live larva (0.02 lpg) in muscle tissue. the low number of larvae recovered is in agreement with findings from studies involving t. britovi (moretti et al. 1997) and t. spiralis and t. pseudospiralis (tomašovičová 1981). no larvae or adult parasites were observed in the body cavity, stomach or gastrointestinal tract of fish euthanised at day 7, 28, 33 or 35 p.i. the minimum infective larval dose required in a specific host is dependant on both parasiteand host characteristics (gottstein et al. 2009). previous studies involving fish experimentally infected with t. pseudospiralis and t. spiralis (tomašovičová 1981), t. britovi (moretti et al. 1997) and t. zimbabwensis and t. papuae (pozio & la rosa 2005) were reported on varying dosages of trichinella larvae administered to fish and no mention was made of the live weight of fish used. the lack of data in respect of the weight of fish and larvae per gram of fish administered in previous studies precludes a comparison to assess the minimum infective dosage needed for t. zimbabwensis to establish in fish. in an experimental study involving t. zimbabwensis and t. papuae, reptiles were successfully infected with an inoculum of 3000 larvae per animal (pozio et al. 2004). this dose might have been greater than inocula used for catfish and tigerfish in this study although information on the weight of animals was not given. previous studies in other poikilothermic hosts, experimentally infected with different trichinella taxa, suggest that temperature plays an integral role in larval survival and/or development in the host (asatrian, movsessian & gevorkian 2000; cristeau & perian 1999; pozio et al. 2004). in this study, water temperatures in ponds where catfish were kept were lower than those reported by tomašovičová (1981) and moretti et al. (1997). temperatures recorded during tigerfish trials in this study were much higher than those reported in the studies conducted by tomašovičová (1981) and moretti et al. (1997) and compared favourably with the lower end of temperatures reported by pozio and la rosa (2005). water temperature in the river was significantly higher compared to the experimental ponds for t1 (p = 0.002), t2g1 (p = 0.0002), t2g2 (p = 0.002), t3g (p = 0.04) and t3g3 (p < 0.0001), but not so for t3g1 (p = 0.07). this precludes any definitive conclusion concerning the influence of temperature on t. zimbabwensis larval development and survival in fish. previous studies also suggested several host-related factors for the failure of t. zimbabwensis to establish in fish and this includes: (1) lack of villi in the small intestine preventing larvae from anchoring to the intestinal wall and resist peristaltic movements, (2) the copious amounts of bile and mucous excreted in the intestine of fish, (3) low body temperature of the host, (4) quick passage of larvae through the intestine and (5) possible destruction of larvae by digestive processes (moretti et al. 1997). trichinella pseudospiralis larvae have been reported to migrate without moulting from the gastrointestinal tract to the body cavity, organs and muscles of fish whilst t. spiralis larvae only migrated to the body cavity of fish (tomašovičová 1981). trichinella britovi larvae migrated without further development and were isolated from the gastrointestinal tract and body cavity of carp (cyprinus carpio) and from the gastrointestinal tract of catfish (ictalurus melas) (moretti et al. 1997). in this study, no positive results could be obtained from the fish during the early stages after infection (day 1, 2 and 7 p.i.) in catfish or day 7 p.i. in tigerfish. from an epidemiological perspective, the negative results from this study in catfish rule out the potential of this fish species as a host for t. zimbabwensis. positive results from tigerfish, although interesting, are inconclusive on whether tigerfish could act as reservoir hosts for t. zimbabwensis. conclusion results from this study suggest that t. zimbabwensis is unable to develop and establish in the african sharp tooth catfish, however, results from tigerfish suggest that some individual fish could, under very specific circumstances, maintain t. zimbabwensis larvae. it is not known whether the parasite can fully develop and reproduce in this host. the paucity of information concerning the minimum infective dosage required to establish t. zimbabwensis infection, the potential influence of host (kapel 1995; la grange et al. 2013; reina, munoz-ojeda & serrano 1996; soule et al. 1989) and parasite characteristics (hurnikova et al. 2004; kapel, webster & gamble 2005) and the potential influence of temperature on larval development and survival in fish precludes a general hypothesis that fish do not play any role in the epidemiology of t. zimbabwensis. future research efforts should focus on additional tropical fish that cohabit with other known hosts of t. zimbabwensis in the gknp to elucidate the role of any specific fish species in the epidemiology of t. zimbabwensis. acknowledgements the authors would like to acknowledge the mpumalanga department of agriculture, rural development, land and environmental affairs for logistical and financial support provided through the chief directorate of veterinary services, and dr j.p. raath and staff of wildlifevets.com and wildlife pharmaceuticals for financial, logistical and technical support provided. competing interests the authors have declared that no competing interests exist. authors’ contributions l.l.j. and s.m. conceived and designed the study. l.l.j. carried out the experiments and analysed the data and drafted the manuscript. all authors were involved in the revision and approval of the final version of the manuscript. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were 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introduction milk yield, stage of lactation and parity of goats may influence the length of the withdrawal period of intramammary antibiotics from milk. these factors were investigated in goats after treatment with three intramammary antibiotic products. the effect of treatment on milk compositional quality (milk fat, total protein and lactose percentages) was investigated. 243 onderstepoort journal of veterinary research, 74:243–249 (2007) intramammary antibiotics in dairy goats: effect of stage of lactation, parity and milk volume on withdrawal periods, and the effect of treatment on milk compositional quality j. karzis1*, e.f. donkin2 and i.m. petzer1 abstract karzis, j., donkin, e.f. & petzer, i.m. 2007 intramammary antibiotics in dairy goats: effect of stage of lactation, parity and milk volume on withdrawal periods, and the effect of treatment on milk compositional quality. onderstepoort journal of veterinary research, 74:243–249 the length of the antibiotic withdrawal period after intramammary treatment was influenced by the milk yield of dairy goats during this trial. shorter withdrawal periods were seen in relatively high yielding dairy goats (production above 1.5 l per day) compared to low producers (less than 1.3 l per day). high yielding goats treated with curaclox lc (norbrook [pharmacia ah]) had a withdrawal period of 42 h, while low yielding goats, treated with the same product, had a withdrawal period of 74 h. the recommended withdrawal period for curaclox lc for use in cattle is 72 h. relatively high yielding goats treated with rilexine 200 lc (logos agvet [virbac]) had a significantly shorter withdrawal period (37 h) than that recommended for use in cattle (96 h). low yielding goats treated with spectrazol milking cow (schering-plough animal health) had a significantly longer (95 h) withdrawal period than that recommended for use in cattle (60 h). withdrawal periods were also influenced by stage of lactation and parity. there was a moderate positive correlation between lactation number and withdrawal period, as measured by tris (r2 = 0.621), and a moderate negative correlation between stage of lactation and withdrawal period (r2 = –0.669). in trials 1, 2 and 3 combined there was a moderate negative correlation between withdrawal period and volume (r2 = –0.511) and a strong positive correlation between withdrawal period and lactation number (r2 = 0.720). the differences in percentage milk fat, protein and lactose before, during and after treatment were not statistically significant except in trial 3 (curaclox lc and rilexine 200 lc) where protein and lactose differed significantly. in trial 2 (spectrazol milking cow) milk fat percentages differed significantly between treatment and control groups as did protein percentages in trial 3. these differences are however, not biologically meaningful. keywords: antibiotic withdrawal periods, dairy goats, lactose, milk fat, milk protein, milk yield, parity, stage of lactation * author to whom correspondence is to be directed. e-mail: jkarzis@mweb.co.za or joannejk@iafrica.com 1 department of production animal studies, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of animal and wildlife sciences, faculty of natural and agricultural sciences, university of pretoria, 0002 south africa accepted for publication 27 march 2007—editor 244 intramammary antibiotics in dairy goats: effects the withdrawal period may be defined as the period that is required following the last medication in order to bring the concentration of the drug to below a tolerable value. the tolerable value of a drug depends on the acceptable daily intake (adi), which is based on pharmacological studies in experimental animals and is the lowest dose that is found to give some dis order, divided by a large safety factor. parameters that have to be studied to establish the adi value are: • the adi and the average amount of food taken by humans. • the maximum residue level (mrl) permissible in foodstuffs of animal origin, such as meat or milk (debackere 1995). many factors influence the elimination of antibiotics after intramammary application. these include the dose administered, the number of udder halves treated, the number of treatments, the milk yield, the condition of the udder, combination of treatments (e.g. anti-inflammatory and antibiotic) and the health status of the animal (le roux 2004). goat milk containing antibiotic residues cannot be used for human consumption as it poses a health hazard, more so due to its often use for food for babies, and it’s affect on the production of cheese. the normal milk fat concentration of most european dairy goat breeds under cold environmental conditions is approximately 3.8 %, which is less than that for nubians and pygmy goats (haenlein 2004). the compositional quality of goat milk for different breeds is given in table 1. materials and methods herds used in trials three trials were conducted. trial 1 and trial 2 were conducted at the faculty of veterinary science, university of pretoria (herd a), while trial 3 was conducted on a commercial goat dairy in the limpopo province of south africa (herd b). trial 1 herd a consisted of 15 lactating multiparous saanen goats, all except one of which were in early lactation; the exception being in late lactation. for details see karzis, donkin & petzer 2007. trial 2 trial 2 was conducted in herd a using 14 lactating multiparous saanen goats, all except one of which were in mid lactation. the exception was in late lactation. for details see karzis et al. 2007. trial 3 trial 3 was conducted in herd b, using 64 saanen, saanen-indigenous crossbreeds and toggenburg goats, all of which were in mid lactation. fifty-seven out of the 64 goats were in their first and the rest in their second lactation. for details see karzis et al. 2007. in all trials, results of clinical udder examinations, milk yield, parity and stage of lactation were considered and selection of experimental animals was done by the principle of pairing. for details see karzis et al. 2007. goats were considered to have a bacterial udder infection, when bacteria were isolated from the milk sample. sampling during trials goats were milked at 12 hourly intervals and foremilk samples were taken from udder halves of all goats for three milkings prior to the first intramammary treatment to establish baseline parameters (karzis 2005). for details see (karzis et al. 2007). table 1 compositional quality of goat milk for different breeds authors breed milk fat % mean + se or sd protein % mean + se or sd lactose % mean + se park 1991 park & humphrey 1986 calderon et al. 1984 zeng & escobar 1996 french alpine and anglo nubian alpine and nubian french alpine alpine and nubian 3.94 + 1.21 (sd) 4.47 + 0.13 (se) 3.40 + 0.09 (se) 4.08 * 3.51 + 0.77 (sd) 3.42 + 0.05 (se) 3.30 + 0.06 (se) 3.20* ** ** ** 4.41 * nubian goats produced a significantly higher (p < 0.05) percentage butterfat and protein than alpine does in the same in vesti gation ** not given 245 j. karzis, e.f. donkin & i.m. petzer a composite milk sample from each udder half was taken before, during and after treatment to determine the percentage milk fat, protein and lactose. antibiotic treatment in trial 1, six untreated goats were used as controls, while eight goats were treated with curaclox lc (norbrook [pharmacia ah]), 75 mg sodium ampicillin and 200 mg sodium cloxacillin per dose. in trial 2, seven goats were untreated controls and seven goats were treated, after each of three consecutive milkings at 12 hourly intervals, with spectrazol milking cow (schering-plough animal health), 250 mg cefuroxime per dose. two products were investigated in trial 3: rilexine (sa) 200 lc (logos agvet [virbac]), 100 mg cephalexin, 100 mg neomycin sulphate and 10 mg prednisolone per dose, and curaclox lc. twenty untreated goats acted as controls and 20 goats were treated, after each of three consecutive milkings at 12 hourly intervals, with rilexine (sa) 200 lc, while 12 goats were treated with curaclox lc and 12 goats served as controls. for details see karzis et al. 2007. laboratory procedures thermo-resistant inhibitory substances the tris test was carried out on all milk samples to determine the presence of antibiotic residues and other heat stable inhibitory substances in the milk (karzis 2005). milk oscan an accredited laboratory using a milk oscan system 4000 (the rhine ruhr group) measured the percentage milk fat, total protein and lactose. data management all data were entered and stored in microsoft excel and data were analysed statistically using the statistical programme genstat (2003). pearson’s coefficient measures the linear relationship between two random variates (–1 < r < 1) (draper & smith 1981). the tabled critical values for determining significance of a correlation coefficient are dependent on the pairs of data in the sample. generally, a coefficient of about + 0.7 or more is regarded as indicating a fairly strong correlation while + 0.9 indicates a very strong correlation (rayner 1969). results effects of yield, stage of lactation and parity on withdrawal periods table 2 showed milk production levels and withdrawal periods as measured by tris for curaclox lc, spectrazol milking cow and rilexine 200lc. statistical analysis of milk fat, protein and lactose table 3 showed milk fat percentages for treatment (t1, t2 and t3) and control groups of goats. table 4 showed protein percentages of milk treatment (t1, t2 and t3) and control groups of goats. table 5 showed lactose percentages of milk for treatment (t1, t2 and t3) and control groups of goats. discussion effects of milk yield, stage of lactation and parity on the length of withdrawal periods goats used in trial 1 and 2 had low milk yields while those used in trial 3 were relatively high producers. there was a strong negative correlation (r2 = 0.701) between withdrawal period and milk yield after use of curaclox lc in trial 1 (karzis 2005). this was possibly because of the dilution factor. a moderate negative correlation (r2 = –0.493) was found between withdrawal period and milk yield in trial 2 table 2 milk production levels and antibiotic withdrawal periods as measured by tris from trials 1, 2 and 3 product trial production level withdrawal periods + sd (h) spectrazol milking cow curaclox lc curaclox lc curaclox lc rilexine 200 lc trial 2 trial 1 trials 1 and 3 trial 3 trial 3 low low low and high high high 95 + 17.23 74 + 19.21 59 + 24.31 42 + 7.08 37 + 9.94 246 intramammary antibiotics in dairy goats: effects after treatment with spectrazol milking cow. however, no linear correlation was found between yield and withdrawal period in trial 3 after treatment with both curaclox lc and rilexine 200 lc. withdrawal periods for curaclox lc were shorter in trial 3 with mostly high producing animals, than in trial 1 or in trial 1 and 3 combined, although there was no linear correlation between withdrawal period and yield. in trials 1, 2 and 3 combined there was a moderate negative correlation between withdrawal period and yield (r2 = –0.511). treatment with curaclox lc (trial 1) and spectrazol milking cow (trial 2) did not affect the milk yield of the goats. there was a moderate positive correlation between lactation number and withdrawal period (r2 = 0.621), a moderate negative correlation between stage of lactation and withdrawal period as measured by tris (r2 = –0.669), and a moderate negative correlation between stage of lactation and lactation number in trials 1 and 3 for curaclox lc. the combined data of trials 1, 2 and 3, however, showed a strong positive correlation between withdrawal period and lactation number (r2 = 0.720). a weak negative linear correlation (r2 = –0.373) was found between yield and lactation number in the combined data of trials 1, 2 and 3 (karzis 2005). table 3 milk fat percentages for treatment (t1, t2 and t3) and control groups of goats. (analysis of an unbalanced design) trial no. and products used treatment groups mean milk fat % at (–12 h) + sd mean milk fat % at (12 h) + sd mean milk fat % at (108 h) + sd f probability between treatments f probability interaction between treatments at different times trial 1; curaclox lc t1 c1 4.29 + 1.52 4.56 + 0.63 4.16 + 1.51 3.99 + 0.79 4.07 + 1.41 3.99 + 0.46 p = 0.984 p = 0.729 trial 2; spectrazol milking cow t2 c2 3.83 + 1.05 3.48 + 0.62 4.05 + 1.14 3.39 + 0.53 3.55 + 0.67 3.28 + 0.78 p = 0.022 p = 0.566 trial 3; curaclox lc (t1) and rilexine 200 lc (t3) c t1 t3 3.53 + 0.63 3.19 + 0.54 3.39 + 0.60 3.39 + 0.61 3.55 + 0.31 3.85 + 0.52 3.72 + 1.10 3.48 + 0.51 3.66 + 0.58 p = 0.320 p = 0.199 trials 1 and 3 c 3.81 + 0.78 3.66 + 0.75 3.80 + 0.95 p = 0.359 p = 0.963 p is significant at the 5 % level (p < 0.05) table 4 protein percentages of milk for treatment (t1, t2 and t3) and control groups of goats. (analysis of an unbalanced design) trial and products used treatment groups mean protein % at (–12 h) + sd mean protein % at (12 h) + sd mean protein % at (108 h) + sd f probability between treatments f probability interaction between treatments at different times trial 1; curaclox lc t1 c1 3.35 + 0.59 3.24 + 0.54 3.21 + 0.44 3.33 + 0.27 3.17 + 0.40 3.18 + 0.38 p = 0.992 p = 0.701 trial 2; spectrazol t2 c2 3.07 + 0.64 3.11 + 0.41 3.04 + 0.44 3.09 + 0.37 3.04 + 0.49 2.99 + 0.45 p = 0.885 p = 0.973 trial 3; curaclox lc (t1) and rilexine (t3) c t1 t3 3.13 + 0.21 3.24 + 0.29 3.30 + 0.25 3.02 + 0.22 3.29 + 0.23 2.77 + 0.44 3.09 + 0.23 3.15 + 0.22 3.34 + 0.98 p = 0.054 p < 0.001 trials 1 and 3; curaclox lc c t1 3.16 + 0.21 3.31 + 0.50 3.16 + 0.22 3.25 + 0.38 3.11 + 0.23 3.16 + 0.34 p = 0.069 p = 0.611 p is significant at the 5 % level (p < 0.05) 247 j. karzis, e.f. donkin & i.m. petzer table 5 lactose percentages of milk for treatment (t1, t2 and t3) and control groups of goats. (analysis of an unbalanced design) trial and products used treatment groups mean lactose % at (–12 h) + sd mean lactose % at (12 h) + sd mean lactose % at (108 h) + sd f probability between treatments f probability interaction between treatments at different times trial 1; curaclox lc t1 c1 4.32 + 0.94 4.22 + 0.43 4.32 + 0.41 4.44 + 0.26 4.52 + 0.27 4.23 + 0.45 p = 0.131 p = 0.156 trial 2; spectrazol milking cow t2 c2 4.33 + 0.71 4.19 + 0.64 4.18 + 0.48 4.25 + 0.49 4.25 + 0.43 4.03 + 0.56 p = 0.426 p = 0.780 trial 3; curaclox lc (t1) and rilexine 200 lc (t3) c t1 t3 4.58 + 0.17 4.69 + 0.22 4.69 + 0.16 4.57 + 0.22 4.42 + 0.20 4.48 + 0.18 4.59 + 0.16 4.60 + 0.28 4.61 + 0.19 p = 0.755 p = 0.001 trials 1 and 3; curaclox lc c t1 4.48 + 0.31 4.45 + 0.78 4.51 + 0.24 4.34 + 0.34 4.48 + 0.32 4.55 + 0.27 p = 0.494 p = 0.474 p is significant at the 5 % level (p < 0.05) this was not the case in a study by dulin, paape, schultze & weinland (1983) where milk yield increased with increasing lactation number. goats treated with curaclox lc and spectrazol milking cow in trials 1 and 2 and which had bacterial udder infections had lower milk yields than goats without bacterial udder infections in trials 1 and 2. goats with bacterial udder infections also showed longer withdrawal periods compared with non-infected goats in the same trials. however, this was not the case in the high and mid yielding goats in trial 3 treated with curaclox lc and rilexine 200 lc. it was therefore evident that the presence of a bacterial udder infection decreased the milk yield in lowyielding goats but not in midand high-yielding goats. analysis of milk fat, protein and lactose milk fat, protein and lactose concentrations were measured three times during each trial, prior to treatment, during treatment, and post treatment when antibiotic residues were no longer present in the milk. milk fat in previous studies moderately strong positive correlations were present between percentage milk fat and percentage protein in goat milk and moderately negative correlations between percentage milk fat and electrical conductivity (park & humphrey 1986; park 1991). in this study, electrical conductivity meas urements were taken, but due to the unreliability of the conductivity meters there was insufficient data for statistical analysis. the percentage milk fat, protein and lactose measured prior to treatment differed significantly between the saanen goats used in trials 1 and 2, and the saanen-toggenburg crossbreeds goats used in trial 3 (tables 3, 4 and 5). the milk fat percentage also depends on stage of lactation and diet (concentrate diet) according to cal deron, peters, smith & franke (1984) and don kin, boya zoglu, els, macgregor, ramsay & lubout (2000). no significant differences were found in the three trials between the milk fat percentage before, during and after treatment. no significant differences were found between the milk fat of goats in the treatment and control groups in trials 1 and 3 and the combined data of trials 1 and 3 for curaclox lc treatment. in trial 3 there was no significant difference between the treatments with curaclox lc (t1) and rilexine 200 lc (t3), and the control group (c) in the percentages of milk fat (table 3). these results indicate that treatment with both curaclox lc (t1) and rilexine 200 lc (t3) did not influence the milk fat percentage. however, a significant difference (p = 0.022) was found between treatment (t2) and control (c2) groups in trial 2 in the percentage of milk fat where spectrazol milking cow had been used (table 3). this could have been due to the low production or the high fat content of the carrier substance of spectrazol milking cow (a succinyllated fatty acid with triglycerides). 248 intramammary antibiotics in dairy goats: effects milk protein the percentage of milk protein is known to differ between goat breeds (table 1). a high concentrate diet has been found to cause milk fat to decrease and that it has no effect on milk protein or total solids (calderon et al. 1984). park (1991) found moderately strong negative correlations between the percentage protein and electrical conductivity (park 1991). donkin et al. (2000) reported that the percentage protein in saanen and saanen-indigenous crossbred goats remains relatively stable in first and second lactations. milk protein percentage before, during and after treatment was found not to be significantly different in trials 1 and 2 or with the combined data from trials 1 and 3 after treatment with curaclox lc. however, there is a significant difference (p < 0.001) between the percentage protein of treatment groups and between the three respective treatment times (table 4). the percentage of protein in milk from goats in the treatment and control groups showed no significant differences in trial 1 (table 4), showing that the treatment with curaclox lc and spectrazol milking cow did not influence the percentage protein in goat milk. mean protein percentage of milk from goats between treatment (t1) and control (c) groups for curaclox lc in trials 1 and 3 are not significantly different (table 4). however, protein percentage is significantly different (p = 0.054) between treatments (t1) using curaclox lc, (t3) rilexine 200 lc and control (c) goats in trial 3 (table 4). this difference might have been caused by nutritional changes on the commercial dairy farm. lactose donkin et al. (2000) found that the percentage lactose does not differ between goats in first and second lactations, but that it does differ between goats in their first lactation. lactose percentages of milk from goats were not significantly different in trials 1 and 2 or from the combined data of trials 1 and 3 after treatment with curaclox lc. this was not the case for trial 3 where there was a significant difference (p = 0.001) between the percentage lactose of milk from goats at the three respective treatment times (table 5). the percentage protein of milk from the goats did not differ significantly in trials 1, 2 and 3, or in the combined data of trials 1 and 3 after treatment with curaclox lc (table 5). it seems therefore that the difference in the percentage lactose between the three respective treatment times seen in trial 3 was not due to the treatment. conclusion the lengths of withdrawal periods after intramammary antibiotic treatment were influenced by milk yield. relatively high producers had a shorter withdrawal period than low producers treated with the same intramammary antibiotic. however, treatment with intramammary antibiotics as such did not affect the yield significantly. future research on withdrawal periods of goat milk treated with intramammary antibiotics should take milk yield into account. withdrawal periods were also affected by stage of lactation and parity. in trials 1, 2 and 3 and in combined data of trials 1 and 3 after the use of curaclox lc, withdrawal periods increased with increasing lactation number. stage of lactation and parity should be taken into account in further studies on somatic cell counts (scc) and withdrawal periods of intramammary antibiotics in goat milk. the differences in percentage milk fat, protein and lactose before, during and after treatment were not statistically significant, except in trial 3 where protein and lactose differed significantly. in trial 2 (spectrazol milking cow) milk fat percentages differed significantly between treatment and control groups, as did protein percentages in trial 3. these differences were, however, not biologically meaningful. breed and stage of lactation of goats affected the percentages of milk fat, protein and lactose of goats. further investigations are necessary to determine the effects of intramammary antibiotic treatment in different goat breeds and at different stages of lactation. acknowledgements we thank the national research foundation for the funding of this research, mr tobie fourie and the staff of the limpopo melkery and the staff of the milk laboratory, onderstepoort and the onderstepoort teaching animal unit, faculty of veterinary science for all their help, and marie smith of the agricultural research council silverton for the statistical analysis. references calderon, i., de peters, e.j., smith, n.e. & franke, a. a. 1984. composition of goat’s milk: changes within and ef249 j. karzis, e.f. donkin & i.m. petzer fects of a high concentration diet. journal of dairy science, 67:1905–1911. debackere, m. 1995. pharmacokinetics and pharmacodynamics of antimicrobials in relation to their residues in milk. proceedings of the symposium on residues of antimicrobial drugs and other inhibitors in milk, kiel, germany, 1995: 41. donkin, e.f., boyazoglu, p.a., els, h.c., macgregor, r.g., ramsay, k.a. & lubout, p.c. 2000. productivity of saanen, south african indigenous and crossbred goats fed a complete feed: preliminary results. proceedings of the 7th inter national conference on goats, france, 2000: 132–135. draper, n. & smith, h. 1981. applied regression analysis, 2nd ed. new york: wiley and sons. dulin, a.m., paape m.j., schultze, w.d. & weinland, b. t. 1983. effect of parity, stage of lactation, and intramammary infection on concentration of somatic cells and cytoplasmic particles in goat milk. journal of dairy science, 66: 2426–2433. genstat® for windows® 2003. introduction, edited by r.w. payne, vsn international. haenlein, g.f.w. 2004. goat milk in human nutrition. small ruminant research 51:155–163. karzis, j. 2005. intramammary antibiotics in dairy goats: withdrawal periods and tissue tolerance. m.sc. thesis, university of pretoria. karzis, j., donkin, e.f. & petzer, i.m. 2007. intramammary antibiotics in dairy goats: withdrawal periods of three intramammary antibiotics compared to recommended withdrawal periods for cattle. onderstepoort journal of veterinary research, 74:217–222. le roux, l. 2004. antibiotika: antibiotiese klassifikasie—kli niese gebruik van antimikrobiale middels (3), livestock health and production review, 6:5–9. park, y.w. 1991. interrelationships between somatic cell counts, electrical conductivity, bacteria counts, percent fat and protein in goat milk. small ruminant research, 5:367–375. park, y.w. & humphrey, r.d. 1986. bacterial cell counts in goat milk and their correlations with somatic cell counts, percent fat and protein. journal of dairy science, 69:32–37. rayner, a.a. 1969. a first course in biometry for agriculture students. pietermaritzburg: university of natal press. zeng, s.s. & escobar, e.n. 1996. effect of breed and milking method on somatic cell count, standard plate count and composition of goat milk. small ruminant research, 19:169–175. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) keitiretse molefe department of animal health, faculty of natural and agricultural science, north-west university, mafikeng, south africa mulunda mwanza department of animal health, faculty of natural and agricultural science, north-west university, mafikeng, south africa citation molefe, k. & mwanza, m., 2019, ‘serum biochemistry in cows of different breeds presented with reproductive conditions’, onderstepoort journal of veterinary research 86(1), a1742. https://doi.org/10.4102/ojvr.v86i1.1742 original research serum biochemistry in cows of different breeds presented with reproductive conditions keitiretse molefe, mulunda mwanza received: 13 feb. 2019; accepted: 28 mar. 2019; published: 29 aug. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract minimising health problems and increasing yield have always been the objectives in livestock agriculture. hence, increases in incidences of reproductive conditions in cattle farming pose a great threat to productivity and impose undesirable economic implications. this study aimed to examine the concentrations of different biochemical compounds in cows with reproductive conditions. seventy-seven blood samples were collected from cows at different rural areas around mafikeng, following cases of downer cow syndrome, dystocia, retained placenta, vaginal prolapse and abortion. means of serum metabolites across the different reproductive conditions were statistically compared using pearson’s chi-square test to determine variations of serum metabolites in cows of different breeds. in mixed breed cows, higher than normal calcium concentrations were observed in downer cow syndrome (25.25 ± 8.47) and dystocia (85.50 ± 8.46) cases. it was also observed that cholesterol concentrations were significantly low in abortion (2.52 ± 0.79), retained placenta (3.18 ± 0.61) and vaginal prolapse (2.37 ± 0.97) cases in afrikaner cows. the study showed that brahman (43.1%) and afrikaner (43.1%) breeds were mostly affected by downer cow syndrome. additionally, the occurrences of downer cow syndrome (53.9%) and abortions (60%) were mostly observed in cows of 1–3 years, in second and first parities, respectively. this study proves that concentrations of calcium, urea or blood urea nitrogen (bun), magnesium and cholesterol are significantly altered in incidences of reproductive conditions in cows of different breeds. it is also shown that serum biochemistry is affected by reproductive conditions in cows of different ages and parity. this data serves as a tool that could be used to enhance research in animal production and reproduction. keywords: animal health; cow; metabolic disorders; nutrition; reproductive performance; serum biochemistry. introduction the use of serum metabolic profiling in veterinary medicine is a significant test in herd health assessment (oetzel 2004). the physiological and nutritional status of cows can be evaluated using the analysis of blood biochemical parameters (ashmawy 2015). irregularities in numerous biochemical factors have been held liable for reproductive failures in cows (bazzano et al. 2016). the cow’s immune capacity during the transition period greatly influences its susceptibility to reproductive and metabolic disorders (azawi 2008). the incidences of reproductive conditions are normally elevated during the transition period (santos, rutigliano & filho 2009). disturbances in the normal function of reproductive processes leading to dystocia can severely affect cattle production (savc, kenny & beltman 2016). associated risk factors and causes of dystocia include direct factors (e.g. uterine torsion and foetal mal-presentations), phenotypic factors (calf birth weight, multiple calving, prenatal mortality, cow pelvic area, cow body weight, body condition at calving and gestation length), non-genetic and genetic factors (gaafar, el-lateif & el-hady 2011; mee 2012; zaborski et al. 2009). dystocia is an undesirable condition because of its impact on cows’ subsequent reproductive performance and negative economic effects on farm production. the estimated cost of dystocia in any parity on average is approximately $24.24, which is approximately r323.78 (south african rand) as per case attendance (dematawewa & berger 1997). vaginal prolapse is another important reproductive health problem seen in cows. it is a condition of the reproductive tract frequently seen in 24-month-old cows, either pregnant or non-pregnant (do nascimento, mello & corrêa 2016). a study of abuom et al. (2012) revealed that the incidences of vaginal prolapse increased during the dry season as a consequence of poor nutrition leading to loss of body condition as well as the peri-vaginal fat, which is important in supporting the uterus and vagina within the pelvic cavity. also, foods containing oestrogenic substances such as subterranean clover pasture, soya-bean meal, mouldy maize and barley may result in a high incidence of vaginal prolapse (noakes, parkinson & england 2001). bovine abortions are complex reproductive conditions that occur between 42 and 280 days of pregnancy which may result from several pathogenic agents such as bacterial, viral, fungal, protozoal and non-infectious agents (anka et al. 2014). the cost of production losses due to abortions is estimated to be about $640.00 (or r8548.64) per abortion case (gädicke, vidal & monti 2010). implementation of precautionary measures such as good health management, sufficient nutrition and other environmental factors affecting reproduction during pregnancy are important aspects to assure reduction in the high cost of animal losses through abortions. the deficiency in secretions of oxytocin, prostaglandins (pgf2α) and serum calcium levels, dystocia and delay in the involution of the uterus may cause retained placenta (akar & yildiz 2005). premature induction of parturition with glucocorticoids and/or prostaglandins, low plasma oestrogen concentration, deficiency in vitamin e and selenium, negative energy balance, hypocalcaemia hormonal disorders and others factors predispose cows to the incidences of retained placenta (jemal 2016). downer cow syndrome is defined as lateral or sternal recumbency that persists longer than 24 hours whereby the animal is unable to rise to a standing position (ménard & thompson 2007:487). the aetiology of recumbency of the animal is not well understood; however, the association of energetic or electrolyte metabolism as well as infectious diseases and trauma may result in this syndrome (guyot et al. 2017). the main causes of downer cow syndrome are hypocalcaemia and energy imbalance (radostits et al. 2000). other factors related to the incidences of downer cow syndrome include post-parturient disorders such as fatty liver, ketosis, metritis, nerve paralysis and mastitis (kimura, reinhardt & goff 2006). the association between nutrition, diseases and reproductive performance has been a subject of increasing interest to producers, veterinarians and nutritional researchers in recent years (martinez et al. 2014; pradhan & nakagoshi 2008). the multifactorial nature of the reproductive conditions seriously limits the effectiveness of treatment methods and increases the risk of misdiagnoses. note that it is difficult to monitor the level of impact reproductive conditions have on cattle production, mainly because of lack of data from diagnostics (thornton 2010). consequently, cattle production efficiency remains in a critically hindered status, as farmers still face challenges in their attempt to reduce reproductive health illnesses. therefore it is necessary to conduct this study in order to bring about a guiding tool of variations in serum metabolites in cows of different breeds presenting with downer cow syndrome, dystocia, retained placenta, vaginal prolapse and abortion. materials and methods study design the study was conducted in the mafikeng area, found in the north-west province in south africa. the geographical coordinates of the study area are 25°51′s and 25°38′e. blood samples were collected from communal farms around mafikeng. samples were collected when cases of reproductive conditions were reported to the north-west university dale beighle centre for animal health studies. a total of 77 blood samples were collected from cows with cases of downer cow syndrome (n = 13), dystocia (n = 14), retained placenta (n = 13), vaginal prolapse (n = 9), and abortion (n = 28). blood was collected from the jugular vein using an 18g double-pointed vacutainer needle into clot activator red stopped tubes (greiner bio-one, kremsmunster, austria; germany). samples were placed on ice and transported to the north-west university animal health laboratory for analysis. serum was obtained by centrifugation (2500 revolutions per minute for 10 minutes) immediately after collection and was stored at -20 °c until assay. the idexx catalyst dx® chemistry analyzer was used for blood biochemistry testing of serum metabolites to determine concentrations of calcium, magnesium, total bilirubin, cholesterol (chol), ammonia, triglycerides, urea, uric acid and aspartate amino-transferase (ast) following the procedure of the manufacturer (idexx laboratories inc 2014, westbrook, me, united states [us]). information about the age, parity and breed of the cows were also recorded. descriptive statistics (frequencies and percentages) were used to determine ages and parities of cows affected by reproductive conditions. the measure of association was performed using pearson’s chi-square test to determine association between cow breeds (afrikaner, brahman, charolaise, mixed, nguni, drakensberger and bonsmara) and blood chemistry (calcium, magnesium, total protein, creatinine kinase, lipase, triglycerides, urea or bun, uric acid, ast, sodium, chloride, potassium, chol, total bilirubin and ammonia) in the different groups (cows experiencing downer cow syndrome, dystocia, retained placenta, vaginal prolapse and abortion) and different concentrations of serum metabolites, and the interaction (reproductive condition × cow breed) is indicated in the results. data were analysed statistically in statistical analysis software (version 20) using the analysis of variances techniques (anova). the results for serum metabolites concentrations were expressed as means ± se. significance levels for all the tests were set at p < 0.05. ethical considerations the study was approved by the animal ethics committee at the north-west university (nwu-00409-18-s5). results this study aimed to examine the concentrations of different biochemical compounds in different breeds of cows with reproductive conditions. the data in this section also shows the distribution of affected cows between ages and parities of affected cows. this study showed significant (p < 0.05) variations regarding the reproductive condition × cow breed interactions seen in concentrations of urea or bun, calcium, ast, total bilirubin, lipase, potassium and total protein (table 1). additionally, the serum concentrations of triglyceride, creatinine kinase, sodium, chloride and total protein did not show a significant difference in association to the conditions (p > 0.05), as indicated in table 1. table 1: p-values obtained after statistical analysis of variance between average serum metabolites concentrations in cows affected by reproductive condition without the breed distinctions and within different breeds. the level of significance within a particular reproductive condition and breed is further elaborated in subsequent tables. in this section the tables indicate the mean variations of different serum metabolites in cows presented with reproductive conditions within a specific breed showing significant differences. table 2 shows significantly high serum mean concentrations of calcium in mixed breed cows which experienced downer cow syndrome (25.25 ± 8.47 mmol/l) and dystocia (85.50 ± 8.46 mmol/l). significant difference in calcium concentrations was not observed in other reproductive conditions. table 2: mean serum calcium (ca) concentrations in different cow breeds experiencing reproductive conditions. the table shows higher mean concentrations of urea in aborting bonsmara cows (18.5 ± 3.04 mmol/l), charolaise cows with downer cow syndrome (10.35 ± 3.04 mmol/l) and afrikaner cows with vaginal prolapses (14.7 ± 2.15 mmol/l), while low mean urea (1.45 ± 3.04 mmol/l) concentrations were seen in brahman cows with dystocia (table 4). table 3: mean serum magnesium concentrations in cows presenting abortion, downer cow syndrome and dystocia in different breeds. table 4: mean serum urea or blood urea nitrogen concentration in different cow breeds experiencing reproductive conditions. in aborting cows, chol mean concentrations were low in afrikaner, bonsmara, brahman, drakensberger and nguni cows (table 5). moreover, cows presenting with downer cow syndrome showed low chol concentrations in afrikaner, brahman and mixed breed cows, as seen in table 5. low chol mean concentrations were also noted in retained placenta and vaginal prolapse in afrikaner cows, as indicated in table 5. other findings revealed that brahman, afrikaner, mixed breed and nguni cows with dystocia had significantly low chol mean concentrations, as shown in table 5. table 5: mean serum cholesterol concentrations in cows experiencing reproductive conditions in different breeds. the above graphical representation indicated abortion (46.15%), retained placenta (44.1%), dystocia (41.18%) and vaginal prolapse (43.5%) cases of cows in the first parities (figure 1), whereas downer cow syndrome (46.15%) and vaginal prolapses (42.10%) mostly occurred in the second parity cows (figure 1). figure 1: parity distribution of cows affected by reproductive conditions parities. the histogram above shows that approximately 53.9% of downer cow syndrome and 60% of abortion cases were seen in cows of ages 3–4 years (figure 2). amongst other conditions, retained placenta (60%), dystocia (60%) and vaginal prolapses (41.2%) occurred in cows of ages 5–6 years. additionally, the frequencies in 7–10 years old cows were very minimal showing low distribution amongst the occurring reproductive conditions (figure 2). figure 2: age association with the occurrence of reproductive conditions in cows. discussion poor reproductive capacity in cow herds has negative implications on farm profitability as financial losses occur through high treatment and replacement costs, which consequently decreases farm income aptitudes (gädicke et al. 2010). constant herd health assessment can help reduce the impact of reproductive losses by minimising the costs of veterinary services. this study aimed to determine differences in the mean concentrations of serum metabolites in cows affected by reproductive conditions. in the current study, concentrations of calcium, urea or bun, ast, total protein, total bilirubin, potassium and lipase significantly (p ≤ 0.05) varied in the breed and reproductive condition interactions (table 1). additionally, no significant differences were seen in concentrations of triglyceride, creatinine kinase, sodium and chloride and total protein did not show significant difference in relation to the incidences of reproductive conditions (p > 0.05). in our study the mean concentrations of calcium indicated significantly high concentrations in cases of both downer cow syndrome (25.25 ± 8.47 mmol/l) and dystocia (85.50 ± 8.46 mmol/l) in mixed breed cows. on the contrary, the incidence of downer cow syndrome has been linked with low calcium levels (benzaquen et al. 2015:186). however, such associations were not seen in this study, because the concentrations of calcium in most of the cows were within the normal ranges (2.0–2.8 mmol/l). similar observations were reported by mcdougall (2001:60–67), where no relationship was established between low blood calcium, reproductive performance and dystocia. high levels of calcium in downer cow syndrome may be interpreted as a result of hormonal imbalance, for instance excessive secretion of parathyroid hormone (pth) causing excessive bone resorption (balamurugan et al. 2017:694), which could explain the increased calcium level in downer cows observed in this study. in downer cow syndrome, low mean concentration of calcium was observed in brahman (1.98 ± 9.76 mmol/l) and charolaise (0.78 ± 11.95 mmol/l) breed cows; however, no significant differences were indicated in the current study. this may be because of a lack of homogeneity in the number of cows within the different breeds, because low calcium is a known indicator for downer cow syndrome (benzaquen et al. 2015:186). a significantly different magnesium concentration below normal was seen in aborting afrikaner cows in this experiment. similar observations were reported by musa, lanyasunya & mukisira (2016:117), indicating a magnesium deficiency in aborting cows. the results also indicated magnesium concentrations with no significant differences (p > 0.05) in aborting cows of brahman and nguni breeds. magnesium mean concentrations in downer cow syndrome and dystocia were significantly high in mixed breed cows. these results agree with those recently reported by adams, ishler & moore (2017:96), indicating that a rise in blood magnesium levels is usually observed in downer cow syndrome because of the decrease in calcium levels. moreover, sufficient calcium levels are required to prevent dystocia and downer cow syndrome, because of its functional characteristics in muscle contraction and relation (goff 2008:45). the present study also showed significantly higher urea mean concentrations in aborting (18.5 ± 3.04 mmol/l) bonsmara cows and downer cow syndrome (10.35 ± 3.04 mmol/l) charolaise cows. our findings agree with previous works reported, showing that high urea concentrations may lead to abortions in cows (rhoads et al. 2009:1986). other observed results indicated low urea levels in dystocia (1.45 ± 3.04 mmol/l) bonsmara cows. cholesterol mean concentrations of aborting cows were significantly low in different breeds experimented in the present study. negative energy balance, poor body condition and low chol concentrations are usually indicators for postpartum health illnesses and poor nutrition status in cows (sepúlveda-varas et al. 2015:e0122317), which could explain the low level of chol in aborting cows. in downer cow syndrome cows, significantly low chol concentrations were noted in afrikaner (2.83 ± 1.12 mmol/l), brahman (2.17 ± 1.12 mmol/l) and mixed breed cows (6.50 ± 0.97 mmol/l), as seen in table 5. civelek et al. (2011:341) reported similar results. these results imply that when calcium levels are decreased, the concentration of chol is likely to drop. significantly low chol mean concentrations were also noted in retained placenta (3.18 ± 0.61 mmol/l) and vaginal prolapse (2.37 ± 0.97 mmol/l) in afrikaner cows, as indicated in table 5. the current results are in agreement with the study of civelek et al. (2011:341) which reported lowered chol level in cows-retained placenta. the results of this study showed that the incidences of dystocia were most frequent in cows of ages 5–6 years and in the first parity (figures 1 and 2). similar data indicating a direct relationship between dystocia, age and parity (more frequent in primiparous than high parity cows) have previously been presented (abera 2017:1–9; mcdougall 2001:60–67). the occurrence of dystocia have dire economic implications on production, as cows become more susceptible to post-partum illnesses, most likely to die soon after parturition and encounter increased calve mortalities (de amicis et al. 2018:104). according to mee (2008:933–101), the cases of downer cow syndrome are mostly seen in older cows. however, in this study, the cases of downer cow syndrome were more prominent in cows of ages 3–4 than in those of 7–10 years (figure 2). similar results have been described, showing that age has an impact on the incidence of metabolic disorders as the level of susceptibility may vary according to age, and moreover, heifers are more susceptible to downer cow syndrome (kutanaee et al. 2014:367). the cases of retained placenta were observed (60%) in cows of ages 5–6 years in their first (44.1%) parity (figures 1 and 2). the results agree with those of sharma et al. (2017:3103) which indicated (40.22%) incidence in primiparous cows. previous studies have also stated that incidences of retained placenta in most cases increase because of downer cow syndrome and dystocia (mcdougall 2001:60–67). this study revealed that vaginal prolapses were most frequent in cows of ages 5–6 years in the first parity (44.1%), as seen in figures 1 and 2. similarly, prolapse of the vagina has been reported to be influenced by cow age and parity (sarma, das & nath 2017:1067). these results therefore suggest that metabolic profile testing is a good indicator for bovine reproductive conditions. conclusion the differences in serum metabolites concentrations were seen in cows of different breeds with reproductive conditions. particularly, variations of serum calcium, magnesium, urea or bun, ast and chol concentrations was observed in cows affected by dystocia, retained placenta, downer cow syndrome, vaginal prolapse and abortion. this study also showed that nguni and bonsmara breeds were the least affected by the reproductive conditions, suggesting that they are the most ideal to use in natural pasture rearing of cows. it is also indicated that age and parity could influence the change in blood chemistry as the cases of reproductive conditions were noted in cows of varying age groups and parities. the information obtained in this study serves as a useful tool for farmers as it can help them make informed decisions in determining the best animals to use for production. acknowledgements the authors express their gratitude to all large animal clinic staff at the north-west university, particularly mr v. mjekula and mr m.f. ijane. the farmers from communal areas around mafikeng are also acknowledged. the north-west university postgraduate bursary and the national research foundation (nrf) for their financial support are also acknowledged. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.m. collected and analysed samples and m.m. conceived and supervised the project. k.m. and m.m. conducted data analysis, interpreted the results and wrote the manuscript. funding information this study was made possible by north-west university postgraduate bursary and the national research foundation. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references abera, d., 2017, ‘management of dystocia cases in the cattle: a review’, journal of reproduction and infertility 8(1), 1–9. https://doi.org/10.5829/idosi.jri.2017.01.09 abuom, t.o., njenga, m.j., wabacha, j.k., tsuma, v.t. & gitau., g.k., 2012, ‘incidence and risk factors of 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zaborski, d., grzesiak, w., szatkowsk, i., dybus, a., muszynska, m. & jedrzejczak, m., 2009, ‘factors affecting dystocia in cattle’, in forsyth (ed.), international handbook of tourism economics, vol. 44, pp. 540–551, edward elgar, england. thekisoe_339-342.indd bovine theileriosis syndromes, which are tick-borne diseases, include east coast fever (ecf) caused by theileria parva, tropical theileriosis caused by theileria annulata and cerebral theileriosis “ormilo” caused by theileria taurotragi (lynen, loomu, mlinga, kessy & nalitolela 2000). in tanzania, bovine trypanosomosis, mainly caused by trypanosoma congolense and t. vivax, ranks second only to ecf in terms of economic impact on the livestock industry (mugittu, silayo, majiwa, kimbita, mutayoba & maselle 2001). loop-mediated isothermal amplification (lamp) is a novel gene amplification method that is rapid, simple and highly sensitive (notomi, okayama, masubuchi, yonekawa, watanabe, amino & hase 2000). lamp reaction can be conducted in 60 minutes using a simple laboratory heat block or water bath for incubation and results can be visualized by either gel electrophoresis or by naked eye (white turbidity in reaction tubes) (mori, nagamine, tomita & notomi 2001). in this study, we evaluated 339 onderstepoort journal of veterinary research, 74:339–342 (2007) research communication preliminary application and evaluation of loop-mediated isothermal amplification (lamp) for detection of bovine theileriosis and trypanosomosis in tanzania o.m.m. thekisoe1, j.d. omolo2, e.s. swai3, k. hayashida1, 4, j. zhang1, c. sugimoto4 and n. inoue1* abstract thekisoe, o.m.m., omolo, j.d., swai, e.s., hayashida, k., zhang, j., sugimoto, c. & inoue, n. 2007. preliminary application and evaluation of loop-mediated isothermal amplification (lamp) for detection of bovine theileriosis and trypanosomosis in tanzania. onderstepoort journal of veterinary research, 74:339–342 the sensitivity of lamp, pcr and microscopy to detect theileria spp. and trypanosoma congolense in field-derived bovine blood samples from tanzania was evaluated and compared. no parasites were detected by microscopy. furthermore, no bovine theileria spp. were detected by lamp and pcr from all the 24 samples collected from arusha. four and one out of 24 samples were positive for theileria congolense infection by lamp and pcr respectively while, 18 and nine out of 40 samples from dar es salaam were positive by lamp and pcr for theileria spp. infection, respectively. although all samples from dar es salaam were negative for trypanosoma congolense infections by pcr, 12 out of 40 samples were lamp positive. whilst pcr is an established gene amplification method for the detection of theileria and trypanosome parasites, this study introduces lamp as an alternative molecular diagnostic tool that could be used in large-scale epidemiological surveys. keywords: bovine theileria spp., lamp, microscopy, pcr, tanzania trypanosoma congolense * author to whom correspondence is to be directed. e-mail: ircpmi@obihiro.ac.jp 1 national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, obihiro, hokkaido, 080-8555, japan 2 ministry of livestock development, department of veterinary services, box 456, dodoma, tanzania 3 veterinary investigation centre, box 1068, arusha, tanzania 4 research center for zoonosis control, hokkaido university, sapporo, hokkaido, 011-0818, japan accepted for publication 13 march 2007—editor 340 loop-mediated isothermal amplifi cation for detection of bovine theileriosis and trypanosomosis in tanzania and compared the sensitivity of lamp, pcr and micro scopy for the detection of bovine theileriosis and trypanosomosis (t. congolense) from cattle samples collected from tanzania’s arusha and dar es salaam farms. forty blood samples blotted on filter papers (fta® card, whatman, uk) and thin blood smears were prepared from blood collected from cattle of unknown age on ten farms near dar es salaam and 24 samples from cattle on seven farms near arusha (animal age ranged between 4 months and 8 years). genomic dna used in lamp primer specificity and sensitivity tests was extracted according to sam brook & russel (2001). the blood samples dried on filter papers were purified according to the manufacturer’s instructions and then used as a dna template. the lamp primer sets used in the current study were designed from t. parva hsp70 (accession no. u40190) and t. congolense 18s rrna (accession no. u22315) using primer explorer v2 (fujitsu, japan), with the following sequences: lamp primer set for t. parva hsp70 fip: 5’-tgg gtt acg ggc ttc ttg gtt tcc tac gtc gca ttc act gac-3’; bip: 5’-att ttc gac gcc aag agg ctc aaa tgg cca gtg ctt cat gtc-3’; f3: 5’-gga aac agg aca acg ccg-3’, and b3: 5’-ccg ttt ggt ccg ttg gta a-3’. lamp primer set for t. congolense 18s rrna fip: 5’-gcg cat gcg tcg gtg tta ttt tcg cgt gtg tgt tca tgt ca-3’; bip: 5’-act ctc ccc cca aaa tgg ttg tcc aag cac gca aat tca cat-3’; f3: 5’-tgt gtg ttt gtc gtg gaa gc-3’, and b3: 5’-att cgt gac cgc gtc aaa-3’. both lamp and polymerase chain reaction (pcr) (f3 and b3 lamp primers were used as the pcr primer pair) were conducted as described by kuboki, inoue, sakurai, di cello, grab, suzuki, sugimoto & igarashi (2003). for pcr, dna was extracted from filter papers using qiagen kit according to the manufacture’s instructions (qiaamp dna mini kit, qiagen®; maryland, usa). lamp primer sets designed from hsp70 of t. parva amplifies the major bovine theileria spp. except t. orientalis dna (fig. 1a) and are highly sensitive for the detection of theileria dna (fig. 1b). these primers can therefore be used as universal primers for gene amplification of bovine theileria spp. dna. lamp primers for t. congolense are also highly sensitive and specific (o.m.m. thekisoe & n. inoue, unpublished data 2006). during the collection of the samples in arusha, no ticks were observed on the sampled animals which probably explains the absence of theileria spp. infections by diagnostic methods used in this study (table 1). there were no antibodies detected by the indirect fluorescent antibody test (ifat) against thei leria spp. in seven serum samples collected from animals in arusha (data not shown). in contrast, the fact that theileria spp. were detected from the dar es salaam samples (table 1) is not surprising as bovine theileria infections are common in this region (msami 2001; swai, french, karimuribo, fitzpatrick, bryant, brown & ogden 2005). it has also been reported that animals which survive t. parva infections remain reservoirs of the parasite (swai et al. 2005). our study revealed a low prevalence of t. congolense infections in arusha (table 1). however, antibodies against t. congolese were detected using enzyme-linked immunosorbent assay elisa (bannai, sakurai, inoue, sugimoto & igarashi 2003) from five out of seven serum samples collected from animals with a previous history of trypanosome infections in arusha (data not shown). there was a significantly higher prevalence of t. congolense infections detected by lamp and pcr in dar es salaam as compared to arusha (table 1). table 1 detection performance of lamp, pcr and thin blood smears method arusha dar es salaam theileria spp. +* (%) t. congolense +* (%) theileria spp. +* (%) t. congolense +* (%) lamp pcr tbs** 0/24 (0.0) 0/24 (0.0) 0/24 (0.0) 4/24 (16.6) 1/24 (4.1) 0/24 (0.0) 18/40 (45) 9/40 (22.5) 0/40 (0.0) 12/40 (30.0) 0/40 (0.0) 0/40 (0.0) +* number of positively detected samples ** thin blood smear 341 o.m.m. thekisoe et al. this study further confirms the presence of t. congolense in sampled areas of tanzania as previously reported (malele, craske, knight, ferris, njiru, hamilton, lehane, lehane & gibson 2003), which is mainly due to wide distribution of different tsetse fly species infesting about 60 % of the country (mugittu et al. 2001). no parasites were detected by microscopic examination of giemsa-stained thin blood smears from all the sampled cattle in both arusha and dar es salaam. in conclusion, lamp showed higher detection sensitivity than pcr and microscopy, as has previously been reported (kuboki et al. 2003). furthermore, lamp can easily amplify dna templates from filter papers since bst dna polymerase used in lamp is not affected by blood-derived tissue components that can inactivate taq dna polymerase used in pcr (grab, lonsdale-eccles & inoue 2005). hence, due to its rapidity, simplicity and high sensitivity, it is suggested that lamp can be a better molecular diagnostic tool for application in large-scale epidemiological studies of theileriosis and trypanosomosis. acknowledgements we are grateful to the farmers in arusha and dar es salaam for their co-operation. we also thank the director of veterinary service, tanzania, for permission to publish this work. this study was made possible by program for promotion of basic research activities for innovative biosciences, probrain, japan, to ni, and in part by a jsps grant-in-aid for scientific research to ni. references bannai, h., sakurai, t., inoue, n., sugimoto, c. & igarashi, i. 2003. cloning and expression of mitochondrial heat shock protein 70 of trypanosoma congolense and potential use as a diagnostic antigen. clinical and diagnostic laboratory immunology, 10:926–933. grab, d.j., lonsdale-eccles, j. & inoue, n. 2005. lamp for tadpoles. nature methods, 2:635. kuboki, n., inoue, n., sakurai, t., di cello, f., grab, d.j., suzuki, h., sugimoto, c. & igarashi, i. 2003. loop-mediated isothermal amplification for detection of african trypanosomes. journal of clinical microbiology, 41: 5517–5524. fig. 1 (a) specificity tests of lamp and pcr (f3, b3) primers for detection of bovine theileria spp. dna. lanes m: 100 bp maker; 1: t. parva; 2: t. annulata; 3: t. taurotragi; 4: t. mutans; 5: t. orientalis; 6: b. bovis; 7: t. brucei; 8: bovine blood. (b) sensitivity of lamp and pcr (f3 and b3) primers for detection of theileria parva dna quantified with a spectrophotometer (smart spec 3000, usa) and serially diluted from 100 ng to 1 fg m 1 2 3 4 5 6 7 8 pcr 181 bp lamp a pcr 181 bp lamp m 100 ng 10 ng 1 ng 100 pg 10 pg 1 pg 100 fg 10 fg 1 fg b 342 loop-mediated isothermal amplifi cation for detection of bovine theileriosis and trypanosomosis in tanzania lynen, g., loomu, p.m., mlinga, n., kessy, v.j. & nalitolela, s. 2000. ormilo in indigenous cattle population of the pastoral community. proceedings of the 17th tanzania veterinary association scientific conference, december 2000, arusha international conference centre, arusha, tanzania. malele, i., craske, l., knight, c., ferris, v., njiru, z., hamilton, p., lehane, s., lehane, m. & gibson, w. 2003. the use of specific and generic primers to identify trypanosome infections of wild tsetse flies in tanzania by pcr. infection, genetics and evolution, 3:271–279. mori, y., nagamine, k., tomita, n. & notomi, t. 2001. detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. biochemical and biophysical research communications, 289:150–154. mugittu, k.n., silayo, r.s., majiwa, p.a.o., kimbita, e. k., mutayoba, b.m. & maselle, r. 2001. application of pcr and dna probes in the characterisation of trypanosomes in the blood of cattle in farms in morogoro, tanzania. veterinary parasitology, 94:105–112. msami, h.m. 2001. an epidemic of east coast fever on a dairy farm in eastern tanzania. preventive veterinary medicine, 49:55–60. notomi, t., okayama, h., masubuchi, h., yonekawa, t., watanabe, k., amino, n. & hase, t. 2000. loop-mediated isothermal amplification of dna. nucleic acids research, 28:e63. sambrook, j. & russell, d.w. 2001. preparation and analysis of eukaryotic genomic dna, in molecular cloning, 3rd ed., edited by j. sambrook & d.w. russell, cold spring harbor, new york: cold spring harbor laboratory press. swai, e.s., french, n.p., karimuribo, e.d., fitzpatrick, j.l., bryant, m.j., brown, p.e. & ogden, n.h. 2005. spatial and management factors associated with exposure of smallholder dairy cattle in tanzania to tick-borne pathogens. international journal for parasitology, 35:1085–1096. abstract introduction materials and methods results discussion acknowledgements references about the author(s) emily p. lane department of research and scientific services, national zoological gardens of south africa, south africa helene brettschneider department of research and scientific services, national zoological gardens of south africa, south africa peter caldwell old chapel veterinary clinic, pretoria, south africa almero oosthuizen department of research and scientific services, national zoological gardens of south africa, south africa desiré l. dalton department of research and scientific services, national zoological gardens of south africa, south africa department of genetics, university of the free state, south africa liza du plessis idexx laboratories (pty) ltd., onderstepoort, south africa johan steyl department of paraclinical science, university of pretoria, south africa antoinette kotze department of research and scientific services, national zoological gardens of south africa, south africa department of genetics, university of the free state, south africa citation lane, e.p., brettschneider, h., caldwell, p., oosthuizen, a., dalton, d.l., du plessis, l. et al., 2016, ‘feline panleukopaenia virus in captive non-domestic felids in south africa’, onderstepoort journal of veterinary research 83(1), a1099. http://dx.doi.org/10.4102/ojvr.v83i1.1099 original research feline panleukopaenia virus in captive non-domestic felids in south africa emily p. lane, helene brettschneider, peter caldwell, almero oosthuizen, desiré l. dalton, liza du plessis, johan steyl, antoinette kotze received: 21 oct. 2015; accepted: 09 dec. 2015; published: 09 june 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract an outbreak of feline panleukopaenia virus (fplv) infection was diagnosed by pathology, electron microscopy and polymerase chain reaction (pcr) in vaccinated captive-bred subadult cheetahs in south africa. subsequent to this disease outbreak, 12 cases of fplv diagnosed on histology were confirmed by pcr in captive african black-footed cat, caracal, cheetah, lion, ocelot and serval. phylogenetic analyses of the viral capsid protein gene on pcr-positive samples, vaccine and national center for biotechnology information (ncbi) reference strains identified a previously unknown strain of fplv, present since at least 2006, that differs from both the inactivated and the modified live vaccine strains. a previously described south african strain from domestic cats and cheetahs was identified in a serval. surveys of fplv strains in south african felids are needed to determine the geographical and host species distribution of this virus. since non-domestic species may be reservoirs of parvoviruses, and since these viruses readily change host specificity, the risks of fplv transmission between captive-bred and free-ranging carnivores and domestic cats and dogs warrant further research. introduction feline panleukopaenia virus (fplv) infection, the first viral disease identified in cats, is one of the most common viral diseases of domestic cats (felis domesticus). fplvs are classified under the family parvoviridae and are related to mink enteritis virus (mev) and canine parvovirus (cpv) (nakamura et al. 2001; steinel et al. 2000; truyen et al. 1996). it is highly contagious, affects both domestic and non-domestic felids and is transmitted by direct and indirect contact with infected animals and their secretions, especially faeces. despite aggressive treatment it can be fatal in less than 24 hours. like all parvoviruses, fplv is extremely resistant and can survive for longer than one year in suitable environments (nakamura et al. 2001). fplv-like viruses show a global distribution, infecting a wide range of felids including the cheetah (acinonyx jubatus) (van vuuren et al. 2000), european wild cat (felis sylvestris sylvestris) (wasieri et al. 2009), lion (panthera leo) (chandran et al. 1993), african wild cat (felis lybica), ocelot (leopardus pardalis), puma (puma concolor), siberian tiger (panthera tigris altaica), and spotted cat (leopardus tigrinus) (filoni et al. 2006). in addition, the virus has been reported in the mink (neovison vison), arctic fox (vulpes lagopus) and raccoon (procyon lotor), and is genetically very similar but distinct from cpv-like viruses from dogs (canis familiaris) and raccoons (filoni et al. 2006; parrish 1995; truyen et al. 1996). cpv is an ancient canid pathogen that re-emerged as a domestic dog pandemic in the late 1970s in europe, as a variant of fplv or a closely-related parvovirus in non-domestic carnivores, most likely through a viral mutation that facilitated previously blocked entry to cells through the transferrin receptor type 1 (allison et al. 2013; belyi, levine & skalka 2010; nakamura et al. 2001; steinel et al. 2001). although the original strain (cpv type 2) did not infect felids, cpv2s subsequently mutated into additional strains (cpv types 2a, 2b and 2c) that are reported to induce disease in cats (hoelzer et al. 2008; parrish et al. 1991; steinel et al. 2000). parvoviruses in non-domestic carnivores may represent a reservoir that periodically spills over into domestic carnivore populations or provide alternate evolutionary pathways that facilitate pandemics (allison et al. 2012, 2013, 2014; belyi et al. 2010). both cpv and fplv typically infect multiple hosts and passage through different hosts may favour selected viral capsid mutations that facilitate changed host specificity (allison et al. 2014). molecular confirmation of fplv in a diseased domestic cat and two diseased captive born cheetahs in 2000 challenged the perception that fplv had disappeared from domestic cats in south africa due to the increased incidence of cpv strains type 2a and b infecting cats in the 1980–1990s (van vuuren et al. 2000). while parvoviral infections have in the past been diagnosed clinically, by electron microscopy and histopathological examination in domestic and non-domestic felids, the advent of molecular diagnostic techniques provides a new opportunity to evaluate the genetic variability of fplv in affected animals. this article reports the clinical features and pathology of an outbreak of fplv in vaccinated captive cheetahs, as well as preliminary findings on the molecular epidemiology of fplv in captive non-domestic felids in south africa. materials and methods sample collection date, type and locality, host species, age, sex and vaccination status as well as pathological disease diagnosis and molecular analysis results are displayed in table 1. in october and november 2012, eleven 6–8-month-old captive-bred cheetah cubs from one cheetah breeding institution were presented to the old chapel veterinary clinic with acute inappetance, lethargy and vomiting. five cubs died and were submitted to the national zoological gardens of south africa (nzg) for necropsy examination. tissue samples collected during necropsy examinations were preserved in 10% buffered formalin for histopathology, processed routinely and stained with haematoxylin-eosin. dna extraction and molecular characterisation were performed on fresh intestine from these cubs (cases 1–5). table 1: sample collection date, type and locality; species, sex, age and vaccination history; diagnosis and molecular analysis of samples tested for of feline panleukopaenia virus (fplv). subsequent to the outbreak in cheetahs, animal carcasses, a rectal swab, formalin-fixed tissues or formalin-fixed paraffin-embedded tissues (ffpe) from various single or multiple cases in captive felids were submitted to the nzg for pathological examination and/or molecular diagnostic testing for fplv infection by the owners, veterinarians and pathologists throughout south africa (cases 6–20). submitted cases of suspected or confirmed fplv based on clinical signs and typical macroscopic and histological findings, or diseases presenting with similar signs, included two caracals (caracal caracal), seven cheetahs, three lions, one ocelot and two servals (leptailurus serval). clinical signs and pathological findings were not available from many of these cases. before the 2012 cheetah outbreak molecular diagnostic confirmation of suspected fplv cases was not readily available in south africa. to investigate prior suspected or confirmed cases of fplv molecular diagnostic testing was performed on archived tissue samples from felids in the nzg biobank (cases a–g), including both suspected and histologically typical fplv cases. cases were from gauteng and north west provinces and included one african black-footed cat (felis nigripes), a domestic cat, three cheetahs and two lions. samples from a puma, a leopard (panthera pardus) and an ocelot in which clinical and histological evidence of fplv was absent (cases i–iii) were included as negative control cases. dna was also extracted from current fellocell® and fel-o-vax® (zoetis; boehringer ingelheim) vaccines. all the samples are archived at the nzg biobank. dna from all the fresh, frozen samples and vaccines were extracted using the zr genomic dnatm tissue miniprep kit (zymo research). dna from ffpe samples were extracted using the masterpure™ complete dna & rna purification kit (epicentre) following the manufacturers specifications. molecular analysis of the parvovirus genome from all the isolates was conducted by amplification of a ~1300 base pair (bp) region of the viral capsid protein (vp2) gene (wasieri et al. 2009). this large fragment was amplified in four polymerase chain reactions (pcrs), in order to reduce the fragment size for ease of amplification especially for ffpe samples. pcr cycling conditions were as follows: initial denaturation at 94 °c for 5 min, five cycles of denaturation at 94 °c for 30 s, annealing at 52 °c for 50 s and extension at 72 °c for 1 min, 10 cycles of denaturation at 94 °c for 30 s, annealing at 50 °c for 50 s and extension at 72 °c for 1 min and 15 cycles of denaturation at 94 °c for 30 s, annealing at 48 °c for 50 s and extension at 72 °c for 60 s. a final extension step of 72 °c for 5 min concluded the cycling. pcr was conducted in a dna-free hood to ensure a lack of contamination, confirmed by the inclusion of a dna-negative control (double distilled water) for each gene region. all the positive amplicons, as determined by gel electrophoresis, were purified using exonuclease i and alkaline phosphatase (thermofisher) treatment, cycle sequenced using the bigdye v3.1 (applied biosystems) chemistry and run on a 3130 genetic analyser (applied biosystems). the resulting sequences were analysed and aligned using the clustalx (thompson et al. 1997) function incorporated in mega5 (tamura et al. 2011) and phylogenetically analysed using distance (neighbor-joining, nj) in mega5 and bayesian methods (ba) in mrbayes v3.1 (huelsenbeck & ronquist 2001). the best-fit model of sequence evolution was selected under the akaike information criterion (aic) in jmodeltest (posada 2008). phylogenetic analyses were performed on a 51-taxon vp2 gene dataset of 29 pcr-positive case samples, two vaccine strains and 30 national center for biotechnology information (ncbi) parvovirus reference strains. results from nj (10 000 bootstrap replicates) and ba (1 million generations) are presented in figure 4. the hky+i model of sequence evolution was chosen as the best-fit model in jmodeltest under the aic. all the sequences generated during this study were submitted to the ncbi website under accession numbers (kp033232–kp033250). results clinical signs were described in detail only in the initial cheetah outbreak (cases 1–5) and included pyrexia, haemorrhagic diathesis (figure 1), hypersalivation, dehydration and inconsistent variably haemorrhagic diarrhoea. some cubs showed polypnoea and marked aggression, thought to be due to pain. five cubs died, despite aggressive treatment with intravenous and subcutaneous fluid replacement, anti-emetics, antibiotics, proton pump inhibitors, gastric cytoprotective agents, immune and appetite stimulants, multivitamins and a single dose of prednisolone (1%, 1 mg/kg). oral electrolytes and small amounts of highly digestible cat food were syringe-fed to the cubs. figure 1: feline panleukopaenia virus infection; cheetah, case 8. on necropsy examination, the cheetah cubs showed similar lesions including variable dehydration, mild acute diffuse tissue congestion, and moderate acute intestinal serosal congestion and haemorrhage (figure 2). variable numbers of petechiae and ecchymoses were present in other organs. intestinal contents were scant, watery and bile stained. the intestinal mucosa was covered in a pale yellow-tan pseudomembrane. figure 2: feline panleukopaenia virus infection; abdomen, cheetah, case 9. severe intestinal serosal congestion and petechiation (*) of the intestinal loops adjacent to the kidney (k). histologically, extensive necrosis of the intestinal epithelium was accompanied by villous collapse, marked hyperplasia of scanty remaining epithelium, and a thick coat of fibrin, necrotic cellular debris and myriad bacteria on the mucosal surface (figure 3). in addition, bone marrow pancytopenia and marked splenic, thymic, peyer’s patch and lymph node lymphoid depletion were present. colonies of bacteria of varied morphology occurred in the capillaries of many tissues associated with variable tissue necrosis. a heavy growth of escherichia coli was obtained from the intestine from case 1 and normal intestinal bacterial flora from case 3. on negative contrast electron microscopy intestinal contents contained small numbers of parvovirus particles (case 1). figure 3: feline panleukopaenia virus infection; intestine, cheetah, case 9. loss of intestinal crypt epithelial cells (arrows) is accompanied by mural haemorrhage (*) and bacterial colonies on the luminal surface (arrowheads). he. the pathological lesions were consistent with fplv infection with secondary bacterial enteritis and septicaemia. additional pathological findings included moderate diffuse acute interstitial pneumonia with moderate multifocal acute pulmonary oedema (cases 1, 3 & 4); multifocal acute moderate necrohemorrhagic bacterial gastritis as well as lingual and gastric candidiasis, mild serosanguinous hydropericardium and hydrothorax and acute moderate segmental renal tubular necrosis (case 2); oesophageal and gastric candidiasis and acute mild multifocal pancreatic necrosis (case 3); and mild acute multifocal necrotising aspiration pneumonia (case 5). all the cubs were in good body condition at the time of death and showed mild to moderate diffuse adrenocortical hyperplasia. phylogenetic analyses of the vp2 gene region of both vaccine strains and viruses from all cases were consistent with fplv (99.99% similarity). all the samples from this study clustered separately from cpv (98% similarity) and clustered in two clades. most of the positive cases (clade 1) clustered closely (99.94% – 100%) with reference samples (eu659111.1, jn867594.1, ab262659.1) from carnivores in japan and the united states of america (usa), and were similar to the fell-o-vax® vaccine. clade 2 is represented in the current dataset by one sample (case 10), which clustered with previously described south african strains of fplv isolated from a domestic cat (1999) and two cheetahs (2000) as well as an fplv strain isolated from a wildcat and a mink in the usa (1995). this strain is 99.99% similar to the fellocell® vaccine (figure 4). figure 4: feline panleukopaenia virus infection; neighbour-joining (nj) tree of dna extracted from south african felid samples, vaccine strains (*) and reference samples. discussion clinical features, pathology, electron microscopy and molecular diagnostic tests confirmed the presence of a previously unreported strain of fplv in the cheetah cubs in the initial outbreak (cases 1–4). this strain was also detected in archived samples from a lion that died in 2006 (case a) and an african black-footed cat that died in 2007 (case b), as well as in samples submitted from other captive felids that died throughout the country after november 2012, including cheetahs (cases 7–9, 16–19), a caracal (cases 15), a lion (case 14), an ocelot (case 12) and a serval (case 6). the testing of archived samples confirms that the strain from clade 1 was present in felids in at least north west province before the initial cheetah outbreak and clade 1 virus subsequently caused disease in unvaccinated felids in gauteng and north west. clade 1 strain apparently co-exists with the previously described strain (van vuuren et al. 2000) from clade 2 at least in the serval (case 10). although clade 1 clustered with samples from japan and the usa, the absence of information on the distribution and phylogeny of fplv in domestic and non-domestic felids in south africa means that this should be interpreted with caution. the testing of more cases of fplv in domestic and non-domestic felids (both captive and free-ranging) is needed to determine the geographical and host species distribution of the virus in south africa and the relationship between these strains and those described from outside the country. this research would be greatly assisted by the provision of more detailed information on clinical features, histological findings and vaccination status of animals from which samples were submitted for pathological or molecular diagnostic samples. cases were, as expected, equally prevalent in male and female animals, and were seen only in animals under eight months of age. too few cases were tested to evaluate seasonal patterns of fplv disease incidence. positive cases were found in animals from five of south africa’s nine provinces; however, more extensive epidemiological surveys are needed to determine whether a geographical pattern is present, or if this was due to cheetah numbers differing between the provinces, or to clinicians and pathologists in these provinces submitting more samples than those from the other provinces. should there be variation in the geographical distribution of the virus, this could inform provincial and national regulations regarding the movement of captive carnivores. strict hygiene measures should in any case be implemented when animal attendants and visitors travel between national and international captive felid institutions. the survival of six of the 11 cheetah cubs in the initial outbreak was attributed to a lower infection load, intense monitoring and early aggressive treatment once the diagnosis had been made. stress due to weaning and translocation may have been a predisposing factor in the variable susceptibility of these cubs to disease. however, fplv can cause a spectrum of disease and the severity and clinical signs of the disease may be altered by the pathogenicity of variable secondary bacterial enteric infections (parrish 1995). prior to the initial outbreak cases of fplv were rarely experienced by the authors in captive carnivores in south africa and molecular diagnostic techniques were not then readily available. the authors are unaware of any reports of the prevalence and strain type of fplv in domestic cats or free-ranging felids in south africa. captive breeding of lion and other carnivores for hunting, ecotourism and conservation has grown significantly in the past decade (lindsey et al. 2012). intensive farming conditions, poor hygiene, a lack of vaccination and the ineffective exclusion of domestic carnivores from enclosures, surroundings and feeding stations may be increasing the incidence of fplv in captive carnivores. the captive breeding of non-domestic carnivores may expose domestic dogs and cats to new variants of the virus, since non-domestic carnivores may be primary reservoirs for parvoviruses, the viruses are readily transmitted between species, and the viruses readily develop viral capsid protein mutations on passage through new hosts (allison et al. 2012, 2013, 2014; belyi et al. 2010). further research is needed into the risks and patterns of fplv transmission between domestic and both captive-bred and free-ranging non-domestic carnivores. the source of the initial outbreak in the cheetahs remains uncertain. however, molecular characterisation of the strain indicated that the disease was not caused by cpv or inadequate inactivation of the inactivated vaccine, nor by unexpected pathogenicity of the live vaccine. although vaccine viability was not tested, the failure of the live vaccine to induce immunity may have played a role in the outbreak. vaccine failure may have been related to the loss of cold chain management during a 2012 transport strike. ante mortem serological tests for fplv antibodies were not available, so the immunity of the affected and non-affected animals could not be determined. apart from the animals that were vaccinated during the transport strike in 2012, all other positive cases occurred in unvaccinated felids. the vaccination of domestic cats prevents disease due to fplv as well as cpv 2b and 2c (chalmers et al. 1999; jack et al. 2014). maternal immunity lasts between 2 and 4.5 months and domestic cats are therefore usually affected at 3–5 months of age. inactivated (killed) vaccines can be used in pregnant queens, kittens under 4 weeks old and debilitated cats (parrish 1995). inactivated vaccines have also been proven to be safe and induce high persistent levels of serum antibodies to fplv in cheetahs (wack et al. 1993). immunisation with modified live vaccines of adult domestic cats is thought to confer lifelong immunity; however, fplv vaccines are not registered for use in other felids and their efficacy against different virus variants, particularly in non-domestic felids, is not well documented. a modified live vaccine induced a significant immune response in tigers but not lions (risi et al. 2012). despite this, vaccine regimens using a combination of inactivated (dams in late pregnancy and cubs at eight and 12 weeks of age) and live vaccines (cubs at 16 weeks of age) reduce cub mortality and have been proven to be largely safe and effective in captive cheetah populations (munson & citino 2005; p. caldwell, personal communication 2015). other cheetah cubs on the same property during and since the 2012 outbreak survived. the clade 1 strain isolated from vaccinated animals was different from the modified live vaccine strain, so disease due to the vaccine strain was ruled out. further research is needed to determine the relationship, if any, between disease in cheetahs, domestic cats and serval caused by clade 2 viral strains. following the 2012 outbreak, control measures employed on the cheetah breeding facility were effective and no further deaths occurred. the enclosures were sprayed several times in the first week using high-pressure delivery systems for parvoclean (kyron), f10 products (health and hygiene [pty] ltd) or swimming pool chlorine (2.67 g/l water). biosecurity measures included staff movement from clean to infected enclosures for feeding and inspections; new and frequently changed cleaning cloths as well as overalls and boots for all staff; hand gels and body sprays of f10 products used when moving between enclosures. positive cases were detected from fresh and frozen tissue, a faecal swab and ffpe intestinal samples from 16 animals clinically and pathologically diagnosed as suffering from fplv or suspected fplv (table 1). three cases without macroscopic and histological evidence of fplv tested negative (cases i–iii). however, eight animals (cases 9, 11, 13, 20, c–f) with histological lesions suggestive of fplv tested negative. intestinal and lymphoid tissue histological lesions are considered pathognomonic for fplv (brown, bake & barker 2007), therefore these may represent false negative test results. unfortunately, immunohistochemical tests for fplv antigen were not available to verify the histological or pcr results. the amount of virus in the intestine may vary temporally, since viral antigen is rarely detectable by immunohistochemistry once clinical signs and intestinal epithelial necrosis are present (brown, bake & barker 2007). nonetheless, fplv dna was detected in many of these clinical cases where intestinal necrosis was present without intestinal viral inclusions. false negatives may also occur when the presence of virus is below the level of detection, when dna is degraded by prolonged storage in either the freezer or in formalin (srinivasan, sedmak & jewel 2002) or through the use of non-buffered formalin as a fixative (gilbert et al. 2007). nonetheless, archived ffpe tissue samples were found in this study to be a valuable test sample. as previously described (errington, jones & sawyer 2014), tissue swabs may be an adequate sample for molecular analysis but since only one swab was tested this should be further evaluated with paired tissue samples. the discordance in the incompletely vaccinated serval diagnosed pathologically with salmonellosis with fplv dna in the intestine could have been caused by typical fplv lesions being obscured by secondary opportunistic bacterial infections or the presence of fplv infection without fplv-induced disease (parrish 1995). in addition, a false positive test result due to inadvertent contamination of the test sample with fplv dna is possible. although infections with viruses of the feline parvovirus subgroup have been reported before in several wild carnivore species, including african felids, this study reveals the first in-depth insight into strain diversity within captive felids in south africa and the first documented cases of fplv infection in the african black-footed cat and caracal. further surveys are needed to determine the geographical and host species distribution as well as the genetic variability of fplv in these species. vaccine failure, inadequate vaccination and stress may be predisposing factors in the susceptibility to disease. retention of the current vaccination programme as well as good hygiene practices at breeding and rearing facilities is recommended for the prevention and control of the disease. acknowledgements we thank the ann van dyk cheetah centre for providing valuable information that helped us understand the epidemiology of this case. in addition to cases handled by the authors, we are grateful to dr katja koeppel of the johannesburg zoo and dr lucia lange of pathcare for submitting samples to the nzg biobank that could be used in this study. the staff of the nzg biobank, pathology section of the department of paraclinical studies, faculty of veterinary science, university of pretoria, the electron microscopy unit of the arc-onderstepoort veterinary institute and molecular diagnostic services provided excellent technical assistance. the costs of the diagnostic tests were covered by core grant allocations of the national zoological gardens of south africa, a national facility of the national research foundation. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions e.p.l. did the necropsy examinations, diagnosed histological lesions of fplv and provided samples for the genetic analysis, and contributed significantly to the writing and editing of the manuscript, h.b. designed and supervised the genetic analysis and contributed significantly to the writing and editing of the manuscript, p.c. submitted affected animals to the nzg for pathology examination and genetic analysis and contributed clinical information to the manuscript, a.o. conducted the genetic analysis and related research, d.l.d. designed and supervised the genetic analysis and edited the manuscript, l.d.p. submitted diagnostic materials to the nzg and edited the manuscript, j.s. submitted diagnostic materials to the nzg and edited the manuscript, and a.k. supervised the project, made sufficient resources available for its completion and edited the manuscript. references allison, a.b., harbison, c.e., pagan, i., stucker, k.m., kaelber, j.t. & brown, j.d. et al., 2012, ‘the role of intermediate hosts in the cross-species transmission and emergence of a pandemic virus’, journal of virology 86(2), 865–872. allison, a.b., kohler, d.j., fox, k.a., brown, j.d., gerhold, r.w. & shearn-bochsler, v.i. et al., 2013, ‘frequent 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discussion conclusion acknowledgements references about the author(s) martin chamai department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda leonard omadang department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda jospeh erume department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda michael ocaido department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda peter oba department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda emmanuel othieno department of pathology, college of health sciences, makerere university, uganda straton bonaventure department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda annah kitibwa department of biomolecular resources and biolaboratory sciences, college of veterinary medicine, animal resources and biosecurity, makerere university, uganda citation chamai, m., omadang, l., erume, j., ocaido, m., oba, p., othieno, e. et al., 2016, ‘identification of echinococcus granulosus strains using polymerase chain reaction–restriction fragment length polymorphism amongst livestock in moroto district, uganda’, onderstepoort journal of veterinary research 83(1), a1068. http://dx.doi.org/10.4102/ojvr.v83i1.1068 original research identification of echinococcus granulosus strains using polymerase chain reaction–restriction fragment length polymorphism amongst livestock in moroto district, uganda martin chamai, leonard omadang, jospeh erume, michael ocaido, peter oba, emmanuel othieno, straton bonaventure, annah kitibwa received: 01 oct. 2015; accepted: 29 dec. 2015; published: 29 july 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract a descriptive study was conducted to identify the different strains of echinococcus granulosus occurring in livestock in moroto district, uganda. echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and sanger dna sequencing. echinococcus granulosus genotypes or strains were identified through use of bioinformatics tools: bioedit, blast and mega6. the major finding of this study was the existence of a limited number of e. granulosus genotypes from cattle, goats, sheep and camels. the most predominant genotype was g1 (96.05%), corresponding to the common sheep strain. to a limited extent (3.95%), the study revealed the existence of echinococcus canadensis g6/7 in three (n = 3) of the e. granulosus–positive samples. no other strains of e. granulosus were identified. it was concluded that the common sheep strain of echinococcus sensu stricto and g6/7 of e. canadensis were responsible for echinococcal disease in moroto district, uganda. introduction echinococcosis in humans is one of the neglected zoonotic diseases. it is caused by metacestodes of the genus echinococcus, with the main definitive host as the dog (eckert et al. 2001). this genus includes small tapeworms of carnivores with larval stages known as hydatid cysts or metacestodes that proliferate in various organs of the intermediate hosts, including humans. the genus echinococcus included four morphologically indistinct species (echinococcus granulosus, echinococcus multilocularis, echinococcus oligarthrus and echinococcus vogeli) until recently, when echinococcus shiquicus and echinococcus felidis were added as the fifth and sixth species (huttner et al. 2008; xiao et al. 2005). currently, there are four forms of hydatidosis that have been deemed taxonomically valid (soulsby 1982). these include cystic echinococcosis (ce) (e. granulosus), alveolar echinococcosis (e. multilocularis) and polycystic echinococcosis (e. vogeli and e. oligarthrus). of these, the most common form is cystic, followed by alveolar, echinococcosis. alveolar echinococcosis is more severe and fatal than ce but sporadic and more difficult to treat because of its characteristic multiple budding and infiltration into organs, including the brain (eckert et al. 2001). ce is prevalent in sub-saharan africa (macpherson & wachira 1997) and is caused by the metacestode of the dog tapeworm e. granulosus. the disease is currently considered an emerging zoonotic disease worldwide (eckert et al. 2001), with an estimated global burden of over 1 million disability-adjusted life-years lost (budke, deplazes & torgerson 2006). this is almost equal to the burden caused by african trypanosomiasis and schistosomiasis (budke, deplazes & torgerson 2006). in china, for example, ce has been worse, with between 60 000 and 1 300 000 people affected, with children accounting for one-third (craig 2005). ce transmission is more intense in remote pastoral communities or regions where veterinary services are poor and where dogs have easy access to animal carcasses and offal. it has been suggested that some of the risk factors responsible for human ce include livestock raising, pastoral life, gender, ethnicity, dog ownership, poor hygiene and low socio-economic status (dowling, abo-shehada & torgerson 2000; larrieu et al. 2002). economically, ce poses a threat to animal farming (thompson 2008), leading to losses because of condemnation of edible organs, reduced meat and milk production, decreased hide and fleece value and decrease in fecundity (polydorou 1981; romazanov 1983). the public health concern is more critical, and as yet, there are no control measures in place. in eastern africa, areas with high prevalence of human ce are said to be focally distributed in kenya, northern tanzania and south sudan, with prevalence of at least 6% amongst nomadic populations (macpherson et al. 1989). there is a significant public health problem amongst pastoralists in the region, especially the turkana and maasai communities in kenya (french & nelson 1982). one of the highest reported incidences of human ce (220/100 000) in the world was in the turkana district of north-western kenya (french & nelson 1982). likewise, in south sudan, human ce is frequent in the extreme south-east of the country at the border with kenya, where prevalence stands at 2% – 3.5% as examined by ultrasound scans (magambo et al. 1996; magambo, zeyhle & wachira 1998). in uganda, inangolet et al. (2010) conducted a study to establish the prevalence of e. granulosus amongst dogs in karamoja and found the prevalence to be 66.3%; however, no information on genotypes was provided. this information provides the background to the study. materials and methods study area the study was conducted between february 2013 and august 2014 in moroto district, sub-region of karamoja. karamoja sub-region is located in north-eastern uganda and comprises seven districts, namely abim, amudat, kaabong, kotido, moroto, nakapiripirit and napak. the coordinates of the town (moroto) are 2°31’48.0”n, 34°40’12.0”e (latitude: 2.5300; longitude: 34.6700). it is bordered in the north by south sudan, in east by turkana region and in the west by pokot district of kenya. moroto district was chosen because of high incidences of ce (inangolet et al. 2010) and its proximity to turkana (figure 1), which once had the world’s highest incidences (macpherson 1998; macpherson et al. 1989). figure 1: map of uganda showing one of echinococcal endemic regions (circled pattern) and administrative sub-counties of moroto district. study design this was a descriptive study where samples were obtained from livestock at moroto municipal council abattoir. details of the species of slaughter livestock (table 1) were recorded including numbers of livestock. upon inspection, infected organs were isolated and stored until a large sample size (n ≥ 20 samples) was reached. samples were collected every 3 months and transported to makerere university molecular biology laboratory for testing. table 1: areas of origin and number of animals that were considered for genotyping of echinoccocus granulosus strains. dna extraction dna extraction from tissue samples was performed as described by nakao, sako and ito (2003), where small sections of the hydatid cysts or single isolated protoscolices were lysed using 20 µl of 0.02 m naoh, heated at 99 °c for 10 minutes and used directly as template. nested polymerase chain reaction amplification first amplification of the nadh dehydrogenase 1 gene (nadh 1 gene) was performed as described by addy et al. (2012) using primers nad a (5’tgt ttt tga gat cag ttc ggt gtg 3’) and nad c (5’cat aat caa acg gag tac gat tag 3’) as forward and reverse primers, respectively. a 25-µl reaction mixture containing 2.5 µl of polymerase chain reaction (pcr) buffer (ph 8.3), 2.0 µl of 2 mm mgcl2, 0.5 µl of 200 µm of each dntp, 0.625 µl of 12.5 pmol of each primer, 0.125 µl of 1.25 u of taq polymerase and 2.0 µl of template dna were subjected to pcr conditions of denaturation at 94 °c for 5 minutes and then 94 °c for 30 seconds. annealing was carried out at 55 °c for 30 seconds, elongation at 72 °c for 1 minute, then final elongation at 72 °c for 5 minutes for 35 cycles. the target product size was 1075 base pairs (bp). a second amplification was carried out under the same conditions using 2.0 µl of amplicon (produced with first amplification) with primers nad b (5’cag ttc ggt gtg ctt ttg ggt ctg 3’) and nad d (5’gag tac gat tag tct cac aca gca 3’) as forward and reverse primers, respectively. the product size for this amplification was 850 bp (addy et al. 2012). restriction fragment length polymorphism fifteen microlitres of the nested pcr amplicons was digested using 0.5 µl of hph i restriction [recognition sequences; 5′…ggtga (n8)*…3′ and 3′…ccact (n7)*…5′] enzyme in presence of 7.5 µl of nuclease-free water and 2.0 µl of 10x buffer b as instructed by the manufacturer (fermentas, gmbh, germany). restriction was performed overnight at 37 °c. the hph i restriction endonuclease produced a staggered cut at nucleotide positions at or ta bp 8 bp or 9 bp away from the recognition sequences. echinococcus granulosus sensu stricto (g1–g3) restriction was expected to produce approximately 425 bp, 320 bp and 204 bp, whilst echinococcus canadensis (g6/7) was expected to produce approximately 425 bp and 107 bp following online restriction mapping. agarose gel electrophoresis successful amplification of e. granulosus nadh 1 gene was determined by electrophoresis on 1.5% agarose gel prepared in 1x tris acetate ethylenediaminetetraacetic acid (tae) buffer containing 1% ethidium bromide, whilst successful digestion of the nadh 1 gene was electrophoresed on 2.0% agarose prepared in 1x tae buffer with 1% ethidium bromide. electrophoresis was performed at 120 v for 45–55 minutes; separated fragments were visualised under uv transillumination. sequencing ten percent (n = 8) of the samples that produced the g1–g3 banding patterns on restriction fragment length polymorphism (rflp) and all the samples (n = 3) that produced g6/7 patterns were sent for sanger dna sequencing assay at inqaba biotech industries ltd, south africa. data analysis gels were photographed and bands manually analysed against positive and negative controls. following dna sequencing, chromatograms were visually analysed using bioedit sequence alignment editor software. genotypes were determined online using the national centre for biotechnology information (ncbi) blast computer programme for homology (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) against published sequences in the genbank. pairwise sequence alignments were made using the online blast programme against reference sequences obtained from the genbank. genetic relatedness of the e. granulosus genotypes were determined using mega6 software. the muscle programme of this software was used for multiple sequence alignments, trimming was carried out and phylogenetic analysis was accomplished using the phylogeny programme of the same software. results a total of 2624 samples from ruminant livestock, including cattle, goats, sheep and camels slaughtered at moroto municipal council abattoir, were examined by a veterinary officer for the presence of hydatid cysts in visceral organs. of the examined animals, 104 that had suspected cysts were sampled (table 1). these samples were subject to pcr-rflp for genotyping e. granulosus strains (addy et al. 2012) and 10% of positive samples for dna sequencing. a majority, 81.2% (n = 85) of the suspected samples, were collected from cattle, 10.6% (n = 11) were from goats, 6.7% (n = 7) were from sheep, whilst only 0.96% (n = 1) was extracted from a camel (table 1). amplification of echinococcus granulosus nadh 1 gene of the suspected 104 samples, successful amplification of the e. granulosus nadh 1 gene occurred in 73.1% (n = 76) of the samples producing a band of approximately 850 bp, whilst 26.9% (n = 28) were not successfully amplified. figure 2 shows a representative gel for the e. granulosus nadh 1 gene pcr products on 1.5% 1x tae agarose gel electrophoresis. figure 2: a 1.5% agarose gel showing echinococcus granulosus nadh 1 gene nested pcr analysis results. genotyping of echinococcus granulosus strains in moroto district by restriction fragment length polymorphism of the e. granulosus nadh 1 gene successfully amplified samples (n = 76), 96.05% (n = 73) were genotyped by rflp as g1–g3, whilst, 3.95% (n = 3) were g6/7. a representative gel following restriction of nested pcr product by hph i was as shown in figure 3. figure 3: a 2.0% agarose gel showing echinococcus granulosus nadh 1 gene rflp analysis results. genotyping of echinococcus granulosus strains in moroto district by sequencing following the inability of rflp genotyping assay to resolve genotypes g1–g3 and g6/7, 10% of the samples that yielded the g1–g3 (n = 8) restriction pattern and all three that produced the g6/7 patterns were subjected to dna sequencing. using a blast search, sequences were compared with published sequences in the ncbi genbank (table 2). table 2: comparison of test sequences with those from the genbank. all the samples (n = 8) that yielded the g1–g3 pattern were homologous to the common sheep g1 strain with maximum identity of 99%. meanwhile, 66.7% (n = 2) of the samples that produced the g6/7 restriction pattern remained unresolved because they showed homology with both g6 and g7 genbank reference sequences at 99/100% maximum sequence identity. one (n = 1) of the sequences that yielded g6/7 rflp pattern was homologous at 99% maximum sequence identity to the g6 strain alone, although with a poor e-score (4e-155). phylogenetic analysis of the 11 samples that were subjected to dna sequencing, 10 produced uniformly sized sequences (≈ 520 bp). only one (mrt049) produced a shorter sequence (≈ 312 bp); it was not used in mega alignment and phylogenetic analysis. therefore, 10 sequences were considered for comparison with other sequences in the ncbi genbank. the criterion for the ncbi sequence selection absolutely relied on the genotype and size of the sequences (> 700 bp). of these, eight sequences belonged to g1 genotype whilst two sequences belonged to g6/7 genotype. table 3 shows sequences selected from ncbi genbank for phylogenetic analysis. table 3: test sequences (mrt) and genbank accession numbers for sequences used in phylogenetic tree construction. genetic relatedness of test isolates with those in genbank was assessed using maximum likelihood method based on the hasegawa-kishino-yano model (hasegawa, kishino & yano 1985) and revealed two large clades. clade a comprised g1s in which the g1 isolates from the present study clustered with those of morocco, china, argentina and india. clade b cluster comprised the g6/7 isolates from the present study along with genotypes g6–10 from japan, china and kazakhstan (figure 4). no published genbank sequences were obtained from east africa countries including uganda. figure 4: molecular phylogenetic analysis of test isolates along with national centre for biotechnology information published sequences by maximum likelihood method. discussion this is the first study to genetically characterise the genotypes of e. granulosus amongst livestock in uganda. our data clearly showed the predominance of e. granulosus genotype g1 (96.05%) corresponding to the common sheep strain, with a minority (3.95%) of cases being e. canadensis (g6/7) genotype. none of the other strains reported elsewhere, including the buffalo strain (g3), the equines strain (g4) and the still poorly characterised cervid strains (g8, g9 and g10) were found in this sample from moroto district. our findings were in agreement with those of addy et al. (2012), who reported the predominance of genotype g1 (almost 100%) in neighbouring kenya, but they also reported the occurrence of e. ortleppi genotype, not found in our study. our findings differed from studies conducted in sudan, which reported predominance of the camel strain (g6) and to a lesser extent (< 26%), the cattle strain (g5) (dinkel et al. 2004; omer et al. 2010). some african camel-rearing countries like algeria, libya and tunisia have also reported the existence and predominance of g1 over g6/7 (abushhewa et al. 2010; farjallah et al. 2007; maillard et al. 2007). this could be attributed to continental globalisation of the common sheep strain (g1) from europe (spain and portugal) to northern african countries, especially libya, algeria and morocco, which are geographical neighbours. these reports indicate regional variations of the e. granulosus genotypes, although the common sheep strain (g1) appears to be predominant. in turkey, for example, snabel et al. (2009) also reported the sheep strain (g1) as the predominant genotype. our data suggest that the e. granulosus genotypes circulating in dogs and livestock in karamoja region could be originating from the neighbouring turkana region of kenya. the borders between these regions are porous, with unrestricted movement of livestock; moreover, the lifestyle of the karamojong and turkana could also be a major factor that explains existence and persistence of echinococcosis in these areas. the lifestyle of these pastoralists is mainly characterised by close interaction with dogs, poor hygiene, insufficient veterinary services and lack of knowledge of disease and its mode(s) of transmission. besides these, unending cattle rustling practices amongst these nomadic groups could also explain the spread of echinococcosis between karamoja sub-region and turkana of kenya. in agreement with eryildiz and sakru (2012), both genotypes (test genotypes) were closely related to other studies. most of the clade a study moroto (mrt) g1 strains shared the same node with other strains, indicating a monophyletic relationship. this meant that the g1 genotypes of other studies were homologous with g1s of the study. similarly, clade b strains, which included the study mrt g6/7, also revealed origin from a common ancestry. although the study revealed a divergence at the basal nodes with reduced bootstrap values in clade b, this could be explained by earlier claims of poor characterisation in the e. canadensis (g6–g10) group, as reported by boubaker et al. (2013). nevertheless, genotypes g8 and g10 in particular showed greater distinctness from the others, which almost rendered the relationship in clade b paraphyletic. this distinctness could be attributed to the fact that both are cervid strains (wild) as compared with the rest of the strains that are domestic. this did not, however, rule out common ancestry with the rest of clade b genotypes. the biggest setback to the study was the absence of published eastern africa genbank sequences, especially for kenya and uganda, because it was believed that moroto echinococcal disease was shared with turkana of kenya, but it was not possible to compare the study findings with other eastern african countries. this therefore presented a missed opportunity for a better understanding of e. granulosus genotype distribution in eastern africa, especially in uganda (moroto), kenya (turkana) and south sudan (toposa). the study was also hindered by the low resolution power of the rflp assay that failed to distinguish between g1–g3 and g6/7 genotypes, although this was circumvented through dna sequencing, which was costly. conclusion the project determined the strains of the dog tapeworm responsible for the echinococcal disease amongst livestock in moroto district (karamoja area) to be the common sheep strain (g1) of echinococcus sensu stricto and e. canadensis (g6/7). no other strains of e. granulosus were identified. our finding of predominance of the g1 genotype in moroto district (karamoja), a genotype identified as the chief causative agent of hydatidosis across all species, calls for institution of strict public health measures that include meat inspection, proper disposal of cystic organs to avoid access by dogs and restriction of movement of animals from one area to another. notably, the study needs to be complemented with studies amongst humans and dogs. this will help to map a linkage between the definitive host, the grazing intermediate ungulates and humans who are adversely affected by this infection compared with other intermediate hosts. this will make understanding of the disease epidemiology better in karamoja, hence better public health mitigation and treatment strategies. acknowledgements we thank cystic echinococcosis in sub-saharan africa research initiative for financially supporting the study and prof. michael ocaido and prof. joseph erume for supervision and guidance. we also thank silver ochwo and our entire m.sc. molecular biology fraternity (2011) for their advice and guidance. special thanks go to erastus mulinge and dr. cecilia mbae of kenya for guidance in molecular work. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors' contributions m.c. developed the proposal, collected samples, performed laboratory analyses, interpreted data and wrote the thesis. l.o. supported proposal development, collected samples and supported thesis writing. j.e. supervised proposal development, performed laboratory analyses and supported thesis writing. m.o. supervised proposal development, performed laboratory analysis and supported thesis writing. p.o. collected samples and performed laboratory analysis. e.o. supported proposal development. s.b. supported laboratory analyses and supported in result interpretation. a.k. performed the laboratory analyses. references abushhewa, m.h., abushhiwa, m.h., nolan, m.j., jex, a.r., campbell, b.e., jabbar, a. et al., 2010, ‘genetic classification of echinococcus granulosus cysts from humans, cattle and camels in libya using mutation scanning-based analysis of mitochondrial loci’, molecular and cellular probes 24, 346–351. addy, f., alakonya, a., wamae, n., magambo, j., mbae, c., mulinge, e. et al., 2012, ‘prevalence and diversity of cystic echinococcosis in livestock in maasailand, kenya’, parasitology research 111(6), 2289–2294. boubaker, g., macchiaroli, n., prada, l., 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‘echinococcus shiquicus n. sp., a taeniid cestode from tibetan fox and plateau pika in china’, international journal for parasitology 35(6), 693–701. horak_215-227.indd introduction reptiles in southern africa are hosts of several species of ixodid ticks (theiler 1962). the majority of these ticks belong to the genus amblyomma, of which nine species, including three species that belonged to the former genus aponomma (now amblyomma), occur in south africa (walker 1991). theiler (1943, 1945a, b) has published illustrations of amblyomma sylvaticum and of the ornate and inornate aponommas. the latter ticks have recently been placed in the genus amblyomma and are now designated amblyomma exornatum, amblyomma latum and amblyomma transversale (klompen, dobson & barker 2002). theiler & salisbury (1959) have illus215 onderstepoort journal of veterinary research, 73:215–227 (2006) parasites of domestic and wild animals in south africa. xlvii. ticks of tortoises and other reptiles i.g. horak1, i.j. mckay2*, b.t. henen3, heloise heyne2, margaretha d. hofmeyr3 and a.l. de villiers4 abstract horak, i.g., mckay, i.j., henen, b.t., heyne, heloise, hofmeyr, margaretha d. & de vil liers, a.l. 2006. parasites of domestic and wild animals in south africa. xlvii. ticks of tortoises and other reptiles. onderstepoort journal of veterinary research, 73:215–227 a total of 586 reptiles, belonging to 35 species and five subspecies, were examined in surveys aimed at determining the species spectrum and geographic distribution of ticks that infest them. of these reptiles 509 were tortoises, 28 monitor or other lizards, and 49 snakes. nine ixodid tick species, of which seven belonged to the genus amblyomma, and one argasid tick, ornithodoros compactus were recovered. seven of the ten tick species are parasites of reptiles. amongst these seven species am blyomma marmoreum was most prevalent and numerous on leopard tortoises, geochelone pardalis; amblyomma nuttalli was present only on bell’s hinged tortoises, kinixys belliana; and most amblyomma sylvaticum were collected from angulate tortoises, chersina angulata. amblyomma exornatum (formerly aponomma exornatum) was only recovered from monitor lizards, varanus spp.; most amblyomma latum (formerly aponomma latum) were from snakes; and a single nymph of amblyomma trans versale (formerly aponomma transversale) was collected from a southern african python, python natalensis. all 30 namaqualand speckled padloper tortoises, homopus signatus signatus, examined were infested with o. compactus. the seasonal occurrence of a. sylvaticum and the geographic distribution of this tick and of a. marmoreum, a. nuttalli, a. exornatum, a. latum and o. compactus are illustrated. keywords: amblyomma spp., argasid tick, geographic distribution, ixodid ticks, ornithodoros compactus, reptiles, seasonality, snakes, tortoises, varanid lizards 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa, and division of parasitology, arc-onderstepoort veterinary institute, onderstepoort, 0110 south africa 2 division of parasitology, arc-onderstepoort veterinary institute, onderstepoort, 0110 south africa. * dr mckay’s present address is: school of geosciences, university of witwaters rand, private bag x3, wits, 2050 south africa 3 chelonian biodiversity and conservation southern africa, de part ment of biodiversity and conservation biology, university of the western cape, private bag x17, bellville, 7535 south africa 4 scientific services, western cape nature conservation board, private bag x5014, stellenbosch, 7599 south africa accepted for publication 12 april 2006—editor 216 parasites of domestic and wild animals in south africa. xlvii trated ticks of ‘the amblyomma marmoreum group’, and colour illustrations of amblyomma hebraeum and a. marmoreum have been published by walker & olwage (1987). a single argasid species, ornithodoros compactus infests tortoises in southern africa, and has been illustrated by walton (1962). theiler (1962) has listed the reptile species and the localities in sub-saharan africa from which ticks of the genera amblyomma and aponomma (now ambly omma) have been collected. norval (1983, 1985) has recorded the hosts and mapped the localities at which amblyomma nuttalli, a. marmoreum, a. exornatum, a. latum and a. transversale have been collected in zimbabwe. walker & schulz (1984) have recorded the numbers of a. hebraeum and a. marmoreum collected from tortoises in the addo elephant national park, eastern cape province, and dower, petney & horak (1988) those collected from leopard tortoises in the andries vosloo kudu reserve and in the thomas baines nature reserve, eastern cape province. the seasonality of a. marmoreum on leopard tortoises in the national zoological gardens, pre toria, gauteng province has been determined by rechav & fielden (1995), and the hosts, seasonality and geographic distribution of the latter tick in south africa have been recorded and illustrated by horak, mckay, heyne & spickett (2006). walton (1962) has listed the tortoise species from which o. compactus has been collected. with the exception of a. hebraeum and o. compactus, the above-mentioned ticks have all been recorded on reptiles imported into the united states of amer ica (usa) from africa (burridge, simmons & allan 2000; burridge 2001; burridge & simmons 2003). this illus trates not only the close association between the ticks and their hosts, but their prolonged periods of attachment, which exceed the length of time from the capture of the reptile in africa, its translocation to a foreign country and its legal or illegal importation into that country. amblyomma marmoreum has in fact adapted to its new habitat and become estab lished at a reptile breeding facility in florida in the usa (allan, simmons & burridge 1998). according to walton (1962) the o. compactus recorded in european zoological gardens, probably arrived there as first stage nymphs on imported tortoises. the purpose of this paper is to record the species spectrum of ticks that infest tortoises and various other reptiles in south africa, and to illustrate the seasonality and geographic distributions of those species for which sufficient data exist. materials and methods the data for this communication are derived from four surveys of ticks parasitizing tortoises and other reptiles. firstly a study conducted specifically on tortoises, chiefly in the southern provinces of south africa, between 1975 and 1981, during which all vis ible ticks were collected, but no record was kept of the number of tortoises that were not infested. sec ondly ticks were collected from tortoises in the west ern and northern cape provinces between 2000 and 2003 as part of a larger investigation on the biol ogy and conservation of these animals. again no record was kept of the number of tortoises that were not infested. thirdly from casual collections between 1981 and 2005 from tortoises and other reptiles, and finally studies on 23 leopard tortoises conducted by dower et al. (1988) and horak & boomker (1998) have been included for completeness’ sake. the vast majority of collections were made from free-living reptiles, with a few made from captive animals. with the exception of the study by dower et al. (1988), in which ticks were recovered after detaching from caged, naturally infested tortoises, ticks were collected manually, or by means of pincers, and preserved in 70 % ethyl alcohol for subsequent identification and counting. the collections made by dower et al. (1988) were exhaustive, while the others concentrated on collecting only the visible and hence more easily accessible ticks. the parasite host tables that follow list the total number of tortoises and other reptiles of each species that were infested irrespective of the tick species and the numbers of animals that were infested with particular species. the localities at which ticks were collected were recorded and their geographic coordinates determined and plotted. the results of the collections pertaining to a. marmoreum have been reported separately (horak et al. 2006) and, with the exception of the tick-host table and geographic distribution map, to which new data have been added, they are not repeated here. large numbers of all stages of development of a. sylvaticum were collected from angulate tortoises during the course of the surveys, and the mean numbers recovered each month, irrespective of the year, have been used to determine the seasonal occurrence of this tick on these animals. the maps illustrating the geographic distributions of the various tick species have been compiled from the coordinates of localities listed by theiler (1962), as well as from those at which ticks were collected in the surveys reported here. 217 i.g. horak et al. we have used branch (1998) as our primary source of reference for the specific and common names of the tortoise and other reptile species examined. these have been listed in the tables and the scientific names will not be repeated in the text. whenever we have been uncertain of the sub-specific identity of a particular tortoise we have used the geographic distribution maps plotted by branch (1998), or the habitat preferences of the various sub-species of tent tortoises (branch 1989), and the field experience of b.t.h. and m.d.h. to arrive at an acceptable solution. when this has not been possible we have used only the specific name of the tortoise. in some cases the animals were identified only as ‘tortoise’ or ‘monitor lizard’ by the collectors and are listed as such in the tables. results and discussion amblyomma hebraeum the hosts of adult a. hebraeum are large herbivorous mammals, whereas the larvae and nymphs can be found on a variety of large and small mammals and on ground-frequenting birds, and the nymphs also on reptiles (norval 1983; horak, macivor, petney & de vos 1987; dower et al. 1988). conse quently, despite the fact that a. hebraeum adults were table 1 ticks collected from various reptile species tick and host species number infested with ticks number infested with spe cies listed number of ticks collected larvae nymphs males females total amblyomma hebraeum leopard tortoise, geochelone pardalis 31* 19 0 686 20 13 719 rock monitor, varanus albigularis albigularis 4* 2 0 33 0 0 33 amblyomma nuttalli bell’s hinged tortoise, kinixys belliana 7 5 0 0 9 0 9 amblyomma exornatum rock monitor, v. a. albigularis 7 7 0 112 191 116 419 water monitor, varanus niloticus 12 12 0 53 104 75 232 “monitor”, varanus spp. 3 3 0 3 17 2 22 amblyomma transversale southern african python, python natalensis 3 1 0 1 0 0 1 hyalomma truncatum leopard tortoise, g. pardalis 82 3 0 0 1 3 4 geometric tortoise, psammobates geometricus 86 1 0 0 1 0 1 rhipicephalus gertrudae geometric tortoise, p. geometricus 86 1 0 0 0 1 1 “tortoise” 9 1 0 0 2 0 2 * = number of animals examined within the distribution range of a. hebraeum 218 parasites of domestic and wild animals in south africa. xlvii table 2 amblyomma marmoreum collected from reptiles host species number infested with ticks number infested with a. mar moreum number of ticks collected larvae nymphs males females total leopard tortoise, g. pardalis 82 80 336 105 545 217 1 203 bell’s hinged tortoise, k. belliana 7 2 0 0 5 3 8 geometric tortoise, p. geometricus 86 68 28 9 64 14 115 serrated tortoise, psammobates oculiferus 11 11 0 1 9 4 14 tent tortoise, psammobates tentorius tentorius 23 22 0 8 21 11 40 namaqualand tent tortoise, psammobates tentorius trimeni 5 1 0 0 1 0 1 bushmanland tent tortoise, psammobates tentorius verroxii 11 2 0 0 4 0 4 tent tortoise, psammobates tentorius 5 1 1 0 0 0 1 angulate tortoise, chersina angulata 138 9 0 6 17 2 25 common padloper, homopus areolatus 87 75 68 65 33 21 187 karoo padloper, homopus boulengeri 2 2 2 5 0 0 7 greater padloper, homopus femoralis 4 4 0 1 4 0 5 southern speckled padloper, homopus signatus cafer 8 2 81 0 1 0 82 namaqualand speckled pad loper, homopus signatus signatus 30 0 0 0 0 0 0 “tortoise” 10 8 7 21 23 10 61 rock monitor, v. a. albigularis 7 3 0 7 1 0 8 “monitor”, varanus spp. 3 1 0 2 2 0 4 southern african python, p. natalensis 3 3 0 11 0 0 11 “python” 1 1 0 0 0 1 1 puff adder, bitis arietans 3 2 0 2 2 0 4 gaboon adder, bitis gabonica 1 1 2 0 0 0 2 common egg eater, dasypeltis scabra 1 1 0 1 0 0 1 boomslang, dispholidus typus 1 1 0 2 0 0 2 total 529 300 525 246 732 283 1 786 219 i.g. horak et al. present on a number of leopard tortoises examined within the tick’s distribution range (table 1), these infestations must be considered opportune, possibly triggered by a. hebraeum or a. marmoreum males already present on the tortoises. attached, engorging a. hebraeum males produce pheromones that, with the carbon dioxide exhaled by their hosts, attract con-specific females, other males and nymphs (norval, yunker & butler 1987). the life stage spectrum of the ticks attached to the 19 infested leopard tortoises seems to indicate that male a. marmoreum may also produce pheromones that can be detected by con-specific ticks as well as by a. hebraeum nymphs and adults. howell, walker & nevill (1978) have illustrated the geographic distribution of a. hebraeum within the boundaries of south africa, and the present records from tortoises all fall within this range. amblyomma marmoreum this tick, colloquially known as the south african tortoise tick, is probably the species most frequently encountered on tortoises throughout south africa (theiler & salisbury 1959; horak et al. 2006). it is the largest ixodid tick in south africa, and engorged females may exceed 25 mm in length. with the exception of angulate and namaqualand speckled padloper tortoises (the latter the smallest tortoise species in the world), a large percentage of individuals within each tortoise species were infested (table 2). it would seem that the larger the species the greater the number of adult ticks it is likely to harbour, and leopard tortoises, the largest of the south african species, carried most adult ticks. amongst the latter was a tortoise infested with 63 male and 105 female ticks. the immature stages, particularly larvae, infest not only reptiles, but also a large variety of mammals and birds (horak et al. 2006). the comparatively small total number of immature ticks collected from the various tortoise species could not sustain the large number of adult ticks collected from these animals. because complete collections were not always made, large numbers of immature ticks were probably overlooked. furthermore, the large number of larvae and fewer nymphs that attach to a variety of warm-blooded hosts, both avian and mammalian (horak et al. 2006), could serve to augment the apparently insufficient numbers of immature ticks collected. theiler & salisbury (1959) mapped the distribution of a. marmoreum in south africa from the geographic coordinates of 49 localities at which collections had been made. horak et al. (2006) added more than 100 localities to that map and we have now added approximately 70 more sites (fig. 1). most collections have been made in the south-western fig. 1 geographic distribution of amblyomma marmoreum in south africa 220 parasites of domestic and wild animals in south africa. xlvii western cape province, the western half of the eastern cape and free state provinces, the eastern regions of the northern cape province, and the northeastern regions of kwazulu-natal and mpumulanga provinces. this distribution closely corresponds to that of several of the tick’s most favoured host species, namely common padloper tortoises in southwestern western and eastern cape provinces, geometric tortoises in south-western western cape prov ince, tent tortoises in western western and northern cape provinces, serrated tortoises in eastern northern cape province, and leopard tortoises in the central, northern and north-eastern regions of south africa (branch 1998). amblyomma nuttalli the difficulty in distinguishing between a. nuttalli and other ticks of the a. marmoreum group has been discussed by theiler & salisbury (1959). one of the hosts frequently infested by adult a. nuttalli is bell’s hinged tortoise, kinixys belliana (hoogstraal 1956; table 1), but other tortoises, monitor lizards and snakes may also be infested (theiler & salisbury 1959; burridge et al. 2000). the immature stages infest the same hosts as the adults, but are also found on birds and mammals (theiler 1962). the tick is widespread in the afrotropical region, but in south africa almost all collections have been made in northeastern kwazulu-natal (theiler 1962; walker 1991; fig. 2). amblyomma sylvaticum theiler (1962) describes a. sylvaticum as “this somewhat rare cape tortoise tick”, an observation refuted by the present surveys, during which, after a. marmoreum, a. sylvaticum was the tick most frequently collected. theiler lists adult ticks on the angulate tortoise, tent tortoise and common padloper as well as on a mole snake, pseudaspis cana. in contrast to a. marmoreum, which infests a wide spectrum of tortoise species, all stages of development of a. syl vaticum would seem to infest angulate tortoises by pref erence (table 3). the number of immature ticks collected from these tortoises could clearly not sustain their adult burdens, and incomplete collections can in part be responsible for this. although we have examined numerous birds and mammals, we have not collected larvae or nymphs of a. sylvaticum from any of them, and it would thus appear as if infestations on other tortoise species and reptiles augment the population of immature ticks (table 3). the considerable differences in the numbers of angulate tortoises examined in the different months of fig. 2 geographic distributions of amblyomma nuttalli, amblyomma sylvaticum and ornithodoros compactus in south africa 221 i.g. horak et al. the year as well as in the tick burdens of individual animals make it difficult to obtain a clear pattern of seasonality for a. sylvaticum. five or fewer tortoises were examined per month in january, march (none examined), june, august and november, and six or more in the other months, with 36 examined during may. to compensate for these inequalities, 3-month running means have been used to illustrate seasonality. with this in mind, most larvae were present from may to november, most nymphs during the period august to january, and most adults from august to february (fig. 3). this pattern of seasonality agrees with the sizes of ticks observed on angulate tortoises during january, may, july and september by b.t.h. and m.d.h. if this indeed were the pattern of seasonality, it would imply that female ticks deposit eggs in summer and that most larvae hatch from early winter to early summer. these larvae would feed and moult to nymphs from midwinter to midsummer, and once the latter had fed and moulted they would table 3 amblyomma sylvaticum collected from various reptile hosts host species number infested with ticks number infested with a. sylvaticum number of ticks collected larvae nymphs males females total geometric tortoise, p. geometricus 86 14 2 3 0 14 19 tent tortoise, p. t. tentorius 23 1 0 0 1 0 1 namaqualand tent tortoise, p. t. trimeni 5 0 0 0 0 0 0 bushmanland tent tortoise, p. t. verroxii 11 1 0 0 2 0 2 tent tortoise, p. tentorius 5 1 3 0 0 0 3 angulate tortoise, c. angulata 138 124 736 249 277 159 1 421 common padloper, h. areolatus 87 4 0 14 2 3 19 southern speckled padloper, h. s. cafer 8 5 0 0 4 1 5 namaqualand speckled padloper, h. s. signatus 30 0 0 0 0 0 0 knox’s desert lizard, meroles knoxi 3 3 62 0 0 0 62 southern spiny agama, agama hispida 1 1 5 0 0 0 5 total 397 154 808 266 286 177 1 537 ��������� ����� ����������� ����� � � � � � � � �� ��� � � ��� ��� ��� ��� ��� ��� � � ��� �� ! � ����� � � � � � � � � � � �� � � � "�� � ��#�$� �%���� fig. 3 seasonal occurrence of amblyomma sylvaticum on angulate tortoises in south africa (3-month running means) 222 parasites of domestic and wild animals in south africa. xlvii maintain the numbers of adult ticks during summer. from the available data we are, however, unable to determine whether the life cycle requires 1 or 2 years to complete, or whether diapause occurs during any particular stage of development. according to theiler (1962), who based her findings on collections made at 12 localities, the distribution of a. sylvaticum is confined to the eastern and western cape provinces and to parts of little namaqualand. we were unable to trace two of theiler’s 12 collection sites, but have now plotted a further 33 localities. from these it is apparent that a. sylvaticum is a strictly south african species, predominantly present in the western regions of the northern and western cape provinces as well as in the southwestern regions of the latter province and of the eastern cape province, with a single outlying record from a tent tortoise in the centre of the northern cape province (fig. 2). infestation was most prevalent on angulate tortoises, southern speckled padloper tortoises and on knox’s desert lizards. angulate tortoises and the desert lizards have a strong association with sandy soils. although southern speckled padloper tortoises occur in rocky terrain, they can be sympatric with angulate tortoises (m.d.h. and b.t.h.) and have sandy soil in their habitat. it is possible that these soils and the micro-environment associated with them provide essential elements for the survival of the free-living stages of a. sylvaticum. this tick and its angulate tortoise hosts are both common in the west coast national park and nearly all tortoises examined by two of us (b.t.h. and m.d.h.) were infested. however, despite examining large numbers of angulate tortoises on dassen island, an island about 10 km southwest of the park, only one tick has been collected from these reptiles. the absence of ticks on this small island might be due to its very salty environment and ephemeral and mostly exotic vegetation. amblyomma sylvaticum has been recorded in the united states on a tortoise imported from south africa (burridge & simmons 2003). amblyomma exornatum no a. exornatum larvae were recovered, but 168 nymphs and 505 adult ticks were collected from 22 monitor lizards (table 1). according to theiler (1945a), hoogstraal (1956) and norval (1985) these large lizards are its preferred hosts. a single rock monitor examined during the present surveys in the national zoological gardens, pretoria, harboured 79 nymphs, 150 males and 94 female ticks. although no adult ticks were collected during october, 14 of fig. 4 geographic distributions of amblyomma exornatum and amblyomma latum in south africa 223 i.g. horak et al. the 21 collections of adult ticks were made during the period september to december (spring to midsummer). thirty-one of the 37 localities at which we have recovered a. exornatum are in the eastern and southeastern half of the country (fig. 4). according to theiler (1962) and norval (1985) it is widespread in south africa and zimbabwe, and theiler suggests that it is probably present across the range of its varanid hosts in these countries. the single record from simonstown in south-west western cape province originates from a monitor lizard that had probably been translocated, as this region lies outside the geographic distribution of these lizards (branch 1998). amblyomma latum this tick, aptly named the snake tick by hoogstraal, infests snakes, and more particularly the larger species (theiler 1945b, 1962; hoogstraal 1956; norval 1985). according to walker (1991) the occasional records from lizards and even from some mammals, represent incidental infestations. the single female collected from a leopard tortoise in the present study would thus seem to be unusual (table 4). amblyomma latum has, however, been collected from two captive yellow-footed tortoises, geochelone denticulata, in the usa. the latter tortoise is native to south america, where this tick does not occur, and the tortoises probably acquired infestation while in captivity (burridge 2001; burridge & simmons 2003). the 32 males, two females, eight nymphs and 76 larvae collected from a black mamba near hector spruit, in the lowveld of mpumalanga province, appear to be one of the largest recorded collections of a. latum taken from a single snake. many of the adult ticks in this collection were attached under scales in and adjacent to a large superficial dorsal wound just posterior to the snake’s head. whether the ticks had caused the lesion, or had attached there because of it, could not be ascertained. according to burridge (2001), adult ticks attach under dorsal and lateral scales on all parts of the body of snakes, with the head being the most common site. in zimbabwe, norval (1985) recovered all developmental stages of a. latum throughout the year, but collected most samples in the warm months when snakes are most active. during the present surveys 28 collections of adult ticks were made during the period october to february (summer) and only 11 from march to september (late summer to spring). the distribution of a. latum in south africa, based on theiler’s (1962) records, to which we have added several localities, is illustrated in fig. 4. most records of this tick emanate from the eastern half of the country, with a single collection in the far south-west from a reptile in a snake park in cape town. amblyomma transversale a single nymph of this tick was collected from a southern african python (table 1), which incidentally was also infested with nymphs of a. marmoreum and an adult a. latum. pythons are apparently the only host species recorded for a. transversale in southern africa (norval 1985; walker 1991). hyalomma truncatum despite our examination of large numbers of tortoises in regions in which h. truncatum is present (howell et al. 1978), only three leopard tortoises and one geometric tortoise were infested with this tick (table 1). these ticks should be considered ‘stragglers’ from a heavily infested environment. the hosts of adult h. truncatum are large herbiv orous mammals (norval 1982; horak, swanepoel & gum mow 2002), to which they attach in the axillary and perianal regions and in the tail brush. rhipicephalus gertrudae this tick is particularly prevalent in the western cape province, and its adults infest herbivores and carnivores (walker, keirans & horak 2000; horak & matthee 2003). the ticks on two tortoises examined during june and august respectively (table 1), must be considered ‘stragglers’ reflecting the tick’s abundance in the western cape province at this time of the year (horak & fourie 1992). ornithodoros compactus this tick has been recorded on the tortoises, testudo oculifera (psammobates oculiferus), testudo verreauxi (psammobates tentorius verroxii), testudo shonlandi (psammobates tentorius verroxii) and homopus femoralis in southern africa (walton 1962), and tortoises would appear to be its only hosts. in the current surveys ticks were collected from six tortoise species, and infestation was most prevalent on namaqualand speckled padloper tortoises. the larvae of o. compactus are not parasitic, but 450 nymphs and 57 adults (70 adults if the 13 unsexed adult ticks are included) were collected from 58 tortoises (table 5). 224 parasites of domestic and wild animals in south africa. xlvii unlike other ornithodoros species, of which the nymphs and adults spend most of their time in the dwellings or burrows of their hosts, or under the surface of the soil, it would seem as if those of o. compactus live on their tortoise hosts. namaqualand speckled padloper tortoises, all of which were infested, prefer rocky terrain, but also have sand in their habitat. these tortoises often rest among the debris and soil under shrubs or between boulders and it is possibly here that they acquire infestation table 4 amblyomma latum collected from various reptile hosts host species number infested with ticks number infested with a. latum number of ticks collected larvae nymphs males females total leopard tortoise, g. pardalis 82 1 0 0 0 1 1 aurora house snake, lamprophis aurora 1 1 0 0 1 0 1 brown house snake, lamprophis fuliginosus 4 4 0 1 3 2 6 black mamba, dendroaspis polylepis 5 5 76 8 81 28 193 snouted cobra, naja annulifera annulifera 5 5 2 1 30 3 36 mozambique spitting cobra, naja mossambica 5 5 0 1 15 0 16 cape (yellow) cobra, naja nivea 1 1 0 0 2 3 5 rinkhals, hemachatus haemachatus 1 1 0 0 7 0 7 puff adder, b. arietans 3 2 0 0 1 1 2 snouted night adder, causus defilippii 2 2 4 2 0 2 8 common night adder, causus rhombeatus 1 1 0 0 0 2 2 cape file snake, mehelya capensis 6 6 1 16 49 9 75 black file snake, mehelya nyassae 1 1 0 3 0 0 3 herald snake, crotaphopeltis hotamboeia 1 1 0 2 0 0 2 shield-nose snake, aspidelaps scutatus 3 3 0 0 7 0 7 southern african python, p. natalensis 3 2 0 0 5 14 19 olive grass snake, psammophis mossambicus 3 3 8 2 4 0 14 striped skink, mabuya striata 1 1 6 0 0 0 6 variable skink, mabuya varia 1 1 3 0 0 0 3 total 129 46 100 36 205 65 406 225 i.g. horak et al. via newly moulted first stage nymphs. these nymphs would have moulted from non-parasitic larvae that had hatched from eggs that had dropped off or were deposited in these protected localities. walton (1962) has suggested that the axillary and perineal skin folds are probably the most suitable sites for the ticks to survive on tortoises. loehr, henen & hofmeyr (2006) found that the ticks tend to use the hind limbs of namaqualand speckled padloper tortoises more frequently than the fore limbs and cervical region, and many move when the tortoises are handled. like other ornithodoros spp. feeding appears to be occasional, and only one male and two female ticks of the 57 adults that we collected appeared to have recently engorged. the distribution of o. compactus is apparently restricted to southern africa south of the zambezi river (walton 1962). in the present surveys most collections were made from tortoises in the semiarid northern cape province (fig. 2). general only 26 of the 591 reptiles examined harboured mixed infestations of two tick species, and only two hosted three species. one of the latter was a rock monitor, which was infested with nymphs of a. hebraeum and of a. marmoreum and nymphs and adults of a. exornatum, and the other a southern african python that harboured nymphs of a. marmoreum, a single a. transversale nymph, and a male a. latum. eighteen leopard tortoises were infested with both a. hebraeum and a. marmoreum, three with a. marmoreum and h. truncatum, and one with a. marmoreum and a nymph of o. compactus. a geometric tortoise harboured a. marmoreum and a female r. gertrudae, and a namaqualand tent tortoise was infested with one male a. marmoreum and 20 o. com pactus nymphs. two rock monitors were infested with a. marmoreum and a. exornatum. it would thus seem that, with the exception of a. hebraeum and a. marmoreum, the ticks infesting reptiles have table 5 ornithodoros compactus collected from various reptile hosts host species number infested with ticks number infested with o. compactus number of ticks collected larvae nymphs males females total leopard tortoise, g. pardalis 51* 2 0 3 0 0 3 geometric tortoise, p. geometricus 86 2 0 26 2 0 28 tent tortoise, p. t. tentorius 23 0 0 0 0 0 0 namaqualand tent tortoise, p. t. trimeni 5 5 0 30 0 0 30 bushmanland tent tortoise, p. t. verroxii 11 8 0 79 2 0 81 tent tortoise, p. tentorius 5 3 0 27 0 0 27 angulate tortoise, c. angulata 138 3 0 9 0 0 9 common padloper, h. areolatus 87 5 0 18 0 0 18 southern speckled padloper, h. s. cafer 8 0 0 0 0 0 0 namaqualand speckled padloper, h. s. signatus 30 30 0 258 12 41 311** total 444 58 0 450 16 41 507** * number of tortoises examined within the apparent distribution range of o. compactus ** plus 13 unsexed adults 226 parasites of domestic and wild animals in south africa. xlvii evolved fairly distinct preferences for individual host species or for hosts belonging to the same genus or grouping. acknowledgements we are indebted to mr p.d. burdett who collected ticks from large numbers of reptiles, and to dr e.h.w. baard, who made these collections available to us. dr w. haacke is thanked for the collections of ticks he made from several reptiles, and dr t.e.j. leuteritz for collecting ticks from tortoises at the tierberg research centre and near clanwilliam. we are grateful to south african national parks and the nature conservation divisions of the western and northern cape provinces’ provincial administrations for permission to collect ticks from reptiles. dr r. williams of the arc-onderstepoort veterinary institute is thanked for compiling the distribution maps. the participation of i.g. horak in this project has been facil itated through the icttd-3, financed by the inter national cooperation program of the european union through coordination action project no. 510561. b.t. henen and m.d. hofmeyr received fund ing towards this project from the national research foundation, the royal society of london and the senate at the university of the western cape. references allan, s.a., simmons, l.-a. & burridge, m.j. 1998. establishment of the tortoise tick amblyomma marmoreum (acari: ixodidae) on a reptile-breeding facility in florida. journal of medical entomology, 35:621–624. branch, w.r. 1989. psammobates tentorius, in the conservation biology of tortoises, iucn species survival commission occasional paper 5, edited by i.r. swingland & m.w. clemens. gland, switzerland: 91–93. branch, w.r. 1998. field guide to snakes and other reptiles of southern africa. cape town: struik publishers. burridge, m.j., simmons, l.-a. & allan, s.a. 2000. introduction of potential heartwater vectors and other exotic ticks into florida on imported reptiles. journal of parasitology, 86: 700–704. burridge, m.j. 2001. ticks (acari: 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(ixodida: ixodidae), and the synonymy of aponomma neumann, 1899 with amblyomma koch, 1844. systematic parasitology, 53:101–107. loehr, v.j.t., henen, b.t. & hofmeyr, m.d. 2006. tick infestations in the namaqualand speckled padloper, homopus signatus signatus (gmelin, 1789). african journal of herpetology (in press). norval, r.a.i. 1982. the ticks of zimbabwe. iv. the genus hyalomma. zimbabwe veterinary journal, 13:2–10. norval, r.a.i. 1983. the ticks of zimbabwe. vii. the genus amblyomma. zimbabwe veterinary journal, 14:292–305. norval, r.a.i. 1985. the ticks of zimbabwe. xi. the genus aponomma. zimbabwe veterinary journal, 16:5–8. norval, r.a.i., yunker, c.e. & butler, j.f. 1987. field sampling of unfed adults of amblyomma hebraeum (koch). experimental and applied acarology, 3:213–217. rechav, y. & fielden, l.j. 1995. seasonal abundance of the tortoise tick amblyomma marmoreum (acari: ixodidae) on the leopard tortoise. journal of medical entomology, 32:161– 165. theiler, gertrud 1943. 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the bont tick, amblyomma hebraeum, from the angulate tortoise, chersina angulata, and the leopard tortoise, geochelone pardalis. onderstepoort journal of veterinary research, 51: 171–173. walker, jane b. & olwage, a. 1987. the tick vectors of cowdria ruminantium (ixodoidea, ixodidae, genus amblyomma) and their distribution. onderstepoort journal of veterinary research, 54:353–379. walker, jane b. 1991. a review of the ixodid ticks (acari: ixodidae) occurring in southern africa. onderstepoort journal of veterinary research, 58:81–105. walker, jane b., keirans, j.e. & horak, i.g. 2000. the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world. cambridge: cambridge academic press. walton, g.a. 1962. the ornithodoros moubata superspecies problem in relation to human relapsing fever epidemiology. symposium of the zoological society of london. no. 6:83– 156. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [878.740 1133.858] >> setpagedevice abstract introduction methods results discussion conclusion acknowledgements references about the author(s) ivan g. horak department of veterinary tropical diseases, university of pretoria, south africa christiaan r. boshoff wild game, gravelotte, south africa david v. cooper ekzn wildlife, st lucia, south africa christoper m. foggin victoria falls wildlife trust, victoria falls, zimbabwe danny govender scientific services, sanparks, skukuza, south africa alan harrison institute of biological and environmental sciences, university of aberdeen, united kingdom guy hausler veterinary wildlife services, sanparks, skukuza, south africa markus hofmeyr veterinary wildlife services, sanparks, skukuza, south africa j. werner kilian okuakuejo rest camp, etosha national park, namibia duncan n. macfadyen department of research and conservation, e oppenheimer & son, south africa pierre j. nel department of economic development, tourism and environmental affairs, bloemfontein, south africa dean peinke eastern cape parks & tourism agency, east london, south africa david squarre wildlife veterinary unit, zambia wildlife authority, zambia david zimmermann veterinary wildlife services, sanparks, port elizabeth, south africa citation horak, i.g., boshoff, c.r., cooper, d.v., foggin, c.m., govender, d., harrison, a. et al., 2017, ‘parasites of domestic and wild animals in south africa. xlix. ticks (acari: ixodidae) infesting white and black rhinoceroses in southern africa’, onderstepoort journal of veterinary research 84(1), a1301. https://doi.org/10.4102/ojvr.v84i1.1301 original research parasites of domestic and wild animals in south africa. xlix. ticks (acari: ixodidae) infesting white and black rhinoceroses in southern africa ivan g. horak, christiaan r. boshoff, david v. cooper, christoper m. foggin, danny govender, alan harrison, guy hausler, markus hofmeyr, j. werner kilian, duncan n. macfadyen, pierre j. nel, dean peinke, david squarre, david zimmermann received: 22 june 2016; accepted: 05 aug. 2016; published: 30 jan. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the objectives of the study were to determine the species composition of ticks infesting white and black rhinoceroses in southern africa as well as the conservation status of those tick species that prefer rhinos as hosts. ticks were collected opportunistically from rhinos that had been immobilised for management purposes, and 447 white rhinoceroses (ceratotherium simum) and 164 black rhinoceroses (diceros bicornis) were sampled in south africa, 61 black rhinos in namibia, 18 white and 12 black rhinos in zimbabwe, and 24 black rhinos in zambia. nineteen tick species were recovered, of which two species, amblyomma rhinocerotis and dermacentor rhinocerinus, prefer rhinos as hosts. a. rhinocerotis was collected only in the north-eastern kwazulu-natal reserves of south africa and is endangered, while d. rhinocerinus is present in these reserves as well as in the kruger national park and surrounding conservancies. eight of the tick species collected from the rhinos are ornate, and seven species are regularly collected from cattle. the species present on rhinos in the eastern, moister reserves of south africa were amongst others amblyomma hebraeum, a. rhinocerotis, d. rhinocerinus, rhipicephalus maculatus, rhipicephalus simus and rhipicephalus zumpti, while those on rhinos in the karoo and the drier western regions, including namibia, were the drought-tolerant species, hyalomma glabrum, hyalomma rufipes, hyalomma truncatum and rhipicephalus gertrudae. the species composition of ticks on rhinoceroses in zambia differed markedly from those of the other southern african countries in that amblyomma sparsum, amblyomma tholloni and amblyomma variegatum accounted for the majority of infestations. introduction historically white rhinoceroses (ceratotherium simum) were present from south africa in the south to the coastal regions of north-eastern africa in the north (skinner & chimimba 2005). however, with the arrival of european settlers in southern africa, as well as hunters, naturalists and travellers, the numbers of white rhinos in this region rapidly dwindled. towards the end of the 19th century the last white rhinos were shot in zimbabwe and botswana, while in south africa their numbers had declined to approximately 20–50 animals in north-eastern kwazulu-natal. the proclamation of the hluhluwe and imfolozi game reserves in 1895 and the mkuze game reserve in 1912 in kwazulu-natal is considered to have rescued them from extinction in south africa (skinner & chimimba 2005). their numbers have steadily increased since then and in 1961 the natal parks board initiated a project on the relocation of rhinoceroses from the reserves under its control to the kruger national park and other state and provincially controlled reserves, as well as to privately owned reserves lying within the rhinos’ former distribution range. although the number of white rhinos in south africa now exceeds 18 000, the ever-increasing rate at which they are being poached may soon surpass their birth rate. the numbers of black rhinoceroses (diceros bicornis) declined as rapidly as those of white rhinoceroses. from a situation in which they were present on the slopes of table mountain in 1652, to where the proclamation of the hluhluwe, imfolozi and mkuze game reserves in kwazulu-natal rescued them from extinction in this country (skinner & chimimba 2005). numbers on the continent are believed to have declined from approximately 100 000 in the early 1960s to only 2410 in 1995. since then careful management and relocations have seen their numbers on the continent increase to 4880 by the end of 2010 (cumming, du toit & stuart 1990; knight, balfour & emslie 2013), with most present in the north-eastern kwazulu-natal reserves, the kruger national park and the etosha national park. both rhinoceros species are infested with a greater array of ornate ixodid ticks than any other mammal species in east and southern africa. most of these are brightly coloured members of the genus amblyomma, and include amblyomma eburneum, amblyomma gemma, amblyomma hebraeum, amblyomma personatum, amblyomma rhinocerotis, amblyomma sparsum, amblyomma tholloni and amblyomma variegatum. rhinos also harbour dermacentor rhinocerinus, an ornate tick that is for all practical purposes a specific parasite of these animals. they are also infested by three of the four rhipicephalus species that are ornate, rhipicephalus humeralis, rhipicephalus maculatus and rhipicephalus pulchellus. a number of tick surveys conducted in africa have included rhinoceroses amongst various other animal species sampled. collections made from black rhinoceroses in tanzania yielded 18 tick species, including six species in the genus amblyomma as well as d. rhinocerinus, and two ornate rhipicephalus spp. (yeoman & walker 1967). twenty-one tick species were identified in collections from black rhinoceroses in kenya; these included seven amblyomma spp., d. rhinocerinus and three ornate rhipicephalus spp. (walker 1974). ten tick species were present in collections made from white and black rhinoceroses in zimbabwe, and three amblyomma spp. and d. rhinocerinus were recovered (norval 1983; norval & colborne 1985). in a checklist of ticks that infest large mammals in the kwazulu-natal reserves in south africa, 11 species were reported on rhinoceroses, these included three amblyomma spp. (one of them doubtful), d. rhinocerinus and r. maculatus (baker & keep 1970). collections from two white and four black rhinoceroses sampled at various localities in south africa yielded nine tick species, including a. hebraeum, d. rhinocerinus and r. maculatus (knapp et al. 1997). it is generally not realised that should rhinoceroses go extinct, a multitude of smaller creatures will also disappear. amongst these are the rhinoceros-specific ticks, a. personatum, a. rhinocerotis, cosmiomma hippopotamensis and d. rhinocerinus, the flies rhinomusca dutoiti, rhinomusca brucei and gyrostigma rhinocerontis, as well as a horde of rhino-specific nematodes and an even greater number of commensal protozoal species. the extinction of rhinos will thus destroy a whole ecosystem of parasites and commensals. with the exception of the study by knapp et al. (1997), no surveys devoted solely to rhinoceroses and the ticks that infest them have been conducted in southern africa. the objective of the present investigation is to address this shortcoming. as a consequence, a number of surveys aimed at determining the species composition of ticks that infest these animals in the sub-continent were initiated. methods participants in the study collected ticks opportunistically from rhinoceroses immobilised for management purposes. particular attention was paid to the ears, the axilla, inner thighs, peri-anal region and the tail brush. although this collection procedure represented the ideal, any ticks collected from rhinos were included in the study. more detailed or complete collections were not possible because of the time these would take, causing additional anaesthetic stress on the animals. ticks collected from each rhino were placed in separate bottles or plastic vials containing 70% ethyl alcohol or undiluted methylated spirits. a label written in pencil, indicating the rhino’s species, its name or identity (if known), gender, the date, the locality at which it was immobilised and the identity of the person responsible for collecting ticks, was placed with the ticks in the bottle or vial. this was not always possible, and several labels were attached to the outside of the containers. the ticks were identified and counted using a stereoscopic microscope. the identities and total numbers of adult ticks collected from white and black rhinoceroses across southern africa are summarised in tabular format. they are also tabulated according to the regions in which they were collected from rhinos within south africa. a separate table has been created for ticks collected in namibia, zimbabwe and tanzania. the regional distributions of the 19 tick species collected from rhinoceroses in southern africa are represented in a mosaic format. the conservation status of the ticks that prefer rhinos as hosts and the introduction of ticks into non-endemic habitats or reintroduction into endemic habitats are discussed, as is the role of rhinos as maintenance hosts of ticks that are vectors of diseases of domestic livestock or wildlife species. ethical considerations no rhinos were immobilised for the sole purpose of collecting ticks. collection was incidental to other management procedures necessitating immobilisation. results south africa the tick species recovered from a total of 465 white and 261 black rhinoceroses across southern africa, and the number of animals infested with each species are summarised in table 1. twelve tick species, including the nymphs of rhipicephalus appendiculatus, were collected from white rhinoceroses, with a. hebraeum present in the majority of collections, followed by d. rhinocerinus, hyalomma truncatum and hyalomma rufipes. sixteen species were recovered from black rhinoceroses, and a. hebraeum was the most commonly collected tick, followed by h. rufipes and h. truncatum. the two rhino species harboured ten tick species in common. table 1: ixodid ticks collected from white and black rhinoceroses in southern africa. the species and numbers of ticks collected from 303 white rhinoceroses in the kruger national park and surrounding conservancies and from nine black rhinoceroses in the park, as well as those collected from 49 white rhinos and 20 black rhinos in the north-eastern kwazulu-natal reserves, are summarised in table 2 and the regions in which the ticks were collected are represented in figure 1. five species were collected from white rhinos and four from black rhinos in the kruger national park. amblyomma hebraeum was present in the majority of collections made from either rhino species, followed by d. rhinocerinus. three white rhinos were infested with a. tholloni, a tick whose adults prefer elephants as hosts. table 2: ixodid ticks collected from rhinoceroses in the kruger national park and surrounding conservancies, and in the north-eastern kwazulu-natal reserves. figure 1: the regional distribution of ticks that infest rhinoceroses in southern africa. five tick species were recovered from the white rhinos in the kwazulu-natal reserves and six from the black rhinos. amblyomma hebraeum was present in the majority of collections followed by r. maculatus on both white and black rhinos, while some animals of both species were infested with the rhinoceros specific ticks a. rhinocerotis and d. rhinocerinus (figure 1). the tick species collected from rhinoceroses in the free state, northern cape province, north west province, western limpopo province, and the central and south-eastern region of the limpopo province and north-eastern gauteng are summarised in table 3. hyalomma rufipes and h. truncatum were the most frequently collected ticks from both rhino species in the free state and northern cape. the collections of a. hebraeum from white rhinoceroses in the free state were made from animals that had recently been introduced into the province from regions in which the tick was present and do not represent established populations. most white rhinos in eastern north west province and western limpopo province were infested with a. hebraeum, and it was the only species collected from black rhinoceroses. table 3: ixodid ticks collected from rhinoceroses in the free state, northern cape province, north-western parks and central region of two northern provinces of south africa. ticks were also collected from a dead white rhinoceros originating from a reserve in the southern central region of limpopo province and presented for necropsy at the faculty of veterinary science, pretoria university, onderstepoort. because it was dead, considerably more ticks were collected from it than from other white rhinos that were temporarily immobilised. ticks were also collected from eight white rhinos in the south-eastern region of limpopo province and two animals in north-eastern gauteng. we consider all these animals to have come from the central region of two of the northern provinces of south africa and the numbers and species of ticks collected from them are summarised in table 3. in addition to a. hebraeum and h. rufipes, they harboured rhipicephalus evertsi evertsi and rhipicephalus follis and the dead animal was infested with a large number of rhipicephalus simus. three of the eleven animals were infested with seven nymphs of r. appendiculatus. the species and numbers of ticks collected from rhinoceroses in the great fish river nature reserve and the addo elephant national park in the eastern cape province, and the mountain zebra and the karoo national parks are summarised in table 4. all but one of the black rhinoceroses examined in the eastern cape province were infested with a. hebraeum, and a substantial number of collections of r. follis and particularly r. simus were also made. the rather rare species, rhipicephalus zumpti, was also present. four of the black rhinoceroses examined in the mountain zebra and karoo national parks were infested with hyalomma glabrum and five with h. truncatum and r. follis. table 4: ixodid ticks collected from black rhinoceroses in the great fish river nature reserve and the addo elephant national park, eastern cape province, and the mountain zebra and karoo national parks. namibia the tick species collected from black rhinoceroses in three regions of namibia are summarised in table 5. the rhinos in the etosha national park were infested with h. rufipes and h. truncatum, the damaraland rhinos with h. rufipes and one animal with the very rare rhipicephalus longiceps, while the rhinos in the hardap nature reserve harboured h. rufipes, h. truncatum and rhipicephalus gertrudae, an assemblage of ticks fairly similar to that on white and black rhinoceroses in the free state and northern cape provinces (figure 1). table 5: ixodid ticks collected from rhinoceroses in namibia, zimbabwe and zambia. zimbabwe the results of collections from rhinos in the malilangwe reserve in south-eastern zimbabwe are summarised in table 5. all the animals were infested with a. hebraeum. a single collection of a. tholloni was made from a black rhinoceros. zambia the tick species present on black rhinoceroses examined in the north luangwa national park in northern province differed substantially from those of the other southern african animals (table 5). amblyomma sparsum was the dominant species with all but one animal being infested. a number of collections of a. tholloni and a. variegatum were also made (figure 1). the zambian rhinos had originally come from the kruger national park, marakele national park and the kwazulu-natal and eastern cape parks. discussion amblyomma gemma the only rhinos infested with a. gemma were those examined in zambia (figure 1). neither theiler (1962) nor walker and olwage (1987) include zambia within the distribution range of a. gemma. in neighbouring tanzania, its distribution is almost entirely limited to semi-arid bush and thicket and by rainfall between 38 mm and 76 mm, with a few records close to the north-eastern border of zambia (yeoman & walker 1967). its collection now from a black rhinoceros in northern province, zambia is possibly a new locality record. the preferred hosts of the adults of a. gemma are large domestic and wild herbivores (theiler 1962; walker 1974; yeoman & walker 1967). yeoman and walker (1967) record collections from four of eight rhinoceroses examined in tanzania, and walker (1974) reports collections from 28 of 54 black rhinoceroses sampled in kenya. considering that only one of the 24 rhinoceroses examined in zambia was infested, it may imply that the distribution of a. gemma in northern zambia is tenuous, or that collections were made during the wrong season. nymphs have been recovered from helmeted guineafowls and cape hares (yeoman & walker 1967). amblyomma hebraeum amblyomma hebraeum is present along the southern and eastern seaboard of south africa, from approximately port elizabeth in the west to southern mozambique in the east. it also occurs in the northern provinces of south africa, south-eastern botswana, and southern and north-western zimbabwe (walker & olwage 1987). the collections from rhinos in the north-eastern, eastern and south-eastern regions of south africa and south-east zimbabwe (figure 1) all lie within its known geographical distribution range (norval 1983; spickett 2013; walker & olwage 1987). it has been introduced into the grassland biome of free state province on white rhinoceroses but is unlikely to survive there (horak et al. 2015). however, should it be introduced into the savanna biome in the north of the same province its establishment there is a distinct possibility. the adults of this brightly coloured tick infest cattle, sheep and goats as well as the larger wildlife species, while its immature stages infest the same hosts as the adults, but also hares, the larger ground frequenting birds and tortoises (dower, petney & horak 1988; horak, golezardy & uys 2007; horak et al. 1987). in zimbabwe, norval (1983) recorded a. hebraeum in 15 of 19 tick collections made from white rhinoceroses, and in the present survey the 30 rhinoceroses examined in that country were all infested. the large proportion of rhinos that were infested in most of the regions in which a. hebraeum occurs, as well as the large numbers of adult ticks that have been collected from rhinos when total collections were made (knapp et al. 1997), is a clear indication that rhinos must be considered as one of the preferred hosts of this species. amblyomma hebraeum is the most effective vector of ehrlichia ruminatium, the causative organism of heartwater in cattle, sheep and goats and some wildlife species (norval & horak 2004). it is also the vector of rickettsia africae, the causative organism of african tick bite fever in humans (kelly 2001). amblyomma rhinocerotis the first two ticks with a south african origin to be described were amblyomma rhinocerotis and amblyomma sylvaticum. amblyomma rhinocerotis had been collected from a rhinoceros at the cape of good hope and a. sylvaticum from angulate tortoises, and both were described by de geer in 1778. by all accounts, the adults of a. rhinocerotis are host-specific parasites of white and black rhinoceroses, and were probably present on these animals in the coastal and wooded inland regions from cape town in the south-west to the kruger national park in the north-east of south africa. however, by the turn of the 19th century a. rhinocerotis and its hosts possibly only survived in the north-eastern region of kwazulu-natal, south africa. subsequent to the review on the ticks infesting larger wildlife in the kwazulu-natal reserves by baker and keep in 1970, there have been no reports, until now, of its presence in south africa. in zimbabwe, duncan (1989) visually estimated that there were between 100 and 500 or more adult a. rhinocerotis on 16 of 18 black rhinoceroses prior to their treatment with an acaricide and relocation from the zambezi valley to safer habitats in the centre of the country. there have been no subsequent reports of its occurrence in that country. in their review of the ixodid ticks which they believe to be endangered, mihalca, gherman and cozma (2011) regard a. rhinocerotis as ‘critically endangered’ if black rhinoceroses were its only hosts within a particular region, whereas on white rhinoceroses it is ‘near threatened’. despite numerous translocations of rhinos from the north-eastern kwazulu-natal reserves, a. rhinocerotis has failed to become established elsewhere, perhaps because rhinos are treated with an acaricide before translocation. its precarious survival even in the kwazulu-natal reserves is highlighted by the fact that only 11 of the 69 rhinoceroses examined there were infested and that only 18 male and 12 female ticks were collected. not one of the 303 white or 9 black rhinoceroses examined in the kruger national park was infested, nor any of the 96 black rhinoceroses in the eastern cape and karoo (figure 1). some of the latter animals were examined in the albany thicket biome, a habitat possibly suitable for the survival of the tick. nor were any of the rhinos examined in namibia, zimbabwe or zambia infested. amblyomma sparsum walker and olwage (1987) have plotted the overall distribution of a. sparsum with most records coming from tanzania and kenya, some from northern and north-western zimbabwe and a single record from south-eastern zambia. according to theiler and salisbury (1959), there is an isolated record of a. sparsum from grootfontein, namibia. walker (1991) elaborates on this record, stating that it was a male tick collected by a government veterinary officer in 1933. no subsequent collections have been made, and we now believe a. sparsum to be extinct in namibia. the adults of a. sparsum infest two remarkably different groups of hosts. the one group comprises tortoises, monitor lizards and the larger species of snakes and the other rhinoceroses and buffaloes (walker 1974; yeoman & walker 1967). norval (1983) recorded a total of 619 male and 175 female a. sparsum in collections from 66 black rhinoceroses in north-western zimbabwe, and duncan (1989) visually estimated there to be between 10 and 50 ticks on 16 of 18 rhinoceroses he examined in the same region. the collection now of a. sparsum from 23 of 24 black rhinoceroses in north luangwa national park, zambia indicates that it is well established there (figure 1). norval and mackenzie (1981) successfully transmitted e. ruminantium to sheep by means of a. sparsum nymphs that had fed as larvae on an infected sheep. however, transmission via adult ticks that had fed either as larvae or as nymphs on infected sheep, failed. amblyomma tholloni according to walker and olwage (1987), the distribution of a. tholloni is linked to that of its preferred host, the african elephant, loxodonta africana in southern, east, and central africa, as well as in some of the southern countries in west africa. sixteen collections of a. tholloni were made from elephants in the kruger national park during the present survey, while in zimbabwe all 29 elephants and 22 of 24 hippopotamuses examined for ticks, were infested (norval 1983). the recovery of a. tholloni from three white rhinoceroses in the kruger national park, a single black rhinoceros in zimbabwe and 14 black rhinoceroses in zambia, implies that it may use rhinos as alternative hosts to elephants and hippopotamuses. mihalca et al. (2011) list a. tholloni as coendangered with its elephant hosts as ‘vulnerable’ to extinction. mackenzie and norval (1980) experimentally transmitted e. ruminantium to sheep by means of a. tholloni nymphs that had been fed as larvae on an infected sheep, and by adult ticks that had been fed as nymphs on infected sheep. because of their preference for elephants, the adults of a. tholloni are unlikely to play a role in the transmission of e. ruminantium in the field. in addition, elephants and domestic livestock very rarely share the same habitat, thus free-living a. tholloni questing for hosts are unlikely to be present in the absence of elephants. however, mackenzie and norval (1980) reported that cattle, sheep and goats at the rekomitji research station situated in a game reserve in the zambezi valley of zimbabwe were frequently infested with a. tholloni larvae and nymphs and that cases of heartwater were recorded in domestic livestock at the research station in the absence of any of the known vectors. amblyomma variegatum the geographical distribution of a. variegatum includes north-western zimbabwe, the zambezi strip in north-eastern namibia, much of zambia and thence northwards into sub-saharan africa (walker & olwage 1987). adult ticks prefer cattle as well as large wild herbivores as hosts, while the immature stages infest the same hosts as the adults, and also hares and the larger species of ground-feeding birds (petney, horak & rechav 1987; theiler 1962). theiler (1962) reports adult ticks on white and black rhinoceroses, while walker (1974) records collections from five of 54 black rhinoceroses in kenya. the large proportion of black rhinoceroses infested with a. variegatum in zambia suggests that these animals are amongst the preferred hosts for adult ticks. amblyomma variegatum is an effective vector of e. ruminantium (norval & horak 2004) and also of r. africae (kelly 2001). cosmiomma hippopotamensis joseph burke, a british naturalist made the first ever collection of c. hippopotamensis from a rhinoceros or a hippopotamus about 20 km to the north-west of pretoria in 1840, and a male tick and female tick from this collection were described by denny in 1843. no collections have been made in south africa since then (apanaskevich et al. 2013). in namibia, bezuidenhout and schneider (1972) made the last recorded collections of c. hippopotamensis in kaokoland in the north-west of the country in 1971. they collected 114 adult ticks from vegetation along footpaths used by rhinoceroses to get to water and successfully fed some of these ticks on a rhinoceros calf. one of the three female ticks that engorged on the calf laid a large batch of fertile eggs from which larvae hatched and these were used in studies to determine the life cycle of the tick (apanaskevich et al. 2013). the translocation of rhinos from kaokoland to the etosha national park and settlement of humans in the north-west of the country has probably contributed to the disappearance of c. hippopotamensis in namibia. we think it is now extinct in both south africa and namibia, and perhaps in africa. in their review of ixodid tick species coendangered with their hosts, mihalca et al. (2011) consider c. hippopotamensis’ status as ‘vulnerable’. dermacentor rhinocerinus only two of the 35 dermacentor species that occur world-wide are present in the afrotropical region, dermacentor circumguttatus and d. rhinocerinus (guglielmone et al. 2014). the adults of d. circumguttatus prefer elephants as hosts and those of d. rhinocerinus prefer white and black rhinoceroses (guglielmone et al. 2014; knapp et al. 1997). dermacentor rhinocerinus was first described by denny in 1843 from a male specimen collected from a black rhinoceros in south africa (keirans 1993). dermacentor rhinocerinus is widespread in africa, but as rhino populations decrease or disappear because of poaching, its distribution range is diminishing (keirans 1993). mihalca et al. (2011) consider d. rhinocerinus to be ‘critically endangered’ should black rhinos be its only hosts, and ‘near threatened’ on white rhinos. with the possible exception of pockets in north-eastern kwazulu-natal, d. rhinocerinus, with its rhino hosts, had probably become extinct in the rest of south africa, including the region now known as the kruger national park, by the end of the 19th and beginning of the 20th centuries. we believe that d. rhinocerinus has been re-introduced into the kruger national park with rhinoceroses from the kwazulu-natal parks (braack et al. 1995). its prevalence on rhinos in the park and surrounding conservancies now appears to be greater than that in the kwazulu-natal nature reserves. the larvae and nymphs of d. rhinocerinus feed on rodents (horak & cohen 2001), for which they probably quest from the soil surface or from the base of tufts of grass. the brightly coloured questing adult ticks are commonly encountered high up on thick grass stems along the verges of roads and game paths, wherever there are dense populations of rhinos in the kruger national park. since 1933, when it was collected from a black rhinoceros at grootfontein, no collections of d. rhinocerinus have been made in namibia (walker 1991). we now consider it to be extinct there. during the 1970’s and 80’s d. rhinocerinus was present in the south-east and south-west of zimbabwe and at several localities in the zambezi valley in the north-west of the country (duncan 1989; norval & colborne 1985). this may no longer be true after the poaching and consequent precautionary translocation of rhinoceroses that has taken place. none of the 30 rhinoceroses examined in south-eastern zimbabwe in the present study, were infested. although d. rhinocerinus was not present amongst the ticks collected from rhinos in zambia, it has previously been reported as occurring there (keirans 1993). hyalomma glabrum hyalomma glabrum, previously referred to as hyalomma marginatum turanicum, was reinstated as a valid species by apanaskevich and horak (2006). it is the most colourful of the three hyalomma species that occur in south africa. in addition to the ivory coloured band that encircles the distal margin of each segment of its legs, the dorsal surface of each segment is coated with a strip of ivory coloured enamelling. this is especially distinct on the segments of the hind legs. hyalomma glabrum is a strictly south african tick and is the only hyalomma sp. whose distribution is confined to the southern hemisphere. it is present in nama karoo and succulent karoo biomes in the eastern, western and northern cape provinces (apanaskevich & horak 2006). it would seem that h. glabrum and h. rufipes are mutually exclusive in their habitat preferences. the adults of h. glabrum have a preference for large herbivores and large numbers have been collected from cape mountain zebras and especially eland in the mountain zebra national park. its immature stages have been collected from scrub hares and birds in the park (horak et al. 1991a). the presence of adult ticks on black rhinoceroses after their re-introduction into the mountain zebra national park and the karoo national park more than a century after their disappearance in the karoo is thus not unexpected. the collections from black rhinoceroses are first records on these animals. hyalomma rufipes hyalomma rufipes is a drought-tolerant species and with the exception of the north-eastern regions of mpumalanga and limpopo provinces, the eastern regions of kwazulu-natal, the eastern free state and the southern regions of the western cape province, it is present throughout south africa (spickett 2013). in agreement with this pattern of distribution, no h. rufipes were collected from rhinoceroses in the kruger national park and surrounding conservancies, nor in the north-eastern parks of kwazulu-natal. hyalomma rufipes was replaced by h. glabrum in the karoo and their occurrence seems to be mutually exclusive (figure 1). hyalomma rufipes was the predominant species in the central and western free state and northern cape province, and was present on black rhinos in all three regions in which collections were made in namibia. the preferred hosts of the adults of h. rufipes are large domestic and wild ruminants, particularly cattle, giraffes and eland (dreyer, fourie & kok 1998; horak et al. 2007), while the immature stages infest cape hares, scrub hares and birds (horak & fourie 1991; van niekerk, fourie & horak 2006). it was the second most prevalent species collected from rhinos in southern africa in the present study (figure 1), and rhinos must be considered as one of the preferred hosts of adult h. rufipes. hyalomma rufipes is a vector of babesia occultans, the causative organism of benign babesiosis in cattle, with infection passing transovarially from one generation of adults to the next (gray & de vos 1981). it is also the most effective vector in south africa of the virus causing crimean-congo haemorrhagic fever in humans (swanepoel et al. 1983). hyalomma truncatum hyalomma trunctum is a drought-tolerant species and is absent in the moist eastern as well as the colder highveld regions of south africa (spickett 2013). it is the only species that was collected in every region in which rhinos were examined (figure 1). a large number of collections were made from rhinos in the free state and northern cape provinces, it was the only hyalomma species collected from rhinos in the kruger national park and the predominant species on animals in the etosha national park. the adults of h. truncatum prefer large herbivores as hosts and large numbers have been collected from giraffes and eland (horak et al. 2007), while the immature stages infest cape hares, scrub hares and murid rodents (horak & fourie 1991; horak et al. 1991a). judging by the large number of rhinos that were infested and that ticks were present on them in every region included in the present study, rhinos must be considered as one of the preferred hosts of the adults of h. truncatum. hyalomma truncatum is a vector of babesia caballi, the cause of equine piroplasmosis, with infection passing transovarially from one generation of adults to the next. the females also produce an epitheliotrophic toxin responsible for sweating sickness in calves (norval & horak 2004). rhipicephalus species the ticks, r. follis, r. gertrudae and r. simus, are similar in appearance but the denseness of punctations on the conscuta of the males and scuta of the females varies (walker, keirans & horak 2000). although there is some overlap, their geographical distributions differ (walker et al. 2000). rhipicephalus follis is moderately punctate and is associated with mountainous terrain mainly east of longitude 24º. rhipicephalus gertrudae is heavily punctate and is present in drier regions west of this longitude and in the winter rainfall regions of the western and northern cape provinces where summers are hot and dry. it is also present in namibia. but for four irregular rows of large punctations, the conscutum of r. simus males is smooth. it is widespread in the northern, eastern and south-eastern regions of south africa. the collections made from rhinoceroses in the various regions reflect the distributions of these three ticks. all of them prefer monogastric animals such as zebras, warthogs and now also rhinoceroses, as well as the larger carnivores as hosts, but buffaloes, elands and cattle may also be infested (horak et al. 2007). their larvae and nymphs prefer murid rodents as hosts (walker et al. 2000). a single collection of r. longiceps was made from a black rhinoceros in namibia. it is the only rhipicephalus species, which, relative to its size, has long mouthparts (walker et al. 2000). its distribution is confined to namibia and angola and in total very few collections have been made (walker et al. 2000). these collections include ticks from warthogs and a giraffe and now also a rhinoceros (horak et al. 1992; horak et al. 1983). rhipicephalus maculatus is the only ornate rhipicephalus species present in southern africa. it is present in a broad strip of coastal mosaic vegetation and adjacent woodlands from durban in kwazulu-natal northwards to somalia (walker et al. 2000). it has short but sturdy mouthparts and its preferred hosts are large mammals with thick hides, such as elephants, rhinoceroses, warthogs, bushpigs and buffaloes (baker & keep 1970; horak, boomker & flamand 1991b; horak et al. 2007; walker et al. 2000). several of these animals do not have dense hair-coats and hence ticks with short mouthparts are prone to be removed by grooming or predation by red-billed oxpeckers, buphagus erythrorhyncus (bezuidenhout & stutterheim 1980). infestation of rhinos with r. appendiculatus nymphs and with r. eversti eversi adults should be regarded as incidental. rhipicephalus neumanni occurs in the drier western regions of south africa and in southern namibia (walker et al. 2000), and both animals that were infested were sampled in the northern cape province. rhipicephalus zumpti is a rather rare species and is present in coastal woodland and adjacent regions in kwazulu-natal and the eastern cape province (walker et al. 2000). it also seems to have a preference for monogastric mammals and fairly large numbers have been collected from bushpigs in north-eastern kwazulu-natal (horak et al. 1991b) and now three males from two black rhinoceroses in the eastern cape province. general although fewer black than white rhinoceroses were sampled for ticks, they were examined in more geographic regions and hence a greater number of tick species were recovered from them. those in zambia alone harboured three species not collected elsewhere. the central and south eastern region of limpopo province combined with northern gauteng constituting the central northern provinces, was the most species-rich with seven species collected from rhinos. most of the ticks that were collected from rhinos in the present study are characterised by one or two features; they are ornate and/or have long or sturdy mouthparts. all the amblyomma spp. collected from the rhinos are ornate and have long mouthparts. their long mouthparts not only ensure a sturdy hold-fast on the thick hides of their hosts but also have the advantage of making them less prone to being removed by grooming or to predation by oxpeckers because of the difficulty with which they are dislodged. their ornamentation possibly alerts oxpeckers to the fact that they will be difficult to remove. dermacentor rhinocerinus is ornate and has medium length, sturdy mouthparts. the three hyalomma spp. have long mouthparts and the legs of h. glabrum are ornate. rhipicephalus longiceps has long mouthparts and r. maculatus is ornate and has short but sturdy mouthparts. moreover, r. maculatus adults probably escape severe predation by attaching amongst the amblyomma spp. on the ventral aspects of their hosts’ bodies. in addition, the colouration on the scuta of r. maculatus females mimics that of a. hebraeum females. rhipicephalus follis, r. gertrudae and r. simus have short mouthparts, but with h. truncatum, attach to the tail and its tip within the tailbrush. conclusion the ticks that infest rhinoceroses in southern africa are generally ornate and have long or robust mouthparts. these comprise six amblyomma species and d. rhinocerinus, h. glabrum and r. maculatus, as well as the now possibly extinct species cosmiomma hippotamensis. amongst these ticks a. rhinocerotis is in danger of extinction in south africa, while d. rhinocerinus is likely to survive in the north-eastern regions of the country for as long as rhinoceroses are also present. the only species that was present in all ten regions in which ticks were collected was h. truncatum, followed by h. rufipes in seven regions. the latter two species and a. hebraeum and a. variegatum are important vectors of disease to domestic livestock. acknowledgements we express our gratitude to all the technicians, rangers, wardens and nature reserve managers, who assisted with tick collections and to dylan smith who assisted with collections in the northern cape province and ortwin aschenborn who collected ticks from rhinos in the hardap nature reserve in namibia. we are also grateful to sanparks and the provincial nature reserve authorities as well as private institutions and individuals for granting permission to make collections from rhinos in the reserves under their control. the participation of the senior author in the project was funded by a grant from the national research foundation. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions i.g.h. was the project leader, identified all the ticks and was responsible for writing the first and final drafts of the manuscript. c.r.b., d.g., g.h. and m.h. collected ticks from rhinos in the kruger national park and its surrounds. m.h. also collected ticks from rhinos in north west province and assisted d.s. with the collection of ticks in zambia. d.v.c. collected ticks in kwazulu-natal, d.p. and d.z. collected ticks in the eastern cape province, p.j.n. collected ticks in the free state, d.n.m. collected ticks in the northern cape province, c.m.f. collected ticks in zimbabwe and j.w.k. collected ticks from rhinos in namibia. a.h. reviewed the data for possible statistical analysis. all authors read and approved the manuscript. references apanaskevich, d.a. & horak, i.g., 2006, ‘the genus hyalomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum delpy, 1949 (acari, ixodidae) as a valid species with a redescription of the adults, the first description of its immature stages and notes on its biology’, onderstepoort journal of veterinary research 73, 1–12. https://dx.doi.org/10.4102/ojvr.v73i1.164 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81–105. walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. walker, j.b. & olwage, a., 1987, ‘the tick vectors of cowdria ruminantium (ixodoidea, ixodidae, genus amblyomma) and their distribution’, onderstepoort journal of veterinary research 54, 353–379. yeoman, g.h. & walker, j.b., 1967, the ixodid ticks of tanzania. a study of the zoogeography of the ixodidae of an east african country, commonwealth institute of entomology, london. abstract introduction materials and methods results ethical considerations discussion conclusion acknowledgements references about the author(s) m. ozkan timurkan department of virology, atatürk university, turkey m. eray alcigir department of pathology, ankara university, turkey citation timurkan, m.o. & alcigir, m.e., 2017, ‘phylogenetic analysis of a partial l1 gene from bovine papillomavirus type 1 isolated from naturally occurring papilloma cases in the northwestern region of turkey’, onderstepoort journal of veterinary research 84(1), a1450. https://doi.org/10.4102/ojvr.v84i1.1450 original research phylogenetic analysis of a partial l1 gene from bovine papillomavirus type 1 isolated from naturally occurring papilloma cases in the northwestern region of turkey m. ozkan timurkan, m. eray alcigir received: 10 mar. 2017; accepted: 16 may 2017; published: 28 june 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study was aimed at the molecular characterisation of bovine papillomavirus type 1 (bpv-1) isolated from papilloma cases in the northwestern region of turkey. bpv-1 is a widely occurring oncogenic virus in cattle and is associated with benign epithelial neoplasia which causes significant economic losses in dairy and beef cattle because of treatment costs. in this study, 29 suspected papilloma specimens were collected from cattle in northwestern turkey. these samples underwent molecular characterisation via the polymerase chain reaction (pcr) and sequencing analysis as well as macroscopic and histopathological examination. the histopathological examinations confirmed papilloma as the main lesion type in the specimens. of the 29 papilloma-like tissue samples that were collected, 11 (i.e. 37.93%) were detected as positive and determined as containing bpv-1 (11 of 11, 100%). using a partial sequence for the l1 gene acquired from genbank, phylogenetic analysis confirmed the presence of bpv-1 and revealed that the infection might have originated in cross bred domestic and imported cattle. this study provides potentially useful information on the origin and spread of this disease. its results can potentially aid in the development of appropriate control measures and therapeutic or vaccination strategies against the bpv-1 strain of bovine papillomatosis. introduction bovine papillomavirus (bpv) has a double-stranded, circular, 8-kb dna genome. bpv displays tropism for mucosal and skin tissues, squamous epithelium and mesenchymal tissue. infection is associated with the development of benign neoplastic lesions (alcigir, atalay vural & timurkan 2016; shafti-keramat et al. 2009). although bpv types are species-specific, bpv-1, bpv-2 and bpv-13 can infect equids as well as cattle and cause the development of tumours in these species (bocaneti et al. 2016). to date, 14 bpv types have been defined and classified into four genera: xipapillomavirus (bpvs 3, 4, 6, 9, 10, 11 and 12), deltapapillomavirus (bpvs 1, 2, 13 and 14), epsilonpapillomavirus (bpvs 5 and 8) and dyoxipapillomavirus (bpv 7) (hamad et al. 2016; lunardi et al. 2013). holstein, simmental, swiss and jersey cattle, as well as each of these breeds crossbred with anatolian black cattle are the main cattle breeds in turkey constituting important sources of dairy and beef products (bor 2014). bpv genotypes cause papillomatosis, a disease which is responsible for significant economic losses because of growth reduction, weight loss and decreased milk production in the infected animals (santos et al. 2016). therefore, greater knowledge of this oncogenic disease would have a great impact on cattle breeding in turkey. the natural carriers and primary source of bpv are cattle. the virus enters the body through scratches or other injuries, and infection spreads via both direct and indirect contact with infected cattle. the infection also appears to spread through contact with contaminated materials, milking machines and semen. other factors, including malnutrition, hormonal imbalances, mutations and long-term exposure to sunlight can increase the risk of infection by causing immunodeficiency (hamad et al. 2016; lindsey et al. 2009). recent epidemiological reports indicate that bpv is prevalent in asian countries such as iraq (hamad et al. 2016) and turkey (ataseven, kanat & ergun 2016; dagalp et al. 2017); european countries such as italy (grindatto et al. 2015); african countries such as nigeria (jeremiah, fagbohun & babalola 2016) and american countries such as brazil (alcântara et al. 2015) and mexico (löhr et al. 2005). this study aimed to identify and characterise the bpv-1 strains prevalent in turkey. this study demonstrates the molecular detection and characterisation of bpv dna from cutaneous warts in some indigenous breeds of cattle in northwestern turkey. materials and methods animals and sample collection within this context, samples were collected from cases diagnosed as papilloma, a national project (ankara university, research scientific projects; project no: 14b0239001), and sample collection continued after the project ended. of all the samples from suspected papilloma cases, 29 were collected by registered veterinarians from several cattle slaughterhouses in various provinces (ankara, bursa and samsun) of northwestern turkey. of the 29 samples, 11 were from anatolian black, holstein, simental and jersey (half-breed) cattle with a papilloma diagnosis. the study sample also included cows with bovine papillomatosis that were brought to private clinics by their owners between march 2014 and november 2016 (table 1). the collected samples had varying diameters (1 cm – 10 cm) and were harvested from different parts of the body (such as the udder and teat, neck, cranial and caudal abdominal region, and also plica genus). each sample was immediately divided into two parts, which were either stored in a deep freezer for subsequent molecular analysis or fixed in 10% neutral buffered formalin for histologic examination (hamad et al. 2016). table 1: bovine papillomavirus samples and their origins. polymerase chain reaction analysis a nucleic acid extraction kit (vivantis technologies sdn bhd, shah alam, selangor, malaysia) was used to extract dna from 11 frozen tissue samples according to the manufacturer’s instructions. a primer set specifically designed for bpv-1 was used (forward 5’ – ggagcgcctgctaactatagga – 3’; reverse 5’ – atctgttgtttgggtggtgac – 3’) to obtain 301 bp dna fragments. the samples were also evaluated in terms of bpv-2 and bpv-4 primer sets (lindsey et al. 2009). the polymerase chain reaction (pcr) amplification was performed following a standard protocol in 100 mm tris hcl (ph 9.0), 500 mm kcl, 1.5 mm mgcl2, 0.2 mm dntp, 0.25 mm of each primer and 2.5 units of taq dna polymerase (thermo fisher scientific, waltham, ma, usa). the pcr cocktail (final reaction volume, 30 µl) was amplified under the following conditions: 35 cycles of denaturation at 94 °c for 60 s, annealing at 50 °c for 60 s and extension at 72 °c for 60 s followed by a final extension at 72 °c for 10 min. the pcr products were detected by electrophoresis on a 1.0% agarose gel containing ethidium bromide, which was placed in a tris-acetate-edta (tae) buffer and run at a constant voltage (120 v) for approximately 25 min. dna was visualised using a vilber loumart gel documentation system (vilber lourmart, marne-la-vallée, france). bovine papillomavirus type 1 sequencing a total of 11 bpv-1-positive specimens, as confirmed by pcr, were selected as representative of the bpv’s geographical distribution and their viral genome type was sequenced. for sequencing analysis, pcr products were purified using a genejet gel extraction kit (thermo fisher scientific, waltham, ma, usa). the cleaned pcr products were sequenced using a big dye terminator v3.1 cycle sequencing kit (applied bio systems, forester city, ca, usa) at the pendik veterinary control institute, i̇stanbul, turkey. sequenced products were run on the applied biosystems abi, 3100 (applied biosystems, forester city, ca, usa). phylogenetic analysis of bovine papillomavirus type 1 the sequences obtained in this study were subjected to phylogenetic analysis. sequences were aligned using bioedit software (version 5.0.6; north carolina state university, raleigh, nc, usa) (hall 1999). to identify evolutionary relationships among the analysed sequences, we performed phylogenetic analysis with a maximum-likelihood phylogenetic tree by using molecular evolutionary genetics analysis (mega) software (version 6.0; tokyo metropolitan university) (tamura et al. 2013). the maximum-likelihood phylogenetic tree was used to generate a general time-reversible model by using a discrete gamma distribution with five rate categories and test of phylogeny was performed using a bootstrap method with 1000 bootstrap replicates. pathomorphological examination eleven bpv-1 positive cases, which included solid masses, were derived from various body parts including the neck (n = 2), plica genus (n = 1), cranial and caudal abdominal region (n = 5) and mammary region (n = 3). papilloma suspected masses were evaluated according to size, weight, elasticity, colour and appearance of the outer and cut sections. all collected samples were processed routinely after fixation in 10% buffered formalin. the samples were embedded in paraffin wax and cut into 4-μm-thick sections. the sections were stained according to haematoxylin-eosin (h&e) staining protocols, then evaluated using a light microscope (leica, dm4000) and photographed with a camera attachment (bioscience, mbf cx9000). results polymerase chain reaction results bpv-1 dna was detected in 11 of the 29 collected papilloma samples. all 29 samples were negative for bpv-2 and bpv-4 dna. the 11 bpv-1-positive samples produced dna fragments for the l1 gene that were 301 bp in length. these fragments were amplified using bpv-1 specific primers. pcr analysis showed that 100% (11/11) of the analysed bovine papillomatoses were induced by bpv-1 in the assessed turkish cattle populations which included both imported breeds and crossbreeds being raised in turkey. bovine papillomavirus type 1 sequencing sequencing analysis was conducted on the l1 gene pcr products amplified from the collected samples and revealed that they were similar and of a single type (bpv-1). the results confirmed the presence of bpv-1, with a 97% sequence identity to the majority of the basic local alignment search tool (blast)-searched bpv-1 sequences. this finding is a genotypic confirmation of the presence of bpv-1 as a primary causative agent of bovine papillomatosis in cattle in turkey. evaluation of phylogenetic analysis a phylogenetic tree was generated using retrieved genome sequences that were deposited in genbank and compared with the sequence (figure 1) of the 11 bpv turkish strains (in table 1 with accession numbers). the aligned sample sequences were classified as bpv-1 (genus deltapapillomavirus). the aligned sequences showed a high percentage sequence identity to the nucleotide sequence of bpv-1 and to the sequence of a bpv strain isolated in japan (accession number: x02346). figure 1: phylogenetic tree showing the identified turkish bpv sequence. the sequence was found to belong to the genus deltapapillomavirus. the sequence was constructed via the maximum likelihood method using mega 6 software. pathomorphological findings macroscopically resected skin samples suspected of papilloma were greyish-white multilobulated outgrowths with a firm consistency (figure 2). some of the skin showed brownish-black pigmentation. the cut sections of the skin warts were dark brownish-black. homogeneous, greyish-white connective tissue was observed just beneath the warts. histopathological analysis revealed that the epidermal part of the tumours comprised mostly hyperplastic spinosum layer cells and other proliferative neoplastic epidermal cells. hydropic or vacuolar degeneration was observed in some hyperplastic spinosum cells, as well as koilocytes that were characterised by enlarged, pale eosinophilic and vacuolated cytoplasm, and pyknotic nuclei with eccentric localisation. this gave rise to a reticular appearance together with epithelial junctions in the form of bridges. in the granular layer, keratohyalin granules of eosinophilic and cytoplasmic particles were attached to those cells. in addition, acanthosis, orthokeratosis and parakeratotic hyperkeratosis were present (figures 3 and 4). in addition, some proliferative activity and vascularization was observed in the connective tissue of papilloma consisting of parenchyma. figure 2: macroscopic appearance of papilloma. figure 3: spinosum layer proliferation (bilateral white arrows), hyperkeratosis (bilateral black arrows), acanthosis (a). figure 4: vacuolar degeneration in spinosum layer cells (black arrows), formation of bridges between epithelial cells because of stretching of tonofibrillary junctions (white arrows). ethical considerations this study was approved and confirmed by the local ethical committee at ankara university, ankara, turkey (number: 53184147/56050, date: 21 october 2013). discussion in this study, pcr was used to analyse viral dna and evaluated histopathology diagnosed cases of bovine papillomatosis to identify and characterise the bpv-1 genotype that exists in turkey and to detect its lesions on cattle skin. genotype-specific primers were used to identify and characterise bpv-1 and findings were confirmed by sequencing and phylogenetic analysis, which are considered to be the most appropriate methods for characterising bpv (grindatto et al. 2015). several studies (grindatto et al. 2015; santos et al. 2016) have been conducted to determine the prevalence of bpv infection in cattle, the most prevalent types of the virus, their geographic distributions and the risk factors involved in the development of skin papillomas. some recent turkish studies (ataseven et al. 2016; tan et al. 2012) described pcr-based molecular typing of bpvs in cattle with cutaneous papillomatosis, but this typing was not confirmed by phylogenetic analyses. in our study, we concluded that infection with several of the bpv types described in the aforementioned studies does not always cause papillomas. sometimes, fibropapilloma and papilloma-like lesions can also occur on the skin of cattle. however, in this study, we found a greater tendency for papilloma development among warts on cattle skin infected with several sub-clusters of bpv-1 with this finding supporting a papilloma diagnosis. this situation was of interest because different bpv-1 strains with a wide geographical distribution are found in turkey. our results indicate that most bpv infections in cattle are caused by bpv with only one genotype. in contrast, other studies have revealed the occurrence of coinfections (grindatto et al. 2015), some with two, three and four bpv genotypes having been reported (lindsey et al. 2009). however, we detected only bpv-1 in our study. this finding might indicate the resistance of the animals to other genotypes, or it may suggest that animals are infected by bpv-1 only once in their lives. bpv-1 dna was detected in 11 samples in our molecular analyses, confirming the presence of bpv-1, which belongs to the genus deltapapillomavirus. phylogenetic analysis showed that the strains were closely clustered according to province. furthermore, the samsun strains, which are part of the same sub-cluster, were closer to the reference strain (accession number: x023467) but the ankara and bursa strains were not. this study revealed that highly pathogenic bpv-1 is widespread in turkey, especially in the cities of ankara, bursa and samsun. this genotype has been found in association with the development of cutaneous papillomatosis as papilloma or fibropapilloma (alcigir et al. 2016). this genotype (bpv-1) has been found in association with the development of cutaneous papillomatosis manifesting as papilloma or fibropapilloma (alcigir et al. 2016). when the strains in our study were grouped together, we discovered two different sub-clusters, namely the ankara strains and the samsun–bursa strains, with each sub-cluster being closely related to each other and similar in terms of nucleotide sequences. we also found some chronological differences and similarities among the collected samples. for instance, the samples from the ankara province were collected in 2014, whereas the samples from the samsun and bursa provinces were collected in 2015 and 2016. amplicons obtained from bpv isolates via pcr were sequenced and comparative sequence analysis showed a 93.9% – 100% identity among the bpv isolates and 95.0% – 99.2% identity between our isolates and the bpv-1 reference strain (x023467) (data not shown). in addition, other papillomaviruses tend to cause papillomas in organs such as the bladder and more frequently, in the oesophagus. these lesions occur more commonly with the solid forms. therefore, it appears likely that that bpv-1 might have an affinity for the skin, resulting in lesions on the skin rather than on the mucosa. to the best of our knowledge, this is the first molecular characterisation of bpv-1 according to geographic distribution in turkey since previous studies did not investigate the presence of bpv-1 in the samsun and bursa provinces. in the current study, we showed that bpv-1 infection is also prevalent in the ankara province, a finding that contributes significantly to the body of knowledge regarding the geographical distribution of the various types of bpv in turkey because a limited number of molecular studies were previously conducted in this region, such as those of dagalp et al. (2017) in the provinces of ankara, hatay, adana, izmir and kars and by ataseven et al. (2016) in adana, hatay and osmaniye (figure 5). we propose that conducting more phylogenetic studies will further our understanding of genetic relationships among the bpv types and their relative prevalence across the different provinces in turkey. identifying these strains and developing regional vaccine programmes are extremely important. figure 5: map of turkey showing location of the studies in black. analysis of bpv-1 isolates collected from the northwestern provinces of turkey revealed a possible relationship with papilloma lesions as indicated by the present study results, or with fibropapilloma lesions as indicated by the results of alcigir et al. (2016). however, further study is warranted to determine the relationship between specific neoplastic lesions and the corresponding strains of bpv-1. in terms of both gross pathology and histopathological findings, this close relationship was present between multiple formations, shapes and epidermal and/or connective tissue patterns of neoplastic tissue in papilloma cases from older cattle. we observed that papilloma masses were mostly encountered in the thin-skinned regions of young cattle. further analysis of statistical data is required to elucidate this close relationship. the results of this study suggest that bpv-1 infections may occur preferentially in thin-skinned areas or after corruption of skin integrity because of trauma such as abrasions in susceptible regions such as the plica genus, abdominal region, udder and teats. therefore, those locations may have a greater predisposition to viral infections in young cattle. accordingly, bpv-1 cutaneous oncogenicity should be further investigated on the basis of the localisation of tumours, cattle age and breed. in addition, it must be determined whether these predispositions are characteristic of anatolian black, holstein (half-breed), jersey (half-breed) as well as other cattle breeds. the papillomavirus, especially bpv-1, might have the ability to evolve between anatolian crossbred herds. however, new studies in this field have enabled the elucidation of molecular and histological differences among the subtypes or strains of the different types of bpv. conclusion in conclusion, to our knowledge, this is the first study to detect and characterise the bpv-1 genotype in turkey. its findings have assisted in detecting bpv in neoplastic tissue by clarifying its interaction with the skin. in the longer term, our findings may also help to reduce the economic losses related to bovine papillomatosis by providing the basis for the implementation of vaccination programmes in northwestern turkey. acknowledgements 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kafkas üniversitesi veteriner fakültesi dergisi 18, 739–744. https://doi.org/10.9775/kvfd.2012.5341 abstract introduction materials and methods results discussion conclusion acknowledgements references appendix 1 about the author(s) ayesha hassim department of veterinary tropical diseases, university of pretoria, south africa edgar h. dekker department of agriculture, forestry and fisheries, south africa charles byaruhanga department of veterinary tropical diseases, university of pretoria, south africa tommy reardon department of agriculture, forestry and fisheries, south africa henriette van heerden department of veterinary tropical diseases, university of pretoria, south africa citation hassim, a., dekker, e.h., byaruhanga, c., reardon, t. & van heerden, h., 2017, ‘a retrospective study of anthrax on the ghaap plateau, northern cape province of south africa, with special reference to the 2007–2008 outbreaks’, onderstepoort journal of veterinary research 84(1), a1414. https://doi.org/10.4102/ojvr.v84i1.1414 original research a retrospective study of anthrax on the ghaap plateau, northern cape province of south africa, with special reference to the 2007–2008 outbreaks ayesha hassim, edgar h. dekker, charles byaruhanga, tommy reardon, henriette van heerden received: 31 dec. 2016; accepted: 04 july 2017; published: 28 sept. 2017 copyright: © 2017. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract anthrax is a zoonotic disease caused by the gram-positive, endospore-forming and soil-borne bacterium bacillus anthracis. when in spore form, the organism can survive in dormancy in the environment for decades. it is a controlled disease of livestock and wild ungulates in south africa. in south africa, the two enzootic regions are the kruger national park and the ghaap plateau in the northern cape province. farms on the plateau span thousands of hectares comprising of wildlife – livestock mixed use farming. in 2007–2008, anthrax outbreaks in the province led to government officials intervening to aid farmers with control measures aimed at preventing further losses. because of the ability of the organism to persist in the environment for prolonged periods, an environmental risk or isolation survey was carried out in 2012 to determine the efficacy of control measures employed during the 2007–2008, anthrax outbreaks. no b. anthracis could be isolated from the old carcass sites, even when bone fragments from the carcasses were still clearly evident. this is an indication that the control measures and protocols were apparently successful in stemming the continuity of spore deposits at previously positive carcass sites. introduction the northern cape province (ncp) is in south africa, situated 30° s, 22° e. it is the largest province in south africa spanning 372 889 km2. a dolomitic escarpment elevates the mid-eastern border of the province, extending 275 km to the south west, known as the ghaap plateau (partridge et al. 2010; smit 1978). the province is divided into two ecological areas, namely the savannah biome in the north-eastern half of the province while the south-western half hosts the rare and more arid nama karoo biome (fourie & roberts 1973). the province contains a number of state, provincial and privately owned wildlife conservancy areas. the ncp also services the kgalagadi transfrontier park bordering botswana and the richtersveld transfrontier park bordering namibia (http://www.sanparks.co.za/conservation). because of this predominating savannah in the eastern part of the province, the majority of the remaining land is utilised for extensive farming of sheep, cattle and mixed farming which includes wild game (http://www.southafrica.info/about/geography/northern-cape.htm). the ncp has alkali, phosphorus deficient soils which lead to pica in grazers and browsers in the form of osteophagia and geophagia (boyazoglu 1973; de vos & turnbull 2004; theiler 1912). this animal behavioural characteristic has resulted in a variety of infections with similar pathologies of which ‘lamsiekte’ (botulism, clostridium botulinum, ‘miltsiekte’ (anthrax, bacillus anthracis) and ‘stijfsiekte’ (three day sickness, rhabdovirus) are amongst the most common (theiler 1912; viljoen, curson & fourie 1928). both anthrax and botulism are caused by soil-borne, spore-forming and toxin-producing bacteria. carcasses are typically observed to have an opisthotonic form with oedema or protruding of the tongue for both diseases (edmonds 1922; theiler 1912; van der lugt et al. 1995). anthrax and botulism have been observed to occur on the same farm, even simultaneously at times and this has been a cause for misdiagnosis in the past (kriek & odendaal 1994; theiler 1912, 1927; viljoen et al. 1928). anthrax is an acute or peracute zoonotic disease predominantly affecting livestock and wild ungulates with episodic spill over to humans and carnivores. it is characterised by oedema, sudden death syndrome, black eschars and haemorrhaging from the orifices (turnbull 2008). the disease is caused by the gram-positive, aerobic and endospore-forming bacterium b. anthracis. vegetative b. anthracis cells have a distinct encapsulated, square ended ‘box-shaped’ appearance on giemsa stained blood smears (hugh-jones & de vos 2002; turnbull 2008), which is a means of distinguishing them from other gram-positive rod shaped bacteria microscopically (theiler 1912). the use of selective media and morphological selection are typically employed for bacteriologic isolation of b. anthracis followed by testing for sensitivity to gamma phage and penicillin as well as verification of virulence factors as added confirmation for the bacterium and differentiation from closely related bacillus cereus sensu lato group organisms (knisely 1966; turnbull 1999). infection of a host can be through ingestion, inhalation or cutaneous. once an infected animal has died, its carcass becomes a potential site of infection for the next host (dragon et al. 2005). anthrax is an world organisation for animal health (oie) reportable disease and opening of carcasses is strictly prohibited (turnbull 2008). sporulation is triggered by nutrient shortages and exposure to oxygen (sterne 1937; turnbull 2008). bacterial spore counts are higher where bloody discharge from the orifices and bodily fluids from the carcass soak the ground (bellan et al. 2013). sporulation also takes place when a carcass is opened by scavengers such as vultures (gyps spp./trigonoceps occipitalis/torgos tracheliotos), crows (corvus spp.), jackal (canis spp.) or hyena (crocuta crocuta) (hugh-jones & de vos 2002; turnbull 2008). blowflies are considered as mechanical vectors of anthrax in the kruger national park (knp) because they feed on a carcass and then deposit b. anthracis laden regurgitate on vegetation around the carcass. this contaminated vegetation may then be a potential source of infection to susceptible kudus (tragelaphus strepsiceros) and other browsers or grazers (blackburn et al. 2014; braack & de vos 1990; de vos & turnbull 2004; hugh-jones & de vos 2002). spores in the environment have been recovered by de vos (1990) from bones during archaeological excavations at a site in knp that were estimated to be 200 ± 50 years old. to reduce such spore inoculum in the environment incineration of anthrax carcasses and burial have been the preferred method and in accordance with who and oie guidelines internationally (turnbull 2008). treating anthrax carcasses with 10% formalin would kill the bacteria, deter scavengers that would open the carcass and decrease spread by flies, but remains controversial because of the health and safety issues related to its handling. turnbull (2008) also proposed covering or wrapping carcasses in plastic or tarpaulins to keep the skin intact and reduce vegetative bacilli through putrefaction and thus anthrax spores in the environment. other control methods include covering the carcass in thorns to deter animals from eating at the site, as well as the use of insecticides to inhibit the role of flies and carrion insects on the carcass (williams & barker 2008; williams & richardson 1984). the two anthrax-enzootic areas in south africa (figure 1) are the ncp and knp (bengis et al. 2002; de vos & turnbull 2004; hugh-jones & de vos 2002; smith et al. 2000). while mandatory vaccination schemes have virtually abolished anthrax in livestock in most of the country, these areas remain enzootic because of the predominance of wildlife conservancies and game farms (gilfoyle 2006; turnbull 2008). a large number of animals usually succumb to the disease before it is noticed because of the short incubation period and the large areas involved. kudus are typical fatalities of anthrax, but in 2008 large numbers of antelope and equids were also affected in the ncp outbreaks. the outbreak gained momentum from 2007, and by the end of march 2008, had resulted in the deaths of thousands of heads of game and economic losses to the farmers of the ghaap plateau amounting to millions of south african rand (visagie 2008). it was the largest recorded outbreak in the region in recent history and the department of agriculture fisheries and forestry in south africa mobilised national and provincial state veterinary services to aid in diagnosis, surveillance and control measures in order to stem the outbreaks (nduli 2009; visagie 2008). the aims of this study were (1) to report the 2007/2008 b. anthracis outbreaks in the ncp, (2) to determine the b. anthracis spore concentrations from bone and environmental samples during the 2007–2008 ncp anthrax outbreaks using bacteriological methods and (3) to compare the spore concentrations from bone and soil collected at the same sites in 2012 to determine the spore endurance and/or efficacy of the control measures employed during the 2007/2008 outbreaks in reducing the inoculum in the environment. figure 1: a modified map of south africa detailing the national parks. the two circled areas represent the anthrax-enzootic areas (kruger national park and the ghaap plateau). materials and methods observations, sample collection and control measures during 2007–2008 anthrax outbreaks the first cases of the 2007–2008 anthrax outbreaks in ncp were reported in november 2007 at doringbult farm (figure 2) after heavy spring rainfall during mid-august – november 2007. later that month, the skeletal remains of one female kudu (nc/17) on grootsalmonsfontein and two female kudus (nc/27 and nc/28) on the adjacent farm dikbosch were recovered, all of which were gestating (eh dekker pers. comm., february 2013; nduli 2009). the first isolated cases of anthrax from kudu were identified in the latter farm dikbosch (figure 2). in february 2008, five more kudu carcasses (both males and females) were discovered on dikbosch and kleinsalmonsfontein farms and by the following month anthrax cases included zebra, wildebeest, impala, sheep and kudu along the length of the ghaap escarpment (figure 2). figure 2: anthrax-positive carcass sites indicating the distribution of susceptible species discovered on farms during the 2007–2008 anthrax outbreaks in the ghaap plateau in the northern cape province, south africa. after a kudu at doringbult was confirmed suspicious for anthrax from a giemsa stained blood smear, more comprehensive samples were collected for diagnostics. knp (skukuza veterinary services) and northern cape province veterinary services visited farms to aid farmers in the diagnosis and control of the outbreak. the condition of the carcasses was documented along with collection of blood smear and bone samples (mandibular, orbital, rib, vertebra, femur and/or pelvic bones if possible). soil samples were collected from under the head, abdomen and tail of the carcass for diagnostic purposes. environmental samples such as crow faeces and bone fragments were collected from carcass sites where evidence of scavenger activity was visible (table 1). louse flies (hippobosca rufipes) were observed and collected from recently dead kudu carcasses at clearwater and klipfontein (table 1, figure 3a). random soil samples were also collected from farms at the top of the plateau and pans at the base of the escarpment to determine spore counts in areas not contaminated by fresh carcasses (table 1). figure 3: (a) louse fly (hippobosca rufipes) collected from an anthrax-positive carcass from clearwater, south africa, during the 2008 outbreaks. (b) site on anthrax-positive carcass where hippobosca rufipes had fed. table 1: anthrax-positive carcasses (based on giemsa stained blood smears) and environmental samples collected from farms along the ghaap plateau in the northern cape province, south africa, during outbreaks in 2007–2008 indicating control measures implemented by farm owners and state veterinary services. control measures included vaccination of livestock; treatment of the carcass sites which involved spraying either 10% chlorine or 10% formalin on the carcasses; burning of the carcasses and covering up each animal with black plastic or tarpaulins to increase bacterial vegetative cell death and limit blowfly and scavenger access to the carcass. table 1 indicates the farms affected by the outbreak based on giemsa stained blood smears as well as carcass condition and observable blowfly, louse fly and crow activity around carcasses. collection of soil and bone samples in 2012 from the 2007–2008 anthrax outbreaks carcass sites in march 2012, samples were collected at each of the anthrax positive sites from farms, along the ghaap plateau, which suffered losses in 2007–2008 (table 2). figure 4 indicates the sites visited. soil samples were also collected from gravel pits, river and stream beds as well as dry pans where water concentrates after rain. the soil sampling followed the topography (water movement) of the land and thus included areas not sampled in 2008 in the search for concentrator sites (see figure 1-a1 using the topography of doringbult game farm as an example). bone samples were taken at sites where bones were available. about 10 of the 48 sites visited yielded bones from the 2007–2008 outbreaks because the black plastic or tarpaulin remnants or charring from cremation sites were indicators of anthrax cases where control measures were implemented in 2008 (figure 5). bacillus anthracis positive controls consisted of two bone samples from the original collection in 2008 (nc/14 and nc/29) that were included as bacteriologic isolation controls. figure 4: soil and bone samples collected (n = 62) in 2012 from farms and carcass sites identified as anthrax-positive by farmers and state veterinary services in 2007–2008, south africa. additional soil samples were taken along water runoff paths following the ghaap escarpment drainage topography (indicated as the diagonal ridge line across the map). figure 5: (a) bones collected in 2012 for isolation of bacillus anthracis from doringbult game farm, south africa (table 1 samples ah56 and ah57). (b) the black plastic tarp covers and remaining kudu bones were clearly visible at this site which was sprayed with 10% chlorine before being covered up with the plastic covers in 2008 (table 1 sample nc/10). table 2: the risk analysis survey of soil and bone sampling of the (2007 and 2008) anthrax-positive carcass sites in 2012. bacterial culture one gram samples of all soil, crow faeces and ground bone samples were placed in a mccartney bottle with 9 ml sterile phosphate buffered saline (pbs) and shaken for at least 2 hrs. samples were heat treated at 65 °c for 25 min to select for spores. a 100 µl of this solution was plated out onto polymyxin edta thallous acetate (pet) agar (plet agar from turnbull 2008) where lysozyme was omitted (e.h. dekker, pers. comm., february 2013) and incubated overnight at 37 °c, then further incubated for 24 hrs when colonies were not apparent. serial 10-fold dilutions for spore counts were made and plated in triplicate for dilutions of 1×10-1 to 1×10-6. the average colony forming units (cfu) of b. anthracis from the 3 plates was calculated. isolates were confirmed as b. anthracis when sensitive to penicillin and γ-phage. the louse flies’ heads were removed and directly plated onto 5% impala blood agar followed by overnight incubation at 37 °c. all white, non-haemolytic and ‘ground glass’ domed colonies were tested with penicillin and γ-phage to confirm b. anthracis. isolation methods employed in 2012 were exactly the same as those for 2008 with the exception that trimethoprim sulphamethoxazole polymyxin blood agar (tsbpa) (turnbull 2008) was used in addition to pet and 5% impala blood agar to maximise isolation success. statistical analyses a cursory examination of the spore data appeared to highlight a disparity in isolate counts from the various bone samples collected. the spore count data from each bone sample were compared to determine if any trends were apparent. analysis of variance (anova) was used to evaluate carcass spore counts followed by tukey’s for pairwise comparison test to compare the significance of spore yields between bone samples. fisher’s exact test was used to determine the significance of carcass effect variables such as locality, species, sex and age because of the small sample size. results isolation and spore counts from 2007–2008 outbreaks the first reported cases, where bone samples were collected for bacteriologic diagnostics, were on dikbosch farm (nc/27 and nc/28) with the isolation of b. anthracis. thereafter, bone and soil samples were collected on six other farms in early 2008 (table 1) where a mean of 6.87×103 cfu/g and 1.57×103 cfu/g spores from bone and soil samples, respectively were collected at all 29 carcass sites (figure 6). a mean of 200 spores was isolated from the heads of the louse flies and 300 spores/g from the crow faeces (skukuza state veterinary services laboratory report). the distribution of carcasses was greater on the top of the escarpment (in terms of carcass dispersal) while being confined to the floodplains at the base of the escarpment (figure 4). although the first kudus discovered in 2007 were all female, by the end of the anthrax outbreak, overall twice as many male carcasses were discovered than female carcasses. the numbers of confirmed carcasses were 39 kudus, 6 zebra, 2 impala and 2 wildebeest that died of anthrax. during the collection of samples during the 2007–2008 outbreaks, various control measures were taken to decrease the inoculum of spores in the environment as indicated in table 1. these control methods included spraying carcasses with disinfectants, incineration and covering the carcasses in plastic. figure 6: isolation spore counts for bone samples collected from various accessible parts of a carcass, as well as soil sampled from beneath the mouth, abdomen and head of carcasses from farms along the northern cape province, ghaap plateau, during the anthrax outbreaks of 2007–2008. the mean spore counts isolated from each bone type, soil location and farm area are indicated in colony forming units per gram. average b. anthracis colony counts from pet and blood agar dilution series plates for the various bone samples can be seen in figure 6. the anova evaluation of the spore counts had a significant p-value of 0.028 between bone types, whereas the tukey’s pairwise comparison test only showed significance when comparing the femur (p = 0.029) and pelvic bones (p = 0.049) with the mandibular bone. the mandibular bone generally produced higher spore counts (figure 6), although, no significance was evidenced by comparison of the mandibular to the rib, vertebrae and orbital bones. according to fisher’s scoring, the locality of the carcasses was also significant with a p-value of 0.022. the mandibular bone yielded the highest mean spore counts at all localities, with higher counts observed in skeletons or carcasses at kleinsalmonsfontein followed by grootsalmonsfontein, vlakfontein, doringbult and dikbosch, respectively (figure 6; figure 2-a1). all of these farms are situated on the top of the ghaap escarpment. isolation from the soil under the carcasses at the mouth, abdomen and tail region produced varied spore counts (table 3). this is reflected in a p-value of 0.504 indicating that there is no significance between the sampling location under the abdomen and tail of the carcass (figure 3-a1). bacillus anthracis spore counts from the soil under the mouth generally yielded higher spore counts accounting for 61% of the mean spores isolated from all the farms (figure 6). there is an agreement of the b. anthracis spore counts between the bone and spore counts in the soil at the location of these bones (mouth, abdomen and tail) (table 3). the mean spore counts under the mouth or head represented 61% of the total isolated from soil, while the mean spore counts in the mandibular (45%) and orbital (21%) bones together accounted for 66% of the total isolated. the rib (13%) with the vertebrae (9%) made up 22% of the mean spores isolated from bone versus the soil under the abdomen which represented 21% of the total soil spores isolated. the femur together with the pelvic bones comprise 12% of total spores from bone versus the soil collected from under the hind of the animal reflecting 19% of the mean (figure 6). table 3: bacillus anthracis spore counts obtained from different isolation media from 1 gram of ground mandibular bones collected during the 2007–2008 anthrax outbreaks in the northern cape province, south africa. random soil samples taken from two pans at doringbult did not yield any b. anthracis spores, whereas random samples collected at the lowest points downhill on doringbult of carcasses nc/01 and nc/04 yielded 5×104 and 1.2×104 spores per gram of soil on pet agar, respectively (figure 1-a1). a soil sample collected at grootsalmonsfontein at the lowest point below carcass nc/18 contained 400 spores per gram of soil. isolations in 2012 the soil and bone samples collected in 2012, yielded a variety of bacillus spp. on the different media employed including bacillus subtilis, b. cereus and bacillus thuringiensis, but contrary to expectation, no b. anthracis was found. the control b. anthracis bone samples nc/14 and nc/29 from the original 2008 outbreaks were positive for the presence of b. anthracis spores as indicated in table 3. similar b. anthracis spore counts were obtained in 2008 and 2012 from the reference bone samples nc/14 and nc/29. discussion the isolation of b. anthracis from samples collected in 2007–2008 demonstrates a unique ecology at each carcass site with a variety of vectors such as water, crows, blowflies and louse flies evidencing activity. there is also a difference in b. anthracis yields between bone types and soil samples collected from the carcass sites during this outbreak. the mandibular bone yielded the highest spore counts and mean spore counts under the mouth or head of the carcass yielded the highest counts from soil. control measures were taken during the 2008 anthrax outbreak seemed to reduce the spore counts as no b. anthracis could be isolated from the soil or bone from the carcass sites in 2012. during the anthrax outbreaks of 2007–2008 in ncp, a disproportionate number of adult male kudus died from the disease. this is in keeping with studies in etosha (lindeque & turnbull 1994). this is notable because kudus are browsers and not dependent on as frequent water intake as their grazing counterparts. kudu seemed to be the most susceptible species which died of anthrax, followed by zebra, impala and wildebeest, all of which except the kudu died on the game conservancies. the carcass distribution is not well represented because state veterinary services only intervened on the farms and not the national parks and nature conservancies where the disease is considered part of the natural ecology. on the other game farms, domestic animals and kudu died in greater numbers during the 2007–2008 anthrax outbreaks in ncp. it has been consistently noted that spore counts in the soil appear to be dependent on the opening of a carcass and soil closest to these openings produce higher contagion concentrations (dragon et al. 2005). this can be clearly seen for soil spore counts taken from under carcasses in doringbult with the highest spore counts obtained from soil under the mouth of 2 female kudu carcasses (figure 5 samples n/c04 and nc/05). both of these carcasses had opened heads. the 2 carcasses did differ in spore counts obtained from soil under the abdomen where nc/04 had a noticeable hole in the abdomen while no such observation was made for nc/05. according to bellan et al. (2013), the spore density at a carcass site is dependent on the vegetative cell concentration at host death coupled with the sporulation efficiency of the bacterium, spore survival and propensity for environmental replication. sporulation is triggered by a paucity of nutrients coupled with exposure to oxygen (koehler 2009; minett & dhanda 1941; sterne 1937; turnbull 2008; van schaik, prigent & fouet 2007). as such, soils contaminated by bacterial laden bodily fluids would contain more spores than an area a distance away from the carcass, which was observed by dragon et al. (2005). the higher spore counts obtained from soil under the mouth of carcasses because of haemorrhagic discharge affirms the findings of bellan et al. (2013), dragon et al. (2005) and our study where high spore count were obtained from carcass heads opened by scavengers, thus providing extravasation fluid as enrichment media to the soil for possible vegetative bacterial amplification in the environment. statistically the mandible, ribs, orbital bone and vertebrae produced equivalent b. anthracis spore counts, although the mean spore counts demonstrated better isolation success rates from the mandibular, rib and orbital bones. in the ncp, crows are often observed to peck out the eyes of a carcass (hugh-jones & de vos 2002). because of the lack of blood clotting (leppla 1984) and the exposure of the haemorrhagic fluid in the orbital sockets to the elements; sporulation would be triggered along with increased contagion deposition in the environment. in knp scavengers like vultures and hyeana are responsible for opening the carcass (hugh-jones & de vos 2002). because of a lack or limiting numbers of scavengers in ncp, crows as well as scavengers like jackals and to a lesser extent hyena and leopards are responsible for opening the carcass. blowflies as well as louse flies have been observed on carcasses in ncp (hugh-jones & de vos 2002). louse flies belong to the family hippoboscidae and are blood-feeding flies. these flies although strong fliers, seldom fly more than a few metres, choosing instead to move to the next closest host when disturbed. they are abundant in the summer months and can be found clustered on the perineal and pubic regions as well as the neck and sides of the animal. they have a long standing association with anthrax transmission amongst cattle (howell, walker & nevill 1978). cattle louse flies were observed on kudu carcasses at sites in clearwater (schmidtsdrif) and dikbosch in ncp during the 2007–2008 outbreaks. the louse flies were easily collected from the kudu carcasses and an average of 200 spores were isolated from the head (mouthparts) of a louse fly. as a blood feeder, it has been suggested that this makes it a potential mechanical vector to its next animal host (de vos & turnbull 2004; howell et al. 1978). this however requires further study. the ability of anthrax spores to persist in the earth for extended periods is because of specific soil and climatic conditions, which provide an environment conducive to its survival (de vos & turnbull 2004; smith et al. 2000). soils rich in organic matter, ample in calcium, prone to alkalinity and with an ambient temperature above 15.5 °c are considered optimal for persistence of spores. the exosporium of the spore is negatively charged (this charge and its strength are ph dependent) whereas humus particles are positively charged and are therefore able to chelate, which then provides environmental stability for the spores (hugh-jones & blackburn 2009). the spores can persist in soil and bone for decades or even centuries under such conditions (de vos 1998; wilson & russell 1964). these humus-spore clumps have the added advantage of being buoyant. during flooding, as was the case of the ncp 2007–2008 anthrax outbreaks, these clumps can be deposited or concentrated at water collection sites once the water evaporates, as the spores are highly resistant to uv radiation (hugh-jones & blackburn 2009; hugh-jones & de vos 2002; vilas-boas et al. 2007). many studies have evaluated the influencing factors on the dissemination and survival of b. anthracis that leads to outbreak trends. these include stocking rates, elevation, soil factors as mentioned above, water content and rainfall (barro et al. 2016; blackburn 2010; chikerema et al. 2013; joyner et al. 2010). steenkamp (2013) elucidated the importance of the role of water and topography in the dissemination of anthrax in knp along with various other studies (pienaar 1960; viljoen et al. 1928). the ghaap is unique in its distribution of natural springs, kimberlite pipes and dolomitic sinkholes which serve to flush the area with groundwater during periods of heavy rainfall and after groundwater recharge (smit 1978). this theory is consistent with the high spore counts observed in water collection pans downhill from carcass sites during the 2007–2008 anthrax outbreaks. it can be argued that b. anthracis survival in the soil and its subsequent exposure to inclement conditions could dilute the spores to a negligible detection limit (smith et al. 2000), however, according to de vos (1998) spores can remain stable in bone for decades if not centuries. the stable spore counts from the stored bone samples isolated in 2012 (table 3) are an example of the spores endurance. the pet agar plates used in this study had spore counts equivalent to the tspba. the lack of b. anthracis spores from bone samples collected in 2012 reinforces the effectiveness of control measures employed during the 2007–2008 outbreaks. because of the employment of a variety of control measures, the efficacy of the individual actions of using chlorine, formalin or the black sails or tarps cannot be determined, nor whether all the measures worked synergistically to reduce the inoculum in the environment. it is unclear to what extent the control measures of the 2007–2008 outbreaks managed to further stem the dissemination of spores through dilution in the environment. bones treated at carcass sites were not a source of infection to animals suffering from pica in later seasons because they did not yield viable spores which could pose a threat of infection (de vos 1998; de vos & turnbull 2004). the reduction of inoculum in the environment and the corresponding reduced spore exposure is paramount in the control of anthrax (de vos & turnbull 2004; turnbull 2008; watson & keir 1994). this was indicated when 2012 isolations from the reference bone samples collected during the outbreak (used as positive controls) had spore counts similar to those enumerated at the time of collection. conclusion there are too many variables at each carcass site that could possibly influence the dissemination of anthrax which serves to complicate our understanding of the disease as seen with the 2007–2008 anthrax outbreaks in ncp. modelling which includes all the factors (host, bacterium, vectors and environment) will provide more insight into the unique ecology of anthrax on the ghaap plateau. carcass bones are a reliable source for the successful isolation of b. anthracis. the mandibular and orbital bones as well as soil beneath the head of a carcass proved to be a prodigious source of viable b. anthracis spores from carcasses collected during the 2007–2008 ncp outbreaks the control measures of burning or spraying carcasses with chlorine or formalin and then covering them with tarp or plastic were applied by farmers on the recommendation of the veterinary services during the 2007–2008 outbreaks. as no spores could be isolated in 2012 from the same sites and bones remaining in the environment, this finding indicates that these control measures had greatly reduced or possibly even eliminated the inoculum at the treated carcass sites. acknowledgements we offer our sincere thanks to the farmers of the northern cape for their hospitality and wholehearted co-operation; such studies would not be possible without you. thanks are also due to all the state veterinary services staff involved for their efforts and data contributions as well as the northern cape provincial government for allowing us to conduct this study. the authors would like to acknowledge and thank the national research foundation (nrf) for funding this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.h. was responsible for experimental work, data capture and data analysis. e.h.d. generated field data in 2008 and facilitated field survey data collection in 2012. t.r. generated field data in 2008. c.b. performed statistical analyses and data vetting. 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n.d., from http://eu.wiley.com/wileycda/wileytitle/productcd-0813825563.html williams, h. & richardson, a., 1984, ‘growth energetics in relation to temperature for larvae of four species of necrophagous flies (diptera: calliphoridae)’, australian journal of ecology 9, 141–152. https://doi.org/10.1111/j.1442-9993.1984.tb01352.x wilson, j.b. & russell, k.e., 1964, ‘isolation of bacillus anthracis from soil stored 60 years’, journal of bacteriology 87, 237–238. appendix 1 figure 1-a1: a closer view of water drainage on doringbult and honey’s corner farms in the ghaap plateau in the northern cape province. the water runoff moves towards doringbult and thereafter to honey’s corner farm where mass alluvial deposition occurs (white areas). figure 2-a1: the mean bacillus anthracis spore counts (in colony forming units per gram) obtained from different bones types in the same carcass on farms afflicted with anthrax in 2008, northern cape, south africa. figure 3-a1: mean bacillus anthracis spore counts in colony forming units per gram obtained from soil under different parts of anthrax-positive carcasses on farms on the ghaap plateau during an outbreak in 2008, northern cape, south africa. abstract background materials and methods results discussion conclusion acknowledgements references about the author(s) bo yu college of veterinary medicine, yangzhou university, yangzhou, china institute of animal husbandry and veterinary medicine, guizhou academy of agricultural sciences, guiyang, china yanan zhang institute of animal husbandry and veterinary medicine, guizhou academy of agricultural sciences, guiyang, china li yang institute of animal husbandry and veterinary medicine, guizhou academy of agricultural sciences, guiyang, china jinge xu institute of animal husbandry and veterinary medicine, guizhou academy of agricultural sciences, guiyang, china shijin bu college of veterinary medicine, yangzhou university, yangzhou, china citation yu, b., zhang, y., yang, l., xu, j. & bu, s., 2021, ‘analysis of antibiotic resistance phenotypes and genes of escherichia coli from healthy swine in guizhou, china’, onderstepoort journal of veterinary research 88(1), a1880. https://doi.org/10.4102/ojvr.v88i1.1880 original research analysis of antibiotic resistance phenotypes and genes of escherichia coli from healthy swine in guizhou, china bo yu, yanan zhang, li yang, jinge xu, shijin bu received: 11 may 2020; accepted: 09 dec. 2020; published: 24 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this study was carried out to investigate the resistance phenotypes and resistance genes of escherichia coli from swine in guizhou, china. a total of 47 e. coli strains isolated between 2013 and 2018 were tested using the kirby–bauer (k–b) method to verify their resistance to 19 common clinical antimicrobials. five classes consisting of 29 resistance genes were detected using polymerase chain reaction. the status regarding extended-spectrum β-lactamase (esbl) and the relationship between esbl ctx-m-type β-lactamase genes and plasmid-mediated quinolone resistance (pmqr) genes were analysed. a total of 46 strains (97.9%) were found to be multidrug resistant. amongst them, 27 strains (57.4%) were resistant to more than eight antimicrobials, and the maximum number of resistant antimicrobial agents was 16. twenty antibiotic resistance genes were detected, including six β-lactamase genes blatem (74.5%), blactx-m-9g (29.8%), bladha (17.0%), blactx-m-1g (10.6%), blashv (8.5%), blaoxa (2.1%), five aminoglycoside-modifying enzyme genes aac(3′)-iv (93.6%), aada1 (78.7%), aada2 (76.6%), aac(3′)-ii c (55.3%), aac(6′)-ib (2.1%) and five amphenicol resistance genes flor (70.2%), cmla (53.2%), cat2 (10.6%), cat1 (6.4%), cmlb (2.1%), three pmqr genes qnrs (55.3%), oqxa (53.2%), qepa (27.7%) and polypeptide resistance gene mcr-1 (40.4%). the detection rate of esbl-positive strains was 80.9% (38/47) and esbl tem-type was the most abundant esbls. the percentage of the pmqr gene in blactx-m-positive strains was high, and the detection rate of blactx-m-9g was the highest in ctx-m type. it is clear that multiple drug resistant e. coli is common in healthy swine in this study. extended-spectrum β-lactamase is very abundant in the e. coli strains isolated from swine and most of them are multiple compound genotypes. keywords: antimicrobial resistance; extended-spectrum β-lactamase; esbls; e. coli from swine; plasmid-mediated quinolone resistance; pmqr; resistance genes. background colibacillosis is a common infectious disease in swine caused by pathogenic escherichia coli, and the main manifestations are sepsis, yellow scour and white scour in piglets. as there are many e. coli serotypes, vaccine-based control is not effective and antimicrobials are the main treatment methods at present (yan & zhao 2018; zang & zhou 2019). with high-dose and wide use of antimicrobials in breeding factories and livestock farms, the multidrug resistant (mdr) problem of bacteria is becoming increasingly serious. especially, the infection caused by extended-spectrum β-lactamase (esbl) producing bacteria increases the difficulty to control and represents a major global public health concern. escherichia coli with ctx-ms are the most common species associated with global esbls (peirano & pitout 2019). currently, ctx-m-15 (forming part of the ctx-m-1 subfamily first described in e. coli in 2001 from india [karim et al. 2001]) is the most frequent ctx-m worldwide. this is closely followed by ctx-m-14 (forming part of the ctx-m-9 subfamily first reported in e. coli, k. pneumoniae and shigella spp. isolated in korea in 2001 [pai et al. 2001]). in the meantime, antibiotic resistance genes (args) carried by e. coli of food animal origin would spread along the food chain and eventually cause harm to the public and human health (gao, han & guan 2017; yin, chen & cao 2018). therefore, it is necessary to monitor the prevalence of drug resistance and args in animals, particularly in swine. this study investigated the prevalence of resistance phenotypes and resistance genes of e. coli isolated from swine in guizhou in recent years, analysed esbl status and the relationship between ctx-m-type β-lactamase genes and plasmid-mediated quinolone resistance (pmqr) genes. the findings would provide cues for rationale use of antimicrobials in veterinary practice to reduce transmission of drug resistance in the animal. materials and methods escherichia coli strains a total of 112 faecal swab samples were collected from 47 large-scale pig farms in 13 counties and cities, including guiyang, anshun, bijie, qianxinan, qiandongnan and qiannan in guizhou province between 2013 and 2018. the samples were taken from diseased piglets with yellow and white scour symptoms, streaked out on mackay medium and cultured at 37 °c for 24 h. pink colonies were picked and purified on luria-bertani (lb) agar plate at 37 °c for 24 h, and the plates were stored at 4 °c for subsequent use. isolated bacterial strains were cultured in lb medium at 37 °c for 24 h and tested according to bergey’s manual of systematic bacteriology. the strains were further classified using 16s ribosomal ribonucleic acid (rrna) sequences, which were sequenced using a pair of universal primers (16s-f, 5′-tgt ggg aac ggc gag tcg gaa tac-3′; 16s-r, 5′-ggg cgc agg gga tga aac tca ac-3′) (shahi, singh & kumar 2013). reagent and culture medium macconkey agar medium, eosin-methylene blue agar medium, lb broth, lb agar medium, casein hydrolysate agar (mh agar, muller-hinton agar), and casein hydrolysate broth (mh broth, muller-hinton broth) were purchased from huankai microbial technology, guangzhou; dna markers and premix taq (ex taq version 2.0 plus dye) were purchased from takara bio, dalian; agarose was purchased from biowest, spain. antimicrobial susceptibility disks were purchased from binhe microorganism reagent, hangzhou and they were used to test susceptibility to beta-lactam antibiotics beta-lactam antibiotics (cefotaxime 30 micrograms [μg], ceftazidime 30 μg, cefoxitin 30 μg, ampicillin 10 μg, amoxicillin-clavulanate 20 μg and 10 μg), aminoglycosides (amikacin 30 μg, gentamicin 10 μg, kanamycin 30 μg, streptomycin 10 μg, neomycin 30 μg), tetracyclines (tetracycline 30 μg, doxycycline 30 μg), amphenicols (chloramphenicol 30 μg, florfenicol 30 μg), fluoroquinolones (ciprofloxacin 5 µg, enrofloxacin 5 μg, levofloxacin 5 μg), polymyxin b (300 units) and sulfisoxazole (250 μg and 300 μg). drug susceptibility test the kirby–bauer method (k–b method) was used to test the susceptibility to antimicrobials. escherichia coli atcc®25922 was used as the quality control strain in antimicrobial susceptibility testing. the breakpoints for tested drugs were used as recommended by the standards and guidelines described by the clsi (2018b: m100) and veterinary clsi (vet01-a4/vet01-s2) (clsi 2018a, 2018b). based on the test, e. coli was classified as susceptible (s), intermediate (i) and resistant (r). test of antibiotic resistance genes extraction of bacterial deoxyribonucleic acid strains stored at −80 °c were inoculated in lb broth for recovery, streaked on eosin-methylene blue agar medium and incubated at 37 °c overnight. fresh single colonies on the eosin-methylene blue plate were inoculated in 3 ml lb broth and cultured at 37 °c on a shaker operated at 200 revolutions per minute (r/min) for 12 h. the culture was then centrifuged at 12 000 r/min for 5 min. bacterial pellet was suspended in 500 μl 1 × te, vortexed and boiled for 10 min. bacterial dna was extracted using tianamp bacterial dna extraction kit (tiangen, china) according to manufacturer’s instruction. polymerase chain reaction polymerase chain reaction (pcr) was used to amplify genes from the e. coli strains, including β-lactamase genes (bladha, blacmy-2, blatem, blashv, blactx-m-1g, blactx-m-9g, blaoxa), aminoglycoside modifying enzyme genes (aac(3’)-ia, aac(3’)-iic, aac(3’)-iv, aac(6’)-ib, aada1, aada2, rmta, rmtb), amphenicol resistance genes (cat1, cat2, cmla, cmlb, flor), pmqr genes (qnra, qnrb, qnrc, qnrd, qnrs, qepa, oqxa, oqxb) and polypeptide resistance gene mcr-1. the primers for pcr are listed in table 2 and were synthesised at sangon biotech, shanghai. polymerase chain reaction amplification was performed in a final volume of 25 μl containing 1 μl bacterial dna, 0.5 μl each of primers, 12.5 μl premix taq (ex taq version 2.0 plus dye). parameters for pcr were: initial denaturation for 10 min at 94 °c, followed by 30 cycles of 45 s at 94 °c, 45 s at annealing temperature and 1 min at 72 °c with a final extension of 10 min at 72 °c. the annealing temperatures for each pair of primers are listed in table 1. the amplified pcr products were separated by electrophoresis on a 1% agarose gel in tris-borate-ethylenediaminetetraacetic acid (edta) buffer (tbe) containing 1% goldview nucleic acid gel stain (solarbio, beijing, china) and monitored in gel documentation unit (biorad laboratories, united states). the amplicons were sequenced at sangon biotech, shanghai. sequence alignments were carried out using blast on ncbi website http://www.ncbi.nlm.nih.gov/ to determine the genes of interest. table 1: primers for polymerase chain reaction. ethical consideration no animal-related tests were involved in this study, and the faecal swab samples were provided by the farm owners who volunteered to participate in the study. the sampling protocols, strain isolation and experiments were conducted in accordance with section 20 of the animal diseases act of 1984 (act no. 35 of 1984), technical guidelines for isolation and identification of animal origin escherichia coli (db51/t 2084–2015) and the declaration of helsinki, and were approved by the animal research ethics committee of institute of animal husbandry and veterinary medicine, the college of veterinary medicine, yangzhou university, yangzhou, china. results isolated escherichia coli strains were multidrug resistant drug susceptibility tests showed that 47 e. coli strains isolated from swine had the highest rates of resistance to tetracycline and doxycycline, which were both 95.7% (n = 45), followed by resistance to sulfaisoxazole (89.4%, n = 42), ceftazidime (70.2%, n = 33), amoxicillin (63.8%, n = 30), chloramphenicol (63.8%, n = 30), ampicillin (61.7%, n = 29), florfenicol (57.4%, n = 27) and kanamycin (53.2%, n = 25). the rates of resistance to enrofloxacin, gentamicin, polymyxin b and cefotaxime were 46.8% (n = 22), 40.4% (n = 19), 38.3% (n = 18) and 31.9% (n = 15), respectively. the strains had relatively lower rates of resistance to ciprofloxacin, levofloxacin, neomycin and streptomycin, which were 29.8% (n = 14), 29.8% (n = 14), 19.1% (n = 9) and 17.0% (n = 8), respectively. in addition, the tests also showed that the strain had a rate of resistance to cefoxitin at only 8.5% (n = 4), and none of them was resistant to amikacin (table 2). table 2: antibiotic resistance of 47 escherichia coli isolates over different years. when analysed based on isolation time (table 2), the rates of resistance to ampicillin, amoxicillin, enrofloxacin and levofloxacin increased from 2013 to 2018, whilst the rate of resistance to kanamycin decreased during the same period. on other hand, the rates of resistance to ceftazidime and cefoxitin remained unchanged from 2013 to 2017, and increased in 2018; the rates of resistance to cefotaxime, gentamicin, streptomycin and neomycin were the highest from 2015 to 2017, and decreased in 2018. the rates of resistance of the 47 strains to florfenicol, chloramphenicol, tetracycline, doxycycline and sulfaisoxazole were higher than 50% from 2013 to 2018. for polymyxin b, the rate of resistance was 14.3% (2/14) in 2018, obviously lower than that from 2013 to 2014 (12.8%) and from 2015 to 2017 (55.6%, 10/18). according to the criteria of multidrug resistance test, all 46 strains tested were resistant to more than three classes of antimicrobial agents, indicating serious multidrug resistance in these strains (figure 1). amongst them, 27 strains were resistant to more than eight antimicrobials, four strains were resistant to 15 antimicrobials, of which, ecp27 resisted to as many as 16 antimicrobials (supplementary table 1). figure 1: distribution of escherichia coil strains resistant to antimicrobials. antibiotic resistance genes were detected in all isolates in this study, 20 args were detected (table 3) using pcr-based assays and verified by sequencing and sequence alignment with blasl. they were generally consistent with respective resistance phenotypes. of which, aac(3′)-iv (93.6%, n = 44), aada1 (78.7%, n = 37), aada2 (76.6%, n = 36), blatem (74.5%, n = 35), flor (70.2%, n = 33), qnrs (55.3%, n = 26), aac(3′)-iic (55.3%, n = 26), cmla (53.2%, n = 25) and oqxa (53.2%, n = 25) were prevalent with higher detection rates. amongst the 47 e. coli strains, 39 strains carried β-lactamase genes with a detection rate of 83.0%, of which, the detection rates of blatem, bladha, blashv and blaoxa were 74.5% (35/47), 17.0% (8/47), 8.5% (4/47) and 2.1% (1/47), respectively. the blacmy-2 gene was not detected, the detection rate of blactx-m-9g in ctx-m was 29.8% (highest) and the detection rate of blactx-m-1g was 10.6%. amongst the strains positive for aminoglycoside-modifying enzyme genes, only one strain was positive for aac(6′)-ib. the detection rates of cmla (53.2%, 25/47) and flor (70.2%, 33/47) were higher, which were the main causes for amphenicols resistance. three pmqr genes qnrs, oqxa and qepa were detected at rates of 55.3% (26/47), 53.2%, (25/47) and 27.7% (13/47), respectively. in addition, the detection rate of the mobilised colistin resistance gene mcr-1 was 40.4%. table 3: detection rate of antibiotic resistance genes in 47 escherichia coli isolates. antibiotic resistance genes were detected in all 47 strains, of which, 28 (59.6%) strains had more than eight args, nine (19.1%) strains had more than 10 args, and ecp27 had 14 args (supplementary table 1). various extended-spectrum β-lactamase genes were present in the isolates amongst the 47 strains tested, 38 (80.9%) strains carried esbls and 20 of them harbored various number of esbls. the most frequent was blatem+blactx-m-9g (26.3%, 10/38); three strains were found to have three esbls genes and ecp27 had four esbls genes blatem, blactx-m-1g, blactx-m-9g and blashv (figure 2, supplementary table 1). figure 2: genetic compositions of extended-spectrum β-lactamase strains. left column: multiple gene strains, right column: single gene strains. various plasmid-mediated quinolone resistance genes were detected in ctx-m positive strains we analysed the pmqr genes in 17 strains carrying blactx-m and the results are shown in figure 3. the pmqr genes were found in 14 strains, including strains with two genes blactx-m-9g+qnrs (two strains), blactx-m-9g+oqxa (two strains), blactx-m-1g+qnrs (one strain) and blactx-m-1g+qepa (one strain), three genes blactx-m-9g+qnrs+oqxa (four strains), blactx-m-9g+qepa+oqxa (one strain), blactx-m-1g+blactx-m-9g+qnrs (one strain) and blactx-m-1g+qnrs+oqxa (one strain) and four genes blactx-m-1g+blactx-m-9g+qnrs+oqxa (one strain), of which, blactx-m-9g+qnrs+oqxa were the most frequent (23.5%, 4/17). figure 3: genetic compositions of ctx-m positive strains. left column: multiple gene strains, right column: single gene strains. discussion we investigated 47 e. coli strains isolated from swine in guizhou from 2013 to 2018 for their resistance to 21 common antimicrobial agents. the results showed serious multidrug resistance in this region. the isolated strains have the highest resistance to the tetracycline antimicrobials, which are comparable to the rates of resistance to tetracycline (97.6%) and doxycycline (89.3%) in e. coli from swine in guizhou in 2018 (wang & tan 2018). the rate of resistance to sulfaisoxazole is 89.4%, which is close to the rate of resistance to sulfonamide antimicrobials in 2018 (kou, liu & tan 2018). the results may be related to the long-term irrational use of these three antibiotics in the prevention and treatment of piglet respiratory diseases and bacterial diarrhoea in pig breeding in guizhou. the strains are also resistant to β-lactam drugs, the rates of resistance to third-generation cephalosporin ceftazidime and cefotaxime were 70.2% and 31.9%, respectively, which were higher than that of second-generation cephalosporin drug cefoxitin (8.5%). this may be attributed to the irrational use of third-generation cephalosporins and the less use of second-generation cephalosporins in the pig farms in guizhou in recent years. for aminoglycoside resistance, the highest rates were resistant to kanamycin and gentamicin, followed by neomycin and streptomycin. however, none of the strain is resistant to amikacin. these results are consistent with earlier reports for guangxi (ge, shi & hu 2019), chongqing (chen & liao 2019), fujian (yang, luo & xie 2018) and southeastern shanxi (shen, kong & guo 2019). this could be because of the less use of amikacin in the pig farms for disease treatment and prevention in guizhou. the rates of resistance to aminoglycoside gentamicin, streptomycin, ciprofloxacin (a fluoroquinolone antimicrobial agent) and polymyxin (a polypeptide antimicrobial agent) are obviously lower than the rates of resistance (gentamicin 81.10%, streptomycin 53.66%, ciprofloxacin 53.05% and polymyxin 51.83%) observed in guizhou in 2016 (cao, tan & liu 2016), whilst the rate of resistance to florfenicol (an amphenicol agent) is obviously higher than previously reported rate (cao et al. 2016). it is likely that the irrational use of fluorophenicol in the pig farms in recent years and the less use of gentamicin, streptomycin and polymyxins are responsible for the situation. at the same time, the reduction in ciprofloxacin resistance rate may be related to china’s ban on the use of four antibiotics in aquaculture from 2016, as well as improved management of livestock form and changes of antimicrobials used and administration method of antimicrobials. from 2 to 14 args are detected in various stains and over a half of the strains are found to have more than eight resistance genes, indicating that the proportion and quantity of args are high in these strains and the drug resistance problem is serious. in this study, five amphenicols resistant genes were detected, of which, flor (70.2%, 33/47) and cmla (53.2%, 25/47) genes are frequent, which are believed to be the main cause of amphenicol resistance in the strains. the detection rates of four aminoglycoside-modifying enzyme genes (aac(3’)-iv, aada1, aada2, aac(3’)-iic) are higher than 50%, and multiple aminoglycoside modifying enzyme genes are detected in the same strains, resulting in high-level resistance to aminoglycosides. in this study, the detection rates of aminoglycosides resistance genes are basically consistent with the resistance phenotypes. however, five aac(3′)-iv positive strains are found susceptible to aminoglycosides, probably because of infective expression of the gene in these strains. mcr-1i is also frequently found in the strains. mcr-1i is present in plasmid and chromosome, but mainly plasmids. plasmids could carry multiple args (zhu 2019). the high detection rate (40.4%) of mcr-1 in this study likely results from overdose use of polymyxins in pig feed in guizhou. at present, the resistance of gram-negative bacteria to β-lactam drugs is increasingly higher, and the most common resistance mechanism is that bacteria can produce three metalloenzymes: esbl, cephalosporinase (ampc enzyme) and carbapenems (meini et al. 2019). the first two enzymes are the main causes for drug resistance of e. coli from livestock and poultry. this study detected five β-lactamase genes (the detection rate is 83.0%), including four esbl genes (blatem, blactx-m, blashv, blaoxa) and one ampc enzyme gene (bladha). there are 38 (80.9%) e. coli strains that produce esbl, and these strains are more frequent to be resistant to various antimicrobials than the other nine strains that do not produce esbls. extended-spectrum β-lactamase -positive strains can produce β-lactamase to hydrolyse β-lactamase antibiotics, it often leads to clinical failure of infection treatment. in the previous reports, ctx-m-type was most prevalent in esbls (ho et al. 2011; jiang, kui & huang 2015; yang, zeng & lin 2019; zeng 2016), but in this study, blatem is the most popular in 38 esbls-positive strains (89.7%, 35/38), and it is basically consistent with the detection rate of 90.51% in guizhou in 2016 (cao et al. 2016), which should be taken seriously. blactx-m-9g is most prevalent amongst ctx-ms in this study (29.8%), conforming to the prevalence situation reported in this country (jiang et al. 2015; yang et al. 2019). amongst 38 bacterial strains carrying esbls genes, eight complex esbls genotypes are detected, of which, the proportion of blatem+blactx-m-9g is the highest (26.3%, 10/38). furthermore, coexistence of blactx-m-1g and blactx-m-9g are found in ecp4 and ecp27, which carried four esbls genes. these findings showed that esbls genotypes are complex in the e. coli strains in guizhou and would result in clinical challenges for infection control. low-resistant pmqr genes conferring quinolone resistances are shown to be closely related to the prevalence of esbls (ma et al. 2009; wu et al. 2007; yang, zhuang & hua 2018), especially qnr/oqxab and blactx-m genes, which may be located on the same plasmid (liu et al. 2014; strahilevitz et al. 2009). in this study, pmqr genes were analysed in 17 e. coli strains and 14 strains were found to have blactx-m with a detection rate of 82.4%, of which, qnrs and oqxa are detected at 58.8% and 52.9% of the strains. the complex genotype blactx-m-9g+qnrs+oqxa is detected in four strains. it is, therefore, likely that the esbls and pmqr genes are located on the same plasmid in the e. coli stains, which would somewhat increase the risk of horizontal transmission of args. previously, it was shown that oqxa and oqxb frequently coexist (yang et al. 2018). however, this coexistence was not observed in our study, and further study is needed to clarify the situation. conclusion extended-spectrum β-lactamase is widespread in e. coli from pigs in guizhou and tem-type is the most prevalent esbls. most of the e. coli strains have multiple esbls. in ctx-m strains, ctx-m-9g is the most frequent; pmqr carriers are often detected in blactx-m-positive strains, and the two genes are likely located on the same plasmids. therefore, the co-transmission needs to be addressed in the future. only oqxa was detected but not oqxb in this study, indicating a need to clarify their relationship. co-existence of blatem+blactx-m-1g+blactx-m-9g in ecp4 and blatem+blactx-m-1g+blactx-m-9g+blashv in ecp27 may allow further investigation of the position and position effect. in addition, over 40% strains are found to contain mcr-1, and the location of the gene needs to be defined. acknowledgements we thank all of the staff at the guizhou hongyu animal husbandry technology development co., ltd, who were involved in the response to this event but are not listed as authors. competing interests the authors have declared that no competing interests exist. authors’ contributions b.y., y.z. and s.b. deigned the study. b.y., y.z., l.y. and s.b. analysed and interpreted the experimental data. y.z., l.y. and j.x. performed the statistical analysis. l.y., j.x. and s.b. were major contributors in writing the manuscript. all authors read and approved the final manuscript. funding information this study was funded by the priority academic program 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origins’, journal of south china agricultural university 22(3), 22–28. zhu, y., 2019, ‘the study on epidemiology and transmission mechanism of polymyxin resistance gene mcr-1 in escherichia coli from animal origin’, chinese academy of agricultural sciences 232(1), 123–127. ker_273-277.qxd introduction it has been shown that certain characteristics of a premature ventricular complex (pvc) can reflect either the presence or the absence of myocardial disease (soloff 1961; schamroth 1980; moulton, medcalf & lazzara 1990). a pvc is the expression of an impulse that arises prematurely in an ectopic focus and can originate in the specialized conduction tissue distal to the bifurcation of his or in the ventricular myocardium itself (soloff 1961; myerburg & kessler 1998). on the 12-lead, surface electrocardiogram (ecg) pvcs are recognized by the premature occurrence of a qrs complex that is abnormal in shape and with a duration that exceeds that of the dominant qrs complex. the t wave of a pvc is opposite in direction to the major deflection of the qrs complex (myerburg & kessler 1998). however, narrow pvcs can occur and have been explained as originating at a point equidistant from each ventricle in the ventricular septum or by arising high in the fascicular system (olgin & zipes 2001). currently, three mechanisms of pvc generation are recognized: enhanced automaticity, triggered activity and re-entry (lerman 2000). pvcs originating in normal myocardium where no pathology is present, have the following characteristics (soloff 1961; schamroth 1980): (a) the qrs amplitude is large and exceeds 20 mm, or higher. (b) the qrs complex does not exceed 120 ms in duration. (c) the qrs deflection has a smooth contour with no notching 273 onderstepoort journal of veterinary research, 72:273–277 (2005) electrocardiographic surrogates of structural myocardial alterations in the dorper sheep heart j. ker1 and e.c. webb2 abstract ker, j. & webb, e.c. 2005. electrocardiographic surrogates of structural myocardial alterations in the dorper sheep heart. onderstepoort journal of veterinary research, 72:273–277 in this study we evaluated the validity of well-known human electrocardiographic markers of myocardial pathology in dorper sheep. these markers include: the duration of the qrs complex of premature ventricular complexes (pvcs), the presence of notching of the qrs complex of pvcs and change of the st-segment of pvcs. it was shown that these three electrocardiographic phenomena correlate with myocardial pathology in the hearts of dorper sheep. we also describe a new electrocardiographic indicator of myocardial pathology, namely an increase in the frequency of cardiac memory t waves as a new electrocardiographic surrogate for myocardial pathology in the hearts of dorper sheep. keywords: cardiac memory t waves, dorper sheep, electrocardiography, myocardial pathology, premature ventricular complexes 1 department of physiology, university of pretoria and pretoria heart hospital, p.o. box 24318, gezina, pretoria, 0031 south africa. e-mail: jker@wol.co.za 2 department of animal and wildlife sciences, faculty of agricultural sciences, university of pretoria, pretoria, 0002 south africa accepted for publication 14 june 2005—editor (d) the st-segment and t wave are opposite in direction to the qrs deflection. (e) the st-segment does not display any isoelectric period. (f) the st-segment blends imperceptibly with the proximal limb of the t wave, so that the two cannot be separated. (g) the t wave has asymmetrical limbs. myocardial pathology can alter these features of “uncomplicated” pvcs and any one or more of the following changes are indicative of “complicated” pvcs, where “complicated” is indicative of the presence of myocardial pathology (soloff 1961; schamroth 1980): (a) the qrs complex is diminished in amplitude. (b) the qrs complex widens and exceeds 120 ms. (c) there is marked notching and irregularity of the qrs complex. (d) the st-segment displays an isoelectric period. (e) the t waves tend to be sharply pointed and symmetrical. (f) the t wave has the same polarity as that of the qrs complex. however, the above data is applicable to the human heart and no such data exists for the dorper sheep heart. in addition, we wanted to know whether cardiac memory t waves can be used as another electrocardiographic indicator of myocardial pathology. cardiac memory is an electrocardiographic phenomenon reflected in the t wave, when t waves of normally conducted beats seem to “remember” the polarity of the qrs complexes of previous abnormally conducted beats (rosenbaum & blanco 1982; rosen 2001). currently, it is still unclear whether cardiac memory t waves can be used as an electrocardiographic warning for myocardial pathology to come (rosen 2001). we have a valid model for the induction of pvcs and cardiac memory in the dorper sheep heart (ker & webb 2003; ker, webb, ker & bekker 2003) and the purpose of this study was to examine the possibility that cardiac memory t waves can be used as an electrocardiographic indicator of myocardial pathology and secondly, whether the characteristics of “complicated” pvcs are true for the dorper sheep heart. materials and methods this study was performed with the approval of and adherence to the guidelines of the pretoria biomedical research centre’s animal use and care committee. in this study, we looked at existing electrocardiographic tracings on ten dorper wethers that were used in our experiments on cardiac memory t waves induced by pvcs, and the structural effects of pvcs on myocardial histology (ker, webb, ker & bekker 2003; ker, webb & van der merwe 2004). in these experiments, pvcs were induced by the guidewire of an arrow central venous pressure catheter set that was advanced into the right ventricle via the left internal jugular vein by the seldinger technique. pvcs induced in this way are capable of inducing cardiac memory t waves and furthermore, it has been shown that dorper hearts exposed to prolonged periods of pvcs in this way develop structural abnormalities of their left ventricles, the ventricle not exposed to the guidewire (ker, webb & van der merwe 2004). whether these left ventricular, myocardial alterations are caused by the pvcs themselves, by the guide-wire, infection, or some other, unknown culprit is not relevant for this study, because we wanted to document changes in the morphology of pvcs, compared between normal and abnormal left ventricles. in this way every wether serves as its own control in the following way: at the start of the study when the left ventricle is structurally normal, the character of pvcs and/or memory t waves should differ from those at the end of the study when the left ventricles are structurally abnormal (the cause of these changes is not relevant, as we wanted to correlate electrocardiographic changes with histologic changes). results the ten wethers were exposed to pvcs for a mean number of 21 days. 274 electrocardiographic surrogates of structural myocardial alterations in dorper sheep heart fig. 1 an example of an uncomplicated pvc occurring in the normal ovine heart during the first day of being subjected to pvcs. the third beat in this tracing from lead v4 is a pvc. note the narrow qrs complex with a duration of 40 ms and the isoelectric st segment. no notching of the qrs complex is present three morphological changes were noted in pvcs when comparing the first with the last day of study. first, the qrs duration of the pvcs increased. secondly, notching appeared in the qrs complexes of the pvcs. lastly, the isoelectric st-segments of the pvcs disappeared (see table 1). see fig. 1 for an example of an uncomplicated pvc and fig. 2 as an example of a pvc displaying these morphological alterations. in order to detect if there is any difference between the early and late occurrence of cardiac memory t waves, the first and last 10 % of pvcs were evaluated in every wether (table 2). the t wave of the first normal beat after every pvc were evaluated in order to determine whether these t waves retained the vector of the previous pvc qrs complex (see fig. 3). histological evaluation of the left ventricles revealed myocarditis in all the specimens, based on myocytolysis and an infiltration of white blood cells into the myocardium (see fig. 4 and 5) (ker, webb & van der merwe 2004). 275 j. ker & e.c. webb table 1 electrocardiographic characteristics of pvcs on the first and last day of study sheep mean qrs mean qrs number of number of number of number of number of number of number duration duration pvcs pvcs pvcs with pvcs with pvcs with pvcs with on first day on last day > 0.06 s > 0.06 s notching notching isoelectric isoelectric (ms) (ms) on first day on last day on first on last day ststday segments segments on first day on last day 1 0.038 0.060 20 20 2 2 44 44 2 0.040 0.070 1 15 0 9 13 2 3 0.042 0.080 8 48 3 3 76 76 4 0.040 0.060 4 16 1 10 13 3 5 0.038 0.080 4 18 1 11 15 4 6 0.040 0.060 2 10 1 7 11 2 7 0.038 0.070 32 130 9 81 110 22 8 0.040 0.070 3 18 2 9 14 3 9 0.038 0.060 3 13 1 8 15 3 10 0.038 0.080 3 30 3 22 25 5 increase in qrs duration: p = 0.013 (paired t-test). or = 13.93 (odds ratio that the qrs duration of pvcs will increase from < 0.06 s on the first day of study to > 0.06 s on the last day of study) notching of qrs complexes: p = 0.034 (paired t-test). or = 11.17 (odds ratio that pvcs will have notching of their qrs complexes on the last day of study) disappearance of isoelectric st-segments: p = 0.031 (paired t-test). or = 12.86 (odds ratio that the isoelectric st-segment of pvcs will disappear on the last day of study) table 2 early versus late cardiac memory t waves sheep number of early number of late number memory t waves memory t waves 1 2 5 2 17 21 3 2 3 4 8 12 5 15 15 6 8 37 7 40 188 8 10 21 9 31 90 10 40 51 p = 0.049 (paired t-test) or = 10.38 (odds ratio that there will be an increase in frequency of cardiac memory t waves between the first and last day of study) fig. 2 the tracing was taken from lead v4 from the same sheep’s tracing illustrated in fig. 1, but this time with 14 days of exposure to pvcs. the changes are possibly indicative of myocardial pathology. the third beat is a pvc. note the notching of the qrs complex. the qrs complex is much broader than that illustrated in fig. 1, with a duration of 80 ms. also note the loss of the st segment discussion on the first day of pvc exposure in the wethers the pvcs had the following characteristics: (a) the qrs duration did not exceed 60 ms. (b) there was no notching of the qrs complex. (c) the st-segment was isoelectric. on the last day of pvc exposure certain changes were noted in the pvcs: (a) the qrs duration prolonged to > 60 ms (b) notching of the qrs complex appeared. (c) the isoelectric st-segment disappeared. it is important to realize that normal dorper wethers were used in these studies. six normal wethers were slaughtered and their left ventricles were subjected to histological examination in order to document the normal histological appearance of the dorper sheep heart (see fig. 6). at the end of these studies all the wethers which had been subjected to pvcs for a mean of 21 days, had an abnormal histological appearance of the left ventricle, indicative of myocarditis. in this way every wether served as its own control—at the beginning of the study when normal wethers entered the study, the pvcs had different characteristics than at the end of the study when myocardial pathology was present. as we want to document that certain changes occurs in pvcs when the left ventricle undergoes structural alteration, the specific cause of these structural changes is not relevant. it is concluded that the following characteristics of pvcs can be used to identify underlying myocardial pathology in the dorper sheep heart: 276 electrocardiographic surrogates of structural myocardial alterations in dorper sheep heart fig. 3 an example of cardiac memory t waves. the third and fifth beats in this tracing are pvcs (broken arrow). note the bifid t waves before the first pvc (arrow), and the inverted t waves after the pvcs (double arrow). the t wave retains the vector of the qrs complex of the pvc, hence the name “cardiac memory” fig. 6 normal dorper heart muscle fig. 4 infiltration of the left ventricular interstitium by mixed inflammatory cells, a feature of myocarditis fig. 5 myocytolysis, another feature of myocarditis, occurs in addition to the inflammatory cell infiltrate in the left ventricle (a) the qrs duration will exceed 60 ms. (b) notching of the qrs complex. (c) disappearance of the st-segment. in addition, we identified a new electrocardiographic surrogate for myocardial pathology—the cardiac memory t wave. it is shown in this study that the true value of using cardiac memory t waves as an electrocardiographic surrogate for structural myocardial alterations in the dorper heart does not lie in an instantaneous electrocardiographic assessment, but in electrocardiographic follow-up, in order to determine if there is an increase in the frequency of cardiac memory t waves. as shown in this study, an increase of at least 42 % in the frequency of cardiac memory t waves, following pvcs is indicative of underlying structural myocardial alterations. although al four pvc changes were statistically significant with p values below 0.05, the increase in qrs duration had the lowest p value (= 0.013) and the increase in cardiac memory t wave frequency had the highest p value (= 0.049). an unexplained phenomenon is the disappearance of the isoelectric st-segments of pvcs at the end of the study, when myocardial pathology is present. this is in contrast to the human heart, where it has been described that the appearance of isoelectric st-segments is a sign of myocardial pathology. this observation is consistent, but unexplained and will need clarification in future studies. it is possible that some metabolic difference between the human and ovine heart may offer an explanation to this observation. references ker, j. & webb, e.c. 2003. ventriculo-atrial conduction in the ovine heart, caused by premature ventricular complexes. onderstepoort journal of veterinary research, 70:107–111. ker, j., webb, e.c., ker, j.a. & bekker, p.a. 2003. the heart remembers: observations of cardiac memory in the dorper sheep heart. onderstepoort journal of veterinary research, 70:299–305. ker, j., webb, e.c. & van der merwe, c.f. 2004. ventricular dyssynchrony as a cause of structural disease in the heart of dorper sheep. onderstepoort journal of veterinary research, 71:197–202. lerman, b.b. 2000. ventricular arrhythmias and sudden death, in cecil textbook of medicine, 21st ed., edited by b.b. lerman. philadelphia: w.b. saunders. moulton, k.p., medcalf, t. & lazzara, r. 1990. premature ventricular complex morphology. a marker for left ventricular structure and function. circulation, 81:1245–1251. myerburg, r.j. & kessler, k.m. 1998. recognition, clinical assessment and management of arrhythmias and conduction disturbances, in hurst’s the heart, 9th ed., edited by r.w. alexander, r.c. schlant & v. fuster. new york: mcgrawhill. olgin, j.e. & zipes, d.p. 2001. specific arrhythmias: diagnosis and treatment, in heart disease. a textbook of cardiovascular medicine, 6th ed., edited by e. braunwald, d.p. zipes & p. libby. philadelphia: w.b. saunders. rosen, m.r. 2001. the heart remembers: clinical implications. lancet, 357:468–471. rosenbaum, m.b. & blanco, h.h. 1982. electrotonic modulation of the t wave and cardiac memory. american journal of cardiology, 50:213–222. schamroth, l. 1980. ventricular extrasystoles, ventricular tachycardia and ventricular fibrillation: clinical electrocardiographic considerations. progress in cardiovascular diseases, 23:13–32. soloff, l.a. 1961. ventricular premature beats diagnostic of myocardial disease. american journal of medical science, 242:315–319. 277 j. ker & e.c. webb article information authors: estelle h. venter1 truuske gerdes2 isabel wright3 johan terblanche2 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa 2department of theriogenology, university of pretoria, south africa 3virology section, onderstepoort veterinary institute, south africa correspondence to: estelle venter email: estelle.venter@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 03 sept. 2010 accepted: 16 nov. 2010 published: 24 feb. 2011 how to cite this article: venter, e.h., gerdes, t., wright, i. & terblanche, j., 2011, ‘an investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos’, onderstepoort journal of veterinary research 78(1): art. #17, 7 pages. doi:10.4102/ojvr.v78i1.17 copyright notice: © 2011. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) an investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos in this original research... open access • abstract • introduction • materials and methods    • experiment 1    • experiment 2    • ethical consideration • results    • experiment 1    • experiment 2 • discussion • conclusion • acknowledgements • references abstract (back to top) bluetongue (bt), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. the causative agent, bluetongue virus (btv), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. the risk of the transmission of btv by bovine embryos is negligible if the embryos are washed according to the international embryo transfer society (iets) protocol. two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to btv. firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to btv-2 and btv-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. in the second experiment, embryos were recovered from sheep at the peak of viraemia. a total of 96 embryos were collected from btv-infected sheep 21 days after infection. in both experiments half the embryos were washed and treated with trypsin according to the iets protocol while the remaining embryos were neither washed nor treated. all were tested for the presence of btv using cell culture techniques. the virus was detected after three passages in bhk-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to btv-4 at a titre of 1 x 103.5. no embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for btv after the standard washing procedure had been followed. the washing procedure of the iets protocol can thus clear sheep embryos infected with btv either in vitro or in vivo. introduction (back to top) bluetongue (bt) is a disease that causes substantial economic losses owing to not only its effect on certain species of animals, particularly sheep, but also its associated impact on industries as a result of international regulations restricting the movement of susceptible livestock and their live germplasm. bt is responsible for direct economic losses on affected farms owing to the high morbidity and mortality. losses in non-endemic regions can be high, with belgium, for example, having lost a quarter of their sheep population during an outbreak caused by bluetongue virus serotype 8 (btv-8) in north-western europe during 2007 (gloster et al. 2008). btv is a member of the genus orbivirus in the family reoviridae. bt was first described in south africa, but has since been recognised in most countries in the tropics and subtropics. since 1999, however, there have been widespread outbreaks of bt in greece, italy, corsica and the balearic islands (spain). cases have also occurred in bulgaria, croatia, macedonia, kosovo and yugoslavia, which are well north of the generally recognised distribution. it appears that the virus spread to these countries from turkey and north africa (purse et al. 2005; mellor et al. 2008). during late september 2000, outbreaks occurred on the spanish island of majorca (after an absence of 40 years) and in october on the french island of corsica. by early december there were additional outbreaks in majorca and minorca (spain): 118 outbreaks involving 14 248 sheep in october and subsequently another 166 outbreaks involving 16 605 animals (sheep, goats and cattle) also occurred (purse et al. 2005; mellor et al. 2008). in october 2006 btv-8 caused severe outbreaks in belgium, italy, spain and northern european countries in particular (wilson & mellor 2008; saegerman, berkvens & mellor 2008). the disease in sheep is characterised by congestion, haemorrhages, cyanosis and ulceration of mucous membranes, particularly those of the buccal cavity. there may be laminitis, myositis and oedema of the head and neck. some data suggest that foetal abnormalities may occur when ewes are infected early in pregnancy. the severity of the disease varies, with the mortality rate in sheep being between 5% and 50%. clinical bt in cattle is rare (< 5% of infected animals). the virus has little, if any, effect on reproduction, but cattle may develop a prolonged viraemia. prenatal infection of sheep and cattle foetuses at an early stage of development may lead to embryonic death. foetuses infected during the later stages of gestation survive and develop antibodies, and are not persistently infected (parsonson 1992). transmission of orbiviruses is by culicoides spp. midges, which are biological vectors. it is thought that the virus can also be transferred from viraemic pregnant dams (sheep and cattle) to their developing foetuses. although btv may be found in the semen of some rams and bulls, it can be isolated only at the time of peak viraemia, its presence apparently being the result of infected blood cells in the semen (osburn 1994). the risk of the transmission of the btv by bovine embryos does exist, but is negligible if the embryos are washed between collection and freezing and are transported according to the protocol set by the international embryo transfer society (iets) (acree et al. 1991; bowen & howard 1984; thomas, singh & hare 1983, 1985). hare et al. (1988) and singh, dulac and henderson (1997) found that btv-11 was not transmitted when sheep embryos collected from infected donors were washed and transferred to uninfected bt-susceptible recipients. gilbert, coubrough and weiss (1987), however, transferred embryos infected with btv-10 directly, without washing, to 13 susceptible recipient ewes. none of the ewes became pregnant and nine of them seroconverted. these results support the hypothesis that the zona pellucida of sheep is ‘stickier’ than that of bovines, which implies that it ‘traps’ the btv. this must undoubtedly influence the nature of the washing procedure for the embryos. there is a constant demand for embryos from south africa for export to other countries. figures presented at the 2003 congress of the south african veterinary specialist embryo group (r. de la ray) demonstrated the demand for embryos from different animal species. in 1998, 7048 sheep embryos were exported mainly to australia and canada, followed by 10 408 the next year, and a further 12 681 in 2000 to australia, canada, china and brazil. this clearly indicates that south africa plays a significant role in the world’s trade in embryos and that this area can be very viable economically. apart from the information mentioned above there is little information on the possible role of sheep embryos in the transmission and spread of btv and the contradictory results that have been obtained to date create a great deal of uncertainty. iets has therefore classified bt in sheep as a category 3 disease (stringfellow & seidel 1998). although the preliminary evidence indicates that the risk of transmission is negligible, provided that embryos are handled according to protocol, iets requires additional in vitro and in vivo experimental data to substantiate the preliminary findings mentioned above. the practical implication for the control of sheep diseases in category 3 is that countries importing sheep embryos no longer rely only on the procedure of washing the embryos before freezing; additional diagnostic tests are required to be performed on donor animals. this has considerable cost implications for both the exporting and the importing countries. owing to the requirement for additional diagnostic tests (e.g. determining the btv status of donors using tissue culture techniques) delivery of consignments of embryos may be delayed considerably. to reclassify bt from an iets category 3 to a category 1 disease (i.e. one that does not require application of any additional tests) one must be able to certify that embryos are free from btv and are therefore suitable for export purposes. to achieve this aim two experiments were undertaken (table 1) to establish (1) whether embryos from btv-free donors that are infected with btv in vitro are virus-free after washing according to the iets protocol, and (2) whether washed and unwashed embryos from btv-infected donors are virus-free when evaluated for the presence of btv with an in vitro virus detection (cell culture) method. materials and methods (back to top) experiment 1 twenty btv-free ewes were purchased and kept in an insect-free animal facility at the faculty of veterinary science, university of pretoria, onderstepoort, south africa. they were confirmed as being negative for btv antibodies before commencement of the experiment. a commercial group-specific competitive enzyme-linked immunosorbent assay (c-elisa) from the institute for animal health, pirbright was used for this purpose according to the standard operating procedure (sop) of the virology section of the onderstepoort veterinary institute.a total of 59 ova, embryos or degenerated embryos were collected from 12 of these ewes according to the sop of the reproduction section of the department of theriogenology, faculty of veterinary science. the ova and degenerated embryos were separately pooled and infected with btv-2 or btv-4 at virus titres of 1 x 102.88 and 1 x 103.5 respectively (table 2). btv-infected embryos were incubated for 1 h at 37 °c in an atmosphere containing 5% co2. half the number of embryos were then washed using an adapted protocol (bioniche complete flush medium with hanks’ salts) and treated with trypsin according to the method set out in the iets manual (stringfellow & seidel 1998). the washing method was, however, adapted by using micropipette tips from a different manufacturer so that no foreign material could become adherent and so be transferred. the bhk cell culture was also centrifuged and the clear supernatant containing the virus was removed and used as exposure medium. the remaining embryos were neither washed nor treated with trypsin. a btv isolation method using bhk-21 cells was performed on all the embryos. aliquots of wash fluids were taken from wells 1, 2 and 10 in each wash plate, as well as from the media in which the embryos were held before (virus bath) and after washing (holding medium). all the specimens were processed separately on bhk-21 cells. embryos were added to 200 µl eagle’s minimum essential medium (emem) (biowhittaker, cambrex) in eppendorf tubes (aec-amersham, johannesburg), homogenised for 30 s and centrifuged for 5 min at 9300 rcf using an eppendorf 5415 r centrifuge. supernatants were aliquoted in 96-well plates in triplicate in 25-µl volumes per well. a 100-µl volume of bhk-21 cells in emem was added and the plates were incubated for 6 days at 37 °c in an incubator containing 5% co2 before reading. the cells were monitored for the cytopathic effects of btv. negative cell cultures were blind-passaged after 7 days for two additional passages. experiment 2 the objective of this experiment was to recover embryos from viraemic donors at the peak of viraemia. the same ewes (n = 16) as for experiment 1 were used, together with two rams and two wethers, and were kept in an insect-free animal facility at the faculty of veterinary science. they were again confirmed negative for btv antibodies by the method as described for experiment 1 before commencement of the current experiment. a btv-4 field isolate, which forms part of the reference collection at the office international des epizooties (oie) reference laboratory (virology section, onderstepoort veterinary institute) and is stored in their antigen bank, was used as inoculum. to prepare the material, two sheep received inoculations of 5 ml – 10 ml of blood stored in ocg (potassium oxalate, glycerol and water) from a field case. blood was collected from them in heparin upon developing a febrile reaction (rectal temperature above 40 °c) 5–9 days post-inoculation (p.i.). this was used as an inoculum to infect the donors, with approximately 200 ml providing sufficient fresh, virulent btv material needed for infection of the ewes. it had a titre of 1 x 107.8 tcid50. the oestrus cycle of the ewes were synchronised. the ewes were superovulated and inseminated with semen obtained from btv-negative rams. each then received an intravenous inoculation comprising 5 ml of the btv-4 inoculum. after the first 2 days of inoculation their rectal temperatures were taken twice daily until day 15 p.i. reactors were bled for btv isolation when their rectal temperature reached or exceeded 40 °c and serum from the donors was assayed for antibodies on days 21 and 28 p.i. two or three embryos per donor were collected from the infected ewes 21 days p.i., with a total of 96 being collected. about 40 of the embryos were washed and treated with trypsin according to the techniques prescribed in the iets manual (stringfellow & seidel 1998), while the others were neither washed nor treated. btv detection techniques were used on all embryos. instead of sonication, a vibrating plunger for eppendorf microcentrifuge tubes was used. embryos were placed in 200 µl emem in eppendorf microcentrifuge tubes and homogenised by a 30-s burst of the vibrating plunger. an individual sterile plunger was used for each specimen.virus was isolated on bhk cells according to the sop of the oie reference laboratory at onderstepoort. a total of 96 embryos, ova and degenerated embryos were recovered from donor ewes, 7 days after they were inoculated with btv. the embryos and their wash fluids as well as the fluids collected from the uterine flushes were cultured directly on bhk-21 cells in 96-well culture plates. live embryos and degenerated embryos were divided into groups of two or three each, which resulted in a total of 107 specimens being obtained for culture. each specimen was inoculated on a monolayer in triplicate and three passages were performed before a final result was recorded. cells were routinely maintained with emem supplemented with 5% foetal calf serum (biowhittaker–adcock ingram, johannesburg). to confirm the presence of btv in the donors, blood samples were collected from them in heparin on the day of flushing. this was processed according to the sop of the australian embryo export protocol. five embryonated chicken eggs were inoculated intravenously per specimen. the eggs were incubated for 7 days at 34.5 °c during each passage. the blood of live embryos in chilled eggs as well as that of recently dead embryos was collected and inoculated into eggs for the second passage. dead embryos, from either of the egg passages were further subjected to two consecutive 7-day passages on chicken embryo reticulum cells (later re-classified as a hamster cell). ethical consideration the donors showed a rise in temperature within 4 days after inoculation. they were clinically sick on the day of embryo recovery. once a temperature of 40 °c or higher was recorded, the ewes were treated with antibiotics and anti-inflammatory and antipyretic drugs immediately. on the seventh day after inoculation (the day of flushing) all the donors had seroconverted and they tested positive for btv using a c-elisa. classical clinical signs of bt were observed within 4 days after inoculation and some ewes had died. results (back to top) experiment 1 results obtained in the investigation where ova, embryos and degenerated embryos that had been infected with btv in vitro were first exposed to virus and then either washed or left unwashed, followed by passaging three times on bhk cells, are demonstrated in table 3. with the exception of three samples, all tested negative for the presence of btv using bhk-21 cell cultures. btv was detected in one wash bath (virus passage 3, bath 1) and two unwashed ova. experiment 2 in this experiment embryos were recovered from viraemic donors at the peak of viraemia, 7 days after infection. donors were inoculated 7 days before embryo recovery (i.e. on day 14 of the superovulatory programme). therefore, the day of embryo recovery coincided with the day of peak fever and viraemia. all the donors revealed a rise in rectal temperature (> 40 °c) from day 4 p.i. and presented with classical signs of bt on the day of embryo flushing. although the field isolate induced the development of only mild clinical signs of bt in the two test animals used to prepare the inoculum and were considered not sufficiently severe as to endanger the lives of the experimental animals, the first evidence of virulence came when the first wether (no. 4741) died 9 days after inoculation, while showing severe torticollis. the second wether (no. 4739), however, recovered uneventfully after showing mild clinical signs of bt. the donor ewes were therefore fully susceptible to bt and the inoculated blood induced the disease. fifteen of them became clinically ill and presented with the classical signs of bt while 12 of them died. one ewe (no. 13) remained unaffected but showed a rectal temperature rise on day 4 p.i. and seroconverted. all the donors had seroconverted by day 7 p.i. post-mortem examinations revealed the classical lesions of bt, including haemorrhages in the wall of the pulmonary artery, which is regarded as pathognomonic for bt. a total of 96 live embryos or degenerated embryos were recovered from the donors. all were suitable for use in the trial. forty-eight were washed while an equivalent number were not washed. btv was not isolated from any of the embryos, uterine flush fluid or selected wash fluids when specimens of these were cultured directly in tissue culture even after all material had been passaged on bhk cells three times. the temperature response, clinical signs and tissue lesions observed during post-mortem examination were typical of btv infection. these, plus the number of chick embryo deaths during intravenous isolation from blood of the donors and the recovery of virus from three spleens prove that these ewes died from btv infections, the identity of the virus subsequently being confirmed as btv-4. the vector-free housing in which the animals were kept was also secure. the antibody titres from the three surviving donor ewes and one wether were determined on days 7, 14, 21, 28 and 36 p.i. these results are reflected in table 4. table 1: a summary of the experimental procedure for establishing the infectivity of btv from in vivo-derived ovine embryos. table 2: the number of embryos, ova and degenerated embryos infected with different titres of btv (experiment 1). discussion (back to top) two south african reference strains of btv, serotypes 2 and 4, were used to infect live ova and degenerated sheep embryos. these strains were selected from the collection held in the btv oie reference laboratory at the onderstepoort veterinary institute, because they are well adapted to laboratory conditions and growth in cell culture, well characterised and have a documented history. the aim of the study was to determine whether the prescribed washing procedure in the iets protocol is sufficient to ‘clean’ infected ovine embryos of btv. extensive investigations over the past 25 years have provided substantial scientific evidence that bovine embryos derived in vivo from an intact zona pellucida do not transmit disease, including bt, provided that the embryos are properly handled in the period between their collection and transfer (stringfellow, givens & waldorp 2004). the oie has adopted the standardised iets protocol (stringfellow & seidel 1998) for treatment of bovine embryos derived in vivo. therefore, bt in cattle appears as a category i disease (caused by a pathogenic agent for which there is sufficient research to show that the risk of transmission via embryos derived in vivo is negligible if the embryos are properly handled between collection and transfer) in appendix 3 of the oie manual and the 1998 iets manual (stringfellow & seidel 1998). unfortunately, in the case of ovine embryos and btv, the same conclusion is not possible because there is insufficient published data in this regard. in the 1998 iets manual (stringfellow & seidel 1998), bt is placed in category 3, in which the preliminary evidence indicates that the risk of transmission is negligible provided that the embryos are properly handled in the period between their collection and transfer, but for which additional in vitro and in vivo experimental data are required to substantiate the preliminary findings. this was identified in 2004 when wrathall et al. (2004) proposed that it be changed to category 2, that is, a disease for which substantial evidence has accrued to show that the risk of transmission is negligible provided that embryos are handled properly between collection and transfer but for which additional transfers (experimental data) are required to verify the existing data. in this study, embryos obtained from superovulated ewes were, after infection with btv, washed and tested for the presence of the virus using a cell culture method. although the iets protocol states that the washing procedure is designed to consistently remove 106–107 logs of virus, a pilot trial during which another embryo washing procedure was compared showed the prescribed washing method to be ineffective. the procedure was then slightly modified for use in the first experiment. firstly, the pipette tips used were changed to consist of an absolutely smooth material so that no material would adhere to them and thus be transferred. secondly, the infected bhk-21 cell culture was centrifuged and the clear supernatant containing the virus was removed and used as the medium in the bath in which the embryos were exposed to the virus. the results obtained by changing the method were superior to those previously obtained and are illustrated in table 3. the efficacy of the 10 washes was clearly demonstrated, and the presence of the virus after three passages in bhk-21 cells was only detected in one wash bath and two unwashed embryos exposed to btv-4 at a titre of 1 x 103.5. in previous work using a low (1 x 102.0) and a high (1 x 106.7) titre of btv, the low-titre-infected unwashed embryos were negative, while the high-titre-infected embryos were positive (terblanche, unpublished information). clearly the btv-4 titre used in this study was just above the cut-off limit and resulted in a positive test. the second objective was to recover embryos from viraemic donors and, after using the iets wash process, to test for the presence of btv. the same ewes were used as donors and after synchronisation they were infected with a field isolate of btv-4. the donors showed a rise in rectal temperature within 4 days of inoculation and embryos were collected from them at the peak of viraemia when they were showing classical clinical signs of bt. evidence for seroconversion was detected on day 7 p.i. by use of a c-elisa (table 4) and the ewes were then flushed. as the ewes showed severe clinical signs they were treated with antibiotics and anti-inflammatory and antipyretic drugs once a rectal temperature of 40 °c or higer was recorded. one ewe died before flushing and 12 of them died within 7 days of surgery. all showed the classical lesions associated with btv infection on post-mortem examination. all embryos and uterine flush fluids obtained from viraemic donors were negative for btv after the standard washing procedure had been followed. all the specimens that were collected were cultured directly on cell cultures. in general, in order to improve the sensitivity of the culturing process, field isolates of btv need to be amplified by growing them in eggs before tissue culture is attempted. it is obvious that in this experiment both the washing and the culturing procedures would have contributed to the uniformly negative result. there is very little information available on the transmission of btv via transfer of ovine embryos. the iets manual, appendix b (stringfellow & seidel 1998) includes two reports dealing with such transfer of ovine embryos and btv [i.e. those of gilbert et al. (1987) and hare et al. (1988)]. singh et al. (1997) have also published data on the transfer of the virus via ovine embryos but reported that they had obtained conflicting results. both hare et al. (1988) and singh et al. (1997) found that embryos from btv-infected donors, when properly washed, were virus-free and could be transferred without infecting recipients. in contrast, they found that when embryos from btv-free donors were artificially exposed to btv-11, the virus remained adherent to the embryos, even after washing. singh et al. (1997) washed embryos according to the iets protocol, but found that all the inoculated embryonated chicken eggs became infected with btv, while only five of 11 samples were positive in bhk-21 cells. this clearly illustrates that the culturing procedure employed for btv isolation is extremely important, unless the virus is well adapted to laboratory conditions. gilbert et al. (1987) transmitted btv-10 to recipient ewes after the embryos that they used had been exposed to the virus for 8 h. they had, however, not used the iets method for the post-exposure washing procedure, but merely transferred the embryos through four different dishes, each of which contained ‘clean’ medium. after the embryos had been transferred, two of the 15 recipients seroconverted. table 3: results from embryos exposed to btv in vitro, washed and passaged three times on bhk-21 cells (experiment 1). table 4: c-elisa serology results for donors inoculated with btv and flushed after 7 days (experiment 2). conclusion (back to top) in this report we have shown that sheep embryos infected in vitro with btv can be cleared of virus by using the washing procedure of the iets protocol, and that embryos derived from sheep infected with a field strain of btv were negative for virus after the washing procedure although wild-type virus isolation techniques were not used. this re-emphasises the fact that the risk of transmission of btv by embryos is negligible if the embryos are handled correctly between collection and transfer. acknowledgements (back to top) the research was funded by the national research foundation, project no. fa2004051100013. we thank prof r.c. tustin for editing the manuscript. references (back to top) acree, j.a., echterkamp, s.e., kappes, s.m., luedke, a.j., holbrook, f.r., pearsonj.e. et al., 1991, ‘failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients and offspring’, theriogenology 36, 689–697. bowen, r.a. & howard, t.h., 1984, ‘transmission of bluetongue virus by intrauterine inoculation or insemination of virus-containing bovine semen’, american journal of veterinary research 45, 1386–1388. gilbert, r.o., coubrough, r.i. & weiss, k.e., 1987, ‘the transmission of bluetongue virus by embryo transfer in sheep’, theriogenology 27, 527–540. gloster, j., burgin, l., witham, c., athanassiadou, m. & mellor, p.s., 2008, ‘bluetongue in the united kingdom and northern europe in 2007 and key issues for 2008’, veterinary record 162, 298–302. hare, w.c., luedke, a.j., thomas, f.c., bowen, r.a., singh, e.l., eaglesome, m.d. et al., 1988, ‘nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep’, american journal of veterinary research 49, 468–472. mellor, p.s., carpenter, s., harrup, l., baylis, m. & mertens, p.p., 2008, ‘bluetongue in europe and the mediterranean basin: history of occurrence prior to 2006’, preventive veterinary medicine 87, 4–20. osburn, b.i., 1994, ‘the impact of bluetongue virus on reproduction’, comparative immunology and microbiology of infectious diseases 17, 189–196. parsonson, i.m., 1992, ‘overview of bluetongue virus infection in sheep’, in t.e. walton & b.i. osburn (eds.), bluetongue, african horsesickness and related orbiviruses, pp. 713–724, crc press, boca raton, florida, usa. purse, b.v., mellor, p.s., rogers, d.j., samuel, a.r., mertens, p.p. & baylis, m., 2005, ‘climate change and the recent emergence of bluetongue in europe’, nature reviews microbiology 3, 171–181. saegerman, c., berkvens, d. & mellor, p.s., 2008, ‘bluetongue epidemiology in the european union’, emerging infectious diseases 14, 539–544. singh, e.l., dulac, g.c. & henderson, j.m., 1997, ‘embryo transfer as a means of controlling viral infections. xv. failure to transmit bluetongue virus through the transfer of embryos from viremic sheep donors’, theriogenology 47, 1205–1214. stringfellow, d.a. & seidel, s.m., 1998, manual of the international embryo transfer society, 3rd edn., iets, savoy, illinois, usa. stringfellow, d.a., givens, m.d. & waldorp, j.g., 2004, ‘biosecurity issues with current and emerging embryo technologies’, reproduction, fertility and development 16, 93–102. thomas, f.c., singh e.l. & hare, w.c.d., 1983, ‘embryo transfer as a means of controlling viral infections. iii. non-transmission of bluetongue virus from viremic cattle’, theriogenology 19, 425–431. thomas, f.c., singh e.l. & hare, w.c.d., 1985, ‘embryo transfer as a means of controlling viral infections. vi. bluetongue virusfree calves from infectious semen’, theriogenology 24, 345–350. wilson, a. & mellor, p., 2008, ‘bluetongue in europe: vectors, epidemiology and climate change’, parasitology research 103, 569–577. wrathall, a.e., simmons, h.a., bowles, d.j. & jones, s., 2004, ‘biosecurity strategies for conserving valuable livestock genetic resources’, reproduction, fertility and development 16, 103–112. uys_339-343.qxd birds are hosts of the immature stages of several ixodid tick species (theiler 1962) and consequently could serve as a source of subsequent adult tick infestation for other hosts. this is particularly so for migrating birds (hoogstraal, kaiser, traylor, gaber & guindy 1961), or species such as helmeted guineafowls, numida meleagris, that on occasion can harbour large numbers of immature ticks (horak & williams 1986; horak, spickett, braack & williams 1991). two species of guineafowls and several species of francolins occur in south africa and of these the ixodid tick burdens of helmeted guineafowls, cape francolins, francolinus capensis, and greywing francolins, francolinus africanus, have been determined (horak & williams 1986; horak et al. 1991; horak & boomker 1998). similar data for crested francolins, francolinus sephaena, or crested guineafowls, guttera pucherani, both of which occur in the northern and north-eastern regions of south africa, have not been collected. the objective of the present study was to determine whether crested francolins, which prefer thick cover and matted vegetation, play the same role as helmeted guineafowls, which prefer thorny scrub and savanna veld, as hosts for the immature stages of ticks of veterinary importance. the species spec339 onderstepoort journal of veterinary research, 72:339–343 (2005) research communication ticks on crested francolins, francolinus sephaena, and on the vegetation on a farm in limpopo province, south africa a.c. uys1 and i.g. horak* department of veterinary tropical diseases, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract uys, a.c. & horak, i.g. 2005. ticks on crested francolins, francolinus sephaena, and on the vegetation on a farm in limpopo province, south africa. onderstepoort journal of veterinary research, 72:339–343 ticks were collected at approximately bi-monthly intervals between june 1996 and june 1997 from crested francolins, francolinus sephaena, and from the vegetation on a mixed cattle and wildlife farm in limpopo province, south africa. the birds were infested with the immature stages of 13 tick species, of which amblyomma hebraeum, amblyomma marmoreum and hyalomma marginatum rufipes were the most numerous and prevalent. ten ixodid tick species were collected from the vegetation, of which the immature stages of rhipicephalus appendiculatus, rhipicephalus (boophilus) decoloratus and rhipicephalus evertsi evertsi were the most numerous. no adult ticks were collected from the birds and only two from the vegetation. the restricted home range of crested francolins implies that they could serve as a source of tick infestation only for other animals within the same habitat as the birds. keywords: crested francolins, francolinus sephaena, free-living ticks, limpopo province, parasitic ticks * author to whom correspondence is to be directed 1 present address: p.o. box 652, newlands, 0049 south africa accepted for publication 22 june 2005—editor trum of ticks on the francolins was also compared with that of free-living ticks on the vegetation at the same locality. the survey was conducted on the farm “sandspruit” (24°38´s, 27°40´e), comprising 655 ha in the valley of a seasonally flowing river along the southern foothills of the waterberg mountain range in limpopo province. annual rainfall varies between 550 mm and 600 mm, mean annual temperature is approximately 20 °c, and the vegetation is classified as sour bushveld and sourish mixed bushveld (acocks 1988). a herd of 140 cows and six bulls ran on the farm, and these were hand-sprayed with a synthetic pyrethroid for the control of ticks at weekly intervals during summer and fortnightly during winter. donkeys and a variety of wild ungulates including warthogs, impalas, greater kudus and grey duikers were also present on the farm. an attempt was made to shoot six crested francolins every second month from june 1996 to june 1997. this, however, was not always possible because of the very dense vegetation resulting from above average rainfall during the preceding year and during the study period, and a total of only 23 birds were collected. the carcass of each bird was placed in a sturdy plastic bag immediately after it had been shot, and the bag was sealed and transported to a field laboratory. here the bird was decapitated approximately 1 cm caudad to the junction between the head and the neck, the wings and the tail were severed from the body and the feathered portion of the carcass was skinned. the legs were severed at the tibiotarsal-tarsometatarsal joint, but left attached to the skin. the head, wings, body with legs attached, and tail were placed separately in plastic jars and sufficient tick-detaching agent (triatix: hoechst roussel vet (pty) ltd) was added to cover the material in each jar. except that the skin and feathers of both wings, both legs and the entire body were examined under a stereoscopic microscope, ticks were otherwise recovered from the material in the jars and identified and counted as described by horak & williams (1986). free-living ticks were collected from the vegetation using the drag-sampling method described by petney & horak (1987), and with the exception of february 1997, when heavy rainfall prevented sampling, this was done every second month from june 1996 to june 1997 in each of four woodland habitats and four open grassland habitats. the birds were infested with the immature stages of 12 ixodid tick species and the larvae of one argasid tick species (table 1). the larvae of a. hebraeum were the most numerous and prevalent followed by those of amblyomma marmoreum and hyalomma marginatum rufipes. the immature stages of only four tick species can be regarded as parasites of the francolins, namely a. hebraeum, a. marmoreum, h. marginatum rufipes and argas sp. the remaining species should be considered accidental infestations or “stragglers”. the highest total burden, comprising 256 larvae and five nymphs, was recorded on a bird examined during april 1997, whereas a bird examined during february 1997 harboured no ticks. the mean tick burdens of the francolins consisted of 88 larvae and three nymphs. the carcasses of 19 of the 23 francolins were processed in a manner such that the attachment distribution of their ticks could be determined; this is summarized in table 2. the majority of a. hebraeum and h. marginatum rufipes larvae were recovered from the heads of the birds, while those of a. marmoreum were collected from the body and wings. ten ixodid tick species were collected from the vegetation, the larvae of rhipicephalus appendiculatus being the most numerous followed by those of rhipicephalus (boophilus) decoloratus and rhipicephalus evertsi evertsi (table 3). although ten of the tick species on the francolins were also present on the vegetation, the proportional species representation in the two tick populations differed considerably. the mean burden of 37 a. hebraeum larvae on the francolins is considerably lower than the mean burdens of 184 and 568 larvae on helmeted guineafowls in the kruger national park, mpumulanga province and the andries vosloo kudu reserve, eastern cape province respectively (horak et al. 1991). however, four francolins each harboured more than 100 larvae, indicating that these birds are also capable of carrying large numbers of ticks. horak & williams (1986) regard helmeted guineafowls as important disseminators of a. hebraeum because of their mobility and the large numbers of immature ticks that they harbour. crested francolins on the other hand with their more restricted home ranges would seem more likely to contribute to the maintenance of localized populations of this tick. this would indeed appear to be the case in that the proportional representation of a. hebraeum on the francolins, with their preference for dense vegetation, was considerably greater than that on the vegetation (tables 1 and 3). 340 ticks on crested francolins, francolinus sephaena, in limpopo province, south africa helmeted guineafowls examined in the valley bushveld of the eastern cape province were infested mainly with the larvae of a. hebraeum and haemaphysalis silacea and lesser numbers of a. marmoreum and small numbers of h. marginatum rufipes (horak & williams 1986). of these 86.9 % were 341 a.c. uys & i.g. horak table 1 ticks collected from 23 crested francolins on the farm “sandspruit”, limpopo province total number of ticks collected proportion no. of birds tick species larvae nymphs total (%) infested amblyomma hebraeum 853 37 890 40.44 20 amblyomma marmoreum 582 10 592 26.90 13 hyalomma marginatum rufipes 507 16 523 23.76 13 hyalomma truncatum 1 0 1 0.05 1 ixodes sp. 1 0 1 0.05 1 rhipicephalus (boophilus) decoloratus 4 0 4 0.18 3 rhipicephalus appendiculatus 7 3 10 0.45 3 rhipicephalus evertsi evertsi 6 0 6 0.27 3 rhipicephalus exophthalmos 3 0 3 0.14 2 rhipicephalus pravus group 8 0 8 0.36 4 rhipicephalus turanicus 7 0 7 0.32 3 rhipicephalus zambeziensis 63 3 66 3.00 10 argas sp. 90 0 90 4.09 4 table 2 the attachment distribution of ticks on 18 crested francolins on the farm “sandspruit”, limpopo province tick species and life number of relative abundance (%) stage ticks head body wings tail amblyomma hebraeum larvae 731 84.5 11.9 3.0 0.6 nymphs 32 46.9 28.1 18.8 6.2 amblyomma marmoreum larvae 578 13.7 32.7 32.7 20.9 nymphs 9 44.5 22.2 22.2 11.1 hyalomma marginatum rufipes larvae 382 91.6 5.8 1.8 0.8 nymphs 10 90.0 10.0 table 3 free-living ticks collected by drag-sampling the vegetation on the farm “sandspruit”, limpopo province total number of ticks collected tick species proportion (%) larvae nymphs total amblyomma hebraeum 111 0 111 7.34 amblyomma marmoreum 10 0 10 0.66 hyalomma marginatum rufipes 13 0 13 0.86 ixodes sp. 2 0 2* 0.13 rhipicephalus (boophilus) decoloratus 363 0 363 24.01 rhipicephalus appendiculatus 470 86 556** 36.77 rhipicephalus evertsi evertsi 356 0 356 23.55 rhipicephalus turanicus 3 0 3 0.20 rhipicephalus zambeziensis 85 13 98 6.48 * plus 1 male ** plus 1 female recovered from the heads and upper necks of the guineafowls, a proportion similar to the 84.5 % now recorded for a. hebraeum larvae on the heads of the francolins (table 2). tortoises, and particularly the leopard tortoise, geochelone pardalis, are the preferred hosts of all stages of development of a. marmoreum (norval 1975). helmeted guineafowls are good hosts of the larvae of this tick, and in the mountain zebra national park in the eastern cape province they harboured a mean burden of 86 larvae with a prevalence of 90 %, compared to eight larvae with a prevalence of 66.7 % on birds in the andries vosloo kudu reserve, and 19 larvae with a prevalence of 75.4 % on guineafowls in the kruger national park (horak & williams 1986; horak et al. 1991). the mean burden of the crested francolins in the present study was 25 larvae with a prevalence of 56.5 %. three of the seven cape francolins examined by horak & boomker (1998) in the bontebok national park harboured a total of 15 larvae and one nymph of this tick, and four of the seven greywing francolins a total of 129 larvae. the heads of the crested francolins were the attachment site least favoured by the larvae of a. marmoreum, with the body and wings harbouring most. this attachment distribution could be because of competition with the larvae of a. hebraeum and of h. marginatum rufipes, both of which prefer the head as an attachment site (table 2). the 26.9 % proportional representation of the immature stages of a. marmoreum on the francolins as opposed to the 0.66 % on the vegetation indicates that larvae are attracted to the birds, or that the habitat most favoured by the birds coincides with that most suitable for the survival of the free-living stages of the tick, or that only a small proportion of larvae are collected from the vegetation by drag-sampling, or that the timing of the drag-sampling did not correspond with the periods of peak larval numbers on the vegetation. the preferred hosts of the immature stages of h. marginatum rufipes are hares, lepus spp. and ground-frequenting birds (rechav, zeederberg & zeller 1987; horak et al. 1991). their presence on the francolins in the present survey is thus not unexpected. as this is a two-host tick the ratio of 31.7 larvae to one nymph on the birds indicates a very poor translation of larvae to nymphs. forty-four larvae and 132 nymphs of h. marginatum rufipes were collected from three of five scrub hares, lepus saxatilis, examined in the same area as the francolins, indicating excellent translation from one life stage to the next on these animals (horak, spickett, braack, penzhorn, bagnall & uys 1995). as with the previous two species there was a considerable difference between the proportional representation of larvae on the francolins (23.76 %) and free-living larvae on the vegetation (0.86 %). the single h. truncatum larva recovered from a bird in june 1996 is considered a “straggler”. the immature stages of the latter tick prefer hares as hosts, and it was the most numerous of the nine tick species collected from five scrub hares examined in the same region as the francolins (horak et al. 1995). the larvae of r. (boophilus) decoloratus were amongst the most numerous of the free-living ticks collected from the vegetation. this tick prefers domestic and wild bovids as hosts (mason & norval 1980), and its presence on any bird must be considered accidental and a reflection of the number of free-living ticks in the environment. the farm on which the present survey was conducted lies in a region of south africa in which the distributions of r. appendiculatus and rhipicephalus zambeziensis overlap (walker, keirans & horak 2000). although the immature stages of r. appendiculatus were considerably more numerous than those of r. zambeziensis on the vegetation the converse was true on the birds. a similar pattern of infestation has been observed on helmeted guineafowls in the kruger national park, where both ticks also occur (horak et al. 1991). equids are the preferred hosts of all stages of development of rhipicephalus evertsi evertsi, but cattle are also good hosts (walker et al. 2000). the large number of larvae collected from the vegetation can be ascribed to the presence of cattle and donkeys on the farm, while those on the francolins are accidental infestations. most records of rhipicephalus exophthalmos emanate from the southern and western regions of south africa and from namibia. scrub hares are preferred hosts of the adults, and with elephant shrews, elephantulus spp., are the only animals to date from which the immature stages have been recovered (walker et al. 2000). scrub hares are also preferred hosts of all stages of development of the rhipicephalus pravus-like tick (horak et al. 1995), of which larvae were collected from four of the francolins. an adult of rhipicephalus turanicus has previously been collected from a scrub hare in the study region (horak et al. 1995), and the recovery of larvae from three francolins in the present survey confirms its presence at this locality. infestation of the birds with the larvae of the aforemen342 ticks on crested francolins, francolinus sephaena, in limpopo province, south africa tioned three ticks should be considered as accidental. four of the 23 birds were infested with the larvae of an argas sp. a similar proportion of 118 helmeted guineafowls examined in the kruger national park were also infested with larvae of a tick belonging to this genus (horak et al. 1991). acknowledgements we thank messrs b.j. sneddon and d.a. uys for their assistance with shooting the francolins and performing the drag-sampling respectively. this research was financed by a grant from the faculty of veterinary science, university of pretoria. references acocks, j.p.h. 1988. veld types of south africa with accompanying veld type map. 3rd ed. (memoirs of the botanical survey of south africa, no. 57). hoogstraal, h., kaiser, m.n., traylor, m.a., gaber, s. & guindy, e. 1961. ticks (ixodoidea) on birds migrating from africa to europe and asia. bulletin of the world health organisation, 24:197–212. horak, i.g. & williams, e.j. 1986. parasites of domestic and wild animals in south africa. xviii. the crowned guineafowl (numida meleagridis), an important host of immature ixodid ticks. onderstepoort journal of veterinary research, 53: 119–122. horak, i.g., spickett, a.m., braack, l.e.o. & williams, e.j. 1991. parasites of domestic and wild animals in south africa. xxvii. ticks on helmeted guineafowls in the eastern cape province and eastern transvaal lowveld. onderstepoort journal of veterinary research, 58:137–143. horak, i.g., spickett, a.m., braack, l.e.o., penzhorn, b.l., bagnall, r.j. & uys, a.c. 1995. parasites of domestic and wild animals in south africa. xxxiii. ixodid ticks on scrub hares in the north-eastern regions of northern and eastern transvaal and of kwazulu-natal. onderstepoort journal of veterinary research, 62:123–131. horak, i.g. & boomker, j. 1998. parasites of domestic and wild animals in south africa. xxxv. ixodid ticks and bot fly larvae in the bontebok national park. onderstepoort journal of veterinary research, 65:205–211. mason, c.a. & norval, r.a.i. 1980. the ticks of zimbabwe. i. the genus boophilus. zimbabwe veterinary journal, 11:36– 43. norval, r.a.i. 1975. studies on the ecology of amblyomma marmoreum koch 1844 (acarina: ixodidae). journal of parasitology, 61:737–742. petney, t.n. & horak, i.g. 1987. the effect of dipping on parasitic and free-living populations of amblyomma hebraeum on a farm and on an adjacent nature reserve. onderstepoort journal of veterinary research, 54:529–533. rechav, y., zeederberg, m.e. & zeller, d.a. 1987. dynamics of african tick (acari: ixodoidea) populations in a natural crimean-congo hemorrhagic fever focus. journal of medical entomology, 24:575–583. theiler, g. 1962. the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region), project s 9958. report to the director of veterinary services, onderstepoort. mimeographed. walker, jane b., keirans, j.e. & horak, i.g. 2000. the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world. cambridge: academic press. 343 a.c. uys & i.g. horak abstract introduction methods results discussion conclusion acknowledgements references about the author(s) samuel mantip department of veterinary tropical diseases, university of pretoria, south africa viral research division, national veterinary research institute, south africa melvyn quan department of veterinary tropical diseases, university of pretoria, south africa david shamaki viral research division, national veterinary research institute, south africa moritz van vuuren department of veterinary tropical diseases, university of pretoria, south africa citation mantip, s., quan, m., shamaki, d. & van vuuren, m., 2016, ‘comparison of nucleotide sequences of recent and previous lineages of peste-des-petits-ruminants viruses of sheep and goats in nigeria’, onderstepoort journal of veterinary research 83(1), a1163. http://dx.doi.org/10.4102/ojvr.v83i1.1163 original research comparison of nucleotide sequences of recent and previous lineages of peste-des-petits-ruminants viruses of sheep and goats in nigeria samuel mantip, melvyn quan, david shamaki, moritz van vuuren received: 31 jan. 2016; accepted: 18 apr. 2016; published: 31 aug. 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract peste-des-petits-ruminants virus (pprv) is a highly contagious, fatal and economically important viral disease of small ruminants that is still endemic and militates against the production of sheep and goats in endemic areas of the world. the aim of this study was to describe the viral strains within the country. this was carried out by collecting tissue and swab samples from sheep and goats in various agro-ecological zones of nigeria. the phylogeny of archived pprv strains or isolates and those circulating and causing recent outbreaks was determined by sequencing of the nucleoprotein (n)-gene. twenty tissue and swab samples from apparently healthy and sick sheep and goats were collected randomly from 18 states, namely 3 states in each of the 6 agro-ecological zones visited. a total of 360 samples were collected. a total of 35 samples of 360 (9.7%) tested positive by reverse transcriptase–polymerase chain reaction, of which 25 were from oculo-nasal swabs and 10 were from tissue samples. neighbour-joining phylogenetic analysis using phylogenetic analysis using parsimony (paup) identified four different lineages, that is, lineages i, ii, iii and iv. interestingly, the nigerian strains described in this study grouped in two separate major lineages, that is, lineages ii and iv. strains from sokoto, oyo, plateau and ondo states grouped according to the historical distribution of pprv together with the nigerian 75/1 strain of lineage ii, while other strains from sokoto, oyo, plateau, akwa-ibom, adamawa, kaduna, lagos, bauchi, niger and kano states grouped together with the east african and asian strains of lineage iv. this finding confirms that both lineage ii and iv strains of pprv are circulating in nigeria. previously, only strains of lineage ii were found to be present in the country. introduction peste-des-petits-ruminants virus (pprv) belongs to the family paramyxoviridae and genus morbillivirus. pprv is a negative-sense, single-stranded, non-segmented ribonucleic acid (rna) virus. it is enveloped, pleomorphic, and ranges in size from 150 nm to 700 nm. six transcriptional units encode eight proteins; the nucleocapsid protein (n), phosphoprotein (p), large polymerase (l), fusion (f), haemagglutinin (h) and the matrix (m) protein and non-structural proteins c and v. peste-des-petits ruminants (ppr) has been mistaken for rinderpest since the fourth century ad (curasson 1932; henning 1956). in 1942, gargadennec and lalanne reported a disease in sheep and goats that resembled rinderpest in the west african country of ivory coast. clinical observations of ppr differed to rinderpest, infected sheep and goats were unable to transmit the disease to in-contact cattle (gargadennec & lalanne 1942). the first reported isolation of the aetiological agent of the disease was in 1962 by gilbert and monier. in nigeria, pprv was successfully isolated in 1975 (taylor 1979). pprv is distributed widely across west, central, east and some parts of north africa, the arabic peninsula, the middle east and asia (nanda et al. 1996). the ppr-endemic areas of the world are also important sheepand goat-rearing regions, and small livestock serve as an important means of providing food and commodities for trade. the production of these economically important animals for poor rural farmers is seriously hampered by the high morbidity caused by pprv. mortality from pprv in a naive flock can be as high as 80% – 90%. ppr manifests with pyrexia, oculo-nasal mucopurulent discharges, stomatitis, enteritis and conjunctivitis (losos et al. 1986). in severe cases, death is caused by secondary bacterial pneumonia or severe dehydration caused by acute diarrhoea. abortion caused by co-infection of both pprv and pestiviruses has been reported (abubakar, ali & khan 2008). because of the highly contagious nature of the disease, it can be transmitted easily to healthy animals through direct contact with the secretions and excretions of infected animals (ezeibe et al. 2008). the virulence of pprv varies from strain to strain, although only one serotype is recognised. pprv is sometimes referred to as a more serious disease of goats than sheep; however, outbreaks affecting both sheep and goat populations have been reported (chauhan et al. 2009; roeder et al. 1994; taylor & abegunde 1979; taylor et al. 2002; singh et al. 2004; wang et al. 2009). in some outbreaks, goats appear not to be affected, while sheep succumb with high rates of morbidity and mortality (yesilbag et al. 2005). the reason for this variability is unclear, but both viral strains and host species are thought to be of importance. different strains of pprv exhibit varied virulence when experimentally infected into the same breed of goat (couacy-hymann et al. 2007), and different breeds of goats have been shown to respond differently to infection with the same virus (diop, sarr & libeau 2005). the current molecular characterisation of pprv strains based on the n-gene divides them into four genetically distinct lineages (banyard et al. 2010). lineage i is represented mainly by west african strains from the 1970s and recent strains from central africa; lineage ii by west african strains from the ivory coast, guinea and burkina faso; lineage iii by isolates from eastern africa, sudan, yemen and oman; lineage iv includes all viruses isolated from recent outbreaks across the arabian peninsula, the middle east and southern asia (and recently across several african territories (banyard et al. 2010). because of the adverse consequences posed by pprv in the production of sheep and goats in nigeria, there is a need to undertake a study of pprv in sheep and goats from recent outbreaks across the different regions of the country. it is important to understand the spread of the pathogen within the country in comparison with other endemic areas of the world. the sequences derived from this study will provide the basis for better understanding of the epidemiology of pprv. the aim of this research project is to compare the sequences of recently occurring pprv strains with previously sequenced pprv isolates in genbank. methods location eighteen states were selected to represent the different agro-ecological zones of the country. these agro-ecological zones were aligned within the six geopolitical zones of the country for ease of reference. at present, there is no comprehensive figure for the population of sheep and goats in nigeria, but in 1983, a population of 22 million goats and 8 million sheep was estimated (federal livestock department, lagos, nigeria). locations where samples were collected were marked and recorded by the use of a gps apparatus (garmin) gpsmap 76csx with sensor. sample collection three states were identified randomly from each of the six agro-ecological zones and 20 samples of either oculo-nasal swabs or tissues (lung, spleen and lymph node) were collected with consent of the animals’ owners from each of the 18 states. swabs were collected from live animals, while tissues were collected from dead animals. the locations for sampling included major towns, their surrounding villages and suburbs. stratified sampling was used in herd or farm sites, grazing sites, market places and house backyard range. sampling was carried out during necropsy on dead animals and from sick animals with clinical signs that resembled ppr. the total number of samples collected was 360, which comprised 169 tissue samples and 191 oculo-nasal swabs. to ensure that the quality of the samples was maintained from the field to the laboratory, samples were always kept on iceblocks in a sample collection flask. preparation of necropsy tissues necropsy tissues were processed based on the standard operating procedure of the ppr laboratory, viral research division, national veterinary research institute (nvri), nigeria. briefly, 0.5 g of lung, spleen and lymph node from the same animal were pooled together and ground with a mortar and pestle to which sterile sand and phosphate-buffered saline had been added. the 10% suspension was centrifuged at 1800 g for 5 min and the supernatant filtered through a 0.22-µm filter to remove bacterial and fungal contaminants. nucleic acid was extracted from the filtrate. the remaining unprocessed tissues were stored at -80 °c for future use. identification of peste-des-petits-ruminants virus by reverse transcriptase-polymerase chain reaction a qiaamp viral rna mini extraction kit® qiagen (hilden, germany) was used to extract total rna from the field samples and from the positive control, according to the manufacturer’s instructions. the positive control was a live attenuated pprv nigeria 75/1 vaccine strain produced at the nvri, nigeria (diallo et al. 1989; taylor 1979). the extracted rna was stored at -80 °c until used. the samples were screened by a one-step reverse transcriptase–polymerase chain reaction (rt-pcr) assay for the detection of pprv nucleic acid using a geneamp® pcr system (lifetech) and the following verso (thermo-scientific®) protocol: 3.5 µl of ultra-pure sterile water, 1.0 µl rt-enhancer, 1.0 µl (10 pmole) of each forward primer ppr-np3 (5’-tctcggaaatcgcctcacagactg) and reverse primer ppr-np4 (5’-cct cct cct ggt cct ggt cct cca gaa tct), 12.5 µl one-step pcr hot-start master mix (2×), 1.0 µl of enzyme-mix and 5.0 µl of rna template in a final volume of 25 µl. the pcr cycling conditions consisted of an initial cycle of 50 °c for 15 min, 95 °c for 15 min, 45 cycles of 95 °c for 30 sec, 55 °c for 30 sec, 72 °c for 30 sec and a final extension at 72 °c for 10 min. the amplified pcr products were analysed by electrophoresis in a 1% tris acetate–edta agarose gel stained with ethidium bromide at a concentration of 1 µg/ml, and run at 120 v for 45 min. the bands were visualised under ultraviolet light and photographed. a 350-basepair (bp) fragment of the n-gene between nucleotides 1232 and 1583 of the n-gene (couacy-hyman et al. 2002; embl accession no. x74443) was amplified. sequencing the amplicons were purified using a qiaquick® pcr purification kit (qiagen®, southern cross biotechnology) according to the manufacturer’s instructions, packaged in dry-ice and sent by air from nvri (vom, nigeria) to inqaba biotec (south africa) for sequencing. amplicons were sequenced directly with a bigdye® terminator v.3.1 cycle sequencing kit (lifetech) according to the manufacturer’s instructions. precipitation of extension products was by the ethanol precipitation protocol of lifetech®. samples were electrophoresed using the spectrumedix genetics analysis system sce 240. phylogenetic analysis sequences were assembled using the staden package (staden, beal & bonfield 2000), aligned with clustalx v.2.1 (larkin et al. 2007) and edited with bioedit v.7.2.3 (hall 1999), and molecular evolutionary genetics analysis v.5.2. additional n sequences obtained from pprv isolated in other endemic countries in the world were obtained from genbank and were selected based on locations and n-gene lineage. a 243-nucleotide fragment of the pprv n-gene (between nucleotides 1307–1549 of sequence genbank accession number l39878) was used for phylogenetic analysis. a general time-reversible model with a gamma-shaped distribution of rate variation across sites (gtr + g) was selected by akaike information criterion in modeltest v3.7 (posada & crandall 1998). a 1000 bootstrap distance analysis was performed in paup* v4b10. results amplicons of the expected 350 bp were obtained by rt-pcr (figure 1). eleven of 35 (31.4%) tissue samples from female caprines were positive, 9 of 35 (25.7%) tissue samples from male caprines were positive, 2 of 35 (5.7%) tissue samples from female ovines were positive, 3 of 35 (8.6%) tissue samples from male ovines were positive, 4 of 35 (11.4%) oculo-nasal swabs from female caprine were positive, 5 of 35 (14.3%) oculo-nasal swabs from male caprines were positive, zero of 35 (0%) oculo-nasal swab from female ovines was negative and 1 of 35 (2.9%) oculo-nasal swabs from male ovines was positive for pprv as indicated in table 1. a total of 35 samples of 360 (9.7%) tested positive by rt-pcr, of which 25 were from tissue samples and 10 were from oculo-nasal swabs (table 2). the nucleotide sequences were deposited into genbank under the accession numbers kf908036–kf908047 (table 3). figure 1: positive reverse transcriptase–polymerase chain reaction of the peste-des-petits-ruminants virus n-gene of swab/homogenate samples is indicated by a 350-bp band. table 1: description of the animals and samples that tested positive for peste-des-petits-ruminants virus by verso one-step reverse transcriptase. tissue samples were pooled. table 2: description of the regions, study areas and period of sample collection, location and numerical number of positives in percentage and total number of samples collected. table 3: national center for biotechnology information genbank accession numbers of sequences described in this study. an unrooted bootstrapped 50% majority-rule consensus cladogram was produced. the sequences from oman ibri and ethiopia of lineage iii grouped together (bootstrap value 100%). sequences from côte d’ivoire, senegal and burkina faso grouped together in lineage i (bootstrap value 100%). the next clade consisted of nigeria 75/1, ghana 78 and mali sequences in lineage ii (bootstrap value 97%). the tree branched to lineage iv, which consisted of indian and israeli sequences (bootstrap value 88%) (figure 2). figure 2: fifty percent majority-rule consensus cladogram of partial unrooted peste-des-petits-ruminants virus n-gene sequences from this study and genbank. the phylogeny was determined by distance analysis in phylogenetic analysis using parsimony. the genetic distances, mean and standard deviation calculated between the nigerian pprv strains and the genbank nigerian strains are summarised in table 4. table 4: genetic distances between peste-des-petits-ruminants virus n genbank nigerian sequences and the recent nigerian sequences, using a general time-reversible model with a gamma-shaped distribution of rate variation across sites (gtr + g). discussion the aim of this study was to ascertain whether there were genetic variations between the present strains and previously isolated strains circulating in the country and to determine the geographical lineage(s) of the studied pprv. ppr was also known in the past in nigeria as ‘kata’ and goat plaque all over the world. in most developing countries including nigeria, small-ruminant farming covers the basic necessities of the majority of peasants. sheep and goats are essential sources of subsistence. they improve the survival of the poor people in these countries. unfortunately, ppr is adversely threatening the production of sheep and goats in endemic areas. ppr is a trans-boundary disease and is part of the group of economically important animal diseases whose outbreaks should be notified to the world organisation for animal health (oie for international des epizooties). in nigeria, because of the high importance of sheep and goats for poor farmers, the control of diseases that have a negative impact on their production is a major goal for programmes aimed at poverty alleviation. sheep and goats are reared in the same fashion in nigeria with slight differences between the savannah north and tropical rain forest zone of the south. the animals are rarely housed or tethered, except in areas such as eastern nigeria where pressure on land is creating competition between crops and livestock, so the latter must be restrained (carew 1982; sempeho 1982). the animals roam freely, requiring minimum investment in housing and feeds, non-labour intensive and are efficient in the utilisation of roughages, farm residues and agro-industrial by-products. in nigeria, sheep and goats are widely distributed all over the country across diverse ecological and climatic zones, with the pattern of ownership cutting across socio-economic status. in nigeria, it has been estimated that sheep and goats provide over 35% of the total animal protein consumed. their hides support the leather industry and at a conservative off-take value of 600 naira for a small ruminant, the total financial value of the small-ruminant industry in nigeria could be put at about 40 billion naira, based on an estimated population of 34.45 million goats and 22.09 million sheep (shamaki 2002). however, disease remains a major limiting factor in their production. the diseases that cause significant deaths in sheep and goats include bacterial and viral pneumonia, parasitic and viral gastro-enteritis, ectoparasitism as well as nutritional and metabolic diseases. among these, ppr is of paramount significance as a disease entity, as it causes both respiratory and gastro-intestinal tract disorders. the expected number of 60 samples per zone and 1 pprv amplicon sequence representation per state with location of sampling mapped by gps coordinates was achieved across the country except for the riverine niger delta of bayelsa state where movement to villages was difficult because of lack of access roads, and northern states because of the insurgence of the boko haram militia. however, sampling was stratified and targeted sheep and goats with classical or sub-clinical signs of fever and depression, ocular and nasal discharges, diarrhoea and emaciation. the sampling was more of a passive surveillance in locations within the study areas, which included the six agro-ecological zones of nigeria, as indicated in table 1. the 35 positive samples out of a total of 360 (9.7%) samples were from all the agro-ecological zones except the south-eastern zone for which no positive results were obtained by rt-pcr, although some farmers in the zone complained of losing all their goats to an epidemic that resembled ppr prior to sampling. livestock are not allowed to roam freely in this region, thereby limiting the chances of cross-transmission of the disease from one flock to another during an outbreak. other possible reasons for the negative results in this area could be because of very low viral load, which could not be detected by the assay, or it could be that the rna was lost during processing. previous findings by wosu et al. (1994) and obi et al. (1983) revealed that ppr was prevalent in the south-east zone of the country. based on the results in tables 1 and 2 and the percentages of the analysed results according to species, sex and breed, it was concluded that pprv in an infected population affects both sheep and goats, male and female animals of both species, and equally affects both the long-legged species of the north and the short-legged species or west african dwarf goats or sheep of the south. fourteen samples collected from the study areas of the north-central region in the month of july, 9 samples from the north-west collected in august, 11 samples from the north-east collected in september, 15 samples from the south-west collected in october, 3 samples from the south-south collected in january tested positive by rt-pcr for pprv, while none collected from south-east in november tested positive. these findings suggest that ppr was prevalent in nigeria during the sampled period. however, a study conducted by wosu et al. (1990) reported that ppr incidence was higher in south-eastern nigeria during the dry hamattan season (december – january) than in the rainy season with a peak in april. sampling before the expected period of high incidence of ppr outbreaks in the south-eastern region could also be the reason for the inability to get positive samples in this zone. cattaneo et al. (1987) suggested that the n-gene of pprv was more abundant in positive tissue samples and couacy-hymann et al. (2002) used the n-gene to detect pprv in suspected samples. targeting of pprv n-gene for pcr gave very promising results. n-gene codes for an internal structural protein and mrnas of the n-gene are the most abundant transcripts of the virus, making it an attractive target for development of a highly sensitive and specific diagnostic assay for pprv (george 2002). based on clinical signs and the detection of pprv using rt-pcr, it was concluded that the disease outbreaks in the present study areas were caused by pprv, although tests for other pathogens were not carried out. the genetic similarity and divergence among the present pprv sequence strains of nigeria and their relatedness to some of the previously described isolates in genbank were analysed. the ncbi blast search revealed that some nigerian pprvs obtained in this study had an identity of 100% for nigeria 75/1 and 95% for nigeria 76/1, and unexpectedly most of the strains recently obtained had 97% – 99% identity to strains from the republic of gabon (97%) and (96%) identity to strains from the republic of iran (96%) (table 3). nucleotide genetic diversity was examined (table 4), and the shortest genetic mean distance ± standard deviation (0.032 ± 0.0187) was between the recent pprv strains of lineage ii ng vacnvri 75/1. this suggests that of the present ppr vaccine produced in nvri, vom is similar to the previous nigerian strains deposited in genbank. even though the vaccine viral strain passed through a series of tissue culture attenuations, the genetic relationship between the field strain nigeria 75/1 and the vaccine strain ng vacnvri 75/1 is still similar, that is, using the (gtr + g), the genetic distance is 0.00415, the mean ± standard deviation is 0.032 ± 0.0187 which are significantly negligible. the longest mean genetic distance (0.0681 and 0.00913) was between lineage ii samples from plateau, sokoto and oyo states. this increase in genetic distance could be because of genetic drift over time. in lineage iv of the recently studied strains, the shortest genetic mean distance (0.0157) was from the niger and plateau states, and the longest genetic mean distance variable (0.0187) was from kaduna and bauchi states. the emergence of these new strains in nigeria is probably because of the introduction of the strains from other lineage iv pprv-endemic countries through trade. based on previous studies of geographical distribution of pprv (couacy-hymann et al. 2002; dhar et al. 2002; ozkul et al. 2002; shaila et al. 1996), all nigerian strains/isolates normally clustered together into a separate branch from all the asian isolates. unexpectedly, the new nigerian strains clustered in two separate lineages (figure 2). strains from sokoto, oyo and plateau states clustered with existing strains from mali-91 (dq840192), senegal-2010 (hq131963) and sierra-leone-2009 (jn802080) of lineage ii. recent strains from ondo state and the nvri vaccine 75/1 isolate clustered with the previous nigeria 75/1 (dq840160), nigeria 75/2 (dq840161), nigeria 75/3 (dq840162) and ghana 78 (dq840166) of lineage ii. in lineage iv, new strains from plateau, bauchi, oyo, sokoto, kano, adamawa, kaduna and bauchi states formed a sub-cluster, and strains from adamawa, bauchi, lagos, kaduna, plateau, niger, oyo and akwa-ibom formed another sub-cluster. the strain from sudan 2008 (hq131921) was closely related to the new nigerian strains compared to other genbank isolates in lineage iv. this finding suggests that both lineage ii and iv strains of pprvs are circulating presently in all the studied agro-ecological zones of nigeria as indicated in figure 3 and as described by woma et al. (2015). despite the fact that the recent strains from lineage iv seem to predominate in most of the study area, there is no clear demarcation of the state or region where these new strains are found. they appear to be spread all over the study area as indicated in figure 3. the introduction of the new strain is probably because of the movement of sheep and goats from the sahel african countries into nigeria and from the northern dry savannah region to the wet forest southern region of the country for grazing during the dry season. trade of animals and animal products from other african countries to nigeria and from one state to another may serve as the major source of transmission of trans-boundary animal diseases including pprv. the presence of the new strain of pprv in the country is not surprising because nigeria is a major hub of animal product consumption in west africa because of its large population. meeting the ever-increasing domestic demand leads to the importation of sheep and goats from the neighbouring sahel countries that raise livestock. low disease monitoring, introduction and re-introduction of many exotic diseases and disease strains have been traced to the animals that entered the country through the porous borders. figure 3: map of nigeria describing the gps location of sampling indicated in red ink and lineages isolated. the data obtained from this study have provided important and additional information on the molecular epidemiology of ppr virus in nigeria. however, the clustering of sequences in different areas indicates that the pprv population in nigeria is more diverse than previously thought and this study provides a better epidemiological picture of pprv in endemic areas of the world, such as nigeria. conclusion the results obtained from this study suggest that ppr still remains endemic with sporadic epidemics in nigeria and other sub-saharan african countries. regular outbreaks in areas where it was considered exotic in the past are conceivably because of an increase in trade and commerce. during this study, molecular characterisation of ppr viruses based on the amplification and sequencing of fragments of the n-gene showed that the pprv from nigeria belonged to two different lineages: ii and iv, with two sub-clusters in lineage ii and two sub-clusters in lineage iv. the new strains were related most closely to isolates from the east african countries of gabon, central republic of africa and sudan via cameroon and chad. however, there was no clear demarcation that separated where these two different strains were found in nigeria, but rather both appeared to be found in all the study areas. this suggests that both strains are endemic and circulating all over the country. considering the wide distribution of ppr in the world, it would be very helpful to institute an investigative and monitory measure to check the movement of animals from one location to another, especially for countries where ppr is still an exotic disease. ppr should be included in the list of differential diagnoses for pneumo-enteritis in small ruminants in non-endemic countries, and sero-surveillance for ppr in countries bordering endemic and affected countries especially in southern africa, should be considered. acknowledgements the authors thank dr mohammed s. ahmed, executive director, nvri vom, for the financial support. the authors also thank the staff and postgraduate students of the department of veterinary tropical disease (dvtd), university of pretoria, south africa, for their friendship and technical support and the entire staff of nvri vom for their moral and technical support. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions m.v.v. is the project leader, d.s., m.v.v. and s.m. were responsible for experimental and project design. s.m. performed most of the experiments. m.q., 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woma, t.y., adombi, c.m., yu, d., qasim, a.m., sabi, a.a., maurice, n.a. et al., 2015, ‘co-circulation of peste-des-petits-ruminants virus asian lineage iv with lineage ii in nigeria’, transbound and emerging diseases 63(3), 235-242. http://dx.doi.org/10.1111/tbed.12387 wosu, l.o., 1994, ‘current status of peste des petits ruminants (ppr) disease in small ruminants. a review article’, stud res vet med 2, 83–90. dutoit_315-321.indd introduction spirocercosis is a canine disease caused by the nem atode spirocerca lupi (rudolphi, 1809) (spiru rida: spirocercidae) (van der merwe, kirberger, clift, williams, keller & naidoo, in press). this is a cosmopolitan parasite, but is found more commonly in the warmer tropical and subtropical regions of the world (bailey 1972). spirocerca lupi is a parasite mainly of domestic dogs, although it also infects other members of the family canidae (mazaki-tovi, baneth, aroch, harrus, kass, ben-ari, zur, aizenberg, bark & lavy 2002). natural infections have been reported in coyotes, wolves, foxes and jackals, which serve as important reservoir hosts (bailey 1972). dung beetles are the main intermediate hosts of this parasitic nematode (bailey 1972). there is a plethora of literature on the clinical, diagnostic and epidemiologic aspects of spirocercosis in dogs, while very few studies have focused on the host-parasite associations between dung beetles and s. lupi (bailey 1972; chhabra & singh 1973, 315 onderstepoort journal of veterinary research, 75:315–321 (2008) prevalence of the dog nematode spirocerca lupi in populations of its intermediate dung beetle host in the tshwane (pretoria) metropole, south africa c.a. du toit1 *, c.h. scholtz1 and w.b. hyman2 abstract du toit, c.a., scholtz, c.h. & hyman, w.b. 2008. prevalence of the dog nematode spirocerca lupi in populations of its intermediate dung beetle host in the pretoria metropole, south africa. onderstepoort journal of veterinary research, 75:315–321 spirocerca lupi (spirurida: spirocercidae) is a cosmopolitan parasite, principally of domestic dogs and dung beetles are its main intermediate hosts. in south africa there has recently been growing concern over the upsurge of reported cases of clinical spirocercosis in dogs, while little is known or understood about the dynamics of the host-parasite associations between dung beetles and this nematode. we determined and compared the prevalence of infection in dung beetles between rural, urban and periurban areas of tshwane (pretoria) metropole. dung beetles were sampled during april and october 2006, at various localities in each of these areas. localities were selected on the basis of being focal areas of high infection with s. lupi in dogs. pig, dog and cow dung-baited pitfall traps were used for sampling the beetles. trap contents were collected 48 h after the traps had been set and only dung beetles were collected from the traps. in total, 453 specimens belonging to 18 species were collected from 63 pitfall traps in all three areas. the numbers of species that were collected varied among the three areas. dung beetles, irrespective of species (18) and numbers (447), predominantly preferred pig dung. the prevalence of dung beetles infected with the larvae of s. lupi varied considerably in the three areas. in the urban area 13.5 % of the dung beetles dissected were infected, while the prevalence of s. lupi in dung beetles in the rural area was 2.3 %. all the dung beetles that were infected with this nematode showed a preference for omnivore (pig and dog) dung. keywords: dung beetle, intermediate host, metropole, prevalence, spirocerca lupi, spirocercosis, tshwane (pretoria) * author to whom correspondence is to be directed. e-mail: cdutoit@zoology.up.ac.za 1 scarab research group, department of zoology and entomol ogy, university of pretoria, pretoria, 0002 south africa 2 vet-pharm cc, p.o. box 60, bredell, 1623 south africa accepted for publication 20 august 2008—editor 316 prevalence of dog nematode spirocerca lupi in dung beetle host in tshwane, south africa 1977). in a study to determine the prevalence of helminth parasites in dogs from a resource-limited urban community in gauteng province, minnaar & kre cek (2001) found that 14.5 % of dogs were infected with this parasite. a similar study by minnaar, krecek & fourie (2002) in a resource-limited periurban community in the free state province showed a prevalence of infection of 13 % in dogs from that re gion. recently, there has been growing concern over the upsurge of reported cases of spirocercosis in dogs in south africa, while little is known or understood about the dynamics of the host-parasite associations between dung beetles and this nematode. in this paper we discuss the prevalence of infection in populations of dung beetles with the larvae of s. lupi in rural, urban and peri-urban areas in the tshwane (pretoria) metropole of south africa. spirocerca lupi larval life cycle intermediate as well as paratenic (transport) hosts are involved in the larval life cycle of this parasite. however, in light of the scope of this paper the discussion is limited to larval development in the coprophagous beetle intermediate host. embryonated s. lupi eggs (l1) are passed in the faeces of the definitive host, whereafter it is ingested by a number of suitable species of coprophagous beetle intermediate hosts (van der merwe et al., in press). the eggs hatch after ingestion and within 2 months the larvae develop into the infective stage (l3). in the intermediate beetle host the infective stage larvae become encysted mainly on the tracheal tubules (mazaki-tovi et al. 2002). the coprophagous beetle, containing l3 larvae is ingested either by a paratenic or definitive host. the final host (dog) may become infected after ingestion of such a coprophagous beetle (van der merwe et al., in press). materials and methods we conducted a pilot study during 2006 in the tshwa ne (pretoria) metropole to determine and compare the prevalence of infection in dung beetles with the larvae of s. lupi between rural, urban and peri-unban areas. the prevalence of infection with this parasite was also compared between dungspecific and non-specific dung beetle species from the same communities. description of the study area the study was conducted in the tshwane (pretoria) metropole north of the magaliesberg range. this mountain range separates the metropole into two large vegetation types: cooler bankenveld (bredenkamp & van rooyen 1998) to the south and warmer sour bushveld and clay thorn bushveld (van rooyen & bredenkamp 1998a, b) to the north. the study area was classified into rural, urban and peri-urban areas, based on characteristics of their individual land use and the potential free-range limits of the dogs within each area. this distinction between areas translated into agricultural smallholdings being classified as rural areas, suburban gardens as being urban areas and resource-limited townships and informal settlements as being peri-urban areas. experimental design dung beetles were sampled during april and october 2006 at various localities in each of these areas. localities were selected on the basis of being focal areas of high infection with s. lupi in dogs. the department of veterinary tropical diseases, university of pretoria provided information concerning the infection rates in dogs from various areas, which they had compiled from reports of necropsies performed at the onderstepoort campus. dung beetles were sampled in three localities per area. in this study the domestic dog was treated as an omnivore. pig, dog and cow dung-baited pitfall traps were used for capturing dung beetles. nine pitfall traps were placed in three transects in each locality. transects were separated by 15-m intervals and each of the three traps per transect were placed 10 m apart. each transect was baited with one of the three different dung types. the plastic buckets used for the traps had a 1 000 mℓ capacity and were 11 cm in diameter and 12 cm deep. traps were sunk into the ground so that the rims of the buckets were level with the soil surface and were filled to about one fifth their volumes with a solution of liquid soap and water to immobilize trapped dung beetles. dung baits were suspended on u-shaped metal wire, placed over the traps. trap contents were collected 48 h after the traps had been set and only dung beetles were collected from the traps. morphospecies were identified and conspecific beetles, collected from the same dung type and area (rural, urban or peri urban), were pooled and stored together in absolute ethanol in labelled jars. the beetles were then positively identified in the laboratory. data collection and analysis a maximum of 20 specimens per species per dung type and locality were dissected. the dung beetles were dissected in distilled water and examined un317 c.a. du toit, c.h. scholtz & w.b. hyman der a stereoscopic microscope for the presence or absence of s. lupi larvae (mönnig 1938). individual beetles were recorded as being either positive or negative for infection. the data for all the localities in an area were combined for statistical analysis. the significance in difference of prevalence of infection between areas was tested using the chisquare test (fowler, cohen & jarvis 1998). the 2x3 contingency table was subdivided (zar 1984) into three 2x2 contingency tables in a series of multiple comparisons between areas. yates’ corrected chisquare tests (fowler et al. 1998) were used to test which areas’ prevalence of infected beetles occurred at relative frequencies significantly different from those of the others. furthermore, fisher exact tests (zar 1984) were performed for all the 2x2 tables that had more than 20 % of their expected frequencies below five. a sequential bonferroni correc tion (rice 1989) was applied for the multiple comparisons. the prevalence of infected dung beetles in each area was calculated (rózsa, reiczigel & majoros 2000) and reported as a percentage. results the results of the sampling effort that took place during april 2006 were omitted from this study, due to the data being insufficient for statistical analysis. however, a sampling protocol was established for the subsequent sampling that was done during october 2006. in total, 453 specimens belonging to 18 species were collected from the 63 pitfall traps in the three areas during october 2006. the numbers of species that were collected varied among the three areas. dung beetles, irrespective of species (18) and numbers (447), predominantly preferred pig dung. only six individuals of three species were collected from pitfall traps baited with dog dung, and no dung beetles were attracted to cattle dung. the rural area, where 11 species were collected, showed the highest species richness, followed by the periurban area, where nine species were collected. the urban area, with only six species collected, had the lowest richness. the prevalence of infection with s. lupi larvae in dung beetles varied considerably in the three areas. in the urban area 13.5 % (7/52) of the dung beetles dissected were infected with the nematode and the number of parasite larvae per beetle varied between 1 and 119 (table 1). prevalence of infection in the rural area was 2.3 % (3/129) (table 2), with the number of larvae per beetle ranging from 1 to 10. no dung beetles collected from the peri-urban area were found to be infected with s. lupi larvae (table 3). the three areas differed significantly from one another with regard to the prevalence of dung beetles infected with s. lupi (chi-square test: χ2 = 16.19, df = 2; p < 0.05) (table 4). the prevalence of infected dung beetles differed significantly between the rural and urban areas (yates’ corrected chi-square test: χ2 = 8.15, df = 1; p < 0.05; fisher exact test: χ2 = 7.61, df = 1; p < 0.05) (table 5), as well as between the urban and periurban areas (yates’ corrected chi-square test: χ2 = 9.94, df = 1; p < 0.05; fisher exact test: χ2 = 9.64, df = 1; p < 0.05) (table 6). however, there was no significant difference in the prevalence of infected dung beetles between the rural and peri-urban areas (yates’ corrected chi-square test: χ2 = 2.49, df = 1; p < 0.05; fisher exact test: χ2 = 1.24, df = 1; p < 0.05) (table 7). the results remained unchanged after a sequential bonferroni correction was applied to the multiple comparisons. all the dung beetles that were infected with this nematode showed a preference for omnivore (pig and dog) dung. table 1 results of the dissection of various dung beetle species from an urban area in the tshwane (pretoria) metropole to investigate the incidence of infection with spirocerca lupi under natural conditions dung beetle species number dissected number positive for s. lupi number of parasite larvae per beetle range average gymnopleurus virens onthophagus ebenus onthophagus pugionatus onthophagus spp. b onthophagus sugillatus onthophagus vinctus 1 6 40 3 1 1 0 1 5 0 1 0 – 9 1– 119 – 105 – – 9.0 37.8 – 105.0 – 318 prevalence of dog nematode spirocerca lupi in dung beetle host in tshwane, south africa dung beetle—spirocerca lupi associations the prevalence of canine spirocercosis varies within its geographical range (mazaki-tovi et al. 2002) and the dung beetle intermediate hosts are widely distributed throughout the distribution area of s. lupi (bailey 1972). this study showed that the prevalence of this parasite in its intermediate dung beetle hosts differs significantly among rural (2.3 %), urban (13.5 %) and peri-urban (0 %) areas in the tshwane (pretoria) metropole. parasites are aggregated across their host pop ulations with the majority of them occurring in the minority of their hosts. moreover, parasite prevalence changes temporally and spatially (wilson, bjornstad, dobson, merler, poglayen, randolph, read & skorping 2002). these heterogeneities arise from the variation between individuals in their exposure to parasite infective stages and by differences in their susceptibility once an infective agent has been encountered (wilson et al. 2002). it seems that the table 2 results of the dissection of various dung beetle species from a rural area in the tshwane (pretoria) metropole to investigate the incidence of infection with spirocerca lupi under natural conditions dung beetle species number dissected number positive for s. lupi number of parasite larvae per beetle range average euonthophagus carbonarius gymnopleurus virens onthophagus aeruginosis onthophagus obtusicornis onthophagus pugionatus onthophagus spp. b onthophagus spp. nr. pullus onthophagus sugillatus onthophagus vinctus sisyphus goryi tiniocellus spinipes 2 6 20 20 21 9 1 22 2 20 6 0 2 0 1 0 0 0 0 0 0 0 – 1– 10 – 9 – – – – – – – – 6.5 – 9.0 – – – – – – – table 3 results of the dissection of various dung beetle species from a peri-urban area in the tshwane (pretoria) metropole to investigate the incidence of infection with spirocerca lupi under natural conditions dung beetle species number dissected number positive for s. lupi number of parasite larvae per beetle range average euoniticellus intermedius liatongus militaris nr. sisyphus ruber onitis alexis onthophagus aeruginosis onthophagus lamelliger onthophagus spp. b onthophagus stellio onthophagus sugillatus 3 2 7 1 11 3 1 21 22 0 0 0 0 0 0 0 0 0 – – – – – – – – – – – – – – – – – – table 4 observed frequencies of uninfected and infected dung beetles from three areas in the tshwane (pretoria) metropole beetles area total rural urban peri-urban uninfected dung beetles infected dung beetles 126 3 45 7 71 0 242 10 total 129 52 71 252 319 c.a. du toit, c.h. scholtz & w.b. hyman prevalence of spirocercosis is influenced by the proximity of the final host to the intermediate hosts, as well as the density of such infected hosts in the environment where they are preyed upon by the definitive host (mazaki-tovi et al. 2002). there are also several selective factors that control beetle associations in dung beetle communities (lumaret, kadiri & bertrand 1992). these factors include the nature of the soil substrate (lumaret et al. 1992), fauna and flora of the specific region, rainfall and temperature (bailey 1972). the widespread use of pesticides in an area might lead to a decrease in the population size of dung beetles, which will lead to a decrease in the prevalence of this parasite in that area (bailey 1972). maximum dung beetle activity is correlated with the onset of the rainy season in many parts of the world. during this season there would be optimal opportunity for a final host to ingest infect ed dung beetles (brodey, thompson, sayer & eugster 1977). conditions for maximum dung beetle activity were suboptimal during october 2006 when sampling took place. although temperatures were constantly above 25 °c, no rain had yet been recorded for any of the localities in the rural, urban or peri-urban areas. the rural area was devoted to mainly small scale livestock and crop production, but sampling sites were always located in patches of natural vegetation, which might explain why the highest number of species (11 species) was collected in that area. although the peri-urban area had the second highest number of recorded species (nine species), sites in this area were heavily polluted by rubbish such as plastic bags, broken glass, paper and biological waste material. furthermore, these sites were mostly ecologically degraded and the vegetation predom inantly alien. the fact that the peri-urban sites had the second highest number of species might be attributable to the ever present and seemingly abundant goats and cattle which roam the area. the urban area had the lowest species number (six) of all three areas. although the majority of gardens in this area are watered throughout the year, they represent a modified environment of which the vegetation is almost exclusively alien. a small patch of natural vegetatable 5 observed frequencies of uninfected and infected dung beetles from a rural and an urban area in the tshwane (pretoria) metropole beetles area total rural urban uninfected dung beetles infected dung beetles 126 3 45 7 171 10 total 129 52 181 table 6 observed frequencies of uninfected and infected dung beetles from an urban and a peri-urban area in the tshwane (pretoria) metropole beetles area total urban peri-urban uninfected dung beetles infected dung beetles 45 7 71 0 116 7 total 52 71 123 table 7 observed frequencies of uninfected and infected dung beetles from a rural and a peri-urban area in the tshwane (pretoria) metropole beetles area total rural peri-urban uninfected dung beetles infected dung beetles 126 3 71 0 197 3 total 129 71 200 320 prevalence of dog nematode spirocerca lupi in dung beetle host in tshwane, south africa tion was found in only one of the urban sites, where a few ostriches were kept. pesticides are also often applied to maintain the integrity and aesthetic value of gardens. the availability of excrement as a food source influences the abundance of dung beetles in a specific area (bailey 1972), although it seems that food is not an important determinant of local species distributions (lumaret et al. 1992). dung beetles show preferences for certain dung types (lumaret et al. 1992). this holds important implications for the prevalence of this parasite in dung beetle populations. dung beetles that are not attracted to the faeces of any of the various definitive hosts might not be good intermediate hosts under natural conditions (bailey 1972). in this study only omnivore dung-specific dung beetles were found to be parasitized by s. lupi larvae. this might be related to the fact that the definitive hosts are mainly domestic dogs and a few other members of the family canidae. there was a high concentration of domestic dogs in the urban area and the sampling sites in the rural area were all close to pig farms. furthermore, owners of properties in the rural area often kept more than three dogs. a sufficient explanation cannot be offered for the absence of herbivore dung-specific or generalist dung beetles from the peri-urban area. the feeding biology of adult dung beetles is not fully understood (holter, scholtz & wardhaugh 2002). miller (1961) attributed the absence of helminth eggs in the digestive tracts of dung beetles to the masticating action of their mandibles. from his experiments he deduced that the mandibles of dung beetles serve the dual purpose of gathering and masticating faecal material and that helminth eggs are destroyed or damaged in this process. however, this does not sufficiently explain the consistent presence of s. lupi parasites in members of certain taxa. in subsequent research holter et al. (2002) and holter & scholtz (2005) have shown that dung beetles do not masticate their food prior to ingestion and determined the size of ingested food particles in different species of dung beetles. in these studies it was shown that dung beetles from various taxa and across different body size classes and ecotypes (tunnellers, rollers and endocoprids) ingest food particles that varied from less than 5 μm to more than 83 μm in diameter. food particles are filtered through filtration channels connected to the molar surface through narrow fissures prior to ingestion (holter et al. 2002). the eggs of s. lupi measure 11–15 x 30–37 μm (mönnig 1938) and this, rather than the masticating action of the mandibles of dung beetles, might explain the absence of parasites in members of certain taxa. thus, dung beetles that can only ingest food particles that are larger than the eggs, will be able to serve as intermediate hosts of this parasite. the prevalence of spirocercosis also varies over relatively short periods of time (bailey 1972). in a study by chhabra & singh (1973) it was shown that the prevalence of infection in beetles increased towards the middle of the breeding season of dung beetles infected in the laboratory. in israel the rate of detection of spirocercosis is significantly higher during the colder months. this might be explained by the seasonality of the main dung beetle intermediate host, onthophagus sellatus, in that country (mazaki-tovi et al. 2002). moreover, if any of the beetles dissected during this study contained parasite eggs or first instar larvae they would have been recorded as being negative for infection with this parasite since they are too small to detect under the light microscope that was used in this study. the developmental time from egg to the infective third instar larva is about 2 months (mazaki-tovi et al. 2002). there seems to be an increased incidence of clinical spirocercosis among dogs in south africa in both urban and rural areas, perhaps due to improved techniques for early diagnosis as well as an increased global multidisciplinary interest in this disease and its agents. this study indicated that the area with the least species diversity had the highest prevalence of infection. thus, the simple classification of areas as rural, urban or peri-urban might not be sufficient and we want to investigate whether there may be a correlation between human population density and the prevalence of infection among dung beetle populations from corresponding areas. therefore, we will determine and compare the prevalence of infection in dung beetles in a specific number of areas of high and low human population densities in two geographical regions [tshwane (pre toria) metropole and grahamstown]. these regions have been selected on the basis of being focal areas of high infection with s. lupi in dogs. it is not known exactly which or how many species of dung beetles transmit this parasite or what the effect of dung preference on susceptible and nonsusceptible dung beetle species is. thus, we want to determine which species of dung beetles in each geographical range are susceptible to infection under natural conditions. moreover, we want to determine whether dung beetle species that are not attracted to the faeces of the definitive host and that 321 c.a. du toit, c.h. scholtz & w.b. hyman may not be good intermediate hosts under natural conditions, are capable of being good intermediate hosts under experimental conditions. conclusions there is an urgent need for better control and preventative measures to be investigated for this disease in dogs. a better understanding of the dynamics of the intermediate host-parasite associations be tween dung beetles and s. lupi under south african conditions may contribute to establishing preventative measures for the spread of this disease as well as finding more effective treatment for spirocercosis in domestic dogs. furthermore, it might aid in identifying objectives and priorities for management for those with a technical interest in the problem, as well as those who might be affected emotionally and economically. acknowledgements we thank the nrf for their funding of this project as well as the university of pretoria for logistical and financial support. furthermore, we thank dr adrian davis for the identification of the dung beetles and dr federico escobar for advice on the experimental design of the project. references bailey, w.s. 1972. spirocerca lupi: a continuing inquiry. journal of parasitology, 58:3–22. bredenkamp, g. & van rooyen, n. 1998. rocky highveld grassland, in vegetation of south africa, lesotho and swaziland, 2nd ed., edited by a.b. low & a.g. robelo. pretoria: department of environmental affairs and tourism. brodey, r.s., thompson, r.g., sayer, p.d. & eugster, b. 1977. spirocerca lupi infection in dogs in kenya. veterinary parasitology, 3:49–59. chhabra, r.c. & singh, k.s. 1973. a study of the life-history of 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j. & majoros, g. 2000. quantifying parasites in samples of hosts. journal of parasitology, 86: 228–232. van der merwe, l.l., kirberger, r.m., clift, s., williams, m., keller, n. & naidoo, v. spirocerca lupi infection in the dog: a review. the veterinary journal (in press). van rooyen, n. & bredenkamp, g. 1998a. clay thorn bushveld, in vegetation of south africa, lesotho and swaziland, 2nd ed., edited by a.b. low & a.g. robelo. pretoria: department of environmental affairs and tourism. van rooyen, n. & bredenkamp, g. 1998b. waterberg moist mountain bushveld, in vegetation of south africa, lesotho and swaziland, 2nd ed., edited by a.b. low & a.g. robelo. pretoria: department of environmental affairs and tourism. wilson, k., bjornstad, o.n., dobson, a.p., merler, s., poglayen, g., randolph, s.e., read, a.f. & skorping, a. 2002. heterogeneities in macroparasite infections: patterns and processes, in the ecology of wildlife diseases, edited by p.j. hudson, a. rizzoli, b.t. grenfell, h. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice http://www.ojvr.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on https:// ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https://ojvr.org 2. in your ‘user home’ [https://ojvr.org/index. php/ojvr/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest(s). 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the onderstepoort journal of veterinary research. please do not hesitate to contact us if you require assistance in performing this task. publisher: publishing@aosis.co.za tel: +27 21 975 2602 tel: 086 1000 381 the editorial team of onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of onderstepoort journal of veterinary research. we appreciate the time taken to perform your review(s) successfully. abdelwahid s. ali abdulrasaq b. nafiu ali bouattour ali halajian andre vosloo ayana dinka barend mans bersissa e. kumsa borden mushonga caryn l. shacklock claude sabeta davies m. pfukenyi dilek muz estelle h. venter frans jongejan gift matope guzmán álvarez hassen kabede henriette van heerden herman m. chambaro isaac phiri jannie crafford john b. muma junita liebenberg kalenzi david kembenya e. migiro kristine coleman maab i. al-farwachi marco romito mary-louise penrith matthew a. adeleke maxwell barson michael chimonyo mohammed el khasmi motlatso t. hlokwe nicola e. collins olena kudlai peter thompson radhwane saidi ricky chazya rolf k. schuster selim a. alarape shahn bisschop sikirullai o. jeje sohail h. khan takeet michael veysel s. ataseven yasushi itoh zeynep karapınar http://www.ojvr.org� https://ojvr.org� https://ojvr.org� https://ojvr.org� https://ojvr.org/index.php/ojvr/user https://ojvr.org/index.php/ojvr/user mailto:publishing@aosis.co.za abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) musafiri karama department of paraclinical sciences, faculty of veterinary science, university of pretoria, onderstepoort, south africa beniamino t. cenci-goga department of paraclinical sciences, faculty of veterinary science, university of pretoria, onderstepoort, south africa department of veterinary medicine, laboratorio di ispezione degli alimenti di origine animale, university of perugia, perugia, italy alice prosperi experimental zooprofilattico institute of lombardy and emilia-romagna ‘bruno ubertini’, brescia, italy department of veterinary medical sciences, university of bologna, bologna, italy eric etter department of production animal studies, faculty of veterinary science, university of pretoria, onderstepoort, south africa centre de coopération internationale en recherche agronomique pour le développement-inra, umr astre baillarguet international campus, university of montpellier, montpellier, france saeed el-ashram college of life science and engineering, foshan university, foshan, china faculty of science, kafr elsheikh university, kafr el sheikh, egypt cheryl mccrindle department of agriculture and animal health, university of south africa, johannesburg, south africa jackson n. ombui department of public health, pharmacology and toxicology, college of agriculture and veterinary sciences, university of nairobi, nairobi, kenya alan kalake gauteng department of agriculture and rural development, gauteng, johannesburg, south africa citation karama, m., cenci-goga, b.t., prosperi, a., etter, e., el-ashram, s., mccrindle, c. et al., 2019, ‘prevalence and risk factors associated with campylobacter spp. occurrence in healthy dogs visiting four rural community veterinary clinics in south africa’, onderstepoort journal of veterinary research 86(1), a1673. https://doi.org/10.4102/ojvr.v86i1.1673 original research prevalence and risk factors associated with campylobacter spp. occurrence in healthy dogs visiting four rural community veterinary clinics in south africa musafiri karama, beniamino t. cenci-goga, alice prosperi, eric etter, saeed el-ashram, cheryl mccrindle, jackson n. ombui, alan kalake received: 23 july 2018; accepted: 17 jan. 2019; published: 28 may 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract reports on the occurrence of campylobacter spp. in dogs in south africa are non-existent. this study investigated the prevalence of campylobacter spp. in 481 dogs visiting four rural community veterinary clinics in south africa. dogs were screened for campylobacter spp. by culture and polymerase chain reaction (pcr), and logistic regression analysis was performed to assess the association between sex, clinic, breed and age and the occurrence of campylobacter spp. in dogs. the prevalence of campylobacter spp. was 41.50% (95% confidence interval [ci], 37.39% – 46.04%). campylobacter jejuni, c. upsaliensis and c. coli were detected in 29.31% (95% ci, 25.42% – 33.54%), 13.10% (95% ci, 10.37% – 16.42%) and 5.41% (95% ci, 3.71% – 7.82%) of dogs, respectively. dogs carrying more than one species of campylobacter spp. accounted for 6.23% (95% ci, 4.40% – 8.78%). campylobacter upsaliensis and c. jejuni were detected in 3.74% (95% ci, 2.37% – 5.86%), whereas c. coli and c. jejuni were found in 2.49% (95% ci, 1.42% – 4.34%) of dogs. age and clinic were the risk factors significantly associated with campylobacter spp. occurrence, while age, breed and clinic were predictors of c. jejuni carriage. furthermore, age was the only risk factor associated with a higher likelihood of carrying c. upsaliensis. the prevalence of campylobacter spp. c. jejuni and c. upsaliensis increased significantly as dogs grew older. in addition, the odds of carrying campylobacter spp. were higher in the staffordshire bull terrier breed compared to crossbreed dogs. in conclusion, this study shows that dogs visiting rural community veterinary clinics in south africa are reservoirs of campylobacter spp. and may be potential sources of campylobacter spp. for humans living in close proximity of the dog populations under study. keywords: dogs; campylobacter spp.; c. jejuni; c. coli; c. upsaliensis; risk factors; south africa. introduction campylobacter spp. are the leading cause of bacterial diarrhoeal diseases globally. around 400–500 million cases of campylobacter infections occur each year globally (friedman et al. 2000). although most human cases of campylobacteriosis are foodborne or waterborne (jacobs-reitsma, lyhs & wagenaar 2008), a number of studies have shown that contact with dogs is a risk factor for human campylobacteriosis (couturier, hale & couturier 2012; damborg et al. 2016; man 2011; mughini gras et al. 2012; neimann et al. 2003; rossi et al. 2008; tenkate & stafford 2001). manifestations of campylobacter infections in humans include mild watery to severe bloody diarrhoea, nausea and vomiting and in some cases life-threatening complications such as guillain-barré syndrome or its variant, miller-fisher syndrome (jacobs et al. 2008). in immunocompromised patients, campylobacter jejuni is an important cause of severe bacteraemia, which may lead to death (tee & mijch 1998). dogs are considered asymptomatic carriers of campylobacter spp. (acke et al. 2009; carbonero et al. 2012; hald & madsen 1997). a number of studies have reported campylobacter spp. carriage rates ranging from 2.7% to 100% in dogs (chaban, ngeleka & hill 2010; hald et al. 2004; tsai et al. 2007). furthermore, clinical cases in dogs with campylobacter spp. as a primary or secondary cause of diarrhoea have also been reported (burnens, angéloz-wick & nicolet 1992; mcorist & browning 1982). healthy and diarrhoeic dogs may harbour one or more campylobacter species including c. jejuni, c. coli and c. upsaliensis (carbonero et al. 2012; chaban et al. 2010; giacomelli et al. 2015; holmberg et al. 2015; parsons et al. 2011). both c. jejuni and c. upsaliensis have been implicated in dog-associated campylobacter spp. infections in humans (bourke, chan & sherman 1998; couturier et al. 2012; nachamkin, allos & ho 1998). zoonotic transmission of campylobacter spp. from dogs occurs through direct contact with infected dogs or dog faeces (damborg et al. 2016; mughini gras et al. 2012). there are suggestions that contact with dogs may be associated with up to 6% of human campylobacteriosis cases (rossi et al. 2008; tenkate & stafford 2001). although dogs are considered an important reservoir of campylobacter spp. (hald & madsen 1997; hald et al. 2004), current data on the prevalence of campylobacter spp. in dogs in south africa and on the african continent are lacking. therefore, the aim of this study was to investigate the prevalence and risk factors associated with campylobacter spp. occurrence in healthy dogs visiting four rural community veterinary clinics in south africa. materials and methods study design, area and population this cross-sectional study was conducted at four rural community veterinary clinics located in gauteng province, south africa (see figure 1). a total of 481 dogs were screened for campylobacter spp. including c. jejuni, c. coli and c. upsaliensis. each dog owner had an identity card on which the following variables were recorded: name of the dog and owner, vaccination status, sex, date of birth and breed. faecal swabs were obtained from all dogs that visited a particular clinic on the sampling day. this study was approved by the animal ethics committee of the university of pretoria (v056-15). figure 1: a map of gauteng province showing the four clinics and/or sites where the samples were collected. sample collection and campylobacter spp. culture methods sterile swabs were used to collect faecal samples from dogs during routine vaccination and deworming campaigns. each clinic was visited once, and one sample was obtained from each animal. for culture and isolation of campylobacter spp., swabs were spread-plated on campy cva agar (brucella agar containing 5% defibrinated sheep blood supplemented with 20 mg of cefoperazone, 10 mg of vancomycin and 2 mg of amphotericin b) (becton, dickinson and company, sparks, md, united states [us]). the inoculated plates were incubated at 37 °c for 48–72 hours in tightly sealed anaerobic system containers in which gaspaktm ez campy system sachets (becton, dickinson and company, sparks, md, us) were placed to create a microaerophilic atmosphere (approximately 6% – 16% oxygen and 2% – 10% carbon dioxide). we used 37 °c to incubate campylobacter spp. instead of the generally used 42 °c to mimic the ‘natural’ growth environmental temperature of campylobacter spp. in the gastrointestinal tract of dogs. furthermore, 37 °c was used to favour the growth of c. upsaliensis, which grows optimally at 37 °c (lastovica & le roux 2001). dna extraction briefly, a sterile inoculating loop was used to harvest colony sweeps from all campy cva plates that showed growth after 48 h – 72 h. a loop-full of colony sweeps was suspended in a 1.5 ml eppendorf tube containing 1 ml of fa buffer (becton, dickinson and company). bacterial suspensions were mixed and washed by vortexing, followed by centrifugation (15 000 g) for 5 minutes. after the first wash and centrifugation cycle, the supernatant was discarded and the bacterial pellet was resuspended in fa buffer (becton, dickinson and company). two additional washes and centrifugation cycles were carried out, after which the pellet was suspended in 500 µl of sterile water, vortexed and the homogeneous cell suspension was boiled to 100 °c for 15 min, then stored at -20 °c for further processing. campylobacter spp. screening a campylobacter spp. specific multiplex polymerase chain reaction (pcr) protocol (forbes & horne 2009) was used to screen dna for campylobacter spp. (c. jejuni, c. coli and c. upsaliensis). primers lpxac. jejuni-acaacttggtgacgatgttgta (klena et al. 2004), lpxac. coli gatagtagacaaataagagagaatmag (forbes & horne 2009) and lpxac. upsaliensis-aagtcgtatattttcytacgcttgtgtg (klena et al. 2004) were used as forward primers. primers lpxa-r1-caatcatgtgcgatatgacaataygccat, lpxa-r2-caatcatga-gcaatatgacaataagccat and lpxar kk2m caatcatgdgcdatatgasaatahgccat were used as reverse primers for c. jejuni, c. coli and c. upsaliensis (klena et al. 2004; forbes & horne 2009). each pcr reaction (25 µl) contained 2.5 µl of 10x thermopol reaction buffer, 2.0 µl of 2.5 mm deoxyribonucleotides triphosphate (dntps), 0.25 µl of 100 mm mgcl2, 1.25 µl of 0.5 µm of each forward primer and 1.25 µl of 0.25 µm of each reverse primer in a 50:50 mixture, 1u of taq dna polymerase (new england biolabs® inc., ipswich, ma, us) and 5 µl of dna template. sterile water was used to top up the reaction volume to 25 µl. campylobacter jejuni atcc 33560, c. coli derived from atcc 33559 (microbiologics, st cloud, mn, us) and an in-house dog c. upsaliensis isolate were used as positive controls, and sterile water was the negative control. all pcr reagents were supplied by new england biolabs, except for the primers, which were supplied by inqaba biotec (inqaba biotec, pretoria, south africa) or integrated dna technologies (idt, san diego, ca, us). polymerase chain reactions were performed in a c1000 touchtm (bio-rad, hercules, ca, us) or a veriti™ (applied biosystems®, foster city, ca, us) thermal cycler. amplified dna was electrophoresed in 2.5% (w/v) agarose gels in tris-acetate-ethylenediaminetetraacetic acid buffer. gels were stained with ethidium bromide (0.05 mg/µl), and amplicons were visualised under ultraviolet light in a gel doc system (bio-rad, hercules, ca, us). campylobacter speciation colony sweeps were obtained from all campy cva plates that were positive for campylobacter spp. on pcr screening, streaked on horse blood agar and incubated at 37 °c for 48 h – 72 h to obtain single colonies. three suspect campylobacter spp. single colonies were taken from each horse blood agar plate with a sterile plastic inoculating loop or swab, spread-plated separately on horse blood agar plates and incubated at 37 °c for 48 h – 72 h to multiply and purify the single colonies. after incubation, pure single colony bacterial sweeps were harvested using a sterile plastic inoculating loop or swab, and the bacterial cells were suspended in 1.5 ml fa buffer in an eppendorf tube. dna was extracted from the single colony sweeps by the boiling method and screened for c. jejuni, c. coli and c. upsaliensis using the aforementioned primers and multiplex pcr protocol (forbes & horne 2009; klena et al. 2004). single colony isolates that were confirmed as c. jejuni, c. coli or c. upsaliensis on pcr were stored at -80 °c in cryovials containing a sterile freezing mixture (70% brucella broth and 30% glycerol). statistical analysis all statistical analyses were performed using the ‘base’, ‘epidisplay’ and ‘aod’ packages of the r software version 3.3.3 (r foundation for statistical computing 2017, vienna, austria, http://www.r-project.org/). the prevalence of campylobacter spp., c. jejuni, c. coli and c. upsaliensis, were computed on 481 dogs using a general linear model considering the error distribution as binomial and the link function ‘logit’ (logistic regression). the following risk factors were tested: sex, breed, clinic, age and number of vaccinations. as a categorical variable, ‘breed’ included crossbreed, molosser, staffordshire bull terrier, toy and other breeds. to prepare for the logistic regression model, the potential relationship or association between the number of vaccinations and the age of the dog was tested. because of non-normality in the distribution of ages in relation to time and number of vaccinations and heterogeneity of variance, an analysis of variance (anova) could not be applied. instead, a general linear model using count data (family = poisson) was used to align the ages of dogs with the number of vaccinations. because dog ages were statistically linked with the number of vaccinations, the number of vaccinations as a risk factor was removed from the logistic regression model and only sex, breed, clinic and age were kept for risk factor analysis. all the records for which sex and breed were not determined or missing (n = 100) were removed from the database, and only 381 dog samples were used in the model. a general linear model (family = binomial) with a full model encompassing all risk factors and all possible interactions was initially performed. using a backward stepwise model selection based on the akaike information criterion (aic), the best model was kept (with the smallest aic). a likelihood ratio test was performed to test the overall significance of each risk factor (multilevel comparison). because age was analysed as a continuous variable, odds ratio (or) and prevalence predictions were not calculated for this risk factor. in terms of levels within each categorical risk factor, for comparison purposes, the risk factor associated with the lowest campylobacter spp. prevalence was used as a reference. adjusted or that took into account all cofounder variables were calculated, and confidence intervals (cis). odds ratio significance was computed using the wald’s test. to predict the prevalence of campylobacter spp. within different categories for each risk factor, the ‘predict’ function was applied on logistic regression results. finally, risk factors for c. jejuni, c. upsaliensis and c. coli were tested using the same approach that was applied for campylobacter spp. risk factor analysis. for all analyses, p-values < 0.05 were considered significant. results descriptive analysis of the 481 dogs that were tested for campylobacter spp. (see table 1), 41.58% (95% ci, 37.39% – 46.04%) were positive for campylobacter spp. the distribution of individual campylobacter species was as follows: c. jejuni (29.31%; 95% ci, 25.42% – 33.54%) was the most frequent species, followed by c. upsaliensis (13.10%; 95% ci, 10.37% – 16.42%) and c. coli (5.41%; 95% ci, 3.71% – 7.82%) (see table 1). similarly, 6.23% (95% ci, 4.40% – 8.78%) of dogs with mixed infections were also detected: c. jejuni + c upsaliensis, 3.74% (95% ci, 2.37% – 5.86%) and c. jejuni + c. coli, 2.49% (95% ci, 1.42% – 4.34%). table 1: prevalence and risk factors of campylobacter spp. according to clinic, breed, sex and age. risk factors a total of 381 dogs were included in the final model. logistic regression showed that clinic, age and breed were significant risk factors for carrying campylobacter spp. (see table 1). no interactions between any of these risk factors were statistically significant in the model. overall, visiting a particular clinic and the age of the dog were significant risk factors for carrying c. jejuni while age was the only risk factor associated with carrying c. upsaliensis. dogs visiting clinics b, c and d were respectively 9.61, 5.68 and 12.3 times more likely to carry campylobacter spp. in comparison to dogs visiting clinic a (reference clinic) (see table 1). in addition, breed was a predictor of campylobacter spp., with the odds of carrying campylobacter spp. significantly higher in the staffordshire bull terrier breed in comparison to crossbreed dogs, which had the lowest prevalence of campylobacter spp. furthermore, the prevalence of campylobacter spp., c. jejuni and c. upsaliensis increased significantly as dogs grew older. discussion to our knowledge, this is the first study reporting on the occurrence of campylobacter spp. in dogs visiting rural community veterinary clinics and the risk factors associated with campylobacter spp. carriage in dogs in south africa. our results showed that 41.5% of dogs carried campylobacter spp., in line with similar studies, which have reported campylobacter spp. prevalence in dogs ranging from 35% to 43% in europe and north america (acke et al. 2009; holmberg et al. 2015; parsons et al. 2010; procter et al. 2014; workman et al. 2005). however, higher prevalence rates of campylobacter spp. of up to 75.5% have been reported in dogs in different countries including sweden, canada, the uk and usa (chaban et al. 2010; engvall et al. 2003; leahy et al. 2017; parsons et al. 2011). variations in campylobacter spp. occurrence rates in dogs have been ascribed to a number of factors, including dog living conditions – whether a dog is confined in a house, a shelter or kennel, or is a stray. higher campylobacter spp. occurrence rates have been reported in dogs living in shelters or kennels and stray dogs (baker, barton & lanser 1999; parsons et al. 2011; procter et al. 2014; tsai et al. 2007; workman et al. 2005) in comparison to in-house dogs. additional factors such as campylobacter spp. culture conditions, including the incubation temperature and atmosphere, as well as antimicrobial supplements that are used for selection of campylobacter spp. in various recovery media or enrichment broths, may also influence campylobacter detection rates (allos & lastovica 2008; aspinall et al. 1996; lastovica & le roux 2001). campylobacter jejuni was the most frequent campylobacter species in dogs, followed by c. upsaliensis and c. coli to a lesser extent. this finding is in agreement with previous studies, which have reported c. jejuni as the most frequent species in dogs compared to other campylobacter species (carbonero et al. 2012; giacomelli et al. 2015; tsai et al. 2007). however, a number of reports have also found c. upsaliensis to be the most frequent species in dogs (acke et al. 2009; chaban et al. 2009; holmberg et al. 2015; parsons et al. 2011; rossi et al. 2008). a number of studies have found that c. upsaliensis was more frequent in dogs confined in household compounds while c. jejuni was more common in stray dogs and shelter or kennel dogs (carbonero et al. 2012; leonard et al. 2011; parsons et al. 2011; procter et al. 2014). the majority of dog owners in this study indicated that their dogs were not housed in fenced yards and dogs were allowed to leave their living premises and freely roam in the neighbourhood, thereby living a ‘semi-stray’ life. the stray nature of dogs sampled in this study may have played a role in the predominance of c. jejuni over c. upsaliensis. roaming, scavenging and hunting behaviours of dogs living a semi-stray life exposes dogs to environments, food and water sources that may favour higher environmental contamination levels with c. jejuni compared to c. upsaliensis, which has been found to be more frequent in dogs living in-house that are fed home-cooked food (leonard et al. 2011). while the role played by c. jejuni in human disease is well recognised globally, of particular interest in this study was the presence of dogs infected with c. upsaliensis. campylobacter upsaliensis has emerged in the last 20 years (bourke et al. 1998) as an important species in dogs worldwide (chaban et al. 2010; engvall et al. 2003; parsons et al. 2010) and a cause of campylobacteriosis in humans (allos & lastovica 2008; couturier et al. 2012; labarca et al. 2002; nakamura et al. 2015). this is the first time c. upsaliensis has been reported in dogs in south africa. this finding is of public health significance as c. upsaliensis has been previously reported as the third most frequent campylobacter species in south africa over a period of 10 years in paediatric patients, accounting for 23% of campylobacter spp. cases (lastovica & engel 2001). carriage of more than one campylobacter species was observed in 6.2% of dogs. dogs with mixed infections carried c. jejuni and c. upsaliensis or c. jejuni and c. coli. dogs carrying multiple campylobacter species may have been exposed to environments and sources that allow these campylobacter species to thrive favourably. similar findings have been reported elsewhere (bojanić et al. 2017; chaban et al. 2010; engvall et al. 2003; hald et al. 2004; koene et al. 2004). a number of studies have recommended the use of more than one campylobacter spp. culture medium to facilitate the isolation and increase the chance of recovering multiple campylobacter species of public health importance from faecal samples (endtz et al. 1991; baker et al. 1999; koene et al. 2004). in the aforementioned studies in which multiple campylobacter species were detected in individual dogs, at least two media were used to isolate campylobacter spp. while evaluation of the sensitivity of the medium used in this study to recover campylobacter spp. is beyond the scope of this investigation, detection of dogs carrying multiple campylobacter species indicates that campy cva agar was a reliable single medium for direct and simultaneous recovery of more than one campylobacter species from dog faeces. concerning the different risk factors that were investigated in this study, our findings showed that the overall prevalence of campylobacter spp. and particularly the prevalence of c. jejuni and c. upsaliensis increased as dogs grew older, with predominance of c. jejuni in dogs younger than 1 year in comparison to dogs older than 1 year. similar studies have reported that dogs less than 1 year old were more likely to be colonised by campylobacter spp. (acke et al. 2009; guest, stephen & price 2007; hald et al. 2004; leahy et al. 2017; parsons et al. 2010; procter et al. 2014; sandberg et al. 2002). the high prevalence of campylobacter spp. in younger dogs may be most probably ascribed to an immature immune system and an underdeveloped enteric microbiota that is unable to outcompete and displace campylobacter spp. in the intestine. this finding was not surprising as the dog population under study was skewed towards a higher number of dogs younger than 1 year (88%) compared to dogs that were older than 1 year. consistent with previous studies, the prevalence of campylobacter spp. was not significantly different in male and female dogs (nair et al. 1985; olson & sandstedt 1987; sandberg et al. 2002; torre & tello 1993). however, breed was overall a predictor for campylobacter carriage, with the staffordshire bull terrier breed more likely to carry campylobacter spp. in comparison to crossbreed dogs. although breed has never been reported as a risk factor for campylobacter spp. occurrence in dogs, this finding may indicate that dogs belonging to the staffy breed, which is a pure breed, may be more susceptible to disease in comparison to crossbreeds, which are generally considered more resistant to disease. visiting a particular clinic was identified as a risk factor for carrying c. jejuni, with dogs visiting clinics b, c and d presenting a higher risk of carrying campylobacter spp. and particularly c. jejuni. while the reasons behind this finding are not clear, the authors postulate that there are yet-unidentified factors such as dog living conditions (in-house vs. stray dogs) that may be favouring a higher occurrence rate of c. jejuni in dogs living in the communities serviced by clinics b, c and d in comparison to clinic a, which had the lowest campylobacter spp. prevalence. conclusion this study provides useful information on the prevalence and risk factors of c. jejuni, c. upsaliensis and c. coli in dogs visiting rural community veterinary clinics in south africa. our results indicate that dogs visiting the veterinary rural community clinics under study are reservoirs and may be an important source of campylobacter spp. for humans. however, a limitation of this study is that the dogs studied were not recruited randomly and the prevalence of campylobacter presented in this study may not be a reflection of the larger dog population of south africa. future epidemiological and characterisation studies comparing dog and human campylobacter spp. isolates are needed to establish the zoonotic potential of campylobacter spp. carried by dogs in south africa. acknowledgements funding from the national research foundation including the south africa–italy and south africa–kenya joint science and technology research cooperation programmes, the faculty of veterinary science, university of pretoria, and the gauteng department of agriculture and rural development is gratefully acknowledged. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ 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of clinical microbiology 43, 2642–2650. https://doi.org/10.1128/jcm.43.6.2642-2650.2005 abstract introduction materials and methods results discussion acknowledgements references about the author(s) eufrásia augusto paraclinic department, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique jescka aleixo paraclinic department, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique florentina d. chilala paraclinic department, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique abel g. chilundo clinic department, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique benígna gaspar paraclinic department, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique custódio g. bila paraclinic department, faculty of veterinary medicine, eduardo mondlane university, maputo, mozambique citation augusto, e., aleixo, j., chilala, f.d., chilundo, a.g., gaspar, b. & bila, c.g., 2022, ‘physical, chemical and microbiological assessments of drinking water of small-layer farms’, onderstepoort journal of veterinary research 89(1), a2067. https://doi.org/10.4102/ojvr.v89i1.2067 original research physical, chemical and microbiological assessments of drinking water of small-layer farms eufrásia augusto, jescka aleixo, florentina d. chilala, abel g. chilundo, benígna gaspar, custódio g. bila received: 21 july 2022; accepted: 06 oct. 2022; published: 28 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract water quality is critical for poultry farming. this study assessed the physical, chemical and microbiological quality of drinking water in small-layer farms in southern mozambique and identified potential risk factors for total coliform (tc) and escherichia coli contamination of drinking water. in 20 farms, 57 samples were collected and examined for ph, nitrate content (nc), nitrite level (nl) and total hardness contents (th). furthermore, tc and e. coli growth were assessed at 37 °c. one hundred per cent of the drinking water was of acceptable quality in terms of ph (6.5–8.5), nc (50 mg/l) and nl (3 mg/l). total hardness contents exceeded the recommended standard in 37.5% of borehole water samples and 91.7% of tap water samples, respectively. total coliform and e. coli were found in 40% and 15% of water samples. tap water samples had the greatest contamination, with tc and e. coli levels of 41.7% and 16.7%, respectively. although not statistically significant, sampling from the beginning of the nipple line (p = 0.101, or = 7.357, 95% confidence interval [ci]: 0.678–79.886) and not cleaning the rearing equipment regularly (p = 0.098, or = 3.966, 95% ci: 0.766–20.280) were factors affecting the tc growth. sampling from the tank water source (p = 0.001, or = 0.005, 95% ci: 0.000–0.121) and borehole water source (or = 13 585) and not cleaning the equipment consistently (p = 0.073, or = 9.682, 95% ci: 0.810–115.68) were all factors affecting e. coli growth. it is concluded that the th and microbiological quality of the drinking water of the study region are inadequate. regular water quality assessments should be incorporated into mozambican layer farm management to limit the potential for health concerns, and farmers should thoroughly clean and disinfect their rearing equipment. contribution: we should incorporate regular water quality assessments into mozambican layer farm management to limit the potential for health concerns, and farmers should thoroughly clean and disinfect their rearing equipment. keywords: water quality; layers; total coliforms; escherichia coli; risk factors; mozambique. introduction water is required for the preservation of important activities, which is why living organisms must consume it to exist (barros, amaral & rossi 2001). water is essential to preserve homeostasis in living organisms, since it takes part in various processes in the body, including food digestion, nutrient absorption and transport, body temperature regulation and metabolite excretion (lehninger, nelson & cox 1993). water is used for irrigation of fields, sanitary management, cleaning, disinfection of facilities and animal consumption in livestock production (barbosa 2013; simoni et al. 2015). water may be obtained from a variety of sources, including springs, shallow wells, deep wells, lakes and streams (amaral 2004). because of open human defecation, inappropriate disposal of animal droppings and dead carcasses, pits built near water sources and manure dumping during the rainy season, surface water sources such as rivers and lakes are more prone to microbial contamination (amaral 2004). water is essential in poultry farming for management, feeding, cleaning and environmental temperature regulation (gama et al. 2008). besides supplementation and disease treatment, water may be used as an electrolyte replacement therapy and a medication and vaccine delivery medium (folorunso, kayonde & onibon 2014; gama 2005). organic matter accumulation in water supply systems, such as reservoirs, drinkers and battery system pipes, may develop during layer bird rearing. algae development, mineral deposition and dirt can result from accumulated organic matter, creating a suitable habitat for the multiplication of microorganisms in the water (folorunso et al. 2014). the development of illnesses, reduced egg production and/or mortality of laying hens can all result from microbial contamination (amaral 2004; cardozo et al. 2015; di martino et al. 2018; el allaoui, rhazi filali & derouich 2016). the use of water of poor physical, chemical or bacteriological quality can adversely affect livestock health and performance (hapke 2000; tabler 2003; travel et al. 2007). when used for watering birds, the use of water of dubious quality negatively interferes with well-being and zootechnical indices and enhances the spread of diseases, causing serious economic losses (tabler 2003). although water quality is a major factor in controlling animal health problems (travel et al. 2006), the quality of drinking water used in the mozambican poultry industry has never been evaluated. it was hypothesised that poor drinking water quality was contributing to mozambique’s high morbidity and mortality rates. as a result, this study aims to evaluate the physical, chemical and microbiological water quality in small-layer farms in southern mozambique, as well as to identify potential risk factors for bacterial contamination of drinking water. materials and methods choice of farm sites the research was conducted in the municipalities of maputo and matola in mozambique’s southern region. the municipalities of maputo and matola, respectively, have seven and 41 districts (figure 1). figure 1: study area, showing (a) mozambique administrative division, (b) maputo province and (c) maputo and matola municipalities. sampling in october 2020, drinking water samples were collected once in each layer farm. layer farms were picked based on convenience and nonprobabilistic sampling, and only immediately available layer farms were chosen, with a preference for small egg producers with less than 1000 layers. using a battery rearing system, three samples were collected from each layer farm: (1) the tank water source, (2) the tank at the beginning of the nipple line and (3) the end of the nipple line. only samples from the water supply and drinkers were taken from farms using the floor-rearing system. water samples were taken in 500 ml sterile bottles using a specified process to avoid contamination (di martino et al. 2018; el allaoui et al. 2016). they were then transferred to the laboratory at 4 °c and processed within 24 h for microbiological analyses and 48 h for chemical and physical analyses. in 20-layer farms, 57 water samples were collected, with 17 using a battery cage rearing system and three using a floor rearing system, respectively. physical and chemical analyses a portable ph meter was used to determine the ph (hach, england). the edta (ethylenediaminetetraacetic acid) titration method was used to determine the level of hardness (apat 2003). ion chromatography was used to determine the levels of nitrates and nitrites (apat 2003). microbiological analysis total coliforms after suitable dilutions, 100 ml of water was filtered to be tested via a 0.45 µm pore size cellulose membrane filter to determine total coliforms (tc) (standing committee of analysts 2002). afterward, the membrane was put on lauryl sulphate agar (oxoid, uk) and incubated for 24 h at 37 °c ± 0.5 °c. if colonies are yellow after primary culture in membrane lauryl sulphate agar (mlsa), water is most likely contaminated with tcs. faecal coliforms and escherichia coli detection the presence of faecal coliforms was detected in the same way as the tcs, with the exception that the incubation was carried out at 44 °c ± 0.5 °c for 24 h. a representative number of the usual colonies were subcultured on nutrient agar medium (oxoid, united kingdom [uk]) at 37 °c ± 1 °c for 24 h based on verified yellow colonies of faecal coliforms. to distinguish faecal coliforms, biochemical approaches were used. the presence of red colouring on the surface of the nutrient agar medium was then used to regulate indole synthesis by adding 0.2 ml – 0.3 ml kovacs reagent (merck millipore, uk). all colonies exhibited a negative oxidase reaction, but a positive indole reaction turned out to be escherichia coli in the end. if tcs or e. coli were found in a sample, it was contaminated. questionnaire survey parallel to the collection of samples for physical, chemical and microbiological analyses, the layer farmers in this study completed a multiple-choice questionnaire validated by the authors of this paper that covered a variety of factors affecting water quality (including the number of birds in production, farmer experience with layer rearing, infrastructures, water sources, hygiene and equipment). the purpose of the questionnaire was to identify potential risk factors for bacterial contamination of drinking water in layers. statistical analysis for statistical analysis, all data were input into a microsoft excel® spreadsheet (microsoft corporation, redmond, washington, united states) (ms excel 2018) and exported to the statistical package for the social sciences (spss®) statistical software version 16 (ibm corporation, armonk, new york, united states) (ibm spss statistics 16). for quantitative variables (ph, hardness, nitrates and nitrites), descriptive statistics were used, including the computation of mean, standard deviation, frequencies and percentages (water source, point of collection, washing facilities, washing equipment, hardness and ph limit). a logistic regression model was used to investigate the risk factors associated with the quality of the water used to water laying hens, with the univariant analysis considering the response dichotomic and dependent variables (presence or absence of tcs and e. coli) and the study’s independent variables (water source, collection point, washing facilities, washing equipment, hardness and ph limit). a multivariate analysis was performed on all variables that were significant in the univariate analysis (p < 0.05) to see if the existing association was because of confounding factors. the odds ratio (or), which was obtained directly from the logistic regression estimates and provided in tables indicating the respective significance, was used to measure the degree of association between the independent and dependent variables. because there is no specific legislation for animal drinking, the physical, chemical and microbiological data from this study were compared with those from the european council directive 98/83/ec on the quality of water meant for human consumption (european commission 1998). ethical considerations ethical review and approval were granted by the scientific board of the faculty of veterinary medicine, eduardo mondlane university, ethical clearance number: 891favet. results physical and chemical analysis the physical, chemical and microbiological data are summarised in table 1 and table 2. the ph values are between 6.5 and 8.5. the concentrations of nitrate and nitrite were 50 mg/l and 3 mg/l, respectively. the values of the hardness parameters ranged from 50 mg/l to 740 mg/l. table 1: chemical and physical analysis of drinking water in 20 farms at three sampling sites: the water source (a), the beginning (b) and the end of the nipple line (c). the data are expressed as median values with standard deviation. table 2: drinking water quality in layer farms supplied with either borehole or tap water in matola and maputo municipalities. microbiological analysis total coliform and e. coli contamination were found in 40% and 15% of the drinking water samples. tap water samples had the greatest contamination, with e. coli and tc levels of 16.7% and 41.7%, respectively (table 2). potential risk factors for water contamination with total coliform and escherichia coli table 3 shows the variables that were deemed risk factors for tc and e. coli in the water given to birds. the multivariate logistic regression model revealed no significant risk factors for the increase of tc in laying hens’ drinking water. table 3: binary regression model of risk factors related to water quality for laying hens. although not statistically significant, the sampling point ‘beginning of the nipple line’ (p = 0.101, or = 7.357, 95% ci: 0.678–79.886), the ‘satisfactory’ ph threshold (p = 0.123, or = 5.605, 95% ci: 0.626–50.192) and the variable ‘no equipment cleaning’ (p = 0.098, or = 3.966, 95% ci: 0.766–20.280) were linked to a higher risk of tc growth in the drinking water of laying hens. when compared with the remaining locations, the probability of e. coli incidence or growth in drinking water for the layers relating to the collection point ‘tank water source’ (p = 0.001, or = 0.005, 95% ci: 0.000–0.121) was statistically significant (beginning and end of nipple lines). e. coli growth is higher in the variable ‘borehole water source’ (or = 13.585). consistent with this discovery, the variable ‘do not clean equipment’ also contributed to the increase in e. coli growth in laying hens’ water (p = 0.073, or = 9.682, 95% ci: 0.810–115.68). discussion according to the authors’ knowledge, this is the first research to examine the physical, chemical and microbiological features of water used by small-layer farmers in southern mozambique, as well as the risk factors related to tc and e. coli. in terms of the ph (6.5–8.5), nitrate content (nc) (50 mg/l) and nitrite content (3 mg/l), 100% of the drinking water given to the layers in the studied area was of satisfactory quality, while total hardness (th) exceeded the recommended standard in 37.5% and 91.7% of water samples collected from boreholes and tap water, respectively. using the ph parameter to evaluate the poultry water quality has sparked debate. according to grizzle, armbrust and saxton (1996) and cardozo et al. (2015), using water with a ph lower or higher than the recommended one (ph 4.8) did not affect poultry performance or water consumption. on the other hand, carter and sneed (1996) noticed a decline in broiler growth and feed conversion when they consumed water with a ph lower than 6. the impact of ph levels on water distribution systems in poultry farming management appears to be critical. low-ph water (between 2 and 4) can cause corrosion in water transport equipment and limit the efficiency of detergents, disinfectants and vaccines (gama 2005). as a result, knowing the ph of the water and, if required, making the appropriate changes is critical when providing these veterinary consumables (gama et al. 2008). high nitrate levels might indicate an overabundance of organic matter in the water supply because of the usage of animal manure or nitrogen-based fertilisers (fonseca 2017). according to gama et al. (2008), excessive amounts of nitrates in drinking water can induce poultry toxicosis by causing methaemoglobin to develop, which cannot transport oxygen to the cells. this toxicosis causes decreased growth and appetite in birds (gama et al. 2008). because nitrates are a by-product of ammonium oxidation or nitrate reduction, their presence in water indicates recent pollution (parron & muniz 2011). nitrite levels (nl) in poultry drinking water have been linked to decreased appetite, growth inhibition and lower laying rates in laying hens (gama 2005). only one layer farm had an unacceptable hardness parameter from tap water supplied to this farm. higher levels of hardness in drinking water can induce an unpleasant taste, reduced water consumption and decreased egg production in layer farming (cardozo et al. 2015). also, increased levels of hardness promote calcium and magnesium deposition in the farm’s water transport tubes, resulting in precipitates that create an excellent environment for biofilm that contaminates the water (cardozo 2012; folorunso et al. 2014). likewise, for the description of lower ph in drinking water, higher hardness negatively affects the efficiency of detergents, disinfectants, medicines and vaccines in poultry farm management (di martino et al. 2018). regardless of the water source, drinking water samples from 40% of the layer farms showed tc growth. when tap water is used, this number increases. the major issue related to tc contamination is a loss of birds’ immunity, which leaves them prone to opportunistic pathogenic infections that can harm poultry productivity and quality (gama 2005). escherichia coli is a bacterium linked to colibacillosis epidemics in birds, which can lead to disease outbreaks. it is significant because it forms 95% of the bacteria that make up the faecal coliforms, the most well-known and studied group of bacteria. (cardozo et al. 2015; gama 2005). poultry farms that used tap water from the public supply network had greater e. coli contamination (16.7%). this finding could be because the public water supply network’s piping system is very old and may have become contaminated with algae, dust and organic material, allowing bacteria to thrive (folorunso et al. 2014). drinking borehole water was discovered to be a risk factor for the growth of tcs and e. coli. borehole water, unlike tap water, is not chemically treated to prevent bacterial contamination. this lack of prevention may have favoured the higher contamination found in borehole drinking water. in this study, water collection from point a (tank water source) was identified as a risk factor for e. coli growth in the layers’ drinking water. in mozambique, small egg farm producers frequently ignore water reservoir hygiene because they are entirely unaware of the necessity for regular cleaning. this conclusion supports the findings of folorunso et al. (2014), who said that water reservoirs can be a source of contamination even if the water is of high quality, especially when replenished without proper cleaning. di martino et al. (2018), on the other hand, observed a contrary outcome, with increased proliferation of e. coli and other microbes in the water collected at point c (nipple nozzle). poor cleaning and disinfection, as well as exposure to birds, rodents and other animals, can all contribute to the contamination of water reservoirs (folorunso et al. 2014). this study concludes that the th content and microbiological quality of the drinking water of the study region are inadequate. therefore, to limit the risk of health problems, frequent water quality assessments should be incorporated into the administration of mozambican layer farms. farmers should also thoroughly clean and disinfect their farming equipment. acknowledgements we would like to acknowledge the farmers for their helpful cooperation. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.a., j.a., f.d.c., 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disclosure’, biological, são paulo 70(1), 43–49. grizzle, j., armbrust, t. & saxton, a., 1996, ‘water quality: the effect of water nitrate and ph on broiler growth performance’, the journal of applied poultry research 5(4), 330–336. https://doi.org/10.1093/japr/5.4.330 hapke, h.j., 2000, ‘effect of drinking water on animal health: toxicologic health risks’, deutsche tierärztliche wochenschrift 107(8), 335–336. lehninger, l., nelson, d.l. & cox, m.m., 1993, principles of biochemistry/water: its effects on dissolved biomolecules, 2nd edn., worth publishers, new york, ny. parron, l. & muniz, d., 2011, manual of sampling procedures and physical-chemical analysis of water, p. 69, embrapa forestry, colombo. simoni, d., volkweis, h., lazzaretti, j., regina, e., boita, d.f. & benetti, f., 2015, ‘microbiological quality of water used in food production by family agro-industries in the municipality of constantina’, electronic journal of environmental management, education and technology 19, 18–36. standing committee of analysts, 2002, ‘the microbiology of drinking water – part 3 practices and procedures for laboratories, viewed 03 august 2020, from https://www.gov.uk/government/publications/standing-committee-of-analysts-sca-blue-books. tabler, g.t., 2003, ‘water intake: a good measure of broiler performance’, avian advise 5(3), 7–9. travel, a., bouvarel, i., chevalier, d. & fulbert, l., 2007, ‘un bon démarrage (0–42 jours) permet de limiter l’apparition d’entérite chez les dindonneaux’, journées de la recherche avicole 7, 84–88. travel, a., bouvarel, i., chevalier, d., fulbert, l., & garnier, s., 2006, ‘conduite d’élevage et pathologies digestives des dindonneaux quels facteurs maîtriser pour limiter l’impact des entérites non spécifiques ? étude de cas. rapport ofival’, viewed 03 august 2020, from https://doczz.fr/doc/1063536/conduite-d-%c3%a9levage-et-pathologies-digestives-des. abolnik_347-351.indd introduction highly pathogenic avian influenza (hpai) is a devastating disease of poultry that has zoonotic potential for humans and is caused by strains of the h5 and h7 subtypes of influenza a viruses (family ortho myxoviridae). the low pathogenicity avian influenza (lpai) virus precursors to hpai viruses are ubiquitous in wild waterfowl and shorebirds and usually only cause asymptomatic infections in these reser voir hosts. once transferred to terrestrial poultry, lpai viruses can convert into hpai (webster, bean, gorman, chambers & kawaoka 1992). although the virulence determinants of avian influenza viruses are multigenic in nature, an insertion of multiple basic amino acids at the cleavage site of the haemagglutinin precursor protein, h0, targets this protein for cleavage by ubiquitous subtilisin-like endonucleases, resulting in rapid systemic spread of the virus. in contrast, lpai viruses contain a monobasic composition at h0 which is targeted by trypsin-like proteases that are limited to cells of the intestinal and respiratory tracts (rott 1979; stienekegröber, vey, angliker, shaw, thomas, roberts, klenk & garten 1992; vey, adler, klenk, rott & garten 1992; wood, mccauley, bashiruddin & alex ander 1993). the amino acid sequence at h0 is thus recognized as an important and reliable molecular virulence marker (oie terrestrial manual 2004). south africa was affected by two separate outbreaks of hpai h5n2 in intensively-farmed ostriches between 2004 and 2006, and intensive serological and virological surveillance was conducted during both outbreaks. of thousands of tracheal swabs or organ samples tested, 46 were positive or suspect positive for the presence of aiv by real-time reverse transcription-pcr (rrt-pcr) or nucleic acid-based sequence assay (nasba) (m. romito, unpublished laboratory data). h5-type viruses were isolated in only 3 (6.5 %) of these cases, viz. the hpai h5n2 virus isolated in the eastern cape outbreak in 2004 (osza04n227), the hpai h5n2 virus isolated in 347 onderstepoort journal of veterinary research, 75:347–351 (2008) research communication a rapid and sensitive real-time reverse transcription pcr for the pathotyping of south african h5n2 avian influenza viruses c. abolnik arc-onderstepoort veterinary institute, private bag x5, onderstepoort, 0110 south africa abstract abolnik, c. 2008. a rapid and sensitive real-time reverse transcription pcr for the pathotyping of south african h5n2 avian influenza viruses. onderstepoort journal of veterinary research, 75:347– 351 a fluorescence resonance energy transfer (fret) real-time reverse-transcription (rrt-pcr) assay was developed that distinguishes stains of south african and european highly pathogenic (hpai) from low pathogenicity (lpai) h5 avian influenza viruses in the absence of virus isolation, irrespective of the length of insertion at the hemagglutinin cleavage site (h0). the assay was used to pathotype h5-type viruses detected by rrt-pcr in ostrich tracheal swabs collected during the 2006 hpai h5n2 outbreak in the western cape province. keywords: avian influenza virus, h5n2, pathotyping, real-time reverse transcription pcr, fret accepted for publication 10 june 2008—editor 348 transcription pcr for the pathotyping of south african h5n2 avian infl uenza viruses the western cape outbreak of 2006 (osza06ai1091) and the lpai h5n2 virus isolated in the same outbreak (osza06ai1160). the isolate osza06ai1091 contained fewer multiple basic amino acids at h0 than did isolate osza04n227, but was clearly hpai according to the oie definition (abolnik 2007b). unfortunately however, the nucleic acids detected by rrt-pcr/ nasba are present in insufficient quantities to allow further molecular characterization (hoffmann, starick, depner, werner & beer 2007; unpublished laboratory data 2007), yet for control purposes there is an urgent need to be able to differentiate between lpai and hpai viruses in ostriches especially where virus isolation has not been successful. at least two other groups have described sensitive rrt-pcr assays for hpai h5 pathotyping, hoewever the sybr green method described by payungporn, chutinimitkul, chaisingh, damrongwantanapokin, nuansrichay, pinyochon, amon sin, donis, theamboonlers & poovorawan (2006) may give false positive results (fernández, gutierrez, sorlozano, romero, soto & ruiz-cabello 2006). in the method of hoffmann and co-workers (2007), a set of hydrolysis probes was described that detects the asian group of hpai h5n1, and the h0 cleavage site sequence of the qinghai strain in particular. however, insertions/substitutions in the h0 cleavage site abolished signal generation. as demonstrated for isolates from in south africa (fig. 1), potential variations in the h0 cleavage site sequence need to be taken into account. therefore a rapid and sensitive rrt-pcr assay was developed to distinguish between south african hpai and lpai h5n2 viruses in the absence of virus isolation that does not rely on the sequence at h0. materials and methods primer and probe design a set of primers (infa_h5_f and infa_h5_r) and three probes (infa_h5_640, infa_h5_fl and infa_ h5_705) were designed for the amplification and detection of a fragment spanning the cleavage site sequence of the h5n2 ha gene (table 1). the lcred640 fluorophore-labelled infa_h5_640 probe was designed to hybridize over the consensus lpai sequence that is conserved within the h5 lineage. the infa_h5_fl binds a sequence adjacent to this, and is labelled at both the 5’ and 3’ ends with fluorescein. the lcred705 fluorophore-labelled infa_h5_ 705 probe binds at the opposite end of infa_h5_fl, in a conserved region within the h5 sub -lineage. in the presence of an lpai sequence, the three probes will bind in tandem and both accep tor probes will be excited by the flourescein donor probe, resulting in table 1 primers and probes used to differentiate between hpai and lpai in this studya primer/probe sequence (5’-3’), -fluorophore nucleotide position (fig. 1) infa_h5_f infa_h5_r infa_h5_640 infa_h5_fl infa_h5_705 gtgccccaaatacgtgaartca ccatctattgcttkktgagtggactc 640-tcctcttgtttctytttgagggacatt-p f-cctccttctataaarcctgctatrgccccaaata-f -pccataccaaccrtctaccatkccttgcc-705 951–972 1169–1143 1029–1067 1069–1102 1105–1133 a primers and probes were designed in collaboration with and manufactured by tib molbiol, eresburgstr. 22-23, d-12103 berlin, germany table 2 results of the h5 rrt-pcr pathotyping assay sample f2(640) cp f3(705) cp pathotype dh20 osza06ai1091 osza06ai1160 ai1133.1 ai1133.11 ai1133.12 ai1133.13 ai1133.15 ai1133.16 ai1133.17 ai1133.23 ai1120.37 ai1149.6 negative control hpai positive control lpai positive control ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab ostrich tracheal swab – – 20.46 – – 26.34 – – – – – – – – 22.34 20.59 – 29.90 26.21 28.83 31.12 – – – 29.80 – – hpai lpai – hpai lpai hpai hpai – – – hpai – 349 c. abolnik f ig . 1 m u lti p le n u cl e o tid e s e q u e n ce a lig n m e n t o f h 5 s tr a in s fo r th e r e g io n s p a n n in g t h e h 0 c le a va g e s ite ( n u m b e ri n g u p st re a m o f th e h 0 c le a va g e s ite is b a se d o n g e n b a n k se q u e n ce d q 8 5 1 5 6 1 [ n o t sh o w n ]) 1 0 2 0 1 0 3 0 1 0 4 0 1 0 5 0 1 0 6 0 1 0 7 0 1 0 8 0 1 0 9 0 1 1 0 0 1 1 1 0 1 1 2 0 1 1 3 0 1 1 4 0 1 1 5 0 . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | o s z a 0 6 a i 1 1 6 0 ( h 5 n 2 ) g c g a c t g g a c t c a g g a a t g t c c c t c a a a g a g a a a c a a g a g g a t t a t t t g g g g c t a t a g c a g g t t t t a t a g a a g g a g g a t g g c a a g g c a t g g t a g a t g g t t g g t a t g g a t a c c a c c a t a g c a a t g a g c a g g o s z a 0 6 a i 1 0 9 1 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a g a a . . . a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . o s z a 0 4 n 2 2 7 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g a a a a a a g a a g a a . . . a . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . a . . . . . . . . . . . . . . . . . . . . . c . . . . . . . a / m a l l a r d / b a v a r i a / 1 / 2 0 0 5 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a / t e a l / i t a l y / 3 8 1 2 / 2 0 0 5 ( h 5 n 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a / m a l l a r d / i t a l y / 3 4 0 1 / 0 5 ( h 5 n 1 ) . . . . . . . . . . . . . . . . . . . . t . . . . . . . . . . . . . . . . . . . . g c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a / m a l l a r d / s w e d e n / 2 / 0 2 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . t . . . . . . . . . . . c . . . . . . . a / m a l l a r d / s w e d e n / 3 9 / 0 2 ( h 5 n 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . c . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . a . . . . a / d u c k / d e n m a r k / 6 5 0 4 7 / 0 4 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . a . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . a / c h i c k e n / i t a l y / 3 1 2 / 1 9 9 7 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a g a a g a a . . . a . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . g . . . . . . . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . a . . . . a / c h i c k e n / i t a l y / 8 / 9 8 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a g a a g a a . . . a . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . g . . . . . . . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . a . . . . a / t u r k e y / e n g / 5 0 9 2 / 9 1 ( h 5 n 1 ) . . . . . . . . . . c . . . a . . c . . . . . . . . . . . . a a a a g a a . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . c . . . . . g . . . . . . . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . . . . . . a / m a l l a r d / s w e d e n / 3 1 / 0 2 ( h 5 n 2 ) . . . . . g . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . a . . . . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . a d u c k / m o n g o l i a / 5 4 / 0 1 ( h 5 n 1 ) . . a . . a . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . c . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . a . a / d u c k / p r i m o r i e / 2 6 3 3 / 0 1 ( h 5 n 3 ) . . a . . a . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . a . a / m a l l a r d / s w e d e n / 8 0 / 0 2 ( h 5 n 9 ) . . a . . g . . g . . . . . . . . . a . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . a . . . . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . a / c h i c k e n / i t a l y / 9 0 9 7 / 9 7 ( h 5 n 9 ) . . . . . . . . g . c . . . . . . c . . . . . . . . . . a g . . g . . . . . . . . . c . . . . . . . . . . c . . . . . . . . c . . c . . . . . g . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . . . . . . a / d u c k / h o k k a i d o / v a c 1 / 0 4 ( h 5 n 1 ) . . a . . a . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . c . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c a . . . . a . a / d u c k / m o n g o l i a / 5 0 0 / 0 1 ( h 5 n 3 ) . . a . . a . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . c . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . a . a / d u c k / m o n g o l i a / 5 4 / 0 1 ( h 5 n 2 ) . . a . . a . . g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . c . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . a . a / d u c k / m o n g o l i a / 5 9 6 / 0 1 ( h 5 n 3 ) . . a . . a . . g . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . t . . . c . . . . . . . . c . . c . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . a . a / d u c k / h o k k a i d o / 4 4 7 / 0 0 ( h 5 n 3 ) . . a . . . . . g . . . . . . . . . . . t . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . . . . a . a / c h i c k e n / f r / 0 3 4 2 6 a / 0 3 ( h 5 n 2 ) . . . . . . . . g . c . . . . . . . . . t . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . g . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . a . . . . a / m a l l a r d / s w e d e n / 2 1 / 0 2 ( h 5 n 2 ) . . . . . . . . g . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . g . . . . . . . . a . . . a . . . . c . . . . . . . . . . . g . . . . . t . . . . . . . . c . . . . . . . a / d u c k / p o t s d a m / 1 4 0 2 6 / 8 6 ( h 5 n 2 ) . . . . . . . . . . . . . . . . . c . . t . . . . . . . . . . . . . . . . . . . . t c . . . . . . . a . . . . . . . . . . . c . . . . . . . . g . . . . . . . . . . . . . . a . . . . . . . . c . . c . . . . . . . . g . . . . . . . . . . . . . . c . . . . . . . a / d u c k / h o n g k o n g / 3 4 2 / 7 8 ( h 5 n 2 ) . . a . . c . . . . . . . . . . . c . . t . . . . . . . . . . . g . . . . . . . . t c . . . . . . . a . . . . . . . . . . . c . . . . . . . . g . . . . . . . . . . . g . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . . . . a . a / s w a n / h o k k a i d o / 6 7 / 9 6 ( h 5 n 3 ) . . . . . . . . . . . . . . a . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . t . . c . . . . . . . . g . . . . . . . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . . . . . . a / p e k i n d u c k / s g p o r e / f 5 9 / 0 4 / 9 8 . . a . . . . . . . . . . . a . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . a . . . . . . . . c . . c . . . . . . . . g . . . . . . . . c . . . . . c . . . . . . . a / d u c k / m l a y s i a / f 1 1 9 3 / 9 7 ( h 5 n 3 ) . . a . . . . . . . . . . . a . . . . . . . . . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . c . . . . . . . . . . . . . . . . . . . . . . . a . . . . . . . . c . . . . . . . . . . . g . . . . . . . . . . . . . . c . . . . . . . 350 transcription pcr for the pathotyping of south african h5n2 avian infl uenza viruses two signals that are detectable at different wavelengths (lightcycler® channels f2 and f3). the presence of insertions or substitutions in the h0 cleavage site of hpai virus will however abolish the binding of infa_h5_640 and only one signal in channel f3 would thus be visible. rna extraction and first-strand cdna synthesis viral rna was extracted from tracheal swabs or infective allantoic fluid with a total nucleic acid isolation kit (roche) using a magnalyser and trizol® ls reagent (gibco, invitrogen), respectively. firststrand cdna synthesis was accomplished by incubating 5 μℓ rna, 2 μℓ random hexamer and 6 μℓ dh20 at 65 °c for 10 min. after snap-cooling in an ice bath for 5 min, 4 μℓ of 5 x rt buffer, 0.5 μℓ of rnase inhibitor, 2 μℓ 10 mm dntps and 0.5 μℓ transcriptor reverse transcriptase® (roche) were added. the reactions were incubated at 55 °c for 30 min. rrt-pcr a lightcycler® faststart dna masterplus hybprobe kit (roche) and lightcycler® 2.0 device were utilized for real-time pcr using 5 μℓ of the cdna in a total volume of 20 μℓ. the temperature profile was used as follows: 10 min at 94 °c, and 45 cycles of 20 s at 95 °c, 15 s at 55 °c, and 20 s at 72 °c. fluoro phore-specific emission data were collected during the annealing step. crossing point (cp) values were calculated with lightcycler 3.5 software using the second derivative maximum method. the assay was applied to ten samples from the recent outbreaks in 2006 that were positive or suspect positive for the presence of h5 virus by rrt-pcr during surveillance but where virus isolation was unsuccessful (table 2). results and discussion the positive controls, osza061160 (lpai) and osza06ai1091 (hpai), produced fluorescent signals in both and in one channel, respectively (ta ble 2). unknown samples ai1133.11, ai1133.13, ai1133.15 and ai1120.37 were hpai positive, where as ai1133.12 was lpai positive. five of the samples gave negative results, which may have been due to rna degradation during prolonged storage. interestingly, lpai and hpai were detected within the same flock (ai1133 samples). osza04n227 (hpai, 2004) did not amplify, due to an unusual sequence deletion detected between nucleotides 1121 and 1134 (fig. 1) (abolnik 2007a). (the assay has been adapted for high throughput on the lightcycler480® system by replacing the lcred710 fluorophore with an lcred610 fluorophore, and use of a one-step rt-pcr kit). in south africa the reemergence of h5n2 in poultry is a constant threat because the lpai strain exists in the wild bird reservoir (abolnik 2007b) but this particular lpai h5n2 lineage is also circulating in the european wild waterfowl population (abolnik 2007a) and therefore after further validation the primer and probe set described here could be used in europe since poultry in that region is also at risk of infection and consequent mutation. i have described here a rapid and sensitive method to pathotype h5-positive swabs where virus isolation has been unsuccessful. this approach of detecting the consensus lpai h0 sequence with abolishment of binding as an indicator of the presence of an hpai strain (provided that adjacent or upstream controls are included) could be adapted for the pathotyping of other lineages of aiv h5 and extended to hydrolysis probe chemistries. acknowledgements i thank hans-henno dorries for assisting with primer/ probe design, rachel maluleke and bontsi marumomochothloane for technical assistance, and truuske gerdes and marco romito for useful discussions. references abolnik, c. 2007a. molecular epidemiology of newcastle disease and avian influenza in south africa. ph.d. thesis. university of pretoria, south africa. http://upetd.up.ac.za/upetd.htm. abolnik, c. 2007b. molecular characterization of h5n2 avian influenza viruses isolated from south african ostriches in 2006. avian diseases, 51:873–879. fernandez, gutierrez, f.j., sorlozano, a., romero, j.m., soto, m.j. & ruiz-cabello, f. 2006. comparison of the sybr green and the hybridization probe format for real-time pcr detection of hhv-6. microbiology research, 161:158–163. hoffmann, b., harder, t., starick, e., depner, k., wer ner, o. & beer, m. 2007. rapid and highly sensitive pathotyping of avian influenza a h5n1 virus using real-time rt-pcr. journal of clinical microbiology, 45:600–603. oie terrestrial manual 2004. highly pathogenic avian influenza. paris: office international des epizooties. payungporn, s., chutinimitkul, s., chaisingh, a., damrongwantanapokin, s., nuansrichay, b., piny ochon, w., amonsin, a., donis, r.o., theamboonlers, a. & poovorawan, y. 2006. discrimination between highly pathogenic and low pathogenic h5 avian influenza a viruses. emerging infectious diseases, 12:700–701. 351 c. abolnik rott, r. 1979. molecular basis of infectivity and pathogenicity of myxoviruses. archives of virology, 59:285–298. stieneke-gröber, a., vey, m., angliker, h., shaw, e., thomas, g., roberts, c., klenk, h-d. & garten, w. 1992. influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin endoprotease. embo journal, 11:2407–2414. vey, m., orlich, m., adler, s., klenk, h-d., rott, r. & gar ten, w. 1992. haemagglutinin activation of pathogenic avian influenza viruses of serotype h7 requires the recognition motif r-x-r/k-r. virology, 188:408–413. webster, r.g., bean, w.j., gorman, o.t., chambers, t.m. & kawaoka, y. 1992. evolution and ecology of influenza a viruses. microbiology reviews, 56:152–179. wood, g.w., mccauley, j.w., bashiruddin, j.b. & alexander, d.j. 1993. deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza a viruses of h5 and h7 subtypes. archives of virology, 130:209–217. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice olwoch_45-72.indd introduction the realization that species distributions are significantly influenced by climate has placed considerable emphasis on the need to acquire information concerning the ways in which the present distributions of organisms will be affected by climate change. in response to this challenge, the issue of vector dis tribution has recently received much attention. nu merous studies (sutherst & maywald 1985; nix 1986; perry, lessard, norval, kundert & kruska 1990; norval, perry & young 1992; rogers & randolph 1993; sutherst, maywald & skarratt 1995; randolph & rogers 1997; estrada-peña 1999; rogers & randolph 2000; randolph 2001, 2002; erasmus, kshatriya, mansell, chown & van jaarsveld 2000; erasmus, van jaarsveld, chown, kshatriya & wessels 2002; olwoch, rautenbach, erasmus, engelbrecht & van jaarsveld 2003; van staden, erasmus, roux, wingfield & van jaarsveld 2004; thomas, cameron, green, bakkenes, beaumont, collingham, erasmus, ferrierra, grainger, hannah, hughes, huntley, van jaarsveld, midgley, miles, ortega-huerta, peterson, 45 onderstepoort journal of veterinary research, 74:45–72 (2007) climate change and the genus rhipicephalus (acari: ixodidae) in africa j.m. olwoch1*, a.s. van jaarsveld2, c.h. scholtz3 and i.g. horak4 abstract olwoch, j.m., van jaarsveld, a.s., scholtz, c.h. & horak, i.g. 2007. climate change and the genus rhipicephalus (acari: ixodidae) in africa. onderstepoort journal of veterinary research, 74:45–72 the suitability of present and future climates for 30 rhipicephalus species in africa are predicted using a simple climate envelope model as well as a division of atmospheric research limited-area model (darlam). darlam’s predictions are compared with the mean outcome from two global circulation models. east africa and south africa are considered the most vulnerable regions on the continent to climate-induced changes in tick distributions and tick-borne diseases. more than 50 % of the species examined show potential range expansion and more than 70 % of this range expansion is found in economically important tick species. more than 20 % of the species experienced range shifts of between 50 and 100 %. there is also an increase in tick species richness in the south-western regions of the sub-continent. actual range alterations due to climate change may be even greater since factors like land degradation and human population increase have not been included in this modelling process. however, these predictions are also subject to the effect that climate change may have on the hosts of the ticks, particularly those that favour a restricted range of hosts. where possible, the anticipated biological implications of the predicted changes are explored. keywords: climate change, rhipicephalus species, sub-saharan africa, tick-borne disease * author to whom correspondence is to be directed. e-mail: jane.olwoch@up.ac.za 1 department of geography, geo-informatics and meteorology, university of pretoria, pretoria, 0002 south africa. 2 cib, department of botany and zoology, stellenbosch university, stellenbosch, 7160 south africa 3 scarab research group, department of zoology and entomology, university of pretoria, pretoria, 0002 south africa 4 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa and department of zoology and entomology, university of the free state, bloemfontein, 9300 south africa accepted for publication 28 september 2006—editor 46 climate change and rhipicephalus (acari: ixodidae) in africa philips & williams 2004) have attempted to predict the distribution of species based on the major environmental factors that would influence this. this approach neither disregards the need for further detailed and comprehensive eco-physiological studies nor does it pretend to predict the future. what it does is define the role of climate as a factor in determining the potential for future vector establishment when all other elements are excluded (sutherst 2003). this paper also accents the necessity of acquiring more detailed information concerning the biology and environmental sensitivities of each species in the light of likely climate change. in the absence of such information, relatively straightforward statistical methods that seek correlations between environmental factors and the presence of animals or plants are likely to remain the best pragmatic approach for exploring the expected future distributions of large numbers of species. this study focuses on climate induced changes likely to occur in the dis tribution of some species of an economically important african arthropod, namely the tick genus rhipi cephalus. the intimate relationship between climate and the requirements of ticks for survival is well documented (tukahirwa 1976; rechav 1981, 1982; short & norval 1981; minshull & norval 1982; norval, walker & colborne 1982; dipeolu 1989; perry et al. 1990; norval et al. 1992; okello-onen, tukahirwa, perry, rowlands, nagda, musisi, heinonen, mwayi & opudaasibo 1999). this has led to several studies using climate as a means of predicting the distributions of african ticks (rogers & randolph 1993; randolph 1993, 2001; randolph & rogers 1997; norval, sutherst, kurki, kerr & gibson 1997; cumming 2000a, b; estrada-peña 2001; olwoch et al. 2003). collectively the results obtained from these works, some of which used data garnered mainly from interpolated climate databases at 25 km resolution (perry et al. 1990) or 6 x 6 km resolutions (cumming 2000b), suggest that accurate predictions of tick distributions at different temporal and spatial scales should be feasible. this approach would be especially useful for predicting the distribution of species in poorly sampled species in poorly sampled regions of africa. the genus rhipicephalus is the fourth largest in the family ixodidae (walker, keirans & horak 2000), and there are 74 species currently recognized. it is essentially an african genus with approximately 63 species recorded only in the afro-tropical region and ten species outside the region. one species, rhipicephalus evertsi evertsi dönitz, 1910, whose distribution was originally confined to the afro-tropical region has now gained a foothold on the arabian peninsula and its distribution is expected to spread even further (walker et al. 2000). only 30 species are included in this paper. they are those whose ecological, life history strategies and climatic requirements are relatively well known and, as two distribution data sets have been used, the current distribution of these ticks are relatively well plotted. the way in which these distributions will be influenced by climate change is poorly documented and forms the essence of this paper. the use of climate-matching models to predict tick distributions one of the earliest climate matching approaches was climex, which calculates the climatic suitability of geographic regions for species using a temperature-dependent growth index moderated by four growth indices: hot, cold, dry and wet (sutherst & maywald 1985). the use of climex in northern australia was considered a great success for predicting the distribution of the tick boophilus microplus (canestrini, 1887), and it was anticipated that this initial success could be translated into predicting distributions of african tick species. however, early studies tended to over-estimate the distribution of b. microplus across africa, and the predicted high incidence of rhipicephalus appendiculatus neumann, 1901 in west africa was at complete variance with the tick’s absence in this region. moreover, when the distributions of amblyomma spp. in africa were modelled using climex, these were found to be dissimilar to their known distributions. these conflicting results led norval, perry, melt zer, kruska & boothroyd (1994) to conclude that the predicted climatic suitability of regions for amblyomma hebraeum koch and amblyomma variegatum fabricius, 1794, is almost the opposite of the actual distribution of these ticks, both in zim ba bwe and in the rest of africa (norval, perry, gebreab & lessard 1991; norval et al. 1992). bioclim was the second climate-based approach employed to model tick distributions (nix 1986; norval et al. 1992). bioclim generates 24 climatic attributes from which annual and seasonal mean conditions, extreme values and intra-year seasonality are derived, for each of a selection of geographic points throughout the distribution range of a tick species. computer-selected thresholds and limits for each of the indices are matched across a geographical grid to predict potential species distribution. this model generally provided a better fit between the predicted and known distributions of r. 47 j.m. olwoch et al. appendiculatus, although at a finer scale the match in some areas of the east african highlands was unsatisfactory (norval et al. 1992). the climate database used was interpolated at an increased resolution (8 km), and this factor alone may explain the improved accuracy when compared to the earlier climex-based attempts. a subsequent logistic regression approach (cumming 2000c), based on inter polated climate and elevation data for africa with a resolution of 25 km (hutchinson, nix, macmahon & ord 1996) achieved even better accuracy. such an approach, however, normally requires the existence of a training data set that includes presence and absence information (estrada-peña 2003). while it is relatively easy to ascertain where ticks have been collected, it is more difficult to confirm the reliability of surveys in which a tick species is cited as not present. consequently the assumption that non-presence always implies absence may limit the application of this modelling approach (estradapeña 2003). the use of an advanced very high-resolution radio meter (avhrr) mounted on the national oceanic and atmospheric administration’s (noaa’s) meteorological satellites was given preference in the 1990s. this instrument allowed the direct detection of environmental factors at an 8 km resolution (lessard et al. 1990). the main predictor in this procedure is the satellite-derived maximum mean monthly normalized difference vegetation index (ndvi). however, this technique was reportedly very complicated when used to predict the distribution of r. appendiculatus (kruska & perry 1991). there are, however, initiatives to revive confidence in the ndvi approach as a predictive tool in research (randolph 2002). the present study used a single species distribution modelling procedure (erasmus et al. 2000), originally developed by jeffree & jeffree (1994, 1996), for predicting species distribution patterns and for evaluating the relative performance of predicted future climate data sets. this model was subsequently modified to accept multivariate inputs to yield probability of presence maps for species (erasmus et al. 2000). when used to predict the contemporary potential distribution of african ticks (olwoch et al. 2003) the model achieved positive predictions of more than 70 % for the four tick species tested. climate data used for predicting african tick distributions the principal sources of climate data for predictive distribution modelling are climate surfaces, generated by interpolating data sampled at varying intensities across a region. consequently, differences between these climate surfaces can usually be attributed to spatial and temporal evenness of the data used for interpolation. most modern interpolation techniques are pattern based and statistically incorporate horizontal as well as vertical (altitudinal) adjustments (hutchinson 1989, 1991; hutchinson & gessler 1994). these climate surfaces are, how ever, relatively smooth because of extensive interpolation between low-resolution point observations. another source of climate data is global circulation models (gcms). these are coupled ocean-atmosphere models that provide three-dimensional simulations of the atmosphere. to date gcms have produced climate data at a horizontal resolution that is generally too coarse for use in predictive species modelling (> 100 x 100 km grid point resolution), especially for species that are habitat specialists or that are influenced by fine-scale environmental gradients. computational requirements usually preclude gcms being run at meso-scale grid resolutions (10–100 km). the present study used a division of atmospheric research limited area model (darlam) as the main climate data set. darlam is a potential alternative source of high-resolution climate data that involves the nesting of a high-resolution limited area model within a gcm over the area of interest (for review see mcgregor 1997). the gcm supplies the limited area model with initial and boundary conditions. with a grid resolution of 10–100 km, the limited area model is able to simulate some of the meso-scale properties of the circulation model. this technique provides a viable fine scale alternative to the use of observed or interpolated climate surfaces or very coarse scale gcms climate surfaces. the resolution attained by this dynamic modelling process is essentially limited by the computing power available to the modellers. the implication of using these datasets for predicting current tick distributions has been assessed by olwoch et al. (2003). in this study, darlam’s future predictions are compared with those obtained by using mean climates from two gcms (cgcm and centre for climate system research/national institute for environ mental studies [ccsr/nies]). materials and methods study area the study area covers sub-saharan africa (fig. 1) and was divided into 3 000 grids cells of 60 x 60 km 48 climate change and rhipicephalus (acari: ixodidae) in africa resolution. this resolution was determined by the darlam climate data. tick data point localities of tick recoveries were obtained from cumming (1999), who compiled the data that he used from various collections of ticks, and from recent collections made by one of us (i.g.h.). combining data sets from different sources frequently compounds identification and distribution errors and for this reason data congruence with walker et al. (2000), who provide well-illustrated distributions of rhipicephalus species, was used to assess data quality in the final compiled dataset. synonyms provided by the latter authors also solve the common dataset problem of referring to one species, but using different names, or referring to a group of species as a single species. the tick species selected for this study belong to the ixodid genus rhipi cephalus. species point localities were assigned to particular 60 km x 60 km grid cells by means of a spatial intersect in arcview gis. a conservative estimate of the accuracy of these point localities is 0.2 ° (g.s. cumming, personal communication 2003) and consequently this approximation is considered reasonable. the rhipicephalus species selected are those whose distribution and life history strategies are rela tively well known and it is our hope that these results will provide a baseline model for future modelling of other tick species. predictive species modelling a simple climate envelope model was used to predict the future distribution of the focal species (erasmus et al. 2000, 2002). the input data comprised 3 000 grid cells of 60 x 60 km size populated with climate variables covering sub-saharan africa. reliable presence records of the selected tick species and the present climate values at these locations were used to construct a climate envelope, using a principal components-type approach. this climate envelope represents the range of climates within which a particular tick is known to occur, and can be interpreted as the realized niche, as defined solely by climate. to arrive at a predicted distribution in a climate change scenario, the existing climate envelope is applied to a climate surface representing future climates, and a new geographical interpretation of distribution is derived [see erasmus et al. (2000, 2002) and olwoch et al. (2003) for a more detailed explanation]. this approach was used as a standardized base for evaluating the relative performances of the darlam and the mean gcm climate data sets, and allows the creation of a probability surface of climate suitability for each species modelled. fig. 1 study area national boundaries km 49 j.m. olwoch et al. darlam present and future climate data the australian commonwealth scientific and industrial research organisation (csiro) developed the high-resolution limited-area model darlam for use in both short-term meso-scale atmospheric studies and longer-term climate simulation experiments (walsh & mcgregor 1995). in the present study ten separate 30-day simulations were performed for both january and july for separate 10-year periods. the periods selected are the 1990s and 2020. the simulations were performed at a horizontal grid resolution of 60 x 60 km using a domain of 100 x 100 grid points that cover sub-saharan africa. the month ly average of the ten simulations constitutes the model climatology for the month. the csiro mark 2 gcm was used to force darlam at its lateral boundaries. the gcm was integrated for the period 1960– 2100, with greenhouse gas forcing corresponding to the a2 sres (special report on emission scenario, issued by the intergovernmental panel on climate change) scenario. engelbrecht, rautenbach, macgregor & katzfey (2002) illustrated that darlam is capable of simulating the regional characteristics of atmospheric variables such as near-surface temperature, lowlevel wind patterns and rainfall over sub-saharan africa with considerable detail. the model does, however, tend to overestimate total rainfall over regions with a steep topography. the darlam simulations were performed at the laboratory for research in atmospheric modelling (lram) at the university of pretoria on a pentium iii workstation with two 550 mhz processors. gcm future climate data the gcm climate data used in this study were downloaded from the ipcc/ddc website. through various stages in arcview gis, the original gcm data were processed to fit the 60 x 60 km resolution of darlam. the canadian global coupled model (cgcm2) was the first gcm climate used in this study. it is based on the earlier cgcm1, but with some improvements aimed at addressing shortcomings identified in the first version. in particular, the ocean mixing parameterization has been changed (gent & mcwilliams 1990), and following flato & hibler (1992) sea-ice dynamics has been included. the version of gcm2 used for control and doubled co2 experiments has ten vertical levels with the lowest prognostic level located at approximately 200 m above the surface. a description of cgcm2 and a comparison, relative to cgcm1, of its response to increasing greenhouse-gas forcing can be found in flato & boer (2001). the climate change projections used in this study are those from the newer ipcc sres a2 scenario. the second gcm model used was developed by the center for climate system research/national institute for environmental studies, (japan) (ccsr/ nies) cgcm (nozawa, emori, numaguti, tsushima, takemura, nakajima, abe-ouchi & kimoto 2001). this model is also based on emission scenarios (sres) of the intergovernmental panel on climate change (ipcc). it is a transient coupled oceanatmosphere model, which was developed to investigate the direct and indirect climate impacts of the anthropogenic sulphate and carbonaceous aerosols in future projections of climate change. the data used here are from the a2 scenarios. direct radiative forcing of the carbonaceous aerosols nearly nullifies that of the sulphate aerosols for all scenarios. estimated total indirect radiative forcing is about –1.3 wm-2 for the a1, b1, and b2 scenarios, and is about –2.0 wm-2 for the a2 scenario in the latter half of the 21st century. global and annual averages of the surface air temperature increase for all scenarios because of the dominance of the radiative forcing of the increased co2. global warming is decelerated with an increase in the anthropogenic sulphate and carbonaceous aerosols, because indirect forcing due to the aerosols has a significant cooling effect. geographical distribution of the surface warming does not depend much on the scenarios. cloud feedback becomes dominant in the latter half of the 21st century, and this introduces further warm ing at the surface. predicting current and future distribution of rhipicephalus species the predicted current distributions were initially obtained using current climate predicted by darlam. this represents a useful comparison between predicted distributions and known records (see olwoch et al. 2003). to obtain predicted future distributions the grid cells are populated with future climate variables. the predicted current distributions were obtained using the predictive species model (erasmus et al. 2000) and six climate variables of current and future mean maximum temperature, mean minimum temperature and mean rainfall of january and july provided by darlam. the predicted future distributions were obtained by using both darlam and the mean gcm climates. the predictive modelling was executed in s-plus (s-plus 2000), while maps of the results were drawn in arcview gis. the resultant potential distribution maps represent the probability values of their suitability for ticks based on climate. 50 climate change and rhipicephalus (acari: ixodidae) in africa analysis of predicted tick range changes a number of analyses were performed to compare the predicted current and future distributions of ticks. these included: (i) analysis of species range expansion (ii) an analysis of range contraction (iii) change in species richness pattern (iv) species range shifts (v) an assessment of overlap between darlam and gcm predicted future distributions. these range changes were initially analysed for the whole study area and subsequently, in some cases, on a regional basis. in the second analysis, ticks were grouped into the following regions depend ing on their principal regional distribution, namely east africa, central africa and southern africa, and a fourth group of ticks that were termed “general” ticks. the east african tick species include rhipicephalus aquatilis walker, keirans & pegram, 1993, rhipi cephalus armatus pocock, 1900, rhipicephalus bequaer ti zumpt, 1949, rhipicephalus carnivoralis walker, 1966, rhipicephalus humeralis rondelli, 1926, rhipicephalus kochi dönitz, 1905, rhipicephalus ma culatus neumann, 1901, rhipicephalus muehlensi zumpt, 1943, rhipicephalus planus neumann, 1907 and rhipicephalus pulchellus gerstäcker, 1873. the central african species include rhipicephalus complanatus neumann, 1911, rhipicephalus compositus neumann, 1897, rhipicephalus dux dönitz, 1910, rhipicephalus longus neumann, 1907, rhipicephalus lunulatus neumann, 1907, rhipicephalus masseyi nuttall & warburton, 1908, rhipicephalus punctatus warburton, 1912, rhipicephalus senegalensis koch, 1844, rhipicephalus supertritus neumann, 1907 and rhipicephalus ziemanni neumann, 1904. the southern african species include ticks of the rhipicephalus capensis group (rhipicephalus capensis koch, 1844; rhipicephalus follis dönitz, 1910 and rhipicephalus gertrudae feldman-muhsam, 1960), rhipicephalus distinctus bedford, 1932, rhipicephalus exopthalmos keirans & walker, 1993, rhipicephalus oculatus neumann, 1901, rhipi cephalus zambeziensis walker, norval & corwin, 1981 and the subspecies rhipicephalus evertsi mimeticus dönitz, 1910. there are also species that have wide ranging distributions that overlap in various regions of africa. these species, termed “general” ticks include r. appendiculatus, r. evertsi evertsi neumann, 1897, rhipicephalus pravus dönitz, 1910 and rhipi cephalus simus koch, 1844. the above groupings are presented to facilitate interpretation of the current findings and do not imply that the ticks placed in particular geographical regions are restricted to these areas, but rather localise their distribution with extensions into neighbouring regions. range expansion and contraction in order to obtain range changes in terms of contractions or expansions, predicted current or future distributions were first obtained. the predicted current or future distributions were taken as the number of grid cells in which the probability of occurrence is equal to or greater than 50 %. the difference in the number of grid cells between the predicted present distribution (dp) and predicted future distribution (df) constitutes distribution range change (dc). these range changes may either represent contractions or expansions. we initially performed this analysis on a sub-saharan scale and later on a regional scale in order to establish which regions in africa would experience greater changes in predicted tick distribution ranges (current and future) and therefore appear more vulnerable to climate change. we analysed the differences between the predicted distributions using the kolmogorov-smirnoff two-sample test. furthermore, we divided the ticks into economically important and unimportant species. a com parison of range changes between the current and future predictions was performed on this latter grouping to assess which of the two groups is more vulnerable to climate change. in all the above analyses we assessed the proportion of species that experienced expanded or contracted range changes and the degree of the predicted expansion/contraction. analysis of change in species richness pattern and degree of range shifts species richness patterns were calculated as the number of species in the predicted current or future distribution per grid cell following erasmus et al. (2002). this analysis was performed for the whole of sub-saharan africa. range shifts were calculated as the number of additional grid cells in the predicted future distribution as a proportion of the current predicted distribution. we used the current predicted distribution instead of current known records because most regions in africa are poorly sampled. 51 j.m. olwoch et al. comparing predicted future distributions of ticks based on climates simulated by darlam and gcm the accuracy of any climate model is as good as the initial conditions that are used to configure it. since there is no climate model that provides an accurate projection of the future, it seemed prudent to use the results from more than one climate model in this study. a comparison was therefore made to assess the differences between the predicted future climate suitability for tick species using a regional climate provided by darlam and a mean of two gcms described above. the analysis was performed on a sub-saharan scale and also on a regional scale. we assessed the degree of proportional overlap between the predicted current distribution and the predicted future distribution (darlam and gcm) by means of the proportional overlap method (prendergast, quinn, lawton, eversham & gibbons 1993; reyers, van jaarsveld & krüger 2000). in this case the proportional overlap was calculated as nc/ns where nc is the number of common grid cells between a pair of areas under comparison and ns is the number of grid cells containing data for both groups or the maximum number of overlapping grid cells possible. results model validation was not performed in this study because the same climate envelope model had previously been subjected to rigorous evaluation using presence-absence data re sulting from a coordinated and systematic survey effort. erasmus et al. (2002) used the distribution records of 34 bird species and tested performance of the model using receiver operator characteristic analyses (fielding & bell 1997). the model performed sig nificantly better than a random model with no discriminatory ability. it also accurately predicted the complete known distributions for 24 of the 34 bird species, using a 20 % sub-sample of the known rec ords (erasmus et al. 2002). this satisfactory documented performance of the model and the relatively good predictions that were obtained when it was used to predict the current distributions of four afri can ticks (olwoch et al. 2003) are sufficient reasons to consider the model adequate for the present study. future climate—darlam the climatological anomalies for the 2020s vs the 1990s as predicted by darlam are depicted in fig. 2. january minimum and maximum temperatures are simulated to increase by more than 2 °c over certain regions of sub-saharan africa. many of the eastern regions are expected to become drier with an associated pattern of higher sea-level pressure, whilst the western subcontinent is expected to bemaximum temperature (°c) minimum temperature (°c) mean sea-level pressure (hpa) rainfall (mm/day) fig. 2 darlam’s climatological anomalies for the 2020s v. 1990s 52 climate change and rhipicephalus (acari: ixodidae) in africa come wetter. an interesting feature of the july anomaly fields is that parts of the central subcontinent are simulated to become cooler and wetter. species distribution changes broad scale range changes (fig. 3a, b) the predicted current and future distributions of the selected rhipicephalus spp. using darlam are provided in figures 10–39. on a sub-saharan scale, the ranges of 46 % of the tick species, namely r. appendiculatus, r. capensis group, r. distinctus, r. humeralis, r. kochi, r. longus, r. masseyi, r. oculatus, r. planus, r. punctatus, r. senegalensis, r. simus, r. zambeziensis and r. ziemanni are predicted to contract. the ranges of 54 % of the species, namely r. aquatilis, r. armatus, r. bequaerti, r. carnivoralis, r. complanatus, r. compositus, r. dux, r. evertsi evertsi, r. evertsi mimeticus, r. exopthalmos, r. lunulatus, r. maculatus, r. muehlensi, r. pravus, r. pulchellus and r. supertritus are predicted to expand over the same period (fig. 3a, b). these results translate into an area expansion of 3 502 800 km2 (12 %) in total tick range with a total reduction of 640 800 km2 (2 %). central african species (fig. 4a, b, 16,17, 19, 25, 26, 28, 34, 35 and 39) fifty-five percent of species in central africa are predicted to show range reductions (r. longus, r. masseyi, r. punctatus, r. senegalensis and r. zie manni) while 45 % (r. complanatus, r. compositus, r. dux and r. lunulatus) are predicted to show range expansions. although the ranges of the majority of tick species are predicted to contract, the total area of contraction is only 19 %, while the total area of expansion by the remaining species is 81 %. the tick species predicted to expand its range most in this region is r. lunulatus with a total expansion of 252 000 km2. east african species (fig. 5a, b, 11, 12, 13, 15, 23, 24, 27, 29, 31 and 33) in east africa 30 % of the species (r. humeralis, r. kochi and r. planus) are predicted to show range contractions, while 70 % (r. aquatilis, r. armatus, r. bequaerti, r. carnivoralis r. maculatus, r. muehlensi and r. pulchellus) are predicted to show range expansions. this translates into a total expansion of 1 760 400 km2 (91 %) with a mere 169 200 km2 (9 %) reduction in total area. the predicted greater expansions are mainly attributable to r. bequaerti which � �� �� ��� �� � �� � �� � �� ��� �� � �� ��� � � � �� ��� �� ��� � �� ��� �� � �� ��� � � �� �� � � �� �� ��� ��� � �� ��� � � �� ��� � � � �� � � � �� �� � �� � � �� �� ��� �� �� �� � �� ��� �� � � ������� �������� ����� ����� ��������������������� �� ����� ����� ����� ��� ��� ��� ��� � �� ��� �� � ��� �� � � ��� �� �� ��� ��� ��� � � ��� �� �� �� � � ��� �� � � �� ��� � ��� �� �� �� � � ��� �� �� � � � ��� �� � ��� �� �� ��� �� � � ��� �� �� ��� �� � � �� ��� �� �� �� �� � ��� �� ��� � ��� �� �� �� � ��� �� �� �� � ��� ��� �� ��� �� ��� ���� ��� � �� � �� � � �� �� ��� �� � �� � �� � �������� ����� ����� ������������������� �� ����� ����� ����� ��� ��� ��� ��� � ������� ������� ������ � ����� ������� ���������� ����� ����� ������������������� �� ������� ����� ������� ���������� ����� ����� ��������������������� �� ������� �� ��� � � �� �� � � �� �� ��� � � � �� � � � �� �� � �� ��� �� � � � ����� ����� ��� ��� ��� ��� � � �� �� ��� �� � �� � �� � � � �� �� ��� �� � �� � �� � ����� ��� ��� ��� ��� � ��� �� �� �� � � ��� �� �� � � � ��� �� � ��� �� ��� � ������� ������ fig. 3 rhipicephalus species in sub-saharan africa that are predicted to show (a) range size contraction and (b) range size expansion fig. 4 rhipicephalus species in central africa that are predicted to show (a) range size contraction and (b) range size expansion 53 j.m. olwoch et al. more than doubles its present range and r. pulchellus, which is predicted to expand its range by some 921 600 km2 (49 %). southern african species (fig. 6a, b, 14, 18, 21, 22, 30 and 38) in southern africa some 66 % (r. capensis group, r. distinctus, r. oculatus and r. zambeziensis) of the tick species are predicted to contract their ranges. although only 33 % (r. evertsi mimeticus and r. exopthalmos) of the ticks are predicted to expand their current ranges, the total range expansion is 439 200 km2 (23 %) while the total range reduction is only 64 800 km2 (1 %). most of the expansion in this region is attributable to r. evertsi mimeticus. “general” tick species (fig. 7a, b, 10, 20, 32 and 36) the “general” ticks are those rhipicephalus species that are widely distributed with current distributions overlapping within various geographical regions of the subcontinent. this does not necessarily mean that these species are not specialists with regard to their ecological requirements, e.g. r. appen diculatus is confined to parts of eastern, central and southeastern africa (walker et al. 2000). it is a species of significant economic importance in africa because it transmits theileria parva, the cause of east coast fever (ecf), which is a major cause of cattle mortality and also causes considerable production losses in cattle in most african countries (okello-onen et al. 1999). the only tick species predicted to contract its range in this group is r. appendiculatus, which is predicted to contract its range by 212 400 km2 (5 %). the ranges of 75 % of the ticks in this category, namely r. evertsi evertsi, r. pravus and r. simus are predicted to expand. the total range expansion in this region is equivalent to 864 000 km2 (7 %). this expansion is mainly associated with r. simus, which is predicted to expand its range by 601 200 km2 (70 %). changes in species richness patterns and range shifts the future climate predicted by darlam will alter the species richness distribution pattern of african rhipicephalus. compared to the current pattern (fig. 8a) the predicted pattern is spatially different and broader (fig. 8b) with encroachment of ticks into new regions. these regions, which include anfig. 5 rhipicephalus species in east africa that are predicted to show (a) range size contraction and (b) range size expansion fig. 6 rhipicephalus species in southern africa that are predicted to show (a) range size contraction and (b) range size expansion ������� �������� ���������� ����� ����� ��������������������� �� ����� ��� ��� ��� ��� � � �� �� ��� �� � �� � �� � ��� ��� ��� �� ��� ��� �� ��� ��� � � ������� �������� ���������� ����� ����� ������������������� �� ����� ��� ��� ��� ��� � � �� �� ��� �� � �� � �� � ��� ��� ����� ��� ��� �� ��� �� ��� ��� ��� ��� � ��� ��� � � ��� ��� ��� � � ��� �� ��� � � ��� �� ��� � � ������� ������ ������� ���� ������ ���������� ����� ����� ��������������������� �� ��� ��� ��� ��� � � �� �� ��� �� � �� � �� � ���� ��� � ���� � � � � ���� ��� ���� � � ���� ���� � ������� ���� ��� ��� ��� ��� ���� ������ ���������� ����� ����� ������������������� �� ��� ��� ��� � � �� �� ��� �� � �� � �� � � ���� ��� ��� �� ������� ������ 54 climate change and rhipicephalus (acari: ixodidae) in africa gola, namibia, botswana and the northern cape province of south africa, are forecast to experience a more than 50 % increase in tick species richness (fig. 8c). this could be related to increased rainfall in these regions, rendering the south-western regions of sub-saharan africa more suitable for ticks. the general west-east shift in species ranges reported by erasmus et al. (2002) is not supported in this study on ticks, in which varying degrees of shift in different directions appears to be the emergent pattern for this taxon. analysis of range shifts further indicates that 80 % of species show less than a 50 % range shift, while 20 % of species show a range displacement of more than a 100 %. the latter species are mainly those that populate the western arid regions of africa (fig. 8). species range changes and tick-borne disease implications thirty percent of the ticks included in this study are classified as economically important because they are vectors of diseases of domestic livestock or other animals (table 1). although r. appendiculatus, the principal rhipicephalus vector of disease in afri ca shows range contraction, the remaining vectors are responsible for 52 % of the predicted range expansion under future climate conditions. the non-vector species are responsible for 48 % of future tick range expansions. predicted future distributions using darlam and gcm climate data the predictions of future climate suitability for ticks when darlam is used are generally more extensive than those generated when using the combined gcm climates. with the exception of four species (r. compositus, r. evertsi mimeticus, r. exopthalmos and r. oculatus), and the r. capensis group, darlam predicts wider ranges of climatic suitability than the combined gcm climate surfaces. darlam’s total predictions are 31 % broader than the gcms. the average difference in the range sizes predicted for darlam and gcm is 511 200 km2. stat is tically there is no significant difference between the predictions by darlam and gcm using a kolmogorov-smirnov test (p > 0.1, n = 30). however, when the climate data simulated by darlam and gcm are analysed for statistical significance, there fig. 7 rhipicephalus species in the “general” ticks that are predicted to show (a) range size contraction and (b) range expansion ������� !"���� �#��������� ����� ����� ������������������� �� ����� ����� ����� ��� ��� ��� ��� � � �� �� ��� �� � �� � �� � �� ��� �� �� �� �� � �� ��� �� � �� � � �� � ������� ������ ������� !"���� �#��������� ����� ����� ��������������������� �� ����� ����� ��� � �� �� ��� �� � �� � �� � ������� ������ � � fig. 8 species richness pattern of rhipicephalus species in subsaharan africa: (a) current; (b) future; and (c) areas with more than 50 % increase 0–4 5–8 9–12 13–16 17–21 national boundaries b a c more than 50 % increase in species richness 55 j.m. olwoch et al. table 1 rhipicephalus spp. and the diseases with which they or the toxins they produce are associated disease and causal agents animal affected vectors east coast fever (theileria parva) cattle r. appendiculatus r. zambeziensis tick toxicosis cattle r. appendiculatus corridor disease or buffalo disease (theileria parva) cattle, buffalo r. appendiculatus r. zambeziensis gall sickness (anaplasma marginale) cattle r. evertsi evertsi r. simus equine biliary fever or equine piloplasmosis (babesia caballi, theileria equi) horses, mules, donkeys r. evertsi evertsi spring lamb paralysis lambs, calves r. evertsi evertsi canine biliary fever or canine tick fever (babesia canis), rickettsiosis (ehlichia canis) dogs r. sanguineus paralysis sheep, lambs, calves r. lunulatus nairobi sheep disease (bunyaviridae) sheep r. pulchellus table 2 proportional overlaps between pairs of grid cells between the predicted ranges from darlam and the ranges predicted using mean values for two gcms “general” tick species darlam 2030 gcm 2030 common grids proportional overlap value (%) r. appendiculatus r. evertsi evertsi r. pravus r. simus 969 1 220 1 300 1 209 858 945 868 858 476 611 621 597 55 65 29 60 east africa r. aquatilis r. armatus r. bequaerti r. carnivoralis r. humeralis r. kochi r. maculatus r. muehlensi r. planus r. pulchellus 33 153 215 205 79 797 425 699 719 841 17 116 40 111 33 491 173 678 413 217 5 29 11 67 12 326 98 315 278 179 29 25 28 60 55 29 56 67 55 25 central africa r. complanatus r. compositus r. dux r. longus r. lunulatus r. masseyi r. punctatus r. senegalensis r. ziemanni 626 699 201 1 020 897 450 458 803 611 595 737* 192 990 518 338 440 622 558 400 443 77 642 308 202 262 415 352 67 60 40 25 28 60 28 56 72 south africa r. capensis group r. distinctus r. evertsi mimeticus r. exopthalmos r. oculatus r. zambeziensis 485 707 423 212 386 299 547* 352 577* 236* 434* 226 309 254 49 116 293 169 56 72 49 15 49 60 bold* = gcm predictions wider than those of darlam 56 climate change and rhipicephalus (acari: ixodidae) in africa fig. 9 box plots based on the kolmogorov-smirnov test that compared climate predicted by darlam and a combined gcm ���������� �� �� �� $� $� $� $� $� �� �� �� �� �� �� �� �� � � � �� � $� �% �� �% �� % � &% &�� ���������� �� �� � �� �� � �� $% $� �% �� �% �� % � ���������� �� �� � � � �� � ����� ����� ��� ��� ��� ��� � &��� � � �� �� ���������� �� �� ���������� �� �� $� �� �� �� �� �� �� �� �� �� �� � � � � � �� �� � ���������� �� �� ����� '�� ��� (�� ��� %�� ��� $�� ��� ��� � &��� � � �� �� �%&(%�) *��* �*��� � 57 j.m. olwoch et al. fig. 10 r. appendiculatus fig. 11 r. aquatilis fig. 10–39 predicted probability distribution of rhipicephalus species using darlam and gcm future climates: (a) darlam; (b) gcm 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability a b 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability a b a b 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability a b 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability fig. 12 r. armatus fig. 13 r. bequaerti 58 climate change and rhipicephalus (acari: ixodidae) in africa fig. 14 r. capensis group fig. 15 r. carnivoralis 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability a b 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability a b a b 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability a b 0.748–1.000 0.499–0.748 0.249–0.499 0.000–0.249 national boundaries probability fig. 16 r. complanatus fig. 17 r. compositus fig. 10–39 continued http://www.ojvr.org open access table of contents review article review of african swine fever outbreaks history in south africa: from 1926 to 2018 ciza a. mushagalusa, eric etter, mary-louise penrith onderstepoort journal of veterinary research | vol 88, no 1 | a1919 | 29 september 2021 review article the spread and antimicrobial resistance of staphylococcus aureus in south african dairy herds – a review joanne karzis, inge-marie petzer, vinny naidoo, edward f. donkin onderstepoort journal of veterinary research | vol 88, no 1 | a1937 | 26 october 2021 research communication case report: control of intestinal nematodes in captive chlorocebus sabaeus katalina cruz, tatiana m. corey, michel vandenplas, maría trelis, antonio osuna, patrick j. kelly onderstepoort journal of veterinary research | vol 88, no 1 | a1903 | 28 may 2021 research communication surveillance of the rabies-related lyssavirus, mokola in non-volant small mammals in south africa william c. mcmahon, jessica coertse, teresa kearney, mark keith, lourens h. swanepoel, wanda markotter onderstepoort journal of veterinary research | vol 88, no 1 | a1911 | 03 august 2021 research communication occurrence of methicillin-resistant staphylococci in the pig-production chain in ibadan, nigeria opeyemi u. lawal, abimbola o. adekanmbi, olawale o. adelowo onderstepoort journal of veterinary research | vol 88, no 1 | a1959 | 30 november 2021 research communication epidemiology and microscopic diagnosis of tuberculosis in pigs and small ruminants slaughtered at bobo-dioulasso abattoir, burkina faso adama sanou, amadou dicko, kadiatou r. sow, arthur djibougou, antoinette kabore, bassirou diarra, arsène k. ouedraogo, dezemon zingue, moumini nouctara, zekiba tarnagda onderstepoort journal of veterinary research | vol 88, no 1 | a1908 | 03 december 2021 research communication updated distribution and host records for the argasid tick ornithodoros (pavlovskyella) zumpti: a potential vector of african swine fever virus in south africa anthony f. craig, livio heath, jan e. crafford, juergen a. richt, robert swanepoel onderstepoort journal of veterinary research | vol 88, no 1 | a1960 | 09 december 2021 72 82 92 97 110 114 120 page i of ii table of contents original research plasmodium berghei-induced malaria decreases pain sensitivity in mice aboyeji l. oyewole, oluwole akinola, bamidele v. owoyele onderstepoort journal of veterinary research | vol 88, no 1 | a1871 | 11 january 2021 original research epidemiology and serological detection of rift valley fever disease in farm animals in southern egypt hassan y.a.h. mahmoud, alsagher o. ali onderstepoort journal of veterinary research | vol 88, no 1 | a1877 | 03 february 2021 original research analysis of antibiotic resistance phenotypes and genes of escherichia coli from healthy swine in guizhou, china bo yu, yanan zhang, li yang, jinge xu, shijin bu onderstepoort journal of veterinary research | vol 88, no 1 | a1880 | 24 february 2021 original research in vitro anti-toxoplasma gondii efficacy of synthesised benzyltriazole derivatives huanping guo, yang gao, david d. n’da, xuenan xuan onderstepoort journal of veterinary research | vol 88, no 1 | a1898 | 11 june 2021 original research spatial distribution and habitat selection of culicoides imicola: the potential vector of bluetongue virus in tunisia ben h. thameur, sghaier soufiène, heni haj ammar, salah hammami onderstepoort journal of veterinary research | vol 88, no 1 | a1861 | 16 august 2021 original research finding of a two-headed green turtle embryo during nest monitoring in baa atoll, maldives stephanie köhnk, rosie brown, amelia liddell onderstepoort journal of veterinary research | vol 88, no 1 | a1940 | 24 august 2021 original research wide circulation of peste des petits ruminants virus in sheep and goats across nigeria samuel e. mantip, anthony sigismeau, maurice nanven, atuman joel, abayomi m. qasim, sada aliyu, ibrahim musa, ogechukwu ezeanyika, ibikunle faramade, garba ahmed, timothy y. woma, david shamaki, genevieve libeau, souaibou farougou, arnaud bataille onderstepoort journal of veterinary research | vol 88, no 1 | a1899 | 07 september 2021 review article the history of the emergence and transmission of human coronaviruses elijah n. mulabbi, robert tweyongyere, denis k. byarugaba onderstepoort journal of veterinary research | vol 88, no 1 | a1872 | 10 february 2021 1 19 24 32 40 49 57 64 vol 88, no 1 (2021) issn: 0030-2465 (print) | issn: 2219-0635 (online)onderstepoort journal of veterinary research http://www.ojvr.org� http://www.ojvr.org open access table of contents correction corrigendum: anthelmintic activity of acetone extracts from south african plants used on egg hatching of haemonchus contortus gerda fouche, bellonah m. sakong, olubukola t. adenubi, elizabeth pauw, tlabo leboho, mbokota c. khosa, kevin w. wellington, jacobus n. eloff onderstepoort journal of veterinary research | vol 88, no 1 | a1949 | 27 july 2021 correction corrigendum: investigation of the acaricidal activity of the acetone and ethanol extracts of 12 south african plants against the adult ticks of rhipicephalus turanicus gerda fouche, bellonah m. sakong, olubukola t. adenubi, jean paul dzoyem, vinny naidoo, tlabo leboho, mbokota c. khosa, kevin w. wellington, jacobus n. eloff onderstepoort journal of veterinary research | vol 88, no 1 | a1950 | 27 july 2021 124 125 correction corrigendum: acaricidal activity of the aqueous and hydroethanolic extracts of 15 south african plants against rhipicephalus turanicus and their toxicity on human liver and kidney cells gerda fouche, olubukola t. adenubi, tlabo leboho, mbokota c. khosa, lyndy j. mcgaw, vinny naidoo, kevin w. wellington, jacobus n. eloff onderstepoort journal of veterinary research | vol 88, no 1 | a1951 | 27 july 2021 reviewer acknowledgements onderstepoort journal of veterinary research | vol 88, no 1 | a1981 | 21 december 2021 126 127 page ii of ii http://www.ojvr.org� abstract acknowledgements references about the author(s) anthony f. craig vectors and vector-borne diseases research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa livio heath agricultural research council-onderstepoort veterinary research transboundary animal diseases laboratory, onderstepoort, south africa jan e. crafford vectors and vector-borne diseases research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa juergen a. richt diagnostic medicine/pathobiology, center of excellence for emerging and zoonotic animal diseases (ceezad), college of veterinary medicine, kansas state university, manhattan, kansas, united states of america robert swanepoel vectors and vector-borne diseases research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation craig, a.f., heath, l., crafford, j.e., richt, j.a. & swanepoel, r., 2021, ‘updated distribution and host records for the argasid tick ornithodoros (pavlovskyella) zumpti: a potential vector of african swine fever virus in south africa’, onderstepoort journal of veterinary research 88(1), a1960. https://doi.org/10.4102/ojvr.v88i1.1960 research communication updated distribution and host records for the argasid tick ornithodoros (pavlovskyella) zumpti: a potential vector of african swine fever virus in south africa anthony f. craig, livio heath, jan e. crafford, juergen a. richt, robert swanepoel received: 10 july 2021; accepted: 22 oct. 2021; published: 09 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract african swine fever virus (asfv) causes a lethal and contagious disease of domestic pigs. in south africa, the virus historically circulated in warthogs and ornithodorid ticks that were only found in warthog burrows in the north of the country. regulations implemented in 1935 to prevent transfer of infected animals or products to the south initially proved effective but from 2016 there have been outbreaks of disease in the south that cannot be traced to transfer of infection from the north. from 1963 there were widespread translocations of warthogs to the south, initially from a source considered to be free of ornithodorid ticks. we undertook to determine whether sylvatic circulation of asfv occurs in the south, including identification of potential new vectors, through testing extralimital warthogs for antibody and ticks for virus. results of testing warthogs for antibody and other species of ticks for virus will be presented separately. here we report finding ornithodoros (pavlovskyella) zumpti ticks in warthog burrows for the first time. this occurred in the eastern cape province (ecp) in 2019. since african swine fever was recognised in the ecp for the first time in 2020 and outbreaks of the disease in domestic pigs continue to occur there, priority should be given to determining the distribution range and vector potential of o. (p.) zumpti for asfv. keywords: african swine fever virus; sylvatic circulation; arbovirus; south africa; extralimital warthogs. the argasid tick ornithodoros (pavlovskyella) zumpti was described in 1953 as a new species from the burrow of a four-striped field mouse (rhabdomys pumilio sensu lato) in the cathcart district of the eastern cape province (ecp) of south africa (heisch & guggisberg 1953). in 1961, the same species was collected approximately 300 km south-west of the type locality from the nest of a saunders’ vlei rat (otomys saundersiae) in kariega district, formerly uitenhage, ecp, during investigation of the bionomics of the tick (fearnhead 1962). more recently, in 2014, o. (p.) zumpti was again found in cathcart district, in a porcupine (hystrix africaeaustralis) burrow, and also in the burrow of a springhare (pedetes capensis) in makhanda district, formerly grahamstown, during a phylogenetic study of the argasidae (see figure 1) (mans et al. 2019). figure 1: map showing the african swine fever (asf) controlled area in south africa, the distribution of extralimital warthogs derived from swanepoel (2016), the sites of asf outbreaks in domestic pigs in the eastern cape province in 2020 and the sites of collection of ornithodoros (pavloskyella) zumpti ticks from host-associated sites as indicated. argasid ticks of the genus ornithodoros serve as biological vectors of african swine fever virus (asfv) that causes lethal disease in domestic pigs. the virus is maintained in the savannah areas of eastern and southern africa in sylvatic circulation between warthogs (phaecochoerus africanus) that develop benign viremic infection and eyeless argasid ticks of the ornithodoros (ornithodoros) moubata complex that live in warthog burrows (penrith, thomson & bastos 2019). warthogs develop intense viremia after infection by ticks whilst still confined to their burrows as young piglets, but naïve adults develop viremia of low intensity after infection, and have not been shown to be contagious for domestic pigs. however, they can convey infected ticks to the environs of piggeries or infected warthog offal may be fed to pigs in swill. the infection is contagious in domestic pigs and spreads without need for the tick vectors (penrith et al. 2019). vector-borne transmission of asfv was first recognised in domestic pigs and ornithodoros (pavlovskyella) erraticus ticks in spain after the virus had accidentally been introduced into europe (sánchez botija 1963). over recent decades, both pig farming and the occurrence of asfv have spread from east to west across africa (penrith et al. 2019) and in 2006 the virus was found in o. (p.) sonrai ticks collected in rodent burrows close to piggeries in senegal although the epidemiological significance of the finding was regarded as uncertain (vial et al. 2007). only members of the subgenus ornithodoros have been identified as vectors of asfv elsewhere in sub-saharan africa. nevertheless, vector competence studies in various countries abroad have established that the ability to transmit asfv is a general property of ticks of both the ornithodoros and pavlovskyella subgenera, but the efficiency of transmission varies with virus isolate and species of tick (pereira de oliveira et al. 2020). in south africa, african swine fever (asf) was originally prevalent in the northern part of the country where domestic pigs had contact with warthogs (penrith et al. 2019). hence, a controlled area was declared in 1935 to include the known distribution range of warthogs at the time (see figure 1) and regulations were implemented to prevent transfer of infected suids or products to the south (de kock, robinson & keppel 1940). the regulations initially proved effective; but from 2016 onwards there have been a series of asf outbreaks in domestic pigs south of the controlled area that cannot be linked to transfer of infection from the north. the department of agriculture, land reform and rural development (dalrrd) in 2020, reported the first known outbreaks of asf in the ecp, and in 2021; the first outbreaks of asf in the western cape province since the disease was eradicated there in 1939 (dalrrd 2020, 2021; janse van rensburg et al. 2020). from 1963 onwards, there were widespread translocations of warthogs from the north to nature reserves and game ranches in the southern half of south africa where the extralimital animals flourished to become an invasive species (see figure 1) (swanepoel 2016). originally, the warthogs were obtained by officials of national and provincial parks from hluhluwe-imfolozi game reserve considered to be free of ornithodorid ticks and asfv, but it has been suggested that later there were multiple unrecorded translocations from other sources to private nature reserves (swanepoel 2016). furthermore, the taxonomy of the afrotropical members of the subgenus ornithodoros was recently revised, including description of new species (bakkes et al. 2018). although information on the distribution ranges and vector capacity of the ticks remains incomplete, the implication is that the potential exists for sylvatic circulation of asfv in ornithodorids and extralimital warthogs to occur beyond the controlled area in south africa. we conducted a survey for evidence of asfv infection in warthogs and ticks outside the controlled area, to be reported separately and in the process encountered new hosts for o. (p.) zumpti ticks. we collected ornithodorid ticks identified as o. (p.) zumpti from warthog burrows in addo elephant national park (addo enp), ecp, in 2019 before asf had been recorded in the province and found no asfv nucleic acid in the ticks. approximately 10% weight per volume (w/v) suspensions were prepared by homogenising pools of 1–2 adults or 5–25 nymphal ticks in phosphate-buffered saline, ph 7.2 and automated nucleic acid extraction was performed with indimag pathogen kits (indical bioscience, germany) using slight modifications to the manufacturer’s instruction. a total of 200 µl tick homogenate supernatant was added to 200 µl al buffer (qiagen, germany), incubated at 70 °c for 10 min and 200 µl of the al lysate added to the extraction buffer. eluates were tested for asfv nucleic acid using the real-time quantitative polymerase chain reaction (qpcr) assay of zsak et al. (2005) as modified by sunwoo et al. (2019). positive and no template controls were included in each pcr run. the ticks were collected in the nyathi (33.25070s, 24.29166e), main camp (33.44492s, 25.78378e) and colchester (33.41170s, 25.78466e) sections of the park from burrows observed over the course of a week to be occupied by warthogs. the same species was also obtained from a small burrow in main camp section (33.44492s, 25.78378e) observed to be occupied by a family of yellow mongooses (cynictis penicillata) (see figure 1). identification of the ticks was based on morphology (heisch & guggisberg 1953), confirmed by the gertrud theiler tick museum of the agricultural research council-onderstepoort veterinary research, onderstepoort, south africa and by amplification and comparison of partial tick mitochondrial 16s ribosomal ribonucleic acid (rrna) gene sequences with genbank data (see figure 2) (bakkes et al. 2018; mans et al. 2019). the o. (p) zumpti sequences fell into three clades with 94% – 96% identity and more extensive sequencing studies are required to determine whether the differences represent within-species variation or novel cryptic species. figure 2: neighbour-joining tree based on partial mitochondrial 16s rrna gene sequence (276 nt) depicting phylogenetic relationships between the 3 ornithodoros (pavlovskyella) zumpti tick pools from the present study (genbank accession numbers in bold) and representative ornithodororos species sequences from genbank. the corresponding gene sequence of amblyomma hebraeum was included as an outlier. percentage bootstrap support values were derived following 10 000 replications. evolutionary analyses were conducted in mega x software. partial cytochrome b sequences of mammalian mitochondrial deoxyribonucleic acid (dna) were determined from suspensions of partially engorged ticks (lah et al. 2015) and compared with sequences in genbank national center for biotechnology information (ncbi) using a blastx search to confirm warthogs as blood meal donors for o. (p.) zumpti. there was insufficient nucleic acid for cytochrome b analysis from ticks recovered from the mongoose burrow. the morphology of o. (p.) zumpti was well described by heisch and guggisberg (1953) but since it is presently the only known representative of the subgenus pavlovskyella in south africa, preliminary identification can be based on macroscopic features in the field. in each instar of their life cycle, o. (p.) zumpti ticks are noticeably smaller than members of the ornithodoros subgenus that occur in warthog burrows. from a dorsal view, ticks of the ornithodoros subgenus have a broadly rounded shape at both the anterior and posterior ends, and are slightly concave bilaterally. o. (p.) zumpti ticks have a bluntly pointed hood that covers the basis capituli at the anterior end, whilst the sides are roughly parallel (see figure 3). figure 3: dorsal and ventral views of adult female ornithodoros (pavlovskyella) zumpti tick. in view of the ongoing outbreaks of the asf in the ecp it should be a priority to determine the distribution range and vector competence of o. (p.) zumpti ticks for asfv and even to investigate whether warthogs and o. (p.) zumpti ticks may already have become involved in circulation of the virus in that province. acknowledgements competing interests the authors declare that no financial or personal relationships have inappropriately influenced them in preparing this manuscript. authors’ contributions a.f.c. planned and performed the study and prepared the manuscript under supervision, as part of a phd project. l.h. provided guidance in conducting laboratory procedures. j.e.c. co-supervised the study and provided guidance in conducting field studies. j.a.r. suggested conducting a research project on african swine fever in south africa, and raised the funds for the research contract. r.s. supervised planning and performance of the study and preparation of the manuscript. ethical considerations research ethics approval was obtained from the animal ethics committee, up, and the research committee of the faculty of veterinary science, up. section 20 approval in terms of the animal diseases act 35 of 1984 was obtained from the director of animal health, department of agriculture, land reform and rural development, south africa. research and materials transfer agreements were concluded with the scientific services committee of the south african national parks board and written informed consent was obtained from the owners of other properties from which samples were received. samples were transported in appropriate biosafety packaging under permit from relevant state veterinarians and laboratory procedures were performed under animal biosafety level 3 biocontainment facility (bsl3) conditions in transboundary animal diseases laboratory (tadl). funding information the project was supported by a research contract from kansas state university and a grant was awarded by the south african agricultural sector education and training authority (agriseta) to the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria (up). we acknowledge the support of national bio and agro-defense facility (nbaf) transition funds from the state of kansas, the national institute of general medical sciences (nigms) of the national institutes of health under award number p20gm130448 and the department of homeland security center of excellence for emerging and zoonotic animal diseases under grant number hshqdc 16-a-b0006 to j.a.r. data availability derived data supporting the findings of this study are available within the article or from the corresponding author, a.f.c., on request. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors, and the publisher. references bakkes, d.k., de klerk, d., latif, a.a. & mans, b.j., 2018, ‘integrative taxonomy of afrotropical ornithodoros (ornithodoros) (acari: ixodida: argasidae)’, ticks and tick borne diseases 9(4), 1006–1037. https://doi.org/10.1016/j.ttbdis.2018.03.024 dalrrd 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https://doi.org/10.3390/vaccines7010012 swanepoel, m., 2016, ‘distribution, utilization and management of the extralimital common warthog phacochoerus africanus in south africa’, doctor of philosophy, conservation, ecology, and entomology thesis, stellenbosch university, stellenbosch, viewed 21 august 2018, from https://scholar.sun.ac.za. vial, l., wieland, b., jori, f., etter, e., dixon, l. & roger, f., 2007, ‘african swine fever virus dna in soft ticks, senegal’, emerging infectious diseases 13(12), 1928–1931. https://doi.org/10.3201/eid1312.071022 zsak, l., borca, m.v., risatti, g.r., zsak, a., french, r.a., lu, z. et al., 2005, ‘preclinical diagnosis of african swine fever in contact-exposed swine by a real-time pcr assay’, journal of clinical microbiology 43(1), 112–119. https://doi.org/10.1128/jcm.43.1.112-119.2005 abstract introduction methodology results discussion conclusion acknowledgements references appendix 1 about the author(s) ayaovi b. yaovi research unit on communicable diseases, polytechnic school of abomey-calavi, university of abomey-calavi, cotonou, benin philippe sessou research unit on communicable diseases, polytechnic school of abomey-calavi, university of abomey-calavi, cotonou, benin aretas b.n. tonouhewa research unit on communicable diseases, polytechnic school of abomey-calavi, university of abomey-calavi, cotonou, benin gildas y.m. hounmanou department of veterinary and animal sciences, university of copenhagen, copenhagen, denmark deborah thomson one health lessons, arlington, virginia, united states of america roger pelle international livestock research institute, nairobi, kenya souaïbou farougou research unit on communicable diseases, polytechnic school of abomey-calavi, university of abomey-calavi, cotonou, benin arindam mitra department of microbiology, adamas university, kolkata, west bengal, india citation yaovi, a.b., sessou, p., tonouhewa, a.b.n., hounmanou, g.y.m., thomson, d., pelle, r. et al., 2022, ‘prevalence of antibiotic-resistant bacteria amongst dogs in africa: a meta-analysis review’, onderstepoort journal of veterinary research 89(1), a1970. https://doi.org/10.4102/ojvr.v89i1.1970 review article prevalence of antibiotic-resistant bacteria amongst dogs in africa: a meta-analysis review ayaovi b. yaovi, philippe sessou, aretas b.n. tonouhewa, gildas y.m. hounmanou, deborah thomson, roger pelle, souaïbou farougou, arindam mitra received: 01 sept. 2021; accepted: 22 feb. 2022; published: 10 oct. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract antimicrobial resistance (amr) is a global public health threat for both human and veterinary medicine. increasing evidence suggests that animals are important sources of amr to humans; however, most of these studies focus on production animals. in order to determine the pattern of amr in pets, mainly in dogs in africa, a meta-analysis was performed with amr studies conducted in african countries and published between january 2000 and january 2021 in four databases: medline (pubmed), scopus, cab abstract and google scholar. seven bacterial strains, namely staphylococcus aureus, escherichia coli, salmonella spp., pseudomonas aeruginosa, streptococcus pyogenes, coagulase-negative staphylococcus (snc) and staphylococcus pseudintermedius were included in this study. a total of 18 out of 234 indexed articles met the study criteria. the results revealed that multiple bacteria were resistant to various commonly used antibiotics including enrofloxacin, ciprofloxacin, gentamicin, amoxicillin, clavulanic acid, cotrimoxazole, streptomycin, tetracycline and chloramphenicol. concerning multidrug resistance, e. coli strains came first with the highest prevalence of 98%, followed by p. aeroginosa (92%) and salmonella spp. (53%). in contrast, the overall prevalence of multidrug resistance was low for s. aureus (18%) and s. pseudintermedius (25%). it is therefore urgent to find, as soon as possible, alternatives to replace these antibiotics, which have become ineffective in controlling these bacteria in dogs in africa. moreover, further metagenomic studies are needed to describe the full resistome and mobilome in dogs regardless of the bacteria. keywords: prevalence; antibiotic resistance; dogs; africa; meta-analysis; antimicrobial resistance. introduction the importance of the bond between people and their pets is increasingly recognised and many owners consider their pets to be a member of their family (wsava 2020). this is particularly the case with dogs that are highly valued amongst all other pets because of their many benefits to humans such as sheep guarding, detection of drugs and explosives, hunting and safety, breeding and companionship (daodu et al. 2017). for years, the dog was the most widely employed scent-detector tool for civilian and military purposes. recently, many studies highlighted the role of canine olfactory ability in the medical field, specifically in detecting different infectious, metabolic and neoplastic conditions including the coronavirus disease 2019 (covid-19) (sakr et al. 2021). trained biodetection dogs are already being used for detecting illicit substances and for forensic purposes. they have also been used for helping to detect cancer (sharun et al. 2021). a study conducted by oliva and johnston (2020) found a buffering effect of dog ownership against loneliness. despite the benefits of dog ownership, dogs are susceptible to many infectious agents responsible for viral diseases such as rabies, parvovirosis, canine distemper and bacterial diseases, namely leptospirosis, pasteurellosis, skin diseases and parasitic illness such as piroplasmosis, and so on (ghasemzadeh & namazi 2015). as a result of their importance, most owners try to take care of their animals to the best of their ability and strive to keep their animals healthy and ‘happy’ (wsava 2020). this has led to a high use of antimicrobials such as antibiotics to treat diseases and injuries in their animals and the use of vaccines for the prevention of certain diseases (gwenzi et al. 2021). antibiotic resistance is a global public health problem that could be responsible for more than 10 million deaths per year and thus become the leading cause of mortality by 2050, with an economic cost of $100 billion if left unchecked (o’neill 2016). the antibiotic agents used are often more closely related to those used in human medicine and antibiotic resistance risks should not be ignored as the close relationship between dog and human subjects presents an opportunity for the two-way transfer of bacterial (commensal and pathogen) or genetic determinants of resistance with associated potential for morbidity and mortality on both sides (argudín et al. 2017; rendle & page 2018). the misuse of antibiotics has been reported in several regions of the world and is at the origin of the antibiotic resistance phenomenon (kavanagh, mitra & basu 2021; le huy et al. 2020). in africa, numerous recent studies in different countries have revealed high prevalence of resistant bacterial strains in dogs (qekwana et al. 2020; zewdu et al. 2019). a survey conducted in nigeria revealed that 82% of dog owners had, at different times, body-to-body contact of less than 50 cm between their face and the dog’s body (daodu et al. 2017). this close contact can promote the exchange of resistant pathogens via saliva, urine, faeces, aerosols, skin and thus amplify the phenomenon of antibiotic resistance in humans and exchange of antibiotic resistance between humans and dogs. given the increase prevalence of antibiotic-resistant bacteria and the risk of bacterial transmission between dogs and their owners, it is important to understand the overall level of antibiotic resistance in the dog population. with this knowledge, veterinarians can then make recommendations to protect the health of both their patients and clients. this meta-analysis aims to assess the overall prevalence of antibiotic-resistant bacterial strains in dogs in africa to develop and implement local and continental control programmes for drug-resistant bacterial strains and their spread. methodology literature search a systematic search was conducted according to the preferred reporting items for systematic reviews and meta-analysis (prisma) protocol items on the phenomenon of antibiotic resistance in companion animals in africa between january 2021 and march 2021. searches were conducted in four databases, medline (pubmed), scopus, cab abstract and google scholar by indexing several keywords in the title and abstract of the articles stored in these different databases. the search terms included ‘companion animals’ and ‘antimicrobial resistance’ and ‘africa’ or ‘dog’ and ‘antimicrobial resistance’ and ‘africa’ or ‘dog’ and ‘antibacterial resistance’ and ‘africa’ or ‘dog’ and ‘antibiotic resistance’ and ‘africa’ or ‘multidrug resistance’ and ‘africa’ or ‘dog’ and these term with each of 54 african countries. all studies (in english or french) on bacterial antimicrobial resistance (amr) in dogs published between january 2000 and january 2021 were included. data extraction after excluding irrelevant studies (non-african countries, antibiotic resistance in other animal species) and duplicates, the remaining studies were fully read in order to extract the data needed for this review: name of the first author, year of publication, country where the study was carried out, sources of samples, sample size, methods of analysis (disc diffusion method or molecular method), number of positive cases, number of bacterial strains tested and prevalence of antibiotic resistant strains. figure 1 presents the flow chart of the process of identifying studies. figure 1: workflow for selection of studies from various databases. statistical analyses data extracted from all selected publications were directly inserted into the excel spreadsheet and subjected to meta-analysis in r version 3.1 (r core team 2016) using the meta and metafor packages (schwarzer 2007; viechtbauer 2010). the mantel-haenszel fixed-effects model (fem) and the der simonian and laird random-effects model (rem) were used to generate an overall estimate of the prevalence of antibiotic resistance. heterogeneity between the different studies was assessed using the i2 statistic of higgins et al. (2003). i2 values of 25%, 50% and 75% were considered to have a low, moderate and high degree of heterogeneity, respectively. when heterogeneity between studies is low, the fixed effect model estimates were considered, whilst the random effect model was used to generate the overall prevalence in case of high heterogeneity. finally, to summarise the results of the meta-analysis, a forest plot showing the different prevalence estimates of multidrug resistance (mdr) bacteria across studies, a confidence interval and an overall prevalence was generated for each of the two models used. ethical considerations this article followed all ethical standards of research without direct contact with human or animal subjects. results characteristics of eligible studies the characteristics of the studies selected for the meta-analysis are presented in table 1. a total of 18 eligible studies were selected for the meta-analysis out of 234 indexed articles. the selected studies took place in countries such as nigeria (8 studies), south africa (3), kenya (2), tanzania (1), ethiopia (2), egypt (1) and tunisia (1) (figure 2). the sources of contamination used in these studies are mainly, in order of importance, rectal, nasal, oral, skin, vaginal, ear canal and wound swabs. the methods used by these authors to study antibiotic resistance are disk diffusion and molecular methods. figure 2: map of countries where antibiotic resistance is studied amongst dogs. table 1: the outline of selected antimicrobial resistance studies in dogs. prevalence of antibiotic-resistant bacterial strains isolated from dogs in africa many bacterial strains isolated from dogs and identified either biochemically or genotypically have been tested for antibiotic efficacy in eligible studies, however only seven strains with major public health impacts are included in this meta-analysis. these are staphylococcus aureus, escherichia coli, salmonella spp., p.aeruginosa, streptococcus pyogenes, coagulase-negative staphylococcus and staphylococcus pseudintermedius. prevalence of antibiotic-resistant staphylococcus aureus strains table 2 shows the overall prevalence of antibiotic-resistant s. aureus isolates from dogs in africa. analysis of this table reveals a higher prevalence (> 52%) of s. aureus isolates resistant to the following antibiotics: nalidixic acid, streptomycin, methicillin, cotrimoxazole, ampicillin, amoxicillin, kanamycin, tetracycline, penicillin g and pefloxacin. these isolates showed a low prevalence of resistance to the other antibiotics tested such as oxacillin, gentamycin, enrofloxacin, norfloxacin, erythromycin and a higher sensitivity to ofloxacin and ciprofloxacin. the prevalence of multidrug resistant strains was 18%. table 2: the overall prevalence of antibiotic resistant staphylococcus aureus strains isolated from dogs in africa. prevalence of antibiotic-resistant escherichia coli strains table 3 shows the overall prevalence of antibiotic resistant e.coli strains isolated from dogs in africa. an analysis of this table shows that the e. coli isolates tested were resistant to cefuroxime, cefotaxime, ceftazidime, ceftriaxone and clindamycin, all with 100% of prevalence. these strains were also resistant to kanamycin, ampicillin, amoxicillin, penicillin g, cefalotin, nalidixic acid, gentamicin, cotrimoxazole, tetracycline, doxycycline, chloramphenicol, nitrofurantoin and tylosin with more than 50% of prevalence. in addition, less than 50% of the strains tested were resistant to the other antibiotics evaluated in the selected studies. beta-lactams, macrolides and related agents, sulfonamides, cyclines, phenicols and nitrofurans are the families in which resistance of escherichia coli strains is most marked. in addition, 98% of isolates are multidrug resistant. table 3: the overall prevalence of antibiotic resistant escherichia coli strains isolated from dogs in africa. prevalence of antibiotic-resistant salmonella strains the overall prevalence of antibiotic resistant salmonella strains isolated from dogs in africa is summarised in table 4. an analysis of this table shows that a significant proportion of the salmonella strains tested were resistant to ampicillin, penicillin g, ceftazidime, cefotaxime, gentamicin, lincomycin, cotrimoxazole, oxytetracycline, streptomycin and vancomycin, with prevalences ranging from 53% to 100%. regarding sulfamethazine, chloramphenicol, tetracycline, cefalotin, doxycycline and amoxicillin and clavulanic acid, less than half of the strains tested were resistant. however, none of the salmonella strains tested were resistant to the quinolone antibiotics, particularly norfloxacin, ciprofloxacin and enrofloxacin. the prevalence of multidrug resistant salmonella strains is 53%. table 4: the overall prevalence of antibiotic resistant salmonella strains isolated from dogs in africa. prevalence of antibiotic-resistant pseudomonas aeruginosa strains table 5 shows the prevalence of antibiotic-resistant p. aeruginosa strains isolated from dogs in africa. from analysis of this table, all p. aeruginosa isolates tested are resistant to ampicillin, amoxicillin, cloxacillin, cephalexin, flucloxacillin, cefuroxime, gentamicin, neomycin, norfloxacin, nalidixic acid, erythromycin, cotrimoxazole and nitrofurantoin. prevalences of more than 50% in p. aeruginosa strains are resistant to amoxicillin and clavulanic acid, penicillin g, carbenicillin, piperacillin, ceftazidime, enrofloxacin, orbifloxacin, lincomycin, tetracycline, doxycycline, chloramphenicol and tylosin. also, there is a low prevalence of p. aeruginosa strains detected to be resistant to imipenem, tobramycin and ciprofloxacin. however, these strains are sensitive to ofloxacin. resistance of p. aeruginosa is marked in all families of antibiotics tested and the prevalence of multidrug resistant strains is 92%. table 5: prevalence of antibiotic-resistant pseudomonas aeruginosa strains isolated from dogs in africa. prevalence of antibiotic-resistant streptococcus pyogenes strains table 6 shows the overall prevalence of antibiotic resistant s. pyogenes strains isolated from dogs in africa. streptococcus. pyogenes isolates are resistant to ampicillin, amoxicillin, penicillin g, flucloxacillin, cefuroxime, streptomycin, neomycin, nalidixic acid, erythromycin, tetracycline, chloramphenicol and nitrofurantoin with an overall prevalence of 100.00%. a high prevalence of norfloxacin-resistant s. pyogenes (87.50%) is also recorded. but these isolates are susceptible to cloxacillin, cephalothin, ciprofloxacin, enrofloxacin and ofloxacin. in addition, resistance in s. pyogenes affected all families of antibiotics tested. table 6: the overall prevalence of antibiotic resistant streptococcus pyogenes strains isolated from dogs in africa. prevalence of antibiotic resistant coagulase negative staphylococcus strains the overall prevalence of antibiotic resistant coagulase negative staphylococcus (scn) strains isolated from dogs in africa is shown in table 7. analysis of this table shows that all isolates tested are resistant to ampicillin, penicillin g, cotrimoxazole and tetracycline. furthermore, high prevalences of resistant scn were also recorded for amoxicillin, amoxicillin and clavulanic acid, cefuroxime, nalidixic acid and chloramphenicol. for methicillin, streptomycin, ceftriaxone, gentamicin and enrofloxacin, less than half of the isolates tested were found to be resistant. in addition, isolates were found to be susceptible to ciprofloxacin, norfloxacin and pefloxacin. table 7: the overall prevalence of antibiotic resistant coagulase negative staphylococcus strains isolated from dogs in africa. prevalence of antibiotic resistant staphylococcus pseudintermedius table 8 shows the overall prevalence of antibiotic-resistant s. pseudintermedius strains isolated from dogs in africa. analysis of this table shows that high prevalences of antibiotic resistant s. pseudintermedius isolates are found only in ampicillin (66.07%), penicillin g (53.19%) and clindamycin (51.79%). on the other hand, these strains are mainly susceptible to tobramycin, teicoplanin, vancomycin and mupirocin. however, a low prevalence of resistant isolates was observed for the other antibiotics tested, with percentages ranging from 1.82% to 29.09%. the prevalence (25.00%) of multidrug resistance was low. table 8: the overall prevalence of antibiotic resistant staphylococcus pseudintermedius strains isolated from dogs in africa. resistance to antibiotics common to the seven strains figure 3 summarises the prevalence of the seven bacterial strains according to the antibiotics they have in common. the figure shows that all strains were resistant to ampicillin and penicillin g. with the exception of p. aeruginosa, which is resistant to enrofloxacin (74.37%), all strains were sensitive to ciprofloxacin and enrofloxacin. only salmonella (80.00%) and p. aeruginosa (60.00%) were resistant to gentamicin. on the other hand, p. aeruginosa (93.83%) and staphylococcus negative coagulase (83.33%) were resistant to amoxicillin and clavulanic acid, whilst for streptomycin and cotrimoxazole, staphylococcus negative coagulase and s. pseudintermedius isolates were found to be weakly resistant. escherichia coli isolates were moderately resistant to streptomycin (40.93%) and s. pyogenes isolates were weakly resistant to cotrimoxazole (12.5%). all strains were resistant to tetracycline, except salmonella (30.19%) and s. pseudintermedius (29.09%). with the exception of s. aureus (25.00%), salmonella (20.00%) and s. pseudintermedius (7.16%), which were sensitive to chloramphenicol, the other strains were resistant to this antibiotic. figure 3: antibiotic resistance of strains from sampled animals. prevalence of multidrug resistant strains in dogs the prevalences of multidrug resistant strains isolated from dogs in africa are presented in figure 4. from the analysis of this table it is obvious that the e. coli strain has the highest prevalence of multidrug resistance followed by streptococcus cn first then p. aeruginosa then s. pyogenes. in contrast, the prevalence is low for s. aureus and s. pseudintermedius (appendix 1). figure 4: prevalence of multidrug resistant strains in dogs in africa. discussion antibiotic resistance is a growing phenomenon in african countries and in companion animals, notably dogs. many antibiotics used in veterinary medicine are similar to those used in human medicine. the present systematic review assessed the overall prevalence of antibiotic-resistant bacterial strains isolated from dogs in africa and found that many strains were multidrug resistant to most common and current used antibiotics (ampicillin, ampicillin/cloxacillin, tetracycline, penicillin, amoxicillin, cotrimoxazole, chloramphenicol and ciprofloxacin) used in humans (okpara et al. 2018). the high resistance of s. aureus strains to ampicillin found in dogs was also observed in humans (100.0%) and pigs (100.0%) in tanzania by katakweba et al. (2016). but for tetracycline, resistance is moderate in humans (45.5%) and pigs (50.0%) in tanzania, whilst it is high in dogs. in the democratic republic of congo, resistance prevalences of 33.00% – 72.00% against tetracycline, 5.00% – 54.00% against cotrimoxazole, 31.00% against gentamicin, 26.00% – 69.00% against erythromycin and 20.00% – 59.00% against ciprofloxacin have been reported in humans (lupande-mwenebitu et al. 2020). our study showed contrasting findings: whilst the results are similar to those obtained in europe by moyaert et al. (2019), the prevalence of penicillin g-resistant isolates in the present analysis (56.23%) is lower than that obtained in europe (65.20%). furthermore, strains from the seven studied african countries were resistant to cotrimoxazole (73.77%) and tetracycline (60.09%), whilst those isolated from european countries were found to be sensitive to the same antibiotics, with low resistance prevalences equal to 0.00% and 13.00%, respectively (moyaert et al. 2019). staphylococcus aureus strains were resistant to nalidixic acid, streptomycin, methicillin, cotrimoxazole, ampicillin, amoxicillin, kanamycin, tetracycline, penicillin g and pefloxacin. with regard to multidrug resistance, the prevalence in dogs in the present study (18.0%) is lower than that recorded in humans in nigeria (68.0%) (ogundipe et al. 2020) and in cats (28.6%) in south africa (qekwana et al. 2017). in this study, e. coli strains were resistant to 18 antibiotics. these were cefuroxime, cefotaxime, ceftazidime, ceftriaxone, clindamycin, kanamycin, ampicillin, amoxicillin, penicillin g, cefalotin, nalidixic acid, gentamicin, cotrimoxazole, tetracycline, doxycycline, chloramphenicol, nitrofurantoin and tylosin. these results differ from those obtained in europe by moyaert et al. (2019), where e. coli strains tested were susceptible to ampicillin, cotrimoxazole and tetracycline, marked by low prevalences of resistance of 36.40%, 21.20% and 18.2%, respectively. however, the sensitivity of e. coli strains to orbifloxacin observed in this study is also reported in the study conducted in europe by ludwig et al. (2016) where the prevalence of resistant isolates was low, 3.7%. furthermore, in the meta-analysis study conducted by emami, javanmardi and pirbonyeh (2020), 73.00% of e. coli strains isolated from pregnant women in africa were resistant to ampicillin and 52.00% of e. coli strains from asia. these prevalences are lower than the prevalence in dogs in the present study (86.49%). compared with gentamicin (30.54%), amoxicillin and clavulanic acid (45.11%) and tetracycline (70.27%), the prevalences of resistant e. coli in dogs are higher than those in humans in africa, which are 23.00%, 44.00% and 60.00%, respectively, but the prevalence of ciprofloxacin resistant e. coli in dogs (6.05%) is very low compared with that in humans (26.00%). regarding antibiotic resistance of salmonella strains, the results are not in agreement with those obtained in europe by bataller et al. (2020) who reported that salmonella serovars are susceptible to ceftazidime, cefotaxime, gentamicin and cotrimoxazole. nevertheless, the results obtained for the sensitivity of salmonella strains to ciprofloxacin are similar to those reported by the same authors. for p. aeruginosa strains, the results observed differ from those obtained in the studies conducted in europe by ludwig et al. (2016), where the prevalence of p. aeruginosa strains resistant to gentamicin and enrofloxacin are low, 18.80% and 18.20%, respectively. regarding coagulase negative staphylococcus strains, the results recorded in this study are contrary to those reported by wedley et al. (2014) who observed the sensitivity of united kingdom (uk) coagulase negative staphylococcus isolates to cotrimoxazole and tetracycline, with resistance prevalences of 22.50% and 27.50%, respectively. in contrast, methicillin, which is effective on african isolates with a low prevalence of resistance (33.33%), was found to be ineffective on uk strains, with a resistance prevalence equal to 100.00% (wedley et al. 2014). as for s. pseudintermedius, the result observed for penicillin g activity on isolates in this study is different from that obtained in europe by moyaert et al. (2019) where tested s. pseudintermedius isolates were susceptible to this antibiotic, with a resistance prevalence equal to 20.0%. however, this result is similar to the one obtained in japan by bardiau et al. (2013), but the prevalence recorded in our study for this antibiotic (53.19%) is lower than that observed in japan (> 95.0%). compared with tetracycline and enrofloxacin, the susceptibility of s. pseudintermedius strains observed in this study is also found in european isolates by moyaert et al. (2019). for clindamycin, the prevalence obtained (51.79%) is lower than that obtained in work carried out in european and north american regions by perreten et al. (2010), where the prevalence of resistant strains is equal to 89.3%. on the other hand, the results for chloramphenicol (7.16%), ciprofloxacin (1.82%), enrofloxacin (8.93%), gentamicin (4.50%), kanamycin (5.85%), streptomycin (1.82%) and tetracycline (29.09%) are contrary to those reported by perreten et al. (2010) who reported resistance of s. pseudintermedius isolates to these antibiotics, with prevalences of 57.30%, 87.40%, 84.50%, 69.90%, 93.20%, 90.30% and 69.90%, respectively. the sensitivity of s. pseudintermedius strains from africa to vancomycin (100%) confirms the results of perreten et al. in japan, s. pseudintermedius strains were found to be resistant to ciprofloxacin, streptomycin, gentamicin, kanamycin, cotrimoxazole, chloramphenicol and tetracycline, with prevalences ranging from about 70.00% to 100.00% (bardiau et al. 2013); this differs from the results obtained in the present study. there are several reasons for the differences in these results: the level of knowledge about antimicrobials and their use, the attitudes of owners towards dog healthcare, the spatial epidemiology of amr, the level of enforcement of existing legislation on antimicrobials in veterinary medicine, and the conditions of access to veterinary services in given regions. in a study conducted in nigeria by okpara et al. (2018), a low level of knowledge on antimicrobials and their use was observed amongst the respondents; 64.4% of the respondents administered antimicrobials to their animals themselves and 60.6% used antimicrobials without veterinary prescriptions. also, more than half of the respondents (51.5%) had never used veterinary services for their animals whilst 23.1% rarely used them. from the same study, 71.3% of the respondents mentioned that they did not have veterinary practices in their communities. this can lead to problems such as misdirection of antimicrobials, overor under-dosing, and poor storage and handling of antimicrobial agents. regarding the spatial epidemiology of amr, the study conducted in south africa by qekwana et al. (2019) revealed the similarity of prescribing practices amongst veterinarians in the study areas. given that the realities of african countries differ from those of other continents in several respects, the differences in prevalences may, therefore, be related to differences in prescribing practices. the lack of surveillance programmes on antimicrobial use, as well as the lack of enforcement of existing legislation with appropriate sanctions for violators in african countries may account for the differences in results. such high prevalences of multidrug resistant strains amongst the investigated microbial strains constitute a real health problem that could lead to therapeutic failures in these animals and humans, especially their owners. indeed, the sharing of the resistance genes of these strains between these animals and their owners could be a source of treatment failure of these targets in case of diseases (feiyang et al. 2021). educating dog owners about the impact of antibiotic resistance on animals and nearby humans is essential. in addition, appropriate integrated programmes to control amr in africa, especially for companion animals, should be implemented. conclusion antibiotic resistance is a serious problem worldwide. this phenomenon is increasing in africa, in pets and particularly in dogs, which are relatively closer companions to humans. all strains investigated are resistant to ampicillin and penicillin g, but are sensitive to ciprofloxacin. in relation to the other antibiotics, the level of resistance varies according to the strain. multidrug resistance is much higher in e. coli, p. aeruginosa and salmonella strains compared with s. aureus and s. pseudintermedius strains. the high prevalence of resistance observed for strains of e. coli, salmonella, p. aeruginosa, s. pyogenes, coagulase-negative staphylococcus, s. aureus and s. pseudintermedius, which are zoonotic strains, should not be overlooked. as more than one of these bacteria can be found in the same individual and the antibiotics used in animals are practically used in humans for the treatment of infections, it is important to use these antibiotics rationally, so that control of one bacterial strain does not lead to resistance in the other. in view of the multidrug resistance observed in each of these strains, it is important and immediate need to implement measures to prevent the exchange of pathogens between dogs and their owners and to apply good hygienic practices and promote alternatives such as appropriate medicinal plants to combat these different strains in africa. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors were responsible for project design. this study was conceptualised and protocols were carried out by p.s., a.m., r.p. and a.b.y. a.b.y., p.s., g.y.m.h. and a.b.n.t. performed database search, data extraction, statistical analysis, and wrote the manuscript. a.b.y., d.t., p.s., r.p. and s.f. performed data curation and quality assessment of each study was completed independently by these authors. all authors provided critical feedback and helped shape the research, analysis and manuscript. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references abdel-moein, k.a., el-hariri, m. & samir, a., 2012, ‘methicillin-resistant staphylococcus aureus: an emerging pathogen of pets in egypt with a public health burden’, transboundary and emerging diseases 59(4), 331–335. 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introduction methods results discussion acknowledgements references about the author(s) edward kariuki department of veterinary services, kenya wildlife service, nairobi, kenya school of biomedical sciences, jomo kenyatta university of agriculture and technology, nairobi, kenya hellen kutima department of zoology, jomo kenyatta university of agriculture and technology, nairobi, kenya michael kock international wildlife veterinary services, greyton, south africa ivan g. horak department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa roaland jooste animal health division, bayer (pty) ltd, isando, johannesburg, south africa luis neves department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation kariuki, e., kutima, h., kock, m., horak, i.g., jooste, r. & neves, l., 2019, ‘ixodid ticks (acari: ixodidae) collected from african savanna elephants (loxodonta africana) and african forest elephants (loxodonta cyclotis)’, onderstepoort journal of veterinary research 86(1), a1781. https://doi.org/10.4102/ojvr.v86i1.1781 research communication ixodid ticks (acari: ixodidae) collected from african savanna elephants (loxodonta africana) and african forest elephants (loxodonta cyclotis) edward kariuki, hellen kutima, michael kock, ivan g. horak, roaland jooste, luis neves received: 18 apr. 2019; accepted: 15 july 2019; published: 06 nov. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract eight ixodid tick species were collected from 173 african savanna elephants (loxodonta africana) in kenya, northern mozambique and zimbabwe, and two species were collected from six african forest elephants (loxodonta cyclotis) in the republic of congo. a new host record is reported for amblyomma eburneum. a list of ticks collected from elephants in various african countries, and stored in the united states national tick collection, is supplied as well as an annotated checklist of the 27 ixodid tick species that have been collected from african elephants. the geographic distributions and alternative hosts of the various tick species collected from elephants are briefly discussed. keywords: african savanna elephants; african forest elephants; ixodid ticks; republic of congo; kenya. introduction despite the african savanna elephants’ iconic status, no surveys dedicated to the ticks of elephants as such have been conducted in africa. hoogstraal (1956) recorded collections of amblyomma tholloni from four elephants in the equatoria province, sudan, and theiler (1962) reported collections of eight amblyomma spp. (two are doubtful records) from elephants in sub-saharan africa. among these, a. tholloni was present in 18 countries from sierra leone and sudan in the north to south africa in the south. theiler also reported collections of dermacentor circumguttatus from elephants in 10 countries in west, central and east africa, from sierra leone in the west to tanzania in the east and northern mozambique in the south. furthermore, she recorded the collection of 10 rhipicephalus spp. from elephants, among which were three of the four ornate rhipicephalus spp.: rhipicephalus humeralis, rhipicephalus maculatus and rhipicephalus pulchellus. unfortunately, theiler did not record the number of elephants infested with each species. yeoman and walker (1967) reported collections from 19 elephants in tanzania along with collections made from numerous other animals, including domestic livestock. the elephants were infested with four tick species and 16 animals were infested with a. tholloni, three with d. circumguttatus and four with rhipicephalus maculatus. in kenya, walker (1974) recorded eight tick species in collections made from 78 elephants. forty-four animals were infested with a. tholloni, 28 with r. humeralis, 12 with r. maculatus and nine with rhipicephalus praetextatus (then referred to as rhipicephalus simus). in addition to a. tholloni and r. maculatus, baker and keep (1970) included rhipicephalus appendiculatus, rhipicephalus muehlensi and r. simus in a checklist of the ticks found on elephants and other large wildlife species in the north-eastern reserves of kwazulu-natal, south africa. amblyomma tholloni was the only species in collections made from 29 elephants in zimbabwe (norval 1983). including the above-mentioned rhipicephalus species, walker, keirans and horak (2000) list 15 rhipicephalus species that have been collected from elephants, among which is the fourth ornate rhipicephalus species, r. dux. in a review of the ticks of the central african republic, uilenberg, estrada-peña and thal (2013) recorded a number of historic as well as recent collections of amblyomma astrion, amblyomma cohaerens, a. tholloni, amblyomma variegatum, d. circumguttatus and rhipicephalus longus from elephants. in namibia, 8 of 45 elephants examined in the etosha national park harboured only hyalomma truncatum, while no ticks were collected from the remaining elephants (turner et al. 2017). in south africa, horak et al. (2018) recorded 32 collections from elephants, of which one animal was infested with a solitary amblyomma hebraeum female, 23 with a. tholloni and eight with r. maculatus. here, we report opportunistic collections made since 1988 from african savanna elephants in kenya, northern mozambique and zimbabwe and from african forest elephants in the republic of congo. we also include a list of tick species collected from elephants and deposited in the united states national tick collection (usntc) as well as an annotated checklist of the tick species collected to date from african elephants. methods the management or translocation of elephants usually necessitates that they are restrained by sedation. between 1988 and 2019, ticks were collected opportunistically from 173 sedated african savanna elephants and six african forest elephants. the animals were examined visually and ticks removed both manually and by using forceps. the ticks from each elephant were placed in individually labelled vials containing 70% ethanol and transported to the laboratory, where they were identified under a stereoscopic microscope using conventional keys and descriptions. no total collections of ticks were made from any of the elephants because of the size of the animal, the shortage of workforce, difficult operating conditions and the usually short period between sedation and resuscitation. the countries of origin and the species and number of ticks collected have been summarised in tabular format. ethical considerations the kenyatta university ethics review committee (ku-erc) reviewed and approved the study protocol. the national commission for science, technology and innovation (nacosti) and the kenya wildlife service (kws) authorised the field study in kenya. results the number of elephants examined in kenya, mozambique, zimbabwe and the democratic republic of congo, and the species and number of ticks collected are summarised in table 1. table 1: species and number of ticks collected from savanna and forest elephants. a total of 128 elephants were examined at 61 localities across kenya and 1964 ticks were collected (table 1). the adults of eight tick species were identified in the collections. two species belonged to the genus amblyomma and six to the genus rhipicephalus. amblyomma tholloni was the most prevalent species and was collected from 88.3% of the elephants. amblyomma eburneum is the first record of this species for african elephants. three ornate species of rhipicephalus are present in kenya: r. humeralis, r. maculatus and r. pulchellus (walker et al. 2000), and all were collected from the elephants. ticks were also collected from a sick elephant in the shimba hills national reserve, kenya. it was infested with large numbers of ticks, and more than 3000 r. maculatus, as well as some a. tholloni, were collected from the posterior surface of a single ear. amblyomma tholloni was collected from all 36 elephants sampled in the reserva nacional do niassa, niassa province, mozambique, and r. maculatus was present in eight of these animals (table 1). the nine animals sampled in zimbabwe were infested only with a. tholloni. six forest elephants examined in the odzala-kokoua national park, republic of congo, were all infested with a. tholloni, while d. circumguttatus was collected from five animals (table 1). the species present in historic collections of ticks from african elephants between 1905 and 1980 and deposited in the usntc are listed in table 2. table 2: ticks collected from african elephants (1905–1980) and deposited in the united states national tick collection. ticks from elephants in 14 african countries were present in the usntc. amblyomma tholloni was collected from elephants in 10 countries, d. circumguttatus in five, r. praetextatus (as r. simus) in three and r. maculatus in two. rhipicephalus humeralis was collected from two elephants in kenya, amblyomma cohaerens from a single animal in uganda and r. pravus from a single animal in somalia. an annotated checklist of tick species recorded on african elephants is presented in table 3. table 3: annotated checklist of the tick species collected from african elephants. a total of 27 ixodid tick species have been collected from african elephants, and eight amblyomma species, 16 rhipicephalus species as well as d. circumguttatus, d. rhinocerinus and hyalomma truncatum have been reported. discussion the discussion will focus on the tick species collected in the current survey. amblyomma species amblyomma eburneum is a relatively rare species. yeoman and walker (1967) recorded it in the eastern province, tanzania, while according to walker (1974), ‘the few records of a. eburneum in kenya are widely scattered’, where it has been found mainly in the centre and south of the country. here, we report the first record on the african elephant. collections have, however, been made from buffaloes in tanzania (yeoman & walker 1967), cattle, black rhinoceroses, buffaloes, an oryx and a warthog in kenya (walker 1974), and buffaloes in mozambique (dias 1993). amblyomma tholloni was present in the majority of collections made from elephants in kenya and tanzania as well as in all the collections made from these animals in zimbabwe (norval 1983; walker 1974; yeoman & walker 1967). the collection of a. tholloni from elephants in kenya, the republic of congo, northern mozambique and zimbabwe in the present study confirms its widespread geographical distribution on african elephants. in zimbabwe, however, norval (1983) recorded a. tholloni on 22 of 24 hippopotamuses from which ticks had been collected. one of the two hippopotamuses we examined in kenya was infested with a. tholloni. ticks were collected from the inside of the lips, the nostrils, an ear and the skinfolds of the neck of this hippopotamus. mackenzie and norval (1980) demonstrated that a. tholloni could transmit ehrlichia ruminatium, the causative organism of heartwater in cattle, sheep and goats in the laboratory. they also noted that cattle, sheep and goats at a research station within a nature reserve in the zambezi valley were often infested with the immature stages of a. tholloni and that cases of heartwater occurred in these animals in the absence of any of the other known tick vectors. dermacentor circumguttatus hoogstraal (1956) regarded d. circumguttatus as an elephant tick and reported that it was present on these animals in west and central africa, extending in the east only to the western regions of uganda and sudan. however, yeoman and walker (1967) reported it in elephants in the far north-western corner of tanzania where it borders uganda. dias (1993) reported 10 males and two females of a subspecies that he referred to as d. circumguttatus cunhasilvai from elephants in the mossurize region of manica province, mozambique. dermacentor circumguttatus has not been collected from this locality since then, nor were any of the elephants examined in the present study in the reserva nacional do niassa, mozambique, infested with it. rhipicephalus species we believe that the occurrence of r. appendiculatus on elephants represents an unusual infestation. rhipicephalus compositus, of which a single collection was made, has been collected from thick-skinned animals, rhinoceroses, warthogs, bush pigs and buffaloes. while a large number of collections have also been made from cattle, the occurrence of this species on an elephant should be regarded as rare (walker et al. 2000). rhipicephalus humeralis is an ornate rhipicephalus species and is only present in mainland tanzania, kenya and somalia (walker et al. 2000). in kenya, it occurs in the coastal region and bushed and wooded grassland in the south and central regions. walker (1974) recorded 16 collections from cattle, 28 from elephants and nine from black rhinoceroses in kenya, with one or two collections from other domestic and wild animals. rhipicephalus maculatus is present in coastal woodland and bush and adjacent inland from north-eastern kwazulu-natal to southern somalia (walker et al. 2000). in addition to the coastal regions of kenya, tanzania and mozambique, it is also present in bushed and wooded grassland in south and central kenya as well as in woodland and bush adjacent to the coastal regions of mozambique and tanzania (walker et al. 2000). it is a large ornate species with stout mouthparts and has been collected from hosts with thick hides: elephants, white and black rhinoceroses, bush pigs and buffaloes (baker & keep 1970; horak et al. 1983, 2017; horak, boomker & flamand 1991; kariuki et al. 2012). rhipicephalus praetextatus is present in central and southern ethiopia and northern somalia (walker et al. 2000). ticks originally identified as r. simus and now presumed to be r. praetextatus have been recorded in southern somalia, and practically throughout kenya and tanzania (walker et al. 2000). walker et al. (2000) reported 12 collections of r. praetextatus (species unconfirmed) from elephants in east africa, and it has subsequently been collected from elephants in the ngorongoro crater, tanzania (fyumagwa et al. 2007). in the current study, r. praetextatus was collected from 11 elephants. rhipicephalus pulchellus is widespread in eritrea, eastern ethiopia, somalia, east of the great rift valley in kenya and north-eastern tanzania (walker et al. 2000). kariuki et al. (2012) collected r. pulchellus from cattle and sympatric buffaloes in south-eastern kenya. this ornate tick species infests a multitude of domestic and wild animals among which walker et al. (2000) reported three african elephants. seven elephants in our study were infested with r. pulchellus. although rhipicephalus dux was not collected in the present survey, walker et al. (2000) have recorded its presence on elephants. it is found in the democratic republic of congo, particularly in the north-east, in rwanda and in north-western uganda (matthysse & colbo 1987). thus, the four ornate rhipicephalus spp. have all been collected from elephants. acknowledgements the authors thank the kenya wildlife service and the national research fund, kenya, for providing resources used for the field work. a grant from the national research foundation (nrf) to i.g. horak made it possible to sponsor the visits of e.k. kariuki to south africa. edward kariuki thanks friends and veterinary students for assistance with collecting ticks. competing interests the authors declare that they have no financial or personal interests that may have inappropriately influenced them in writing this article. authors’ contributions all authors read and approved the manuscript. the first draft of the manuscript was prepared by e.k., i.g.h. and l.n. e.k., h.k., m.k. r.j. collected the ticks and e.k., h.k., l.n. and i.g.h. identified them. funding information this study was financially supported by the kenya wildlife service and the national research foundation (nrf) of south africa grant to i.g. horak. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer any opinion, finding and conclusion or recommendation expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references aeschlimann, a., 1967, ‘biologie et écologie des tiques (ixodoidea) de côte d’ivoire’, acta tropica 24, 281–405. baker, m.k. & keep, m.e., 1970, ‘checklist of the ticks found on the larger game animals in the natal game reserves’, lammergeyer 12, 41–47. dias, j.a.t.s., 1993, ‘some data concerning the ticks (acarina-ixodoidea) presently known in mozambique’, garcia de orta, séries zoologica, lisboa 18(1/2), 27–48. fyumagwa, r.d., runyoro, v., horak, i.g. & hoare, r., 2007, ‘ecology and control of ticks as disease vectors in wildlife of the ngorongoro crater, tanzania’, south african journal of wildlife research 37(1), 79–90. https://doi.org/10.3957/0379-4369-37.1.79 hoogstraal, h., 1956, african ixodoidea. i. ticks of the sudan (with special reference to equatoria province and with preliminary reviews of the genera boophilus, margaropus and hyalomma), p. 1101, research report nm 005.050.29.07, department of the navy bureau of medicine and surgery, washington, dc. horak, i.g., boomker, j. & flamand, j.r.b., 1991, ‘ixodid ticks and lice infesting red duikers and bushpigs in north-eastern natal’, onderstepoort journal of veterinary research 58(1), 281–284. horak, i.g., boshoff, c.r., cooper, d.v., foggin, c.m., govender, d., harrison, a. et al., 2017, ‘parasites of domestic and wild animals in south africa. xlix. ticks (acari: ixodidae) infesting white and black rhinoceroses in southern africa’, onderstepoort journal of veterinary research 84(1), e1–e11. https://doi.org/10.4102/ojvr.v84i1.1301 horak, i.g., heyne, h., williams, r., gallivan, g.j., spickett, a.m., bezuidenhout, j.d. & estrada-peña, a., 2018, the ticks (acari: ixodidae) of southern africa, springer international publishing ag, basel, switzerland. horak, i.g., potgieter, f.t., walker, j.b., de vos, v. & boomker, j., 1983, ‘the ixodid tick burdens of various large ruminant species in south african nature reserves’, onderstepoort journal of veterinary research 50, 221–228. kariuki, e.k., penzhorn b.l., horak i.g., 2012, ‘ticks (acari: ixodidae) infesting cattle and african buffaloes in the tsavo conservation area, kenya’, onderstepoort journal of veterinary research 79(1), 437–440 https://doi.org/10.4102/ojvr.v79i1.437 mackenzie, p.k.i. & norval, r.a.i., 1980, ‘the transmission of cowdria ruminantium by amblyomma tholloni’, veterinary parasitology 7(3), 265–268. https://doi.org/10.1016/0304-4017(80)90030-8 matthysse, j.g. & colbo, m.h., 1987, the ixodid ticks of uganda, p. 426, entomological society of america, college park, md. norval, r.a.i., 1983, ‘the ticks of zimbabwe. vii. the genus amblyomma’, zimbabwe veterinary journal 14, 3–18. theiler, g., 1962, the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region), p. 260, project s 9958, report to the director of veterinary services, onderstepoort (mimeographed). turner, w.c., küsters, m., versfeld, w. & horak, i.g., 2017, ‘ixodid tick diversity on wild mammals, birds and reptiles in and around etosha national park, namibia’, african journal of ecology 55(4), 714–721. https://doi.org/10.1111/aje.12369 uilenberg, g., estrada-peña, a. & thal, j., 2013, ‘ticks of the central african republic’, experimental and applied acarology 60(1), 1–40. https://doi.org/10.1007/s10493-012-9605-2 walker, j.b., 1974, the ixodid ticks of kenya: a review of present knowledge of their hosts and distribution, bcommonwealth institute of entomology, london. walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. yeoman, g.h. & walker, j.b., 1967, the ixodid ticks of tanzania. a study of the zoogeography of the ixodidae of an east african country, commonwealth institute of entomology, london. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) stephanie köhnk olive ridley project, bramhall, stockport, cheshire, united kingdom morphology lab, center of natural history, university of hamburg, hamburg, germany rosie brown olive ridley project, bramhall, stockport, cheshire, united kingdom marine turtle rescue centre, coco palm dhuni kolhu, maldives amelia liddell olive ridley project, bramhall, stockport, cheshire, united kingdom marine turtle rescue centre, coco palm dhuni kolhu, maldives citation köhnk, s., brown, r. & liddell, a., 2021, ‘finding of a two-headed green turtle embryo during nest monitoring in baa atoll, maldives’, onderstepoort journal of veterinary research 88(1), a1940. https://doi.org/10.4102/ojvr.v88i1.1940 original research finding of a two-headed green turtle embryo during nest monitoring in baa atoll, maldives stephanie köhnk, rosie brown, amelia liddell received: 23 feb. 2021; accepted: 25 jun. 2021; published: 24 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract green sea turtles are one of the two species of marine turtles known to nest in the maldives. the prevalent time of nesting seems to be inconsistent throughout the island nation. in this study, sea turtle nesting activity was monitored on the island of coco palm dhuni kolhu in baa atoll over a period of 12 months. a total of 13 nests were confirmed with a median hatching success rate of 89.58% as ascertained by nest excavation. in one of the nests, a severely deformed hatchling with polycephaly, an opening in the neck area and a lordotic spine was found, and we investigated in detail with radiographic images and a necropsy. our findings support the importance of consistent nesting activity and nest monitoring efforts in the country as a basis for conservation efforts. keywords: case report; chelonia mydas; congenital malformation; polycephaly; sea turtle; nesting; indian ocean. introduction congenital malformations in wildlife are only reported sporadically, but they have been found in fishes (dethelfsen, von westerhagen & cameron 1996; hevia-homazábal, pasten-marambio & vega 2011), amphibians (blaustein & johnson 2003; henle et al. 2012) and reptiles (frye 1991; sant’anna et al. 2013), birds (ohlendorf et al. 1986; pourlis 2011) and mammals (rojas-lleonart, silveira-prade & sotero-delgado 2011; stills & bullock 1981). in sea turtles, various case reports from different populations all over the world are known (see, e.g., bárcenas-ibarra et al. 2015; caldwell 1959; carswell & lewis 2002; craven et al. 2019; dodd 1988; drennen 1990; eckert et al. 2012; ehrhart & witherington 1987; fowler 1979; gularte 2000; hughes, bass & mentis 1967; kaska & downie 1999; reichart 1993; rhodin, pritchard & mittermeier 1984). malformations can range in severity from benign scute abnormalities to detrimental conditions such as ectromelia or deformities of the spine (bárcenas-ibarra et al. 2015; dodd 1988; miller 1982; rhodin et al. 1984). early reports of developmental abnormalities date back to the 19th century, when agassiz (1857) described cases of scute irregularities. this is the most common abnormality observed in sea turtles (see dodd 1988 for a summary), which is not detrimental on its own because adult turtles with abnormal scute patterns are spotted regularly (bentley et al. 2021; brongersma 1968; kobayashi et al. 2017a; türkozan, illgaz & sak 2001). more serious abnormalities can include twinning, deformities of the eyes, the jaw or the entire body and carapace, a reduction or absence of limbs, dior polycephaly as well as leucism and albinism (bárcenas-ibarra et al. 2015; caldwell 1959; carswell & lewis 2002; dodd 1988; drennen 1990; eckert et al. 2012; ehrhart & witherington 1987; fowler 1979; gularte 2000; hughes et al. 1967; ingle et al. 2021; kaska & downie 1999; reichart 1993; rhodin et al. 1984), which have been documented in loggerheads, hawksbills, green turtles, leatherbacks and olive ridleys. the prevalence of congenital malformations can differ between sea turtle populations but overview studies are currently still rare (see e.g. bárcenas-ibarra et al. 2015; calderón peña & azanza ricardo 2021; craven et al. 2019; fowler 1979; kaska, downie & furness 2000; peters, verhoeven & strijbosch 1994). previous case reports from the indian ocean include a study from diamond (1976) on hawksbill turtles in the seychelles in which the author found two cases of deformed hatchlings. these showed a transverse medial fold across the body and, though viable at the time of hatching, did not survive past 5 and 9 weeks, respectively. other studies described instances of twinning in sri lanka (deraniyagala 1930, 1932; hewavisenthi 1989), thailand (junchompoo, penpian & tarkoolrangsi 2013), malaysia (chan 1985) and mozambique (louro & pereira 2009), as well as supernumerary suprapygal bones in loggerheads in sri lanka (deraniyagala 1939) and kyphosis in green turtles in indonesia (rhodin et al. 1984). in the maldives, an island nation in the northern indian ocean, two species of sea turtles are known to nest regularly: green and hawksbill turtles (ahmed et al. 2020; ali & shimal 2016; hudgins et al. 2017). nesting is spread over the entire length of the country and occurs all year round with hatching success rates between 33.7% and 96.8% (hudgins et al. 2017). no general information or overview study on unhatched eggs, embryo or hatchling malformations is available for the maldives at the time of this publication. herein, we report on sea turtle nesting activity in 2020 in coco palm dhuni kolhu (cpdk), baa atoll, maldives, including the case of an embryo with congenital malformations. materials and methods sea turtle nesting activity was recorded on cpdk, baa atoll, maldives throughout the year (in agreement with the guidelines of the environmental protection agency of the maldives, research permits epa/2018/psr-t03 and epa/2020/psr-t06). nesting attempts and true nests were recorded, and nests were monitored throughout the incubation period until hatching occurred. nests were excavated, and the nests’ contents were analysed at least 2 days after the emergence of the first hatchling, to allow for natural hatching of any stragglers. variation in excavation times resulted from coronavirus disease 2019 (covid-19)related safety measures and organisational restrictions. any unhatched eggs were opened by hand, pinching the eggshell between two fingers and pulling apart to reveal the egg contents, which were then classified according to the criteria specified in table 1 and reburied at the site of the nest. table 1: categorisation criteria for unhatched eggs examined during nest excavations. the single malformed embryo was found in one of the nests and was further studied to determine the extent of the malformation as best as possible. the embryo was intact within its egg, in a normal foetal position (back flippers curled in a cranial direction, following the curve of the egg; front flippers curled in a medial direction, coming to rest in contact with and cranial to the back flippers). it had reached a more advanced stage of development (stage 4) but had not survived to the point of pipping. radiographic images were obtained with a veterinary x-rays high frequency diagnostic unit machine in four standard views; dorsoventral, lateral (right and left), craniocaudal and ventrodorsal, followed by a standard necropsy (work 2000) to characterise any duplication of internal organs and the points of fusion. no further tissue samples were taken for histological or genetic evaluation. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results nesting activity in 2020, we recorded 13 false crawls (nesting attempts) and 13 confirmed nests on the island cpdk between april and october. all false crawls, failed nest attempts and nests were identified as green turtle (chelonia mydas) activities with confirmed nests restricted to june to september (table 2). table 2: recorded false crawl and confirmed nesting events on coco palm dhuni kolhu in 2020. egg counts ranged between 30 and 142 eggs in confirmed nests (median = 113; n = 12) and hatching success rate was 40.14% – 93.62% (median success rate of 89.58%; n = 12, s.d. = 16.57%) after an average of 59 days of incubation. the unhatched eggs in the 12 excavated nests were mostly completely undeveloped, showing no signs of embryo development (for details, also see table 2). in a nest laid on 10 august 2020, that was estimated to have hatched on 09 october 2020 and excavated on 15 october 2020, a malformed embryo was found (figure 1). figure 1: hatchling with polycephaly after it was removed from the egg. (a) ventral view, prominent yolk sac present. arrow indicating the fused eyelid. (b) anterior-dorsal view. point of fusion on the head indicated by black arrow, dorso-cervical opening indicated by the white arrow. the two-headed specimen this embryo represents a case of polycephaly, with the heads incompletely separated down the midline (figure 1a). externally, the heads fused at the supraorbital scale and caudal to the rhamphotheca (figure 1). both heads shared a medial canthus of the eyelid, but each eye had a distinct globe. additionally, external deformations included the presence of an opening in the dorsal cervical region (figure 1b) and a lordotic region in the spine and carapace (figure 2a and c). figure 2: radiographic images of the malformed hatchling. (a) positioning of the hatchling for lateral image acquisition – spinal deformation clearly visible (arrow). (b) ventral view – yolk sac visible and fusion of the two skulls indicated by arrow. (c) lateral view – lordotic region of the spin indicated by arrow. (d) dorsal view – point of fusion of the two necks indicated by arrow. radiographic images indicated the fusion of the skulls at approximately the level of the post orbital bones (figure 2b). each head had its own cervical vertebrae down to approximately the level of cervical vertebrae 3 and 4, where they fused to continue down into a relatively normal single embryo body (figure 2d), apart from the lordosis at approximately the level of the first and second thoracic vertebrae (figure 2c). because of the presence of a significant yolk sac and the small size of the embryo (straight carapace length = 38.4 mm, straight carapace width = 35.4 mm and snout vent length left head = 54.6 mm), the coelomic contents were indistinct on the radiographic images, but the axial and appendicular skeletons were more easily distinguishable because of the higher density of bone – and therefore greater absorption of x-rays – than soft tissue (fat, muscle and viscera). all four flippers were anatomically normal in all respects, as confirmed during the following necropsy. the necropsy demonstrated that the embryo had a normal coelomic cavity, with all organs present and with no duplication, aplasia or abnormalities (figure 3). all organs had a normal appearance, size, colour and texture based on visual assessment. at the approximate level of the thoracic inlet, the oesophagus and trachea split to serve each separate head, which each had a distinct and normally formed oral cavity, nares and glottis. the cause for the opening on the dorsal surface of the neck was unclear as the anatomical features were hard to distinguish during dissection. the fusion or lack, thereof, of the brain was not assessed because of the lack of appropriate tools to adequately expose the brain without destroying it in the process. figure 3: necropsy image of the hatchling with plastron removed and pectoral muscles reflected cranially showing normal organ development without duplications below the neck. all organs present and visually assessed as normal in appearance, size, colour and texture. h = heart, i = intestine, l = liver, p = pectoral muscle, s = stomach, t = trachea. discussion nesting and hatching success on coco palm dhuni kolhu in general, nests recorded in 2020 on cpdk showed a relatively high median hatching success rate of almost 90%. as embryonic development can reflect the environmental conditions during incubation (see e.g. bailey et al. 2018; booth 2017; kaska & downie 1999; kobayashi et al. 2017b; miller 1985; packard 1991; rafferty & reina 2012), the high hatching success rate in this case can indicate good incubation conditions on the island. reptile nests are highly sensitive to specific temperatures and humidity (deeming 2004; fuentes, limpus & hamann 2011; segura & cajade 2010). successful nests again point towards great incubation conditions for sea turtle nests on cpdk. comparable hatching rates have been documented in the entire country (hudgins et al. 2017), showing that beach conditions in the maldives are currently well-suited for sea turtle nests, providing these nests are left to develop undisturbed. previous studies worldwide showed a reduction in hatching success rates in nests that were relocated (ahles & milton 2016; eckert & eckert 1990; hudgins et al. 2017; pintus et al. 2006). in this study, four nests were relocated because of closeness to the high water mark. this was done within 12 h after oviposition in three cases, as careful movement of eggs is less likely to be detrimental within that time frame (parmenter 1980). the exception was one nest (table 2, number 19), which was unexpectedly washed out at a late point in incubation during a bad weather event. the decision was made to relocate the nest at the later point in development, because of the direct risk of losing the nest. the combination of a large number of false crawls and no confirmed nests in the first two months of activity may point towards a variability in fine-scale natal homing, which is known from other green turtle populations (nishizawa et al. 2011; shamblin et al. 2020). turtles could be nesting on neighbouring islands within a certain distance, and additional studies are needed to determine the extent of natal homing ranges in green turtles in the maldives. congenital malformations apart from benign scute abnormalities, the most common aberrations from a normal developmental pattern include changes to the limbs, followed by changes to the head (craven et al. 2019), the latter being the case presented here. according to miller (1985), skull development starts as early as 6–11 days after incubation, pointing towards the development of the malformation of the presented embryo in the earliest stages of incubation. any errors occurring in the first half of development, within the first 30 days, respectively, can have both congenital and environmental factors as an origin (bárcenas-ibarra et al. 2015; dodd 1988; drennen 1990) and often prove to be fatal. this leaves the exact cause of the observed abnormalities to speculate in many cases, as in the one presented here. the malformed embryo found on cpdk died inside the egg, as would be expected as more serious deformities, such as polycephaly in combination with a cervical opening, are often fatal prior to hatching or even pipping (miller 1985). the opening in the neck likely represents an incomplete fusion of the spinal canal or an associated defect of the neural crest. together with the presence of two heads, this might be caused by terminal bifurcation of the notochord during neurulation, as documented in other vertebrates (spemann & mangold 1924; twigg & wilikie 2015), or because of the incomplete separation of early embryos (see wu et al., 2002 for a review). similar to general hatching success, environmental conditions can influence the chance of abnormalities in development with temperature and humidity playing a prominent role. according to mcgehee (1979), incubation at overall lower temperatures (< 30 °c) can increase the chance of hatchling deformities. most likely though, it is not a low temperature alone that triggers aberrant development but a combination with other environmental or hereditary factors (miller 1982), similar to associated causes reported in snakes (wallach 2007). as the maldives have comparably stable high temperatures year-round, the influence of low temperature on the development of the hatchling is considered unlikely, but sand temperature measurements should be taken to confirm this assumption. additionally, various components such as polycyclic aromatic hydrocarbons or mercury have been identified as possible agents changing deoxyribonucleic acid (dna) methylation patterns and thus triggering abnormal development (bárcenas-ibarra et al. 2015; see martín-del-campo, sifuentes-romero & garcías-gasca 2019; martín-del-campo et al. 2021 for a summary). the prevalence of such components in sea turtle eggs in the maldives is currently unknown. conclusion as the maldives is a rather widespread country with many small individual islands, systematic evaluation of sea turtle nesting activity is still problematic. for accurate estimates of natal homing ranges of individual females and thus possible population management units within the nesting green turtle population of the maldives, consistent monitoring efforts need to be extended. inclusion of islands with known nesting activity as well as their neighbouring islands will be necessary for the quantification of the resident nesting population and its internal structure. monitoring and nest excavation efforts are also essential for evaluating the overall frequency of congenital malformations, which are known from sea turtle populations worldwide but seem to appear at different frequencies within different populations. consistent monitoring is needed as a basis to investigate environmental factors, causes and triggers for such malformations, which is especially relevant in the maldives in the light of continued island and beach development all over the country. acknowledgements the olive ridley project would like to thank its partners at coco palm dhuni kolhu for hosting the rescue centre and staff on the island, which facilitated the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.l. and r.b. collected the nesting data, and a.l. performed the veterinary examination of the embryo. s.k. conceptualised the study and wrote the manuscript. all authors contributed to editing the manuscript to its final form in the preparation for publication. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. 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cattle keeper group alicia cloete, cornelia gerstenberg, natalie mayet, stefano tempia onderstepoort journal of veterinary research | vol 86, no 1 | a1671 | 18 february 2019 original research sheep enteric cestodes and their influence on clinical indicators used in targeted selective treatments against gastrointestinal nematodes salah meradi, jacques cabaret, bourhane bentounsi onderstepoort journal of veterinary research | vol 86, no 1 | a1648 | 28 february 2019 original research vitamin d status in dogs with babesiosis eran dvir, chantal rosa, ian handel, richard j. mellanby, johan p. schoeman onderstepoort journal of veterinary research | vol 86, no 1 | a1644 | 28 march 2019 original research prevalence of gastrointestinal helminths and parasites in smallholder pigs reared in the central free state province ifeoma c. nwafor, hester roberts, pieter fourie onderstepoort journal of veterinary research | vol 86, no 1 | a1687 | 11 april 2019 original research risk of establishment of canine leishmaniasis infection through the import of dogs into south africa abdalla a. latif, bonginkosi nkabinde, brian peba, olivier matthee, ronel pienaar, antoinette josemans, daniel marumo, karien labuschagne, nada a. abdelatief, andreas krüger, ben j. mans onderstepoort journal of veterinary research | vol 86, no 1 | a1634 | 28 may 2019 original research prevalence and risk factors associated with campylobacter spp. occurrence in healthy dogs visiting four rural community veterinary clinics in south africa musafiri karama, beniamino t. cenci-goga, alice prosperi, eric etter, saeed el-ashram, cheryl mccrindle, jackson n. ombui, alan kalake onderstepoort journal of veterinary research | vol 86, no 1 | a1673 | 28 may 2019 original research the effects of extraction techniques and quantitative determination of oxalates in nerium oleander and feeds kedibone g. kgosana onderstepoort journal of veterinary research | vol 86, no 1 | a1611 | 29 may 2019 original research african animal 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| 22 august 2019 113 121 129 140 149 161 168 176 original research molecular diagnosis of acute and chronic infection of trypanosoma evansi in experimental male and female mice tahani s. behour, shawky m. aboelhadid, wahid m. mousa, adel s. amin, saeed a. el-ashram onderstepoort journal of veterinary research | vol 86, no 1 | a1638 | 26 august 2019 original research molecular and restriction fragment length polymorphism analysis of canine parvovirus 2 (cpv-2) in dogs in southeast anatolia, turkey pelin f. polat, adem şahan, gürbüz aksoy, mehmet o. timurkan, ender dinçer onderstepoort journal of veterinary research | vol 86, no 1 | a1734 | 27 august 2019 original research serum biochemistry in cows of different breeds presented with reproductive conditions keitiretse molefe, mulunda mwanza onderstepoort journal of veterinary research | vol 86, no 1 | a1742 | 29 august 2019 original research phylogenetic studies of larval digenean trematodes from freshwater snails and fish species in the proximity of tshwane metropolitan, south africa esmey b. moema, pieter h. king, johnny n. rakgole onderstepoort journal of veterinary research | vol 86, no 1 | a1729 | 17 september 2019 original research injectable organic and inorganic selenium in dairy cows – effects on milk, blood and somatic cell count levels gert m. ferreira, inge-marie petzer onderstepoort journal of veterinary research | vol 86, no 1 | a1664 | 08 october 2019 original research first-time detection of bovine viral diarrhoea virus, bvdv-1, in cattle in botswana sara lysholm, solomon s. ramabu, mikael berg, jonas j. wensman onderstepoort journal of veterinary research | vol 86, no 1 | a1764 | 09 october 2019 original research rift valley fever virus in small ruminants in the democratic republic of the congo georges m. tshilenge, mfumu l.k mulumba, gerald misinzo, rob noad, william g. dundon onderstepoort journal of veterinary research | vol 86, no 1 | a1737 | 10 october 2019 original research using genetic and phenetic markers to assess population isolation within the southernmost tsetse fly belt in africa chantel j. de beer, gert j. venter, marc j.b. vreysen, fernando c. mulandane, luis neves, sihle mdluli, otto koekemoer onderstepoort journal of veterinary research | vol 86, no 1 | a1768 | 16 october 2019 original research seasonal activity of ticks infesting domestic dogs in bejaia province, northern algeria rosa kebbi, mohamed nait-mouloud, lila hassissen, abdelhanine ayad onderstepoort journal of veterinary research | vol 86, no 1 | a1755 | 17 october 2019 185 195 203 210 217 225 232 237 245 page ii of iii http://www.ojvr.org open access table of contents iii original research antimicrobial usage in pig production: effects on escherichia coli virulence profiles and antimicrobial resistance rukayya h. abubakar, evelyn madoroba, oluwawemimo adebowale, olubunmi g. fasanmi, folorunso o. fasina onderstepoort journal of veterinary research | vol 86, no 1 | a1743 | 31 october 2019 original research seroprevalence and associated risk factors of toxoplasma gondii infection in domestic animals in southeastern south africa whatmore m. tagwireyi, eric etter, luis neves onderstepoort journal of veterinary research | vol 86, no 1 | a1688 | 05 november 2019 251 262 reviewer acknowledgement onderstepoort journal of veterinary research | vol 86, no 1 | a1836 | 11 december 2019 268 page iii of iii abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) annelize jonker tuberculosis and brucellosis research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa peter n. thompson department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa anita l. michel tuberculosis and brucellosis research programme, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation jonker, a., thompson, p.n. & michel, a.l., 2023, ‘approaches to increase recovery of bacterial and fungal abortion agents in domestic ruminants’, onderstepoort journal of veterinary research 90(1), a2010. https://doi.org/10.4102/ojvr.v90i1.2010 original research approaches to increase recovery of bacterial and fungal abortion agents in domestic ruminants annelize jonker, peter n. thompson, anita l. michel received: 05 mar. 2022; accepted: 17 aug. 2022; published: 11 jan. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract abortions in domestic ruminants cause significant economic losses to farmers. determining the cause of an abortion is important for control efforts, but it can be challenging. all available diagnostic methods in the bacteriology laboratory should be employed in every case due to the many limiting factors (autolysis, lack of history, range of samples) that complicate the investigation process. the purpose of this study was to determine whether the recovery of diagnostically significant isolates from domestic ruminant abortion cases could be increased through the use of a combination of the existing aerobic culture and brucella selective method with methods that are commonly recommended in the literature reporting abortion investigations. these methods are examination of wet preparations and impression smears stained by the modified ziehl–neelsen method, anaerobic, microaerophilic, leptospira, mycoplasma and fungal culture. samples of placenta and aborted foetuses from 135 routine clinical abortion cases of cattle (n = 88), sheep (n = 25) and goats (n = 22) were analysed by the new combination of methods. in 46 cases, bacteria were identified as aetiological agents and in one case a fungus. isolation of brucella species increased to 7.4% over two years compared with the previous 10 years (7.3%), as well as campylobacter jejuni (n = 2) and rhizopus species (n = 1). salmonella species (5.9%) and trueperella pyogenes (4.4%) were also isolated more often. in conclusion, the approach was effective in removing test selection bias in the bacteriology laboratory. the importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by increased isolation of brucella abortus and isolation of c. jejuni. the combination of methods that yielded the most clinically relevant isolates was aerobic, microaerophilic, brucella and fungal cultures. keywords: abortion; culture; brucella; campylobacter; bovine; ovine; caprine; ruminants. introduction abortions in domestic ruminants cause significant economic losses to farmers worldwide (borel et al. 2014). abortion is defined as the expulsion of the foetus from the uterus before it is full term and viable (baumgartner 2015). diagnosis of the cause of an abortion plays an important role in the subsequent mitigation and control efforts (borel et al. 2014). a diagnosis depends not only on the detection of a bacterial/fungal agent but also on finding macroand/or microscopic lesions indicative of bacterial/fungal infection in foetal tissues, which can be challenging (clothier & anderson 2016; wheelhouse & dagleish 2014). autolysis is one of the most important factors the laboratory diagnostician has to contend with, because it masks lesions and the agent of abortion may be outcompeted by contaminating bacteria (kirkbride 1986). foetuses may already be autolytic by the time they are aborted because of an infection process or maceration (clune et al. 2021; givens & marley 2008). other major stumbling blocks tend to be lack of history and incomplete range of samples received at the laboratory (holler 2012; wheelhouse & dagleish 2014). limited test selection, a cost-reducing measure, often aimed at detecting the most common agents or those that can be zoonotic can drastically reduce the chances of reaching a diagnosis (borel et al. 2014; schnydrig et al. 2017). placenta and abomasal fluid samples are most likely to yield the organism of interest (clothier & anderson 2016; holler 2012). agents of abortion that only affect the placenta such as chlamydia species, coxiella burnetii, as well as bacillus licheniformis and some fungi, may not be detected if the placenta is not included (clothier & anderson 2016; wheelhouse & dagleish 2014). fungal infections may result in macroscopic lesions in the placenta but not in the foetus (borel et al. 2014). kidney and liver samples are useful for the detection of leptospira interrogans (eds. markey et al. 2013; mgode et al. 2015). the use of dark field microscopy of wet mounts as well as impression smears was reported by several studies. kirkbride (1992) as well as macías-rioseco et al. (2020) used dark field microscopy to screen for motile bacteria, such as campylobacter, in abomasal fluid. impression smears stained by the modified ziehl–neelsen method (mzn) were included in routine abortion investigations although the method was shown to have limited sensitivity and specificity (borel et al. 2014; schnydrig et al. 2017). in a south african report of chlamydiosis in a beef herd, ehret et al. (1975) mentioned the use of the modified ziehl–neelsen method. conventional culture methods vary between laboratories, different species of animals and countries. bacterial isolation employing aerobic, as well as microaerophilic culture was commonly reported (agerholm et al. 2006; campero et al. 2003; clothier & anderson 2016; schnydrig et al. 2017). aerobic culture on agar plates with 5% blood in a 5% co2 atmosphere is sufficient to isolate most bacteria (borel et al. 2014). addition of microaerophilic culture increases sensitivity for bacteria that are sensitive to oxygen (borel et al. 2014). anaerobic bacteria such as fusobacterium necrophorum, fusobacterium nucleatum and bacteroides fragilis have been reported in cases of ruminant abortion on rare occasions (agerholm et al. 2006; syrjälä et al. 2007), which may be ascribed to the fact that anaerobic culture is not commonly included in abortion panels (borel et al. 2014). schnydrig et al. (2017) included selective media for brucella, campylobacter, listeria and fungi to increase the chances of isolating these microorganisms by suppressing the growth of contaminating bacteria. a number of bacterial agents of abortion are contagious and/or become endemic in herds. brucella abortus (b. abortus) is common and brucella melitensis (b. melitensis) as well as brucella ovis (b. ovis) are isolated sporadically in south africa (kolo et al. 2019; matle et al. 2021; mbizeni 2015). campylobacter fetus ssp. venerealis, campylobacter fetus ssp. fetus, campylobacter jejuni and arcobacter species are occasionally implicated in cases of abortion in south africa (bath et al. 2013; coetzer & oberem 2018), while campylobacter species were more commonly reported as causes of abortion in argentina and new zealand (campero et al. 2003; west et al. 2002). the families chlamydiaceae, waddliaceae, parachlamydiaceae and rhabdochlamydiaceae are of significance in contagious abortions (borel et al. 2014). however, members of these families fall in the difficult to culture category, because they do not grow on conventional growth media (vidal et al. 2017). serological studies indicate that chlamydia abortus plays a role in ruminant abortions in south africa (ndou & dlamini 2012; sri jeyakumar 2001). l. interrogans serovars have been implicated in bovine abortion in south africa and other countries such as the united states (clothier & anderson 2016; herr et al. 1982; knudtson & kirkbride 1992). several salmonella subspecies and serovars are associated with abortion in ruminants, for example, salmonella enterica subspecies diarizonae, salmonella enterica subspecies enterica serovar dublin and salmonella enterica subspecies enterica serovar abortusovis (eds. markey et al. 2013). salmonella enterica subspecies enterica serovar dublin is a known cause of abortions in cattle in south africa (coetzer & oberem 2018). opportunistic bacterial agents of abortion may be commensals of mucous membranes or may be common in the environment, for example, bacillus spp., escherichia coli (e. coli), histophilus somni, pasteurella spp., staphylococcus spp., streptococcus spp., trueperella pyogenes (t. pyogenes) and mycoplasma species. abortions caused by these bacteria are sporadic, but their occurrence is widespread (clothier & anderson 2016). mycotic or fungal abortion is caused by opportunistic fungal pathogens that are normally present as saprophytes in the environment of the animal. fungi most commonly associated with abortion are aspergillus species and candida species (anderson 2007; holler 2012; mee 2020). additional herd information and history such as the number of abortions, the number of pregnant animals, clinical signs in dams and changes in husbandry are useful (borel et al. 2014). number of abortions per number of pregnant animals (abortion rate) is a useful guideline to determine whether an investigation is indicated or not. abortion rates less than 2% are usually not seen as an indication for investigation (menzies 2011). most abortions are sporadic with less than 5% of pregnant animals aborting (givens & marley 2008). however, it must be borne in mind that an abortion rate of between 2% and 5% may be an indication that an endemic infectious agent is present (menzies 2011). additional information must be interpreted with caution as it can be misleading. for example, aetiological agents should not be ruled out simply because animals were vaccinated as vaccination does not result in complete immunity (kirkbride 1986). moreover, unvaccinated, infected animals could have been introduced into a herd prior to an abortion event. the aim of this study was to analyse samples of placenta and aborted foetuses from cattle, sheep and goats by means of a set combination of conventional culture and identification methods for bacteria and fungi. the objective was to determine whether more diagnostically significant isolates could be cultured if anaerobic, campylobacter, leptospira, mycoplasma and fungal culture were added to the existing aerobic and brucella selective culture and if applying the new combination of methods to every abortion case could remove test selection bias. materials and methods routine diagnostic samples from products of abortion from cattle, sheep and goats submitted to the bacteriology laboratory of the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria over a two-year period (september 2017 to september 2019) were included in the study. samples were accepted from the pathology section at the faculty of veterinary science, regional government laboratories, private laboratories, private veterinarians or state veterinarians. criteria for selection was abortion meaning a foetus expelled from the uterus before it was full term and viable (baumgartner 2015). indications that the calf/lamb/kid was born alive such as aerated lungs, colostrum in the abomasum or worn golden slippers were criteria for exclusion. one or more foetuses submitted as a single case were recorded as a single case, except when different agents were recovered from the different foetuses. samples accepted were any of the following: placenta, stomach content, lung, liver, spleen and kidney from aborted foetuses. samples were accepted whether they were in good or poor condition. sample size was calculated as 235 foetuses for this descriptive study (survey toolbox-www.epitools.ausvet.com.au) (fosgate 2009); therefore, the aim was to analyse samples from 200 to 300 foetuses. conventional microbiological analyses the methods that were previously applied to placenta and foetus samples in the bacteriology laboratory at the dvtd were impression smears stained by the gram and modified ziehl–neelsen methods as well as aerobic culture, brucella selective culture and fungal culture. brucella selective culture and fungal culture were performed on request. for the purpose of this study, wet mounts, microaerophilic culture, anaerobic culture, mycoplasma selective culture and leptospira selective culture were added. direct smears wet mounts were made of stomach content samples, if included in abortion cases, and examined by dark field microscopy for campylobacter species, leptospira species and other motile bacteria. impression smears were made of all placenta and foetus samples, heat fixed and stained by gram and mzn methods (eds. markey et al. 2013). aerobic culture all samples were inoculated on columbia blood agar with 5% horse blood (cba) (selecta-media, thermofischer, gauteng, south africa), macconkey agar (mcc) (without crystal violet) (selecta-media, thermofischer, gauteng, south africa) and xylose-lactose-desoxycolate medium (xld) (oxoid cm0469b, thermofischer, gauteng, south africa). inoculated cba plates were incubated in a 5% carbon dioxide (co2) atmosphere at 37 ºc ± 1 ºc and mcc and xld plates were incubated in normal air at 37 ºc ± 1 ºc. cba plates were examined daily for 10 days. mcc plates were examined after 24 h. primary identification consisted of gram stain as well as catalase and oxidase tests. the outcome of these tests determined carbohydrate combinations for secondary identification (eds. markey et al. 2013). anaerobic culture only stomach content was inoculated on pre-reduced cba and incubated anaerobically at 37 ºc ± 1 ºc for 5 days in an anaerobic workstation (bactron anaerobic chamber, united scientific, gauteng, south africa). if stomach content was not received, this culture method was not performed. an anaerobic detector strip (oxoid, thermoscientific, gauteng, south africa) was used to confirm anaerobiosis. primary identification consisted of gram stain, catalase and oxidase tests. depending on the outcome of these tests, isolates were identified by mastring (mast id, davies diagnostics, gauteng, south africa) or rapid id 32a (biomerieux, gauteng, south africa) (eds. markey et al. 2013). brucella selective culture in addition to aerobic culture, placenta, stomach content and/or lung were inoculated on farrell medium (oxoid cm0169 with supplement (sr0083a), thermoscientific, gauteng, south africa). if these samples were not received, this culture method was not performed. one plate of farrell medium was inoculated with an in-house b. abortus control culture for quality control purposes. plates were incubated in co2 at 37 °c ± 1 °c for 10 days and examined daily. cultures were regarded as negative when no characteristic growth was obtained by day 10. suspect colonies were subcultured onto two cba plates and one mcc plate. one cba plate was incubated in co2, the other cba plate was incubated in normal air together with the mcc plate. primary identification included a gram stain, oxidase, mzn stain and a urease slant (selecta-media, thermoscientific, gauteng, south africa) (woah 2016). presumptive brucella species identified were submitted to the arc-ovr for confirmation and phenotyping. salmonella selective culture in addition to direct culture for salmonella, placenta, stomach content and organ samples were inoculated on rappaport-vassiliades broth (rv) (oxoid cm 0866b, thermoscientific, gauteng, south africa) and incubated at 42 °c ± 1 °c in normal air. after 24 h, the broth culture was inoculated on xld medium (oxoid cm0469b, thermoscientific, gauteng, south africa) and incubated at 37 °c ± 1 °c in normal air. primary identification included a gram stain, catalase, oxidase and spot indole tests. api® 10s (biomerieux, gauteng, south africa) was used for secondary identification (eds. markey et al. 2013). if no characteristic colonies were seen, the culture was regarded as negative. salmonella isolates were serotyped according to the white-kauffman-le minor scheme (grimont & weil 2007). mycoplasma selective culture in addition to aerobic culture, stomach content, placenta and lung were inoculated directly on a plate of mycoplasma medium (oxoid cm0401 & sr0059, thermoscientific, gauteng, south africa) and in mycoplasma broth (oxoid cm0403 & sr0059, thermoscientific, gauteng, south africa). if these samples were not received, this culture method was not performed. the broth was incubated in normal air at 37 ºc ± 1 ºc and plated onto mycoplasma medium after 24 h. plates were incubated in co2 at 37 ºc ± 1 ºc; examined at 48 and 96 h after inoculation and thereafter once a week. if no characteristic growth was obtained after 14 days’ incubation, the culture was regarded as negative. suspect cultures were identified to genus level as described by markey et al. (eds. 2013). campylobacter culture placenta, stomach content and liver were inoculated on cba. if none of these samples were received, this culture method was not performed. inoculated plates were incubated in a microaerophilic atmosphere (6% o2, 10% co2 and 84% n) (campygen, oxoid ltd, thermoscientific, gauteng, south africa) in anaerobic jars (oxoid ltd, thermoscientific, gauteng, south africa) at 37 ºc ± 1 ºc for five days. the plates were examined after 72 h and again at the end of five days. if no suspect cultures were noted by day five, plates were discarded as negative. primary identification included gram stain, catalase and oxidase tests. secondary identification included growth in a microaerophilic atmosphere at 42 ºc, growth at 37 ºc in normal air, growth at 22 ºc, growth in the presence of 1% glycine, production of h2s in triple sugar iron medium (tsi) (selecta-media, thermoscientific, gauteng, south africa) and hippurate hydrolysis (remel, thermoscientific, gauteng south africa) (eds. markey et al. 2013). charcoal-cefoperazone-dextrose-agar (ccda) (selecta media, thermoscientific, gauteng, south africa) was inoculated with placenta, stomach content and liver samples from cases that yielded campylobacter isolates on cba. the campylobacter isolates from these cases and a control strain, c. jejuni (atcc 33560) were also inoculated on ccda. the inoculated medium was incubated in a microaerophilic atmosphere at 37 ºc ± 1 ºc for five days. the plates were examined for suspect colonies after 72 h and again after five days. if no suspect colonies were observed by day five, plates were discarded as negative. primary and secondary identification of suspect colonies was performed as described above. leptospira culture one tube of leptospira ellinghausen–mccullough–johnson–harris (emjh) medium (difco 279410 & supplement 279510, becton dickinson, gauteng, south africa) was inoculated with 1 g kidney or liver. if none of these samples were received, this culture method was not performed. cultures were incubated at 30 ºc ± 1 ºc in normal air and examined weekly for 13 weeks. contaminated cultures were subcultured into a new tube of emjh medium with 5’-fluorouracil (sigma, gauteng, south africa). turbidity and/or the formation of dinger’s ring as well as characteristic morphology on dark-field microscopy was regarded as presumptive positive. if no turbidity is evident by week 13, cultures were regarded as negative (eds. markey et al. 2013). presumptive positive cultures were submitted to the agricultural research council-onderstepoort veterinary research (arc-ovr) for polymerase chain reaction (pcr) confirmation. fungal culture stomach content and placenta (as well as skin lesions, if submitted) were inoculated on potato dextrose agar (selecta-media, thermoscientific, gauteng, south africa). if none of these samples were received, this culture method was not performed. the plates were incubated at 37 ºc for five days. plates were examined twice a week. a sticky tape colony smear was made of suspect colonies and stained by lactophenol cotton blue stain (merk, gauteng, south africa). fungi were identified by colony morphology. yeasts were identified by gram stain and api® 20c aux (biomerieux, gauteng, south africa) (eds. markey et al. 2013). supplementary information data such as pathology lesions, immunohistochemical staining results and pcr results were extracted from diagnostic reports for supplementary information. the methodologies for these procedures were, however, not available. herd history additional production data such as farming system, feeding system, number of pregnant animals, number of abortions and vaccination status were extracted from pathology reports. data capture and analysis data were captured in an excel spreadsheet from which summary tables were created. the aim was to investigate patterns with respect to diagnostic rate, completeness of submission, condition of samples, methods used and season. diagnostic rate was calculated by dividing the number of confirmed diagnoses by the total number of submissions and multiplying by 100. the result was expressed as a percentage. a confirmed diagnosis was defined as a diagnosis made by the pathologist taking into account the pathological lesion and the bacterial or fungal isolate. abortion rate was calculated by dividing the number of abortions by the number of pregnant animals in a herd (if available) and multiplying by 100. the result was expressed as a percentage. abortion rate is used to determine the significance of the number of abortions in a herd. data were exported into a statistical program (epi info 7™ 7.2.3.1, centers for disease control and prevention). percentages with 95% confidence intervals (ci) were calculated, and multivariate logistic regression was used to investigate associations between completeness of submission, condition of samples, confirmed diagnosis and season. ethical considerations ethical clearance to conduct this study was obtained from the university of pretoria, faculty of veterinary science research ethics and animal ethics committee. (no. v092-17 & v070-21). results number of cases the calculated number of 235 abortion cases was not reached because of lower-than-expected submission rates during 2018 and 2019. samples from 135 abortion cases were received for bacterial and fungal culture. eighty-eight were bovine, 25 were ovine and 22 were caprine cases. forty-six complete submissions (foetus and placenta) were received: 26 bovine, 11 ovine and nine caprine cases. eighty-nine submissions were incomplete: 85 cases of foetus only and four cases of placenta only. most samples (n = 100) were considered to be good quality and 35 were poor quality (table 1). table 1: diagnosis and diagnostic rate (with 95% confidence intervals) compared to the completeness of submissions as well as quality of samples. diagnostic rate overall diagnostic rate for cases submitted to the bacteriology laboratory was 42.2% (95% ci: 46, 69). diagnostic rate per animal species was 36 of 88 bovine cases (32.9%, 95% ci: 27, 46), 14 of 25 ovine cases (48.0%, 95% ci: 9, 19) and seven of 22 caprine cases (27.3%, 95% ci: 3, 12). bovine cases were most often received (88/135). complete submissions (foetus and placenta) had the highest diagnostic rate overall at 43.5% (95% ci: 13, 27) although this was not the case for all species. diagnostic rate for complete bovine cases was 10 of 26 (38.5%, 95% ci: 5, 15) and for incomplete cases 24 of 62 (38.7%, 95% ci: 16, 32). diagnostic rate for complete ovine cases was five of 11 (45.5%, 95% ci: 2, 8) and for incomplete cases seven of 14 (50%, 95% ci: 3, 11). the diagnostic rate for complete caprine cases was three of nine (33.3%, 95% ci: 1, 6) and for incomplete cases, three of 13 (23%, 95% ci: 1, 7). submissions where the placenta was included (n = 50) had a diagnostic rate of 42.0% (95% ci: 14, 28). in 47 of 57 submissions where a diagnosis was made, bacteria or fungi were implicated (table 1). most cases were submitted in winter (n = 59) followed by spring (n = 34). most b. abortus-positive cases (n = 8) and t. pyogenes-positive cases (n = 6) were submitted in winter and spring. multivariate logistic regression analysis was performed to investigate the association between diagnosis and degree of autolysis, completeness of submission as well as season (table 2). table 2: multivariate regression analysis investigating association between diagnosis and degree of autolysis, completeness of submission and season. the odds ratio and ci for autolysis, complete submission and season are > 1, but p > 0.05. this indicates that there is no significant association between diagnosis and degree of autolysis, complete submission (inclusion of placenta) or season. no wet mounts were positive for motile bacteria. modified ziehl–neelsen stained impression smears were positive for small, partially acid-fast coccobacilli in six of 10 cases (60%, 95% ci: 3, 9) that yielded b. abortus-positive cultures. in addition, three cases that were positive for partially acid-fast rods on stained impression smears were pcr positive for c. burnetii. no chlamydiales-positive cases were positive for partially acid-fast bacteria on impression smears. eighty-five potentially abortifacient isolates were cultured from 135 submissions. bacteria were most common (73/85). twelve isolates were endemic/contagious bacteria and 73 were environmental or commensal bacteria that cause sporadic abortions. twelve isolates were fungi. in 46 cases, bacteria were identified as aetiological agents and in one case a fungus (table 3). b. abortus was the most common of the endemic/contagious bacteria at 10/12 isolates. it was also one of the most common bacterial isolates overall (10/71). table 3: significant bacterial and fungal isolates from products of abortion detected by impression smears stained by the modified ziehl–neelsen method, conventional culture, immunohistochemistry and pcr. submissions were received from six of nine provinces of south africa. farms of origin were mainly in gauteng province (n = 40) followed by north west province (n = 30) and mpumalanga (n = 20) (table 2). b. abortus was isolated from bovine submissions from north west, free state, mpumalanga and northern cape provinces. c. jejuni was isolated from ovine and caprine submissions from gauteng province. campylobacter fetus was isolated from submissions from the free state and north west provinces. t. pyogenes was isolated from bovine cases from gauteng, north west, eastern cape and free state provinces. a number of co-infections (n = 12) were identified, notably b. licheniformis and rhizopus species, b. abortus biovar i and salmonella species, c. abortus and chlamydia pecorum as well as chlamydia species and enterobacteriaceae. eighteen cases were recorded where there was pathological evidence of an infectious process, but no potential pathogen was isolated. most cases had placental lesions (n = 12), necrotic placentitis (n = 6), necrotic placentitis with vasculitis (n = 2) or placentitis (n = 4). additional production-related history (farming system, livestock species, vaccinations, feeding system, herd size, number of abortions) was reported for 50 cases (37%). in 38 of these cases, farming systems were recorded. farming systems were mostly meat production (n = 30) and dairy (n = 9). cattle (n = 19) and goats (n = 9) were the most common livestock species. in 12 cases, vaccinations were reported. five of these cases included brucella vaccination. two cases included c. abortus vaccination. feeding systems were mostly pasture based with supplementary feeding (n = 6). one owner reported feeding silage the quality of which was sometimes poor. nine owners reported the number of abortions and number of pregnant animals or abortion percentage. abortion percentage ranged from 1% to 75%. two owners reported abortions over time: nine abortions in five days and four abortions in two days. vaccination history was recorded in 12 cases. a wide range of vaccines were used many of which do not have the prevention of abortion as their primary application. vaccines for the prevention of abortion were brucella s19 and rb51 recorded in five cases, as well as enzootic abortion vaccine (c. abortus) recorded in two cases. abortions in these seven cases were because of agents other than those vaccinated against indicating that the vaccines were effective or that animals were not exposed to the agents the vaccines are protective against. discussion in this study, the standard diagnostic approach for abortion investigations in this laboratory, namely impression smears stained by the gram and modified ziehl-neelsen methods, aerobic culture with the addition of brucella culture if requested, was challenged. we investigated the effect of adding anaerobic, brucella, campylobacter, mycoplasma and leptospira culture methods and applying the combination of methods to every submission of foetus and placenta samples. the objectives were to determine whether the extended combination of conventional culture methods could increase the recovery of bacterial and fungal causes of abortions. in addition, pathological lesions as well as results of other methods of detection employed by pathologists in case investigations such as pcr and immunohistochemistry (ihc) were recorded. the number of submissions per year in this study; 83 (2018) and 72 (2019) increased markedly compared to submissions recorded by a retrospective study (2006–2016) by jonker and michel (2021) where submissions varied between 11 and 55 per year. the increased number of submissions was probably because of this research project where the conventional bacteriology fees were subsidised. despite this, submissions were less than the statistically calculated sample size for the study and still biased toward a bacterial or fungal aetiology for the following reasons. notification of abortion is not compulsory in south africa. products of abortion are only submitted for investigation when the field diagnostician and the farmer need a diagnosis, and the farmer can afford the fees. fees for abortion investigation can be considerable, so cost cutting measures are commonly employed leading to test selection bias. only samples that are highly suspect for a bacterial or fungal infection will be submitted to a bacteriology laboratory. this approach is contrary to recommendations by kirkbride (1992) and borel et al. (2014) that all diagnostic methods available should be employed in every abortion case to ensure the best possible chance of a diagnosis. number of submissions decreased as distance increased. most submissions were received from farms in gauteng province, which is closest to the veterinary faculty at onderstepoort, followed by neighbouring north west and mpumalanga provinces. this is probably because of increased costs related to the transport of products of abortion and other veterinary laboratories in closer proximity. inclusion of additional methods resulted in two c. jejuni isolates that grew only in a microaerophilic atmosphere and would not have been isolated by the previous options for culture. detection of brucella improved from 7.3% over 10 years (jonker & michel 2021) to 7.4% over two years. anaerobic culture did not yield isolates that could be implicated as causes of abortion. leptospira cultures did not yield isolates. anaerobes and leptospira are labile and could die before samples reach the laboratory leading to false negative results when culture is attempted. molecular methods should be investigated as an alternative for the detection of these bacteria as genetic material can still be detected after the death of an organism. agerholm et al. (2006) reported f. necrophorum as the second most common aetiology in their ovine study. in their study, f. necrophorum was never cultured but was detected by 16s rrna gene pcr after bacteria associated with inflammation was noted during the histological examination. the overall diagnostic rate of 42.2% in this study was higher than the 35.1% found in our previous retrospective study (2006–2016) (jonker & michel 2021). of the cases where a diagnosis was made 82.5% had a bacterial/fungal cause. this was expected because products of abortion are only submitted when a bacterial or fungal infection is suspected. the diagnostic rate for ovine cases (48.0%) was highest followed by bovine cases (32.9%). this phenomenon is probably related to a number of submissions for the different species. as previously reported, the availability of placenta appeared to positively influence the diagnostic rate. in this study, placentas were received in 37% cases, an improvement over 22.2% of placentas recorded in the retrospective study (jonker & michel 2021). this is probably because of continued efforts to emphasise the importance of submission of placenta in reports to clients. however, similar to the retrospective study, no statistically significant association (p < 0.05) was found between the overall diagnostic rate and completeness of submission. this is probably because of the small number of cases in these two studies. the significance of a bacterial isolate is indicated by compatible history, isolation in large numbers in pure or almost pure culture from one or more than one sample, as well as histopathological findings that correlate with the isolate (anderson et al. 1990; borel et al. 2014; kirkbride 1993). determination of the significance of a fungal isolate is similar to the additional requirement that fungal hyphae must be visualised microscopically in tissues (borel et al. 2014; campero et al. 2003). contamination by bacteria or fungi results in a mixed culture without any dominant isolates (kirkbride 1986). b. abortus was the most common isolate reported overall, as well as from three provinces: north west, free state and mpumalanga. in this study, 7.4% cases were positive for b. abortus. b. melitensis was not isolated. modified ziehl–neelsen-stained impression smears were negative in 40.0% of brucella culture-positive cases confirming insufficient sensitivity of mzn-stained smears as reported by schnydrig et al. (2017). b. abortus was also the most common isolate (7.3%) reported previously in south africa (jonker & michel 2021). in this study, campylobacter species was only isolated in two cases of bovine, one case of ovine and one case of caprine abortion. only c. jejuni could be cultured on ccda. although campylobacter species did not appear to play a significant role in abortion the findings clearly highlight the importance of including a nonselective culture medium in campylobacter culture. in contrast with this study, campylobacter species was reported as a significant cause of abortion in countries in the northern hemisphere (the netherlands and the united kingdom) as well as argentina in the southern hemisphere where it was the second most common aetiology in bovine cases (campero et al. 2003; carson 2017; van engelen et al. 2014). vaccine can be used to control c. fetus subspecies venerealis infection. therefore, it is important to differentiate campylobacter species from one another although phenotypic differentiation of species in this family does present challenges (van der graaff-van bloois 2014). salmonella species (5.9%) and t. pyogenes (4.4%) were implicated more commonly as agents of abortion compared with the retrospective study by jonker and michel (2021). the reason for the increased isolation of salmonella species could be the reduction of test selection bias in this study. in this study, similar to several other studies, a number of co-infections were recorded, such as b. licheniformis and rhizopus species, b. abortus biovar i and salmonella species as well as c. abortus and e. coli. a south african study by schutte et al. (1976) reported co-infection of c. burnetii and c. psittaci. schnydrig et al. (2017) reported 31.2% cases with co-infections. campero et al. (2003) reported a case of b. abortus and campylobacter species co-infection. agerholm et al. (2006) reported a dual infection of f. necrophorum and l. monocytogenes in an ovine foetus. the role of bacteria in dual infections needs further investigation as it is not clear whether both agents cause the abortion in such cases. in 12 cases (8.8%), evidence of an infectious process was seen macroand/or microscopically, but no agent of abortion was isolated. this is a reduction of cases from 14.6% previously recorded by jonker and michel (2021). application of an extended combination of diagnostic methods to all cases probably contributed to this improvement. more brucella sp. were detected by adding brucella selective culture. c. jejuni was detected by microaerophilic culture. mycoplasma species was detected by mycoplasma selective culture. salmonella was detected by aerobic culture in cases where the client only requested chlamydia detection. increased use of ihc and pcr methods also lead to increased detection of chlamydia sp. and c. burnetii. conclusion the new combination of conventional culture methods applied to all abortion cases only served to remove test selection bias in the bacteriology laboratory. nonetheless, the importance of performing an in-depth study on the products of abortion by means of an extensive, combination of conventional culture methods was emphasised by an increase in the number of b. abortus and c. jejuni isolations. some of these isolates would have been missed if the culture in a microaerophilic atmosphere and brucella selective culture was not included. the combination of methods that yielded the highest number of significant isolates was aerobic, microaerophilic, brucella and fungal culture. recommendations further investigation is recommended to extend the use of such methods to detect and identify difficult to culture agents of abortion to species level. b. abortus was the most common agent of abortion isolated in this laboratory over a period of two years. because of the common occurrence of brucellosis, the recommendation is that all laboratories in south africa employ brucella species detection. acknowledgements the authors would like to thank ms d landman, mr e kapp and ms t lukhele of the dvtd bacteriology laboratory for assistance with the analysis of samples. the authors also acknowledge the pathology section of the department of paraclinical studies, faculty of veterinary sciences, university of pretoria and idexx veterinary laboratories for permission to include data from pathology case reports. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.j. was involved in conceptualising project, analysis of samples, writing article, procurement of funding and administration of project. p.n.t. was involved in supervising and assistance with statistical analysis. a.m. was a study leader involved in the critical review and editing of manuscript. funding information the 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woah (world organisation for animal health), 2016, ‘brucellosis (brucella abortus, b. melitensis and b. suis) (infection with b abortus, b. melitensis and b. suis)’, in manual of diagnostic tests and vaccines for terrestrial animals, viewed 20 september 2021, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.01.04_brucellosis.pdf. abstract introduction research method and design results discussion conclusion acknowledgements references about the author(s) sara lysholm department of clinical sciences, faculty of veterinary medicine and animal science, swedish university of agricultural sciences, uppsala, sweden solomon s. ramabu department of animal science and production, botswana university of agriculture and natural resources, gaborone, botswana mikael berg department of biomedical sciences and veterinary public health, faculty of veterinary medicine and animal science, swedish university of agricultural sciences, uppsala, sweden jonas j. wensman department of clinical sciences, faculty of veterinary medicine and animal science, swedish university of agricultural sciences, uppsala, sweden citation lysholm, s., ramabu, s.s., berg, m. & wensman, j.j., 2019, ‘first-time detection of bovine viral diarrhoea virus, bvdv-1, in cattle in botswana’, onderstepoort journal of veterinary research 86(1), a1764. https://doi.org/10.4102/ojvr.v86i1.1764 original research first-time detection of bovine viral diarrhoea virus, bvdv-1, in cattle in botswana sara lysholm, solomon s. ramabu, mikael berg, jonas j. wensman received: 14 mar. 2019; accepted: 22 may 2019; published: 09 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract infectious diseases are serious constraints for improving livestock productivity. bovine viral diarrhoea virus (bvdv) is a virus causing grave economic losses throughout the cattle producing world. infection is often not apparent, but the virus can also cause respiratory signs, diarrhoea, reproductive problems and immunosuppression. risk factors for disease transmission include, but are not limited to, herd size, animal trade and grazing on communal pastures. several prevalence studies have been conducted in southern africa, but in botswana the occurrence is largely unknown. in this study, blood samples were obtained from 100 goats from three villages around the capital city, gaborone. also, 364 blood samples from cattle around gaborone, collected as part of another study, were analysed. the detected antibody prevalence was 0% in goats and 53.6% in cattle when using a competitive enzyme-linked immunoassay. three animals from two different herds were positive for viral nucleic acids on polymerase chain reaction. the two herds with viraemic animals had significantly higher antibody prevalence compared to the other herds. also, two of the detected viruses were sequenced and found to be most similar to bvdv-1a. to the authors’ knowledge, this is the first time that sequencing has been performed on bvdv isolated in botswana. keywords: bovine viral diarrhoea virus; bvdv-1; cattle; botswana; infectious diseases. introduction livestock play a vital role in alleviating poverty and hunger for numerous farmers around the world, for example, in countries such as botswana in southern africa, where 81.5% of the cattle and 95% of the goats are kept by traditional farmers (statistics botswana 2016). it is a well-known fact that in order to optimise livestock performance, it is crucial to maintain a good animal health status. a major obstacle in this regard is infectious diseases. bovine viral diarrhoea virus (bvdv) is a positive-sense single-stranded ribonucleic acid (rna) virus in the genus pestivirus, which can have a grave exacerbating effect on animal health and productivity (houe, pedersen & meyling 1993). there are two different genotypes, bvdv-1 and -2, and within these several subgenotypes, exists (fulton et al. 2003; vilcek et al. 2001). cattle are considered the primary host, but serological evidence of infection has also been found in, for example, sheep, goats and eland antelope (broaddus et al. 2007; torsson et al. 2017; vilcek et al. 2000). bovine viral diarrhoea virus infection is in most instances subclinical, but the virus can also cause respiratory signs, diarrhoea, reproductive failure, congenital malformations, pyrexia, depression, inappetence, nasal discharge and erosion of oral mucosa (walz 2015). the virus is also known to cause leukopenia and immunosuppression which, in turn, renders the animal more susceptible to secondary infections. however, the most serious outcome of infection is the development of persistently infected foetuses (walz 2015). bovine viral diarrhoea virus is present in the majority of countries and seroprevalence is often 60% or more in endemic regions (houe & meyling 1991). differences in seroprevalence are dependent on various factors, for example, on trade routines, usage of communal pastures as well as vaccination practises (houe 1999). antibody prevalence is generally higher in adults compared to young animals (houe & meyling 1991; hyera, liess & frey 1991; mishra et al. 2009; nigussie et al. 2010; torsson et al. 2017), in large than in small herds (almedida et al. 2013; graham et al. 2013; mockeliuniene et al. 2004; sarrazin et al. 2013) and is often elevated in areas where cattle density is high (saa et al. 2012). for small ruminants, regular contact with cattle has been shown to constitute a risk factor (mishra et al. 2009). beside this, seropositivity is significantly higher in herds with persistently infected animals compared to herds with only transient infection (houe & meyling 1991). in herds with persistent infection, where the animals are kept under close confinement, antibody prevalence is usually 90% or more by the time that the persistently infected calf has reached 3–4 months of age (houe et al. 1993). the prevalence of persistently infected animals in endemic areas is also highly variable but is generally around 1–2% (houe & meyling 1991). several prevalence studies have been conducted in southern africa. in south africa, prevalences ranging from 37% to 100% in cattle have been detected (ferreira, lourens & van vuuren 2000; njiro et al. 2011; ularamu et al. 2013). in a namibian study in the late 1980s, 49% of cattle, 9% of sheep and 5% of goats had neutralising antibodies to bvdv (depner, hubschle & liess 1991b). on the kafue flats in zambia in 1987, an antibody prevalence of 76.2% in cattle was found (ghirotti et al. 1991). in tanzania, a 34% seroprevalence in cattle, 32.1% in sheep and 24.9% in goats have been observed (hyera et al. 1991). more recently, torsson et al. (2017) found 3.9% and 1.7% calculated true prevalence in sheep and goats in tanzania. in botswana, however, knowledge of bvdv prevalence is limited and dated. in the 1970s, two cattle herds with clinical signs indicative of bvdv infection were investigated, and an antibody prevalence of 42% and 70% found. also, testing was conducted in a nearby village without clinical signs of bvdv, which detected a 19% seropositivity (hunter & carmichael 1975). in another study in 1973, 88 out of 100 botswana cattle were seropositive for bvdv (theodoris, boshoff & botha 1973, referred by depner et al. 1991b). several different genotypes of bvdv have been detected in africa and bvdv-1a is the genotype most frequently found (yesilbag, alpay & becher 2017). to the authors’ knowledge, sequencing of bvdv has never been undertaken in botswana. hence, the aim of this study was to study the seroprevalence of bvdv in cattle and goats around gabarone, and to identify circulating genotypes. research method and design study design blood samples from 100 goats were collected in three villages situated in or just outside of gaborone, botswana, namely modipane, kopong and gakuto. samples were obtained in september 2016, which is at the end of the dry season. five smallholder farmers in modipane, three in kopong and three in gakuto were chosen for the study. inclusion criteria for farmers were residing in the village of choice, owning an appropriate number of adult goats, that is, 20–30 goats, which is equal to the average herd size in botswana (statistics botswana 2016), as well as on consenting to participate in the study. ten goats from each herd were sampled in all flocks except two in which the total number of adults was less than 20. as these two herds were located immediately adjacent to each other and the animals usually grazed together, they were considered as one epidemiological unit, and sampling hence divided between the two, that is, five goats from each herd. only adult goats (> 1 year old) were selected and both sexes were represented; however, as all flocks consisted of more female goats than male goats, no effort was made to sample both sexes evenly. selection of goats was randomised by catching all adults in a flock but only sampling every second or every third animal, depending on the size of the flock. jugular blood samples were obtained using a closed vacutainer system and serum tubes (bd vacutainer®, franklin lakes, new jersey, united states [us]). a short description of each animal was recorded, as well as an assessment of their body condition score (bcs). body condition score was estimated by inspecting the goats visually as well as palpating the lumbar and sternal area, and then assigning them a number from 1 to 5, one being extremely thin, five obese and three ideal. after sampling, the tubes were left standing in room temperature to allow sera to separate. serum was transferred and then stored at -20 °c until further use. in addition, 364 blood samples from cattle (> 6 months) previously obtained in october 2014 to march 2015, that is, the botswana warm season, were tested (ramabu et al. 2018). samples were taken within an approximate 150 km radius of gaborone from both beef and dairy cattle. nine different farms, five dairy and four beef, were chosen for the study. in dairy farms, samples were obtained from the whole herd, whereas in beef farms, the farmer selected a paddock and samples were then taken there. approximately 75% – 100% of the animals at each farm or enclosure were sampled. after sampling, the test tubes were left standing in order to allow serum to separate. serum was then placed in cryotubes and stored at -20 °c until further use. antibody enzyme-linked immunoassay for detection of antibodies, a competitive enzyme-linked immunoassay (elisa) was used (sensitivity 98.9%, specificity 100% on bovine samples according to the manufacturer, data for goats not available; idexx bvdv/md/bdv p80 protein antibody test kit, idexx, hoofddorp, the netherlands). the test was used according to the instructions provided by the manufacturer. the absorbance values were measured at 450 nm (thermo scientific multiskan fc elisa reader, thermo scientific, vantaa, finland). then, the validity of the positive and negative controls was calculated. for invalid assays, the assay was not repeated because of shortage of material. percentage inhibition was computed by dividing the sample absorbance value with the mean of the negative control, and then multiplying the result with 100. samples with a higher value than 50% were considered negative and lower than 40% were considered positive. samples with a value between 40% and 50% were denoted as doubtful. antigen enzyme-linked immunoassay sera negative on antibody elisa (ab-elisa) were subsequently analysed with antigen elisa (ag-elisa) (sensitivity approaching 100%, specificity 99.7% according to the manufacturer; idexx bovine viral diarrhea virus antigen test kit/serum plus, idexx). the test was performed according to the instructions provided by the manufacturer. the plate absorbance was measured at 450 nm (thermo scientific multiskan fc elisa reader, thermo scientific, vantaa, finland). validity of the negative and positive control was calculated. percentage inhibition was computed by subtracting the mean of the negative control from the absorbance value of the samples. the result was interpreted as negative if the difference was less than 0.3, but positive if it was more than 0.3. ribonucleic acid isolation ribonucleic acid was isolated from samples positive for antigen on ag-elisa, as well as from serum samples negative for antibodies and antigen in herds with a seroprevalence of 50% or more. unfortunately, the remaining sample volume was too low in some samples and hence only 26 samples were subjected to rna extraction. the procedure was performed according to the instructions provided by the manufacturer (macherey-nagel viral rna isolation nucleospin rna virus kit, macherey-nagel, düren, germany). the isolated rna was stored at -20 °c until further use. find the agent cards all samples subjected to rna isolation were subsequently applied to whatman find the agent (fta) cards (sigma-aldrich, saint-louis, missouri, us) to enable transportation. from each sample, 125 µl of serum, 30 µl rna-isolate and 10 µl polymerase chain reaction (pcr) product were applied. reverse transcriptase polymerase chain reaction polymerase chain reaction was subsequently performed on the pcr product that had been applied to fta cards. the cards were placed on a punch pad and 2 mm punches were obtained using a harris micro punch (sigma-aldrich). one punch from each sample was then subjected to elution. in this step, the punch was placed in a microcentrifuge tube, containing 51 µl rna-processing buffer (50 µl buffer containing 10 mm tris-hcl and 0.1 mm ethylenediamine tetraacetic acid, and 1 µl dithiothreitol) and was subsequently left to incubate for 15 minutes, after which the punch was removed. polymerase chain reaction was then performed on both the eluate as well as on unprocessed punches directly included in the pcr tube during the reaction (applied biosystems agpath-id one-step rt-pcr reagents kit, applied biosystems/thermo scientific, foster city, california, us). the pcr reaction volume was 25 µl, containing, 12.5 µl 2x rt-pcr-buffer, 1 µl 25x rt-pcr enzyme mix, 4.5 µl nuclease-free water, 5 µl of template or nuclease-free water for the negative control and 0.4 µm of each of the primers opes13a and opes14a. the primers amplify a 296 base pairs (bp) pcr – product of the 5′ non-coding region (ncr) (elvander et al. 1998). the thermocycler (proflex pcr system, applied biosystems, foster city) was set to the following profile: reverse transcription at 45 °c for 10 min, followed by an inactivation or initial denaturation step at 95 °c for 10 min, and 40 cycles of 95 °c for 15 seconds, 50 °c for 1 min and 72 °c for 1 min, with a final extension step at 72 °c for 7 min. polymerase chain reaction products were analysed by gel electrophoresis. deoxyribonucleic acid extraction from gels and deoxyribonucleic acid sequencing amplified pcr products were extracted from excised gel bands and purified in accordance with manufacturer’s instructions (thermo scientific genejet gel extraction kit, thermo scientific). samples were subsequently sent to macrogen lab in the netherlands for sanger sequencing. sequence data were then entered into the ncbi database for analysis. statistical analyses all statistical analyses were performed in rstudio version 1.1.383. ethical considerations oral consent was obtained from the goat farmers who participated in the study. firstly, the farmer was informed about the outline and goals of the study, as well as the potential benefits and hazards involved. farmers were informed that participation was voluntary and they could choose to withdraw their consent at any time. data protection was achieved through a coding system only known and understood by the people directly involved in the study. the goats used were subjected to blood sampling with vacutainer needles. if an animal proved difficult to obtain blood from, another goat was chosen instead. no animal was subjected to more than two sampling attempts. results antibody detection all sampled goats were negative for antibodies to bvdv, and hence, no further testing was performed. in tested cattle, 53.6% (195/364) were seropositive for border disease virus (bdv) or bvdv on ab-elisa on an individual level. seroprevalence within the tested herds ranged from 16.7% to 97.9% (table 1). all herds (100%) had at least one seropositive animal. seroprevalence in the sampled dairy farms was 50.9%, and 55.8 % in beef farms (table 2). difference between dairy and beef was not statistically significant (p = 0.40, chi-squared test). table 1: the sampled cattle farms and their seroprevalence of bovine viral diarrhoea virus antibodies. table 2: seroprevalences in dairy and beef cattle and their associated confidence interval. a total of 20 samples (5.5%) got results denoted as ‘doubtful’, according to the cut-off values provided by the manufacturer, and they were considered as negative in the analysis. antigen and virus detection one sample was positive on ag-elisa, which is equivalent to an antigen prevalence of 0.27% (1/364). on subsequent pcr runs, the sample positive on ag-elisa only resulted in weak bands, even though the procedure was repeated several times to get a more distinct result. in addition, two other samples that were negative on ag-elisa were pcr-positive. the sample that was positive on ag-elisa as well as weakly positive on pcr originated from gaborone 3 which is the herd that had the highest seroprevalence (97.9%). the two samples that were only pcr-positive originated from gaborone 1 which had the second highest seroprevalence (88.1%). seroprevalences in gaborone 1 and 3 were statistically significantly higher compared to all other herds with logistic regression performed in rstudio using the package lme4, with village and herd controlled for in the analysis (p-value < 0.001) (bates et al. 2015). genetic sequencing the pcr products from two of the viraemic animals, originating from the same herd, were sent for sequencing. the two products were 338 and 337 base pairs in length. the sequences were identical to each other and most similar to genotype bvdv-1a. the segments showed high sequence resemblance with several bvdv-1 strains in the national center for biotechnology information (ncbi) database. thetop hit, when sorted after maximum score, was usmarc-53875 (workman et al. 2015) (table 3), followed by two nadl strains (vassilev & donis 2000). the top hits originating from the african continent were five strains from mozambique isolated in 1991 and 1992, and three strains isolated in south africa (baule et al. 1997). these african strains were also the top hits when sorted after identity. table 3: information from the national center for biotechnology information blast consolidated into a table. discussion because of the potentially severe consequences of bvdv introduction into a herd, achieving control of the virus is desirable. to accomplish this, knowledge of pathogen prevalence is crucial. in this study, the antibody prevalence in cattle was 53.5% and the viral prevalence 0.83%, as assessed by ab-elisa and pcr, respectively. according to houe and meyling (1991), antibody and antigen prevalence in endemic regions is usually 60% or more and 1% – 2% or more, respectively. however, these numbers vary greatly with differences in management strategies, for example, trade routines and usage of communal pastures. previous studies conducted in southern africa have found antibody prevalences ranging from approximately 10% – 50% in asymptomatic cattle (depner et al. 1991b; handel et al. 2011; hyera et al. 1991; nigussie et al. 2010; njiro et al. 2011). these comparatively low prevalences are perhaps more likely because of differences in management strategies, for example, less intensive production systems, rather than the virus not being endemic in the region. it is also important to take into consideration that the study design was not randomised. the selection of both villages and farmers was convenience-based, and therefore, caution should be exercised when extrapolating the results to the target population. however, as the result in this study is comparable to prevalence studies conducted in other countries in southern africa, the authors believe them to be representable. the cattle samples analysed in this study were obtained as part of another research project (ramabu et al. 2018). because of this, it was not recorded whether the farmers vaccinated their animals for bvdv. it can therefore not be excluded that the detected antibody prevalence is because of vaccination rather than natural infection. however, in gaborone 1 and 3, antigen-positive animals were found, which indicates active infection. the seroprevalences in these herds were 97.9% and 88.1%, respectively. rigorous vaccination strategies can give rise to such high percentages, but as viral antigen and nucleic acids also were detected, contact with viraemic individuals is a more plausible cause. the ab-elisa that was used in this study detects antibodies for both bvdv and the closely related bdv, which is also capable of infecting both cattle and goats (nettleton 1990). because of this, it cannot be said for certain whether seropositive animals had antibodies to bvdv or bdv. however, viruses from two of the viraemic animals were sequenced as bvdv-1a. the third was positive on ag-elisa in addition to pcr, which is specific for bvdv. therefore, the observed seroprevalences in these two herds are probably because of either the presence of persistently infected animals or active infection with bvdv. also, to the authors’ knowledge, bdv has never been detected in southern africa. however, this may be because of a lack of surveys and/or failure of detection, rather than the virus being absent in the region. when utilising the ab-elisa, the negative control was continuously invalid. reportedly, this was a common problem with the elisa reader that was used for the analysis. however, for the ag-elisa, the same elisa reader was utilised without similar problems. both procedures were performed by the same person and using the same basic techniques. the problem with the negative control in the ab-elisa may have resulted in antibody prevalence values lower than the actual value. to calculate the percentage of inhibition, the sample absorbance value was divided with the mean of the two negative controls, and then multiplied with 100. therefore, with low negative control values, the percentage of inhibition value may get misleadingly high and therefore falsely classify samples as negative or doubtful when they were, in fact, positive. ideally, when an ag-positive animal is detected, testing should be repeated after 3–4 weeks to differentiate between acute (transient) and persistent infections. because of the time-lapse between sampling and analysis, it was not possible in this study. here, ag-elisa was only performed on seronegative animals in herds with at least one seropositive animal to identify persistently infected animals, characterised by being viraemic without seroconversion. however, an animal can be immunotolerant, and hence seronegative to one strain, but seropositive to a heterologous strain because of vaccination or natural infection. because of this, it is possible that some viraemic animals were missed. however, according to lindberg and alenius (1999), this risk is likely negligible. six of the antibody negative samples were not subjected to ag-elisa and pcr because the sample volumes were too low. five of these samples originated from lobatse 2, where seroprevalence was 60%, and one from ramatlabama 1, where prevalence was 25.5%. according to houe et al. (1993), seroprevalence in herds with persistent infection is usually 90% or more. it is therefore unlikely that any of these individuals were persistently infected. also, there was not enough available material to perform rna isolation and pcr on all of the seronegative samples. therefore, rna isolation and pcr were only performed on antibody negative animals in herds with an antibody prevalence of 50% or more. however, within-herd seroprevalence is influenced by a number of factors, for example, management strategies, such as contact between different groups of animals. as this information was not available for the tested cattle herds, a lower cut-off value was chosen to minimise the risk of a viraemic animal escaping identification. in this study, amplicon obtained from two pcrs was sequenced, and found to belong to the bvdv-1 genotype, which according to previous studies is more common in africa than bvdv-2 (baule et al. 1997; emran et al. 2014; kabongo, baule & van vuuren 2003; thabti et al. 2005; ularamu et al. 2013; van vuuren 2005; vilcek et al. 2000; yesilbag et al. 2017). for example, in 2013, 82.5% of samples obtained from feedlots in various parts of south africa and namibia belonged to bvdv-1, and 17.5% to bvdv-2 (ularamu et al. 2013). the two isolates sequenced in this study were identical to each other, which is not surprising, given the fact that they originated from animals in the same herd. the two segments were similar to a high number of sequences in the ncbi nucleotide database besides the ones discussed here. this is not surprising either because the sequenced part of the genome, that is, the 5′ncr, is a highly conserved region (bauermann et al. 2013; van vuuren 2005). the sequence that shared the most similarities, according to the ncbi nucleotide database, when sorted after maximum score, is usmarc-53875, followed by two nadl strains (vassilev & donis 2000) (table 3). nadl is a reference strain for bvdv-1a that originates from north america (vassilev & donis 2000). the most similar sequences from the african continent originate from mozambique and south africa. according to a review by yesilbag et al. (2017), bvdv-1a is the bvdv variant most often detected on the african continent. in the goats sampled in this study, no evidence of antibodies to bvdv or bdv could be found. previous studies conducted in other african countries have found seroprevalences of 5% in neighbouring namibia (depner et al. 1991b). in tanzania, different studies have recorded prevalences ranging from 1.7% to 24.9% (hyera et al. 1991; torsson et al. 2017). all goats sampled in this study originated from herds around gaborone, which is the same region as the viraemic cattle were found. because of this, it is unlikely that the 0% seroprevalence is because of bvdv not existing in the area. it is more likely that the goats were insufficiently exposed to the virus to develop antibodies. also, seroprevalence in goats is in general considerably lower compared to cattle. this is probably because persistent infection is an unusual occurrence in this species (bachofen et al. 2013; broaddus et al. 2007; depner et al. 1991a; passler et al. 2014). there is a great need of further research on bvdv in botswana. more knowledge is needed on the implications of bvdv in these settings and its economic significance to smallholder farmers. such studies should not only focus on the direct effects of bvdv but also on its role as an immunosuppressing co-pathogen in, for example, respiratory and enteric diseases. also, more extensive knowledge is needed on prevalence, as well as local risk factors for transmission, because this knowledge is imperative when establishing efficient and cost-effective measures for viral control. last but not least, there is a great need for extended knowledge regarding control methods applicable to the local settings. this coupled together could lead to a reduced incidence of bvdv, and thereby not only improve animal health and welfare, but also increase profits for farmers in botswana. conclusion seroprevalence of bvdv in and around gaborone was 0.0% in goats and 53.6% in cattle. the seroprevalence was 50.9% in dairy cattle and 55.8% in beef cattle; however, this difference was not statistically significant. the two herds from where the three viraemic animals originated had significantly higher seroprevalence compared to the other herds. also, two of the detected viruses belonged to the genotype bvdv-1a. acknowledgements the authors would also like to extend their sincere gratitude to the staff at the botswana university of agriculture and natural resources (buan) for all their help, and to all farmers who took part in this research. competing interests there are no competing interests. authors’ contributions s.l. performed field and laboratory work, analysed and interpreted the data, and drafted the manuscript. s.s.r. conceived, designed and supervised the study, facilitated field work and critically revised the manuscript. m.b. designed and supervised the study, interpreted the data and drafted themanuscript. j.j.w. conceived, designed and supervised the study, interpreted the data and drafted the manuscript. all authors approved the final version of the manuscript. funding information this project was sponsored by the swedish governmental agency swedish international development cooperation agency (sida) through a minor field study (mfs) scholarship, as well as a scholarship from the faculty of veterinary medicine scholarship fund (slu.ua 2016.3.1.5-914). the project was also funded by the swedish research council (grant no. 348-2014-4293). data availability statement all data will be available in the swedish university of agricultural sciences (slu) database tilda. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references bachofen, c., vogt, h-r., stalder, h., mathys, t., zanoni, r., hilbe, m. et al., 2013, ‘persistent infections after natural transmission of bovine viral diarrhoea virus from cattle to goats and among goats’, veterinary research 44(1), 32. https://doi.org/10.1186/1297-9716-44-32 bauermann, f., ridpath, j., weiblen, r. & flores, e., 2013, ‘hobi-like viruses: an emerging group of pestiviruses’, journal of veterinary diagnostic investigation 25(1), 6–15. https://doi.org/10.1177/1040638712473103 baule, c., van vuuren, m., lowings, j. & bélak, s., 1997, ‘genetic heterogeneity of bovine viral diarrhoea viruses in southern africa’, virus research 52(2), 205–220. bates, d., maechler, m., bolker, b. & walker, s., 2015, ‘fitting linear mixed-effects models using lme4’, journal of statistical software 67(1), 1–48. https://doi.org/10.18637/jss.v067.i01 broaddus, c., holyoak, r., dawson, l., step, d., funk, r. & kapil, s., 2007, ‘transmission of bovine viral diarrhea virus to adult goats from persistently infected cattle’, journal of veterinary diagnostic investigation 19(5), 545–548. https://doi.org/10.1177/104063870701900514 depner k., hübschle o.j.b., liess, b. 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infections of immature amphistomes in the small intestines of immunologically incompetent hosts (horak 1967; dutt 1980; gupta 1993). the disease is characterised by sporadic epidemics of acute parasitic gastroenteritis and loss of production associated with high mortality and morbidity particularly in young stock (butler & yeoman 1962; boray 1969; horak 1971; singh & lakra 1971; dutt 1980). 321 onderstepoort journal of veterinary research, 72:321–332 (2005) a macroand light microscopical study of the pathology of calicophoron microbothrium infection in experimentally infected cattle m. mavenyengwa1, s. mukaratirwa1, m. obwolo1 and j. monrad2 abstract mavenyengwa, m., mukaratirwa, s., obwolo, m.j. & monrad, j. 2005. a macroand light microscopical study of the pathology of calicophoron microbothrium infection in experimentally infected cattle. onderstepoort journal of veterinary research, 72:321–332 twelve tuli weaner steers aged 1 year were randomly subdivided into three groups of four animals and infected with different doses of calicophoron microbothrium metacercariae. each animal in group i received a low dose (ld) of 5 000 metacercariae, group ii a medium dose (md) of 15 000 metacercariae, group iii a high dose (hd) of 25 000 metacercariae and one additional animal was kept as an uninfected control (c). after infection, one animal from each group was slaughtered on day 28, 42, 56 and 84 post infection (pi) and samples from the ileum, jejunum, duodenum, abomasum and the rumen were collected for histopathological and cytological examination. on day 28 pi, the gross pathological lesions observed in the duodenum of the ld and the md animals were similar and comprised duodenal thickening, corrugation, hyperaemia, petechiation and ulceration. in the hd animal the duodenal lesions were similar but more severe. the abomasal folds were severely oedematous in the md group and nearly occluded the abomasal lumen. moderate oedema of the abomasal folds was also present in the ld and hd animals. the gross pathological lesions regressed in all the infected groups with increasing age of infection and had disappeared completely by day 56 pi. on day 28 pi the histopathological lesions in the duodenum and jejunum of the ld and md groups were similar, comprising subtotal villous atrophy, hyperplasia of brunner’s glands and peyer’s patches and moderate infiltration of eosinophils, mast cells and a few globule leukocytes, basophils and lymphocytes in the lamina propria. the hd group had total villous atrophy, severe hyperplasia and cystic dilatation of brunner’s glands, which had expanded to cover the entire submucosa. on day 42 pi the histopathological lesions were still present in the md and the hd groups comprising subtotal villous atrophy and hyperplasia of brunner’s glands. heavy infiltrations of eosinophils, moderate amounts of mast cells and a few basophils, globule leukocytes and lymphocytes were still present in the lamina propria of all three groups. on day 56 pi, a few glands were still cystic in the md and the hd groups. moderate cell infiltrations were still present in the lamina propria of all the three groups and by day 84 pi complete regeneration had occurred in all animals. keywords: calicophoron microbothrium, cattle, pathology, intestinal tract, metacercariae, rumen, tuli 1 faculty of veterinary science, paraclinical veterinary studies, university of zimbabwe, harare, zimbabwe 2 danish centre for experimental parasitology, frederiksberg, copenhagen, denmark accepted for publication 5 july 2005—editor infection follows ingestion of herbage heavily contaminated with amphistome metacercariae (dutt 1980; boray 1985). the adult ruminal amphistomes are not associated with clinical disease and have been described as commensals living on ruminal contents of the host (boray 1985; cheruiyot & wamae 1988). the amphistome species responsible for the majority of outbreaks of immature amphistomosis in ruminants and their related snail intermediate hosts have been recorded in many countries (dinnik & dinnik 1962; dinnik 1964; horak 1971; dutt 1980; over 1982; boray 1985; eduardo 1982a, 1983, 1988; sey 1991; mukaratirwa, siegismund, kristensen & chandiwana 1996; mukaratirwa, kristensen, siegismund & chandiwana 1998; chingwena, mukaratirwa, kristensen & chimbari 2002; madzingira, mukaratirwa, pandey & dorny 2002). in zimbabwe, amphistome species recovered from domestic and wild ruminants include calicophoron calicophorum, calicophoron microbothrium, calicophoron clavula, calicophoron phillerouxi, calicophoron sukumum, calicophoron raja, paramphistomum cervi, cotylophoron cotylophorum and cotylophoron jacksoni (eduardo 1982a, 1983, 1988; jooste 1989; sey 1991; madzingira et al. 2002). in southern africa, including zimbabwe, c. microbothrium, with the snail species bulinus tropicus as the main intermediate host, has a wide distribution and has been incriminated in the majority of outbreaks of immature amphistomosis in ruminants (dinnik & dinnik 1962; swart & reinecke 1962a; dinnik 1964, 1965; horak 1971; over 1982; chingwena et al. 2002). the clinical and pathological manifestations of amphistomosis have been studied in both field and experimental infections particularly in sheep and goats (horak & clark 1963; deorani & katiyar 1967; horak 1967; boray 1969; panda & misra 1980; singh, sahai & jha 1984; rolfe, boray & collins 1994). studies in cattle are limited and information on the clinical and pathological manifestations of the disease has been derived mainly from field outbreaks (butler & yeoman 1962; singh & lakra 1971; kelly & henderson 1973) and no experimental infections have been conducted to follow the course of the disease and establish its pathogenesis. the objective of this study was to describe the pathology of c. microbothrium in experimentally infected cattle. materials and methods study area and selection of experimental animals the study was conducted at grassland research station, a state owned farm in marondera about 80 km south east of harare, zimbabwe. the farm is amphistome-free and mainly raises tuli cattle for commercial beef production. it is subdivided into paddocks where cattle can graze and have access to clean borehole water provided in troughs. prior to the commencement of the study, faecal samples were collected from 30 1-year-old tuli weaner steers for coprological examination using the sedimentation technique and the modified mcmaster technique (maff 1986) to screen for trematode and nematode infection respectively. thirteen animals were randomly selected for the study from the parasite-free weaner steers, on the basis of age and mass. the animals selected were allowed to graze in paddocks and had access to clean borehole water provided in concrete troughs and were dewormed every month using 10 % fenbendazole. identification of c. microbothrium and harvesting of metacercariae adult b. tropicus naturally infected with c. microbothrium were collected from a single natural habitat and maintained under laboratory conditions for periodic shedding of metacercariae. the identity of the c. microbothrium metacercariae was confirmed through the infection of a single sheep and recovery of mature amphistomes from the rumen for identification. the amphistomes were identified as c. microbothrium using the methods described by eduardo (1982b, 1983) and gupta (1993). for mass production of metacercariae and to avoid the ingestion of the shed metacercariae by the snails, small nylon gauze bags with an aperture size of 2 mm, each capable of holding 20 snails, were used to confine snails during shedding. the open end of each snail bag was stapled close. each bag was immersed in individual plastic containers holding 500 ml of dechlorinated pond water and exposed to direct sunlight for 3 h. the cercariae encysted at the water meniscus. shedding was induced every 2 days whereafter the snails were placed back in the dark aquaria. the metacercariae were stored in individual plastic containers with dechlorinated pond water added to just below the level of the band of metacercariae. they were kept at a temperature of between 24–25 °c for a minimum of 322 pathology of calicophoron microbothrium infection in experimentally infected cattle 6 days before they were harvested with a fine toothbrush and dosed to the experimental animals. infection of experimental animals the numbers of metacercariae dosed to cattle in this study were an extrapolation from rolfe, boray, nichols & collins (1991) and metacercaria viability studies conducted by swart & reinecke (1962b), which showed that less than 50 % of metacercariae administered to ruminants survive. three dosing regimens were therefore designed: each animal in group i received 5 000 metacercariae and acted as the low dose group (ld), in group ii each animal received 15 000 metacercariae representing the medium dose group (md) and in group iii each animal received 25 000 metacercariae representing the high dose group (hd). the dose for individual animals was estimated by counting the metacercaria in several aliquots, as described by rolfe et al. (1994) and was administered by direct inoculation into the rumen using a trocar and canula. one animal remained as an uninfected control (c). the animals were monitored daily for the development of clinical signs. one animal from groups i–iii was randomly selected, stunned and slaughtered on day 28, 42, 56 and 84 post infection (pi). the control animal was slaughtered on day 28 and used as a reference for comparing pathological lesions. sample collection from slaughtered animals a midline incision from the xyphoid cartilage to the inguinal region was made and the rumen and intestines were exposed by cutting through and reflecting the abdominal and paracostal muscles on either side of the midline incision. the small intestines of each animal slaughtered were separated from the rest of the organs with minimal manipulation. starting from the distal end of the jejunum, one metre long small intestinal loops were tied at both ends, cut and placed in labelled plastic containers for fluke recovery. this was followed by the collection of samples of the intestines for histopathological examination. the samples were rinsed in physiological saline to remove excess faecal material before each segment was preserved in bouin’s fixative. samples were also removed from the cranial ruminal pillar and the fundus of the abomasum and preserved in bouin’s fixative fluid. fluke recovery to determine the distribution of flukes established along the small intestines, the intestinal segments were scraped to the level of the muscular tunic using glass slides to recover the embedded flukes. the contents of the abomasum, omasum and the reticulum were washed into separate containers and serially sieved under pressure through 2400 µm and 850 µm sieves, respectively. the intestinal mucosal scrapings were similarly sieved and the flukes were collected after sedimentation at the bottom of a jar of 10 l capacity. excess water was decanted and the recovered flukes were preserved in 70 % alcohol for identification and enumeration. gross pathology at each necropsy the intestinal portions were opened separately, examined for gross pathological lesions before their mucosal surfaces were scraped to remove embedded flukes (boray 1969). in assessing the gross pathology of the small intestine, the duodenum, proximal jejunum, distal jejunum and the ileum were demarcated on the basis of their distances from the pylorus. the duodenum constituted the first meter from the pylorus, the proximal jejunum extended for 3 m from the distal end of the duodenum while the distal jejunum extended for 4 m from the distal end of the proximal jejunum. the ileum extended up to the ileo-cecal junction. a checklist of gross pathological lesions was drawn up based on the severity of abomasal oedema, intestinal wall thickening, mucosal corrugation, hyperaemia, petechiation and ulceration. the severity of the gross pathological lesions present were compared between the different experimental groups. histopathology the samples preserved in bouin’s fluid were embedded in paraffin wax. triplicate 4 µm thick sections were cut from each segment of the small intestines, abomasum and rumen and each section stained with haematoxylin and eosin (h&e), toluidine blueferric sulphate-ferrioxamine b or giemsa-chromotrope stain. the toluidine blue stain was used to differentiate mast cells while the giemsa-chromotrope stain was used to differentiate eosinophils and basophils. the h&e stain was used to stain globule leukocytes and the evaluation of general histopathology of the gastrointestinal regions. similar to the assessment of the gross pathology, a checklist of histopathological lesions was drawn up based on the severity of villous atrophy, cryptal hyperplasia, goblet cell hyperplasia, dilatation of brunner’s glands and hyperplasia of peyer’s patches. the histopathological lesions observed were compared between the different experimental groups. 323 m. mavenyengwa et al. data analysis the villous crypt unit method for counting eosinophils, mast cells, basophils and globule leukocytes as described by miller & jarret (1971) was used. for each intestinal segment, three intact villous crypt units were identified and the different cell types estimated. the populations of tissue eosinophils, mast cells, globule leukocytes and basophils in the different intestinal sections were estimated using a four point scale (0, +, ++, +++). results clinical signs animals in the ld and c groups remained clinically normal during the study period. an effortless and voluminous foul smelling projectile diarrhoea developed in three of the four animals in the md group on day 21 pi and persisted for 3 days in two of the animals. in the third animal, the diarrhoea persisted for a week and the animal became inappetant, dehydrated, emaciated, lagged behind the group and never fully recovered until it was slaughtered on day 56 pi. no clinical signs were evident in the fourth animal in the group. in the hd group, a non-projectile diarrhoea developed in all animals. the diarrhoea developed on day 21 pi and persisted for 3 days before all the animals recovered. thereafter no clinical signs or changes in body condition were observed in any of the animals until day 84 pi when the last animal in the group was slaughtered. fluke recovery and distribution the distribution patterns of flukes recovered from the various gastrointestinal sites among the different experimental groups on day 28 pi are presented in fig. 1. in the ld group, 781 (15.6 %) of juvenile flukes were recovered from the different regions of the gut of the animal slaughtered on day 28 pi. of the recovered flukes, 22.4 % were from the proximal third of the duodenum, 7.9 % from the abomasum and the remaining 69.7 % from the rumen along the oesophageal groove and the cranial pillar of the rumen. in the md group, 4 044 (27 %) of juvenile flukes were recovered from the various intestinal sites of the animal slaughtered on day 28 pi. of the flukes present, 8.3 % occurred in the proximal third of the duodenum, 4.9 % in the abomasum and the remaining 86.8 % were recovered along the oesophageal groove and the cranial pillar of the rumen. in the hd group, 965 (4 %) of juvenile flukes were recovered from the various gut sites the animal slaughtered on day 28 pi. of the flukes present, 44.4 % were recovered from the proximal half of the duodenum, 35.3 % from the abomasum, 10.4 % from the omasum and the remaining 10 % from the oesophageal groove and the cranial pillar of the rumen. all the flukes were recovered from the cranial pillars of the rumen of all the animals slaughtered on days 42, 56 and 84 pi, and the percentage that had become established in the slaughtered animals is presented in fig. 2. the percentage of flukes recovered at each slaughter occasion varied between animals, with recovery rates ranging from 4 % to 56 %. 324 pathology of calicophoron microbothrium infection in experimentally infected cattle � �� �� �� �� �� �� � � �� ����� �� ����� ������ ����� �������� ���� ����� � � � � � �� � �� �� � �� � �� �� � � ��� �� �������� �� ������ �� � �� �� �� �� �� �� � � ����� ���� ����� � � � � � �� � �� �� � �� � �� �� � � ��� �� �������� �� ������ �� �� �� � fig. 1 distribution patterns of recovered calicophoron microbothrium (%) in cattle infected with different doses of metacercariae and slaughtered on day 28 post infection fig. 2 percentage establishment of calicophoron microbothrium recovered from the rumen of cattle infected with different doses of metacercariae and slaughtered on day 42, 56 and 84 post infection pathology gross pathology the gross pathological lesions of the gut observed in all the infected groups at different stages of infection were confined to the abomasum, the duodenum and the jejunum and are summarized in table 1. histopathology the histopathological lesions of the gut observed in all the infected groups at different stages of infection were confined to the duodenum and the jejunum and were characterized by villous atrophy, goblet cell hyperplasia, cystic dilatation of brunner’s glands and hyperplasia of peyer’s patches. there was subtotal villous atrophy in the duodenum of the ld animal slaughtered on day 28 pi. the villous atrophy was characterized by fusion of villi, transformation of absorptive enterocytes into low cuboidal, and moderate hyperplasia of the crypts which were elongate and emptying into the intestinal lumen. the goblet cells were hyperplastic and occurred in large numbers in the crypts and along epithelia of the villi. necrotic debris and free-lying flukes were observed in the lumen. the lamina propria was moderately infiltrated by eosinophils, mast cells and a few globule leukocytes, basophils and lymphocytes. in the submucosa brunner’s glands were hyperplastic, and occupied nearly half of the submucosal area. some cystic glands were surrounded by eosinophils and a few lymphocytes. the peyer’s patches were hyperplastic and expanded into the mucosal area in some sections. in the jejunum, subtotal villous atrophy characterized by shortening and blunting of the villi with elongate crypts emptying into the intestinal lumen was present. the lamina propria was mildly infiltrated by eosinophils, mast cells and low levels of globule leukocytes and basophils. the duodenal and jejunal lesions in the md animal were similar to those in the ld animal slaughtered on day 28 pi. however, infiltration of eosinophils and mast cells into the lamina propria tended to be heavier. in the hd animal, brunner’s glands were hyperplastic and cystic and had expanded to cover the entire submucosal area on day 28 pi. the cystic glands were surrounded by an intense eosinophilic cell reaction and a few lymphocytes. total villous atrophy characterized by severe shortening of the villi and elongate crypts opening into the duodenal lumen was evident. goblet cell and peyer’s patch hyperplasia were present and comparable to those in the ld and md animals. the lamina propria in both the duodenum and the proximal jejunum was heavily infiltrated by eosinophils, moderate amounts of mast cells and a few basophils, globule leukocytes and lymphocytes. the histopathological lesions in the distal jejunum were similar to those in the ld and md animals slaughtered at the same time. on day 42 pi the duodenal glands of the ld animal were hyperplastic, non-cystic and contained little mucin. moderate villous atrophy and goblet cell hyperplasia were still present. the lamina propria was moderately infiltrated by eosinophils, mast cells, lymphocytes and a few globule leukocytes while basophils were not present. similar cell infiltrations were present in the jejunum. in the md animal, the submucosal area of the duodenum was expanded to three times the size of the control animal and half the space was occupied by the hyperplastic brunner’s glands. some of the glands were cystic and contained mucin. an intense eosinophilic cell reaction was present in the submucosa and around the cystic glands. subtotal villous atrophy and mild goblet cell hyperplasia was still present along the crypts and epithelia of the villi. the lamina propria was heavily infiltrated by eosinophils, mast cells and low numbers of globule leukocytes and basophils. in the jejunum, subtotal villous atrophy was still present. the lamina propria was heavily infiltrated by eosinophils, moderate amounts of mast cells and lymphocytes and low numbers of globule leukocytes. in the hd animal, the duodenal and jejunal lesions were similar to the md animal. however, infiltration of eosinophils and mast cells into the lamina propria was moderate and comparable to the ld animal. on day 56 pi, the villi were elongate and leaf-like with normal epithelia in both the duodenum and jejunum of the ld, md and the hd animals. some brunner’s glands were still cystic in the md and hd animals. goblet cell numbers had reduced to minimal levels in the majority of sections, while regenerative fibrous tissue deposition became evident in the lamina propria and in the submucosal area of all animals. the lamina propria of the md animal was, however, still infiltrated by large numbers of eosinophils, lymphocytes and moderate numbers of mast cells in comparison to the ld and the hd animals. on day 84 pi, the villi in both the duodenum and the jejunum of all infected animals were elongate and 325 m. mavenyengwa et al. 326 pathology of calicophoron microbothrium infection in experimentally infected cattle days pi group gut site main gross lesions 28 ld abomasum mild oedema of abomasal folds present but only appreciated on cut surfaces md severe oedema of abomasal folds present, abomasal folds swol len and nearly occluded the lumen hd moderate oedema of abomasal folds present, abomasal folds moderately swollen and could be easily flipped over c no gross lesions observed ld, md duodenum and duodenal wall thickened and corrugated. height from muscularis jejunum mucosa to tip of mucosal rugae varied from 8 mm in the proximal duodenum to 4 mm in the distal end. the duodenal and jejunal contents were watery and mucoid hd duodenal wall severely thickened and corrugated. height from muscularis mucosa to tip of mucosal rugae varied from 10 mm in the proximal duodenum to 4 mm in the distal end. the duodenal and jejunal contents similar to the ld and md groups c no lesions detected. duodenal wall thickening and corrugation absent. height from muscularis mucosa to tip of mucosal rugae varied from 3 mm in the proximal duodenum to 2 mm in the distal distal end. duodenal and jejunal contents normal 42 ld, c abomasum no lesions detected md, hd mild oedema of abomasal folds present but only appreciated on cut surfaces ld, md, hd duodenum and mild thickening and corrugation of duodenal wall. height from jejunum muscularis mucosa to tip of mucosal rugae varied from 5 mm in the proximal duodenum to 3 mm in the distal end. duodenal and jejunal contents viscous with mucus floccules present c no lesions detected 56 ld, hd, c abomasum no lesions detected md mild oedema of abomasal folds present but only appreciated on cut surfaces ld, md, hd duodenum and mild thickening and corrugation of duodenal wall. height from jejunum muscularis mucosa to tip of mucosal rugae varied from 5 mm in the proximal duodenum to 3 mm in the distal end. duodenal and jejunal contents viscous with mucus floccules present c no lesions detected 84 ld, md, abomasum, no lesions detected hd, c duodenum and jejunum md rumen anterior fragmentation of papillary tips, flukes firmly attached to the base pillar of the ruminal papillae. foci of papillae inhabited by the flukes were pale and some of the papillae had fallen off the ruminal wall leaving non-ulcerated surface patches ld, hd some papillae had fragmented tips but maintained their normal colour c no lesions detected ld = low dose md = medium dose hd = high dose c = control table 1 summary of gross pathological lesions of the abomasum, duodenum and the jejunum of groups of cattle experimentally infected with different doses of calicophoron microbothrium and slaughtered on different days post infection covered with normal epithelium. the lamina propria was infiltrated with mainly lymphocytes, fibrocytes and a few eosinophils. brunner’s glands were normal in the ld and md animals while in the hd animal a few were still cystic and surrounded by lymphocytes. histopathological lesions of the rumen were evident on days 56 and 84 pi in the md animals and were characterized by thickening and vacuolation of the stratum corneum. in areas where the papillae had fallen off due to parasite feeding and anchorage, microabcesses were present confined to the stratum corneum. a mild cell reaction comprising scattered eosinophils and a few lymphocytes was present around the affected areas at the dermal-epidermal junction. no significant histopathological lesions were seen in the rumen of the ld, hd and the control animals at any stage of infection. discussion the pathology of amphistome infection has been systematically studied in goats and sheep (horak & clark 1963; singh et al. 1984; rolfe et al. 1994) in which experimental infections were conducted and the disease followed until after patency. besides a few reports which describe the pathology of amphi stomes in cattle following acute field outbreaks (butler & yeoman 1962; singh & lakra 1971; kelly & henderson 1973) this study is the first in cattle, where periodic tissue alterations following infection with different doses of c. microbothrium metacercariae are described. the clinical signs exhibited by the infected cattle in the present study are similar to experimental and field observations made in other reports (butler & yeoman 1962; horak & clark 1963; horak 1967; deorani & katiyar 1967; boray 1969; singh & lakra 1971; panda & misra 1980; rolfe et al. 1994; pillai & alikutty 1995). however, polydypsia as described in sheep by horak & clark (1963) and by boray (1969) was not observed and could have been missed since the study animals were not constantly under observation. the occurrence of clinical amphistomosis and subsequent pathology in ruminants is dependent on the dose, the virulence and the establishment levels of the infecting metacercaria in the small intestine of the host (horak 1966, 1967; rolfe et al. 1994). the doses of metacercariae used in this study managed to reproduce the clinical disease. the parasite establishment rates obtained for the ld and md groups are comparable to those obtained by horak (1967) in sheep and induced the expected clinical trends of the disease. however, parasite establishment in the hd group was low and could be due to either complete failure of parasites to establish or overcrowding of the immature amphistomes in the small intestine and subsequent expulsion of unattached parasites by the host during the diarrhoeic phase of the infection (boray 1969). the highest concentration of immature amphistomes was recovered from the small intestines of the hd group making the later theory more likely. it is also evident that fluke migration to the rumen was slow in the hd group and only a small proportion of the flukes had migrated to the rumen by day 28 pi. similar trends have been observed in sheep and cattle heavily infected with immature amphistomes (horak 1967; boray 1971; rolfe et al. 1994). in this study, the gross pathological lesions were confined to the jejunum, duodenum and the abomasum of all infected groups and this is related to the migratory habits of the immature flukes which upon establishment in the duodenum migrate to the rumen through the abomasum and omasum causing tissue destruction (boray 1969; singh & lakra 1971; rolfe et al. 1994). similar lesion distribution has also been observed in cattle (butler & yeoman 1962; singh & lakra 1971; kelly & henderson 1973) and in sheep (horak & clark 1963; deorani & katiyar 1967; horak 1967; boray 1969). the intensity of lesions appeared correlated with the establishment rate and subsequent migration of immature flukes to the rumen. large numbers of immature flukes in the duodenum have been reported to cause overcrowding and competition among the parasites which results in delayed migration and exacerbates the disease (horak 1967; boray 1971; rolfe et al. 1994). at the same time, poorly attached flukes, particularly those which excyst beyond the duodenal area, are ejected (horak 1971). this scenario probably explains the massive duodenal pathology observed in the hd group compared to the other infected groups. although overall fluke establishment was low in the hd group, the highest number of immature flukes was recorded in the duodenum of this group. the duodenal and abomasal changes observed in the infected groups in this study are similar to observations made by butler & yeoman (1962), singh & lakra (1971) and kelly & henderson (1973) in cattle. the immature flukes cause tissue destruction by 327 m. mavenyengwa et al. anchoring and burrowing into the intestinal mucosa during migration to the submucosa for sojournment in brunner’s glands (deorani & katiyar 1967; boray 1969; singh & lakra 1971). the lesions were present until day 42 pi after which they regressed and by day 84 pi no appreciable differences in gross pathology could be detected between the infected and the control groups. the disappearance of the lesions coincided with the completion of migration of the immature amphistomes from the duodenum to the rumen, confirming earlier reports that the pathogenic mechanisms of amphistomes are related to duodenal damage caused the by immature flukes (horak 1971). mature amphistomes are considered non-pathogenic in the rumen (cheruiyot & wamae 1988), but the finding of ruminal lesions in this study seem to suggest that when present in large numbers they may cause disease directly or indirectly through systemic absorption of ruminal microflora via necrotic papillary ends. however, the dose of metacercariae used in this study appeared insufficient to cause clinical ruminal dysfunction. the ruminal lesions observed in this study have also been described in sheep and goats (mukherjee & deorani 1962; singh et al. 1984; rolfe et al. 1994). the ruminal lesions were pronounced on day 84 pi particularly in the md group from which a large number of flukes were recovered. this is probably attributable to the attainment of maturity and the crowding effect at a single predilection site by the flukes (horak 1971). histopathological examination of the duodenal mucosa did not reveal any flukes embedded in brunner’s glands in any of the infected groups as has been observed in sheep and goats (varma 1961; rolfe et al. 1994). brunner’s glands provide an ideal environment for fluke growth until they become mature enough to withstand the acid environment in the abomasum during migration to the rumen (deorani & katiyar 1967; boray 1969; deorani & jain 1969). brunner’s glands were, however, cystic with an atrophied basal epithelium suggesting prior occupation. some flukes, however, could be seen in the mucosal tissue probably migrating to the duodenal surface. the absence of flukes embedded in brunner’s glands is probably related to the design of the study in which the animals were slaughtered late in the course of the infection after parasitic migration to the rumen had commenced. there was glandular enlargement with severe villous atrophy characterized by destruction of absorptive enterocytes, fusion of villi and subsequent cryptal hyperplasia as reported in sheep (rolfe et al. 1994). villous atrophy characterized by villous and cryptal epithelial differentiation, hyperplasia and loss of function is a known feature of helminth infections and its mechanisms are obscure but could be related to immunological mechanisms against gastrointestinal parasites (miller 1984). similarly, with amphistome infections, the mechanisms of villous atrophy are unknown but have been ascribed to either traumatic destruction of tissues or enzymatic dissolution of the mucosa by the migrating flukes (singh & lakra 1971). villous atrophy together with cystic dilatation of brunner’s glands, hyperplasia of lymphoid follicles and marked mixed cellular infiltration into the lamina propria were probably responsible for the duodenal thickening and corrugation of the mucosa observed grossly in this study. the severity of the lesion appeared correlated with the magnitude of the dose of metacercariae as lesion intensity tended to increase with increasing dose and this could be related to the different levels of sensitization that occur following metacercaria excystment and subsequent migration to the rumen. in this study, the migration of immature flukes into the duodenal submucosa induced an acute inflammatory reaction characterized by an infiltration of eosinophils, mast cells, basophils, globule leukocytes, lymphocytes and macrophages into the mucosal parenchyma. the goblet cell population also increased and the cells appeared active. similar responses have been observed as a feature of gastrointestinal nematode infection in sheep (huntley, patterson, mackellar, jackson, stevenson & coop 1995; pfeffer, douch, shaw, gatehouse, rabel, green, shirer, jonas & bisset 1996; winter, wright & lee 1997). the increase in the number of the various cell types infiltrating the intestinal mucosa during helminth infections has been associated with the development of resistance to parasitic infections, but the mechanisms of the resistance remain unclear (huntley et al. 1995). the response of eosinophils, mast cells and goblet cells to c. microbothrium infection occurred at the same time since their kinetic pattern appeared similar. the cells were seen in significant numbers in the mucosa from day 28 pi, peaked on day 42 pi and were maintained at more or less this level till day 56 when they subsided in all the infected groups. this cellular response coincided with the migration of parasites from the duodenum to the rumen. a severe infiltration of tissue eosinophils was observed in the mucosa and the submucosa around the dilated brunner’s glands. a similar eosinophil infiltration pattern has been observed in sheep and 328 pathology of calicophoron microbothrium infection in experimentally infected cattle goats (deorani & katiyar 1967). tissue eosinophilia has been reported to be a feature of helminth infections (rainbird, macmillan & meeusen 1998). the eosinophils, following activation by th2 cellderived interleukin-3 and interleukin-5 and through the mediation of parasite-induced ige, igg and complement c3b (butterworth 1984; rainbird et al. 1998 ) can bind to the parasite surface and degranulate to release potent biological molecules including major basic protein, eosinophilic cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase and toxic oxygen-derived metabolites such as superoxide anions, hydroxyl radicals, hydrogen peroxide and singlet oxygen (mclaren, mckean, olsson, venge & kay 1981; butterworth 1984; jones 1993; behm & ovington 2000; falcone, pritchard & gibbs 2001). the biological products released form a potent helminthicidal system believed to be responsible for parasite damage (spry 1988; behm & ovington 2000). considering the eosinophil antiparasite mechanisms as hypothesized by several workers (mclaren et al. 1981; butterworth 1984; jones 1993; behm & ovington 2000; falcone et al. 2001) to be valid, the presence of high numbers of eosinophils in early infection in the duodenum in this study, would be regarded as part of the host defence mechanism against c. microbothrium infection. despite the presence of severe lesions in the duodenum of the hd group the mucosal eosinophilic cell infiltration was not as heavy in this group on day 42 pi as compared to the md group. it might be possible that the majority of the eosinophils could have been lost through desquamation and exfoliation during the antiparasite reaction that followed. once in tissues the eosinophils can survive for several weeks (spry 1988; behm & ovington 2000) and this might probably explain their continued presence in the duodenum despite the completed migration of the parasite to the rumen. intestinal mastocytosis has been associated with helminth infections in sheep (miller 1984; huntley, newlands & miller 1984; huntley 1992) and similar to the eosinophils, the role of mastocytes remains unclear. in this study, the mucosal mast cell infiltration peaked at the same time as the eosinophils, showing a similar kinetic pattern. such a kinetic pattern would be expected as the two cells are interrelated in their functional roles during helminth expulsion. the mast cells, following activation by th2 cellderived interleukin-3 and interleukin-5 and crosslinkage of parasite antigens to mast cell surface bound ige receptors (lee, swieter, & befus 1986; falcone et al. 2001; kawakami & galli 2002) degranulate and release vasoactive amines including histamin, serotonin, proteases and leukotrines c, d and e, collectively known as slow reactive substances of anaphylaxis (srs-a) (lee et al. 1986). degranulation of mast cells provokes a type-i hypersensitivity reaction characterized by smooth muscle contraction and increased vascular permeability and subsequent influx of fluids into the gut making the environment hostile to the parasite (miller 1992; falcone et al. 2001). in this study the goblet cell population also increased at the same time as the mast cells and eosinophils increased. goblet cell hyperplasia has been reported in the intestines of parasitized animals, particularly in sheep (miller 1984) and the mucin component of their secretory products intermingles with other epithelial secretory products like iga and ige antibodies resulting in an increase in the viscosity of intestinal contents. the increased viscosity fixes the parasite for antibody binding and subsequent lethal attack by the effector cells (miller 1984). in this study, increased fluidity and viscocity of the gut contents were observed till day 42 pi and could be related to the activities of the mast and goblet cell populations. such mechanisms would be expected to be important in the rejection of immature amphistomes excysting in the distal jejunum and ileum where the environment is unsuitable for growth and subsequent migration to the rumen. the origin of the globule leukocyte is controversial (gregory 1979), but it has been reported to occur in high numbers in sheep infected with gastrointestinal helminths (miller 1984; huntley 1992). in this study the globule leukocyte levels had a kinetic pattern similar to those of the mast cell and the eosinophils, but in lower intensities. the levels of the globule leukocyte cell infiltration seem to suggest that while the globule leukocyte might be important in sheep where it is associated with the development of resistance to parasitic infections (douch, harrison, elliott, buchanan, & greer 1986; douch & morum 1993; stankiewicz, jonas, douch, rabel, bisset, & cabaj 1993; rolfe et al. 1994; pfeffer et al. 1996) the same cannot be said for cattle infected with amphistomes. the globule leukocyte kinetic pattern resembled that of mast cells. this could be expected considering the theory of its derivation as described by huntley et al. (1984) where the globule leukocyte is derived from the mast cell. while the composition of the granular contents of the globule leukocyte is more or less similar to that of the mast cell granules, the mechanisms of how the globule leukocyte is involved in resistance are unknown. 329 m. mavenyengwa et al. some authors, however, have linked the globule leukocyte mechanism of action to a type-i hypersensitivity reaction (stankiewicz et al. 1993). like the globule leukocyte, basophil numbers were found in low numbers and they appeared to be of minor importance in cattle infected with c. microbothrium. presently there is very little evidence that basophils are involved in ruminant defence mechanisms against gastrointestinal parasites. however, basophils have been seen in large numbers in guinea pigs infected with trichostrongylus colubriformis (rothwell & dineen 1972; rothwell & love 1975). although the number of experimental animals used in the present study was limited, the study has demonstrated the chronology of pathological lesions which occur in cattle experimentally infected with different doses of c. microbothrium. the chronology of pathological lesions observed in the present study are similar to those described in goats experimentally infected with paramphistomum cervi (singh et al. 1984) and in sheep experimentally infected with p. ichikawai (rolfe et al. 1994). however, this study might not have mimicked field conditions where infection gradually builds up resulting in variations of the lesions induced by the parasite. acknowledgements danida through the enreca-livestock helminths research project is acknowledged for the provision of funds. thanks also goes to all support staff in the department of paraclinical veterinary studies, university of zimbabwe, for their assistance with the collection of samples, and to the staff in the histology section of the central veterinary laboratory for the production of tissue slides and the staff at blair research 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onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of onderstepoort journal of veterinary research. we appreciate the time taken to perform your review(s) successfully. alan kemp anna baauw annelise botes armanda d. bastos benson c. iweriebor caryn l. shacklock chantel j. de beer colleen t. downs darshana morar davies m. pfukenyi deon bakkes donofrio gaetano emilia bagnicka emmanuel s. swai ezekiel omoshaba faez f. abdullah faith ebhodaghe felix nchu francesco mira gian m. cosseddu harold salant hassan y.a.h. mahmoud hussain makun jelena gotić joyce siwila khalid m. gaafar krishnamoorthy paramanandham kristina roesel lia s. rotherham manal s. ismail marc drillich marietjie venter marshet a. mitiku matthias nemeth michael chimonyo mithilesh singh mohammed el khasmi motlatso t. hlokwe mpho w. phoofolo nicola beesley nompumelelo shange patrick unger paul k. njuki paul o. odeniran rajinder saini remo lobetti renata piccinini ricky chazya rolf k. schuster saber raeghi siyavuya bulani snorre stuen sohail h. khan solomon n. karshima tatjana sitt 00 acknowledgement to reviewers http://www.ojvr.org� https://ojvr.org https://ojvr.org https://ojvr.org https://ojvr.org/index.php/ojvr/user https://ojvr.org/index.php/ojvr/user mailto:publishing@aosis.co.za abstract introduction amphistome species infecting ruminants in east and southern africa snail intermediate hosts epidemiological features of amphistome infections in ruminants in east and southern africa impact on production diagnosis control conclusions acknowledgements references about the author(s) davies m. pfukenyi faculty of veterinary science, university of zimbabwe, zimbabwe school of life sciences, university of kwazulu-natal, south africa samson mukaratirwa school of life sciences, university of kwazulu-natal, south africa citation pfukenyi, d.m. & mukaratirwa, s., 2018, ‘amphistome infections in domestic and wild ruminants in east and southern africa: a review’, onderstepoort journal of veterinary research 85(1), a1584. https://doi.org/10.4102/ojvr.v85i1.1584 review article amphistome infections in domestic and wild ruminants in east and southern africa: a review davies m. pfukenyi, samson mukaratirwa received: 02 nov. 2017; accepted: 11 sept. 2018; published: 18 oct. 2018 copyright: © 2018. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in this article, the main amphistome species infecting domestic and wild ruminants in east and southern africa, their snail intermediate hosts and epidemiological features are reviewed and discussed. twenty-six amphistome species belonging to nine genera from three families occur in domestic and wild ruminants in the region under review and over 70% of them belong to the genera calicophoron, carmyerius and cotylophoron. of the amphistome species, 76.9% are shared between domestic and wild ruminant hosts – an important observation when considering the different options for control. seven freshwater snail species belonging to four genera from two families act as intermediate hosts of the identified amphistome species, with the genus bulinus contributing 57% of the snail species. some of the snails are intermediate hosts of amphistome species belonging to the same genus or to different genera; a phenomenon not yet fully elucidated as some snails are reported to be naturally infected with amphistome cercariae of unidentified species. only nine (34.6%, 9/26) of the amphistome species have known snail intermediate hosts, while most (65.4%, 17/26) have unknown hosts. species of intermediate hosts and the potential of the flukes to infect these hosts, the biological potential of the snail hosts, the definitive hosts management systems and their grazing habits are considered to be the main factors influencing the epidemiology of amphistomosis. based on the epidemiological features of amphistome infections, various practical control options are discussed. further research is necessary to determine amphistome–snail associations, develop diagnostic tests that can detect prepatent infections in the definitive host, determine the burden and economic importance of amphistomosis in domestic and wild ruminants and the efficacy of different anthelmintics in the treatment of patent infections. introduction amphistomosis is a disease of domestic and wild ruminants caused by digenetic trematodes of the superfamily paramphistomoidea fischoeder, 1901 (lotfy et al. 2010). the superfamily has a cosmopolitan distribution and is composed of hundreds of species belonging to 12 families (jones 2005). given their ubiquity and their abundance within hosts, it seems likely that the importance of these flukes is underestimated globally (lotfy et al. 2010). various species of the different paramphistomoid families, especially members of paramphistomidae and gastrothylacidae, cause amphistomosis among ruminants. the disease is caused by a severe infection with immature flukes in the small intestines of immunologically incompetent hosts. the amphistomes are responsible for lower nutrition conversion and result in a loss of weight and/or a decrease in milk production, causing great economic losses (horak 1971). however, most reports on the disease do not quote the responsible amphistome species as they are difficult to identify from a systematic point of view (horak 1971). calicophoron microbothrium is probably the biggest cause of this disease in africa (dinnik 1964a). knowledge of the different amphistome species infecting domestic and wild ruminants facilitates a better understanding of the amphistome–host associations and the epidemiology of the disease. a wide range of gastropods belonging to the genera bulinus müller 1781, biomphalaria preston 1910, ceratophallus brown and mandahl-barth 1973 and galba müller 1774 act as the intermediate hosts of amphistomes in africa (dinnik 1961, 1965; dinnik & dinnik 1954; southgate et al. 1989; wright, southgate & howard 1979). the prevalence of snail-borne diseases such as amphistomosis is influenced by both the abundance of infected definitive hosts and the abundance and efficiency of the snail intermediate hosts. hence, the epidemiology and seasonal patterns of infection with amphistomes is determined to a large extent by the availability of the snail intermediate hosts and the grazing habits of the definitive hosts (horak 1971; rolfe et al. 1991). information on the snail hosts of different amphistome species is essential as knowledge of the amphistome–snail associations has an influence on amphistomosis epidemiology and control. in this review, to avoid confusion, genera of parasites and snail hosts have been abbreviated using the first three letters of the genus name and these include: for amphistomes – bilatorchis (bil.), calicophoron (cal.), carmyerius (car.), choerocotyloides (cho.), cotylophoron (cot.), gastrothylax (gas.), gigantocotyle (gig.), orthocoelium (ort.) and stephanopharynx (ste.) and for snail hosts – biomphalaria (bio.), bulinus (bul.) and ceratophallus (cer.). the authorities of the digenean families and species and that of the snail species referred to in this review can be found in table 1. table 1: checklist of amphistome species and their ruminant and snail intermediate hosts reported in east and southern african countries. in this paper, we review the information available to date on amphistome species infecting domestic and wild ruminants in east and southern african countries, the snail intermediate hosts, as well as the epidemiology of amphistomosis and available control options. amphistome species infecting ruminants in east and southern africa reported amphistome species and their respective domestic and wild ruminant hosts in east and southern african countries are shown in table 1. data show that the documented species belong to four families: choerocotyloididae, gastrothylacidae, paramphistomidae and stephanopharyngidae, and nine genera: one from choerocotyloididae (choerocotyloides prudhoe, yeh & khalil 1964), two from gastrothylacidae (carmyerius stiles & goldberger 1910 and gastrothylax poirier 1883), five from paramphistomidae (bilatorchis fischoeder, 1901, calicophoron näsmark 1937, cotylophoron stiles & goldberger 1910, gigantocotyle näsmark 1937 and orthocoelium [stiles & goldberger 1910] price & mcintosh 1953) and one from stephanopharyngidae (stephanopharynx fischoeder 1901). twenty-six species occur in domestic and wild ruminants in the area under review. seventy-seven per cent of them (20/26) belong to calicophoron, carmyerius and cotylophoron with the genus calicophoron accounting for approximately 35% of the species, followed by carmyerius (27%) and cotylophoron (15%). seventy-five per cent (9/12) of the known calicophoron species and more than 40% of carmyerius (43.8%, 7/16) and cotylophoron (57.1%, 4/7) species occur in the area under review. however, less than 40% of gastrothylax (33.3%, 1/3), gigantocotyle (25%, 1/4) and orthocoelium (9.1%, 1/11) known species occur in ruminants in east and southern africa. most of the calicophoron species have a wider distribution with respect to countries where reported compared with species of the other genera. calicophoron microbothrium has the widest distribution followed by cot. cotylophorum and cal. raja. calicophoroncalicophorum, cal. phillerouxi, cal. sukari, cal. fuelleborni and cal. spatiosus also have a wider distribution compared with the rest of the other species. seven species; bil. papillogenitalis, cal. daubneyi, car. bubalis, car. dollfusi, cho. onotragi, cot. macrosphinctris and ort. scoliocoelium had the narrowest distribution, being reported in only one country each. the majority of the species (76.9%) are shared between domestic and wild ruminant hosts and approximately 12% of them have not been documented in domestic ruminants as yet (bil. papillogenitalis, car. bubalis and cot. macrosphinctris), while another 12% (3/26) have not been reported in wild ruminants (cal. daubneyi, car. dollfusi and ort. scoliocoelium). approximately 85% (22/26) are found in domestic and 88% (23/26) in wild ruminants. all the species recorded in domestic ruminants occur in cattle with cal. daubneyi, car. dollfusi and ort. scoliocoelium documented in this ruminant host only. sheep are hosts to 55% (12/22), while goats are hosts to 50% of the species reported in domestic ruminants. half (11/22) of the species have been reported in all the domestic ruminants with most (63.6%, 7/11) of them being calicophoron species. the range of wild ruminant hosts varies for the different species. cotylophoron cotylophorum has the highest wild ruminant host range, 19 host species belonging to 10 genera followed by cal. raja, 18 host species belonging to 12 genera, cal. microbothrium, 17 host species belonging to 11 genera and car. spatiosus, 10 host species belonging to 10 genera. calicophoron sukari has the lowest wild ruminant host range, with only one wild ruminant host, that is, the buffalo. of the wild ruminant hosts, buffaloes are hosts to 78% (18/23) of the amphistome species recorded in wild ruminants followed by the waterbuck (52.2%, 12/23), the kafue lechwe (47.8%, 11/23), the roan antelope (47.8%, 11/23) and the hartebeest (43.4%, 10/23). the blue wildebeest, bushbuck, eland, impala, kudu and sable antelope are also hosts to more than 25% of the amphistome species documented in wild ruminant hosts. mixed farming systems of cattle and game, particularly antelope, have become an important agricultural activity in most east and southern african countries. in addition, there has been the creation of transfrontier conservation areas (tfcas) involving many african countries, particularly in southern africa, resulting in increased livestock–wildlife interface areas. therefore, domestic and wild animals are coming into ever more intimate contact in many interface areas, particularly in rural areas at the edges of the tfcas and in farms practising mixed cattle and game farming, thus promoting the possibility of parasite exchange. these observations are important when considering the different options for their control. for instance, phiri et al. (2011) observed that the host range of many helminths found in the kafue lechwe is broad and they could serve as a potentially stable source of infection to domestic animals such as goats and cattle. hence, issues concerning livestock management and conservation may arise. snail intermediate hosts table 1 shows the reported intermediate snail hosts of different amphistome species recorded in the study areas under review. data show that seven snail species – bio. pfeifferi, bul. forskalii, bul. globosus, bul. nasutus, bul. tropicus and cer. natalensis all planorbidae rafinesque 1815 and galba truncatula belonging to lymnaeidae rafinesque 1815 – are so far confirmed intermediate hosts of identified amphistome species. the genus bulinus contributes 57% (4/7) of the confirmed snail intermediate hosts, while the remaining genera contribute one species each. the data also show that some snail species are intermediate hosts of amphistome species belonging to the same genus, for example, bul. forskalii (cal. microbothrium and cal. phillerouxi), bul. tropicus (cal. calicophorum and cal. microbothrium) and cer. natalensis (car. exoporus and car. mancupatus) or amphistome species belonging to different genera, for example, bul. globosus (cal. microbothrium and car. parvipapillatus) and cer. natalensis (car. exoporus, car. mancupatus and ort. scoliocoelium). the capacity of the various snail species to act as intermediate hosts for paramphistomoids has not been fully elucidated yet, as some of the snail hosts have been reported to be naturally infected with amphistome cercariae of unidentified species (chingwena et al. 2002a; dinnik 1961; loker, moyo & gardner 1981; lotfy et al. 2010; mukaratirwa et al. 1998; pfukenyi et al. 2005a; wright et al. 1979). adult amphistomes are difficult to identify using their anatomical and morphological features as they have thick robust bodies in which the internal organs are difficult to characterise (jones 1990). as amphistomes in snail hosts are in their larval stages, species identification is made even more difficult. hence, the technical difficulties in making precise species identifications impact greatly on the better understanding of amphistome–snail associations. however, lotfy et al. (2010) demonstrated its2 as a good molecular marker for amphistome identification, which can be used to identify both adult amphistome and cercariae to species level. the current review shows that only nine (34.6%, 9/26) amphistome species (cal. calicophorum, cal. daubneyi, cal. microbothrium, cal. phillerouxi, cal. sukari, car. exoporus, car. mancupatus, car. parvipapillatus and ort. scoliocoelium) have known snail hosts. except for cal. microbothrium, presently with four known snail hosts (bul. forskalii, bul. globosus, bul. nasutus and bul. tropicus) in east and southern africa, all the other species have one known snail host each. in addition, bio. pfeifferi and melanoides tuberculata are known experimentally to serve as snail hosts of this parasite (chingwena et al. 2002b). calicophoron microbothrium is widely distributed in the areas under review and the wide range of its snail hosts probably supports its reported broad geographical distribution. data under review show that most of the known amphistome species have unknown snail hosts (65.4%, 17/26). the snail hosts of four calicophoron species (cal.bothriophoron, cal. clavula, cal. raja and cal. sukumum) are currently not known. in tanzania, amphistome cercariae of unidentified calicophoron species were recorded in bul. forskalii (lotfy et al. 2010). lotfy et al. (2010) suggested that besides cal. phillerouxi, which is known from bul. forskalii, two other calicophoron species known in tanzania, cal. bothriophoron and cal. sukumum with unknown snail hosts, cannot be ruled out. in the east african region, bul. abyssinicus is reported as the intermediate host of cal. clavula in somalia (sobrero 1962). dinnik and hammond (1968) suggested bul. globosus as a likely snail host of cal. raja as it is experimentally proven to be susceptible to infection. four carmyerius species with presently unknown snail hosts are car. bubalis, car. dollfusi, car. gregarious and car. spatiosus. amphistome cercariae belonging to an unidentified species of the family gastrothylacidae were recorded from cer. natalensis in kenya (lotfy et al. 2010). the genus carmyerius is one of four genera belonging to the family gastrothylacidae. hence, besides car. mancupatus and car. exoporus already known from this snail host, one other carmyerius species known in kenya, car. spatiosus with unknown snail hosts, cannot be ruled out (lotfy et al. 2010). besides cer. natalensis, wright et al. (1979) also suggested bul. forskalii as likely snail hosts of car. spatiosus in zambia. to date, all four cotylophoron species have unknown snail hosts. the other species with unknown snail hosts are bil. papillogenitalis, cho. onotragi, gas. crumenifer, gig. symmeri and ste. compactus. however, even though not yet confirmed, bul. forskalii has been speculated to act as the intermediate host of ste. compactus (dinnik 1965). epidemiological features of amphistome infections in ruminants in east and southern africa the epidemiology and prevalence of amphistomosis depend on several factors. these include the species of definitive and intermediate hosts (rolfe et al. 1991), the potential of the flukes to infect these hosts (dinnik 1964a; dinnik & dinnik 1954; horak 1967), the topography and biological potential of the snail hosts (dinnik 1964a; horak 1971; rolfe et al. 1991; swart & reinecke 1962a, 1962b), the definitive hosts’ management systems and their grazing habits as well as climate (rolfe et al. 1991). data on amphistome infection prevalence are scarce for the reviewed countries and are currently only available from six countries (table 2). the prevalence data are based on coprology and fluke counts with most studies having conducted in cattle. because of difficulties in amphistome species identification, specific species prevalence data are lacking. prevalence studies are limited for goats, sheep and wild ruminants. the available data show a high prevalence in the kafue lechwe, but low prevalence rate in goats and sheep. in cattle, the coprological prevalence varies from 23.7% to 86.5%, while it varies from 25.5% to 96% on fluke counts, the high prevalence perhaps being explained by the fact that amphistome infection in ruminants is commonly because of several species. in the highlands of kenya, cal. microbothrium, cal. daubneyi and cal. jacksoni were recovered from a single animal and in few cases cal. sukari, car. exoporus and car. mancupatus were present as well (dinnik 1964a). another combination of six species (cal. microbothrium, cal. phillerouxi, cal. raja, cot. cotylophorum, car. parvipapillatus and ste. compactus) was found in an ox in zambia (dinnik 1964a). amphistomes recovered in slaughtered cattle were a combination of cal. microbothrium and cot. jacksoni in tanzania (keyyu et al. 2006). infections with different amphistome species are also reported in zimbabwe in cattle (dube et al. 2004; dube & tizauone 2014) and in sheep and goats (dube, masanganise & dube 2010). in addition, most amphistome species (85%) are shared between domestic and wild ruminant hosts (table 1), providing a potentially stable source of infection among the ruminant animals. furthermore, the availability of a wide range of the snail hosts (table 1) with high biological potential also increases the successful propagation of amphistomes in the environment leading to increased infection exposure in ruminants. limited routine anthelmintic treatment, particularly in rural communities who practice communal grazing, and the lack of effective drugs against amphistomes are also possible explanations for the high prevalence in domestic ruminants. an increase in the prevalence of amphistome infections has been reported in western europe (foster et al. 2008; mage et al. 2002; murphy et al. 2008; toolan et al. 2015). besides an improvement in quality of diagnosis, the increase has also been attributed to the absence of an effective anthelmintic against amphistome infections (mage et al. 2002). table 2: prevalence of amphistomes in ruminants in east and southern african countries based on faecal egg and fluke counts. studies on animal-breed predisposition to amphistome infection are limited. indigenous cattle breeds were observed to have a significantly higher prevalence and intensity than the exotic breeds and crosses in kenya and uganda (howell 2011; kanyari, kagira & mhoma 2010). in tanzania, the maasai zebu cattle had a significantly higher prevalence than the iringa red cattle; however, the numbers of animals involved were too small for any meaningful interpretations to be made (nzalawahe et al. 2015). literature reports a variable effect of sex in domestic ruminants. despite females tending to record higher prevalences than males, the associations were not significant (kanyari et al. 2009, 2010; keyyu et al. 2006; phiri, chota & phiri 2007a; phiri, phiri & monrad 2006). however, pfukenyi et al. (2005a) observed significantly higher prevalences in pregnant and lactating cows compared with bulls, oxen and dry cows. similarly, howell (2011) reported a significantly higher prevalence in female cattle compared with males. the differences between sexes could probably be related to grazing patterns, sex hormones and treatment regimes. adult domestic ruminants are reported to have a significantly higher prevalence compared with young animals (howell 2011; kanyari et al. 2009, 2010; keyyu et al. 2005, 2006; nzalawahe et al. 2014; pfukenyi et al. 2005a; phiri et al. 2007a; vassilev 1999). this is attributed to a long exposure time in adults leading to immunity against the pathogenic effects of immature amphistomes but still having the mature ones maintaining their high egg production capacity (horak 1971). the resistance to amphistome re-infection in cattle was demonstrated clinically (horak 1967) with no simultaneous studies on the cellular effector systems that characterise the acquired resistance. mavenyengwa et al. (2008) showed that the resistance to cal. microbothrium re-infection in cattle involves eosinophils and mast cells that are targeted at immature flukes. epidemiologically, adults act as a constant source of infection, but clinical amphistomosis remains a problem for young animals, with adults grazing the same pastures exhibiting no clinical effects despite continued egg production (boray 1959, 1969; butler & yeoman 1962; rolfe et al. 1991). amphistomes may survive for up to two years in the definitive hosts, providing a virtually constant source of infection for successive generations of snail hosts. in rural areas, the grazing management of young and adult animals may differ, where young animals graze around farms or homesteads while adults are trekked long distances to valleys, flood plains or swampy areas where they are exposed to high metacercariae-contaminated pastures (keyyu et al. 2005, 2006). animal grazing area and/or habitat is significantly associated with prevalence and intensity of amphistomes in domestic ruminants (howell 2011; kanyari et al. 2009, 2010; keyyu et al. 2005, 2006; nzalawahe et al. 2014, 2015; pfukenyi et al. 2005a; phiri et al. 2006). the prevalence is highest in animals grazing in areas characterised by wetlands or swampy or marshy grazing areas where the distribution of suitable snail habitats is widespread. for instance, the highveld region in zimbabwe, characterised by wet/swampy grazing areas where distribution of snail habitats is widespread, is associated with a higher prevalence compared with the lowveld which is characterised by dry land grazing with a focal distribution of snail habitats (pfukenyi et al. 2005a, 2005b). similarly, the presence of wetlands and high livestock density in the cattle grazing areas of the zambian western and southern provinces is associated with an increased risk of acquiring amphistome infections (phiri et al. 2006) and the same observations have been reported in kenya (kanyari et al. 2009, 2010), tanzania (keyyu et al. 2005, 2006; nzalawahe et al. 2015) and uganda (howell 2011). in tanzania, traditional communal grazing areas exhibited the highest prevalence of amphistomes compared with other sectors and this is attributed to heavy contamination of the habitats with eggs where intermediate host snails breed, because of high stocking densities with subsequent heavy metacercarial density on vegetation grazed by the animals especially during the dry season (keyyu et al. 2005, 2006). villages practising irrigation of crops are associated with high amphistome infection rates in tanzania (nzalawahe et al. 2014) as this provides favourable ecological conditions for growth of snail hosts and development of trematode larval stages. the prevalence in domestic ruminants as measured by coprology follows a seasonal pattern with an increase towards the end of the dry season and a peak during the wet months of the year (keyyu et al. 2005; pfukenyi et al. 2005a; phiri et al. 2007a; reinecke 1983; vatta & krecek 2002). outbreaks of acute clinical amphistomosis because of immature flukes are usually confined to the drier months of the year (boray 1969; butler & yeoman 1962; dinnik 1964a; horak 1967, 1971; rolfe et al. 1991; vassilev 1999). towards the end of the rainy season and onset of the dry season, conditions in permanent water sources become favourable for an increase in the number of the snail intermediate hosts, reaching their peak during the mid-to-end of the dry season (chingwena et al. 2002a; pfukenyi et al. 2005a, 2005b; phiri et al. 2007b). as the snail hosts are extremely adaptable and prolific breeders, this ensures their widespread availability as well as heavy shedding of cercariae which encyst on vegetation surrounding the habitats (dinnik 1964a). the proportion of infected snails increases from the end of the rainy season into the dry season (chingwena et al. 2002a; pfukenyi et al. 2005a). a combination of high snail numbers, asexual multiplication of the fluke in infected snails and survival of snails in suitable environments for several months may result in shedding of large numbers of cercariae. during this period, the infective metacercariae are spread over pastures surrounding permanent water sources where they can survive for several months. this coincides with pasture areas being narrowed around permanent water sources or wetland environments where animal concentration becomes high (pfukenyi et al. 2005b). contamination rates of these areas are increased, resulting in more snails being infected and high numbers of metacercariae on surrounding herbage, leading in turn to acute infections of animals with amphistomes. thus, a build-up of immature flukes occurs, accounting for clinical amphistomosis outbreaks and low amphistome prevalence as measured by coprology. the outbreaks are common in ruminants that graze in marshy or swampy areas and are usually confined to the dry season. however, on irrigated pastures, moisture is often adequate for the survival of snail hosts and metacercariae, and hence outbreaks can occur throughout the year. in tanzania, some villages practising year-round zero-grazing had high levels of amphistome infections attributed to the acquisition of cattle fodder from irrigation canals and swamps contaminated with metacercariae (nzalawahe et al. 2014). development of amphistomes into adults takes 5–9 months (dinnik & dinnik 1962) and the prepatent period is 56–89 days (dinnik & dinnik 1962; horak 1971). five to 9 months after infection, the immature flukes become fully mature and this would lead to high faecal egg production and thus, account for the high prevalence during the rainy season as measured by coprology. during this period, abundant grazing and alternative water sources are available. hence, drinking from and grazing around infected permanent water sources is greatly reduced. furthermore, snail habitats and pastures are constantly flooded, and thus snails and the parasitic free-living stages are regularly flushed (pfukenyi et al. 2005b). in summary, the intermediate and definitive hosts acquire most of the infection during the beginning and/or middle of the dry season. this results in immature fluke infections and clinical amphistomosis during the dry season and patent (mature fluke) infections during the wet months and at the end of the dry season. however, the timing may vary depending on location, length of the rainy season and the grazing habits of the animals (pfukenyi et al. 2005b). impact on production adult flukes are not associated with clinical amphistomosis (mavenyengwa, mukaratirwa & monrad 2010). however, in heavy infections they have been hypothesised to cause weakness, recurrent ruminal tympany, ruminal atony, weight loss, anaemia and production losses (anuracpreeda, wanichanon & sobhon 2008). they are also reported to be associated with inflammation of the mucosa and mucoid diarrhoea (rolfe & boray 1993). based on coprology, poor body condition is reported to be significantly associated with high amphistome prevalence in cattle (kanyari et al. 2010). a similar observation was noted in small ruminants (kanyari et al. 2009), but the association was not significant. cattle infected with more than 500 adult amphistomes had a significant reduction in final carcass mass when compared with controls (marchand 1984; dube & tizauone 2014). the concurrent infection of amphistomes with other parasites known to depress growth rate such as strongyles (kanyari et al. 2010), fasciola species (kanyari et al. 2010; keyyu et al. 2006; nzalawahe et al. 2014; phiri et al. 2006; yabe et al. 2008) and moniezia species (kanyari et al. 2010) is a likely explanation of the significant association between poor body condition and amphistome infections. however, further studies on the effect of adult amphistomes on production are required. clinical amphistomosis is caused by the immature flukes that lodge in the first 3 m of the small intestine (mavenyengwa et al. 2010). the occurrence of clinical amphistomosis and subsequent clinical pathology in ruminants is dependent on the dose, pathogenicity of the species and the level of establishment of the metacercariae in the host’s small intestine (horak 1967; mavenyengwa et al. 2010). in ruminants, the disease is characterised by anorexia, anaemia, submandibular oedema, and hypoproteinemia, foul-smelling fetid diarrhoea, general weakness, polydipsia, and a reduction in feed conversion, weight and milk production and mortality in young animals (boray 1969; horak 1966; mavenyengwa et al. 2010; mohan 2011; pillai & alikutty 1995; rolfe, boray & collins 1994; spencer, fraser & chang 1996). together with gastrointestinal nematodes, amphistome infection in cows can reduce milk production by approximately 0.4 l/day – 3 l/day (mohan 2011; spencer et al. 1996). anthelmintic treatment of dairy cows infected with gastrointestinal nematodes (oxfendazole) and amphistomes (oxyclozanide) resulted in a significant increase in milk production, averaging 0.4 l/day (spencer et al. 1996). the reduction in milk yield during clinical amphistomosis is associated with fetid diarrhoea (mohan 2011). mohan (2011) also reported anoestrus during clinical amphistomosis, while a functional obstruction or paralytic ileus of the intestine because of severe amphistomosis was reported in a cow (yogeshpriya et al. 2011). despite their ubiquity and abundance, as well as an increase in their prevalence in domestic ruminants, the economic importance of amphistome infections is not yet fully known and is likely to be underestimated in eastern and southern africa – an area which requires further studies. diagnosis diagnosis of amphistomes in live animals is still dependent on faecal detection of eggs (rieu et al. 2007) and this method only detects the presence of adult rumen fluke infection (malrait et al. 2015). the filtration technique with sieves and sedimentation is the most accurate method to identify eggs in faeces (horak 1971). using contrast stains such as methylene blue or methyl green to distinguish amphistome eggs from fasciola ova is advisable. one drawback of this diagnostic method is that, in acute infections, it is highly probable not to find eggs or only very few as this is usually associated with massive infection with immature flukes (horak 1971). the agreement between a modified mcmaster method and necroscopic diagnosis of amphistome infection is reported to be high (rieu et al. 2007) with no significant differences being observed between the two methods. the modified mcmaster method showed a significant association between eggs per gram (epg) counts and parasite burden; more than 100 epg indicated the presence of more than 100 adult amphistomes in the rumen and/or reticulum (rieu et al. 2007). similarly, the mini-flotac is a reliable method of assessing the presence of adult amphistome infection with both sensitivity and specificity being above 0.9 (malrait et al. 2015). a good correlation was found between faecal egg count (fec) and estimated rumen fluke burden with a fec > 200, indicating the presence of more than 200 adult rumen flukes in the rumen and/or reticulum (malrait et al. 2015). the adult worms are difficult to identify to species level because most have thick robust bodies in which the internal organs are difficult to see. even by using histological techniques, species identification is still problematic (lotfy et al. 2010). as the flukes responsible for disease are sexually immature, specific identification is made even more difficult and the diagnosis has to rely on the dubious procedure of identifying a few adult worms, which may be present in the rumen of the animal (horak 1971). because of these problems, pcr-based techniques providing rdna its2 sequences have proven to be reliable tools to identify amphistome species and to determine their phylogenetic relationships (itagaki et al. 2003; rinaldi et al. 2005). using cercariae and rediae from snail hosts and adult flukes obtained from slaughterhouses, lotfy et al. (2010) confirmed its2 as a good molecular marker for amphistome identification that can also be used to determine phylogenetic and amphistome–snail associations. the clinical diagnosis of amphistomosis remains challenging as immunological techniques are usually not conclusive (horak 1967, 1971). croposcopic examination cannot be used for the early diagnosis of clinical amphistomosis which is vital for prompt treatment before considerable damages and economic losses are incurred. for the identification of immature flukes, the recommended method is to mix approximately 10 g of faeces with 100 ml – 200 ml of water (horak 1971). the mixture is allowed to stand for 5 min, followed by decanting any supernatant fluid and then repeating the procedure four to five times. young flukes, resembling small white or pink rice grains, will be seen after pouring the sediment on a black surface for examination (horak 1971). in dead animals, postmortem, pathological and clinical pathological findings combined with the presence of immature flukes in the affected intestines would be confirmative. the gross pathological, histopathological and clinical pathological findings are as described in the literature (horak 1966, 1967, 1971; horak & clarke 1963; mavenyengwa et al. 2005, 2008, 2010; pillai & alikutty 1995). an indirect elisa performed to detect coproantigens in faecal supernatants of 100 cattle known to be infected with gas. crumenifer had a sensitivity of 74% (kandasamy & devada 2011). generally, the sensitivity of the indirect elisa ranges from 74% to 86% and its specificity from 79% to 90% (hassan et al. 2005; kandasamy & devada 2011; salib et al. 2015; sanchis et al. 2012; shivjot et al. 2009). however, shivjot et al. (2009) reported a very low specificity of 23.7%. the elisa was shown to be more specific and accurate but less sensitive than western blotting for the diagnosis of amphistome infections in cattle and buffaloes (salib et al. 2015). results indicate the feasibility of elisa for the detection of coproantigens of amphistome infections, especially for the diagnosis of immature amphistomosis where faecal examination may not reveal eggs. control the available epidemiological information on amphistomes of ruminants in the area under review can be used to design appropriate integrated control measures. options available for the control of amphistome infections are mainly based on chemical treatment, non-chemical management practices and immunological control. chemical treatment chemical control involves treatment with a product that is effective against both adult and immature flukes. oxyclozanide given twice, three days apart, has a high efficacy against both adult and juvenile amphistomes (rolfe & boray 1987) and a high anthelmintic performance in cattle (arias et al. 2013; rolfe & boray 1987; spencer et al. 1996) and small ruminants (paraud et al. 2009; rolfe & boray 1988; sanabria et al. 2014). studies in tanzania showed a reduced efficacy of levamisole–oxyclozanide combination against amphistomes in cattle (keyyu et al. 2008) and this is of great concern as they are the commonly available drugs in the country. however, levamisole is widely used to treat nematode infections in livestock and it is not intended as treatment against trematodes. when given orally at a higher dosage (10 mg/kg), closantel has a high efficacy against mature flukes (arias et al. 2013). however, treatment of mature flukes with intra-ruminally (rolfe & boray 1993) or subcutaneously administered (malrait et al. 2015) closantel is not effective. in countries where oxyclozanide is unavailable, the use of closantel to treat against mature flukes is recommended. when administered at high doses (50 mg/kg and 100 mg/kg), niclosamide has 94% – 99% efficacy against immature amphistomes (rolfe & boray 1987). even though it is not of direct benefit to the animal, treatment against mature amphistomes will prevent egg laying and thus reduce pasture contamination (horak 1971), while treatment against the immature flukes will reduce the impact of the disease. during the rainy season, mature amphistomes are expected and anthelmintic treatment with drugs effective against adult flukes is indicated. the strategic anthelmintic treatment against mature amphistomes should be given in adult animals at the end of the rainy season (pfukenyi et al. 2005a, 2005b) or beginning of the dry season (keyyu et al. 2005) to reduce the opportunity for snail infections. the timing of this treatment is dependent on local factors, length of the rainy season and the grazing habits of the animals. where possible, adult animals targeted for treatment should have high levels of infection based on coprology. depending on availability, oxyclozanide or closantel can be administered during this period to treat against mature amphistomes. disease epidemiology indicates that large burdens of immature amphistomes are expected during the dry season. as adult animals are resistant to the pathogenic effects of the migrating immature amphistomes, the target for treatment would be young animals being exposed to the infection for the first time (pfukenyi et al. 2005a). hence, the first anthelmintic treatment can be administered in young animals during the mid-dry season period when maximum migration of immature amphistomes starting 3–4 weeks after infection in the early dry season is expected. to remove potentially high burdens of immature amphistomes acquired later in the dry season, a second treatment could be given towards the end of the dry season (pfukenyi et al. 2005a). oxyclozanide or niclosamide can be administered during this period to treat against immature amphistomes. in communal areas, animals are communally grazed and for optimum benefits, the recommended anthelmintic treatments should be well organised and preferably done at the same time within a village. where cattle are dipped for the control of ticks, dip tank facilities where all animals are gathered during dipping sessions could be used for organised fluke control (pfukenyi et al. 2005a, 2005b). the efficacy of medicinal plant extracts against amphistomes has recently been evaluated. the ethanol extract of punica granatum l. (lythraceae), commonly known as pomegranate, is highly effective against amphistomes in naturally infected sheep (lalhmingchhuanmawii, veerakumari & raman 2014). the authors concluded that the plant extract could be successfully used as an anthelmintic to treat amphistomes in domestic ruminants. similarly, an aqueous extract of acacia concinna (willd.) dc. (fabaceae) significantly reduced egg counts of amphistomes in naturally infected sheep and also restored the haemato-biochemical profile to normal in extract-treated sheep (priya, veerakumari & raman 2013). however, efficacy of the p. granatum and a. concinna extracts was not established in immature amphistomes. other studies have also shown medicinal plants extracts to be effective against amphistomes (elango & rahuman 2011; kamaraj et al. 2010). chemical control of the snail hosts through application of molluscicides such as niclosamide may also be done. to achieve cost-effective control, this type of control should be done during the peak transmission period to reduce numbers of infected snails and cercarial shedding. thus, the application could be done during the mid-dry and towards the end of the dry season (pfukenyi et al. 2005b). the application is practical and economical in areas where snail habitats are focal and not widespread, but regular application may be necessary because of the rapid recovery of the snail populations during brief periods of favourable conditions. however, molluscicide application causes environmental pollution and also kills non-targeted aquatic organisms (roberts & suhardono 1996). immunological control hafeez and rao (1981) showed that the lifespan and pathogenicity of amphistomes developing from gamma irradiated (2 or 3 krad) metacercariae were greatly reduced with the higher irradiation dose resulting in the complete absence of the flukes in infected animals. single vaccination of kids and lambs with 3000 irradiated (2 or 3 krad) metacercariae stimulated a significant degree of resistance against challenge and the resistance was more pronounced in the group vaccinated with a higher irradiation dose (hafeez & rao 1981). earlier, horak (1967) successfully immunied sheep, goats and cattle against massive artificial infections with cal. microbothrium. the animals were given immunising infections with at least 40 000 metacercariae and later challenged with larger doses of metacercariae (horak 1967). cattle were the most suitable subjects for immunisation with the immunity being effective for at least a year post-immunisation (horak 1967, 1971). mavenyengwa et al. (2008) demonstrated that cattle acquire resistance to amphistome infection. this resistance is targeted at immature amphistomes and it involves eosinophils and mast cells. however, despite promising immunisation results, the mass production of snail hosts and metacercariae remains a challenge and a major limiting factor (horak 1967, 1971; mavenyengwa et al. 2006; swart & reinecke 1962a, 1962b). thus, the success of a large-scale immunisation program is dependent on a viable metacercariae mass production system. non-chemical control the best preventive method against amphistome infections is to keep domestic ruminants from infected pastures (pfukenyi et al. 2005b). fencing-off or drainage of wetlands or marshy/swampy areas and provision of clean pastures and cercariae-free water in troughs are advised (roberts & suhardono 1996). similarly, habitat management through vegetation clearance is also effective in controlling the snails (woolhouse & chandiwana 1990). however, habitat management and complete separation of stock from snail habitats are only practical and economical where the snail habitats are focal and not widespread (pfukenyi et al. 2005b). these control methods are not feasible in communal grazing areas. it is also important to repair any leaks in dams and water troughs as they can create an ideal habitat for the snail hosts. conclusions twenty-six amphistome species belonging to nine genera from three families occur in domestic and wild ruminants in the area under review and seven snail species belonging to four genera from two families act as their intermediate hosts. eighty-five per cent of the amphistome species are shared between domestic and wild ruminant hosts. some snails are intermediate hosts of amphistome species belonging to the same genus or to different genera – a phenomenon not yet fully elucidated. only nine (34.6%) of the amphistome species have known snail intermediate hosts, while most (65.4%) have unknown hosts. the epidemiology of amphistomosis depends on the species of definitive and intermediate hosts and the potential of the flukes to infect these hosts, the topography and biological potential of the snail hosts, the management systems of the definitive host and their grazing habits and climatic factors. based on current epidemiological information, the strategic anthelmintic treatment against mature amphistomes should be given in adult animals at the end of the rainy or early dry season. the anthelmintic treatment in young animals against immature amphistomes should be administered during the mid-dry and towards the end of the dry season. further research is necessary to determine the economic importance of amphistomosis, amphistome–snail associations, efficacy of different anthelmintics and to develop diagnostic tests that can detect prepatent infections in the definitive host. acknowledgements the authors would like to thank the anonymous reviewers whose suggestions improved the manuscript. 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mellor, boorman & baylis, 2000; meiswinkel, venter & nevill 2004). in sub-saharan africa, they are of interest primarily as vectors of bluetongue virus (btv) and african horse sickness virus (ahsv), which cause two devastating diseases of ovinae and equidae, respectively. while the impact of both these viruses in south africa is limited, because of high endemic immunity in those hosts in which they commonly cause disease and the use of polyvalent live attenuated vaccines, sporadic and severe outbreaks of these diseases still occur (meiswinkel et al. 2004). outbreaks of african horse sickness (ahs) in 1999 and 2004 in the surveillance zone of the declared ahs-free area in the stellenbosch district had considerable financial implications and on both occasions led to a 2-year embargo on the export of horses from south africa (venter, koekemoer & paweska 2006). of the approximately 120 species of culicoides identified in southern africa (meiswinkel, unpublished data 1996), the vast majority remain uninvestigated as potential vectors of disease. however, culicoides imicola kiefer, 1913 and culicoides bolitinos meiswinkel, 1989 two species that are closely associated with both livestock and game (meiswinkel 1989, 1995) have been identified as primary candidates for this role. both ahsv and btv have been shown to replicate under laboratory conditions to what are presumed to be transmissible levels in these spe343 onderstepoort journal of veterinary research, 74:343–347 (2007) research communication culicoides biting midges at the national zoological gardens of south africa k. labuschagne1, l.j. gerber2, i. espie3 and s. carpenter4 abstract labuschagne, k., gerber, l.j., espie, i. & carpenter, s. 2007. culicoides biting midges at the national zoological gardens of south africa. onderstepoort journal of veterinary research, 74: 343–347 culicoides biting midges (diptera: ceratopogonidae) are responsible for the transmission of a large number of pathogens to livestock and wild animals. in this study the presence of the genus, using light traps based at four different sites within the national zoological gardens of south africa, was investigated during 2002–2004. in total, 37 species were recorded, including large numbers of culicoides imicola kieffer, 1913, which is responsible for the transmission of economically important arboviruses in south africa, europe, middle and far east. these results are discussed with reference to the wider culicoides fauna in the onderstepoort area of south africa, their vector competence as well as biosecurity at the national zoological gardens. keywords: abundance, biosecurity, culicoides, light trap, national zoological gardens, survey ¹ agricultural research council, onderstepoort veterinary institute, division of entomology, private bag x05, onderste poort, 0110 south africa. e-mail: labuschagnek@arc.agric.za 2 tshwane university of technology, department biomedical sciences, private bag x680, pretoria, 0001 south africa 3 national zoological gardens of south africa, boom street, p.o. box 754, pretoria, 0001 south africa 4 institute of animal health, pirbright laboratory, pirbright, surrey, gu24-0nf uk accepted for publication 19 march 2007—editor 344 culicoides biting midges at the national zoological gardens of south africa cies (venter, paweska, van dijk, mellor & tabachnick 1998; venter, graham & hamblin 2000). in this paper, the results of a light trap-based survey carried out over 2 years in which the culicoides spp. occurring in the zoo is presented and their biosecurity risk is defined. given the movement of animals as part of breeding programmes or loans into countries with a greater clinical disease risk from e.g. bluetongue (bt) or ahs than that in south africa, biosecurity practices are important to minimize the risk of accidentally importing these viruses/ diseases as happened following the importation of ahsvinfected zebras from namibia into spain (lubroth 1988). additionally, methods to minimize the numbers of culicoides breeding in this habitat are suggested. the national zoological gardens is located in central pretoria (25°45’ s; 28°11’ e) and its inhabitants are isolated from other game and large livestock in the general area by the surrounding urban development, although poultry and some small livestock such as goats are kept. the zoo covers an area of approximately 80 ha and houses over 100 exotic and indigenous species of mammals and over 160 bird species. the survey was carried out from august 2002 to august 2004 using 220v black light downdraught light traps (meiswinkel et al. 2004) placed at four sites on the premises of the zoo (fig. 1). the sites were selected in close proximity of animal species possessing the potential of filarial, protozoa and viral transmission (karstad 1979; meiswinkel et al. 2004). the sites chosen were at the cape buffalo (syncerus caffer), african elephant (loxodonta africana), white rhinoceros (ceratotherium simum)/ black-faced impala (aepyceros melampus petersi) enclosures and the walk-through aviary. light traps were operated once weekly from dusk until dawn. collections were made into a 1 % savlon (containing clorhexidane gluconate 0.3 g/100 ml and cetrimide 3.0 g/100 ml [johnson & johnson, sa]) -water solution and transferred the following morning to 80 % alcohol for storage purposes. all the collections containing more than 500 culicoides specimens were sub-sampled using the method of van ark & meiswinkel (1992). culicoides was identified to species level by examination of wing markings (meiswinkel 1996), with the exception of the 150 m n s w o fig. 1 plan of the national zoological gardens of south africa, pretoria, south africa showing placement of light traps. (1) cape buffalo enclosure, (2) african elephant enclosure, (3) walk-through aviary and (4) white rhinoceros/black-faced impala enclosure culicoides nigripennis carter, ingram and macfie, 1920 and culicoides accraensis carter, ingram and macfie, 1920 species complexes which are extreme ly difficult to separate accurately based on their mor phology. in order to prevent a bias in numbers of the different species at each site, comparisons of the number and species present were made only between those days when all four traps were in operation. ninety samples from each site were compared and used for analysis. in this subset of 360 samples a total of 478 040 midges was collected and identified, representing a total of 33 species (table 1). the trap based at the elephant enclosure caught the greatest total number of culicoides, representing some 28 different species, followed by the white rhinoceros/black-faced impala site (29 species), the buffalo site (17 species) and the aviary (12 species) (table 1). pro portionately, c. imicola was by far the most common ly trapped species, constituting more than 95 % of all midges caught at the buffalo and elephant sites and 67 % at the white rhinoceros/blackfaced impala site, where culi coides cor345 k. labuschagne et al. nutus de meillon, 1937 was also comparatively abundant (19 %) (table 1). the avi ary site, how ever, was dominated by both culicoides leucostictus kieffer, 1911 (48 %) and c. nigripennis sensu lata (43 %) with c. imicola comprising only 1.5 % of the total catch. this highlighted the host preferences of c. imi cola for larger mammals and increases the capacity of this species being a vector of ahs and bt viruses. the abundance and diversity of the culicoides fauna found at the national zoological gardens closely correlates with the results of a similar earlier study near livestock in the onderstepoort area (25°39’ s; 28°11’ e) north of pretoria (venter, meiswinkel, nevill & edwards 1996a). as in the case at onderstepoort, the presence of substantial populations of c. imicola is likely to pose the most easily identifiable threat from a biosecurity point of view, especially as light-trap catches tend to represent only a small proportion of the populations sampled. as larvae, this species primarily utilizes a wide range of organically enriched habitats and population numbers could potentially be reduced by chemical treatment (apperson 1975; holbrook & agun 1984), or rapid removal (bishop, mckenzie, spohr & barchia 2005) or dilution of animal waste. the efficiency of habitat removal techniques for african species of table 1 culicoides spp. recovered from 360 black light trap collections in the national zoological gardens, south africa during august 2002 to august 2004 species mean (maximum) no. culicoides caught/trap night buffalo elephant aviary rhinoceros/impala c. imicola 203.7 (8 027) 3 033.0 (65 520) 0.1 (10) 1 272.4 (18 288) c. enderleini 9.1 (462) 47.4 (1 386) 91.8 (1 703) c. bolitinos 3.9 (165) 9.5 (86) < 0.1 (1) 5.1 (103) c. cornutus 1.1 (29) 4.3 (54) 0.1 (2) 363.3 (4 699) c. zuluensis 1.0 (35) 9.9 (75) 18.2 (184) c. nigripennis s.l. 0.8 (24) 9.7 (109) 3.8 (268) 40.2 (650) c. nivosus 0.5 (7) 11.3 (126) 0.2 (5) 21.9 (490) c. leucostictus 0.3 (4) 33.3 (325) 4.3 (38) 58.7 (755) c. pycnostictus 0.3 (8) 20.2 (254) 0.3 (6) 19.5 (209) c. magnus 0.1 (3) 0.9 (15) 0.1 (4) c. subschultzei 0.1 (3) 0.2 (10) 2.1 (54) c. brucei 0.1 (3) 0.6 (10) 0.2 (22) c. gulbenkiani 0.1 (5) < 0.1 (2) < 0.1 (1) c. nevilli 0.1 (2) 0.6 (33) 0.5 (31) c. engubandei < 0.1 (1) < 0.1 (1) 0.3 (8) c. huambensis < 0.1 (1) c. tropicalis < 0.1 (1) 0.3 (10) 0.1 (5) c. sp. # 54 0.3 (8) 0.2 (14) c. sp. # 94 < 0.1 (3) < 0.1 (3) c. accraensis s.l. 0.9 (52) 0.1 (1) < 0.1 (5) c. albopunctatus < 0.1 (2) < 0.1 (5) c. nr. angolensis < 0.1 (3) c. bedfordi 0.4 (13) < 0.1 (1) 0.5 (10) c. coarctatus 0.2 (14) 0.1 (8) c. dekeyseri < 0.1 (4) c. eriodendroni < 0.1 (2) c. exspectator 0.4 (7) 0.4 (15) c. sp. # 107 < 0.1 c. loxodontis < 0.1 (1) c. neavei 0.5 (10) 0.4 (14) c. ravus 0.1 (10) < 0.1 (3) 0.3 (31) c. schultzei < 0.1 (1) c. similis 0.5 (10) < 0.1 (2) 0.6 (15) total 221.0 3 184.5 8.9 1 897.1 346 culicoides biting midges at the national zoological gardens of south africa culicoides is unknown. in the case of this isolated habitat, modification to the habitat could be considered. this must, however, be an ongoing process given that re-colonization of areas from other parts of the zoo, or from beyond the zoo’s boundaries (suburban areas), would appear to be likely given the similarities between the culicoides fauna in the zoo and that of the local (onderstepoort) area. chemical treatment can be effective but unfortunately non-target organisms are often also killed (holbrook & agun 1984). the biosecurity risk posed by species of culicoides other than c. imicola present in the zoo remains unclear. while c. bolitinos is known to possess a substantially greater vector competence for both btv and ahsv than c. imicola (venter et al. 1998, 2000), it was found in relatively low abundance, and should be relatively easy to control by more rapid removal (bishop et al. 2005) or treatment of dung (standfast, muller & wilson 1984) from animal holding areas as it is known to breed directly in the dung of large herbivores (meiswinkel 1989). the vector competency for arboviruses of culicoides enderleini cornet & brunhes, 1994, the second most common species at the buffalo and elephant sites and culicoides zulu ensis de meillon, 1936, is currently unknown though it was shown that c. zuluensis is susceptible to oral infection with the live attenuated vaccine strains of ahsv (paweska, prinsloo & venter 2003). these species are common and widespread across south africa and although the former appears to utilize a wide range of hosts (braverman & phelps 1981; meiswinkel et al. 2004), which may mitigate against it being an efficient vector, it was identified as a potential risk in a previous study (venter, nevill, van der linde 1996b). also of interest are the large numbers of c. cornutus at the white rhinoceros/black-faced impala enclosure. little is known of this species’ vector competence for arboviruses or other pathogens, its host preference or its larval habitat requirements. however, c. cornutus is very closely related to culicoides sonorensis wirth and jones, 1957, the vector of btv in north america and c. cornutus is often the dominant species collected in the bt enzootic area of kenya (glick 1990). there fore, this species remains an unknown risk in terms of disease transmission. of the remaining species, c. nigripennis sensu lata dominated the aviary fauna, but was also found in smaller numbers around all the mammal-based traps. again, little is known regarding this group’s competency for transmitting arboviruses. however, their apparent host preference for birds (or at least a very wide host preference) would appear to preclude them from representing a major biosecurity risk for the transmission of mammalian arboviruses, although not of protozoa. at least ten species of protozoa are transmitted by culicoides spp. to birds (linley 1985). imports of sub-clinically infected zoo animals into areas with susceptible hosts for arboviruses have caused outbreaks of a wide range of diseases caused by arboviruses, e.g. the outbreak of ahs in spain from 1987–1990, which was initiated by the importation of infected zebras (lubroth 1988). the difficulty of detecting diseases, such as bt and ahs at ports of entry into countries has emphasized the need for routine screening and quarantine. however, these procedures are expensive but quarantine procedures could possibly be reduced, especially when captive bred stock is being relocated by reducing the chances of infection with certain arboviruses in the first place, through the methods suggested in this communication. the major difficulty in accomplishing this process is ensuring that the methods employed in vector control remain ongoing, which, barring the most isolated cases, will require considerable effort in endemic areas of arbovirus distribution. acknowledgements the authors thank the personnel of the national zoological gardens for all their support and assistance. the survey was funded by the onderstepoort veterinary institute (arc-ovi) and the european union (eu contract: qlk2-2001-01722 on the development of a safe, efficacious bluetongue virus vaccination strategy for europe). we thank karin kappmeier green and gert venter for helpful comments on drafts of this manuscript. references apperson, c.s. 1975. biological activity of insecticides against culicoides variipennis (coquillett) (diptera: ceratopogonidae). proceedings of the 43rd annual conference of the mosquito control association: 118–119. bishop, a.l., mckenzie, h.j., spohr, l.j. & barchia, i.m. 2005. interactions between dung beetles (coleoptera: scarabaeidae) and the arbovirus vector culicoides brevitarsis kieffer (diptera: ceratopogonidae). australian journal of entomology, 44:8996. braverman, y. & phelps, r.j. 1981. species composition in samples of culicoides (diptera: ceratopogonidae) collected near salisbury, zimbabwe in 1976–77. journal of the entomological society of southern africa, 44:315–323. 347 k. labuschagne et al. glick, j.j. 1990. culicoides biting midges (diptera: cerato po gonidae) of kenya. journal of medical entomology, 27:85–195. holbrook, f.r. & agun, s.k. 1984. field trials of pesticides to control larval culicoides variipennis (ceratopogonidae). mosquito news, 44:233–236. karstad, l. 1979. a partly annotated bibliography on infections, parasites, and diseases of african wild animals. international development research centre, canada: 1–111. linley, j.r. 1985. biting midges (diptera: ceratopogonidae) as vectors of nonviral animal pathogens. journal of medical entomology, 22:589–599. lubroth, j. 1988. african horse sickness and the epizootic in spain 1987. equine practise, 10:26–33. meiswinkel, r. 1989. afrotropical culicoides: a redescription of c. (avaritia) imicola kieffer, 1913 (diptera: ceratopogo nidae) with description of the closely allied c. (a) bolitinos sp. nov. reared from dung of the african buffalo, blue wildebeest and cattle in south africa. onderstepoort journal of veter inary research, 56:23–39. meiswinkel, r. 1995. afrotropical culicoides: biosystematics of the imicola group, subgenus avaritia (diptera: cerato pogonidae). with special reference to the epidemiology of african horse sickness. m.sc. thesis, university of pretoria. meiswinkel, r., venter, g.j. & nevill, e.m. 2004. vectors: culicoides spp., in infectious diseases of livestock, 2nd ed, edited by j.a.w. coetzer & r.c. tustin. cape town: oxford university press. mellor, p.s., boorman, j. & baylis, m. 2000. culicoides biting midges: their role as arbovirus vectors. annual review of entomology, 45:307–340. paweska, j.t., prinsloo, s., venter, g.j. 2003. oral susceptibility of south african culicoides species to live-attenuated serotype-specific vaccine strains of african horse sickness virus (ahsv). medical and veterinary entomology, 15: 436–447. standfast, h.a., muller, m.j. & wilson, d.d. 1984. mortality of culicoides brevitarsis (diptera: ceratopogonidae) fed on cattle treated with ivermectin. journal of medical entomology, 77:419–421. van ark, h. & meiswinkel, r. 1992. sub-sampling of large light trap catches of culicoides (diptera: ceratopogonidae). onderstepoort journal of veterinary research, 59:183–189. venter, g.j., graham, s.d. & hamblin, c. 2000. african horse sickness epidemiology: vector competence of south african culicoides species for african horse sickness virus serotypes 3, 5 and 8. medical and veterinary entomology, 14:245–250. venter, g.j., koekemoer, j.j.o. &. paweska, j.t. 2006. investigations on outbreaks of african horse sickness in the surveillance zone of south africa. revue scienfique et technique, office international des epizooties, 25:1097–1109. venter, g.j., meiswinkel, r., nevill, e.m. & edwardes, m. 1996a. culicoides (diptera: ceratopogonidae) associated with livestock in the onderstepoort area, gauteng, south africa as determined by light-trap collections. onderstepoort journal of veterinary research, 63:315–325. venter, g.j., nevill, e.m. & van der linde, t.c. de k. 1996b. geographical distribution and relative abundance of stock-associated culicoides species (diptera: ceratopogonidae) in southern africa, in relation to their potential as viral vectors. onderstepoort journal of veterinary research, 63: 25–38. venter, g.j., paweska, j.t., van dijk, a.a., mellor, p.s. & tabachnick, w.j. 1998. vector competence of culicoides bolitinos and c. imicola (diptera: ceratopogonidae) for south african bluetongue virus serotypes 1, 3 and 4. medical and veterinary entomology, 12:101–108. sabeta_pp95-100.qxd introduction rabies virus (rv) is the prototype member of the lyssavirus genus, rhabdoviridae family, of the order mononegavirales (wunner, larson, dietzschold & smith 1988; tordo & kouknetzoff 1993). rabies virus possesses a non-segmented, negative-stranded rna genome of ~12 kb in length. the viral genome contains information for five proteins: nucleoprotein (n), matrix protein (m), phosphoprotein (p), glycoprotein (g) and polymerase (l). epidemiologically, rv is found virtually worldwide and is perpetuated in several domestic and wild carnivore species. in the southern african countries of zimbabwe and south africa, rabies virus exists as two epidemiologically separate lineages (referred to as canid and mongoose rabies biotypes), which has been confirmed by antigenic and genetic studies (foggin 1988; king, meredith & thomson 1993, 1994; von teichman, thomson, meredith & nel 1995; nel, jacobs, jaftha & meredith 1997). prior to the era of antigenic and genetic tools for studying the epidemiology of rabies, the existence of the two rabies biotypes was postulated from historical records and case surveillance data. in this paper, we demonstrate the use of reverse transcription polymerase chain reaction (rt-pcr) for confirming a clinical diagnosis of rabies infection in a 2-year-old thoroughbred colt imported in july 2003, from golden acres, harare in zimbabwe to the ashburton training centre, south africa (see fig. 1 for approximate locations of the two sites). 95 onderstepoort journal of veterinary research, 72:95–100 (2005) research communication importation of canid rabies in a horse relocated from zimbabwe to south africa c.t. sabeta1 and j.l. randles2 abstract sabeta, c.t. & randles, j.l. 2005. importation of canid rabies in a horse relocated from zimbabwe to south africa. onderstepoort journal of veterinary research, 72:95–100 in july 2003 a 2-year-old thoroughbred colt was imported from harare, zimbabwe to the ashburton training centre, pietermaritzburg, south africa. five months after importation, the colt presented with clinical signs suggestive of rabies: it was uncoordinated, showed muscle tremors and was biting at itself. brain tissue was submitted for analysis and the clinical diagnosis was confirmed by the fluorescent antibody test and reverse-transcription polymerase chain reaction (rt-pcr). phylogenetic analysis of the nucleotide sequence of the cytoplasmic domain of the glycoprotein and the g-l intergenic region of the rabies virus confirmed it to be an infection with a canid rabies virus, originating from an area in zimbabwe endemic for the domestic dog (canis familiaris) and side-striped jackal (canis adustus) rabies. keywords: canid rabies biotype, epidemiology, lyssavirus, rabies virus, south africa, zimbabwe 1 onderstepoort veterinary institute, rabies unit, private bag x05, onderstepoort, 0110 south africa e-mail: sabetac@arc.agric.za 2 rabies diagnostics unit, allerton provincial laboratory, private bag x2, cascades, pietermaritzburg, 3202 south africa accepted for publication 20 july 2004—editor 96 canid rabies in horse relocated from zimbabwe to south africa �������� � ����� ��� � ����� � � ��������� �� ������� ���� ���� ������ ����������� ��� fig. 1 map of zimbabwe and south africa showing the approximate geographical locations of harare (where the 2-yearold colt originated) and pietermaritzburg (where it subsequently developed clinical signs compatible with rabies) table 1 virus isolates included in the phylogenetic comparison lab. ref species locality and country of origin year accession no. no. isolated 22642a jackal (c. adustus)b glendale, zimbabwe 1994 af177088 21467 caninec goromonzi, zimbabwe 1993 af177066 21111 jackal (c. adustus) bromley, zimbabwe 1992 af177083 19649 jackal (c. adustus) marondera, zimbabwe 1991 ay605006 28498 jackal (c. adustus) marondera, zimbabwe 2002 ay605041 23357 jackal (c. adustus) wedza, zimbabwe 1995 af177090 24465 canine wedza cld, zimbabwe 1996 af177074 23367 jackal (c. adustus) marondera, zimbabwe 1995 af177093 24299 canine musikavanhu cl, zimbabwe 1996 af177073 23652 canine mutare, zimbabwe 1995 af177071 a03/646a equine pietermaritzburg, south africa 2003 ay605005 29263 lion (panthera leo) marondera, zimbabwe 2003 ay605014 24505 canine gutu, zimbabwe 1996 af177075 16387 canine zhombe cl, zimbabwe 1986 af177057 16254 canine odzi, zimbabwe 1986 af177055 22547 canine kumutsenzere, zimbabwe 1994 af177070 16347 canine troutbeck, nyanga, zimbabwe 1986 af177056 21869 canine nyakasoro, pfungwe cl, zimbabwe 1993 af177069 21057 canine muzarabani cl, zimbabwe 1992 af177064 20519 canine lower gweru, zimbabwe 1992 af177060 a00/413 canine pietermaritzburg, south africa 2000 ay605042 a90/57 canine durban, south africa 1990 af177101 a90/352 canine durban, south africa 1990 af177100 a95/755 canine amanzimtoti, south africa 1995 af303081 28522 jackal (c. mesomelas) gwanda, zimbabwe 2002 ay605038 23374 jackal (c. mesomelas) bulawayo, zimbabwe 1995 af177091 21579 jackal (c. mesomelas) tsholotsho cl, zimbabwe 1993 af177086 17722 jackal (c. mesomelas) gwanda, zimbabwe 1988 af177079 20034 canine bvumba, mutare, zimbabwe 1991 af177059 16838 canine shangani, zimbabwe 1987 af177058 27792 jackal (c. mesomelas) fort rixon, zimbabwe 2001 ay605032 27890 jackal (c. mesomelas) insiza, zimbabwe 2001 ay605039 19671 civettictis civetta rusape, zimbabwe 1991 af304188 a laboratory reference numbers: isolates from zimbabwe use the harare central veterinary laboratory rabies reference; isolates prefixed by “a” indicate the allerton provincial veterinary laboratory reference numbers, pietermaritzburg b where the jackal species was not definitively identified at collection, the species zone that it originated in, and hence the principal host, is given in parenthesis (see bingham et al. 1999b) c we use the term “canine” to refer to the domestic dog, canis familiaris d cl stands for communal lands 97 c.t. sabeta & j.l. randles the colt presented with clinical signs suggestive of rabies on 6 december 2003. it was uncoordinated, manifested muscle tremors and was biting at itself. it was subsequently admitted to the summerfield equine hospital on the same day where it was examined by a veterinary surgeon. it was euthanased the following day and brain tissue was submitted for rabies testing to the allerton provincial laboratory in pietermaritzburg, kwazulu-natal. although there was no recorded history of a dog bite and no skin wounds were detected, the brain sample tested positive by the fluorescent antibody test (fat). the rabies virus (laboratory reference no. a03/646) was then subjected to molecular characterization in order to establish its origin. materials and methods for molecular characterisation of the rabies virus (a03/646), it was decided to target the variable glycoprotein gene. rt-pcr and dna sequence analyses were carried out at the rabies unit, onderstepoort veterinary institute, pretoria as described previously (von teichman et al. 1995; sabeta, bingham & nel 2003). an amplicon of the expected size (~850 bp) was obtained with the g/l primer set and purified with a wizard pcr cleanup system (promega, usa). an aliquot (~50 ng) of the purified amplicon was sequenced using the bigdye terminator system (perkin elmer) with the same primer pair (g/l) as in the previous rt-pcr steps. a consensus sequence of the equine rabies virus was assembled after comparison of the forward and reverse sequences with sequence navigator software (pe applied biosytems). phylogenetic analysis of the nucleotide sequence of the rabies virus together with nucleotide sequences of other virus isolates from our database (table 1) was done using the phylip software package (phylogeny inference package). in brief, a multiple alignment of a 592-nucleotide region of the nucleotide sequences included in the analysis was generated with clustalw (thompson, higgins & thompson 1994). distance calculations were done using the kimura 2-parameter model for evolutionary rate (kimura 1980). for construction of the phylogenetic trees, the neighbour joining (nj) method combined with a 1 000 bootstrap iterations was used (saitou & nei 1987; hills & bull 1993). the program treeview was used to display the graphical output of the tree (fig. 2) (page 1996). results and discussion both the direct immunofluorescent antibody test (fat) and rt-pcr confirmed the rabies infection. the rabies virus was successfully amplified by the g/l primer pair to yield an expected product of approximately 850 bp (not shown). nucleotide sequencing of the purified pcr amplicon and phylogenetic comparison with other nucleotide sequences from our database of zimbabwean and south african rabies g-l nucleotide sequences demonstrated it to be a canid rabies virus (genotype 1) originating from zimbabwe (see fig. 2). despite the close genetic relatedness of the canid rabies virus isolates compared here [mean sequence homology of 96.3 % calculated in mega (kumar, tamura, jakobsen & nei 2001)], the rabies virus (a03/646) was found to cluster with zimbabwean canid rabies virus isolates (cluster 1) endemic within the domestic dog (canis familiaris) and sidestriped jackal (canis adustus) (bingham, foggin, wandeler & hill 1999a; bingham, foggin, wandeler & hill 1999b; sabeta et al. 2003). cluster 1 was statistically supported with a bootstrap value of 98 %. cluster 2 is composed of virus isolates exclusively from domestic dogs in north-eastern zimbabwe. clusters 1 and 2 consist of zimbabwean viral isolates and can be distinguished from isolates from domestic dogs in kwazulu-natal (cluster 3), the region in which the colt developed clinical rabies 5 months after it had been imported from harare. cluster 4 is made up of viruses exclusively from black-backed jackal (canis mesomelas) and cluster 5 from both domestic dogs and c. mesomelas. both clusters 4 and 5 represent viral isolates that are associated with rabies cycles in the c. mesomelas-zone of southern zimbabwe and northern south africa (sabeta et al. 2003). rabies in horses is a relatively uncommon disease and transmission of the disease from horses to humans is rare (green, smith, vernau & beacock 1992). however, a potential risk to veterinarians, horse handlers and horse owners does exist and should be emphasized. given the high incidence of rabies in zimbabwe and in the sub-region as a whole, it would be advisable for horse owners and those in the horse industry alike to vaccinate their animals against rabies. the absence of wounds in the 2-year-old colt does not exclude inoculation by a bite from a rabid animal, because had bite wounds been present, they might have been very small and puncture-like and were therefore not detected, or they could have healed prior to the development of clinical signs. the rabies virus isolate investigated 98 canid rabies in horse relocated from zimbabwe to south africa � !" � #"!$ � ### �#%!"% � &"%& � ""!� � '!!$ � ''�$ � " %%%& � '!� &%%% � �� % !' � "��� &% �#!'&$&&$' %! �#! �" � �"$ �#!'"$ � ��#% � #&!% � #��$ ���("#' �%�(�$ �%�('� �%�($�� %%#�� � &� � ''$" � #�$% �#$$ %' % � ��'" �#!&'& � $$% � $&%� %$ )* � #%!$# � " ' # ��'(!"! fig. 2 neighbour joining tree showing the phylogenetic position of the equine rabies virus (a03/646) described in the paper. the inferred phylogeny is based on sequence comparison of the cytoplasmic domain of the glycoprotein and the g-l intergenic regions of the virus isolates. included in the phylogenetic tree are: a typical zimbabwean mongoose rabies virus (ci19671), typical canid rabies viruses from zimbabwe (j22642, d21467, j21111, j19649, j28948, j23357, d24465, j23667, d24299, d23652, lion29263, d24505, d16387, d16254, d22547, d16347, d21869, d21057, d20519, j28522, j23374, j21579, j17722, d20034, d16838, j27792, j27890) and south africa (a00/413, a90/57, a90/352, a95/755) (see table 1 for epidemiological information of these isolates). the bootstrap values are shown on the branches. the horizontal branch lengths are proportional to the similarity of sequences within and between clusters, with the scale bar indicating nucleotide substitutions per site. vertical lines are for clarity of presentation only and the sequence of pv was included as an outgroup and described here would most likely have been transmitted as a result of a dog bite. this is highly probable considering that the canid rabies variant is widespread and prevalent, making it the most threatening variant for humans and domestic carnivore species alike (bingham et al. 1999a). this calls for stricter import/export regulations for animals in transit in order to diminish the spread of rabies across regional borders. although horses are known to be moderately susceptible to rabies infection, long incubation periods of up to 5 months, as shown in this case, may be unusual. the clinical signs of rabies are variable in horses and generally appear after an incubation period of 2–6 weeks, although in some it may be up to 3 months (beran 1981). however, rabies virus has been isolated from the saliva of a dog that survived 20 months post-exposure to the virus (green et al. 1992). although it has been shown that rabies virus remain at the site of a bite for most of the incubation period, which is generally 10–90 days, longer incubation periods have been described by smith, fishbean, rupprecht & clark (1991). this investigation illustrates the usefulness of rtpcr as a tool for studying animal viral diseases such as rabies. furthermore, it increases our understanding of epidemiological relationships of lyssaviruses not only through establishing their genetic relationships but also by providing us with information on the origin, geographical distribution and paths of transmission of the various strains of the virus (brown 1994). several molecular epidemiological studies of rabies have been conducted in the southern african subregion (nel, thomson & von teichman 1993; von teichman et al. 1995; nel et al. 1997; sabeta et al. 2003). further molecular studies of lyssaviruses in countries in the sub-region that include botswana, namibia, mozambique, swaziland, lesotho and zambia should be encouraged, and are already being realized. these studies are essential in order to elucidate the dynamics of rabies in the sub-region. an expansion of our current nucleotide sequence database of rabies viruses would thus be useful for tracing the routes of infection as illustrated by the equine case described here and also for establishing concrete measures to control the disease. acknowledgements this investigation was made possible by funding from the international foundation of sciences (ifs) (grant #b3179-1). we thank prof. berndt klingeborn for critically reading this manuscript. references beran, g.w. 1981. rabies and infections by rabies-related viruses, in crc handbook series on zoonoses, section b. viral zoonoses, vol ii, edited by g.w. beran. boca raton, florida: crc press. bingham, j., foggin, c.m., wandeler, a.i. & hill, f.w.g. 1999a. the epidemiology of rabies in zimbabwe. 1. rabies in dogs (canis familiaris). onderstepoort journal of veterinary research, 66:1–10. bingham, j., foggin, c.m., wandeler, a.i. & hill, f.w.g. 1999b. the epidemiology of rabies in zimbabwe. 1. rabies in jackals (canis adustus and canis mesomelas). onderstepoort journal of veterinary research, 66:11–23. brown, a.j. 1994. methods of evolutionary analysis of viral sequences, in evolutionary biology of viruses, edited by s.s. morse. new york: raven press. foggin, c.m. 1988. rabies and rabies-related viruses in zimbabwe: historical, virological and ecological aspects. ph.d. thesis, university of zimbabwe. green, s.l., smith, l., vernau, w. & beacock, s.m. 1992. rabies in horses: 21 cases (1970–1990). journal of the american veterinary medical association, 200:1133–1137. hills, d.m. & bull, j.j. 1993. an empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. systematic biology, 42:182–192. kimura, m. 1980. a simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. journal of molecular evolution, 16: 111–120. king, a.a., meredith, c.d. & thomson, g.r. 1993. canid and viverrid viruses in south africa. onderstepoort journal of veterinary research, 60:295–299. king, a.a., meredith, c.d. & thomson, g.r. 1994. the biology of southern african lyssavirus variants, in lyssaviruses, edited by c.e. rupprecht, b. dietzshold & h. koprowski. berlin: springer-verlag publishers. kumar, s., tamura, k., jakobsen, i.b. & nei, m. 2001. mega2: molecular evolutionary genetics analysis software, arizona state university, tempe, arizona, usa. nel, l.h., thomson, g.r. & von teichman, b.f. 1993. molecular epidemiology of rabies virus in south africa. onderstepoort journal of veterinary research, 60:301–306. nel, l.h., jacobs, j., jaftha, j. & meredith, c. 1997. natural spillover of a distinctly canidae-associated biotype of rabies virus in an expanded wildlife host range in southern africa. virus genes, 15:79–82. page, r.d.m. 1996. treeview: an application to display phylogenetic trees on personal computers. computer applications in the biosciences, 12:83–88. sabeta, c.t., bingham, j. & nel, l.h. 2003. molecular epidemiology of canid rabies in zimbabwe and south africa. virus research, 91:203–211. saitou, n. & nei, m. 1987. the neighbour-joining method: a new method for reconstructing phylogenetic trees. molecular biology and evolution, 4:406–425. smith, j.s., fishbean, d.b., rupprecht, c.e. & clark, k. 1991. unexplained rabies in three immigrants in the united states. the new england journal of medicine, 166:205–211. 99 c.t. sabeta & j.l. randles thompson, j.d., higgins, d.g. & gibson, t.j. 1994. clustalw: improving the sensitivity of progressive multiple alignment through sequence weighting, position-specific gap penalties and weight matrix choice. nucleic acids research, 22:4673–4680. tordo, n. & kouknetzoff, a. 1993. the rabies virus genome: an overview. onderstepoort journal of veterinary research, 60:263–269. von teichman, b.f., thomson, g.r., meredith, c.d. & nel, l.h. 1995. molecular epidemiology of rabies virus in south africa: evidence for two distinct virus groups. journal of general virology, 76:73–82. wunner, w.h., larson, j.k., dietzschold, b. & smith, c.l. 1988. the molecular biology of rabies viruses. reviews of infectious diseases, 10:771–784. 100 canid rabies in horse relocated from zimbabwe to south africa bezuidenhout_175-180.qxd introduction the gross, light and electron microscopic structure of the respiratory air sacs of the fowl are well documented (carlson & beggs 1973). a simple epithelium resting on a basal lamina supported by a layer of fibro-elastic connective tissue lines the respiratory surface of the air sacs. the epithelium varies from simple squamous to a ciliated columnar epithelium with goblet cells. air sacs of birds are prone to infection by various microorganisms, including fungi, bacteria and viruses. some microorganisms or their toxins need to attach to glycoproteins or glycolipids on cell surfaces in order to cause disease, e.g. the s glycoproteins of the infectious bronchitis virus bind to neuraminic acid-containing glycans (schultze, cavanagh & herrler 1992). the relative expression of glycoconjugates is important because of the significant role membrane glycoconjugates appear to play in respiratory host defenses (lasky 1992; abdi, kobzik, li & mentzer 1995). the diversity of glycoconjugates and the application of lectin histochemistry is extensively reviewed by spicer & schulte (1992). most lectins are multimeric proteins that share the common property of binding to defined sugar structures (spicer & schulte 1992). the assumed monosaccharide specificity of a lectin can be very different from the actual complex oligosaccharide(s) it recognizes in histochemical preparations. because of the specificity that 175 onderstepoort journal of veterinary research, 72:175–180 (2005) a lectin histochemical study of the thoracic respiratory air sacs of the fowl a.j. bezuidenhout department of biomedical sciences, college of veterinary medicine cornell university, ithaca, new york 14850 abstract bezuidenhout, a.j. 2005. a lectin histochemical study of the thoracic respiratory air sacs of the fowl. onderstepoort journal of veterinary research, 72:175–180 the lectin-binding characteristics of the epithelial lining of the thoracic air sacs of the chicken were determined. con a, lca and psa bound to the apical membrane as well as to the cytoplasm distal to the nucleus of the surface epithelium, indicated the presence of α-linked mannose as well as nacetylchitobiose-linked α-fucose residues in the glycoproteins. gsl i bound to the apical membrane and cytoplasm distal to the nucleus, but not to the cilia of the epithelium, where-as mpl, dba and rca120 bound to the apical membrane, cilia and cytoplasm, indicated the presence of α-linked nacetylgalactosamine residues. however, neither sja or sba showed any binding, indicating the absence of β anomers of galactosyl (β1.3)n-acetylgalactosamine and β-linked n-acetylgalactosamine residues. uea i bound to the apical membrane and cilia, as well as to the cytoplasm of a few cells, indicated the presence of α-linked fucose residues. pna bound to the apical membrane of some, but not all, surface epithelium cells, indicated the presence of galactosyl (β1.3)n-acetylgalactosamine residues. wga bound to the apical membrane and cilia, as well as to the cytoplasm of a few cells, indicated the presence of neuraminic acid residues. keywords: fowl, lectin binding, thoracic air sacs accepted for publication 15 april 2005—editor each lectin has toward a particular carbohydrate structure, even oligosaccharides with identical sugar compositions can be distinguished. some lectins will bind only to structures with mannose or glucose residues, while others may recognize only galactose residues. some lectins require that the particular sugar be in a terminal non-reducing position in the oligosaccharide, while others can bind to sugars within the oligosaccharide chain. some lectins do not distinguish between α or β anomers while others require not only the correct anomeric structure, but also a specific sequence of sugars for binding. some glycoproteins have terminal sialic acid residues that can block lectin binding and require desialylation before binding. no data could be found in the existing literature on the lectin-binding glycoproteins or glycolipids of the epithelium lining the respiratory surface of air sacs in chickens. the present study was undertaken to determine the lectin-binding characteristics of the thoracic air sacs of the chicken using lectin histochemical techniques. materials and methods source and type of birds for this study a total of 25 specific pathogen free (spf) white leghorn chickens were obtained from the poultry research farm, cornell university, ithaca, new york. the birds were clinically healthy, of both sexes, 42–84 days old and weighed 140– 900 g. collection of the air sac membranes the birds were euthanased with co2 in a chamber designed for this purpose. after euthanasia they were placed in dorsal recumbency, the skin over the ventral abdomen and thorax was removed and the hips dislocated to provide stability in dorsal recumbency. the ventral abdominal wall was incised just caudal to the sternum to open the ventral hepatic peritoneal cavities. from here the incision was extended cranially along the lateral borders of the pectoral muscles, transecting the sternal ribs close to their junctions with the sternum. both clavicles and coracoids were cut and the sternum reflected cranially. the ventral abdominal wall was removed and the gizzard freed from its attachments to facilitate access to the thoracic air sacs. the medial wall of the thoracic air sacs was visualized by cutting the left and right hepatic ligaments and reflecting the liver and gut medially. the septum separating the cranial and caudal thoracic air sacs was located and a small transverse incision was made over the septum along their ventral borders. the septum was then incised along its entire length to create one cavity consisting of both the cranial and caudal thoracic air sacs. a thin (2 mm thick) stainless steel ring was passed through the incision and placed so as to include the medial wall of both cranial and caudal air sacs. a thick (3 mm) stainless steel ring was placed on the peritoneal surface of the air sac and aligned with the thin ring. the two rings were clamped together with a modified hemostat and the membrane was removed by cutting the tissue along the outer circumference of the rings. the rings were then clamped in a small binder clamp and the hemostat removed. the entire specimen (clamp, rings and membrane) was placed in either 10 % phosphatebuffered formalin or bouin’s fixative. this ensured that the membranes did not shrink during fixing and processing. processing of specimens for light microscopy for paraffin wax embedding the tissues were dehydrated through a graded series of ethanol, cleared in propar (anatech ltd., lake rd., battle creek, mi 49015) and infiltrated with paraffin wax. after infiltration the membrane was cut from the rings, embedded in paraffin wax and sectioned at 4 µm on a rotary microtome. paraffin wax-embedded sections were mounted on slides coated with poly-l-lysine (vector laboratories, burlingame, california). lectins used thirteen biotinylated lectins (kit i and kit ii, vector laboratories) were screened. these lectins were obtained from the plants maclura pomifera (mpl), dolichos biflorus (dba), glycine maxi (sba), arachis hypogaea (pna), ricinus communis (rca), triticum vulgaris (wga), ulex europaeas (uea), concanavalin a (con a), griffonia simplicifolia (gsl), pisum sativum (psa), lens culinaris (lca) and sophora japonica agglutinin (sja), and wheat germ agglutinin (wga). optimal dilution concentration for each lectin was determined and varied from 5–50 µg/ml. lectin staining tissue sections were de-paraffinated in xylene and re-hydrated through a graded series of ethanol. endogenous peroxidase activity was blocked by incubation of sections for 10 min at room temperature in 0.5 % (v/v) hydrogen peroxide in methanol. after rinsing the sections in 0.01 mol phosphate buffer 176 thoracic respiratory air sacs of the fowl solution (pbs), the specific biotinylated lectin at the appropriate dilution in pbs was added to cover the tissue sections, and then incubated at 37 °c in a humid chamber for 1 h. negative controls for each tissue were incubated with buffer only, and for binding specificity the lectins were blocked with their inhibitory sugars. the sections were washed with pbs and covered with pre-diluted streptavidin-peroxidase conjugate (vector laboratories) for 15 min at room temperature in a humid chamber. the sections were then washed six times with pbs and incubated with aminoethyl carbazole (aec) substrate (zymed laboratories inc., san francisco, california) for 2–20 min at room temperature. colour development was monitored under the microscope. sections were washed in distilled water to stop further colour development, and then counterstained with haematoxylin. all sections were examined under an olympus bh-2 light microscope and relevant areas photographed. lectin staining was recorded as positive or negative. results and discussion the results of staining with various lectins are summarized in table i. processing paraffin wax embedded tissues for light microscopy requires removing the wax from the tissues prior to staining. this process also removes most of the glycolipids (brooks, leathem & schumacher 1997) that are therefore excluded in the results of this study. it is generally agreed that a squamous epithelium lines the air sacs, and that many of the cells contain surfactant in the form of myeloid inclusions. welldefined areas of ciliated cuboidal to columnar cells, as well as goblet cells are randomly distributed over the surface of the membrane (lucas 1970; smith, meier, lamke, neill & box 1986). the results of the present study showed that areas of squamous epithelium reacted evenly for all the lectins, except for sja and sba that did not show any positive binding, irrespective of the concentration of the lectin that was used. due to the very attenuated nature of the squamous epithelium it was not possible to distinguish between staining of the apical membrane and staining of the cytoplasm distal to the nucleus. contrary to the squamous epithelium, areas of columnar and ciliated epithelium did not react uniformly with all the lectins that showed positive binding. some lectins bound to the apical membrane of ciliated and non-ciliated cells, while others only bound to the apical membrane of non-ciliated cells. binding to the cytoplasm also varied between the different lectins. the lectins con a, lca and psa bind to a wide variety of membrane glycoproteins that have a core structure that includes a-linked mannose. lca rec177 a.j. bezuidenhout table 1 results of lectin binding lectin binding sugar specificity blocking sugar mpl 6.25 µg/ml positive n-acetyl galactosamine d+ galactose uea 50 µg/ml positive fucose fucose pna 50 µg/ml positive galactose d+ galactose rca 50 µg/ml positive galactose and nd+ galactose acetylgalactosamine con a 50 µg/l positive glucose and mannose methyl mannoside and methyl glucoside dba 25 µg/ml positive n-acetyl galactosamine n-acetyl galactosamine gsl i 25 µg/ml positive galactose and ngalactose and/or n-acetyl acetylgalactosamine galactosamine did not block wga 12.5 µg/ml positive n-acetylglucosamine chitobiose psa 12.5 µg/ml positive glucose and mannose n-methyl mannoside and methyl glucoside lca 12.5 µg/ml positive glucose and mannose n-methyl mannoside and methyl glucoside sba up to 100 µg/ml negative n-acetyl galactosamine sja up to 100 µg/ml negative n-acetyl galactosamine ognizes additional sugars as part of the receptor structure and is therefore more specific than con a. psa is almost identical to lca and binds to α-linked mannose with an n-acetylchitobiose-linked α-fucose residue included in the receptor sequence (spicer & schulte 1992; brooks et al. 1997). all three lectins 178 thoracic respiratory air sacs of the fowl a b c d e f g h i j k l require ca and mg cations for binding. con a (fig. 1l), lca (fig. 1a) and psa (fig. 1b) showed strong binding to the apical membrane, and to a lesser degree to the cytoplasm distal to the nucleus of the surface epithelium of the thoracic air sacs. this would indicate the presence of a-linked mannose, as well as α-linked mannose with an n-acetylchitobiose-linked α-fucose residue in the glycoproteins of the apical membrane and in the cytoplasm of the epithelium. the cytoplasm of the surface epithelium contains large amounts of glycogen (carlson & beggs 1973). therefore the affinity of the cytoplasm for con a is probably due to the presence of glucose residues in the glycogen (pedini, ceccarelli & gargiulo 1994; nadel 2003). n-methyl mannoside and methyl glucoside effectively blocked binding of the three lectins. the lectins gsl i, mpl, rca120 sja, sba and dba preferentially bind to α-linked n-acetylgalactosamine residues of glycoproteins (spicer & schulte 1992). gsl i (fig. 1h) showed binding to the apical membrane and to the cytoplasm distal to the nucleus of a few cells, but did not bind to the cilia of the epithelium. mpl (fig. 1k), dba (fig. 1g) and rca120 (fig. 1e) showed binding to the apical membrane and cilia, as well as to the cytoplasm proximal to the nucleus of some cells. neither sja (fig. 1c) nor sba (fig. 1d) showed any binding to the epithelium. this may be due to the fact that sja preferentially binds to β anomers of galactosyl (β1.3)n-acetylgalactosamine and that sba preferentially binds to oligosaccharide structures with terminal α or β-linked nacetylgalactosamine residues (spicer & schulte 1992). this would indicate that a-linked n-acetylgalactosamine residues are present on the glycoproteins of the epithelium, but that it is neither present as β anomers, nor as terminal α or β residues. binding of gsl i, mpl, dba and rca120 could effectively be blocked with galactose. uea i binds to glycoproteins and glycolipids containing α-linked fucose residues at a branch or terminal position on the glycoprotein (spicer & schulte 1992). the lectin showed positive binding to the apical membrane and cilia of the epithelium (fig. 1i). a few cells also showed positive binding to the cytoplasm distal to the nucleus. this would indicate that the glycoproteins of the apical membrane and the cytoplasm of some cells contain α-linked fucose residues. binding could effectively be blocked with fucose. pna binds specifically to galactosyl (β1.3)n-acetylgalactosamine (t-antigen) present in many glycoconjugates in soluble and membrane associated glycoproteins (lotan, skutelsky, danon & sharon 1975). the lectin showed positive binding to the apical membrane and cytoplasm of some cells of the epithelium, but not to all of them (fig. 1f). this would indicate that the specific receptor is not present on all surface-lining cells. binding could effectively be blocked with galactose. wga preferentially binds to oligosaccharides containing terminal dimers and trimers of n-acetylglucosamine or chitobiose, and can also interact with some glycoproteins via sialic (neuraminic) acid residues (spicer & schulte 1992). the lectin showed binding to the apical membrane and cilia, as well as to the cytoplasm of a few cells (fig. 1j). this would 179 a.j. bezuidenhout fig. 1 results of lectin binding a lca bound to the apical membrane and cytoplasm distal to the nucleus of the surface epithelium b psa bound to the apical membrane and cytoplasm distal to the nucleus of the surface epithelium c sja showed no affinity to any part of the surface epithelium d sba showed no affinity to any part of the surface epithelium e rca 120 bound to the apical membrane, cilia and cytoplasm distal to the nucleus of the surface epithelium f pna bound to the apical membrane and cytoplasm of some, but not all, cells g dba bound to the apical membrane and cilia, and to the cytoplasm of some of the surface epithelium h gsl bound to the apical membrane, but not to the cilia of the surface epithelium i uea i bound to the apical membrane and cilia, as well as to the cytoplasm distal to the nucleus of some cells of the surface epithelium j wga bound to the apical membrane and cilia of the surface epithelium k mpl bound to the apical membrane and cilia, as well as to the cytoplasm distal to the nucleus of some surface epithelial cells l con a bound to the apical membrane of the surface epithelium indicate that the glycoproteins of the apical membrane and cytoplasm of some cells contain dimers and trimers of n-acetylglucosamine or chitobiose, or sialic acid residues as part of their structure. binding was effectively blocked with chitin hydrolysate. in conclusion, most cells of the epithelium lining the respiratory surface of the thoracic air sacs of the fowl showed an affinity to a wide variety of lectins. this indicated that the apical cell membrane and the cytoplasm distal to the nucleus contained α-linked mannose, α-linked mannose with an n-acetylchitobiose-linked α-fucose, α-linked n-acetylgalactosamine, α-linked fucose, galactosyl (β1.3)n-acetylgalactosamine and dimers and trimers of n-acetylglucosamine or chitobiose, or sialic acid residues as part of their glycoconjugate structure. this information will be useful in further studies of air sacculitis in poultry. references abdi, k., kobzik, l., li, x. & mentzer, s.j. 1995. expression of membrane glycoconjugates on the sheep lung endothelium. laboratory investigation, 72:445–452. brooks, s.a., leathem, a.j.c. & schumacher, u. (eds) 1997. lectin histochemistry, a concise practical handbook. oxford: bios scientific publishers ltd. carlson, h.c. & beggs, e.c. 1973. ultrastructure of the abdominal air sac of the fowl. research in veterinary science, 14:148–150. lasky, l.a. 1992. selectins: interpreters of cell-specific carbohydrate information during inflammation. science, 258:964– 969. lotan, h., skutelsky, e., danon, d. & sharon, n. 1975. the purification, composition, and specificity of the anti-t lectin from peanut (arachis hypogaea). journal of biological chemistry, 250:8518–8523. lucas, a.m. 1970. avian functional anatomic problems. federation proceedings, 29(5):1641–1648. nadel, e. 2003. metabolism and nutrition, in medical physiology, edited by w.f. boron & e.l. boulpaep. philadephia: saunders. pedini, v., ceccarelli, p. & gargiulo, a.m. 1994. glycoconjugates in the mandibular salivary gland of adult dogs revealed by lectin histochemistry. research in veterinary science, 57:353–357. schultze, b., cavanagh, d. & herrler, g. 1992. neuramindase treatment of avian infectious bronchitis coronavirus reveals a hemagglutinating activity that is dependent on sialic acid-containing receptors on erythrocytes. virology, 189:792–794. smith, j.h., meier, j.l., lamke, c., neill, p.j. & box, e.d. 1986. microscopic and submicroscopic anatomy of the parabronchi, air sacs and respiratory space of the budgerigar (melopsittacus undulatus). american journal of anatomy, 177:221–242. spicer, s.s. & schulte, b.a. 1992. diversity of cell glycoconjugates shown histochemically: a perspective. journal of histochemistry and cytochemistry, 40:1–38. 180 thoracic respiratory air sacs of the fowl junker_27-36.indd 27 onderstepoort journal of veterinary research, 73:27–36 (2006) a check-list of the pentastomid parasites of crocodilians and freshwater chelonians k. junker and j. boomker* department of veterinary tropical diseases, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract junker, k. & boomker, j. 2006. a check-list of the pentastomid parasites of crocodilians and freshwater chelonians. onderstepoort journal of veterinary research, 73:27–36 based on published records and own data a summary is given of the geographical distribution of the currently known species of pentastomid parasites infecting crocodiles and alligators, as well as freshwater chelonians. a brief generic diagnosis is provided for each genus. fourteen out of the currently 23 living crocodilian species have been recorded as being host to one or more pentastomes. out of the 32 pentastome species six are considered species inquirendae. presently, six genera of crocodilian pentastomes, agema, alofia, leiperia, sebekia, selfia and subtriquetra are recognized. african crocodiles harbour eight pentastome species, six of which have been recorded from the nile crocodile, crocodylus niloticus. three species belong to the genus sebekia, alofia being represented by two and leiperia by only one species. two species, alofia parva and agema silvaepalustris, occur in the dwarf crocodile, osteolaemus tetraspis, and the slender-snouted crocodile, crocodylus cataphractus, exclusively, but a single sebekia species is shared with the nile crocodile. the genus agema is endemic to the african region. infective stages of the pentastome sub triquetra rileyi, thought to utilize nile crocodiles as final hosts, have been recovered only from fishes. the largest number of pentastome species is found in the australasian region. of these, the indo-pacific croc odile, crocodylus porosus, harbours seven, representing the genera alofia, sebekia, lei peria and selfia. selfia is exclusive to the latter host. the genus subtriquetra has been reported from “indian crocodiles”, a term possibly referring to either crocodylus palustris, crocodylus porosus or gavi alis gangeticus. ten species of pentastomes parasitizing the crocodilian genera alligator, caiman, crocodylus and melanosuchus have been recorded from the neotropical region including the southern states of the north american continent. the two most wide-spread pentastome genera, alofia and sebekia, have been recorded together with representatives of the genus sub triquetra and immature and larval forms of leiperia. to date the two monospecific genera, pelonia, from two terrapin species, pelusios sinuatus and pelomedusa subrufa, in south africa, and diesingia from hydraspis geoffroyana and hydromedusa tectifera in south america, are the only chelonian pentastomes recovered world-wide. a possible exception is the crocodilian pentastome sebekia mississippiensis which can reach maturity in exper imentally infected terrapins. keywords: agema, alligator, alofia, caiman, crocodylus, diesingia, gavialis, hydraspis, hydromedusa, leiperia, melanosuchus, pelomedusa, pelonia, pentastomida, phrynops, sebekia, selfia, subtriquetra, terrapins * author to whom correspondence is to be directed. e-mail: jboomker@op.up.ac.za accepted for publication 1 august 2005—editor 28 check-list of pentastomid parasites of crocodilians and freshwater chelonians introduction the pentastomid parasites of chelonians and crocodilians are currently divided into the family sebekidae and subtriquetridae. the former comprises seven gen era, namely, agema, alofia, selfia, sebekia, leiperia, diesingia and pelonia. while the first four genera inhabit the lungs and bronchioles of the croco dilian host, leiperia occurs in the trachea and bronchi (riley, spratt & winch 1990; riley 1994; riley & huch zermeyer 1996; riley, hill & huch zermeyer 1997). diesingia and pelonia parasitize the lungs of chelonian final hosts (junker & boomker 2002; junker, riley & boomker 2003). a single member of sebekia, sebekia mississippiensis, might be able to reach maturity in chelonians, too, but as yet no mature specimens have been collected from naturally infected hosts (dukes, shealy & rogers 1971). members of the monogeneric family sub triquetridae inhabit the nasopharynx of their crocodilian final hosts, but subtriquetra rileyi, of which currently only infective larvae have been recovered, needs verification (winch & riley 1986a; junker, boomker & booyse 1998). during the past 10 years renewed progress has been made as regards the taxonomy of crocodilian pentastomes. the older genera, alofia, sebekia and leiperia, have been revised, and examination of new material has led to the description of several new genera and species (riley 1994; riley et al. 1990, 1997; riley & huchzermeyer 1996; junker et al. 1998). however, there is a dearth of data concerning the chelonian pentastomids. in order to provide a quick reference tool, this checklist consists of two parts, following the example of sambon (1922). the first part lists the parasites under their scientific names, their synonyms and their authorities. a short generic diagnosis precedes each genus and the parasites are grouped according to the geographic distribution of their respective hosts. the list starts with africa, followed by australasia. south and north america are listed last. the second part of the check-list alphabetically lists the hosts and their synonyms, and, also in alphabetical order, their respective parasites. the nomenclature and synonyms of the crocodilian and chelonian hosts are according to getz (2002). only references dealing with mature pentastomes are included in the check-list, but for completeness’ sake the intermediate hosts of pentastome species of which only larval forms are known are listed. parasite/host check-list of the pentastomida family sebekidae sambon, 1922 genus agema riley, hill & huchzermeyer, 1997 generic diagnosis: overall shape typical of smaller members of the sebekidae; males claviform, females with more uniform diameter and conical posterior terminating in a small blunt point; adult hooks smooth; blades smoothly curved without abrupt right-angled bend near to base; blade on anterior hook pair larger than that of posterior pair; fulcrum without cowl; mouth ovoid and sides of cadre united anteriorly and posteriorly by segments of chitin which appear as two crescents; copulatory spicules delicate and elongate; basal section without a hooked collar (riley et al. 1997). africa 1. agema silvaepalustris riley, hill & huchzermeyer, 1997 crocodylus cataphractus riley, hill & huchzermeyer (1997), republic of the congo osteolaemus tetraspis riley, hill & huchzermeyer (1997), republic of the congo genus alofia giglioli, 1922 generic diagnosis: size small; body banana-shaped; hooks large with long, narrow, slightly curved blade and a slender base; absence of chitinous formation at the base of the anterior hook; chitinous buccal cadre large and u-shaped; intestine as in sebekia; found in crocodiles (fain 1961 in riley 1994); caudal extremity of female bluntly rounded, often swollen into a bulb (riley 1994); hooks usually smooth (rarely with patches of minute spines [see alofia nilotici and alofia parva (riley & huchzermeyer 1995a, b)], blades finely canaliculated, bent through almost a right angle at the base; peg-like extension of oral cadre projects into oesophagus; copulatory spicules with double-hooked collar on the shorter of the two anterior extensions (riley & huchzermeyer 1995a, b; junker, boomker & bolton 1999). africa 1. alofia nilotici riley & huchzermeyer, 1995 crocodylus niloticus 29 k. junker & j.boomker riley & huchzermeyer (1995a), bo t swana junker, boomker & bolton (1999), south africa 2. alofia parva riley & huchzermeyer, 1995 crocodylus cataphractus riley & huchzermeyer (1995b, 2000), repub lic of the congo osteolaemus tetraspis osborni riley & huchzermeyer (1995b, 2000), republic of the congo 3. alofia simpsoni riley, 1994 unknown crocodilian riley (1994), ghana crocodylus niloticus riley & huchzermeyer (1995a), botswana junker, boomker & bolton (1999), south africa australasian region 4. alofia ginae giglioli, 1922 unknown crocodilian sambon (1922), samoa most probably crocodylus porosus, as it is the only crocodilian whose range extends as far as fiji in the pacific ocean and it is known to have colonized many small islands as far as nearly 1 000 km from land (ross 1989). crocodylus porosus (?) riley (1994), philippines the collector did not specify the host, but riley (1994) concludes it to be c. porosus, as the only other philippinian crocodile, crocodylus mindorensis, does not occur in the region from which the parasites were recovered. 5. alofia merki giglioli, 1922 sebekia merki heymons, 1941 unknown crocodilian sambon (1922), samoa most probably crocodylus porosus, as it is the only crocodilian whose range extends as far as fiji in the pacific ocean and it is known to have colonized many small islands as far as nearly 1 000 km from land (ross 1989). crocodylus porosus riley (1994), northern territory, australia riley (1994), philippines 6. alofia indica (von linstow, 1906) hett, 1924, species inquirenda gavialis gangeticus hett (1924), india south & north american region 7. alofia platycephala (lohrmann, 1889) giglioli, 1922 pentastomum platycephalum lohrmann, 1889; porocephalus platycephalus shipley, 1898; reighardia platycephala sambon, 1910 unknown crocodilian lohrmann (1889), south america caiman crocodilus self & rego (1985), brazil caiman latirostris heymons (1941), paraguay comment: hirst (1922) described alofia adriatica (hirst, 1922) giglioli, 1922 from an unknown host from the adriatic. as crocodilians do not occur in the adriatic this species will have to remain species inquirenda until further material becomes available. genus leiperia sambon, 1922 generic diagnosis: female with spirally coiled abdomen; broad bands of chloride cells; hooks smooth, flat-topped with sharply curved blade; oral cadre vshaped with large anterior flanges, numerous pores around the pharynx; copulatory spicules heavily chitinized with complex internal supporting structures, shorter of the two anterior extensions forms a smooth collar (double in l. australiensis) around the longer spatulate extension; cirrus tip modified into a flattened trumpet of longitudinally-striated chitin (riley & huchzermeyer 1996; junker, boomker, swa nepoel & taraschewski 2000). africa 1. leiperia cincinnalis (vaney & sambon, 1910) sambon, 1922 reighardia cincinnalis vaney & sambon, 1910; 30 check-list of pentastomid parasites of crocodilians and freshwater chelonians porocephalus nematoides de beauchamp, 1918 crocodylus cataphractus fain (1961), central africa: infective larva crocodylus niloticus vaney & sambon (1910), uganda sambon (1922), zimbabwe rodhain & vuylsteke (1932), democratic republic of the congo junker, boomker, swanepoel & tara schewski (2000), south africa australasian region 2. leiperia australiensis riley & huchzermeyer, 1996 crocodylus johnsoni riley & huchzermeyer (1996), northern ter ritory, australia crocodylus porosus riley & huchzermeyer (1996), northern territory, australia south & north american region 3. leiperia gracilis diesing, 1936, species inquirenda pentastoma gracile diesing, 1836 (partim); penta stomum gracile leidy, 1856; pentastoma gracilis parona, 1891; porocephalus gracilis shipley, 1898; porocephalus crocodili wheeler, 1915 (par tim); leiperia neotropica heymons & vitz thum, 1935 immature and larval forms were recovered from the following hosts, but adults have not been collected. alligator mississippiensis leidy (1856, in sambon 1922), locality unknown, north america crocodylus acutus heymons (1935), south america caiman crocodilus heymons (1935), brazil genus sebekia sambon, 1922 generic diagnosis: body short and squat with 58– 94 compressed annuli; lateral lines conspicuous; cephalothorax small, wedge-shaped and projecting nipple-like from the abdomen, ventral surface continuous with that of the abdomen; mouth subterminal and shaped like an inverted ‘u’; oral cadre oval to elongate, highly variable in shape, without long, parallel sides and generally united anteriorly; hooks small, equal or subequal, claw-shaped, with convex or flat dorsal surface; all hooks spiny (rarely only the anterior pair); all fulcra often with spinous anterior extension (rarely only the anterior pair); hook barb curved, strongly united and continuous with shank; spicules generally obpyriform, with one or two fine sclerotized rods supporting membranous region distally. parasites of the lungs of crocodilians, rarely of chelonians (riley et al. 1990). africa 1. sebekia cesarisi giglioli, 1922 crocodylus sp. sambon (1922), africa crocodylus niloticus riley & huchzermeyer (1995a), botswana junker, boomker & bolton (1999), south africa 2. sebekia okavangoensis riley & huchzermeyer, 1995 sebekia cesarisi riley, spratt & winch, 1990 crocodylus cataphractus riley & huchzermeyer (2000), republic of the congo crocodylus niloticus riley & huchzermeyer (1995a), botswana junker, boomker & bolton (1999), south africa osteolaemus tetraspis riley & huchzermeyer (2000), republic of the congo 3. sebekia wedli giglioli, 1922 pentastoma oxycephalum var. minor wedli, 1861; sebekia oxycephala self & rego, 1985 crocodylus niloticus devos (1939), democratic republic of the congo riley, spratt & winch (1990), uganda riley & huchzermeyer (1995a), botswana junker, boomker & bolton (1999), south africa 31 k. junker & j.boomker australasian region 4. sebekia johnstoni riley, spratt & winch, 1990 crocodylus johnsoni riley, spratt & winch (1990), northern territory, australia crocodylus porosus riley, spratt & winch (1990), northern territory, australia 5. sebekia multiannulata riley, spratt & winch, 1990 crocodylus johnsoni riley, spratt & winch (1990), northern territory, australia crocodylus porosus riley, spratt & winch (1990), northern territory, australia 6. sebekia purdieae riley, spratt & winch, 1990 crocodylus porosus riley, spratt & winch (1990), northern territory, australia 7. sebekia jubini (vaney & sambon, 1910) sambon, 1922, species inquirenda porocephalus jubini vaney & sambon, 1910 crocodylus siamensis sambon (1922), locality unknown, southeast asia 8. sebekia novaeguineae riley, spratt & winch, 1990 crocodylus novaeguineae riley, spratt & winch (1990), papua new guinea south & north american region 9. sebekia acuminata travassos, 1924, species inquirenda unknown crocodilian travassos (1924), brazil 10. sebekia divestei giglioli, 1922 crocodylus acutus sambon (1922), locality unknown, neotropical region 11. sebekia microhamus self & rego, 1985 caiman crocodilus self & rego (1985), brazil 12. sebekia mississippiensis overstreet, self & vliet, 1985 pentastoma oxycephalum diesing, 1836 (partim); pentastomum gracile (syn. leiperia gracilis) leidy, 1856 alligator mississippiensis deakins (1971), usa hazen, aho, murphy, esch & schmidt (1978), usa overstreet, self & vliet (1985), usa 13. sebekia oxycephala (diesing, 1836) sambon, 1922 pentastoma proboscideum rudolphi, 1819 (partim); pentastoma oxycephalum diesing, 1836 (partim); pentastoma gracile diesing, 1836 (partim); pentastomum oxycephalum diesing, 1850 (partim); pentastomum gracile diesing, 1850 (par tim); pentastomum heterodontis leuckart, 1860; pentastomum oxycephalum chatin, 1882; porocephalus oxycephalus stiles, 1893; pentastoma proboscideum crocodili scleropis rudolphi (shipley in sambon 1922); reighardia oxycephala vaney & sambon, 1910; porocephalus crocodili wheeler, 1913 (partim); sebekia oxycephala sambon, 1922 (partim); bdukus ichthyius holl, 1929; leiperia heterodontis heymons & vitzthum, 1935; sebekia crocodili heymons & vitzthum, 1935 alligator mississippiensis sambon (1922), locality unknown caiman crocodilus sambon (1922), locality unknown winch & riley (1986b), trinidad, south america caiman latirostris heymons (1941), locality unknown crocodylus acutus sambon (1922), locality unknown 14. sebekia samboni travassos, 1924, species inquirenda unknown crocodilian travassos (1924), brazil 15. sebekia trinitatis riley, spratt & winch, 1990 32 check-list of pentastomid parasites of crocodilians and freshwater chelonians caiman crocodilus riley, spratt & winch (1990), trinidad, south america genus selfia riley, 1994 generic diagnosis: size small, cephalothorax minute in comparison with diameter of the abdomen; 78–82 well defined annuli; abdomen strongly curled ventrally; caudal extremity of female abruptly tapered to blunt point; hooks very small, with tiny blade only slightly offset from transversely creased and folded shank; rear of anterior hooks enveloped by soft, spinous cowl which forms an extension of the fulcrum; buccal cadre somewhat variable in shape, being oval to more u-shaped, but lacking parallel sides; copulatory spicule of male like that of alofia (riley 1994). australasian region 1. selfia porosus riley, 1994 crocodylus porosus riley (1994), northern territory, australia genus diesingia sambon, 1922 generic diagnosis: hooks smooth, flat-topped, with sharply curved blades; fulcra with anterior cowl-like extension, extension smooth in posterior and spiny in anterior fulcra; oral cadre open anteriorly with an oesphageal peg similar to that in alofia; copulatory spicule with cowry shell-shaped base, the short, ventral extension is transformed into a structure resembling the collembolan fulcrum, and is connected to the base by a joint (junker, riley & boomker 2003). south & north american region 1. diesingia megastoma (diesing, 1836) sambon, 1922 pentastoma megastomum diesing, 1836; pen tastomum megastomum leuckart, 1860; poro cephalus megastomus shipley, 1898; sebekia mega stoma travassos, 1923; sebekia crocodili hey mons & vitzthum, 1935; diesingia megastoma heymons; 1941; butantanella megastoma da fonseca & ruiz, 1956; sebekia megastoma self & rego, 1985 phrynops geoffroanus diesing (1836), brazil hydromedusa tectifera da fonseca & ruiz (1956), brazil genus pelonia junker & boomker, 2002 generic diagnosis: hooks smooth, dorsally convex, with sharply bent blades, fulcra without extensions; oral cadre more or less u-shaped, closed anteriorly by delicate chitinous fibres; copulatory spicules almost identical to those of sebekia wedli, with cowry shell-shaped base and the short anterior extension ending in a smooth collar, the long spatulate extension carries small chitinous teeth (junker & boomker 2002). africa 1. pelonia africana junker & boomker, 2002 pelomedusa subrufa junker & boomker (2002), south africa pelusios sinuatus junker & boomker (2002), south africa family subtriquetridae fain, 1961 genus subtriquetra sambon, 1922 generic diagnosis: body elliptical, ventrally flattened and dorsally dome shaped with flattened margins; hooks simple, slender and sharply pointed, disposed in a curved line; oral cadre rounded (fain 1961; winch & riley 1986a; junker et al. 1998). africa 1. subtriquetra rileyi junker, boomker & booyse, 1998 infective larvae: tilapia rendalli swierstrae junker, boomker & booyse (1998), south africa oreochromis mossambicus junker, boomker & booyse (1998), south africa australasian region 2. subtriquetra megacephala (baird, 1853) sambon, 1922 pentastoma megacephalum baird, 1853; porocephalus megacephalus shipley, 1898 crocodylus palustris sambon (1922), sunderbunds, india crocodylus palustris, crocodylus porosus or 33 k. junker & j.boomker gavialis gangeticus? (“sangor crocodile”) sambon (1922), bengal, india 3. subtriquetra shipleyi hett, 1924 crocodylus palustris crocodylus porosus or gavialis gangeticus ? (“indian crocodile”) hett (1924), india south & north american region 4. subtriquetra subtriquetra (diesing, 1836) pentastoma proboscideum bresmer, 1824 (partim); pentastoma subtriquetrum diesing, 1836; pen tastomum subtriquetrum diesing, 1850; penta stomum pusillum diesing, 1856; linguatula subtriquetra raillet, 1883; linguatula pusilla ship ley, 1898 caiman crocodilus sambon (1922), south america winch & riley (1986a), trinidad, south america melanosuchus niger sambon (1922), south america host/parasite check-list of the pentastomida crocodylia family alligatoridae (cuvier, 1807) (alligators and caimans) genus alligator cuvier, 1807 1. alligator mississippiensis (daudin, 1801) daudin, 1802 (american alligator) crocodilus mississipiensis daudin, 1801 leiperia gracilis, species inquirenda, larval forms only sebekia mississippiensis sebekia oxycephala genus caiman spix, 1825 1. caiman crocodilus (linnaeus, 1758) (common or spectacled caiman) lacerta crocodilus linnaeus, 1758; caiman sclerops schneider, 1801 (fide medem 1981); perosuchus fuscus cope, 1868; alligator (jacare) chi apasius bocourt, 1876 alofia platycephala leiperia gracilis, species inquirenda, larval forms sebekia microhamus sebekia oxycephala sebekia trinitatis subtriquetra subtriquetra 2. caiman latirostris (daudin, 1801) (broad-snouted caiman) crocodilus latirostris daudin, 1801; caiman fissipes spix, 1825; champsa fissipes wagler, 1828 (fide hoogmoed & gruber, 1983); alligator cynocephalus duméril & bibron, 1836; jacare latirostris gray, 1862; alligator latirostris boulanger, 1886; jacaretinga latirostris vaillant, 1898 alofia platycephala sebekia oxycephala genus melanosuchus gray, 1862 1. melanosuchus niger (spix, 1825) (black caiman) caiman niger spix, 1825 subtriquetra subtriquetra family crocodylidae (cuvier, 1807) (crocodiles) subfamily crocodylinae (cuvier, 1807) genus crocodylus laurenti, 1768 1. crocodylus acutus (cuvier, 1807) (american crocodile) crocodilus acutus cuvier, 1807 leiperia gracilis, species inquirenda, larval forms only sebekia divestei sebekia oxycephala 2. crocodylus cataphractus cuvier, 1825 (slendersnouted crocodile) crocodilus cataphractus falconer, 1846 agema silvaepalustris alofia parva leiperia cincinnalis, infective larva sebekia okavangoensis 34 check-list of pentastomid parasites of crocodilians and freshwater chelonians 3. crocodylus johnsoni krefft, 1873 (australian freshwater crocodile) tomistoma kreffti gray in krefft, 1873 (nomen nudum); crocodilus (philas) johnstoni gray, 1874; crocodylus johnstoni cogger, 2000 leiperia australiensis sebekia johnstoni sebekia multiannulata 4. crocodylus niloticus laurenti, 1768 (nile crocodile) crocodilus vulgaris cuvier, 1807; crocodilus multi scutatus rüppell in cretzschmar, 1826; croco di lus marginatus geoffroy, 1827; crocodilus mada gascariensis grandidier, 1872; crocodilus vul garis var. madagascariensis boettger, 1877 alofia nilotici alofia simpsoni leiperia cincinnalis sebekia cesarisi sebekia okavangoensis sebekia wedli 5. crocodylus novaeguineae schmidt, 1928 (new guinea crocodile) sebekia novaeguineae 6. crocodylus palustris lesson, 1831 (mugger, marsh crocodile) subtriquetra megacephala subtriquetra shipleyi (“indian crocodile”) 7. crocodylus porosus schneider, 1801 (indopacific or saltwater crocodile) crocodylus natans meyer, 1795; crocodilus porosus schneider, 1801; crocodilus oopholis schneider, 1801; crocodilus biporcatus cuvier, 1807; crocodilus biporcatus raninus müller & schlegel, 1844; oopholis pondicherianus gray, 1862; crocodylus porosus australis deraniyagala, 1953; crocodylus porosus minikanna deran iya gala, 1953 alofia ginae (possibly crocodylus mindorensis, but distributionally unlikely) alofia merki leiperia australiensis sebekia johnstoni sebekia multiannulata sebekia purdieae selfia porosus subtriquetra shipleyi (“indian crocodile”) 8. crocodylus siamensis schneider, 1801 (siamese crocodile) crocodilus galeatus cuvier, 1807 sebekia jubini, species inquirenda genus osteolaemus cope, 1861 1. osteolaemus tetraspis cope, 1861 (dwarf crocodile) crocodilus frontatus murray, 1862; halcrosia afzelii lilljeborg, 1867; halcrosia nigra gray, 1867; halcrosia nigra gray, 1870; osteoblepharon osborni schmidt, 1919; osteolaemus tetraspis tetraspis wermuth & mertens, 1961 agema silvaepalustris alofia parva sebekia okavangoensis family gavialidae adams, 1854 (gharials) genus gavialis oppel, 1811 1. gavialis gangeticus (gmelin, 1789) (gharial) lacerta gangetica gmelin, 1789 subtriquetra megacephala (“indian crocodile”) subtriquetra shipleyi (“indian crocodile”) chelonia suborder pleurodira (side-necked turtles) family pelomedusidae cope, 1868 genus pelomedusa wagler, 1830 1. pelomedusa subrufa (lacépède, 1788) (cape ter ra pin) testudo subrufa lacépède, 1788; testudo galeata schoepff, 1792; testudo badia donndorf, 1798; testudo rubicunda suckow, 1798; emys olivacea schweigger, 1812 (non emys olivacea gray, 1855); pentonyx capensis duméril & bibron, 1835; pentonyx gehafie rüppell, 1835; pentonix americana cornalia, 1849; pelomedusa mozambica peters (nomen nudum) in gray 1855 (?); pelomedusa mossambicensis peters (nomen nudum) in lichtenstein 1856; pelomedusa 35 k. junker & j.boomker nigra gray, 1863; pelomedusa gasconi rochebrune, 1884; pelomedusa galeata boulanger, 1889; pelomedusa galeata var. disjuncta vaillant & grandidier, 1910; pelomedusa galeata orangensis hewitt, 1935; pelomedusa galeata devilliersi hewitt, 1935; pelomedusa galeata dama ren sis hewitt, 1935; pelomedusa subrufa wett steini mertens, 1937; testudo emys arabica n.-ehrenberg in stresemann 1954 pelonia africana genus pelusios wagler, 1830 1. pelusios sinuatus (smith, 1838) (serrated hinged terrapin, african serrated mud turtle) sternotherus sinuatus smith, 1838; sternotherus dentatus peters, 1848 (nomen nudum); sternothaerus sinuatus boulanger, 1889; sternothaerus bottegi boulanger, 1895; pelusios sinuatus zuluensis hewitt, 1927; pelusios sinuatus leptus hewitt, 1927 pelonia africana family chelidae gray, 1825 (snake-necked turtles) genus hydromedusa wagler, 1830 1. hydromedusa tectifera cope, 1869 [1870] (south american snake-necked turtle, uruguay snakenecked turtle) hydromedusa platanensis gray, 1873; hydro med usa wagleri günther, 1884 diesingia megastoma genus phrynops wagler, 1830 1. phrynops geoffroanus (schweigger, 1812) gorzula & señaris, 1999 (geoffroy’s side-necked turtle) emys geoffroana schweigger, 1812; emys geoffreana schweigger, 1812 (fide boulanger, 1886); emys depressa merrem, 1820 (non emys depressa spix, 1824); emys viridis spix, 1824 (?); emys geoffroyana gray, 1831; platemys geoffreana duméril & bibron, 1835; platemys neu wiedii duméril & bibron, 1835; platemys waglerii duméril & bibron, 1835; platemys tuberosa peters, 1870; platemys geoffroyana boulanger, 1886; hydraspis geoffroyana boulanger, 1889; hydraspis wagleri boulanger, 1889; hydraspis tuberosa boulanger, 1889; hydraspis boulangeri bohls, 1895; phrynops geoffroana mertens et al., 1934; phrynops geoffroana geoffroana müller, 1939; phrynops tuberosa mertens et al., 1934; phrynops geoffroana tuberosa müller, 1939 diesingia megastoma references da fonseca, f. & ruiz, j.m. 1956. was ist eigentlich pentastoma megastomum diesing, 1836? (porocephalida, porocephalidae). senckenbergiana biologica, 37:469–485. deakins, d.e. 1971. pentastomes from blackbeard island, georgia, with notes on north american pentastomes. journal of parasitology, 57:1197. devos, r. 1939. deux porocephales des crocodiles de luapula. annales de la société belge de médecine tropicale, 19:193– 195. diesing, k.m. 1836. versuch der monographie der gattung pentastoma. annalen des wiener museums der natur geschich te, 1:1–32. dukes, g.h., shealy, r.m. & rogers, w.a. 1971. sebekia oxycephala (pentastomida) in largemouth bass from lake st. john, concordia parish, louisiana. journal of para sitology, 57:1028. fain, a. 1961. pentastomides de l’ afrique centrale. annales du musée royale de l’ afrique centrale, série 8, 92:1–115. getz, w. 2002. reptilia. http://srs.embl-heidelberg.de:8000/ hazen, t.c., aho, j.m., murphy, t.m., esch, g.w. & schmidt, g.d. 1978. the parasite fauna of the american alligator (alligator mississippiensis) in south carolina. journal of wildlife diseases, 14:435–439. hett, m.l. 1924. on the family linguatulidae. proceedings of the zoological society of london, 1924: 107–159. heymons, r. 1935. pentastomida, in bronns klassen und ordnungen des tierreichs, vol. 5. leipzig: akademische verlagsgesellschaft. heymons, r. 1941. beiträge zur systematik der pentastomiden vi—die arten der gattung alofia im vergleich mit sebekia. zeitschrift für parasitenkunde, 12:419–432. hirst, s. 1922. on a new linguatulid from the adriatic. annals and magazine of natural history, 10:289. junker, k. & boomker, j. 2002. description of pelonia africana n. g., n. sp. (pentastomida: sebekidae) from the lungs of pelomedusa subrufa and pelusios sinuatus (chelonia) in south africa. onderstepoort journal of veterinary research, 69:53–59. junker, k., boomker, j. & bolton, l. 1999. pentastomid infections in the nile crocodile (crocodylus niloticus) in the kruger national park, south africa, with a description of the males of alofia simpsoni. onderstepoort journal of veterinary research, 66:65–71. junker, k., boomker, j. & booyse, d.g. 1998. pentastomid infections in cichlid fishes in the kruger national park and the description of the infective larva of subtriquetra rileyi n. sp.. onderstepoort journal of veterinary research, 65:159–167. junker, k., boomker, j., swanepoel, d. & tara schewski, h. 2000. leiperia cincinnalis sambon, 1922 from nile crocodiles crocodylus niloticus in the kruger national park, south africa, with a description of the male. systematic parasitology, 47:29–41. 36 check-list of pentastomid parasites of crocodilians and freshwater chelonians junker, k., riley, j. & boomker, j. 2003. redescription of diesingia megastoma (diesing, 1836) sambon, 1922, a penta stomid parasite from the south american terrapin hydromedusa tectifera. systematic parasitology, 56:211–218. lohrmann, e. 1889. untersuchungen über den anatomischen bau der pentastomen. archiv für naturgeschichte, 1:303– 337. overstreet, r.m., self, j.t. & vliet, k.a. 1985. the pentastomid sebekia mississippiensis sp. n. in the american alligator and other hosts. proceedings of the helminthological society of washington, 52:266–277. riley, j. 1994. a revision of the genus alofia giglioli, 1922 and a description of a new monotypic genus, selfia: two genera of pentastomid parasites (porocephalida: sebekidae) inhabiting the bronchioles of the marine crocodile crocodylus porosus and other crocodilians. systematic parasitology, 29: 23–41. riley, j., hill, g.f. & huchzermeyer, f.w. 1997. a description of agema, a new monotypic pentastomid genus from the lungs of the african dwarf and slender-snouted croco diles. systematic parasitology, 37:207–217. riley, j. & huchzermeyer, f.w. 1995a. description of four species of pentastomid parasites belonging to the genera alofia giglioli, 1922 and sebekia sambon, 1922, from a single nile crocodile cocodylus niloticus from botswana. systematic parasitology, 31:221–238. riley, j. & huchzermeyer, f.w. 1995b. pentastomid parasites of the family sebekidae fain, 1961 in west african dwarf crocodiles osteolaemus tetraspis cope, 1851 from the congo, with a description of alofia parva n. sp. onderstepoort journal of veterinary research, 62:151–162. riley, j. & huchzermeyer, f.w. 1996. a reassessment of the pentastomid genus leiperia sambon, 1922 with a description of a new species from both the indopacific crocodile crocodylus porosus and johnston’s crocodile c. johnstoni in australia. systematic parasitology, 34:53–66. riley, j. & huchzermeyer, f.w. 2000. diet and lung parasites of swamp forest dwarf crocodiles (osteolaemus tetraspis osborni) in the northern congo republic. copeia, 2: 582–586. riley, j., spratt, d.m. & winch, j.m. 1990. a revision of the genus sebekia sambon, 1922 (pentastomida) from crocodilians with descriptions of five new species. systematic parasitology, 16:1–25. rodhain, j. & vuylsteke, c. 1932. contribution à l’ étude des porocéphales des crocodiles africains. révue de zoologie et de botanique africaines, 23:1–11. ross, c.a (ed.). 1989. crocodiles and alligators, london: meere hurst press. sambon, l.w. 1922. a synopsis of the family linguatulidae. journal of tropical medicine and hygiene, 25:188–206; 391–428. self, j.t. & rego, a.a. 1985. reassessments and revisions of certain genera and species of the family sebekidae (pen tastomida) including a description of sebekia microhamus n. sp.. systematic parasitology, 7:33–41. travassos, l. 1924. sebekia du poumon des crocodiles d’amerique. comptes rendus hebdomaires des séances et mémoires de la société de biologie, 90:239–240. vaney, c. & sambon, l.w. 1910. preliminary notes on three new species of tongue worms (linguatulidae) in the collection of the “museum d’histoire naturelle”, paris. transactions of the royal society of tropical medicine and hygiene, 3: 129–134. winch, j.m. & riley, j. 1986a. studies on the behaviour, and development in fish, of subtriquetra subtriquetra: a uniquely free-living pentastomid larva from a crocodilian. parasitology, 93:81–98. winch, j.m. & riley, j. 1986b. morphogenesis of larval sebekia oxycephala (pentastomida) from a south american crocodilian (caiman sclerops) in experimentally infected fish. zeitschrift für parasitenkunde, 72:251–264. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 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/namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [1200 1200] /pagesize [595.276 841.890] >> setpagedevice junker_115-128.indd introduction the genus tetrameres creplin, 1846 are cosmopolitan parasites, infecting a variety of aquatic and terrestrial avian hosts. females are usually located in the proventricular glands, and the males are found free in the lumen of the proventriculus (ander son 1992). several tetrameres species have been recorded from the african continent, of which tetrameres fissispina (diesing, 1861) travassos, 1914 that parasitises ducks, pigeons and domestic chickens and tetrameres americana cram, 1927 that parasitises domestic chickens, turkeys and quails are the most commonly reported ones (permin, magwisha, kassuku, nansen, bisgaard, frandsen & gibbons 1997; poulsen, permin, hindsbo, yelifari, nansen & bloch 2000). tetrameres coccinea (seurat, 1914) travassos, 1914 from the greater flamingo, phoenicopterus ruber, linnaeus, 1758, cattle egret, bubulcus ibis (linnaeus, 1758) and eurasian spoonbill, platalea leucorodia linnaeus, 1758, as well as tetrameres lhuillieri (seurat, 1918) from the rock partridge, alectoris graeca (meisner, 1804) and the stock pigeon, columba oenas linnaeus, 1758 were recorded from algeria (yamaguti 1961). tetrameres nouveli (seurat, 1914) travassos, 1914 was present in the blackwinged stilt, himantopus himantopus (linnaeus, 1758) in algeria (yamaguti 1961), and in nigeria tetra meres plectropteri thwaite 1926 was found in the spur-winged goose, plectropterus gambensis (lin naeus, 1766) (yamaguti 1961). 115 onderstepoort journal of veterinary research, 74:115–128 (2007) tetrameres numida n. sp. (nematoda: tetrameridae) from helmeted guineafowls, numida meleagris (linnaeus, 1758), in south africa k. junker and j. boomker* department of veterinary tropical diseases, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract junker, k. & boomker, j. 2007. tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls, numida meleagris (linnaeus, 1758), in south africa. onderstepoort journal of veterinary research, 74:115–128 tetrameres numida n. sp. from the proventriculus of helmeted guineafowls, numida meleagris, in south africa is described from eight male and four female specimens. the new species shares some characteristics with other tetrameres species, but can be differentiated by a unique combination of characters. it bears two rows of cuticular spines extending over the whole length of the body and possesses two spicules. the left spicule measures 1 699–2 304 μm and the right one 106–170 μm. caudal spines are arranged in three ventral and three lateral pairs and the tail is 257–297 μm long. diagnostic criteria of some of the previously described species of the genus tetrameres from africa and other parts of the world have been compiled from the literature and are included here. keywords: helmeted guineafowls, nematodes, tetrameres numida * author to whom correspondence is to be directed. e-mail: joop.boomker@up.ac.za accepted for publication date 4 april 2007—editor 116 tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls in south africa both tetrameres paradisea ortlepp, 1932 and tetrameres prozeskyi (ortlepp, 1964) were described from south african hosts. tetrameres paradisea was recovered from a stanley’s crane, anthropoides paradisea (lichtenstein, 1793) (ortlepp 1932), and t. prozeskyi occurred in red-billed hornbills, tockus erythrorhynchus (temminck, 1823) and southern yellow-billed hornbills, tockus leucomelas (lichtenstein, 1842) (= tockus flavirostris leucomelas), respectively (ortlepp 1964). previous records of tetrameres spp. from guineafowls pertain mostly to studies in north and west africa, tetrameres fissispina being recorded from helmeted guineafowls in these countries (fabiyi 1972; vercruysse, harris, bray, nagalo, pangui & gibson 1985). appleton (1983) found tetrameres sp. females in crested guineafowls, guttera edouardi (hartlaub, 1867) (= guttera pucherani), in natal (now kwazulu-natal province), south africa, but because males were not present, the species could not be determined. we here describe a new species of the genus tetrameres from helmeted guineafowls in south africa for which we propose the name tetrameres numida n. sp. with regards to the classification of the genus tetrameres we have followed that of chabaud (1975), placing the genus into the subfamily tetramerinae railliet, 1915 within the family tetrameridae travassos, 1914, which is one of four families comprising the superfamily habronematoidea. at the time the genus had been divided into the subgenera tetrameres s. str., gynaecophila gubanov, 1950, petrow i meres chertkova, 1953 and gubernacules rasheed, 1960. chabaud (1975), arguing that this division could lead to errors and bore little phylogenetic significance, chose not to retain these, but divided the genus tetrameres into the two subgenera tetrameres (tetrameres) creplin, 1846 and tetrameres (micro tetrameres) travassos, 1915. in the light of new findings, especially concerning the morphology of adults and larval stages of these two subspecies, anderson (1992), while retaining their position within the subfamiliy, recognized tetrameres creplin, 1846 and microtetrameres travassos, 1915 as two distinct genera, a generic classification that had been suggested by skrjabin (1969). we adopt his view in the present paper. material and methods fifteen helmeted guineafowls, numida meleagris (linnaeus, 1758), were collected on a farm 60 km to the west of musina (messina), limpopo province, south africa (22°22.139’ s, 29°30.399’ e) between july 2005 and november 2006. ten of these were mature guineafowls and five were young birds, about 6–10 months old (siegfried 1966). eight male tetrameres sp. were recovered from the proventriculus, where they occurred free in the lumen and four females were dissected from the proventricular glands. two guineafowls harboured a single male each, two hosts harboured two and three males respectively, and from a single host one male and four females were recovered. all hosts were mature guineafowls. the worms were fixed in 70 % ethanol and cleared in lactophenol for identification. all measurements, unless otherwise indicated, are in micrometres. description tetrameres numida n. sp. (fig. 1–3; tables 1, 2) with characters of the genus. sexual dimorphism marked. male: body elongated, tapering towards both ends, posteriorly to a tail with a short, pointed tip. cuticle with fine transverse striation and longitudinal cuticular grooves. total length 4.3–4.5 mm; maximum width 0.16–0.17 mm. inconspicuous lateral alae extending down the length of the body; parallell to these run two lateral rows of cuticular spines (fig. 2f). one row of spines is situated dorsally, the second row ventrally to the lateral alae (fig. 1b). a pair of deirids with apical spines is situated at approximately the height of the second pair of cuticular spines at a distance of 139–204 from the apex (fig. 1b). cuticular spines start at 93–154 from the apex, numbering approximately 42–47 per row. the nerve ring and excretory pore are 215–284 and 236–331 from the anterior extremity, respectively. the excretory pore is slightly posterior to the nerve ring. the triangular mouth is bounded by a pair of trilobed pseudolabia. the inner surface of each lobe carries two to four tooth-like processes. the precise number is difficult to assess in our specimens (fig. 1a, 2a). depth of buccal capsule 16–28, inner diameter 8– 11. oesophagus divided into muscular and glandular portion, 232–401 and 734–984, respectively. total length of oesophagus 1 023–1 318. spicules unequal and dissimilar. right spicule tubular, with slight bend and spatulate tip, 106–170 long (fig. 1c, 2d). left spicule long and thin, trough-shaped, with spatulate tip. shaft slightly twisted at 100–120 from proximal end. total length 1 699–2 304 (fig. 1d–f, 2c, 2e). a gubernaculum is absent. tail 117 k. junker & j. boomker fig. 1 tetrameres numida n. sp. male. a. apical view of the trilobed pseudolabia surrounding the triangular mouth. note the toothlike processes (scale bar = 10 μm). b. ventro-lateral view of the anterior end (scale bar = 100 μm). c. ventral aspect of the posterior end (scale bar = 100 μm). d. lateral view of the proximal end of the left spicule showing the slight twist (scale bar = 100 μm). e. ventral view of the proximal end of the left spicule (scale bar = 100 μm). f. distal end of the left spicule, ventral view (scale bar = 100 μm). female. g. complete female (scale bar = 1 mm). h. anterior extremity (scale bar = 100 μm) g h f e d c ba 118 tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls in south africa length 257–297. six pairs of caudal spines, three pairs each in two ventral and two lateral rows, respectively. one or two additional ventral spines may be present (fig. 1c). female: specimens in situ red. a minute head and tail of regular nematode shape, but often twisted or bent, emerge at opposite sides from the central part of the body which is distinctly globular and slightly bent along the axis (fig. 1g–h, 3a, 3c). the cuticle bears marked transverse striation and four longitudinal cuticular grooves. the latter divide the body into four segments of which the two segments following the outer curve are slightly longer (fig. 1g). much of the internal detail is obscured by the eggfig. 2 tetrameres numida n. sp. male. a. head, apical view. b. anterior extremity, ventral view. c. left spicule, anterior end. d. posterior extremity with right spicule and distal tip of left spicule. e. tip of left spicule. f. body spines (see arrow) fe dc ba 119 k. junker & j. boomker filled uterus coils surrounding a large sacular intestine. body length 4.2–5.3 mm, maximum width 2.6– 3.5 mm. the following measurements were derived from a single specimen: the deirids are at 179 and 190 and the nerve ring at 215 from the apex, respectively. the excretory pore could not be located. depth of buccal capsule 23, inner diameter 7. muscular part of oesophagus 333, the distal part of the glandular oesophagus obscured by the uterus. eggs are elongate with near parallel sides, polar filaments were not seen (fig. 3d). eggs containing fully developed larvae are 56–59 long and 31–34 wide. anus and vulva appeared to be confined in body folds. emerging from the last body fold is a tail approximately 336 long with a simple tip. specific diagnosis: tetrameres numida is differentiated from other members of the genus, by the possession of two rows of somatic spines and the arrangement of its caudal spines in two ventral and two lateral rows with usually three pairs of spines each, although deviation might occur. a short right and a long left spicule are present, ranging from 106–131 and from 1 699–2 304 in length, respectively. host: numida meleagris (linnaeus, 1758), hel meted guineafowl. site: males occur free in the lumen of the proventriculus, females are situated in the proventricular glands. locality: musina (messina), limpopo province, south africa (22°22.139’ s, 29°30.399’ e). etymology: the specific epithet numida refers to the host. types deposited in the national collection of animal helminths at the onderstepoort veterinary institute, pretoria, south africa. holotype male: t.2191, alotype female: t.2192, paratype males: t.2193– t.2195. fig. 3 tetrameres numida n. sp. female. a. three whole specimens, approximately 4 mm in length. note the globular shape. b. anterior extremity. c. posterior end. note the digested blood showing as dark smudge. d. egg containing fully developed larva dc ba 120 tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls in south africa t a b l e 1 t h e m o rp h o lo g ic a l ch a ra ct e ri st ic s o f t e tr a m e re s n u m id a s p . n . m a le s fr o m h e lm e te d g u in e a fo w ls , co m p a re d t o t e tr a m e re s p a ra d is e a o rt le p p , 1 9 3 2 a n d t o t e tr a m e re s p ro ze sk yi ( o rt le p p , 1 9 6 4 ), a ll d e sc ri b e d f ro m s o u th a fr ic a n h o st s. a ll m e a su re m e n ts in m ic ro m e tr e s u n le ss o th e rw is e in d ic a te d m o rp h o lo g ic a l c ri te ri a g f m 1 /n 4 t .2 1 9 1 t .2 1 9 3 t .2 1 9 4 t .2 1 9 5 g f m 1 1 /1 g f m 1 2 /1 t e tr a m e re s p a ra d is e a t e tr a m e re s p ro ze s k y i s o u rc e t h is p a p e r t h is p a p e r t h is p a p e r t h is p a p e r t h is p a p e r t h is p a p e r t h is p a p e r o rt le p p ( 1 9 3 2 ) o rt le p p ( 1 9 6 4 ) b o d y le n g th ( m m ) 4 .3 4 .4 4 .4 4 .3 4 .3 n 4 .5 5 .8 1 .3 – 2 .4 b o d y w id th m a xi m u m n n 1 6 0 1 6 0 1 6 4 1 7 0 1 6 2 1 4 0 6 0 -7 0 d is ta n ce a p e x to f ir st s o m a tic s p in e n 1 2 6 & 1 1 7 9 6 & 1 0 0 1 0 2 & 9 3 1 0 5 & 9 4 1 3 1 & 1 5 4 9 6 & 1 1 3 n n d is ta n ce a p e x to d e ir id s n 1 7 4 & 1 8 0 1 3 9 & 1 4 9 1 7 9 & 1 7 2 1 6 5 & 1 7 7 1 7 4 & 1 8 1 1 7 5 & 2 0 4 8 5 ~ 5 0 – 6 0 d is ta n ce a p e x to n e rv e r in g n 2 5 6 2 1 5 2 3 4 2 4 4 2 8 4 2 6 4 n ~ 1 5 0 – 1 6 0 d is ta n ce a p e x to e xc re to ry p o re 2 6 8 3 0 7 2 3 6 2 8 7 2 9 6 3 3 1 3 1 6 n n d e p th o f b u cc a l c a p su le 2 2 2 5 2 8 2 3 2 1 2 2 1 6 2 5 5 .0 – 7 .0 w id th o f b u cc a l c a p su le ( in n e r) n 1 0 1 0 8 8 1 1 8 1 2 1 1 .0 – 1 3 .0 m u sc u la r o e so p h a g u s n 3 5 1 3 0 4 2 3 2 2 6 0 4 0 1 4 0 0 3 1 0 1 6 0 – 2 1 0 g la n d u la r o e so p h a g u s n 7 3 4 7 6 9 9 8 4 7 8 1 8 1 2 9 1 8 9 0 0 3 0 0 – 4 0 0 o e so p h a g u s to ta l l e n g th n 1 0 8 5 1 0 7 3 1 2 1 6 1 0 2 3 1 2 1 3 1 3 1 8 1 2 1 0 n l e n g th o f ta il 2 8 4 2 9 7 2 8 7 2 5 7 2 9 6 n 2 9 0 1 1 5 1 4 0 – 1 6 0 l e n g th o f ri g h t sp ic u le 1 3 1 1 3 0 1 0 6 1 1 0 1 3 1 1 2 0 1 7 0 a b se n t u su a lly a b se n tb l e n g th o f le ft s p ic u le 1 9 8 8 2 1 0 3 2 3 0 4 2 1 6 9 1 6 9 9 n 2 2 0 4 6 9 0 ; 5 0 4 – 6 2 6 a 2 3 0 – 2 6 0 n d a ta n o t a va ila b le a r a n g e g iv e n b y m o llh a g e n ( 1 9 7 6 ) in c re m o n te e t a l. (2 0 0 1 ) b a r ig h t sp ic u le w a s p re se n t in t h re e o f m o re t h a n 3 0 m a le s 121 k. junker & j. boomker t a b l e 2 a c o m p a ri so n o f m o rp h o lo g ic a l c h a ra ct e ri st ic s o f so m e s p e ci e s o f th e g e n u s t e tr a m e re s c re p lin , 1 8 4 6 s p e c ie s b o d y le n g th o f m a le ( m m ) n u m b e r o f ro w s o f s o m a ti c s p in e s l e n g th o f ro w s o f s o m a ti c s p in e s n u m b e r o f s p ic u le s s p ic u le l e n g th ( m m ) a rr a n g e m e n t o f c a u d a l s p in e s o r p a p il la e p o la r fi la m e n ts o n e g g s s o u rc e t e tr a m e re s a m e ri ca n a c ra m , 1 9 2 7 5 – 5 .5 4 n 2 l e ft : 0 .2 9 – 0 .3 1 ; ri g h t: 0 .1 – 0 .1 3 5 v e n tr a l p a ir s, n o la te ra l p a ir s n s ch m id t (1 9 6 2 ); g ib b o n s e t a l. (1 9 9 6 ) t e tr a m e re s a ra lie n si s e fim o v & r ijo w a , 1 9 3 9 2 .5 5 4 w h o le b o d y le n g th 2 l o n g : 0 .9 1 3 ; s h o rt : 0 .2 2 2 v e n tr a l p a ir s a n d 2 su b la te ra l r o w s w ith 6 a n d 7 s p in e s, re sp e ci tv e ly . t w o la te ra l t a il p a p ill a e a ls o p re se n t n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s a u st ra lis j o h n st o n & m a w so n , 1 9 4 1 7 .8 – 9 .0 2 w h o le b o d y le n g th 2 l o n g : 5 .8 – 6 .3 ; sh o rt : 0 .8 5 t o 6 s m a ll sp in e s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s b iz iu ra e j o h n st o n & m a w so n , 1 9 4 1 4 .2 – 4 .4 4 w h o le b o d y le n g th 2 l o n g : 0 .2 5 – 0 .2 6 ; sh o rt : 0 .0 7 n n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s ca lid ri s m a w so n , 1 9 6 8 2 .2 – 2 .5 4 /2 4 r o w s a n te ri o rl y, f ro m g la n d u la r o e so p h a g u s o n w a rd s o n ly 2 2 l e ft : 0 .7 5 – 1 .0 ; ri g h t: 0 .0 8 – 0 .0 9 5 v e n tr a l p a ir s, 2 la te ra l p a ir s o n ly m a le s kn o w n m a w so n ( 1 9 6 8 ) t e tr a m e re s ca rd in a lis q u e n tin & b a rr e , 1 9 7 6 4 .2 – 4 .9 5 2 w h o le b o d y le n g th 2 l e ft : 0 .3 6 5 – 0 .4 0 0 ; ri g h t: 0 .0 6 5 – 0 .0 8 5 a 4 – 5 p a ir s o f p o st cl o a ca l s p in e s p re se n t q u e n tin & b a rr e (1 9 7 6 ) t e tr a m e re s cl a d o rh yn ch i m a w so n , 1 9 6 8 2 .0 – 2 .9 4 w h o le b o d y le n g th 1 l e ft : 1 .0 – 1 .3 7 3 s u b ve n tr a l p a ir s, 3 su b la te ra l p a ir s p re se n t m a w so n ( 1 9 6 8 ); p e n ce e t a l. (1 9 7 5 ); c re m o n te e t a l. (2 0 0 1 ) t e tr a m e re s co lo ra d e n si s s ch m id t, 1 9 6 2 2 .0 5 4 w h o le b o d y le n g th 2 l e ft : 0 .7 7 7 ; ri g h t: 0 .0 6 7 4 v e n tr a l p a ir s, 3 la te ra l p a ir s p re se n t s ch m id t (1 9 6 2 ) t e tr a m e re s co n fu sa t ra va ss o s, 1 9 1 9 4 .0 – 5 .0 4 n 2 l o n g : 0 .2 9 1 ; sh o rt : 0 .0 6 8 3 v e n tr a l p a ir s, 3 la te ra l p a ir s s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s co rd o n ife re n s r a sh e e d , 1 9 6 0 n 4 n n l e ft s p ic u le : 0 .4 0 n n p e n ce e t a l. (1 9 7 5 ) t e tr a m e re s cr a m i s w a le s, 1 9 3 6 2 .9 – 4 4 n 2 l e ft : 0 .2 7 – 0 .3 5 ; ri g h t: 0 .1 3 6 – 0 .1 8 5 n n s ch m id t (1 9 6 2 ); g ib b o n s e t a l. (1 9 9 6 ) t e tr a m e re s cr a m i a si a tic a r yj ik o v, 1 9 6 3 3 .2 5 – 3 .6 4 w h o le b o d y le n g th 2 l o n g : 0 .2 3 8 – 0 .2 5 4 ; sh o rt : 0 .0 9 9 – 0 .1 0 6 5 v e n tr a l p a ir s, 3 la te ra l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) 122 tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls in south africa s p e c ie s b o d y le n g th o f m a le ( m m ) n u m b e r o f ro w s o f s o m a ti c s p in e s l e n g th o f ro w s o f s o m a ti c s p in e s n u m b e r o f s p ic u le s s p ic u le l e n g th ( m m ) a rr a n g e m e n t o f c a u d a l s p in e s o r p a p il la e p o la r fi la m e n ts o n e g g s s o u rc e t e tr a m e re s cy g n i r yj ik o v & k o zl o v, 1 9 6 0 n 4 n 2 l e ft : a b o u t o n e h a lf th e le n g th o f th a t o f t . tin a m ic o la 3 r o w s o f 5 c a u d a l p a p ill a e n p e n ce e t a l. (1 9 7 5 ) t e tr a m e re s d u b ia t ra va ss o s, 1 9 1 7 b 1 .3 5 – 2 .2 8 4 /2 d o rs o la te ra l r o w s re a ch o n ly t h e le ve l o f th e p o st e ri o r e n d o f th e g la n d u la r o e so p h a g u s 2 l o n g : 0 .7 1 – 0 .7 7 ; sh o rt : 0 .0 6 – 0 .0 8 4 v e n tr a l p a ir s, 3 la te ra l p a ir s p re se n t m a m a e v (1 9 5 9 ) ci te d b y s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s fe rm in i v ig u e ra s, 1 9 3 5 2 .5 n n 2 l o n g : 0 .0 7 3 ; sh o rt : 0 .0 2 3 3 p a ir s o f p o st cl o a ca l sp in e s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s fis si sp in a (d ie si n g , 1 8 6 1 ) t ra va ss o s, 1 9 1 4 3 .0 – 6 .0 n n 2 l e ft : 0 .8 2 – 1 .5 ; ri g h t: 0 .2 8 – 0 .4 9 8 p a ir s o f p o st a n a l sp in e s n g ib b o n s e t a l. (1 9 9 6 ) 3 .2 – 3 .9 4 n 2 l o n g : 0 .3 7 – 0 .4 9 ; sh o rt : 0 .1 6 5 – 0 .1 9 8 3 v e n tr a l p a ir s, 5 la te ra l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s g a le ri cu la tu s o sc h m a ri n , 1 9 5 6 3 .4 4 w h o le b o d y le n g th 2 l o n g e r: 0 .4 5 0 ; sh o rt : 0 .0 8 6 p re se n t n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s g ig a s t ra va ss o s, 1 9 1 9 7 .5 4 w h o le b o d y le n g th 2 l o n g : 0 .7 4 ; sh o rt : 0 .0 1 6 t a il p a p ill a e h a ve n o t b e e n f o u n d n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s g lo b o sa ( v o n l in st o w , 1 8 7 9 ) 3 .6 – 3 .7 5 4 w h o le b o d y le n g th , sp a r s e r in p o st e ri o r h a lf 2 /1 l o n g : 0 .3 ; s h o rt sp ic u le r u d im e n ta ry s m a ll sp in e s p o st e ri o r to c lo a ca n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s g ru si s h u m a ko vi ts h , 1 9 4 6 3 .4 5 – 4 .4 0 2 2 d is tin ct r o w s, b u t sp in e s sc a tt e re d a n te ri o r to n e rv e r in g a n d p o st e ri o r to a n u s 1 0 .6 3 8 – 0 .7 8 3 s e ve ra l i rr e g u la r ro w s o f sp in e s n s kr ja b in & s o b o le v (1 9 6 3 ); b u sh e t a l. (1 9 7 3 ); p e n ce e t a l. (1 9 7 5 ) t e tr a m e re s g u b a n o vi s h ig in , 1 9 5 7 6 .6 7 2 w h o le b o d y le n g th , st a rt in g a t tr a n si tio n fr o m m u sc u la r to g la n d u la r o e so p h a g u s 2 l o n g : 3 .9 9 6 ; sh o rt : 0 .1 3 1 4 v e n tr a l p a ir s o f co n ic a l p a p ill a e , 3 la te ra l p a ir s o f st a lk e d p a p ill a e n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s h a g e n b e ck i t ra va ss o s & v o g e ls a n g , 1 9 3 0 3 .1 – 3 .4 2 ? r o w s o f cu tic u la r sp in e s a lo n g la te ra l fie ld s (2 r o w s ill u st ra te d ) l o n g s p ic u le : t h in a n d e n d in g a s a s p u r, p ro xi m a l 0 .0 7 – 0 .0 8 tw is te d . s h o rt s p ic u le 0 .0 3 2 – 0 .0 4 4 v e n tr a l p a ir s, 2 la te ra l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s lh u ill ie ri ( s e u ra t, 1 9 1 8 ) n 4 n 1 0 .4 8 n p re se n t o rt le p p ( 1 9 6 4 ) t a b l e 2 (c o n t. ) 123 k. junker & j. boomker s p e c ie s b o d y le n g th o f m a le ( m m ) n u m b e r o f ro w s o f s o m a ti c s p in e s l e n g th o f ro w s o f s o m a ti c s p in e s n u m b e r o f s p ic u le s s p ic u le l e n g th ( m m ) a rr a n g e m e n t o f c a u d a l s p in e s o r p a p il la e p o la r fi la m e n ts o n e g g s s o u rc e t e tr a m e re s lo b ib yc is m a w so n , 1 9 6 8 1 .5 4 /2 4 r o w s a n te ri o rl y, f ro m n e rv e r in g o n w a rd s o n ly 2 1 l e ft : 0 .7 3 6 s u b ve n tr a l p a ir s o n ly m a le kn o w n m a w so n ( 1 9 6 8 ) t e tr a m e re s m e g a p h a sm id ia ta c re m o n te , d ig ia n i, b a la & n a vo n e ( 2 0 0 1 ) 1 .9 4 – 2 .0 3 4 w h o le b o d y le n g th 1 l e ft : 0 .9 6 – 1 .2 2 6 s u b ve n tr a l p a ir s, 2 la te ra l p a ir s n c re m o n te e t a l. (2 0 0 1 ) t e tr a m e re s m ic ro p e n is t ra va ss o s, 1 9 1 5 4 .0 – 5 .0 2 w h o le b o d y le n g th 2 l o n g : 0 .3 5 5 ; sh o rt : 0 .0 5 6 2 v e n tr a l p a ir s n o rt le p p ( 1 9 3 2 ); s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s m ic ro sp in o sa v ig u e ra s, 1 9 3 5 3 .0 2 w h o le b o d y le n g th 2 l o n g : 1 .1 3 5 ; sh o rt : 0 .0 6 5 5 v e n tr a l p a ir s a b se n t s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s m o h te d a i b h a le ra o a n d r a o , 1 9 4 4 4 .2 7 – 5 .8 4 /2 s u b m e d ia n s p in e s e n d p o st e ri o r to m id d le o f g la n d u la r o e so p h a g u s 2 l o n g : 0 .3 9 7 – 0 .4 3 0 ; sh o rt : 0 .1 4 2 – 0 .1 6 0 5 s u b ve n tr a l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s n o u ve li (s e u ra t, 1 9 1 4 ) 1 .0 – 2 .4 4 w h o le b o d y le n g th 1 l e ft : 3 5 0 – 5 8 0 c 3 o r 4 s u b ve n tr a l p a ir s, 2 o r 3 s u b la te ra l p a ir s p re se n t o rt le p p ( 1 9 3 2 ); m a w so n ( 1 9 6 8 ); c re m o n te e t a l. (2 0 0 1 ) 2 .1 6 4 w h o le b o d y le n g th 1 0 .4 8 0 ; se co n d s p ic u le ru d im e n ta ry ( s e u ra t 1 9 1 4 , ci te d b y s kr ja b in & s o b o le v 1 9 6 3 ) 4 v e n ra l a n d 3 l a te ra l p a ir s ill u st ra te d ; a cc o rd in g t o t e xt 2 p a p ill a e i n p o st e ri o r th ir d o f ta il p re se n t s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s n u m e n ii m a m a e v, 1 9 5 9 1 .6 4 – 2 .4 4 /2 d o rs o la te ra l r o w s re a ch o n ly t h e le ve l o f th e p o st e ri o r p a rt o f th e o e so p h a g u s 2 l o n g : 1 .0 8 – 1 .2 4 ; sh o rt : 0 .0 8 – 0 .1 0 4 v e n tr a l p a ir s, 3 la te ra l p a ir s a b se n t s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s n u m id a n . sp . 4 .3 – 4 .4 2 w h o le b o d y le n g th 2 l e ft : 1 .6 9 9 – 2 .3 0 4 ; ri g h t: 0 .1 0 6 – 0 .1 3 1 3 v e n tr a l p a ir s, 3 la te ra l p a ir s a b se n t t h is p a p e r t e tr a m e re s o xy la b ia tu s o sc h m a ri n , 1 9 5 6 5 .0 n w h o le b o d y le n g th 2 l o n g : 0 .9 4 0 ; sh o rt : 0 .1 2 5 e xt e n d p o st e ri o rl y to m id d le o f ta il, g e tt in g ve ry s m a ll n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s p a ra a ra lie n si s o sc h m a ri n , 1 9 5 6 1 .7 1 4 w h o le b o d y le n g th 1 0 .4 0 5 – 0 .4 2 0 n n s kr ja b in & s o b o le v (1 9 6 3 ); m a w so n ( 1 9 6 8 ); m o llh a g e n ( 1 9 7 6 ) in c re m o n te e t a l. (2 0 0 1 ) t a b l e 2 (c o n t. ) 124 tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls in south africa s p e c ie s b o d y le n g th o f m a le ( m m ) n u m b e r o f ro w s o f s o m a ti c s p in e s l e n g th o f ro w s o f s o m a ti c s p in e s n u m b e r o f s p ic u le s s p ic u le l e n g th ( m m ) a rr a n g e m e n t o f c a u d a l s p in e s o r p a p il la e p o la r fi la m e n ts o n e g g s s o u rc e t e tr a m e re s p a ra d is e a o rt le p p , 1 9 3 2 5 .8 2 w h o le b o d y le n g th 1 l e ft : 0 .6 9 d 3 v e n tr a l p a ir s, 3 d o rs o -e xt e rn a l p a ir s a b se n t o rt le p p ( 1 9 3 2 ) t e tr a m e re s p a ra d o xa (d ie si n g , 1 8 3 5 ) 1 2 – 1 5 2 n 2 l o n g : 3 .0 o r lo n g e r ; sh o rt : 0 .4 8 0 d ra sh e ( 1 8 8 4 ) ill u st ra te d a v e ry s m a ll p a ir o f ve n tr a l p a p ill a e a n d 3 a n d 4 la te ra l p a p ill a e r e sp e ct iv e ly n s kr ja b in & s o b o le v (1 9 6 3 ), d ra sh e ( 1 8 8 4 ) ci te d b y s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s p a tt e rs o n i c ra m , 1 9 3 3 4 .2 – 4 .6 2 w h o le b o d y le n g th 1 1 .2 – 1 .5 n n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s p a u ci sp in a s a n d g ro u n d , 1 9 2 8 n 2 f e w , o n ly in p o st e ri o r 2 /3 2 l e ft : 0 .3 2 8 – 0 .3 7 1 ; ri g h t: 0 .0 1 2 – 0 .1 5 4 e 3 c a u d a l p a p ill a e n b u sh e t a l. (1 9 7 3 ); q u e n tin & b a rr e ( 1 9 7 6 ) 3 .1 – 4 .5 1 1 r o w in m e d ia n ve n tr a l f ie ld , n o t m o re th a n 2 5 s p in e s, o n ly in p o st 2 /3 2 l o n g : 0 .3 2 8 – 0 .3 7 1 ; sh o rt : 0 .1 5 4 3 c a u d a l p a p ill a e n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s p a vl o vs ki i i yg is , 1 9 6 5 n 4 n 1 n 4 v e n tr a l p a ir s, 4 la te ra l p a ir s n p e n ce e t a l. (1 9 7 5 ) t e tr a m e re s p a vo n is t sc h e rt ko va , 1 9 5 3 4 .7 n ir re g u la r a n d d e n se a n te ri o rl y, in m id d le a n d p o st e ri o r p a rt a lm o st in vi si b le 2 l o n g : 0 .4 3 ; sh o rt : 0 .1 0 5 4 r o w s o f sp in e s, a n d 3 p a p ill a e : 1 la te ra l p a ir , 1 u n p a ir m e d ia n p a p ill a n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s p h a e n ic o p te ru s a li, 1 9 7 0 n 4 n 2 n n n p e n ce e t a l. (1 9 7 5 ) t e tr a m e re s p le ct ro p te ri t h w a ite , 1 9 2 6 n n n n l e ft : 0 .8 5 n n o rt le p p ( 1 9 6 4 ) t e tr a m e re s p ro ze sk yi (o rt le p p , 1 9 6 4 ) 1 .3 – 2 .4 4 w h o le b o d y le n g th 1 l e ft : 0 .2 3 – 0 .2 6 f 3 v e n tr a l p a ir s, 3 la te ra l p a ir sg n o rt le p p ( 1 9 6 4 ) t e tr a m e re s p u ch o vi g u sh a n sk a ja , 1 9 4 9 3 .8 6 – 4 .3 3 9 2 w h o le b o d y le n g th 1 0 .3 0 7 – 0 .3 0 9 ; se co n d sp ic u le r u d im e n ta ry : 0 .0 0 8 n n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e rs r yj ik o vi c h u a n , 1 9 6 1 4 .5 4 w h o le b o d y le n g th 2 l o n g : 0 .2 0 8 ; sh o rt : 0 .0 6 2 4 v e n tr a l p a ir s, 3 la te ra l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s sa kh a ro w i p e tr o w , 1 9 2 6 9 .4 7 4 n 2 l e ft : 0 .1 9 5 ; ri g h t: 1 .0 2 1 n n s kr ja b in & s o b o le v (1 9 6 3 ) t a b l e 2 (c o n t. ) 125 k. junker & j. boomker s p e c ie s b o d y le n g th o f m a le ( m m ) n u m b e r o f ro w s o f s o m a ti c s p in e s l e n g th o f ro w s o f s o m a ti c s p in e s n u m b e r o f s p ic u le s s p ic u le l e n g th ( m m ) a rr a n g e m e n t o f c a u d a l s p in e s o r p a p il la e p o la r fi la m e n ts o n e g g s s o u rc e t e tr a m e re s sc o lo p a ci d is m a w so n , 1 9 6 8 1 .0 6 – 1 .8 4 /2 4 r o w s a n te ri o rl y, f ro m e n d o f o e so p h a g u s o n ly 2 r o w s 2 l e ft :0 .7 0 – 0 .8 5 ; ri g h t: 0 .0 7 – 0 .1 0 5 4 s u b ve n tr a l p a ir s, 3 su b la te ra l p a ir s p re se n t m a w so n ( 1 9 6 8 ) t e tr a m e re s so m a te ri a e r yj ik o v, 1 9 6 3 4 .8 4 n o s p in e s in t h e m id d le p a rt o f th e b o d y 2 l o n g : 0 .5 7 6 ; sh o rt : 0 .0 8 6 5 v e n tr a l p a ir s, 4 la te ra l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s sp ir o sp ic u lu m p in to & v in ce n te , 1 9 9 5 2 .5 2 – 4 .0 6 n t h in ly d is p e rs e d a n d p o o rl y d e ve lo p e d 2 l e ft : 0 .8 2 – 1 .0 8 ; ri g h t: n n n p in to & v ic e n te (1 9 9 5 ) t e tr a m e re s sk rj a b in i p a n o w a , 1 9 2 6 2 .6 4 w h o le b o d y le n g th 2 l o n g : 1 .5 4 3 ; sh o rt : 0 .1 0 3 n o t fo u n d n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s te tr ic a t ra va ss o s, 1 9 1 7 2 .6 4 d is sa p e a r n e a r la st q u a rt e r o f b o d y le n g th 2 l o n g : 0 .2 ; sh o rt : 0 .0 2 2 4 la te ra l p a ir s, 4 su b la te ra l p a ir s n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s tim o p h e e w o i t ra va ss o s, 1 9 5 0 4 .7 n w h o le b o d y le n g th 2 l o n g : 0 .4 2 1 ; sh o rt : 0 .1 8 9 n n s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s tin a m ic o la p e n ce , m o llh a g e n & p re st w o o d , 1 9 7 5 6 .5 2 4 v e n tr a l r o w s w h o le b o d y le n g th , d o rs a l ro w s e n d 1 .0 2 m m fr o m a p e x 2 l e ft : 2 .2 6 ; ri g h t: 0 .2 0 7 5 s u b ve n tr a l p a ir s, 3 ve n tr o -l a te ra l p a ir s a b se n t p e n ce e t a l. (1 9 7 5 ) t e tr a m e re s u xo ri u s m a m a e v, 1 9 5 9 n 4 n 2 l e ft : 2 .1 – 2 .3 h ; ri g h t: 0 .0 8 8 4 v e n tr o la te ra l p a ir s, 2 su b d o rs a l p a ir s a b se n t m a m a e v (1 9 5 9 ); p e n ce e t a l.( 1 9 7 5 ) 4 .7 6 – 5 .0 4 /2 d o rs o la te ra l r o w s re a ch o n ly t h e b e g in n in g o f th e g la n d u la r o e so p h a g u s 2 l o n g : 2 .1 – 2 .2 4 ; sh o rt : 0 .0 8 6 – 0 .0 8 8 4 v e n tr o la te ra l p a ir s, 2 su b d o rs a l p a ir s a b se n t s kr ja b in & s o b o le v (1 9 6 3 ) t e tr a m e re s vi e tn a m e n si s f a n th e v ie t, 1 9 6 8 n 4 n 2 l e ft : 1 .2 8 ; ri g h t: 0 .1 4 8 5 v e n tr a l p a ir s (l a te ra l a b se n t) n f a n t h e v ie t (1 9 6 8 ) in h e l m in th o lo g ic a l a b st ra ct s (1 9 7 0 ), p e n ce e t a l. (1 9 7 5 ) t a b l e 2 (c o n t. ) n n o in fo rm a tio n a t o u r d is p o sa l a t h e o ri g in a l r e a d s 6 5 -3 5 0 μ m . w e c o n si d e r th is a t yp in g e rr o r a n d in cl u d e t h e r a n g e o f si n g le m e a su re m e n ts p ro vi d e d b y q u e n tin & b a rr e ( 1 9 7 6 ) b s kr ja b in & s o b o le v (1 9 6 3 ) a ls o i n cl u d e a d e sc ri p tio n a ft e r c ra m ( 1 9 2 7 ), w h ic h d iff e rs s lig h tly f ro m th a t o f m a m a e v (1 9 5 9 ) c c re m o n te e t a l. (2 0 0 1 ) g iv e a r a n g e o f 0 .3 1 2 – 0 .5 8 7 m m d c re m o n te e t a l. (2 0 0 1 ) q u o te m o llh a g e n ( 1 9 7 6 ) g iv in g a r a n g e o f 0 .5 0 4 – 0 .6 2 6 m m e t h e le n g th p ro vi d e d b y q u e n tin & b a rr e ( 1 9 7 6 ) is 1 2 – 1 5 4 μ m . w e c o n si d e r th is a n e rr o r. s kr ja b in & s o b o le v g iv e t h e w id th o f th e r ig h t sp ic u le a s 1 2 μ m f a cc o rd in g t o o rt le p p ( 1 9 6 4 ) in t h re e o f a b o u t 3 0 m a le s a r ig h t sp ic u le w a s p re se n t g c re m o n te e t a l. (2 0 0 1 ) q u o te m o llh a g e n ( 1 9 7 6 ) a s t . p ro ze sk yi h a vi n g v a ry in g c a u d a l p a p ill a e ( 3 /0 , 3 /3 , 4 /1 , 4 /2 ) h c a lc u la te d f ro m a 1 :2 4 t o 1 :2 6 r a tio b e tw e e n r ig h t a n d le ft s p ic u le 126 tetrameres numida n. sp. (nematoda: tetrameridae) from hel meted guineafowls in south africa discussion some of the main morphological characteristics of many of the species belonging to the genus tetrameres are listed in table 2. of the tetrameres species with two rows of cuticular spines, tetrameres pattersoni cram, 1933, t. paradisea and tetrameres grusi shumakovitsh, 1946 have only one spicule and the spicule measurements of the latter two species differ distinctly from those in our specimens (ortlepp 1932; schmidt 1962; bush, pence & forrester 1973). tetrameres gubanovi shigin, 1957 bears two rows of body spines, but has seven pairs of caudal papillae (pence et al. 1975), as opposed to six pairs of caudal spines in t. numida n. sp. the use of the term caudal spines or caudal papillae is not always clear. pence et al. (1975) use the term caudal papillae for several species in their publication. they list t. paradisea as well as t. prozeskyi as having caudal papillae, but in the original descriptions ortlepp (1932, 1964) clearly refers to cuticular spines. thus, pence et al. (1975) seem to use the term indiscriminately. mawson (1968), however, describes t. nouveli as having caudal spines, but points out that in tetrameres lobibycis mawson, 1968 the spines are more like elongate papillae, and refers to tetrameres calidris mawson, 1968 and tetrameres scolopacidis mawson, 1968 as having papillae. the left spicules of tetrameres cardinalis quentin & barre, 1976 and tetrameres paucispina sandground, 1928 are much shorter than those measured in our specimens (quentin & barre 1976). tetrameres micropenis travassos, 1915 has been recovered from ciconiiform hosts, nyctanassa violacea (linnaeus, 1758) and cochlearius cochlearia (linnaeus, 1766) (yamaguti 1961), whose geographic distribution is restricted to north and south america (lepage 2006). tetrameres fissispina has been recorded from guineafowls in africa (fabiyi 1972; vercruysse et al. 1985) and, like t. americana, has a high prevalence in domestic chickens, whose nematode fauna is similar to that of guineafowls (mukaratirwa, hove, es mann, hoj, permin & nansen 2001; magwisha, kassuku, kyvsgaard & permin 2002). tetrameres fissispina distinguishes itself from the new species by its shorter spicules and the larger number of caudal spines. tetrameres americana differs not only in the spicule size and the number and arrangement of caudal spines, but also in its four rows of somatic spines (schmidt 1962; gibbons, jones & khalil 1996). the head of the female and the apical view of the head of the male of t. numida n. sp. most closely resemble tetrameres tinamicola pence, mollhagen & prestwood, 1975. the authors of the latter species describe the male head as possessing a triangular mouth surrounded by a pair of trilobed structures originating from the inner surface of the pseudolabia. each lobe bears a pair of tooth-like processes in t. tinamicola. similar processes can be seen in our specimens, but it is difficult to determine their exact number. however, there seem to be three or four per lobe. pronounced lateral alae, as illustrated by pence et al. (1975), were not found in our specimens. moreover, t. tinamicola has a total of four rows of cuticular spines and the deirids are without apical spines. while the length of the left spicule of both species is similar, the right spicule of t. numida is only approximately half the length of t. tinamicola. ortlepp (1932) described the buccal capsule of t. paradisea as having trilobed structures showing two to three bright refringent markings towards its posterior border. this, as well as other features of our specimens such as the transverse grooves anterior to the cloaca and the size of the spines, appeared so similar to t. paradisea that we initially considered assigning them to t. paradisea, especially in view of the fact that both were recovered from south african hosts. close examination has nevertheless revealed distinct differences between the two. tetrameres paradisea possesses a single spicule, whereas in our males two spicules are consistently present. while the arrangement of caudal spines is nearly identical and both carry three pairs of ventral and three pairs of externo-dorsal or lateral spines, the tail of t. paradisea is considerably shorter than that of our specimens (see table 1). ortlepp (1932) described and illustrated two rows of body spines found in t. paradisea and he uses this criterion to distinguish his species from tetrameres nouveli which he lists as possessing four rows of spines. cremonte, digiani, bala & navone (2001) record t. paradisea as having four rows of spines, but cite mollhagen (1976) as describing the dorsal rows of spines as very short, ending at 94–155 from the anterior end. when comparing t. paradisea to t. prozeskyi, ortlepp (1964) lists the length of the left spicule of the former species as 0.48 mm, but his original description of t. paradisea (ortlepp, 1932) clearly states the length of the spicule as 0.69 mm. we list t. pro127 k. junker & j. boomker zeskyi as monospicular, which differentiates it from our bispicular specimens. as regards t. prozeskyi it should be borne in mind that ortlepp (1964) found a well-chitinized right spicule in three of the more than 30 males he examined. in the summary of the description of tetrameres cardinalis quentin & barre, 1976, the range of the length of the right spicule is given as 65–350 μm (quentin & barre 1976). as this seems erroneous, we decided to include the range provided in the same paper, namely 365–400, in table 2. similarly, we consider the first measurement these authors provide for the short spicule of t. paucispina as incorrect and believe it should read 120 instead of 12. apart from t. numida n. sp., only t. tinamicola and tetrameres uxorius mamaev, 1959 have a left spicule that reaches 2 mm in length, while in the remaining tetrameres spp. the long spicule usually does not exceed 1 mm (mamaev 1959; pence et al. 1975). relative to body length, however, there are other species with long spicules, such as t. lobibycis where the single spicule reaches about half of the body length (1.5 mm) and t. scolopacidis where the spicule length reaches almost two thirds of the body length (1.06–1.8 mm) (mawson 1968). to our knowledge, tetrameres phaenicopterus ali, 1970 is the only member of the genus tetrameres possessing a gubernaculum (pence et al. 1975) and tetrameres greeni mawson, 1979 is unique in the genus tetrameres in that it has caudal alae (mawson 1979). tetrameres spirospiculum pinto & vicente, 1995 is distinguished from our specimens and all the other species of tetrameres by the spiral shaped distal end of the longer of its two spicules (pinto & vicente 1995). the numbers of t. numida n. sp. recovered from the guineafowl hosts from musina (messina) were low, and the parasite was only found in the older birds, being absent in young adults. while it is possible that guineafowls are not the main host for this parasite, we attribute the low intensity of infection to the fact that the area had been experiencing a severe drought during the past years. this would decrease the survival rates of nematode eggs while at the same time causing the numbers of possible intermediate hosts necessary for the completion of the life-cycle to decline. while differences in the immune status between guineafowls of different age might play a role in the intensity of infection, we believe that the presence of t. numida n. sp. in older hosts simply reflects the increased possibility of prior exposure to the parasite as a function of time. acknowledgements the authors are indebted to dr s. sokolov of the institute of parasitology, russian academy of science, moscow, for obtaining the extensive chapter on the genus tetrameres in “principles of nematodology xi” (skrjabin & sobolev 1963) and to dr d.a. apanaskevich, georgia southern university, for the translation from the original russian into english. the authors thank mr k. meyer and mr m. storm, the previous and current owners of the farm sandown, musina (messina), respectively, for placing the guineafowls at our disposal and mr h.e. hattingh, university of limpopo, for collecting them. dr w.j. luus-powell, university of limpopo, has kindly facilitated the co-operation. ms d.t. durand, university of pretoria, photographed the three complete females of t. numida. this research was made possible through a claude leon foundation postdoctoral fellowship grant to the first author. references anderson, r.c. 1992. nematode parasites of vertebrates, their development and transmission, 1st ed. wallingford and new york: cab international. appleton, c.c. 1983. tetrameriasis in crested guineafowl from natal. ostrich, 54:238–240. bergan, j.f., radomski, a.a., pence, d.b. & rhodes, o.e. jr. 1994. tetrameres (petrowimeres) striata in ducks. journal of wildlife diseases, 30:351–358. bush, a.o., pence, d.b. & forrester, d.j. 1973. tetrameres (gynaecophila) williamsi sp. n. 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(ed.). 1969. key to parasitic nematodes. vol. 1, spirurata and filariata. translated and edited by m. raveh, 1991. leiden: e.j. brill publishing company. skrjabin, k.i. & sobolev, a.a. 1963. principles of nematodology xi. spirurata of animals and man and the diseases caused by them part i (spiruroidea). moscow: izdatelstv akademii nauk sssr (russian). vercruysse, j., harris, e.a., bray, r.a., nagalo, m., pangui, m. & gibson, d.i. 1985. a survey of gastrointestinal helminths of the common helmet guinea fowl (numida meleagris galeata) in burkina faso. avian diseases, 29:742– 745. yamaguti, s. 1961. systema helminthum. the nematodes of vertebrates. vol. iii, parts i & ii. new york: interscience publishers. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) rungano magadu production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa peter n. thompson production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa citation magadu, r. & thompson, p.n., 2023, ‘seroprevalence and factors associated with coxiella burnetii exposure in goats in moretele’, onderstepoort journal of veterinary research 90(1), a2071. https://doi.org/10.4102/ojvr.v90i1.2071 original research seroprevalence and factors associated with coxiella burnetii exposure in goats in moretele rungano magadu, peter n. thompson received: 03 aug. 2022; accepted: 09 feb. 2023; published: 04 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract infection with coxiella burnetii causes significant economic impact and poses zoonotic risk to people exposed to livestock, yet few studies in south africa have assessed seroprevalence of c. burnetii infection and no information is available for goats. very little information is available regarding risk factors and outcomes of c. burnetii infection in peri-urban farming areas where widespread mixing of ruminants occurs. this study estimated the seroprevalence of c. burnetii infection among communally farmed goats in an area adjacent to the densely populated gauteng province. sera were collected from 216 goats in 39 herds, and questionnaires were completed to establish management practices as potential risk factors. c. burnetii antibody testing was done by elisa. thirty two out of 216 goats tested positive for c. burnetii antibodies and the overall seroprevalence, adjusted for sampling weights and clustering, was 18.4% (95% confidence interval [ci]: 12.2% – 23.5%). the intraclass correlation coefficient was 0.06, indicating low-to-moderate clustering. multiple logistic regression showed age was significantly associated with seropositivity, with higher seroprevalence in animals ≥ 19 months old (26%) than animals ≤ 6 months old (6%) (odds ratio [or]: 6.6; p = 0.010). we concluded c. burnetii infection is common in goats in moretele and a potential cause of abortion in goats and poses the potential zoonotic disease risk. contribution: despite the threats posed on animal health and productivity, scant information is published on c. burnetii in south africa. this research established preliminary estimates of c. burnetii seroprevalence. the research is original from a south african perspective, relevant to africa and focused on infectious disease in livestock. keywords: seroprevalence; coxiella burnetii; goats; elisa; risk factors; zoonosis. introduction q fever is a zoonotic disease caused by the small gram-negative coccobacillus, coxiella burnetii, an obligate intracellular bacteria belonging to the order legionellales and bacterial family coxiellaceae (arricau-bouvery & rodolakis 2005; menadi et al. 2020). c. burnetii infection is endemic worldwide, except in new zealand, and affects a wide range of domestic and wild mammals, fish and some arthropods (huang et al. 2021; karagul, malal & akar 2019; kazar 2005). domestic ruminants such as cattle, goats and sheep are considered the reservoirs of human infection and infected animals shed c. burnetii in the faeces, urine, milk and vaginal discharge (guatteo et al. 2011; menadi et al. 2020). more than 40 species of ticks have been shown to be capable of multiplying c. burnetii in their git mucosal cells and then passing out viable bacteria in their faeces (knap et al. 2019; körner et al. 2020). ticks are thought to play an important role in the maintenance of infection within domestic and wildlife hosts and infection rates of up to 10% have been reported in some studies (bellabidi et al. 2020; körner et al. 2020). animal vaccines against c. burnetii are licensed for use in some european countries but are not registered or used in south africa and most of africa (bauer et al. 2021; rahaman et al. 2019). c. burnetii infections of animals are mostly subclinical but when clinical disease occurs, it is characterised by abortions, particularly in sheep and goats, sometimes on an epidemic scale, and by stillbirths, premature birth and birth of weak offspring (madariaga et al. 2003; woldehiwet 2004). in infected ewes and nanny goats, c. burnetii infection can cause infertility and metritis, while infected cows can develop mastitis due to the pathogen (menadi et al. 2020). affected sheep shed the organism transiently, and they experience higher rates of abortions than cattle (waag 2007). in humans, about 60% of c. burnetii infections are associated with asymptomatic disease or mild, self-limiting, flu-like symptoms with occasional complications such as endocarditis, meningitis and atypical pneumonia (njeru et al. 2016). c. burnetii infection in animals is of particular importance in developing countries due to poor tick control and mixing of ruminant herds in communal pastures (vanderburg et al. 2014), which amplify the risk for animals becoming infected. it has been shown that people who are positive for human immunodeficiency virus (hiv) are at higher risk of developing severe signs and symptoms of q fever (patil & regunath 2021) and as south africa has the highest population of people living with hiv in the world, with 7.7 million hiv-positive people (parker et al. 2020) out of a population of about 58 million (de micco et al. 2022), the potential impact of q fever in humans can be high. the consumption of unpasteurised dairy products, which is widespread in south african communal areas, is one of the transmission modes of c. burnetii infection from animals to humans (gale et al. 2015). despite the effects of c. burnetii infection on productivity in ruminants and the associated human health risk, very little has been done to assess infection rates in animal and human populations, especially in south african peri-urban communal farming areas. a study of c. burnetii seroprevalence in cattle in gauteng province (formerly transvaal province) estimated c. burnetii antibody prevalence at 8% (gummow, poerstamper & herr 1987) and adesiyun et al. (2020) reported a seroprevalence of 38% in cattle in a rural area of south africa at a wildlife-livestock interface. however, there are no reports on c. burnetii seroprevalence in south africa for goats, which are very commonly kept in communal and peri-urban areas throughout south africa due to their hardy nature. in south africa, there is a lack of active surveillance programmes and a lack of epidemiological data, and c. burnetii infection in animals is not listed as a controlled or notifiable disease according to the animal diseases act of south africa (act 35 of 1984) (mangena et al. 2021). elsewhere in africa, c. burnetii infection in animals and humans has been documented in countries such as ghana, cote d’ivoire, togo and burkina faso but, as in south africa, there are very few active surveillance programmes and hence epidemiological data on infections in humans and animals are sparse (johnson et al. 2019). as it is a neglected zoonotic disease with potentially serious effects on human health and animal economics, c. burnetii surveys are necessary to establish the impact on human and animal populations, particularly in communal areas. the objective of this study was to estimate the prevalence of exposure to c. burnetii among the goat population in the villages of the moretele communal farming area, a peri-urban area adjacent to the densely populated province of gauteng, south africa. in addition, associated risk factors for seropositivity and potential outcomes of exposure to c. burnetii were also assessed. materials and methods study area the study area was located between latitudes 25.142°s to 25.285°s and longitudes 27.970°e to 28.253°e. eight villages in the north west province, near the boundary of limpopo and gauteng provinces of south africa, were selected for sampling (figure 1). the villages are located in the floodplain catchment areas of the moretele, apies and tshwane rivers. the terrain in the study area is mainly flat, semi-arid and the predominant vegetation types are sour bushveld, savannah and springbokvlakte thornveld (dynamic intergrated geo-environmental services 2012). the average annual rainfall in the study area is 565 mm, with rain falling in the summer months between october and march, and the annual average temperature is 25 °c (accuweather 2022; dynamic intergrated geo-environmental services 2012). the human population density in moretele is about 818/km2 (statssa 2022). the study area is peri-urban, located about 30 km north-west of hammanskraal township and 20 km north of soshanguve, which is the largest township in pretoria (tshwane). most livestock owners in the area have small herds of cattle and/or goats and make use of communal pastures and drinking points, resulting in frequent mixing of animals from different herds. there is generally minimal veterinary intervention, mostly comprising vaccinations by state veterinary services personnel. figure 1: map of study area on the boundary between gauteng and the north west province, south africa, showing the sampling locations of goats tested for c. burnetii antibodies. grey lines indicate roads; yellow squares indicate villages; red dots indicate properties on which c. burnetii-positive goats were found and black dots are c. burnetii-negative properties. study design and sampling the study was conducted in december 2018 and was designed to estimate the seroprevalence of c. burnetii in goats using multi-stage random sampling. information on goat population distribution by household was obtained from state-employed animal health technicians responsible for animal health in the study area. only villages where several goat owners owned five goats or more were selected. within selected villages, random sampling of households by a hat draw method was followed by random sampling of goats within households after obtaining written informed consent from each farmer. sample size calculation sample size was calculated based on the equation for a sample size to estimate prevalence with 95% confidence in an infinite population assuming simple random sampling (thrusfield 2007): where n is the required sample size, pexp is the expected prevalence and d is the desired absolute precision. a minimum of 110 goats were calculated for sampling based on an expected prevalence of 20% and a desired precision of 7.5%. the design effect (deff) for clustering was calculated as follows: where m is the cluster size and ρ is the intracluster correlation coefficient (icc) (bennett et al. 1991), set at 0.2 as it is unlikely to exceed this value for most infectious diseases (otte & gumm 1997). for an average cluster size (m) of five goats sampled per household, deff was calculated as 1.8 and multiplied by the sample size for simple random sampling to give an adjusted minimum required sample size of 198 goats, to be sampled from 40 households. blood sample collection ten millilitre of blood from each goat was collected by jugular venepuncture with 20-gauge needle into a vacutainer serum tube. samples were transported on ice in a cooler box to the laboratory where they were centrifuged at 1500 g for 10 min at room temperature to separate. the serum samples were stored at -20 °c until serological testing. collection of data a questionnaire was used to collect data from goat owners. the questionnaire collected information in two parts with the first part being individual goat-specific information such as age, sex, breed, history of kidding, history of abortion and the origin of each goat sampled. flock-specific information made up the second part of the questionnaire and obtained information such as flock abortions in the previous 12 months, deaths in the previous 12 months and management practices such as dipping of goats, routine buying and selling of goats and the use of injectable tetracyclines in the goat flocks. laboratory testing testing for c. burnetii antibodies was done using the lsivettm ruminant q fever – serum/milk enzyme-linked immunosorbent assay (elisa) (priocheck, leylstad, the netherlands), which is an indirect elisa kit for the detection of phase 1 and phase 2 anti-c. burnetii antibodies in ruminant serum or milk. the kit was used according to the manufacturer’s recommendations. validations performed by inra and life technologies report a diagnostic sensitivity (se) of 87% and diagnostic specificity (sp) of 100% for this test (de oliveira et al. 2018). for each sample, the sample/positive (s/p) ratio was calculated as: where odsample was the optical density of each sample tested, odnc was the average optical density of the negative control and odpc was the average optical density of the positive control. the c. burnetii antibody titre was then calculated as titre = s/p × 100. the results were interpreted as follows: ≤ 40 was negative, 40–100 was mild positive, 100–200 was moderate positive and > 200 was strong positive. statistical analysis for the analysis, mild, moderate and strong positives were all regarded as positive results. age was categorised into terciles (≤ 6 months, 7–19 months, > 19 months). prevalence of c. burnetii antibodies and 95% confidence intervals was calculated and adjusted for sampling weights and clustering using the svy (survey) commands in stata 14 (statacorp, college station, texas, united states). prevalences and confidence intervals were then also adjusted for the reported diagnostic se and sp of the elisa using the formula (rogan & gladen 1978): where tp is true prevalence and ap is apparent prevalence. the icc (ρ) was calculated as: where k is the number of herds, yi+ is the number of seropositive animals in herd i, ni is the number of animals tested in herd i and p is the overall unadjusted seroprevalence. univariate analysis of potential risk factors for seropositivity was done by cross-tabulation and the fisher’s exact test. factors associated with a positive test outcome at p < 0.2 were selected for inclusion in a multivariable logistic regression model, which was then developed by backward elimination until variables remaining in the model were significant at p < 0.05. herd size was included as a random effect and adjustment for sampling weights and clustering in the multistage survey design was done using the svy command in stata®. univariate associations between seropositivity and its potential consequences, such as abortions, were assessed using logistic regression and odds ratios were calculated. statistical significance was assessed at p < 0.05. ethical considerations the research protocol was approved by the university of pretoria animal ethics committee (approval number v001-18), the university of pretoria faculty of humanities research ethics committee (approval number gw20170928hs) and the department of agriculture, forestry and fisheries (approval numbers 12/11/1/1/6 and 12/5/1). results a total of 216 goats belonging to 39 goat owners (average of 5.5 goats per herd) were sampled across the eight villages. the total number of c. burnetii antibody-positive goats was 32/216, with estimated animal-level prevalence, after adjustment for sampling weights, clustering and diagnostic se and sp, of 18.4% (95% ci: 12.2% – 23.5%). seropositive goats were found in 20/39 household herds, giving an estimated herd prevalence of 51% (95% ci: 35% – 68%). the intraclass correlation co-efficient (icc) was 0.06, indicating low to moderate clustering of c. burnetii seropositivity within the tested goat herds. univariate associations (p < 0.2) of potential predictor variables with c. burnetii seropositivity were found for age category (p = 0.001) and breed (p = 0.159) (table 1). these variables were therefore selected for inclusion in the multivariable model. table 1: factors associated with seropositivity to coxiella burnetiii amongst communally-farmed goats in moretele district: univariate analysis. for the multivariable analysis, due to small category sizes, breed was re-categorised as boergoat, mixed breed and other (included kalahari red, angora and saanen). in the final model (table 2), the odds of seropositivity increased with age, being 6.6 times greater in goats > 19 months than in those ≤ 6 months (95% ci: 1.6–26.7; p = 0.010). prevalence also varied by breed, with the lowest odds of seropositivity in boergoats and the highest in ‘other’ (p = 0.033). table 2: mixed-effects logistic regression model of factors associated with coxiella burnetii seropositivity. univariate analysis of potential consequences of infection (table 3) showed that goats with a history of abortion were significantly more likely to be c. burnetii-seropositive (or: 4.6; 95% ci: 1.1–20.2; p = 0.042). in addition, goats in herds that had experienced more than two abortions in the past 12 months tended to have greater odds of seropositivity than goats in herds that had experienced no abortions (or: 2.9; 95% ci: 0.9–6.8; p = 0.071). table 3: potential consequences of coxiella burnetii infection in communally-farmed goats in moretele district and their association with seropositivity to c. burnetii. discussion this study is the first to document seroprevalence of c. burnetii in goats in south africa, showing widespread exposure to the pathogen in the study area, with estimated animal-level and herd-level seroprevalences of 18.4% and 51%, respectively. this finding is of public health significance, since it indicates frequent exposure to an important zoonotic pathogen amongst livestock in close proximity to densely-populated urban areas. muleme et al. (2017) reported an animal seroprevalence of 25% – 43% for c. burnetii within herds of dairy goats in victoria, australia in a study conducted during an outbreak between 2012 and 2014. this higher seroprevalence is likely due to more intensive farming systems in dairy goats, with higher population density and concentrated kidding seasons, which are the highest risk periods for uninfected animals becoming infected by infected placentas and uterine fluids (muleme et al. 2017). a study in sheep and goats in northern egypt showed an overall animal seroprevalence of c. burnetii of 15% – 20%, similar to the current study, and the authors suggest that the seroprevalence of c. burnetii is generally higher in goats than in sheep (selim et al. 2018). the similarity in seroprevalence could be due to similarities in management practices and climatic conditions, with low rainfall, hot temperatures and possibly abundance of ticks being common to the two study areas. moretele communal area, as with other rural communities, has most goats on communal grazing and this results in widespread mixing of goat herds, spread of infection between herds and a high herd seroprevalence, compared to private farms with no herd mixing. however, the herd seroprevalence of c. burnetii in this study (51%) was lower than the 80% reported from northern jordan (lafi et al. 2020). the higher farm level seroprevalence in jordan could be explained by the arid nature of pastures in jordan where the nomadic goats have to walk long distances to find food and also, the more frequent mixing of goat herds in these sparse pastures leads to greater horizontal transmission. in contrast, herd-level seroprevalence was only 8.0% for intensively-farmed dairy goat herds in a study in victoria, australia (tan 2018), where there was likely absence of mixing of goat herds and more frequent use of acaricides. a study in communal areas in senegal, where wc. burnetii deoxyribonucleic acid (dna) was assayed in ticks, showed infection rates of up to 37.6% of the sampled ticks (mediannikov et al. 2010). we postulate that high tick infection rates could have contributed to the higher herd seroprevalence seen in this study, compared to those by tan (2018). another study in algeria, though in cattle, by menadi et al. (2020) reported a herd seroprevalence of 45.6%, similar to the 51% found in this study. although this was in cattle, this is possibly due to similarities in animals mixing in communal pastures, similar tick control practices and sharing communal water sources as well as closely related climatic conditions of algeria and semi-arid moretele. there was evidence that increasing age was significantly associated with increased odds of seropositivity and animals > 19 months of age were 6.6 times more likely to test positive for c. burnetii antibodies than were animals < 6 months of age. this age association was also reported in an egyptian study where animals > 4 years of age had higher seroprevalence than younger ones (klemmer et al. 2018) as well as in another study in kurdistan province of iran where animals 3 years and older were more likely to test positive (fakour, jamali & ahmadi 2021). it is to be expected that, as with most infectious diseases, the seroprevalence will increase with age due to cumulative exposure to c. burnetii over time. in this study, there was no significant association of c. burnetii seropositivity with herd size. this contrasts with the findings of rizzo et al. (2016) in north-west italy, where the seroprevalence in sheep and goat herds larger than 12 goats was about three times higher than in smaller herds. rizzo et al. (2016) postulate this could be due to crowding which amplifies the risk of horizontal transmission of c. burnetii. in our study, larger herd size did not necessarily mean that there was more crowding at pasture, due to the absence of fencing in the extensive grazing system on communal pastures. the icc of 0.06 demonstrates low to moderate clustering of c. burnetii within the tested goat herds. coxie lla burnetii is a contagious pathogen, therefore, the presence of one infected goat likely means several other goats in the same herd are also infected, and clustering is to be expected. communal grazing, over and above the risk of aerosol transmission, pasture contamination with faecal material and urine, and vaginal discharge contaminated with c. burnetii, also carries the risk of infected ticks moving within and between goat herds (angelakis & raoult 2010). because in communal systems transmission occurs easily between herds, clustering of contagious diseases within herds is likely to be less pronounced than where herds are kept separate, resulting in only low to moderate clustering, consistent with our finding. in contrast to our study, adesiyun et al. (2020) reported a much higher degree of clustering of c. burnetii seropositivity in communally farmed cattle at dip tanks adjacent to the kruger national park (icc = 0.57). the difference can be ascribed to the fact that, in that study, clustering was assessed at the dip tank level, not at the level of herds within dip tanks, and animals from different dip tanks are very unlikely to mix, compared with animals from different herds within communal areas. this study showed an association between seropositivity for c. burnetii and the occurrence of abortion, at both the individual and the herd level. this confirms the importance of c. burnetii as an abortigenic agent that could have significant economic impact in livestock herds. it should be considered as a possible differential diagnosis when investigating single or outbreaks of abortions in ruminant animals in south africa, and precautions should be taken to prevent zoonotic transmission when handling abortion products, as for other important zoonotic abortigenic agents that occur in south africa, such as brucellosis and rift valley fever. conclusion c. burnetii infection is widespread in the moretele municipality of south africa and is present in more than 50% of goat herds. it is likely that the seroprevalence is similar in many other communal areas throughout the country, due to similarities in management practices, as well as the widespread transport of goats between different parts of the country. further studies in south africa in cattle, sheep and goats, and in people that are occupationally at risk should be done to determine the true extent of infection with c. burnetii in animals and the occupational risk of infection for humans. studies into the prevalence of c. burnetii in cases of abortion, mastitis and metritis should be designed to assess the impact of disease on various production systems for cattle, sheep and goats. once comprehensive data on disease prevalence and impacts in south african livestock farming is elucidated, optimal management practices to mitigate against the spread of c. burnetii withinin intensive and extensive animal husbandry systems can be suggested. these management practices may include effective tick control, purchasing animals from herds that regularly test and minimising mixing of herds, although some of these are not always easily applicable in communal areas. awareness campaigns should be conducted to educate farmers on the risks of c. burnetii, appropriate protective measures to prevent human exposure, and the benefits of controlling this infection in animals. acknowledgements the authors acknowledge, with gratitude, all the farmers who assisted in making the study possible by availing their animals for testing and providing information in the questionnaires. we also thank the animal health technicians in moretele communal area, masethe maime and conny kaotsane, for assistance in identifying goat-owning households for the study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions r.m. and p.n.t. contributed to the study’s conception and design. funding of the study was done by p.n.t., while the material preparation, data and sample collection were performed by r.m. data analysis were performed by p.n.t. the first draft of the manuscript was written by r.m., and both authors read and approved the final manuscript. funding information this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data availability the data generated by this study are available on reasonable request from the corresponding author, r.m. disclaimer the views expressed within this article are solely the authors and do not reflect the opinions and beliefs of the university of pretoria and its affiliates. references accuweather, 2022, accuweather, viewed 15 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the only known methods of con­ trol as there is presently no vaccine available to com­ bat the disease (penrith, thomson & bastos 2004). asf outbreaks have severely affected the pig in­ dustry in southern, east and west africa in recent years (penrith et al. 2004; penrith, lopes pereira, lopes da silva, quembo, nhamusso & banze 2007) decimating pig stocks on both subsistence and commercial farms. the natural arthropod host, a long­lived, eyeless, warthog­burrow­dwelling argasid tick of the genus 385 onderstepoort journal of veterinary research, 76:385–392 (2009) molecular monitoring of african swine fever virus using surveys targeted at adult ornithodoros ticks: a re-evaluation of mkuze game reserve, south africa l.f. arnot1, 2, j.t. du toit1, 3 and a.d.s. bastos1, 4* abstract arnot, l.f., du toit, j.t. & bastos, a.d.s. 2009. molecular monitoring of african swine fever virus using surveys targeted at adult ornithodoros ticks: a re­evaluation of mkuze game reserve, south africa. onderstepoort journal of veterinary research, 76:385–392 the mkuze game reserve (mgr), in north­eastern kwazulu­natal province, south africa is an african swine fever virus (asf) controlled area. in a survey conducted in 1978, asf prevalence in warthogs and ornithodoros ticks in mgr was determined to be 2 % and 0.06 %, respectively. these values, acknowledged as being unusually low compared to other east and southern african asf­ positive sylvatic­cycle host populations, have not been assessed since. the availability of a sensitive pcr­based virus detection method, developed specifically for the sylvatic tampan host, prompted a re­evaluation of asf virus (asfv) prevalence in mgr ticks. of the 98 warthog burrows inspected for ornithodoros presence, 59 (60.2 %) were found to contain tampans and tick sampling was signifi­ cantly male­biased. whilst gender sampling­bias is not unusual, the 27 % increase in infestation rate of warthog burrows since the 1978 survey is noteworthy as it anticipates a concomitant increase in asfv prevalence, particularly in light of the high proportion (75 %) of adult ticks sampled. however, despite dna integrity being confirmed by internal control amplification of the host 16s gene, pcr screening failed to detect asfv. these results suggest that asfv has either disappeared from mgr or if present, is localized, occurring at exceptionally low levels. further extensive surveys are required to establish the asfv status of sylvatic hosts in this controlled area. keywords: african swine fever virus, mkuze game reserve, ornithodoros porcinus, pcr, warthog * author to whom correspondence is to be directed. e­mail: ad­ bastos@zoology.up.ac.za 1 mammal research institute, department of zoology and ento­ mology, university of pretoria, pretoria, 0002 south africa 2 current address: department of paraclinical sciences, fac­ ulty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 3 current address: department of wildland resources, utah state university, logan, ut 84322­5230, usa 4 arc­onderstepoort veterinary institute, private bag x05, on derstepoort, 0110 south africa accepted for publication 13 april 2009—editor 386 molecular monitoring of african swine fever virus: re­evaluation of mkuze game reserve, south africa ornithodoros (kleiboeker & scoles 2001), is the most important maintenance host and vector of the asf virus (asfv) in africa (plowright, parker & peirce 1969; thomson 1985). the distribution of asf in southern africa is determined largely by the distribution of this sylvatic cycle soft tick and its pre­ ferred vertebrate host, the common warthog phaco­ choerus africanus (thomson 1985). in the inverte­ brate tick host, the virus can be transmitted sexually, trans­ovarially and trans­stadially (reviewed by klei­ boeker & scoles 2001). infection rates increase with increasing size and age of the tick, and females dis­ play higher infection rates than males, due most likely to directional sexual transmission of the virus from males to females (plowright 1977). in the ab­ sence of an infectious vertebrate blood meal, the virus can be maintained for periods of up to 15 months in the sylvatic ornithodoros tick (plowright, perry, pierce & parker 1970) and possibly even in­ definitely (plowright 1977). whilst the virus can per­ sist for prolonged periods in infected ticks, which are considered to be the hosts of primary impor­ tance in asf epidemiology, warthogs play an impor­ tant role in amplifying the virus in tick colonies. this ostensibly occurs at those times when virus levels in viraemic neonate warthogs surpass the threshold level required to infect ticks (thomson 1985; plow­ right et al. 1994). as warthogs are seasonal breed­ ers in southern africa, the proposed viral transmis­ sion between the vertebrate and invertebrate hosts is cyclical, coinciding with the warthog farrowing sea son that runs from november to december (cum ming 2005). asfv seroprevalence in large asf­infected warthog populations is generally high, ranging from 50–100 %, while the presence of virus in extensively surveyed east african tick popula­ tions is substantially lower, ranging from 0.26 to 5.03 % in adult ticks (plowright 1977). a study was undertaken in 1978 to assess the asf status in warthog and ornithodoros sylvatic cycle hosts in mkuze game reserve (mgr), south africa (thomson, gainaru, lewis, biggs, nevill, van der pypekamp, gerber, esterhuysen, bengis, bezuiden­ hout & condy 1983; thomson 1985). in this study, the asfv infection rate for 5018 ticks was deter­ mined by inoculating pig leucocyte cultures with pooled ground tick suspensions (3–5 adult females, 6–8 adult males and > 10 nymphs; thomson et al. 1983). following blind passage, three haemadsorp­ tion­(had)­negative isolates, of reduced virulence for domestic pigs, were obtained, corresponding to a 0.06 % infection rate. this is 23­fold lower than the asfv infection rate in ornithodoros in the kruger national park (knp), south africa (thomson et al. 1983). similarly, asfv seroprevalence in warthogs in mgr was unusually low (2 %) on immuno­electro­ osmophoresis (ieop) compared to the 94 % on ieop for knp, with elisa seroprevalence esti­ mates being 6 % and 85 %, respectively. the mgr prevalence levels were the lowest documented for the six asf­positive southern african localities eval­ uated (thomson et al. 1983), and remain the lowest reported thus far for an asf­positive region in which substantial numbers of both sylvatic hosts co­occur. the exceptionally low asfv prevalence in mgr, availability of a sensitive pcr­based method for de­ termining infection rates in ticks (bastos, arnot, jacq uier & maree, 2009), lack of large­scale surveys since 1978 and the recent incorporation of mgr into the greater st lucia wetland park, prompted a re­evaluation of the asfv status in the sylvatic host that is of primary importance in asf epidemiology, the warthog­burrow­dwelling ornitho doros tampan. materials and methods study area and warthog census data the mgr is a 37 000 ha conservation area situated in north­eastern kwazulu­natal (kzn) province, south africa and constitutes the north­western spur of the greater st lucia wetland park. the park is flanked to the north and south by the high­preva­ lence asf and zero­prevalence warthog populations of the knp and hluhluwe­imfolozi park (hip), re­ spectively. in 1984, the number of warthog estimated to be present in the northern (main) part of the re­ serve (fig. 1) was 630, while during the 2002 game census conducted by the kzn nature conservation service in the same part of the reserve, the warthog population was estimated to be in the vicinity of 1 000 individuals (craig mulqueeny, personal com­ munication 2002). tick collection a total of 98 warthog burrows throughout the reserve were sampled for tampans (fig. 1). the exact posi­ tion of each burrow was recorded using a hand held gps unit. loose soil was removed from the burrows with a spade and sieved over a white plastic sheet, using a stainless­steel labotec test sieve, with aper­ tures of 3 350 µm. the plastic sheet facilitated the de tection of small ticks that passed through the sieve. ticks were transported to the university of pretoria in sealed containers, under a permit issued by the department of agricultural technical services, and stored at –70 °c until they were required for anal­ 387 l.f. arnot, j.t. du toit & a.d.s. bastos ysis. the species designation ornithodoros porci­ nus for ticks collected is used here in accordance with the results of a molecular phylogenetic analysis of geographical diversity in the tick host of asfv (bas tos et al. 2009). this analysis confirmed the monophyly of o. porcinus but revealed a lack of support for the o.p. porcinus and o.p. domesticus sub­species designations. the ventral and dorsal fig. 1 map of mkuze game reserve depicting the distribution of the 98 warthog burrows (m1–m98) investigated for the presence of ornithodoros key phinda resource reserve makasa isimangaliso (st lucia) wetland park ornithodoros infested burrows burrows accommodation hides roads rivers nsumo pan mkuze game reserve 388 molecular monitoring of african swine fever virus: re­evaluation of mkuze game reserve, south africa surfaces of all ticks were photographed and they were visually sexed using the guidelines of walton (1962), and classed as male, female or nymphs. a two sample t­test was used to test for any difference in the overall occurrence of male and female ticks across all samples collected in mgr, using genstat version 4.2 (lane & payne 1996). molecular analyses dna was extracted using a silica­guanidine thiocy­ anate technique from 10 % w/v ground tick suspen­ sions, prepared as described previously (bastos et al. 2009). asfv genome presence was evaluated using a duplex pcr approach that, in addition to be ing confirmatory for the virus, is also informative with respect to host taxonomy and asfv genotype (bastos et al. 2009). false negative results, due either to a failed pcr or a failed dna extraction, are precluded through amplification of the tick mitochon­ drial 16s gene, which acts as an internal control. positive and negative controls were always includ­ ed to ensure reaction efficiency and freedom from contamination of the reagents used. to determine the genetic relatedness of the three mgr 1978 viruses to each other and to previously characterised, regional viruses, nucleotide sequenc­ es corresponding to the c­terminal end of the p72 gene were generated as previously described (bas­ tos, penrith, cruciere, edrich, hutchings, roger, cou­ acy­hymann & thomson 2003). in addition, three south african tampan isolates sampled between 1979 and 1987, were characterised. sequences generated by a manual sequencing approach were submitted to genbank (www.ncbi.nlm.nih.gov) un­ der accession numbers fj455835 to fj455840, and were complemented with homologous reference sequence data available for each of the 14 southern african genotypes described to date (bastos et al. 2003; boshoff, bastos, gerber & vosloo 2007). a molecular phylogeny was inferred in mega4 (ta­ mura, dudley, nei & kumar 2007) using the neigh­ bour­joining algorithm with nodal support being as­ sessed by 10 000 bootstrap replications. results burrow infestation rate and sex ratios of adult ticks tampans were collected from 59 of the 98 burrows inspected, which corresponds to a 60.2 % tick infes­ tation rate. of the 348 ticks collected, 88 (25 %) were nymphs and 161 of the 260 adult ticks (62 %) were males. this 62:38 male:female bias was sta­ tistically significant (p < 0.01; two­sample t­test). screening for asfv genome presence all 348 ticks were screened for the presence of asfv using the sylvatic tick duplex pcr (bastos et al. 2009). none of these 348 ticks amplified the ex­ pected 478 bp asfv p72 gene target. however, with the exception of the negative controls, all reac­ tions amplified the expected 313 bp 16s host mito­ chondrial gene target of the internal control, thereby confirming template dna and reaction integrity and precluding the possibility of false negative results. retrospective genetic analysis of the 1979 mkuze tick isolates nucleotide sequencing of the c­terminal end of the p72 gene revealed that the three mgr tick viruses isolated in 1978 were identical to each other and also to two viruses that caused outbreaks of asf in domestic pigs in 1973 and 1975 in south africa (fig. 2). phylogenetic analysis of the homologous 401 nucleotide (nt) dataset identified the mgr viruses as belonging to genotype xx, whilst three additional south african tampan viruses clustered within south­ ern african, domestic pig genotypes iii and xxii. none of the tick viruses were unique across the gene region sequenced, with each being identical to a domestic pig virus (fig. 2). discussion the two notable differences between the 1978 and 2002 surveys in the mgr with respect to the syl­ vatic hosts of asfv, relate to changes in burrow infestation as well as warthog numbers. o. porcinus were found in 59 out of the 98 burrows surveyed (60 % infestation rate) in 2002, whilst thomson (1985) reported that ticks were retrieved from 13 out of the 40 burrows (33 %) surveyed in 1978 in mgr. these results indicate that tick populations were present in 27 % more burrows in 2002 than in 1978, probably due to the estimated 59 % increase in warthog numbers from 1984 to 2002. the marked increase in warthog numbers, together with the ob­ servation that 10.8 % and 47 % of warthogs sam­ pled outside their burrows in namibia and in south africa, respectively, were infested with ornithodoros nymphs (horak, biggs, hanssen & hanssen, 1983; horak, boomker, de vos & potgieter, 1988), lends credence to the proposed role played by warthogs in asf epidemiology, as disseminators of the inver­ 389 l.f. arnot, j.t. du toit & a.d.s. bastos tebrate tick host (thomson 1985). an increase in asfv prevalence could thus be anticipated in mgr because of the increase in warthog numbers and concomitant increase in burrow infestation rates and resulting tick­warthog contacts. this was not, however, the case. instead, none of the 348 ticks screened using a molecular approach developed specifically for asfv screening of ornithodoros por­ cinus (bastos et al. 2009) harboured the virus. analysis of the c­terminal p72 gene region revealed that the three mgr viruses isolated from pooled spec/207 spec/205 spec/209 ang/70 zam/02/1 nam/01/80 genotype i genotype xviizim/92/1 moz60-98 mad/1/98 genotype ii bot/1/99 spec257 (1993) ellisras pig spec140 warmbaths tampan (1987) spec57 thabazimbi tampan (1985) genotype iii genotype ivrsa/1/99w spec/120 rsa/03/96 genotype xix lillie (1973) pig gr22-6 mkuze tampan (1978) gr21-23 mkuze tampan (1978) 24823 (1975) pig gr21-11 mkuze tampan (1978) genotype xx spec/53 rsa/01/96 genotype xxi moz/1960 moz/1979 genotype v moz/94/1 moz/94/8 genotype vi gr44a2t knp tampan (1979) spec/245 (1992) pig genotype xxii spec/154 spec/260 rsa/1/98 genotype vii genotype xviiinam/01/95 genotype xi kab/62 genotype viiimoza-98 genotype xv tan/1/01 genotype xvi tan/2003/1 genotype ix uga/1/95 genotype x uga/3/9510 69 8 9 9 8 6 6 9 5 96 50 5 6 7 73 79 6 0.005 fig. 2 genetic relatedness of the three mkuze game reserve tampan viruses isolated in 1978 (indicated in bold with grey shading) and three additional south african tampan viruses (denoted by grey shading alone), based on c­terminal p72 gene sequence analysis nodal support ≥ 50 % from 10 000 bootstrap replicates is indicated next to each node and the previously defined genotypes recovered following neighbor­joining analysis of the homologous 401 nt dataset, are shown to the right of the relevant lineage 67 54 50 96 79 73 61 79 64 64 91 88 96 57 99 69 89 100 0.005 390 molecular monitoring of african swine fever virus: re­evaluation of mkuze game reserve, south africa ticks sampled in 1978 were identical to each other and to two historical, genotype xx domestic pig strains, recovered from asf outbreaks in south africa, in 1973 and 1975 (fig. 2). these mgr vi­ ruses were, however, distinct from the three other south african tampan isolates, with the kruger na­ tional park (knp) tampan virus clustering within genotype xxii and the thabazimbi and warmbaths (bela bela) tick viruses being assigned to genotype iii. the two domestic pig viruses, lillie and 24823, shown to be identical to the mgr tick viruses, were also had­negative, with isolate 24823 having re­ duced virulence in domestic pigs (pini 1977). these results are consistent with the observations of thom­ son and co­workers (1983) that the three mkuze vi­ ruses were unusual for their non­haemadsorbing characteristic and reduced virulence in domestic pigs. other atypical features of the asf­status of the mgr sylvatic hosts are the unusually low sero­ prevalence of the vertebrate (warthog) host (2 % and 6 % on ieop and elisa, respectively). in par­ ticular, it was noted that only one of three warthog sub­yearlings from the same sounder, was found to be seropositive to asf (thomson et al. 1983). this unexpected result indicates that the specificity/ accuracy of asf serological tests in warthogs is possibly sub­optimal and underscores the need to investigate and validate tests specifically for sylvatic vertebrate hosts (jori & bastos 2009). transformation of the asfv incidence by sex and size class of ornithodoros ticks sampled at three southern african localities (table 4 of thomson et al. 1983) revealed that at all localities, more adult males than females were recovered, with the aver­ age male:female ratio being 61:39. the 62:38 ratio recorded in the 2002 mgr survey is therefore con­ sistent with these previous observations of male bias. however, differences in the size classes were pronounced with substantially more nymphs than adults being collected in earlier surveys (pini 1977; thomson et al. 1983). with the exception of knp, 6.7 more nymphs than adults (13 % adults: 87 % nymphs) on average, were collected from tick pop­ ulations sampled in the 1970s by thomson and co­ workers (1983). in contrast, sampling was skewed towards adults (75:25 adults:nymphs) in the 2002 survey due most likely to differences in the aperture size of the sieves used in the 1978 versus 2002 sur­ vey. this higher proportion of adults to nymphs in the 2002 sample is relevant as a nine­fold higher asfv infection rate was observed in adult ticks ver­ sus nymphs (2.5 % versus 0.27 %), on average, in three southern africa tick populations based on data provided in table 4 of the thomson et al. (1983) study. whereas it may be tempting to attribute the nega­ tive results obtained from the 2002 survey to inad­ equate sampling, as just 348 ticks were screened compared to 5018 in the 1978 survey, a number of important differences should be borne in mind. first, warthog numbers and burrow infestation rates were 59 % and 27 % higher during the 2002 survey. second, an almost six­fold higher proportion of adult ticks were collected in 2002 compared to 1978. third, pcr was found to be 1.4 times more sensi­ tive than asfv isolation from ornithodoros ticks in europe following first­round amplification alone (bas to, portugal, nix, cartaxeiro, boinas, dixon, leitão & martins 2006). this result is in keeping with those from studies on other viruses which found that pcr was between 1.4 and 1.5 times more sensitive than virus isolation (risatti, holinka, lu, kutish, callahan, nelson, brea tió & borca 2005; wang, o’keefe, orr, loth, banks, wakeley, west, card, ibata, van maanen, thoren, isaksson & kerkhofs 2008). therefore, even if the increase in warthog numbers and burrow infestations is discounted and only differential, adult tick infection rate (nine­fold higher in adults versus nymphs) and the 1.4­fold higher sensitivity afforded by pcr screening are considered, it can be determined that in spite of the smaller sample size, a five­fold increase (from 0.06 to 0.30 %) in asfv prevalence in the 2002 mgr tick sample was a reasonable expectation. the neg ative result, despite more burrows and propor­ tionally more adults being screened using a pcr­ based approach that precludes false negatives, makes it highly improbable that any asfv­positive tick colonies went undetected in the 2002 survey. this then begs the question as to whether asfv still exists within the mgr. if the virus still persists then it must be extremely localized and restricted to burrows that were not surveyed in our study. as asf is a controlled disease of economic signifi­ cance, it has major implications for the development of pig industries in northern kzn and also impacts on trade in indigenous swine. any change in asfv infection of sylvatic hosts in mgr would require re­ assessment of the continued inclusion of mgr with­ in the asf control area. this pcr­based pilot study has highlighted the possible disappearance of the virus from mgr and the value of performing adult ornithodoros tick targeted molecular surveys. it has also indentified a need for further, more extensive studies to establish beyond doubt whether asfv still persists within the greater mgr area. 391 l.f. arnot, j.t. du toit & a.d.s. bastos conclusion ninety­eight burrows examined for the presence of o. porcinus in mgr yielded 348 o. porcinus ticks in 59 burrows. despite a 27 % increase in burrow in­ festation and 59 % increase in warthog density since 1978, a nearly six­fold increase in proportion­ al sampling of adult ticks versus nymphs, and em­ ployment of a sensitive pcr screening method, no evidence of asfv genome presence could be found. it is therefore possible that asfv no longer exists within mgr and that if it does, that the asf infection rate is extremely low and restricted to a small number of warthog burrows. we propose that molecular screening of adult ticks is reliable and cost­effective for evaluating asfv control area sta­ tus and for conducting asfv surveillance in areas of particular concern. acknowledgements we are grateful to warren mccall for field assist­ ance, stephan swanepoel for providing virus isola­ tion information and ingrid booysen for preparing the mkuze map. financial support for the mkuze tick survey was provided through an rdp and thuthuka grant awarded to adsb by the university of pretoria and the national research foundation of south africa. references basto, a.p., portugal, r.s., nix, r.j., cartaxeiro, c., boinas, f., dixon, l.k., leitão, a. & martins, c. 2006. development of a nested pcr and its internal control for the detection of african swine 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the detection of bovine herpesvirus 1 in ex­ tended bovine semen. veterinary microbiology, 126:11–19. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) jeanette m. wentzel hans hoheisen research station, faculty of veterinary science, university of pretoria, pretoria, south africa department of veterinary tropical disease, faculty of veterinary science, university of pretoria, pretoria, south africa louise j. biggs department of production animals, faculty of veterinary science, university of pretoria, pretoria, south africa moritz van vuuren department of veterinary tropical disease, faculty of veterinary science, university of pretoria, pretoria, south africa citation wentzel, j.m., biggs, l.j. & van vuuren, m., 2022, ‘comparing the minimum inhibitory and mutant prevention concentrations of selected antibiotics against animal isolates of pasteurella multocida and salmonella typhimurium’, onderstepoort journal of veterinary research 89(1), a1955. https://doi.org/10.4102/ojvr.v89i1.1955 original research comparing the minimum inhibitory and mutant prevention concentrations of selected antibiotics against animal isolates of pasteurella multocida and salmonella typhimurium jeanette m. wentzel, louise j. biggs, moritz van vuuren received: 18 june 2021; accepted: 18 oct. 2021; published: 10 jan. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract historically, the use of antibiotics was not well regulated in veterinary medicine. the emergence of antibiotic resistance (abr) in pathogenic bacteria in human and veterinary medicine has driven the need for greater antibiotic stewardship. the preservation of certain antibiotic classes for use exclusively in humans, especially in cases of multidrug resistance, has highlighted the need for veterinarians to reduce its use and redefine dosage regimens of antibiotics to ensure efficacy and guard against the development of abr pathogens. the minimum inhibitory concentration (mic), the lowest concentration of an antibiotic drug that will prevent the growth of a bacterium, is recognised as a method to assist in antibiotic dosage determination. minimum inhibitory concentrations sometimes fail to deal with first-step mutants in bacterial populations; therefore dosing regimens based solely on mic can lead to the development of abr. the mutant prevention concentration (mpc) is the minimum inhibitory antibiotic concentration of the most resistant first-step mutant. mutant prevention concentration determination as a complementary and sometimes preferable alternative to mic determination for veterinarians when managing bacterial pathogens. the results of this study focused on livestock pathogens and antibiotics used to treat them, which had a mic value of 0.25 µg/ml for enrofloxacin against all 27 isolates of salmonella typhimurium. the mpc values were 0.50 µg/ml, with the exception of five isolates that had mpc values of 4.00 µg/ml. the mpc test yielded 65.52% (18 isolates) salmonella isolates with florfenicol mics in the sensitive range, while 11 isolates were in the resistant range. seventeen isolates (58.62%) of pasteurella multocida had mic values in the susceptible range and 41.38% (12 isolates) had an intermediate mic value. mutant prevention concentration determinations as done in this study is effective for the antibiotic treatment of bacterial infections and minimising the development of resistance. the mpc method can be used to better control to prevent the development of antibiotic drug resistance used in animals. keywords: minimum inhibitory concentration; mic; mutant prevention concentration; mpc; animals; salmonella; pasteurella. introduction the growing problem of antibiotic resistance (abr) is of global concern, with many multidrug resistant bacteria now listed in human medicine. this issue is becoming increasingly relevant in veterinary medicine also, with the risk of resistance genes being transferred between pathogens of humans and animals through various routes and the increasing limitations on antibiotic use in animals, especially food-producing animals. various research projects have focused on the role and perspective of the veterinarian in the fight against resistance to antibiotics and its responsible use, including the methods to determine antibiotic selection and dosage (fortané 2019; martinez et al. 2014; trek diagnostic systems 2005). it must be borne in mind that resistance to antibiotics while not occurring in every instance of their use can drive resistance when treatments are not fully effective in eliminating bacterial infections (gebru et al. 2011; jaganath, schaaf & donald 2017). alternative tests to minimum inhibitory concentration (mic) determinations and ongoing improvements to determine the most effective dose to treat animals are essential (martinez et al. 2014). research has shown that the mutant prevention concentration (mpc) addresses the limitations of mics in situations such as persistent bacterial infections, where standard dosing is ineffective. this would therefore benefit veterinarians involved in regular antibiotic use situations, such as herd management plans in intensive animal rearing systems. (firsov et al. 2013). the mic testing is currently the most commonly used method in diagnostic laboratories to determine the resistance of bacteria to certain antibiotics. the mic method determines the lowest concentration (in µg/ml) of an antibiotic that inhibits the growth of a given strain of bacteria and shows the interaction between the drug and the pathogen (martinez et al. 2014). minimum inhibitory concentrations can, however, prove ineffective where there is a high rate of mutations in specific organisms such as tuberculosis (jaganath et al. 2017). with the mpc method, a higher inoculum size (108 coli forming unit (cfu)/ml) is used to block the growth of the least susceptible bacteria present (coyner 2012). the mpc values are defined as the antibiotic concentration at which 100% eradication of isolates occurs. the usefulness of mpc lies in the application to calculate the potency of antibiotics along with the comparison to determine the efficacy of different antibiotics against single-step resistant mutants, noting the incidence of resistant mutants (rodrı́guez et al. 2004). when selecting antibiotic type and dose, the use of the mpc method will assist in reducing the bacterial load and will also prevent selective amplification of resistant populations more specifically than mic determinations (coyner 2012). the application of mpc values can contribute to a reduction in bacterial resistance, improve therapeutic outcomes and assist responsible use of antibiotics (gebru et al. 2011). with the increasing importance of resistant bacteria and preserving certain antibiotics for their treatment, veterinarians are under pressure to use antibiotics responsibly, especially in food-producing species. in intensive farming situations with large cohorts of a single species repeatedly being reared in the same environment, careful and targeted antibiotic use is crucial to prevent the emergence and persistence of resistant bacteria. evidence such as that provided by mpcs can be used by veterinarians to make long-term bacterial disease management plans and help educate farmers regarding the importance of responsible use of antibiotics. the aim of this study was to illustrate mpc determination as a complementary and sometimes preferable alternative to mic determination for veterinarians when managing bacterial pathogens. the test results can contribute to the database of mpc values for application in the treatment of livestock. materials and methods sampling and storage isolates of salmonella typhimurium and pasteurella multocida from specimens obtained from the department of veterinary tropical diseases, university of pretoria, idexx laboratories, disease control africa, stellenbosch provincial veterinary laboratory, pathcare veterinary laboratories and vetdiagnostix were all confirmed and bio-banked on beads (cryobank®, thermo fischer) at –70 ºc until it could be processed for mic and mpc tests (wentzel 2013) (see table 1). table 1: demographics on the source for each isolate. biochemical identification of isolates isolate confirmation of either p. multocida or s. typhimurium was done with biochemical assays (wentzel 2013) (see table 2) (songer & post 2005; quinn, carter & carter 1994) or the vitek® system (supplied by biomerieux, vitek 2xl, france). table 2: assays for salmonella typhimurium and pasteurella multocida isolation and confirmation. antibiotic susceptibility methods minimum inhibition concentration the mic method was done in 96-well microplates (trek diagnostic systems 2005). this quantitative method used breakpoint values to categorise an organism as either a sensitive or a resistant category (blondeau et al. 2007). the mic plate preparation was as described in the clinical laboratory standards institute (clsi) m31-28 guidelines (watts et al. 2008). the mic broth microdilution method was done as per manufacturer instructions of the commercially produced equine (equi) and bopof (bovine and porcine specific formulary containing fda approved food animal compounds) sensititre® mic plate (sensititre plates®, trek diagnostics, united kingdom) (trek diagnostic systems 2005). the antibiotic dilution ranges were oxytetracycline/florfenicol at 0.5 µg/ml – 8.0 µg/ml on the bopof mic plate and florfenicol at 0.25 µg/ml – 8.0 µg/ml and 0.25 µg/ml – 2.0 µg/ml enrofloxacin (trek diagnostic systems 2005). lysed horse blood was added to the bopof plates to improve the visual readings of the p. multocida reactions. the mic dilution of s. typhimurium was determined using equi plates. all samples were tested in duplicate. mutant prevention concentration the mpc method as described by blondeau (2009a) was used to determine the mpc values for the s. typhimurium isolates (see figure 1). the mpc detection method utilised agar plates with different concentrations of antibiotic drugs to each plate (i.e. agar dilution method) (blondeau 2009b), enabling testing one isolate against various antibiotic concentrations in the same time frame. it differs from mic in that mic tests are done at 105 cfu/ml bacterial concentrations, whereas the mpc determination is done at a bacterial concentration of 109 cfu/ml (blondeau 2009a). figure 1: adapted mutant prevention concentration method plate – with c being the control, the other each section an antibiotic dilution. enrofloxacin and oxytetracycline stock solution were prepared in water, while florfenicol was prepared within methanol and stored in a refrigerator (wentzel 2013). double serial dilutions of each stock solution were made, starting each dilution at the lowest mic value obtained (wentzel 2013). the working concentration of each antibiotic (enrofloxacin-fluka, 17849, oxytetracycline-sigma, 04638 and florfenicol-sigma, f1427) was calculated and used at different concentrations within the mueller hinton (mh) agar (oxoid cm 0337) (wentzel 2013). todd hewitt broth (oxoid, cm 0189) was used as enrichment to culture the p. multocida isolates replacing mh broth (quinn et al. 1994). besides this exception, the method was used as per literature (blondeau 2009a). the concentration of each isolate was measured against mcfarland no. 9 standard (biomerieux, france) with a spectrophotometer (densicheck®, biomerieux) to ensure the density was 109 cfu/ml (wentzel 2013). plates were examined after 24 h of incubation at 37 °c for growth with the highest antibiotic concentration with no bacterial growth being the mpc value (µg per ml) (blondeau et al. 2007). on each plate one quarter was left inoculated with no antibiotic dilution added, this quarter acted as control (see figure 1). efficacy determination calculations calculated pharmacodynamic/pharmacokinetic values were determined as an indication of bacterial inhibition and effective treatment for each antibiotic (blondeau et al. 2004). the effective treatment of each antibiotic was determined using the formula area under the curve (auc)/mic, with a desired ratio of > 125 for gram-negative and 30–50 for gram-positive organisms for optimal efficacy (hesje, tillotson & blondeau 2007). each antibiotic’s bacterial inhibition was determined with the formula of cmax (drug concentration)/mic and auc/mic (blondeau et al. 2004). these calculations were made using the drug concentrations in the index of veterinary specialities (ivs) (swan 2005) compared to the data obtained for this study. ethical considerations approval to conduct the study was received from the university of pretoria, onderstepoort faculty of veterinary science (v063/11). results the enrofloxacin mic values of 27 isolates of s. typhimurium were all 0.25 µg/ml, while all mpc values was 0.5 µg/ml, with the exception of five strains being 4.0 µg/ml (wentzel 2013). the mpc test yielded 18 (65.52%) isolates sensitive to florfenicol, while 11 (34.48%) isolates were resistant to florfenicol. the mic/mpc ratio of six isolates was either similar or varied by only one dilution (wentzel 2013). seventeen (58.62%) isolates of p. multocida had susceptible mic values and 12 (41.38%) isolates had an intermediate value, while 16 (55.17%) of the isolates yielded a resistant mpc value to oxytetracycline while five isolates had an mic/mpc ratio of 0 (wentzel 2013) (see tables 3 and 4). table 3: minimum inhibitory concentration and mutant prevention concentration results obtained during current study. table 4: combined summaries of the pharmacodynamic/pharmacokinetic data for the results obtained for pasteurella multocida and salmonella typhimurium using reference values from previous research. discussion general the clinical breakpoints published in the clsi guideline refer to the pharmacodynamic and pharmacokinetic attributes of isolates (boothe 2006). each clinical breakpoint with respect to the mic is useful to treat clinical infections but is different from the epidemiological cut-off value that is often lower than the clinical breakpoint. minimum inhibition concentrations results are divided into three groups being (1) sensitive, (2) intermediate or (3) resistant (silley, bywater & simjee 2006). the susceptibility breakpoint of enrofloxacin for animal pathogens is ≤ 0.5 µg/ml, and the resistance breakpoint is ≥ 4.0 µg/ml (boothe 2006). none of the mic values in the current study were resistant. the susceptibility breakpoint of florfenicol for animal pathogens is ≤ 2.0 µg/ml, and the resistance breakpoint is ≥ 8.0 µg/ml (boothe 2006). none of the mic values for florfenicol were categorised as resistant during the study. the breakpoint for resistance of oxytetracycline for animal pathogens is ≥ 16 µg/ml, while the susceptibility breakpoint is ≤ 4 µg/ml (boothe 2006), 12 of the mic values in the current study were intermediate and remaining susceptible (wentzel 2013). these breakpoints were used as the reference range in this study. the closer the obtained value to the breakpoint for resistance, the higher the chance of treatment contributing to the development of resistance to the specific antibiotic (silley et al. 2006). the mic and mpc values were used to calculate the pharmacokinetic/pharmacodynamic (pk/pd) parameters. the mpc values in the pd/pk parameter calculation were unknown at the time of the study, and this requires further research (wentzel 2013). the infection site and dose influence the pk/pd parameters such as auc (a measure of the total amount of antibiotic drug present over a specific time interval), t > mic and cmax (hesje et al. 2007). therefore, this study used the values obtained from previously documented studies because it supported the results of the mic and mpc tests. previous research indicates that the cmax/mic must have a value of 8–12, to be clinically eff ective and to reduce the development of resistance (hesje et al. 2007). the auc/mic should be > 125 to have a positive clinical response and minimise abr from developing. the auc/mpc50 calculation had a value of ≥ 22, being gram-negative organism, indicating that this treatment can reduce the development of resistance (hesje et al. 2007). all of the s. typhimurium isolates originated from clinical cases. no official clinical breakpoints exist for enrofloxacin use in animals against s. typhimurium; therefore the clinical human breakpoints for s. typhimurium and enrofloxacin were used as a guideline in the interpretation of the results. the enrofloxacin clinical reference range is 0.5 µg/ml – 4.0 µg/ml (watts et al. 2008). the mic50 value of enrofloxacin during this study for s. typhimurium was 0.25 µg/ml. this suggests that treating the horses with enrofloxacin was likely adequate when veterinarians use it off label. the few results obtained from this study indicate that enrofloxacin use has not been abused by the equine industry of south africa to date. the mpc testing measured the mic with the most resistant sub population (gianvecchio et al. 2019) so the s. typhimurium isolates with a low mpc50 value for enrofloxacin showed the efficacy of the antibiotic against the bacteria. the enrofloxacin use even off label is common in horses; the veterinarians usually treat a horse using similar doses as cattle (boeckh et al. 2001). the results obtained from the s. typhimurium isolates were confirmed with results of previous studies. studies showed mpc50 values with a four-fold increase from the mic50. during this study, the mpc90 concentration was 4 µg/ml, thus a 16-fold increase from the mic90. the mpc values above the mpc50 will block both susceptible and mutant bacterial growth; alternatively this can be an indication of second-step mutations (blondeau & fitch 2019). it is important to know that the mpc will block only the least susceptible bacteria and that it is independent of the mechanism of resistance (blondeau et al. 2001). amongst the s. typhimurium isolates, there were five strains with mpc values above the mpc50 value (wentzel 2013). the mutant selection window (msw) shows the correlation between the mic50 and mpc50 values and indicates the effectiveness of the treatment/dosing. this is the concentration where the selective amplification of the organism occurs and where resistant populations can develop (drlica 2003). additionally, time-dependent antibiotics that stay within the msw such as oxytetracycline promote the chances of resistance (drlica 2003). twenty-two (81.48%) of the s. typhimurium isolates treated with enrofloxacin yielded results similar to the mic50 and mpc50 values (wentzel 2013). isolates included samples from surveillance programmes (44.82%) and clinical cases (55.17%) for the testing of florfenicol and oxytetracycline against p. multocida, all isolates had mic value that was sensitive to florfenicol (wentzel 2013). the clinical reference range for florfenicol against p. multocida infections is 2 µg/ml – 8 µg/ml. eleven of the isolates had mic values below the mic50 and eight were suspected (mic50 of 0.5 µg/ml). the mic of florfenicol for p. multocida was within the range when using either the mic50 (0.50 µg/ml) or mic90 (< 2.00 µg/ml) as calculated in this study (wentzel 2013). therefore, the treatment of these animals with standard doses of florfenicol suffering from infections with these isolates will be within the therapeutic reference range of the antibiotic. during the current study, the mean mic concentration of florfenicol for p. multocida was slightly higher at 0.50 µg/ml, while the hörmansdorfer and bauer (1998) study found the mic values for p. multocida as 0.47 µg/ml for cattle and 0.51 µg/ml for pig strains (hörmansdorfer & bauer 1998). ten p. multocida isolates had an mic50 value of 2.00 µg/ml and an mic90 of 4.00 µg/ml for florfenicol, with an mic50 for oxytetracycline of 0.25 µg/ml and the mic90 of 32.00 µg/ml (sweeney, brumbaugh & watts 2008). the clinical therapeutic reference range of oxytetracycline for p. multocida is 4 µg/ml – 16 µg/ml (blondeau & fitch 2019). the mic50 value of 2 µg/ml is below the clinical reference range of the antibiotic; 16 isolates (55.17% of samples tested) had mic values below the clinical breakpoint. therefore, no resistance was present within these isolates. a single isolate had an mic value of 4 µg/ml, which is below the reference range, six (20.69% of the samples) of the isolates had an mic90 of 8 µg/ml, while six isolates had an mic > 8 µg/ml. these results were expected since oxytetracycline is the most commonly used antibiotic drug in cattle in south africa (van et al. 2020). these mic90 values are above the clinical breakpoint for resistance, indicating that the treatment of these animals will normally be unsuccessful. previous research with bovine respiratory disease-causing organisms in cattle had mic90 values of florfenicol and oxytetracycline against p. multocida of 0.5 µg/ml and 1.0 µg/ml, respectively (giguere & tessman 2011). the mic90 values in the current study were 2 µg/ml and > 8 µg/ml, respectively, both these values are much higher than the reference range as per clsi guidelines. the mpc results of the study compared the results obtained with the clinical reference range representing an mpc50 of 2 µg/ml and an mpc90 of > 32 µg/ml for florfenicol (blondeau & fitch 2019). the mpc50 results against p. multocida were < 2 µg/ml for 16 (62.07% of isolates); these are below the clinical breakpoint for florfenicol. there were 11 isolates of p. multocida with mpc values above the clinical reference range of florfenicol. the mpc90 concentration represents an alternative to the mic50 values in this study and using higher dosages to exceed the mpc90 will theoretically be a more effective treatment regimen to minimise resistance development (blondeau et al. 2007). prior to treatment with these higher concentrations (mpc90 values), the treatment must be proven safe, as it can be toxic depending on the antibiotic used. the results for oxytetracycline against p. multocida showed an mpc50 value of 16 µg/ml for 16 (55.17% of the tested) isolates. treating animals to reach an mpc50 value of 16 µg/ml will be within the clinical reference range of the organism. in this study, both mpc50 and mpc90 values were 16 µg/ml. none of the p. multocida isolates exposed to oxytetracycline had mpc values above the mpc50. this creates the need for susceptibility methods such as mpc, which can determine drug concentrations that will kill first-step mutants. the safety of this concentration should be determined first before used for therapy. during the current study, p. multocida had two isolates with similar mic50 and mpc50 values for oxytetracycline, and a single isolate had mic and mpc values within the msw. none of the p. multocida isolates exposed to florfenicol fell between the mic50 and mpc50 values. the closer the mic:mpc ratio is to each other, the higher the suitability of the antibiotic (zhao & drlica 2001). the mic50 and mpc50 ratios for enrofloxacin against s. typhimurium was 0.25:< 0.50 and 0.50:< 2.00 for florfenicol against p. multocida, as such the dosages used will be suitable for treatment. the mic50:mpc50 ratio of 2:> 16 for oxytetracycline against p. multocida as such indicating that treatment at much higher dosages may be needed that might lead to toxicity at the required effective concentration. the mic50:mpc50 ratio in this study is similar to the clinical reference range for oxytetracycline. the higher mpc values than the mic values were expected. the mic90:mpc90 ratio for enrofloxacin against s. typhimirium was < 0.25:4.00, a 16-fold difference. the mic90:mpc90 ratio for florfenicol against p. multocida was 2.00:> 32.00, a 16-fold difference, and the mic90:mpc90 ratio of > 8:6 for oxytetracycline represents a twofold difference. enrofloxacin is not registered for use in horses, in south africa; thus the pk/pd parameters could not be calculated (swan 2005). pasteurella multocida isolates responded to florfenicol and were measured with the pd/pk parameters. the auc/mic value of 283.56 and an auc/mic90 value of 70.89 indicated that the treatment will be effective to ensure a positive clinical response; unfortunately the auc/mic value of 56 for p. multocida isolates exposed to oxytetracycline indicated that treatment would be unsuccessful in these animals (wentzel 2013). the cmax/mic result showed that florfenicol at the mic50 will minimise resistance with a value of 9.38. the oxytetracycline mic50 concentration will not prevent resistance in the p. multocida organisms with a value of 2.85 (wentzel 2013). limitations included the initial visual reading of the mic results; however, after consultation with trek, adding lysed horse blood to the mh broth before adding the inoculum to the 96-well plates made the reading of the results much easier. the mic has published known errors with the reading of mic results, and these include fading end-points (no distinct end-points) or skips (a well with no growth, between wells that have growth) (trek diagnostic systems 2005). these samples were retested in duplicate to confirm the results during this study. conclusion distinctions could be made between the mic50, mic90 and the mpc50, mpc90 for each antibiotic. applying both methods can be useful for the treatment of highly resistant bacteria and should be investigated further to be more readily available to practitioners. the laboratory interprets the mic results and provides the information to the practitioner and represents a potentially less toxic and cheaper dosing strategy than mpc. antibiotic susceptibility testing by means of mic determinations as done in this study is used for the effective antibiotic treatment of bacterial infections and minimising the development of resistance. the mpc method can be used to better control to prevent the development of antibiotic drug resistance used in animals. acknowledgements this article forms part of the published thesis of j.m.w. submitted in partial fulfilment of the requirements for the degree of magister scientiae (veterinary tropical diseases) in the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, south africa, entitled ‘a comparative study of the minimum inhibitory and mutant prevention concentrations of florfenicol and oxytetracycline for animal isolates of pasteurella multocida and salmonella typhimurium’. supervisor: m.v.v., 2012. refer to link: https://repository.up.ac.za/handle/2263/26219. the authors also wish to thank dr. helena rampf for assisting with the article editing and english edits as well as dr. john frean for assisting with writing and editing on the article. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.v.v. supervised the research and assisted with planning and test development, funding and writing of the article. l.j.b. was the production animal specialist and antibiotics (field specialist) and assisted with the applicability of results, writing of the article. j.m.w. was the student and was responsible for testing, results and writing of the article. funding information the authors received no financial support for the research, authorship and/or publication of this article. data availability the raw data is available on the university of pretoria repository as part of the m. thesis at https://repository.up.ac.za/handle/2263/26219. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors, and the publisher. references blondeau, j.m., 2009a, ‘new concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach’, veterinary dermatology 20(5–6), 383–396. https://doi.org/10.1111/j.1365-3164.2009.00856.x blondeau, j.m., 2009b, stat international companion animal: steps to antimicrobial therapy, north american 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ruminants characterized by acute heart failure without the development of premonitory signs 4–8 weeks after the ingestion of certain rubiaceous plants. grossly, extra-cardiac signs of heart failure are seen in many, but not all, cases. a diagnosis of gousiekte is traditionally confirmed by the presence of appropriate microscopical lesions in the myocardium, namely loss of myofibres, replacement fibrosis and lymphocytic infiltrates of varying intensity, especially in the subendocardial zone of the apex and left ventricular free wall (newsholme & coetzer 1984; kellerman, coetzer & naudé 1988). smit (1959) noted that some naturally poisoned animals showed more acute changes than those described above, with degeneration of myofibres as the principle lesion. marked deviations from the “typical” lesions have also been reported in certain experimental cases. hurter, naudé, adelaar, smit & codd (1972) described fairly severe focal degeneration of myocardial fibres, accompanied by congestion, oedema and small haemorrhages. they attributed these more acute lesions to the high dosage of plant material administered to the animals. furthermore, deaths of animals without 219 onderstepoort journal of veterinary research, 72:219–230 (2005) a study of the pathology and pathogenesis of the myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants l. prozesky1, s.s. bastianello2, n. fourie3 and r.a. schultz4 abstract prozesky, l., bastianello, s.s., fourie, n. & schultz, r.a. 2005. a study of the pathology and pathogenesis of the myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants. onderstepoort journal of veterinary research, 72:219–230 myocardial lesions were studied in sheep in which gousiekte was induced by experimental dosage of pachystigma pygmaeum, fadogia homblei or pavetta harborii. the single most consistent diagnostic histological feature in 33 animals was hypertrophy of myocardial fibres in the subendocardial region. fibrosis in the subendocardial region of the apex or left ventricular wall was often scarce or absent in animals with a short latent period, and was not always prominent even in sheep with an intermediate or long latent period. the presence or absence of fibrosis cannot therefore be used to confirm or exclude gousiekte, particularly in cases with shorter latent periods. light microscopical and ultrastructural lesions in sheep with gousiekte correspond to a large extent to changes reported in humans with dilated cardiomyopathy of unknown cause. it appears that the myocardial lesions in gousiekte represent a final common pathway of cellular damage rather than a manifestation of a specific type of heart disease. the predilection for hypertrophy of myofibres in the subendocardial region is probably related to diminished perfusion that potentiates the primary myocardial dysfunction. keywords: cardiotoxicosis, dilated cardiomyopathy, fadogia homblei, gousiekte, pachystigma pygmaeum, pathogenesis, pathology, pavetta harborii, ruminants, south africa 1 department of paraclinical sciences, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: leon.prozesky@up.ac.za 2 gribbles vetlab, 33 flemington street, glenside, sa 5065, australia 3 intervet, private bag x2026, isando, 1600 south africa 4 division of toxicology. onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa accepted for publication 20 april 2005—editor notable lesions were reported in dosing trials by adelaar, terblanche & smit (1966) and anderson & naudé (onderstepoort veterinary institute, unpublished data 1959–1962) and were erroneously deemed negative for gousiekte. since a diagnosis of the disease can be confirmed only by histopathological examination, it is imperative to describe the full spectrum of lesions of gousiekte so that even “atypical” disease can be diagnosed accurately. the need for accurate diagnosis of gousiekte is especially pressing because the toxicity of chemical fractions of the plants can be estimated only by dosing them to ruminants. if the unusual lesions seen occasionally under experimental conditions cannot be identified as caused by gousiekte, the danger exists that toxic fractions could be discarded unwittingly. there appears to be a close similarity in the pattern of myocardial fibrosis in animals that die of gousiekte and that in humans with dilated cardiomyopathy. the latter is regarded as a syndrome in which a variety of aetiological factors such as viral infections, toxic agents, chronic alcohol abuse and genetic factors have been implicated (weekes, wheeler, yan, weil, eschenhagen, scholtysik & dunn 1999). unverferth (1985) identified fibrosis of increasing severity from epito endocardium in patients with dilated cardiomyopathy. the aim of this study was, firstly, to investigate the effect of duration of latency on the nature of the myocardial histopathological lesions in gousiekte and, secondly, to characterize lesion patterns in animals with short, medium (intermediate) and long latent periods respectively, latent period being defined as the elapsed time between first exposure of the animal to toxic plant material and death of the animal. the need to explain mortality of sheep without the development of “typical” gousiekte lesions was a further factor that prompted the study. materials and methods series 1 thirty-three sheep of different sexes and ages were dosed separately in unrelated trials with varying amounts of the rubiaceous plant, pachystigma pygmaeum, fadogia homblei, pavetta harborii or an extract of p. harborii (table 1). the trials from which the data were extracted were carried out over several years and are described in detail by fourie (1994) and fourie, schultz, prozesky, kellerman & labuschagne (1989). all 33 animals either died or were slaughtered in extremis between 27 and 57 days after the commencement of dosing. at necropsy, samples of various organs and two to three tissue samples from the left free ventricular wall were collected in 10 % buffered formalin. they were routinely processed for histopathological examination and sections were stained with haematoxylin and eosin (he) and masson’s trichrome stain for collagen (anon. 1968). series 2 specimens were collected from the free left ventricular wall from seven of the 33 sheep in series 1 and from two others (table 2) for transmission electron microscopy. cubes (0.5–1.0 mm) were cut and fixed in 2.5 % glutaraldehyde (ph 7.2–7.4) for 24 h. selected blocks were post-fixed in 2 % osmium tetroxide for 1 h, dehydrated in a graded ethanol series (50–100 %), passed through propylene oxide as the intermediate solvent, and embedded in embed 812. thick (1–2 micron) sections were cut for tissue orientation and stained with toluidine blue. thin sections from selected blocks were stained at room temperature for 20 min in a saturated aqueous solution of uranyl acetate, rinsed and post-stained for 3 min in reynold’s lead citrate. results gross pathology sheep in series 1 were divided into three groups on the basis of data from field and experimental cases on the length of the latent period. sheep with a short latent period (< 35 days) were placed in group a, those with an intermediate period (35–41 days) in group b, and those with a long period (> 41 days) in group c (table 3). pulmonary oedema was present in 24 (73 %) of the sheep, hydropericardium in 20 (61 %) and hydrothorax in 12 (36 %) (fig. 1 and 2). ascites was evident in three (9 %) cases and generalised congestion in five (15 %). seven of the eight cases of ascites and/or generalised congestion occurred in sheep with a long latent period. other lesions noted were mild and included nephrosis, hepatomegaly, splenomegaly and oedema of the mediastinum, mesenterium, abomasum and the wall of the gall bladder. in 22 (66 %) sheep, one or more of three cardiac lesions, namely dilatation (subjectively evaluated) (fig. 3), subendocardial fibrosis (pallor) or myocardial mottling, were present. seventeen sheep (51 %) exhibited dilatation or subendocardial pallor (fibrosis) and only 5 (15 %) myocardial mottling. 220 myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants 221 l. prozesky et al. table 1 sheep examined in series 1 after dosing with different gousiekte plants sheep no. sexa ageb initial live dried material dosing total dose last duration of mass dosedc regimen (kg) dosing experiment (kg) (g/kgxday) (day) (day) 1-4172 w l 22 p. pygmaeum 13.6 x 20 6.30 26 27 2-6713 w l 32 p. harborii ex 0.57 x 14 8.00 17 27 3-5077 w 4t 29 p. pygmaeum 10.3 x 31 9.30 30 31 4-4982 w l 31 p. pygmaeum 10 x 23 7.13 30 31 5-3874 e l 22 p. pygmaeum 10 x 30 6.60 34 34 6-6248 r 2t 38 f. homblei 10 x 22 8.36 29 34 7-5231 w a 60 p. harborii 10 x 20 12.00 19 34 8-3885 e l 27 p. pygmaeum 10 x 30 8.10 34 35 9-3727 w 2t 35 p. pygmaeum 15 x 6 9.12 34 35 10-5217 w a 53 p. harborii 10 x 35 18.55 34 35 11-3899 e l 25 p. pygmaeum 10 x 30 7.50 34 36 12-3890 e l 33 p. pygmaeum 10 x 30 9.90 34 38 13-6346 r l 36 f. homblei 10 x 22 7.92 29 40 14-5051 w 4t 29 p. pygmaeum 10.3 x 31 9.30 30 42 15-3904 e l 27 p. pygmaeum 10 x 30 8.10 34 42 16-4977 w l 35 p. pygmaeum 10 x 30 10.50 39 42 17-6317 e 4t 39 f. homblei 10 x 22 8.58 29 42 18-4983 w l 31 p. pygmaeum 10 x 31 9.61 42 43 19-5109 e 4t 34 f. homblei 10 x 40 13.60 40 44 20-5068 w 2t 24 p. pygmaeum 12.2 x 25 7.50 25 45 21-6375 e 2t 34 f. homblei 10 x 22 7.48 29 45 22-6363 e 4t 40 f. homblei 10 x 22 8.80 29 45 23-4915 r l 41 p. pygmaeum 10 x 19 7.79 24 45 24-5256 w a 59 p. harborii 10 x 20 11.80 27 50 25-4984 w l 25 p. pygmaeum 10 x 31 7.75 42 51 26-4979 w l 28 p. pygmaeum 10 x 31 8.68 42 51 27-6232 w 4t 31 p. pygmaeum 10 x 34 10.54 44 51 28-5244 w a 56 p. harborii 10 x 34 19.04 45 54 29-3708 r l 59 p. harborii 10 x 32 18.88 44 54 30-6258 r l 36 f. homblei 10 x 22 7.92 29 57 31-6314 e 2t 34 f. homblei 10 x 22 7.48 29 57 32-6364 e 4t 34 f. homblei 10 x 22 7.48 29 57 33-6386 e 4t 32 f. homblei 10 x 22 7.04 29 57 a r = ram e = ewe w = wether b a = adult 2t = 2 tooth 4t = 4 tooth l = lamb c ex = extract 1 2 fig. 1 hydropericardium in a sheep that died after a long latent period fig. 2 note the lung oedema and hydrothorax 222 myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants table 2 sheep examined in series 2 after dosing with different gousiekte plants sheep no. initial dried material period dosed dose total dose duration of live mass dosed (g/kg x day) (kg) experiment (days) (kg) (days) and fate 1 26.5 p. pygmaeum 34 10 x 30 7.95 36 killed in extremis 2 25 p. pygmaeum 34 10 x 30 7.50 36 killed in extremis 3 38 f. homblei 30 10 x 22 8.36 34 died 4 36 f. homblei 29 10 x 22 7.92 40 killed in extremis 5 34 f. homblei 29 10 x 22 7.48 57 died 6 32 f. homblei 29 10 x 22 7.04 57 died 7 34 f. homblei 29 10 x 22 7.48 57 died 8 36 f. homblei 30 10 x 22 7.92 57 killed in extremis 9 27 p. pygmaeum 24 10 x 19 8.32 72 10 10 x 7 killed in extremis 8 8 x 6 table 3 macroscopic pathological features in 33 sheep in series 1 number of animals affected macroscopic pathological feature group aa group bb group cc total percentage (n = 7) (n = 6) (n = 20) (n = 33) per feature pulmonary oedema 4 5 15 24 73 hydropericardium 5 5 10 20 61 hydrothorax 2 2 8 12 36 generalized congestion 1 0 4 5 15 ascites 0 0 3 3 9 cardiac dilatation 1 1 7 9 27 heart left ventricular subendo0 0 8 8 24 cardial pallor (fibrosis) myocardial mottling 1 0 4 5 15 a short latent period < 35 days b intermediate latent period 35–41 days c long latent period > 41 days histopathology the pertinent histopathological features in the left ventricle of sheep dosed with gousiekte plants are tabulated in table 4. the lesions were located primarily in the subendocardial zone, the four most characteristic features being hypertrophy or atrophy of fibres, focal necrosis and foci of replacement fibrosis (fig. 4–6). amongst the other changes noted were foci of round cell infiltration (fig. 7); focal to diffuse endocardial thickening; pallor and vacuolation of the purkinje fibres; and myocardial arterial or arteriolar lesions, characterized by medial oedema and hypertrophy accompanied by perivascular accumulation of unidentified cells. focal to diffuse areas of hypertrophy in the inner third of the myocardium were recorded in all the sheep. hypertrophy was mainly mild in nature and multifocal in distribution in those with short latent periods (< 5 weeks) and diffuse, moderate or moderately severe in those with intermediate (5–6 weeks) or long latent periods (> 6 weeks). foci of replacement fibrosis were present in 30 (91 %) of the sheep. sheep with short latent periods generally had small, indistinct, scattered foci of fibrosis. in 4 sheep with long latent periods, the fibrosis was extensive and in 15 animals varied from moderate to severe. in sheep with intermediate latent periods, the extent of fibrosis was variable. focal areas of coagulative necrosis were evident in 26 (79 %) sheep. vacuolated fibres with perinuclear myocytolysis were often evident closely associated with coagulated fibres. in sheep with short latent periods, the foci were usually small and distributed throughout the left ventricular wall. in sheep with longer latent periods, the foci were scattered in the midand subepicardial zones or occurred in close proximity to the fibrotic areas in the subendocardial zone. in sheep with intermediate latent periods necrotic foci were numerous. atrophy of myocardial fibres was present in 19 (58 %) sheep and was generally focal, involving individual or small groups of fibres. in four sheep with long latent periods, extensive tracts of atrophic fibres were present in the subendocardial zone. in two sheep having short latent periods, atrophy was evident diffusely throughout the myocardium. foci of round cell infiltration were recorded in all the sheep. the foci were generally small, contained few cells and were composed mainly of small lympho223 l. prozesky et al. 3 4 5 6 fig. 3 enlargement of the heart due to dilation of both ventricles fig. 4 fibre hypertrophy with nuclear hyperplasia (arrow). he, x 200 fig. 5 diffuse atrophy of fibres throughout the myocardium in a sheep with a short latent period. he, x 100 fig. 6 multifocal necrosis (arrow) with replacement fibrosis (star). masson’s trichrome x100 cytes, except for three sheep with long latent periods, where the foci were prominent and contained moderate to large numbers of cells. the foci were widely distributed throughout the interstitium, especially perivascularly, in all cases. in the sheep with the long latent periods, the majority of foci were found closely associated with the areas of fibrosis and necrosis in the subendocardial zone. focal to diffuse, mild to moderate thickening of the endocardium was evident in 27 (82 %) cases. in sheep with short or intermediate latent periods, it was usually mild and either focal or diffuse in nature. in contrast, in sheep with a long latent period, all but two exhibited diffuse, moderate to severe thickening of the endocardium. in 26 (79 %) sheep the purkinje fibres were pale staining, and many contained single or multiple, indistinct to prominent vacuoles. examination of the myocardial arteries and arterioles disclosed vacuolation of the tunica media and a perivascular accumulation of cells of an indeterminate origin in 20 (61 %) cases. hypertrophy of the tunica media of these vessels was evident in 23 (70 %) sheep. the hypertrophy was usually mild and in only four cases was it prominent. transmission electron microscopy a common finding in degenerative myocardial cells was myofibrillar loss that varied significantly in degree from cell to cell within the same animal. single or multiple sarcomeres within a myofibril were affected. mild myofibril loss was characterized by widening of the perinuclear myofibrillar free zone, whereas more advanced loss resulted in large myofibril-free areas of cytoplasm in the central part of the myofibre, with a few intact peripheral myofibrils below the sarco224 myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants table 4 histopathological lesions in the hearts of 33 sheep in series 1 histopathological features group of animals total percentage features distribution aa bb cc per feature n = 7 n = 6 n = 20 n = 33 myofibre nuclear hypertrophyd diffuse 7 6 20 33 100 myofibre hypertrophy in focal 5 1 0 33 100 subendocardial myocardiume diffuse 2 5 20 focal disorganization of myofibres in – 7 5 19 31 94 subendocardial myocardiumf replacement fibrosis scattered foci 6 5 15 30 91 extensive tracts 0 0 4 focal areas of coagulative/ necrosis/ scattered foci 6 6 14 26 79 myocytolysis of fibres myofibre atrophy focal 4 3 10 19 58 diffuse 2 0 0 foci of round cell infiltration scattered foci 7 7 20 33 100 endocardial thickening focal 2 1 0 27 82 diffuse 4 2 18 purkinje fibre pallor and/or – 7 4 15 26 79 vacuolation medial oedema/ – 3 4 13 20 61 myocardial perivascular cellular vessels infiltration medial hypertrophy – 4 5 14 23 70 a short latent period < 35 days b intermediate latent period 35–41 days c long latent period > 41 days d, e, f all represent features of myocardial hypertrophy lemma. degenerative fibrils had a frayed appearance with a preferential loss of thin (actin) filaments (fig. 8 and 9). a common finding in degenerative cells was the presence of fine tangled (interwoven) fibrillar masses representing disintegrated myofilaments, often intermingled with cellular organelles (fig. 10 and 11). myocardial cells with advanced myofibrillar loss were atrophic (fig. 12), with a decreased cell diameter, whereas cells with a moderate degree of myofibrillar loss maintained a normal cell diameter as a result of the proliferation of other cytoplasmic components, particularly mitochondria. scattered z bands were irregularly thickened and often fragmented, with streaking of the affected z band material into the surrounding myofibrillar tissue and cytoplasm (fig. 12). cells with more advanced degenerative changes were characterized by the selective proliferation of certain organelles, namely mitochondria (fig. 13 and 14) and sarcoplasmic reticulum (fig. 15), and the deposition of material in areas previously occupied by myofibrils. this material included glycogen, homogenous residual bodies (suspected lipid), electron dense bodies, myelin figures of unknown origin, or a fine fibrillar matrix (lysed myofilaments). mitochondria varied considerably in size and shape and showed various alterations including pyknosis, the formation of concentric cristae and an increase in the size, number and density of dense granules. rupture of swollen cristae was frequently noted and varied in extent from lysis of a few cristae to complete loss of cristae, resulting in an empty external mitochondrial membrane or the accumulation of moderately electron dense material replacing the internal structure of the mitochondria. there were indications that damage to mitochondria on occasion resulted in the formation of concentric layers of electron dense membrane material (myelin figures). medium electron dense material, which was enclosed by a single or double membrane or was intermingled with dissociated fibrils and cellular organelles, was occasionally noted in degenerative cells. this was considered a form of intracellular oedema. 225 l. prozesky et al. 9 10 7 8 fig. 7 multifocal small foci of predominantly lymphocytic infiltration (arrow) were present in all the sheep. masson’s trichrome x100 fig. 8 degenerative fibrils had a frayed appearance (star) fig. 9 note disintegrated myofilaments in cross section (arrow) fig. 10 disintegrated myofilaments (arrow) were often intermingled with cellular organelles, e.g. mitochondria. intercalated disc (star) changes in the sarcoplasmic reticulum included dilatation and proliferation. the latter was particularly notable in areas of myofibrillar loss and was often seen in conjunction with mitochondrial proliferation and the accumulation of glycogen deposits. occasional degenerative cells were noted in association with dilated transverse tubules. the most striking intercalated disc abnormality noted was a significant increase in length resulting from the development of complex folds. the folded discs were associated with cells that exhibited a wide spectrum of degenerative changes, particularly disintegration of myofilaments ranging from mild to severe. many of the affected discs exhibited multiple small areas of separation of the two opposing unit membranes (fig. 13). complete separation between cells at the level of the intercalated disc was also noted. the intercellular space between degenerative cells was often distended and contained numerous membrane bound empty spaces that varied in size and shape, ranging from circular to oval or pleomorphic. some of these structures were attached to the sarcolemma and appeared to represent excessive folding of it (fig. 10). similar structures were also present in the subjacent sarcolemmal sarcoplasma. discussion certain gross lesions are suggestive of gousiekte as the cause of death. these include signs of left-sided heart failure, such as pulmonary oedema, hydropericardium and hydrothorax, and specific cardiac lesions, particularly cardiac dilatation and subendocardial fibrosis. the presence of pulmonary oedema, hydropericardium and/or hydrothorax in most of the sheep in this study suggests that gousiekte causes left-sided heart failure. features suggestive of congestive (biventricular) heart failure, such as generalized congestion and/or ascites, were less common. seven 226 myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants 11 12 13 14 fig. 11 cross section of myofibre. note complex folding of intercalated disc (star) fig. 12 myocardial cells with advanced myofibrillar loss were atrophic. note streaking of z-band material (star) and mitochondria proliferation (arrow) fig. 13 disintegrated myofibre. note mitochondria (arrow) and sarcoplasmic reticulum proliferation (arrow head) fig. 14 myofibre with advanced myofibrillar loss due to myofilament disintegration (star). note mitochondria proliferation (arrow) and sarcoplasmic reticulum proliferation (arrow head) of the eight sheep with either ascites or generalized congestion had long latent periods, and the myocardial lesions were widespread, stretching from the apex and left free ventricular wall to the septum and right free ventricular wall. this suggests that congestive heart failure only occurs in cases where the pathological process extends beyond the initial predilection site in the left ventricle. cardiac dilatation, subendocardial fibrosis or myocardial mottling were evident in two thirds of the sheep examined in this study. of the various histopathological lesions recorded, the single most consistent diagnostic feature of gousiekte recorded in this series was hypertrophy of myocardial fibres in the subendocardial region of the left ventricular wall. in cases with a short latent period (< 5 weeks), the diagnosis was based on the presence of mild to moderately hypertrophic fibres in the subendocardial myocardium, with small, scattered foci of necrosis and, occasionally, focal areas of fibrosis. a few of the cases with a short latent period exhibited diffuse myocardial atrophy and focal hypertrophy in the absence of necrosis or fibrosis. cases with a longer latent period (> 5 weeks) were more typical of gousiekte and were characterized by diffuse, moderate to severe, subendocardial myocardial fibre hypertrophy, coalescent focal to extensive fibrosis, and multifocal necrosis. in rare instances, fibrosis was absent even in cases with intermediate or long latent periods. in the past, histopathological confirmation of gousiekte has been based on the presence of moderate to severe coalescent or extensive fibrosis in the subendocardial myocardium of the apex or free wall of the left ventricle (kellerman et al. 1988). however, in six out of seven sheep with short latent periods in this study, fibrosis was lacking or not prominent, and even in a few sheep with an intermediate or long latent period, fibrosis was absent. the presence or absence of fibrosis on its own cannot therefore be used as a single diagnostic criterion for the confirmation or exclusion of gousiekte, particularly in cases with shorter latent periods. focal areas of necrosis were seen in the majority of cases of gousiekte, regardless of whether the latent period was short, intermediate or long. this feature, together with the presence of hypertrophy as discussed above, can be regarded as a significant microscopic lesion for the histopathological diagnosis of gousiekte, especially in cases where fibrosis is minimal and focal in nature or completely absent. myocardial fibre atrophy, although mostly focal in nature and present in only 58 % of cases, should be regarded as one of the diagnostic features of gousiekte. it was especially significant in a few cases with a short latent period, where it occurred diffusely in the myocardium in the absence of multifocal fibrosis or prominent myofibre hypertrophy. in the majority of cases, however, myocardial fibre atrophy was usually focal in nature and involved only individual or small groups of fibres. occasionally diffuse atrophy can be the most striking histological feature in field cases of gousiekte (prozesky, fourie, neser & nel 1988). round cell infiltrates in the myocardium have in the past been regarded as a feature of gousiekte (smit 1959). although present in all cases with long latent periods in this study, these infiltrations were only prominent in three sheep. round cells occurred in small indistinct foci consisting of a few cells in the myocardial interstitium, especially around blood vessels or in association with foci of fibrosis or necrosis. the small number of cells and the association thereof with myocardial necrosis or fibrosis imply that they accumulate in response to necrosis of myofibres. foci of round cell infiltrates may be present in a variety of cardiac conditions and should not be regarded as a specific diagnostic feature of gousiekte. other features that may be present, such as endocardial thickening, pallor and vacuolation of the purkinje fibres, and arterial lesions, which were seen in cases with longer latent periods, were all regarded as secondary. the most striking ultrastructural degenerative lesions noted in this study included loss of myofibrils, involving particularly thin filaments; deposition of glycogen and residual bodies and selective proliferation of organelles, such as mitochondria and sarcoplasmic reticulum, in areas previously occupied by myofibrils; variation in size and shape of mitochondria with increase in size, number and density of dense granules, and swelling and rupture of cristae; increase in length of the intercalated discs and development of complex folding; excessive folding of the sarcolemma; and irregular thickening and fragmentation of z bands. advanced myocardial degeneration was characterized by complete loss of myofibrils with loss of intercellular connections. to a large extent the light microscopical and ultrastructural lesions described in sheep with gousiekte in this study and a previous report (schutte, els, booyens & pienaar 1984) correspond with changes reported in humans with dilated cardiomyopathy of unknown cause (unverferth 1985). unverferth (1985) identified fibrosis of increasing severity extending 227 l. prozesky et al. from the epito the endocardium in patients with dilated cardiomyopathy. in his study fibrosis was more prominent in the left side of the interventricular septum compared to the right. the fibrosis in the subendocardium of the left ventricular free wall was identical to that in the subendocardium on the left side of the interventricular septum, whereas the right ventricular free wall did not demonstrate a gradation in the degree of fibrosis across the wall . a predilection for subendocardial fibrosis may be attributed to the following causes: cellular hypertrophy, aetiological factors, wall stress and an abnormal oxygen supply-to-demand ratio (unverferth 1985). the active principle contained by plants inducing gousiekte has been isolated (fourie 1994; fourie, erasmus, schultz & prozesky 1995) and identified as pavetamine (vleggaar, university of pretoria, unpublished data 1997). pavetamine was found to be an inhibiter of myocardial protein synthesis in rats (schultz, fourie, basson, labuschagne & prozesky 2001). the effect of pavetamine on the heart was sustained for at least 48 h, in contrast to the other organs that were either unaffected or recovered rapidly (schultz et al. 2001). the term protein turnover is used to describe the continuous synthesis and degradation of muscle protein in the body, including that of myocardium (swick & song 1974; earl, laurent, everett, bonnin & sparrow 1978). in the rat, dog, fowl and mouse it was found that the average protein turnover rate of cardiac muscle was more rapid and that of skeletal muscle slower (earl et al. 1978). in the rat a ten-fold difference in turnover rate between the ventricles and tensor fasciae latae was found using 3h-leucine. swick & song (1974) reported a six-fold difference in turnover rate between myosin from cardiac and skeletal muscle in the rat. schultz et al. (2001) postulated that while the body is breaking down myocardial protein in its ongoing turnover, pavetamine in experimental rats inhibits the formation of new myocardial protein. depending on the half-life of the affected cardiac protein, a point will be reached where breakdown of myocardial contractile protein exceeds synthesis, to the extent that heart failure will occur. in bovine hereditary dilated cardiomyopathy a number of proteins are significantly reduced (weekes et al. 1999). many of these proteins are found exclusively in the mitochondria, suggesting that in congestive heart failure the myocardium is unable to provide sufficient energy to cope with the increased mechanical stresses. dilated cardiomyopathy in humans and animals is characterized by impaired systolic function with reduced ejection fraction and increased end-systolic blood pressure, as the heart adapts to maintain a normal stroke volume (dec & fuster 1994; weekes et al. 1999). an increase in the cardiac pulmonary flow index (cpfi) (van der walt & van rooyen 1977; fourie et al. 1989) and an increase in the serum aspartate transaminase (ast) (fourie et al. 1989; fourie 1994) are reliable clinical and physio-pathological indicators in sheep of cardiac damage in gousiekte. an increase in the cpfi is attributed to a decrease in both stroke volume and pumping efficiency of the left ventricle relative to the right ventricle, resulting in an increase in the ventricular filling pressure and pulmonary blood volume (pretorius, terblanche, van der walt & van ryssen 1973; van der walt & van rooyen 1977; van rooyen, van der walt, joubert & lotter 1984). tachycardia and an increase in the cpfi are commonplace in animals with gousiekte during the later stages of the disease (van der walt & van rooyen 1977; van der walt, van rooyen, cilliers, van ryssen & van aarde 1981), i.e. after 35 days (fourie et al. 1989). both these factors interfere with adequate myocardial perfusion. this will further interfere with myocardial perfusion, most notably subendocardial tissue, because of an increased intraventricular pressure that is readily transmitted to principally subendocardial coronary arteries (unverferth 1985). transmural distribution of coronary blood flow in the wall of the heart has been studied by various workers (rudolph & heyman 1967). in dogs, the subendocardial tissue has 20 % greater oxygen consumption per unit weight than subepicardial tissue (weiss, neubauer, lipp & sinha 1978). there is overwhelming evidence in humans that subendocardial myofibres demonstrate greater ischaemic damage than subepicardial myofibres (unverferth 1985). blood flow to the subendocardium normally occurs in diastole, whereas subepicardial flow is maintained in systole and diastole (rouleau, boerboom & surjadhana 1979). tachycardia thus reduces diastolic and myocardial perfusion, particularly subendocardial perfusion, and increases oxygen demand (unverferth 1985). furthermore, any substantial increase in oxygen demand is met by an increase in coronary blood flow by alterations in coronary vascular resistance. dilated cardiomyopathy is characterized by a low cardiac output and a low coronary blood flow (weiss, ellis, sciacca, johnson, schmidt & cannon 1976) and can therefore not meet this demand. from a pathophysiological viewpoint, sheep with gousiekte with a long latent period demonstrate several characteristics that increase the risk of myocardial ischaemia of, in particular, the subendocardial tissue. 228 myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants considering the available information, the following pathogenesis of the myocardial lesions in animals with gousiekte is postulated. following exposure to gousiekte plants, pavetamine inhibits the formation of new myocardial protein. a point is reached where breakdown of myocardial contractile protein exceeds synthesis to the extent that contractibility of myofibres is affected and heart failure occurs. the majority of animals die without clinical signs of congestive heart failure. congestive heart failure can, however, occur in animals with a long latent period, and in these animals various degrees of dilated cardiomyopathy and concomitant, primarily subendocardial, fibrosis of the left free ventricular wall and the left side of the interventricular septum is evident. it is hypothesised that the pattern of subendocardial fibrosis is the result of a self-perpetuating cycle resulting from diminished myocardial perfusion, most notably of the subendocardial tissue. inadequate perfusion may therefore potentiate the primary myocardial dysfunction. the heart has a limited range of responses to injury, and the response will vary according to the nature, duration and severity of the insult (newsholme 1982; kumar, cotran & robbins 2003). the most common myofibre changes seen in animals with gousiekte include varying degrees of hypertrophy, atrophy, necrosis and fibrosis. the myocardial changes in animals with gousiekte therefore appear to represent a final common pathway of cellular damage rather than a manifestation of a specific type of heart disease. there is no evidence that pavetamine selectively affects certain myocardial fibres, and the predilection for hypertrophy of myofibres in the subendocardial region is probably related to the diminished perfusion that potentiates the primary myocardial dysfunction in this region. references adelaar, t.f., terblanche, m. & smit, j.d. 1966. a report on negative experiments with ferric chloride as a prophylactic agent against gousiekte. journal of the south african veterinary medical association, 37:199–201. anon. 1968. manual of histological staining methods, 3rd ed. armed forces institute of pathology, washington dc, new york: mc graw-hill. dec, g.w. & fuster, v. 1994. idiopathic dilated cardiomyopathy. new england journal of medicine, 331:1564–1575. earl, c.a., laurent, g.j., everett, a.w., bonnin, c.m. & sparrow, m.p. 1978. turnover rates of muscle protein in cardiac and skeletal muscles of dog, fowl, rat and mouse. australian journal of experimental biology and medical science. 56:265–277. fourie, n. 1994. isolation of the cardiotoxin from gousiekteinducing plants and investigation of the pathogenesis and diagnosis of the disease. ph.d. thesis, university of pretoria. fourie. n., erasmus, g.l., schultz, r.a. & prozesky, l. 1995. isolation of the toxin responsible for gousiekte, a plant induced cardiomyopathy of ruminants in southern africa. onderstepoort journal of veterinary research, 62: 77–87 fourie, n., schultz, r. anitra, prozesky, l., kellerman, t.s. & labuschagne leonie. 1989. clinical pathological changes in gousiekte, a plant-induced cardiotoxicosis of ruminants. onderstepoort journal of veterinary research, 56:73–80. hurter, l.r., naude, t.w., adelaar, t.f., smit, j.d. & codd, l.e. 1972. ingestion of the plant fadogia monticola robyns as an additional cause of gousiekte in ruminants. onderstepoort journal of veterinary research, 39:71–82. kellerman, t.s., coetzer, j.a.w. & naudé, t.w. 1988. gousiekte, in plant poisonings and mycotoxicoses of livestock in southern africa. cape town: oxford university press. kumar, v., cotran, r. & robbins, s.l. 2003. robbins basic pathology, 7th ed. philadelphia, london, toronto, montreal, sydney & tokyo: w.b. saunders company. newsholme, s.j. 1982. reaction patterns in myocardium in response to injury. journal of the south african veterinary association, 53:52–59. newsholme, s.j. & coetzer, j.a.w. 1984. myocardial pathology of domestic ruminants in southern africa. journal of the south african veterinary association, 55:89–96. pretorius, p.j., terblanche, m., van der walt, j.d. & van ryssen, j.c.j. 1973. cardiac failure in ruminants caused by gousiekte, in cardiomyopathies. vol. 2, edited by e. bajusz & g. rona. baltimore: university park press. prozesky, l., fourie, n., neser, j.a. & nel, p.w. 1988. a field outbreak in 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diagnose van gousiekte. journal of the south african veterinary medical association, 30:447–450. swick, r.w. & song, h. 1974. turnover of various muscle proteins. journal of animal science, 38:1150–1157. unverferth, d. 1985. dilated cardiomyopathy. mount kisco, new york: futura publishing company, inc. van der walt, j.j. & van rooyen, j.m. 1977. use of technetium-99 m to determine haemodynamic changes during the development of ventricular failure with gousiekte. south african medical journal, 52:375. van der walt, j.j., van rooyen, j.m., cilliers, g.d., van ryssen, c.j. & van aarde, m.n. 1981. ratio of car229 l. prozesky et al. diopulmonary blood volume to stroke volume as an index of cardiac function in animals and in man. cardiovascular research, 15:580–587 van rooyen, j.m., van der walt, j.j., joubert, h. & lötter, a.p. 1984. die beheer van haemodinamiese veranderinge tydens gousiekte. proceedings pharmacology and physiology congress, potchefstroom, south africa: 207– 208. weekes, j., wheeler, c.h., yan, j., weil, j., eschenhagen, t., scholtysik, g. & dunn, m.j. 1999. bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. electrophoresis, 20:898–906. weiss, h.b., ellis, k., sciacca, r.r., johnson, l.l., schmidt, d.h. & cannon, p.j. 1976. myocardial blood flow in congestive and hypertrophic cardiomyopathy. circulation research, 54:484–494. weiss, h.r., neubauer, j.a., lipp, j.a. & sinha, a.k. 1978. quantitative determination of regional oxygen consumption in the dog heart. circulation research, 42:394–401. 230 myocardial lesions in gousiekte, a plant-induced cardiotoxicosis of ruminants fourie_293-301.qxd introduction the ixodid ticks that infest elephant shrews in southern africa have been recorded by theiler (1962), who listed 14 species, and reviewed by fourie, du toit, kok & horak (1995), who list 22 species. the ticks that infest these small mammals are of more than purely academic interest in that rock elephant shrews, elephantulus myurus, are the preferred hosts of the immature stages of three ticks capable of inducing paralysis in domestic animals (fourie, horak & van den heever 1992a; fourie, horak, kok & van zyl 2002). these ticks are ixodes rubicundus, responsible for massive annual mortality amongst sheep (spickett & heyne 1988), rhipicentor nuttalli that can cause paralysis in dogs (perchman 1976; norval & colborne 1985), and rhipicephalus warburtoni (then referred to as a tick belonging to the rhipicephalus pravus group) that has been incriminated in paralysis in goat kids (fourie, horak & marais 1988a). the hosts of the immature and adult stages of i. rubicundus have been recorded by stampa (1959) and horak, moolman & fourie (1987a), and the role of e. myurus in the life cycle of this tick, colloquially 293 onderstepoort journal of veterinary research, 72:293–301 (2005) elephant shrews as hosts of immature ixodid ticks l.j. fourie1, i.g. horak2 and p.f. woodall3 abstract fourie, l.j., horak, i.g. & woodall, p.f. 2005. elephant shrews as hosts of immature ixodid ticks. onderstepoort journal of veterinary research, 72:293–301 two hundred and seventy-three elephant shrews, consisting of 193 elephantulus myurus, 67 elephantulus edwardii and 13 animals belonging to other species, were examined for ixodid ticks at 18 localities in south africa and namibia. the immature stages of ixodes rubicundus, rhipicentor nuttalli, rhipicephalus warburtoni and a rhipicephalus pravus-like tick were the most numerous of the 18 tick species recovered. substantial numbers of immature rhipicephalus arnoldi, rhipicephalus distinctus and rhipicephalus exophthalmos were also collected from elephant shrews at particular localities. larvae of i. rubicundus were most numerous on e. myurus in free state province from april to july and nymphs from june to october. larvae of r. nuttalli were most numerous on these animals during april, may, august and september, and nymphs in february and from april to august. the immature stages of r. warburtoni were collected from e. myurus only in free state province, and larvae were generally most numerous from december to august and nymphs from april to october. keywords: elephant shrews, ixodes rubicundus, ixodid ticks, macroscelids, rhipicentor nuttalli, rhipicephalus warburtoni ! department of zoology and entomology, university of the free state, bloemfontein, 9301 south africa. e-mail: fourie@clinvet.com 2 department of zoology and entomology, university of the free state, bloemfontein, 9301 south africa and department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa 3 school of veterinary science, the university of queensland, brisbane, 4072 australia accepted for publication 14 june 2005—editor known as the karoo paralysis tick, has been highlighted by du toit, fourie & horak (1994a, b) and fourie & horak (1994). the hosts in zimbabwe of adult r. nuttalli have been listed by norval & colborne (1985), while in south africa the hosts of both adult and immature ticks have been recorded by fourie et al. (2002), who established the details of its life cycle. walker, keirans & horak (2000) have summarized the host records obtained by fourie and his co-workers for r. warburtoni during extensive surveys they conducted in free state province between 1985 and 1991. fourie et al. (1992a, 2002) have also documented the seasonal occurrence of the immature stages of the latter three ticks on e. myurus. the economic importance of these ticks and the pivotal role of e. myurus in their life cycles prompted further surveys on these animals and on other species of elephant shrews, and the objective of this study was to determine both the species composition and seasonality of ticks that infest them. fleas were also collected during the surveys and have been recorded by beaucournu, horak & fourie (2003). materials and methods ticks were collected by one of two methods from elephant shrews. in the first e. myurus were trapped in box traps each month from august 1994 to june 1995 on the farm spes bona in central free state province. they were transported to the laboratory and individually caged above trays from which engorged larvae or nymphs that had detached were collected daily. once all larvae and nymphs had detached, the elephant shrews were returned to their original habitats on the farm and released. the following month’s trapping commenced a week after this release so that if the same shrews were recaptured they would have had adequate time to become re-infested with ticks. despite intensive efforts no elephant shrews were trapped during july 1995. 294 elephant shrews as hosts of ixodid ticks table 1 elephant shrews and the localities at which they were examined for ixodid ticks elephant shrew species province or locality and coordinates common name scientific name (no. examined) short-snouted elephant shrew elephantulus brachyrynchus (4) pilanesberg nr (25°15´s, 27°05´e) cape elephant shrew elephantulus edwardii (67) gifberg (31°47´s, 18°40´e) clanwilliam (32°11´s, 18°54´e) bushveld elephant shrew elephantulus intufi (1) erongo (21°27´s, 15°52´e) (namibia) rock elephant shrew elephantulus myurus (193) free state province môreson (28°48´s, 27°13´e) spes bona (29°07´s, 26°04´e) wolvekop (29°27´s, 26°40´e) langberg (29°36´s, 25°28´e) preezfontein (29°50´s, 25°23´e) welbedacht dam (29°51´s, 26°53´e) tussen-die-riviere nr (30°29´s, 26°15´e) northern provinces mapungubwe (22°12´s, 29°21´e) pafuri (23°27´s, 31°19´e) (knp) gustav kliengbiel nr (25°06´s, 30°31´e) pilanesberg nr (25°15´s, 27°05´e) pretoria district (25°45´s, 28°12´e) smith´s rock elephant shrew elephantulus rupestris (6) avis (22°35´s, 17°07´e) (namibia) beaufort west (32°21´s, 22°35´e) short-eared elephant shrew macroscelides proboscideus (1) victoria west (31°24´s, 23°07´e) four-toed elephant shrew petrodromus tetradactylus (1) pafuri (23°27´s, 31°19´e) (knp) nr= nature reserve knp= kruger national park the detached, engorged larvae and nymphs were washed in water to remove traces of urine and prevent the growth of fungi and were maintained at 20 °c and a relative humidity of approximately 90 % in permanent darkness. once moulting was completed the various species were identified from the newly emerged nymphs or adults and counted. in the second method elephant shrews were captured in box traps, euthanased and their carcasses processed for the recovery of ticks as described by fourie et al. (1992a). the seasonal occurrence of the immature stages of i. rubicundus, r. nuttalli and r. warburtoni was determined from the average monthly tick burdens of e. myurus examined within free state province, the only province in which ticks were collected from elephant shrews during every month of the year. results and discussion the species of elephant shrews examined, and the localities at which they were trapped are summarized in table 1. tick collections were made at 18 localities from 273 elephant shrews belonging to seven species. the ticks collected from the elephant shrews are summarized in tables 2–8. 295 l.j. fourie, i.g. horak & p.f. woodall table 2 ixodid ticks collected from 66 elephantulus edwardii on the farm gifberg (31°47´s, 18°40´e) in the western cape province total number of ticks collected number of tick species larvae nymphs total infested animals haemaphysalis hyracophila 1 2 3 2 haemaphysalis leachi 1 0 1 1 ixodes rubicundus 1 962 679 2 641 33 rhipicephalus arnoldi 0 1 1 1 rhipicephalus distinctus 15 1 16 11 rhipicephalus exophthalmos 53 1 54 3 rhipicephalus gertrudae 6 4 10 5 rhipicephalus sp. 20 1 21 9 table 3 ixodid ticks collected from 54 elephantulus myurus on the farm langberg (29°36´s, 25°28´e) in free state province total number of ticks collected number of tick species larvae nymphs total infested animals amblyomma marmoreum 5 1 6 4 haemaphysalis hyracophila 0 1 1 1 hyalomma truncatum 1 0 1 1 ixodes rubicundus 1 168 353 1 521 42 rhipicephalus evertsi evertsi 1 1 2 2 rhipicephalus warburtoni 1 862 672 2 534 54 table 4 ixodid ticks collected from 86* elephantulus myurus on the farms spes bona (29°07´s, 26°04´e) and wolvekop (29°27´s, 26°40´e) in free state province total number of ticks collected number of tick species larvae nymphs total infested animals haemaphysalis hyracophila 1 0 1 1 ixodes rubicundus** 146 538 684 37 rhipicentor nuttalli 361 75 436 54 rhipicephalus arnoldi 151 50 201 45 rhipicephalus distinctus 4 1 5 4 rhipicephalus warburtoni 2 162 609 2 771 70 * = although ticks were collected from 86 animals, only those from 72 animals were identified ** = engorged larvae and nymphs of i. rubicundus that detached from 21 heavily infested animals were used for experimental purposes without their numbers being recorded eighteen tick species were recovered and the immature stages of i. rubicundus, r. nuttalli, r. warburtoni and a rhipicephalus pravus-like tick were the most numerous of these. however, substantial numbers of immature rhipicephalus arnoldi, rhipicephalus distinctus and rhipicephalus exopthalmos were also collected from elephant shrews at particular localities. ixodes rubicundus large numbers of larvae and nymphs of i. rubicundus were collected from e. myurus in free state province and from elephantulus edwardii in western cape province in the present study. stampa (1959) collected approximately 200 larvae and nymphs from nine elephantulus spp. in the new bethesda region of the eastern cape province, and more than 2 600 from 76 smith’s red rock rabbits, pronolagus rupestris, at the same locality. on the other hand, horak & fourie (1991) recovered only six larvae from 28 red rock rabbits they examined on two farms in south-western free state province, whereas 132 rock elephant shrews examined on the same farms harboured 4 331 larvae and nymphs (fourie et al. 1992a). it would thus appear that these small mammals supplant each other as hosts for the immature stages of i. rubicundus depending upon locality and environment. larvae were most numerous on rock elephant shrews in free state province from april to july and 296 elephant shrews as hosts of ixodid ticks table 5 ixodid ticks collected from 24 elephantulus myurus on the farm môreson (28°48´s, 27°13´e) in free state province total number of ticks collected number of tick species larvae nymphs total infested animals hyalomma marginatum rufipes 1 0 1 1 ixodes rubicundus 13 87 100 14 rhipicentor nuttalli 147 70 217 22 rhipicephalus distinctus 2 0 2 2 rhipicephalus warburtoni 171 69 240 20 table 6 ixodid ticks collected from 8 elephantulus myurus at various localities in free state province number of ticks collected infested coordinates of tick species larvae nymphs total animals collection localities hyalomma truncatum 0 8 8 1 (30°29´s, 26°15´e) ixodes rubicundus 155 117 272 6 (29°50´s, 25°23´e) (29°51´s, 26°53´e) (30°29´s, 26°15´e) rhipicephalus evertsi evertsi 1 0 1 1 (29°51´s, 26°53´e) rhipicephalus warburtoni 421 47 468 7 (29°50´s, 25°23´e) (29°51´s, 26°53´e) (30°29´s, 26°15´e) table 7 ixodid ticks collected from 21 elephantulus myurus in the four northern provinces of south africa number of ticks collected infested coordinates of tick species larvae nymphs total animals collection localities ixodes bakeri 0 0 1 female 1 (25°06´s, 30°31´e) rhipicentor nuttalli 176 117 293 13 (23°27´s, 31°19´e) (25°45´s, 28°12´e) rhipicephalus pravus group 679 158 837 13 (23°27´s, 31°19´e) (25°15´s, 27°05´e) (25°45´s, 28°12´e) nymphs from june to october (fig. 1a), matching the seasonal occurrence recorded by fourie et al. (1992a) on these animals in south-western free state province during 1985. the 4-month difference in peak activity between larvae and nymphs (april to august) agrees with the observations of stampa (1959) and fourie et al. (1992a) and represents the moulting period of detached larvae and their subsequent attachment to hosts as newly moulted nymphs. the vast majority of larvae that infest elephant shrews during autumn and early winter originate from eggs deposited by females during the winter of the previous year. these eggs over-summer in a state of diapause and larvae only hatch in autumn (fourie & horak 1994). engorged nymphs that detach from elephant shrews in late winter and spring also over-summer in a state of diapause and only moult to adults during late summer and autumn of the following year (fourie & horak 1994). the life cycle thus takes 2 years to complete, with the immature stages present during the cooler months of the first year and the adults during the same months of the second year. stampa (1959) collected adult i. rubicundus from several wild carnivores in the new bethesda region, and horak et al. (1987a) have suggested that caracals, caracal caracal, are one of the preferred hosts of this tick. subsequent collections from wild carnivores as well as from domestic dogs indicate that several species are suitable hosts for adult i. rubicundus (horak, braack, fourie & walker 2000; jacobs, fourie, kok & horak 2001; horak & matthee 2003). wild bovids that frequent mountain slopes, namely mountain reedbuck, redunca fulvorufula, grey rhebok, pelea capreolus, and eland, taurotragus oryx, as well as domestic sheep on mountainous pastures, also serve as hosts for adult ticks (stampa 1959; fourie et al. 1988b; horak, fourie, novellie & williams 1991). paralysis in sheep and goats is positively correlated with the intensity of infestation of female i. rubicundus and is observed during the cooler months of the year (fourie, petney, horak & de jager 1989; fourie, horak & van zyl 1992b). rhipicentor nuttalli the genus rhipicentor contains only two species, rhipicentor bicornis and rhipicentor nuttalli, whose distributions are restricted to africa (theiler 1961, 1962). r. nuttalli has been recorded in the eastern and western cape, free state, gauteng, northwest and limpopo provinces, south africa (theiler 1961; du toit 1993). in namibia it occurs north of windhoek, and it is also present in botswana and zimbabwe (theiler 1961; norval & colborne 1985). in this study its immature stages were collected from e. myurus in central free state province, northern gauteng province and in northern and northeastern limpopo province, and from e. edwardii in south-western western cape province and elephan297 l.j. fourie, i.g. horak & p.f. woodall table 8 ixodid ticks collected from various species of elephant shrews at several localities elephant shrew species locality tick species (no. examined) (no. of infested animals) elephantulus brachyrynchus (4) pilanesberg, hyalomma truncatum 1 larva (1) north west province ixodes sp. 1 nymph (1) rhipicephalus pravus group 48 nymphs (4) elephantulus edwardii (1) clanwilliam, ixodes rubicundus 2 nymphs (1) western cape province rhipicentor nuttalli 7 nymphs (1) elephantulus intufi (1) erongo, namibia rhipicentor nuttalli 3 nymphs (1) elephantulus rupestris (1) beaufort west, rhipicephalus sp. 8 larvae, 11 nymphs (1) western cape province elephantulus rupestris (5) avis, rhipicephalus exophthalmos 1 larva, 35 nymphs (5) namibia rhipicephalus sp. 1 nymph (1) macroscelides proboscideus (1) victoria west, rhipicephalus exophthalmos 51 larvae, 15 nymphs (1) northern cape province petrodromus tetradactylus (1) pafuri, haemaphysalis leachi 2 nymphs (1) limpopo province rhipicephalus kochi 1 larva, 74 nymphs (1) tulus intufi at erongo, namibia. r. bicornis occurs in the northern regions of south africa and in zimbabwe, northern namibia and zambia, and is widely distributed in central africa (theiler 1961; walker 1991). the hosts of the immature stages of r. nuttalli were unknown until du toit (1993) collected larvae and nymphs from e. myurus in free state province. adult r. nuttalli has been collected from domestic dogs and several wild carnivore species as well as from the south african hedgehog, atelerix frontalis and porcupine, hystrix africaeaustralis (theiler 1961; walker 1991; horak et al. 2000). norval & colborne (1985) state that whereas r. nuttalli is known in south africa as the hedgehog tick, their findings in zimbabwe indicate that the adults are principally parasites of leopards, panthera pardus, and that its distribution is restricted to the rocky habitats normally favoured by these animals. the large number of e. myurus infested with r. nuttalli in the present study confirms the observation by du toit (1993) that they are hosts of the immature stages of this tick. the collection of nymphs from e. edwardii and e. intufi suggests that these animals, and possibly other species of elephant shrews, are also suitable hosts for the immature stages of this tick, particularly in those regions in which e. myurus does not occur. although immature ticks were present on e. myurus throughout the year, the majority of larvae were collected in april and may and during august and september and nymphs in february and from april to august (fig. 1b). this differs somewhat from the findings of fourie et al. (2002), also in free state province, that larvae were generally most numerous during the period march to september, and nymphs during may to october. theiler (1962) noted that adult ticks are common on dogs in the clanwilliam district in the western cape province and in the omaruru district, namibia during late summer. in gauteng and free state provinces adult ticks have been collected from wild carnivores in september, october and december (horak et al. 2000). in zimbabwe, however, norval & colborne (1985) found that adult ticks were present throughout the year. rhipicephalus arnoldi this tick utilizes smith’s red rock rabbits as hosts for all its developmental stages (horak et al. 1991; horak & fourie 1991), but large numbers of immature ticks have also been collected from rock das298 elephant shrews as hosts of ixodid ticks ��� ��� ��� �� �� �� �� � � � � �� � � � � � � �� �� �� � � � �� �� �� � � � �� � � ��� � � �� ��� ��� ��� ��� ��� ��� � �� ������� �� �� ��������� �� � � � � �� � � � � � � �� �� �� � � � �� �� �� � � �� �� � � � � �� � � ��� � � �� ��� ��� ��� ��� ��� ��� � �� ����� ���� �� �������������� �� ����� ����� ���� � ������������ �� ! �� "� �� #� �� �� � � � � �� � � � � � � �� �� �� � � � �� �� �� � � � �� � � ��� � � �� ��� ��� ��� ��� ��� ��� � �� $ �� � ��%�&' fig. 1 the seasonal occurrence of the immature stages of (a) ixodes rubicundus, (b) rhipicentor nuttalli, and (c) rhipicephalus warburtoni on rock elephant shrews in free state province, south africa sies, procavia capensis (horak & fourie 1986; horak et al. 1991). it has been suggested by horak & fourie (1986) that, even if those on rock dassies are taken into account, the low ratio of immature to adult ticks implies that some other animal could also harbour considerable numbers of immature ticks of this species. it would now appear as if rock elephant shrews might fulfil this role in free state province. as their common names imply rock dassies, red rock rabbits and rock elephant shrews inhabit the same rocky habitats (skinner & smithers 1990), and since they all have dense pelts it is not surprising that they host some tick species in common. however, whereas rock dassies are also hosts of adult haemaphysalis hyracophila and adult r. distinctus, and red rock rabbits of adult r. arnoldi (horak & fourie 1986, 1991; horak et al. 1991), rock elephant shrews harbour only the immature stages of these species. rhipicephalus distinctus this is a tick of rock dassies (horak & fourie 1986; walker et al. 2000), and sympatry between these animals and elephant shrews probably contributed to its presence on 11 e. edwardii in the western cape province (table 2) and on six e. myurus in free state province (tables 4 and 5). rhipicephalus exophthalmos two e. edwardii examined in the western cape province were infested with larvae and one with a nymph of r. exophthalmos (table 2). the immature stages of this tick were also present on elephantulus rupestris at avis, namibia, and on the single macroscelides proboscideus examined at victoria west, northern cape province (table 8). scrub hares, lepus saxatilis, which incidentally are also hosts of adult r. exophthalmos, are the only other animals recorded as hosts for the immature stages of this tick (walker et al. 2000). theiler (1962) lists nearly all elephant shrew species as hosts of the immature stages of a closely related species, namely rhipicephalus oculatus. it is, however, possible that several of her records refer to r. exophthalmos, which has only recently been described (keirans, walker, horak & heyne 1993). rhipicephalus gertrudae the adults of r. gertrudae feed on domestic and wild ruminants and carnivores and its immature stages on murid rodents (walker et al. 2000; horak & matthee 2003) and the presence of its immature stages on e. edwardii in the western cape province is possibly accidental (table 2). this tick occurs in the southern, central and western regions of south africa and in namibia (walker et al. 2000). rhipicephalus kochi although widespread elsewhere in east africa, r. kochi has been collected only in the far north-east of limpopo and kwazulu-natal provinces in south africa. the preferred hosts of all stages of development are medium-sized and larger antelopes and scrub hares (walker et al. 2000). judging by the single larva and large number of nymphs collected from the single four-toed elephant shrew examined at pafuri in north-eastern limpopo province (table 8), these animals appear to be good hosts of the immature stages of r. kochi. rhipicephalus pravus group the immature stages of this r. pravus-like tick were present on e. myurus in northern gauteng province and north-eastern limpopo province, and its nymphs on e. myurus and elephantulus brachyrynchus in north-west province (tables 7 and 8). all its parasitic stages have been collected from scrub hares at shingwedzi and pafuri in the kruger national park in north-eastern limpopo province, and on farms in the north-west of this province (horak, spickett, braack & penzhorn 1993; horak, spickett, braack, penzhorn, bagnall & uys 1995). walker et al. (2000) place r. arnoldi, r. exophthalmos, r. kochi, r. oculatus, r. pravus and r. warburtoni in the r. pravus group of ticks on the morphological characters of their immature stages and suggest that these stages favour macroscelids as hosts. the validity of their grouping is supported by the present study in which, with the exception of those of r. oculatus, the immature stages of all these ticks were collected from elephant shrews. rhipicephalus warburtoni this tick has only recently been described (walker et al. 2000). it was previously variously referred to as an atypical strain of rhipicephalus appendiculatus (theiler 1949); as rhipicephalus pravus (theiler 1962); as an r. pravus-like tick (fourie et al. 1988a, b); as an rhipicephalus punctatus-like tick (fourie & horak 1990); and as rhipicephalus punctatus in several publications by fourie & horak and their coworkers. the preferred hosts of the adults are domestic and wild ruminants and hares, and elephant shrews and hares are the preferred hosts of the immature stages (fourie et al. 1992a; walker et al. 2000). its distribution is virtually confined to the 299 l.j. fourie, i.g. horak & p.f. woodall central and southern regions of free state province (walker et al. 2000), and its presence on 171 of the 182 e. myurus examined in this province is thus not unexpected (tables 3–6). larvae were generally most numerous from december to august and nymphs from april to october (fig. 1c). with the exception of april, when larval numbers were exceptionally high, this pattern of seasonality is fairly similar to that recorded by fourie et al. (1992a). most adults of this species are present on angora goats in south-western free state province from september or november to february (fourie & horak 1991), and it was during this period that paralysis was observed in goat kids (fourie et al. 1988a). other species the immature stages of the remaining tick species should all be considered as “stragglers”. adult amblyomma marmoreum infests tortoises and its immature stages are found on these animals as well as ground-frequenting birds, hares, carnivores and ruminants (norval 1975; horak, macivor, petney & de vos 1987b). adult and immature h. hyracophila are parasites of rock dassies (horak & fourie 1986), whereas adults of haemaphysalis leachi are parasites of carnivores and its immature stages of murid rodents (norval 1984; fourie et al. 1992a). however, the latter may also occur on the same hosts as the adults (horak & matthee 2003). adult hyalomma marginatum rufipes and hyalomma truncatum infest large herbivorous animals and 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the sahara (ethiopian region). project s 9958. report to the director of veterinary services, onderstepoort. mimeographed. walker, jane b. 1991. a review of the ixodid ticks (acari: ixodidae) occurring in southern africa. onderstepoort journal of veterinary research, 58:81–105. walker, jane b., keirans, j.e. & horak, i.g. 2000. the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world. cambridge: cambridge academic press. 301 l.j. fourie, i.g. horak & p.f. woodall abstract introduction material and methods results discussion conclusion acknowledgements references about the author(s) rosa kebbi department of environment biological sciences, faculty of nature and life sciences, university of bejaia, bejaia, algeria mohamed nait-mouloud department of environment biological sciences, faculty of nature and life sciences, university of bejaia, bejaia, algeria lila hassissen private veterinary practice, sidi-ahmed district, bejaia, algeria abdelhanine ayad department of environment biological sciences, faculty of nature and life sciences, university of bejaia, bejaia, algeria citation kebbi, r., nait-mouloud, m., hassissen, l. & ayad, a., 2019, ‘seasonal activity of ticks infesting domestic dogs in bejaia province, northern algeria’, onderstepoort journal of veterinary research 86(1), a1755. https://doi.org/10.4102/ojvr.v86i1.1755 original research seasonal activity of ticks infesting domestic dogs in bejaia province, northern algeria rosa kebbi, mohamed nait-mouloud, lila hassissen, abdelhanine ayad received: 27 feb. 2019; accepted: 15 may 2019; published: 17 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this epidemiological study aimed to determine the species of tick infestation in dogs, their prevalence and dynamic in the bejaia province, northeastern algeria. a total of 631 dogs were examined from different localities of the bejaia province between march 2016 and february 2017. of the 631 examined dogs, 15% were infested with one or more tick species. a total of 339 adult ticks were collected and identified, including 199 male tick species and 140 female tick species. our results revealed that most of these were rhipicephalus species, with rhipicephalus sanguineus (51.32%) being the most prevalent followed by rhipicephalus bursa (35.1%) and rhipicephalus turanicus (12.98%). ixodes ricinus represented only 0.6% of all ticks collected. the highest infested seasons were spring (22.55%) and summer (22.54%) and the lowest infested seasons were autumn (8.62%) and winter ( 0.9%). there is no significant difference between the sex of the animal and the prevalence of infestation (p = 0.837). also, the prevalence of infestation by ticks in young animals was higher than that in adult animals (p = 0.550). a significant difference between the prevalence of infestation and animal breed was observed (p = 0.042). this study is the first epidemiological investigation conducted on the prevalence of hard ticks infesting domestic dogs in bejaia (northeastern algeria) based on conventional methods. it is therefore necessary to implement an effective tick control strategy during infestation periods in order to prevent vector-borne diseases. keywords: prevalence; dynamic; ticks; dogs; bejaia province. introduction after mosquitoes, ticks are the second most important obligate haematophagous arthropods that parasitise all classes of vertebrates for their blood meal in almost all regions of the world, particularly in africa (laamri et al. 2012; xhaxhiu et al. 2009). they can also crucially transmit to animals, especially dogs, a large number of protozoa and bacteria, such as babesia, theileria and anaplasma spp. (gray et al. 2013; marquez-jimenez et al. 2005). the seasonal dynamics of ticks is likely to affect the transmission of pathogens. several ecological factors can influence the survival and development of ticks, especially temperature, relative humidity and vegetation cover (sahibi & rhalem 2007). all tick species are significant disease vectors and the increased incidence of these diseases is mainly attributed to climate change that affects ticks directly or indirectly (gray et al. 2009). ticks are highly climate-sensitive arthropods, and all stages of their life cycle depend on a complex combination of climatic variables. the presence of hosts and vegetation greatly modulate the dynamics of their populations. however, vegetation is a major modifier of local climatic conditions, to which ticks must adapt for their development and survival (estrada-peña, ayllón & de la fuente 2012). dogs are the most commonly owned companion pets worldwide (moriello 2003). they are the most successful canids, adapted to human habituation, which contribute to the physical, social and emotional well-being of their owners (dohoo et al. 1998). it is possible that dogs carry ticks in the domestic environment and transmit these to humans, which may constitute a major concern for public health (dantas-torres, chomel & otranto 2012). in algeria, very few studies have been conducted on the vectorial role of ticks that infest dogs, except the inventory carried out by matallah et al. (2013). in terms of biodiversity and specific biology, ixodidea fauna is not sufficiently known in algeria (meddour & meddour 2006). because of certain geographical specifications and the probable presence of different types of ticks infesting companion animals, epidemiology studies of ticks in dogs are extremely important to plan the approaches of pest management. this study aimed at determining the species of tick infestation in dogs, their prevalence and dynamics by performing an epidemiological study in the bejaia province (northeastern algeria). material and methods study area and dogs the study was carried out in the bejaia province of algeria (36°43’n, 5°04’e) (figure 1) from march 2016 to february 2017. the province has four distinct seasons: winter (january to march), spring (april to june), summer (july to september) and autumn (october to december). the annual rainfall in the region during the study period ranged from 679 mm to 821 mm. the mean maximum summer temperature was 29.9 °c (august) and the mean minimum winter temperature was 6.4 °c (january) during the study period (table 1). figure 1: map of the study area, bejaia (northern algeria, latitude 36°43’n and longitude 5°04’e). table 1: mean ± standard deviation, minimum and maximum value of temperature, rainfall and humidity per month in the bejaia area, algeria (2016–2017). a total of 631 dogs were selected randomly from different habitats (home and farmhouse) and localities of the bejaia province. all dogs were presented to a veterinary clinic for different reasons (care, vaccinations, etc.). a dermatological examination was performed by veterinary practitioners and all observations were recorded for each dog examined throughout the study. a complete examination of the skin, visually and by palpation, was done for the presence of ticks. the age of the animals ranged between a few days and over 15 years, with mixed breeds. tick collection and identification all ticks were removed carefully to ensure that the mouthparts remained intact. the ticks collected were preserved in individually labelled plastic containers containing 70% ethanol. tick identification was carried out using standard methods at the laboratory of animal biology, university of bejaia. each tick was identified using a stereomicroscope (motic, st-37c-2loo) according to the standard morphological identification keys (meddour & meddour 2006; walker et al. 2014). statistics analysis prevalence was calculated as the ratio between the number of infested dogs and the total number of examined dogs. abundance was determined as the ratio between the total number of parasitic species and the total number of examined dogs. infestation intensity was calculated as the ratio between the total number of parasitic species in hosts and the number of infested hosts. statistical analyses were carried out using the r software version 3.4.4 (http://www.r-project.org/). tick infestation was analysed using the following factors of variation: sex (male and female), age (young: ≤ 12 months; adult: > 1 year), breed (german shepherd, rottweiler, american staffordshire terrier, belgian shepherd, dogo argentino, french pointer and others) and season (spring, summer, autumn and winter). statistical analysis was performed using a negative binomial general linear model (function ‘glm.nb’ in library ‘mass’ in r). tukey’s post-hoc test was performed using the ‘glht’ function from the multcomp package. the values were statistically different when the p-value was < 0.05. ethical considerations ethical clearance to conduct the study was obtained from the scientific committee of the faculty of nature and life sciences, university of bejaia. results of the 631 examined dogs, 15% were infested with one or more tick species. a total of 339 mature ticks were collected and identified, including 199 male and 140 female tick species. the overall prevalence of infestation was found to be 15% (94/631) during clinical examination. in this study, two tick genera, rhipicephalus and ixodes, were identified based on the external morphological characteristics (table 2). our results revealed that among the rhipicephalus species, rhipicephalus sanguineus was the most prevalent (51.32%), followed by r. bursa (35.1%) and r. turanicus (12.98%). ixodes ricinus only represented 0.6% of all ticks collected. the ears and neck are the preferred attachment sites of ticks, with a prevalence of 55.8% and 22.2%, respectively. mixed infestations with more than one tick species were recorded in the majority of dogs (table 3). table 2: number (male and female) and prevalence of various tick species in dogs in the bejaia province, northeastern algeria, between march 2016 and february 2017. table 3: prevalence of infested dogs in the bejaia province, northeastern algeria, with mixed tick infestations. table 4 illustrates the seasonal variation of tick infestation in dogs in the bejaia province. the total tick prevalence peaked during spring and summer (22.55% and 22.54%, respectively) and the lowest prevalence occurred during winter (0.9%) (p < 0.001). the abundance and high intensity of ticks were observed in spring (1.04 and 4.58, respectively) and summer (0.61 and 2.71, respectively), but the lowest abundance and intensity were observed in winter (2.00 and 0.01, respectively) (p < 0.05). the monthly variation in the prevalence of infested animals because of r. sanguineus, r. bursa, r. turanicus and i. ricinus is shown in figure 2. the prevalence rates recorded of infested dogs by r. sanguineus, r. bursa and r. turanicus were very high during april and may. as regards to i. ricinus, a prevalence rate was observed only in december. figure 2: seasonal abundance of ticks (rhipicephalus sanguineus, rhipicephalus bursa, rhipicephalus turanicus and ixodes ricinus) found on owned dogs in the bejaia province (2016–2017). table 4: seasonal variation of tick infestation of dogs in the bejaia province, northeastern algeria. the variation of prevalence of infestation by sex, age and breed is shown in table 5. there was no significant difference between the sex of the animal and the prevalence of infestation (p = 0.837) and the age of the animal and the prevalence of infestation (p = 0.550). conversely, the prevalence of infestation by ticks in young animals (≤ 1 year of age) was higher than that in adult animals (> 1 year of age) (p = 0.550). tick prevalence differed significantly between dog breeds (p = 0.042). table 5: the variation of the prevalence of tick infestation in dogs with related risk factors (sex, age, breed and season) in the bejaia province. discussion ectoparasitic infestation is widespread in wild and domestic animals worldwide, several of which are responsible for diseases (krčmar et al. 2014; ranju et al. 2012). in algeria, several surveys have been conducted to study the population of ticks parasitic in cattle (aouadi et al. 2017; boucheikhchoukh et al. 2018; kouidria et al. 2018); however, few structured and published reports are available relating to ticks infesting dogs. the present epidemiological study, extending over 1 year, was conducted to evaluate the spectrum of tick species involved, the levels of infestations and the seasonal dynamic of these ectoparasites. this study is the first on ticks infesting domestic dogs in the bejaia province (northeastern algeria). our results revealed that the domestic dogs are infested by a variety of tick species, with one or more tick species per infested dog as described previously (ebrahimzade, fattahi & mohammad 2016; estrada-peña et al. 2017; latrofa et al. 2017; maurelli et al. 2018). the overall prevalence of infestation was slightly low, which could be a result of difficulties in detecting smaller tick life stages (larvae and nymphs) during clinical examination. studies performed in different countries have shown that the prevalence of infestation by ticks is significantly variable. the prevalence observed in the current study is higher compared to studies from iran (8.6%) (ebrahimzade, fattahi & mohammad 2016) and albania (3.5%) (kumsa & mekonnen 2011). however, it is substantially lower than that reported by matallah et al. (2013) in the souk-ahras and el-kala areas, northeastern algeria (63% and 37 %, respectively). the low rate observed in this study may be explained by the fact that dogs are better maintained by the use of acaricidal treatments. the prevalence rates suggests that these ticks present a real major health problem for domestic dogs and their owners as supported by several studies (kumsa & mekonnen 2011; rinaldi et al. 2007). based on the identification keys from morphological characteristics that are approved for african countries, the current results describe four tick species, with a predominance r. sanguineus. this finding is in agreement with the previous reports (benredjem et al. 2014; bessas et al. 2016; dahmani et al. 2015; leulmi et al. 2016). a similar finding of the predominance of this tick species in infested dogs has been reported by matallah et al. (2013) from northeastern algeria. in 2011, mosallanejad, alborzi and katvandi (2011) observed the same tick species in companion dogs in the ahvaz district, southwestern iran. studies performed in different countries have shown that the number of tick species is highly variable. bryson et al. (2000) recorded six species of ticks from dogs belonging to people in resource-poor communities in south africa. also, de mato et al. (2008) identified nine species of ixodid ticks from dogs in mozambique. in ethiopia, kumsa and mekonnen (2011) identified two species of ticks, namely, amblyomma spp. and haemaphysalis leachi. the variation reported in previous studies might be attributed to different factors, such as geographical locations, climatic conditions and management practices (krčmar et al. 2014). additionally, indirect effects of climate change will impact the number of infected ticks by affecting vegetation (gray et al. 2009). the correlation between positive cases of vector-borne disease and their geographic distribution, as well as potential risk factors (age, sex, breed, type of dog, habitat and prophylactic treatments), was evaluated previously (mircean et al. 2012). also, lindgren, tälleklin and polfeldt (2000) concluded that the relatively mild climate of the 1990s in sweden was probably one of the primary reasons for the observed increase in density and geographic range of i. ricinus ticks. in addition, similar preferred attachment sites of ticks in dogs were found in previous studies (foldvari & farkas 2005; krčmar et al. 2014) compared to our data. rhipicephalus sanguineus, known as the kennel tick, is the most widespread tick in dogs; however, it can also be found in cattle (walker et al. 2014). our results are in agreement with reports by several researchers (de mato et al. 2008; horak & matthee 2003; neves & horak 2004). dantas-torres and otranto (2017) noted that the life cycle of r. sanguineus is adapted to artificial structures such as human habitations and dog kennels. according to walker et al. (2014), r. bursa is one of the common rhipicephalus species in northern africa, and their preferred hosts are sheep, goats, cattle and horses. however, in this study, the presence of r. bursa could be explained by the fact that the dogs were in contact with domestic ruminants, for example, farm dogs. on the other hand, the adult r. turanicus infests a variety of hosts such as cats, sheep, goats and wild carnivores, while immature stages of the species never infest dogs (horak et al. 2000). our result revealed two cases of i. ricinus ticks collected from companion dogs in the bejaia area, algeria. in north africa, it is known that the ixodid tick species infest livestock, and their adults are present in large numbers only on livestock (walker et al. 2014). however, i. ricinus has been observed in more than 300 host species, including mammals, birds and reptiles (gern & humair 2002). it is considered as a potential vector of many disease agents (farkas 2002). this result could be explained by the fact that dogs would have been infested in the wild. according to walker et al. (2014), all stages of the development of i. ricinus climb in vegetation for transfer to the host. moreover, the most favourable conditions for the development of i. ricinus are in temperatures that are relatively cold and high levels of humidity. note that the only cases of i. ricinus was found in december. in this survey, the rhipicephalus species collected from companion dogs has shown mainly spring activity, while i. ricinus has a winter activity. this could be ascribed to a variety of climatic conditions in this study region. it has been reported that r. sanguineus was present in various maghreb countries of different bioclimatic zones, in which the adult ticks have a seasonal activity from march to november, with a peak activity in may, and it was absent in winter (bouattour 2002). moreover, in other studies, the presence of r. sanguineus was reported from june to september (leulmi 2012). in morocco, morel (2000) revealed that the seasonal dynamic of r. bursa was unimodal, with a peak in activity during the warm season ranging from march to september; this finding is similar to the results of the present study. likewise, the r. turanicus tick species appears in march and disappears in july (tsatsaris et al. 2016). as shown in figure 2, r. turanicus has been collected between april and november, with a peak infestation in april and may. concerning the activity period of i. ricinus, it agrees with the results reported previously, that is, autumn–winter (bouattour, darghouth & daoued 1999). in contrast, i. ricinus developed in bimodal mode in the temperate countries, with intense and low activity peaks during spring and autumn, respectively. in the uk, the maximum abundance of i. ricinus has been observed in april and may and their stages show a lesser resurgence in numbers in late summer (dobson & randolph 2011). in the present study, the overall prevalence of dogs infested by different male tick species was higher than female tick species; however, this was not statistically significant (p = 0.837). similarly, ebrahimzade, fattahi and mohammad (2016) reported no statistically significant difference in the tick burden between the sexes living in the same ecological environments. in addition, rodriguez-vivas et al. (2003) suggested that both sexes are susceptible to ectoparasite infestation. in contrast, mosallanejad et al. (2011) found a significant difference between male and female tick species infested by ectoparasites (35.82% vs. 20.33%, respectively). the proportion of infestation caused by the total number of ticks varied between two age groups of dogs. it was the highest in young and the lowest in adult dogs (p = 0.55). studies have shown that the susceptibility of tick infestation is greater in young animals than in adult animals (moghaddar, shorigeh & gastrodashty 2001; raut et al. 2006). the influence of age has also been reported by a study in tunisia, where the frequency of infestation of adult cattle was higher than that of young animals (bouattour 2001). these results could be because of the immunity system of infected animals which is less developed at an early age (dillard, saari & anttila 2007). there is a significant difference between the breed of animal and the prevalence of tick infestation (p = 0.042). in contrast, no significant effect to bovine (benchikh-elfegoun et al. 2007) and dog (lorusso et al. 2010) breeds on tick infestation was observed in other studies. on the other hand, a previous study reported that dog breeds have a direct influence on the infestation rate (silveira, passos & ribeiro 2009). statistical analysis revealed that german shepherd breeds are the most infested compared to other breeds examined in this study. smith et al. (2011) found that dogs with medium hair length were twice as likely to have ticks than dogs with short hair (p < 0.001). dogs characterised by long hair could provide a conducive environment for tick survival compared to short-haired dogs. it is easy to detect and remove the ticks in the short-haired dogs than those with long hair. also, short-haired dogs may be more effective in self-grooming and may remove ticks themselves. conclusion this study is the first epidemiological investigation conducted on the species of ticks, their prevalence and dynamic in infesting domestic dogs in the bejaia province (northeastern algeria) based on conventional methods. our finding showed the presence of four species infesting dogs, with r. sanguineus being the most abundant. the low frequency of ticks in the study area raises concerns about the endemic presence of certain diseases transmitted to dogs. it is therefore necessary to implement an effective tick control strategy during infestation periods in order to prevent vector-borne diseases. also it is recommended that studies regarding the dynamics of hard ticks in other regions should be conducted to complement the knowledge of ixodidea fauna in algeria. acknowledgements the authors gratefully acknowledge all veterinary colleagues (dr l. hassissen, dr i. debbouze, dr n. djermoune and dr h. ghalem) for the help during the sampling and for their hospitality in veterinary practice. competing interests the authors declare that they have no potential conflict of interest. authors’ contributions r.k. carried out the experimental work and wrote the manuscript. m.n.-m. carried out the statistical analysis. l.h. contributed towards the sampling process. a.a. designed and supervised the experimental study and reviewed the manuscript. all authors have read and approved the final version of the manuscript. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit 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https://doi.org/10.1016/j.vetpar.2009.01.028 smith, f.d., ballantyne, r., morgan, e.r. & wall, r., 2011, ‘prevalence, distribution and risk associated with tick infestation of dogs in great britain’, medical and veterinary entomology 25(4), 377–384. https://doi.org/10.1111/j.1365-2915.2011.00954.x tsatsaris, a., chochlakis, d., papadopoulos, b., petsa, a., georgalis, l., angelakis, e. et al., 2016, ‘species composition, distribution, ecological preference and host association of ticks in cyprus’, experimental and applied acarology 70(4), 523–542. https://doi.org/10.1007/s10493-016-0091-9 walker, a.r., bouattour, a., camicas, j.l., estrada-peña, a., horak, i.j., latif, a.a. et al., 2014, ticks of domestic animals in africa: a guide to identification of species, bioscience reports, edinburgh. wikipedia, the free encyclopedia, municipalities of the province of bejaia, viewed 25 march 2017, from https://fr.wikipedia.org/wiki/communes_de_la_wilaya_de_b%c3%a9ja%c3%afa. xhaxhiu, d., kusi, i., rapti, d., visser, m., knaus, m., lindner, t. et al., 2009, ‘ectoparasites of dogs and cats in albania’, parasitology research 105(6), 1577–1587. https://doi.org/10.1007/s00436-009-1591-x bragg_pp1-6.qxd introduction infectious coryza (ic), caused by the bacterium haemophilus paragallinarum, was first recorded in south africa in 1968 on a multi-age layer farm (buys 1982). the first recorded use of a vaccine against h. paragallinarum in south africa was in 1975 (buys 1982), which appeared to effectively control the disease. however, in the mid 1980s, increasing vaccination failures and outbreaks of ic were reported in south africa bragg, coetzee & verschoor (1996) undertook a serological survey of nad-dependent h. paragallinarum isolates collected over a 30-year period, using the haemagglutination inhibition test first described by kume, sawata, nakase & matsumoto (1983) and modified by blackall, eaves & rogers (1990a). they demonstrated that there had been a significant change in the incidence of the four different south african serovars over a 30-year period from the early 1970s (before the use of vaccines) to the mid 1990s. they demonstrated that the incidence of the nad-dependent serovar a-1 and c-2 isolates had decreased, while the incidence of 1 onderstepoort journal of veterinary research, 72:1–6 (2005) effects of differences in virulence of different serovars of haemophilus paragallinarum on perceived vaccine efficacy r.r. bragg department of microbial, biochemical and food biotechnology, university of the free state p.o. box 339, bloemfontein, 9300 south africa. e-mail: braggrr.sci.@mail.uovs.ac.za abstract bragg, r.r. 2005. effects of differences in virulence of different serovars of haemophilus paragallinarum on perceived vaccine efficacy. onderstepoort journal of veterinary research, 72:1–6 the virulence of four south african field isolates of nad-dependent haemophilus paragallinarum and two field isolates of nad-independent h. paragallinarum has previously been tested in unvaccinated chickens. in this study, the disease profiles caused by the nad-dependent isolates of h. paragallinarum in vaccinated chickens were studied. it was shown that the clinical signs induced in the vaccinated chickens were substantially less severe than were those in unvaccinated chickens, as was expected. however, due to the high virulence of the serovar c-3 isolates, clinical signs in the vaccinated chickens challenged with this isolate were still detected. these were as severe as those occurring in unvaccinated chickens challenged with serovar b-1 isolates. although the clinical signs induced in unvaccinated birds challenged with serovar a-1 were more severe than those occurring when vaccinated birds were challenged with serovar c-3, the overall disease profiles were similar. substantial clinical signs were recorded in vaccinated birds challenged with serovar c-3. this could be interpreted as vaccination failure if the disease profile obtained in unvaccinated birds is not considered. it was found that a high level of protection was provided by this vaccine against challenge by serovar c-3. the high virulence of this serovar resulted in the development of clinical signs in vaccinated birds. these findings could possibly explain the large number of so-called vaccination failures that are reported in south africa. keywords: haemophilus paragallinarum, immunity, infectious coryza, nad-independent isolates accepted for publication 1 july 2004—editor serovar c-3 had increased significantly. serovar c-3 has only been reported in south africa (kume et al. 1983; blackall, eavers & aus 1990b; bragg et al. 1996) and recently in zimbabwe (bragg 2002a). none of the vaccines used at that stage contained serovar c-3 and this prompted bragg et al. (1996) to suggest that the observed changes in incidence were due to the fact that none of the vaccines used contained serovar c-3 and they suggested that there was a need for a local vaccine. bragg (2002b, c) investigated the virulence of naturally occurring nad-dependent isolates of h. paragallinarum and naturally occurring nad-independent isolates of h. paragallinarum in unvaccinated chickens. in these experiments it was demonstrated that the serovar c-3 isolate of nad-dependent h. paragallinarum was highly virulent. the virulence of serovar c-2 was also found to be very high, although lower than that of the serovar c-3 isolate (bragg 2002b). the virulence of serovar a-1 was lower than expected (bragg 2002b), while the low virulence of the south african isolate of serogroup b confirmed the results of yamaguchi, blackall, takigami, iritani & hayashi (1990). when considering the naturally occurring nadindependent isolates, which were first recorded in south africa in the early 1990s (horner, bishop & haw 1992; mouahid, bisgaard, morley, mutters & mannheim 1992; bragg, coetzee & verschoor 1993; horner, bishop, jarvis & coetzee 1995; bragg, greyling & verschoor 1997), it was demonstrated that the virulence of the serogroup a isolates was similar. however, a marked difference in virulence was recorded between the nad-dependent serovar c-3 isolate and the nad-independent isolate. the nad-independent isolate showed much lower virulence. taole, albertyn, van heerden & bragg (2002) demonstrated that laboratory manipulation of naturally occurring nad-dependent serovar c-3 isolates with a plasmid from naturally occurring nad-independent isolates resulted in a lowering of the virulence of the transformed strain. bragg (2004a) demonstrated that the clinical signs caused in vaccinated and unvaccinated chickens challenged with a nad-independent strain of h. paragallinarum were statistically similar, suggesting possible immune evasion by the nad-independent variants. this is in direct contradiction to the finding of jacobs & van der werf (2000) who demonstrated that their vaccine provided protection against the nad-independent isolates of h. paragallinarum. it is important to note that different challenge models were used in the different studies. using the challenge model described by bragg (2002b) in which a numerical score can be given to the clinical signs and a disease profile and mean disease score can be calculated, it would be interesting to investigate the effects that prior vaccination has on the ability of the different serovars to cause disease. the high virulence of the serovar c-3 isolates found by bragg (2002b) raises the question of whether the increased incidence of these isolates in the 1990s (bragg et al. 1996) was due to the use of incorrect strains in the vaccine as proposed by bragg et al. (1996), or due to the elevated virulence of naddependent serovar c-3 isolates as demonstrated by bragg (2002b). the objective of the present investigation was to obtain information pertaining to these questions. materials and methods chickens a total of 80 unvaccinated commercial layer chickens were obtained from a supplier of point-of-lay chickens. these chickens were obtained at 11 weeks of age before they were vaccinated against ic. they were obtained from a farm which had no previous history of ic and were housed in isolated layer facilities at the university of the free state until they were used in the experiments. when the birds were 12 weeks of age, 40 of them were randomly selected and vaccinated with an experimental vaccine that contained isolates of all four south african serovars (a-1, b-1, c-2 and c-3) of h. paragallinarum in aluminum hydroxide which had been specifically selected based on ability to agglutinate red blood cells (bragg, unpublished data 2003). they were each vaccinated subcutaneously with 0.5 ml of the vaccine and subsequently revaccinated at 16 weeks of age with the same experimental vaccine. both the vaccinated and unvaccinated chickens remained in the facilities until they were 25 weeks of age and were producing eggs. when 25 weeks of age, the vaccinated and unvaccinated chickens were randomly assigned to one of four groups, each of which consisted of ten vaccinated and ten unvaccinated chickens. they were then placed into layer cages in the isolation facilities of the university in such a way that all ten birds of each group were in a row of five cages. a total of two chickens were placed into each cage. the vaccinated chickens were placed on one side of the 2 differences in virulence of serovars of haemophilus paragallinarum on perceived vaccine efficacy battery of cages, while the unvaccinated chickens were placed on the opposite side. challenge exposure methods the chickens were challenged with one of the four different serovars of nad-dependent h. paragallinarum isolates according to the methods described by bragg (2002b), without modification. basically, one chicken in the middle cage of the row of five cages was selected and challenged by means of an intra-sinus injection of 0.1 ml of the challenge bacterium. the remaining birds in the group were then infected via an in-contact natural challenge route. the severity of the clinical signs in the birds were recorded and scored for a 20-day period according to the methods described by bragg (2002b). the nad-dependent isolates used for challenge purposes were 49 (a-1), 58 (b-1), 7 (c-2) and 46 (c-3) and were the same isolates used in the production of the experimental vaccine. all the isolates used have previously been described and serotyped and are the same isolates which were used for the establishment of virulence (bragg 2002b). results the results of the scoring of the clinical signs were used to produce a graphic representation of the course of the disease in the vaccinated and unvaccinated chickens. the disease profiles of birds challenged with the nad-dependent serovar a-1 isolate of h. paragallinarum are given in fig. 1, while those of the birds challenged with serovar b-1, c-2 and c3 are reflected in fig. 2, 3 and 4, respectively. a summary of the mean disease scores, as calculated from the disease profiles in vaccinated and unvaccinated chickens challenged with the different serovars appears in table 1, as is the maximum disease score for each of the groups of chickens. the duration of the infection, as calculated from fig.1–4 as the last day on which clinical signs were recorded, is also presented in table 1. the percentage protection, as calculated from the mean disease scores of vaccinated chickens versus that of unvaccinated chickens, as well as the result of the statistical analyses of the disease profiles are given in table 1. a comparison of the clinical disease profile obtained when vaccinated chickens were challenged with serovar c-3 and the unvaccinated profile of chickens challenged with serovar a-1 can be seen in fig. 5. discussion it is evident from the data recorded (fig. 1–4) that distinct statistically significant (table 1) differences in the disease profiles for the vaccinated and unvaccinated chickens were obtained. similar virulence patterns to those reported by bragg (2002b) for nad-dependent strains of h. paragallinarum were recorded in the unvaccinated chickens, although the mean disease scores in this experiment were found to be higher for all the isolates. 3 r.r. bragg � � � � �� � �� �� � � � � � � � � ��� ��� ��� ��� ��� ��� ��� ��� ��� � � �� �� ��� �� �� �� ��� ����������� � � � � � � � � �� �� �� �� �� �� �� �� �� ��� �� � � � � �� � �� �� � � � � � � � � ��� ��� ��� ��� ��� ��� ��� ��� ��� � �� ��� ����������� � � � � � � � � �� �� �� �� �� �� �� �� �� ��� �� � �� �� ��� �� �� ������ ���� �������� ����! �����"#���� �� ��� ���$�� % �� ��� #� &����"���� # ��'���"� #������ ��(%���� ����������� ���� ������ ���� �������� ����! �����"#���� �� ��� ���$�� % �� ��� #� &����"���� # ��'���"� #������ ��)%���� ����������� ���� the levels of protection provided by the experimental vaccine ranged from 67.5 % in the vaccinated chickens challenged with serovar b-1 to 85 % in the birds challenged with serovar a-1 (table 1). the levels of protection obtained when the birds were challenged with the serogroup c isolates were found to be 73.4 % for serovar c-2 and 75.7 % for serovar c-3. these levels of protection are similar to those obtained using the challenge model developed by bragg (2002b) when using commercially available ic vaccines in south africa (data not shown). rimler, davis & page (1977) also found protection levels between 80 % and 100 % when chickens vaccinated with serogroup a isolates were challenged with serogroup a isolates, thus demonstrating that the protection levels obtained with this experimental vaccine are within the acceptable protection range for inactivated bacterial vaccines. when considering the clinical signs manifested by vaccinated chickens challenged with the different serovars of h. paragallinarum, it is noted that substantial clinical signs were still recorded. when they were challenged with serovar a-1, the mean disease score obtained over the 20-day period was only 0.17 with a maximum daily disease score of 1, as compared to a mean disease score of 1.13 with a maximum daily disease score of 3.4 obtained in unvaccinated chickens. this translates into an 85 % level of protection. however, during the peak reaction following challenge, a maximum mean daily disease score of 1 was found in the vaccinated group of chickens. there were thus clinical signs of ic in these chickens, in spite of the fact that 85 % protection was obtained with the vaccine. it has been demonstrated in this experiment and previously by bragg (2002b) that the serotype b 4 differences in virulence of serovars of haemophilus paragallinarum on perceived vaccine efficacy � � � � �� � �� �� � � � � � � � � ��� ��� ��� ��� ��� ��� ��� ��� ��� � � �� �� ��� �� �� �� ��� ����������� � � � � � � � � �� �� �� �� �� �� �� �� �� ��� �� � � � � �� � �� �� � � � � � � � � ��� ��� ��� ��� ��� ��� ��� ��� ��� � � �� �� ��� �� �� �� ��� ����������� � � � � � � � � �� �� �� �� �� �� �� �� �� ��� �� ������ ���� �������� ����! �����"#���� �� ��� ���$�� % �� ��� #� &����"���� # ��'���"� #������ ��*%���� ����������� ���� ������ ���� �������� ����! �����"#���� �� ��� ���$�� % �� ��� #� &����"���� # ��'���"� #������ ��*%���� ����������� ���� table 1 mean disease scores, maximum daily disease score, duration of clinical signs and percentage protection obtained in vaccinated and unvaccinated chickens challenged with the different serovars of h. paragallinarum isolate vaccinated mean* max ** duration# in days % protection significance a-1 no 1.13 3.4 13 a-1 yes 0.17 1.0 6 85.00 p = 0.00016 b-1 no 0.46 2.4 8 b-1 yes 0.13 0.8 6 67.50 p = 0.04919 c-2 no 1.28 3.6 14 c-2 yes 0.34 2.2 7 73.40 p = 0.00348 c-3 no 1.85 4.2 15 c-3 yes 0.45 1.6 11 75.70 p = 0.00036 * = mean disease score ** = maximum daily disease score # = duration of clinical signs isolate in south africa is of low virulence. when vaccinated chickens were challenged with serovar b-1, clinical signs were still apparent despite a 67.5 % level of protection that was provided by the vaccine. it has also been established in this experiment and by (bragg 2004b) that the serogroup c isolates are of high virulence. the clinical signs of ic that developed in vaccinated chickens challenged with serovar c-3, where there was 75.7 % protection, were substantial. a mean disease score of 0.45 was found with the maximum mean daily disease score of 3.6 (table 1) in the vaccinated chickens. the overall disease profiles obtained in this challenge experiment were similar to those obtained when unvaccinated chickens were challenged with serovar b-1 or serovar a-1 (fig. 5). if the clinical signs that developed in vaccinated birds challenged with the c-3 serovar are merely observed, but are not compared to those obtained in the unvaccinated birds challenged with the same isolate, it could be postulated that the vaccine did not provide protection. this hypothesis would be even further strengthened if the clinical signs in the vaccinated chickens challenged with c-3 are compared to those obtained in unvaccinated birds challenged with serovar a-1 or b-1, in which the clinical signs were of similar intensity. evaluations of the efficacy of an ic vaccine based on challenge models other than the one used in this experiment might lead to confusing results. jacobs & van der werf (2000) state that the vaccine used in their experiment provided protection against the different serovars of nad-independent h. paragallinarum found in south africa. bragg (2004a), on the other hand, suggested that the nad-independent strains are capable of evading the immune system. these conflicting results could be a result of the low virulence of the nad-independent isolates. bragg (2002c) found that the naturally occurring nad-independent isolates are of low virulence. taole et al. (2002) were able to convert virulent nad-dependent serovar c-3 isolates to nad-independent isolates using a plasmid and they demonstrated that the conversion of the virulent nad-dependent isolate to nad independence significantly reduced the virulence of the isolate. if a vaccine challenge model is based purely on the observation of clinical signs and does not score the clinical signs, it would seem that the vaccine used provides good protection, as suggested by jacobs & van der werf (2000) when, in fact, the isolate used for challenge is merely of very low virulence, as demonstrated by bragg (2002c) and taole et al. (2002). conclusions without making use of the challenge model developed by bragg (2000b) in which the clinical signs are scored and the mean daily disease score for the group of chickens is plotted graphically, a misconception of the levels of protection afforded by a particular vaccine against a specific challenge can be made. the early work on the ic problem in south africa was based on the assumption that the vaccines used at the time were not providing protection against the serovar c-3 isolate because of the number of serious “vaccine breaks” which were investigated and from which serovar c-3 isolates were made. with the development of this challenge model, it was established that the virulence of the different serovars has a profound impact on the perceived vaccine efficacy. it has been demonstrated in this experiment that the vaccine used provided high levels of protection. however, the virulence of the serogroup c isolates induced substantial clinical signs in the vaccinated chickens. these clinical signs were due to the virulence of the isolates and not to vaccine failure as was previously proposed. the findings in this experiment suggest that it will not be possible to completely control the ic problem in south africa through the use of inactivated vaccines alone. bragg (2004b) has suggested a continual disinfection programme that can be used in conjunction with a vaccination programme to reduce the impact of ic to the poultry producer. another 5 r.r. bragg � � � � �� � �� �� � � � � � � � � ��� ��� ��� ��� ��� ��� ��� ��� ��� � �� ��� ����������� � � � � � � � � �� �� �� �� �� �� �� �� �� ��� �� ��� �� ���(%� ��� �� ���)%� � �� ���*%� ������ ���� ��� ����� ��� �! ����� "#��� � �� ��� #� &��� "���� # ��'��� "� #� ����� �� *%�� ��� $�� �� �� #� &����"���� # ��'���"� #������ ��(%�����)%���� ����������� ���� possible approach in the control ic is through the use of live vaccines. acknowledgements i am grateful to dr freek potgieter of the animal house of the university of the free state for his assistance with the housing of the birds. i am also grateful to the national research foundation for funding aspects of this work. references blackall, p.j., eaves, l.e. & rogers, d.g. 1990a. proposal of a new serovar and altered nomenclature for haemophilus paragallinarum in the kume hemaglutinin scheme. journal of clinical microbiology, 28:1185–1187. blackall, p.j., eavers, e.l. & aus, g. 1990b. serotyping of haemophilus paragallinarum by the page scheme: comparison of the use of agglutination and hemagglutination-inhibition tests. avian diseases, 34:643–645. bragg, r.r., coetzee, l. & verschoor, j.a. 1993. plasmid encoded nad independence in some south african isolates of haemophilus paragallinarum. onderstepoort journal of veterinary research, 60:147–152. bragg, r.r., coetzee, l. & verschoor, j.a. 1996. changes in the incidence of the different serovars of haemophilus paragallinarum in south africa: a possible explanation for vaccination failures. onderstepoort journal of veterinary research, 63:217–226. bragg, r.r., greyling, j.m. & verschoor, j.a. 1997. isolation and identification of nad-independent bacteria from chickens with symptoms of infectious coryza. avian pathology, 26:595–606. bragg, r.r. 2002a. isolation of serovar c-3 haemophilus paragallinarum from zimbabwe: a further indication of the need for local vaccines against infectious coryza. onderstepoort journal of veterinary research, 69:129–132. bragg, r.r. 2002b. virulence of south african isolates of haemophilus paragallinarum. part 1: nad-dependent field isolates. onderstepoort journal of veterinary research, 69: 163–169. bragg, r.r. 2002c. virulence of south african isolates of haemophilus paragallinarum. part 2: naturally occurring nad-independent field isolates. onderstepoort journal of veterinary research, 69:171–175. bragg, r.r. 2004a. evidence of evasion of protective immunity by nad-independent isolates of haemophilus paragallinarum in poultry. onderstepoort journal of veterinary research, 71:53–58. bragg, r.r. 2004b. limitation of the spread and impact of infectious coryza through the use of a continuous disinfection programme. onderstepoort journal of veterinary research, 71:1–8. buys, s.b. 1982. die bereiding van ‘n bakterien teen haemophilus paragallinarum besmetting, geskik vir suid-afrikaanse omstandighede. m.med.vet (bact.) thesis. faculty of veterinary science, university of pretoria. horner, r.f., bishop, g.c. & haw, c. 1992. an upper respiratory disease of commercial chickens resembling infectious coryza, but caused by a v factor independent bacterium. avian pathology, 21:421–427 horner, r.f., bishop, g.c., jarvis, c.j. & coetzee, t.h.t. 1995. nad (v-factor)-independent and typical haemophilus paragallinarum infecting commercial chickens: a five year field study. avian pathology, 24:453–463. jacobs, a.a. & van der werf, j. 2000. efficacy of a commercially available coryza vaccine against challenge with recent south african nad-independent isolates of haemophilus paragallinarum in chickens. journal of the south african veterinary association, 71:109–110. kume, k., sawata, a., nakase, y. & matsumoto, m. 1983. serological classification of haemophilus paragallinarum with a hemagglutinin scheme. journal of clinical microbiology, 17:985–964. mouahid, m., bisgaard, m., morley, a.j., mutters, r. & mannheim, w. 1992. occurrence of v-factor (nad) independent strains of haemophilus paragallinarum. veterinary microbiology, 31:363–368. rimler, r.b., davis, r.b., page, r.k. 1977. infectious coryza: cross-protection studies, using seven strains of haemophilus paragallinarum. american journal of veterinary research, 38:1587–1598. taole, m., albertyn, j., van heerden, e. & bragg, r.r. 2002. virulence of south african isolates of haemophilus paragallinarum. part 3: experimentally produced nadindependent isolate. onderstepoort journal of veterinary research, 69:189–196. yamaguchi, t., blackall, p.j., takigami, s., iritani, y. & hayashi, y. 1990. pathogenicity and serovar-specific hemagglutinating antigens of haemophilus paragallinarum serovar b strains. avian diseases, 34:964–968. 6 differences in virulence of serovars of haemophilus paragallinarum on perceived vaccine efficacy abstract introduction materials and methods blocking enzyme-linked immunosorbent assay technical basis of the test test procedure interpretation of the test result data management and analysis discussion conclusion acknowledgements references about the author(s) hassan y.a.h. mahmoud department of animal medicine, division of infectious disease, faculty of veterinary medicine, south valley university, qena, egypt alsagher o. ali department of animal medicine, division of infectious disease, faculty of veterinary medicine, south valley university, qena, egypt citation mahmoud, h.y.a.h. & ali, a.o., 2021, ‘epidemiology and serological detection of rift valley fever disease in farm animals in southern egypt’, onderstepoort journal of veterinary research 88(1), a1877. https://doi.org/10.4102/ojvr.v88i1.1877 original research epidemiology and serological detection of rift valley fever disease in farm animals in southern egypt hassan y.a.h. mahmoud, alsagher o. ali received: 25 apr. 2020; accepted: 25 sept. 2020; published: 03 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in this study, the serological surveillance of rift valley fever virus (rvfv) in southern egypt was carried out for 460 serum samples collected from domestic animals (unvaccinated), including cattle, sheep, goat, camel and donkey reared in three different provinces (qena, luxor and aswan). enzyme linked immunosorbent assay (elisa) was used to detect rvfv antibodies. the results showed that 97 out of 460 animals were positive by using blocking elisa. the percentage of rvfv infection in cattle, sheep, goat, camel and donkey was 5.55%, 65.21%, 14.44%, 20.65% and 0%, respectively. geographical distribution and breeding system were taken into consideration for rvfv infection in these animals. the most prevalent type of infection was identified in intensive breeding farms systems (27.63%), and then in individual breeding systems (11.68%). qena had a higher infection rate of rvfv (23.55%), in comparison to aswan and luxor (20.65% and 14.14%, respectively). marked seroprevalence recorded in this study indicates a high incidence of infection in sheep (65.21%) and camel (20.65%); this necessitates the application of more effective strategies to control these types of infections in egypt. this study provides a concise picture about the rvfv disease in southern egypt. we need more similar studies targeted to clarify the reliable epidemiological status of rvfv disease in southern egypt and other localities. keywords: farm animals; rvfv; southern egypt; epidemiology; camel; infectious diseases. introduction rift valley fever (rvf) is a vector-borne viral disease caused by rift valley fever virus (rvfv), a member of the bunyaviridae family and phlebovirus genus that primarily affects domestic ruminants, causing large epizootics with high mortality rates in young animals and abortions in affected dams (mansfield et al. 2015; shabani et al. 2015). the virus was discovered in 1930 during an outbreak that affected livestock in east africa (daubney & hudson 1931; boushab et al. 2015). six years later, rvfv antibodies were found in human sera from southern sudan (findlay et al. 1936). the primary major human epidemic occurred in 1951 in south africa (tolou et al. 2009). since then, multiple outbreaks were reported in several parts of africa and the middle east, notably in egypt (1977, 2003) (faye et al. 2007; sissoko et al. 2009), kenya (1997–1998, 2006–2007) (faye et al. 2010), saudi arabia and yemen (2000–2001) (al-hazmi et al. 2003; nabeth et al. 2001), sudan (2007, 2010) (hassan et al. 2011; nabeth et al. 2001), mayotte (2008) (sissoko et al. 2009) and mauritania (1987, 1993–1994, 1998, 2003, 2010, 2012) (boushab et al. 2015; faye et al. 2007; nabeth et al. 2001). the disease occurrence follows the unusual trend of heavy rainfall leading to flooding, resulting in providing a conducive environment for dormant mosquito eggs infected by rvfv to hatch and become predominant mosquito populations that transmit virus to animals and subsequently from animals to humans (shabani et al. 2015). however, currently, there is no proof for person-to-person transmission of rvfv (rakotoarivelo et al. 2011; shabani et al. 2015). in humans, rvfv infection is typically asymptomatic or causes influenza-like illness accompanied by fever and headache but occasionally leads to serious complications, such as haemorrhagic syndromes, retinitis, encephalitis and death (adam, karsany & adam 2010; al-hazmi et al. 2003; mansfield et al. 2015; shabani et al. 2015). during the late 1980s, a new extension of the geographic range of rvfv into western africa was detected. in 2000, rvfv caused an epidemic in saudi arabia and yemen, which was the first time that rvfv was detected outside africa (balkhy & memish 2003; madani et al. 2003). various wildlife species including buffalo and camel have been shown to be seropositive in endemic areas, suggesting a role for these animals in the virus life cycle (britch et al. 2013). rift valley fever virus (rvfv) could also be transmitted to different mosquito species that function as linking vectors to other wild and farm animals and to humans, which can cause more amplification of the transmission cycle (martin et al. 2008). rift valley fever disease (rvfd) is a vector-borne viral disease of domestic ruminants characterised by widespread abortions and infant deaths, and flu-like symptoms. it is endemic in egypt, kingdom of saudi arabia and yemen. outbreak occurs during periods of high downfall or in the environments supporting the proliferation of rvfv-infected dipteran vectors. rvfv is classified as a category (a) priority infective agent by the national institute of allergy and infectious diseases, showing the possibility to cause social disturbances and requiring public health awareness. rvfv is the third most dangerous animal threat according to the united states department of agriculture animal and plant health inspection service. rift valley fever virus is a sensible danger that spreads to new geographic areas through the movement of virus-carrying vectors confined within aircrafts and ship cargo holds and the supporting ecological conditions. vector transmission is poorly understood because of lack of information regarding mosquito ecologies, increasing the risk of entrance of rvfv to a new area. materials and methods animals and geographic locations a total of 460 serum samples were collected from apparently and clinically healthy animals, including cattle, sheep, goat, camel and donkey of different age, sex, breeding system and location. serum samples were collected from animals during the period from may 2017 to june 2019, randomly in different villages in aswan, qena and luxor governorates in southern egypt (figure 1), from individual owners and smallholder farms located in a similar environmental and husbandry conditions, characterised by hot and dry weather. in addition, there were 20 sheep that could not be classified as part of the individual or intensive breeding system. figure 1: map of egypt showing the location of qena, luxor and aswan governates, where the samples were collected. clinical examination clinical examination was performed for animals physically, including breed, age and sex; body condition, temperature, respiratory rate and mucous membranes were examined and recorded for individual animals in this study. blood sampling blood samples were collected through vein puncture from each animal in glass tubes without anticoagulant, and serum was separated by centrifugation and then stored at −20 °c until use. blocking enzyme-linked immunosorbent assay detection of specific antibodies to the n-protein of rvfv antibodies does not depend on the animal species and can therefore be used with sera samples of all species affected by rvfv, according to the manufacturer’s instructions (fvr.k.3/5 ingezim rift valley fever™ compac (immunologiay genetica aplicada s.a, madrid, spain). technical basis of the test the recombinant n-protein of the rvfv was used as the antigen, which is used to bind to a polystyrene plate, and after incubating the sera samples, a rvfv-specific monoclonal antibody (mab) was added. if the sample contains antibodies specific of the virus, they will not allow the binding of the labelled mab to the antigen, whereas if it does not contain specific antibodies, the mab will bind to the antigen, which is coating the plate. by washing the plate to eliminate all non-fixed material, the presence or absence of labelled mab can be detected by adding the substrate, which, in the presence of the peroxidase, will develop a colorimetric reaction. if there has been colour development, it means that the conjugate has bound to the antigen, and therefore the sample is negative. on the other hand, if the sample contains specific antibodies to rvfv, they will block the binding of the conjugate and there will be no colour development. test procedure before starting the test, all reagents should be in room temperature (20 °c – 25 °c) for 20 min. after that add 80 µl of the diluent fluid to each well and then add 20 μl of each sample in every well to be assayed. add 100 μl of positive control (pc) and negative control (nc) to their respective wells, and then seal the plate and incubate for 45 min at 22 °c – 25 °c. wash the plate for four times and then add 100 μl of the ready conjugate to each well, and seal the plate and incubate for 30 min at 22 °c – 25 °c. wash the plate for four times and add 100 μl of the substrate solution to each well and keep the plate at room temperature for 10 min, and then add 100 μl of the stop solution to each well and read the optical density (od) of each well at 405 nm. interpretation of the test the test is considered valid when the od of the nc is higher than 0.8, and the pc is lower than 0.25. calculate the inhibition percentage (ip) of each sample as follows: ip of sample = 100 – ([sample od/nc od] × 100), samples will be considered positive when the ip is ≥ 45%, samples will be considered negative when the ip is ≤ 40%, and samples with an ip between 40% and 45% should be considered as uncertain. ethical consideration the study was reviewed and approved by research code of ethics at the south valley university (rcoe-svu). result sera samples were collected from 460 animals, including cattle, sheep, goat, camel and donkey in south egypt from three governorates (aswan, luxor and qena) (table 1). investigated sera of animals revealed that the seropositive state of animals against rvfv was 21.08% (97/460), in overall samples, and, that for each species, cattle, sheep, goat, camel and donkey were 5.55% (5/92), 65.21% (60/92), 14.44% (13/92) and 0% (0/92), respectively (table 2). epidemiological status was established to clarify the influence of age, sex, location and management system (individual/intensive farming system). it revealed that qena had a higher infection rate (23.55%) than aswan and luxor, 20.65% and 14.14%, respectively (table 3). marked infection was recorded in sheep (65.21%) and camels (20.65%), more than goats (14.44%), cattle (5.55%) and donkeys (0.0%). table 1: classification of animals according to geographical distribution, age, sex and breeding system. table 2: infection rate of rift valley fever in cattle, sheep, goat, camel and donkey. table 3: rift valley fever infection in animals regard to location, age, sex and breeding system. data management and analysis the data was collected and analysed using microsoft 2016 excel (http://www.microsoft.com/en-eg/download/details.aspx?id=50283) discussion arboviruses are one amongst the re-emerging pathogens that recently produce unhealthiness everywhere. one-hundred and fifty arboviruses are documented to cause sickness in humans, and the majority is animal diseases, supported by exceeding transmission cycle between arthropods as vectors and vertebrate creature reservoirs as the main amplifying hosts. rvfv is one amongst the foremost aggressive migrating arboviruses. it is transmitted in an enzootic cycle amongst wildlife and mosquitoes, besides wild and farm animals (figueiredo 2007). camel is believed to be resistant to rvfv under clinical conditions, with no apparent infection (swanepoel & coetzer 2004). however, clinical signs together with fever and abortion in approximately 10% of pregnant she-camels were determined in free-ranging herds during the 2006–2007 outbreak in kenya (munyua et al. 2010). proof of rvfv circulation amongst domestic camels has also been reported (munyua et al. 2010; nabeth et al. 2001; olaleye, tomori & schmitz 1996). the current study revealed that the seroprevalence of rvfv in camels was 21.11%; this result is considered as a high percentage of seropositivity, indicating camels act as reservoir for maintenance of rvfv and high precautions should be applied as camels are used in egypt for meat and milk production and in aswan and luxor for entertainment. the routes of spreading detected to date appear to be in parallel with the migration routes of camels. therefore, there is some smart proof that viremic, non-symptomatic infected camels transported the virus to egypt and probably conjointly to the peninsula (hoogstraal et al. 1979). horses appear to be resistant to rvfv (swanepoel & coetzer 2004), and experimental infections didn’t result in symptoms or viremia (daubney & hudson 1931). however, within the lower nile river, rvfv antibodies are detected in horses and donkeys (eisa 1984; meegan, hoogstraal & moussa 1979). moreover, in nigeria, rvfv antibodies were detected in horses using a complement fixation test (olaleye et al. 1989; olaleye et al. 1996), and the virus was isolated in nigeria from sheep around 1959; however, this wasn’t related to an epizootic event (adeyeye, ekong & pilau 2011), and serological results in this time that were obtained from zebras (equus burchelli) were negative. this result may clarify that equine might not support rvfv. in this study, the seroprevalence of rvfv in donkey was 0%, indicating that donkeys in egypt do not maintain rvfv. the result from the examination of cattle, sheep and goat sera revealed that 5 (5.55%), 60 (65.21%) and 13 (14.44%), respectively, were seropositive for rvfv. according to the geographical distribution, qena had a higher prevalence of rvfv (23.55%) than aswan (20.65%) and luxor (14.14%). in regard to the breeding system, there is a higher prevalence of seropositivity in intensive breeding system (27.63%) than individual breeding system (11.68%). the result for farm animals in the southern part of egypt leads us to indicate that there is no risk factor either from the breeding system or geographic location in the southern part of egypt. this may be because there is no high difference in temperature and climate in this area. the geographic distribution of rvf has changed widely from its discovery in 1931 (daubney & hudson 1931). rift valley fever disease started in eastern africa, then it emerged throughout africa as well as to madagascar and the southern part of egypt. the first step in the control of rvfv disease is early detection of the disease in animals by applying active police roles for investigation and herd monitoring (davies & martin 2006). in addition, there is transovarian transmission in arthropod and it is a crucial mechanism for the persistence of rvfv in endemic areas (linthicum et al. 1988). but transovarian transmission alone is not enough to control of rvfv endemicity over long periods. it should be investigation of different host, including wild animals and domestic mammals, between the large-scale of outbreak. conclusion this study provides valuable data on the high prevalence of rvfv in sheep and camel in southern egypt; this will assist in the development of prevention and control strategies for the disease. the high prevalence of rvfv antibodies in camel might be the principal factor limiting the livestock industry in egypt. more research and effort are needed from governmental and non-governmental authorities to minimise the economic losses caused by rvfv, in addition to the zoonotic risks from rvfv infections. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions both authors contributed equally to this work. funding 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2004, ‘rift valley fever’, in j.a.w. coetzer, g.r. thomson & r.c. tustin (eds.), infectious diseases of livestock, pp. 1037–1070, oxford university press, oxford. mcgaw_129-134.qxd introduction gunnera is the only genus of the family gunneraceae, which includes 30–40 herbaceous species, mostly distributed in the southern hemisphere (wanntorp, wanntorp, oxelman & kãllersjö 2001). the greatest diversity is found in south and central america. a few species are also found in hawaii, new zealand, tasmania, africa and the malay archipelago (wanntorp et al. 2001). gunnera perpensa l. is widely distributed in the southern, eastern and northern parts of south africa and northwards into tropical africa (van wyk, van oudtshoorn & gericke 1997). it is a vigorous perennial herb that is restricted to moist habitats and grows in marshy areas and along river banks. it has large, round, pumpkin-like leaves and fleshy, pink tuberous rhizomes (van wyk & gericke 2000). the plant is known in south africa by various names including ugobho (zulu), qobo (sotho), rambola-vhadzimu (venda), river pumpkin (english) and rivierpampoen (afrikaans) (van wyk et al. 1997). 129 onderstepoort journal of veterinary research, 72:129–134 (2005) is the use of gunnera perpensa extracts in endometritis related to antibacterial activity? l.j. mcgaw1, r. gehring2, l. katsoulis3 and j.n. eloff1* abstract mcgaw, l.j., gehring, r., katsoulis, l. & eloff, j.n. 2005. is the use of gunnera perpensa extracts in endometritis related to antibacterial activity? onderstepoort journal of veterinary research, 72:129–134 rhizome extracts of gunnera perpensa are used in traditional remedies in south africa to treat endometritis both in humans and animals. an investigation was undertaken to determine whether this plant possesses antibacterial activity, which may explain its efficacy. gunnera perpensa rhizome extracts were prepared serially with solvents of increasing polarity and tested for antibacterial activity. test bacteria included the gram-positive enterococcus faecalis and staphylococcus aureus and the gram-negative escherichia coli and pseudomonas aeruginosa. a moderate to weak level of antibacterial activity in most of the extracts resulted, with the best minimal inhibitory concentration (mic) value of 2.61 mg ml–1 shown by the acetone extract against s. aureus. the extracts were also submitted to the brine shrimp assay to detect possible toxic or pharmacological effects. all the extracts were lethal to the brine shrimp larvae at a concentration of 5 mg ml–1. the acetone extract was extremely toxic at 1 mg ml–1, with some toxicity evident at 0.1 mg ml–1. the remainder of the extracts generally displayed little activity at concentrations lower than 5 mg ml–1. in summary, the results indicate that although the extracts demonstrated a level of pharmacological activity, the relatively weak antibacterial activity is unlikely to justify the use of g. perpensa rhizomes in the traditional treatment of endometritis. rather, the slightly antibacterial nature of the rhizomes may contribute to an additive effect, along with their known uterotonic activity, to the overall efficacy of the preparation. keywords: antibacterial, brine shrimp, endometritis, ethnoveterinary, gunnera perpensa, traditional medicine * author to whom correspondence is to be directed. e-mail: kobus.eloff@up.ac.za 1 programme for phytomedicine, department of paraclinical sciences, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, 0002 south africa 3 department of pharmacology, university of witwatersrand, johannesburg, 4000 south africa accepted for publication 19 january 2005—editor infusions or decoctions of the rhizome of g. perpensa are used by black south african women to induce or augment labour and as a postnatal medication to expedite the expulsion of the after-birth (hutchings, scott, lewis & cunningham 1996; kaido, veale, havlik & rama 1997; van wyk et al. 1997; van wyk & gericke 2000). decoctions are also administered to animals by the zulu and southern sotho to assist the expulsion of the placenta (watt & breyer-brandwijk 1962). according to anecdotal reports, this herbal remedy is used to treat cattle by subsistence farmers and also by commercial farmers of the underberg region of kwazulunatal, south africa to assist in the expulsion of fetal membranes, and in the treatment and prevention of endometritis (t. collins, unpublished data 2002). conventional treatment of retained foetal membranes and endometritis in cattle includes manual removal of the membranes, stimulation of uterine contraction and stimulation of uterine defences as well as topical and systemic antibacterial therapy (kudlác 1991). the use of g. perpensa for treating humans and animals may therefore be due to antibacterial effects. in a preliminary screening, mcgaw, jäger & van staden (2000) reported antibacterial activity of the aqueous rhizome extract against one bacterial pathogen, the gram-positive staphylococcus aureus. however, no activity was demonstrated against bacillus subtilis (gram-positive) or the gramnegative bacteria e. coli and klebsiella pneumoniae. the chemistry of gunnera species appears not to have been thoroughly studied, although watt & breyer-brandwijk (1962) mention the presence of a bitter principle, celastrin, in g. perpensa. doyle & scogin (1988) detected caffeic acid, ellagic acid, ellagitannins and quercetin in gunnera densiflora and gunnera manicata. alkaloids, iridoids, proanthocyanidins, saponins and sedoheptulose were not present in these species (doyle & scogin 1988). the compounds present in these two gunnera species that have antibacterial activity, are caffeic acid (thiem & goslinska 2004), ellagic acid (ohemeng, schwender, fu & barrett 1993), ellagitannins (nishizawa, nakata, kishida, ayer & browne 1990) and quercetin (basile, sorbo, giordano, ricciardi, ferrara, montesano, castaldo cobianchi, vuotto & ferrara 2000). regarding pharmacological activity, kaido et al. (1997) reported on the uterotonic activity of aqueous root decoctions of g. perpensa. joseph & mafatle (1994) considered the effects of intraperitoneal administration of the aqueous extracts of rhizomes of the plant on the maintenance of pregnancy and parturition in rats, and discovered that the extract had a significant deleterious effect on foetuses. the authors postulated that the presence of celastrin or an unknown chemical in the extract was sufficient to kill the unborn rats, but was not enough to kill the adults (joseph & mafatle 1994). antithrombin activity has been detailed in a related species, gunnera tinctoria (de medeiros, macedo, contancia, nguyen, cunningham & miles 2000). recently, khan, peter, mackenzie, katsoulis, gehring, munro, van heerden & drewes (2004) isolated a major constituent, z-venusol, along with the minor components pyrogallol, succinic acid, lactic acid, and the trimethyl ether of ellagic acid glucoside from the aqueous extract of g. perpensa rhizomes. these authors demonstrated the stimulation of a direct contractile response of isolated rat uterus by the aqueous rhizome extract, which also induced continuous uterine contractility after removal of all additives from the organ bath (khan et al. 2004). interestingly, venusol did not trigger the contractile response but induced the state of continuous contractility after flushing of the organ bath (khan et al. 2004). because endometritis is an important economic disease in stock farming, and resource-poor farmers frequently do not have access to orthodox veterinary medicine, we examined the biological activity of g. perpensa in more depth as part of an ethnoveterinary medicine project. materials and methods collection of plant material gunnera perpensa l. rhizomes were collected in may 2002 from the underberg region of kwazulunatal. they were washed, air-dried and cut into small pieces of c. 1 cm3 using an electric band saw. the material was then milled in a rotomill (macsalab) and passed through a 1 mm mesh. preparation of extracts the dried powder (35.7 g) was extracted by vigorous shaking for 15 min with 150 ml hexane. the extract was removed by vacuum filtration through whatman no. 1 filter paper using a buchner funnel. the extraction process was repeated on the marc until the extract had little colour. extraction was continued on the marc in a serial manner using dichloromethane, acetone, methanol and 20 % ethanol in water (ethanol/water). the extracts were subsequently dried using a rotary vacuum evaporator (büchi) at 40 °c. 130 gunnera perpensa extracts in endometritis ´ ´ antibacterial screening the dried plant extracts were dissolved to a known concentration. the acetone and ethanol/water extracts were dissolved in acetone and water respectively, while the remainder of the extracts were suspended in a small volume of dimethyl sulfoxide (dmso) and made up to the required concentration with water. the test organisms in the investigation of antibacterial activity included two gram-positive bacteria, enterococcus faecalis (atcc 29212) and staphylococcus aureus (atcc 29213), and two gram-negative species, pseudomonas aeruginosa (atcc 27853) and escherichia coli (atcc 35219). the specific strains used are recommended for antibacterial activity testing by the national committee for clinical laboratory standards (nccls 1990). these bacteria are responsible for most nosocomial diseases in hospitals (sacho & schoub 1993). the tetrazolium violet serial dilution microplate method of eloff (1998) was used to evaluate the antibacterial activity of the extracts. this method allows the calculation of minimal inhibitory concentration (mic) values for active plant extracts against bacterial species. freshly inoculated bacterial cultures in müller-hinton (mh) broth were incubated overnight at 37 °c. the cultures were diluted (1:100) with mh broth before use in the assay. a twofold serial dilution of plant extract (100 µl) was prepared in 96-well microtitre plates, and 100 µl bacterial culture was added to each well. the presence of bacterial growth was detected by adding to each well 40 µl p-iodonitrotetrazolium violet (int, 0.2 mg ml–1) that is reduced to a red-coloured formazan by respiring bacteria. when there was a substantial decrease in colour 1 hour after addition of the int, the concentration in that well was taken to be the mic of the plant extract against the particular bacterial strain. the antibiotic neomycin was included as a standard in each assay, and appropriate negative solvent controls were included. the direct thin-layer chromatography (tlc) bioautography assay (hamburger & cordell 1987) was employed to determine the number of antibacterial compounds and their rf values in the gunnera extracts. in this assay, bacterial cultures were incubated in mh broth overnight at 37 °c, centrifuged, and the resulting pellet resuspended in fresh mh broth. the bacteria were sprayed onto a thoroughly dried tlc chromatogram and incubated overnight at 37 °c. a solution of int (2 mg ml–1) was sprayed on the plate and incubated again at 37 °c for approximately an hour. white spots appearing on a pink background of bacterial growth indicated the presence of antibacterial compounds on the chromatogram. screening for toxicity the possible toxicity of the extracts was investigated using the brine shrimp (artemia salina) microwell cytotoxicity bioassay (solís, wright, anderson, gupta & phillipson 1993). brine shrimp eggs were obtained from a local pet shop and hatched in artificial seawater (3.8 g nacl per 100 ml distilled h2o). after 48 h, the phototropic nauplii (first stage larvae) were collected using a pasteur pipette. the plant extracts were diluted to concentrations of 0.1, 1, 5 and 10 mg ml–1 with the artificial seawater. for each concentration, the plant extract solution was placed in two replicate wells of a 96-well microtitre plate. a suspension of nauplii (100 µl, containing approximately 10–15 nauplii) was added to each well. the microplate was covered and incubated for 24 h at room temperature. the number of dead and live nauplii in each well was counted using a dissecting microscope. if control deaths occurred, the percent death values were corrected using abbott’s formula: corrected mortality percentage: m – m x 100 s m= mean percentage of dead larvae in treated tubes m= mean percentage of dead larvae in controls s = mean percentage of living organisms in controls podophyllotoxin (sigma) at a concentration of 5 µg ml–1 was used as a positive control and solvent blanks were included in each assay. results and discussion antibacterial activity the mic values for the different bacterial species varied from 2.6–12.5 mg ml–1 (table 1). the mic value was higher than 12.5 mg ml–1 for all the extracts on p. aeruginosa and for the hexane extract on e. coli. mcgaw et al. (2000) reported that ethanol and water extracts of g. perpensa displayed antibacterial activity against s. aureus, with mic values of 3.13 and 0.78 mg ml–1, respectively. these extracts 131 l.j. mcgaw et al. were prepared using separate aliquots of plant material, in contrast to the present investigation where the extracts were prepared serially with the same dried, ground plant material. the first extracting solvent used was hexane followed by the other solvents with increasing polarity on the same plant material. this may partially explain the absence of activity in the 20 % ethanol extract as the preceding solvents possibly removed some of the antibacterial compounds. the extract prepared using acetone had the highest antibacterial activity against the gram-positive bacteria. gram-positive species are generally more susceptible than gram-negative species to plant extracts (vlietinck, van hoof, totté, lasure, van den berghe, rwangabo & mvukiyumwami 1995), and this was also true in our experiments. according to the mic level of 8 mg ml–1 to denote antibacterial activity of plant extracts suggested by fabry, okemo & ansorg (1998) most extracts were inactive or had borderline antibacterial activity. the magnitude of the antibacterial activity depends not only on the mic value but also on the total quantity of the specific extract obtained from the plant. the total activity can be calculated from the quantity in mg extracted from 1 g divided by the mic in mg ml–1 (eloff 2000). the resultant value reflects the volume in ml to which the antibacterial compound can be diluted and still inhibit the growth of 132 gunnera perpensa extracts in endometritis fig. 1 gunnera perpensa extracts separated on a tlc plate a: solvent system bea (benzene:ethyl acetate:ammonia = 18:2:0.2) and sprayed with e. coli. b: solvent system cef (chloroform:ethyl acetate:formic acid = 10:8:2) and sprayed with s. aureus. extracts: h = hexane, d = dichloromethane, ac = acetone, m = methanol, ew = ethanol/water (20 %) h d ac m ew h d ac m ew a b table 1 average mic values for g. perpensa extracts (serial extraction) escherichia coli enterococcus pseudomonas staphylococcus faecalis aeruginosa aureus extracting solvent mg extracted mica total mic total mic total mic total from 1 g mg ml–1 activity mg ml–1 activity mg ml–1 activity mg ml–1 activity ml g–1 ml g–1 ml g–1 ml g–1 hexane 1.46 –b – 12.5 0.12 – – 6.25 0.23 dichloromethane 3.78 12.5 0.30 5.21 0.73 – – 6.25 0.60 acetone 69.38 12.5 5.55 4.17 16.64 – – 2.61 26.58 methanol 54.63 6.25 8.74 3.13 17.45 – – 3.13 17.45 ethanol/water 21.50 – – – – – – – – (20 %) neomycin (x 10–3) 1.56 6.25 25 0.78 a mic is taken as last concentration of plant extract to completely inhibit bacterial growth (= mlc, minimum lethal concentration) b mic > 12.5 mg ml–1 the test organism. the highest total activity value of 26.6 ml g–1 for the acetone extract on s. aureus compares poorly with values of 182 found for combretum erythrophyllum extracts on s. aureus and e. coli (eloff 1999) and of values higher than 258 ml g–1 for cassine peragua var. peragua (eloff 2000). the bioautography plates are depicted in fig. 1. the serial extraction led to some fractionation of the antibacterial compounds in g. perpensa (fig. 1). the presence of antibacterial compounds on the bioautogram with activity against e. coli and s. aureus supports the results obtained in the microtitre plate assay, with the hexane, dichloromethane and acetone extracts showing antibacterial activity. one compound with major activity (dichloromethane extract: rf = 0.28) and at least three compounds with minor activity (dichloromethane extract: rf = 0.65; hexane extract: rf = 0.31, rf = 0.48) against e. coli are present. the growth of s. aureus was inhibited strongly by at least four compounds (dichloromethane extract: rf = 0.32, rf = 0.46, rf = 0.63, rf = 0.77). in all cases the active compounds were relatively non-polar. it has been shown that g. densiflora and g. manicata contain caffeic acid, ellagic acid, ellagitannins and quercetin (doyle & scogin 1988), all of which are known to have antibacterial activity. if a chemotaxonomic relationship exists between g. perpensa and these two related species, it is possible for similar compounds to be responsible for the antibacterial activity of g. perpensa. brine shrimp assay the bioassay against brine shrimp larvae (nauplii) has been employed to test for the presence of toxic substances, and also as a means of facilitating the isolation of biologically active substances (maclaughlin 1991, cited by solís et al. 1993). this assay can be used to evaluate plants for pharmacological activity, taking into account the principle that pharmacology is merely toxicology at a lower dose (vlietinck & apers 2001). all of the gunnera extracts studied were toxic at a concentration of 10 mg ml–1 (table 2). at 1 and 0.1 mg ml–1, the hexane and dichloromethane extracts had no effect on the nauplii. this indicates that highly non-polar compounds from g. perpensa rhizomes are not toxic to brine shrimp nauplii. the acetone extract appeared to be the most toxic, as at a concentration of 1 mg ml–1, no nauplii survived. in relation to results obtained for other plant extracts, the g. perpensa extracts are not regarded as being highly toxic. for instance, wanyoike, chhabra, lang’at-thoruwa & omar (2004) reported toxic effects against brine shrimp larvae by a selection of kenyan medicinal plant extracts at concentrations of less than 0.03 mg ml–1. further tests should be performed to evaluate the potential mammalian toxicity of g. perpensa extracts, since toxicity against brine shrimp larvae is merely a preliminary indication of the likely presence of toxic compounds in an extract. nevertheless, the toxic level for brine shrimp larvae is probably considerably higher than the therapeutic dose used on animals. conclusions gunnera perpensa contains several compounds with antibacterial activity against e. coli and s. aureus. based on the mic values and total activity, it appears unlikely that the purported efficacy of g. perpensa extracts could be due solely to an antibacterial effect. the total antibacterial activity of the extracts was substantially lower than values found for many other plants. if one considers that aqueous extracts are used by subsistence level farmers and that the bioactive compounds according to the bioautography results were all relatively non-polar compounds this also indicates that efficacy can probably not be ascribed to antibacterial effects of the plant extract. on the other hand mcgaw et al. (2000) found that water extracts had a higher activity than ethanol extracts and thus in this study it may be that first extracting plant material with non133 l.j. mcgaw et al. table 2 percentage mortality of gunnera perpensa extracts against artemia salina (brine shrimp) larvae percentage mortality at various concentrations of extract extract 0.1 mg ml–1 1 mg ml–1 5 mg ml–1 10 mg ml–1 hexane 0 0 14 24 dichloromethane 0 0 92 100 acetone 24 100 100 100 methanol 12 69 100 100 ethanol/water 17 36 100 100 polar extractants may have inhibited the solubility or activity of antibacterial compounds present in the g. perpensa rhizomes. in the serial extraction procedure, acetone and methanol may have already extracted the more polar active compounds in the rhizomes before the ethanol/water extract was prepared. the application of gunnera perpensa in treating endometritis and related ailments may therefore largely be due to its previously reported uterotonic activity (kaido et al. 1997), rather 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of five kenyan medicinal plants. journal of ethnopharmacology, 90:129–133. watt, j.m. & breyer-brandwijk, m.g. 1962. the medicinal and poisonous plants of southern and eastern africa. london: e. s. livingstone. 134 gunnera perpensa extracts in endometritis ´ ´ ojvr 87-1_2020_contents.indd http://www.ojvr.org open access table of contents original research failure of diplodiatoxin to induce diplodiosis in juvenile goats christo j. botha, louis g.j. ackerman, mxolisi g. masango, luke f. arnot onderstepoort journal of veterinary research | vol 87, no 1 | a1712 | 05 march 2020 original research seroprevalence and associated risk factors of rift valley fever in cattle and selected wildlife species at the livestock/wildlife interface areas of gonarezhou national park, zimbabwe masimba ndengu, gift matope, musavengana tivapasi, davies m. pfukenyi, catherine cetre-sossah, michel de garine-wichatitsky onderstepoort journal of veterinary research | vol 87, no 1 | a1731 | 08 april 2020 original research in vitro propagation and genome sequencing of three ‘atypical’ ehrlichia ruminantium isolates junita liebenberg, helena c. steyn, antoinette i. josemans, erika faber, erich zweygarth onderstepoort journal of veterinary research | vol 87, no 1 | a1769 | 24 june 2020 original research genetic diversity of ehrlichia ruminantium field strains from selected farms in south africa helena c. steyn, alri pretorius onderstepoort journal of veterinary research | vol 87, no 1 | a1741 | 25 june 2020 original research antibiotic resistance profile of gram-negative bacteria isolated from lafenwa abattoir effluent and its receiving water (ogun river) in abeokuta, ogun state, nigeria samuel n. akpan, olubusola a. odeniyi, oluwawemimo o. adebowale, selim a. alarape, olanike k. adeyemo onderstepoort journal of veterinary research | vol 87, no 1 | a1854 | 15 september 2020 original research study on the prevalence and genetic diversity of eimeria species from broilers and free-range chickens in kwazulu-natal province, south africa abiodun j. fatoba, oliver t. zishiri, damer p. blake, sunday o. peters, jeffrey lebepe, samson mukaratirwa, matthew a. adeleke onderstepoort journal of veterinary research | vol 87, no 1 | a1837 | 17 september 2020 original research protective efficacy of inactivated newcastle disease virus vaccines prepared in two different oil-based adjuvants oday a. aljumaili, muhammad b. bello, swee k. yeap, abdul r. omar, aini ideris onderstepoort journal of veterinary research | vol 87, no 1 | a1865 | 28 september 2020 original research implications of a conserved region of bluetongue virus protein vp2 in cross-neutralisation of bluetongue virus serotypes shiva j. jyothi, sunil r. patil, narasimha y. reddy, rao p. panduranga, uma madala, gnana m. prakash, kalyani putty onderstepoort journal of veterinary research | vol 87, no 1 | a1816 | 08 october 2020 69 73 80 94 106 112 122 129 page i of ii table of contents review article challenges for controlling bovine tuberculosis in south africa luke f. arnot, anita michel onderstepoort journal of veterinary research | vol 87, no 1 | a1690 | 27 february 2020 review article a review of listeria monocytogenes from meat and meat products: epidemiology, virulence factors, antimicrobial resistance and diagnosis itumeleng matle, khanyisile r. mbatha, evelyn madoroba onderstepoort journal of veterinary research | vol 87, no 1 | a1869 | 09 october 2020 review article zoonotic origins and animal hosts of coronaviruses causing human disease pandemics: a review abdalla a. latif, samson mukaratirwa onderstepoort journal of veterinary research | vol 87, no 1 | a1895 | 21 december 2020 research communication survey of the knowledge, attitude and perceptions on bovine tuberculosis in mnisi community, mpumalanga, south africa rudo marange, darshana morar-leather, folorunso o. fasina onderstepoort journal of veterinary research | vol 87, no 1 | a1808 | 30 july 2020 research communication experimental infection of tigerfish (hydrocynus vittatus) and african sharp tooth catfish (clarias gariepinus) with trichinella zimbabwensis louis j. la grange, samson mukaratirwa onderstepoort journal of veterinary research | vol 87, no 1 | a1876 | 05 november 2020 original research an assessment of bovine herpes virus 4 as a causative agent in abortions and neonatal death seval b. dağalp, ali r. babaoglu, firat doğan, touraj a. farzani, feray alkan onderstepoort journal of veterinary research | vol 87, no 1 | a1761 | 06 february 2020 original research validating a non-invasive technique for monitoring physiological stress in the samango monkey juan scheun, adrian s.w. tordiffe, kirsten wimberger, andre ganswindt onderstepoort journal of veterinary research | vol 87, no 1 | a1720 | 27 february 2020 original research investigating selective media for optimal isolation of brucella spp. in south africa maphuti b. ledwaba, okechukwu c. ndumnego, itumeleng matle, awoke k. gelaw, henriette van heerden onderstepoort journal of veterinary research | vol 87, no 1 | a1792 | 04 march 2020 1 9 29 38 42 47 52 60 vol 87, no 1 (2020) issn: 0030-2465 (print) | issn: 2219-0635 (online)onderstepoort journal of veterinary research http://www.ojvr.org open access table of contents original research detection and characterisation of sheep-associated malignant catarrhal fever infection from ruminants by using tegument and gb gene sequences of ovhv-2 tuba ç. oğuzoğlu, seçkin salar, ece adıgüzel, cansu demirden, onur ülgenalp onderstepoort journal of veterinary research | vol 87, no 1 | a1886 | 11 november 2020 original research a report on the housing vervet monkeys adjacent to domestic cats as a means of environmental enrichment john k. chipangura, andre ganswindt, vinny naidoo onderstepoort journal of veterinary research | vol 87, no 1 | a1870 | 26 november 2020 original research detection of peste des petits ruminants virus in pneumonic lungs from apparently healthy sheep and goats slaughtered at al-hasaheisa slaughterhouse, gezira state, central sudan alaa e.m. alhussain, nahid a.s. abdalla, sana i. mohammed, mihad hakeem, ihsan h. ahmed, nussieba a. osman onderstepoort journal of veterinary research | vol 87, no 1 | a1892 | 17 december 2020 135 139 145 original research seroprevalence and risk factors for trypanosoma evansi, the causative agent of surra, in the dromedary camel (camelus dromedarius) population in southeastern algeria mohammed h. benaissa, nora mimoune, younes bentria, tahar kernif, abdelaziz boukhelkhal, curtis r. youngs, rachid kaidi, bernard faye, youcef halis onderstepoort journal of veterinary research | vol 87, no 1 | a1891 | 21 december 2020 reviewer acknowledgement onderstepoort journal of veterinary research | vol 87, no 1 | a1912 | 20 december 2020 151 160 page ii of ii mahan_119-128.qxd introduction canine monocytic ehrlichiosis caused by ehrlichia canis is an infectious, non-contagious, tick-transmitted disease of dogs. it has a worldwide distribution, except australia and new zealand, and closely follows the distribution of the tick vectors, rhipicephalus sanguineus and dermacentor variabilis (donatien & lestoquard 1935; lewis, hill & ristic 1978; keefe, holland, salyer & ristic 1982; kelly 2000). clinically, the disease is characterized by three successive phases, the acute, the subclinical and the chronic phase (kelly 2000). haematological changes are evident in all phases and are characterized by thrombocytopenia, mild anaemia and variable leukocyte responses (walker, rundquist, 119 onderstepoort journal of veterinary research, 72:119–128 (2005) a preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated ehrlichia canis organisms sunita mahan1, p.j. kelly1, 2 and s.m. mahan3* abstract mahan sunita, kelly, p.j. & mahan, s.m. 2005. a preliminary study to evaluate the immune responses induced by immunization of dogs with inactivated ehrlichia canis organisms. onderstepoort journal of veterinary research, 72:119–128 ehrlichia canis is an intracellular pathogen that causes canine monocytic ehrlichiosis. although the role of antibody responses cannot be discounted, control of this intracellular pathogen is expected to be by cell mediated immune responses. the immune responses in dogs immunized with inactivated e. canis organisms in combination with quil a were evaluated. immunization provoked strong humoral and cellular immune responses, which were demonstrable by western blotting and lymphocyte proliferation assays. by western blotting antibodies to several immunodominant e. canis proteins were detected in serum from immunized dogs and antibody titres increased after each immunization. the complement of immunogenic proteins recognized by the antisera were similar to those recognized in serum from infected dogs. upon challenge with live e. canis, rapid anamnestic humoral responses were detected in the serum of immunized dogs and primary antibody responses were detected in the serum from control dogs. following immunization, a lymphocyte proliferative response (cellular immunity) was detected in peripheral blood mononuclear cells (pbmns) of immunized dogs upon stimulation with e. canis antigens. these responses were absent from non-immunized control dogs until after infection with live e. canis, when antigen specific-lymphocyte proliferation responses were also detected in the pbmns of the control dogs. it can be thus concluded that immunization against canine monocytic ehrlichiosis may be feasible. however, the immunization regimen needs to be optimized and a detailed investigation needs to be done to determine if this regimen can prevent development of acute and chronic disease. keywords: ehrlichia canis, canine monocytic ehrlichiosis, immune responses, immunization * author to whom correspondence is to be directed. university of florida heartwater control research program, postnet suite 294, private bag x06, waterkloof, pretoria, 0145 south africa. e-mail: sumanmah@mweb.co.za 1 faculty of veterinary medicine, university of zimbabwe, mt. pleasant, harare, zimbabwe 2 current address: ross university school of veterinary medicine, west farm, basseterre, st. kitts, west indies 3 uf/usaid/sadc heartwater research project, central veterinary diagnostic and research laboratory, p.o. box cy 551, causeway, harare, zimbabwe accepted for publication 10 january 2005—editor taylor, wilson, andrews, barck, hogge, huxoll, hildebrandt & nims 1970; amyx, huxoll, zeiler & hildebrandt 1971; seamer & snape 1972). the acute phase usually resolves spontaneously into a subclinical phase, followed by the chronic phase in dogs which are unable to mount an effective immune response against the parasite. the chronic phase may be mild or severe depending on the strain of the parasite, age and breed of the animal, as well as the presence of concurrent disease. the chronic severe form is characterized by weight loss, pale mucous membranes, abdominal tenderness and bleeding tendencies with epistaxis reported in 30– 50 % of the cases (greene & harvey 1984; kelly 2000). it has been reported that german shepherds are more prone to the chronic severe form of the disease, associated with specific and non-specific immunosupression induced by the e. canis infection (huxoll, amyx, hemelt, hildebrandt, nims & gouchenour 1972; nyindo, huxoll, ristic, kakoma, brown, carson & stephenson 1980; ristic & holland 1993). immunization against e. canis infection is not available at present. current methods of disease prevention include tick control by routine use of acaricides and prophylactic treatment with tetracycline. treatment with tetracycline, doxycycline and imidocarb dipropionate is effective against e. canis infection, although this is not without limitations, especially in the chronic severe form of the disease. there is one anecdotal report of unsuccessful immunization of dogs against canine ehrlichiosis with inactivated cell culture-derived e. canis antigens using an adjuvant that enhances humoral immunity (ristic & holland 1993). although both humoral and cell mediated immune responses are provoked during infection, it is documented that humoral immunity plays little role in protection against e. canis infection in vivo. it is generally recognized that cell mediated immunity is important in protection against intracellular parasites. this would also apply to e. canis because of its intracellular location (kakoma, carson, ristic, huxoll, stephenson & nyindo 1977; nyindo et al. 1980; ristic & holland 1993). previous reports have highlighted that immunization against phylogenetically related organisms such as ehrlichia ruminantium and ehrlichia risticii, using inactivated organisms can induce protection against live challenge (rikihisa 1991; dame, mahan & yowell 1992; van vliet, jongejan & zeijst 1992; martinez, maillard, coisne, sheikboudou & bensaid 1994; mahan, andrew, tebele, burridge & barbet 1995; martinez, perez, sheikboudou, debus & bensaid 1996; mahan, kumbula, burridge & barbet 1998; dumler, barbet, bekker, dasch, palmer, ray, rikihisa & rurangirwa 2001; mahan, smith, kumbula, burridge & barbet 2001). in this study, we demonstrate that immunization of german shepherd dogs (gsds) with inactivated e. canis organisms in combination with quil a induces humoral and cellular immune responses which exert a suppressive effect on rickettsaemia following live e. canis challenge. materials and methods propagation of e. canis in dh82 canine macrophages ehrlichia canis (oklahoma strain) was cultured in dh82 cells in eagles minimal essential medium (emem, earles base), containing 10 % fetal calf serum, 0.292 g/l glutamine (gibco), 25 mm sodium bicarbonate and 25 mm hepes. the cultures were grown in 75 cm2 tissue culture flasks (costar 3275, ma, usa) at 37 °c. the medium was changed twice a week until more than 80 % of the cells were infected as determined by evaluation of giemsastained cytospin preparations of cells in the supernatant. at this stage, cells were either harvested for live challenge of dogs by gently tapping the flask or fresh medium was added to the existing medium twice weekly until all the cells were infected and the dh82 cell monolayer had disintegrated. the contents of these flasks were frozen at –80 °c for subsequent preparation of e. canis antigen for use in western blotting or lymphocyte proliferation tests or for purification of dna which was used in polymerase chain reaction (pcr) assays. preparation of e. canis antigen the frozen e. canis infected dh82 cell cultures were thawed, sonicated and centrifuged at low speed (1 000 x g) to remove cellular debris and to release e. canis. the supernatants were placed over an equal volume of 25 % sucrose in phosphate buffered saline (pbs) and centrifuged at 7 500 x g for half an hour. the resultant pellet containing the e. canis organisms was washed three times in pbs and resuspended in pbs. the protein concentration of this stock antigen was estimated by the lowry method (lowry, rosebrough, farr & randall 1951), using bovine serum albumin (bsa) in pbs as the standard. for immunization, freshly harvested live e. canis organisms were inactivated with an equal volume of 120 immune responses induced by immunization of dogs with inactivated ehrlichia canis organisms 0.8 % β-propiolactone in sterile pbs on ice for 2 h and frozen overnight at –20 °c as described by mahan et al. (1995) for inactivation of e. ruminantium. inactivation of the organisms was confirmed by staining with 6-carboxy-fluoresceine-diacetate (6-cfda; sigma, st. louis, mo, usa) which only stains live cells. final confirmation of inactivation of the organisms was achieved by inoculating 10 µl of the final immunogen into a centrifugation-shell vial (sterilin, feltham, england) containing dh82 cells. these cultures were incubated at 37 °c for 4 weeks, when the cover slip in the shell vial was harvested and examined under the microscope for e. canis organisms after staining with modified wright’s stain. a lack of recovery of infected dh82 cells in these vials was proof that the organisms had been inactivated adequately. positive control cultures were also set up which were inoculated with normal viable organisms from which infected dh82 cells were recovered. immunization of experimental dogs initially, ten healthy 6 to 8-month-old gsds (two groups of five dogs: control and infected) based at the zimbabwe republic police (zrp) dog training school, harare (in 1996/7), were committed to this study with authorization from the relevant zrp officials. all the dogs had been previously vaccinated against canine parvovirus, rabies, distemper, hepatitis and leptospirosis infections as per the recommendation of the individual vaccine manufacturers and were dewormed on a monthly basis. an effective ectoparasite control programme involving weekly dipping with acaricides was in place at the zrp kennels. pre-immunization screening (clinical, haematological and biochemical) demonstrated that all dogs were healthy (data not shown). however, after commencement of the experiment, only two control dogs were available, because the other three contracted parvovirus infection and died. since there were no replacements available, one group containing five immunized and the other two control dogs remained. the dogs were bled by jugular venipuncture for analysis of serum, which was stored at –20 °c, for antibodies to e. canis. all the dogs were found to have had no previous exposure to e. canis, since they were sero-negative by western blotting based on the absence of reactivity with the immunodominant 27 kda protein and other immunodominant e. canis proteins (brouqui, dumler, raoult & walker 1992; mahan, tebele, mukwedeya, semu, nyathi, wassink, kelly, peter & barbet 1993). the five dogs, alka, duke, spider, zita and zulu, were immunized three times subcutaneously with the inactivated vaccine, at 2-week intervals. each dose of the inactivated e. canis vaccine was prepared in 1 ml of sterile pbs containing 200 µg of e. canis antigen mixed with 100 µg of quil a adjuvant (superfos, denmark). the two control dogs, nita and nondo, were inoculated at the same time with 100 µg of quil a in 1 ml of pbs. immunological monitoring pre-immunization (day 0), post-immunization (2 weeks after each immunization), pre-challenge (45 days after third immunization) and post-challenge (1, 3, 11 and 37 weeks after challenge) antibody responses to e. canis antigens were monitored by western blotting as described previously (mahan et al. 1993). ehrlichia canis antigen (20–30 µg per lane) was electrophoretically separated on 12 % sodium dodecyl sulphate-polyacrylamide gels (sdspage) using the laemmli system (laemmli 1970). the resolved antigens were electro-transferred to nitrocellulose membranes and blocked for 3 h at room temperature in washing buffer 1 ph 8.0 (tris buffered saline [tbs: 0.1 m tris hcl and 0.9 % nacl] containing 0.25 % gelatine and 0.25 % tween 20). the membranes were reacted overnight with pre-immunization sera diluted in the blocking buffer at 1/100; post-immunization sera at 1/100–1/8 000 and post challenge at 1/100–1/32 000. the reactions of the antisera were developed by incubation with horse radish peroxidase labelled protein g, followed by 4cn peroxidase substrate (purchased from kirkegaard and perry, maryland, usa). the results of the western blots were photographed using a polaroid mp4 land camera and polaroid 57 speed film. the positive serum sample that was used in these western blots was from a dog experimentally infected previously with e. canis (oklahoma strain). the negative control serum was from a dog which had never been exposed to e. canis. to verify the presence of cell-mediated immune responses, lymphocyte proliferation tests (lpts) were performed 4 days before challenge on two immunized dogs (duke and zulu) and one control dog (nondo). the assay was repeated 7 weeks post-challenge on the same two immunized dogs (duke and zulu) and both control dogs (nita and nondo). briefly, 20 ml of blood were collected from the jugular vein of each dog into vacutainer tubes containing (edta), centrifuged at 4 000 x g for 15 min. the buffy coat was harvested and made up to 5 ml with sterile pbs. this diluted buffy coat was gently layered on 4 ml of histopaque (sigma diagnostics, st. louis, mo, usa) and centrifuged for 30 121 s. mahan, p.j. kelly & s.m. mahan min at 400 x g at room temperature to recover peripheral blood mononuclear cells (pbmns). the interface containing the pbmns was harvested, mixed with 10 ml of sterile alsever’s solution (0.55 g citric acid, 20.5 g d-glucose, 4.2 g nacl and 8 g trisodium citrate in 1 l of distilled water) and centrifuged at 450 x g for 10 min. the resulting pellet was washed three times in 50 ml of alsever’s solution (180 x g, 10 min each) to eliminate platelets. the final pellet containing pbmns was re-suspended in 0.5 ml of pbs and the cell viability assessed with the trypan blue exclusion stain. the concentration of the cells was adjusted to 2 x106/ml with rpmi complete medium (supplemented with 10 % foetal calf serum, 25 mm hepes, 50 µg gentamicin sulphate, 27 mm sodium carbonate, 5 x10–5 m 2mercaptoethanol). aliquots of pbmns (2 x105 cells in 100 µl) from each dog were dispensed into ubottomed 96 well tissue culture plates (costar, ma, usa), and stimulated in duplicate with 5, 10 or 20 µg antigen (pre-challenge tests) and 10 µg e. canis antigen (post-challenge tests). the same e. canis antigen was used for all lpts. negative control cultures contained wells of cells without antigen and positive control cultures contained 5 µg of concanavalin a which had been previously shown to be the optimal concentration for inducing proliferation in pbmns (data not shown). the plates were incubated at 37°c in 5 % co2 in air atmosphere for 4 days when 0.5 µci of 3h-thymidine (amersham, england) was added to each well and incubated overnight. after harvesting the cells with a phd cell harvester (model 200a, cambridge technology inc., watertown, ma), the incorporated radioactivity was counted in a beckman ls 6500 scintillation counter and expressed as counts per minute. challenge of dogs with live e. canis immunized and control group dogs were challenged intravenously 45 days after the last immunization, with 107 e. canis (oklahoma strain)-infected dh82 cells, suspended in 5 ml of media. viability of the infected cells was confirmed by trypan blue exclusion stain. this challenge dose was not pre-tested due to lack of additional uninfected dogs. however, this e. canis oklahoma infected cell culture challenge dose was chosen based on studies reported by iqbal & rikihisa (1994) who had shown that it induced mild but significant clinical and haematologic changes consistent with canine monocytic ehrlichia infections and resulted in establishment of chronic infection in which e. canis could be detected for a long period after the infection had been initiated. isolation of e. canis from peripheral blood of infected dogs to determine if the immune responses induced by immunization would suppress the rickettsaemia, isolation of e. canis was attempted from the immunized and control dogs during the first 2 weeks after challenge with live e. canis. twenty millilitres of blood in edta vacutainer tubes were obtained aseptically from each dog on days 9 and 14 after live e. canis challenge. peripheral blood mononuclear cells were isolated from these samples as described above for lpts. after the final wash, the cells were resuspended in1 ml of emem without antibiotics and 0.5 ml of the cell suspension was inoculated onto centrifugation-shell vials with confluent layers of dh82 cells. fresh medium (1 ml) was added to each vial and the vials incubated at 37 °c. media was changed twice a week for 4 weeks when cover slips were harvested, air-dried, fixed in methanol, stained with modified wright’s stain and examined under the microscope for the presence of e. canis organisms. detection of e. canis in peripheral blood of challenged dogs by pcr to demonstrate that all the dogs had been successfully challenged with e. canis, a 16s e. canis-specific pcr was conducted on pbmns of all dogs. the latter were isolated from each dog on days 4, 9, 14, 22 and 30 after live e. canis challenge as described above. for pcr, 0.5 ml of the final cell suspension was used. the suspended cells were placed in eppendorf tubes and centrifuged at 12 000 x g for 30 seconds. the supernatant was removed and the cells resuspended in 1 ml of saponin lysis buffer (0.22 % nacl, 0.015 % saponin and 1 mm edta) which was added to lyse any remaining erythrocytes which might inhibit the pcr (panaccio & lew 1991). after 2 min at room temperature, the tubes were centrifuged at 12 000 x g for 30 s and the saponin lysis buffer was removed. after two further washes in 1 ml of sterile pbs, the cell pellet was resuspended in 100 µl of proteinase k buffer (pk buffer; 0.1m tris hcl [ph 7.5] and 0.15 m nacl, 1 % sds) and stored at –20 °c. dna was obtained from the isolated buffy coats as follows. the cells were thawed at 37 °c and freeze-thawed twice to disrupt the cells. after further addition of 100 µl of pk buffer, the cell debris was digested with 10 µl of lysozyme (10 mg/ml) for 1 h at 37 °c and then further digested overnight with 10 µl of proteinase k enzyme (10 mg/ml) at 37 °c, followed by 1 h at 56 °c. dna extraction was performed by the phe122 immune responses induced by immunization of dogs with inactivated ehrlichia canis organisms nol-chloroform method (maniatis, fritsh & sambrook 1982). the extracted dna was transferred to sterile tubes and stored at –20 °c until pcr was performed. ehrlichia canis dna was extracted from infected dh82 cell cultures as described above and used to validate the e. canis pcr and as a positive control for subsequent test pcrs. a pcr assay was conducted using ehrlichia general primers, he3 (5’ggtaccgtcattatcttccc-3’) and he2 (5’gtggcagacgggtgagtaatgc-3’) as an internal control for the assay. these 16s ribosomal dna (16s rdna) general primers amplify dna from organisms in the ehrlichia group (e. canis, e. chaffenssis, e. ruminantium (savadye, kelly & mahan 1998; peter, deem, barbet, norval, simbi, kelly & mahan 1995; peter, barbet, alleman, simbi, burridge & mahan 2000; dumler et al. 2001) and an unknown ehrlichial agent found in zimbabwe in non-heartwater areas (savadye, kelly & mahan 1998). the e. canis specific primer used in combination with he2, was he(c) (5’caattatttatagcctctggctatagg-3’), and this combination of primers amplifies dna specifically from e. canis (peter et al. 1995). the ehrlichia general primers and the e. canis specific primers amplify a 350 base pair dna fragment by pcr. ehrlichia ruminantium pcr primers, ab128 and ab129 (mahan, waghela, mcguire, rurangirwa, wassink & barbet 1992), were used to test for specificity of the pcr conditions and to ensure that there was no contamination with e. ruminantium dna, which is routinely grown at the laboratory where these experiments were conducted. these primers amplify a 279 base pair dna fragment. the e. canis and e. ruminantium pcr assays were performed as described previously (savadye, kelly & mahan 1998; peter et al. 1995; 2000). the specificity of the pcr assay was validated by amplifying a 350 bp dna fragment from e. canis dna but not from e. ruminantium dna, a phylogenetically closely related species, using the 16s rdna primers specific for detection of e. canis. further evidence of the assay’s specificity and its optimal operation was obtained by the use of general 16s rdna ehrlichia primers and e. ruminantium specific primers on the same template dna samples. this combination of primers amplified 350 bp and 279 bp fragments from the e. ruminantium dna, but only the 350 bp fragment from the e. canis dna sample (data not shown). to improve the detection of the amplified dna, southern blotting followed by hybridization to the 32p dctp labelled e. canis specific 350 bp dna fragment was performed. the result of this hybridization was visualized by autoradiography (mahan et al. 1992). the 350 bp dna fragment (used as probe) was amplified from genomic e. canis dna, excised from a 0.8 % low melting temperature agarose gel and purified by a standard phenol:chloroform extraction method (maniatis et al. 1982). the purified dna fragment was labelled with 32p dctp by the random primer extension method (boehringer mannheim). results responses to immunization clinical and haematological responses the dogs in the immunized and control groups developed an oedematous swelling at the site of injection which fluctuated in size from approximately 5–25 mm, but subsided by 4 weeks post-immunization. similar swellings were also noted in the control dogs that had been injected with quil a alone. there were no other clinical or haematological changes that could be attributed to immunization in any of the dogs (data not shown). detection of e. canis specific antibody and lymphocyte proliferation responses the kinetics of antibody titre development and reactions following the various immunizations, was determined by western blotting. serum from all e. canis immunized dogs reacted with the immunodominant 27 kda protein and lower and higher molecular mass proteins of e. canis (fig. 1a). the antibody recognition patterns of e. canis proteins of the serum from immunized dogs was similar to the pattern of the positive control serum used in the western blotting assay and of infected dogs after challenge (see fig. 1b). the antibody titres and the antibody binding signals increased markedly after each immunization (fig. 1b), but had decreased during the period leading to challenge with live e. canis (fig. 2). fig. 1a shows that the two nonimmunized control dogs, nita and nondo, remained sero-negative throughout the immunization period, but strongly sero-converted following challenge with live e. canis (fig. 1b and 2). cell mediated immune responses were only tested in two of the immunized dogs, duke and zulu, as representatives of this group. proliferative responses of their respective pbmns to e. canis antigens were detected (fig. 3), which were much higher than those of control cultures, and of the non-immu123 s. mahan, p.j. kelly & s.m. mahan 124 immune responses induced by immunization of dogs with inactivated ehrlichia canis organisms + – ni no alka duke spider zita zulu control immunized dogs 200 97 69 46 30 21 14 kda – + nita nondo alka duke spider zita zulu control immunized dogs 200 97 69 46 30 21 kda fig. 1a western blots to evaluate e. canis specific antibody levels in the sera of the two control dogs, nita (ni) and nondo (no), and the five immunized dogs, alka, duke, spider, zita, zulu, 2 weeks post third immunization. lane 1, positive control, lane 2, negative control; lane 1 for each dog is a pre-immunization serum reaction (1/100); lanes 2 and 3 for the control dogs are postimmunization reactions at 1/100 and 1/1 000; lanes 2–6 for the immunized dogs are post-immunization reactions at 1/100, 1/1 000, 1/2 000, 1/4 000, 1/8 000. molecular mass markers are shown on the left in kda fig. 1b western blots to demonstrate the rise in e. canis specific antibody titres in the sera of the two control dogs, nita and nondo, and the five immunized dogs, alka, duke, spider, zita, zulu, 37 weeks post-challenge. lane 1, negative control; lane 2, positive control. the five lanes for the two control and immunized dogs show serum reactions at dilutions of 1/100, 1/1 000, 1/2 000, 1/4 000 and 1/8 000. molecular mas markers are shown on the right in kda ��������� ��� ���� ���� ��� � �� �� � ��� � ���� � � �� � � � � � �� � � �� � � � � ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� �� �� �� � � � �� �� �� ���� ������ fig. 2 kinetics of antibody titres determined by western blots before and after immunization or challenge with e. canis. following immunization antibody titres increased and after challenge anamnestic and primary antibody responses were detected in the immunized and control dogs, respectively nized dog, nondo. the pbmns of the other three immunized dogs were not tested in lpt. the positive control concanavalin a induced strong proliferation in these pbmns (data not shown) confirming the culture conditions were satisfactory for induction of proliferative cellular responses. immunological responses of the dogs after challenge with live e. canis at the time of challenge, the antibody titres in the immunized dogs had decreased (fig. 2). the live e. canis challenge induced rapid anamnestic antibody responses in the five immunized dogs. one week post-challenge, the antibody titres in the immunized dogs increased dramatically to levels between 1/4 000 to1/16 000 while only one control dog (nita) had developed a detectable antibody titre of 1/100. at 3 weeks post-challenge, antibody titres ranged from 1/8 000 to 1/16 000 in all immunized and control dogs, and by 11 weeks post-challenge, they were higher (fig. 2). western blot analyses performed on sera obtained at 37 weeks post-challenge from all dogs demonstrated reactions with all the immunogenic e. canis proteins (fig. 1b). cell mediated immune responses demonstrated by lpts were detected in pbmns from zulu and duke (immunized) and nondo and nita (controls) at 7 weeks post-challenge. a proliferative response was detected to e. canis proteins in all animals (fig. 4) and in the control dogs represent their primary exposure to e. canis and coincided with high titred antibody responses detected by western blotting. having demonstrated that humoral and cellular responses were present after immunization, its effect on rickettsaemia was analyzed. immunization seemed to suppress the development of rickettsaemia following challenge with live e. canis. this statement is supported by the fact that it was not possible to isolate e. canis from the pbmns of the challenged immunized dogs in cell culture on days 9 and 14 post-challenge. in contrast, isolation of e. canis organisms was successfully achieved from the pbmns of the two control dogs on day 9 (nita and nondo) and on day 14 (only nita). confirmation that all dogs were challenged with live e. canis organisms was achieved by detection of e. canis in the blood by a 16s e. canis-specific pcr assay (data not shown), and by the fact that anamnestic and primary immune responses were detected in 125 s. mahan, p.j. kelly & s.m. mahan ������ ��� � �� �� �� �� �� �� � � � � � �� � � � �� �� � � � � � � � � ��� �������� � ������������� �������������� �������������� fig. 3 pre-challenge lymphocyte proliferation tests performed with pbmns of the immunized dogs, duke and zulu, and the control dog, nondo. pbmns of each dog were co-cultured with e. canis antigens and proliferation was only detected in pbmns of immunized dogs in the presence of e. canis antigen at 5, 10 or 20 µg of antigen fig. 4 post-challenge lymphocyte proliferation tests performed with pbmns of the immunized dogs, duke and zulu, and the control dogs, nondo and nita. pbmns of each dog were co-cultured with e. canis antigens and proliferation was detected in pbmns of all dogs in the presence of e. canis antigen at 10 µg of antigen ������ ���� ����� ����� ����� � � � � � �� � � � �� �� � � � � � � � � ���� �� ��� � ���� �������� � �������������� ��� the immunized and control dogs after challenge, respectively. discussion the major objective of this experiment was to detect immune responses of dogs to immunization with inactivated e. canis in conjunction with quil a. quil a was included as an adjuvant since it is a potent stimulator of both humoral and cell mediated immune responses and causes minimal side effects (gupta & siber 1995). strong humoral responses were induced in the immunized dogs to various e. canis proteins. the complement of proteins recognized by serum of immunized dogs was generally similar to that recognized after infection with live e. canis and as reported by others (nyindo, kakoma & hansen 1991; brouqui et al. 1992; mahan et al. 1993). the antibodies detected by western blotting were of the igg class since horseradish peroxidase conjugated protein g only binds with igg and was used as the second step reagent (eliasson, andersson, olsson, wigzell & uhlen 1989). such antibody responses following immunization were, however, short lived as titres declined from between 1/4 000 and 1/8 000 to 1/100 and 1/1 000 at the time of challenge, 6 weeks after the third immunization (fig. 2). the decline in antibody titres was probably attributed to a short exposure to e. canis proteins during the immunization period, since the organisms used were inactivated and hence persisted for a short period in the dogs. infection with e. canis on the other hand caused persistent antibody production which were detectable for up to 37 weeks, that being the end of the study. however, immunization induced b cell memory responses, since, following challenge with live e. canis, rapid anamnestic antibody responses were detected. antibody titres in the immunized dogs increased from pre-challenge levels of 1/100 –1/1 000 to 1/8 000–1/16 000 1 week post-challenge (fig. 2). in contrast, following e. canis infection, the rise in antibody titres was more gradual in the control dogs and was indicative of a primary exposure. the antibody titres in these control dogs only reached those of the immunized group by 3 weeks post-challenge. the protective role of antibodies to e. canis in vivo is uncertain because dogs become fully susceptible to reinfection with homologous e. canis after treatment with tetracyclines despite the presence of high antibody titres (amyx et al. 1971; buhles, huxoll & ristic1974). in vitro, however, antibodies against e. canis have been shown to suppress growth of e. canis organisms (lewis et al. 1978). cell mediated immune responses were induced in the immunized dogs. peripheral blood mononuclear cells from the immunized dogs proliferated when stimulated with e. canis antigen in lpts. these assays are frequently used for detection of t cell activation and indicate incorporation of 3h-thymidine into the newly synthesized dna of lymphocytes proliferating following antigen stimulation (mwangi, mahan, nyanjui, taracha & mckeever 1998; mwangi, mckeever, nyanjui, barbet & mahan 2002). phenotypic characterization of the responding cell populations or their cytokine responses in the lpts was not done to define cell populations involved or their respective cytokine responses to antigen stimulation. cell mediated immune responses are considered critical for the control of infections by intracellular pathogens since the intracellular location of the organisms renders them inaccessible to circulating antibodies. there are two reports on the induction of cmi responses in dogs infected with e. canis. kakoma et al. (1977) demonstrated cytotoxicity against monocytes from dogs infected with e. canis using autologous lymphocytes. nyindo et al. (1980) used leukocyte migration inhibition tests to demonstrate that dogs infected with e. canis produced a migration inhibition factor. proliferative cellular responses to e. canis antigens were also present in the two control dogs following the challenge with live e. canis, demonstrating that infection induced activation of cellular immunity, although the nature of this immunity was not fully explored. these proliferation assay results are in contrast with results obtained from experiments where pbmns from bovines immune to infection with e. ruminantium were stimulated with lysate antigens. peripheral blood mononuclear cells from such animals did not proliferate in the presence of antigen lysate and only proliferated when e. ruminantium-infected autologous endothelial cells or monocytes were used for stimulation (mwangi et al. 1998). these observations represent different pathways of induction of immunity to infection to the respective pathogens in their specific hosts. the immune responses induced by immunization appeared to inhibit rickettsaemia. while isolation of e. canis from the mononuclear cells of both unvaccinated control dogs, nita and nondo was possible, cultures from all the immunized dogs were negative. this preliminary study shows that immunization of dogs with inactivated e. canis in conjunction with quil a induces strong humoral and cell mediated immune responses. immunization appears to inhib126 immune responses induced by immunization of dogs with inactivated ehrlichia canis organisms it rickettsaemia resulting from challenge with live e. canis and this may be of benefit in the control of canine monocytic ehrlichiosis as tick vectors mainly become infected during the acute phase of the disease when rickettsaemia is relatively high (lewis et al. 1978). further studies are required to optimize the immunization regimen in a larger number of dogs and to determine the level and type of immune protection induced against virulent, heterologous e. canis challenge. acknowledgements we thank the zimbabwe republic police for allowing us use of their dogs for this study. we also thank barbara byrom, bigboy simbi and shalt semu of the uf/usaid/ sadc heartwater research project and kylie wray of the faculty of veterinary science, university of zimbabwe for technical assistance at various stages of the study. references amyx, h.a., huxoll, d.l., zeiler, d.c. & hildebrandt, p.k. 1971. therapeutic and prophylactic value of tetracycline in dogs infected with the agent of tropical canine pancytopenia. american journal of veterinary research, 159: 1429–1432. brouqui, p., dumler, j.s., raoult, d. & walker, d.h. 1992. antigenic characterisation of ehrlichiae: protein immunoblotting of ehrlichia canis, ehrlichia sennetsu and ehrlichia risticii. journal of clinical microbiology, 30:1062– 1066. buhles, w.c., huxoll, d.l. & ristic, m. 1974. tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis 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inactivated ehrlichia canis organisms introduction livestock are very essential to the economies of many developing countries of the world. for the resource-limited poor farming communities, livestock perform multiple functions, which are imperative for the livelihoods of the rural households. the eastern cape province of south africa is particularly known for its vast number of livestock. of the various species of livestock raised by the rural farmers, cattle are undoubtedly most valued (tapson 1983). the number of cattle in the eastern cape as at may 2007 was estimated to be above 3 million, or approximately 23 % of the south african total (anonymous 2008). poor health conditions of livestock, however, constitute an important factor preventing them from achieving optimal productivity (alawa, jokthan & akut 2002). apart from various infectious diseases, of significant importance among livestock health problems are wounds and myiasis. wounds have been defined by livestock farmers in south africa to include sores, abscesses, warts and inflamed skin lesions (luseba, elgorashi, ntloedibe & van staden 2007). myiasis remains an unresolved problem for animal production and is responsible for severe economic losses through poor hide quality, reduced weight gain, loss of fertility and reduction in the production of milk (otranto & stevens 2002). animals may also die from infection of such lesions, as well as from loss of blood and tissue fluids. treatment of cattle wounds and myiasis is therefore 393 onderstepoort journal of veterinary research, 76:393–397 (2009) traditional remedies used for the treatment of cattle wounds and myiasis in amatola basin, eastern cape province, south africa o.t. soyelu1 and p.j. masika2* abstract soyelu, o.t. & masika, p.j. 2009. traditional remedies used for the treatment of cattle wounds and myiasis in amatola basin, eastern cape province, south africa. onderstepoort journal of vet er­ inary research, 76:393–397 a questionnaire survey was carried out to document traditional remedies used in the treatment of cattle wounds and myiasis in amatola basin. eighty-three cattle farmers from nine villages were interviewed. the majority of respondents (67.9 %) use traditional remedies for treatment of these conditions. however, 25.9 % of them use a combination of traditional remedies with conventional medicines. the survey revealed that 13 medicinal plants belonging to 11 families were used. aloe ferox, prunus persica and phytolacca heptandra were, however, the most commonly used. leaves are the most frequently used plant part, often prepared as an infusion. other non-plant remedies used include disinfectants, antiseptics as well as diesel and petrol. manual removal of maggots from wounds was also practise and treatment is usually continued until the wound heals. keywords: cattle, medicinal plants, myiasis, remedies, wounds * author to whom correspondence is to be directed. e-mail: pmasika@ufh.ac.za 1 department of animal science, faculty of science and agriculture, university of fort hare, private bag x1314, alice, 5700 south africa 2 agricultural and rural development research institute (ardri), university of fort hare, private bag x1314, alice, 5700 south africa accepted for publication 16 april 2009—editor 394 traditional remedies used for treatment of cattle wounds and myiasis in amatola basin, south africa considered vital in order to improve and increase productivity. in recent years, the cost of conventional medicines for use in animals has escalated and they have thus become unaffordable to most livestock raisers (njoroge & bussmann 2006). traditional remedies and practices, the ethno-veterinary medicines (evms), are now sometimes the only available alternative to expensive or unavailable modern forms of health care (luseba & van der merwe 2006). the world health organization (who 2002, cited by kiringe 2006) attributed the popular use of traditional medicine among rural communities to it being affordable, readily accessible and culturally acceptable. the use of evms in the treatment of animal health problems, including wounds, has been in practice for a long time in the eastern cape province (masika, sonandi & van averbeke 1997; van wyk, van oudts hoorn & gericke 1997). an ethnobotanical study by grierson & afolayan (1999) documented 38 plant species frequently used for human wound treatment in the province. however, traditional remedies for the treatment of livestock wounds and myiasis have not been documented. this fact underlines the urgency in documenting and preserving traditional knowledge before it is lost forever (tabuti, dhillion & lye 2003). therefore, the main objective of this study was to document the traditional remedies and practices used for the treatment of cattle wounds and myiasis by cattle owners in the amatola basin. materials and methods study area the amatola basin is a remote area in the amathole district municipality of the eastern cape province, south africa. it is an area comprising 13 villages and is bounded to the north by the amatola mountain range. the major part of the basin is well suited for extensive livestock production; therefore, livestock constitute the main component of the farming enterprise among the inhabitants of the amatola basin (tapson 1983). methodology a questionnaire survey was carried out in nine villages selected randomly out of the 13 villages in amatola basin. a total of 83 structured questionnaires were administered by personal interviews with cattle farmers using the snowball sampling technique (singh, pandey & aggarwal 2007). two cattle farmers identified in each of the selected villages were interviewed. thereafter they assisted in identifying other respondents and the cycle continued until the end of the survey period. questionnaires were so structured to obtain information on the various traditional remedies and practices used for the treatment of cattle wounds and myiasis in the study area. samples of plants used were collected with the help of the farmers and voucher specimens were deposited in the giffen herbarium at the university of fort hare. the data were analysed to obtain frequencies using the statistical analyses system, version 8.0 (sas 1999) for microsoft wind ows. results and discussion the results of the survey indicated that wounds and myiasis were serious health problems of cattle in the study area. a great majority of the interviewed farmers reported incidences of wounds and myiasis (84.3 % and 92.5 %, respectively) in their herds. this could be because of the high level of parasitic infestations (particularly ticks) and poor management practices. farmers attributed cattle wounds to various causes such as tick bites (56.6 %), castration (18.9 %), abscesses (13.2 %) and wire-cuts (9.4 %), as well as from fighting among cohorts (1.89 %). thus, tick bite is the main cause of cattle wounds, predisposing animals to abscesses and fly larvae infestations (kaufman, koehler & butler 2006; moyo & masika 2009). similar results were also reported among the tsonga people in limpopo province of south africa (luseba & van der merwe 2006). the high level of tick infestation in the ama tola basin is strongly associated with ineffective and poorly managed dipping systems. respondents indicated that there is no structured or routine maintenance programme in place to ensure the proper management of the diptanks. about a half of the respondents (53.2 %) attributed the cause of myiasis to be unidentified blue-flies, which lay their eggs on the wounds of the animals. the rest did not know what its cause is. respondents were able to diagnose myiasis when the animal wounds got bigger and drained blood-tinged fluid having a foul smell. the most damaging myiasiscausing flies (chrysomya spp., cochliomyia spp.) deposit their eggs in and around wounds. their larvae crawl into the wound, and burrow deep into the underlying tissues to feed. they develop through three larval stages (hall 2004), causing extensive local tissue damage. chrysomya chloropyga has been reported to be a primary and secondary cause of sheep myiasis in south africa and lucilia cuprina 395 o.t. soyelu & p.j. masika is responsible for the condition known as “blowfly strike” of sheep in a number of countries including south africa (hall 1991). however, there is inadequate data on the myiasis-causing flies of cattle and the direct economic losses that can be attributed to these flies in south africa. resource-limited livestock farmers are often faced with the problem of low income and limited access to veterinary services. therefore, for the management of animal health problems and also to improve productivity, they often depend on traditional knowledge (meyer, afolayan, taylor & engelbrecht 1996). table 1 list of plants used for the treatment cattle wounds and myiasis, plant part used and method of preparation reported by the respondents in amatola basin, eastern cape family and scientific name local name plant part method of preparation asphodelaceae aloe ferox mill ikhala leaves (1) infusion used as a wash for wounds (2) juice applied as dressing for wounds asteraceae haplocarpha scaposa harv. isikhali root decoction used as a wash for wounds fabaceae calpurnia aurea (aiton) benth umbethu umhlahlampethu leaves infusion used as a lotion for wounds. effective against maggot-infested wounds acacia karroo hayne umnga leaves crush leaves and mix with madubula schotia latifolia jacq. umgxam bark decoction used as a wash for wounds lamiaceae plectranthus ambiguous (bolus) codd irhajojo leaves stem (1) infusion used as a wash for wounds (2) sap applied to wounds. effective against maggot-infested wounds phytolaccaceae phytolacca heptandra retz. umnyanja leaves infusion/decoction used as a wash for maggotinfested wounds pittosporaceae pittosporum viridiflorum sims umkwenkwe root decoction used as a wash for wounds ptaeroxylaceae ptaeroxylon obliquum (thunb.) radlk umthathi leaves bark (1) leaf decoction used as a wash for wounds (2) crush bark and mix with used oil to form paste rosaceae prunus persica (l.) batsch. ipesika leaves infusion used as a wash for wounds. effective against maggot-infested wounds sapindaceae hippobromus pauciflorus (l.f.) radlk ulatile leaves infusion used as a wash for wounds solanaceae solanum sodomeum umthuma leaves fruits (1) infusion used as a wash for wounds (2) crush fruits and add fat to form paste tiliaceae grewia occidentalis l.f. umnqabasa leaves twigs infusion used as a lotion for wound 396 traditional remedies used for treatment of cattle wounds and myiasis in amatola basin, south africa in this study, most of the respondents (67.9 %) use traditional remedies to treat cattle wounds and myiasis. some of these farmers (25.9 %), however, com bine the use of traditional remedies with the orthodox medicines. this practice is believed to result into a more effective remedy than using either of the remedies alone. in addition, the combination of remedies is perceived to hasten the healing process thus giving a better result. masika, van averbeke & sonandi (2000) made a similar observation in a survey among livestock farmers in the central eastern cape province. in many parts of the world, the majority of ethnoveterinary practices are based on the use of medicinal plants available in the respective localities as compared to the other traditional remedies (dilshad, najeeb-ur-rehmana, iqbal, muhammad, iqbal & ahmed 2008). a total of 13 plant species belonging to 11 families (table 1) were reported to be used in the study area for the treatment of cattle wounds and myiasis. the most frequently used of these plants are aloe ferox mill. (25.3 %), prunus persica (l.) batsch. (25.3 %) and phytolacca heptandra retz. (24.1 %). results from the present study showed that farmers use different plant parts for the treatment of cattle wounds and myiasis. leaves were reported as the most frequently used plant part constituting 52.9 % of the herbal preparations followed in descending order by the bark (17.6 %), roots and stem (11.8 % each) and fruit (5.9 %). herbal remedies are prepared mainly by soaking crushed plant material in water (infusion) but sometimes it was boiled (decoction). others are prepared as paste and, in some instances, sap from plant parts is squeezed out and applied as a dressing for wounds. another observation was that more than one method of preparation or plant part could be used in the preparation of these remedies (table 1). all respondents applied herbal remedies topically on animal wounds, as this is the preferred form of administration employed in the treatment of skin diseases and various forms of skin conditions (tabuti et al. 2003). the survey also revealed five non-plant remedies used by the cattle farmers in the study area. ‘madubu la’ (phenols, cresols and xylenols; board man bros, stutterheim), a commercial household disinfectant, is the most popularly used product (90.6 %) among the non-plant remedies. the other four products are dettol (4.8 % chloroxylenol, 9 % terpineol; reckitt benckiser, south africa), used by 6.3 % of the respondents, ‘zifozonke’ (potassium permanganate), used by 18.8 % of the farmers, and petrol and diesel (hydrocarbons) by 12.5 %. ‘madubula’, dettol and ‘zifozonke’ are usually diluted in water before use as a wash for animal wounds. thereafter, some farmers may decide to apply either petrol or diesel to the affected portion of the animal skin. farmers claimed that ‘madubula’, dettol and ‘zifozonke’ hasten the healing process while petrol and diesel are believed to have the power to expel maggots that are deeply situated in the wounds. however, the rationale behind the acclaimed effectiveness of nonplant materials could be attributed to the disinfectant properties of ‘madubula’ and ‘zifozonke’ as well as the antiseptic properties of dettol. petrol and diesel have also been reported to possibly have an antifeedant effect on the maggots in wounds (farooq, iqbal, mushtaq, muhammad, iqbal & arshad 2008). the repelling odour of the petroleum products, in addition to their anti-feedant property, is believed to lead to starvation and eventual death of maggots. farmers also manually remove maggots from wounds with their bare hands or by using a tablespoon before the application of the remedies. this practice is considered as a faster way of getting rid of the invading maggots in the wounds. respondents prefer and still use traditional remedies because of their greater availability, easy accessibility, low costs and apparent effectiveness. all the medicinal plants, except the peach, prunus per­ sica, used for the treatment of cattle wounds and myiases are locally available in the bushveld around the study area. the peach is the only plant species reported to be cultivated and was grown by 21.1 % of the respondents, mainly because of its edible fruit. apart from the aforementioned reasons, respondents also prefer using traditional remedies because they believe the plants are safe to use and that they do not have any side effects on their animals. in addition, there is no need for withdrawal periods before the consumption of meat from animals treated with herbal remedies (luseba et al. 2007). in general, knowledge of the respondents on ethnoveterinary remedies was acquired mainly from their parents or grandparents (65.1 %) and neighbours (25 %); only a few learn from their friends and extension agents. it was observed that indigenous knowledge resides mainly with the older generation, as the majority of respondents (85.5 %) were aged 50 years or older. parents often verbally transfer their knowledge of traditional remedies to their children. however, the younger generation are no longer keen on learning or using this knowledge (van der merwe, swan & botha 2001). the major concern here is that information given orally might become distorted, and that the cycle of oral transfer from 397 o.t. soyelu & p.j. masika one generation to another might soon be broken. hence, there is need for proper documentation, which will also encourage scientific validation of the efficacy of traditional remedies. the results of this survey add to our understanding of the various innovations and practices that farmers put into use in order to ensure the health of their livestock. medicinal plants used for the treatment of livestock ailments are believed to possess pharmacologically active compounds (coetzee 2000). however, there is limited information in literature on many of the plants documented in this study, especially, regarding their wound healing abilities. information generated from this study will therefore form a basis for further validation of the efficacy, mode of action and safety of traditional remedies used in wound and myiasis treatment. acknowledgements the authors are grateful to the national research foundation (nrf) of south africa for funding this research, and to the people of amatola basin for their cooperation and willingness to share their knowledge. references alawa, j.p., jokthan, g.e. & akut, k. 2002. ethnoveterinary medical practice for ruminants in the subhumid zone of northern nigeria. preventive veterinary medicine, 54:79–90. anonymous 2008. estimated cattle numbers for 2007. pretoria: department of agriculture, south africa. coetzee, e. 2000. the magic of muti (medicinal) plants. farm­ er’s weekly, 14:26–29. dilshad, s.m.r., najeeb-ur-rehmana, iqbal, z., muhammad, g., iqbal, a. & ahmed, n. 2008. an inventory of the ethnoveterinary practices for reproductive disorders in cattle and buffaloes, sargodha district of pakistan. journal of eth­ no pharmacology, 117:393–402. farooq, z., iqbal, z., mushtaq, s., muhammad, g., iqbal, m.z. & arshad, m. 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72:189–196. van wyk, b.e., van oudtshoorn, b. & gericke, n. 1997. medicinal plants of south africa. pretoria: briza publications. abstract introduction materials and methods results and discussion acknowledgements references about the author(s) rudo marange department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, pretoria, south africa darshana morar-leather department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, pretoria, south africa folorunso o. fasina department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, pretoria, south africa food and agriculture organization of the united nations, dar es salaam, united republic of tanzania citation marange, r., morar-leather, d. & fasina, f.o., 2020, ‘survey of the knowledge, attitude and perceptions on bovine tuberculosis in mnisi community, mpumalanga, south africa’, onderstepoort journal of veterinary research 87(1), a1808. https://doi.org/10.4102/ojvr.v87i1.1808 research communication survey of the knowledge, attitude and perceptions on bovine tuberculosis in mnisi community, mpumalanga, south africa rudo marange, darshana morar-leather, folorunso o. fasina received: 27 aug. 2019; accepted: 11 may 2020; published: 30 july 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract tuberculosis (tb) is a global health concern of zoonotic importance, and mycobacterium bovis and mycobacterium tuberculosis are the most common causes of tb in animals and humans, respectively. integral to tb control strategies are the communities affected by this epidemic. tuberculosis awareness by the community is an effective tb control strategy as education empowers people to make informed choices with regard to mitigating tb risk factors in their daily lives. we conducted a knowledge, attitude and perceptions survey in mnisi pastoral community in south africa using a semi-structured questionnaire to evaluate the level of bovine tb (btb) awareness, and provided informed feedback to the community on the outcome of the study. although participants were aware of tb, the knowledge of the zoonotic potential of btb and about susceptible hosts was limited. the study findings showed knowledge gaps regarding common risk factors, including coughing while herding cattle, unsupervised/uninspected communal slaughter and improper disposal of infected meat. in contrast, it was noted that the majority of participants discarded meat with visible lesions and consumed pasteurised milk; thus, the risk of tb transmission via the ingestion route is low. tuberculosis knowledge gaps were evident in the community, and public health and veterinary authorities need to improve relationships with stakeholders and implement awareness programmes that use a one health approach. keywords: bovine tuberculosis; awareness; mpumalanga; surveillance; zoonosis introduction mycobacterium bovis is the causal agent of bovine tuberculosis (btb), an infectious and chronic disease in animals and humans (neill, skuce & pollock 2005). mycobacterium bovis threatens wildlife conservation and the economy, and zoonotic tb appears aggravated in developing countries because of the poor veterinary infrastructures, paucity of funds for disease prevention and controls (etter et al. 2006; olea-popelka et al. 2017). in low-to-middle-income countries (lmics) in africa, btb is prevalent in cattle populations (ayele et al. 2004) and impacts animal production negatively (e.g. carcass condemnations, decreased milk yields, low meat yield because of emaciation and poor reproductive performances). such production losses or a single animal death has far-reaching economic and sociocultural consequences on subsistence farmers in resource-poor communities (olea-popelka et al. 2017). bovine tb has been implicated in the increasing cases of human health problems in africa, such as the human immunodeficiency virus (hiv) pandemic (cosivi et al. 1998), and an increased human tb because of mycobacterium tuberculosis may suggest a concomitant increase in human infections with m. bovis. in lmics, human–animal interactions are intensive, with close cohabitation and poor hygiene standards during handling of animal products (ameni et al. 2007). while drinking raw milk and eating undercooked meat are obvious risk factors for the transmission, traditional practices such as sharing of dwellings with animals at night and plastering houses with cow dung are inadvertent risk for btb transmission (katale et al. 2012). addressing the identified risk factors is sometimes complicated by cultural resistance (olea-popelka et al. 2017). attempting to change norms, such as plastering houses with cow dung, without giving cheaper more accessible alternatives would be met with resistance. for example, suggesting cement as an alternative building material comes with high cost and is not a feasible alternative to the already impoverished communities. because livestock disease monitoring, control and eradication activities should use multi-partner, multi-disciplinary approaches and should be farmer-centred, policy-makers should take cognisance of farmers’ awareness levels and perceptions on btb in planning mitigations and control strategies (ameni et al. 2007). materials and methods mnisi is located in the north-eastern corner of bushbuckridge municipal area of mpumalanga province of south africa (24.8398°s, 31.0464°e), with approximately 12 832 cattle and 15 diptanks (musoke et al. 2015). based on the records from the mpumalanga veterinary service, a total of 1447 livestock farmers were identified and divided amongst the 15 diptanks. using a simple ballot system, five of the diptanks were selected. sample size was calculated using the following formula: sample size where population size (for finite population correction factor – fpc) (n): 552 farmers; hypothesised % frequency of outcome factor in the population (p): 50% ±5; confidence limits as % of 100(absolute ±%)(d): 5%; design effect (for cluster surveys-deff): 1. using proportional representations, 127 farmers were recruited for questionnaire administration (minimum of 20 and maximum of 30 farmers per diptank’s catchment area). all questions were prepared in english and translated into xitsonga, the lingua franca of the community. data were captured, filtered and analysed for descriptive statistics using microsoft excel®. ethical consideration the project was approved by the university of pretoria animal ethics committee (aec) (certificate number v116-16) and the faculty of health sciences research ethics committee (certificate number 374/2016). informed consent was obtained from all the participants who were briefed about their right to withdraw from the study if they wished so and guaranteed confidentiality. results and discussion a total of 110 participants completed questionnaire (table 1). approximately 94% of participants were aware of tb. mpumalanga has the second highest hiv prevalence in south africa at 35.1% (zuurendonk 2014), and the level of awareness could probably be because of the hiv/aids and tb awareness programmes and mass media campaigns. however, disease-specific knowledge was not matched with awareness level. as 61% of the participants associated tb with humans only, it is inferred that many are unaware of its zoonotic potential. this gap creates an inadvertent risk of zoonotic infection to humans who live in close contact and interact with cattle and goats. in ethiopia, 84% of studied participants were unaware of transmission routes and risk factors associated with tb (tschopp et al. 2015). similar studies in india revealed that poor communities that have high tb prevalences with little exposure to mass media also have little knowledge of factors affecting tb risk and transmission (sreeramareddy, harsha kumar & arokiasamy 2013). table 1: mnisi community tuberculosis questionnaire results. more than half of the participants in our study would herd animals while coughing persistently. a proportion of the human population can be asymptomatic carriers of m. bovis (green 2016), and many cough cases in rural populace go uninvestigated unless they become life threatening (matebesi, meulemans & timmerman 2005). in addition, human origin m. tuberculosis has been isolated in cow’s milk, indicating the probable human–cattle transmission (regassa, medhin & ameni 2008). targeted tb awareness campaigns on the knowledge of transmission and risks to promote behavioural changes that discourage such occupationally risky behaviour will be pertinent to promote healthcare-seeking behaviour (sreeramareddy et al. 2013). only 34% of the respondents herded cattle and goats together. low inter-herd risk of disease transmission between goats and cattle has been confirmed in the pastoralist communities of ethiopia (tschopp et al. 2015). communal slaughter remains a notable risk factor for plausible spread of zoonotic tb. ninety-nine per cent of the community members confirmed to have carried out communal slaughter without the supervision of any veterinary health officials, and approximately 28% will consume the meat at home with family members, while more than 70% of them even share the meat with the public. with such practices, a single case of tb in a slaughtered animal or any other rapidly spreading infectious disease like anthrax has the potential of reaching to a significant proportion of the community and may exacerbate communal tb spread. approximately 92% of the participants have seen tb-like lesions before in slaughtered animals, and almost 100% are at risk of infection through sharing, purchase and home consumption of uninspected meat. given that communal slaughter might continue for the foreseeable future because of poverty, salvage slaughter, inability of rural farmers to compete fairly in the formal markets and lesser intensity of veterinary service delivery in rural areas, incentivised self-reporting should be promoted with additional benefits of improved case reporting and reduced burden of zoonoses. the frequency of slaughter per household in this survey is however low. typically, in south africa and other developing countries, a reluctance to slaughter large ruminants is linked to wealth preservation based on heads of cattle owned (green 2016). only 2.7% of participants would ignore and cook meat with tb-like lesions. this affirmed earlier findings, which stated that most farmers in the mnisi community discard meat with visible abnormalities (musoke 2016). it is noteworthy that the majority of the participants understood the dangers of eating meat with apparent lesions, yet only 38% of respondents would throw out the whole carcass. of the respondents that will dispose the potentially infected carcasses, 20% opted for discarding in bins or feeding the meat to the dogs, a method that significantly increases the risk of exposure and communal transmission of m. bovis. scavengers and carnivores can contract tb from meat sources and from recycled environmental contamination and human sources (millán et al. 2008; moravkova et al. 2011; thoen et al. 2009). the risk of acquired infection from drinking infected raw milk in the mnisi community is low as only 2.7% of the participants drink raw milk irregularly. this contradicts popular belief that people in rural areas in lmics are at risk of contracting m. bovis through drinking of unpasteurised milk (ingestion route) (musoke 2016). the reasons for this good practice were not ascertained in this study but the ready accessibility of pasteurised milk, powdered milk and coffee creamers in petty trading stores, tuckshops or spaza shops, may be linked to this observation. the respondents suggested to improve veterinary services through the provision of more facilities and extensive repairs of current infrastructure. certain individuals displayed a clear disconnect and some level of mistrust between the community and veterinary services. such opinions or perceptions include top-down approach to policy formulation or implementation, veterinary officials sometimes carry out procedures on livestock without explaining the basis and purpose of the procedures to owners and lack of adequate feedback from earlier studies conducted in the community. in britain, breakdown in relationships between the state authorities and farmers was linked with differential farmer attitudes and views to most effective scientifically based interventions and the practicality of state recommendations on btb (cowie et al. 2014). such discordance will negatively impact the implementation of and compliance with recommendations on eradication. the multiple roles of state authorities, such as policy-makers, enforcers and advisors, to farmers on various issues sometimes put them in conflicting positions; notwithstanding, efforts must be intensified to create harmony and mutual trust amongst all stakeholders for btb control. in this instance, a feedback session was held to educate farmers about tb knowledge gaps identified during the study. the farmers appreciated the good practice of providing feedback to communities where studies are conducted and requested more actions to cultivate trust between researchers and farmers. acknowledgements the authors would like to acknowledge and appreciate the people and institutions for their input and support. this project would not have been possible without the support of the mnisi community, university of pretoria projects facilitation team including jacques van rooyen, ilana van wyk and jeanette wentzel, and the environmental monitors who translated and facilitated engagements with the community. they express their sincere thanks to the mpumalanga state veterinary services including dr bjorn reininghaus, solly mokone (the animal health controller) and all the technicians for giving them access to their operational data and allowing them to conduct the study during their dipping sessions. competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. funding information the authors are grateful to the belgian directorate general for development co-operation framework fa4 (itm/decd) and the national research foundation incentive funding for financing this research. data availability statement the authors confirm that the data supporting the findings of this study are available within the article [and/or] its supplementary materials. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the 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16. https://doi.org/10.1186/1471-2334-13-16 thoen, c.o., lobue, p.a., enarson, d.a., kaneene, j.b. & de kantor, i.n., 2009, ‘tuberculosis: a re-emerging disease in animals and humans’, veterinaria italiana 45(1), 135–181. tschopp, r., bekele, s., moti, t., young, d. & aseffa, a., 2015, ‘brucellosis and bovine tuberculosis prevalence in livestock from pastoralist communities adjacent to awash national park, ethiopia’, preventive veterinary medicine 120(2), 187–194. https://doi.org/10.1016/j.prevetmed.2015.03.004 zuurendonk, r.h.m., 2014, ‘the prevalence and risk factors of cryptosporidium infection among children in the mnisi community, south africa’, master’s thesis, utrecht university, utrecht, the netherlands. spotswood_135-143.qxd introduction anaemia is a common presentation in humans and animals. in a joint statement issued by the world health organisation (who) and the united nations children’s fund (unicef) on the who website in 2004, the number of anaemic people worldwide was estimated at 2 billion, with approximately 50 % of these attributed to iron deficiency (who/unicef 2004) this is seen by the who as a major public health problem: not only does iron deficient anaemia have dramatic effects on human health , such as increased maternal and child mortality, but it also has more insidious consequences, such as retardation in cognitive and physical development in children and reduced work productivity in adults. this document emphasised the multifactorial aetiology of anaemia: these vary from blood loss; helminth infections (particularly hookworm and schistosomosis); toxicosis (such as rodenticide and drug-induced); tuberculosis; hiv/aids; hereditary; chronic renal failure; immune mediated and blood parasites such as falciparum malaria in people. the last is very important with 300–500 million clinical cases of malaria occurring worldwide each year with approximately 2 million of them being fatal (artavanis-tsakonas, tongren & riley 2003). 135 onderstepoort journal of veterinary research, 72:135–143 (2005) a canine model of normovolaemic acute anaemia t.c. spotswood, r.m. kirberger, l.m.p.k. koma, f. reyers and l. van der merwe department of companion animal clinical studies, faculty of veterinary science, university of pretoria private bag x04,onderstepoort, 0110 south africa abstract spotswood, t.c., kirberger, r.m., koma, l.m.p.k., reyers, f. & van der merwe, l. 2005. a canine model of normovolaemic acute anaemia. onderstepoort journal of veterinary research, 72:135–143 the objective was to develop a non-terminal, acute normovolaemic anaemia model in dogs that has minimal effects on patient well-being. eleven normal beagle dogs were used. about 20 % of the circulating blood volume was removed from the jugular vein 1–3 times per day over a 3–4 day period until a haematocrit (ht) of 13–17 % was obtained. normovolaemia was maintained by replacing the volume deficit of the red blood cells with ringer’s lactate and re-infusing the plasma. full blood count and ht were monitored twice daily. the 13–17 % ht was reached within 3–4 days with the number of phlebotomies ranging from four to seven. the model was primarily developed to determine echocardiographic values as well as doppler abdominal splanchnic blood flow parameters in anaemic dogs as part of a study that will compare these results to similar studies in babesiosis-induced anaemia. the model may also be useful in the evaluation of the pathophysiology of anaemia in dogs or as a model for anaemia in humans. keywords: anaemia model, dog, normovolaemia accepted for publication 31 march 2005—editor in dogs, immune-mediated haemolytic anaemia, particularly the idiopathic form (reimer, troy & warnick 1999), is the most common cause except in countries such as like south africa where canine babesiosis is caused by a particularly severe strain of parasite (babesia canis rossi) and can account for up to 12 % of all cases presented to veterinary practices (reyers, leisewitz, lobetti, milner, jacobson & van zyl 1998). many of these patients suffer from severe anaemia necessitating hospitalization and may die if untreated. the role of anaemia and haemodynamic changes in the pathophysiology of diseases such as babesiosis has not been clearly elucidated and is being investigated in our department. the research programme prompted the inter alia development of this model in an attempt to create a normotensive, normovolaemic anaemic state. in addition, it is intended to use the model to assess the effect of red cell reduction and the associated reduction in viscosity, at selectable levels of anaemia, during the recovery period, on a number of parameters (such as blood flow and erythrocyte regeneration) without the possible added effect of parasite-host generated inflammatory, haemodynamic and potential parasite-associated toxic suppression. numerous anaemia models have been developed in a variety of species. models for human malaria have used japanese monkeys infected with plasmodium coatneyi (kawai, aikawa & kano 1993) and squirrel monkeys infected with plasmodium falciparum (contamin, behr, mercereau-puijalon & michel 2000). erythrocyte metabolism has been studied in an acute blood loss model in the horse (smith & agar 1976). the horses were bled at 16 ml/kg daily and reached a haematocrit (ht) of < 15 % after 3–4 days. the circulatory effects of anaemia induced hypoxia have also been studied in pigs (schou, perez de sa, sigurdardottir, roscher, jonkarker & werner 1996). a number of models have been developed in rats to study iron deficiency anaemia. these include the animals being fed iron deficient diets (gambling, charania, hannah, antipatis, lea & mcardle 2002) or undergoing phlebotomy at regular intervals (bhargave & gabbe 1984). in dogs, experimental anaemia models have mainly been developed for human cardiovascular studies (fowler & holmes 1971; fowler & holmes 1975; szlyk, king, jennings, cain & chapler 1984). invasive experimental techniques in animals have documented a hyperdynamic cardiovascular response to severe acute normovolaemic anaemia (haemodilution) characterized by increased cardiac output and reduced systemic vascular resistance (fowler, franch & bloom 1956; vatner, higgins & franklin 1972). in dogs and rats, invasive methods have been used to compute cardiac output and systemic vascular resistance in order to demonstrate the hyperdynamic state in anaemia (donald, ferguson & milburn 1968). the methods include cardiac and great blood vessel catheterization with indicator dilution, pressure transducers or electromagnetic flow meters and intraoperative echocardiography and indwelling doppler flow meters (donald et al. 1968). these studies did not always document exactly how the model was created. in some cases the dogs were euthanased at the end of the trial (habler, kleen, podtschaske, hutter, tiede, kemming, welte, corso & messmer 1996) but in others nothing was stated on experimental animal survival. (fowler et al. 1956; fowler & holmes 1971, 1975). in these studies normovolaemia was maintained by a variety of methods. exchange transfusions were performed with a range of molecular weights and concentrations of dextran solutions (mainly 6 % to simulate the viscosity of plasma and maintain oncotic pressure) (fowler et al.1956; fowler & holmes 1975; szlyk et al. 1984; fahim & singh 1992), dextran in physiologic saline (fowler & holmes 1971), hydroxyethyl starch (habler et al. 1996), ringer’s lactate (geha 1976) and harvested plasma with dextran (vatner et al. 1972). in most of these models the experimental data were recorded immediately after the exchange infusion while the dogs were under general anaesthesia with their haemodynamic parameters rigorously controlled. an experimental anaemia model using non-anesthetized dogs has been developed (lobetti, reyers & nesbit 1996), specifically for veterinary related research, but these dogs were euthanased afterwards. in humans, acute normovolaemic haemodilution (anh) is a technique used to eliminate or decrease the need for homologous blood transfusions in surgical patients (stehling & zauder 1991). the advantages of anh include avoiding disease transmission and transfusion reactions, and the acquisition of fresh autologous blood for transfusion (stehling & zauder 1991). reduction in red blood cell (rbc) loss during surgery is limited: for example, a patient with an ht of 40 % who loses a litre of blood loses 400 ml of rbcs. by inducing anh and reducing the ht to 25 % preoperatively, the patient will only lose 250 ml of rbcs per litre of blood loss. in practice, anh has been used extensively in elderly patients and small children, especially for cardiac surgery (stehling & zauder 1991). briefly, the tech136 canine model of normovolaemic acute anaemia nique of anh involves the removal of blood before surgery and its simultaneous replacement with a crystalloid or colloid. several formulae for the calculation of the volume of blood to be removed for achieving the desired ht in humans have been developed (bourke & smith 1974; gross 1983), including the following one (bourke & smith 1974): lt = v (ln ho – ln ht) where lt = blood removed v = circulating blood volume ln = natural logarithm ho = initial ht ht = final ht. this formula takes into account the progressive haemodilution and therefore the exponential reduction of the red blood cell number per unit volume blood during extraction. the investigators showed that their formula was accurate in clinical practice in predicting the final ht, with a standard deviation of only 1.46 volume percent of the mean difference between actual and predicted ht. to the best of the authors’ knowledge, this predictive formula has not been verified in dogs. the objectives of this experiment were to develop and describe, in detail, a non-lethal canine normovolaemic acute anaemia model (ht range 13–17 %) that has minimal effects on patient well-being. developing such a model would permit, inter alia, the evaluation of the effect of anaemia on echocardiographic and duplex doppler parameters of abdominal splanchnic blood flows as well as the marrow response to varying degrees of anaemia that could be applied in studies on the pathophysiology of babesiosis-induced anaemia and possibly apply correction factors to some of these parameters, based on the degree of anaemia. the animal use and care committee of the faculty of veterinary science at the university of pretoria, approved this study (reference 36-5-562). materials and methods animals one intact male, three neutered males and seven non-pregnant female beagle dogs, on loan from the onderstepoort veterinary academic research unit (ovaru), were used. the ages of the dogs were between 2 and 3 years. the general health status of each dog was evaluated by a complete physical, faecal and urine examination performed 2–4 weeks before the trial. in addition, peripheral blood smear examinations (for blood parasites), full blood count (fbc), ht and biochemical profiles for kidney and liver function, liver cell integrity and electrolytes were performed. these included serum levels of total proteins, albumin, urea, creatinine, sodium, potassium, total and ionised calcium, phosphate, alanine amino transferase and alkaline phosphatase. all dogs then underwent echocardiography, general abdominal ultrasonography and thoracic radiography. only dogs in good physical condition, and clinically healthy with the above findings within normal limits were included. all dogs were then dewormed and received a long acting ectoparaciticide. for the active trial period, the dogs were transferred to and housed at the onderstepoort veterinary academic hospital. they were fed a high protein and calorie commercial dog food. on day 0, prior to the first phlebotomy, and before the first (baseline) experimental ultrasonographic examinations, the physical examination, peripheral blood smear, fbc and ht were repeated. the biochemical profiles listed above but excluding total calcium, phosphate and urea were also repeated. peripheral blood smear examinations were conducted weekly to ensure that haematozoon parasites did not contribute to, or interfere with, the experimentally induced anaemia. habitus and appetite were monitored daily on a scale from level 1 to level 4. for habitus, 1 was severely depressed and moribund ranging to 4 which was bright and active. for appetite, 1 was anorexic and ranged to 4 for a good appetite. every morning during the trial the rectal temperature and heart rate were monitored and a physical examination was performed. the study was self-controlled with each dog being compared with its own pre-trial and day 0 ht data. sedation was used for bleeding uncooperative dogs. the use and choice of sedatives depended on the temperament of the dog and clinician preference, and was one of the following: a diazepam (pax, bayer isando, rsa) and morphine sulphate [bodene (pty) ltd., port elizabeth, rsa] combination; an acepromazine (aceprom 2, bayer isando, rsa) and butorphenol (torbugesic, fort dodge animal health, fort dodge, iowa, usa) combination; and medetomidine (domitor, novartis, kempton park, rsa) which was reversed with atipamazole (antisedan, novartis, kempton park, rsa) after phlebotomy. induction of anaemia the phlebotomy procedure was a modification of the method previously described (lobetti et al. 137 t.c. spotswood et al. 1996). it has been shown that 20 % of the estimated blood volume may be safely removed at one time without inducing shock (knottenbelt & mackin 1998). based on the assumption that circulating blood volume is about 90 ml/kg in the dog (knottenbelt & mackin 1998), the formula for the maximum volume of blood that could be removed is: blood volume to be removed (ml) = 20 % x mass (kg) x 90 multiple phlebotomies were performed at least 4 h apart on a daily basis until an ht of 14–17 % was obtained. the blood was collected in a fenwal® triple blood-pack with adsol red cell preservation solution (adcock ingram, aeroton, rsa) by a vacuum technique using a suction pressure of 125– 175 mm hg. a proportion of the 70 ml citrate anticoagulant in the bag was removed just prior to phlebotomy to maintain the correct ratio with the volume of blood collected. a cephalic over-the-needle catheter was placed in the cephalic vein prior to phlebotomy to ensure venous access for re-infusion and possible emergency treatment. catheter venous access was maintained for the duration of the active bleeding phase of the trial, and catheters were replaced after 3 days. the dogs were manually restrained in lateral recumbency with the head and neck in moderate extension. at least two people (usually three) were required for the phlebotomy, two for restraint of the dog (one holding the head and one the limbs), and the third to insert the needle into the jugular vein and control the needle. the jugular vein region was aseptically prepared. if a subcutaneous haematoma developed, an attempt was made to draw blood from a different site on the same vein. as a last resort the contralateral vein was used for collection. the onderstepoort academic hospital blood bank vacuum collection technique weighed the collecting bag blood during collection allowing reasonably accurate determination of the removed blood volume. the volume of blood to mass of blood ratio was assumed to be 1 ml:1g, for practical purposes. however, the sg of blood is 1.053 therefore the ratio of blood volume:blood mass is 1:1.05, for example, 200 g of blood = 190 ml of blood (mathews 1998). maintaining normovolaemic status to ensure normovolaemia after bleeding, the volume deficit of the packed cells (determined from multiplying the ht by the volume removed) was accounted for by infusing an equal amount of ringer’s lactate solution, using a 15 drop/ml blood infusion administration set connected to the cephalic catheter, immediately after completion of phlebotomy. the blood bag was then centrifuged for 10 min in a refrigerated sigma® automatic centrifuge at 4 °c and 3 500 revolutions per minute. after centrifuging, the plasma was separated from the packed cells and sealed, and the adsol preservative added to the remaining packed red blood cells. the plasma was then immediately infused back into the same dog over a period of an hour, simultaneously with the ringer’s, if this was not yet finished. the packed cell bag was labelled and stored at 4 °c for the duration of the study in the event that it was needed for re-infusion. the packed cells were discarded at the end of the project as the blood type had not been established and thus precluded its use in other dogs. the individual animal’s response to the phlebotomy was monitored by the ht readings prior to each bleed. once the predetermined ht range of between 14–17 % was reached, and verified by the laboratory-calculated value, dogs were allowed to recover naturally from their anaemia with daily ht monitoring. additional procedures microhaematocrit pcv was monitored twice daily just prior to phlebotomy and daily or twice daily (if recovery was more rapid than anticipated) during the recovery phase. the results were compared with the analyser-calculated value when an accurate value was necessary, such as when haemodynamic data were to be captured. the ht was done as close to the same time of day as possible. each dog was weighed daily during the trial, and then later whenever experimental data were captured. in the first week of the study, one dog was introduced to the trial followed by two dogs each additional week. this limitation was to accommodate the additional experimental procedures that had to be performed on each dog. these were an echocardiographic examination, which was completed within 45–60 min, followed by an abdominal splanchnic vascular doppler examination lasting 90–120 min. at the end of the trial each animal, having recovered sufficiently from the induced anaemia, was returned to ovaru. all echocardiographic and doppler parameters were measured immediately before and after induction of severe, acute anaemia (ht 14–17 %). measurements were repeated during recovery in moderate chronic (ht 25–27 %) and mild chronic anaemia (ht 31–37 %). a fbc was repeated in each ht range. 138 canine model of normovolaemic acute anaemia collected data were also applied retrospectively to the theoretical haemodilution equation, described by bourke & smith (1974) to evaluate its accuracy in this group of dogs. the total volumes of the actual extracted blood were compared with the theoretically-predicted volumes calculated from bourke and smith’s (1974) equation for the same final ht. results the body mass median, mean, standard deviation (sd) and range of the 11 beagles were 11.3, 11.9 (1.8) and 9.5–15.2 kg, respectively. on day 0, 33 % of dogs were sedated, on day 1, 76 %, on day 2, 74 % and on day 3, 60 %. dogs 3 and 7 that received a morphine diazepam combination were judged to be nauseous and were treated effectively with metaclopramide (clopamon, pharmacare, port elizabeth, rsa). in dog 10 an apparent adverse reaction to the medetomidine occurred on the third day of bleeding. the dog collapsed towards the end of the phlebotomy, approximately 20 min after medetomidine injection. on immediate medetomidine reversal with atipamazole, the dog recovered uneventfully. the process of phlebotomy together with the laboratory work took about 40 min (15–20 min bleeding and 20 min plasma preparation). the infusion of ringer’s lactate solution took about 20 min, which at times ran concurrently with the plasma. plasma infusion took about 60 min, which was slow due to its high viscosity, and during this period dogs not receiving their own plasma were monitored for possible transfusion reactions in (schneider 2000). eight dogs were bled once, two dogs twice and one dog three times on the first day. this variation was purely for convenience to get the dogs into the system. on the second day all of them were bled twice with the exception of one that was only bled once. on the third day eight dogs were bled twice and the remaining three dogs had attained the required ht by the afternoon and were thus bled only in the morning. on the fourth day, five dogs were bled once. the number of phlebotomies required to obtain the optimal ht ranged from 4–7 with a mean (sd) of 5.3 (0.9). the mean (sd) and range of removed blood volume (in ml) on day 1 was 226 (32) and 190–304; day 2 was 225 (36.6) and 90–304; day 3 was 219 (43.1) and 114–290; and day 4 was 211 (43.7) and 171–285. the low volume of 114 ml removed in one of the dogs on day 3 was due to the sedation collapse described above. complications of the procedure were minimal. two dogs (dog 4 once and dog 5 twice) received donor plasma instead of their own plasma. on one occasion in each dog this was due to an electrical power failure that caused a delay in re-infusion and in the other to rupture of the blood collecting bag in the centrifuge. dog 4 developed a mild facial oedema which was not treated, and which resolved spontaneously within 36 h. body temperature remained normal throughout the trial. heart rate increased from a mean (sd) on day 0 of 96 (13.2) (n = 9) to a peak of 132 (14.7) on day 2 and decreased to 128 (13.6) (n = 10) on day 4. habitus remained at level 4 in seven dogs throughout the trial. dog 8 had a level 3 on the last day it was bled as well as on the next day. the remaining dogs (dogs 2, 4, 6 and 10) had a level 3 on the day after the final bleed. two of these dogs had other problems that could have accounted for the mild depression; dog 4 developed facial oedema and dog 10 a lame hind leg of uncertain aetiology on that day. appetite remained at level 4 in eight of the dogs. dogs 6 and 9 had slightly reduced appetites on the last day of phlebotomy. dog 6 ate only a small amount on the following day. dog 1 ate nothing on the second and third days after bleeding but this was believed to be due to a change in food consistency. the effect of phlebotomy on the ht as well as during the dogs’ recovery over the next 10 days is illustrated in fig. 1. the percentage differences between the actual and predicted volumes using bourke & smith’s (1974) haemodilution equation to achieve the final haematocrit are summarized (fig. 2). the standard deviation of the percentage difference between actual and predicted volumes in this group of dogs was 17.4 % volume. discussion this experimental anaemia model provides a technique to produce acute anaemia over a period of 3–4 days in dogs with minimal side effects. sedation was only required in a third of the dogs on day 0, but was increased up to 75 % of the dogs on the remaining days as the dogs became aware that they were going to be bled. one of the dogs that collapsed towards the end of the phlebotomy, after medetomidine injection, was judged to have acute cardiac output failure. this is likely to have been due to a combination of the sedative and anaemic state of the animal. on immediate reversal of the medetomidine, the dog recovered uneventfully. medetomidine was used on this occasion in this dog as 139 t.c. spotswood et al. it was particularly uncooperative, and the other sedation protocols, having been used with previous phlebotomies, were judged to be inadequate. medetomidine is not recommended for use as a sedative in this anaemia model, and uncooperative or fractious dogs should rather be excluded from trials. the phlebotomy and re-infusion process could be completed within 90 min if the ringer’s infusion took place at the same time as the separating of the plasma. plasma infusion could be done while the next dog was being bled and thus with adequate labour, 4–6 dogs could be bled in a single day. in this study a maximum of two dogs were bled per day as the same workers had to perform other experimental procedures during the acute anaemia and recovery stages. the induction of anaemia and procedures performed up to recovery to mild chronic anaemia thus took a month for the group (2 weeks for each dog). 140 canine model of normovolaemic acute anaemia fig. 1 the mean ht value of all dogs measured on any particular day relative to the first phlebotomy being set as day 0. during the first 4 days ht readings were often taken twice daily and the afternoon readings have been plotted as “half-days”. the pre-trial “screening” data were all assigned to day 7 for convenience, although some were conducted more than one week before the “active trial”. the error bars represent 2 se on either side of the mean values �� �� �� �� �� �� �� �� �� �� � � �� �� �� �� �� � � � � � � � � �� �� �� �� �� �� � � �� �� �� �� � � � � � �� � � � �� ���� ���� � ��� ���������� fig. 2 the percentage difference between the actual and predicted volumes of blood by the haemodilution equation of bourke and smith (1974) to achieve the final ht for each dog. a percentage difference of 0 would indicate that the actual and predicted blood volumes were the same. a positive percentage means that the actual blood volume was in excess of the predicted blood volume, while a negative percentage means that the actual volume was less than the volume predicted ��� ������ �� � �� � � �� � � �� � � � � �� �� � � � � � � � �� � � ����� ����� ����� ����� ��� ���� ���� ���� � � � � � � � �� �� the blood volume was removed acutely and directly from the central compartment of the beagles. fluid replacement with ringer’s lactate, equal in volume to the calculated red blood cell volume removed, was administered in the first 30 min after phlebotomy. the plasma was transfused after 30– 60 min, often together with the ringer’s lactate as a colloid as well as for volume replacement. a minimum of 4 h was allowed between phlebotomies to allow for normal trans-membrane fluid movements. under normal conditions, starlings forces across the extra-cellular membranes cause approximately 80 % of transfused isotonic crystalloids to filter into the interstitium within 1 h (mathews 1998). the value of replacing the lost red blood cell mass with crystalloids is thus debatable but it did give volume support in the immediate post-phlebotomy phase while the plasma was being prepared for re-infusion. autoplasma re-infusion appears to be preferable as the one dog that received donor plasma developed mild facial oedema, indicating a mild type 1 hypersensitivity reaction against donor proteins (hohenhaus 2000). the true volume deficit created by red cell removal, assuming an initial ht of 45 % and volume collection of 200 ml, was only 9 % and would probably have been compensated for by normal fluid redistribution between compartments during normal water intake and albumin production by the liver. as dogs became progressively more anaemic the effective red blood cell mass volume deficit would become progressively less significant due to the compounding effects of the iatrogenic anaemia. the decision not to use colloids as post collection replacement fluids was based on the above premise as well as the fact that the oncotic effect would have been cumulative as the half-life of 6 % hetastarch (haes-steril, fresenius kabi, bad homburg, germany), the volume replacement fluid of choice at our institution, is 25 h (rudloff & kirby 1997). this would have had a marked effect when blood was being drawn daily and even twice daily and would have interfered with natural fluid homeostatic compensatory mechanisms causing a degree of haemodilution (rudloff & kirby 1997). the ideal post collection transfusion would have been own plasma plus the red cell volume of a mixture of donor plasma and 0.9 % saline to a concentration of 30 g/l this would have been laborious and unnecessary considering the small volumes involved and the ability of the body to compensate for these changes. in this model, echocardiographic and doppler parameters were measured several hours after re-infusion in unsedated dogs and thus blood volumes should have reinstated their fluid equilibrium. the microhaematocrit technique to measure pcv was selected to monitor the anaemia on a day-today basis because of its ease of use, low cost, convenience (such as after hours monitoring), and the rapid acquisition of results. for data recording it was, however, rejected early in the study in favour of the analyser-calculated reading (calculated automatically from the mean cell volume and the rbc number) due to discrepancies of up to 2.4 % higher pcv when compared with the analyser-calculated ht. although the microhaematocrit method was very useful in monitoring the anaemia during the trial it is recommended that the automated cell counter values be used to verify the exact ht when necessary. it is believed that the discrepancy between the values using the two methods was due to inter-equipment variation which needs to be investigated further. from the ht data (fig. 1) it can be seen that the pretrial screening ht values and the values obtained on the day that (but before) phlebotomy commenced, were essentially the same and well within the reference range of 37–55 % (meinkoth & clinkenbeard 2000). the anaemia model reproducibly produced a decline of ht to its nadir (at approximately 15 %) by the 4th and 5th days, which are similar to the anaemia experimental model described in the horse (smith & agar 1976). by comparing the actual and predicted volumes of blood for the final ht in this small group of dogs (fig. 2), it would appear that the theoretical haemodilution equation described (bourke & smith 1974) is unsuitable for clinical application in dogs. more work would be needed to establish an equivalent predictive equation in this species. until then, careful monitoring of the ht during the bleeding phase is recommended. recovery, from day 5 was progressive and essentially linear, for the next week (until an ht of 30 % was achieved by day 11). it would appear that after that level of recovery had been reached, the stimulus for regeneration abated somewhat and the recovery tailed off quite markedly. the original trial design did not include monitoring beyond 2 weeks, but one dog was fortuitously sampled on day 43 and was found to have an ht of 42 %. the recovery rate was within the range that had been anticipated but the plateau after day 11 was unexpected as it had been assumed that a plateau would be reached at an ht in the 35–40 % range. 141 t.c. spotswood et al. the current model was adapted from one used to determine the effect of hypoxia in the development of nephropathy in the dog (lobetti et al. 1996). this acute anaemia model can be used in many other studies such as the effect of haematinics or cardiovascular drugs, acute and subacute blood loss (e.g. gastric ulceration), evaluating the effects of various degrees of anaemia on splanchnic doppler blood flow, the usefulness of cell-counter-derived erythrocyte parameters (red cell distribution width and machine-generated reticulocyte counts) in haemorrhagic and immune-mediated anaemias as well as studies on canine babesiosis as a model for falciparum malaria in humans. acknowledgements we gratefully acknowledge the assistance of sr k. hastie for handling and care of the dogs, ms e. myburg and g. pretorius for laboratory work and sr m. watts for assisting with the phlebotomy and processing of the blood. this trial was supported by the faculty of veterinary science, university of pretoria research fund and merial south africa (pty) ltd. references artavanis-tsakonas, k., tongren, j.e. & riley, e.m. 2003. the war between the malaria parasite and the immune system: immunity, immunoregulation and immunopathology. clinical experimental immunology, 133:145–152. bhargave, m. & gabbe, e.e. 1984. chronic experimental iron losses in rats not leading to overt iron deficiency: a model for the regulation of the whole-body iron balance in an iron-replete 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africa bellonah m. sakong department of paraclinical sciences, university of pretoria, south africa olubukola t. adenubi department of paraclinical sciences, university of pretoria, south africa elizabeth pauw department of paraclinical sciences, university of pretoria, south africa tlabo leboho csir biosciences, pretoria, south africa mbokota c. khosa agricultural research council – tropical and subtropical crops, nelspruit, south africa kevin w. wellington csir biosciences, pretoria, south africa jacobus n. eloff department of paraclinical sciences, university of pretoria, south africa citation fouche, g., sakong, b.m., adenubi, o.t., pauw, e., leboho, t., khosa, m.c. et al., 2021, ‘corrigendum: anthelmintic activity of acetone extracts from south african plants used on egg hatching of haemonchus contortus’, onderstepoort journal of veterinary research 88(1), a1949. https://doi.org/10.4102/ojvr.v88i1.1949 note: doi of original article published: http://dx.doi.org/10.4102/ojvr.v83i1.1164 correction corrigendum: anthelmintic activity of acetone extracts from south african plants used on egg hatching of haemonchus contortus gerda fouche, bellonah m. sakong, olubukola t. adenubi, elizabeth pauw, tlabo leboho, mbokota c. khosa, kevin w. wellington, jacobus n. eloff published: 27 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published version of this article, fouche, g., sakong, b.m., adenubi, o.t., pauw, e., leboho, t., wellington, k.w. et al., 2016, ‘anthelmintic activity of acetone extracts from south african plants used on egg hatching of haemonchus contortus’, onderstepoort journal of veterinary research 83(1), a1164. https://doi.org/10.4102/ojvr.v83i1.1164, the sixth author, mbokota c. khosa, was omitted from the ‘authors’ and ‘affiliations’ sections. the indicated author should be added as the sixth author, and the following affiliation should be added as his affiliation: agricultural research council – tropical and subtropical crops, nelspruit, south africa. the authors’ contributions section is hereby update to: authors’ contributions g.f. conceptualised the study. m.c.k. was involved in the collection of some of the plant material and in the preparation of the extracts used in the biological screening assays. g.f., k.w.w. and t.l. carried out the literature search and plant selection. t.l. prepared the plant extracts. j.n.e. and e.p. arranged for sheep to be infected, collected the eggs and guided the study. b.m.s. performed the egg hatch assay on the extracts. m.c.k. was also involved in the fractionation and isolation process in the natural product chemistry laboratory. o.t.a. screened the extracts for toxicity on vero cells. k.w.w. wrote the first draft of the manuscript. this correction does not alter the study’s findings of significance or the overall interpretation of the study results. the authors apologise for any inconvenience caused. abstract introduction materials and methods results discussion acknowledgements references about the author(s) tuba ç. oğuzoğlu department of virology, faculty of veterinary medicine, ankara university, ankara, turkey seçkin salar department of obstetric and gnynecology, faculty of veterinary medicine, ankara university, ankara, turkey ece adıgüzel department of virology, faculty of veterinary medicine, ankara university, ankara, turkey cansu demirden department of virology, faculty of veterinary medicine, ankara university, ankara, turkey onur ülgenalp department of virology, faculty of veterinary medicine, ankara university, ankara, turkey citation oğuzoğlu, t.ç., salar, s., adıgüzel, e., demirden, c. & ülgenalp, o., 2020, ‘detection and characterisation of sheep-associated malignant catarrhal fever infection from ruminants by using tegument and gb gene sequences of ovhv-2’, onderstepoort journal of veterinary research 87(1), a1886. https://doi.org/10.4102/ojvr.v87i1.1886 original research detection and characterisation of sheep-associated malignant catarrhal fever infection from ruminants by using tegument and gb gene sequences of ovhv-2 tuba ç. oğuzoğlu, seçkin salar, ece adıgüzel, cansu demirden, onur ülgenalp received: 29 may 2020; accepted: 07 sept. 2020; published: 11 nov. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract in this study, positive blood and organ samples were obtained from different mixed herds of sheep and cattle against ovine herpesvirus 2 (ovhv-2) infection. target-positive dna was sequenced and compared with worldwide distributed ovhv-2 sequences. tegument gene (422 base pairs) and glycoprotein b (gb) gene (2800 base pairs) amplicons of ovhv-2 genome were used for understanding of epidemiology of malignant catarrhal fever (mcf) infection in turkey. the results of nucleotide sequencing of polymerase chain reaction (pcr) products indicated presence of sheep-associated form for mcf infection in turkey. although the obtained sequences were genetically different from each other, it was found that genetic variations were limited. keywords: malignant catarrhal fever; tegument gene; gb gene; ovhv-2; turkey. introduction known as a fatal infection, malignant catarrhal fever (mcf) affects susceptible cattle, wild ruminants and pigs (plowright, ferris & scott 1960). two causative agents for mcf are predominantly described, one of them carried by sheep infected with ovine herpesvirus 2 (ovhv-2) (called sheep-associated mcf) and the other carried by wildebeest (connochaetes species) containing alcelaphine herpesvirus 1 (alhv-1) (called wildebeest-associated form) (roizman 1992; russel, stewart & haig 2009). to date, more than 10 identified causative viruses have been associated with this infection in domestic and wildlife ruminants (crawford et al. 2002; nthiwa et al. 2019; seeley et al. 2018). the clinicopathological findings of mcf in affected animals are not clear about differential diagnoses. recently, new forms, neurological syndrome and systemic necrotising vasculitis, have been described that make determining clinical pictures and diagnosing the disease difficult (martins et al. 2017; pesavento et al. 2019). famous findings have described high fever, generalised lymphadenopathy, keratoconjunctivitis, corneal oedema and opacity, but these symptoms are not always evident (plowright 1990). the tegument gene ovhv-2 is encoded by open reading frame (orf) 33 and orf 75 and used for the purpose of detection of ovhv-2 dna and phylogenetic analysis based on sequence data (baxter et al. 1993). the glycoprotein b (gb) gene of ovhv-2 is the most conserved herpes virus protein, encoded by orf 8 (pereira 1994). glycoprotein b plays a role in virus entry to a host organism and spread between cells. by using this on primers of the gb gene, it is possible to investigate the geographical localisation amongst the sequence variations of ovhv-2 strains (dunowska et al. 2001) or evaluate vaccine targets for alhv-1/ovhv-2 chimeric viruses (cunha et al. 2016). the present report describes the detection of an mcf infection circulating in cattle and sheep in turkey. a molecular comparison between cattle and sheep strains was made using the tegument and gb gene sequences of ovhv-2 to explain the origin of the infection. materials and methods in total, eight animals from three different farms were sampled in this study. whole-blood sample was taken from dairy cattle with clinical symptoms. additionally, blood samples were taken from five sheep located close to the cattle boxes at the same farm. one of 50 lactating cows that contracted the disease showed the main clinical symptoms of persistent high fever (> 41 °c), dyspnea, nasal and ocular discharges, conjunctival and scleral hyperemia and keratoconjunctivitis with corneal opacity (figure 1a). the cow was separated 5 days after the first symptoms of the disease were detected. five of 145 sheep housed in the same barn showed signs of high fever (> 40.5 °c), scleral hyperemia and nasal and ocular discharges, and they were separated from the flock (figure 1b). figure 1: (a) clinical signs of an affected cow; (b) clinical signs of affected sheep. additionally, a cow and a sheep from different farms in the same village were sampled at different time intervals. at the farm where the cow was sampled, there were 80 sheep, 100 yearlings and 32 cattle. for this farm, a veterinarian reported that six cattle had died. in addition, sheep deaths were reported from a sheep-only farm in the same village, so a lung sample was taken from a dead sheep by the veterinarian and sent to the laboratory for diagnosis. viral dna was extracted from sampling materials using the phenol:chloroform:isoamlyalchohol (25:24:1) method described by sambrook, fritsch and maniatis (1989). for detection of infection, polymerase chain reactions (pcrs) were performed using the primers 556/775/555 that code the tegument gene of ovhv-2 as previously described by baxter et al. (1993) for hemi-nested pcr. the first round yielded a 422-base-pair (bp) pcr product, and the nested round yielded a 238-bp pcr product. secondly, the orf 8 primer pair (91/116) was used to amplify the ovhv-2 gb gene described by dunowska et al. (2001) for molecular characterisation. the pcr amplifications were modified for annealing temperatures (55 °c for 556/775/555 primers, 50 °c for 91/116 primers). polymerase chain reaction products were visualised by agarose gel electrophoresis. positive amplicons were purified and sequenced commercially. phylogenetic analyses were performed using the bioedit (hall 1999) and mega (tamura et al. 2013) software programmes. the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. the evolutionary distances were computed using the kimura 2-parameter method and are in the units of the number of base substitutions per site. ethical consideration the authors confirm that ethical clearance was not required for the study. results one cow from the first farm was found positive for mcf by using the tegument gene primers of ovhv-2. cattle and sheep were being kept together in this farm. additionally, in two blood samples from five sheep obtained from the same farm, dna of ovhv-2 was found. all three positive sequences for the tegument gene were closely related to each other (figure 2a). this result indicates the presence of an infection transferring to cattle by contact with infected sheep. figure 2: phylogenetic analyses by using tegument (a) and gb (b) gene sequences of ovine herpesvirus 2 strains in this study. the tegument gene sequences of samples from a sheep and a cow taken from the different farms in the same region were very close to each other, just as in the first farm. it was concluded that the infection in this village occurred as a result of transmission by contact between nearby farms. all five tegument gene sequences in this study were closely related to each other. whilst gb gene primers reacted only with bovine samples, tegument gene primers reacted in both ovine and bovine samples. bovine-originated ovhv-2 viruses in this study were in separate phylogenetic branches as they came from different geographical areas, yet both of them were affected by the group of ovine-derived herpes viruses (figure 2b). the amplicons obtained from positive samples for the gb gene of ovhv-2 were ~2800 bp. comparison of deduced amino acid sequences from gb gene between our mcf strains and the reference strains obtained from genbank has shown that the s3 strain had more amino acid substitution changes than the malatya sample (ms) (data not shown). comparing the gb gene sequences of turkish mcf strains, the identity rates were estimated at 99.3% by using clustal w algorithm (data not shown). the similarity rates between turkish mcf strains and other reference strains (dq198083, ay839756, kc577352, kr063167, af385441, mh065713) from genbank ranged from 98.6% to 99.1% (data not shown). similarly, the divergence percentage between two turkish mcf strains was found to be 0.7%; and with other strains from genbank, it was found to range from 0.9% to 1.4%. discussion serological (yeşilbağ 2007) and virological studies (yazıcı et al. 2006; yildirim et al. 2012) generally report the presence and surveying of mcf infections in the asymptomatic carrier and affected animals in turkey. the first goal of this study was to detect the dna of ovhv-2 in diagnostic samples from suspected animals infected with mcf. the second goal, the purpose of determining the origin of the virus, was to make a comparison between cattle and sheep ovhv-2 sequences obtained from affected animals. in this study, we compared the tegument gene and gb gene sequences of ovhv-2 viruses (figures 1 and 2), which were obtained from cattle and sheep from two different geographical regions in turkey. the first positive results belonged to the samples taken from one cow and five sheep in the same farm, and they all presented suspicious clinical findings related to mcf. the cow and the two sheep were found to be positive for ovhv-2 in pcr. the other positive results were acquired from a single sheep and a single cow that were sampled at different time intervals from different farms. our results show that the viruses obtained from the same regions or the same farm match each other, and the disease could be identified as sheep-associated mcf. according to the phylogenetic analysis of the tegument gene, mss that were taken from a cow and a sheep in the same village but different farms were closely related to the ovhv-2 sequences obtained from canada, norway and uk samples. they were also very similar to each other. the other three tegument-gene-positive samples in this study belonged to one cow and two sheep in the same farm. they were also quite close to each other. interestingly, in the phylogenetic tree, these samples were located closer to other tegument gene sequences (from kars) obtained from another study in turkey (yildirim et al. 2012) than other samples in the current study. generally, the genetic identity amongst turkish mcf samples and from those of other parts of the world has seen no significant genomic variations. we were able to compare the tegument gene sequences because the amplicons were obtained by using the mentioned gene primers. interestingly, only positive samples that were bovine originated could be evaluated in terms of gb genes, whilst sheep-originated samples did not react with gb gene primers. it is concluded that this situation is important for laboratory diagnosis; hence, we recommend using the tegument gene instead of the gb gene in pcr for mcf diagnostics. malignant catarrhal fever is a fatal disease for cattle, the last hosts of this infection. although experimental vaccine studies have been reported for cattle (haig et al. 2008; lankester et al. 2016; russel et al. 2012), there is no commercial product available in common use because of an insufficient antibody response in terms of intensity and duration. prevention of mcf has classically focused on control of transmission of ovhv-2 between sheep and cattle. by separating sheep from susceptible species like cattle, the infection can be limited (li et al. 1999). although there is known to be a sheep-originated mcf infection in turkey, there is a necessity to investigate different viruses that can be obtained from different animal species (sheep, goats, cattle and wild ruminants). thus, we believe that the acquisition of viruses that are adapted to cell culture, which are potential vaccine candidates, will allow us to fight mcf infections. in conclusion, ohv-2 tegument gene sequences obtained from cattle and sheep in this study were very closely related. this finding supports the hypothesis that subclinically infected sheep are the source of the infectious virus for susceptible dairy cattle. although limitation of sheep and cattle rearing together is important to prevent this infection, we believe that prophylactic protection measures may be more preferable in mixed herds. in this context, we believe that there will be a need for field isolates 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turkey’, tropical animal health and production 39, 363–368. https://doi.org/10.1007/s11250-007-9024-2 yildirim, y., bilge-dağalp, s., yılmaz, v. & faraji-majarashin, a., 2012, ‘molecular characterisation of ovine herpesvirus type 2 (ovhv-2) in turkey’, acta veterinaria hungarica 60(4), 521–527. https://doi.org/10.1556/avet.2012.046 meintjes_165-170.qxd introduction the return of urea via the blood and saliva to the gastrointestinal tract of ruminants provides a valuable source of nitrogen to rumen microbes for protein synthesis (godwin & williams 1986). therefore, the role of the kidney in conserving urea particularly during periods of low nitrogen intake, e.g. during drought conditions, is of immense importance in the ruminant. renal function during times of nitrogen shortage changes in such a way as to significantly decrease urea loss via the urine. this was found to be achieved both through a decrease in glomerular fil165 onderstepoort journal of veterinary research, 71:165–170 (2004) changes in the renal handling of urea in sheep on a low protein diet exposed to saline drinking water r.a. meintjes and h. engelbrecht department of anatomy and physiology, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract meintjes, r.a. & engelbrecht, h. 2004. changes in the renal handling of urea in sheep on a low protein diet exposed to saline drinking water. onderstepoort journal of veterinary research, 71: 165–170 previous trials have demonstrated that sheep on a low protein diet and free access to water, and sheep dosed with boluses of nacl intraruminally also with free access to water, showed decreases in urea loss via the urine compared to control animals. we monitored urea excretion in sheep on a relatively poor protein diet when they were exposed to saline drinking water, i.e. they were unable to vary their intake of nacl:water. sheep on isotonic saline drinking water (phase 3) excreted significantly more urea via the urine (284 mm/day) compared to phase 1 when they were on non-saline drinking water (urea excretion = 230 mm/day) and phase 2 when they were on half isotonic saline drinking water (urea excretion = 244 mm/day).this finding was explained by the high glomerular filtration rate (gfr) 91.9 l/day, compared to 82.4 l/day (phase 1) and 77.9 l/day (phase 2), together with a significantly raised fractional excretion of urea (feurea) (51.1 %) during this phase, and was in spite of the significantly lower plasma concentrations of urea in phase 3 compared to phase 1. the feurea probably results from the osmotic diuresis caused by the salt. there were indications of a raised plasma antidiuretic hormone (adh) concentration and this would have opposed urea loss, as adh promotes urea reabsorption. however, this adh effect was probably counteracted to some extent by a low plasma angiotensin ii concentration, for which again there were indications, inhibiting urea reabsorption during the phases of salt loading. as atrial natriuretic peptide both increases gfr and decrease sodium reabsorption from the tubule, it was probably instrumental in causing the increase in gfr and the increase in the fractional excretion of sodium (fena). keywords: kidney, low protein diet, saline drinking water, sheep, urea accepted for publication 27 january 2004—editor tration rate (gfr) and a decrease in the fractional excretion of urea (feurea) (leng, szanyiova & boda 1985). van der walt, boomker, meintjes & shultheiss (1999), although unable to demonstrate any change in gfr in sheep on a low protein diet, showed an almost 50 % reduction in the feurea. during times of water depletion there is also a reduction in the amount of urea lost via the kidneys. this has been ascribed mainly to a reduction in gfr and therefore less urea being made available for excretion rather than to a decrease in the feurea (leng, szanyiova, varady & boda 1987). in this respect it was later established that restriction of water intake for 3 days results in an upgrading of urea transporter mrna in the inner stripe of the outer medulla (shayakul, smith, mackenzie, lee, brown & hediger 2000). the intraruminal infusion of nacl at doses in excess of 1 250 mm/day in sheep with free access to drinking water, resulted in a decline in renal urea loss (godwin & williams 1986). although the saline water had the effect of increasing gfr, effective renal plasma flow (erpf) and even the feurea, less urea was excreted in the urine. this finding was ascribed solely to a lower concentration of urea in the plasma during the phase of nacl supplementation than during the control phase. in the trial referred to above, the sheep were able to regulate the ratio of water:nacl taken in, as they were given free access to non-saline drinking water. the question, however, arises as to how the kidney would handle urea in a situation where animals on a high salt intake are unable to vary the ratio of water:salt. under such conditions, the need to excrete excess nacl while at the same time retaining sufficient water to prevent dehydration, requires definite changes in kidney function. these changes may or may not favour the retention of urea by the kidney. in many parts of the world, e.g. in the vicinity of large salt pans, animals have access to only salt-laden water, i.e. they are faced with a large intake of salt and are unable to vary the ratio of salt: water taken in. if, in addition, as is often the case, their nitrogen (protein) intake is also low, the renal handling of urea becomes critical. this issue, i.e. the effect of saline drinking water together with a low protein diet on the renal handling of urea was investigated in the trial reported here. material and methods six german merino wethers (body mass 30–35 kg) were individually housed in metabolic crates in a room where the ambient temperature was kept at 22 °c. the crates were equipped for the collection of urine into refrigerated containers. the sheep were fitted with faecal bags for the collection of faeces so as to minimize contamination of the urine. throughout the trial the animals were fed a diet containing 8 % protein in the form of eragrostis teff hay at the rate of 2 % of their body mass per day (van der walt et al. 1999). the trial consisted of three phases, each lasting 7 days: phase 1 fresh water was made available ad libitum. phase 2 half isotonic saline (4.5 g nacl/l water) was offered ad libitum as the sole source of drinking water. phase 3 isotonic saline (9 g nacl/l water) was offered ad libitum as the sole source of drinking water. during each phase the animals were allowed 4 days to adapt to the drinking water before samples were taken on a daily basis for 3 consecutive days for analyses. everyday at 08:30 the feed and water intakes and urine output of each sheep were recorded and a sample of urine was retained for later analysis. blood samples were collected by venipuncture into heparin and edta for later analysis. the following analyses were carried out on both urine and blood samples: 1. urea concentration using a method described by fawcett & scott (1960) for plasma and urine 2. creatinine concentration using the jaffé method (plasma was first deproteinated). 3. na+ and k+ concentrations by the ion-specific electrode method (eschweiler ecolyte electrolyte analyser). furthermore, haematocrits of the blood samples were determined by centrifugation (using microhaematocrit tubes) and total plasma protein was estimated using a refractometer (bellingham & stanley ltd., tunbridge wells, england). from the analytical data the following derived values were calculated: 1. glomerular filtration rate. this was taken to be equal to the plasma clearance of endogenous creatinine, on the assumption that creatinine is neither absorbed nor secreted by the renal tubule in the sheep (nawaz & shah 1984; bastl, rudnick & nairns 1985). 166 renal handling of urea in sheep on low protein diet exposed to saline drinking water 2. fractional excretion of urea (feurea) (bastl et al. 1985). 3. fractional excretion of sodium (fena) (bastl et al. 1985). 4. plasma clearance of urea (curea) (bastl et al. 1985). 5. urea filtered per day (ureafiltd) 6. electrolyte-free water reabsorption/excretion (tch2o) (rose 1986). tch2o = uvol (u[na] + u[k] – 1) (l/day) (pl[na] + pl[k]) where u[na] and u[k] = the urine concentrations of na + and k+ respectively, and pl[na] and pl[k] = the plasma concentrations of na+ and k+ respectively. statistical methods: as, within a phase, there was no valid reason for the readings obtained for the variables to differ from day to day, a one way repeated measure analysis of variance (anova) test was applied to the values. if having established that between the phases there was a significant difference (at the p < 0.05 confidence level) in a variable, duncan’s multiple comparison test was used to indicate which phases in fact differed from each other. the ncss – pass 2000 programme was used. results there was a significant increase in water intake as the water source changed from fresh water to half isotonic saline water and then again with the change to isotonic saline (see table 1). the average daily water intakes for the respective phases resulted in average daily nacl intakes (via the drinking water) of 0 mm/day (phase 1), 304 mm/day (phase 2) and 801 mm/day (phase 3). the volume of urine voided per day corresponded to the increase in water intake with each phase. so, too, did the amount of sodium excreted via the urine and the fractional excretion of sodium (table 1). both plasma sodium and urea concentrations decreased significantly with the change to saline drinking water, although no further change was recorded with the further increase in salt loading (phase 3). plasma potassium concentration increased from when fresh to half isotonic drinking water was offered, and increased even further with the change from the latter to isotonic drinking water. the haematocrit and total plasma protein content remained unchanged throughout all three phases, indicating that there was neither haemoconcentration nor haemodilution with the various treatments. the amount of urea filtered per day was significantly lower in phase 2 compared to phases 1 and 3. this was due to the relatively low gfr together with a low plasma urea concentration. the amount of urea excreted via the urine increased significantly when the sheep were offered isotonic saline (phase 3). similarly, gfr rose significantly during the same phase. the fractional excretion of urea increased significantly with the change to saline drinking water, there being no difference between phases 2 and 3. both the plasma clearance of urea and the electrolyte-free water reabsorbed increased significantly from phase 1 to phase 2 and then again from phase 2 to phase 3. in fact, tch2o changed from excretion (negative value) during the control 167 r.a. meintjes & h. engelbrecht table 1 daily drinking water intake (h2o in) and urine variables, viz. volume (uvol), sodium concentration (u[na]), potassium concentration (u[k]), sodium excreted via the urine (na ex ur) and fractional excretion of sodium (fena) for sheep on fresh water (phase 1), half isotonic saline (phase 2) and isotonic saline (phase 3). mean values (sd); n = 6 are given variable phase 1 phase 2 phase 3 h20 in (l/day) 3.192 a (0.284) 3.660b (0.447) 5.008c (0.537) uvol (l/day) 0.526 a (0.140) 0.907b (0.402) 2.258c (0.529) u[na] (mm/l) 16 a (13) 220b (48) 273c (43) u[k] (mm/l) 106 a (7) 109a (22) 79b (22) na ex ur (mm/day) 10a (11) 193b (62) 597c (96) fena (%) 0.061 a (0.094) 1.866b (0.709) 4.539c (0.867) abc values in rows with different superscripts differ significantly from each other at the p < 0.05 level phase to reabsorption (positive value) during the saline loading phases. discussion in sheep 24–50 % of filtered urea is normally excreted by the kidneys in the urine (bickhardt & dungelhoef 1994). the remainder is reabsorbed by a process of simple diffusion by the renal tubular epithelium. the diffusion gradient favouring urea reabsorption does not exist to begin with, i.e. on the arrival of the filtrate in a particular section of the nephron tubule, the concentration of urea in the filtrate only rises above that in the plasma following water reabsorption (costanzo 1998). it follows therefore that urea can only be reabsorbed in those parts of the nephron where water is first reabsorbed, viz. the proximal convoluted tubule, the descending limb of henlé’s loop and the collecting duct (wang, butler, nielsen, nielsen, knepper & masilamani 2002). urea reabsorbed from the inner medullary collecting duct contributes to the hypertonicity of the inner medulla interstitium. much of this urea reenters the thin part of the ascending limb of henlé’s loop to recycle in the distal half of the nephron tubule (imai & kokko 1974). different types of urea transporter proteins have been identified, viz. ut1 transporters in the terminal inner medullary collecting duct and ut2 and ut3 transporters in the thin limb of henlé’s loop, and in the descending vasae rectae respectively (tsukaguchi, shayakul, berber & hediger 1988) . in the current trial, urea loss via the urine was significantly higher when the nacl intake of a sheep reached an average of about 800 mm/day (phase 3), compared to phases 1 and 2. this was most likely due to two factors working together. firstly, the significantly greater gfr during phase 3 (91.9 l/day) compared to phases 1 (82.4 l/day) and 2 (77.9 l/day). this had the effect of making as much urea available to the nephron tubule during phase 3 compared to phase 1, in spite of the lower urea con168 renal handling of urea in sheep on low protein diet exposed to saline drinking water table 2 plasma concentrations of sodium (pl[na]), potassium (pl[k]), urea (pl[urea]) and total proteins (tpp) as well as the haematocrit (hct) for sheep on fresh water (phase 1), half isotonic saline (phase 2) and isotonic saline (phase 3). mean values (sd); n = 6 are given variable phase 1 phase 2 phase 3 pl[na] (mm/l) 146 a (7) 137b (1) 137b (1) pl[k] (mm/l) 2.34 a (0.27) 3.04b (0.49) 6.08c (0.56) pl[urea] (mm/l) 6.8 a (0.7) 6.0b (1.0) 6.1b (1.0) tpp (g/l) 55a (7) 53a (7) 54a (4) hct (%) 22a (4) 21a (3) 22a (3) abc values in rows with different superscripts differ significantly from each other at the p < 0.05 level table 3 urine urea concentrations (u[urea]), urea excreted daily via the urine (ur exd), glomerular filtration rate (gfr), urea filtered (urea filtd), fractional excretion of urea (feurea), plasma clearance of urea (curea) and electrolyte-free water reabsorbed (tch2o) for sheep on fresh water (phase 1), half isotonic saline (phase 2) and isotonic saline (phase 3). mean values (sd); n = 6 are given variable phase 1 phase 2 phase 3 u[urea] (mm/l) 484 a (96) 304b (98) 139c (44) ur exd (mm/day) 230a (27) 244a (31) 284b (47) gfr (l/day) 82.4a (16.3) 77.9a (10.8) 91.9b (19.8) urea filtd (mm/day) 559a (61) 451b (64) 569a (143) feurea (%) 41.6 a (6) 53.9b (12.5) 51.1b (11.6) curea (l/day) 34 a (6.1) 41.5b (4.9) 47.6c (5.0) tch2o (l/day) –0.07 a (0.06) 1.01b (0.24) 2.19c (0.55) abc values in rows with different superscripts differ significantly from each other at the p < 0.05 level centration in the plasma (6.1 mm in phase 3 compared to 6.8 mm during phase 1). secondly, the fractional excretion of urea was significantly higher during phase 3 than in the control phase. during phase 2, although the fractional excretion of urea was also significantly higher than in the control phase (and similar to that in phase 3), the amount of urea excreted was similar to that of the control phase because of a similar gfr to the control phase together with a significantly lower plasma urea concentration than in the control phase, both factors favouring less urea finding its way into bowman’s capsule in phase 2 than in phase 3 (see ureafiltd in table 3). in the experiment by godwin & williams (1986), salt loading also led to a significant decrease in plasma urea concentration (from about 6 mm at zero salt loading to 0.8 mm at a nacl load of 2 000 mm/day). this was in spite of a significant reduction in urinary urea loss with salt loading. no explanation was offered for this finding, but it is also reflected in phase 2 of the current trial, viz. a significant reduction in plasma urea concentration compared to the control phase, while urinary excretion of urea was similar during these two phases. it is possible that the delivery of urea to the rumen is enhanced by the intake of saline drinking water, and that this may have an effect on plasma urea concentrations or possibly under conditions of excess salt intake, the kidney adjusts the ratio of urea to sodium in the medullary interstitium in favour of the former. godwin & williams (1986) found that gfr only rose significantly once a level of 1 500 mm nacl/day was infused intraruminally. in the current trial, a daily intake of 800 mm nacl was sufficient to raise the gfr. the differences obtained between the two trials are probably accounted for by the different methods of salt loading, viz. different salt:water intakes. during the phases of salt loading, natriuresis was obligatory for homeostasis. there was also a need to excrete the excess water taken in when saline water was the only source of drinking water available. accordingly, the fractional excretion of sodium rose significantly from phase 1 to phase 2 and again from phase 2 to phase 3. as atrial natriuretic peptide (anp) has a potent effect on increasing the fena, it is highly likely that plasma concentrations of this hormone were also raised and accounted for the increases in fena seen in this trial (sonnenburg 1990). although urine volume also increased in parallel with the excretion of salt, the electrolyte-free water reabsorption also increased. as electrolyte-free water reabsorption is largely governed by antidiuretic hormone (adh) (rose 1986), the increase in this variable observed with salt loading in the trial, suggests a concurrent increase in antidiuretic hormone concentration in the plasma. urea uptake via the ut1 inner medullary collecting duct is stimulated by adh (tsukaguchi et al. 1988). it is reasonable that increased urea reabsorption brought about by the higher levels of adh in fact neutralized to some degree the increase in fractional excretion of urea observed with salt loading in this trial. however, saline loading also brought about significant increases in natriuresis. this, taken together with the significant decreases in plasma sodium concentration and increases in plasma potassium concentration, points to inhibition of the reninangiotensin-aldosterone axis (raa) (reid, morris & ganong 1978). as the effect of adh on urea transport (ut1 transporter) is augmented by angiotensin ii (kato, klein, zhang & sands 2000), the amount of urea reabsorption stimulated by adh was probably minimized in this case, because of simultaneously low levels of circulating angiotensin ii. to conclude, in this trial we showed an increase in the urinary excretion of urea in sheep on a low protein diet with isotonic saline as the sole source of drinking water. this finding is in contrast to the decreased loss of urinary urea in sheep on low protein diets on ad lib. non-saline water (van der walt et al. 1999), and in salt-loaded sheep which were able to adjust their intake of water:salt (godwin & williams 1986). in this trial, where the animals were on a fixed intake of water:salt, the effect of the increased loss of urea probably contributed to the lower urea concentration in the plasma of the animals during phase 3 (although some other mechanism must also have been operating here if one considers the low plasma urea concentration in the absence of increased urea loss in phase 2). taking the greatly increased flow rate of urine into account with saline loading, the increase in urinary loss of urea, although significant, was not that much greater than that in control animals. this was probably due to the effect of a raised adh plasma concentration (evidenced by the increase in electrolyte-free water reabsorption), which would increase urea reabsorption in the inner medulla. this effect was presumably neutralized to some extent by low concentrations of angiotensin ii (evidenced by the changing patterns in na+ and k+ concentrations in the plasma and the natriuresis with 169 r.a. meintjes & h. engelbrecht salt loading). the increases in fena are explained by a probable rise in plasma atrial natriuretic peptide (anp) (sonnenburg 1990). acknowledgement we wish to thank the national research foundation of south africa for funding this trial. references bastl, c.p., rudnick, m.r. & nairns, r.g. 1985. assessment of renal function: characteristics of the functional and organic forms of acute renal failure, in the kidney: physiology and pathophysiology, edited by d.w. seldin & g. giebisch. new york: raven press. bickhardt, k. & dungelhoef, r. 1994. clinical studies of kidney function in sheep. i. methods and reference values of healthy animals. deutsche tierarzliche wochenschrifte, 101: 463–466. costanzo, l.s. 1998. renal physiology, in physiology. philadelphia: w.b. saunders. fawcett, j.k. & scott, j.g. 1960. a rapid precise method for the determination of urea. journal of clinical pathology, 13: 156–159. godwin, i.r. & williams, v.j. 1986. effects of intraruminal sodium chloride infusion on rumen and renal nitrogen and electrolyte dynamics in sheep. british journal of nutrition, 56:379–394. imai, m. & kokko, j.p. 1974. sodium chloride, urea, and water transport in the thin ascending limb of henlé. generation of osmotic gradients by passive diffusion of solutes. journal of clinical investigation, 53:393–402. kato, a., klein, j.d., zhang, c. & sands, j.m. 2000. angiotensin ii increases vasopressin – stimulated 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vasopressin in brattleboro rats. american journal of physiology, 278:f620–f627. sonnenberg, h. 1990 mechanisms of release and renal action of atrial natriuretic peptide. acta physiologica scandenavica: 138, suppl. 591:80–87. tsukaguchi, h., shayakul, c., berber, u.v. & hediger, m.a. 1988. urea transporters in the kidney: molecular analysis and contribution to the urea concentrating process. american journal of physiology, 275:f319–f324. van der walt, j.g., boomker, e.a., meintjes, r.a. & schultheiss, w.a. 1999. effect of water intake on the nitrogen balance of sheep fed a low or a medium protein diet. south african journal of animal science, 29:105–109. wang, x., butler, k., nielsen, j., nielsen, s., knepper, m.a. & masilamani, s. 2002. decreased abundance of collecting duct urea transporters (uta-1 and uta-3) with extracellular fluid volume expansion. american 170 renal handling of urea in sheep on low protein diet exposed to saline drinking water sebei_87-92.qxd introduction lice are divided into sucking and biting species (anoplura: linognathidae and ischnocera: trichodectidae). both biting and sucking lice are ectoparasites that adversely affect low productivity in goats (pandita & ram 1990). the actual loss resulting from louse infestation of goats is not easy to quantify but it was estimated to be ud$800 000 in the usa (steelman 1976). zumpt (1970) describes losses in sheep during winter on the highveld regions in south africa and o’callaghan, beveridge, barton & mcevan (1989) describe the effects of experimental infestations with linognathus vituli on undernourished calves. unhygienic conditions, increased population density and inadequate housing promote the spread of louse infestations. infestation can result in anaemia, hypoproteinaemia, secondary infestation, nutritional deficiencies and reduced vigour as well as in bacterial or fungal infections of bite wounds (howell, walker & nevill 1978; pandita & ram 1990; oberem & schröder 1993). in south africa infestations with linognathus africanus (the blue sucking louse of goats) and bovicola limbatus (the red biting louse of goats) have been described in a commercial herd of angora goats in the eastern cape province (horak, mcivor & greeff 2001). scanning electron microscopic descriptions of lice have rarely been made. previously morphological 87 onderstepoort journal of veterinary research, 71:87–92 (2004) use of scanning electron microscopy to confirm the identity of lice infesting communally grazed goat herds p.j. sebei1, c.m.e. mccrindle1, e.d. green2 and m.l. turner3 abstract sebei, p.j., mccrindle, c.m.e., green, e.d. & turner, m.l. 2004. use of scanning electron microscopy to confirm the identity of lice infesting communally grazed goat herds. onderstepoort journal of veterinary research, 71:87–92 lice have been described on goats in commercial farming systems in south africa but not from flocks on communal grazing. during a longitudinal survey on the causes of goat kid mortality, conducted in jericho district, north west province, lice were collected from communally grazed indigenous goats. these lice were prepared for and viewed by scanning electron microscopy, and micromorphological taxonomic details are described. three species of lice were found in the study area and identified as bovicola caprae, bovicola limbatus and linognathus africanus. sucking and biting lice were found in ten of the 12 herds of goats examined. lice were found on both mature goats and kids. bovicola caprae and l. africanus were the most common biting and sucking lice respectively in all herds examined. scanning electron microscopy revealed additional features which aided in the identification of the louse species. photomicrographs were more accurate aids to identification than the line drawings in the literature and facilitated identification using dissecting microscope. keywords: bovicola spp., goat, lice, linognathus africanus, scanning electron microscope 1 department of paraclinical sciences, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of anatomy, medical university of southern africa, box 232, medunsa, 0204 south africa 3 electron microscope unit, medical university of southern africa, box 232, medunsa, 0204 south africa accepted for publication 23 october 2003—editor features used for the taxonomic differentiation of lice species were described using the standard light microscope (green, turner & sebei 2001; turner, baker & marais 2002). light microscopy does not show in sufficient detail the morphology required for their classification. because the taxonomic differences between bovicola caprae and b. limbatus are based on the structure of their genital organs it has led to difficulty in identifying these louse species from the field. materials and methods during a longitudinal survey on the causes of mortalities in goat kids in the jericho district, north west province, south africa (sebei 2002), live lice were collected from 12 herds of communally grazed indigenous type goats. as lice can easily be seen with the naked eye when the fleece is parted (zumpt 1970) goats were visually and manually examined for external parasites in the coat of the animal. areas sampled were the dorsal neck from behind the ear, tail area, back and rump, flanks, the dewlap/brisket area, buttock and the midline laterally (price & graham, 1997). live lice were collected by means of forceps dabbed in 70 % alcohol and were then preserved in 70 % alcohol. fixed specimens were inspected under a dissection microscope. undamaged adult males and females were selected for scanning electron microscopy (sem), which has successfully been used for the identification of lice (green & baker 1996; mccrindle, green & bryson 1999; turner et al. 2002). lice were processed for sem using the method described by turner et al. (2002). a structured interview with the farmers was conducted in order to evaluate the stock management practices (including dipping) they used. results it was found that 83 % (n = 10) of the herds investigated (n = 12) were infested with biting and/or sucking lice. three species of lice were found, namely b. caprae, b. limbatus and l. africanus (table 1). lice were found on both mature goats and kids. bovicola caprae and l. africanus were the most common biting and sucking lice respectively in the herds examined. five species of lice have been described on goats throughout the world (price & graham 1997), but no records could be found of simultaneous infestations by three or more species. horak et al. (2001) have found mixed infestations of b. limbatus and l. africanus. in this study two species of biting lice (fig. 1 and 2) and one species of sucking louse (fig. 3) infested one herd, and all three species were collected from individual goats in the herd. for epidemiological studies it was necessary to accurately identify these three species. o’callaghan et al. (1989) specifically mentioned difficulties in identifying species on goats using the line-drawings made from light microscopy that are available in the literature. distinguishing micro-morphological features used for species identification under sem are shown (fig. 1–12). the sucking louse was identified as l. africanus by the following characteristics which distinguish it from linognathus stenopsis (o’callaghan et al. 1989). the shape of the head, with the prominent bulging ocular points posterior to the antenna, is typical and was confirmed by sem (fig. 3), together with the rounded female gonopods (fig. 5) (ferris 1951; kim, pratt & stojanovich 1986). the female terminal gonopod of l. africanus is rounded and lacks the “tooth-like” process described for l. stenopsis (kim et al. 1986). the gonopods of the male included a pair of terminal tubercles with setae (fig. 6) and are characteristic of l. africanus (price & graham 1997). differentiating between the bovicola spp. was more difficult as the adult females are difficult to separate (price & graham 1997). none of the bovicola spp. were densely covered with setae or large enough to be identified as bovicola crassipes which is one of the three bovicola spp. infesting goats (ledger 1980). under the stereoscopic microscope the males of b. caprae and b. limbatus were only distinguishable by the number of sclerotized sternites (ledger 1980). all the paratergites of both sexes of 88 lice infesting communally grazed goat herds table 1 distribution of lice in different herd farmer bovicola bovicola linognathus code caprae limbata africanus 1 negative negative negative 2 positive negative positive 3 positive negative negative 4 negative negative positive 5 positive positive positive 6 positive positive positive 7 positive negative positive 8 positive negative positive 9 positive positive positive 10 positive negative positive 11 negative negative negative 12 positive positive positive 89 p.j. sebei et al. fig. 1 ventral surface of bovicola caprae attached to a hair by paired mandibles (*) fig. 2 anteroventral surface of bovicola limbatus showing mandibles (*) and tarsal claws fig. 3 ventral view adult female linognathus africanus showing prominent ocular processes (<<) on head and legs ending in large single claws fig. 4 dorsal view of adult male linognathus africanus showing ocular processes (<<) on the head and pseudopenis (black arrow on abdomen) fig. 5 ventral view of the female gonopods of linognathus africanus which lacks the apical tooth at (*) as found in linognathus stenopsis fig. 6 ventral view of male gonopods of linognathus africanus showing the tubercles (*) and pseudopenis (p) 90 lice infesting communally grazed goat herds fig. 7 dorsal view of male bovicola limbatus showing the conical male gonopods (g) fig. 8 enlarged view of the conical male gonopods of bovicola limbatus showing the characteristic long setae fig. 9 dorsal view of male bovicola caprae showing the conical male gonopods (g) fig. 10 enlarged view of the conical male gonopods of bovicola caprae showing the terminal flaps (t) covered with short setae closing the genital opening (o) fig. 11 enlarged view of the antennal sensilla coeloconica of bovicola caprae showing the two pore organs with the tuft organs (p) and two plates organs enclosed in a single plate (*) fig. 12 enlarged view of the antennal sensilla coeloconica of bovicola caprae showing the two pore organs with the tuft organs (p) and three plate organs each enclosed in their own plates (*) b. caprae were well-sclerotized and brown in colour while in b. limbatus only the anterior two paratergites were sclerotized in both sexes. these paratergites were not previously identifiable from the line drawings published in the literature (werneck 1950). however, once accurate micro-morphology was determined by sem, the two species could easily be differentiated under the stereoscopic microscope. according to price & graham (1997) b. caprae and b. limbatus can be differentiated only by examining the genitalia. the male gonopods of both species, when viewed under low magnification, are conical in shape (fig. 7–9). this was confirmed with sem. the conical gonopods of b. limbatus have scattered long setae (fig. 8) while the posterior end of the gonopods of b. caprae are densely covered with short setae (fig. 10). the distinctive characteristic of b. caprae is the presence of two terminal flaps, which close over the genital opening (fig. 10). the antennal sensilla coeloconica were similar in both species with two pore organs each containing a tuft organ (fig. 11–12). the three adjacent plate organs, however, were different. in b. limbatus the anterior three plate organs share one plate (fig. 11) while the two plate organs in b. caprae are each surrounded by their own plate (fig. 12) (ferris 1951; miller & frederick 1971). discussion the main aim of this investigation was to study by sem the micro-morphological features which could aid in identification or differentiation of louse species found on communally grazed goat herds. micrographs illustrating taxonomic morphological differences between b. limbatus, b. caprae and l. africanus show details of micro-morphology not obvious under the light microscope. the differences between morphological features seen in micrographs obtained from sem and the line drawings seen in the literature could be attributed to the difference in the resolution power of light and electron microscopes (green et al. 2001; turner et al. 2002), and sem thus allowed for more accurate species identification. once positively identified by sem, it was found that specimens of b. caprae could also be distinguished from b. limbatus under the stereoscopic microscope on the basis of colour. this will be useful for field studies of the epidemiology of two the species. micrographs obtained using sem can be used to update the literature references used for the identification of these lice species. many of the line drawings, which are currently the only taxonomic references, were published in obscure journals, textbooks and memoirs of many years ago (werneck 1950; ferris 1951; kim et al. 1986). although the three goat louse species identified in this study have been recorded from commercial goat production systems, this is the first record from goats in a communal grazing system. according to seddon (1967) lice infestation occurs more frequently on young animals. this was also the case in this study. probably as a result of better identification of lice species due to use of sem, it was found that all three species could infest a single animal,. mixed infestations, by all three species of lice, were also found in a single herd. no records could be found in the literature that mixed infestations by different species of red biting lice occur. this may be due to the limited resolution obtained by light microscopy and the fact that morphological differences can only be distinguished on male specimens. acknowledgements we extend our sincere gratitude to the farmers for allowing us to handle their goats and collect specimens from them and also for their patience and hospitality during the survey. the national research foundation is thanked for financial support. references ferris, g.f. 1951. the sucking lice. volume 1. memoirs of the pacific coast entomological society. san francisco: pacific coast entomological society, 223–225. green, e.d. & baker, c. 1996. observations on the micromorphology of tropical rat mite ornithonyssus bacoti (hirst) as revealed by scanning microscopy. journal of the south african veterinary association, 67:128–132. green, e.d., turner, m.l. & sebei, p.j. 2001. functional morphology of the goat biting louse (bovicola caprae). proceedings of the microscopy society of southern africa, 31: 62. horak, i.g., macivor, k.m. de f. & greef, c.j. 2001. parasites of domestic and wild animals in south africa. xxxix. helminth and arthropod parasites of angora goats in the southern karoo. onderstepoort journal of veterinary research, 68:27–35. howell, c.j., walker, j.b. & nevill, e.m. 1978. ticks, mites and insects infesting domestic animals in south africa. part 1. descriptions and biology. department of agricultural technical services, republic of south africa (science bulletin, no. 393). kim, k.c., pratt, h.d. & stojanovich, c.j. 1986. the sucking lice of north america: an illustrated manual. university park and london: pennsylvania state university press. 91 p.j. sebei et al. ledger, j.a. 1980. the arthropod parasites of vertebrates in africa south of the sahara. volume iv. phthiraptera (insecta). publications of the south african institute for medical research, no. 56:255–257. mccrindle, c.m.e., green, e.d. & bryson, n.r. 1999. a primary animal health care approach to treatment and control of flea (ctenocephalides felis) infestation in indigenous goats kept on communal grazing. journal of the south african veterinary association, 70:21–24. miller, j.r. & frederick, h. 1971. antennal sensilla coeloconica of pedicinus obtusus obtusus. annals of the entomological society of america, 64:973–975. oberem, p.t. & schröder, j. 1993. ectoand endoparasites, in livestock production systems—principles and practice, edited by c. maree & n.h. casey. brooklyn: agridevelopment foundation: 334–360. o’callaghan, m.g., beveridge, i., barton, m.a. & mcevan, d.r. 1989. recognition of the sucking louse, linognathus africanus, on goats. australian veterinary journal, 66:228–229. pandita, n.n. & ram, s. 1990. control of ectoparasitic infestation in country goats. small ruminant research, 3:403–412. price, m.a. & graham, o.h. 1997. chewing and sucking lice as parasites of mammals and birds. united states department of agriculture (agricultural research service technical bulletin, no.1849). sebei, p.j. 2002. the assessment of some factors influencing the survival of kids in a small-scale communal goat production system. msc thesis, university of pretoria. seddon, h.r. 1967. diseases of domestic animals in australia, part 2, arthropod infestations (flies, lice and fleas), 2nd ed. commonwealth of australia (service publications, no. 6). steelman, c.d. 1976. effects of external and internal arthropods parasites on domestic livestock production. annual review of entomology, 21:155–178. turner, m.l., baker, c. & marais, r. 2002. scanning electron microscopical investigation of the waterbuck louse bovicola (syn. damalinia) hilli found at the rietvlei nature reserve near pretoria. koedoe, 45:59–63. werneck, f.l. 1950. os malófagos de mamíferos, parte ii: ischnocera (continuaçao de trichodectidae) e rhynchopthirina. memorias do instituto oswaldo cruz, 5:1–207 zumpt, g.f. 1970. observations on red lice (damalinia ovis) infestations in sheep on the transvaal highveld. journal of the south african veterinary medical association, 41:315– 317. 92 lice infesting communally grazed goat herds abstract introduction research methods and design study population laboratory procedures investigation of cow risk factors statistical analysis results discussion prevalence and species identification of non-aureus staphylococci strain typing interherd relationship of non-aureus staphylococci species farming systems somatic cell counts parity days in milk conclusion acknowledgements references about the author(s) inge-marie petzer department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa christiaan labuschagne inqaba biotechnical industries, pretoria, south africa lufuno phophi department of paraclinical science, faculty of veterinary science, university of pretoria, pretoria, south africa joanne karzis department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa citation petzer, i-m., labuschagne, c., phophi, l. & karzis, j., 2022, ‘species identification and cow risks of non-aureus staphylococci from south african dairy herds’, onderstepoort journal of veterinary research 89(1), a2021. https://doi.org/10.4102/ojvr.v89i1.2021 note: additional supporting information may be found in the online version of this article as online appendix 1. original research species identification and cow risks of non-aureus staphylococci from south african dairy herds inge-marie petzer, christiaan labuschagne, lufuno phophi, joanne karzis received: 23 mar. 2022; accepted: 14 june 2022; published: 27 july 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract detailed information on specific species of non-aureus staphylococci (nas) has become a necessity for effective udder health control programs in south africa. the main objective of this preliminary study was to identify the different nas species and strains present in dairy herds in south africa using a cost-effective method. a further objective was to investigate the effects of cow risk factors and farming systems on the nas isolates identified. a total of 214 nas, isolated from milk collected from 17 south african dairy herds, were identified using three diagnostic tests (api staph test, maldi-tof and 16s rrna). there was a good observed agreement between the maldi-tof and 16s rrna sequencing (92.2%) and a poor observed agreement between the maldi-tof and api staph (25.7%). the genetic relatedness within species was investigated in 128 of these isolates using random polymorphic amplified deoxyribonucleic acid (dna) (rapd), verified by multilocus sequence typing (mlst), and phylogenetic analysis and cow risk factors were investigated on species level. the main nas species isolated were staphylococcus chromogenes (75.2%), staphylococcus epidermidis (9.4%) and staphylococcus haemolyticus (8.9%). the rapd test identified 34 staphylococcus chromogenes, 13 staphylococcus epidermidis and nine staphylococcus haemolyticus strains, indicating genetic diversity amongst strains and herds. the presence of nas intramammary infections was found to be significantly related to the farming systems, composite cow milk somatic cell count (scc), parity and days in milk (dim). significantly more nas were isolated from primiparous and from older cows. this knowledge could assist with the management of nas on dairy farms. keywords: molecular epidemiology; nas species identification; genetic diversity; cow risk factors; intramammary infection; dairy cows; south africa introduction mastitis remains the disease that is the biggest economic challenge amongst dairy cattle in developed countries (geary et al. 2012; pyörälä & taponen 2009). the subclinical form of the disease is considered the most important; it is increasing compared to clinical mastitis and is estimated to be responsible for more than 80% of economic mastitis losses (giesecke, du preez & petzer 1994; shim, shanks & morin 2004), due to long-term reduction in milk quality and milk production during a cow’s lifetime. in many well-managed dairy herds, a decrease in the prevalence of intramammary infection (imi) caused by contagious major pathogens is experienced whilst a higher proportion of subclinical or mild clinical infections caused by non-aureus staphylococci (nas) (previously called coagulase-negative staphylococci) is present (petzer et al. 2017). during a retrospective study, using a data set of both clinical and subclinical mastitis isolates from 830 south african commercial dairy farms, bacteria were isolated from 33.9% of 95 228 quarter milk samples examined. of the isolates, 12.4% were major gram-positive (6.8% staphylococcus aureus, 2.1% streptococcus agalactiae, 2.5% streptococcus uberis and 1.0% streptococcus dysgalactiae), 0.3% major gram-negative (mainly escherichia coli and klebsiella spp.), 19.2% nas and 2.0% other (petzer et al. 2017). during the period 2018–2020, nas were isolated from 76.5% (n = 59 274) of all bacteria tested at the milk laboratory of the university of pretoria from composite cow milk samples. thirty-five percent of the milk samples from which the nas bacteria were isolated had a somatic cell count (scc) in excess of 200 000 cells/ml milk, whilst 19.2% had an scc of more than 500 000 cells/ml milk (pers. comm., milk laboratory, pretoria, 2021). this data suggested that nas is the most prevalent group of pathogens isolated from milk in south africa, similar to other countries (piessens et al. 2011; sampinon et al. 2009; taponen et al. 2006), and was the motivation for this study. although nas were treated as a homogeneous bacterial group in udder health, 53 different nas species have been identified since 2017, and 26 thereof were isolated from bovine milk (condas et al. 2017; de buck et al. 2021). different nas vary between different species in their epidemiology, pathogenicity, virulence, ecology and host adaptation, antimicrobial resistance profiles (de buck et al. 2021; de visscher et al. 2014; vanderhaeghen et al. 2014, 2015) and even between different strains within the same nas species (piccart et al. 2016; piessens et al. 2012). non-aureus staphylococci have distinct relationships within the microbiome of the udder and may also have protective effects against other mastitis pathogens (de buck et al. 2021). the main ecological niches for nas are skin and mucous membranes (taponen et al. 2006). in cattle, nas are frequently isolated from the cow’s milk, hair coat, teat skin (de visscher et al. 2014; piessens et al. 2011) and faeces (wuytack et al. 2017).the most common nas isolated from bovine milk are s. chromogenes, s. epidermidis, s. simulans, s. haemolyticus and s. xylosus (dalen et al. 2017; vanderhaeghen et al. 2015). most nas imi, but especially s. chromogenes, seem to persist for 149.4 days or longer (range: 63.0–329.8 days), contrary to general beliefs (piessens et al. 2011; suprѐ et al. 2011). this indicates the adaption of nas for survival in udder parenchyma. staphylococci spp. are still identified based on colony morphology, haemolysis patterns, gram staining, catalase and coagulase production. new knowledge regarding coagulase production of nas species has complicated such basic identification. other methods used in species identification of nas include the api staph and vitek commercial kits (biomerieux), pulsed-field gel electrophoresis (pfge), polymerase chain reaction (pcr), internal transcribed spacer (its)-pcr, maldi-tof mass spectrometry, malsequencing-based identification systems of the 16s rrna, hsp60, tuf, soda, and rpob genes, multilocus sequence typing (mlst) and now whole genome sequencing (wgs) (de buck et al. 2017; vanderhaeghen et al. 2015). randomly amplified polymorphic dna (rapd)-pcr is a simple and rapid technique used in epidemiological studies that can be performed with low quantities of dna to identify genetic variation and establish strain-specific fingerprints (babalola 2003; neela et al. 2005; olive & bean 1999; rabouam et al. 1999). non-aureus staphylococci are also classified according to their distribution, either in the cow’s environment or in cow’s milk. the size of a pan genome compared with a core genome is often an indication of adaptation of an organism to a specific niche (de buck et al. 2017). current knowledge suggests that s. chromogenes is a bovine opportunistic pathogen (vanderhaeghen et al. 2015) and s. epidermidis a human-adapted species, and both species are rarely found in the environment (piessens et al. 2011; suprѐ et al. 2011). staphylococcus haemolyticus appears to be opportunistic, as it occupies a variety of habitats (de visscher et al. 2017; vanderhaeghen et al. 2015). the most frequently isolated nas from the teat skin and apex was s. chromogenes (taponen, björkroth & pyörälä 2008; thorberg et al. 2009). there was no known study in south africa that identifies the specific nas species or strains, despite the high prevalence of nas as a potential mastitis-causing pathogen. this motivated this research. it became necessary to discover their epidemiology in order to identify the true source and risk of imi in bovine mastitis. this preliminary study was performed to fill a gap in knowledge. the main objective of this nas study in south africa was to identify the foremost nas species up to strain level that were isolated from milk samples, using a cost-effective method which could be used in future routine diagnosis. a further objective was to investigate the effects of some cow factors and farming systems on these nas isolates. a better understanding of nas in south africa and added knowledge on the effects of such cow factors on different nas species can provide valuable information for proactive udder health management. research methods and design the study was approved by the university of pretoria animal ethics committee (reference number v046–18) as well as the research ethics committee (reference number rec024–18). study population individual cow milk samples were used from 17 dairy herds which submitted samples of all lactating cows in the herd for routine analysis. sampling took place from april to november 2017, and the number of lactating cows per herd used in the study varied from 165 to 1493. after performing routine microbiology and cytology on the composite cow milk samples per herd, a total of 2140 nas isolates were isolated from 11 608 samples examined. information on the farming systems, days in milk (dim) and parity were obtained from the records of the producers of each herd (afimilk or delpro management systems). ten percent of nas isolates, per herd, were selected at random for this study, accounting for the 214 isolates. therefore, these nas isolates were from cows with clinical or subclinical mastitis or with intramammary nas infections. parity and dim of cows were recorded at sampling. breeds were holstein friesland, jersey and crossbreeds, and they varied between and within herds. laboratory procedures initial bacteria isolation was performed at the south african national accreditation system (sanas) accredited milk laboratory (university of pretoria, faculty of veterinary science, onderstepoort). the classical microbiological methods were performed (national mastitis council 2017), and initial identification of staphylococci was performed phenotypically using classic microbiology such as colony morphology, pigmentation, haemolysis, catalase, staphylase–coagulase, maltose and potassium hydroxide tests (petzer et al. 2017). pure colonies from these nas isolates, after initial phenotypic identification, were frozen at −80 °c until required. somatic cell count was performed by fluoro-opto-electronic means using a fossomatic fc (rhine ruhr, wendywood, south africa). non-aureus staphylococci species identification three diagnostic methods were compared for accuracy and relative cost-effectiveness for the identification of nas species. the stored nas isolates were revived and tested using the following methods: matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (maldi tof), api staph test and 16 s rrna sequencing. matrix assisted laser desorption/ionisation time-of-flight mass spectrometry the fresh isolates were identified using the maldi-tof, bruker daltonics, bremen, germany (department of plant and soil sciences, university of pretoria, south africa). the maldi biolayer 3.0 software with an integrated pattern-matching algorithm was used to compare generated peak lists against the reference library (van dyk et al. 2016). 16s rrna test genomic deoxyribonucleic acid (dna) was extracted from pure cultures using the quick-dna™ fungal/bacterial miniprep kit (zymo research, catalogue no. d6005). the 16s recombinant dna (rdna) target region was amplified using onetaq® quick-load® 2x master mix (neb, catalogue no. m0486) with the primers 27-f (5-agagtttgatcmtggctcag-3) and 1492-r (5-cggttaccttgttacgactt-3) (lane 1991). the pcr products were run on a gel and gel-extracted with the zymoclean™ gel dna recovery kit (zymo research, catalogue no. d4001). the extracted fragments were sequenced in the forward and reverse direction (nimagen, brilliantdye™ terminator cycle sequencing kit v3.1, brd3-100/1000) and purified (zymo research, zr-96 dna sequencing clean-up kit™, catalogue no. d4050). the purified fragments were analysed on the abi 3500xl and 3730xl genetic analyzers (applied biosystems, thermofisher scientific). clc bio main workbench version 7.3 was used to analyse the .ab1 files generated, and species identification was obtained by a blast search (ncbi). analytical profile index staph test the analytical profile index (api) staph test was performed according to manufacturer’s specifications and read after 24 (± 2 h). the staphylococcus aureus atcc 25923 and staphylococcus epidermidis atcc 12228 served as reference strains for quality control purposes. strain typing random polymorphic amplified dna analysis was performed, and preliminary phylogenetic trees were drawn on the three major groups of nas species identified, consisting of s. chromogenes (n = 88), s. epidermidis (n = 15) and s. haemolyticus (n = 14), as the other nas species identified from the remaining 11 isolates of the total 214 nas isolate subset were very low in numbers (n = 1 or 2 each). the rapd method was chosen due to economic constraints for this study, which consisted of a relatively large number of samples. the rapd fingerprints were amplified using onetaq® quick-load® 2x master mix (neb,catalogue no. m0486) with the primers b0043-17 (5-gcgatcccca-3), s260 (5-acagccccca-3) and s33 (5-cagcacccac-3) (idil & seyis bilkay 2014) containing 0.5 µg/ml ethidium bromide (vwr life science, united states [us]) and visualised under uv light by gel doc (cleaver scientific, uk). the rapd-pcr banding patterns generated were analysed manually and each informative band scored as (1) for presence or (0) for absence. cluster analyses were carried out and dendrograms were created using the unweighted pair-group method with averaging (upgma) in mega x. on the gels, a ladder was run in the first and last lane of each tier. in cases where all samples did not fit on a single gel, random samples were run again on subsequent gels to confirm patterns. random samples were also repeated within each pcr set to verify results. in addition, mlst was performed on a subset of samples as an added verification of the rapd-pcr analysis. seven s. chromogenes isolates (nas-128, nas-163, nas-49, nas-141, nas-143, nas-97, nas-16) were selected and six housekeeping genes (arcc, dnaj, fumc, glpf, hutu, menf) sequenced for each (huebner et al. 2021). the relationships were inferred by using the maximum likelihood method (ml) and jukes–cantor model (jukes & cantor 1996) in mega x (kumar et al. 2018). the percentage of trees in which the associated taxa clustered together was shown next to the branches. the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. there was a total of 3637 positions in the final dataset. investigation of cow risk factors the number of nas isolates in total and of each nas species identified were compared between herds. this information was summarised in a table (appendix 1, table s3); statistical analysis could not be performed per herd due to low sample numbers. the effects of farming systems (total mixed ration [tmr] and pasture-based systems), as well as cow factors (parity, dim and scc level) on the total number of nas isolates were tested. individually identified nas species and strains were too few for reliable statistical testing. statistical analysis data were captured in excel, checked and cleaned. the one sample binomial test (procedure bntest) was used to test the probability of success under a binomial distribution (freund, mohr & wilson 2010). the proportion calculated was the overall observed species agreement between the two tests in each case. this overall observed species agreement was carried out between the 16s rrna sequencing method (reference method) and the maldi-tof test and between the 16s rrna sequencing method and the api test. individually identified nas species were too few for reliable statistical testing; thus, a one-way chi-square test was used for equal proportions per group on total number of isolates for farming systems, scc categories, parity and dim. if the probability level (under null hypothesis) was p > 0.05, then the proportions of strain categories did not differ at the 5% level (freund et al. 2010). data were analysed using the statistical software genstat® (vsn international 2019). results identification of non-aureus staphylococci there was good overall probability of agreement of 0.973 between the 16s rrna and the maldi-tof identification methods, with an exact probability, p < 0.001 at a 95% confidence interval (ci) for proportion (0.9313, 0.9925) (appendix 1, table s1). however, there was poor overall probability of agreement of 0.261 between the 16s rrna and the api identification methods, with an exact probability p < 0.001 at a 95% ci for proportion (0.1906, 0.3408) (appendix 1, table s2). this poor observational agreement between 16s rrna and api methods showed the same diagnosis for only 9/16, 19/86 and 3/18 for s. epidermidis, s. chromogenes and s. haemolyticus, respectively (appendix 1, table s2). the most prevalent nas species isolated from milk originating from 17 south african dairy herds using the maldi-tof results were staphylococcus chromogenes (75.2%), followed by s. epidermidis (9.4%), s. haemolyticus (8.9%), s. simulans (2.8%), s. xylosus (2.3%) and one isolate each from s. auricularis, s. sciuri, s. hyicus and s. hominis. staphylococcus chromogenes was the most prevalent isolate in all but two herds, in which equal numbers of s. chromogenes and s. epidermidis were isolated (appendix 1, table s3). staphylococcus chromogenes embodied 45.5% – 100.0% of nas per herd, for the herds tested (appendix 1, table s3). staphylococcus epidermidis and s. haemolyticus were both the second most prevalent nas species, occurring in eight herds each (appendix 1, table s3). the three foremost groups of nas species, s. chromogenes (n = 88), s. epidermidis (n = 15) and s. haemolyticus (n = 14), were examined by rapd-pcr with three random 10-mer primers to assess relative genetic relationships. amplification revealed polymorphic bands ranging from 200 base pairs (bp) to 3000 bp. isolates were scored across seven bands for primer b0043-17, nine bands for primer s33 and seven bands for primer s260. all three primer banding patterns were combined for each sample to create the dendrograms for each species (figures 1–3). the further mlst analysis performed on a subset of seven s. chromogenes isolates indicated the same relationships observed for the rapd analysis, supporting the results of the rapd-pcr method. figure 1: random polymorphic amplified dna dendrogram of 88 staphylococcus chromogenes using simple-match similarity matrix clustered by the unweighted pair-group with arithmetic mean. letters of the alphabet indicate different herds, whilst the nas number is the identification of the isolate tested. the dendrograms were created using the unweighted pair-group method, and the numbers on each branch indicate similarity between. figure 2: random polymorphic amplified dna dendrogram of 15 staphylococcus epidermidis. letters of the alphabet indicate different herds, whilst the non-aureus staphylococci sample numbers identify a non-aureus staphylococcus isolate. figure 3: random polymorphic amplified dna dendrogram of 14 staphylococcus haemolyticus. letters of the alphabet indicate different herds, whilst the nas number is the sample number. cow risk factors the number of nas isolates differed per herd, for example herd l (total samples tested = 401) having only 20 isolates, from which two were selected, and herd n (total samples tested = 550) having 40 nas isolates with 4 tested, of which only one nas species was isolated (appendix 1, table s3). a significant (p < 0.001) larger proportion of the total nas isolates originated from pasture-based herds (70.95%) rather than from tmr based herds (29.05%) (table 1). table 1: the total number and percentage of non-aureus staphylococci isolates from milk samples in pasture-based and total mixed ration farming systems. the proportions of the total nas isolates varied significantly between level of scc (p < 0.001) (table 2). somatic cell count levels of nas isolates were not consistent (table 2). all four cases of clinical mastitis identified were caused by s. chromogenes. table 2: the total number and percentage of non-aureus staphylococcus isolates from milk samples, per cow somatic cell count categories. the proportions of the total nas isolates per parity (lactation number) were significantly different (p < 0.001) (table 3). non-aureus staphylococci were predominantly isolated from primiparous cows (36.71%) and from third-lactation and older cows (37.68%), whilst 15.46% and 10.14% were isolated from secondand third-lactation cows respectively (table 3). table 3: the total number and percentage of non-aureus staphylococcus isolates from milk samples, per parity (lactation numbers). the proportions of the total nas isolates per dim or stages of lactation were significantly different (p < 0.001) (table 4). all nas species tested were more frequently isolated from cows in late lactation (200 plus dim) and the least frequently isolated from those in early lactation. on species level, 57.14% s. chromogenes, 60.00% s. epidermidis, 68.42% s. haemolyticus, and 71.43% other nas species were isolated from cows in late lactation (table 4). table 4: the total number and percentage of non-aureus staphylococcus isolates from milk samples per days in milk/stages of lactation. discussion staphylococci are the most commonly isolated bacteria from milk of dairy cows (condas et al. 2017). when performing routine mastitis diagnosis, staphylococci are usually divided into nas and coagulase-positive staphylococci (s. aureus) (taponen & pyörälä 2009). according to zadoks and watts (2009), the accurate identification of nas at species level cannot be provided reliably by classical bacteriology using phenotypic and biochemical criteria alone. prevalence and species identification of non-aureus staphylococci a broad variation in colony morphology and colour were observed in 214 nas isolates originating from 17 south african commercial dairy herds, decreasing the discriminative power of this method of identification and species typing. the maldi-tof method was selected as the preferred method for routine diagnosis of nas to species level in south africa, as its results compared favourably with those of the 16s rrna sequencing method (reference method used), and it was the most cost-effective test in south africa at the time. there was good overall probability of agreement of 0.973 between the 16s rrna and the maldi-tof identification methods, with an exact probability p < 0.001 at a 95% ci for proportion (0.9313, 0.9925). this can be seen by the diagonal pattern highlighted in the table of agreement (table 2), showing the same diagnosis for 16/17, 99/100 and 16/17 for s. epidermidis, s. chromogenes and s. haemolyticusi, respectively. although very few numbers were isolated of s. hyicus, s. sciuri, s. simulans and s. xylosus, this table showed that both the 16s rrna and maldi-tof method picked up these species (table 2). however, there was poor overall probability of agreement of 0.261 between the 16s rrna and the api identification methods, with an exact probability p < 0.001 at a 95% ci for proportion (0.1906, 0.3408) (appendix 1, table s1). this finding agreed with other studies which showed that phenotypic tests (api staph id 32 and staph-zym) were inaccurate in species identification of nas from bovine milk samples, and genotypic methods were shown to have higher typing ability and accuracy in the identification of bovine nas (de buck et al. 2021; sampimon et al. 2009; taponen & pyörälä 2009). api staph has also been shown to have a moderate to low performance in goat nas identification (koop et al. 2012). the overall predominant nas species in this study was s. chromogenes, followed by s. epidermidis and s. haemolyticus. staphylococcus simulans and s. xylosus were isolated at lower frequencies and s. hominis, s. hyicus and s. sciuri in < 0.5% (appendix 1, table s3). the results of this study relate to the findings of piessens et al. (2011) that identified s. chromogenes as the most prominent nas and s. epidermidis, s haemolyticus and s. simulans as frequently isolated by other studies (dalen et al. 2017; vanderhaeghen et al. 2015). the three species of nas isolates identified in this study agreed with the findings from other studies conducted in countries such as canada, belgium (4 studies), finland, the netherlands, the united states, poland and argentina (condas et al. 2017; de visscher et al. 2016, 2017; jenkins et al. 2019; valckenier et al. 2021). the prevalence of each species of nas in this study (appendix 1, table s3) differed from that found in a study performed in switzerland, which found that the most prevalent nas species were s. xylosus (35%), s. vitulinus (10%) and s. chromogenes (7%), whereas s. chromogenes (5%), s. xylosus (5%) and s. haemolyticus (4%) isolates were least prevalent (jenkins et al. 2019). less prevalent nas species such as s. simulans and s. xylosus were isolated in higher frequencies by suprѐ et al. (2011). staphylococcus chromogenes was the most predominant species isolated from the 17 south african herds and may complicate basic microbiology diagnostics, as its coagulase results are inconstant (wuytack et al. 2017). similar to this study, other studies also used 16s rrna sequencing to identify nas species successfully (casaes nunes et al. 2016). as shown in figures 1–3 of this study, previous similar studies also determined evolutionary relationships of nas strains by the construction of dendrograms (travesari et al. 2019). the different nas species and strains in this study indicated that these herds contained genetically diverse strains, which agreed with the results of two other studies which also showed genetic diversity within strains and between herds (koop et al. 2012; naushad et al. 2019). strain typing great diversity in genotypes of the 88 s. chromogenes isolates was indicated, with 34 different strains in total and 17 groups of closely related strains (figure 1). group one consisted of 13 identical strains and a further strain which was different but closely related to group one (figure 1). the largest cluster of 11 isolates originated from four different herds. the maximum likelihood phylogeny, drawn from sequencing data for the subset of 7 s. chromogenes isolates on which mlst analysis was performed, reflected the same relationships observed from the rapd-pcr analysis. this mlst analysis was performed in order to validate the rapd dendrograms which otherwise may include possible bias, as explained in the study by van belkum et al. (2001), which compared the accuracy of strain typing between various genetic methods. in both analyses, isolates nas-128 and nas-163 as well as nas-48 and nas-97 fell together as the same strain, whilst nas-141 fell closer to these two clusters than nas-16 and nas-143, which fell on a separate branch and were more dissimilar compared to the other isolates. this showed similar genetic diversity to the study by jenkins et al. (2019), in which 26 different strains were found out of a total of 48 isolates for s. chromogenes. genetic diversity amongst the 15 s. epidermidis isolates was evident as 13 potentially different strains (figure 2) were present. the s. epidermidis formed two clusters of similar and one dissimilar isolate (figure 2). these findings were also similar to those of jenkins et al. (2019), who found that amongst the 13 s. epidermidis isolates tested, each of them was a different strain. the 14 s. haemolyticus isolates consisted of nine different strains, indicated by two closely related clusters as well as two dissimilar isolates (figure 3). these results were also similar to the results obtained by jenkins et al. (2019) for s. haemolyticus, which showed wide genetic diversity with the strains clustered into six major groups and multiple subgroups. the rapd-pcr analysis indicated that several strains of s. chromogenes, s. epidermidis and s. haemolyticus were isolated, with similar strains originating from different herds as well as different strains being isolated from the same herd (figures 1–3). the different nas species and strains isolated from south african dairy herds showed broad genetic diversity, which agreed with the results of a study in the united states of america (jenkins et al. 2019) and with a review study showing similar worldwide results (de buck et al. 2021). interherd relationship of non-aureus staphylococci species the number of isolates per herd, as indicated in appendix 1, table s3, were 10% of the total number of nas isolated per herd. the number of nas species per herd was too low for statistical analysis on the interherd relationship (appendix 1, table s3). however, the table indicated different nas species per herd, and s. chromogenes was the most prominent in all herds (appendix 1, table s3). this agreed with the study by travesari et al. (2019), which also found differences in nas species distribution between herds. similar to other studies (de buck et al. 2021; idil & seyis bilkay 2014; jukes & cantor 1996; naushad et al. 2019; piessens et al. 2011), the results of this study showed wide diversity not only between nas species but also between different nas strains within the same species for s. chromogenes, s. epidermidis and s. haemolyticus, both amongst different herds and within the same herd (appendix 1, table s3 and figures 1–3). this suggested a potentially large natural reservoir of nas species and strains, suggesting that cow risk factors on a herd level may be involved in the establishment of particular species in a dairy herd (de buck et al. 2017; piessens et al. 2011). the same authors (piessens et al. 2011) showed that the primary reservoirs of the nas species that cause imi varied. staphylococcus chromogenes and s. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. however, for s. haemolyticus and s. simulans, the environment was found as a reservoir, suggesting that imi with these species were possibly environmental in origin (piessens et al. 2011). farming systems a significantly larger proportion (p < 0.001) of the total nas isolates originated from pasture-based herds (71.0%) than from tmr-based herds (29%) (table 1). proportionally more s. chromogenes was isolated from pasture-based (75.16%) than tmr herds (24.84%), whilst s. haemolyticus was more prevalent among tmr herds (table 1). strains of s. chromogenes seemed to vary both within and between herds and farming systems (appendix 1, table s3 and figure 1). these findings were in general agreement with those of taponen et al. (2016). a study by jenkins et al. (2019) showed a variation in geographical distribution of the different nas species, and the three major species that occurred independent of geographical region with the highest frequencies were s. chromogenes, s. haemolyticus and s. simulans. somatic cell counts the proportions of the total nas isolates per scc group were significantly different (p < 0.001) (table 2). this finding agreed with a comparison observed between the average scc of milk samples from which specific nas species were isolated and the number of exo-enzyme, host evasion and iron uptake genes these species carried (condas et al. 2017; naushad et al. 2019). in addition, these studies showed interesting associations between virulence genes identified in nas, with marked differences in the strength of these associations between isolates with low scc and clinical mastitis nas isolates (de buck et al. 2021 naushad et al. 2019). however, the scc of different nas species were not consistent, which could have been attributed to many of the other factors affecting scc. thus, the different nas species and strains did not show significant trends in scc (table 2). there is increased pressure to achieve lower bulk tank scc levels and in turn benefit from the associated milk quality premiums (sears & mccarthy 2003). although most subclinical nas imi are not treated, they are the most prevalent bacteria isolated from milk samples in south africa (petzer et al. 2017) and other countries (de buck et al. 2017; monteiro et al. 2017). the amount of nas isolates identified in south africa has increased from 6.2% to 38.0% of milk samples when all lactating cows in a herd are cultured (petzer et al. 2017). the scc level of the nas positive composite cow milk samples varied considerably, from 2000 cells/ml milk to exceeding one million cells/ml milk. herds with 35% nas imi may have had scc distribution of more than 91% of cows with scc below 250 000 cell/ml milk, whilst in other herds with similar bulk tank scc levels, there was 10.0% nas imi and 45.0% of cows had scc at levels below 250 000 cells/ml milk. non-aureus staphylococci are known to increase the scc threefold or fourfold and decrease the quality of milk (leitner & blum 2017; taponen & pyörälä 2009). bacterial cure rate for nas imi treated with antimicrobials was significantly higher (86.0%) than in non-treated quarters (46.0%). however, antimicrobial resistance was more common for nas than for s. aureus (taponen et al. 2006). there was a need for a cost-effective method of identification of nas species that could form part of the current routine diagnostic portion of a proactive udder health program to investigate one of the reasons why there was such a large variation in scc with nas imi. parity the proportions of the total nas isolates per lactation number (parity) were significantly different (p < 0.001) (table 3). the findings that nas were isolated more frequently from first lactation cows (36.71%) and older cows (≥ 3 lactations) (37.68%) were different from other studies. sampimon et al. (2009) and tenhagen et al. (2009) found nas to be more common in primiparous than in multiparous cows. staphylococcus chromogenes was more prominent in primiparous cows and in cows in their second lactation, whilst similar to the findings of sampimon et al. (2009), s. epidermidis and s. haemolyticus were more frequently isolated from the older cows (≥ 3 lactations). days in milk the proportions of the total nas isolates per lactation days group or dim were significantly different (p < 0.001) (table 4). all nas species in this study were more frequently isolated from cows in late lactation (200 days plus) and the least from early lactation cows (table 4). these findings were in contrast to those of jenkins et al. (2019), which found that fewer numbers of s. chromogenes, s. haemolyticus and s. epidermidis were isolated from cows in late lactations compared to early lactations, although these were not significantly different. conclusion this preliminary study for south africa identified nas to species level using three different methods. there was a good observed agreement between species identification of maldi-tof and 16s rrna sequencing and a poor observed agreement with the phenotypic api staph test. the maldi-tof was found to be the most cost-effective method to be used in future for routine nas species identification under south african conditions. the overall predominant nas species isolated in high frequencies from south african dairy herds were s. chromogenes, s. epidermidis and s. haemolyticus. the rapd-pcr analysis, validated by mlst for strain level identification of nas, showed wide genetic diversity of various strains of s. chromogenes, s. epidermidis and s. haemolyticus isolated between different herds and within the same herd. no clear patterns of origin of particular strains could be deduced, which highlighted the extensive genetic diversity of the nas isolated from herds in this study. there was a significantly larger proportion of the total nas isolates originating from pasture-based than from tmr herds. the proportions of the total nas isolates varied significantly between the level of scc, parity and dim. knowledge of the effect of these cow factors on nas can assist in the improvement of udder health management of dairy herds affected by nas in south africa. acknowledgements the authors would like to thank the staff of the milk laboratory, faculty of veterinary science, university of pretoria; mrs corrie watermeyer and mrs fredah konaite; miss renette badenhorst, ms ofentse nckako, ms nodolly mmema and mr kgomotso malekane. the authors also thank the national research foundation (nrf) of south africa for its funding support for the maldi-tof biotyper (grant-specific unique reference number (uid) 74426), prof. lise korsten, ms zamma zulu and the staff of the university of pretoria’s department of plant and soil sciences. the authors would also like to thank mrs marie smith for her assistance with the statistical analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions conceptualisation, i.m.p., j.k. and c.l.; methodology, i.m.p., j.k., c.l. and l.p.; software, i.m.p., j.k. and c.l.; validation, c.l.; formal analysis, i.m.p., j.k. and c.l.; investigation, i.m.p., l.p.; resources, l.p. and i.m.p.; data curation, j.k., i.m.p. and l.p.; writing – original draft preparation, j.k. and i.m.p.; writing – review and editing, i.m.p., j.k. and c.l.; visualisation, i.m.p. and j.k.; supervision, i.m.p.; project administration, j.k.; funding acquisition, i.m.p. and j.k. all authors have read and agreed to the published version of the article. ethical considerations the study was approved by the university of pretoria animal ethics committee (ref. no. v046–18) as well as the research ethics committee (ref. no. rec024–18). funding information this research was partially funded by the national research foundation of south africa (grant no. 120319) and milk sa (grant no. prj 0212). data availability data supporting the findings of this study are available from the corresponding author, i.-m.p., on request. disclaimer the views expressed in the submitted article are the authors’ own and not an official position of the institution or funder. references babalola, o.o., 2003, ‘molecular techniques: an overview of methods for the detection of bacteria’, african journal of biotechnology 2(12), 710–713. https://doi.org/10.5897/ajb2003.000-1127 casaes nunes, r.s., pires de souza, c., pereira, k.s., del aguila, e.m. & flosi paschoalin, v.m., 2016, ‘identification and molecular phylogeny of 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helmeted guineafowls, numida meleagris (linnaeus, 1758), are distributed throughout most of south africa and almost the entire african continent (del hoyo, elliot & sargatal 1994). studies to elucidate the helminth fauna of these hosts in south africa have been undertaken by saayman (1966), crowe (1977) and verster & ptasinska-kloryga (1987), but were restricted to the eastern cape, the northern cape and gauteng provinces. although relatively wide-spread in africa, crested guineafowls, guttera edouardi (hartlaub, 1867), are scarce and have a limited distribution within south africa. they occur in the limpopo, north west, mpumalanga and kwazulu-natal provinces and are listed as rare or accidental in gauteng province (hockey, dean & ryan 2005; lepage 2007). to date our knowledge concerning their helminth fauna is virtually non-existent. ortlepp (1937, 1938a,b, 1963) reported on the cestode and nematode parasites of guineafowls of southern africa present in the national collection of animal helminths, formerly known as the onderstepoort helminthological collection, or material made 265 onderstepoort journal of veterinary research, 74:265–280 (2007) helminths of guineafowls in limpopo province, south africa k. junker and j. boomker* department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, o110 south africa abstract junker, k. & boomker, j. 2007. helminths of guineafowls in limpopo province, south africa. onder stepoort journal of veterinary research, 74:265–280 between july 2005 and november 2006 the gastro-intestinal helminths of 15 helmeted guineafowls and a single crested guineafowl from musina, limpopo province were examined, and in july and august 2005 helminths were collected from five helmeted guineafowls from mokopane in the same province. the acanthocephalan mediorhynchus gallinarum, the cestodes abuladzugnia gutterae, davainea nana, hymenolepis cantaniana, numidella numida, octopetalum numida, ortleppolepis multiuncinata, porogynia paronai, raillietina angusta, raillietina pintneri, raillietina steinhardti and raillieti na sp. and the nematodes ascaridia numidae, cyrnea parroti, gongylonema congolense, hadjelia truncata, sicarius caudatus, subulura dentigera, subulura suctoria, subulura sp., tetrameres numida and an unidentified subulurid were recovered. a single trematode species, dicrocoelium macrostomum, was present in the liver. mediorhynchus gallinarum, a. gutterae, o. multiuncinata, h. truncata and s. caudatus are recorded for the first time from helmeted guineafowls, as well as from south africa. south africa is a new geographic record for d. macrostomum, g. congolense and d. nana. subulura suctoria, g. congolense and h. truncata from the crested guineafowl constitute new hostparasite associations. keywords: acanthocephalans, cestodes, guineafowls, guttera edouardi, nematodes, numida meleagris, trematodes * author to whom correspondence is to be directed. e-mail: joop.boomker@up.ac.za accepted for publication 13 april 2007—editor 266 helminths of guineafowls in limpopo province, south africa available to him by various collectors. he described several new species of cestodes and nematodes and added numerous parasites to the host-parasite list of guineafowls in south africa. his reseach, however, was of a taxonomic nature and the material at his disposal represented incidental findings rather than complete collections. in this paper we present data obtained from 16 birds, including a single crested guineafowl, at musina, lim popo province, and from five helmeted guineafowls at mokopane, limpopo province, south africa. material and methods in july and august 2005 a total of five helmeted guineafowls were sampled in the vicinity of mokopane (potgietersrus), limpopo province. a complete helminth recovery was not possible, but some of the worms present in the small intestine of three of the birds, the complete caeca of one of them and part of the intestinal and caecal contents of another were collected and fixed separately in 70 % ethanol. in july 2005 and in may, july and november 2006, three, five, three and four helmeted guineafowls (eight males and seven females) were collected on a farm approximately 60 km west of musina (messina), limpopo province (22°22’ s, 29°30’ e, altitude 700–800 m). the vegetation type in the study area is classified as mopani veld (acocks 1988). the birds were aged according to the criteria established by siegfried (1966) and in total ten adults and five juveniles were collected. the juveniles were between six and ten months old (siegfried 1966). in november 2006 a single adult female crested guineafowl, found moribund in a wire snare, was made available to us for examination. the carcasses of the birds were opened according to standard techniques for necropsies of chickens, and the viscera removed. the trachea was opened and macroscopically examined for helminths. the crop, proventriculus, gizzard, small intestine and caecum/colon were separated and individually washed over a 150 μm sieve. the livers of nine helmeted guineafowls and the single crested guineafowl were sliced into 5 mm wide sections and incubated in phosphate-buffered saline at 40° c for 30 min. subsequently, the slices together with the saline were washed over a 150 μm sieve. the gastrointestinal and liver residues left on the sieves, as well as the organs themselves were fixed separately in 70 % ethanol and transported to the laboratory at onderstepoort. each sample was examined under a stereoscopic microscope and the helminths removed. cestodes were stained in haematoxylin and mounted in canada balsam or mounted and cleared in hoyer’s medium. acanthocephalans were cleared in hoyer’s medium and studied as temporary mounts in the same medium. all nematodes were cleared in lactophenol for identification. the ecological terms are used in accordance with the definitions of margolis, esch, holmes, kuris & schad (1982). results all the guineafowls were infected and all were concurrently parasitized by acanthocephalans, cestodes and nematodes. data on the prevalence, intensity and habitat preference of the parasites from the helmeted guineafowls in musina are presented in tables 1 and 2. five of the nine hosts (55.6 %), whose livers were examined, harboured dicrocoelium macrostomum, the intensity of infection ranging from 8 to 182 flukes. in addition, the livers of three of the nine birds yielded five, 11 and five young specimens of porogynia paronai. these had the typical three circles of large hammershaped rostellar hooks and small, unarmed suckers. no differential development could be seen in any of the proglottids of the short strobilae which ranged from 2.3 to 3.8 mm (n = 5) in length. the scolices were 689–746 μm wide and the rostella were 261– 329 μm wide. birds from mokopane yielded the nematodes subulura suctoria, subulura dentigera and ascaridia numidae and seven cestodes, namely ortleppolepis multiuncinata, porogynia paronai, raillietina steinhardti, raillietina pintneri, raillietina sp., numidella numida and octopetalum numida. subulura dentigera and s. suctoria were co-specific in the two hosts from mokopane. one of these harboured a total of 579 nematodes consisting of 142 male and 159 female s. suctoria, 134 male and 126 female s. dentigera and 18 immature suctoria spp. these nematodes were suspended freely in the contents of the posterior saccate part of the caeca, virtually occupying the entire lumen (fig. 2d). eight of the 15 helmeted guineafowls from musina harboured s. dentigera and s. suctoria concurrently, and in all these hosts s. suctoria by far outnumbered 267 k. junker & j. boomker t a b l e 1 t h e s ite p re fe re n ce , p re va le n ce a n d i n te n si ty o f in fe ct io n o f a ca n th o ce p h a la n s a n d c e st o d e s co lle ct e d f ro m 1 5 h e lm e te d g u in e a fo w ls i n l im p o p o p ro vi n ce , s o u th a fr ic a . a d d iti o n a l d a ta o n g u in e a fo w l h e lm in th s in s o u th e rn a fr ic a f ro m v a ri o u s a u th o rs a re in cl u d e d f o r co m p a ri so n p a ra s it e t h is p a p e r v e rs te r & p ta s in s k a -k lo ry g a (1 9 8 7 ) s a a y m a n ( 1 9 6 6 ) c ro w e (1 9 7 7 ) o rt le p p (1 9 6 3 ) s it e p re v a le n c e (% ) in te n s it y p re v a le n c e (% ) in te n s it y p re v a le n c e (% ) in te n s it y p re s e n c e p re s e n c e m e a n ( ± s d ) r a n g e m e a n r a n g e m e a n r a n g e a c a n th o c e p h a la n s m e d io rh yn ch u s g a lli n a ru m m e d io rh yn ch u s n u m id a e m e d io rh yn ch u s ta e n ia tu s s i s i s i 1 0 0 – – 5 5 .7 ( ± 7 8 .3 ) – – 2 – 2 3 1 – – – – 2 7 – – 1 .7 – – 0 – 2 2 – 3 9 – – 1 1 .5 – – ? – 2 7 – – – + – – – c e s to d e s a b u la d zu g n ia g u tt e ra e a b u la d zu g n ia t ra n sv a a le n si s d a va in e a n a n a h ym e n o le p is c a n ta n ia n a n u m id e lla n u m id a o ct o p e ta lu m n u m id a o rt le p p o le p is m u lti u n ci n a ta p a ro n ie lla s p .a p o ro g yn ia p a ro n a i r a ill ie tin a a n g u st a r a ill ie tin a p in tn e ri r a ill ie tin a s te in h a rd ti r a ill ie tin a s p . r a ill ie tin a s p .a s kr ja b in ia d e w e ti s i s i s i s i s i s i s i s i s i s i s i s i s i s i s i 8 0 – 3 3 4 0 6 7 6 7 8 7 – 4 7 5 3 8 0 5 3 7 3 – – 1 1 .7 ( ± 8 .2 ) – 5 .8 ( ± 4 .4 ) 4 2 .7 ( ± 7 0 .4 ) 5 5 .9 ( ± 7 2 .7 ) 9 1 .9 ( ± 1 1 0 .7 ) 9 .3 ( ± 5 .2 ) – 1 2 .3 ( ± 1 3 .3 ) 1 0 .3 ( ± 7 .9 ) 5 .3 ( ± 3 .9 ) 4 9 .0 ( ± 6 0 .2 ) 1 5 .8 ( ± 8 .8 ) – – 1 – 2 8 – 1 – 1 0 1 – 1 2 4 1 – 1 4 4 1 – 3 6 0 2 – 1 4 – 5 – 3 9 1 – 2 5 2 – 1 2 4 – 1 3 7 6 – 2 8 – – – – – – 2 9 4 8 – 2 5 – 8 4 4 3 1 – 3 5 – – – – – 1 .8 8 – 1 .5 – < 1 .0 3 .9 1 .9 – 2 .7 – – – – – 0 – 4 2 0 – 7 2 – 0 – 1 7 – 0 – 2 1 0 – 4 5 0 – 2 0 – 0 – 1 7 – – – – – 4 7 7 5 – – 7 5 – 3 6 – – – – – – – – 8 .7 1 6 .0 – – ? – 6 .3 – – – – – – – – ? – 1 4 ? – – ? – 5 – 3 – 2 7 – – – – – – – – – + – – + – + – – – – + + + – + + + – + + + + – – + a l is te d b y v e rs te r & p ta si n sk a -k lo ry g a (1 9 8 7 ) a s a n e w s p e ci e s, b u t w e re n o t su b se q u e n tly d e sc ri b e d s i = s m a ll in te st in e 268 helminths of guineafowls in limpopo province, south africa t a b l e 2 t h e s ite p re fe re n ce , p re va le n ce a n d in te n si ty o f in fe ct io n o f n e m a to d e s co lle ct e d f ro m 1 5 h e lm e te d g u in e a fo w ls in l im p o p o p ro vi n ce , s o u th a fr ic a . a d d iti o n a l d a ta o n g u in e a fo w l n e m a to d e s in s o u th e rn a fr ic a fr o m v a ri o u s a u th o rs a re in cl u d e d f o r co m p a ri so n n e m a to d e s t h is p a p e r v e rs te r & p ta s in s k a -k lo ry g a (1 9 8 7 ) s a a y m a n ( 1 9 6 6 ) c ro w e (1 9 7 7 ) o rt le p p (1 9 3 7 , 1 9 3 8 b , 1 9 6 4 )b s it e p re v a le n c e (% ) in te n s it y p re v a le n c e (% ) in te n s it y p re v a le n c e (% ) in te n s it y p re s e n c e p re s e n c e m e a n ( ± s d ) r a n g e m e a n r a n g e m e a n r a n g e a sc a ri d ia g a lli a sc a ri d ia n u m id a e c yr n e a p a rr o ti d is p h a ry n x n a su ta g o n g yl o n e m a c o n g o le n se g o n g yl o n e m a in g lu vi co la h a d je lia in e rm is h a d je lia t ru n ca ta h e te ra ki s g a lli n a ru m s ic a ri u s ca u d a tu s s u b u lu ra d e n tig e ra s u b u lu ra s u ct o ri a s u b u lu ra s p . u n id e n tif ie d s u b u lu ri d t e tr a m e re s n u m id a s i s i g iz p ro v c ro p c ro p g iz g iz c a e ca g iz , s i c a e ca c a e ca c a e ca s i p ro v – 6 1 0 0 – 4 0 – – 5 3 – 5 3 5 3 1 0 0 4 0 1 3 3 3 – 4 .0 a 1 3 .8 ( ± 1 8 .2 ) – 2 3 .0 ( ± 2 2 .0 ) – – 1 .6 ( ± 0 .5 ) – 2 .1 ( ± 1 .7 ) 1 5 .9 ( ± 1 3 .4 ) 5 3 6 .3 ( ± 5 8 9 .2 ) 4 4 .0 ( ± 6 5 .4 ) 2 .5 ( ± 0 .7 ) 2 .4 ( ± 1 .7 ) – 4 a 2 – 7 5 – 2 – 6 1 – – 1 – 2 – 1 – 6 1 – 3 1 9 – 2 2 1 4 1 – 1 7 0 2 – 3 1 – 5 2 1 3 1 3 1 0 – – – – 4 – 6 2 3 1 0 – – < 1 < 1 < 1 1 .8 – – – – < 1 – 1 .3 < 1 < 1 – – 0 – 2 0 – 1 9 0 – 1 6 0 – 5 9 – – – – 0 – 2 – 0 – 5 4 0 – 4 0 0 – 4 – – 6 4 – – – – – – – ? – – – – – – 5 .4 – – – – – – – 1 4 8 – – – – – – ? – 9 – – – – – – – ? – 2 5 7 – – – – – – – – – – – – – – – – + + – – – – + + – – + + – + – + + – – – a o n ly a s in g le h o st h a rb o u re d t h is p a ra si te b in cl u d in g u n p u b lis h e d r e co rd s o f o rt le p p c ite d in v e rs te r & p ta si n sk a -k lo ry g a ( 1 9 8 7 ) s i = s m a ll in te st in e g iz = g iz za rd p ro v = p ro ve n tr ic u lu s 269 k. junker & j. boomker s. dentigera, the ratio ranging from 4.5:1 to 53:1. in the remaining hosts only s. suctoria was present (fig. 2e, f). the crested guineafowl harboured a single acanthocephalan species, mediorhynchus gallinarum (n = 48), five species of cestodes, namely abuladzugnia gutterae (n = 1), n. numida (n = 114), o. numida (n = 57), o. multiuncinata (n = 1) and p. paronai (n = 52), as well as three species of nematodes, s. suctoria (n = 260), gongylonema congolense (n = 56) and hadjelia truncata (n = 2), representing a total of 591 helminths. fig. 1 a, b. cyrnea parroti male. a. anterior end. b. posterior end. c, d. gongylonema congolense. c. anterior extremity of female, ventral view. the arrow points to the excretory pore. d. posterior extremity of male. the inset illustrates the barbed tip of the long spicule. e, f. hadjelia truncata male. e. ventral view of anterior extremity. f. lateral view of anterior extremity fe dc ba 270 helminths of guineafowls in limpopo province, south africa our finding of m. gallinarum, a. gutterae, h. multiuncinata, h. truncata and sicarius caudatus in hel meted guineafowls in south africa constitutes new host associations, as well as new geographic records for these parasites. dicrocoelium macrostomum, g. congolense and davainea nana are recorded in south africa for the first time, and the crested guineafowl is a new host for the nematodes s. suctoria, g. congolense and h. truncata. despite the generally high helminth burdens, the helmeted guineafowls were in good physical condifig. 2 a, b, c. sicarius caudatus. a. anterior extremity of male. the deirids are marked by arrows. b. posterior extremity of female. note the finger-like protruberances (arrow) at the tip of the tail. c. posterior extremity of male. d. distal part of guineafowl caecum filled with subulura spp. e. subulura dentigera female, anterior part. the arrow indicates the cuticular denticles as described by ortlepp (1937); x 400. f. subulura suctoria female, anterior part; x 400 fe dc ba 271 k. junker & j. boomker tion and no obvious lesions were associated with the presence of helminths. the crop mucosa of a single bird from musina had an inflamed appearance. this, however, did not seem to be related to g. congolense living in shallow tunnels under the crop lining, but rather to the presence of numerous thorny seeds of tribulus terrestris. taxonomic remarks cyrnea parroti seurat, 1917 (table 3; fig. 1a, b) ortlepp (1938b) described habronema numidae from helmeted guineafowls in malawi, south africa and swaziland. this nematode has subsequently been included in the genus cyrnea seurat, 1914, but it is still listed under its original name in yamaguti (1961) as well as in verster & ptasinska-kloryga (1987). in his work on the habronematinae, chabaud (1958) divided the genus cyrnea into two subgenera, procyrnea chabaud, 1958 and cyrnea chabaud, 1958, which he later raised to genus level (chabaud 1975). following an in-depth study of the cephalic structures, he synonymized cyrnea (cyrnea) numidae (ortlepp, 1938) with cyrnea (cyrnea) parroti seurat, 1917 (chabaud 1958). specimens from our hosts mounted en face show the same arrangement of submedian lobes and simple lips as illustrated for c. parroti by chabaud (1958) and otherwise conform well with the description and measurements supplied by ortlepp (1938b) for c. numidae. the range of measurements in our specimens was, however, generally wider than that provided by the latter author (table 3). ortlepp (1938b) himself stated that his new species most closely resembled cyrnea eurycerca and c. parroti and that the arrangement of the caudal papillae in the males as well as the spicules were very similar. gongylonema congolense fain, 1955 (table 4; fig. 1c, d) this parasite was first described by fain (1955a) from domestic chickens, a single duck, cairina moschata domestica and from n. meleagris from the democratic republic of the congo and rwanda. sub sequently it has been recorded from n. meleagris in burundi, nigeria, ethiopia, ghana and burkina faso (fain & thienpont 1958; fabiyi 1972; graber 1976; hodasi 1976; vercruysse, harris, bray, nagalo, pangui & gibson 1985). one of the main morphological characteristics of this species is the hook situated at a distance of about 50 μm from the distal tip of the left spicule (fain 1955a) (fig. 1d). the hook itself carries three fine barbs. in our specimens the barbed hook of the tip of the left spicule was often difficult to see, but in specimens where the distance could be determined it varied from 31 to 46 μm. it is not always easy to judge whether the left spicule is intact or damaged, which could lead to measuring errors. there are, however, sufficient other characteristics, such as the gubernaculum, the extent and arrangement of the cuticular plaques (fig. 1c), as well as the length of the right spicule to differentiate g. congolense from other species utilizing avian hosts (fain 1955a). while our specimens fit in well with fain’s (1955a, b) description of g. congolense, we have not been able to confirm that the excretory pore opens on a transversally elongated plaque as was described by him. in our specimens it would seem that the two median ventral longitudinal rows of plaques are interrupted, leaving a plaque-free zone immediately anterior and posterior to the excretory pore (fig. 1c). measurements of our specimens and those of fain (1955a) taken from guineafowl hosts are presented in table 4. these indicate that there is little geographic variation in the morphology of g. congolense from the same host species. hadjelia truncata (creplin, 1825) (table 5; fig. 1e, f) the most obvious differences between h. truncata and sympatric specimens of c. parroti are the position of the vulva and the winged appearance of the lips of h. truncata in ventral view (fig. 1e, f). in h. truncata the vulva is distinctly anterior and positioned in front of the posterior end of the oesophagus. these characteristics are in accordance with the generic diagnosis of hadjelia provided by yamaguti (1961). measurements of the specimens from the guineafowls fall well within the range of measurements provided by ortlepp (1964) for hadjelia inermis (gedoelst, 1919) (table 5). hadjelia inermis had been synonymized with h. truncata by chabaud & campana (1950), and ortlepp (1964) commented on this, but chose to retain the former species. he lists his own measurements for h. inermis collected from redand yellow-billed hornbills from south africa, together with measurements for h. inermis taken from gedoelst (1919) and for h. inermis and h. truncata as provided by cram (1927, cited by ortlepp 272 helminths of guineafowls in limpopo province, south africa t a b l e 3 t h e m a in m o rp h o lo g ic a l c ri te ri a o f c yr n e a p a rr o ti s e u ra t, 1 9 1 7 f ro m h e lm e te d g u in e a fo w ls . t h e r a n g e o f m e a su re m e n ts is p ro vi d e d . a ll m e a su re m e n ts in m ic ro m e tr e s u n le ss o th e rw is e s ta te d s o u rc e p re s e n t s tu d y o rt le p p ( 1 9 3 8 b ) m o rp h o lo g ic a l c ri te ri a m a le s ( n = 6 ) f e m a le s ( n = 4 ) m a le s f e m a le s b o d y le n g th ( m m ) m a xi m u m w id th d is ta n ce a p e x to n e rv e r in g d is ta n ce a p e x to d e ir id s d is ta n ce a p e x to e xc re to ry p o re d e p th o f b u cc a l c a p su le w id th o f b u cc a l c a p su le ( in n e r) m u sc u la r o e so p h a g u s g la n d u la r o e so p h a g u s o e so p h a g u s to ta l l e n g th l e n g th o f ta il d is ta n ce v u lv a t o p o st e ri o r e n d e g g le n g th x e g g w id th l e n g th o f ri g h t sp ic u le l e n g th o f le ft s p ic u le l e n g th o f g u b e rn a cu lu m l e n g th o f ca u d a l a la e 9 – 1 1 2 2 9 – 2 7 4 1 8 7 – 2 6 2 2 2 0 – 3 7 0 2 2 0 – 3 6 2 2 9 – 3 6 1 0 – 1 5 3 0 4 – 3 9 3 2 2 3 4 – 2 5 2 6 2 2 8 4 – 2 8 3 0 1 2 0 – 1 9 3 – – 4 1 0 – 5 1 0 a 8 3 4 – 1 3 5 4 a 6 3 – 8 4 a 4 3 7 – 6 1 8 1 1 – 1 6 2 3 2 – 3 8 0 2 3 7 – 2 6 3 3 0 9 – 3 6 4 3 1 1 – 3 5 7 3 4 – 4 5 1 2 – 1 6 – – 2 0 3 9 – 3 0 5 6 1 2 8 – 1 5 0 6 6 1 – 8 9 7 4 5 – 4 6 x 2 5 – 2 7 – – – – 1 1 – 1 3 1 8 0 – 2 1 0 2 1 0 – 2 4 0 – 2 5 0 – 2 9 0 3 0 1 0 2 7 0 – 3 0 0 1 7 0 0 – 2 0 0 0 – – – – 4 2 0 – 4 3 8 1 0 8 0 – 1 1 1 0 7 0 4 2 0 – 5 2 0 1 8 – 1 9 3 0 0 – 3 6 0 2 1 0 – 2 4 0 – 2 5 0 – 2 9 0 3 6 1 2 3 3 0 2 4 0 0 – 2 6 0 0 – 1 2 6 – 1 3 0 ~ 7 5 0 4 2 – 4 5 x 2 4 – – – – a m e a su re m e n ts o f th e s p ic u le s a n d t h e g u b e rn a cu lu m a re d e ri ve d f ro m t e n m a le s 273 k. junker & j. boomker t a b l e 4 t h e m a in m o rp h o lo g ic a l c ri te ri a o f g o n g yl o n e m a c o n g o le n se f a in , 1 9 5 5 m a le s fr o m h e lm e te d g u in e a fo w ls fr o m s o u th a fr ic a ( p re se n t st u d y, g f m /n r e p re se n ts o u r sp e ci m e n n u m b e r) , a n d f ro m t h e d e m o cr a tic r e p u b lic o f th e c o n g o a n d r w a n d a ( f a in 1 9 5 5 a ). a ll m e a su re m e n ts in m ic ro m e tr e s u n le ss o th e rw is e s ta te d s o u rc e p re s e n t s tu d y f a in ( 1 9 5 5 b ) m o rp h o lo g ic a l c ri te ri a g f m 3 /n 1 g f m 1 /n 1 6 g f m 1 /n 1 7 g f m 1 /n 2 1 g f m 1 /n 2 2 g f m 1 /n 2 3 g f m 1 /n 2 4 g f m 1 /n 2 5 (n = 5 ) b o d y le n g th ( m m ) m a xi m u m w id th d is ta n ce a p e x to d e ir id s d is ta n ce a p e x to n e rv e r in g d is ta n ce a p e x to e xc re to ry p o re d is ta n ce a p e x to e n d o f p la q u e s d is ta n ce a p e x to c e rv ic a l a ile s d e p th o f b u cc a l c a p su le m u sc u la r o e so p h a g u s g la n d u la r o e so p h a g u s o e so p h a g u s to ta l l e n g th l e n g th o f ta il c a u d a l a la e ( le ft ; ri g h t) l e n g th o f g u b e rn a cu lu m l e n g th o f ri g h t sp ic u le l e n g th o f le ft s p ic u le ( m m ) 1 7 2 6 6 8 4 2 1 0 3 0 5 4 4 0 1 0 2 ; 1 1 0 3 1 – – 4 1 2 5 2 0 7 6 0 0 ( le ft ) 8 7 9 8 8 .7 1 5 2 2 8 7 0 ; 8 6 1 9 0 3 0 3 3 8 5 1 1 8 – 3 6 2 2 9 6 7 3 4 0 7 1 7 3 – 7 3 1 0 1 – 1 4 2 4 4 1 0 7 2 3 2 3 5 5 4 8 4 1 5 3 3 0 2 1 6 3 9 3 4 4 1 5 0 1 8 3 – 8 5 7 9 4 .8 1 3 2 1 5 8 8 1 8 8 3 2 2 4 7 0 1 0 7 ; 1 2 3 3 0 3 0 4 3 0 7 5 3 3 7 9 1 7 0 – 8 7 8 6 5 .5 – – 9 8 2 2 4 3 4 0 – – – 4 3 1 3 8 7 9 4 3 1 0 – – 8 2 9 9 7 .4 1 4 2 3 0 1 0 9 ; 1 0 4 2 2 3 3 6 2 4 8 6 1 5 0 ; 1 4 1 3 4 3 8 7 3 4 1 7 3 8 0 4 1 9 7 – – 9 8 5 .5 1 7 2 4 7 9 6 ; 8 3 1 9 1 3 3 7 5 0 0 1 2 5 ; 1 2 3 3 1 3 3 2 3 5 2 3 3 8 5 3 2 0 2 6 3 0 ; 5 8 3 8 0 1 0 0 8 .1 – 2 3 0 – – – – – – – – – 1 6 5 – 8 0 9 4 8 .2 1 2 – 2 4 1 7 0 – 2 0 0 8 5 – 1 2 5 1 9 6 – 2 3 5 3 1 0 – 3 5 0 4 5 0 – 4 7 5 1 2 5 – 1 7 5 3 0 – 4 5 2 9 0 – 4 0 0 2 5 2 0 – 3 9 2 0 – 1 8 5 – 2 0 0 5 7 5 – 7 0 0 ; 4 5 0 – 5 0 0 6 8 – 8 5 1 0 4 – 1 4 0 7 – 1 1 274 helminths of guineafowls in limpopo province, south africa t a b l e 5 t h e m a in m o rp h o lo g ic a l c ri te ri a o f h a d je lia t ru n ca ta ( c re p lin , 1 8 2 5 ) fr o m h e lm e te d g u in e a fo w ls . t h e r a n g e o f m e a su re m e n ts is p ro vi d e d . a ll m e a su re m e n ts in m ic ro m e tr e s u n le ss o th e rw is e s ta te d s o u rc e p re s e n t s tu d y g e d o e ls t (1 9 1 9 ) o rt le p p ( 1 9 6 4 ) m o rp h o lo g ic a l c ri te ri a m a le s f e m a le s m a le s f e m a le s m a le s f e m a le s g f m 9 /1 g f m /1 1 g f m 1 /1 0 g f m 1 /1 4 g f m 6 /1 b o d y le n g th ( m m ) m a xi m u m w id th d is ta n ce a p e x to n e rv e r in g d is ta n ce a p e x to d e ir id s d is ta n ce a p e x to e xc re t. p o re d e p th o f b u cc a l c a p su le w id th o f b u cc a l c a p su le ( in n e r) m u sc u la r o e so p h a g u s g la n d u la r o e so p h a g u s o e so p h a g u s to ta l l e n g th d is ta n ce a p e x to v u lv a l e n g th o f ta il e g g le n g th x e g g w id th l e n g th o f le ft s p ic u le l e n g th o f ri g h t sp ic u le 7 1 6 0 2 0 8 2 3 7 ; 2 3 9 2 7 5 4 4 5 3 6 9 1 7 5 0 2 1 1 9 – – – 1 3 4 6 2 7 1 8 1 4 5 2 1 2 2 3 8 ; 2 3 1 2 5 9 4 2 7 3 9 7 1 9 2 7 2 3 2 4 – – – 1 4 3 4 2 5 4 1 0 2 0 9 1 8 5 2 0 6 ; 2 0 4 2 3 4 3 9 5 3 5 8 1 9 4 8 2 3 0 6 1 6 9 8 1 3 8 5 0 x 3 2 – – – 2 1 7 1 5 9 1 6 0 ; 1 6 1 1 7 9 4 0 7 3 4 6 2 0 7 6 2 4 2 2 1 6 9 1 – 5 3 x 3 5 – – 1 1 1 4 0 – 2 5 7 ; 2 5 9 2 9 0 4 1 6 4 9 5 1 9 8 8 2 4 8 3 2 2 3 8 1 2 1 – – – 6 .1 – 6 .4 5 1 4 0 – 1 4 4 1 8 0 – 2 1 5 2 1 0 – 2 6 0 2 2 0 – 2 7 5 – – – – 2 0 0 0 – 1 2 0 – 1 6 0 0 – 1 9 0 0 2 0 0 1 8 – 2 1 .8 2 4 0 – 2 6 0 2 6 0 – 2 7 5 3 3 0 3 6 0 – – – – 2 4 0 0 – 3 6 0 0 1 8 6 0 – 2 9 7 0 9 0 – 1 2 0 5 4 – 5 7 x 3 0 – 3 2 – – 6 – 7 – – – – 4 0 – 5 0 1 0 2 3 0 – 2 8 0 1 9 0 0 – 2 2 0 0 2 1 3 0 – 2 4 8 0 – 1 2 0 – 1 4 0 – 1 2 0 0 – 1 5 0 0 2 1 5 – 2 8 0 1 7 – 1 9 – – – – 4 7 – 5 2 1 2 4 0 0 – 4 5 0 2 0 0 0 – 2 3 0 0 2 4 0 0 – 2 7 5 0 2 2 0 0 – 2 5 0 0 1 1 0 – 1 2 0 3 2 – 3 7 x 2 5 – 2 7 – – 275 k. junker & j. boomker t a b l e 6 m o rp h o lo g ic a l c ri te ri a o f s ic a ri u s d ip te ru m ( p o p o va , 1 9 2 7 ), s ic a ri u s h o o p o e s h a rm a , 1 9 7 1 , s ic a ri u s ca u d a tu s q u e n tin & w e rt h e im , 1 9 7 5 a n d s ic a ri u s re n a ta e c a n cr in i, b a lb o & i o ri , 1 9 9 1 d e sc ri b e d f ro m a vi a n h o st s. a ll m e a su re m e n ts in m ic ro m e tr e s u n le ss o th e rw is e s ta te d s o u rc e a li ( 1 9 6 1 ) s h a rm a ( 1 9 7 1 ) q u e n ti n & w e rt h e im (1 9 7 5 ) p re s e n t s tu d y c a n c ri n i, b a lb o & i o ri (1 9 9 1 ) m o rp h o lo g ic a l c ri te ri a s ic a ri u s d ip te ru m s ic a ri u s h o o p o e s ic a ri u s c a u d a tu s s ic a ri u s c a u d a tu s s ic a ri u s r e n a ta e m a le f e m a le m a le f e m a le m a le f e m a le m a le f e m a le m a le f e m a le b o d y le n g th ( m m ) 1 0 .2 – 1 1 .9 1 2 .9 – 1 6 .1 6 .7 – 9 .4 1 1 .2 – 1 8 .2 7 .3 1 3 .3 5 .9 8 .3 4 .9 7 .3 d is ta n ce a p e x to d e ir id s 7 0 – 7 4 a 8 0 – 9 0 a 6 0 – 7 0 5 0 – 6 0 8 5 8 5 6 3 ; 6 8 7 8 ; 8 7 6 8 ; 7 0 6 5 ; 7 5 d is ta n ce a p e x to n e rv e ri n g 1 8 0 – 2 0 0 2 4 0 – 2 6 0 1 1 0 – 1 4 0 1 6 5 – 2 1 0 2 1 5 2 5 0 2 0 8 2 2 6 – 2 2 7 1 5 0 1 6 5 d is ta n ce a p e x to e xc re t. p o re 2 4 0 – 2 6 0 2 8 0 – 3 2 0 1 3 0 – 1 8 0 1 8 0 – 2 2 0 2 7 0 3 1 0 – 2 7 8 – 2 9 8 – – d e p th o f b u cc a l c a p su le 4 3 – 4 5 5 2 – 5 8 1 4 – 1 7 2 5 2 8 3 8 2 8 3 1 – 3 3 2 5 2 5 m u sc u la r o e so p h a g u s 5 1 0 – 5 3 0 5 6 0 – 6 1 0 2 4 0 – 3 2 0 3 2 0 – 3 8 0 3 8 0 4 1 0 – – 2 5 0 2 3 6 g la n d u la r o e so p h a g u s 2 8 8 0 – 2 9 2 0 3 1 6 0 – 3 9 1 0 2 8 0 0 – 3 0 4 0 3 2 0 0 – 3 6 0 0 2 1 7 0 2 9 0 0 – – 1 9 5 0 2 3 6 5 o e so p h a g u s to ta l le n g th 3 4 0 0 – 3 5 0 0 3 7 0 0 – 4 5 0 0 – – 2 5 5 0 3 3 1 0 2 5 3 5 2 7 2 2 2 2 0 0 2 6 0 1 l e n g th o f ta il 2 1 0 1 8 5 – 2 1 0 1 7 6 – 2 0 8 1 6 7 – 2 5 6 1 9 0 2 5 0 1 6 1 1 6 8 – 1 1 0 l e n g th o f ri g h t sp ic u le 9 3 – 1 6 0 – 4 4 0 – 5 6 0 – 1 7 0 – 1 9 0 – 1 7 1 – 1 7 5 – l e n g th o f le ft s p ic u le 6 2 0 – 6 9 0 – 4 7 0 – 6 0 0 – 4 0 0 – 4 5 0 – 4 1 3 – 3 6 0 – d is ta n ce v u lv a t o t ip o f ta il – 5 0 0 0 – – – 4 9 5 0 – 2 9 6 0 – 2 8 0 0 e g g le n g th – 3 8 – 4 0 – 3 3 – 4 6 – 4 3 – 3 8 – 3 7 – 4 0 e g g w id th – 3 0 – 3 7 – 2 9 – 3 9 – 3 0 – 2 9 – 2 5 – 2 7 m a x. w id th o f a la e – – – – 4 5 4 5 3 6 4 0 – 5 0 – – e xt e n si o n a la e w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y w h o le b o d y a c e rv ic a l p a p ill a e in a li (1 9 6 1 ) in te rp re te d h e re a s d e ir id s 276 helminths of guineafowls in limpopo province, south africa 1964) (table 5). as we have not been able to examine the type specimens of either species, we have chosen to adopt the conclusion of the in-depth morphological study of chabaud & campana (1950). the single complete specimen in our collection appears slightly shorter than previously described ones. the depth of the buccal cavity of our specimens corresponds with the lower range of the pharynx sizes provided by ortlepp (1964) and all three authors he quoted. the oesophagus length is relatively uniform in all the sources quoted by ortlepp (1964). in our specimens the total length of the oesophagus only reached 2.1 and 1.9 mm which, considering that these specimens are short, does not seem extraordinary. the egg size is very similar to that of h. inermis as recorded by gedoelst (1919) and cram (1927, cited by ortlepp 1964), but larger than given by ortlepp (1964). ortlepp (1964) pointed out that this was the only noteworthy difference between his specimens and those described by gedoelst (1919). according to chabaud (1958) the genus hadjelia has been described from numerous birds, especially coraciiformes, from europe, asia and africa. data pertaining to african hosts mainly list bucerotiformes (ortlepp 1964) and we are aware of only one reference to hadjelia from galliform birds, namely hadjelia lhuillieri seurat, 1916 from alectoris barbara (= caccabis petrosa from algeria in seurat 1916). incidentally, chabaud (1958) speculated that the latter species might be identical with h. truncata, but thought that the characteristics of the single known female specimen of h. lhuillieri were not sufficient to draw a final conclusion. sicarius caudatus quentin & wertheim, 1975 (table 6; fig. 2a, b, c) four species of the genus sicarius are known from avian hosts, namely sicarius dipterum (popova, 1927), sicarius hoopoe sharma, 1971, sicarius caudatus quentin & wertheim, 1975 and sicarius renatae cancrini, balbo & iori, 1991. the left spicule of s. dipterum is distinctly longer (660–670 μm) than that of our specimens, whereas the subequal spicules of s. hoopoe are 440–560 and 470–600 μm in length (cancrini, balbo & iori 1991). our specimens best fit the description of s. caudatus, as they have six pairs of caudal papillae as opposed to the eight pairs of s. renatae (cancrini et al. 1991). according to quentin & wertheim (1975) the deirids in s. caudatus are situated at the origin of the lateral alae. in some of our specimens, we have observed the same arrangement, but in one male and one female the right and left deirids emerge 11 and 27 μm, and 17 and 37 μm anterior to the origin of the alae (fig. 2a). we have too little material to comment on the significance of this observation. quentin & wertheim (1975) describe the cuticular processes in the tail of s. caudatus as atrophied, the tail consisting merely of a smooth stump, which at best has rugged edges. our specimens possess about seven distinct, albeit short, cuticular extensions similar to those illustrated by cancrini et al. (1991) for s. renatae (fig. 2b). despite these differences we have allocated our specimens to s. caudatus. apart from the original description and their inclusion in some taxonomic reviews (chabaud 1958; ali 1961), we have not found any other references to s. caudatus in the literature. the measurements of the specimens collected during this study are included in table 6. discussion despite the fact that various studies on the helminths of guineafowls in south africa have been conducted, direct comparisons between the results of these studies are not always possible, as they had different objectives. ortlepp (1937, 1938a, b, 1963) studied the helminths of all the organs and the entire alimentary canal, but his work was of a taxonomic nature, based on incidental findings, and presented no epidemiological data. crowe (1977) listed the helminth species recovered from the small intestine, caeca and rectum of guineafowls, but in his subsequent analysis grouped them as acanthocephalans, cestodes and nematodes respectively. the two studies providing data on the prevalence and intensity of the helminths are those of saayman (1966) and verster & ptasinska-kloryga (1987). however, saayman (1966) only examined the intestinal tract and verster & ptasinska-kloryga (1987) collected helminths from the gizzard, intestine and caeca. thus, their data on species richness would not reflect worms located in e.g. the crop or proventriculus. the study conducted on guineafowls in burkina faso by vercruysse et al. (1985) lends itself best to comparison with ours, as they examined the complete alimentary tract, including the crop and proventriculus. of the total of 13 helminth species collected by these authors, eight species coincide with species recovered from our hosts. if the single acanthocephalan present in the birds from burkina faso 277 k. junker & j. boomker is taken into account, this number will increase by one. vercruysse et al. (1985) record the acanthocephalan mediorhynchus selengensis, which has been synonymized with m. gallinarum by schmidt & kuntz (1977), and the nematodes cyrnea parroti, s. suctoria, g. congolense and a. numidae, which are also recorded in this study. in addition to these species, vercruysse et al. (1985) recorded the cestode cotugnia digonopora and the nematodes eucoleus annulatus, t. fissispina and dispharynx spiralis. nematodes cyrnea with the exception of c. parroti, helminths were recovered from their usual predilection sites. according to anderson (1992) members of the genus cyrnea occur in the proventriculus of birds and he records cyrnea colini in the wall of the proventriculus near the gizzard of bobwhite quails. we did not recover c. parroti from the proventriculus, but in all infected guineafowls the parasites were situated under the gizzard lining and could only be seen after the horny layer had been removed. there seemed, however, to be a preference for the proventricular-gizzard isthmus as described for cyrnea neeli from wild turkeys in the south-eastern united states (davidson, hon & forrester 1977). similarly, c. parroti recovered from helmeted guineafowls in burkina fasso were also present in the gizzard (vercuysse et al. 1985). subulura the genus subulura has a wide distribution in gallinaceaous birds on the african continent and records exist from zimbabwe, tanzania, ghana, nigeria and somalia (nicholls, bailey, gibbons, jones & samour 1995; nfor, ajanusi, agbede & esievo 1999; poulsen, permin, hindsbo, yelifari, nansen & bloch 2000; permin, esmann, hoj, hove & mukaratirwa 2002; magwisha, kassuku, kyvsgaard & permin 2002). however, the genus is not restricted to the african continent and, according to yamaguti (1961) is a cosmopolitan species. ortlepp (1937) recovered s. suctoria in association with s. dentigera from guineafowls from various regions in south africa and swaziland and concluded that the two species had a wide distribution. contrary to our findings, he found s. dentigera to be far more abundant than s. suctoria. verster & ptasinska-kloryga (1987) collected helminths from 48 guineafowls in the vicinity of pretoria. subulura suctoria was present in 11 and s. denti gera in three of the hosts examined. from these and our own results it is apparent that the two species, s. suctoria and s. dentigera often share the same habitat. it is difficult to judge from our data whether these two species are interactive and compete for the same resources. if so, s. suctoria would seem the stronger competitor as it consistently occurred in higher numbers than s. dentigera. however, the numbers of s. dentigera were not greater in hosts with relatively low burdens of s. suctoria, but rather the numbers of s. dentigera were low in these hosts as well. it is possible, that this association is similar to the major-minor species concept, as seen with theladorsagia circumcincta and theladorsagia davtiani in sheep and goats. a literature study confirms the dominance of s. suctoria in guineafowls and vercruysse et al. (1985) recorded a 100 % prevalence of s. suctoria from 103 helmeted guineafowls in burkina faso. in addition to being the most prevalent nematode, these authors also found s. suctoria to be one of the most numerous parasites (26–1 071 worms per host). subulura dentigera was not reported from these hosts. ascaridia numidae ascaridia numidae is another nematode commonly encountered in helmeted guineafowls and has been recorded from various geographic localities in africa. the prevalence and intensity of this parasite varies greatly from 98.1 % with a range of intensity from 1 to 1 452 in hosts in burkina faso (vercruysse et al. 1985) and 86.7 % with intensities ranging from 1 to 504 in birds in ghana (hodasi 1976) to a low prevalence of 13 % with a maximum of 19 worms per host in south africa (verster & ptasinska-kloryga 1987). in the present study a. numidae was present in a single host only. gongylonema both hodasi (1976) and vercruysse et al. (1985), record g. congolense from hosts they examined, with a prevalence of 48.9 and 73.8 %, respectively. this indicates that g. congolense not only forms a regular part of the helminth community of guineafowls in south africa, but throughout the african continent. with the exception of gongylonema ingluvicola allegedly recorded by ortlepp (“1937, 1938, unpublished records” cited by verster & ptasinskakloryga 1987), the absence of this genus in previous reports on helminths of guineafowls in south 278 helminths of guineafowls in limpopo province, south africa africa, is most likely due to the fact that earlier authors did not examine the crop of the hosts in their studies. tetrameres while tetrameres numida was recovered in low numbers from the musina guineafowls, none of the more com monly reported species of this genus was present in our material. a second species, which has been recorded from guineafowls and is also a common para site of domestic chickens, is tetrameres fissispina diesing, 1861. vercruysse et al. (1985) re port a 48.5 % prevalence and an intensity of infection ranging from 1 to 146 worms per host from helmeted guineafowls in burkina faso, and 23.3 % of 126 helmeted guineafowls in nigeria were infected with t. fissispina (fabiyi 1972). in ghana the prevalence of infection in the same host was 8.9 % with a mean worm burden of 2.8, ranging from one to eight. young scavenging chickens in ghana had a prevalence of t. fissispina of 58 % (poulsen et al. 2000). we are aware of a single record of three females of t. fissispina from a single helmeted guineafowl in south africa (le roux 1926), and the same author reports a high percentage of infection (78 %) in 60 domestic chickens in the same country. the proventriculus of a single, heavily infected host contained a minimum of 150 females (le roux 1926). a third species commonly infecting domestic chickens, namely tetrameres americana, which had a 60 and 62 % prevalence in adult chickens in tanzania and zimbabwe, respectively (per min, magwisha, kassuku, nansen, bisgaard, frand sen & gibbons 1997; permin et al. 2002), has not yet been recorded from guineafowls. from the literature cited above it would appear that the prevalence of the genus tetrameres is slightly higher in domestic chickens than in helmeted guineafowls. since the data above concerning the domestic chickens above pertain to free-ranging or scavenging chickens, guineafowls and domestic fowls probably had an equal chance of exposure to the parasite. whether the higher infection rates in chickens are a result of higher host densities or whether guineafowls are generally more resistant towards helminth infections remains speculation. trematodes the literature contains few reports of trematodes from guineafowl hosts, but a number of trematodes have been listed as parasitizing not only the digestive tract and urogenital system, but also the respiratory system of domestic fowls (soulsby 1968). to our knowledge the only published records of trematodes from the liver of guineafowls are those of d. macrostomum, that occurs in the gall bladder and bile ducts of n. meleagris (= n. ptilorhyncha) in egypt (lesbouyries 1941) and lutztrema sp. from the gallbladder of helmeted guineafowls in ghana (hodasi 1976). the former parasite has also been found in the liver of helmeted guineafowls in the kruger national park (horak 2007, personal communication) and was present in the musina hosts. the results of this study and unpublished data of horak (2007) suggest that d. macrostomum is not uncommon in south african guineafowls and can reach high intensities in individual hosts. postharmostomum gallinum has been reported from the gastro-intestinal tract from crested guineafowls in pakistan (khan, khan & rayaz 1984). hodasi (1969, 1976) collected postharmostomum ntowi and episthmium ghanense and episthmium africanus from the gastro-intestinal tracts of domestic chickens. intensities and prevalences were low and the latter author concluded that trematodes were rare parasites in gallinaceous birds. the fact that the intermediate hosts of trematodes are mainly molluscs or rarely annelids (gibbons, jones & khalil 1996), both of which are typically associated with moist environmental conditions, might well explain why trematodes played a minor role as parasites of the guineafowls in our dry study area. cestodes porogynia the presence of young stages of porogynia paronai in the liver of infected hosts is unusual. hodasi (1976), however, recovered adult cotugnia meleagridis from the small intestine of helmeted guineafowls in ghana, and recorded numerous young forms of this parasite from the host’s gallbladder. since the life cycle of porogynia is not known, one can only speculate on the presence of immatures in the liver. during the normal course of cestode development in avian hosts, the cysticercoid is freed from the arthropod intermediate host in the intestine as a result of mechanical and chemical actions. subsequently, the scolex evaginates and the cysticercoid attaches itself to the gut wall (reid 1962). the fact that young p. paronai were recovered from the liver of three birds and in relatively high numbers, in addition to 279 k. junker & j. boomker their uniform stage of development, suggests that their presence is not a result of post-mortem migration. whether the newly freed cysticercoid, assuming that an arthropod is the intermediate host, migrates up the common bile duct to mature to a certain stage, before leaving the liver to resume its final maturation in the small intestine, or whether we have observed aberrant migration of juvenile stages will remain speculation until the development of p. paronai can be studied in more detail. abuladzugnia interestingly, the cestode a. gutterae, which was com mon in the guineafowls examined by us was not found in any of the previous surveys. ortlepp (1963) originally described this species as cotugnia gutterae from three specimens that had been collected from crested guineafowls in mozambique. since then there seem to have been no further records of this parasite. spasskii (1973) created the genus abuladzugnia to accommodate a. gutterae and another of ortlepp’s (1963) species formerly described as cotugnia transvaalensis. conclusion the above findings suggest, that despite geographical variation in the prevalence and intensity of individual helminth species, probably caused by environmental conditions, such as temperature, rainfall and soil conditions, the helminth community of guineafowls in africa is composed of a relatively stable body of core and secondary species enriched by satellite species. the latter probably depend on local conditions and can be influenced by abiotic conditions, but also the presence or absence of certain intermediate hosts and other terrestrial birds which may serve as reservoir hosts for certain parasites. we interpret the relative uniformity in the helminth community of helmeted guineafowls in africa as flowing 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[new genera of cyclophyllidean cestodes.] parazity zhivotnykh i rastenii, 9:38–48 [in russian]. vercruysse, j., harris, e.a., bray, r.a., nagalo, m., pangui, m. & gibson, d.i. 1985. a survey of gastrointestinal helminths of the common helmet guinea fowl (numida meleagris galeata) in burkina faso. avian diseases, 29:742– 745. verster, a. & ptasinska-kloryga, y. 1987. helminths of helmeted guineafowl in southern africa. south african journal of wildlife research, supplement i: 36–38. yamaguti, s. 1961. the nematodes of vertebrates, vol. iii, part i & ii, in systema helminthum. new york: interscience publishers. swai_211-217.qxd introduction cattle ticks of the genus boophilus are the only vectors of babesia bovis infection (mahoney 1979), and of these, boophilus microplus larvae have been singled out as the most efficient transmitting agents of babesia bovis in areas were this infection exists (riek 1966). the classic epidemiological model of babesiosis in cattle is based on the long lasting immunity induced by primary infection in calves up to 7–9 months old (riek 1968) when they are naturally resistant to the clinical effects of primary infection. in those herds subjected to babesia bovis infections are from 0.0005 to 0.005 infective bites/ head/day, are in endemic instability, while the risk is low, below or above those limit (mahoney & ross 1972). in this article we present the results of a cross-sectional study estimation of the prevalence of babesia bovis antibodies in dairy cattle on the two contrasting and diverse smallholder dairying regions in tanzania where boophilus microplus is often thought not to exist (lynen, bakuname & sanka 1999). the objective were to quantify the occurrence of babesia bovis through a serological survey, estimate the rate of infection of it and explore the possible relationship between the it and some animal/ farm level variables. 211 onderstepoort journal of veterinary research, 71:211–217 (2004) cross-sectional estimation of babesia bovis antibody prevalence in cattle in two contrasting dairying areas in tanzania e.s. swai1, e.d. karimuribo2, n.p. french3, n.h. ogden4, j. fitzpatrick5, d. kambarage2 and m.j. bryant6 abstract swai, e.s., karimuribo, e.d., french, n.p., ogden, n.h., fitzpatrick, j., kambarage, d. & bryant, m.j. 2004. cross sectional estimation of babesia bovis antibody prevalence in cattle in two contrasting dairy areas in tanzania. onderstepoort journal of veterinary research, 71: 211–217 the crude prevalence of antibodies to babesia bovis infection in cattle was estimated by serology using indirect elisa during the period january to april, 1999. sera were obtained from 1 395 dairy cattle (of all ages, sexes and breeds) on smallholder farms, the majority being kept under a zero grazing regime. the crude prevalence of antibodies to babesia bovis was 6 % for tanga and 12 % for iringa. the forces of infection based on the age sero-prevalence profile, were estimated at six for iringa and four for tanga per 100 cattle years-risk, respectively. using random effect logistic regression as the analytical method, the factors (variables) of age, source of animals and geographic location were hypothesised to be associated with sero-positivity of babesia bovis in the two regions. keywords: babesia bovis, dairy cattle, epidemiology, force of infection, sero-prevalence, smallholder, tanzania 1 veterinary investigation centre, box 1068 arusha, tanzania. e-mail: emasw@yahoo.co.uk 2 faculty of veterinary medicine, sokoine university of agriculture, morogoro, tanzania 3 department of veterinary clinical science and animal husbandry, university of liverpool, uk 4 department of veterinary pre-clinical science, university of liverpool, uk 5 department of veterinary clinical studies, university of glasgow, glasgow, uk 6 department of agriculture, university of reading, uk accepted for publication 23 february 2004—editor materials and methods area and study population the study was carried out in five administrative districts of tanga region and two in the iringa region of tanzania. tanga region is situated on the north eastern corner of tanzania lying between longitude 36 and 38e and latitude 4 and 6s. the region has heterogeneous physical and climatic features varying from hot, humid coastal lowlands in the east to the cool usambara mountains in the north and semiarid plain in the southwest. there are two rainy seasons, the long rains occurring between march and may and the short rains occurring between september and november. rainfall varies widely from 500 mm in semi-arid areas to 1 400 mm in coastal areas and up to 2 000 mm in some inland mountain areas. daytime temperatures vary from 23–28 °c during the cool season (may to september) to 30–33 °c during the hot season (december to march). iringa region is one of three in the southern highland zone of tanzania and lies between latitude 7 and 8s and longitude 35 and 36e. the region lies between 1 340 and 2 090 m above sea level. rainfall is annually bimodal and ranges between 600 and 1 600 mm per annum, with most rain falling between march and june and occasional light rain between august and september. in both regions, type of cattle kept includes bos taurus breed (friesian, ayrshire, jersey, simmental) and crosses of these breeds with bos indicus breeds (tanzania shorthorn zebu, boran and sahiwal). study design a sample size of farms and animals was estimated using epi-info version 6.04b (cdc, atlanta, usa) in order to provide 80 % power, with a confidence of α = 0.05, to estimate disease prevalence and detect associations between dependent and independent variables (gitau, mcdermott, walter-toews, lissemore, osumo & muruki 1994; french & tyrer 1997). the farms in each study region were randomly (epiinfo version 6.04b) selected in october 1998, from a sampling frame of 3 001 and 500 (in tanga and in iringa, respectively) using the databases of the tanga and iringa dairy development projects. the dutch and swiss governments have been supporting dairy schemes in tanga and iringa regions, respectively. over the last 15 years (1983–1998) of support, huge data bases have been generated. as the antibody prevalence was not known a priori, a 50 % prevalence was assumed when calculating the total number of farms required for the study, with a 10 % allowable error. the farms in both study sites were estimated (on previous experience) to have an average of three to four dairy stock of any age, breed or sex. a farm sample size of 200 in each study region was considered necessary to provide between 600 and 800 animals for the study and to allow for a “design effect” of 2.0. farms having more than ten animals were excluded from the selection process because such herds are not considered as “smallholder” enterprises (tanga dairy development programme 1999). questionnaire design and administration one person in each region collected most farmlevel and some animal-level data using a structured questionnaire, which was completed by the smallholder on all the selected farms on a single visit. the questionnaire was designed to comprise mostly closed ended (categorical) questions to ease data processing, minimize variation and improve precision of responses (thrusfield 2000). questionnaire administration and collection of data from animals were carried out by two separate teams, during the period of january to april 1999, in the two regions. although comparisons are made between the two regions, the data were not combined for analyses to allow any unexplained variation due to variations in the precise way that data were collected to be estimated. the information gathered concerned farm and animal events that had occurred during 1998 and included cattle location, source (homebred or brought-in), mode of acquisition of dairy stock, sex, level of exotic blood (filial generation), breed codes, age and housing practices, as well as whether or not a system of zero-grazing was practised of if the cattle had been allowed to graze on pastures in the 3 months prior to sampling. the detailed variables studied have been described by swai (2002). the responses to many of these questions were investigated as explanatory variables in the analyses of sero-conversion to babesia bovis. collection of sera and their analysis during the visit to each farm, blood samples were collected by jugular venipuncture into 10 ml “vacutainer” tubes (becton dickson, uk) from all animals on the farm. these were labelled and transported in a refrigerated cool box to local laboratories where aliquots of sera were obtained by centrifugation at 3 000 g for 20 min after which they were then stored 212 babesia bovis antibody prevalence in cattle in tanzania at –20 °c at sokoine university of agriculture (sua) prior to dispatch in refrigerated containers to the international livestock research institute (ilri) in nairobi, kenya where they were subjected to indirect enzyme-linked immunosorbent assay (elisa) in order to evaluate the level of antibodies to babesia bovis (katende, goddeeris, nkonge, morzaria & musoke 1990; katende,toye, skilton, nene, morzaria & musoke 1998). the elisa has shown a sensitivity of 99 % and a specificity of 98 % (katende et al. 1998). the results were expressed as percent positivity (pp) values of optical densities (wright, nilsson, van rooij, lelenta & jeggo 1993), relative to that of a strongly positive control serum. each test serum sample was analysed in duplicate and that of the control sera in triplicate. the threshold level of pp for positivity was 25 % for babesia bovis. statistical methods descriptive statistics for the animaland farm-level explanatory variables examined in the study were developed using epi-info, version 6.04d. the relationships between explanatory variables and outcome response (sero-conversion to babesia bovis) were investigated in two steps by logistic regression (using egret for windows version 2.0, seattle, usa) with “farm” as a random effect because animals on one farm may not have been statistically independent of one another (kristula, curtis, galligan & bartholomew 1992). in the first step, the relationships between each explanatory and outcome variable were individually investigated. in the second step, any variables that were significantly associated at the p < 0.25 level were included in multivariable models producing, by forwards and backwards substitution and elimination, the most parsimonious models in which all explanatory variables remained significant at the p < 0.05 level. the criteria for inclusion and exclusion were a change of in deviance significant at the 5 % level according to the maximum likelihood ratio test—chi square distribution. forces of infection were estimated from age seroprevalence profiles using maximum likelihood methods (mlm) in excel (microsoft, usa) with solver add-in (thrusfield 2000). assuming a stable population size and age structure and a constant force of infection across all age groups, the log likelihood was derived using the following formula: where ri = number of sero-positive in group i, ni = number tested in age group i and λ = the force of infection results farm participation all selected 200 farms from each of tanga and iringa regions were visited and farmers interviewed during the period of january 1999 to april 1999 (a 100 % response rate). in tanga, a total of 697 animals kept on 185 (92.5 %) farms were examined. fifteen farms (7.5 %) had no animals during the actual survey period. in iringa, a total of 698 animals from 195 (97.5 %) farms were examined. three farms (1.5 %) had no animals and two farms (1 %) could not be sampled because the owner could not be traced. the number of animals examined per herd was 3 ranged from 1 to 9 animals. the distribution of cattle amongst categories of each variable investigated is summarised in table 1. serological responses to babesia bovis the crude sero-prevalence of antibodies to babesia bovis was 6 % (4.7, 8.6) and 12 % (9.6, 14.7) for tanga and iringa, respectively (table 2). the estimated force of infection was 0.04 and 0.06 per animal years-risk for tanga and iringa, respectively. the graphical forces of infection are shown in fig.1 and 2. the estimated force of infection was slightly higher in iringa than in tanga. the results of the final logistic regression model are detailed in tables 3 and 4. in both tanga and iringa regions sero-prevalence was significantly greater in grazed animals than in zero-grazed animals. in iringa region animals located in iringa urban district were significantly more likely to be sero-positive for babesia bovis than animals on farms in iringa rural district (or = 5.83, p = 0.010). in both regions cattle brought onto the farms were significantly more likely to be sero-positive than animals born on-farm (or = 2.43, p = 0.035 and or = 3.1, p = 0.016, respectively for iringa and tanga regions). discussion the prevalence of babesia bovis infection as reflected by elisa appears to be comparatively higher in iringa (12 %) than in tanga (6 %). however, the 213 e.s. swai et al. loglikelihood l = ∑ ri lne–λi + (ni – ri) ln (1 – e–λi) a i = 1 214 babesia bovis antibody prevalence in cattle in tanzania table 1 the proportions of cattle in each category of each variable investigated during the study no. of animals (%) variable categories iringa tanga animal-level variables sex male 182 (26) 146 (21) female 516 (74) 551 (79) source of animal homebred 406 (58) 436 (63) brought-in 292 (41) 261 (37) filial generation f1 350 (50) 217 (31) f2 347 (49) 459 (66) f3 1 (0.1) 21 (3) breed codes ayrshire cross 403 (58) 169 (24) friesian cross 305 (44) 604 (86) jersey cross 1 (0.1) 12 (2) simmental cross 0 5 (1) sahiwal cross 0 12 (2) tshz cross 150 (22) 541 (77) boran cross 549 (78) 121 (17) age < 3 years 440 (63) 396 (57) 3 to < 6 years 165 (24) 214 (31) > 6 years 93 (13) 87 (12) grazing history in 1998 zero grazing 489 (70) 631 (90) semi/free grazing 209 (30) 66 (10) farm-level variables farm classification peri-urban 109 (16) 117 (17) urban 391 (56) 318 (46) rural 198 (28) 262 (37) district (iringa) iringa urban 461 (66) na iringa rural 237 (34) district (tanga) tanga na 341 (48.8) muheza 185 (26.5) pangani 26 (4) korogwe 64 (9) lushoto 81 (12) mode of acquisition bought cash 471 (67) 268 (38) credit (hit) 117 (17) 356 (51) others (gift) 110 (16) 73 (11) table 2 the prevalence (with +/– 95 % confidence intervals) of cattle sero-positive for babesia bovis in the study regions (january to april 1999) prevalence 95 % ci region no tested no positive (%) lower upper tanga 697 42 6 4.7 8.6 iringa 698 84 12 9.6 14.7 estimated prevalences were low when compared to the findings of 88 % by woodford, jones, rae, boid & bell-saikyi (1990) in pemba, tanzania; over 60% by perez, herrero, jimenez, carpenter & buening (1994) in costa rica; and 73 % of dairy cattle by maloo, thorpe, kioo, ngumi, rowland & perry (2001) in coastal kenya. there was also evidence of wide spread of this pathogen within the studied districts, particularly in iringa. the distribution of babesia bovis infection was limited to some districts in tanga. this distribution pattern may suggest that a situation of “endemic instability” prevails and that a similar distribution of the vector ticks (boophilus spp.) occurs in the district where the babesia infection is known to exist. the low serological prevalence for babesia bovis infection may be due to its poor infectivity for babesia (mahoney & mirre 1971) and subsequently the occurrence of lower inoculation rates in susceptible cattle (mahoney 1979). the lower observed inoculation rates (force of infection), in the light of these findings, may predict an enhanced tick resistance (mahoney 1979) or a genetically based immunity to babesia bovis characteristic of zebu crosses, which results in low inoculation rates of this babesia parasite. as in other studies, in both tanga and iringa regions, a history of recent grazing prior to sampling was 215 e.s. swai et al. table 3 final logistic binomial multiple regression for sero-prevalence of babesia bovis in dairy cattle, tanga, tanzania (january to april 1999) factor β(se) or lower to upper wald p likelihood ratio p 95 % ci constant –4.33 (0.56) grazing vs zero grazing 1.84 (0.57) 6.34 2.05–19.56 0.001 < 0.001 brought vs homebred 1.14 (0.47) 3.15 1.23–8.03 0.016 < 0.001 random term 1.44 (0.38) table 4 final logistic binomial multiple regression for sero-prevalence of babesia bovis in dairy cattle, iringa, tanzania (january to april 1999) factor β(se) or lower to upper wald p likelihood ratio p 95 % ci constant –5.57 (0.82) grazing vs zero grazing 1.29 (0.48) 3.65 1.40–9.49 0.007 < 0.001 brought vs homebred 0.88 (0.42) 2.43 1.06–5.56 0.035 < 0.001 iringa urban vs iringa rural 1.76 (0.68) 3.83 1.51–22.53 0.010 0.032 age centred 0.008 (0.006) 0.062 random term 2.06 (0.40) fig. 1 force of infection estimates for babesia bovis infection in dairy cattle—iringa, tanzania (january to april 1999) fig. 2 force of infection estimates for babesia bovis infection in dairy cattle—tanga, tanzania (january to april 1999) �������� �������� ���������������������� � � �� �� �� � � � � � � ��������� � � � �� � � � �� � � � � � � � � �� � � �� �� �� � � �� �� �� � � � � � � ��� ��� ��� ��� � ��� ��� ��� ��� � � � � � � � � � �� � � � � � � � � � �� �� � ��� ��� ��� ��� � ��� ��� ��� ��� �������� �������� ���������������������� � � �� �� �� � � � � � � � � �� � � � �� � � � � � � � � �� � � �� �� �� � � �� �� �� � � � � � � ��������� associated with a significantly higher likelihood of an animal being sero-positive to babesia bovis infection, compared to zero-grazed animals (maloo et al. 2001). sero-prevalence varied with the mode of acquisition of the animals. accounting for age, brought-in animals were more likely to be sero-positive for babesia bovis than were homebred animals. this would be consistent with the fact that most dairy stock available for sale in local markets are pregnant heifers from specialized large-scale crossbreeding ranches where they are mostly grazed rather than housed (swai 2002). allowing for variations in sero-prevalence related to age and management factors, such as grazing, geographic variation in sero-prevalence of babesia bovis infection was also determined. this variation was more conspicuous in the iringa study region with the odds of infection being three fold for cattle situated in iringa urban areas when compared to that in the cattle in the iringa rural district. this implies that there must be geographical variations in either the density of tick vectors or the prevalence of babesia bovis infection in host-seeking ticks, or both. variations in sero-prevalence may partly be owing to cattle immune responses (mahoney 1979). geographic variation in sero-prevalence was more uniform and generally higher in the iringa region. babesiosis has not been reported to be a major clinical problem in both study regions (tddp 1999; southern highland dairy development programme 1995) but there might be underestimations of clinical babesiosis in these regions. a diagnosis of bracken poisoning is frequently made by farmers and extension workers when cattle show clinical signs of “red water” (haemoglobinuria or haematuria) and in the light of the findings in this survey, some or many of these cases may, in fact, be misdiagnosed cases of babesiosis. an increased trend of sero-positivity associated with age was evident in this study. these findings are consistent with previous reports (hugh-jones, busch & jones 1988) which showed young animals to be more resistant to primary infections. we decided a priori to allow for intraherd correlation in the analysis by incorporating “farm” as a randomeffect term in all models. however in common with similar studies (gitau et al. 1994), there was little evidence of clustering at the level of farm, probably owing to the small number of animals per farm (mcdermott & schukken 1994) conclusion the results of this study can be summarized as follows: • sero-prevalence of antibodies to babesia bovis was low in smallholder dairy cattle in the study regions, most likely due to zero-grazing management, but there was evidence of a wide distribution in both the study regions. • consistent with this the force of infection varied geographically. it was, however, comparatively low and the likelihood of a bovine encountering the infection increased significantly with age. • farmer reporting of grazing significantly increased the likelihood of contact of cattle with infective ticks. • the source of animals, particularly recently purchased animals, may be more likely to have encountered and recovered from babesia bovis infection. acknowledgements we thank the government of uk through dfid\ nrrd animal health research programme for financing this work which formed part of a study for the ph.d. degree on the epidemiology of tick-borne diseases in small-scale farming systems in tanzania. the generous cooperation of smallholder farmers, extension staff and laboratory personnel at ilri/sua is also acknowledged. our thanks are extended to the director of veterinary service, tanzania for permission to publish this work. references cytel software corporation 1999. statistics and epidemiology research corporation, ver. 2.0. seattle, usa. epi-info 1996. epi-info. centre for disease control, version 6.04d, atlanta, usa and geneva, switzerland. french, n.p. & tyrer, j. 1997. birth and death of cattle on small scale dairy farms in zimbabwe. society of veterinary epidemiology and preventive medicine, chester. poster presentation. gitau, g.k., mcdermott, j.j., waltner-toews, d., lissemore, k.d., osumo, j.m. & muriuki, d. 1994. factors influencing calf morbidity and mortality in smallholder dairy farms in kiambu district, kenya. preventive veterinary medicine, 21:167–177. hugh-jones, m.e., busch, r.c. & jones, f. 1988. seroprevalence survey of anaplasma marginale card–test reactors in louisiana, usa, cattle. preventive veterinary medicine, 6:143–153. 216 babesia bovis antibody prevalence in cattle in tanzania katende, j.m., goddeeris, b.m., nkonge, c.g., morzaria, s.p & musoke, a.j. 1990. identification of a theileria antigen for use in an antibody and antigen detection elisa. parasite immunology, 12:419–433. katende, j.m., toye, p., skilton, r.a., nene, v., morzaria, s.p & musoke, a.j. 1998. an elisa for detection of t. parva antibodies using recombinant immunodominant molecule. parasitology research, 84:408–416. kristula, m.a., curtis, c.r., galligan, d.t & bartholomew, r.w. 1992. use of a repeated measures logistic regression model to predict chronic mastitis in dairy cows. preventive veterinary medicine, 14:57–68. lynen, g., bakuname, c. & sanka, p. 1999. tick and tbd survey in northern regions of tanzania. proceedings of tanzania veterinary association conference, arusha, december, 1999. mahoney, d.f. & mirre, g.b. 1971. annals of tropical medicine and parasitology, 65:309–317. mahoney, d.f. & ross, d.r. 1972. bovine babesiosis: computer simulation of babesia argentina rates in bos taurus cattle. annals of tropical medicine and parasitology, 68: 385–392. mahoney, d.f. 1979. babesiosis in domestic animals, in parasitic protozoa, vol. iv, edited by j.p. kreier. new york: academic press. maloo, s.h., thorpe, w., kioo, g., ngumi, p., rowland, g.j. & perry, b.d. 2001. sero-prevalence of vector transmitted infections of small-holder dairy cattle in coastal kenya. preventive veterinary medicine, 52:1–16. mcdermott, j.j & schukken, y.h. 1994. a review of the methods used to adjust fro cluster effects in explanatory epidemiological studies of animal populations. preventive veterinary medicine, 18:155–173. perez, e., herrero, m.v., jimenez, c., carpenter, t.e. & buening, g.b. 1994. epidemiology of bovine anaplasmosis and babesiosis in costa rica. preventive veterinary medicine, 20:23–31. riek, r.f. 1966. the life cycle of babesia argentina in the tick vector boophilus microplus. australian journal of agricultural research, 17:247–254 riek, r.f. 1968. babesiosis, in infection blood disease of man and animals, edited by d.f. weinmann & m. ristic. new york: academic press. southern highland dairy development programme. 1995. annual progress report, iringa. swai, e.s. 2002. epidemiological studies of tick borne diseases in small scale dairy farming systems in tanzania. ph.d. thesis, university of reading. tanga dairy development programme 1999. annual progress report, tanga. thrusfield, m. 2000. veterinary epidemiology. uk, blackwell science woodford, j.s., jones, t.w., rae, p.f., boid, r. & bellsaikyi 1990. sero-epidemiological studies of bovine babesiosis on pemba island, tanzania. veterinary parasitology, 37:175–184. wright, p.f., nilsson, f., van rooij, e.m.a., lelenta, m. & jeggo, m.h. 1993. standardisation and validation of enzyme linkage immunosorbent assay technique for detection of antibody in infectious disease diagnosis. revue scientifique technique office international des epizooties, 12: 435–450. 217 e.s. swai et al. abstract introduction methodology results and discussion conclusion acknowledgements references about the author(s) abdalla a. latif school of life sciences, college of agriculture, engineering and science, university of kwazulu-natal, westville campus, durban, south africa samson mukaratirwa school of life sciences, college of agriculture, engineering and science, university of kwazulu-natal, westville campus, durban, south africa one health centre for zoonoses and tropical veterinary medicine, school of veterinary medicine, ross university, st kitts, basseterre, west indies citation latif, a.a. & mukaratirwa, s., 2020, ‘zoonotic origins and animal hosts of coronaviruses causing human disease pandemics: a review’, onderstepoort journal of veterinary research 87(1), a1895. https://doi.org/10.4102/ojvr.v87i1.1895 review article zoonotic origins and animal hosts of coronaviruses causing human disease pandemics: a review abdalla a. latif, samson mukaratirwa received: 21 july 2020; accepted: 14 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the first known severe disease caused by a coronavirus (cov) in humans emerged with the severe acute respiratory syndrome (sars) epidemic in china, which killed 774 people during its 2002/2003 outbreak. the middle east respiratory syndrome (mers) was the second human fatal disease, which started in 2012 in saudi arabia and resulted in 858 fatalities. in december 2019, a new virus, sars-cov-2 (covid-19), originating from china, began generating headlines worldwide because of the unprecedented speed of its transmission; 5.2 million people were infected and 338 480 had been reported dead from december 2019 to may 2020. these human coronaviruses are believed to have an animal origin and had reached humans through species jump. coronaviruses are well known for their high frequency of recombination and high mutation rates, allowing them to adapt to new hosts and ecological niches. this review summarises existing information on what is currently known on the role of wild and domesticated animals and discussions on whether they are the natural reservoir/amplifiers hosts or incidental hosts of covs. results of experimental infection and transmission using different wild, domesticated and pet animals are also reviewed. the need for a one health approach in implementing measures and practices is highlighted to improve human health and reduce the emergence of pandemics from these zoonotic viruses. keywords: coronaviruses; humans; zoonosis; animal hosts; one health. introduction coronaviruses (covs) are a group of large single-stranded ribonucleic acid (rna) viruses, belong to the order nidovirales, family coronaviridae, and are classified based on the difference in protein sequences into four genera: alpha, beta, gamma and delta covs (woo et al. 2012). the morphology represents crown-like spikes on the outer surface of the virus seen under electron microscope, thus acquiring the name coronavirus. the evolution model suggests that bat cov serves as gene sources of alphaand beta-covs, whilst wild bird cov serves as gene sources of gamma and delta covs (wang & zhang 2016; woo et al. 2012). the beta-covs include severe acute respiratory syndrome coronavirus (sars-cov), the middle east respiratory syndrome coronavirus (mers-cov) and severe acute respiratory syndrome coronavirus-2 (sars-cov-2) (chan et al. 2013). coronavirsues of each genus are found in diverse animal species, including horses, cattle, pigs, dogs, cats, birds, rabbits, rats and ferrets and other wildlife animals, and some are infective in humans (chan et al. 2013). they cause mild to severe diseases in animals and humans. clinical signs and symptoms include respiratory, enteric, hepatic, renal and neurological manifestations, amongst other forms of the disease. it remains unclear as to how covs of each species have evolved, but different evolution models have been proposed, including a model where the bat covs serve as a gene source of all covs (cui, fang & zheng-li 2019). coronavirsues have constantly crossed species barriers and some have emerged as important human pathogens (cui et al. 2019). coronavirus species that cause important diseases in animals include avian infectious bronchitis virus, transmissible gastroenteritis virus in pigs, porcine epidemic diarrhoea virus and swine acute diarrhoea syndrome cov. the first known severe disease caused by a cov in humans emerged with the aevere acute respiratory syndrome (sars) epidemic in china (guan et al. 2003; kuiken et al. 2003; zhou et al. 2020), which killed 774 people during its 2002/2003 outbreak. the middle east respiratory syndrome (mers) was the second human disease, which started in 2012 in saudi arabia, resulting in 858 fatalities (van boheemen et al. 2012; zaki et al. 2012). in december 2019, a new virus began to spread worldwide because of the high rate of its transmission; 5.2 million got infected and 338 480 died from december 2019 to may 2020 (who 2020). the disease is caused by an infection with a novel coronavirus sars-cov-2 and was named covid-19, which stands for cov disease 2019 (guan et al. 2003; zhou et al. 2020). coronaviruses have high mutation rates which allow them to adapt to new hosts and ecological niche (cui et al. 2019). this review article discusses the animal origin and hosts of human coronaviruses (hcovs), summarising existing information on what is currently known on the role of wildlife animals, domesticated and pet animals, and whether they are the natural reservoir hosts, intermediate hosts or incidental hosts of covs. it is important to determine the host range of the hcovs for prevention and control purposes, risk assessments, development of diagnostic techniques, vaccines and therapeutics. the interdisciplinarity nature of a one health approach presents an ideal opportunity to deal with such complex pandemics engaging professionals from different disciplines, including medical professionals, veterinary professionals, environmental health and social sciences (kelly et al. 2017). methodology the search focus of this review was on the zoonotic origin and susceptibility of pet and wildlife animals to hcovs. literature was accessed from google scholar and medline/pubmed and the inclusion criteria in our literature search covered the period from the first occurrence of hcov pandemic in 2002 to the current covid-19 pandemic till september 2020, and included the following: the role of wild, domesticated and pet animals in the maintenance and transmission of the covs (search terms: ‘coronavirus transmission’, ‘wildlife’, ‘companion animals’, ‘livestock’) results from experimental infection and transmission using different wild, domesticated and pet animals (search terms: ‘coronavirus transmission’, ‘experimental infection’ and ‘laboratory animals’). the potential of pet and wildlife animals acquiring cov infection from infected persons who have had close contact with such animals (search terms: ‘coronavirus natural transmission’ and ‘human–pet animals/wildlife transmission’). for this review, a reservoir host is defined as one in which the infectious agent normally lives and multiplies and is, therefore, a common source of infection to other animals; thus, it is frequently an ancestral/natural host. it harbours hcov permanently and the virus is usually well adapted and non-pathogenic to the host. because the infectious agent frequently depends upon the natural host for its long-term existence, the host is also called a maintenance host (thrusfield 2005). an intermediate host is a new host, which becomes infected with hcov from the natural/reservoir host before or at the time of its introduction to humans. the virus may be amplified in the intermediate host and in this case serves as the zoonotic source of human infection (thrusfield 2005). an amplifier host is one in which the virus multiplies rapidly to high levels, providing an important source of infection. such an animal, because of temporal increase in its population size, may suddenly increase the magnitude of the infecting virus (thrusfield 2005). incidental (dead-end or accidental) host is defined as a host that does not usually transmit the virus to other animals (thrusfield 2005). the aspects of hcovs transmission, pathogenesis, genomic and vaccines studies, control and socioeconomics were excluded. ethical consideration this article followed all ethical standards for a research without direct contact with human or animal subjects. results and discussion severe acute respiratory syndrome search results showed reports which revealed that early infected human cases of sars in 2002 and 2003 had history of contact with live game animal markets or handling meat for consumption in the guangdong province of southern china (zhong et al. 2003). the epidemiological linkage of the initial human cases to wild game animals suggested that sars-cov is zoonotic in origin (guan et al. 2003). natural reservoir host following wide surveillance using next-generation sequencing performed in wild animals, over 200 novel covs have been identified in bats, and 35% of the bat virome sequenced is composed of covs (banerjee et al. 2019; woo et al. 2010). generally, covs do not cause apparently overt disease in bats and this was confirmed following experimental infection. they have been identified as natural reservoir hosts for a number of highly lethal viral diseases in human and domestic animals (fan et al. 2019). bats are one of the oldest mammalian groups with a unique immune system, which allows them to be asymptomatic carriers of these viruses (allocati et al. 2016; o’shea et al. 2014). in addition, they have a long lifespan and can fly across large geographical regions. bats are known as major evolutionary reservoirs and ecological drivers of covs diversity that may spill over to humans and cause disease (banerjee et al. 2019; martina et al. 2003; tu et al. 2004). the contact rate amongst bats and between bats and humans or domestic animals is a major contributing factor to zoonotic virus transmission. the novel covs related to human sars-cov (sars-rcovs) were discovered in horseshoe bats (genus rhinolophus) in china and hong kong (lau et al. 2005; martina et al. 2003; tu et al. 2004). these sars-rcovs showed a genome sequence identity of 88% – 90% amongst the group and 87% – 92% identity to human or palm civet sars-cov isolates (ge et al. 2013; li et al. 2005). intermediate hosts investigations were carried out on seven wild and one domestic animal species in a live animal market in southern china that supplied meat in restaurants in guangdong province (guan et al. 2003). of the apparently healthy masked palm civets (paguma larvata), which came from several different owners, four out of six tested positive for sars-like virus. one raccoon dog (nyctereutes procyonoides) was found to have the virus in faeces. sera from the palm civets and the raccoon dog had neutralising antibody to the animal cov (tu et al. 2004). similarly, sera from people who recovered from sars inhibited the growth of virus from the civets (lau et al. 2005). this cross-reaction is a strong indication that these viruses are similar and that these animals serve as intermediate hosts. moreover, the full-length genome sequence of the animal cov (s cov) had 99.8% homology to the human sars-cov, indicating that they are closely related (guan et al. 2003; lau et al. 2010; skowronski et al. 2005). masked palm civets that had not been exposed to live animal markets tested negative for sars-cov, implying that palm civets serve as the intermediate amplifying host but not the natural reservoir of sarscov (cheng et al. 2007; song et al. 2005; ye et al. 2020). experimental animal infection palm civets (paguma larvata) were shown to be susceptible and exhibited symptoms after experimental infection by sars-cov (guan et al. 2003). chinese ferret badgers (melogale moschata), raccoon dogs and domestic cats (felis domesticus) were also susceptible to infection by sars-cov (martina et al. 2003). cats remained asymptomatic, although some of the infected ferrets died of the disease. a primate model using cynomolgus macaques (macaca fascicularis), which showed clinical and pathological features with some similarities to those found in humans, proved the theory (koch’s postulate) that sars-cov is the causative agent of sars (cheng et al. 2007; fouchier et al. 2003; martina et al. 2003). golden syrian hamsters (mesocricetus auratus) were found suitable for immune-prophylaxis and treatment studies as clinical signs were accompanied by high levels of viral titres and histopathological lesions (roberts et al. 2005). similarly, inoculated common marmosets generally had mild clinical disease and histopathological changes of pneumonia with extrapulmonary dissemination and high levels of viral replication in affected tissues (greenough et al. 2005). pigs and chickens are not susceptible to sars-cov (weingartl et al. 2004). an important observation of these experimental animal infections is the diverse range of mammalian species that are susceptible to infection by sars-cov, which again demonstrated that sars-cov is highly capable of jumping interspecies barriers and is an excellent candidate as an emerging or re-emerging zoonotic pathogen (cui et al. 2019). these findings suggest that the wild animal markets provided a venue for the animal sars cov-like virus to amplify and transmit to new hosts, including humans. a mass cull of civet cats was undertaken for disease control in china in early 2004 following re-emergence of human cases with a possible link to these animals (skowronski et al. 2005). it is not, however, clear whether any single animal species or different species of these animals is the intermediate host in the wild (cheng et al. 2007; vijayanand, wilkins & woodhead 2004). these results also throw light on the animal origin and evolution of sars-cov at the beginning of the sars epidemic. middle east respiratory syndrome the mers caused by a novel cov (mers-cov) was first isolated in the kingdom of saudi arabia in 2012 (cotton et al. 2014; zaki et al. 2012). natural reservoir host middle east respiratory syndrome-related covs have been found in several bat families, including vespertilionidae, molossidae, nyteridae and emballonuridae from the middle east, africa, the americas, asia and europe (anthony et al. 2013; fan et al. 2019; ithete et al. 2013; memish et al. 2013; mohd, al-tawfiq & memish 2016). a cov from egyptian tomb bats, taphozous perforates, showed 100% nucleotide identity to virus from the human index case patient (memish et al. 2013). ithete et al. (2013) reported the identification of a south african bat-derived cov from neoromicia zuluensis bat that has the closet phylogenetic relationship with mers-cov, termed pml/2011. this pml/2011 differed from mers-cov by only one amino acid exchange and is the closest to mers-cov. a cov with 96.5% amino acid identity to mers-cov was found in a nyctinomops laticaudatus bat from mexico (anthony et al. 2013). nycteris gambiensis, from ghana, was found to carry 2c beta-covs (mohd et al. 2016). the same authors screened pipistrellus bat species from europe, where 14.7% were found to carry the 2c beta-cov. both 2c beta-covs isolated are genetically very closely related to mers-cov (mohd et al. 2016). all types of bat mers-related covs have been identified in the vespertilionidae bat family and in multiple species in china (fan et al. 2019). the mers coronavirus infection in humans related to human–camel contact has only occurred in saudi arabia and not in any other continent. overall, this relatedness of the cov found in different bat species indicates that mers-cov originated from bats (annan et al. 2013; cui et al. 2019). intermediate host dromedary camels have been found to harbour the same mers-cov and mers-cov-like antibodies as humans and to shed the virus in high numbers in secretions from the upper respiratory tract (adney et al. 2014). cross-reactive antibodies to mers-cov have been found in dromedary camels in oman, canary islands and egypt (perera et al. 2013; reusken et al. 2013). in addition, a fatal case of human mers-cov infection was transmitted through contact with an infected camel with rhinorrhoea, and the full genome sequence of the isolates from the patient and the camel was identical (azhar et al. 2014). it has been reported that a high proportion of dromedaries at a slaughterhouse shed nasal mers-cov, with a high risk of human exposure and potential of driving the epidemic (farag et al. 2015). the detection of mers-cov-specific antibodies with high prevalence in camels from the middle east, africa and asia, the high genetic stability of the virus in camel populations and the high genetic identity between human and camel strains point to the evidence that mers-cov is perfectly adapted to camels and has been spread freely in this domesticated animal for a long time (muller et al. 2014). camels experimentally infected with mers-cov showed only mild symptoms but with massive virus shedding through respiratory route and faecal-oral route. middle east respiratory syndrome coronavirus adapts in camels and maintains long-term endemicity and, therefore, the intermediate host can be defined as a natural secondary reservoir host with a spillover human infection (ye et al. 2020). approximately 55% of primary mers-cov cases are caused as a result of direct contact with dromedary camels or camel products (conzade et al. 2018). experimental animal infection several animal species have been experimentally infected with mers-cov, including rhesus macaques, cynomolgus macaques, marmosets, ferrets, mice, syrian hamsters, rabbits and dromedary camels (adney et al. 2014; falzarano et al. 2014; yao et al. 2014). the outcome and development of lower respiratory tract disease varied in these animal models. rhesus macaques were infected with mers-cov using intratracheal inoculation. the infected monkeys showed clinical signs of the disease (mild to moderate respiratory disease) with virus replication in lungs, lymph nodes, upper respiratory tract, histological lesions, and neutralising antibody production, indicating that this monkey model is suitable for studies of mers-cov infection (yao et al. 2014). the infection of marmosets revealed broncho-interstitial pneumonia and viral antigen detected in the lungs and had viral titres a 1000-fold higher than that in rhesus macaques (falzarano et al. 2014; kuiken et al. 2003; skariyachan et al. 2019). the clinical symptoms in the common marmoset model were mild to severe respiratory disease (falzarano et al. 2014; skariyachan et al. 2019). new zealand white rabbits have been proved to shed mers-cov from their upper respiratory tract, but remained asymptomatic after mers-cov inoculation (skariyachan et al. 2019; zaki et al. 2012). infected transgenic mice showed severe respiratory disease, weight loss and 100% mortality (agrawal et al. 2015). jamaican fruit bats infected with mers-cov had the virus detected in their respiratory and intestinal tracts for 9 days, with no overt signs of the disease (munster et al. 2016). there is no evidence of sustained human-to-human transmission in the community and no evidence of airborne transmission as main routes of transmission from all information available from recent mers-cov cases (who 2019b). in epidemiological terms, mers-cov was not considered of pandemic potential (table 1). cases resulting from animal-to-human transmission will continue to occur and will eventually lead to limited community transmission within households. infected camels shed mers-cov through nasal and eye discharge, faeces, milk, meat and urine. such camels may not show any signs of infection, which places a great risk in any contacts made with camels that can potentially infect humans. consistent application of adequate prevention and control measures has been used to prevent virus transmission (who 2019b). table 1: summary of epidemiological data for severe acute respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus 2. severe acute respiratory syndrome coronavirus 2 the ongoing novel coronavirus outbreak is the third highly infectious disease emerging at the animal–human interface, causing considerable concern and disruption as it spreads across the world (murdoch & french 2020). the index cases were epidemiologically linked to seafood and live wholesale animal market in wuhan, hubei province, china, in november 2019. the world case fatality rate for covid-19 is 6.9%, higher than that of the influenza h1n1 2009 pandemic (< 1%) and is low compared to 10% for sars-cov and 34.5% for mers-cov (table 1). the who (2020) declared the outbreak as a public health emergency of international concern (pandemic) on 30 january 2020. natural reservoir host genomic analysis revealed that sars-cov-2 is phylogenetically related to severe acute respiratory syndrome-like (sars-like) bat viruses; therefore, bats could be the possible primary reservoir (hu et al. 2017; zhou et al. 2020). severe acute respiratory syndrome-coronavirus 2 belongs to the species sars-rcov together with sars-cov from humans and sars-rcovs from horseshoe bats (gorbalenya et al. 2020; wu et al. 2020), concluding that sars-cov-2 has a bat origin. according to genome sequences available so far, the virus most closely related (96.2% of nucleotide sequence identity) to sars-cov-2 is strain batcovratg13 identified from a bat, rhinolophus affinis, from yunnan province, china, followed by sars-rcovs identified from pangolins (tang et al. 2020). intermediate host the pangolin (manis javanica) was found to harbour a cov, beta-covs, which is strikingly homologous to sars-cov-2. this indicates that pangolins might serve as one of the intermediate hosts or that the pangolin beta-covs could contribute gene fragments to the final version of sars-cov-2 (contini et al. 2020). scientists have been working to identify the source of sars-cov-2 (cyranoski 2020; lam et al. 2020; lau et al. 2020; xiao et al. 2020), and high-throughput sequencing of pangolins tissues obtained before the occurrence of covid-19 pandemic revealed the presence of cov sequences that fall into the sars-cov-2 lineage (lam et al. 2020). the genome sequence of one virus isolate had very high similarity (99.83% – 99.92%) to genomic organisations to sars-cov-2. another study suggested that sars-cov-2 might be a recombinant virus, with its genome backbone evolved from yunnan bat-virus-like sarsr-covs and from the pangolin-virus-like sarsr-covs (cyranoski 2020). the discovery of multiple lineages of pangolin cov and their similarity with sarscov-2 suggest that pangolins should be considered as possible intermediate hosts in the emergence of novel covs and should be removed from wet markets to prevent zoonotic transmission. incidental hosts cats: previous reports have shown that sars-cov can infect cats (martina et al. 2003), implying that they might also be susceptible to sars-cov-2. chinese researchers investigated the serological prevalence of sars-cov-2 in pet cats after the outbreak by an indirect enzyme-linked immunosorbent assay (elisa) and virus neutralisation test (zhang et al. 2020). the results indicated that 14.7% (15/102) of the cat blood samples collected after the outbreak tested positive for the presence of antibodies specific for sars-cov-2. three cats owned by covid-19-affected patients had the highest levels of antibodies and this might suggest a cat–human transmission of sars-cov-2 and further studies are urgently needed to confirm this. human–pet cat transmission has been reported from several countries. a pet cat in spain whose owner had recently died of covid-19 tested positive for sars-cov-2 by reverse transcription-polymerase chain reaction (rt-pcr) (american veterinary medical association 2020; segalésa et al. 2020). in france, sars-cov-2 rna was detected in samples collected from a cat whose owner was suspected to have covid-19. a second sars-cov-2-positive cat had developed clinical signs of respiratory disease, including cough, and was examined several times. the nasopharyngeal sample was positive for sars-cov-2 (american veterinary medical association 2020; fritz et al. 2021). two pet cats with confirmed cases of sars-cov-2 infection in new york had clinical signs of a mild respiratory illness. the owner of the first cat assumed to be an asymptomatic carrier and the second cat owner had tested positive for covid-19 before the cat became ill (american veterinary medical association 2020; newman et al. 2020). in belgium, sars-cov-2 rna was detected by rt-pcr and high-throughput sequencing pcr in the faeces and vomit of a cat with digestive and respiratory clinical signs. the cat was owned by a person confirmed to be infected with sars-cov-2 (american veterinary medical association 2020; brown 2020). using rt-pcr, a cat in germany was confirmed to be infected with sars-cov-2. the owner had died of covid-19 (american veterinary medical association 2020). zoo tigers and lions: the united states department of agriculture’s (usda) national veterinary services laboratories confirmed that a 4-year-old malayan tiger at new york’s bronx zoo tested sars-cov-2 positive (usda 2020). this is the first report of a tiger being infected with covid-19. samples from the tiger were taken and tested after several lions and tigers at the zoo showed symptoms of respiratory illness and decreased appetite. however, only samples from the tiger were taken for diagnosis. dogs: dogs belonging to covid-19-infected owners were screened for sars-cov-2 during the outbreak in china (sit et al. 2020). two out of 15 dogs from households with confirmed human cases of covid-19 were found to be infected using quantitative rt–pcr, serology, sequencing the viral genome, and in one dog, virus isolation. severe acute respiratory syndrome-coronavirus 2 rna was detected from nasal and oral swabs. both dogs had seroconverted using neutralisation assays. viral genetic sequences of viruses from the two dogs were identical to the virus detected in the respective human cases. the animals remained asymptomatic during quarantine. the results suggest that there are instances of human-to-animal transmission of sars-cov-2, although it is unclear whether infected dogs can transmit the virus to other animals or to humans. minks: four mink farms have been reported positive for sars-cov-2 in the netherlands since the start of the outbreak (oreshkova et al. 2020; american veterinary medical association 2020), and there are concerns about the potential for widespread transmission of the virus. the affected farms had reported an increased incidence of gastrointestinal and respiratory disease, including fatal infections, with pregnant minks developing more serious disease with increased mortality. the disease was confirmed in sick minks by sars-cov-2-specific pcr assay. it was believed that the virus was transmitted to minks by covid-19-infected farm caretakers; however, mink-to-mink transmission could have spread the virus within each farm. experimental animal infection ferrets: the chinese ferret badgers (melogale moschata) was infected with sars-cov and showed clinical signs and efficient transmission between infected and naïve controls (roberts et al. 2005). based on these results, ferrets were considered as suitable animal models for sars-cov-2, and hence studies to use ferrets as an experimental animal model to evaluate treatments and vaccines for the disease in humans were performed by kim et al. (2020). the authors have shown in a comprehensive study that ferrets can be infected experimentally with sars-cov-2, can shed virus in body discharges and transmit the infection to other naïve ferrets. high levels of viral rna were detected in nasal washes of all infected ferrets, between 2 and 8 days after covid-19 inoculation. three naive ferrets were placed in contact with the infected ones, and they all became infected, showing that the virus is transmissible between ferrets. ferrets appear to be susceptible to infection but less so to disease and no mortality. infectious virus was detected from the nasal washes of all ferrets but not from the rectal swabs. antibodies against sars-cov-2 were detected in all ferrets by an elisa and a neutralisation assay test. ferrets have already been used as models in influenza studies (bouvier 2015), and several laboratories have started covid-19 research using these highly susceptible animals. cats: in an infectivity study carried out under laboratory conditions (shi et al. 2020), viral rna was detected in the upper and lower respiratory tracts and in the small intestine of two cats that were euthanised on days 3 and 6 post-infection. infectious virus was detected in the respiratory system, and droplet transmission had occurred between cats. all virus-inoculated cats and one exposed cat had developed antibody tested by elisa and neutralisation assays. in sars-cov-2 direct contact transmission study (halfmann et al. 2020), three domestic cats were inoculated with the virus and each was co-housed with a naïve cat 1 day after inoculation of the virus. nasal and rectal swab specimens were taken daily and tested for infectious virus on vero-e6 cells. the virus was detectable in all three inoculated cats and the three co-housed cats. all the cats remained asymptomatic and all had high igg antibody titres. dogs: in a sars-cov-2 infectivity and direct transmission study under controlled conditions (shi et al. 2020), five 3-month-old dogs were inoculated intra-nasally and co-housed with two uninoculated dogs. pharyngeal and rectal swab specimens were collected from days 2 to 14 post-infection. viral rna was positive in the rectal swabs; however, infectious virus and viral rna were not detected in any swabs, organs or tissues collected from a euthanised positive dog. only two dogs out of four of the inoculated dogs had positive antibody titres, whilst the two contact dogs tested seronegative. these results concluded that dogs are less susceptible to sars-cov-2. other domestic and wildlife animals: susceptibility of pigs, chickens and ducks to sars-cov-2 was investigated by using the virus intra-nasal inoculation methodology as that used to assess cats and dogs (shi et al. 2020). infected animals were housed in the same room with their uninfected species to investigate the transmission of the virus. viral rna was not detected in any swabs collected from the inoculated animals or from naïve contact animals. viral rna was not detected in all organs examined in euthanised pigs and all the animals were seronegative. these results indicate that pigs, chickens and ducks are not susceptible to sars-cov-2. one health approach to control cov disease 2019 pandemic: to prevent the spread of the novel covid-19, the chinese government banned the trade in wild animals. a similar measure was also introduced in 2003 for sars, but this control measure was not sustained. continuous surveillance of mammals is essential for a better understanding of the ecology of covs and for the prevention of animal-to-human transmission of the disease (zhou et al. 2020). with reports of positive transmission of sars-cov-2 from humans to domestic cats and to tigers and lions at the bronx zoo (usda 2020), coupled with these experimental data showing the ease of transmission between domestic cats, there is a public health need to recognise and further investigate the potential chain of human–cat–human transmission. this is of importance, given the potential for sarscov-2 transmission between family members in households with cats whilst living under ‘lockdown’ regulations. moreover, cats may be passive intermediate host of sars-cov-2 because infected cats may not show any appreciable symptoms that might be recognised by their owners. given the need to stop the covid19 pandemic through various mechanisms, including breaking transmission chains, a better understanding of the role cats and other domesticated animals may play in the transmission of sars-cov-2 to humans is needed and emphasises the importance of closer collaboration amongst various scientific disciplines and stakeholders following the one health approach. conclusion three pandemics caused by the highly pathogenic hcov of zoonotic origin occurred during 2002, 2012 and 2019. table 1 summarises the epidemiological data for sars-cov, mers-cov and sars-cov-2. in the case of mers-cov, there is no evidence of sustained human-to-human transmission in the community and no evidence of airborne transmission as main routes of transmission (who 2019a). in contrast, the diversity of animal species susceptible to sars-cov and sars-cov-2 strongly suggests a jump of these viruses to cross species barrier. based on current information, the three hcovs that are presumed to have originated from their natural reservoir hosts, the bats and their intermediate hosts have also been identified. the intermediate hosts for sars-cov proven to date are the masked palm civets and raccoon dogs. this pandemic was controlled by the elimination of the intermediate host, palm civets, from live markets in china. middle east respiratory syndrome coronavirus closely related to bat cov were identified in countries in four continents, which implied that the natural reservoir host originated from bats. however, none of these geographical areas investigated, except for saudi arabia, is known for mers-cov primary infection in humans. the high prevalence of antibody titres, the high genetic stability of the virus and the high genetic identity between camel and human strain are all evidence that mers-cov is perfectly adapted and maintained amongst camels over the years. therefore, camels are intermediate hosts in this situation and can be referred to as natural secondary reservoir hosts. a phylogenetic analysis showed that sars-cov-19 significantly clustered with a sequence from the bat sars-like cov isolated in 2015 and that the transmission chain began from the bat (benvenuto et al. 2020). severe acute respiratory syndrome-coronavirus 2-related covs were identified in malayan pangolins, which suggests that pangolins should be considered as possible intermediate hosts in the emergence of sars-cov-19. incidental infections, experimental infectivity and transmission findings from laboratory studies have shown that, of the animal species screened and investigated so far, cats are the most susceptible species for covid-19. public health and veterinary services should work together using a one health approach to share information and conduct a risk assessment when a person with covid-19 reports being in contact with companion animals or other animals. it is recommended that rt-pcr should be used to test oral, nasal and faecal/rectal samples of such animals. however, there is no conclusive evidence that domestic and wildlife animals can actively transmit sars-cov-2 to humans. on the other hand, scientific research and epidemiological investigations are yet to start. acknowledgements competing interests the authors have declared that no competing interests exist. authors’ contributions all authors contributed equally to this work. funding information this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer 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(r.) leachi by hoogstraal (1958), and his designation of a neotype, stimulated taxonomic studies of ticks belonging to this cluster of species. during the 1970s and 1980s camicas and hoogstraal and their co-workers elucidated taxonomic problems associated with this group and described or re-established a number 181 onderstepoort journal of veterinary research, 74:181–208 (2007) redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haemaphysalis (rhipistoma) leachi (audouin, 1826) (ixodida, ixodidae) d.a. apanaskevich1, i.g. horak2 and j-l. camicas3 abstract apanaskevich, d.a., horak, i.g. & camicas, j-l. 2007. redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haemaphysalis (rhipistoma) leachi (audouin, 1826) (ixodida, ixodidae). onderstepoort journal of veterinary research, 74:181–208 koch (1844) originally described only the male of haemaphysalis (rhipistoma) elliptica (koch, 1844), which he named rhipistoma ellipticum. for the past century, however, this name has been considered a junior synonym of haemaphysalis (rhipistoma) leachi (audouin, 1826), or a nomen nudum. we redescribe here the male and larva of h. (r.) elliptica and describe the female and nymph for the first time. our redescription is based on the male holotype, plus numerous specimens from southern and east africa. the adults of this tick parasitize domestic and wild carnivores, and the immature stages infest rodents in these regions. for comparative purposes redescriptions of all parasitic stages of h. (r.) leachi are provided. it parasitizes the same hosts as h. (r.) elliptica in egypt, and in northeastern, central, west and east africa. keywords: descriptions, geographic distribution, haemaphysalis (rhipistoma) elliptica, haema physalis (rhipistoma) leachi, hosts 1 united states national tick collection, institute of arthro podology and parasitology, georgia southern university, statesboro, georgia 30460-8056, united states of america previously department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa e-mail: dapanaskevich@georgiasouthern.edu 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa; division of parasitology, onderstepoort vet erinary institute, onderstepoort, 0110 south africa; and dep artment of zoology and entomology, university of the free state, bloemfontein, 9300 south africa 3 laboratory of medical acarology, centre ird, montpellier, france accepted for publication 7 march 2007—editor 182 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) of species. hoogstraal & kim (1985) consolidated the accumulated data on haemaphysalis koch, 1844 and on the subgenus rhipistoma koch, 1844 as well as on the h. (r.) leachi group. they placed these ticks in three subgroups, namely h. (r.) leachi, haem a physalis (rhipistoma) pedetes and haemaphysalis (rhipistoma) spinulosa. camicas, hervy, adam & morel (1998) concurred with this decision and updated the species composition of the three subgroups. the h. (r.) leachi subgroup now consisted of five species, namely h. (r.) elliptica (koch, 1844), h. (r.) leachi (audouin, 1826), haema phy salis (rhipistoma) moreli camicas, hoogstraal & el kam mah, 1972, haemaphysalis (rhipistoma) paraleachi camicas, hoogstraal & el kammah, 1983, and haemaphysalis (rhipistoma) puncta leachi cami cas, hoogstraal & el kammah, 1973. the h. (r.) pedetes subgroup contained two species, viz. h. (r.) pedetes hoogstraal, 1972 and haemaphysalis (rhipistoma) zumpti hoogstraal & el kammah, 1974, while the h. (r.) spinulosa subgroup incorporated four species, namely haema physalis (rhipistoma) muhsamae santos dias, 1954, haema physa lis (rhipistoma) norvali hoog straal & wassef, 1983, h. (r.) spinulosa neumann, 1906 and haemaphysalis (rhipistoma) subterra hoog straal, el kammah & camicas, 1992. there are only two synonyms for species within the h. (r.) leachi group, and these are haemaphysalis leachi var. humerosoides theiler, 1943, that has been syn onymized with h. (r.) leachi, and haemaphysalis ethiopica santos dias, 1958, that has been synony mized with h. (r.) spinulosa. camicas et al. (1998), in their review of the ticks of the world, created two problems within the taxonomy of the h. (r.) leachi group by re-establishing two names, namely h. (r.) elliptica and h. (r.) muhsamae. the present paper addresses the taxonomic status of h. (r.) elliptica, while that of h. (r.) muhsamae, which for several decades has been considered a junior synonym of h. (r.) spinulosa, will be tackled in a future communication. koch (1844) originally described haemaphysalis (rhipistoma) elliptica (koch, 1844) as rhipistoma ellipticum. neumann (1897) placed this species in the genus haemaphysalis koch, 1844 and synonymized it with h. (r.) leachi (audouin, 1826). thereafter the majority of tick taxonomists considered h. (r.) elliptica to be a junior synonym of h. (r.) leachi, or a nomen nudum (nuttall & warburton 1915; camicas et al. 1972). little more than a century later camicas et al. (1998) re-established this taxon, but gave no reasons for their decision, thus begging the question, is h. (r.) elliptica a valid taxon or not? after an exhaustive study of many collections of haemaphysalis that had been identified as h. (r.) leachi, and a comparison of these ticks with true h. (r.) leachi from north africa and with the holotype specimen of h. (r.) elliptica, we concluded that many of the southern and east african ticks previously identified as h. (r.) leachi are actually h. (r.) elliptica. furthermore, these studies enabled us to delimit the geographic distributions of both ticks. we here redescribe the male [the first description is given by koch (1844), under the name rhipistoma ellipticum], and the larva [the first description is given by bedford (1934), under the name haemaphysalis leachi], and describe the female and nymph of h. (r.) elliptica for the first time. for comparative purposes we have also redescribed all stages of development of h. (r.) leachi. material examined the material examined is summarized in tables 1, 2 and 3. specimens from south africa and mozambique were studied by igh, or by igh and daa, and the remainder were examined by daa. because of difficulties experienced in the identification of specimens we used the following material for the present study: (i) all primary identifications have been based on males. (ii) with the exception of collections from egypt and south africa, collections containing only females have been excluded. (iii) females in collections containing males of two or more species have been excluded. (iv) the immature stages that we have studied come only from laboratory-reared specimens from allopatric localities within the distribution ranges of the two ticks, namely south africa for h. (r.) elliptica, and egypt and the central african republic for h. (r.) leachi. the records of jlc have not been included because they need to be rechecked in relation to the new characters that we have found. the descriptions of the adults of various haema physalis species by hoogstraal and his co-authors are characterized by the use of proportions between measurements of particular structures, mainly those of the gnathosoma. however, we could not find any exact description of the scheme of measurements taken by hoogstraal and his co-workers, who gave only brief explanations in the texts. the exact features or structures between which some of the meas183 d.a. apanaskevich, i.g. horak & j-l. camicas urements were made are for the most part quite easily recognizable, but for several they are not. con sequently, we have taken those measurements that we consider are the most suitable for describing the species. except for the measurements for which an explanation is given in the text, a scheme of the measurements that we have taken is illustrated in fig. 1. because the larva and nymph have sometimes been inadequately described or not described at all, our set of measurements for them does not differ substantially from that used for these stages of development of previously described species. for the adults we tried to follow hoogstraal’s format so that our measurements would at least approximate those that had been used before. measurements for the male conscutum and female scutum and their total lengths are given in millimetres (mm), and those for the immature stages in micrometres (μm). the measurements are arranged as follows: minimum – maximum (average ± standard deviation, n = number of specimens measured). when measuring the dorsal and ventral spurs on palpal segments ii and iii, it must be noted that they are not in the same plane as the gnathosoma as they are directed either dorsally or ventrally. config. 1 scheme of measurements for haemaphysalis. a, nymph, gnathosoma dorsally: a-e—combined palpal width, b-d – width of basis capituli, c-e – width of palp; b, nymph, gnathosoma ventrally: a-b – length of hypostome, a-c – length of gnathosoma, d-e – width of hypostome, f-g – length of palp; c, nymph, scutum: a-b – width, c-d – length; d, male, gnathosoma dorsally: ad – width of palpal segment ii, a-g – combined palpal width, b-e – width of palpal segment iii, c-f – width of basis capituli, h-j – length of palpal segment iii, i-k – length of palpal segment ii, k-m – length of basis capituli, l-m – length of dorsal cornua; e, nymph, leg i: a-b – length of genu, c-d – width of genu a a b c d e a b c d e a b c d f a b c d g f g h i e j k l m b c d e a b c d 184 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) sequently, the shape and the length of these spurs vary according to the plane along which they are observed. daa’s illustrations of the gnathosoma of the larvae and nymphs are based on slide-mounted specimens, but because of the differences in planes even in these preparations, the spurs on the palpal segments are in reality longer than illustrated. this observation has been verified by scanning electron microscopy. furthermore, in order to simplify identification for persons who may in future examine these species we have attempted to use a minimum of poorly defined diagnostic characters. haemaphysalis (rhipistoma) elliptica (koch, 1844) the south african carnivore haemaphysalid (fig. 2–7) synonym haemaphysalis leachi humerosoides theiler, 1943 sensu theiler, 1943. the collection lot (no. 2754), identified as haemaphysalis leachi var. humerosoides by g. theiler, contains nine vials. in the catalogue listing the specimens in the onderstepoort veterinary institute tick collection the first vial (i) is marked as “type”: i (13 ♂, 17 ♀) – bilene, macia [mozambique], 25.v.1940, peaf collection, xiii, banino. according to its label, the second vial may also contain specimens of the original type series: ii (10 ♂, 15 ♀) – angonia, massoane [mozambique], 12.vii.1940, peaf collection, xxv, banino. daa and igh have identified all the specimens in these vials as h. (r.) elliptica. the other vials contain various ticks of the h. leachi group as well as rhipicephalus koch, 1844 collected from localities in africa at a later stage. holotype male, cape of good hope (western cape province, south africa), deposited in the natural history museum of berlin, berlin, germany; collection no. zmb 1099. this specimen has been examined by all of us and studied by daa and jlc. description and redescription male (fig. 2a–c, 3a–f) length from palpal apices to posterior margin of conscutum 2.41–3.54 (3.00 ± 0.19, n = 323); breadth of conscutum (at widest point) 1.19–1.75 (1.47 ± 0.10, n = 322); ratio 1.78–2.32 (2.05 ± 0.10, n = 319). colour reddish brown. conscutum (fig. 2a–c): ca 1.9 times as long as broad; margins slightly convex, broadest at level of fig. 2 haemaphysalis elliptica, male, a, conscutum. bar = 1 mm; b, c, left posterior half of conscutum . bar = 1 mm. all setation is omitted a b c 185 d.a. apanaskevich, i.g. horak & j-l. camicas spiracular plates, smoothly rounded posteriorly. cerv ical pits narrow, deep, converging. cervical grooves indistinct, short, shallow, diverging. lateral grooves deep, distinct, extend to anterior 1/4 of scutal length; enclose first or first and second festoons. punctations dense, medium-sized, discrete, relatively deep. festoons number 11. genital structures (fig. 3a): as illustrated. spiracular plates (fig. 3b): variable in fig. 3 haemaphysalis elliptica, male, a, genital structures: apron and postgenital sclerite. bar = 200 μm; b, spiracular plate (a – anterior; d – dorsal). bar = 200 μm; c, gnathosoma dorsally. bar = 200 μm; d, gnathosoma ventrally. bar = 200 μm; e, hypostome. bar = 100 μm; f, coxae. bar = 500 μm. all setation is omitted f d c e a b a d 186 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) size, usually slightly broader than long; suboval; dorsal projection triangular. capitulum (fig. 3c, d): basis capituli dorsally ca. 1.7 times as broad as long; lateral margins diverging anteriorly; cornua elongately triangular, apices rounded, ca. 1/3 as long as length of basis capituli; ventrally as illustrated. palps broadly salient (leachi type); combined breadth ca. 1.9 times breadth of basis capituli. segment ii ca. 1.7 times as broad as long; dorsomedian margin of segment ii gradually widening anteriorly at level of its mid-length; posterodorsal spur large, triangular; posteroventral spur large, triangular, with straight lateral margin. segment iii ca. 1.6 times as broad as long; ca. 1/2 the length of segment ii; ventral spur of segment iii narrowly elongate, u-shaped apex at level of anterior 1/4 of length of segment ii. hypostome (fig. 3e): slightly shorter than palps; dental formula 4/4; denticles in subequal-length files of 6 or 7. coxae (fig. 3f): i to iv each with a short, subtriangular, more or less bluntly pointed spur, extending somewhat beyond coxal margin; spur of coxae iv usually subequal to that of coxae iii. female (fig. 4, 5a–f) length from palpal apices to posterior margin of scutum 1.42–1.92 (1.73 ± 0.10, n = 131); breadth of scutum (at widest point) 0.82–1.14 (1.02 ± 0.06, n = 133); ratio 1.50–1.86 (1.70 ± 0.07, n = 131). scutum (fig. 4): ca. 1.3 times as long as broad; anterior margins diverging for anterior 1/5 of total length, subparallel 1/5 of the length, thence gradually converging, bluntly rounded posteriorly; slight postero-lateral angles. cervical grooves narrow arcs extending 2/3 of total scutal length. punctations moderately dense, denser on lateral fields, absent in cervical grooves; medium-sized, discrete, relative ly deep. posterior lip of genital aperture (fig. 5a): broadly u-shaped. spiracular plates (fig. 5b): varying in size; irregularly suboval or subcircular; dorsal projection short, broadly triangular. capitulum (fig. 5c, d): basis capituli dorsally ca. 2.4 times as broad as long; external margins diverging anteriorly; cornua short, broadly triangular, bluntly pointed, ca. 1/6 as long as the length of the of basis capituli; porose areas elongate-oval, tilted inwards, moderate size, widely spaced. basis capituli ventrally as illustrated. palps broadly salient (leachi type); combined breadth ca. 1.6 times breadth of basis capituli. segment ii ca. 1.4 times as broad as long; dorsomedian margin of segment ii gradually widening anteriorly at level of its midlength; posterodorsal spur large, triangular; posterolateral margin straight; posteroventral spur reduced to short rounded projection or curve. segment iii ca. 1.2 times as broad as long; ca. 0.7 times as long as segment ii; ventral spur of segment iii narrowly u-shaped, elongate, apex at level of anterior 1/3 of length of segment ii. hypostome (fig. 5e): nearly as long as palps; dental formula 4/4; denticles usually in files of 9 or 10. coxae (fig. 5f): i to iv each with a short, subtriangular, more or less bluntly pointed spur, extending somewhat beyond coxal margin; spur on coxae iv subequal to that of coxae iii. nymph (fig. 6a–e) length (unengorged) from palpal apices to posterior body margin 1 366–1 683 (1 543 ± 77.93, n = 32); breadth of idiosoma (at widest point) 756–988 (896 ± 62.33, n = 32); ratio 1.60–1.85 (1.73 ± 0.06, n = 32). scutum (fig. 6a): length 431–510 (472 ± 20.95, n = 32), breadth 421–549 (427 ± 27.71, n = 32), ratio 0.92–1.08 (1.00 ± 0.04, n = 32); irregularly circular. spiracular plates (fig. 6b): suboval. fig. 4 haemaphysalis elliptica, female, scutum. bar = 1 mm. all setation is omitted 187 d.a. apanaskevich, i.g. horak & j-l. camicas capitulum (fig. 6c, d): length 240–284 (265 ± 11.91, n = 32), breadth (palps combined) 336–402 (371 ± 16.97, n = 32), ratio 0.69–0.79 (0.71 ± 0.004, n = 32). basis capituli dorsally subrectangular; cornua slight bulges; ventrally as illustrated. palps: length 167–198 (182 ± 8.69, n = 32), breadth 147–181 fig. 5 haemaphysalis elliptica, female, a, genital structures: posterior lip of the genital aperture and vestibular part of vagina. bar = 200 μm; b, spiracular plate (a – anterior; d – dorsal). bar = 200 μm; c, gnathosoma dorsally. bar = 200 μm; d, gnathosoma ventrally. bar = 200 μm; e, hypostome. bar = 100 μm; f, coxae. bar = 500 μm. all setation is omitted f d c e a b a d 188 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) fig. 6 haemaphysalis elliptica, nymph, a, scutum. bar = 200 μm; b, spiracular plate (a – anterior; d – dorsal). bar = 50 μm; c, gnathosoma dorsally. bar = 200 μm; d, gnathosoma ventrally. bar = 200 μm; e, coxae. bar = 200 μm. setation of palpal segment iv is omitted ab c d e a d 189 d.a. apanaskevich, i.g. horak & j-l. camicas (160 ± 7.77, n = 32), ratio 1.08–1.21 (1.14 ± 0.03, n = 32); broadly salient; anterolateral margin slightly concave. dorsomedian margin of segment ii gradually widening anteriorly at level of its midlength; dorsal spur moderate; ventral spur large, broad; lateral margin of spur slightly concave. ventral spur of segment iii distinct, broadly triangular, with sharp apex. hypostome (fig. 6d): length 97–116 (107 ± 5.47, n = 32), breadth 40–48 (45 ± 2.05, n = 32), ratio 2.20– 2.67 (2.39 ± 0.10, n = 32); nearly as long as palps; dental formula 2/2; denticles in files of 7 to 9 (usually 8). fig. 7 haemaphysalis elliptica, larva, a, scutum. bar = 200 μm; b, gnathosoma dorsally. bar = 100 μm; c, gnathosoma ventrally. bar = 100 μm; d, coxae. bar = 100 μm. setation of palpal segment iv is omitted a b c d 199 d.a. apanaskevich, i.g. horak & j-l. camicas n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* ♂ ♀ i. g . h o ra k c o ll e c ti o n ( s o u th a fr ic a & m o za m b iq u e ) (c o n t. ) 2 4 2 6 3 7 2 0 c a n is f a m ili a ri s g ra h a m st o w n 3 3 1 9 s , 2 6 3 2 e ( h o ra k e t a l. 1 9 8 7 ) a u g 1 9 8 3 t o j u l 1 9 8 6 o w n e rs 8 7 2 6 9 c a n is f a m ili a ri s b o sc h ko p , n o rt h w e st p ro vi n ce o ct 1 9 9 9 t o m a y 2 0 0 0 z . v. d . m e rw e 6 9 c a n is f a m ili a ri s l u a la , 1 7 4 4 s , 3 6 1 5 e , z a m b e zi a p ro v. m o za m b iq u e (n e ve s e t a l. 2 0 0 4 ) g . b e st e r 2 2 c a n is f a m ili a ri s x a ix a i, 2 5 0 6 s , 3 3 3 4 e g a za p ro v. m o za m b iq u e (n e ve s e t a l. 2 0 0 4 ) g . b e st e r 7 0 6 5 d o m e st ic c a ts v e t. c lin ic s te lle n b o sc h 3 3 5 5 s , 1 8 5 0 e ( h o ra k & m a tt h e e 2 0 0 3 ) o ct 2 0 0 0 t o d e c 2 0 0 2 a ss is ta n t c a ts 6 0 4 6 6 3 d o m e st ic c a t p re to ri a s e p 2 0 0 3 t o m a y 2 0 0 6 n . d o n ki n c a ts 3 1 c a n is m e so m e la s (1 ) k n p ( h o ra k e t a l. 2 0 0 0 ) i. g . h o ra k 4 5 c a n is m e so m e la s (1 ) v e ke e rd e vl e i, f re e s ta te ( h o ra k e t a l. 2 0 0 0 ) 1 5 9 3 4 a ci n o n yx ju b a tu s (3 ) k n p ( h o ra k e t a l. 2 0 0 0 ) i. g . h o ra k 2 1 9 8 8 3 1 p a n th e ra le o ( 1 9 ) k n p ( h o ra k e t a l. 2 0 0 0 ) i. g . h o ra k 6 3 2 6 p a n th e ra p a rd u s (3 ) k n p ( h o ra k e t a l. 2 0 0 0 ) i. g . h o ra k 1 6 p a n th e ra p a rd u s (1 ) r o o d e p la a t d a m , g a u te n g ( h o ra k e t a l. 2 0 0 0 ) 8 5 2 3 c iv e tt ic tis c iv e tt a ( 4 ) k n p ( h o ra k e t a l. 2 0 0 0 ) i. g . h o ra k 8 7 p a ra h ya e n a b ru n n e a ( 1 ) b o n a cc o rd , g a u te n g ( h o ra k e t a l. 2 0 0 0 ) i. g . h o ra k * a ll th e c o lle ct io n n u m b e rs a re t h o se o f sp e ci m e n s in t h e u n ite d s ta te s n a tio n a l t ic k c o lle ct io n + r e a re d s p e ci m e n s t a b l e 1 (c o n t. ) 200 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) t a b l e 2 h a e m a p h ys a lis ( r h ip is to m a ) le a ch i ( a u d o u in , 1 8 2 6 ), m a te ri a l e xa m in e d n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* ♂ ♀ c a m e ro o n 1 d o m e st ic d o g 2 6 d e c 1 9 4 9 0 9 4 7 1 6 c e n tr a l a fr ic a n r e p u b li c 4 4 p a n th e ra le o b a m in g u i b a n g o ra n , b a m in g u i, g a za o j. t h a l 0 9 6 9 8 8 2 3 d o m e st ic d o g l a t o p ia 1 2 a u g 1 9 6 9 g . u ile n b e rg 0 9 6 9 9 1 3 1 8 d o m e st ic d o g n a n a n a m b e re , b o u a r 2 3 j u l 1 9 6 9 m . g ir e t 0 9 6 9 8 2 6 1 6 d o m e st ic d o g n a n a n a m b e re , b o u a r 2 4 j u l 1 9 6 9 m . g ir e t 0 9 6 9 7 7 2 2 3 1 d o m e st ic d o g n a n a n a m b e re , b o u a r l a te a u g 1 9 6 9 m . g ir e t 0 9 6 9 9 0 1 2 4 6 d o m e st ic d o g n a n a n a m b e re , b o u a r a u g 1 9 6 9 m . g ir e t 0 9 6 9 9 6 1 6 5 1 d o m e st ic d o g n a n a n a m b e re , b o u a r a u g 1 9 6 9 m . g ir e t 0 9 6 9 8 7 1 2 d o m e st ic c a t n a n a n a m b e re , b o u a r 2 7 j u l 1 9 6 9 m . g ir e t 0 9 6 9 9 5 3 2 d o m e st ic c a t n a n a n a m b e re , b o u a r a u g 1 9 6 9 m . g ir e t 0 9 6 9 8 5 2 4 d o m e st ic c a t n a n a n a m b e re , b o u a r 0 7 j a n 1 9 7 0 r . l a co tt e 0 9 6 9 8 4 8 4 d o m e st ic c a t n a n a n a m b e re , b o u a r 1 1 j a n 1 9 7 0 r . l a co tt e 0 9 6 9 8 6 1 5 d o m e st ic c a t n a n a n a m b e re , b o u a r 1 8 j a n 1 9 7 0 g . u ile n b e rg 0 9 6 9 9 3 1 3 5 c iv e tt ic tis c iv e tt a 1 4 a p r 1 9 7 1 j. t h a l 0 9 7 1 4 9 2 8 7 p a n th e ra le o 2 6 f e b 1 9 7 1 j. t h a l 0 9 7 1 5 0 d e m o c ra ti c r e p u b li c o f c o n g o 1 1 p a n th e ra le o b o d io 1 5 d e c 1 9 5 0 0 8 6 2 6 4 6 1 g e n e tt a m a cu la ta g a ra m b a p a rk 1 6 s e p 1 9 5 1 h . d e s a e g e r 0 3 6 7 1 1 2 p a n th e ra p a rd u s k a sa i, l u lu a b u rg 1 9 2 5 c . w a e rt 0 4 6 7 1 7 1 k a ta n g a ( n ), m u h ilo 1 9 6 6 j. b a fo rt 1 2 3 7 7 1 1 3 c ro cu ta c ro cu ta 1 2 a p r 1 9 5 1 p . s ch o e m a ke r 0 3 7 4 6 1 1 9 2 l e p ta ilu ru s se rv a l 1 7 a u g 1 9 5 1 j. v . 0 3 7 4 5 4 8 3 l e p ta ilu ru s se rv a l 2 9 s e p 1 9 5 1 h . d e s a e g e r 0 3 7 4 5 2 e g y p t 1 f e lis c h a u s a le xa n d ri a , a m ir iy a r o a d 2 9 j a n 1 9 6 5 i. h e lm y, d . o sb o rn 0 7 8 7 8 9 5 f e lis c h a u s a le xa n d ri a , 1 4 k m s w o f a le xa n d ri a 0 9 n o v 1 9 6 5 i. h e lm y, d . o sb o rn 0 7 8 7 9 1 3 5 6 v u lp e s vu lp e s b e n i s u e f, b e n i s u e f, a l h a ka m n a h 2 0 j a n 1 9 8 2 l o ca l h u n te r 1 2 3 7 6 9 2 3 a rv ic a n th is n ilo tic u s n e st d a q a h liy a , a g a , m in sh a t e l i kh w a 1 2 n o v 1 9 5 3 h . h o o g st ra a l 0 7 8 7 3 2 4 v u lp e s vu lp e s d a q a h liy a , v ill a g e o f t a n b o u l ( 5 m ile s w o f s im b ill a w e in ) 2 4 f e b 1 9 4 7 0 2 5 4 8 0 201 d.a. apanaskevich, i.g. horak & j-l. camicas n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* ♂ ♀ e g y p t (c o n t. ) 1 f e lis s ilv e st ri s (= ly b ic a ) e l w a d i e l g e d e e d , d a kh la o a si s, m u t 2 6 a p r 1 9 7 4 i. h e lm y, s . t a w fik 0 9 4 3 6 6 1 9 c a n is a u re u s f a iy u m , f a iy u m 1 5 a u g 1 9 5 6 h . h o o g st ra a l 0 7 8 7 6 0 1 c a n is a u re u s f a iy u m , f a iy u m ( n e a r) 0 2 f e b 1 9 5 5 h . h o o g st ra a l 0 7 8 7 5 6 4 v u lp e s vu lp e s f a iy u m , ib sh a w a i, a b u d in q a sh 0 9 f e b 1 9 8 3 l o ca l h u n te r 1 2 3 7 6 8 1 2 3 v u lp e s vu lp e s f a iy u m , ib sh a w a i, e l n a zl a 0 9 f e b 1 9 8 3 l o ca l h u n te r 1 2 3 7 7 3 2 4 c a n is a u re u s f a iy u m , ib sh a w a i, q a sr e l g ib a li 0 9 f e b 1 9 8 3 l o ca l h u n te r 1 2 1 7 5 6 1 v u lp e s vu lp e s f a iy u m , k o m o sh im 0 6 f e b 1 9 4 8 h . h o o g st ra a l 0 7 8 7 5 9 1 f e lis c h a u s f a iy u m , k o m o sh im ( 1 m ile n o rt h o f) 2 8 d e c 1 9 5 3 h . h o o g st ra a l 0 7 8 7 6 1 1 1 f e lis c h a u s f a iy u m , t a m iy a , f a n u s 1 2 a p r 1 9 5 4 h . h o o g st ra a l 0 7 8 7 6 3 5 f e lis c h a u s f a iy u m , t a m iy a , f a n u s 1 2 a p r 1 9 5 4 h . h o o g st ra a l 0 7 8 7 6 4 5 4 f e lis c h a u s f a iy u m , t a m iy a , f a n u s 1 2 a p r 1 9 5 4 h . h o o g st ra a l 0 3 4 5 8 3 1 g iz a , a iy a t, k a fr a m m a r h . h o o g st ra a l 0 7 8 7 5 5 2 2 a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , b e n i m a g d u l 1 4 j u n 1 9 5 3 h . h o o g st ra a l 0 7 8 7 3 7 6 f e lis c h a u s g iz a , im b a b a , e l b a ra g il 0 4 n o v 1 9 5 8 h . h o o g st ra a l 0 7 8 7 8 4 1 2 v u lp e s vu lp e s g iz a , im b a b a , g iz za ya 3 0 a p r 1 9 5 4 h . h o o g st ra a l 0 7 8 7 8 5 5 5 a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , k ir d a sa 1 4 f e b 1 9 5 7 h . h o o g st ra a l 0 7 8 7 3 0 3 3 a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , k ir d a sa 1 4 j u n 1 9 5 3 h . h o o g st ra a l 0 7 8 7 3 8 9 + 1 1 + a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , k ir d a sa 2 8 – 2 9 j u n 1 9 5 3 h . h o o g st ra a l 0 7 8 7 3 6 1 a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , k ir d a sa 1 5 j u l 1 9 5 3 h . h o o g st ra a l 0 7 8 7 4 1 1 + 2 + a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , k ir d a sa 1 4 j u n 1 9 5 3 h . h o o g st ra a l 0 5 6 7 5 7 1 a rv ic a n th is n ilo tic u s n e st g iz a , im b a b a , k ir d a sa 0 3 j a n 1 9 5 5 h . h o o g st ra a l 0 7 8 7 3 1 1 v u lp e s vu lp e s g iz a , im b a b a , t a n a sh 1 4 j a n 1 9 6 0 h . h o o g st ra a l 0 7 8 7 6 5 2 a rv ic a n th is n ilo tic u s b u rr o w m in iy a , m a g h a g h a , m a g h a g h a 1 2 m a r 1 9 5 2 h . h o o g st ra a l 0 7 8 7 4 4 1 v u lp e s vu lp e s m in iy a , m a g h a g h a , s a ft 0 1 j a n 1 9 7 9 l o ca l h u n te r 1 2 3 7 7 2 1 c a n is a u re u s m in iy a , m in ya 1 1 j a n 1 9 8 1 l o ca l h u n te r 1 2 3 7 6 7 1 3 7 f e lis c h a u s q a ly u b iy a , e l a m a r e l k u b ra 1 5 j a n 1 9 5 4 h . h o o g st ra a l 0 7 8 7 5 7 4 2 c a n is a u re u s q a ly u b iy a , q a ly u b iy a , s a n a tir , e zb e t ih sa n 0 3 f e b 1 9 6 6 i. h e lm y, d . o sb o rn 0 7 8 7 9 0 4 f e lis c h a u s q e n a , is n a , w a d i n a ss im 0 7 a p r 1 9 5 3 h . h o o g st ra a l 0 7 8 7 6 2 e th io p ia 1 v e g e ta tio n g a m o -g o fa , a rb a m in e h 1 4 j a n 1 9 6 6 h . h o o g st ra a l 0 9 2 7 3 5 5 1 c o lo b u s p o ly ko m o s h a re r, h ir n a v a lle y, d ir e d a w a c o lle g e ( 8 0 k m w o f) 2 1 s e p 1 9 6 2 b . g la ss 0 9 2 7 3 0 1 c a n is m e so m e la s h a re r, r d . b e tw e e n d a ca ta a n d e re r r iv e rs b . g la ss 0 9 2 7 3 2 t a b l e 2 (c o n t. ) 202 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* ♂ ♀ e th io p ia ( c o n t. ) 9 1 ic h n e u m ia a lb ic a u d a h a re r, r d . b e tw e e n d a ca ta a n d e re r r iv e rs 0 4 j u l 1 9 6 2 b . g la ss 0 9 2 7 3 1 1 2 d o m e st ic c a t ilu b a b o r, g a m b e lla j. s . a sh 0 9 2 7 4 1 1 h u m a n ilu b a b o r, g a m b e lla 3 0 j u l 1 9 7 2 j. s . a sh 0 9 2 7 4 2 1 7 1 0 c iv e tt ic tis c iv e tt a k e fa , m e za n t e fa ri 1 0 m a r 1 9 8 0 h .k . l a ll 1 2 3 7 6 0 7 3 v u lp e s sp . k e fa , s o ko ru /d e ke a u g 1 9 7 9 h .k . l a ll 1 2 3 7 7 0 1 9 h ya e n a h ya e n a r o ck v a lle y b . g la ss 0 9 2 7 3 3 1 3 d o m e st ic d o g s h a sh a m a n i 2 5 j u n 1 9 6 1 l .w . t e lle r 0 9 2 7 2 6 1 h u m a n s h o a , a d d is a b a b a 1 1 m a r 1 9 7 0 j. s . a sh 0 9 2 7 4 3 1 “b la ck -t a ile d m o n g o o se ” s h o a , k o ka 2 5 f e b 1 9 7 3 r . t ra u b , j. s . a sh 0 9 2 7 4 7 2 2 1 1 l e p ta ilu ru s se rv a l s h o a , l a ke l a n g o n o r d ( 1 0 m ile s n o f) 2 8 d e c 1 9 7 4 l . s h o ld t 0 9 2 7 4 6 k e n y a 1 6 2 c iv e tt ic tis c iv e tt a c e n tr a l, m u ra n g a , m itu b ir i 0 8 f e b 1 9 5 3 d .g . m a ci n n e s 0 9 5 0 6 6 l ib e ri a 1 1 d o m e st ic d o g b o la h im 1 9 3 0 t . k o lb e 0 4 6 7 1 6 8 5 d o m e st ic c a t h a rb e l r . f o x 0 8 7 9 2 3 m a li 6 4 5 c iv e tt ic tis c iv e tt a k a ye s, n io ro d u s a h e l, l o ra k b a n c 1 2 f e b 1 9 5 6 0 8 4 5 7 8 1 4 2 l e p ta ilu ru s se rv a l k a ye s, n io ro d u s a h e l, l o ra k b a n c 0 6 f e b 1 9 5 6 0 8 9 5 7 9 3 p a n th e ra p a rd u s s ik a ss o , s ik a ss o 2 0 a u g 1 9 5 4 0 8 9 5 7 6 s e n e g a l 1 l e p ta ilu ru s se rv a l c a sa m a n ce , b ig n o n a 2 1 o ct 1 9 4 5 f ro m p .c . m o re l 0 8 8 2 7 8 1 v e g e ta tio n s a n g a lk a m 0 5 a p r 1 9 4 5 l . k a rt m a n 0 2 1 7 9 3 3 f e lis s ilv e st ri s ( = ly b ic a ) s e n e g a l o ri e n ta l, n io ko lo k o b a , b a d i m a r 1 9 5 7 f ro m p .c . m o re l 0 8 8 2 7 7 7 c iv e tt ic tis c iv e tt a s e n e g a l o ri e n ta l, n io ko lo k o b a f e b 1 9 5 6 f ro m p .c . m o re l 0 8 8 2 7 6 9 3 9 l e p ta ilu ru s se rv a l t h ie s, m b o u r, s a n d ia ra 1 8 m a y 1 9 5 6 f ro m p .c . m o re l 0 8 8 2 7 9 s u d a n 1 h ya e n a h ya e n a b a h r e l g h a za l, t ir o l 2 1 j a n 1 9 5 5 e .t .m . r e id 0 9 3 0 1 2 1 3 d o m e st ic c a t b a h r e l g h a za l, w a u o ct 1 9 5 3 s .v .s . 0 9 3 0 2 5 2 1 a rv ic a n th is s p . b u rr o w e q u a to ri a , ju b a , ju b a 1 0 d e c 1 9 5 2 0 9 3 0 1 3 8 c iv e tt ic tis c iv e tt a e q u a to ri a , t o ri t, o b b o 0 9 a p r 1 9 5 0 h . h o o g st ra a l 0 9 3 0 3 1 t a b l e 2 (c o n t. ) 203 d.a. apanaskevich, i.g. horak & j-l. camicas n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* ♂ ♀ s u d a n ( c o n t. ) 1 3 5 c a n is a u re u s e q u a to ri a , t o ri t, t o ri t 2 5 n o v 1 9 4 9 h . h o o g st ra a l 0 9 4 9 5 9 1 4 c a n is a u re u s e q u a to ri a , t o ri t, t o ri t 0 6 d e c 1 9 4 9 0 9 3 0 1 4 2 2 2 c a n is a u re u s e q u a to ri a , t o ri t, t o ri t 0 2 d e c 1 9 4 9 0 9 3 0 1 6 1 c a n is a u re u s e q u a to ri a , t o ri t, t o ri t 0 6 d e c 1 9 4 9 h . h o o g st ra a l 0 9 3 0 1 5 1 1 c iv e tt ic tis c iv e tt a e q u a to ri a , t o ri t, t o ri t 0 8 f e b 1 9 5 1 h . h o o g st ra a l 0 9 3 0 2 3 1 4 d o m e st ic d o g e q u a to ri a , t o ri t, t o ri t 0 4 j a n 1 9 5 2 0 9 3 0 2 4 1 m u n g o s m u n g o e q u a to ri a , t o ri t, t o ri t 1 3 a p r 1 9 5 0 0 9 3 0 1 1 6 p a n th e ra le o e q u a to ri a , t o ri t, t o ri t 1 5 m a r 1 9 5 2 j. o w e n 0 9 3 0 2 8 1 4 3 c a n is m e so m e la s e q u a to ri a , u b o 0 8 m a y 1 9 4 8 0 9 3 0 1 8 1 c a n is a d u st u s u p p e r n ile , m a la ka l ( n e a r) 1 4 m a r 1 9 6 4 h . h o o g st ra a l, s . g a b e r 0 9 4 9 6 0 2 c a n is a d u st u s u p p e r n ile , m a la ka l, m a la ka l ( 7 m ile s n o f) 0 1 f e b 1 9 6 2 h . h o o g st ra a l, s . g a b e r 0 9 4 9 6 1 8 p a n th e ra le o u p p e r n ile , 4 0 m i s o f m a la ka l, a b w o n g 2 7 f e b 1 9 6 1 h . h o o g st ra a l 0 9 2 9 7 8 1 c a n is a d u st u s u p p e r n ile , p a lo ic h , g e lh a k f o re st 2 3 m a y 1 9 6 2 h . h o o g st ra a l, s . g a b e r 0 9 2 8 8 1 1 f e lis s ilv e st ri s (= ly b ic a ) u p p e r n ile , p a lo ic h , p a lo ic h ( 5 m ile s n o f) 2 7 f e b 1 9 6 2 h . h o o g st ra a l, s . g a b e r 0 9 4 9 6 2 2 f e lis s ilv e st ri s u p p e r n ile , p a lo ic h , t ir ( n e a r) 2 3 f e b 1 9 6 1 h . h o o g st ra a l, s . g a b e r 0 9 2 9 7 3 2 1 l e p ta ilu ru s se rv a l u p p e r n ile , p a lo ic h , t ir ( n e a r) 0 7 f e b 1 9 6 1 h . h o o g st ra a l, s . g a b e r 0 9 4 9 5 8 t a n za n ia 1 c a n is m e so m e la s m a ra , s e re n g e ti p la in s, s e ro n e ra 2 5 s e p 1 9 7 4 d . s ch m id t 0 9 5 2 0 4 1 c o n n o ch a e te s ta u ri n u s y e id a , s w a m p s 0 4 o ct 1 9 3 8 l .r . p a d d o ck 1 1 5 7 7 4 u g a n d a 1 1 1 p a n th e ra le o a n ko le , n ya b u sh o zi 2 2 o ct 1 9 6 5 j. m a tt h ys se 1 2 3 7 6 3 1 2 p a n th e ra le o a n ko le , n ya b u sh o zi 0 9 o ct 1 9 6 5 j. m a tt h ys se 1 2 0 3 6 7 7 6 p a n th e ra p a rd u s a n ko le , m b a ra ra 0 9 a u g 1 9 6 5 j. m a tt h ys se 1 2 3 7 6 2 2 p a n th e ra le o a n ko le , m b a ra ra ( 3 5 m ile s s w o f) 1 4 a p r 1 9 6 2 m a jo r p ri ce 0 8 9 5 4 2 2 d o m e st ic d o g b a g a n d a , e n te b b e 2 7 a p r 1 9 6 6 j. m a tt h ys se 0 5 3 8 2 6 7 1 d o m e st ic d o g k a ra m o ja , k a m p a la 0 5 f e b 1 9 4 0 g .h .e . h o p ki n s 1 2 0 3 6 5 3 p a n th e ra p a rd u s k a ra m o ja , m o ro to , l a b w o r 1 6 a u g 1 9 6 5 j. m a tt h ys se 1 2 3 7 6 4 t a b l e 2 (c o n t. ) 204 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* ♂ ♀ u g a n d a ( c o n t. ) 2 k ig e zi , n a ka b a n d e 2 5 o ct 1 9 4 0 1 2 0 3 6 6 1 1 d o m e st ic d o g p a co ta , a sw a , a ch o li 2 1 j u n 1 9 6 6 j. m a tt h ys se 0 5 3 8 3 5 1 0 2 p a n th e ra le o r u w e n zo ri n a tio n a l p a rk 3 1 j u l 1 9 6 9 m .h . w o o d fo rd 0 8 8 6 3 4 3 p a n th e ra le o r u w e n zo ri n a tio n a l p a rk , m w e ya 1 9 j u n 1 9 7 4 m .h . w o o d fo rd 1 2 0 3 6 3 2 1 c iv e tt ic tis c iv e tt a w . m e n g o , e n te b b e a p r 1 9 7 9 m .n . k a is e r 1 2 3 7 6 1 z a m b ia 1 0 1 7 p a n th e ra le o s o u th e rn , n a m w a la 1 9 a u g 1 9 5 1 j. g . m a tt h ys se 0 9 1 2 9 4 1 1 0 d o m e st ic d o g b a ro ts e la n d , k a la b o ( e o f) a p r 1 9 6 5 r .g . ja p p 0 9 1 2 9 7 1 1 f e lis s ilv e st ri s (= ly b ic u s) b a ro ts e la n d , k a la b o ( e o f) 3 0 d e c 1 9 6 4 r .g . ja p p 0 9 1 2 9 6 1 m a st o m ys n a ta le n si s m t. m a ku lu 1 6 j u n 1 9 7 0 m .h . c o lb o 0 9 1 3 0 0 6 c iv e tt ic tis c iv e tt a e a st e rn , l u n d a zi , c h ib e m b e p o n to o n ( 1 0 m i n o f) 0 7 a u g 1 9 6 2 g . c o rb e t, j. i n g le s 0 9 1 3 0 4 1 5 1 c iv e tt ic tis c iv e tt a l o ch in va r ju l 1 9 5 9 1 2 3 7 6 5 1 p o ta m o ch o e ru s p o rc u s s u sa c a m p , c h ip a ta , e a st e rn 2 4 j u l 1 9 8 0 s .g .a . w e a k 1 2 3 7 6 6 z im b a b w e 1 6 c iv e tt ic tis c iv e tt a m a sh o n a la n d s o u th , s a lis b u ry , a tla n tic a f o u n d a tio n r e se a rc h s ta tio n 0 7 j u l 1 9 7 7 r .a .i . n o rv a l 1 0 7 2 7 2 1 3 c iv e tt ic tis c iv e tt a m a sh o n a la n d s o u th , s a lis b u ry , c a lg a ry f a rm 1 6 m a y 1 9 7 7 r .a .i . n o rv a l 1 0 7 0 9 6 3 c iv e tt ic tis c iv e tt a m a sh o n a la n d s o u th , s a lis b u ry , c a lg a ry f a rm 1 4 f e b 1 9 7 7 r .a .i . n o rv a l 1 2 1 5 9 9 6 l e p ta ilu ru s se rv a l m a sh o n a la n d s o u th , s a lis b u ry , c a lg a ry f a rm 0 9 f e b 1 9 7 7 r .a .i . n o rv a l 1 2 1 6 0 3 7 f e lis s ilv e st ri s (= li b yc a ) m a sh o n a la n d s o u th , s a lis b u ry , m a zo e v e te ri n a ry f a rm 0 6 s e p 1 9 7 6 r .a .i . n o rv a l 1 0 3 0 4 7 8 9 l e p ta ilu ru s se rv a l m a sh o n a la n d s o u th , s in o ia 2 5 m a y 1 9 7 7 r .a .i . n o rv a l 1 2 1 5 9 5 * a ll th e c o lle ct io n n u m b e rs a re t h o se o f sp e ci m e n s in t h e u n ite d s ta te s n a tio n a l t ic k c o lle ct io n + r e a re d s p e ci m e n s fr o m e n g o rg e d n ym p h s fo u n d in a rv ic a n th is n ilo tic u s n e st s t a b l e 2 (c o n t. ) 205 d.a. apanaskevich, i.g. horak & j-l. camicas t a b l e 3 h a e m a p h ys a lis ( r h ip is to m a ) e lli p tic a a n d h a e m a p h ys a lis ( r h ip is to m a ) le a ch i, im m a tu re s ta g e s e xa m in e d n o . o f ti c k s h o s t l o c a li ty d a te o f c o ll e c ti o n c o ll e c to r c o ll e c ti o n n o .* n l h a e m a p h y s a li s ( r h ip is to m a ) e ll ip ti c a 1 6 + 3 0 + s o u th a fr ic a ig h 1 6 + 3 0 + d o m e st ic d o g s o u th a fr ic a , p re to ri a 1 3 j u l 2 0 0 5 k . ju n ke r ig h 3 0 + a ci n o n yx ju b a tu s s o u th a fr ic a , h o e d sp ru it 2 1 f e b 2 0 0 6 i. h u b m e r ig h h a e m a p h y s a li s ( r h ip is to m a ) le a c h i 8 + d o m e st ic d o g c e n tr a l a fr ic a n r e p u b lic , n a n a n a m b e re , b o u a r 1 0 o ct 1 9 6 9 t o 1 4 j a n 1 9 7 0 r . l a co tt e 0 9 6 9 9 2 3 0 + d o m e st ic d o g c e n tr a l a fr ic a n r e p u b lic , n a n a n a m b e re , b o u a r 1 6 o ct t o 1 0 d e c 1 9 6 9 r . l a co tt e 0 9 6 9 7 9 3 0 + d o m e st ic d o g c e n tr a l a fr ic a n r e p u b lic , n a n a n a m b e re , b o u a r 0 2 a u g t o 0 3 s e p 1 9 6 9 m . g ir e t 0 9 6 9 8 0 3 0 + d o m e st ic d o g e g yp t, e l w a d i e l g e d e e d , d a kh la o a si s, e l h in d a w 2 0 n o v 1 9 7 2 i. h e lm y 0 7 8 8 0 1 2 + a rv ic a n th is n ilo tic u s n e st e g yp t, g iz a , im b a b a , a u si m s e p 1 9 5 3 h . h o o g st ra a l 0 7 8 7 3 4 3 0 + v u lp e s vu lp e s e g yp t, g iz a , im b a b a , s a ft e l l a b a n 0 6 m a r 1 9 5 8 h . h o o g st ra a l 0 7 8 7 4 9 * a ll th e c o lle ct io n n u m b e rs a re t h o se o f sp e ci m e n s in t h e u n ite d s ta te s n a tio n a l t ic k c o lle ct io n + r e a re d s p e ci m e n s 206 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) table 4 differential diagnosis between haemaphysalis (rhipistoma) elliptica and haemaphysalis (rhipistoma) leachi haemaphysalis (r.) elliptica haemaphysalis (r.) leachi male (fig. 2, 3, 8 and 9 ) 1. longer and broader tick: – length (from palpal apices to posterior margin of conscutum) avg. 3.00 mm – width (of conscutum) avg. 1.47 mm – ratio length to width avg. 2.05 1. shorter and more slender tick: – length (from palpal apices to posterior margin of conscutum) avg. 2.45 mm – width (of conscutum) avg. 1.06 mm – ratio length to width avg. 2.30 2. one or two of first festoons enclosed by marginal groove 2. two or three of first festoons enclosed by marginal groove 3. dorsally median margin of palpal segment ii gradually widening anteriorly from the segment’s mid-length 3. dorsally median margin of palpal segment ii abruptly widening anteriorly from the segment’s mid-length 4. lateral margin of ventral spur on palpal segment ii straight 4. lateral margin of ventral spur on palpal segment ii concave female (fig. 4, 5, 10 and 11) 1. longer and broader tick: – length (from palpal apices to posterior margin of scutum) avg. 1.73 mm – width (of scutum) avg. 1.02 mm – ratio length to width avg. 1.70 1. shorter and more slender tick: – length (from palpal apices to posterior margin of scutum) avg. 1.53 mm – width (of scutum) avg. 0.84 mm – ratio length to width avg. 1.81 2. dorsal cornua shorter, approximately 1/6 length of basis capituli 2. dorsal cornua longer, approximately 1/4 length of basis capituli 3. dorsally median margin of palpal segment ii gradually widening anteriorly at segment’s mid-length 3. dorsally median margin of palpal segment ii abruptly widening anteriorly at segment’s mid-length nymph (fig. 6 and 12) 1. larger (see description) 1. smaller (see description) 2. dorsally median margin of palpal segment ii widening grad ually anteriorly 2. dorsally median margin of palpal segment ii widening sharply anteriorly 3. ventral spur of palpal segment ii broad 3. ventral spur of palpal segment ii narrow 4. denticles of hypostome in files of 7 to 9 (usually 8) 4. denticles of hypostome in files of 5 or 6 larva (fig. 7 and 13) 1. larger (see description) 1. smaller (see description) 2. ventral spur of palpal segment ii broad 2. ventral spur of palpal segment ii narrow 3. ventral spur of palpal segment iii indistinct, fold-like 3. ventral spur of palpal segment iii distinct, triangular 4. denticles in files of 7 or 8 4. denticles in files of 4 to 6 (usually 5) remarks on identification our study has shown that h. (r.) elliptica is an independent species belonging to the h. (r.) leachi subgroup and that it is clearly distinguishable from h. (r.) leachi. however, the value of diagnostic characters varies from stage to stage. the easiest stage to distinguish is the larval. all the larval characters that we have chosen clearly differentiate the larvae of h. (r.) elliptica from those of h. (r.) leachi. males of h. (r.) elliptica are also quite easily distinguishable from those of h. (r.) leachi. the main differentiating characters are the shape of the lateral margin of the ventral spur on palpal segment ii and total size of the ticks. the most obvious character for nymphs is the number of denticles per file on the hypostome, and total size. females are the most difficult to distinguish. the main characters are total size and the size of the dorsal cornua, and because both characters have a metric value, difficult specimens do not have to be excluded during routine examinations. the females of most closely related spe cies within the h. (r.) leachi group are difficult to distinguish interspecifically. however, because of the morphological stability of haemaphysalis species, the size of various structures is of consid207 d.a. apanaskevich, i.g. horak & j-l. camicas erable value for discriminating between all the parasitic stages of closely related species. finally, unpublished molecular data confirm our opinion on the species independency of h. (r.) elliptica. distribution and hosts haemaphysalis (r.) elliptica is present in east and southern africa, and daa and igh have recorded it in the democratic republic of congo, kenya, mozam bique, south africa, tanzania, uganda, zambia and zimbabwe (table 1). jlc adds ethiopia, malawi and rwanda to this list. haemaphysalis (r.) leachi has chiefly been recorded from north (egypt) and east africa south to the north of zimbabwe. judging by collection data this species is probably quite common in central africa. a few collections have been made in west africa. daa and igh record this species from cameroon, central african republic, democratic republic of congo, egypt, ethiopia, kenya, liberia, mali, senegal, sudan, tanzania, uganda, zambia and zimbabwe (table 2). jlc adds burundi, chad and guinea to this list. both species share a large area of sympatry in east africa. the hosts of adult h. (r.) elliptica are various carnivore species, amongst which are the domestic dog, domestic cat, lion, panthera leo, and leopard, panthera pardus (table 1). the hosts of the immature stages are diverse rodent species, and they may very occasionally be present on the same hosts as the adults. the hosts of adult h. (r.) leachi are similar to those of h. (r.) elliptica, namely domestic and wild carnivores (table 2). the immature stages use various rodents and other small mammals as hosts. it will, however, only be possible to determine the actual host range of the immature stages of h. (r.) elliptica and h. (r.) leachi once a taxonomic revision of the whole h. (r.) leachi group has been completed. the adults of both species have been found in a number of collections taken from a single host. both geographic and host sympatry indirectly confirm the specific independency of h. (r.) elliptica and h. (r.) leachi. disease relationships haemaphysalis (r.) elliptica (then referred to as h. leachi) is the vector of babesia canis rossi, the cause of virulent babesiosis in domestic dogs in south africa (lewis, penzhorn, lopez-rebollar & de waal 1996). we are, however, unable to find any records of h. (r.) leachi transmitting babesia canis in egypt. in south africa, h. (r.) elliptica (as h. leachi) has been recorded as transmitting rickettsia conori, resulting in tick bite fever in humans (gear 1954). possibly because of its preference for carnivores, adult h. (r.) elliptica (then recorded as h. leachi) is one of the tick species most frequently collected from humans working in the field (horak, fourie, heyne, walker & needham 2002). acknowledgements we are most grateful to dr j. dunlop, natural history museum of berlin, germany, for the loan of koch’s type specimen of h. (r.) elliptica. dr j.h. oliver jr and dr l. beati, georgia southern university, united states of america, granted permission and assistance to daa, while on a visit to the usa, to examine ticks in the united states national tick collection. dr a. latif and ms h. heyne, onderstepoort veterinary institute, south africa, granted permission and assistance to daa to study the type series of h. leachi var. humerosoides in the gertrud theiler tick museum. dr j.b. walker kindly spent several hours discussing our ideas on the relationship of the two species. this study was made possible by the award of a postdoctoral fellowship to daa by the claude leon foundation. a visit to the us national tick collection by daa and the publication of this study have been facilitated through the integrated consortium on ticks and tick-borne diseases (icttd3), financed by the international cooperation program of the european union through coordination action project no. 510561. references bedford, g.a.h. 1934. south african ticks. part i. onderstepoort journal of veterinary science and animal industry, 2:49– 99. camicas, j.-l., hoogstraal, h. & el kammah, k.m. 1972. notes on african haemaphysalis ticks. viii. h. (rhipistoma) moreli sp. n., a carnivore parasite of the h. (r.) leachi group (ixodoidea: ixodidae). journal of parasitology, 58:1185– 1196. camicas, j.-l., hoogstraal, h. & el kammah, k.m. 1973. notes on african haemaphysalis ticks. xi. h. (rhipistoma) punctaleachi sp. n., a parasite of west african forest carnivores (ixodoidea: ixodidae). journal of parasitology, 59:563– 568. camicas, j.-l., hoogstraal, h. & el kammah, k.m. 1983. notes on african haemaphysalis ticks. xiv. description of adults of h. (rhipistoma) paraleachi sp. n., a carnivore para208 redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844) site of the h. 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journal of veterinary research, 59:285–292. theiler, gertrud 1962. the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region). project s 9958. report to the director of veterinary services, onderstepoort. 260 pp. mimeographed. theiler, gertrud 1943. notes on the ticks off domestic stock from portuguese east africa. estação. anti-malárica de lourenço marques: imprensa nacional de moçambique. abstract introduction case report results and discussion conclusion acknowledgements references about the author(s) katalina cruz faculty of veterinary medicine, ross university school of veterinary medicine, basseterre, st kitts and nevis, west indies department of parasitology, faculty of sciences, institute of biotechnology, biochemistry and molecular parasitology, university of granada, granada, spain tatiana m. corey st kitts biomedical research foundation and virscio, st kitts and nevis, lower bourreyaeu, west indies michel vandenplas faculty of veterinary medicine, ross university school of veterinary medicine, basseterre, st kitts and nevis, west indies maría trelis department of parasitology, faculty of pharmacy, university of valencia, valencia, spain research unit on endocrinology, nutrition and clinical dietetics, health research institute la fe, valencia, spain antonio osuna department of parasitology, faculty of sciences, institute of biotechnology, biochemistry and molecular parasitology, university of granada, granada, spain patrick j. kelly faculty of veterinary medicine, ross university school of veterinary medicine, basseterre, st kitts and nevis, west indies citation cruz, k., corey, t.m., vandenplas, m., trelis, m., osuna, a. & kelly, p.j., 2021, ‘case report: control of intestinal nematodes in captive chlorocebus sabaeus’, onderstepoort journal of veterinary research 88(1), a1903. https://doi.org/10.4102/ojvr.v88i1.1903 research communication case report: control of intestinal nematodes in captive chlorocebus sabaeus katalina cruz, tatiana m. corey, michel vandenplas, maría trelis, antonio osuna, patrick j. kelly received: 09 sept. 2020; accepted: 12 apr. 2021; published: 28 may 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract there are limited data on the efficacy of antiparasitic treatments and husbandry methods to control nematode infections in captive populations of african green monkeys (agms), chlorocebus sabaeus. in faecal egg count (fec) tests, 10 of the 11 (91%) adult male agms captured from the large feral population on the island of st kitts had evidence of nematode infections, mostly capillaria (8/11, 73%), trichuris trichiura (7/11, 64%) and strongylid species (7/11, 64%) specifically (hookworm and trichostrongylus, 50/50), but also strongyloides fuelleborni (1/11, 9%). when kept in individual cages with cleaning and feeding regimens to prevent reinfections and treated concurrently with ivermectin (300 µg/kg, given subcutaneously) and albendazole (10 mg/kg, given orally) daily for 3 days, 60% (6/10) of the agms were negative at a follow-up fec at 3 months and by fec and necropsy at the end of the study 5–8 months later. one monkey appeared to have been reinfected with t. trichiura after being negative by fec at 3 months post-treatment. four agms were positive for t. trichiura at the 3 month fec follow-up but were negative at the end of the study after one further treatment regimen. although initially being cleared of capillaria following treatment, three agms were found to be infected at the end of the study. the ivermectin and albendazole treatment regimen coupled with good husbandry practices to prevent reinfections effectively controlled nematode infections in captive agms. keywords: capillaria; trichuris; hookworm; trichostrongylus; strongyloides; albendazole; ivermectin; captive; african green monkeys. introduction chlorocebus sabaeus, african green monkeys (agms) of the cercopithecidae family, widely found in africa, were introduced onto the caribbean island of st kitts in the 17th century (mcguire 1974). there are limited published data on the parasitic burden of the vast (perhaps 50 000; dore, gallagher & mill 2021 – unpublished) feral population of agms, which commonly come into contact with people and may play a role in the epidemiology of human zoonotic parasitic infections common on the island (berger 2020). incomplete caribbean studies have reported island agms to be infected with trichuris trichiura (yao et al. 2018), strongyloides sp. (gallagher et al. 2019; ritchie et al. 1967), primasubulura sp. (cameron 1930) and schistosoma mansoni (cameron 1928). there are limited data on effective anthelmintic treatments for agms and husbandry recommendations to control parasitic infections in captive research animals. nonhuman primate research facilities require effective parasite treatment and prevention protocols to minimise infections that may confound research outcomes and cause unnecessary stress, which lowers the quality of life of study animals. ritchie et al. (1967) reported that thiabendazole [two 50 mg/kg doses, po 14 days apart] significantly reduced or eliminated strongyloides eggs in the faeces. kagira et al. (2011) reported that a 3-day regimen of albendazole (7 mg/kg, po) and ivermectin (300 µm/kg, sq) reduced the faecal egg counts (fec) of t. trichiura and strongyles consistently by 100% after 7, 14 and 28 days. chapman et al. (2016) reported that two doses of ivermectin (300 µg/kg, po) 5 days apart resulted in a 100% cure rate for nematodes in wild agms, with no eggs found in the faeces 1 month post-treatment. unfortunately, the evaluation of nematode infections is challenging because of inadequate data on the drug efficacy in agms and the lack of studies investigating prolonged follow-up, which is needed to detect confounding factors (autoinfection, reinfection, larval transplacental or colostrum transmission, dormant stages and autoinfection resulting from parthenogenesis) (reichard et al. 2017). furthermore, fecs can be unreliable, and studies that enable necropsy evaluations are relevant in the absence of a ‘gold standard’ (geary et al. 2010). in this report, we provide a more complete description of the nematode parasites present in agms on st kitts and evaluate a treatment protocol and husbandry practices to control infections in captive agms. case report animals and facilities the st kitts biomedical research foundation (skbrf) and translational research affiliate virscio, inc. are american association of accredited laboratory animal care (aaalac)-accredited biomedical research facilities for integrated preclinical research and development with a multidisciplinary non-human primate focus. the facility houses around 1000 c. sabaeus study animals according to regulations in the guide for the care and use of laboratory animals (national research council of the national academies 2011). the convenience sample of apparently healthy agms used in our study were enrolled in other terminal studies that had been approved by the institutional animal care and use committee (approval number ac18175) and conducted in 2019. these studies did not involve procedures that would have influenced the parasitological status of the animals. eleven wild-caught adult male agms were housed in individual stainless steel cages that meet non-human primate requirements (national research council of the national academies 2011) for the duration of their study period. the cages were washed down with water twice daily, and every 2 weeks they were sanitised using a combination of steam (82 °c) and a quaternary-based disinfectant (consume eco-lyzer, consume nature’s way, spartan chemical company inc., ohio, maumee, united states [us]), or an accelerated hydrogen peroxide foam (peroxigard, virox technologies inc., oakville, ontario, canada). adenosine triphosphate bioluminescence testing of randomly selected dry surfaces was used to determine the sanitation effectiveness. the main food provided was monkey chow (envigo teklad 8773 primate biscuits, indiana, indianapolis, us) once a day supplemented with seasonal produce sourced locally. the produce was provided for both nutrition and as an enrichment activity for the animals’ well-being, and it was washed first if evidently soiled. water, provided ad libitum with lixit water valves, was filtered (neo-pure ps-27097-05 9 3/4 inch, 5 microns, neologic solutions, greenville, south carolina, us) and treated with ultraviolet light (sanitron ultraviolet water purifiers, atlantic ultraviolet corporation, hauppauge, new york, us). the water lines were sanitised monthly with a sodium hypochlorite flush delivered by an edstrom automated watering system (avidity science, waterford, wisconsin, us). treatments the deworming regimen was based on a report from kenya (kagira et al. 2011) and consisted of ivermectin (300 µg/kg, sq; noromectin, norbrook, newry, co. down, northern ireland) and albendazole (ival) (10 mg/kg, po by nasogastric or orogastric gavage; valbazen, zoetis, kalamazoo, michigan, us) daily for 3 days, following chemical restraint (ketamine hcl, 8 mg/kg, bioniche pharma usa llc, us, and xylazine, 1.6 mg/kg, anased, akorn inc., lake forest, illinois, us). the animals were returned to their cages and monitored by cage-side observation twice a day for the duration of the study period. no side effects were observed post-treatment. the treatment was repeated if an animal had a positive fec result during the routine 3-month screening. monitoring of treatments day 0 and 3 month follow-up fecs were carried out on faeces collected directly from the cages. a final fec and counts of adult t. trichiura in the large intestine were performed at routine necropsies 6 or 8 months after the initial ival treatment. for the recuperation of adult nematodes, the large intestine was carefully examined whilst removing the ingesta with a circular rubbing motion, washed with 0.9% saline, filtered through a 100-µm sieve and examined for nematodes under a stereomicroscope (7× – 10× magnification). faecal analysis and morphologic identification for fecs, aliquots (2 g) of uncontaminated faeces were collected from the cage’s under-tray and analysed within 48 h using the double centrifugation technique (5 min, 500 g) with sheather’s sugar solution (spg 1.28). eggs were identified to the species level whenever possible using described morphological characteristics (modrý et al. 2015) (table 1 and figure 1). figure 1: morphologic identification of nematode egg species found (measurements in micrometres). table 1: morphologic identification guidelines followed for egg identification. ethical considerations the african green monkeys used in our study were enrolled in other terminal studies that had been approved by the institutional animal care and use committee of the st kitts biomedical research foundation. approval to conduct the study was also obtained from st. kitts biomedical research foundation, virscio and axion research foundation, biomedical research of the central nervous system (iacuc ac18175). results and discussion ten of the eleven (91%) wild-caught adult c. sabaeus study subjects, all male, employed in the study were infected with a variety of nematodes: capillaria (8/11, 73%), t. trichiura (7/11, 64%), strongylids (7/11, 64%; hookworms and trichostrongylus [50/50]) and strongyloides fuelleborni (s. fuelleborni) (1/11, 9%) (tables 2 and 3). whilst the latter species have been described in agms in africa previously (chapman et al. 2016; gallagher et al. 2019; yao et al. 2018), our finding of capillaria on the island is of note as eggs of this genus have not previously been reported in the faeces of agms. table 2: faecal egg count results and adult trichuris trichiura found at necropsy in animals treated with ivermectin and albendazole. table 3: faecal egg count results and adult trichuris trichiura found at necropsy in animals treated with ivermectin and albendazole. infections, then, might have only been acquired in the 300 years since agms were introduced onto st kitts. unfortunately, we could not determine the capillaria species involved as egg morphology alone cannot be used for speciation, and local experience is that no adults have been reported in the necropsy findings of agms on the island of st. kitts. overall, the ival treatment regimen together with the husbandry measures in place in our study were relatively successful at controlling nematode infections in captive agms. following a single ival treatment, 6/10 (60%) of the infected agms were negative for nematodes 6–8 months later as confirmed by fec and at necropsy (table 2). a second ival treatment given to the four agms with persistent t. trichiura infections 3 months after the first treatment, resulted in the elimination of the t. trichiura 3–5 months later as evidenced by negative fecs and necropsy findings (table 3). although apparently cleared of capillaria following the first ival treatment, three of these four agms had capillaria eggs in their faeces at necropsy 3–5 months after a second treatment. the apparent treatment failures with t. trichiura and capillaria described above warrant further comment. we suspect that the four agms that had t. trichiura eggs 3 months after the first treatment with ival most likely had infections in the prepatent period, which might exceed 12 weeks in humans (bundy & cooper 1989). it is also possible that the animals became infected whilst in captivity, which is the most likely explanation for the single agm (animal 7; table 2) that was negative for t. trichiura 3 months after the first ival treatment but was positive for adults and eggs at necropsy after 8 months. trichuris eggs can remain inactive on surfaces for years (bundy & cooper 1989; reichard et al. 2017), and although the agm cages in our study were cleaned and disinfected regularly, trichuris eggs are difficult to inactivate (bundy & cooper 1989). it is of note that six of these seven animals that were negative for fec 3 months after the one ival treatment were still negative 6–8 months post-treatment. this indicates that the hygiene measures in use in our facility were mostly effective at preventing reinfection. how four agms became positive for capillaria following the apparent resolution of infection after the first ival treatment is unclear; without being able to determine the infecting specie/s, it is not possible to determine whether these agms had been in the prepatent period during treatment, were autoinfected, reinfected or had false-negative test results previously. the main problem with the ival treatment regimen is the requirement for three consecutive sedations for albendazole administration; such repeated dosing, however, is needed for effective ovicidal and larvicidal activity (martin, robertson & bjorn 1997). further, this also enabled three administrations of ivermectin, which helps overcome the problem that single doses perhaps only temporarily inhibit the motion and feeding of nematodes instead of causing their death or expulsion (martin et al. 1997). conclusion african green monkeys can harbour several nematode species, and effective anthelmintic protocols are needed to ensure the optimal health and well-being of animals in captivity and to prevent zoonotic infections in handlers and local people. the ival treatment protocol and husbandry practices we describe would appear to be effective in controlling nematode infections in captive agms. acknowledgements the authors thank the st kitts biomedical research foundation and virscio for providing the samples that were analysed for this case report and monica gozalbo monfort and shamara welcome for their technical assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions k.c. planned the report, assisted with sample collection, performed the data entry and analysis, and took the lead in writing the manuscript. t.m.c. planned the report, supervised the treatments and sample collection, and critically reviewed the manuscript. m.v., a.o. and m.t. contributed to the student supervision and critically reviewed the manuscript. p.j.k. conceived and planned the report, supervised the execution of the study, and contributed to the interpretation of the results and writing of the manuscript. funding information funding was provided by the ross university school of veterinary medicine via the one health center for zoonoses and tropical veterinary medicine. data availability the authors confirm that the data supporting the findings of this study are available within the article. the raw data were generated at the st kitts biomedical research foundation and virscio. derived data supporting the findings of this study are available from the corresponding author (k.c.) on request. disclaimer the views expressed in this report are the authors’ own and not an official position of the institution or funder. references berger, d.s., 2020, infectious diseases of saint kitts and nevis, gideon informatics inc., los angeles, ca. bundy, d.a.p. & cooper, e.s., 1989, ‘trichuris and trichuriasis in humans’, advances in parasitology 28, 107–173. https://doi.org/10.1016/s0065-308x(08)60332-2 cameron, t.w.m., 1928, ‘a new definitive host for schistosoma mansoni’, journal of helminthology 6(4), 219–222. https://doi.org/10.1017/s0022149x00002364 cameron, t.w.m., 1930, ‘the species of subulura molin in primates’, journal of helminthology 8(1), 49. https://doi.org/10.1017/s0022149x00002510 chapman, c.a., friant, s., godfrey, k., liu, c., sakar, d., schoof, v.a.m. et al., 2016, ‘social behaviours and networks of vervet monkeys are influenced by gastrointestinal parasites’, plos one 11(8), e0161113. https://doi.org/10.1371/journal.pone.0161113 dore, k.m., gallagher, c. & mill, c.a., 2021, ‘estimation of green monkey population size in st. kitts using telemetry and home range analysis’, unpublished manuscript. gallagher, c., beierschmitt, a., cruz, k., choo, j. & ketzis, j., 2019, ‘should monkeys wash their hands and feet: a pilot-study on sources of zoonotic parasite exposure’, one health 7, 100088. https://doi.org/10.1016/j.onehlt.2019.100088 geary, t.g., woo, k., mccarthy, j.s., mackenzie, c.d., horton, j., prichard, r.k. et al., 2010, ‘unresolved issues in anthelmintic pharmacology for helminthiases of humans’, international journal for parasitology 40(1), 1–13. https://doi.org/10.1016/j.ijpara.2009.11.001 kagira, j.m., mulei, i., oluoch, g., waititu, k., maingi, n. & ngotho, m., 2011, ‘high efficacy of combined albendazole and ivermectin treatment against gastrointestinal nematodes in vervet monkeys and baboons’, scandinavian journal of laboratory animal science 38(3), 187–193. martin, r.j., robertson, a.p. & bjorn, h., 1997, ‘target sites of anthelmintics’, parasitology 114(suppl 7), s111–s124. https://doi.org/10.1017/s0031182097001029 mcguire, m., 1974, ‘the st. kitts vervet’, contributions to primatology, vol. 1, karger, basel. modrý, d., petrželková, k.j., kalousová, b. & hasegawa, h., 2015, parasites of african great apes, atlas of coproscopic diagnostics, hpi-lab, brno. reichard, m.v., thomas, j.e., chavez-suarez, m., cullin, c.o., white, g.l., wydysh, e.c. et al., 2017, ‘pilot study to assess the efficacy of ivermectin and fenbendazole for treating captive-born olive baboons (papio anubis) coinfected with strongyloides fülleborni and trichuris trichiura’, journal of the american association for laboratory animal science 56(1), 52–56. ritchie, l.s., knight, w.b., oliver-gonzalez, j., frick, l.p., morris, j.m. & croker, w.l., 1967, ‘schistosoma mansoni infections in cercopithecus sabaeus monkeys’, the journal of parasitology 53(6), 1217–1224. https://doi.org/10.2307/3276683 national research council of the national academies, 2011, guide for the care and use of laboratory animals, 8th edn., national academies press, washington, dc. yao, c., walkush, j., shim, d., cruz, k. & ketzis, j., 2018, ‘molecular species identification of trichuris trichiura in african green monkey on st. kitts, west indies’, veterinary parasitology: regional studies and reports 11, 22–26. https://doi.org/10.1016/j.vprsr.2017.11.004 emikpe_pp101-103.qxd indigenous chickens (gallus gallus domesticus) are raised in many subsistence households in africa and asia (spradbrow 1993). they serve as an important source of animal protein to the rural poor (gueye 1998). most of them are kept in free-range systems and scavenge for food. it is widely believed that they act as potential reservoirs for important poultry diseases (bouzoubaa, lemainguer & bell 1992). various investigations have been carried out on important poultry diseases affecting indigenous chickens in africa: viral (adene 1983; mushi, binta, chabo, hera, thibanyene & mkana 2001; ohore, ozegbe, emikpe & okojie 2003), mycoplasmal (mushi, binta, chabo, mathiao & ndebele 1999), bacterial (mdegela, msoffe, waihenya, kasanya, mkambo, minga & olsen 2002; ohore, ozegbe, emikpe & oluwayelu 2002) and parasitic infections (magbool, ahmed & raza 1998) but there is a dearth of information on chicken infectious anaemia (wicht & maharaj 1998) especially in indigenous chickens. chicken infectious anaemia (cia) is a circoviral disease of young chickens characterized by aplastic anaemia, generalized lymphoid atroply and subsequent immunosuppression (bulow & schat 1997). it has been widely reported in major chicken-producing countries (yuasa, taniguchi & yoshida 1979; bulow, fuchs, vielitz & landgraf 1983; rosenberger & cloud 1989; mcnulty, connor, mcneilly, mclaughlin & kirkpatrick 1990; firth & imai 1990; zhou, shen, yang, han, wei, xiao & zhou 1997) and is associated with high morbidity and relatively low mortality (mcnulty, connor, mcneilly, kirkpatrick & mcferran 1988) except with simultaneous infections of marek’s disease virus or infectious bursal disease virus (yuasa, taniguchi, noguchi & yoshida 1980). 101 onderstepoort journal of veterinary research, 72:101–103 (2005) research communication serological evidence of chicken anaemia virus infection in nigerian indigenous chickens b.o. emikpe*, d.o. oluwayelu, o.g. ohore, o.a. oladele and a.t. oladokun faculty of veterinary medicine, university of ibadan, ibadan, nigeria abstract emikpe, b.o., oluwayelu, d.o., ohore, o.g., oladele, o.a. & oladokun, a.t. 2005. serological evidence of chicken anaemia virus infection in nigerian indigenous chickens. onderstepoort journal of veterinary research, 72:101–103 serum samples from 20 out of 180 (11.1 %) apparently healthy nigerian indigenous chickens were negative for antibodies against chicken anaemia virus using the enzyme-linked immunosorbent assay (elisa). of the 160 positive sera (88.9 %), 12 (7.5 %) had titres ranging from 1 500–3 000, 46 (28.8 %) had titres from 3 000–5 000 while 102 (63.8 %) had titres between 5 000–11 000. the overall mean titre value was 5 845 ± 2 402. this appears to be evidence of a natural outbreak of the infection since the chickens had no history of vaccination against any poultry disease. keywords: antibodies, chicken infectious anaemia, enzyme-linked immunosorbent assay * author to whom correspondence is to be directed. e-mail: banabis2001@yahoo.com accepted for publication 19 january 2005—editor a total of 180 apparently healthy nigerian indigenous chickens aged between 4 and 12 months from four communities—apata, odo-ona, university of ibadan and yemetu—in ibadan, oyo state, nigeria were used. ibadan, located in the southwest of nigeria, has the highest commercial poultry activity in the country with indigenous birds transported in from the different geographic zones of the country. many residents of the city keep small flocks of indigenous chickens, mainly extensively, for domestic use. blood samples were collected from 36 flocks, with one of five birds being randomly selected per household and bled via the jugular vein. serum samples were dispensed in 0.5 ml aliquots in tubes and stored at –20 °c until used. antibodies to chicken anaemia virus (cav), if present, were detected and quantified using the enzymelinked immunosorbent assay as described by mcnulty et al. (1988) and todd, mackie, mawhinney, connor, mcneilly & mcnulty (1990) using commercial kits (kirkgaard & perry laboratory, usa). a universal microplate reader (biotek instrument inc. highland park, united states of america) read the plates at 405 nm wavelength. according to the manufacturer, a titre of 1 500 and above was regarded as positive. a point prevalence of 88.9 % (160/180) was obtained in the chickens. in the four locations, the prevalence ranged from 80– 96 %. the mean titre values for positive reactors were 5 546.6 ± 2 541.4 for the university, 4 649.7 ± 1 880.3 for yemetu, 6 948 ± 3 093 for odo-ona and 6 245.8 ± 2 093.5 for apata communities, respectively, while the overall mean titre value was 5 845 ± 2 402. the mean titre distribution of positive samples for cav antibody revealed that 56.7% (102/180) had a titre of more than 5 000 while 25.5 % (46/180) had titres between 3 000 and 5 000 and 6.7 % had titres from 1 500–3 000. there are no routine vaccination programmes against cia in commercial or indigenous chicken production systems in nigeria. recently, owoade et al. (2004) reported the serologic detection of cia in commercial poultry in nigeria. the present study revealed a high prevalence of cav infection in indigenous chickens in ibadan, nigeria. a higher percentage (63.8 %) of the chickens had titres greater than 5 000, which is quite remarkable and is an indication of repeated exposure to the virus. the titres obtained in this study could only have been acquired from natural infection as the chickens had not been vaccinated against any disease. the minimum age of the birds was 4 months. therefore, the presence of maternally-derived antibodies (ma) can be ruled out because in chickens these wane by 3–4 weeks of age (mcnulty et al. 1988). this is the first report on the prevalence of antibodies to cav in nigerian indigenous chickens. the possibility of false positive results is extremely low since elisa is highly specific and sensitive and has minimal non-specific binding reaction (todd et al. 1990). the uncontrolled movement of these chickens as well as their scavenging nature predisposes them to cav infection as the virus is known to persist in the environment (bulow & schat 1997). the high prevalence (88.9 %) of antibodies against cav obtained in this study, coupled with the earlier report of cav antibody detection in commercial chickens, indicates the possible emergence of a relatively new disease problem in the nigerian poultry industry. efforts to isolate and characterize the virus from nigerian chickens are therefore imperative. references adene, d.f. 1983. serological survey of marek’s disease in exotic and local chickens in nigeria. tropical veterinarian, 1: 138–140. bouzoubaa k., lemainguer, k. & bell, j.g. 1992. village chickens as a reservoir of salmonella pullorum and salmonella gallinarum in morocco. preventive veterinary medicine, 12:95–100. bulow, v.v., fuchs, b., vielitz, e. & landgraf, h. 1983. early mortality syndrome of chickens after dual infection with marek’s disease virus (mdv) and chicken anemia agent (caa). zentralblatt für veterinärmedizin, b 30:742– 750. bulow, v.v. & schat, k.a. 1997. chicken infectious anemia, in diseases of poultry, edited by b.w. calnek & h.j. barnes. ames, iowa: iowa state university press. firth, g.a. & imai, k. 1990. isolation of chicken anaemia agent from australian poultry. australian veterinary journal, 67: 301–302. gueye, e.f. 1998. village egg and fowl meat production in africa. world poultry science journal, 54:73–87. magbool, a., ahmed, m. & raza, a. 1998. prevalence of helminth parasites of poultry under different managemental conditions. journal faculty of veterinary medicine university of tehran, 53:102–103. mcnulty, m.s., connor, t.j., mcneilly, f., kirkpatrick, k.s. & mcferran, j.b. 1988. a serological survey of domestic poultry in the united kingdom for antibody to chicken anaemia agent. avian pathology 17:315–324. mcnulty, m.s., connor, t.j., mcneilly, f., mclaughlin, m.f. & kirkpatrick, k.s. 1990. preliminary characterization of isolates of chicken anaemia agent from the united kingdom. avian pathology, 19:67–73. mdegela, r.h., msoffe, p.l.m., waihenya, r.w., kasanya, j.c., mkambo, m.m.a., minga, u.m. & olsen, 102 chicken anaemia virus infection in nigerian indigenous chickens j.e. 2002. comparative pathogenesis of experimental infection with salmonella gallinarum in local and commercial chickens. tropical animal health & production, 34:195–204. mushi, e.z., binta, m.g., chabo, r.g., mathiao, m. & ndebele, r.t. 1999. detection of mycoplasma gallisepticum & mycoplasma synoviae antibodies in the sera of indigenous chickens by rapid serum agglutination test at minopane gaborone, botswana. onderstepoort journal of veterinary research, 66:333–334. mushi, e.z., binta, m.g., chabo, r.g., hera, j.m.k., thibanyene, k.m. & mkana, j. 2001. antibodies to newcastle disease virus in the sera of indigenous chicken in oodi, kgatleng, botswana. onderstepoort journal of veterinary research, 68:69–70. ohore, o.g., ozegbe, p.c., emikpe, b.o. & okojie, v.e. 2003. a survey of antibodies to newcastle disease virus in apparently healthy adult nigerian indigenous chickens (gallus gallus domesticus) in ibadan using elisa. african journal of clinical and experimental microbiology, 3:38–40. ohore, o.g., ozegbe, p.c., emikpe, b.o. & oluwayelu, d.o. 2002. the prevalence of fowl typhoid antibodies in indigenous nigerian chickens (gallus gallus domesticus). bulletin of animal health & production in africa, 50:63–65. owoade, a.a., oluwayelu, d.o., faghohun, o.a, ammerlaan, w., mulders, m.n. & muller, c.p. 2004. serologic evidence of chicken infectious anemia in commercial chicken flocks in southwest nigeria. avian diseases, 48:202–205. rosenberger, j.k. & cloud, s.s. 1989. the isolation and characterization of chicken anemia agent (caa) from broilers in the united states. avian diseases, 33:707–713. spradbrow, p.b. 1993. newcastle disease in village chickens. poultry science review, 5:57–96. todd, d., mackie, d.p., mawhinney, k.a., connor, t.j., mcneilly, f. & mcnulty, m.s. 1990. development of an enzyme-linked immunosorbent assay to detect serum antibody to chicken anemia agent. avian diseases, 34:359– 363. wicht, j.v. & maharaj, s.b. 1993. chicken anaemia agent in south africa. the veterinary record, 133:147–148. yuasa, n., taniguchi, t. & yoshida, i. 1979. isolation and some characteristics of an agent inducing anemia in chicks. avian diseases, 23:366–385. yuasa, n., taniguchi, t., noguchi, t. & yoshida, i. 1980. effect of infectious bursal disease virus infection on incidence of anemia by chicken anemia agent. avian diseases, 24:202–209. zhou, w., shen, b., yang, b., han, s., wel, l., xiao, b. & zhou, j. 1997. isolation and identification of chicken infectious anemia virus in china. avian diseases, 41:361–364. 103 b.o. emikpe et al. garijo_153œ156.qxd introduction the interest in the parasitoses of wildlife must be related both to the increase in the number of game reserves and to the potential transmission of these parasites to domestic animals. most of the available information is related to artiodactyla, but little is known about that concerning giraffes (round 1968; pester & laurence 1974; boomker, horak & de vos 1986; krecek, boomker, penzhorn & scheepers 1990; fukomoto, uchida, ohbayashi, ikebe & sasano 1996). the study of animals that have died in accidents or from other causes provides useful opportunities for advancing the knowledge of the parasites of artiodactylids. this is of particular importance in the case of the giraffe, a species which is considered to be significant in european zoos. this study provides data of parasites found in this species living in spain and describes the presence of worms not usually found in giraffes. materials and methods during november 2002, a 4-year-old pregnant (3rd month of gestation) female cape giraffe (giraffa camelopardalis giraffa) died from an unknown cause in the aitana zoo, alicante, in the southeast of spain. no clinical signs were observed. the feeding of the giraffes in the zoo is based on quercus rotundifolia, daphne gnidium and pinus halepensis and lucerne is supplied in limited quantities. both natural and artificial water resources are available to all animals. 153 onderstepoort journal of veterinary research, 70:153–156 (2004) research communication helminths in a giraffe (giraffa ccamelopardalis ggiraffa) from a zoo in spain m.m. garijo, j.m. ortiz and m.r. ruiz de ibáñez department of parasitology, faculty of veterinary science, university of murcia 30100 espinardo, murcia, spain abstract garijo, m.m., ortiz, j.m. & ruiz de ybáñez, m.r. 2004. helminths in a giraffe (giraffa camelopardalis giraffa) from a zoo in spain. onderstepoort journal of veterinary research, 71:153–156 a pregnant female cape giraffe (giraffa camelopardalis giraffa) died from an unknown cause in the aitana zoo, alicante, spain. neither clinical signs nor macroscopic lesions were observed at necropsy. the alimentary tract was removed and examined for parasites. a total of 2 724 nematodes were found, including camelostrongylus mentulatus, trichostrongylus axei, ostertagia ostertagi, teladorsagia circumcincta, teladorsagia trifurcata, marshallagia marshalli, trichostrongylus vitrinus, trichostrongylus colubriformis, spiculopteragia asymmetrica and trichuris giraffae. only t. giraffae and c. mentulatus have been previously reported from giraffes. the other nematodes are common in mouflons, fallow and red deer, which can usually be found in the same paddock as the giraffes in aitana zoo. although its occurrence is unusual in this host, c. mentulatus was the most abundant nematode in our giraffe. this parasite has been related to disease, and even death, in several wild ruminants. keywords: gastrointestinal nematodes, giraffa camelopardalis giraffa, helminths, spain accepted for publication 10 november 2003—editor the dead animal was sent to the veterinary science faculty, university of murcia, where it was necropsied. no macroscopic lesions were observed. the animal was in good condition as adipose tissue was present around the small and large intestines and the kidneys. the alimentary tract was removed and examined for parasites according to the method described by ortiz, ruiz de ybáñez, garijo, goyena, espeso, abaigar & cano 2001. in brief, the abomasum, small and large intestines were processed separately by scraping the mucosal surfaces and washing the material so gained as well as the gut contents through a series of mesh screens, the final one of which had apertures 0.3 mm in size. the material remaining on the sieves was collected separately and fixed in 10 % formalin. each sample, divided into smaller portions, was examined under a stereoscopic microscope and the nematodes present were removed. adult male parasites were cleared in lactophenol for 48 h, and identified with the aid of the keys of skrjabin, shikhobalova, schulz, popova, boev & delyaure (1961) and durette-desset (1983, 1989). representative specimens of the nematodes recovered are deposited in the faculty of veterinary science, university of murcia’s parasite collection. results and discussion a total of 2 724 nematodes were found (1 158 males and 1 566 females). ten nematode species were recovered and neither trematodes nor cestodes were found. the abomasum yielded a total of 1 109 helminths (593 females and 516 males). the study of the males gave the following distribution: camelostrongylus mentulatus 284 trichostrongylus axei 101 ostertagia ostertagi 84 teladorsagia circumcincta 35 marshallagia marshalli 4 trichostrongylus vitrinus 3 spiculopteragia asymmetrica 3 teladorsagia trifurcata 2 the small intestine harboured 1 601 nematodes, 967 females and 634 males, comprising 587 t. vitrinus and 47 trichostrongylus colubriformis. fourteen worms were found in the large intestine (six females and eight males). all those males were identified as trichuris giraffae. little information is available in the literature on the parasites of giraffes, and all refer to those recovered from animals living in national parks. krecek et al. (1990), in a study on six animals in the etosha national park, namibia, recorded parasite loads ranging from 18 to 531. these numbers are much lower than those obtained in the present work. it should be mentioned that keeping the animals in paddocks, as the giraffe in this study was, makes re-infection easier. on the other hand, boomker et al. (1986) recorded 2 621 and 19 157 helminths, respectively, in two giraffes in the kruger national park, south africa, a much larger number than recorded for the animals from etosha. krecek et al. (1990) attributed these differences to the low rainfall at etosha national park. of the helminths recovered in our study only t. giraffae and c. mentulatus are reported as occurring in giraffe (round 1968; fukumoto et al. 1996). the remaining species are common nematodes in other ruminant species but constitute the first records for giraffe. under natural conditions giraffes are exclusively browsers (dorst & dandelot 1972). however, the nematodes found in the giraffe in this study are considered to be mainly those of grazers. the manner in which the animals are maintained in the zoo is probably the reason for this. the zoo can be considered as a type of safari park as the animals are kept in very large paddocks. in addition, they can even change paddocks if they wish to do so. in this respect mouflons, ovis musimon, are frequently found in the paddocks of other animal species. restricting animals to game reserves and parks facilitates infections between different animal species, and, in addition, many helminths of antelope are not very host specific (boomker et al. 1986). although the feeding habits of giraffes, in general, makes it difficult to acquire helminths of other ruminant species, boomker et al. (1986) consider that they could acquire these when forced to graze. in the aitana zoo lucerne is provided to the herbivores and is placed on the ground. this procedure could explain the presence of nematodes considered uncommon in giraffes, but which are frequently seen in other species in the zoo such as mouflons, fallow deer, dama dama and red deer, cervus elaphus. other gastrointestinal parasites previously recorded in giraffes, i.e. moniezia expansa (pester & laurence 1974), hydatid cysts (kelly, boray & dixon 1968; krecek et al. 1990), haemonchus mitchelli (sachs et al. 1973; krecek et al. 1990), skrjabinema spp. (krecek et al. 1990) or parabronema skrjabini (boomker et al. 1986; krecek et al. 1990), were not found in the present study. 154 helminths in a giraffe in spain camelostrongylus mentulatus was the most abundant nematode species in our giraffe. its occurrence is unusual and it has only once been referred to in giraffe in japan. as fukomoto et al. (1996) suggested, the infection might have been present in other ruminant species kept in the same paddock of the zoo. camelostrongylus mentulatus has been found in many domestic and wild ruminant species such as camels, camelus dromedarius, llamas, lama glama, and red deer (averbeck, scholtthauer & hinueber 1981; kock 1986; flach & sewell 1987; alani & yahay 1993; el azazy 1995; molina, gutiérrez, rodríguez-ponce, viera & hernández 1997; rossi & ferroglio 2001). no information is available concerning the pathology of c. mentulatus infections in wild ruminants but disease caused by it has been described in sheep, camels, oryx, oryx gazella, blackbuck, antilope cervicapra, giraffe and thompson’s gazelles, gazella thomsoni. the cardinal sings were diarrhoea and weight loss. there is one report of c. mentulatus infection which might have had a fatal outcome: this was in a dorcas gazelle, gazella dorcas neglecta, in which a severe abomasitis occurred when hypobiotic larvae of the parasite emerged from mucosal nodules (wisser, tscherner & jantschke 2001). however, the absence of clinical signs and an advanced state of decomposition of the alimentary tract of this animal made it impossible to determine whether the c. mentulatus infection was the actual cause of the death. cross-infection betwen different species of wild ruminants with the trichostrongylid nematodes found in this study is possible, as is cross-infection between domestic and wild ruminants. live wild animals in game reserves are not always strictly monitored for helminth infection. the current report emphasizes the need for surveillance and control of parasites as a means of improving the health of sheltered animals. acknowledgements we thank mr juan daniel sevila vant soldt and mrs rebeca atencia from the aitana zoo for their cooperation and allowing us to examine the giraffe. references alani, a.j. & yahay, h.q. 1993. observations on the epidemiology of intestinal helminths in sheep in ninerah province (iraq). international journal of animal science, 8:51– 55. averbeck, g.a., scholtthauer, j.c & hinueber, i.g. 1981. camelostrongylus mentulatus infection in a camel (camelus dromedarius): a case report. journal of zoo animal medicine, 12:24–26. boomker, j., horak, i.g. & de vos, v. 1986. the helminth parasites of various artiodactylids from some south african nature reserves. onderstepoort journal of veterinary research, 53:93–102. dorst, j. & dandelot, p. 1972. a field guide to the larger mammals of africa. london: collins. durette-desset, m.-c. 1983. keys to genera of the superfamily trichostrongyloidea, in cih keys to the nematode parasites of vertebrates, no. 10, edited by r.c. anderson & a.g. chabaud. commonwealth agricultural bureaux, farnham royal, bucks. durette-desset, m.-c. 1989. nomenclature proposée pour les espèces décrites dans la sous-famille des ostertagiinae lopez-neyra, 1947. annales de parasitologie humaine et comparée, 64:356–373. el-azazy, o.m.e. 1995. seasonal changes and inhibited development of the abomasal nematodes of sheep and goats in saudi arabia. veterinary parasitology, 58:91–98. flach, e.j. & sewell, m.m.h. 1987. gastrointestinal nematodiasis in blackbuck (antilope cervicapra) at edinburgh zoo. journal of zoo animal medicine, 18:56–61. fukomoto, s., uchida, t., ohbayashi, m., ikebe, y. & sasano, s. 1996. a new host record of camelostrongylus mentulatus (nematoda: trichostrongyloidea) from the abomasum of a giraffe at a zoo in japan. journal of veterinary medicine science, 58:1223–1225. kelly, j.d., boray, j.c. & dixon, r.t. 1968. fertile hydatid cysts in the giraffe. australian veterinary journal, 44:525. kock, r.a. 1986. enteric nematode infestation in thomson’s gazelles, gazella thomsoni, at whipsnade park, the zoological society of london. journal of zoo animal medicine, 17:61–64. krecek, r.c., boomker, j., penzhorn, b.l. & scheepers, l. 1990. internal parasites of giraffes (giraffa camelopardalis angolensis) from etosha national park, namibia. journal of wildlife diseases, 26:395–397. molina, j.m., gutiérrez, a.c., rodríguez-ponce, e., viera, j.a. & hernández, s. 1997. abomasal nematodes in goats from the subtropical island of grand canary (spain). veterinary parasitology, 28:259–270. ortiz, j., ruiz de ybáñez, m.r., garijo, m.m., goyena, m., espeso, g., abaigar, t. & cano, m. 2001. abomasal and small intestinal nematodes from captive gazelles in spain. journal of helminthology, 75:363–365. pester, f.r.n. & laurence, b.r. 1974. the parasite load of some african game animals. journal of zoology, 174:397– 406. rossi, l. & ferroglio, e. 2001. camelostrongylus mentulatus in a roe deer from the italian western alps. the veterinary record, 149:335. round, m.c. 1968. check list of the helminth parasites of african mammals of the orders carnivora, tubulidentata, proboscidea, hyracoidea, artiodactyla and perissodactyla. technical communication no. 38, commonwealth bureau of helminthology, st. albans, england. sachs, r., gibbons, l.m. & lweno, m.f. 1973. species of haemonchus from domestic and wild ruminants in tanzania, east africa, including a description of h. dinniki n. sp. zeitschrift für tropenmedizin und parasitologie 24:467–475. 155 m.m. garijo et al. skrjabin, k.i., shikhobalova, n.p., schulz, r.s., popova, t.i., boev, s.n. & delyaure, s.l. 1961. key to parasitic nematodes, vol. iii. strongylata. academy of sciences of the u.s.s.r. jerusalem: israel program for scientific translation. wisser, j., tscherner, w. & jantschke, b. 2001. infestation with camelostrongylus mentulatus in dorcas gazelles (gazella dorcas neglecta) caused lethal abomasitis. verhandlungsberichte erkrankungen zootierie, 40:81–86. 156 helminths in a giraffe in spain magona_231-237.qxd introduction tsetse flies (glossina spp.) are estimated to infest 106 400 km2, 50 % of the entire surface area of uganda and 2.2 million head of cattle of the estimated national cattle population of 5.4 million are at risk of trypanosomosis (chizyuka 1998). it is estimated that tsetse infestation and incidence of trypanosomosis has prevented the keeping of an extra 3.3 million head of cattle in uganda (chizyuka 1998). existing reports indicate the prevalence of trypanosomosis in cattle in uganda to be 11.9 % under the intensive dairy system and 25 % under the communal grazing systems (magona & mayende 2002). in the past, control of tsetse and both human and animal trypanosomosis in southeast uganda was achieved successfully through a donor-funded large-scale deployment of insecticide-impregnated traps (lancien, muguwa, lannes & bouvier 1990) integrated with limited application of pour-on and chemotherapy against animal trypanosomosis (magona, okuna, katabazi, okoth, omollo, mayende & 231 onderstepoort journal of veterinary research, 71:231–237 (2004) protective efficacy of isometamidium chloride and diminazene aceturate against natural trypanosoma brucei, trypanosoma congolense and trypanosoma vivax infections in cattle under a suppressed tsetse population in uganda j.w. magona, j.s.p. mayende, r. okiria and n.m. okuna livestock health research institute, p.o. box 96, tororo, uganda. e-mail: liridir@naro-ug.org abstract magona, j.w., mayende, j.s.p., okiria, r. & okuna, n.m. 2004. protective efficacy of isometamidium chloride and diminazene aceturate against natural trypanosoma brucei, trypanosoma congolense and trypanosoma vivax infections in cattle under a suppressed tsetse population in uganda. onderstepoort journal of veterinary research, 71:231–237 the protective efficacy of isometamidium chloride (ismm) and diminazene aceturate (dim) against trypanosoma brucei, trypanosoma congolense and trypanosoma vivax infections in cattle under a suppressed tsetse population was assessed in southeast uganda. a total of 66 and 57 trypanosome-infected cattle were treated with ismm and dim, respectively together with 177 trypanosomefree animals not treated were followed for 12 months, checked every 4 weeks. there was no statistical difference in the mean time to infection with any trypanosome species in animals treated with ismm or dim. however, the mean time to trypanosome infection was significantly longer for treated animals than controls. the mean time to infection with each of the three trypanosome species differed significantly, with the average time to t. vivax infection the lowest, followed by t. congolense and then t. brucei. the protective efficacy of dim was as good as that of ismm; implying curative treatments against trypanosomosis are sufficient for combination with tsetse control. isometamidium chloride or dim had the highest impact on t. brucei and t. congolense infections in cattle. keywords: diminazene aceturate, isometamidium chloride, protective efficacy, suppressed tsetse population accepted for publication 12 march 2004—editor drabile 1998). direct application of deltamethrin pour-on on cattle has been used to successfully control glossina fuscipes fuscipes in southeast uganda (magona et al. 1998; okiria, okuna, magona & mayende 2002). control of animal trypanosomosis in uganda has relied on the use of both diminazene aceturate (dim) and isometamidium chloride (ismm) for the last 30 years (mwambu & mayende 1971; rushigajiki, mayende, guloba & wilson 1986; okuna, magona, okiria & mayende 1993a; okuna, magona & mayende 1993b). these trypanocidal drugs in many african countries such as uganda are reported to be popular with livestock owners and veterinarians probably because they are affordable and effective (geerts & holmes 1999). repeated use of trypanocidal drugs has led to emergence of drug resistance. strains of trypanosomes resistant to diminazene aceturate have been reported in uganda (mwambu & mayende 1971; matovu, iten, enyaru, schmid, lubega, brun & kaminsky 1997). however, latest reports by olila, mcdermott, eisler, mitema, patzelt, clausen, poetzsch, zessin, mehlitz & peregrine (2002), revealed diminazene aceturate and isometamidium chloride were still effective against trypanosome isolates obtained from some sites in southeast uganda, where farmers routinely used trypanocidal treatments against animal trypanosomosis. despite this optimistic report, it is imperative to guard against the emergence of trypanocidal resistance. integration of tsetse control and trypanocidal drug use has been determined to be an effective method to counteract either single or multiple drug resistance (fox, mmbando, fox & wilson 1993; leak, peregrine, mulatu, rowlands, d’ieteren 1996) and is recommended for the prevention of emergence of trypanocidal drug resistance (geerts & holmes 1999). in either large-scale or farmer-level animal trypanosomosis campaigns involving integration of vector control with trypanocidal drug use, project managers or farmers often face difficulties in deciding whether to select a curative drug—diminazene aceturate— or a prophylactic one—isometamidium chloride—to combine with vector control. field reports on the protective efficacy of either diminazene aceturate or isometamidium chloride under suppressed tsetse populations are rare. in this paper a study conducted in southeast uganda to evaluate the protective efficacy of diminazene aceturate and isometamidium chloride combined with application of deltamethrin pour-on is described. materials and methods study area the study was conducted at masaba in busia district, southeast uganda. location, farming system, climate and vegetation of the study area have been described previously (okiria et al. 2002). glossina f. fuscipes is the main tsetse species in the area, but glossina pallidipes also exists in low densities (magona, katabazi, olaho-mukani, mayende & walubengo 1997; okiria et al. 2002). the main tsetse control method in the study area was deltamethrin pour-on applied on cattle (okiria et al. 2002). this tsetse control programme maintained the tsetse density in the study area between 0 and 0.5 flies per trap per day (okiria et al. 2002)—a situation herein described as a suppressed tsetse population. cattle three hundred zebu cattle of all ages and both sexes kept under a communal grazing system were used in the study. sixty-six of the cattle composed of 26 naturally infected with trypanosoma brucei, 14 infected with trypanosoma congolense and 26 infected with trypanosoma vivax were ear-tagged and treated with ismm by intramuscular injection at a dosage rate of 0.5 mg/kg body mass. another group of 57 cattle consisted of 24 infected with t. brucei, 15 infected with t. congolense and 18 infected with t. vivax were ear-tagged and treated with dim by intramuscular injection at a dosage rate of 7.0 mg/kg body mass. a group of 177 trypanosome-free cattle belonging to the same herds and kept under the same challenge of trypanosomosis than the treated cattle were left untreated but included in the study as controls. there was a similar composition of animals according age groups and sex between the treated and control groups. all the 300 cattle were examined every 4 weeks for a period of 12 months for trypanosome infections, using the buffy coat technique (murray, murray & mcintyre 1977). trypanosomes were specifically identified under a standard microscope. cattle that became re-infected were treated with dim at a dosage rate of 7.0 mg/kg body mass. data analysis for purposes of data analysis, an animal was excluded as soon as it became re-infected to avoid being counted again with an infection of a different trypanosome species. survival analysis was used 232 isometamidium chloride and diminazene against trypanosoma infections in cattle in uganda to compare the protective efficacy of ismm and dim to infection with different trypanosome species. all analyses were performed in glim, version 6, using the method outlined by crawley (1993). since not all animals became infected during the course of the study, a censoring indicator was assigned to each individual: animals that became infected were scored as 1, and those that remained trypanosomefree on the last parasitological examination were scored 0. survival time was defined as either the month parasites were first detected (for infected animals) or the month of last parasitological examination (for non-infected animals). the censoring indicator (1/0 score) was set as the response variable and assigned poisson errors. a log-linear hazard model was fitted to the data with natural log of survival time declared as an offset. treatment regimen was fitted to the model as a factorial explanatory variable with three levels, namely 1 for controls, 2 for ismm-treated animals, and 3 for those treated with dim. significant difference between the ismm and dim groups was tested by combing levels 2 and 3 of the treatment regimen variable, and comparing the increase in deviance to the value in the chi-square table with one degree of freedom. if the grouping of ismm and dim animals into a single treated group was achieved without a significant loss of explanatory power, the model simplification was accepted. difference between treated animals (ismm and dim-treated animals combined) and controls was tested by combining levels: grouping treated and control animals together and comparing the change in deviance to the chisquare table with one degree of freedom. the survival analysis was carried out for each trypanosome species (t. brucei, t. congolense and t. vivax) separately. a similar procedure was used to test differences between the survival times to infection with different species of trypanosome in control animals. the explanatory variable was trypanosome species with three levels, 1 for t. brucei, 2 for t. congolense and 3 for t. vivax). the log-linear hazard model assumed a constant hazard function, which is equivalent to the force of infection, which is the instantaneous rate at which animals acquire trypanosome infections. the reciprocal of the hazard function is the mean time to infection, which is reported, together with 95 % confidence intervals, for the significant groupings from the different survival analyses. results infections with mixed trypanosome species were rarely detected in the experimental cattle. of the 177 control animals, 55 were infected with at least one trypanosome species, 16 with t. brucei only, 13 with t. congolense only, and 26 with t. vivax only during the first infections. over the entire 12 months 21 t. brucei (21/177), 36 t. congolense (36/177) and 133 t. vivax infections (133/177) were detected among the control cattle. among the 66 treated with ismm, there were no re-infections in animals that originally had t. brucei infections (0/26), five re-infections for those that had t. congolense (5/14) and eight re-infections in those that had t. vivax infections (8/26) over a period of 12 months. among the 57 treated with dim, there were two re-infections in those that had t. brucei infections (2/24), one re-infection in those that had t. congolense infections (1/15) and four re-infections in those that had t. vivax infections (4/18). the mean time to infection with each of the three trypanosome species is shown in table 1. the mean time to infections of the various trypanosome species differed significantly (χ2 = 166.30, d.f. = 2, p < 0.01), with the average time to t. vivax infection being lowest, followed by t. congolense and then t. brucei infection. there was no statistical difference in the mean time to infection with any trypanosome in animals treated with ismm or dim (χ2 = 0.943, d.f. = 1, p > 0.05), and the mean time to infection was significantly 233 j.w. magona et al. table 1 the mean time, in months, to infection (with 95 % confidence intervals) for control and treated cattle (ismm and dim groups combined) in under a suppressed tsetse population in southeast uganda estimated from the survival analyses mean months to infection (95 % confidence intervals) trypanosome species controls treated t. brucei 83.9 (54.7–128.7) 667.1 (170.2–2614.8) t. congolense 48.5 (35.0–67.2) 224.5 (101.7–495.9) t. vivax 9.2 (7.7–10.9) 83.9 (60.6–187.7) any trypanosome species 5.1 (4.5–5.9) 62.9 (40.8–96.9) longer for treated animals than controls (χ2 = 205.6, d.f. = 1, p < 0.01). no statistical difference in the mean time to infection with t. brucei in animals treated with ismm or dim (χ2 = 3.13, d.f. = 1, p > 0.05) was noted and the mean time to infection was also significantly longer for treated animals than controls (χ2 = 13.46, d.f. = 1, p < 0.01). there was also no statistical difference in the mean time to infection with t. congolense in animals treated with ismm or dim (χ2 = 2.36, d.f. = 1, p > 0.05), and the mean time to infection was signifi234 isometamidium chloride and diminazene against trypanosoma infections in cattle in uganda fig. 2 proportion of control (filled squares), ismm-treated (stars) and dim-treated (open circles) cattle free of t. congolense infections under a suppressed tsetse population in southeast uganda fig. 3 proportion of control (filled squares), ismm-treated (stars) and dim-treated (open circles) cattle free of t. vivax infections under a suppressed tsetse population in southeast uganda fig. 4 proportion of control (filled squares), ismm-treated (stars) and dim-treated (open circles) cattle free of infections with any trypanosome species under a suppressed tsetse population in southeast uganda fig. 1 proportion of control (filled squares), ismm-treated (stars) and dim-treated (open circles) cattle free of t. brucei infections under a suppressed tsetse population in southeast uganda � ��� ��� ��� ��� ��� � � � � � �� �� � �� � � �� �� � �� � � � � � � � �� � ��� �������� � ��� ��� ��� ��� ��� � � � � � �� �� � �� � � �� �� � �� � � � � � � � �� � ��� �������� � ��� ��� ��� ��� ��� � � � � � �� �� � �� � � �� �� � �� � � � � � � � �� � ��� �������� � ��� ��� ��� ��� ��� � � � � � �� �� � �� � � �� �� � �� � � � � � � � �� � ��� �������� cantly longer for treated animals than controls (χ2 = 16.73, d.f. = 1, p < 0.01). there was no statistical difference in the mean time to infection with t. vivax in animals treated with ismm or dim (χ2 = 0.851, d.f. = 1, p > 0.05). the mean time to infection was significantly longer for treated animals than controls (χ2 = 124.2, d.f. = 1, p > 0.01). the proportion of t. brucei-infected cattle that remained free of re-infection after either ismm or dim treatment and the untreated cattle that remained free of t. brucei infection is shown in fig. 1. the ismm-treated group had the highest proportion of cattle free of t. brucei re-infection, followed by the dim-treated group and then the untreated group. the proportion of t. congolense-infected cattle that remained free of re-infection after either ismm or dim treatment and the untreated cattle that remained free of t. congolense infection is shown in fig. 2. both the ismm-treated and dim-treated groups had a high and similar proportion of cattle free of t. congolense re-infection, which was higher than that of the untreated group. but the untreated group had a reasonably high proportion of cattle free of t. congolense infection. the proportion of t. vivax-infected cattle that remained free of re-infection after either ismm or dim treatment and the untreated cattle that remained free of t. vivax infection is shown in fig. 3. although both ismm-treated and dim-treated groups had a high and similar proportion of cattle that were free of t. vivax re-infection, the untreated group had a very low proportion of cattle (20 %) that remained free of t. vivax infection. in fig. 4 the proportion of trypanosome-infected cattle in general that remained free of re-infection after either ismm or dim treatment, and the untreated group that remained free of infection with any trypanosome species is shown. both ismm and dimtreated groups had a high and similar proportion of cattle that remained free of re-infection with any trypanosome species, but after approximately 11 months, all untreated cattle had been infected at least once with any trypanosome species. discussion the selection of infected animals for the ismmtreated and dim-treated groups might have introduced an interfering factor such as possible development of immunity by animals because of recent trypanosomal infections, however, infected animals were used for purposing of assessing the efficacy of the two trypanocidal drugs, ismm and dim under field conditions. infections with mixed trypanosome species were rarely detected probably because of differences in the sensitivity of the different trypanosome species to trypanocidal drugs and being affected differently by vector control. the mean time to infections of the various trypanosome species differed significantly, with the average time to t. vivax infection being lowest, followed by t. congolense and then t. brucei infection. this finding on the mean time to infection of the various trypanosome species reflects the developmental periods for the various trypanosome species in the tsetse vector, being shortest for t. vivax hence allowing its infection rate to build up faster during control programmes than those for t. congolense and t. brucei (magona, greiner & mehlitz 2000; okiria et al. 2002). other field studies conducted in southeast uganda have reported a drastic decline of t. brucei infection in cattle following treatment with dim at a dosage rate of 7 mg/kg body mass (rushigajik et al. 1986). there was no statistical difference in the mean time to infection with t. brucei, t. congolense or t. vivax in animals treated with ismm or dim, but for all three the trypanosome species the mean time to infection was significantly longer for treated animals than controls. the general practice in the use of both ismm and dim in the control of animal trypanosomosis, involves treatment of animals with ismm every 3 months because the prophylactic period is expected to last for this period, while dim is used as a curative drug against trypanosomosis whenever there are cases. diminazene aceturate is used to clear trypanosome reservoir infection in animals but it is expected to protect animals for only 2 weeks. it is evident from this study that combination of diminazene’s excellent property of clearing reservoir infection in animals when the dosage of 7.0 mg/kg body mass is used with vector control attains a protective efficacy against any trypanosome species, which is as long as that achieved when the isometamidium chloride is used at the dosage of 0.5 mg/kg body mass and combined with vector control. this phenomenon could probably be attributed to the interplay of the reduced tsetse challenge and disease transmission due to vector control, and possible developed of immunity in cattle that have had recent trypanosomal infection but treated as well the effect of drug prophylaxis. these findings imply, as regards protective efficacy, either diminazene aceturate or isometamidium chloride can be select235 j.w. magona et al. ed for combination with vector control. under such a trypanosomosis control strategy, selection of the trypanocide has to be based on the cost of the drug and the targeted animal health improvement rather than the protective efficacy. the ismm-treated group had the highest proportion of cattle free of t. brucei re-infection, followed by the dim-treated group and then the untreated group, which implies that t. brucei infection was more sensitive to ismm than to dim. other workers have also reported that t. brucei isolates from southeast uganda are sensitive to dim and ismm (olila et al. 2002). both the ismm-treated and dim-treated groups had a high and similar proportion of cattle free of t. congolense re-infection, which was higher than that of the untreated group. but the untreated group had a reasonably high proportion of cattle free of t. congolense infection. it is evident that t. congolense infections were equally sensitive to both of the trypanocidal drugs ismm and dim. although both ismm-treated and dim-treated groups had a high and similar proportion of cattle that were free of re-infection with trypanosomes, the untreated group had a very low proportion of cattle (20 %) that remained free of t. vivax infection. trypanosoma vivax isolates from southeast uganda have been reported to be sensitive to ismm and dim (olila et al. 2002), but, during control programmes involving either chemotherapy or vector control in southeast uganda, t. vivax infections in cattle have been observed to predominate over other trypanosome species (magona et al. 1998; magona et al. 2000; okiria et al. 2002) due to its shorter developmental period (4–5 days) in the tsetse vector than that of other trypanosome species t. brucei or t. congolense. both the ismm-treated and dim-treated groups had a high and similar proportion of cattle that remained free of re-infection with any trypanosome species, but after approximately 11 months, all untreated cattle had been infected at least once with any trypanosome species. this implies that despite the suppressed tsetse population, substantial transmission of trypanosomosis especially due to t. vivax was possible. nevertheless, both ismm and dim maintained a satisfactory protective efficacy. these findings and the fact that there are only a small number of technical or anecdotal reports of the emergence of drug-resistant trypanosome populations in cattle in uganda suggest that both ismm and dim are still effective against animal trypanosomosis. even the few existing reports have either reported ineffectiveness of trypanocidal drugs for animal use on human infective trypanosomes (matovu et al. 1997; enyaru, matovu, lubega & kaminsky 1998) or in one genuine incidence involving dim, resistance was attributed to cross-resistance to dim by probable quinapyramine-resistant trypanosome strains (mwambu & mayende 1971). this limited occurrence of drug resistance to both ismm and dim in uganda could probably be because of the long tradition of integrating both trypanocidal drugs use with vector control (lancien et al. 1990; ndyabahinduka 1993; magona et al. 1998) that has been advocated for over many years. other studies on farmer control programmes against animal trypanosomosis in mbarara district of uganda, involving the application of deltamethrin pour-on combined with the routine use of ismm have also revealed good efficacy of trypanocidal drugs (okuna, unpublished data 2003). in conclusion, a combination of trypanocidal drug use and vector control in the control of animal trypanosomosis, as demonstrated by these findings and others published elsewhere (fox et al. 1993; leak et al. 1996), has substantial potential in minimizing the occurrence of drug-resistant trypanosome strains. it is evident from this study that in such a control strategy either dim (curative) or ismm (prophylactic) could be used. the highest impact of this control strategy seems to be on t. brucei and t. congolense infections in cattle in southeast uganda. these results confirm the effectiveness of the trypanocidal drugs ismm and dim against trypanosome infections despite the many years of their use in the fight against animal trypanosomosis in southeast uganda. acknowledgements we thank abbas guloba, michael oretum and geoffrey odyek for their technical assistance and dr paul coleman of london school of tropical medicine and hygiene for his assistance in data analysis. we are grateful to the world health organization for financially supporting this study. this study is published with the permission of the director, livestock health research institute, tororo, uganda. references chizyuka, g. 1998. fao liaison officers summary report, harare, october 1998. 236 isometamidium chloride and diminazene against trypanosoma infections in cattle in uganda crawley, m.j. 1993. glim for ecologists. london: blackwell scientific publications. enyaru, j.c.k., matovu, e., lubega, g.w. & kaminsky, r. 1998. response of a t. b. rhodesiense stock with reduced drug susceptibility in vitro to treatment in mice and cattle. acta tropica, 69:261–269. fox, r., mmbando, s.o., fox, m.s. & wilson, a. 1993. effect on herd health and productivity of controlling tsetse and trypanosomosis by applying deltamethrin to cattle. tropical animal health and production, 25:203–213. geerts, s. & holmes, p.h. 1999. drug management and parasite resistance in animal trypanosomosis in africa. paat position paper 1999. lancien, j., muguwa, j., lannes, c. & bouvier, j.b. 1990. tsetse and human trypanosomiasis challenge in south-eastern uganda. insect science and its applications, 11:411– 416. leak, s.g.a., peregrine, a.s., mulatu, w., rowlands, g.j. & d’ieteren, g. 1996. use of insecticide-impregnated targets for the control of tsetse flies (glossina spp.) and trypanosomiasis occurring in cattle in an area of south-west ethiopia with a high prevalence of drug-resistant trypanosomes. tropical 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and control, kampala, uganda, 1993, 117:100–102. okiria, r., okuna, n.m., magona, j.w., mayende, j.s.p. 2002. sustainability of tsetse control by subsequent treatment of 10% of a previously treated ugandan cattle population with 1% w/v deltamethrin. tropical animal health and production, 34:105–114. okuna, n.m., magona, j., okiria, r. & mayende, j.s.p. 1993a. observation, in the field, on a herd of trypanosome infected cattle treated with isometamidium chloride or diminazene aceturate and kept under deltamethrin cover in tororo district. proceedings of the 22nd meeting of the international scientific council for trypanosomiasis research and control, kampala, uganda, 1993, 117:174. okuna, n.m., magona, j. & mayende, j.s.p. 1993b. preliminary observations on the impact of trypanosomiasis on sheep and goats in an enzootic area in uganda and need for prophylactic treatment. proceedings of the 22nd meeting of the international scientific council for trypanosomiasis research and control, kampala, uganda, 1993, 117:176. olila, d., mcdermott, j.j., eisler, m.c., mitema, e.s., patzelt, r.j., clausen, p.-h., poetzsch, c.j., zessin, k.-h., mehlitz, d. & peregrine, a.s. 2002. drug sensitivity of trypanosome population from cattle in a periurban dairy production system in uganda. acta tropica, 84: 19–30. rushigajiki, p.k.b, mayende, j.s.p, guloba, a. & wilson, a.j. 1986. maintenance of a herd of breeding cattle in an area of trypanosome challenge. a study on the chemotherapy of bovine trypanosomiasis at lugala, uganda from 1972 and 1978. bulletin of animal health and production in africa, 34:149–155. 237 j.w. magona et al. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) leon prozesky department of paraclinical sciences, university of pretoria, south africa johan neser department of paraclinical sciences, university of pretoria, south africa heinz meissner department of paraclinical sciences, university of pretoria, south africa kenneth botha department of paraclinical sciences, university of pretoria, south africa lubbe jacobs lubern animal feeds, hartswater, south africa craig shepstone department of paraclinical sciences, university of pretoria, south africa hannes viljoen animal nutrition and health, centurion, south africa hinner köster kaonna investments (pty) ltd, pretoria, south africa chris de brouwer department of agriculture, north west province, south africa jan van zyl farmer north west province gerjan van der veen department of paraclinical sciences, university of pretoria, south africa citation prozesky, l., neser, j., meissner, h., botha, k., jacobs, l., shepstone, c. et al., 2016, ‘preliminary report on osteochondrosis in cattle in the north-western parts of south africa’, onderstepoort journal of veterinary research 83(1), a1083. http://dx.doi.org/10.4102/ojvr.v83i1.1083 original research preliminary report on osteochondrosis in cattle in the north-western parts of south africa leon prozesky, johan neser, heinz meissner, kenneth botha, lubbe jacobs, craig shepstone, hannes viljoen, hinner köster, chris de brouwer, jan van zyl, gerjan van der veen received: 06 oct. 2015; accepted: 09 dec. 2015; published: 27 july 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the north-western part of south africa, in particular, is well known for mineral imbalances. aphosphorosis, resulting in rickets and osteomalacia, received a lot of attention at the turn of the nineteenth century (1882–1912). this was followed in 1997 by research on vryburg hepatosis, another area-specific mineral imbalance–related disease in young calves reared on manganese-rich soil derived from the weathering of dolomitic (carbonate) rock formations. in 1982, a totally new syndrome (osteochondrosis) manifested in, amongst others, areas in south africa where aphosphorosis was rife. osteochondrosis was also identified in the south-western parts of namibia as well as southern botswana and other areas in south africa. osteochondrosis has a multifactorial aetiology and this study focused on the role of minerals, particularly phosphorus, in the development of the disease. a significant improvement in the clinical signs in experimental animals and a reduction of osteochondrosis occurred on farms where animals received bioavailable trace minerals and phosphorus as part of a balanced lick. an increase in the occurrence of the disease on farms during severe drought conditions in 2012–2013 prompted researchers to investigate the possible role of chronic metabolic acidosis in the pathogenesis of the disease. introduction since the 1800s, the north-western parts of south africa, particularly the north west and northern cape provinces, have been known as an area characterised by aphosphorosis (‘stiff sickness’) in cattle (theiler 1912; theiler et al. 1927), caused by a phosphorus deficiency that resulted in osteomalacia. affected animals often developed botulism because of the consumption of bones contaminated with clostridium botulinum (theiler, du toit & malan 1937; theiler et al. 1927). the bone meal and coarse salt licks introduced to prevent osteomalacia and botulism seemed to have resolved the osteomalacia or rickets syndrome in the north-western parts of the country. however, in 1982, farmers and local veterinarians started reporting lameness accompanied by swelling of the stifle joint in cattle. initially, the lameness seemed to affect only a small number of show animals and it was also seen in phase d tested bulls, but the incidence steadily increased, and in 2004, farmers reported an incidence that ranged from 2% to 20%. in some instances up to 40% of animals were affected. the syndrome was found to be present in cattle of all age groups and classes (both commercial and non-commercial), and apparently in most breeds of cattle farmed in that area. afrikaner cattle seemed to be more resistant to this condition. the only factor in common amongst affected animals was the geographical area in which they lived. necropsies performed on 21 severely affected cattle revealed chronic lesions that enabled a diagnosis of osteochondrosis to be made. osteochondrosis is a common and important joint disorder that occurs in humans and many animal species, particularly pigs, horses and dogs (bradley & dandy 1989). it is defined as a focal disturbance of endochondral ossification and has a multifactorial aetiology (ekman & carlsson 1998; gerber et al. 1999; schenk & goodnight 1996; ytrehus, carlson & ekman 2007). whilst existing evidence supports the importance of genetic and conformational factors (ytrehus et al. 2007), a number of possible aetiologies and predisposing factors, such as over-nutrition, rapid growth, genetics (experimentally proven in pigs [hittmeier et al. 2006; ytrehus et al. 2004]), ischaemia, excess dietary calcium, hormonal influences and trauma, have been proposed (trostel, mclaughlin & pool 2002; wooderd 1997). in human medicine, osteochondrosis has been defined as an idiopathic condition characterised by disorderliness of endochondral ossification (siffert 1981). as early as 1978, it was suggested that the pathophysiology of osteochondrosis is essentially the same in all species, including humans (olsson 1987; olsson & reiland 1978). although formation of fragile cartilage, failure of chondrocyte differentiation, subchondral bone necrosis and failure of blood supply to the growth cartilage have all been proposed as an initial step in the pathogenesis, the recent literature strongly supports failure of blood supply to growth cartilage as being the most likely (ytrehus et al. 2004, 2007). localised failure of endochondral ossification ensues, which typically results in necrosis of affected cartilage. it can involve the physis or the articular–epiphyseal cartilage (hill, sutton & thompson 1998; reiland 1978; trostel et al. 2002). mild trauma to the necrotic cartilage may result in cleft formation of the affected cartilage, with release of cartilage degeneration products into the joint causing a synovitis evident clinically as joint effusion and pain with lameness (trostel et al. 2002). in pigs, the lesion is slightly different in that there is no evidence of necrosis of cartilage, but rather a focal failure of mineralisation of hypertrophic cartilage cells (wooderd 1997). in dogs, osteochondrosis lesions are typically bilateral (trostel et al. 2002). osteochondrosis in cattle is found in all types of husbandry systems, including feedlots (davies & munro 1999; heinola et al. 2006; jensen et al. 1981; reiland et al. 1978), pure-bred beef (dutra, carlsten & ekman 1999), dairy (trostle et al. 1997) and animals grazing rangelands (hill et al. 1998). cattle affected with this disorder develop effusions in the weight-bearing joints, in particular the femoro-tibial (stifle) joint, associated with inflammation and pain, causing lameness of varying degrees. as a result, animals are unable to walk long distances for grazing, have decreased feed intake, have decreased milk production and a loss in body condition, and bulls have decreased mating ability (persson, soderquist & ekman 2007). animals are often eventually slaughtered as a result of severe lameness and loss of condition. because, in the north west province, outbreak cattle of multiple breeds were affected, and the area is characterised by mineral imbalances, samples of liver and rib were subjected to mineral analysis together with samples taken from healthy control cattle and water samples from the affected area. a limited feeding trial on seven cattle of the same group as the necropsied cattle was undertaken. based on the results obtained, various studies were subsequently undertaken to examine the possible role of mineral imbalances in the syndrome, the details of which will be published elsewhere. the study describes the pathology of the lesions observed at necropsy on which the diagnosis of osteochondrosis is based. the other important syndromes caused by mineral imbalances in the same area are discussed, and the results of the mineral analysis and pilot feeding trial are briefly summarised. materials and methods during june 2004, 28 clinically affected female adult animals (2–7 years old), representing various breeds, were donated by farmers from the north west province to the university of pretoria for research purposes. the animals showed lameness and articular swelling that was most prominent in the stifle joint. from the time of arrival, animals were fed only good quality eragrostis sp. hay without any mineral supplement (lick). pathological examinations necropsies were conducted on 21 animals (5 animals 2–3 years old and 16 animals 4–6 years old). all the joints of the limbs were opened and examined, as well as multiple vertebral joints, and lesions were described and photographed. bone samples were taken for histopathological examination, and bone and liver samples were taken for mineral analysis (to be described in detail elsewhere). feeding trial the remaining seven animals (three 2–3 years old and four 4–6 years old) with less severe lameness and swelling of the stifle joints were fed on a commercial production supplement (maxitech, afgri) at a recommended daily intake of 1.5 kg per animal per day for a period of 3 months, from august to october 2004. an additional supply of a commercial 6% phosphorus supplement (sukrafos) containing macroand micro-minerals was also fed for the first month at a daily intake of 80 g per animal per day. eragrostis sp. hay was supplied ad libitum. water supplied by the local municipality was also freely available. the animals were slaughtered and necropsied at the end of the trial. results pathology lesions involving the articular cartilage were present in all the animals in various joints (including the atlanto-axial, tibio-tarsal, scapular-humeral and femoral-pelvic joints), but were consistently most prominent in the femoro-tibial joints, often evident clinically as a joint effusion (figure 1). the macroscopic and microscopic lesions associated with osteochondrosis are well documented, and the lesions noted in the experimental cattle did not differ from those reported (hill et al. 1998; reiland 1978; ytrehus et al. 2007). for the purpose of this article, the lesions are briefly summarised. figure 1: joint effusion of the femoro-tibial joint in a clinically affected animal with osteochondrosis. there was notable variation in the extent of the lesions. the opposing articular cartilage surfaces lacked the normal lustre and had a mildly eroded appearance. in the stifle joints, lesions on the medial and lateral condyles ranged from shallow clefts to several converging fissures, to multifocal to coalescing ulcerations forming grooves and irregular craters in the thickened cartilage (figure 2) that sometimes formed subchondral cysts containing necrotic material. the cysts were surrounded by well-vascularised fibrous connective tissue (figure 3). severe degenerative lesions extended to the menisci (figure 4). figure 2: irregular crater in the right femur condyle. figure 3: subchondral cysts containing necrotic material and surrounded by well-vascularised fibrous connective tissue. figure 4: severe degenerative chondral lesions extending to the menisci. ulceration of the articular cartilage often resulted in an exposed, irregular connective tissue base. abnormal trabecular pattern of the growth plate was noted in young animals, whereas in some older animals, lesions in the growth plate indicative of endochondral failure were characterised by the presence of a cyst approximately 10 mm – 15 mm in diameter at the level of the growth plate (figures 5 and 6). the joint spaces contained recently detached flaps of degenerative cartilage or osteochondral bodies. in all the animals, the joint capsules showed mild to moderate fibrous thickening, with hyperplasia of the stratum synoviale. joints contained copious amounts of synovial fluid, which appeared macroscopically normal. figure 5: abnormal trabecular pattern of the growth plate was noted in young animals. figure 6: cyst at the level of the growth plate (older animal). representative lesions on the distal femur were selected to demonstrate the histopathological lesions. lesions were noted in the entire articular–epiphyseal cartilage complex, involving both the avascular articular cartilage and the vascular epiphyseal cartilage, and extended into the underlying subchondral bone (figure 7). multifocal necrosis was present in the vascularised epiphyseal cartilage, and the latter was covered by a thick necrotic cartilage layer. clefts extended from the articular surface to the subchondral bone and contained multifocal necrosis and well-vascularised connective tissue (granulation tissue) that sometimes extended into the ulcerated articular cartilage complex. figure 7: extensive necrosis of the articular–epiphyseal cartilage complex extending to the subchondral bone. feeding trial after a period of 3 months it was agreed by several observers, including two experienced farmers from the north west province, that a significant improvement in the degree of external swelling of the most severely affected stifle joints had occurred and that this was accompanied clinically by a reduction in the degree of lameness, particularly in the younger animals. all the animals also showed an increase in body condition score; at the start of the trial, they had a score of 2 and all improved to a score of 3. body mass also increased by an average of approximately 43 kg per animal. at necropsy, joint fluid samples were collected and tested negative for bacteria and mycoplasmas. lesions in the joints and bones were similar to those described in the slaughtered animals, except that in the younger animals, the degenerative hyaline cartilage was replaced by irregular fibrous cartilage (figure 8). figure 8: note irregular fibrous articular cartilage covering the femur condyle. discussion the pathological lesions confirmed a diagnosis of osteochondrosis. the current study confirmed that failure of endochondral ossification resulting in necrosis of either the physis or the articular–epiphyseal cartilage was central in the pathogenesis of the lesions, as proposed by ytrehus et al. (2004) and ytrehus et al. (2007). to the best of our knowledge, the high incidence of osteochondrosis in cattle in south africa since 1982, particularly in, although not confined to, the north-western parts of the country, represents the largest outbreak of this condition ever recorded in cattle. it has had a major impact on beef production in the affected areas. it is therefore important that the cause of the problem should be established in order to determine how to manage it. even though the clinical signs and macroand microscopic lesions associated with osteochondrosis are well documented, to a large extent the aetiology remains controversial. problems associated with the study of osteochondrosis include, amongst others, the multifactorial aetiology proposed for the condition, a lack of parameters to measure the improvement in affected animals (e.g. following a change in the diet because of the relatively slow regenerative capacity of cartilage and bone, which are the main tissues affected) and the difficulty in producing clinical cases experimentally. whilst strong evidence has been adduced for breed and conformation being influential in the development of osteochondrosis (ytrehus et al. 2007), it seems probable that other factors may play a role in the present outbreak. although apparent sparing of the afrikaner breed of cattle as well as a probable conformational link have been observed in the outbreak, the wide range of breeds and ages affected and the geographical distribution of the outbreak are suggestive of additional contributory factors, some of which, in particular nutritional factors, may be of major importance. from a geological point of view, the north-western parts of south africa, the south-western parts of namibia, as well as southern botswana where osteochondrosis was reported are characterised by the presence of superficial dolomitic (carbonate) rock formations. the area also has a history of well-documented syndromes in cattle that are demonstrably because of mineral deficiencies or imbalance. the best known of these is osteomalacia resulting from aphosphorosis, described by theiler (1912), which also results in lameness and became known as ‘stiff-sickness’ (stijfziekte). it was therefore important to establish whether the outbreak that started in 1982 was a manifestation of aphosphorosis or a new condition. stiff sickness and botulism (lamziekte) were diseases of cattle commonly found in the north-western parts of south africa and assumed to be two different manifestations of the same disease. it was soon suspected, and later demonstrated, to be linked to a phosphate deficiency (theiler 1912; theiler et al. 1937), and in 1919 theiler discovered and later proved that botulism was a disease distinct from osteomalacia and was caused by toxins released from c. botulinum present in bones the cattle were ingesting as a result of pica (theiler et al. 1927). osteomalacia is essentially defective mineralisation of osteoid in adult animals. its pathogenesis is very similar to that of rickets in young animals. in rickets, there is abnormal endochondral ossification of the cartilage growth plates as well as defective bone formation at sites of bone remodelling. in osteomalacia, because the growth plates have closed, only sites of bone remodelling are affected. the essential lesion is defective mineralisation of the cartilage matrix of growth plates (rickets) and defective mineralisation of osteoid (both diseases). osteomalacia and rickets are manifestations of one of a number of possible nutritional abnormalities, viz. aphosphorosis, vitamin d deficiency, calcium deficiency or calcium:phosphorus imbalance (thompson 2007). the lesions are variable, and a diagnosis may therefore be difficult. in rickets, the forelimbs are often bowed and the ends of the long bones at the growth plates are enlarged, as are the costochondral junctions. in severe cases of rickets, pathological fractures are common. in osteomalacia, the bones are weaker than normal, resulting in excessive bone matrix deposition where stresses and strains on the bones are greatest. as in rickets, the bones fracture easily. macroscopically, the cortices of the bones are thin, spongy and soft. kyphosis or lordosis is often noted. the thorax is often narrowed and flattened, and the sternum may be prominent (thompson 2007; wooderd 1997). historically, osteomalacia occurred only on certain farms, where it was endemic every year at more or less the same time. affected animals had a severe craving for bones. young growing, pregnant animals or cows with calves at foot were most commonly affected, and the condition was most prevalent when the grazing was poor, for example, during times of drought. clinically affected animals appeared to experience severe pain and showed signs of arthritis and laminitis, and in young animals, signs of rickets were noted. animals showed a shifting lameness, lying down frequently. they walked with their backs arched and their hind legs tucked up underneath the abdomen and placed their weight on their heels, which resulted in abnormal hoof growth (theiler 1912). pica noted in animals with stiff sickness was proven experimentally to be associated directly with aphosphorosis. the main stimulus attracting phosphorus-deficient cattle to bones is olfactory, with old bones being their first choice. phosphorus-deficient cattle are not attracted to other animal products such as fat, meat or blood. an intravenous injection of sodium phosphate was found to abolish the cravings for bones within minutes (mcdowell 1996). provision of bone meal and coarse salt licks resolved the problem of osteomalacia and botulism in the north-western parts of the country. following the outbreak of bovine spongiform encephalopathy in the united kingdom in 1985, the use of bone meal in south africa as a feed supplement was banned, but supplementation of animals with a high phosphorus–containing lick (p18) continues to prevent cattle from developing osteomalacia at the research station where the original research on the condition was carried out (c. de brouwer, pers. comm., 2014). vryburg hepatosis is an area-specific mineral imbalance–related disease. the name is derived from the district in the north west province in south africa where most of the cases originated and the characteristic histopathological changes were observed in the livers of affected animals (neser et al. 1997). the condition occurs in young calves 7–14 days old reared on manganese-rich soil derived from the weathering of dolomitic (carbonate) rock formations in the northern cape and north west provinces. affected animals develop severe subacute to chronic cholangiohepatitis with icterus and most untreated animals succumb to the disease. the disease manifests clinically in young calves as geophagia, and affected animals sometimes also lick iron poles. geophagia is followed by constipation, dehydration and death within 7–10 days. on severely affected farms, 50% – 75% of calves may develop geophagia, with a mortality rate close to 100% in untreated cases. liver analysis revealed high manganese levels, ranging from 10 ppm to 1800 ppm wet mass (normal range 2 ppm – 3 ppm wet mass). grazing plants from affected farms had higher levels of manganese and a higher ratio of manganese–to-iron than plants from unaffected farms (neser et al. 1997). manganese interferes with iron and cobalt absorption from the gastrointestinal tract (hurley & keen 1986; thompson & valberg 1972). treatment of calves by injection with commercial iron-dextran compounds and vitamin b12 at 1–2 days after birth and repeated at 14 days of age has a significant preventative effect. furthermore, in endemic areas, geophagia can be prevented by rearing calves in enclosures covered with a thick layer of dung. vryburg hepatosis was experimentally induced by dosing a calf and two lambs with relatively high levels of manganese oxide (elsenbroek & neser 2002; neser et al. 1997). the diagnosis of osteochondrosis indicated that the current outbreak likely had a different aetiology from osteomalacia. a survey of affected farms revealed that factors associated with the current outbreak included the following: geographical area, breed (fast-growing breeds were more severely affected than slow-growing breeds), sex (male animals were more severely affected than female animals), age (weaner’s were more affected than adult animals), conformation of the animals (cattle with structural bone deformities, e.g. straight hocks were more at risk), nutritional status of the animals, management systems (calving throughout the year compared to fixed calving seasons) and ongoing selection pressures on animals (shorter calving intervals and heavier weaning weights). it was, however, unclear whether any of these were the primary cause of the condition or whether they merely exacerbated it. that the conditions were linked to the area was strengthened when enquiries further afield revealed that the condition was also present in certain geologically similar parts of namibia and botswana. given the apparent geological link and the fact that an association between nutrition and cattle diseases has been demonstrated in the area, possible involvement of mineral imbalances warranted further investigation, although the involvement of dietary factors was not supported by evidence presented in a recent review (ytrehus et al. 2007). nevertheless, osteochondrosis has been associated with a copper deficiency in several species, including deer, bison and horses. the lesions are caused either by a primary copper deficiency or by exposure to factors that inhibit copper absorption or metabolism (e.g. zinc, cadmium or inorganic sulphates) (hurtig et al. 1993). these authors further suggested a relationship between low copper intake in fast-growing horses, inferior collagen quality, biomechanically weak cartilage and bone, and lesions of osteochondrosis dissecans. osteochondrosis associated with a copper deficiency was also reported in new zealand in young wapiti cross red deer hybrids (audigé et al. 1995; thompson et al. 1994; wilson & grace 2001). however, there was no evidence of copper deficiency in the current study. davies and munro (1999) described an outbreak of osteochondrosis in bull beef cattle following failure to provide dietary mineral and vitamin supplementation. analysis of the metacarpal bone from two bulls revealed adequate magnesium, phosphorus and bone ash, but a slightly low calcium concentration. the vitamin a concentration was also low. dietary analysis suggested inadequate calcium, sodium and copper intake and mild deficiency of vitamins a, d and e. a balanced mineral and vitamin supplement was added to the diet when it became clear that the supplement had been omitted. a gradual clinical improvement was seen in the majority of the animals, and after 2–3 weeks, the growth rate and coat quality had improved significantly. this outbreak provides evidence that in some cases a mineral and vitamin imbalance is a likely contributing factor to the development of osteochondrosis in growing cattle. calcium deficiency, with a distorted calcium-to-phosphate ratio, was associated with an outbreak of osteoarthritis in fattening bulls that was probably osteochondrosis. calcium deficiency caused more serious lesions in the age group 5–8 months than in the age group 12–18 months. osteoarthritis lesions occurred in more than 80% of the animals with a calcium-deficient diet (heinola et al. 2006). similarly, other authors have reported osteochondrosis-type lesions in feedlots and bull stations where the feed given was thought to be one of the causative factors (jensen et al. 1981; reiland et al. 1978; trostle et al. 1998; weisbrode et al. 1982). the results of the preliminary investigation using samples of liver and rib from affected cattle as well as from cattle from an unaffected herd within the area of the outbreak and an unaffected herd outside the outbreak area revealed that the cattle had low magnesium levels and some of the cattle had low manganese and/or phosphorus levels at the time of testing. water samples collected from 27 affected farms revealed high levels of bromine in all samples, with nickel, lead and selenium often in elevated concentrations. the very high water hardness values suggest that scaling and poor palatability may pose some problems, whilst contributions to acid–base balance aspects may also be relevant. it is, however, realised that mineral analysis of liver, bone and water samples from farms where osteochondrosis is rife must be analysed on an ongoing basis to compare results during different times of the year, and particularly during periods of drought, which has a significant influence on the composition of the water. however, preliminary observations including the feeding trial indicate that the primary cause of osteochondrosis in southern africa may be a deficiency or imbalance in mineral intake. conclusion extensive on-farm trials after the completion of this study have confirmed that supplementation with balanced minerals, bioavailable phosphorus and vitamins has a significant impact on preventing and curing the condition in the field. commercial mineral supplements have been formulated for use in affected areas. an upsurge of the condition during a drought in 2012/2013 has implicated metabolic acidosis as a possible complicating factor, as described in previous studies (barzel & jowsey 1969; bushinsky 2001; green & kleeman 1991; kraut et al. 1986; sutton, wong & dirks 1997). acid–base balance is a state of equilibrium between acidity and alkalinity of the body fluids, also called hydrogen ion balance. the body’s acid–base balance is normally tightly regulated by buffering agents and mechanisms related to the respiratory, renal and skeletal systems, keeping the arterial blood ph between 7.38 and 7.42 (cunningham & klein 2007). investigations into this aspect are being pursued. these studies will be described in detail in future publications. acknowledgements competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions l.p. was the project leader. j.n. performed some of the experiments. h.m. assisted with the project design. k.b., c.s. and g.v.d.v. were postgraduate students at the university of pretoria and performed most of the experiments. h.v., h.k., c.d.b. and l.j. assisted with the experimental design. j.v.z. farmer, vryburg, made his cattle available for the trial and assisted with the experiments. references audigé, l., wilson, p.r., morris, r.s. & davidson, g.w., 1995, ‘osteochondrosis, skeletal abnormalities and enzootic ataxia associated with copper deficiency in a farmed red deer (cervus elaphus) herd’, new zealand veterinary journal 43, 70–76. barzel, u.s. & jowsey, j., 1969, ‘the 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and pathogenesis of osteochondrosis’, veterinary pathology 44, 429–448. ytrehus, b., carlson, c.s., lundeheim, n., mathisen, l., reinholt, f.p., teige, j. et al., 2004, ‘vascularisation and osteochondrosis of the epiphyseal growth cartilage of the distal femur in pigs – development with age, growth rate, weight and joint shape’, bone 34, 454–465. abstract introduction methods results discussion acknowledgements references appendix 1 about the author(s) aboyeji l. oyewole department of physiology, neuroscience and inflammation unit, college of health sciences, faculty of basic medical sciences, university of ilorin, ilorin, nigeria bioresearch hub laboratory, ilorin, nigeria oluwole akinola department of anatomy, college of health sciences, faculty of basic medical sciences, university of ilorin, ilorin, nigeria bamidele v. owoyele department of physiology, neuroscience and inflammation unit, college of health sciences, faculty of basic medical sciences, university of ilorin, ilorin, nigeria citation oyewole, a.l., akinola, o. & owoyele, b.v., 2021, ‘plasmodium berghei-induced malaria decreases pain sensitivity in mice’, onderstepoort journal of veterinary research 88(1), a1871. https://doi.org/10.4102/ojvr.v88i1.1871 original research plasmodium berghei-induced malaria decreases pain sensitivity in mice aboyeji l. oyewole, oluwole akinola, bamidele v. owoyele received: 09 apr. 2020; accepted: 10 sept. 2020; published: 11 jan. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract various types of pain were reported by people with plasmodium falciparum and were mostly attributed to a symptom of malarial infection. neural processes of pain sensation during malarial infection and their contributions to malaria-related death are poorly understood. thus, these form the focus of this study. swiss mice used for this study were randomly divided into two groups. animals in the first group (pb-infected group) were inoculated with plasmodium berghei to induce malaria whilst the other group (intact group) was not infected. formalin test was used to assess pain sensitivity in both groups and using various antagonists, the possible mechanism for deviation in pain sensitivity was probed. also, plasma and brain samples collected from animals in both groups were subjected to biochemical and/or histological studies. the results showed that pb-infected mice exhibited diminished pain-related behaviours to noxious chemical. the observed parasite-induced analgesia appeared to be synergistically mediated via µ-opioid, α2 and 5ht2a receptors. when varied drugs capable of decreasing pain threshold (pro-nociceptive drugs) were used, the survival rate was not significantly different in the pb-infected mice. this showed little or no contribution of the pain processing system to malaria-related death. also, using an anti-cd68 antibody, there was no immunopositive cell in the brain to attribute the observed effects to cerebral malaria. although in the haematoxylin and eosin-stained tissues, there were mild morphological changes in the motor and anterior cingulate cortices. in conclusion, the pain symptom was remarkably decreased in the animal model for malaria, and thus, the model may not be appropriate for investigating malaria-linked pain as reported in humans. this is the first report showing that at a critical point, the malaria parasite caused pain-relieving effects in swiss mice. keywords: inflammatory pain; malaria; infection pain; behaviour; opioidergic; serotoninergic. introduction the world malaria report 2018 estimated the global tally of new malaria cases and death as at 2016 to be 216 000 000 and 445 000, respectively (who 2018). despite several up-scaling interventions, increased hotspots for malaria transmission, zoonotic malaria transmission, emergence and re-emergence of resistant plasmodium parasites have continued to amplify malaria prevalence globally. at a specific parasitic density, nociceptors are either depolarised, hyperpolarised or sensitised, and this consequently results in a localised or generalised alteration in pain perception. whilst the previous infection inflammation-induced pain dogma still holds, recent findings showed certain pathogens and their products can directly activate nociceptors. in the infection inflammation-induced pain pathway, pathogens and their products are known to have prototypic molecular pattern that stimulate toll-like receptor which in turn activate biosynthesis and release of pro-inflammatory cytokines (miller et al. 2009; schedlowski, engler & grigoleit 2014). apart from the cytokines (interleukins, tumour necrosis factor and neurotrophins) (burnstock 2009), mediators like prostaglandins, histamine, purines, serotonin and bradykinins are also released primarily to recruit immune cells to the site of injury or infection. however, peripheral nociceptor terminals via their receptors and ion channels are sensitised by these mediators, consequently resulting in increasing pain sensation (pinho-ribeiro et al. 2017). although this pathway was believed for years as the sole genesis of pain during infection, emerging studies, however, showed a direct pathogen-induced pain at a specific density in parasitaemic infection. in bacterial infection, for instance, lipopolysaccharide via its lipid a anchored moiety component can directly stimulate transient receptor potential ankyrin 1 ion channel on nociceptors to produce pain (kashem et al. 2015). also, a gram-positive bacterium like staphylococcus aureus produces α-haemolysin that stimulates nociceptors. this forms the basis for thermal and mechanical allodynia accompanying staphylococcus aureus infection (tran et al. 2013). also, n-formyl peptides (the metabolic by-products of all bacteria) stimulate g protein-coupled nociceptors (formyl peptide receptor 1) to elicit pain sensation (tran et al. 2013). viral and fungal pathogens are also not exempted from the direct-infection-induced pain. although the mechanism behind nociceptive stimulation remains unclear, herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus infections triggered varied pain sensations whilst fungal pathogen candida albicans was also associated with pain thrush possibly caused by calcium influx into nociceptors (nagel & don 2014; tong et al. 2010). in human malarial infection, haemolysis of parasitised red blood cells releases parasites, their allied endotoxins (haemozoin and parasite deoxyribonucleic acid) and other cellular components that trigger toll-like receptor 9 stimulation (parroche et al. 2007) and tumour necrosis factor secretion (wassmer et al. 2015). these physiological processes are known to activate the infection-inflammation-induced pain pathway (miller et al. 2009; pinho-ribeiro et al. 2017; schedlowski et al. 2014), an observation corroborated by various clinical case reviews where malaria is documented to present with fever, chills, vomiting and various pains such as headache, myalgia, abdominal pain or back pain (reisinger et al. 2005; sarma & kumar 1998). despite reported body pain in malaria patients, there is scanty information on malaria-related pain in animal model for malaria. also, there is no report yet on contribution or association of malaria-related pain sensitivity to malaria-linked mortality. although severe anaemia and cerebral malaria are known to be the typical pathological hallmarks that lead to death in untreated malaria infection, functional data from the study of pain are, however, shifting the aetiological causes of most mortalities to pain. torrance and co-workers associated severe chronic pain to increase mortality irrespective of socio-demographic factors (torrance et al. 2010). this study was designed to investigate pain sensitivity in malarial infection with a view to improve our understanding on handling noxious sensation during malaria attack. against our assumption, we observed that increased pain sensitivity reported in human was absent in animal-infected with malaria. in fact, pain sensitivity continued to decline as parasitaemia level increased in the swiss mice used. methods animals and drugs mice and parasites orders for swiss mice of the same progeny were placed at central animal facility (caf) in the university of ilorin, nigeria, several weeks before the commencement of these experiments. the same progeny set of animals was used to eliminate possible genetic bias. the mice used were 8to 12-week-old male that had been subsequently backcrossed for at least five generations. male mice were adopted to eliminate reported sex difference in pain sensation test, although this variation appeared to be absent in pb-infected mice but was substantial in the control, intact group. the mice were moved from caf where they were bred to resident in the college of health sciences animals facility, university of ilorin, nigeria, where they were allowed to habituate to experimental conditions and handling before these studies. animals were randomly divided into two groups of plasmodium berghei (pb)-infected and intact (uninfected) mice. the chloroquine-resistant pb used was provided by the malaria research laboratories, institute for advance medical research and training, college of medicine, university of ibadan, nigeria. experimental mice were inoculated by intraperitoneal (i.p.) injection of 1 × 107 parasitised erythrocytes from donor mice of 18% – 20% parasitaemia level. plasmodium berghei strain was used because the model has gained wider scientific acceptance for its reproducibility and excellent mimicking of life cycle, structure, physiology and other crucial aspects of humans’ malarial parasites. it is one of the malarial parasites that infect mammals (murine rodents) other than humans. percentage parasitaemia was assessed by estimating the percentage of parasitised-red blood cells in giemsa-stained thin blood smears using a light microscope. behavioural tests were carried out in the intact and infected mice within days 3 and 5 of the experiment. at the end of the behavioural study, the animals were anaesthetised with ketamine hydrochloride (50 mg/kg; im) and a thoracoabdominal incision was performed to expose the heart. one ml nipro disposable syringe with 27-gauge needle was used for cardiac puncture and collection of blood. the blood was put in the heparinised eppendorf tube and centrifuged for 15 min at 3000 rpm. the plasma was extracted into a plain eppendorf tube, kept at -20 °c until use and was used to determine selected biochemical parameters. following blood collection, the mice were transcardially perfused with 20 ml ice-cold phosphate-buffered saline (pbs) and the brains were quickly extracted, homogenised with 0.25 m sucrose solution and centrifuged at 3000 rpm for 15 min. the supernatant was aspirated into plain eppendorf tubes and used to assay some brain-biochemical factors. drugs hydralazine hydrochloride (mkbs4863v), ketanserin (+)-tartrate salt (mkbr2179v), propranolol hydrochloride (bcbl8710v), phentolamine hydrochloride (085m4021v) and yohimbine hydrochloride (bcbm8231v) were procured from sigma-aldrich, st. louis, missouri, united states (us). the drugs were prepared in pbs prior to their i.p. administrations at doses of 5 mg/kg, 0.3 mg/kg, 2 mg/kg, 10 mg/kg and 10 mg/kg, respectively. the infected animals were observed until erythrocyte-stage of pb was established (this coincided with day 3 post-inoculation) and treatments with the drugs commenced immediately. the treatment followed a 6-dose regimen of 0, 8, 24, 36, 48 and 60 h for both intact and pb-infected mice. thirty minutes after the last treatments on day 5, relevant behavioural and biochemical tests were conducted. naloxone hydrochloride (samarth life sciences pvt. ltd., mumbai, india (inlxb1602)) injection (400 µg/ml) was administered via i.p. at a dose of 5 mg/kg whilst the controls were given 5 ml/kg of pbs. treatments were carried out twice daily (12-h interval), starting on day 3 post-i.p. inoculation of pb. thirty minutes after last treatments on day 5, pain-related behavioural responses were evaluated in all animals. relevance of the drugs to the study almost all the aforementioned drugs are antagonists meant to probe the contribution of precise receptors to malaria-related nociception and mortality. briefly, hydralazine hydrochloride was used to inhibit monoamine reuptake whilst ketanserin (+)-tartrate salt, propranolol hydrochloride, phentolamine hydrochloride and yohimbine hydrochloride were, respectively, used to block serotonin receptor type 2, all beta adrenergic receptors, all alpha adrenergic receptors and alpha receptor type 2. also, naloxone hydrochloride was used to block opioid receptors. assessment of weight, blood glucose and haematological parameters body weight the weight of each mouse was measured using a camry digital scale (camry, zhongshan, china). the scale sensitivity is 0.1 gram and has a maximum capacity to measure 2 kg. the mice were measured by placing each mouse on the centre-top of the scale. to avoid unwanted movement, the mouse was put in a light-weight perforated carton, covered and placed on the scale. prior to this, the carton was tared to zero. the display weight was recorded in gram (g). fasting blood glucose glucose level was assessed with an accu-chek active glucometer (roche diabetes care gmbh, mannheim, germany). glucose level was assessed from a drop of tail vein blood on the accu-chek active test strip inserted into the meter. value was displayed in mg/dl. haematological parameters this was performed using a sysmex kx-21n automated haematology analyer (sysmex america, inc., mundelein, us). about 0.8 ± 0.3 ml of blood sample was collected via cardiac puncture from each mouse into the tri-potassium ethylenediamine tetra-acetic acid (k3edta)-coated sample bottle. the whole blood volume needed is 50 µl, and detection principle for blood elements is the direct current detection method with automatic discriminators that separate the cell populations based on complex algorithms. for haemoglobin and haematocrit, the detection principles are non-cyanide method and cumulative pulse height detection method, respectively. sysmex cell counters have an exceeding uncompromised precision and accuracy regardless of the samples’ quantities or concentrations. kits serotonin and noradrenaline enzyme-linked immunosorbent assay (elisa) kits were from elabscience biotechnology co., ltd. wuhan, china, and were used to evaluate the level of these neurotransmitters in the plasma and brain. briefly, 50 µl of sample or standard was added to a microtiter plate that had been pre-coated separately with serotonin and noradrenaline. 50 µl of biotinylated detection antibody (ab) specific for either serotonin or noradrenaline was added to each well. the microtiter plate with its contents was incubated for 45 min at 37 °c. at the expiration of incubation time, excess conjugate and unbounded sample or standard were washed from the plate. this was immediately followed by addition of 100 µl of horseradish peroxidase to each microplate well and then incubated for 30 min at 37 °c. the incubation was followed by washing of the plate and addition of 90 µl of tmb substrate solution to each well. the enzyme-substrate reaction was allowed for 15 min and was stopped with sulphuric acid solution. the change in colour was quantified using a plate reader at a wavelength of 450 nm. the concentration of serotonin and noradrenaline was determined by comparing the absorbance to the standard curves. enzymes protein was measured as described by lowry et al. (1951); (lowry et al. 1951) using bovine serum albumin as the standard. activities of catalase, superoxide dismutase and reduced glutathione were evaluated using methods described by beers and sizer (1952) (beers & sizer 1952), misra and fridovich (1972) (misra & fridovich 1972) and jollow et al. (1974) (jollow et al. 1974), respectively. however, activities of ca2+ – mg2+ atpase and na+ – k+ atpase were assessed using graham and jack (1990) and jarrett and penniston (1977) methods, respectively (graham & jack 1990; jarrett & penniston 1977). evaluation of sensory pain sensation formalin pain test pain and its related behavioural response were evaluated using formalin test (hunskaar & hole 1987). briefly, 20 µl of 2.5% formalin in pbs was injected into the plantar surface of the right hind paw. each mouse was placed in a round transparent plastic (20 cm in diameter) or open field maze and videotaped for 50 min post-injection of formalin. the total time spent licking the injected hind paw (behavioural response to pain) was documented and used to quantify pain perception. in this model, increased pain behavioural response (paw licking, shaking and lifting) means increase in pain sensitivity or perception and vice versa. this interpretation is symmetrical to the one in the literature and its history of ethological validity remains consistent for decades (hunskaar, fasmer & hole 1985; tabata-imai, inoue & mori 2014; yin et al. 2016). this formalin pain model is preferred as it needed only one stimulus to elicit both neurogenic and inflammatory pain. the first 5 min after injection of formalin was taken as early phase (neurogenic pain) whilst the period between 20 min and 50 min post-injection was recorded as late phase (inflammatory pain). data were extracted in quadruplicate by trained observers blinded to the experimental design, and average values were calculated. foot stamp test immediately after the formalin test, the two hind limbs (formalin-injected and intact paw) were dipped into yellow and blue ink-soaked sponges, respectively. the mice were permitted to walk individually on a white paper placed in their respective resident cage for 2 min (nagae, hiraga & yoneda 2007). pain perception in mice was reported as % usage of formalin-injected foot, as shown below. von frey test: mechanical sensitivity was evaluated by measuring the threshold required for the withdrawal of the right hind paw (lambert, mallos & zagami 2009) against von frey filament. summarily, the ventral surface of hind paw was stabbed with the filament using feeble but enough pressure to cause bucking. the filament was removed after 5 s for any unresponsive mouse. for responsive animals, the value (calibration) on von frey filament was accepted as withdrawal threshold if the hind paw is withdrawn at minimum of five times from seven stimuli applied. cold plate test: each mouse was put in a perforated restriction glass chamber arranged on a thin glass surface platform. the restriction chambers were separated with blinds to prevent the animals from seeing each other. after acclimatisation, ice-pellet was applied on the right hind paw until the mouse exhibited pain-related behaviours such as lifting or moving away the affected limb. time taken to display any of these behaviours is taken as the end-point. the procedure was repeated three times with 2-min interval, and final mean value was documented for each mouse (kohan et al. 2002). assessment of motor function and pathological changes in the brain open field maze the motor function of the mice was assessed using a (40 cm × 40 cm × 40 cm) tiled floor 16 square cells open field maze. it was first described in 1932 and popularly used in the evaluation of anxiety in rodent. open field test can be adapted to assess nonanxiogenic phenomena such as memory, exploration and locomotor activity (deacon 2006). mouse was introduced to the centre square cell of the maze with the mouse not facing the handler. each mouse explored the novel environment for 5 min, and all activities displaced in the maze were captured by a mounted video camera just above the paradigm. as described by seibenhener and wooten (2015), the following indices for motor function were extracted by four persons blinded to the experiment and, average values of indices: ambulatory activity, rearing-time and frequency were recorded. similar or increased horizontal (ambulation) and vertical (rearing) locomotive behaviour with intact animals suggest motor competence (bichler et al. 2013; choi et al. 2017). assessment of pathological changes in the frontal cortex after pain test on day 4, mice were anaesthetised, perfused successively with cold pbs and 4% pbs-paraformaldehyde via cardiac puncture. brains were then removed, immersed in 4% pbs-paraformaldehyde and kept for 24 h at 4 °c. thereafter, tissues were processed using ascending grades of ethanol, xylene (for clearing) and paraffin wax (for embedding). frontal cortex was sectioned (5µm; mk 1110 rotary microtome) and stained with cresyl fast violet (cfv) and haematoxylin and eosin (h&e). also, immunohistochemistry was carried out on the sections of frontal cortex using anti-mouse-cd68 (1:100; bio-rad, united kingdom). basically, sectioned tissues were deparaffinised, underwent antigen retrieval and subjected to cd68 antibody interaction. photomicrographs of varied stained tissues were captured using the zeiss axiostar plus light microscope. statistical analysis unpaired t-tests, one-way anova with bonferroni’s post hoc test and log-rank (kaplan-meier) test were used. ibm spss statistics (software version 24, armonk, new york, us) and prism (graphpad software version 7.0, la jolla, california, us) were used for analyses. ethical consideration all procedures used were in accordance with the approval from the university ethical review committee (approval number: uerc/asn/2016/485), university of ilorin, ilorin, nigeria. these techniques were in harmony with relevant institutional guidelines and regulations for animal experiment and welfare. also, these guidelines are in strict compliance with the applicable section of national institutes of health guides (nih publications no. 8023, revised 1978) for the care and use of laboratory animals. results data of interest were assessed (at day 4 post-pb inoculation except otherwise stated) in quadruplicate, and the averages were accepted as value for each parameter. results as presented were from one experiment representative of two experiments performed. pb-induced malaria causes alteration in body weight, fasting blood glucose and haematological parameters in swiss mice intact animals gained significant weight when their initial weights were compared with their final weights. for pb-infected mice, there was a decrease in the final body weight compared to initial weights (figure 1a), although this was not significantly different. however, when weight difference was compared, there was a significant decrease in the body weight of pb-infected mice compared to intact mice (figure 1b). also, the fasting blood glucose level on day 5 significantly decreased in pb-infected mice when compared to intact mice (figure 1c). figure 1: body weight and blood glucose following pb infection in swiss mice. (a) final body weight compared with initial weight. (b) body weight difference preand post-infection periods. (c) fasting blood glucose level at day 5. similarly, red blood cell count (figure 2a), haemoglobin level (figure 2b) and platelet cell count (figure 2c) decreased significantly in pb-infected mice compared to intact mice. however, the white blood cell count (figure 2d), lymphocytes count (figure 2e) and platelet large cell ratio (figure 2f) were significantly increased in pb-infected mice compared to intact mice. figure 2: the haematological parameters of pb-infected and intact (uninfected) mice. (a) red blood cells count. (b) haemoglobin level. (c) platelets count. (d) white blood cells count. (e) lymphocytes count. (f) platelet large cell ratio. pb-infection attenuates pain perception pain as one of the common symptoms of malaria in human was probed in an animal model for malaria. on day 3 post-parasite inoculation, pain sensitivity was not statistically different in the infected mice compared to intact mice (figures 3a and b). on day 4, however, there was a significant decrease in sensitivity to neurogenic and inflammatory pain in the pb-infected mice compared to intact animals (figures 3c and d). this decrease in pain sensitivity was more striking on day 5 probably because the percentage parasitaemia was becoming more life-threatening. precisely, there was a significant decrease in pain perception in pb-infected animals compared to intact mice (figures 3e and f) during both early and late phase of formalin test. to investigate the trend of pain perception within the 3 days of purview, the ratio of behavioural responses to formalin pain in pb-infected and intact mice was plotted and it showed a decline in perception curves for neurogenic and inflammatory pain (figures 3g and h). the percentages of parasitaemia within these days were 12.54% (day 3), 20.35% (day 4) and 39.11% (day 5); (figure 3i). figure 3: pb-induced analgesia in mice. (a) and (b) time spent on paw licking during the early (0–5 min) and late (20–50 min) phase of formalin test on day 3 post-pb inoculation (early phase and late phase), respectively. (c) and (d) time spent on paw licking during the early (0–5 min) and late (20–50 min) phase of formalin test on day 4 post-pb inoculation (early phase and late phase), respectively. (e) and (f) time spent on paw licking during the early (0–5 min) and late (20–50 min) phase of formalin test on day 5 post-pb inoculation (early phase and late phase), respectively. (g) and (h) successive trend of pain sensitivity during the neurogenic and inflammatory pain (early phase pain perception and late phase pain perception), respectively. (i) percentage parasitaemia level on days 3, 4 and 5. to ascertain that the observed analgesic-like effect recorded in pb-infected mice was not because of compromised motor function, motor activities were simultaneously assessed with formalin pain test on open field maze at day 4 post-pb inoculation. although ambulation (horizontal movement; figure 4a) and rearing behaviours (vertical movement; figures 4b and c) were significantly decreased in pb-infected mice compared to intact mice during the early phase of formalin test (0–5 min), these behaviours improved better at the late pain phase (20–50 min), showing that the motor function in the infected mice (figures 4d, e and f) was relatively viable. also, a significant (p < 0.05) increase in ‘latent period’ (duration between injection of formalin and the first paw-licking behaviour) was observed in the pb-infected animals compared to the intact ones (figure 5a). again, both early (figures 5b and c) and late (figures 5d and e) pain-related behaviours (paw licking time and frequency) decreased significantly in pb-infected animals compared to intact mice, corroborating the earlier observations. to affirm the observed ameliorated pain perception, foot stamp test was carried out. when the plantar surface of the hind paw is injected with formalin solution (1% – 5%), a mouse with normal sensory perception uses more of its foot-tip than the full footprint (figure 5f). the full footprint count for the injected limb compared to the intact limb was not significantly different in the pb-infected mice (figure 5f). in the intact mice, there was a significant decrease in the full footprint counts of the injected foot (figure 5g) compared to intact foot. between the two groups however, pb-infected mice exhibited a significant increase in full-foot (injected paw/intact paw) count compared to the intact mice (figure 5h). lastly, we attempted to investigate whether decreased pain sensitivity was limited to noxious chemical only, and von frey test and cold-plate test were carried out. the results also showed a significant decrease in pain perception to noxious mechanical and thermal stimuli in pb-infected mice compared to the intact animals (appendix 1, figures 1-a1[a] and 1-a1[b]). figure 4: motor behavioural indices in pb-induced malaria. (a) number of line crossing during early phase of formalin test. (b) and (c) time spent and frequency of rearing during the neurogenic pain phase of formalin test, respectively. (d) number of line crossing during late (20–50 min) phase of formalin test. (e) and (f) time spent and frequency of rearing during the inflammatory pain (late) phase of formalin test, respectively. figure 5: pb-induced malaria ameliorates chronic pain perception. (a) ‘latent period’: time taken to make the first paw lick behaviour after formalin injection. (b) early phase paw-licking (0–5 min) time during formalin test. (c) frequency of paw licking during the early phase. (d) late phase paw licking (20–50 min) duration during formalin test. (e) frequency of paw licking during the late phase. (f) number of full footprint of injected paws in pb-infected mice. (g) number of full footprint of injected paws in intact mice. (h) tolerability for using injected paw to intact paw. when propranolol and phentolamine hydrochloride were administered to probe adrenergic involvement, similar pattern of diminished pain sensitivity was exhibited during early and late phase of formalin test in the pb-infected mice compared to intact mice (figures 6a–h). also, treatment with yohimbine hydrochloride (α2 – antagonist) demonstrated similar decrease in pain-related behaviours except that paw licking time at the early phase was not significantly different in pb-infected mice compared to intact animals (figures 6i–l). serotoninergic probing with ketanserin hydrochloride (5-ht2ar antagonist) showed similar significant decrease in early phase pain behaviour in the pb-infected mice (figures 6m and n) compared to intact animals but pain sensitivity at the late phase was not significantly different in pb-infected mice compared to intact animals (figures 6o and p). however, the analgesic-like effect in pb-infected mice was blocked following naloxone treatment (figures 7a–d). this made us to assume that naloxone probably had anti-malarial effect. our investigation of this assumption showed almost no difference in percentage parasitaemia between pb-infected mice and naloxone-treated animals (figure 7e). figure 6: role of adrenergic and serotoninergic system in pb-induced analgesia. (a) and (b) time spent and frequency of paw licking at the early phase (0–5 min) of formalin test in propranolol hydrochloride-treated mice, respectively. (c) and (d) time spent and frequency of paw licking during the late phase (20–50 min) of formalin test in propranolol hydrochloride-treated mice, respectively. (e) and (f) time spent and frequency of paw licking at the early phase (0–5 min) of formalin test in phentolamine hydrochloride-treated mice, respectively. (g) and (h) time spent and frequency of paw licking at the late phase (20–50 min) of formalin test in phentolamine hydrochloride-treated mice, respectively. (i) and (j) time spent and frequency of paw licking during the early phase (0–5 min) of formalin test in yohimbine hydrochloride-treated mice, respectively. (k) and (l) time spent and frequency of paw licking during the late phase (20–50 min) of formalin test in yohimbine hydrochloride-treated mice, respectively. (m) and (n) time spent and frequency of paw licking at the early phase (0–5 min) of formalin test in ketanserin hydrochloride-treated mice, respectively. (o) and (p) time spent and frequency of paw licking during the late phase (20–50 min) of formalin test in ketanserin hydrochloride-treated mice, respectively. figure 7: impact of naloxone on pain sensitivity and survival time in pb-infected mice. (a) and (b) time used for paw licking and its frequency during the early phase (0–5 min) of formalin test, respectively. (c) and (d) time spent on paw licking and its frequency during the late phase (20–50 min) of formalin test. (e) percentage parasitaemia level. (f) survival time in pb-infected mice. pain has little or no contribution to malaria-related death to investigate contribution of pain system in malaria-related mortality, various drugs known to interfere with pain processing system were used to investigate animals’ survival time. there was no significant difference in the survival time between animals treated with acetaminophen, pentazocine, ibuprofen, phentolamine and naloxone compared with placebo (pbs)-treated pb-infected group. however, about 20% of hydralazine-treated animals survived till day 9, about 48 h after the death of animals in other groups (figure 7f). pb-induced malaria elicits changes in selected atp-hydrolytic enzymes, neurotransmitters and oxidative enzymes in swiss mice we then investigated enzymes necessary for the utilisation of metabolic energy in the brain of the experimental mice. na-k atpase and ca-mg atpase were not significantly altered in pb-infected mice compared to that of intact mice (figures 8a and b). the protein level was not different either (figure 8c) but there was a biological increase in the brain serotonin level for the pb-infected mice compared to intact mice (figure 8d); this, however, is not statistically different. also, the noradrenaline level in the pb-infected mice was not significantly different compared with the intact mice (figure 8e). in the plasma, serotonin level in the pb-infected mice increased significantly compared with the intact animals (figure 9a). also, catalase and glutathione peroxidase activities decreased significantly in pb-infected mice compared to intact mice (figures 9b and c). plasma noradrenaline, protein level and superoxide dismutase activity in pb-infected mice were not significantly different compared with the intact mice (figures 9d, e and f). figure 8: biochemical changes in the brain of pb-infected swiss mice. (a) and (b) brain activities of na+ – k+ atpase and ca2+ – mg2+ atpase, respectively. (c) total brain protein level. (d) and (e) levels of serotonin and noradrenaline in the brain, respectively. figure 9: changes in neurotransmitter and antioxidant enzymes in the plasma of pb-infected swiss mice. (a) plasma serotonin level. (b) and (c) activities of catalase and glutathione peroxidase, respectively. (d) plasma noradrenaline level. (e) total plasma protein level. (f) superoxide dismutase activity. pb induces slight morphological change in the brain to exclude the possibility that experimentally induced cerebral malaria was responsible for the observed difference in pain sensitivity on day 4, sections of the brains were surveyed for activated microglia using antibody to macrophage marker cd68. following injury or alterations in their parenchyma microenvironment, resident microglia in healthy brain are rapidly activated and assumed typical morphological thickening with rounded amoeboid shape. areas involved in pain, fear (cingulate cortices) and locomotor (primary motor cortex) were primarily evaluated. in each three representative mouse, cd68 revealing macrophages were undetectable either along the borders of four serially sectioned frontal cortices or within the core areas of cingulate cortices (figures 10a–d) and primary motor cortex (figures 10e–f). absence of this transmembrane glycoprotein suggested that both intact and pb-infected mice enjoyed a relatively quiescent microenvironment. cell morphology was also examined using haematoxylin and eosin-stained sections. cingulate cortices (figures 11a–d) and primary motor cortex (figures 11e–f) stained intensely and showed a mixture of smallto medium-sized neurons. the neuronal cells appeared to have symmetrical cell distribution and cytoarchitecture. sections from intact mice exhibited copious intact cells with centralised nuclei (yellow arrow) in contrast to sections from infected mice where fewer cells appeared ghost-like (black arrow) with nuclei pushed to the periphery. all tissues (figures 11a–f) showed fewer resting microglia (white arrow). nuclei of resting microglia are recognised with their dark stain, distinguished rod-, elongatedor ‘cigar’-shaped and usually irregularly contoured. cresyl fast violet-stained sections showed prominent cell bodies (black arrow) that signify abundant ribosomes attached on rough endoplasmic reticulum in the cingulate cortices (figures 12a–d) of intact and pb-infected animals, although nissl substance in cingulate cortex area 2 of pb-infected mice (figure 12d) appeared pale (red arrow). also, primary motor area in the intact animals (figure 12e) showed copious cell bodies (black arrow) compared to the pb-infected (figure 12f) mice where fewer pyramidal cell bodies appeared faintly stained and assumed round-shaped (red arrow). figure 10: microglial immunostaining within the frontal part of the cerebral cortex. (a) and (b) anti-cd68 immunostaining sections of cingulate cortex area 1 in intact and pb-infected mice, respectively. (c) and (d) staining cingulate cortex area 2 with anti-cd68 antibody in intact and pb-infected mice, respectively. (e) and (f) respective immunostaining of the primary motor area with anti-cd68 antibody in intact and pb-infected mice. there was no immunopositive cell in all the parts of the brain investigated with the marker for cerebral malaria. figure 11: neuronal histochemistry of cerebral cortex. (a) and (b) haematoxylin and eosin-stained sections of cingulate cortex area 1 in intact and pb-infected mice, respectively. (c) and (d) neural cells distribution in cingulate cortex area 2 in intact and pb-infected mice, respectively. (e) and (f) morphology of neural cells at the primary motor area of intact and pb-infected mice in haematoxylin and eosin-stained sections, respectively. despite both pb-infected and intact mice appeared to have symmetrical cytoarchitecture and cell distribution, several nerve cells appeared fading with centralised nuclei (yellow arrow). there are also few neural cells that appeared ghost-like (black arrow) with peripheral nuclei, yet most of the microglia present (white arrow) are not activated. figure 12: neuronal population within the midline of brain. (a) and (b) cresyl violet-stained sections of cingulate cortex area 1 in intact and pb-infected mice, respectively. (c) and (d) nissl substance distribution in cresyl violet-stained sections of cingulate cortex area 2 in intact and pb-infected mice, respectively. (e) and (f) primary motor cortex in intact and pb-infected mice stained with cresyl violet, respectively. note, almost all the cell bodies stained remarkably (black arrow) although few of them appeared pale and assumed round-shaped (red arrow). discussion the present study established pb-induced analgesic-like effect in an animal model for malaria during life-threatening stage. this analgesic-like effect was enhanced with daily increase in parasitaemia level, and it was shown by a striking diminished pain-related behaviours. the effect seems to have central synergistic contributions of α2, µ-opioid and 5ht2a receptors. this assertion was drawn from the facts that when antagonists of α2, µ-opioid and 5ht2a receptor were used, there was increase in pain-related behaviours (that is, improved in pain sensitivity) in the pb-infected mice during the neurogenic (early) and or inflammatory (late) phase of formalin pain test compared to intact animals. precisely, nociceptive behaviour in pb-infected mice was improved with naloxone (µ-opioid antagonist; both early and late phases), yohimbine hydrochloride (α2 antagonist; early phase) and ketanserin hydrochloride (5ht2a antagonist; late phase). also, results from this study showed little or no contribution of the pain processing system on malaria-related death in infected mice. survival time was similar to mice treated with drugs capable of dampening or enhancing pain perception. this study is important as it captures the neglected spectrum of neural physiology that precedes experimental cerebral malaria and death in animal model for malaria. despite accumulated evidence that showed close similarity between animal models for malaria and malaria presentation in humans, the present findings showed that neural handling of sensory (pain) sensation in this animal model contrasts the one obtainable in humans. also, malaria infection abolished reported sex-difference responses to noxious stimuli in mice. evidence-based studies demonstrated that parasitic infection can both increase and decrease pain perception (huang et al. 2016; kavaliers & colwell 1993). rodent-specific malarial parasite, plasmodium berghei, consistently showed a decreased response to neurogenic and inflammatory pain with daily proliferation in mice. this contrasts obtainable claims in human studies where malaria-linked pain was widely reported (sarma & kumar 1998; reisinger et al. 2005; zaki 2010), and pb parasites or their products at a critical stage mimicked analgesic effects in mice. the observed reduction in response to pain sensation seems to be associated with parasitaemia level. infectious parasite-induced analgesic-like effect was also reported in eimeria vermiformis and schistosoma mansoni infection (fiore et al. 1996; kavaliers & colwell 1993, 1994). the critical point when parasitic anti-nociception begins in this animal model for malarial infection seems to fall between > 12.54% and ≤ 20.35% parasitaemia level. this was drawn from this study based on the fact that on day 3 post-pb infection (at 12.54% parasitaemia level), pain-related behavioural responses to noxious formalin were not significantly different in pb-infected mice compared to the intact mice. but 24 h later (day 4, at 20.35% parasitaemia level), response to noxious chemical (2.5% formalin) decreased in pb-infected mice relative to the intact mice. this critical point (> 12.54% and ≤ 20.35% parasitaemia level) was limited by inability to establish the specific time and parasitaemia level at which the decline in pain sensitivity commenced. notwithstanding, the critical period was hypothesised to coincide with experimental cerebral malaria in infected animals. the hypothesis was tested via investigating the presence of cd68 immunopositive cells in the brain sections of pb-infected mice and also by examining other morphological alterations in h&eand cfv-stained sections. cd68 revealing activated macrophages was unexpressed either in intact or pb-infected mice. resting microglia were noted in all h&e-stained sections (intact and pb-infected), and there was no remarkable morphological or cytoarchitecture variation that could be linked to experimental cerebral malaria. although there was slight dispersed nissl substance in the cingulate cortex area 2 and primary motor cortex of pb-infected mice, the stained cell bodies still showed the presence of sufficient amounts of nissl body. for the present study, pain-related behaviours are defined as different measurable series of well-coordinated, repetitive and reproducible muscular exertions displayed in response to noxious stimuli. hence, these muscular exertions-linked behaviours can be affected in animals with compromised motor function. to ascertain viability of motor function in the infected mice, pain test was carried out in a novel open field arena. the maze has been reported to be useful in mood and motor function test (albert & istvan 2014). our finding is in conflict with few claims in the literature where increased locomotion was associated with suppressed pain perception (i.e. decreased pain sensitivity) (carstens & moberg 2000; sheahan et al. 2017). specifically, despite increase in pain sensitivity (suppressed pain) in pb-infected mice, early phase vertical (rearing) and horizontal (ambulation) locomotor activities were significantly decreased compared to the intact animals. however, during the late phase (20–50 min) of the formalin test, the intact animals spent much of their time on paw licking as against the pb-infected mice; thus, the horizontal and vertical locomotor activities in the pb-infected animals improved and were not significantly different compared to the intact mice. together, these results showed that the motor function in the pb-infected mice was still viable but might not be at its optimal performance capacity. also, histological evaluation of primary motor area showed minor changes that could not be described as dysfunctional morphological alterations. these findings reinforce the claim for partial competence of the motor command generating area in the pb-infected mice. again, decreased pain sensitivity to noxious chemical (formalin) was re-demonstrated in mice and similar declined pain sensitivity was established in mechanical and thermal pain test (see appendix 1, figures 1-a1[a] and 1-a1[b]). the malaria-induced analgesic-like effect was, however, abolished in naloxone-treated mice without beneficial effect on the percentage parasitaemia level, thus showing possible involvement of opioidergic receptor. naloxone blocks opioid receptors (chen, chen & mao 2014), and it enhances response to pain when administered at high doses in animals (mena, mathur & nayar 1996) or human beings (levine, gordon & field 1979). despite compelling reports on the roles of adrenergic system in pain modulation via its α and β receptors (carroll, mackey & gaeta 2007; takashi et al. 2017), antagonists of these receptors could not reverse the observed pb-linked analgesia. instead, the nonselective beta-receptor antagonist seemed to have further ‘hyperpolarised’ the pain system by almost eliminating early phase response to pain stimulus in infected mice and completely abolished pain-related behaviours during the late phase of formalin test. to the best of our knowledge, we could not find similar report in humans. it will be good to investigate whether people managing their high blood pressure with propranolol do experience a remarkable decrease in pain sensitivity during malaria attack. however, propranolol had been shown to decrease pain via its actions on β receptors in humans and rodents (exposto et al. 2015; petersen et al. 2018; schweinhardt et al. 2013). unlike the action of propranolol on β receptors, the alpha (α) receptors component of adrenergic system showed minimal or no effect on the observed analgesia when phentolamine hydrochloride (nonselective α-receptor antagonist) and yohimbine hydrochloride (α2-receptor antagonist) were used. the action of α2-receptor blocker in the amelioration of pain sensation as previously reported in animal studies (pertovaara 1993; takashi et al. 2017) was recorded in pb-infected mice at the early phase of formalin test but not during the late phase probably because of the short half-life of the antagonist. also, pb-induced analgesia became perturbed at the late phase when 5-ht2a receptor antagonist was used, suggesting that α2and 5-ht2a-receptor might contribute to the anti-nociceptive-like effects. why 5-ht2a receptor antagonist used could not elicit similar effect during the early phase of formalin test is not known. however, action of the 5-ht2a receptor in pain modulation remains controversial. compelling evidence showed that the 5-ht2a receptor could enhance (abbott, hong & blier 1996; cervantes-duran, rocha-gonzalez & granados-soto 2013) or ameliorate (honda et al. 2006; li et al. 2013) pain perception in humans and rodents. it is, therefore, possible that synergistic effects of pb parasites and/or their products on opioidergic, adrenergic and serotoninergic receptors in the host led to increased non-responsiveness to noxious chemical during a critical stage of pb infection in mice. the present study also showed a result on delayed perception of pain in the infected animals upon injection of formalin, a sharp contrast to what was observed in intact mice. profound suppression of pain sensation to its minimum level without major alteration of the motor function demonstrated that only neural circuitry for pain seemed to be affected by the parasites and/or their products. the possibility of affected circuitry contributing to malaria-linked mortality cannot be overlooked. investigating this, our results showed that there was no significant difference in the survival time for the infected animals treated with drugs capable of causing alteration in pain sensitivity or perception compared to placebo-treated mice. however, the monoamine-reuptake inhibitor (hydralazine hydrochloride) appeared to increase survival time in the hydralazine-treated mice compared to the placebo group. the survival of about 20% of these mice for extra hours could not be described as remarkable, and data were not sufficient to extrapolate any possible usefulness in preventing malaria-related death. despite the fact that naloxone treatments blocked pb-induced analgesic-like effect, there was no difference in the survival time relative to the placebo group. combined, pain processing system contributes little or no input on malaria-related death in mice. also, evaluation of basic secretory and metabolic activities of the whole brain cells showed that protein level, noradrenaline and activities na+ – k+ atpase and ca2+ – mg2+ atpase were not significantly different in the pb-infected mice compared to the intact mice. the level of serotonin was, however, increased in the brain of pb-infected mice, although this was not significantly different compared to intact animals but it suggested possible elevation in the biosynthesis of this neurotransmitter. the action of serotonin in the pain modulatory system was claimed to be either attenuation or facilitation of pain signalling (rahman et al. 2011; viguier et al. 2013) and either effect can be abolished with serotonin receptor antagonists. based on the activity of oxidative enzymes evaluated in the plasma, infected animals appeared to have decreased antioxidant enzymes activities compared to the intact mice. also, total plasma protein and noradrenaline levels were not statistically different in the pb-infected mice relative to intact animals. again, plasma serotonin was increased in the pb-infected mice compared to intact animals. the increased plasma serotonin might possibly be from lysed platelet cells because estimated platelet cell count was significantly decreased in the pb-infected mice compared to the intact mice. in conclusion, the present studies attempted to elucidate the influence of host-pb interactions on pain sensitivity and its contribution to malaria-related death. the study showed that the nociceptive processing system of malaria-infected mice has little or no input in malaria-related mortality, and pb-induced malaria elicited analgesic-like effect (decreased pain-related behaviours to noxious stimulus) during critical stage in the host animals. the parasites capacity to interfere with pain sensation appeared to be orchestrated via µ-opioid, α2 and 5ht2a receptor as blockage of these receptors elicited increased pain-linked behaviours (at early and/or late phase of formalin test) to noxious stimulus in pb-infected swiss mice. owing to all these evidence-based observations, animal model for malaria may not be suitable for investigating malaria-related pain as reported in humans. acknowledgements the authors wish to thank o. akinola for generous donation of pb, and also the following, a. alli-oluwafuyi, a.b. nafiu and f.a. sulaimon for their advice during laboratory work as well as useful criticism of draft manuscript. competing interests the authors have declared that no competing interests exist. authors’ contributions a.l.o. performed the experiments, analysed data and wrote the manuscript. o.a. supervised the histological studies section, contributed to writing and reviewing of the manuscript. b.v.o. conceived the ideas, supervised the experiments, acquired and analysed data and wrote the manuscript. funding information this study was funded by institution based research fund (uil/credit/30, from tetfund, nigeria), staff development award (uil/sse/pf/5358) from the university of ilorin, nigeria and aau small grant for thesis and desertification (pc/6) from the association of african universities. data availability statement all data will be made available on request directed to the corresponding author. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily 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(a) latency to withdraw paw during cold plate test. (b) withdrawal threshold in von frey test. mugunieri_279-284.qxd introduction dairy production in kenya is dominated by smallholder farmers who contribute about 85 % of total milk produced (peeler & omore 1997). it is estimated that the dairy herd in kenya comprises about 3 million head of cattle, of which 2.5 million are owned by smallholders numbering about 650 000 (moa 1996). this implies that this sector employs many people who derive a regular source of income and have a balanced nutrition. the sector is a catalyst for economic development and, when compared to other agricultural activities, it has higher financial returns (mcintire, bourzat & pingali 1992). dairy cattle production constraints have been extensively described by various authors (stotz 1983; moll, lohding & young 1984; maloo, thorpe, ngumi & perry 1994; peeler & omore 1997). these studies have identified livestock diseases as one of the major constraints to dairy production in kenya. major diseases cited are tick-borne diseases (tbds) and trypanosomosis. some field studies have attempted to ascertain the actual incidence and impact of tbds on dairy production (gitau, o’callaghan, mcdermott, omore, odima, mulei & kilungu 1994; maloo et al. 1994). however, there exists only very limited information that measures the losses associated with trypanosomosis and the benefits that can accrue after its effective control (mbogoh & mukhebi 1997). this study provides some information on the losses associated with trypanosomosis among dairy farmers in western kenya which could be used in designing sustainable strategies for managing the disease in future control programmes in this region. 279 onderstepoort journal of veterinary research, 72:279–284 (2005) association of trypanosomosis risk with dairy cattle production in western kenya g.l. mugunieri* and g.o. matete kari – trypanosomosis research center, p.o. box 362, kikuyu 00902, kenya abstract mugunieri, g.l. & matete, g.o. 2005. association of trypanosomosis risk with dairy cattle production in western kenya. onderstepoort journal of veterinary research, 72:279–284 dairy cattle reared in western kenya are exposed to medium to high levels of trypanosomosis risk. the social background, farm characteristics and dairy cattle productivity of 90 and 30 randomly selected farmers from mediumand high-risk trypanosomosis areas, respectively, were compared. all the 120 farmers were visited between july and august 2002. data analysis was performed using descriptive statistics and analysis of variance. the results showed that increased trypanosomosis risk represented by an increase in disease prevalence in cattle of 1 % to 20 % decreased the density of dairy cattle by 53 % and increased the calving interval from 14 to 25 months. the increased risk was also associated with a significant increase in cattle mortalities and in a lactation period of 257 to 300 days. it was concluded that removal of the trypanosomosis constraint on dairy production would lead to expansion of dairying since the domestic demand for dairy products is expected to increase. keywords: dairy cattle, kenya, production, trypanosomosis risk * author to whom correspondence is to be addressed. e-mail: ketri@africaonline.co.ke accepted for publication 14 june 2005—editor materials and methods study area the study region was stratified into two zones having different levels of trypanosomosis risk based on the prevalence of the disease in cattle for the period 1996–1999. it was assumed that the trypanosomosis risk prevailing between these years influenced production decisions of the subsequent years under study. following the outbreak of bovine trypanosomosis in western kenya in the late 1980s (angus 1996), the kenya trypanosomiasis research institute (ketri) initiated routine trypanosomosis surveillance studies in this region, under a larger programme that was funded by the department for international development (dfid) of the united kingdom (ketri 1994). under this programme cattle, randomly sampled in selected villages on a quarterly basis, were screened for the presence of trypanosome parasites, using the standardized protocols for the microhaematocrit centrifugation-buffy coat technique, and thick and film blood smears (woo 1969). on the basis of this data, this investigation categorized the study region into zones of medium and, from which farmers were sampled for data collection. a sampling frame consisting of dairy farmers in each zone was constructed with the assistance of local animal health and provincial administration staff and village elders. the medium risk zone consisted of three administrative divisions with an estimated area of about 377 km2 and had 238 dairy farmers. the high-risk zone comprised one administrative division with an area of about 92 km2 and had 39 dairy farmers. from this list of farmers, 120 were randomly sampled, 90 being from the mediumrisk zone and 30 from the high-risk area. the sample size was determined by resource availability. information on each of these trypanosomosis risk zones is given in table 1. data collection each of the 120 farms was visited once between july and august 2002. during the visit, a comprehensive pre-tested questionnaire was administered. data were collected on the individual farmer and farm characteristics, cattle nutrition and grazing management, farm enterprises, farm inputs and revenues, and general livestock management issues. a detailed account of each commodity produced on the farm listing all inputs and their costs, outputs and prices was tabulated. data on the management practices were verified by physical inspection of assets and farm records at the time of the interview. all the farmers under survey were enrolled under the national dairy development project (nddp), and farm kept records detailing milk production, fertility performance and mortalities. data analysis descriptive statistics were used to describe and summarize dairy cattle density and productivity indicators. analysis of variance was used to compare productivity of livestock under different levels of trypanosomosis risk. productivity analysis was based on the premise that improved livestock health management may increase productivity of cattle (card 1989; mugunieri, omiti & irungu 2004). the impact of trypanosomosis risk on the success that cattle keepers have achieved while engaging in dairy production can be obtained by: (a) comparing the peak milk output per animal under different trypanosomosis risk all other factors being equal between the two zones, the peak milk output per animal was taken as a proxy for productivity of animals reared by farmers under varying trypanosomosis risk levels. the measurements of milk production were obtained from the records kept by farmers, all of whom were enrolled under the nddp. the peak milk output average (for all animals kept) was compared across the two trypanosomosis risk levels. (b) calculating the calving rate under different trypanosomosis risk conditions this study estimated the ratio of births to the number of adult females in the herd in each calendar year between 1999 and 2002. this was named the “calving rate” (cr). cr = lb/af, where cr = calving rate, lb = number of births in a specified period and af = number of adult females in the herd in a specified time period. (c) comparing dairy cattle densities under different trypanosomosis risk the number of dairy animals per farmer was estimated over a period of 3 years (1999, 2000 and 2001). the average of the annual means for the 3 years expressed as total livestock units (tlu) was taken as the average number of dairy animals per farmer. the computation of the tlu was based on figures in ilca (1990). this was then multiplied by the total number of farmers engaging in dairying to yield the approximate total number of dairy animals for each region. the ratio between this computed value and size (area) of the region in km2 gave an 280 association of trypanosomosis risk with dairy cattle production in kenya indicator of the dairy cattle density (dcd) per km2 for each trypanosomosis risk region. results and discussion farmer and farm characteristics under varying trypanosomosis risk the farmer and farm characteristics across the two trypanosomosis risk zones are given in table 2. farmers were on average 47 years old and owned 6.6 acres of land. there were no significant differences in distances to the nearest market and artificial insemination or breeding bull services across the two trypanosomosis risk zones. there were no significant differences in the number of visits made to attend to tbds. there was also 100 % positive response with regard to use of acaricides in the management of ticks. this implied that tbds may not have been confounding factors in variation in productivity and practices between the two risk zones. trypanosomosis treatment visits were also not significantly different between the two risk areas, but there appeared to have been a more rigorous application of insecticides for vector control in the high-risk areas. it was difficult to compare the costs incurred in treatment of these ailments as farmers were served by different cadres of veterinary personnel who charged different fees even for similar recorded cases. the predominant grazing system practised in the high-risk trypanosomosis area was a small-scale, intensive grazing system, alternatively called the zero-grazing system. this system was used by 28 % and 87 % of farmers in the mediumand highrisk areas, respectively. the remainder of the farmers used the semi-intensive system that is also called the paddock-grazing management (peeler & omore 1997). the zero-grazing system was developed in kenya to solve the constraint of land scarcity and improve milk production (de jong 1996). it is a capital and labour intensive system where exotic breeds and their crosses are kept under intensive grazing, mainly for commercial dairy purposes. animals are confined in housing units where they are stall fed with fodder cut from the field. manufactured feeds are also widely used, especially at milking. on the other hand, under the paddock system, exotic cattle breeds and their crosses graze on natural pastures usually supplemented with cultivated fodder. purchased inputs such as manufactured feeds and veterinary services are widely employed in the production process. it appears that increased trypanosomosis risk necessitated investments in capital281 g.l. mugunieri & g.o. matete table 1 summary information on study divisions division year villages mean number of average annual ascribed dairy farmers (size km2) sampled per cattle sampled per prevalence trypanosomosis population visit* visit (%) risk size malakisi 1996 4 48.7 (24.39) 0.25 (0.50) medium 82 (101.4) 1997 4 73.5 (7.76) 4.75 (5.62) 1998 3 57.7 (14.84) 2.67 (1.15) 1999 4 70.0 (14.10) 6.75 (1.71) matayos 1996 4 59.2 (18.94) 4.50 (4.20) medium 78 (173.7) 1997 4 55.5 (24.37) 3.00 (2.16) 1998 4 53.7 (26.92) 5.75 (2.87) 1999 4 105.2 (42.18) 3.00 (1.15) amagoro 1996 4 64.5 (19.82) 21.00 (19.35) high 39 (91.7) 1997 8 61.2 (32.65) 19.50 (11.86) 1998 8 52.4 (24.30) 18.00 (12.52) 1999 8 68.6 (27.34) 26.00 (15.06) funyula 1996 3 83.3 (51.14) 4.00 (3.46) medium 70 (101.4) 1997 4 38.7 (21.53) 7.00 (2.71) 1998 4+ 50.0 4.50 (4.12) 1999 4 100.0 (49.21) 3.75 (1.26) values in parenthesis are standard deviations sources: angus 1996; ketri 1999 * four visits made per year + data for one visit only intensive grazing systems in the form of construction of dairy cattle housing units, when an opportunity existed to use the less capital-intensive system by utilizing the most abundant resource (land). the use of the zero-grazing system when land was not a constraint, acted as an insurance against increased production risk by minimizing animal-tsetse contact. this implied that dairying became an enterprise for households with higher income, which was supported by the fact that off-farm income of dairy farmers in high-risk trypanosomosis areas was significantly higher. dairy cattle density, calving rate and milk productivity the average dairy herd size was 3.2 animals (sd = 2.4) (table 3), computed as the three-year average of the annual means that were derived for the years 1999, 2000 and 2001. the herd consisted mainly of mature cows, heifers and calves. the dcd was 1.5 tlu in the mediumrisk areas compared to 0.98 tlu in the high-risk zone. the mean peak milk output per animal (computed as the mean of peak milk yield attained per cattle) was significantly higher (p < 0.05) for the high-risk trypanosomosis area. based on the computation of the calving rate, an average of 0.8 births per female per year was recorded for the mediumrisk trypanosomosis areas as compared to 0.5 births per female per year for the high-risk areas. this translated to a calving interval of 14 months for the medium-risk as compared to 25 months for the highrisk areas. consequently, the average lactation period for cattle in the medium-risk area was significant282 association of trypanosomosis risk with dairy cattle production in kenya table 2 farm and farmer characteristics under varying trypanosomosis risk trypanosomosis risk characteristic (means/frequency) medium risk (n = 90) high risk (n = 30) land size (acres) 7.86 7.80 household size (number) 8.43 11.03 crop gross margin per acre per year 427 503 off-farm income per month (kshs) 2 083* 8 303* pasture feed size (acres) 1.76 1.36 years of experience in dairying 8.90 5.80 mature female dairy animals 2.45+ 1.80+ household head age (years) 50.7 46.7 tbds treatment visits (2001/02) 3.06 3.83 trypanosomosis treatment visits (2001/02) 3.42 3.43 trypanocidal drugs use (% frequency of farmers who used dugs) 58 63 insecticide applied on cattle (% frequency of farmers who used pour-on) 28 60 values with the same superscript are significantly different between groups (p = 0.05) table 3 relation between dairy cattle density indicators and trypanosomosis risk level of trypanosomosis risk characteristic medium high size of region (km2) 539.3 91.7 households in the region (no.) 34 951 7 617 population in the region 165 103 33 954 population density (persons per km2) 306.1 370.3 dairying households (approximate) 230 39 mean hh dairy cattle numbers (3-year average for means) 3.56* (sd + 2.5; n = 93) 2.30* (sd + 2.10; n = 29) dairy animals (approximate) 819 90 dairy cattle density (dcd) (no. per km2) 1.5 0.98 household undertaking dairying (%) 0.70 0.50 peak milk output (l/day) 7.3* (sd + 4.0) 9.6* (sd + 4.6) lactation period (days) 257.64+ (sd + 32.95) 300+ (sd + 73.93) calving ratio (cr) 0.83* (sd + 0.25) 0.47* (sd + 0.30) deaths per farmer per year (3-year average) 0.009* (sd + 0.05) 0.052* (sd + 0.12) values with the same superscript are significantly different between groups (p = 0.05) ly shorter. the average mortalities per farmer were significantly higher in the high-risk area. the differences in the peak milk output per day attained between the two trypanosomosis risk zones could be due to two factors. this could be a proxy of the quality of animals reared between the two zones. alternatively, the quality of animals could have been the same but differences in productivity could be due to the intensive production systems employed by farmers in the high-risk area. the longer lactation period and higher mortality rates in the high-risk areas may have been due to the impact of the disease. this is in concordance to the knowledge that increase in trypanosomosis risk often prolongs the calving interval due to abortions, early embryonic deaths and silent heat (swallow 2000). other factors that might influence the calving interval, such as physical access to breeding services (artificial insemination and bulls), were not significantly different. conclusion the dairy sector in kenya is the most developed in eastern and central africa, making the country self sufficient in major dairy products, except in periods of drought (omiti & muma 2001). however, it is projected that by the year 2010, demand for dairy products will outstrip supply if urgent measures to sustain growth in production are not undertaken (peeler & omore 1997). expanding production in areas with potential that are currently under-exploited provides one of the avenues to increase production. trypanosomosis is one of the major constraints limiting production in these areas. this study has shown that trypanosomosis risk alters the grazing management system used and makes dairy farming expensive. increased trypanosomosis risk represented by an increased prevalence of 1 % to 20 % decreased the density of dairy animals by about 53 % and was associated with an increase of calving interval from about 14 to 25 months among the dairy farmers. the most common method used for the control of tsetse flies by dairy farmers was insecticide application (pour-on preparations) on their cattle. trypanocidal drugs were the main control method used in management of the disease. farmers stocked these drugs, which animal health personnel used for treating their animals. the private use of drugs to control trypanosomosis has become popular among the farmers in most trypanosomosis-endemic areas of kenya (ilrad 1994; mugunieri & murilla 2003). in addition, the use of insecticide-treated cattle has also become very common, unlike the use of artificial baits (traps and targets), which have not gained popularity in the absence of concerted support from governmental or non-governmental organizations (eisler, torr, machila, coleman & morton 2002). it is suggested that this contrast reflects, at least in part, the differences in public and private benefits. on the one hand, use of drugs confers an entirely private benefit, which is immediately effective even when a single farmer treats a single cow. on the other, the benefits of artificial baits are largely public and only realized when large numbers of individuals work in a concerted manner over a large area for a relatively long period. application of insecticides on cattle has a mix of public and private benefits. capitalizing on the perceived private and visible benefits of insecticide-treated cattle; this approach can be promoted for wide application to complement the use of drugs in the management of trypanosomosis. it is recommended that appropriate means of promoting its use by farmers and rural communities should be devised by the government. this will not only ameliorate the risk imposed by trypanosomosis on farmers currently undertaking dairying, but will see expansion of the enterprise to ensure that the country remains self-sufficient in the dairy products. acknowledgements the world bank under the kenya agricultural research institute-agricultural research fund program funded this research. this article is published with permission of the center director, kari-trc. references angus, s.d. 1996. the epidemiology of trypanosomosis in village livestock in an endemic sleeping sickness area of western kenya. ph.d. thesis, university of glasgow. card [coordinated agricultural and rural development] 1989. small ruminants in zimbabwe, a review of functions, research and proposals for communal lands. discussion paper. masvingo, zimbabwe: belmont press. de jong, r. 1996. dairy 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investigations. reports from busia, bungoma, siaya and transmara districts. muguga (technical team report, no. 1 of 1999). maloo, s.h., thorpe, w., ngumi, p.n. & perry, b.d. 1994. systematic epidemiological studies to identify and resolve constraints to smallholder dairy production in coastal lowlands of kenya. the kenya veterinarian, 18:359. mbogoh, s.g. & mukhebi, a.w. 1997. evaluation of the role of, programme and resource requirements of the socioeconomics unit of ketri. consultancy report. ketri/oda trypanosomosis research project phase iii. ketri, kikuyu. mcintire, j., bourzat, d. & pingali, p. 1992. crops livestock interaction in sub-saharan africa. washington dc: world bank. moa [ministry of agriculture] 1996. annual report. department of animal production. hill plaza, nairobi. moll, g., lohding, a. & young, a.s. 1984. epidemiology of theileriosis in the transmara division, kenya: husbandry and disease background and preliminary investigations on theileriosis in calves. preventive veterinary medicine, 2:801–831. mugunieri, g.l. & murilla, g.a. 2003. resistance to trypanocidal drugs—suggestions from field survey on drug use in kwale district, kenya. ondestepoort journal of veterinary research, 70:29–36. mugunieri, g.l., omiti, j.m. & irungu, p. 2004. performance of community-based animal health workers in the delivery of livestock health services. tropical animal health and production, 36:523–535. omiti, j.m. & muma, m. 2000. policy and institutional strategies to commercialize the dairy sector in kenya. nairobi: institute of policy analysis and research (ipar) (occasional paper, no. 006/2000). peeler, e.j. & omore, a.o. 1997. manual of livestock production systems in kenya, 2nd ed. kari/dfid national agricultural research project ii. kikuyu, kenya: national veterinary research centre. stotz, d. 1983. production techniques and economics of smallholder livestock production systems in kenya: farm management handbook of kenya. nairobi: ministry of livestock development. swallow, b.m. 2000. impacts of trypanosomosis on african agriculture. programme against african trypanosomiasis (paat). rome: fao (technical and scientific series, 2). woo, p.t.k. 1969. the haematocrit centrifugation for the detection of trypanosomes in blood. canadian journal of zoology, 47:921–923. 284 association of trypanosomosis risk with dairy cattle production in kenya abstract introduction method results discussion conclusion acknowledgements references about the author(s) ciza a. mushagalusa department of animal production, faculty of agriculture, université evangélique en afrique, bukavu, the democratic republic of the congo eric etter department of production animals studies, faculty of veterinary sciences, university of pretoria, pretoria, south africaumr astre (animals, health, territories, risks, ecosystems), cirad, petit-bourg, france mary-louise penrith department of veterinary tropical diseases, faculty of veterinary sciences, university of pretoria, pretoria, south africa citation mushagalusa, c.a., etter, e. & penrith, m-l., 2021, ‘review of african swine fever outbreaks history in south africa: from 1926 to 2018’, onderstepoort journal of veterinary research 88(1), a1919. https://doi.org/10.4102/ojvr.v88i1.1919 review article review of african swine fever outbreaks history in south africa: from 1926 to 2018 ciza a. mushagalusa, eric etter, mary-louise penrith received: 25 nov. 2020; accepted: 25 june 2021; published: 29 sept. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the article reviews the outbreaks and distribution of african swine fever (asf) in south africa since the first probable outbreak that occurred in the koedoesrand ward in 1926. retrospective data on the asf outbreaks in south africa were obtained from the world organisation for animal health (oie) disease database and the south african veterinary services annual reports in addition to published articles and online sources. south africa has experienced many outbreaks that can be divided into 2 time periods: the period before the development of the oie diseases database (1993) and the period after. more than 141 outbreaks of asf were reported during the first period. since the development of oie disease database, 72 outbreaks directly involving 2968 cases, 2187 dead and 2358 killed pigs mainly in smallholder pig farms were reported. the median number of cases for a given asf outbreak is 17, but in 50% of outbreaks no pigs were killed for prevention. the most important asf outbreak was reported in april 2014 in the greater zeerust district (north west province) involving 326 cases and 1462 killed pigs. however, the outbreak with highest mortality involving 250 pigs was reported in 2016 (free state province). according to phylogenetic analysis, nine p72 genotypes (i, iii, iv, vii, viii, xix, xx, xxi and xxii) have been identified in south africa. season-wise, more outbreaks were recorded during summer. it was also observed that the oie disease database could contain errors that would have been introduced through compiled forms at country level. spatiotemporal studies on asf outbreaks in south africa are therefore required in order to assess statistically and quantitatively the clustering of outbreaks over space and time. keywords: african swine fever; distribution; history; outbreak; transmission; domestic pig. introduction in africa, livestock contributes considerably to the national agricultural economy. according to the department of agriculture, forestry and fisheries (daff) (2012) report, in south africa livestock constitutes about half of the agricultural output (48%), and represents the vast majority of meat requirement (85%), with the pork industry representing specifically 2.15% of the primary agriculture sector (boettiger 2000; munzhelele et al. 2016). despite increases in livestock numbers in africa, the deficit between the supply and demand for animal products remains large because it requires changes in production systems, especially the development of cleanliness and good hygiene that are difficult to maintain as livestock numbers increase (madzimure et al. 2015). in this situation, animal overcrowding can compromise the immune system through the stress it causes and furthermore facilitates the transmission of infectious diseases (madzimure et al. 2015). consequences can be catastrophic, and the result is a strong imperative to provide proof that animals and their products are safe and comply with international trade standards to avoid spread of emerging and transboundary animal diseases (de klerk 2012). intensification of production systems that will result from the increased demand for livestock products as well as the impending impacts of climate change have evoked concern worldwide. notably because of more than 600 million smallholder-farmers worldwide whose livelihoods depend directly on their livestock production (thornton et al. 2006). in south africa, the dualism of the agricultural sector between a sophisticated commercial sector and a subsistence sector represented by small-scale production with low capital investment has created an imbalanced distribution of resources and support services (antwi & seahlodi 2011; madzimure et al. 2013). in 2010–2011, the south african pig population was approximately 125 000 sows, mostly being held in the commercial sector (approximately 100 000 sows), but with a significant part (25 000 sows) held in small-scale farms (mokoele et al. 2014). this dualism is determinant in the spread of animal diseases, as early disease detection and the enhanced biosecurity needed to control them will be difficult in small-scale sector because of lack of resources. it has been found that all african swine fever (asf) outbreaks reported in south african domestic pigs during the period covered by this study occurred in smallholder pig farms or at least in relatively small enterprises with poor management and low biosecurity (mokoele et al. 2014). african swine fever is a fatal, haemorrhagic, highly contagious viral disease with serious global implications, caused by a large cytoplasmic double-stranded deoxyribonucleic acid (dna) arbovirus, named the african swine fever virus (asfv), belonging to the genus asfivirus, family asfarviridae (alonso et al. 2018). in 1935, the northern parts of the limpopo, mpumalanga, north west and kwazulu-natal provinces were proclaimed as an asf control area on the basis of the distribution of the asf epidemiological sylvatic cycle. so far 24 different genotypes of this virus have been described to be circulating in africa, and 9 genotypes have been identified in south africa based on the p72 sequencing (achenbach et al. 2017; bastos et al. 2003; boshoff et al. 2007; quembo et al. 2018). because of its case fatality rate approaching 100% for peracute and acute forms of the disease, its transboundary nature, the trade restrictions in affected regions and the fear it instigates in affected communities, many authors consider it as the most dangerous swine disease (chenais et al. 2017; costard et al. 2009; hess 1971; penrith & vosloo 2009; plowright et al. 1994; vial et al. 2007). the most devastating effects are observed among small scale or poorer pig producers. constrained with financial and resource limitations, they are not able to ensure basic biosecurity or appropriate prevention and control measures (edelsten & chinombo 1995). because of the lack of compensation schemes, restarting production activities after outbreaks is often difficult and this can consequently lead to the loss of the pig population, which can impact negatively on food security (costard et al. 2009). in order to examine the context in which asf outbreaks occur in domestic pigs, reviewing asf outbreak history and clustering their distribution is a priority as it has been found that prevention programmes’ effectiveness varies over time and space. to achieve identification of asf patterns, the knowledge of its history, epidemiology, and the identification of risk factors for disease occurrence are essential (vergne et al. 2016). mapping disease outbreak distribution represents an important tool for planning health campaigns, surveillance, and interventions, as it facilitates the identification of transmission high-risk areas and orientates control activities (mott et al. 1995). therefore, this study aimed to explore asf outbreaks history in south africa in order to develop appropriate control measures and prevention strategies. method data collection retrospective data on asf outbreaks were obtained from the oie disease database (from october 1993 to may 2018), oie reports, reviewed published articles, online sources, the veterinary services annual reports (data before 1993), and personal communications with officials involved in outbreaks control and eradication. the world organisation for animal health (oie) diseases database comprised the year and the month of outbreak, the province, the state veterinary area, the district, the species, the number of outbreaks, cases, dead and killed animals. data analysis descriptive analysis was performed on asf outbreaks in order to describe their spatial and temporal (seasonal) distribution. seasonal comparison was done using a chi-square test with a significance level of p < 0.05. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results distribution of warthogs and ornithodoros ticks in 1935, the northern parts of the limpopo, mpumalanga, north west and kwazulu-natal provinces were proclaimed an asf control area on the basis of the distribution of the asf epidemiological sylvatic cycle. this sylvatic cycle is defined by asfv transmission between the common warthog (phacochoerus africanus) and burrow-inhabiting ticks of the ornithodoros moubata complex (ixodoidea: argasidae) during ticks’ feeding and the repeated asf outbreaks in domestic pigs (chenais et al. 2017; penrith & vosloo 2009; sánchez-vizcaíno et al. 2015). ticks are infected by asfv during the short viraemic phase of infection in warthog piglets, while the transmission between ticks may be sexual, transovarial and transstadial (jori et al. 2013; plowright et al. 1970; thomson 1985). despite long-term persistence, asfv is genetically stable in host ticks, which are the intermediate vector between warthog and domestic pigs or other warthogs (plowright et al. 1970). distribution of african swine fever virus genes in south africa in south africa, bastos et al. (2003) and boshoff et al. (2007) identified seven p72 genotypes out of the 24 genotypes recovered in africa clustered in three lineages (table 1). genotypes iii and vii, which have a transboundary distribution (northern south africa and neighbouring botswana) and iv, xix, xx, xxi and xxii, are found in several regions in africa. phylogenetic analyses have shown links between virus variants that have caused epidemics that are not related in time (spec/53, spec/120, spec/125, spec/251 and rsa/2/96) and differentiated between viruses previously thought to be part of a single outbreak (example: rsa/1/95 and rsa/5/95), describing the genotype-richness of asfv in south africa following the designation used by bastos et al. (2003), lubisi et al. (2005) and boshoff et al. (2007). in addition, two more p72 genotypes (i and viii) of asfv were isolated from sylvatic hosts (ticks) in the kruger national park (knp). the genotype i was isolated in 1978 and 1981 (zsak et al. 2005); while the genotype viii was isolated in 2001 (janse van rensburg et al. 2020). table 1: distribution of the african swine fever viruses and genes characterised in south africa. review of previous african swine fever disease outbreaks in south africa in the frame of this study, the history of asf in south africa is divided into two periods: the period before the development of disease database (oie data) in 1993 and from october 1993 up to may 2018, period covered by the oie data. african swine fever disease outbreaks in south africa before 1993 figure 1 illustrates local, metropolitan municipalities and consequently provinces probably affected by african swine fever before the development of the oie diseases database in 1993. figure 1: distribution of probable swine fever in south africa before the development of the organisation for animal health diseases database in 1993. the limpopo province in the endemic region was the main region affected by swine fever. outbreaks of ‘swine fever’ reported from the western cape province between the turn of the century and 1918 were attributed to classical swine fever (csf), the only known ‘swine fever’ at that time. this diagnosis was supported by arnold theiler’s investigations in 1905 (de kock, robinson & keppel 1940; penrith 2013). the first asf outbreak in south africa was reported in the northern part of the country, koedoesrand ward, specifically in the vicinity of maasstroom, in august 1926 (steyn 1928). according to farmers, at that period it was impossible to raise pigs because of that disease, affecting more free roaming pigs of all ages than animals kept in sties, especially on farms with a large number of warthogs in potential proximity to the pigs. in eastern europe, it has been shown that wild boar and free-roaming pigs’ direct contact when scavenging and/or inhabiting similar ranges contributes significantly to the spreading of asf (melletti & meijaard 2017). however, the situation involving the sylvatic cycle in africa differs from that in europe, because transmission between eurasian wild boars is direct owing to the fact that they are conspecific with domestic pigs and equally susceptible to the disease, and dead wild boars have proven a potent source of infection (beltrán-alcrudo et al. 2017), whereas warthogs do not develop clinical signs of asf or die of it and transmission to pigs depends on the ticks. (jori et al. 2013). steyn (1928) stated that this disease was often a sudden attack and infected animals were characterised by dullness, heavy breathing, grinding of teeth, and foaming in the mouth. steyn found that this ‘koedoesrand disease’ had a number of similarities with the east african swine fever described by montgomery (1921), such as the incubation period, symptoms and post-mortem lesions, probable association with warthogs, and the immunity developed by affected pigs. a number of asf outbreaks, very similar to the disease described by steyn (1928) and walker (1933), were reported in the 1930s in western cape province and former transvaal region (encompassing the current gauteng, limpopo, mpumalanga, the main part of north west and a tiny portion of kwazulu-natal provinces). these outbreaks were attributed to pig movements and associated with the presence of warthogs in the north-eastern part of south africa that was proclaimed as ‘a swine fever control area’ in 1935. in the control area, strict control measures are applied and pig movements are restricted. a number of outbreaks occurred in this period, but were mostly recurrences on the same farms. some of the reported outbreaks, according to de kock et al. (1940), were: lombardy estate outbreak, where the owner lost all his 30 pigs, was characterised by high fever followed by death in two to three days; cairngorm estate outbreak, probably infected by the previous farm but this has never been confirmed; savoy estate outbreak, in february 1934, when 50 pigs died within a few days most of time 12–24 h after presenting symptoms and sometimes without symptoms. these outbreaks occurred in the gauteng province. from the experience gained from previous outbreaks, routine examination was introduced in the northern transvaal region. in the year 1934–1935, no outbreak was reported in this region; in 1935–1936, one outbreak was reported in potgietersrus, two in the waterberg, and three in zoutpansberg. in 1937, two outbreaks occurred in the pietersburg district. most of the pigs (85%) in this region were not enclosed while up to 25% were not controlled at all and ran free, which facilitated contacts with wild suids reservoirs that resulted in infection (de kock et al. 1940). in the western cape province, an outbreak was reported at the imperial cold storage company, gouda, tulbagh district, in pigs destined for slaughter in october 1933. these were believed to have been infected by pigs coming from johannesburg. the gouda outbreak was probably the source of outbreaks observed on onverwacht farm and in wellington. towards the end of 1934, another outbreak where about 300 pigs died occurred in retreat, a suburb of cape town. in the year 1934–1935, several outbreaks were observed and the disease became widespread and quarantine measures with prohibition of movements of pigs was introduced in a large part of the country, an area of about 600 square miles. another outbreak was observed in the piquetberg area (de kock et al. 1940). in 1936, five outbreaks were reported in the western cape (two in wellington, one in caledon, one in franschhoek and one in worcester). the caledon outbreak extended from elgin to villiersdorp. free-ranging pig movements, the local custom of distributing pork products and the sharing of production tools were the key contributing drivers to the spreading of the disease (de kock et al. 1940; penrith 2013). in order to control its spread, buffer zones were created that enabled the identification of many unsuspected cases (de kock et al. 1940). in 1939, two linked outbreaks were observed in the western cape province, piquetberg district. the first occurred in a farm where swine fever was previously reported in 1934, but it was probably sourced from another farm where an outbreak occurred a few months earlier. in this latter farm, it was suggested that carriers probably existed and one of them had been sent to the first farm a few months previously (de kock et al. 1940). eblé et al. (2019) confirmed that clinically healthy pigs experimentally infected with a moderately virulent virus may be a source of new acute infections in a proportion of exposed pigs up to 55 days post infection, but there is little evidence that such pigs contribute to maintenance of the asf virus in domestic pig populations (petrov et al. 2018; ståhl et al. 2019). from 1933 up to december 1934. about 11 000 pigs were affected, of which 72.7% were lost. pigs were often preventively slaughtered before many had died, so that in 82 outbreaks there were only about 328 direct deaths from the disease itself. from december 1934 to june 1935, about 3.610 pigs were destroyed. although disease outbreaks reported between 1933 and 1939 were confirmed as being swine fever, but were not clearly defined as classical african swine fever. in fact at that time most people, including de kock et al. (1940), thought that the virus that caused asf was likely to be a particularly virulent csf virus. they were closely related to the ‘east african swine fever’ described by montgomery in 1921, and an association with warthogs in the north-eastern part of south africa was apparent in the asf control area (de kock et al. 1940; penrith 2013). african swine fever outbreaks were confirmed since 1973 as stated by bastos et al. (2003) and boshoff et al. (2007). in the 1950s. three asf outbreaks were reported mainly in the northern transvaal (northern province, now limpopo) between 1953 and 1956. a dead pig near a farmhouse was suspected of being the source of infection, but annual reports did not give precision of the district or their exact location. in the 1960s, only one outbreak was reported. in this outbreak, 23 pigs were found infected with asfv and 49 pigs were slaughtered as a preventive measure. it occurred in 1961–1962 in the district of soutpansberg (in the current limpopo province). in the 1970s, 26 outbreaks were reported in the districts of giyani, ritavi, seshego, bolobedu, pietersburg, letaba, thabazimbi in the current limpopo province, white river (in the current mpumalanga province) and rustenburg (in the current north west province) state veterinary areas. seventeen farms were affected in 1973–1974 after a long period of apparent asf absence (12 years since 1962). although it is possible that there were no cases, it is likely that some people would not have reported the loss of a small number of pigs. in the 1980s, 7 outbreaks were reported in the transvaal province (concentrated in the current limpopo province) region. one outbreak occurred respectively in each of the following: the former transvaal (1981–1982), but the exact district of occurrence was not determined by the annual report of veterinary services, letaba district (1985–1986), waterberg (1985–1986), potgietersrus district (1986–1987), nylstroom (1988–1989) and two outbreaks in the district of ellisras (1989–1990). in the 1990s, just before the development of the oie disease database in 1993, two outbreaks occurred in thabazimbi (1992–1993) and soutpansberg (1992–1993) districts (both in the current limpopo province). rapid detection, determination of the extent of contamination and elimination or disinfection of contaminated pigs and contact herds with payment of compensation were the measures applied to control outbreaks in south africa. all probable asf outbreaks reported before the oie diseases database are presented in table 2. table 2: probable african swine fever outbreaks reported before the development of the world organisation for animal health diseases database in 1993. african swine fever outbreaks distribution in south africa from 1993 up to may 2018 since 1993, daff has reported laboratory-confirmed asf outbreaks to the oie, with their date (year and month), location (district, province), and number of killed pigs, case morbidity and mortality data. in order to preserve the anonymity of the farm, a jamming system is used for gps coordinates. figure 2 illustrates local, metropolitan municipalities and provinces where asf outbreaks have been reported to the oie from october 1993 to may 2018. figure 2: distribution of municipalities where african swine fever outbreaks have been reported in south africa from 1993 to 2018. african swine fever outbreaks occurred mainly in limpopo province, with 35 outbreaks (48.6%), and other northern south african provinces such as gauteng, mpumalanga, north west and free state provinces, representing respectively 8.3 (6 outbreaks), 12.5 (9 outbreaks), 8.3 (6 outbreaks) and 15.8% (11 outbreaks) of outbreaks. in this interval, one outbreak was reported in knp and four in the northern cape province. it seems that this incidence of asf in the knp is a result of the change in the geocoding of the information done by daff, as no outbreaks inside the parks are likely because there are no domestic pigs inside them. alternatively, this reported outbreak to the oie in the park maybe an occasional test of a positive killed warthog, as the asf virus can sometimes be detected in lymph nodes. african swine fever antibodies have been found in warthogs in several regions in eastern and southern africa that coincide with the presence of infected ticks (jori et al. 2013). in endemic zones, up to 100% of warthogs may carry asfv antibodies (beltrán-alcrudo et al. 2017). all reported asf outbreaks in south africa occurred in the following local municipalities: lephalale (11), mangaung (8), delmas (7), thabazimbi (6), lesedi (5), musina (5), dikgatlong (3), ba-phalaborwa (3), makhado (3), bela-bela (3), ramotshere moiloa (3), letsemeng (2), maruleng (2), tswaing (2), naledi govan mbeki (1) and naledi emalahleni (1), nama khoi (1), modimolle (1), mogalakwena (1) and mamusa (1). thus, daff reported 72 confirmed outbreaks directly involving 2968 cases, 2187 dead and 2358 killed pigs, mainly in smallholder pig farms with low biosecurity practices. in table 3, errors introduced in compiling forms at country level are presented. what one would expect in an outbreak of asf, particularly in a non-endemic area, would be that the number of dead pigs would be close or equal to the number of cases but not more, because each dead pig would also represent a case. the number of pigs killed can be any number, usually it is the number of susceptible pigs minus the dead pigs if that is provided, but it could be whatever number the vets decided (depending on the chosen policy) should be removed from the population. the most likely explanation for the discrepancy would be that the person filling in the form put the dead and killed pigs in the wrong columns. thus, to estimate the total number of cases and dead, we have swapped the values corresponding to these columns. table 3: errors observed in the african swine fever outbreaks reported to the world organisation for animal health between october 1993 and may 2018. summary of african swine fever outbreaks in south africa between 1993 and 2018 the median number of cases for a given asf outbreak is 17, while it is 10 and 0 respectively for dead and killed pigs (table 4). for a given asf outbreak, about 25% of cases involved fewer than 6 pigs, while about 25% involved more than 42 pigs. in case of dead pigs, there were fewer than 4 in about 25% of cases, while in about 25% more than 34 pigs died. in addition, we observed that in 50% of outbreaks no pig was killed for prevention. according to the oie database, the most important asf outbreak in south africa was reported to the oie in april 2014 in the north west province, greater zeerust district. it involved 326 cases and 1462 pigs were killed, while the asf outbreaks with the most dead pigs involved 250 pigs in 2016, in the free state province. table 4: summary of cases, dead and killed pigs by african swine fever outbreaks in south africa. seasonal distribution of african swine fever outbreaks in south africa the seasonal distribution of outbreaks in south africa indicated that asf outbreaks have been reported in all seasons in both the control and free asf zone as illustrated by figure 3. the chi-square test has shown a significant difference (p = 0.0054) between the proportions of outbreaks in each season. the summer period (december-february) is the season with the highest number of outbreaks, where 29 outbreaks were reported in both controlled and free zones, followed by the winter period (june-august), where 21 outbreaks were reported, and autumn (march-may) and spring (september-november) periods where 11 outbreaks each were reported. before 2012 and thus within the controlled area there was no seasonal difference. figure 3: seasonal distribution of african swine fever outbreaks in south africa from 1993 to 2018. according to the oie disease database, before 2012 outbreaks were not reported beyond the demarcation line of asf control area. unfortunately, the oie database used in the frame of this study did not report two spill-overs in the 1990s to farms just outside the red line (control area limit), in both cases affecting a single farm with few pigs, and easily traced back to the control area (magadla et al. 2016; penrith, pers. comm., 2018). one outbreak was linked to a boar that was incubating asf being brought to a farm just outside the control zone to mate a sow that got sick and died there. the maximum number of outbreaks for a single year was observed outside the control area, when 15 outbreaks were reported in mpumalanga (9) and gauteng (6). we have observed that outbreaks have tended to occur in the free area since 2012 where they were not previously reported. discussion in order to understand the context in which asf outbreaks occur in domestic pigs, all outbreaks reported to the oie from 1993 up to 2018 have been analysed. in addition, all probable asf outbreaks that occurred in south africa before the development of the oie diseases database in 1993 were summarised from 1926 when the first probable cases of african swine fever were recorded. thus, according to de kock et al. (1940) and south african veterinary services annual reports from 1954 up to 1993, more than 149 swine fever outbreaks were recorded mainly in the northern part of the country that was then defined as the ‘asf control area’ but these outbreaks were not clearly defined as classical or african swine fever. this control area was defined according to the local distribution of the infected natural host (penrith & vosloo 2009). surveys of ectoparasites of warthogs in both north-eastern south africa and namibia have shown that nymphs of ornithodoros moubata complex ticks can be found on warthogs in fairly large numbers (boomker et al. 1991; horak et al. 1983, 1988). regarding the low probability of direct transmission between warthogs and domestic pigs, the transmission from warthogs to pigs is ascribed to infected ticks transported from the warthog’s burrow to pig’s premises. since 1993, daff has reported 72 confirmed outbreaks directly involving 2968 cases, 2187 dead and 2358 killed pigs. however, these numbers should be treated with caution as they depend upon the time the outbreak was reported to the oie and errors can be made in the compiling of the forms at country level. as an illustration, we can consider the outbreak that occurred in nama khoi, northern cape province on 23 may 2018 (outside the asf control area) and resolved on 10 december 2018. according to the oie disease database, this outbreak affected 14 pigs (14 cases, 14 dead, none killed). this is contrary to what is mentioned in the final report published by the oie in the frame of the wahis (oie’s web-based platform, world animal health information system) project suggesting that this outbreak was declared resolved on 10 december 2018 and involved 100 animals (susceptible) instead of 14, including 23 cases, 23 deaths and 77 animals killed and disposed of (modisane 2018). this situation was also observed in reporting the asf outbreaks considered as the first large scale outbreaks outside of the control area in ekhurhuleni and lesedi municipalities that occurred in january 2012. in these outbreaks, the oie disease database did not report culled animals, while geertsma, mpofu and walters (2012) reported 44 culled animals in ekhurhuleni and 121 in lesedi in 5 outbreaks. in addition, the outbreak that was reported by geertsma et al. (2012) in the pretoria state veterinary area (schietpoort farm) in lesedi in january 2012 affecting 5 pigs (5 cases, 3 dead) was not reported to the oie. our results showed that the 2012 outbreaks were the first outbreaks reported outside the control area but the official reports did not include the two minor outbreaks in the 1990s involving single farms described above, one of which occurred in 1996 and was mentioned by magadla et al. (2016). the development of the new oie-wahis should check this discrepancy in order to correct them. concerning errors made in the compiling of the forms at country level, 5 errors were observed where the number of dead pigs by asf was greater that the number of cases, which does not make sense unless the cases represent only the confirmed cases (the ones that were sampled and sent to the laboratory for diagnosis), but this scenario seems less probable than a simple error. results obtained in the frame of this work showed that the season has a significant effect on the number of outbreaks despite the fact that they have been reported in all seasons. the summer is the season with the highest number of outbreaks mainly after 2012. it appears logical as more pigs move and are slaughtered around christmas time, and this seasonal effect is more likely to be linked to pig’s movements rather than climatic and environmental conditions (penrith & vosloo 2009). in addition, arnot, du toit and bastos (2009) suggested that ‘late spring to early summer’ is recognised as the farrowing period for warthogs, with increased viraemia in neonatal warthogs in burrows. it is suspected that some of the spring and early summer outbreaks are related to very dry conditions before the start of the rainy season. much of limpopo is fairly marginal climatically, and when warthogs are hungry and thirsty they are more likely to scavenge around pig farms. this would probably not have much of a pattern because the rains are not always late and droughts are fairly irregular, but most likely to occur when the dry season (winter) is prolonged. these outbreaks occurred mainly in the northern south african provinces (limpopo, gauteng, mpumalanga, north west and free state) and seem qualitatively clustered in that region despite the fact that since 2012 more and more outbreaks have been reported outside the control area. according to magadla et al. (2016), other reasons that could explain the clustering of outbreaks could be the distribution of ‘small scale pig farms and socio-cultural and religious practices that influence pig movement and contact with warthogs and infected tampans’. african swine fever outbreaks were reported mostly in relatively small piggeries with conditions of poor management and low biosecurity (mokoele et al. 2014). in both prior to and post 1993 periods, commercial and small-scale production systems with low capital have been observed in south africa (antwi & seahlodi 2011). according to krecek et al. (2008), 25% of pigs in south africa belong to so-called emerging pig producers or smallholder pig farms. unfortunately, multiple factors constrain the development and production of smallholder pigs such as disease outbreaks and pig husbandry practices in communal production and free-range systems (antwi & seahlodi 2011). in smallholder farms, bad hygienic conditions associated with poor biosecurity encourage disease occurrence and spread (renaudeau 2009). their negative effect is accentuated by failure to identify diseases early, high prices of the medication, and lack of experience of extension services, veterinarians and preventive healthcare that result in a high rate of mortality (tekle, tesfay & kifleyohannes 2013). in addition, smallholder farmers utilise risky methods that consist of borrowing boars for breeding from relatives, neighbours, or allow sows to roam freely to be serviced or use boars bought at auction or untested boars (munzhelele et al. 2016). feeding of swill from kitchen and restaurant waste have been reported in smallholder pig farming in south africa and other african countries, which can result in transmission of diseases such as asf (mokoele et al. 2014). considering the increasing numbers of pigs kept, this disease is becoming very important as in the absence of an existing vaccine, the culling of susceptible animals at detection of an outbreak remains the main measure to control asf outbreaks, and compensation for culled pigs is no longer a policy in south africa. thus, better management strategies including improvement of pig husbandry by confined systems and marketing for outbreak prevention become essential (penrith 2013; penrith et al. 2013). conclusion african swine fever remains a serious threat to the pig production sector worldwide. this study explored the history of asf in south african pig production systems. in south africa, there is a need to remain aware that asf control measures should no longer be seen as the sole responsibility of the veterinary services, and that this disease is only a matter for asf control zone, but a matter for all. all pig producers must take the necessary precautions to ensure that their own herds are not infected. as the success of any disease prevention and/or control programme depends on a better understanding of its epidemiology, further analysis of the available data is needed to clearly understand the distribution patterns of asf disease. spatio-temporal studies of asf in south africa are therefore needed to statistically and quantitatively assess the clustering of outbreaks in space and time. acknowledgements the authors would like to thank the belgian directorate general for development co-operation framework agreement (3-iii dgd/itm 2014–2016) (project 920108; collaborative mstah itm/up) awarded to the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions all authors contributed to the identification of research questions, the critical review of the results, and approved the final version of the manuscript, e.e. and m.l.p. provided the data needed to conduct this study and the valuable knowledge with regard to asf in south africa. m.c.a. summarised results and wrote the initial version of the manuscript. funding information ‘unravelling the effect of contact networks & socio-economic factors in the emergence of infectious diseases at the wild-domestic interface’ project funded by the ecology and evolution of infectious diseases program, grant no. 2019-67015-28981 from the united states department of agriculture national institute of food and agriculture. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions 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csir biosciences, pretoria, south africa jacobus n. elof department of paraclinical sciences, university of pretoria, south africa citation fouche, g., sakong, b.m., adenubi, o.t., pauw, e., leboho, t., wellington, k.w. et al., 2016, ‘anthelmintic activity of acetone extracts from south african plants used on egg hatching of haemonchus contortus’, onderstepoort journal of veterinary research 83(1), a1164. http://dx.doi.org/10.4102/ojvr.v83i1.1164 original research anthelmintic activity of acetone extracts from south african plants used on egg hatching of haemonchus contortus gerda fouche, bellonah m. sakong, olubukola t. adenubi, elizabeth pauw, tlabo leboho, kevin w. wellington, jacobus n. elof received: 02 feb. 2016; accepted: 12 may 2016; published: 28 july 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the nematode, haemonchus contortus, is responsible for major economic losses in the livestock industry. the management of parasites such as h. contortus has been through the use of synthetic parasiticides. this has resulted in the presence of residues in meat and milk, which affects food safety. the development of resistance to available anthelmintics coupled with their high cost has further complicated matters. this has led to the investigation of alternative methods to manage nematodes, including the use of plants and plant extracts as a potential source of novel anthelmintics. acetone extracts were prepared from 15 south african plant species and their anthelmintic activity determined using the egg hatch assay (eha). the leaf extract of cleome gynandra had the best inhibitory activity (68% ± 3%) at a concentration of 2.5 mg/ml, followed by the stem extract of maerua angolensis (65% ± 5%). the extracts had a relatively low toxicity on vero cells determined by the mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cellular assay. introduction livestock production in tropical and developing countries is severely hampered by gastrointestinal parasites (adejinmi & harrison 1997; hounzangbe-adote et al. 2005). the gastrointestinal parasitic nematode, haemonchus contortus, also known as the barber pole worm, resides in the gut of sheep and other livestock. it accounts for about 80% of the global parasite afflictions of diseased animals (arosemena et al. 1999) and is notorious for its high pathogenicity (angulo-cubillán et al. 2010). in small ruminants, gastrointestinal nematodes have traditionally been managed by the use of synthetic anthelmintic compounds (mendoza de gives et al. 1998). the systematic application of anthelmintic drugs, in an effort to manage infections produced by h. contortus, has led to the emergence of resistant strains (akhtar et al. 2000; prichard 1994). there have been reports of parasite resistance to anthelmintic drugs in many countries (melo, bevilaqua & reis 2009; schnyder et al. 2005), and multiple anthelmintic resistance has reached extreme levels (torres-acosta et al. 2012). certain drugs may also cause problems such as food residues and environmental pollution (hammond, fielding & bishop 1997). this global problem has caused severe losses in productivity and is also the main restricting factor for the livestock sector (melo et al. 2009; waller 1994). novel alternative methods are thus required. research has been conducted on plant species as alternative anthelmintics to manage gastrointestinal infections in small ruminants (adamu, naidoo & eloff 2013; batista et al. 1999; slomp et al. 2009). the use of anthelmintic plant extracts may be sustainable and environmentally acceptable and could provide an alternative to synthetic anthelmintics. furthermore, anthelmintic plant extracts have a mixture of active principles that could act in synergy, yielding the anthelmintic effect and limit the development of resistance. this differs from commercial drugs, which usually have only one molecule acting on the parasite when not administered as a combination formulation. resistance is therefore likely to develop more slowly in the natural product. the aim of this study was to determine the in vitro anthelmintic action of acetone extracts from 15 south african plant species used traditionally to control parasites such as h. contortus using the egg hatch assay (eha). we used only one concentration as a first step in selecting plant species for in-depth follow-up research. the toxicity of the acetone extracts was also determined against vero cells. materials and methods plant material collection fifteen plants [aloe rupestris baker, antizoma angustifolia (burch.) miers ex harv., calpurnia aurea ssp. aurea (aiton) benth., senna italica subsp. arachoides (burch.) lock, cissus quadrangularis l., clematis brachiata thunb., cleome gynandra l., ficus sycomorus l., hypoxis rigidula baker var rigidula, maerua angolensis dc, monsonia angustifolia e. mey. ex a. rich., pelargonium luridium (andrews) sweet, schkuhria pinnata (lam.) kuntze ex thell., sclerocarya birrea (a. rich.) hochst and tabernaemontana elegans stapf.] were selected on the basis of available literature and ethno-veterinary usage over many years at council for scientific and industrial research (csir) (unpublished data). these plants were collected from different locations in south africa during the summer season. production of dried, ground plant material plant material was dried in an oven at 30 °c – 60 °c followed by grinding to fine particles using a hammer mill. preparation of the acetone extracts the acetone extract was prepared by adding 200 ml of acetone to 20 g of each ground plant material that was stirred for 1 h. the extract was decanted and filtered, and the residue was re-extracted with the same volume of acetone once again for 1 h; the third time, the same volume of acetone was used but the mixture was stirred overnight. the extracts were combined and the acetone evaporated on a rotary evaporator. the yield that was obtained for each of the plant species is shown in table 1. table 1: the plant and plant part used for the solvent extraction, plant family, solvent, the mass and percentage yield of extract obtained. egg recovery and preparation the method used was based on the world association for the advancement of veterinary parasitology (waavp) guidelines described by adamu et al. (2013). briefly, h. contortus eggs were collected from sheep that were housed indoors on a concrete floor. approximately 10 g – 15 g of sheep faecal pellets were crushed in water to form a slurry and cleared of organic debris by serially filtering it through sieves of pore sizes 150 µm, 63 µm and 20 µm. the eggs were collected on a 20-µm sieve and washed off with a 40% sugar solution (density 1.18) into 50-ml centrifuge tubes. the tubes were then centrifuged for 5 min at 1000 rpm to separate the floating eggs from other debris. the supernatant was decanted on a 20-µm sieve, and the eggs were washed off with water and collected in a 500-ml container. the concentration of eggs in the egg suspension was determined by counting the eggs using a microscope and a mcmaster. the egg concentration was subsequently brought to a final concentration of 100 eggs per 200 µl. to avoid proliferation of fungi, 5 µg amphotericin b solution (sigma, germany) was added per millilitre of egg suspension. egg hatch assay the in vitro eha was based on the procedure described by adamu et al. (2013), which is based on the method described by coles et al. (1992). the egg suspension (200 µl) containing ~100 fresh eggs were distributed into each well of a 24-flat-bottomed microtitre plate. the same volume (200 µl) of the dried plant extract (5 mg/ml) dissolved in solvent was added to each well. albendazole was used as positive control, and the solvents were used as the negative controls. albendazole was dissolved in 5% dimethyl sulphoxide (dmso) in water and evaluated at various concentrations (0.008 μg/ml – 25 μg/ml). the plates were incubated for 48 h at 27 °c at 70% relative humidity. the experiment was replicated three times for each extract on the same plate. after incubation, the hatched larvae and unhatched eggs were counted using an inverted microscope under 20 × magnification. the percentage inhibition of egg hatching was calculated using the formula of bizimenyera et al. (2006): egg hatch inhibition (%) = 100 (1 – number of larvae/number of larvae and eggs in water control)    [eqn 1] the results of the eha are shown in table 2. table 2: mean inhibition percentages of the acetone extracts (2.5 mg/ml) on egg hatching of haemonchus contortus from sheep and the toxicity values (lc50) against vero cells. determining the toxicity of the plant extracts the toxicity of the plant extracts were determined by using the method employed by adamu et al. (2013): vero african green monkey kidney cells were obtained from a confluent monolayer and then trypsinised and seeded (0.5 × 103 cells/well) in a 96-well microtitre plate. this was followed by incubation overnight at 37 °c in 200 μl of 5% minimal essential medium (mem, highveld biological, south africa) and supplemented with 0.1% gentamicin (virbacr) and 5% foetal calf serum (adcock-ingram) (adamu et al. 2013). the media were replaced with 200 μl of the extracts (1 mg/ml, 0.1 mg/ml, 0.01 mg/ml and 0.001 mg/ml) after 24 h and incubated for another 5 days. viability of cells was determined using the tetrazolium-based colorimetric mtt assay (3-5-dimethyl thiazol-2-yl-2, 5-diphenyl tetrazolium bromide) as described by mosmann (1983) (adamu et al. 2013). basically, the medium in each well was removed, replaced with fresh medium and 30 μl (5 mg/ml) mtt in phosphate-buffered saline (pbs) followed by incubation for 4 h. the medium was then removed before washing the cells with pbs and before the addition of dmso (50 μl) to dissolve any formazan crystals (adamu et al. 2013). a versamax microplate reader at 570 nm (path length 1 cm) was used to measure the absorbance of the wells. doxorubicin was used as a positive control and tested at different concentrations. the negative control was a well containing cells without an extract. the percentage of cell viability was calculated relative to the pure growth. the lc50 value was calculated by determining the concentration of each plant extract resulting in 50% reduction of absorbance compared to untreated cells. tests on the concentration of each extract were carried out in triplicate, and each experiment was repeated three times. the lc50 results are expressed as the mean ± standard deviation (s.d.) of the three replicates. a plant extract having an lc50 value > 20 μg/ml has an acceptable level of toxicity, whilst a value < 20 μg/ml is regarded as toxic (kuete & efferth 2010). data analysis excel for windows 7 was used to record the results produced in this study. kinetica 5.0 (thermo) using a sigmoid inhibitory model was used to calculate the lc50 values. the results are presented as the mean lc50 and the standard deviation of the mean. results extraction of plant material acetone was selected as extractant because it has many advantages over other generally used extractants (eloff 1998). the extraction yield was very similar for the different plant species and plant parts and varied from 5% to 6% (table 1). effect of different solvents on haemonchus contortus egg hatching the dried extracts did not dissolve in water, and therefore the use of acetone, dmso and tween 80 on the hatching of eggs. the final concentration of the solvents in the well was 50.0%, 25.0%, 12.5%, 6.3% and 3.1%. even 3.1%, the lowest concentration of acetone, inhibited 94.0% egg hatching. the lowest concentration of tween 80 inhibited 19.0% egg hatching. dmso concentrations of 25.0% and higher led to 100.0% inhibition of egg hatching. the best results were obtained with 3.1% and 6.3% dmso leading to 13.0% and 19.0% inhibition of egg hatching, respectively. the results for the negative controls of water and pbs were 13.0% and 11.0%, indicating that 3.1% dmso did not have a marked effect on the egg hatching. the lower degree of hatching with the negative controls probably indicates damage to the eggs during processing. consequently, the extracts were dissolved in 6.0% dmso leading to a 3.0% final concentration after adding the same volume of egg suspension. determining the inhibitory activity of the plant extracts on haemonchus contortus egg hatching the two negative controls also had a degree of activity on the egg hatching. the results are presented in table 2 as the mean egg hatch inhibition (%) and the standard deviation of the mean. at 2.5 mg/ml, extracts of c. gynandra (leaves), m. angolensis (stem), m. angustifolia (whole plant) and s. italica subsp. arachoides (roots, leaves and fruit) had a mean inhibition rate of between 55% and 68% which was much higher than the water and dmso negative controls. our choice of 2.5 mg/ml appeared to be a good concentration because only four species had what would have been an lc50 in the order of 2.5 mg/ml. albendazole, the positive control, recorded 100% inhibition at the lowest concentration, 0.008 µg/ml. the extract of c. brachiata (whole plant) had the lowest activity (11%), which was even lower than the water control group (16%). the activity of the plant extracts was so much lower than that of albendazole that the feasibility of using plant extracts can be questioned. plant extracts may be more active in the larval development assay (lda). determining the cytotoxicity of the plant extracts the tetrazolium-based (mtt) colorimetric assay (mosmann 1983) was used to determine the viability of vero african green monkey kidney cells in the presence of each of the plant extracts and the results are shown in table 2. from these results, it is apparent that none of the plant acetone extracts were as toxic as doxorubicin (2.97 µg/ml ± 0.016 µg/ml = 5.12 µm ± 0.028 µm). the leaf extract of c. gynandra was the least toxic (lc50 = 553.61 µg/ml ± 18.83 µg/ml) followed by the stem extract of c. aurea (lc50 = 223.97 µg/ml ± 5.4 µg/ml), the fruit extract of s. birrea (lc50 = 214.79 µg/ml ± 14 µg/ml), the stem extract of m. angolensis (lc50 = 180.64 µg/ml ± 3.5 µg/ml) and the bark and stem extract of f. sycomorus (lc50 = 172.94 µg/ml ± 8.91 μg/ml). the whole plant extract of p. luridum (lc50 = 30.58 μg/ml ± 3.40 μg/ml) was the most toxic of all the plants against vero cells. discussion the eha is an in vitro assay used to evaluate the anthelmintic activities of natural products. the capacity to reduce egg hatching could help to modulate the risk of parasitism by limiting the infectivity of pastures grazed by ruminants (max 2010). the aim of this study was to determine the inhibitory activity of the acetone extracts of 15 plant species on egg hatching of h. contortus in order to select the most promising plant species that could control the nematodes in the animal gut for further study. in previous studies, it was found that aqueous extracts contained few compounds, had very low biological activity (eloff, famakin & katerere 2005; kotze & eloff 2002) and had low or negligible anthelmintic activity (bizimenyera et al. 2006; worku, franco & miller 2009). acetone was therefore selected as an appropriate extractant because it is miscible with organic and aqueous solvents, non-toxic to bacteria and fungi, and also has the capacity to extract a wide range of polar compounds (eloff 1998). as shown in table 2, it is evident that the extracts of four plant species had anthelmintic activity (inhibitory activity above 50%) at the concentration tested. cleome gynandra leaf extracts had the best anthelmintic activity with an egg hatch inhibition of 68% ± 3% and low toxicity (lc50 = 553.61 µg/ml ± 18.83 µg/ml) on vero cells. our results are in agreement with that of other researchers who have also reported on the anthelmintic activity of c. gynandra. two authors (jadhav, ghawate & bhamber 2011; thenmozhi et al. 2014) used the unverified assumption that the indian adult earthworm (pheretima posthuma) could be used as a model for the activity of c. gynandra (syn. gynandropsis pentaphylla) extracts against intestinal roundworm parasites of human beings because of its anatomical and physiological resemblance. they also used a physiologically non-relevant high concentration of 25 mg/ml and concluded that these extracts had potent anthelmintic activity when it killed earthworms after 53 min without examining any helminths. sowunmi and afolayan (2015) also did a phytochemical analysis of the acetone extract of different parts of c. gynandra. the polyphenolic contents of the various parts of the plant were significantly high. leaf acetone extracts of c. gynandra had the highest concentration of total phenolics (126.79 mg/g ± 0.55 mg/g), flavonoids (40.58 mg/g ± 0.06 mg/g) and flavanols (42.41 mg/g ± 0.05 mg/g), whilst the stem extract had the highest amount of proanthocyanidins (419.01 mg/g ± 0.67 mg/g) compared to the leaves (403.29 mg/g ± 0.89 mg/g) and fruits (107.18 mg/g ± 0.59 mg/g). the low concentration of saponins and alkaloids suggests that this plant may have low toxicity (sowunmi & afolayan 2015). this suggestion is supported by the low toxicity we observed against vero cells (lc50 = 553.61 µg/ml ± 18.83 µg/ml). alcohol and aqueous extracts from the leaves of cleome viscosa linn were also investigated for their anthelmintic activity against the adult indian earthworm, p. posthuma, as well as ascaridia galli. three concentrations (50 mg/ml, 100 mg/ml and 150 mg/ml) of each extract were studied, which entailed the determination of time of paralysis and time of death of the worm. both the extracts had significant anthelmintic activity at the highest concentration of 150 mg/ml. the water leaf extract had weaker activity than the methanolic leaf extract, and both extracts caused paralysis and death of worms. phytochemical screening of the methanol extract showed that anthraquinone glycosides, phenolic compounds and steroids were present in c. viscosa linn, whilst in the aqueous extract glycosides and phenolic compounds were present. flavonoids were identified as being one of the chemical constituents amongst the phenolic compounds in the crude extracts. polyphenolic compounds are known for their anthelmintic activity (kaushik, katiyar & sen 1974; lal et al. 1976; szewezuk, mongelli & pomilio 2003). synthetic phenolic anthelmintics such as niclosamide, oxyclozanide and bithionol interfere with energy generation in helminth parasites by uncoupling oxidative phosphorylation (bate-smith 1962; martin 1997; tandon et al. 1997). the phenolic content may therefore have produced similar activity in the extracts of c. viscosa linn and c. gynandra. the second best egg hatch inhibition of 65% ± 5% was by m. angolensis (stem) extract that had low toxicity (lc50 = 180.64 µg/ml ± 3.5 µg/ml) on vero cells. phytochemical screening of the methanolic extract of the stem bark found that glycosides, tannins, saponins, terpenes, flavonoids, carbohydrates, proteins and alkaloids were present in m. angolensis (ayo et al. 2013; meda et al. 2013; pl@ntuse). these compounds could also be present in the acetone stem extract tested in this study. it has been reported that maerua edulis (gilg and gilg-ben.) dewolf and maerua subcordata (gilg) dewolf have been used in traditional anthelmintic remedies in kenya to treat sheep infected with h. contortus (gakuya 2001). in this study, aqueous extract from both unground and ground material of each plant material was prepared using boiling water. twenty-one clinically healthy sheep of mixed breeds and sexes were randomly allocated to four treatment groups, four animals each. faecal egg counts were performed for all the sheep. it was found that the crude extracts could control helminthoses to a reasonable extent and maintain the animal at a clinically healthy state. the brine shrimp assay was used to detect bioactivity in the water, chloroform and methanol extracts of m. subcordata and m. edulis. the chloroform extract was the most toxic to the brine shrimps compared to the water and methanol extracts (gakuya 2001). monsonia angustifolia (whole plant) had an egg hatch inhibition of 56% ± 6% and also had low toxicity on vero cells (lc50 = 120.37 µg/ml ± 4.06 µg/ml). five compounds identified as aryl naphthalene lignans (5-methoxyjusticidin a, justicidin a, chinensinaphthol, retrochinensinaphthol methyl ether and suchilactone) were isolated during the fractionation of the organic (methanol-dichloromethane) extract of m. angustifolia (khorombi 2006). lignans are a group of naturally occurring phenolic compounds. the drug podophyllum, a lignan, is obtained from the dried root and rhizomes of two species of podophyllum (berberidaceae), the american species podophyllum peltatum and the indian species podophyllum hexandrum (podophyllum emodi). the european settlers reported using the root extensively, particularly as a cathartic and anthelmintic (konuklugil 1995). thus, the anthelmintic activity of m. angustifolia may be attributed to the lignans. senna italica subsp. arachoides (burch.) lock (roots, leaves, fruit) had an egg hatch inhibition of 55% ± 13% and had a higher toxicity on vero cells (lc50 = 46.31 μg/ml ± 2.89 μg/ml) than that of c. gynandra (leaves), m. angolensis (stem) and m. angustifolia (whole plant) which also had anthelmintic activity. aloe rupestris (leaves) only had an egg inhibition of 47% ± 7% against h. contortus and low toxicity (lc50 = 63.46 ± 11.00) against vero cells. it has been reported that other aloe species such as aloe ferox can affect h. contortus of goats negatively (maphosa et al. 2010). the amino acids, saponins and sterols in a. ferox can disturb protein structure and therefore affect the growth and repair of the nematode body (mabusela, stephen & botha 1990). tabernaemontana elegans (leaves) also only had an egg inhibition of 47% ± 7% and low toxicity (lc50 = 32.35 ± 0.88) against vero cells. in guadeloupe (french west indies), another species, tabernaemontana citrifolia, has traditionally been used as an anthelmintic preparation for ruminants (marie-magdeleine et al. 2010). marie-magdeleine et al. (2010) prepared aqueous, methanolic and dichloromethane extracts from the fruit, leaves and roots of t. citrifolia for testing on four developmental stages of h. contortus. the eha, the lda, the l3 migration inhibition assay (lmi) and the adult worm motility assay (awm) were employed in the testing. from the tests it was apparent that there were significant effects for the different parts of t. citrifolia when compared to the negative control and that the differences depended on the parasitic stage. the efficacies on the larval development of h. contortus ranged from 88.9% to 99.8% for fruit, from 72.1% to 83.8% for roots and from 33.5% to 85.0% for leaves. for the methanolic extract a dose-dependent effect was observed. alkaloid compounds are present in the different parts of t. citrifolia and may be responsible for the observed anthelmintic activity against h. contortus (marie-magdeleine et al. 2010). another species, tabernaemontana coronaria, was also investigated for anthelmintic activity against the indian adult earthworm p. posthuma. it was found that the ethanolic extract of the leaves had potent anthelmintic activity (pushpa et al. 2011). anthelmintic activity has also been documented for other plant species where an organic extract was also used in the determination of the anthelmintic activity (ademola & eloff 2010, 2011; monteiro et al. 2010). adamu et al. (2013) reported that heteromorpha trifoliate, leucosidia sericea and maesa lanceolata had 100% inhibition, whilst clausenia anisata had 80% inhibition at a concentration of 3.13 mg/ml in the eha. several other plant species have been documented as having anthelmintic activity. these are lantana camara (verbenaceae), tagetes minuta (asteraceae), mentha villosa (lamiaceae) (albuquerque et al. 2007) and alpinia zerumbet (zingiberaceae) (almeida 1993). in this study, extracts of c. gynandra (leaves), m. angolensis (stem), m. angustifolia (whole plant) and s. italica subsp. arachoides (roots, leaves and fruit) only had inhibition activity between 55% and 68% at 2.5 mg/ml. higher inhibitory activity for these extracts may have been obtained if they had also been evaluated at 3.13 mg/ml. the kidney is one of the main sites of excretion in animals, and therefore, renal cells in culture were used as an indicator of toxicity for this study. these cells were explicitly chosen because of the favoured blood supply of the kidney and their high metabolic capacity. the results of the toxicity study were encouraging because the extracts had low toxicity (lc50 > 20 µg/ml) against vero cells. the determination of cellular toxicity is valuable because it will give a good indication of whether in vitro toxicity is also an indicator of in vivo toxicity. the results of this study are significant because egg hatch inhibition is an important method by which pasture contamination by animals can be reduced during grazing. thus, there is the possibility that the plant material in this study can be administered as a feed to control helminths, although the concentrations required for efficacy were very high. overall, the use of these botanicals could contribute to a helminth control programme. conclusion the acetone extracts of c. gynandra, m. angolensis, m. angustifolia and s. italica subsp. arachoides have some anthelmintic activity against h. contortus egg hatching. the activity of each of these extracts was lower than that of the positive control, albendazole. the most promising plant species is c. gynandra, which may be further studied to identify the active constituents responsible for anthelmintic activity. acknowledgements the authors thank the technology innovation agency (tia) of south africa for financial support and professor lyndy mcgaw for informative discussions about the toxicity of plant extracts on vero cells. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions g.f. conceptualised the study. g.f., k.w.w. and t.l. did the literature search and plant selection. t.l. prepared the plant extracts. j.n.e. and e.p. arranged for sheep to be infected, collected the eggs and guided the study. b.m.s. did the egg hatch assay on the extracts. o.t.a. screened the extracts for toxicity on vero cells. k.w.w. wrote the first draft of the manuscript. references adamu, m., naidoo, v. & eloff, j.n., 2013, ‘efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in south africa on egg hatching and larval development of haemonchus contortus’, bmc veterinary research 9, 38. http://dx.doi.org/10.1186/1746-6148-9-38 adejinmi, j.o. & harrison, l.j.s., 1997, ‘parasitic nematodes of domestic ruminants in nigeria: impact on ruminant production and control’, tropical veterinarian 15, 137–148. ademola, i.o. & eloff, j.n., 2010, ‘in vitro anthelmintic activity of combretum molle (r. br. ex g. don) (combretaceae) against haemonchus contortus ova and larvae’, veterinary parasitology 169, 198–203. http://dx.doi.org/10.1016/j.vetpar.2009.12.036 ademola, i.o. & eloff, j.n., 2011, ‘anthelmintic activity of acetone extract and fractions of vernonia amygdalina against haemonchus contortus eggs and larvae’, tropical animal health and production 43, 521–527. http://dx.doi.org/10.1007/s11250-010-9727-7 akhtar, m.s., igbal, z., khan, m.n. & lateef, m., 2000, ‘anthelmintic activity of medicinal plants with particular reference to their use in animals in the indo–pakistan subcontinent’, small ruminants research 38, 99–107. http://dx.doi.org/10.1016/s0921-4488(00)00163-2 albuquerque, u.p., medeiros, p.m., almeida, a.l., monteiro, j.m., neto, e.m.f.l., melo, j.g. et al., 2007, ‘medicinal plants of the caatinga (semi-arid) vegetation of ne brazil: a quantitative approach’, journal of ethnopharmacology 114, 325–354. http://dx.doi.org/10.1016/j.jep.2007.08.017 almeida, e.r. 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‘evaluation of the activity of plant extracts in boer goats’, american journal of animal and veterinary sciences 4, 2–79. http://dx.doi.org/10.3844/ajavsp.2009.72.79 abstract introduction materials and methods results discussion conclusion acknowledgements references appendix 1 about the author(s) jean-yves ekra sacids foundation for one health, africa centre of excellence for infectious diseases of humans and animals in east and southern africa, morogoro, tanzania, united republic of tanzania department of microbiology, parasitology and biotechnology, college of veterinary medicine and biomedical sciences, sokoine university of agriculture, morogoro, united republic of tanzania edouard k. n’goran département de biochimie-génétique, unité de formation et de recherche (ufr) des sciences biologiques, université peleforo gon coulibaly, korhogo, côte d’ivoire léonard e.g. mboera sacids foundation for one health, africa centre of excellence for infectious diseases of humans and animals in east and southern africa, morogoro, tanzania, united republic of tanzania eliakunda m. mafie department of microbiology, parasitology and biotechnology, college of veterinary medicine and biomedical sciences, sokoine university of agriculture, morogoro, united republic of tanzania citation ekra, j-y., n’goran, e.k., mboera, l.e.g. & mafie, e.m., 2023, ‘prevalence of bovine trypanosomiasis in côte d’ivoire: systematic review and meta-analysis’, onderstepoort journal of veterinary research 90(1), a2069. https://doi.org/10.4102/ojvr.v90i1.2069 review article prevalence of bovine trypanosomiasis in côte d’ivoire: systematic review and meta-analysis jean-yves ekra, edouard k. n’goran, léonard e.g. mboera, eliakunda m. mafie received: 28 july 2022; accepted: 23 jan. 2023; published: 27 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract bovine trypanosomiasis is a parasitic disease caused by protozoans of the genus trypanosoma. the disease cause economic losses in livestock production. in order to determine the status of research on this disease in côte d’ivoire, we used the systematic review method and meta-analysis. three electronics databases, namely google scholar, pubmed and crossref were used to search for publications on trypanosomiasis prevalence that met our inclusion criteria. twenty five articles were identified, 11 of which met the inclusion criteria. bovine trypanosomiasis prevalence of 2.99% (95% confidence interval [ci]: 2.96% – 3.01%) to 25.28% (95% ci: 25.17% – 25.38%) were recorded between 1960 and 2021. the analyses showed that the most infected regions were the bagoue 11.26% (95% ci: 11.25% – 11.27%), bounkani 14.94% (95% ci: 14.93% – 14.95%), gbeke 10.34% (95% ci: 10.33% – 10.35%), marahoue 13.79% (95% ci: 13.78% – 13.80%), poro 8.50% (95% ci: 8.49% – 8.51%), and tchologo 11.83% (95% ci: 11.82% – 11.84%).the most sensitive diagnostic method used was the polymerase chain reaction (pcr). the species of trypanosomes diagnosed were typanosoma vivax 4.99% (95% ci: 4.97% – 5.01%), t. congolense 1.51% (95% ci: 1.49% – 1.52%), and t. brucei 0.61% (95% ci: 0.59% – 0.62%). despite some variation, the prevalence of bovine trypanosomiasis in côte d’ivoire caused mainly by t. vivax has increased in the years between 1977 and 2017. efforts to control tsetse and other mechanical vectors should also be put in place to minimize its transmission. contribution: the authors studied the prevalence of bovine trypanosomiasis using the systematic review method and ma in order to determine the status of research on this disease in côte d’ivoire. keywords: bovine trypanosomiasis; species; distribution; cote d’ivoire; meta-analysis; prevalence. introduction trypanosomes are flagellate unicellular protozoa belonging to the family trypanosomatidae and the genus trypanosoma (aubry 2012). many of the species that cause both animal and human trypanosomiasis belong to the genus trypanosoma (mekata et al. 2013). african animal and human trypanosomiasis are transmitted by tsetse flies of the genus glossina. nevertheless, some species have evolved to adapt to mechanical transmission, which has let them to spread beyond africa’s tsetse zone, resulting in the ‘nagana’ cattle wasting disease. trypanosomes can be mechanically transmitted by hematophagous flies of the genera stomoxys and tabanus (bafort, kageruka & timperman 1973). the ability of the parasites to remain in the vector’s mouth cavity is necessary for mechanical transmission. therefore, the success of parasite transmission increases with the length of the blood-sucking gap of the vector between an infected and an uninfected animal (desquesnes et al. 2013). the pathogens responsible for the most important trypanosomal infections in livestock are trypanosoma congolense (mackenzie et al. 1978) (savannah group), trypanosoma vivax (ziemann 1905) and trypanosoma brucei (difonzo & bordia 1998). in africa, trypanosomes are mainly found in the humid or subhumid areas (sané et al. 2000) estimated to cover about 10 million km2. trypanosomal infections in this zone constitute a major constraint to livestock development. indeed, there are nearly 50 million cattle and about 70 million small ruminants continuously exposed to trypanosomiasis risk (paylhard 2019; fandamu et al. 2005). typanosomiasis has a significant socio-economic impact and limits animal productivity in africa with direct and indirect losses estimated at $4.5 billion (de la rocque 2001, 2003). côte d’ivoire is one of the most affected countries (douati, kupper & kotia 1986). this is due to its vegetation, climate and hydrographic network which constitute an ecosystem favourable to the development of tsetse flies, the vectors of this disease. despite current control measures, the prevalence of this disease is between 11% and 21% in the north of côte d’ivoire. the most affected areas are the departments of korhogo, boundiali and ferkessédougou. despite its impact on the economy and animal health, bovine trypanosomiasis in côte d’ivoire has been disregarded in terms of control interventions (sehi 2019). furthermore, the military and political crises have devastated what little had been achieved in vector control. although bovine trypanosomiasis has been studied in côte d’ivoire, the epidemiology of the disease remains poorly understood. in order to revitalise the fight against bovine trypanosomiasis, an inventory of the disease burden is critical. in order to give a general overview of the epidemiology of bovine trypanosomiasis in côte d’ivoire, this systematic review and meta-analysis (ma) were conducted. specifically, to analyse the trypanosomes species distribution; evaluate methods of detection and determine the prevalence of the disease. materials and methods search strategy and inclusion criteria following the guidelines in the preferred reporting items for systematic reviews and meta-analyses (prisma) guide, the systematic review (sr) and ma were carried out (moher et al. 2009). inclusion and exclusion standards were established in accordance with the study’s objectives. to find articles reporting the detection of trypanosome infection in cattle in côte d’ivoire between 1960 and 2021, a thorough search was performed. the databases of google scholar, pubmed and crossref were searched for relevant literature. the next search term was entered: the title, abstract and keywords, when appropriate, used the terms ‘bovine trypanosomiasis prevalence’ or ‘bovine trypanosomiasis and côte d’ivoire’. regarding language and publication date, there were no limitations (the last search was performed on 17 july 2021). also included were additional manual searches of author collections of pertinent peer-reviewed articles. for the purpose of choosing preliminary studies, rayyan qcri (cambridge, united states and doha, qatar) (ouzzani et al. 2016) was used. articles from other databases were imported via publish or perish (harzing 2007). articles from pubmed were imported directly into rayyan qcri. references that were identical in terms of location, numerator, denominator and study period were eliminated. after a preliminary title and abstract screening, the complete texts of articles that were considered to be potentially relevant were examined. the bibliographies of the articles that were included were examined to locate additional pertinent and qualified articles. inclusion and exclusion criteria relevant literature was searched, evaluated and analysed according to the prisma statement (harzing 2007; kamioka 2019). inclusion and exclusion criteria, as well as search syntaxes, were predefined. initially, references were selected based on their titles. emphasis was placed on articles describing the epidemiology of bovine trypanosomiasis in côte d’ivoire, without limiting the date of publication. a previously stated inclusion criteria were established to include articles that offered relevant quantitative information on the epidemiology of bovine trypanosomiasis. the references retained after the initial selection were analysed according to the abstract. abstracts without full text, review articles, redundant information and studies that only describe the diagnosis of bovine trypanosomiasis were excluded. the full-text file of the publication was included if the data in the abstract were insufficient for evaluation. the following inclusion criteria were used to examine full-text publications: (1) a publication should contain information on cattle in côte d’ivoire; (2) contain information on the prevalence incidence and distribution of trypanosome species in cattle in côte d’ivoire. the following causes contributed to the exclusion of full-text publications: (1) unspecified diagnostic test; (2) sample source not specified; (3) review articles; (4) articles reporting data published elsewhere; (5) cases reports based only on clinical signs; (6) epidemic reports without a lab confirmation and (7) reports of prevalence in any diagnostic test experimentation. publications with pertinent epidemiological data were included in the quantitative ma to determine the overall prevalence. information such as cattle breeds, trypanosomes species responsible for the infections, sample size, method of diagnosis and prevalence were included in the analysis. prevalence estimation was performed after diagnostic tests categorisation. thus, three categories were used to group the diagnostic tests: (1) parasitological methods such as wet blood smears, stained blood smears and microhematocrit concentration; (2) serological tests using antibody-based enzyme-linked immunosorbent assay (ab-elisa) and antigen-based enzyme-linked immunosorbent assay (ag-elisa) and (3) molecular tests including ordinary polymerase chain reaction (pcr) with various primers. data extraction reference information such as author’s name, title and publication year were recorded in the database. the following data were extracted from the included publications: study area (department, region), periods of sample collection, cattle breeds, sample size, origin of the sample collected, diagnostic method used, number of positives, prevalence. prevalence of trypanosomiasis was defined as the frequency of infection with t. congolense, t. vivax and t. brucei in a given population at a given time. for articles that only reported the total number of animals sampled and the prevalence, the number of positives was estimated. similarly, prevalence was estimated for article that reported only the number of samples and the number of positives. case reports containing the required information, with the exception of prevalence data, were also included in the analysis. all data were recorded in an ms excel spreadsheet. statistical analysis the data analysis was performed in several steps. firstly, the mean prevalence was estimated by summing the number of cases in all studies included, divided by the total number of cattle sampled. if the year of data collection was not specified in the research, the publication year minus one (n−1) was considered. secondly, the estimation of the total prevalence of bovine trypanosomiasis in the general sample size and their 95% confidence intervals (cis) were evaluated by a random-effects. in these studies, heterogeneity was assessed by cochran’s q-test and the inverse of variance index (i). the variance index (i-squared) reflects the percentage of overall variation between studies due to heterogeneity and not by chance. i-squared values of 25%, 50% and 75%, respectively, indicate low, moderate and high degrees of heterogeneity. i-squared values of 0% imply that no heterogeneity was detected. on a standardised scale, q is the weight of squares. it is presented as a p-value, with low values suggesting heterogeneity. subgroup analyses were carried out to identify potential sources of study heterogeneity. the study area (region and department), sample size, cattle breed, blood source, diagnostic method and trypanosome species were variables considered in the subgroup analysis. a funnel plot and an egger plot allowed us to examine by visualisation the bias between studies. in addition, the statistical significance of bias was tested with egger’s regression skewness test (egger et al. 1997). data were analysed using comprehensive meta-analysis software version 3.3.070 (biostat, englewood, united states [us]). using quantum gis version 2.0.1 (open source geospatial foundation, boston, us) a map detailing the trypanosome species found at the research sites was developed. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. results literature selection a total of 1902 potential articles (figure 1) were obtained from the literature searches. after reviewing titles, abstracts and excluding repeated articles from the database, for full text review, 176 articles were chosen. after examination of these articles based on our selection criteria, we obtained 25 articles that were eligible for systematic review. of these 25 articles, 11 were selected to make the ma. figure 1: preferred reporting items for systematic reviews and meta-analyses flow diagram of studies’ screening and selection. geographical distribution of bovine trypanosomiasis the studies included in this review were conducted between 1979 and 2016 in 26 departments grouped into 12 regions (bafing, bagoue, bere, bounkani, gkeke, hambol, kabadougou, marahoue, poro, tchologo, tompki and worodougou). sample sizes ranged from 87 to 3040 animals with infection prevalence ranging from 2.99 % (95% ci: 2.88% – 3.09%) to 25.28% (95% ci: 25.17% – 25.38%). with i-squared and p-value of 98.86 and p < 0.001, the prevalence of trypanosomiasis in cattle in côte d’ivoire was heterogeneous. details of the studies are summarised in table 1. table 1: studies included in the quantitative synthesis. the spatial distribution of prevalence is illustrated in figure 2, which shows that studies on the geographical distribution of bovine trypanosomiasis were mainly conducted in the northern, northwestern, northeastern and central regions of côte d’ivoire. the highest prevalence rates were observed in the departments of bouna 14.94% (95% ci: 14.93% – 14.95%), beoumi 14.68% (95% ci: 14.67% – 314.69%), bonon 14.29% (95% ci: 14.28% – 14.29%), sinfra 13.46% (95% ci: 13.45% – 13.47%), boundiali 11.55% (95% ci: 11.54% – 11.56%), ferkessedougou 11% (95% ci: 10.99% – 11.01%), korhogo 8.69% (95% ci: 8.68% – 8.70%) and m’bemgue 8.36% (95% ci: 8.35% – 8.37%). figure 2: distribution of the trypanosome species circulating in cattle in côte d’ivoire based on studies published between 1960 and 2021 included in the systematic review. analysis and regression of data extracted from 11 studies included in the meta-analysis the prevalence estimates of the 11 studies included in the ma are shown in table 1-a1, table 2-a1 and table 3-a1. heterogeneity was observed both at the level of the studies and in the subgroups defined as factors of variation. these subgroups or factors are region, department, method of diagnosis, breed of cattle and trypanosome species responsible for the infections. the subgroup analysis revealed large variations in prevalence and stable meta-regression models with z = -11.13; p < 0.001. the ma shows that the most infected regions were bagouée, bounkani, gbeke, marahoue, poro and tchologo with prevalence of 11.26% (95% ci: 11.25% – 11.27%), 14.94% (95% ci: 14.93% – 14.95%), 10.34% (95% ci: 10.33% – 10.35%), 13.79% (95% ci: 13.78% – 13.79%), 8.50% (95% ci: 8.49% – 8.51%) and 11.83% (95% ci: 11.82% – 11.84%), respectively. through these analyses, the most sensitive diagnostic method was the pcr technique. parasitological diagnostic techniques (giemsa and woo techniques) were more sensitive than elisa test. these different techniques confirm the presence of trypanosome species such as t. vivax, t. congolense and t. brucei. the observed prevalence was 4.99% (95% ci: 4.97% – 5.01%), 1.51% (95% ci: 1.49% – 1.52%) and 0.61% (95% ci: 0.59% – 0.62%), respectively. regarding cattle breeds, the ma revealed that the least infected breed was the taurine breed baoulé with a prevalence of 1.62% (95% ci: 1.60% – 1.64%). the taurine breeds (n’dama and baoulé) were less infected compared with the zébu and méré breeds whose prevalence rates were 3.67% (95% ci: 3.65% – 3.69%) and 3.50% (95% ci: 3.48% – 3.52%), respectively (table 3-a1). the meta-regression (figure 3 and figure 4) by survey year (considered as continuous variable) indicated a significant increase of the prevalence of bovine trypanosomiasis (z = -11.13; q = 191.05; degree of freedon [df] = 10; p < 0.001). figure 3 and figure 4 show the scatter plot of the regression of mean prevalence with survey year. the p-value for the survey year was < 0.05, indicating a stable pattern over time and space. figure 3: prevalence regression by years of different studies. figure 4: plots of precision (1/standard error) by estimation of prevalence. discussion this study presents a systematic review of bovine trypanosomiasis prevalence and its pathogens in côte d’ivoire. the analyses indicate that bovine trypanosomiasis prevalence in côte d’ivoire has significantly increased over the years. the increase in prevalence could be explained by several factors including limited number of studies and political prioritisation of the eradication of human african trypanosomiasis over bovine trypanosomiasis. since the first outbreak of the african trypanosomiasis, 11 studies over a period of 50 years have been conducted on bovine trypanosomiasis and the majority of these studies were concentrated in the last 18 years. control and eradication of animal trypanosomiasis have also been hampered by the various military-political crises in côte d’ivoire since 2000s. tsetse fly control efforts have not been successful in these areas, unlike in areas endemic to human trypanosomiasis that were under state control during these crises (kambiré et al. 2012). the highest prevalence was found in the northern part of côte d’ivoire. these results are consistent with the findings of n’djetchi et al. (2017). this zone is partly the major livestock area because of its vegetation and climate that favour livestock activity and in this area the animals have a role in the socio-cultural development and also a value (sokouri et al. 2014). this territory is crossed by the four major rivers, which are the bandama, cavally, sassandra and comoé. these rivers are bordered by gallery forests (acapovi-yao et al. 2016; camus 1981), which are known to be preferred sites for tsetse fly development (bokar 1997; sokouri et al. 2014). this area has several small dense forests, where cultural rites are conducted. these small forests are off-limits and constitute a favourable biotope for the proliferation of tsetse flies. the incriminated vectors are glossina palpalis palpalis (robineau-desvoidy 1830), g. palpalis gambiensis (vanderplanck 1949) and g. tachninoides (westwood 1830) (acapovi-yao et al. 2010; gouteux & millet 1984). according to some authors (djohan et al. 2015; jamonneau et al. 2003), g. palpalis gambiensis and g. tachninoides are the vectors responsible for the transmission of animal trypanosomiasis. the high prevalence in the northern zone could be because of incorrect use of trypanocides, which promotes the development of trypanosome resistance (simo et al. 2020). trypanosoma congolense, t. vivax and t. brucei are commonly found in the 12 regions of sudanese zone of côte d’ivoire (solano et al. 1997) and are considered to be the most important pathogens in trypanosoma infections in these regions (acapovi-yao et al. 2016; poivey et al. 1983; yao et al. 2020). their presence may be related to the aforementioned specific tsetse species that are found in cattle in these farming areas mainly concentrated in the northern part of côte d’ivoire, which is the savannah zone. the majority of studies included in this review have used parasitological methods. these methods are dependent on a high parasitemia in infected animals and on a morphological observation, which require expertise and experience of a technician making the observations (hailu 2019). irrespective of the technique used, pcr proved to be the most sensitive. the technique was validated using blood samples from experimentally infected animals, revealing its higher sensitivity and specificity when compared with parasitological techniques (cox et al. 2005; solano et al. 1999). this confirms the place of molecular techniques as the best means of diagnosis and identification of microorganisms. the most predominant species of trypanosoma was t. vivax. similar observations have also been reported in burkina faso (desquesnes & dia 2003). this might be as a result of the transmission of t. vivax by other mechanical vectors as cattle are subjected to biting flies (tabanids and stomoxys) while grazing. according to desquesnes et al. (2022), t. vivax geographical distribution is the whole of sub-saharan africa, the south and central american and iran. t. congolense savannah group is the agent responsible for the most severe form of animal trypanosomiasis (bengaly et al. 2002). the analyses showed that the taurine breeds (indigenous) are less parasitized than méré (cross-breed) and zebu breeds. other authors (yao et al. 2020) reported similar results; this is because of an inherited trypanotolerance trait in taurines. indeed, thanks to this genetic predisposition, these animals remain productive in these endemic areas. with the méré, a breed resulting from cross-breeding taurines and zebu, the objective was to create a more hardy breed with this trypanotolerant trait. unfortunately, high prevalence of trypanosomiasis among the mérés suggests otherwise (sokouri et al. 2009). conclusion this work represents the first attempt of a systematic review of bovine trypanosomiasis in côte d’ivoire. this review of systematic and ma provides comprehensive information on geographic distribution, infected cattle breeds and prevalence of bovine trypanosomiasis. the findings indicate variation in estimated prevalence depending on the diagnostic test used. estimated bovine trypanosomiasis prevalence is high; however, variability was observed between departments and survey years. based on the economic importance of the disease in livestock production, its detection in cattle is of great concern. it is recommended that pcr, which was the most sensitive technique, should be used in diagnosis and to facilitate control. risk of transmission of trypanosomiasis in cattle in côte d’ivoire remain high. considering the high prevalence of trypanosomiasis in crossbred cattle, crossbreeding in farms should be controlled. as t. vivax was one of the most important trypanosome species detected. in addition to regular tsetse control campaigns in the country, efforts to control other mechanical vectors should also be put in place. acknowledgements the authors are indebted to mr bamba zie, a student at the peleforo gon coulibaly university of korhogo, for his help in the design of the trypanosome species distribution map. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.-y.e. conducted the analysis, developed the maps and drafted the manuscript. e.k.n. made corrections and adjusted the ideas and gave suggestions for data management. l.e.g.m. read and made corrections and suggestions. e.m.m. supervised the work, made corrections, provided ideas for the development of the map and approved the final manuscript. funding information this work is supported by the partnership for skills in applied sciences, engineering and technology (paset) – regional scholarship and innovation fund (rsif). data availability the authors confirm that the data supporting the findings of this 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karamoko, y., soro, d., faustin, z., bi, z., joel, a. et al., 2017, ‘prevalence of trypanosoma, babesia and anaplasma in cattle reared in the north of côte d’ivoire’, international journal of biosciences 10(2), 21–28. https://doi.org/10.12692/ijb/10.2.21-28 appendix 1 table 1-a1: pooled prevalence estimates of bovine trypanosomiasis by region and department. table 2-a1: pooled prevalence estimates of bovine trypanosomiasis respect by diagnostic methods. table 3-a1: pooled prevalence of bovine trypanosomiasis, stratified by bovine breed and trypanosome species. mahan_99-105.qxd introduction heartwater is an acute infectious disease caused by ehrlichia (cowdria) ruminantium and is transmitted to domestic and wild ruminants by amblyomma ticks (walker & olwage 1987). in susceptible domestic ruminants, heartwater can cause severe losses through high mortality rates of up to 90 % (mahan, smith, kumbula, burridge & barbet 2001). animals which recover from primary infection become asymptomatic carriers of e. ruminantium and serve as reservoirs of infection for ticks (andrew 99 onderstepoort journal of veterinary research, 71:99–105 (2004) the pcs20 pcr assay for ehrlichia rruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the united states of america s.m. mahan1*, b.h. simbi1 and m.j. burridge2 abstract mahan, s.m., simbi, b.h. & burridge, m.j. 2004. the pcs20 pcr assay for ehrlichia ruminantium does not cross-react with the novel deer ehrlichial agent found in white-tailed deer in the united states of america. onderstepoort journal of veterinary research, 71:99–105 white-tailed deer are susceptible to heartwater (ehrlichia [cowdria] ruminantium infection) and are likely to suffer high mortality if the disease spreads to the united states. it is vital, therefore, to validate a highly specific and sensitive detection method for e. ruminantium infection that can be reliably used in testing white-tailed deer, which are reservoirs of antigenically or genetically related agents such as ehrlichia chaffeensis, anaplasma (ehrlichia) phagocytophilum (hge agent) and ehrlichia ewingii. recently, a novel but as yet unnamed ehrlichial species, the white-tailed deer ehrlichia (wtde), has been discovered in deer populations in the united states. although the significance of wtde as a pathogen is unknown at present, it can be distinguished from other ehrlichia spp. based on 16s rrna gene sequence analysis. in this study it was differentiated from e. ruminantium by the use of the pcs20 pcr assay which has high specificity and sensitivity for the detection of e. ruminantium. this assay did not amplify dna from the wtde dna samples isolated from deer resident in florida, georgia and missouri, but amplified the specific 279 bp fragment from e. ruminantium dna. the specificity of the pcs20 pcr assay for e. ruminantium was confirmed by southern hybridization. similarly, the 16s pcr primers (nested) that amplify a specific 405–412 bp fragment from the wtde dna samples, did not amplify any product from e. ruminantium dna. this result demonstrates that it would be possible to differentiate between e. ruminantium and the novel wtde agent found in white tailed deer by applying the two respective pcr assays followed by southern hybridizations. since the pcs20 pcr assay also does not amplify any dna products from e. chaffeensis or ehrlichia canis dna, it is therefore the method of choice for the detection of e. ruminantium in these deer and other animal hosts. keywords: ehrlichia (cowdria) ruminantium, pcr, pcs20 sequence, white-tailed deer, white-tailed deer ehrlichia * author to whom correspondence is to be directed current address: university of florida heartwater control research program, postnet suite 294, private bag x06, waterkloof, pretoria, 0145 south africa e-mail: sumanmah@mweb.co.za 1 uf/usaid/sadc heartwater research project, central veterinary research and diagnostic laboratory, p.o. box cy 551, causeway, harare, zimbabwe 2 department of pathobiology, university of florida, p.o. box 110880, gainesville, fl. 32610 usa accepted for publication 23 october 2003—editor & norval 1989; peter, burridge & mahan 2002). several wild game species are affected by e. ruminantium. some wild animal species are refractory to e. ruminantium infection, while some serve as carriers and others, such as white-tailed deer (odocoileus virginianus), springbok (antidorcas marsupialis) and kafue lechwe (kobus leche kafuensis) suffer high mortality when infected experimentally or naturally (camus, barré, martinez & uilenberg 1996; peter et al. 2002). the role played by wild animal species in the epidemiology of heartwater is still not fully understood. nevertheless, it has been shown that e. ruminantium can be maintained in amblyomma hebraeum ticks found exclusively in the kruger national park in south africa where there has been no contact between the wild game species and cattle for at least 40 years (peter, bryson, perry, o’callaghan, medley, smith, mlambo, horak, burridge & mahan 1999), indicating that the ticks maintain their infection by feeding on infected wild animal species found in the park. in the united states of america (usa), the whitetailed deer population plays a key role in the epidemiology of ehrlichial infections which cause serious illnesses in humans (dawson, stallknecht, howerth, warner, biggie, davidson, lockhart, nettles, olson & childs 1994; ewing, dawson, kocan, barker, warner, panciera, fox, kocan & blouin 1995; lockhart, davidson, stallknecht, dawson & howerth 1997). white-tailed deer are reservoirs of ehrlichia chaffeensis the causative agent of human monocytic ehrlichiosis, anaplasma (ehrlichia) phagocytophilum (dawson, warner, baker, ewing, stallknecht, davidson, kocan, lockhart & olson, 1996), the cause of human granulocytic ehrlichiosis (previously known as the hge agent), ehrlichia ewingii, one of the causative agents of canine granulocytotropic ehrlichiosis and also causes infections in humans (yabsley, varela, tate, dugan, stallknecht, little & davidson 2002) and a novel ehrlichial organism, white-tailed deer ehrlichia (wtde). the wtde which is distinct from the other ehrlichial agents based on 16s rrna sequences (dawson et al. 1996; brandsma, little, lockhart, davidson, stallknecht & dawson 1999), is closely related to anaplasma (ehrlichia) phagocytophila genogroup, which also includes ehrlichia equi, ehrlichia platys and the hge agent, also based on 16s analysis (little, dawson, lockhart, stallknecht, warner & davidson 1997). heartwater, at present, does not occur in the usa but if it did invade the country, white-tailed deer would be a susceptible target population. it is, therefore, important to differentiate infections caused by the various ehrlichial agents. serological assays available for detection of antibodies to e. ruminantium also detect crossreactions due to other ehrlichiae hence compromising interpretations of serological results (mahan, barbet, tebele, nyathi, wassink, semu, peter & kelly 1993). consequently, it is critical to have an assay that can be used with confidence for the detection and differentiation of e. ruminantium infection from infections caused by other agents. the pcs20 pcr assay has been shown to be highly specific and sensitive for detection of e. ruminantium dna in ticks and in clinically ill animals (mahan, waghela, mcguire, rurangirwa, wassink & barbet 1992; mahan et al. 1993; peter, deem, barbet, norval, simbi, kelly & mahan 1995; allsopp, hattingh, vogel & allsopp 1999; peter, barbet, alleman, simbi, burridge & mahan 2000). in addition, it has previously been demonstrated that the pcs20 pcr assay does not amplify any dna products from e. chaffeensis and ehrlichia canis dna (peter et al. 1995, 2000), the infectious agents most closely related to e. ruminantium by 16s rdna analysis and by analysis of genes encoding the outer membrane proteins (reddy, sulsona, barbet, mahan, burridge & alleman 1998; sulsona, mahan & barbet 1999; yu, mcbride, zhang & walker 2000; ohashi, rikihisa & unver 2001). the pcs20 pcr assay was used to differentiate between the wtde agent and e. ruminantium. materials and methods white-tailed deer ehrlichia and e. rruminantium dna blood samples from white-tailed deer infected with the wtde were collected from deer located in georgia, missouri and florida in usa, and dna was prepared as described previously (little et al. 1997). these dna samples were a kind donation from dr susan little of university of georgia. ehrlichia ruminantium dna was extracted from bovine endothelial cell cultures infected with the crystal springs strain (byrom, yunker, donovan & smith 1991). briefly, infected bovine endothelial cell cultures were spun at low speed (400 x g) to pellet large cellular material; this was followed by highspeed centrifugation (30 000 x g) of the supernatants to pellet the organisms. the organisms were resuspended in phosphate buffered saline (pbs) and purified through discontinuous percol gradients ranging from 0–40 % in pbs to separate them from bovine cellular materials at 400 x g. the purified organisms were harvested from the top 100 erlichial agent in white-tailed deer in usa layer (0 %) and washed twice with pbs by centrifugation at 30 000 x g and thereafter resuspended in fresh pbs. dna was extracted from the organisms by the qiagen technique as recommended by the manufacturers (qiagen genomic dna extraction kit, qiagen corp, valencia. ca). pcr assay to detect white-tailed deer ehrlichia dna a diagnostic nested pcr was performed using an established protocol (little et al. 1997). the pcr primers were kindly provided by dr susan little. a primary reaction using external general ehrlichia 16s primers ecb and ecc on white-tailed deer or e. ruminantium dna was conducted as previously described (little et al. 1997). a secondary reaction on the primary pcr product was carried out using internal primers dga and ga1ur specific to the wtde (little et al. 1997). for the primary amplification, a 50 µl reaction mixture containing 5 µl of dna, 10 mm tris cl (ph 8.3), 50 mm kcl, 0.001 % gelatine, 2.5 mm mgcl2, 200 µm each of deoxynucleoside triphosphates datp, dctp, dgtp and dttp, 1.25 u taq dna polymerase (perkin elmer corporation, norwalk, connecticut, usa), and 1 µm each of primers ecb (5’cgtattaccgcggctgctggca-3’), and ecc (5’-agaacgaacgctggcggcaagcc-3’), was overlaid with 50 µl of mineral oil (sigma co., st louis, missouri, usa). amplification was done by denaturation at 94 °c for 1 min, annealing at 45 °c for 2 min and extension at 72 °c for 2 min. a total of 40 cycles were performed followed by an automatic extension for 10 min at 72 °c. the secondary nested pcr was performed using 1 µl of the product from the primary reaction mixed as above but with 5 µm each of the primers dga 5’ttatctctgtagcttgctacg-3’ and gaiur 5’gagtttgccgggacttcttct-3’. the reaction was amplified for 30 cycles of 1 min denaturation at 94 °c, 2 min annealing at 55 °c and 2 min extension at 72 °c. the products from the second nested pcr reactions were analyzed by electrophoresis on a 1.5 % agarose gel and viewed with uv light illumination and photography after staining with ethidium bromide. the pcr products were transferred onto a nylon membrane (nen life science products, boston, massachusetts) and hybridized with a 32plabelled specific pcs20 dna probe or the 16s wtde amplified dna fragment as probe, as described previously (mahan et al. 1992; peter et al. 2000). the results of the hybridizations were evaluated by autoradiography. in all the pcr assays, a reagent blank control (no template dna) and a respective positive control (wtde or e. ruminantium dna) reaction were included. the e. rruminantium-specific pcs20 pcr assay a nested pcr, specific for e. ruminantium, was also performed to amplify dna from e. ruminantium and wtde-infected deer dna samples. in a primary reaction, primers u24 (5’tttccctatgatacagaaggtaac-3’) and l24 (5’-aaagcaaggattgtgatctggacc-3’) were used to amplify from e. ruminantium dna (350 bp dna fragment) and from wtde dna. in the second or nested pcr assay with primers ab 128 (5’actagtagaaattgcacaatctat-3’) and ab129 (5’-tgataacttggtgcgggaaatcctt-3’), a 279 bp e. ruminantium-specific dna fragment was amplified as described previously (mahan et al. 1992; peter et al. 2000). each pcr reaction contained a volume of 50 µl containing the following components: 5 µl of template dna, 10 mm tris ph 8.3, 50 mm kcl, 0.001 % gelatine, 3.0 mm mgcl2, 0.5 µm each of the primers l24 and u24 (genosys biotechnologies, inc. the woodlands, texas, usa), 200 µm of deoxynucleoside triphosphates datp, dctp, dgtp and dttp, and 1.25 u taq polymerase. denaturation of dna was done at 94 °c for 1 min, annealing of primers was at 55 °c for 1 min and extension at 72 °c for 2 min. after 45 cycles, an automatic 10 min extension at 72 °c was followed by a soak at 4 °c. in the second or nested reaction, 1 µl of the primary product was used in a 50 µl reaction mix with the same reaction and amplification conditions as the primary reaction but with the primers: ab 128 and ab 129. in all the pcr reactions, a reagent blank control (no template dna) and a respective positive control was included. the products from the second nested pcr reactions were analyzed by electrophoresis on a 1.5 % agarose gel and viewed with uv light illumination and photography after staining with ethidium bromide. the pcr products were transferred onto nylon membrane (nen life science products, boston. ma) and hybridized with a 32p-labelled specific pcs20 dna probe or the 16s wtde-amplified dna fragment as probe, as described above. the results of the hybridizations were evaluated by autoradiography. results the nested wtde 16s specific primers dga and gaiur amplified a dna product of 405–412 bp 101 s.m. mahan, b.h. simbi & m.j. burridge from the deer samples containing wtde dna from florida, georgia and missouri, but not from e. ruminantium dna (fig. 1). the general ehrlichia primers ecb and ecc amplified a product from the e. ruminantium dna which was of higher molecular weight than the product from the nested pcr on wtde dna, and appeared as a diffuse band when visualized by agar gel electrophoresis following the nested pcr (fig. 1, lane 8). in comparison, the (nested) primers ab 128 and ab 129 specific for the pcs20 sequence of e. ruminantium, amplified the 279 bp fragment from e. ruminantium dna but not from the wtde-infected deer dna samples originating from florida, georgia and missouri (fig. 1). further confirmation of the specificity of the two pcr reactions was done by southern hybridization. the labelled pcs20 probe of e. ruminantium hybridized only to the products from the pcs20 pcr assays on e. ruminantium dna and not to any of the samples which were amplified from wtde dna. the 405–412 bp probe of wtde hybridized only to the products from the nested pcr done with the wtde primers on wtde dna and not to products from e. ruminantium dna (fig. 2a and b). discussion white-tailed deer play an important role in the epidemiology of infections which affect humans, as they are reservoirs of e. chaffeensis, e. ewingii and anaplasma (ehrlichia) phagocytophilum (the hge agent) (little, stallknecht, lockhart, dawson & davidson 1998). the role, if any, of the recently identified wtde in the pathogenesis of disease remains to be determined because, it has not yet been isolated and characterized in-vitro. however, based on the 16s sequences, the wtde has been shown to be unique and different from other ehrlichial agents so far characterized, but it is closely allied to e. phagocytophila genogroup, which includes e. platys, e. equi and anaplasma (ehrlichia) phagocytophilum (the hge agent) (dawson et al. 1996). the phylogenetic classification of the ehrlichia and rickettsia spp. has been recently revised based on sequence similarities of the16s gene, the citrate synthase gene, the groel sequences and genes encoding structural outer membrane proteins of these organisms that were previously classified 102 erlichial agent in white-tailed deer in usa 405–412 bp fig. 1 a pcs20 pcr assay for e. ruminantium does not detect dna from white tailed deer ehrlichia. agarose gel electrophoresis of pcr products with primers specific for wtde show amplification of a dna product of 405–412 bp from wtde dna (lanes 1, 3, 4, 5 and 6) but not from e. ruminantium crystal springs strain dna (lane 8). lanes 11 and 18 represent the results of the e. ruminantium-specific pcr assay. no amplification was detected when the pcs20 pcr was done on wtde dna (lanes 13–16) but amplification of a 279 bp fragment was seen with e. ruminantium dna (lanes 11 and 18). lane m is the 123 bp dna markers. wtde dna samples are from white tailed deer from the following locations: lanes 1 and 6 are os 82 (ossabaw island georgia), lane 3 is mo (missouri), lane 4 is florida 2 and lane 5 is florida 1. lanes 2 and 17 are reagent blanks and 7, 9, 10, 12 and 17 are empty m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 m 279 bp under different families or tribes (dumler, barbet, bekker, dasch, jongejan, palmer, ray, rikihisa & rurangirwa 2001). due to its genetic similarity to e. chaffeensis and e. canis, cowdria ruminantium has been placed in the genus ehrlichia and renamed ehrlichia ruminantium. this genus now includes e. ruminantium, e. chaffeensis, e. canis, e. ewingii, and ehrlichia muris. because of the close genetic and structural relationship of these ehrlichia spp., and their related epidemiology, it is important to differentiate them from each other using reliable detection assays. serological assays are unable to differentiate definitively between e. ruminantium and several of the related ehrlichial agents because of marked cross-reactions between immunogenic proteins of these organisms. the pcr assay has proved to be more specific and sensitive in differentiating these related organisms. the pcs20 pcr assay, which is specific for e. ruminantium, is the method of choice for its detection and differentiation from the other ehrlichial agents (peter et al. 1995, 2000; allsopp et al. 1999). it was also able to differentiate between the wtde organism and e. ruminantium in this study. this was shown by both agarose gel electrophoresis and southern hybridizations. the nested pcr assay for the wtde was also found to be specific. however, the fragment that was amplified after the primary pcr using ecb and ecc primers on e. ruminantium might be confused as being a true amplification, and hence southern hybridization with the 405–412 bp fragment of wtde dna as probe would assist in determining whether the agent being detected is e. ruminantium or the wtde agent. the pcs20 pcr assay would be the method of choice for the detection of e. ruminantium infections in the white tailed deer, if heartwater did spread to the usa. this assay will also not detect infections caused by other related ehrlichial agents and, hence, can be applied reliably for surveillance of heartwater in animals and vectors. the risks of the introduction of heartwater onto the american mainland associated with animal movements are increasing (burridge, simmons, peter & 103 s.m. mahan, b.h. simbi & m.j. burridge 405–412 bp fig. 2a southern hybridization of pcs20 32p-labelled probe with the pcr products as in fig. 1, to show positive hybridization of the pcs20 e. ruminantium probe to the 279 bp products from e. ruminantium dna. no hybridization occurred with the wtde dna samples fig. 2b southern hybridization of 405–412 bp wtde pcr product 32p-labelled as probe with the pcr products as in fig. 1 and show positive hybridization of the probe to the wtde dna samples but not to the samples from e. ruminantium dna 1 2 3 54 6 7 8 9 10 11 12 13 14 15 16 17 18 279 bp a b mahan 2002). one measure to reduce these risks is the use of improved tests, such as the pcr assay, for the screening of animals to prevent the entry of carriers of e. ruminantium. for these tests to be used reliably, it is important that they do not produce cross-reactions with native ehrlichial parasites. this report demonstrates that the pcs20 pcr assay for e. ruminantium does not cross-react with the novel wtde organism, making it a valuable tool in monitoring and prevention of introduction of heartwater into the usa and elsewhere. acknowledgements this study was funded by the united states agency for international development grant number lag1328-g-00-3030-00 to the university of florida. we thank dr susan little for the donation of the samples infected with the wtde and the pcr primers specific for wtde. references allsopp, m.t.e.p., hattingh, c.m., vogel, s.w. & allsopp, b.a. 1999. evaluation of 16s, map 1 and pcs20 probes for the detection of cowdria and ehrlichia species. epidemiology and infection, 122:323–328. andrew, h.r. &. norval, r.a.i. 1989. the carrier status of sheep, cattle and african buffalo recovered from heartwater. veterinary parasitology, 34:261–266. brandsma, a.r., little, s.e., lockhart, j.m., davidson, w.r., stallknecht, d.e. & dawson, j.e. 1999. novel ehrlichia organism (rickettsiales: ehrlichiae) in white-tailed deer associated with lone star tick (acari: ixodidae) parasitism. journal of medical entomology, 36:190– 194. burridge, m.j., simmons, l.a., peter, t.f. & mahan, s.m. 2002. increasing risks of introduction of heartwater onto the american mainland associated with animal movements. annals of new york academy of sciences, 969:269– 274. byrom, b., yunker, c.e., donovan, p.l. & smith, g.e. 1991. in vitro isolation of cowdria ruminantium from plasma of infected ruminants. veterinary microbiology, 26:263–268. camus e., barré, n., martinez, d. & uilenberg, g. 1996. heartwater (cowdriosis). a review, 2nd ed. paris: office international des epizooties. dawson, j.e., stallknecht, d.e., howerth, e.w., warner, c.k., biggie, k., davidson, w.r., lockhart, j.m., nettles v.f., olson, j.g. & childs, j.e.1994. susceptibility of white-tailed deer (odocoileus virginianus) to infection with ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis. journal of clinical microbiology, 32:2725–2728. dawson, j.e., warner, c.k., baker, v., ewing, s.a., stallknecht, d.e., davidson, w.r., kocan, a.a., lockhart, j.m. & olson, j.g. 1996. ehrlichia-like 16s rrna sequence from wild white-tailed deer (odocoileus virginianus). journal of parasitology, 82:52–58. dumler, j.s., barbet, a.f., bekker, c., dasch, g.a., jongejan, f., palmer, g.h. , ray, s.c., rikihisa, y. & rurangirwa, f. 2001. reorganization of genera in the families rickettsiaceae and anaplasmataceae in the order rickettsiales: unification of some species of ehrlichia with anaplasma, cowdria and ehrlichia with neorickettsia, descriptions of six new species combinations and designation of ehrlichia equi and “hge agent” as subjective synonyms of ehrlichia phagocytophila. international journal of systematic & evolutionary microbiology, 51:2145–2165. ewing, s.a., dawson, j.e., kocan, a.a., barker, c.k., warner, c.k., panciera, r.j., fox, j.c., kocan, k.m. & blouin, e.f. 1995. experimental transmission of ehrlichia chaffeensis (rickettsiales: ehrlichiae) among whitetailed deer by amblyomma americanum (acari: ixodidae). journal of medical entomology, 32:368–374. little, s.e., dawson j.e., lockhart, j.m., stallknecht, d.e., warner, c.k. & davidson, w.r. 1997. development and use of specific polymerase reaction for the detection of an organism resembling ehrlichia sp. in white tailed deer. journal of wildlife diseases, 33:246–253. little, s.e., stallknecht, d.e., lockhart, j.m., dawson, j.e. & davidson, w.r. 1998. natural coinfection of a white-tailed deer (odocoileus virginianus) population with three ehrlichia spp. journal of parasitology, 84:897–901. lockhart, j.m., davidson, w.r., stallknecht, d.e., dawson j.e. & howerth, e.w. 1997. isolation of ehrlichia chaffeensis from white-tailed deer (odocoileus virginianus) confirms their role as natural reservoir hosts. journal of clinical microbiology 35:1681–1686. mahan, s.m., waghela, s.d., mcguire, t.c., rurangirwa, f.r., wassink, l.a. & barbet, a.f. 1992. a cloned dna probe for cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep. journal of clinical microbiology, 30:981–986. mahan, s.m., barbet, a.f., tebele, n., nyathi, c., wassink, l.a., semu, s., peter, t. & kelly, p.j. 1993. an immunoblotting diagnostic assay for heartwater based on the immunodominant 32kd protein of cowdria ruminantium detects also false positives in field sera. journal of clinical microbiology 31:2729–2737. mahan, s.m., smith, g.e., kumbula, d., burridge, m.j. & barbet, a.f. 2001. reduction in mortality from heartwater in cattle, sheep and goats exposed to field challenge using an inactivated vaccine. veterinary parasitology, 97: 295–308. ohashi, n., rikihisa, y. & unver, a. 2001. analysis of transcriptionally active gene clusters of major outer membrane protein multigene family in ehrlichia canis and e. chaffeensis. infection and immunity, 69:2083–2091. peter, t.f., deem, s.l., barbet, a.f., norval, r.a.i., simbi, b.h., kelly, p.j. & mahan, s.m. 1995. development and evaluation of a polymerase chain reaction assay for detection of low levels of cowdria ruminantium infection in amblyomma ticks not detected by dna probe. journal of clinical microbiology, 33:166–172. peter, t. f., bryson, n.r., perry, b.d., o’callaghan, c.j., medley, g.f., smith, g.e., mlambo, g., horak, i.g., burridge, m.j. & mahan, s.m. 1999. cowdria ruminantium infection in ticks in the kruger national park. the veterinary record, 145:304–307. peter, t.f., barbet, a.f., alleman, a.r., simbi, b.h., burridge, m.j. & mahan, s.m. 2000. detection of the agent of heartwater, cowdria ruminantium, in amblyomma ticks by pcr: validation and application of the assay to field ticks. journal of clinical microbiology, 38:1539–1544. 104 erlichial agent in white-tailed deer in usa peter, t.f., burridge, m.j. & mahan, s.m. 2002. ehrlichia (cowdria) ruminantium infection (heartwater) in wild animals. trends in parasitology 18:214–218. reddy, g.r., sulsona, c.r., barbet, a.f., mahan, s.m., burridge, m.j. & alleman, a.r. 1998. molecular characterization of a 28 kda surface antigen gene family of the tribe ehrlichiae. biochemical and biophysical research communications, 247:636–643. sulsona, c.r., mahan, s.m. & barbet, a.f. 1999. the map1 gene of cowdria ruminantium is a member of a multigene family containing both conserved and variable genes. biochemical and biophysical research communications, 257:300–305. walker, j.b. & olwage, a. 1987. the tick vectors of cowdria ruminantium (ixodoidea, ixodidae, genus amblyomma) and their distribution. onderstepoort journal of veterinary research, 54:353–379. yabsley, m.j., varela, a.s., tate, c.m., dugan, v.g., stallknecht, d.e., little, s.e. & davidson, w.r. 2002. ehrlichia ewingii infection in white-tailed deer (odocoileus virginanus). emerging infectious diseases. 8:668– 671. yu, x., mcbride, j.w., zhang, x. & walker, d.h. 2000. characterization of the complete transcriptionally active ehrlichia chaffeensis 28kda outer membrane protein multigene family. gene, 248:59–68. 105 s.m. mahan, b.h. simbi & m.j. burridge horak_9-14.qxd introduction for the past 30 years ectoparasites have been collected from a variety of wild carnivores in surveys aimed at determining the species composition of the arthropods that infest them. the tick species recovered from carnivores in two of these surveys have been recorded by horak, jacot guillarmod, moolman & de vos (1987) and horak, braack, fourie & walker (2000), as have the ticks, mites, fleas and lice infesting yellow mongooses, cynictis penicillata (horak, chaparro, beaucournu & louw 1999). this paper lists the remaining fleas collected during the surveys. materials and methods the procedures followed for the collection of ectoparasites have been described by horak et al. (2000) and, but for certain details, will not be repeated here. the material collected by vigorous scrubbing of the skins of the dead animals was preserved in 10 % formalin until it was examined microscopically and the ectoparasites so detected were transferred to 70 % alcohol. the prior storage in formalin made specific identification of delicate structures of some fleas impossible, and consequently if there was any doubt as to the identity of a flea it was excluded from our findings. the carnivore species and the localities at which they were examined are listed in table 1. in addition, we received fleas collected from a domestic dog in johannesburg, gauteng province. 9 onderstepoort journal of veterinary research, 71:9–14 (2004) parasites of domestic and wild animals in south africa. xliv. fleas (insecta: siphonaptera: pulicidae) collected from 15 carnivore species i.g. horak1, j.-c. beaucournu2 and l.e.o. braack3 abstract horak, i.g., beaucournu, j.-c. & braack, l.e.o. 2004. parasites of domestic and wild animals in south africa. xliv. fleas (insecta: siphonaptera: pulicidae) collected from 15 carnivore species. onderstepoort journal of veterinary research, 71:9–14 fleas were collected from 61 wild carnivores belonging to 13 species in various nature reserves and on farms, two feral domestic cats in a nature reserve and a domestic dog in the city of johannesburg. eleven flea species, including two subspecies of one of these, belonging to six genera were recovered. amongst these only ctenocephalides felis felis and ctenocephalides felis strongylus are considered specific parasites of carnivores. the remaining ten species normally infest the prey animals of the various carnivores. keywords: carnivores, fleas, pulicidae, siphonaptera, south africa 1 department of zoology and entomology, university of the free state, po box 339, bloemfontein, 9300 south africa 2 universite de rennes, faculté de médecine, parasitologie et zoologie appliquée, 2, avenue du professeur léon bernard, cs 34317-35043, rennes cedex, france 3 scientific services, south african national parks, kruger national park, skukuza, 1350 south africa. present address: p.o. box 2550, brooklyn square, 0075 south africa accepted for publication 1 september 2003—editor results and discussion besides the single domestic dog, fleas were collected from 13 species of wild carnivore as well as from two feral domestic cats. eleven flea species and two subspecies of one of these were recovered, and the various hosts and their flea burdens are summarized in table 2. pulicidae archaeopsyllinae genus: ctenocephalides five taxa of this flea occur in south africa and three of these were collected, namely ctenocephalides damarensis, ctenocephalides felis felis and ctenocephalides felis strongylus. of the remaining two, ctenocephalides canis is not only rare, but is essentially a parasite of palearctic carnivores (in south africa only the domestic dog fulfils this requirement). moreover, it and c. felis strongylus that infests dogs in this country have often been confused. the other, ctenocephalides connatus, is a parasite of yellow mongooses, suricates, suricata suricatta and ground squirrels, xerus inauris (de meillon, davis & hardy 1961; segerman 1995; horak et al. 1999). as we examined only one suricate its absence is not surprising. besides infestation of several animals by the inevitable “stragglers”, the numerous collections of c. damarensis confirm that the preferred host of this flea, the scrub hare, lepus saxatilis, constitutes a regular prey item for several carnivore species. as far as we can ascertain this is the first time that this flea has been recorded from acinonyx jubatus, caracal caracal, felis catus (feral), panthera leo, panthera pardus, ichneumia albicauda, proteles cristatus, mellivora capensis, civittictis civetta and genetta tigrina. extensive surveys of ectoparasites infesting l. saxatilis have been conducted at six widely separated localities in south africa (louw, horak & braack 1993; louw, horak, horak & braack 1995). of the 560 scrub hares examined 449 were infested with c. damarensis, which at the time were identified as c. felis damarensis, and the mean burden of infested animals was 12.6 fleas. beaucournu & ménier (1998) have, however, demonstrated that these fleas are not a subspecies of c. felis but a wellfounded species, namely c. damarensis. furthermore, we have removed ten fleas of this species from flannel strips used for the collection of free-living ticks from the vegetation in the kruger national park (knp). thus, not only are a large percentage of prey animals infested with c. damarensis, but there are also several off-host “stragglers” present in the environment, resulting in the high prevalence of infestation on carnivores. some fleas continue to 10 parasites of domestic and wild animals in south africa. xliv table 1 carnivores and the localities at which they were examined for fleas carnivore species no. examined locality or province common name scientific name domestic dog canis familiaris 1 johannesburg, gauteng black-backed jackal canis mesomelas 5 knp, central free state, north-western northern cape, eastern cape cheetah acinonyx jubatus 3 knp caracal caracal caracal 52 south-western free state, north-western northern cape, eastern cape feral domestic cats felis catus 7 knp african wild cat felis lybica 3 knp, south-western free state, north-western northern cape lion panthera leo 23 knp, north-eastern kwazulu-natal leopard panthera pardus 5 knp, gauteng white-tailed mongoose ichneumia albicauda 2 knp meercat suricata suricatta 1 north-western northern cape spotted hyaena crocuta crocuta 9 knp, north-eastern kwazulu-natal aardwolf proteles cristatus 2 south-western free state, eastern cape honey badger mellivora capensis 9 knp, northern northern cape, western cape civet cat civettictis civetta 6 knp, north-eastern mpumalanga large-spotted genet genetta tigrina 8 knp, north-eastern mpumalanga knp = kruger national park live, and even thrive, on accidentally infested felid or viverrid hosts. in europe ceratophyllus sciurorum, a very common flea of squirrels, survives on secondarily infested mustelids (smit 1966; beaucournu 1973, 1982). the only c. felis felis identified in the present study were five males and 12 females taken from a maltese poodle, which lived with its owners in an apartment in johannesburg. we have been unable to determine the origin of these fleas. ctenocephalides felis strongylus is widespread in south africa, but is only common on domestic dogs in rural areas. the taxonomic features accepted by beaucournu & ménier (1998) for this species do not permit differentiation between its females and those of c. damarensis. hence the numbers of females listed for the two species may not be exact. ctenocephalides felis strongylus is found only exceptionally on wild hosts, implying that the latter are secondarily infested, probably by “stragglers” from domestic dogs (segerman 1995). its rarity on wild hosts may, however, be artificial rather than actual, in that it has often been confused with other ctenocephalides spp., particularly c. damarensis as discussed above. only three collections of c. 11 i.g. horak, j-c. beaucournu & l.e.o. braack table 2 fleas collected from wild carnivores, feral domestic cats and a domestic dog in south africa species, gender and number of fleas collected (no. of hosts infested) host species no. ctenocephalides spp. echidnophaga spp. procaviopsylla spp. other species examined male female male female male female male female c. damarensis e. larina p. creusae m. sjoestedti c. mesomelas 5 0 2 (1) 0 1 (1) a. jubatus 3 1 4 (1) 1 2 (1) 0 1 (1) c. caracal 52 22 44 (11) 0 1 (1) 9 13 (5) f. catus 7 3 4 (2) p. angolensis f. lybica 3 1 2 (1) 1 0 (1) p. leo 23 8 17 (5) 3 6 (4) 0 1 (1) p. pardus 5 1 5 (1) 1 1 (1) i. albicauda 2 3 14 (1) c. crocuta 9 1 1 (1) p. cristatus 2 1 3 (1) e. gallinacea s. caffer m. capensis 9 1 6 (1) 1 4 (1) 0 6 (3) c. civetta 6 8 12 (3) x. brasiliensis g. tigrina 8 5 4 (2) 3 2 (1) c. felis felis c. familiaris 1 5 12 (1) c. felis strongylus p. divergens c. caracal 52 7 5 (3) 7 28 (10) 5 11 (3) e. bradyta s. suricatta 1 1 2 (1) felis strongylus were made; all from caracals in the eastern cape province, two in the coastal region and one in the karoo. pulicinae genus: echidnophaga echidnophaga gallinacea and echidnophaga larina do not normally infest carnivores, but represent infestations acquired from their prey. echidnophaga bradyta on the other hand has secondarily adapted to certain carnivores, in particular s. suricatta and c. penicillata (segerman 1995; horak et al. 1999). a new species, echidnophaga suricatta, that specifically infests s. suricatta has, however, recently been described (hastriter 2000). echidnophaga gallinacea is primarily a parasite of rodents, but has also been found on a number of other small mammals and on birds (segerman 1995). no e. gallinacea were, however, recovered from 92 rodents examined over a period of 2 years in the southern regions of the knp (braack, horak, jordaan, segerman & louw 1996). warthogs, phacochoerus africanus, are the preferred hosts of e. larina and 46 of 51 warthogs examined in the knp were infested with a total of 12 932 e. larina and phacopsylla inexpectata (the latter at the time identified as echidnophaga inexpectata) (beaucournu & horak 1994). the fleas belonging to these two species could not be counted separately because we were unable to dislodge many of them from their firm attachment in the skin on the softer undersides of the warthogs and they had to be counted in situ (horak, boomker, de vos & potgieter 1988). cheetahs, lions, leopards and hyaenas probably acquire infestation with e. larina when catching and devouring warthogs, whereas jackals could become infested either from catching young warthogs, or scavenging on the remains of warthogs killed by the larger carnivores, or even while sheltering in recently vacated warthog burrows. xenopsyllinae genus: procaviopsylla fleas of this genus are parasites of hyraxes, and secondarily of the predators of these animals. the three species of procaviopsylla present in south africa (procaviopsylla angolensis, procaviopsylla creusae and procaviopsylla divergens) are all represented in our collections. in south africa they parasitize the rock hyrax, procavia capensis, but according to de meillon et al. (1961) it is possible that specimens of p. angolensis that they studied came from dendrohyrax (= heterohyrax) brucei. the distribution of the carnivores infested in the present study, namely caracals, jackals and feral cats overlaps that of hyraxes. a total of 2 197 p. creusae were collected from 76 of 77 rock hyraxes examined in the mountain zebra national park, eastern cape province (horak & fourie 1986), and some of the caracals in the present study came from this park. however, to state “in fact, the caracal is the type host of this flea” as was done by horak & fourie (1986) is misleading as to the identity of the preferred host. although p. creusae was originally collected and described from caracals, these animals are certainly not the preferred hosts. haeselbarth, segerman & zumpt (1966) stated “this is the common parasite of the rock dassie (procavia capensis)…only a few stragglers have been found on rodents, the caracal lynx (felis caracal, the type host) and even the pied starling (spreo bicolor)”. the designation of a type host, which is not also the preferred host is a common problem, particularly with fleas. genus: xenopsylla with rare exceptions fleas of this genus infest rodents. thirty-one of 46 aethomys chrysophilus examined in the knp were infested with a total of 170 xenopsylla brasiliensis and 23 of 46 tatera leucogaster with a total of 140 xenopsylla frayi (braack et al. 1996). the five x. brasiliensis collected from one of the large spotted genets represent a secondary infestation of a predator by the fleas of its prey. genus: synosternus host-specificity in fleas of this genus varies according to species. their preferred hosts are rodents, lagomorphs and medium-sized carnivores such as viverrids, small felids and wild dogs. the principal host of synosternus caffer is the springhare, pedetes capensis. although springhares are widespread in south africa the distribution of s. caffer is restricted to the more western arid regions of the country (segerman 1995). the only animal on which we found fleas of this species was the honey badger, mellivora capensis, which is an omnivore. it would usually not be capable of catching a springhare unless it had dug open their burrows and caught their young, thus acquiring infestation from its prey or otherwise via “straggling” by fleas within 12 parasites of domestic and wild animals in south africa. xliv the burrow. the presence of s. caffer on eight of 20 yellow mongooses examined in the semi-arid region of kuruman in the northern cape province (horak et al. 1999), can be attributed to “straggling” because springhare warrens are frequently used as shelters by various mongoose species (skinner & smithers 1990). moeopsyllinae genus: moeopsylla this genus contains the single species moeopsylla sjoestedti, a specific parasite of warthogs (segerman 1995). it could thus secondarily infest large carnivores such as lions and leopards that prey upon these animals. a total of 143 fleas of this species were collected from 23 of 51 warthogs examined in the knp (horak et al. 1988). general unlike the holarctic region, where fleas of the genus chaetopsylla (vermipsyllidae) specifically infest carnivores, it would appear that, with the exception of certain ctenocephalides species and perhaps e. suricatta, fleas that infest carnivores within the afro-tropical region are not specific parasites of these animals. a similar situation pertains in the oriental region, where only ctenocephalides orientis specifically infests carnivores. in the neotropical region, however, certain fleas of the family rhopalopsyllidae, or the subfamily rhopalopsyllinae are frequently found on carnivores, and their host specificity appears extensive, with various families and genera of fleas infesting different families and genera of hosts. a significant contribution to our understanding of the fleas of the southern african region can be attributed to research on the vectors of plague (de meillon et al. 1961; segerman 1995), in the epidemiology of which carnivores and their specific fleas are of generally little direct importance. however, in this context their “accidental” flea fauna may be of greater significance. the presence on a predator of a rodent-specific flea, of which the preferred host is presumably absent from a region, should encourage mammalogists to continue searching for the host in that region. acknowledgements we are indebted to the south african national parks for placing the laboratory facilities at skukuza and the skins of the animals examined in the kruger national park at our disposal. we are most grateful to the following persons for assisting with the collection of fleas from these skins or from those of other carnivores: colleen begg, lientjie cohen, santa meyer, renadè bartlett, louise horak, ulrike zieger, keith begg, roy bengis, andrew cauldwell, gerbert grohs, bernd hey, johan sithole, andré uys and eddie williams, and to dr joyce segerman for sending us the fleas from the domestic dog. references beaucournu, j.-c. 1973. notes sur les siphonaptères parasites de carnivores en france. annales de parasitologie humaine et comparée, 48:497–516. beaucournu, j.-c. 1982. les siphonaptères et leurs hôtes: rapports phylétiques, convergences et déviations. deuxième symposium sur la spécificité parasitaire des parasites de vertébrés, paris 1981, mémoires du muséum national d’histoire naturelle, (n.s.), série a, zoologie, 123:203–208. beaucournu, j.-c. & horak, i.g. 1994. phacopsylla gen. nov. for echidnophaga inexpectata smit, 1950 (siphonaptera, pulicidae). journal of african zoology, 108:133–141. beaucournu, j.-c. & ménier, karine 1998. le genre ctenocephalides stiles & collins, 1930 (siphonaptera, pulicidae). parasite, 5:3–16. braack, l.e.o., horak, i.g., jordaan, leonora c., segerman, joyce & louw, j.p. 1996. the comparative host status of red veld rats (aethomys chrysophilus) and bushveld gerbils (tatera leucogaster) for epifaunal arthropods in the southern kruger national park, south africa. onderstepoort journal of veterinary research, 63:149–158. de meillon, b., davis, d. & hardy, f. 1961. plague in southern africa. vol. 1. the siphonaptera (excluding ischnopsyllidae). pretoria: government printer. haeselbarth, e., segerman, j. & zumpt, f. 1966. the arthropod parasites of vertebrates in africa south of the sahara (ethiopian region). vol. iii. (insecta excl. phthiraptera). johannesburg: publications of the south african institute for medical research, no. 52. hastriter, m.w. 2000. echidnophaga suricatta (siphonaptera: pulicidae), a new species of flea from the northern cape province, south africa. african zoology, 35:77–83. horak, i.g. & fourie, l.j. 1986. parasites of domestic and wild animals in south africa. xix. ixodid ticks and fleas on rock dassies (procavia capensis) in the mountain zebra national park. onderstepoort journal of veterinary research, 53:123–126. horak, i.g., jacot guillarmod, amy, moolman, l.c. & de vos, v. 1987. parasites of domestic and wild animals in south africa. xxii. ixodid ticks on domestic dogs and on wild carnivores. onderstepoort journal of veterinary research, 54:573–580. horak, i.g., boomker, j., de vos, v. & potgieter, f.t. 1988. parasites of domestic and wild animals in south africa. xxiii. helminth and arthropod parasites of warthogs, phacochoerus aethiopicus, in the eastern transvaal lowveld. onderstepoort journal of veterinary research, 55: 145–152. 13 i.g. horak, j-c. beaucournu & l.e.o. braack horak, i.g., chaparro, f., beaucournu, j.-c. & louw, j.p. 1999. parasites of domestic and wild animals in south africa. xxxvi. arthropod parasites of yellow mongooses, cynictis penicillata (g. cuvier, 1829). onderstepoort journal of veterinary research, 66:33–38. horak, i.g., braack, l.e.o., fourie, l.j. & walker, jane b. 2000. parasites of domestic and wild animals in south africa. xxxviii. ixodid ticks collected from 23 wild carnivore species. onderstepoort journal of veterinary research, 67:239–250. louw, j.p., horak, i.g. & braack, l.e.o. 1993. fleas and lice on scrub hares (lepus saxatilis) in south africa. onderstepoort journal of veterinary research, 60:95–101. louw, j.p., horak, i.g., horak, m. louise & braack, l.e.o. 1995. fleas, lice and mites on scrub hares (lepus saxatilis) in northern and eastern transvaal and in kwazulu-natal, south africa. onderstepoort journal of veterinary research, 62:133–137. segerman, joyce 1995. siphonaptera of southern africa. handbook for the identification of fleas. johannesburg: publications of the south african institute for medical research, no. 57. skinner, j.d. & smithers, r.h.n. 1990. mammals of the southern african subregion. pretoria: university of pretoria. smit, f.g.a.m. 1966. siphonaptera. insecta helvetica, catalogus 1: 1–107. 14 parasites of domestic and wild animals in south africa. xliv flaoyen_181-183.qxd introduction since the 1960s there has been an increased awareness in developed countries of the effects of pollutants on the environment. a similar awareness has in recent years been created in developing countries. hepatic and renal concentrations of copper and other trace elements in hippopotami living in and adjacent to the kafue and luangwa rivers in zambia have previously been reported (mwase, almli, sivertsen, musonda & flåøyen 2002). samples of the luangwa hippopotami were collected at mfuwe in southern luangwa national park. this location was chosen as the river, and thus the area, was presumably uncontaminated with heavy metals and regarded as a reference site. the concentrations of copper and other essential elements were similar to those reported in normal domestic and wild ruminants, and there was no indication of increased concentration of trace elements due to pollution from the mining areas in the copperbelt. organochlorines (ocs) such as organochlorine pesticides (ocps) and polychlorinated biphenyls (pcbs) are other groups of pollutants that might cause harmful effects on the environment. as mfuwe is far from intensive farming areas and extensive use of pesticides, and from industrial areas, tissue samples collected from the hippopotami were considered suitable for measuring background levels of ocps and pcbs. materials and methods subcutaneous fat was collected from 14 male hippopotami at mfuwe in southern luangwa national park, zambia. the samples were taken from the 181 onderstepoort journal of veterinary research, 72:181–183 (2005) research communication pesticide residues in adipose tissue from hippopotami (hippopotamus amphibius l) living in and adjacent to the luangwa river in zambia a. flåøyen1, a. polder2, m. mwase3, b. almli1 & m.m. musonda3 abstract flåøyen, a., polder, a., mwase, m., almli, b. & musonda, m.m. 2005. pesticide residues in adipose tissue from hippopotami (hippopotamus amphibius l) living in and adjacent to the luangwa river in zambia. onderstepoort journal of veterinary research, 72:181–183 the concentration of organochlorines (ocs) such as organochlorine pesticides and polychlorinated biphenyls were measured in adipose tissue collected from 14 male hippopotami at mfuwe in the southern part of the luangwa national park, zambia. the samples contained low levels of ocs, and the concentrations of ocs were comparable to or lower than reported for wild herbivores studied in other parts of the world. keywords: hippopotami, organochlorines, polychlorinated biphenyls, pesticides 1 national veterinary institute; p.o. box 8156 dep., n-0033 oslo, norway 2 norwegian school of veterinary science, p.o. box 8146 dep., n-0033 oslo, norway 3 university of zambia, samora machel school of veterinary medicine, p.o. box 32379, lusaka, zambia accepted for publication 24 january 2005—editor same animals that were included in the study by mwase et al. (2002) on heavy metal and trace element concentrations. the age of the animals ranged from 12–40 years and the median age was 22 years. the adipose tissues were collected using clean stainless steel equipment, placed in clean polypropylene containers and stored below 0 °c. during transportation to norway, the samples were kept frozen on dry ice. prior to the chemical analyses the samples were homogenized in a blender. concentrations of hexachlorobenzene (hcb), the sum of hexachlorocyclohexane (hch) (α-, βand γhch), the sum of chlordanes (chls) (oxychlordane, cis-chlordane and transnonachlor), the sum of ddts (p,p’-ddt, p,p’-dde, o,p’-ddd and p,p’ddd), mirex, and the sum of 34 pcbs (iupac nos. 28, 31, 47, 52, 56, 66, 74, 87, 99, 101, 105, 110, 114, 118, 128, 136, 137, 138, 141, 149, 151, 153, 156, 157, 170, 180, 183, 187, 189, 194, 196, 199, 206 and 209) were measured by accredited methods (ns-en iso/iec 17025, norwegian accreditation) at the laboratory for environmental toxicology of the norwegian school of veterinary science in oslo, norway. the extraction, lipid clean-up and gc-ecd analyses were done according to accepted methods (brevik 1978, bernhoft, wiik & skaare 1997). pcbs 29, 112 and 207 were used as internal standards. quantification limits for the individual ocps and pcbs were 0.02–0.12 ng/g in subcutaneous tissue. the percentages of recoveries were in the range of 98–144 %. results very low concentrations of ocs were detected in the tissue from the animals studied (table 1). the dominating ocs were the sum of ddts, followed by the sum of pcbs, hcb, the sum of chls, mirex and the sum of hchs. gamma-hch was the main contributor to sum hchs with 96 %, while α-hch was only detected in two samples, and β-hch was not present in any of the samples. the concentrations of o,p’-ddd were below the detection limits, whereas p,p’-dde and p,p’-ddt contributed 48 % and 49 % to the sum of ddts, respectively. of the 35 pcbs tested for only nine were detected in the samples analysed. pcb 138, 153, 156, 170, 180 were detected in all 14 samples, pcb 118 and pcb 149 in 12 of the 14 samples, and pcb 105 and pcb 157 were present in 10 of the 14 samples. pcb 153 was the dominating pcb congener contributing 23 % to the sum of pcb. the percentage fat varied considerably between the samples. discussion hippopotami are herbivores, which, in general, are known to biomagnify only low levels of ocs in their tissues (thomas, tracey, marshall & norstrom 1992). the results obtained in this study were in agreement with those of herbivores living in other parts of the world. hcb, sum hchs, sum chls, sum ddts, sum pcbs have previously been found in concentrations of 1.34, 0.24, 0.31, 1.33 and 7.90 µg/kg in fat of lambs grazing uncultivated pastures in greenland (amap 2004). corresponding levels of hcb, sum ddts and sum pcbs in roe deer from northern italy have been found to be 1.1, 7.3 and 10.9 µg/kg fat, respectively (naso, zaccaroni, perrone, ferrante, severino, stracciari & lucisano 2004). the high ratio of ddt/dde (median 1.55) found in this study indicates that pp-ddt still is used for insect vector control in the area. it is concluded that hippopotami in the south luwanga valley area are exposed to very low levels of organochlorines, and the concentrations of ocs 182 pesticide residues in hippopotami (hippopotamus amphibius l) living in zambia table 1 concentrations of pesticide residues in fat (µg/kg wet weight (ww) and µg/kg fat weight (fw) from 14 male hippopotami collected at mfuwe in the southern luangwa national park, zambia during october 1998 analyte median range median range µg/kg ww µg/kg ww µg/kg fw µg/kg fw fat content % 64 5.1–87 hcb 0.60 0.17–3.2 1.4 0.74– 4.8 sum hch 0.09 0.04–0.14 0.18 0.05–1.3 sum chlordanes 0.41 0.31–5.2 0.68 0.52–7.2 sum ddt 2.25 1.8–18 4.4 2.7–36 ratio ddt/dde 1.7 0.08–5.0 1.7 0.08–5.0 mirex 0.13 n.d.a–0.49 0.24 0.12–0.73 sum pcb 1.7 0.76–1.5 1.8 1.3–28 a n.d. = not detected (one animal) are comparable to or lower than reported for wild herbivores studied in other parts of the world. acknowledgements the authors wish to thank mr h.k. mwima, mr k. changwe, mr c. kashitu, ms m. chunduma and mr c. nagwata for assisting with the sampling in zambia and ms l.t. torp for technical assistance in the laboratory. we also thank nufu-norwegian council for higher education’s programme for development research and education for financing the study references amap. 2004. amap assessment 2002: persistent organic pollutants in the arctic. oslo: arctic monitoring and assessment programme (amap). bernhoft, a., wiig, ø. & skaare, j.u. 1997. organochlorines in polar bears (ursus maritimus) at svalbard. environmental pollution, 95:159–175. brevik, e.m. 1997. gas chromatographic method for the determination of organochlorine pesticides in human milk. bulletin of environmental contamination and toxicology, 19: 281–286. mwase, m., almli, b., sivertsen, t., musonda, m.m. & flåøyen, a. 2002. hepatic and renal concentrations of copper and other trace elements in hippopotami (hippopotamus amphibius l) living in and adjacent to the kafue and luangwa rivers in zambia. onderstepoort journal of veterinary research, 69:207–214. naso, b., zaccaroni, a., perrone, d., ferrante, m.c., severino, l., stracciari, g.l. & lucisano, a. 2004. organochlorine pesticides and polychlorinated biphenyls in european roe deer capreolus capreolus resident in a protected area in northern italy. science of the total environment, 328:83–93 thomas, d.j. tracey, b., marshall h. & norstrom, r.j. 1992: arctic terrestrial ecosystem contamination. science of the total environment, 122:135–164. 183 a. flåøyen et al. bragg_53-58.qxd introduction nad-independent variants of haemophilus paragallinarum have been isolated from chickens suffering from infectious coryza (ic) in south africa since 1989 (mouahid, bisgaard, morley, mutters & mannheim 1992; horner, bishop & haw 1992; bragg, coetzee & verschoor 1993a). bragg et al. (1993a) established that the genes encoding for nad independence appear to be car53 onderstepoort journal of veterinary research, 71:53–58 (2004) evidence of possible evasion of protective immunity by nad-independent isolates of haemophilus paragallinarum in poultry r.r. bragg department of microbial, biochemical and food biotechnology, university of the free state, p.o. box 399, bloemfontein, 9300 south africa. e-mail: braggrr@sci.uovs.ac.za abstract bragg, r.r. 2004. evidence of possible evasion of protective immunity by nad-independent isolates of haemophilus paragallinarum in poultry. onderstepoort journal of veterinary research, 71:53–58 an indication of the ability of nad-independent variants of haemophilus paragallinarum to evade the immune system has been obtained from data obtained from several experiments. firstly, it was noted that there was a difference in the serovar distribution between the nad-dependent isolates in the 1990s and the nad-independent isolates, as there was a significant decrease in the incidence of serogroup a nad-dependent isolates. this can possibly be attributed to the extensive use of vaccines. on the other hand, most of the earlier nad-independent isolates were serovar a. this is a possible indication of evasion of the protective immunity by the nad-independent isolates. further evidence of possible evasion of the protective immunity was obtained from results obtained when different isolates, both nad dependent and nad independent, were tested with a panel of monoclonal antibodies (mabs). the v1 mab reaction pattern was only seen in the reference strain 0083 among all of the nad-dependent isolates tested in south africa. this mab was, however, found to react with some of the nad-independent isolates. furthermore, the isolation of nad-dependent isolates in australia which react with the v1 mab also suggest possible evasion of the protective immunity by the nad-independent isolates as no vaccines containing strain 0083 are used in australia. in order to investigate the hypothesis of immune-evasion by nad-independent h. paragallinarum, vaccinated and unvaccinated chickens were challenged with a nad-independent serogroup c isolate. as a control, chickens were also challenged with nad-dependent h. paragallinarum of the same serogroup. the results obtained indicate that there is no significant difference in the disease profiles obtained in vaccinated and unvaccinated chickens challenged with the nad-independent isolate, thus providing further evidence of evasion of the productivity immunity by the nad-independent isolates. the ability of the nad-independent isolates to evade the immune system suggests that a different vaccination strategy, or alternative control methods may be needed for the control of ic caused by these isolates. keywords: haemophilus paragallinarum, immunity, infectious coryza, nad independent accepted for publication 17 october 2003—editor ried on a transferable plasmid and they succeeded in transforming reference strains of nad-dependent h. paragallinarum into nad-independent isolates. bragg (2002b) demonstrated that the nad-independent isolates appear to be less virulent that the nad-dependent isolates of the same serovar. taole, albertyn, van heerden & bragg (2002) demonstrated that experimentally produced nad-independent isolates made from virulent serovar c-3 isolates were less virulent that the wild type naddependent isolates. bragg, coetzee & verschoor (1996) established that there has been a significant change in the serovar distribution of nad-dependent h. paragallinarum isolates since the 1970s. they established that in the 1990s, the incidence of serovar a-1 nad-dependent h. paragallinarum had decreases from 34 % in the 1970s to 5 % in the 1990s and suggested that this decrease was a result of the intensive use of vaccines, all of which contain serogroup a isolates. on the other hand, miflin, horner, blackall, chen, bishop, morrow, yamaguchi & iritani (1994) reported that all of the nad-independent south african isolates which they had received were serogroup a isolates. these isolates were, however, collected from a localized area of south africa. bragg, greyling & verschoor (1997) reported on a much wider collection of nad-independent isolates and they also isolated serogroup c nad-independent isolates for the first time, and they indicated that the incidence of serogroup a nad-independent isolates was 58 %. one possible explanation for the difference in the incidence of the serogroup a isolates among the nad-dependent and nad-independent isolates could possibly be the evasion of protective immunity by the nad-independent isolates. bragg et al. (1996) suggested that the decrease in the incidence of serogroup a nad-dependent isolates could be as a result of intensive use of vaccines, all of which contain a serogroup a isolate. the fact that a large number of nad-independent isolates were found to be serogroup a at a time when the incidence of serogroup a isolates amongst the nad-dependent strains was decreasing, suggests that the nad-independent isolates might be able to evade the protective immunity in the birds. further indications of the possible evasion of the protective immunity by nad-independent isolates of h. paragallinarum was obtained from monoclonal antibody studies performed by bragg, coetzee & verschoor (1993b) and bragg, gunter, coetzee & verschoor 1997b) who investigated the possibilities of using a panel of mabs established by verschoor, coetzee & visser (1989) for the serotyping of h. paragallinarum. although these mabs detected certain antigens in h. paragallinarum (either protein or lipopolysaccharide in nature) (bragg et al. 1997b), they could not be used for any meaningful serological classification of h. paragallinarum. however, during this work, large numbers of isolates of h. paragallinarum, both nad dependent and nad independent, were examined with this panel of mabs (bragg et al. 1993b, 1997b). one mab was of particular interest. this was the mab termed v1, which detected a lipopolysaccharide of between 13.8–14 kda (bragg et al. 1997b). this mab was found to react only with the reference strain 0083. none of the south african nad-dependent field isolates of h. paragallinarum reacted with this mab. a small sample of nad-dependent field isolates of h. paragallinarum (kindly supplied by dr p. j. blackall, department of primary industries, brisbane, australia) were tested with these mabs. one of the wild type australian field isolates (as well as the mutant derived from it) reacted with the v1 mab (bragg et al. 1997b). when examining the nad-independent isolates, 12.5 % of the isolates were found to react with the v1 mab (bragg et al. 1997b). this was in sharp contrast to the situation in the nad-dependent isolates. in south africa, many of the registered ic vaccines contain isolate 0083. in australia, no imported ic vaccines have been used and all of the ic vaccines used contain australian isolates (blackall 1991; blackall & reid 1987; blackall, eaves, rogers & firth 1992). thus, strain 0083 has not been used in vaccines in australia (blackall, personal communication 1997). the isolation of naddependent field isolates in australia which express the antigen detected by the v1 mab, and the high incidence of nad-independent field isolates reacting with this mab, suggested a possible immuneevasion by the nad-independent isolates. until recently, one of the problems faced when attempting to verify the hypothesis of immune-evasion by the nad-independent isolates was the existence of a suitable challenge model where the protective effects of a vaccine could be accurately measured. bragg (2002a) established a new challenge model for ic that facilitates the comparison of the virulence of different isolates and also allows for the accurate comparison of the efficacy of different vaccines. 54 immunity by nad-independent isolates haemophilus paragallinarum in poultry this challenge model is based largely on the various challenge models used by various authors (bragg 2002a), but in this model a numerical score is given to the clinical signs. the numerical scores can be used to construct a graphic representation of the disease profile over a 20-day period. by superimposing the disease profiles of two different isolates, or from two different vaccination programmes, comparisons can be established. this data also lends itself to statistical analysis. this challenge model is used to confirm the hypothesis of immune-evasion by nad-independent isolates of h. paragallinarum. materials and methods twenty unvaccinated commercial layer chickens were obtained from a supplier of point-of-lay chickens. these chickens were obtained at 11 weeks of age before they were vaccinated against ic. these chickens were collected from a site with no previous history of ic and were housed in disinfected layer facilities until they were used in the experiments. another 20 unvaccinated commercial layer chickens were obtained from the same flock and were vaccinated with an experimental vaccine containing south african isolates of serovars a-1, b-1, c-2 and c-3 h. paragallinarum when at 12 weeks of age. they were re-vaccinated at 16 weeks of age. both vaccinated and unvaccinated chickens remained in the facilities until they were 25 weeks of age and were producing eggs. the protective capability of this vaccine was previously verified and will be reported on separately. all four different serovars of h. paragallinarum have been included in the experimental vaccine as it has been well established that there is limited cross protection between the different serogroups (rimler, davis & page 1977; kume, sawata & nakase 1980a, b). when they were 25 weeks of age, the vaccinated and unvaccinated chickens were subdivided into two groups of ten vaccinated and two groups of ten unvaccinated chickens. they were placed in cages of a battery in such a way such that ten chickens were placed into the top five cages (two chickens per cage) and ten chickens in the bottom row of cages. the vaccinated chickens were placed in the cages on one side of the battery, while the unvaccinated chickens were placed in those on the other side. one group of the vaccinated and one group of the unvaccinated birds was challenged with strain 1343 [a south african field isolate of nad-independent h. paragallinarum (bragg 2002b)] while the remaining two groups were challenged with strain 46 c-3 [an nad-dependent field isolate of h. paragallinarum (bragg 2002a)] according to the methods used by bragg (2002a). the clinical signs in the birds were recorded and scored for a 20-day period. the severity of the clinical signs was given a numerical value according to the methods described by bragg (2002a) and disease profiles were plotted. results the results of the scoring of the clinical signs in the groups that were challenged with nad-dependent serovar c-3 were used to produce a graphic representation of the course of the disease which is given in fig. 1. similarly the disease profiles obtained in the chickens challenged with the nad-independent serovar c-3 are given in fig. 2. mean disease scores for each of the different experimental groups, as calculated from the disease profiles, are reflected in table 1. a statistical analysis of the disease profiles obtained was performed using a t test and the results obtained are shown in table 1. 55 r.r. bragg table 1 summary of the disease scores and profiles obtained when vaccinated and unvaccinated chickens challenged with serovar c-3 isolates of nad-dependent and nad-independent isolates of h. paragallinarum isolate vaccinated mean disease score significance highest score duration (days)# c-3 (dep*) yes 0.45 0.000358 1.6 12 c-3 (dep) no 1.85 4.2 15 c-3 (indep**) yes 0.375 0.389 1.2 15 c-3 (indep) no 0.415 1.3 19 # calculated as the last day on which clinical signs were seen in the group of chickens * nad dependent ** nad independent discussion it can be seen from this experiment that chickens which were vaccinated with an experimental vaccine were not protected against challenge with an nad-independent isolate of h. paragallinarum. it was also demonstrated in this experiment that the experimental vaccine used in this experiment provided protection to the nad-dependent isolate of the same serovar. it is well known that protection afforded by a h. paragallinarum vaccine is dependent of the serovar used to make the vaccine (rimler et al. 1977; kume et al. 1980a, b). the use of the experimental challenge model proposed by bragg (2002a) has shown that the nadindependent isolates have the ability to evade the immune system. it can be seen from fig. 2 that the overall diseases profiles in the vaccinated and unvaccinated chickens are similar. it was found that there is not a significant difference between the two profiles (p = 0.389). it is evident from the graph in fig. 2 that the development of clinical signs in the vaccinated birds was delayed when compared to that of unvaccinated birds and that the duration of clinical signs was shorter in the vaccinated birds. however, the highest daily disease scores of the two groups of birds were found to be very similar (table 1). this is in contrast to the results obtained when the birds were challenged with the naddependent isolates. the data obtained revealed that there is a significant difference (p = 0.000358) between the disease profiles obtained for the vaccinated and unvaccinated birds. these results indicate that the vaccine used in this experiment provided protection when the birds were challenged with the nad-dependent serovar c-3 isolate. 56 immunity by nad-independent isolates haemophilus paragallinarum in poultry ���������� ���� �� � � � � � � � � � �� �� �� �� �� �� �� �� �� � � �� ������ ���� ������ �� �� ��� �� ��� �� ��� �� ��� � � � �� �� � � � � �� � �� ���������� ���� �� � � � �� �� � � � � �� � �� �� �� �� �� � ��� ��� � � � � � � � � � �� �� �� �� �� �� �� �� �� � � �� ������ ���� ������ fig. 1 disease profiles of vaccinated and unvaccinated chickens challenged with an naddependent serovar c-3 isolate of h. paragallinarum fig. 2 disease profiles of vaccinated and unvaccinated chickens challenged with an nadindependent serovar c-3 isolate of h. paragallinarum these findings strongly suggest that the nad-independent isolate was capable of evading the protective immunity induced in the vaccinated birds and support the hypothesis of immune-evasion by nad-independent strains which was postulated from results obtained from previously described work. one possible explanation for the ability of the nadindependent isolates to evade the immune system could be that the acquisition of the genes for nad independence, which appear to be plasmid mediated and allow h. paragallinarum to use other niches in the sinuses of the chicken. it is possible that the nad-dependent isolates need to colonize areas in the sinus with a rich blood supply, thus allowing for the drawing of the needed nad from the circulating blood stream. however, if the birds had been vaccinated with inactivated bacterins against h. paragallinarum, the circulating blood would also have contained specific antibodies against the different serovars of h. paragallinarum (depending on the composition of the vaccine). if the acquisition of nad independence is freeing the bacteria from their dependence on drawing nad for the blood supply, this might allow them to grow in other areas of the sinuses. it would be interesting to investigate the locations of nad-dependent and nad-independent strains of h. paragallinarum in the sinuses of experimentally infected chickens. conclusions it would appear, from the results obtained in this experiment that the nad-independent isolates appeared to have been able to evade the immune system in the vaccinated chickens. if this hypothesis is correct, there is a need for an alternative vaccination strategy, or some other disease control strategy, for the control of ic caused by the nadindependent variants, such as the stimulation of local immunity in the upper respiratory tract of the chickens. other disease control methods could include improved biosecurity through the use of nontoxic disinfectants as described recently by bragg (2004). fortunately, it would appear that the nadindependent isolates of serogroup c h. paragallinarum are less virulent that the nad-dependent strains (bragg 2002b; taole et al. 2002). acknowledgements i am are grateful to the national research foundation (gun no. 2053243) for funding aspects of this work. we are also grateful to avimune (pty) ltd. for supplying the monoclonal antibodies for the work performed previously. references blackall, p.j. 1991. an evaluation of the cross-protection afforded by inactivated infectious coryza vaccines. australian veterinary journal, 68:266–267. blackall, p.j. & reid, g.g. 1987. further efficacy studies on inactivated aluminum-hydroxide adsorbed vaccines against infectious coryza. avian diseases, 31:527–532. blackall, p.j., eaves, l.e., rogers, d.g. & firth, g. 1992. an evaluation of inactivated infectious coryza vaccines containing a double emulsion adjuvant system. avian diseases, 36:632–636. bragg, r.r. 2002a. virulence of south african isolates of haemophilus paragallinarum. part 1. nad-dependent field isolates. onderstepoort journal of veterinary research, 69: 63–169. bragg, r.r. 2002b. virulence of south african isolates of haemophilus paragallinarum. part 2. naturally occurring nad-independent field isolates. onderstepoort journal of veterinary research, 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characterization of vfactor (nad)-independent haemophilus paragallinarum. avian diseases, 39:304–308. mouahid, m., bisgaard, m., morley, a.j., mutters, r. & mannheim, w. 1992. occurrence of v-factor (nad) independent isolates of haemophilus paragallinarum. veterinary microbiology, 31:363–368. rimler, r.b., davis, r.b. & page, r.k. 1977. infectious coryza: cross-protection studies, using seven strains of haemophilus paragallinarum. american journal of veterinary research, 38:1587–1598. taole, m., albertyn, j., van heerden, e. & bragg, r.r. 2002. virulence of south african isolates of haemophilus paragallinarum. part 3: experimentally produced nadindependent isolate. onderstepoort journal of veterinary research, 69:89–196. verschoor, j.a., coetzee, l. & visser, l. 1989. monoclonal antibodies characterization of two field strains of haemophilus paragallinarum isolated from vaccinated layer hens. avian diseases, 33:219–225. 58 immunity by nad-independent isolates haemophilus paragallinarum in poultry abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) agnes t. laleye department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa national veterinary research institute, vom, nigeria modupeore adeyemi department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa celia abolnik department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa citation laleye, a.t., adeyemi, m. & abolnik, c., 2022, ‘propagation of avian influenza virus in embryonated ostrich eggs’, onderstepoort journal of veterinary research 89(1), a2011. https://doi.org/10.4102/ojvr.v89i1.2011 original research propagation of avian influenza virus in embryonated ostrich eggs agnes t. laleye, modupeore adeyemi, celia abolnik received: 07 mar. 2022; accepted: 28 june 2022; published: 08 dec. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract influenza a viruses (iavs) are typically isolated and cultured by successive passages using 9to 11-day-old embryonated chicken eggs (eces) and in 14-day old eces for virus mutational studies. real-time reverse transcription-polymerase chain reaction tests (rt-pcrs) are commonly used for iav diagnosis, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus. efforts at isolating ostrich-origin iavs from rt-pcr positive specimens using eces have often been unsuccessful, raising the possibility of a species bottleneck, whereby ostrich-adapted iavs may not readily infect and replicate in eces, yet the capacity of an ostrich embryo to support the replication of influenza viruses has not been previously demonstrated. this study describes an optimised method for h5 and h7 subtype iav isolation and propagation in 28-day old embryonated ostrich eggs (eoes), the biological equivalent of 14-day old eces. the viability of eoes transported from breeding sites could be maximised by pre-incubating the eggs for 12 to 14 days prior to long-distance transportation. this method applied to studies for ostrich-adapted virus isolation and in ovo studies will enable better understanding of the virus-host interaction in ostriches and the emergence of potentially zoonotic diseases. keywords: avian influenza virus; propagation; ostrich; embryonated eggs; protocol. introduction avian influenza (ai) is a notifiable disease caused by influenza a viruses (iavs) in the family orthomyxoviridae (oie 2017). influenza a viruses have a wide host range, including birds and mammals, and are of zoonotic importance (oie 2017). two pathotypes of iavs have been described – the severe, highly pathogenic avian influenza (hpai) and low-pathogenicity ai (lpai), which is associated with mild respiratory disease (alexander 2000; oie 2017). the first outbreak of ai in ostriches, reported in the oudtshoorn area in south africa in the early 1980s, was caused by an h7n1 lpai virus with a low pathogenicity index in chickens (allwright et al. 1993). subsequent infections of ostriches with lpai viruses continued to be reported, including h1n2, h5n9 lpai, h6n1, h6n8, h7n1 lpai, h7n7 lpai, h9n2 and h10n1 (abolnik et al. 2016; olivier 2006). ostriches are also susceptible to infection with hpai viruses, although such infections generally cause mild clinical signs (abolnik et al. 2009, 2012, 2016; de benedictis et al. 2007; manvell et al. 2005). interestingly, outbreaks of h7 lpai among young flocks were observed to have presented with more severe clinical signs and higher mortalities in the ostriches than h5n2 hpai outbreaks (abolnik et al. 2009). outbreaks of h5n2 hpai in ostriches occurred in 2004 in the eastern cape province and in 2006 and 2011 in the western cape province of south africa (abolnik et al. 2012; van helden et al. 2016), with devastating economic impacts on the south african ostrich industry through control measures and trade restrictions. the h5n2 outbreaks were highly localised, with hpai emerging from lpai virus precursors in the ostriches (abolnik 2007; abolnik et al. 2012, 2016), compared with the outbreaks caused post-2017 by clade 2.3.4.4 h5nx hpai viruses introduced by wild migratory birds from the northern hemisphere (abolnik et al. 2019). influenza a viruses are typically isolated and cultured by successive passages using 9to 11-day-old embryonated chicken eggs (eces), which is considered the gold standard for virus detection by the oie (word organization for animal health 2017; spackman & killian 2014). in recent times, sensitive nucleic acid detection assays like real-time reverse transcription-polymerase chain reaction tests (rt-pcrs) are more commonly used for diagnostic purposes, but virus isolation remains invaluable in terms of its high sensitivity, providing viable isolates for further studies and the ability to distinguish between viable and nonviable virus (burrell, howard & murphy 2017). efforts at isolating ostrich-origin iavs from rt-pcr positive specimens using eces have often been unsuccessful (abolnik et al. 2016), for various possible reasons. for example, a recent study found that bacteria present in the ostrich trachea have antiviral effects on iav, which may partially explain the failure to isolate iavs from rt-pcr positive tracheal swabs (abolnik et al. 2021). however, the possibility of a species bottleneck remains, whereby ostrich-adapted iavs may not readily infect and replicate in chicken eces. if the latter is true, then ostrich-origin iavs may replicate more readily in ostrich embryos without the need for prior adaptation. previously, molecular markers arising from specific mutations in different segments of the iav genome have been shown to be responsible for adaptation of avian viruses to mammalian hosts (gabriel, herwig & klenk 2008; hatta et al. 2001; subbarao, london & murphy 1993). the average weight of an ostrich egg, at 1600 g, is around 25 times that of a chicken’s, and the shell thickness of an ostrich egg varies from 1.5 mm to 2.2 mm compared to the chicken egg shell, which has a thickness of 0.3 mm to 0.4 mm (kokoszyński 2017). the embryonic development of ostriches is comparable to that of chickens; the incubation period of ostriches is exactly double that of chickens, and any particular stage of ostrich embryonic development can be obtained by reference to the corresponding developmental stage in the chicken embryo (deeming 1997; brand et al. 2017). intensive ostrich farming started in the late 1800s (shanawany 1995), and artificial incubation conditions for ostrich eggs have been well studied (brand et al. 2017). the hatchability of artificially incubated ostrich eggs is normally between 50% and 60% of all the fertile eggs that were set (van schalkwyk et al. 2000), while that of chicken eggs ranges between 80% and 95%, depending on a number of variables including size, weight and duration of storage (ayeni et al. 2020). the transmission dynamics and pathogenesis of iav in ostriches are relatively poorly understood in comparison to other poultry, because in vivo studies require high bio-containment animal facilities. ostriches are large and potentially dangerous as adults, and birds of any age (but particularly chicks) are prone to stress and are difficult to manage indoors. fourteen-day-old eces have been used with success as an alternative to live chickens to passage iavs and study the emergence of hpai from lpai precursors (laleye & abolnik 2020; seekings et al. 2020), but the growth of iavs or any other viruses has not been explored in ostrich eggs before. this study aimed at developing and optimising a method for propagating iavs in embryonated ostrich eggs (eoes), as a possible substitute to the use of sentient ostriches for experimental purposes and as an alternative method to facilitate the recovery of viable ostrich-origin viruses in cases where other isolation methods are unsuccessful. materials and methods ostrich egg source and transport batches of fresh eoes with an anticipated fertility rate of 50% – 60% (van schalkwyk et al. 2000) were purchased from klein karoo international (kki) research laboratory in oudtshoorn, south africa. eggs were sourced from ostrich farms that were certified ai-free by the department of agriculture, forestry and fisheries. these farms are tested monthly by serological methods to prove freedom of infection. in total, 102 ostrich eggs were used in the study. the university of pretoria, faculty of veterinary science in pretoria, gauteng province, where the study was performed is located 1207 km from the kki research laboratory, western cape province, and the closest airports for air freight are located in johannesburg and george, respectively. the distance over which the eoes had to be transported and still remain viable presented a significant challenge; therefore, different approaches for safe transport were investigated. in each case, the eggs were individually encased in several layers of plastic bubble wrap and placed into cardboard boxes filled with shredded paper for insulation. boxes were clearly marked as ‘fragile’. method 1: air freight with a commercial courier service. the package was collected and delivered door-to-door by the courier company. method 2: road freight with meat trucks. the package was transported by a truck shipping packaged ostrich meat for international export purposes from oudtshoorn to or tambo international airport in johannesburg, where it was personally collected on arrival. method 3: air transport as hand luggage. personnel from the kki laboratory travelled by air to or tambo international airport in johannesburg, where the package was collected. the eoes were first incubated at the kki laboratory for 12 to 14 days prior to packaging and transport at recommended temperature and humidity in an incubation chamber. egg handling and incubation egg storage all experiments were conducted in the poultry biosafety level 3 (bsl 3) facility at the faculty of veterinary science. upon receipt, eggs were left undisturbed for a period of 12 h, usually overnight, to allow them to stabilise. thereafter, the eggs were candled using a portable hand-held led egg candling lamp (ecotao, south africa) to determine the air-cell position, which was marked on the shell surface with a pencil or permanent marker. unincubated eggs that were not used immediately were stored in padded plastic crates, placed in an isolation room in the bsl 3 facility that was separated from the area used for working with live virus. the room temperature was maintained at 18 °c with the air conditioning unit in accordance with the specified optimal temperature (15 °c to 20 °c) for the storage of fertile ostrich eggs (swart, rahn & de kock 1987; van schalkwyk et al. 2000). to maintain the required relative humidity of 75% to 80%, bowls of water were placed in the room. the room temperature and relative humidity were monitored with a digital temp-hygrometer monitor (pmi, south africa). the eggs were stored for a maximum of seven days before incubation, with the air-cell positioned horizontally and the eggs turned manually at least once a day. setting eggs in the incubator eggs stored at 18 °c were allowed to acclimatise for at least 12 h at room temperature (25 °c) before setting the incubator, to avoid an undesirable increase in humidity inside the incubator, which may potentially enhance growth and multiplication of microbes (brand et al. 2014). an sh1700 model incubator (surehatch, brackenfell, south africa) was used in this study. the sh1700 model incubator has an internal volume of 1.28 m3, digital temperature control, and an automatic egg turner that rotates through 90° every hour. the incubator was customised with crates for ostrich eggs, with a capacity for 30 eggs. eggs were set with the air-cell up from day one. temperature and the relative humidity were set at 36 °c (± 0.5 °c) and 28%, respectively (brand et al. 2014), with daily monitoring and recording on a log. eggs were candled daily and those containing dead embryos were removed and discarded. after every incubation batch, the interior of the incubator was cleaned and disinfected with 10% f10 sc veterinary disinfectant solution. viruses two ostrich-origin lpaiv originally isolated in eces, a/ostrich/south africa/325863/2015 (h5n2) and a/ostrich/south africa/ord/2012 (h7n1), were used in the study. the viruses were propagated in specific pathogen-free eces (avifarms pty [ltd], pretoria, south africa), and eid50 titres were determined according to the method of reed and muench (1938). at the time of the experiments, the stocks had been passaged three times in eces and the virus concentration given at 106 50% egg infectious doses (eid50)/0.1 ml, prescribed viral dose for challenge studies in avian species (li et al. 2016). avian influenza virus propagation in embryonated ostrich eggs at the 28th day of incubation, viable eoes were removed from the incubator and placed in the biosafety cabinet for the inoculation process. the egg shell surface was disinfected with 70% ethanol and left for a few minutes to dry. a dremel 4000 drill (dremel, south africa) was used to carefully drill an inoculating hole of 1 mm – 2 mm diameter in the egg shell. using a 1 ml syringe with 21 gauge (g) x1” needle, 0.5 ml of allantoic fluid containing 106 eid50/ 0.1 ml of virus was inoculated into the allantoic sac of 3 eoes each by inserting the entire needle vertically through the inoculating hole. inoculating holes were sealed with adhesive stickers. inoculated eoes were incubated for 3–5 days per passage, under the same conditions as uninoculated eggs, but without turning, and were candled every 24 h. at the end of the incubation period, the eggs were chilled overnight at 4 °c. the allantoic fluid as well as embryonic tissues were collected and tested for haemagglutinating activity (ha) using 1% chicken red blood cells according to the standard procedure (oie 2017), and bacteriological tests on blood agar (ba). tissues collected from the embryos included trachea, heart, liver, spleen, lungs, intestine, kidneys and brain. the embryonic tissues were pooled and homogenised in antibiotic solution containing 50 mg gentamycin (virbac) and 100 mg enrofloxacin (baytril) per litre, using a silent crusher m homogenizer (heildoph schwabach, germany). the homogenates were clarified at 1000 × g and supernatant collected for ha and bacteriological tests. bacteria-free allantoic fluids with ha activity were aliquoted and stored at −80 °c for further analysis, while the supernatant of homogenised embryonic tissues were used for the subsequent passage. virus detection viral rnas were extracted from ha-positive allantoic fluids and supernatants of homogenised tissue using trizol reagent (gibco, invitrogen) according to the manufacturer’s recommended procedure, and tested for iav m-gene presence with vetmax-plus multiplex one step rt-pcr kit (applied biosystems, ca, united states [us]) and previously described primers and probes (spackman et al. 2003). the reactions included 2 µl pcr-grade water, 6 µl 2× rt-pcr buffer, 0.5 µl rt enzyme mix, 0.5 µl of 10 pmol/µl of each primer, 0.15 µl of 5 pmol/µl of the probe and 3 µl of extracted rna or positive control rna or nuclease-free water as a no template control. the reactions were performed in an applied biosystems steponeplus rt-pcr system (life technologies, carlsbad, ca, us) with the following cycling conditions: 1 cycle of 48 °c for 10 min; 1 cycle of 95 °c for 10 min; 40 cycles of amplification at 95 °c for 15 s and 53 °c for 45 s (data capture point). ethical considerations ethical clearance for the study was obtained from the animal ethics committee of the university of pretoria (project number v010-17). results effect of mode of long-distance transport on embryonated ostrich egg viability transporting eoes by air freight with a commercial courier resulted in a high proportion of broken eggs and poor viability of the remaining intact eggs (table 1), presumably due to rough handling and fluctuating temperature through transportation process. in the first delivery batch, five of the 12 eggs were broken on arrival, and of the seven intact eggs, only one developed a viable embryo at the end of the 28-day incubation period. the second batch transported by a different courier arrived with all 12 eggs broken. table 1: effect of mode of long-distance transport on embryonated ostrich egg viability. transporting eoes by road freight with the ostrich meat trucks substantially minimised handling and temperature fluctuations, and although a slight improvement on method one was noted, eoe viability was still poor. between 41.7% and 66.7% of eggs arriving in four batches were confirmed as nonviable by day six of incubation, and of those that survived, only 8.3% to 33.3% were viable at 28 days. transporting eoes as hand luggage by air yielded the best result. of 18 eggs received in three batches of six eggs each, 15 (83%) were viable at day 28 of incubation and could be used in the subsequent experiments. passage and isolation of influenza a viruses in embryonated ostrich eggs four successive passages were completed with the h5n2 lpai virus in the 28-day old eoes, whereas five passages were completed with the h7n1 lpai virus and the allantoic fluids as well as the supernatants of homogenised embryonic tissues from each passage were tested for ha activity and by real-time rt-pcr (table 2). the ha titres for h5n2-infected allantoic fluids increased with each passage from 25 to 28, whereas the titres for the corresponding tissue homogenates increased from 22 to 24. higher ha titres in the allantoic fluids compared with the embryos are consistent with the site of replication in the egg of lpai viruses. low pathogenicity avian influenza viruses replicate in the cells of the choriallantoic membrane and virus particles are released during budding into the allantoic fluids (rott et al. 1980). on ha, allantoic fluids containing the h5n2 lpai virus showed complete haemagglutination, whereas embryonic tissue homogenates displayed incomplete haemagglutination (data not shown), consistent with the alfs having higher virus titres than the embryonic tissues. similar ha patterns were observed with the h7n1 lpai virus, but the titres were generally higher ranging from 28 to 210 in the allantoic fluids. in the tissue homogenates, passage 1 had a higher ha titre of 25, but from passage 2 onwards the values ranged from 23 and 24. table 2: detection of influenza a viruses passaged in 28-day old embryonated ostrich eggs. real-time pcr results confirmed that the haemagglutinating phenotype was caused by the presence of iav in all samples. the average cycle threshold (ct) values for h5n2 virus-infected allantoic fluids and embryonic tissue homogenates were 16.18 and 22.90 respectively, whereas those for the h7n1 virus were 15.90 and 22.70, respectively. discussion embryonated ostrich eggs have the potential to be developed as in ovo passage models and for the selective propagation of iavs that are ostrich-adapted; therefore, in this study the methods for transporting, incubating and inoculating these large eggs were described. previously described challenges associated with the artificial incubation of ostrich eggs which include poor hatchability (± 45%), high rates of infertility (± 20%) and substantive in-shell deaths (± 30%), compared to that of other domestic poultry birds (brand et al. 2014) were confirmed in this study. at the outset of the study, it was discovered that despite proper packaging to protect the eggs, the use of commercial air and road freight services either resulted in broken or damaged eggs or poor viability. the best method for long-distance transport of eoes, although very expensive, was for a person to carry them as hand luggage by airline. it was also found that pre-incubating the eggs prior for 12 to 14 days prior to transport substantially improved the chances of incubating viable embryos to 28 days. designing experiments that involve the use of eoes should take the timing of the ostrich breeding system into consideration. as opposed to specific pathogen-free chicken eggs, which may be obtained any time as required, ostrich eggs are only available during their breeding season, which coincides with increased photoperiod and may vary with altitude and latitude (bertram 1979; ipek & sahan 2004; smith et al. 1995). breeding peaks around early spring in the northern hemisphere; whereas in the southern africa, production spans june (mid-winter) to january (summer) (lambrechts 2004). the authors recently demonstrated, using 14-day eces, that passaging the same ostrich-origin h5n2 and h7n1 lpai strains resulted in the appearance of the hpai variants after 11 passages and seven passages for h5n2 and h7n1, respectively (laleye & abolnik 2020). the emergence of both h5 and h7 hpai viruses from lpai precursors in chickens is demonstrated in field outbreaks, in vivo and in ovo studies (laleye & abolnik 2020). in ostriches, h5n2 hpai that emerged from lpai precursors in field outbreaks are reported (abolnik et al. 2016), but it is still unknown whether mutation to hpai can occur with the h7 subtype. field evidence suggests that h7 lpai viruses are incapable of mutating to hpai as h7n1 and/or h7n7 viruses have circulated in ostriches in south africa and italy on numerous occasions, sometimes for many months, without any evidence of conversion, and h7 hpai has never been reported in ostriches in any country (abolnik et al. 2009, 2016; manvell et al. 2005). future work could entail passaging the ostrich-origin h7n1 lpai virus in 28-day eoes to determine the risk of hpai emergence. conclusion this study was the first to describe the methods for long-distance transport, incubation, inoculation and passage of iavs in 28-day eoes. the methods could be applied to isolate ostrich-adapted viruses and in ovo studies aimed at better understanding of the virus–host interaction in ostriches and the emergence of potentially zoonotic diseases. acknowledgements virus strains isolated by deltamune laboratories (pty) ltd south africa were used with permission. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.t.l. and m.a. performed the experimental work. c.a. conceptualised and designed the 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https://doi.org/10.1080/00071660086394 abstract introduction materials and methods results discussion conclusion acknowledgements references appendix 1 about the author(s) shima e. abdalla antimicrobial research unit, college of health sciences, university of kwazulu-natal, durban, south africa akebe l.k. abia antimicrobial research unit, college of health sciences, university of kwazulu-natal, durban, south africa daniel g. amoako antimicrobial research unit, college of health sciences, university of kwazulu-natal, durban, south africa keith perrett epidemiology section, kwazulu-natal agriculture and rural development-veterinary service, pietermaritzburg, south africa linda a. bester biomedical resource unit, college of health sciences, university of kwazulu-natal, durban, south africa sabiha y. essack antimicrobial research unit, college of health sciences, university of kwazulu-natal, durban, south africa citation abdalla, s.e., abia, a.l.k., amoako, d.g., perrett, k., bester, l.a. & essack, s.y., 2022, ‘food animals as reservoirs and potential sources of multidrug-resistant diarrheagenic e. coli pathotypes: focus on intensive pig farming in south africa’, onderstepoort journal of veterinary research 89(1), a1963. https://doi.org/10.4102/ojvr.v89i1.1963 original research food animals as reservoirs and potential sources of multidrug-resistant diarrheagenic e. coli pathotypes: focus on intensive pig farming in south africa shima e. abdalla, akebe l.k. abia, daniel g. amoako, keith perrett, linda a. bester, sabiha y. essack received: 13 july 2021; accepted: 10 nov. 2021; published: 20 jan. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: diarrheagenic e. coli (dec) strains are a major cause of diarrheal diseases in both developed and developing countries. healthy asymptomatic animals may be reservoirs of zoonotic dec, which may enter the food chain via the weak points in hygiene practices. aim: we investigated the prevalence of dec along the pig production continuum from farm-to-fork. methods: a total of 417 samples were collected from specific points along the pig production system, that is, farm, transport, abattoir and food. e. coli was isolated and enumerated using colilert. ten isolates from each quanti-tray were selected randomly and phenotypically identified using eosin methylene blue agar selective media. real-time polymerase chain reaction (pcr) was used to confirm the species and to classify them into the various diarrheagenic pathotypes. antimicrobial susceptibility was determined against a panel of 20 antibiotics using the kirby-bauer disk diffusion method and eucast guideline. results: the final sample size consisted of 1044 isolates, of which 45.40% (474/1044) were dec and 73% (762/1044) were multidrug-resistant. enteroinvasive e. coli (eiec) was the most predominant dec at all the sampling sites. conclusion: the presence of dec in food animal production environments and food of animal origin could serve as reservoirs for transmitting these bacteria to humans, especially in occupationally exposed workers and via food. adherence to good hygienic practices along the pig production continuum is essential for mitigating the risk of transmission and infection, and ensuring food safety. keywords: diarrheagenic e. coli; multidrug resistance; intensive pig farming; farm-to-fork; low-income countries; south africa; multiple-antibiotic resistance index; biosecurity. introduction intensive pig production can be defined as raising a large number of animals on limited land (mennerat et al. 2010) to increase profits and ensure sustainability in meat production (noya et al. 2017). in some countries, intensive pig farms are further integrated with meat-processing industries, forming supply chains (davies 2011). additionally, intensive pig production is practised under strict conditions such as temperature control, reduction of contact between animals and waste, improvement of effluent treatment, parturition control with human intervention and the use of vaccines. however, these measures are inadequate to achieve ideal sanitary conditions (alustiza et al. 2012), and livestock still act as intermediary or amplifier hosts of pathogens transmitted to humans (jones et al. 2013). to prevent the introduction and spread of infectious diseases in pig production, biosecure environments and strict hygiene conditions have been adopted (julio pinto & santiago urcelay 2003). biosecurity prevents direct and indirect disease transmission between animals from the same and between different batches or farms (sahlstrom et al. 2014). within meat processing plants, pathogens can easily be transferred to meat from the animals’ gastrointestinal tract, environment and meat handlers’ hands, especially under poor sanitary conditions (ncoko, jaja & oguttu 2020). although most eschericheia coli (e. coli) strains are commensals and live harmlessly in the colon of humans and other animals, several pathogenic e. coli strains cause intestinal and extraintestinal diseases in healthy and immunocompromised humans (gomes et al. 2016) and animals. these pathogenic e. coli strains carry several different virulence factors, controlled by genes located on chromosomes, plasmids or phages (borges et al. 2012). pathogenic e. coli can cause different dieases and affect both gastriontestinal and extraintestinal sites. several gastointestinal e. coli pathotypes contribute to diarrhea (croxen et al. 2013). diarrheagenic e. coli (dec) strains are considered major causes of diarrheal diseases in developed and developing countries (aijuka et al. 2018; estrada-garcia & navarro-garcia 2012). foodborne diseases resulting from the consumption of food contaminated by dec have been recognised amongst the most challenging health issues worldwide (galli et al. 2016). members of the dec group are classified into six pathotypes, that is, enteroaggregative e. coli (eaec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec), enteroinvasive e. coli (eiec), diffusely aggregative e. coli (daec) and enterohemorrhagic/shiga toxin-producing e. coli (ehiec/ stec) (acosta et al. 2016). the epecis associated with infantile and persistent diarrhea. it is mainly detected by the presence of the eae gene (ochoa & contreras 2011). the etec is an important cause of diarrhea in children and travellers (isidean et al. 2011). it is characterised by the presence of a heat-stable (st) and/or heat-labile (lt) enterotoxin gene, encoding colonisation factors and toxin production (gomes et al. 2016). this pathotype remains a problem for humans, pigs and calves. it causes diarrhea in neonatal and recently weaned piglets, which is considered one of the most important diseases affecting pig farming economically (melkebeek, goddeeris & cox 2013). the eiec causes dysentery in humans, is closely related to shigella (hosseini nave et al. 2016), and can be distinguished from other e. coli by the detection of the ipah gene (van den beld & reubsaet 2012). the eaec has been associated with persistent diarrhea in children, travellers and humans with immunodeficiency virus infections; it induces chronic inflammation in the absence of dysentery (okhuysen & dupont 2010). the eaec is characterised by the transcriptional activator encoding aggr gene (wang et al. 2017). the ehec/stec infections range from mild to severe, complicated, bloody diarrhea and haemolytic uremic syndrome (friesema et al. 2011). this pathotype produces one or more types of shiga toxin (stx1 and/or stx2) (dias et al. 2016). the daec pathotypes consist of a heterogeneous group of organisms with variable virulence factors that may play a role in causing sporadic diarrheal illnesses, particularly in paediatric patients (wang et al. 2017), and are detected by the presence of the daad gene (riveros et al. 2017). apart from causing infections, e. coli pathotypes have been reported globally for their resistance to numerous antibiotics, including those used in clinical medicine. the escalation of antibiotic resistance in pathogenic bacteria is a major global public health concern (fernandez, bert & nicolas-chanoine 2016) because it has led to prolonged illness and higher treatment failure rates (van den honert, gouws & hoffman 2018). this concern is further exacerbated by the emergence of multidrug-resistant (mdr) bacteria, resulting in reduced treatment options for the infections they cause (lammie & hughes 2016). the mdr e. coli has been increasingly isolated from livestock and animal products (kallau et al. 2018). this increase has been attributed to increased antibiotic use and sub-optimal biosecurity programmes on farms (mbelle et al. 2019). human exposure to mdr dec could lead to disease outbreaks with severe adverse public health consequences. knowledge of the prevalence and distribution of these mdr pathotypes within the pig production continuum is therefore imperative. we investigated the prevalence of mdr dec pathotypes in a pig production continuum using a farm-to-fork approach. such information could help identify areas needing attention across the continuum, to prevent the spread of these infectious agents to humans through both meat products and environmental exposure. materials and methods study design, sample collection and enumeration of e. coli the study was a longitudinal study conducted over 18 weeks (september 2018 – january 2019) covering the farm-to-fork pig production continuum. it included sampling on the farm, transport system (truck) and attached abattoir, using the guidelines of the world health organization advisory group on integrated surveillance of antimicrobial resistance (who-agisar) (who 2017). a total of 417 samples were collected and processed as previously described (abdalla et al. 2021). samples consisted of faeces, litter and slurry (farm), swabs of transport vehicles, and ceacal, carcass swabs and final meat cut swabs (abattoir). briefly, the defined substrate colilerttm-18 system from idexx (idexx laboratories (pty) ltd., johannesburg, south africa) was used to detect and quantify e. coli according to manufacturer instructions. all processed samples were incubated for 18–24 h at 37 °c and examined under uv light for fluorescence. e. coli confirmation and detection of pathotypes pure e. coli strains were obtained from fluorescent quanti-tray wells as previously described (abia, ubomba-jaswa & momba 2015). ten isolates were randomly selected from each of the 417 samples and phenotypically identified on eosin methylene blue agar (himedia laboratories pvt. ltd., mumbai, india). the isolates were further streaked on nutrient agar (neogen, lansing, michigan, united states) and incubated at 37 °c for 24 h. deoxyribonucleic acid (dna) was extracted from the isolates using the boiling method (amoako et al. 2019). real-time polymerase chain reactions were used to confirm e. coli and determine the various dec pathotypes targeting specific genes (appendix 1). the reaction mixtures and thermal cycling conditions were as previously described (abia et al. 2015), except for the master mix where the luna® universal qpcr master mix (new england biolabs, ipswich, massachusetts, united states) was used in this study. after the final extension step, a melt curve was generated and analysed as previously described (molechan et al. 2019). all reactions were performed on a quantstudiotm 5 (thermofischer scientific, bedford, ma, usa). the dna from reference e. coli strains was used as positive controls (table 1-a1), whilst the reaction mixture with no dna (replaced with nuclease-free water) was used as a no template control. all controls were obtained in-house from the antimicrobial research unit microbial bank. antibiotic susceptibility testing the antibiotic susceptibility profiles of the confirmed diarrheagenic isolates were determined against 20 antibiotics using the disk diffusion method on muller hinton agar (oxoid, basingstoke, hampshire, england) as previously described (abdalla et al. 2021). the e. coli atcc® 25922 was used for quality control. the diameters of the zones of inhibition were measured and interpreted according to the european committee on antimicrobial susceptibility testing breakpoints (eucast 2017). the clinical and laboratory standards institute guidelines (clsi) were used (clsi 2017) for antibiotics that did not have published breakpoints in the eucast guidelines. all the antibiotic discs were purchased from oxoid (basingstoke, hampshire, england). isolates showing resistance to ≥ 1 agent in > 3 distinct antibiotic classes were considered as mdr (amoako et al. 2016). a multiple-antibiotic resistance index (mari) was calculated for these isolates to ascertain whether these isolates originated from high antibiotic use environments (abdalla et al. 2021). statistical analysis the data were analysed using the statistical package for the social science (spss) version 26 (ibm corporation, armonk, new york, united states). descriptive statistics were used to describe the frequency of dec along the pork production chain. the statistical significance of the differences in counts and dec prevalence between different sources was determined using the one-way analysis of variance (anova) with tukey’s honestly significant difference (hsd) post-hoc test. a p-value < 0.05 was considered statistically significant. ethical considerations ethical approval was obtained from the animal research ethics committee (arec 073/016pd) and the biomedical research ethics committee (bca444/16) of the university of kwazulu-natal. a section 20a permit (12/11/1/5) was further obtained from the south african national department of agriculture, forestry and fisheries. results mean e. coli count per sampling site the e. coli was isolated from all the samples collected in this study. the mean e. coli concentrations per sampling site are shown in figure 1. the highest mean e. coli count was recorded in the faecal samples (1.59 × 106 mpn/100 ml), whilst the lowest was recorded in truck samples. there was an overall statistically significant difference (p = 0.002) between the e. coli counts from the different sampling points (table 2-a1). the post-hoc analysis revealed a statistically significant difference in the overall e. coli count between the farm and truck (p = 0.045), and the farm and abattoir (p = 0.004). however, no statistically significant difference was observed between the truck and abattoir e. coli counts (p = 0.183). figure 1: e. coli counts along the pig production continuum farm (round 1–7), truck, abattoir. error bars represent 5% error. identification of e. coli pathotypes a total of 1044 isolates were confirmed as e. coli using real-time pcr. pathotyping using pathotype-specific genes showed that 45.40% (474) of the isolates carried at least one of the virulence genes tested (table 1). the highest percentage of dec was isolated on the farm, whilst the lowest was found at the transport (truck). the eiec at 19% was the most prevalent pathotype, whilst ehec was the least prevalent (0.1%). table 1: distribution of diarrheagenic e. coli pathotypes along the farm-to-fork continuum. like with the abundance of e. coli across the continuum, there was an overall statistically significant difference (p = 0.000) between the prevalence of the dec pathotypes from the different sampling points (table 3-a1). similarly, tukey’s hsd post-hoc analysis of the dec pathotypes prevalence was statistically significantly different between the farm and truck (p = 0.000), farm and abattoir (p = 0.039) and truck and abattoir (p = 0.006) (table-3a1). antibiotic susceptibility profiles only 1% (5 isolates) of the total dec was susceptible to all the antibiotics been tested. overall, the highest resistance was against tetracycline 90.5% (429), whilst all the isolates were susceptible to meropenem (figure 2); the percentage resistance to each antibiotic differed by source (figure 3). figure 2: overall antibiotic resistance in diarrheagenic e. coli pathotypes across the pig production chain. figure 3: percentage resistance to each antibiotic differed by source. the mdr was detected in 73.84% (350/474) of the total isolates, most of which were from the farm (92.29%; 323/350). most of these isolates (69.71%; 244/350) recorded a mari above 0.2 (figure 4). the highest mari was 0.9 (resistance to 18/20 antibiotics tested), recorded by an ehec strain on the farm (table 4-a1). figure 4: the multiple antibiotic resistance (mar) indices of e. coli sampled across different sources (farm, water, truck and abattoir). discussion this study investigated the prevalence of dec pathotypes and their antibiotic resistance profiles in intensive pig farming in umgungundlovu district, kwazulu-natal, south africa, using the farm-to-fork approach. the e. coli was recovered from all the samples along the pig production continuum. of the total number of 1044 e. coli isolates constituting the sample size across the continuum, 45% were dec, with eiec being the most prevalent pathotype. also, 99% of the isolates were resistant to at least one of the antibiotics tested, 73.84% were mdr, most recording a mari greater than 0.2. enumeration of e. coli across the continuum the enumertion results reported in this study are lower than those previously reported in an earlier study in south africa (king & schmidt 2017) and bulgaria (petkov et al. 2006). however, the reported results in the current study were higher than those reported in a study involving 13 pigs farms in australia (chinivasagam et al. 2004). despite the discrepancies between our study and the previous study, the e. coli abundance in the faeces observed in these studies was expected, as the human and animal intestines serve as reservoirs (jafari, aslani & bouzari 2012). unlike most studies that focus on farms or slaughterhouses, the present study used a farm-to-fork approach, ensuring adequate evaluation of the presence and abundance of e. coli along the entire continuum. thus, although e. coli was statistically significantly most abundant in the farm, substantial numbers were also recorded at the abattoir, whilst the lowest e. coli counts were found in the truck samples. the presence of e. coli at the abattoir revealed that although the scalding and singeing method has a significant impact in lowering the e. coli abundance in the carcass, it is still not enough to eliminate contamination during processing (wu et al. 2009), especially during evisceration, as previously highlighted by warriner et al. (2002). whilst the abundance of e. coli in the faecal samples may not be considered a significant threat to human health, the considerable numbers in the meat portions could represent a meat safety challenge that needs to be addressed by ensuring that more efficient hygienic practices are applied to prevent packaging of contaminated meat. it has been demonstrated that meat protein is a potential source of bacterial foodborne disease because of its high protein content, water activity and approximately neutral ph, allowing the proliferation of bacteria (jaja, green & muchenje 2018). prevalence of diarrheagenic e. coli pathotypes the dec pathotypes continue to be a major health threat globally because of the disease outbreak-causing potentials. thus, identifying all possible reservoirs of these pathotypes is essential to ensure public health. in the current study, the highest prevalence of dec was found on the farm. this was logical because the samples isolated from the farm were predominantly faecal. also, the number of samples isolated from the farm was much higher than those isolated from the truck and abattoir, increasing the chances of isolating the pathotypes at farm level. the prevalence of dec in the truck after the loading of pigs was higher, indicating their possible faecal sources. although different dec pathotypes, including epec, have been implicated in colibacillosis in pigs, etec is the most frequently isolated pathotype (garcía-meniño et al. 2018). the etec coli is a major cause of severe diarrhea in suckling and weaning animals, and is a cause of significant losses in the african pig industry (kagambega et al. 2012; kylla et al. 2019). the relatively low prevalence of epec and etec could be because this study was conducted on healthy animals, and no incidence of diseased pigs was recorded throughout the study. apart from epec and etec, the other dec pathotypes were also recorded in the current study, albeit at low prevalences (table 1). this indicates that pigs constitute a significant reservoir of dec pathotypes that could cause human infections (borges et al. 2012; kagambega et al. 2012). interestingly, although eiec is the most detected pathotype in the present study, this pathotype is rarely reported in pigs. also, this pathotype was the only pathotype that was spread across all the sampling points in the continuum. although this dominant occurrence could not be explained, it is important to note that this pathotype is highly implicated in bacillary dysentery in humans, particularly in low-income countries (pasqua et al. 2017). it has also been implicated in foodborne disease outbreaks even in highly industrialised countries with improved hygiene and sanitation, such as the united states (venkitanarayanan & doyle 2003). therefore, its detection across the continuum, including substantially at the abattoir, calls for stringent implementation of hygienic protocols in intensive pig farm systems. we also detected ehec (1%), including o157:h7 (3%), at the farm level. despite their low prevalence, this percentage was still significant because, although most pathogenic e. coli are infective at high doses, ehec requires only ten cells to be infective (limayem & martin 2014). antibiotic susceptibility profiles the growing problem of antibiotic resistance has become a significant public health concern (tanih et al. 2015). in south africa, sulphonamides, tetracyclines, macrolides, ionophores, quinoxalines, glycolipids, polypeptides, streptogramins, oligosaccharides, phosphonic acid, nitroimidazoles and polymeric compounds are registered in various dosage for veterinary use and freely available to farmers for prophylaxis, metaphylaxis, growth promotion and infection treatment (eagar & naidoo 2017). this could explain the antibiotic resistance profiles observed, with most isolates showing resistance to tetracycline, chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole (figure 2a). the incidence of drug-resistant dec pathotypes revealed that pork might pose a public health risk. although tigecycline and imipenem recorded low resistance rates in the current study, they are still alarming because they are considered last-resort antibiotics for human use. the high percentage of mdr isolates obtained in the current study suggest that these organisms were exposed to high antibiotic use environments. this is substantiated by the fact that most of the mdr isolates recorded maris greater than 0.2. a mari greater or equal to 0.2 indicated potential high-risk as the organisms probably originate from environments where antibiotics are extensively used (teshome et al. 2020). diarrhea caused by foodborne pathogens such as dec pathotypes is an important cause of death, especially in children in lowand middle-income countries (jaja et al. 2018). the rise in mdr dec aggravates the situation because mdr strains are known to limit treatment options. in order to minimise the risk and prevent microbial contamination along the food production chain, good hygiene manufacturing practice (ghmp) and hazard analysis critical control points (haccp) practices should be implemented, in addition to appropriate retail and consumer meat handling and processing (galli et al. 2016). in south africa, meat safety control is a shared responsibility between the department of agriculture, land reform and rural development (dalrrd) and the department of health (doh). approaches to meat safety control include farm-to-fork control with the publication of haccp regulations. conclusion the presence of mdr dec pathotypes in animal food production environments and food of animal origin indicates that these animals serve as reservoirs and potential sources of these pathogenic organisms that could be transmitted to humans. this is especially in occupationally exposed workers and through consumption of undercooked pork. adherence to good hygienic practices along the pig production continuum and thorough cooking are essential for mitigating the risk of transmission and infection and ensuring food safety. acknowledgements we would like to acknowledge managers and workers at both the farm and abattoir for their helpful cooperation. competing interests s.y.e. is the chairperson of the global respiratory infection partnership and a member of the global hygiene council, both sponsored by an unrestricted educational grant from reckitt and benckiser ltd., united kingdom. all other authors have no competing interests. authors’ contributions all authors contributed toward the conceptualisation, validation, writing, review, and editing of the article. s.e.a. contributed to the article methodology, investigations and original draft preparation. s.y.e contributed to project administration, resources, and funding. s.e.a., a.l.k.a. and d.g.a. contributed to the formal analysis of the article. s.y.e., a.l.k.a., d.g.a. and l.a.b. were responsible for supervision. all authors have read and agreed to the published version of the manuscript. funding information the research was funded by the who advisory group on integrated surveillance of antimicrobial resistance (agisar) research project: ‘triangulation of antibiotic resistance from humans, the food chain and associated environments – a one health project’ (reference identity: 204517), south african research chair initiative of the department of science and technology and national research foundation of south africa (grant number: 98342), the south african medical research council (samrc) and uk medical research council and the college of health sciences, university of kwazulu-natal, south africa. data availability the authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials. disclaimer any opinion, finding and conclusion or recommendation expressed in this material are those of the authors and do not necessarily reflect the views of the 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who, 2017, integrated surveillance of antimicrobial resistance in foodborne bacteria: application of a one health approach: guidance from the who advisory group on integrated surveillanec of antimicrobial resistance (agisar), world health organization, geneva. wu, s., dalsgaard, a., vieira, a.r., emborg, h.-d. & jensen, l.b., 2009, ‘prevalence of tetracycline resistance and genotypic analysis of populations of escherichia coli from animals, carcasses and cuts processed at a pig slaughterhouse’, international journal of food microbiology 135(3), 254–259. https://doi.org/10.1016/j.ijfoodmicro.2009.07.027 appendix 1 table 1-a1: primers and controls used for pathotypes identification. table 2-a1: statistical difference between abundance of e. coli at the different sampling points. table 3-a1: prevalence of diarrheagenic e. coli along the pig production continuum. table 4-a1: multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. table 4-a1 (continues...): multiple-antibiotic resistance index for all diarrheagenic e. coli pathotypes along the farm-to-fork continuum. abstract introduction materials and methods results discussion acknowledgements references about the author(s) chantel j. de beer department of epidemiology, parasites and vectors, agricultural research council, onderstepoort veterinary research (arc-ovr), pretoria, south africa gert j. venter department of epidemiology, parasites and vectors, agricultural research council, onderstepoort veterinary research (arc-ovr), pretoria, south africa department of veterinary and tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa marc j.b. vreysen insect pest control laboratory, joint fao/iaea division of nuclear techniques in food and agriculture, seibersdorf, austria fernando c. mulandane biotechnology centre, eduardo mondlane university, maputo, mozambique luis neves department of veterinary and tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa biotechnology centre, eduardo mondlane university, maputo, mozambique sihle mdluli department of veterinary services, epidemiology unit, mbabane, eswatini otto koekemoer department of epidemiology, parasites and vectors, agricultural research council, onderstepoort veterinary research (arc-ovr), pretoria, south africa department of veterinary and tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation de beer, c.j., venter, g.j., vreysen, m.j.b., mulandane, f.c., neves, l., mdluli, s., et al., 2019, ‘using genetic and phenetic markers to assess population isolation within the southernmost tsetse fly belt in africa ’, onderstepoort journal of veterinary research 86(1), a1768. https://doi.org/10.4102/ojvr.v86i1.1768 original research using genetic and phenetic markers to assess population isolation within the southernmost tsetse fly belt in africa chantel j. de beer, gert j. venter, marc j.b. vreysen, fernando c. mulandane, luis neves, sihle mdluli, otto koekemoer received: 22 mar. 2019; accepted: 12 july 2019; published: 16 oct. 2019 copyright: © 2019. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the effective control of tsetse flies (diptera; glossinidae), the biological vectors of trypanosome parasites that cause human african trypanosomosis and african animal trypanosomosis throughout sub-saharan africa, is crucial for the development of productive livestock systems. the degree of genetic isolation of the targeted populations, which indicate reinvasion potential from uncontrolled areas, will be critical to establish a control strategy. molecular and morphometrics markers were used to assess the degree of genetic isolation between seemingly fragmented populations of glossina brevipalpis newstead and glossina austeni newstead present in south africa. these populations were also compared with flies from adjacent areas in mozambique and eswatini. for the molecular markers, deoxyribonucleic acid was extracted, a r16s2 polymerase chain reaction (pcr) was performed and the pcr product sequenced. nine landmarks were used for the morphometrics study as defined by vein intersections in the right wings of female flies. generalised procrustes analyses and regression on centroid size were used to determine the cartesian coordinates for comparison between populations. both methods indicated an absence of significant barriers to gene flow between the g. brevipalpis and g. austeni populations of south africa and southern mozambique. sustainable control can only be achieved if implemented following an area-wide management approach against the entire g. brevipalpis and g. austeni populations of south africa and southern mozambique. limited gene flow detected between the g. austeni population from eswatini and that of south africa or mozambique may imply that these two populations are in the proses of becoming isolated. keywords: glossina brevipalpis; glossina austeni; geometric morphometrics; mitochondrial dna; veterinary entomology. introduction african animal trypanosomosis is endemic in south africa with an estimated 350 000 cattle at risk in the rural north east of the kwazulu-natal province (kappmeier, nevill & bagnall 1998; kappmeier green, potgieter & vreysen 2007). the sustainable control of tsetse flies (diptera; glossinidae), the vectors of the trypanosome parasites (kinetoplastida; trypanosomatidae), therefore remains a priority. presently, two species of tsetse flies, glossina brevipalpis newstead and glossina austeni newstead, are found in an area of about 16 000 km2 in the north eastern part of the kwazulu-natal province in south africa. the infested area stretches from about 10 km south of the mfolozi river in the south, for approximately 190 km, to the border of mozambique in the north, and from the indian ocean coast in the east for 80 km to the hluhluwe-imfolozi park in the west (de beer et al. 2016a; kappmeier green et al. 2007). these south african populations of g. austeni and g. brevipalpis, extending into the matutuine district (maputo province) of mozambique, represent the southern most distribution of tsetse flies in africa (sigauque et al. 2000). the matutuine district borders in the south with the north eastern part of the kwazulu-natal province in south africa. the northern limit is the boane and namaacha districts of mozambique. in the east, it borders with the indian ocean and in the west with eswatini. while, to date, no g. brevipalpis has been sampled in eswatini, low numbers of g. austeni were trapped in 2008. these flies were only found in the mlawula nature reserve, situated in the north east of the country (saini & simarro 2008). this reserve extends into the mbuluzi game reserve in the north west, the simunye nature reserve in the south and hlane royal national park further to the south west. in the east, the reserve borders with the lebombo mountains (elevation 776 m), an 800-km-long narrow range of mountains that stretch from hluhluwe in the kwazulu-natal province in the south to punda maria in the limpopo province in south africa in the north parallel with the mozambican border. determining the exact geographical limits of tsetse fly populations and the level of genetic interactions between these populations on mainland africa remains challenging. in contrast to previous surveys in south africa that indicated g. brevipalpis to be confined to two distinct bands in the north and south of the infested area in the kwazulu-natal province (kappmeier green et al. 2007), thorough surveys (de beer et al. 2016a) showed that both g. brevipalpis and g. austeni appear to be more continuously distributed throughout the area. in view of its relatively low abundance in the area between the two bands, it would be important to elucidate the genetic distance between the g. brevipalpis populations in the north and the south to develop an appropriate control strategy. similarly, the low abundance and patchy distribution of g. austeni in this area may result in the development of localised genetically isolated populations. although g. austeni, and perhaps also g. brevipalpis, is continuously distributed throughout north eastern kwazulu-natal province, these populations are, on a micro-ecological scale, often confined to pockets of dense vegetation (esterhuizen 2007). this, in addition to the relatively sedentary behaviour of the species, may favour the development of potential genetically isolated populations. it is known that habitat fragmentation can lead to slight but significant variations in localised environmental conditions to which tsetse populations will adapt physiologically and demographically thereby affecting tsetse–trypanosome interactions and hence trypanosomosis risk (mweempwa et al. 2015). eradication of a tsetse population will in most cases only be sustainable if the control programme follows an area-wide integrated pest management (aw-ipm) approach, that is, a strategy that targets the entire pest population (hendrichs et al. 2005; klassen 2005). such an approach is fairly straightforward if the targeted population represents a geographically well-defined population with no gene flow from neighbouring populations, or if a rolling-carpet approach is implemented (hendrichs et al. 2005). the rolling-carpet approach is dynamic as the basic operational phases (pre-intervention, population reduction, release of sterile insects and maintenance of pest status) are carried out simultaneously in a phased manner (hendrichs et al. 2005). kappmeier green et al. (2007) proposed an aw-ipm strategy that included a sterile insect technique (sit) component based on distribution data of the fly populations prior to 1999 (kappmeier green 2002). the recent sampling of g. brevipalpis in areas where they were considered absent before requires a revisit of the proposed strategy. in the present study, available molecular and phenetic (geometric morphometrics) markers were used to determine the level of genetic isolation between the southern and northern tsetse fly pockets in south africa. in addition, the south african populations were compared with flies collected in the adjacent areas in southern mozambique and eswatini. the level of genetic isolation between the various populations within south africa and between these three countries will provide an approximation of the invasion potential of flies from neighbouring areas (solano et al. 2010) into the controlled areas. this knowledge will be a prerequisite for the development of the most appropriate control strategy, that is, whether these populations can be tackled separately or in sequence or whether they constitute one population. materials and methods tsetse fly sampling tsetse flies were collected with odour-baited h-traps (kappmeier 2000; kappmeier & nevill 1999; kappmeier green et al. 2007) in 15 sites for molecular marker analyses and in 12 sites for morphometric marker analyses (figure 1). in south africa, flies were sampled in 12 and 10 sites for the molecular and morphometric marker analyses, respectively (figure 1). in southern mozambique, flies were collected in two sites, east and west of the maputo river, for molecular marker analyses (figure 1). only flies collected east of the maputo river were subjected to morphometric analyses (figure 1). in eswatini, flies were collected only in the mlawula nature reserve as they are confined to this area (figure 1). these sites comprised the entire geographical distribution area of tsetse flies in the region. in the traps, flies were guided to plastic collection bottles that contained a 20% ethanol solution to which savlon® (johnson & johnson, pharmedica laboratories [pty] ltd., east london, south africa) (0.4 ml/l) and formalin (0.4 ml/l) were added. the sampled flies were removed from the collection bottles and preserved in 95% ethanol until analysed. figure 1: sites used for the collection of tsetse flies for comparative molecular and geometric morphometrics in eswatini: (1) mlawula nature reserve, mozambique, (2) west and (3) east of the maputo river and south africa (4) ndumu, (5) tembe, (6) kosi bay, (7) mbazana, (8) mkuzi, (9) lower mkuzi, (10) phinda, (11) false bay park, (12) hluhluwe-imfolozi park north, (13) hluhluwe-imfolozi park south, (14) boomerang and (15) st lucia. at sites 8, 13 and 21 flies were used only for molecular analyses. glossina austeni and g. brevipalpis from laboratory colonies maintained at the agricultural research council-onderstepoort veterinary research (arc-ovr), pretoria, south africa, were included for the genetic analyses as out-groups. the colonies of g. austeni and g. brevipalpis at the arc-ovr originate from laboratory colonies at the vector and vector-borne diseases research institute, tanga, tanzania, and the fao/iaea insect pest control laboratory in seibersdorf, austria, respectively (de beer, venter & vreysen 2016b). the colonies in tanga and seibersdorf were originally established from material collected in 1982 on the unguja island of zanzibar and kenya in east africa (de beer et al. 2016b). as such these out-groups represent genetically isolated populations which were bred in isolation for nearly 36 years. deoxyribonucleic acid extraction deoxyribonucleic acid (dna) was extracted from ethanol preserved g. brevipalpis and g. austeni specimens. after removal from the ethanol, the flies were briefly air-dried and rehydrated in phosphate buffered saline (pbs). the wings and legs of the flies were removed, and the body crushed in fresh pbs using a micro-pestle in a 1.5 ml polypropylene tube. after centrifugation, 200 µl of the supernatant suspension was used for dna extraction with a dneasy blood & tissue kit (qiagen, johannesburg, south africa). the purified dna was suspended in 100 µl of the supplied elution buffer and either processed immediately or stored at −20 °c. mitochondrial locus amplification and sequencing the n1-j-12585 [5′ggt ccc tta cga att tga ata tat cct 3′] and lr-n-12866 [5′aca tga tct gag ttc aaa ccg g3′] primer pairs (simon et al. 1994) were used to amplify the mitochondrial 16s-2 ribosomal ribonucleic acid (rrna) locus from 2 to 5 µl of the extracted dna as described previously (krafsur, marquez & ouma 2008). the amplification products were verified by gel electrophoresis and sequenced using the same primers as those used for the amplification. the sequencing reactions contained around 10 ng of template dna and 3.2 pmol of the primer made up to a final volume of 12 µl with ultra-pure h2o. sequencing was performed on a 3100 genetic analyser (applied biosystems, south africa (pty) ltd, johannesburg) using the big dye terminator v 3.1 cycle sequencing kit (applied biosystems, south africa (pty) ltd). morphometric analysis for morphometric analysis, the right wings of 40 females of each species per site were used. lower numbers of flies were available from mozambique (n = 36), kosi bay (n = 31) and false bay park (n = 37) for g. brevipalpis and mozambique (n = 14) and the hluhluwe-imfolozi park (n = 13) for g. austeni. the right wings of 345 g. brevipalpis and 346 g. austeni females were removed, dry mounted between two microscope slides (patterson & schofield 2005) and photographed using a dino x lite digital microscope (idcp b.v, naarden, the netherlands). nine landmarks (cartesian coordinates) were defined by vein intersections (figure 2) using the collection of coordinates (coo) programme of the collection of landmark for identification and characterisation (clic) software package (dujardin, kaba & henry 2010). figure 2: glossina austeni slide-mounted wing indicating the nine landmarks as defined by vein intersections. data analysis deoxyribonucleic acid sequences were aligned with clustal omega (sievers et al. 2011), keeping all default parameters. the alignment files were exported in fasta format and used as input for mega7 (kumar, stecher & tamura 2016) to construct the phylogenetic trees and ibdws (jensen, bohonak & kelley 2005) to estimate the fixation index (fst values) from haploid genetic distance data, according to the methods used by weir (1990). gaps at the end of sequences were treated as missing data. phylogenetic trees were constructed from the aligned sequences using the maximum likelihood algorithm with a substitution model and 500 bootstrap replications (tamura & nei 1993). the model of nucleotide substitution that best fits the data set was determined using the maximum likelihood model selection function in mega7 software (kumar et al. 2016). separate trees, using 16s rrna-gene sequence data, were generated for each species. morphometric analyses of the cartesian coordinates were carried out using the morphoj integrated software package (klingenberg 2011). the cartesian coordinates were subjected to a generalised procrustes analysis (rohlf 1996) and variations in wing shape (partial warps) were determined by procrustes superposition through generalised least squares (rohlf 1999). through procrustes superimposition analyses, the wings were scaled to the same size, transposed to the same centre of gravity and orientated to provide the minimum sum of squared distances between corresponding landmarks. principal component analyses of the shape variables using morphoj (klingenberg 2011) provided 14 partial warps. differences in wing shape were determined by canonical shape dissimilarity (klingenberg 2011). in addition, the centroid size, as an indicator of size variation, was determined by the square root of the sum of the squared distances of all landmarks from the centroid (bookstein 1991). centroid size was analysed using the statistical software graphpad instat (version 3.00, 2003). a one-way analysis of variance (anova) was used to assess significant differences between the means of the centroid size (p value < 0.05 was considered significant) of wings of flies collected from the various sites. the data were normally distributed, standard (parametric) methods were used and the tukey’s test was applied. multivariate regression of partial warps on size was used to estimate the residual allometry and the statistical significance was estimated by the 10 000 runs permutation tests (klingenberg 2011). ethical considerations materials used in the study posed no health risk to researchers and no vertebrate animals were involved. the study was conducted as part of a project funded by the department of science and technology on national assets (p10000035) at the arc-ovr (agricultural research council, onderstepoort veterinary research) in collaboration with the food and agriculture organization (fao) or international atomic energy agency (iaea) division of nuclear techniques in food and agriculture and the department of technical cooperation of the iaea under project raf 5069. permission to carry out research in terms of section 20 of the animal diseases act south africa (act no. 35 of 1984) has been granted for tsetse fly collection (ref 12/11/1/1/9). results mitochondrial deoxyribonucleic acid analysis after quality checks, mitochondrial dna (mtdna) sequences from 79 g. austeni and 116 g. brevipalpis specimens were used in multiple alignments, which included sequences from four g. austeni and two g. brevipalpis from the tsetse colonies as out-groups. the phylogenetic trees (figure 3), constructed from the aligned sequences, showed minimum variation. figure 3: phylogenetic trees constructed from partial sequence analysis of the 16s subunit 2 ribosomal ribonucleic acid gene of glossina brevipalpis (a) and glossina austeni (b) from eswatini (esw), mozambique (moz e; moz w) and south africa [ndumu (ndu), tembe (tem), kosi bay (kos), mbazana (mba), mkuzi (mk), lower mkuzi (umb), phinda (pi), false bay park (mfp), hluhluwe-imfolozi park north (hip), hluhluwe-imfolozi park south (hp), boomerang (mfk), st lucia (st)]. colony (col) flies of both species were used as out-groups. red and blue groupings indicate the south africa-southern mozambique and eswatini haplotypes, respectively. arrows indicate specimens carrying contrasting haplotypes from their group. the phylogeny of g. brevipalpis from south africa and southern mozambique did not show any significant branching or grouping of specimens, with the exception of the colony flies (figure 3a). for both species, a prominent mozambique south africa genotype was evident with only minor substructuring of isolates. the branches or groupings did not correlate with geographic origin and the nodes had very low bootstrap support values (p-distances < 0.01). the only exceptions were the colony flies and g. austeni from eswatini which formed a distinct phylogenetic grouping (figure 3b). the eswatini g. austeni, however, did not form a monophyletic group as two specimens carried the south africa-southern mozambique haplotype and one specimen (esw-550) had a unique haplotype (figure 3b). additionally, one specimen from southern mozambique (moz e-12) carried the eswatini haplotype (figure 3b). for g. austeni, 198 sites out of 349 in the sequence alignments were variable and the haplotypic diversity was 0.106 overall, and 0.054 when the out-group consisting of the colonised east african specimens was excluded. the fst values indicate a great level of genetic differentiation between the g. austeni populations from south africa and eswatini (0.22), whereas the level of genetic differentiation was moderate between the populations of southern mozambique and eswatini (0.15). morphometric analysis the shape of the right wings of female g. brevipalpis and g. austeni was analysed to assess the degree of genetic isolation between the populations within south africa as well as between the south african and the southern mozambique populations. the analysis for the g. austeni populations was extended to include the population in eswatini. the allometric effect was removed so that shape could be analysed independently. the two species were evaluated separately. the principal component analyses indicated that the first two discriminant factors (shape components) accounted for 71% and 69% of the variance for g. brevipalpis and g. austeni, respectively. these discriminant factors indicated that there was no clear wing shape separation between the g. brevipalpis populations collected from the sites in south africa and southern mozambique (figure 4a). the multivariate regression of the first relative warp against centroid size (100 000 permutation rounds) was also not significant (p = 0.30), indicating that there was no residual allometry. figure 4: the distribution of glossina brevipalpis (a) and glossina austeni (b) female right wing shape in the morphospace defined by the first two canonical variants; flies were collected from different sites. in contrast, the observed residual allometry for g. austeni, as the multivariate regression was significant (p = 0.01), might indicate an environmental effect. there was no separation within the first discriminant factors between the g. austeni collected from the different sites in south africa as well as those from southern mozambique (figure 4b). in addition, no shape separation was observed between g. austeni collected from eswatini, southern mozambique or south africa (figure 4b). the only exception includes flies collected at st lucia and eswatini. this is explained by the 228 km distance between these two sites, located at the boundaries of the study area (figure 1). the wing size variation as indicated by the isometric estimator known as the average wing centroid size was determined for both species. for g. brevipalpis, this ranged from 1512 ± 35 for flies collected from the hluhluwe-imfolozi park to 1568 ± 35 for flies from st lucia (figures 1 and 5a). the most significant differences in wing centroid size were found between the populations of the hluhluwe-imfolozi park and southern mozambique (p < 0.01), ndumu (p < 0.05), kosi bay (p < 0.05) and st lucia (p < 0.01) (figure 5a). significant differences in wing centroid size were also observed between the g. brevipalpis populations sampled in southern mozambique and boomerang (p < 0.05), tembe versus st lucia (p < 0.01) and boomerang versus st lucia (p < 0.01) (figure 5a). figure 5: centroid size variations of the right wings of female glossina brevipalpis (a) and g. austeni (b) according to localities. each box shows the group median separating the 25th and 75th quartiles, the capped bars indicate maximum and minimum values and circles indicate the outliers. boxes followed by a different letter indicate that the centroid size was significantly different at the 5% level. within south africa, wing centroid size was significantly different (p < 0.01) between g. austeni from phinda (916 ± 26) and st lucia (941 ± 52) (figure 5b). similarly, the average female wing centroid size of the g. austeni population from eswatini (892 ± 37) was significantly different (p < 0.01) from that of the flies from southern mozambique and south africa (figure 5b). this indicates a longitudinal (coast to interior) trend in wing size for both species. flies collected at the coastal sites were, on average, bigger than flies collected in the interior (figure 5). a trend in wing size, although to a lesser extent, was also observed in a latitudinal direction with bigger flies, on average, collected in the south (figure 5). discussion the southern mozambique populations of g. brevipalpis and g. austeni extend into south africa and eswatini (g. austeni) (saini & simarro 2008; sigauque et al. 2000). the confinement of flies to areas with suitable vegetation (i.e. protected areas and game parks) linked to their relatively sedentary behaviour and habitat fragmentation in the area might have created opportunities for these populations to become genetically isolated. molecular and/or morphometric markers have been used in the past to estimate gene flow between subpopulations as an indirect measure of dispersal (bouyer et al. 2007, 2009; camara et al. 2006; gooding & krafsur 2005; solano et al. 2000, 2009). geometric wing morphometry in conjunction with microsatellite analyses showed that the populations of glossina palpalis gambiensis that vanderplank found on the loos islands, 5 km off the coast of guinea, were genetically isolated from two populations on mainland guinea (camara et al. 2006). similarly, a study using microsatellite and mtdna markers in conjunction with morphometrics showed that the g. p. gambiensis population of the niayes in senegal was genetically isolated from the main tsetse belt 120 km away in the eastern part of the country (solano et al. 2010). the mtdna sequences as obtained with the 16srrna gene marker used in the present study were sensitive enough to discriminate between g. austeni populations from south africa-southern mozambique and those of eswatini. there was, however, low variability within and between tsetse fly populations of both species from south africa and southern mozambique. the presence of a south africa-southern mozambique haplotype specimen collected from eswatini and vice versa (two specimens) indicate limited gene flow and potential incomplete isolation (figure 3). this isolation might be driven by the geographical distance between the sites, the physical barrier represented by the lebombo mountains and regular bush fires occurring in the mlawula nature reserve in eswatini. although the fst values were potentially skewed by the unequal numbers in each of the populations, the phylogenetic analysis seems to indicate that there is probably more migration of g. austeni between eswatini and southern mozambique than between eswatini and south africa, which is plausible, given the locations and geography of the sampling sites (figure 1). morphometric markers after procrustes superposition indicated a lack of any significant differentiation in wing shape between populations within south africa, southern mozambique and eswatini. in congruence with the molecular analyses, wing shape variations indicated that the g. austeni from eswatini was to some extent isolated from the south africa-southern mozambique populations. the significant differences obtained in wing centroid size for both species collected at various sites within south africa as well as in neighbouring countries are the result of variation in environmental condition between sites. as indicated by de beer (2016), the temperature and humidity fluctuations were more pronounced in the interior, for example, ndumu than at the coast, for example, st lucia. the mean temperature in the interior ranged from 27 °c in the hot months to 15.5 °c in the colder months, and the mean relative humidity from 80% in the rainy to 50% in the dry season (de beer 2016). the mean temperature at the coast ranged from 26 to 17 °c and the mean relative humidity from 99% to 68% (de beer 2016). this indicates that populations at the coast are subjected to less pronounced fluctuations in environmental conditions than individuals collected in the interior. although comparable results were obtained with morphometric analyses and those using mtdna, both methods have limitations. the use of microsatellite markers, which have proved to be very useful and accurate to study isolation between populations of the same tsetse species (kaba et al. 2012), was not possible as these markers were not developed at the time of the study. we may therefore have missed out the information on real isolation between populations that may have occurred recently, because of a lack of variability and, hence, sensitivity of the 16srrna gene marker. in the evaluation of these results, it must be taken into consideration that mtdna is a very slow evolving fragment of the dna (galtier et al. 2009) and that the lack of differentiation, as obtained in the present study, could be because of low sensitivity of the marker chosen. in addition, the marker is maternally inherited and may or may not reflect patterns of nuclear differentiation (galtier et al. 2009). the current data prompt a more conclusive analysis, using microsatellites, which is currently underway, to confirm or refute the present results. it still needs to be confirmed whether the tsetse populations that encompass south africa, southern mozambique and eswatini are isolated from the main tsetse fly belt north of maputo. it is assumed that this belt starts approximately 500 km further north in central mozambique south of the save river (dias 1961; mulandane 2013). the presence of trypanosoma congolense positive cattle, diagnosed during routine surveillance programmes in the area between maputo and the save river, is a strong indication of the presence of tsetse flies as cyclical vectors. this area needs to be surveyed in detail to confirm the presence or absence of tsetse files. should flies be sampled, these need to be included in the planned genetic study using microsatellites. the degree of genetic isolation of the southern african population from those further north in africa also needs to be determined. within the known limitations of the used markers, the data of this study seem to provide some evidence of the absence of significant barriers to gene flow between the populations in south africa and southern mozambique and justify the need for further investigation. this seems to imply that the g. brevipalpis and g. austeni populations of south africa and southern mozambique can be considered homogenous and that localised control (e.g. only in south africa) may not be sustainable because of reinvasion from uncontrolled neighbouring areas. should the data be confirmed by the ongoing microsatellite work, this would entail the need for an aw-ipm approach against the entire tsetse fly belt of south africa and southern mozambique. the entire area should be controlled simultaneously or in a sequential way using temporary barriers of impregnated traps and/or targets between tsetse free areas following the ‘rolling-carpet’ principle (vreysen et al. 2007). acknowledgements the authors thank their co-workers in the epidemiology, parasites and vectors programme as well as the staff members at the kwazulu-natal tsetse research station for their valuable assistance. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.j.d.-b., g.j.v., o.k., l.n., s.m. and f.c.m. conceptualised and designed the study. 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data analysis results discussion acknowledgements references appendix 1 about the author(s) maphuti b. ledwaba department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa okechukwu c. ndumnego africa health research institute, durban, south africa itumeleng matle department of bacteriology, agricultural research council – onderstepoort veterinary research institute, onderstepoort, south africa awoke k. gelaw department of bacteriology, agricultural research council – onderstepoort veterinary research institute, onderstepoort, south africa henriette van heerden department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation ledwaba, m.b., ndumnego, o.c., matle, i., gelaw, a.k. & van heerden, h., 2020, ‘investigating selective media for optimal isolation of brucella spp. in south africa’, onderstepoort journal of veterinary research 87(1), a1792. https://doi.org/10.4102/ojvr.v87i1.1792 original research investigating selective media for optimal isolation of brucella spp. in south africa maphuti b. ledwaba, okechukwu c. ndumnego, itumeleng matle, awoke k. gelaw, henriette van heerden received: 21 may 2019; accepted: 01 oct. 2019; published: 04 mar. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract bovine brucellosis in south africa is caused mainly by brucella abortus biovar (bv.) 1 and less frequently by b. abortus bv. 2. bacterial isolation is regarded as the gold standard for diagnosis of brucella species; however, it is not very sensitive. the aim of this study was to determine the selective medium with optimum antibiotic composition that will allow the growth of brucella species (spp.) while inhibiting moulds, yeast and most, if not all, gram-negative contaminants in south africa. in the controlled experiment, modified agrifood research and technology center of aragon (cita) medium (mcita) seemed to be the optimum selective medium for isolation of brucella spp. as compared with farrell’s medium (fm) and modified thayer martin (mtm), while fm inhibited the growth of most fungal and bacterial contaminants. mean comparison between the three media used to culture b. abortus resulted in lower mean difference ranging from 0 to 2.33. in case of brucella ovis, high mean difference was obtained when comparing fm with mcita (10.33) and mtm (12). however, the mean differences of 0.67 and 1.67 were obtained when comparing mcita and mtm media used to, respectively, culture pasteurised and raw milk spiked with b. ovis. further optimisation at the agricultural research council – onderstepoort veterinary research institute resulted in a comparable performance between fm and mcita; however, mcita allowed optimal growth of the fastidious b. ovis, which is generally inhibited on fm. generally, mcita seemed to be the optimum selective medium for isolation of brucella spp., while fm inhibits the growth of most fungal and bacterial contaminants. thus, veterinary laboratories can use mcita and/or fm but should take into consideration the detection of factious brucella isolated in the country or region. keywords: bovine brucellosis; selective media; bacterial isolation. introduction bovine brucellosis is a widespread zoonotic infection affecting animals and humans. it is of veterinary, public health and economic importance, as it causes substantial losses of livestock, thus affecting global trade and the livelihood of communities relying on their animals for survival (maudlin, eisler & welburn 2009; seimenis 2008). the disease is primarily caused by biovars of brucella abortus, and less frequently by brucella suis and brucella melitensis, which are intracellular organisms and members of the genus brucella (oie 2016). the above-mentioned three species (spp.) as well as brucella canis, brucella neotomae and brucella ovis are classical brucella species listed in the 1980 approved lists of bacterial names (corbel & brinley-morgan 1982; oie 2016). other members of the genus include the atypical brucella ceti and brucella pinnipedialis (foster et al. 2007), brucella microti (scholz et al. 2008), brucella inopinata (scholz et al. 2010), brucella papionis from baboons (papio spp.) and brucella vulpis from red foxes (scholz & vergnaud 2013; scholz et al. 2016; whatmore et al. 2014). in south africa, previous reports showed that b. abortus biovar (bv.) 1 causes 90% of infection cases in cattle, while the remaining 10% of the cases is because of b. abortus bv. 2 (bishop et al. 1994; godfroid et al. 2004). however, b. melitensis has been reported in slaughtered cattle in a recent study (kolo et al. 2018). brucella control and eradication programmes should involve diagnostic assays that will address the development and transmissions of the causal organisms while taking into account local regulations and the suspected species occurring in the country (christopher & upadyaya 2014; nicoletti 1984). brucellosis has no clinical feature that allows accurate diagnosis except for abortion, which also occurs in other diseases like heartwater, salmonellosis, rift valley fever, leptospirosis and others (diaz, casanova, ariza & moriyón 2011; ferreira et al. 2003). moreover, precise diagnosis of the disease is made difficult by several factors that include varying incubation periods, type of specimens, sensitivity and specificity of tests (especially serological assays), and multiple testing to confirm status of the herd/animals. therefore, isolation of the organism is still regarded as the gold standard, compared with other techniques (mcgiven 2003; oie 2016), irrespective of the fact that cultures are not always sensitive, and it is time-consuming to culture and complete the phenotypic traits involved in identifying the bacteria because brucella spp. are slow-growing organisms (corbel & brinley-morgan 1982; poester et al. 2010). lack of resources also have a negative impact on diagnosis because isolation of brucella bv. species can only be done in an appropriate biosafety laboratory (biosafety level 2+ and above). samples for the isolation of brucella are mostly chosen based on the observed symptoms. they usually include stomach contents of the aborted foetuses, foetal membranes, vaginal secretions, milk, hygroma fluids, tissue samples from the udder, mammary and genital lymphs, and others. in animals, brucella organisms can be obtained mostly from the infected placenta as well as from milk and lymph nodes, whereas in humans it can be isolated from blood, urine and cerebrospinal fluid (bishop et al. 1994; corbel 1997; poester et al. 2010). a broad spectrum of culture media is available to grow brucella bv. species and all have their own advantages and disadvantages. biphasic media and broth are usually preferred when culturing liquid specimens, whereas solid media with 2.5% agar can be used to grow other specimens (castaneda 1947; poester et al. 2010). selective media are recommended and used for the isolation of the slow-growing brucella spp. because they have the capacity to inhibit the growth of most commensal and environmental bacteria (de miguel et al. 2011; farrell 1974; marin et al. 1996). colonies of brucella spp. usually appear after 2–30 days of incubation, and these are transparent with smooth surfaces and intact borders (alton et al. 1988; da silva mol et al. 2012). veterinary diagnostic laboratories in south africa and many other countries use farrell’s medium (fm) to culture brucella from field samples. moreover, the office international des epizooties (oie) recommends the use of both fm and modified thayer martin (mtm) as the nalidixic acid, and bacitracin in fm is inhibitory for b. ovis and some b. melitensis and b. abortus biovars (marin et al. 1996). farrell’s medium was developed for the isolation of b. abortus from contaminated samples (farrell 1974), while thayer martin medium was developed in 1964 for the isolation of neisseria gonorrhoeae and neisseria meningitidis (thayer & martin 1964). the latter medium was later modified after the withdrawal of ristocetin, which was used to inhibit the growth of gram-positive bacteria (thayer & martin 1966). it is important to use a selective media that allow the growth of the more fastidious brucella bv. species like b. abortus bv. 2, b. ovis and b. canis. previous studies showed that the concentration of some of the antibiotics contained in various selective media can inhibit the growth of some strains of b. ovis, b. melitensis and b. abortus bv. 2, 3 and 4 (corbel & macmillan 1998; marin et al. 1996; oie 2016). de miguel et al. (2011) reported agrifood research and technology center of aragon (cita) medium, which is more sensitive than both mtm and fm for isolation of all smooth brucella species while inhibiting most contaminant microorganisms. the oie (2016) recommended the use of fm with either mtm or cita for maximal isolation of brucellae. the main aim of this study was to determine the medium composed of applicable antibiotic concentrations that will allow and support the growth of brucella spp. causing brucellosis in south africa while inhibiting the growth of moulds, yeast and other gram-negative contaminants. materials and methods samples freeze-dried b. abortus bv. 2 strain 2534/15 and b. ovis strain rc48 were obtained from agricultural research council-onderstepoort veterinary research institute (arc-ovr) storage collection and cultured on blood agar at 37° c in a 10% carbon dioxide incubator. the two species were opted for use in the controlled study because they are fastidious as reported in corner and alton (1982). the freshly grown colonies of the above-mentioned strains were harvested and suspended in phosphate-buffered saline (pbs) and used in a controlled experiment. various samples brought in from different provinces of south africa for routine diagnostic screening at arc-ovr were used to further test agrifood research and technology center of aragon (cita) medium mcita in conjunction with fm in collaboration with the general bacteriology section in the organisation. both media were also used to isolate brucella from milk collected from the seropositive animals during the study. comparison of three different media in a controlled experiment farrell’s medium, mtm and cita media were used in a controlled study to determine the optimum selective medium for isolation of brucella from field samples. gonococcus (gc) agar base and blood agar base no. 2 (thermo scientific™ oxoid™, gauteng, south africa), foetal bovine serum (highveld biologicals, johannesburg, south africa) and the required antibiotics (sigma-aldrich; johannesburg, south africa) were used for the preparation of both mtm and cita (table 1), as described in a previous report by de miguel et al. (2011), whereas fm was obtained from selecta media, johannesburg, south africa. raw and pasteurised milk were obtained from a nearby dairy farm. the milk (9 ml) was spiked with 1 ml of bacterial suspension prepared by suspending freshly grown b. abortus bv. 2 strain 2534/15 and b. ovis strain rc48 strains in pbs and adjusted to an initial concentration of 1 × 105 colony-forming units (cfu)/ml. the spiked milk was then diluted to 1:10, 1:100 and 1:1000 concentrations from which 0.1 ml/plate of each was spread and cultured on fm, mtm and cita media in triplicate. unspiked raw and pasteurised milk were also inoculated on all the three media as negative controls to monitor any contamination. table 1: compositions and enhancements of brucella selective media (farrell’s medium, modified thayer martin and cita) collated in the study. plates were incubated at 37 °c in a 5% – 10% carbon dioxide incubator and monitored every day for any bacterial or contaminant growth for up to 6 days, and the experiment was terminated because of high number of fungi and contaminants and thus too numerous to count (tntc). therefore, the composition of cita and mtm was slightly modified by introducing natamycin to reduce the growth of contaminants and referred to as modified modified thayer martin (mtm) and modified cita (mcita). although it was noted that the agar bases used in the media involved were different, they were not changed. that was because the main focus was to optimise the antifungals/antibacterials to reduce the growth of contaminants, which negatively affect the growth of brucella spp., which are slow-growing pathogens isolated from field or clinical samples that are heavily contaminated in most cases. the controlled experiment was repeated using mcita, mtm and fm (table 1), as described above. the number of suspect brucella cfu, contaminants cfu and contaminated plates because of fungi was recorded over 10 days of incubation at 37 °c in 5% – 10% carbon dioxide incubator. isolation of brucella from the diagnostic samples using modified agrifood research and technology center of aragon and farrell’s medium the performance of mcita medium was further validated under field condition in collaboration with arc-ovr, which uses fm for the isolation of brucella from field samples submitted for routine screening. based on the overall performance of mtm medium in the controlled experiment, it was not considered for use in the isolation of the bacteria from field samples. both media were simultaneously used to culture bovine tissue samples such as aborted foetuses (abomasal fluids, liver, spleen), lymph nodes, mammary glands, spleen, uterus, tonsils, as well as semen, hygromas and fluids. samples used were submitted between january and september 2017, mostly from gauteng and north west provinces of south africa. all the tissue samples were homogenised using mortar and pestle method and the equal sample amounts were cultured in duplicate on mcita and fm. plates were incubated as described above and monitored every 2 days from days 2 to 12 following the arc-ovr standard operating procedure (sop). milk from the seropositive animals sampled from two farms involved in the study was also cultured at the arc-ovr bacteriology laboratory and biotyped at arc-ovr. culture was considered positive when at least one cfu of brucella was isolated. the plates were examined every 2 days for suspect brucella cfu over the period of 12 days. any visible suspected brucella colonies were subcultured, typed and identified using standard microbiology procedures (oie 2016). briefly, brucella isolates were biotyped based on their colony morphology, reaction to oxidase, urease and catalase tests, production of hydrogen sulphide, agglutination on anti-brucella mono-specific sera abortus and melitensis, growth in the absence of carbon dioxide and growth in the presence of basic fuchsin and thionin dyes. moreover, lysis by different phages (tb, wb, fi and iz1) as well as inhibition by erythritol (1000 µg) and antibiotics (streptomycin 10 µg, penicillin g 10 units and rifampicin 30 µg) (tbilisi; weybridge; izatnagar1; berkeley2) (according to the oie terrestrial manual 2016) were also performed. brucella abortus bv. 1 culture was used as a control in all the phenotypic tests. data analysis the average (mean) and standard deviation of cfu/ml at each dilution inoculated on mcita, mtm and fm were determined on microsoft excel. statistical comparison of the means was performed using one-way analysis of variance (anova) with a fisher’s least significant difference (lsd) post hoc test in xlstat (statistical software for excel) version 2018.6. ethical considerations the experimental protocols were approved by the animal experiments and ethics committee of the university of pretoria (v096-15 aec approval) and the section 20 approval obtained from daff (sdah-epidem 15012613530_ section 20) for the use of animals and animal products. results comparison of three different media (farrell’s medium, modified thayer martin and agrifood research and technology center of aragon) in a controlled experiment growth was observed from day 2 on the mtm plates inoculated with raw spiked and unspiked milk, but none of the colonies were suspected to be brucella. tiny suspect brucella colonies were observed from day 3 on all mtm, fm and cita media plates inoculated with 1:10 dilution suspension. irrespective of a high number of contaminants on mtm plates, suspect brucella colonies grew on this medium at a lower rate (figure 1; figure 1-a1). brucella ovis displayed poor growth rate on fm compared with b. abortus bv. 2 (figure 1). on cita, both strains grew at a lower rate as well; however, the number of contaminants observed was lower than those on mtm (figure 1). the experiment was terminated on day 6 because of overgrowth of fungi on most plates inoculated with raw milk, thus making it impossible to count and differentiate brucella-specific bacterial colonies. figure 1: total colony-forming unit per millilitre (colony-forming unit/ml of both brucella and contaminants) count over a period of 6 days of inoculated milk samples with brucella abortus biovar 2, brucella ovis and unspiked. the colony-forming units are the average at day 6 of the 0.1 ml of 1:10, 1:100 and 1:1000 dilutions of pasteurised and raw milk spiked with brucella abortus bv. 2 strain 2534/15 or brucella ovis strain rc48 as well as unspiked pasteurised and raw milk inoculated on fm, modified thayer martin and cita media after being incubated at 37 °c in 5% – 10% co2. the experiment was terminated because of overgrowth of fungi on most plates inoculated with raw milk, thus making it impossible to count brucella-specific bacterial colonies. comparison using farrell’s media and the optimised modified thayer martin and modified agrifood research and technology center of aragon cita and mtm were modified by introducing natamycin (20 mg/l) (table 1) to enhance the performance of both media, especially decreasing the growth rate of fungal and bacterial contaminants on both media, as they make it difficult to isolate slow-growing brucella colonies. using the mcita, fm and mtm, all the control plates inoculated with unspiked pasteurised milk did not show any growth throughout the experiment (table 1-a1). modified mtm medium still showed a higher number of contaminants as the initial experiment before optimisation; however, there was an improvement with mcita, and a few contaminants were recorded as shown in table 1-a1. the performance of fm was consistent throughout the study, and the only setback with this medium was the inhibition of b. ovis, as shown in previous studies as well. in terms of brucella growth, suspect colonies were visible from day 2 on the mcita and fm as compared with day 3 with mtm, which had the highest bacterial growth rate but mostly being contaminants (table 1-a1). the cfu mean comparison showed that the sensitivity of all the media used was comparable for the isolation of b. abortus bv. 2 in pasteurised and raw milk, with the mean difference ranging from 0 to 2.33 (figure 2; table 2-a1). moreover, cfu mean ± standard deviation (sd) of b. ovis strain rc48 was generally lower in fm, irrespective of the milk type (1.3 ± 1.5 in pasteurised and 0.33 ± 0.57 in raw milk), which resulted in high mean difference when compared with mcita (10.33) and mtm (12) (figure 2; table 2-a1). however, both mcita and mtm media showed a mean difference of 0.67 and 1.67 when, respectively, using pasteurised and raw milk spiked with b. ovis. figure 2: comparison of the colony-forming unit/ml (mean) from all the plates of modified cita, modified thayer martin and farrell’s medium inoculated with pasteurised or raw milk spiked with brucella abortus biovar 2 strain 2534/15 and brucella ovis strain rc48; performed using one-way analysis of variance with a fisher’s least significant difference. testing modified cita and farrell’s medium on diagnostic samples the mcita was further investigated in collaboration with arc-ovr bacteriology section using samples received for diagnostic screening. different sample types (table 2) were inoculated on fm and mcita and the plates were, respectively, observed on days 2, 4, 6, 8 and 10 for any bacterial growth. suspect, tiny brucella colonies were visible from as early as day 2 on both media. suspect brucella colonies growing on mcita did not grow when transferred or subcultured on fm. the performance rate of both media did not vary and the incubation period was similar; however, b. ovis grew very well on mcita as compared with fm, as reported in previous studies. farrell’s medium inhibited the growth of most contaminants as compared with mcita, irrespective of the inhibition of b. ovis. all suspect isolates were subcultured, typed and identified as b abortus bv. 1, b. abortus bv. 2, b. abortus vaccine s19 and b. ovis (table 2). table 2: list of bovine diagnostic samples, type as well as biotyping identity and the days when suspect brucella colonies of each sample were initially observed on both cita with modifications and farrell’s medium. discussion isolation of brucella bacterium is still considered the gold standard in the diagnosis of the disease because it is the only test allowing a definite diagnosis presently; however, the availability of tests that allows direct isolation of deoxyribonucleic acid (dna) from tissue and liquid specimens is considered as a convenient alternative. in this study, we conducted a controlled experiment to determine a selective media that will allow a justifiable performance in the isolation of all brucella spp. it was shown that cita medium allows the growth of majority of brucella spp. including the fastidious b. abortus bv. 2 and b. ovis, whereas the growth of the latter species was suppressed in fm. slight modification of cita medium (mcita) was necessary because of the observed low inhibition rate of contaminants, especially on plates inoculated with raw milk. a higher isolation rate for b. abortus bv. 2 and b. ovis was observed with mcita compared with fm. amphotericin b and nystatin were used in cita at a respective concentration of 4 and 17.7 mg/l, to control fungal infection (de miguel et al. 2011). however, this concentration could not be increased because of the risk of toxicity that may inhibit the growth of brucella species, especially b. abortus bv. 2 and 4 (farrell & robertson 1967). thus, natamycin was introduced as an alternative supplementary fungal inhibitor because there is no reported synergy between this antifungal and the other two (amphotericin b and nystatin) used in the media, as shown in previous studies (ghannoum & rice 1999; te welscher et al. 2008, welscher 2012). natamycin, a polyene antifungal antibiotic produced by streptomyces natalensis, is highly active against various fungi and yeasts such as histoplasma capsulatum, aspergillus niger, candida albicans and others (struyk et al. 1957–1958). it is used as fungi growth inhibitor because it is able to bind specifically to ergosterol, thus inhibiting the functioning of the membrane transport protein (te welscher et al. 2008, 2012). nystatin, filipin and amphotericin b disrupt the permeability of the membrane and the cytoplasmic features of the pathogen by interacting with the steroids available in the membrane (de miguel et al. 2011; te welscher et al. 2008). in this study, the inhibition of fungal growth by natamycin was shown by the performance of fm, which inhibited the growth of most fungal contaminants throughout the study in spite of the medium inhibiting the growth of b. ovis as well. previous studies have shown that amphotericin b and natamycin have different modes of action even though they both belong to the macrolide polyene class of antibiotics (ciesielski et al. 2016; lalitha et al. 2010). furthermore, te welscher et al. (2008) also reported that natamycin, filipin and nystatin have different mode of action as well. based on their findings, we suggested that it may be the reason why the use of nystatin, natamycin and amphotericin b together in one media did not show any negative effect on bacterial growth in this study. natamycin showed very high minimum inhibiting concentration (mic) against most bacteria as compared with fungi because the sterol-lacking membrane available in bacteria enables them to tolerate this antibiotic (de boer 1988). furthermore, ghannoum and rice (1999) indicated that there is a significant variation between the structures of the bacteria and fungi; thus, antifungals and antibacterials target the features or functions of ergosterol and the contaminant fungal organisms, respectively. in a report by oostendorp (1981), it was indicated that natamycin lacks acute toxicity and the minimum ld50 was found to be 2.5 mg/kg – 4.5 mg/kg in animal studies. adding natamycin to mtm and cita made a crucial difference because most contaminants were inhibited as compared with when using the original media, even though cita medium performed much better than mtm. in spite of the slight modification, findings in our study correspond with previous studies (de miguel et al. 2011; de nardi júnior et al. 2015; vicente et al. 2014), where the medium also displayed higher efficacy in supporting the growth of brucella spp. as compared with other media used in those studies. the varying performance of cita observed in this study and previous reports might be because of different field conditions between the countries tested and south africa (de miguel et al. 2011; de nardi júnior et al. 2015; vicente et al. 2014), thus indicating the necessity of optimising the media in the diagnosis of brucellosis while using local samples. the use of a selective medium that could suppress fungal, yeast and bacterial contaminant growth has been previously reported to improve isolation of brucella spp. (de miguel et al. 2011; farrell 1974; marin et al. 1996). farrell’s medium is evidently the most used medium for the isolation of brucella spp. however, the concentration of nalidixic acid and bacitracin in this medium inhibits the growth of other brucella spp. like b. ovis, b. abortus bv. 2 and other biotypes of b. melitensis (marin et al. 1996). therefore, the oie recommends the use of two media preferably fm and mtm simultaneously to overcome the setback effected by these antibiotics. this setback led to the development of various selective media ever since fm was first reported (de miguel et al. 2011; ewalt et al. 1983; hornsby et al. 2000). cita is one of the developed media (de miguel et al. 2011), and it is well recommended for isolation of brucella spp. by the oie (2016) because of the reliable efficiency reported in several studies (de miguel et al. 2011; vicente et al. 2014). the use of b. abortus bv. 2 and b. ovis strain in the controlled experiment was because of their fastidious growth and reports from previous studies, suggesting that a selective medium optimal for the growth of these species will also allow the growth of less fastidious brucella spp. (corner & alton 1982). moreover, isolation of brucella from various sample types and different field conditions during validation of the optimised cita indicated that the media can be used for isolation of brucella from a wide range of sample types and environmental conditions. recent reports indicated that pathogens express most of their genes based on the environmental and distinct conditions as well as the accessible nutritional sources (wareth, melzer & neubauer 2017). the above-mentioned authors also suspect that pathogens can undergo some mutations to adapt to the distinct pressure in the laboratory, irrespective of their natural environment encounters, but this needs to be further investigated. raw milk serves as a favourable environment and source of nutrients for majority of fungal and commensal bacteria species (delavenne et al. 2011), negatively affecting the isolation of the slower-growing brucella species. as indicated in the results, the growth rate of both b. abortus bv. 2 and b. ovis in raw milk was lower than pasteurised milk in all the three media. this corresponds with a previous study by falenski et al. (2011), which indicated that the initial concentration of 5 × 107 cfu/ml of b. abortus 1119-3 added to raw milk dropped to 2 × 107 cfu/ml within 4 days in contrast to when added to ultra high temperature (uht)-milk, which showed an increased range of 1.5 × 108 – 7.2 × 108 cfu/ml from 2 to 46 days, respectively. in spite of the evidence that brucella pathogens are difficult to grow and time-consuming because of the phenotypic processes involved in biotyping, isolation of these bacteria is still observed as the gold standard in the diagnosis of brucellosis (oie 2016). in addition, it is not certain whether brucella growth will be obtained during culturing in spite of its specificity and even if the sample is from a known positive animal with a chronic infection that does not shed the bacteria (capparelli et al. 2009); thus, in this study, we used different sample types from one animal to improve isolation as well to avoid missing any possible co-infection. alton et al. (1988) indicated that a standard basal media could also be used to isolate the majority of brucella spp. nonetheless, this is not practical for the initial isolation of brucellae from field clinical samples. this may be because of various factors like sample collection, transportation, storage, available expertise, availability of viable bacteria in samples and so on (corner & alton 1982). based on the sample type as well as the distance between the collection site and the laboratory, it is of utmost importance to store and transport the samples send for routine screening/diagnosis properly to avoid contamination or sample spoilage (alton et al. 1988). brucella isolation requires expertise and it is laborious; hence, it is necessary to further investigate any suspect colony observed on each plate with staining methods to avoid misdiagnosing the bacteria (oie 2016). staining methods are inexpensive, rapid and user-friendly tests used for the presumptive identification of brucella suspect colonies. however, other gram-negative bacteria like coxiella burnetii and chlamydia psittaci can be morphologically equivalent to brucella spp. on the most commonly used modified ziehl-neelsen staining (poester et al. 2010; stamp et al. 1950). tilak et al. (2016) also reported misidentification of five brevundimonas diminuta isolates as brucella spp. using stamp’s modified ziehl-neelsen staining because they also stained pink against the blue background. typically, brucella can be easily detected in endemic areas as compared with areas with low infection rate where some of the cases might be overlooked (jimenez de bagues et al. 1991). therefore, the use of an efficient and reliable medium can overcome the disadvantages encountered with the various sample types and their conditions. in this study, cita with slight modification has proven to be more sensitive and regarded as the medium of choice in the isolation of all brucella spp., as indicated in previous studies. even though the optimisation and modification done on the formulation of cita medium were not major, the efficacy of this medium was improved when culturing field and diagnostic samples in this study. thus, the use of this medium, or in conjunction with fm, may improve the overall isolation of these intracellular, fastidious and slow-growing pathogens. acknowledgements the authors thank the gauteng department of agriculture and rural development (gdard), national research foundation (nrf), agriseta and the university of pretoria for the support and funding of the study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.b.l. performed the experiments, analysed the data and wrote the article. m.i. and k.a. provided resources and samples at arc-ovr, while n.o.c. and h.v.h. are study leaders. funding information this work was supported financially by the gauteng department of agriculture and rural development (gdard), national research foundation (nrf), cdc, agriseta and the university of pretoria. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the 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infection’, international journal of systematic and evolutionary microbiology 60(pt 4), 801–808. https://doi.org/10.1099/ijs.0.011148-0 scholz, h.c. & vergnaud, g., 2013, ‘molecular characterisation of brucella species’, revue scientifique et technique (international office of epizootics) 32(1), 1. https://doi.org/10.20506/rst.32.1.2189 scholz, h.c., revilla-fernandez, s., al dahouk, s., hammerl, j.a., zygmunt, m.s., cloeckaert, a. et al., 2016, ‘brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (vulpes vulpes)’, international journal of systematic and evolutionary microbiology 66(5), 2090–2098. https://doi.org/10.1099/ijsem.0.000998 seimenis, a.m., 2008, ‘the spread of zoonoses and other infectious diseases through the international trade of animals and animal products’, veterinaria italiana 44(4), 591–599. https://www.izs.it/vet_italiana. stamp, j.t., mcewen, a.d., watt, j.a.a. & nisbet, d.i., 1950, ‘enzootic abortion in ewes: trans-mission of the disease’, veterinary record 62(17), 251–254. https://doi.org/10.1136/vr.62.17.251 struyk, a.p., hoette, i., drost, g., waisvisz, j.m., van eek, t. & hoogerheide, j.c., 1957–1958, ‘pimaricin, a new antifungal antibiotic’, antibiotics annual 5, 878–885. pmid: 13521908. te welscher, y.m., ten napel, h.h., balagué, m.m., souza, c.m., riezman, h., de kruijff, b. et al., 2008, ‘natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane’, the journal of biological chemistry 283(10), 6393–6401. https://doi.org/10.1074/jbc.m707821200 te welscher, y.m., van leeuwen, m.r., de kruijff, b., dijksterhuis, j. & breukink, e., 2012, ‘polyene antibiotic that inhibits membrane transport proteins’, proceedings of the national academy of sciences of the united states of america 109(28), 11156–11159. https://doi.org/10.1073/pnas.1203375109 thayer, j.d. & martin, j.e., 1964, ‘a selective medium for the cultivation of n. gonorrhoeae and n. meningitidis’, public health reports 79, 49–57. https://doi.org/10.2307/4592051 thayer, j.d. & martin, j.e., 1966, ‘improved medium selective for cultivation of n. gonorrhoeae and n. meningitidis’, public health reports 81(6), 559–562. https://doi.org/10.2307/4592771 tilak, k., eshwara, v.k., tellapragada, c. & mukhopadhyay, c., 2016, ‘stamp’s modified ziehl–neelsen staining for brucella: beware of the first impressions’, indian journal of medical microbiology 34(4), 561–562. https://doi.org/10.4103/0255-0857.195363 vicente, a.f., antunes, j.m.a.p., lara, g.h.b., mioni, m.s.r., allendorf, s.d., peres, m.g. et al., 2014, ‘evaluation of three formulations of culture media for isolation of brucella spp. regarding their ability to inhibit the growth of contaminating organisms’, biomed research international 2014, 702072. https://doi.org/10.1155/2014/702072 wareth, g., melzer, f. & neubauer, h., 2017, ‘in brucella: selective pressure may turn some genes on instead of default off position’, medical hypothesis 130, 29–31. https://doi.org/10.1016/j.mehy.2017.04.006 whatmore, a.m., davison, n., cloeckaert, a., al dahouk, s., zygmunt, m.s., brew, s.d. et al., 2014, ‘brucella pappi sp. nov. isolated from baboons (papio spp.)’, international journal of systematic and evolutionary microbiology 64(pt 12), 4120–4128. https://doi.org/10.1099/ijs.0.065482-0 appendix 1 table 1-a1: total colony-forming unit per plate (cfu/plate) for 1:10 dilution of all three media over a period of 12 days. ojvr_v89_i1_contents.indd http://www.ojvr.org open access table of contents original research antibody response to raboral vr-g® oral rabies vaccine in captive and free-ranging black-backed jackals (canis mesomelas) katja n. koeppel, peter geertsma, brian f. kuhn, ockert l. van schalkwyk, peter n. thompson onderstepoort journal of veterinary research | vol 89, no 1 | a1975 | 10 february 2022 original research protective effects of methanolic leaf extracts of monanthotaxis caffra against aflatoxin b1-induced hepatotoxicity in rats rhulani makhuvele, kenn foubert, nina hermans, luc pieters, luc verschaeve, esam elgorashi onderstepoort journal of veterinary research | vol 89, no 1 | a1968 | 23 march 2022 original research effect of cytochrome p450 inhibition on toxicity of diclofenac in chickens: unravelling toxicity in gyps vultures sara locke, vinny naidoo, ibrahim hassan, neil duncan onderstepoort journal of veterinary research | vol 89, no 1 | a1978 | 14 june 2022 original research species identification and cow risks of non-aureus staphylococci from south african dairy herds inge-marie petzer, christiaan labuschagne, lufuno phophi, joanne karzis onderstepoort journal of veterinary research | vol 89, no 1 | a2021 | 27 july 2022 original research barriers to vaccine use in small ruminants and poultry in tanzania sitira williams, isabella endacott, abel b. ekiri, mirende kichuki, mariana dineva, erika galipo, vadim alexeenko, ruth alafiatayo, erik mijten, gabriel varga, alasdair j.c. cook onderstepoort journal of veterinary research | vol 89, no 1 | a2007 | 04 august 2022 original research seroprevalence of infectious bronchitis virus and avian reovirus in free backyard chickens sonia c. pinto, jescka aleixo, kleidy camela, abel g. chilundo, custódio g. bila onderstepoort journal of veterinary research | vol 89, no 1 | a2042 | 11 november 2022 original research propagation of avian influenza virus in embryonated ostrich eggs agnes t. laleye, modupeore adeyemi, celia abolnik onderstepoort journal of veterinary research | vol 89, no 1 | a2011 | 08 december 2022 reviewer acknowledgement onderstepoort journal of veterinary research | vol 89, no 1 | a2086 | 19 december 2022 56 65 71 79 89 100 104 110 page i of i table of contents review article prevalence of antibiotic-resistant bacteria amongst dogs in africa: a meta-analysis review ayaovi b. yaovi, philippe sessou, aretas b.n. tonouhewa, gildas y.m. hounmanou, deborah thomson, roger pelle, souaïbou farougou, arindam mitra onderstepoort journal of veterinary research | vol 89, no 1 | a1970 | 10 october 2022 original research comparing the minimum inhibitory and mutant prevention concentrations of selected antibiotics against animal isolates of pasteurella multocida and salmonella typhimurium jeanette m. wentzel, louise j. biggs, moritz van vuuren onderstepoort journal of veterinary research | vol 89, no 1 | a1955 | 10 january 2022 original research characterisation and antibiotic resistance of yersinia enterocolitica from various meat categories, south africa emmanuel m. seakamela, letlhogonolo diseko, dikeledi malatji, lavhelesani makhado, mmatau motau, kudakwashe jambwa, kudakwashe magwedere, nombasa ntushelo, itumeleng matle onderstepoort journal of veterinary research | vol 89, no 1 | a2006 | 07 november 2022 original research physical, chemical and microbiological assessments of drinking water of small-layer farms eufrásia augusto, jescka aleixo, florentina d. chilala, abel g. chilundo, benígna gaspar, custódio g. bila onderstepoort journal of veterinary research | vol 89, no 1 | a2067 | 28 november 2022 original research food animals as reservoirs and potential sources of multidrug-resistant diarrheagenic e. coli pathotypes: focus on intensive pig farming in south africa shima e. abdalla, akebe l.k. abia, daniel g. amoako, keith perrett, linda a. bester, sabiha y. essack onderstepoort journal of veterinary research | vol 89, no 1 | a1963 | 20 january 2022 original research human and animal fasciolosis: coprological survey in narok, baringo and kisumu counties, kenya cornelius k. kipyegen, charles i. muleke, elick o. otachi onderstepoort journal of veterinary research | vol 89, no 1 | a1954 | 31 january 2022 1 13 20 31 37 50 vol 89, no 1 (2022) issn: 0030-2465 (print) | issn: 2219-0635 (online)onderstepoort journal of veterinary research authors’ contributions about the author(s) gerda fouche council for scientific and industrial research (csir) biosciences, pretoria, south africa bellonah m. sakong department of paraclinical sciences, university of pretoria, south africa olubukola t. adenubi department of paraclinical sciences, university of pretoria, south africa jean paul dzoyem department of paraclinical sciences, university of pretoria, south africa vinny naidoo biomedical research center, university of pretoria, south africa tlabo leboho council for scientific and industrial research (csir) biosciences, pretoria, south africa mbokota c. khosa agricultural research council – tropical and subtropical crops, nelspruit, south africa kevin w. wellington council for scientific and industrial research (csir) biosciences, pretoria, south africa jacobus n. eloff department of paraclinical sciences, university of pretoria, south africa citation fouche, g., sakong, b.m., adenubi, o.t., dzoyem, j.p., naidoo, v., leboho, t. et al., 2021, ‘corrigendum: investigation of the acaricidal activity of the acetone and ethanol extracts of 12 south african plants against the adult ticks of rhipicephalus turanicus’, onderstepoort journal of veterinary research 88(1), a1950. https://doi.org/10.4102/ojvr.v88i1.1950 note: doi of original article published: https://doi.org/10.4102/ojvr.v84i1.1523 correction corrigendum: investigation of the acaricidal activity of the acetone and ethanol extracts of 12 south african plants against the adult ticks of rhipicephalus turanicus gerda fouche, bellonah m. sakong, olubukola t. adenubi, jean paul dzoyem, vinny naidoo, tlabo leboho, mbokota c. khosa, kevin w. wellington, jacobus n. eloff published: 27 july 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. in the published version of this article, fouche, g., sakong, b.m., adenubi, o.t., dzoyem, j.p., naidoo, v., leboho, t. et al., 2017, ‘investigation of the acaricidal activity of the acetone and ethanol extracts of 12 south african plants against the adult ticks of rhipicephalus turanicus’, onderstepoort journal of veterinary research 84(1), a1523. https://doi.org/10.4102/ojvr.v84i1.1523, the seventh author, mbokota c. khosa, was omitted from the ‘authors’ and ‘affiliations’ sections. the indicated author should be added as the seventh author, and the following affiliation should be added as his affiliation: agricultural research council – tropical and subtropical crops, nelspruit, south africa. the authors’ contributions section is hereby update to: authors’ contributions g.f. conceptualised the study. m.c.k. was involved in the collection of some of the plant material and in the preparation of the extracts used in the biological screening assays. g.f., k.w.w. and t.l. carried out the literature search and plant selection. t.l. prepared the plant extracts. j.n.e. conceptualised the study in a joint application and supervised the students and postdoctoral fellows. v.n. supervised determination of acaricidal activity. j.p.d. supervised the determination of cytotoxicity. b.m.s. determined vero cell toxicity. m.c.k. was also involved in the fractionation and isolation process in the natural product chemistry laboratory. o.t.a. determined the acaricidal activity against adult ticks of r. turanicus. k.w.w. wrote the first draft of the manuscript. salih_319-326.qxd introduction eleven genera of ticks and 63 tick species infesting a variety of animal species, including birds and reptiles, have been identified in the sudan (hoogstraal 1956). according to hoogstraal (1956), karrar, kaiser & hoogstraal (1963) and osman, el hussein, neima & abdulla (1982) ticks infesting livestock in the sudan are mainly hyalomma anatolicum anatolicum, hyalomma dromedarii, hyalomma marginatum rufipes, hyalomma impressum, hyalomma impeltatum, hyalomma truncatum, rhipicephalus evertsi evertsi, rhipicephalus sanguineus group, rhipicephalus simus group, rhipicephalus appendiculatus, boophilus decoloratus, boophilus annulatus, amblyomma lepidum and amblyomma variegatum. although different species of ticks and the diseases they transmit occur in different ecological regions of 319 onderstepoort journal of veterinary research, 71:319–326 (2004) preliminary survey of ticks (acari: ixodidae) on cattle in northern sudan d.a. salih1*, s.m. hassan2, a.m. el hussein1 and f. jongejan3 abstract salih, d.a., hassan, s.m., el hussein, a.m. & jongejan, f. 2004. preliminary survey of ticks (acari: ixodidae) on cattle in northern sudan. onderstepoort journal of veterinary research, 71: 319–326 in a cross sectional survey conducted during the period june 2001 to july 2002, the geographical distribution of ticks on cattle in the sudan was determined. seventeen locations were surveyed from northern, central, eastern, western, blue nile and white nile provinces. total body collections of ticks were made from 20 cattle at each location. four tick genera and 11 species were identified. the tick species collected included amblyomma lepidum, amblyomma variegatum, boophilus decoloratus, hyalomma anatolicum anatolicum, hyalomma dromedarii, hyalomma impeltatum, hyalomma marginatum rufipes, hyalomma truncatum, rhipicephalus evertsi evertsi, rhipicephalus sanguineus group and rhipicephalus simus simus. major ecological changes have occurred due to extensive animal movement, deforestation, desertification and establishment of large mechanized agricultural schemes. these factors have certainly affected the distribution of ticks and tick-borne diseases in the sudan. the absence of a. variegatum and a. lepidum in northern sudan was not surprising, since these tick species are known to survive in humid areas and not in the desert and semi-desert areas of northern sudan. the absence of b. annulatus in northern and central sudan is in accordance with the finding that this tick species is restricted to the southern parts of the central sudan. the presence of h. anatolicum anatolicum in um benin in relatively high abundance is an interesting finding. the present finding may indicate that the southern limit of this species has changed and moved southwards to latitude 13° n. it is concluded that major changes in tick distribution have taken place in the sudan. keywords: cattle, preliminary survey, sudan, ticks * author to whom correspondence is to be directed. e-mail: diaeldin2000@hotmail.com 1 central veterinary research laboratories, al amarat, p.o. box 8067, khartoum, sudan 2 department of parasitology, faculty of veterinary medicine, university of khartoum, p.o. box 32 khartoum north, sudan 3 utrecht university, faculty of veterinary medicine, division of parasitology and tropical veterinary medicine, p.o. box 80165, 3508td utrecht, the netherlands, and department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa accepted for publication 11 june 2004—editor the world, their impact on animal production is similar in nature and importance (fao 1984). despite considerable progress made in combating ticks and tick-borne diseases, the latter seem to be increasing both in prevalence and severity. tick-borne diseases are responsible for hundreds of million us dollar losses per year in tropical and temperate areas where they pose a problem. in the sudan no accurate economic evaluations have been made. however, economic losses in khartoum state alone due to tropical theileriosis have been estimated to be between 4 and 6 million us$ (latif 1994). this cross-sectional survey was designed to provide additional data on the current distribution of major tick species associated with cattle in northern sudan. materials and methods study area the study area extended from the latitudes 11°78’– 19°61’ n and longitudes 22°45’–37°2’ e; this area is desert in the north and savannah in the south. ticks were collected from cattle in atbara and el damer in the northern province and from khartoum, central province. in the blue nile province, the locations selected were madani, sennar, um benin and damazin and in the white nile province, el dieum, rabak and kosti. el obied, nyala, fasher and genina represented the western province. from the eastern province localities in gadarif, kassala and port sudan were surveyed (fig. 1). collection of ticks at each location, ticks were collected from cattle on at least four farms that were kept separate. on each farm indigenous (zebu) and cross (indigenous × friesian) bred animals were selected. the age groups of cattle on each farm sampled were: those less than 1 year, 1–2 years, 2–3 years, 3–4 years and more than 4 years. cattle sampled were of different sexes and they were maintained under three managemental systems: intensive (zero grazing), extensive (out-grazing) and semi-intensive. total body collections of ticks were made from cattle using a pair of blunt forceps. prior to collection, each animal was cast and restrained. the ticks collected were kept separately according to body site in labelled universal bottles containing 70 % ethanol. on each label geographical information was recorded indicating location, animal number, site of 320 preliminary survey of ticks (acari: ixodidae) on cattle in northern sudan ����� ��� � � ��������� � ���� � ������� � �� ������� ������ � � ���� � ������ � ������ ������ ��� ����� � ����� ������ ������ ������ � � � � ������ � !"" #�$"" �� %������� &����� ����'�� ����� ������( fig. 1 map of the sudan showing localities where samples were collected the body and date of collection. in the laboratory the ticks were identified according to hoogstraal (1956), hoogstraal & kaiser (1959) and okello-onen, hassan & essuman (1999). statistical analysis data collected on the ticks associated with cattle in the various localities were subjected to the appropriate general linear model (glm) procedure of statistical analysis using the statistical analysis system (sas) package. the sas was used to perform analysis of variance (anova) while mean separations were performed using the ryan-einot-gabrielwelsch (regw) multiple range test (day & quinn 1989). results four genera and 11 species of ticks were identified during the survey period. the tick species were a. lepidum, a. variegatum, b. decoloratus, h. anatolicum anatolicum, h. dromedarii, h. impeltatum, h. marginatum rufipes, h. truncatum, r. evertsi evertsi, r. sanguineus group and r. simus simus. most of the ticks were found in the upper perineum (i.e. from the base of tail, around the anus and around the vulva of female animals). the anova, mean squares, r-squares, coefficient of variance and f values are shown in table 1. in general, the mean number of ticks per head of cattle was relatively low, ranging from (5.8 ± 0.63) in port sudan to (19.6 ± 2.25) in khartoum (table 2). with the exception of boophilus spp., males outnumbered females of all tick species. there were no significant differences (p > 0.05) between mean number of ticks per head of cattle in the zero grazing, out-grazing and semi-intensive systems. data on distribution, highest and lowest mean, and abundance are given separately for each species as follows: amblyomma lepidum this east african bont tick species was found in all locations, with the exception of atbara, el damer and khartoum and um benin. it was recorded along the white nile from el dieum to rabak. in western province this tick species was prevalent in all locations, while in eastern province it was recorded in gadarif and port sudan, but not in kassala. the highest mean total body collection of ticks was recorded in gadarif (3.3 ± 0.87), while the lowest mean was recorded in el dieum (0.4 ± 0.22)(table 2). there was a significant difference (p < 0.05) between the mean number of ticks collected in gadarif, madani, el obied and damazin in comparison with other locations. but there were no significant differences (p > 0.05) between the numbers of ticks collected in madani, el obied and damazin (table 2). amblyomma variegatum the tropical bont tick was only found in kosti, el obied and nyala. the highest mean total body count of ticks was recorded in el obied (1.2 ± 0.42), while the lowest mean was recorded in nyala (0.5 ± 0.22) (table 2). there was a significant difference (p < 0.05) in the mean total body count of ticks in el obied in comparison with nyala and kosti, while there was no significant difference (p > 0.05) between the counts in nyala and kosti (table 2). boophilus decoloratus the blue tick was only recorded in madani and rabak. there was no significant difference (p > 0.05) between the mean total body count of ticks in madani (1.6 ± 0.311) and in rabak (1.5 ± 0.0.34) (table 2). hyalomma anatolicum anatolicum this xerophilic tick was the most abundant species in atbara, el damer and khartoum. in port sudan it was only collected from a single host. the highest mean was recorded in khartoum (6.6 ± 1.17) while the lowest mean was in port sudan (1.1 ± 0.28) (table 2). there was a significant difference (p < 0.05) between the mean total body count of ticks in khartoum in comparison with other locations. however, there was no significant difference (p > 0.05) between the mean number of ticks collected from khartoum compared to atbara, el damer and madani (table 2). there was also no significant difference (p > 0.05) between the mean numbers of ticks collected in sennar, um benin, kosti and kassala (table 2). hyalomma dromedarii the camel tick was collected in all locations with the exception of kosti and kassala. the highest mean total body count of ticks was recorded in el damer (3.6 ± 0.97), while the lowest mean was recorded in damazin and fasher (0.5 ± 0.22) (table 2). there was a significant difference (p < 0.05) between the mean numbers of ticks collected in el damer in comparison with other locations. on the other hand, 321 d.a. salih et al. t a b l e 2 m e a n s (± s e ) o f to ta l b o d y co lle ct io n o f t ic ks f ro m c a tt le in d iff e re n t lo ca lit ie s in t h e s u d a n d u ri n g t h e y e a rs 2 0 0 1 a n d 2 0 0 2 * l o ca tio n s a . le p id u m a . va ri e g a tu m b . d e co lo ra tu s h . a . a n a to lic u m h . d ro m e d a ri i h . im p e lta tu m h . m . ru fip e s h . tr u n ca tu m r . e . e ve rt si r . s. s im u s r . sa n g u in e u s g ro u p t o ta l a tb a ra 0 d 0 b 0 b 4 .5 ± 1 .3 1 a b c 2 .7 ± 0 .4 9 a b c 2 .1 ± 0 .4 5 a 4 .1 ± 0 .7 a 0 c 4 .0 ± 0 .8 6 b 0 b 2 .7 ± 0 .5 1 a b c 2 0 .1 ± 1 .8 2 a e l d a m e r 0 d 0 b 0 b 5 .2 ± 1 .3 2 a b 3 .6 ± 0 .9 7 a 0 c 5 .7 ± 0 .5 2 a 0 c 2 .0 ± 0 .5 2 b cd e 0 b 3 .0 ± 0 .5 9 a b c 1 9 .5 ± 2 .3 2 a b k h a rt o u m 0 d 0 b 0 b 6 .6 ± 1 .1 7 a 2 .9 ± 0 .9 8 a b c 0 c 2 .8 ± 0 .6 1 a 0 c 2 .3 ± 0 .6 8 b cd e 0 b 5 .0 ± 0 .8 6 a 1 9 .6 ± 2 .2 5 a b m a d a n i 2 .0 ± 0 .4 4 a b c 0 b 1 .6 ± 0 .3 1 a 5 .2 ± 1 .5 a b 1 .2 ± 0 .3 9 a b cd 0 c 4 .9 ± 0 .6 7 a 0 c 1 .1 ± 0 .3 1 cd e 0 b 1 .1 ± 0 .2 7 cd 1 7 .1 ± 1 .9 a b s e n n a r 0 .8 ± 0 .4 1 cd 0 b 0 b 2 .0 ± 0 .3 3 b cd 1 .3 ± 0 .2 6 a b cd 0 c 2 .4 ± 0 .4 a 0 c 0 .7 ± 0 .3 3 e d 0 b 0 d 7 .2 ± 0 .9 6 b e f u m b e n in 0 d 0 b 0 b 2 .4 ± 0 .7 b cd 1 .7 ± 0 .2 1 a b cd 0 c 4 .8 ± 0 .7 1 a 0 c 0 e 0 b 1 .2 ± 0 .2 cd 1 0 .1 ± 1 .2 3 b cd e f d a m a zi n 2 .8 ± 0 .4 7 a b 0 b 0 b 0 d 0 .5 ± 0 .2 2 cd 0 c 5 .6 ± 0 .8 7 a 0 c 3 .8 ± 0 .8 1 b c 0 b 1 .6 ± 0 .3 b cd 1 4 .3 ± 1 .4 3 a b cd g a d a ri f 3 .3 ± 0 .8 7 a 0 b 0 b 0 d 1 .3 ± 0 .1 5 a b cd 0 c 4 .2 ± 0 .5 7 a 0 c 3 .4 ± 1 .3 6 b cd 0 b 0 .7 ± 0 .3 3 cd 1 2 .9 ± 2 .3 a b cd e k a ss a la 0 d 0 b 0 b 2 .1 ± 0 .5 3 b cd 0 d 0 c 4 .0 ± 0 .7 9 a 0 c 1 .7 ± 0 .6 3 b cd e 0 b 3 .8 ± 0 .9 8 a b 1 1 .6 ± 1 .3 4 a b cd e f p o rt s u d a n 0 .6 ± 0 .2 2 cd 0 b 0 b 1 .1 ± 0 .2 8 d 0 .7 ± 0 .2 1 b cd 0 c 2 .7 ± 0 .5 6 a 0 c 0 .7 ± 0 .2 6 e d 0 b 0 d 5 .8 ± 0 .6 3 f e l d ie u m 0 .4 ± 0 .2 2 cd 0 b 0 b 1 .4 ± 0 .3 4 cd 1 .0 3 ± 0 .3 a b cd 0 .8 ± 0 .2 5 b c 3 .6 ± 0 .8 5 a 0 .4 ± 0 .1 6 c 0 e 0 .9 ± 0 .3 5 a 1 .6 ± 0 .4 2 b cd 1 0 .4 ± 1 .4 8 b cd e f r a b a k 1 .3 ± 0 .3 7 b cd 0 b 1 .5 ± 0 .3 4 a 1 .3 ± 0 .5 8 cd 3 .0 ± 0 .8 8 a b 0 c 5 .1 ± 0 .7 5 a 0 c 0 e 0 b 2 .4 ± 1 .0 2 b cd 1 4 .6 ± 1 .4 5 a b c k o st i 0 .7 ± 0 .2 1 cd 0 .7 ± 0 .4 2 a b 0 b 1 .9 ± 0 .4 6 b cd 0 d 1 .3 ± 0 .4 7 a b 3 .4 ± 0 .5 4 a 1 .9 ± 0 .4 8 a b 0 .4 ± 0 .2 2 e 0 b 1 .0 ± 0 .2 9 cd 1 1 .3 ± 1 .5 1 a b cd e f e l o b ie d 1 .9 ± 0 .4 3 a b c 1 .2 ± 0 .4 2 a 0 b 0 d 2 .1 ± 0 .5 2 a b cd 0 c 2 .8 ± 0 .5 1 a 0 c 6 .7 ± 0 .7 3 a 0 b 0 d 1 4 .7 ± 1 .3 4 a b c n ya la 0 .7 ± 0 .4 2 cd 0 .5 ± 0 .2 2 b 0 b 0 d 1 .0 ± 0 .5 6 b cd 0 c 2 .5 ± 0 .5 4 a 0 .5 ± 0 .2 6 c 0 .4 0 b 2 .7 ± 0 .5 6 a b c 8 .3 ± 1 .6 9 d e f f a sh e r 0 .6 ± 0 .2 2 cd 0 b 0 b 0 d 0 .5 ± 0 .2 2 cd 0 c 4 .2 ± 0 .7 8 a 1 .0 ± 0 .5 2 b c 1 .5 ± 0 .5 6 b cd e 0 b 0 .9 ± 0 .4 1 cd 8 .7 ± 1 .7 cd e f g e n in a 1 .2 ± 0 .4 2 b cd 0 b 0 b 0 d 1 .2 ± 0 .3 9 a b cd 1 .5 ± 0 .6 2 a b 4 .0 ± 0 .7 5 a 2 .7 ± 0 .5 6 a 1 .0 ± 0 .4 7 d e 0 b 1 .4 ± 0 .5 4 b cd 1 3 .2 ± 2 .0 5 a b cd e n u m b e r o f o b se rv a tio n s = 2 0 in e a ch lo ca tio n m e a n s (± s e ) fo llo w e d b y th e s a m e le tt e rs in e a ch c o lu m n a re n o t si g n ifi ca n tly d iff e re n t a t 5 % le ve l b a se d o n r ya n ’s q t e st ( r e g w q ) * c o lle ct io n w a s ca rr ie d o u t o n ce in e a ch lo ca tio n 322 preliminary survey of ticks (acari: ixodidae) on cattle in northern sudan t a b l e 1 a n o v a m e a n s q u a re s (m e ), r -s q u a re s (r ), c o e ff ic ie n t o f v a ri a n ce ( c v ) a n d f v a lu e s (f ) fo r to ta l b o d y co lle ct io n s o f tic k sp e ci e s fr o m c a tt le i n d iff e re n t lo ca lit ie s in t h e s u d a n d u ri n g t h e y e a rs 2 0 0 1 a n d 2 0 0 2 s o u rc e o f va ri a tio n m e r c v f a . le p id u m 0 .1 2 0 .5 7 1 1 .9 1 1 2 .7 0 * a . va ri e g a tu m 0 .0 2 0 .4 0 7 .9 0 6 .4 2 * b . d e co lo ra tu s 0 .0 5 0 .8 2 4 .7 6 4 4 .9 4 * h . a . a n a to lic u m 0 .2 3 0 .6 5 1 2 .8 9 1 7 .9 7 * h . d ro m e d a ri i 0 .1 0 0 .4 7 1 2 .4 5 8 .6 4 * h . im p e lta tu m 0 .0 7 0 .6 4 8 .6 0 2 .2 5 * h . m . ru fip e s 0 .0 2 0 .1 9 9 .4 6 1 7 .1 6 * h . tr u n ca tu m 0 .0 8 0 .6 7 8 .7 9 1 9 .2 2 * r . e . e ve rt si 0 .1 7 0 .4 9 1 5 .4 7 9 .3 3 * r . sa n g u in e u s g ro u p 0 .1 4 0 .5 4 1 2 .5 2 1 1 .3 4 * r . s. s im u s 0 .0 0 9 0 .4 5 4 .8 3 8 .1 1 * d e g re e s o f fr e e d o m = 1 6 * p < 0 .0 0 1 there was a significant difference (p < 0.05) in the mean numbers of tick collected in damazin and fasher and the rest of the locations (table 2). hyalomma impeltatum kratz’s hyalomma was recorded in atbara with the highest mean numbers of ticks (2.1 ± 0.45), while the lowest mean recorded in el dieum (0.8 ± 0.25) (table 2). there was a significant difference (p < 0.05) in the mean number of ticks collected in atbara in comparison with other locations. there was no significant difference (p > 0.05), however, between the numbers of ticks collected in atbara, genina and kosti; which were significantly different (p < 0.05) from those in el dieum (table 2). hyalomma marginatum rufipes the hairy hyalomma tick was found in all locations. the highest mean total of these ticks was recorded in el damer (5.7 ± 0.52), with the lowest mean in sennar (2.4 ± 0.4)(table 2). there were no significant differences (p > 0.05) in the mean numbers of ticks collected from all locations (table 2). hyalomma truncatum the highest mean number of ticks of this species was recorded in genina (2.7 ± 0.56), while the lowest mean was recorded in el dieum (0.4 ± 0.16) (table 2). this species was not recorded in northern, central and blue nile provinces. the presence of this tick species was significantly different (p < 0.05) in genina in comparison with other locations. there were no significant differences (p > 0.05) between the mean numbers of ticks collected in el dieum, nyala and fasher (table 2). rhipicephalus evertsi evertsi the red-legged tick was present in all locations with the exception of um benin, el dieum and rabak. the highest mean total body count of ticks was recorded in el obied (6.7 ± 0.73), while the lowest mean was recorded in kosti and nyala (0.4 ± 0.22) (table 2). there was a significant difference (p < 0.05) in the mean number of ticks collected in el obied in comparison with other locations. there were, however, no significant differences in numbers (p > 0.05) between those collected in el damer, khartoum, kassala and fasher (table 2). rhipicephalus sanguineus group this tick species was not recorded in el obied, port sudan and sennar locations, but it was present in the other locations (table 2). the highest mean total body counts were recorded in khartoum (5.0 ± 0.86), while the lowest was recorded in gadarif (0.7 ± 0.33). the presence of this tick species was significantly different (p > 0.05) in khartoum in comparison with other locations. there were no significant differences (p > 0.05) between madani, um benin, damazin, rabak, kosti, fasher and genina (table 2). rhipicephalus simus simus this tick species was recorded only in el dieum where the mean number of ticks collected was 0.9 ± 0.35 (table 2). discussion since the previous surveys on ticks in the sudan conducted by hoogstraal (1956), karrar et al. (1963), osman, ibrahim & osman (1977) osman et al. (1982), fao (1983) and jongejan, zivkovic, pegram, tatchell, fison, latif & paine (1987), major ecological changes have occurred due to extensive animal movement, deforestation, desertification and the establishment of large mechanized agricultural schemes (hassan & osman 2003). these factors have certainly affected the distribution of ticks and tick-borne diseases in the sudan. as a result knowledge on tick-borne disease prevalence throughout the sudan is fragmented (el hussein, mohammed, osman & osman 1991; el-imam 2003). this crosssectional survey was designed to provide a further understanding of the current distribution of the major ticks species in the sudan. from the knowledge of the distribution of tick vectors at a given location, the potential distribution of diseases they transmit can be broadly predicted (fao 1984). the prevalence of the 11 tick species (table 2) recorded during this study is an indication of the prevalence of the most important tick-borne diseases (tbds) in the sudan. although studies on tbds were not carried out in this study, the possibility of their presence cannot be ruled out since the most potential tick vectors transmitting other tbds were reported in the sudan in 1983 (fao 1983). the tick species identified in this survey are generally similar to those reported by hoogstraal (1956), karrar et al. (1963), fao (1983), jongejan et al. (1987) and el-imam (2003). the absence of a. variegatum and a. lepidum in northern sudan is not surprising, since these tick species are known to survive in humid areas (jongejan et al. 1987; os323 d.a. salih et al. man & hassan 2003) but not in desert and semidesert areas such as northern sudan (hoogstraal 1956). it is not surprising that the presence of a. lepidum in gadarif represented the highest mean total body collection among the locations that were investigated, as the area is known to be favourable for the propagation of this tick species (karrar et al. 1963). although this tick species has not previously been reported from el dieum, a low level of infestation was recorded during this investigation. movement of tick-infested cattle from kordofan and the upper nile state, where this tick species occurs (hoogstraal 1956) as well as migrating birds may have introduced this species into el dieum (hoogstraal 1961). the same interpretation may apply to port sudan, where a. lepidum was recorded on one indigenous domestic bovine introduced from halfa el gadida, where a. lepidum is prevalent (karrar et al. 1963). no a. variegatum was encountered on cattle during the period of the present investigation in kassala. this may be attributed to the seasonal activity of this tick species, as this survey was conducted in april in the kassala area. osman et al. (1982) reported that a. lepidum reached peak activity between august and december in the rainy season in kordofan. moreover, attempts by the local farmers to control ticks by the indiscriminate use of acaricides may have caused fluctuations in tick populations (fao 1984). the absence of b. annulatus in northern and central sudan is in accordance with the finding that this tick species is restricted to the southern parts of central sudan (fao 1983). only small numbers of b. decoloratus ticks were recorded during this study in madani and rabak (table 2). the presence of h. a. anatolicum in northern and central provinces implies that t. annulata is likely to be endemic in these areas (el ghali & el hussein 1995; elhaj & hamid 2003). this tick species has the ability to establish itself in animal housing in order to complete its life cycle (fao 1984). all the animals surveyed in atbara, el damer and khartoum were kept under an intensive managemental system (closed system) in which sheds are constructed of local building materials. sites such as mud walls with cracks, and water and food containers could be used by ticks as shelters, which allow them to survive adverse environmental conditions. the presence of h. a. anatolicum in um benin in relatively high abundance (2.4 ± 0.7) (table 2) is an interesting finding. according to the publication of the fao (1983) only one female of this tick species was collected from um benin. in the present survey, greater numbers of ticks of both sexes were encountered. this finding may indicate that the limit of this tick species has moved southwards to latitude 13° n. this may be attributed to ecological and other changes that have occurred since 1983 as a result of drought, animal movements, deforestation, desertification and establishment of large irrigation schemes. however, this finding needs to be verified by conducting an intensive survey, which includes all the seasons of the year. the low level of h. dromedarii recorded in this study in the khartoum area may be a reflection of the fact that cattle do not frequently come in contact with camels in this area, since h. dromedarii is well known as a camel tick (hoogstraal 1956). rhipicephalus e. evertsi and the r. sanguineus group, though present in most of the locations investigated, are unlikely to play a significant role in the epidemiology of tropical theileriosis. in previous studies in the sudan, large numbers of r. sanguineus have been reported on livestock in darfour province (osman 1978) and in kassala province (karrar et al. 1963). balfour in 1911 (cited by hoogstraal 1956) mentioned that r. e. evertsi was introduced into the khartoum area on cattle from malakal in southern sudan. this tick species and r. sanguineus were found to infest sheep, goats, dogs, birds, horses and donkeys (hoogstraal 1956). both tick species are now established in khartoum (kheir & majid 1999). the population density of the species identified was variable in the locations in which the survey was conducted (table 2). the highest tick count per animal was encountered in khartoum (19.6 ± 2.25), while the lowest tick count was recorded in port sudan (5.8 ± 0.63) (table 2). these differences may be attributed to the managemental systems, host density, vegetation cover and other ecological factors. the greater number of ticks in khartoum than in other locations is probably due to the dense livestock population in the area, which would have led to a high pick-up rate of ticks by cattle that are always kept in pens. cattle in the other locations are not confined in pens because of the nomadic life of cattle owners. the lower infestation rates in western province may be attributed to the nature of the grazing pastures, which are relatively open and dry with little vegetation cover. the nomadic system of livestock production in western sudan enhances control of tick populations as grazing areas are naturally spelt (jongejan et al. 1987). the minority of the tick species (four out of the 11) (table 2) was present in nearly all locations. however, the occurrence of certain species was very 324 preliminary survey of ticks (acari: ixodidae) on cattle in northern sudan high in some areas and very low in others. for example, a. lepidum, which is the most dominant species in damazin (table 2), was less prevalent in fasher. this may be attributed to the type of animal husbandry practical and a favourable humid environment in areas around the blue nile river. similarly, the highest mean number of h. impeltatum was recorded in atbara and the lowest mean number in el dieum (table 2). this tick is mainly restricted to the dry zone and its immature stages are commonly associated with desert rodents (hoogstraal 1956). the absence of any tick species in any given period does not mean that it is absent from an area, as it can reappear when optimum climatic conditions prevail (osman et al. 1982). furthermore, the distribution limits of ticks are not always constant and are determined by a complicated interplay of factors such as climate, vegetation cover, host density, host susceptibility, host resistance, grazing habits and types of animal husbandry (tatchell & easton 1986; punyua, latif, nokoe & capstick 1991; sonenshine & mather 1994). the overall infestation rate of tick species in the present study was much lower than was expected if it is compared with the findings of jongejan et al. (1987). certain environmental and managemental factors might have been responsible for the lower tick burdens. these include the high ambient temperatures and low relative humidities, which prevailed during the period of the study, which may have affected the breeding abilities of the ticks. application of chemical acaricides as sprays or hand dressings was practised in some locations, and despite its occasional and improper use, it still gives a degree of control of ticks on the cattle. the introduction of hens as a means of biological control (hassan, dipeolu, amoo & odhiambo 1991; hassan, dipeolu & munyinyi 1992), as practised in many locations, might also have resulted in reduced tick numbers. another factor that could contribute to a low level of ticks infestation, especially in a closed system (zero grazing), is the regular removal of dung, and free-living stages of ticks are collected and burnt along with the litter. in conclusion, it must be emphasized that this is an ad hoc survey, aimed at determining the tick species associated with cattle in the sudan. in order to re-map tick prevalence and distribution as reported by hoogstraal (1956), a large scale and systematic survey which includes ticks on all livestock and wildlife species in the country should be conducted. acknowledgements this research was partially supported by the international atomic energy agency (iaea) through project no. sud/05/027 entitled “control of ticks and tick-borne diseases”. this work is published with the kind permission of the director-general, animal resources research corporation. references day, r.w. & quinn, g.p. 1989. comparison of treatment after an analysis of variance in ecology. ecological monographs, 59:433–463. el ghali, a.a. & el hussein, a.m. 1995. diseases of livestock in el damer province, el nile state, sudan: a two-year retrospective study. sudan journal of veterinary science and animal husbandry, 34:37–45. el hussein, a.m., mohammed, s.a., osman, a.k. & osman, o.m. 1991. a preliminary survey of blood parasites and brucellosis in dairy cattle in northern state. sudan journal of veterinary research, 10:51–56. elhaj, m.t. & hamid, m.e. 2003. haematological and serum chemistry studies on crossbred calves infected with theileria species. sudan journal of veterinary science and animal husbandry, 42:128–140. el-imam, a.h. 2003. ecological studies on ticks (acari: ixodidae) infesting cattle in kosti province. sudan journal of veterinary science and animal husbandry, 42:62–71. fao. 1983. ticks and tick-borne diseases control. the sudan: tick ecology and tick control, technical report no. 1, ag: cp/sud/024/den, rome: food and agricultural organization of the united nations. fao. 1984. tick and tick-borne diseases control. a practical field manual. vol. i and ii. rome: food and agricultural organization of the united nations. hassan, s.m. & osman, a.m. 2003. ecological and biological factors determining population dynamics of ixodid ticks: a review. sudan journal of veterinary science and animal husbandry, 42:16–44. hassan, s.m., dipeolu, o.o. & munyinyi, d.m. 1992. influence of exposure period and management methods on the effectiveness of chickens as predators of ticks infesting cattle. veterinary parasitology, 43:301–309. hassan, s.m., dipeolu, o.o., amoo, a.o. & odhiambo, t.r. 1991. predation on livestock ticks by chicken. veterinary parasitology, 38:199–204. hoogstraal, h. 1956. african ixodoidea. i. ticks of the sudan (with special reference to equatoria province and with preliminary reviews of the genera boophilus, margaropus, and hyalomma). department of the navy, bureau of medicine and surgery. washington, d.c., usa. hoogstraal, h. & kaiser, m.n. 1959. observations on egyptian hyalomma ticks (ixodoidea: ixodidae). 5: biological notes and differences in identity of h. anatolicum and its subspecies anatolicum koch and excavatum koch among russian and other workers identity of h. lusitanicum koch. entomological society of america, 52:243–261. hoogstraal, h. 1961. migrating birds and their ectoparasite in relation to disease. east africa medical journal, 38:221– 226. jongejan, f., zivkovic, d., pegram, r.g., tatchell, r.j., fison, t., latif, a.a. & paine, g. 1987. ticks (acari: ixodidae) of the blue and white nile ecosystems in the 325 d.a. salih et al. sudan with particular reference to the rhipicephalus sanguineus group. experimental and applied acarology, 3:331–346. karrar, g., kaiser, m.n. & hoogstraal, h. 1963. ecology and host-relationships of ticks (ixodoidea) infesting domestic animals in kassala province, sudan, with special reference to amblyomma lepidum donitz. bulletin of entomological research, 54:509–522. kheir, s.m. & majid, a.a. 1999. ticks, tsetse and biting flies of importance in sudan: a review. sudan journal of veterinary science and animal husbandry, 38:137–142. latif, a.a. 1994. economic losses in exotic breeds of cattle due to theileriosis in the sudan, in tropical theileriosis in the sudan, edited by a.m. atelmanan & s.m. kheir. proceedings of a workshop held at the sudan veterinary association residence, khartoum, sudan, 4–5 may, 1994. okello-onen, j., hassan, s.m. & essuman, s. 1999. taxonomy of african ticks: an identification manual. nairobi, kenya: icipe science press. osman, a.m. & hassan, s.m. 2003. the ecology and distribution of the east africa tick amblyomma lepidum: a review. sudan journal of veterinary science and animal husbandry, 42:45–53. osman, o.m. 1978. preliminary notes on the distribution of ticks (acarina. ixodidae) in darfour province, sudan. bulletin of animal health and production in africa, 26:329–333. osman, o.m., el hussein, a.m., neima, a. & abdulla, h.s. 1982. ecological studies on ticks (acarina: ixodidae) of kordofan region, sudan. bulletin of animal health and production in africa, 30:45–53. osman, o.m., ibrahim, a.m. & osman, a.g. 1977. some observations on ticks of sedentary cattle in southern darfour province. proceedings of the 8th veterinary association conference, khartoum, sudan: 82–92. punyua, d.k., latif, a.a., nokoe, s. & capstick, p.b. 1991. tick (acari: ixodidae) infestations on zebu cattle in western kenya: seasonal dynamics of four species of ticks on traditionally managed cattle. journal of medical entomology, 28:630–636. sonenshine, d.e. & mather, t.n. 1994. ecological dynamics of tick-borne zoonoses. oxford: oxford university press. tatchell, r.j. & easton, e. 1986. tick (acari: ixodidae) ecological studies in tanzania. bulletin of entomological research, 76:229–246. 326 preliminary survey of ticks (acari: ixodidae) on cattle in northern sudan abstract introduction materials and method results discussion conclusion acknowledgements references about the author(s) sara locke department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa vinny naidoo department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa ibrahim hassan department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa neil duncan department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa citation locke, s., naidoo, v., hassan, i. & duncan, n., 2022, ‘effect of cytochrome p450 inhibition on toxicity of diclofenac in chickens: unravelling toxicity in gyps vultures’, onderstepoort journal of veterinary research 89(1), a1978. https://doi.org/10.4102/ojvr.v89i1.1978 original research effect of cytochrome p450 inhibition on toxicity of diclofenac in chickens: unravelling toxicity in gyps vultures sara locke, vinny naidoo, ibrahim hassan, neil duncan received: 06 oct. 2021; accepted: 20 apr. 2022; published: 14 june 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract diclofenac was responsible for the decimation of gyps vulture species on the indian subcontinent during the 1980s and 1990s. gyps vultures are extremely sensitive (the lethal dose 50 [ld50] ~ 0.1 mg/kg – 0.2 mg/kg), with toxicity appearing to be linked to metabolic deficiency, demonstrated by the long t1/2 (~12 h – 17 h). this is in striking comparison to the domestic chicken (gallus gallus domesticus), in which the ld50 is ~10 mg/kg and the t1/2 is ~1 h. the phase 1 cytochrome p450 (cyp) 2c subfamily has been cited as a possible reason for metabolic deficiency. the aim of this study was to determine if cyp2c9 homolog pharmacogenomic differences amongst avian species is driving diclofenac toxicity in gyps vultures. we exposed each of 10 cyp-inhibited test group chickens to a unique dose of diclofenac (as per the organisation for economic co-operation and development [oecd] toxicity testing guidelines) and compared the toxicity and pharmacokinetic results to control group birds that received no cyp inhibitor. although no differences were noted in the ld50 values for each group (11.92 mg/kg in the cyp-inhibited test group and 11.58 mg/kg in the control group), the pharmacokinetic profile of the test group was suggestive of partial inhibition of cyp metabolism. evaluation of the metabolite peaks produced also suggested partial metabolic inhibition in test group birds, as they produced lower amounts of metabolites for one of the three peaks demonstrated and had higher diclofenac exposure. this pilot study supports the hypothesis that cyp metabolism is varied amongst bird species and may explain the higher resilience to diclofenac in the chicken versus vultures. keywords: diclofenac; toxicity; vulture; cytochrome p450; pharmacokinetics; chicken. introduction the use of chemicals to exert a biological effect has long been understood and utilised by humans. today, it can be little disputed that the use of modern chemical compounds in the human and veterinary sector has had profound influence on health and disease management, agriculture and the economy at large. the impact of pharmaceuticals on the environment has, on several occasions, been the price we have paid for these advancements. a historical yet infamous example is the pesticide dichlorodiphenyltrichloroethane (ddt). whilst controlling crop-destroying insects in the united states (us) and assisting with mitigating the spread of diseases like malaria in africa, ddt had devastating effects on other wildlife species, most notably raptorial birds of prey such as the peregrine falcon (falco peregrinus) and the bald eagle (haliaeetus leucocephalus) (köhler & triebskorn 2013; rattner 2009). in the veterinary context, animal pharmaceuticals have not been exempted from their share of impact on the environment, and one of the biggest impacts of any veterinary drug has been that caused by the non-steroidal anti-inflammatory drug (nsaid) diclofenac. used for the relief of pain and inflammation in domestic ruminants on the indian subcontinent, diclofenac decimated populations of gyps vulture species in just 20 years, the so-called ‘asian vulture crisis’ (prakash et al. 2007). these birds seemed to be susceptible to the drug at a low ld50 of 0.1 mg/kg – 0.2 mg/kg (swan et al. 2006), with exposure resulting from a single engorgement at a carcass. in comparison, the ld50 in the domestic chicken (gallus gallus domesticus) was 9.8 mg/kg (naidoo et al. 2007) and as high as 616 mg/kg in the pigeon (columba livia domestica) (hassan et al. 2018). the difference in ld50 also appeared to be correlated with the pharmacokinetic parameter t1/2 or half-life of elimination, suggesting deficient metabolism as a cause of increased sensitivity. the t1/2 is long in gyps vultures (> 7 h) and short in the chicken and pigeon (< 7 h). a plausible explanation for these striking interspecies differences could lie in the avian clade evolutionary history. whilst birds appear to have evolved from only three major groups (the palaeognathae, the galloanserae and the neoaves) (watanabe et al. 2013), they diverged greatly over the millennia into the thousands of species present today, and their ability to metabolise xenobiotic compounds most likely diverged concurrently in accordance with difference of diet and habitat (hutchinson et al. 2014; thomas 2007; watanabe et al. 2013). xenobiotic metabolism is divided into phase 1 and phase 2 reactions. these enzymatic processes serve, for the most part, to reduce biological activity of a compound and facilitate its excretion by the body (brunton, hilal-dandan & knollmann 2018). of the groups of enzymes responsible for these phases, the cytochrome p450 (cyp) superfamily of phase 1 reactions is by far the largest and most diverse across animal species (brunton et al. 2018; hutchinson et al. 2014; konstandi, johnson & lang 2014). as cyp enzymes are renowned for interspecies pharmacogenomic variation and appear to be active in gyps vultures, they have been cited as the most likely cause of diclofenac metabolic deficiency in these birds (hassan et al. 2018; fourie et al. 2015; naidoo et al. 2010, 2018). in humans, diclofenac is largely metabolised by phase 1 cyp2c9 activity (brunton et al. 2018; daly et al. 2007; leemann, transon & dayer 1993). homologous cyp2c9 enzymes have also been found to be present in the domestic chicken (kawalek et al. 2006; shafi et al. 2015; watanabe et al. 2013). in the case of gyps vultures, it is hypothesised that their sensitivity to diclofenac may be as a result of a homologous cyp2c9 deficiency (hutchinson et al. 2014; naidoo et al. 2010, 2011). whilst there is speculation that an underlying cyp enzyme deficiency is responsible for the toxic effect of the drug because of zero order metabolism, the absence of the enzyme is not easy to prove as absence can be a true or a false result. an alternate manner of proving the role of a specific enzyme in toxicity would be to take a species where the metabolism is known to be present and inhibit it. if a deficiency defines toxicity, inhibiting the naturally present system should create the same level of toxicity as for the species deficient in it. a useful feature of cyp enzymes is that they are subject to inhibition of activity by chemical means. this feature can be exploited; inhibition of a specific enzyme by one drug should alter the pharmacokinetics of another drug that is a substrate for the same enzyme. the primary aim of this study was to ascertain the impact of cyp2c9 inhibition in the chicken in terms of pharmacokinetic profiles, ld50 and metabolites formed. materials and method animal housing and care thirty day-old ross broiler chickens (gallus gallus domesticus) weighing an average of 45.13 g were purchased for the study. the birds were housed at the university of pretoria biomedical research facility (upbrc), faculty of veterinary science. the birds were housed on wood shavings on a light-dark cycle of 10 h/14 h, with reducing temperatures from 30.6 °c to 19 °c and humidity fluctuating between 20% and 60%. the birds had ad lib access to food (poultry standard ration, according to age) and municipal potable water, and they were individually identified with unique wing-tags. at five weeks of age, the birds were allocated to the two treatment groups to achieve a group size of ten. the study protocol was approved by the university of pretoria animal ethics committee (aec) and the faculty of veterinary science research ethics committee (rec). study design the study design followed the oecd guidelines for acute avian oral toxicity testing (oecd 2016), with the test group on diclofenac sodium (panamor 75®, aspen pharmacare holdings) and fluconazole (diflucan®, pfizer) and the control group on diclofenac sodium alone (n = 10). fluconazole was used as the cyp2c9 inhibitor (brunton et al. 2018) at 15 mg/kg (carpenter & marion 2018), intra-peritoneally (i/p) for three days prior to the start of diclofenac dosing in the test group. the test and control groups were subsequently dosed with diclofenac sodium. each bird within a group was dosed with a unique dose, spaced around the working ld50 of 9.8 mg/kg (naidoo et al. 2007) on a log scale, by intravenous (iv) injection, according to the oecd maximum likelihood estimation method (oecd 2016). the specific doses were 3.36 mg/kg, 4.26 mg/kg, 5.40 mg/kg, 6.86 mg/kg, 8.70 mg/kg, 11.04 mg/kg, 14.00 mg/kg, 17.77 mg/kg, 22.55 mg/kg and 28.61 mg/kg. following the administration of diclofenac, the birds were monitored for a 15-day period. birds still alive after 15 days were terminated by sedation and carbon dioxide asphyxiation. sampling blood samples were drawn from the wing veins of the birds using 21 gage (g) needles attached to preheparinised 3 ml syringes at 0.0 h, 0.25 h, 0.5 h, 1.0 h and 2.0 h after dosing. samples were transferred into prelabelled plasma tubes and centrifuged within 1 h of sampling at 1660 × g and 25 °c for 15 min. samples were split for uric acid and diclofenac analysis and were then stored at –80 °c. scheduled and unscheduled deaths were submitted to the university of pretoria paraclinical department, section of veterinary pathology, for full gross and histological evaluation. where needed, samples were collected in 10% buffered formalin, sectioned and stained via haematoxylin & eosin (h&e) technique and subjected to histopathologic examination. drug analysis samples were analysed at the university of pretoria, veterinary, paraclinical department, toxicology and pharmacology laboratory. diclofenac sodium, 4′-hydroxydiclofenac, 5′-hydroxydiclofenac and fluconazole analytical standards were purchased from sigma aldrich (merck, south africa). in short, 200 μl of plasma was placed in a 2-ml tube, followed sequentially by 400 μl of diethyl ether and 400 μl of potassium dihydrogen phosphate (0.3 m and ph 3.5). after mixing, tubes were then centrifuged for 20 min at 5878 × g and 4 °c and placed in an ice bath of methanol and co2 for 3 min, which allowed the organic layer to be separated from the aqueous phase. the separated organic phase was dried under nitrogen flow at 50 °c until dry. for the high-performance liquid chromatography (hplc) analysis, 400 μl of the mobile phase, consisting of a 42.5:57.5 ratio of sodium dihydrogen phosphate (0.05 m and ph 4.86–4.88): acetonitrile, was added to dissolve the dried sample residue. a beckman system gold analyser with a 32 karat™ software package, a diode array detector (dad) 168, an autosampler module 508 and a programmable solvent module 126 (beckman instruments, fullerton, california, us) was used for the hplc analysis with a 250 mm × 4.6 mm, 5 μ base deactivated silica (bds) hypersil phenyl column. a mobile phase of ratio 42.5:57.5 sodium dihydrogen phosphate (0.05 m and ph 4.86–4.88) : acetonitrile was used for both diclofenac and metabolite detection. for analysis, 30 μl was injected at 1 μl/min, and detection at 275 nm. the gradient method of analysis, with a total runtime of 8 min, was used to gradually decrease the ratio of sodium dihydrogen phosphate and acetonitrile from its starting ratio to 20:80 over a period of 3 min. the mean retention time for diclofenac and the 4′and 5′-hydroxydiclofenac metabolite combination was 4.51 min and 3.45 min, respectively. the standard calibration curve showed an r² value > 0.99. the limit of detection (lod) and limit of quantification (loq) were 0.195 μg/ml and 0.396 μg/ml, respectively, for diclofenac, with a range of 0.718 μg – 25.000 μg. for the 4′and 5′-hydroxydiclofenac metabolites, the lod and loq were both 0.095 μg/ml, with a range of 0.095 μg – 3.125 μg. diclofenac pharmacokinetic and statistical analysis concentration-time data generated following hplc analysis was dose equalised to 1 mg/kg and evaluated by non-compartmental modelling (kinetica 5.1, thermo scientific). the pharmacokinetic parameters maximum plasma concentration (cmax) and time to maximum plasma concentration (tmax) were read off the curve. area under the plasma concentration versus time curve to the last quantifiable time point (auclast) and area under the moment curve (aumc) were calculated using the linear trapezoidal rule as follows: and where c is the diclofenac concentration and t is the time. area under the plasma curve extrapolated to infinity was calculated as: where is the last measured concentration and β is the terminal elimination portion of the curve gradient, on a natural logarithmic scale. the half-life of elimination (t1/2) was calculated as: whole plasma clearance (cl) was calculated as: volume of distribution (vd) was calculated as: where cp0 was the calculated concentration at time 0. the mean residence time (mrt) was calculated as: uric acid analysis plasma samples were evaluated by the university of pretoria, companion animal clinical studies department, clinical pathology laboratory. samples were analysed on a cobas integra 400 plus analyser (roche diagnostics, mannheim, germany), using the uricase enzyme colorimetric test, by measuring the absorbance of the red colour intensity of quinone-diimine dye formed at the end of the reaction at 552 nm. the reference range used for normal chicken uric acid concentration was based on ross et al. (1978) and wilson and miles (1988). uric acid auclast values were calculated using the linear trapezoidal rule. baseline corrections per scheduled time points (difference between 0 h) were also calculated. data analysis the ld50 was determined using the maximum likelihood estimator (mle) probit model (oecd 2016) using a chi-square goodness-of-fit model. microsoft® excel workbook sequential design calculator (sedec) was used for these calculations. for the pharmacokinetic data, results were presented as mean and standard deviation. parameters were also subjected to univariate analysis of variance (anova) analysis. uric acid concentrations, uric acid auclast values and baseline corrections were compared using a student’s t-test. for the metabolite analysis, three distinct peaks, suspected to be metabolites (not visible in blank plasma), were seen on hplc analysis. because of inability to separate standard 4′-hydroxydiclofenac and 5′-hydroxydiclofenac peaks on calibration and extreme variation in profiles produced between birds, metabolite peaks were analysed and compared by first calculating the dose equalised auclast values for each peak, based on the peak height readings (response) over time. these values were compared to the diclofenac peak dose equalised auclast values, per treatment group and mortality status. the metabolite auclast results were further subject to binary logistic regression to check for relationship and significance thereof between the extent of exposure and mortality. the treatment groups were compared by subjecting the means of the ratio of diclofenac : metabolite auclast peak to a student’s t-test. all statistical analysis was in statistical package for social sciences (spss) version 1.0.0.95 statistical software (ibm, new york, ny, us). ethical considerations this study was approved by the animal ethics committee (aec) of the university of pretoria (number: v078-18). results clinical signs and mortalities in the test group, the two birds receiving the highest dose died immediately after diclofenac dosing, from what we believe to be acute cardiovascular collapse. two birds (dosed at 14.00 mg/kg and 6.86 mg/kg body weight [bw]) died within approximately 48 h of dosing. these birds showed expected typical signs of diclofenac toxicity: lethargy and depression, anorexia and unwillingness to move. the remaining birds showed no obvious signs of intoxication. the ld50 was calculated at 11.92 mg/kg bw, with the 95% confidence intervals of 3.87 mg/kg and 61.87 mg/kg and a probability of 0.56. in the control group birds, one bird (dosed at 5.40 mg/kg bw) died 15 min after dosing, also from what was likely cardiovascular collapse. a second bird (dosed at 28.61 mg/kg bw) died approximately 7 h after dosing with minimal symptoms, whilst a third (dosed at 17.70 mg/kg bw) died at approximately 28 h with expected typical signs. a fourth bird (dosed at 22.55 mg/kg bw) was euthanised at approximately 56 h after dosing. the remaining birds showed no obvious signs of intoxication. the ld50 for diclofenac in control birds was calculated at 11.52 mg/kg bw, with the 95% confidence intervals of 3.31 mg/kg and 78.62 mg/kg and a probability of 0.78. pathology the birds that died within 15 min of treatment, irrespective of the group, showed no gross or histopathological lesions. for the other birds that died within 56 h, irrespective of the group, there was a mild to marked urate deposition on all serosal surfaces and in varying degrees in the pericardium, epicardium and air sacs. urate crystals were also seen on the liver and spleen and in the joints and tendon sheaths of the legs (figure 1). histopathologically, there was mild to marked widespread cell injury and necrosis of the renal tubular epithelium, with dilatation of the damaged tubules and marked deposition of globule urates within the lumens of the damaged tubules. cell changes ranged from increased eosinophilia to cell membrane disruption and sloughing into the lumen, with spicules of uric acid visible within the eosinophilic masses. in some birds there were many urate tophi showing giant cell aggregates at the periphery. figure 1: gross pathology findings in a test group bird that succumbed approximately 48 h following intravenous administration of diclofenac. (a) visceral gout with uric acid accumulation on the serosal surface of the liver and pericardium (white arrows), (b) nephrosis of the kidneys evident as pale and yellowish (black arrow) and (c) uric acid accumulation in the tibeo-tarsal joint indicative of articular gout (blue arrow). diclofenac pharmacokinetic analysis the pharmacokinetic parameters per treatment group and mortality status are presented in table 1, and the mean plasma concentrations versus time profiles are presented in figure 2. the majority of the birds showed the expected i/v curve of linear depletion from a maximum concentration at the first point of sampling. one bird from the test group (dosed at 8.70 mg/kg bw) had a 2 h outlier reading which was impossibly high, with the result that we omitted the data point from the pharmacokinetic (pk) and statistical calculations. another bird from the test group (dosed at 6.86 mg/kg bw) had a very high 15 min reading. the latter was consequently omitted entirely from the anova calculations but included in the descriptive statistics in table 1. three birds, two from the control group and one from the test group, showed absorptive components, likely because of accidental subcutaneous dosing. the mean cmax and tmax values were 0.51 mg/ml and 0.38 h, respectively. figure 2: mean diclofenac plasma concentration time curves by treatment group, dose corrected to 1 mg/kg. table 1: pharmacokinetic parameters for diclofenac in test and control birds following intravenous diclofenac exposure. presented as geometric means (gm) on dose-corrected parameters, with the standard error of the mean (sem). analysis of variance showed no significant difference between groups for cmax, auclast or t1/2, although the values for cmax and auclast were, on average, higher for the test group, driven largely by the values from the birds which died (table 1 and figure 2). whilst the mean t1/2 value for the test group was lower than that of the control group (also driven largely by values from the birds that died), this must be interpreted with caution as we were unable to get values for the two highest-dosed birds in the test group because of early mortality. as evidence of the impact this may have had, the corresponding birds in the control group receiving the highest doses had long t1/2 values (7.01 h and 2.75 h). metabolite analysis when evaluating metabolite peak auclast values, there was no statistically significant relationship between the extent of metabolite exposure and mortality nor between dose and metabolite production. there was also no statistically significant difference in exposure between treatment groups. when comparing the two treatment groups (both dead and alive birds), the test birds produced higher auclast metabolite values compared to the control birds (table 2). the exception to this was peak 3 in the birds that died, which was notably lower in the test birds (value in bold in table 2). the mean dose-corrected diclofenac peak auclast reading was also considerably higher in the dead birds (value in bold in table 2). a different picture was present when comparing birds that died with those that survived across both treatment groups (table 2); the birds that died produced lower auclast metabolite values for all three metabolite peaks across both groups. the exception was peak 3, where the mean auclast values were almost identical between dead and alive birds within the control group. table 2: mean dose-corrected area under the curve to the last measured (quantifiable) time point values for each metabolite peak and diclofenac per treatment group. uric acid analysis there were no significant differences between the treatment groups when comparing the mean auclast values or baseline corrections over the scheduled sampling points of 0.25 h, 0.5 h, 1.0 h and 2.0 h post-dosing. all birds (test and control) showed a relatively uniform uric acid concentration at 0 h of sampling, before exposure to diclofenac, and within the reference range as described by ross et al. (1978) and wilson and miles (1988). because of ethical limitations regarding the volume of blood that could be drawn per bird, it was not possible to evaluate uric acid trends further than 2 h post-dosing. discussion diclofenac has been responsible for the mass mortality of three species of asian vultures. despite the causality being well established, much still needs to be understood about the mechanism of toxicity and why gyps vultures were so susceptible. at present there is a fair body of literature that suggests a pharmacokinetic and metabolic reason as the underlying driver. pharmacokinetic data from studies involving diclofenac administration in various other species of birds (hassan et al. 2018; naidoo et al. 2007, 2011; rattner et al. 2008; swan et al. 2006), suggest an association between mortality and pk parameters that reflect the length of exposure: t1/2 and mrt. in most cases, birds with longer t1/2 and mrt values died compared to birds that had shorter values. this correlation is most striking in gyps vulture species studied, where the t1/2 values are 12.24 h and 16.78 h and the mrt values are 15.11 h and 26.10 h for the cape griffon vulture (cgv) and the african white-backed vulture (awbv), respectively (naidoo et al. 2007). these values are despite a dose equal to that given to the chickens in the study by naidoo et al. (2007) and much lower than those given to most of the other bird species studied. other notable differences in their pharmacokinetics are in the auclast values, which are much higher, and the cl values, which are much lower than in other bird species. the pk picture is suggestive of zero-order kinetic metabolism in these gyps vultures, or saturation of the intrinsic enzyme metabolising capability. the specific phase 1 cyp enzyme that metabolises the majority of diclofenac in the human liver is cyp2c9, producing the major and minor metabolites 4′and 3′-hydroxydiclofenac, respectively (bort et al. 1999; leemann et al. 1993). a further minor 5′-hydroxylation metabolite is produced through additional cyp enzymes, including cyp3a4, cyp2c8, cyp2c18 and cyp2c19 (bort et al. 1999; shen et al. 1999). from the evidence available, it is reasonable to conclude that metabolism in other mammals is also because of the activity of a member of the cyp2c subfamily. for instance, in the rat and dog, the cyp2c9 homologous enzymes are cyp2c21 and cyp2b11 respectively (shen et al. 1997; shou et al. 2003). in the rat, the same metabolites are produced as in humans, that is, 3′-, 4′and 5′-hydroxydiclofenac (shen et al. 1997). although much less is known about avian cyp enzymes and their role in diclofenac metabolism (hunter, mahmood & martinez 2008), the domestic chicken remains the best-studied species. it is now known that, as in humans, the cyp2 family is predominant in the chicken, and the cyp2c homologs are currently known as cyp2c8/9, cyp2c18, cyp2c23a, cyp2c23b and cyp2c45 (kawalek et al. 2006; shang, jiang & deng 2013; watanabe et al. 2013). furthermore, the cyp2c8/9 homolog has been shown to produce 3′-, 4′and 5′-hydroxydiclofenac in broiler chickens (joseph et al. 2006). by inhibiting cyp2c9 homologs in a test group of domestic chickens (a species where the enzyme is known to function in diclofenac metabolism) with an azole inhibitor, we compared the pk parameters obtained with those from control birds which received only diclofenac. this was in an attempt to ascertain if the length of exposure and related toxicity in vultures may be because of cyp2c subfamily metabolic deficiency. since the drug is a phase 1 inhibitor, it was expected that it would result in an increase in exposure to diclofenac and reduction in exposure to metabolites. the chicken was specifically chosen since it has a defined ld50 of 10 mg/kg (naidoo et al. 2007), which whilst lower than other bird species, is still higher than for old-world vultures. for this study, two groups of birds were treated at the range of doses required to establish the ld50. the study was able to confirm that the ld50 was in the region of 10 mg/kg as previously found in other studies. more so, deaths were confirmed to once again occur with a consistent clinical and pathological picture (hassan et al. 2018; naidoo et al. 2007; oaks & watson 2011; swan et al. 2006). an unexpected finding was the death of the birds within 15 min in both groups. with the absence of pathology, the cause of death is possibly attributed to normal cardiac electrical disturbance, such as atrial fibrillation or flutter; this has been documented in humans, even at low doses of diclofenac (schmidt, sørensen & pedersen 2018). all other birds that succumbed died within an expected timeframe of 48 h after dosing, with the exception of a bird from the negative control group, which was euthanised for humane reasons at 56 h post-dosing. gross pathological signs were also typical, being serosal surface deposition of uric acid crystals and varying degrees of nephrosis. the original aim of the study was to determine if a cyp inhibitor would make diclofenac more lethal. from the results, it was evident that this was not the case and is likely an indication of insufficient inhibition of the enzyme, either from too low a dose or too short a duration. for the chosen dose, this was the listed dose for birds and was a convenient dose to administer intraperitoneally. the absence of a statistical difference between treatment groups, either for the pk parameters or for comparison of the diclofenac:metabolite ratio peaks, may also be as a result of sample size used – only eight birds from the test group and nine from the control group. we also acknowledge a shortcoming in the study design in that different individuals were used between treatment groups, and therefore inter-subject variation is a complicating factor; that is, as this was an ld50 toxicity study, it was not possible to use a crossover study design. evident from the study is the large coefficient of variation in the obtained pharmacokinetic parameters, which is indicative of a large intrasubject variability. this is not surprising as the cyp activity of various individuals is well known to vary, with some individuals being rapid metabolisers and others slow metabolisers (brunton et al. 2018; o’brien, chan & silber 2004; paulson et al. 1999). thus, a more valid indicator of effect in this study is in the pk trends and in the difference between the alive and dead animal, both within and between groups. despite a failure to indicate statistical significance, a trend was starting to emerge to suggest that some cyp inhibition resulted. the test group of chickens which received a cyp inhibitor demonstrated a mean pk curve suggestive of metabolic inhibition when compared to the control group, evident in the geometric mean values for auclast (0.5 μg/ml*h vs 0.4 μg/ml*h), cmax (0.61 μg/ml vs 0.41 μg/ml) and clearance (1.52 l/h*kg vs 1.59 l/h*kg). birds with suppressed metabolism would be expected to have a higher auclast. clearance (based on auclast) can be expressed per clearing organ through the following equation: where q is the blood flow to the organ and e represents the ‘extraction ratio’ or percentage of drug removed from the blood by that organ during a single passage (toutain & bousquet-mélou 2004). as it was not expected that changes in blood flow to metabolising organs would impact clearance in this study (naidoo & swan 2009), clearance would be related to the extraction ratio, e. with this being dependent on the intrinsic metabolising capability of an organ system, the lower geometric mean clearance for the cyp-inhibited test group of birds suggests capacity limited metabolism as a function of cyp enzyme inhibition as a plausible cause in support of the auclast values seen. when the within-groups effects are compared, the pk differences become more pronounced. in dead test group birds, there was higher exposure to diclofenac compared to dead control group birds (table 1), as evidenced by the higher geomean auclast (0.87 μg/ml*h vs. 0.31 μg/ml*h) and cmax values (1.24 μg/ml vs. 0.24 μg/ml) and lower cl values (0.88 l/h*kg vs. 0.32 l/h*kg). the presence of data from the two highest-dosed birds (both died within 15 min of dosing) in the test group may have increased the reflected difference in exposure between groups further and may be a factor in why t1/2 and mrt values are higher in dead control birds (the two highest-dosed birds contributed to the higher values for these parameters), where one would have expected them to be higher in test birds. from a metabolite aspect, the dead birds had lower ratios of metabolite to diclofenac than those that survived. this would support the assertion that cyp enzymes are involved with metabolism and that lower activity will result in death. whilst not the intention of the study, this confirmed earlier findings that toxicity is driven by metabolic capacity. an unexpected finding from this study was seen when comparing the metabolites between the two groups. one would have expected the cyp enzyme inhibition (test) group to have lower metabolites. this was not the case, and the opposite was evident. as mentioned above, this once again indicates that cyp inhibition did not occur significantly. more so, it shows how variable the cyp enzyme is in a species. this result may also explain why the chicken is more resilient than the vulture; that is, the higher degree of natural variation in cyp metabolism would explain the higher ld50 in the chicken in comparison to the vulture. conclusion whilst there was no significant difference in the ld50 for chickens given a known cyp2c9 inhibitor and those not, the mean pk curve and the ratio of hplc diclofenac to metabolite peaks for the dead birds dosed with the inhibitor were suggestive of partial inhibition of cyp2c functioning playing a role in toxicity. to confirm this, a larger sample size would be needed to cater for the large intrasubject variability seen. acknowledgements the authors wish to thank and acknowledge the involvement of the following personnel at the university of pretoria: mrs antonette van wyk, mrs ilse janse van rensberg and mr humbelani ratshibanda from the upbrc for their involvement with rearing the chickens for this project and assistance during the actual study; mrs fransie lottering and mrs madelyn de wet for organising purchase orders and other administrative functions involved with the project; ms arina ferreira, mr williams mokgojane and mr rexton ramuageli from the pharmacology and toxicology laboratory for assisting with lab equipment and the hplc machine. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.l. conceptualised the study, implemented the study protocol, analysed the samples, participated in data analysis and collated the first draft of the manuscript. v.n. conceptualised this project, arranged funding, participated in the implementation of the study, ran the pharmacokinetic data analysis and assisted with reviewing and editing of the manuscript. v.n. was also the master of science supervisor of s.l. n.d. conducted the gross and microscopic pathological examinations. i.h. directed and assisted with the hplc pharmacokinetic drug analysis. funding information this study was funded by the university of pretoria, department of paraclinical studies. data availability (1) the original thesis with associated raw data is available on the university of pretoria repository, using the following handle; https://repository.up.ac.za/handle/2263/75858. (2) the following figures have associated raw data: table 1, table 2 and figure 2. 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group have shown unexpectedly high numbers of helminth parasites in yellowfish species in the vaal dam. the high number of helminth parasites can be attributed to bothriocephalus acheilognathi which has been introduced with cyprinid fish into south africa from asia (brandt, van as, schoonbee & hamilton-attwell 1981). the asian tapeworm, originally a parasite of the chinese grass carp (ctenopharyngodon idella valenciennes, 1844) and the silver carp (hypothalmichthys molitrix valenciennes, 1844) in the southern parts of china (boomker, huchzermeyer & naudé 1980), has spread rapidly to other countries by means of infected fish (pool 1987) and has adapted itself successfully to the common carp (cyprinus carpio linnaeus, 1758) (boomker et al. 1980). in south africa, bothriocephalid parasites have been found in various dams and freshwater systems in 207 onderstepoort journal of veterinary research, 72:207–217 (2005) aspects of the ecology of the asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in the vaal dam, south africa a. bertasso and a. avenant-oldewage department of zoology, university of johannesburg*, p.o. box 524, auckland park, 2006 south africa abstract bertasso, a. & avenant-oldewage, a. 2005. aspects of the ecology of the asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in the vaal dam in south africa. onderstepoort journal of veterinary research, 72:207–217 seasonal surveys were conducted at the vaal dam between april 2000 and january 2001. twenty smallmouth yellowfish (labeobarbus aeneus) and 20 largemouth yellowfish (labeobarbus kimberleyensis) were collected with the aid of gill nets. surface water quality variables were included. the cestodes were identified as either bothriocephalus acheilognathi yamaguti, 1934 or “other cestode spp.”. the majority (99.8 %) of the cestodes found in both yellowfish species were identified as b. acheilognathi (asian tapeworm). the prevalence, mean intensity and abundance of b. acheilognathi in both yellowfish species were calculated. ecological parameters including species specificity, seasonality, gender specificity and relationships between fish size and the asian tapeworm prevalence were also included. in this study, b. acheilognathi preferred l. kimberleyensis over l. aeneus although a low intensity was observed in smallmouth yellowfish. furthermore, the infection (in terms of prevalence, abundance and mean intensity) in largemouth yellowfish was markedly higher. seasonal patterns observed in the asian tapeworm’s infection of smallmouth yellowfish are attributed to breeding and subsequent feeding patterns of this fish species with relatively high infections recorded in winter and spring. for l. kimberleyensis no explanation can be given regarding the seasonal patterns observed for the mean intensity and abundance of b. acheilognathi. the maximum and minimum mean intensity and abundance values in largemouth yellowfish were recorded in autumn and spring, respectively. in addition, the prevalence of b. acheilognathi was consistently high in all four seasons. keywords: asian tapeworm, bothriocephalus acheilognathi, labeobarbus aeneus, labeobarbus kimberleyensis, seasonal prevalence, yellowfish * formerly rand afrikaans university accepted for publication 15 april 2005—editor gauteng province. localities in south africa where b. acheilognathi has been found include the komatipoort area (mpumalanga province) (boomker et al. 1980), marble hall (mpumalanga province) (brandt et al. 1981), boskop dam (kwazulu-natal province) (van as, schoonbee & brandt 1981), hartbeespoort dam (north west province), piet gouws dam (limpopo province) (mashego 1982), olifants river (limpopo province) (mashego 1982), glen alpine dam (limpopo province) (mashego 1982) and the vaal dam (gauteng province) (mashego 1982). the tapeworm’s presence in most of the localities mentioned above can be attributed to the supply of common carp fry to commercial farmers (boomker et al. 1980). however, according to mashego (1982) its presence in the vaal dam cannot be accounted for. brandt et al. (1981) mentioned the possibility that this tapeworm was imported into south africa with the common carp as long ago as 1859 or with the dinkelsbühl aischgrund variety of the common carp in 1952. cyprinus carpio has been found in the vaal dam but its introduction date is uncertain. bothriocephalus acheilognathi was originally described as three different species from wild fish in japan (as b. acheilognathi yamaguti, 1934 and bothriocephalus opsariichthydis yamaguti, 1934) and from grass carp (c. idella) from south china (as bothriocephalus gowkongensis yeh, 1955) (paperna 1996). these three species were later recognised as being identical (körting 1975; molnár 1977) with the name b. acheilognathi taking priority. pool (1988) believes that the three species, b. acheilognathi, bothriocephalus kivuensis and bothriocephalus aegyptiacus, are identical, with b. acheilognathi having priority. various authorities (as cited by pool & chubb 1985 and pool 1988) noted that bothriocephalus opsariichthydis yamaguti, 1934, bothriocephalus fluviatilis yamaguti, 1952, b. gowkongensis yeh, 1955, bothriocephalus phoxini molnár, 1968 and schyzocotyle fluviatilis akhmerov, 1960 are the same species as b. acheilognathi yamaguti, 1934. pool & chubb (1985) concluded that there is only one bothriocephalus species parasitizing cyprinid fish and suggested the continued use of the name b. acheilognathi. cestodes in this study were identified as either b. acheilognathi or “other cestode spp.”. the purpose of this article is to provide a brief description of the parasite’s infection, seasonality, gender specificity and species (host) specificity in the two yellowfish species sampled. a comparison between the parasite’s infection in labeobarbus aeneus and its infection in labeobarbus kimberleyensis is also included. materials and methods study location four surveys were conducted in the vaal dam at groot eiland, also known as rau island—one per season, namely in april 2000 (early autumn), june 2000 (winter), october 2000 (late spring) and january 2001 (summer). the vaal dam is located in the upper reaches of the vaal river. water quality water quality data for the duration of the project were obtained from the rand water board, and were collected during routine monitoring activities made by the rand water board. standard techniques were used by the rand water board to analyse the water samples. for some months various parameters were not measured, the reasons being that sampling and/ or measuring instruments were either not in working order or being serviced. the following surface water variables were included: ph, temperature, electrical conductivity, dissolved oxygen and light penetration. collection of fish and cestodes a field laboratory was set up for each survey. the fish species collected during the four surveys were l. aeneus (smallmouth yellowfish) and l. kimberleyensis (largemouth yellowfish). the fish were identified based on the size of the snout as suggested by skelton (2001). twenty largemouth and 20 smallmouth yellowfish per survey were collected using gill nets consisting of four sections with varying stretched mesh sizes of 90, 110 and 130 mm, respectively. they were weighed (in grams) and measured (fork length in millimetres). the fish were killed by severing the spinal cord behind the head and were subsequently dissected by making an insertion from the anus towards the head. once they had been dissected, the intestines were removed and placed in a normal saline solution in petri dishes for examination. methods described by khalil (1991) were used for processing the platyhelminth parasites found during the surveys. parasites were collected as soon as possible after the death of the fish to prevent any deterioration. the intestines were pulled open carefully using two sharp tweezers to ensure that the cestodes were kept intact. each cestode was carefully and slowly dislodged from the intes208 asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in south africa tinal wall, ensuring that it remained intact. they were transferred to a clean sampling bottle containing normal saline solution, which was then shaken vigorously for a few minutes to dislodge debris and induce muscle fatigue in the helminths, which in turn, deters strong contraction of the scolices and relaxes them. while swirling the sampling bottle, an equal amount (equal to the amount of saline solution already present in the sampling bottle) of a hot alcohol-formaldehyde-acetic acid (afa) solution was added to kill and fix the specimens. specimens were then stored in 70 % alcohol. identification of cestodes the cestodes were stained with grenacher’s borax armine stain (pantin 1964) and identified. statistical analyses all specimens were counted and the totals obtained were used for statistical analyses which were conducted by the university statistical consultation services. prevalence, abundance and mean intensity of b. acheilognathi were calculated per season for each fish species. infection statistics were calculated by making use of the definitions set by margolis, esch, holmes, kurtis & schad (1982) and bush, lafferty, lotz & shostak (1997). data was analysed to determine the seasonality (using anova and then scheffe or dunnet t3) and species and gender specificity (using pearson chisquare test and t-tests) of b. acheilognathi. a comparison between the infections in the two fish species was done (using the t-test). in addition, the infection of b. acheilognathi (intensity) in each fish species was compared to the size (fork length) of the fish sampled. regression analysis was used to determine if any correlations existed. digital micrographs of stained specimens were taken using a zeiss axioplan 2 imaging microscope. results water quality surface water variables and data obtained in this study are presented in table 1. identification of cestodes according to mashego (1982), the classification of the bothriocephalid worms is based primarily on the shape of the scolex. pool (1984) concluded from his study on b. acheilognathi that the identification of adults should be based on the heart-shaped scolex and prominent square apical disc. the identification of the bothriocephalid cestodes in this study was therefore based on these characteristics. specimens found in the current study were compared to sketches provided by various authorities (yamaguti 1934; yeh 1955; molnár & murai 1973; mashego 1982) as well as the diagnosis of b. acheilognathi (as b. gowkongensis) by yeh (1955), as cited by paperna (1996). micrographs and sketches of b. acheilognathi collected during the four surveys are presented in fig. 1. when reviewing available sketches it is the authors’ opinion that the scolex of specimens from the current study compared fairly well with many of the bothriocephalus species that have a heart-shaped scolex (fig. 1a). as mentioned in paperna (1996), the eggs are operculated (fig. 1f) and the vitellaria laterally scattered (fig. 1d). according to the diagnosis given in paperna (1996), mature and gravid segments vary in breadth and length. however, in this study this was not the case. most segments were broader than they were long (fig. 1b and c). we however, disagree with the system of labelling the reproductive system as proposed by yamaguti (1934), and consider that, after reviewing the available literature, it should be as indicated in fig. 1e. 209 a. bertasso & a. avenant-oldewage table 1 summary of water quality variables recorded seasonally at the vaal dam survey ph temperature electrical dissolved secchi disc conductivity oxygen readings (ph units) (°c) (ms/m) (mg/l o2) (cm) autumn (april 2000) 7.60 – 17.00 – – winter (june2000) 7.34 19.90 16.00 12.50 30.00 spring (october 2000) 8.00 21.40 17.00 – 28.00 summer (january 2001) 8.26 25.00 23.00 6.30 28.00 – no data available 210 asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in south africa fig. 1 micrographs and sketches of bothriocephalus acheilognathi collected during the four seasons of the survey in the vaal dam a heart-shaped scolex with bothria b mature proglottid with reproductive organs c young adult proglottid d vitellaria scattered e sketch of a young adult proglottid f operculated egg abbreviations: cs – cirrus sac; ut – uterus; ov – ovary; vit – vitellaria; t – testes; vd – vas deferens bothria testes gental atrium cirrus sac uterus ovary vitellaria egg coils of vas deferens ovary testestestes uterine coils cirrus sac a b c d e f parasite numbers the tapeworms encountered in this study were grouped as either b. acheilognathi or “other cestode spp.”. the number of b. acheilognathi and other cestode spp. collected during the four seasons of the survey is tabulated in table 2. of the 160 fish sampled, only 19 out of 80 l. aeneus harboured b. acheilognathi while none harboured other cestode spp.; and 68 out of 80 l. kimberleyensis harboured b. acheilognathi while only six harboured other cestode spp. infection statistics of b. acheilognathi the percentage of hosts (prevalence) infected with b. acheilognathi, and its intensity (mean intensity) and abundance (relative density) in both l. aeneus and l. kimberleyensis are illustrated graphically in fig. 2, 3 and 4, respectively. a statistical comparison (t-test) of the two fish species in terms of b. acheilognathi prevalence, abundance and mean intensity is also included to determine whether or not there are significant differences between fish species. 211 a. bertasso & a. avenant-oldewage table 2 number of cestodes collected from labeobarbus aeneus and labeobarbus kimberleyensis at the vaal dam during the four seasons labeobarbus aeneus (n = 80) labeobarbus kimberleyensis (n = 80) survey b. acheilognathi other cestode spp. b. acheilognathi other cestode spp. autumn (april 2000) 4 0 3 417 4 winter (june2000) 298 0 1 651 0 spring (october 2000) 256 0 1 120 1 summer (january 2001) 24 0 2 040 3 total 582 0 8 216 8 total cestodes 582 8 228 ��� �� �� �� �� �� �� � � �� � �� ��� ��� ������ ������ � �� � � � � � � � � ����� ��� ����� ������ � ��� �� ��� ����� �� ��� ����� ����������� �� ��� �!���" ����������� �� ������� ��� �!���" ����������� �� ��� � ��#��� ����������� �� ������� ��� � ��#��� ��� ��� ��� � � ��� �� �� �� � � �� ��� ��� ������ ������ � � � � � � � ����� ��� ����� ������ � ��� �� ��� ����� �� ��� ����� ����������� �� ��� �!���" ����������� �� ������� ��� �!���" ����������� �� ��� � ��#��� ����������� �� ������� ��� � ��#��� fig. 2 graph depicting the prevalence of bothriocephalus acheilognathi in labeobarbus aeneus and labeobarbus kimberleyensis during the four seasons fig. 3 graph depicting the abundance (relative density) of bothriocephalus acheilognathi in labeobarbus aeneus and labeobarbus kimberleyensis during the four seasons of the survey prevalence the prevalence of b. acheilognathi in l. kimberleyensis was relatively constant over all seasons, ranging from 80–90 %, whereas the prevalence in l. aeneus was fairly constant (10–15 %) in autumn, spring and summer followed by a considerable increase in winter (55 %) (fig. 2). when comparing its prevalence in the two fish species, that in l. kimberleyensis was considerably higher. statistical analyses indicate that, there was a significant difference (t-test, p = 0.001) in its prevalence in the two fish species. abundance (relative density) during all surveys, the abundance of the asian tapeworm was considerably higher in l. kimberleyensis (fig. 3). in this fish species, abundance values ranged from 55.0 (spring) to 170.9 (autumn) while in l. aeneus these values ranged from 0.2 (autumn) to 14.9 (winter). the following seasonal trend was observed for largemouth yellowfish: values decreased from autumn to winter and then again in spring. the latter was followed by an increase in summer. for smallmouth yellowfish the opposite trend was observed. values increased from autumn to winter and were followed by a decrease in spring and a further decrease in summer. statistical analyses indicate that there was a significant difference (t-test, p = 0.011) between the relative densities of b. acheilognathi in the two fish species sampled. mean intensity excluding spring, the infection was considerably more intense in l. kimberleyensis (fig. 4). the highest value recorded for l. kimberleyensis was 213.6 in autumn and the lowest value was 68.8 recorded in spring. during the remaining seasons the mean intensity decreased to 102.2 and 85.8 in summer and winter respectively. similarly, the mean intensities in l. aeneus differed considerably from 2.00 in autumn to 78.7 in spring. during winter and summer a mean intensity of 27.1 and 4.3, respectively, was recorded. statistical analyses indicate that, there could be a significant difference between the mean intensities of b. acheilognathi in the two fish species (t-test, p value was slightly above 0.05 at 0.053) but due to either the sample size being too small or the variance being too big this cannot be said for certain. ecological parameters gender specificity in both fish species, there were no significant differences (t-test, p values > 0.05) in the average number of b. acheilognathi found in males and females even though the average number in males and females differed. in l. aeneus the average numbers of b. acheilognathi in male and in female fish were 12.85 and 2.70, respectively. in l. kimberleyensis the average numbers were 91.43 and 107.42, respectively. in addition, the presence or absence of b. acheilognathi is not dependent on the gender of fish species (pearson chi-square test; p values for both fish species > 0.05). similar numbers of male (7) and female (12) l. aeneus and male (37) and female (31) l. kimberleyensis were found to harbour b. acheilognathi. in l. aeneus, the prevalence of b. acheilognathi in males and females was 0.21 and 0.26, respectively. similarly, in l. kimberleyensis the prevalence of b. acheilognathi was 0.84 in males and 0.86 in females. seasonality in l. aeneus, the highest number of b. acheilognathi were observed during the winter survey while in l. kimberleyensis the highest number was observed during the spring survey (table 2). from 212 asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in south africa �� �� ��� ��� �� � � � � �� �� � �� � �� ��� ��� ������ ������ ����� ��� ����� ������ � ��� �� ��� ����� �� ��� ����� ����������� �� ��� �!���" ����������� �� ������� ��� �!���" ����������� �� ��� � ��#��� ����������� �� ������� ��� � ��#��� fig. 4 graph depicting the mean intensity of bothriocephalus acheilognathi in labeobarbus aeneus and labeobarbus kimberleyensis during the four seasons the statistical analyses (pearson chi-square test) performed on the data, the presence/absence of b. acheilognathi in l. aeneus was dependent on the season (p = 0.002) with the highest number of infected fish (11) being in those caught in winter and the lowest (2) being in autumn. this was not the case for l. kimberleyensis. similar numbers of l. kimberleyensis (between 16 and 18) were infected with b. acheilognathi during all four seasons of the survey. however, when conducting the anova test to determine if there were significant differences between seasons, the results show that there were significant (p = 0.003) seasonal differences based on the number of b. acheilognathi found in l. kimberleyensis. post hoc tests were then undertaken to distinguish which of the seasons differed significantly. the results of the statistical analyses (dunnet t3 test) showed that autumn and spring differed significantly with a p value of 0.011. no significant seasonal differences were determined in the number of b. acheilognathi found in l. aeneus. species specificity when comparing fish species, l. kimberleyensis harboured a higher number of b. acheilognathi than l. aeneus (table 2). the number of b. acheilognathi found in l. kimberleyensis totalled 8 216, while l. aeneus only had a total of 582 b. acheilognathi in the same number of hosts (80). in addition, a higher number of l. kimberleyensis (68) were infected with b. acheilognathi than l. aeneus (19). from the statistical analyses (pearson chi-square) performed when data is pooled according to fish species, the presence/absence of b. acheilognathi was highly dependent on fish species, p values being 0.000. size specificity as can be seen in fig. 5, no correlations were observed between the sizes (fork lengths) of yellowfish (both smallmouth and largemouth) sampled and the number of asian tapeworms recorded. discussion exceptionally high numbers of b. acheilognathi and low numbers (an insignificant amount) of other cestode spp. were found in the yellowfish sampled. as highlighted by khan & thulin (1991), parasites are a natural part of the aquatic community and their distribution and abundance are potentially either directly or indirectly affected by a number of biotic and abiotic factors. 213 a. bertasso & a. avenant-oldewage ��� ��� �� �� �� �� ��� ��� �� � �� ������������ ��� � � � � � �� � �� � � �� �� � � � � �� � � � � �� � � � �� � ����������� �� ��� ������� ������ ������� ������� ���������������� ����������������� � $�����!�%�&��� � ��� ��� �� �� �� �� ��� ��� �� � �� ������������ ��� � � � � � �� � �� � � �� �� � � � � �� � � � � �� � � � �� � ����������� �� ������� ��� ������� ������� ����������������������������������� ���������� � $�����!��&��� ' fig. 5 graphs showing that there was no correlation between the size (fork length) of labeobarbus aeneus (a) and labeobarbus kimberleyensis (b) sampled during the four seasons and the number of bothriocephalus acheilognathi. the statistically calculated p value is included infection the infection of b. acheilognathi (in terms of prevalence, abundance and mean intensity) in l. kimberleyensis was greater than that observed in l. aeneus. noticeably higher numbers of this tapeworm were observed in largemouth yellowfish and a noticeably higher number of this fish species was infected. similar prevalence and mean intensity values to those obtained in this study were recorded for l. aeneus and l. kimberleyensis in a separate preliminary study conducted by nickanor, reynecke, avenant-oldewage & mashego (2002) in the vaal dam during 2001. the infection statistics indicate that both prevalence and abundance of b. acheilognathi differed significantly (statistically) between the fish species with l. kimberleyensis exhibiting higher values. the mean intensity of b. acheilognathi was higher in l. kimberleyensis for most seasons, except in spring when the opposite was the case. in spring a small number of l. aeneus were infected with a relatively high number of tapeworms thereby increasing the mean intensity. it is possible that the mean intensities of the fish species differ significantly but this is uncertain in this study because either the sample size was too small or the variance was too big to distinguish between the fish species. it would be expected that the high numbers (and subsequently high prevalence, abundance and mean intensity) of b. acheilognathi found in l. kimberleyensis in this study are linked to the life cycle of the tapeworm as the transmission of parasite to host is via an intermediate host eaten by the fish (paperna 1996). körting (1975) mentioned that the intermediate host of the asian tapeworm for carp is a crustacean and that a number of crustaceans can act as intermediate hosts. when considering the food preference of largemouth yellowfish, it is found that it initially feeds on insects and crustaceans, but once it reaches a fork length of more than 300 mm it feeds on other fish (skelton 2001). although the majority of infected largemouth yellowfish collected during the survey varied in fork length between 360 and 420 mm, it is possible that infected crustaceans are eaten occasionally, resulting in an infection in l. kimberleyensis. it was unexpected, however, to find an infection as heavy as that obtained in this study. when considering the infection of b. acheilognathi (in terms of prevalence, mean intensity and abundance) in relation to fish size (fork length), no correlations were recorded in l. kimberleyensis (fig. 5). it is important to note that l. kimberleyensis was the first recorded host of b. acheilognathi (as b. gowkongensis) in south africa in 1978 (brandt et al. 1981). the type of crustacean or copepod acting as an intermediate host for b. acheilognathi for this fish species should be determined. this opportunistic tapeworm has already adapted successfully to the common carp, cyprinus carpio (körting 1974; boomker et al. 1980) in south african waters. this carp species feeds on a range of plant and animal matter (skelton 2001) and, more specifically, carp fry, which tend to be more heavily infected with the tapeworm, feed on zooplankton (boomker et al. 1980). a better understanding of the intermediate host could explain the high infection of b. acheilognathi in l. kimberleyensis. other authorities, such as marcogliese & esch (1989) and williams & jones (1994), mention that metacestodes use planktonic or benthic copepods as intermediate hosts. if this is the case, then l. aeneus should be the preferred host as this fish species is broadly omnivorous with zooplankton, benthic invertebrates, vegetation, algae and detritus forming the major food of the species (dörgeloh 1985; skelton 2001). although l. aeneus is not the preferred host (nickanor et al. 2002; this study), it still becomes infected (although low) with b. acheilognathi. poulin (1998) mentioned that parasites such as the asian tapeworm that enter their host through ingestion, cannot prevent non-host species from eating the infected intermediate hosts. this method of transmission (ingestion) enables more host species to become infected. another reason why both fish species are infected with this tapeworm could be that the intermediate copepod host species can vary considerably. various genera of copepods have been found to be compatible intermediate hosts (williams & jones 1994; paperna 1996) and in this case it is possible that a larger crustacean, such as a crab, acts as a paratenic host—this would explain the enigma behind the higher infection observed in largemouth yellowfish. in a study conducted by de leon, garcia-prieto, leon-regagnon & choudhury (2000) in mexico it was found that helminth communities were generally more abundant in carnivorous fish species than in herbivores and detritivores. this matter needs to be researched further in order to gain a better understanding of infections by the asian tapeworm. seasonal trends in l. aeneus, the prevalence of b. acheilognathi was fairly constant except in winter when noticeably higher numbers of smallmouth yellowfish were infected. prevalence values in winter were approximately four to five times higher than in the remain214 asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in south africa ing seasons (fig. 2). this could be due to a change in feeding regime, but during the four seasonal surveys it has been found that l. aeneus fed well (bertasso 2004). körting (1974) indicated that early spring, when plankton grows, is likely to be a significant season in terms of seasonal incidence and infective period. this was not the case, however, in l. aeneus in this study. in spring, the prevalence was fairly low. the reason could be that more infected food (copepods) were available in that particular winter than is usually the case. temperature data recorded during the winter of this study (table 1) were higher than in a previous separate study conducted in the vaal dam (crafford 2000). in winter visibility (light penetration) in the vaal dam was at its maximum although in the remaining three seasons similar (slightly lower) values were exhibited (table 1). it can be assumed that the finding of an increased prevalence in winter is exceptional and was caused by an external unknown factor. statistical analyses indicated that, as a result of this high prevalence in winter, the presence of b. acheilognathi in l. aeneus is dependent on the season. in l. kimberleyensis, no trend was observed for prevalence of the asian tapeworm throughout the four seasons (fig. 2); a similar, very high number of fish being infected throughout the year. abundance values in l. aeneus exhibited a pattern similar to the prevalence values observed in the same fish species (fig. 3). values increased considerably from autumn to winter followed by a decline in spring and a further decline in summer. it is assumed that this seasonal trend is related to the breeding and subsequent feeding patterns of l. aeneus. feeding habits of the host account for a large percentage of the variation in the total number of parasites per host species (williams & jones 1994). the number of parasites in a host would depend on how much the host eats and whether the food is infected. in winter, the fish eat enough food to sustain them through the breeding season, which lasts from spring through to late summer (skelton 2001) and explains the decrease in abundance recorded in spring and summer. as soon as the breeding season ends fish start eating again. this results in an increase in abundance from autumn to winter. in l. kimberleyensis, the seasonal trend in abundance could not be attributed to the fish’s breeding patterns. largemouth yellowfish breed in mid to late summer (skelton 2001) which would mean that abundance values should be higher in winter and spring, but nevertheless the opposite was observed (fig. 3). a sharp decrease in abundance values was recorded from autumn to winter after which abundance values remained relatively constant decreasing slightly in spring but increasing again in summer. this seasonal trend could be related to changes in the amount of food and subsequently infected food available. in autumn the high abundance relative to the other three seasons was due to the considerably higher number of b. acheilognathi found in the l. kimberleyensis in the survey. a similar pattern to that recorded for the abundance of b. acheilognathi in l. kimberleyensis was recorded for the mean intensity (fig. 4). this is due to the fact that most of the fish sampled during the four seasons were infected with b. acheilognathi. the mean intensity in l. aeneus also exhibited a similar pattern to that of the abundance in l. aeneus, except that the mean intensity peaked in spring rather than in winter (fig. 4). mean intensities in the smallmouth yellowfish increased considerably from autumn to winter followed by a significant increase (three times that of winter) in spring after which the intensity decreased in summer to an intensity similar to that recorded in autumn. in spring, the mean intensity recorded in l. aeneus was even higher than that recorded in l. kimberleyensis. opposite seasonal trends were recorded for the mean intensity of the two fish species. statistical analysis indicates that the occurrence of b. acheilognathi in smallmouth yellowfish is dependent on the season with the highest number of fish infected in winter. however, no statistically meaningful differences were observed in the intensity of b. acheilognathi found during each season. the opposite is true for the largemouth yellowfish sampled in this study. statistical analysis indicates that the occurrence of b. acheilognathi was not dependent on the season, even though there were significant statistical differences between the intensity of b. acheilognathi recorded in autumn and that recorded in spring. fish gender and species specificity statistical analysis indicates that the presence of b. acheilognathi is highly dependent on the species of fish with l. kimberleyensis, as mentioned above, having the highest infection of the two yellowfish species. poulin (1998) noted that high host specificity could be an artefact of inadequate sampling but in this study 20 fish per species per season were collected which cancels out this possibility. in addition, various studies conducted in the vaal and olifants river systems, in which cestode endoparasites have been incorporated, have revealed either 215 a. bertasso & a. avenant-oldewage an absence of cestodes or low infections of them in a range of fish species, namely clarias gariepinus (marx 1996; crafford 2000; watson 2001), labeobarbus marequensis (watson 2001), oreochromis mossambicus (watson 2001), labeo capensis and labeo umbratus (groenewald 2000). when pooling the data according to the sex of the fish, the tapeworms exhibited no preference for male or female fish. similar numbers of male and female l. kimberleyensis were infected. even though there were noticeably lower numbers of infected female smallmouth yellowfish when compared to males there was no dependency (statistically) on fish gender. size specificity in both yellowfish species sampled no correlations were observed between fish size and asian tapeworm infection (fig. 5). the statistical p values were closer to 0 than 1. conclusion in this study, the majority of the tapeworms were identified as b. acheilognathi based on the heartshaped scolex and presence of bothria. this was achieved after comparing the specimens collected with the descriptions and sketches in the literature. bothriocephalus acheilognathi in this study was species (host) specific with a considerably higher infection (in terms of prevalence, abundance and mean intensity) recorded in l. kimberleyensis (largemouth yellowfish). the reason for this has still not been determined. the asian tapeworm in this study was not fish-gender specific. seasonal trends were observed for prevalence, abundance and mean intensity of the tapeworm in l. aeneus although statistical analyses indicate that no significant differences existed between seasons. seasonal trends in smallmouth yellowfish were attributed to breeding and subsequent feeding patterns of the fish. in l. kimberleyensis, no seasonal trend was recorded for prevalence. the abundance and mean intensities of b. acheilognathi, however, varied seasonally with the highest value being recorded in autumn and the lowest value in spring. statistical analyses indicate that there was a significant difference between the presence of b. acheilognathi in these two seasons. the reason for the seasonal trends observed in largemouth yellowfish cannot be explained. further research on largemouth yellowfish concentrating on factors such as post-spawning migrations of the host, schooling behaviour, age of host, reproductive behaviour, host feeding behaviour, host hormone levels/state of maturity, immunological response of host, availability of infected intermediate hosts as food, site of infection, negative interaction between parasites (as outlined by williams & jones 1994) should be conducted to provide an explanation as to why there are seasonal variations in b. acheilognathi’s infections in this fish species. acknowledgements we thank the national research fund (nrf) and rau sasol fund for providing money and prof. s.n. mashego for assistance in identifying the tapeworms. references bertasso, a. 2004. ecological parameters of selected helminth species in labeobarbus aeneus and labeobarbus kimberleyensis in the vaal dam, and an evaluation of their influence on indicators of environmental health. m.sc. dissertation, rand afrikaans university. boomker, j., huchzermeyer, f.w. & naudé, t.w. 1980. bothriocephalosis in the common carp in the eastern transvaal. journal of the south african veterinary association, 51: 263–264. brandt, f. de w., van as, j.g., schoonbee, h.j. & hamilton-attwell, v.l. 1981. the occurrence and treatment of bothriocephalus in the common carp, cyprinus carpio in fish ponds with notes on its presence in the largemouth yellowfish barbus kimberleyensis from the vaal dam, transvaal. water sa, 7:35–42. bush, a.o., lafferty, k.d., lotz, j.m. & shostak, a.w. 1997. parasitology meets ecology on its own terms: margolis et al. revisited. journal of parasitology, 83:575–583. crafford, d. 2000. application of a fish health assessment index and associated parasite index on clarias gariepinus (sharptooth catfish) in the vaal river system, with reference to heavy metals. m.sc. dissertation, rand afrikaans university. de leon, g.p.p., garcia-prieto, l., leon-regagnon, v. & choudhury, a. 2000. helminth communities of native and introduced fishes in lake patzcuaro, michoacan, mexico. journal of fish biology, 57:303–325. dörgeloh, w. 1985. food selection and competition for food among three fish species, salmo giardneri, barbus aeneus and clarias gariepinus. south african journal of science, 81: 693. groenewald, m. 2000. bioaccumulation of metals and the general health of fish from the vaal dam and vaal river barrage. m.sc. dissertation, rand afrikaans university. khalil, l. 1991. techniques for identification and investigative helminthology. st albans: international institute of parasitology. khan, r.a. & thulin, j. 1991. influence of pollution on parasites of aquatic animals. advances in parasitology, 30:201– 238. 216 asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 in yellowfish in south africa körting, w. 1974. bothriocephalosis of the carp. veterinary medical review, 2:165–171. körting, w. 1975. larval development of bothriocephalus sp. (cestoda: pseudophyllidea) from carp (cyprinus carpio l.) in germany. journal of fish biology, 7:727–733. marcogliese, d.j. & esch, g.w. 1989. experimental and natural infection of planktonic and benthic copepods by the asian tapeworm, bothriocephalus acheilognathi. proceedings of the helminthology society of washington, 56:151– 155. margolis, l., esch, g.w., holmes, j.c., kurtis, a.m. & schad, g.a. 1982. the use of ecological terms in parasitology (report of an ad hoc committee of the american society of parasitologists). journal of parasitology, 68:131– 133. marx, h.m. 1996. evaluation of a health assessment index with reference to metal bioaccumulation in clarias gariepinus and aspects of the biology of the parasite lamproglena clariae. m.sc. thesis, rand afrikaans university. mashego, s.n. 1982. a seasonal investigation of the helminth parasites of barbus species in water bodies in lebowa and venda, south africa. ph.d. thesis, university of the north. molnár, k. 1977. on the synonyms of bothriocephalus acheilognathi yamaguti, 1934. parasitologia hungarica, 10: 61–62 molnár, k. & murai, é. 1973. morphological studies on bothriocephalus gowkongensis yeh, 1955 and b. phoxini molnár, 1968 (cestoda, pseudophyllidea). parasitologia hungarica, 6:99–108. nickanor, n., reynecke, d.p., avenant-oldewage, a. & mashego, s.n. 2002. a comparative study of stomach and intestine contents in barbus aeneus and barbus kimberleyensis in the vaal dam to clarify variance in tapeworm infestation. journal of south african veterinary association, 73(3)142–159. pantin, c.f.a. 1964. notes on microscopical techniques for zoologists. cambridge: cambridge university press. paperna, i. 1996. parasites, infections and diseases of fish in africa—an update. rome: fao (cifa technical paper, no. 31). pool, d. 1984. a scanning electron microscope study of the life cycle of bothriocephalus acheilognathi yamaguti, 1934. journal of fish biology, 25:361–364. pool, d.w. & chubb, j.c. 1985. a critical scanning electron microscope study of the scolex of bothriocephalus acheilognathi yamaguti 1934, with a review of the taxonomic history of the genus bothriocephalus parasitizing cyprinid fish. systematic parasitology, 7:199–211. pool, d.w. 1987. a note on the synonymy of bothriocephalus acheilognathi yamaguti 1934, b. aegyptiacus ryšavý and moravec, 1975 and b. kivuensis baer and fain, 1958. parasitology research, 73:146–150. pool, d.w. 1988. an experimental study of the biology of bothriocephalus acheilognathi yamaguti 1934 (cestoda: pseadophyllidea). abstract of thesis, university of liverpool. poulin, r. 1998. evolutionary ecology of parasites: from individuals to communities. london: chapman & hall. skelton, p. 1993. a complete guide to the freshwater fishes of southern africa. halfway house: southern book publishers. skelton, p. 2001. a complete guide to the freshwater fishes of southern africa, 2nd ed. halfway house: southern book publishers. van as, j.g., schoonbee, h.j. & brandt, f. de w. 1981. further records of the occurrence of bothriocephalus (cestoda: pseudophyllidea) in the transvaal. south african journal of science, 77:343. watson, r. 2001. the evaluation of a fish health assessment index as a biomonitoring tool for heavy metal contamination in the olifants river catchment area. ph.d. thesis, rand afrikaans university. williams, h. & jones, a. 1994. parasitic worms of fish. london: taylor & francis. yamaguti, s. 1934. studies on the helminth fauna of japan. part 4: cestodes of fish. japanese journal of zoology, 6:1– 112. yeh, l.s. 1955. on a new tapeworm bothriocephalus gowkongensis n. sp. (cestoda: bothriocephalidae) from freshwater fish in china. acta zoologica sinica, 7:69–74. 217 a. bertasso & a. avenant-oldewage horak_291-306.qxd introduction during the past 40 years large tracts of land along the southern coast of the eastern cape province, south africa, stretching from approximately east london (33°01’ s, 27°53’ e) in the east to humansdorp (34°02’ s, 24°46’ e) in the west, have been planted to kikuyu grass (pennisetum clandestinum). this is a temperate zone with frost-free winters and 291 onderstepoort journal of veterinary research, 71:291–306 (2004) parasites of domestic and wild animals in south africa. xlv. helminths of dairy calves on dry-land kikuyu grass pastures in the eastern cape province i.g. horak1, ursula evans2 and r.e. purnell3 abstract horak, i.g., evans, ursula & purnell, r.e. 2004. parasites of domestic and wild animals in south africa. xlv. helminths of dairy calves on dry-land kikuyu grass pastures in the eastern cape province. onderstepoort journal of veterinary research, 71:291–306 successive pairs of approximately 4-month-old friesland bull calves, raised under worm-free conditions, were exposed to helminth infection for 14 days on dry-land kikuyu grass pastures at 28-day to monthly intervals, on a coastal farm in a non-seasonal rainfall region of the eastern cape province. with the exception of one pair of calves exposed for 28 days, this procedure was repeated for 28 consecutive months from december 1982 to march 1985. the day after removal from the pastures one calf of each pair was slaughtered and processed for helminth recovery and the other 21 days later. both members of the last four pairs of calves were killed 21 days after removal from the pastures. sixteen nematode species were recovered from the calves, and infection with ostertagia ostertagi was the most intense and prevalent, followed by cooperia oncophora. the calves acquired the greatest number of nematodes from the pastures from june to october of the first year and from june to august of the second year of the survey. few worms were recovered from the tracer calves examined from november or december to march or april in each year of the survey. the seasonal patterns of infection with cooperia spp., haemonchus placei, nematodirus helvetianus, oesophagostomum spp., o. ostertagi and trichostrongylus axei were all similar and were negatively correlated to atmospheric temperature and evaporation. slight to moderate arrest in the development of fourth stage larvae occurred from july to september in cooperia spp., april to july in h. placei, and august to october in o. ostertagi and trichostrongylus spp. during the first year of the survey. too few worms were present in the second year to determine a seasonal pattern of arrest. species survival during the hot and windy summer months appeared to be achieved via a combination of arrested larval development and an ageing residual population of adult worms in the host, and a small extant population of infective larvae on the pastures. keywords: cooperia spp., eastern cape province, haemonchus placei, kikuyu grass pastures, nematodirus helvetianus, oesophagostomum spp., ostertagia ostertagi, seasonality, tracer calves, trichostrongylus axei 1 department of zoology and entomology, university of the free state, p.o. box 339, bloemfontein, 9300 south africa 2 5 diana close, constantia, 7800 south africa 3 pfizer central research, sandwich, kent, england present address: cherry trees, wootton close, petham, ct4 5we, england accepted for publication 7 april 2004—editor non-seasonal rainfall. the summers are hot and windy and often interspersed with short droughts. the dry-land kikuyu grass pastures are usually heavily fertilized and are highly productive, and because the grass is not suitable for making hay it is best utilized by grazing animals, particularly dairy cows and replacement heifers. the stocking rate is high and most dairy farmers consider nematode parasites a constraint to the raising of stock. several surveys of helminths parasitizing small domestic ruminants on farms in the eastern cape province and adjacent southern regions of the western cape province have been conducted. rossiter (1964) and muller (1968) have examined sheep on coastal farms, barrow (1964) and horak (2003) sheep on inland farms, and boomker, horak & macivor (1989), fivaz, horak & williams (1990) and horak, knight & williams (1991) have done so for goats on inland farms. no such studies have been conducted on the helminths of cattle in this region. this paper describes the seasonal patterns of helminth infection in dairy calves on a coastal farm near alexandria. the research was conducted a number of years ago, but for various reasons was not published. despite the passage of time the results remain unique as this is the first survey of helminths in cattle in this region, and the seasonal intensity of infection differs considerably from that recorded in cattle elsewhere in south africa. the ixodid ticks recovered from the survey animals have been reported separately (horak 1999). materials and methods study site the farm “glen dye” (33°45’ s, 26°29’ e; alt. 137 m) is situated approximately 3 km from the coast south of alexandria, eastern cape province, and at the time of the survey was 232 ha in extent. with the exception of patches of indigenous coastal forest, retained for windbreaks and shade, virtually the entire farm has been planted to kikuyu grass and divided into paddocks approximately 1 ha in size. these paddocks are not irrigated, but are heavily fertilized, and are used mainly as grazing for dairy cattle. rainfall is non-seasonal and the long-term mean annual total is 830 mm, virtually equally distributed between the first and second halves of the year. total annual precipitation is supplemented by heavy coastal mist. at the time of the survey the farm was stocked with approximately 200 friesland cows, 90 replacement heifers, 30 beef cattle and 40 mutton-breed sheep. four hectares of pasture on a south facing incline were divided into four 1 ha fenced paddocks and allocated to the study. prior to fencing this particular bit of pasture had been grazed by cattle and horses. subsequent to fencing five friesland calves were rotated through the four camps, and on occasion dairy cows also, to ensure their contamination with worm eggs. all cattle were removed during november 1982 and on 29 november 1982, 15 9month-old friesland heifer calves were placed in one of the four paddocks and thereafter rotated through them. this ensured that the camps were evenly seeded with helminth eggs. none of the heifers was treated with an anthelmintic during their sojourn on the pastures. the heifers were artificially inseminated and removed from the paddocks shortly before each one was due to calve. the first heifers were taken off during may and the last during september 1984. faecal worm egg counts on 29 november 1982 and 4 january 1983, and at approximately 4-weekly intervals thereafter until january 1984, faeces were collected from each heifer for faecal worm egg counts. once the faeces required for individual egg counts had been weighed the remainder were pooled, thoroughly mixed and a single faecal culture made for larval differentiation (reinecke 1973). nematodirus and trichuris eggs were counted separately from those of the other nematodes. the eggs of the other nematodes were allocated to the various genera in proportion to the third stage infective larvae harvested from the faecal cultures. larval collections from herbage on 29 november 1982, and on 4 january 1983 and at 4-weekly intervals thereafter until january 1984, when sampling ceased, herbage was collected during the morning in a w-pattern from 144 approximately equidistant points in the four survey paddocks. four separate pinches of grass, taken at soil level, were collected by hand in the vicinity of each of these points and the samples so collected placed in a large plastic bag, which was sealed and transported to the laboratory at grahamstown. at the laboratory the herbage was weighed, thoroughly mixed and 250 g, consisting of pinches of grass taken from the total, was placed in a bucket 292 parasites of domestic and wild animals in south africa. xlv with 10 l of water and 1 ml of tween 80. the remainder was put in a large container, and water and tween 80 were added at the rate of 10 l per 250 g of herbage and 1 ml per 10 l of water respectively. the herbage was left to soak overnight and removed the following morning, at the same time squeezing out water trapped in it. the grass from the larger container was discarded, while that from the bucket was placed on a tray in a drying-oven at 56 °c. the remaining contents of both containers were allowed to sediment overnight and the supernatent fluid was discarded. the sediment was passed through a sieve with 38 µm apertures and the material retained on the sieve was baermannized overnight through a sieve with 300 µm apertures. the liquid drawn from the baermann funnel the following morning was allowed to sediment overnight and the supernatent fluid was discarded. third stage infective larvae in a representative sample of the sediment were counted and converted to larvae per kg of dry herbage. survey animals at regular intervals friesland bull calves, approximately 1 week old, were removed from their dams and raised until the age of approximately 4 months in individual pens, with expanded metal floors, which were cleaned daily. these conditions were intended to prevent the calves acquiring infection with helminths. at 4-weekly intervals two of the older calves were each drenched with albendazole (valbazen: pfizer animal health) at 15 mg/kg body mass to eliminate nematodes they may have acquired during rearing, and once every 28 days from 3 december 1982 to 3 january 1984, and thereafter once every calendar month from 13 february 1984 to 5 march 1985, 29 separate pairs of these calves were placed on the pastures with the 15 heifers for a period of 14 days. in december 1984 the calves remained on the pastures for 28 days. during september 1984 the last of the heifers were removed from the experimental paddocks and in order to make use of the excess herbage that now became available, other cattle were allowed to graze these plots with the tracer calves. none of the tracer calves was treated with an anthelmintic while on the pastures or thereafter. at the conclusion of their periods of exposure the calves were transported to grahamstown, and on the following day one was killed and its gastrointestinal tract and its contents processed for helminth recovery (day 0 tracer). the other calf was housed indoors in a cage with a steel-mesh floor and maintained under conditions intended to preclude the acquisition of further helminth infection. this calf was killed 21 days after it had been taken off the pastures (day 21 tracer) and its lungs and its gastro-intestinal tract and contents processed for the recovery of helminths. the last four pairs of calves were all slaughtered 21 days after their respective periods of exposure. necropsy procedure the contents of the abomasa and small intestines of the day 0 tracer calves were washed over sieves with 38 µm apertures and their caecal and colonic contents over sieves with 150 µm apertures. the mucosae of all these organs were subjected to pepsin/hcl digestion (reinecke 1973) and washed over sieves with 38 µm apertures. all material retained in the sieves was collected and preserved with formalin. since no worms had been recovered from the digested material of the first 12 day 0 tracer calves examined, digestion of the mucosae of the caecum and colon was discontinued. the contents of the abomasa and the digests of the abomasal mucosae of the day 21 tracer calves were washed over sieves with 38 µm apertures, and the contents of their small and large intestines over sieves with 150 µm apertures. no digests were made of the mucosae of the latter organs. the trachea and the bronchi of the right lungs of these calves were slit open, and the lungs thoroughly washed. the washings were sieved over a sieve with 150 µm apertures and the sieve contents examined for lungworms. helminth identification and counts adult helminths in two or three 1/32nd to 1/128th representative samples of the processed material were identified to genus level and counted under a stereoscopic microscope. the size of the aliquots was decreased to between 1/200th and 1/500th in eight of the calves when the nematodes initially seen in a larger aliquot were deemed to be particularly numerous. all male worms in the aliquots were collected, and the posterior ends of the larger species, namely h. placei and n. helvetianus, and the anterior and posterior ends of the oesophagostomum spp., were cut off to facilitate examination. after the bursae and spicules of all the male worms and the anterior ends of the oesophagostomum spp. had been cleared in warm lactophenol, they were specifically identified under a standard binocular microscope. the female worms encountered were allocated to species in proportion to the number of males identified within 293 i.g. horak, ursula evans & r.e. purnell that genus and species. third and fourth stage larvae were also counted. they were collected from the aliquots, placed in a drop of water and covered with a cover-slip before identification under a standard binocular microscope using the descriptions by the authors listed in reinecke (1973). after removal of the representative samples from the processed ingesta of the day 21 tracer calves the remainder of the small intestinal contents was examined macroscopically for cestodes and that of the large intestine for adult oesophagostomum and trichuris species. this was not done for the day 0 tracer calves as their helminths were still immature and hence difficult to see with the naked eye. the 28-day intervals between the slaughter of the various day 0 tracer calves exposed from december 1982 to january 1984 resulted in two of these calves being killed during august 1983, one on 3 august and the other on 31 august. to accommodate the worm burdens of these calves separately the month of august 1983 appears twice in the figures depicting worm burdens. climatological data daily minimum and maximum atmospheric temperatures, rainfall, evaporation, daily hours of sunshine and wind-run (km/24 h) were measured on “glen dye”. results and discussion climate the greatest mean temperatures, evaporation and wind-run and longest hours of sunlight were generally recorded from november to april (fig. 1). rain fell during every month of the survey with monthly totals in excess of 100 mm during july 1983, june 1984 and january 1985. heifers the differential faecal worm egg counts of the 15 heifers are summarized in table 1. the 35-day interval between the first worm egg counts and the second and the approximately 28-day intervals thereafter resulted in two sets of counts being done during january and march 1983 and none in december 1982 and february 1983. larvae of six nematode genera were recovered from the faecal cultures. mean faecal worm egg counts rose from november 1982 and reached a peak from late march to august 1983. they declined thereafter to their lowest levels in december 1983 and january 1984 (fig. 2). although the larvae of bunostomum sp. were present in the faecal cultures of the heifers on four occasions, no worms of this genus were recovered from any of the tracer calves. this is possibly because of the pulmonary migration of bunostomum sp. larvae and its protracted life cycle (reinecke 1983). the output of cooperia spp. eggs by the heifers was never very high, but effectively dropped to zero from october 1983, while those of haemonchus, oesophagostomum and ostertagia decreased from november onwards. these decreases are probably age-associated (reinecke 1983) and occurred as the heifers reached 2 years of age during november 1983. however, the egg output of trichostrongylus spp. remained fairly constant throughout 1983. eggs of moniezia benedini were present in the faeces of the heifers from january to april and again in september 1983. the seasonal pattern of egg excretion by this cestode corresponds closely to the acquisition of infection noted in the tracer calves examined during 1983 (fig. 10). herbage larval counts the numbers of larvae recovered per kg of dry herbage and the mean faecal worm egg counts of the heifers are graphically illustrated in fig. 2. the heifers deposited nematode eggs on the pastures from december 1982, but the first larvae were collected from the herbage only in april 1983 and reached a peak during september and october. no larvae were recovered from the herbage during july 1983 and january 1984, and no herbage samples were taken during december 1983. tracer calves the total number of helminths of each species collected from the 29 pairs of tracer calves and their prevalence in these animals are summarized in table 2. sixteen nematode species, of which cooperia oncophora and ostertagia ostertagi were the most numerous and prevalent, were recovered from the tracer calves. moniezia benedeni was the only cestode present. with the exception of september 1983 when large numbers of larvae were collected from the herbage and large numbers of helminths were recovered from the tracer calves, the numbers of nematodes in the tracer calves did not match the seasonal 294 parasites of domestic and wild animals in south africa. xlv 295 i.g. horak, ursula evans & r.e. purnell fig. 1 climate on the farm "glen dye", eastern cape province. a) mean minimum and maximum atmospheric temperatures, b) total monthly rainfall and mean monthly evaporation, and c) mean daily hours of sunshine and wind run (km/24 h) occurrence of larvae on the herbage, but rather followed the faecal worm egg counts of the heifers (fig. 2 and 3). although herbage collections ceased in january 1984, the seasonal pattern of infection in the tracer calves exposed thereafter indicated that acquisition of infection followed a pattern fairly similar to that recorded during 1983. the markedly lower and shorter peak in intensity of infection in the tracer calves during 1984, can be ascribed to the fact that the 15 heifers used to “seed” the pasture with infection were by now a year older and had become relatively immune to infection and hence voided considerably fewer worm eggs in their faeces (table 1). the animals with which the heifers were replaced during september 1984 were adult cows, and thus also unlikely to have contributed much to pasture contamination. excluding the day 0 tracer calves slaughtered on 3 and 31 august 1983 respectively, in both of which the intensity of infection was very high, and the day 0 tracers slaughtered during june and july 1984, both of which were purging, the intensity of infection of the day 0 and the day 21 tracer calves was generally very similar. the intensity of infection in both calves in each pair exceeded 29 000 worms during june and 69 000 worms from july to october 1983. during 1984 the burdens of both calves of each pair exposed from june to august exceeded 10 000 worms. the burdens of the tracer calves exposed from december 1982 to march 1983, december 1983 to may 1984, and september 1984 to march 1985 were always modest and never exceeded 3 500 worms. there appeared to be a distinctly negative correlation between the peaks in seasonal occurrence of the nematodes acquired from the pastures by the tracer calves and the mean monthly atmospheric temperatures and evaporation (fig. 1a and fig. 3). the seasonal pattern of infection in the calves is similar to that recorded by muller (1968) in sheep on artificial pastures close to the coast in the southern western cape province. he found that conditions for the acquisition of infection of all nematode species were optimal during autumn, winter and early spring, and stated that the relatively low level of infection during the summer “is in all probability the result of light pasture contamination combined � � � � � � �� �� �� �� �� �� �� �� �� � � ��� �� ����� �� ����� ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � � ��������� �� ����� ��������� �� ����� ������� ���!� "�� ���� � � � �� �� �� � � � � � � � �� �� � � � � ��� �#� ��� �#� ��� #� � ������ !� ����� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � ��� $������� �%��& � ��� $� "�� ���� ��%��& '(��!�������!�� �� � �� �� �)�� � � � � � � � � � � � � � � � � � � � � � � � � � � � � ������ �������� ���� �� ����� ���� ��� $�� �� � �� �� �)�� (��!���� � � � � " �� � � � � # � $� % � *�� ��� ��� ��� ��� ��� ��� �� � � � � � � � �� & � � � �� � � � � � �� ' � � �# �* �� �� + # * � � 296 parasites of domestic and wild animals in south africa. xlv table 1 differential faecal worm egg counts of 15 friesland heifers on kikuyu grass pastures on the farm “glen dye”, eastern cape province average number of eggs per g of faeces calculated from larvae eggs only identified during faecal recovered in faecal cultures worm egg counts date bun coop haem oes ost trich total nemato trichuris moniezia 1982 29 nov 0 22 0 0 34 11 67 0 0 0 1983 04 jan 0 46 78 17 38 33 212 4 0 0 31 jan 46 12 7 116 54 33 268 0 0 pos 01 mar 1 14 2 10 31 19 77 0 0 pos 29 mar 0 22 41 36 67 21 187 0 7 pos 27 apr 0 25 169 32 101 33 360 0 3 pos 24 may 5 11 54 1 19 40 130 0 0 0 21 jun 0 60 76 39 76 76 327 0 0 0 19 jul 0 9 97 41 32 51 230 0 0 0 16 aug 3 12 59 96 32 45 247 0 0 0 13 sep 0 12 14 2 50 45 123 0 0 pos 11 oct 0 0 12 25 17 59 113 0 0 0 08 nov 0 0 3 1 13 63 80 0 0 0 06 dec 0 0 2 7 10 42 61 0 0 0 1984 03 jan 0 1 6 3 8 18 36 0 0 0 bun = bunostomum coop = cooperia haem = haemonchus oes = oesophagostomum ost = ostertagia trich = trichostrongylus nemato = nematodirus ( ��� )) ���!���� ���" � ��"� ��+�� ����� ����� ,�� #�� ��� � ( � � � � $# ' � �& � � �� � ' � ��� ��� ��� ��� ��� �� �� �� � � � ) � � � �� �� � � � �' ' �$ ' � � � �� �� � ������ � � � � � � � � � � � �� � ������ !� ����� ���� �� *�����& ���% -��"� ./)�!�$�� �0�) 1)) .)��� � + � � � �� �� � � � � ��� ��� ��� ��� ��� �� � 2 �� �� ��� ! �0��! ����!� "�� ���� � � � �� � � � ) � � � � � � �� � � � � #�� ��� *�� ��� ��� ��� � ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � �� � � � � � � � � �� ��$������� � ��$�������� � 1"�� ���� � fig. 2 three point moving mean faecal worm egg counts of 15 untreated heifers and mean numbers of infective nematode larvae per kg of dry herbage on the farm "glen dye", eastern cape province. (two sets of egg counts and larval collections in january and in march 1983, and none in december 1982 and february 1983) fig. 3 mean monthly evaporation on the pastures and the seasonality of nematode infection in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 with the deleterious effects of intense sunlight, heat and evaporation on the free-living stages.” these results are similar to those obtained by williams, knox, baumann, snider, kimball & hoerner (1983) for a similar spectrum of nematode species in tracer calves exposed on artificial pastures contaminated by yearling cattle in the temperate red river valley region of louisiana, united states of america. tracer calves grazing lucerne pastures in western argentina also acquired scant infection during summer, whereas considerably larger burdens were recorded in autumn-grazed calves (suarez, busetti & fort 1992). unfortunately no calves were exposed during winter in the latter study, thus precluding further comparisons. urquhart, armour, duncan, dunn & jennings (1987) state that several environmental factors that affect the microhabitat of the free-living stages of parasitic nematodes, are vital for their development and survival. moderate temperatures and high humidity favour development, while cool temperatures prolong survival. according to these authors microclimatic humidity depends not only on rainfall and temperature, but also on other elements such as soil structure, vegetation type and drainage. on “glen dye” mild winter temperatures and the lush nature of the kikuyu pastures after good rainfall probably ensured adequate temperature and humidity for larval development and survival during the cooler months of the year. however, rotational grazing of the pastures prevented the formation of a significant “mat” between the soil and the herbage, which, according to urquhart et al. (1987), holds a permanent store of moisture in which the relative humidity remains high even after weeks of drought. the absence of a “mat” coupled with the high rate of evaporation, induced by long hours of sunlight and high wind-run, and the sandy nature of the soil, all contributed towards low microclimatic humidity in the summer months, and hence the paucity of infective larvae. the adult and larval burdens of cooperia spp., haemonchus placei, nematodirus helvetianus, oesophagostomum spp., o. ostertagi and trichostrongylus spp. of the tracer calves are graphically illustrated in fig. 4–9. 297 i.g. horak, ursula evans & r.e. purnell table 2 helminths collected from 58 tracer bull calves on kikuyu grass pastures on the farm “glen dye”, eastern cape province total number of helminths nematode species mean burden (% of 3rd stage 4th stage adults total animals infected) larvae larvae cooperia spp. 7 948 212 717 + 220 665 3 805 (55) cooperia macmasteri – – 56 004 56 004 966 (59) cooperia oncophora – – 235 724 235 724 4 064 (76) cooperia punctata – – 8 952 8 952 154 (41) cooperia spatulata – – 10 531 10 531 182 (36) dictyocaulus viviparus 0 0 10 10 0.2 (3) haemonchus placei 10 590 29 120 12 359 52 069 898 (74) nematodirus helvetianus 1 688 4 695 7 450 13 833 238 (60) oesophagostomum spp. 1 369 3 371 + 4 740 82 (52) oesophagostomum radiatum – – 419 419 7 (38) oesophagostomum venulosum – – 347 347 6 (14) ostertagia ostertagi 89 959 344 298 391 094 825 351 14 230 (98) strongyloides sp. 15 586 946 0 16 532 285 (35) trichostrongylus spp. 2 580 29 280 + 31 860 549 (57) trichostrongylus axei – – 54 246 54 246 935 (52) trichostrongylus colubriformis – – 220 220 4 (5) trichostrongylus falculatus – – 12 12 0.2 (2) trichostrongylus rugatus – – 325 325 6 (9) trichuris sp. 19 (immatures) 419 438 8 (41) total 1 532 278 26 419 (100) cestode species scolices total moniezia benedeni 43 43 0.7 (22.4) + = adults of these genera identified specifically – = 3rd and 4th stage larvae of these species identified only to generic level cooperia spp. cooperia oncophora followed by cooperia macmasteri were the most numerous and prevalent of the four cooperia spp. recovered. in earlier surveys in cattle in the hot and semi-arid regions in the north and north-west of south africa, in sub-tropical regions in the north-east and on the cool highveld, cooperia pectinata and cooperia punctata were the most numerous, if not the only species present (reinecke 1960b, horak 1978; horak & louw 1978; malan, reinecke & roper 1982; dreyer, fourie & kok 1999; louw 1999). in this survey, however, only small numbers of c. punctata and no c. pectinata were recovered. both c. pectinata and c. punctata and also cooperia spatulata have previously been recorded in sheep on artificial pastures close to the coast in the south-eastern western cape province (muller 1968). the seasonal pattern of infection of cooperia spp. larvae and those of the adults of the four cooperia spp. were very similar (fig. 4). during 1983 burdens in excess of 23 000 worms comprising all developmental stages of this genus were recorded in calves exposed from 22 june to 14 september. in both 1983 and 1984 the largest worm burdens were present in calves exposed during july, with the single largest burden of 117 952 worms present in the day 0 tracer calf exposed on 20 july and slaughtered on 3 august 1983, compared to the highest burden of only 3 110 worms in one of the four tracer calves exposed during july and august 1984. the development of third and fourth stage cooperia spp. larvae to fourth stage larvae and adult worms is evident from the burdens of successive pairs of day 0 and day 21 tracer calves (fig. 4a–c). the fourth stage larvae still present in the day 21 tracers represent worms that have been arrested in their development. however, arrested development was not a prominent feature in this genus, with the highest proportion accounting for only 17 % of the total cooperia spp. burden in the day 21 tracer calf exposed during september 1983 (early spring). the low percentage of arrested larvae is similar to that observed by smeal, fraser & robinson (1980b) in calves on pastures on the northern tablelands and on the north coast of new 298 parasites of domestic and wild animals in south africa. xlv fig. 4 seasonality of a) cooperia spp. larvae, b) adult cooperia mcmasteri, and c) adult cooperia oncophora in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 ! � ) � � �� � � �� �� " � �� � *����� ������ ������ ������ ������ ������ ������ ����� � 3������ ���������� ��4 ������ �������� ���� �� ����� ���� ��$�������� ��%-*& ��$������� ��%-��5�-*& � � � � � � � � � � � � � � � � � � � � � � � � � � �� � ! � ) � � � � � �� " � �� � ������ ������ +���� #���� *���� ����� � �!� ����������� ������ ������ �������� ���� �� ����� ���� ��$�������� ���$������� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � ! � ) � � � � � �� " � �� � ������ *����� ������ ������ ������ � �!� ������������ ������ ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��$������� � ��$�������� � ' south wales, australia. they recorded large burdens of cooperia spp. in calves on pastures that had been contaminated in autumn, but with a single exception of 32.4 %, arrested larvae never exceeded 15 % of the total burden. a considerable number of adult worms of all four cooperia spp. were already present in the day 0 tracer calves killed 15 days after their first exposure on the pastures (fig. 4b and c). this can be attributed to the fourth larval moult taking place as soon as 8 days after infection in both c. pectinata and c. punctata (keith 1967; reinecke 1973), and hence probably also in other cooperia spp. that infect cattle. dictyocaulus viviparus in south africa infection with d. viviparus is confined to animals in the western cape province and in isolated foci in the mist-belt of the drakensberg in kwazulunatal and mpumalanga provinces. it is common in cattle on irrigated pastures on the highveld of gauteng province (reinecke 1983). small numbers have also been recovered from blue wildebeest, connochaetes taurinus, in a hot lowveld environment (horak, de vos & brown 1983). the high rate of evaporation on the dry-land pastures in this survey probably had a severely limiting effect on the survival of free-living larvae and hence the small numbers of worms in the tracer calves. haemonchus placei the highest intensity of infection was recorded in tracer calves examined from june to october 1983 and during june 1984. adult worms were already present in the day 0 tracer calves 15 days after their first exposure to infestation on the pastures. the day 21 tracers removed from the pastures in late august and during september 1983 harboured no h. placei whatsoever (fig. 5), but both were infected with large numbers of immature and adult o. ostertagi and trichostrongylus axei (fig. 8 and 9). it could be that a density-dependent reaction with elimination of h. placei had occurred as postulated by muller (1968) for the interaction that took place between ostertagia spp., t. axei and h. contortus, to the detriment of the latter species, in sheep on coastal pastures in the western cape province. a comparison between the burdens of third and fourth stage h. placei larvae of the day 0 tracer calves with those of their day 21 partners shows that arrest in larval development was most prevalent in infection acquired during autumn and winter in 1983, and declined during late winter and spring (fig. 5a). the intensity of infection in 1984 was too low to ascertain a reliable seasonal trend in larval arrest. a similar autumn and winter pattern in both the acquisition of infection and arrest in larval development has been recorded in calves grazed on pastures contaminated with haemonchus spp. during autumn on the sub-tropical north coast of new south wales (smeal et al. 1980b). in south africa arrest in larval development exceeded 90 % in haemonchus spp. in tracer calves grazed on irrigated pastures on the highveld of gauteng 299 i.g. horak, ursula evans & r.e. purnell ! � ) � � �� � � �� �� " � �� � #���� ����� *���� ����� ����� ����� � 3������ ���� � ������� �� � ������ �������� ���� �� ����� ���� ��$�������� ��%-*&��$������� ��%-��5�-*& � � � � � � � � � � � � � � � � � � � � � � � � � � � �� �!� ����� � ������� �� ! � ) � � � � � �� " � �� � ����� ����� ����� ����� ��� � ������ �������� ���� �� ����� ���� ��$�������� ���$������� � � � � � � � � � � � � � � � � � � � �� � � � � � � � � � � fig. 5 seasonality of a) immature and b) adult haemonchus placei in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 province between april or may and october (horak & louw 1978). although their worm burdens were very small it also appeared to be present from april to july in h. placei in cattle on the eastern free state highveld (dreyer et al. 1999). the winters in the latter regions are dry and cold with frequent frosts. in the southern bushveld of limpopo province, 100 % arrest in development was recorded in h. placei in tracer calves grazing natural vegetation from april to july (horak 1978), while no worms of this species were picked up from the pastures from august to october. in a separate survey in this province schröder (1979) noted that no worms of this species were acquired from the pastures from may to october. the winters in this region are dry and warm. it would thus appear that haemonchus spp. employ the same strategy of arrested larval development in order to survive within the host during winters that are either cold and dry or warm and dry. in the present study, however, the summer months seemed to be the least favourable for the acquisition of infective larvae from the pastures. this could have been caused by a failure of the larvae to survive, or because with increasing age the 15 heifers became more resistant to infection and excreted fewer nematode eggs with their faeces, or because the long hours of summer sun, increased wind run, with accompanying high evaporation on the southerly sloping pasture, dried the outer layer of faeces thus trapping infective larvae in dung-pats as described by reinecke (1960a). whatever the reason, the proportion of arrested larvae in the h. placei infections acquired by the tracer calves before summer was on the decline, and this did not appear to be a major strategy for over-summering on “glen dye”. nematodirus helvetianus compared to the number of records for other nematodes parasitizing cattle in south africa there are few for n. helvetianus. reinecke (1983) states, “although this parasite is rarely diagnosed in south africa, it occurs in calves in the western cape province, eastern cape province and kwazulu-natal”. he also said that its eggs are seldom detected in the faeces of calves older than 5 months. nematodirus helvetianus would thus appear to be a parasite of young animals in the moist, temperate climates of the coastal provinces of south africa. this assumption is supported by the findings of reinecke (1960b), horak (1978), horak & louw (1978), schröder (1979), malan et al. (1982) and fourie & horak (1990), none of who recovered worms of this species from cattle examined on inland farms. eggs of nematodirus sp. were only present in the faeces of the heifers that grazed the survey camps with the tracer calves during january 1983 (when the heifers were 10 months old). in contrast, with the exception of two, every tracer calf exposed between december 1982 and december 1983 acquired infection with n. helvetianus. however, smeal, robinson & fraser (1980a), working in the central coastal region of new south wales, australia, recovered no worms of this genus from calves grazing pastures on which small numbers of nematodirus spp. larvae were present. 300 parasites of domestic and wild animals in south africa. xlv 3������ ��� ����������������� �� ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��$������� ��%-��5�-*& ��$�������� ��%-*& ! � ) � � �� � � �� �� " � �� � ��#�� ����� +�� *�� � � ! � ) � � � � � �� " � �� � ��#�� ����� +�� *�� � �!� ���� ����������������� �� ������ �������� ���� �� ����� ���� ��$�������� ���$������� � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � fig. 6 seasonality of a) immature and b) adult nematodirus helvetianus in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 the increase in the size of worm burdens in the tracer calves examined during autumn and winter and their decline in those exposed in summer (fig. 6), are opposite to the findings of eysker & van miltenburg (1988) at utrecht, in the netherlands. they recovered between 19 500 and 34 700 n. helvetianus from tracer calves grazing experimental paddocks during august (summer), compared to 7 900– 20 000 worms from those grazing these paddocks during october (autumn). these differences confirm the importance of local environmental factors in determining the seasonality of this and other nematodes. according to herlich (1954) and reinecke (1973) the fourth moult in n. helvetianus takes place between 14 and 16 days after infection. judging by the number of adult worms already present in the day 0 tracer calves slaughtered 15 days after their first exposure to infection on the pastures, the fourth moult seems to have occurred sooner than the 14th day in at least some of these worms. oesophagostomum spp. oesophagostomum radiatum is a parasite of cattle and is distributed throughout south africa (horak 1981a) from the arid north-west (reinecke 1960b) to the subtropical north and north-east (horak 1978; schröder 1979; malan et al. 1982), the coastal regions of kwazulu natal in the east (hobbs 1961) and the non-seasonal rainfall regions of the south (table 2). oesophagostomum venulosum is present only in the southern regions of the eastern and western cape provinces (horak 1981a), where it infects cattle, sheep and goats (muller 1968; reinecke, kirkpatrick, swart, kriel & frank 1987; reinecke & louw 1989; horak et al. 1991; horak 2003; table 2). in contrast to most other species, infection with oesophagostomum spp. in the tracer calves was as intense during autumn and winter of the second year of the survey as it was in the first (fig. 7). most infection with these species was acquired in april and from june to october 1983 and from may to july 1984, the cooler months of the year. during the third year of the survey, however, adult o. radiatum was also present in all but one of the eight tracer calves examined from december 1984 to march 1985 (summer). at vryburg in the northern cape province reinecke (1960b) recorded the highest egg counts and intensity of infection of o. radiatum during winter, but he suggested that the calves had actually become infected during summer and autumn. tracer calves at nylsvley in limpopo province acquired the highest intensity of infection from june to january (horak 1978), whereas in north-eastern mpumalanga province most infection was picked up between november and february (malan et al. 1982). the winter prevalence of o. venulosum in the calves agrees with that in tracer lambs on coastal pastures in the southern western cape province, where it was most numerous from march to september (muller 1968). in the south-western region of the latter 301 i.g. horak, ursula evans & r.e. purnell 3������ ������������� � � ��4 � ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��$�������� ��%-*&��$������� ��%-��6�-*& ! � ) � � �� � � �� �� " � �� � +�� #�� *�� ��� � ! � ) � � � � � �� " � �� � ��� ��� ��� ��� �� �� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ������ �������� ���� �� ����� ���� ����� ����� ���������� �!� �������������� � � ��� � fig. 7 seasonality of a) oesophagostomum spp. larvae, and b) adult oesophagostomum radiatum and oesophagostomum venulosum in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 province it was most numerous in sheep on kikuyu pastures from june to september (reinecke et al. 1987). the fourth larval moult of o. radiatum takes place 19 days after infection (andrews & maldonado 1941). consequently a high proportion of fourth stage larvae, even 21 days after removal of the calves from the pastures, cannot be considered as evidence of arrested development. this also explains the absence of adult worms in every day 0 tracer calf. the same probably also applies to o. venulosum. ostertagia ostertagi more o. ostertagi were recovered from the tracer calves than all other gastro-intestinal nematodes combined (table 2), making it the most important helminth species infecting cattle on coastal pastures in the eastern cape province. horak (1981a) suggested that the geographic distribution of o. ostertagi is limited to the eastern and western cape provinces, and the northern inland surveys of reinecke (1960b), horak (1978), horak & louw (1978) schröder (1979) and malan et al. (1982), in which no o. ostertagi were encountered, and that of dreyer et al. (1999) in the eastern free state, in which a single tracer calf was infested, would seem to support this distribution pattern. however, all 14 calves examined by louw (1999) on the eastern highveld of mpumalanga province in the north of the country were infected and harboured mean burdens of slightly fewer than 500 worms. furthermore tsotetsi & mbati (2003) recovered o. ostertagi larvae from the faeces of cattle on the north-eastern highveld of free state province. the latter findings indicate that a revision of the geographic distribution of this nematode in south africa is necessary. intensity of infection peaked in the tracer calves from july to october 1983 and from june to august 1984 (fig. 8), but was considerably lower during the latter period than in the former. infection acquired from the pasture was negligible during the three summers of the survey. although this pattern of seasonality is similar to that recorded by williams et al. (1983) in tracer calves exposed on artificial pastures in the temperate red river valley region of louisiana, united states of america, the proportions of arrested larvae differ. in louisiana these larvae increased from 5.0 % to 79.4 % of the o. ostertagi burdens from late winter to late spring, while on “glen dye” they increased from 2.7 % in late winter to 42.3 % in spring. however, at the same time as the development of some larvae was arrested, others rapidly developed to adult worms, and 15 days after their first exposure to infection during august and september 1983 the day 0 tracer calves harboured 5 625 and 8 125 adult o. ostertagi respectively. a total of 2 674 third stage larvae were recovered from the various day 21 tracer calves, implying that a small proportion of o. ostertagi larvae may be arrested in this stage of development as demonstrated by eysker (1978) for trichostrongylus spp. in sheep. 302 parasites of domestic and wild animals in south africa. xlv �!� ���������������������� � ! � ) � � � � � �� " � �� � ������� +����� #����� �*����� ������ �� ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��$�������� ���$������� � ! � ) � � �� � � �� �� " � �� � �#����� ������� +����� *����� � ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��$�������� ��%-*&��$������� ��%-��5�-*& 3������ ��������������������� � fig. 8 seasonality of a) immature and b) adult ostertagia ostertagi in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 strongyloides sp. third and fourth stage larvae of this genus were identified by the characteristic appearance of their oesophagi. the absence of adult worms reflects either a state of immunity or of host incompatibility. trichostrongylus spp. with the exception of t. axei, worms belonging to this genus are not important parasites of cattle. in surveys conducted on cattle in the eastern highveld regions of the free state and mpumalanga provinces t. axei was the most numerous nematode recovered (dreyer et al. 1999; louw 1999). in this survey t. axei comprised 99 % of the total number of adult trichostrongylus spp. (table 2), and it can safely be assumed that the larvae of this species accounted for a similar proportion of the immature worms. if this assumption is correct only o. ostertagi and c. oncophora exceeded t. axei in numbers in the present survey. the peaks in intensity of infection of adult t. axei were similar in both years of the survey, but their duration differed, extending from august to october in 1983 and from june to july in 1984 (fig. 9). arrested fourth stage larvae comprised 9.6–28.8 % of the trichostrongylus spp. burdens in the day 21 tracer calves examined from july to october 1983, and 44.3 % in the calf examined in june 1984. these calves all harboured more than 20 000 adult o. ostertagi and four of the five more than 7 000 adult t. axei, and consequently retardation in the development of the larvae may in part have been density dependent (smeal et al. 1980b). third stage larvae were recovered from only one of the day 21 tracer calves. the fourth larval moult in t. axei takes place 10–14 days after infection (douvres 1957), and yet less than 0.3 % of the worms in the day 0 tracer calves were adult. with the exception of o. radiatum, in which the fourth moult takes place 19 days after infection, a considerably greater proportion of the worms of all other species were already adult in the day 0 tracer calves. it would thus seem that irrespective of the magnitude of the worm burden there was some delay in the development of t. axei to adulthood. trichostrongylus colubriformis, trichostrongylus rugatus and trichostrongylus falculatus are parasites of sheep, and the latter two may be common in these animals in the southern regions of the eastern and western cape provinces (barrow 1964; rossiter 1964; muller 1968; reinecke et al. 1987; horak 2003). trichuris sp. slightly more than 40 % of the calves were infected with worms of this genus and the presence of adult worms in the day 0 tracer calves indicates that at least some of these worms had been acquired before the calves were exposed on the pastures. it also implies that the double therapeutic dose of albendazole used to clear the calves of infection before exposure had not been effective against this nematode. 303 i.g. horak, ursula evans & r.e. purnell �!� ����� ������ ���������� � ������ �������� ���� �� ����� ���� �� � � � � � � � � � � � � � � � � � � � � � � � � �� � ��$������� � ��$�������� � ! � ) � � � � � �� " � �� � ������ ������ +���� #���� *���� ����� � 3������ ���� ������ ����� � ������ �������� ���� �� ����� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� ��$������� ��%-��5�-*& ��$�������� ��%-*& ! � ) � � �� � � �� �� " � �� � ������ +���� �#���� *���� ����� �� fig. 9 seasonality of a) trichostrongylus spp. larvae, and b) adult trichostrongylus axei in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 moniezia benedeni this cestode infects cattle and a number of wild bovids, and is especially a parasite of younger animals (horak et al. 1983; reinecke 1983). in contrast to the nematodes, m. benedeni appeared to favour the summer months and it was present in tracer calves from january to june in the first year of the survey, during november in the second year and from december to february in the third year (fig. 10). this pattern of seasonality can probably be coupled to the slower development of cysticercoids in their oribatid mite intermediate hosts during the cooler winter months compared to their more rapid development to infectivity during the warmer summer months (kuznetsov 1970). general the survey commenced during december 1982 on the assumption that, as in previous studies in south africa, cattle on dry-land pasture, or on natural grazing, acquire most nematode infection during mid and late summer (hobbs 1961; horak 1978; schröder 1979; malan et al. 1982). this did not materialise and the highest intensities of infection occurred from late autumn to spring, a pattern similar to that recorded for haemonchus spp. on irrigated pastures on the gauteng highveld (horak & louw 1978). however, in the gauteng survey intensities of infection remained comparatively high during summer, whereas in this study they were generally negligible. the survival of parasitic nematodes during seasons when external climatic conditions are unfavourable for the development of their free-living stages, is frequently accomplished by arrest in the development of third or fourth stage larvae in the host animal (michel 1974; eysker 1978; horak 1981b). with the exception of h. placei, in which arrested development in the fourth larval stage occurred in autumn and winter, early in the season of helminth acquisition, this phenomenon was generally observed in late winter or spring, late in the season of nematode acquisition in this study. it was never a prominent feature despite the fact that the external climate during the summer months seemed to be a limiting factor for the survival or acquisition of the free-living infective stages. survival of the various nematode species during these months was thus probably achieved via a combination of strategies. amongst these are a moderate degree of arrested larval development in the host, a residual but ageing population of adult worms, possible survival of larvae in dung-pats on the pasture (reinecke 1960a), and possible survival of larvae in the soil which then only become available on the herbage during the cooler autumn months (reinecke & louw 1989). acknowledgements we are most grateful to messrs john and simon matthews for making the experimental heifers, experimental grazing plots and other facilities on their farm “glen dye” available to us for the duration of the survey. the assistance of mr j. white with managing the cattle on the farm, messrs m.m. knight and e.j. williams with processing the carcasses for helminth recovery, and of dr j.a. van wyk with reviewing the manuscript is greatly appreciated. this research was wholly financed by a generous grant from pfizer central research. references andrews, j.s. & maldonado, j.f. 1941. the life history of oesohagostomum radiatum, the common nodular worm of cattle. research bulletin of the puerto rico university agricultural experiment station, no. 2. barrow, d.b. 1964. the epizootiology of nematode parasites of sheep in the border area. onderstepoort journal of veterinary research, 31:151–162. boomker, j., horak, i.g. & macivor, k.m. de f. 1989. helminth parasites of grysbok, common duikers and angora and boer goats in the valley bushveld in the eastern cape province. onderstepoort journal of veterinary research, 56:165–172. douvres, f.w. 1957. the morphogenesis of the parasitic stages of trichostrongylus axei and trichostrongylus colu304 parasites of domestic and wild animals in south africa. xlv ! � ) � � � � � �� � � � �� �� , # � � ������ �������� ���� �� ����� ���� ��$�������� � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� �� �������� ��� � fig. 10 seasonality of moniezia benedeni in tracer calves on the farm "glen dye", eastern cape province. two sets of calves were examined during august 1983 briformis nematode parasites of cattle. proceedings of the helminthological society of washington, 24:4–14. dreyer, karin, fourie, l.j. & kok, d.j. 1999. gastrointestinal parasites of cattle in the communal grazing system of botshabelo in the free state. onderstepoort journal of veterinary research, 66:145–149. eysker, m. 1978. inhibition of the development of trichostrongylus spp. as third stage larvae in sheep. veterinary parasitology, 4:29–33. eysker, m. & van miltenburg, l. 1988. epidemiological patterns of gastrointestinal and lung helminth infections in grazing calves in the netherlands. veterinary parasitology, 29:29–39. fivaz, b.h., horak, i.g. & williams, e.j. 1990. helminth and arthropod parasites of angora goats on irrigated kikuyu grass pastures in the eastern cape province. journal of the south african veterinary association, 61:112–116. fourie, l.j. & horak, i.g. 1990. parasites of cattle in the south-western orange free state. journal of the south african veterinary association, 61:27–28. herlich, h. 1954. the life history of nematodirus helvetianus, may, 1920, a nematode parasite in cattle. journal of parasitology, 40:60–70. hobbs, w.b. 1961. helminth research in south africa. i. seasonal incidence of nematode parasites in cattle in the natal coastal area. journal of the south african veterinary medical association, 32:151–155. horak, i.g. & louw, j.p. 1978. parasites of domestic and wild animals in south africa. vi. helminths in calves on irrigated pastures on the transvaal highveld. onderstepoort journal of veterinary research, 45:23–28. horak, i.g. 1978. parasites of domestic and wild animals in south africa. xi. helminths in cattle on natural pastures in the northern transvaal. onderstepoort journal of veterinary research, 45:229–234. horak, i.g. 1981a. host specificity and the distribution of the helminth parasites of sheep, cattle, impala and blesbok according to climate. journal of the south african veterinary association, 52:201–206. horak, i.g. 1981b. the similarity between arrested development in parasitic nematodes and diapause in insects. journal of the south african veterinary association, 52:299–303. horak, i.g., de vos, v. & brown, moira r. 1983. parasites of domestic and wild animals in south africa. xvi. helminths and arthropod parasites of blue and black wildebeest (connochaetes taurinus and connochaetes gnou). onderstepoort journal of veterinary research, 50:243–255. horak, i.g., knight, m.m. & williams, e.j. 1991. parasites of domestic and wild animals in south africa. xxviii. helminth and arthropod parasites of angora goats and kids in valley bushveld. onderstepoort journal of veterinary research, 58:253–260. horak, i.g. 1999. parasites of domestic and wild animals in south africa. xxxvii. ixodid ticks on cattle on kikuyu grass pastures and in valley bushveld in the eastern cape province. onderstepoort journal of veterinary research, 66: 175–184. horak, i.g. 2003. parasites of domestic and wild animals in south africa. xlii. helminths of sheep on four farms in the eastern cape province. onderstepoort journal of veterinary research, 70:175–186. keith, r.k. 1967. the life history of cooperia pectinata ransom. australian journal of zoology, 15:739–744. kuznetsov, m.i. 1970. development times of moniezia cysticercoids in scheloribates laevigatus under natural conditions, in oribatids and their role in the processes of soil formation, edited by e.m. bulanova-zakhvatkina et al. vilnius: akademiya nauk litovskoi srr, 223–227. (abstract helminthological abstracts, series a, 40:3935). louw, j.p. 1999. the helminths of ranch calves in the northeastern mountain grassland of south africa. onderstepoort journal of veterinary research, 66:335–338. malan, f.s., reinecke, r.k. & roper, nancy a. 1982. the seasonal incidence of helminth parasites of cattle in the eastern transvaal lowveld. journal of the south african veterinary association, 53:179–184. michel, j.f. 1974. arrested development of nematodes and some related phenomena. advances in parasitology, 12: 279–366. muller, g.l. 1968. the epizootiology of helminth infestation of sheep in the south-western cape. onderstepoort journal of veterinary research, 35:159–194. reinecke, r.k. 1960a. the rôle of infested dung in the transmission of nematode parasites of cattle. journal of the south african veterinary medical association, 31:45–52. reinecke, r.k. 1960b. a field study of some nematode parasites of bovines in a semi-arid area, with special reference to their biology and possible methods of prophylaxis. onderstepoort journal of veterinary research, 28:365–464. reinecke, r.k. 1973. the larval anthelmintic test in ruminants. pretoria: department of agricultural technical services. (technical communication, no. 106). reinecke, r.k. 1983. veterinary helminthology. durban & pretoria: butterworths publishers (pty) ltd. reinecke, r.k., kirkpatrick, r., swart, lydia, kriel, anna m.d. & frank, f. 1987. parasites in sheep grazing on kikuyu (pennisetum clandestinum) pastures in the winter-rainfall region. onderstepoort journal of veterinary research, 54:27–38. reinecke, r.k. & louw, j.p. 1989. overberg research projects. i. the epidemiology of parasitic nematodes in ewes, suckling lambs and weaners. journal of the south african veterinary association, 60:176–185. rossiter, l.w. 1964. the epizootiology of nematode parasites of sheep in the coastal area of the eastern province. onderstepoort journal of veterinary research, 31:143–150. schröder, j. 1979. the seasonal incidence of helminth parasites of cattle in the northern transvaal bushveld. journal of the south african veterinary association, 50:23–27. smeal, m.g., robinson, g.g. & fraser, g.c. 1980a. seasonal availability of nematode larvae on pastures grazed by cattle in new south wales. australian veterinary journal, 56:74–79. smeal, m.g., fraser, g.c. & robinson, g.g. 1980b. seasonal changes in the structure of nematode populations of cattle in new south wales in relation to inhibited larval development. australian veterinary journal, 56:80–86. suarez, v.h., busetti, m.r. & fort, m.c. 1992. epidemiology and effects of nematode infections on beef cow-calf systems of argentina’s western pampas. veterinary parasitology, 42: 73–81. tsotetsi, a.m. & mbati, p.a. 2003. parasitic helminths of veterinary importance in cattle, sheep and goats on communal farms in the northeastern free state, south africa. journal of the south african veterinary association, 74:45–48. urquhart, g.m., armour, j., duncan, j.l., dunn, a.m. & jennings, f.w. 1987. veterinary parasitology. new york: churchill livingstone inc. 305 i.g. horak, ursula evans & r.e. purnell williams, j.c., knox, j.w., baumann, b.a., snider, t.g., kimball, m.d. & hoerner, t.j. 1983. seasonal changes of gastrointestinal nematode populations in yearling beef cattle in louisiana with emphasis on prevalence of inhibition in ostertagia ostertagi. international journal for parasitology, 13:133–143. 306 parasites of domestic and wild animals in south africa. xlv polat_77-79.qxd introduction a number of clinical and nutritional disorders can be assessed by the use of blood biochemistry. non-specific avian values are inadequate because biochemical and hematological status is a reflection of many factors including species, age, management and nutrition (bowes, julian & stirtzinger 1989; okotie-eboh, bailey, hicks & kubena 1992; aydin, ak, galip & zaugg 2003). serum protein fractions can be separated by electrophoresis which shows that albumin is the largest protein fraction in normal avian serum. avian albumin is similar in structure to mammalian albumin. the globulin component of avian serum protein is composed of separate alpha (α), beta (β) and gamma (γ) fractions. relative and total amounts of serum protein fractions are affected by infections, inflammation, and nutritional and physiological status, and are therefore important health indicators. serum globulin and immunoglobulin levels can be valuable indicators of the ability of animals to perform the activities of daily living, and may be useful predictors of subclinical diseases (bell & freeman 1971; nozaki, nohara, ashitomi, zukeran, inafuku, akisaka & suzuki 1998). lipids are transported in the blood as components of lipoproteins, and therefore, changes in plasma lipids should be reflected in the amount and distribution of lipoproteins. lipid synthesized is transported to adipose tissue mainly in the form of very low density lipoproteins (whitehead, hood, heard & pym 1984). the aim of this study was to determine for reference purposes serum albumin, α1-globulin, α2-globulin, β-globulin and γ-globulin, α-lipoprotein (high density lipo77 onderstepoort journal of veterinary research, 71:77–79 (2004) research communication reference serum protein and lipoprotein fractions of ostriches (struthio ccamelus) in turkey u. polat1, m. cetin1, o. turkyilmaz2 and a. yalcin1 abstract polat, u., cetin, m., turkyilmaz, o. & yalcin, a. 2004. reference serum protein and lipoprotein fractions of ostriches (struthio camelus) in turkey. onderstepoort journal of veterinary research, 71:77–79 the aim of this study was to determine for reference purposes the values of serum albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin, and α-lipoprotein (high density lipoprotein), pre-β-lipoprotein (very low density lipoprotein) and β-lipoprotein (low density lipoprotein) fractions of normal ostriches (struthio camelus) in turkey. five male and five female ostriches, 18 months old, were used. all the ostriches were fed on a diet that contained 15.14 % crude protein and 2 950 kcal/kg of metabolizable energy. the serum protein and lipoprotein fractions were measured using agarose gel electrophoresis. the fractions were found to be 60.96 % albumin, 0.24 % α1-globulin, 15.91 % α2-globulin, 13.34 % β-globulin, 9.55 % γ-globulin, 53.77 % hdl, 0.60 % vldl and 48.09 % ldl. keywords: lipoprotein fraction, ostrich, protein fraction, struthio camelus 1 university of uludag, faculty of veterinary, department of biochemistry, bursa 16059 turkey 2 bornova veterinary control and research institute, izmir, turkey accepted for publication 16 october 2003—editor protein (hdl)), pre-β-lipoprotein (very low density lipoprotein (vldl)) and α-lipoprotein (low density lipoprotein (ldl)) fractions of normal ostriches (struthio camelus) in turkey. materials and methods the ostriches used in the study were obtained from the ostrich raising unit, faculty of agriculture, uludag university, which is situated in the bursa region of western turkey. five male and five female ostriches, 18 months old, were used as the study material. all the ostriches were fed a diet that contained 15.14 % crude protein and 2 950 kcal/kg of metabolizable energy (table 1). water was supplied ad libitum. the heads of the birds were covered with a hood to facilitate handling (spinu, spinu & degen 1999). blood samples were taken from a wing vein using vacutainer® blood collection tubes and were transported in an ice chest to a laboratory for analyses. the samples were centrifuged at 3 000 rpm for 5 min and the serum fraction so obtained was separated and stored at –20 °c for later analysis. the composition of the feed of the ostriches was analyzed by the weende method (a.o.a.c 1984). serum albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin and, α-lipoprotein, pre-β-lipoprotein and β-lipoprotein fractions were measured using agarose gel electrophoresis (rep, helena-laboratories). results and discussion to our knowledge, very little information is available on the protein and lipoprotein fractions of ostriches. changes in the constituents of the individual protein-lipoprotein fractions and changes in the relative amounts of each fraction in serum occur in many illnesses and nutrition-related problems (wedler, prokpo, kunzi, meyer, stocker & burgi 1998). the present study provides a picture of serum protein and lipoprotein fractions in healthy ostriches maintained under intensive conditions. the composition of the feed of the ostriches used in the study is shown in table 1 and, serum protein and lipoprotein fractions in table 2. albumin is the most abundant of the serum proteins. in animals it constitutes between 35 % and 50 % of the total serum proteins, in contrast to humans and nonhuman primates in which albumin accounts for 60–67 % of the total (kaneko 1989). there are species differences in albumin migration in electrophoretic analysis. cockatiel albumin migrates to a position equivalent to chicken α-globulins, while the migration of cockatiel pre-albumin is similar to that of chicken albumin (archer & battison 1997). decreased albumin concentration has been determined in birds suffering from maldigestion, malabsorption and protein-losing enteropathy. other causes of hypoalbuminaemia include protein-losing nephropathy and liver failure (wilson, greenacre & howerth 1999; harr 2002). while bradley, naude & oelofsen (1985) reported that the albumin fraction of ostriches was 71 %, in the present study was found to be 60.96 % (table 2). the reason for this discrepancy may be the result of differences in such factors as diet, physiological status and age. the α-globulin fraction is the most rapidly migrating of all the globulins, and in most species, except in ruminants, it migrates as an α1 (fast) and an α2 (slow) fraction. in general, the α1-globulins are smaller than the α2, but there appears to be no functional separation between the two fractions (kaneko 1989). the α1and α2-globulin fractions in this study were 0.24 and 15.94 % respectively (table 2). serum α1-globulin and α2-globulin fractions in human were reported by wijnen & van dieijen-visser (1996) to be 21 and 19 %. 78 reference serum protein and lipoprotein fractions of ostriches in turkey table 1 composition of mixed feed and nutrient matters of ostriches composition of mixed composition of nutrient feed (%) matters (%) barley 29.5 dry matter 89.37 oat 29.5 organic matter 81.86 wheat 19.5 ether extracts 3.22 soybean meal 19.5 crude fiber 7.57 limestone 1.0 crude protein 15.14 salt 0.5 ash 7.51 vitamin-mineral nitrogen free extra premix 0.5 matters 55.93 total 100.00 metabolic energy (kcal/kg) 2 950 table 2 serum protein and lipoprotein fractions* of the ostrich protein and lipoprotein fractions (%) x ± s.d. albumin 60.96 ± 2.40 α1-globulin 0.24 ± 0.13 α2-globulin 15.91 ± 2.56 β-globulin 13.34 ± 3.83 γ-globulin 9.55 ± 1.44 hdl 53.77 ± 9.55 vldl 0.60 ± 0.14 ldl 48.09 ± 6.99 n = 10 * the figures of the ten birds were combined and averaged the β-globulins trail the α2 fraction and similarly migrate as β1 and β2 fractions in most domestic animals, except ruminants. important proteins of this fraction are complement, haemopexin, transferrin, ferritin and c-reactive protein (kaneko 1989). the β-globulin fraction in the ostriches in the current study was found to be only one fraction. in this study, β-globulin and γ-globulin fractions were reported as 13.34 and 9.55 % (table 2). these fractions in human reported by wijnen & van dieijen-visser (1996) were higher than the data in the present study. the γ-globulin fraction in humans was found to be 21 % (nozaki et al. 1998). in normal chicken plasma, almost 75 % of lipoprotein is hdl, about 10 % is vldl and the remainder is ldl. therefore, although triglycerides constitute a much greater percentage of vldl than hdl, hdl is the major carrier of plasma triglycerides. also, more than 70 % of the plasma cholesterol of chickens is found in hdl (bell & freeman 1971; gould & siegel 1985). hdl, vldl and ldl fractions in this study were found to be 53.77, 0.60 and 48.09 % respectively. lipoprotein fractions in chickens were reported by gould & siegel (1985) to be hdl 74 %, vldl 7 % and ldl 19 %. references a.o.a.c. 1984. official methods of analysis, 14th ed. washington, dc: association of official analytical chemists. archer, f.j. & battison, a.l. 1997. differences in electrophoresis patterns between plasma albumins of the cockatiel (nymphicus hollandicus) and the chicken (gallus gallus domesticus). avian pathology, 26:865–870. aydin, c., ak, i., galip, n. & zaugg, s.n. 2003. the effects of dietary protein levels on some hematological and production parameters of breeding ostriches (struthio camelus). indian veterinary journal, 80:5. bell, d.j. & freeman, b.m. 1971. physiology and biochemistry of the domestic fowl, vol. 3. london & new york: academic press. bowes, v.a., julian r.j. & stirtzinger, t. 1989. comparison of serum biochemical profiles of male broilers with female broilers and white leghorn chickens. canadian journal of veterinary research, 53:7–11. bradley, g., naude, r.j. & oelofsen, w. 1985. the isolation and characterization of serum albumin from the ostrich (struthio camelus). comparative biochemistry and physiology b, 82:829–835. gould, n.r. & siegel, h.s. 1985. serum lipoproteins in chickens after administration of adrenocorticotropin or exposure to high temperature. poultry science, 64:567–574. harr k.e. 2002. clinical chemistry of companion avian species: a review. veterinary clinical pathology, 31:140–151. kaneko, j.j. 1989. clinical biochemistry of domestic animals, 4th ed. boston: academic press. nozaki, h., nohara, y., ashitomi, i., zukeran, r., inafuku, t., akisaka, m. & suzuki, m. 1998. serum globulin levels and activities of daily living in centenarians. nippon ronen igakkai zasshi, 35:680–685. okotie-eboh, g., bailey, c.a., hicks, k.d. & kubena, l.f. 1992. reference serum biochemical values for emus and ostriches. american journal of veterinary research, 53: 1765–1768. spinu, m., spinu, o. & degen, a.a. 1999. hematological and immunological variables in a domesticated and wild subspecies of ostrich. british poultry science, 40:613–618. wedler, v., prokpo, s., kunzi, w., meyer, v.e., stocker, r. & burgi, u. 1998. tracking dysproteinemia in thermal injuries using serum protein electrophoresis. annals of burns and fire disasters, 9:222–227. whitehead, c.c., hood, r.l., heard, g.s. & pym, r.a. 1984. comparison of plasma very low density lipoproteins and lipogenic enzymes as predictors of fat content and food conversion efficiency in selected lines of broiler chickens. british poultry science, 25:277–286. wilson, g.h., greenacre, c.b.& howerth, e.w. 1999. ascaridosis in a group of psittacine birds. journal of avian medicine and surgery, 13:32–39. wijnen, p.a. & van dieijen-visser, m.p. 1996. capillary electrophoresis of serum proteins. reproducibility, comparison with agarose gel electrophoresis and a review of the literature. european journal of clinical chemistry and clinical biochemistry, 34:535–545. 79 u. polat et al. vdmerwe_309-314.qxd introduction game ranching is becoming a lucrative industry in southern africa, leading to a number of wildlife species being kept in areas other than their natural habitat. management interventions such as the use of acaricides are required to maintain the health of these animals, to reduce stress levels, physical damage inflicted by ectoparasites and transmission of tick-borne diseases, especially when game are kept under semi-intensive conditions. many game animals are often relocated to other game reserves for various reasons. the presence of tuberculosis and other diseases in african buffaloes in certain areas has led to their relocation to various game reserves to participate in disease-free breeding programmes. translocation and export of game animals requires complete cleaning from tick infestations (hamel & van amelsfoort 1985). three trials on various game species are discussed in this paper. a pour-on acaricide formulation consisting of amitraz and cypermethrin (both at 1 % m/v), was evaluated against naturally occurring infestations of ticks on eland (taurotragus oryx), african buffaloes (syncerus caffer) and blesbok (dam309 onderstepoort journal of veterinary research, 72:309–314 (2005) acaricide efficiency of amitraz/cypermethrin and abamectin pour-on preparations in game j.s. van der merwe1*, f.j. smit2, a.m. durand3, l.p. krüger4 and l.m. michael5 abstract van der merwe, j.s., smit, f.j., durand, a.m., krüger, l.p. & michael, l.m. 2005. acaricide efficiency of amitraz/cypermethrin and abamectin pour-on preparations in game. onderstepoort journal of veterinary research, 72:309–314 the efficacy of an amitraz/cypermethrin pour-on preparation (1 % w/v each) was tested against natural tick infestations of buffaloes, eland and blesbok in three separate trials. the eland were also treated with a 0.02 % abamectin (w/v) acaricidal pour-on preparation. the amitraz/cypermethrin pour-on was effective against amblyomma hebraeum, rhipicephalus evertsi evertsi, rhipicephalus appendiculatus and hyalomma marginatum rufipes on the buffaloes. both acaricides were effective against r. appendiculatus and rhipicephalus (boophilus) decoloratus in the eland. the amitraz/cypermethrin acaricide was effective against r. (boophilus) decoloratus in the blesbok. ticks can cause damage to the skins, secondary infections, abscesses, anaemia, loss of condition, tick toxicosis and act as vectors of infectious diseases. introduction of hosts and/or ticks from endemic to non-endemic areas because of translocation of game, may lead to severe losses. the pouron acaricides tested were effective against natural tick infestations and should always be used according to the manufacturer’s instructions and efficacy claims. keywords: abamectin, acaricides, african buffalo, amitraz, blesbok, cypermethrin, eland, game, pour-on, tick infestation * author to whom correspondence is to be directed. e-mail: kvdmerwe@virbac.co.za 1 virbac rsa, private bag x115, halfway house, 1685 south africa 2 p.o. box 4511, mokopane, 0600 south africa 3 p.o. box 18348, pretoria north, 0116 south africa 4 animal improvement institute, irene, private bag x2, irene, 0062 south africa 5 8 trevor street, murrayfield, pretoria, 0184 south africa accepted for publication 20 june 2005—editor aliscus dorcas phillipsi). a second pour-on acaricide formulation, consisting of 0.002 % abamectin (m/v), was also tested on eland. materials and methods animals that appeared healthy and showed suitable visible infestations of external parasites were selected for the trials. no ectoparasite treatment was administered for at least 3 weeks prior to commencement of the study. the animals were not ranked and were not allocated according to any criteria, but were treated at random to reduce handling stress. the doses recommended were the same as recommended for domestic stock. the study animals were not destined to enter the food chain, therefore a withdrawal period was not applicable. at the end of each study the animals involved were relocated back to the game reserve. no statistical analysis was conducted during any of the trials. the efficacy of the acaricide(s) was calculated by using the formula: % control = (c – t) c x 100 where: c = mean number of ticks on day 0 t = mean number of ticks on day 7 no adverse circumstances occurred during the study period to affect the quality or integrity of the data or study in any of the three trials. buffalo trial the efficacy of a pour-on acaricide, containing 1 % (w/v) each of amitraz and cypermethrin, was evaluated against natural infestations of ectoparasites of african buffaloes located at the mabalingwe nature reserve in the limpopo province of south africa. sixteen buffalo of either sex and older than 8 months were involved in this study. natural infestations of amblyomma spp., rhipicephalus spp. and hyalomma spp. were observed on the buffaloes. the study design is given in table 1. the buffaloes were allocated to two groups, each consisting of eight animals. group t1 was the treated group and received the pour-on acaricide along the dorsal midline area (or as near as possible) from the withers to the tubae coxae using plastic syringes at a dosage rate of 0.1 ml product per kg body mass. the dosage rate for domestic stock recommended by the manufacturer was used, as the same tick species occur on buffaloes and domestic stock. group t2 was the untreated group. the body mass of the buffaloes in the trial varied between 140 and 350 kg. animals were weighed by means of a weight band while being restrained in a handling crush. ticks were counted on day 0 (before treatment) and on day 7 (after treatment), while the animals were restrained in the crush. the animals were housed in a boma consisting of two holding pens with dimensions of 100 m x 100 m. individuals of the two groups were not in contact with each other. eland trial this trial was conducted to evaluate the effectiveness of two pour-on formulations in the control of natural infestations of the following ectoparasites in eland: amblyomma hebraeum, rhipicephalus evertsi evertsi, rhipicephalus appendiculatus and rhipicephalus (boophilus) decoloratus. the eland were relocated from a game farm to the inkwenkwezi game park, both situated in the eastern cape province. they were allowed to acclimatise for 7 days before commencement of the experimental phase. the 17 study animals, of either sex, were tranquillized during handling. they weighed between 155 and 471 kg, appeared healthy and had not received treatment against ectoparasites for at least 3 weeks prior to commencement of the trial. the study design is given in table 2. the animals were divided into three groups, i.e. two treatment groups t1 and t2 consisting of six animals and a control group (untreated) consisting of five animals. two pour-on acaricide formulations were evaluated. the first consisted of a mixture of amitraz and cypermethrin (both at 1 % m/v) and was applied at a dosage rate of 0.1 ml/kg body mass. the second consisted of abamectin (at 0.002 % m/v) and was applied at a dosage rate of 0.1 mg/kg body mass. the eland were weighed by means of a weight band after they had been tranquillized and their ticks counted. the required dose was applied along the dorsal midline area (or as near as possible) from the withers to the tubae coxae using plastic disposable syringes. the animals were treated on day 0 after the ticks were counted. ticks were again counted on day 7. the eland were housed in a boma consisting of two pens each with dimensions of 9 m x 9 m, the sides of the pens were covered with plastic sheeting to reduce stress levels. they were all contained in the same boma and were fed lucerne, guava leaves and grass hay. water was available ad libitum. blesbok trial the purpose of this trial was to evaluate the efficacy of an amitraz/cypermethrin acaricidal pour-on in blesbok rams naturally infested with field strains of ticks. twelve animals, approximately 2 years of age, were captured over a period of 2 days. ticks were 310 acaricide efficiency in game counted prior to treatment and after treatment on day 7. the first six animals captured were allocated to the untreated control group and the next six animals to the treatment group. the experimental design is given in table 3. the animals were allowed to acquire a natural tick infestation in the game reserve. the area is regarded as a high tick environment. the blesbok were darted using an opioid agonist to tranquillize them. tick counts were conducted, the animals were weighed and the treatment group received the treatment while they were still tranquillized. the treatment group (t1) received the same pour-on formulation as was used on the buffaloes at a dosage rate of 0.1 ml/kg body mass and was applied by means of a plastic syringe, in one line from the withers to the base of the tail. the control group (t2) was left untreated. the dosage rate is the same as recommended for cattle as similar tick populations occur on both blesbok and cattle. ticks were counted macroscopically on individual animals and identified to species level. the animals were kept in a boma consisting of two holding pens with dimensions of approximately 9 m x 9 m. a hinged gate between the two pens was available to allow animals to be removed from the sedated animals during tick counts. the boma and the gate were completely covered with plastic sheeting on the inside for protection against injury. the two groups of animals were maintained together in the boma for the duration of the study for practical reasons. the animals were fed a ration of guava leaves, grass and lucerne. water was available ad libitum. results none of the animals involved in the trials showed any adverse effects to the acaricide(s) evaluated during the three trials. buffalo trial the results obtained are given in table 4. the amitraz/cypermethrin acaricide was 94.6 % effective against a. hebraeum, 100 % effective against r. evertsi evertsi, 99.9 % effective against r. appendiculatus and 100 % effective against h. marginatum rufipes. this pour-on acaricide is therefore regarded as effective against these tick species on buffaloes, because of the > 80 % efficacy. eland trial the results obtained are given in table 5. the amitraz/cypermethrin pour-on acaricide showed 86.11 % efficacy against r. appendiculatus and 98.1 % efficacy against r. (boophilus) decoloratus. the abamectin pour-on was 87.48 % effective against r. appendiculatus and 88.1 % effective against r. (boophilus) decoloratus. lower than 80 % efficacy against a. hebraeum and r. evertsi evertsi was found for both acaricide pour-on formulations in the eland. these two acaricides can only be regarded 311 j.s. van der merwe et al. table 1 experimental design of the buffalo trial group no. of animals treatment application route dose rate (ml/kg body mass) t1 8 amitraz/cypermethrin pour-on percutaneous 0.1 t2 8 untreated control not applicable not applicable table 2 experimental design of the eland trial group no. of animals treatment application route dose rate (ml/kg body mass) t1 6 amitraz/cypermethrin pour-on percutaneous 0.1 t2 6 abamectin pour-on percutaneous 0.1 t3 5 untreated control not applicable not applicable table 3 experimental design of the blesbok trial group no. of animals treatment application route dose rate (ml/kg body mass) t1 6 amitraz/cypermethrin pour-on percutaneous 0.1 t2 6 untreated control not applicable not applicable table 5 percentage efficacies of two pour-on acaricides against ticks of eland type of tick amitraz/cypermethrin pour-on abamectin pour-on amblyomma hebraeum 60.23 38.12 rhipicephalus evertsi evertsi 68.98 61.18 rhipicephalus appendiculatus 86.11* 87.48* rhipicephalus (boophilus) decoloratus 98.10* 88.10* * more than 80 % efficacy = effective table 7 list of diseases and their vectors (adapted from howell, walker & nevill 1983 to include only ticks relevant to this paper) disease and disease-causing organism susceptible animals vectors heartwater [ehrlichia (cowdria) cattle, sheep, goats amblyomma hebraeum ruminantum)] redwater (babesia bigemina) cattle rhipicephalus (boophilus) decoloratus gallsickness (anaplasma marginale) cattle rhipicephalus (boophilus) decoloratus corridor disease (theileria lawrencei) cattle, buffalo rhipicephalus appendiculatus theileriosis (theileria species) cattle amblyomma hebraeum rhipicephalus evertsi rhipicephalus appendiculatus biliary fever (babesia equi) horses rhipicephalus evertsi spirochaetosis (borrelia theileri) cattle, sheep, goats, horses, mules, rhipicephalus (boophilus) decoloratus donkeys rhipicephalus evertsi tick bite fever (rickettsia conori) humans amblyomma hebraeum hyalomma marginatum rufipes rhipicephalus evertsi crimean-congo haemorrhagic fever humans main vectors: hyalomma marginatum virus (cchfv) (rechav, zeederberg rufipes & zeller 1987) hyalomma truncatum cchfv also found in: rhipicephalus evertsi evertsi 312 acaricide efficiency in game table 4 percentage efficacy of a pour-on acaricide against ticks on the buffaloes type of tick amitraz/cypermethrin pour-on amblyomma hebraeum 94.6* rhipicephalus evertsi evertsi 100.0* rhipicephalus appendiculatus 99.9* hyalomma marginatum rufipes 100.0* * more than 80 % efficacy = effective table 6 percentage efficacy of a pour-on acaricide against ticks of blesbok type of tick amitraz/cypermethrin pour-on amblyomma hebraeum 17.00 rhipicephalus evertsi evertsi 55.30 rhipicephalus (boophilus) decoloratus 83.40* * more than 80 % efficacy = effective table 8 list of diseases caused by tick toxins and their vectors (adapted from howell et al. 1983 to include ticks relevant to this paper) disease susceptible animal vectors spring lamb paralysis lambs, calves rhipicephalus evertsi evertsi tick toxicosis cattle rhipicephalus appendiculatus as effective against r. appendiculatus and r. (boophilus) decoloratus in eland. a reduction in the tick numbers of the control animals was obtained, which can be ascribed to contamination with the acaricide from the treated animals as they were housed in the same pens. blesbok trial an efficacy of 83.4 % was obtained against r. (boophilus) decoloratus, 55.3 % against r. evertsi evertsi and 17 % against a. hebraeum. the results obtained are given in table 6. the blesbok had very low tick infestations and the results are therefore inconclusive. an effective rating against r. (boophilus) decoloratus is applicable because of the > 80 % efficacy shown. discussion ticks are important in domesticated animals and game in south africa. problems can range from slight irritation, to lesions which cause damage to skins and hides, and to the fact that they act as vectors for certain infectious diseases, such as corridor disease and heartwater. some of the diseases and their vectors applicable to the tick populations relevant to this paper are given in table 7. diseases caused by tick toxins from ticks relevant to this paper are shown in table 8. (howell, walker & nevill 1983). in both cases the information was adapted to include only the tick species found on the game during the three studies discussed in this paper. the growth in the game industry in south africa, combined with the translocation of game to areas where they do not normally occur and keeping of game under semi-intensive conditions have led to the role of ectoparasites, such as ticks, becoming more important. ticks can cause direct and indirect damage in these animals. blood loss, high tick burdens and toxicosis caused by certain ticks can bring about a decline in their health. the skin lesions caused by ticks can have implications for the game trophy, hunting tourism and taxidermy industries. wounds can also lead to entry of secondary organisms that can cause abscesses or other infections. tick bites can damage auricles and teats of animals. tick infestations of game may lead to loss of production and even death, because of tick toxicosis, metabolic disturbances, anaemia, secondary infections of tick wounds and transfer of blood parasites by ticks (horak 1980; lightfoot & norval 1981, as cited by zieger, horak, cauldwell, uys & bothma 1998). old, sick and young animals have a lower resistance and are therefore more susceptible to the above problems caused by ticks because of high tick burdens (heyne, personal communication 2004). studies conducted on various types of game indicate that a healthy animal is better able to withstand the effects of ticks than an injured or ill animal (boomker & horak 2002). hamel & van amelsfoort (1985) found an eland in an emaciated and anaemic condition associated with a high tick and eland-specific sucking lice (lignognathus taurotragus) burden. the ticks they found were r. (boophilus) decoloratus, r. evertsi evertsi, and a. hebraeum, which occurred on different sites of the body, according to species preferences. considerable risks are associated with translocation of wild ruminants from heartwater-endemic areas to heartwater-free areas, which have large populations of susceptible domestic and wild ruminant hosts and tick species capable of transmitting the disease (peter, anderson, burridge & mahan 1998). these authors demonstrated a carrier state for ehrlichia (cowdria) ruminantium in eland, giraffes (giraffa camelopardalis), kudus (tragelaphus strepsiceros) and blue wildebeest (connochaetes taurinus). the vector used to transmit the organism to naive ruminants during their study was a. hebraeum. wild ruminants seem to play an important role in the epidemiology of heartwater by acting as reservoirs of e. (cowdria) ruminantium infection. andrew & norval (1989) have shown that sheep, cattle and african buffaloes remain carriers of heartwater for long periods after recovery from the disease. the possibility that other ruminants may also be carriers of heartwater exist and further research is required to elucidate this possibility. paralyses caused by the karoo paralysis tick, ixodes rubicundus, in gemsbok (oryx gazella) was studied by fourie & vrahimis (1989). it would seem that 313 j.s. van der merwe et al. gemsbok with a high tick burden are more likely to become paralysed than those with a low tick burden. this toxicosis seems to be age-related. adult gemsbok are either more resistant to the toxin, or their tick burdens are not high enough to cause paralysis. high mortality rates in the subadult group suggest that mortalities induced by the karoo paralysis tick can retard population growth. this may also be true of other conditions caused by ticks. the suitability of an area for the introduction of a new game species should not be determined only from a grazing perspective, but harmful host-parasite associations should also be considered (fourie & horak 1987, as cited by fourie & vrahimis 1989). game endemic to a specific region are normally not seriously affected by tick-borne parasites and diseases. if hosts and/or ticks are introduced through their translocation to non-endemic areas, severe losses may occur (lightfoot & norval 1981, as cited by zieger et al. 1998). from the above it is clear that the presence of ticks on game species such as buffaloes, eland and blesbok can have severe implications. the use of management interventions such as acaricides to reduce tick burden is essential. the pour-on acaricides tested were both effective against natural infestations of ticks occurring in buffaloes, eland and blesbok and should be used according to the manufacturer’s recommendations and efficacy claims. references andrew, h.r. & norval, r.a.i. 1989. the carrier status of sheep, cattle and african buffalo recovered from heartwater. veterinary parasitology, 34:261–266. boomker, j. & horak, i.g. 2002. ectoand endoparasites, in game ranch management, 4th ed., edited by j. du p. bothma. pretoria: van schaik publishers. fourie, l.j. & vrahimis, s. 1989. tick-induced paralysis and mortality of gemsbok. south african journal of wildlife research, 19:118–121. hamel, h.d. & van amelsfoort, a. 1985. tick infestation and its treatment in an eland antelope (case report). veterinary medical review, 2:152–157. horak, i.g. 1980. control of parasites in antelope in small game reserves. journal of the south african veterinary association, 51:17–19. howell, c.j., walker, jane b. & nevill, e.m. 1983. bosluise, myte en insekte van huisdiere in suid-afrika. 1. beskrywing en biologie. [pretoria:] departement van landbou-tegniese dienste (wetenskaplike pamflet, no. 393). peter, t.f., anderson, e.c., burridge, m.j. & mahan, s.m. 1998. demonstration of a carrier state for cowdria ruminantium in wild ruminants from africa. journal of wildlife diseases, 34:567–575. rechav, y., zeederberg, m.e. & zeller, d.a. 1987. dynamics of african tick (acari: ixodoidea) populations in a natural crimean-congo hemorrhagic fever focus. journal of medical entomology, 24:574–583. zieger, u., horak, i.g., cauldwell, a.e., uys, a.c. & bothma, j. du p. 1998. the effect of chemical tick control on cattle on free-living ixodid ticks and on ticks parasitic on sympatric impala in the central province, zambia. south african journal of wildlife research, 28:10–15. 314 acaricide efficiency in game 443 onderstepoort journal of veterinary research, 76:443–448 (2009) the brody effect induced by premature ventricular complexes in the ovine heart j. ker1*, e.c. webb2 and d. van papendorp1 abstract ker, j. webb, e.c. & van papendorp, d. 2009. the brody effect induced by premature ventricular complexes in the ovine heart. onderstepoort journal of veterinary research, 76:443–448 the qrs response of the electrocardiogram to bleeding has been a source of interest to the physiologist for more than a century. studies in the dog, cat and chicken have shown a reduction in qrs amplitude in response to bleeding. this effect has been explained by the so-called brody effect, in which the intraventricular mass of blood acts as a conducting medium, augmenting radial conduction, thus resulting in the subsequent reduction in qrs amplitude in conditions where the intraventricular mass of blood is reduced. the aim of this study was to evaluate whether the brody effect will be present in the ovine heart and, furthermore, to evaluate if the right and left ventricles will demonstrate the same qrs change if the brody effect is indeed present. this study clearly demonstrated that the brody effect is present in the ovine heart. furthermore, two unique aspects emanating from this study are firstly the fact that this is the first study to show that premature ventricular complexes are able to induce the brody effect and, secondly that there is a very clear difference in the response of the right and the left ventricles when the brody effect is induced in the ovine heart. keywords: brody effect; ovine heart; qrs amplitude; premature ventricular complex introduction the variations in the qrs amplitude of the electrocardiogram, caused by alterations in ventricular filling, have been a source of interest to the physiologist since 1910 (brody 1956; nelson, lange, hecht, carlisle & ruby 1956; ishikawa, berson & pipberger 1971; manoach, gitter, grossman & varon 1971; manoach 2000). the experiments in which ventricular filling has been increased and decreased were described in both poikilotherms and homeotherms (manoach, gitter, grossman & varon 1972). in all these experiments, ventricular filling was decreased directly by bleeding or clamping of the inferior vena cava, or indirectly by compression of the heart via the induction of a pericardial effusion, or increased directly via the infusion of blood and other fluids or indirectly via clamping of the aorta. in both poikilotherms and homeotherms the qrs amplitude of the electrocardiogram changes during bleeding—leading to emptying of the ventricle—or filling of the ventricle (manoach et al. 1972). studies in homeotherms (normal mammals, specifically cats and dogs, as well as non-mammals, such as chickens) have shown that a decrease in ventricular filling causes a reduction in the amplitude of * author to whom correspondence is to be directed. e-mail: jker@wol.co.za 1 department of physiology, faculty of medicine, university of pretoria, south africa 2 department of animal and wildlife sciences, faculty of natural and agricultural sciences, university of pretoria, south africa accepted for publication 16 april 2009—editor 444 brody effect induced by premature ventricular complexes in ovine heart the qrs complex while increased ventricular filling causes an increase in the amplitude of the qrs complex (manoach, gitter, grossman & varon 1972; manoach, varon, grossman, gitter & sroka 1971; manoach, gitter, grossman, varon & gassne 1971). schwan 1928 (cited by brody 1956) stated that the conductivities of the lung and myocardium are almost the same, but that the conductivity of intraventricular blood is approximately ten times that of the surrounding tissue and thus that the intraventricular blood mass will exert an effect upon electrical potentials generated within the myocardium. brody (1956) examined this effect and concluded that the net effect of the intracavitary blood mass on the cardiac dipole is to augment the radial components and to reduce the tangential components, thus explaining why bleeding will lead to a reduction in the qrs amplitude in the mammalian heart. since the original study by brody (1956) this phenomenon became known as the brody effect. this effect results from the lower resistivity of the intraventricular blood mass as compared to the surrounding tissues, in essence causing a short-circuiting effect (ishikawa 1976). however, the qrs amplitude increases after bleeding in frogs, turtles and lizards (poikilotherms) (mano ach et al. 1971; pipberger, ishikawa & berson 1972). this opposite qrs reaction to bleeding in homeotherms and poikilotherms is ascribed to phylogenetic differences (manoach et al. 1972). however, especially in light of the brody effect, this opposite reaction has never been properly explained in the literature to date. there is ample experimental evidence for the brody effect from a number of other researchers (angelakos & gokhan 1963; horan, andreae & yoffee 1961; nelson et al. 1956). more interestingly, and poorly examined until now, are the different qrs responses of the right and left ventricles to bleeding. angelakos & gokhan (1963), in their experiments on dogs, noted a decrease in amplitude of left ventricular qrs complexes to bleeding, as expected. however, they also noted an opposite effect over the right ventricle—this makes physiological sense as right ventricular activation is predominantly tangential (pipberger et al. 1972). other means of altering left ventricular volume and inducing the brody effect, such as rapid atrial pacing, have also been described (daniels, iskandrian, hakki, kane, bemis, horowitz, greenspan & segal 1984). the purpose of this study was to identify the existence of the brody effect in the ovine heart. no evidence of any description of the brody effect in the ovine heart could be found in the literature. fur thermore, we specifically sought to examine the possibility that the right and left ventricle will show a different qrs response during the brody effect. premature ventricular complexes (pvcs) were chosen to reduce left and right ventricular volumes, in order to try and induce the brody effect and to document any possible difference between right and left ventricular qrs amplitude responses. a premature ventricular complex (pvc) is the expression of an impulse that arises prematurely in an ectopic ventricular focus and can originate in the specialized conduction tissue distal to the bifurcation of the bundle of his or in the ventricular myocardium itself (schamroth 1980; myerburg & kessler 1998). as a pvc by definition arises prematurely, the premature contraction of the ventricle will impair the diastolic filling and thus volume, as the diastolic time interval is shortened and will thus lead to a lower ventricular volume. the premature impulse, whether it originates from the left or right ventricle, will be propagated to the other ventricle, therefore both ven tricles will contract prematurely, resulting in a lowering of both ventricular volumes. it was postulated that the brody effect in the ovine heart could be demonstrated in this way, and it was hoped that these results would shed light on a possible different qrs response from the right and left ventricles. materials and methods this study was performed with the approval of, and adherence to, the guidelines of the pretoria biomed ical research centre`s animal use and care committee. a clinically normal dorper wether, aged 10 months and weighing 35 kg was used for this study. the sheep was fed lucerne hay ad libitum and received 300 g per day of pelleted concentrate (10 mj me/kg dm with 14 % crude protein) and had free access to water at all times. after an overnight fast, the wether was sedated by an intramuscular dose of ketamine hydrochloride (brevinaze, manufactured by intramed) at a dose of 100 mg and placed in the left lateral decubitus position. it was then placed under continuous electrocardiographic monitoring as follows: einthoven`s triangle was moved from the frontal to the sagittal plane, as described before by schultz & pretorius (schulz & pretorius 1972), by moving the standard and unipolar limb electrodes as follows: 445 j. ker, e.c. webb & d. van papendorp (a) avr moved from the right forelimb to the head between the ears; (b) avl moved from the left forelimb to the sacrum; (c) avf moved from the left hindlimb to the sternal angle; and (d) the earth electrode was placed on the right hindleg, just above the hock. the six precordial leads were placed as follows, as described before by ker & webb (2003): (a) v1 placed 7 cm to the right of the sternal angle; (b) v2 placed 7 cm to the left of the sternal angle; (c) v3 placed 4.5 cm below and 1 cm to the left of v2; (d) v4 placed 4.5 cm below and 1 cm to the left of v3; (e) v5 placed 4.5 cm below and 1 cm to the left of v4; and (f) v6 placed 4.5 cm below and 1 cm to the left of v5. meditrace 200 (mtd), disposable ecg conductive, adhesive electrodes were used. the skin areas where the ecg electrodes were placed were clipped and the attached electrodes were secured with super glue (by bostik). premature ventricular complexes were then induced from the left ventricular apex using the seldinger technique a spring-wire guide, diameter 0.81 mm and length 60 cm, was advanced into the left ventricular apex via the right internal carotid artery under fluoroscopic guidance. for a period of 20 min the spring-wire guide was moved manually in order to induce pvcs from the left ventricular apex. twelve-lead electrocardiograms were continuously recorded during this period. results fig. 1 shows the 12-lead electrocardiogram of the sedated wether in the left, lateral decubitus position, before induction of pvcs. note the negative polarity of the qrs complex in lead v1, with an amplitude of 0.4 mv, and also the negative polarity of the qrs complex in lead v6, with an amplitude of 1.0 mv. fig. 2 shows the 12-lead electrocardiogram at the start of pvc induction. note the 3 pvcs in lead v1. they are easily recognized by their positive qrs fig. 1 the normal 12-lead electrocardiogram, before the induction of pvcs 446 brody effect induced by premature ventricular complexes in ovine heart fig. 2 note the 3 pvcs with a positive qrs polarity in lead v1 and a negative polarity in lead v6 fig. 3 the 12-lead electrocardiogram, demonstrating the appearance of pvcs in leads i, ii, iii, avr, avl and avf 447 j. ker, e.c. webb & d. van papendorp polarity, as opposed to the rest of the normal beats. in addition, note the same 3 pvcs in lead v6. here they have a negative qrs polarity with a much greater amplitude than that of the normal beats. this is a very important observation, as it indicates that the chosen position for lead v1 is ideal for evaluating the right ventricle and that the chosen position for lead v6 is ideal for evaluating the left ventricle: the pvc originates from the apex of the left ventricle, thus left ventricular activation initiates and propagates to the right ventricle. this process occurs in the direction of lead v1, thus the positive qrs polarity and occurs in a direction away from lead v6, thus the negative qrs polarity. fig. 3 is the 12-lead electrocardiogram demonstrating the appearance of pvcs in leads i, ii, iii, avr, avl and avf. pvcs are now induced at a higher frequency. fig. 4 records the 12-lead electrocardiogram after exactly 28 min of pvcs. note the striking changes in qrs amplitude in leads v1 and v6. in lead v1 the qrs amplitude is still negative, but at a much greater amplitude at 1.9 mv. in lead v6 the qrs amplitude is positive with an amplitude of 0.8 mv. discussion it was possible to demonstrate the brody effect in the ovine heart, by inducing pvcs in order to reduce the intraventricular volumes. the left ventricle responded as described in other homeotherms (e.g. cats, dogs and chickens) by a reduction in qrs amplitude. as the qrs amplitude in lead v6 changed from a negative polarity with an amplitude of 1.0 mv to a positive amplitude of 0.8 mv, this can be regarded as a reduction of 1.8 mv [0.8 mv – (–1.0 mv) = 1.8 mv]. the right ventricle responded by an increase in qrs amplitude as described by angelakos & gokhan (1963); in this case from –0.4 mv to –1.9 mv, thus an increase of 1.5 mv. it is concluded that the ovine heart is a valid model for the study of the brody effect, and, in addition this is also the first report of the brody effect in the ovine heart. there is a paucity of data on the brody effect in recent literature and it is still unsatisfactorily explained why there is a different response to a reduction in ventricular volume between the poikilothermic and homeothermic heart. fig. 4 this is the exact same 12-lead electrocardiogram after 28 min of pvcs. note the increased amplitude in lead v1 and the change in polarity of the qrs complex in lead v6 448 brody effect induced by premature ventricular complexes in ovine heart the uniqueness of this study is the fact that it is the first study in the literature which documents that pvcs are a valid method of inducing the brody effect. references angelakos, e.t. & gokhan, n. 1963. influence of venous inflow volume on the magnitude of the qrs potentials in vivo. cardiologia, 42:337. brody, d.a. 1956. a theoretical analysis of intracavitary blood mass influence on the heart lead relationship. circulation re­ search, 4:731–738. daniels, s., iskandrian, a,s., hakki, a.h., kane, s.a., bemis, c., horowitz, l.n., greenspan, a.m. & segal, b.l. 1984. correlation between changes in r wave amplitude and left ventricular volume induced by rapid atrial pacing. american heart journal, 107:711–717. horan, l.g., andreae, r.l. & yoffee, h.f. 1961. the effect of intracavitary carbon dioxide on surface potentials in the intact canine chest. american heart journal, 61:504. ishikawa, k., berson, a.s. & pipberger, h.v. 1971. electrocardiographic changes due to cardiac enlargement. amer­ i can heart journal, 81:635. ishikawa, k. 1976. influence of alterations in the hematocrit upon the p wave and the qrs complex. chest, 69:762–768. ker, j & webb, e.c. 2003. ventriculo-atrial conduction in the ovine heart, caused by premature ventricular complexes. onderstepoort journal of veterinary research, 70:107–111. manoach, m. 2000. on brody effect and increase in qrs amplitude. journal of cardiovascular electrophysiology, 11: 833. manoach, m., gitter, s., grossman, e. & varon, d. 1971. some considerations regarding the importance of blood, heart and tissue conductivity with regards to qrs amplitude changes after hemorrhage. american heart journal, 31:726–728. manoach, m., varon, d., grossman, e., gitter, s. & sroka, m. 1971. influence of bleeding on the qrs amplitude in adult chickens and chick embryo. israel journal of medical science, 7:708. manoach, m., gitter, s., grossman, e., varon, d. & gassne, s. 1971. influence of hemorrhage on the qrs complex of the electrocardiogram. american heart journal, 82:55–61. manoach, m., gitter, s., grossman, e. & varon, d. 1972. the relation between the conductivity of the blood and the body tissue and the amplitude of the qrs during heart filling and pericardial compression in the cat. american heart journal, 84:72–75. myerburg, r.j. & kessler, k.m. 1998. recognition, clinical assessment and management of arrhythmias and conduction disturbances, in hurst`s the heart. new york: mcgrawhill. nelson, c.v., lange, r.l., hecht, h.h., carlisle, r.p. & ruby, a.s. 1956. effect of intracardiac blood and of fluids of different conductivities on the magnitude of surface vectors. circulation, 14:977. pipberger, h.v., ishikawa, k. & berson. 1972. reply: let ter to the editor. american heart journal, 83:295–297. schamroth, l. 1980. ventricular extrasystoles, ventricular tachy cardia and ventricular fibrillation: clinical electrocardiographic considerations. progress in cardiovascular dis­ eases, 23:13–32. schultz, r.a. & pretorius, p.j. 1972. an electrocardiographic study of normal sheep using a modified technique. onderstepoort journal of veterinary research, 39:97–106. article information authors: dirk g. booyse1 burk a. dehority2 affiliations: 1department of anatomy and physiology, university of pretoria, south africa 2department of animal sciences, ohio state university, usa correspondence to: dirk booyse email: dbooyse@op.up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 02 mar. 2011 accepted: 23 may 2011 published: 12 oct. 2011 how to cite this article: booyse, d.g. & dehority, b.a., 2011, ‘protozoa and digestive tract parameters of the impala’, onderstepoort journal of veterinary research 78(1), art. #327, 5 pages. doi:10.4102/ojvr.v78i1.327 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) protozoa and digestive tract parameters of the impala in this original research... open access • abstract • introduction • materials and methods    • animals    • feed    • measurements    • protozoa • results and discussion    • protozoa    • physiological parameters • conclusion • acknowledgements    • authors’ contributions • references abstract (back to top) intestinal contents were collected from eight impala at three different localities during the winter hunting season (2005–2009), as well as from another 24 animals from a one-year trial at a game farm called ditholo (2003–2004). gas production, protozoa counts and several other physiological parameters were measured from both rumen and caecum or colon contents. only higher ophryoscolecid and isotrichidae species of protozoa were counted and identified. ostracodinium gracile was present in all 32 impala. eudiplodinium maggii was present in 31 animals and eudiplodinium impalae and epidinium (either ecaudatum or caudatum) in 30 animals. dasytricha ruminantium was present in only 11 of the impala. concentrations of protozoa were correlated with the season of sample collection and highly correlated with the animals living on the game farm. gas production (ml/g of wet rumen ingesta) was weakly correlated with protozoa concentration but not with the season of collection. introduction (back to top) a total of 13 protozoan species have previously been reported from the rumen of the impala. dogiel (1925) listed six protozoan species from impala in kenya and later, in 1932, he observed five species in uganda. four species were the same in both locations and the fifth belonged to the same genus but was identified as a different species (table 1). in south africa, van der wath and myburgh (1941) reported the presence of three species in impala rumens and later kleynhans (1977) recorded the presence of 11 species. four of the species observed by kleynhans, isotricha prostoma, entodinium parvum, eudiplodinium maggii and dasytricha ruminantium, were new host records for impala (see table 1). dehority and odenyo (2003) identified protozoa in impala from kenya only to the generic level and did not find any genera in which species had not previously been observed. apart from the counting and identification of protozoa, several other parameters of the digestive tract of the impala (rumen, small intestine, caecum and colon) were also investigated and are reported here for the first time. these include ph and temperature of contents, length of organ, contents weight per organ, gas production, dry matter and density. materials and methods (back to top) animals one female animal was collected from a farm called karoobult, 100 km north-west of pretoria (24°43’49.45”s, 27°33’22.88”e), in july 2009, five animals (three male and two female) were from the loskop dam nature reserve in mpumalanga province, 200 km east of pretoria (25°25’51.91”s, 29°19’39.80”e), and two male animals were from the rietvlei nature reserve, 50 km south of pretoria (25°52’41.17”s, 28°16’17.83”e). in addition, 24 animals (all male) were from a one-year trial from the ditholo game farm, 60 km north of pretoria (25°19’43.95”s, 28°19’04.34”e). feed impala are mixed feeders and therefore graze as well as browse. feed available to the impala consists of vegetation types called bushvelds. a bushveld can be described as a heavily grassed plain, dotted by dense clusters of trees and tall shrubs. the grasses found here are generally tall and turn yellow or brown in winter, which is the dry season throughout most of south africa. sour bushveld consists of grazing fields, mostly comprising grasses like eragrostis curvula, eragrostis chloromelas, hyparrhenia hirta and cymbopogon excavatus. these grasses are fast-growing plants in the summer, but die rapidly in winter, turning hard and tasteless. sour grass species can, however, recover quite rapidly from overgrazing. sweet bushveld generally consists of grasses that grow slowly in summer but tend to stay highly nutritious and palatable during the dry season (winter). sweet grass species include digitaria eriantha, panicum maximum and urochloa mosambicensis. sweet grasses recover very slowly from heavy impacts like overgrazing. mixed bushveld is a field composed of both sour and sweet grasses. the veld type in the rietvlei and ditholo areas is described as a sourish mixed bushveld, whilst karoobult represents sour bushveld and the vegetation from the loskop area is described as a mixed bushveld. in the spring and summer, impala both feed on grass (eragrostis) and browse on trees like acacia karoo and acacia caffra. however, in the winter they are forced to ‘low graze’ on grass, as little or no browse is available. measurements after shooting, the animals were weighed, the digestive tract removed and the different anatomical sections (rumen, small intestines, caecum and colon) were ligated before separation. each section was weighed and the temperature, ph and length of the organs were measured and noted. weights were measured with an electronic scale capable of measuring up to two decimal places and ph was measured with a portable, battery-powered ph meter (eutech, model ec-ph-10/01n, singapore). organ content weights were estimated by weighing the organ full and empty. samples for fermentation studies (200 ml) were placed in fermentation vessels and gas production was measured every minute by means of a glass syringe (fitted with a 20-gauge stainless steel needle), inserted through the rubber stopper. measurements were taken over a period of 45 min. the exact weight of the samples was determined after complete fermentation. protozoa a large sample of ingesta was taken from the rumen and the fluid squeezed out into a 40-ml plastic specimen bottle. for the caecum and colon samples, small aliquots were taken at random from the contents and poured into the 40-ml container. these samples were then transferred to larger marked containers and 100 ml 70% methanol was added to each. back at the laboratory, the protozoa samples were washed through sieves as described previously (booyse, boomker & dehority 2010). in essence, the entire sample was washed through a set of sieves with an inner and outer chamber. the sample was poured into the inner sieve (pore size 110 μm) and washed with water. particulate matter and protozoa passed through to the outer sieve, which had a pore size of 37 μm. the outer sieve was fitted with a draining tap to allow the contents to be drained into a bottle. the washed sample was then allowed to stand for 30 min and the volume adjusted back to 40 ml by decanting the excess water. two drops of brilliant green stain were added to each of the 40-ml samples, which were then allowed to stand for 24 h (dehority 1984). three aliquots (0.1 ml each) from each sample were placed onto separate microscope slides and fitted with a cover slip. protozoa on each slide were counted using a standard light microscope fitted with a panasonic digital camera (model gp220). concentrations were determined by multiplying the mean of all three counts by 10, thus giving a count per millilitre. results and discussion (back to top) protozoa initial examination of the protozoa samples revealed – unexpectedly – a complete absence of entodinium species. subsequent investigation revealed that these species were washed out through the 37-µm pores. the samples were washed primarily to remove most of the larger plant debris, which interferes with microscopical observation. because all the samples had been washed, we were able to enumerate only isotrichidae and the subfamilies diplodiniinae and ophryoscolecinae in the family ophryoscolecidae (larger ophryoscolecid protozoa). however, we were able to shoot an additional impala, collect rumen contents, stain and count protozoa in a 0.1-ml sample directly, without washing through sieves. our examination revealed the presence of numerous entodinium species in this last animal, which were identified and included with our previous data in table 1. table 1: rumen protozoa observed from impala (aepyceros melampus). four earlier reports on protozoa in the impala are also included in table 1 for comparison. to date, 13 species of protozoa have been observed in the impala rumen. in south africa, 11 of these were reported by kleynhans in 1977, nine in the present study and three by van der wath and myburgh in 1941. dogiel reported a total of five species from impala in kenya (1925) and uganda (1932). table 2 reflects the occurrence and concentration of d. ruminantium and the larger ophryoscolecids (five species) as grouped by the collection site. ostracodinium gracile was present in all 32 animals, e. maggii was present in 31 animals, whilst eudiplodinium impalae and epidinium caudatum were found in 30. in contrast, epidinium ecaudatum was present in only 16 animals and dasytricha species were found in nine. thus, the majority of the larger ophryoscolecids were represented by four species, namely o. gracile, e. maggii, e. impalae and ep. caudatum. the total concentration of all six species averaged about 1.6 x 104 per ml in the 24 animals from ditholo, 0.32 x 104 per ml in the five from loskop, 1.38 x 104 per ml in the two impala from rietvlei and 0.28 x 104 per ml in the single animal from karoobult. table 2: occurrence and concentration of dasytricha ruminantium and larger ophryoscolecid protozoa in rumen contents of the impala. the caecum and colon contents were mixed, washed as described above, and examined microscopically for protozoa. the results in table 3 show that only very low concentrations were present in the ingesta from these organs. presumably, the protozoa would be digested as they move through the abomasum and small intestine; however, some cells are obviously able to pass through unharmed. it is possible that they are embedded in larger food particles and are not exposed to the acidic and enzymatic conditions in those organs. these concentrations are considerably higher than what one author (b.a.d.) has previously observed in domestic ruminants (unpublished). table 3: occurrence and concentration of dasytricha ruminantium and larger ophryoscolecid protozoa in the caecum or colon contents of impala. a possible relationship between the time (i.e. the month when the animal was harvested) and the concentration of the larger protozoa was investigated, taking all 32 animals into consideration. a correlation coefficient (r) of 0.65 was obtained (r2 = 0.42). however, when only the 24 animals from a single location (ditholo) were used, the correlation coefficient was 0.865 (r2 = 0.75). figure 1 shows the regression of protozoa concentration for all 32 animals, according to time of harvesting. the first sample was obtained in december and labeled as 0, followed by january as 1, february as 2, etc. this was based on the assumption that the start of the summer growing season would begin in december, when pasture and browse were low, and that feed would become increasingly available during the following months. figure 1: regression of concentration of the larger protozoa in impala rumen contents according to the month of collection. it is also of interest that only two genera of the subfamily diplodiniinae, eudiplodinium and ostracodinium, have been found in the impala. species of diplodinium, metadinium, enoploplastron, elytroplastron and polyplastron occur in many of the other wild ruminants in africa (dehority & odenyo 2003; dogiel 1925, 1932; kleynhans & van hoven 1975; van hoven 1975, 1978, 1983). physiological parameters table 4 presents information on the collection site, age, sex, organ ph, weight of the organ contents, percentage dry matter, density and gas production from organ contents, incubated in vitro (gas production was not measured with small intestine ingesta). except for the animal from karoobult and the two from rietvlei, rumen ph values averaged below 6.0. this value would be expected in grazing ruminants, as demonstrated in the study by dehority and tirabasso (2001), where sheep were fed 1, 6, or 24 times per day. rumen dry matter values fell within the normal reported range of 10% – 13% (dehority 2003). table 4: physiological parameters of the impala digestive tract and gas production by rumen, caecum and colon contents. the aim of measuring gas production was to evaluate whether this parameter was associated with protozoal concentrations. when gas production was correlated with numbers of larger protozoa, the correlation coefficient was 0.31, indicating little or no association. this is not entirely unexpected, because a number of factors would be involved. probably the most critical would be the amount of available substrate in the rumen contents, regardless of the concentration of protozoa and bacteria. gas production from rumen, caecum and colon contents was greater in the animals on the game farm, suggesting that more substrate was available in the ingesta from these animals than from those living in the bushveld. in future studies it would be desirable to measure fermentation capacity as described by el-shazley and hungate (1965), where additional substrate is added. temperature of organ contents ranged from 30 ºc to 41 ºc for the rumen, between 11 ºc and 28 ºc for the small intestine, between 17 ºc and 31 ºc for the caecum and between 13 ºc and 30 ºc for the colon. length of the organs ranged from 281 mm to 570 mm for the rumen, 6218 mm to 13 852 mm for the small intestine, 162 mm to 652 mm for the caecum, and 190 mm to 1411 mm for the colon (data not shown). conclusion (back to top) the number of genera and species of protozoa occurring in the impala is limited compared to other african wild ruminants and domesticated cattle and sheep (booyse & dehority in press; dehority & odenyo 2003; dogiel 1932; kleynhans & van hoven 1976; van hoven 1975, 1978, 1983). only four species of entodinium, two genera of diplodiniinae and two species of epidinium have been observed. acknowledgements (back to top) authors’ contributions d.g.b. collected the samples, was responsible for all the measurements and conducted the microscopic studies. b.a.d. assisted with identification of the species, compilation of the data and writing the manuscript. references (back to top) booyse, d.g. & dehority, b.a., in press, ‘rumen protozoa in south african sheep with a summary of the worldwide distribution of sheep protozoa’, onderstepoort journal of veterinary research. pmid:2628563 booyse, d.g., boomker, e.a. & dehority, b.a., 2010, ‘protozoa in the digestive tract of wild herbivores in south africa. i: warthogs (phacochoerus aethiopicus)’, zootaxa 2492, 63–68. dehority, b.a., 1984, ‘evaluation of sub-sampling and fixation procedures used for counting rumen protozoa’, applied and environmental microbiology 48, 182–185. pmid:6476828, pmid:240360 dehority, b.a., 2003, rumen microbiology, nottingham university press, nottingham, uk. dehority, b.a. & odenyo, a.a., 2003, ‘influence of diet on the rumen protozoal fauna of indigenous african wild ruminants’, journal of eukaryotic microbiology 50, 220–223. dehority, b.a. & tirabasso, p.a., 2001, ‘effect of feeding frequency on bacterial and fungal concentrations, ph and other parameters in the rumen’, journal of animal science 79, 2908–2912. pmid:11768121 dogiel, v., 1925, ‘nouveaux infusoires de la famille des ophryoscolécidés parasites d’antilopes africaines’ [new infusoria of the family of ophryoscolecid parasites in african antelope], annales de parasitologie 2, 116–142. dogiel, v., 1932, ‘beschreibung einiger neuer vertreter der familie ophryoscolecidae aus afrikanischen antilopen nebst revision der infusorienfauna afrikanischer wiederkäuer‘ [description of some new representatives of the family ophryoscolecidae from african antelope together with a revision of the infusorial fauna of african ruminants], archiv für protistenkunde 77, 92–107. el-shazley, k. & hungate, r.e., 1965, ‘fermentation capacity as a measure of net growth of rumen organisms’, applied microbiology 13, 62–69. pmid:14269247, pmid:1058191 kleynhans, c.j., 1977, ‘the rumen protozoa of the impala (aepyceros melampus) lichtenstein, and the kudu (tragelaphus strepsiceros) pallas’, phd thesis, department of zoology, university of pretoria. kleynhans, c.j. & van hoven, w., 1976, ‘rumen protozoa of the giraffe with a description of two new species’, east african wildlife journal 14, 203–214. van der wath, j.g. & myburgh, s.j., 1941, ‘studies on the alimentary tract of merino sheep in south africa. vi. the role of infusoria in ruminal digestion with some remarks on ruminant bacteria’, onderstepoort journal of veterinary science and animal industry 17, 61–85. van hoven, w., 1975, ‘rumen ciliates of the tsessebe (damaliscus lunatus lunatus) in south africa’, journal of protozoology 22, 457–462. pmid:811788 van hoven, w., 1978, ‘development and seasonal changes in the rumen protozoan population in young blesbok (damaliscus dorcas phillipsi harper 1939)’, south african journal of wildlife research 8, 127–130. van hoven, w., 1983, ‘rumen ciliates with descriptions of two new species from three african reedbuck species’, journal of protozoology 30, 688–691. horak_255-261.qxd introduction a large number of surveys have focused on the role of small mammals as hosts of the immature stages of ixodid ticks in south africa. the accent has been mainly on murid rodents (rechav 1982; howell, petney & horak 1989; horak, fourie, novellie & williams 1991; fourie, horak & van den heever 1992; braack, horak, jordaan, segerman & louw 1996; horak & boomker 1998; horak & cohen 2001; petney, horak, howell & meyer 2004), but elephant shrews (stampa 1959; fourie et al. 1992; fourie, horak, kok & van zyl 2002), hares and rabbits (stampa 1959; horak, sheppey, knight & beuthin 1986; horak & fourie 1991; horak et al. 1991; horak, spickett, braack & penzhorn 1993; horak, spickett, braack, penzhorn, bagnall & uys 1995; macivor & horak 2003), rock dassies (horak & fourie 1986; horak et al. 1991), and small carnivores (horak, chaparro, beaucournu & louw 1999; horak, braack, fourie & walker 2000) have also been examined. the adults of some of the tick species that infest these small animals as larvae or nymphs are important vectors of disease or toxins to domestic livestock, whereas others are of little or no economic consequence. thus murid rodents are among the preferred hosts of the immature stages of haemaphysalis leachi, whose adults parasitize and transmit canine babesiosis to domestic dogs (lewis, penzhorn, lopez-rebollar & de waal 1996), and of 255 onderstepoort journal of veterinary research, 72:255–261 (2005) small mammals as hosts of immature ixodid ticks i.g. horak1, 2, l.j. fourie2 and l.e.o. braack3 abstract horak, i.g., fourie, l.j. & braack, l.e.o. 2005. small mammals as hosts of immature ixodid ticks. onderstepoort journal of veterinary research, 72:255–261 two hundred and twenty-five small mammals belonging to 16 species were examined for ticks in free state, mpumalanga and limpopo provinces, south africa, and 18 ixodid tick species, of which two could only be identified to genus level, were recovered. scrub hares, lepus saxatilis, and cape hares, lepus capensis, harboured the largest number of tick species. in free state province namaqua rock mice, aethomys namaquensis, and four-striped grass mice, rhabdomys pumilio, were good hosts of the immature stages of haemaphysalis leachi and rhipicephalus gertrudae, while in mpumalanga and limpopo provinces red veld rats, aethomys chrysophilus, namaqua rock mice and natal multimammate mice, mastomys natalensis were good hosts of h. leachi and rhipicephalus simus. haemaphysalis leachi was the only tick recovered from animals in all three provinces. keywords: immature ixodid ticks, haemaphysalis leachi, rhipicephalus gertrudae, rhipicephalus simus, small mammals, south africa 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa. e-mail: ivan.horak@up.ac.za 2 department of zoology and entomology, university of the free state, bloemfontein, 9301 south africa 3 scientific services, south african national parks, kruger national park, skukuza, 1350 south africa. present address: p.o. box 2550, brooklyn square, 0075 south africa accepted for publication 14 june 2005—editor rhipicephalus simus, whose adults parasitize cattle, horses and domestic dogs and transmit anaplasmosis to cattle and produce a toxin causing paralysis in calves and lambs (walker, keirans & horak 2000). rock elephant shrews, elephantulus myurus, are the hosts most favoured by the immature stages of ixodes rubicundus and rhipicephalus warburtoni, whose adults cause paralysis in sheep and goats (stampa 1959; walker et al. 2000), and scrub hares, lepus saxatilis, by the immature stages of hyalomma marginatum rufipes and hyalomma truncatum, whose adults are parasites of domestic cattle, sheep and goats. the former tick transmits anaplasmosis and babesia occultans to cattle, and the latter secretes a toxin that is the cause of sweating sickness in these animals (walker, bouattour, camicas, estrada-peña, horak, latif, pegram & preston 2003). the objective of the present paper is to present recent data on the ixodid ticks that infest small mammals in three of the nine provinces of south africa, and to supply the geographic coordinates of the localities at which the ticks were collected for future mapping purposes. materials and methods two hundred and twenty-five small mammals, belonging to 16 species, were collected in free state, mpumalanga and limpopo provinces, south africa and processed for tick recovery as described by horak et al. (1986). one hundred and sixty-three of these animals belonging to 10 species were examined in free state province and 62 belonging to 11 species were examined in mpumalanga and limpopo provinces (table 1). results and discussion the ticks collected in free state province are summarized in tables 2 and 3, and those from animals in the north-eastern regions of mpumalanga and limpopo provinces in tables 4 and 5. twelve tick species, of which two could be identified only to genus level, were recovered in free state province and nine in mpumalanga and limpopo provinces. only h. leachi was collected in all three provinces, while h. truncatum and rhipicephalus evertsi evertsi were collected in two, namely free state and mpumalanga provinces. scrub hares, l. saxatilis, and cape hares, lepus capensis, were infested with the largest number of tick species. in free state province namaqua rock mice, aethomys namaquensis, and four-striped grass mice, rhabdomys pumilio, were good hosts of the immature stages of h. leachi and rhipicephalus gertrudae, while in mpumalanga and limpopo provinces red veld rats, aethomys chrysophilus, namaqua rock mice and natal multimammate mice, mastomys natalensis, were good hosts of h. leachi and r. simus, a tick anagolous to r. gertrudae. dermacentor rhinocerinus the hosts of the immature stages of this tick were unknown until horak & cohen (2001) collected larvae and nymphs from rodents in mpumalanga province. for the sake of completeness we have repeated their findings here because other tick species, recovered from the same hosts, were not listed in the earlier publication. dermacentor rhinocerinus is a host-specific parasite of rhinoceroses (keirans 1993), of which there are several in the mthethomusha reserve, mpumalanga province. these animals must have been the source of infestation reflected in the burdens of immature ticks of five of the 38 rodents examined in the reserve (table 4). there are rhinoceroses in the willem pretorius nature reserve in free state province, but it is not known whether d. rhinocerinus is also present. haemaphysalis leachi domestic dogs and the larger wild felids are the preferred hosts of adult h. leachi, while its immature stages occur mainly on murid rodents (norval 1984; braack et al. 1996; horak et al. 2000). its widespread distribution in the present survey confirms norval’s (1984) assertion that provided hosts for its adults and immature stages are present, almost any locality in southern africa can be regarded as suitable. furthermore the large range of carnivore and rodent species parasitized by its adult and immature stages, respectively (norval 1984; horak et al. 2000; tables 2 and 4) and their widespread distribution, ensure that should h. leachi be introduced into a region it would readily become established. this assumption is corroborated by its extensive but discontinuous distribution in south africa (howell, walker & nevill 1978). haemaphysalis leachi transmits babesia canis, the cause of canine babesiosis in domestic dogs in south africa (lewis et al. 1996). judging by the host preferences of the adult and immature stages of the tick it would seem that transmission mostly takes place via the adults with infection passing transovarially from adult ticks of the previous generation without it being lost when the immature stages feed on rodents. 256 small mammals as hosts of immature ixodid ticks 257 i.g. horak, l.j. fourie & l.e.o. braack table 1 small mammals and the localities at which they were examined for ixodid ticks small mammal species province, locality and coordinatescommon name scientific name no. examined tree squirrel paraxerus cepapi 2 limpopo, pafuri (23°27´s, 31°19´e) (knp) short-tailed pouched saccostomys 1 free state, tussen-die-riviere nr (30°29´s, 26°15´e) mouse campestris 3 limpopo, pafuri (23°27´s, 31°19´e) (knp) bushveld gerbil tatera leucogaster 5 free state, sandveld nr (27°38´s 25°42´e) 4 mpumalanga, mtethomusha nr (25°29´s, 31°17´e) 1 mpumalanga, pretoriuskop (25°10´s, 31°16´e) (knp) red veld rat aethomys 10 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) chrysophilus 1 mpumalanga, berg-en-dal (25°25´s, 31°27´e) (knp) 8 limpopo, pafuri (23°27´s, 31°19´e) (knp) namaqua rock mouse aethomys 17 free state, “preezfontein” (29°50´s, 25°23´e) namaquensis 21 free state, tussen-die-riviere nr (30°29´s, 26°15´e) 34 free state, welbedacht dam (29°51´s, 26°53´e) 5 free state, zandbult (locality unknown) 5 free state, wolhuterskop (28°14´s, 28°18´e) 15 free state, willem pretorius nr (28°19´s, 27°15´e) 6 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) 1 limpopo, tshalungwa (22°33´s, 31°05´e) (knp) 3 limpopo, pafuri (23°27´s, 31°19´e) (knp) single-striped mouse lemniscomys rosalia 1 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) multimammate mouse mastomys coucha 10 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) natal multimammate mastomys natalensis 6 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) mouse 1 limpopo, dube station (knp) 1 limpopo, mashikiri (22°35´s, 31°11´e) (knp) 1 limpopo, pafuri knp (23°27´s, 31°19´e) pigmy mouse mus minutoides 2 free state, welbedacht dam (29°51´s, 26°53´e) black rat rattus rattus 1 free state, sandveld nr (27°38´s 25°42´e) 1 mpumalanga, skukuza (24°58´s, 31°36´e) (knp) four-striped grass rhabdomys pumilio 7 free state, tussen-die-riviere nr (30°29´s, 26°15´e) mouse 5 free state, platberg (28°16´s, 29°10´e) 4 free state, welbedacht dam (29°51´s, 26°53´e) 7 free state, zandbult (locality unknown) 9 free state, wolhuterskop (28°14´s, 28°18´e) 4 free state, golden gate np (28°31´s, 28°37´e) angoni swamp rat otomys angoniensis 1 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) swamp rat otomys irroratus 1 free state, golden gate np (28°31´s, 28°37´e) spring hare pedetes capensis 14 free state, sandveld nr (29°51´s, 26°53´e) cape hare lepus capensis 1 free state, tussen-die-riviere nr (30°29´s, 26°15´e) 2 free state, willem pretorius nr (28°19´s, 27°15´e) scrub hare lepus saxatilis 1 free state, tussen-die-riviere nr (30°29´s, 26°15´e) 2 free state, willem pretorius nr (28°19´s, 27°15´e) 1 mpumalanga, mthethomusha nr (25°29´s, 31°17´e) knp= kruger national park np = national park nr = nature reserve 258 small mammals as hosts of immature ixodid ticks table 2 ticks, other than rhipicephalus species, on small mammals in free state province no. examined number of ticks recovered tick and host species (no. infested) larvae nymphs total amblyomma marmoreum lepus saxatilis 3 (1) 4 17 21 haemaphysalis leachi aethomys namaquensis 97 (38) 63 35 98 rhabdomys pumilio 36 (9) 72 5 77 hyalomma marginatum rhabdomys pumilio 36 (1) 1 0 1 lepus capensis 3 (1) 12 6 18 lepus saxatilis 3 (2) 4 9 13 hyalomma truncatum aethomys namaquensis 97 (2) 2 0 2 rhabdomys pumilio 36 (1) 1 0 1 tatera leucogaster 5 (1) 4 0 4 lepus capensis 3 (2) 4 7 11 lepus saxatilis 3 (2) 33 39 72 ixodes rubicundus aethomys namaquensis 97 (6) 16 2 18 lepus saxatilis 3 (1) 0 1 1 ixodes sp. aethomys namaquensis 97 (8) 2 7 9 rhabdomys pumilio 36 (2) 1 1 2 margaropus winthemi rhabdomys pumilio 36 (1) 1 0 1 lepus saxatilis 3 (1) 4 1 5 table 3 rhipicephalus species on small mammals in free state province no. examined number of ticks recovered tick and host species (no. infested) larvae nymphs total rhipicephalus evertsi evertsi aethomys namaquensis 97 (2) 2 0 2 tatera leucogaster 5 (1) 1 0 1 lepus capensis 3 (2) 6 17 23 lepus saxatilis 3 (2) 31 61 92 rhipicephalus gertrudae aethomys namaquensis 97 (29) 99 48 147 rhabdomys pumilio 36 (11) 50 6 56 rhipicephalus lounsburyi rhabdomys pumilio 36 (1) 1 1 2 rhipicephalus warburtoni aethomys namaquensis 97 (11) 39 0 39 pedetes capensis 14 (14) 0 34 34 lepus capensis 3 (1) 6 0 6 + 3 %% lepus saxatilis 3 (3) 22 1 23 + 7 %% rhipicephalus sp. aethomys namaquensis 97 (2) 1 1 2 rhabdomys pumilio 36 (1) 1 0 1 tatera leucogaster 5 (3) 2 12 14 table 4 ticks, other than rhipicephalus species, on small mammals in north-eastern mpumalanga and limpopo provinces no. examined number of ticks recovered tick and host species (no. infested) larvae nymphs total amblyomma hebraeum mastomys natalensis 9 (1) 1 0 1 dermacentor rhinocerinus aethomys chrysophilus 19 (1) 1 0 1 mastomys natalensis 9 (1) 0 1 1 tatera leucogaster 5 (3) 3 2 5 haemaphysalis leachi aethomys chrysophilus 19 (11) 17 29 46 aethomys namaquensis 10 (6) 17 7 24 mastomys natalensis 9 (2) 0 8 8 mastomys coucha 10 (2) 3 1 4 saccostomys campestris 3 (3) 1 4 5 paraxerus cepapi 2 (1) 2 0 2 hyalomma truncatum lepus saxatilis 1 (1) 0 14 14 rhipicephalus (boophilus) decoloratus aethomys chrysophilus 19 (1) 1 0 1 hyalomma species the immature stages of h. marginatum rufipes and h. marginatum turanicum infest cape hares, scrub hares and ground frequenting birds, and those of h. truncatum hares, gerbils and murid rodents (rechav, zeederberg & zeller 1987; horak & fourie 1991; horak et al. 1991; braack et al. 1996). the distributions of h. marginatum rufipes and h. marginatum turanicum overlap in the southern free state (howell et al. 1978), and as we are unable to differentiate between their immature stages we have assigned 259 i.g. horak, l.j. fourie & l.e.o. braack table 5 rhipicephalus species on small mammals in north-eastern mpumalanga and limpopo provinces no. examined number of ticks recovered tick and host species (no. infested) larvae nymphs total rhipicephalus appendiculatus mastomys natalensis 9 (1) 1 0 1 lepus saxatilis 1 (1) 0 6 6 rhipicephalus evertsi evertsi lepus saxatilis 1 (1) 0 5 5 rhipicephalus simus aethomys chrysophilus 19 (15) 106 48 154 aethomys namaquensis 10 (10) 112 15 127 mastomys natalensis 9 (5) 101 9 110 mastomys coucha 10 (1) 1 0 1 otomys angoniensis 1 (1) 11 10 21 lemniscomys rosalia 1 (1) 9 3 12 saccostomys campestris 3 (1) 1 0 1 rattus rattus 1 (1) 1 0 1 tatera leucogaster 5 (2) 2 1 3 paraxerus cepapi 2 (2) 11 1 12 rhipicephalus zambeziensis paraxerus cepapi 2 (2) 11 18 29 only the specific epithet marginatum to the ticks collected from hares and a four-striped grass mouse examined in this region. both hare species were infested with the larvae and nymphs of h. truncatum and three rodent species with only the larvae of this tick. the immature stages of the three hyalomma ticks are present on their preferred hosts from early autumn to early summer (horak et al. 1991; 1993; horak & fourie 1991), and their presence in any study would thus be influenced by the season in which host animals are examined. rhipicephalus evertsi evertsi the small number of rodents infested, and then only with larvae of this two-host tick, indicates that in contrast to hares, on which both larvae and nymphs were present, they are not good hosts. although a large variety of domestic and wild ruminants are infested with r. evertsi evertsi, the preferred hosts of all stages of development are domestic and wild equids (walker et al. 2000). rhipicephalus gertrudae this tick replaces r. simus in the winter rainfall regions of south-western western cape province and in the more arid regions of this province and northern cape province as well as in the central and western regions of free state province (walker et al. 2000). its adult and immature stages have much the same host preference as r. simus, but in addition, the adults seem to favour primates, including humans (brain & bohrmann 1992; walker et al. 2000; horak, fourie, heyne, walker & needham 2002). the recovery of its larvae and nymphs only from a. namaquensis and r. pumilio and not from other small mammals within its distribution range does not necessarily reflect a host preference for these species, but may be influenced by the localities and seasons in which the mice were examined. rhipicephalus lounsburyi the higher mountainous regions of the eastern cape province are one of the preferred habitats of this tick (walker et al. 2000). it has not previously been recorded in free state province, but platberg, the locality at which it was collected from a fourstriped grass mouse, forms part of the same mountain range in which it has been collected in the eastern cape province. the adults attach around the feet of their antelope and sheep hosts, while the only known host of its immature stages is a fourstriped grass mouse, from which a single nymph was collected (walker et al. 2000). rhipicephalus simus of all species collected in the two northern provinces, the immature stages of r. simus has the largest host range, and judging by the numbers of larvae and the fact that nymphs also were recovered, most of the small mammal species examined could be considered as suitable hosts. although cattle are frequently infested, the adults are parasites of the larger carnivore species, including domestic dogs, and of large monogastric animals such as zebras, rhinoceroses and warthogs (horak et al. 2000; walker et al. 2000). rhipicephalus warburtoni the distribution of r. warburtoni is virtually confined to free state province (walker et al. 2000). all stages of development prefer hares as hosts, while the adults are found on domestic and wild ruminants and the immature stages on rock elephant shrews (walker et al. 2000). its presence on all the spring hares, pedetes capensis, and on some of the namaqua rock mice examined indicates that these animals may also be suitable hosts. the adults, that at the time were identified as belonging to a species similar to rhipicephalus pravus, produce a toxin causing paralysis in goat kids in the spring and early summer (fourie, horak & marais 1988). other species with the possible exception of those on scrub hares, we consider the remaining species to be accidental parasites or “stragglers” on the small mammals. the immature stages of amblyomma hebraeum and amblyomma marmoreum parasitize scrub hares and small carnivores (horak et al. 1995; horak et al. 2000), but are hardly ever found on rodents (howell et al. 1989; horak et al. 1991; braack et al. 1996; petney et al. 2004). the immature stages of i. rubicundus prefer rock elephant shrews and smith’s red rock rabbits, pronolagus rupestris (stampa 1959; fourie et al. 1992), and the one-host ticks, margaropus winthemi and rhipicephalus (boophilus) decoloratus, are parasites of large herbivores (howell et al. 1978). acknowledgements we are most grateful to the farmers, landowners and nature reserve managers for permission to collect small mammals on the properties under their jurisdiction. the assistance of mr e.j. williams, ms m. cohen and mr j. sithole with the collection of ticks from many of the animals is greatly appreciated. 260 small mammals as hosts of immature ixodid ticks references braack, l.e.o., horak, i.g., jordaan, leonora c., segerman, joyce & louw, j.p. 1996. the comparative host status of red veld rats (aethomys chrysophilus) and bushveld gerbils (tatera leucogaster) for epifaunal arthropods in the southern kruger national park, south africa. onderstepoort journal of veterinary research, 63:149–158. 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l.e.o. braack abstract introduction materials and methods results discussion acknowledgements references about the author(s) safa e.m. ali department of pathology, parasitology and microbiology, college of veterinary medicine, sudan university of science and technology, khartoum-north, sudan equine-specialty center, global veterinary services and agriculture, doha city, qatar yassin a.m. ahmed department of pathology, parasitology and microbiology, college of veterinary medicine, sudan university of science and technology, khartoum-north, sudan nokhbat alnawadir veterinary pharmacy, al duwadimi, saudi arabia alwia a. osman general directorate of animal health & epizootics diseases control, ministry of animal resources, khartoum, sudan omiema a. gamal eldin general directorate of animal health & epizootics diseases control, ministry of animal resources, khartoum, sudan nussieba a. osman department of pathology, parasitology and microbiology, college of veterinary medicine, sudan university of science and technology, khartoum-north, sudan citation ali, s.e.m., ahmed, y.a.m., osman, a.a., gamal eldin, o.a. & osman, n.a., 2023, ‘prevalence of peste des petits ruminants virus antibodies in sheep and goats sera from central-western sudan’, onderstepoort journal of veterinary research 90(1), a2074. https://doi.org/10.4102/ojvr.v90i1.2074 original research prevalence of peste des petits ruminants virus antibodies in sheep and goats sera from central-western sudan safa e.m. ali, yassin a.m. ahmed, alwia a. osman, omiema a. gamal eldin, nussieba a. osman received: 23 aug. 2022; accepted: 16 nov. 2022; published: 28 feb. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract clinical signs suggestive of peste des petits ruminants (ppr) involved herds of small ruminants, which were described elsewhere in sudan. peste des petits ruminants was confirmed using an immunocapture elisa (ic-elisa) assay in samples of infected and dead animals in areas of outbreaks. therefore, to update information regarding the current situation and for assessment of the serological prevalence of ppr in small ruminants mingled at central and western sudan during 2018–2019, 368 sera were collected from sheep (325 sera) and goats (43 sera) with different ages and breeds. these sera included 186 sera (173 sheep and 13 goats) from white nile state and 182 sera (152 sheep and 30 goats) from kordofan states. competitive elisa demonstrated higher prevalence of pprv antibodies of 88.9%, 90.7% and 88.6% in both sheep and goats, goats, and sheep sera, respectively. moreover, 100%, 94.7% and 78.5% seroprevalence values were demonstrated in south kordofan, north kordofan and white nile states. the higher seroprevalence values detected in sera of unvaccinated sheep and goats indicated the wide exposure of these animals to pprv and presence of protection following ppr viral infection. the findings of the study indicated that ppr is endemic in the surveyed areas of sudan. contribution: the study will contribute effectively to the global eradication programme of ppr organised by the world organization for animal health (woah, formerly oie) and food and agriculture organization (fao). to completely eliminate ppr from sudan by 2030, local efforts should be directed towards effectively and wholly vaccinating small ruminants using pprv vaccine especially in routes of seasonal animal’s movement and shared grazing areas. keywords: peste des petits ruminants; ppr; pprv; sudan; sheep; goats; antibodies; seroprevalence. introduction peste des petits ruminants (ppr), also known as sheep and goat plague, is an acute, highly contagious and fatal viral disease causing high mortality rates in naïve populations of domestic small ruminants and some wildlife species (diallo & libeau 2014; office international des epizooties [oie] 2021). in endemic regions, the economic is seriously affected by the presence of ppr through limiting the trade, export and import of new animal breeds, the development of intensive livestock production and availability of protein for human consumption (banyard et al. 2010; singh et al. 2014). peste des petits ruminants is caused by peste des petits ruminant virus (pprv), a member of the small ruminant morbillivirus species in the morbillivirus genus of the paramyxoviridae family (amarasinghe et al. 2019; maes et al. 2019). pest des petits ruminants, a fatal disease of goats with high mortality, was first described in the ivory coast (cote d’ivoire) in west africa (gargadennec & lalanne 1942). peste des petits ruminants affects mainly lambs and kids causing severe mortality (ozkul et al. 2002). depending upon the extent of the predisposing factors and virulence of the virus strain, the severity of the disease can vary as peracute, acute and subacute; however, usually the course of ppr runs as an acute (braide 1981; kulkarni et al. 1996; obi et al. 1983; oie 2021). in the early 1970s, the first outbreak of the disease in sheep and goats was in south gadaref, eastern sudan (elhag ali 1973). based on the clinical signs appeared in the affected animals, the disease was first misdiagnosed as rinderpest (rp); however, later pprvs were isolated and the existence of ppr in the country was documented (elhag ali & taylor 1984). subsequently, outbreaks of the disease were reported in two areas in central sudan; sinnar in 1971 and mieliq in 1972 (elhag ali & taylor 1984). during the 1990s, ppr outbreaks continued to occur in sheep and goats in central sudan, specifically in gezira state (el hassan et al. 1994) and khartoum state (zeidan 1994). serological surveys of ppr performed among small ruminants indicated existence of the disease in all areas investigated (abdalla et al. 2012; el amin & hassan 1999; haroun et al. 2002; ishag et al. 2015; osman et al. 2009, 2018; rasheed 1992; saeed et al. 2010, 2017; saeed, abdel-aziz & gumaa 2018; salih et al. 2014; shuaib et al. 2014). currently, ppr is endemic in the country with outbreaks occurring regularly in small ruminants leading to significant economic losses (osman et al. 2018; saeed et al. 2017). in sudan, at first sheep and goats were known as the only hosts of ppr. however, lately camels and gazelles were described as possible unusual hosts (asil et al. 2019; khalafalla et al. 2010). in sheep and goat populations with high risk of ppr viral infection, the disease is controlled by focused vaccinations followed by mass vaccination campaigns. in sudan, the live-attenuated ppr vaccine originated from nigeria 75/1 strain (diallo et al. 1989) is produced locally for vaccination of small ruminants in the field on yearly basis (fadol & el hussein 2004). despite implementation of the vaccination programme, ppr outbreaks were constantly reported in sudan, on an annual basis, indicating persistence of the disease. this might be due to application of the ppr vaccine in restricted areas in the country. in response to several suggestive ppr outbreaks occurred lately in the country, the study was designed to update information regarding the current situation, investigate the presence and determine the prevalence of pprv antibodies among sheep and goat flocks mingled at the central (white nile state) and western (kordofan states) sudan (figure 1). these were accomplished by serological detection of antibodies against pprv in sera of small ruminants from these areas. figure 1: locations of serological investigations of peste des petits ruminants in white nile and kordofan states, sudan. materials and methods study area white nile state white nile state, situated in the central area of sudan (figure 1), has an area of 39 701 km2 (2008 estimate). rabak is the capital of the state. it is administratively subdivided into four districts, ‘ed dueim, al gutaina, kosti and al jabalien’, which is subdivided into nine localities: ‘ed dueim, al gutaina, kosti, rabak, al jabalien, tendulti, um remta, alsalaam and guli’ (figure 2). white nile state is bordered by khartoum, gezira and sinnar states in the east, north kordofan (nk) in the north, south kordofan (sk) in the west and the republic of south sudan (rss) in the south. figure 2: districts and localities of serological investigations of peste des petits ruminants in white nile, north kordofan and south kordofan states. state capitals are presented. kordofan states kordofan (kurdufan) states, which covers an area of 376 145 km2 (146 932 miles2) (2011 estimate), is composed of three states: ‘north-, southand west kordofan’ (figure 1). it is largely an undulating plain, with the nuba mountains in the southeast quarter. during the dry season, the area is desert but it is fertile during the rainy season from june to september. north kordofan (nk) has an area of 185 302 km2 and consists mostly of large grazing areas. el-obeid is the capital of the state. it is divided into eight localities ‘sodari, jabrat el sheikh, bara, west bara, umm dam, shickan, el rahad and om ruwaba’ (figure 2). north kordofan is bordered by northern state in the north, north darfur in the west, west and south kordofan in the south, and khartoum and white nile in the east. south kordofan (sk), centred on the nuba hills, has an area of 158 355 km2. the state capital is kadugli. south kordofan is divided into 5 areas, ‘dilling, rashad, abu gebaiha, talodi and kadugli’, which are further subdivided into 16 localities ‘al qoz, dilling, habila, rashad, alabasia, eltadamon, delami, abu kershola, kadogli, heiban, um derin, buram, talodi, ghdeer, abu gebaiha and al leri’. south kordofan is bordered by nk in the north, west kordofan in the west, white nile in the east, and rss in the east and the south (figure 2). sample collection and preparation during 2018–2019, a total of 368 sera from sheep (325 sera) and goats (43 sera) were collected from three states of sudan, namely white nile (central sudan), nk and sk (western sudan) (table 1, figure 1). table 1: sheep and goat sera collected from white nile and kordofan states in sudan during 2018–2019. a total of 186 sera from sheep (n = 173) and goats (n = 13) were collected from three districts in ed dueim and rabak localities of the white nile state in sudan during october 2018 – november 2018. these sera were collected from ead aljam, ed dueim locality (102 sera, 92 sheep and 10 goats); gezira um jaar, ed dueim locality (47 sera, 44 sheep and 3 goats) and gezira aba, rabak locality (37 sheep sera) (table 1, figure 2). in addition, a total of 182 sera from sheep (n = 152) and goats (n = 30) were collected from nk and sk states in western sudan during 2018 and 2019. during 2018, 19 sheep and goat sera were collected from two districts in two localities: para, para locality (9 goat sera) and elobied, shickan locality (10 sheep sera) in nk state. during 2019, 163 sera from sheep (n = 142) and goats (n = 21) were collected from altartar districts in eltadamon locality, sk state (table 1, figure 2). these sera were originated from sedentary uninfected, nonvaccinated sheep and goats, above one year of age and of different local breeds. all sera were collected from private farms in which permissions were obtained from animal owners before initiation of sample collection. of note, animal owners declared the complete absence of any vaccination practices against ppr in sheep and goat herds in these areas. blood samples were collected aseptically from animals, and sera were prepared following the standard procedure as described previously (osman et al. 2018) and stored at −20 °c. competitive-enzyme linked immunosorbent assay a competitive screening elisa kit ‘id screen® ppr competition kit for the detection of antibodies against ppr in sheep and goats serum and plasma’ developed by cirad-emvt, fao reference laboratory for ppr in montpellier, france (idvet innovative diagnostics, france), was used for testing of these sera following the manufacturer’s instructions. ethical considerations an approval to conduct the study was obtained from the sudan university of science and technology institutional ethics committee (dsr – iec – 02-2-2017). results description of the clinical disease in sheep and goats and outbreaks investigations between october 2018 and november 2018, outbreaks suggestive of ppr involved sheep and goats in three different districts in two localities, ead aljam in ed dueim locality, gezira um jaar in ed dueim locality and gezira aba in rabak locality, of the white nile state (central sudan) (figure 2). during 2018, two outbreaks suggestive of ppr occurred mainly in sheep and goats and were reported in two districts in two different localities, para in para locality and elobied in shickan locality, of the nk state (western sudan). in 2019, another two outbreaks suggestive of ppr were reported in one district, altartar in eltadamon locality, of the sk state (western sudan) (figure 2). in all these outbreaks, both infected sheep and goats showed clinical signs described normally in suspected cases of ppr including dullness, depression, dry muzzle, sudden onset of pyrexia, anorexia, nasal and ocular discharges, mouth lesions, stomatitis, emaciation and diarrhoea. respiratory signs, dyspnea, rapid and laboured breathing associated with pneumonia were also observed. these cases were associated with higher morbidity and mortality rates and in some cases preceded to sudden death which was mostly observed among young animals. for confirmation of the clinical diagnosis, samples from infected and dead animals were collected by the veterinary authorities in areas of outbreaks. peste des petits ruminants was confirmed by testing these samples at the central veterinary research laboratory (cvrl), soba, khartoum, using an immunocapture elisa (ic-elisa) assay. after ppr had been diagnosed and confirmed as the leading cause of these outbreaks, a mass vaccination campaign was undertaken by the local veterinary authorities in these states, in order to control the disease. the overall seroprevalence of peste des petits ruminants virus antibodies in both sheep and goat sera as demonstrated by the c-elisa assay, 327/368 of the tested sheep and goat sera were positive with 88.9% overall seroprevalence of pprv antibodies. on the species basis, the higher overall seroprevalence of pprv antibodies was observed among goats (90.7%, 39/43 sera) rather than sheep (88.6%, 288/325 sera) (table 2). table 2: the overall seroprevalence of peste des petits ruminants virus antibodies, in sheep and goat sera, as demonstrated by competitive-enzyme linked immunosorbent assay. the overall seroprevalence of peste des petits ruminants virus antibodies in both sheep and goat sera in surveyed states of sudan within the surveyed states of sudan, the highest overall seroprevalence of pprv antibodies in both sheep and goat sera was demonstrated in sk state (100%, 163/163), followed by nk state (94.7%, 18/19) and the least seroprevalence was detected in the white nile state (78.5%, 146/186) (table 3). table 3: the overall seroprevalence of peste des petits ruminants virus antibodies in both sheep and goat sera in surveyed states of sudan. seroprevalence of peste des petits ruminants virus antibodies in sheep or goat sera in surveyed states of sudan considering the species basis within the surveyed states of sudan, among sheep, the highest overall seroprevalence of pprv antibodies was demonstrated in both sk (100%, 142/142) and nk (100%, 10/10) states whereas the least seroprevalence was demonstrated in white nile state (78.6%, 136/173) (table 4). table 4: seroprevalence of peste des petits ruminants virus antibodies in sheep or goat sera in surveyed states of sudan. considering the species basis within the surveyed states of sudan, among goats, the highest overall seroprevalence of pprv antibodies was demonstrated in sk state (100%, 21/21), followed by nk state (88.9%, 8/9) whereas the least seroprevalence was demonstrated in white nile state (76.9%, 10/13) (table 4). seroprevalence of peste des petits ruminants virus antibodies in sheep and goat sera within districts and localities of the white nile state within districts and localities of the white nile state (central sudan), the highest seroprevalence of pprv antibodies in sheep and goat sera was demonstrated in gezira um jaar, ed dueim locality (93.6%, 44/47) followed by ead aljam, ed dueim locality (76.5%, 78/102) and the least prevalence was presented in gezira aba, rabak locality (64.9%, 24/37) (table 5). table 5: seroprevalence of peste des petits ruminants virus antibodies in sheep and goat sera within districts and localities of the white nile state. considering the species basis, within sheep, the highest seroprevalence of pprv antibodies was demonstrated in gezira um jaar, ed dueim locality (93.2%, 41/44), followed by ead aljam, ed dueim locality (77.2%, 71/92), and finally the least seroprevalence was presented in gezira aba, rabak locality (64.9%, 24/37) (table 5). considering the species basis, within goats, the highest seroprevalence of pprv antibodies was demonstrated in gezira um jaar, ed dueim locality (100%, 3/3) while the least seroprevalence was presented in ead aljam, ed dueim locality (70%, 7/10) (table 5). seroprevalence of peste des petits ruminants virus antibodies in sheep and goat sera within districts and localities of kordofan states within districts and localities of nk and sk states (western sudan), the highest overall seroprevalence of pprv antibodies in sheep and goat sera was demonstrated in both altartar, eltadamon locality, sk state (100%, 163/163) and in elobied, shickan locality, nk state (100%, 10/10), whereas the least seroprevalence was demonstrated in para, para locality, nk state (88.9%, 8/9) (table 6). table 6: seroprevalence of peste des petits ruminants virus antibodies in sheep and goat sera within districts and localities of kordofan states. considering the species under study, within sheep, the highest seroprevalence of pprv antibodies was demonstrated in both altartar, eltadamon locality, sk state (100%, 142/142) and elobied, shickan locality, nk state (100%, 10/10) (table 6). considering the species under study, within goats, the highest seroprevalence of pprv antibodies was demonstrated in altartar, eltadamon locality, sk state (100%, 21/21) and the least seroprevalence was detected in para, para locality, nk state (88.9%, 8/9) (table 6). discussion since its first recognition at the early 1970s in sudan (elhag ali 1973), outbreaks of ppr were continually reported elsewhere in small ruminants (osman et al. 2018; saeed et al. 2010; 2017), camels (khalafalla et al. 2010; kwiatek et al. 2011) and lately in gazelles (asil et al., 2019). the disease has becomes more endemic covering a wide belt in sudan and the entire african continent (kwiatek et al. 2011; oie 2021). in response to several suggestive ppr outbreaks involving sheep and goats in white nile and kordofan states during 2018 and 2019, the study aimed to investigate and update information regarding the presence of ppr and for assessment of the serological prevalence of pprv antibodies in these areas. serological surveys performed earlier in the country demonstrated the prevalence of the disease in sheep and goats in the central areas, specifically in gezira, khartoum, white nile, sinnar and blue nile states (abdalla et al., 2012; haroun et al. 2002; ishag et al. 2015; osman et al. 2009, 2018; saeed et al. 2010, 2017). likewise, many sero-surveys performed in sheep and goats demonstrated the prevalence of pprv antibodies in western sudan, in both kordofan and darfur states (abdalla et al., 2012; el amin & hassan, 1999; haroun et al. 2002; osman et al. 2009; rasheed 1992; saeed et al. 2010, 2017; salih et al., 2014; shuaib et al. 2014). according to the annual reports of the world organization for animal health (woah, formerly oie), ppr outbreaks were reported regularly in the surveyed states during the last 10 years (oie world animal health information system [oie-wahis] 2020). in this study, 88.9% overall seroprevalence of pprv antibodies was demonstrated when sheep and goat sera were tested by c-elisa assay. in a similar study conducted during 2016–2017 in north and central sudan, lower overall antibodies seroprevalence (80.9%) was demonstrated among sheep and goats using c-elisa (osman et al. 2018). the study by saeed et al. (2017) demonstrated much lower overall seroprevalence of pprv (49.4%) among different animal species. these results appear much lower than the prevalence achieved in this study. in this study, goat sera yielded the higher overall seroprevalence of pprv antibodies (90.7%) compared with sheep sera (88.6%). a relatively lower overall seroprevalence in sheep (84.5%) and goats (66.1%) was demonstrated by osman et al. (2018). the study by saeed et al. (2017) demonstrated much lower seroprevalence of pprv of 67.1% and 48.2% among sheep and goats, respectively. similarly, 67.2% and 55.6% seroprevalence was achieved from sheep and goats, respectively (saeed et al. 2010). the results of all previous studies appear much lower than the prevalence achieved in the present study. moreover, 100%, 94.7% and 78.5% overall seroprevalences of pprv antibodies in both sheep and goat sera were demonstrated in sk, nk and white nile states, respectively. the higher seroprevalence values achieved indicated the wide distribution of the disease in different localities of kordofan states; however, the incidence of the disease is higher in sk compared with nk; this is possibly due to its increased rate of seasonal animal movement. due to its location, most of small ruminant herds in nk state are vaccinated regularly during vaccination campaigns organised by the local veterinary authorities, thus may explain the lower seroprevalence values detected. in a similar study conducted during 2016 – 2017, 88.5% and 48.4% overall antibodies seroprevalence was demonstrated among sheep and goats in gezira and khartoum states (central sudan), respectively (osman et al. 2018). this result appears much lower than the prevalence achieved in this study. among sheep, 100%, 100% and 78.6% overall seroprevalence of pprv antibodies was demonstrated in sk, nk and white nile states, respectively. similarly, among goats, 100%, 88.9% and 76.9% overall seroprevalence of pprv antibodies was demonstrated in sk, nk and white nile states, respectively. in contrast, lower overall seroprevalence (74%) was demonstrated among sheep in both kordofan and kassala states (shuaib et al. 2014). likewise, a much lower overall seroprevalence (68.1%) was detected in sheep sera collected from darfur states in western sudan (saeed et al. 2017). these results appear much higher compared with 54% seroprevalence value demonstrated during 2008–2009 in white nile state (ishag et al. 2015). of note, a very lower seroprevalence value (39.8%) was demonstrated among unvaccinated sheep and goats in nk state in sudan (salih et al. 2014). the results of all previous studies appear much lower than the prevalence achieved in the present study. in the white nile state (central sudan), 93.6%, 76.5% and 64.9% seroprevalence of pprv antibodies in small ruminants sera was demonstrated in gezira um jaar, ed dueim locality; ead aljam, ed dueim locality; and gezira aba, rabak locality, respectively. among sheep, 93.2%, 77.2% and 64.9% seroprevalence of pprv antibodies was demonstrated in gezira um jaar, ed dueim locality; ead aljam, ed dueim locality; and gezira aba, rabak locality, respectively. among goats, 100% and 70% seroprevalence of pprv antibodies was demonstrated in gezira um jaar, ed dueim locality and ead aljam, ed dueim locality, respectively. generally, slightly higher seroprevalence values, among small ruminants, were achieved in this study than previously demonstrated. the reason for the higher seroprevalence among sheep than in goats is that higher fatalities were observed among goats. in kordofan states (western sudan), 100%, 100% and 88.9% seroprevalence of pprv antibodies in small ruminants sera was demonstrated in altartar, eltadamon locality, sk state; elobied, shickan locality, nk state; and para, para locality, nk state, respectively. among sheep, 100% and 100% seroprevalence of pprv antibodies was demonstrated in altartar, eltadamon locality, sk state and elobied, shickan locality, nk state, respectively. among goats, 100% and 88.9% seroprevalence of pprv antibodies was demonstrated in altartar, eltadamon locality, sk state and para, para locality, nk state, respectively. in contrary, lower seroprevalence of pprv (74.5%) was demonstrated among sheep in nk state (shuaib et al. 2014). in other study, lower seroprevalence (58.3%) in sheep and (38.8%) in goats was demonstrated in kordofan states during 2008–2010 (saeed et al. 2017). these results appear much lower than seroprevalence values achieved in this study. the findings of the study indicated the occurrence of ppr in mixed herds of sheep and goats in the investigated areas. the higher seroprevalence values achieved, although small ruminants had never been vaccinated against ppr, indicated the wide exposure of these animals to pprv. our suspicion of pprv infection, due to appearance of typical clinical signs of ppr in infected small ruminants in areas of outbreaks, was confirmed by laboratory diagnosis using ic-elisa. the continual presence of ppr outbreaks in different areas in sudan might influence the currently established global eradication programme of the disease which was launched by the woah, formerly oie, and the food and agriculture organization (fao). animal herds in these areas have a special pattern of seasonal movement towards the south at the beginning of the hot summer season and backwards at the beginning of autumn. some animals may cross the country borders entering the neighbouring countries such as the republic of south sudan where it is staying there for many months and escape vaccination which takes place during this period. in fact, ppr is endemic in all african countries bordering sudan. sharing grazing areas with infected small ruminants and other animal species in neighbouring countries could contribute to appearance of outbreaks with new pprv strains or lineages via contact with infected small ruminants. to prevent spread of the disease and presence of new outbreaks, it is recommended that the vaccination campaigns must reach all herd sectors. acknowledgements we are profoundly thankful to the staff of the ministry of agriculture and animal resources in white nile state and elobied veterinary research laboratory, north kordofan state, for their constant help and support during field work. we are grateful to the staff of the elisa laboratory, general directorate of animal health and epizootics diseases control, federal ministry of animal resources, khartoum, for their technical support and providing laboratory facilities. we are grateful to dr. selma kamal ahmed, epidemiology department, central veterinary research laboratory, soba, khartoum, sudan, for drawing the map of the sudan. competing interests the authors have declared that no competing interest exists. authors’ contributions s.e.m.a. and y.a.m.a. were responsible for sample collection and preparations, performed the c-elisa assay, data analysis and wrote the drafted manuscript. the authors contributed equally to this work as joint first authors. a.a.o. and o.a.g.e. performed the c-elisa assay with s.e.m.a. and y.a.m.a. n.a.o supervised and designed the study, performed data analysis and results interpretation, prepared the draft and final manuscript. funding information the research in this study was funded by a research project for n.a.o from the republic of the sudan, sudanese ministry of higher education and scientific research, commission of scientific research and innovation (grant no. 2017/972). data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the views of their institutions. references abdalla, a.s., majok, a.a., el malik, k.h. & ali, a.s., 2012, ‘sero-prevalence of peste des petits ruminants virus (pprv) in small ruminants in blue nile, gadaref and north kordofan states of sudan’, journal of public health and epidemiology 4(3), 56–64. https://doi.org/10.5897/jphe11.213 amarasinghe, g.k., ayllón, m.a., bào, y., basler, c.f., bavari, s., blasdell, k.r. et al., 2019, ‘taxonomy of the order mononegavirales: update 2019’, archives of virology 164(7), 1967–1980. https://doi.org/10.1007/s00705-019-04247-4 asil, r.m., ludlow, m., ballal, a., alsarraj, s., ali, w.h., mohamed, b.a. et al., 2019, ‘first detection and 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s. & sinha, d.k., 2014, ‘estimation of economic losses due to peste de petits ruminants in small ruminants in india’, veterinary world 7(4), 194–199. https://doi.org/10.14202/vetworld.2014.194-199 zeidan, m.i., 1994, ‘diagnosis and distribution of peste des petits ruminants in small ruminants in khartoum state during 1992–1994’, thesis for master degree, university of khartoum, sudan. article information author: ivan g. horak1,2 shaun welman3 stacey l. hallam3 heike lutermann4 nomakwezi mzilikazi3 affiliation: 1department of veterinary tropical diseases, university of pretoria, south africa 2department of zoology and entomology, university of the free state, south africa 3department of zoology, nelson mandela metropolitan university, south africa 4department of zoology and entomology, university of pretoria, south africa correspondence to: ivan horak email: ivan.horak@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 01 nov. 2010 accepted: 12 dec. 2010 published: 17 mar. 2011 how to cite this article: horak, i.g., welman, s., hallam, s.l., lutermann, h. & mzilikazi, n., 2011, ‘ticks of four-toed elephant shrews and southern african hedgehogs’, onderstepoort journal of veterinary research 78(1): art. #243, 3 pages. doi:10.4102/ojvr.v78i1.243 copyright notice: © 2011. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) ticks of four-toed elephant shrews and southern african hedgehogs in this original research... open access • abstract • introduction • materials and methods    • ethical considerations • results and discussion    • four-toed elephant shrews    • southern african hedgehogs • conclusion • acknowledgements • references abstract (back to top) several studies on ticks infesting small mammals, including elephant shrews, have been conducted in south africa; however, these studies have included only a single four-toed elephant shrew and no hedgehogs. this study thus aimed to identify and quantify the ixodid ticks infesting four-toed elephant shrews and southern african hedgehogs. four-toed elephant shrews (petrodromus tetradactylus) were trapped in dense shrub undergrowth in a nature reserve in north-eastern kwazulu-natal. they were separately housed, first in cages and later in glass terraria fitted with wire-mesh bases to allow detached ticks to fall through for collection. southern african hedgehogs (atelerix frontalis) were hand caught on a farm in the eastern region of the northern cape province and all visible ticks were collected by means of tweezers while the animals were anaesthetised. the ticks from each animal were preserved separately in 70% ethanol for later identification and counting. the immature stages of five ixodid tick species were collected from the elephant shrews, of which rhipicephalus muehlensi was the most common. it has not been recorded previously on any species of elephant shrew. three ixodid tick species were collected from the hedgehogs. large numbers of adult haemaphysalis colesbergensis, which has not been encountered previously on hedgehogs, were collected from these animals. four-toed elephant shrews are good hosts of the larvae and nymphs of r. muehlensi, and southern african hedgehogs are good hosts of adult h. colesbergensis. introduction (back to top) several papers regarding the role of small mammals as hosts of immature ixodid ticks have been published in south africa during the past two decades (braack et al. 1996; horak & cohen 2001; matthee et al. 2007; petney et al. 2004). rock elephant shrews (elephantulus myurus), and to a lesser extent cape elephant shrews (elephantulus edwardii), dominate the tick-associated literature for elephant shrews (fourie, horak & van den heever 1992; fourie et al. 2002; fourie, horak & woodall 2005). this is primarily because they are the preferred hosts of the immature stages of tick species that affect commercially important livestock (fourie et al. 2005). however, with the exception of a single four-toed elephant shrew (petrodromus tetradactylus) examined in north-eastern mpumalanga province (fourie et al. 2005), no tick collections have been made from these animals or from southern african hedgehogs (atelerix frontalis), another small insectivorous species, during the past 20 years. to date, the immature stages of only two ixodid tick species have been recorded on four-toed elephant shrews in south africa (theiler 1962); however, the immature stages of a further nine species have been collected from them in zambia (macleod 1970) and one in tanzania (clifford, walker & keirans 1973). small numbers of several tick species have been collected from southern african hedgehogs (theiler 1962; walker, keirans & horak 2000), but, with the exception of a small number of species, all of these should be considered accidental infestations and simply a reflection of the abundance of the free-living stages of ticks in a particular region. the opportunity arose to collect ticks from these two small mammal species during a study on the metabolic rate of four-toed elephant shrews in north-eastern kwazulu-natal and a similar study on southern african hedgehogs in the eastern region of the northern cape province, south africa. here we report on the ixodid tick species collected from these animals. materials and methods (back to top) several four-toed elephant shrews were caught at bonamanzi game park (28°03’47.17’’s, 32°18’06. 79’’e) in walk-in wire-mesh traps (60 cm x 25 cm x 25 cm) during april 2009. trapping occurred in dense shrub undergrowth where the animals’ characteristic runways could be identified. the nine captured animals were subsequently housed separately in cages (56 cm x 34 cm x 20 cm) each fitted with a tray to collect any detached ticks, and later in glass terraria (60 cm x 30 cm x 30 cm) fitted with wire-mesh bases to allow detached ticks to fall through. detached ticks were collected on a daily basis and each animal’s were preserved separately in 70% ethanol in internally labelled bottles. the ticks were identified to species level and counted using a stereoscopic microscope. the hedgehogs were caught by hand on and around the farm plaatfontein (31°01.693’s, 23°45.993’e). all visible ticks were collected with tweezers from the 12 anaesthetised animals (immediately after implantation of body temperature data loggers). the same collection procedures as for ticks on the elephant shrews were then followed. ethical considerations all procedures adhered to the code of animal experimentation adopted by the nelson mandela metropolitan university. ezemvelo kzn wildlife issued a permit for the capture and transport of elephant shrews (permit number 1462/2009) and the northern cape department of tourism, environment and conservation issued a permit for the capture of hedgehogs (permit number flora 036/2009). results and discussion (back to top) four-toed elephant shrews a total of 4007 immature ticks, belonging to five species, were collected from the nine animals, which were all infested (table 1). the majority of these were larvae and nymphs of rhipicephalus muehlensi, of which a large proportion were engorged. a few engorged nymphs that had escaped immersion in alcohol moulted into adults, thus confirming the species identification. despite the region in which the study was conducted encompassing part of the geographic distributions of rhipicephalus appendiculatus and rhipicephalus maculatus, only a single larva of each of these species was recovered. in view of these findings, as well as for comparative purposes, the results of previous surveys on ticks infesting nyalas (tragelaphus angasii) and scrub hares (lepus saxatilis) in north-eastern kwazulu-natal (horak et al. 1995a; horak, boomker & flamand 1995b) have been summarised in table 2. table 1: species and number of ticks collected from nine four-toed elephant shrews in north-eastern kwazulu-natal. large numbers of r. appendiculatus, r. maculatus and r. muehlensi have previously been collected from nyalas, indicating not only that they are good hosts but also that the geographic distributions of the three tick species overlap in the study region (table 2). similarly, scrub hares have been found to be infested with fair numbers of the immature stages of these ticks, but the four-toed elephant shrews harboured only a single larva of each of r. appendiculatus and r. maculatus, and large numbers of larvae and nymphs of r. muehlensi. table 2: host status of four-toed elephant shrews for three sympatric rhipicephalus species in north-eastern kwazulu-natal. walker et al. (2000) have placed r. muehlensi and r. maculatus within the r. appendiculatus group of ticks based on the morphology of their immature stages. this group consists of 11 species. the immature stages of each of these 11 species may infest scrub hares, but only those of r. appendiculatus have been collected, and then only in very small numbers, from elephant shrews (macleod 1970; theiler 1962). the present collection of a single larva of each of r. appendiculatus and r. maculatus, in an environment in which these ticks abound, confirms that elephant shrews are not favoured hosts of the immature stages of ticks in this group. the collection of large numbers of r. muehlensi larvae and nymphs from four-toed elephant shrews is thus remarkable. furthermore, the large numbers of engorged larvae and nymphs which were recovered, together with the fact that some of the nymphs had moulted to adults, imply that four-toed elephant shrews are excellent hosts of the immature stages of r. muehlensi. this host preference resembles that of the immature stages of the six species comprising the rhipicephalus pravus group of ticks, all of which use elephant shrews as hosts (clifford et al. 1973; fourie et al. 2005; macleod 1970; theiler 1962; walker et al. 2000). of the 79 nyalas examined in north-eastern kwazulu-natal, 16 harboured all stages of development of haemaphysalis silacea (horak et al. 1995b). this tick has never been recorded on an elephant shrew. its presence in this study, albeit in small numbers, on five of the nine animals examined, suggests that four-toed elephant shrews can serve as hosts of the tick’s immature stages. larvae and nymphs of the haemaphysalis elliptica group of ticks have been collected in small numbers from rock elephant shrews (fourie et al. 1992), and now also from four-toed elephant shrews. their preferred hosts are, however, murid rodents (braack et al. 1996; fourie et al. 1992; matthee et al. 2007). southern african hedgehogs a total of 204 ticks belonging to three species were collected from the 12 examined animals, of which 11 were infested (table 3). the majority of the ticks were adult haemaphysalis colesbergensis, of which a large proportion of the females were engorged. an old male that harboured 55 male and 43 female h. colesbergensis was among the 11 infested animals. h. colesbergensis has been described only recently (apanaskevich & horak 2008) and is named after the town colesberg (in the eastern region of the northern cape province) where adult ticks have been collected from a dog, domestic cats, caracals (caracal caracal) and an african wildcat (felis silvestris). their tick burdens are summarised in table 4 for comparative purposes. table 3: species and number of ticks collected from 12 southern african hedge-hogs in the eastern region of the northern cape province. table 4: hosts and number of haemaphysalis colesbergensis collected from carnivores near colesberg in the eastern region of the northern cape province. the numbers now recovered from the hedgehogs and the fact that several females were engorged indicate that these small mammals should also be considered preferred hosts of h. colesbergensis. its presence on southern african hedgehogs is not entirely unexpected, as hedgehogs seem to be one of the preferred hosts of ticks belonging to this genus. in fact, haemaphysalis norvali, a species that appears to be specific to hedgehogs, has been described in zimbabwe (hoogstraal & wassef 1983). to our knowledge h. norvali is not present in south africa. a larva and five nymphs of the south african tortoise tick, amblyomma marmoreum, were collected from four of the hedgehogs. the immature stages of a. marmoreum infest an extremely wide range of domestic and wild mammals and birds in south africa (horak et al. 2006) and hedgehogs are no exception (theiler 1962). although none were recovered now, the adults of rhipicentor nuttalli also infest hedgehogs (fourie et al. 2002; norval & colborne 1985; theiler 1962) and several wild carnivores, of which the larger felids appear to be the hosts of choice (horak, heyne & donkin 2010; norval & colborne 1985). the immature stages of r. nuttalli infest elephant shrews, of which the rock elephant shrew and the cape elephant shrew are the most favoured (fourie et al. 2002, 2005). conclusion (back to top) four-toed elephant shrews are a new host record for the larvae and nymphs of r. muehlensi, a tick of which all stages of development infest nyalas in north-eastern kwazulu-natal. large numbers of adult h. colesbergensis may infest southern african hedgehogs and several species of carnivores in the eastern region of the northern cape province. the hedgehogs constitute a new host record for this tick species. acknowledgements (back to top) we thank the staff at bonamanzi game park for their hospitality and assistance. thanks are also due to ezemvelo kzn wildlife and the northern cape department of tourism, environment and conservation for the capture and transport permits. we are most grateful to mr and mrs j. theron, on whose farm the hedgehogs were collected. this study was financed by the national research foundation of south africa, the oppenheimer memorial trust and the nelson mandela metropolitan university. references (back to top) apanaskevich, d.a. & horak, i.g., 2008, ‘two new species of african haemaphysalis ticks (acari: ixodidae), carnivore parasites of the h. (rhipistoma) leachi group’, journal of parasitology 94, 594–607. doi:10.1645/ge-1374.1, doi:10.1645/ge-1374r.1, pmid:18605788. braack, l.e.o., horak, i.g., jordaan, l.c., segerman, j. & louw, j.p., 1996, ‘the comparative host status of red veld rats (aethomys chrysophilus) and bushveld gerbils (tatera leucogaster) for epifaunal arthropods in the southern kruger national park, south africa’, onderstepoort journal of veterinary research 63, 149–158. clifford, c.m., walker, j.b. & keirans, j.e., 1973, ‘clarification of the status of rhipicephalus kochi dönitz, 1905 (ixodoidea, ixodidae)’, onderstepoort journal of veterinary research 50, 77–89. fourie, l.j., horak, i.g. & van den heever, j.j., 1992, ‘the relative host status of rock elephant shrews elephantulus myurus and namaqua rock mice aethomys namaquensis for economically important ticks’, south african journal of zoology 27, 108–114. fourie, l.j., horak, i.g., kok, d.j. & van zyl, w., 2002, ‘hosts, seasonal occurrence and life cycle of rhipicentor nuttalli (acari: ixodidae)’, onderstepoort journal of veterinary research 69, 177–187. fourie, l.j., horak, i.g. & woodall, p.f., 2005, ‘elephant shrews as hosts of immature ixodid ticks’, onderstepoort journal of veterinary research 72, 293–301. hoogstraal, h. & wassef, h.y., 1983, ‘notes on african haemaphysalis ticks. xv. h. (rhipistoma) norvali sp. n., a hedgehog parasite of the h. (r.) spinulosa group in zimbabwe (acarina: ixodidae)’, onderstepoort journal of veterinary research 50, 183–189. horak, i.g., spickett, a.m., braack, l.e.o., penzhorn, b.l., bagnall, r.j. & uys, a.c., 1995a, ‘parasites of domestic and wild animals in south africa. xxxiii. ixodid ticks on scrub hares in the north-eastern regions of northern and eastern transvaal and of kwazulu-natal’, onderstepoort journal of veterinary research 62, 123–131. horak, i.g., boomker, j. & flamand, j.r.b., 1995b, ‘parasites of domestic and wild animals in south africa. xxxiv. arthropod parasites of nyalas in north-eastern kwazulu-natal’, onderstepoort journal of veterinary research 62, 171–179. horak, i.g. & cohen, m., 2001, ‘hosts of the immature stages of the rhinoceros tick, dermacentor rhinocerinus (acari, ixodidae)’, onderstepoort journal of veterinary research 68, 75–77. horak, i.g., mckay, i.j., heyne, h. & spickett, a.m., 2006, ‘hosts, seasonality and geographic distribution of the south african tortoise tick, amblyomma marmoreum’, onderstepoort journal of veterinary research 73, 13–25. horak i.g., heyne, h. & donkin, e.f., 2010, ‘parasites of domestic and wild animals in south africa. xlviii. ticks (acari: ixodoidea) infesting domestic cats and wild felids’, onderstepoort journal of veterinary research 77(1): art. #3, 7 pages. doi:10.4102/ojvr.v77i1.3. macleod, j., 1970, ‘tick infestation patterns in the southern province of zambia’, bulletin of entomological research 60, 253–274. doi:10.1017s0007485300040773. matthee, s., horak, i.g., beacournu, j.c., durden, l.a., ueckermann, e.a. & mcgeoch, m.a., 2007, ‘epifaunistic arthropod parasites of the four-striped mouse, rhabdomys pumilio, in the western cape province, south africa’, journal of parasitology 93, 47–59. doi:10.1645/ge-819r2.1 , pmid:17436941. norval, r.a.i. & colborne, j., 1985, ‘the ticks of zimbabwe. x. the genera dermacentor and rhipicentor ’, zimbabwe veterinary journal 16, 1–4. petney, t.n., horak, i.g., howell, d.j. & meyer, s., 2004, ‘striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks’, onderstepoort journal of veterinary research 71, 313–318. theiler, g., 1962, the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region), division of veterinary services, onderstepoort, (mimeographed). walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. article information authors: inge-marie petzer1 joanne karzis1 theodorus j. van der schans1 johanna c. watermeyer1 norman mitchell-innes2 stephanie eloff1 geoffrey t. fosgate1 affiliations: 1department of production animal studies, university of pretoria, onderstepoort, south africa2herd husbandry help cc, irene, south africa correspondence to: inge-marie petzer postal address: private bag x04, onderstepoort 0110, south africa dates: received: 20 may 2011 accepted: 28 sept. 2011 published: 10 feb. 2012 how to cite this article: petzer, i.m., karzis, j., van der schans, t.j., watermeyer, j.c., mitchell-innes, n., eloff, s. & fosgate, g.t., 2012, ‘comparing effects of freezing at -196 °c and -20 °c on the viability of mastitis pathogens’, onderstepoort journal of veterinary research 79(1), art. #343, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.343 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. comparing effects of freezing at -196 °c and -20 °c on the viability of mastitis pathogens in this original research... open access • abstract • introduction • materials and methods    • sample preparation    • experimental procedure       • sample schedule       • thawing of samples       • identification of mastitogenic bacteria       • recording of bacterial growth       • statistical analysis • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the aim of this study was to compare the effects of cryopreservation at approximately -196 °c in liquid nitrogen (n) and freezing at approximately -20 °c in a freezer, on the viability and survival of eight different mastitogenic bacteria inoculated in milk. bacteria were frozen at approximately -20 °c in a freezer and cryopreserved at approximately -196 °c in liquid nitrogen. an effective preservation method was needed for follow-up samples from cows identified in the south african national milk recording scheme (nmrs) with somatic cell counts above 250 000 cells/ml milk. the organisation responsible for sample collection of the nmrs milk samples also provides producers with liquid nitrogen for their semen flasks at the collection sites. this existing mode of storage and transport could therefore be utilised.ten samples of each organism were thawed and cultured bi-weekly until week 18 for both temperature treatments. an additional sampling was performed at week 30 for samples frozen at approximately -20 °c. freezing and cryopreservation did not impair subsequent isolation of streptococcus dysgalactiae, streptococcus uberis, enterococcus faecalis, staphylococcus aureus (sth) (phage type lytic group iii) or sta. aureus (sta) (phage typed, other than lytic group iii). survival was indicated by the isolation of bacteria from samples, and viability by the strength of growth of the bacteria isolated. the survival of streptococcus agalactiae decreased after week 12 and escherichia coli after week 16 of freezing, but both organisms survived under cryogenic preservation until week 18. coagulase-negative staphylococci survived until week 18 for both freezing and cryogenic preservation. both storage methods could thus contribute to the improvement of a pro-active approach towards udder health management in south african dairy herds. introduction top ↑ detection and identification of pathogens in milk has become a valuable tool for monitoring the udder health of modern dairy cows. culturing of milk from cases with increased somatic cell counts (above 250 000 cells/ml) can also be used to assist producers in a pro-active approach to herd udder health.freezing can be lethal to many living systems; whilst preserving some cell structures, it disrupts others and the ability of micro-organisms to withstand freezing might differ (mazur 1970). variable results have been reported by different authors who assessed the survival and viability of mastitogenic organisms in milk at temperatures of -20 °c and -80 °c. whilst some studies (bashandy & heider 1979; luedecke et al. 1971; murdough, deitz & pankey 1996; pankey et al. 1987; schukken et al. 1989) showed that freezing at -20 °c and -80 °c had no effect on the recovery of mastitogenic bacteria, others showed significant increases in the recovery of similar organisms (hubackova & rysanek 2007; schukken et al. 1989; sol et al. 2002; villanueva, tyler & thurmond 1991). little has been reported about the effect of freezing and storing on streptococcus dysgalactiae, streptococcus uberis, enterococcus faecalis and coagulase-negative staphylococci. farrat (1980) demonstrated that freezing at -80 °c yielded higher survival rates of bacteria than freezing at -20 °c. little, however, is known about the overall survival and viability differences of various mastitogenic bacteria when frozen and when cryopreserved. the objective of this study was to compare the two different freezing temperatures (approximately -20 °c and approximately -196 °c) on the survival and viability of eight mastitogenic bacteria commonly isolated from milk samples of south african dairy herds. the evaluated organisms were streptococcus agalactiae, str. dysgalactiae, str. uberis, coagulase-negative staphylococci, e. faecalis, escherichia coli and two groups of staphylococcus aureus differentiated by means of phage typing. materials and methods top ↑ two methods of preservation were investigated and full cream uht (ultra-high-temperature processing) milk was selected as the diluent to standardise bacterial concentrations. milk samples where scc increased to more than 250 000 cells/ml, and not only milk from cows with clinical mastitis, were to be examined. a chest freezer was used in this study to simulate field conditions (schukken et al. 1989). sample preparation eight mastitogenic bacteria namely, sta. aureus (phage type, lytic group iii), sta. aureus (phage type, other than lytic group iii), str. agalactiae, str. dysgalactiae, str. uberis, e. faecalis, coagulase-negative staphylococci and e. coli were obtained from field cases of mastitis just prior to commencing this study. these eight bacteria were re-cultured from primary culture plates and incubated for a period of 24 hours to obtain pure cultures. negative control plates were used in all cases to verify plate sterility.colonies of sta. aureus were mixed thoroughly in physiological saline to obtain a 0.5 mcfarland standard that is equal to a 1.5 x 108 cfu/ml bacterial suspension. a volume of 5 ml of this bacterial suspension was added to 480 ml of full cream uht milk and mixed thoroughly by using a sterile magnetic stirrer prior to, and whilst making, aliquots. aliquots of 1.5 ml each were pipetted into 201 marked eppendorf tubes [1.5 ml eppendorf micro test tubes, merck (pty) ltd, midrand south africa] of which 100 were meant for cryopreservation and 100 for freezing. the content of the remaining 1 tube was plated out on columbia agar and incubated for 24 h at 37 °c ± 1 °c to verify growth. this process was repeated for each organism in this trial. ten tubes of each of the eight organisms were packed together and sealed in plastic bags (for freezing in a chest freezer) and separate cryogenic canisters (for cryopreservation in liquid nitrogen) to ensure minimum disturbance of the remaining samples on test days, on removal of test samples from the freezer and nitrogen tank, respectively. in order to ensure that sample tubes remained in the cryogenic canisters, cotton wool was packed firmly on top of the samples in each of the cryogenic containers, to act as a stopper. all cryogenic canisters were fully submerged, without any adaptation period, into the liquid phase of a 14 l liquid nitrogen flask filled to 75% capacity for transportation purposes to the organisation, 30 km away. at the organisation, the cryogenic canisters were put into a large liquid nitrogen tank, in excess of 3 m3 and/or a gross weight of 3000 kg (used for storing semen for long periods of time at the organisation), where the samples remained until required for testing. this organisation specialises in storing large quantities of semen over long periods of time. the vacuum insulated tanks were filled twice a week with 30 kg to 50 kg liquid nitrogen as required and kept at a temperature range of -192 °c to -196 °c. daily monitoring of the liquid level of the tanks is carried out to ensure that all samples remain fully submerged. on each test day, one cryogenic container with all the test samples for that day, was removed by a senior employee of the organisation from the liquid nitrogen tank, and placed directly in a 6 l liquid nitrogen flask filled to 75% capacity for transportation to the milk laboratory. the 10 sealed plastic bags containing the eppendorf tubes with samples, were stored under identical environmental conditions. the plastic bags containing the samples were placed next to each other in a freezer basket in the centre of the freezer (frostline, k.i.c. manufacturing, model number: of 355) at approximately -20 °c at the milk laboratory of the department of production animal studies, onderstepoort, university of pretoria. the temperature of this freezer was verified with a digital thermometer and probe (manufactured by hanna, model no: hi93530, serial no: 33948956, calibrated by inter cal test and measurement centre, 907 richards drive, halfway house, midrand). this freezer was opened not more than twice a week for short periods of time. on each of the test days, 1 of the sealed plastic bags containing samples was removed from the chest freezer. experimental procedure sample schedule all the samples were prepared, frozen and cryopreserved on day 0 of the trial. the first testing commenced at week 2 after freezing and continued every 2 weeks thereafter until week 18 for both the samples, frozen and cryopreserved. one additional batch of frozen samples was analysed at week 30 in order to establish extended survival and viability, as this is the method used most commonly in practice. thawing of samples test day samples from both frozen and cryopreserved batches were thawed at room temperature. the tops of all the eppendorf tubes of cryopreserved samples were immediately covered with a hdpe (high density poly-ethylene) sheet to prevent the lids of the eppendorf tubes from popping when taken from the cryogenic canisters, and placed into a micro test tube rack. the samples, of which the lids did pop, were not processed because of possible contamination, which resulted in missing numbers of liquid nitrogen samples. the eppendorf tubes used were suitable for sample storage between -80 °c and -100 °c, and not -196 °c, hence the popping of lids at thawing. identification of mastitogenic bacteria with each batch of columbia agar base plates made, control plates are incubated for 24 h ± 1 h at 37 °c ± 1 °c and evaluated for sterility prior to use. test milk samples were thawed and plated on columbia agar base plates [oxoid, supplied by quantum biotechnologies (pty) ltd, ferndale, south africa] with 5% defibrinated bovine blood. for each group of bacteria (10 samples) at each test period, one uncultured plate was included as a negative control. test plates were incubated for 24–48 hours at 37 °c ± 1 °c. isolated bacteria were identified in accordance with standard laboratory milk culture methodology based on colony morphology, haemolysis, catalase, koh test and gram staining. additional tests included a strepkit [latex agglutination test from oxoid supplied by quantum biotechnologies (pty) ltd, ferndale, south africa], staphylase test [quantum biotechnologies (pty) ltd, ferndale, south africa] and the api 20e kit [biomerieux south africa (pty) ltd, 7 malibongwe street, fontainebleau, johannesburg, gauteng 2194, south africa], (sandholm et al. 1995; karzis 2005). recording of bacterial growth bacterial viability was recorded and based on the strength of bacterial growth. when there were less than 10 colonies, the actual number of colonies was recorded (figure 1); otherwise the area of growth was used as a semi-quantitative criterion for viability. scores of 1+, 2+ and 3+ were allocated when growth was evident on ⅓, ⅔, or the complete area of inoculation respectively (figure 2). figure 1: columbia agar base plate, showing six colonies, where the actual number of colonies was recorded (for less than 10 colonies present). figure 2: columbia agar base plates showing growth scores. statistical analysis statistical analysis was performed by using the fisher exact test and reported as a two-sided p-value with significance set as p < 0.05. results top ↑ in some of the 10 batches of cryopreserved samples, less than 10 samples were examined because of sample loss that resulted from the popping of lids of eppendorf tubes. streptococcus dysgalactiae, str. uberis and sta. aureus (sta and sth) bacteria could be isolated from all samples stored, showing a growth score of 3+ for the full duration of the study at both temperatures. table 1: comparison between the survival of streptococcus agalactiae (sag) and escherichia coli (eco) for frozen and cryopreserved samples, for weeks 14–18. all bacterial samples survived for str. agalactiae and e. coli for samples frozen and cryopreserved from week 2 to week 12. differences in survival of the two bacteria between the two preservation methods only occurred from week 14 to week 18 (table 1). there was a significant difference between the survival of str. agalactiae samples that were frozen and those that were cryopreserved, for week 14 and week 18 (p = 0.033). there was no significant difference (table 1) between the survival of the e. coli samples that were frozen and those that were cryopreserved.although coagulase-negative staphylococci survived in all the samples up to week 18 from both temperature treatments, a decrease in viability was observed after 4 weeks of cryopreservation (figure 3) and 8 weeks of freezing (figure 4). in the cryopreserved samples, growth of bacteria scored at 3+ and 2+ were present in all the samples up to week 12 and at week 16. at week 14 coagulase-negative staphylococci were isolated from 83% of samples and at week 18 from 88%. growths scored at 3+ and 2+ were present only until week 8 in all samples frozen at approximately -20 °c, in 70% of samples at weeks 10, 12 and 18, and in 80% of samples at week 14, and in 50% of samples at week 16 of freezing. at week 30, coagulase-negative staphylococci could only be isolated from 50% of the samples frozen and only with a score of 1+ growth. figure 3: percentage survival and viability of coagulase-negative staphylococci for cryopreserved samples. figure 4: percentage survival and viability of coagulase-negative staphylococci for frozen samples. streptococcus agalactiae was isolated from all samples until week 18 and week 12 from cryopreserved and frozen samples, respectively, and from only 50% of samples frozen after week 12.cryopreserved e. faecalis survived in all the samples showing 3+ growth until week 12 and at week 18, whilst 20% of e. faecalis did not survive at week 8. during week 14 and week 16 viability decreased, showing a growth of 2+ in 20% and 11% of cryopreserved samples, respectively (figure 5). enterococcus faecalis survived and remained viable in all frozen samples with a score of 3+ growth until week 30, except for week 12 where viability decreased and 10% of cultures showed a score of 2+ growth. figure 5: percentage survival and viability of enterococcus faecalis for cryopreserved samples. lower survival rates were observed in e. coli at week 14 and week 18 in 10% of cryopreserved samples. escherichia coli survived in all frozen samples up to week 16, but only from 50% of samples at weeks 18 and 30. at week 12 only 20% of the cultures decreased in viability, showing a score of 2+ growth (figure 6). figure 6: percentage survival and viability of escherichia coli for frozen samples. discussion top ↑ the ability of the organisms to withstand temperatures varied, but in general better survival and viability of bacteria were observed in cryopreserved samples in comparison to frozen samples. the rate at which bacteria are frozen is important, as slow freezing can cause cell wall collapse because of dehydration, or because of the formation of ice crystals which expand and in doing so destroy the cell walls (ed. harris 1954). the entire cell content might contract and become concentrated causing the acid-base ratio to be altered and the salt concentration to rise to levels that are potentially lethal to cells. a rapid freezing process helps to prevent these destructive events (ed. harris 1954). although coagulase-negative staphylococci survived from both temperature treatments until the end of the study, there was a difference in viability. these findings are in agreement with those of luedecke et al. (1971), which also showed a decrease in colony count during 70 days of storage. this, however, differed from findings of schukken et al. (1989), and hubackova and rysanek (2007), who detected increases in staphylococcus epidermidis numbers from as soon as 7 days after freezing and which increased with storage periods. luedecke et al. (1971) and murdough et al. (1996), however, found no effect on the viability of sta. epidermidis after freezing. in this study no changes were observed in the survival and viability of sta. aureus (both sta and sth) after cryopreservation for 18 weeks and freezing for 30 weeks, which agrees with results recorded by other researchers (bashandy & heider 1979; luedecke et al. 1971; murdough et al. 1996; pankey et al. 1987; schukken et al. 1989) who reported that freezing at temperatures of -20 °c and -80 °c had no effect on the recovery of sta. aureus. sol et al. (2002) even reported an increase in sta. aureus numbers during freezing. there was 100% survival of e. coli up to week 16 for frozen samples, and week 12 for cryopreserved samples. the viability of frozen samples decreased at week 12, however, whilst no decrease in viability was observed for cryopreserved samples. these findings are in agreement with the report of murdough et al. (1996), but contradict numerous other studies that found significant declines in the isolation of e. coli from as early as 72 hours after freezing (hubackova & rysanek 2007; pankey et al. 1987; schukken et al. 1989; sol et al. 2002; storper, ziv & saran 1982). an observation was made during this study that e. coli colonies took more than double the time to react with 5% koh from weeks 10 and 12 than for frozen and cryopreserved samples, respectively. results from literature on the survival of str. agalactiae have been varied. the results obtained in this study for frozen samples are in agreement with studies of luedecke et al. (1971); bashandy and heider (1979), and storper et al. (1982), who all observed a significant decrease in survival over time. villanueva et al. (1991) on the other hand, found a 2.50-fold increase in colony numbers when stored at -20 °c for 23 days, whilst sol et al. (2002) found that str. agalactiae could be isolated from more samples if incubated at 37 °c after freezing. murdough et al. (1996) showed no differences in the survival of str. agalactiae after being frozen for 6 weeks. in cryopreserved samples there was no observed decrease in either the survival or the viability of str. agalactiae. this study found no difference in the survival and viability of str. uberis and str. dysgalactiae between frozen and cryopreserved samples over time, and only slight differences for e. faecalis. these findings are in agreement with those of murdough et al. (1996) who detected no significant changes in the survival of str. dysgalactiae and str. uberis numbers. results of bashandy and heider (1979); storper et al. (1982) and pankey et al. (1987), however, found a significant decrease in the survival of non-agalactiae streptococcal isolates that were recovered from milk samples stored at temperatures of -18 °c to -70 °c. staphylococcus aureus (sta and sth), str. dysgalactiae and str. uberis were frozen and cryopreserved for 18 and 30 weeks respectively, without a reduction in survival and viability. although coagulase-negative staphylococci survived until week 18 for frozen and cryopreserved samples, viability decreased after week 8 for frozen samples, and week 2 for cryopreserved samples. in the case of e. coli, cryopreservation had a slight advantage of survival for 2 weeks longer compared to samples that were frozen at approximately -20 °c in a freezer. streptococcus agalactiae had the largest variation in survival between frozen and cryopreserved samples after week 12, namely 100% survival in cryopreserved samples, and only 50% in frozen samples. this study has indicated that milk can be frozen for at least 12 weeks and cryopreserved for up to 18 weeks, for the eight most commonly isolated mastitogenic organisms in south africa. the process of thawing cryopreserved samples, however, in this study caused sample loss through the use of eppendorf tubes, which were suitable for storage between -80 °c and -100 °c and not for cryopreservation. the use of suitable containers for cryopreservation will prevent loss of samples during thawing. both freezing and cryopreservation can contribute to improving pro-active udder health management. acknowledgements top ↑ the authors of this paper thank the cryogenic facility at taurus, irene, south africa for the use of their facilities in storing milk samples in liquid nitrogen. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions i.m.p. (university of pretoria) was the project leader. i.m.p. (university of pretoria), j.c.w. (university of pretoria) and t.j.v.d.s. (university of pretoria) were responsible for experimental and project design. n.m.i. (herd husbandry help cc) assisted with the storage of samples in liquid nitrogen (-196 ºc) by making facilities available. s.e. (university of pretoria) assisted with data capturing. j.k. (university of pretoria) and i.m.p. (university of pretoria) were responsible for the literature review, the writing-up of the article, and g.t.f. (university of pretoria) assisted with data utilisation and statistical analysis. references top ↑ bashandy, e.y. & heider, l.e., 1979, ‘the effect of freezing milk samples on the cultural results’, zentralblatt fϋr veterinärmed reihe 26, 1–6. farrat, j., 1980, ‘freeze-thaw injuries in living cells’, international journal of refrigeration 3, 191. http://dx.doi.org/10.1016/0140-7007(80)90045-6 harris, r.j.c. (ed.), 1954, biological applications of freezing and drying, academic press incorporated, new york. hubackova, m. & rysanek, d., 2007, ‘effects of freezing milk samples on the recovery of alimentary pathogens and indicator microorganisms’, acta veterinaria brunensis 76, 301–307. http://dx.doi.org/10.2754/avb200776020301 karzis, j., 2005, ‘intramammary antibiotics in dairy goats: withdrawal periods and tissue tolerance’, msc thesis, dept. of production animal studies, university of pretoria. luedecke, l.o., forster, l., williams, k. & hillers, j.k., 1971, ‘effect of freezing and storage at -20 °c on survival of mastitis pathogens’, journal of dairy science 55, 417–418. http://dx.doi.org/10.3168/jds.s0022-0302(72)85509-7 mazur, p., 1970, ‘cryobiology: the freezing of biological systems’, science 168, 939–949. http://dx.doi.org/10.1126/science.168.3934.939, pmid:5462399 murdough, p.a., deitz, k.e. & pankey, j.w., 1996, ‘effects of freezing on the viability of nine pathogens from quarters with subclinical mastitis’, journal of dairy science 79, 334–336. http://dx.doi.org/10.3168/jds.s0022-0302(96)76368-3 murray, p.r., baron, e.j., jorgensen, j.h., landry, m.l. & pfaller, m.a., 2007, manual of clinical microbiology, 9th edn., asm press, washington dc. pankey, j.w., wadsworth, j.k., metha, k.h. & murdough, p.a., 1987, ‘effects of storage on viability of mastitis pathogens’, journal of dairy science 70(1), 132. schukken, y.h., smit, j.a.h., grommers, f.j., vandegeer, d. & brand, a., 1989, ‘effects of freezing on bacteriologic culturing on mastitis milk samples’, journal of dairy science 72, 1900–1906. http://dx.doi.org/10.3168/jds.s0022-0302(89)79309-7 sandholm, m., honkanen-buzalski, t., kaartinen, l. & pyörälä, s., 1995, the bovine udder and mastitis, university of helsinki, faculty of veterinary medicine, helsinki. sol, j., sampimon, o.c., hartmane, e. & barkema, h.w., 2002, ‘effects of preculture freezing and incubation on bacteriologic isolation from subclinical mastitis samples’, veterinary microbiology 85, 241–249. http://dx.doi.org/10.1016/s0378-1135(01)00507-7 storper, m., ziv, g. & saran, a., 1982, ‘effect of storing milk samples at -18 °c on the viability of certain udder pathogens’, refuah vetterinarith 39, 1–2. villanueva, m.r., tyler, j.w. & thurmond, m.c., 1991, ‘recovery of streptococcus agalactiae and staphylococcus aureus from fresh and frozen bovine milk’, journal of the american veterinary medical association 198(8), 1398–1400. pmid:2061157 ker_197-202.qxd introduction in 1925 wiggers observed that, if ventricular activation proceeds from the ventricular epicardium instead of from the atrium, the consequence will be a lower peak left ventricular pressure as well as a lower dp/dt. he also concluded that the normal sequence of ventricular activation is essential for optimum ventricular function (varna & camm 2001). in the mammalian heart, cardiac activation is initiated in the atria and proceeds to the ventricles via the specialized conduction system (scs). this scs consists of the his bundle, the left and right bundle branches with their major fascicles and the peripheral purkinje network (van dam 1989). the terminal ramifications of this purkinje network merge into the ventricular myocardium at sites of purkinje-myocardial coupling. proximal to these sites the scs has no functional connections with the myocardium because of a thin, collagenous sheath (van dam 1989). ventricular dyssynchrony can be defined as an abnormality of the normal, organized electromechanical coupling of the ventricles and this disturbance is the consequence of an interventricular conduction delay (aranda & schofield 2002). ventricular dyssynchrony is not an uncommon clinical problem and has numerous causes, such as left bundle branch 197 onderstepoort journal of veterinary research, 71:197–202 (2004) ventricular dyssynchrony as a cause of structural disease in the heart of dorper sheep j. ker1, e.c. webb2 and c.f. van der merwe3 abstract ker, j., webb, e.c. & van der merwe, c.f. 2004. ventricular dyssynchrony as a cause of structural disease in the heart of dorper sheep. onderstepoort journal of veterinary research, 71: 197–202 ventricular dyssynchrony is a disturbance of the normal, organized electromechanical coupling of the two ventricles. this condition has many causes, such as left bundle branch block, ventricular preexcitation, right ventricular pacing and right ventricular premature ventricular complexes (pvcs). ventricular dyssynchrony has many adverse haemodynamic effects on the left ventricle and we wanted to know whether these adverse haemodynamic effects might have any structural consequences on the left ventricles of such hearts. six healthy dorper wethers were subjected to numerous right ventricular pvcs to induce ventricular dyssynchrony in order to determine whether any structural consequences will occur in the left ventricles of these hearts. myocarditis in the musculature of the left ventricles of all six these hearts was seen. keywords: dorper sheep, premature ventricular complexes, ventricular dyssynchrony 1 department of physiology, faculty of medicine, university of pretoria, p.o. box 24318, gezina, pretoria, 0031 south africa. e-mail: james.ker@med.up.ac.za 2 department of animal and wildlife sciences, faculty of natural and agricultural sciences, university of pretoria, pretoria, 0002 south africa 3 department of microscopy and microanalysis, university of pretoria, pretoria, 0002 south africa accepted for publication 28 january 2004—editor block (littmann & symanski 2000), right ventricular pacing (badke & boinay 1980; grover & glantz 1983; dicarlo & morady 1987; rosenqvist & isaaz 1991), ventricular pre-excitation (sutherland & kukulski 2000) and premature ventricular complexes (sutherland & kukulski 2000). during periods of ventricular dyssynchrony, right ventricular activation begins and is completed before the initiation of left ventricular activation (dunn 1987). right ventricular depolarization is then followed by a reversed activation sequence of the interventricular septum, proceeding from right to left, which is then followed by a parallel, rather than radial, spread of depolarization towards the left ventricular free wall (dunn 1987). this delayed activation of the left ventricle, more specifically the lateral wall of the left ventricle, has several important haemodynamic consequences (aranda & schofield 2002): • there is delayed contraction of the posteromedial papillary muscle, which leads to systolic mitral regurgitation. • the delayed activation of the left ventricle leads to a delay in the onset of early diastolic ventricular filling, but atrial activation proceeds normally. the result is that early, passive diastolic filling of the left ventricle and atrial contraction occur simultaneously. this has been demonstrated by the merging of the e and a waves on mitral doppler inflow patterns in these hearts. • atrial activation during the early, passive diastolic filling phase of the left ventricle decreases the total transmitral flow which results in a diminished preload of the left ventricle. • the delayed activation of the posteromedial papillary muscle also leads to diastolic mitral regurgitation. • the interventricular septum is a crucial structure for the maintenance of interventricular dependance. however, with delayed activation of the left ventricular lateral wall, left ventricular systolic pressure will be increased well after depolarization of the interventricular septum is completed. the consequence is septal dyskinesis, with the septum moving away from the left ventricular wall during left ventricular contraction. the result is a decreased septal contribution to left ventricular stroke volume. ventricular dyssynchrony is a common clinical problem and has been shown to occur in approximately 30 % of humans with chronic heart failure, both of ischaemic and idiopathic origin (barold 2001; abraham & fisher 2002). the objective of this investigation was to determine if ventricular dyssynchrony per se can cause structural heart disease, instead of it always being considered a consequence of such structural disease of the heart. materials and methods six clinically normal dorper wethers, all between the ages of 9 and 12 months and weighing between 35 and 40 kg, were used in this study. they were fed lucerne hay ad libitum, received 300 g per day of pelleted concentrate (10 mj me/kg dm with 14 % crude protein) and had free access to water at all times. right ventricular premature ventricular complexes (pvcs) were used to induce ventricular dyssynchrony. right ventricular pvcs were induced in each wether as previously described by ker & webb (2003): a spring-wire guide, diameter 0.81 mm and length 600 mm, was advanced into the right ventricle via the left internal jugular vein, using the seldinger technique. the position of the wire was confirmed in every case by an x-ray. right ventricular pvcs were induced by mechanical movement of the spring-wire guide inside the right ventricle. these right ventricular pvcs were induced on a daily basis in every wether for variable periods. thus pvcs were induced in sheep 1 for 15 days, in sheep 2 for 36 days, in sheep 3 for 28 days, in sheep 4 for 16 days, in sheep 5 for 53 days and in sheep 6 for 34 days. on completion of each series of pvcs, each wether was subsequently slaughtered and their hearts subjected to histological examination. only the musculature of the left ventricles were examined histologically as the right ventricles were subjected to mechanical trauma by the spring-wire guides used to induce the right ventricular pvcs and therefore some histological changes in the musculature of the right ventricles are to be expected. however, because the left ventricle is isolated from the right by the interventricular septum the wire will have no direct histological effects on the left ventricle. left ventricular dissection • the musculature of each left ventricle (lv) was dissected into three regions: two transverse in198 ventricular dyssynhrony in heart of dorper sheep cisions were made, one at the level of the base and the other at the level of the apex of the posteromedial papillary muscle. this divides the lv into three regions: base, mid-region and apex. • each of these three regions were then dissected into four parts: anterior, posterior, septal and lateral. • in this way every lv was dissected into 12 pieces, representing the musculature of the entire lv, which were subsequently all subjected to histological examination. • these 12 segments were numbered as follows: a = anterior part of base b = anterior part of mid region c = anterior part of apex d = septal part of base e = septal part of mid region f = septal part of apex g = lateral part of base h = lateral part of mid region i = lateral part of apex j = posterior part of base k = posterior part of mid region l = posterior part of apex histological evaluation tissue blocks from the 12 sites were fixed in 10 % buffered formalin and paraffin-embedded sections for light microscopy were prepared using routine histological procedures. they were stained with hematoxylin and eosin (he). all the sections were evaluated for the following: 1. myocardial cellular abnormalities and/or 2. myocardial interstitial abnormalities. histologic categories on the basis of the morphological findings each of the 12 specimens was assigned to one of four categories: 1. normal 2. myocardial cellular abnormalities 3. myocardial interstitial abnormalities 4. both myocardial cellular and interstitial abnormalities. the heart of a normal dorper wether, of similar age and mass, served as a histological control (see fig. 1). results no sheep in this study showed any signs of infection as a result of the spring-wire guide during the entire study period. the induction of pvcs was possible throughout the study period in all the animals. histologic abnormalities as compared to the histological control (fig. 1) we observed histological changes in all six of the experimental animals. these changes consisted of both myocardial cellular and interstitial abnormalities in the musculature of the left ventricle (table 1). according to the dallas criteria (hare & baughman 1994; kühl, noutsias, seeberg & schultheiss 1996; pisani, taylor & mason 1997; feldman & mcnamara 2000) these observed myocardial cellular and interstitial changes are indicative of myocarditis. relation between the pvc load and number of abnormal left ventricular segments we found no relation between the pvc load, as well as the number of days subjected to pvcs, and the number of abnormal left ventricular segments (table 2). location of left ventricular histologic abnormalities the location of these histologic abnormalities in the left ventricles of the experimental animals was evenly distributed between the anterior, posterior, septal and lateral wall (table 3). 199 j. ker, e.c. webb & c.f. van der merwe fig. 1 longitudinal section through the musculature of the left ventricle of a normal dorper wether heart 200 ventricular dyssynhrony in heart of dorper sheep table 1 histologic outcomes sheep histologic abnormalities in left ventricle 1 myocardial interstitial abnormalities* in segments c, d, e, f, g, h, i, j, k 2 myocardial interstitial abnormalities in segments b, c, d, e, i, j, k, l myocardial cellular abnormalities** in segments b, e, k fibrosis in segments b, k 3 myocardial interstitial abnormalities in segments a, b, d, g, k myocardial cellular abnormalities in segments a, d, g 4 myocardial cellular abnormalities in segments a, c 5 myocardial interstitial abnormalities in segments a, b, d, e, h, i, k myocardial cellular abnormalities in segment a fibrosis in segments d, f, i 6 myocardial interstitial abnormalities in segments d, i * myocardial interstitial abnormalities consist of an infiltration of white blood cells (> 5 wbcs/high power field) ** myocardial cellular abnormalities consist of myocytolysis table 2 days subjected to pvcs, pvc load and number of abnormal left ventricular segments days subjected pvc load* number of abnormal to pvcs left ventricular segments 53 902 8 36 575 8 34 908 2 28 371 5 16 1 887 2 15 221 9 * the spring-wire guides were left in situ in the right ventricles and electrocardiography was done once daily. therefore, the number of actual pvcs may be much higher than the number of pvcs counted fig. 2 infiltration of the left ventricular interstitium by a mixed inflammatory cell infiltrate, a feature of myocarditis fig. 3 myocytolysis in the left ventricle, another feature of myocarditis table 3 location of left ventricular histologic abnormalities location of abnormal number of abnormal left left ventricular segments ventricular segments (total of 6) anterior 5 posterior 4 septal 5 lateral 5 discussion any alteration in the normal sequence of left ventricular activation, thereby leading to interventricular dyssynchrony, has several important consequences. these include haemodynamic, myocardial and metabolic disturbances (aranda & schofield 2002; van oosterhout, prinzen, arts, schreuder, vanagt, cleutjens & reneman 1998; zanco, desideri, mobilia, carguel, milan, celegon, buchberger & ferlin 2000). firstly, haemodynamic consequences are mainly due to delayed activation of the lateral wall of the left ventricle. these include systolic and diastolic mitral regurgitation, a diminished preload of the left ventricle and septal dyskinesis causing a decreased septal contribution to left ventricular stroke volume (aranda & schofield 2002). secondly, myocardial consequences have been described (van oosterhout, prinzen, arts, schreuder, vanagt, cleutjens & reneman 1998). asynchronous activation of the left ventricle induces asymmetrical left ventricular hypertrophy. when there is a period of asynchronous activation of the left ventricle, as induced by right ventricular pacing or pvcs, this causes regional differences in the workload of the left ventricle. the workload is lower in early than in late-activated regions and as a result the early-activated regions become thinner and the late-activated regions become thicker. thirdly, metabolic consequences have been described as well (zanco, desideri, mobilia, cargnel, milan, celegon, buchberger & ferlin 2000). there is a reduction of septal glucose uptake and metabolism in the left ventricle during periods of ventricular dyssynchrony. according to the dallas criteria (hare & baughman 1994; kühl, noutsias, seeberg & schultheiss 1996; pisani, taylor & mason 1997; feldman & mcnamara 2000) all six of the experimental animals developed myocarditis. myocarditis has many causes (feldman & mcnamara 2000). some of these include infectious agents, physical agents, drugs, heavy metals, systemic diseases and various miscellaneous causes, such as snakeand spider bites (feldman & mcnamara 2000). furthermore, myocarditis consists of various histopathologic subclasses and these include a lymphocytic, eosinophilic, neutrophilic, giant cell and a granulomatous type (pisani, taylor & mason 1997). might it be possible that there is also an immunological consequence of ventricular dyssynchrony, that presents with myocardial inflammation, as seen in our study? a search of the medline database from 1966 to the present revealed only two studies where myocarditis occurred in situations of ventricular dyssynchrony. basso, corrado, rossi & thiene (2001) examined the hearts of eight human patients who died from wolff-parkinson-white (wpw) syndrome and found isolated atrial myocarditis in four (50 %) of these hearts. this syndrome is a condition in which strands of atrial-like muscle form atrioventricular bypass tracts which activate the right ventricle causing right ventricular activation to precede left ventricular activation. it is therefore a cause of ventricular dyssynchrony. the syndrome is a congenital disorder and therefore its relationship to myocarditis is either coincidental or possibly causal. because pvcs do not constitute a congenital disorder, but rather a complication of various cardiac and non-cardiac disorders, its possible relation to myocarditis is more complex. biase, chiddo, caruso, tritto, marchese & rizzon (1992) examined ventricular endomyocardial biopsies from 26 human patients with pvcs and found acute myocarditis in 7 % and borderline myocarditis in 3.5 %. however, it is not possible to determine from this data whether pvcs are a consequence or a cause of myocarditis. we found in this study that the induction of right ventricular pvcs consistently led to the development of myocarditis of the left ventricle in all of the experimental animals. an interesting finding is that there was no relation between the pvc load and the number of abnormal left ventricular segments (see table 2). as stated before, the spring-wire guides were left in situ in the right ventricles and electrocardiography was done once daily. it is therefore possible that the actual number of pvcs may be higher in sheep with less days exposed to pvcs. alternatively, it is possible that the critical time period for the development of myocarditis is 2 weeks or less and/or that the critical number of pvcs needed to induce myocarditis is 221 or less. another interesting finding is the distribution of myocardial inflammation (table 2). the histological manifestations of myocarditis are evenly distributed among the anterior, posterior, septal and lateral walls of the left ventricle of all the experimental animals. in conclusion, myocarditis was detected in the left ventricles in all of the experimental animals. in this study this was a diffuse process, which involved all 201 j. ker, e.c. webb & c.f. van der merwe four walls of the left ventricle. we conclude that right ventricular pvcs, a cause of ventricular dyssynchrony, may be a cause of myocarditis in the dorper wether and that pvcs are not always a complication of myocarditis but that there is a possible causal relationship. therefore, we propose a fourth consequence of ventricular dyssynchrony, an alteration of the immune system, leading to myocarditis. references abraham, w.t. & fisher, w.g. 2002. cardiac resynchronization in chronic heart failure. new england journal of medicine, 346:1845–1853. aranda, j.m. & schofield, r.s. 2002. ventricular dyssynchrony in dilated cardiomyopathy: the role of biventricular pacing in the treatment of congestive heart failure. clinical cardiology, 25:357–362. badke, f.r. & boinay, p. 1980. effects of ventricular pacing on regional left ventricular performance in the dog. american journal of physiology, 238:h858–867. barold, s.s. 2001. what is cardiac resynchronization therapy? american journal of medicine, 111:224–232. basso, c., corrado, d., rossi, l. & thiene, g. 2001. ventricular pre-excitation in children and young adults. atrial myocarditis as a 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electrocardiology.theory and practice in health and disease, edited by p.w macfarlane. new york: pergamon press. van oosterhout, m.f.m., prinzen, f.w., arts, t., schreuder, j.j., vanagt, w.y.r., cleutjens, j.p.m. & reneman, r.s. 1998. asynchronous electrical activation induces asymmetrical hypertrophy of the left ventricular wall. circulation, 98:588–595. varma, c. & camm, a.j. 2001. pacing for heart failure. lancet, 357:1277–1283. zanco, p., desideri, a., mobilia, g., cargnel, s., milan, e., celegon, l., buchberger, r. & ferlin, g. 2000. effects of left bundle branch block on myocardial fdg pet in patients without significant coronary artery stenoses. journal of nuclear medicine, 41:973–977. 202 ventricular dyssynhrony in heart of dorper sheep abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) ben h. thameur ministry of agriculture of tunisia, general directorate of veterinary services, crda nabeul, tunisia sghaier soufiène tunisian veterinary research institute, r djebel el akhdhar errabta, tunisia heni haj ammar ministry of agriculture of tunisia, general directorate of veterinary services, rue alain savary, tunisia salah hammami ecole nationale de médecine vétérinaire de sidi thabet, iresa, university of manouba, sidi thabet, tunisia citation thameur, b.h., soufiène, s., haj ammar, h. & hammami, s., 2021, ‘spatial distribution and habitat selection of culicoides imicola: the potential vector of bluetongue virus in tunisia’, onderstepoort journal of veterinary research 88(1), a1861. https://doi.org/10.4102/ojvr.v88i1.1861 original research spatial distribution and habitat selection of culicoides imicola: the potential vector of bluetongue virus in tunisia ben h. thameur, sghaier soufiène, heni haj ammar, salah hammami received: 02 mar. 2020; accepted: 09 dec. 2020; published: 16 aug. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the increasing threat of vector-borne diseases (vbds) represents a great challenge to those who manage public and animal health. determining the spatial distribution of arthropod vector species is an essential step in studying the risk of transmission of a vector-borne pathogen (vbp) and in estimating risk levels of vbd. risk maps allow better targeting surveillance and help in designing control measures. we aimed to study the geographical distribution of culicoides imicola, the main competent vector of bluetongue virus (btv) in sheep in tunisia. fifty-three records covering the whole distribution range of c.imicola in tunisia were obtained during a 2-year field entomological survey (august 2017 – january 2018 and august 2018 – january 2019). the ecological niche of c. imicola is described using ecological-niche factor analysis (enfa) and mahalanobis distances factor analysis (madifa). an environmental suitability map (esm) was developed by maxent software to map the optimal habitat under the current climate background. the maxent model was highly accurate with a statistically significant area under curve (auc) value of 0.941. the location of the potential distribution of c. imicola is predicted in specified regions of tunisia. our findings can be applied in various ways such as surveillance and control program of btv in tunisia. keywords: tunisia; bluetongue virus; c. imicola; enfa; ecological-niche factor analysis; madifa; mahalanobis distances factor analysis; ecological niche model; maxent. introduction the spread of disease vectors and the growing number of vector-borne diseases (vbds) pose a major threat to human and animal health. despite considerable success in controlling communicable diseases, vbds have caused and still cause damage on different continents. bluetongue (bt), an emerging and re-emerging vbd, is caused by double-stranded ribonucleic acid (rna) orbiviruses of the reoviridae family. bluetongue virus (btv) infects domestic and wild ruminants and it is transmitted by biting midges of the genus culicoides (diptera: ceratopogonidae). between 1999 and 2002, btv serotype 2 epizootics have occurred in tunisia (ben fredj et al. 2003; hammami 2004). in 2006 and 2009, tunisia’s general directorate of veterinary services (dgsv) warned of incursion of two additional serotypes: btv1 and btv4, successively (lorusso et al. 2013; sghaier et al. 2014). in 2016, a novel bt serotype 3 was reported (lorusso et al. 2018). genome sequencing revealed two unrelated western strains of btv-3, one circulating in cap-bon (northwest of tunisia) and neighbouring areas, and the other circulating nearby the border with libya. culicoides imicola is the main potential vector of bt in tunisia (hammami 2004; sghaier et al. 2014). determining the spatial distribution of arthropod vector species is an essential step in studying the risk of transmission of a vector-borne pathogen (vbp) and in estimating the level of risk. geospatial technologies are commonly used to evaluate vector patterns or human or animal case distributions and to estimate the risk of disease transmission based on entomological, epidemiological and environmental factors. diverse modelling methods have been developed and are commonly used to predict the geographic range of a species given presence-only (po) occurrence data and environmental variables assumed to influence its distribution (baasch et al. 2010; bateman, vanderwal & johnson 2011; güthlin et al. 2011). in our study, we used two complementary protocol analyses: ecological niche factor analysis (enfa; hirzel et al. 2002) and mahalanobis distances factor analysis (madifa; calenge & basille 2008), to explore the relationship between the ecological niche and the availability of habitats of c. imicola. after selecting the variables associated with the ecological niche of c. imicola, we used the maximum entropy (maxent) approach to model c. imicola presence and mapping habitat suitability (hs) (phillips, anderson & schapire 2006). we expect this approach to prove to be an effective tool to assess and visualise the risk of the establishment and the spread of bt in tunisia. materials and methods presence points a large entomological monitoring was carried out over the entire territory of tunisia between 2017 and 2019 to describe the diversity, distribution and seasonal dynamics of culicoides in relation with bt cases. seventy-two sites were monitored during two periods; 34 sites from august 2017 to january 2018 and 38 sites from august 2018 to january 2019. two-night catches per site were performed monthly using black ultraviolet (uv)-light traps manufactured by the onderstepoort veterinary institute (ovi, south africa). insects were collected in a beaker containing 200 millilitres (ml) – 250 ml of water and 3–4 drops of detergent, as a wetting agent. each catch was transported to the laboratory, then covered and preserved in 70% ethanol for further study. culicoides imicola were identified according to wing patterns, using stereoscopic microscope and taxonomic keys and subsequently confirmed by mounting specimens on microscope slides (delécolle 1985). culicoides. imicola is identified in 53 sites (presence points, white dots in figure 6). bioclimatic variable to determine which environmental variables most influence the distribution of c. imicola, the main vector of bt in tunisia, we included 12 bioclimatic variables in our model (table 1). the selection of environmental and climatic predictors used in the analysis was based on the literature review and association already proved with the disease (calvete et al. 2009; ciss et al. 2019; van doninck et al. 2014). the bioclimatic data, with a spatial resolution of 30 arc seconds (~1 kilometres [km]), were downloaded from the world climate website (http://www.worldclim.org/current) and averaged over a 50 year period between 1950 and 2000 at the same spatial resolution. first worldclim data files are converted to esri ascii format in diva-gis (http://www.diva-gis.org) and trimmed in the region of tunisia. the final step was to convert the trimmed grid files to the esri ascii format. the files are then ready to be used in r-studio and in maxent software. table 1: bioclimatic variables used in the model. modelling habitat suitability we used two complementary model analyses; enfa and madifa, to explore the relationship between the ecological niche of c. imicola and the availability of habitats (calenge & basille 2008). the enfa and madifa multivariate analyses were performed using the functions ‘enfa’ and ‘madifa’ of the package adehabitaths (calenge 2006) available in the open-source software r (r development core team 2009). the enfa analyses the data in relation to the environment, that is, what is available for the species; the madifa focuses on the habitat, that is, what the species uses. in enfa, the components have direct ecological meaning. the first factor is the marginality: the direction in which the species niche differs at most from the available conditions in the global area. the higher the absolute value of marginality, the more the species habitat differs from the study area. a positive marginality means that the species prefers higher-than-mean values on the ecological variable. specialisation factors indicate how restricted the species’ niche is in relation to the study area. the madifa, in contrast to the enfa, locates the directions in ecological space for which the niche is narrowest relative to the available environment (calenge & basille 2008). it combines species marginality and specialisation into a unique measure of hs. the two methods make it possible to predict the potential habitat for the species and to build environmental suitability maps (esm) (basille et al. 2008). after selecting environmental predictors associated with the ecological niche of c. imicola, we used the maxent approach to model c. imicola presence and mapping esm. maximum entropy model: mapping of the optimal regions for culicoides imicola in this study, we used the maximum entropy model (maxent version 3.4.1 [phillip et al. 2006]; http://www.cs.princeton.edu/wschapire/maxent/) because it has been shown to perform better with small sample sizes compared with other modelling methods (elith et al. 2006; kumar & stohlgren 2009; pearson et al. 2007). maximum entropy uses po data to predict the distribution of a species based on the theory of maximum entropy. the programme attempts to estimate a probability distribution of species occurrence that is closest to uniform whilst still subject to environmental constraints (elith et al. 2011). maximum entropy takes a list of species presence locations as input, often called po data, as well as a set of environmental predictors across a user-defined landscape that is divided into grid cells. from this landscape, maxent extracts a sample of background locations that it contrasts against the presence locations. presence is unknown at back-ground locations. the relative occurrence rate (ror) is the relative probability that a cell is contained in a collection of presence samples. the ror corresponds to maxent’s raw output. maximum entropy can be used to predict the probability of presence only by using a transformation of the ror, called logistic output (phillips & dudik 2008). maximum entropy predicts rors as a function of the environmental predictors at that location. these rors sum to unity across the landscape because the denominator is a sum of the rors over all grid cells in the study (called normalisation). normalisation ensures that the occurrence rates are in fact rors. to reduce multi-collinearity amongst the bioclimatic variables used in maxent, highly correlated variables (r ≥ 0.85 pearson correlation coefficient) were eliminated from further models (graham 2003). jackknife analyses were performed to determine variables which, when omitted, reduce the model reliability. we used the area under the receiving operator curve (auc) to evaluate model performance. the value of auc ranges from 0 to 1. an auc value of 0.50 indicates that the model did not perform better than random, whereas a value of 1.0 indicates perfect discrimination (swets 1988). the model with the highest auc value was considered the best performer. results environmental factors associated with the collection data as illustrated by the enfa (figure 1) the centroid of the ecological niche was far from the centroid of the available habitat, meaning that used and available distributions differed with respect to their mean (marginality, first axis) and variance (specialisation, second axis). the enfa method provides for our presence data an overall marginality of m = 7.706, showing that c. imicola habitat is very different from the mean habitat of the study area (c. imicola presence needs specific ecological conditions) and an overall specialisation of s = 2.663, which means that we found c. imicola in a small range of conditions. if we observe the marginality coefficients of every eco-geographical variable (table 2), bio4, bio18, bio5, bio6 and bio10 correlate with the marginality axis at 39%, 38%, 32%, 27% and 26%, respectively. positive values mean that the values of variables inside site areas are greater than values in the whole study area. bio 10 correlates with the first specialisation axis (71%, table 2, figure 1). bio4 and bio6 also correlate with this axis but to a lesser extent showing 42% and 23%, respectively. the values of specialisation factors indicate that c. imicola sites have a small range of bio10, bio4 and bio6 (table 2). figure 1: results of the ecological-niche factor analysis for c. imicola in tunisia. table 2: correlations (%) between variables and axes for the ecological niche factor analysis (marginality and 1st axe of specialisation) and the mahalanobis distances factor analysis (1st and 2nd axes). the ecological-niche factor analysis results displayed by the marginality axis (x-axis) and first specialisation axis (y-axis). marginality was strong as the dot representing the centroid of the distribution of used units shifted from the origin of the axes, the centroid of the distribution of available weights. the polygons correspond to the minimum convex polygons of the distributions of available (light grey area) and used (dark grey area) resource units. the polygon of the used habitat (realised niche) was narrow in its extent on the y-axis meaning that specialisation was also high. the direction and length of the arrows are a metric of the contribution of the variables to marginality and specialisation. the results of madifa identified additional significant ecological variables in the study area, as it identified the directions of the ecological space where mahalanobis distances were the largest, corresponding to the environmental conditions scarcely used by c. imicola. as illustrated by the scatter niche of madifa (figure 2), the used points are distributed in the restricted area of the factorial axes found by the analysis. this confirms that through analysis an interesting direction could be identified. the black dots (used habitats) are significantly offset on the first and second axis from the centre of the grey area and occupy part of the middle right sector of the available habitat. the first axis (abscissa) of madifa was strongly correlated with bio12, bio10 and bio16 and the second axis (ordinate) was strongly correlated with bio16, bio12 and bio10 (table 2, figure 2). these variables affect the position of the availability niche in relation to the used niche for the first and second factors, respectively. figure 2: scatter niche diagram of the cloud of available (grey circle) and used (black squares) points on the first two axes of the mahalanobis distances factor analysis. the black points correspond to the ecological variables used by the c. imicola. grid lines (separated by a distance of 0.2) can be used to measure the correlations between ecogeographical variables and significant factors (i.e. abscissa and ordinate axes) on the graph for each analysis. the results of enfa and madifa are complementary as the marginality axis of the enfa and the first component of the madifa were significantly correlated (rho = 0.689) (figure 3). figure 3: correlation between the first axis of the mahalanobis distances factor analysis and the marginality axis of the ecological niche factor analysis. during this procedure, variables that did not contribute to the ecological niche or highly correlated variables (with a coefficient of correlation higher than 0.95), were not used in the statistical analyses in maxent. maximum entropy model: mapping of the optimal regions for culicoides imicola the reduction of predictor variables resulted in the inclusion of only height variables for maxent model. table 3 gives estimates of relative contributions of the environmental variables to the maxent model. table 3: relative contributions of the environmental variables to the maximum entropy model. the maxent model for c. imicola probability distribution in tunisia provided satisfactory results, with an auc value of 0.941 (±0.001), which is higher than 0.5 of a random model (figure 4). precipitation of warmest quarter (bio18) contributed most to the model, followed by the mean temperature of warmest quarter (bio10) and the annual precipitation (bio12). the cumulative contribution of these three factors is 86% (table 3). figure 4: the receiver operating characteristic of the predicted model. the results of the jackknife test of variables’ contribution are shown in figure 5. bio18 provided very high gains (> 1.0) when used independently, indicating that this bioclimatic variable contained more useful information in itself than the other variables did. bio10 and bio12 had moderate gain when used independently (0.8 > gain < 1). other variables including bio16 and bio4 had low gains when used in isolation; they did not contain much information by themselves. figure 5: the jackknife test for evaluating the relative importance of environmental variables for c. imicola distribution in tunisia (the regularised training gain describes show much better the maxent distribution fits the presence data compared with a uniform distribution. the dark blue bars indicate that the gain from using each variable in isolation. the light blue bars indicate the gain lost by removing the single variable from the full model, and the red bar indicates the gain using all of the variables). current suitable areas for culicoides imicola based on the major environmental variables that modulate distribution of c. imicola, suitable habitats for this species were predicted in tunisia. this is a representation of the maxent model for c. imicola. warmer colours show areas that have better predicted conditions (figure 6). figure 6: maximum entropy model for c. imicola probability suitable area presence in tunisia (white dots show the presence locations used for training). discussion studies of the regional climatic niche of c. imicola have been conducted to determine its distribution in many countries using correlative modelling methods (guichard et al. 2014). however, our study is the first to model the climatic niche of c. imicola, based on its general distribution in tunisia. we employed two complementary model analyses: enfa and madifa, to explore the relationship between the ecological niche of c. imicola and the availability of habitats (calenge & basille 2008). the enfa and the madifa statistical tools use po data (basille et al. 2008; hirzel et al. 2002). the enfa and madifa can be suitable and efficient with data for which absences are unreliable or unknown (hirzel et al. 2002). furthermore, it is not necessary to know the limiting factors of the species but just to have an idea of which parameters could influence the species establishment and maintenance. our results suggest that the ecological niche of c. imicola is very different from the available habitat. the most important predictors driving the distribution of c. imicola in tunisia were related to the precipitation of warmest quarter (bio18) (61%), followed by mean temperature of warmest quarter (bio10) and the annual precipitation (bio12). the cumulative contribution of these three factors is 86%. during the warmer months (june–september), the precipitation was the most influential factor determining the distribution of c. imicola in tunisia. field observations indicate that the population of c. imicola peaks in the september–october period and it seems that the summer rainfall has a direct impact on this population. this is in accordance with the observations from sghaier et al. (2017) who noted that c. imicola peaks in tunisia in october and that the maximum of bt cases in sheep in tunisia are recorded in october and november (oie 2018). precipitation may influence distribution through an impact on the availability of breeding sites. ducheyne et al. (2013) demonstrated that precipitation, especially summer rain-fall (june–september) was the most influential factor determining c. imicola distribution in spain. this is in agreement with the observations made by calvete et al. (2008, 2009) who noted that the coefficient of variation and the total amount of precipitation significantly influenced the presence of c. imicola (calvete et al. 2008, 2009). in relation to precipitation values, more complex considerations should be made. in fact, although precipitation values may be used as predictors for species’ presence. culicoides imicola populations peaked at the end of the rainy warm season (september–october). the fact that mean rainfall was negatively associated with c. imicola abundances could confirm that rainfall can inhibit the activity of some culicoides imicola species as shown by murray (1991). the second important predictor driving the distribution of c. imicola in tunisia is related to the mean temperature of warmest quarter (bio10). this variable is negatively correlated to species occurrence. high temperatures combined with elevated dryness (typical from certain areas of central and south of tunisia) are fatal for culicoides species. in general, temperature is known to influence greatly the development and survival of all stages of the life cycle of culicoides species (verhoef, venter & weldon 2014), driving the dynamics and distribution of many culicoides species (paweska, venter & mellor 2002). temperature not only influences flight activity but also significantly affects the development time of immature stages, vector competence and adult survival. modelling attempts often rely on a mean temperature threshold of 12.5 °c to predict presence or absence of c. imicola (peters et al. 2011; wittmann, mellor & baylis 2001) as established by purse et al. (2007). statistical analysis of our data suggested that c. imicola was significantly more abundant in coastal areas at lowers altitudes. the same observations were reported in sardinia where c. imicola and culicoides newsteadi occur more frequently in coastal areas, whereas culicoides obsoletus and culicoides pulicaris are restricted to more mountainous central areas (ramilo et al. 2012). the coastal mediterranean zone exhibited the coolest maximum temperatures during summer. the low probability of occurrence observed in central and south regions in tunisia is probably because of the very dry climate and hot temperatures registered in this region, especially in summer. the presence of permanently arable land with water sources nearby is favourable for this species development in the summer. however, high temperatures combined with elevated dryness (typical from certain areas of central and south of tunisia) are fatal for culicoides species. it is important to keep in mind that there might be other variables that cannot be captured by satellite imagery and that may have an influence on the occurrence of these species on a local scale, such as soil conditions (affecting breeding sites) and farming practices. it is assumed that crop irrigation practice is a supporting factor for c. imicola presence. high-risk areas are shown in the governorate of sidi bouzid, where most of bt cases have been reported in tunisia (oie 2018). the effect of crop irrigation practice as a risk factor of c. imicola presence in tunisia should be further investigated. culicoides imicola shows some preference for different trees and, again for drier environments, probably used for breeding. this species breeds in areas where sunny surfaces prevail together with low vegetation (ippoliti et al. 2013). culicoides imicola avoid areas covered by forest complex (conte et al. 2007; meiswinkel, venter & nevill 2004). the negative influence of some permanent crops (olive groves) in c. imicola occurrence shows that this species may have preferences when choosing the best vegetation for breeding, oviposition and for larval and pupae development, which must be further evaluated. the high abundance of c. imicola is most likely linked to its preference for breeding in areas where soil is moist and nutrient-rich and with full exposure to sunlight, characteristics, which are more commonly found in the centre and coastal areas of tunisia. this hypothesis should be further investigated. conclusion we present here the potential ecological niche for c. imicola in tunisia. the map presented here can be used to determine areas where the vector can be found. this would facilitate the implementation of control and surveillance programmes by tunisian veterinary authorities. it is important to note that the high abundance of c. imicola in some regions of tunisia underlines the real risk of spreading a new disease. thus, it is important to improve our understanding of climatic factors in c. imicola activity influencing its distribution and seasonal pattern. further studies are needed to continue the monitoring of other potential vectors in an attempt to limit the potential incursion and spread of the disease in other regions of tunisia. we expect this approach to be promising in predicting the potential distribution of other arthropod vectors and can be an effective tool to assess and visualise the risk of the establishment and spread of vbd. acknowledgements the authors would like to thank thomas balenghien for his help in sending traps via the project vectornet. we would like to acknowledge the veterinarians from region for their collaboration and procurement of c. imicola. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions b.h.t., s.h. and s.s. conceived and designed the study. b.h.t. wrote the article. b.h.t., s.h., s.s and h.h.a. reviewed the article. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. funding information this study was partially funded by the eu project ‘understanding pathogen, livestock, environment interactions involving bluetongue’ (pale-blu, h2020-sfs-2016-2) and the blue-med project ‘a novel integrated and sustainable approach to monitor and control bluetongue spread in the mediterranean basin’. this work has been written in the framework of the project prima: bluemed, funded by the ministry of higher education and scientific research, tunisia. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references baasch, d.m., tyre, a.j., millspaugh, j.j., 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technique oie 20(3), 731–736. https://doi.org/10.20506/rst.20.3.1306 kalemaz_145-151.qxd introduction bovine tuberculosis (btb) caused by mycobacterium bovis in african buffaloes (syncerus caffer) in the queen elizabeth national park (qenp) was first detected in the early 1960s (thurlbeck, butas, mankiewicz & laws 1965). the first tuberculosis survey was performed in the qenp buffaloes in 1967. in this survey btb was found in 38 % of 64 buffaloes selected because of poor body condition and 10 % of 52 buffaloes that were randomly selected (woodford 1982a). in this study it was concluded that btb in the buffaloes had originated from cattle, because this disease had been documented in cattle close to or bordering the park and infected buffaloes were only found in those sectors with a buffalo/cattle interface. the maramagambo forest was thought to have been an adequate barrier to prevent the spread of btb into sector a of the park, which is without a buffalo/cattle interface (woodford 1982a). it was also concluded that btb had spilled over from the buffaloes into warthogs since 9 % of a sample of 90 warthogs in sectors d and e were found to be positive (woodford 1982b). bovine tuberculosis in the 145 onderstepoort journal of veterinary research, 72:145–151 (2005) a preliminary investigation of tuberculosis and other diseases in african buffalo (syncerus caffer) in queen elizabeth national park, uganda g. kalema-zikusoka1, r.g. bengis2, a.l. michel3 and m.h. woodford4 abstract kalema-zikusoka, g., bengis, r.g., michel, a.l. & woodford, m.h. 2005. a survey of tuberculosis and other diseases in african buffalo (syncerus caffer) in queen elizabeth national park, uganda. onderstepoort journal of veterinary research, 72:145–151 a survey to determine the prevalence of bovine tuberculosis caused by mycobacterium bovis and certain other infectious diseases was conducted on 42 free-ranging african buffaloes, (syncerus caffer) from may to june 1997 in the queen elizabeth national park, uganda. using the gamma interferon test, exposure to m. bovis was detected in 21.6 % of the buffaloes. one dead buffalo and an emaciated warthog (phacochoerus aethiopicus) that was euthanased, were necropsied; both had miliary granulomas from which m. bovis was isolated. none of the buffaloes sampled in sector a of the park, which has no cattle interface, tested positive for bovine tuberculosis (btb) exposure. the prevalence and distribution of btb does not appear to have changed significantly since the 1960s, but this may be due to fluxes in the buffalo population. serological testing for foot-and-mouth disease (fmd) demonstrated positive exposure of 57.1 % of the buffaloes sampled, with types a, o and sat 1–3, which is the first known report of fmd antibodies to a and o types in free ranging african buffaloes. foot-and-mouth disease virus types sat 1 and sat 3 were isolated from buffalo probang samples. two percent of the buffaloes had been exposed to brucellosis. none of the buffaloes tested had antibodies to rinderpest, leptospirosis or q fever. keywords: african buffalo, bovine tuberculosis, foot-and-mouth disease, syncerus caffer 1 conservation through public health, plot 39, babiiha avenue, p.o. box 10950, kampala, uganda 2 directorate of animal health, p.o. box 12, skukuza, 1350 south africa 3 onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 4 iucn veterinary specialist group accepted for publication 31 march 2005—editor kruger national park (knp) in south africa is also believed to have originated from cattle (de vos, bengis, kriek, michel, keet, raath & huchzermeyer 2001; bengis, kock & fischer 2002) and the disease in buffaloes has subsequently been reported to have spilled over into lions, leopards, kudus, baboons and other species (keet, kriek, penrith, michel & huchzermeyer 1996; bengis 1999; keet, kriek, bengis, grobler & michel 2000; keet, kriek, bengis & michel 2001). in the 1970s and 1980s, a 76.7 % reduction of the qenp buffalo population occurred as a result of severe poaching during the civil wars (eltringham & woodford 1973; anon. 1996). this study was initiated to determine whether the reduction in buffalo numbers in the 1970s and 1980s had any effect on the btb prevalence reported in the earlier study, which took place in the late 1960s (woodford 1982a). in the present study in the buffaloes, foot-and-mouth disease (fmd), rinderpest, brucellosis, leptospirosis and q fever, which are important infectious diseases of livestock, were also investigated. materials and methods study site queen elizabeth national park is a protected area bordering lakes george and edward and contains the kazinga channel that connects the two lakes (fig. 1). the park is unfenced and surrounding reserves have village enclaves in which there are cattle. the northern and southern parts are separated by the maramagambo forest of 320 km2, which appears to form a barrier for migration of buffaloes between the northern and southern sectors of the park (woodford 1982a). the northern part is separated further into sectors b, c, d, e and f (fig. 2). sector a is separated from sector b by the incomplete barrier of the maramagambo forest, sector b is separated from sector c by the complete barrier of the kazinga channel, sector c is separated from sector d incompletely by the settlement at katwe village and by the crater area. immobilization techniques and sampling locations the buffaloes were darted from a four-wheel drive vehicle using a daninject dart gun firing 1.5 ml daninject darts. the anesthetic cocktail contained 7– 8 mg etorphine (kyron, south africa) plus 60–70 mg xylazine (kyron, south africa). after sampling, the sedative effects of the cocktail on the buffaloes were reversed by the intravenous administration of 14– 18 mg diprenorphine (kyron, south africa) and 60– 70 mg yohimbine (kyron, south africa), respectively. forty-two buffaloes were sampled from 18 different breeding herds and several bull groups. sampling was randomized in that the first animal in the herd that offered a clear shot was darted, two to three animals were sampled in each herd. there was some bias towards older animals since calves and yearlings were rarely sampled. sampled buffalo herds were spatially distributed north and south of the kazinga channel in sectors d and b, respectively, and also from sector a (ishasha) south of the maramagambo forest. samples blood and oesophageal-pharyngeal probang samples were collected from 42 buffaloes. the blood was collected from the jugular vein or tail vein in heparinized and clot tubes. the probang samples were placed in sterile phosphate buffered saline (pbs) and stored in liquid nitrogen until further analysis at the onderstepoort veterinary institute (ovi), south africa. blood in the heparinized tubes was divided into three 1.5 ml aliquots and each aliquot was incubated within 8 h of collection in three different reagents. the first aliquot was incubated in 30 µl of bovine tuberculin purified protein derivative (ppd) (id-lelystad, the netherlands), the second aliquot in 60 µl of avian tuberculin ppd (id-lelystad, the netherlands) and the third aliquot in a control of 30 µl of phosphate buffered saline (pbs) (onderstepoort veterinary institute, south africa) for 20– 28 h. after incubation, plasma was collected in cryovails and stored in liquid nitrogen until further analysis at the ovi tuberculosis laboratory in south africa. gamma interferon (ifn) assay for tuberculosis the commercially available bovigam assay (csl, australia) was used according to the manufacturer’s instructions for the execution of this test. a sandwich enzyme-linked immunosorbent assay (elisa) was performed as described by rothel, jones, corner, cox & wood (1990). unbound material was removed by washing after incubation. horseradish peroxidase labeled anti-bovine ifn gamma was added. unreacted conjugate was removed by washing after incubation. an enzyme substrate was added. the rate of conversion of this enzyme substrate was proportional to the amount of bound inter146 tuberculosis and other diseases in african buffalo (syncerus caffer) in uganda feron (ifn)-gamma. the amount of colour development was estimated spectrophotometrically after termination of the reaction. test for foot-and-mouth disease the oesophageal-pharyngeal probang sample from each buffalo was prepared for the isolation of fmd virus by grinding it in a mortar with a pestle and adding sufficient phosphate buffered saline to make a 10 % tissue suspension. this was inoculated in 1 ml amounts into ten tubes containing cultures of primary porcine kidney cells, which were then incubated at 37 °c for 48 h. cultures were observed daily for cytopathic effects. cultures not showing cytopathic effects after 48 h were incubated for a further 48 h. supernatants from affected cultures were used to type the viral isolate by antigen capture elisa using the method described by roeder & le blanc smith (1987). 147 g. kalema-zikusoka et al. ����� ���� � ���� ������ �� � �� ������������ ��� ���������� ��������� ����� ����� �� ��� ��!� ���� ��"#"# ��� ���$��������� ������� ����$ �����!���� �����# #��#��# ��� ��� ��!������ "��� �%� �������� � ����� � ������ ���� ������� & '&&%� � � ��(����� ������� ���� ���������! ��� ��)�# ��� ���������� �������� ��������� � � �� ���� � � *#� ������� � fig. 1 location plan serology a competitive antibody elisa test and a liquid phase blocking elisa test were used to test for exposure to rinderpest (anderson, barrett & scott 1997) and fmd (hamblin, barnett & hedger 1986), respectively. an indirect immunofluoresence assay (ifa) (to, htwe, kako, kim, yamaguchi, fukushi & hirai 1998) was used to detect antibodies to coxiella burnetii (q fever). serum agglutination (adesiyun & cazabon 1996) and complement fixation tests (jones, hendricks & berman 1963) were used to detect antibodies to brucella abortus. microscopic agglutination tests (mat) and serum neutralization tests as described by leighton & kuiken (2001) were used to determine exposure to leptospira serotypes. postmortem examinations permission was also obtained to euthanase three emaciated animals for study. a female warthog, one buffalo cow and one male baboon were euthanased using suxamethonium chloride (scoline, kyron, 148 tuberculosis and other diseases in african buffalo (syncerus caffer) in uganda # + + � , � � � ������ �� -../0 ,������ � � �� ������ � � ����� � -..10 ������� ���� ������������ �!������ ��%�!������ ����� ���� " ��� �� ����� ���������!� ������ � ��( ��� �� �� �� �� � '2/ 23 0 #���� �� #� �� �� � �� � � fig. 2 queen elizabeth national park, study sectors south africa). a dead buffalo cow found intact, which was estimated to have been dead for less than 12 h, was also necropsied. samples of lesions in these animals were collected and frozen for mycobacterial culture as described previously (bengis, kriek, keet, raath, de vos & huchzermeyer 1996) and additional samples were placed in 10 % formaldehyde for histopathological examination at the ovi. results all the darted buffaloes were in good bodily condition with no overt clinical signs of btb. no ungulates with overt clinical signs suggestive of rinderpest were observed in the park during this study. buffalo sampling was restricted to sectors a, b and d because buffalo herds living in the vicinity of human communities in sectors c and e were difficult to approach within darting range as a result of poaching, which occurs on a significant scale. eight (21.6 %) out of 38 buffaloes were positive reactors to m. bovis on the gamma ifn test, which took into consideration m. avium and the positive control. four of the samples were not interpretable. the dead buffalo and the warthog that was euthanased had diffuse miliary granulomas present in many lymph nodes, liver and lungs from which m. bovis was isolated. the emaciated buffalo was not suffering from tuberculosis and the loss of bodily condition was attributable to severe dental attrition. the emaciated baboon that was euthanased and necropsied had granulomatous lung lesions, but m. bovis was not isolated from them. histopathologically, the lung lesions were shown to be caused by a trematode, probably a paragonimus sp. (georgi 1985). the prevalence of fmd antibody was 57.14 %. positive animals had antibodies to one or more of the viral types. of these positive buffaloes, 83.3 % had antibodies to sat 1, 29.2 % to sat 2, 33.3 % to sat 3, 20.8 % to type a and 58.3 % to type o. a sat 1 type virus was isolated from two probang samples, and sat 3 type virus from another. one of the 42 buffaloes reacted positive in the serum agglutination and complement fixation tests for b. abortus. a second buffalo was a weak positive reactor in these two tests and was considered suspect. the remaining 40 animals tested negative for brucellosis. all the buffaloes tested were serologically negative for rinderpest, q fever and leptospira serotypes. discussion when comparing the results of the current btb survey with the survey conducted in the 1960s, it is important to note that the results of the earlier survey were based on lesions found at post-mortem examinations and histopathology with culture of a certain percentage of samples (woodford 1982a). mycobacterium bovis was responsible for 12 out of 14 infections typed from buffaloes and for two out of six typed from warthogs. atypical mycobacteria were responsible for the balance of infections typed and was thought to be from fish, frogs or birds (woodford 1982a, 1982b). therefore, the 1960 study results are rough approximations of the actual btb prevalence. recent studies, however, have found an excellent correlation between necropsy and culture findings of culled buffaloes and the blood-based gamma ifn test used in this survey (grobler, michel, de klerk & bengis 2002). in both buffalo surveys conducted in the 1960s and this current one mainly mature buffaloes were sampled to increase the likelihood of detecting infected animals (bengis 1999, de vos et al. 2001). the current survey demonstrated that the btb prevalence in qenp buffaloes has not changed significantly over 30 years, and that spillover infection from buffaloes into warthogs is still occurring. however, this study did not investigate the possibility of warthogs being maintenance hosts of btb. one of the reasons why the tb prevalence may not have changed significantly over 30 years is that extensive poaching of buffaloes and other large mammals, including elephants, occurred during the period of ugandan civil wars in the 1970s and 1980s. the buffalo numbers decreased by 76.7 %, i.e. from 18 000 to 4 200 (eltringham & woodford 1973, anonymous 1996). if it is assumed that btb transmission is density dependent, then this population decrease may have reduced the rate of spatial spread of the disease within and between the buffalo herds. the population has now grown to over 10 000 animals (richard lamprey, personal communication 2002) and, although the prevalence appears to have remained the same, it may have been tracking the population densities, and is now increasing progressively, as has been reported from the knp (de vos et al. 2001). the african buffalo, being a wild bovid with similar habits to cattle, as well as filling a similar ecological niche, may be a good indicator species for monitoring certain cattle associated diseases such as btb and rinderpest. cattle, in turn, may be considered sentinels for detecting certain afro-endemic dis149 g. kalema-zikusoka et al. eases in buffaloes. cattle still graze illegally in the park and use water holes in the interior and the periphery of qenp, creating a diffuse interface for them to mix with buffaloes, with the potential of bidirectional transmission of infectious diseases. bovine tuberculosis is a chronic erosive disease, but because it does not have a high clinical profile in cattle, it is not considered to be as important in uganda as the epidemic viral diseases of ruminant cattle such as fmd and rinderpest which may cause severe morbidity and mortality in cattle (bengis et al. 2002). in africa, buffaloes are known maintenance hosts of fmd viral types sat 1, sat 2 and sat 3 (hedger 1972; sutmoller, thomson, hargreaves, foggin & anderson 2000). the results of this survey show that the qenp buffaloes are no exception regarding these types. however, the finding of antibodies to types a and o was surprising, as this has not been previously recorded in free-ranging african buffalo populations (thomson, vosloo & bastos 2003). the buffalo pharyngeo-oesophageal probang samples, however, only yielded sat 1 and sat 3 types. there remains a possibility that some of the qenp buffaloes sampled had cross-reactions to the fmd a and o types (vosloo, personal communication 2003). genome sequencing studies on these buffalo isolates demonstrated that the uganda sat 1 buffalo strain from this survey was distinct from sat 1 strains of buffaloes in zambia, malawi, namibia, zimbabwe, botswana, swaziland, knp and other parts of south africa (thomson et al. 2003). cattle in the village enclaves around qenp suffer from sporadic fmd outbreaks, almost on a yearly basis (joram tumushabe, personal communication 2002), causing morbidity, and in some instances, mortality. molecular epidemiological studies to characterize the strains infecting the cattle and to compare them with buffalo strains would help to identify fmd vaccine candidates, which could be used to protect these cattle. cattle and buffalo isolates may possibly even one day yield one of the afro-alien types (a, o, c and asia 1). the low prevalence of brucellosis reactors in the qenp buffaloes could indicate that they are not maintaining the disease, or possibly have limited exposure from a low prevalence cattle source. however, it has been reported that if brucellosis cannot be maintained in bovidae populations, another brucella species other than brucella abortus may be involved, emphasizing the importance of brucellosis bacteriology and ruling out contact with other brucella spp. infected domestic livestock. the prevalence of brucellosis in the cattle at the interface needs to be examined further, because, as with btb, brucellosis is also of public health significance and humans can get infected through consumption of raw milk. the low prevalence of brucellosis reactors could also be attributed to the lack of sensitivity of the serum agglutination and complement fixation tests (cft) employed in the survey. when the tests were performed in 1997, the cft was the most sensitive test and recommended as the confirmatory test for brucellosis. however, a more recent indirect elisa test appears to be more sensitive than the cft (paweska et al. 2002) and would be good to use for future surveys. the absence of antibodies to rinderpest in the qenp buffaloes sampled is in agreement with previous findings. rinderpest has not been reported from this part of uganda since the 1930s (plowright, laws & rampton 1964). there are also no published reports to date of outbreaks of leptospirosis or q fever in livestock or humans, even though the causative organisms are rather ubiquitous in nature. unfortunately, buffaloes at the periphery of the park that were more likely to mix with cattle could not be sampled in this survey. this area has been subject to severe poaching and the buffaloes were consequently nervous and difficult to approach with a vehicle. follow-up studies are necessary to investigate the prevalence of significant ruminant diseases at this buffalo/livestock interface, and to compare results with cattle that live further away from the park in order to determine the direction of disease transmission between these two closely related species. education of cattle keepers and park staff on the risks of disease transmission between buffaloes and livestock could potentially reduce the historical resentment of cattle keepers to not being allowed to legally graze their animals in this park since it was gazetted in 1952. acknowledgements we thank the uganda wildlife authority and the south african national directorate of animal health for permission and logistical assistance to carry out this study, the food and agricultural organization, care for the wild, makerere university faculty of veterinary medicine (muk/fvm) and uganda government veterinary services for funds and logistical assistance, dr eric edroma (uwa executive director), dr ludwig siefert (senior lecturer from muk/ fvm), abdullah latiff (chief park warden of qenp), marcello onen, tom friday and joseph machati 150 tuberculosis and other diseases in african buffalo (syncerus caffer) in uganda (field assistants and ranger from qenp) for logistical and technical assistance, and dr suzanne kennedy-stoskopf from north carolina state university for editorial assistance and comments on the manuscript. references adesiyun, a.a. & cazabon, e.p. 1996. seroprevalences of brucellosis, q fever and toxoplasmosis in slaughter livestock in trinidad. revue d’élevage et de médecine vétérinaire des pays tropicaux, 49:28–30. anderson, j., barrett, t. & scott, g.r. 1997. manual on the diagnosis of rinderpest, 3rd ed. rome: fao. anonymous 1996. a survey of the wildlife protected areas of uganda, phase i: september 1995 – january 1996. report to the uganda government by the ministry of tourism, wildlife and antiquities. bengis, r.g., kriek, n.p.j., keet, d.f., raath, j.p., de vos, v. & huchzermeyer, h. 1996. an outbreak of bovine tuberculosis in a free-living african buffalo (syncerus caffer—sparrman) population in the kruger national park: a preliminary report. onderstepoort journal of veterinary research, 63:15–18. bengis, r.g. 1999. tuberculosis in free-ranging mammals, in zoo and wild animal medicine, current therapy 4, edited by m.e. fowler. philadelphia, pennsylvania: w. b. saunders company. bengis, r.g., kock, r.a. & fischer, j. 2002. infectious animal diseases: the wildlife/livestock interface. revue scientific et technique, 21:53–65. de vos, v., bengis, r.g., kriek, n.p.j., michel, a., keet, d.f., raath, j.p. & huchzermeyer, h.f. 2001. the epidemiology of tuberculosis in free-ranging african buffalo (syncerus caffer) in the kruger national park, south africa. onderstepoort journal of veterinary research, 68:119–130. eltringham, s. & woodford, m.h. 1973. the numbers and distribution of buffalo in the rwenzori national park, uganda. east african wildlife journal, 11:151–164. georgi, j.r. 1985. parasitology for veterinarians. philadelphia, pennsylvania: w. b. saunders company. grobler, d.g., michel, a.l., de klerk, l. & bengis, r.g. 2002. the gamma interferon test: its usefulness in a bovine tuberculosis survey in african buffaloes (syncerus caffer) in the kruger national park. onderstepoort journal of veterinary research, 69:221–227. hamblin, c., barnett, i.t.r. & hedger, r.s. 1986. a new enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against foot-and-mouth disease virus. journal of immunological methods, 93:115–121. hedger, r.s. 1972. foot-and-mouth disease and the african buffalo (syncerus caffer). journal of comparative pathology, 82:19–28. jones, l.m., hendricks, j.r. & berman, d.t. 1963. the standardization and use of the complement-fixation test for the diagnosis of bovine brucellosis with a review of the literature. american journal of veterinary research, 24:1143– 1151. keet, d.f., kriek, n.p.j., penrith, m.l., michel, a. & huchzermeyer, h. 1996. tuberculosis in buffaloes (syncerus caffer) in the kruger national park: spread of the disease to other species. onderstepoort journal of veterinary research, 63:239–244. keet, d.f., kriek, n.p.j., bengis, r.g., grobler, d.g. & michel, a. 2000. the rise and fall of tuberculosis in a freeranging chacma baboon troop in the kruger national park. onderstepoort journal of veterinary research, 67:115–122. keet, d.f., kriek, n.p.j., bengis, r.g. & michel, a.l. 2001. tuberculosis in kudus (tragelaphus strepsiceros) in the kruger national park. onderstepoort journal of veterinary research, 68:225–230. leighton, f.a. & kuiken, t. 2001. bacterial infections, leptospirosis, in infectious diseases of wild mammals, 3rd ed., edited by e.s. williams & i.k. barker. ames, iowa: iowa state university press. plowright, w., laws, r.m. & rampton, c.s. 1964. serological evidence for the susceptibility of the hippopotamus (hippopotamus amphibius linnaeus) to natural infection with rinderpest virus. journal of hygiene (london), 62: 329–336. roeder, p.l. & le blanc smith, p.m. 1987. detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay, a sensitive, rapid and reliable technique for primary diagnosis. research in veterinary science, 43:225–232. rothel, j.s., jones, s.l., corner, l.a., cox, j.c. & wood, p.r. 1990. a sandwich enzyme immunoassay for bovine interferon gamma and its use for the detection of tuberculosis in cattle. australian veterinary journal, 67:134– 137. sutmoller, p., thomson, g.r., hargreaves, s.k., foggin, c.m. & anderson, e.c. 2000. the foot-andmouth disease risk posed by african buffalo within wildlife conservancies to the cattle industry of zimbabwe. preventive veterinary medicine, 44:43–60. to, h., htwe, k.k., kako, n., kim, h.j., yamaguchi, t., fukushi, h. & hirai, k. 1998. prevalence of coxiella burnetti in dairy cattle with reproductive disorders. journal of veterinary medical science, 60:859–861. thomson, g.r., vosloo, w. & bastos, a.d.s. 2003. footand-mouth disease in wildlife. virus research, 91:145–161. thurlbeck, w.m., butas, c.a., mankiewicz, e.m. & laws, r.m. 1965. chronic pulmonary disease in wild buffalo (syncerus caffer) in uganda. american review of respiratory diseases, 92:801–805. woodford, m.h. 1982a. tuberculosis in wildlife in the ruwenzori national park, uganda (part i). tropical animal health and production, 14:81–88. woodford, m.h. 1982b. tuberculosis in wildlife in the ruwenzori national park, uganda (part ii). tropical animal health and production, 14:155–160. 151 g. kalema-zikusoka et al. nganga_219-226.qxd introduction gastrointestinal helminth parasitism is one of the major animal health problems facing ruminant livestock production in kenya with losses that may be high owing to both clinical and chronic sub-clinical infections (allonby & urquhart 1975; carles 1993; githigia, thamsborg, munyua & maingi 2001; nginyi, duncan, mellor, stear, wanyangu, bain & gatongi 2001). for the necessary rational and sustainable control of these helminths, comprehensive knowledge of the epidemiology of the parasites in relation to specific climatic, management and production environments is crucial (barger 1999). since the various regions of kenya differ widely as regards, breeds of sheep and production systems, studies carried out in one area may not apply to another. most previous studies in this country were on the woolled breeds of sheep in the high rainfall areas in the rift valley (mbaria, mcdermott, kyule, 219 onderstepoort journal of veterinary research, 71:219–226 (2004) epidemiology of gastrointestinal helminths infections in dorper sheep in a semi-arid area of kenya c.j. ng’ang’a*, n. maingi, w.k. munyua and p.w.n. kanyari department of veterinary pathology, microbiology and parasitology, faculty of veterinary medicine university of nairobi, p.o. box 29053, 00625 nairobi, kenya abstract ng’ang’a, c.j., maingi, n., munyua, w.k. & kanyari, p.w.n. 2004. epidemiology of gastrointestinal helminths infections in dorper sheep in a semi-arid area of kenya. onderstepoort journal of veterinary research, 71:219–226 a survey on the prevalence and intensity of infection with gastrointestinal helminths of dorper sheep in relation to age and weather factors was carried out on a ranch in kajiado district, a semi-arid area of kenya for a period of 13 months (may 1999 to may 2000). faecal samples from lambs (3 months to 1 year), yearlings (1–2 years) and adult breeding ewes (2–4 years) were examined for helminth egg output and helminth genus composition at 3-week intervals. the results indicated that the prevalence of strongyle and tapeworms infections were highest for lambs, followed by the adult breeding ewes and then for the yearlings. in all age groups the proportions of infected animals were higher during the wet season than in the dry season for both nematodes and tapeworms. the mean strongyle egg counts were higher during the dry season for lambs, but were higher during the wet season for the other age groups. mixed strongyle infections were detected, with trichostrongylus (55 %), haemonchus (28 %), cooperia (10.5 %) and oesophagostomum (6.5 %) being the most frequently encountered genera throughout the study period. the trends in strongyle faecal egg counts indicated the occurrence of hypobiosis, with resumption of development towards the end of the dry season and at the onset of the short rains in october and november. self-cure was also observed in september and november in all age groups, although less frequently in lactating ewes. the prevalence and intensities of infection with gastrointestinal helminths in this area appeared to be influenced by the age of the host and weather factors. keywords: dorper sheep, gastrointestinal helminths, hypobiosis, self-cure, semi-arid region * author to whom correspondence is to be directed. e-mail: danida@alphanet.co.ke or vetpath@uonbi.ac.ke accepted for publication 9 february 2004—editor gichanga & manyole 1995) and in the central highlands (maingi, gichohi, munyua, gathuma & thamsborg 1997; nginyi et al. 2001). extensive studies in the semi-arid areas have only been carried out on red maasai and woolled breeds in naivasha (allonby & urquhart 1975; preston & allonby 1979a, b; gatongi, prichard, ranjan, gathuma, munyua, cheruyot & scott 1998). the objective of the present study was therefore to investigate the prevalence and intensity of infection with gastrointestinal nematodes of dorper sheep in relation to age of host and weather factors in kajiado district in a semi-arid area of kenya. materials and methods study area the study was carried out at the maasai rural training centre ranch in isinya division of kajiado district. the area lies at an altitude of approximately 1 500 m to1 850 m above sea level and receives an average annual rainfall of about 600 mm. the rainfall is bimodal, with long rains falling between march and may and short rains from october to december. the mean annual temperatures ranges from 18–20 °c with a mean minimum of 12–14 °c and a mean maximum of 24–26 °c, with little variation between seasons. experimental animals and treatments the study was carried out between may 1999 and may 2000 with sheep of three age groups, namely lambs (3 months to 1 year), yearlings (1–2 years) and adult breeding ewes (2–4 years). each group consisted of 20 female sheep randomly selected from the flock on the ranch. on 5th may 1999, all the animals were weighed, ear tagged and given a single dose of albendazole (valbazen®, novartis east africa ltd, nairobi) at a dosage rate of 5 mg/kg body mass. no further anthelmintic treatments were given except for salvage treatments, which were administered to animals showing clinical signs of helminthosis and those with over 7 000 eggs per gram of faeces. the ewes lambed between october and november 1999 and the lambs were weaned in march 2000. in january 2000 new lambs were recruited at the age of 3 months and the previous ones moved to the group of yearlings. faecal sampling and processing faecal samples were collected at 3-weekly intervals from the rectum of individual animals and stored at 4 °c until examined. nematode egg counts per gram of faeces (epg) were done and the presence of tapeworm eggs noted for each sample, using a modified mcmaster technique (whitlock 1948) at a sensitivity of 100 eggs, and saturated sodium chloride as the floatation solution. a sedimentation technique was used to detect fasciola eggs and a modified bearmann method to search for lungworm larvae (hansen & perry 1994). faecal culture on each sampling occasion, faecal samples for all age groups were pooled, incubated for 10 days at 27 °c, nematode larvae recovered and at least 100 identified per sample based on morphology and size as described by reinecke (1983) and in the maff (1986) manual. pastures sampling and recovery of infective larvae herbage samples were collected at 3-weekly intervals around the paddocks where the sheep were grazing. the w-transect and the infectivity levels determined as described by hansen & perry (1994) was used. infectivity levels were expressed as the number of larvae (l3) per kg dry herbage. the pastures mainly consisted of themeda triandra, pennisetum meziannim and setaria spp., and had been grazed by sheep for the last 5 years. statistical analysis strongyle egg counts and herbage larval counts were logarithmically transformed [log (x + 10)] to normalize their distribution, and analysis of variance (anova) performed in a microsoft excel program. comparisons were made between age groups (lambs, yearlings and breeding ewes) and seasons (wet and dry). to exclude the effects of treatment, all the data for may 1999 and data from all animals given salvage treatment was excluded from the analysis. results rainfall distribution and temperatures the long-term monthly mean rainfall distribution pattern for the study area, the monthly total rainfall and the number of wet days recorded per month between may 1999 and may 2000 are shown in fig. 1. although the total of 292 mm of rainfall recorded was less than half of the long-term mean of 658 mm, 220 gastrointestinal helminths infections in dorper sheep in kenya that during the short rainy season from october to december 1999 (228 mm) was higher than the longterm mean (176 mm), in contrast to the long rainy season from march to may 2000 when only 18 % of the long term mean fell (52 mm vs 285 mm). in fig. 2 the mean monthly long-term minimum and maximum air temperature for the study area and the minimum and maximum air temperatures recorded 221 c.j. ng’ang’a et al. ������ � � � � ��� � � � � �� �� �� �� ��� ��� ��� ��� � � � � � �� �� �� �� � � � � �� �� �� � � �� � �� � �� �� � ��� ��� ��� ��� ��� �� ��� ��� ��� �� ��������� ����! ���� ��"�#� $�%�%&�%%�' ����! ���� ��"�#� �� �%%% �� �� ���� ������ �# ��"� (� ) �� �%%% �� �� ���� fig. 1 long-term monthly rainfall (1969–1998), the total monthly rainfall and the number of rainy days per month (may 1999 to may 2000) for isinya division in kajiado district � * �� �* �� �* +� +* �� �� � ��� ��� ��� ��� ��� �� ��� ��� ��� �� ������ � � � � � �� �� �� � �� � ��������� ���� �"�, ����, ��������� ���� ��-, ����, �"�, ����, �� �%%% �� �� ���� ��-, ����, �� �%%% �� �� ���� fig. 2 long-term mean minimum and maximum monthly temperatures (1969–1998) and the minimum and maximum monthly temperatures (may 1999 to may 2000) for isinya division in kajiado district from may 1999 to may 2000 are graphically illustrated. faecal egg counts no fasciola eggs or lungworm larvae were detected over the study period. the arithmetic mean epg for all the three age groups of sheep were nil posttreatment in may 1999. three major peaks developed one from july to august 1999, one from october to november 1999 and another from january to april 2000. between the peaks, two major troughs in epg occurred, in september and in november to december 1999. the mean epg over the entire sampling period were highest for the lambs (geometric mean 762) followed by the adult ewes (472) and lowest for the yearlings (394). the mean counts were higher during the dry season (june to september 1999 and january to february 2000) for lambs, but higher during the wet season (november to december 1999 and march to may 2000) for the other age groups. the proportions of faecal samples from lambs, yearlings and adult breeding ewes that were found positive for strongyle and tapeworm eggs during the wet and the dry seasons are shown in table 1. the majority of animals in all three age groups (85– 92 %) were shedding strongyle eggs during both the dry and the wet seasons. the prevalence of strongyle and tapeworm eggs was however, higher during the wet seasons for all age groups of sheep. the prevalence for both strongyle and tapeworms was highest for the lambs, followed by the adult ewes and was lowest for the yearlings. differential larval counts trichostrongylus, haemonchus, cooperia and oesophagostomum were recovered in that order of 222 gastrointestinal helminths infections in dorper sheep in kenya table 1 the proportion of faecal samples for dorper lambs, yearlings and breeding ewes that were found to be positive for strongyle and tapeworm eggs in kajiado district of kenya during the period may 1999 to may 2000 percentage of positive samples seasons parasite eggs lambs yearlings adult breeding ewes dry strongyle 90.3 85.9 84.6 tapeworms 48.4 21.2 25.4 wet strongyle 92.3 89.7 92.3 tapeworms 65.2 35.6 40.2 table 2 the mean distribution of genera of gastrointestinal nematodes in faecal cultures from dorper sheep during the wet and the dry season season nematode genera dry (%) wet (%) trichostrongylus 61 49 haemonchus 24 32 cooperia 12 9 oesophagostomum 3 9 strongyloides 0 1 table 3 the number of dorper lambs, yearling and adults given salvage treatment based on clinical manifestation of nematodosis and egg counts (over 7 000 epg) between may 1999 and may 2000 treatment basis lambs yearlings adults trichostrongylosis (july to september) 5 2 1 others 1 0 0 haemonchosis (july to september) 2 2 1 others 1 1 1 over 7 000 epg 3 0 2 223 c.j. ng’ang’a et al. .� / �������"�� �# ! ���"��"� ����� 0�� / 0�������� �� / �� #����� 1 / 1���"�� � / ����"�� )��)������) 2��� "��) 3���("�� �4�) � � � �*� *�� 5*� � ��� � �*� � *�� � 5*� � ��� � �*� � *�� � 5*� + ��� + �*� + *�� �� �� .� 0�� ������ 6 %% 6 �� �6 ���6 �� �6 ���6 ���6 %% 6 %% �6 %% �6 %% �6 %% �6 %%�6 %% 1 � � � � �� �� � � �� �� � � � � �� �� � � � � � � � � � � � � � � � � � � � fig. 3 arithmetic mean strongyle eggs per gram (epg) of faeces for dorper lambs, yearlings and adult breeding ewes recorded every 3 weeks in a semi-arid area of kajiado district during the period may 1999 to may 2000 � �*� *�� 5*� � ��� � �*� � *�� � 5*� � ��� � �*� � *�� � 5*� ������ � � �� � � � �� � � � �� ! � � � �� 6 %% 6 �� �6 ���6 �� �6 ���6 ���6 %% 6 %% �6 %% �6 %% �6 %% �6 %%�6 %% � � � � � � � � � � � � � � � � � � fig. 4 number of strongyle larvae per kg of dry herbage recovered every 3 weeks from pastures grazed by dorper sheep in a semi-arid area of kajiado district during the period may 1999 to may 2000 occurrence throughout the year (table 2). strongyloides was occasionally encountered during the wet season. there was a sudden increase in the proportion of haemonchus from 18 % in september 1999 to 45 % in october 1999 and a decrease in trichostrongylus spp. from 76 % to 40 % over the same period. herbage larval counts larval counts rose during the wet seasons, closely following the peaks in rainfall in december 1999 and april 2000, with no l3 being detectable during the dry season from july 1999 to october 1999 (fig. 4). salvage treatments during the study period, 12 lambs, five yearlings and five adult breeding ewes received salvage treatment based on faecal egg counts and clinical signs of helminthosis which were mostly diarrhoea, unthriftiness and bottle jaw. in all age groups, most clinical cases of helminthosis and salvage treatments occurred between july and september during the dry season, where 87 % were as a result of trichostrongylosis. during the wet season most treatments were made in october and november, where 62 % were as a result of haemonchosis (table 3). discussion the results of the present study confirmed that age and physiological status of the host influence the susceptibility to helminth infections. the prevalence of infection, the strongyle egg counts and the number of animals that received salvage treatments were highest in the lambs and lowest in the yearlings, probably owing to immunological hypo-responsiveness in lambs (watson & gill 1991; colditz, watson, gray & wilson 1996), compared to developing immunity as the animals age. on the other hand, acquired immunity to nematode infection tends to be lost in late pregnancy and in lactation (barger 1993). most of the adult ewes used in this study were pregnant between june and october 1999 and lactating between october 1999 and march 2000. the peak in strongyle faecal egg output observed from july to august 1999 probably commenced from residual larval population on the pastures (fig. 4). however, relatively low numbers of l3 were available on the pastures at the time, and it is possible that maturation of hypobiotic haemonchus contortus may have played a role, as suggested also by gatongi et al. (1998 in sheep and goats in the semiarid area of naivasha and by where they were accompanied by gatongi et al. (2001) in goats in the marginal low potential areas of thika district. the sudden increase in the proportions of haemonchus in faecal cultures in october and november 1999 further suggests that hypobiosis played a role. hypobiosis is a state of arrested development during adverse conditions, with resumption of development when field conditions improve (gibbs 1968). in the tropics, hypobiosis usually occurs during the dry season and the resumption of development occurs towards the end of the dry season or at the onset of the rains (ogunsusi 1979; jacquiet, colas, cabaret, dia, cheikh & thiam 1995; gatongi et al. 1998), much as in the present study and that of githigia et al. (2001) in goats. in contrast, gatongi et al. (1998) observed the resumption of development in sheep and goats in a semi-arid area of naivasha targeted towards the long rains. troughs in faecal egg counts in september and november/december 1999 were considered to be the result of self-cure. similar observations were made in a semi-arid area of naivasha in merino and red maasai breeds (preston & allonby 1979b). in grazing animals, the self-cure is commonly observed after rains when the intake of infective larvae provides the stimulus for resident worms to be eliminated and tends to occur in nearly all the sheep in the flock (allonby & urquhart 1973). the phenomenon may also occur in sheep on lush pastures in the absence of re-infection. this may be as a result of an “anthelmintic substance” or an allergic substance in freshly growing grass or owing to physiological alterations in the abomasum (allonby & urquhart 1973). the spectrum of the self-cure may range from merely the temporary suppression of egg-laying to complete expulsion of the adult worm burden (allonby & urquhart 1973). in the present study, the self-cure that occurred in november to december may be attributed to reinfection by larvae and or the consumption of lush pastures that resulted from the short rains. however, none of these factors could directly be associated with the phenomenon in september as this was a dry month and not conducive for pasture growth and development of the infective larvae. similar observations were made in naivasha where the red maasai sheep self-cured during the dry season in the absence of fresh pastures (preston & allonby 1979a). the self-cure observed at this time might therefore have resulted from of a temporary cessation in egg production by the parasites. 224 gastrointestinal helminths infections in dorper sheep in kenya most clinical cases of helminthosis and salvage treatments in this study occurred between july and november and is attributed to two main reasons: firstly, from july to october, when the animals were in poor body condition and therefore highly susceptible to helminthosis, the pastures were mostly dry and of low nutritional value; and secondly, the resumption of development of hypobiotic larvae of haemonchus towards the end of the dry season and at the onset of the short rains (october to november), as also observed by gatongi et al. (1998) in sheep and goats towards the end of the dry season and at the onset of the long rains under the semi-arid environment of naivasha. the finding that trichostrongylus spp. were most prevalent in the study area differed from that of gatongi et al. (1998) and maingi et al. (1997) who reported a higher prevalence for haemonchus in sheep in the semi-arid area of naivasha and the high rainfall area of nyandarua district in kenya, respectively. this can be attributed to the fact that trichostrongylus colubriformis free-living stages are more resistant to desiccation than haemonchus and some other species (banks, singh, barger, pratap & le jambre 1990). this conclusion is supported by the fact that animals were treated with albendazole at the start of the trial and that the re-infection that followed occurred during the dry period which was favourable to the more resilient trichostrongylus species. the dominance was further enhanced by the inadequate long rains of march to may 2000. the results of the study indicate that control of gastrointestinal helminths in the study area should aim at reducing the impact of helminthosis in all age groups. the lambs should be protected from the adverse effects of infection until they acquire immunity and the control of infections in the other age groups be targeted towards reduction of pasture contamination based on the rainfall distribution pattern and the reproductive status of the adult ewes. acknowledgements this study was financed through the food and agriculture organization (fao) technical cooperation programme projected no. fao-tcp/ken/8822 (integrated sustainable parasite control in ruminants in mixed farming systems in kenya) at the university of nairobi. the authors thank the management of the maasai rural training centre ranch for allowing unlimited access to their animals and facilities. the technical assistance of messrs h.e. weda and r.o. otieno is also appreciated. references allonby, e.w. & urquhart, g.m. 1973. self-cure of haemonchus contortus under field conditions. parasitology, 66:43–53. allonby, e.w. & urquhart, g.m. 1975. the epidemiology and pathogenic significance of haemonchus in a merino flock in east africa. veterinary parasitology, 1:129–143. banks, d.j.d., singh, r., barger, i.a., pratap, b. & le jambre, l.f. 1990. development and survival of infective larvae of haemonchus contortus and trichostrongylus colubriformis on pastures in a tropical environment. international journal for parasitology, 20:155–160. barger, i.a. 1999. the role of epidemiological knowledge and grazing management for helminth control in small ruminants. international journal for parasitology, 29:41– 47. carles, a.b. 1993. helminthosis as a constraint to livestock productivity in kenya, in progress towards control of helminths in kenya, edited by j.a. onyango-abuje, r.k. bain, s.w. wanyangu & m.a. ihiga. nairobi, kenya: kari/oda. colditz, i.g., watson, d.l., gray, g.d. & eady, s.j. 1996. some relationships between age, immune responsiveness and resistance to parasites in ruminants. international journal for parasitology, 26:869–877. gatongi, p.m., prichard, r.k., ranjan, s., gathuma, j.m., munyua, w.k., cheruyot, h. & scott, m.e. 1998. hypobiosis of haemonchus contortus in natural infections of sheep and goats in a semi-arid area of kenya. veterinary parasitology, 77:49–61. gibbs, h.c. 1968. some factors involved in the “spring rise” phenomena in sheep, in the reaction of the host to parasitism, edited by e.j.l. soulsby. ng. marburg/lahn: alwert. githigia, s.m., thamsborg, s.m., munyua, w.k. & maingi, n. 2001. impact of gastrointestinal helminths on production in goats in kenya. small ruminant research, 42:21–29. hansen, j. & perry, b. 1994. the epidemiology, diagnosis and control of gastrointestinal parasites of ruminants in africa. ilrad, nairobi kenya. jacquiet, p., colas, f., cabaret, j., dia, m.l., cheikh, d. & thiam, a. 1995. dry areas: an example of seasonal evolution in helminth infection of sheep and goats in southern mauritania. veterinary parasitology, 56:137–148. maff. 1986. manual of veterinary parasitological techniques, ministry of agriculture, fisheries and food, london: hmso (technical bulletin, no. 18). maingi, n., gichohi, v.m., munyua, w.k., gathuma, j.m. & thamsborg, s.m. 1997. the epidemiology of nematodes and liver fluke infections in sheep in nyandarua district of kenya. bulletin of animal health and production in africa, 45:27–34. mbaria, j.m., mcdermott, j.m., kyule, m.n., gichanga, e.j. & manyole, p.n. 1995. prevalence of helminths and helminth control practices in molo division, nakuru district, kenya. bulletin of animal health and production in africa, 43:35– 44. nginyi, j.m., duncan, j.l., mellor, d.j., stear, m.j., wanyangu, s.w., bain, r.k. & gatongi, p.m. 2001. epidemiology of parasitic gastrointestinal nematode infections of ruminants on smallholder farms in central kenya. research in veterinary science, 70:33–39. ogunsusi, r.a. 1979. termination of arrested development of trichostrongylids in northern nigeria. research in veterinary science, 26:189–192. 225 c.j. ng’ang’a et al. preston, j.m. & allonby, e.w. 1979a. the influence of breed on the susceptibility of sheep to haemonchus contortus infection in kenya. research in veterinary science, 26: 134–139. preston, j.m. & allonby, e.w. 1979b. the influence of haemoglobin phenotype on the susceptibility of sheep to haemonchus contortus infection in kenya. research in veterinary science, 26:140–144. reinecke, r.k. 1983. veterinary helminthology. durban: butterworths. watson, d.l. 1991. effects of weaning on antibody responses and nematode parasitism in merino lambs. research in veterinary science, 51:128–132. watson, d.l. & gill, h.s. 1991. post natal ontogeny of immunological responsiveness in merino sheep. research in veterinary science, 51:88–93. whitlock, h.v. 1948. some modifications of the mcmaster helminth egg counting technique and apparatus. journal of the council of science and industrial research of australia, 51:177–180. 226 gastrointestinal helminths infections in dorper sheep in kenya maichomo_257-261.qxd introduction gastrointestinal (git) parasites constitute a major variable in livestock production systems, particularly in sheep and goats where these infections are the single greatest constraint to production in the tropics (mukhebi, shavulimo, ruvuna & rurangirwa 1985). in kenya, haemonchosis causes an annual loss of us$ 26 million in sheep and goats, while returns could be increased by as much as 470 % by controlling the disease (allonby 1975; mukhebi et al. 1985). in most agro-climatic zones (acz) in kenya, haemonchosis has been shown to be the leading cause of mortality in small ruminants (kanyari 1993; kagira & kanyari 2001). the most important genera of nematodes in kenya are haemonchus, trichostrongylus, and oesophagostomum (gatongi 1995; mwendia 1996). losses due to git parasitism can be categorized as direct or indirect. direct losses are due to acute illness and death, forced premature slaughter and rejection of parts of the carcass at meat inspection in abattoirs. acute parasitic conditions can be recognized and affected animals are generally treated 257 onderstepoort journal of veterinary research, 71:257–261 (2004) the point prevalence of gastro-intestinal parasites in calves, sheep and goats in magadi division, south-western kenya m.w. maichomo1, j.m. kagira1 and t. walker2 abstract maichomo, m.w., kagira, j.m. & walker, t. 2004. the prevalence of gastro-intestinal parasites in calves, sheep and goats in magadi division, south-western kenya. onderstepoort journal of veterinary research, 71:257–261 helminths cause great economic loss in livestock in africa, and can be categorized as either direct or indirect losses. arid and semi-arid lands (asal) in kenya comprise 71 % of total land area and harbour the largest population of cattle, sheep and goats. however, little information on the distribution and impact of gastro-intestinal (git) parasitism in these animals is available. this survey was conducted to establish the prevalence of git parasites infecting calves, sheep and goats and their relative importance in magadi division, which is semi-arid. faecal samples were obtained directly from the rectum of 109 calves, 133 goats and 20 sheep and submitted to the laboratory for faecal worm egg counts, and coccidial oocysts examination using a modified mcmaster method. the significance of differences in mean egg count per gram (epg) between animal species and herds (farms) were assessed using analysis of variance. the overall prevalence of nematodes in the calves, sheep and goats was 69.2 %, 80 % and 82 %, respectively. about 10 % of sheep and goats had epgs higher than 1 000, the remainder having light to moderate infections. the overall prevalence of coccidial oocysts in calves, sheep and goats was 30 %, 44 % and 45 %, respectively. poor productivity in asal areas, where nutrition is often poor, is likely to be pronounced in the presence of parasite infections. these findings indicate that viable internal parasite control should be implemented in the study area in order to increase the productivity of the livestock there. keywords: cattle, coccidia, gastro-intestinal parasites, goats, helminthosis, sheep 1 kenya trypanosomiasis research institute, p.o. box 362, kikuyu, kenya 2 university of georgia, college of veterinary medicine, athens, georgia, 30602, united states of america accepted for publication 15 march 2004—editor 258 by the farmer and thus direct losses can be avoided. in contrast, sub-clinical levels of parasitism cause indirect and more subtle losses and do not induce obvious clinical signs (barger, siale, banks & le jambre 1994). this type of parasitism is regarded as the most important cause of economic loss, as it is a flock or herd problem, unlike the acute syndrome, and tends to affect animals on a low plane of nutrition as well as lactating does and ewes. subclinical levels of parasitism reduce potential productivity of sheep and reduce feed conversion (ndarathi, waghela & semenye 1989; cobon & o’sullivan 1992; suttle 1994). it is difficult to assess the economic effects of such subclinical infections but it is important to quantify them. in africa, ruminants are kept mainly for their meat, milk and skins. the production systems are diverse and are mainly dependent on the local climate. in pastoral areas of kenya, the main breeds of livestock are of the indigenous type, which includes zebu cattle, red maasai and blackhead somali sheep, and the east african goat. the productivity of these animals is limited mainly by the seasonal availability of feed and diseases (semenye 1987; baker, lahlou-kassi, rege, reynolds, bekele, mukasa-mugerwa & rey 1992). up-to-date information is lacking on the magnitude of git parasites in livestock maintained in traditional grazing systems in the magadi division of kenya, a semi arid region. changing socio-cultural values among members of the maasai community in the magadi division have induced changes in their traditional lifestyle in that they now keep fewer but high-producing animals, which have been improved by cross breeding. however, due to greater susceptibility to parasites, the production of such “improved” crosses may be more severely affected by sub-clinical infection than in the case of the indigenous breeds, unless parasites are controlled through a well-managed herd health program. the present survey was undertaken to determine the prevalence and intensity of internal parasites in calves, sheep and goats in magadi division, south-western kenya, inhabited by the maasai community. materials and methods study area the magadi division is in the kajiado district of the rift valley province, which is located 170 km southwest of nairobi. the district lies between latitudes 1°10’–3°10’ s; 36°5’–37°55’ e. it occupies an area of 22 106 km² with an altitude that ranges from 650– 1 500 m above sea level. the region is mainly semiarid with mean temperature ranges of 18–38 °c. the mean annual rainfall is 440 mm and is distributed over two rainy seasons, which consists of the “long rains”, from march to may and the “short rains”, from october to december. the vegetation types include: (i) tall grassland on deep alluvial soils (ii) tussocky annual grassland on deep alluvial soils (iii) shrubland on stony brecchia soils (iv) riverine and seasonally flooded woodland (agnew 1996). animal sampling and data collection a cross-sectional survey of 109 calves, 20 sheep and 133 goats from nine randomly selected herds (munii, mpesi, lendulo, nkedienye, timoi, kinoya, lempaine, sarara and sadera) in olkiramatian and shompole communal group ranches (which border each other) of the magadi division was done for two months from may to june 2002. all calves and half of the sheep and goats (randomly selected) in each herd were sampled only once. all calves were under 1 year of age while the sheep and goats were a mixture of weaners and adults. faecal samples were obtained from the rectum and submitted to a laboratory for the determination of the number of nematode eggs per gram (epg) of faeces, using a modified mcmaster technique (maff 1986). the presence of oocysts and moniezia spp. ova was also recorded. rainfall, temperature and humidity values were recorded daily from january to june 2002. statistical analysis data obtained was subjected to a univariate analysis of variance (anova) to establish whether or not there were significant differences in mean (µ) epg of the animals. results climatic data from january to june 2002, 565.2 mm of rainfall as recorded. the mean monthly temperature ranged between 21.7 °c and 34 °c and relative humidity from 56 % in the morning to 35 % in the afternoon. gastro-intestinal parasites in calves, sheep and goats in kenya prevalence of nematodes calves (fig. 1) a total of 109 calves were sampled. the overall mean prevalence of nematodes was 69.2 %. trichostrongylid eggs were more common (59.6 %) than strongyloides (27.4 %). munii’s herd had the highest mean epg of 250 while mpesi’s had the lowest mean epg of 23 (table 1). goats (fig. 2) a total of 133 goats were sampled. the highest prevalence was recorded in the animals in the munii and sarara herds while it was lowest in those in the kinoya herd. the overall prevalence of nematodes was 82 %. trichostrongylids and strongyloides affected 72 % and 43 % of the goats, respectively. mean epg values were highest in munii and lowest in kinoya herds (table 1). sheep since only 20 sheep were sampled, the prevalence of nematodes was not assessed per herd. however, the overall flock prevalence was 80 %, with a mean epg of 326. trichostrongylids and strongyloides affected 75 % and 45 % of the sheep, respectively. 259 m.w. maichomo, j.m. kagira & t. walker ����� ������ � ��� � ������� ��� � ���� ������� ����� � �� � � � � � � � � ���� �� �� �� �� � ���� �� �� �� �� � � �� � � � � � � � � ����� ��� �� ��� ���� ������ � ������� ������ ������ �������� ������� � ��� ! �"� ������ ��������� �� �������� ������� � ��� ! �"� ������ ��������� �� fig.1 prevalence of nematodes in calves from five herds in magadi division fig. 2 prevalence of nematodes in six herds of goats in magadi division � � �� � �� � � �#� �� $� �� %� �� �� � �&�� �$&�'' $��&����� (����� �� ������� ������������ ������������������������� ��� � � �� � �� � � �$� �� %� �� �� � )�����* �"��!�� � � % � )�����* �"��!�� fig. 3 percentage of animals shedding various ranges of nematode eggs fig. 4 percentage of animals infected by a given number of parasites 260 gastro-intestinal parasites in calves, sheep and goats in kenya intensity of nematodes (fig. 3) most animals were lightly infected with ranges between 25 and 499 epg. some sheep and goats, however, did have an epg above 1000. other git parasites coccidial oocysts and moniezia spp. ova were also observed in some of the faecal samples. the overall prevalence of oocysts in the calves, goats and sheep was 30 %, 44 % and 45 %, respectively. the coccidial oocysts were not identified to genus level. moniezia spp. eggs, which were also not identified to species level, were observed in 8 %, 10 % and 5 % in the cattle, sheep and goats, respectively. mixed infections the percentage of animals infected by a given number of git parasites, namely trichostrongylid, strongyloides, moniezia spp. and coccidia are shown in fig. 4. most of the animals were infected with one or two species of parasites. statistical analysis significant differences between the numbers of epg of trichostrongylid and strongyloides were obtained (p = 0.000) with the former being higher (µ = 314, range 0–6 300) than the latter (µ = 106, range 0– 7 500). anova results indicated that there were host differences in trichostrongylid epg levels (p = 0.006, µ = 122 in calves, 469 in goats and 320 in sheep), as well as sex differences in the animals with higher values in males (p = 0.0068). the hostlevel difference for strongyloides epg levels were also significant (p = 0.0656), with goats having higher burdens (µ = 173) than calves (µ = 117.5) and sheep (µ = 20.8). the munii and nkedienye herds, located in wetter areas, had larger numbers of animals infected with git parasites. discussion livestock keeping is the most viable and self-sustaining enterprise in the pastoral community of magadi division and is therefore their most important means of livelihood. for effective management of animal helminths, information on their epidemiology is essential (herlich 1978). a previous study on helminthoses in small ruminants in this region (mwendia 1996) revealed a lower mean epg level in the same season (after long rains) than those obtained in the present study. this could be due to the changing lifestyle of the local maasai people from nomadic to sedentary, with emphasis on quality and economical beef production, necessitated by an increase in the human population and cost of living. indeed, the prevalence of nematodes on herds in sedentary herds (munii and nkedienye) was higher than those of nomadic ones. the increase in helminthoses that was found could also be explained by an increase in the rainfall, being 562.6 mm in the first 6 months of 2002 compared to a total of 330 mm within the same period in 1994 (mwendia 1996). however, faecal egg counts that were obtained in sheep in the present study were low compared to those reported in sheep in the highlands (maingi 1996). the reason for this is believed to be the high temperatures and dry conditions in the pastoral areas, which lead to desiccation of nematode eggs and larvae. inadequate nutrition, however, which is common in this area, may exacerbate the course of nematode infection. the animals are generally malnourished and suffer from other diseases, and are thus not resistant to nematode infection. although the nematode eggs in this study were not identified to species level, haemonchus contortus and trichostrongylus colubriformis are considered to be the parasites most responsible for helminthoses in small ruminants in highlands and pastoral areas of kenya, respectively (gatongi 1995; maingi, 1996; mwendia 1996). sheep and goats had higher burdens when compared to calves. cattle have been shown to have low nematode burdens up to 10 months of age, while the heaviest burdens are detected in 1.5 to 3-yearold animals, thereafter declining to even lower burdens (kaufman & pfister 1990). in the current study, most of the calves were about 1 year old. since the study area has a high prevalence of trypanosomosis (roderick, 1995), it is possible that animals suftable 1 mean epg of total nematodes in calves, sheep and goats from different herds in magadi division mean epg herds calves sheep goats kinoya – 25 86 lempaine – 451 205 lendulo 49 – – mpesi 23 – – munii 250 – 1 118 nkedienye 208 – 114 sadera – – 780 sarara – 502 537 timoi 55 – – 261 m.w. maichomo, j.m. kagira & t. walker fering from both helminthoses and trypanosomosis will be more seriously affected. studies in cattle and goats have shown that the pathogenicity of h. contortus is increased in animals with concomitant experimental infection with trypanosoma congolense (griffin, allonby & preston 1981; kaufman, dwinger, hallebeck, van dijk & pfister 1992). mixed parasitic infections in hosts is the rule rather than the exception in most grazing systems as was demonstrated by the concurrent presence of up to four parasite genera. the occurrence of mixed infections may lead to decreased production due to the synergistic effect of each parasite, although this depends on the proportion of pathogenic species. the prevalence of coccidia in herds sampled ranged between 30 % and 45 %. this is comparable to reports by kanyari (1993) in sheep and goats. some eimeria species are considered pathogenic to the three host species studied. although coccidiosis is regarded as a disease of housed or confined animals, lack of grazing pasture during the dry season may increase the “stocking rate” of animals on the few pastures remaining, a situation that may lead to a proliferation of oocysts. it is concluded that gastrointestinal parasites that can cause economic losses are present in livestock in the study area. further studies are needed to establish the seasonal epidemiology of these parasites and how they interact with trypanosomes in affecting production of ruminants. such information will be important in designing an integrated control program for these parasites. acknowledgements we thank messrs ken otieno, vincent malonza, njoroge kisiango and tiberius marete for their technical assistance. the maasai farmers at nguruman allowed us to use their animals and the director, ketri, gave permission to publish this work. references agnew, a.d.q. 1996. the graze and browse resource at olkiramatian group ranch. fourth report to ketri/nri on landscape/vegetation monitoring at olkiramatian group ranch. report to the oda on a visit to the kenya trypanosomosis research institute (nguruman station), may 1996. oda: london. allonby, e.w. 1975. investigation of small-stock diseases in kenya. interim technical report, sheep and goats development project. rome: food and agriculture organization of the united nations. baker, r.l., lahlou-kassi, a., rege, j.e.o., reynolds, l., bekele, t., mukasa-mugerwa, e. & rey, b. 1992. a review of genetic resistance to endoparasites in small ruminants and an outline of ilca’s research programme in this area. proceedings of the 10th scientific workshop of the small ruminant collaborative research support program, nairobi, kenya, sr-crsp. nairobi, kenya. barger, i.a., siale, k., banks, d.j.d. & le jambre, l.f. 1994. rotational grazing for control of gastrointestinal nematodes of goats in a wet tropical environment. veterinary parasitology, 53:109–116. cobon, d.h. & o’sullivan, b.m. 1992. effects of haemonchus contortus on productivity of ewes, lambs and weaners in a semi-arid environment. journal of agricultural science, 118:245–248. gatongi, p.m. 1995. the epidemiology and control of gastrointestinal nematodes of small ruminants in a semi-arid area of kenya with emphasis on hypobiosis of haemonchus contortus. ph.d. thesis, mcgill university, canada. griffin, l., allonby, e.w. & preston, j.m. 1981. the interactions of trypanosoma congolense and haemonchus contortus infections in two breeds of goats. i. parasitology. journal of comparative pathology, 91:85–95. herlich, h. 1978. the importance of helminth infections in ruminants. world animal health review, 26:26–29. kanyari, p.w.n. 1993. the relationship between coccidian and helminth infections in sheep and goats in kenya. veterinary parasitology, 51:137–141. kagira, j.m. & kanyari, p.w.n. 2001. the role of parasitic diseases as causes of mortality in small ruminants in a highpotential farming area in central kenya. journal of the south african veterinary association, 72:147–149. kaufmann, j. & pfister, k. 1990. the seasonal epidemiology of gastrointestinal nematodes in ndama cattle in gambia. veterinary parasitology, 37:45–54 kaufmann, j., dwinger, r.h., hallebeck, a., van dijk, b. & pfister, k. 1992. the interaction of trypanosoma congolense and haemonchus contortus in trypanotolerant cattle. veterinary parasitology, 43:157–170 maff. 1986. ministry of agriculture, fisheries and food. manual of veterinary parasitological techniques, 3rd ed. reference book 418. london: her majesty’s stationery office. maingi, e.n. 1996. anthelmintic resistance, epidemiology and control of gastrointestinal nematodes of small ruminant in nyandarua district of kenya and in denmark. ph.d. thesis, university of nairobi. mukhebi, a., shavulimo, r.s., ruvuna, f. & rurangirwa, f. 1985. economics on internal parasitic control among goats in western kenya. proceedings of the 4th small ruminant collaborative support program (sr-crsp) scientific workshop. ilrad, nairobi, kenya, march 1985. mwendia, c.m. 1996. productivity and disease constraints of small ruminants in maasailand, kajiado district, kenya. ph.d. thesis, university of reading. ndarathi, c.m., waghela, s. & semenye, p.p. 1989. helminthiasis in maasai ranches in kenya. bulletin of animal health and production in africa, 37:205–208. roderick, s. 1995. pastoralist cattle productivity in a tsetse infested area of south west kenya. ph.d. thesis, university of reading. semenye, p.p. 1987. factors influencing maasai cattle productivity in kajiado district, kenya. ph.d. thesis, university of nairobi. suttle, n.f. 1994. seasonal infections and nutritional status. proceedings of the nutritional society of england and scotland, 53:545–555. article information authors: nkululeko nyangiwe1 sindisile goni1 louis p. hervé-claude2 inga ruddat2 ivan g. horak3,4 affiliations: 1döhne agricultural development institute, stutterheim, south africa 2institute of biometry, epidemiology and information processing, university of veterinary medicine of hannover, germany 3department of veterinary tropical diseases, university of pretoria, south africa 4department of zoology and entomology, university of the free state, south africa correspondence to: ivan horak email: ivan.horak@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 07 feb. 2011 accepted: 14 may 2011 published: 07 oct. 2011 how to cite this article: nyangiwe, n., goni, s., hervé-claude, l.p., ruddat, i. & horak, i.g., 2011, ‘ticks on pastures and on two breeds of cattle in the eastern cape province, south africa’, onderstepoort journal of veterinary research 78(1), art. #320, 9 pages. doi:10.4102/ojvr.v78i1.320 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) ticks on pastures and on two breeds of cattle in the eastern cape province, south africa in this original research... open access • abstract • introduction • materials and methods • results    • climate    • questing ticks    • parasitic ticks • discussion    • general    • amblyomma hebraeum    • hyalomma rufipes    • rhipicephalus appendiculatus    • rhipicephalus decoloratus    • rhipicephalus evertsi evertsi    • rhipicephalus follis    • rhipicephalus microplus    • less commonly collected species • conclusion • acknowledgements    • authors’ contributions • references abstract (back to top) many studies on the population dynamics of questing ticks on pastures and of parasitic ticks on cattle have been conducted. few, however, have attempted to link the two in a single study. this study aimed to assess the population dynamics of questing ixodid ticks on pastures and of adult ticks on two breeds of cattle with different levels of susceptibility to tick infestation on the same pastures. between january 2005 and december 2009 questing ixodid ticks were collected monthlyfrom natural pastures at the döhne agricultural development institute and at the adjacent campagna production system in the amahlathi district, eastern cape province, south africa. between february 2007 and january 2010 adult ticks were collected monthly from bonsmara and nguni cattle grazing these pastures. ten tick species were collected from the pastures and 12 from the cattle. significantly more questing larvae of rhipicephalus appendiculatus, rhipicephalus decoloratus, rhipicephalus evertsi evertsi and rhipicephalus microplus were recovered from the pastures grazed by bonsmara cattle than from those grazed by nguni cattle (p ≤ 0.05). significantly more adult hyalomma rufipes, rhipicephalus follis, r. appendiculatus, r. decoloratus, r. evertsi evertsi and r. microplus were collected from the bonsmara cattle than from the nguni cattle (p ≤ 0.05). the study showed that nguni cattle are less susceptible to tick infestation than are bonsmara cattle and fewerquesting ticks are collected from pastures grazed by nguni cattle than by bonsmara cattle. introduction (back to top) at least 75 ixodid tick species are present in south africa (walker 1991), but only four of these are important vectors of disease in cattle. they are amblyomma hebraeum, vector of ehrlichia ruminantium, the causative organism of heartwater; rhipicephalus appendiculatus, vector of theileria parva, the causative organism of east coast fever; rhipicephalus decoloratus, vector of babesia bigemina, the causative organism of african redwater; and rhipicephalus microplus, vector of both babesia bovis, the causative organism of asiatic redwater, and b. bigemina (norval & horak 2004). amblyomma hebraeum, r. appendiculatus and r. decoloratus are indigenous to south africa, whilst it is generally accepted that r. microplus was introduced into the country on cattle imported from madagascar after the rinderpest epidemic (hoogstraal 1956). some breeds of cattle, and specifically nguni cattle, have the ability to acquire resistance to tick infestation (spickett et al. 1989). the döhne agricultural development institute, which includes the adjacent campagna production system, eastern cape province, south africa, has maintained separate herds ofbonsmara and nguni cattle for several years. both r. decoloratus and r. microplus are present on these animals. the set-up at the döhne institute thus presents an excellent opportunity to compare the resistance of bonsmara and nguni cattle to natural tick infestations and to explore the dynamics of r. decoloratus and r. microplus, as well as other tick species, on these animals and on the pastures. our study had several objectives. firstly, we aimed to establish the species composition of free-living ticks questing for hosts on the pastures at döhne and campagna, and of adult ticks on cattle grazing the pastures at the two localities. secondly, the study aimed to determine whether the number of questing ticks onthe pastures grazed by bonsmara cattle and the number of adult ticks on these animals differed from that on the pastures grazed by nguni cattle and the number of adult ticks on the latter animals. thirdly, we aimed to determine the seasonality of questing ticks on the pastures and of adult ticks on the cattle. materials and methods (back to top) the study was conducted at the döhne agricultural development institute (hereafter referred to as döhne) and at the adjacent campagna production system (hereafter referred to as campagna), both located in the amahlathi district, eastern cape province. the term döhne complex will be used when referring to both.döhne (32°31’s, 27°28’e) comprises an area of 992 ha and its vegetation is defined as döhne sourveld (acocks 1988). the dominant grass species are eragrostis curvula, eragrostis plana, sporobolus africanus and themeda triandra. campagna (32°29’s, 27°29’e) is 248 ha in extent and its vegetation is characterised by an open, treed savanna with a moderate shrub and grass cover and is classified as eastern province thornveld (acocks 1988). in addition to the four major grass species atdöhne, the grazing on campagna consists of andropogon appendiculatus and heteropogon contortus. a herd of about 46 bonsmara cattle, which included 16 replacement heifers, rotationally grazed five camps of natural veld at döhne. the camps varied in size(from 12.7 ha to 17.0 ha), with a total surface area of 72 ha. a herd of about 64 nguni cattle, of which 24 were replacement heifers, similarly grazed five camps of between 9.9 ha and 14.7 ha, with a total surface area of 65.3 ha. at campagna a herd of approximately 30 bonsmara cattle, of which seven were replacement heifers, rotationally grazed five camps of between 10.9 ha and 16.2 ha, with a total surface area of 69.4 ha. when grazing was inadequate the animals were fed lucerne hay and winter and summer licks were provided ad libitum. because of a sudden spate of mortalities amongst the bonsmara cattle a few years earlier, due probably tobabesia bovis infection, they were treated with the acaricide flumethrin (drastic deadline, bayer animal health) at least once a month, administered as a pour-on. the nguni cattle were treated only when adult ticks became visible. ten flannel strips (100 cm x 10 cm) were attached next to each other on a 120-cm-long wooden spar with velcro tape and the two ends of a twine harness were tied to the tips of the spar. an operator collected ticks questing for hosts from the vegetation by dragging the spar, using the twine harness, across the vegetation over adistance of 100 m (spickett et al. 1992). drags were always repeated six times at approximately 50-m intervals in camps grazed by the respective cattle breeds. after each drag, the ticks on the flannel strips were collected using forceps and stored in 70% ethanol in internally labelled glass vials. drag-sampling of the vegetation was performed monthly between january 2005 and december 2009. with a few exceptions sampling was performed during the fourth week of each month so thatit coincided with the weighing and dipping of the cattle grazing the pastures. drags were not performed over dew-laden grass early in the morning or immediately after rain, as this would wet the flannel strips and decrease their efficacy for picking up questing ticks. during the five years of the study a total of 1080 drags were made and all the ticks collected were identified, classed by life stage and counted under a stereoscopic microscope. the aggregate numbers of larvae collected permonth were used to illustrate graphically the seasonal abundance of the most numerous tick species. from february 2007 to january 2010, adult ticks were collected monthly from one side (including half the head and one ear) and from the whole of the upperperineum and tail-brush of three bonsmara and three nguni cattle at döhne and three bonsmara cattle at campagna. all the ticks collected were identified, classed by gender, and counted under a stereoscopic microscope. the aggregate numbers of adult ticks collected per month were used to illustrate graphically the seasonality of the most numerous species. maximum and minimum atmospheric temperatures, rainfall and relative humidity were measured daily at döhne. the tick counts were entered into a microsoft excel database and all analyses were performed using sas (sas institute inc. 2006, edition 9.1.3). because the data were not normally distributed, owing to a large number of zero counts which makes log transformation in the search for normality unreliable, non-parametric tests(kruskal–wallis and wilcoxon) were performed (altman 1991) to determine significant differences between groups. the groups comprised ticks collected from döhne pastures and bonsmara cattle on these pastures, ticks from döhne pastures and nguni cattle on these pastures, and ticks from the pastures and bonsmara cattle at campagna. analyses were performed for all species of questing and parasitic ticks whenever there were adequate numbers. a significance level of α = 0.05 was used for each comparison. similar methodology was used to compare tick numbers (both questing and parasitic) between years. the kruskal–wallis test was conducted to determine general fluctuations. wilcoxon tests were used for pair-wise year comparisons. to control the overall significance level of α = 0.05 bonferroni–holm adjustment for p-values was applied (holm 1979). spearman correlation coefficients (dohoo, martin & stryhn 2010) were calculated to determinethe relationship between questing and parasitic tick numbers, and monthly and annual rainfall. results (back to top) climate maximum and minimum atmospheric temperatures, annual rainfall and relative humidity are graphically illustrated in figure 1a, b and c. the highest mean temperatures were recorded during each february of the survey, whilst the lowest were recorded in various months between may and september. high maximumtemperatures were sustained for six months during the summer of 2008/2009. most rain fell during the period october–march, with a marked decrease in precipitation in 2009. relative humidity was generally the lowest during the mid-winter months of june and july and highest from early to late summer (october or november to march or april). figure 1: (a) mean monthly maximum and minimum temperatures, (b) monthly rainfall and (c) mean monthly relative humidity at döhne. questing ticks the species and total numbers of questing ticks collected from the vegetation at döhne and campagna are summarised in table 1. ten tick species were collectedfrom the vegetation and the total number of questing larvae collected from the pastures grazed by bonsmara cattle at döhne was significantly different from the number collected from the pastures grazed by nguni cattle (p < 0.001). the numbers of r. appendiculatus, r. decoloratus, rhipicephalus evertsi evertsi and r. microplus larvae collected from the pastures grazed by bonsmara cattle at döhne were significantly different from those collected from pastures grazed by nguni cattle(p ≤ 0.05). in all cases the tick counts associated with bonsmara cattle were larger. the numbers of r. appendiculatus, r. decoloratus and r. microplus larvae collected from the pastures grazed by bonsmara cattle at döhne were also significantly greater than those collected from the pastures at campagna (p ≤ 0.05). the numbers of larvae of the five major tick species collected annually from the pastures at döhne and campagna are summarised in table 2. during one or more years of the survey the annual total numbers of larvae of each of these species exhibited significant fluctuations (p ≤ 0.05) when compared with the numbers collected in any other year. only one a. hebraeum larva was recovered during the first three years of the survey, but numbers increased substantially thereafter. the number of r. appendiculatus larvae collected during 2007 was substantially lower than in the previous years, but increased thereafter, whereas its nymphs and the larvae of r. evertsi evertsi exhibited a decreasing trend during the study period. significantly fewer r. decoloratus larvae were collected during 2006 than in any other year of the survey, whilst slight annual fluctuations in the numbers of r. microplus larvae were observed. questing r. appendiculatus larvae reached peak numbers in april, followed by a smaller peak in august. nymphs peaked in october (figure 2a). larvae of r. decoloratus and r. microplus were most abundant during august (figure 2b), whilst r. evertsi evertsi larvae were most abundant during january, with successivelysmaller peaks in june and november, respectively (figure 2c). the total numbers of r. evertsi evertsi larvae collected during each year of the survey appeared to be positively but not significantly (p = 0.5) correlated with annual rainfall (figure 3). it should be noted, however, that this calculation was based on only five observations. table 1: species composition of ticks questing for hosts from the pastures grazed by bonsmara or nguni cattle at döhne or by bonsmara cattle at campagna (2005–2009). table 2: questing immature ticks of the five major species on the pastures at döhne and campagna during each year of the survey (2005–2009). figure 2: composite seasonal abundance of questing (a) rhipicephalus appendiculatus larvae and nymphs, (b) rhipicephalus decoloratus and rhipicephalus microplus larvae, and (c) rhipicephalus evertsi evertsi larvae on the pastures at döhne and campagna (2005–2009). figure 3: annual rainfall and numbers of questing rhipicephalus evertsi evertsi larvae collected at döhne and campagna (2005–2009). parasitic ticks the species and numbers of ticks collected from the cattle at döhne and campagna are summarised in table 3. adult ticks belonging to 12 species were collectedand the total number of ticks collected from bonsmara cattle at döhne significantly exceeded the number collected from nguni cattle (p < 0.001). greater numbers of r. appendiculatus, r. decoloratus, r. evertsi evertsi, rhipicephalus follis and r. microplus were collected from the bonsmara cattle at döhne than from the nguni cattle (p ≤ 0.05). a. hebraeum was collected only from the bonsmara cattle at campagna and these animals also harboured the vast majority of hyalomma rufipes. bonsmara cattle at döhne carried significantly fewer h. rufipes and significantly more r. microplus than bonsmara cattle at campagna (p ≤ 0.05). the total numbers of ticks of the seven most abundant species collected annually from the bonsmara and nguni cattle at döhne and the bonsmara cattle atcampagna are summarised in table 4. only two a. hebraeum adults were collected in 2007, but considerably more were collected in 2008 and 2009. the numbers of h. rufipes and r. microplus fluctuated only slightly (but not significantly) during the three years of the study, whereas significant annual differences were observed in the numbers of the remaining tick species. moreover, r. evertsi evertsi showed a significant increase and r. follis a significant decrease in numbers. the increasein annual numbers of adult r. evertsi evertsi seemed to be marginally inversely correlated with annual rainfall (figure 4), with a correlation coefficient of –0.50 for aggregated yearly rainfall and –0.01 for monthly rainfall data. the correlations were, however, not significant. the decrease in annual numbers of r. follis seemed to be marginally positively correlated with annual rainfall (figure 4), with a correlation coefficient of 0.50 for aggregated yearly rainfall and 0.29 for monthly rainfall.also these correlations proved to be non-significant. the seasonal abundances of the six most numerous tick species collected from the cattle at döhne and campagna are graphically illustrated in figure 5. adult h. rufipes reached peak numbers in midsummer and late summer, whilst r. appendiculatus numbers peaked in spring and late summer (figure 5a). the numbers of r. decoloratus and r. microplus declined erratically from january to august, increased in september and declined towards november, followed by a marked increase in december (figure 5b). the greatest numbers of r. evertsi evertsi were present from january to march and in september and the fewest from may to august, whereas most r. follis were collected during august (figure 5c). table 3: adult ticks collected from bonsmara and nguni cattle at döhne, and bonsmara cattle at campagna (february 2007 to january 2010). table 4: number of adult ticks collected during each 12-month period from six bonsmara cattle and three nguni cattle (february 2007 to january 2010). figure 4: annual rainfall and numbers of adult rhipicephalus evertsi evertsi and rhipicephalus follis on cattle at döhne and campagna (february 2007 – january 2010). figure 5: composite seasonal abundance of adult (a) hyalomma rufipes and rhipicephalus appendiculatus, (b) rhipicephalus decoloratus and rhipicephalus microplus, and (c) rhipicephalus evertsi evertsi and rhipicephalus follis on cattle at döhne and campagna (february 2007 – january 2010). discussion (back to top) general rechav (1987) and rechav, dauth and els (1990) have demonstrated that under field conditions in south africa, brahman cattle are more resistant to tick infestation than hereford or simmentaler cattle. spickett et al. (1989) have shown that nguni cattle are more resistant to natural tick challenge than bonsmara or hereford cattleand rechav and kostrzewski (1991) have reported that nguni cattle were more resistant to infestation by r. decoloratus than the five other breeds of cattle in their study. despite the fact that the bonsmara cattle in the present study were regularly treated with an acaricide, and the nguni cattle only when adult ticks became visible, the bonsmara cattle harboured significantly greater numbers of adult ticks than the nguni cattle, confirming the findings of spickett et al. (1989). the largernumbers of questing larvae recovered from the pastures grazed by bonsmara cattle can thus be construed as a consequence of the greater number of adult ticks on these animals when compared with the numbers of larvae on the pastures grazed by nguni cattle. the small numbers of questing ticks collected from the vegetation and parasitic ticks from cattle are probably a consequence of the regular application of acaricides to cattle at döhne and campagna. despite these small numbers, a total of 13 species were represented. in comparison, only six ixodid tick species were collected during 2004 and 2005 from vegetation and cattle at 72 dip-tanks in rural farming areas east of döhne (horak et al. 2009). although the number of ticks collected ateach occasion in the present study was low, aggregate monthly and annual trends could be determined because the survey was conducted over several years. the immature stages of haemaphysalis elliptica, r. follis and rhipicephalus simus all use rodents as hosts (petney et al. 2004) and h. rufipes use hares and birds (horak &fourie 1991; van niekerk, fourie & horak 2006). adult h. elliptica use carnivores and large felids as hosts and the adults of the other three ticks use cattle and other large hosts. engorged female ticks detach from these hosts wherever they may roam, feed or rest, and then seek out a sheltered environment to lay their eggs. very few larvae of these species are usually collected from the vegetation and in the absence of another explanation it must be assumed that they quest for their smallmammal hosts from the soil surface or from the base of the vegetation. amblyomma hebraeum the first appearance of an a. hebraeum larva on the pastures in 2006 and the presence of a small number of adult ticks on cattle in 2007 may be the result of the introduction of engorged immature ticks on birds, particularly helmeted guineafowls and francolins, from neighbouring farms. these birds may harbour exceptionally large numbers of a. hebraeum larvae and lesser numbers of nymphs (horak et al. 1991a; uys & horak 2005). the long-term (1969–2001) mean annual rainfall at döhne is 773 mm, although, with the exception of 1992 when only 382 mm was recorded, the 490 mm recorded in 2009 is the lowest for the past 40 years. estrada-peña, horak and petney (2008) noted that more intense periods of drought in the highveld of zimbabwe may leadto an expansion of the distribution of a. hebraeum in this region, whereas increased temperatures may have the same consequence in the lowveld. the treed-savanna habitat at campagna constitutes a suitable vegetational environment for all stages of development of a. hebraeum. moreover, the lower than normal rainfall in the last year of the study, coupled with sustained high summer temperatures, may have contributed to an optimal environment in which a small existing population of tickscould flourish or in which introduced ticks could become established. larvae and adult ticks were collected from the vegetation and cattle at campagna, but only larvae were collected from the pastures at döhne. howell, walker andnevill (1978) state that a. hebraeum cannot survive in open grasslands, and horak, gallivan and spickett (2011) collected fewest questing a. hebraeum larvae from grasslands compared to woodlands and gullies in a treed savanna locality in the kruger national park. the open grass pastures at döhne would thus seem unlikely to sustain a. hebraeum; nevertheless, some ticks must have completed their life cycles there, hence the presence of questing larvae. hyalomma rufipes this is a two-host tick and its immature stages infest hares and ground-feeding birds (horak & fourie 1991; van niekerk et al. 2006). because most larvae are likely toquest for their relatively small hosts from the soil surface, few were collected by dragging. campagna provided a suitable environment for all stages of development of h. rufipes, whereas the open pastures of döhne did not. the small number of adult ticks collected from bonsmara cattle at döhne each year could represent an existing population or be the consequence of engorged nymphs transported there on birds. the summer abundance of adult h. rufipes agrees with that observed oncattle in limpopo province and in the south-western free state (fourie & horak 1990; schröder 1980). rhipicephalus appendiculatus r. appendiculatus is a three-host species, which, in south africa, completes a single life cycle annually. in valley bushveld, approximately 60 km south-west of döhne, larvae of r. appendiculatus were most abundant on cattle and kudus from late summer to mid-winter, whilst nymphs were found in spring and adults from early tolate summer (horak et al. 1992; horak 1999). this pattern of seasonality is not unlike that observed for free-living larvae and nymphs on the pastures and parasitic adult ticks on the cattle at the döhne complex. theiler (1962) stated that r. appendiculatus is consistently absent in open grasslands, whilst short et al. (1989) reported reduced survival of the free-living stages of r. appendiculatus in short grass habitats in zimbabwe. this does not appear to be the case at the döhne complex, where slightly more r. appendiculatus larvae were collected from the open grass pastures and adult ticks from the bonsmara cattle at döhne than from the vegetation and cattle in the treed savanna habitat ofcampagna. rhipicephalus decoloratus r. decoloratus is a one-host species and consequently only larvae are expected to quest for hosts from the vegetation; however, a single male was also collected from a drag sample. this phenomenon will be discussed in greater detail for r. microplus. the spring rise in the number of questing larvae, accompanied by an increase in the number of adult ticks, would appear to be the impetus required to maintain the parasitic life cycle for the remainder of the year. the decline in numbers inthe late summer probably results from resistance to infestation acquired by the cattle and hence fewer females detaching and laying eggs, thus resulting in fewer questing larvae. similar findings on questing larvae have been recorded at two localities in the kruger national park (horak et al. 2011). rhipicephalus evertsi evertsi r. evertsi evertsi is a two-host tick with one of the most widespread distributions of any tick species in sub-saharan africa (walker, keirans & horak 2000). with the exception of the arid northern cape province and northern western cape province it is present throughout south africa (howell et al. 1978). in an open, treedsavanna in the south of the kruger national park, most r. evertsi evertsi larvae were collected in grassland compared to woodland and gully habitats (gallivan et al. 2011). it infests all domestic and wild bovids and equids, with hares also being effective hosts of the immature stages (walker et al. 2000). despite the species’ widespread distribution and extensive host range, adult ticks are never recovered in large numbers. r. evertsi evertsi can complete more than one life cycle annually and thus more than one peak in the abundance of immature and adult ticks can occur in a single year. the pattern of summer, winter and spring peaks in the numbers of questing larvae collected from the pastures at döhne (figure 5c) is similar to that found forticks on sheep on a highveld farm in the eastern free state (horak, williams & van schalkwyk 1991b). the decrease in numbers of questing larvae on the pastures at döhne with decreasing rainfall, whilst the numbers of adult ticks on cattle increased is difficult to explain. rhipicephalus follis this tick has a strictly south african distribution and occurs in hilly or mountainous terrain in the east of the country, from the southern and south-eastern coastal regions to the escarpment in the mpumalanga province (walker et al. 2000). the adults prefer large herbivorous animals as hosts, whilst the immature stages prefermurid rodents. large numbers of adult ticks have been collected from elands in the mountain zebra national park near cradock (horak et al. 1991c), and immature ticks from four-striped mice in the thomas baines reserve near grahamstown (petney et al. 2004). adult ticks appear to be present throughout the year (figure 5c), as also found by horak et al. (1991c). few larvae were collected from the vegetation, probably because they quest for their murid rodent hosts from the soil surfaceor from the base of the vegetation. the decrease in numbers of r. follis adults over time was probably associated with a decrease in rodent populations during a period of below-average rainfall. rhipicephalus microplus one of the first documented records of r. microplus in south africa is that made by howard (1908). he referred to it as margaropus annulatus australis, but his illustrations clearly depict r. microplus. howard recorded the species’ presence at king william’s town (approximately 50 km south-east of döhne) and in the southern districts of the cape colony. theiler (1962) cites dönitz as stating that this species (as boophilus annulatus) seems to be displacing r. decoloratus in south africa inland from the coast. the distribution of r. microplus described by howell et al. (1978) is discontinuous and extends from the southern regions of the western cape province along the coast and adjacent inland regions to north-eastern kwazulu-natal, with scattered localities in the northern provinces. since thenits distribution has expanded remarkably, usually at the expense of r. decoloratus, and this phenomenon has been particularly noticeable in the eastern region of the eastern cape province (horak et al. 2009). reasons for this displacement have been suggested by horak et al. (2009). both species are, however, present at döhne and although r. microplus is predominant, r. decoloratus appears to have maintained its status over the past five years.theoretically, only larvae of this one-host tick should quest for hosts from the vegetation. however, 11 male ticks were also collected from the drag cloths, implying that they must have detached shortly before or after moulting and were now questing from the vegetation for a second host. mason and norval (1981) mention that norval (unpublished data) had collected adult male ticks from drag samples and they then demonstrated that larvae and adult males of r. microplus could transfer from infested to uninfested cattle under field conditions. larvae of r. microplus were the most numerous of all species collected from the vegetation at the döhne complex and significantly greater numbers (p < 0.001) were collected from the pastures grazed by bonsmara cattle than from those grazed by nguni cattle. similarly, the numbers of adult ticks collected from the bonsmara cattle differed significantly (p < 0.001) from those collected from the nguni cattle. less commonly collected species haemaphysalis aciculifer is present in patches along the south-eastern, southern and eastern coastline and adjacent inland regions of south africa. its immaturestages infest rodents and birds and its adults infest various antelope species (horak & boomker 1998). h. elliptica, previously referred to as haemaphysalis leachi, parasitises domestic dogs and cats and the larger wild felids (apanaskevich, horak & camicas 2007) and its presence on the pastures and cattle in this study are probably a consequence of the odd dog or feral cat traversing the camps. haemaphysalis silacea is essentially a tick of the valley bushveld and surroundingwell-wooded regions of the eastern cape province, where all stages parasitise kudus and cattle (horak et al. 1992; horak 1999). the few ticks collected at döhne probably represent an eastern fringe of its distribution. ticks of the ixodes pilosus group are present along the south-western coast and adjacent areas of the western and eastern cape provinces, extending to the southern coastal and adjacent inland regions of kwazulu-natal (howell et al. 1978). rhipicephalus lounsburyi is a strictly south african species and is present in the more mountainous and hilly regions of the eastern cape province (walker et al. 2000). the geographic distribution of r. simus is more extensive than that of r. follis and although their distributions overlap (walker et al. 2000), one or the other is usually more numerous at a particular locality, depending on the environment. conclusion (back to top) at a locality in the eastern cape province, south africa, where 13 species of ixodid ticks were present, nguni cattle proved to be less susceptible to infestation withadult ticks than bonsmara cattle. fewer questing ticks were also collected from pastures grazed by nguni cattle than from those grazed by bonsmara cattle. acknowledgements (back to top) we thank the eastern cape department of agriculture for permission to conduct the study at döhne and campagna and for approving the participation of messrs nyangiwe and goni in the project. ritha wentzel of the agricultural research council kindly provided us with the climatological data. the study was funded by the university of pretoria, bayer animal health, the national research foundation and the institute for tropical medicine, belgium. authors’ contributions n.n. and i.g.h. planned the survey; n.n. was the project leader and collected the ticks and assisted with the manuscript; s.g. assisted n.n. with the tickcollections; l.p.h-c. and i.r. performed statistical calculations and wrote the statistical part of the manuscript; i.g.h. identified the ticks and wrote the manuscript. references (back to top) acocks, j.p.h., 1988, veld types of south africa with accompanying veld type map, 3rd edn., botanical research institute, department 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parasitology 8, 185–188. doi:10.1016/0304-4017(81)90046-7 norval, r.a.i. & horak, i.g., 2004, ‘vectors: ticks’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, pp. 3–42, oxford university press, cape town. petney, t.n., horak, i.g., howell, d.j. & meyer, s., 2004, ‘striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks’, onderstepoort journal of veterinary research 71, 313–318. pmid:15732458 rechav, y., 1987, ‘resistance of brahman and hereford cattle to african ticks with reference to serum gamma globulin levels and blood composition’, experimental and applied acarology 3, 219–232. doi:10.1007/bf01270458 rechav, y., dauth, j. & els, d.a., 1990, ‘resistance of brahman and simmentaler cattle to southern african ticks’, onderstepoort journal of veterinary research 57, 7–12. pmid:2339000 rechav, y. & kostrzewski, m.w., 1991, ‘relative resistance of six cattle breeds to the tick boophilus decoloratus in south africa’, 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veterinary research 59, 285–292. pmid:1297958 theiler, g., 1962, ‘the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region)’, division of veterinary services, onderstepoort (mimeographed). uys, a.c. & horak, i.g., 2005, ‘ticks on crested francolins, francolinus sephaena, and on the vegetation on a farm in limpopo province, south africa’, onderstepoort journal of veterinary research 72, 339–343. pmid:16562738 van niekerk, j., fourie, l.j. & horak, i.g., 2006, ‘birds as hosts of immature ixodid ticks in free state province, south africa’, onderstepoort journal of veterinary research 73, 123–130. pmid:16958263 walker, j.b., 1991, ‘a review of the ixodid ticks (acari, ixodidae) occurring in southern africa’, onderstepoort journal of veterinary research 58, 81−105. pmid:1881661 walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. article information authors: arthur m. spickett1 i. heloise heyne1 roy williams2 affiliations: 1parasites, vectors and vector-borne diseases programme, arc-onderstepoort veterinary institute, south africa 2veterinary geographic information systems programme, arc-onderstepoort veterinary institute, south africa correspondence to: arthur spickett email: spickettam@arc.agric.za postal address: private bag x05, onderstepoort 0110, south africa dates: received: 12 nov. 2010 accepted: 12 jan. 2011 published: 14 sept. 2011 republished: 09 nov. 2011 how to cite this article: spickett, a.m., heyne, i.h. & williams, r., 2011, ‘survey of the livestock ticks of the north west province, south africa’, onderstepoort journal of veterinary research 78(1), art. #305, 12 pages. doi:10.4102/ojvr.v78i1.305 note: this article was republished with the corrected name of ingard h. heyne. copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) survey of the livestock ticks of the north west province, south africa in this original research... open access • abstract • introduction • materials and methods    • study locality    • survey design and execution • results and discussion    • number of ticks, species composition and collections    • tick species distribution and seasonality       • amblyomma hebraeum       • hyalomma rufipes       • hyalomma truncatum       • rhipicephalus appendiculatus       • rhipicephalus (boophilus) decoloratus       • rhipicephalus (boophilus) microplus       • rhipicephalus evertsi evertsi       • rhipicephalus evertsi mimeticus       • rhipicephalus simus    • tick-borne disease serology • conclusion • acknowledgements • references abstract (back to top) ticks, as vectors of disease and damage agents, impact directly and indirectly on the economy of the livestock industry in southern africa. this study surveyed the occurrence and distribution of ticks infesting livestock across the north west province, south africa. during three phases in consecutive years, officers of the provincial veterinary department collected specimens monthly from livestock hosts at specified sites across the province. data analysis constituted the fourth phase of the study. a total of 1090 collections from 265 sites yielded 42 566 tick specimens, comprising 22 different tick species (18 ixodids, 4 argasids). the specimens represent all of the major tick vectors of disease that occur in south africa. the major tick-borne diseases (i.e. heartwater, both african and asiatic bovine babesiosis and anaplasmosis) were found to be prevalent mainly in the north-eastern region of the province, which also displayed the highest tick species diversity. the central region appears transitory to some of the major vectors. although some tick species were contained within specific regions, others were widespread across the province. associated serology data show that most herds sampled in areas endemic for babesiosis and anaplasmosis in the north-eastern region are endemically unstable and at risk to these tick-borne diseases should vector control measures become ineffective. introduction (back to top) ixodid ticks are economically of the most important external parasites of livestock in the tropical and sub-tropical parts of the world, including south africa. heavy infestation can cause loss of blood, reduce live weight gain rates and lower milk yield, whilst the long-mouthed ticks downgrade the quality of the hides and cause secondary infections (de castro 1997). they are also recognised worldwide as major vectors of a number of disease-causing pathogens in humans and domestic and game animals, including arboviruses, rickettsiae, spirochaetes and parasitic protozoa. they act as reservoirs and/or multipliers of these organisms and transmit a number of important tick-borne diseases such as heartwater, bovine babesiosis, anaplasmosis and theileriosis. some ixodid ticks also produce toxins that cause paralysis in sheep (spickett & heyne 1988) and sweating sickness in calves (jongejan & uilenberg 1994). ticks therefore impact both directly (by virtue of heavy infestations) and indirectly (through the transmission of tick-borne diseases) at an economical level as well as in a social context. at the macro-economic level, exports and the commercial production of protein may be affected and at the micro-economic level the subsistence economy of the resource-poor farmer may be at risk. whilst little actual data exist, it was estimated already some 30 years ago that mortality losses attributed to tick-borne diseases, together with acaricide and vaccine costs, were in the region of r70 million per annum (van rensburg 1981), whilst a panel of the food and agriculture organization placed total losses in south africa at r550 million per annum in 1985. however, these are estimates at best and many resources would have to be harnessed in order to determine more accurate figures regarding the economic impact of ticks. only one study – on heartwater, which is probably the most important tick-borne disease in south africa – has been attempted (minjauw 2005), with total annual losses estimated at r189.6 million. paramount to assessing the impact of vectors and disease agents are the identification of the species involved, their distribution and their association with disease. long-term research since the time of arnold theiler in the early 20th century, including the last comprehensive tick survey in south africa by gertrud theiler in 1957 (theiler 1962), has placed south africa in the fortunate position of having a wide knowledge of ticks and their associated disease epidemiologies. these findings are documented in wide-ranging publications, with the most important disease-oriented knowledge being condensed into two publications (coetzer & tustin 2004; walker, keirans & horak 2000). in the south african context, some 90 ixodid and 25 argasid tick species have been identified. of these, 35 are normally associated with domestic animals and approximately 15 are considered of economic importance. they transmit, or are associated with, some 23 diseases or health syndromes (norval & horak 2004). in the context of ticks and tick-borne diseases affecting livestock resources, the directorate of veterinary services in the north west province (department of agriculture), commissioned a survey to determine tick species presence, their seasonal occurrence, contribution to livestock diseases and distribution across the province. materials and methods (back to top) study locality the survey was conducted over a total area of 116 320 km² in the north west province of south africa (between approximately 24°15’s, 22°30’e and 28°15’s, 28°30’e). the area may, according to acocks (1988), be sectored roughly into three main regions based on vegetation (figure 1). the north-eastern region consists of bushveld (mixed, sour and sourish-mixed bushveld, the latter interspersed with bankenveld and turf thornveld). the central region has an equally mixed vegetation, consisting of dry and sandy cymbopogon–themeda veld with sourish-mixed bushveld in its northern reaches. the western, more arid region of the province consists mainly of kalahari thornveld. figure 1: vegetation types of the north west province of south africa, according to acocks, j.p.h., 1988, veld types of south africa with accompanying veld type map, 3rd edn., department of agriculture and water supply, pretoria. the climate of the province is characterised by well-defined seasons: hot summers and cool, sunny winters. the climate and rainfall vary from the more mountainous and wetter eastern region to the drier, semi-desert plains of the kalahari in the west. the rainy season usually falls between october and march. rainfall is highly variable, with regard to both time and region. on average, the western part of the province receives less than 300 mm rainfall per annum, the central part around 550 mm and the eastern and south-eastern parts receive more than 600 mm. there are wide seasonal and daily variations in temperature. summers are very hot (daily average maximum temperatures of 32 ºc in january) and winters are mild to cold (average daily minimum in july is 1 ºc). the far western part is arid, encompassing the eastern portion of the kalahari desert. the central part of the province is typically semi-arid, whilst the eastern region is predominantly temperate. agriculture is the second most important economic activity. the eastern, wetter region of the province largely supports livestock and crop farming, whilst the central and southern regions are dominated by wheat and maize farming. livestock and game farming occur in the semi-arid western region of the province. tourism is considered to have a major growth potential as the province has a rich natural heritage and a network of well-managed, malaria-free national and provincial parks and nature reserves, which boast the ’big five’. according to the department of agriculture, forestry and fisheries (2004) livestock production revolves around the estimated 1.77 million cattle, 729 000 sheep and 753 000 goats in the province. approximately 43% of cattle, 46% of sheep and 47% of goats are owned by the communal sector. the majority of cattle are found in the north-eastern region (42%), with 34% and 24% being in the central and western regions, respectively. sheep and goats are farmed predominantly in the western region (44% and 46%, respectively), whilst only to a limited extent in the north-eastern and central regions (sheep: 29% and 27%, respectively; goats: 34% and 20%, respectively). it is this considerable livestock resource that could be adversely affected by ticks and tick-borne diseases. survey design and execution the survey was conducted in four phases over a number of years since 2001. the first three phases included monthly tick collections from identified sites in the north-eastern, central and western regions during three consecutive years (figure 2). figure 2: state veterinarian areas in the north-eastern, central and western regions that served as collection sites for the three-phase tick collection survey in the north west province, south africa. collection sites were selected from 1:250 000 charts and subsequently between four and 41 collection sites, depending on spatial distribution, logistics and collectors’ time constraints, were assigned to specific areas in each of the three regions (table 1). areas considered marginal for specific tick species were included in the selected sites. a total of 114 sites were selected in the north-eastern region (collection phase 1), 116 in the central region (collection phase 2) and 61 in the western region (collection phase 3). as comprehensive coverage as possible of the province was sought to allow for tick distribution mapping. collection sites were not assigned to the immediate north-east and north-west of bloemhof (central region) owing to crop farming predominating in these areas. table 1: sampling areas in three regions of the north west province, south africa. during each collection phase, nominated animal health technicians from each region received in-house training in basic tick identification, collection and specimen handling before collection commenced at their specified sites. the fourth phase (performed at the onderstepoort veterinary institute [ovi]) involved species identification, database acquisition, distribution mapping using arcgis (esri) software and reporting. ticks were collected off one side of livestock hosts – mainly cattle, goats and sheep – using forceps. each collection was placed in 70% alcohol in a specimen bottle for dispatch to the ovi for identification and recording. only one collector, in the bray area, consistently sampled chicken coops. owing to prior training collectors were able to perform basic tick species identification in situ, so that as wide a range of species present on the animal could be collected. to gain data on species seasonality, monthly collections from most sites were attempted. tick collections were, however, disrupted to some extent during the second phase (central region) because of a swine fever outbreak in the eastern cape, which required secondment of personnel to assist with containment operations. in some areas of the north-eastern region, a high incidence of both rhipicephalus (boophilus) decoloratus and rhipicephalus (boophilus) microplus, the vectors of bovine babesiosis, was evident. a standard immunofluorescent antibody test (joyner et al. 1972) and a competitive inhibition enzyme-linked immunosorbent assay (ndung’u et al. 1995; visser et al. 1992) were used to test blood samples for antibodies to babesia bovis and babesia bigemina, and anaplasma marginale, respectively. sera collected from 13 properties in the affected areas (from at least 30 cattle per herd, 9–12 months of age) were tested to determine the antibody status of these herds to african and asiatic babesiosis and anaplasmosis. results and discussion (back to top) number of ticks, species composition and collections a total of 42 566 ticks (from 1190 collections across 265 collection sites) were received for identification. the breakdown of collections is as follows: (1) north-eastern region: 21 045 specimens from 429 collections (2) central region: 10 391 specimens from 398 collections (3) western region: 11 130 from 363 collections. owing to the collection method employed almost all the ticks received were adults (97.9%), with roughly equal numbers of males and females. the immatures (nymphs = 1.8%; larvae = 0.3%), mostly rhipicephalus evertsi evertsi and four nymphs of otobius megnini, were collected more by chance than intention from the ears of cattle. altogether 22 tick species were identified (18 from six ixodidae genera; four from three argasidae genera). the main tick species were all hard ticks (ixodidae) and included r. evertsi evertsi (31.5%), hyalomma rufipes (22.4%), amblyomma hebraeum (17.3%), r. (b.) decoloratus (12.5%), rhipicephalus appendiculatus (11.2%), hyalomma truncatum (2.3%), r. (b.) microplus (1.6%), rhipicephalus evertsi mimeticus (0.5%) and rhipicephalus simus (0.21%). other ticks of lesser importance or those collected in low numbers or during only a few collections, as well as the argasidae collections, are as shown in table 2. table 2: ixodid ticks of lesser importance and argasid ticks identified from each of the three collection regions. monthly collections over a period of 10–12 months were received from 70 collection sites, which allowed the seasonal occurrence of the major tick vectors to be determined. monthly collections from a further 175 collection sites were received over a period varying from 1 to 9 months. tick species distribution and seasonality the distribution patterns and seasonality of the major tick species collected are described below. amblyomma hebraeum according to norval and horak (2004), amblyomma hebraeum is present in grassed bushveld and wooded savanna regions. in the present survey, a. hebraeum accounted for 17.3% of the collected ticks and appears well established in the mixed and sourish-mixed bushveld areas of the north west province (figure 3). it is present mainly in the north-eastern and northern portion (mixed bushveld) of the central region, with singular intrusions into the kalahari thornveld south-east of vryburg and west of mafikeng. the latter distribution appears to be established populations because they were consistently collected during the summer months at these sites. figure 3: the geographic distribution of amblyomma hebraeum in the north west province, south africa. adult ticks prefer large hosts such as cattle and large wild ruminants, but also infest sheep and goats (horak et al. 1987; walker et al. 2003). in this survey 90.8% of a. hebraeum specimens were recovered from cattle, 4.9% from goats and 3.3% from sheep (1.0% were not stipulated). the adults of this species were present throughout the year, with a high incidence in both the north-eastern and central regions during november and december (summer), similar to observations by rechav (1982) for the eastern cape. higher numbers of adults were found in the central region between march and may (autumn) than in the north-eastern region during these months. hyalomma rufipes h. rufipes (previously the subspecies hyalomma marginatum rufipes [apanaskevich & horak 2008]) is a vector of the crimean–congo haemorrhagic fever virus. it is the most widely distributed species of this tick genus in south africa, occurring in the greater part of the country (norval & horak 2004). it was found distributed widely over the whole of the north west province, with it being absent from only eight of the collection sites (figure 4). the life cycle may be completed in one year and peak numbers of adults were evident in the summer months (november–february). figure 4: the geographic distribution of hyalomma rufipes in the north west province, south africa. h. rufipes accounted for 22.4% of the total number of specimens collected in this survey. of these, 97.2% were from cattle, 1.5% from sheep and 0.5% were found on goats (0.8% were not assigned a host). hyalomma truncatum h. truncatum is widely distributed over the whole of the north west province (figure 5) and was present in all the ecological zones sampled. of the adult h. truncatum specimens collected, 89.9% were from cattle, 0.8% from sheep and 8.5% from goats; 0.8% were not assigned a host. h. truncatum accounted for only 2.3% of the total number of specimens collected. some strains of this species transmit a toxin that causes an epitheliotrophic condition called sweating sickness, which affects young calves in particular (neitz 1959). the tendency for adults to attach preferentially in the tail switch of cattle (where it is easily overlooked during sampling) may explain why this tick was not collected at more of the collection sites. its seasonal occurrence in the north west province confirms its annual life cycle, with adults reaching peak numbers between january and march. figure 5: the geographic distribution of hyalomma truncatum in the north west province, south africa. rhipicephalus appendiculatus r. appendiculatus is restricted to the higher-rainfall, eastern regions of south africa (walker et al. 2000) and according to theiler (1962) does not occur in open grassland without bush; it prefers tall grass interspersed with trees. accordingly, this species was found well established in the north-eastern region of the province and in the bushveld areas of the northern reaches of the central region around mafikeng (figure 6). the remainder of the province, especially the western region, appears to be ecologically unsuited to the establishment of this tick species. figure 6: the geographic distribution of rhipicephalus appendiculatus in the north west province, south africa. this species accounted for 11.2% of the specimens collected. of the total number of adults collected, 86.9% were from cattle, 7.4% from goats and 5.0% from sheep; the remaining 0.7% were not assigned a host. adult activity commenced only during the summer months (between december and march), as reported for the eastern cape by rechav (1982). rhipicephalus (boophilus) decoloratus the ticks we have chosen to name r. (b.) decoloratus and r. (b.) microplus were originally known as boophilus decoloratus and boophilus microplus, respectively. however, based on molecular evidence (beati & keirans 2001; murrell, campbell & barker 2000), they were included in a 2002 world list of valid tick names as r. (b.) decoloratus and r. (b.) microplus (horak, camicas & keirans 2002). guglielmone et al. (2010) have subsequently omitted the subgenus and these ticks appear in their list of valid tick names as r. decoloratus and r. microplus. we chose to include the subgenus in our nomenclature. r. (b.) decoloratus represented 12.5% of the collections in this survey, with 98.1% of specimens being collected from cattle, only 0.5% from sheep and 0.3% from goats. the remaining 1.1% were not assigned hosts. this distribution confirms its host preference for large ungulates (mason & norval 1980). the species normally occurs in temperate savanna regions, typically in grassand woodland areas used by cattle, and tends to be absent in drier areas (walker et al. 2003). it is well established in the grassed bushveld biomes (especially the sourish-mixed bushveld) of the north-eastern region and northern reaches of the central region of the north west province, around rustenburg, zeerust, mafikeng and potchefstroom (figure 7). the tick also inhabits seemingly suitable habitats (probably created by irrigated land use) in the cymbopogon–themeda veld near bloemhof and the kalahari thornveld near kudumane and vryburg in the western region. apparently, suitably high temperatures – well above the developmental threshold of 10 ºc (spickett & heyne 1990) – and host availability yielded high numbers of this one-host species, even during the winter months. peak numbers were evident in december and january and again from march to july. since r. (b.) decoloratus is a vector of b. bigemina, african babesiosis may therefore be a threat throughout the year. figure 7: the geographic distribution of rhipicephalus (boophilus) decoloratus in the north west province, south africa. rhipicephalus (boophilus) microplus r. (b.) microplus, as r. (b.) decoloratus, inhabits savanna climatic regions in wooded grasslands used as cattle pasture (walker et al. 2003). r. (b.) microplus (figure 8) was found to have a more limited distribution compared to that of r. (b.) decoloratus, being confined to the sourish-mixed bushveld of the north-eastern region (around rustenburg, north-east of potchefstroom and north of zeerust). the species was also found in an isolated pocket near bloemhof, probably introduced by cattle and finding suitable habitats here. figure 8: the geographic distribution of rhipicephalus (boophilus) microplus in the north west province, south africa. cattle are considered the only effective hosts of r. (b.) microplus (mason & norval 1980), to the extent that this species is absent in game parks, where no cattle occur (horak et al. 1986). recently, goats have been implicated as alternate hosts for this species, but in the presence of cattle in order to maintain populations (nyangiwe & horak 2007). in the present survey 81.8% of r. (b.) microplus specimens were collected from cattle and, significantly, a relatively high 7.7% were found on goats. none were found on sheep. the remaining 10.5% of the collected r. (b.) microplus specimens were not assigned a host by the collectors, but were most probably collected off cattle. the seasonality of r. (b.) microplus was similar to that of r. (b.) decoloratus, with peak numbers occurring during the autumn months of april and may. rhipicephalus evertsi evertsi r. evertsi evertsi is widely distributed and common on livestock throughout much of africa, occurring in desert, steppe, savanna and temperate climatic regions (walker et al. 2003). it has the most widespread distribution of species in the genus rhipicephalus in africa and has an extensive host range (walker et al. 2000). it was the most commonly collected species in the north west province (31.5% of all specimens), being distributed over the whole province and absent at only two collection sites (figure 9). in this survey, the adults that were collected came from cattle (79.1%), goats (15.9%) and sheep (3.9%), whilst the remaining 1.1% were unaccounted for as to host. peak activity was recorded early in autumn (may), although high numbers were also recorded during the summer months. figure 9: the geographic distribution of rhipicephalus evertsi evertsi in the north west province, south africa. rhipicephalus evertsi mimeticus although of minimal economic importance, this species is interesting in that it is recorded as occurring in the arid regions of angola, namibia and botswana (theiler 1962). with regard to morphology and biology it is very similar to its subspecies r. evertsi evertsi and can also cause paralysis in sheep, mainly lambs, owing to secreting a toxin in the saliva whilst feeding (gothe 1999). this species has infiltrated the north west province, with suitable habitat conditions enabling it to establish populations in the western region (figure 10), possibly by means of sheep introduced from botswana or namibia, and thus likely displacing r. evertsi evertsi at these localities. recurring monthly collections of relatively high numbers off sheep from these sites confirmed the establishment of viable r. evertsi mimeticus populations. being subspecies, r. evertsi mimeticus and r. evertsi evertsi should interbreed, yet both were found to be morphologically distinct and could easily be distinguished taxonomically at the same localities, which may indicate some degree of speciation. this observation needs further investigation. figure 10: the geographic distribution of rhipicephalus eversti mimeticus in the north west province, south africa. rhipicephalus simus this species establishes in regions with a savanna climate and is never encountered in high numbers (norval & mason 1981; walker et al. 2000). it is well established in the bushveld areas of the north-eastern region of the province to which its distribution is confined (figure 11). of the r. simus collected, 71.9% were from cattle, 4.0% from sheep and 18.5% from goats. this species was also recovered from dogs (4.0%), whilst 1.6% were not assigned a host by collectors. figure 11: the geographic distribution of rhipicephalus simus in the north west province, south africa. tick-borne disease serology as shown in figures 7 and 8, r. (b.) decoloratus and r. (b.) microplus occurred sympatrically at some localities in the north-eastern region of the province. both species are associated with the transmission of the causative organism of anaplasmosis (ana. marginale) as well as the organisms causing bovine babesiosis (babesia bigemina and babesia bovis) (de vos & potgieter 1994). the antibody status of cattle herds to these tick-borne pathogens in the areas of sympatric distribution of these two tick vector species, as well as the presence (or absence) of the two tick vector species is shown in table 3. table 3: serology results from livestock at various localities and associated presence or absence of vectors. the results indicate that only one property (commiesiedrift) is in a state of endemic stability (norval et al. 1983) to b. bigemina (100%), whereby sufficient infected vector ticks (r. [b.] decoloratus) are present to transmit the pathogen such that all the animals show an antibody response indicative of disease immunity. all other properties appear to experience endemic instability to both b. bigemina and b. bovis, strongly suggesting that intensive chemical control is practiced on these properties, thereby reducing vector challenge and, subsequently, pathogen transmission. on the endemically unstable properties, more than 40% of the cattle show no antibodies to the causative organisms and are thus fully susceptible and at risk to babesiosis should they be challenged by infected ticks. none of the animals on six properties showed antibodies to b. bigemina despite the presence of the tick vector, possibly owing to uninfected ticks (unlikely) or extremely stringent tick control practices. these animals could be completely susceptible to bovine babesiosis should they be challenged by infected ticks. r. (b.) microplus was absent on five of these properties and, as expected, none of the animals tested positive for b. bovis antibodies. however, on one of the properties (leeukraal) on which no animals tested positive for b. bigemina in the presence of the tick vector, 50% of the animals tested positive for b. bovis in the presence of the vector tick. the latter case is difficult to explain: it is possibile that r. (b.) microplus, as a vector, presented a much higher challenge than did r. (b.) decoloratus and that antibody manifestation to b. bigemina was subsequently lost. on two properties, respectively 28% and 40% of the animals showed antibody titres to b. bovis, indicative of r. (b.) microplus infestation, although this vector tick was not collected at the time of the survey. conclusion (back to top) this study was conducted to survey the occurrence and distribution of ticks infesting livestock in the north west province of south africa. the survey entailed the monthly collection of tick specimens from livestock hosts at specified sites in the north-eastern, central and western regions of the province. tick specimens were subsequently identified and the distributions of the major species plotted. according to this survey, livestock in this province harbour 22 tick species (18 ixodids; 4 argasids). the major tick-borne diseases were prevalent mainly in the north-eastern region, which also displayed the highest tick species diversity. the vectors of corridor disease (buffalo-associated theileria parva), namely r. appendiculatus and rhipicephalus zambeziensis were present in the north-eastern region of the province, which indicates that care should be exercised in the introduction of corridor-infected buffalo in these regions. the central region appears transitory to the major vectors a. hebraeum and r. (b.) decoloratus, whilst the two hyalomma vectors of crimean–congo haemorrhagic fever virus are widespread over the whole province. the north-western area (bray) of the western region has been infiltrated with r. evertsi mimeticus, a species considered to be non-endemic to south africa. most herds sampled for serology in areas endemic for babesiosis and anaplasmosis in the north-eastern region are endemically unstable and at risk to these tick-borne diseases should vector control measures become ineffective. acknowledgements (back to top) this study was funded by the directorate of veterinary services of the north west province (department of agriculture). we wish to thank the directorate for their wholehearted and unstinting support for this survey, at managerial and operational level. special thanks go to the many officers directly involved in the collections. references (back to top) acocks, j.p.h., 1988, veld types of south africa with accompanying veld type map, 3rd edn., department of agriculture and water supply, pretoria. apanaskevich, d.a. & horak, i.g., 2008, ‘the genus hyalomma koch, 1844. v. re-evaluation of the taxonomic rank of taxa comprising the h. (euhyalomma) marginatum group of species (acari: ixododidae) with redescription of all parasitic stages and notes on biology’, international journal of acarology 34, 13−42. doi:10.1080/01647950808683704 beati, l. & keirans, j.e., 2001, ‘analysis of the systematic relationships among ticks of the genera rhipicephalus and boophilus (acari: ixodidae) based on mitochondrial 12s ribosomal dna gene sequences and morphological characters’, journal of parasitology 87, 32−48. doi:10.2307/3285173, pmid:11227901 coetzer, j.a.w. & tustin, r.c., 2004, infectious diseases of livestock, oxford university press, cape town. de castro, j.j., 1997, ‘sustainable tick and tickborne disease control in livestock improvement in developing countries’, veterinary parasitology 71, 77−97. doi:10.1016/s0304-4017(97)00033-2 de vos, a.j. & potgieter, f.t., 1994, ‘bovine babesiosis’, in j.a.w. coetzer, g.r. thomson & r.c. tustin (eds.), infectious diseases of livestock with special reference to southern africa, pp. 278–294, oxford university press, cape town. department of agriculture, forestry and fisheries, 2004, livestock statistics 2004, department of agriculture, forestry and fisheries, pretoria. gothe, r., 1999, zeckentoxikosen [tick toxicoses], hieronymus buchenproduktion, munchen. guglielmone, a.a., robbins, r.g., apanaskevich, d.a., petney, t.n., estrada-peña, a., horak, i.g. et al., 2010, ‘the argasidae, ixodidae and nuttalliellidae (acari: ixodida) of the world: a list of valid species names’, zootaxa 2528, 1−28. horak, i.g., sheppey, k., knight, m.m. & beuthin, c.l., 1986, ‘parasites of domestic and wild animals in south africa. xxi. arthropod parasites of vaal ribbok, bontebok and scrub hares in the western cape province’, onderstepoort journal of veterinary research 53, 187−197. horak, i.g., macivor, k.m., petney, t.n. & de vos, v., 1987, ‘some avian and mammalian hosts of amblyomma hebraeum and amblyomma marmoreum (acari, ixodidae)’, onderstepoort journal of veterinary research 54, 397−403. horak, i.g., camicas, j.-l. & keirans, j.e., 2002, ‘the argasidae, ixodidae and nuttalliellidae (acari: ixodida): a world list of valid tick names’, experimental and applied acarology 28, 27−54. doi:10.1023/a:1025381712339 jongejan, f. & uilenberg, g., 1994, ‘ticks and control methods’, revue scientifique et technique de office international des epizooties 13, 1201−1226. joyner, l.p., donnely, j., payner, r. & brocklesby, d.w., 1972, ‘the indirect fluorescent antibody test for the differentiation of infections with babesia divergens or babesia major’, research in veterinary science 13, 515–518. pmid:4567732 mason, c.a. & norval, r.a.i., 1980, ‘the ticks of zimbabwe. i. the genus boophilus’, zimbabwe veterinary journal 11, 36−43. minjauw, b., 2005, ‘the economic impact of heartwater (cowdria ruminantium infection) in the republic of south africa, and of its control through the use of new inactivated vaccines’, draft country report, uf/usaid/sadc heartwater research project, washington, dc. murrell, a.n., campbell, j.h. & barker, s.c., 2000, ‘phylogenetic analysis of the rhipicephaline ticks indicates that the genus rhipicephalus is paraphyletic’, molecular phylogenetics and evolution 16, 1−7. doi:10.1006/mpev.2000.0762, pmid:10877935 ndung’u, l.w., aguirre, c., rurangirwa, f.r., mcelwain, t.f., mcguire, t.c., knowles, d.p. et al., 1995, ‘detection of anaplasma ovis infection in goats by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay’, journal of clinical microbiology 33, 675–679. neitz, w.o., 1959, ‘sweating sickness: the present state of our knowledge’, onderstepoort journal of veterinary research 23, 3−38. norval, r.a.i. & mason, c.a., 1981, ‘the ticks of zimbabwe. ii. the life cycle, distribution and hosts of rhipicephalus simus koch, 1844’, zimbabwe veterinary journal 12, 2−9. norval, r.a.i. & horak, i.g., 2004, ‘vectors: ticks’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, 2nd edn., pp. 3−42, oxford university press, cape town. norval, r.a.i., fivaz, b.h., lawrence, j.a. & daillecourt, t., 1983, ‘epidemiology of tick-borne diseases of cattle in zimbabwe. i. babesiosis’, tropical animal health and production 15, 87–94. doi:10.1007/bf02239802, pmid:6868134 nyangiwe, n. & horak, i.g., 2007, ‘goats as alternative hosts of cattle ticks’, onderstepoort journal of veterinary research 74, 1−7. pmid:17708147 rechav, y., 1982, ‘dynamics of tick populations (acari: ixodidae) in the eastern cape province of south africa’, journal of medical entomology 19, 679−700. pmid:7154025 spickett, a.m. & heyne, h., 1988, ‘a survey of karoo tick paralysis in south africa’, onderstepoort journal of veterinary research 55, 89−92. pmid:3393373 spickett, a.m. & heyne, h., 1990, ‘the prehatch period and larval survival of boophilus decoloratus (koch, 1844) (acarina, ixodidae) under natural conditions in the transvaal, south africa’, onderstepoort journal of veterinary research 57, 95−98. pmid:2339003 theiler, g., 1962, ‘the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region), project s 9958’, report to the director of veterinary services, onderstepoort. van rensburg, s.j.j., 1981, ‘the importance and need for tick control – what it means to the industry’, in g.b. whitehead & j.d. gibson (eds.), tick biology and control: international conference proceedings, grahamstown, south africa, january 27–29, 1981, pp. 119–122. visser, e.s., mcguire, t.c., palmer, g.h., davis, w.c., shkap, v., pipano, e. et al., 1992, ‘the anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized anaplasma species’, infection and immunity 60, 5139–5144. pmid:1280624, pmid:258289 walker, a.r., bouattour, j.-l., camicas, a., estrada-pena, i.g., horak, i.g., latif, a.a. et al., 2003, ticks of domestic animals in africa: a guide to identification of species, bioscience reports, edinburgh. walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. durand_185-187.qxd introduction pastures are continually contaminated by the freeliving stages of various species of parasitic nematodes which infect domestic livestock. the resistance of one such parasite, haemonchus contortus, to anthelmintics in south africa and other parts of the world, led to the formulation of alternative strategies for its control. among these was the use of nematophagous fungi such as duddingtonia flagrans for biological control of infective larvae on pastures. chlamydospores of d. flagrans, fed to livestock, survive the digestive processes and are viable when voided in the faeces. parts of the fungal mycelium growing from germinating chlamydospores become modified to form three-dimensional adhesive nets which trap and then feed on nematode larvae. on contaminated pastures, d. flagrans has been shown to be effective in reducing the number of infective larvae of h. contortus and trichostrongylus colubriformis from sheep, ostertagia ostertagi from calves, strongyles from horses, and oesophagostomum dentatum and hyostrongylus rubidus from pigs (larsen 2000). the aim of this study was to find a south african strain of d. flagrans adapted to local environmental 185 onderstepoort journal of veterinary research, 72:185–187 (2005) research communication survey of nematophagous fungi in south africa d.t. durand1*, h.m. boshoff2, l.m. michael3 and r.c. krecek4 abstract durand, d.t., boshoff, h.m., michael, l.m. & krecek, r.c. 2005. survey of nematophagous fungi in south africa. onderstepoort journal of veterinary research, 72:185–187 three hundred and eighty-four samples of leaf litter, soil, faeces from domestic and game animals, compost and aqueous cultures of infective nematode larvae contaminated with unidentified fungi were plated out on water agar, baited with pure infective larvae of haemonchus contortus, incubated and examined for the presence of nematophagous fungi. duddingtonia flagrans was isolated from five samples, and 73 samples were positive for other nematophagous fungi. keywords: arthrobotrys oligospora, duddingtonia flagrans, haemonchus contortus, nematophagous fungi * author to whom correspondence is to be directed 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. present address: p.o. box 81989, doornpoort, 0017 south africa 3 division of parasitology, agricultural research council, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa. present address: bayer (pty) ltd, p.o. box 143, isando, 1600 south africa 4 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. present address: department of zoology and entomology, faculty of natural and agricultural sciences, university of pretoria, pretoria, 0002 south africa accepted for publication 24 january 2005—editor conditions that could be used in an integrated worm control programme. in addition (although not the primary aim), the occurrence of other nematophagous fungi in the study area was also recorded. materials and methods the modified sprinkling technique of larsen, wolstrup, henriksen, dackman, grønvold & nansen (1991) was used for the isolation of nematophagous fungi. soil, faeces, leaf litter and compost samples collected from five provinces in south africa (gauteng, mpumalanga, north west, limpopo and free state) were sprinkled in a cross configuration on individual plates of 2 % water agar containing 0.02 % tetracycline hydrochloride to suppress bacterial growth. approximately 3 ml of aqueous cultures of infective nematode larvae contaminated with unidentified fungi were pipetted onto the surface of plates containing the same medium. the plates were incubated at 26 °c, baited at least twice a week for 3 weeks with approximately 3 500 infective h. contortus larvae from a pure aqueous culture and examined for signs of nematophagous activity every 2–3 days. the conidia and chlamydospores of some nematophagous isolates were photographed using light microscopy or scanning electron microscopy (sem). others were measured and, in some cases, drawn to facilitate identification. identification was based on the descriptions of type strains (de hoog 1985; rubner 1996). the criteria included the type of structure used to trap larvae, the shape, size and numbers of conidia, the configuration of the conidiophore, and the presence or absence of chlamydospores in mature fungal cultures. conidia growing from trapped larvae on water agar plates were transferred by means of sterile glass inoculation needles to corn meal agar (difco® laboratories) plates, to purify the isolate. pure cultures of six isolates of nematophagous fungi were sent to the “centraalbureau voor schimmelcultures” in the netherlands for expert identification, to either confirm our findings or identify isolates that were problematic. conidia and chlamydospores of d. flagrans were prepared for sem as follows: 1 cm square blocks of corn meal agar cultures were vapour-fixed in osmium tetroxide for 12 h and air-dried for 2 days. the air-dried blocks were mounted on aluminium stubs, sputter coated with gold and viewed in a philips xl 20 scanning electron microscope. results five isolates of d. flagrans and 73 isolates of other nematophagous fungi were obtained from 384 cultures of soil, faeces, compost, leaf litter and aqueous suspensions of infective larvae contaminated with unidentified fungi. the samples were collected within a 500 km radius of pretoria, south africa. the localities included agricultural land, bushveld, urban gardens, nature reserves and a commercial compost processing plant. the most common nematophagous fungus isolated was arthrobotrys oligospora. the other nematophagous fungi isolated were arthrobotrys superba, arthrobotrys dactyloides, arthrobotrys botryospora, arthrobotrys scaphoides and monacrosporium gephyropagum. two isolates of d. flagrans were isolated from compost and three from leaf litter. 186 nematophagous fungi in south africa fig. 1 sem of conidia from a south african isolate of duddingtonia flagrans fig. 2 sem of a chlamydospore from a south african isolate of duddingtonia flagrans discussion twenty percent of the samples cultured were positive for nematophagous fungi and d. flagrans has for the first time been isolated in africa. this provides the opportunity to compare the local fungus with strains from elsewhere in the world as regards their efficacy in reducing infective parasitic larvae on pastures. the doses of d. flagrans chlamydospores required to significantly reduce the number of infective larvae contaminating pasture, are in the order of 105–106 chlamydospores per kilogram body mass per day (peña, miller, fontenot, gillespie & larsen 2002) and the residual effect is limited to 2 days (waller, knox & faedo 2001). locally isolated strains of d. flagrans would need to be cultured in bulk to obtain sufficient quantities of chlamydospores for further studies under african conditions. duddingtonia flagrans grows well on cereals and approximately 250 000 chlamydospores have been counted on the surface of a single grain of barley (grønvold, wolstrup, nansen, henriksen, larsen & bresciani 1993). it has been shown that there is very little genetic variation in isolates of d. flagrans from denmark, the united kingdom, france, germany, the united states of america, australia, malaysia and india (faedo 2001). faedo (2001) suggests that it is possible that d. flagrans from countries in europe was introduced to asia and australia in exported livestock. the south african strains of d. flagrans had not yet been isolated at the time of faedo’s study but the same hypothesis applies. chlamydospores from a strain of d. flagrans isolated in denmark have been produced commercially. this survey has demonstrated the presence of local strains of d. flagrans in south africa and together with the limited genetic variation between isolates from different parts of the world, should address any concerns about importing a foreign fungus into this country. recent studies in australia have been focused on finding a practical method of delivery of d. flagrans chlamydospores to livestock, for use by large scale commercial farmers (waller, faedo & ellis, 2001; waller, knox & faedo 2001). acknowledgements the authors wish to thank the following people and organizations: dr michael larsen and dr kobus van der merwe for their help and encouragement, ms d. josling for the sem preparations and photographs, just nature organics, onderstepoort and mr hans heilgendorff of the national botanical gardens (pretoria) for their cooperation, the food and agricultural organization, u.n, virbac rsa, and the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, for financial support. references de hoog, g.s. (ed.) 1985. taxonomy of the dactylaria complex, iv–vi. studies in mycology, 26:1–122. faedo, m. 2001. growth, trapping and genetic diversity of duddingtonia flagrans—a biological control agent of free-living larval stages of ruminant parasitic nematodes. ph.d thesis, danish centre for experimental parasitology, the royal veterinary and agricultural university, denmark. grønvold, j., wolstrup, j., nansen, p., henriksen, s.a., larsen, m. & bresciani, j. 1993. biological control of nematode parasites in cattle with nematode-trapping fungi: a survey of danish studies.veterinary parasitology, 48:311–325. larsen, m. 2000. prospects for controlling animal parasitic nematodes by predacious micro fungi. parasitology, 120: 121– 131. larsen, m., wolstrup, j., henriksen, s.a., dackman, c., grønvold, j. & nansen, p. 1991. in vitro stress selection of nematophagous fungi for biocontrol of parasitic nematodes in ruminants. journal of helminthology, 65:193– 200. peña, m.t., miller, j.e., fontenot, m.e., gillespie, a. & larsen, m. 2002. evaluation of duddingtonia flagrans in reducing infective larvae of haemonchus contortus in feces of sheep. veterinary parasitology, 103:259–265. rubner, a. 1996. revision of predacious hyphomycetes in the dactylella-monacrosporium complex. studies in mycology, 39:1–134. waller, p.j., faedo, m. & ellis, k. 2001. the potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: towards the development of a fungal controlled release device. veterinary parasitology, 102:299–308. waller, p.j., knox, m.r. & faedo, m. 2001. the potential of nematophagous fungi to control the free-living stages of nematode parasites of sheep: feeding and block studies with duddingtonia flagrans. veterinary parasitology, 102:321– 330. 187 d.t. durand et al. nel_239-246.qxd introduction antimicrobial drugs have been used in animals since shortly after their introduction to human medicine. they have been of great benefit to animals in terms of alleviation of suffering and to humans in helping to meet the growing demand for animal protein and in controlling the agents of potentially serious zoonoses (martel, chaslus-dancla, coudert, poumart & lafont 1995). however, through the increasing use of antimicrobial drugs in humans, animals, fish and crops, an antimicrobial resistance problem has been created that is rapidly moving internationally to the forefront of public health concerns (anon. 1999) with numerous governmental and non-gov239 onderstepoort journal of veterinary research, 71:239–246 (2004) towards the establishment and standardization of a veterinary antimicrobial resistance surveillance and monitoring programme in south africa h. nel1, m. van vuuren1 and g.e. swan2 abstract nel, h., van vuuren, m. & swan, g.e. 2004. towards the establishment and standardization of a veterinary antimicrobial resistance surveillance and monitoring programme in south africa. onderstepoort journal of veterinary research, 71:239–246 the objective of this study was to establish a repeatable, standardized laboratory procedure for monitoring the development of antimicrobial resistance in bacteria isolated from animals and food of animal origin in south africa, with reagents prepared in-house. the emergence of resistance and the spread of resistant bacteria can be limited by implementing a veterinary antimicrobial drug policy, in which inter alia systematic monitoring and prudent use play essential roles. the bacteria included in this study represented three different categories, namely zoonotic bacteria (salmonella), indicator bacteria (escherichia coli, enterococcus faecalis and enterococcus faecium) and veterinary pathogens (mannheimia haemolytica). thirty isolates of each species were collected with the aim of standardizing the laboratory methodology for a future national veterinary surveillance and monitoring programme. susceptibility to ten selected antimicrobial drugs was determined by means of minimum inhibitory concentrations (mics) using the microdilution method. the method according to the national committee for clinical laboratory standards was used as the standard. multi-well plates containing varying dilutions of antimicrobial drugs and prepared in-house for mic determinations, yielded repeatable results. storage of plates for 2 months at –70 °c did not influence results meaningfully. within this limited sample of bacteria, mic results did not indicate meaningful resistance against any of the ten selected antimicrobial drugs. the findings of the study will be used to establish a national veterinary antimicrobial resistance surveillance and monitoring programme in south africa. to allow for international comparison of data, harmonisation of the surveillance and monitoring programme in accordance with global trends is encouraged. ideally it should be combined with a programme monitoring the quantities of antimicrobial drugs used. the aim is to contribute to slowing down the emergence of resistance and the problems associated with this phenomenon by means of the rational use of antimicrobial drugs. keywords: antimicrobial resistance, harmonisation, minimum inhibitory concentration, monitoring 1 department of veterinary tropical diseases, faculty of veterinary science, private bag x04 onderstepoort, 0110 south africa 2 department of paraclinical sciences, faculty of veterinary science, private bag x04, onderstepoort, 0110 south africa accepted for publication 7 april 2004—editor ernmental organizations being involved (caprioli, busani, martel & helmuth 2000). the role and impact of the use of antimicrobial drugs in animals on the development of bacterial resistance against these drugs have not clearly been delineated. while there is general concern about the emergence of antimicrobial resistance, the important aspect for the animal industries is the concern about the human health aspects of antimicrobial resistance that result from the use of antimicrobial drugs in animals, particularly their use for growth enhancement and disease prophylaxis and the relative volumes of antimicrobial drugs used in animals vs those used in humans. two critical issues that linked the use of antimicrobial drugs in animals and public health concerns were the use of antimicrobial drugs as digestive enhancing drugs (growth promoters) and work done in denmark that demonstrated the emergence of vancomycin-resistant enterococci and its association with the use of a glycopeptide antibiotic, avoparcin in animals (woodford 1998). more recently, the potential impact of resistant campylobacter infections in humans owing to fluoroquinolone use in chickens also contributed to concerns relating to the use of antimicrobial drugs in food producing animals. it is therefore necessary that the risk of development of resistance be considered by the animal industries and that surveillance for resistance and guidelines for the prudent use of antimicrobial drugs be established. antimicrobial drugs are considered essential for controlling bacterial infection and are among the most commonly used drugs in veterinary medicine. to safeguard the efficacy of their use in veterinary medicine and to minimize possible public health risks, the emergence of resistance and the spread of resistant bacteria must be limited by implementing a veterinary antibiotic policy in which, inter alia, systematic monitoring for the development of resistance and prudent use of such drugs play essential roles (martel et al. 1995; anon. 1999). the monitoring of antimicrobial resistance in bacteria from animal sources in south africa is in its infancy. however, the veterinary profession in this country recognizes it as an emerging problem and seeks to address the problem by developing and implementing a national antimicrobial strategy for the use of antimicrobials in animals which inter alia will include the need for a standardized monitoring programme. the ultimate goal for a national antimicrobial strategy is to prolong the efficacy of existing and new antimicrobial agents that are needed to control both human and animal infectious diseases and to minimize infections with zoonotic pathogens in humans (tollefson, angulo, fedorka-cray 1998). the objectives of the study were to establish a repeatable, standardized laboratory procedure that can be used for monitoring the development of antimicrobial resistance in bacteria isolated from animals and food of animal origin in south africa, and to make recommendations for the practical implementation of a monitoring programme that can provide information on a national scale. materials and methods specimens specimens for the isolation of bacteria and stored isolates of bacteria were obtained from the bacteriology laboratory and poultry reference laboratory, department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, the onderstepoort veterinary institute and du buisson, bruinette & kramer, medical pathologists, pretoria. in addition, lung specimens were obtained from various cattle feedlots in south africa. thirty isolates of each species of bacterium identified for possible inclusion in a future surveillance programme were collected and stored for testing. they included representatives of zoonotic pathogens (salmonella), indicator bacteria of animal origin (escherichia coli, enterococcus faecium and enterococcus faecalis) and a veterinary pathogen (mannheimia haemolytica). some strains of e. faecalis of human origin were also included to make up the selected number of thirty isolates per bacterial species. the specimens were randomly collected but formal randomization was not carried out. when isolates of bacteria were received from participating laboratories, they were examined for purity and viability, and their identification confirmed by performing relevant tests. pure strains of overnight growth of each organism were inoculated into brain heart infusion broth (ca milsch), transferred to sterile vials and stored at –70 °c. microdilution susceptibility test direct phenotypic susceptibility testing in which the lowest concentration of an antimicrobial drug that can effectively inhibit bacterial cell division is determined, was used. sterile, plastic, microdilution plates with round wells (sterilab), each containing 0.05 ml of cation-adjusted mueller-hinton broth (ca milsch), was used. enhanced growth and greater consisten240 veterinary antimicrobial resistance surveillance and monitoring programme in south africa cy was obtained when testing mannheimia haemolytica by supplementing the mueller-hinton broth with 0.1 ml inactivated bovine serum prior to inoculation. the inoculum was prepared by emulsifying bacterial colonies in a tube containing 5 ml of muellerhinton broth and standardizing the concentrations spectrophotometrically. each well of a freshly prepared or stored plate was inoculated with 0.05 ml of inoculum within 15 min after it was standardized. plates were incubated at 35 °c for 16–20 h in an aerobic incubator in stacks of not more than four plates to ensure that even incubation temperatures were kept. antimicrobial drugs the plates containing the varying dilutions of the antimicrobial drugs for testing were prepared inhouse. the document entitled performance standards for antimicrobial disk and dilution susceptibil241 h. nel, m. van vuuren & g.e. swan table 1 minimum inhibitory concentrations (mic) recorded for antimicrobial drugs tested against the different isolates antimicrobial organism mic results in µg/ml enrofloxacin salmonella < 0.03 – 2 e. coli < 0.03 – > 4 m. haemolytica < 0.03 – 0.5 e. faecalis, e. faecium 0.125 – > 4 oxytetracycline salmonella 2 – > 64 e. coli 4 – > 64 m. haemolytica < 0.5 – > 64 e. faecalis, e. faecium < 0.5 – > 64 gentamicin salmonella 0.5 – > 32 e. coli 0.5 – 8 m. haemolytica 0.5 – 8 e. faecalis, e. faecium 1 – 32 florfenicol salmonella 4 – > 16 e. coli 4 – > 16 m. haemolytica 0.5 – 4 e. faecalis, e. faecium 1 – 4 amoxycillin salmonella < 0.5 – > 32 e. coli < 0.5 – > 32 m. haemolytica < 0.5 – 16 e. faecalis, e. faecium < 0.5 – 4 neomycin salmonella < 1 – 32 e. coli < 1 – 128 m. haemolytica 2 – 32 e. faecalis, e. faecium 8 – > 128 tilmicosin salmonella 64 – > 64 e. coli 64 – > 64 m. haemolytica < 0.5 – 32 e. faecalis, e. faecium < 0.5 – > 64 trimethoprim/ salmonella < 0.125 – > 16 sulfamethoxazole e. coli < 0.125 – > 16 m. haemolytica < 0.125 e. faecalis, e. faecium < 0.125 – 1 sulfadimethoxine salmonella < 0.125 – > 512 e. coli 32 – > 512 m. haemolytica < 4 – 256 e. faecalis, e. faecium < 4 – > 512 cephalexin salmonella 4 – > 8 e. coli 4 – > 8 m. haemolytica < 0.125 – 4 e. faecalis, e. faecium 0.5 – > 8 ity tests for bacteria isolated from animals was used as the guidance document (national committee for clinical laboratory standards 1999). briefly, antimicrobial stock solutions were prepared by weighing the powders and dissolving them to yield the required concentrations based on the potency of the respective antimicrobial drugs. some of the drugs had to be dissolved in solvents other than water. in those cases only enough solvent was used to solubilize the antimicrobial powder to give a translucent solution. it was diluted further to the final stock concentration with water or the appropriate diluent. aliquots of 1 ml of the stock solution were dispensed into sterile 2 ml eppendorf tubes, sealed and stored at –70 °c. vials were removed when needed and used the same day. any unused drugs were discarded at the end of the day. two-fold dilutions were used to dilute the antimicrobial drugs and their concentration ranges were determined by published breakpoints for the different antimicrobial drugs. the concentration ranges of the different doubling dilutions in µg/ml were as follows: enrofloxacin (0.03–4), oxytetracycline (0.5–64), gentamicin (0.25–32), florfenicol (0.125–16), amoxycillin (0.5–32), neomycin (1–128), tilmicosin (0.5–64), trimethoprim/sulfamethoxazole (0.125/304–16/2.4), sulfadimethoxine (4–512) and cephalexin (0.125– 8). for each antimicrobial concentration in the dilution range, aliquots of 0.05 ml were dispensed to the corresponding wells in each plate. quality control the following reference strains of bacteria obtained from the american type culture collection were used: escherichia coli atcc 25922, pseudomonas aeruginosa atcc 27853, enterococcus faecalis atcc 29212, and staphylococcus aureus atcc 29213. the antimicrobial susceptibility of reference organisms was tested on each occasion that a new batch of microdilution plates was prepared. when the minimum inhibitory concentrations (mics) of the reference strains did not fall between the ranges according to the requirements of the national committee for clinical laboratory standards (nccls), the plates were discarded. the results of these tests were compared with the expected values given by the nccls in table 4 of document m31-a, for accuracy. results were recorded on a quality-control record sheet. other control procedures included growth control, purity control and inoculum control (sahm & washington 1991; nccls 1999). repeatability susceptibility tests were done twice for each individual bacterial organism to determine the inter-plate variation of microdilution plates. in addition, prepared plates were stored frozen at –70 °c until required for use. as the plates were filled, they were stacked in groups of five plates and covered with an adhesive seal. in this way, each tray fitted on top of the other tightly enough to provide a cover that minimizes evaporation and contamination. each stack was then sealed in a plastic bag. these were thawed and tested 2 months after freezing to determine the influence of storage temperature on the repeatability of the test. prior to inoculation, the plates were thawed at room temperature for approximately 1 h before use. results the mic range of each organism tested against each antimicrobial drug is indicated in table 1. 242 veterinary antimicrobial resistance surveillance and monitoring programme in south africa table 2 repeatability results of duplicate testing of selected antimicrobial drugs against four bacterial species percentage of repeat analyses within one dilution antimicrobial drug salmonella e. coli mannheimia e. faecalis, haemolytica e. faecium enrofloxacin 93.33 100.0 90.0 100.0 oxytetracycline 93.33 93.33 90.0 86.67 gentamicin 96.67 93.33 100.0 96.67 florfenicol 96.67 100.0 100.0 100.0 amoxycillin 90.0 90.0 96.67 100.0 neomycin 100.0 96.67 100.0 93.33 tilmicosin 100.0 100.0 100.0 93.33 trimethoprim/sulfa 96.67 90.0 100.0 100.0 sulfadimethoxine 100.0 90.0 96.67 96.67 cephalexin 100.0 100.0 100.0 100.0 repeatability of test table 2 shows the repeatability in percentage of the duplicate test results, allowing a difference in mic values corresponding to one log2 dilution step. all the antimicrobial drugs, except for oxytetracycline, when tested against e. faecalis and e. faecium gave a repeatability percentage of 90 % and higher. an overall agreement of 96.5 % was obtained between the 120 specimens and panel of antimicrobial drugs. effect of storage the results given in table 3 represent the data collected after testing the reference strains on plates that were stored at –70 °c for 2 months. discussion the microdilution minimal inhibitory concentration antimicrobial susceptibility test was chosen as the test of choice for future antimicrobial resistance surveillance and monitoring because it overcomes several limitations of the disk diffusion test. it is a quantitative determination of the degree of susceptibility, not dependent on subjective interpretation, measurement of zones of growth inhibition or extrapolation of mic values from zone sizes. the precise amount of antimicrobial drug required to inhibit bacterial growth can be determined (fales, morehouse, mittal, bean-knudsen, nelson, kintner, turk, turk, brown & shaw 1989). the mic is generally a reproducible quantitative characteristic of the bacterial isolate that can be measured readily (prescott & baggot 1985). in addition, quantitative susceptibility testing using the microdilution method is preferred when testing bacteria of frequently unpredictable susceptibility, such as salmonella and e. coli. it is also the method of choice when testing bacteria that have developed multiple drug resistance (baggot 1998). the guidelines of the nccls were used as the reference method for preparing the varying dilutions of antimicrobial drugs and determining the minimum inhibitory concentrations. it is the quantitative method used by most countries for the determination of susceptibility of bacteria. the nccls has also developed protocols for susceptibility testing of bacteria of animal origin and the determination of the interpretive criteria. the world organisation for animal health (oie) (anon. 1999) has endorsed the use of nccls standards and guidelines (franklin, acar, anthony, gupta, nicholls, tamura, thompson, threlfall, vose, van vuuren, white, wegener & costarrica 2001). using the microdilution method standardized by the nccls will contribute to harmonisation of laboratory methodologies and allow more meaningful comparisons of data. values obtained for each of the ten antimicrobial agents used against each isolate were generally 243 h. nel, m. van vuuren & g.e. swan table 3 minimum inhibitory concentrations of reference strains tested after plates were stored frozen for 2 months staphylococcus aureus enterococcus faecalis escherichia coli pseudomonas aeruginosa atcc 29213 atcc 29212 atcc 25922 atcc 27853 drugs control resultsb control results control results control results limitsa limits limits limits 1 0.03–0.12 0.06 0.12–1 0.25 0.008–0.03 < 0.03 1–4 4 2 0.25–1 0.5 8–32 8 0.05–2 2 8–32 8 3 0.12–1 0.5 4–16 16 0.25–1 1 0.5–2 2 4 2–8 2 2–8 2 2–8 8 > 16 > 16 5 0.25–1 0.5 0.5–2 0.5 2–8 4 – – 6 1–4 2 16–64 16 1–4 2 – – 7 1–4 1 > 32 32 > 64 > 64 > 16 > 16 8 < 0.5/9.5 0.5 < 0.5/0.9 < 0.125 < 0.5/9.5 < 0.125 8/152– 32 32/608 9 32–128 32 32–128 32 8–32 8 – – 10 0.12–0.5 0.5 – – 4–16 4 – – a accepted quality control ranges of mics (µg/ml) for reference strains, derived from nccls, table 4, document m31-a, vol. 19, no. 11, june 1999 b results (µg/ml) after storage for 2 months at –70 °c (1) enrofloxacin; (2) oxytetracycline; (3) gentamicin; (4) florfenicol; (5) amoxycillin; (6) neomycin; (7) tilmicosin; (8) trimethoprim/sulfamethoxazole; (9) sulfadimethoxine; (10) cephalexin similar to those reported in related studies (post, cole & raleigh 1991; burrows, morton & fales 1993; watts, salmon, yancey, nersession & kounev 1993; hirsh & jang 1994; bengtsson, franklin, greko, karlsson & wallen 2000). however, different mic values were obtained for neomycin against e. coli and salmonella. some of the results fell within comparable ranges, but a high percentage of mics were also found to be < 1 µg/ml. a particularly large deviation was found with sulfadimethoxine against m. haemolytica. the highest recorded mic value in this study was > 256 µg/ml whilst the greatest distribution of mics were obtained at a dilution of < 64 µg/ml and 128 µg/ml. this could be owing to the use of samples from diagnostic submissions in this study, whereas comparative studies made use of specimens taken from healthy animals. the results of microdilution susceptibility testing are considered to be satisfactory if the results of individual tests vary no more than plus or minus one serial dilution. the results of the two replicate tests in this project revealed agreement within one dilution for most organisms tested. to determine whether repeatability is adequate, it is considered necessary to calculate the coefficients of variation (sd of replicates divided by mean of replicates) (jacobson 1998). however, to do these calculations continuous data are required but not discrete data as was the case in this study. this is owing to the fact that the results are also considered to be concordant when the independent tests vary by one serial dilution. using the standardized method of the nccls, with all the quality control measures, the results showed 90 % comparability, except for oxytetracycline that showed 86.67 % comparability. the data thus indicated a high degree of correlation between the two sets of test results. stability was shown for plates prepared in-house and frozen for a period of 2 months. therefore, microtitre plates containing the antimicrobial drugs in suspension can be safely stored at –70 °c for at least 2 months following preparation. the results point to the fact that custom-made microdilution plates are as good as commercially produced plates provided that universally accepted quality control strains are always included. in this regard, the atcc control strains are satisfactory as far as susceptible strains are concerned. however, national reference laboratories should also strive to measure results against resistant strains. most clinical specimens from which the bacterial species used in this study were isolated were diagnostic submissions and the results must therefore be interpreted with caution. bacteria from these types of submissions tend to be species from severe and/or recurrent clinical cases and some may possess some degree of antimicrobial resistance. thus, the prevalence of resistant strains may be overestimated and may not reflect the resistance situation in the animal population as a whole (bengtsson et al. 2000). the number of bacterial species used in this study was relatively small and cannot be considered as representative of resistance patterns for the bacteria tested. based on knowledge of antimicrobial drug use in production animals in south africa, a future surveillance programme should ideally include specimens from adult cattle and calves, slaughter pigs, broiler chickens and layer hens. in addition, it is recommended that active surveillance as opposed to passive surveillance be conducted. the former represents a sampling scheme where the nature and number of specimens are defined and collection takes place at regular intervals to assure consistency. the structure of a veterinary antimicrobial surveillance and monitoring programme will of necessity have a different emphasis when compared with similar programmes for humans. the amount of data regularly generated in human hospitals is uncommon in the veterinary field, probably because a variety of animal species must be included. veterinary antimicrobial surveillance and monitoring programmes should aim at detecting the level of resistance in bacteria isolated both from animals and animal-derived foods, at evaluating the risks of the use of antimicrobial drugs in animals and at quantifying the impact of these findings on human health (moreno, dominguez, teshager, herrero & porrero 2000). specimen collection can include diagnostic submissions that are readily available, but the emphasis should be on field specimens taken from healthy animals, food of animal origin and abattoirs. all specimens collected should preferably be from food producing animals, and sampling done randomly to avoid bias. specimens can be collected weekly, monthly, or otherwise as required. the oie guidance document (franklin et al. 2001) is not prescriptive about the exact number of specimens to be collected. thus, it is recommended that an optimal number of specimens as determined by available resources be collected and included to give a true reflection and valid statistical results. the oie has recommended certain bacterial species that could be included in surveillance programmes. these are grouped under the following 244 veterinary antimicrobial resistance surveillance and monitoring programme in south africa three major categories of organisms (franklin et al. 2001): • veterinary pathogens: pasteurella spp., haemophilus somnus, actinobacillus pleuropneumoniae, e. coli, salmonella serotypes, staphylococcus aureus, streptococcus spp. from udder specimens, streptococcus suis, brachyspira spp. and aeromonas and vibrio spp. from fish specimens. • zoonotic bacteria: salmonella typhimurium, salmonella enteritidis, campylobacter jejuni, campylobacter coli and an enterohaemorrhagic e. coli such as o157. • commensal/indicator bacteria: e. coli and enterococci especially, e. faecium. the latter bacteria are known to be indicators of antimicrobial resistance and should be obtained from healthy animals. for a national surveillance and monitoring programme in south africa, it is recommended that the bacterial species tested in this study are included. they represent the three major categories of organisms and, in the developmental stages of the programme, will not provide a formidable challenge in terms of isolation and identification. additional organisms and types of specimens can be included when available resources and capacity make it possible. for example, it may be worthy of consideration to restrict salmonella monitoring to bovine and porcine specimens and include campylobacter spp. isolations from poultry specimens. the antimicrobial drugs included in this study are similarly recommended for the initial phase of the programme. they represent most of the important antimicrobial classes used in animals and humans. the selection of two aminoglycosides within the panel of antimicrobials was based on the gastrointestinal specific action of neomycin compared to systemic antibacterial indications of gentamicin (prescott 2000). the difference in the aminoglycoside-modifying enzymes in r plasmid-bearing bacteria between neomycin and gentamicin was also considered (lambert & o’ grady 1992). explosive outbreaks of nosocomial infections caused by gentamicin-resistant bacteria of many species have been reported in human hospitals (prescott 2000). neomycin plasmid-mediated resistance is relatively common in enteric commensals and pathogens but less common among other opportunist pathogens. other drugs can be considered for inclusion at later stages, for example antimicrobial drugs that are used in humans and not in animals (e.g. glycopeptides and streptogramins). it would be ideal to develop antimicrobial panels for each category of bacteria to be tested. antimicrobial panels can also be designed specifically for gram-negative and gram-positive bacteria or for each bacterial species tested in the surveillance programme (aarestrup, bager, jensen, madsen, meyling & wegener 1998; moreno et al. 2000; aarestrup 2000; franklin et al. 2001). information on the consumption of antimicrobial drugs used in different animal species is also needed to assess the impact on the occurrence of resistance and to determine where and for which infections most antimicrobial drugs are used. a key use of surveillance data is to relate this to antimicrobial use so that where resistance emerges, appropriate feedback exists and measures can be taken by regulatory authorities to address use patterns that may be contributing to the emergence of resistance. cautious, rational, quantitatively and qualitatively adjusted use of antimicrobial drugs in human and veterinary medicine will at least slow down the emergence of resistance and the problems associated with this phenomenon (aarestrup 2000; cohen & tartasky 1997). at present, information on antimicrobial resistance in bacteria from animals is lacking in most countries. a structured surveillance and monitoring programme for antimicrobial resistance in south africa will contribute to the detection of emerging resistance problems at the earliest possible stage. references aarestrup, f.m., bager, f., jensen, n.e., madsen, m., meyling, a. & wegener, h.c. 1998. surveillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in denmark. apmis, 106:606–622. aarestrup, f.m. 2000. occurrence, selection and spread of resistance to antimicrobial agents used for growth promotion for food animals in denmark. apmis 108(101 suppl): 5s– 48s. anonymous 1999. office international des epizooties. the use of antibiotics in animals—ensuring the protection of public health. summary and recommendations from the european scientific conference, paris, france, 24– 26 march 1999. bengtsson, b., franklin, a., greko, c., karlsson, m., & wallen, c. 2000. swedish veterinary antimicrobial resistance monitoring. uppsala, sweden: wikströms. baggot, j.d. 1998. antimicrobial selection, administration and dosage. journal of the south african veterinary association, 69:174–185. burrows, g.e., morton, r.j. & fales, w.h. 1993. microdilution antimicrobial susceptibilities of selected gram-negative veterinary bacterial isolates. journal of veterinary diagnostic investigation, 5:541–547. 245 h. nel, m. van vuuren & g.e. swan caprioli, a., busani, l., martel, j.l. & helmuth, r. 2000. monitoring of antibiotic resistance in bacteria of animal origin: epidemiological and microbiological methodologies. international journal of antimicrobial agents, 14:295–301. cohen, f., & tartasky, d. 1997. microbial resistance to drug therapy: a review. american journal of infection control, 25: 51–64. fales, w.h., morehouse, l.g., mittal, k.r., beanknudsen, c., nelson, s.l, kintner, l.d., turk, j.r., turk, m.a., brown, t.p. & shaw, d.p. 1989. antimicrobial susceptibility and serotypes of actinobacillus (haemophilus) pleuropneumoniae recovered from missouri swine. journal of veterinary diagnostic investigation, 1:16– 19. franklin, a., acar, j., anthony, f., gupta, r., nicholls, t., tamura, y., thompson, s., threlfall, e.j., vose, d., van vuuren, m., white, d.g., wegener, h.c. & costarrica, m.l. 2001. antimicrobial resistance: harmonization of national antimicrobial resistance monitoring and surveillance programmes in animals and animal derived food. revue scientifique et technique, 20:859–870. hirsh, d.c. & jang, s.s. 1994. antimicrobial susceptibility of selected infectious bacterial agents obtained from dogs. journal of the american animal hospital association, 30:487– 494. jacobson, r.h. 1998. validation of serological assays for diagnosis of infectious diseases. revue scientifique et technique, 17:469–486. lambert, h.p. & o’ grady, f.w. (eds) 1992. antibiotic and chemotherapy, 6th ed., edinburgh: churchill livingstone. martel, j.l., chaslus-dancla, e., coudert, m., poumart, f. & lafont, j.p. 1995. survey of antimicrobial resistance in bacterial isolates from diseased cattle in france. microbial drug resistance, 1:273–283. moreno, m.a., dominguez, l., teshager, t., herrero, i.a. & porrero, m.c. 2000. antibiotic resistance monitoring: the spanish programme. international journal of antimicrobial agents, 14: 285–290. national committee for clinical laboratory standards 1999. performance standards for antimicrobial disk and dilution tests for bacteria isolated from animals: approved standard. document m31-a, vol.19, no. 11, june 1999. post, k.w., cole, n.a. & raleigh, r.h. 1991. in vitro antimicrobial susceptibility of pasteurella haemolytica and pasteurella multocida recovered from cattle with bovine respiratory disease complex. journal of veterinary diagnostic investigation, 3:124–126. prescott, m.a. & baggot, j.d. 1985. antimicrobial susceptibility testing and antimicrobial drug dosage. journal of the american veterinary medical association, 187:363–368. prescott, j.f. 2000. aminoglycosides and aminocyclitols, in antimicrobial therapy in veterinary medicine, edited by j.f. prescott, j.d. baggot & r.d. walker, 3rd ed. ames: iowa state university press. sahm, d.f. & washington, j.a. 1991. antibacterial susceptibility tests: dilution methods, in manual of clinical microbiology, edited by a. balows, w.j. hausler, k.l. hermann, h.d. isenberg & h.j. shadomy, 5th ed. american society for microbiology, washington, d.c. tollefson, l., angulo, f.j. & fedorka-cray, p.j. 1998. national surveillance for antibiotic resistance in zoonotic enteric pathogens. the veterinary clinics of north america: food animal practice, 14:141–150. watts, j.l., salmon, s.a., yancey, r.j., nersession, b. & kounev, z.v. 1993. minimum inhibitory concentrations of bacteria isolated from ducks with septicemia and airsacculitis. journal of veterinary diagnostic investigation, 5:625– 628. woodford, n. 1998. glycopeptide-resistant enterococci: a decade of experience. journal of medical microbiology, 47: 849–62. 246 veterinary antimicrobial resistance surveillance and monitoring programme in south africa 2194matete_pp317-323.qxd introduction human trypanosomosis occurs in 36 african counties that lie between latitudes 14 ° north and 29 ° south. western kenya is an area endemic for human trypanosomosis (sleeping sickness), which is a cause for public health concern wherever it occurs. current estimates of the parasitological prevalence of trypanosoma brucei in animals in western kenya stand between 2 % and 5 % but may rise beyond 10 % during outbreaks with three to five cases of sleeping sickness in humans (matete, unpublished data 2002). the disease prevalence, however, increases beyond 70 % during epidemics involving over 10 sleeping sickness cases (welburn, picozzi, fevre, coleman, oditt, carrington & maudlin 2001). trypanosomosis of dogs was first described in 1908 (bevan 1913). it has been established that dogs are susceptible to various trypanosomes including t. brucei brucei, t. brucei rhodesiense, t. brucei gambiense, trypanosoma congolense, tryp317 onderstepoort journal of veterinary research, 70:317–323 (2003) occurrence, clinical manifestation and the epidemiological implications of naturally occurring canine trypanosomosis in western kenya g.o. matete* kenya trypanosomiasis research institute p.o. box 362, kikuyu, kenya abstract matete, g.o. 2003. occurrence, clinical manifestation and the epidemiological implications of naturally occurring canine trypanosomosis in western kenya. onderstepoort journal of veterinary research, 70:317–323 domestic dogs were screened for trypanosoma brucei infection using the haematocrit centrifugation technique as part of routine active surveillance exercises in the busia and teso districts of kenya. the purpose was to assess the role of dogs as sentinels for the occurrence of human sleeping sickness. out of 200 dogs screened, five were found to be infected at the various test sites. these five succumbed to the disease within four weeks, and exhibited a distinct and pronounced corneal opacity before death. blood from two naturally infected dogs were tested for the presence of the serum resistance associated (sra) gene and one tested positive, confirming it as human infective (t. brucei rhodesiense) prevalence (0.5 %). it is considered that the occurrence of this clinical sign could be used as an early warning prediction of future outbreaks. this type of prediction could form an integral part of an indigenous technical knowledge set in areas lying at the edges of the tsetse (glossina) belts where t. brucei is the main trypanosome species that affects dogs. the occurrence of corneal opacity in dogs could indicate a rise in the levels of t. brucei a proportion of which could be human infective t. b. rhodesiense circulating in the population early enough before disease outbreak occurs. it is thought that during sleeping sickness epidemics the domestic dog will be the first casualty rapidly succumbing to disease long before it is noticed in man. prompt prediction of disease outbreaks would thus enable early interventions that would reduce the morbidity, mortality and the general economic losses associated with sleeping sickness to be instituted. keywords: canine, epidemiology, sleeping sickness, trypanosomosis * e-mail: georgematete2002@yahoo.com accepted for publication 24 june 2003—editor anosoma evansi and trypanosoma cruzi (morrison, murray, sayer & preston 1983). the clinical signs of disease have been described by morrison et al. (1983) and kaggwa, munyua & mugera (1983; 1984), and the pathology by kaggwa, munyua & mugera (1988). gibson & gashumba (1983) described dogs as carriers of t. b. rhodesiense in uganda. in nigeria, trypanosomosis in dogs is a common occurrence in the t. b. gambiense belt, where omamegbe, orajaka & omehelu (1984) reported the ocular, lymphoadenopathic and meningial forms as the three distinct forms of disease. dogs are an integral part of the lives of livestock farmers in western kenya, an endemic area for t. brucei rhodesiense. blind dogs that eventually die within a few weeks of the start of clinical signs have been associated with potential outbreaks of sleeping sickness in humans by the local communities living within the tsetse belt of kenya and uganda. they therefore bring their dogs for screening during active surveillance programmes (oditt, personal communication 2000). transmission mainly occurs around the watering sites and is sustained in animal-tsetse-animal cycle by glossina pallidipes. the infection is then spread to humans by glossina fuscipes fuscipes. efforts to utilize current markers, like minisatellites, have been able to distinguish between ugandan t. b. rhodesiense and t. b. brucei (macleod, twedie, welburn, maudlin, turner & tait 2000). the ectopic expression of human serum resistance (sra), a 743 bp-molecular mass gene product allows t. b. rhodesiense to survive exposure to normal human serum. the presence of the gene product correlates to human serum resistance and offers the prospect of a simple one-step diagnostic kit for t. b. rhodesiense (xong, vanhamme, chamekh, chifwembe, van den abbeele, pays, van meirvene, hamers, de baetselier & pays 1998; welburn et al. 2001). the screening of the sra gene has been shown to distinguish between t. b. rhodesiense and t. b. brucei in non-human hosts (welburn et al. 2001). treatment of dogs is not always successful, and break-through infection after isometamidium prohylaxis occurs in some (lopes-pereira, lopes-pereira, escrivao, schwalbach & pinto 1997). melarsoprol (aventis pharma) is the drug of choice in the chemotherapeutic management of the late stage-t. b. rhodesiense sleeping sickness in humans. a 10day treatment protocol has been investigated for t. b. gambiense in angola, and it is hoped that this will reduce the duration of confinement from 42 days to just below 14 days (blum & burri 2002). however, there are no reports on the use of melarsoprol in dogs. in the current study a short-course treatment with melarsoprol was attempted on one naturally infected dog. the occurrence, clinical manifestations and epidemiological implications of canine trypanosomosis in t. b. rhodesiense sleeping sickness endemic areas are discussed in detail. an attempt was also made to validate dogs as carriers of t. b. rhodesiense by testing their infective trypanosomes for the presence of the sra gene. material and methods the study was undertaken in teso and busia districts that fall within the lake victoria basin of western kenya. these districts cover an area of 1 819 km2 and their altitude ranges from 1 128 m in the extreme south to 1 463 m above sea level in central and north teso. the districts have a mean annual rainfall of approximately 1 270 mm, which is bimodal in distribution. maximum temperatures range between 26 °c and 30 °c while the mean minimum temperature range between 14 °c and 22°c. the tsetse fly species present in the area are g. f. fuscipes and g. pallidipes in riverine and savannah habitats, respectively. most of the uncultivated land is presently encroached by lantana camara and digithonia spp. thickets that form good breeding grounds for tsetse flies. the investigation was carried out over a 1-year period, between april 2001 and march 2002, as part of a regular active surveillance programme. the administrative locational/sub-locational maps were used to select locations for purposive samplings based on the previous occurrence of sleeping sickness and reports of deaths in cattle. after site selection, a double randomisation was done from a complete list of villages within the locations to ensure an even distribution of sampling sites. a geographical positioning system (gps) was used to mark the exact locations of blind dogs. these sites were obochun, koruruma, busia township, budubusi and rwambwa. a questionnaire was used to determine the historical features of the disease. at the same time humans and cattle were also screened, and for comparative purposes, the apparent density of tsetse flies was determined. five methods were used to detect trypanosomes in the blood of dogs, which was collected from the jugular vein. these were the microscopic examination of wet smears and giemsa-stained thin blood smears, the haematocrit centrifugation technique (woo 1969), buffy coat examination (murray, mur318 canine trypanosomosis in western kenya ray & mcintyre 1977) and a spinal tap for cerebrospinal fluid (csf). haematological analysis for packed cell volume and haemoglobin concentration was done from blood drawn into capillary tubes. examination of the morphological features of trypanosomes was made using the giemsa-stained thin smears, and their motility in wet preparations. parasites were then subjected to blood incubation and an infectivity test to determine their infectivity for humans (rickman & robson 1970; brun & jenni 1987). one dog, whose owner consented to treatment with a 10-day course of melarsoprol at 2.2 mg/kg body mass, was treated intravenously at 9:00 daily though either the radial or femoral veins. the dog was subsequently euthanased 1 week after the termination of treatment on the instructions of the owner and an extensive postmortem examination was carried out. samples from the eyes and brain were taken and preserved in 10 % formalin, and tissue sections cut, stained with haematoxylin and eosin according to routine techniques. during the necropsy, impression smears were made from the cerebellum and stained with giemsa. the polymerase chain reaction (pcr) and sra gene probes were used to test for presence of t. b. rhodesiense in two dogs from the two sleeping sickness focus of kenya. the two isolates compared were ketri 3119 isolated from south nyanza and ketri 3739 isolated at obochun in teso district in 2001. samples were prepared for standard pcr cycling and screening for presence of t. brucei sl., was done as described by de greef, inberrechts, matthyssens, van meirvenne & hamers (1989) and artama, agey, & donelson (1992), and sra gene detection was done according to gibson, backhouse & griffiths (2002). results a total of 200 dogs of indeterminate breed were sampled during the study of which five were positive for t. brucei, giving a total prevalence of 2.5 %. four dogs (2.0 %) had t. b. brucei, while t. b. rhodesiense was present in one (0.5 %). the comparative infection rate in man was estimated at 0.5 %, in cattle 10.2 % and 0.2 % in tsetse flies. most respondents to the questionnaire indicated that dogs had died after becoming blind, with conservative mortality estimated at 34 % within the previous three years. a variety of non-specific clinical signs were noted in the five infected dogs. these included depression, sleepiness, pale mucous membranes, lethargy, anorexia, mass loss and a rough hair coat. other signs were recumbence, enlarged superficial lymph nodes, dyspnoea and fever with a mean of 39.8 °c. neurological observations included dullness and lack of coordination manifesting as paraplegia, bilateral hyperflexic patellar reflexes, lack of conscious proprioception in the pelvic limbs and marginally depressed hopping reflexes of the forelimb. corneal opacity, conjunctivitis with a mucopurrulent discharge, uveitis, haemorrhage and turbidity in the anterior chamber, and loss of vision were also noted. however, the predominant sign was varying degrees of bilateral cloudiness of the cornea due to the turbid aqueous humour (fig. 1). oedema of the front limbs, head and neck was also observed. progressive mental deterioration similar to the aggressive behaviour associated with rabies, together with lethargy and mania, were the historical observations obtained from the questionnaire. wet blood films in the five infected dogs were teeming with trypanosomes and haematology indicated a leukocytosis as a result of neutrophilia and lymphocytosis. moderate normocytic normochromic anaemia with reduced packed cell volume, haemoglobin values and red blood cells (rbc) counts were also observed. the cerebrospinal fluid of the one dog was still positive for trypanosomes, as were the brain impression smears. the numbers of lymphocytes in the csf were elevated. following treatment of one dog with melarsoprol, trypanosomes disappeared in blood within 24 h. in 5 days a gradual but progressive clearing of the uvea was observed, which was followed by a complete recovery of the eyesight by 10 days after the first treatment (fig. 2). the dog gained in strength, began to feed and became more active. although it responded clinically and trypanosomes were absent in the csf, they were later found in the brain impression smears. the dog was then destroyed at day 17. at necropsy the body was slightly emaciated due to anorexia, evidenced by prominence of dorsal vertebral and scapular spines. in addition the dog exhibited moderate cerebral meningitis and oedema, and scattered haemorrhages were present in the meninges. cellular infiltration of the leptomeninges with macrophages, neutrophils, plasma cells and lymphocytes was observed. moderate encephalitis manifested by the presence of perivacular cuffing, which was particularly prominent in the brain stem and cerebellum, was evident. astrocyte and meningial hyperplasia was also present. the front limbs were oedematous with a muco-purulent discharge at the radio-ulnar junction on the left. these were presumed to be septic decubitus wounds due 319 g.o. matete 320 canine trypanosomosis in western kenya fig. 2 the same dog as in fig. 1 after complete recovery of the eyesight fig. 1 naturally infected dog showing corneal opacity ����� ���� ����� ��� �� �������������������� � � ����������� �� ������� ��� �� � ����������������� �� ��������� ����� ������������� �������� ���� ���� table 1 response of dog stabilates to biit and sra gene probe number date isolated biit pcr (t. brucei sl.) sra gene ketri 37381 5/11/01 + + +* ketri 37391 31/5/01 + + + ketri 31192 south nyanza – + –* key 1 isolate 3739 was a sub culture of 3738 passages in mice; sra gene were shown to be conserved 2 isolate was from lambwe valley a different focus of sleeping sickness * isolates tested by pcr and by sra gene probes few due to cost all five dogs were biit positive fig. 3 model of the prevailing situation of trypanosomiasis in western kenya. when the prevailing levels of t. b. rhodesiense exceed 10 % they undergo a clonal expansion to induce an outbreak or even epidemic. the same situation can arise when t. brucei exceed 50 % in a population, then a switch to t. b. rhodesiense occurs and these then undergo a clonal expansion 321 g.o. matete to long-standing sleepiness. about 50 ml of strawcoloured fluid occurred in the abdomen and hydrothorax was also observed. polymerase chain reaction was able to identify the two isolates as being t. brucei sl. the sra gene probe differentiated ketri 3119 as t. b. brucei and ketri 3739 as t. b. rhodesiense. the isolate ketri 3739 was also resistant to human serum and multiplied when inoculated in mice after incubation in normal human serum (table 1.). discussion although it has been established that dogs are carriers of t. b. rhodesiense data remains inadequate due to the use of techniques of low sensitivity. the infection rate estimated at 0.5 % in man, 10.2 % in cattle and 0.2 % in tsetse in the current study compared well with the finding in the busoga focus of 0.6 % in man, 10.5 % in cattle and 0.2 % in tsetse flies reported by maudlin, welburn, gashumba, okuna & kalunda (1990). in an endemic situation as evidenced in western kenya human infective trypanosomes (t. b. rhodesiense) are difficult to detect due to their low prevalence. macleod, tait & turner (2001) have already shown that a clonal expansion of t. b rhodesiense occurs in the field. this could be an important determinant of trypanosome diversity except where clones have acquired the ability to infect humans. they report that when t. b. brucei acquires ability to infect humans it then spreads rapidly through the human population from the single source event. it is now postulated that tsetse flies feed on dogs at levels approximate to the 9 % observed in cattle and that human infective trypanosomes circulate in tsetse at sub infective doses within the endemic areas. this is in agreement with anon (1991), that animal sentinel systems, particularly dogs, have been used as surrogates to human exposure to environmental risks or to monitor complex environments. an outbreak of sleeping sickness would thus occur when either the prevalence of t. b. rhodesiense is at least 10 %, or rapidly transforms itself into an epidemic when the prevalence exceeds 50 %. the same situation could arise when proportion of t. b. brucei circulating in the population is 50 %. this is sufficient to enable a switch between human serum sensitive and human serum resistant forms in adequate numbers to cause an outbreak. the findings of rickman & kolala (1980), de greef et al. (1989) and macleod et al. (2001) are consistent with the postulates of this model. an extensive study using molecular techniques may provide confirmation to the scenario suggested (fig. 3). it was clear from this model that time and space were important determinants of both the genetic diversity of trypanosomes circulating in the area, the course of the disease and its final outcome. the clinical signs and pathology observed in the current study were similar to those described by kaggwa et al. (1983; 1984) and morrison et al. (1983) for both experimental and natural infections. the predominant clinical sign in all the cases was the blue/white cloudiness of the cornea in all the dogs. this prevalence of corneal opacity of 2.5 % in t. b. rhodesiense sleeping sickness areas differs from the 52.8 % obtained by omamegbe et al. (1984) in t. b. gambiense areas of nigeria. the description is similar to that described by sayer, morrison, preston, hird, price & murray (1979). the opacity was bilateral in all the dogs studied. corneal opacity is, however, rarely noted in humans (apted 1970), and is attributed to the descent of parasites along the optic artery from the brain with the subsequent induction of immune complexes into the aqueous humour. these complexes form granular deposits within the aqueous, which progressively cloud the cornea by adhering to its inner surface and thus hinder vision. this is in agreement with one of the hypotheses of the immune reaction described by lambert, berney & kazyumba (1981). other presenting signs, such as oedema of the limbs, emaciation, loss of muscle mass, hydrothorax and ascites, were similar to those described by morrison et al. (1983). the nervous signs exhibited by the dogs were similar to those noted by berger, palmer, hodges & hall (1991) in infections of t. cruzi. these changes in behaviour could be attributed to meningial invasion with subsequent inflammation and oedema. the invasion is induced by proteases such as trypanopains, oligopeptidases and cell surface associated acid phosphates produced by the parasites (lonsdale-eccles & grab 2002). these result in a host immune response with a large numbers of lymphocytes and plasma cells forming immune complexes of igm, morula cells and t-helper cells within the brain (zola, wassuoumbou-loubienga, goma & mouanga-yidika 1994). brain impression smears in this study indicated localization of trypanosomes in the brain, which was similar to findings of ikede & losos (1972), and is suggested as the cause of most treatment failures. the changes noted in the cerebrospinal fluid were an indication of a non-suppurative inflammation similar to that observed in t. cruzi by berger et al. (1991). the pronounced corneal opacity in the dog could be used as a preliminary sentinel sign for the transmission of future sleeping sickness outbreaks as part of the indigenous technical knowledge. in areas where the occurrence of t. vivax predominates over that of t. congolense, t. brucei becomes the only causative agent for trypanosomosis in dogs. the disease in dogs within such areas may therefore be attributed to a rise in the circulating levels of t. brucei in the animal population. there is circumstantial evidence that t. brucei in dogs could therefore be useful in predicting the occurrence of sleeping sickness outbreaks in western kenya. the sporadic and very low disease prevalence in dogs within the area closely reflects the situation occurring in humans. dogs have similarly been used as sentinel animals to monitor the transmission of t. cruzi in rural areas in argentina (castanera, lauricella, chuit & gurtler 1998). when dogs are used as sentinel animals, the occurrence of corneal opacity would indicate a rise in levels of t. brucei circulating in the population. if molecular tools, e.g. sra gene probes, are then used to confirm the parasites in dogs as t. b. rhodesiense, sleeping sickness will be detected before outbreaks occur in humans. the dog may in itself have little relevance as a reservoir for the human infective forms of trypanosomes considering the relatively acute phase of the disease with death occurring between 2 weeks and 1 month after the onset of clinical signs. the clinical signs manifest more rapidly that it does in man where it is reported to take between 3 weeks and 6 months (odiit, kansiime & enyaru 1997). the finding of the sra gene in trypanosomes infecting a dog in this study confirms the ability of dogs to carry the human infective trypanosomes. it is noteworthy that the isolate from kenya had been obtained from obochun, an area that neighbours the most recent site of an outbreak of sleeping sickness in uganda (unpublished liri reports 2001). since t. b. brucei is able to switch from human serum sensitive to human serum resistant forms, the course of the disease in the dog should therefore be studied from the beginning of outbreaks to the epidemic phase to see if this hypothesis holds. prompt prediction of disease outbreaks would thus enable early interventions to be instituted that would reduce the morbidity and mortality of sleeping sickness as well as the general economic losses associated with it. complete recovery of sight was obtained in ten days compared to reports of 45 days in t. evansi infections as described by vershney, varsheny & dwivedi (1998). it is possible that with the cure of trypanosomes, the opacity could then resolve spontaneously. further tests are required to validate the use of melarsoprol in the treatment of valuable dogs, e.g. bomb or drug sniffers and guard dogs with trypanosomosis. acknowledgements i am grateful for the excellent technical assistance by staff of ketri alupe 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(abstract in pubmed). welburn, s.c., picozzi, k., fevre, e.m., coleman, p.g., oditt, m., carrington, m. & maudlin, i. 2001. identification of human-infective trypanosomes in animal reservoir of sleeping sickness in uganda by means of serum resistance associated (sra) gene. lancet, 358:2017–19. woo, p.k.t., 1969. the haematocrit centrifugation for the detection of trypanosomes in blood. canadian journal of zoology, 47:921–923. xong, h., vanhamme, l., chamekh, m., chifwembe, c.e., van den abbeele, j., pays, a., van meirvene, n., hamers, r., de baetselier, p. & pays, e. 1998. a vsg expression site-associated gene confers resistance to human serum in trypanosoma rhodesiense. cell, 95:839– 846. zola, j.m., wassuoumbou-loubienga, s., goma, g.c. & mouanga-yidika, g. 1994. meningio-encephalitite aigue a trypanosoma brucei gambiense revelee par un oedeme papillaire. bulletin of society of pathology and exobiology, 87:312–314. 323 g.o. matete abstract listeria species human listeriosis pathogenicity of listeria monocytogenes virulence factors of listeria monocytogenes epidemiology of human listeriosis legislations relating to listeria monocytogenes in food occurrence of listeria monocytogenes in meat value chain molecular methods of detection treatment antimicrobial resistance in listeria monocytogenes conclusion acknowledgements references about the author(s) itumeleng matle bacteriology division, agricultural research council – onderstepoort veterinary research, onderstepoort, pretoria, south africa department of agriculture and animal health, university of south africa, science campus, florida, south africa khanyisile r. mbatha department of agriculture and animal health, university of south africa, science campus, florida, south africa evelyn madoroba department of biochemistry and microbiology, university of zululand, kwadlangezwa, south africa citation matle, i., mbatha, k.r. & madoroba, e., 2020, ‘a review of listeria monocytogenes from meat and meat products: epidemiology, virulence factors, antimicrobial resistance and diagnosis’, onderstepoort journal of veterinary research 87(1), a1869. https://doi.org/10.4102/ojvr.v87i1.1869 review article a review of listeria monocytogenes from meat and meat products: epidemiology, virulence factors, antimicrobial resistance and diagnosis itumeleng matle, khanyisile r. mbatha, evelyn madoroba received: 31 mar. 2020; accepted: 04 aug. 2020; published: 09 oct. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract listeria monocytogenes is a zoonotic food-borne pathogen that is associated with serious public health and economic implications. in animals, l. monocytogenes can be associated with clinical listeriosis, which is characterised by symptoms such as abortion, encephalitis and septicaemia. in human beings, listeriosis symptoms include encephalitis, septicaemia and meningitis. in addition, listeriosis may cause gastroenteric symptoms in human beings and still births or spontaneous abortions in pregnant women. in the last few years, a number of reported outbreaks and sporadic cases associated with consumption of contaminated meat and meat products with l. monocytogenes have increased in developing countries. a variety of virulence factors play a role in the pathogenicity of l. monocytogenes. this zoonotic pathogen can be diagnosed using both classical microbiological techniques and molecular-based methods. there is limited information about l. monocytogenes recovered from meat and meat products in african countries. this review strives to: (1) provide information on prevalence and control measures of l. monocytogenes along the meat value chain, (2) describe the epidemiology of l. monocytogenes (3) provide an overview of different methods for detection and typing of l. monocytogenes for epidemiological, regulatory and trading purposes and (4) discuss the pathogenicity, virulence traits and antimicrobial resistance profiles of l. monocytogenes. keyword: listeria monocytogenes; meat and meat products; epidemiology; virulence factors; diagnosis; antimicrobial resistance. listeria species listeria species (spp.) are short gram-positive rods (nyarko & donnelly 2015) that belong to the phylum firmicutes, class bacilli, order bacillales (jadhav 2015). the listeria spp. are facultatively anaerobic, non-spore forming, about 0.5 µm in width and 1 µm – 1.5 µm in length (wieczorek, dmowska & osek 2012) and belong taxonomically to the clostridium-bacillus-lactobacillus sub-branch with brochothrix thermosphacta (schmid et al. 2014). listeria spp. are generally motile because of peritrichous flagella at temperature range of 24 °c – 28 °c but non-motile above 30 °c (indrawattana et al. 2011). these species are also catalase positive; however, exceptions have been reported (cepeda et al. 2006). listeria spp. are oxidase, urea and indole negative and hydrolyse aesculin (de vasconcelos byrne et al. 2016). listeria spp. also have the ability to tolerate salt conditions (nacl) up to 20% (weight/volume [w/v]) and grows in a ph range of 4.4–9.6 (holch et al. 2013). the growth temperature for these species ranges from –0.4 °c to 45 °c, with an optimum growth temperature of 37 °c and can survive at relatively low water activities (aw < 0.90) (liu 2006). these growth conditions contribute to their versatility to grow and survive under extreme environmental conditions posed at food-processing facilities and become a serious problem for food industry (ducey et al. 2006). the genus listeria consists of 20 low guanine (g) + cytosine (c) (38%) content species, which includes l. monocytogenes, l. marthii, l. innocua, l. welshimeri, l. seeligeri, l. costaricensis, l. ivanovii, l. grayi, l. rocourtiae, l. fleischmannii, l. newyorkensis, l. weihenstephanensis, l. floridensis, l. aquatica, l. thailandensis, l. cornellensis, l. riparia, l. booriae, l. goaensis and l. grandensis (den bakker et al. 2010). this classification of listeria spp. is based on different analysis assays including 16s ribosomal ribonucleic acid (rrna), deoxyribonucleic acid (dna) sequencing information and multilocus enzyme analysis. it has also been proved useful for surveillance and epidemiological purposes in outbreaks linked to food-borne listeriosis (chasseignaux et al. 2001). out of the species of listeria identified thus far, only l. monocytogenes can cause infection in both humans and animals, whereas l. ivanovii is pathogenic to animals, predominantly in ruminants and has rarely been implicated with human infections (gouin et al. 1994; orsi, bakker and wiedmann 2011; zhang, jayarao & knabel 2004). however, l. seeligeri and l. welshimeri have been reported as agents of sporadic cases of human listeriosis (gouin et al. 1994). listeria spp. are phenotypically very similar but can be differentiated by biochemical tests including haemolysis test, acid production from d-xylose, l-rhamnose, mannitol, motility and alpha methy-d mannoside (orsi et al. 2011). the christie–atkins–munch-peterson test can also be useful for differentiation of listeria spp. this test works on the principle to test for haemolysis enhancement in the presence of staphylococcus aureus (fda et al. 2008). listeria spp. are mostly environmental contaminants with primary habitation of soil. however, these species have also been found in water, sewage and decaying vegetation (willaarts, pardo & de la mora 2013). listeria spp. have been found in a variety of animals including ruminants, birds, marine life, insects, ticks and crustaceans (van vuuren 2001). these species have been isolated from 1% to 7% of intestinal content of healthy animals (borucki & call 2003); thus animals can serve as carriers. human beings are also carriers of listeria spp. as these were isolated from 5% to 10% of stools of healthy human adults and 1.3% of the younger people (churchill, lee & hall 2006). as a result of their ubiquitous and resilient character, listeria spp. have the capacity to enter the food supply chain and contaminate a wide variety of food products (leong, alvarez-ordóñez & jordan 2014). listeria monocytogenes listeria monocytogenes was first described by hülphers in 1910 from the necrotic liver of a rabbit in sweden and named bacillus hepatis (carvalho, sousa & cabanes 2014; hülphers 1911). murray isolated a similar bacterium in 1926 as a causative agent of an epizootic in rabbits and guinea pigs in research laboratories of cambridge, united kingdom, and named bacterium monocytogenes (lekkas 2016; murray, webb & swann 1926; rantsiou et al. 2008). a year later (1927), pirie also isolated a bacterium corresponding to the description given by hülphers and murray from wild gerbils in south africa (sa) (jemal 2014; mitchell, pirie & ingram 1927). the bacterium was named listerellahepatolytica in honour of british surgeon, lord joseph lister, the father of antisepsis (gray & killinger 1966). however, it was until 1940, that the present name, l. monocytogenes was recognised (lamont & sobel 2011). listeria monocytogenes was first isolated in humans by nyfeldt in 1929, and in the same year gill also described the illness in sheep called circling diseases caused by l. monocytogenes (gill et al. 1937; nyfeldt 1929). listeria monocytogenes was then recognised as pathogen that caused sporadic human infections and was mainly associated with workers encountering diseased animals (lamont & sobel 2011). in the 1980s, after several outbreaks including vacherin mont d’or in switzerland in 1983–1987 and improperly pasteurised milk in the united states (us) in 1983, that was when interest for the pathogen amongst food manufacturers started to emerge (klumpp & loessner 2013; lekkas 2016). since then, l. monocytogenes outbreaks have been linked to consumption of contaminated foods, which include dairy products, meat products, seafood products and vegetables (loman 2012; ragon et al. 2008; zuber et al. 2019). listeria monocytogenes is amongst the dangerous bacterial food-borne pathogens in the world, which cause severe human diseases (maertens de noordhout et al. 2014). this pathogen has been divided into 13 serotypes (½a, ½b, ½c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, 7) based on somatic and flagellar antigens (dhama et al. 2015). these serotypes are further grouped into four genetic diversity lineages (i–iv). these lineages consist of specific serotypes; lineage i harbours serotypes ½b, 3b, 4b, 4d, 4e and 7. serotypes ½b and 4b within lineage i encode listeriolysin s virulence factor, which is not present in other lineages (orsi et al. 2011). lineage ii contains serotypes ½a, ½c, 3a and 3c and often harbours several plasmids that are resistant to heavy metals (dhama et al. 2015). serotypes 4b, ½a, 4a and 4c belong to lineage iii and 4a, 4c and atypical 4b serotypes have been characterised as lineage iv isolates (haase et al. 2014). lineage iii and iv serotypes are rarely isolated and have distinctive genetic and phenotypic characteristics but occur mostly in ruminants (camargo, woodward & nero 2016). human listeriosis listeriosis is a zoonotic disease that is mainly acquired through consumption of contaminated food by l. monocytogenes (hilliard et al. 2018). other possible routes of contamination for humans include direct contact with infected animals and environments (vázquez-boland et al. 2001). uncommon occurrence of listeriosis has also been reported in human beings in the form of endocarditis, hepatitis, myocarditis, arteritis, pneumonia, sinusitis, conjunctivitis, ophthalmitis and joint infections (amato et al. 2017). the incidences of listeriosis are very low in general population, but it remains a major and deadly food-borne disease with hospitalisation rate of over 95% (scallan et al. 2011). this disease occurs in specific segments of the population, which are the elderly, pregnant women, unborn babies and immunocompromised people such as those suffering from acquired immune deficiency syndrome (aids) or cancer or those who have undergone organ transplants (maertens de noordhout et al. 2014). listeriosis is characterised by a wide spectrum of infections, which are categorised into two forms, namely severe invasive listeriosis and non-invasive febrile gastroenteritis (buchanan et al. 2017). invasive listeriosis mostly occurs in immunocompromised individuals and manifests itself as sepsis, meningitis, endocarditis, encephalitis, meningoencephalitis, septicaemia and brain infection (doganay 2003). the brain infection because of listeriosis in immunocompromised adults is responsible for 22% fatalities, whereas endocarditis occurs in 10% of adults (mizuno et al. 2007). pregnant woman have 17-fold increased risk of contracting invasive listeriosis, and this infection mostly occurs in the third trimester (mateus et al. 2013). listeriosis in pregnant women is generally associated with flu-like symptoms with or without gastrointestinal problem (doganay 2003). however, the consequences of foetus or newborn infection are extremely severe, which includes abortion, premature birth, pneumonia and meningitis (indrawattana et al. 2011). invasive listeriosis is responsible for over 90% of hospitalisation and between 20% and 30% case fatality rate (leong et al. 2014), making it one of the most serious food-borne diseases. non-invasive gastroenteritis can manifest in immunocompetent adults and usually causes atypical meningitis, septicaemia and febrile gastroenteritis characterised by fever and watery diarrhoea lasting for 2–3 days, which is often accompanied by headache and backache (mateus et al. 2013). these symptoms are usually self-limiting and can be resolved within a short period without seeking any medical attention, subsequently leading to undiagnosed and under reporting of cases (matle 2016). in addition, the health workers are unlikely to report condition associated with non-invasive listeriosis because of their less severe manifestation when individuals seek medical attention. this poses a challenge to the health care system to maintain the knowledge and diagnose listeriosis cases especially in the developing world (maertens de noordhout et al. 2014). the expression of both forms of listeriosis depends on the age of the individual, immune status of the individual, infectious dose and mode of infection, virulence of strain ingested and physiological stage (poimenidou et al. 2018). the clinical signs of this disease often appear after a long incubation time (1 day – 70 days), which make epidemiological source tracing very difficult (buchanan et al. 2017). goulet et al. (2008) reported that incubation periods are largely influenced by clinical forms of the disease, with the longest incubation periods observed in pregnancy cases (median 27.5 days), followed by the central nervous system infection (median 9 days), sepsis (median 2 days) and febrile gastrointestinal disease (median 24 h). the estimated infective dose for listeriosis to occur in susceptible population is 0.1 to 10 million colony-forming units (cfu), whereas in healthy individual it is 10 to 100 million cfu (angelo et al. 2017). a study performed by nappi et al. (2005) indicated that molecular characterisation of l. monocytogenes using serotyping allowed for the identification of the serotypes ½a, ½b and 4b as the predominate causative agents of listeriosis in humans. furthermore, several studies showed that most human listeriosis outbreaks have been associated with l. monocytogenes serotype 4b, suggesting specific virulence properties in this serotype (de cesare et al. 2001; salcedo et al. 2003; vasconcelos et al. 2008). serotyping of l. monocytogenes isolates have also shown that serotypes belonging to antigenic group ½ (½a, ½b, ½c) have been over-represented in food isolates with sharp increase in clinical human isolates (vázquez-boland et al. 2001). no association have been established between certain forms of listeriosis and specific serotypes, but studies suggest a link between perinatal listeriosis and serotypes ½a and 4b (soni et al. 2014). in europe and north america, most human listeriosis cases over the past 20 years (2000–2010) involved serotype 4b and it was shown to be over-represented in perinatal listeriosis (lacroix et al. 2014). the four defined lineages (i, ii, iii and iv) of the l. monocytogenes, lineage i and ii isolates have been associated with the majority of human listeriosis outbreaks and concurrent sporadic cases in the world (schmitz-esser et al. 2015). this suggests that serotypes harboured by these lineages have an increased virulence or a better adaptation to the human host (corde et al. 2018). the distribution of these lineages varies by region with lineage ii isolates mostly common amongst human listeriosis cases in europe (martín et al. 2014), compared with the us where lineage i strains seem to be predominant amongst human listeriosis cases (roberts et al. 2018). the difference in geographical distribution of these lineages is linked to epidemic clones (ecs). epidemic clones are closely related strains, associated with several geographically and temporally distinct listeriosis outbreaks (chen & knabel 2007). these ecs are also implicated in many human listeriosis outbreaks and contribute significantly to sporadic cases worldwide (scortti et al. 2018). the eci and ecii were each responsible for repeated human listeriosis in the us and europe (mammina et al. 2013). the eciii caused a multistate outbreak associated with contaminated turkey (chen & knabel 2007). many food-borne listeriosis outbreaks have been linked to diverse food products, but different types of meat have been implicated in major human listeriosis outbreaks worldwide. table 1 gives an overview of the listeriosis outbreaks associated with meat products during 1987–2018. the first laboratory-confirmed invasive case of listeriosis associated with meat products occurred in 1988 because of consumption of contaminated turkey franks (schwartz et al. 2018). since then, the vast majority of meat products were involved in listeriosis outbreaks or sporadic cases and included processed, vacuum-packaged meat products (cases et al. 2016; chen et al. 2017), pork tongue (bozzuto, ruggieri & molinari 2010), sausages (cases et al. 2016) and polony (smith et al. 2019). the largest documented outbreaks of listeriosis in sa occurred between 2017 and 2018. this outbreak was associated with consumption of polony, which is a ready-to-eat (rte) meat product and serotype 4b was the predominant isolate (smith et al. 2019). table 1: major food-borne listeriosis outbreaks because of meat products in the world. pathogenicity of listeria monocytogenes the success of l. monocytogenes to induce infection is because of the ability to promote its own internalisation through host cells (carvalho et al. 2014). this pathogen has the capacity to pass three important barriers in the human host, namely the intestinal epithelium, the blood–brain barrier and the placenta and subsequently disseminate to other organs (chen et al. 2009). the infection process of host cell by l. monocytogenes involved several different stages: adhesion and invasion of host cells, internalisation by host cells, lysis of vacuole, intracellular multiplication and intercellular spread to the adjacent cell (vazquez-boland et al. 2001). upon ingestion through contaminated food, l. monocytogenes survives exposure to high acidity, bile salts, non-specific inflammatory attacks and proteolytic enzymes from the host system (jeyaletchumi et al. 2012). having survived this stage, l. monocytogenes adheres to and enters both phagocytic and non-phagocytic cells of the host through the assistance of surface proteins called internalin (carvalho et al. 2014). the phagocyte cells possess mechanisms that are used to destroy ingested bacteria; therefore, the ability of l. monocytogenes to survive within these cells contributes to its pathogenicity (wilson et al. 2018). after adhering to the epithelial tissue of the gastrointestinal tract through assistance of internalin proteins, l. monocytogenes is internalised by the macrophages in a primary phagosomal vacuole. after it has been internalised, l. monocytogenes escapes from phagosomal vacuole through the assistance of cytolysin called listeriolysin o (llo) and phosphatidylinositol-specific phospholipase (plca). the pathogen then replicates in the cytoplasm because of sufficient nutrients from the host. an actin-based motility machinery of the host cell facilitates intracellular movement of the organism across the cytoplasm to neighbouring cells (neves et al. 2013), thus spreading the infection without re-exposure to host extracellular immune surveillance (vazquez-boland et al. 2001). bacterial surface protein called actin polymerisation protein (acta) was identified as the molecular determinant for intracellular movement of the l. monocytogenes within the cytoplasm (bonazzi, lecuit & cossart 2009). upon being internalised by neighbouring cells, l. monocytogenes is confined in a double-membrane vacuole from which it escapes with the assistance of llo and plcb to restart its life cycle as has been shown in figure 1. figure 1: pathogenesis and virulence genes involved in listeriosis infection in human cells. virulence factors of listeria monocytogenes listeria monocytogenes consists of a large group of virulence factors that contribute to its pathogenicity and act in various steps of host infection cycle (jeyaletchumi et al. 2012). the majority of virulence determinants of l. monocytogenes are clustered along the chromosome in genomic islands or listeria pathogenicity island-1 (lipi-1) (denes et al. 2014). however, lipi-3 and lipi-4 have also been identified through whole genome sequencings (wgss) to carry important virulence factors of l. monocytogenes. the lipi-1 contains six virulence factors, which include internalin (hain et al. 2012), phosphatidylinositol-specific phospholipase (gilmour et al. 2010), actin polymerisation protein and metalloprotease (encoded by mpl) (jeyaletchumi et al. 2012). the expression of these genes is controlled by the positive regulatory factor a (prfa) (rabinovich et al. 2012). internalin adhesion and invasion of host cell by l. monocytogenes is the first step in intracellular life cycle. these steps are important for l. monocytogenes to cause disease in the host and are primarily mediated by two subfamilies of internalin proteins (vazquez-boland et al. 2001). the first subfamily is large surface proteins (70–80 kda), such as inla and inlb that attach to the bacterial cell through their c-terminal regions (bonazzi et al. 2009). the second group is the smaller sized surface proteins (25 kda – 30 kda) such as inlc, inld, inle, inlf, inlg and inlh that lack the c-terminal cell-wall anchor region (vazquez-boland et al. 2001). the adhesion of l. monocytogenes to the host and internalisation into a membrane bound vacuole is facilitated by inla and inlb genes, which are encoded by inlab operon (hain et al. 2012; hernandez-milian & payeras-cifre 2014; jeyaletchumi et al. 2012). the inla is responsible for facilitating the binding between l. monocytogenes and the host adhesion protein e-cadherin for invasion of epithelial cells (vazquez-boland et al. 2001). an e-cadherin is a protein expressed on the surface of enterocytes (lekkas 2016). this binding promotes local cytoskeletal rearrangements in the host cell to stimulate the uptake of l. monocytogenes by epithelial cells. the inlb binds the cellular receptor met, a tyrosine kinase protein, which is also the endogenous ligand of the hepatocyte growth factor (bonazzi et al. 2009). this binding allows l. monocytogenes to enter into a much wider range of host–cell types such as hepatocytes, fibroblasts and epithelioid cells (sabet et al. 2005). the differences in expression of inla and inlb are associated with poor invasion (werbrouck et al. 2006) and mutations in inla, which results in low invasion ability (cases et al. 2016). other proteins of the internalin family include inlc and inlj that are involved in post-intestinal dissemination of l. monocytogenes infection (pournajaf et al. 2016). an inlc is produced after the l. monocytogenes has entered the host cell and functions to interact with ikb kinase (ikkα), which in turn prevents activation of nuclear factor-κb (nf-κb), a proinflammatory pathway (liu et al. 2007) to dampen the host’s innate responses (cases et al. 2016). an inlj protein was discovered, as proteins that assist l. monocytogenes to cross the intestinal barrier of the host cell (cases et al. 2016). listeriolysin o and phospholipases once l. monocytogenes invades the host cell, it is trapped in the single-layer membrane vacuole. however, later in the infectious process, the bacteria are surrounded by a double-membrane vacuole (yu et al. 2018). escaping from both layers of vacuole membranes is important for an effective infection, and failure to escape the membrane results in an infection that is removed fast from the tissues (pushkareva & ermolaeva 2010). the haemolysin (hly), gene, is responsible for producing a pore-forming surface toxin called llo, which is required for lysis of vacuole membranes and the release of l. monocytogenes into cytoplasm (kyoui et al. 2014). the absence of llo equals avirulent strains of l. monocytogenes as the bacterium will not reach the cytoplasm (pushkareva & ermolaeva 2010); thus, it can be said that llo is secreted by all virulent strains of l. monocytogenes. the llo is highly affected by the environmental ph with higher levels of expression observed under acidic ph levels (ph < 6) and lower activity levels observed at neutral ph (pushkareva & ermolaeva 2010). listeriolysin s is another virulence factor that was identified by cotter et al. (2008). this virulence factor is located in lipi3 and it is regarded as secondary haemolysin, which is specifically found only on lineage i strains of l. monocytogenes. this second haemolysin is only induced under oxidative stress conditions, contributes to virulence of the pathogen as assessed by murine (mice and rats) and human polymorphonuclear neutrophil-based studies and is similar to the peptide streptolysin s produced by streptococcus (cotter et al. 2008). listeria monocytogenes also secretes phosphatidylinositol phospholipase c that are involved in the lysis of vacuole membranes. two phospholipases that assist in lysis of vacuole membranes are plca (gouin et al. 1994) and plcb (vazquez-boland et al. 2001). studies have showed that plca assists in the escape of l. monocytogenes from the primary vacuole, whereas plcb is active during cell-to-cell spread of the bacteria (doyle et al. 2013). maturation of plcb is dependent on a zinc metalloprotease, which is encoded by mpl gene. metalloprotease also assists hly, plcb and plca to disrupt the primary vacuoles after host cell invasion (o’connor et al. 2010). actin polymerising protein listeria monocytogenes reaches the cytoplasm after lyses of the vacuole and subsequently moves to infect other cells (chatterjee et al. 2006). it achieves this by a surface protein called acta that induces polymerisation of globular actin molecules to actin filaments. the filaments then facilitate movement of l. monocytogenes both interand intra-cellularly (klumpp & loessner 2013). therefore, acta is also required for l. monocytogenes pathogenicity (vázquez-boland et al. 2001), as it is critical in cytoplasmic movement of this pathogen. in a study performed by doyle et al. (2013), using mice to compare the virulence of l. monocytogenes serotypes, the researchers observed lower virulence in serotypes 4a, 4c, 4d and 4e. the lower virulence in those serotypes was attributed to the production of lower levels of the acta protein with actin tail formation. serotypes 4a, 4c, 4d and 4e are considered avirulent strains because of lack of lower levels of acta protein (doyle et al. 2013). invasion-associated protein (protein p60) the invasion-associated protein (iap) is an extracellular protein p60 that is encoded by iap gene (camejo et al. 2011). protein p60 is common amongst listeria spp. and regarded as an essential murine hydrolase enzyme that facilitates septum separation during the final stage of cell division (yu et al. 2018). it is also involved in adherence of l. monocytogenes to the host cell and plays an important role in virulence and pathogenicity of this bacterium (quendera et al. 2016). positive regulatory factor a expression of prfa is controlled in different ways either by prfa itself or by an alternative sigma factor σb (duroux et al. 2015). the temperature has an influence on the production of virulence factors because the secondary structure of untranslated prfa-mrna is temperature dependent (revazishvili et al. 2004). at 30 °c, the shine-dalgarno sequence is blocked and the ribosomes are not able to bind and translate the sequence. as a result of the positive feedback mechanism, only a small amount of prfa is therefore transcribed (duroux et al. 2015). at 37 °c, the secondary structure has changed, which results in translation of prfa-mrna followed by synthesis of prfa and results in a higher amount of transcribed prfa. the prfa is the primary regulator of the expression of the virulence factors present in the virulence gene cluster (figure 2), but other proteins act as virulence gene regulators too. the virr is a response regulator critical for l. monocytogenes virulence (ragon et al. 2008). the genes regulated by virr encode abc transporters, proteins involved in resistance to human defences in staphylococcus aureus and cell wall modification proteins (ragon et al. 2008). the alternative sigma factor σb is an overall regulator of the expression of several genes in response to several types of stresses, and it also regulates the expression of prfa and thereby the expression of virulence factors. the prfa is the primary regulator of the expression of the virulence factors present in the virulence gene cluster, but other proteins act as virulence gene regulators as well (ryan et al. 2010). figure 2: organisation of the central virulence gene cluster of listeria monocytogenes. epidemiology of human listeriosis the cases of human listeriosis and the number of high profile outbreaks that resulted in many deaths had significantly increased in many countries (de castro et al. 2012). the increase is mainly because of changing consumption behaviours, as many individuals consume rte foods (mateus et al. 2013). furthermore, globalisation of food trade and demographic changes such as increase in susceptible populations because of ageing and existence of other immune-compromising infections have augmented the risk of listeriosis (wang et al. 2012). the introduction of sequencing methodologies for detection and typing of listeriosis outbreaks has also led to increased number of cases being reported (zuber et al. 2019). epidemiological surveillance studies showed that human listeriosis is mostly reported in high-income and industrialised countries because of proper surveillance system for food-borne diseases (grace 2015). table 2 shows the overall incidence of listeriosis per 100 000 people in different developed countries. surveillance of human listeriosis performed by european union (eu) between 2006 and 2012 on 18 member states reported a notification rate of 0.41 cases per 100 000 population (food and authority 2013). the highest notification rates were observed in finland, spain and denmark with a hospitalisation rate of 91.6% on an average (food and authority 2013). the surveillance also reported an increased incidence of listeriosis in greece (0.3), sweden (0.6), norwary (1.0), france (0.6) and scandinavia (0.2). this trend is accounted for by increased cases in the population older than 60 years and the higher consumption of smoked fish in these countries (food and authority 2013). furthermore, the surveillance reported 198 deaths because of listeriosis from 18 member states. furthermore, the eu performed wide baseline survey in 26 member states between 2010 and 2011 to determine the prevalence of l. monocytogenes in food products at retail outlets (food and authority 2013). a total of 13 088 food samples including smoked or gravad fish (3053), meat products (3530) and cheese types (3452) were tested for the presence of l. monocytogenes. the eu prevalence of l. monocytogenes varied amongst different food products with the highest recorded in fish (10.4%), followed by meat products (2.07%) and cheese (0.47%) (food and authority 2013). table 2: incidences of listeriosis in different countries. the notification rate of human listeriosis in the us was reported to be approximately 0.3 cases per 100 000 population (todd & notermans 2011). this rate was similar to that reported in canada and new zealand, which was lower compared with europe (food and authority 2013). in the us, approximately 1 600 individuals get listeriosis annually with 21% case fatality rate (todd & notermans 2011). almost all the fatalities were reported in high-risk groups, such as older adults, pregnant women and people who were immunocompromised. the foodborne disease active surveillance network (foodnet) had a surveillance system in 10 states of america for laboratory-confirmed cases of food-borne diseases (rip 2011). this system revealed that there is a greater likelihood of being hospitalised from illness caused by listeria than any other food-borne pathogen in the us (rip 2011). the ozfoodnet which is a working group established by the australian government and department of health and ageing to survey listeriosis reported an annual (2001–2010) incidence rate of 0.3 cases per 100 000 population in australia (popovic, heron & covacin 2014). according to the japan nosocomial infections surveillance, approximately 135–201 cases of human listeriosis occurred annually between 2008 and 2011 (miya et al. 2015), which suggests an incidence rate that is equivalent to 1.40 cases per 100 000 populations. cases of human listeriosis have also been reported in other asian countries such as taiwan that reported 48 cases of listeriosis between 1996 and 2018 (laksanalamai et al. 2014), whereas china reported 479 cases over a period of 46 years (1964–2010) (jadhav 2015). the world health organization (who) evaluated the median rate of listeriosis in different regions in 2010 and estimated that the incidence of listeriosis in africa was 0.1 cases per 100 000 populations (carp-cǎrare, vlad-sabie & floriştean 2013). however, the data in this evaluation were limited as many african countries neither report nor monitor the incidence of listeriosis because of lack or absence of targeted surveillance programmes and reporting systems (grace 2015; todd & notermans 2011). despite the poor surveillance programmes and lack of epidemiological data to establish a comprehensive incidence rate of human listeriosis in africa, there are a number of studies that reported the occurrence of l. monocytogenes in foods from african countries (table 3) (bouayad & hamdi 2012; derra et al. 2013; el-shenawy et al. 2011; matle et al. 2019). table 3: prevalence of listeria monocytogenes in foods in africa. in sa, an average of 60 to 80 laboratory-confirmed listeriosis cases were reported annually before 2017 (smith et al. 2019). this suggests an incidence rate of 0.1 cases per 100 000 populations, which is in agreement with that reported by who. however, in 2017, an increase in laboratory-confirmed cases of listeriosis was reported by national institute for communicable diseases (nicd), which was linked to an outbreak. in 2018, the nicd had identified a total of 1024 laboratory-confirmed human cases across all nine provinces of sa that resulted in 216 deaths. the majority of cases were reported in the gauteng province (59%) followed by the western cape (13%) and kwazulu-natal (7%) provinces. these cases were mostly reported in vulnerable groups, which includes infants (≤ 28 days), hiv-positive individuals, the elderly (> 65 years) and pregnant mothers (smith et al. 2019). the agricultural research council of sa performed a comprehensive national baseline survey across nine provinces of sa including major ports of entry between 2014 and 2016 to determine the occurrence of l. monocytogenes in meat and meat products in abattoirs, meat-processing plants and retail outlets. meat samples (n = 2017) consisting of raw meat, processed meat and rte meat were analysed for l. monocytogenes. the occurrence of l. monocytogenes in meat destined for the south african market varied between imported (12.4%) and domestic (15.0%) meat samples, with the highest proportion reported in processed meat (19.5%), followed by rte (13.5%) meat products and raw (10.1%) meat (matle et al. 2019). legislations relating to listeria monocytogenes in food food-borne pathogens including l. monocytogenes remain a serious threat to public health and a significant impediment to socio-economic development worldwide (grace 2015). many countries responded to the threat posed by food-borne pathogens through implementation of strict regulations for microbiological standards or criteria in relation to contamination of food products (strydom 2015). microbiological criteria for food safety refer to the guidelines that are used to determine if a food product(s) is/are acceptable for human consumption based on the bacterial load of specific micro-organism on the food. the microbiological standards vary amongst different countries; however, they are guided by international standards such as international commission on microbiological specifications for food (icmsf) and codex alimentarius commission (cac) (rip 2011). the icmsf is a voluntary advisory organisation, which sets standards and methods regarding the presence of micro-organisms in food. the icmsf states that food sample testing can be used for evaluation of safety and quality of food and to assess, validate the efficacy of microbial control measure such as hazard analysis and critical control point (rip 2011). the icmsf recommends maximum of 100 cfu/g of l. monocytogenes in food at the time of consumption for non-risk consumers. the cac is a set of food standards, guidelines and codes of practice produced with the aim of protecting consumer health and facilitating international trade (rip 2011). compliance with the cac standards is voluntary, but many governments and non-government institutions use the cac guidelines as the basis for legislation and microbiological standards. the cac standard on l. monocytogenes applies to only rte foods, which divides them into three categories based on the ability of food product to support the growth of this pathogen (food and authority 2013). microbiological criterion for l. monocytogenes in rte foods belonging to the first category does not need to be determined. therefore, microbiological criteria according to cac have been set for rte food products belonging to the second and third categories. the second group represents rte foods that do not support the growth of l. monocytogenes, whereas the third group applies to the products that can support the growth of this pathogen. the acceptable level of 100 cfu/g has been set for food in the second category, whereas zero tolerance policy (absence of l. monocytogenes in 25 g of food) for products in the third category (obaidat, salman & lafi 2015). the european microbiological standard for l. monocytogenes in rte food products is in accordance with cac recommendations (food and authority 2013). the standard requires the absence of l. monocytogenes in rte food products that are intended for infant consumption or as a medical food and in the food that can support the growth of this pathogen. however, in rte food products that cannot support the growth of l. monocytogenes, the level < 100 cfu/g is required during the shelf life of those foods. canada, australia and new zealand adopted microbiological standards similar to those of europe as recommended by cac (jadhav 2015). the us has zero tolerance policy for l. monocytogenes in rte food products and food-processing facilities and failure to comply is considered a serious offence (piet et al. 2016). in sa, meat safety is an important part of public health linking agriculture to health. the responsibility of ensuring meat safety is shared by two main national departments, namely the department of agriculture, forestry & fisheries (daff) and the department of health (doh). the daff exercises authority over farms, feedlots and abattoirs and is mandated to administer the animal diseases act, act 35 of 1984 and the meat safety act, act 40 of 2000 (magwedere, songabe & dziva 2015). as soon as the meat leaves the abattoir supervision of distribution, retail and marketing falls in the hands of the doh, which is entrusted with the foodstuffs, cosmetics and disinfectants act (fcda), act 54 of 1972 (as amended by act 39 of 2007). section 2 (b) (i) of the fcda does not allow any individual at food premises registered under this act to handle meat from an animal slaughtered in contravention of the meat safety act. the fcda forbids selling, manufacturing or importing for sale, any foodstuff that is contaminated, tainted or decayed or is in terms of any regulation deemed to be, detrimental to human health. however, these regulations are salient on microbiological criterion for l. monocytogenes in meat (magwedere et al. 2015). the only microbiological criterion available for testing of meat in sa is the south africa national standard (sans): 885:2011, which is non-mandatory and allows a maximum of 100 cfu/g for l. monocytogenes in rte processed meat products. occurrence of listeria monocytogenes in meat value chain listeria monocytogenes has been isolated from many different environments such as agricultural soil and vegetation (susana et al. 2017), food-processing facilities (martín et al. 2014) and retail outlets (henri et al. 2016). therefore, several sources have been identified as possible routes for contamination of food and subsequently transmission of l. monocytogenes to human beings. contamination of meat and meat products with l. monocytogenes is a complex process that links primary food production (farm, feedlots) to retail outlets (figure 3). figure 3: implicated routes of transmission for listeria monocytogenes infection to humans. listeria monocytogenes in animal farm in the farm or feedlots, l. monocytogenes is mostly found in soil as natural inhabitant but often at relatively low numbers (o’connor et al. 2010). listeria monocytogenes can survive in the soil especially in agricultural soil for months and even grow (rip 2011). sauders et al. (2012) reported a prevalence range from 8.7% to 51.4% for l. monocytogenes in agricultural soil, whereas in non-agricultural soils the prevalence ranged between 15.2% and 43.2%. this suggests that the soil serves as the principal reservoir of l. monocytogenes, where meat-producing animals are exposed to this pathogen through interaction with the natural environment. contaminated soil dust has also been found to contain this pathogen, suggesting that the animal can acquire l. monocytogenes through air and subsequently transmit it to human beings through the meat value chain (korthals et al. 2008). several studies have associated animal listeriosis with consumption of contaminated silage by l. monocytogenes (korthals et al. 2008; harakeh et al. 2009; parihar 2004; werbrouck et al. 2006). silages are a traditional feed used to conserve animals during forage shortage because of seasonal changes in dry season (zhu, gooneratne & hussain 2017). therefore, a poorly prepared silage can support the growth of l. monocytogenes that can subsequently infect animals (nightingale et al. 2004). faeces shed by infected animals along with silage are vehicles for primary infection in ruminants and reintroducing of l. monocytogenes into the environment (lekkas 2016). asymptomatic shedding of l. monocytogenes by infected animals can also transmit this pathogen to human beings through food (piet et al. 2016). other sources of l. monocytogenes in a farm include poor animal husbandry, natural water and wastewater source. linke et al. (2014) found that natural water and wastewater sources near farming communities harbour large quantities of l. monocytogenes and may serve as sources of animal contamination. as l. monocytogenes is found naturally in the environment, pathogen control measures should therefore start from farm level up until the meat reaches the consumers’ table. pre-harvest pathogen control includes all measures and management practices at farm level to reduce the probability of having pathogens in animals and final meat products (nørrung & buncic 2008). this should aim to minimise sources, access, levels and transfer of contaminants to the animal. technologies used to reduce pathogen levels in animals include diet manipulation (supplements), effective biosecurity and optimum animal welfare (sofos & geornaras 2010). proper animal management practices such as provision of clean water and feed and proper waste management to limit spreading of pathogens into the environment also help to reduce pathogen levels in animals (buncic et al. 2014). however, it is difficult for farmers to control pathogens at this level because of lack of knowledge, resources and money in some cases. listeria monocytogenes in food-processing facilities listeria monocytogenes has also been reported in different meat-processing facilities such as abattoirs, meat-processing plants and butcheries (carpentier & cerf 2011). several studies suggest that animals presented for slaughter are an important source of initial contamination of meat-processing facilities with l. monocytogenes (autio et al. 2000; churchill et al. 2006; lekkas 2016; nigtingale et al. 2004). autio et al. (2000) characterised different l. monocytogenes strains isolated from environmental samples in a pig farm and carcasses at abattoirs using pulsed-field gel electrophoresis (pfge). the same pulsotypes were detected in a pig farm and carcasses, indicating that l. monocytogenes from farm might have contaminated the carcasses during production and processing cycle. furthermore, nel, van vuuren and swan (2004) indicated that animals presented for slaughter often harbour large quantities of micro-organisms including l. monocytogenes on the external surface and hooves, which can be introduced in processing facilities if proper hygiene measures are not followed. in addition to animals, contamination may also enter the meat-processing facility through raw material and personnel carrying l. monocytogenes (el-shenawy et al. 2011; pava-ripoll et al. 2012; thévenot, dernburg & vernozy-rozand 2006). floors, floor drains, racks and rollers are also reported to be sources of contamination in meat-processing facilities (thévenot et al. 2006). once l. monocytogenes enters the processing facilities it is unlikely to be eradicated if proper monitoring programmes are not intensive as its survival is influenced by several complex factors (carpentier & cerf 2011). the contributing factors to survival of l. monocytogenes include the ability to proliferate under harsh stress conditions such as low temperature, ph and osmotic stress (takahashi et al. 2014), resistance to sanitation agents and formation of biofilms (carpentier & cerf 2011). meat-processing facilities use cold storage (4 °c) to reduce the proliferation of bacteria on meat. although this process is effective against many bacteria, it supports the growth of l. monocytogenes as this bacterium has the ability to survive at low temperature. the presence of cold shock proteins and sigma factor σb, encoded by sigb, in listeria spp., help them to survive in low temperature and osmotic pressure (leong et al. 2014). the occurrence of l. monocytogenes in meat-processing environment may also increase or decrease because of sanitation (carpentier & cerf 2011). sanitation is a process that consists of cleaning protocols, followed by disinfection. proper cleaning and sanitation procedures decrease the occurrence of l. monocytogenes in a meat-processing facility. however, this pathogen is often found even after cleaning. it shows the persistence of some strains and, on many occasions, the insufficiency of the cleaning (cruz & fletcher 2011). during the cleaning process, detergents are used to remove microbial agent on food-processing areas. however, this process is hampered by the presence of harbourage sites within the facility and concentration of sanitation (lourenço, neves & brito 2009). harbourage sites are areas where the sanitation agents do not reach correctly, and as a result of this it allows the proliferation of l. monocytogenes. several studies showed that l. monocytogenes has the ability to resist commonly used sanitation agents in food-processing facilities (bremer, monk & butler 2002; fouladynezhad et al. 2013; pan et al. 2006). these studies also reported that the resistance of l. monocytogenes to sanitising agents in food-processing facilities is because of the formation of biofilms and they subsequently persist in facility for several years. in fact, these studies showed that l. monocytogenes could form biofilms on surfaces such as polyethylene, polyvinyl chloride, glass and stainless steel in food-processing facilities. the formation of biofilms are influenced by characteristics of strains, physical and chemical properties of the substrate for attachment, growth phase of the bacteria, temperature, growth media and the presence of other microorganisms (carpentier & cerf 2011). listeria monocytogenes in retail level the detection of l. monocytogenes in meat and meat products at retail level does not mean that contamination occurred in the retail environment (sauders et al. 2016). however, cross-contamination from surfaces, equipment and workers and persistence of strains have been identified as the main source of l. monocytogenes in retail products. gombas et al. (2003) reported that meat products handled, sliced and packaged at retail outlets have high levels of l. monocytogenes than in products pre-packed at the abattoir. however, this depends on the level of hygienic practice followed by food handlers. personal hygiene of meat handlers and proper sanitisation of contact surfaces and utensils are important to prevent cross-contamination or recontamination in retail outlets (bogere & baluka 2014). storage temperatures should also be controlled to inhibit multiplication, surviving and growth of existing pathogens. listeria monocytogenes in meat and meat products several studies have reported the existence of l. monocytogenes in meat and meat products originating from different animal species including game (lambertz et al. 2012; kramarenko et al. 2013; who & world organisation for animal health 2014). in a study performed by vitas and garcia-jalon (2004) in spain that examined 396 meat product samples collected from 55 small meat-processing units identified l. monocytogenes in 34.9% of minced pork and beef meat products and in 36.1% of poultry meat. in sweden, a survey of 507 heat-treated rte meat product samples from 110 municipalities were analysed for l. monocytogenes. this pathogen was detected in 61% of heat-treated ham meat products, followed by 12% of turkey, 9% of roast beef and 7% of sausage (lambertz et al. 2012). in estonia, a survey baseline conducted over 10 years (2008–2010) indicated that l. monocytogenes was prevalent in 18.7% of raw meat and raw meat products, and 2% in rte meat products collected from various food-processing facilities (kramarenko et al. 2013). in ireland, rte meat samples analysed between 2013 and 2014 identified l. monocytogenes in 4.2% of meat products collected from poultry (leong et al. 2014). ismaiel, ali and enan (2014) reported l. monocytogenes in beef carcasses, raw lean beef, frozen chicken meat and camel meat. dhanashree et al. (2003) and okutani et al. (2004) isolated l. monocytogenes in various meat products in india and japan, respectively. matle et al. (2019) reported l. monocytogenes in various meat and meat products including raw intact meat (10.1%), raw processed meat (19.5%) and rte meat products (13.5%) collected from cattle, pork, sheep, game and poultry in sa. diagnosis of listeria monocytogenes identification of l. monocytogenes is extremely important for prevention and disease control. the method used for detection and isolation of l. monocytogenes has evolved over the years from cold enrichment technique to conventional and molecular methods (who & world organisation for animal health 2014). conventional methods several conventional methods have been developed for isolation and identification of l. monocytogenes in food samples. the conventional method of isolation of l. monocytogenes includes antibody-based tests, enzyme-linked immunosorbent assay, culture-based methods and immune-capture techniques (välimaa, tilsala-timisjärvi & virtanen 2015). out of these methods, culture-based tests are usually preferred for many reasons such as being sensitive, cheap and they remain the ‘gold standards’ compared with other methods that are validated (barajas et al. 2019). in addition, pure colonies of the targeted organisms obtained by culture-based assays are useful for epidemiological surveillance and outbreak management purposes (who & world organisation for animal health 2014). the drawbacks of culture-based methods include low resolution regarding distinguishing bacterial strains. furthermore, these methods are laborious (leong et al. 2014) and phenotypic changes because of environmental selection, contaminating bacteria and atypical reactions by atypical strains can provide false-negative results (välimaa et al. 2015). the isolation and identification of l. monocytogenes using culture-based methods involve the use of selective agents and enrichment procedure. the purpose of the selective agents is to inhibit other competing microflora whilst the enrichment procedure allows the increase of l. monocytogenes to detectable levels and the recovery of injured or stressed cells (chen et al. 2017). there are three commonly used culture-based methods (figures 4–6) for isolation of l. monocytogenes in foods because of international regulations and requirements. these methods include the international standard (iso), the united states department of agriculture (usda) and one-broth listeria method (gasanov, hughes & hansbro 2005; gómez et al. 2013; zhang et al. 2004). although these methods are internationally recommended and accepted for testing of a wide variety of food matrices, they must be used in accordance with their scope. figure 4: the illustration of international standard method. figure 5: the illustration of united states department of agriculture method. figure 6: the illustration of one-broth method. the iso 11290 standard is recommended for isolation of l. monocytogenes in a large variety of food and feed products as well as environmental samples (liu et al. 2007). the usda method is recommended for the detection of listeria spp. in meat and poultry food products and environmental swabs (jeyaletchumi et al. 2012). one broth listeria method that has been approved by the association française de normalisation (afnor) is used for isolation of listeria spp. from dairy food products, meat, seafood and vegetables (gasanov et al. 2005). all above-mentioned methods involve a series of primary and secondary enrichments of the samples in selective broth media. the iso 11290 method utilises half fraser broth and fraser broth for primary and secondary enrichment, respectively. the usda method uses a two-step enrichment in university of vermont media, whereas the one broth listeria method uses a one-step enrichment in listeria broth, which takes 2 days to produce results as opposed to the 5 and 4 days needed by iso and usda methods, respectively (leong et al. 2017). these enrichment media contain different selective agents including cycloheximide, colistin, cefotetan, fosfomycin, lithium chloride, nalidixic acid, acriflavine, phenylethanol, ceftazidime, polymyxin b and moxalactam (jadhav 2015). these antibiotics inhibit mostly the growth of gram-negative bacteria that are often present as competitors in food samples. the mechanism of inhibition varies amongst these antibiotics, which includes inhibition of protein synthesis (cycloheximide, fosfomycin and nalidixic acid), disruption of the outer cell membrane of bacteria (colistin) and beta-lactamase (cefotetan) amongst others. following primary and secondary enrichment, the broth is generally plated onto selective or differential media. the iso 11290 recommends the use of oxford and palcam agar for detection and isolation of l. monocytogenes. the usda uses chromogenic media such as agar listeria ottaviani and agosti and rapid-l. mono (leong et al. 2017), whereas one broth listeria method requires the use of listeria brilliance green agar. these media are typically dependent on the β-glucosidase activity of listeria, which cleaves the chromogenic substrate producing blue or green colonies. lecithin present in the agar is hydrolysed by phospholipase enzyme synthesised only by l. monocytogenes, leading to the formation of opaque halos around their colonies (jeyaletchumi et al. 2012). presumptive listerial colonies on selective agar are confirmed by rapid tests and on biochemical properties such as gram stain, catalase test, motility test, ability to produce haemolysis on blood agar plates, christie–atkins–munch-peterson test with rhodococcusequi and staphylococcus aureus and carbohydrate utilisation tests. molecular methods of detection polymerase chain reaction-based methods for detection of listeria monocytogenes • conventional polymerase chain reaction the polymerase chain reaction (pcr) method has been used extensively for the detection of l. monocytogenes. conventional pcr targets the most common and specific genes of l. monocytogenes such as hly, inla, inlb, iap, plca, plcb, 16s and 23s rrna genes and dth-18 delayed type hypersensitivity protein (jadhav 2015). the conventional pcr techniques are used more frequently than cultural procedures as they are simple and can provide quick results (jeyaletchumi et al. 2012). however, this pcr cannot distinguish between live or dead cells or viable but not culturable cells (truter 2015), metabolically injured, stressed cells or reliably detect low levels of l. monocytogenes (jadhav 2015). therefore, a positive sample through conventional pcr does not necessarily mean that the organism is alive and in required concentrations, which makes that organism a public health risk (quendera et al. 2016). multiplex polymerase chain reaction multiplex pcr is another pcr-based method, which allows the detection of multiple strains from the same species or multiple pathogens in a sample simultaneously (chen et al. 2017). the detection specificity of this method depends on the specific binding of the primer pair to the target sequence of the micro-organism. ryu et al. (2013) developed a multiplex pcr method that can distinguish between five different listeria spp. including l. monocytogenes by targeting different genes for each species. multiplex pcr can detect between 1 and 100 cfu/ml listeria (jadhav 2015); however, similar to conventional pcr it can overestimate the presence of the pathogen because it cannot distinguish between live and dead cells. real-time polymerase chain reaction real-time pcr differs from other pcr techniques because the amplicon is observed as it accumulates. the procedure monitors the accumulation of fluorescence levels, which in turn depend on the amount of the accumulated pcr product. the fluorescent molecule can be either a target-specific probe labelled with a fluorescent dye together with a quencher molecule or can be a non-specific dna-binding dye. the method is highly sensitive, can detect and trace amounts of target dna, can be automated and has the ability to quantify bacterial load without any post-pcr handling. however, its disadvantages are that primer dimers can show fluorescence, it is highly dependent on primer concentration and design and it requires stringent quality controls. subtyping of listeria monocytogenes subtyping is a process that is used to discriminate amongst different bacterial strains that belong to the same species (jeyaletchumi et al. 2012). subtyping procedures are very useful for source identification and tracking of individual strains of l. monocytogenes that are involved in listeriosis outbreaks and to determine the population genetics, taxonomy and epidemiology of this pathogen (doumith et al. 2004). there are many subtyping methods, which are broadly grouped as phenotypic and genotypic; however, not all will be discussed in detail in the current study, as they are not within the scope of this review. those methods are listed in table 4. table 4: advantages and disadvantages of subtyping techniques used for listeria monocytogenes strains. phenotypic subtyping methods serotyping: serotyping is the first method used to differentiate l. monocytogenes strains from each other based on antigen–antibodies reaction (ntivuguruzwa 2016). listeria monocytogenes serotyping is performed using the slide agglutination method that characterise l. monocytogenes into 13 serotypes (½a, ½b, ½c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7) using unique combinations from somatic (o) and flagellar (h) surface antigens (antisera) reaction (doumith et al. 2004). the value of this method in epidemiological studies is very limited because of poor discriminative powers (gasanov et al. 2005). serotyping is also associated with many drawbacks including failure to provide consistent or reliable and repeatable results, and it measures the phenotypic characteristics of l. monocytogenes, which are often subjected to change and does not always accurately reflect the genotype of a micro-organism (liu 2006). in addition, serotyping is time consuming, difficult and requires high-quality antisera (shaker & hassanien 2015). antigen sharing between l. monocytogenes and l. seeligeri may lead to detection of incorrect serotype (liu 2006). genetic sub-typing methods polymerase chain reaction serogroup multiplex polymerase chain reaction assays: to overcome the limitations of slide agglutination and enzyme-linked immunosorbent assay (elisa) serotyping, a multiplex pcr-based method was then introduced by borucki and call (2003) for serotyping l. monocytogenes into five pcr serogroups; iia corresponded to serotypes ½a and 3a; iic to ½c and 3c; iib to ½b, 3b and 7; and ivb to 4b, 4d and 4e (doumith et al. 2004). this method uses primer pairs that target five genes (lmo0737, lmo1118, orf2110 and orf2819 and prs) to characterise l. monocytogenes strains and assign those pcr serogroups (doumith et al. 2004; nho et al. 2015). however, this assay could not distinguish between ½a and 3a, ½c and 3c, ½b, 3b and 7, 4a and 4c and between 4b, 4d and 4e, but as 3a, 3c, 3b, 7, 4a, 4c, 4d and 4e are rarely involved in human listeriosis, this approach was considered to be suitable for rapid detection of serotypes. although this method is quick, robust and easy to implement, it has limited discriminatory power, thus providing poor resolution for epidemiological typing (nho et al. 2015). pulsed-field gel electrophoresis: the pfge is considered as ‘gold standard’ subtyping method for source tracking and epidemiologic investigations of infection caused by l. monocytogenes. this is because of its high discrimination power, robustness and reproducibility (martín et al. 2014). however, it is time consuming and labour intensive, usually taking 2–3 days to complete and requires equipment of relatively high cost (li et al. 2017). in pfge, the genomic dna is digested (generally using asci and apai enzymes) with infrequent cutting endonucleases, which results in the generation of fewer fragments with high molecular mass (kalpana & muriana 2002). these fragments can be separated on the basis of their size using pfge (ruppitsch et al. 2015). the pfge usually separates dna fragments of less than 50 kb, as dna fragments above 50 kb produce large and diffused bands (jersek et al. 1999). in addition, it uses electric fields of alternating direction, which cause the dna fragments to continuously change direction. this results in the resolution of high molecular weight dna into separate bands (hopkins, arnold & threlfall 2007). multiple-locus variable number of tandem repeat analysis: the multiple-locus variable number of tandem repeat analysis (mlva) works on the principle to detect variation in the number of tandem repeats (vntrs) at a specific locus in the genome dna. the vntrs are short segments of dna that have variable copy numbers that can be determined by performing pcr amplification. the size of the pcr product is then analysed on agarose gels and/or by dna-sequencing systems. the pcr product sizes are then used to determine the number of repeats in each region (volpe sperry et al. 2008). therefore, by combining the size differences from several repeat loci regions, a multidigit, strain-specific code (profile) can be acquired, and these profiles can therefore be used for cluster analysis (møretrø, langsrud and heir 2013; murphy et al. 2007). the mlva gained acceptance as subtyping method of bacterial isolates because of its robustness, rapidness and high discriminatory power (camargo et al. 2016). many authors suggested that mlva can be a useful tool for the characterisation of l. monocytogenes strains and that it represents an attractive first-line screening method to epidemiological investigations and listeriosis surveillance (chenal-francisque et al. 2015; lindstedt et al. 2008; murphy et al. 2007). however, the main drawback of mlva is the lack of reproducibility and its results cannot be compared amongst different laboratories. in addition, vntrs are unstable and can even undergo change during routine laboratory subculturing and therefore affect the reproducibility of the mlva method. multilocus sequence typing: different multilocus sequence typing (mlst) methods have been described for l. monocytogenes typing (den bakker et al. 2013; roberts et al. 2018). the mlst depends on multiple gene fragments or genes to differentiate between subtypes (jeyaletchumi et al. 2012). the method relies on amplification of seven loci from housekeeping genes that are analysed for nucleotide differences. this method has been shown to be more discriminatory when compared against the gold standard for typing of l. monocytogenes pfge method (leong et al. 2017). furthermore, mlst is considered an expensive and time-consuming method because it requires numerous sequencing reactions per isolate and cannot be multiplexed and it does not have enough discriminatory power for 4b l. monocytogenes serotypes (jadhav 2015), which are amongst the serotypes often implicated in outbreaks. direct interrogation of single-nucleotide polymorphisms (snps) could offer a more efficient alternative for dna sequence-based subtyping based on the fact that the majority of sites sequenced for mlst are invariant (ducey et al. 2006). whole-genome sequencing: despite the fact that the phenotypic and molecular methodologies mentioned above have numerous advantages, those techniques, however, give limited information about the pathogenic organism because of discriminatory capacity (lekkas 2016). the wgs has the power to overcome this hurdle and group isolates into epidemiologically relevant groups. the wgs has been recommended as new gold standard for subtyping of l. monocytogenes strain associated with outbreak (fox et al. 2016). this technique has greatly improved since its inception, with the reduction in process time and the introduction of high-throughput next-generation sequencing technology. furthermore, the technology is becoming cheaper and more user-friendly. in addition to epidemiological data, wgs can provide rapid generation of whole-genome sequence data that can help to identify targets that could be used to develop assays (lekkas 2016). although the use of wgs provides valuable information that was not available previously in such a short time period, there are still major barriers that need to be addressed before these techniques can be incorporated in food-borne pathogen detection (lekkas 2016). the need for computer platforms that are operator friendly, powerful enough to handle the massive data bases that are created and are easily interpreted still exists. in addition, there is no consensus on how these data will be stored or used by regulatory authorities such as fda and cdc during inspections or outbreak investigations. it is almost certain that within the volume of data collected there will be some sequence data that might be misconstrued as indicating a health hazard (eruteya & odunfa 2014). furthermore, limitations of the wgs approach are the need for highly trained bioinformatics professionals that smaller companies will not be able to afford, lack of standardised and validated protocols like the ones already existing for pfge, lack of reference databases and lastly a large investment of resources that also have their own limitations (orsi et al. 2011). in addition, food-borne pathogens are usually found at very low numbers, which poses its own limitations for epidemiological studies (lekkas 2016). treatment treatment of human listeriosis can be a challenging task as l. monocytogenes may invade almost all cell types (dhama et al. 2015). furthermore, the treatment of human listeriosis is often ineffective because of long incubation period of l. monocytogenes, which makes the treatment period to vary according to the level of the infection. however, antibiotics have been used to treat human listeriosis successfully for a very long time (al-nabulsi et al. 2015). listeria monocytogenes is generally susceptible to the majority of antibiotics, but cephalosporin, fosfomycin and fluoroquinolones are not active against this pathogen (noll, kleta & al 2018). the intrinsic resistance of l. monocytogenes against these antibiotics is because of lack or low affinity of enzyme catalysing the final step of cell wall synthesis (al-nabulsi et al. 2015). the antibiotic of choice for treating human listeriosis is ampicillin or penicillin g in combination with an aminoglycoside such as gentamicin. trimethoprim in combination with a sulfonamide, such as sulfamethoxazole-co-trimoxazole, is considered second choice of therapy (kovacevic et al. 2013). furthermore, tetracycline, erythromycin and vancomycin have been used to treat human listeriosis (rip 2011). however, evolution of bacteria towards resistance has been considerably accelerated in l. monocytogenes (moreno et al. 2014). antimicrobial resistance in listeria monocytogenes the acceleration in antimicrobial resistance of l. monocytogenes is linked to selective pressure exerted by over-prescription of drugs in clinical settings and their heavy use as promoters for growth in farm animals and increased global trade and travel, which favour the spread of antimicrobial resistance between countries and continents (moreno et al. 2014; zhang et al. 2004). resistant l. monocytogenes strains have been reported against first-line antibiotics. gentamicin-resistant clinical strains of l. monocytogenes were reported (walsh et al. 2001). listeria monocytogenes strain resistant to ampicillin was identified in the us (verraes et al. 2013). listeria monocytogenes resistant to streptomycin, erythromycin, kanamycin, sulfonamide and rifampin were also reported in clinical isolates in different countries (moreno et al. 2014; zhang et al. 2004). multiple drug resistance has also been observed in strains isolated from foods and environmental samples across the world (morobe et al. 2009). a study conducted in northern ireland (walsh et al. 2001) showed 0.6% of l. monocytogenes from retail foods. furthermore, antimicrobial resistance of l. monocytogenes isolated from food and animal sources (n = 167) in the us was determined. the resistance to ciprofloxacin, tetracycline, sulfonamide and nalidixic acid were 1.8%, 9%, 73% and 100%, respectively (zhang et al. 2004). mechanisms of antibiotic resistance in listeria monocytogenes listeria monocytogenes becomes resistant to antimicrobial agents through acquisition of three types of movable genetic elements, namely self-transferable plasmids, mobilisable plasmids and conjugative transposons (moreno et al. 2014). efflux pumps were reported to be associated with fluoroquinolone resistance in listeria (wilson et al. 2018). however, there is an increase in reports of l. monocytogenes spontaneously acquiring resistant genes through mutations (moreno et al. 2014). mutations that occur in the promoter or operator coding regions can lead to overexpression of the endogenous genes such as those that encode for antimicrobial inactivating enzymes like the β-lactamase ampcgene (siu et al. 2003). point mutations that occur in genes encoding for antimicrobial target regions can result in a target site that is resistant to the antimicrobial activity. such a mutation was seen in the gyrase gene, whose mutation led to the expression of a fluoroquinolone-resistant gyrase enzyme (hopkins et al. 2007). antibiotic resistance mediated by conjugation conjugation is the process of transfer of genetic material, which occurs between living bacterial cells that are in direct contact (verraes et al. 2013). conjugation is the major mechanism used by l. monocytogenes strains to acquire antimicrobial resistance. enterococci and streptococci, in particular, represent a reservoir of resistance genes for l. monocytogenes. the gastrointestinal tract of humans is considered the most probable site where the acquisition by listeria spp. of conjugative plasmids and transposons from enterococcus–streptococcus takes place (wilson et al. 2018). charpentier and courvalin (1999) reported that a broad host-range of plasmid pip510 and pamß1 initially found in streptococcus agalactiae and enterococcus faecalis, respectively, encoding resistance to chloramphenicol, macrolides, lincosamides erythromycin and streptogramins can be transferred by conjugation to l. monocytogenes. the tn916, a broad host-range conjugative transposon that is primarily found in enterococcus faecalis can also be conjugated from e. faecalis to l. innocua (walsh et al. 2001). conjugative transfer of the tn916-related transposon tn1545, initially found in streptococcus pneumoniae was obtained from e. faecalis to l. monocytogenes in vitro and in vivo (mata, baquero & pe 2000). conjugative plasmids and transposons originating from enterococcus–streptococcus responsible for the emergence of resistance to tetracycline and chloramphenicol in l. monocytogenes have been reported (walsh et al. 2001). active efflux of antibiotics efflux mechanisms in l. monocytogenes was first reported in 2000 (mata et al. 2000). the sequence of mdrl (multidrug efflux transporter of listeria) protein is highly homological to the sequence of protein yfmo, a putative chromosomal multidrug efflux transporter of bacillus subtilis. an allele-substituted mutant of this gene in l. monocytogenes failed to pump out ethidium bromide and presented increased susceptibility to macrolides, cefotaxime and heavy metals. efflux pump lde (listeria drug efflux) is associated with fluoroquinolone resistance in clinical isolates of l. monocytogenes in france (verraes et al. 2013). the lde protein showed 44% homology with pmra (pneumoniae multidrug resistance) of streptococcus pneumoniae, which belongs to the major facilitator superfamily of secondary multidrug transporters. the insertional inactivation of the gene lde resulted in increased susceptibility of fluoroquinolones in l. monocytogenes (verraes et al. 2013). alternative methods to control listeria monocytogenes different alternative methods and therapies have been explored to reduce the presence of l. monocytogenes in foods, as there are few therapeutic options because of rapid development of antimicrobial resistance in this pathogen. the use of bacteriophage as biocontrol for l. monocytogenes and bacteriocins and essential oils in food, food-processing plants and humans has been reported (klumpp & loessner 2013; soni et al. 2014). the use of probiotics has also been reported to inhibit growth of l. monocytogenes as they enhanced host immunity (dhama et al. 2015). bacteriophages are viruses that can kill bacteria and were found as candidates for biocontrol of l. monocytogenes in meat and meat products during processing and packaging (strydom 2015). bacteriophages showed a high degree of specificity to lysis of l. monocytogenes strains without detrimental effects on normal microflora of the ultimate consumer and other desired bacteria in the food (dhama et al. 2015). furthermore, bacteriophages can self-perpetuate and they are stable during long cold storage (strydom 2015). this suggests bacteriophages will be active against l. monocytogenes post-processing. based on these desirable attributes, several commercial bacteriophage-based products have been developed for biocontrol of l. monocytogenes in food (leong et al. 2017), which includes phage lm-103a, phage lmp-102a, (strydom 2015), ply511 phage, listshield, listexp-100 and listextm (dhama et al. 2015). these products have been reported to function effectively against l. monocytogenes in foods and food facilities in different countries. for example, listexp-100 and listshield are used to control l. monocytogenes in the netherlands and the us, respectively (dhama et al. 2015). however, other countries such as sa still do not permit the use of bacteriophage-based products in food products and food-processing plants (strydom 2015). bacteriocins are ribosomally synthesised antimicrobial peptides that can disrupt the integrity of the target cell membrane through forming pore on the membrane. the bacteriocins such as nisin have been used in meat products to inhibit the growth of l. monocytogenes (rahimi et al. 2012). bacteriocins have the potential to inhibit a wide variety of unrelated species or only closely related species. recently nisin has been reported against l. monocytogenes and limited data have been generated to complete the understanding of the potential use for other bacteriocins for biocontrol of food-borne pathogens (leong et al. 2017). furthermore, essential oils from plant extracts have potential antimicrobial properties, which are suggested to reduce the survival of l. monocytogenes in various products. these essential oils include thyme, rosemary and oregano (hilliard et al. 2018) and those from cinnamomum cuspidatum and cinnamomum crassinervium (dhama et al. 2015). conclusion listeria monocytogenes is amongst major food-borne pathogen in the world that has commanded most research and surveillance attention from government agencies and food industry over the last few years. furthermore, methods for isolation, detection, identification and subtyping for l. monocytogenes from food products have increased rapidly with wgs as the new gold standard for typing of this pathogen. despite the extensive research and development on l. monocytogenes, outbreaks associated with this pathogen continue to be reported and are exacerbated by a high number of susceptible individuals in most countries. however, there are no data on prevalence of l. monocytogenes from most african countries that are considered to have a significant population that is immunocompromised because of hiv, tb, malaria and other infectious diseases associated with poverty. therefore, targeted surveillance programmes are necessary in those countries not only to determine prevalence but also for the development of regulations and microbiological criterion. in addition, increases in antibiotic resistance amongst l. monocytogenes strains are in line with a worldwide pattern of an increasing prevalence of antibiotic resistance amongst food-borne pathogens. alternative therapies such as bacteriophages, bacteriocins and essential oil have been explored and show promising results. acknowledgements the following organisations and individuals are acknowledged for their contributions: department of agriculture, land reform and rural development – directorate: veterinary public health for project funding and the use of data for this study. the officials from the department of agriculture, forestry, and fisheries – directorate: veterinary public health (lizzy molele, pauline modibane, maphaseka mosia, mavis phaswane and maruping ntsatsi) for the field collection of samples for this study and mphane molefe for authorising funding allocation and the approval of the study. the authors are grateful to the agricultural research council: onderstepoort veterinary research for providing all research facilities. competing interests the authors have declared that no competing interests exist. authors’ contributions i.m. conceived part of the review and wrote the manuscript. k.r.m. supervised part of the project and reviewed the article. e.m. conceived the project, obtained funding for the project, designed the study, supervised sample collection and data capture and reviewed the article. funding information the study was funded by the department of agriculture, land reform and rural development (dalrrd) under project number 21.1.1/vph-01/ovi. ethical consideration the authors confirm that ethical clearance was not required for the study. data availability statement data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.’ references al-nabulsi, 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djordjevic, v., 2019, ‘molecular characterization of listeria monocytogenes isolates from a smallscale meat processor in montenegro, 2011–2014’, journal of food microbiology 79(2019), 116–122. https://doi.org/10.1016/j.fm.2018.12.005 abstract introduction materials and methods results discussion acknowledgements references about the author(s) samuel e. mantip communicable disease research unit, polytechnic school of abomey-calavi, university of abomey-calavi, contonou, benin virology division, national veterinary research institute, vom, nigeria anthony sigismeau cirad, umr astre, montpellier, france astre, university montpellier, cirad, inra, montpellier, france maurice nanven virology division, national veterinary research institute, vom, nigeria atuman joel virology division, national veterinary research institute, vom, nigeria abayomi m. qasim virology division, national veterinary research institute, vom, nigeria sada aliyu virology division, national veterinary research institute, vom, nigeria ibrahim musa virology division, national veterinary research institute, vom, nigeria ogechukwu ezeanyika virology division, national veterinary research institute, vom, nigeria ibikunle faramade virology division, national veterinary research institute, vom, nigeria garba ahmed virology division, national veterinary research institute, vom, nigeria timothy y. woma virology division, national veterinary research institute, vom, nigeria david shamaki virology division, national veterinary research institute, vom, nigeria genevieve libeau cirad, umr astre, montpellier, france astre, university montpellier, cirad, inra, montpellier, france souaibou farougou communicable disease research unit, polytechnic school of abomey-calavi, university of abomey-calavi, contonou, benin arnaud bataille cirad, umr astre, montpellier, france astre, university montpellier, cirad, inra, montpellier, france citation mantip, s.e., sigismeau, a., nanven, m., joel, a., qasim, a.m., aliyu, s. et al., 2021 ‘wide circulation of peste des petits ruminants virus in sheep and goats across nigeria’, onderstepoort journal of veterinary research 88(1), a1899. https://doi.org/10.4102/ojvr.v88i1.1899 original research wide circulation of peste des petits ruminants virus in sheep and goats across nigeria samuel e. mantip, anthony sigismeau, maurice nanven, atuman joel, abayomi m. qasim, sada aliyu, ibrahim musa, ogechukwu ezeanyika, ibikunle faramade, garba ahmed, timothy y. woma, david shamaki, genevieve libeau, souaibou farougou, arnaud bataille received: 05 aug. 2020; accepted: 15 june 2021; published: 07 sept. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract peste des petits ruminants (ppr) is a highly contagious viral disease that mainly affects goats and sheep in asia, africa and the middle east, and threatens europe [r.e.1]. the disease is endemic on the african continent, particularly in west africa, and is a major factor driving food insecurity in low-income populations. the aim of this research study was to carry out surveillance, genetic characterisation and isolation of recently circulating ppr viruses (pprv) in sheep and goats from the six agro-ecological zones of nigeria. a total of 268 post-mortem tissue samples of lung and mesenteric ganglia were collected from clinically suspected sheep and goats in 18 different states, of which five never previously sampled. the presence of pprv was confirmed using a reverse-transcription coupled with a polymerase chain reaction (rt-pcr) assay. a total of 72 samples, 17 sheep (6%) and 55 goats (21%), were found to be ppr positive. positive samples were distributed in almost all states, except kano, where ppr was detected in previous studies. the pprv-positive samples were further confirmed by sequencing or virus isolation in areas where the infection had never previously been detected. these results confirm the active circulation of pprv across all six agro-ecological zones of nigeria, and consequently, the need for introducing strict measures for the control and prevention of the disease in the country. keywords: peste des petits ruminants; nigeria; transboundary animal disease; virus isolation; rt-pcr; morbillivirus; sequencing. introduction peste des petits ruminants (ppr) is an acute, highly contagious viral disease mainly affecting goats and sheep. the disease was first reported in 1942 by gargadennec and lalanne (1942) in sheep and goats that resembled rinderpest in the west african country of ivory coast. however, the ppr virus (pprv) was not isolated until 1967 (bourdin & laurent-vautier 1968). the pprv belongs to the family paramyxoviridae and genus morbillivirus (gibbs et al. 1979), with taxonomic name small ruminant morbillivirus (amarsingh et al. 2019). this genus currently includes seven known members: measles virus, pprv, canine distemper virus, seal distemper virus, cetacean morbillivirus, feline morbillivirus and rinderpest virus. in addition to the endemic presence of ppr in sub-saharan africa in the past decades, in recent years, field data and laboratory findings have confirmed the dramatic southward spread of ppr, affecting gabon, the democratic republic of congo, somalia, kenya and tanzania (swai et al. 2009). an outbreak of ppr infection was reported in angola for the first time in october 2012 and in zambia in july 2015 (baron et al. 2016). the risk of introduction of ppr is now high in neighbouring countries with major sheep and goat populations, such as the republic of south africa and mozambique. in addition to africa, ppr infection has been reported in many asian countries, including china. after an initial identification in tibet in 2007 (wang et al. 2009), a major ppr epizootic was reported there in 2013–2014 (bao et al. 2017). as ppr has an economic impact, it is on the list of notifiable diseases to the international office of epizootics (oie, world animal health organization, https://www.oie.int) in the event of emergence. it is now the subject of a worldwide eradication campaign by oie and fao-un (food and agriculture organization of the united nations; fao & oie 2015). pprv transmission is only horizontal, by contact, which can be direct or indirect. indirect transmission of the virus can be through ingestion of food or contaminated water or aerosolised transmission (libeau et al. 2014). goats are usually more sensitive than sheep, which have a higher recovery rate (khan et al. 2009). symptoms include fever, oculo-nasal discharge, diarrhea, dyspnoea, and effusion of the epithelium of the oral and nasal mucous membranes. nasal and ocular discharges become mucopurulent, which causes a faint odour (wohlsein & saliki 2006). the rate of pprv-related morbidity in a flock can reach 100%, and mortality is estimated at 90% (singh et al. 2009). pprv is classified into four genetically distinct lines, ‘lineage i’ to ‘lineage iv’, and all four are currently circulating in africa (mantip et al. 2016). in nigeria, sheep and goats are widespread across different ecological and climatic zones of the country. it is estimated that these animals provide more than 35% of total animal protein consumption in the country (mantip et al. 2016). the total industrial value of small ruminants in nigeria is around 40 billion naira (~€100 million (euros), shamaki 2002). the results obtained in previous studies suggest that ppr remains an endemic disease in the country, accompanied by sporadic epidemics. strains of pprv of both lineages ii and iv have been found to circulate in nigeria in 2012. however, areas where the disease was considered as being exotic in the past could be the places today of regular epidemics because of an increase in trade and commerce. the aim of this research study was to provide an update on ppr circulation and epidemiology in sheep and goats across nigeria using molecular diagnostics and virus isolation. materials and methods study area the study area included all six agro-ecological zones of nigeria: north-central, north-west, north-east, south-west, south-east and south-south. in order to obtain a representative sample of sheep and goat flocks in the country, samples were collected in three states in each agro-ecological zone. sampling was conducted between 2017 and 2018 in 18 states: bauchi, adamawa, taraba, kano, kebbi, katsina, plateau, kwara, benue, oyo, osun, ogun, abia, anambra, enugu, akwa ibom, cross river and rivers (figure 1 and table 1). figure 1: sampling locations in different states of nigeria over the period 2017–2018. table 1: list of peste des petits ruminants viruses-positive samples and partial n gene sequences obtained during this study. sampling was carried out in each of these states by moving from one market place to another, with markets having been judged to be the most important location for sampling in each state. target sampling size was 60 samples per zone and 20 locations per state. the sampling location was mapped using gps coordinates. goats and sheep suspected of being infected by ppr were identified based on clinical symptoms, which included fever over 40 °c, weight loss, ocular-nasal discharge and diarrhea. veterinarians belonging to the nigerian national veterinary services conducted the field studies in accordance with local legislation. the suspected animals were purchased from their owners, systematically euthanised, and an autopsy was performed to collect tissue samples. in addition, samples were collected from slaughterhouses located near the markets. the tissue samples collected from most animals had visible traces of diarrhea and oculo-nasal discharges on their carcasses. the tissue samples were immediately packed with ice and ice accumulators, and transported to the ppr laboratory of the national veterinary research institute, vom, plateau state, nigeria, and stored in a freezer at –70 °c before the sampling team moved on to another state. the samples were subsequently shipped to cirad, montpellier, france for further laboratory analyses. molecular detection of the virus nucleic acid tissue samples were cut into pieces and ground in 3 ml of minimum essential medium (mem) by vortexing with 0.2 µm glass beads. the swabs were placed in 1 ml mem and vortexed. in all cases, the sample suspensions were centrifuged for 3 min at 1000 g and the supernatant was collected. total ribonucleic acid (rna) was extracted with an extraction robot kingfishertm and id kit genetm mag universal extraction (idvet, france), as described by the manufacturer. the molecular diagnosis of ppr for each sample was performed by reverse-transcription coupled with a polymerisation chain reaction (rt-pcr) that uses the pair of primers np3 (5’-gtc-tcg-gaa-atc-gcc-tca-cag-act-3’) and np4 (5’-cct-cct-cct-ggt-cct-cca-gaa-tct-3’) that specifically target the partial n gene (couacy-hymann et al. 2002). the rt-pcr was performed using quantabio qtxt xlt one-step rt-pcr kit, following manufacturer’s instruction. the rt-pcr mixture (25 µl) was composed of 5 µl rna, 12.5 µl of one-step tough-mix (2x), 1.5 µl of np3 10 µm, 1.5 µl of np4 10 µm, 1 µl of qscript xlt 1-step rt 25x, 3.5 µl of h2o and 5 µl of nucleic acids from each sample. the rt-pcr cycle programme consisted of rt for 20 min at 48 °c, denaturation for 3 min at 94 °c; 40 cycles of 15 s at 94 °c, 30 s at 60 °c and final extension for 1 min at 72 °c. the presence of pcr product of the expected size (355 bp) was controlled by 1.5% agarose gel migration. the fragment size of our amplicon was monitored using a 100-bp molecular weight marker after illumination under uv radiation or blue light. phylogenetic analysis when positive pcr products were obtained from areas where ppr had never previously been detected (kebbi, katsina, rivers, enugu and abia states; see the results section), one positive sample per state was selected based on the brightness of the amplicon on the gel, and sent to genewiz (united kingdom) for purification and sequencing in both forward and reverse directions. the sequences were submitted to the ncbi genbank database (table 1). forward and reverse deoxyribonucleic acid (dna) sequences were assembled using bioedit and trimmed to remove poor-quality portions of the sequences (final size = 255 base pairs [bp]). corrected sequences were aligned with a dataset of pprv n gene sequences publicly available in genbank using mega 6 representatives of the four genetic lineages. the phylogenetic tree was constructed using the maximum likelihood method implemented in mega 6, with node supports evaluated by bootstrap analyses with 1000 replicates. cell culture and virus isolation virus was isolated from field samples on canine histiocytic sarcoma (chs) cells using the same five pcr positive samples sent for sequencing. chs cells are cv1 cells (monkey cell line) modified to express slam receptors (‘signaling lymphocytic activation molecule’), or cd150 of goats, which are highly sensitive to pprv growth (adombi et al. 2011). these cells, which are very sensitive to pprv infection, were used to isolate the virus from field samples. five samples from different states that showed strong pcr amplification results were selected for the isolation attempt. aliquots (150 ml) of viral solution contained in the homogenate samples were inoculated into the chs cell mat seeded at a minimum confluence of 70% in 25 cm2 flasks. the flask was gently shaken at 15-min intervals for 1 h. culture medium was composed of mem, foetal calf serum (fcs), an antibiotic and antimycotic was added. antibiotics and antimycotics help to protect cells from potential contamination by pathogens (bacteria, fungi, etc.) that may be present in the sample. the infected flasks were incubated with 5% co2 at 37 °c. the cytopathogenic effect (cpe) occurs after varying intervals because of the different viral loads in the samples. incubation was stopped when the cpe reached 80% (figure 3), and the flasks were subjected to cycles of freezing (–80 °c) and thawing (at room temperature or ambient temperature). these cycles cause the cells to burst and release viral particles. culture medium that contained dead cells and viral particles was collected and centrifuged. the supernatant of the culture medium was used to extract the total nucleic acids as explained above. some samples showed bacterial or fungal contamination during the first isolation attempt. a second attempt, including an additional filtration step and the addition of a second antibiotic, was, therefore, necessary. the size of the filter pores used was 0.45 mm. the bacteria are all retained by pores of this size. the second antibiotic used was gentamycin. subsequently, the presence of the virus in the medium was confirmed by collecting and testing the cell culture supernatant by rt-pcr. ethical considerations approval to conduct the study was received from the ethics committee: national veterinary research institute, animal use and care committee (aucc, nvri, vom; reference no. aec/02/87/20). approval was also received from the university of abomey-calavi, benin republic. results total nucleic acids were extracted from a total of 268 samples taken from 18 different states. amplification of the target gene by rt-pcr using the pair of pprv primers was successful for 73 samples (27%; table 1). at least one sample was positive for pprv of samples from 17 states: plateau, benue, kwara, bauchi, adamawa, taraba, katsina, kebbi, enugu, abia, anambra, oyo, ondo, osun, rivers, cross river and akwa-ibom. none of the samples were found to be positive amongst the 20 samples from kano state (table 1). amongst the positive samples, 17 came from sheep (6%) and 55 from goats (21%). the sequence of the partial n gene was successfully obtained from one sample each collected from the states of kebbi, katsina, river, enugu and abia. phylogenetic analysis showed that all five samples sequenced in this study belonged to lineage iv and clustered with a strain collected in nigeria in 2013 (figure 2; woma et al. 2016) (r.e.4). the cpe was observed in only one out of the five samples used in the isolation attempt, which included the sample from the state of kastina. the presence of pprv in the cell culture was confirmed by rt-pcr (figure 3). figure 2: phylogenetic tree of the partial n gene of peste des petits ruminants viruses strains derived from samples in nigeria and publicly available sequences. the sequences obtained in this study are indicated with a black dot. the numbers at the nodes are bootstrap values obtained from 1000 replicates are shown if > 50%. figure 3: canine histiocytic sarcoma cells at 100% confluence observed under a light microscope, ×100 magnification, when (a) non-infected or (b) with cytopathic effect (cpe) because of peste des petits ruminants viruses infection. discussion since they found that ppr as a disease in sheep and goats for the first time in nigeria in 1973, and the eventual successful isolation of the virus in 1975 (taylor & abegunde 1979), the disease has remained a major threat for small ruminant production and has had negative impacts on food security, with ppr being the most economically important livestock disease in nigeria. even though ppr has been of concern in the nigerian livestock sector for a long time now, surprisingly few studies have been carried out to understand the molecular epidemiology of the disease in nigeria. additionally, the few studies that were conducted did not get as far as isolating and sequencing the isolates. exceptions concern the virus detected and isolated in 1975 (taylor & abegunde 1979) and the molecular virus detection in 2002 (shamaki 2002) that were found to cluster within lineage ii. the results of this study provide evidence for the continued circulation of ppr across all six agro-ecological zones of nigeria. the presence of pprv was detected by n-gene-based rt-pcr in 27% (72/268) of random geo-spatially suspected samples, proving the circulation of pprv in the study areas. notably, we provide the first molecular confirmation of the presence of pprv in the south-east agro-ecological zone. in a similar previous study, a total of 35 samples out of 360 (9.7%) tested positive using rt-pcr, of which 25 were from oculo-nasal swabs and 10 were from tissue samples (mantip et al. 2016). similar previous studies using clinical cases in ethiopia reported a positivity rate of 46.4% using rt-pcr (alemu et al. 2019). in morocco, a similar study reported a ppr positivity rate of 44.4% (16/36) using rt-pcr and a positivity rate of 80% in sudan (kwiatek et al. 2011). the presence of pprv was also confirmed in 33.3% (7/21) and 51.2% (17/33) of clinical samples tested in algeria and in north-central state in nigeria, respectively, using a set of primers specific to the f gene of the pprv (de nardi et al. 2012). in northern and eastern tanzania, the pprv genome was also detected in 29.6% and 31.1% of the goats tested (kgotlele et al. 2014). however, it has been shown that the level of positivity can be influenced by the type of sample used for diagnosis, the stage of infection and the type of gene targeted for rt-pcr (luka et al. 2012). such comparisons consequently only provide limited information to assess the rt-pcr technique or to provide details on the prevalence of the disease. furthermore, as the data are provided by studies conducted in difficult field environments, 100% success is not feasible for the following reasons: (1) samples are not collected at the right time, as the locations of the outbreak are usually reached too late for antigen–gene detection, that is, the window for pcr diagnosis is too narrow; (2) according to these publications, the samples are stored differently: the shorter the harvest campaign, the faster the samples will be kept at the right temperature; (3) viral loads fluctuate depending on the host species and breed, as well as on the virulence of the pprv strain, as demonstrated in experimental (couacy-hymann et al. 2007; couacy-hymann et al. 2009) and natural infections. this study revealed a significantly higher rate of pprv infection in goats than in sheep samples with rt-pcr, with 17 positive samples obtained from sheep (6%) compared with 55 (21%) from goats. the pprv exhibits more or less similar levels of virulence in sheep and goats; however, goats are more severely affected, whilst sheep generally suffer from a milder form (lefevre & diallo 1990) or rarely suffer from a clinical disease (fakri et al. 2017; el hag ali & taylor 1984; roeder et al. 1994). nevertheless, high mortality rates were reported in sheep during an outbreak by taylor (1984) who hypothesised that sheep possess innate resistance to the clinical effects of the disease; however, that occasional field strains can overcome this resistance resulting in high mortality rates (el hag ali & taylor 1984). breed may also affect the outcome of pprv infection and its epidemiology; guinean breeds (west african dwarf, iogoon, kindi and djallonke) are known to be highly susceptible (lefevre & diallo 1990). british breeds exhibited severe clinical reactions when experimentally infected, whereas sudanese breeds failed to develop a characteristic clinical response (el hag ali & taylor 1984). it has been suggested that pprv be isolated from field samples in cell culture for further identification (fao & oie 2015; lefevre et al. 1990). this study showed that the inoculation, isolation and propagation of pprv in chs-20 cells succeeded from the first passage of one of the selected positive samples, with the cpe characteristic in agreement with that described by the world organisation for animal health (oie 2019; adombi et al. 2011). the presence of the virus in the chs-20 medium was confirmed by collecting and testing the cell culture supernatant by rt-pcr. molecular characterisation of the circulating strains by phylogenetic analysis using the n gene is the most accurate way of identifying the lineage genetics belonging to new strains. this tool is important in understanding the epidemiology of pprv and in tracking outbreaks in ppr-prone and endemic countries. such information helps to establish the diversity and circulation of strains in the field, thereby tracing the space–time origin of pprv and making it possible to estimate the risk of its introduction in the herd (libeau et al. 2014). this study presents the first molecular confirmation of ppr infection in the states of kebbi and kastina (north-west), abia and enugu (south-east), and rivers (south-south) in nigeria. the sequences obtained showed that the samples tested belong to lineage iv. previous studies showed that both lineages ii and iv were circulating in neighbouring states in 2010-2012 (mantip et al. 2016; woma et al. 2016). further sequencing of ppr-positive samples is needed to assess whether lineage ii is still circulating in any of these five states. the successful molecular findings and virus isolation in this study confirm the active presence of pprv infections amongst populations of sheep and goats in all six agro-ecological zones, suggesting that ppr is currently endemic virtually everywhere in nigeria. we, therefore, recommend systematic vaccination with thorough investigation of outbreaks and surveillance to contain outbreaks within the affected locations, states and regions of the country. we also recommend strengthening of the surveillance system, with emphasis on early detection, in epidemiologically closely linked administrative units (local government areas) to which the disease could potentially spread. such interventions should be in line with broader regional and national control programmes for ppr in africa. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.e.m., a.b., g.l., d.s. and s.f. conceived and planned the experiments. s.e.m., m.n., a.j., a.m.q., s.a., i.m., o.e., i.f. and g.a. collected the samples. s.e.m. and a.s. performed laboratory analyses. s.e.m., a.s. and a.b. analysed the results. s.e.m., a.s., a.b., d.s., t.y.w., g.l. and s.f. interpreted the results. s.e.m. and a.b. wrote the first draft of the manuscript. all co-authors critically revised and approved the final version of the manuscript. funding information this research article was funded by the european commission directorate general for health and food safety (grant no. si2.756606) awarded to the european union reference laboratory for peste des petits ruminants (eurl-ppr) and by a grant from european commission (development cooperation instruments) awarded to the project ‘eu support to livestock disease surveillance knowledge integration – lidiski’ (food/2019/410-957) in the framework of the development of smart innovation through research in the agriculture (desira) programme. data availability the data supporting the findings of this study are freely available in genbank at https://www.ncbi.nlm.nih.gov/ (accession numbers: mt193235, mt193237, mt193247, mt193249 and mt193250). disclaimer the views expressed in the article are those of the authors and not an official position of the institution or funder. references adombi, c.m., lelenta, m., lamien, c.e., shamaki, d., koffi, y.m., traore, a. et al., 2011, ‘monkey cv1 cell line expressing the sheep-goat slam protein: a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens’, journal of virological methods 173(2), 306–313. https://doi.org/10.1016/j.jviromet.2011.02.024 alemu, b., gari, g., libeau, g., kwiatek, o., kidane, m., belayneh, r. et al., 2019, ‘molecular 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and goats’, research in veterinary sciences 26(1), 94–96. https://doi.org/10.1016/s0034-5288(20)30949-8 wohlsein, p. & saliki, j., 2006, ‘rinderpest and plague of small ruminants – the diseases’, in t. barret, p-p. pastoret & w.p. taylor (eds), rinderpest and peste of small ruminants,academic press, pp. 68–v. https://doi.org/10.1016/b978-012088385-1/50034-4 woma, t.y., adombi, c.m., yu, d., qasim, a.m.m., sabi, a.a., maurice, n.a. et al., 2016, ‘co-circulation of peste-des-petits-ruminants virus asian lineage iv with lineage ii in nigeria’, transboundary and emerging diseases 63(3), 235–242. https://doi.org/10.1111/tbed.12387 abstract introduction research methods and design results discussion conclusion acknowledgements references about the author(s) ghada a. ibrahim bacteriology department, agriculture research center (arc), animal health research institute, ismailia, egypt ahmed m. salah-eldein wildlife and zoo department, faculty of veterinary medicine, suez canal university, ismailia, egypt mayasar i. al-zaban department of biology, college of science, princess nourah bint abdulrahman university, riyadh, saudi arabia amal s.a. el-oksh biotechnology department, reference lab of quality control of poultry production (rlqp), agriculture research center (arc), animal health research institute, sharkia, egypt elsayyad m. ahmed department of virology, animal health, research institute (ahri), agricultural research center (arc), giza, egypt doaa s. farid department of environmental protection, faculty of environmental agricultural sciences, arish university, el-arish, egypt enas m. saad wildlife and zoo department, faculty of veterinary medicine, suez canal university, ismailia, egypt citation ibrahim, g.a., salah-eldein, a.m., al-zaban, m.i., el-oksh, a.s., ahmed, e.m., farid, d.s. et al., 2023, ‘monitoring the genetic variation of some escherichia coli strains in wild birds and cattle’, onderstepoort journal of veterinary research 90(1), a2085. https://doi.org/10.4102/ojvr.v90i1.2085 original research monitoring the genetic variation of some escherichia coli strains in wild birds and cattle ghada a. ibrahim, ahmed m. salah-eldein, mayasar i. al-zaban, amal s.a. el-oksh, elsayyad m. ahmed, doaa s. farid, enas m. saad received: 13 oct. 2022; accepted: 01 june 2023; published: 26 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract to date, there is limited data about the genetic relationship of escherichia coli between wild birds and cattle because these birds act as silent vectors for many zoonotic bacteria. this study aimed to elucidate the role of rooming wild birds in the vicinity of cattle farm in transmission of the same pathogenic e. coli variants, identifying their virulence, resistance traits and genetic similarities of fimh virulence gene. about 240 faecal/cloacal swabs were collected from both species and examined bacteriologically. escherichia coli was yielded in 45.8% and 32.5%, respectively, of examined cattle and wild birds. the most prevalent detected e. coli serovar was o26. high tetracycline and chloramphenicol resistance were recorded; however, gentamycin and ciprofloxacin exhibited the highest sensitivity rates. polymerase chain reaction (pcr) conserved genotypic resistance (teta and blactx-m) and virulence attributes (fimh, stx1, eaea and ompa) of e. coli isolates were discussed in detail. the fimh gene revealed 100% sequence similarity when comparing with different e. coli isolates globally and locally. finally, a close genetic association of e. coli with both wild birds and cattle was detected, thus strengthening its role in the dissemination of the infection via environment. prevention and conservative policy should be carried as e. coli constitute enormous significant zoonotic risks to livestock and animal workers. also, further studies to the whole genome sequencing of fimh, other virulence and resistance genes of e. coli are recommended trying to limit the possibilities of co-infection and transfer among different species. contribution: the current study recorded updated data about the critical infectious role of wild birds to livestock, including cattle farms in egypt. it also delivered some recommendations for good hygienic practices in cattle farms which must be implemented for handling animal manure. keywords: e. coli; wild birds; cattle; virulence genes; resistant genes; pcr; antibiotics; sequencing. introduction wildlife constitutes a magnificent network that might spread a number of potentially significant bacterial diseases with their antimicrobial-resistance traits among livestock animals and infrastructure because of high bird travel and mobilisation in multiple countries during different seasons (arnold et al. 2016). migratory and non-migratory wild birds could serve as the main transmitter for the infection with the family enterobacteriaceae specifically escherichia coli spp. (shobrak & abo-amer 2014). escherichia coli is a gram-negative environmental bacterium that commensally inhabits the intestinal tract of warm-blooded animals and birds. however, these bacteria could cohabit with other bacteria forming the commensal gut microbiota; some e. coli variants might develop virulent characteristics producing the disease (umpiérrez et al. 2021). hence, the antimicrobial resistance problems could extremely extend to transfer the antibiotic-resistant genes among different species via plasmid or transposon (borges et al. 2017). for antimicrobial resistance in the veterinary field, e. coli resistance was consistently the highest (fahim et al. 2019). furthermore, e. coli had also been regarded as a reliable indicator bacteria for tracking antibiotic resistance in domestic and wild animals. however, enteropathogenic (epec) and shiga toxigenic e. coli (stec) are categorised under diarrheagenic e. coli, and the intestinal microbiota of wild birds could include both serovars with their multi-drug resistance attributes (borges et al. 2017). moreover, the precise genetic bacterial profile enables more knowledge for the most disseminating genotypes to distinguish its potential links to virulence factors that could infect many animals and free-living birds (skarżyńska et al. 2021). as wildlife could play the main role in the microbial spreading in cattle farms, the mechanisms of this bacterial spreading among species in the farm environment are poorly understood (tormoehlen et al. 2019). the current investigation was conducted to estimate the likelihood of e. coli infections transfer by wild birds that were frequently in contact with cattle and identifying the interrelationship between both species in farms in egypt. also, to study the most virulence and antibiotic resistance traits of the e. coli spp. isolates with a special regard to partial-genome resistance of fimh gene. research methods and design sampling a total of 240 rectal and cloacal swab samples were collected from cattle species (n = 60 apparently healthy, n = 60 diarrhoeic cattle) and 120 from different resident wild birds from ismailia governorate, egypt. cows and buffaloes were included in this study of native breeds. resident free-living wild birds (hooded crow [n = 21], cattle egret [n = 18], spur-winged plover [n = 24], pied kingfisher [n = 21], green bee-eater [n = 24] and stone curlew [n = 12]) were captured with nets nearby these cattle farms and released after swabbing. animals were housed in open yards surrounded by a fence, partially covered with sheds and muddy floors. all cattle rectal and bird cloacal swabs were collected aseptically and transferred immediately under hygienic measures to the laboratories of the animal health research institute (ahri) ismailia branch in ice thermal boxes for bacteriological examination, serological and polymerase chain reaction (pcr) identification. bacterial cultivation and identification all cattle rectal and bird cloacal swabs were enriched overnight onto buffered peptone broth and then incubated at 37 °c under aerobic conditions. subsequently, the cultures were isolated (on macconkey, emb and sheep blood agar plates) and identified using conventional techniques (quinn et al. 2002). furthermore, the pathogenicity of each one of e. coli recovered isolates was evaluated separately onto congo red agar plate medium then they were kept overnight at 37 °c. the cultures were kept at room temperature for 48 h (singh et al. 2014). after 48 h at room temperature, congo red-positive pathogenic e. coli isolates appeared with a brick red colour while non-pathogenic ones were colourless (saha et al. 2020). serological identification the recovered e. coli isolates were serotyped based on their ‘o’ antigen according to the manual of the reference lab for veterinary quality control on poultry production, animal health research institute, dokki, egypt. the identified isolates were preserved in tryptone broth 1% with adding glycerol to a final concentration of 15%. the tubes were kept at −20 °c for further analysis. antimicrobial susceptibility testing the purified e. coli isolates were characterised for antibiotic susceptibility on mueller–hinton agar plates by the disc diffusion method using 11 different antibiotic discs (tetracycline, chloramphenicol, amoxiclavulinic acid, penicillin, piperacillin, streptomycin, fosfomycin, gentamycin, danofloxacin ciprofloxacin and cefepime), which were selected according to the panel of antibiotics of interest to the dairy industry and public health in our country. the results were interpreted using the standard guidelines of the clinical and laboratory standards institute (clsi 2020). polymerase chain reaction screening of virulence and resistant traits of escherichia coli spp. following the manufacturer’s instructions of qiaamp dna mini kit (qiagen, germany), the genomic deoxyribonucleic acid (dna) of e. coli bacterial isolates was extracted. the pcr was performed using thermal profiles and oligonucleotides primers (table 1) and a 25 μl reaction volume containing; 12.5 μl master mix (emeraldamp max pcr, takara, japan), 1 μl (20 pmol) from each forward and reverse primer (metabion, germany), 5.5 μl dnase free water and 5 μl dna template. the amplified genes fragments were visualised in ethidium bromide-stained agarose gel 1.5% against 100 base pair (bp) dna ladder (genedirex, taiwan) in 1× tris-borate edta (tbe) buffer at 100 voltage (v) per 30 min and then recorded using the syngene gel documentation system. all reactions included a negative (non-template) and positive control of reference e. coli strains supplied by ahri, dokki, giza, egypt. table 1: primer sequences and amplicon sizes of target genes. gene sequencing of fimh gene two retrieved e. coli isolates one from diarrhoeic cattle and the other from hooded crow were selected for genotyping of the fimh gene. qiaquick® gel extraction kit (qiagen, germany) was used to purify the pcr products. the sequence reactions were performed using the bigdyetm terminator version 3.1 cycle sequencing kit (thermo fisher scientific, united states [us]) and then were analysed using the 3130 genetic analyzer (applied biosystems™, us). the obtained sequences were trimmed, consensus generated and analysed using the uniport ugene software version 43.0 (okonechnikov et al. 2012). sequences were investigated using the national center for biotechnology information (ncbi) online basic local alignment tool (blast at https://blast.ncbi.nlm.nih.gov/blast.cgi). for phylogenetic analysis, fimh gene sequences from different sources and origins including egypt were selected and retrieved from the genbank in fatsa format. sequence alignments (msa) were performed using muscle algorism with the same software multiple (okonechnikov et al. 2012). iq-tree was used to construct the phylogenetic tree (nguyen et al. 2014) using the maximum likelihood method, best model finder and 1000 bootstrap replicates for both nucleotide and protein sequences. the constructed trees were annotated using the interactive tree of life (itol) (letunic & bork 2021). results phenotypic characterisation of the recovered escherichia coli isolates in cattle and wild birds the suspected e. coli colonies revealed a pink colouration on macconkey agar medium, metallic green sheen on emb and some (enterohaemorrhagic) strains gave haemolysis on blood agar. microscopically, the isolates appear as gram-negative rod-shaped motile bacilli. also, their pathogenicity was confirmed on congo red medium (gave positive orange or bright red colonies). biochemically, they all were identified and confirmed as e. coli spp. the isolation rate of escherichia coli in cattle and wild birds bacterial examination of 120 rectal swabs of both apparently healthy and diarrhoeic cattle revealed that e. coli was recovered from 55 of 120 (45.8%) animals. it was detected in 17 of 60 (28.3%) apparently healthy animals; however, it was isolated from 38 of 60 (63.3%) diarrhoeic animals. moreover, in wild birds, 39 (32.5%) were positive for e. coli, of which 18 of 21 (85.7%) were isolated from the hooded crow, 10 of 18 (55.6%) from cattle egret, pied kingfisher 3 of 24 (12.5%), spur-winged plover 3 of 21 (14.3%) and 5 of 12 (41.7%) from stone curlew (table 2). table 2: the prevalence of different escherichia coli serotypes in cattle and wild birds. serological identification of recovered isolates serological identification revealed different serotypes, according to o-antigen in which the most predominant serotype was o26 (n = 21/94, 22.4%), followed by o114 (n = 20/94, 21.3%), o128 (n = 9/94, 9.6%), o125 (n = 17/94, 18.1%), o111 (n = 11/94, 11.7%), o78 (n = 7/94, 7.4%), o55 (n = 7/94, 7.4%) and o44 (n = 2/94, 2.1%). enterohaemorrhagic (ehec) e. coli serovars were screened in this study in 38 of 94 (40.4%), 16 of 94 (17%) were enterotoxigenic (etec), 29 of 94 (30.9%) were epec and 11 of 94 (11.7%) were enteroaggregative (eaec) as shown in table 2. antibiotic resistance pattern most examined e. coli isolates demonstrated high multidrug resistance levels against tetracycline and chloramphenicol (95.7% and 93.6%), respectively, followed by piperacillin, penicillin and streptomycin (90.4%, 88.3% and 88%), respectively. a moderate resistance level was recorded against both fosfomycin (54.3%) and amoxiclav (47.9%). meanwhile, gentamycin, cefepime, ciprofloxacin and danofloxacin recorded the lowest resistance rates (9.6% and 10.6%, 12.8% and 26.6%), respectively, as shown in table 3. table 3: phenotypic characterisation of antibiotic resistance profile of escherichia coli isolates. polymerase chain reaction investigations of virulence and resistant attributes of the recovered isolates polymerase chain reaction screening of the virulence genes of most 10 multi-drug resistant (mdr) e. coli isolates (figure 1a–f) revealed that fimh and ompa genes were detected in all isolates 10 of 10 (100%) (figure 1a and b), while the eaea gene was detected in 1 of 10 (10%) only of total isolates (figure 1c), stx1 gene was demonstrated in 6 of 10 (60%) of mdr isolates (figure 1d), pcr failed to detect hly and stx2 genes in the examined e. coli isolates. the presence of teta and blactx-m genes of tetracycline and penicillin β-lactamase inhibitors resistance was confirmed in all 10 of 10 (100%) of mdr tested e. coli isolates (figure 1e and f) as data shown in table 4. figure 1: the electrophoretic gel pattern of mdr genes from e. coli isolates: (a): lane 1–10 positive for fimh at 508 bp, (b): lane 1–10 positive for ompa at 919 bp, (c): lane 1 positive for eaea at 248 bp, (d): lane 1, 2, 3, 4, 5, 6, 7 positive for stx1 at 614 bp, (e): lane 1–10 positive for teta at 576 bp, (f): lane 1–10 positive for blactx-m at 593 bp. table 4: polymerase chain reaction amplification results of different virulence and resistant genes of isolates. phylogenetic analysis of fimh virulence gene the fimh was detected in all examined isolates with the conventional pcr technique. fimh gene sequences from two selected wild birds (hooded crow) and cattle isolates were examined and then documented in the gene bank database and assigned the accession numbers (on239271 and on239272), respectively. the phylogenetic analysis of the fimh gene selected from two isolates revealed 99% nucleotides identity between their sequences (cattle and wild bird isolates, figure 2). only three single nucleotides polymorphism (snp) in cattle isolates were shown at positions 192.291,348, but these snps did not show any effect at amino acid (a.a) level because they were identical (100% identity, figure 3). both cattle and wild bird isolates showed 100% homology with most sequences retrieved from the genbank from egypt and geographical locations also from different isolation sources including different animal species, food and environmental source (table 5). these translated a.a phylogeny results confirmed the high conservation level of the sequenced fimh gene for both retrieved egyptian e. coli isolates and globally (figure 4). figure 2: the maximum likelihood (ml) phylogenetic tree for partially nucleotide sequenced fimh gene of e. coli isolated from wild bird and cattle. the tree was constructed in iq-tree with model finders. the accession number and a brief genbank id are assigned to each retrieved sequence from different l isolation sources and origins. the two isolates are highlighted in red with a green background, various clades are designated with different colours, and the numbers above the branches are the branch length. the bootstrap values were computed from 1000 bootstrap repeats and branch length as visualised by itol. the tree’s roots are in the middle. figure 3: the msa for partial nucleotide fimh gene fragment of the identified escherichia coli isolates compared with other isolates and strains retrieved from the gene bank visualised by ugen programme. the dot (.) represents identity, while a single alphabet highlights the differences among aligned sequences. figure 4: rooted maximum likelihood phylogenetic tree for a translated protein of fimh gene escherichia coli sequences obtained from wild bird and cattle isolates. the branch colour represents the homology between protein sequences. rings from inner to outer: the first ring (origin) represents the geographical location of selected isolates, the second ring (source) is for the sample source of isolates, the branch length, and bootstrap values computed from 1000 bootstrap repeats are also visualised. table 5: source modifier tabulates for fimh gene isolates and strains sequences retrieved from genbank for alignment, phylogenetic analysis, and tree construction for isolates from wilds birds and cattle in egypt. discussion many studies implicated the crucial role of wild birds in the pathogenesis of e. coli spp. in livestock animals (fadel, afifi & al-qabili 2017; fahim et al. 2019). however, its pathogenesis in these birds was still unclear. diarrhoeagenic strains of e. coli were isolated from different wild bird species including migratory and non-migratory (ahmed et al. 2019), passeriformes, columbiformes and pelecaniformes (sanches et al. 2017). escherichia coli also was isolated from birds of prey, waterfowls and passerines. farm animals could infect wild birds or vice versa (fadel et al. 2017). they infiltrated animal enclosures in search of water and food hence infecting them with different pathogens or even acquiring the infection from these animals. moreover, the feeding practice of cattle in open yards could result in the accumulation of their manure and so the attraction of these wild birds to those farms (medhanie et al. 2015). the present article stated that e. coli was detected higher (45.8%) from all examined apparently healthy and diarrhoeic cattle than from different wild birds (32.5%). for apparently healthy cattle, it was detected in 17 of 60 (28.3%); however, it was isolated from 38 of 60 (63.3%) of diarrhoeic animals. however, from different wild bird species, 18 of 21 isolates were obtained from the hooded crow (85.7%), 9 of 18 (50%) cattle egret, pied kingfisher (n = 3/24, 12.5%), spur-winged plover (n = 3/21, 14.3%), 6 of 12 (50%) from stone curlew. in the same way, recent studies discussed the propagation rate of e. coli in wild birds, cattle and their environment in which it was found in a range of 17% – 47% in the faeces samples of wild birds (house sparrows, red-winged blackbirds, european starling and brown-headed cowbirds) despite its percentage was recorded higher in cattle farms (tormoehlen et al. 2019). also, 478 positive e. coli samples of migratory birds were reported in china from a total of 1387 (34.7%) faecal, cloacal and throat samples (yuan et al. 2021). ahmed et al. (2019) isolated e. coli from 60% and 45% of examined hooded crows and cattle egrets in egypt, respectively. however, a higher rate of e. coli was recovered from the faeces of wild birds (70%) than migratory waterfowls (33.3%) (fahim et al. 2019). from a different point of view, a large number of e. coli were isolated from egret wild birds than from cattle in the same study (fashae et al. 2021) this variation in the isolation rate in different studies might be because of either insensitivity testing method or other anonymous agents (ballem et al. 2021). moreover, e. coli isolates were recorded also in the united states (21% of beef cattle and 13% of dairy cattle) (venegas-vargas et al. 2016). also, 112 of 409 positive e. coli isolates were retrieved from cattle in portugal with a prevalence of 27.4%, and 133 stec isolates were identified (ballem et al. 2021). in addition, 106 and 29 e. coli isolates were yielded from 77 diarrheic and in-contact calves (awad et al. 2020). from a serological view, the most predominant type of e. coli in this study was o26 (n = 26/94), followed by o114, o128, o125, o111, o78, o55 and o44 (table 2). corresponding results were reported by (mahmoud et al. 2015; navarro-gonzalez et al. 2020) in which different subtypes of e. coli o26, o55, o111, o124, o119, o114, o26, o44 and o163 were recorded. antibiotic-resistant bacteria could pose a rising hazard to global public health and accompanied environmental contamination problems (who 2017). the regulation (ec) no. 1831/2003 of the european parliament and of the council of 22 september 2003 on additives for use in animal nutrition banned the use of growth-promoting antimicrobials in animal production. as a result of the diversity in ecological niches, the migratory birds act as reservoirs and transporters of antibiotic-resistant bacteria and consequently play a significant epidemiological role in the dissemination of antibiotic-resistant genes (args) (cao et al. 2020). these birds could carry args during migration leading to the dissemination of mdr bacteria and args through the environment (yuan et al. 2021). the presented information in this study displayed mdr phenomena of the yielded isolates because they showed high antimicrobial resistances against tetracycline and chloramphenicol with prevalence rates of 95.7% and 93.6%, respectively, followed by piperacillin, penicillin and streptomycin (90.4%, 88.3% and 88%), respectively. meanwhile, gentamycin, cefepime, ciprofloxacin and danofloxacin were highly sensitive where the lowest resistance was recorded (9.6% and 10.6%, 12.8% and 26.6%), respectively, as shown in table 3. these results might be of good importance in management routines for cattle farms to control the spread of antimicrobial resistance. similar to this, the wild bird e. coli isolates exhibited bacterial resistance in many studies in the last years. escherichia coli isolates showed great resistance to penicillin g, piperacillin, tetracycline, cotrimoxazole, ampicillin and nitrofurantoin (shinde et al. 2020). also, 376 recovered e. coli isolates from hooded and white-naped cranes in japan were found resistant to oxytetracycline, ampicillin and nalidixic acid antibiotics. a high resistance level was also recorded against tetracycline followed by sulfamethoxazole, ampicillin, trimethoprim and ciprofloxacin in most e. coli isolates (suenaga et al. 2019). furthermore, 87 of 88 egret’s and 53 of 55 cattle e. coli isolates were found to have mdr against more than one antimicrobial. tetracycline resistance was highest in e. coli isolates from egret birds (n = 85/87), further followed by streptomycin (n = 69/87) and ciprofloxacin resistance (n = 38/87). for cattle e. coli isolates multiple authors reported mdr patterns of e. coli isolates (iweriebor et al. 2015, mahmoud et al. 2020). the mdr phenomena are of great concern because the resistant strain could be transmitted to humans by consumption of either milk or food carrying antibiotic-resistant bacteria, which could lead to the acquisition of antibiotic-resistant infections (geletu, usmael & ibrahim 2022), and also could be transmitted to accompanying animals and their offspring (roca-saavedra et al. 2018). it was previously documented that resistant strains selected during an antimicrobial treatment last for a long time in the intestinal tract when this treatment ceases. in addition, these resistant strains could modify animal health. the results in this study were consistent with geletu et al. (2022) who revealed that tetracycline (80%) was the drug that most e. coli isolates from dairy cattle were extremely resistant to, followed by ceftriaxone and vancomycin (83%). however, gentamycin (90%) and nitrofurantoin (70%) were the most sensitive drugs, respectively. additionally, tetracycline resistance (n = 47/53) was the most often seen phenotype in cow cefotaxime-resistant e. coli, followed by streptomycin (n = 46/53) and ciprofloxacin (n = 17/53) resistance (fashae et al. 2021). moreover, mahmoud et al. (2015) recorded the e. coli. resistance against oxytetracycline and ampicillin in cattle samples. moreover, the most responsive medications, nevertheless, were ceftiofur (40%) and lincospectine (56.6%), followed by danofloxacin (56.6%), enrofloxacin (40%) and danofloxacine (56.6%). furthermore, sulfamethoxazole, ampicillin, trimethoprim and ciprofloxacin were the antibiotics with the highest rates of resistance in e. coli isolates, followed by tetracycline (hang et al. 2019). in the present study, the highest resistance levels of e. coli isolates might be because of the non-judicious use of antibiotics on a cattle farm. also, this high antimicrobial resistance of e. coli isolates in cattle might confer a selective advantage towards intestinal colonisation, which might itself increase the faecal shedding of antimicrobial-resistant e. coli (harkins, mcallister & reynolds 2020). studying the genotypic virulence attributes of the isolated bacterial species was applied by conventional pcr technique. our results indicated that the fimh gene, one of the virulence genes involved in bacterial adhesion, was found to be present in 10 of 10 (100%) of the tested e. coli. also, identical findings were recorded by nüesch-inderbinen (et al. 2018) who discovered that the fimh gene was present in all (100%) of their isolates. in the present study, eaea (attaching and effacing virulence factor) was detected in 1 of 10 (10%) of tested e. coli isolates, despite this finding complied with a study by sanches et al. (2017) in which eaea gene was found in a rate of 5.74%. moreover, mohamed and sayed (2017) implied that eaea gene was exhibited in 43.75% of yielded e. coli isolates. these results disagreed with the finding by nüesch-inderbinen et al. (2018) who recorded that eaea gene was not present in any of the studied e. coli isolates. depending on the retrieved data in this study, pcr confirmed the positivity of the ompa gene (outer membrane protein a) virulence gene in 10 of 10 (100%) of e. coli isolates. the frequency of detected ompa gene in our study was much higher than another study in which this gene was detected in (82%) of e. coli isolates (ammar et al. 2015). while the presence of virulence factors such as shiga toxin (stx1 and stx2) and α-haemolysin (hly) of e. coli is pivotal for suggesting the increased pathogenicity of these strains, serogroups are still crucial for identifying potential diseases. in this study, the prevalence of stx1 virulence gene was 6 of 10 (60%), while (stx2 and hly) genes failed to be detected. in accordance with the recorded result, nasef, el oksh and ibrahim (2017) detected stx1, stx2 and hly (45%, 65% and 80%), respectively. studying the antimicrobial genotypic attributes of the isolates was applied by pcr to investigate the presence of tetracycline and penicillin β-lactams resistance gene (teta and blactx-m), the results indicated positive detection in all 10 of 10 (100% for each) of the tested e. coli. similar outcomes were reported by (fashae et al. 2021) who detected the blactx-m gene in 83.3% of mdr e. coli isolates. furthermore, according to gholami-ahangaran et al. (2021), all e. coli isolates from faecal samples of pet birds included the teta gene. the phylogenetic analysis of the fimh gene of two selected e. coli isolates from both resident free-living wild birds and cattle, which was in contact, demonstrated a high conservation level of the gene at (a.a) level as previously proved by vandemaele, hensen and goddeeris (2004). the homology was 100% with gene sequence from different resources including avian, cattle, birds, pig and from food sources such as milk and ice cream and also from environmental sources such as water, sewage and farm soil prove the potential role of wild birds as a reservoir for e. coli having mdr genes. this was concordant with results mentioned by nabil et al. (2020). however, the detected snps between both examined samples represented no change at protein level proves the common source nature of pathogens suggesting the possible role of wild birds to contaminate water sources and disseminating the infection to cattle farms (fahim et al. 2019). also, our findings could highlight the public health concern of presence of wild birds carrying e. coli with mdr genes in contact with dairy cattle farms and its surrounding environment that could transmit infection to human through the food chain. conclusion this study reported updated data about the critical infectious role of faecal matter of wild birds to cattle farm. highly virulent and resistant pathogenic serovar of e. coli could be disseminated towards different animal species triggering several diseases, threatening their health and impairing the animal farm economy. hence, strict recommendations for animal manure with good hygienic practices in cattle farms should be implicated. also, one health approach should be implicated to inherit the dispersion of multiple antimicrobial resistance phenomena. furthermore, more advanced sequencing approaches should be studied on the whole genome level for such bacteria to indicate the interrelationship of virulent and resistant genes in different animal, human and wild bird species. acknowledgements the authors would like to thank princess nourah bint abdulrahman university, riyadh, saudi arabia, for funding and financial support. the authors also thank prof. dr. abdelazeem algammal, professor of microbiology, faculty of veterinary medicine, suez canal university, for his help in reviewing and editing this work. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions g.a.i. conducted the laboratory investigations, writing of the original draft and editing. a.m.s.-e. assisted with the article methodology, sample collection and reviewing. m.a.-z. assisted with the conceptualisation, supervision, review and editing. a.s.e.o. conducted laboratory investigations and writing of the original draft. e.m.a. conducted gene sequencing analysis, writing and editing the original draft. d.s.f. assisted with methodology, data interpretation and editing of the original draft. e.m.s. assisted with the conceptualisation, sample collection and editing of the original draft. all authors read and approved the final manuscript. ethical considerations no experimental animals were used in this study. all the procedures of the study were adapted according to the ethical and humane principles of the ethics and animal experimentation committee of suez canal university (approval no. 2022007). all the diagnostic methods and laboratory work were conducted according to isolation, biosafety, and quality standards of ahri, agricultural research council, dokki, giza, egypt. funding information this study 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henrik christensen2 kurt handberg3 uswege minga4 john e. olsen2 affiliations: 1department of virology, ministry of livestock development, tanzania 2department of veterinary disease biology, university of copenhagen, denmark 3department of clinical microbiology, university hospital of aarhus, denmark 4faculty of science, technology and environmental studies, open university of tanzania, tanzania correspondence to: henrik christensen email: hech@life.ku.dk postal address: department of veterinary disease biology, university of copenhagen, stigbøjlen 4, 1870 frederksberg c, denmark dates: received: 20 dec. 2010 accepted: 08 apr. 2011 published: 30 sept. 2011 how to cite this article: yongolo, m.g., christensen, h., handberg, k., minga, u. & olsen, j.e., 2011, ‘on the origin and diversity of newcastle disease virus in tanzania’, onderstepoort journal of veterinary research 78(1), art. #312, 8 pages. doi:10.4102/ojvr.v78i1.312 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) on the origin and diversity of newcastle disease virus in tanzania in this original research... open access • abstract • introduction • materials and methods    • materials       • viral rna extraction    • primers and reverse transcriptase polymerase chain reaction    • sequence analysis, alignment and phylogenetic analysis • results • discussion • conclusion • references abstract (back to top) free-range rural chickens (frcs) dominate the poultry industry in developing countries and chickens are exposed to multi-host infections, including newcastle disease virus (ndv). the knowledge about the characteristics of ndv from frcs is limited. this study investigated the persistence, spread and risks of ndv from frcs. ndv isolates (n = 21) from unvaccinated frcs in tanzania were characterised by conventional intracerebral pathogenicity index (icpi) and sequence analysis of a partial region of the deduced fusion protein encompassing the cleavage site. results showed that five isolates were screened as lentogenic, nine as mesogenic and six as velogenic. phylogenetic analysis of the 21 isolates compared to reference sequences revealed three, four, nine and five isolates in g enotypes 1, 2, 3c and 4a, respectively. genotype 3c also included published sequences of tanzanian isolates obtained from exotic birds and chicken isolates from uganda. the analysis showed that ndv were persistently present among chicken populations and possibly spread through live chicken markets or migration of wild birds. differences in amino acid sequences detected around the cleavage site separated the isolates in six types. however, cleavage site pattern could not fully differentiate mesogenic isolates from velogenic isolates. introduction (back to top) free-range rural chickens (frcs) populations dominate the poultry industry in developing countries (permin & hansen 1998; regmi 2001). the management of frc allows interactions between chickens and ducks, pigeons, wild birds and other animals. this predisposes chickens to multi-host infections, including newcastle disease virus (ndv). moreover, frc flocks and populations are composed of multi-age chickens, which are usually not vaccinated. over 250 species of birds are reported to be susceptible to ndv infection (alexander 2001). chickens are the most susceptible species and infections of susceptible birds with virulent ndv isolates lead to severe disease and heavy stock losses (alexander 2001). thus, newcastle disease (nd) is apparently responsible for major losses in frc populations (spradbrow 1993). it is also of economical importance in commercial poultry production and trade worldwide (alexander 2001). major outbreaks have been reported since nd was reported for the first time (lomniczi et al. 1998). newcastle disease occurs sporadically among frcs, with seasonal epidemics and up to 100% mortality in unvaccinated flocks (spradbrow 2000; yongolo, machangu & minga 2002). whereas much has been reported on ndv contributing to outbreaks in commercial production, knowledge is limited about the ndv circulating and causing nd oubreaks in frc populations. ndv are categorised as avian paramyxovirus serotype 1 (apmv-1) and belongs to the genus avulavirus, subfamily paramyxovirinae in the family paramyxoviridae (eds. van regenmortel et al. 2000). the apmv-1 serotype has a negative-sense, single-stranded rna genome composed of six genes coding for rna-directed rna polymerase, haemagglutinin-neuraminidase protein, fusion protein (f), matrix protein, phosphoprotein and nucleoprotein (philips, samson & emmerson 1998). fusion protein synthesised as a precursor f0 must be proteolytically cleaved to the peptides f1 and f2 for fusion activity to occur between virus and host. this separation occurs between amino acid residues 116 and 117, with postion 117 being the first amino acid for the f1 fusion polypeptide. the sequence 111ggrqgr|l117 of an avirulent isolate turned virulent when converted to 111grrqrr|f117 (peeters et al. 1999) and the peptide sequence at the cleavage site can be used to predict virulence. virulence of ndv is conventionally estimated by determining the intracerebral pathogenicity index (icpi), intravenous pathogenicity index and the mean death time in day-old chicks, 6-week-old chicks and embryonated chicken eggs (eces) of between 9 and 11 days old, respectively (office international des epizooties 2009). results from these three methods cannot differentiate between strains and used alone they have very little epidemiological significance. pathotyping by such methods can only classify ndv a highly (velogenic), moderately (mesogenic) and slightly (lentogenic) pathogenic. however, strain differentiation and epidemiological data can be achieved through restriction enzyme analysis, cleavage site mapping and sequence analysis of the fusion protein gene (ballagi-pordány et al. 1996). based on these methods, eight genotypes have been revealed to have been involved in major epidemics (yu et al. 2001). the genotypes have recently been reclassified and 16 new genotypes are now recognised (aldous et al. 2003). of the recent genotypes, novel genotypes viib, viid and viii (5b, 5d and 3d according to aldous et al. 2003) were identified from outbreaks in southern africa, asia and europe (abolnik et al. 2004; herczeg et al. 1999; li et al. 2001; lomniczi et al. 1998; yang et al. 1999). in this study, 21 ndv isolates from tanzanian frcs and ducks, with no history of vaccination against nd (yongolo 1996), were characterised to gain molecular epidemiological information. however, the characteristics of ndv affecting frcs that could influence endemicity were not addressed or documented (yongolo 1996). the aim of this study was to demonstrate ndv persistence, spread and infection frc to further understanding of the epidemiology of nd in frcs and possible risks to the commercial poultry industry. materials and methods (back to top) materials the sources of virus are shown in table 1. these isolates were samples taken from tanzanian frcs between 1994 and 1995 at monthly intervals. they were confirmed and deposited at the central veterinary laboratories agency, weybridge, surrey (uk) and deposited at the sokoine university of agriculture, morogoro, tanzania (yongolo 1996). all isolates were passaged three times in eces (between 9 and 11 days old) before use. of the 21 isolates, one had been isolated from live chicken markets in the mbeya region of tanzania and the rest from village chicken in the regions morogoro and tabora. one isolate was obtained from a duck kept with chickens in the same flock (figure 1). table 1: list of newcastle disease virus isolates investigated. figure 1: map of tanzania with indication of the three regions from where isolates were received. before rna extraction, all isolates were passaged in 10-day-old eces and the presence of ndv in the allantoic harvests was detected following standard procedures of haemagglutination (ha) and ha inhibition test according to the commission of the european communities (1992). the virulence of each isolate after the third passage was estimated by determining the icpi in day-old chicks according to standards of the office international des epizooties (oie 2009). viral rna extraction viral rna was extracted directly from the allantoic fluid harvested from ndv-infected eces. virus extraction was performed at the danish veterinary laboratory in aarhus, denmark. rna was isolated using the qiagen rneasy mini kit rt-pcr system (qiagen, hilden, germany) and expand (roche diagnostics, hvidovre, denmark) according to the protocol provided with the qiagen rneasy® kit. cells in 400 µl of the allantoic fluid were lysed and dnase inactivated using 284 µl rtl buffer that contained guanidine isothiocyanate (gitc), with subsequent addition of 6 µl of β-mercaptoethanol. the spin column was washed using the two washing buffers, rw1 and rpe, provided in the kit and followed by centrifugation. elution was performed with 50 µl rnase-free water and the elute was preserved at –20 °c until use. primers and reverse transcriptase polymerase chain reaction primer sequences, originally designed to amplify the f protein gene between position 148 and 535 to yield an expected product 388 nucleotides long, were obtained from jørgensen et al. (1999). the procedure for single-tube reverse transcriptase polymerase chain reaction (rt-pcr) was carried out according to jørgensen et al. (1999) using mgcl2at a concentration of 1.5 mm. all reagents were handled on ice and the enzyme was added just before transferring the reagents to the pcr machine (ptc-200, mj research, watertown, massachusetts). initial reverse transcription was performed for 30 min at 50 °c and denaturation was then carried out for 2 min at 94 °c. ten cycles, entailing denaturation for 30 s at 94 °c, annealing for 30 s at 58 °c and elongation for 1 min at 68 °c, were performed. after 10 cycles, an additional 25 were performed with the elongation time extended by 5 s in each cycle. finally, elongation was performed for 7 min at 68 °c. after rt-pcr, dna was extracted from a sliced agarose gel (stained with ethidium bromide) using the qiagen ii gel extraction kit as described by the qiaex ii handbook (qiagen, hilden, germany). the elute was centrifuged and the dna-containing supernatant was stored at –20 °c until use. sequencing of the rt-pcr-derived nucleotide sequences was determined by cycle sequencing (sears et al. 1992), using the amplitaq fs dye terminator kit and the 373a automatic sequencer (applied biosystems, foster city, california). the primers ndv-2 and ndv-3 (jørgensen et al. 1999) used during pcr amplification were also used in sequencing. the conditions for performing dna sequencing and data evaluation were as recommended by applied biosystems. sequence analysis, alignment and phylogenetic analysis sequence analysis was performed on 17 isolates from frcs and a duck. sequences were compared to reference sequences from other parts of the world, selected to represent the 16 genotypes reported to date (aldous et al. 2003). further selection of sequences was based on the blast search programme (altschul et al. 1997) in the genbank database (benson et al. 2004) in order to identify closely related sequences. only sequences with clear indication of isolate identity, host and country of origin were included. six sequences previously determined from tanzanian isolates (aldous et al. 2003; seal et al. 1998) and all sequences described for the african continent were also included. the region of the f protein gene between position 231 and 374 (b1 isolate, accession number nc_002617) was aligned with representatives of published genotypes resulting in 180 positions for comparison. pair-wise sequence comparisons were performed using emboss (water) (http://www.ebi.ac.uk). the phylogenetic analysis was perfomed by neighbour joining analysis by clustaxx (thompson et al. 1997) and the tree visualised by mega4 (tamura et al. 2007). results (back to top) results from the virulence assessment in day-old chicks showed the icpi to be between 0.0 and 2.0 (table 1). icpi intervals of 0.00–0.70, 0.71–1.20 and 1.21–2.00 were used to classify the virus as lentogenic, mesogenic or velogenic, respectively. the phylogenetic tree is shown in figure 2. ndv isolates from tanzania were asigned to four of the 16 genotypes defined earlier (aldous et al. 2003). the three isolates classified as genotype 1 (table 2 and figure 2) showed 98.4–99.2% nucleotide similarity. four isolates, including the only one from the mbeya region and the reference strain b1, were described as belonging to genotype 2 (table 2 and figure 2). the isolates of this group investigated in the current study showed 97.4–99.6% dna sequence similarity. blast search showed nearly 100 sequences with high similarity to this genotype 2, including more versions of the lasota strain (data not shown). table 2: protein signatures of the f gene cleavage site of newcastle disease virus compared to published cleavage sites of slightly virulent and virulent newcastle disease virus. figure 2: neighbour-joining-based phylogeny of newcastle disease virus isolates from tanzania with reference to published genotypes based on partial nucleotide sequence comparison of the f gene. nine isolates belonged to genotype 3c (table 1 and figure 2). the isolates showed 90.6–100% dna sequence similarity. isolates mg6.33c and tb1.34 even shared sequences, as did mg10.4c and mg10.5c (table 2). the only duck isolate was included in this genotype. the ugandan outbreak strain also belonged to this group and showed the highest similarity to isolate mg10.5c (97.9%). the presence of identical sequences in different regions of tanzania, as well as the high similarity to the strain from uganda, indicates rapid spread of ndv. isolate 6324/95 sampled in 1995 from wild birds represented in the investigation of seal et al. (1998) also belonged to this group. five tanzanian isolates were grouped as belonging to genotype 4a (table 2 and figure 2). the isolates showed 96.6–99.0% dna sequence similarity. isolate mg3.35c was isolated in march 1995, while mg6.9c, mg6.16c and mg6.30c were isolated in june 1995. mg3.35c may have been the progenitor ndv for the june isolates. this group showed only little similarity to other sequences deposited in the nucleotide databases (< 92%), as determined by the blast search. the predicted amino acid sequences of the fusion protein gene region (positions 231–420) were aligned. the variations observed for the tanzanian isolates are shown in table 2. all isolates for genotypes 1 and 2 showed leucine at position 117 of the cleavage site, whereas genotypes 3c and 4a sequences showed phenylalanine at that position. discussion (back to top) according to aldous and alexander (2001) ndv is an apmv-1 with an icpi of 0.7 or higher or with at least three basic amino acids between positions 113 and 116 and phenylalanine at position 117. most of the apmv-1 virus isolated in the present study fit this definition, except for three isolates (mg10.3c, mg7.10c, tb9.26c) with an icpi of 0.5 or lower and leucine at position 117 (see tables 1 and 2). the ndv isolates from tanzania could be classified as genotypes 1, 2, 3c and 4a according to the aldous et al. (2003) designations. genotype 2 viruses are known to be vaccine strains mainly isolated from the usa (herczeg et al. 1999; liang et al. 2002). the lasota strain, which is the commonly used type of vaccine in tanzania, could have been the progenitor virus for some of the isolates involved in disease. the study showed that isolates belonging to genotype 3c were dominant and involved in nd outbreaks amongst frcs in tanzania and probably also other east african countries. the nine isolates were velogenic or mesogenic and isolated from apparently healthy or sick birds (table 1). otim et al. (2004) identified a member of genotype 3c from nd outbreaks in uganda, which may be related to those from tanzania in the current investigation. this would confirm that poultry move between uganda and tanzania. however, other sources for these isolates may also exist, because lomniczi et al. (1998) reported that old hungarian isolates belong to this group. members of this group have also been reported in the usa, mexico (see figure 2) and germany, dating back to the 1970s (wehmann et al. 2003). the isolates reported by seal et al. (1998) from finch and parakeet also belonged to this group. unfortunately, abolnik et al. (2004) sequenced another partial region than we did and sequences could therefore not be compared phylogenetically. based on the comparison of labelled groups, group 3c (old v) was found in tanzania but does seem to have been present in south africa. groups iv, viib, viic, viid and viii, in turn, were found in south africa but not in tanzania. the presence of genotype 1 ndv was reported in tanzania for the first time. it is apparent from this study that genotype 1 ndv are still prevalent. members of genotype 1, together with those of genotypes 2, 3a and 3b (aldous et al. 2003), have been reported to be involved in the first panzootic outbreaks (herczeg et al. 1999; yu et al. 2001). because genotype 1 ndv were involved in the first panzootic outbreak, which started in the mid-1920s in other parts of the world, and the recovery of related ndv in tanzania in 1995, old genotypes are possibly still circulating worldwide, together with new genotypes. it is well known from previous findings that genotype 1 viruses are mainly lentogenic (collins, bashiruddin & alexander 1993; peeters et al. 1999; king & seal 1998). all three genotype 1 isolates had leucine at position 117, which is found only in low-virulence ndv (collins, govey & alexander 2003; kant et al. 1997; oberdörfer & werner 1998; seal, king & bennett 1995), whereas all virulent ndv have phenylalanine at position 117 (collins et al. 1993). genotype 1 comprised representative isolates from the two sampled regions of tanzania. it is not known when genotype 1 viruses could have been introduced into tanzania. isolate v4 could possibly have been introduced into tanzania as a thermostable vaccine for research purposes in 1992, 1995 and 1996 (foster et al. 1999). given the geographical location and the short time since v4 was introduced in tanzania it is most unlikely that it is the progenitor virus for this group. on the other hand, v4 may have spread from neighbouring malawi, where extensive field trials using the thermostable v4 had been carried out to control nd in intensive and smallholder village chicken flocks before 1987 (sagild & haresnape 1987). the absence of genotypes 5a, 5b, 5c, 5d, 3d (aldous et al. 2003) viruses in tanzania indicates that ndv, which caused devastating outbreaks of velogenic nd from 1993 to 1995 in southern african countries, did not reach tanzania or were not isolated (herczeg et al. 1999). recent outbreaks from nigeria were found to belong to genotypes 5f/g/h and 3a in addition to 1 and 2 (snoeck et al. 2009); however, members of these groups were not identified from tanzania. we can conclude that no particular unique types of ndv exist in tanzania, but that the current types observed probably have descended from past outbreak strains in other parts of the world as well as from vaccine strains. no temporal correlation was found between isolates sampled in morogoro, tabora or mbeya. at morogoro and tabora all four genotypes were sampled; however, most isolates were obtained from morogoro in june 1995 and they belonged to three genotypes (1, 3c and 4a). the study has shown that ndv is endemic to tanzania, because ndv was isolated from village and markets in different geographical locations at different times of the year. our study also showed that the different isolates from the chickens and ducks showed different levels of virulence and that multiple lineages were circulating amongst domestic birds. this is important because such birds could be the source of potentially virulent ndv to frcs and commercial poultry worldwide. the presence of different pathotypes was confirmed by icpi and the presence of variable amino acid sequences at the cleavage site. the presence of variable pathotypes in the field may complicate disease outcome and diagnosis. therefore, mere isolation of ndv cannot be used for nd diagnosis or for determining the prevalence of nd unless characterisation and clinical disease are correlated. the persistent presense of ndv in frcs is important because they predispose chickens to secondary and opportunistic upper respiratory bacterial infections (such as from escherichia coli). therefore, frequent programmed vaccination is recommended. this will guarantee protection of most chickens in an endemic situation. enforcement of biosecurity may be the best alternative, but this is not feasible in the free-range system with its low input and low output management system, as is commonly practiced in tanzania and other developing countries. genetic variations amongst ndv in frcs were established in this study. these results showed an epidemiological relationship between ndv from frcs of tanzania to ndv reported in other countries. accorrding to tsai et al. (2000), viral divergence of 0.2–0.9% indicates that the viruses are genetically highly homogenous and similar. in the present study, more than 99% similarity was found amongst some members of all genotypes and even between isolates from different regions (morogoro and tabora). dna similarity of up to 100% amongst some isolates found in the two regions studied and the phylogenetic tree analysis provided evidence that the isolates had a common origin. however, results in this study showed that no group of viruses was found exclusively in tabora, although a specific group of viruses was found only in morogoro. isolates from tabora may therefore have had the same ancestor as some of the morogoro isolates. the one-way type of relationship is attributed to the one-way marketing of live chickens from western regions to coastal regions of tanzania. ndv could have spread from tabora to morogoro through live bird marketing, which is commonly practised (mlozi et al. 2003). therefore, live bird marketing may be the main vehicle for spreading nd in tanzania. several amino acid sequence differences were detected in the studied isolates, specifically differences with regard to the fusion protein cleavage site. the differences correlated with virulence. most results showed correlation between genotype f cleavage sequence and virulence for both slightly virulent and virulent ndv isolates. these results are in agreement with what was reported by yu et al. (2001). lack of correlation between icpi values and the cleavage site motif, together with the findings on amino acid substitutions around the cleavage site, which leads to ndv with changed virulence, means that the actual virulence characteristics of an isolate cannot be determined by the cleavage site motif alone. consequently, analysis of cleavage site motifs can be used only qualitatively to determine the virulence of ndv isolates. therefore, the icpi cannot be replaced in totality by analysis of the cleavage site. furthermore, these results show that fusion protein gene cleavage site is not the only factor that determines virulence. just as for the avian influenza virus (alexander 2000; tollis & di trani 2002) hn protein precursor also plays a role in the virulence of ndv (cobaleda et al. 2002) and findings by huang et al. (2003) show v protein of ndv significantly contribute to the virus virulence as an alpha/beta interferon antagonist. thus, although f cleavage site is the major determinant of virulence, other factors also contribute to actual virus virulence. changes of ndv from avirulent to virulent was previously suspected from two outbreaks of nd amongst poultry in ireland (alexander et al. 1992). the causative ndv were velogenic and very similar, antigenically and genetically, to avirulent viruses isolated from feral waterfowls, suggesting that velogenic viruses arose from avirulent ndv from wild birds (collins et al. 1993; collins et al. 1998). furthermore, recent outbreaks in australia (1998–2001) revealed genetic resemblance of virulent ndv to field lentogenic isolates (gould et al. 2001). in both field observations and the recent experimental study by shengquing et al. (2002), step-by-step amino acid substitution was demonstrated. the management of frcs allows for unlimited contact with other hosts including wild birds found to be infected mainly with lentogenic ndv, with the exception of few species (takakuwa et al. 1998). this means that avirulent and lentogenic ndv isolates with avirulent genes have the potential to become velogenic after transmission to and repeated passage in chickens. exposure to ndv and repeated passage are highest in frcs compared to commercial chickens. therefore, control of nd in frc populations is important to reduce the chances of generating unselected populations of different ndv. the presence of isolates in frcs with drifting virulence suggests that virulent isolates could emerge from lentogenic vaccine viruses. these results have important implications for the use of live virus vaccine in the control of nd amongst rural frcs as well as commercial chickens. although there are no reports of cases of virulent ndv arising from the current live vaccines, future plans about nd control should take the possibility into consideration, especially with regard to nd control in rural frc populations. it is recommended that old and new vaccines should be carefully selected and, when introduced, monitored to detect their conversion to virulence. conclusion (back to top) in conclusion, investigation of ndv isolated from frcs in tanzania showed presence of lentogenic, mesogenic and velogenic types, which phylogenetic analysis of a partial region of the fusion protein gene allocated to genotypes 1, 2, 3c and 4a. genotype 3c also included published sequences of tanzanian isolates obtained from exotic birds and chicken isolates from uganda. the analysis showed that ndv were persistently present among chicken populations and possibly spread through live chicken markets or migration of wild birds. references (back to top) abolnik, c., horner, r.f., bisschop, s.p., parker, m.e., romito, m. & viljoen, g.j.a., 2004, ‘a 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3department of tropical diseases, university of pretoria, south africa 4school of veterinary and biomedical sciences, james cook university, australia correspondence to: mohamed sirdar postal address: private bag x04, onderstepoort 0110, south africa dates: received: 14 july 2011 accepted: 03 nov. 2011 published: 16 apr. 2012 how to cite this article: sirdar, m.m., picard, j., bisschop, s., jambalang, a.r. & gummow, b., 2012, ‘a survey of antimicrobial residues in table eggs in khartoum state, sudan, 2007–2008’, onderstepoort journal of veterinary research 79(1), art. #360, 9 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.360 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a survey of antimicrobial residues in table eggs in khartoum state, sudan, 2007–2008 in this original research... open access • abstract • introduction • materials and methods    • study design    • laboratory analysis    • data analysis    • spatial analysis • results • discussion    • comparison of khartoum state antimicrobial residue results with the results of other countries in africa • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the risk to consumers of antimicrobial residues in table eggs produced in khartoum state, sudan, was studied. all producing layer farms (n = 175) in the state were sampled in april, june and august 2008. a total of 933 eggs from 335 layer houses were screened for antimicrobial residues by using the growth inhibition of geobacillus stearothermophilus var. calidolactis in-house test. a high proportion of layer farms (72% in april, 61% in june and 66% in august) and layer houses (63% april, 59% in june and 61% in august) were found to have antimicrobial residues, with no significant difference in prevalence (p = 0.57) between study periods. the study showed that the consumer was at constant risk of exposure to antimicrobial residues in table eggs. the paper discusses reasons for the high prevalence of antimicrobial residues in sudanese eggs and its implications, and makes recommendations to address this important public health problem. introduction top ↑ the sudanese poultry industry is located principally in khartoum state which is the source for 90% of the country’s production. the total poultry population in sudan is estimated at 45 million. the commercial sector comprises 30 million chickens of which 20 million are layer hens. it contributes 45% of the agricultural income of the state, whilst the latter (agricultural income) contributes 7% of the total income (ministry of agriculture, animal resource and irrigation 2005).antimicrobials are used by the poultry industry to enhance growth and feed efficiency and to reduce bacterial disease (donoghue 2003). in layer hens, antimicrobials are used only to treat and to prevent bacterial infections. antimicrobial classes used to treat poultry are similar to those used in human medicine and include aminoglycosides, tetracyclines, beta-lactams, quinolones, macrolides, polypeptides, amphenicols and sulphonamides (stolker & brinkman 2005). antimicrobial residues may have a direct toxic effect on consumers, for example allergic reactions in hypersensitive individuals (dayan 1993; ormerod, reid & main 1987; woodward 1991). it has become necessary, therefore, that regulations are in place to ensure that antimicrobial residues are not present in animal products for human consumption at levels that may affect human health detrimentally. internationally recognised organisations such as the world health organisation (who), food and agriculture organisation (fao), veterinary medicine directorate (vmd) of the european union (eu), as well as the food and drug administration in the usa (fda), have set maximum tolerance levels or acceptable daily intakes (adis) for humans, and withholding times for pharmacologically active substances, including antimicrobial agents, prior to marketing (al-ghamdi et al. 2000). surveillance systems should be in place in conjunction with these regulations to ensure that these standards are met and that analyses can detect antimicrobials at less than the maximum residue levels. sudan currently has neither regulations to govern the use of antimicrobials or the maximum allowable antimicrobial concentrations in food, nor does sudan have any systems to monitor the presence of antimicrobial residues in animal products. the objective of this study was, therefore, to investigate the risk to consumers of antimicrobial residues in commercial layer eggs in khartoum state, sudan, given the lack of regulations. materials and methods top ↑ study design the study was conducted in khartoum state where 90% of the country’s eggs are produced. khartoum state is divided into three localities, namely khartoum, khartoum north (bahry) and omdurman, which made up the sampling frame for the study (figure 1). most of these farms were situated close to the nile river and consisted predominantly of smallholdings that made use of traditional open-houses. egg samples were collected in all of these localities in april (mid-summer), june (the start of the rainy season) and august (the rainy season) 2008 to determine whether the season has any influence on the presence of antimicrobial residues in eggs. three eggs were collected from each house: one close to the entrance, one in the center and one at the far end of each house, regardless of the number of houses on the farm. if one or more eggs from that house had antimicrobial residues, the house was considered as positive, thus increasing the sensitivity of the survey. if one or more houses were found to contain antimicrobial residues, the farm was considered as positive. it was assumed that mass in-water or mass in-feed treatment of houses was the method of choice when treating layers and therefore that antimicrobial residues should be equally present in all the eggs in the house at a specific time. figure 1: khartoum state map showing the three localities of the study area and the layer farm density in the state for 2007/2008. each of the periods of egg collection was analysed as an independent survey because not all the farms were available for sampling for the different sampling periods. the reasons for this will be discussed later. laboratory analysis each egg sample was labeled with a unique number that corresponded to a data set that contained the date of collection, farm origin and the house number. the eggs were transported in carton storage trays at room temperature and were processed within 72 hours of collection. in a clean, dust-free room, the eggs were sprayed with 70% ethanol, allowed to air-dry and the tip of the shell at the air-sac was removed with scissors by using an aseptic technique. the contents were then poured into whirlpak® plastic bags (nasco, usa) labeled with the unique identifier number, farm origin, poultry house number and the date of collection and stored in a freezer (-18 °c) until processed.after defrosting, each sample was homogenised with a stomacher lab-blender 400 (seward medical, united kingdom) and 100 µl was added to 900 µl of nutrient broth (labretoria, south africa) containing 1% glucose (merck chemicals, south africa), 0.002% phenol red (merck chemicals, south africa) and approximately 105 colony forming units/ml of viable geobacillus stearothermophillus var. calidolactis attc 7953 (merck chemicals, south africa). the test was carried out in triplicate to increase sensitivity and to detect operator error. a control containing egg contents from hens that were known to be untreated with antimicrobials was used to check the viability of the organism and to determine the end-point of incubation. samples were incubated in a water bath (labotec, south africa) at 80 ºc for 10 minutes to destroy lysozyme and other protein inhibitors, as well as to stimulate the germination of g. stearothermophilus spores. the samples were then incubated at 65 ºc for 2–4 hours, depending on when the growth control changed from red to yellow. a colour change from red to yellow indicated glucose fermentation, whereas retention of the red colour indicated an inhibition of growth and hence the presence of inhibitors. data analysis data were collated and analysed with the microsoft excel (microsoft corporation, usa, 2003) spreadsheet program, and the comparison of the proportion of layer houses with antimicrobial residues in the three periods of collection were analysed with the survey toolbox (animal health service, australia) statistical package.the sampling unit for determining the prevalence of residues within khartoum state was the poultry house. a farm was considered as positive if one or more houses on the farm were found to contain eggs with antimicrobial residues and a house was considered as positive if one or more eggs contained detectable antimicrobial residues. the apparent prevalence (pˆ) derived from the survey is the number of farms with detectable antibiotic residues divided by the total number of farms sampled. the apparent prevalence was calculated separately for each period of collection. based on published results of a similar test, the sensitivity (se) of the diagnostic test was assumed to be 72.5% and the specificity (sp) as 98% (gaudin et al. 2009). the 95% confidence interval for the true prevalence (p) was calculated according to cameron and baldock (1998) by using the variance estimate [var(pˆ)] of the apparent prevalence (pˆ) as follows: where (pˆ) is the apparent prevalence, se denotes sensitivity, sp represents specificity, and n indicates the sample size. the confidence interval for the true prevalence (p) is then: spatial analysis the mapping program, google earth version 4.3, was used to identify the farms and areas of sampling and to record the coordinates of all the farms sampled in khartoum state. the main challenge was to record the coordinates of each farm sampled because they were clustered in each area with a difference of only seconds, or even fractions of a second, between them. all coordinates were entered into the spreadsheet program, excel (microsoft corporation, usa, 2003). the excel file was converted for use in arcview 9.3 (esri redlands 2009). africa, sudan and khartoum state shape files were downloaded from www. maplibrary.org (map maker trust 2009).maps of khartoum state that show the sampling locations, farm density, farms sampled and the prevalence of antibiotic residues for each period of collection, were created by using arcview 9.3, as stated above. results top ↑ prior to sampling (january 2008), a census was carried out of all the poultry farms in khartoum state with the aim of repeat sampling of the same farms. the census showed that there were 252 layer farms, containing 764 houses, in the state. a proportion of the eggs sampled in april had noticeable bacterial contamination when they arrived at the laboratory in south africa, and consequently could not be analysed. the poor state of these samples was caused by an unexpected delay when the samples were couriered from sudan to south africa and, in addition, the samples were delayed at the airport for irradiation before clearance. originally 290 eggs were sampled in april 2008; however, the delays and preservation difficulties in the importation of the egg contents to south africa meant that 111 eggs had to be discarded, which left a total of 43 farms and 68 houses from which eggs could be processed. in total, 933 eggs were analysed from 335 layer houses on 175 farms of which 43 farms (68 houses) were sampled in april 2008, 79 farms (154 houses) in june 2008, and 53 farms (113 houses) in august of the same year. of the 933 eggs, 197 (21%) were collected in april 2008, 427 (46%) in june, and 309 (33%) in august 2009. the areas sampled in the survey and the number of houses in each area surveyed, as well as the number of houses that were found to have eggs with antimicrobial residues, and the 95% confidence intervals for the true prevalence of houses affected, for the three sampling periods, have been tabulated (table 1). table 1: number of houses sampled in 2008 and found positive for antimicrobial residues in each area sampled with 95% confidence interval for the true prevalence of houses with residues for each period sampled given below. the areas sampled in the survey in relation to the number of farms in each area surveyed, as well as the number of farms that were found to have houses with antimicrobial residues, and the 95% confidence intervals for the true prevalence of farms affected, for the three sampling periods, were tabulated as well (table 2). table 2: number of farms sampled in 2008 and found positive for antimicrobial residues in each area sampled with 95% confidence interval for the true prevalence of farms with residues for each period sampled given below. the spatial distribution of antimicrobial residues for each sampling period is shown (figure 2, figure 3 and figure 4, respectively). of the farms sampled in april 2008, 72% had antimicrobial residues in them, whilst 63% of the layer houses in total had antimicrobial residues (table 1 and table 2). the 95% confidence interval for the true prevalence of antimicrobials in april on farms was 64% – 80%, and for houses 57% – 70%. figure 2: spatial distribution of antimicrobial residues in farms in khartoum state surveyed in april 2008. figure 3: spatial distribution of antimicrobial residues in khartoum state farms surveyed in june 2008. figure 4: spatial distribution of antimicrobial residues in khartoum state farms surveyed in august 2008. farms sampled in june 2008, showed that 61% of the farms had antimicrobial residues. the proportion of layer houses with antimicrobial residues from farms with antimicrobial residues was 59% (table 1 and table 2). the 95% confidence interval for the true prevalence of antimicrobials in june on farms was 54% – 67%, and for houses 55% – 64%. on those farms with antimicrobial residues, an average of 90% of layer houses contained eggs with antimicrobial residues.in august 2008, 66% of the farms had antimicrobial residues in their eggs, whilst antimicrobial residues were detected in 61% of the layer houses (table 1 and table 2). the 95% confidence interval for the true prevalence of antimicrobials in august on farms was 59% – 74%, and for houses 56% – 66%. on those farms with antimicrobial residues, an average of 83% of layer houses contained eggs with antimicrobial residues. no difference in prevalence between sampling periods could be shown in khartoum state farms or layer houses (p-valuefarms = 0.57 and p-valuelayer houses = 0.88). a comparison of the proportion of layer farms with antimicrobial residues in each area, for each period of collection (april, june and august), showed that only ‘el-selait’, and ‘el-tibna & zakiab’, had significant differences between the three periods with a p-value of 0.02 and 0.08, respectively (figure 2, figure 3 and figure 4). discussion top ↑ only 175 farms (335 layer houses) participated in the survey in all three periods of collection because small farms were constantly changing their production status from a layer to a broiler set-up, or rearing pullets up to point-of-lay during the survey, which meant that not all farms, as reflected in the census, could be included in each sampling period. another reason for not being able to sample all farms was that some owners were reluctant to participate in the survey, because of perceived biosecurity risks or a leaking of production information to competitors. this resulted in the inclusion of new farms or the exclusion of some farms in subsequent samplings; therefore, it was decided to regard each sampling period as an independent survey. this may have resulted in some bias in the subsequent prevalence results but should not have impacted materially on the observed trends. the june sampling had the highest number of sampled farms, followed by the august sampling. fewer farms were available in august because of increased feed prices, which forced small producers to leave the business by either selling their flocks as spent hens or depopulating their flocks. in addition to that, august is the rainy season in sudan, which results in damage to the inadequate poultry housing so that farmers do not keep laying flocks in this season. one reason for the result of the lowest number of productive farms in april is that most farmers start to rear pullets in winter (january and february), which means that their birds only come into lay in june. there were some farms with only one house with antimicrobial residues, whilst the rest were negative. this is expected when antimicrobials are used therapeutically as only those houses with diseased birds will be supplied with medicated water. it was observed also that sometimes, within a layer house, one egg sample may be positive and the other two samples negative even though the study design assumed that all hens in a house were treated at the same time and thus that all eggs in a house would contain residues. the reason for this anomaly could be that eggs were collected shortly (within a day) after the withdrawal period of the drug treatment stopped, which resulted in very low concentrations in some eggs on the same farm. another reason may be that, as most medications are either administered via the water or the feed, hens will vary in their uptake, absorption and secretion of antibiotic. on-farm factors that would have played a role as well are individual medications of birds, uneven distribution of the medicated feed and water, and the use of too low concentrations or aged antibiotics, which may have affected the sensitivity of the test. the screening test used also varies in its ability to detect antimicrobials. for example, it could detect the fluoroquinolones, penicillins, macrolides, tetracyclines, gentamicin and trimethoprim, at less than, or equal to, the maximum residue limit (mrl), but varied in its ability to detect the sulphonamides and fosfomycin (jambalang 2011). a high prevalence of antimicrobial residues in april was detected in farms (table 2) in shambat (88%), jurayf gharb (89%), kalakilah & dikhainat (75%), el-tibna & zakiab (67%), and el-selait (67%). these areas are characterised by a high density of farms, which increases the risk of spread of diseases between farms, so that farms would use antimicrobials more readily. there are also more broiler farms amongst the layer farms in this region. el-mazalat had no antibiotic residues; however, only one open-house farm from a total of three farms was sampled in this area. in spite of recording the lowest prevalence of antimicrobial residues in eggs in june, 41% of the areas screened had a prevalence of 80% or above. all the farms sampled in kuku, halfay al muluk, el-haj usif & el-shigla, butri, sowba gharb, tyba al-hasanab, and el-shigailab had antimicrobial residues in their eggs. this could be related to the fact that even though 22% of the all layer houses in the state were sampled in june 2008, only 9.4% of farms sampled were in areas of highest layer house densities (halfay al muluk, as-saqqay and el-tibna & zakiab). eid babiker, el-salam and abo rof had no antimicrobial residue; however, the sample size was not large enough to declare these areas free from antimicrobial residues because only one house was sampled per area. this makes it difficult to interpret the true prevalence of antimicrobial residues, which could change if all farms in these areas were productive. of the farms sampled in august, 66% showed antimicrobial residues in the eggs, which were slightly higher than the june sampling and less than the april sampling. the same trend was observed when results were compared by poultry house, rather than by farm. a high prevalence (100%) of antimicrobial residue was seen in el-haj usif & el-shigla, elfaki hashim, kuku, el-selait, sawba gharb, el-mazalat, kadaro & droshab kabbashi and jurayf gharb. all the above mentioned areas were located along the nile, and layer houses were affected by the moist environment. el-samrab was the only area that did not have antimicrobial residues. there was no significant difference (p = 0.57) in the overall number of farms or layer houses (p = 0.88) with antimicrobial residues between the three periods of collection. this was surprising as it was expected that the antimicrobial use would be the highest during the rainy season (june–october) when infectious diseases in birds would be expected to peak as a result of insufficient housing to cope with the high rainfall and high humidity within these houses. there was a significant difference, however, between individual sampled areas in the three collection periods. ’el-selait‘, and ’el-tibna & zakiab‘, showed a significant difference between all three periods of collection (p = 0.02) and (p = 0.08), respectively. the difference was mainly because of the absence of antimicrobial residues in the samples collected from ’el-tibna & zakiab‘ in the august collection. the results for as-saqqay (p = 0.09), kadaro & droshab (p = 0.05), and el-selait (p = 0.006), differed significantly between june and august. it is seen that only 1 out of 7 samples was positive in the samples collected from as-saqqay, and half of the 18 samples collected from el-selait showed antimicrobial residues in them in the june collection, whilst all samples collected in august in these areas were positive. it is apparent, therefore, that most farmers use antimicrobials throughout the entire production cycle. antimicrobials in layers are only administered therapeutically or to prevent disease, and consequently indicates that there is a high level of infectious disease on most farms. infectious disease on smallholdings are common because of the close proximity of other farms, poor biosecurity, a hot climate and inadequate housing which allows pathogens to survive and can stress the birds. furthermore, the lack of understanding of the effects of antimicrobial residues on human consumers and the lack regulations related to mrls contributed to the high use of antimicrobials. comparison of khartoum state antimicrobial residue results with the results of other countries in africa published data on antimicrobial residues in eggs, or even in chicken meat, is scarce. this section highlights some results of surveys carried out in several countries, including results of surveys carried out on chicken meat, for comparative purposes.available data suggests that antimicrobial residues may be present in a large proportion of poultry products in developing countries, especially in africa, the middle east and south america. similar to our results, al-ghamdi et al. (2000), who used the high-performance liquid chromatography (hplc) method, reported an antimicrobial residue prevalence of 69.7% in chicken meat and 60% in eggs sampled from the eastern province of saudi arabia. a study conducted at tehran, iran, in 2006 by salehzadeh et al. (2006), using hplc, found that 95% of the 86 poultry farms tested had oxytetracycline residues in muscle, liver and kidney samples that were above the mrls. nonga et al. (2009) carried out a study to assess antimicrobial residues in commercial chicken eggs in morogoro municipality in tanzania. the study showed that all eggs sampled (70 eggs) and analysed with the delvotest kit, were positive for antimicrobial residues. a study conducted in senegal that made use of a variety of tests, including hplc, revealed that 20% of the poultry farms sampled in 2001–2002 had antimicrobial residues in their meat products and 43% of the meat samples in 2003 had antimicrobial residues in them (bada alambedji et al. 2008). a study conducted in nigeria (kabir et al. 2004) found antimicrobial residues in 1% of the eggs sampled (200 eggs), and in 21.8% of 378 slaughtered broilers, using a disc diffusion microbial inhibition test with b. cereus and micrococcus luteus. this test, however, is less sensitive to some classes of antibiotics than, for example, the delvotest (nonga et al. 2009). a study conducted in gauteng, south africa, using the same microbial screening test as the study in khartoum state, showed that 9.1% (6.4–12.5 ci) of the eggs tested were positive for antimicrobial residues (jambalang 2011). by using a commercial scintigraphic test known as charm ii, adesiyun et al. (2005) showed that the prevalence of antimicrobial residues in eggs in trinidad was from eggs collected from farms (6.5%), from markets (16.1%) and from supermarkets (15%). both these countries have surveillance systems in place, but testing is selective. in a study conducted in kuwait to assess the prevalence of antimicrobial residues in eggs, tissue and feed samples also using the charm ii system, showed that all eggs sampled in a shopping center (222) were negative for antimicrobial residues. this study showed that the surveillance systems used in kuwait for monitoring and applying the standards of antimicrobial residues in food is well-conducted and highly efficient (alomirah et al. 2007). the results of this study do not vary much from the results of other developing countries, even though the methods of analysis and sampling schemes are different. many north african countries have a serious problem of antimicrobial residues in food available for human consumers. much of this is a consequence of poor regulation and/or ignorance of the consequences of antimicrobials to human health. conclusion top ↑ in conclusion, the control of veterinary antibiotics used to ensure safer animal food products is needed in developing countries. observation of drug withdrawal periods and extension programmes for farmers will be highly beneficial. alternative practices such as vaccinations may reduce the use of antibiotics in poultry, the presence of antimicrobial residues and the development of drug resistant bacteria. this study serves as the first scientific evidence of the contamination of eggs by antimicrobial residues in sudan. although the concern of antimicrobial residues is an international issue and public health problem in eggs or food of animal origin in general, the study shows that there is a very high prevalence of antimicrobial residues in table eggs served for human consumption in sudan. these findings revealed that all consumers in sudan are probably at risk and urgent attention is needed. at present in sudan, there are no routine programmes for monitoring antimicrobial residues in poultry products or in animal derived food. it is therefore recommended that the ministry of animal resources and fisheries and the national standardization and metrology organization, who are responsible for the antimicrobial residues aspect in sudan, need to construct comprehensive and well-designed regulations for antibiotic use in animals, set standards and limits for residues, monitor and survey products, and enforce compliance to ensure that only safe food is marketed and that consumers are protected. this study can serve as a baseline for the sudanese authorities to monitor future intervention programmes. acknowledgements top ↑ the authors thank the south african national research foundation (nrf) and the malawian islamic zakaat fund (izf) for their financial assistance for this project. the work in this study was conducted as an approved research project (no. v047/07) of the university of pretoria, south africa, and met the ethical requirements for such research. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions this work was conducted by m.m.s. (national cooperative corporation) as part of his msc in epidemiology at the university of pretoria. m.m.s. (national cooperative corporation) was involved in all aspects of the project and carried out the field work for the project and some of the laboratory analyses. the principle supervisor for this project was b.g. (university of pretoria) who was responsible for the design and management of the project and the guidance of m.m.s. (national cooperative corporation) in all aspects of the project, but particularly for the epidemiology (data and spatial analysis). j.p. (university of pretoria) acted as a co-supervisor and provided assistance and input principally into the microbiological components of the project (laboratory analysis). all three of these authors contributed extensively to the writing of this article. a.j. (university of pretoria) assisted with some of the laboratory analyses and s.b. (university of pretoria) acted as a co-supervisor to m.m.s. (national cooperative corporation), as well as a poultry consultant. references top ↑ adesiyun, a., offiah, n., lashley, v., seepersadsingh, n., rodrigo, s. & georges, k., 2005, `prevalence of antimicrobial residues in table eggs in trinidad’, journal of food protection 68, 1501–1505. pmid:16013396 al-ghamdi, m., al-mustafa, z., el-morsy, f., al-faky, a., haider, i. & essa, h., 2000, ‘residues of tetracycline compounds in poultry products in the eastern province of saudi arabia’, public health 114, 300–304. http://dx.doi.org/10.1016/s0033-3506(00)00350-4, pmid:10962596 alomirah, h., al-mazeedi, h., al-zenki, s., al-faili, b., al-foudary, m., abuzaid, a-h. et al., 2007, ‘prevalence of antimicrobial resides in eggs, tissues and feed samples in the state of kuwait’, arab gulf journal of scientific research 25, 130–137. bada alambedji, r., akakpo, a.j., teko agbo, a., chataigner, b., stevens, a. & garin, b., 2008, ‘control of residues: example of antimicrobials in food in senegal’, conference on veterinary medicinal products in africa, dakar, 25–27 march. cameron, a.r. & baldock, f.c., 1998, ‘a new probability formula for surveys to substantiate freedom from disease’, preventive veterinary medicine 34, 1–17. http://dx.doi.org/10.1016/s0167-5877(97)00081-0 dayan, a.d., 1993, ‘allergy to antimicrobial residues in food: assessment of the risk to man’, veterinary microbiology 35, 213–226. http://dx.doi.org/10.1016/0378-1135(93)90146-x donoghue, d.j., 2003, ‘antibiotic residues in poultry tissues and eggs: human health concerns?’, poultry science 82, 618–621. pmid:12710482 gaudin, v., hedou, c., rault, a., sanders, p. & verdon, e., 2009, ‘comparative study of three screening tests, two microbiological tube tests, and a multi-sulphonamide elisa kit for the detection of antimicrobial and sulphonamide residues in eggs’, food additives and contaminants a 26, 427–440. http://dx.doi.org/10.1080/02652030802527626 jambalang, a., 2011, ‘the development and validation of a bacteriological screening test for antimicrobial residues in eggs’, msc dissertation, dept. of tropical diseases, university of pretoria. kabir, j., umoh, v.j., audu-okoh, e., umoh, j.u. & kwaga, j.k.p., 2004, ‘veterinary drug use in poultry farms and determination of antimicrobial drug residues in commercial eggs and slaughtered chicken in kaduna state, nigeria’, food control 15, 99–105. http://dx.doi.org/10.1016/s0956-7135(03)00020-3 ministry of agriculture, animal resource and irrigation, khartoum, sudan, unpublished data, 2005. nonga, h.e., simon, c., karimuribo, e.d. & mdegela, r.h., 2009, ‘assessment of antimicrobial usage and residues in commercial chicken eggs from small holder poultry keepers in morogoro municipality, tanzania’, zoonoses and public health 57, 339–344. pmid:19486498 ormerod, a.d., reid, t.m. & main, r.a., 1987, ‘penicillin in milk: its importance in urticaria’, clinical allergy 17, 229–234. http://dx.doi.org/10.1111/j.1365-2222.1987.tb02007.x salehzadeh, f., madani, r., salehzadeh, a., rokni, n. & golchinefar, f., 2006, ‘oxytetracycline residue in chicken tissues from tehran slaughterhouses in iran’, pakistan journal of nutrition 5, 377–381. http://dx.doi.org/10.3923/pjn.2006.377.381 stolker, a.a. & brinkman, u.a., 2005, ‘analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animals – a review’, journal of chromatography 1067, 15–53. http://dx.doi.org/10.1016/j.chroma.2005.02.037, pmid:15844509 thrusfield, m., 2005, veterinary epidemiology, 3rd edn., blackwell science ltd, oxford, uk. woodward, k.n., 1991, ‘hypersensitivity in humans and exposure to veterinary drugs’, veterinary and human toxicology 33, 168–172. pmid:2035248 naude_pp23-37.qxd 23 onderstepoort journal of veterinary research, 72:23–37 (2005) claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised cyperus esculentus (nut sedge) on the highveld of south africa t.w. naudè1, c.j. botha2, j.h. vorster3, c. roux4, e.j. van der linde4, s.i. van der walt5, g.e. rottinghaus6, l. van jaarsveld7 and a.n. lawrence8 abstract naudé, t.w., botha, c.j., vorster, j.h., roux, c., van der linde, e.j., van der walt, s.i., rottinghaus, g.e., van jaarsveld, l. & lawrence, a.n. 2005. claviceps cyperi, a new cause of severe ergotism in dairy cattle consuming maize silage and teff hay contaminated with ergotised cyperus esculentus (nut sedge) on the highveld of south africa. onderstepoort journal of veterinary research, 72:23–37 during december/january 1996/97 typical summer syndrome (hyperthermia and a 30 % drop in milk yield) occurred in succession in two holstein dairy herds (n = 240 and n = 150 milking cows, respectively) on the south african highveld. these farms are situated in the midst of the prime maize and dairy farming areas of south africa where this condition had never been diagnosed before. the individual components of the concentrate on both farms were negative for ergot alkaloids. endophytic fungi and/or ergot infestation of teff and other grasses fed to the cows were then suspected of being involved, but neither endophytes nor ergot alkaloids could be implicated from these sources. by measuring the serum prolactin levels of groups of sheep (n = 5) fed the first farm’s total mixed ration (tmr) or its three individual fibre components for a period of 11 days, the source of the ergot alkaloids was identified. a statistically significant decrease in the level of this hormone occurred only in the group on maize silage (which constituted 28 % on dry matter base of the tmr). the involvement of the maize silage was further chemically confirmed by the high levels of total ergot alkaloids, predominantly ergocryptine, found by lc-ms in the silage as well as in the tmr (115–975 ppb and 65–300 ppb, respectively). the ergot alkaloid content (mainly ergocryptine) of the maize silage on the second affected farm was 875 ppb. withdrawal of contaminated silage resulted in gradual recovery of stock on both farms. nut sedge (cyperus esculentus and cyperus rotundus of the family cyperaceae) has a world-wide distribution and is a common weed in annual crops, and can be parasitized by claviceps cyperi. careful examination of the maize silage from both farms revealed that it was heavily contaminated 1 division of toxicology, arc-onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa present address: section of toxicology, department of paraclinical sciences, faculty for veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: theuns.naude@up.ac.za 2 section of toxicology, department of paraclinical sciences, faculty for veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 3 division of pathology, arc-onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 4 mycology unit, division of biosystematics, arc-plant protection research institute, queenswood, pretoria, 0121 south africa 5 arc-animal improvement institute, private bag x2, irene, pretoria, 0062 south africa 6 veterinary medical diagnostic laboratory, university of missouri, columbia, mo 65211, usa 7 private practitioner, balfour, mpumalanga, south africa 8 farmer, greylingstad, mpumalanga, south africa accepted for publication 9 june 2004—editor introduction ergot alkaloid chemistry and pathophysiology chemically two types of ergot alkaloids are recognised (garner, rottinghaus, cornell & testereci 1993) both basically consisting of d-lysergic acid with an amide binding at c8: ergoclavines (simple lysergic acid amides, e.g. ergonovine) and ergopeptines (with three amino acids in a cyclol ring structure, e.g. ergotamine [fig. 1]). all these alkaloids may induce problems in animal production and are produced by fungi of the clavicipitaceae infecting members of the poaceae (cereals and grasses). the claviceps tribe, of which claviceps purpurea is a prime example, infects the florets of various cereals and grasses producing overwintering sclerotia (ergots) in the maturing stages of the plants. these contain highly toxic levels of especially ergopeptine alkaloids. the balansiae tribe, on the other hand, are endophytic (intercellular) fungi rendering the grass itself toxic due to all of these alkaloids. of these neotyphodium (= acremonium) coenophialum (morganjones & gams) glenn, c.w. bacon & hanlin, infecting tall fescue grass (festuca elatior l.) and producing primarily ergovaline, is the most important example of veterinary significance and the cause of the “fescue foot” syndrome (bacon, lyons, porter & robbins 1986; cheek, 1998). the mechanisms of action of ergot alkaloids are varied and complex—some are completely unrelated, and some are even mutually antagonistic. they appear to result from their action as partial agonists or antagonists at α-adrenergic, dopaminergic and tryptaminergic receptors. the ergopeptine alkaloids cause prolonged constriction of both arteries and veins peripherally by α-adrenergic blockade which is more pronounced in post capillary than precapillary vessels. in addition, it can also damage the capillary endothelium with consequent development of gangrene (hoffman & lefkovitz 1996; peroutka 1996). the dopamine agonist action directly activates lactotrope d2-dopamine receptors leading to inhibition of spontaneous and trh-induced release 24 claviceps cyperi, a new cause of severe ergotism in dairy cattle on the highveld of south africa with nut sedge and that it contained minute sclerotia, identified as those of claviceps cyperi, originating from the latter. nut sedge was abundant on both farms and it is believed that late seasonal rain had resulted in mature, heavily ergotised nut sedge being cut with the silage. claviceps cyperi sclerotia, collected on the affected fields in the following autumn contained 3 600–4 000 ppm ergocryptine. that the dominant alkaloid produced by this particular fungus was indeed ergocryptine, was confirmed by negative ion chemical ionization ms/ms. in one further outbreak in another holstein herd, teff hay contaminated with ergotised nut sedge and containing 1 200 ppb alkaloids, was incriminated as the cause of the condition. this is the first report of bovine ergotism not associated with the poaceae infected with claviceps purpureum or endophytes but with the family cyperaceae and this particular fungal phytopathogen. keywords: claviceps cyperi, cyperus esculentus, dairy cattle, ergotism � � � �� � � �� � �� �� �������� � �� � � �� � � � � � �������� �� � � �� �� � � � � � ��� � � � � �������� ���� � ���� ����� � ��� ����� ��� �� � ��� � � �� � � fig. 1 the structures of (a) d-lysergic acid (b) ergonovine, a typical amide of the latter and (c) ergotamine and ergocryptine, typical cyclol ergopeptine alkaloids of prolactin (ascoli & segaloff 1996) with consequently profound effects on lactation and reproduction. in “summer syndrome” of cattle hyperpyrexia (due to cutaneous vasoconstriction and thus inability of animals to dissipate heat) and a precipitous drop in milk production occurs particularly in high producing dairy cows. ergotism in south africa this has only once been reported in the winter rainfall area of south africa (schneider, miles, garthwaite,van halderen, wessels & lategan 1996) and resulted from claviceps purpurea sclerotia originating from annual rye grass (lolium hybrids) growing as weeds in barley lands. the ergots were concentrated in barley screenings and were inadvertently incorporated in a commercial dairy concentrate eventually affecting some 3 000 cows. the signs were typically those of summer syndrome and calves that had been exposed to high doses of the infected material developed the gangrenous form of ergotism. the latter form of ergotism has also recently been diagnosed in south africa (botha, naudé, moroe & rottinghaus 2004). highveld ergotism the central plateau grass veld of south africa occurring at an altitude of ca 1 000 m above sea level and having a relatively high annual rainfall, is known as the highveld and is the prime maize and dairy area of the country. it is also relatively free from the more serious tick-borne diseases and plant poisonings of cattle. maize silage is regarded as a safe feed and is extensively produced and utilized in both dairy and feedlot farming. apart from the transient and temporary deleterious effects of mouldy maize silage (mainly related to unpalatability) no other frank toxicity has been recorded in south africa. ergotism in any form had never been reported from this area. during mid-summer 1996/97 three outbreaks of typical summer syndrome in dairy cows were clinically diagnosed and confirmed chemically in the area and the sequence of events is described as they chronologically occurred. the first was at greylingstad (28°52” e; 26°47” s) and will be designated as the first outbreak and was investigated on 20 december 1996 (day 0 of this outbreak). the next was near memel (27°29” s; 29°47” e), designated as the second outbreak, which was investigated 8 weeks after the first outbreak. the third was near vrede (29°06” e; 27°33”), designated as the third outbreak, and was investigated 3 months after the initial outbreak. all the farms are situated within a radius of ca 70 km of each other with the first ca 140 km from the arconderstepoort veterinary institute (ovi) from where the investigation was instigated and co-ordinated by the diagnostic unit of the division of toxicology. materials and methods these varied from farm to farm. the detailed techniques undertaken during the first outbreak are described, whereas those that were used during the other two outbreaks were merely confirmatory and basically only of a chemical nature unless otherwise stated. clinical pathology venous blood was collected in heparinized and non-heparinized tubes under vacuum from ten clinically affected cows in all three outbreaks for routine chemical pathological analyses. these included the haematocrit, haemoglobin concentration, serum γgt-, astand ck-activities, and urea, creatinine, sodium, potassium, calcium, phosphate and copper values. mycology and endophyte examination the first phase of this investigation consisted of the examination of pasture grasses for possible endophytes; this was done only during the first outbreak. it was undertaken at the ovi and the stellenbosch regional veterinary laboratory on fresh eragrostis tef and eragrostis curvula as well as the grasses of other species in the holding camps. for this purpose sections of the plants were stained for subsequent light microscopy using the rose-bengal staining technique of saha, jackson & johnson-cicalese (1988). material was also despatched to the toxinology and food safety research group of the ruakura agricultural research station, hamilton, new zealand where it was examined by elisa for the presence of toxigenic endophytes and related ergot alkaloids by the technique of garthwaite, sprosen, briggs, collin & towers (1994). the second phase consisted of mycologically confirming the identity of the causative organism on nut sedge (vide infra). 25 t.w. naudé et al. necropsy and histopathology no mortalities occurred and only one examination was undertaken on one badly affected holstein cow in the first outbreak that had markedly lost condition. the animal was slaughtered at an abattoir on day 0 and specimens of all major organs were collected in buffered 10 % formalin, processed according to standard procedures and stained with haematoxylin and eosin for histopathological examination. chemical analyses analysis for gossypol was done at the division of toxicology of the ovi according to the method of pons & hoffpauiar (1957). analyses for ergot alkaloids were initially performed at meadows cape in paarl, western cape province, south africa where extractions were done according to the technique of scott, lombaert, pellaers, bachler & lappi (1992) and the high performance liquid chromatography (hplc) by the method of rottinghaus, schultz, ross & hill (1993). a waters 600 hplc system with a 470 fluorescence detector and a 717 auto sampler and a phenomex luna c18 column (0.15 x 4.6 m) were used. subsequently, milled feed or basic chloroform extracts of it were sent by courier to the veterinary medical diagnostic laboratory, university of missouri, columbia, usa for ergopeptine analysis by the hplc technique described by rottinghaus et al. (1993). samples were extracted with alkaline chloroform, filtered and applied to ergosil cleanup columns (analtech, newark, de). following elution of pigments with acetone:chloroform (8:2), the ergopeptine alkaloids were eluted with methanol and analysed by hplc with fluorescence detection. confirmation of ergopeptine alkaloids was obtained by treatment of the sample with 0.2 % acetic acid and re-examination by hplc for the -inine isomers. the hplc system consisted of a perkin elmer lc 250 pump, a perkin elmer iss200 autosampler, with detection on a hitachi f-1200 fluorescence detector. a phenomenex luna c18 column (0.15 x 4.6 m) was used with a mobile phase of acetonitrile: water (35:65) with 200 mg ammonium carbonate per liter. the identity of the α-ergocryptine was unequivocally confirmed by further silica gel/organic binder cleanup and tandem mass spectroscopy (finnigan/mat tsq 70 tandem mass spectrometer [ms/ms]) at the us department of agriculture, national veterinary services laboratories, ames, iowa, usa, according to the technique of rottinghaus et al. (1993). briefly, chloroform extracts were processed through silica gel/organic binder cleanup columns described above. a portion of the extract was applied to a direct exposure probe, the solvent was allowed to evaporate and the probe was inserted into the mass spectrometer source. the ms/ms was operated in the negative chemical ionization mode with methane as the reagent gas and argon as the collision gas. daughter ions were collected for the ergocryptine parent ion m/z 308. typical daughter spectra were obtained with base peak m/z 209 as described by plattner, yates & porter (1983) and rottinghaus et al. (1993). prolactin (prl) determination this was only done during the first outbreak. it consisted of a trial using sheep in an attempt to determine the source of the ergot. five groups (n = 5) of randomly selected, ca 1-year-old mutton merino ewes were given an exclusive diet of the suspect feed components of the milk cows for 11 days. the groups received the total mixed ration (tmr), teff hay (eragrostis tef), eragrostis curvula hay, a mixture of grasses growing in the holding pens of the cows or the maize silage used on the farm. all rations were fed ad lib and the animals had free access to drinking water. blood was collected by jugular venipuncture on days 0, 4, 7 and 11 and the serum separated by centrifugation at 4 °c and stored at –20 °c for subsequent examination. blood was similarly collected from the median caudal (coxygeal) vein, ca 4 h after milking from 20 cows with the most pronounced clinical signs of hyperthermia on day 1 of this outbreak (21/12/1996) and again on day 29 (18/1/1997) from 19 animals with the most dramatic drop in milk production. the serum was stored at –20 °c until analyzed. to compare the prl levels to those of unexposed animals, a set of determinations was also done on the serum of a group of normal holstein cows (n = 8) from the dairy herd of the arc-animal nutrition and animal products institute, irene. the animals were fed a similar diet and were at approximately the same stage of lactation. prolactin concentrations were determined by a double antibody radio-immunoassay (hunter & greenwood 1964) as optimized by van zyl (1988). the antigen and antiserum were supplied by the national institute of arthritis, diabetes and digestive and kidney diseases (niaddk), maryland, usa. 26 claviceps cyperi, a new cause of severe ergotism in dairy cattle on the highveld of south africa the radioligand assay data were calculated with a computer programme based on the (logit-log) theory of rodbard & frazier (1975). the correlation coefficient of the standard curve was always greater than –0.99. the results were statistically analysed by multiple regression analysis with the aid of statistica for windows, release 5.1 (f) of 1997. history and results of investigation of the three outbreaks first outbreak this occurred on a long established, well-managed dairy farm near greylingstad with a 240 holstein cow herd (210 in milk and 500 head in toto) in mid summer 1996. typical summer syndrome, viz. hyperthermia, open-mouthed breathing (fig. 2), shade seeking (even on overcast days), lying down in damp, muddy areas, wading into a dam up to the neck, even at night (fig. 3) and a 33 % drop in milk production had been occurring since the beginning of december. these signs were only evident in cows in milk of which the majority were involved. an increasing incidence of infertility became apparent. it subsequently transpired that the problem had followed the opening and use of a new bunker of maize silage. milk cows were on a total mixed ration (tmr) and had access to small holding camps with limited natural grasses mainly kikuyu (pennisetum clandestinum) and cynodon dactylon and smaller numbers of eleusine indica, eragrostis plana and bromus catharticus. the tmr consisted of the following: 20 kg (dry matter base) maize silage (of the previous season) 1.5 kg teff or eragrostis curvula hay 4.5 kg soda maize (naoh-treated maize) 3 kg lucerne hay 1.5 kg hominy chop (maize bran and germ) 2.0 kg cotton seed 2.5 kg molasses 3.8 kg high protein concentrate (hpc) 51 % soyabean oil cake 25 % cotton oil cake fish meal, gluten, molasses urea, salt and minerals. the ration consisted of 52 % maize silage as fed and this, the hay and soda maize originated from the farm. the other components and hpc were obtained from a local commercial feed company. a tentative diagnosis of ergotism was made as there was no history of any other causative agents of hyperthermia such as haloginated salicylanilides or pentachlorophenol which uncouples oxidative phosphorilation (osweiler, carson, buck & van gelder 1985) being used. the gossypol content of the ration was considered but the most probable cause was thought to be ergotism and the source first suspected was the brought-in components 27 t.w. naudé et al. fig. 2 open-mouthed breathing as seen in the “summer syndrome” fig. 3 cows wading into a dam to cool off such as the hpc. the possibility of ergotised or endophyte-infected grass also had to be considered. results chemical pathological blood analyses these revealed no specific changes indicating any specific cause of the syndrome. examination for ergotised or endophyte infected grass the only sclerotia found on members of the poaceae on this farm were on natural paspalum (paspalum dilatatum) grass growing in particularly the moister, shaded areas of the natural grazing. as teff and e. curvula were specifically cultivated for and formed a significant ingredient of the cows’ ration, these two grass species (despite the fact that they had never previously been implicated in endophytic ergotism) had to be considered. endophytic hyphae were indeed present in these two grass species examined according to the technique of saha et al. (1988) and our findings were confirmed by the regional veterinary labotatory at stellenbosch (a. van halderen & j. wessels, personal communication 1997). the ruakura agricultural research station, however, found the teff and eragrostis curvula seed negative for neotyphodium-like endophyte mycelium by elisa and microscopy (garthwaite 1997), and excluded lysergol-containing endophytic ergot alkaloids and paxilline (a precursor of the toxic lolitrems, a further indicator of toxic endophytes) by elisa (garthwaite et al. 1994) from all seed and vegetative material of teff and e. curvula submitted to them (i. garthwaite & c.c. miles, agresearch, ruakura, new zealand, personal communication 1997). the holding camps’ grasses, p. clandestinum, c. dactylon, e. indica, e. plana and b. catharticus were also examined locally for endophytes but with negative results. gossypol analysis results levels in the feed (0.02–0.08 %) and in the cotton seed (0.8 % free gossypol) were within normal limits. ergot alkaloid analyses the maize, hpc, and all brought-in components of the ration were tested by both meadows, cape and the university of misssouri with negative results. all except one of the grass specimens, including teff and e. curvula seed, were consistently devoid of any ergot alkaloids at both laboratories, the one exception being, at that stage inexplicable, a positive result, viz. a specimen of unsifted teff seed which meadows, cape found to have a strong peak consistent with ergocryptine. this was later confirmed by the university of misssouri and quantified by hplc to contain a total of 580 ppb of ergocryptine. analyses of two batches of the tmr and dried silage sent in december 1996 and on 7/1/1997 to paarl were negative for these alkaloids. on day 55 (13 february1998) some 8 weeks after the condition was diagnosed clinically, no positive chemical results were yet available. however, the prolactin results, indicating that the silage was indeed the cause were available (vide infra) and this, in conjunction with the second outbreak (vide infra) in which the owner had already withdrawn the batch of silage that had twice been associated with the syndrome, lead to withdrawal of the silage from the cows’ ration at this first outbreak. the first positive analytical hplc results were received on day 59 and these, together with subsequently data received from the university of missouri on four batches of tmr and/or silage, are reflected in table 1. an example of a typical chromatogram of the alkaloids found on extraction of the samples by hplc appears in fig. 4. removal of the incriminated silage from the ration resulted in the gradual abatement of the clinical signs of intoxication and return of milk production to pre-exposure levels as reflected in fig. 5. after the 2 months’ exposure a definite reddish tinge to the hair of the black patches of the holsteins was evident in a number of animals. severe problems with infertility were still experienced for some months more and a number of animals had to be culled for this reason. results of prolactin determinations the results of these tests only became available on day 52, 7 weeks after the initial clinical diagnosis of ergotism was made. in the sheep trial a statistically significant drop in prl level only occurred in the maize silage group (fig. 6). the prl data of the holstein cows are reflected in table 2. 28 claviceps cyperi, a new cause of severe ergotism in dairy cattle on the highveld of south africa 29 t.w. naudé et al. table 1 results of chemical analyses of different batches of maize silage and tmr date collected feed component ergot alkaloid level 7/1/1997 tmr 300 ppb ergot alkaloids: ergosine 10 ppb ergocornine 31 ppb ergocrystine 33 ppb ergotamine 34 ppb ergocryptine 200 ppb maize silage ergocryptine 530 ppb (other alkaloids negligible) 8/1/1997* tmr 30 ppb ergocryptine maize silage 50 ppb ergocryptine 15/1/1997 maize silage negative 21/1/1997 tmr 65 ppm ergocryptine maize silage 235 ppb ergocryptine * chloroform extract tmr = total mixed ration � � � �� � � � � � �� � �� � � � � �� � �� � � � � � � ��� � � � �� � �� � �� � � � � ����� ! " # $� $% � � �� � �� � �� � � � � ����� ! " # $� $% fig. 4 a typical chromatograms of a claviceps cyperi sclerotial extract containing the different ergopeptine alkaloids (left) in relation to that of a standard mixture (right). note the exceptionally high ergocryptine peak table 2 decrease in milk yield and serum prolactin concentration of affected and normal holstein cows in the first outbreak group date average % drop in milk average serum prolactin yield (range) in ng/ml (range) most severely clinically affected cows (n = 20) day 1 23.55 (+10–72) 0.804 (0–1.28) cows with most severe drop in milk yield (n = 19) day 29 47.70 (33–61) 0.194 (0–0.717) control group* (n = 7) 3/2/1997 not applicable 2.096 (1.1–4.45) * the prl values of an eighth cow were exceptionally high, possibly due to stress, and were not considered determination of the causative fungus on day 59 the origin of the syndrome had been unequivocally determined as being the maize silage but no reason why it was positive was evident. various weed grass spp growing in the maize fields in the year subsequent to the outbreak (1997) were inspected but no sclerotia could be found on any. the only ergotisation of maize ever described is that of claviceps gigantea by fuentes, de lourdes de la isla, ullstrup & roderiquez in 1964. this fungus with its exceptionally large sclerotia on maize, had, however, never been seen in south africa (fucikovsky & moreno 1971). the breakthrough occurred on day 59 when one of the authors (cr) remembered seeing ergotised nut sedge in the national collection of fungi of south africa (prem) in pretoria. it became apparent that the nut sedges, cyperus esculentus and cyperus rotundus, which grow widely in maize fields in many countries, could be ergotised and that this had been described in south africa by loveless in 1967 (vide infra). examination of specimens from this herbarium revealed the typical appearance of the small sclerotia. subsequent careful scrutiny of the dried, sifted silage fed to the cows of the first outbreak (and which contained numerous yellow nut sedge seed heads), the typical sclerotia could be demonstrated (fig. 7). a few weeks later the yellow nut sedge weeds growing in the maize fields at all three sites of the outbreaks investigated, were starting to ripen and found to be infected with this particular fungus. that this was indeed the cause of the syndrome was unequivocally proved by chemical determination of the ergot alkaloid levels in extracts of claviceps cyperi sclerotia itself collected on sedges in the maize fields at the sites of the first two of the three outbreaks that had been investigated (table 3) botanical data on yellow nut sedge, cyperus esculentus var. esculentus l. although both yellow and red nut sedge are subject to ergotisation, only yellow nut sedge was encountered on the farms involved in the three outbreaks. yellow nut sedge, (fig. 8), is a fairly robust, stoliniferous, perennial herb growing up to 0.7 m high. the stolons are covered with scales and end in tubers. annual spikelets are brown or rust-coloured (versus reddish in c. rotundus) and obtuse. the glumes are 2.2–2.6 mm long with raised reddish-brown nerves almost to the margin and with an uncoloured marginal border. the plant flowers from november to april (retief & herman 1997). cyperus esculentus var. esculentus l. is widespread in africa south of the sahara, iran, yemen, australia, cambodia and thailand where it can cause serious problems in especially annual crops when it grows as a weed in cultivated fields. other varieties occur in the usa, canada, south america and europe where they can similarly cause problems (holm, plucknett, pancho & herberger 1977). mycological data on claviceps cyperi historical review claviceps nigricans was the first species of this genus described by tulasne (1853) on members of the cyperaceae, viz. on the genera eleocharis and scirpus. doidge (1950) listed undetermined claviceps spp. on both c. esculentus and c. rotundus. these had been collected at different localities in and around pretoria during the period 1940–1944. loveless, an expert on claviceps, studied this material that had been deposited in the national collection of fungi, pretoria and described a new species, claviceps cyperi in 1967. it was subsequently never reported again until the outbreaks of bovine ergotism described here occurred in 1996/7. life cycle claviceps cyperi on yellow nut sedge (cyperus esculentus) produces dark greyish-brown sclerotia which turn black when becoming older (fig. 9). these sclerotia are similar to but much smaller than those formed by claviceps purpurea on rye and other members of the poaceae (gramineae). most are 6–8 mm long by 1 mm wide but specimens of up to 12 mm in length have also been found. they are generally straight but the longer sclerotia are slightly curved (fig. 10) however, in addition, numerous 30 claviceps cyperi, a new cause of severe ergotism in dairy cattle on the highveld of south africa table 3 ergocryptine levels of sclerotia collected in maize lands in 1997(note that levels are, in contrast to all the other chemical determinations mentioned, given in ppm and not ppb ) ergocryptine origin of claviceps cyperi sclerotia levels (ppm) in sclerotia first outbreak (greylingstad) 3 600 second outbreak (memel) 4 000 recovered from silage in second outbreak 36.5 third outbreak (vrede) 4 200 31 t.w. naudé et al. � � � �� � �� � � �� � � & ��'(% )�� &��'(* �� & � $��+� �,�+� �,�+�����-��������.��/.�� �!.$�.(% 0" 0� 0! �# �% �" �� ! � " % # $! $� ! $ � 0 " , % * ��� �� ����� � �� �� � �� � � � � � � � �� � �� � � � � � �� �� ���� fig. 5 change in average milk yield in relation to feeding and withdrawal of incriminated maize silage in the first outbreak. the date of the first visit to the farm is indicated by the arrow fig. 6 drop in serum prolactin concentration (mean " sd) in five sheep ewes fed the suspect maize silage in the first outbreak. the regression curve for the dependant variable prl is described by y = 3.870606 – 0.378650 x days, f(1,18) = 4.7684 (p < 0.04247), i.e. there is a 95.8 % certainty that the slope of y is not equal to zero fig. 7 the small, black, rod-like claviceps cyperi sclerotia in dried maize silage (compare with fig. 10) fig. 8 cyperus esculentus, yellow nut sedge fig. 9 a flower head of cyperus esculentus showing a large, curved sclerotium of claviceps cyperi fig. 10 variation in size and shape of claviceps cyperi sclerotia (left) in comparison with rice grains (centre) and tagetes minuta, khaki bush, seeds (right) small, immature microsclerotia (3–5 mm long) may also be present. sclerotia are formed as the overwintering phase of the fungus when the nut sedge seed ripens and are able to survive extremely low temperatures. the extent of infection may vary from year to year, depending on temperature and rainfall. sclerotia (which represent the sexual phase of the fungus) drop onto the ground where they have a resting or overwintering period. in the subsequent growth season, when conditions are favourable, they germinate to form stromata containing spores (ascospores) which are disseminated by rain, wind and insects, and infect young newly formed florets of the host plant. the spores then germinate and form a mycelial mass which, on its turn, gives rise to asexual conidia. this constitutes the start of the asexual or sphacelial stage of the fungus during which a sticky, sugary substance (“honeydew”), containing numerous conidia, is formed and continues to form during most of the summer. the “honey dew” is attractive to insects which then transmit the conidia to other florets infecting them. this generally coincides with the production of pollen by the nut sedge. the mycelial mass eventually forms the “sphacelium” which replaces the ovule of the floret and a sclerotium is formed. no seed is produced by the infected floret. another fungus (cladosporum sp.) grows profusely on the “honey dew”, covering parts of the inflorescence with its mycelium and spores, giving it a black, sooty appearance and is a very obvious characteristic of infection. “honeydew” is also well-known on sorghum (sorghum bicolor subsp. arundinaceum) and paspalum where it is respectively formed by claviceps sorghi and claviceps paspali. morphology the morphology of the sexual phase of the life cycle has been fully described by loveless (1967). when a sclerotium germinates, a stroma consisting of a stipe (stem) with capitulum (head) is formed. the capitulum is at the upper end of the stipe, very similar to a mushroom. this structure is, however, very small and inconspicuous. the stipe may be up to 6 mm long and the capitulum only 1 mm in diameter. when sectioned, the different perithecia are clearly seen embedded within the capitulum. inside the perithecia are the spore-carrying sacs called the asci. these asci contain needle-shaped ascospores. the ascospores have several septa at which they may separate with each fragment having the ability to inflect a floret of the nut sedge host plant. the conidia are small and narrowly elliptic. the asexual stage resulting in conidia has now also been cultured in vitro and fully described by van der linde (2003). the presently known distribution of c. cyperi appears in fig. 11. despite the world-wide distribution of cyperus esculentus and c. rotundus their infection with claviceps cyperi has not been recorded outside south africa. it is a characteristic of claviceps spp. that although the whole infected host plant may contain toxin, its sclerotia are the particularly dangerously toxic components of the fungus as they contain the highest concentration of toxins (in this particular case ergocryptine). exposure of animals to the sclerotial (overwintering) phase must, therefore, be avoided. discussion it took almost 2 months from the first visit and the tentative diagnosis of ergotism on this farm to confirm the diagnosis and to determine the origin of the intoxication. this was only made possible by a concerted team effort by veterinarians, mycologists, animal scientists, chemists and the farmer. that there was a certain degree of luck involved in this relatively rapid confirmation of the diagnosis is clear from the analytical results reflected in table 1. to make the silage green maize plants as well as any weeds present, were mechanically cut about 80 mm above ground level. the density of the weed infestation, including nut sedge, on the land varied considerably from area to area. loads of freshly-cut material were uniformly spread over the entire length of a particular bunker in consecutive layers. consequently, maize originating from different areas of the land contaminated with different degrees of weeds and ergotised nut sedge was, therefore, present and haphazardly distributed in the final ensiled product. the ergot alkaloid concentration, therefore, would be expected to vary considerably in the bunker. this explains the fact that silage varied in its toxicity or lack of it (table 1). it was indeed fortunate that the silage fed to the sheep in one of the groups in the prolactin determination experiment contained enough alkaloid to lower their serum prolactin levels. this was of cardinal significance in the determination of the origin of the ergot intoxication. the negative response of the group receiving the silage-containing tmr is probably due to the dilution effect of the other constituents. 32 claviceps cyperi, a new cause of severe ergotism in dairy cattle on the highveld of south africa the apparent illogical finding that an unsifted teff seed sample was positive for ergocryptine could have been a serious “red herring” in the determination of the diagnosis at that stage of the investigation. this finding is, however, consistent with the fact that teff is an annual and, therefore, lands on which it is grown are as prone as maize lands to nut sedge infestation and thus to ergot contamination. the third outbreak investigated (vide infra) was undoubtedly due to nut sedge contaminated teff hay. the rather slow recovery and return of milk yield and degree of infertility experienced in the cows of this particular herd (fig. 3) can probably be ascribed to the prolonged exposure of more than 2 months to the alkaloids. second outbreak this outbreak of claviceps cyperi ergotism occurred on a well-established and managed dairy farm near memel which was visited on 11 february 1997. there were 150 holstein cows in production and 317 animals in toto. milk production was 10 % above the country’s average. typical summer syndrome with 70 % of cows being affected, occurred. they manifested hyper pyrexia (once even breaking through an electric fence to get to a small stream), dyspnoea and polypnoea (which prompted testing and treatment for infectious bovine rhinotraecheitis and inoculation for bovine viral diarrhoea). a concomitant drop in milk production, was also experienced on two occasions, the first in august 1996 and the second in january 1997. the first had occurred when maize silage from a specific bunker made the previous year was fed to them. clinical signs abated on cessation of its use. the second commenced when this silage was again used as feed, milk production decreasing from 2 750 to 1 969 l/day (32 %) over 30 days. as had occurred in the first outbreak, seasonal late rains had resulted in making the lands inaccessible for early cutting of maize for ensiling. the farmer and his attending veterinarian had recognised the association of the syndrome with the silage and the farmer had ceased feeding it just prior to our visit. 33 t.w. naudé et al. fig. 11 the distribution of claviceps cyperi in relation to the main maize producing area of south africa during the outbreak the cows received a tmr consisting of the following: 5 kg silage (dry matter base, wet 15 kg) 2 kg cotton seed 4 kg dry lucerne 2–3 kg e. curvula hay 11 kg of a commercial dairy meal. on withdrawal of the silage the clinical signs had abated. milk production had increased dramatically after 1 month and had returned to almost normal after 2 months. two cows had, however, suffered from necrosis and eventual loss of the tips of their tails. more than normal problems with infertility in general were still being experienced. the hair coats of many animals had a definite reddish tinge. specimens as in the first outbreak ten cows were bled for routine clinical haematological examination and specimens of the dairy meal and lucerne (brought-in feeds) and the locally produced hay and silage, were collected for chemical analysis. extracts of the latter specimens were forwarded to the university of missouri for examination for ergot alkaloids. results clinical pathology as experienced in the first outbreak the haematological examinations revealed no remarkable deviations from normal. chemical analyses the analyses for ergot alkaloids performed on extracts of the maize silage, e. curvula hay, dairy concentrate, cotton seed and lucerne forwarded to the university of missouri, revealed that only the maize silage contained the alkaloids as is reflected in table 4. discussion a diagnosis of ergotism was evident and the syndrome was clearly associated with the feeding of the silage made in 1996. what was extraordinary in this case was that in the analyses for ergot alkaloids, apart from ergocryptine in the silage, an hplc peak corresponding to ergocornine was evident, a finding that did not occur at the first (vide supra) and third outbreaks (vide infra). on a subsequent visit, 1 month later, ergotised nut sedge was found to be abundant in the maize lands and actual sclerotia in the silage could be demonstrated with ease to the farmer by taking a handful of silage from the face of the bunker and spreading it on a white background. this was the only farm where necrosis of the tail tips occurred in some of the cows. the relatively rapid return of the cows to pre-exposure status can probably be ascribed to the short exposure period to the toxic silage. third outbreak this occurred from january to march 1997 in a mixed herd of milk cows comprising 120 holsteins and ten jerseys. signs of heat stress were observed in the holsteins (but not the jerseys) which consisted of dyspnoea, polypnoea and salivation which were particularly evident after minor exercise. attempts to cool off by lying in muddy pools and even in the drinking trough were also manifested. a drop in milk production of 14 % in january, 26 % in february and 18 % in march was also experienced. a reddish tinge to the black hair of some of the holsteins was evident. the problem had followed a change in ration in late november 1996 when the ration was made up as follows: 550 kg of self mixed concentrate 150 kg e. curvula hay 100 kg teff hay 200 kg dried maize silage. when silage was no longer available it was replaced in the ration by: 50 kg gluten 50 kg molasses. the cows also had access to e. curvula and digitaria eriantha pastures. in december 1996 the ration was changed to: 550 kg of the concentrate 250 kg of teff hay. 34 claviceps cyperi, a new cause of severe ergotism in dairy cattle on the highveld of south africa table 4 ergot alkaloid level of maize silage in the second outbreak sample ergot alkaloid analysis maize silage 680 ppb ergocryptine 295 ppb ergocornine in addition to the two grass pastures, the cows also had access to feed sorghum grazing. the basic change was that the teff concentration in the ration had changed from 10–31 % and within 3 weeks the clinical sign of summer syndrome had set in. the farmer had suspected the teff and on its withdrawal the clinical signs gradually abated and milk production returned to normal. blood was collected for clinical pathological examination from ten of the most severely affected cows. extracts of feed specimens, as reflected in table 5, were submitted to the universitry of missouri for ergot alkaloid determination. examination of the large round bales of teff hay which was incriminated, revealed that it was severely contaminated with claviceps cyperi-infected cyperus esculentus. numerous sclerotia were present. the maize fields and feed sorghum and teff lands were all infested with heavily ergotised nut sedge. the land on which e. curvula was growing appeared to be free of it. results of investigations clinical pathology as in the other two outbreaks this revealed no obvious deviations from the normal. chemical analyses extracts of milled teff hay, e. curvula hay, dried feed sorghum hay and dairy concentrate revealed that only the teff and e. curvula hay contained appreciable quantities of ergot alkaloids as summarised in table 5. discussion just like maize, teff is an annual crop and, consequently, prone to nut-sedge weed infestation. if such nut sedge is, in addition, ergotised and the teff is cut when the nut sedge is seeding and the c. cyperi forming sclerotia for overwintering, it may present an ergot alkaloid concentration which may be problematic in stock. in this particular instance it is clear that this feed component was responsible for the outbreak. on inspection this particular farm also had severely ergotised nut sedge infestation in all annual crops. even the e. curvula, which is a perennial grass and thus not prone to infestation after the first year and which appeared to be free of the weed, contained ergocryptine at a level of 80 ppb. general discussion although c. cyperi was first described by loveless as long ago as 1967 (loveless 1967) it was not known to be toxic until the investigation described here. nut sedges, particularly c. esculentus and in south africa to a lesser extent c. rotundus (c. archer, national botanical institute, pretoria, personal communication 2001), are troublesome weeds which occur in cultivated lands on which annual crops are grown and any feed contaminated with them, when ergotised, are potentially toxic. both species of nut sedge have a world-wide distribution but to date infection with c. cyperi has only been described in south africa. the infection is not very conspicuous as the sclerotia are very small and, consequently, the infection might have been missed elsewhere. an important indicator of possible ergot infection is a black, sooty cladosporum infection of ripening nut sedge seed heads which precedes the appearance of the claviceps sclerotia. a novel finding of these outbreaks of ergotism was that α-ergocryptine (fig. 1), chemically confirmed by ms/ms, was the dominant alkaloid produced by c. cyperi. this alkaloid is usually a minor component of the alkaloids produced by claviceps purpureum and neotyphodium coenophialum. in the second outbreak a strong peak corresponding to ergocornine was detected in the silage but unfortunately the identity was not confirmed by ms/ms. the fact that several specimens of silage from the first outbreak submitted for chemical analysis were negative for ergopeptine alkaloids can be ascribed to various factors such as an uneven distribution of ergotised c. esculentus on the maize lands with a consequent uneven distribution in the silage bunker. furthermore, silage was dried on cement floors before despatch for analysis or extraction of ergopeptine alkaloid and at the outset particular care to pick up the fine material (possibly containing more sclerotia than the rest) and including this in the final specimen, was not taken. this might have resulted in false negative or low alkaloid levels. the finding 35 t.w. naudé et al. table 5 ergopeptine levels of feed in the third outbreak sample ergot alkaloid analysis milled teff hay 1 200 ppb ergocryptine eragrostis curvula hay 80 ppb ergocryptine that sclerotia recovered from silage where the alkaloids had been leaching out of them for months, still contained 36 500 ppb ergocryptine, supports this assumption (table 3). the sheep prolactin test, where the animals were fed silage as it came from the bunker for 11 days only, showed up the silage as the culprit and was an important indicator of the source. in retrospect, the success of the experiment was probably rather fortunate. the causative fungus has undoubtedly been present in south africa for many years. maize silage and teff have always constituted significant components of stock feed and it can only be speculated whether the syndrome in cattle fed these feedstuffs has or has not previously occurred. if it has, then a specific aetiological diagnosis was not made and reported, nor was its significance realized. the exceptionally high intake of infected silage by the cows during the first outbreak, the very high concentration of alkaloids in the silage in the second outbreak and in the teff at the third outbreak, certainly contributed to the manifestation of the syndrome. efforts should be directed at minimising ergot contamination of stock feed as the pathological effects of these alkaloids are, as with all poisons, dose dependant. in the years subsequent to the investigation reported here, the problem has been recognised by veterinarians at an early stage, i.e. as soon as milk production decreases and early clinical signs of summer syndrome appear. on withdrawal of the contaminated feed from the animals the signs abate. there is, however, reason for concern as it is possible that exposure of stock to lower levels of ergot alkaloids does not manifest as typical summer syndrome and might result in undetected lowered milk production, fertility and production. deep cultivation of lands (versus the minimum tillage concept currently in use) will result in burying ripe sclerotia and will therefore reduce infection of the florets of nut sedge the following season. maize growing on patchy areas in lands where heavy infestation of ripe nut sedge seeds occur, should not be used for silage production. hand weeding such areas before the maize is cut can be considered. another preventative strategy is to cut the maize plants at a higher level than the nut sedge heads—this has been used with success. the herbicide halosulfuron (“servien”, novartis) is expensive but is registered for post emergent control of cyperus spp. in maize lands (grobler et al. 2000). it impedes the development of floret and seed formation of nut sedge (and, consequently, ergot production) and can, therefore, be used selectively prior to the cutting of maize plants for ensiling to ensure that ripe, seeding nut sedge with its concomitant ergot contamination is not included in the final product. halosulfuron is not registered for use in teff lands but in commercial enterprises in which seed of e. tef is produced and it is most difficult to separate from that of teff. it is used with success in conjunction with 2.4-d to control annual weeds. however, when teff hay is produced for commercial purposes, use of such weedicide adds ca r 50/ton to the cost of production and the disadvantage of the presence of c. cyperi ergots in the last cutting of hay (when ergot formation is maximal) must be weighed up against the expense. teff is used particularly for the feeding of equids and in the case of breeding mares (which are particularly susceptible to ergot poisoning [cheek 1998]) this extra expense will certainly be justified. contaminated teff should only be fed to stock after careful evaluation of the risk/benefit ratio. acknowledgements assistance from the following instances and individuals is greatly appreciated: • the toxinology & food safety research group, ruakura research centre, hamilton, new zealand for elisa exclusion of endophytes and related toxins from the hay grasses • frank ross of the united states department of animal and plant health inspection service for the tandem mass (ms-ms) confirmation of ergocryptine • meadows cape, and in particular mr y. geyer, for the initial exclusion of the ergot alkaloids in the hpc of the first outbreak and for finding the unsifted teff seed specimen from this farm positive for 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(agric.) thesis, university of the orange free state. 37 t.w. naudé et al. abstract introduction research methods and design results and discussion acknowledgements references about the author(s) tülin g. gökmen department of microbiology, ceyhan veterinary faculty, cukurova university, adana, turkey hatice yazgan department of food hygiene and technology, ceyhan veterinary faculty, cukurova university, adana, turkey yıldız özdemir microbiology laboratory, adana city hospital, adana, turkey sedat sevin department of pharmacology and toxicology, veterinary faculty, ankara university, ankara, turkey nevin turut microbiology laboratory, veterinary control institute, adana, turkey şifa karahan microbiology laboratory, veterinary control institute, adana, turkey funda eşki department of obstetrics and gynecology, veterinary faculty, cukurova university, adana, turkey i̇brahim kıvrak department of chemistry and chemical processing technologies/cosmetic technology program, muğla vocational school, muğla sıtkı koçman university, muğla, turkey osman sezer parasitology laboratory, veterinary control institute, adana, turkey armağan e. ütük department of parasitology, ceyhan veterinary faculty, cukurova university, adana, turkey citation gökmen, t.g., yazgan, h., özdemir, y., sevin, s., turut, n., karahan, ş. et al., 2023, ‘chemical composition and antibacterial activity of bee venom against multi-drug resistant pathogens’, onderstepoort journal of veterinary research 90(1), a2097. https://doi.org/10.4102/ojvr.v90i1.2097 original research chemical composition and antibacterial activity of bee venom against multi-drug resistant pathogens tülin g. gökmen, hatice yazgan, yıldız özdemir, sedat sevin, nevin turut, şifa karahan, funda eşki, i̇brahim kıvrak, osman sezer, armağan e. ütük received: 21 dec. 2022; accepted: 01 june 2023; published: 20 july 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract bee venom with an antimicrobial effect is a powerful natural product. one of the most important areas where new antimicrobials are needed is in the prevention and control of multi-drug resistant pathogens. today, antibacterial products used to treat multi-drug resistant pathogen infections in hospitals and healthcare facilities are insufficient to prevent colonisation and spread, and new products are needed. the aim of the study is to investigate the antibacterial effect of the bee venom (bv), a natural substance, on the species of methicillin resistant staphylococcus aureus, vancomycin resistant enterococcus faecalis, carbapenem resistant escherichia coli, carbapenem resistant klebsiella pneumoniae and carbapenem resistant acinetobacter baumannii. as a result of this study, it was found that mic90 and mbc90 values ranged from 6.25 μg/ml – 12.5 μg/ml and numbers of bacteria decreased by 4–6 logs within 1–24 h for multi-drug resistant pathogens. in particular, vancomycin resistant enterococcus faecalis isolate decreased 6 log cfu/ml at 50 μg/ml and 100 μg/ml concentrations in the first hour. the effective bacterial inhibition rate of bee venom suggests that it could be a potential antibacterial agent for multi-drug resistant pathogens. contribution: the treatment options of antibiotic-resistant pathogens are a major problem in both veterinary and human medicine fields. we have detected a high antibacterial effect against these agents in this bee venom study, which is a natural product. apitherapy is a fashionable treatment method all over the world and is used in many areas of health. bee venom is also a product that can be used as a drug or disinfectant raw material and can fill the natural product gap that can be used against resistant bacteria. keywords: bee venom; multi-drug resistant pathogens; antibacterial activity; microdilution method; bioproduct. introduction multi-drug resistant (mdr) pathogens are an important global problem in the world. they seriously threaten public health in the basic areas such as food and health sector. generally, the environment in which mdr pathogens originates are hospitals and cause most health-associated infections (allegranzi & pittet 2007). the centers for disease control and prevention (cdc) estimate that pathogens have affected 1.7 million people in the united states (us) and have resulted in the death of 99 000 people (haque et al. 2018). although protection and control programmes are used against these infections, the effect is not sufficient. the increase of mdr pathogens complicates the control of infections, because of the irrational use of antibiotics, the decrease in the effectiveness of last option antibiotics in the treatment, and increased resistance to disinfectants (machowska & stålsby lundborg 2018). for this reason, natural products, that have not previously been in contact with mdr pathogens and have no potential for resistance development, can be an important antibacterial option. to date, many natural antibacterial products have been tested for mdr pathogens, and even various bee products have been tried (dinkoy 2017). however, there is a very limited number of studies on bee venom (bv); specifically on the most common and important species such as meticillin resistant staphylococcus aureus (mrsa), vancomycin resistant enterococcus faecalis (vre), carbapenem resistant escherichia coli, klebsiella pneumoniae, and acinetobacter baumannii. bee venom is an antibacterial mixture against gram-positive and negative bacteria and contains various peptides, amines, phospholipids, volatile compounds, aminocytes, sugars and enzymes (carpena et al. 2020). in particular, melittin, apamin and phospholipase c are the most important components. the fact that bv contains active ingredient that causes cell membrane pore formation and membrane phospholipid destruction makes it an important antibacterial bioproduct (funayama et al. 2012). in this study, the aim was to evaluate the antibacterial activity of bv; a natural product, as an option to be used for the control of mdr gram-positive and gram-negative pathogens, which are the biggest problem in terms of morbidity, mortality and economic aspects. to fulfil this, minimal inhibition concentration (mic) and minimal bactericidal concentration (mbc) as well as the chemical composition of honey bv were assessed. research methods and design collection of bee venom samples of bv were collected by the bv collector (beesas ar, turkey) from apis mellifera anatoliaca during the citrus honey season between may 2021 and june 2021. after collection, the bv was scraped with a scalpel on glass plates and stored under room conditions for 8 h and placed in the freezer at −18 °c (gökmen et al. 2023). determination of content and compositions of bee venom the amount of apamin, phospholipase-a2 and melittin components in bv was analysed by high-performance liquid chromatography (hplc) variable wavelength detector (vwd) (agilent 1260 series). infinitylab poroshell c18 ec-c18 (4.6 mm × 50 mm, 2.7 micron) column was used for separation. while the optimum separation temperature was 20 °c, the column flow rate was 1 ml/min. apamin (sigma-a1289), phospholipase a2 (sigma-p9279) and melittin (sigma m2272) standard solutions were prepared at 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml concentrations. the buffer a (0.1% trifluoroacetic acid [tfa] in water [h2o]) and the buffer b (0.1% tfa in acetonitril) were used as mobile phases. the gradian programme has been optimised for peak holding times. absorbance measurements were made at 218 nm (gokmen et al. 2023). bacterial strains in the current study, meticillin resistant s. aureus (mrsa), vancomycin resistant e. faecalis (vre), carbapenem resistant e. coli (crec), carbapenem resistant k. pneumoniae (crkp) and carbapenem resistant a. baumannii (crab) strains were used from the cukurova university, ceyhan veterinary faculty, department of microbiology (adana, turkey). multi-drug resistant pathogens were identified by vitek®2 identification system (biomerieux). detection of antibiotic resistant by disc diffusion method kirby-bauer disk diffusion method was used to determine the antibiotic sensitivity profiles of mdr pathogens (bauer et al. 1966). various discs applied in all species-specific routine antibiograms were used. antibiotic resistance profiles are shown in figure 1. figure 1: kirby-bauer disc diffusion test for multi-drug resistant pathogens. determination of minimal inhibition concentration and minimal bactericidal concentration value of bee venom bee venom mic and mbc were detected by using a microdilution method (clsi 2015). the concentrations of 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml were prepared and added to the wells in the microplates with the muller hinton broth (mhb) and bv. for the five mdr pathogens, in each well, bacterial density was set to 2 cfu/ml × 106 cfu/ml. wells containing mhb and bacterial suspension were used as positive control, only bv and mhb wells were used as negative control. the absorbance of the microplate, which was incubated at 37 °c during the night, was read at 560 nm and 620 nm. according to the absorbance values on microplate, the mic value was determined for each mdr pathogen. later, mbc values were determined by overcoming the samples in the microplate to the müller–hinton agar (mha). time-kill assays the time-kill assay of bv against all tested bacteria was evaluated at their mic according to the previous method with minor modifications (chuesian et al. 2019). different time intervals (0 h, 1 h, 3 h, 6 h, 12 h and 24 h) were applied in the assay. multi-drug resistant pathogens suspension at 0.5 mcfarland turbidity (1.5 cfu/ml × 108 cfu/ml) was inoculated in mediums containing mic value and the previous and next concentrations of the detected mic value. the bacteria-bv mixture was incubated for 0 h, 1 h, 3 h, 6 h, 12 h and 24 h at 37 °c. point one mililitre of each bacterial suspension was spread on mha agar plate at each time point and incubated at 37 °c for 24 h. after incubation bacterial colonies were counted for time-kill assay curve. ethical considerations the study was approved on 04 april 2022 by the ethics committee of adana veterinary control institute, adana, turkey (approval no: 04/04/2022-1/227). results and discussion bee venom consists of 88% water, and in the dry matter there are various peptides such as apamin, histamine, hyaluronidase, phospholipase a (pla) (wehbe et al. 2019). it is known that the composition of bv contains about 18 different bioactive substances (carpena et al. 2020). in this study, the main compounds detected in bv were melittin and pla at proportions of 70.49% and 13.51%, respectively, and the other compound was apamin at 3.85%. although bv has different ingredients, the high amount of some substances may be very meaningful in terms of antimicrobial activity. mellittin causes pore in the membrane at levels of 50% and 60% (sonmez et al. 2022; tanuğur-samancı & kekeçoğlu 2019). in addition, the proportion of pla, which ensures the destruction of phospholipids in the membrane structure, is between 10.00% and 20.95% in a qualified bv (tanuğur-samancı & kekeçoğlu 2019). higher concentrations of pla and melittin in the current study may be a good indicator for a high quality bv (figure 2, table 1). figure 2: time-killing assay graphics for multi-drug resistant pathogens (a) gram-positive multi-drug resistance bacteria, (b) gram-negative multi-drug resistance bacteria. table 1: the concentrations of components of bee venom (±s.d.). in this study, the authors aimed to determine the antibacterial activity of bv on the isolates that we identified as mdr by kirby bauer disc diffusion method. therefore, we used the microdilution method and determined mic and mbc values for mdr pathogens. microdilution method was applied to the microplates using 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml concentrations of bv. absorbances at 560 nm and 620 nm wavelengths were measured with an elisa reader. it was determined that the mic90 and mbc90 values of the pathogens varied between 6.25 μg/ml and 12.5 μg/ml (table 2). table 2: microdilution and time-killing assay at various concentrations for multi-drug resistant pathogens. in the current study, we evaluated two species mdr gram-positive bacteria, which are important problems in healthcare services. in a previous study, mic and mbc values of bv were determined as 0.085 μg/ml and 0.11 μg/ml, and 0.10 μg/ml and 0.14 μg/ml, respectively (han et al. 2016). kong et al. 2020 determined the mic value of bv as 15.6 μg/ml for mrsa. the mic values of melittin against mrsa, were between 0.125 μg/ml and 32 μg/ml (choi et al. 2015; lima et al. 2021; marques pereira et al. 2020). also, we determined the mic and mbc values as 6.25 μg/ml against vre isolate. al-ani et al. (2015) reported that the mic values of bv and mellitin were 100 μg/ml and 500 μg/ml, respectively. the authors evaluated the mic and mbc values of three mdr gram-negative bacteria. carbapenem resistant a. baumannii is a very persistent bacteris found in intensive care units. minimal inhibition concentration and mbc values were observed as 6.25 μg/ml for bv in this study. in various studies, the mic value of bv was determined as 31.25 mg/ml and the mic values of melittin was 0.5 μg/ml – 1 μg/ml and 8 μg/ml – 16 μg/ml (al-safar et al. 2018; askari et al. 2021; bardbari et al. 2018). carbapenem resistant k. pneumoniae causes pneumonia and urinary tract infections with high morbidity and mortality in hospitals. the authors determined mic and mbc values as 12.5 μg/ml for bv. the mic value of melittin was reported as 32 μg/ml for k. pneumoniae carrying the kpc gene (askari et al. 2021). also, e. coli is one of the most important pathogens in sepsis. in the current study, mic and mbc values were determined as 6.25 μg/ml for bv. however, there is no study on the antibacterial activity of whole bv on carbapenem resistant k. pneumoniae and e. coli. in the time-kill analysis, inhibition time was investigated at different concentrations of bv. multi-drug resistant isolates which reproduced in mbh containing 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml and 50 μg/ml bv were inoculated and evaluated at mha 0 h, 1 h, 3 h, 6 h, 12 h and 24 h. it was observed that the time killing periods varied between 1 h and 24 h and the concentration were ranging between 6.25 μg/ml and 12.5 μg/ml (table 2, figure 2). the time-killing assay was applied to all bacteria. bacteria were grouped as gram-positive and gram-negative. the mic and mbc values for mrsa and vre isolates were determined as 6.25 μg/ml against gram-positive bacteria. the inhibition time point of mrsa isolate was 12 h at 6.25 μg/ml. it was determined that s. aureus usa300 reduced 8 log cfu/ml at 100 μg/ml bv concentration in 1 h (choi et al. 2015). han et al. (2016) reported the, mic values for two mrsa isolates as 0.17 μg/ml and 0.85 μg/ml and the amount of mrsa decreased by 3 log cfu/ml. similarly, to the current study, bv reduced the amount of mrsa by 6 log cfu/ml for 100 μg/ml concentration at 1 h. inhibition time point of vre isolate was 3 h at 6.25 μg/ml. interestingly, in the current study, as soon as the vre contacted bv at 50 μg/ml and 100 μg/ml, the growth curve decreased by 6 log cfu/ml in 0 h. the decrease of 6 log cfu/ml in 0 h was an indication that vre was inhibited as soon as it contacted with bv. to the best of the authors’ knowledge this is the first study about the application of whole bv time-killing assay to vre isolate. the inhibition time points at all bv concentrations for vre isolate were shorter than mrsa isolate. the mic and mbc values for carbepenem resistant e. coli and a. baumannii isolates were determined as 6.25 μg/ml against gram-negative bacteria. the inhibition time points of carbapenem resistant a. baumannii and e. coli were 3 h and 6 h, at the concentration of 6.25 μg/ml. the inhibition times of these two pathogens were 1 h at concentrations of 12.5 μg/ml, 25 μg/ml and 50 μg/ml. the mic and mbc values were 12.5 μg/ml for carbapenem resistant k. pneumoniae. the inhibition times of bv were determined as 12 h, 6 h and 3 h, respectively at the concentration of 12.5 μg/ml, 25 μg/ml and 50 μg/ml (figure 2). carbapenem resistant k. pneumoniae, had higher mic value of bv (12.5 μg/ml) and inhibition time (24 h) in the time-killing assay. also, it was inhibited at the concentrations of 50 μg/ml and 100 μg/ml in 3 h. as a result, bv showed a strong antibacterial activity against mdr pathogens, which are difficult to treat and eradicate in areas such as hospitals, veterinary clinics and food sectors. despite this powerful antibacterial activity, it is a natural product that should be approached carefully in human and animals as it can cause allergic reactions. bee venom can be used as a disinfectant in the human and animal hospitals, veterinary clinics, dental clinics, nursing homes and food industry. however, more studies need to be done for determining its disinfecting dose. acknowledgements the authors are thankful to the adana veterinary control institute. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.g.g., h.y., f.e. and a.e.ü. conceived this research and designed experiments; t.g.g., h.y., y.ö., f.e., n.t., ş.k., s.s., i̇.k., o.s. and a.e.ü. performed experiments and analyses; t.g.g. and h.y. wrote the article and all authors participated in the revisions of it. all authors read and approved the final article. funding information this research was financially supported by the scientific research projects unit of cukurova university with the project number of tay-2022-14855, adana, turkey. the authors would like to thank 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and microbiological contamination of anatolian bee venom’, plos one 16(7), e0255161. https://doi.org/10.1371/journal.pone.0255161 wehbe, r., frangieh, j., rima, m., el obeid, d., sabatier, j.m. & fajloun, z., 2019, ‘bee venom: overview of main compounds and bioactivities for therapeutic interests’, molecules 24(16), 2997. https://doi.org/10.3390/molecules24162997 abstract introduction methods review findings conclusion acknowledgements references about the author(s) joanne karzis department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa inge-marie petzer department of production animal studies, faculty of veterinary science, university of pretoria, pretoria, south africa vinny naidoo department of paraclinical sciences, faculty of veterinary science, university of pretoria, pretoria, south africa edward f. donkin department of animal and wildlife sciences, faculty of agricultural sciences, university of pretoria, pretoria, south africa citation karzis, j., petzer, i-m., naidoo, v. & donkin, e.f., 2021, ‘the spread and antimicrobial resistance of staphylococcus aureus in south african dairy herds – a review’, onderstepoort journal of veterinary research 88(1), a1937. https://doi.org/10.4102/ojvr.v88i1.1937 review article the spread and antimicrobial resistance of staphylococcus aureus in south african dairy herds – a review joanne karzis, inge-marie petzer, vinny naidoo, edward f. donkin received: 09 feb. 2021; accepted: 23 aug. 2021; published: 26 oct. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract staphylococcus aureus is internationally recognised as a principal agent of mastitis and the foremost reason for economic loss in the dairy industry. the limited data available on organism-specific antibiotic resistance surveillance in dairy cattle have stimulated the need for such a review article. the objective of this study was to review relevant literature on antimicrobial resistance of mastitis-causing staphylococci isolated from dairy cows in south africa compared to other countries. factors relating to the incidence of mastitis and treatment strategies in terms of the one health concept and food security were included. the web of science (all databases) and relevant websites were used, and articles not written in english were excluded. the incidence of mastitis varied between south africa and other countries. antimicrobial resistance patterns caused by s. aureus also varied in regions within southern africa and those of other countries although some similarities were shown. antimicrobial resistance differed between s. aureus bacteria that were maltose positive and negative (an emerging pathogen). the results highlighted the importance of the availability of organism-specific surveillance data of the incidence of mastitis and antibiotic resistance for specific countries and within similar climatic conditions. accurate knowledge about whether a specific pathogen is resistant to an antibiotic within a certain climate, country, area or farm should reduce the incidence of unnecessary or incorrect treatment with antibiotics. this should enable dairy farmers to deal with these organisms in a more effective manner. therefore such research should be ongoing. keywords: mastitis; economic loss; dairy; cattle; antibiotic; resistance. introduction mastitis is a major endemic disease of dairy cattle alongside respiratory disease. it remains the disease in the developed countries that cause most economic losses (geary et al. 2012). host-adapted mastitis intramammary infections (imi) are transmitted from cow to cow. there are several mastitis pathogens for which the primary reservoir is the udder of the cow and the prevalence of imi because of these pathogens is significant. the coagulase-positive staphylococci are of the most common pathogens in this category, especially staphylococcus aureus (s. aureus) (nickerson, owens & boddie 1995). the udder is the primary reservoir of s. aureus, and this organism is transmitted during milking, although a proportion of heifers that enter the milking herd may already be infected with s. aureus (nickerson et al. 1995). this suggests routes of transmission other than milking equipment, hands of milkers’ and udder cloths used during milking. management and control of s. aureus in a dairy herd require insight of the reservoirs of infection and modes of transmission (rainard et al. 2017). modern molecular techniques have recently been used to identify different strains of the organism, and these tools are likely to improve the understanding of s. aureus epidemiology in dairy herds (leuenberger et al. 2019). there is only a small selection of intramammary antibiotics available in south africa, which are mainly products based on ampicillin and cloxacillin. antibiotic usage has been shown to correlate with the emergence and maintenance of antibiotic-resistant traits within pathogenic strains in ruminants (ben zakour et al. 2008). dairy cattle producers are particularly affected because of the negative impact resistant strains have on milk production and economics (bean, williamson & cursons 2004). host-adapted mastitis is controlled with improvements in hygiene and disinfection aimed at disrupting the transmission of the organism from one cow to another, as well as the elimination of infected cows via antibiotic treatment or culling (barkema, schukken & zadoks 2006). the ability to treat mastitis effectively depends not only on the efficacy of the active ingredient of the antibiotic but also on many aspects of management, hygiene, cow immunity, application of the intramammary product and other factors such as pharmacodynamics and udder parenchyma damage. bacterial susceptibility to an antibiotic, as determined by susceptibility testing, can aid in the appropriate selection of an antibiotic for the treatment of mastitis in dairy cows (brînda 2009), but this is not necessarily an indication of treatment success. the knowledge regarding whether a specific pathogen is resistant to an antibiotic within a certain climate, country, area or farm should reduce the incidence of unnecessary or incorrect treatment with antibiotics. the objectives of this study were to review and collate a comprehensive set of literature from south africa on staphylococci (including s. aureus, other coagulase-positive staphylococci and methicillin-resistant [meca] s. aureus [mrsa]) causing mastitis in dairy cows and to compare this information to that from other countries. factors relating to the incidence of mastitis and to treatment strategies in terms of the one health concept were included. an additional aim was to compare literature on antibiotic resistance of the coagulase positive staphylococci in south africa with that reported from other countries. methods outline of bibliographic search: full articles from scientific journals, conference papers, proceedings and agricultural reports were considered. when conference papers, reports and proceedings are included, then publication bias is not ruled out. studies that were considered were cohort, cross sectional, case control as well as analytical studies. each article was then further studied by obtaining the full text where possible to discover where the study was done and under which conditions that are important to know for mastitis-causing organisms. the time period searched was from 1947 to 2020. literature from all over the world was included for comparison with south african data. in adherence with the limitation of a number of references permitted for review articles, earlier literature was excluded, when recent literature was available. articles excluded were those who did not comply with the above criteria, as well as duplicated articles and articles not presented in english. the web of science (all databases) was used. these databases were selected for the comprehensive collection of data on agricultural and medical information related to mastitis and antibiotic resistance in dairy cows. the present research was done using search terms singly and then combining the different search terms of the various sub-headings throughout this review for analysis. review findings mastitis subclinical mastitis was defined as an infected quarter with a somatic cell count (scc) ≥ 200 000 cells/ml (hillerton 1999) or a composite milk samples with scc ≥ 150 000 cells/ml (petzer et al. 2017), in the absence of clinical changes to milk. quarter milk samples with an scc of ≤100.000 cells/ml milk from which no microorganisms were isolated and without a history of recent infection are considered to be normal (petzer et al. 2017). causes of mastitis bacterial species and occurrence specific knowledge of mastitogenic pathogens is important to manage the disease. the pathogenicity of an organism to cause mastitis mainly determines the degree of importance. moreover, the interactions of the environment, the host and agent also are important. some studies from norway, new zealand, north america, denmark, england, ireland, the netherlands, germany, portugal, france, beijing, south africa, ethiopia and rwanda, between 1975 and 2018, have shown that s. aureus, at that time, may have been the most prevalent cause of mastitis with estimates that 7% – 40% of all cows were infected (bakken 1981; elliott, tattersfield & brookbanks 1975; fox & gay 1993; holmes & zadocks 2011; mekonnen et al. 2017; mpatswenumugabo et al. 2017; schmidt, kock & ehlers 2017; wang et al. 2018). petzer et al. (2009) investigated milk and udder secretions from lactating and dry cows tested for the presence of micro-organisms in a study undertaken from 1996 to 2009 in south africa. the results from lactating cows (n = 379 000) and the dry cows (n = 11 946) revealed that non aureus staphylococci (nas) previously known as coagulase-negative staphylococci (cns) were by far the most abundant bacteria frequently isolated at 61.71%, followed by s. aureus (17.28%) and αβ haemolytic s. aureus (sth) (7.81%) and smaller percentages of all other mastitis-causing bacteria. similar percentages of nas, s. aureus, sth and other organisms were isolated from both lactating and dry cows (petzer et al. 2009). in south africa from 2011 to january 2020, the number of milk producers decreased from 2686 producers to 1164, causing many herds to be amalgamated, therefore with an increased risk for spread of contagious organisms in the absence of adequate biosecurity (sarrazin et al. 2014; sayers et al. 2013). the average herd size increased from 167 (2010) to 459 (october 2019) (milksa 2020). the udder health management system as a whole has improved as these larger herds are mainly of well-managed large commercial dairy herds (milksa 2020). coagulase-positive staphylococci: staphylococcus aureus: staphylococcus aureus is the most universal host adapted mastitis pathogen in many dairies, even though the prevalence has decreased because of improved milking hygiene and widely implemented mastitis control strategies (hillerton 1999). staphylococcus aureus nevertheless has remained a very challenging and important mastitogenic pathogen in south africa (petzer at al. 2009, 2012). petzer et al. (2017) reported a prevalence of 9.05% imi caused by s. aureus in south african dairy herds (2001–2015), whilst a prevalence of 10% – 40% was reported in other countries (liu et al. 2017). chronic carrier cows are the major source of infection, shedding the s. aureus bacteria intermittently, and this may limit the life and milk production potential of the infected quarter (mellenberger & kirk 2001). the design and function of the milking machine and the milking parlour and the resultant milking routine can also predispose cows to mastitis (dodd & neave 1970). flies have also been implicated in the transfer of s. aureus from one animal to another (mellenberger & kirk 2001). udder infection with s. aureus often leads to fibrosis, abscesses and recurring clinical mastitis (mellenberger & kirk 2001). staphylococcus aureus is especially difficult to manage within a herd, and it can be fatal to the cow (mellenberger & kirk 2001). these bacteria have the ability to avoid phagocytosis by producing a polysaccharide mucous biofilm around themselves, leading to poor penetration of the antibiotic during treatment (parul et al. 2019). in this manner, such bacteria are also further shielded from the defence mechanisms within the cells of the cow (mellenberger & kirk 2001). a study by monistero et al. (2018) evaluated 120 isolates from four continents and eight different countries: in europe (germany and italy), in north america (new york state and brazil), in south america (argentina and columbia) and in africa (south africa and tunisia). however, only new variants of existing genotypes were detected for five of these seven countries other than south africa participating in the study, including gtiv, gtivi (argentina), gtaoi, gtaoii (colombia), gtaqi, gtbni, gtbnii, gtbyi(brazil), gtcv, gtiv (new york state) and gtrxiii (italy) (monistero et al. 2018). new genotypes (gtar, gtbz and gtca) were observed for the south african strains. in contrast to the colombia, in the argentinian strain, only one contained the sak gene. this showed an apparently clear dissociation from the strains isolated from humans (monistero et al. 2018). cosandey et al. (2016) described s. aureus genotype b (gtb) only in italy, whilst s. aureus genotype r (clr) and s. aureus genotype c (clc) were identified in many places in europe, america and africa. genotype clr, which represents a large cluster containing 13 variants, was detected in all eight countries studied except for brazil (monistero et al. 2020). cosandey et al. (2016) have shown that s. aureus clr and clc clusters are exclusive to dairy cattle and the spread thereof probably started many years ago, when cattle were moved from europe to other countries (monistero et al. 2018). similarly, the new genotype was found in south africa by monistero et al. (2018) who demonstrated the large variety of s. aureus genotypes in dairy cattle worldwide. this finding has suggested that monitoring these variations may assist with the reduction of spreading of mastitis organisms, as different genotypes are found and identified in different areas (monistero et al. 2018). αβ haemolytic staphylococcus aureus lytic group iii: during 1989, a distinct αβ haemolytic s. aureus was isolated in south africa from milk samples collected from a herd with 300 cows in milk (petzer et al. 2009). these bacteria all belonged to the phage type, lytic group iii, whilst all other s. aureus isolated from milk from other herds were either from lytic group i or ii. a nasal swab was taken from the owner of this herd, and a veterinarian who had chronic sinusitis and s. aureus (lytic group iii) (sth) was isolated. following on this case, numerous other isolates of sth were obtained mainly from milkers over the next 10 years (petzer et al. 2009). another south african study of s. aureus from dairy herds in bloemfontein in 1985 had also isolated s. aureus (lytic group iii) (sth), believed to be of human origin (swartz, jooste & novello 1985). the percentage of sth isolated from milk of south african cows increased to 20%, which was more than the 13% of s. aureus (lytic groups i and ii) isolated in 1999 (petzer at al. 2009). what is of concern is that the sth seems to be more pathogenic compared to the other s. aureus bacteria. of the bacteria isolated from quarters with mastitis, 67.1% of them were sth isolates, whilst 52.4% of them were from the other s. aureus isolates (petzer et al. 2009). the possible effect of anthroponosis of pathogens from immunosuppressed individuals to cows should not be ignored and warrants further investigation (petzer et al. 2009; schmidt, kock & ehlers 2015). methicillin-resistant staphylococcus aureus in humans and animals: methicillin-resistant s. aureus (mrsa) bacteria have an acquired gene that renders them resistant to methicillin and basically to all other beta-lactam antibiotics (perovic et al. 2006). methicillin-resistant s. aureus is a main source of healthcare-associated, community-associated and livestock-associated infections (perovic et al. 2006). methicillin-resistant s. aureus is of serious concern in human medicine and an emerging concern in veterinary medicine (garcía-álvarez et al. 2011). milk samples from south african dairy cattle have tested negative for the meca (gene typically indicating methicillin resistance) on the polymerase chain reaction (pcr) but showed phenotypic mrsa using the cefoxitin disc (badenhorst, karzis & petzer 2014). this could have been the strain of s. aureus now found to carry a homologue of the meca gene now known as the mecc gene (garcía-álvarez et al. 2011). staphylococcus aureus is transmitted amongst humans, amongst animals, from humans to animals and from animals to humans when in close contact (juhász-kaszanyitzky et al. 2007). horizontal transmission amongst humans usually happens via direct contact from hands of infected people or from contaminated food (perovic et al. 2006). maltose-negative staphylococcus aureus: an emerging udder pathogen: staphylococci, which include s. aureus and the staphylococcus pseudintermedius and staphylococcus intermedius groups, are responsible for the most clinical diseases in veterinary medicine and are coagulase positive (sasaki et al. 2007). more recently, based on the nucleotide sequence analysis of the soda and hsp60 genes, isolates that were maltose negative and previously identified (phenotypic characteristics) as staphylococcus intermedius were reclassified into three clusters, namely: staphylococcus intermedius, staphylococcus pseudintermedius and staphylococcus delphini (sasaki et al. 2007). whilst there have been studies on multi-drug resistant s. pseudintermedius isolated from dogs, cats and horses (oguttu, qekwana & odoi 2017; van duijkeren et al. 2011), little work has been undertaken on s. pseudintermedius causing mastitis in dairy cattle. suspected staphylococcus pseudintermedius was initially isolated from one large commercial dairy herd in south africa in 2005 and 2006, which had been found to have a large number of s. aureus with low scc < 100 000 cells/ml milk (karzis et al. 2020b). further identification of these maltose-negative staphylococci using matrix assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry and 16s ribosomal ribonucleic acid (rrna) found these organisms to be s. aureus strain stain type (st) 2992, an identical strain of single origin, isolated repeatedly over time (karzis et al. 2020b). although both the mala and malr genes were present, they were not expressed phenotypically. this may have been attributed to the abnormal stop codon present on the mala gene (gen bank accession number: mn531305) (karzis et al. 2020b). this newly identified maltose-negative s. aureus seemed to have low virulence, similar to that of non-aureus staphylococci, which is different to that of maltose-positive s. aureus (karzis et al. 2020b). factors affecting the incidence of mastitis in cows: cow factors factors affecting the incidence of mastitis in cows can include anatomical factors such as udder suspension, teat canal, internal blood supply, lymphatic system and previous udder damage, from physical trauma, or damaged parenchyma or previous infections and even including high yields (sordillo 2009). the immune system of dairy cows the defence of the mammary gland can be divided into two prominent categories: the innate immunity that is not specific and prevalent in early lactation and acquired or specific immunity, which recognises specific factors of a pathogen (sordillo 2009). cell-mediated immune responses (cmir) and antibody-mediated immune responses (amir) are used as indicators of the adaptive immune responses of livestock (heriazon et al. 2009). intramammary infections provoke inflammatory reactions leading to elevated scc and activate bacteriostatic enzymes in the milk (bruckmaier & meyer 2005). health of dairy cows is influenced by an interaction between the innate components of the immune system and an inflammatory response, which can sometimes be more destructive than helpful (bertoni, minuti & trevisi 2015). infections, as well as metabolic and parasitic diseases, should, therefore, be avoided as far as possible during the peri-parturient period of dairy cows (bertoni et al. 2015). the latter can have a negative impact on the dry matter intake, energy efficiency, liver function, fertility and milk yield (bertoni et al. 2015). antimicrobials antimicrobial use has become part of both human and animal living (lewis 2013). as a result of antimicrobial use in disease control, maintenance of health and applications in the agricultural and environmental sectors, drug resistance has resulted, which now threatens the efficacy of these medicines (department of health & department of agriculture, forestry and fisheries [daff] 2018). the first commercially available antibacterial was prontosil, a sulphonamide, discovered in 1932, introduced in 1936, and resistance was first observed in 1942 (lewis 2013). many people credit alexander fleming for developing the first true antibiotic in 1928. this was described as a term invented by selman waksman who described it as a compound that was either produced or derived from microorganisms that could kill or hinder the growth of another microorganism (waksman 1947). penicillin was discovered in 1928 and introduced in 1938, and resistance was first observed in 1945 (lewis 2013). the drugs available for intramammary remedies used in south africa are mainly limited to cloxacillin and ampicillin combinations in the past, with cephalosporin products introduced into the market more recently. currently, there are only two dry cow and four lactating cow remedies available for use in south africa (july 2021). antibiotics antibiotic classes: antibiotics are divided into classes and then further into subclasses. the main antibiotic classes used for the treatment of mastitis are beta lactams, tetracyclines, quinolones, sulphonamides, amphenicols, aminoglycosides, macrolides, lincosamides, polypeptides and pleuromutilins (gualerzi et al. 2014). there are also many antibiotic subclasses, which are allocated in no consistent manner for some drugs, for example, the beta lactams that are penicillins and cephalosporins. the penicillins are divided by synthesis (natural, biosynthetic and semi-synthetic), penicillinase resistance or duration of action, and the cephalosporins are divided into classes (gualerzi et al. 2014). antibiotic action: the difference between bactericidal agents (which kills the organism) and bacteriostatic agents (which inhibits growth temporarily) has been determined under defined laboratory settings and is dependent on the specific agent and bacteria (pankey & sabath 2004). for the most effective clinical action, the results from in vitro studies need to be combined with relevant pharmacokinetic and pharmacodynamics data to offer a better prediction of efficacy in vivo (pankey & sabath 2004). for pharmacokinetic information, the ability to maintain relevant plasma and tissue concentrations is important. for pharmacodynamics information, the action of the antimicrobial drugs needs to be determined in relation to time or concentration. the term ‘time dependent’ is indicated by the extent and rate at which microorganisms are killed and will remain unchanged and will not be dependent on the maximum antimicrobial concentration, whilst the minimum inhibitory concentration (mic) is maintained for a specified time (t > mic). the term ‘concentration dependent’ indicates the extent to which microorganisms are killed, depending on the antimicrobial concentration in relation to the mic, for example, fluoroquinolones and aminoglycosides (cmax: mic and au24c:mic) (van bambeke & tulkens 2001). antibiotic spectrum: the effect of antibiotics can be bactericidal or bacteriostatic for either a small (narrow spectrum) or larger group (broad spectrum) of pathogens (apua glossary 2019). intrinsic and acquired resistance: when bacteria pose inherent resistance to a specific antibiotic, it is able to resist the action of that antibiotic because of its functional characteristics or intrinsic structure. it is important to have knowledge of such intrinsic resistance of pathogens to avoid unsuitable and ineffective therapies (cox & wright 2013). legislation and the use of antibiotics in south africa antibiotics, including intramammary mastitis remedies, are registered in south africa under two separate acts, namely: medicines and related substances act as amended (act 101 of 1965) for scheduled medicines that are only available on prescription from veterinarians and the fertilizers, farm feeds, agricultural remedies and stock remedies act (act 36 of 1947) where antibiotics and stock remedies can be sold over the counter without prescription. there is much evidence to suggest that dairy producers often treat cows with antibiotics symptomatically or without confirmation, and these actions may lead to the ongoing or emerging resistance to antibiotics (burgos, ellington & varela 2005). antibiotic use in animals: the previous daff and the south african animal health association (saaha) reported on antimicrobial use in animals. from 2014 to 2015, growth promoters (62%), tetracyclines (17%) and macrolides (11%) were mostly used. the growth promoters used were mainly flavophospholipol (flavomycin), ionophores, zinc bacitracin, olaquindox and tylosin and are antibiotics not used in human health (national department of health 2018). antib iotic use in humans: the use of antibiotics in human medicine in south africa during 2015 was found to be 21 149 standard units per 1000/population (one standard unit is equivalent to an injection or one tablet). this is a high usage compared to various other countries. the use of broad-spectrum penicillin was 1.3–3.3 times higher than in other brics (brazil, india, russia and china) countries. however, the use was 0.8 times lower than that in the united kingdom (uk) or united states (us) (national department of health 2018). antibiotic resistance the infectious diseases society of america highlighted six groups of pathogens, which are commonly associated with antimicrobial resistance: klebsiella pneumoniae, s. aureus, enterococcus faecalis and enterococcus faecium, acinetobacter baumannii, pseudomonas aeruginosa and escherichia coli (e. coli) (eskape) (national department of health 2018; santajit & indrawattana 2016). of the eskape pathogens, s. aureus is the most common contagious pathogen associated with mastitis (hillerton 1999), followed by e. coli and klebsiella pneumonia. most of the eskape pathogens are multidrug resistant, and they are primarily responsible for nosocomial infections around the globe (santajit & indrawattana 2016). antimicrobial resistance information from surveillance data in blood cultures of humans for eskape pathogens in south africa (national department of health 2018) indicated that resistance levels for s. aureus had decreased from 36% to 23% over the previous 6 years (national department of health 2018). at present, there are limited data available in south africa on antibiotic resistance surveillance in dairy cattle (petzer et al. 2012; schmidt et al. 2015; van vuuren, pickard & greyling 2007), which has highlighted the need for more detailed information. at the present time, the main national centre for biotechnology information (ncbi) database, containing the main bacterial genome sequences, has predicted the number of potential resistance genes to be in excess of 20 000 of nearly 400 different types (davies & davies 2010). a recent world health organization (who) report has forewarned that resistance to antibiotics is real and an international threat (who 2020), which affects human and animal health care and has an important bearing on the agricultural industry. according to a report by the food and drug association (fda), approximately 80% of all antibiotics used in the usa went to livestock production (usfda 2016). despite recent efforts of many international health organisations (shryock & richwine 2010) to control and withdraw antibiotic use in animal husbandry, new antibiotic resistance continues to emerge. in germany, denmark and sweden, consumption of antimicrobial agents used for animals was reported to have decreased in recent years (koch 2013). contrary to the above findings, the netherlands reported that following the ban on growth promoters in 2006, the use of antibiotics for disease treatment increased (maron, smith & nachman 2013). in 2005, the european union (eu) voted for the total ban of antibiotics as growth promoters in feed animals, whose restriction was effective from 01 january 2006 (eu 2005). overall, the dairy industry is one of the major contributors to worldwide antibiotic usage and information on the trends of susceptibility and resistance in host adapted pathogens such as s. aureus is critical. the european medicines agency (ema) and the european food safety authority (efsa) have published a joint opinion in which they maintained that the use of antimicrobials in animals needs to be rethought, reduced and replaced (international dairy federation 2017). the efsa has supported the restricted use of antimicrobials in animals for only treatment of infectious diseases. the south african national veterinary surveillance and monitoring programme for resistance to antimicrobial drugs (sanvad) has previously found far lower resistance levels than expected in their surveillance (van vuuren et al. 2007). ten per cent resistance to three antibiotics was found, with lower resistance to mastitis remedies. nevertheless, 80% of s. pseudintermedius that were isolated from infections in dogs and pyoderma were resistant to amoxicillin and 20% were resistant to first-generation cephalosporins. petzer et al. (2007) found resistance rates of 23% for tetracyclines, 37% for ampicillin and 45% for penicillin in s. aureus isolates from milk samples (henton et al. 2011). until 2011, data on antibiotics used in livestock production in south africa were limited as pharmaceutical companies were not forthcoming with this information and two different acts controlled these medicines (henton et al. 2011). however, a more recent ministerial report has been more transparent on the antimicrobials used between 2014 and 2015 and has estimated the use of antibiotics at a ratio of 26% – 74% for animals and humans, respectively. these results varied distinctly from those published from china, india and the usa, where a much higher ratio was indicated for animal use (national department of health 2018). staphylococcus aureus antibiotic resistance in south africa: schmidt et al. (2015) found in a limited study conducted in kwazulu-natal that 48% of s. aureus isolates were resistant to beta-lactams. a study by karzis et al. (2019) agreed with this relatively high resistance to penicillin and described a few significant differences in antibiotic resistance between the different seasons and between the provinces of south africa. it is a concern that with the exception of cefuroxime, for all of the tested antibiotics, in most provinces, the predicted prevalence of resistance was above 50%. the lowest predicted prevalence of resistance for all antibiotics, except for cephalosporins, was in kwazulu-natal province during spring. the least resistance was predicted for cephalosporins in gauteng province during winter. the reasons for these differences were obscure (karzis et al. 2019). a study by fox et al. (1995) performed at pullman university (us) also found that season, herd and location played significant roles in the prevalence of imi and was similar to the study by karzis et al. (2019). a study by monistero et al. (2020) that investigated the distribution of antimicrobial resistance genes of s. aureus in six countries found that the south african s. aureus isolates had the highest complete or partial resistance for spiramycin (100%) and for erythromycin (36.4%) (monistero et al. 2020). there were 11 isolates analysed of which three (27.3%) were assessed phenotypically to be resistant to ampicillin and penicillin, whilst only one had phenotypic β-lactamase activity initially, but a second isolate showed intermediate resistance to lincomycin. this study by monistero et al. (2020) tested for the presence of six resistant genes, and ermb and blaz were isolated both at a frequency of 36.4% from the south african strains and seven (63.6%) isolates possessed the β-hemolysin (hlb) gene. the sea and sak immune evasion cluster (iec) genes were present in 90.9% and 100%, respectively, in the south african strains, whilst one strain also contained the sodium channel (snc) gene strain. in line with the virulence, the trends in antimicrobial resistance for the maltose-negative s. aureus st 2992 (karzis et al. 2020b) also showed a closer similarity with the data for non-aureus staphylococci than for the maltose-positive s. aureus (karzis et al. 2020a). unexpectedly, the maltose-negative displayed more resistance (p < 0.001) than the maltose-positive s. aureus isolates to most of the antimicrobials tested using the mic method (karzis et al. 2020a). staphylococcus aureus resistance in other countries: the isolates from argentina and germany also showed some resistance to erythromycin and lincomycin (monistero et al. 2020). amongst the antimicrobial resistance genes that were investigated in the us strains, the hlb gene was the most abundant (88.2%), the sea gene was present in 52.9% and blaz in 41.2%, whilst the erm genes and the sak and scn were not detected (monistero et al. 2020). from the italian isolates, 10 were indicated with penicillin resistance, nine (52.9%) were resistant to ampicillin and in the nine isolates, phenotypic β-lactamase activity was identified (monistero et al. 2020). only one isolate from argentina was phenotypically assessed as resistant to both ampicillin and penicillin, whilst two (12.5%) were β-lactamase positive based on the nitrocefin method (monistero et al. 2020). one isolate from germany was resistant to penicillin, oxacillin and ampicillin, and three were resistant to cephalosporins (cefoperazone, cefazolin and cefquinome). two other isolates from germany were resistant to sulfamethoxazole/trimethoprim and one had intermediate resistance to cefoperazone (monistero et al. 2020). a study performed in tennessee found 82 (34.3%) (n = 239) s. aureus isolates, with resistance to at least one of the 10 antimicrobials tested (abdi et al. 2018). there were dominating clonal patterns amongst isolates, with resistant isolates belonging to two main pulse field gel electrophoresis (pfge) types (abdi et al. 2018). the prevalence of antibiotic resistance was mostly for tetracycline and varied within and between farms over time (abdi et al. 2018). in a study performed in ethiopia, except for penicillin, tetracycline and sulphamethoxazole/trimethoprim, resistance to other drugs was rare (kalayu et al. 2020). staphylococcus aureus resistance in south africa compared to that of other countries: the resistance of the maltose-positive s. aureus observed in a south african study (karzis et al. 2020a) was in agreement with that reported in other studies in denmark and new zealand (salmon et al. 1998). the relatively high resistance rates of s. aureus to penicillin and tetracycline in both the south african studies (karzis et al. 2020a; schmidt et al. 2015) also corresponded with the findings of a recent study in ethiopia (kalayu et al. 2020). similarly, as in south africa, most isolates from the united states (new york state), argentina and germany showed resistance to spiramycin (monistero et al. 2020). in contrast to isolates from south africa, ethiopia and the united states of america, spiramycin and/or penicillin were not effective in 58% of italian isolates (monistero et al. 2020). although the ethiopian study did not test for spiramycin resistance, very high resistance to penicillin was shown (kalayu et al. 2020). studies in both animal and human medicine identified pan and multidrug resistance in s. aureus isolates in other countries (haran et al. 2011; karzis et al. 2020a). the importance of antimicrobial resistance in one health approach the one health initiative encourages interdisciplinary collaboration amongst the human, animal and environmental sectors globally. it is based on the view that such an interdependence will advance future health care and impact positively on longevity and quality of life (essack 2018). thus, a one health approach is required in order to understand and control antimicrobial resistance (who 2020). according to essack (2018), bacterial resistance to multiple antibiotic classes is increasing, and strains are exhibiting multiple resistant mechanisms. resistance genes as well as virulence factors are carried on different portable genetic elements, which are capable of exchange amongst and between bacteria in animals, humans and the environment (essack 2018). implications and recommendations this study summarises the latest and most relevant data available on mastitis causing staphylococci in south africa and abroad on the emergence, spread and antibiotic resistance. this review concentrates mainly on coagulase-positive staphylococci. the most recent south african studies have been reported in the context of the wider field of mastitis and antibiotic resistance worldwide and also in terms of the one health concept, which links the animal, human and environmental components of the literature. one of the key outcomes of this study has shown the importance of the availability of antibiotic resistance surveillance data, specifically for s. aureus and other mastitis causing staphylococci. in particular, such pathogens would be the traditional maltose-positive s. aureus and the emerging pathogen maltose-negative s. aureus. the accurate identification of antibiotic resistance trends and correct treatment should also be likely to reduce the risk of the development of multi-drug-resistant organisms. such information about the antibiotic resistance trends should enable dairy farmers to deal with these organisms in a more effective manner in the different regions of southern africa and worldwide in different countries and under various climatic conditions, without having to perform extensive testing each time. the availability of antibiotic resistance surveillance data for s. aureus in dairy cattle is essential in the context of general public health, by facilitating a safe dairy industry, controlling the incidence of antibiotic resistance in dairy cows and contributing to food security. this might include the need to take account of the possibility of zoonosis (transmission from animals to humans) and anthroponosis (transmission from humans to animals) of antibiotic-resistant bacteria. mastitis and antibiotic treatment have great financial implications for the milk producer when assessing the cost of treatment and also the cost incurred in discarding the milk for the duration of the withdrawal period. thus, more information on the effective and prudent use of antibiotics and the effective management of mastitogenic pathogens by intramammary treatments should reduce expenses for the producers and improve their profitability and sustainability. the development and implementation of a national antimicrobial resistance strategy framework (narsf) are main steps towards containing the threat of antimicrobial resistance in animal and human health in south africa (daff 2018). the responsibility for reducing antibiotic resistance requires a collective effort, and therefore global partnerships need to be strengthened. one of the south african research studies showed the importance of good udder health management, which led to a reduced incidence of antimicrobial resistance of s. aureus infections in 20 well-managed herds studied over an 11-year period (isolates: n = 5942). this programme was the main factor resulting in the difference in the management of antibiotic resistance between south africa and other countries. in subsequent studies in south africa and in other countries, research has implied that additional factors such as environmental factors, seasonal and regional differences also had an effect on antimicrobial resistance and the efficacy of antimicrobial drugs. however, there are many more factors, which need to be considered in future research. thus, the existing research shows the importance of the collaboration of the animal-human-environmental interfaces of the one health concept. this review has shown not only some similarities but also many variations in antibiotic resistance patterns of s. aureus in regions of southern africa and other countries. this highlights the importance of organism specific information in each specific region and under similar climatic conditions, in order for such information to be able to be applied in practice. this is why the existence of organism-specific antibiotic surveillance data in all countries is so important and should be continued as ongoing studies and part of the one health concept. the differences in antimicrobial resistance patterns between the maltose-positive and -negative s. aureus and other staphylococci have been emphasised. this is valuable information for both veterinarians and producers, in order to adapt management procedures, and for development of prudent treatment protocols to manage the disease in an effective manner. this review has focussed on the importance of individual bacterial strains in antimicrobial resistance. the antimicrobial resistance of maltose-negative s. aureus st 2992 was higher than that of maltose-positive s. aureus, and it was more multi-drug resistant. future research is necessary on the incidence of antibiotic resistance in dairy herds in south africa and to identify and evaluate other mastitis causing pathogens, such as non-aureus staphylococci, streptococcus uberis, streptococcus agalactiae, streptococcus dysgalactiae and gram-negative bacteria. conclusion mastitis is the disease in dairy cows that is responsible for the greatest economic loss. its incidence is influenced by the individual cow, the micro-organisms, the environment and management. mastitis caused by s. aureus is still an important problem in udder health in south african dairy herds. this review has shown not only some similarities but also a great variation in antibiotic resistance of s. aureus between different regions of southern africa and other countries. a difference of antibiotic resistance was also shown between maltose-positive and maltose-negative s. aureus (emerging pathogen) and between different levels of management. all this information highlights the importance of good udder health management and organism-specific antibiotic resistance data, for each specific country or situation and under similar climatic conditions. this is essential in order to be able to apply the appropriate management correctly. in future, it is vital that such studies should be continued in an ongoing programme, in order to be able to add to and improve the current surveillance data available for s. aureus in dairy cattle worldwide. in south africa, there have been only limited data available on antibiotic resistance surveillance in dairy cattle, which has stimulated the need for more detailed information to be generated through appropriate research. this knowledge will be valuable to veterinarians and producers when using antibiotics and for the treatment of dairy cattle in different regions and to assist in developing the policy on access and use of antibiotics. resistance to antibiotics is the biggest threat to public health and has the ability to impact society negatively. however, the focus worldwide and in south africa is now on prudent use of antibiotics, which should have an improved effectiveness, reducing the likelihood of any consequent resistance. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions j.k. conceptualised and wrote the article. e.f.d., v.n. and i.-m.p. assisted in conceptualising and writing the article as well as reviewing the manuscript. i.-m.p. assisted with all udder health-related portions and v.n. assisted with the pharmacology-related aspects. ethical considerations this article followed all ethical standards for research without direct contact with human or animal subjects. funding information this work is based on the research supported in part by the national research foundation of south africa (grant number: 120319). the opinions, findings, conclusions and/or recommendations expressed in this publication are the author(s) alone. data availability data sharing is not applicable to this article as no new data were created or analysed in this study. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references abdi, r.d., gillespie, b.e., vaughn, j., merril, c., headrick, s.i., 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2705–2714. https://doi.org/10.1093/jac/dkr367 van vuuren, m., pickard, j. & greyling, j., 2007, south african national veterinary surveillance and monitoring programme for resistance to antimicrobial drugs (sanvad), department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria. waksman, s.a., 1947, ‘what is an antibiotic or an antibiotic substance?’, mycologia 39(5), 565–569. https://doi.org/10.1080/00275514.1947.12017635 wang, w., lin, x., jiang, t., peng, z., xu, j., yi, l. et al., 2018, ‘prevalence and characterization of staphylococcus aureus cultured from raw milk taken from dairy cows with mastitis in beijing, china’, frontiers in microbiology 22(9), 1123. https://doi.org/10.3389/fmicb.2018.01123 world health organization (who), 2020, global antimicrobial resistance and use surveillance system (glass) report – early implementation 2020, geneva, viewed 28 july 2020, from http://www.who.int/health-topics/antimicrobial-resistance. phalatsi_327-331.qxd introduction the blue tick, rhipicephalus (boophilus) decoloratus, is one of the most widely distributed ixodid ticks in south africa (howell, walker & nevill 1978), and is the principal vector of babesia bigemina, the causative organism of african redwater in cattle (de vos & potgieter 1994). it has consequently been the subject of several laboratory and field studies (londt 1974, 1977; robertson 1981; spickett & heyne 1990; horak, spickett & braack 2000). its larvae are most numerous on the vegetation in the eastern cape province during the period february to june (rechav 1982), while infestation on cattle in this province and in kwazulu-natal is most intense from spring or early summer to autumn or early winter (robertson 1981; rechav 1982; baker, ducasse, sutherst & maywald 1989). however, in a recent study in the eastern region of free state province, an area not previously surveyed, r. (b.) decoloratus was most numerous on cattle from late summer to mid-winter, a period during which average daily temperatures decreased from 18–8 °c, with the largest numbers present in the coldest month, namely june (dreyer, fourie & kok 1998). the present study was designed to determine the preference of r. (b.) decoloratus larvae for vertical substrates of various lengths in the laboratory, as well as their hatching and survival under field condi327 onderstepoort journal of veterinary research, 71:327–331 (2004) research communication larval biology of rhipicephalus (boophilus) decoloratus (acarina: ixodidae) in free state province, south africa m.s. phalatsi1,2, l.j. fourie1* and i.g. horak1 abstract phalatsi, m.s., fourie, l.j. & horak, i.g. 2004. larval biology of rhipicephalus (boophilus) decoloratus (acarina: ixodidae) in free state province, south africa. onderstepoort journal of veterinary research, 71:327–331 the objective of this study was to determine certain aspects of the biology of rhipicephalus (boophilus) decoloratus larvae under laboratory and field conditions. larvae allowed 48 h to select a vertical questing substrate preferred 90 cm rods in length to those of 60 or 30 cm, while in a separate experiment migration from rods 5 cm or 25 cm in length to rods 45 cm in length continued between 48 h and 72 h after larval release. hatching of the larval progeny of engorged female ticks exposed to ambient field temperatures during the period june to august, occurred synchronously during the third or fourth week of november. with a single exception, larvae that hatched during november and between april and july survived for 38 days or longer, while those that hatched from december to march survived for 31 days or less. questing larvae were present on vegetation throughout the year, with most being recovered during january and february. parasitic larvae were present on cattle from october to may with most being collected during january and february. keywords: free state province, larval survival, parasitic larvae, questing larvae, rhipicephalus (boophilus) decoloratus, seasonality * author to whom correspondence is to be directed 1 department of zoology and entomology, university of the free state, bloemfontein, 9301 south africa 2 present address: p.o. box 195, maseru, lesotho accepted for publication 29 march 2004—editor tions. in addition, the relationship between the numbers of larvae on the vegetation and those parasitic on cattle was examined. laboratory identical numbers of 1.5 mm diameter copper rods (30, 60, or 90 cm long) were equally spaced vertically in alternating sequence in a square metal arena (16 x 16 x 6 cm) with a wooden base covered with moist vermiculite. fifty milligrams of 7-day-old r. (b.) decoloratus larvae were released into the centre of the arena, and prevented from escaping by double-sided adhesive tape along the edges of the square. the arena was incubated for 48 h at 24 ± 2 °c with a photoperiod of 14 h light and 10 h dark, after which the larvae on each rod were collected and counted and the counts for rods of identical length pooled. within 48 h of release 57.5 % of unfed larvae clustered at the tips of the 90 cm long vertical rods compared to 10.1 % on the tips of the 30 cm long rods. in a follow-on study rod lengths of 5, 25 and 45 cm were used and observations were made 24, 48 and 72 h after the release of 50 mg of larvae into the centre of the arena. at each occasion the numbers of larvae on the rods were categorized on a subjective scale without their being disturbed. larvae observed at 24 h intervals over a 72 h period continued to migrate from 5 cm and 25 cm long rods to rods 45 cm in length. the heights at which adult ticks quest on the vegetation may relate to the size of their preferred hosts (fourie, kok & van zyl 1991; goddard 1992). the preference of r. (b.) decoloratus larvae for taller substrates in our experiments possibly reflects an adaptation by this life stage to expedite infestation of their favoured hosts, which are generally large animals such as domestic cattle, wild bovids and domestic and wild equids (mason & norval 1980). the presence of questing larvae on the tips of tall grass would facilitate their contact with the chest and abdomen of these animals. 328 larval biology of rhipicephalus (boophilus) decoloratus (acarina: ixodidae) in free state, south africa table 1 pre-oviposition period, oviposition period and subsequent survival of the larval progeny of engorged female rhipicephalus (boophilus) decoloratus exposed to ambient outdoor climatic conditions month of tick pre-oviposition oviposition period date of first larval larval survival placement period (days) (days) hatching (days) march 1998 11 30 eggs dried out before eclosion april 31 31 eggs dried out before eclosion may no oviposition took place june 74 8 21 nov. 1998 56 july 33 58 20 nov. 1998 44 august 17 70 23 nov. 1998 38 september no engorged female ticks collected october 20 22 22 dec. 1998 31 november 8 23 12 jan. 1999 30 december 8 19 19 jan. 1999 24 january 1999 7 24 4 mar. 1999 26 february 21 27 5 apr. 1999 84 march 7 29 2 may 1999 56 april 36 21 20 jul. 1999 42 may 89 13 eggs dried out and no larvae hatched june 67 15 eggs dried out and no larvae hatched july no engorged female ticks collected august 20 53 19 nov 1999 28 free-living ticks when procurable, six engorged female r. (b.) decoloratus, each weighing more than 150 mg, were collected at monthly intervals from march 1998 to august 1999 from cattle at botshabelo (29°15’ s, 26°43’ e), a semi-urban township 55 km east of the city of bloemfontein, free state province. these ticks were placed in separate 15 x 30 mm cylindrical vials, gauze-stoppered at both ends, and exposed to prevailing climatic conditions in a field on the premises of the university of the free state in bloemfontein. the vials were placed approximately 2 cm deep in the soil and were loosely covered with a mixture of grass litter and sand. a 1 m squarewire mesh cage was positioned over the placement site to prevent rodents from disturbing the vials, which were examined daily for the onset of oviposition, egg eclosion and survival of larvae. larval survival was calculated as the period between eclosion and the date on which all larvae were considered dead. atmospheric temperature and rainfall were measured on the campus of the university. the pre-oviposition periods of female ticks exposed during the cooler autumn and winter months (april to july) exceeded 30 days, while those exposed from november to january and during march were 8 days or less (table 1). the first larval progeny of ticks exposed from june to august 1998 hatched more or less synchronously from 20–23 november 1998, and those of ticks exposed during august 1999 hatched on 19 november 1999. larvae that hatched during november 1998 and from april to july 1999 survived for the greatest length of time. less than 3 mm of rain per month was measured from june to august and none during september. londt (1974, 1977) noted that the pre-oviposition and oviposition periods of r. (b.) decoloratus females and the incubation period of their eggs were temperature dependent, becoming shorter as the temperature increased, whereas the effects of humidity appeared to be negligible. he recorded mean pre-oviposition periods of 37–42 days for females placed in a stevenson’s screen from late july to september when calculated mean daily temperatures varied between 13.0 and 14.5 °c, compared to 28 days for females maintained at 15 °c in the laboratory, decreasing to 5 days or less for ticks maintained at 25 °c or higher. robertson (1981) exposed engorged female ticks in stevenson’s screens on two coastal farms in the eastern cape province over a period of 45 consecutive months. the pre-oviposition periods of these ticks varied between approximately 8 days for ticks exposed during january to 28 days for those exposed during june, and the incubation periods of their eggs varied from approximately 41 days for females exposed during january to 134 days for those exposed during june. the mean maximum survival times of larvae hatching from these eggs varied between 175–245 days, with no clear seasonal pattern of survival. she stated, “while these data highlight the tremendous survival capacity of these larvae, their survival under true field conditions may be considerably reduced. it should also be remembered that maximum survival periods have been recorded with no attention being paid to the rate of mortality, or the ability of the ageing larvae to attach and feed.” spickett & heyne (1990) placed engorged female r. (b.) decoloratus in gauze containers at the base of grass stems in a field covered throughout the year with approximately 50–100 cm tall indigenous grass. they found that high temperature at the onset of oviposition and rapid temperature accumulation thereafter during the summer months resulted in an incubation period as short as 40 days for eggs laid in december, whereas a low initial temperature and slow temperature accumulation thereafter lead to an incubation period of 138 days for eggs laid during august. they suggested that the critical minimum temperature below which hatching does not take place appears to be 10 °c. larvae survived for approximately 10–35 weeks after hatching and the duration of the survival period was inversely dependent on increased temperature accumulation. in their experiments, larvae that hatched during december and january survived for the shortest period, while larvae that hatched during the third week of april, just prior to the onset of low winter temperatures, survived the longest. the viability of surviving larvae, determined by percentage attachment to cattle, was remarkably good, and not much shorter than their total survival period. the maximum survival time of 84 days for larvae exposed to the prevailing climatic conditions in the present study is thus considerably shorter than that recorded by other authors. questing larvae small-scale cattle farmers residing at botshabelo make communal use of natural pastures along the modder river on the western periphery of the township. at monthly intervals from september 1998 to august 1999 questing tick larvae were collected by 329 m.s. phalatsi, l.j. fourie & i.g. horak dragging ten weighted flannel strips (each 100 x 10 cm), attached adjacent to each other on a wooden spar, over the vegetation alongside the river. on each occasion a single 100 m long drag-sample was taken between 10:00 and 12:00 from the vegetation of three localities along the river. the larvae so collected were identified and counted under a stereoscopic microscope and only the data pertaining to r. (b.) decoloratus were used. larvae were collected from the vegetation during each month of the survey, with peak numbers present during january and february 1999 and very few from may to august 1999 (fig. 1). the peak in larval numbers on the vegetation near botshabelo occurred about 1 month earlier than that recorded on vegetation at three localities in the eastern cape province (rechav 1982) and 2–3 months later than that recorded in the lowveld of mpumalanga province, where temperatures are considerably higher than in the eastern free state (horak et al. 2000). the decline in larval numbers on the vegetation near botshabelo from march onwards could have resulted from depletion by attachment to grazing cattle, accidental ingestion by these animals, predation, or age-related mortality, and was probably due to a combination of these. their increase on the vegetation in september can be ascribed to the hatching of eggs laid by females that had detached during march or april (late summer and autumn) in response to a rise in temperature in spring (robertson 1981). parasitic larvae at approximately 4-week intervals from september 1998 to august 1999 ticks were collected from ten cattle of mixed-breed and older than 1 year, belonging to a single owner at botshabelo. except when an animal was sold or had died, and replaced by the owner with another of similar age, the same animals were examined throughout the study. no acaricides were used on the animals during the study period. ticks were collected from 20 x 20 cm areas of the skin, delineated by a wire square, on the neck, abdomen and iguinal region of each animal. the left and right hand sides of the cattle were sampled in alternate months. after the collection of all visible ticks from one of these areas it was combed with a flea-comb followed by thorough scraping with a serrated edged knife blade. at the laboratory the ticks were separated from the hair under a stereoscopic microscope, identified and counted. for the purpose of this study only the numbers of r. (b.) decoloratus collected were considered. although larvae were collected from the vegetation throughout the year, they were recovered from cattle only from october to may, with a peak in intensity of infestation during january and february 1999 (fig. 1). there was otherwise a significant similarity in the seasonality of larvae collected from the animals and from the vegetation (p < 0.01) (pearson correlation co-efficient test). adult ticks were present throughout the year. the peak in total numbers of r. (b.) decoloratus on the cattle during april, 2 months after peak numbers of larvae had been collected from them and from the vegetation may signify a slight delay in development on the host during the cooler months. this would imply that although larvae were still being acquired in small numbers, the adults from earlier infestations were not detaching at the same tempo. acknowledgements our sincere thanks go to ellie van dalen and eddie williams for their technical and field support, respectively. mr phalatsi greatly appreciates the encouragement he received from his administrative senior mr sefika. the fieldwork at botshabelo would not have been possible without the enthusiastic cooperation of the cattle owners, mr bistolo and mr thami. 330 larval biology of rhipicephalus (boophilus) decoloratus (acarina: ixodidae) in free state, south africa ����� ����� ��� ��� ��� ��� � � �� �� �� � � � � � � � � � � � � � � ����������� �� ������ ����������� � ��� ���� ���� � � � � � � � � �� � �� �� � � ��� ��� ��� ��� ��� ��� � �������������������������� ��������������������� ������������������������� fig. 1 the seasonal occurrence of larvae of rhipicephalus (boophilus) decoloratus on vegetation and on cattle in an eastern region of free state province references baker, maureen k., ducasse, f.b.w., sutherst, r.w. & maywald, g.f. 1989. the seasonal tick populations on traditional and commercial cattle grazed at four altitudes in natal. journal of the south african veterinary association, 60:95–101. de vos, a.j. & potgieter, f.t. 1994. bovine babesiosis, in infectious diseases of livestock with special reference to southern africa, edited by j.a.w. coetzer, g.r. thomson & r.c. tustin. cape town: oxford university press. dreyer, karin, fourie, l.j. & kok, d.j. 1998. tick diversity, abundance and seasonal dynamics in a resource-poor urban environment in the free state province. onderstepoort journal of veterinary research, 65:305–316. fourie, l.j., kok, o.b. & van zyl, j.m. 1991. spatial distribution of the karoo paralysis tick ixodes rubicundus (acari: ixodidae) within a false upper karoo veld type. experimental and applied acarology, 11:37–49. goddard, j. 1992. ecological studies of adult ixodes scapularis in central mississippi: questing activity in relation to time of year, vegetation type, and meteorologic conditions. journal of medical entomology, 29:501–506. horak, i.g., spickett, a.m. & braack, l.e.o. 2000. fluctuations in the abundance of boophilus decoloratus and three rhipicephalus species on vegetation during eleven consecutive years. proceedings of the third international conference on ticks and tick-borne pathogens: into the 21st century, edited by m. kazimírová, m. labuda & p.a. nuttall. bratislava: slovak academy of sciences, 1999: 247–251. howell, c.j., walker, jane b. & nevill, e.m. 1978. ticks, mites and insects infesting domestic animals in south africa. part 1. descriptions and biology. pretoria: department of agricultural technical services, republic of south africa. (science bulletin, no. 393). londt, j.g.h. 1974. the preoviposition period of boophilus decoloratus (koch 1844) (acarina: ixodidae). journal of the entomological society of southern africa, 37:405–412. londt, j.g.h. 1977. oviposition and incubation in boophilus decoloratus (koch, 1844) (acarina: ixodidae). onderstepoort journal of veterinary research, 44:13–20. mason, c.a. & norval, r.a.i. 1980. the ticks of zimbabwe. i. the genus boophilus. zimbabwe veterinary journal, 11:36– 43. rechav, y. 1982. dynamics of tick populations (acari: ixodidae) in the eastern cape province of south africa. journal of medical entomology, 19:679–700. robertson, wendy d. 1981. a four year study of the seasonal fluctuations in the occurrence of the blue tick boophilus decoloratus (koch) in the coastal regions of the eastern cape. proceedings of an international conference on tick biology and control, grahamstown, 1981: 199–204. spickett, a.m. & heyne, heloise. 1990. the prehatch period and larval survival of boophilus decoloratus (koch, 1844) (acarina: ixodidae) under natural conditions in the transvaal, south africa. onderstepoort journal of veterinary research, 57:95–98. 331 m.s. phalatsi, l.j. fourie & i.g. horak durden_59-66.qxd introduction a large number of surveys on the arthropod parasites, particularly ticks, of impalas, aepyceros melampus, have been conducted in southern africa (for review see horak, gallivan, braack, boomker & de vos 2003). the collection techniques employed in a number of these surveys were sufficiently sensitive to collect lice and the louse burdens of impalas have been recorded by horak (1982), horak, boomker, kingsley & de vos (1983), van dyk & mckenzie (1992), matthee, horak & meltzer (1998) and horak et al. (2003). in other surveys the presence of lice has been recorded incidentally. the four louse species (two chewing lice in the genus damalinia and two sucking lice in the genus linognathus) known to infest impalas have been listed by ledger (1980), and were recovered in most of the surveys, but in some of them an unknown linognathus sp. was encountered. on examination these lice appeared to be the same as those collected during october 1965 from an impala at kamazeu, zimbabwe (then rhodesia), and those collected by j.a. ledger during august 1968 from an impala at skukuza in the kruger national park (knp), south africa. weisser (1975) described these lice from an entire female and the terminalia of the male in an unpublished monograph on the linognathidae. several thousand specimens of this louse, designated only as linognathus sp., have been collected from impalas in the knp by horak et al. (2003). the same louse has been collected from impalas examined on a privately owned 59 onderstepoort journal of veterinary research, 71:59–66 (2004) linognathus wweisseri n. sp. (phthiraptera: linognathidae) of impalas, aepyceros mmelampus: description and biology l.a. durden1 and i.g. horak2 abstract durden, l.a. & horak, i.g. 2004. linognathus weisseri n. sp. (phthiraptera: linognathidae) of impalas, aepyceros melampus: description and biology. onderstepoort journal of veterinary research, 71:59–66 the adult male and female and first instar nymph of the sucking louse linognathus weisseri n. sp. are described. this louse was collected from impalas, aepyceros melampus, at three localities in limpopo province, and at three in mpumalanga province, south africa. although it usually accounted for only a small proportion of the total louse burden, its overall prevalence exceeded 27 %. its prevalence on adult male impalas (9 %) was significantly lower (p = 0.004) than that on adult females (39 %), but did not differ among age classes. however, the intensity of l. weisseri infestation was higher on lambs than on yearlings and adults, and peaked on impalas in late winter to early summer. five species of lice are now known to parasitize impalas and a key for distinguishing adults of these species is included. keywords: aepyceros melampus, impalas, lice, linognathus weisseri, phthiraptera 1 department of biology and institute of arthropodology and parasitology, box 8042, georgia southern university, statesboro, ga 30460, united states of america 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa accepted for publication 17 october 2003—editor game ranch by matthee et al. (1998). in this paper the adults and first instar nymph of this louse are described and illustrated, and its biology, garnered from matthee et al. (1998) and horak et al. (2003) as well as other sources, outlined. materials and methods the impalas from which the louse was collected had been processed for ectoparasite recovery as described for greater kudus, tragelaphus strepsiceros, by horak, boomker, spickett & de vos (1992), and the lice were recovered from the processed material as described by horak et al. (2003). the localities at which linognathus weisseri was collected and its prevalence on impalas at these localities are summarized in tabular format and its seasonal occurrence is graphically illustrated. descriptive format taxonomic descriptions follow the format of musser & durden (2002) with modifications specific to linognathus used by weisser (1975) and weisser & ledger (1977). names of setae are from kim & ludwig (1978); these names are spelt out when first used (with abbreviations provided in parentheses), and subsequently abbreviated. description male (fig. 1): length of holotype, 1.32 mm; mean, 1.32 mm; range, 1.26–1.36 mm (n = 6). head, thorax and abdomen moderately sclerotized. head longer than wide, rounded, with blunt anterior apex. three apical head setae (aphs), two dorsal pre-antennal head setae (dpahs), two supra-antennal head setae (spaths), two sutural head setae (shs), two dorsal posterior central head setae (dpochs), one dorsal marginal head seta (dmhs), two to three ventral pre-antennal head setae (vpahs), and one ventral principal head seta (vphs) on each side; vphs moderate in length. antennae 5-segmented, relatively long and slender, with basal segment wider than long and wider than second segment. thorax broader than head with curved margins widening posteriorly; thoracic sternal plate absent; mesothoracic spiracle sub-circular, 0.04 mm in diameter; dorsal principal thoracic seta (dpts) fairly short (0.085 mm) with one (rarely two) small dorsal mesothoracic seta (dmss) between spiracle and dpts on each side; one short dorsal prothoracic seta (dpts) on each side. legs long and relatively narrow; mid-legs and associated claws larger and more robust than fore-legs; hind-legs slightly larger than mid-legs; hind coxae sub-triangular; fore and mid coxae as depicted in fig. 1a. abdomen wider than thorax with curved margins and fine crenulations on dorsal and ventral surfaces as shown in fig. 1a. paratergal plates, tergites and sternites (except sub-genital plate) absent as is typical for linognathus; six moderately-sized spiracles 60 linognathus weisseri n. sp. (phthiraptera: linognathidae) of impalas fig. 1 linognathus weisseri, n. sp., male. a. dorso-ventral view. b. genitalia. both scale bars = 0.1 mm on each side; dorsal marginal abdominal setae (dmas) associated with third spiracle consisting of one long and one moderately long (but clearly shorter) seta; dmas associated with fourth to sixth spiracles each consisting of two long setae; other abdominal setae short to moderate in length (except for four long posterior setae ventrally) and more or less arranged in 12 ventral rows (anterior to subgenital plate) and 14 dorsal rows. dorsal setal rows consisting of the following approximate numbers of setae from anterior to posterior: 4, 6, 6, 12, 10, 5, 10, 6, 12–14, 6, 12–14, 16–18, 2, and 14–18. most dorsal setae are dorsal central abdominal setae (dcas), but rows 3–5 also have dorsal lateral abdominal setae (dlas) as shown in fig. 1a. ventral setal rows anterior to sub-genital plate consisting of the following approximate numbers of setae from anterior to posterior: 2, 2, 4, 4, 4, 6, 4, 6–7, 6, 10–11, 4, and 2. most ventral abdominal setae are ventral central abdominal setae (vcas) but some in rows 6, 8 and 10, are ventral lateral abdominal setae (vlas) as shown in fig. 1a. two groups of two to three short dlas lateral to subgenital plate on each side. genitalia: v-shaped sub-genital plate with irregular anterior and lateral margins, and central elliptical lacuna enclosing two elongate setae (fig. 1a). basal apodeme about twice as long as parameres, well sclerotized, constricted medio-laterally, and with posterior fork (fig. 1b); parameres curved, broad anteriorly and tapering posteriorly, with distinct surface patterning (fig. 1b); endomere large and elliptical; pseudopenis relatively long, distinctly extending beyond apices of parameres. a pair of lateral sclerites bearing numerous setae is present near the apex on segment ix. female (fig. 2): length of allotype, 1.68 mm; mean, 1.75 mm; range, 1.60–1.88 mm (n = 10). head, thorax and abdomen as in male with the following exceptions: head with lateral margins relatively straight and parallel; two dorsal anterior head setae (danhs) on each side. thorax less constricted than in male at junction with abdomen giving the overall body an elliptical shape (fig. 2a). dpts length, 0.128 mm. abdomen: dmas associated with third spiracle consisting of one short seta and one of moderate length; abdominal setae more or less arranged in 12 rows ventrally (anterior to gonopods) and 17 rows dorsally although anterior dorsal rows are difficult to distinguish (fig. 2a). dorsal setal rows consisting of the following approximate numbers of setae from anterior to posterior: 2, 6, 12, 10, 6, 12, 6, 12, 10, 6, 10, 6, 10, 6, 10, 12–14; most dorsal setae are dcas but rows 4, 6, 8, 11, 13, and 15 also have dlas as shown in fig. 2a. a few dcas and dlas are elongate (fig. 2a). ventral setal rows anterior to gonopods consisting of the following approximate numbers of setae from anterior to posterior: 4, 4, 4, 6, 4, 12, 4, 12, 4, 12, 6, 8, 6. most ventral setae are vcas but rows 4, 6, 8, and 10 61 l.a. durden & i.g. horak fig. 2 linognathus weisseri, n. sp., female. a. dorso-ventral view. b. genitalia. both scale bars = 0.1 mm also have vlas as shown in fig. 2a. two to four short vlas on each side at base of gonopods on segment viii. genitalia (fig. 2b): sub-genital plate sub-rectangular and bearing 10–16 small setae; gonopods on abdominal segment viii finger-like with sclerotized lateral margins and rounded apices, each bearing seven to nine setae that are short medially and increase to moderate length posterolaterally; row of tiny setae situated medially between bases of these gonopods; gonopods on abdominal segment ix situated terminally and tapering to form forficulate apical lobes each bearing a row (possibly two separate rows) of hair-like setae. first instar nymph (fig. 3): mean length, 1.09 mm; range, 1.02–1.20 mm (n = 3). head moderately sclerotized; thorax and abdomen lightly sclerotized and with surface sculpting as shown for abdomen of male (fig. 1a). head shaped as in male but with small, pointed, antero-lateral projection immediately posterior to antennal insertion on each side. dorsal head setae as in male. ventral head with three aphs and one vphs on each side. thorax distinctly wider than head, with mesothoracic spiracle (0.03 mm in diameter) and one vpts (0.084 mm in length) on each side. abdomen at widest point slightly wider than thorax; six moderately-sized spiracles, three pairs of long dmas posteriorly (each pair borne on a protruding area of integument), and one pair of moderately long dmas anterior to these on each side. dorsally, with nine pairs of dcas of moderate length; ventrally, with one pair of short anterior vcas and three pairs of posterior vcas of moderate length. specimens examined holotype male, allotype female and 17 paratypes (five males, nine females, three first instar nymphs) ex adult female aepyceros melampus (lichtenstein), nwaswitshaka river bridge, skukuza (24°58’s, 31°36’e), kruger national park, mpumalanga province, south africa; 24 november 1982; coll.: i. g. horak. deposition of types holotype male, allotype female and one paratype nymph (accession number tm 2031126) deposited in the u.s. national museum of natural history, washington dc. paratypes will also be deposited in the natural history museum, london. etymology we have pleasure in naming this species after dr christian f. weisser who first described the entire female and the male genitalia of this species in his dissertation but did not publish the description. we greatly appreciate dr weisser’s encouragement to proceed with this description. synonymy linognathus melampi weisser, 1975; this is an unpublished manuscript name. weisser (1975) stated that a female holotype, a male allotype (two slides) and another four females and two males (four slides) of l. melampi had been deposited in the south african institute of medical research (saimr) collection under “rev. (sic) no. l 232” and “l 231” respectively, but we have been unable to locate these specimens. diagnosis linognathus weisseri is a fairly small and distinct member of its genus. it can be easily distinguished 62 linognathus weisseri n. sp. (phthiraptera: linognathidae) of impalas fig. 3 linognathus weisseri, n. sp., first instar nymph. dorsoventral view. scale bar = 0.1 mm from linognathus elblae, linognathus breviceps and linognathus nesotragi by the absence of hookshaped sclerotized margins on the forehead. it can be distinguished from other sucking lice on impalas by the absence of a thoracic sternal plate (which is present in linognathus aepycerus), the almost parallel post-antennal margins of the head (which are distinctly bulging in l. aepycerus), the slender body shape (which is globular in linognathus nevilli) and the relatively small spiracles (which are large in l. nevilli). the shape of the subgenital plates and genitalia in both sexes further distinguish l. weisseri from other described species of linognathus including those found on impalas. key to adult lice parasitizing impalas 1a. head wider than thorax (chewing lice, damalinia spp.) . .2 1b. head narrower than thorax (sucking lice, linognathus spp.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 2a. head sub-triangular and elongate (about twice as long as wide) with small apical notch . . . . . . .damalinia elongata 2b. head squarish and short (about 1.2 times long as wide) with tapering anterior and posterior lateral margins and large apical notch . . . . . . . . . . . . . . .damalinia aepyceros 3a. body slender (abdomen not more than twice as wide as thorax at widest point), abdominal spiracles small . . . . .4 3b. body globular (abdomen 2.5–3 times as wide as thorax at widest point), abdominal spiracles large . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . linognathus nevilli 4a. thoracic sternal plate present, post-antennal head margin distinctly convex . . . . . . . . . . . . . .linognathus aepycerus 4b. thoracic sternal plate absent, post-antennal head margin nearly straight or mildly convex . . . .linognathus weisseri results and discussion linognathus weisseri was collected from impalas at five of the six localities at which impalas were examined within the kruger national park (knp), and was also present on impalas on letaba ranch, a private game farm in north-eastern limpopo province, south africa (table 1). although l. weisseri usually accounted for only a small percentage of the total number of lice collected, it was common on impalas, with an overall prevalence of 28.8 % on animals examined in the knp and 17.8 % on animals on letaba ranch. in contrast l. aepycerus and l. nevilli were present in all localities in the limpopo and mpumalanga provinces at which impalas were examined, with the former having an overall prevalence rate of 79.4 % and the latter 55.9 %. the prevalence and intensity of infestation of l. weisseri on impalas in the knp did not differ between the skukuza and mbiyamiti regions in 1980/ 81, or between skukuza and pafuri in 1992/93 (p = 0.35). at skukuza the prevalence did not differ between 1980/81 and 1992/93 (p = 0.74) but the intensity of infestation was higher in 1980/81 than in 1992/93 (p = 0.01). the prevalence was considerably lower (p = 0.004) on adult male impalas (9 %) than on adult females (39 %), and none were collected from the adult males at crocodile bridge. the prevalence and intensity of infestation of l. aepycerus on the same animals did not differ significantly between the sexes (p > 0.16), whereas those of l. nevilli did (p < 0.006) (horak et al. 2003). 63 l.a. durden & i.g. horak ����� � � � � �� � � �� � � �� � � ���� � � � � ���� � � � ���� � � � ����� � �� � �� �� �� �� ��������� �� � �� fig. 4 seasonal pattern of intensity of infestation of linognathus weisseri on impalas at skukuza and in the mbiyamiti regions, kruger national park, south africa during 1980/81. the monthly sample ranged from 4–13 impalas 64 linognathus weisseri n. sp. (phthiraptera: linognathidae) of impalas t a b l e 1 li no gn at hu s w ei ss er ic o lle ct e d f ro m im p a la s a t va ri o u s lo ca lit ie s in s o u th a fr ic a n o . e xa m in e d n o . o f l. w ei ss er ic o lle ct e d p ro p o rt io n l o ca lit y c o o rd in a te s p ro vi n ce p e ri o d o f to ta l l o u se (n o . in fe st e d ) n ym p h s a d u lts t o ta l b u rd e n ( % ) n yl sv le y 2 4 °2 9 ’s , 2 8 °4 2 ’e l im p o p o 1 9 7 5 /7 6 3 6 (0 ) 0 0 0 0 l e ta b a r a n ch 2 3 °4 0 ’s , 3 1 °0 4 ’e l im p o p o 1 9 9 4 /9 5 4 5 (8 ) 2 5 4 2 9 0 .5 8 p a fu ri 2 2 °2 7 ’s , 3 1 °1 9 ’e l im p o p o ( k n p ) 1 9 8 0 – 1 9 9 3 2 5 (5 ) 4 0 3 0 7 0 1 .3 n to m e n i p a n s 2 3 °2 6 ’s , 3 1 °1 4 ’e l im p o p o ( k n p ) ju ly 1 9 8 2 2 (2 ) 1 0 4 1 4 1 6 .2 8 s ku ku za 2 4 °5 8 ’s , 3 1 °3 6 ’e m p u m a la n g a ( k n p ) 1 9 8 0 – 1 9 9 3 1 3 9 (5 0 ) 1 9 6 4 1 5 6 9 3 5 3 3 3 .4 6 m b iy a m iti 2 5 °1 7 ’s , 3 1 °3 2 ’e m p u m a la n g a ( k n p ) 1 9 8 0 /8 1 6 0 (1 1 ) 2 4 0 1 7 2 4 1 2 3 .8 1 c ro co d ile b ri d g e 2 5 °2 2 ’s , 3 1 °5 4 ’e m p u m a la n g a ( k n p ) 1 9 8 0 /8 1 1 2 (0 ) 0 0 0 0 l o w e r s a b ie 2 5 °0 6 ’s , 3 1 °5 5 ’e m p u m a la n g a ( k n p ) o ct o b e r 1 9 8 2 1 (1 ) 8 0 0 8 0 5 .4 3 s t l u ci a g a m e p a rk 2 8 °2 3 ’s , 3 2 °3 0 ’e k w a z u lu -n a ta l m a y 1 9 8 4 2 (0 ) 0 0 0 0 the prevalence of l. weisseri did not differ among age classes (p = 0.44), but the intensity of infestation was higher on lambs than on yearlings and adults (p = 0.01). two impala lambs, less than 2 weeks of age were examined, and one was already infested with l. weisseri. both month-old lambs examined were infested. the distribution of l. weisseri on the bodies of 40 impalas, on which it could be determined, is summarized in table 2. most lice were present on the necks, trunks, upper legs and tails of the impalas. unfortunately the collecting procedure followed by horak et al. (2003) did not allow for the determination of a more accurately defined distribution pattern. although the collection procedure of matthee et al. (1998) does permit for such a determination, they collected too few l. weisseri to ascertain its preferred body surface locality. in contrast l. aepycerus is present on the head, neck, body, legs and tail of impalas, with no specific preferred locality, whereas l. nevilli has a pronounced preference for the hind legs (matthee et al. 1998). the intensity of infestation of l. weisseri, determined from the burdens of the 123 impalas examined in sets of 4–13 animals at monthly intervals at skukuza and in the mbiyamiti region, peaked from august to november (fig. 4). that of l. aepycerus on the same animals peaked from july to october, and although there was no clear pattern of seasonality for l. nevilli, most were collected during march, april, august, october and november (horak et al. 2003). although no l. weisseri were collected from january to june 1980, it was present on impalas examined during some of these months in 1981 and in 1992/93. not only does the morphology of l. weisseri differ from that of l. aepycerus and l. nevilli, the two other anopluran lice that infest impalas, but its biology also differs, thus confirming its identity as a valid separate species. acknowledgements we thank dr christian weisser, university of heidelberg, germany, for encouraging us to formally describe this new species, and the south african national parks for placing the impalas and the facilities in the knp at our disposal. our thanks also go to dr jim gallivan of ottowa, canada for statistical processing of the data. references horak, i.g. 1982. parasites of domestic and wild animals in south africa. xv. the seasonal prevalence of ectoparasites on impala and cattle in the northern transvaal. onderstepoort journal of veterinary research, 49:85–93. horak, i.g., boomker, j., kingsley, shirley a. & de vos, v. 1983. the efficacy of ivermectin against helminth and arthropod parasites of impala. journal of the south african veterinary association, 54:251–253. horak, i.g., boomker, j., spickett, a.m. & de vos, v. 1992. parasites of domestic and wild animals in south africa. xxx. ectoparasites of kudus in the eastern transvaal lowveld and the eastern cape province. onderstepoort journal of veterinary research, 59:259–273. horak, i.g., gallivan, g.j., braack, l.e.o., boomker, j. & de vos, v. 2003. parasites of domestic and wild animals in south africa. xli. arthropod parasites of impalas, aepyceros melampus in the kruger national park. onderstepoort journal of veterinary research, 70:131–163. kim, k.c. & ludwig, h.w. 1978. the family classification of the anoplura. systematic entomology, 3:249–284. ledger, j.a. 1980. the arthropod parasites of vertebrates in africa south of the sahara. iv. phthiraptera (insecta). johannesburg: the south african institute for medical research (publication no. 56). matthee, sonja, horak, i.g. & meltzer, d.g.a. 1998. the distribution and seasonal changes of louse populations on impala aepyceros melampus. south african journal of wildlife research, 28:22–25. 65 l.a. durden & i.g. horak table 2 distribution of linognathus spp. on the bodies of impalas total number (proportion %) of lice recovered louse species no. of impalas examined head and ears neck, trunk, upper lower legs and feet legs and tail linognathus weisseri 40 104 (3.2 %) 3 147 (96.6 %) 8 (0.2 %) linognathus aepycerus* 45 349 (18.5 %) 1 041 (55.1 %) ** 498 (26.4 %) *** linognathus nevilli* 45 15 (0.6 %) 224 (9.5 %) ** 2 130 (89.9 %) *** * adapted from matthee et al. (1998) ** neck, trunk and tail *** legs and feet musser, g.g. & durden, l.a. 2002. sulawesi rodents: description of a new genus and species of murinae (muridae, rodentia) and its parasitic new species of sucking louse (insecta, anoplura). american museum novitates, 3368:1– 50. van dyk, p.j. & mckenzie, a.a. 1992. an evaluation of the effectivity of the scrub technique in quantitative ectoparasite ecology. experimental and applied acarology, 15:271–283. weisser, c.f. 1975. a monograph of the linognathidae, anoplura, insecta. (excluding the genus prolinognathus). unpublished ph.d. dissertation, university of heidelberg. weisser, c.f. & ledger, j.a. 1977. two new linognathus (phthiraptera: linognathidae) from roan and nyala (bovidae) in southern africa. journal of the entomological society of south africa, 40:283–289. 66 linognathus weisseri n. sp. (phthiraptera: linognathidae) of impalas abstract introduction material and methods study methods data analysis discussion conclusion acknowledgements references about the author(s) adama sanou department of biomedical, centre muraz, bobo-dioulasso, burkina faso training and research unit in science and technology, nazi boni university, bobo-dioulasso, burkina faso amadou dicko department of biomedical sciences, centre muraz, bobo dioulasso, burkina faso kadiatou r. sow department of biomedical sciences, centre muraz, bobo dioulasso, burkina faso faculty of medicine and dentistry (fmos), university of sciences, techniques and technologies of bamako (usttb), bamako, mali arthur djibougou department of biomedical, centre muraz, bobo-dioulasso, burkina faso antoinette kabore department of biomedical sciences, centre muraz, bobo dioulasso, burkina faso bassirou diarra university of sciences, techniques and technologies of bamako, bamako, mali arsène k. ouedraogo department of biomedical sciences, centre muraz, bobo dioulasso, burkina faso dezemon zingue department of biomedical sciences, centre muraz, bobo dioulasso, burkina faso moumini nouctara department of biomedical sciences, centre muraz, bobo dioulasso, burkina faso zekiba tarnagda training and research unit in science and technology, nazi boni university, bobo-dioulasso, burkina faso citation sanou, a., dicko, a., sow, k.r., djibougou, a., kabore, a., diarra, b. et al., 2021, ‘epidemiology and microscopic diagnosis of tuberculosis in pigs and small ruminants slaughtered at bobo-dioulasso abattoir, burkina faso’, onderstepoort journal of veterinary research 88(1), a1908. https://doi.org/10.4102/ojvr.v88i1.1908 research communication epidemiology and microscopic diagnosis of tuberculosis in pigs and small ruminants slaughtered at bobo-dioulasso abattoir, burkina faso adama sanou, amadou dicko, kadiatou r. sow, arthur djibougou, antoinette kabore, bassirou diarra, arsène k. ouedraogo, dezemon zingue, moumini nouctara, zekiba tarnagda received: 25 sept. 2020; accepted: 03 june 2021; published: 03 dec. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract bovine tuberculosis (btb) is a zoonotic, infectious, chronic and contagious disease, caused by mycobacterium bovis that mainly affects cattle. this pathology has a negative impact on animals and animal products trade. unfortunately, in burkina faso where agriculture and livestock sectors represent around 80% of the socio-economic activities, the real situation of the disease is not well known especially in small ruminants and swine. thus, our study focused on both the epidemiology and the microbiological diagnosis of tuberculosis (tb) in small ruminants and pigs slaughtered at bobo-dioulasso abattoir. a prospective study was conducted between august 2017 and december 2017. epidemiological data collection was performed during routine meat inspection; moreover, samples were taken and transported to the bacteriology laboratory of centre muraz for microbiological analyses. this diagnosis consisted in search of acid fast bacilli (afb) using the hot ziehl–neelsen staining. out of a total of 14 648 small ruminants and 2430 pigs slaughtered during the study period, 156 and 17 had lesions suggestive of btb with prevalence of 1.07% and 0.7%, respectively. females and those between 2 and 4 years old were mainly infected. the most affected organs were: lungs, liver, spleen and lymph nodes. finally, microscopy revealed 43.35% (75/173) of positive cases for afb. these results confirm the presence of btb in small ruminants and pigs in burkina faso. efforts must still be made in the fight against this zoonosis in order to limit its economic and public health impacts. keyword: tuberculosis; small ruminants; pigs; routine inspection; bobo-dioulasso. introduction livestock plays a central role in the economy of the sahel part of africa, which is illustrated as an excellent region for animal breeding. it is indeed one of the main economic activities on which the regional populations are dependent as a source of food and monetary income (csao/ocde 2008). this sector is unfortunately under constant pressure of various parasitic, viral and bacterial pathologies. amongst these, bovine tuberculosis (btb) is one of the most difficult to control because of its insidious and chronic nature. bovine tuberculosis is a bacterial infectious disease caused by mycobacterium bovis (m. bovis), whose main victims are not only domestic cattle but it also affects other animals (domestic and wild) and human (cosivi et al. 1995; csao/ocde 2008). it is considered a major disease for international trade of live animals and animal products, and is on the list of notifiable diseases of the world organisation for animal health (oie). consequently, several developed countries have implemented campaigns to eliminate m. bovis from the cattle herd or at least to control its spread. the success of these control programmes has often been mitigated. indeed, the fact that m. bovis infects a wide host range can complicate attempts to control or eradicate the disease in cattle. however, with multiple efforts to control it, the disease is rare in developed countries. also, the introduction of pasteurisation considerably reduced public health risks in many countries, but the disease continues to cause production losses where it is poorly controlled. regrettably, diseases although eradicated from parts of europe such as foot and mouth disease, rabies, btb, remain enzootic in many developing countries. in africa, in 1998 only seven countries (algeria, burkina faso, cameroon, morocco, namibia, south africa, and zambia) out of 55 had a btb control programme in cattle herds, using tuberculin tests and post-mortem inspection for the surveillance of this disease (cosivi et al. 1998). currently, programmes are implemented in most african countries but remain ineffective because of not only the transhumance movements of animals from areas where the measures are not applied at all but also the lack of synergy between countries involved in the fight. in the sahel, more precisely in burkina faso, small ruminants and cattle breeding is practiced extensively and depends mostly on the availability of natural grazing and water. their availability or abundance are linked to the annual rainfall, which is very often insufficient, thus forcing breeders and their livestock to move from arid regions to more humid ones. in addition, the existence of priority enzootic diseases, such as contagious bovine pleuropneumonia (cbpp), foot-and-mouth disease, which decimate among cattle, and the lack of means and the insidious nature of btb have led to continuous neglect of this disease in the country. the government meat inspection regulations however, provide for regular checks of animal carcasses before consumption. thus, the only information available most often comes from suspicions during routine meat inspection at slaughterhouses and slaughter areas. nevertheless, the existence of btb has been known for a very long time in this country, where the highest rates from all french west african countries were observed in slaughterhouses (regnoult 1963). nonetheless, several studies on btb have only concentrated on cattle, and there are few data available on small ruminants and pigs despite their numerical and economic importance. thus, the study aims to provide epidemiological and microbiological data on btb in small ruminants and pigs slaughtered at the bobo-dioulasso abattoir to contribute to public health and btb control improvement in the country. material and methods type, period and study sites this was a prospective cross-sectional study that took place at bobo-dioulasso abattoir (hauts bassins region, burkina faso) between august 2017 and december 2017. the study settings also included centre muraz, where the laboratory analyses were carried out. in addition, the animals (small ruminants and pigs) studied potentially originate from different parts of the country. sampling we did an exhaustive sampling. our study focused on cases of organ seizures for suspected tb during the study period. the samples consisted of tissues from suspected organs such as lymph nodes (prescapular, pulmonary, hepatic, mandibular, retropharyngeal …), lungs, spleen, liver, udders …, following routine meat inspection at the slaughterhouse. data collection data collection was carried out using a sheet developed for this study. the data collected were epidemiological and bio-clinical information, in particular the registration number of the suspected animal, the type of seizure, age, sex and anatomo-pathological aspects of the carcass (carcass appearance, lesions location). this information was collected through observation by veterinary technicians during meat inspection. study methods sample processing sampling and transport at bobo-dioulasso abattoir, animals are mostly slaughtered daily from midnight to 6 am and during the day to ensure permanence. the carcasses were inspected by veterinary doctors and officers. pieces of organs from suspect animals were transported at 4 °c to centre muraz for microbiological analyses. three bio-clinical samples per suspected animal were taken to increase the probability of btb detection. sample pre-processing pre-treatment consistent of samples decontamination organ pieces and grinding. the samples taken were previously cleaned with sterile distilled water before treatment using the saeng and costil method (delafosse, traore & kone 1995). for the pre-treatment itself, the suspected piece was first deposited in a sterile petri dish and then cut with a scissor in a sterile manner. secondly, the tissue fragments were finely ground in a sterile mortar in presence of sterile sea sand. then 10 ml of sterile distilled water was added to the homogenate and the mixture was collected in a sterile 50 ml falcon tube. finally, after 5 min decanting, a 2 ml volume of supernatant was recovered and placed in another sterile 50 ml falcon tube, and then processed according to petroff method (palomino & portaels 1998) for decontamination in agreement with the following procedure: step 1: a total of 10 ml of naoh were added to the 2 ml of recovered supernatant and the mixture was vigorously mixed with a vortex for 30 s, then for 20 min on the kahn agitator. step 2: the suspension was then completed with sterile distilled water until an average volume of 45 ml and mixed on a vortex for 30 s. step 3: subsequently, the suspension was centrifuged for the first time at 3000 revolutions per minute (rpm) for 20 min. step 3 was repeated 3 times. step 4: after removal of the supernatant, a volume of 1.5 ml of sterile distilled water was added to the pellet and the tube was vigorously vortexed to until obtained an homogenous mixture, ready for microbiological analysis. microbiological analysis methods one part of the pellet was used for microscopical analysis using ziehl–neelsen staining so as to detect the presence of acid fast bacilli (afb). in order to have optimal conditions, all the slides to be used must be sterilised with a fire source (typically a spirit lamp) applying the heat on both faces for a few seconds. a certain volume of the sample is extracted and spread on the slide making a thin uniform barely transparent smear with a sterile bacteriological sampling loop. once this is done, the spread is fixed with a little heat from the spirit lamp, applied (only) under the slide before undergoing staining using the hot ziehl–neelsen method. data analysis a double entry was used to record data using sphinx plus2 v5 software where appropriate, descriptive parameters such as sums, percentages and fractions were then computed. statistical analysis was performed using epi info version 7.2.2.6. logistic regression was used to assess the relationship between lab results and animal characteristics. the p < 0.05 value was considered statistically significant. results frequencies of suspected tuberculosis lesions out of 14 648 total small ruminants and 2430 pigs slaughtered during the study period, 156 and 17 had tb suspected lesions giving prevalence of 1.07% and 0.70%, respectively, and an overall prevalence of 1.01% (see table 1). table 1: frequency of suspicious lesions in small ruminants and pigs slaughtered at the bobo-dioulasso abattoir from august to december 2017. distribution of tuberculosis suspect animals by age and sex of the 173 suspected btb carcasses, 141 (81.5%) were females and 32 (18.5%) were males, that is, a sex ratio of 0.23 (32/141) (see table 2). the age of the animals were between 1 year and 5 years as shown in figure 1. figure 1: repartition of tuberculosis suspected animals by age. table 2: sex ratio in small ruminants and pigs suspected of tuberculosis at the abattoir from august 2017 to december 2017. location of bovine tuberculosis lesions of the 173 suspected animals, 95.38% presented localised lesions to some organs (lungs, liver, spleen, lymph nodes, …) against 4.62% with generalised lesions throughout the carcass. the most affected organ was the lung with a proportion of 36.07%. however, there were sub-populations specificities because the liver was the most affected organ for pigs. for affected organs and the relative numbers refer to figure 2. figure 2: distribution of suspicious bovine tuberculosis lesions by organ in small ruminants (sheep and goats) and pigs at bobo-dioulasso slaughterhouse. proportion of suspected bovine tuberculosis cases based on microscopic examination amongst the 173 carcasses with suspicious lesions, microscopy revealed the presence of btb in 75 (43.35%) samples. table 3 shows the microscopy positive results by animal species. table 3: microscopy results of the different suspected carcasses (sheep, goats and pigs). risk factor analyses the results of multivariate analysis of laboratory results according to animal characteristics are shown in figure 3. this shows that the infection is neither related to age nor to the animal species. figure 3: multivariate analysis of laboratory results according to animal characteristics. discussion the objective of this study was to explore the full breadth of btb in small ruminants and swine populations in burkina faso, suspecting its presence based on its frequency in cattle slaughtered and routinely inspected in burkina faso slaughterhouses. the present study indicates the existence of btb in burkina faso with a proportion of 1.01% of lesions suggestive of this pathology in small ruminants and swine inspected. the presence of btb in small ruminants and pigs is not surprising; indeed, evidence of btb existence dates back to the colonial period based on single cervical intradermal test in cattle (sere 1966). in addition, btb cases have been reported regularly in cattle in many sites of this country and specifically at bobo-dioulasso slaughterhouse based on routine meat inspection (delafosse et al. 1995; tarnagda et al. 2014; vekemans et al. 1999), sometimes with probable transmission between animals (sanou et al. 2014). regular contacts between different animal species on breeding areas may then explain the circulation of the bacillus and persistence of the disease between and within the different animal sub-populations, respectively (gelalcha, zewude & ameni 2019). in the present study, the prevalence of the disease was higher in small ruminants compared with pigs, with respective prevalence of 1.07% and 0.70%. our results were higher than the 0.03% found in goats in nigeria in 2016 (danbirni et al. 2016) and 0.15% and 0.07%, respectively, in sheep and goats found in togo 8 years earlier (kulo & seme 2008). the rates observed elsewhere in africa are higher than those observed in this study. indeed, the prevalence of btb was 6.03% in goats in algeria in 2011 (naima et al. 2011), and 5.80% in ethiopia in pigs in 2013 (arega, conraths & ameni 2013). higher prevalence has also been reported in europe. indeed, it was 10.00% in goats and 3.20% in sheep in spain in 2018 (vidal et al. 2018) and 4.90% in pigs in italy the same year (amato et al. 2018). the present study in addition to confirming the btb existence in small ruminants and swine populations in burkina faso, suggests a slight decrease in its prevalence in pigs compared with the data reported (2.150%) 50 years ago (gidel et al. 1969). the difference in prevalence between our study and those of these authors could be explained by several factors related mainly to farming practices and conditions. the production system with the differences in animal breeding, animal populations density, aeration conditions and introduction of new animals are all factors that influence btb prevalence. thus, within the same geographic region, the prevalence of btb can vary significantly from one area to another. regarding parameters such as age and sex of slaughtered animals, they are not routinely collected by veterinary officers. the data collected during the present study were only related to suspected btb cases. they show that suspected animals were mainly females and between 2 years and 4 years of age even if sex, age are not significantly associated with the prevalence. previous studies have shown a link between sex and btb occurrence (lamireou 2014). these studies found that the disease incidence and seizures were significantly higher in females than males. this could probably be because of hormonal influence, gestation, parturition and lactation, which weaken female’s immune system and thus increase her vulnerability to diseases (kudi et al. 1997). in addition, more females than males are generally slaughtered routinely because the demand for males is affected by religious and cultural events such as christmas, muslim holidays and baptisms and marriages. most of males are kept for slaughter during these periods. as far as age is concerned, it is not generally a risk factor. when it is associated with environmental factors production system (intensive breeding) which favour bacteria circulation, the probability of btb germ infection increases, thus the prevalence can be higher in older animals (boukary et al. 2011). however, age can also lead to a physiological decrease in immunity and therefore constitutes an immunosuppressive factor. regarding the lesions extent that caused carcass seizures during routine inspection, most were located on a given organ. lung damage is the leading cause of btb seizure in sub-saharan africa (kulo & seme 2008; naima et al. 2011). our study confirmed that lungs represent the main localisation for mycobacteria with a frequency of 36.07%. this is also explained by mycobacteria tropism, which infect the lungs first in most cases before spreading to other organs in accordance with air route as btb’s main transmission mode (ullah et al. 2019). these results are similar to those obtained in sudan (aljameel, mohammed & bakhiet 2017) however, other transmission routes such as buccopharyngeal also play a non-negligible role with more than 15% of cases. this can explain the percentage of other lesion types frequently encountered in slaughterhouses such as lymph node reactions, lesions in liver, spleen, etc. the same observation has been made by other authors (amato et al. 2018). these tissues are generally amongst the most affected during routine examination carried out by veterinary inspectors, and thus may contain caseous nodules, usually reflecting chronic btb infection (amato et al. 2018). the liver was the most involved organ in swine, and this may be because of confusion between btb lesions and those of porcine cysticercosis, which is very common in the study area. on the economic level, seizures, especially total ones (0.047%), still constitute a considerable loss for butchers because they receive no compensation. as a result, some pet owners strongly protest and express their outrage for seizure decisions. this total seizure measure would lead to increase in clandestine slaughter as reported by veterinary services. this practice promotes btb transmission to humans, mainly by handling infected carcasses in poor hygienic conditions (sa’idu et al. 2015). the proportion of suspected btb positive lesions in microscopy was 43.35%. despite the microscopic examination low sensitivity, our study found a prevalence above the 13.33% found in algeria in goats (naima et al. 2011) and 10.89% found in democratic republic of congo (luboya et al. 2017). however, the prevalence obtained in the present study was below 76.1% found in cattle in sudan (aljameel et al. 2015). as tb microscopy is most often focused on diagnosis of human cases, the afb search in animal samples requires patience and special attention because of the morphological dissimilarities between the different types of tb bacilli, human and bovine. conclusion this study showed the btb occurrence in small ruminants and pigs slaughtered at bobo-dioulasso abattoir with a prevalence of 1.01% of suspicious lesions. the study found that lungs damage was the leading cause of btb seizure at bobo-dioulasso slaughterhouse. also, the female and old animals were the most infected. additional control efforts are required for more effective control of this zoonosis in burkina faso. acknowledgements the authors would like to thank the regional directorate of animal resources, the bobo-dioulasso slaughterhouse administration, veterinary technicians and centre muraz laboratory workers for technical support. they would also like to thank the nazi boni university for financial support. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions s.a., the main author, was involved in study design, data collection, laboratory work, data analysis and article write-up. a.d. was involved in study design, data collection and data analysis and article write-up. m.n., a.k.o., a.k. and k.r.s. were involved in data collection and laboratory work and article write-up. b.d., d.z., a.d. and z.t. were involved in article write-up. ethical considerations our study obtained authorisation from the ministry of animal and halieutic resources in burkina faso. we also ensured the confidentiality of information provided by the butchers, and the use of the obtained results was limited to the strict exploitation related to the study objectives. this study is also part of the routine diagnosis of btb at bobo-dioulasso slaughterhouse. given that, the routine diagnosis of btb is mandatory, thus no additional informed consent from the owners of animals was required. funding information the authors received financial support from the nazi boni university. data availability the authors confirm that the data supporting the findings of this study are available within the article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references aljameel, m.a., abdel wahab, m.b., fayza, a.o., el tigani, a.e. & abdellatif, m.m., 2015, ‘occurrence of bovine tuberculosis at nyala abattoirs in south darfur state, sudan’, revue d’élevage et de médecine vétérinaire des pays tropicaux 67(2), 61. https://doi.org/10.19182/remvt.10185 aljameel, m.a., mohammed, g.e. & bakhiet, a.o., 2017, ‘tuberculosis in sheep and goats: pathological characteristics based on abattoir study in south darfur state, sudan tuberculosis in sheep and goats: pathological characteristics based on abattoir study in south darfur state, 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et al., 2011, ‘tuberculosis in cattle and goats in the north of algeria’, veterinary research 4(4), 100–103. palomino, j.c. & portaels, f., 1998, ‘effects of decontamination methods and culture conditions on viability of mycobacterium ulcerans in the bactec system’, the journal of clinical microbiology 36, 402–408. https://doi.org/10.1128/jcm.36.2.402-408.1998 regnoult, m.g., 1963, ‘animal tuberculosis in west african territories of french expression’, rev pathol gen physiol clin 63, 1093–1115. sa’idu, a.s., okolocha, e.c., dzikwi, a.a., gamawa, a.a., ibrahim, s., kwaga, j.k.p. et al., 2015, ‘public health implications and risk factors assessment of mycobacterium bovis infections among abattoir personnel in bauchi state, nigeria’, journal of veterinary medicine 2015, 1–5. https://doi.org/10.1155/2015/718193 sanou, a., tarnagda, z., kanyala, e., zingué, d., nouctara, m., ganamé, z. et al., 2014, ‘mycobacterium bovis in burkina faso: epidemiologic and genetic links between human and 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microbiology and infection 5(10), 617–621. https://doi.org/10.1111/j.1469-0691.1999.tb00418.x vidal, e., grasa, m., perálvarez, t., martín, m., mercader, i., pérez de val, b., 2018, ‘transmission of tuberculosis caused by mycobacterium caprae between dairy sheep and goats’, small ruminant research 158, 22–25. https://doi.org/10.1016/j.smallrumres.2017.11.010 mukaratirwa_203-206.qxd introduction philophthalmids are small trematodes of the family philophthalmidae occurring in the eye (conjuctival sac) of birds (kingston 1984). although several species have been reported (yamaguti 1934; ching 1961; nollen & murray 1978; radev, kanev & gold 2000) the family is typified by the species, philophthalmus gralli, commonly known as the “oriental eye-fluke”. philopthalmus gralli was first described from the conjuctival sac of the domestic chicken in hanoi, viet nam, by mathis & leger (1910). it has since been reported in chickens in hawaii (ching 1961), in captive-reared ostriches in florida, usa, (greve & harrison 1980) and in chickens, peafowl, turkeys, ducks, and geese in indochina and formosa (kingston 1984). human infections have also been reported (dissanaike & bilimoria 1958; mimori, hirai, kifune & inada 1982). the eggs or miracidia from the infected birds are eliminated from the avian host into the environment by direct contact of the eyes, nasal and oral passages with water while drinking (alicata 1962). the freshwater snail species, tarebia granifera and melanoides tuberculata have been reported to be the intermediate hosts (nollen & murray 1978). detailed information on the development of p. gralli in t. granifera mauiensis and the domestic chicken has been described by alicata (1962). the clinical signs and lesions associated with attachment of flukes to the conjuctivae are congestion 203 onderstepoort journal of veterinary research, 72:203–206 (2005) first report of an outbreak of the oriental eye-fluke, philophthalmus gralli (mathis & leger 1910), in commercially reared ostriches (struthio camelus) in zimbabwe s. mukaratirwa*, thokozani hove, z.m. cindzi, d.b. maononga, m. taruvinga and elizabeth matenga department of paraclinical veterinary studies, faculty of veterinary science, p.o. box mp 167, mount pleasant, harare, zimbabwe abstract mukaratirwa, s., hove, thokozani, cindzi, z.m., maononga, d.b., taruvinga, m. & matenga, elizabeth. 2005. first report of a field outbreak of the oriental eye-fluke, philophthalmus gralli, in commercially reared ostriches (struthio camelus) in zimbabwe. onderstepoort journal of veterinary research, 72:203–206 a total of 17 commercially reared ostriches (struthio camelus) from msengi farm, chinhoyi, zimbabwe, observed with swollen eyes, severe conjunctivitis and constant lacrimation accompanied by a purulent exudate, were restrained for further clinical examination. some of the birds were semi-blind with severe loss of body condition. when examined, tiny organisms were observed attached to the nictitating membranes and the conjuctival sacs of both eyes. the organisms were identified as philophthalmus gralli, the “oriental eye-fluke” and melanoides tuberculata, a prosobranch snail, was confirmed as the intermediate host through natural and experimental infection. to the best of our knowledge this is the first record of the oriental eye-fluke infection in birds in zimbabwe and africa and extends its known geographical range. keywords: melanoides tuberculata, oriental eye fluke, ostrich, philophthalmus gralli, zimbabwe * e-mail: smukarati@vet.uz.ac.zw accepted for publication 8 july 2003—editor and erosion in chickens (kingston 1984), conjunctivitis with constant lacrimation in ostriches (greve & harrison 1980), redness and irritation of the eye, swelling of the semilunar fold and proliferation of papillae in the palpebral conjuctiva of man (dissanaike & bilimoria 1958; mimori et al. 1982). in this paper, a field outbreak of the “oriental eyefluke”, p. gralli, in commercially reared ostriches in zimbabwe is described. as far as is known, this is the first report of the “oriental eye-fluke” infection in birds in africa. history the first case of the eye-fluke infection was detected at msengi farm in mashonaland west province, zimbabwe during clinical examination of an emaciated semi-blind female breeder ostrich reported by the farm attendant to have worms in the eyes. the owner of the farm indicated that the problem had commenced around april/may 2001 and only the breeders were affected. the affected birds were kept in paddocks with access to a perennial river that forms a pool in the paddocks. the owner first noticed the problem when the water level in the dam was low and only the breeder ostriches that had access to the dam were affected while other farm animals, including cattle and sheep with access to the same pool were unaffected. the affected birds showed excessive lacrimation, swollen eyelids and in some, a purulent exudate discharging from the eyes. materials and methods study animals at the time of our visit, the farm was habouring 37 grower and 17 breeder ostriches kept in separate paddocks but only the latter were in a paddock with access to the dam mentioned above. some of the birds in the paddocks were grazing along the edges of the pool as well as drinking its water. wild birds such as egyptian geese (alopochen aegyptiacus) and white storks (ciconia ciconia) were observed along the banks of the dam intermingling with the ostriches. all the ostriches on the farm were restrained and examined for the presence of the eyefluke. parasite and snail collection for identification flukes were carefully removed from the eyes of the heavily infected birds, using a blunt forceps, and were stored in physiological saline before being transferred, within 2 h, to the parasitology laboratory at the faculty of veterinary science, university of zimbabwe, harare. a portion of the flukes was fixed in 70 % alcohol. the remainder of the flukes were incubated at 37 °c for 1 h in order to induce them to lay eggs as well as the hatching of eggs so that snails could be infected with the released miracidia (alicata 1962). flukes fixed in 70 % alcohol were stained with aceticalum carmine according to the method described by gibbons, jones & khalil (1996) and were identified according to greve & harrison (1980). using a scoop made from a kitchen sieve supported on an iron frame mounted on a 1.5 m long wooden handle, snails were sampled from the edge of the pool where ostriches frequented when drinking water. the live snails were identified according to brown & kristensen (1989). shedding of the snails was induced by exposing live snails to artificial illumination for 2 h as described by frandsen & christensen (1984). snails not shedding cercariae were dissected to determine whether they were infected with larval stages of p. gralli (alicata 1962). infection of laboratory-bred melanoides tuberculata fifteen young laboratory-bred f1 generation of m. tuberculata with a mean shell height of 14 mm, obtained from blair research laboratory, harare were infected by exposing them for 1 h in water containing newly emerged miracidia from the flukes, as described by alicata (1962). following exposure, the snails were transferred to a 2 l-capacity glass aquarium with sand and filamentous green algae. they were maintained at 27 °c in a regime of 12 h light and 12 h darkness in filtered pond water which was changed twice a week. they were fed dried lettuce and commercial trout pellets. on day 92 and succeeding days to day 98 postexposure the snails were periodically observed under a dissecting microscope for the shedding of cercariae of p. gralli by exposing them to artificial illumination for 1 h. surviving snails were dissected 98 days post-exposure to determine whether they were infected with larval stages of p. gralli (alicata 1962). results and discussion the majority of the affected birds showed severe conjunctivitis, constant lacrimation and swollen eyelids (fig. 1). in some, the lacrimation was accom204 oriental eye-fluke, philophthalmus gralli (mathis & leger 1910), in ostriches (struthio camelus) in zimbabwe panied by a thick purulent exudate. heavily infected birds kept their eyes closed and appeared to have lost body condition (fig. 2). seventeen breeder ostriches from the same paddock were confirmed as infected with the worms. physical examination of the semi-blind ostrich revealed numerous tiny organisms attached to the nictitating membrane and conjuctival sac of both eyes (fig. 3). preliminary examination of the specimen of the organism revealed morphological features resembling those of a digenic trematode. in fresh mounts of the parasite, non-operculated eggs, some containing fully developed miracidia, were observed in the uterus. both eyes of 17 breeder ostriches in the same paddock were infected with flukes. the fluke was identified as p. gralli (see fig. 4) based on the following salient morphological features: strong ventral sucker with ventral sucker/oral sucker ratio of approximately 1:1.5, cirrus sac extending beyond the ventral sucker, vitelline glands tubular to follicular and occupying the majority of the distance between the anterior testis and ventral sucker and spherical testis arranged in tandem. ching (1961) who placed great emphasis on the morphology of the vitelline gland for species determination in the genus philophthalmus, described the vitellaria for p. gralli as “tubular with a small number of follicles”. similar vitellaria were seen in adult flukes from this outbreak. 205 s. mukaratirwa et al. fig. 1 emaciated and semi-blind ostrich with eyes infected with philophthalmus gralli fig. 2 swollen eyelids due to heavy infection with philophthalmus gralli fig. 3 philophthalmus gralli (arrow) attached to the conjuctival sac of an ostrich eye fig. 4 adult philophthalmus gralli from the eye of an ostrich. x 3.2. cirrus extends beyond the ventral sucker (a), strong ventral sucker with ventral sucker/oral sucker ratio approximately 1:1.5, vitelline glands tubular to follicular (b) and spherical testis arranged in tandem (c) 1 2 3 4 table 1 prevalence of philopthalmus gralli in field and laboratory-bred melanoides tuberculata source of melanoides no. of snails no. shedding no. with larval stages prevalence of tuberculata collected*/infected§ p. gralli cercariae of p. gralli infection (%) field 45* 0 11 24.4 laboratory-bred 15§ 6 9 100 live snails collected from the edges of the pool were identified as m. tuberculata, which has been reported as a natural intermediate host of p. gralli (nollen & murray 1978). a few empty shells were identified as those of bulinus globosus. results of natural and experimental infection of m. tuberculata with p. gralli are shown in table 1. eleven out of 20 m. tuberculata collected from the field had daughter rediae, granddaughter rediae and well developed cercariae compatible with those of p. gralli as described by alicata (1962). no snails from this group were shedding cercariae. six of the 15 snails kept in the laboratory released p. gralli cercariae and the remaining nine had larval stages, giving a 100 % infection rate in the experimentally infected laboratory-bred group of snails. according to kingston (1984), the genus philophthalmus, apart from p. gralli, comprises several other species reported from asia, the former ussr, usa and europe but not from africa. this is the first reported outbreak of the “oriental eye fluke”, p. gralli, in zimbabwe and africa as far as is known by the authors and extends the known geographical range of p. gralli. cases of ocular infection of captive-reared ostriches with p. gralli in florida, usa have been reported by greve & harrison (1980) and the clinical signs observed were similar to those observed in this outbreak. the origin of the infection reported by greve & harrison (1980) was never established, although it was presumed that the fluke was introduced either through importation of birds for exhibit in a texas zoo (nollen & murray 1978) or through wild migratory birds in an area where the freshwater snail intermediate hosts, t. granifera and m. tuberculata, were present (greve & harrison 1980). information gathered from the present outbreak seems to indicate that the introduction of p. gralli in zimbabwe was through wild migratory birds, possibly white stork. the storks were observed at the locality together with the ostriches in the presence of an ideal biotope for the breeding of the natural intermediate host snail, m. tuberculata. the local aquatic wild birds, especially the egyptian geese, commonly frequenting the locality might have played a role in maintaining the cycle and ostriches might have become infected accidentally whilst grazing or drinking water contaminated with p. gralli metacercariae. it is highly unlikely that the fluke was introduced into zimbabwe through importation of ostriches as zimbabwe does not generally import live ostriches and those on the farm on which the outbreak occurred were acquired locally. the possibility of the fluke spreading in zimbabwe depends on the availability of the intermediate host snail, a stable biotope where the intermediate host snails can breed, and also direct contact of the final host with the snail biotope infected with metacercariae. acknowledgements the authors thank the farm attendants at msengi farm for their assistance in restraining the birds and the blair research laboratory for providing snails for the experimental infections. references alicata, j.e. 1962. life cycle and development of philophthalmus gralli in the intermediate and final hosts. journal of parasitology, 64:47–54. brown, d.s. & kristensen, t.k. 1989. a field guide to african freshwater snails, southern african species. danish bilharziasis laboratory, publication no. 383. ching, h.l. 1961. the development and morphological evaluation of philophthalmus gralli mathis and leger, 1910 with a comparison of species of philophthalmus looss, 1899. proceedings of the helminthological society of washington, 28: 130–138. dissanaike, a.s. & bilimoria, d.p. 1958. on an infection of a human eye with philophthalmus sp. in ceylon. journal of helminthology: 115–118. frandsen, f. & christensen, n. 1984. an introductory guide to the identification of cercariae from african freshwater snails with special reference to cercariae of medical and veterinary importance. acta tropica, 41:181–202. gibbons, l.m., jones, a. & khalil, l.f. 1996. manual of the 8th international training course on identification of helminth parasites of economic importance. international institute of parasitology. london: cab international. greve, j.h. & harrison, g.j. 1980. conjuctivitis caused by eye flukes in captive-reared ostriches. journal of the american veterinary association, 177:909–910. kingston, n. 1984. trematodes, in diseases of poultry, edited by m.s. hofstad. ames: iowa university press. mathis, c. & leger, m. 1910. douve oculaire de la poule. bulletin societe pathologie exotique, 3:245–251. mimori, t., hirai, h., kifune, t. & inada, k. 1982. philophthalmus sp. (trematoda) in a human eye. american journal of tropical medicine and hygiene, 31:859–861. nollen, p.m. & murray, h.d. 1978. philophthalmus gralli: identification, growth characteristics and treatment of an oriental eye fluke of birds introduced into the continental united states. journal of parasitology, 64:178–180. radev, v., kanev, i. & gold, d. 2000. life cycle and identification of an eye-fluke from israel transmitted by melanoides tuberculata. journal of parasitology, 86:773–776. yamaguti, s. 1934. studies on the helminth fauna of japan. part 3. avian trematodes, ii. japanese journal of zoology, 5: 543–583. 206 oriental eye-fluke, philophthalmus gralli (mathis & leger 1910), in ostriches (struthio camelus) in zimbabwe putterrill_263-277_lowres.qxd introduction the tongue of crocodylia has been discussed in a number of publications, mainly in regard to the presence of lingual glands and their participation in salt secretion (dunson 1976, 1982; kochva 1978; taplin & grigg 1981; taplin, grigg, harlow, ellis & dunson 1982; mazzotti & dunson 1984; taplin 1984, 1988; taplin, grigg & beard 1985; taplin & loveridge 1988; chen, tang, wei & zhang 1989; steel 1989; taylor, franklin & grigg 1995). these papers, however, give very little information on the basic structure of the tongue of crocodylids and are concerned primarily with physiological studies regarding salt balance, tolerance to saline conditions and salt secretion. although some morphological information has been presented (gaupp 1888, cited by röse 1893; reese 1913; taguchi 1920; gabe & saint girons 1969; luppa 1977; taplin & grigg 1981; minnich 1982; grigg & gans 1993), only reese (1913) and taguchi (1920) provide any 263 onderstepoort journal of veterinary research, 71:263–277 (2004) general morphology of the oral cavity of the nile crocodile, crocodylus niloticus (laurenti, 1768). ii. the tongue j.f. putterill1* and j.t. soley1 abstract putterill, j.f. & soley, j.t. 2004. general morphology of the oral cavity of the nile crocodile, crocodylus niloticus (laurenti, 1768). ii. tongue. onderstepoort journal of veterinary research, 71: 263–277 the heads of nine 2.5 to 3-year-old nile crocodiles (crocodylus niloticus) were obtained from a commercial farm where crocodiles are raised for their skins and meat. the animals from which these specimens were obtained appeared clinically healthy at the time they were slaughtered. a description of the macroscopic and microscopic features of the tongue of the nile crocodile is presented and the results are compared with published information on this species and other crocodylia. the histological features are supplemented by information supplied by scanning electron microscopy. macroscopic features of interest were the dome shaped structures grouped in a triangular formation on the posterior two-thirds of the dorsum of the tongue. these structures were identified by light microscopy to contain well-developed branched, coiled tubular glands and associated lymphoid tissue. other histological features included a lightly keratinised stratified squamous surface epithelium supported by a thick layer of irregular dense fibrous connective tissue. deep to this region was a clearly demarcated adipose tissue core with a dense mass of striated lingual musculature. localised thickenings were present in the epithelium which were associated with ellipsoid intra-epithelial structures resembling taste buds. keywords: crocodylus niloticus, histology, morphology, nile crocodile, oral cavity, scanning electron microscopy, tongue * author to whom correspondence is to be directed. present address: electron microscopy unit, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa. e-mail: putterillj@arc.agric.za 1 department of anatomy and physiology, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa accepted for publication 16 march 2004—editor meaningful histological descriptions of the crocodilian tongue in general. some detail of the lingual glands of crocodylus porosus and alligator mississippiensis, including ultrastructural features, has also been presented (taplin & grigg 1981; minnich 1982; taplin 1988; grigg & gans 1993). in view of the lack of information regarding the general histological features of the crocodilian tongue, and considering the economic importance of the nile crocodile as a farmed animal in southern africa, this paper details the morphological features of the tongue of the nile crocodile (crocodylus niloticus) and compares the findings with published information on other crocodylids. materials and methods experimental animals the heads of nine 2.5 to 3-year-old nile crocodiles (crocodylus niloticus) were obtained from a commercial farm where crocodiles are raised for their skins and meat. the lengths of the animals sampled ranged from 1.2–1.5 m and the animals were clinically healthy at the time they were slaughtered. they were killed by shooting them in the brain at close range using a .22 calibre rifle. after the carcasses had been skinned and eviscerated the heads were removed and immersion-fixed in a large volume of 10 % phosphate-buffered formalin in plastic buckets for 2 h after which the tongues were removed from six of the heads and processed for light microscopy as detailed below. the remaining three heads with tongues in situ were further immersionfixed for a minimum period of 48 h. care was taken to avoid trapping air in the oral cavity by wedging a small block of wood in the angle of the mouth prior to immersion in the fixative. topography the three formalin-fixed heads with tongues in situ were used for a description of the gross anatomical features and topographical relationships of the structures in the oral cavity. macrophotographs were recorded digitally using a nikon coolpix 995 (nikon, tokyo, japan) digital camera or on 35 mm film using a chinon x-7 (chinon, tokyo, japan) single lens reflex camera. the tongues of these heads were also examined and micrographed using a stereomicroscope (wild m-400 photomakroskop, heerbrugg, switzerland) to obtain higher magnification micrographs of specific topographical features. a dried skull from a 5-year-old (approximate age) specimen was used to provide supporting evidence for the anatomical description. light microscopy as noted above, the tongues were removed from six heads after a short fixation period of 2 h. this pre-fixation was deemed necessary to firm the relatively soft tissue of the organ prior to it being cut into the various segments for light microscopy and scanning electron microscopy. these tongues were freed from the mandible by cutting through the peripheral membrane and the tissue mass at the base of the tongue. the ventral half of the tongue was removed and discarded and the remaining dorsal surface was divided longitudinally into left and right halves down the midline. identical transverse sections from the regions indicated in fig. 1 were taken from each half of the tongue for light microscopy (lm) and scanning electron microscopy (sem), respectively. both sets of samples were fixed for a minimum period of 48 h in fresh 10 % buffered formalin. lm samples were dehydrated through 70, 80, 96 and 2x 100 % ethanol and further processed through 50 : 50 ethanol : xylol, 2x xylol and 2x paraffin wax (60–120 min per step) using a shandon model 2le automatic tissue processor (shandon, pittsburgh, pa, usa). tissue samples were finally embedded manually into paraffin wax in brass moulds. sections were cut at 4–6 µm, stained with haematoxylin and eosin (h&e) (luna 1968) or periodic acid-schiff (pas) (pearse 1985) and viewed and micrographed using a reichert polyvar (reichert, austria) compound light microscope fitted with a differential interference contrast (dic) prism. scanning electron microscopy the samples of the tongue obtained as indicated above and which had been fixed in 10 % phosphatebuffered formalin for a minimum of 48 h, were rinsed for several hours in water to remove traces of phosphate buffer. these samples were routinely dehydrated through an ascending ethanol series (50, 70, 90, 95 and 3x 100 %, 60 min per step) and critical point dried from 100 % ethanol through liquid-co2 in a polaron critical point drier (polaron, watford, england). the samples were then mounted onto brass or aluminium viewing stubs (to expose the epithelial surface) with a conductive paste (carbon dag) and sputter coated with gold using a balzers 020 sputter coater (balzers union, liechtenstein). specimens were viewed and microtographed using a hitachi s-2500 scanning electron microscope (hitachi, tokyo, japan) operated at 8 kv. 264 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). ii results macroscopic features the floor of the oral cavity was formed by the tongue and a wide, rostral mucosal plate continuous with the gingiva. the relatively long tongue was roughly triangular in shape, being much broader caudally than at its tip (fig. 1). it occupied the greater part of the floor of the oral cavity (apart from the rostral plate over the symphysis of the dentary bones) and was bordered peripherally by a loose, highly folded, continuous, fibrous membrane. this membrane attached the ventro-lateral aspect of the tongue to the ventro-medial aspect of the mandible. the membrane was more complexly folded around the rostral tip of the tongue. in the specimens examined the tip of the tongue was always pulled away (in a caudal direction) from the apex formed by the rostral fusion of the paired dentary bones, revealing the highly folded membrane attaching the tongue to the mandible (putterill & soley 2003). pigmentation varied amongst specimens examined and where present, occurred peripherally in the loosely folded membrane and on occasion, superficially on the surface of the tongue (fig. 1 and 2). pigmentation was also often evident at the angle of the tongue and the oral surface of the ventral fold of the gular valve which also formed the ventro-caudal border of the oral cavity. the dorsal surface of the tongue displayed numbers of large, dome-shaped structures which were concentrated in a triangular formation on the posterior two-thirds of the dorsum of the tongue (fig. 2). each of these elevated structures revealed a centrally positioned, darkly pigmented opening (fig. 4) which on histology proved to represent the duct opening of large branched coiled tubular glands situated deeply below the tongue surface (fig. 13). stereomicroscopy revealed small, nipple-like surface extensions scattered between the duct openings (fig. 4). smaller, more discrete, non-pigmented units were observed on the lateral and rostral aspects of the tongue surface (fig. 2). two isolated concentrations of similar structures were situated at the caudo-lateral aspect of the tongue, at the base of the ventral fold of the gular valve (fig. 3). the non-pigmented units differed from the more obvious pigmented units in that they did not display a duct opening. the centrally positioned structure typical of these units represented a flattened disc of thickened epithelium which on lm was observed to house structures resembling taste buds (see below). the tip of the tongue displayed deep transverse and oblique grooves which possibly represented a shrinkage or fixation artefact (fig. 1). the surface of the tongue was generally creamy-yellow in colour and had a slightly spongy texture, even in the fixed specimens. light microscopy cross-sections of the rostral region of the tongue revealed a relatively thin, lightly keratinised stratified squamous epithelium supported by a thick layer of irregular dense fibrous connective tissue (fig. 5). the epithelium on the dorsum of the tongue displayed a few shallow folds, but these rapidly increased in number and complexity towards the lateral borders of the tongue. the epithelial folds were supported by the underlying connective tissue and primary, secondary and occasionally tertiary folds could be distinguished. the epithelium in the highly folded lateral zone appeared more heavily keratinised and a distinct layer of desquamated cells (stratum disjunctum) was generally evident (fig. 10). at the ventro-lateral aspect of the tongue the epithelium, supported by a substantial layer of more loosely arranged irregular dense connective tissue, was reflected laterally towards the medial surface of the mandible with which it was continuous. the loosely arranged, highly folded membrane thus formed, connected the ventro-lateral aspects of the tongue to the mandible. deep to the connective tissue layer was a clearly demarcated adipose tissue core containing large adipocytes, strands of fibrous connective tissue and variably sized bundles of striated muscle fibres (fig. 5 and 9). a large plexus of blood and lymphatic vessels was situated between the irregular dense connective tissue and the adipose tissue core. a large vascular and lymphatic plexus was also situated at the angle of the tongue and the ventral fold of the gular valve. these vessels were sandwiched between the thick sub-epithelial connective tissue layer and the adipose tissue core (fig. 9). more caudal cross-sections of the tongue displayed a similar arrangement of the basic tissue layers outlined above. in addition, however, a dense mass of striated lingual musculature was evident ventral to the adipose core, and large branched coiled tubular glands associated with prominent duct openings characterised the dorsal sub-epithelial layer of connective tissue (fig. 14). the lightly keratinised stratified squamous epithelium varied in thickness (3–6 cell layers) and, in pas-stained sections, was seen to rest on a promi265 j.f. putterill & j.t. soley nent basement membrane. the shape, appearance and orientation of the cells and their nuclei were similar to that described previously for the palate and gingivae (putterill & soley 2003). although the epithelium was relatively thin, it displayed the features typical of stratified squamous epithelia in general. the stratum spinosum was poorly developed in the thinner regions of the epithelium and was 266 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). ii only clearly defined in the areas of localised epithelial thickening associated with glandular openings and specialised sensory structures (fig. 6, 7, 11– 13). in addition to ubiquitous epithelial folds, the epithelial lining of the tongue demonstrated randomly distributed localised thickenings associated with ellipsoid intra-epithelial structures resembling taste buds (fig. 7). the epithelial specialisations were positioned level with, or slightly elevated above, the adjoining epithelial surface, the increase in thickness being accommodated at the expense of the underlying connective tissue layer (fig. 6 and 7). the most noticeable features of the specialisations were an increase in the number of layers of the stratum spinosum and the vertical orientation of cells towards the centre of the epithelial concentration. the vertically positioned cells formed structures with features characteristic of taste buds (fig. 7) similar to those observed in the palate and gingivae (putterill & soley 2003). the underlying connective tissue contained conspicuously less collagen fibres and formed a conical or cup-shaped zone associated with medullated nerve fibres. in some instances the modified connective tissue zone revealed a centrally positioned cell-rich mass. some of the cells morphologically resembled fibroblasts while others were large, round or oval cells with a spherical nucleus. in a few areas the round cells appeared swollen and were arranged in poorly defined vertical rows. small lymphocytic aggregations were often associated with the epithelial specialisations (fig. 6 and 11). the taste receptors appeared to be most numerous on the dorso-lateral aspects of the tongue and also in the epithelium lining the lateral wall of the organ. not all sections of the epithelial thickenings, however, revealed the intraepithelial, ellipsoid structures and pacinian-like corpuscles were occasionally seen in the vicinity of the modified connective tissue core, particularly towards the tip of the tongue. this may indicate that some of the specialisations represent touch or pressure receptors in addition to the more commonly encountered taste receptors (see sem results below). it is also possible that individual sections did not always pass through the centrally positioned taste bud, creating the erroneous impression that it was absent. immediately beneath the epithelium was a narrow layer of fine connective tissue followed by a diffuse yet prominent layer containing melanin pigment (fig. 12 and 13). the melanin appeared to be concentrated around capillaries present in this region and was also observed in the deeper parts of the connective tissue stroma in the vicinity of larger blood vessels and nerves. the sub-epithelial capillary supply appeared relatively sparse. however, in addition to the vascular and lymphatic plexuses mentioned above, numerous large blood and lymphatic vessels and attendant medullated and nonmedullated nerves were observed throughout the deeper lying layer of irregular dense connective tissue. the caudal two-thirds of the tongue was dominated by the presence of large collections of glandular tissue (lingual salivary glands) and masses of associated lymphoid tissue (fig. 13–16). the more centrally positioned glands were generally larger and more complex than those situated more laterally. in oblique or transverse sections the glandular tissue exhibited features typical of compound tubular glands and appeared to be composed of lobes and lobules separated by tracts of connective tissue (fig. 15 and 16). when sectioned in the longitudinal plane, however, it was obvious that the glands were branched, coiled tubular in nature with each branch 267 j.f. putterill & j.t. soley fig. 1 macrophotograph of the mandible with the tongue in situ showing the sampling sites for histology of the tongue (a–d). the fibrous membrane attaching the tongue to the mandible is also obvious in places (white arrows). the rostral dentary shelf (rds) is indicated in the region above the symphysis of the dentary bones. the glottis (gt) and laryngeal mound (lm) are shown in situ on the floor of the pharyngeal cavity and the ventral fold (vf) of the gular valve is seen separating the ventral aspects of the oral and pharyngeal cavities. p = pigmented area. formalin fixed specimen. x 0.5 fig. 2 macrophotograph of the surface of the tongue showing the triangular arrangement of the glandular region (indicated by the dotted line) situated in the posterior two-thirds of the tongue and stretching from approximately m 2 to m 6/7 (not indicated in photograph). the pigmented gland openings are clearly visible. note the less conspicuous, low-profiled units (black arrowheads) situated laterally to the glandular region. the rectangular area is enlarged in fig. 4. p = pigmented area. formalin-fixed specimen. x 1 fig. 3 macrophotograph of the caudo-lateral region of the tongue showing a discrete, triangular region of low-profiled surface units at the base of the ventral fold (vf) of the gular valve. m 7 = molar 7. formalin-fixed specimen. x 1.2 fig. 4 stereomicrograph of the darkly pigmented openings to salivary glands (block arrows) on the surface of the tongue. small nipple-like surface extensions (arrowheads) are scattered between the openings. formalin-fixed specimen. x 6.5 268 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). ii leading to an elaborately coiled secretory endpiece. each secretory endpiece formed a distinct glandular unit. the various branches were linked to a single secretory duct which opened onto the dorsum of the tongue. the main secretory duct was lined by a thick stratified squamous epithelium originating from the stratum germinativum of the surface epithelium with which it was continuous (fig. 13). the epithelial cells, however, were vertically oriented with the final layer lining the duct appearing typically columnar in nature (fig. 17). the epithelium of the main duct thinned rapidly to form a relatively narrow tube lined by three to four layers of cells before dividing from the surface inwards into a number of smaller branches. these branches were lined by a simple columnar epithelium which was continuous with the 269 j.f. putterill & j.t. soley fig. 5 cross-section of the rostral part of the tongue illustrating the lateral aspect. note the increase in the number of folds on the lateral surface, the thick layer of irregular dense subepithelial connective tissue (ct) and the adipose tissue core (a-c) (approximately following the dotted outline) containing scattered bundles of striated muscle fibres (arrowheads). the blocked area shows an epithelial specialisation enlarged in fig. 6. h&e stain. bar = 2 mm fig. 6 an enlargement of the epithelial specialisation blocked in fig. 5. note the thickened epithelium, the characteristic, pale, conical zone of modified subepithelial connective tissue (outlined) and the closely associated lymphocytic aggregations (asterisks). the arrows indicate the groove/trough often seen surrounding these structures. h&e stain. bar = 500 µm fig. 7 modification of the thickened epithelium of a specialisation to form an enlarged oval core reminiscent of a taste bud. note the vertical orientation of the core cells (arrows) and the more substantial stratum spinosum surrounding the taste bud. vacuolation is apparent in the superficial region of the receptor (arrowheads). h&e stain. bar = 50 µm fig. 8 enlargement of the epithelial covering of the tongue surface showing the single layer of vertically oriented cells forming the stratum basale (1), the narrow stratum spinosum (2), the darkly staining stratum granulosum (asterisk) and the superficial stratum corneum (3) which shows signs of desquamation (arrow). the epithelium is supported by irregular dense connective tissue (star). h&e stain. bar = 50 µm fig. 9 caudal limit of the tongue where it abuts the ventral fold (vf) of the gular valve. note the large lymphatic (l) and vascular (bv) plexus in this region as well as the underlying adipose tissue core (a-c) of the tongue. h&e stain. bar = 1 mm fig.10 lateral wall of the tongue illustrating the highly folded nature of the surface and the greater degree of desquamation of the surface layers of the epithelium in this region (arrows). h&e stain. bar = 250 µm fig. 11 lm micrograph of an oblique section through the secretory duct of an underlying salivary gland. note the taste bud (block arrow) situated in the wall of the pore opening onto the tongue surface. oblique sections of the duct (arrowheads) can be seen. lymphocytic aggregations (asterisks) are closely associated with the wall of the secretory duct and the secretory pore (see also fig. 13). h&e stain. bar = 500 µm fig. 12 enlargement of a typical taste bud located in the duct of a salivary gland. note the vertical orientation of the epithelial cells (arrows) and the more substantial stratum spinosum surrounding the taste bud. at the base of the specialisation an apparent nerve plexus (arrowhead) is seen to penetrate below the epithelium into the melanin layer (star). h&e stain. bar = 100 µm 270 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). ii coiled secretory endpieces of the gland. the point of exit of the secretory duct onto the surface of the tongue was marked by a distinct depression or pore. intra-epithelial structures resembling taste buds were often located within the wall of the pore (fig. 11–13). the secretory portions of the glands were coiled tubular in nature and lined by a simple columnar epithelium. the nuclei of the secretory cells were generally round or oval and although situated towards the base of the cell, were not compressed. pas-positive granules were identified in cells lining some tubular profiles, generally towards the periphery of the glands, while most of the secretory tubules showed a light staining reaction or none at all. the tubular lumen was wide and individual secretory profiles were separated by delicate bands of connective tissue (fig. 18). each individual secretory unit was separated from neighbouring units by relatively wide tracts of connective tissue (fig. 15 and 16). the individual units of the lingual salivary glands were generally situated in the deeper region of the sub-epithelial connective tissue, close to the border of the adipose tissue core (fig. 14 and 15). between the glandular tissue and the surface of the tongue, and initially associated with the secretory duct, were large aggregations of lymphoid tissue which formed lingual tonsils (fig. 11 and 13). although the lymphoid masses displayed diffuse infiltration into the thickened stratified epithelium of the secretory duct, obliteration of the epithelium was not observed (fig. 13). loosely arranged collections of lymphoid tissue were also sometimes associated with the glandular tissue itself. the glandular tissue was richly supplied with blood vessels and the vascular and lymphatic plexuses present at the junction of the irregular dense connective tissue and adipose tissue layers were often positioned close to the glandular units (fig. 14). nonmedullated nerves were associated with the glandular tissue. scanning electron microscopy scanning electron microscopy of the tongue surface confirmed the lm observation that the dorsum of the tongue was partially composed of a number of clearly demarcated round to angular units. the various units appeared to be elevated and were surrounded by deep, sometimes wide, clefts which accentuated individual units. the exaggerated division of the units was attributed to shrinkage caused by the drying process for sem. the round to polygonal-shaped units occupying the triangular glandular region situated in the posterior two-thirds of the tongue (fig. 2) exhibited a relatively low profile and smooth surface features. a centrally positioned pore which was frequently filled with debris/glandular secretions, and which was shown by lm to represent the opening of the main secretory duct of the underlying lingual salivary glands, was the most prominent feature. a number of grooves (probably representing epithelial folds) radiated outwards from the pore. the grooves varied in length and occasionally extended the entire diameter of the unit (fig. 19 and inset fig. 19). situated towards the periphery of the units were small, spherical, low-profiled, dome-shaped protrusions (fig. 19 and inset fig. 19) which appeared to represent the nipple-like sur271 j.f. putterill & j.t. soley fig. 13 low magnification of the secretory duct of a deeper lying salivary gland. the duct opens onto the tongue surface by means of a pore (arrow). the duct is not entirely visible due to the slightly oblique plane of the section towards its base. note the lymphoid aggregation (l) associated with the duct. a taste bud (block arrow) is observed towards the opening of the duct (see also fig. 12). note the thin, but prominent layer of melanin pigmentation (arrowhead) immediately below the epithelium. a portion of the secretory units (stars) of a salivary gland are also visible in the micrograph. h&e stain. bar = 500 µm fig. 14 secretory units (stars) of two lingual salivary glands separated by a well-developed vascular and lymphatic plexus (arrowheads). the glandular tissue is positioned deep within the subepithelial connective tissue, close to the adipose tissue core (a-c). striated lingual muscle fibres (m). h&e stain. bar = 2 mm. fig. 15 an example of a typical lingual salivary gland. note the separation of the glandular tissue into lobules/secretory units (stars) by wide tracts of connective tissue (arrowheads). the main secretory duct does not appear in the section although some lymphoid tissue (l) is visible. a-c = adipose tissue core. pas-stain. bar = 1 mm. fig.16 a lobule/secretory unit of a lingual salivary gland. secondary or tertiary branches (asterisks) of the main secretory duct, displaying a wide lumen, can be observed in the rectangle (enlarged in fig. 17). wide connective tissue tracts (arrowheads) can be seen between the lobules of the salivary gland. h&e stain. bar = 500 µm fig.17 higher magnification of the rectangle in fig. 16 showing the double columnar nature of the duct epithelium (double-headed arrow) and the wide lumen of the ducts (d). h&e stain. bar = 1 mm fig.18 higher magnification of a group of secretory tubules of the branched coiled tubular units of the gland demonstrating the pas-positive granules (arrowheads) observed in the apical cytoplasm of the cells. however, this was not a consistent feature. note the wide lumen (lu) of the tubules. pas-stain. bar = 50 µm 272 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). ii face extensions seen by stereomicroscopy (fig. 4). occasional flattened discs (see below) with a random distribution were also observed. more discrete surface units were observed laterally on the tongue surface (fig. 2 and 20) and also in the smaller paired triangular areas situated caudo-laterally at the base of the tongue (fig. 3 and 21). the surface of the discrete units presented a rough, corrugated appearance with epithelial folds of variable depth creating a mosaic-like pattern (fig. 21). centrally, and occasionally eccentrically, positioned on each unit were clearly defined, flattened, disc-shaped structures. more than one disc-shaped structure was seen on some units (fig. 19 and 21). at the centre of each disc was a small pore, which in many cases was obscured by cell debris and/or bacteria. in pores not filled with debris, a variable number of short processes were observed to protrude from the opening. the processes often displayed clavate tips (fig. 23 and 24). based on lm observations, the pores with their processes would appear to represent the superficial component of the taste buds located within localised epithelial specialisations (fig. 7, 11–13). the pore itself was surrounded by concentric arrangements of desquamating surface cells (fig. 22 and 23). individual cells were polygonal in shape and displayed a complicated array of microridges, giving the surface a sponge-like appearance (fig. 23 and 24). occasional domeshaped structures similar to those described in the glandular area were sometimes observed. discussion the present study confirmed the basic histological features reported by both taguchi (1920) in alligator sinensis, crocodylus vulgaris, which represents c. niloticus (king & burke 1997) and c. porosus, and reese (1913) in the “florida alligator” (a. mississippiensis). reese (1910) noted that the dorsum of the tongue is covered with small, evenly distributed papillae. this observation was confirmed by shimada, sato, yokoi, kitagawa, tezuka, & ishii (1990) in a. mississippiensis who described cone-shaped filiform papillae spread across the dorsal surface of the tongue. the small, nipple-like surface projections (fig. 4) seen in the nile crocodile probably represent the papillae observed in the american alligator. the folded nature of the tongue surface revealed in this study was also confirmed in a. mississippiensis (reese 1913) and in the species studied by taguchi (1920). both reese (1913) and taguchi (1920) described the covering of the tongue as a keratinised stratified squamous epithelium which thickened appreciably towards the body and base of the tongue. in the specimens examined during the present study the epithelium, although typically keratinised stratified squamous in structure, appeared thin and uniform in thickness throughout all regions of the tongue except for localised thickenings containing taste buds and at the mouths of the ducts of the lingual salivary glands. to what extent the diet of commercial pellets on which the specimens of c. niloticus were raised may have influenced the thickness of the epithelial lining of the tongue could not be determined. the sequence of supporting connective tissue layers observed in the tongue of c. niloticus, namely, the thick layer of irregular dense fibrous connective tissue and the deeply positioned adipose tissue core was also confirmed in the study of taguchi (1920). according to taguchi the layer of fibrous 273 j.f. putterill & j.t. soley fig. 19 a surface unit found on the glandular area of the tongue. note the large, centrally positioned gland opening (asterisk), the radiating epithelial grooves (arrowhead) and the dome-shaped structures (block arrows). bar = 100 µm inset: overview of another surface unit demonstrating longer epithelial grooves (arrowheads) and similar features (gland opening—asterisk; dome-shaped structure—block arrow) to those seen in the main micrograph. bar = 500 µm fig. 20 typical surface units found in the mid-lateral region of the tongue showing a mosaic pattern of grooves. a flat, disc-shaped structure (possible taste receptor—encircled) and raised, dome-shaped structures (possible pressure receptors—stars) are present. bar = 250 µm inset: enlargement of the dome-shaped structure indicated by the outlined arrow. bar = 100 µm fig. 21 a group of caudo-laterally situated surface units (see fig. 3) showing flat disc-shaped structures (arrowheads). two of these discs are situated on the same unit. bar = 250 µm fig. 22 enlargement of a disc-shaped structure from the caudo-lateral aspect of the tongue showing the central pore (arrow) and the concentric rings of loosened, desquamating surface epithelial cells (arrowheads). bar = 100 µm fig. 23 higher magnification of the central pore to show the protruding cellular processes (arrowhead). note the spongy appearance of the surface of the surrounding epithelial cells. bar = 10 µm fig. 24 an oblique view of clavate cellular processes (arrowhead) which protrude from the pore for only a short distance above the surface. the surrounding epithelial cells display a complex arrangement of microridges. bar = 5 µm connective tissue contained numerous muscle fibres which extended close to the surface of the tongue as well as into the core of adipose tissue. in the nile crocodile the muscle fibres were confined to the adipose tissue core. it is of interest that reese (1913) makes mention of neither an adipose tissue core nor the muscle fibres in a. mississippiensis. however, taguchi (1920) did note that the layer of adipose tissue was practically absent in c. porosus. the muscle tissue housed within the tongue is described by taguchi (1920) as the musculus hyoglossus. gnanamuthu (1937) supports this observation and notes that the “m. hyoglossus of crocodilus [palustris] is peculiar in being the sole muscle of the tongue”. the findings of gnanamuthu (1937) also support the observation in the nile crocodile that the muscle fibres decrease in number towards the tip of the tongue. taguchi (1920) observed that mucosal folds divide the surface of the tongue into small, demarcated fields or units. this was confirmed in the present study both macroscopically (fig. 2) and more graphically by sem (fig. 19 to 22). based on surface details provided by sem, different types of units could be identified. the large units typically situated on the posterior two-thirds of the tongue displayed a large central pore, radiating surface grooves and scattered, peripherally situated domeshaped protrusions. the more discrete surface units occupying the lateral and caudo-lateral regions of the tongue were characterised by the presence of numerous flattened, disc-shaped structures at the centre of which was a small pore. when correlated with lm observations, these structures appear to represent the superficial component of specialised taste receptors. these observations have not previously been reported. taguchi (1920) in his study of three crocodilian species, mentions the presence of lingual taste buds but provides no further detail other than that they are situated between the epithelial cells in the stratum spinosum. taste buds in the nile crocodile are associated with localised epithelial thickenings located throughout the tongue, particularly in the lateral and caudo-lateral aspects of the organ. these epithelial thickenings appear to represent the flattened, disc-shaped structures seen by sem. the presence of short processes seen emanating from the central pore of these structures in sem preparations lends further support to this observation. taste buds were also seen to be concentrated in the thickened epithelium forming the surface openings of the lingual salivary glands. the morphology of the lingual taste buds was similar to that reported for taste buds in the palate and gingivae of the nile crocodile (putterill & soley 2003) and reflects the features previously described by bath (1905, 1906) and taguchi (1920) in these regions of the oral cavity. the concentration of cells observed in the modified connective tissue zone beneath the taste papillae of the tongue has been mentioned in earlier publications. bath, who identified taste buds in the oral cavity of c. niloticus, was of the opinion that these cells represented supportive and protective elements for the nerve fibres supplying the taste buds (bath 1905, 1906). in contrast, hulanicka (1913), in a study of the nerve endings in the tongue, palate and the skin of two crocodylids (c. niloticus and a. lucius [= m. mississippiensis]), concluded that these “supportive” cells were in fact more typical of tactile cells. this observation was based on the morphological characteristics and arrangement of the cells and their relationship to the nerve fibres supplying the taste bud. the “tactile” cells were innervated by branches of the fibres supplying the taste bud. although this study did not examine the distribution of nerve endings in the tongue, the arrangement of the connective tissue elements in the stroma beneath the epithelial specialisations was essentially similar to that described by hulanicka (1913). this would imply that the epithelial specialisations with their modified connective tissue zone possibly function as both taste and tactile receptors. hulanicka (1913) also describes a variety of tactile corpuscles in the crocodilian tongue, including structures similar to the corpuscles of krause. as this study did not specifically address nerve fibre distribution, the authors cannot confirm the presence of the various types of tactile structures. however, structures reminiscent of pacinian corpuscles were regularly identified towards the tip of the tongue, often in association with epithelial specialisations apparently devoid of taste buds. earlier literature describing the tongue of crocodylids (röse 1893; bath 1905, 1906; reese 1913; taguchi 1920; woerdeman 1920; luppa 1977) mentioned the fact that glandular tissue occurred in the specimens examined. reese (1913) studied the histology of the enteron of a. mississippiensis and described the glandular tissue in the tongue as “... probably mucous or slime secreting” and the structure of the glands as being compound tubular alveolar. taguchi (1920) made a comparative histological study of the digestive tract, including the tongue, of three crocodilian species (a. sinensis, c. niloti274 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). ii cus and c. porosus) and described lingual glands. these glands, identified by taguchi (1920) as being of the branched tubular type, appear to be typical of the glandular tissue observed during this study. although taguchi (1920) did not specify a mucous secretion as such, he did state that the secretory cells appeared to be filled with fine granular material. none of the earlier studies, however, mentioned the role of lingual glands in salt secretion. in more recent studies the tongues of the marine or estuarine crocodile (c. porosus) and the american crocodile (crocodylus acutus) have received most attention, seemingly due to the animals’ salt water or estuarine habitat and the implication of the lingual glands in salt secretion during osmoregulation or electrolyte homeostasis (taplin & grigg 1981). steel (1989) reported that the marine or estuarine crocodile (c. porosus) is “quite at home in a marine environment” and “ranges far out into the open ocean.” various authors have noted that the tongue of c. porosus contains 40 complex tubular salt glands that secrete a concentrated solution of nacl when the species is in a saline environment (taplin & grigg 1981; taplin et al. 1982; taplin et al. 1985; taplin 1988; grigg & gans 1993). taplin & grigg (1981) describe the glands in this species as compound tubular glands having “a broad, shallow duct which leads down to a series of smaller ducts that branch repeatedly into lobules of the gland. the larger branches are lined with columnar to cuboidal epithelium which passes into squamous epithelium in the smaller branches. each lobule of the gland is densely packed with branching secretory tubules lined almost entirely by columnar epithelial cells.” according to taplin (1988), the ultrastructural features of the salt secreting glands revealed by transmission electron microscopy were the “characteristically complex interdigitations of lateral cell membranes, expanded intercellular spaces, abundant mitochondria, and extensive network of blood vessels and unmyelinated nerve fibres.” chen et al. (1989) examined the lingual glands of the chinese alligator, alligator sinensis, and reported that they were either simple tubular or complex acinotubular in nature. they concluded that the glands, which number about 100 throughout the tongue, appeared in the posterior two-thirds of the tongue and further stated that they functioned as salt secreting glands as well as serving to lubricate food. this investigation on the nile crocodile identified glandular tissue in a triangular area occupying the posterior two-thirds of the tongue. each lingual salivary gland was demarcated by a raised, domed unit with a centrally positioned, pigmented pore which indicated the opening of the secretory duct (fig. 1, 2, 4 and 13). there were approximately 40 pores present which is similar in number to those seen in the saltwater crocodile by taplin & grigg (1981). however, in species of alligatorinae, many more pores are present with 100 being reported in a. sinensis by chen et al. (1989) and 200 or more in a. mississippiensis (taplin et al. 1982). taplin & grigg (1981) and taplin et al. (1985) used methacholine chloride to stimulate lingual gland secretion in various species of crocodylinae (crocodylus acutus, c. johnsoni, c. acutus, c. palustris, c. cataphractus, c. niloticus and osteolaemus tetraspis), alligatorinae (alligator mississippiensis and caiman crocodilus) and in gavialis gangeticus (family gavialidae), and reported that in all instances the animals possessed functional lingual glands, but that only the crocodylinae secreted significant concentrations of na+, indicating the presence of salt secreting glands. these lingual salt secreting glands open through pores in the posterior region of the tongue and secrete a solution of na+ and cl– at concentrations similar to that of seawater (taplin & grigg 1981). steel (1989) also confirmed that “alligators and caimans do have minute pores on the back of their tongues and in the palatal epithelia around the buccal valve, but it would seem that these are primarily salivary glands, although some na+ and k+ is excreted at a low rate.” taplin (1988) and grigg & gans (1993) note that salt glands have not been found in any members of the alligatoridae, and a study of captive a. mississippiensis suggests that this species cannot maintain homeostasis in hyperosmotic water (lauren 1985). some contradictory evidence has been provided by chen et al. (1989) who noted that the lingual glands of a. sinensis function as salt secreting glands and also serve to lubricate the passage of food. steel (1989) postulated that “this subfamily (crocodylinae) originated in a marine environment and subsequently colonised a freshwater habitat, or conversely, that all crocodylid subfamilies were of freshwater origin but only the crocodylinae acquired a saltwater adaption.” bellairs (1989) also suggested that modern crocodylines had ancestors with “markedly marine habits” and cites taplin (1984). from illustrations presented by taplin & grigg (1981), minnich (1982), taplin (1988) and grigg & gans (1993), it may be deduced that the pores seen by them in the estuarine crocodile represent the region of glandular tissue observed in c. niloticus during this study. no physiological examination of the glands in the 275 j.f. putterill & j.t. soley tongue of c. niloticus was carried out during this study and it is therefore not possible to unequivocally identify them as “salt secreting”. however, the histological structure of the lingual glands observed in the nile crocodile is similar to that described in c. porosus, despite the difference in classification of the glands as “branched coiled tubular” (c. niloticus—this study) or “compound tubular glands” (c. porosus—taplin & grigg 1981). a noteworthy difference was that the ducts connecting the main secretory duct to the secretory units was lined by a simple columnar epithelium in the nile crocodile as opposed to the simple squamous epithelium identified in c. porosus (taplin & grigg 1981). it was noted in the present study that lymphoid tissue was closely associated with the glandular tissue, particularly with the large ducts linking the glands to the surface of the tongue. a similar observation was made by taguchi (1920) in the three species he examined (a. sinensis, c. niloticus and c. porosus) and he described lymph follicles in close proximity to the secretory ducts of the lingual glands as well as lymphocytic penetration of the epithelial layer of the ducts. these lymphoid aggregations prompted taguchi (1920) to state that they “remind one of tongue tonsilli of higher animals.” varying degrees of lymphocytic infiltration of the main secretory duct was observed in the present study and the amount and location of the lymphoid tissue would warrant reference to these structures as lingual tonsils. acknowledgements the authors are indebted to mr and mrs j-g. kuhlmann of izintaba croco farm, de wildt, north west province, south africa, who supplied the material used in this study. the authors also gratefully acknowledge the skilful assistance of the histology laboratories of the pathology division of the onderstepoort veterinary institute and the pathology section, department of paraclinical sciences, faculty of veterinary science, university of pretoria. the advice and guidance given by dr f.w. huchzermeyer is gratefully acknowledged. references bath, w. 1905. über das vorkommen von geschmacksorganen in der mundhöhle von crocodilus niloticus laur. 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(english summary, okajimas folia anatomica japonica, 66:375–392). steel, r. 1989. anatomy of a living fossil, in crocodiles. london: christopher helm. taguchi, h. 1920. beiträge zur kenntnis über die feinere struktur der eingeweideorgane der krokodile. mitteilungen aus der medizinischen fakultät der kaiserlichen universität zu tokyo, 25:119–188. taplin, l. 1984. evolution and zoogeography of crocodilians: a new look at an ancient order, in vertebrate evolution and zoogeography in australia, edited by m. archer & g. clayton. perth: hesperian press. taplin, l.e. 1988. osmoregulation in crocodilians. biological reviews, 63:333–377. taplin, l.e. & grigg, g.c. 1981. salt glands in the tongue of the estuarine crocodile crocodylus porosus. science, 212: 1045–1047. taplin, l.e., grigg, g.c. & beard, l.a. 1985. salt gland function in fresh water crocodiles: evidence for a marine phase in eusuchian evolution?, in biology of australasian frogs and reptiles, edited by g.c. grigg, r. shine & h. ehmann. sydney: surrey beatty & sons. taplin, l.e., grigg, g.c., harlow, p., ellis, t.m. & dunson, w.a. 1982. lingual salt glands in crocodylus acutus and c. johnsoni and their absence from alligator mississippiensis and caiman crocodilus. journal of comparative physiology b, 149:43–47. taplin, l.e. & loveridge, j.p. 1988. nile crocodiles, crocodylus niloticus, and estuarine crocodiles, crocodylus porosus, show similar osmoregulatory responses on exposure to seawater. comparative biochemistry and physiology a, 89:443–448. taylor, g.c., franklin, c.e. & grigg, g.c. 1995. salt loading stimulates secretion by the lingual salt glands in unrestrained crocodylus porosus. journal of experimental zoology, 272:490–495. woerdeman, m.w. 1920. über die gaumendrüsen der krokodile. anatomische anzeiger, 53:345–352. 277 j.f. putterill & j.t. soley article information authors: fahdel m. housawi1 eltayb m. abuelzein1 ahmed a. gamee1 adel i. alafaleq1 affiliations: 1college of veterinary medicine and animal husbandry, king faisal university, saudi arabia correspondence to: eltayb abuelzein postal address: po box 80216, jeddah 21589, saudi arabia dates: received: 19 dec. 2011 accepted: 16 may 2012 published: 07 aug. 2012 how to cite this article: housawi, f.m., abuelzein, e.m., gamee, a.a. & alafaleq, a.i., 2012, ‘comparative study on three locally developed live orf virus vaccines for sheep in saudi arabia’, onderstepoort journal of veterinary research 79(1), art. #397, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.397 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. comparative study on three locally developed live orf virus vaccines for sheep in saudi arabia in this orginal research... open access • abstract • introduction • materials and methods    • virus seed stock    • study population    • vaccines    • the challenge virus    • study design    • the elisa    • statistical analysis    • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the epidemiology of orf virus infection in saudi arabia (sa) has been researched since 1990. the results obtained during this period indicate that the disease is widespread, has great economic impact and that no vaccine has been used against it. the present study compares the immunogenicity and protective efficacy of three locally developed live orf virus vaccines. two of them differ in their passage history in vero cell culture and the third was used as a virulent virus in glycerine buffer. to the best of the authors’ knowledge, no similar comparative study has been conducted in the middle east utilising three types of vaccines prepared from the same virus strain. selection of the candidate seed orf virus and performance of the quality control tests were as laid out by the oie for veterinary vaccine production. the vaccine seed virus was a field orf virus isolated from a previous orf outbreak in saudi arabia. a simple novel formula was developed to calculate the rate of reduction in the healing time (rht %) in the challenged sheep. this allowed direct comparison of the efficacy of the three types of vaccines employed in the present study. the efficacy of each vaccine was tested on a cohort of local noemi sheep. introduction top ↑ orf is a contagious zoonotic disease of sheep and goats. the causative agent is a parapoxvirus of the family poxviridae. the main lesions are usually found on the lips and around the mouth. sometimes, lesions can spread to the eyes, nostrils and teats (abuelzein & housawi 2009). under certain circumstances, the disease can take a generalised form throughout the body (abuelzein & housawi 1997). orf is distributed worldwide and can cause great economic losses to sheep production (robinson & balassu 1981).clinically, orf infection has been known to exist in saudi arabia for decades, but the first isolation of the virus from a disease outbreak in sheep and goats was made in 1990 (housawi et al. 1991). from that time, up-to-date, different clinical forms of orf infection, with various severities, have been reported (abelxein & housawi 1997, 2009; gameel et al. 1995; housawi et al. 1991). a serological survey revealed that the disease was widespread amongst sheep and goats in the country(housawi et al. 1992). in spite of the wide distribution of orf in saudi arabia, no vaccine has so far been used for its control. this paper describes a comparative study on three locally developed live vaccines prepared from a local field orf virus with the intention of finding one suitable to be recommended as a candidate vaccine to be used in the kingdom. efficacies of the individual vaccines are compared and the results are discussed. materials and methods top ↑ virus seed stock the seed vaccine orf virus (hou/sa/97) was selected from two field orf viruses which caused fulminant orf outbreaks in sheep and goats in 1995 and 1997. they were obtained from our collection, which was stored at −86 °c in glycerol buffered saline at ph 7.4. characterisation studies and selection of the seed virus followed the guidelines of the oie (oie 2008), which have previously been reported (housawi 2008).in the present study, scab material containing the seed virus was made up as a 30% suspension (w/v) in phosphate buffered saline (pbs) ph 7.4. following centrifugation at 377 × g for 15 min, the supernatant fluid was collected and antibiotics (procured through the saudi branch of sigma-aldrich) were added at a concentration of 100 iu/ml penicillin, 1 mg/ml streptomycin and 50 iu/ml mycostatin. the supernatant fluid was used to inoculate monolayers of vero cell culture, as described by housawi et al. (1991). when the cytopathic effect (cpe) involved 90% of the cell monolayer, the cells were harvested and stored in 0.5 ml aliquots at −86 °c. study population orf-seronegative indigenous noemi sheep, aged six months old, were used in the study. they were procured from a farm with no history of orf infection and were kept in complete isolation from other animals to avoid natural orf infection. they were provided with feed and water until used in the experiments.the ethical approval to use the sheep was provided by the ethical committee of the faculty of veterinary medicine and animal husbandry, king faisal university, saudi arabia. vaccines three types of live orf vaccines were prepared, namely, a live scab vaccine (lsv), vero cell culture passage 20 vaccine (p20v) and vero passage 75 vaccine (p75v). for preparation of the live scab vaccine (lsv), the ‘master seed virus’ was inoculated into five sheep, as described by housawi et al. (1993). the sheep were observed daily until the development of the orf lesions. the scab material was collected and stored at −86 °c until used for preparation of the lsv vaccine. the scab material was made up as a 50% (w/v) suspension in pbs ph 7.4, processed as described for the virus seed stock and stored at −86 °c until titrated in monolayers of vero cells (housawi et al. 1991). the tissue culture infective dose 50 (tcid50) was calculated following the method of read and muench (1938). the virus suspension was adjusted to contain 106 tcid50/ml in pbs-glycerol (50% v/v). the formulated vaccine was stored at 4 ºc. to prepare the p20v vaccine, the seed virus was serially passaged in monolayers of the vero monkey kidney cell culture (housawi et al. 1993). the 20th passage (p20v) was harvested and titrated in vero cell monolayers as above. aluminium hydroxide gel (sigma-aldrich, saudi branch) was then added at a concentration of 1.6 mg/ml and the final titre was adjusted to 106 tcid50 /ml. the vaccine was stored at 4 °c until used. the same procedure which was used for preparation of the p20v above was adopted for preparation and formulation of the p75v vaccine, except that the seed virus was serially passaged 75 times in vero cell culture. the challenge virus scab material from the experimentally infected sheep as described above, was made into a 50% (w/v) suspension in pbs ph 7.4, centrifuged at 377 × g for 15 min, and the supernatant fluid was collected, processed as before and 0.5 ml aliquots were stored at −86 °c until used in the challenge experiments. study design the experimental sheep were divided into three groups (n = 30) and each group was allocated to one of the respective vaccines. a further fifteen sheep were kept as unvaccinated controls in the challenge experiments.the group of 30 sheep used in the lsv vaccine experiments was subdivided into two equal groups of 15 sheep, a and b. both groups were subdivided into three subgroups of five sheep each, a1, a2 and a3, and b1, b2 and b3. the three a-subgroups received only a primary dose of the lsv, whilst the b-subgroups received the primary dose and a booster dose after a month. the same procedure of creating groups and subgroups was followed for the p20v and p75v vaccines, with the same numbers of sheep for each group. the designations for the p20v groups were d and e, and those for the p75v subgroups were g and f. the subgroups were d1, d2 and d3; e1, e2 and e3; f1, f2 and f3; and g1, g2 and g3. group d received only the primary p20v vaccine dose and group e received the p20v vaccine dose and a booster dose a month later. group g received only the primary p75v vaccine dose and group f the primary p75v vaccine dose followed by a booster dose a month later. the 15 control sheep were also subdivided into three equal sub-groups and each sub-group was used as a control at a challenging point. table 1 shows the vaccination schedule. all sheep of all the subgroups, except the controls, were vaccinated by single scarification (3 cm – 4 cm) on the inner thigh of the right hind limb, and 40 µl of the vaccine suspension was applied. the vaccinated sheep were observed daily and clinical changes at the sites of vaccination were recorded (nettleton et al. 1996). each of the sheep of all sub-groups, whether they received the primary dose only or the primary dose and a booster or were the controls, were challenged and infected in the manner described above. to evaluate the degree of protection in each challenged sheep (nettleton et al. 1996), daily clinical observations were performed on the sites of scarification from the first day following scarification until the scabs had dropped. accordingly, the mean healing time (mht) was calculated. each member of the research team (4 members) took daily readings and the mean was calculated. the criterion for complete healing was judged by presence of a smooth skin surface after the scabs had dropped. before complete healing, the scab is usually strongly adherent to the lesion and its forcible removal at the inner thigh of the sheep is not easy. forcible mechanical removal of a scab leads to abrasion and possible bleeding, which can easily be seen. in this study, none of the scabs in the experimental sheep was exposed to forcible mechanical detachment before complete healing and shedding. the mean reduction time (mrt) per cent, of the orf lesions in the challenged sheep, as compared to the unvaccinated control sheep, was calculated by the following formula: [eqn 1] where mrt % is the mean reduction time %, c is the mean healing time (days) in the unvaccinated control sheep and v is the mean healing time (days) in the vaccinated sheep. the elisa the reference orf antigen used in the elisa was provided by the moredun institute, uk. it was used as 1% nonindet p-40 orf specific extract (nettleton et al 1996). the specific reference sheep anti-orf serum, the non-immune sheep serum collected from orf-free sheep and the mock lamb muscle antigen for elisa were also provided by the moredun institute, uk. the test sheep sera were collected weekly from each sheep subgroup following the primary vaccination or booster. sera were also collected weekly from the sheep following each challenge (after 6, 12 and 18 months respectively). the sera were inactivated at 56 ºc for 30 min and stored at −20 ºc until used in the elisa. an indirect elisa was employed for the detection of humoral antibodies using the above-mentioned reagents. all volumes of reactants were 50 µl per well, incubations at all stages were made at 37 ºc, (except for the substrate which was incubated at room temperature 22 ºc). washing was by flooding and emptying the wells three times with pbs-tween (0.01% tween 20, sigma-aldrich, saudi branch). the elisa plates were coated with the reference orf antigen, incubated for two hours and washed. the test sera were diluted as required in pbs-t + 2% ovalbumin (sigma-aldrich, saudi branch) and incubated for one hour and the plates were washed. donkey anti-sheep igg conjugated to horseradish peroxidase (sigma-aldrich, saudi branch) was added following instructions of the manufacturers, incubated for one hour and washed. the substrate was prepared by adding a 30 mg tablet of orthophenyl diamine (sigma-aldrich, saudi branch) to 75 ml distilled water followed by 40 µl of hydrogen peroxide just before use. the substrate was added and the plates were incubated at room temperature for 10 min in the dark. the reaction was stopped by adding 1 m sulphuric acid and plates were read in an elisa reader (dynatec co.) at 450 nm and the results were interpreted as instructed by the manufacturers. statistical analysis following each challenge point (after 6, 12 and 18 months), the anova, in conjunction with a post hoc test, was used to evaluate the difference in protection between all the vaccinated groups and also between the vaccinated groups and the unvaccinated controls. table 1: vaccination schedule of the experimental sheep. table 2: challenging schedule of the vaccinated sheep and the unvaccinated controls. ethical considerations ethical approval for use of the experimental sheep and all protocols in this study were obtained from the ethical committee of the faculty of veterinary medicine & animal husbandry, king faisal university, saudi arabia. results top ↑ regardless of the vaccine type, typical stages of orf infection, viz., erythema, papule, pustule and scab formation, were seen following primary vaccination or the booster dose in all vaccinated sheep. however, some overlap was observed between the different stages of infection. table 3a shows the clinical changes at the site of vaccination following primary vaccination. table 3b shows that, with all three types of vaccines, the duration of healing following the booster dose was shorter than that following the primary dose only. tables 4, 5 and 6 show the anova and post hoc statistical analysis results of the different groups receiving the different vaccines and challenged after 6, 12 and 18 months. the results indicate highly significant differences between the groups at the 0.05 level and 95% confidence interval (p < 0.05). highly significant differences (p < 0.05) were also obtained between the vaccinated and unvaccinated control groups of sheep. figure 1 illustrates the mrt % values, following each of the 3 challenges in the vaccinated sheep. following the six months’ challenge of the groups that received one dose of the relevant vaccine, the mrt % value was highest for the p20v (33.3%), followed by that for the p75v (30%) and the lsv group (16.7%) respectively. following the 12 months’ challenge, the highest mrt % value was scored for the sheep that received the p20v (30%), followed by those that received the lsv (26.7%) and the lowest was scored by the p75v (16.7%) group. following the 18 months’ challenge, the mrt % values were 33.3%, 16.7% and 13.3% for the p20v, lsv and p75v respectively. figure 2 illustrates the mrt % values for the boostered sheep at each challenge point. at the 6 months’ challenge, the mrt % values were 43.3%, 33.3% and 30% for the p20v, lsv and the p75v respectively. at the challenge point after 12 months, the mrt % values were 36.7% for the p20v and 26.7% for both the lsv and p75v. following the challenge point at 18 months, the mrt % values were 33.3%, 16.7% and 13.3% for the p20v, lsv and p75v respectively. figure 3 represents a typical pattern of elisa values reflecting the serum antibody levels following challenges (as exemplified by the p20v vaccinated sheep following the 6 months’ challenge). the optical density (od) values shown on the y-axis represent net values obtained by subtracting the background reactivity of the pre-immune serum. all samples were measured in duplicate. as seen in figure 3, the mean od value at the day of challenge (day zero) was high (2.93 ± 0.12) then dropped to 2.02 ± 0.15 on day 7 post challenge (pc), (most probably due to neutralisation by the challenge virus). on day 14 pc, the od level rose to 2.83 ± 0.13, then there was a slight drop by day 21 pc (2.52 ± 0.15) and it remained almost at that level until day 28 pc (2.51 ± 0.14). the challenged unvaccinated control sheep were elisa seronegative at the time of challenge but seroconverted after challenge. the od values started rising from day 7 pc to reach a high level by day 28 pc. table 3a: mean clinical changes at the site of scarification after primary vaccination. table 3b: mean clinical changes at the site of scarification after the booster dose. table 4: anova results for all the vaccinated and unvaccinated sheep groups following the 6 month challenge. table 5: anova results for all the vaccinated and unvaccinated sheep groups following the 12 month challenge. table 6: anova results for all the vaccinated and unvaccinated sheep groups following the 18 month challenge. figure 1: percentage reduction in the mean healing time following challenge of the sheep that received a single vaccine dose only. figure 2: percentage reduction in the mean healing time following challenge of the sheep that received the booster vaccine dose. figure 3: typical pattern of elisa mean values in sheep sera following the 6 months challenge. sera were diluted at 1/50 dilution. the optical density (od) values shown on the y-axis are net-values obtained by subtracting the background reactivity of the control non-immune serum. discussion top ↑ unlike other animal viral vaccines, which may confer full protection to the vaccinated animal, orf vaccines do not give 100% protection. therefore, the main purpose behind orf vaccination is to protect vaccinated animals from the severe effects of the field virus and to offer appreciable reduction in the duration of the clinical disease. the present study was undertaken to develop a local efficacious vaccine to be used in saudi arabia, where orf infection is widespread. accordingly, three types of live vaccines were developed from a local field orf virus so as to choose the most efficacious to be recommended for use in the field. to the best of the authors’ knowledge, no similar comparative study, utilising three types of vaccine prepared from the same virus strain, has been conducted in the middle east nor in the developing world. each of the developed vaccines gave clinical responses at the site of vaccination (indicating viability), induced production of humoral antibodies and conferred some degree of protection. to evaluate the ability of the vaccine to minimise the time of healing following challenge, a simple formula was developed in this study to calculate the rate of mean reduction in the healing time (mrt %) following challenge. this exercise enabled direct comparison of the efficacy of the three types of vaccines. comparing the efficacy of the three types of vaccines, it could be seen that p20v gave the best results. it induced highly significant reduction in disease duration following the three challenge points. it is clear that the twenty passages of the orf virus in vero cell culture were satisfactory for the production of a safe and efficacious p20v orf vaccine under our local conditions. it is rather difficult to explain why this occurs, but it is likely that at this passage level the virus has lost its high virulence but can maintain its immunogenicity to confer good protection. generally speaking, the results for the three types of vaccine indicate that protection in the boosted sheep was better than in those that received only one dose of the vaccine, although no improvement was seen in the p75v results. published data indicate that orf protection is predominantly cell mediated. however, humoral antibodies are also vital in the activation of the killer cell (antibody dependent cytotoxic cell – adcc), which is an important branch of the cell-mediated immunity. the present study has illustrated that the three types of vaccine induced the production of humoral antibodies following primary vaccination of the sheep and resulted in an amnesic response following the booster dose. conclusion top ↑ this study involved the development of three different orf vaccines from the same virus isolate, for the first time in the middle east and probably elsewhere.two of the vaccines were prepared by passaging at different levels in vero cell culture; and the third was used as a virulent virus in glycerine buffer saline. the p20v was found to be the most efficacious of the three vaccines, and is recommended for use in the field in saudi arabia. in the present study, a novel formula was also developed to calculate the healing time as a percentage of that seen in control animals instead of scoring it in days. this will enable researchers in the field of orf vaccinology to compare their results directly. acknowledgements top ↑ the authors would like to thank king abdulaziz city for science and technology for grant number at-19-6 and for their ever continuous encouragement. we also would like to thank dr p. nettleton for provision of the reference elisa antigen and antiserum. the technical assistance of mr a. khars and the kind help of dr nahla khames in the statistical analysis are appreciated. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions f.m.h. (king faisal university) was the principal investigator. all authors participated in the project design and brain storming discussions. e.m.a. (king faisal university) prepared the vaccines. e.m.a. (king faisal university) and f.m.h. (king faisal university) performed the elisa experiments. a.a.g. (king faisal university) was the pathologist of the team. he inoculated the sheep with the different vaccines. a.a.g. (king faisal university), e.m.a. (king faisal university), f.m.h. (king faisal university) and a.i.a. (king faisal university) participated in reading of the vaccination & challenge results and subsequent follow-up. e.m.a. (king faisal university), f.m.h. (king faisal university), a.a.g. (king faisal university) and a.i.a. (king faisal university) wrote the manuscript. e.m.a. (king faisal university) is the corresponding author. references top ↑ abuelzein, e. & housawi, f., 1997, ‘severe long-lasting contagious ecthyma infection in a goat’s kid’, journal of veterinary medicine series b 44, 561–564. http://dx.doi.org/10.1111/j.1439-0450.1997.tb01008.x abuelzein, e. & housawi, f., 2009, ‘drastic cutaneous multi-focal orf infection in goats, causing severe dysfunctioning’, revue scientifique et technique (oie) 28, 1025–1029. pmid:20462159 andrewes, c. & horstman, d., 1949, ‘the susceptibility of viruses to ethy1 ether’, journal of general microbiology 3, 290–292. pmid:18149942 gameel, a., abuelzein, e., housawi, f., agib, a. & ibrahim, a., 1995, ‘clinico-pathological observations on naturally occurring contagious ecthyma in lambs in saudi arabia’, revue d’elevage et de médecine vétérinaire des pays tropicaux 48, 233–235. pmid:8745744 housawi, f., 2008, ‘characterization of candidate seed orf viruses to be used as vaccine in sheep and goats in saudi arabia’, scientific journal king faisal university (basic sciences) 9, 137–146. housawi, f. & abuelzein, e., 1991, ‘orf infection following ear tagging in goats’, revue d’elevage et de médecine vétérinaire des pays tropicaux 44, 277–278. housawi, f., abuelzein, e., amin, m. & al afaleq, a., 1991, ‘orf infection in sheep and goats in saudi arabia’, veterinary record 128, 550–551. http://dx.doi.org/10.1136/vr.128.23.550, pmid:1909477 housawi, f., abuelzein, e., al afaleq, a. & amin, m., 1992, ‘serosurveillance for orf antibodies in sheep and goats in saudi arabia employing the elisa technique’, journal of comparative pathology 106, 153–158. http://dx.doi.org/10.1016/0021-9975(92)90044-u housawi, f., abuelzein, e., gameel, a. & al afaleq, a., 1993, ‘a close comparative study on the response of sheep and goats to experimental orf infection’, journal of veterinary medicine series b 40, 272–282. http://dx.doi.org/10.1111/j.1439-0450.1993.tb00138.x marklew, s., 1995, ‘assessment of cell-culture grown on virus vaccines in sheep’, in m. schwyzer (ed.), immunology viral infections: proceedings of the 3rd congress european society, veterinary virology, september 4–7, 1995, pp. 305–309. nettleton, p., brebner, j., pow, t., gilray, j., bell, g. & reid, h., 1996, ‘tissue culture propagated orf virus vaccine protects lambs from orf virus challenge’, veterinary record 138, 184–186. http://dx.doi.org/10.1136/vr.138.8.184, pmid:8677620 office international epizooties (oie), 2008, ‘bovine viral diarrhoea’, manual of standards for diagnostic tests and vaccines , chapter x.5, p. 18, from http://www.oie.int reed, l. & muench, g., 1938, ‘a simple method of estimating fifty percent point’, american journal of hygiene 27, 493. robinson, a. & balassu, t., 1981, ‘contagious pustular dermatitis (orf)’, veterinary bulletin 51, 771–782. talhouk, r. & elzein, e., 1986, ‘characterization of a cell culture adapted goat pox virus’, journal of veterinary medicine series b 33, 543–551. http://dx.doi.org/10.1111/j.1439-0450.1986.tb00066.x cherenet_307-312.qxd introduction bovine trypanosomosis is a serious constraint to agricultural production in extensive areas of the tsetse-infested ethiopian lowlands (slingenbergh 1972). tsetse flies are found mainly in the southern and western parts of the country (langridge 1976). as a result of the trypanosomosis threat, a large proportion of the livestock population is found in the highlands that are assumed to be tsetse-free. although livestock trypanosomosis is a well-known constraint to livestock production in ethiopia little attention has been paid to the trypanosomosis situation in the amhara region in the north-western part of the country. nevertheless, livestock are the backbone of the rural economy in this region where approximately one third of ethiopia’s cattle, sheep and goat population is found. to clarify the trypanosomosis situation a survey was conducted to determine not only the prevalence of trypanosome infections in cattle in a tsetse-infested area and a tsetse-free area of the amhara region but also the seasonal changes in the prevalence of the disease. 307 onderstepoort journal of veterinary research, 71:307–312 (2004) seasonal prevalence of bovine trypanosomosis in a tsetse-infested zone and a tsetse-free zone of the amhara region, north-west ethiopia t. cherenet, r.a. sani1, j.m. panandam2, s. nadzr1, n. speybroeck3 and p. van den bossche3,4,* abstract cherenet, t., sani, r.a., panandam, j.m., nadzr, s., speybroeck, n. & van den bossche, p. 2004. seasonal prevalence of bovine trypanosomosis in a tsetse-infested zone and a tsetse-free zone of the amhara region, north-west ethiopia. onderstepoort journal of veterinary research, 71:307–312 during a period of four consecutive years, trypanosomosis surveys were conducted in a tsetseinfested and tsetse-free area of the amhara region of north-west ethiopia. in each study area randomly selected communal cattle were sampled and their blood was investigated using parasitological diagnostic methods. at the same time the population of biting flies was sampled. the monthly average prevalence of trypanosome infections in cattle did not differ significantly between study areas. in both study areas, the prevalence of trypanosome infections was highest during the long rainy season. trypanosome infections were mainly due to trypanosoma vivax and they significantly reduced the average packed cell volume and the body condition of the animals. the monthly prevalence of infection was correlated with the density of biting flies, such as tabanidae and stomoxys spp., in the preceding month suggesting an important role of mechanical transmission in the epidemiology of trypanosomosis in both areas. keywords: biting flies, bovine trypanosomosis, epidemiology, ethiopia, prevalence * author to whom correspondence should be addressed 1 faculty of veterinary medicine and 2 faculty of agriculture, university putra malaysia, 43400 serdang, malaysia 3 institute of tropical medicine, veterinary department, nationalestraat 155, 2000 antwerp, belgium 4 faculty of veterinary sciences, department of veterinary tropical diseases, private bag x04, onderstepoort, 0110 south africa accepted for publication 7 april 2004—editor material and methods study areas the study areas were located in the amhara region of north-west ethiopia. the region covers an area of approximately 170 000 km2 and is divided into 11 administrative zones. it is characterized by high plateaux, mountains and broad valleys. the elevation varies from about 700–4 600 m above sea level. the steep terrain characterising much of the mountainous areas limits agricultural development. the altitude of the study areas ranges from 700–2 500 m above sea level. the climate in the study areas can be divided into a short rainy season or “belg” between late february and early may and a longer, more reliable rainy season or “kiremt” from june to october. the study areas are densely populated with an estimated average human population density of about 100 persons per km2. more than 90 % of the population live in rural areas and practice subsistence, rainfed cropping combined with extensive grazing of livestock. approximately 30 % of the land area is cultivated, 12 % is used for grazing, 19 % is forest and bush, 26 % is unproductive land and 13 % is classified as un-utilizable land (boped 2000). part of the amhara region is tsetse-infested. glossina morsitans submorsitans and glossina tachinoides are present (langridge 1976). three tsetseinfested administrative zones, west gojjam, east gojjam and awi, were included in this study. samples were collected from cattle from the guanga, ankesha, dangla, bure and denbecha districts in this zone which are located on the edge of the tsetsebelt. the two tsetse-free study areas are located in the north and south gonder administrative zones. cattle were sampled in the gonder-zuria, fogera, libo-kemekem, este, chellga, metema and bassoliben districts. the tsetse-free study areas are located about 500 km north of the tsetse-belt of the amhara region at an altitude above 1800 m. chemotherapy is the only trypanosomosis control method used in the study areas. veterinary supervision is inadequate, which may lead to the inappropriate use of drugs or use of drugs of substandard quality. sample selection the cattle in the tsetse-free and tsetse-infested study areas are indigenous zebus (fogera breed). the surveys were conducted between 1997 and 2001. ten villages were identified in each district and visited at 4 month intervals each year. during each sampling, about 40 heads of communal cattle were selected in each village using a multistage random sampling technique (putt, shaw, woods, tyler & james 1988). sampling method the parasitological diagnostic tests used were those described by paris, murray & mcodimba (1982). blood was collected from an ear vein into heparinized microhaematocrit centrifuge capillary tubes and onto glass slides in order to make thick and thin blood smears. the capillary tubes were sealed with “cristaseal” (hawksley) and centrifuged immediately in a microhaematocrit centrifuge for 5 min at 9 000 rpm. after centrifugation, the packed cell volume (pcv) was determined. animals with a pcv < 24 % were considered to be anaemic. the buffy coat and the uppermost layer of red blood cells in each specimen were extracted, placed onto a microscope slide and examined under a phase contrast microscope with a x 40 objective lens for the presence of motile trypanosomes. the thick and thin blood smears were stained with giemsa and examined under a light microscope using a x 100 oil immersion objective lens. at the time of sampling, the owner, age and sex of each animal were recorded and its body condition was scored using the method described by nicholson & butterworth (1986). entomological survey the population of biting flies was monitored using unbaited biconical (challier, eyraud, lafaye & laveissière 1977) and ngu traps (brightwell, dransfield, kyorku, golder, tarimo & munga 1987). three traps were deployed in representative habitats in the tsetse-infested and tsetse-free study sites for 5 days each month. records were kept of the fly species and number and sex of each species captured in each trap during each month. the monthly mean index of apparent abundance (iaa) of biting flies in the two study sites was calculated as the mean number of biting flies (males and females) captured per trap per month. statistical analyses statistical analyses were carried out in stata 7 (statacorp. 2001). use was made of logistic regression and regression analysis. the relationship between the prevalence of trypanosome infection and the iaa of biting flies was determined after arc308 bovine trypanosomosis in amhara region, north-west ethiopia sine transformation. the pcv data were compared using the non-parametric kruskal-wallis equality of population rank test. results between may 1997 and december 2001, a total of 7 079 head of cattle (3 888 in the tsetse-free area and 3 191 in the tsetse-infested study area) were sampled. a total of 501 animals (262 in the tsetsefree area and 239 in the tsetse-infested area) were found to be infected with trypanosomes. the average parasitological prevalence was 6.6 % and 7.3 % in the tsetse-free and tsetse-infested study area, respectively. the parasitological prevalence of trypanosomosis differed significantly between months (p < 0.0001) but did not differ between the tsetsefree study area and the tsetse-infested study area (p = 0.57). differences between years were small and data were pooled for further analyses. the parasitological prevalence was highest during the long rainy season (june to october) (fig. 1). the majority of the infected animals (262 or 100 % in the tsetse-free and 223 or 93.3 % in the tsetseinfested area) was infected with trypanosoma vivax. a total of 450 animals were infected only with t. vivax and the remainder were co-infected with either trypanosoma congolense (29) or trypanosoma brucei (6) (table 1). trypanosoma brucei was not detected in the tsetse-free area (table 1). trypanosoma congolense accounted for 4.2 % of the trypanosome infections in the tsetse-free area and 10.0 % of the trypanosome infections in the tsetseinfested area (table 1). 309 t. cherenet et al. ����� � � � � � � � � � � � � � �� �� � � � � � �� � � � � � � � � �� �� � �� � � � � � � � � � ����� � � � � � � � � � � � �� � �� � �� �� � � � � � � � � � � � � � fig. 1 monthly average prevalence of trypanosome infections in cattle in the tsetse-infested ( ) study area and tsetse-free ( ) study area of the amhara region of north western ethiopia fig. 2 monthly average pcv of parasitologically positive ( ) and negative ( ) cattle table 1 monthly total number of cattle with t. vivax, t. congolense, t. brucei or mixed infection in the tsetse-free study area and tsetse-infested study area of the amhara region of ethiopia t. vivax t. congolense t. brucei mixed t.c./t.v. mixed t.b./t.v. month free infested free infested free infested free infested free infested jan 28 19 1 1 1 feb 26 10 mar 18 20 apr 36 5 may 16 7 jun 21 30 1 jul 11 7 1 aug 1 6 1 sep 22 32 1 11 11 2 oct 16 26 4 4 1 nov 30 24 6 1 dec 26 13 1 1 1 1 the prevalence of trypanosome infections did not differ between age categories (p = 0.77) or sexes (p = 0.09). the average pcv of the animals infected with trypanosomes was 21.1 ± 3.4 % and 20.9 ± 2.5 % in the tsetse-free area and tsetse-infested study area, respectively. it was significantly lower (p = 0.001) than the average pcv of the animals that were parasitologically negative (28.1 ± 5.2 % and 26.9 ± 5.2 % for the tsetse-free area and tsetseinfested study area, respectively) (fig. 2). the body condition score of cattle infected with trypanosomes was significantly lower (p = 0.012) than that of the parasitologically negative animals (fig. 3). throughout the entomological surveillance period, no tsetse flies were captured in the tsetse-free area. glossina m. submorsitans and g. tachinoides were present in the tsetse-infested areas. haematopota maculosifacies, hippobosca maculata, stomoxys calcitrans, chrysops streptobalius and tabanus atrimanus were biting flies captured in both the tsetsefree area and the tsetse-infested area. the iaa of biting flies (including tsetse flies) showed a clear seasonal trend with high iaa during the long rainy season (fig. 4). the monthly iaa of biting flies correlated significantly (p = 0.023) with the prevalence of trypanosome infections in the same month. discussion the results suggest that trypanosomosis is an important disease of cattle in the amhara region of north-west ethiopia. taking into account the low sensitivity of the parasitological diagnostic methods and the uncontrolled use of trypanocidal drugs, the real prevalence of infection is probably substantially higher. the average overall prevalence of infection in the tsetse-infested study area was comparable to the overall prevalence of infection in the tsetse-free study area but lower than the prevalences reported from other tsetse-infested regions in ethiopia (abebe & jobre 1996; kidanemariam, hadgu & sahle 2002). this is attributed to the location of our sampling sites. sampling was conducted on the edge of the tsetsebelt where the density and thus tsetse challenge could be expected to be lower. the average prevalence of trypanosome infections in the tsetse-free study area was comparable to the one observed by abebe & jobre (1996) in the gojjam and gonder highlands. 310 bovine trypanosomosis in amhara region, north-west ethiopia ��������� ����� � � � �� � � �� �� � � � � � �� � �� � � � � � � � � � � � � � �� �� � � �� � � �� � � �� � � � � � � � ����� � � � � � � � � � � � � � ����� � � � � � � � � � � � � � �� � � �� �� � � � �� �� � � � � � �� �� �� � �� �� fig. 3 proportion of the total number of cattle, infected ( ) or not-infected ( ) with trypanosomes, belonging to different body condition scoring categories fig. 4 monthly average index of apparent abundance of biting flies in the tsetse-free ( ) study area and tsetseinfested ( ) study area (a) and monthly proportion of tsetse flies ( ) and other biting flies ( ) in captures in the tsetse-infested study areas (b) longitudinal studies conducted in the tsetse-infested ghibe valley (south-west ethiopia) have shown that the incidence of trypanosome infections in cattle is highest during the dry season months of october to february, coinciding with the period of higher density of tsetse (leak, mulatu, authie, d’ ieteren, peregrine, rowlands & trail 1993). in our study sites, the prevalence of trypanosome infections increased substantially during the long rainy season (june to october) and remained high during the early dry season (november). outbreaks of acute t. vivax in ethiopian cattle have been attributed to increased rainfall (roeder, scott & pegram 1984). d’amico, gouteux, le gall & cuisance (1996) demonstrated a high level of interaction between cattle and biting flies during the rainy season. the correlation between the prevalence of trypanosome infections and the iaa of biting flies also suggests a close link between trypanosomosis challenge and the number of biting flies. the relative epidemiological importance of the various species of biting flies present in the study area will be the subject of another study. the majority of the trypanosome infections in both study areas were due to t. vivax. this high proportion of t. vivax infections is in accordance with observations made in other tsetse-free areas of ethiopia (roeder et al. 1984; abebe & jobre 1996) and elsewhere (jones & davila 2001). the high proportion of t. vivax infections in the tsetse-infested area contrasts sharply with trypanosome species prevalence data from other tsetse-infested regions of ethiopia where t. congolense is the most prevalent trypanosome species in cattle (leak et al. 1993; abebe & jobre 1996). the high proportion of t. vivax infections in cattle sampled in our tsetse-infested areas is attributed to the location of the study sites which are situated on the edge of the tsetse-belt. ecological conditions for tsetse on the edge of a flybelt are usually less favourable resulting in a high mortality rate of tsetses and favouring the transmission of trypanosome species with a short developmental cycle such as t. vivax. furthermore, the role of mechanical transmission of t. vivax in such areas cannot be underestimated. similar conclusions were drawn by kidanemariam et al. (2002) who conducted surveys along the edge of the tsetse-belt in southern ethiopia. although it has been shown that t. congolense and t. brucei can be transmitted mechanically, the transmission rate is usually low (mihok, maramba, munyoki & kagoiya 1995). hence, the t. congolense cases detected in the tsetse-free study area are attributed to cattle that were imported from the tsetse-infested into the tsetse-free area rather than mechanical transmission of this trypanosome species. the presence of trypanosome infections resulted in a significant decline in pcv and body condition score. other factors such as malnutrition or other diseases may also affect the pcv and body condition. however, it is unlikely that the impact of those additional factors differs greatly between the parasitologically positive and negative animals. hence, relative comparisons between the pcv and body conditions score of parasitological positive and negative animals can be made. from the present study it can thus be concluded that trypanosomosis is an important disease and a potential threat in affecting the health and productivity of cattle in the economically important amhara region of north-west ethiopia. acknowledgements the authors acknowledge prof. dr getachew abebe of the addis abeba university, faculty of veterinary medicine for his field supervision and assistance. the work presented in this paper was funded by the ethiopian agricultural research organisation and the amhara regional agricultural institute and bureau of agriculture of the region. all staff of the genetics laboratory of the university putra malaysia and, in particular, dr p. kalaiselvi are thanked for their assistance. the bahir dar regional veterinary laboratory staff and drs solomon, ato habtamu and ato sahile are thanked for their assistance. references abebe, g. & jobre, y. 1996. trypanosomosis: a threat to cattle production in ethiopia. revue de médecine vétérinaire, 147:897–902. boped (2000). amhara regional 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parasitaire et génétique des populations, institut pasteur d’algérie, algiers, algeria abdelaziz boukhelkhal scientific and technical research centre for arid areas (crstra), biophysical station, nezla, touggourt, algeria curtis r. youngs animal science department, iowa state university, ames, united states of america rachid kaidi institute of veterinary sciences, lbra, university of blida, soumaa, blida, algeria bernard faye umr selmet, cirad-es, montpellier, france youcef halis scientific and technical research centre for arid areas (crstra), biophysical station, nezla, touggourt, algeria citation benaissa, m.h., mimoune, n., bentria, y., kernif, t., boukhelkhal, a., youngs, c.r. et al., 2020, ‘seroprevalence and risk factors for trypanosoma evansi, the causative agent of surra, in the dromedary camel (camelus dromedarius) population in southeastern algeria’, onderstepoort journal of veterinary research 87(1), a1891. https://doi.org/10.4102/ojvr.v87i1.1891 original research seroprevalence and risk factors for trypanosoma evansi, the causative agent of surra, in the dromedary camel (camelus dromedarius) population in southeastern algeria mohammed h. benaissa, nora mimoune, younes bentria, tahar kernif, abdelaziz boukhelkhal, curtis r. youngs, rachid kaidi, bernard faye, youcef halis received: 21 june 2020; accepted: 14 oct. 2020; published: 21 dec. 2020 copyright: © 2020. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract surra, caused by trypanosoma evansi, is a re-emerging animal trypanosomosis, which is of special concern for camel-rearing regions of africa and asia. surra decreases milk yield, lessens animal body condition score and reduces market value of exported animals resulting in substantial economic losses. a cross-sectional seroprevalence study of dromedary camels was conducted in algeria, and major risk factors associated with infection were identified by collecting data on animal characteristics and herd management practices. the seroprevalence of t. evansi infection was determined in sera of 865 camels from 82 herds located in eastern algeria using an antibody test (card agglutination test for trypanosomiasis – catt/t. evansi). individual and herd seroprevalence were 49.5% and 73.2%, respectively, indicating substantial exposure of camels to t. evansi in the four districts studied. five significant risk factors for t. evansi hemoparasite infection were identified: geographical area, herd size, husbandry system, accessibility to natural water sources and type of watering. there was no association between breed, sex or age with t. evansi infection. results of this study provide baseline information that will be useful for launching control programmes in the region and potentially elsewhere. keywords: seroprevalence; trypanosoma evansi; camelus dromedarius; camels; algeria. introduction trypanosomiasis is a significant animal and human health problem worldwide. human african trypanosomiasis (sleeping sickness) is caused by infestation with the flagellate protozoan trypanosoma brucei, whereas animal trypanosomiasis is caused by different species and subspecies of trypanosoma (t. congolense, t. vivax, t. brucei brucei and t. brucei evansi). amongst all of the trypanosomiases, ‘surra’ (caused by trypanosoma brucei evansi) has the widest host range and the broadest geographical distribution. it is transmitted mechanically by the bite of infected tabanid flies; family: tabanidae (desquesnes et al. 2009); genus: tabanus, muscidae stomoxys, haematopota, lyperosia spp. and chrysops spp. (luckins 1998), or infected vampire bats (stoco et al. 2017). transmission occasionally occurs iatrogenically through herd identification and vaccination procedures (gutierrez et al. 2005), and oral transmission was confirmed recently in a mouse model (mandal et al. 2017). trypanosomosis serves as an important constraint to camel production because it substantially reduces animal productivity, thus inducing economic losses. it is a major endemic disease problem throughout central and south america, africa and asia (gutierrez et al. 2000). infection with t. brucei evansi (hereafter, t. evansi) reduces market value of exported animals, decreases milk yield and lessens animal body condition score, resulting in more than $223 million loss to the camel industry (salah, robertson & mohamed 2015). in addition to causing the aforementioned losses, t. evansi is the most important cause of infectious abortions in camels in the middle east and africa (boushaki et al. 2019; more et al. 2017). camels with clinical disease can present progressive emaciation, severe anaemia, reproductive problems (because of infertility, abortions and stillbirths), and eventually death (desquesnes et al. 2013). in algeria, t. evansi was first detected in 1903 in infected camels (sergent & sergent 1905). since that time, few studies have been performed on livestock, and most were conducted in a limited geographical area with a limited number of samples (benfodil et al. 2020; bennoune et al. 2013; boushaki et al. 2019); those studies concluded only that the hemoparasite can be detected in blood samples. limited data are available currently concerning the epidemiology and distribution of the t. evansi parasite in algeria. to gauge levels of endemic stability, it is important to assess the seroprevalence of t. evansi and to characterise local variations in risk factors. to help fill the scientific knowledge gap regarding this important disease, the present study was conducted with the objectives of determining the seroprevalence of t. evansi and investigating potential risk factors for infection with t. evansi in the one-humped camel (camelus dromedarius) population in eastern algeria. materials and methods study area this study was carried out in four districts (biskra, el-oued, ouargla and ghardaia) in eastern algeria. these provinces are located at 00204–00735 e and 28°32–34°56 n (figure 1). this region is considered as one of the most significant camel-rearing areas in algeria, and it is a region where camel milk is increasingly commercialised and consumed. the climate of this region is arid and is characterised by long, hot summers and short, mild winters. figure 1: distribution of trypanosoma evansi seropositivity in dromedary camels in four districts in southeastern algeria. numbers in red indicate the number of camels seropositive for trypanosoma evansi as a proportion of animals tested in the biskra, el-oued, ghardaia and ouargla districts. data collection data were collected using a paper-based questionnaire given to herd owners by trained investigators. the study questionnaire focused on 23 potential explanatory variables grouped by: (1) pastoralists’ socio-demographic characteristics (gender, age, occupation and formal education); (2) individual camel data (age class [< 1 year, 1–3 years, 4–9 years, 10–15 years and > 15 years; ages were assigned based on dental wear and owner information], sex [male, female], breed [sahraoui, tergui]); and (3) herd data (location [district], herd size [small: < 10 head, medium: 11–30 head, large: > 30 head], the introduction of new camels bought at a livestock market [yes or no], presence of other domestic animal species [sheep, goats, cattle; yes or no], epidemiological characteristics [occurrence of abortions in the herd], exchange of bulls for mating [yes or no] and animal management system). sample size determination the minimal sample size required for this study was determined using the random sampling method (thrusfield 2018), considering an expected prevalence of 35% (based on unpublished survey data collected in 2008 in seven districts in algeria) with 5% precision at the 95% confidence level. the formula used for calculation of sample size was: where n = number of sample size, p = expected prevalence and d2 = absolute precision. although the minimum sample size calculated for this study was 350 animals, we increased the number of samples collected to improve the degree of accuracy and to account for some potential sample losses. sample collection and processing a total of 865 blood samples were collected in a randomised manner. a total of 10 ml of blood was collected by jugular venipuncture. blood samples remained at room temperature for 3–5 h to allow clotting, followed by centrifugation for 15 min at 3000 revolution per minute (rpm). the serum fraction was extracted with a micropipette, and serum samples were frozen at −20 °c until analysis. sera were tested for the presence of anti-t. evansi antibodies using a commercially available direct card agglutination test (card agglutination test [catt] for trypanosomiasis, t. evansi kit, institute of tropical medicine, antwerp, belgium) according to the manufacturer’s instructions. approximately, 45 µl of test reagent was transferred onto the test card and was mixed with an equal volume of the test sera diluted 1:4 with catt buffer. the test card was rotated for 5 min at 70 rpm, after which time the reaction was checked carefully for agglutination. positive reaction (evidence of agglutination) was determined by the quantitative presence of blue-colored granules (bajyana-songa & hamers 1988). individuals with a positive catt-p titer _1:4 result (+ = weak, ++ = moderate, +++ = strong) were considered serologically positive. statistical analysis pearson’s chi-square test or fisher’s exact probability test were applied to test for significant associations between potential risk factors and the outcome variable (status of trypanosoma seropositivity in camels) in a univariate analysis. multivariate logistic regression models were carried out using all variables showing a tendency towards statistical significance (p value £0.25). the logistic regression model was developed with a step-wise forward approach using a likelihood ratio test at each step with a p value of 0.10 as the significance level for removal or retention of the variable. in the final model, any variable with a p value < 0.05 was considered statistically significant. model fit analysis was assessed with the hosmer and lemeshow goodness-of-fit test. all statistical analyses were performed using the statistical package for social sciences (spss) version 17.0 (spss inc., chicago, il, united states [us]). ethical consideration animal sampling was implemented with the approval of the national veterinary authorities and was exclusively done by veterinarians; the animals were blood-sampled without suffering and were subsequently released. farmers in each zone gave verbal consent to participate in our study and gave permission for the blood sample collection from camels on their property. we followed the european guidelines (european directives eu 86/609-ste123 and 2010/63/eu) for animal handling. results individual level antibodies against t. evansi were detected in 428 of the 865 individual camels tested, giving an individual seroprevalence rate of 49.5% (95% confidence interval [ci]: 46.1% – 52.9%). at the herd level, 60 of the 82 herds tested had at least one seropositive animal, giving a herd seroprevalence rate of 73.2% (95% ci: 62.2% – 82.4%). results of the univariate analysis (chi-square) revealed that individual animal seroprevalence for t. evansi was affected by geographical area, herd size, husbandry system, accessibility to natural water sources and type of watering (table 1). contrarily, no differences (p > 0.05) were demonstrated in trypanosomosis seroprevalence because of genetic sex, breed or animal age. table 1: factors associated with animal-level seroprevalence of antibodies against trypanosoma evansi for dromedary camel populations of southeastern algeria. there was a very strong association between t. evansi seroprevalence and geographical area (χ2 = 4.80, p < 0.0001). the highest infection rate (62.5%, 125/200) was found in ghardaia (northern part of the sahara region), whereas the district of ouargla had the lowest infection rate (27.5%, 55/200). the analysis showed that camels raised in large herds had a higher (p < 0.007) incidence of t. evansi infection compared with those raised in small herds. regarding the multivariate logistic regression analyses (table 2), three variables remained in the final model: (1) herd size, (2) husbandry system and (3) water source. the odds of t. evansi infection were 2.2 times higher amongst large camel farms (p = 0.007, odds ratio [or] = 2.159; 95% ci = 1262–3693) compared with small herds. herds with more than 50 camels were 2.1 times more likely to have a seropositive animal than herds with fewer than 10 head. the presence of natural sources of water in nearby grazing areas was associated with an increased risk of t. evansi infection (or = 9.415; 95% ci = 1.256–70, 583). animals in a semi-intensive management system were more likely to be infected (or = 2.95; 95% ci: 1.878–4.645) than extensively managed animals. table 2: risk factors for individual-level infection with trypanosoma evansi amongst dromedary camel populations in southeastern algeria. herd level the univariate herd level analysis revealed five factors associated with t. evansi seropositivity (table 3): geographical location (p = 0.004), husbandry system (p = 0.038), history of abortion (p = 0.005), herd size (p = 0.010), and nearby natural water sources (p < 0.05; table 3). trypanosoma evansi seroprevalence was higher (p < 0.001) in ghardaia, biskra and el-oued than in ouargla. in contrast, no association was found between t. evansi seropositivity of the herd and contact with other camel herds, husbandry system (nomadic) or introduction of purchased camels (table 3). table 3: factors associated with animal-level seroprevalence of antibodies against trypanosoma evansi for dromedary camel populations of southeastern algeria. the multivariate logistic regression model showed that the main risk factor associated with t. evansi infection in camel herds was geographical location (table 4). the likelihood of detecting t. evansi antibodies was 11 times higher in the district of ghardaia (or = 11.25; 95% ci [2.00–63.19]), 7.5 times higher in el-oued (or = 7.50; 95% ci [2.02–27.86]) and 6 times higher in biskra (or = 6.00; 95% ci [1.27–28.25]) than in the district of ouargla (table 4). table 4: risk factors for herd-level infection with trypanosoma evansi amongst dromedary camel populations in southeastern algeria. discussion to our knowledge, few studies have been performed in algeria aimed at assessing risk factors associated with t. evansi infection of dromedary camels. our results, which document high seroprevalence rates at the individual and herd levels, could ultimately guide the design and implementation of enhanced surveillance programmes, control measures and prevention strategies for t. evansi not only in eastern algeria but throughout major camel producing regions of the world. in algeria, t. evansi infection in camels was confirmed for the first time more than a century ago when parasitological examinations discovered that 10% of 282 camels were infected (sergent & sergent 1905). outbreaks of trypanosomosis in dromedary herds associated with mortalities and abortions have been documented (boushaki et al. 2019). the t. evansi seropositivity rate in our study (49.5%) was higher than previous serological studies conducted in algeria by benfodil et al. (2020) and boushaki et al. (2019), who reported seroprevalence up to 32.4%, and 9.9%, respectively. the higher seroprevalence in the present study could be because of the differences in the topography of the study areas, age of tested animals, proportion of males versus females, sample size, environmental conditions during the sampling period, or differences in diagnostic methods. however, the t. evansi seroprevalence rate in our study (49.5%) is highly consistent with the findings from studies conducted in other developing countries that revealed t. evansi seroprevalence rates of 46% in burkina faso (dia 2006), 45.9% in kenya (njiru et al. 2004), 43.8% in saudi arabia (el-wathig & faye 2013), 43.5% in egypt (abdel-rady 2008) and 47.7% in pakistan (tehseen et al. 2015). one study in somalia reported a higher t. evansi seroprevalence rate (56.4%) than that found in our study (baumann & zessin 1992). thus, we interpret our results as being in line with results from several studies conducted in other camel-producing regions of the world. researchers in various african and middle eastern countries found lower t. evansi seroprevalence rates than ours. one study in jordan found a 33% infection rate (al-rawashdeh et al. 1999) whilst in eastern chad a 30.5% seroprevalence rate, detected using the catt test, was reported (delafosse & doutoum 2004). even lower rates of t. evansi seroprevalence were reported in the african nations of ethiopia (between 18.2% and 24.9%; aregawi et al. 2015; bogale, kelemework & chanie 2012; fikru et al. 2015; hagos et al. 2009), mauritania (24%, dia et al. 1997), niger (12%, pacholek et al. 2000) and tanzania (8.3%, njiru et al. 2002). others reports of low t. evansi seroprevalence came from uae (10.67%, chaudhary & iqbal 2000), iran (10%, zarif-fard & hashemi-fesharki 2000) and pakistan (4%, hasan et al. 2006). the heterogeneity between seroprevalence rates in different parts of the world is likely because of the differences in density of camel rearing, animal husbandry systems, climatic conditions, density of mechanical vectors, local herd management practices, study sample sizes, as well as cut-off values and sensitivity differences in the serological tests employed. moreover, in our study location, poor-quality veterinary service, divagation and migration of camels to humid area and northland areas in search of feed (benaissa et al. 2012) might have additionally contributed to the high seroprevalence. under such production conditions, it will be important to educate camel farmers how to mitigate risks of t. evansi infection. at the herd level, our study demonstrated wide distribution of camel trypanosomosis. indeed, 73.2% of herds (60 of 82 tested herds) had at least one seropositive animal. our overall herd seroprevalence is comparable to the 80% herd t. evansi seroprevalence reported recently in somalia (mohamed et al. 2020). our study documents a high prevalence of this disease-causing parasite and corroborates field observations of substantial waves of mortality and abortion reported by camel farmers and veterinarians in the region. the high herd-level seroprevalence supports field reports of marked financial impacts of this disease and indicates the need to develop field-practical control and prevention programmes. although few studies have addressed herd-level seroprevalence, our seropositivity result was much higher than the 42.3% reported earlier in algeria (boushaki 2007). given the span of time between studies, our study highlights the continued circulation of the parasite within herds in the region as well as its endemicity. in uncontrolled situations, herd-level seroprevalence could reach as high as 94.9% (delafosse & doutoum 2004). in fact, two important risk factors identified at the individual level in the former study (contact between herds and introduction of new camels into the herd) support that infection can easily spread from one herd to the other. no effect of camel age on t. evansi seropositivity was observed in our study. our finding agrees with some surveys (boushaki et al. 2019; pathak & khanna 1995; shah et al. 2004), but differs from those of other research groups who reported increased seroprevalence with increasing age (atarhouch et al. 2003; bogale et al. 2012; dia et al. 1997; eshetu, desta & amare 2013; gutierrez et al. 2000; mirshekar, yakhchali & shariati-sharifi 2017; olani et al. 2016; tehseen et al. 2015). in contrast to the above findings, one study in ethiopia revealed higher infection rates in younger camels than in older adult camels (lemecha, lidetu & hussein 2008). these conflicting results suggest that future investigations need to be designed to specifically test the hypothesis of age effects on t. evansi seropositivity. the risk of contracting trypanosomosis in semi-intensive and intensive systems was approximately three and two times higher, respectively, than in extensive management systems in our study. this result agrees with previous reports that husbandry systems and practices have a great impact on circulation of t. evansi (dia et al. 1997). serological surveys conducted in sudan detected a higher prevalence of t. evansi in nomadic camels than in camels managed in an agropastoral system (elamin, el bashir & saeed 1998). camel husbandry systems are frequently characterised by a remarkably high level of animal mobility and exchange between farms and areas, and even between countries (e.g., algeria, tunisia, libya, mali, and niger). these highly common movements (controlled or uncontrolled) of camels could explain the wide distribution of t. evansi infection. an association between camel seropositivity for t. evansi and herd history of abortion was observed in our study. this finding is in good agreement with a recent epidemiological study where abortion was associated with t. evansi seropositivity (boushaki et al. 2019). this result was not unexpected, as abortions induced by t. evansi have been reported in other species. unfortunately, data concerning the incidence of abortions and other productivity measures are frequently lacking in camel-dependent livestock production settings. herd t. evansi seropositivity rates varied greatly between geographical areas. the highest risk of camel trypanosomosis was observed in ghardaia (a region with relatively cold climate). ghardaia and biskra, situated in the northern algerian sahara, have an abundance of naturally occurring water sources that are associated with higher rates of t. evansi seropositivity. although our study area belongs to the arid climate stage, it remains rich in natural water resources, with several rivers as well as temporary lakes and wadis appearing during the rainy season. we interpret our results to mean that these natural water sources likely enhance the spread of trypanosomosis through the development and proliferation of tabanids, which are indiscriminate in the transmission of t. evansi. because these vectors seem subservient to standing water (ponds and lakes), regions with wetlands and saline lakes can be identified as higher risk areas as reported recently in a study in seven others provinces of southern algeria (boushaki et al. 2019). worldwide, several authors have reported considerable differences in the seroprevalence of t. evansi in different geographical areas within the same country (aregawi et al. 2015; bogale et al., 2012; delafosse & doutoum 2004; dia et al. 1997; mirshekar et al. 2017; olani et al. 2016; salah, robertson & mohamed 2019; tehseen et al. 2015). in most cases, these within country differences can be explained by the observed variation in ecological factors (e.g., pastoral camel densities, hottest, most humid and wettest districts in the region) that affect vector densities and therefore infection prevalence (hagos et al. 2009). interestingly, geographical variation was not considered in some recent animal-level studies of trypanosoma seroprevalence (ghaemi, zavarib & jannati pirouz 2019; hassan-kadle et al. 2019; salah et al. 2019). the geographical distribution of the parasite and vectors, and thus the occurrence of trypanosomosis, is largely dependent on the nature of the agro-ecological environment. trypanosomosis, therefore, is regarded as endemic in certain regions (holt et al. 2016). the climate is thought to permit and promote high densities of vectors such as the tabanides fly (james et al. 1985). seropositivity for t. evansi infection was not associated with breed in our study. this result is in contrast with the studies that identified breed as a risk factor for t. evansi infection (e.g., in chad; delafosse & doutoum 2004). it seems likely, however, that the confounding effects of breed and hair colour make interpretation of any potential breed effect difficult. animals with dark hair (‘zerga’) were three times more likely to be infected than camels with other colours of hair coat (boushaki et al. 2019). similarly, white-coloured camels in saudi arabia were more likely to be infected with t. evansi than animals with a dark coat (el-wathig et al., 2016). further complicating the interpretation of potential breed effects is the notion that various colour coat phenotypes may belong to the same genotypes (almathen, mwaracharo & hanotte 2012; cherifi et al. 2017). it should be noted that some other studies showed that camels were equally susceptible to trypanosoma infection regardless of breed (pathak & khanna 1995). no difference in t. evansi seropositivity was observed between male and female camels in our study. there are conflicting reports in the literature, however, regarding the effect of sex. some studies reported that trypanosoma infection rate was higher for females than males (dia et al. 1997; shah et al. 2004; sobhy et al. 2017). contrarily, one study in mali (ndoutamia et al. 1999) found higher rates of infection amongst males – a finding supported by a kenyan study where trypanosoma infection rates were 2.6 times higher in males than females (njiru et al. 2004). these conflicting results may be explained by interactions with other factors, disproportionate sampling of males versus females or differential application of husbandry practices to male versus female camels. in many countries, male camels are housed in a pen or tied when females go to the rangelands for grazing. this differential management approach may explain apparent sex differences rather than there being an actual biological predisposition to infection linked to camel sex. serologic testing is an important method for detecting parasitic infections. the direct card agglutination test (catt test) is a validated test that is considered a rapid tool of choice for detection of t. evansi infection in camels (dia et al. 1997; ngaira et al. 2003), buffalos (davison et al. 1999) and cattle (reid & copeman 2003). the catt test has been the test of choice for field investigations, despite the low sensitivity reported in some studies: 65.5% (ngaira et al. 2003) and 68.6% (njiru et al. 2004). the t. evansi direct card agglutination test (catt) uses latex agglutination of beads coated with recombinant antigens rotat 1.2 vsg (manual 2012). moreover, other reports (benfodil et al. 2020; diall et al. 1994; gutierrez et al. 2000; pathak et al. 1997) have shown a moderate to strong agreement between catt and other diagnostic methods such as trypanosome lysis (tl), wcl elisa and rotat 1.2 type a elisa. recent validation studies on serologic tests for t. evansi infection in camels and other ruminants showed the degree of concordance was highest between tl and t. evansi catt (k = 0.784), thus confirming the interest of using t. evansi catt for serodiagnosis of surra in the field (benfodil et al. 2020). because of its mechanical means of transmission, t. evansi may be disseminated through a wide range of vectors. the multitude of potential vectors allowed trypanosoma to become the most widespread pathogens in tropical and subtropical areas (hoare 1972). although t. evansi is not pathogenic to humans, human infestation by this parasite was first reported in india (joshi et al. 2005). a follow-up study (shegokar et al. 2006) reported a 22.7% t. evansi seroprevalence in humans in india. this finding is probably the consequence of frequent exposure of humans to t. evansi-infected livestock. camel farmers can experience considerable economic losses because of t. evansi infections because they result in outbreaks of abortions (and subsequent loss of replacement breeding stock or meat animals), decreased milk production, loss of body condition, decline of production efficiency and increased mortality. additional financial losses occur because of premature culling of sick animals and biannual treatments administered to t. evansi seropositive animals. these devastating financial impacts point out the need to establish effective surveillance, control and prevention programmes. conditions in which contact with a vector is enhanced, such as sedentary camels sharing a common water source in an oasis or contact between animals and target potential reservoirs, are common in camel rearing (elamin et al. 1998). in the particular case of camel husbandry in africa, nomadism is characterised by migration during the dry season to areas with bioclimatic areas or conditions favouring survival of the vectors (e.g., woodlands, valleys, permanent water). nomadism, therefore, is an important risk factor (baumann & zessin, 1992; delafosse & doutoum, 2004; dia et al. 1997; ngaira et al. 2003). in the present study, t. evansi seroprevalence in individual camels in large camel herds reached 52%, which corresponded to a risk of infection that was 2.1 times higher than camels from small herds. this higher risk is probably because of the fact that, in large farms, the infection pressure is stronger with more frequent contact between infected and non-infected animals. our results agree with a study in chad, which revealed that the largest herds were associated with a higher risk of trypanosomosis (delafosse & doutoum 2004). the source of water (e.g., rivers versus artificial wells) was a major risk factor for exposure to t. evansi in our study. herds whose primary water source was a river had greater rates of infection than herds that drank from wells. also, the presence of a dense canopy and permanent ponds create ideal breeding conditions for various species of tabanids, thus promoting spread of the disease (doutoum et al. 2002). where feasible, camel farmers should reduce access to naturally occurring water sources and instead provide well water. conclusion dromedary camels in eastern algeria showed high rates of seropositivity for t. evansi at both the individual and herd levels. at the individual level, the three main risk factors for trypanosomosis were herd size, animal management system and accessibility to naturally occurring sources of water (rivers, lakes). herd seroprevalence varied to a much lesser extent because of geographical location (district). these findings stress the importance of an integrated disease surveillance system for t. evansi. preventive measures are needed to improve the health status of camel herds and to reduce the economic losses associated with outbreaks of camel trypanosomosis (which include abortions, cachexia, decreased productivity and mortality). preventive measures may include surveillance of animal movements, particularly in rural areas. studies employing more sensitive molecular diagnostic techniques, as well as epidemiological surveys to estimate seroprevalence at a national level, are needed to develop an efficient control strategy to avoid an endemic state of disease. in the context of global climate change, trypanosomiasis has been recognised as a re-emerging disease and a serious and economically important health issue with particular interest for africa and asia. acknowledgements we gratefully acknowledge dr nedjima abderrahmane, morsli ismail and ninouh med kamel for assisting with the collection of blood samples, and we express sincere thanks to mimouni fatima zohra, absa oumelkheir and bensedik imane for performing serology. competing interests the authors have declared that no competing interests exist. authors’ contributions b.m.h., k.r. and b.f. conceived and designed the study and performed the laboratory investigations. b.m.h. and b.y. contributed to field work, sample collection, epidemiological analysis and performed the laboratory investigations. b.m.h., b.a. and h.y. analysed the data. b.m.h., c.y., m.n. and k.t. contributed to the literature review, writing and editing of the manuscript. funding information the general directorate of scientific research and technological development (dgrsdt) and scientific and technical research center for arid areas (crstra) provided funding the project, including scientific and technical support for laboratory analyses; 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in the usa (droual, bickford, charlton, cooper & channing 1990). the causative agent a. paragallinarum, (blackall, christensen, beckenham, blackall & bisgaard 2005) is a bacterial organism 299 onderstepoort journal of veterinary research, 76:299–309 (2009) study on the efficacy and safety of different antigens and oil formulations of infectious coryza vaccines containing an nad-independent strain of avibacterium paragallinarum b. dungu1*, b. brett1, r. macdonald1, s. deville2, l. dupuis2, j. theron3 and r.r. bragg4 abstract dungu, b., brett, b., macdonald, r., deville, s., dupuis, l., theron, j. & bragg, r.r. 2009. study on the efficacy and safety of different oil formulations of infectious coryza vaccines containing a nad-independent strain of avibacterium paragallinarum. onderstepoort journal of veterinary research, 76:299–309 the present study was designed to assess and compare three different formulations of the new onderstepoort infectious coryza (ic) quadrivalent vaccine, which contain an nad-independent strain of avibacterium paragallinarum (previously known as haemophilus paragallinarum), and a commercial ic vaccine, not containing an nad-independent strain, for their safety and ability to protect chickens of varying ages against virulent challenges with four different serovars of a. paragallinarum, including the nad-independent strain of the c-3 serovar. four groups of 140 chickens each were vaccinated at the age of 17 weeks and revaccinated at the age of 19 weeks with each of the four vaccine formulations. a similar sized group of non-vaccinated chickens was used as control. two rounds of challenge were conducted: a group of chicken in each vaccination group was challenged between 31 and 35 weeks of age, while another group was challenged between 51 and 55 weeks of age. the “in-contact” challenge model was used in this experiment. for each vaccination group, the four challenge strains representing four local serovars were used in each challenge round. the efficacy of the vaccines was compared based on overall protection levels obtained and the duration of protection. the safety of the different vaccines was determined by the severity of post-vaccination reactions. the need for the incorporation of the nad-independent strain in the vaccine was evidenced by the low protection level against nad-independent challenge recorded in the group of birds vaccinated with the commercial vaccine. the results obtained confirmed not only the variation in virulence of different south african serovars, with serovar c-3 being the most virulent and serovar b having almost no virulence but also the age related increase in susceptibility. the importance of a suitable formulation of the vaccine is discussed. keywords: avibacterium paragallinarum, c-3 serovar, chickens, coryza vaccine, nad-independent strain * author to whom correspondence is to be directed. present address: galvmed, edinburgh eh26 opz scotland, uk. e-mail: baptiste.dungu@galvmed.org 1 onderstepoort biological products, private bag x07, onderstepoort, 0110 south africa 2 seppic, tour kupka c-7, bld franck kupka, 92039 paris, france 3 department of microbiology and plant pathology, university of pretoria, pretoria, 0002 south africa 4 university of the free state, p.o. box 339, bloemfontein, 9300 south africa accepted for publication 19 april 2009—editor 300 infectious coryza vaccines containing an nad-independent strain of avibacterium paragallinarum that can be either nad (or v-factor) dependent or independent (mouahid, bisgaard, morley, mut ters & mannheim 1992; bragg, coetzee & verschoor 1993) for growth in vitro. three serogroups are recognised, namely a, b and c (page 1962), with up to four serovars within serogroup a and c (kume, sa wata, nakai & matsumoto 1983; blackall, eaves & rogers 1990). the role played by nad-independent sero vars has been extensively studied in south afri ca (miflin, horner, blackall, chen, bishop, mor row, ya ma guchi & iritani 1995; bragg, coetzee & ver schoor 1996), where they have been shown to be the most dominant serovars in certain provinces such as kwazulu-natal. since first recorded in south africa in the late 1960s (buys 1982), ic has occurred widely in different parts of the country. despite the introduction of a vaccine in the mid-1970s, which seemed to decrease the incidence of the disease, more outbreaks were recorded in the 1980s. studies conducted by bragg et al. (1996) demonstrated a significant shift in the incidence of the four serovars occurring in the country over a 30-year period. serovar c-3 had become predominant, representing 73 % of isolated serovars in the 1990s, mainly in the period following the introduction of the vaccine, which did not include c-3. this work and the work by other researchers demonstrated and confirmed the importance of including locally occurring serovars in the vaccine for effective control (terzolo, sandoval & gonzalez pondal 1997; blackall 1999). inactivated multivalent vaccines are used worldwide for the control of ic, most of them comprising serovars of serogroups a, b and c (blackall1999). as for most inactivated vaccines, the adjuvant used for the formulation of the vaccine plays a critical role in defining the type of protection afforded by the antigen and the duration of immunity. most ic vaccines are formulated with an oil emulsion. ideally the vaccine should provide effective protection while being safe and having the least effect on chicken productivity. a long-lasting immunity is therefore preferred in order to avoid re-vaccinating layers during their production period. water-in-oil emulsions are thus commonly used as they are known to induce strong and long-term immunity (aucouturier, dupuis & ganne 2001). one of their main disadvantages, however, is the possibility of local reactions when used with a crude antigen (dupuis, ascarateil, aucouturier & ganne 2006). double oil emulsions, consisting of a water-in-oil-in-water emulsion are usually safer and also suitable for poultry (dupuis et al. 2006). depending on the type of formulations, type of emulsifying agent, the surfactant used and many other factors, different responses can be obtained with different formulation that will affect the protection ability of the vaccine, hence the need for in vivo evaluations. an effective combination of a relevant vaccine strain and suitable adjuvant are critical for ensuring that the vaccine provides optimal protection. since its introduction in the mid-1970s the onderstepoort biological products (obp) ic vaccine has been adjusted to include the main serovars occurring in the country. the latest vaccine, coryzaplus vaccine, includes a c-3 nad-independent strain togeth er with locally occurring serovar a and c isolates. the present study was designed to evaluate the efficacy and safety of alternative experimental oil vaccines formulated with the same vaccine antigens as the obp coryzaplus ic vaccine but using different oil adjuvants. the safety and ability of these formulations to provide long term protection to layers kept throughout a production cycle were compared to those of an international ic commercial vaccine registered in south africa. material and methods vaccines the obp coryzaplus vaccine was used in the study, together with two experimental oil vaccines formulated with the same vaccine antigens as the obp vaccine and a commercial vaccine, referred to as “coma”. the obp coryzaplus vaccine is a waterin-oil emulsion of formalin-inactivated a. paragalli­ na rum strains. it is a quadrivalent vaccine containing serotypes a and c (2 strains) and a c-3 nadindependent vaccine strain. the same strains were used to formulate two experimental vaccines: one with the water-in-oil-in-water double emulsion montanide™ isa 206 vg (seppic) and the other with the water-in-oil emulsion, montanide™ isa 70 vg (seppic). the commercial vaccine is a water-in-oil emulsion containing serovar a, b and c, but not the nad-independent strain. the emulsions of the experimental vaccines were evaluated at the seppic vaccine department laboratory in castres (france) for physical and chemical properties using the kf values, the aspect, the particle size and the stability at 4 °c, room temperature 301 b. dungu et al. and 37 °c up to 1 month. they were compared to a placebo emulsions made of a saline solution. chickens in order to ensure homogeneity among the chickens to be studied, 750 fertilized eggs were procured and incubated simultaneously at the agriculture research council (arc) poultry research centre at glen agriculture college, situated near bloem fontein in the free state province, south africa. hatched chicks were kept for 16 weeks before being placed randomly into groups of 70 birds each. two of the groups, totalling 140 chicks, were used for each vac cine, and the same number for non-vaccinated controls. for the challenge experiment, specific groups of birds were transferred to isolated layer facilities in the animal house of the university of the free state, bloemfontein (ufs). this experiment was performed with the approval of the ufs ethics committee under project number 04/04. bacterial isolates used for challenge the challenge strains used in this experiment represented all three recognised serogroups of a. para­ gallinarum and major strains known to occur in south africa. serovar c-3 (tongaat) strain of a. par­ a gallinarum, known to be highly virulent and very prevalent in south africa was used, in parallel with strain c-2 (serovar c-2), isolate 0083 (serogroup a), strain b (serogroup b) and the nad-independent strain 1750 (serovar c-3). the purity of each of the challenge strains was established by plating out the challenge bacteria onto bta plates, streaking with staphylococcus aureus and incubating at 37 °c. vaccination and challenge experiments chickens in the four study groups were vaccinated at 17 weeks of age, and boosted 3 weeks later. the birds were vaccinated by the subcutaneous injection of 0.5 mℓ of the respective vaccines during both vaccination procedures. all vaccinations were conducted by the same operator. the safety of each vaccine was assessed by evaluating local reactions in vaccinated chickens during the first week postinoculation. this evaluation was conducted on a number of chickens randomly selected in each group, during the first and the second vaccination. post vaccination signs recorded were principally swellings of different sizes. they were graded as “none”, when there was no detectable swelling, “mild” for a small lesion and “severe” when there was a prominent swelling, sometimes affecting the general health of the chicken. two rounds of challenges were conducted. the first round of challenges with virulent strains of a. para­ gallinarum was conducted when the birds were between 31 and 35 weeks of age (from 15 weeks post vaccination). the second round of challenge, conducted in the remaining unchallenged vaccinated chickens, took place when they were between 51 and 55 weeks of age. as controls, unvaccinated chickens of similar age were included in all challenge experiments. the challenge method used was according to the “in-contact” challenge model established by bragg (2002a), in which one bird in a group of ten birds is directly challenged by intra-sinus injection with 0.1 mℓ of a bacterial suspension. the remaining birds in the group are challenged through natural in-contact route as the ten birds in each group are in adjoining cages with a communal water supply. the clinical signs were recorded and scored over the 20 days post-challenge observation period, according to the method described by bragg (2002a), and used to calculate percentage protection according to the above method. due to space constraints at the animal laboratory facility of the ufs, each round of vaccination was divided into two phases. for the first round of challenge (at week 31 of age), the first phase involved the tongaat strain, the c-2 strain and the 0083 strain. the cage were then cleaned, disinfected and left empty for a week before conducting the second phase with the nad-independent strain and the serogroup b strain. for the second round, the first phase (week 51 of age) involved the tongaat and the c-2 strains, while the second phase involved strain 0083 and the nad-independent strain 1750. results stability of the experimental ic oil vaccines tables 1a and 1b summarize the results obtained for the physical characteristic evaluation and stability of the two experimental vaccines formulated with montanide™ isa 206 vg and montanide™ isa 70 vg respectively. each one of the two experimental coryza vaccines had comparable physical characteristics to the equivalent seppic placebo in terms 302 infectious coryza vaccines containing an nad-independent strain of avibacterium paragallinarum of homogeneity and particle size. they all were also stable for a month at 4 °c and room temperature. post-vaccinal safety of the vaccines vaccination reactions were recorded during the first week post-vaccination in all groups, following the first and second vaccination. the results of the vaccination reaction observations are given in table 2 and fig. 1 and 2. after the first vaccination, no vaccination reactions were seen in the control group (which were not vaccinated) and in the birds vaccinated with the montanide™ isa 206 vg vaccine. the most severe clinical signs were observed in the birds vaccinated with the obp ic vaccine and the montanide™ isa 70 vg formulation. after the second vaccination, no reactions were observed in the control group or in the birds vaccinated with the montanide™ isa 206 vg formulation. the worst vaccination reactions were seen in the group of birds vaccinated with the coma vaccine, with 20 % of the birds showing very severe vaccination reactions characterized by subcutaneous swelling of varying degree. in some cases, the vaccination reactions were so severe that these birds were removed from the experiment for ethical reasons as their general health condition was seriously deteriorating. table 1a comparative evaluation of the coryza special vaccine formulated with isa 206 adjuvant, and a seppic placebo emulsified in the same adjuvant. karl fisher method (kf) defines the emulsion water content while d (v) is the mean of particles diameter type of emulsion kf ( %) d (v, 0.5) d (v, 0.9) in µm stability after 15 days 4 °c/rt/37 °c stability after one month 4 °c/rt/37 °c seppic placebo emulsion w/o/w 47.5 0.26 0.62 ok/ok/deph ok/ok/deph coryza isa 206 vaccine w/o/w 49.0 0.24 0.42 ok/ok/br ok/ok/br table 1b comparative evaluation of the coryza special vaccine formulated with isa 70 adjuvant and a seppic placebo emulsified in the same adjuvant. karl fisher method (kf) defines the emulsion water content while d (v) is the mean of particles diameter type of emulsion kf ( %) d (v, 0.5) d (v, 0.9) in µm stability after 15 days 4 °c/rt/37 °c stability 1 month 4 °c/rt/37 °c seppic placebo emulsion w/o 29.0 0.33 0.84 ok/ok/ok ok/ok/ok coryza isa 70 vaccine w/o 29.8 0.26 0.55 ok/ok/ok ok/ok/ok 303 b. dungu et al. control coma isa 206 obp ic isa 70 no signs 0 4 0 11 9 mild 0 24 8 38 30 severe 100 72 92 51 61 100 90 80 70 60 50 40 30 20 10 0 p er ce n ta g e fig. 1 graphic representation of the percentage of vaccination reactions obtained after the first vaccination with the different vaccines used in this experiment control coma isa 206 obp ic isa 70 no signs 100 52 100 84 97 mild 0 28 0 3 2 severe 0 20 0 12 1 100 90 80 70 60 50 40 30 20 10 0 p er ce n ta g e fig. 2 graphic representation of the vaccination reactions obtained after the second vaccination with the different vaccines used in this experiment table 2 the number of birds showing vaccination reactions for each of the different vaccines vaccine 1st vaccination 2nd vaccination no. birds no mild severe no. birds no mild severe control 60 60 (100 %) 0 0 60 60 (100 %) 0 0 coma 50 36 (72 %) 12 (24 %) 2 (4 %) 83 43 (52 %) 23 (28 %) 17 (20 %) isa 206 50 46 (92 %) 4 (8 %) 0 (0 %) 78 78 (100 %) 0 0 obp ic 45 23 (51 %) 17 (38 %) 5 (11 %) 122 103 (84 %) 4 (3 %) 15 (12 %) isa 70 46 28 (61 %) 14 (30 %) 4 (9 %) 154 149 (97 %) 3 (2 %) 2 (1 %) 304 infectious coryza vaccines containing an nad-independent strain of avibacterium paragallinarum 1: tongaat c3 strain vaccine mean score % protection control coma isa 206 obp isa 70 1.010 0.191 0.360 0.150 0.130 – 81.1 64.4 85.1 87.1 2: tongaat c-2 strain vaccine mean score % protection control coma isa 206 obp isa 70 0.58 0.04 0.09 0.29 0.00 – 93.1 84.5 50.0 100.0 3: a-1 isolate 0083 vaccine mean score % protection control coma isa 206 obp isa 70 0.55 0.19 0.17 0.20 0.26 – 65.5 69.1 63.6 52.7 4: nad-independent c-3 isolate 1750 vaccine mean score % protection control coma isa 206 obp isa 70 0.15 0.125 0.04 0.04 0.08 – 16.7 73.3 73.3 46.7 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp com a isa 206 control 0.0 0.5 1.0 1.5 2.0 2.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp com a isa 206 control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp com a isa 206 control 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp com a isa 206 control table 3a first vaccination: graphic representation of the mean daily disease scores obtained from each of the vaccines for chickens challenged with different a. paragallinarum challenge strains and table summarizing the corresponding mean lesion score and percentage protection—the tongaat (c-3) strain (1); c-2 strain (2); the a-1 isolate 0083 (3); and the nadindependent c-3 isolate 1750 (4) 305 b. dungu et al. 1: tongaat c3 strain vaccine mean score % protection control coma isa 206 obp isa 70 1.65 0.595 0.065 0.245 0.055 – 64.0 96.1 85.2 96.7 2: tongaat c-2 strain vaccine mean score % protection control coma isa 206 obp isa 70 0.27 0.14 0.195 0.10 0.10 – 48.1 27.8 63.0 63.0 3: a-1 isolate 0083 vaccine mean score % protection control coma isa 206 obp isa 70 0.130 0.075 0.090 0.090 0.080 – 42.3 30.8 30.8 38.5 4: nad-independent c-3 isolate 1750 vaccine mean score % protection control coma isa 206 obp isa 70 0.56 0.13 0.24 0.01 0.03 – 76.8 57.1 98.2 94.6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 isa 206 obp coma control 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp coma isa 206 control 0.0 0.1 0.2 0.3 0.4 0.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp coma isa 206 control 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 isa 70 obp coma isa 206 control table 3b second vaccination: graphic representation of the daily protection scores provided by each of the vaccines for chickens challenged with different a. paragallinarum challenge strains and table summarizing the corresponding mean lesion score and percentage protection—the tongaat (c-3) strain (1); c-2 strain (2); the a-1 isolate 0083 (3); and the nadindependent c-3 isolate 1750 (4) 306 infectious coryza vaccines containing an nad-independent strain of avibacterium paragallinarum efficacy of the vaccine formulations to different serovar after the first challenges the mean daily disease and protection scores for each of the groups of birds are summarized in tables 3a and b for the first and second challenge rounds presented below. no clinical signs occurred in birds challenged with the strain of serovar b, thus the efficacy of the vaccine against serogroup b could not be determined. the percentage protection calculated from the disease challenge and a comparison of the mean disease scores in the first and second rounds are presented in table 4. discussion vaccine reactions of varying degrees occurred in all groups after the first vaccination, irrespective of the type of vaccine. the least severe vaccination reactions were seen in the chickens which received the montanide™ isa 206 vg formulation, which can be attributed to the fact that this was the only formulation in which the continuous phase is aqueous (water-in-oil-in-water), therefore causing the least inflammatory local reaction as compared to water in oil emulsions (jansen, hofmans, theelen, manders & schijns 2006). after the second round of vaccination, the birds vaccinated with the coma vaccine showed the most severe vaccination reactions: a total of 14 birds were removed from the experiment for ethical reasons based on the severity of their reactions. the two experimental formulations induced vaccination reactions in only 3 % of the birds with the montanide™ isa 70 vg formulation and none of the birds inoculated with the montanide™ isa 206 vg formulation. overall, the montanide™ isa formulations showed the least number of local postvaccination reactions. a quadrivalent vaccine needs a potent adjuvant to induce protection. the reactogenic properties of the inactivated bacteria used as antigen in the present vaccines require a safe adjuvant. the acceptable balance between efficacy and safety is obtained through the use of a specific adjuvant formulation. montanide™ isa adjuvants are based on a homogenous ready to use mix of purified mineral oils and refined oleic esters of anhydrous mannitol of vegetable origin. emulsifying properties of specific surfac tants can only be obtained through strict synthesis parameters, and have a direct impact on the vaccine safety and efficacy (stone 1988). furthermore, surfactants used in montanide™ formulations are manufactured in dedicated equipment which avoids any cross contamination. the hydrophilic parts of these amphiphile molecules are made of mannitol sugar known to have better injectability than sorbitol-based surfactants. adjuvant formulations of this quality do not induce adverse reactions observed when multipurpose industrial sorbitan oleate-based formulation (tween/span adjuvant formulation) is used. the montanide™ isa 206 vg renders a water-in-oil-in-water emulsion in a one step process, giving a very fluid vaccine when containing 50 % of adjuvant. the montanide™ isa 70 vg renders a water-in-oil emulsion containing 70 % of adjuvant. in the later case, the antigenic media are entrapped in the oily phase. the different antigenic release profiles (aucouturier et al. 2001) and the ratio of adjuvant to antigenic media can explain the perfect safety profile obtained with montanide™ isa 206 vg. indeed, aqueous-based formulations are quickly eliminated from the injection site while water-in-oil table 4 comparison of all of the vaccines based on percentage protection against the nad-dependent, nad-independent strain and for all of the challenge strains used (excluding serovar b-1, which did not result in any clinical signs when used to challenge chickens in this experiment, including the unvaccinated controls). mean protections obtained for the first and second round of challenges for all of the vaccines and challenged strains used in this experiment are also included challenge coma obp isa 206 isa 70 1st 2nd 1st 2nd 1st 2nd 1st 2nd c-3 81.1 64.0 85.1 85.2 64.4 96.1 87.1 96.7 c-2 93.1 48.1 50.0 63.0 84.5 27.8 100.0 63.0 a-1 65.5 42.3 63.6 30.8 69.1 30.8 54.7 38.5 mean nad-dependent protection 79.9 51.5 66.2 59.7 72.7 51.6 80.6 66.1 nad-independent c-3 16.7 76.8 73.3 98.2 73.3 57.1 46.7 94.6 mean overall protection (all isolate) 64.1 64.2 68.0 78.9 72.8 54.3 72.1 80.3 307 b. dungu et al. emulsions ensure a long-lasting release of the antigens (jansen et al. 2005). the virulence of different challenge strains could be assessed through the monitoring of clinical scores in the unvaccinated control animals. the tongaat c-3 strain demonstrated the highest virulence, followed by the c-2 and the a-1 strains. serovar b did not induce any adverse reactions. the high clinical score recorded with serovar c-3 (1.01 and 1.65 after the first and the second challenge, respectively) is consistent with previous results (bragg 2002a) and confirms the association of the c-3 serovars with most severe outbreaks in south africa. the lack or poor virulence of south african serovar b has also been recorded previously (bragg 2002a.) good levels of protection were, however, recorded for the c-3 serovar for all the vaccine formulations, at both challenges, which strengthen the value of vaccination for the control of ic in south africa. one of the main objectives of this experiment was to investigate the duration of protection provided by the different vaccines. in order to do this, the chickens were kept until they were older than 50 weeks. vaccinated birds were then challenged with the same challenge strains as those used at approximately 30 weeks of age. the results of the second challenge with serovar c-3 in unvaccinated birds showed an increase of the mean disease score from 1.01 in the first challenge to 1.65 in the second challenge, indicating a possible worsening in bird susceptibility to the bacterium associated with increasing age. a similar increase in clinical signs as the birds aged was also recorded with the nadindependent c-3 strain, while the opposite was observed with the c-2 and the a-1 strains. further work is required to confirm these observations. differences of occurrence of ic in different age groups have been reported elsewhere with variations between different countries and production systems (blackall 1999) who reported on a study of village chickens in thailand where it was reported that ic was the most common cause of death in chickens less than 2 months old and in those over 6 months of age. while the protection level with the coma vaccine decreased from 81.1 % to 64 % from the first to the second challenge, the obp coryzaplus vaccine generated an unchanged level of protection (85.1 % for the first challenge and 85.2 % in the second challenge). the protection levels were found to increase to 90 % when the montanide™ isa 70 vg and montanide™ isa 206 vg formulations were used. the montanide™ isa 70 vg vaccine generated the best protection overall to all challenge strains, closely followed by the ovi vaccine. this can be attributed to the similarity in their formulation, water-in-oil and the inclusion of the nad-independent c-3 strain. montanide™ isa 70 vg also showed the best safety results. larger field experiments are required to confirm the findings of the present study. during the first round of challenge experiments, the clinical signs associated with ic in the unvaccinated control birds challenged with the tongaat strain (c-3) were very severe. the levels of protection obtained when the different vaccinated groups of birds were challenged with serovar c-3 ranged from 64.4 % (isa 206) to 87.1 % for the montanide™ isa 70 vg formulation. the clinical signs obtained when unvaccinated birds were challenged with the serovar c-2 strain were less severe than those obtained when the unvaccinated birds were challenged with serovar c-3. these results confirm previous findings by bragg (2002a). the levels of protection obtained by the different vaccines against the serovar c-2 challenge ranged from 50.0 % for the obp vaccine to 100 % for the isa 70 formulation. when unvaccinated birds were challenged with strain 0083 (serovar a-1), the clinical signs were less severe than those obtained in previous challenge experiments. the levels of protection obtained against challenge with serovar a-1 ranged from 52.7 % for montanide™ isa 70 vg and 69.1 % obtained with the montanide™ isa 206 vg formulation. when unvaccinated birds were challenged with serogroup b, no clinical signs were seen. previous work has demonstrated that the south african serogroup b strains are of very low virulence (bragg 2002b), but in those experiments, some clinical signs were recorded. the fact that no clinical signs were seen in the unvaccinated birds makes any comparison of the protection impossible. the levels of virulence recorded with the different serovars correspond to the findings in south african strains of a. paragallinarum recorded by bragg (2002a). the ability to protect birds through vaccination confirms the need for well-structured vaccination programmes. the birds were also challenged with an nad-independent strain of a. paragallinarum. in previous experiments, the virulence of both naturally occurring nad-independent strains (bragg 2002b) and ex308 infectious coryza vaccines containing an nad-independent strain of avibacterium paragallinarum perimentally produced nad-independent strains (taole et al. 2002) were found to be of low virulence. it has also been demonstrated that the levels of protection obtained when vaccinated birds were challenged with the nad-independent strains were very low (bragg 2004). in the present study the prevalence of clinical signs noted in the unvaccinated birds was indeed very low: a mean disease score of only 0.15 was obtained in the unvaccinated birds challenged with the nad-independent serovar c-3 strain as compared to a mean disease score of 1.01 obtained with the nad-dependent serovar c-3 strain of a. paragallinarum. the need for the inclusion of a nad-independent strain in the vaccine was shown by the low protection level afforded by the commercial vaccine coma, which lacks such a strain. the protection afforded by the commercial vaccine to a challenge with the nad-independent strain improved however in late challenge, possibly as a result of an improved crossprotection over time with the other strains in the vaccine. these findings are in contrast to those of jacobs et al. (2000) who demonstrated that the nobilis coryza vaccine (intervet international bv) provided protection against the nad-independent strains in 9-week-old chickens vaccinated 2 weeks earlier, despite the fact that it does not include an nadindependent vaccine antigen. bragg (2004), on the other hand, demonstrated that there was evidence of immune evasion by the nad-independent strains when an experimental vaccine only containing naddependent strains was used to vaccinate the birds. the difference between the finding of jacobs et al. (2000) and bragg (2004) could be attributed to the different challenge models used. bragg et al. (2002b) demonstrated that the naturally occurring nad-in de pendent strains are of low virulence. taole et al. (2002) showed that there is a substantial decrease in virulence when nad-dependent strains of a. par­ agallinarum are experimentally converted to nadindependent strains. this low virulence level could account for the perceived efficacy of a vaccine against the nad-independent strains as reported by jacobs et al. (2000) given the high prevalence of nad-independent serovars in south africa, their involvement in a number of outbreaks throughout the country, and previous observation of the limited ability of the nad-dependent vaccine strain to protect birds against them, it is critical that vaccines in use in the country contain nad-independent strains. the two experimental vaccines, as well as the obp coryzaplus vaccines contained an nad-independent strain of a. paragallinarum, while the coma vaccine did not. protection levels of only 16.7 % were recorded with coma vaccine while it was between 46.7 % (montanide™ isa 70 vg) and 73.3 % (montanide™ isa 206 vg and obp) for the vaccines containing an nad-independent strain. these data clearly indicate the ability of a vaccine containing an nad-independent strain to protect against these strains. in a previous study conducted in south africa it was demonstrated that a commercial vaccine without an nad-independent strain generated good protection against local nad-independent c-3 strain (jacobs et al. 2000). the fact that the chickens used in the experiment were vaccinated at 3 and 7 weeks of age and challenged 2 weeks later could explain the good level of protection recorded. in the present study the chickens were vaccinated and challenged much later (vaccination at 17 and 20 weeks, challenge at 33 or 55 weeks of age), in order to evaluate the level of protection at different stages of the productive life of layers. the results of the present experiment are certainly much closer to the layer production system and suggest the need for the use of a vaccine that includes nad-independent strains in areas where nad-independent variants are known to occur. the low virulence recorded for the nadindependent strains discussed above could also contribute to this discrepancy. the results obtained in the present study confirm the variation in the virulence of different serovars occurring in south africa, with serovar c-3 being the most virulent and serovar b having almost no virulence. the results indicate an age related increase in susceptibility to infection, as illustrated in the increased disease scores. the study highlights the importance of a suitable vaccine formulation that generates protection throughout the productive life of chickens and the need for incorporation of local dominant strains in the vaccine, including nadindependent strains. references aucouturier j., dupuis l. & ganne v. 2001. adjuvants de signed for veterinary and human vaccines. vaccine, 19: 2666–2672. blackall, p.j. 1999. infectious coryza: overview of the disease and new diagnostic options. clinical microbiological review, 12:627-32. blackall, p.j., eaves, l.e. & rogers d.g. 1990. proposal of a new serovar and altered nomenclature for haemophilus paragallinarum in the kume hemagglutinin scheme. journal of clinical microbiology, 28:1185–1187. blackall, p.j., christensen, h., beckenham, t., blackall, l.l. & bisgaard, m. 2005. reclassification of pas teur­ ella gallinarum, haemophilus paragallinarum, pas teur ella avi um and pasteurella volantium as avibacterium gall i na rum gen. nov. comb. nov., avibacterium paragallina rum comb. 309 b. dungu et al. nov., avibacterium avium comb. nov. and avibacterium vol­ antium comb. nov. international journal of systematic and evolutionary microbiology, 55:353–362. bragg, r.r. 2002a. virulence of south african isolates of haemophilus paragallinarum. part 1: nad-dependent field isolates. onderstepoort journal of veterinary research, 69: 163–169. bragg, r.r. 2002b. virulence of south african isolates of haemophilus paragallinarum. part 2: naturally occurring nad-independent field isolates. onderstepoort journal of veterinary research, 69:171–175. bragg, r.r., coetzee, l. & verschoor, j.a. 1993. plasmid encoded nad independence in some south african isolates of haemophilus paragallinarum. onderstepoort jour nal of veterinary research, 60:147–152. bragg, r.r., coetzee l. & verschoor, j.a. 1996. changes in the incidences of the different serovars of haemophilus paragallinarum in south africa; a possible explanation of vaccination failures. onderstepoort journal of veterinary research, 63:217–226. bragg, r.r. 2004. evidence of possible evasion of protective immunity by nad-independent isolates of haemophilus par­ agallinarum in poultry. onderstepoort journal of veterinary research, 71:53–58. buys, s.b. 1982. die bereiding van bakterieë teen haemophilus paragallinarum besmetting, geskik vir suid-afrikaanse omstandighede. m.med.vet. thesis, university of pretoria. droual, r., bickford a.a., charlton b.r., cooper g.l. & channing, s.e. 1990. infectious coryza in meat chickens in the san joaquin valley of california. avian dis­ eases, 34:1009–1016. dupuis, l., ascarateil, s., aucouturier, j. & ganne, v. 2006. seppic vaccine adjuvants for poultry. annals of the new york academy of science, 1081:202-205. horner, r.f., bishop, g.c., jarvis, c.j. & coetzee, t.h.t. 1995. nad (v-factor)-independent and typical hae m­ ophilus paragallinarum infecting commercial chickens: a five year field study. avian pathology, 24:453–463. jacobs, a.a. & van der werf, j. 2000. efficacy of a commercially available coryza vaccine against challenge with recent south african nad-independent isolates of haemophilus paragallinarum in chickens. journal of the south african vet­ erinary association, 71:109–110. jansen, t., hofmans, m.p.m., theelen, m.j.g., manders, f. & schijns v.e.j.c. 2006. structureand oil typebased efficacy of emulsion adjuvants. vaccine, 24:5400– 5405. jansen, t., hofmans, m.p.m., theelen, m.j.g. & schijns, v.e.j.c. 2005. structure-activity relations of water-in-oil vaccine formulations and induced antigen-specific antibody responses vaccine, 23:1053–1060. kume, k., sawata, a., nakai, t. & matsumoto, ? 1983. serological classification of haemophilus paragallinarum with a hemagglutinin system. journal of clinical microbiology, 17: 958–964. miflin, j.k., horner, r.f., blackall, p.j., chen, x., bishop, g.c., morrow, c.j., yamaguchi, t. &. iri tani, y. 1995. phenotypic and molecular characterization of v-factor (nad)-independ ent haemophilus paragallinarum. avian diseases, 39:304–308. mouahid, m., bisgaard, m., morley, a.j., mutters, r. & mannheim, w. 1992. occurrence of v-factor (nad) independent strains of haemophilus paragallinarum. veterinary microbiology, 31:363–368. page, l.a. 1962. haemophilus infections in chickens: i. characteristics of 12 haemophilus isolates recovered from diseased chickens. american journal of veterinary research, 23:85–95. taole, m., albertyn, j., van heerden, e. & bragg, r.r. 2002. virulence of south african isolates of haemophilus paragallinarum. part 3: experimentally produced nad-independent isolates. onderstepoort journal of veterinary res­ earch, 69:189–196. terzolo, h.r., sandoval, v.e. & gonzalez pondal, f. 1997. evaluation of inactivated infectious coryza vaccines in chickens challenged by serovar b strains of haemophilus paragallinarum. avian pathology, 26:365–376. stone, h.d.1988. optimization of hydrophile-lipophile balance for improved efficacy of newcastle disease and avian influenza oil-emulsion vaccines. avian diseases, 32:68–73. article information authors: etheresia pretorius1 jeané olivier1 hester m. oberholzer1 wendy j. van der spuy1 affiliations: 1department of anatomy, university of pretoria, south africa correspondence to: etheresia pretorius email: resia.pretorius@up.ac.za postal address: po box 2034, pretoria 0001, south africa dates: received: 02 nov. 2010 accepted: 16 feb. 2011 published: 07 july 2011 how to cite this article: pretorius, e., olivier, j., oberholzer, h.m. & van der spuy, w.j., 2011, ‘scanning electron microscopy investigation of fibrin networks after thermal injury’, onderstepoort journal of veterinary research 78(1), art. #244, 4 pages. doi:10.4102/ojvr.v78i1.244 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: (online) scanning electron microscopy investigation of fibrin networks after thermal injury in this original research... open access • abstract • introduction • materials and methods    • choice of burn wound model    • burn wound creation    • preparation of fibrin clots    • preparation of washed fibrin clot for scanning electron microscopy • results and discussion • conclusion • references abstract (back to top) injury due to burning is known to impact on coagulation and haemostasis by disturbing the coagulation cascade and is also associated with impaired fibrinolysis. also, venous thrombosis, pulmonary embolism and hypercoagulability are common during thermal injury. using a wistar albino rat model, we investigated in this study whether burn injury affects the ultrastructure of the fibrin networks. a typical fibrin network will contain mostly major, thick fibres with minor, thin fibres distributed amongst them. we found that the clot architecture changes after burn injury, showing more prominent minor, thin fibres in a netted appearance. also, the clot showed areas of matted fibrin. we suggest that the thrombotic events associated with burn injury are due to the thickened and netlike areas formed when thrombin activates the coagulation cascade. this is due to impaired fibrinolysis activities, causing the resulting fibrin clots not to be successfully disseminated. small fragments of these netted, clumped areas may therefore break loose and lead to thrombotic events after burn injuries. the current study therefore provided morphological evidence for thrombotic events associated with burn injury.> introduction (back to top) injury due to burning is known to impact on coagulation and haemostasis (lavrentieva et al. 2008) by inducing subclinical disseminated intravascular coagulation. fibrinolysis, in particular, is affected, with fibrinolytic plasma parameters showing an activation of fibrinolysis 2 hours after burn wound creation, followed by suppression in fibrinolysis after 24 hours and up to 10 days post-burn (fang, ding & kong 1997). such suppression of fibrinolysis may protect fibrin deposited in the wound, which could be important in wound healing. fang et al. (1997) suggested that the suppressive factors of fibrinolysis may be due to enhanced activities of proteins such as plasminogen activation inhibitor-1 (pai-1) and alpha 2-antiplasmin after injury. also, burn injury causes an acquired deficiency of the plasma protein antithrombin iii (atiii), a natural anticoagulant that leads to a high incidence of hypercoagulability and prevalence to thrombosis in patients (kowal-vern et al. 2000). atiii is a natural anticoagulant and the most important inhibitor of blood coagulation owing to its effect on thrombin (kowal-vern et al. 2000; penner 1995). venous thrombosis is associated with low plasma levels of atiii known to occur in burn patients; also, pulmonary embolism, hypercoagulability, adult respiratory distress syndrome, infection and multiple organ failure are common in thermal injury (darling et al. 1996; gando, tedo & kubota 1992; geerts 1994; kowal-vern et al. 2000; kowal-vern et al. 1992; penner 1995). ravindranath et al. (2004) also found that plasma tissue factor pathway inhibitor (tfpi) levels are significantly decreased 24 h after a burn, while thrombin activatable fibrinolytic inhibitor (tafi) levels are significantly increased 24 h and 72 h after a burn.the question that now arises is whether burn injury has an impact on fibrin network architecture in circulating plasma, and whether a local burn injury will change the clot ultrastructure. a wistar rat burn wound model was subsequently used to study the ultrastructure of fibrin networks. materials and methods (back to top) choice of burn wound model fifteen wistar albino rats (between 200 g and 250 g each), which were maintained at the university of pretoria biomedical research centre, were used in this study (three untreated controls and 12 burn injury animals). the rats were fasted for 12 hours before thermal injury; however, they had free access to water. the rats were kept in a room with a 12-hour alternating light and dark cycle and room temperature was kept constant at 22 °c. the animals were fed a standard rat diet, provided with water ad libitum and were housed in individual polycarbonate cages. all experimental protocols complied with the requirements of the university of pretoria animal use and care committee (ethical clearance number: h021/08). burn wound creation on the day of burn wound creation, the rats were anaesthetised with isoflurane and directly injected with the analgesic tramadoltm. wound creation commenced only 15 min after injection of the analgesic to allow it to take effect. the dorsum of each rat was shaved and then exposed to a 1 cm x 1 cm brass block, pre-heated to 95 °c in a hot water bath, for 10 s. the brass block was placed on the dorsum of the rats using gravity only, which resulted in partial thickness skin burns. a physiological saline solution was then administrated intraperitoneally (25 mg/kg) to prevent dehydration of the animal. immediately after thermal injury, all wounds were dressed with a primary gauze dressing and opsitetm as the secondary dressing. to prevent the rats from interfering with the healing process the secondary dressing was covered with a third bandage dressing and fastened with elastoplast around the edges. all animals received another injection of tramadol 1 h after thermal injury. in addition, all animals received a four-hourly subcutaneous injection of analgesics for the remainder of the study. preparation of fibrin clots on day 7, during termination, blood was drawn from each animal and 11 µl of citrate was added to every 100 µl of blood drawn. the blood from each animal was kept separately and studied individually. blood was centrifuged at 1250 rpm for 2 min to obtain platelet-rich plasma (prp).thrombin (provided by the south african national blood services) was used to prepare fibrin clots (pretorius, ekpo & smit 2007; pretorius, oberholzer & smit 2009a; pretorius et al. 2009b). the thrombin (20 u/ml) was prepared in a biological buffer containing 0.2% human serum albumin. when thrombin is added to prp, fibrinogen is converted to fibrin and intracellular platelet components; for example, transforming growth factor, platelet-derived growth factor and fibroblastic growth factor are released into the coagulum. a volume of 10 µl of mouse prp was mixed with 10 µl thrombin. the prp–thrombin mix was immediately transferred by pipette to a 0.2-µm millipore membrane to form the coagulum (fibrin clot). the millipore membrane was then placed in a petri dish on filter paper dampened with phosphate-buffered saline (pbs) (to create a humid environment) and kept at 37 °c for 10 min. the coagula-containing membranes were then washed (by placing them in pbs and magnetically stirring for 20 min) to remove any blood proteins trapped within the fibrin network. preparation of washed fibrin clot for scanning electron microscopy washed fibrin clots were fixed in 2.5% gluteraldehyde in dulbecco’s pbs (0.075 m) at a ph of 7.4 for 1 h. each fibrin clot was rinsed three times in phosphate buffer for 5 min before being fixed for 1 h with 1% osmium tetraoxide. the samples were rinsed three times for 5 min with distilled water and were then dehydrated serially in 30%, 50%, 70% and 90% ethanol, and three times with 100% ethanol. the scanning electron microscopy procedures were completed by drying the samples with hexamethyldisilazane (araujo et al. 2003) before being mounted, followed by coating samples with ruthenium tetraoxide and examining the tissue with a zeiss ultra plus feg scanning electron microscope. results and discussion (back to top) burn injury results in an inflammatory response and there is a synergistic response between the resulting inflammation and coagulation systems (park et al. 2008; sherwood & toliver-kinsky 2004). white blood cell distribution changes during the inflammatory response. as mentioned previously, haemostasis changes during burn injury. after burn injury there is a brief hypocoagulation phase, followed by hypercoagulation owing to intensified procoagulant activity. these steps, in turn, result in depression of fibrinolysis (kirillov & alekaeva 1975). both thrombotic and fibrinolytic pathways are directly triggered proportionally to the extent of the burn injury (bartlett et al. 1981; opal 2000; wells, sissons & hasleton 1984). although this has been researched thoroughly in the past, we do not know what the impact of burn injury is on the ultrastructure of the fibrin networks. from previous research it is known that inflammation causes fibrin networks to present with a changed morphology (oberholzer, vieira & pretorius 2009; pretorius et al. 2007). typically, control fibrin networks consist of major, thick fibres forming the bulk of the clot, with minor, thin fibres sparsely distributed among them (pretorius et al. 2009a; pretorius et al. 2009b). we also know that this arrangement is found in humans, rabbits and mice. however, the thickness of rodent fibrin differs considerably from that of humans (pretorius et al. 2009b). inflammation in humans and mice has previously been shown to look similar, although the fibre thickness varies. pretorius and oberholzer (2009) showed that inflammation in humans and mice show major, thick fibres covered by a thin, matted layer of minor, thin fibres. in the current study, control rat fibrin networks were studied and were found to appear similar to those of balb/c mice; major, thick fibres with minor fibres distributed in between (figure 1). in the burn wound injury animals in this study, fibrin fibres showed a typical inflammatory profile 7 days after injury (figure 2a–c). figure 2a shows major, thick fibres (thick, white arrow) and more prominent minor, thin fibres (thin, white arrows), as well as areas where fibrin has a netted appearance (black arrow). figure 2b shows areas of the clot where thickened, matted fibrin is present (white arrow). some areas of the clot also have a netted appearance (figure 2c: white arrows). this changed morphology suggests that thermal injury affects not only the local area where the wound was created, but also the broader inflammatory processes and ultimately coagulation and haemostasis. ravindranath et al. (2004) mentioned that burn injury disturbs the coagulation cascade and thrombotic process in the procoagulant pathway by impairing fibrinolysis. also, venous thrombosis, pulmonary embolism and hypercoagulability are common in thermal injury (darling et al. 1996; gando et al. 1992; geerts 1994; kowal-vern et al. 2000; kowal-vern et al. 1992; penner 1995). here we suggest that the thrombotic events associated with burn injury are due to the thickened, netlike areas formed when thrombin activates the coagulation cascade. because of impaired fibrinolysis activities, the resulting fibrin clots can then not be disseminated successfully. small fragments of these netted, clumped areas may therefore break loose and, owing to already insufficient fibrinolysis activity, cause thrombotic events after burn injuries. figure 1: control rat fibrin network. figure 2: rat fibrin network after thermal injury (day 7). conclusion (back to top) this study investigated whether burn injury affects the ultrastructure of fibrin networks. results obtained with a rat burn wound model showed morphological changes to the structure of fibrin clots that formed after burn injury. these changes are likely due to impaired fibrinolysis, which explains thrombotic events associated with burn injury. references (back to top) araujo, j.c., téran, f.c., oliveira, r.a., nour, e.a.a., montenegro, m.a.p., campos, j.r. et al., 2003, ‘comparison of hexamethyldisilazane and critical point drying treatments for sem analysis of anaerobic biofilms and granular sludge’, journal of electron microscopy 52(4), 429–433. doi:10.1093/jmicro/52.4.429, pmid:14599106 bartlett, r.h., fong, s.v., maruggo, g., hardeman, t. & anderson, v., 1981, ‘coagulation and platelets changes after thermal injury in man’, burns 7, 370–377. doi:10.1016/0305-4179(81)90013-9 darling, g.e., keresteci, m.a., ibanez, d., pugash, r.a., peters, w.j. & neligan, p.c., 1996, ‘pulmonary complications in inhalation injuries with associated cutaneous burn’, journal of traumatic injury infection and critical care 40, 83–89. doi:10.1097/00005373-199601000-00016 fang, p., ding, r. & kong, l., 1997, ‘the changes of fibrinolysis in plasma and wounds in rats inflicted with deep partial thickness burns’, zhonghua zheng xing shao shang wai ke za zhi 13(4), 259–263. pmid:10452009 gando, s., tedo, i. & kubota, m., 1992, ‘post-trauma coagulation and fibrinolysis’, critical care medicine 20, 594–600. doi:10.1097/00003246-199205000-00009, pmid:1533358 geerts, w.h., 1994, ‘thromboembolism in trauma: the problem and its prevention’, new england journal of medicine 331, 1601–1606. doi:10.1056/nejm199412153312401, pmid:7969340 kirillov, m.m. & alekaeva, l.d., 1975, ‘changes in the blood coagulation system in patients with mechanical trauma and burns’, problemy gematologii i perelivaniia krovi 20(6), 15–18. pmid:1135183 kowal-vern, a., mcgill, v., walenga, j.m. & gamelli, r.l., 2000, ‘antithrombin iii concentrate in the acute phase of thermal injury’, burns 26(1), 97–101. doi:10.1016/s0305-4179(99)00099-6 kowal-vern, a., walenga, j.m., gamelli, r.l., hoppensteadt, d., sharp-pucci, m.m. & schumacher, h.r., 1992, ‘the effect of burn wound size on hemostasis: a correlation of the hemostatic changes to the clinical state’, journal of traumatic injury infection and critical care 33, 50–56. doi:10.1097/00005373-199207000-00011 lavrentieva, a., kontakiotis, t., bitzani, m., papaioannou-gaki, g., parlapani, a., thomareis, o. et al., 2008, ‘early coagulation disorders after severe burn injury: impact on mortality’, intensive care medicine 34(4), 700–706. doi:10.1007/s00134-007-0976-5, pmid:18193192 oberholzer, h.m., vieira, w.a. & pretorius, e., 2009, ‘investigating the effect of the homeopathic immunomodulator, modul8®, on blood count, bronchial lavage and fibrin ultrastructure on experimental asthmatic balb/c mice’, international journal of morphology 27(3), 955–963. opal, s.m., 2000, ‘phylogenetic and functional relationships between coagulation and the innate immune response’, critical care medicine 28(9), 77–80. doi:10.1097/00003246-200009001-00017, pmid:11007204 park, m.s., salinas, j., wade, c.e., wang, j., martini, w., pusateri, a.e. et al., 2008, ‘combining early coagulation and inflammatory status improves prediction of mortality in burned and non-burned trauma patients’, journal of trauma 64(2), 188–194. doi:10.1097/ta.0b013e318160a5a3, pmid:18376164 penner, j., 1995, ‘antithrombin deficiency in special clinical syndromes – part ii: trauma/burns,’ seminars in hematology 32, 42–48. pmid:8821209 pretorius, e. & oberholzer, h.m., 2009, ‘ultrastructural changes of platelets and fibrin networks in human asthma: a qualitative case study’, blood coagulation and fibrinolysis 20(2), 146–149. doi:10.1097/mbc.0b013e328325549a pretorius, e., ekpo, o.e. & smit, e., 2007, ‘comparative ultrastructural analyses of platelets and fibrin networks using the murine model of asthma’, experimental and toxicologic pathology 59(2), 105–114. doi:10.1016/j.etp.2007.02.011, pmid:17600694 pretorius, e., oberholzer, h.m. & smit, e., 2009a, ‘ultrastructure of platelets and fibrin networks of asthmatic mice exposed to selenium and withania somnifera’, anatomical science international 84(3), 210–217. doi:10.1007/s12565-008-0010-1, pmid:19214657 pretorius, e., vieira, w.a., oberholzer, n. & auer, r.e.j., 2009b, ‘comparative scanning electron microscopy of platelets and fibrin networks of humans and different animals’, international journal of morphology 27(1), 69–76. ravindranath, t.m., goto, m., demir, m., tobu, m., kujawski, m.f., hoppensteadt, d. et al., 2004, ‘tissue factor pathway inhibitor and thrombin activatable fibrinolytic inhibitor plasma levels following burn and septic injuries in rats’, clinical and applied thrombosis/hemostasis 10, 379–385. doi:10.1177/107602960401000411, pmid:15497025 sherwood, e.r. & toliver-kinsky, t., 2004, ‘mechanisms of the inflammatory response’, best practice and research: clinical anaesthesiology 18(3), 385–405. doi:10.1016/jbpa.2003.12.002 wells, s., sissons, m. & hasleton, p.s., 1984, ‘quantitation of pulmonary megakaryocytes and fibrin thrombi in patients dying from burns’, histopathology 8, 517–527. doi:10.1111/j.1365-2559.1984.tb02361.x, pmid:6735362 2191putterill_pp281-297.qxd introduction the morphology and microscopic anatomy of the reptilian oral cavity has received much attention in the literature (for a review see luppa 1977), with most studies concentrating on the description and location of glandular tissue, taste receptors and epithelial specialisation of the region. attention has also been given to the embryological and evolutionary development of these specialisations. similarly, most studies on the oral cavity of crocodilians have concentrated on specific morphological features of this region. röse (1893) reported the presence of glandular tissue situated in pits between the teeth of the maxilla (glandulae palatinae) in crocodylus porosus and described the embryological development of these glands. woerdeman (1920) reviewed earlier literature (ca. 1888 to 1914) on the subject and emphasised discrepancies amongst the authors regarding the presence or absence of oral glands in crocodylia. farenholz (1937) reported two areas in the palate in which glands occur, viz., median palatine 281 onderstepoort journal of veterinary research, 70:281–297 (2003) general morphology of the oral cavity of the nile crocodile, crocodylus niloticus (laurenti, 1768). i. palate and gingivae j.f. putterill1* and j.t. soley2 abstract putterill, j.f. & soley, j.t. 2003. general morphology of the oral cavity of the nile crocodile, crocodylus niloticus (laurenti, 1768). i. palate and gingivae. onderstepoort journal of veterinary research, 70:281–297 the heads of nine 2.5 to 3-year-old nile crocodiles (crocodylus niloticus) were obtained from a commercial farm where crocodiles are raised for their skins and meat. the animals from which these specimens originated were clinically healthy at the time they were slaughtered. a detailed description of the macroscopic and microscopic features of the palate and gingivae of the nile crocodile is presented and the results are compared with published information on this species and other crocodylia. the histological features are supplemented by information supplied by scanning electron microscopy. macroscopic features of interest are the small conical process situated at the base of the first two incisors of the maxilla, the distribution of cobbled units on the palate, and the broad dentary shelf forming the rostral aspect of the mandible. histologically the palate and gingivae did not differ significantly from each other and both regions showed a presence of pacinian-type corpuscles. two types of sensory structures (taste receptors and pressure receptors) were identified in the regions examined, both involving modification of the epithelium and the underlying connective tissue. keywords: crocodylus niloticus, histology, morphology, nile crocodile, oral cavity, scanning electron microscopy * author to whom correspondence is to be directed 1 electron microscopy unit, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa. e-mail: john@moon.ovi.ac.za 2 department of anatomy and physiology, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa accepted for publication 12 may 2003—editor glands found only in caiman spp. and small glands at the median aspect of the maxillary teeth, found in caiman spp. and alligator mississippiensis. however, taguchi (1920) found glandular tissue “in the submucosa of the caudal part of the palate and the oral surface of the velum”. kochva (1978) extensively described glandular tissue in reptiles, but only fleetingly refers to the crocodylia. bath (1905, 1906) described the histology of taste receptors in the oral cavity, pharynx and oesophagus of crocodilus niloticus (sic.) and alligator mississipiensis (sic.), finding no clear distinction between those seen in these species and those of higher animals. luppa (1977), who generalised his description of the histological composition of the reptilian oral cavity, stated that “taste buds were scattered throughout the oral epithelium in reptiles and that in lacerta they were most numerous laterally and on the palatal folds.” hulanicka (1913) investigated the innervation of the tongue, palate and the skin of crocodylus niloticus and alligator lucius and described five different nerve endings in the regions studied. alligator lucius represents a. mississippiensis (f.w. huchzermeyer, personal communication 2002). fuchs (1908, cited by barge 1937) postulated the formation of the secondary palate in the crocodylia and compared this formation to other reptiles, concluding that the secondary palate of crocodiles was unique amongst the reptiles. barge (1937) described the embryological development and phylogeny of the secondary palate in crocodiles. ferguson (1979) investigated the developmental mechanisms in normal and abnormal palate formation in the american alligator (a. mississippiensis) and concluded that the crocodylia showed characteristics which were part mammalian and part reptilian, a unique combination which made them a useful model to study palatogenesis. dentition in crocodylia has also received much attention in the literature. of note is the paper by poole (1961) who described tooth replacement in c. niloticus, the studies by westergaard & ferguson (1986, 1987) who described the development of dentition in hatchling and juvenile a. mississippiensis, and the article by kieser, klapsidis, law & marion (1993) who examined heterodonty and patterns of tooth replacement in c. niloticus. edmund (1962, 1969) also made a major contribution to studies on dentition in the reptilia, including the crocodylia, describing the sequence and rate of tooth replacement in these reptiles. although detailed descriptions of specific components of the crocodilian oral cavity have been presented, only a few studies have reported on the general histological features of this region. reese (1913) studied the histology of the enteron of the “florida alligator”, which included the oral cavity. reese’s description, however, concerned histological differences between hibernating and feeding, captive animals. taguchi (1920) compared similar regions of the oral cavity to those examined by reese (1913) in three species of crocodylia, namely, alligator sinensis, krokodilus porosus and krokodilus vulgaris. the latter is believed to represent the nile crocodile, crocodylus niloticus (see http:// www.flmnh.ufl.edu/natsci/herpetology/turtcroclist/ chklst2.htm). throughout this paper, and pertaining only to taguchi (1920), “krokodilus” is referred to as “crocodylus” and “k. vulgaris” as “c. niloticus”. in chiasson’s (1962) publication on the anatomy of the alligator, components of the oral cavity (palate and tongue) are briefly mentioned without any further detail being given. similarly parsons & cameron (1977), who examined the relief of the gastro-intestinal tract of the reptilia, including the crocodylia, only start their description from the oesophagus and do not describe the morphology of the oral and pharyngeal cavities. in view of the paucity of information concerning the general histological features of this part of the upper digestive tract, this paper presents a general topographical description of the oral cavity as well as the macroscopic and microscopic features of the palate and gingivae of the nile crocodile, crocodylus niloticus (laurenti, 1768) and compares the results with published information on this species and other crocodylia. the histological features are supplemented by information supplied by scanning electron microscopy (sem). morphological features of the tongue will be presented in another paper. materials and methods experimental animals the heads of nine 2.5 to 3-year-old nile crocodiles were obtained from a commercial farm where crocodiles are raised for their skins and meat. the lengths of the animals sampled ranged from 1.2– 1.5 m and they were clinically healthy at the time they were slaughtered. the animals were killed by shooting them in the brain at close range using a .22 calibre rifle. after the carcasses had been skinned and eviscerated the heads were removed 282 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i and immersion-fixed in a large volume of 10 % phosphate-buffered formalin in plastic buckets for a minimum period of 48 h. care was taken to exclude air from the oral cavity by wedging a small block of wood in the angle of the mouth prior to immersion in the fixative. samples from the palate and gingivae were taken from the heads and processed for light microscopy (lm) and scanning electron microscopy (sem) according to the procedures detailed below. topography prior to sampling, all nine heads were utilised for a description of the gross anatomical features and topographical relationships of the structures in the oral cavity. macrophotographs were recorded digitally using a nikon coolpix 995 (nikon, tokyo, japan) digital camera or on 35 mm film using a chinon x-7 (chinon, tokyo, japan) single lens reflex camera, respectively. the oral cavities of these heads were also examined and micrographed using a stereomicroscope (wild m-400 photomakroskop, heerbrugg, switzerland) to obtain higher magnification micrographs of specific topographical features. a dried skull from a 5-year-old (approximate age) specimen was used to confirm the position and naming of teeth in the maxilla and mandible as well as to provide supporting evidence for the anatomical description. teeth were named and numbered according to kieser et al. (1993). light microscopy samples of the gingiva from the mandible were removed from the various regions indicated in fig. 1 and were based on the position of the incisor and canine teeth. the portion of gingiva caudal to the indicated regions, i.e. the region involving the molar teeth, was too firmly attached to the underlying bone to permit suitable samples to be taken. the mucosa of the palate was also sampled according to the dental arrangement of the teeth, i.e. from regions i 1 to i 5, c 1 to c 5 and m 1 to m 8 as shown in fig. 2. as the gingiva of the maxilla appeared macroscopically to be continuous with the palate, these specimens were removed together with the samples of the palate. a similar set of specimens (adjacent tissue) from all the indicated regions of the mandible and palate was taken at the same time for sem examination. samples for lm were dehydrated through 70, 80, 96 and 2x 100 % ethanol and further processed through 50 : 50 ethanol : xylol, 2x xylol and 2x paraffin wax (60–120 min per step) using a shandon model 2le automatic tissue processor (shandon, pittsburgh, pa, usa). tissue samples were finally embedded manually into paraffin wax in brass moulds. sections were cut at 4–6 µm, stained with haematoxylin and eosin (h&e) (luna 1968) or periodic acid-schiff (pas) (pearse 1985) and viewed and micrographed using a reichert polyvar (reichert, austria) compound light microscope fitted with a differential interference contrast (dic) prism. scanning electron microscopy the samples of the gingivae and palate obtained as indicated above and which had been fixed in 10 % phosphate-buffered formalin for a minimum of 48 h were subsequently rinsed for several hours in water to remove traces of phosphate buffer. these samples were routinely dehydrated through an ascending ethanol series (50, 70, 90, 95 and 3x 100 %— 60 min per step) and critical point dried from 100 % ethanol through liquid-co2 in a polaron critical point drier (polaron, watford, england). the samples were then mounted onto brass or aluminium viewing stubs (to expose the epithelial surface) with a conductive paste (carbon dag) and sputter coated with gold using a balzers 020 sputter coater (balzers union, liechtenstein). specimens were viewed and photographed using a hitachi s-2500 scanning electron microscope (hitachi, tokyo, japan) operated at 8 kv. results macroscopic features the oral cavity had the form of an isosceles triangle (fig. 1 and 2) and was dorso-ventrally flattened, severely limiting the space within the cavity. the roof of the cavity was formed exclusively by the palate and the indistinct gingiva with which it was continuous. the caudal limit of the roof was demarcated by the notched dorsal component of the gular valve, whereas the rostral limit of the palate was occasionally characterised by the presence of two deep pits which accommodated the first two incisors of the mandible (fig. 2). between the two pits (or at the base of the two i 1 teeth) was a small, rigid, conical process (fig. 2) which emerged from a low-profiled ridge above the anterior palatine foramen. the tip of this process was housed within a shallow depression in the mandible at the base of the first two mandibular incisors (fig. 1 and 3). the surface of the palate had a cobbled appearance 283 j.f. putterill & j.t. soley 284 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i (fig. 2) due to the presence of numerous, raised, cobble-like structures. the cobbles on the rostral two-thirds of the palate were large, whereas those occupying the caudo-lateral aspects of the palate were smaller, had a lower profile, but were densely arranged. between the latter two regions were paired elliptical areas, devoid of cobbles, and which merged medially along the midline of the palate (fig. 2). these smooth areas corresponded to the positioning of the left and right posterior palatine foraminae which were formed by the caudal edges of the maxillary, the lateral edges of the palatine, a small region of the rostral edge of the pterygoid and the medial edge of the transpalatine bones. along the midline of the palate were a series of closely positioned cobbles forming a clearly defined median ridge. this ridge extended from the conical process mentioned above to the base of the dorsal fold of the gular valve. however, the part of the ridge dividing the two smooth elliptical areas above the posterior palatine foraminae was less distinct in nature. the base of the palate adjacent to the dorsal fold of the gular valve displayed a variable number of transverse mucosal folds which closely followed the contours of the dorsal fold across its entire breadth (fig. 2). 285 j.f. putterill & j.t. soley fig. 1 macrophotograph of the mandible with the tongue in situ showing the dental arrangement of the incisor (i 1–3), canine (c 1–5) and molar (m 1–7) teeth, demarcated by the stippled lines. the sampling sites for histology of the gingivae (d and e) are also indicated. the rostral dentary shelf is indicated in the region above the symphysis of the dentary bones. the black arrowhead indicates the position of the shallow depression which houses the small, rigid, conically formed process, situated at the base of the i 1 teeth, seen in fig. 2. the glottis (gt) and laryngeal mound (lm) are shown in situ on the floor of the pharyngeal cavity and the ventral fold (vf) of the gular valve is seen separating the ventral aspects of the oral and pharyngeal cavities. formalin fixed specimen. x 0.75 fig. 2 macrophotograph of the maxilla and palate showing the dental arrangement of the incisor (i 1–5), canine (c 1–5) and molar (m 1–8) teeth, demarcated by the stippled lines. the smooth area of the palate, demarcated by the black arrows, lies above the left and right posterior palatine foraminae. the small, rigid, conical process, situated at the base of the i 1 teeth, is indicated by the arrowhead. also note the smooth zone of mucosa forming the gingiva adjacent to the teeth (asterisks) which stretches from approximately i 5 to m 8 on both sides of the maxilla. the common opening of the internal nares (in) is seen on the roof of the pharyngeal cavity as well as the dorsal fold (df) of the gular valve which separates the dorsal aspects of the oral and pharyngeal cavities. samples of the mucosa of the palate and gingiva were removed from regions a, b and c as indicated on the photograph. formalin fixed specimen. x 0.75 fig. 3 macrophotograph of the rostral portion of the mandible (i 1 to c 5). the gingiva is seen to have a slightly cobbled appearance towards the rostral tip above the dentary symphysis. the black arrow indicates a shallow depression which houses the small, rigid, conically-shaped maxillary process seen in fig. 2. the blocked arrow shows a hole made by the hook in the abattoir when the carcass was suspended during the evisceration process. i 1 = incisor 1; c 1, c 5 = canine 1 and 5. formalin-fixed specimen. x 1.2 the gingiva of the maxilla was continuous with the palate and could practically be considered to be part of it (fig. 2). a relatively wide (4–5 mm), clearly demarcated zone of smooth mucosa (possibly representing the palatal aspect of the gingiva) separated the cobbled portion of the palate from the maxillary teeth, from approximately c 3 to m 8. from approximately c 2 rostrally, the surface of the gingiva also had a cobbled appearance similar to that of the palate and the boundary between the latter and the gingiva was not clearly defined. the teeth of the maxilla reflected the dental formula described by kieser et al. (1993) and were carried in the premaxillary and maxillary bones. in the occluded mouth, the teeth of the maxilla were accommodated in grooves to the outside of the mandible, between the teeth of the lower jaw. the tips of the teeth of the mandible were accommodated in pits situated between the teeth of the maxilla, with the exception of c i, which was accommodated in a maxillary notch and remained visible when the jaws were closed. the floor of the oral cavity was formed by the tongue and a wide, rostral mucosal plate continuous with the gingiva (fig. 1 and 3). this plate represented the mucosa-covered surface of the widened rostral tips of the paired dentary bones of the mandible where they met at the dentary symphysis. this plate extended from the rostrally-positioned first two incisors to a point approximately midway between c 1 and c 2 (fig. 1 and 3). the relatively long tongue was roughly triangular in shape, being much broader caudally than at its tip (fig. 1). it occupied the greater part of the floor of the oral cavity (apart from the rostral plate over the symphysis of the dentary bones) and was bordered peripherally by a loose, highly folded, continuous, fibrous membrane (fig. 1 and 3). the gingiva of the mandible was more clearly defined than that of the maxilla, having a low profiled, cobbled appearance from approximately c 3 rostrally. there was close attachment of the gingiva to the mandibular (dentary and splenial) bones, especially in the region of c 3 (or c 4) to m 7 (or m 8). the rostral tip of the dentary bones formed a broad shelf or plateau (the rostral dentary shelf) which was divided medially by the dentary symphysis. in this region the gingiva had a slightly spongy texture, although the surface also had a cobbled appearance (fig. 1 and 3). the teeth of the mandible were carried in the paired dentary bones and also reflected the dental formula described by kieser et al. (1993) (see fig. 1). from m 4 (or m 5) to m 7 (or m 8) the dentary bone and the medially situated splenial bone were in close association, although the mandibular teeth were clearly housed in the dentary bone. light microscopy the palate sections of the palate stained with h&e revealed a keratinised stratified squamous epithelium of variable thickness in all the regions examined. the stratum basale was composed of a single layer of cuboidal to columnar cells resting on a basement membrane. the basement membrane was most obvious in pas-stained sections and varied in prominence from conspicuous to barely visible. the nuclei of the basal layer of cells were pale, vesicular and round to oval in shape (fig. 4a). where oval, the nuclei were oriented vertically to the surface of the epithelium. the stratum spinosum consisted of 3–6 layers of cells. the cells adjacent to the stratum basale were cuboidal in shape, while the more superficial cells were horizontally flattened. all the cells of this layer displayed the characteristic inter-linking cytoplasmic bridges connecting the individual components. the nuclei of these cells resembled those of the stratum basale. a thin (3–4 layers) stratum granulosum was present above the stratum spinosum. cells in this layer were spindle shaped or flattened and oriented horizontally. the nuclei were pycnotic, flattened and oriented in the same plane as the cells, while the cytoplasm was filled with strongly basophilic-staining keratohyaline granules (fig. 4a). the stratum corneum varied in thickness and was composed of a number of compressed layers of cells in which no nuclei were apparent (fig. 4a). in some areas, particularly towards the gingiva of the teeth and in convoluted regions of the epithelium, a stratum disjunctum consisting of a loose layer of keratinised cells was present (fig. 4b). the epithelium was supported by a thick layer of irregular dense connective tissue with prominent bundles of variably oriented collagen fibres being the most prominent feature (fig. 4a, b, d and e). sandwiched between the deeper regions of the irregular dense supporting connective tissue and the periostium of the palatine bones was a welldeveloped plexus of blood vessels, lymphatics and nerves (medullated and non-medullated). deeply situated striated muscle bundles were noted only in the region of the posterior palatine foraminae, 286 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i 287 j.f. putterill & j.t. soley stretching from the posterior third of the palate to the base of the dorsal gular fold. no other muscular tissue was observed. immediately beneath the basement membrane was a thin layer of fine connective tissue which in places displayed a vacuolated, spongy appearance. this region demonstrated a rich capillary blood supply which was intimately associated with the overlying epithelium. no glandular tissue was ever observed in any of the specimens during histological examination of the palate. lymphocytic aggregations were also not apparent in these sections. melanocytes were observed in the connective tissue a short distance beneath the stratum basale, but never within the epithelial layer. the cells were typically dendritic in nature and displayed large numbers of brown to black melanin granules (melanosomes). the melanocytes were concentrated around the capillary plexus beneath the epithelium and also around the larger blood vessels and nerves more deeply positioned within the connective tissue stroma. in some areas the melanocytes formed a diffuse but definite layer beneath the epithelium. the presence of melanin varied amongst individual specimens examined and in some cases it was found to be entirely absent. mast cells occurred either singly or in small groups throughout the connective tissue layer with concentrations of five or more cells sometimes being observed. the mast cells were large, round and often observed in the vicinity of blood vessels. the pale, round to oval vesicular nucleus was centrally positioned within the cytoplasmic mass which displayed small fine, evenly distributed basophilic granules. prominent pacinian-like corpuscles were randomly scattered throughout the connective tissue layer a short distance beneath the epithelium (fig. 4b and c). these structures typically consisted of a variable number of connective tissue lamellae surrounding an inner core representing the terminal portion of the innervating nerve. the corpuscle was surrounded by a prominent, dense connective tissue capsule (fig. 4c) and large medullated nerves were observed in the vicinity of the corpuscles. three types of surface specialisations were observed in the sections studied. the first type comprised small pointed elevations of the epithelial lining supported by a core of fine connective tissue (fig. 4a). in some instances these elevations presented as a series of small localised projections giving the surface of the palate a scalloped appearance. these structures probably represented the epithelial folds observed by sem (see below). the remaining two types of specialised structures were characterised by modification of both the epithelium and the underlying connective tissue. both structures (fig. 4d and e) displayed a localised thickening of the epithelium due mainly to an increase in the number of layers of the stratum spinosum. the keratinised layer in the region of the epithelial thickening was generally thinner than that of the adjacent tissue. the localised epithelial thickenings were most commonly found in the form of an elevated, domeshaped structure due primarily to the presence of a diffuse, ellipsoid or conical-shaped mass of loosely arranged connective tissue situated immediately beneath the epithelium (fig. 4d). these regions were more lightly stained (h&e-stain) than the surrounding connective tissue (due to a reduction in size and number of the collagen bundles) and caused localised protrusion of the overlying epithelium into the mouth cavity. the morphological features of the specialised regions varied. in some instances the diffuse connective tissue core contained a basophilic cell-rich mass situated immediately adjacent to the basal lamina. in other regions, the connective tissue core displayed a paucity of cells, possibly due to the plane of section. associated with the modified regions of connective tissue were pacinian-like corpuscles which were either found in or adjacent to this zone. nerve tissue featured prominently within the modified connective tissue and large medullated nerves and blood vessels were observed entering/leaving at the base of the connective tissue core. the dome-shaped specialisations were distributed throughout the palate but appeared to be more numerous on the rostral aspect up to the rostral border of the posterior palatine foraminae (see fig. 2). a small number of localised epithelial thickenings appeared flattened in contrast to the dome-shaped structures and were either positioned level with the adjoining epithelial surface or slightly raised above it. the floor of these epithelial specialisations jutted into the underlying connective tissue layer. epithelial cells towards the middle of the specialisation adopted a vertical orientation, forming a large elliptical structure reminiscent of a taste bud (fig. 4e and f). some of the vertically inclined cells revealed dense, somewhat elongated nuclei, particularly towards the periphery of the elliptical structure, and were similar in appearance to the supporting cells of the mammalian taste-bud. similarly inclined cells with more vesicular nuclei were seen among the supporting cells and may have represented neuro-epithelial cells. a modified connective tissue 288 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i core similar to that seen beneath the dome-shaped structures was also evident but did not appear to be specifically associated with pacinian-like corpuscles. attendant medullated nerves, however, were much in evidence. the taste receptors described above appeared to be concentrated on the more lateral aspects of the palate although they were occasionally encountered towards the midline. sem examination confirmed the cobbled appearance of the palate seen macroscopically. it should be noted, however, that individual variation existed in the specimens examined regarding the prominence of the cobbling. each clearly demarcated cobbled unit displayed a centrally positioned domeshaped structure or papilla surrounded by an expanse of loosely attached surface epithelial cells. desquamation of these cells was particularly obvious at the perimeter of the dome-shaped structure (fig. 5b). in much of the palate (roughly corresponding to the surface in contact with the dorsum of the tongue) the epithelial surface surrounding the papillae was thrown into a number of conspicuous folds which branched and anastomosed (fig. 5a). the folds displayed a rostro-caudal or slightly oblique alignment. however, towards the periphery of the palate bordering the gingivae and the dorsal fold of the gular valve, as well as in the smooth region of the palate overlaying the posterior palatine foraminae, the cobbled units displayed a featureless surface around the domed papillae. some of the domed papillae revealed a small centrally positioned depression and radiating grooves (fig. 5c). these structures appeared to occur more commonly in the rostro-lateral regions of the palate. all regions of the palate were characterised by distinct desquamation of the superficial cells of the epithelium. this phenomenon was possibly accentuated by the critical point drying (cpd) process used for sem sample preparation. cracking of the epithelial layer was evident in some specimens examined (see fig. 5c) and was also attributed to the cpd process. higher magnification of the epithelial surface using sem imaging revealed the typical polygonal outline of the individual cells, although the borders were not always clearly demarcated. the keratinised surfaces had a coarse, matted appearance (fig. 5d). the gingivae the composition and structure of the gingival mucosa was similar in general appearance to that of the palate, although some variations in structure were apparent. the epithelial surface was more undulating than that of the palate, with occasional elevated structures protruding from the surface (fig. 6a, c and d). the epithelium itself was thinner than that of the palate, with the stratum corneum and stratum disjunctum forming the most prominent layers. the stratum spinosum was extremely thin and only obvious in regions of localised thickening. below the basement membrane was a thin layer of vacuolated, spongy connective tissue which was continuous with a thick layer of irregular dense connective tissue. 289 j.f. putterill & j.t. soley fig. 4 histological features of the palate a a pointed elevation of the epithelial lining of the palate supported by subepithelial connective tissue (ct). stratum basale (1); stratum spinosum (2); thin stratum granulosum (asterisk); superficial stratum corneum (3). h&e-stain. bar = 100 µm b randomly scattered pacinian-like corpuscles (arrowhead) were located in the subepithelial connective tissue and often associated with epithelial specialisations. note signs of desquamation of the superficial layers of the stratum corneum (arrow). h&e-stain. bar = 100 µm c typical pacinian-like corpuscle displaying concentric lamellae around an inner core (arrowhead). note the thick, fibrous connective tissue capsule surrounding the corpuscle. h&e-stain. bar = 50 µm d light micrograph of an elevated, dome-shaped specialisation commonly found in the rostral and lateral regions of the palate showing a pale connective tissue mass (outlined area) below the epithelium. note the localised thickening of the epithelium and the dark, sub-epithelial cellular mass (arrowhead) h&e-stain. bar = 500 µm e a flattened epithelial specialisation typically found in the mid-, lateral and posterior regions of the palate. note the dense, dark cellular mass (arrowhead) in the deeper connective tissue associated with these receptors and the arrangement of the epithelial cells to form a structure similar to that of a taste bud (rectangle). h&e-stain. bar = 250 µm f higher magnification of the epithelial specialisation shown in the rectangle in fig. 4e. note the translucent circular structure (arrow) filled with fine fibrillar material. it was not possible to determine the function of this type of corpuscle, but it was thought that they might be associated with taste or osmoreception. h&e-stain. bar = 100 µm 290 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i the occurrence, appearance and organisation of mast cells, melanocytes, vascular and nerve plexuses was similar to that seen in the palate. surface specialisations similar to those seen in the palate were evident in the gingiva, namely, the small, pointed epithelial elevations and the larger specialised structures displaying modification of the epithelium and the underlying connective tissue. these structures were particularly obvious at the rostral aspect of the mandible although they were found throughout the gingivae. the small epithelial projections probably represent the conical processes seen by sem (see fig. 6d). the raised, domeshaped structures with a thickened epithelium were, as in the palate, associated with pacinian-like corpuscles situated in the vicinity of the modified connective tissue core. however, the pacinian-like corpuscles appeared to be more abundant in the gingivae, with three to four sometimes being associated with each specialisation. the thickened, non-elevated epithelial specialisations typically also displayed structures resembling taste buds. the “taste bud” was generally situated in the centre of the thickened epithelial lining, although pairs of “taste buds” were sometimes observed (fig. 6b and e). sem of the gingiva revealed a series of raised, dome-shaped structures each of which was surrounded by two concentric rows of smaller, raised conical projections (fig. 6d). these structural units appeared most concentrated on the shelf above the dentary symphysis (rostral dentary shelf) of the mandible and showed smaller concentrations at the base of each tooth (see fig. 6c), from i 1 to c 5, but progressively reduced in numbers caudally on the lingual surfaces of the dentary and splenial bones. the gingiva of the maxilla also displayed a reduced number of these structural units. situated between some of these units were small, flattened and slightly depressed, circular areas, often displaying a centrally situated pore (fig. 6d). pairs of closely associated pores were also occasionally seen (fig. 6e). higher magnification of the flattened discs sometimes showed a mass of fimbriae protruding from the pore (fig. 6e and f). the pores are believed to represent the opening on the surface of the underlying “taste buds” and were sometimes difficult to observe by sem due to occlusion of the pore by cellular debris. the flattened areas did not occur constantly between the domed units described above and also did not appear to be arranged in any sequence or pattern, but their occurrence was most common on the rostral dentary shelf. on occasion they also occurred isolated from any other epithelial specialisations, although they displayed similar morphological features. desquamation of the surface cells was much in evidence and the surface features of the cells were similar to those seen in the palate (see fig. 5d). examination of fresh specimens from the palate and gingiva showed that the cobbled units described above did not display epithelial folding as prominently as formalin-fixed or critical point dried specimens. this phenomenon was presumed to be associated with the shrinking effect of fixation and the processing of the tissue samples for sem observation. discussion meaningful gross morphological descriptions of the oral cavity of the crocodylia are not available in the literature and it appears that only specific speciali291 j.f. putterill & j.t. soley fig. 5 sem features of the palate a sem photograph of a polygonal-shaped cobbled unit (arrowheads) typical of the surface of the palate. note the centrally positioned dome-shaped structure (papilla—circled) and the grooved appearance of the surrounding epithelial surface. bar = 250 µm b higher magnification of the slightly convex, circular structure circled in fig. 5a, the surface of which appears featureless. note the epithelial desquamation (possibly accentuated by sem processing) around the perimeter of the papilla (arrowheads). bar = 50 µm c a convexly raised papilla with a central depression (asterisk) with radiating grooves (black arrowheads). these papillae were observed in the rostro-lateral regions of the palate. light desquamation is also apparent (white arrowheads). the block arrows indicate artefacts (cracking of epithelium) probably caused during sem processing (critical point drying). bar = 100 µm d higher magnification of the typical cell-surface features seen throughout the palate. the arrows indicate the boundaries of a characteristic polygonal-shaped cell. note the complex pattern of microridges on the cell surface. bar = 2 µm 292 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i sations and structures (glands, dentition, the development and structure of the palate and osteology) have been described. although illustrated in a number of papers, the cobbled appearance of the epithelium of the palate and parts of the gingivae have not drawn any comment by the authors. certainly, the small, rigid, conically shaped process (fig. 2) which emerged from a low-profiled ridge above the anterior palatine foramen has not been described. the true structure and function of this process is unknown and was not specifically examined in this study, but may well prove an interesting topic for further investigation. similarly, the description of the clearly defined median ridge, comprising a series of closely positioned cobbles along the midline of the palate, appears also to have drawn no attention from previous authors. the presence of a broad dentary shelf forming the rostral aspect of the mandible (see fig. 1a and 3), and which is richly supplied with sensory structures, is likewise not specifically mentioned in the literature. in his generalised description of the reptilian oral cavity, luppa (1977) noted that within the oral cavity of the reptilia, the epithelium showed considerable regional and specific variation and that within a single species, compound squamous epithelium, ciliated epithelium, goblet cells and simple non-ciliated columnar epithelium may be found. this study revealed that the epithelium of the oral cavity (including the surface of the tongue) varied little in structure except for the lining of the oral aspect of the dorsal and ventral folds of the gular valve. throughout the oral cavity the epithelium was a lightly keratinised stratified squamous epithelium which showed slight localised variation in thickness. a relatively thin stratified squamous epithelium lined all aspects of the palate. however, towards the base of the dorsal fold and on the oral surface of the ventral fold of the gular valve, there was a sharp transition from the lightly keratinised epithelium to a thick, non-keratinised stratified squamous epithelium with prominent epithelial and connective tissue papillae (personal observation). throughout the palate, the epithelium was supported by a thick layer of irregular dense connective tissue at the base of which, adjacent to the periostium of the palatal bones, were well-developed plexuses of blood vessels, lymphatic vessels and nerves. adjacent to the basement membrane was a layer of melanocytes. variation in density was apparent amongst specimens examined and in some cases no melanin appeared to be present. this region is similarly described by taguchi (1920) who also mentions a scattered presence of “pigmented cells”, presumably melanin containing cells. the general composition of the epithelium of the gingivae appeared very similar to that of the palate, although the gingivae had a more undulating surface. the epithelium itself was slightly thinner than that of the palate, with the stratum corneum and stratum disjunctum forming the most prominent layers, particularly in the immediate vicinity of the teeth. 293 j.f. putterill & j.t. soley fig. 6 morphological features of the gingivae a light micrograph of a raised sensory unit commonly found in the rostral region of the mandible showing a pale connective tissue mass (outlined area) below the epithelium. two pacinian-like corpuscles (arrowheads) are closely associated with the base of the modified region of connective tissue. h&e stain. bar = 500 µm b light micrograph of paired structures (arrowheads) resembling taste buds within a flattened epithelial specialisation. this type of specialisation was generally found in the rostral and mid-lateral gingivae of the mandible. note the pale connective tissue mass (outlined area) similar to that shown in fig. 6a below the specialisation. a diffuse, basophilic cell-rich mass (asterisk) is observed within the connective tissue. h&e stain. bar = 500 µm c stereomicrograph of epithelial specialisations (arrows) in the rostral region of the gingiva of the mandible. the specialisations were normally situated close to teeth (white arrowhead). fresh specimen. bar = 500 µm d sem photograph of a group of epithelial specialisations, similarly situated to those shown in fig. 6c. each domeshaped papilla (shown in sagittal section in fig. 6a) is in turn surrounded by rosettes of conical epithelial projections. note the flattened circular area (arrowhead) in the centre of the group of papillae, indicating the flattened type of epithelial specialisation seen in sagittal section in fig. 6b. bar = 500 µm e sem photograph of paired pores (arrows) associated with the taste buds contained within flattened epithelial specialisations similar to those shown in fig. 6b and 6d. bar = 10 µm f higher magnification sem photograph of one of the sensory pores seen in fig. 6e showing exposed fimbriae. bar = 5 µm the occurrence of glands and the presence of taste receptors (sensory neuro-epithelial cells, [luppa 1977] or “schmeckzellen” of krause [1922, cited by luppa 1977]) appear to dominate descriptions amongst authors who have examined the histology or morphology of the oral cavity of crocodiles. kochva (1978) extensively describes glandular tissue in reptiles, but only fleetingly refers to the crocodylia (caiman spp., a. mississippiensis and c. niloticus) noting only that “a cursory examination of some slides of crocodylus niloticus reveals no sublingual glands”. in an earlier study woerdeman (1920) observed that the oral cavity of reptiles was highly glandular, but that crocodiles appeared to be an exception and that various authors had reported the absence of glandular tissue. woerdeman (1920) also reviewed earlier literature (ca. 1888 to 1914) on the subject and emphasised discrepancies amongst the authors regarding the presence or absence of oral glands in crocodylia. gaupp (1888, cited by woerdeman 1920) described the presence of small glandulae linguales but concluded that glandulae sublinguales and glandulae palatinae were absent. stannius (no reference, cited by woerdeman 1920) stated that crocodiles did not have any salivary glands. gegenbaur (1901, cited by woerdeman 1920) reported the absence of labial glands in crocodiles. schimkewitsch (1910, cited by woerdeman 1920) however, describes medial and lateral glandular groups in the palate of crocodiles. these glandulae palatinae, according to schimkewitsch (1910), were the equivalent of the intermaxillary glands in amphibians. farenholz (1937) reported two areas in the palate in which glands occur, viz., median palatine glands, found only in caiman and small glands at the median aspect of the maxillary teeth, found in caiman spp. and a. mississippiensis. woerdeman (1920), while investigating tooth development of crocodylus, found that the development of glands, previously described by röse (1893), was closely associated with the development of the dental system. these glands were observed to open into pits situated between the maxillary teeth and into which fit the tips of the mandibular teeth. the glands are medially situated in the pits and are surrounded by soft connective tissue and covered by a stratified squamous epithelium. the region between the maxillary teeth (i.e., the pits into which the tips of the mandibular teeth fit) was not examined during this study and it is thus not possible to comment on the presence or absence of any glandular tissue in this region. however, taguchi (1920) found glandular tissue “in the submucosa of the caudal part of the palate and the oral surface of the velum” in all three species he examined and described the glands as being branched tubulo-alveolar mucous glands. this statement indicates that taguchi found two clear zones of glands, albeit in close proximity to each other. this investigation clearly indicated that there was no glandular tissue in the palate “proper” and that glandular tissue (as described by taguchi 1920) only occurred on the oral surface of the dorsal fold of the gular valve (personal observation). although dentition was only superficially examined in the nile crocodile during this study, it became important when sampling methods of the palate and gingivae of the lower and upper jaws were considered. teeth were therefore named and numbered according to kieser et al. (1993), who concluded that the nile crocodile was heterodont and had five premaxillary incisor, five canine and six or more post canine (molar) teeth in the maxilla. the dental arrangement in the mandible was three premandibular incisor, five canine and six or more post canine (molar) teeth. the teeth of the mandible were accommodated in the paired dentary bones which united at the rostral, elongated dentary symphysis (see iordansky 1973 for osteology of the crocodilian skull). each tooth emerged from its own alveolus in the dentary bone. the caudal region of the splenial bone, situated medially to the dentary bone, was in close association to molar teeth m 4 to m 7 (or m 8), but did not form part of the accommodation of the teeth in the jaw. this is in contrast to the findings of chiasson (1962) who examined the alligator and stated that the dentary bone “bears the first 14–15 teeth in individual alveoli on each side, the remaining 5–6 teeth being held in a common groove between the dentary and splenial bones.” the teeth of the maxilla were similarly accommodated in individual alveoli in the premaxilla and maxillary bones, which also formed the major portion of the palate. chiasson (1962) stated that in the alligator there were “15 to 16 maxillary teeth on each side. the first few of these are held in individual alveolar sockets but the posterior series are set side by side in a common groove.” pressure receptors are noted by pooley & gans (1976) to occur “between the teeth and (in) the jaws” of the nile crocodile and that they function to gauge the intensity of a bite. they do not, however, give any histological description of these receptors, but do state that similar receptors are found in mammals, including humans. this investigation revealed that lamellated, pacinian-like corpuscles were fre294 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i quently observed in the palate and the gingivae (see fig. 4b, c and 6a). these corpuscles were often associated with dome-shaped epithelial specialisations, the latter structures appearing more numerous in the lateral and rostral regions of the palate and in the gingiva covering the rostral dentary shelf (fig. 1 and 3). similar raised structures have been described by von düring (1973, 1974) on the cranial scales of caiman crocodilus. these cranial touch papillae or integumentary touch papillae are also associated with structures resembling mammalian pacinian corpuscles and the illustrations presented in the papers of andres & von düring (1973), von düring (1973) and von düring & miller (1979) reveal lamellated structures similar to those observed in the oral cavity of c. niloticus in the present study. in contrast, jackson, butler & youson (1996) described slightly convex integumentary sense organs (isos), which only occur on postcranial scales of crocodylids and gavialids and which are not associated with pacinian-like corpuscles. despite the absence of the pacinian-type corpuscles, the micrographs (lm and sem) and illustrations presented by jackson et al. (1996) of the isos reveal certain similarities to the dome-shaped structures described in the present study, namely, the domed, low profiled elevation of the epithelial component of both structures and the underlying pale-staining zone of connective tissue (h&estain). jackson et al. (1996) refer to the modified connective tissue component of the isos as a “diffuse pocket in the dermis” and that it contains fluid. they further note that this diffuse pocket contains very few collagen fibres in comparison to the surrounding connective tissue, an observation also made in the oral cavity in this study. based on their structural characteristics, von düring (1973, 1974) described the touch papillae as being mechanosensory in nature. conclusions drawn by jackson et al. (1996) were that the isos identified by them possibly had a mechanosensory or chemosensory function and that further physiological studies would have to be performed to determine their true function. based on these comments it would appear that the dome-shaped structures associated with pacinian-like corpuscles found in the oral cavity of the nile crocodile function as pressure receptors. hulanicka (1913) examined the nerve endings in the tongue, palate and the skin of two species of crocodile, viz., c. niloticus (eight young specimens examined, 25–45 cm in length) and a. mississippiensis (three young specimens examined, 65–110 cm in length) and described five types of nerve endings in the tongue, palate and the abdominal skin. these were free nerve endings (in the palate), touch cells (in the stroma of the tongue, dermis of the skin, stroma of the stomach, chin and jaws), tactile papillae (in the skin and in the mucosa of the tongue and palate), tactile corpuscles (in the stroma of the tongue, the palate and in the dermis of the skin) and taste buds as described by bath (1905, 1906). hulanicka (1913) described two types of tactile papillae in the mucosa of the palate; the first type was small and pointed and covered by a relatively thin epithelial layer, while the second type was much larger, rounded at the tip and covered by a thicker epithelial layer than that of the pointed papillae. this description corresponds broadly with the pointed (conical) processes and the dome-shaped structures respectively, identified in the present study. the tactile corpuscles identified and illustrated by hulanicka (1913), however, bear little resemblance to the pacinian-like corpuscles observed in our specimens. hulanicka (1913) does note that the size and distribution of the sensory structures change with the age of the specimen. to what extent the larger (older) crocodiles examined in this study (compared to the relatively small specimens described by hulanicka) reflect age related differences, could not be determined. although the specific innervation of the oral cavity was not examined during this study, it was found, as described above, that the palate and gingivae were rich in paciniantype corpuscles. it is of interest to note that the diagrammatic representation of sensory nerve endings and the distribution of nerves in tactile papillae in the palate of a. mississippiensis presented by hulanicka (1913), correspond to the general form of the light-staining connective tissue cores underlying the sensory epithelial structures seen in micrographs presented in this study (see fig. 4d and 6a, b). luppa (1977), who generalised his description of the histological composition of the reptilian oral cavity, reported that taste buds were scattered throughout the epithelium of the oral cavity in reptiles and that in lacerta the taste buds were most numerous laterally and on the palatal folds. luppa (1977) further reported that reptilian taste buds showed no significant differences in their cellular composition from those of mammals and that sensory neuro-epithelial cells (= schmeckzellen of krause 1922, cited by luppa 1977) and supportive cells occurred in both mammals and reptilians. bath (1905, 1906) studied the structure as well as the distribution of taste receptors in the nile crocodile and the alligator, and reported taste receptors 295 j.f. putterill & j.t. soley towards the back of the oral cavity and in the pharyngeal cavity and upper region of the oesophagus of c. niloticus. taguchi (1920) identified small numbers of taste buds in the palate of c. niloticus and c. porosus but not in a. sinensis. sensory structures observed during this study in the epithelium of the palate and gingivae, and presumed to be taste buds, were of similar morphology to those described by bath (1905, 1906) and taguchi (1920). hulanicka (1913), however, disputed some of the findings of bath (1905, 1906) regarding the structure of taste receptors in the species he examined, specifically the association between support cells and the nerve fibres innervating the taste bud. in this study the taste buds displayed typical longitudinally oriented supportive and neuro-epithelial cells and were observed to be associated with medullated nerve concentrations situated in the connective tissue at the base of the taste receptors. in addition, this study graphically illustrated by sem the cuticular processes of the neuro-epithelial cells where they emerged through the taste pore (see fig. 6e and f). although they occurred throughout the palate and gingivae, taste receptors were less common than the ubiquitous pressure receptors. however, the presence of both types of sensory receptors in the palate and gingivae points to the important functional role played by both components of the oral cavity in monitoring taste and pressure. acknowledgements the authors are indebted to mr and mrs kuhlmann of izintaba croco farm, de wildt, northwest province, south africa, who supplied the material used in this study. the authors also gratefully acknowledge the skilful assistance of the histology laboratories of the pathology division of the onderstepoort veterinary institute and the pathology section, department of paraclinical sciences, faculty of veterinary science, university of pretoria. the advice and guidance given by dr f.w. huchzermeyer is gratefully acknowledged. references andres, k.h. & von düring, m. 1973. morphology of cutaneous receptors, in handbook of sensory physiology, edited by h. autrum, r. jung, w.r. loewenstein, d.m. mackay & h.l. teuber. berlin: springer verlag. barge, j.a.j. 1937. mundhöhlendach und gaumen, in handbuch der vergleichenden anatomie der wirbeltiere, edited by l. bolk, e. göppert, e. kallius & w. lubosch. berlin: urban & schwarzenberg. bath, w. 1905. über das vorkommen von geschmacksorganen in der mundhöhle von crocodilus niloticus laur. (abstract, zoologischer anzeiger, 29:352–353). bath, w. 1906. die geschmacksorgane der vögel und krokodile. archiv für biontologie gesellschaft naturforschender freunde zu berlin, 1:1–47. chiasson, r.b. 1962. laboratory anatomy of the alligator. dubuque, iowa: wm.c. brown company publishers. edmund, a.g. 1962. sequence and rate of tooth replacement in the crocodilia. contributions. life sciences division, royal ontario museum, toronto, 56:1–42. edmund, a.g. 1969. dentition, in biology of the reptilia, morphology c, edited by c. gans, a.a. bellairs & t.s. parsons. london: academic press. farenholz, c. 1937. drüsen der mundhöhle, in handbuch der vergleichenden anatomie der wirbeltiere, edited by l. bolk, e. göppert, e. kallius & w. lubosch. berlin: urban & schwarzenberg. ferguson, m.w.j. 1979. the american alligator (alligator mississippiensis): a new model for investigating developmental mechanisms in normal and abnormal palate formation. medical hypotheses, 5:1079–1090. http://www.flmnh.ufl.edu/natsci/herpetology/turtcroclist/chklst2.h tm. crocodile specialist group. reference to krocodilus vulgaris cuvier 1807 (=crocodylus niloticus laurenti, 1768). hulanicka, r. 1913. recherches sur les terminaisons nerveuses dans la langue, le palais, et la peau du crocodile. archives de zoologie expérimentale et générale, 53:1–14. iordansky, n.n. 1973. the skull of the crocodilia, in biology of the reptilia, morphology d, edited by c. gans & t. parsons. london: academic press: 4:201–262. jackson, k., butler, d.g. & youson, j.h. 1996. morphology and ultrastructure of possible integumentary sense organs in the estuarine crocodile (crocodylus porosus). journal of morphology, 229:315–324. kieser, j.a., klapsidis, c., law, l. & marion, m. 1993. heterodonty and patterns of tooth replacement in crocodylus niloticus. journal of morphology, 218:195–201. kochva, e. 1978. oral glands of the reptilia, in biology of the reptilia, physiology b, edited by c. gans & t.s. parsons. london: academic press: 8:43–161. luna, l.g. (ed.) 1968. manual of histologic staining methods of the armed forces institute of pathology, 3rd ed. american registry of pathology. new york: mcgraw-hill book company. luppa, h. 1977. histology of the digestive tract, in biology of the reptilia, morphology e, edited by c. gans & t.s. parsons. london: academic press. parsons, t.s. & cameron, j.e. 1977. internal relief of the digestive tract, in biology of the reptilia, morphology e, edited by c. gans & t.s. parsons. london: academic press. pearse, a.g.e. 1985. histochemistry. theoretical and applied. analytical technology, 4th ed. 2, edinburgh: churchill livingston. poole, d.f.g. 1961. notes on tooth replacement in the nile crocodile, crocodylus niloticus. proceedings of the zoological society of london, 136:131–160. pooley, a.c. & gans, c. 1976. the nile crocodile. scientific american, 234:114–124. reese, a.m. 1913. the histology of the enteron of the florida alligator. anatomical record, 7:105–129. 296 oral cavity of nile crocodile, crocodylus niloticus (laurenti, 1768). i röse, c. 1893. über die nasendrüse und die gaumendrüsen von crocodilus porosus. anatomischer anzeiger, 8:745–751. taguchi, h. 1920. beiträge zur kenntnis über die feinere struktur der eingeweideorgane der krokodile. mitteilungen aus der medizinischen fakultät der kaiserlichen universität zu tokyo, 25:119–188 von düring, m. 1973. the ultrastructure of lamellated mechanoreceptors in the skin of reptiles. zeitschrift für anatomie und entwicklungsgeschichte, 143:81–94. von düring, m. 1974. the ultrastructure of cutaneous receptors in the skin of caiman crocodilus. abhandlungen rheinisch-westfaelische akademie der wissenschaften, 53: 123–134. von düring, m. & miller, m.r. 1979. sensory nerve endings of the skin and deeper structures, in biology of the reptilia, neurology a, edited by c. gans, r.g. northcutt & p. ulinski. london: academic press. westergaard, b. & ferguson, m.w.j. 1986. development of dentition in alligator mississippiensis: early development of the lower jaw. journal of zoology, 210:575–597. westergaard, b. & ferguson, m.w.j. 1987. development of dentition in alligator mississippiensis: later development in the lower jaws of hatchlings and young juveniles. journal of zoology, 212:191–222. woerdeman, m.w. 1920. über die gaumendrüsen der krokodile. anatomische anzeiger, 53:345–352. 297 j.f. putterill & j.t. soley article information authors: ivan g. horak1 heike lutermann2 katarina medger2 dmitry a. apanaskevich3 conrad a. matthee4 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa2department of zoology and entomology, university of pretoria, south africa 3united states national tick collection, georgia southern university, usa 4department of botany and zoology, stellenbosch university, south africa correspondence to: ivan horak postal address: private bag x04, onderstepoort 0110, south africa dates: received: 18 jan. 2012 accepted: 23 jan. 2012 published: 02 feb. 2012 how to cite this article: horak i.g., lutermann, h., medger, k., apanaskevich, d.a. & matthee, c.a., 2012, ‘natural hosts of the larvae of nuttalliella sp. (n. namaqua?) (acari: nuttalliellidae)’, onderstepoort journal of veterinary research 79(1), art. #405, 2 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.405 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. natural hosts of the larvae of nuttalliella sp. (n. namaqua?) (acari: nuttalliellidae) in this research communication... open access • abstract • communication • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the first collection of unengorged and fully engorged larvae of nuttalliella sp. (n. namaqua?) from the murid rodents micaelamys namaquensis, aethomys chrysophilus and acomys spinosissimus in limpopo province and from m. namaquensis in the northern cape province, south africa, is documented. a total of nine larvae were collected from two m. namaquensis in the soutpansberg mountain range in the limpopo province during april 2009. during the last week of september 2011, 221 larvae were collected from rodents at the same locality and 10 of 48 m. namaquensis, 6 of 12 ae. chrysophilus and 3 of 14 ac. spinosissimus were infested. one of the m. namaquensis harboured 53 larvae. five larvae were collected from two m. namaquensis in the northern cape province. total genomic dna was extracted from two larvae and a region of the 18s rrna gene was sequenced for these. blastn searches revealed similarity between these specimens and the nuttalliella sequences published on genbank. communication top ↑ the collection of larvae, nymphs and females of nuttalliella namaqua during 2011, after many years in which none had been detected, has revived interest in the morphology, hosts and biology of this unusual tick (mans et al. 2011). nymphs and females from this collection have successfully been fed on laboratory-infested lizards (mans et al. 2011).various papers on the role of murid rodents as hosts of immature ixodid ticks in south africa have recently been published (matthee et al. 2007; matthee et al. 2010; petney et al. 2004). the present communication records their role as hosts of the larvae of nuttalliella sp. (n. namaqua?). two studies were conducted in goro game reserve (22°58’s, 29°25’e) located in the soutpansberg mountain range, limpopo province, south africa. both studies focused on various aspects of the biology of rock elephant shrews (elephantulus myurus), and included collecting ticks from these small mammals. animals were trapped with baited sherman live traps set overnight on rocky outcrops. in addition to the elephant shrews, a number of murid rodents were trapped and also examined for ticks. animals were collected during april and may 2009, and during the last week of september 2011. the animals were collected under permit number cpm-333-00002 issued by the department of environmental affairs, limpopo province, south africa, and all procedures were approved by the animal ethics committee of the university of pretoria (ec028-07, v052-11). a third study was conducted during 2009 on the farm plaatfontein (31°02’s, 23°46’e) in the eastern region of the northern cape province. it focused on collecting biological data and also ticks from southern african hedgehogs, (atelerix frontalis) (horak et al. 2011), under permit flora 036/2009 issued by the northern cape department of tourism, environment and conservation. however, a number of murid rodents were also trapped and examined for ticks. after removal from the traps the body of each animal was searched for ticks, and particular attention was given to the ear margins, legs and the base of the tail, where ticks aggregated. the rest of the body was searched by back-combing the fur. all ticks were removed using fine-tipped forceps and placed in 70% ethanol, after which the host animals were released. in the first study, five nuttalliella sp. larvae were collected during april 2009 from a namaqua rock mouse (micaelamys namaquensis) and four from another. two of the nine larvae were engorged. these are the first recorded collections of the larvae of this tick from host animals. not one of the 58 rock elephant shrews, or remaining 141 rock mice or 8 spiny mice (acomys spinosissimus) examined at the same time was infested. in the second study, 221 unengorged and engorged nuttalliella sp. larvae were collected. of these, 154 larvae were recovered from 10 of 48 m. namaquensis examined, 58 from 6 of 12 red veld rats (aethomys chrysophilus), and 9 larvae from 3 of 14 ac. spinosissimus (table 1). one of the m. namaquensis harboured a total of 53 larvae, whilst the comparatively large number of larvae collected from the 6 ae. chrysophilus and the fact that 50% of the 12 examined were infested could imply that they are preferred hosts in limpopo province. alternatively, but not mutually exclusive, this may be attributed to the greater body size of the ae. chrysophilus (72.7 g ± 3.3, n = 12) compared to the m. namaquensis (39.0 g ± 0.9, n = 48). none of the 8 rock elephant shrews captured at the same time was infested. in the third study a total of five unengorged larvae were collected from two of an undisclosed number of m. namaquensis in the northern cape province. not one of the 12 hedgehogs examined at the same locality was infested. table 1: larvae of nuttalliella sp. (n. namaqua?) collected from small mammals in limpopo province, south africa. because the larvae of n. namaqua have not previously been collected from natural hosts, nor been described, we relied on morphological features resembling those described for the females to arrive at a diagnosis. as an adjunct to identification of the larvae on taxonomic features, total genomic dna from two larvae collected from m. namaquensis was extracted using the qiagen, dneasy® blood and tissue kit. polymerase chain reaction and sequencing was performed on the 18s rrna gene fragment using conserved universal primers. amplifications were performed following standard pcr protocols and cycle-sequencing reactions were performed using bigdye chemistry. products were analysed on an automated sequencer (abi 3730 xl dna analyzer, applied biosystems). a 466 base-pair region of the 18s rrna gene was obtained for both larvae, and these sequences were aligned to the n. namaqua sequence on genbank (mans et al. 2011; jf751071.1). the region sequenced corresponds to positions 1093–1571 of the published sequence. the sequences generated for the two larvae in our study were identical. blastn searches on genbank revealed 99.58% similarity (476/478) between the newly sampled individuals (genbank access number jq424828) and the 18s rrna nuttalliella sequence published on genbank. further support for the authenticity of our identification of the larvae was obtained by doing a genbank blastn search against the entire database. the next closest alternative match to nuttalliella would be the genus ixodes, but in this instance the level of differentiation is more than 4% (614/640 identities between the larval sequences and that of ixodes persulcatus: ay274888.1). conclusion top ↑ the larvae of a nuttalliellid tick, nuttalliella sp. (n. namaqua?) can now be added to the immature stages of a large variety of ixodid tick species, which by preference feed on murid rodents. acknowledgements top ↑ we thank the management and staff of the goro game reserve for permission to collect animals in the reserve and their continuous support. stacey hallam is thanked for collecting the ticks from mice in the northern cape province. the sequences were generated by lee-gavin williams and primers were provided by anne ropiquet. km acknowledges a doctoral grant from the national research foundation (nrf), hl a research fellowship from the university of pretoria and igh funding from the university of pretoria and the nrf. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions i.g.h. (university of pretoria) identified the ticks with the assistance of d.a.a. (georgia southern university), and also compiled the manuscript. h.l. (university of pretoria) and k.m. (university of pretoria) collected the ticks and did the statistical work, and c.a.m. (stellenbosch university) was responsible for the molecular analysis. references top ↑ horak, i.g., welman, s., hallam, s.l., lutermann, h. & mzilikazi, n., 2011, ‘ticks of four-toed elephant shrews and southern african hedgehogs’, onderstepoort journal of veterinary research 78(1), art. #243, 3 pages. http://dx.doi.org/10.4102/ojvr.v78i1.243 mans, b.j., de klerk, d., pienaar, r. & latif, a.a., 2011, ‘nuttalliella namaqua: a living fossil and closest relative to the ancestral tick lineage: implications for the evolution of blood-feeding in ticks’, plos one 6(8): e23675. http://dx.doi.org/10.1371/journal pone.0023675 matthee, s., horak, i.g., beacournu, j.c., durden, l.a., ueckermann, e.a. & mcgeoch, m.a., 2007, ‘epifaunistic arthropod parasites of the four-striped mouse, rhabdomys pumilio, in the western cape province, south africa’, journal of parasitology 93, 47–59. http://dx.doi.org/10.1645/ge-819r2.1, pmid:17436941 matthee, s., horak, i.g., van der mescht, l., ueckermann, e.a. & radloff, f.g.t., 2010, ‘ectoparasite diversity on rodents at de hoop nature reserve, western cape province’, african zoology 45, 213–224. http://dx.doi.org/10.3377/004.045.0222 petney, t.n., horak, i.g., howell, d.j. & meyer, s., 2004, ‘striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks’, onderstepoort journal of veterinary research 71, 313–318. pmid:15732458 combrink_113-117.qxd introduction the south african bivalent unfrozen redwater vaccine containing babesia bigemina and babesia bovis infected blood was sold until april 1998, when it was finally discontinued and replaced by the two frozen monovalent african (babesia bigemina) and asiatic (babesia bovis) redwater vaccines. diminazene aceturate has been shown to be highly effective against b. bigemina at levels from 0.5 to 3.0 mg/kg (kutler 1981). therefore, a third (1.16 mg/kg) of the prescribed dose of the antibabesial drug diminazene has long been used to block-treat the unfrozen redwater vaccine reactions on day 7 with no known adverse effects to the organisms or to the development of protective immunity in the animal (de waal 1996). the attenuated organisms used in the unfrozen redwater vaccine are susceptible for longer periods to the residual effects of diminazene and imidocarb dipropionate than the virulent field strains (f.t. potgieter & m.p. combrink, unpublished observations 1982). in addition, the inhibitory effect, especially of imidocarb, is more pronounced in reactions following the administra113 onderstepoort journal of veterinary research, 71:113–117 (2004) effect of diminazene block treatment on live redwater vaccine reactions m.p. combrink1 and p.c. troskie parasitology division, onderstepoort veterinary institute private bag x05, onderstepoort, 0110 south africa abstract combrink, m.p. & troskie, p.c. 2004. effect of diminazene block treatment on live redwater vaccine reactions. onderstepoort journal of veterinary research, 71:113–117 one third of the manufacturer’s prescribed dose of diminazene has long been used to block treat the south african unfrozen babesia bigemina and babesia bovis (redwater) vaccine reactions, with no known adverse effects. it is known that the inhibitory effect of antibabesial drugs is more pronounced in animals inoculated with the frozen vaccine than those with the unfrozen vaccine. reports of vaccine failures in some animals in which diminazene was used for block treatment of the reactions following inoculation with frozen south african redwater vaccine led us to reinvestigate the required waiting period before treatment and the reduced dose necessary for successful treatment and development of immunity. results from febrile reactions in cattle following vaccination indicated day 7 as the optimal day for administering block treatment. treatment of b. bigemina vaccine reactions in cattle on day 7 at a level of 0.35 mg/kg (1/10 fraction of the normal dose) diminazene killed all the parasites while b. bovis vaccine parasites survived treatment using diminazene at levels between 0.35 mg/kg and 1.16 mg/kg. however, various other factors, such as the degree of natural resistance of different cattle breeds and individual animals, the accuracy of diminazene content according to the manufacturer’s label claim and the accuracy of the drug dose administered, all influence the successful immunization of animals. consequently block treating of babesia vaccines with diminazene on day 7 after vaccination is not recommended. keywords: babesia bigemina, babesia bovis, babesiosis, diminazene, live redwater vaccine 1 e-mail: mike@moon.ovi.ac.za accepted for publication 10 november 2003—editor tion of the frozen south african b. bovis and b. bigemina vaccines than in those of the unfrozen vaccine (combrink, troskie & de waal 2002). in a recent study on 19 different brands of diminazene obtained from 11 african countries, it was found that approximately one out of every three samples tested fell outside the ± 10 % tolerance limit of the manufacturer’s label claim on the content of diminazene aceturate (tettey, atsriku, chizyuka & slingenberg 2002). based on these findings and reports of vaccine failures in some animals in which diminazene was used for the block treatment of vaccine reactions (combrink, personal observations 2001), it was decided to reinvestigate the duration of the interval between vaccination and treatment as well as the optimum dosage rate of diminazene necessary for successful treatment and development of immunity. materials and methods animals fifty-four fully susceptible 6to 12-month-old intact ayershire and friesian cattle, procured as calves and raised in quarantine stables, were used in a trial to determine the waiting period required before administering diminazene after inoculation with the frozen vaccine. the animals were selected randomly and were divided into two groups (table 1). the efficacy of using reduced dosage rates of diminazene to treat frozen vaccine reactions was determined in eight fully susceptible 20to 30-month-old splenectomized hereford cattle born and bred in the stables of the parasitology division of the onderstepoort veterinary institute. they were divided into four groups of two animals each (table 2). the effect of using reduced dosage levels of diminazene to block-treat frozen vaccine reactions was assessed using twenty-four fully susceptible 12to 22-month-old intact hereford and friesian cattle born and bred in the stables of the parasitology division. the animals were placed into six experimental, two treated and two untreated control groups (table 3). all the animals were housed under tick-free conditions before and for the duration of the experiments. drug administration, vaccination and heterologous challenge accurately determined reduced doses of commercially available diminazene (berenil rtu, intervet sa) were administered to those experimental animals indicated in tables 2 and 3. the frozen babesia vaccines that were used contained the b. bovis south african “s” strain (de vos 1978; callow, mellors & mcgreggor 1979) and the b. bigemina australian “g” strain (dalgleish, callow, mellors & mcgreggor 1981; de vos, combrink & bessenger 1982). the vaccines were thawed by placing them directly from liquid nitrogen storage onto melting ice in which they were kept for 4 h before intramuscular inoculation of 1 ml volumes into those experimental animals indicated in tables 1, 2 and 3. the frozen heterologous blood stabilates that were used to challenge the cattle were the b. bovis south african “f” strain ( de vos 1978) and the b. bigemina south african unmodified “p” strain (de vos et al. 1982). at 120 days post vaccination, these frozen stabilates were thawed directly in a container with water at 37 °c and administered intramuscularly, in quantities that contained 5 x 107 parasites before freezing, into those animals indicated in table 3. monitoring redwater reactions rectal temperatures, haematocrit levels and blood smears were monitored daily (de waal & potgieter 1987). antibodies against babesia were determined in sera collected before and 30 days after vaccination or challenge, using the indirect fluorescent antibody test technique (gray & de vos 1981). evaluation of infectivity and reactions the criteria used to evaluate successful infectivity were either a positive blood smear diagnosis or positive seroconversion following vaccination or challenge. evaluation of the vaccine or challenge reactions was based on a total reaction index score determined during the reaction period for each animal, by adding one point scored for every 1 % parasitaemia, 1 % decline in packed red blood cell volume (de vos 1978) and 1 °c in total temperature rise above the mean pre-inoculation normal temperature of the animal (combrink, de waal & troskie 1997). five points were scored for other clinical signs and ten points for every antibabesial drug treatment, blood transfusion and death. the unpaired t-test was used to determine whether results obtained for the mean total reaction indexes of the vaccinated and challenged groups differed significantly (p < 0.05). 114 diminazene block treatment on live redwater vaccine reactions results and discussion start of vaccine temperature reactions results from febrile responses in 12 of the 28 b. bigemina vaccinated animals of group 1 (table 1), indicated day 7 as the mean starting time of temperature rise > 39.5 °c. as some of the temperature reactions to b. bovis in group 2 animals began as early as day 6 and due to the fact that the two vaccines are genarally inoculated simultaneously, it was decided that the waiting period required before block treating of vaccine reactions should remain at 7 days. none of the animals in the two groups showed any clinical signs of disease. treatment efficacy of reduced diminazene doses results obtained in this study using 0.5 and 0.35 mg/kg doses of diminazene to treat b. bigemina and b. bovis vaccine reactions in the splenectomized cattle proved to be quite effective at parasitaemia levels higher than those which normally prevail in intact animals on day 7 after vaccination. none of the animals in the four groups required any additional treatment (table 2). effect of block treatment on the development of immunity babesia bigemina no parasites could be demonstrated in the blood smears of the experimental animals in groups 1, 2 and 3 after treatment of vaccine reactions on day 7 with 1.16, 0.58 or 0.35 mg/kg doses of diminazene and none of the animals seroconverted (table 3). attempts to detect the organisms by subinoculating 100 ml blood from each individual animal into susceptible splenectomized animals also proved negative. heterologous challenge of experimental animals produced clinical disease requiring treatment in 50 % of the animals. this contributed to significant differences between challenge and vaccination reaction indices, indicating no or inadequate immune response to the killed vaccine parasites. both the vaccinated untreated control animals (group 4) were positive on blood smear examination and serology, and showed no difference between mean challenge and vaccination reaction index results. none of the heterologous strain-infected animals of the unvaccinated treated control group 5 developed clinical signs that required treatment and the resulting mean reaction index also showed no significant differences when compared to those obtained for the vaccinated control and experimental groups. nevertheless, all other results clearly indicate that block treatment with diminazene on day 7 of the b. bigemina vaccine reaction cannot be recommended. babesia bovis parasites could still be demonstrated in the blood smears of all of the experimental animals in groups 115 m.p. combrink & p.c. troskie table 1 start of temperature reactions in cattle vaccinated with the live frozen babesia bigemina and babesia bovis blood vaccines vaccine group no. of no. of animals with no. of animals mean start time of tempno. animals blood smear positive showing temperature erature reactions > 39,5 °c reactions > 39,5 °c (day ± sd) babesia bigemina 1 28 28/28 12/28 7.08 ± 3.26 babesia bovis 2 26 26/26 11/26 10.55 ± 4.87 table 2 efficacy of 0.50 and 0.35 mg/kg diminazene as treatment dose of babesia bigemina and babesia bovis frozen vaccine reactions in splenectomized cattle vaccine group no. of diminazene dosage mean parasitaemia recovered animals not no. animals mg/kg (fraction of at treatment requiring additional normal dose) (% ± sd) treatment babesia bigemina 1 2 0.50 (1/7) 1.6 ± 1.13 2/2 2 2 0.35 (1/10) 0.8 ± 0.14 2/2 babesia bovis 3 2 0.50 (1/7) 3.2 ± 3.11 2/2 4 2 0.35 (1/10) 0.9 ± 0.14 2/2 116 diminazene block treatment on live redwater vaccine reactions t a b l e 3 e ff e ct o f 1 .1 6 , 0 .5 8 a n d 0 .3 5 m g /k g d o se s o f d im in a ze n e a d m in is te re d t o in ta ct c a tt le o n d a y 7 a ft e r va cc in a tio n w ith t h e li ve f ro ze n b ab es ia b ig em in a a n d b ab es ia b ov is v a cci n e s v a cc in e s tr a in h e te ro lo g o u s st ra in p a ra si te g ro u p n o . o f d im in a ze n e a n im a ls d o sa g e m g /k g b lo o d s m e a r b lo o d s m e a r g ro u p r e a ct io n in d e x b lo o d s m e a r g ro u p r e a ct io n in d e x (f ra ct io n o f p o si tiv e o n a n d s e ro lo g y m e a n ± s d * * a n d s e ro lo g y m e a n ± s d * * n o rm a l d o se ) d a y 7 p o si tiv e p o st p o si tiv e d a y 7 b ab es ia 1 2 1 .1 6 ( 1 /3 ) 1 /2 0 /2 * 1 8 .7 5 ± 2 .6 2 a b 2 /2 # 1 6 2 .2 5 ± 4 8 .5 9 a cd bi ge m in a 2 2 0 .5 8 ( 1 /6 ) 0 /2 0 /2 * 2 0 .4 5 ± 1 0 .2 5 a b 2 /2 # 2 1 1 8 .3 0 ± 2 7 .7 2 c e 3 6 0 .3 5 ( 1 /1 0 ) 2 /6 0 /6 * 1 1 .7 3 ± 7 .0 5 b 6 /6 # 2 4 2 .0 7 ± 1 0 .6 8 d f 4 2 u n tr e a te d 1 /2 2 /2 2 1 .6 0 ± 2 .2 6 a b 2 /2 1 3 .4 2 ± 5 .6 9 a b 5 2 1 .1 6 ( 1 /3 ) u n va cc in a te d – – 2 /2 3 3 .7 0 ± 1 3 .0 1 a cd e f b ab es ia 6 2 1 .1 6 ( 1 /3 ) 0 /2 2 /2 2 2 .5 0 ± 4 .7 4 a 2 /2 2 5 .3 8 ± 3 .3 4 a bo vi s 7 2 0 .5 8 ( 1 /6 ) 1 /2 2 /2 2 3 .0 5 ± 6 .1 5 a 2 /2 3 0 .9 0 ± 7 .8 5 a c 8 2 0 .3 5 ( 1 /1 0 ) 2 /2 2 /2 2 4 .0 2 ± 1 5 .6 9 a b 2 /2 5 4 .8 2 ± 2 6 .1 3 a c 9 2 u n tr e a te d 0 /2 2 /2 2 3 .3 5 ± 2 .4 7 a 2 /2 9 .7 9 ± 4 .2 9 a 1 0 2 1 .1 6 ( 1 /3 ) u n va cc in a te d – – 2 /2 4 8 .6 6 ± 1 .7 7 b c * n e g a tiv e f o r su b in o cu la tio n o f b lo o d t o s u sc e p tib le s p le n e ct o m iz e d c a tt le ** m e a n s w ith d iff e re n t su p e rs cr ip ts w ith in a g ro u p a re s ig n ifi ca n tly ( p < 0 .0 5 ) d iff e re n t # n u m b e r o f a n im a ls in g ro u p s h o w in g c lin ic a l d is e a se t h a t re q u ir e d t re a tm e n t 6, 7 and 8 after treatment of vaccine reactions on day 7 with 1.16, 0.58 or 0.35 mg/kg doses of diminazene and all seroconverted (table 3). none of these, nor the animals in control group 9 required any treatment during the heterologous challenge reaction and there were no significant differences to be found between the respective challenge and vaccination reaction index results, indicating adequate protection. however, various factors, such as the degree of natural resistance of different cattle breeds and individual animals to babesia parasites, the infectivity of frozen vaccine being less predictable than that of unfrozen vaccine (due to the death of parasites during freezing and thawing), different procedures of vaccine administration, the accuracy of diminazene content according manufacturer’s label claim and the accuracy of the drug dose administered, all influence the successful immunization of animals. consequently the block treating of b. bovis on day 7 of the vaccine reaction is also not recommended. references callow, l.l., mellors, l.t. & mcgreggor, w. 1979. reduction in virulence of babesia bovis due to rapid passage in splenectomized cattle. international journal for parasitology, 9:333–338. combrink, m.p., de waal, d.t. & troskie, p.c. 1997. evaluation of a 3 ml heartwater (cowdriosis) infective blood vaccine dose. onderstepoort journal of veterinary research, 64:309–311. combrink, m.p., troskie, p.c. & de waal, d.t. 2002. residual effect of antibabesial drugs on the live redwater blood vaccines. annals of the new york acadamy of sciences, 969:169–173. dalgliesh, r.j., callow, l.l., mellors, l.t. & mcgreggor, w. 1981. development of a highly infective babesia bigemina vaccine of reduced virulence. australian veterinary journal, 57:8–11. de vos, a.j. 1978. immunogenicity and pathogenicity of three south african strains of babesia bovis in bos indicus cattle. onderstepoort journal of veterinary research, 45:19–124. de vos, a.j., combrink, m.p. & bessenger, r. 1982. babesia bigemina vaccine: comparison of the efficacy and safety of australian and south african strains under experimental conditions in south africa. onderstepoort journal of veterinary research, 49:155–158. de waal, d.t. & potgieter, f.t. 1987. the transstadial transmission of babesia caballi by rhipicephalus evertsi evertsi. onderstepoort journal of veterinary research, 54: 655–656. de waal, d.t. 1996. vaccination against babesiosis. acta parasitologica turcica, 20 (supplement 1): 487–500. gray, s.j. & de vos, a.j. 1981. studies on a bovine babesia transmitted by hyalomma marginatum rufipes koch, 1844. onderstepoort journal of veterinary research, 48:215–223. kutler, k.l. 1981. chemotherapy of babesioses: a review, in babesioses, edited by m. ristic & j.p. kreier. new york: academic press. tettey, j., atsriku, c., chizyuka, g. & slingenberg, j. 2002. non-conformance of diminazene preparations to manufacturer’s label claims: an extra factor in the development of parasite resistance. icptv newsletter, 5:24–25. 117 m.p. combrink & p.c. troskie kondiah_263-265.qxd psittacine beak and feather disease (pbfd) was first described in the mid-1970s and is readily recognizable in wild and captive psittacine species in australia (raidal, sabine & cross 1993a; raidal, firth & cross 1993b). the causative agent, beak and feather disease virus (bfdv), belongs to the genus circovirus in the family circoviridae and is closely related to porcine circovirus in the same genus and chicken anaemia virus which belongs to the genus gyrovirus within the same family. bfdv is between 14 and 17 nm in diameter, is of icosahedral or spherical symmetry and possesses a circular single-stranded dna genome between 1992 and 2018 nucleotides (ypelaar, bassami, wilcox & raidal 1999; bassami, ypelaar, berryman, wilcox & raidal 2001; ritchie, anderson & lambert 2003). the disease is characterized by progressive symmetric feather dystrophy and loss and the development of beak deformities (ritchie, niagro, latimer, lukert, steffens, rakich & pritchard 1990; raidal et al. 1993a; sanada & sanada 2000). birds eventually die from secondary infections resulting from immunosuppression. 263 onderstepoort journal of veterinary research, 72:263–265 (2005) research communication beak and feather disease virus haemagglutinating activity using erythrocytes from african grey parrots and brown-headed parrots k. kondiah, j. albertyn and r.r. bragg* department of microbial, biochemical and food biotechnology, university of the free state p.o. box 339, bloemfontein, 9300 south africa abstract kondiah, k., albertyn, j. & bragg, r.r. 2005. psittacine beak and feather disease virus haemagglutinating activity using erythrocytes from african grey parrots and brown-headed parrots. onderstepoort journal of veterinary research, 72:263–265 psittacine beak and feather disease (pbfd) is a common viral disease of wild and captive psittacine birds characterized by symmetric feather loss and beak deformities. the causative agent, beak and feather disease virus (bfdv), is a small, circular single-stranded dna virus that belongs to the genus circovirus. bfdv can be detected by pcr or the use of haemagglutination (ha) and haemagglutination inhibition (hi) assays that detect antigen and antibodies respectively. erythrocytes from a limited number of psittacine species of australian origin can be used in these tests. in south africa, the high cost of these birds makes them difficult to obtain for experimental purposes. investigation into the use of erythrocytes from african grey parrots and brown-headed parrots yielded positive results showing the haemagglutinating activity of their erythrocytes with purified bfdv obtained from confirmed clinical cases of the disease. the ha activity was further confirmed by the demonstration of hi using bfdv antiserum from three different african grey parrots previously exposed to the virus and not showing clinical signs of the disease. keywords: beak and feather disease virus, bfdv, haemagglutination, parrots * author to whom correspondence is to be directed. e-mail braggrr.sci@mail.uovs.ac.za accepted for publication 11 april 2005—editor diagnosis of pbfd relies on the detection of nucleic acid using the polymerase chain reaction (pcr) or the detection of antigen by haemagglutination (ha) and antibodies by the haemagglutination inhibition (hi) assay. in south africa, only the pcr test is currently available for the diagnosis of pbfd. studies have shown the ability of bfdv to haemagglutinate erythrocytes from goffin’s cockatoos (cacatua goffini), some guinea pigs and galahs (eolophus roseicapillus) (raidal et al. 1993a). other erythrocytes also reported to have bfdv haemagglutinating activity including those from the domestic goose (anser anser) (sexton, penhale, plant & pass 1994), eastern slender-billed corella (cacatua tenuirostris), some sulphur-crested cockatoos (cacatua galerita), gang gang cockatoo (callocephalon fimbriatum), major mitchell’s cockatoo (cacatua leadbeateri) (soares, guimaraes & durigon 1998), salmon-crested cockatoo (cacatua moluccensis), umbrella cockatoo (cacatua alba) and cockatiel (leptolophus hollandicus) (sanada & sanada 2000). however, it has been suggested that erythrocyte suitability varies amongst species and also amongst individuals (sanada & sanada 2000). most of these birds are difficult to obtain in south africa for experimental purposes because of their high cost, therefore an alternative source of erythrocytes was sought out for use in the ha and hi assays. blood samples for the purpose of this study were obtained from two different african psittacine species, african grey parrots (psittacus erithacus erithacus) and brown-headed parrots (poicephalus cryptoxanthus). blood from three african grey parrots that had continuously tested negative for pbfd by pcr over a two-year period and from five brown-headed parrots that had also tested negative for pbfd by pcr was obtained by puncturing a wing vein and stored at 4 °c in an equal amount of alsever’s solution. blood from the african grey parrots was pooled into a single fraction and that of the brown-headed parrots into a second fraction. the fractions were washed three times in phosphate buffered saline (pbs) and stored as a 25 % erythrocyte stock solution at 4 °c. a working solution of 0.75 % erythrocytes was freshly made before the start of each ha assay. blood was stored for a maximum of 7 days unless haemolysis was observed. purification of bfdv was performed by adapting the method used by raidal et al. (1993b). a 10 % suspension of organs obtained from an african grey parrot, in pbs with added sodium dodecyl sulphate (0.5 %) was homogenized at 4 °c until the organs were well blended (braun hand blender). the homogenate was filtered and centrifuged at 6 500 rpm for 10 min at 4 °c (beckman model j221 centrifuge, ja 20 rotor). sodium chloride and solid polyethylene glycol (peg) were added to the supernatant to a final concentration of 1 m nacl and 10 % peg. the mixture was stirred at 4 °c for an hour and centrifuged at 9 500 rpm for 20 min at 4 °c. the supernatant was discarded and the pellet resuspended in a minimal volume of pbs. cesium chloride (cscl) was added to a concentration of 0.46 g/ml and centrifuged (beckman l8-55 ultracentrifuge, sw 28 rotor; 25 000 rpms, at 4 °c for 24 h) in cscl gradients to recover the purified virus. presence of the virus in the pellet was confirmed by pcr according to the methods described by albertyn, tajbhai & bragg (2004) without any modifications. bfdv dna, which was previously amplified from birds with confirmed cases of pbfd, was used as a positive control in the pcr test. the ha tests were performed in 96 well plates as described by ritchie, niagro, latimer, steffens, pesti & lukert (1991) with a slight modification. serial twofold dilutions of purified bfdv were made in 50 µl of pbs from an original undiluted solution. an equal volume of 0.75 % erythrocytes from each test species was added to the wells and the plate incubated at 25 °c for 45 min. results were visualized and haemagglutination found up to a 1:2 dilution for each test species. to confirm these results, hi assays were performed simultaneously with the ha assays. african grey parrot anti-bfdv serum (original undiluted solution) from three individuals, which had survived infection and had been in constant contact with infected birds for more than 12 months, was serially diluted in 50 µl of pbs. fifty microlitres of undiluted purified virus was added to each well and the plate incubated at 25 °c for 30 min. fifty microlitres of 0.75 % erythrocytes from each test species were added and the plate incubated at 25 °c for a further 45 min. negative controls were also performed and consisted of wells with pbs and erythrocytes only. haemagglutination inhibition was observed by the formation of buttons at the bottom of the wells. haemagglutination was observed in the first two wells, representing a maximum titre of 4 ha units, for each test species in the ha assay and upon the addition of anti-bfdv serum, haemagglutination inhibition was demonstrated. the low concentration of the virus attributed to haemagglutination being 264 psittacine beak and feather disease virus haemagglutinating activity using erythrocytes from parrots observed only up to a one in two dilution but this activity was confirmed by the hi assay using three different anti-bfdv sera. thus, bfdv haemagglutinating activity was found using the erythrocytes of both african grey parrots and brown-headed parrots. in addition, erythrocytes obtained from a single african grey parrot (results not shown) showed similar results for both the ha and hi assays. the establishment that erythrocytes from more readily available species of birds can be used to perform ha and hi assays will greatly assist in the implementation of an alternative diagnostic test to pcr for the detection of pbfd in captive psittacine birds in south africa. it is interesting to note that bfdv is capable of agglutinating erythrocytes collected from australian birds (raidal et al. 1993a; sanada & sanada 2000) and african birds (this study). however, a study by soares et al. (1998) found no haemagglutinating activity of bfdv using erythrocytes from red-shouldered macaws (ara nobilis), orange-winged amazon parrots (amazona amazonica), blue and gold macaws (ara ararauna) and blue-fronted amazon parrots (amazona aestiva) which are all south american birds. african and australian birds are considered to be highly susceptible to bfdv while south american birds are generally regarded as being more resistant to infection by this virus. it is interesting to speculate if the ability of bfdv to agglutinate erythrocytes is related to the pathogenicity of the virus in a particular species of bird. acknowledgements we thank dr freek potgieter and his staff of the animal house of the university of the free state for their assistance with the care of the birds and expertise in the handling of laboratory animals. references albertyn, j., tajbhai, k.m. & bragg, r.r. 2004. psittacine beak and feather disease virus in budgerigars and ringneck parakeets in south africa. onderstepoort journal of veterinary research, 71:29–34. bassami, m.r., ypelaar, i., berryman, d., wilcox, g.e. & raidal, s.r. 2001. genetic diversity of beak and feather disease virus detected in psittacine species in australia. virology, 279:392–400. raidal, s.r., sabine, m. & cross, g.m. 1993a. laboratory diagnosis of psittacine beak and feather disease by haemagglutination and haemagglutination inhibition. australian veterinary journal, 70:133–137. raidal, s.r., firth, g.a. & cross, g.m. 1993b. vaccination and challenge studies with psittacine beak and feather disease virus. australian veterinary journal, 70:437–441. ritchie, b.w., niagro, f.d., latimer, k.s., lukert, p.d., steffens, w.l., rakich, p.m. & pritchard, n. 1990. ultrastructural, protein composition, and antigenic comparison of psittacine beak and feather disease virus purified from four genera of psittacine birds. journal of wildlife diseases, 26:196–203. ritchie, b.w., niagro, f.d., latimer, k.s., steffens, w.l., pesti, d. & lukert, p.d. 1991. hemagglutination by psittacine beak and feather disease virus and use of hemagglutination inhibition for detection of antibodies against the virus. american journal of veterinary research, 52:1810–1815. ritchie, p.a., anderson, i.l. & lambert, d.m. 2003. evidence for specificity of psittacine beak and feather disease viruses among avian hosts. virology, 306:109–115. sanada, n. & sanada, y. 2000. the sensitivities of various erythrocytes in a haemagglutination assay for the detection of psittacine beak and feather disease virus. journal of veterinary medicine b, 47:441–443. sexton, n., penhale, w.j., plant, s.l. & pass, d.a. 1994. use of goose red blood cells for detection of infection with psittacine beak and feather disease virus by haemagglutination and haemagglutination inhibition. australian veterinary journal, 71:345–347. soares, p., guimaraes, m.b. & durigon, e.l. 1998. the haemagglutination spectrum of psittacine beak and feather disease virus in brazilian psittacine birds. http://www.vet.uga.edu/ivcvm/1998/soares/soares.htm. ypelaar, i., bassami, m.r., wilcox, g.e. & raidal, s.r. 1999. a universal polymerase chain reaction for the detection of psittacine beak and feather disease virus. veterinary microbiology, 68:141–148. 265 k. kondiah, j. albertyn & r.r. bragg ahmed_239-243.qxd introduction river nile province in northern sudan covers an area of about 124 000 km2. the province has borders with egypt to the north, the red sea and kassala provinces to the east, khartoum province to the south and the northern province to the west. the numbers of animals in river nile province are modest compared to other regions of the country. sheep and goats constitute the majority of the animal wealth with an estimated 953 000 and 1 149 000 head, respectively (anon. 2002). they are usually raised as small mixed flocks along the nile river and the banks of the seasonal atbara river. river nile province is part of the so-called diseasefree zone of northern sudan, where major contagious diseases such as peste des petits ruminants, sheep pox, and foot-and-mouth disease are rare. the area is used for fattening sheep and as a quarantine of animals for export before they are shipped to neighbouring countries. however, clinical cases of malignant ovine theileriosis are reported throughout the year in this area, but are most prevalent during the hot and dry season between march and may (el ghali & el hussein 1995). very little work has been carried out on the ticks infesting small ruminants in the sudan (osman 1997). we therefore conducted the present study to shed some light on the tick fauna in this part of the country. 239 onderstepoort journal of veterinary research, 72:239–243 (2005) some observations on ticks (acari: ixodidae) infesting sheep in river nile province of northern sudan b.m. ahmed1, a.m. el hussein2* and a.o. el khider1 abstract ahmed, b.m., el hussein, a.m. & el khider, a.o. 2005. some observations on ticks (acari: ixodidae) infesting sheep in river nile province of northern sudan. onderstepoort journal of veterinary research, 72:239–243 five species of ixodid ticks were found in a cross-sectional survey in which 200 sheep were examined for ticks in river nile province, sudan. hyalomma anatolicum anatolicum was the predominant species (73.6 %), whereas ticks belonging to the rhipicephalus sanguineus group (14.7 %), rhipicephalus evertsi evertsi (9.1 %), rhipicephalus simus (2 %) and hyalomma dromedarii (0.5 %) were also found. the mean tick load was 11.2 per animal. in a subsequent longitudinal survey ticks were collected on a monthly basis from eight sentinel sheep that were introduced into the area. it was found that h. a. anatolicum almost disappeared during the hot period between april and august, whereas it’s highest numbers were present in winter between november and february. it is concluded that there is only one generation of h. a. anatolicum per year, which may explain the yearround appearance of clinical cases of malignant ovine theileriosis indicating endemic instability of this disease in river nile province. keywords: northern sudan, sheep, survey, ticks * author to whom correspondence is to be directed. e-mail: abdelhussein@hotmail.com 1 faculty of education, nile valley university, p.o. box 136 atbara, sudan 2 central veterinary research laboratories, p.o. box 8067 alamarat, khartoum, sudan accepted for publication 21 june 2005—editor materials and methods study area river nile province lies within the tropical zone and occupies both desert and semi-desert areas. the most important climatic feature of the province is the occurrence of a long dry season extending for approximately eight months from november to june, while the rainy season is restricted to the months of july to october with rainfall occurring mostly in august. the average annual rainfall ranges between zero in the northern part of the province, to around 100 mm in the southern part. the hot season extends from march to october with may and june usually being the hottest months when daily maximum temperatures can be well above 40 °c. the cold dry season extends from november to february, january being the coldest month. the vegetation of the state is poor and sparse. in the desert zones, it is virtually absent except along the banks of the nile river and water courses where ephemeral herbs and grasses occur after the occasional rainfall. in the basins and along the nile banks acacia spp. are found. in the southern part the vegetation is typical of a semi-desert region, and is dominated by acacia toritilis. other trees are acacia seyal, balanites aegyptica and ziziphus spinachristi. ground shrubs include calotropis procera, caparis decidua, lyptadenia pyrotechnica and cassia spp. the dominant grasses are aristida mutabilis and cympopogon nervatus (anderson 1948, harrison & jackson 1958). cross-sectional survey whole body tick collections were made from 200 randomly selected sheep in 15 different localities in the southern part of river nile province. the ticks were preserved in 70 % ethanol and identified according to the keys provided by hoogstraal (1956). longitudinal survey eight indigenous sheep between 6–12 months old were obtained from a local market and subsequently introduced into el akad village, located at the east bank of the nile river near atbara. the animals were monitored as sentinel animals to study the dynamics of the local tick population. the sheep were kept as one flock under traditional husbandry adjacent to village herds of cattle and flocks of sheep and goats. during daytime they were allowed to mix with village animals while grazing on residual agricultural crops grown along the banks of the nile river. additional feed was provided to the animals upon return to their sheds at sunset. the sheep were completely deticked at monthly intervals for 18 months between october 1996 and march 1998. the ticks were preserved in 70 % ethanol, labelled and identified as above. results cross-sectional survey an average of 11.2 ticks per animal were collected from the 200 sheep. the majority of the ticks (1 506) were adults and 723 were nymphs; only four larvae were collected (table 1). the range and mean tick load per animal of h. a. anatolicum was found to be significantly higher (p < 0.001) than that of all other tick species together. males usually outnumbered females except for r. simus (eight males and 11 females). hyalomma a. anatolicum was found on 59.5 % of the animals examined, whereas r. sanguineus group, r. e. evertsi and r. simus were found on 24 %, 31.5 % and 15.5 % of the animals, respectively. hyalomma dromedarii was found on only two animals (1 %) (table 1). longitudinal survey only h. a. anatolicum and r. sanguineus group ticks were found on sentinel sheep at el akad village. hyalomma a. anatolicum was the predominant species, comprising 92 % of the total number of ticks collected, while r. sanguineus group ticks represented only 8 %. only one female of r. e. evertsi was collected. with the exception of one rhipicephalus spp. nymph, all immature ticks collected (1 963 nymphs and 1 673 larvae) were hyalomma ticks. the seasonal activity of h. a. anatolicum ticks is shown in table 2 and fig. 1. adults of h. a. anatolicum were found to be active throughout the year, but they were most prevalent during the period september to march (late rainy season to early summer). peak infestations occurred during november and a second peak occurred in march. a drastic decrease in tick numbers was observed from may to august (mid-summer to early rainy season). the mean h. a. anatolicum tick load was relatively high (11.6) during winter (november to february) decreasing to 10.6 during summer (march-june) with a further decrease to 7.3 during the rainy season (july to october). a significant difference (p < 0.05) in mean tick load was found only between winter and the rainy season. 240 ticks (acari: ixodidae) infesting sheep in northern sudan 241 b.m. ahmed, a.m. el hussein & a.o. el khider table 1 prevalence of different tick species infesting sheep in river nile province. figures in parenthesis represent numbers of animals infested tick species no. of adults male/female percentage average no. of tick/animal collected of total tick burden range mean ± se h. a. anatolicum 1 108 (119) 766/342 73.57 1–60 9.31 ± 0.97 r. sanguineus group 222 (48) 170/52 14.74 1–22 4.63 ± 0.70 r. evertsi evertsi 137 (63) 97/40 9.10 1–5 2.17 ± 0.19 r. simus 31 (17) 8/11 2.06 1–5 1.82 ± 0.30 h. dromedarii 8 (2) 5/3 0.53 1–7 4.00 ± 3.00 table 2 monthly variations of h. a. anatolicum found on sheep at el-akad area adults immatures (larvae and nymphae) month total mean total mean oct. 1996 31 3.9 58 7.3 nov. 43 5.4 572 71.5 dec. 97 12.1 28 3.5 jan. 91 11.4 16 2 feb. 10 1.3 181 22.6 mar. 107 13.4 4 0.5 apr. 55 6.9 0 0 may 1 0.1 0 0 june 11 1.4 0 0 july 14 1.8 0 0 aug. 7 0.9 0 0 sep. 91 11.4 331 41.3 oct. 121 15.1 570 71.3 nov. 163 20.4 368 46 dec. 125 15.6 135 16.9 jan. 113 14.1 766 95.4 feb. 100 12.5 189 23.6 mar. 1998 251 31.4 112 14 total 1 431 9.9 3 330 23.1 fig. 1 the monthly activity of adults and immature stages of hyalomma a. anatolicum on sheep in el-akad area � � � � �� � � � � � �� � � � � �� � � � � � � � ��� ��� �� �� �� �� � � � � � � � � � � � ���� ���� � ���� ���� ��������� the immature stages of hyalomma spp. (presumably h. a. anatolicum) showed a pronounced activity during the same period as adult h. a. anatolicum (september to march), but they completely disappeared from the animals between april and august (fig. 1). in general, males of h. a. anatolicum outnumbered female ticks throughout the study period (table 1). the mean number of h. a. anatolicum ticks per animal decreased during months when the maximum temperature exceeded 40 °c and also in months when the minimum temperature was below 15 °c (table 2, fig. 1). discussion thirty-five species of ticks are known to infest sheep and goats in the sudan (osman 1997). during the present investigation, five species of ticks were found to infest sheep in river nile province, northern sudan. these were (in order of abundance), h. a. anatolicum, r. sanguineus group, r. e. evertsi, r. simus and h. dromedarii. these species were previously reported on sheep from northern sudan (hoogstraal 1956) and from khartoum province in central sudan (jongejan, zivkovic, pegram, tatchell, fison, latif & paine 1987). in general, the mean number of ticks per sheep (11.2) was comparable to those reported by jongejan et al. (1987) (11.5) and karrar, kaiser & hoogstraal (1963) (13.0), and higher than the 6.7 reported by mohamed (1999). jongejan et al. (1987) indicated that the mean tick load varied between 4.2 in khartoum province and 15.9 in blue nile province, where more favourable conditions for survival and reproduction of ticks exist. it is clear that the ecological conditions of river nile province do not allow for survival and reproduction of several other species that do infest sheep e.g. amblyomma lepidum, amblyomma variegatum, hyalomma spp. and boophilus spp. present in other regions of the country (karrar et al. 1963; osman, el hussein, ahmed & abdulla 1982). male ticks represented the majority for all species collected except for r. simus. this confirms the results of jongejan et al. (1987) who reported that male ticks usually outnumbered females of all species infesting cattle except for boophilus spp. in central and southern sudan. hyalomma a. anatolicum has been described by many workers as a tick adapted to harsh environments (hoogstraal 1956; jongejan et al. 1987). this xerophilic species is the most abundant tick in khartoum province (desert scrub zone in central sudan) (jongejan et al. 1987; mohamed 1999). further south, its distribution was limited to north of latitude 14 °. hyalomma impeltatum, on the other hand, is the most common tick of sheep in the arid zone of kordofan (western sudan) (osman et al. 1982). in the present study, h. a. anatolicum appeared to be the most successful tick on sheep in this arid zone. this may be explained by the fact that it can easily adapt to man-made microenvironments, which are to be found in the agricultural areas along the nile river banks. this may also explain recent reports of this species in areas further south in blue and white nile provinces where intensive farming using indigenous and crossbred animals is now practiced (jongejan et al. 1987; el imam 2003). jongejan et al. (1987) indicated that different species of the r. sanguineus group may inhabit different ecological zones in the sudan. thus, whereas rhipicephalus camicasi was restricted to northern sudan, rhipicephalus turanicus and rhipicephalus guilhoni occurred in southern sudan, and rhipicephalus bergeoni occurred only in eastern sudan adjacent to the sub-highland ecological zone of ethiopia (jongejan et al. 1987). rhipicephalus sanguineus group was found primarily on sheep in eastern sudan (karrar et al. 1963) and in the semiarid and savannah zones in kordofan in western sudan (osman et al. 1982). rhipicephalus sanguineus group ticks collected in river nile province may represent different species (most probably r. camicasi) with different ecological and biological properties. rhipicephalus simus (or more correctly, the r. simus group [horak & walker 2003]) only occasionally infests sheep in sudan (karrar et al. 1963; osman et al. 1982) whereas r. e. evertsi is more common on this host (karrar et al. 1963). both species were found on sheep in the present study area but in low numbers. hyalomma dromedarii was collected only on two animals in the present investigation. since camels are rare in agricultural areas along the nile river in this province this may contribute to the rarity of this species on sheep. however, similar findings were reported by karrar et al. (1963) in eastern sudan, where camels are abundant indicating that sheep are not a preferred host for this species. in this study, the adults of h. a. anatolicum were active throughout the year. however, the results indicate that there are some critical months during which development ceased or was very low. the adults were abundant on sheep during the period 242 ticks (acari: ixodidae) infesting sheep in northern sudan between september and march (late rainy season to winter) when the average monthly temperature ranged between 21 °c and 36 °c. on the other hand, the number of adults ticks was very low during the hot months (april to august) when the maximum temperature exceeded 40 °c and the average monthly temperature ranged between 31.5 °c and 36.5 °c. this high temperature in combination with low relative humidity generally below 50 % may cause mortality or suppress tick activity. this is especially true for the immature ticks that completely disappeared during the same period, put resumed activity when temperatures decreased (september to march). similar results were reported by mohamed (1999) in khartoum province in the semi-arid zone. hyalomma a. anatolicum adults and immatures were collected on sheep throughout the year except during december and january (winter) when adult ticks disappeared, and during january when no immatures were found (mohamed 1999). it appears from the present study that there are two peaks of tick activity, one in january and one in march. however, during the second peak that occurred in march no immatures were found as a result of the high environmental temperature during this time of the year. el ghali & el hussein (1995) reported the year-round occurrence of clinical cases of malignant ovine theileriosis with outbreaks occurring annually (ahmed, el hussein, el ghali & salih 2003), indicating endemic instability of the disease in river nile province. the generally low adult h. a. anatolicum burden and the great fluctuation in their numbers reported here, in addition to their low infection rate (6.2 %) with the parasite theileria lestoquardi (ahmed et al. 2003) and the relative inefficiency of immature stages (nymphs) in transmitting the infection (hooshmandrad & hawa 1973) may not ensure continuous and high transmission rates that are required to create endemic stability in this area. hence, susceptible populations of sheep are always available for the disease to appear. discussion on the seasonal dynamics of r. sanguineus group is impossible because the numbers encountered during the present study were too low. however, in this study, few specimens or none were reported during most of the year except for the marked increase during march (1997). osman 1978, osman et al. (1982), jongejan et al. (1987) and mohamed (1999) reported a marked increase in the population of this tick during and after the rainy season (june to october) in darfur, kordofan, blue and white nile, and khartoum states in the sudan. acknowledgements the authors express their gratitude to prof. frans jongejan, utrecht university for valuable comments on the manuscript. this paper is published by the kind permission of the director of animal resources research cooperation, sudan. references ahemd, b.m., el hussein, a.m., el ghali, a. & salih, d.a. 2003. some studies on the epidemiology of malignant ovine theileriosis in river nile state, northern sudan. journal of animal and veterinary advances, 2:681–685. anderson, f.w. 1948. the vegetation of the sudan, in agriculture in the sudan. oxford: oxford university press. anonymous. 2002. annual report. department of planning and economics. federal ministry of animal resources and fishers, khartoum sudan. el ghali, a. & el hussein, a.m. 1995. diseases of livestock in eddamer province: a two year retrospective study. sudan journal of veterinary science and animal husbandry, 34: 37–45. el imam, a.h. 2003. ecological studies on ticks (acari: ixodidae) infesting cattle in kosti province, sudan. sudan journal of veterinary science and animal husbandry, 42:62–71. harrison, m.n. & jackson, j.k. 1958. ecological classification of the vegetation of the sudan. ministry of agriculture, forests bulletin sudan, 2. hoogstraal, h. 1956. african ixodoidea i. ticks of the sudan (with special reference to equatorial province and with preliminary review of genera boophilus, margaropus and hyalomma) washington dc: us navy. hooshmand-rad, p. & hawa, n.j. 1973. transmission of theileria hirci in sheep by hyalomma anatolicum anatolicum. bulletin of animal health and production, 5:103–109. horak, i.g. & walker, j.b. 2003. the taxonomy, distribution and biology of rhipicephalus mushsame, r. praetextatus, r. simus and r. gertrudae. sudan journal of veterinary science and animal husbandry, 42:100–104. jongejan, f., zivkovic, d., pegram, r.g., tatchell, r.j., fison t., latif, a. & paine, g. 1987. ticks (acari: ixodidae) of the blue and white nile ecosystems in the sudan with particular reference to the rhipicephalus sanguineus group. experimental and applied acarology, 3:331– 346. karrar, g., kaiser, m.n. & hoogstraal, h. 1963. ecology and host-relationships of ticks (ixodoidea) infesting domestic animals in kassala province, sudan, with special reference to ambylomma lepidum donitz. bulletin of entomological research, 54:509–522. mohamed, i.a. 1999. pathogenicity of theileria lestoquardi in susceptible sheep under laboratory conditions. m.v.sc. thesis, university of khartoum. osman, a.m. 1997. ticks infesting sheep and goats in the sudan. parasitologia, 39:139–1482. osman, o.m. 1978. preliminary notes on the distribution of ticks (acarina. ixodidae) in darfour province, sudan. bulletin of animal health and production in africa, 26:329–333. osman, o.m., el hussein, a.m., ahmed, n., & abdulla, h.s. 1982. ecological studies on ticks (acari: ixodidae) of kordofan region, sudan. bulletin of animal health and production, 14:151–154. 243 b.m. ahmed, a.m. el hussein & a.o. el khider schultz_189-201.qxd introduction cardiac glycoside-containing plants have a worldwide distribution, yet poisoning of stock with these plants is of significance only in southern africa, where they collectively cause the most important plant-associated poisoning in the region (kellerman, coetzer & naudé 1988; kellerman, naudé & fourie 1996). south african plants contain two types of cardiac glycosides, viz. cardenolides and bufadienolides. of these, the cardenolide-containing plants such as acokanthera oppositifolia, adenium multiflorum and gomphocarpus fruticosus are of lesser veterinary importance, because they are seldom ingested by stock. the veterinary important cardiac glycoside-containing plants have bufadienolides as their active principles, and poisoning by them may be either acute or chronic. amongst those plants that cause acute poisoning, tulp (moraea pallida, moraea miniata and moraea polystachya), slangkop (drimia spp., formerly urginea) (germishuizen & meyer 2003) and witstorm (thesium lineatum) are the most notable. in both bufadienolide and cardenolide poisoning the respiratory, cardiovascular, gastrointestinal and nervous systems are affected (kellerman et al. 1988, 1996). joubert & schultz (1982a, b and c) demonstrated that activated charcoal is an effective treatment for plant-induced cardiac glycoside poisoning of live189 onderstepoort journal of veterinary research, 72:189–201 (2005) the role of fluorescence polarization immuno-assay in the diagnosis of plant-induced cardiac glycoside poisoning livestock in south africa r.a. schultz1, t.s. kellerman1, 2 and h. van den berg1 abstract schultz, r.a., kellerman, t.s. & van den berg, h. 2005. the role of fluorescence polarization immunoassay in the diagnosis of plant-induced cardiac glycoside poisoning of livestock in south africa. onderstepoort journal of veterinary research, 72:189–201 poisoning with cardiac glycoside-containing plants is collectively the most important plant-associated poisoning of livestock in southern africa. as a diagnosis of this significant poisoning is currently based on circumstantial evidence, a practical chemical procedure indicating the presence of cardiac glycosides in plants and animal specimens would be of considerable benefit. the fluorescence polarization immunoassay (fpia) method, used to determine digoxin plasma levels in humans and dogs, was adapted to estimate cardiac glycoside levels in known cardiac-glycoside-containing plants as well as in the rumen and organs of dosed sheep. positive fpia values were obtained with bufadienolide-containing plants, while negative results were obtained with plants not known to contain cardiac glycosides. the fpia has aided in the diagnosis of cardiac glycoside poisoning in livestock and game in 30 outbreaks examined at the division of toxicology, onderstepoort veterinary institute. each outbreak is briefly described. as a result of this assay, a better understanding of cardiac glycoside poisoning has been reached. keywords: bufadienolide, cardenolide, cardiac glycoside poisoning, ruminants, toxic plants 1 division of toxicology, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 2 department of paraclinical sciences, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa accepted for publication 11 april 2005—editor stock. early diagnosis is most important as animals exhibiting advanced cardiac and respiratory dysfunction are high risk cases and handling while treating them may aggravate their conditions. the diagnosis of cardiac glycoside poisoning is currently based on circumstantial evidence, such as the presence of cardiac glycoside-containing plants that have been ingested, clinical signs and necropsy features consistent with heart failure (kellerman et al. 1988). the diagnoses of field intoxication in the laboratory have been hampered by the diversity of cardiac glycosides and their aglycones in the various plants. several attempts have accordingly been made to develop a functional direct or indirect chemical method for demonstrating cardiac glycosides in plants and specimens of animal tissues, i.e.: • the rubidium test was time-consuming and not suitable for routine use (bourdon & mercier 1969). • thin layer chromatography (tlc) (mcvann, havlik, joubert & monteagudo 1992), high performance liquid chromatography (hplc) and qualitative nuclear magnetic resonance (nmr) studies (h.d. brandt, medical university of southern africa, personal communication 1993), in which in all cases the lack of standards hampered the interpretation of the results. • competitive radioimmunoassay (ria) with antibodies of broad specificity to cardiac glycosides to screen plants and animal specimens for the presence of immunoreactive cardiac glycosides (radford, gillies, hinds & duffy 1986). • cheung, hinds & duffy (1989) observed a good correlation between ria and that of the abbott tdx analyzer, used with digoxin ii reagents, for cardiac glycoside-containing tissue samples. this fluorescence polarization immunoassay (fpia) is an existing medical technique for demonstrating cardenolides in human serum based on antigen/antibody reaction and competitive binding to the commercially available fluorophore for digoxin. some of the phytogenous bufadienolides and cardenolides in southern africa were investigated in our laboratory by modified methods to extract (bourdon & mercier 1969) and test for cross immunity with fpia against the commercially available fluorophore for digoxin (cheung et al. 1989). the aim of the study was to evaluate the role of fpia in the diagnosis of plant-induced cardiac glycoside poisoning of livestock in south africa. materials and methods experimental cases plants fresh or shade-dried leaves, stems and bulbs of various plants were milled or blended for fpia (tables 1–4) or for dosing to sheep (tables 6–8). some of the plants were associated with poisonings while others were collected for determination of normal fpia values. animals thirty-one milk-tooth to full-mouth merino and dorper sheep of various sexes and body mass varying between 19 and 82 kg, were dosed with the dried/ fresh plant material (tables 6–8). background levels were estimated in non-intoxicated animals (table 5). extractions two grams each of plant material, rumenal/stomach contents, minced liver and kidney were homogenized (sorvall omni-mixer, stainless steel chamber) with 40 ml water. these samples, or 40 ml aliquots of clear rumenal fluid, were acidified with 10 drops of concentrated hydrochloric acid. after addition of 80 ml dichloromethane (merck, ar), samples were shaken for 30 min and centrifuged at 3 000 rpm for 30 min in a 250 ml screw-top polypropylene bottle. the supernatant water and emulsion phases were discarded. to the organic phase was added 40 ml water and 10 drops of concentrated ammonia and the resultant mixture was shaken and centrifuged as before. the supernatant fluid was discarded. the cleaned-up organic phase was then filtered (whatman no. 1) through sodium sulphate (merck, ar anhydrous) and the solvent evaporated on a heidolph rotovap at 50 °c. the deposit was dissolved in 2 ml methanol at room temperature with the aid of an ultrasonic water bath (elma transonic 420) and passed through a 0.45 µm polypropylene filter. a 100 µl aliquot of the filtrate was mixed with 1 ml serum of a healthy sheep. a control serum sample was also submitted. the presence of cardiac glycosides in the serum was demonstrated by fpia using an abbott tdx analyzer (abbott laboratories, north chicago, usa) and a digoxin ii kit (abbott laboratories diagnostic division). the cardenolide or bufadienolide equivalents of digoxin are expressed in nmol/l of serum. 190 fluorescence polarization immuno-assay in diagnosis of cardiac glycoside poisoning livestock in south africa field cases the same procedures for extraction and fpia, as for experimentally-induced cardiac glycoside poisonings, were followed. results experimental cases normal values in plants bufadienolide-containing plants the results of the fpia values in plant leaves, stems and bulbs are summarized in table 1. high values of bufadienolide were recorded in two species of the genus moraea: that for dry leaves of m. pallida being 22 230 (n = 7) and for fresh leaves of moraea marlothii 12 440 (n = 1). much lower values were recorded in other members of this genus, i.e. moraea simulans, m. polystachya, moraea tripetala and moraea stricta. low levels of bufadienolides were detected in the fresh leaves of drimia spp. which are collectively known as “slangkop”, while those of the fresh bulbs were higher. with the notable exceptions of kalanchoe lanceolata, tylecodon reticulatus and dried cotyledon orbiculata the values for members of the crassulaceae 191 r.a. schultz, t.s. kellerman & h. van den berg table 1 fpia values in bufadienolide-containing plants fpia (nmol/l) plant leaves or other plant parts as indicated value/range mean no. iridaceae moraea marlothii 12 440 1 moraea pallida 1 976–47 000 (dried) 22 230 7 moraea polystachya 56–208 (dried) 105 8 moraea simulans 72 (fresh) 1 moraea stricta 796 1 moraea tripetala 300 (dried) 1 hyacinthaceae drimia altissima 10–12 11 2 drimia altissima bulb 60 1 drimia delagoensis 104–106 105 2 drimia sanguinea 28 1 drimia sanguinea bulbs 72–5 460 777 12 crassulaceae bryophyllum delagoense 8 (fresh) 1 bryophyllum delagoense stems 16 1 cotyledon orbiculata 11–110 (fresh) 52 9 cotyledon orbiculata 2 100 (dried) 1 kalanchoe lanceolata 592–802 697 2 kalanchoe rotundifolia 3–436 (fresh) 156 3 kalanchoe thyrsiflora 96–168 (fresh) 142 3 kalanchoe crenata 80–96 (fresh) 88 2 tylecodon paniculatus 90 (fresh) 1 tylecodon reticulatus 22 800 (fresh) 1 tylecodon ventricosus 2–41 (fresh) 17 7 tylecodon ventricosus stems 50 1 tylecodon wallichii 1–57 (fresh) 38 4 tylecodon wallichii stems 76 2 santalaceae thesium lineatum 4–102 (dried) 37 3 thesium triflorum 3 1 192 fluorescence polarization immuno-assay in diagnosis of cardiac glycoside poisoning livestock in south africa table 2 fpia values in cardenolide-containing plants fpia (nmol/l) plant leaves (fresh) leaves (dried) value no. value mean no. apocynaceae acokanthera oblongifolia 2 908 1 5 416 1 adenium boehmianum 99 600 1 acokanthera oblongifolia 2 908 1 5 416 1 gomphocarpus fruticosus 3 928 1 5 220–9 280 7 250 2 nerium oleander 7 980 1 4 200–8 630 6 415 2 table 3 fpia values in plants not known to contain cardiac glycosides fpia (nmol/l) plant leaves berries/seeds value/mean no. value no. cucumis myriocarpus 5 1 euphorbia gariepina 0 1 ficus macrophylla 1 1 1.4 1 gnidia burchellii 0 1 ledebouria spp. 0 1 melia azedarach (syringa) 4 1 8 1 merwilla plumbea formerly scilla natalensis 3 2 ornithogalum prasinum 2 1 ornithoglossum viride 4 2 persea americana (avocado pear) 30 1 senecio latifolius 12 1 sophora japonica 1.4 1 0 1 terminalia sericea (silver cluster-leaf) 0.1 3 table 4 fpia values in plants parasitic on cardenolide-containing plants and plants not known to contain cardiac glycosides parasitic plant fpia (nmol/l) host plant fpia (nmol/l) tapinanthus quequensis 8 030 nerium oleander 8 630 (loranthaceae) (apocynaceae) tapinanthus quequensis 0 acacia caffra not done (loranthaceae) (fabaceae) tapinanthus quequensis 0 euclea crispa not done (loranthaceae) (ebenaceae) viscum rotundifolium 1 642 nerium oleander 4 200 (viscaceae) (apocynaceae) viscum rotundifolium 11 ziziphus mucronata 4 (viscaceae) (rhamnaceae) viscum verrucosum 5 acacia karroo not done (viscaceae) (fabaceae) viscum combreticola 8 croton spp. not done (viscaceae) (euphorbiaceae) (collectively known as “plakkies”) were relatively low. little difference was found in the values obtained in the leaves and the stems of tylecodon ventricosus and tylecodon wallichii. cardenolide-containing plants apart from m. pallida, m. marlothii and t. reticulatus (table 1), the values obtained for cardenolide-containing plants, in the one or two specimens of each species examined, seemed generally to be higher (2 908–99 600 nmol/l) than those of the bufadienolide-containing plants (tables 1 and 2). the highest value (99 600 nmol/l, n = 1) was recorded in the fresh leaves of adenium boehmianum (table 2). plants not known to contain cardiac glycosides nil values or extremely low (negligible) levels of “cardiac glycoside” were registered (table 3). parasitic plants parasitic plants collected on nerium oleander had noticeably higher values than either their counterparts or related species growing on plants not known to contain cardiac glycosides (table 4). fpia in experimentally poisoned animals the background fpia levels in the serum and organs of non-intoxicated animals are given in table 5. serum noticeable elevations with high fpia values were recorded in the sera of animals 1–2 h after they were dosed with m. pallida and drimia sanguinea. similar high values were not registered in the sera of sheep to which m. polystachya had been administered or in those dosed with members of the crassulaceae (tables 6–8). rumenal contents, kidney and liver the highest fpia values were recorded in the rumenal contents followed by the kidneys and liver, of sheep poisoned by m. pallida (table 6). the fpia values in the sheep that received fresh bulbs of d. sanguinea, though still noteworthy, were of a lesser order than those of m. pallida. small to moderate elevations were still discernable in the liver and kidneys 24–72 h after dosing with d. sanguinea (table 7). much lower fpia levels were recorded in rumenal contents and kidneys of the two sheep poisoned with m. polystachya (table 6). comments on experimental cases the findings of this investigation should be interpreted in the light of the small number of plants and animals examined (tables 1–8). the fpia values of tulp were variable with m. pallida and m. marlothii in general having higher values than the other moraea spp. (table 1). this finding is of considerable diagnostic importance as m. pallida (yellow tulp) is the species most often incriminated in poisoning of stock (kellerman et al. 1996). to the best of our knowledge m. marlothii, m. stricta and m. tripetala have not been incriminated in poisoning of animals, although there is circumstantial evidence that m. tripetala might have poisoned a human (naudé, kellerman & schultz, unpublished data 1995). the fpia levels in the fresh leaves of slangkop were of the same order as those of plakkies but considerably lower than that of tulp (table 1). the fact that fresh bulbs of the various drimia spp. have higher values than the leaves does not affect the incidence of poisoning as only the latter and flowering spikes are eaten by stock. note that the fpia of flowering stems of drimia altissima (12 nmol/l)—the part of the plant supposedly eaten most often by stock—did not materially differ from that of the leaves (10 nmol/l) in the one specimen examined. the relatively high fpia values in d. sanguinea may be fortuitous from a diagnostic point of view, as this species is responsible for the most “slangkop” deaths. with the exception of t. reticulatus, the other plakkies (bryophyllum, cotyledon, kalanchoe and tylecodon spp.) tested registered relatively low values. as can be expected, dessication increased fpia values: fresh leaves of c. orbiculata (44 nmol/l) after dessication registered a c. 50-fold increase (2 100 nmol/l). conspicuously higher fpia values were obtained in cardenolide-containing plants than in those containing bufadienolides. levels in parasitic plants growing on a cardenolide-containing plant and other noncardiac glycoside-containing plants were comparable to their host plants and is an important factor in the diagnosis of cardiac glycoside poisoning. in plants not known to contain cardiac glycosides very low fpia levels were recorded, only persia 193 r.a. schultz, t.s. kellerman & h. van den berg table 6 fpia values in sheep dosed with dried tulp fpia (nmol/l) sheep plant activated fate (g/kg) charcoal serum rumen liver kidney treatment contents interim (h) time (h) 0 1 2 4 6 20 44 moraea pallida 1 0.75 7 0.1 0.8 1.1 1.1 discharged 2 0.75 20 0.3 0.6 1.6 1.8 0.7 discharged 3 1 400 9 68 died 5 h 4 1.25 4 0.1 1 1.5 1.8 discharged 5 1.25 5 4.5 4.6 discharged 6 1.25 43a 4 41 died 3 h 58b 7 1.25 10a 30 21 died c. 6 h 64b 8 2 0.5 2.7 2.4 450 16 died 2 h 9 2 0 2.3 3.1 480 40 died 2 h 10 2 0 4.4 1 120 26 died within 2 h 11 5 24 died 2 h moraea polystachya 1 5 0.1 0.6 0.4 0.5 13 6 18 died within 24 h 2 5 0.1 0.3 0.6 0.3 3 4 8 died within 24 h a fluid (rumen liquor) b fibre americana (avocado pear) and senecio latifolius registering above 10 nmol/l (table 3). some species in the families hyacinthaceae and crassulaceae also tend to have low levels but it is possible that false negative results can be recorded. it seems probable, however, that high levels found in plants by this method are always an indication of the presence of cardiac glycosides. the higher levels in the wide range of m. pallida and t. reticulatus samples can be explained as being caused by the effect of diluting the samples. to remain within the range of the tdx analyser, dilution of the plant extracts were necessary but the results were non-linear. dilutions were necessary when analysing extracts of cardiac glycoside-containing plants that had a very good cross reactivity with digoxin, which affected the upper levels of the range in these plants. when comparing high levels this must also be borne in mind. from these limited experimental findings it would seem that the possibility of diagnosing m. pallida poisoning is better than in the case of other tulp or slangkop species (tables 6 and 7). the somewhat low fpia values found in the organs of animals poisoned by d. sanguinea probably reflect the low values in the leaves and/or the long interval before clinical signs set in. similarly, in m. polystachya poi194 fluorescence polarization immuno-assay in diagnosis of cardiac glycoside poisoning livestock in south africa table 5 fpia values in non-intoxicated animals fpia (nmol/l) animal serum stomach/rumenal contents liver kidney mean range no. mean range no. mean range no. mean range no. cattle 1.4 0.4–3 6 4.0 0–7 (30) 11 3.2 0–10 12 sheep 0.2 0–2.4 21 0.03 0–0.2 6 1.1 0–4 6 0.3 0–2 12 dogs 0.5 0–5 10 0 0 10 0.5 0–3 10 ostriches 1 0–2 2 2 0–4 2 0 0 2 table 7 fpia in sheep dosed with fresh slangkop (d. sanguinea) fpia (nmol/l) sheep plant activated fate (g/kg) charcoal serum rumen liver kidney treatment contents interim (h) time (h) 0 1 2 4 24 1 1.5 5 8 12 died 72 h 2 2 30 0.7 1.9 2.6 2.5 1.6 discharged 3 2.5 13.5 0 3 2.2 0.6 discharged 4 2.5 24 0.6 1.8 2.3 2.3 discharged 5 2.5 15 12 euthanased 24 h 6 3 30 2.4 2.1 2.8 2.9 1.3 discharged 7 4 3 3 2.6 1.7 4 8 23 euthanased 48 h 8 5 2.1 3.3 3.4 2.4 3.4 6 7 died within 48 h 9 5 0.2 2.3 3 1.4 1.5 2 6 4 died within 48 h 10 5 0.1 2.8 3.4 2.4 2.4 2 8 16 died within 48 h 11 5 96 31 18 died within 24 h soning, the relatively low levels in the organs reflect those in the leaves. the animal organ of choice for analysis depends on the interval between ingestion of the poisonous plant by the animal and collection of the sample. in acute intoxication a sample of the rumenal contents appears to be the best one to take but if the animals have shown clinical signs for several days, the liver and kidneys are probably preferable (tables 6 and 7). elevated fpia values were recorded in the sera of most animals poisoned by cardiac glycoside-containing plants except the plakkies (tables 6 and 8). most of the determinations were carried out on the sera 1–4 h after dosing, but indications are that these high values can persist for 24 h or longer. 195 r.a. schultz, t.s. kellerman & h. van den berg table 8 fpia values in sheep dosed with plakkies (fresh leaves and stems) fpia (nmol/l) sheep plant activated fate g/kg charcoal serum treatment interim (h) time (h) 0 1 2 3 6 24 48 tylecodon ventricosusa 1 2.5 0 0 0 0 0 died day 3 5 10 2 10 23 0 0.11 0 0 0 krimpsiekte 48 day 6 tylecodon wallichii 1 2 0 0 0 0 0 discharged 2 7.5 48 0 0 0 0 0 0 died day 4 cotyledon orbiculata 1 5 0 0.24 0 0 0 0 discharged 2 5 0 0 0 0 discharged 3 10 0.17 0.13 0.15 0.16 0.13 discharged a botha, kellerman, schultz, erasmus, vleggaar & retief 1998 meaningfully elevated values were registered in the sera both of animals that died and those that survived. owing to lack of numbers, however, the effect of treatment with charcoal could not be determined. field cases (history included) cattle outbreak 1 in july 1993 a recumbent bovine in the vicinity of vrede in the eastern free state province was diagnosed as having been poisoned by tulp. the veterinarian described the clinical signs of the animal, which had been sick for a day, as “typical” for tulp poisoning. despite treatment with activated charcoal, the animal died the same night. a fpia value of 4.3 nmol/l in the rumenal contents, in comparison with values in control animals (0.4–3, table 5), supported the diagnosis. outbreak 2 during august 1993 16 heifers (220–250 kg) near vrede developed signs of tulp poisoning after eating the plant. three of them died before, and seven after, treatment with activated charcoal. according to the veterinarian, the clinical signs varied from slight ataxia to difficulty in rising and the virtual inability to walk. their hearts were arrhythmic and they were constipated. fpia values of 292 nmol/l in the rumenal contents, 37.4 nmol/l in the liver and 6.2 nmol/l in a kidney of one of the affected animals were consistent with tulp poisoning. outbreak 3 in august 1993, again in the vrede district, 13 heifers died overnight. two had showed pareses of the hindquarters. the camp in which they grazed was infested with tulp that had been eaten. fpia values in the rumenal content of 44 nmol/l, in the liver of 40 nmol/l and a kidney of 20 nmol/l attested to the heifers having died of tulp poisoning. outbreak 4 five yearling cattle died and six were sick when a herd of 107 head accidentally grazed a heavily tulp infested camp near bronkhorstspruit in gauteng province during september 1993. a large number of the cattle were said to have developed diarrhoea. fragments of tulp leaves could be identified in the rumenal contents of two of the three carcasses brought to the onderstepoort veterinary institute for post mortem examination. the most notable histopathological lesions were moderate oedema and degeneration of myocardial cells accompanied by diffuse foci of myocardial necrosis. some of the fpia values in the carcasses examined (rumen 265 and 3.7 nmol/l; liver 2 and 10 nmol/l; kidney 8 and 2 nmol/l in two of the animals respectively) suggested that the cattle had died of tulp poisoning. outbreak 5 in october 1993 two cattle died suddenly in a camp containing dichapetalum cymosum (gifblaar) near naboomspruit (mookgopong), limpopo province. no significant lesions could be found on post mortem examination and fragments of d. cymosum were not present in the rumen. however, fpia values of 1.4 nmol/l in the rumen, 6 nmol/l in the liver and 14 nmol/l in the kidneys indicated that the animals had been poisoned by a cardiac glycoside-containing plant. outbreak 6 one cow out of 106 died shortly after being translocated to the ellisras (lephalale) district, limpopo province, in november 1993. the fpia values of the liver (10 nmol/l), kidney (8 nmol/l) and rumenal contents (0.3 nmol/l) incriminated d. sanguinea poisoning as possible cause of death. outbreak 7 six heifers died and 25 out of a herd of 134 became sick soon after being introduced into a camp at louis trichardt (makhado), limpopo province, during march 1994. the heifers had replaced cows that had grazed the pasture without mishap for 2 months. the affected animals showed typical signs of cardiac glycoside poisoning; namely, weakness of the hindquarters, stilted gait and diarrhoea. two fell down and had convulsions before they died. apart from rumenal stasis, the necropsy findings on one animal were essentially negative. no poisonous plants could be identified in the pasture. when other causes of death, such as poisoning with pesticides and arsenic, were eliminated, fpia was performed on the sera of nine clinically affected heifers. a high value of 38.1 nmol/l in the serum of one justified a putative diagnosis of cardiac glycoside poisoning. the diagnosis was supported by a fpia value of 12 nmol/l in the liver of a heifer that died. outbreak 8 two cattle became sick and three died out of a herd of 37 during june 1994 on veld infested with tulp 196 fluorescence polarization immuno-assay in diagnosis of cardiac glycoside poisoning livestock in south africa near wonderfontein in mpumalanga province. the affected animals were weak, ataxic and had softer faeces than the rest of the herd. although the cause of death in the heifer presented for necropsy was complicated by pasteurella-pneumonia, the fpia values in her organs were elevated, namely, kidney, 20 nmol/l; rumenal contents, 6 nmol/l; and liver, 4 nmol/l. in the light of this a putative diagnosis of tulp poisoning was made. outbreak 9 four cattle died suddenly and ten became sick in a mixed herd comprising 180 head aged between 8 months to 3 years near rehoboth, namibia, in january 1995. the clinical signs were consistent with those of heart failure and the animals had diarrhoea. their pasture was infested with blue tulp (m. polystachya) which had been eaten. the fpia values in one of the animals (rumenal contents, 18 nmol/l; kidney, 14 nmol/l; liver, 3 nmol/l) corroborated the diagnosis of tulp poisoning. outbreak 10 during april 1995, 50 calves aged 6–7 months and 17 heifers out of a mixed herd of 120 cattle near fochville, gauteng province developed severe watery diarrhoea. the worst affected calves (25) and all the heifers were treated with activated charcoal. seven of the treated calves, nevertheless, died. none of the cows was affected. according to the owner, who was unacquainted with tulp, the pasture contained milkweed (gomphocarpus sp.) which had not been grazed. it must be pointed out that, although gomphocarpus spp. contain cardenolides they are unpalatable and seldom eaten. the fpia values in a dead calf (liver, 12 nmol/l; kidney, 7 nmol/l; rumenal contents, 5 nmol/l) supported a putative diagnosis of cardiac glycoside poisoning. outbreak 11 two cattle died suddenly in july 1995 while grazing in a mountain camp infested with t. lineatum near carnarvon, northern cape province. as t. lineatum plant material from the supposedly toxic camp registered fpia values of 102 nmol/l, cardiac glycoside poisoning must be considered as a possible cause of death. outbreak 12 in july 1995, 11 mature cattle in the molteno district, eastern cape province, died acutely after showing signs such as swaying gait, constipation and aggression. the dead cattle were found in a gorge where they had sought shelter from the cold. the ground was covered with snow and the tulp in the camp showed evidence of being eaten. a diagnosis of tulp poisoning was supported by a fpia value of 9 nmol/l in the rumenal contents of one of the dead animals. outbreak 13 late in september 1995, near ventersburg, free state province, three weaned calves aged 9 months died in a camp containing mainly sprouting green grass, but a portion of it had been cultivated and was sparsely covered by sorghum regrowth and young wheat. the affected animals had manifested ataxia, paralysis, watery diarrhoea and dehydration before death. examination of the rumenal contents of the dead calves did not reveal fragments of tulp leaves. mature cows that had grazed in the camp for weeks were not affected. tulp poisoning was suspected, but no tulp plants except those growing under bushes, which were beyond the reach of the calves. a diagnosis of tulp poisoning was confirmed by high fpia values in the rumenal contents (43 nmol/l), although values in the kidney (3 nmol/l) and liver (4 nmol/l) were low. outbreak 14 seven brahman bull calves out of 50 developed signs of cardiac glycoside poisoning near mooinooi, north west province, during october 1995. six of these died. the clinical signs included weakness of the hindquarters and sudden death while being driven. according to the attendant veterinarian necropsy revealed severe heart damage, congestion and oedema of the lungs and hyperaemia of the digestive tract. a tentative diagnosis of cardiac glycoside poisoning or poisoning with gifblaar was made. although the fpia value in the rumenal contents was low (0.4 nmol/l), that of a kidney was sufficiently high (14 nmol/l) to support a diagnosis of cardiac glycoside poisoning. outbreak 15 in november 1995, rumenal contents and kidney from the decomposed carcass of a cow that had died suddenly near vryburg, northern cape province, were submitted for examination. although neither of the plants was in evidence, poisoning with slangkop or blue tulp was suspected. high fpia values of 20 nmol/l in the rumenal contents and 12 nmol/l in a kidney confirmed the suspicion. 197 r.a. schultz, t.s. kellerman & h. van den berg outbreak 16 in early february 1996, a number of mature cattle became sick and 12 out of 250 died over a period of 8 days near potgietersrus (mokopane), limpopo province, after showing clinical signs of colic, a tendency to lie down when driven and paralysis, especially of the hind quarters. the lesions at necropsy were non-specific but included congestion and oedema of the lungs, epicardial haemorrhages and congestion of the gastro-intestinal tract. the animals had recently been introduced into the “toxic” camp, which contained abundant green grass. a fpia value of 13 nmol/l in the liver of one of the affected animals supported a diagnosis of cardiac glycoside poisoning. sheep and goats outbreak 17 during august 1994 a number of ewes grazing on a harvested maize land near vrede, free state province, died acutely without obvious signs of illness. the non-specific necropsy findings included rumenal stasis, reddening of the gut and, in some, evidence of diarrhoea. a high fpia value of 52 nmol/l in the specimen of rumenal contents from one animal submitted supported the diagnosis of tulp poisoning made by the local veterinarian. however, the fpia value in the liver of this animal was zero. outbreak 18 thirty out of 200 goats died after passing through a marshy area infested with tulp near rehoboth, namibia, in august 1994. clinical signs, consistent with tulp poisoning, such as posterior pareses and muscle tremors, were described. the fpia values in the organs of one of the goats (rumenal contents, 14 nmol/l; a kidney, 4 nmol/l; liver, 0 nmol/l) agreed with a diagnosis of tulp poisoning. outbreak 19 one goat out of 300 died and another became sick in late november 1994 near windhoek, namibia. a tentative diagnosis of cardiac glycoside poisoning was made on the strength of the clinical signs of diarrhoea, muscular spasms and bloat. slightly elevated fpia values in a kidney (6 nmol/l) supported the diagnosis, although those in the rumenal contents (0.3 nmol/l) and liver (0 nmol/l) did not. outbreak 20 twenty-five ewes died on tulp-infested veld near trompsburg, free state province in may 1995. apart hyperaemia of the rumenal wall, the necropsies on two ewes did not reveal significant lesions. a moderately high fpia level of 1.1 nmol/l in the rumenal contents was consistent with cardiac glycoside poisoning. outbreak 21 in july 1995 an unspecified number of sheep died near vrede, free state province, while grazing on a harvested maize land infested with tulp. the owner did not know whether tulp or maize (acidosis, diplodiosis) was responsible for the death of his sheep. a fpia value of 4 nmol/l in the rumenal contents supported the diagnosis of tulp poisoning. outbreak 22 twenty-five out of 90 lambs aged 3–6 weeks died suddenly in august 1995 on a pasture heavily infested with tulp near fauresmith, free state province. the veterinarian suspected pulpy kidney disease, but, as their dams when pregnant had been vaccinated, requested that tests for pesticides and cardiac glycosides be done. moderately high fpia values of 1.5 nmol/l in the rumenal contents and 4 nmol/l in the liver implicated cardiac glycosides as the cause of death of the lambs. outbreak 23 necropsies were performed on three of 15 goats that died on kaalplaas, the ovi experimental farm, between march and september 1995. one, a kid aged 5 months, had been born on the farm, while the other two had been introduced from elsewhere about 10 months earlier. the post mortem features of the animals, which had died suddenly without clinical signs having been seen, were consistent with cardiac glycoside poisoning. investigation of the paddocks revealed the presence of two cardiac glycoside-containing plants, milkweed (gomphocarpus fruticosus, fpia 3 928 nmol/l ) and a tulp (m. stricta, fpia 796 nmol/l), but only the milkweed showed signs of having been eaten. elevations of fpia values in two of the animals (rumenal contents, 3–4 nmol/l; liver, 2–8 nmol/l and kidney, 6 nmol/l) supported a diagnosis of cardiac glycoside poisoning. donkeys outbreak 24 during september 1995, three donkeys were found dead near harrismith, free state province. extracardial changes of heart failure such as foam in the 198 fluorescence polarization immuno-assay in diagnosis of cardiac glycoside poisoning livestock in south africa trachea and bronchi and oedema of the lungs were noted on post mortem examination of one of them. moderately elevated fpia values in the stomach contents (4 nmol/l) and liver (3 nmol/l) suggested that it might have died from cardiac glycoside poisoning. horses outbreak 25 in october 1995 two mares near nottingham road, kwazulu-natal province, died of gas colic while several others refused to eat their rations of lucerne (alfalfa) hay. examination of the hay revealed that it was heavily contaminated with an unidentified tulp species. the fpia value of dry tulp leaves from the bales of lucerne hay that were incriminated was 22 040 nmol/l. a putative diagnosis of tulp poisoning was made. outbreak 26 after failing to demonstrate ionophore antibiotics in the feed of horses suffering from colic near windhoek, namibia, a sample of the feed was subjected to fpia. three of the worst affected horses had died within 7 h of developing clinical signs. after a value of 293 nmol/l in the feed was obtained it was examined microscopically, revealing tulp fragments with a fpia value of 2 000 nmol/l. the findings strongly suggested that the colic was the result of cardiac glycoside poisoning. other animals outbreak 27: suni antelope thirteen suni antelope died suddenly over a period in a breeding camp at skukuza, kruger national park, mpumalanga and limpopo provinces, in may 1994. the post mortem features, congestion and oedema of the lungs, presence of ulcers in the abomasum and subserosal haemorrhages in the wall of the rumen, resembled cardiac glycoside poisoning. inspection of the camp revealed large numbers of k. lanceolata (592–802 nmol/l) which had been grazed. the deaths ceased after the antelope were moved from the toxic camp. the fpia values in the frozen organs (liver, 1–1.2 nmol/l; kidney 0–2.4 nmol/l) of two of the sunis supported the diagnosis of k. lanceolata poisoning. outbreak 28: cape fur seal a sub-adult seal died acutely of apparent heart failure shortly after being moved to a new pen in the pretoria zoo during november 1993. the lungs were severely oedematous and the stomach was filled with plant material identified as remnants of ficus macrophylla. fpia values of 18.6 nmol/l in the stomach content and 2.6 nmol/l in the kidney supported a diagnosis of possible cardiac glycoside poisoning. a list of the plants in or about the pen included f. macrophylla (fpia 1–1.4 nmol/l); sophora japonica (0–1.4 nmol/l), of which the seeds might have fallen in the water; brachychiton sp., of which the flowers littered the floor; and duranta repens, of which the fruit might have been eaten. populus spp. and robinia pseudoacacia (black locust) were also present, but out of reach of the seal and was thus uninvolved in the poisoning. although the source could not be determined, a putative diagnosis of cardiac glycoside poisoning was made. outbreak 29: ostriches four incidents of suspected cardiac glycoside poisoning of ostriches were investigated between 1993 and 1995. in the first incident, a necropsy was performed on the last of six ostriches to die out of a flock of ten. the bird succumbed soon after being found with its head on the ground. apart from oedema of the head and upper neck, no significant lesions were found on post mortem examination, but elevated fpia values in the liver (20 nmol/l) and a kidney (8 nmol/l) suggested cardiac glycoside poisoning. however, inspection of the trampled camp in which the birds had been held for about a year failed to reveal cardiac glycoside-containing plants. a putative diagnosis of cardiac glycoside poisoning was nevertheless made. in the second incident, necropsies were performed on three birds that had died out of a flock of 20 breeding pairs. the affected flock had recently been translocated from another farm into a camp, the vegetation of which had been burnt but the grass had begun to sprout. the fpia values in the stomachs of the dead ostriches were 2–38 nmol/l, in the livers 4– 10 nmol/l and the kidneys 0–14 nmol/l. milkweed (gomphocarpus sp.) collected in the camp registered fpia values of 1 608–3 928 nmol/l in newly sprouted leaves, 1 700 nmol/l in dry stems and 1 600 nmol/l in mature seeds. despite the isolation of velogenic newcastle disease (vnd) virus from the dead birds, a diagnosis of milkweed poisoning was made. in the third incident, the fpia value in the liver of the second month-old ostrich chick to die within 2 days on a farm near pretoria were 24 nmol/l. the stomach contents were negative. since no patho199 r.a. schultz, t.s. kellerman & h. van den berg genic organisms could be isolated from the tissues of this bird, a diagnosis of cardiac glycoside poisoning was made. in the fourth incident, three 7-week-old chicks with nervous signs were presented for necropsy. in addition to megabacteriosis and the isolation of vnd virus, the fpia values of the stomach contents were elevated (stomach contents, 2–44 nmol/l; liver 0–4 nmol/l). a diagnosis of vnd complicated by megabacteriosis and suspected cardiac glycoside poisoning was made. outbreak 30: dog a dog was necropsied that had died within 15 min of playing with a toad (m. williams, personal communication 1996). the owner killed the toad, which was subsequently identified as schismaderma carens or red toad by w.d. haacke, transvaal museum of natural history, pretoria. elevated fpia values of the stomach contents of the dog (30 nmol/l) and the dorsal skin glands of the red toad (60 nmol/l) strongly suggested cardiac glycoside poisoning of the dog. it is known that bufonidae secretes a bufadienolide. schismaderma carens is the only schismaderma spp. among the south african bufonidae, the others being 11 bufo spp. (pantanowitz, naudé & leisewitz 1998). discussion the diversity of cardiac glycosides and their aglycones in various plants has hampered the laboratory diagnoses of cardiac glycoside poisoning in livestock. the advantage of fpia is the broad cross immunity of the commercially available digoxin fluorophore to the large variety of cardiac glycosides found in southern african plant species. in order to eliminate certain chemical compounds in the samples that may interfere with the cross immunity, a simple extraction method is used prior to the assay. as a result of this assay, a better understanding of cardiac glycoside poisoning has been reached. cardiac glycoside values (digoxin-equivalent expressed in nmol/l) were estimated in plants known to contain cardenolides or bufadienolides and plants not known to contain cardiac glycosides. interestingly, parasitic plants not known to be poisonous were found to give positive levels when growing on cardenolide-containing plants. tulp (moraea spp.), most often incriminated in acute cardiac glycoside poisoning of animals had the highest fpia levels which is a significant finding, as this increases the usefulness of the assay as a diagnostic tool. in the sheep experimentally poisoned by cardiac glycoside-containing plants, elevated levels were obtained in blood, liver, kidneys and rumenal contents. approximately 30 field cases, mainly involving cattle, were investigated and successfully diagnosed as cardiac glycoside-related by recording fpia levels in the stomach/rumenal contents, liver and/or kidneys. apart from ruminants, intoxication of horses could also be linked to tulp in their feed, and that of ostriches to milkweed. additionally, the method was used to confirm cardiac glycoside intoxication in a dog that had played with a toad. fpia, coupled with circumstantial evidence, allows early diagnosis of cardiac glycoside poisoning, making early treatment possible. it is particularly important to make an early diagnosis as in animals suffering from advanced cardiac and respiratory dysfunction, the stress of dosing activated charcoal to them often leads to fatal heart failure. in spite of the relative limited number of cases examined, the value of fpia in the diagnosis of plantinduced cardiac glycoside poisoning of livestock in southern africa has been established. the assay is now being successfully applied in the routine diagnosis of this form of poisoning in our laboratory. this test would also be useful in the diagnosis of cardiac glycoside poisoning caused by the inadvertent overdosage of traditional herbal medicines containing plants such as d. sanguinea (joubert & mathibe 1989; foukaridis, osuch, mathibe & tsipa 1995). acknowledgements we acknowledge the assistance given by technical staff (division of toxicology, arc-ovi) and prof. l. prozesky and his staff (formerly of the division of pathology, arc-ovi). our gratitude is extended to veterinarians referring cases of suspected cardiac glycoside poisoning to our laboratory. references botha, c.j., kellerman, t.s., schultz, r. anitra, erasmus, g.l., vleggaar, r. & retief, elizabeth 1998. krimpsiekte in a sheep following a single dose of tylecodon ventricosus (burm.f.) toelken and the isolation of tyledoside d from this plant species. onderstepoort journal of veterinary research, 65:17–23. bourdon, r. & mercier, m. 1969. dosage des hétérosides cardiotoniques dans les liquides bioloqiques par spectrophotométrie d’absorption atomique. annales de biologie clinique, 27:651–657. cheung, k., hinds, j.a. & duffy, p. 1989. detection of poisoning by plant-origin cardiac glycoside with the abbott tdx analyzer. clinical chemistry, 35:295–297. 200 fluorescence polarization immuno-assay in diagnosis of cardiac glycoside poisoning livestock in south africa foukaridis, g.n., osuch, e., mathibe, l. & tsipa, p. 1995. the ethnopharmacology and toxicology of urginea sanguinea in the pretoria area. journal of ethnopharmacology, 49:77–79. germishuizen, g. & meyer, n.l. (eds) 2003. plants of southern africa: an annotated checklist. pretoria: national botanical institute (strelitzia 14). joubert, j.p.j. & schultz, r.a. 1982a. the treatment of urginea sanguinea schinz poisoning in sheep with activated charcoal and potassium chloride. journal of the south african veterinary association, 53:25–28. joubert, j.p.j. & schultz, r.a. 1982b. the treatment of moraea polystachya (thunb) ker-gawl (cardiac glycoside) poisoning in sheep and cattle with activated charcoal and potassium chloride. journal of the south african veterinary association, 53:249–253. joubert, j.p.j. & schultz, r.a. 1982c. the minimal effective dose of activated charcoal in the treatment of sheep poisoned with the cardiac glycoside plant moraea polystachya (thunb) ker-gawl. journal of the south african veterinary association, 53:265–266. joubert, p.h. & mathibe, l. 1989. acute poisoning in developing countries. adverse drug reactions and acute poisoning reviews, 8:165–178. kellerman, t.s., coetzer, j.a.w. & naudé, t.w. 1988. plant poisoning and mycotoxicoses of livestock in southern africa. cape town: oxford university press. kellerman, t.s., naudé, t.w. & fourie, n. 1996. the distribution, diagnoses and estimated economic impact of plant poisonings and mycotoxicoses in south africa. onderstepoort journal of veterinary research, 63:65–90. mcvann, a., havlik, i., joubert, p.h. & monteagudo, f.s.e. 1992. cardiac glycoside poisoning involved in deaths from traditional medicines. south african medical journal, 81:139–141. pantanowitz, l., naudé, t.w. & leisewitz, a. 1998. noxious toads and frogs of south africa. south african medical journal, 88:1408–1413. radford, d.j., gillies, a.d., hinds, j.a. & duffy, p. 1986. naturally occurring cardiac glycosides. the medical journal of australia, 144:540–544. 201 r.a. schultz, t.s. kellerman & h. van den berg oyedele_227-230.qxd introduction canine distemper, caused by canine distemper virus (cdv) of the genus morbillivirus, is the most important viral disease of dogs that is characterized by high morbidity and mortality in unvaccinated canine populations worldwide. it is a highly infectious, acute or subacute febrile disease of dogs and other carnivores which has been known since 1760 (fenner, gibbs, murphy, rott, studdert & white 1993). the advent of preventive vaccination programmes has gone a long way to reduce the incidence of the disease in recent years (mccaw, thompson, tate, bonderer & chen 1998). according to horst (1975), the most effective method of controlling canine distemper is mass vaccination of dogs and other carnivores. most commercial canine distemper vaccines are made from the onderstepoort strain of the virus isolated in south africa (yoshida, shin, iwatsuki, gemma, miyashita, tomonaga, hirayama, mikami & kai 1999). however, strains different from the onderstepoort strain have also been isolated (iwatsuki, miyashita, yoshida, gemma, shim & mori 1999; wakasa, iwatsuki, ohashi, nakamura & kai 2000). in nigeria, commercially available cdv vaccines are marketed and administered in combination with hepatitis, leptospirosis, parvovirus and parainfluenza vaccines (dhlp+p combined vaccine). it is recommended that puppies be given a series of vaccinations to stimulate active immunity as maternally derived immunity declines. this should then 227 onderstepoort journal of veterinary research, 71:227–230 (2004) protective levels of canine distemper virus antibody in an urban dog population using plaque reduction neutralization test o.i. oyedele1*, d.o. oluwayelu2, s.i.b. cadmus2, s.o. odemuyiwa1 and f.d. adu1 abstract oyedele, o.i., oluwayelu, d.o., cadmus, s.i.b., odemuyiwa, s.o. & adu, f.d. 2004. protective levels of canine distemper virus antibody in an urban dog population using plaque reduction neutralization test. onderstepoort journal of veterinary research, 71:227–230 blood samples from 50 dogs were collected at three veterinary clinics in ibadan and abuja, nigeria and the serum from each sample was evaluated serologically for neutralizing antibodies against canine distemper virus (cdv) by the highly sensitive plaque reduction (prn) neutralization assay. thirteen dogs had plaque reduction neutralization titres of 0–100, seven had titres of 100–1 000 while 30 had titres ranging from 1 000–6 000. the prn titres of vaccinated dogs were found to be significantly higher than unvaccinated dogs. the widespread use of the highly reproducible prn test for the evaluation of antibody response to cdv may be very important in the generation of international cdv positive serum standards that should help to improve pre-and post-vaccination testing of dogs worldwide. keywords: antibodies, canine distemper, dogs, plaque reduction neutralization * author to whom correspondence is to be directed. present address: c/o dr o.o. oyedele, school of anatomical sciences, faculty of health sciences, university of the witwatersrand, johannesburg, 2001 south africa 1 department of virology, university college hospital, ibadan, nigeria 2 faculty of veterinary medicine, university of ibadan, ibadan, nigeria accepted for publication 8 july 2003—editor be followed by annual revaccination to maintain immunity (greene 1990). however, the usefulness of annual revaccination of vaccinated dogs is still widely debated. smith (1995) has suggested that a more cost-effective and beneficial approach is to first measure serum antibody titres to determine if revaccination is necessary. the availability and widespread use of attenuated virus vaccines appears to have controlled the disease in most countries. in a routine survey of veterinary clinics in nigeria the prevalence of canine distemper was reported to have become infrequent (oyedele, unpublished data 2001). although previous studies have positively correlated cdv-neutralizing antibody levels with immunity in dogs (greene 1990), the reproducibility and predictive values of serum neutralization test and its usefulness in the quantitation of antibody response have been very controversial (soma, ishii, hara, yamamoto, yoshida, kinoshita & nomura 2001). conversely, several studies have shown that the plaque reduction neutralization (prn) test is more sensitive, specific and reproducible for the quantitation of antibody responses to many viral infections, including canine distemper (shope 1990; lee, cohen, hand & nokes 1999; soma et al. 2001). this study was therefore designed to determine the prevalence of neutralizing antibodies to cdv in dogs in the population in order to evaluate the cdv vaccination programme in nigeria and determine risk of infection using the highly sensitive and reproducible prn test. materials and methods sample collection blood samples were collected from 50 dogs at three veterinary clinics, namely the state veterinary clinic in ibadan and two private veterinary clinics in ibadan and abuja which are major urban centres in nigeria. the dogs were bled by standard venipuncture and serum obtained and stored frozen at –20 °c until analyzed. the records of vaccination, if any, were obtained from the owners of the dogs and verified from clinic records. all dogs with unverifiable vaccination records were considered as unvaccinated and were so grouped. canine distemper virus stock canine distemper virus (onderstepoort strain) was obtained from the laboratory of dr c.p. muller of the immunology division, laboratoire national de sante, luxembourg. the virus was seeded at low multiplicity of infection (moi 0.001) on a confluent vero cell monolayer. culture supernatant collected at peak cytopathic effect was centrifuged at 1 200 g for 5 min at 4 °c and filtered through a 0.2 µm syringe filter (millipore, bedford, massachusetts, usa) to remove cellular debris. infective virus titre was determined by plaque assay and virus aliquots were stored at –70 °c until used. plaque reduction neutralization assay the prn assay was performed as has been described by ho & babuik (1979) with some minor modifications. briefly, serum specimens were heatinactivated at 56 °c for 30 min and serially diluted starting at 1:4 in eagle’s minimum essential medium (emem) in 96-well plates (12 µl per well). a standard inoculum of cdv was added to each well [12 µl containing 50–70 plaque-forming units (pfu)] and incubated at 37 °c in air with 5 % co2 for 180 min. the virus-serum mixtures were then added to duplicate vero cell monolayers when approximately 90 % confluent in 24-well plates (100 µl per well) and incubated at 37 °c in 5 % co2 for 60 min. the inocula were then removed and the monolayers were covered with an overlay of 2 % carboxymethyl cellulose in 2x emem (1 ml per well). the plates were incubated for 4 days at 37 °c in air with 5 % co2. the monolayers were stained with neutral red and plaques counted visually on day 5. the prn values were determined using the karber formula (ballew 1986) and the serum titre required to reduce the number of pfu by 90 % was determined. results twenty-three of the 50 dogs that were tested had verifiable vaccination records while 27 of them did not. of the 23 vaccinated dogs, only four (17.4 %) had prn antibody titres of < 1:100, three (13.0 %) had titres less than 1:1 000, and 16 (69.6 %) had prn titres > 1:1 000. of the 27 dogs with no vaccination records, nine (33.3 %) had prn titres < 1:100, four (14.8 %) had titres < 1:1 000, while 14 (51.9 %) had titres > 1:1 000. a one-tailed student t test done on the mean prn titre of the two groups gave a p value < 0.020882. this shows a significant difference in the prn values of the two groups. considering a titre of < 1:100 as the arbitrary cut-off for protection, vaccinated dogs had a much higher protective titre than the 228 canine distemper virus antibody in urban dog population unvaccinated groups. of the 27 unvaccinated dogs, 14 (51.9 %) had prn titres higher than 1:1 000. this is a rather high figure in unvaccinated dogs and is more than likely an indication of previous exposure to a wild type strain. there is a decline in the prn titres of dogs less than 5 months of age and from 9 months, when they are usually given a second dose of vaccine, there is an increase in prn titre. this increase was sustained throughout the age groups from 9 months to 3 years. discussion serological testing for the prevalence of cdv antibodies has not been done on a routine basis in nigeria but the cdv (combined) vaccine has been used for several years. we have shown in this study that 37 (54 %) of 50 dogs tested had antibodies to cdv, using an arbitrary prn titre > 100 as a cut-off for sero-positivity. in addition, using sero-positivity as a measure of protection, we showed that of 23 vaccinated dogs, four had specific cdv prn titres less than the minimum protective level. the report of von messling, harder, moenning, rautenberg, nolte & hass (1999) suggested that a virus-neutralizing titre of 1:100 correlated with protection against cdv infection. on the basis of this suggestion, only four of 23 vaccinated dogs had specific prn titres less than the minimum protective level, indicating that they were not protected following vaccination. suboptimal titres or loss of infectivity of vaccine virus owing to electrical power failures or faults in handling, transportation and storage of vaccines are probable reasons for this observation. it is noteworthy that these four dogs fall within the 3–6 months age range. in the second group of dogs with no vaccination record, ten out of 27 had prn titres < 1:100. this shows a significant difference in the vaccinated group as compared to the unvaccinated group. high prn titres of > 1 000 were recorded in 14 of the 27 unvaccinated dogs. these high antibody titres in unvaccinated dogs are an indication of previous exposure to canine distemper virus and this would most likely be a wild type strain. however, these results are being interpreted with caution since it may be very difficult to reasonably correlate titres obtained using the prn test and virus neutralization with protection from infection following exposure (twark & dodds 2000; palmquist 2000). nonetheless, a widespread use of the prn test in serological surveys of cdv infection will provide more reproducible data that can be used to generate a bank of internationally standardized cdv-neutralizing sera in future. follow-up prospective investigation of cdv infection in the dogs examined in the present report will go a long 229 o.i. oyedele et al. table 1 history and prn antibody titres of vaccinated dogs s/no. sample no. age (in months) sex breed prn titre 1 n188 3 m alsation* 1 573.9 2 n182 4 f alsation 3.0 3 n205 4 m alsation 73.3 4 n247 4 m alsation 0.0 5 n185 5 f alsation 5 066.0 6 n230 5 m alsation 2 339.0 7 cd009 6 m cross 27.3 8 cd007 9 f alsation 5 559.7 9 cd008 9 m alsation 4 377.3 10 n238 10 f alsation 5 559.7 11 cd002 11 f alsation + labrador 5 066.0 12 1016/573 12 m alsation 2 344.5 13 n263 12 f alsation 910.8 14 cd010 12 m alsation 1 723.2 15 527 15 m alsation 4 740.5 16 559 15 f alsation 4 150.8 17 cd012 15 f alsation 774.7 18 old313 21 f alsation 5 202.4 19 l/024 30 f alsation 1 637.9 20 704 32 f alsation 5 066.0 21 old051 34 f terrier 1 374.8 22 cd004 36 f alsation 724.9 23 cd011 36 m alsation 3 492.4 * alsation = german shepherd way to help establish the minimum protective prn test titres that may be important in such international effort. further serological studies are also necessary to fully understand the epizootiology of cdv in nigeria and better evaluate the effectiveness of current vaccination schedules. references ballew, h.c. 1986. neutralization, in clinical virology manual, edited by s. specter & g.j. lancz. new york: elsevier science. fenner, f.j., gibbs, e.p.j., murphy, f.a., rott, r., studdert, m.j., white, d.o. 1993. veterinary virology. 2nd ed. london: academic press. greene, c.e. 1990. immunoprophylaxis and immunotherapy, in infectious diseases of the dog and cat, edited by c.e. greene. philadelphia: w.b. saunders. ho, c.k. & babiuk, l.a. 1979. a new plaque system for canine distemper: characteristics of the green strain of canine distemper virus. canadian journal of microbiology, 25:680–685. horst, j.c. 1975. diseases of dogs. new york: pergamon press. iwatsuki, k., miyashita, n., yoshida, e., gemma, t., shim, y.s. & mori, t. 1999. molecular and phylogenetic analyses of the haemagglutinin (h) proteins of field isolates of canine distemper virus from naturally infected dogs. journal of general virology, 78:373–380. lee, m.s., cohen, b., hand, j. & nokes, d.j. 1999. a simplified and standardized neutralization enzyme immunoassay for the quantification of measles neutralizing antibody. journal of virological methods, 78:209–217. mccaw, d.l., thompson, m., tate, d., bonderer, a. & chen, y. 1998. serum distemper virus and parvovirus antibody titres among dogs brought to a veterinary hospital for revaccination. journal of the american veterinary medical association, 213:72–75. palmquist, r. 2000. opposing views of revaccination strategies. journal of the american veterinary medical association, 217:1789–1791. shope, r.e. 1990. antigen and antibody detection and update on the diagnosis of dengue. southeast asian journal of tropical medicine & public health, 21:642–645. smith, c.a. 1995. current concepts: are we vaccinating too much? journal of the american veterinary medical association, 207: 421–425. soma, t., ishii, h., hara, m., yamamoto, s., yoshida, t., kinoshita, t. & nomura, k. 2001. comparison of immunoperoxidase plaque staining and neutralizing tests for canine distemper virus. veterinary research communications, 25:311–325. twark, l. & dodds, w.j. 2000. clinical use of serum parvovirus and distemper virus antibody titers for determining revaccination strategies in healthy dogs. journal of the american veterinary medical association, 217:1021–1024. von messling, v., harder, t.c., moenning, v., rautenberg, p., nolte, i. & hass, l. 1999. rapid and sensitive detection of immunoglobulin m (igm) and igg antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay. journal of clinical microbiology, 37:1049–1056. wakasa, c., iwatsuki, k., ohashi, k., nakamura, k. & kai, c. 2000. sequence analysis of the genes encoding the phosphoprotein of recent isolates of canine distemper virus in japan. journal of veterinary medical sciences, 62:97– 101. yoshida, e., shin, y.s., iwatsuki, k., gemma, t., miyashita, n., tomonaga, k., hirayama, n., mikami, t. & kai, c. 1999. epitopes and nuclear localization analysis of canine distemper virus nucleocapsid protein by expression of its deletion mutants. veterinary microbiology, 66:313–320. 230 canine distemper virus antibody in urban dog population albertyn_29-34_web.qxd introduction psittacine beak and feather disease (pbfd) is a viral disease that was first discovered in 1975 in cockatoos in australia. it is caused by the psittacine beak and feather disease virus (pbfdv) which is a small (14–18 nm diameter), icosahedral, nonenveloped virus with a circular single-stranded dna (ssdna) genome that has been characterised and placed in the family circoviridae (raidal, firth & cross 1993; studdert 1993; pass, plant & sexton 1994; sexton, penhale, plant & pass 1994; ramis, latimer, gibert & campagnoli 1998; ypelaar, bassami, wilcox & raidal 1999; todd 2000). the disease occurs predominantly in sulphur-crested cockatoos (cacatua galerita) but also in budgerigars (melopsittacus undulatus) and other psittacine birds. characteristic clinical signs include symmetric feather dystrophy and loss and deformity of the beak in some species (ramis et al. 1998). birds infected with pbfdv usually die as a result of secondary infections, which support the view that they are immunosuppressed; bursal and thymic lesions are consistently detected (todd 2000). histopathology has been used for the routine diagnosis of pbfd, but more recently, polymerase chain reaction (pcr) and haemagglutination (ha) tests have been described for the detection of pbfdv and haemagglutination inhibition (hi) for the detection of antibodies to pbfdv (riddoch, raidal, & cross 1996). since all attempts to cultivate pbfdv 29 onderstepoort journal of veterinary research, 71:29–34 (2004) psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in south africa j. albertyn, k.m. tajbhai and r.r. bragg* department of microbial, biochemical and food biotechnology, university of the free state p.o. box 339, bloemfontein, 9300 south africa abstract albertyn, j., tajbhai, k.m. & bragg, r.r. 2003. psittacine beak and feather disease virus in budgerigars and ring-neck parakeets in south africa. onderstepoort journal of veterinary research, 71:29–34 psittacine beak and feather disease (pbfd) is a common disease of the psittacine species and is caused by the psittacine beak and feather disease virus (pbfdv). in this study the occurrence of the disease in ring-neck parakeets and budgerigars in south africa suffering from feathering problems, using polymerase chain reaction as a diagnostic test was investigated. the genetic variation between viral isolates was also studied. results indicate that pbfdv can be attributed to being the cause of feathering problems in some of the ring-neck parakeets and budgerigars in south africa. genetic variation of isolates occurs between species and individuals. a cheap and easy to use method of blood sample collection on filter paper for diagnostic purposes was also evaluated. it proved to be less stressful to the birds and did not inhibit further processes. keywords: budgerigars, genetic variation, psittacine beak and feather disease virus, ring-neck parakeets, south africa * author to whom correspondence is to be directed. e-mail: braggrr@sci.uovs.ac.za accepted for publication 9 september 2003—editor have so far been unsuccessful, the diagnosis of the disease must be confirmed by the demonstration of viral antigen or viral nucleic acid (todd 2000). bassami, ypelaar, berryman, wilcox & raidal (2001) studied the genetic diversity of pbfdv and concluded that there was no evidence that distinctly different genotypes occur although various differences due to small deletions or insertions are present in the genomes of the isolates. they stated that the significance of these differences is unknown but that there is little evidence to support a relationship between genetic variation and regional distribution of isolates, or that there are differences in pathogenicity, antigenicity or any physiochemical characteristics of the virus. there is no known cure for pbfd but a vaccine consisting of inactivated pbfdv in a double-oil emulsion adjuvant system (doe vaccine) has been investigated (raidal et al. 1993; raidal & cross 1994), and its use can be a safe and effective aid for controlling the disease if it is combined with other management procedures, such as good biosecurity. in the disease, avian polyoma, caused by the avian polyomavirus (apv), clinical and pathological signs similar to those of pbfd are manifested. it has been known to occur in concurrent outbreaks with pbfd although documentation of these occurrences is limited (latimer, niagro, campagnoli, ritchie, pesti & steffens 1993; ramis et al. 1998). differentiation between the two diseases is difficult as the macroand microscopical pathology of the two diseases is similar; diagnosis of avian polyoma disease is performed using techniques such as dna in situ hybridization or pcr (ramis et al. 1998). bird breeders in south africa have been reporting feathering problems in budgerigars and have suspected that this may be due to apv infection. however, bragg (2002) reported that pbfdv is widespread in parrots of south africa breeders and suggested that the feathering problems in the budgerigars could be due to pbfdv infection and not apv infection. this study was undertaken to determine if the occurrence of pbfd in budgerigars and ring-neck parakeets in south africa exists using pcr as an identification tool. it was also undertaken to determine if there is any genetic variation between the isolates. a simple method for collecting blood samples on filter paper was also evaluated for its use in downstream processes for example the extraction of viral dna followed by pcr. materials and methods a total of seventeen birds were obtained from breeders in south africa that showed feathering problems indicative of pbfdv infection. blood samples were collected in heparin lithium tubes from six ring-neck parakeets in bloemfontein, five budgerigars in pretoria and five in cradock, south africa (table 1). in addition, the effectiveness of blood sample collection on filter paper was evaluated. a strip of whatman no.1 filter paper of approximately 5 mm in width and 40 mm in length was sterilized by u.v. radiation. a wing vein of one ring-neck parakeet (p-blm-4) was pricked with a sterile 0.33 mm diameter needle and four separate strips of filter paper was used to absorb blood resulting in the coverage of a blood-soaked area of approximately 5 x 5 mm in size at one end of the filter paper. the filter papers were air-dried vertically at room temperature followed by viral dna extraction from the samples after 24, 48, 72 and 96 h. in addition, a blood spot sample from a ring-neck parakeet in george, south africa (p-geo) that was submitted on a commercially available specimen collection paper was similarly processed after one month of storage at –20 °c. viral dna was extracted from the blood samples using the qiaamp® dna blood mini kit and dried blood spot samples were processed using the qiaamp® dna mini kit according to the manufacturer’s instructions (qiagen). the pcr amplifica30 psittacine beak and feather disease virus in south africa table 1 blood samples obtained from species and sources sample species source p-blm-1 parakeet bloemfontein p-blm-2 parakeet bloemfontein p-blm-3 parakeet bloemfontein p-blm-4 parakeet bloemfontein p-blm-5 parakeet bloemfontein p-blm-6 parakeet bloemfontein p-geo parakeet george b-pta-1 budgerigar pretoria b-pta-2 budgerigar pretoria b-pta-3 budgerigar pretoria b-pta-4 budgerigar pretoria b-pta-5 budgerigar pretoria b-cra-1 budgerigar cradock b-cra-2 budgerigar cradock b-cra-3 budgerigar cradock b-cra-4 budgerigar cradock b-cra-5 budgerigar cradock tion was performed using a set of oligonucleotide primers [pb f1 (aaccctacagacggcgag-3’) and pb r1 (5’-gtcacagtcctccttgtacc-3’)] obtained from literature (ypelaar et al. 1999; bassami et al. 2001), amplifying a region of open reading frame 1 with a predicted size of 717 bp. amplification was carried out according to ypelaar et al. (1999) with a slight modification as follows; denaturation was carried out at 94 °c and annealing was performed at 55 °c. a second set of primers [pb f2 (5’-aaccatgccgtccaagga-3’) and pb r2 (5’-tatcagtaattgatggggtggg-3’)] was designed to amplify the complete region of open reading frame 1 of the pbfdv genome (genbank, accession number af080560) resulting in an amplicon with a predicted size of 876 bp. amplified products were identified by electrophoresis on 0.8 % agarose gels and visualized under uv illumination. both primer sets produced amplicons of the expected size and those obtained with the primer set pb f1 and pb r1 were used in subsequent reactions. restriction digests were performed to determine if there was any genetic difference between the isolates by means of digestion with the restriction endonuclease haeiii. the digest products were identified by electrophoresis on 2.5 % low melting point agarose gels and visualized under uv illumination together with a 50 bp step ladder (promega) as a molecular weight marker. due to the possibility of different amplified products from samples b-cra-1, b-cra-2, b-cra-4 and b-cra-5, these amplicons were cloned into pgem™ teasy vector system (promega), small scale plasmid isolation were performed, the inserts removed from two randomly selected clones and digested with haeiii. the products were again visualized by electrophoresis. results all samples obtained from the ring-neck parakeets and budgerigars (except for sample b-cra-3) produced pcr amplicons of the expected size of approximately 700 bp, using primer set pb f1 and pb r1. sample b-cra-3, that consistently produced a negative result, was further used as a negative control the positive samples amplified with primer set pb f2 and pb r2 also produced fragments of the expected size of approximately 850 bp (data not shown). amplicons obtained using primer set pb f1 and pb r1 were submitted to restriction digestion using haeiii. the digest profile was similar for all the ringneck parakeet samples obtained from bloemfontein with approximate sizes of 450 bp, 250 bp and a third smaller fragment of approximately 50 bp (fig. 1, lanes 2–7). the profile for sample p-geo differed in size from the other ring-neck parakeet samples yielding fragments of approximate sizes 460 bp, 250 bp and less than 50 bp (fig. 1, lane 8). samples obtained from the budgerigars in pretoria (b-pta-1, b-pta-2, b-pta-3, and b-pta-5) had similar digest profiles with fragments of approximately 410 bp, 225 bp and 50 bp in size (fig. 2, lanes 2–4, 6), except for an additional fragment observed in sample b-pta-4 with an approximate size of 370 bp (fig. 2, lane 5). 31 j. albertyn, k.m. tajbhai & r.r. bragg 450 bp 250 bp 150 bp 50 bp 1 2 3 4 5 6 7 8 9 fig. 1 restriction digest profile for the samples obtained from ring-neck parakeets using the restriction enzyme haeiii. the digest products were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (promega) [lanes 1 and 9]. samples pblm-1, p-blm-2, p-blm-3, p-blm-4, p-blm-5, p-blm6 and p-geo were loaded in lanes 2–8 respectively. arrows on the right hand side indicate the discrepancy found of the ~50 bp band between the six samples obtained from bloemfontein (upper arrow) in comparison to the sample obtained from george (lower arrow) 32 psittacine beak and feather disease virus in south africa 1 2 3 4 5 6 7 450 bp 250 bp 150 bp 50 bp fig. 2 restriction digest profile for the samples obtained from budgerigars (pretoria) using the restriction enzyme haeiii. the digest products were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (promega) [lanes 1 and 7]. the samples bpta-1, b-pta-2, b-pta-3, bpta-4 and b-pta-5 (lanes 2–6 respectively) gave similar profiles with sample b-pta-4 containing an additional fragment of approximately 370 bp in size. the fragments below the 50 bp marker were light in intensity and therefore more difficult to observe 1 2 3 4 5 6 7 8 9 10 450 bp 250 bp 150 bp 50 bp 1 2 3 4 5 6 450 bp 250 bp 150 bp 50 bp fig. 3 restriction digest profile for the samples obtained from budgerigars (cradock) using the restriction enzyme haeiii showing multiple fragments. the digest products were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (promega) [lanes 1 and 6] as a molecular mass marker. the samples bcra-1, b-cra-2, b-cra-4 and b-cra-5 were loaded in lanes 2–5 respectively fig. 4 restriction digest profile for two randomly picked clones from each sample obtained from budgerigars (cradock) using the restriction enzyme haeiii. the digest products were loaded onto a 2.5 % agarose gel together with a 50 bp step ladder (promega) [lanes 1 and 10] as a molecular mass marker. loading was performed as follows, bcra-1 [lanes 2 and 3], b-cra-2 [lanes 4 and 5] b-cra-4 [lanes 6 and 7] and b-cra-5 [lanes 8 and 9] the digest profile obtained for the samples obtained from budgerigars in cradock continuously resulted in an unusual profile (fig. 3) that led to the possibility that more than one pcr amplicon (i.e. amplicons from genetically different viral isolates) of approximately 700 bp was present. these amplicons were cloned into the vector pgem™ teasy, inserts were purified from two randomly selected clones for each sample, and digested with haeiii (fig. 4). clones from b-cra-1 (fig. 4, lanes 2 and 3) were similar in profile to each other but different from b-cra-2 and b-cra-4 [fig. 4, lanes 4–7 respectively], which in turn shared a similar profile. sample b-cra-5 [fig. 4, lanes 8 and 9] had a different profile for each clone; with one clone (fig. 4, lane 8) sharing a similar profile with samples b-cra-2 and b-cra 4 and the second clone (fig. 4, lane 9) sharing a profile with that of bcra-1 this profile indicates that at least two genetically different pbfdv isolates were present in these birds and, more importantly, that at least one bird contained both of these genetically different isolates. the samples collected on filter paper all produced amplicons of the expected size (approximately 700 bp) and all amplicons, including the blood sample that was processed after one month of storage at –20 °c, showed the same intensity (fig. 5). none of the products showed any non-specific amplification indicating that the filter paper collection methods is an easy and cost effective way to collect and process blood samples. discussion the pcr primer set pb f1 and pb r1 and primer set pb f2 and pb r2 amplified the expected products in all the samples tested except for one budgerigar that tested negative. the pcr amplicons obtained by amplification using primer set pb f2 and pb r2 were not used in further processes although this primer set also resulted in reproducible results. the reason for designing these primers and performing amplifications using them was to attempt to amplify the entire orf1, which will be important for future studies and the potential development of a subunit vaccine. the variation in the digest profiles between species and individuals suggests genetic variation in the sequences of the amplified region but it is still unknown if these variances indicate any differences in antigenicity or pathogenicity. profiles for all the samples obtained from the different regions in the country were not similar to each other even though they were from the same species (budgerigars from pretoria and cradock). there may be a possibility that there is a relationship between regional distribution and genetic variation but this study does not give enough evidence to confirm or refute this. bassami et al. (2001) reported that there is a possibility that adaptation of particular genotypes to specific species may occur and that regional differences in strains may develop; they suggested that these differences would have a significant implication of the development of a universal pcr assay that would detect all strains of pbfdv. the genetic variation between these birds lends an important view in the production of a vaccine. if there are significant differences in the antigenicity or pathogenicity and other physiochemical characteristics of these isolates then it is important to develop a vaccine that can effectively protect a bird against any isolate or isolates that may infect it at any par33 j. albertyn, k.m. tajbhai & r.r. bragg 1 2 3 4 5 6 7 8 450 bp 250 bp 150 bp 50 bp fig. 5 pcr amplified products of pbfdv (pblm-4) from samples collected on filter paper, followed by dna extraction after 24 [lane 2], 48 [lane 3], 72 [lane 4] and 96 h [lane 5]. lane 6 contains the pcr product of a sample (pgeo) that was received on commercially available specimen collection paper and processed after 1 month. lanes 1 and 8, contains a molecular mass marker (λ ecor1/hindiii), and lane 7 a negative control from a sample that was shown to be negative (bcra-3) ticular time. in the related porcine circovirus (pcv) two distinct genotypes have been identified (todd 2000) and it is possible that similar genotypic differences have occurred in pbfdv. the pcr tests performed were reproducible and confirm the occurrence of pbfdv infection in the ring-neck parakeets and budgerigars in the regions from where the samples were obtained. however, it was difficult to rule out the possibility of apv infection in the birds tested. although a pcr test using primers to amplify the region coding for protein vpi of apv was performed and negative results were obtained, these results are not conclusive due to the lack of a positive control. a positive control was difficult to obtain as there were no reported cases of apv infection in south africa at the time of study. this study then does not exclude the possibility of a concurrent infection of pbfdv and apv in the birds tested. the method evaluated in this study for the collection of blood using filter paper was successful and did not have any negative effects on downstream processes. the results were reproducible and the pcr amplification was comparable to that performed using frozen blood samples. this method is simple, easy, does not require large amounts of blood (thus less stressful to the birds) and the samples do not require the use of anticoagulants, do not have to be frozen and are easy to transport. most importantly however, it does not affect downstream processes. with all these advantages it is evident that the implementation of this method of sample collection is a suitable replacement to the more traditional methods of blood collection. in conclusion, this study has shown the occurrence of pbfdv infection in ring-neck parakeets and budgerigars in south africa by establishing and optimising a reliable pcr diagnostic test with reproducible results. the primers that were designed during this project were successful in producing the amplicon of expected size when used in the pcr test. genetic variation between isolates from ring-neck parakeets and budgerigars is evident as well as genetic differences between isolates within individuals of the same species. however, the significance of these differences to pbfdv antigenicity, pathogenicity and other physiochemical characteristics must be investigated if an effective vaccine is to be produced. blood sample collection on filter paper is easy to use, is less stressful to the birds and does not impact negatively on further processes. acknowledgements we appreciate the help of michel labuschagne of the university of the free state in the practical work. we are grateful to sam rosslee, pierre swart and g. b. swart for supplying blood samples and to dr f. potgieter of the university of the free state for his assistance with the care of the birds. references bassami, m.r., ypelaar, l., berryman, d., wilcox, g.e. & raidal, s.r. 2001. genetic diversity of beak and feather disease virus detected in psittacine species in australia. virology, 279:392–400. bragg, r. 2002. feather disorders in budgerigars in south africa. the budgerigar society of south africa, quarterly bulletin, 6–7. latimer, k.s., niagro, f.d., campagnoli, r.p., ritchie, b.w., pesti, d.a. & steffens, w.l. 1993. diagnosis of concurrent avian polyomavirus and psittacine beak and feather disease virus infections using dna probes. journal of the association of avian veterinarians, 7:141–146. pass, d.a., plant, s.l. & sexton, n. 1994. natural infection of wild doves (streptopelia senegalensis) with the virus of psittacine beak and feather disease. australian veterinary journal, 71:307–308. raidal, s.r., firth, g.a. & cross, g.m. 1993. vaccination and challenge studies with psittacine beak and feather disease virus. australian veterinary journal, 70:437–441. raidal, s.r. & cross, g.m. 1994. control by vaccination of psittacine beak and feather disease in a mixed flock of agapornis spp. australian veterinary practice, 24:178–180. ramis, a., latimer, k.s., gibert, x. & campagnoli, r. 1998. a concurrent outbreak of psittacine beak and feather disease virus, and avian polyomavirus infection in budgerigars (melopsittacus undulatus). avian pathology, 27:43–50. riddoch, p.a., raidal, s.r. & cross, g.m. 1996. psittacine circovirus antibody detection and an update on the methods for diagnosis of psittacine beak and feather disease. australian veterinary practice, 26:134–139. sexton, n., penhale, w.j., plant, s.l. & pass, d.a. 1994. use of goose red blood cells for detection of infection with psittacine beak and feather disease virus by haemagglutination and haemagglutination inhibition. australian veterinary journal, 71:345–347. studdert, m.j. 1993. circoviridae: new viruses of pigs, parrots and chickens. australian veterinary journal, 70:1221– 122. todd, d. 2000. circoviruses: immunosuppressive threats to avian species: a review. avian pathology, 29:373–394. ypelaar, i., bassami, m.r., wilcox, g.e. & raidal, s.r. 1999. a universal polymerase chain reaction for the detection of psittacine beak and feather disease virus. veterinary microbiology, 68:141–148. 34 psittacine beak and feather disease virus in south africa article information authors: fredrick m. kivaria1 angolwisye m. kapaga2 gabriel k. mbassa3 paul f. mtui4 rhombe j. wani5 affiliations: 1national epidemiology section, ministry of livestock and fisheries development, tanzania2central veterinary laboratory, dar es salaam, tanzania 3faculty of veterinary medicine, sokoine university of agriculture, tanzania 4veterinary investigation centre, arusha, tanzania 5ministry of animal resources and fisheries, government of south sudan, south sudan correspondence to: fredrick kivaria postal address: po box 9152, dar es salaam, tanzania dates: received: 09 jan. 2012 accepted: 11 may 2012 published: 03 sept. 2012 how to cite this article: kivaria, f.m., kapaga, a.m., mbassa, g.k., mtui, p.f. & wani, r.j., 2012, ‘epidemiological perspectives of ticks and tick-borne diseases in south sudan: cross-sectional survey results’, onderstepoort journal of veterinary research 79(1), art. #400, 10 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.400 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. epidemiological perspectives of ticks and tick-borne diseases in south sudan: cross-sectional survey results in this original research... open access • abstract • introduction • materials and methods    • sample size    • clinical and laboratory investigation    • tick counts and tick infection proportions    • serological tests    • characterisation of epidemiological states of tick-borne diseases    • molecular characterisation of theileria parva isolates from south sudan    • ethical considerations • results    • temperature, lymph node scores, biopsies and blood smears    • theileria parva, theileria mutans, anaplasm marginale and babesia bigemina antibody patterns    • tick infestation patterns    • epidemiological states of theileria parva, theileria mutans, anaplasma marginale and babesia bigemina infections    • molecular characterisation of theileria parva    • survey of herdsmen • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a cross-sectional study was conducted between september and october 2010 in five states of south sudan that were selected on the basis of the perceived risk of tick-borne diseases. the purpose was to investigate epidemiological parameters of tick-borne diseases in south sudan and their uses in future control strategies. a total of 805 calves were assessed by clinical, microscopic and serological examination and tick counts. the indirect enzyme-linked immuno-sorbent assay (elisa) was used to detect antibodies to theileria parva, theileria mutans, anaplasma marginale and babesian bigemina. sero-conversion risks for t. parva and t. mutans were 27.3% and 31.3% respectively, whilst the risk was 57.6% and 52.8% for a. marginale and b. bigemina, respectively. major tick species identified include rhipicephalus appendiculatus, rhipicephalus decoloratus, rhipicephalus microplus, amblyomma variegatum, and rhipicephalus evertsi. there was great variation (p ≤ 0.001) in the number of all these ticks, both between herds in a state and between calves in an individual herd. the low and intermediate sero-conversion risks observed in the study states suggest that immunisation against east coast fever (ecf) is justified. fortunately, three major genotypes that were identified by applying polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) analysis on the p104 to the blood samples and t. parva muguga, matched very well with t. parva kiambu 5 and t. parva muguga; therefore the muguga cocktail can be used for the immunisation of cattle in south sudan. however, prospective studies are required to develop optimal control measures for tick-borne diseases under different ecological and husbandry practices in south sudan. introduction top ↑ infection with theileria parva causes the disease, east coast fever (ecf), also known as bovine theileriosis. the disease is endemic to the region stretching from the democratic republic of the congo to tanzania, from south sudan to south africa; with a heterogeneous distribution and prevalence strongly related to its vector dynamics, host susceptibility, grazing system and tick-control practices (kivaria 2007; marufu et al. 2010; rubaire-akiiki et al. 2006). in all affected areas, ecf has a considerable epidemiological and economic significance; in tanzania; the disease is the main cause of reported cattle deaths and was estimated to account for 68% of the 364 million usd annual total losses resulting from tick-borne diseases (kivaria 2006). in unvaccinated zebu calves raised by pastoralists, ecf is responsible for an annual mortality risk of 40% – 80% (kivaria 2006). the first outbreak of bovine theileriosis in sudan was reported in 1950 (hoogstraal 1956). after the 1950-outbreak, the disease was not reported again until 1981. since then many outbreaks of ecf have been reported in southern sudan (julla, tingwa & kwajok 1989; julla 1993). in 1983, an outbreak was reported in palotaka, nimule and juba township with calf mortalities estimated at 80% – 100% (julla 1985).unpublished archives indicate that the disease has been restricted previously to the equatorial region of south sudan, probably because of its proximity to ecf-infected zones in kenya, uganda, and north-east congo, coupled with the suitable climatic conditions for the survival of the vector. during the two decades of civil war in south sudan, pastoralists from jonglei state were displaced and moved southward with their cattle to ecf-infected areas in central equatoria state. the gradual introduction of these naïve animals into the infected zone generated some degree of immunity against the disease and the animals were able to survive the tick and parasite challenge. after the comprehensive peace agreement (cpa) in 2005, dinka bor of jonglei state started moving northward with huge numbers of livestock. in june 2008, the first outbreak of ecf was reported in an area called lek yek in bor town, and thereafter, a cycle of outbreaks followed and continued throughout 2009 and 2010, affecting animal production and health as well as the food security of many households whose livelihoods are dependent mainly on livestock. results of several tick-borne diseases (tbd) studies in smallholder dairy and pastoral livestock farming systems in eastern africa indicated that tbds’ sero-prevalence risk differs across agro-ecological zones and grazing systems (bazarusanga et al. 2007; maloo et al. 2001; rubaire-akiiki et al. 2004; swai et al. 2005). the impetus to carry out this cross-sectional study was borne out of the desire to characterise the epidemiological parameters (sero-conversion risk, morbidity and mortality risks) and to document the dynamics of tick infestation of cattle and the infection patterns of bovine theileriosis, babesiosis and anaplasmosis, because these are very important factors to consider when planning production system-specific control strategies of tbd. in this paper we use these factors to characterise the epidemiological states of ecf and other tick-borne diseases, namely anaplasmosis and babesiosis in entral equatoria (ce), eastern equatoria (ee), jonglei (jng), lakes (lks) and western equatoria (we) states of south sudan. in addition, molecular characterisation of t. parva isolates was performed, and this information was combined to assess the current and potential tbd status in each state as well as the implications for future control strategies. materials and methods top ↑ sample size this cross-sectional study was designed and implemented between september and october 2010. one methodological challenge faced during the execution of this survey was the lack of information on husbandry practices and estimated herd level prevalence of tick-borne diseases. deem et al. (1993) suggested that a sample size of 10–20 pastoral herds, that contained an average of 10 calves per herd, would be adequate across a range of epidemiological states, with sample sizes at the top of the range required in epidemiologically unstable areas. based on these results, it was decided to select 20 cattle camps randomly from each state and six calves from each selected camp. a three-stage sampling was employed. payams or divisions were purposefully selected first, based on the perceived risk of tick-borne disease. it was followed by a random selection of cattle camps or boma, and then animals were randomly selected from each cattle camp. the resulting final sample size therefore depended on the number of camps and calves sampled from each state and cattle camps, respectively. the geographical location of the five states, counties and the cattle camps investigated during the study period, have been depicted (figure 1). figure 1: geographical location of the five states, counties and the cattle camps investigated during the cross-sectional survey (september 2010 – october 2010). the target age group was calves of age 4–18 months. aging was performed based on the herdsman-derived information and on the absence of permanent incisors. all herdsmen were questioned on the occurrence of tick-borne diseases, herd size, calf mortality, descriptions of clinical signs observed in calves that had died, acaricide usage, treatment, distance travelled to water and grazing, and the general descriptions of tick problems present in each herd. clinical and laboratory investigation each animal was subjected to clinical examination, including the measurement of rectal temperature. a temperature ≥39.5 °c in adults (> 12 months) and a temperature ≥40 °c in calves (≤ 12 months) were considered a febrile reaction. with regard to the palpation of the pre-scapula lymph nodes, the sizes were scored as + (normal); ++ (slightly enlarged) and +++ (grossly enlarged). in this study, lymph node enlargement of (+) were defined as originating from animals free of theileriosis, whilst palpation results of ≥ ++ were classified as evidence of theileriosis. blood smears and lymph node biopsies were taken to demonstrate the presence of the causing organism. smears were stained with giemsa and microscopically examined under 40 x and 100 x oil immersion objectives. tick counts and tick infection proportions tick infestation was estimated by counting the number of adult ticks (male, female, engorged and non-engorged) on the body of each calf examined. ticks are not randomly distributed on the animal body but rather restricted to a few predilection sites; therefore we preferred to count all the ticks on one side of the animal body instead of making counts per square meter. the ticks on one side of the body of each calf were counted and categorised into species; visual identification was carried out during the tick counts, based on the guidelines described by walker et al. (2003).adult rhipicephalus appendiculatus that were partially engorged and thus potentially with developing sporoblast stages of t. parva, were dissected to remove the salivary glands for examination of infection with t. parva, as described by buscher and otim (1986). tick infections were determined through direct microscopy after staining the tick salivary glands with schiff’s (fuelgen’s) reagent to identify infected acini. the intensity of infection was subsequently estimated by counting the number of infected acini. serological tests blood was collected from each calf in one 10-ml plain vacutainer tube (becton dickinson vacutainer systems, uk) by jugular venepuncture. tubes were labelled and verified before drawing the blood from the calves. after collection, blood samples were stored in a car refrigerator until sera could be separated (usually within 24 hours). the sera were separated by centrifugation at 3000 x g for 20 min and divided into aliquots and stored in freezers (-20 °c) in the laboratory.enzyme-linked immuno-sorbent assays (elisas) were used to detect antibodies to t. parva, theileria mutans, anaplasma marginale and babesian bigemina. polymorphic immune-dominant molecule (pim) recombinant antigen was used as described by katende et al. (1998) for the detection of t. parva antibodies. tests for antibodies to t. mutans and b. bigemina have been described by katende et al. (1990, 1998). each elisa test plate included predetermined positive and negative control sera. optical density (od) readings from the reference highly positive control sera were used to compute the per cent positivity (pp) for the test sera (wright et al. 1993). per cent positivity for test serum was expressed as the per cent of the test serum od (at optimum dilution of 1:200) divided by the mean od reading derived from the strong positive control serum on the linear curve (from a curve of od against the reciprocal of serial dilutions) (katende et al. 1998; wright et al. 1993). for t. parva and t. mutans, a sample was considered positive if the pp value equalled 20 or higher, whilst the cut-off point was 15 for a. marginale and b. bigemina. the risk of exposure of animals to t. parva was estimated by the sero-conversion risk. the sero-conversion risk was computed as the number of animals sero-converted divided by the total number of animals tested. tick identification, microscopic and serological work were conducted at the veterinary investigation centre in arusha, tanzania. characterisation of epidemiological states of tick-borne diseases the epidemiological state of tick-borne diseases in an area can be considered as either endemically stable or unstable. for babesiosis and anaplasmosis, antibody prevalence has been used as an indicator of the existence of endemic stability, and thus the biological evidence of the need for vaccination or for no intervention (de vos 1992; mahoney 1977). in broad terms, there is a strong correlation between high antibody prevalence to these infections and endemic stability, and low antibody prevalence and endemic instability. this phenomenon also exists with t. parva, but it is by no means absolute and it is complicated by various factors. endemic stability is assessed based on a combination of indicators (norval, perry & young 1992) including (1) antibody prevalence, (2) disease incidence, (3) case-fatality proportion and (4) age group affected (norval et al. 1992; perry & young 1995). tick-infection proportions are also considered helpful (perry et al. 1992). according to norval et al. (1992), an endemically stable state is characterised by high antibody prevalence, low disease incidence and case-fatality proportion, and a rapid acquisition of infection in young calves. an endemically unstable state is characterised by the opposite, that is, lower antibody prevalence, high disease incidence and case-fatality proportion and primary infections that occur in all age groups. these criteria were used to characterise the endemic stability of the five states investigated. molecular characterisation of theileria parva isolates from south sudan lymph node biopsy and blood samples collected from cattle were used as the source of deoxyribonucleic acid (dna) for polymerase chain reaction (pcr) analysis. genomic dna was extracted from blood samples received from southern sudan. deoxyribonucleic acid was also extracted from a stabilate of t. parva muguga (muguga, kiambu 5, serengeti transformed) obtained from the ministry of livestock and fisheries development, dar es salaam, and used as control-dna for the processes in the characterisation. the dna extraction and pcr assays were conducted as described by bishop (2001). ethical considerations field observations were conducted and samples collected from animals. no experiments were performed on the animals and the animals used in the collection of samples for the study were treated humanely. results top ↑ a total of 822 calves from the 5 states, 16 counties, 29 payams and 34 cattle camps were investigated; 805 calves had complete observations, whilst 17 calves did not provide all the information, and were therefore omitted from further analyses. of the 805 calves investigated, 50.4% were female calves, whilst 49.6% were male calves; the mean age for female calves was 9.34 months and for male calves 8.98 months. the mean age of the studied calves was 9.16 months, whilst minimum and maximum ages were 0.75 months and 24.00 months, respectively. temperature, lymph node scores, biopsies and blood smears the average temperature was 38.0 °c ± 0.16se, the median was 38.20 °c and the mode was 41 °c; there was no significant (x2 = 6.342) statistical difference in the distribution of temperature recordings amongst the states.a total of 805 pre-scapula lymph nodes were examined, of which 45.0% were positive, with reactions of ++ or higher; 55.0% of the 805 pre-scapula lymph nodes showed a negative reaction of +. palpation results of ++ and +++ accounted for 34.6% and 10.4%, of the palpated lymph nodes respectively. of the 362 grossly enlarged pre-scapula lymph nodes, 3.0% (11) were positive for schizonts; the remaining smears were negative. theileria parva piroplasms were detected in 32 corresponding blood smears, and 6 out of the 32 t. parva positive blood smears were a mixed infection between t. parva and a. marginale. blood parasites were detected in 8.9% (72) of the 805 blood smears examined; the haemo-parasites detected include t. parva (26), a. marginale (38), t. parva and a. marginale (6), b. bigemina and a. marginale (1), and trypanosome species (1). because of the small number of haemo-parasites detected, no statistical comparisons were made for the distribution of these parasites by states; the distribution of these haemo-parasites by states has been summarised (table 1). table 1: the distribution of the haemo-parasites detected in five states of south sudan during a cross-sectional survey (september 2010 – october 2010). theileria parva, theileria mutans, anaplasm marginale and babesia bigemina antibody patterns serological results indicated that 27.3% (220) of the 805 calves screened have sero-converted against t. parva, and 72.7% (585) were negative, whilst for t. mutans 31.3% (252) and 68.7% (553) of the 805 calves screened were sero-positive and negative, respectively. on the other hand, 57.6% (464) and 52.8% (425) of the 805 calves were sero-positive for a. marginale and b. bigemina antibodies, respectively. the mean antibody sero-prevalence with standard errors for t. parva, t. mutans, a. marginale and b. bigemina by states are shown (figure 2). central equatoria state was significantly associated with higher sero-prevalence compared with other states. figure 2: mean serum antibody sero-prevalence of 805 calves investigated during the cross-sectional survey (september 2010 – october 2010). the relationship between the calf age and sero-prevalence (figure 3a–d) shows that the majortiy of the calves are born with very little (negative sign) maternal antibodies to the major tick-borne parasites studied. they sero-convert, however, by the age of 3 months, the age at which, according to the herdsmen, calves go out for grazing. however, the acquired antibodies quickly decay with advancing age, in such a way that the majority of the calves have less than 15% sero-prevalence at 11 months of age. figure 3: relationship between sero-prevalence for (a) theileria parva, (b) theileria mutans, (c) anaplasma marginale and (d) babesia bigemina, and calf age (in months) from five states of south sudan (september 2010 – october 2010). tick infestation patterns the occurrence and distribution of ticks in the study states have been listed (table 2). the highest number of tick counts was made in the eastern equatoria and central equatoria states. the most prevalent ticks were r. appendiculatus and amblyoma variegatum and these were observed in eastern equatoria and central equatoria states, respectively. it is further shown (table 2) that infestation by ticks (total ticks and individual species) significantly (p ≤ 0.001) varied with states, especially in eastern equatoria and central equatoria states; there was also great variation in the number of ticks between calves in an individual herd. no ticks were collected from the western equatoria state. the numbers of r. appendiculatus dissected, the proportion infected with t. parva and the intensity of those infections, have been listed (table 3). the prevalence of infections in dissected ticks was in the range 25% – 28%. table 2: variation in the total number of ticks and the individual tick species on calves in the study states, during the cross-sectional survey (september 2010 – october 2010). table 3: prevalence and intensity of theileria parva infections in non-engorged adult rhipicephalus appendiculatus collected from calves in five states of south sudan (september 2010 – october 2010). epidemiological states of theileria parva, theileria mutans, anaplasma marginale and babesia bigemina infections enzyme-linked immuno-sorbent assays results for t. mutans and b. bigemina have been summarised (table 4) and these results were used to classify the five states for endemic stability. tabulated values show that all states are endemically unstable to t. mutans, but moderately stable to b. bigemina. no morbidity and mortality risk data for t. mutans and b. bigemina were available. the estimates of indicators used to classify the five states for endemic stability to ecf (table 5) and anaplasmosis (table 6) have been listed. all strata were judged to be endemically unstable; however, the degree of their instability varied. we assessed the relative stability of each state over the range of endemic stability or instability (tables 5 and 6). table 4: distribution of theileria mutans and babesia bigemina sero-prevalence by states. table 5: criteria used to characterise endemic stability to bovine theileriosis in the five states studied in south sudan. table 6: criteria used to characterise endemic stability to anaplasma marginale in the five states of south sudan. molecular characterisation of theileria parva the profiles generated by applying polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) analysis on the p104 to the samples and t. parva muguga were obtained (table 7). three major genotypes were identified. the first genotype appeared exactly the same as t. parva muguga and t. parva kiambu 5 at p104 locus with two bands at approximately 220 bp and 280 bp. the second genotype had a lower band at 150 bp, and the higher was similar to t. parva muguga at 280 bp. the final genotype had a band similar to t. parva muguga at 280 bp and a higher band at about 400 bp. table 7: band sizes (bp) of different polymerase chain reaction deoxyribonucleic acid from samples examined for p104 allele. survey of herdsmen interviews with the herdsmen revealed that they were not willing to provide information about their animals. however, the following direct observations were made. the study states are suitable for many types of agricultural activities, including livestock keeping. the lush pastures, huge trees and other types of vegetation indicate abundant rainfall. this was confirmed through the consultative discussions with the state veterinarians and herdsmen who indicated that only 3–4 months of the year are dry. this also means that many parts of the country are suitable habitat for the ticks and their respective diseases. nearly all the herds observed were zebu breeds and the animals were generally in good health. animals graze communally in designated areas and only separated in the evenings into herds belonging to different families. members of the same family keep their animals together at night for ease of guarding them. it was of great epidemiological interest to note that about 90% of the herdsmen met, and even some of the livestock field personnel, did not associate ecf with ticks. instead, they viewed ticks as a major problem only because of their direct physical effects on the animals. this was considered an instrumental factor in the planning of future tick and tick-borne diseases control interventions.despite the general lack of information on tbd, however, there are some indications that livestock keepers are aware of the existence of ecf and other tbd (the majority of herdsmen described clinical signs compatible with those of ecf in their calves), and they have perfected traditional methods of treating the diseases. a standard method of treatment observed during the field investigation, is the burning of lymph nodes with a hot iron and the cauterisation of them with caustic juice derived from a plant. farmers reported that this treatment is effective when applied early in the course of the disease. however, it was difficult to substantiate whether the recovery is an outcome of the treatment or because of an inborn resistance of the calves to the diseases. indigenous cattle are known to develop resistance to the vector tick and to tick-borne diseases (norval et al. 1992). the fact that the case fatality rate from ecf was reported to be so high (table 5), puts into question the herdsmen’s interpretation of the effectiveness of the treatment. currently, control of tick-borne diseases is minimal and limited to irregular use of acaricides (mainly to control tick infestation; herdsmen reportedly used acaricides when sufficient funds became available) and chemotherapy. the herdsmen also control ticks by the application of cattle urine mixed with ash. the main constraints to proper control were found to be a lack of infrastructure (service providers, dipping facilities and drug outlets). there was also a general lack of information on livestock husbandry, for instance, the failure of herdsmen to associate ticks with ecf. generally there is very limited infrastructure in south sudan to support large scale livestock interventions. there are, for example, only two dip tanks (one in central equatoria, and one in western bahr elghazal state) and about eight (central equatoria [2], eastern equatoria [2], unit [2] and warrap sate [2]) functional checkpoints and one quarantine facility in eastern equatoria, in the country. apart from the active network of community animal health workers, there are very few skilled personnel, and communication and input markets are also lacking. the majority of the herdsmen reported a lack of input and extension services as constraints to livestock keeping. discussion top ↑ the objective of this study was to investigate epidemiological parameters that may influence the development of tick and tick-borne disease control strategy for south sudan. the study areas were purposefully selected based on the perceived ticks and tick-borne disease risk. in pastoral production systems, calves and other young animals are usually left to graze near the homestead; hence they were considered to be resident and the survey findings are therefore a true picture of the existing tick and tick-borne disease situation in the given area. results obtained by deem et al. (1993) suggest that, when sero-prevalence results from cross-sectional studies are used to determine the epidemiological status of tick-borne diseases in a given production system, it is important that sero-prevalence is assessed in homogenous systems if they are to be interpreted. such homogenous systems should incorporate the same cattle type (in terms of their susceptibility), the appropriate age-group, sampled at the appropriate time of year and within appropriate strata of the ecological cline. however, this requirement is more important if herd sizes are very small, as in the zero-grazing smallholder herds; in this study larger pastoral herds were used, and given the communal grazing and husbandry practices whereby, at one point in time, almost the entire national herd congregate along the nile river during the dry spell. from an epidemiological perspective, the study population was therefore assumed to be a single, homogenous, free mixing host population. it was not possible to establish the ecf specific case fatality risk reliably. however, consultative discussions with the state veterinarians and the producers in the study areas indicate that case fatality risk caused by ecf as high as 80% – 100% occurs, depending on the time of the year. these estimates compared very well with the results of a recent unpublished socio-economic study in the same areas, but the estimates are very high compared to what would be expected in the east africa region where calf mortality ascribable to tick-borne diseases range between 30% and 50%, to as low as 2% – 3% (norval et al. 1992), depending on the different tick-control practices and the existing endemic situation. however, ecf specific case fatality risk ranging between 50% and 100% are not uncommon in the region. cases of death are common especially in high-susceptible exotic breeds, such as bos taurus, and their crosses with indigenous breeds, but the indigenous breeds are also at risk, particularly in endemically unstable or epidemic areas (norval et al. 1992). data suggest that the majority of the calves are born with very little or no (the negative sign) maternal antibodies to the major tick-borne parasites studied (figure 3), but they sero-convert by the age of 3 months, the age at which, according to the questionnaire results, they go out for grazing. however, the acquired antibodies quickly decay with advancing age; in such a way that the majority of the calves have less than 15% sero-prevalence at 11 months. this could be ascribed to several reasons. firstly, the majority of dams are sero-negative to the common tick-borne diseases. secondly, there may be a low tick challenge and if the tick challenge is good, then it is likely that several factors such as the number, viability, and infectivity of sporozoites within the salivary glands, pathogen and t. parva strain differences, and the duration of tick feeding could interplay to influence the ability of the r. appendiculatus to acquire t. parva and the transmission of t. parva from infected ticks to cattle. in endemic areas the majority of infected ticks likely to become infected by feeding on carrier and clinically affected cattle (medley, perry & young 1993), but the findings recorded (table 1) and the presence of schizonts in only 3.11% of the study population suggest that the subsequent instars would then possibly inoculate relatively fewer parasites, reducing the overall tick-infectivity level. this is probably corroborated by the recent findings by ogden et al. (2003) that the level of parasitaemia could affect the rate of acquisition of t. parva by ticks and the subsequent cattle challenge. however, it is worth noting that, in a given cattle production system, the level of infection challenge will depend on classical risk factors such as climatic suitability for ticks, grazing practices and range (and thus mixing with other potentially infected cattle), tick-control practices and host susceptibility. thirdly, herdsmen in the study areas use cattle urine mixed with ash (the ammonia in urine suffocates the ticks, whilst ash is used as a binding material) to remove ticks from animals. this practice interrupts tick challenge and may also contribute to the observations recorded (see figure 3). fourthly, the relatively high risk levels, particularly of anaplasmosis, could result in early and high calf mortality which could also influence the resulting sero-prevalence patterns in the population, as calves do not live long enough to develop immunity. the observed high level of tick infection (table 3) is probably because of the fulminating theileriosis, and indeed the ticks with high infection prevalence in jonglei state came from a cattle camp that reportedly had an outbreak of ecf just before the survey. the high prevalence of tick infection is far above the recorded levels of 2.6% and 2.3% – 14.2% in more endemic areas of tanzania and kenya (ogden et al. 2003; oura et al. 2004). variations in sero-prevalence in the study states suggest that tick-borne diseases exist in different epidemiological states, so that morbidity and mortality may fluctuate from season to season and from herd to herd. this study, as in other previous studies in the east african region (gitau et al. 1997; rubaire-akiiki et al. 2004, 2006), demonstrated the crucial influence of farm circumstances, grazing systems and agro-ecological zones on the variation in tick and tick-borne diseases risk, both spatially and temporally. thus, herdsmen and the veterinarians in south sudan need to pay close attention to varying ecf challenge in making decisions on ecf and other tbd control. the overall sero-prevalence of 27.30% and 31.30% for t. parva and t. mutans respectively, suggest that endemic instability exists in the study population and an outsized proportion of the population is likely to succumb to clinical theileriosis. on the other hand the overall sero-prevalence of 57.60% and 52.80% for a. marginale and b. bigemina is suggestive of a state of medium endemic stability. it has been suggested that high sero-conversion risks are associated with little or no clinical cases of tick-borne diseases (norval et al. 1992). however, the high reports of clinical cases (tables 5 and 6), indicate that this may not be the case, and clearly sero-prevalence alone is not a reliable indicator of endemic stability. the results of this study have important implications for future ecf and other tick-borne disease control in south sudan. in our opinion, herdsmen and veterinarians in south sudan should aim for managing tick populations and tick-borne diseases within economically acceptable limits, in which the risks of tick-borne diseases outbreak are minimal; this can be achieved by ensuring that cattle are immune. immunity may be maintained by simply ensuring that tick numbers are high enough to perpetuate endemic stability. any control programme should therefore aim to provide sufficient tick control to increase cattle productivity, whilst not reducing tick numbers to the extent that endemic stability is adversely affected. where endemic stability is not possible (as a result of factors affecting transmission of parasites from either clinical cases or carrier animals) vaccination may be required as a disease control measure. the low and intermediate sero-conversion risks observed (table 5) in the study states suggest that ecf immunisation of the study population is justified. fortunately, the similarity (table 7) between the southern sudan and the muguga t. parva isolates implies that the muguga cocktail vaccine can be used in south sudan. with immunisation there will be a greater likelihood of the development of endemic stability; wider immunisation coverage combined with modified acaricide control strategies to allow sufficient tick challenge probably offers the best prospect for establishing and maintaining endemic stability in the southern sudan cattle population. the relatively high sero-prevalence of b. bigemina (table 4) and a. marginale (table 6) suggests that a reduction in the frequency of acaricide application following ecf immunisation in the study states would therefore not be expected to result in an increased incidence of these diseases. it is quite clear from this study that attention will have to be paid to variations in tick and tbd risk, both spatially (tick and tbd dynamics change over relatively short geographical distances) and temporally (seasonally and secularly), to develop optimal combinations of control strategies for tbd under various husbandry and ecological conditions. in the short term, any control strategy to be adopted should be supported by strategically located quarantine or holding grounds with dipping facilities, and enacting and enforcing appropriate legislations that promote good husbandry practices by the herdsmen. it was of epidemiological interest to note that about 90% of the herdsmen and community animal health workers could not associate the presence of ticks with the occurrence of tbd; therefore, for a successful control strategy, an element of public awareness through extension services should feature strongly, and such a strategy should be supported in addition by a strong epidemiological surveillance and laboratory confirmation of the suspected cases. conclusion top ↑ the purpose of this study was to investigate epidemiological parameters of tick-borne diseases in five states of south sudan, and their uses in future control strategies. the results contribute to the epidemiological understanding that bovine theileriosis, anaplasmosis and babesiosis are endemic and exist in an unstable state. the t. parva isolates from south sudan matched very well with t. parva kiambu and t. parva muguga, and consequently the muguga cocktail can be used for immunisation of cattle in south sudan. acknowledgements top ↑ we are grateful to the herdsmen who gave their time and allowed us to sample their animals. the help and participation of staff in the department of veterinary services of the ministry of animal resources and fisheries of the government of south sudan, is highly appreciated. this survey was funded by the livestock epidemio-surveillance project southern sub-project coordinated by vétérinaires sans frontières, belgium. competing interests none of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. authors’ contributions f.m.k. (ministry of livestock and fisheries development) was the project leader and was responsible for the study design and data analysis. g.k.m. (sokoine university of agriculture) and p.f.m. (veterinary investigation centre, arusha) carried out the laboratory work, and a.m.k. (central veterinary laboratory, dar es salaam) and r.j.w. 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(eds.), 1992, the epidemiology of theileriosis in africa, london, academic press. ogden, n.h., gwakisa, p., swai, e., french, n.p., fitzpatrick, j., kambarage, d. et al., 2003, ‘evaluation of pcr to detect theileria parva in field collected tick and bovine samples in tanzania’, veterinary parasitology 112, 117–183. http://dx.doi.org/10.1016/s0304-4017(02)00448-x oura, c.a., bishop, r.p., wampande, e.m., lubega, g.w. & tait, a., 2004, ‘application of a reverse line blot assay to the study of haemoparasites in cattle in uganda’, international journal of parasitilogy 34, 603–613. http://dx.doi.org/10.1016/j.ijpara.2003.12.012, pmid:15064125 perry, b.d. & young, a.s., 1995, ‘the past and future roles of epidemiology and economics in the control of tick-borne diseases of livestock in africa: the case of theileriosis’, preventive veterinary medicine 25, 107–120. http://dx.doi.org/10.1016/0167-5877(95)00546-3 perry, b.d., deem, s.l., medley, g.f., morzaria, s.p. & young, a.s., 1992, ‘the ecology of theileria parva infections of cattle and the development of endemic stability’, in u.g. munderloh & t.j. kurtti (eds.), proceedings of the 1st international conference on tick-borne pathogens at the host-vector interface: an agenda for research, september 15–18, 1992, pp. 1–7. rubaire-akiiki, c.m., okello-onen, j., musunga, d., kabagambe, e.d., vaarst, m., okello, d. et al., 2006, ‘effect of agro-ecological zone and grazing system on incidence of east coast fever in calves in mbale and sironko districts of eastern uganda’, preventive veterinary medicine 75, 251–266. http://dx.doi.org/10.1016/j.prevetmed.2006.04.015, pmid:16797092 rubaire-akiiki, c., okello-onen, j., nasinyama, g.w., vaarst, m., kabagambe, e.k., mwayi, w. et al., 2004, ‘the prevalence of serum antibodies to tick-borne infections in mbale district, uganda: the effect of agro-ecological zone, grazing management and age of cattle’, journal of insect science 4, viewed n.d., from http://insectscience.org/4.8/ swai, e.s., french, n.p., karimuribo, e.d., fitzpatrick, j.l., bryant, m.j., brown, p.e. et al., 2005, ‘spatial and management factors associated with exposure of smallholder dairy cattle in tanzania to tick-borne pathogens’, international journal of parasitology 35, 1085–1096. http://dx.doi.org/10.1016/j.ijpara.2005.04.006, pmid:16023121 walker, a.r., bouattour, a., camicas, j.-l., estrada-pena, a., horak, i.g., latif, a.a. et al., 2003, ticks of domestic animals in africa, bioscience reports, the university of edinburgh, scotland, u.k. wright, p.f., nilsson, f., van rooij, e.m.a., lelenta, m. & jeggo, m.h., 1993, ‘standardisation and validation of enzyme-linked immuno-sorbent assay techniques for the detection of antibody in infectious disease diagnosis’, scientific and technical review (oie) 12, 435–450. pmid:8400384 http://www.ojvr.org open access page 1 of 1 reviewer acknowledgement acknowledgement to reviewers in an effort to facilitate the selection of appropriate peer reviewers for the onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on https:// ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a user. in order to be considered, please email submissions@ojvr.org indicating your intention to register as a reviewer for the journal. to access your details on the website, you will need to follow these steps: 1. log into the online journal at https://ojvr.org 2. in your ‘user home’ [https://ojvr.org/index. php/ojvr/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest(s). 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to the onderstepoort 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have been inadvertently left out. these individuals provided their services to the journal as a reviewer from 01 october 2021 to 30 september 2022. abdalla a. latif abel b. ekiri akin kirbas alaster samkange amare e. gemeda andrea spickett annelise botes bruno mathieu claude sabeta djamel khelef edward c. webb elias k. kassaye esam elgorashi evelyn madoroba gerry swan hanaa m. fadel james madzimure james outa junita liebenberg karien labuschagne lia s. rotherham maab i. al-farwachi motlatso t. hlokwe ndichu maingi ntelekwane g. khasapane ovokeroye abafe paul fandamu ravendra p. chauhan rungano magadu samson mukaratirwa samuel e. mantip sherin r. rouby somayeh namroodi stefan s. ciornei sudesh kumar tlabo leboho whatmore m. tagwireyi http://www.ojvr.org� https://ojvr.org� https://ojvr.org� mailto:submissions@ojvr.org https://ojvr.org� https://ojvr.org/index.php/ojvr/user https://ojvr.org/index.php/ojvr/user mailto:publishing@aosis.co.za mushi_247-249.qxd introduction utilization of yolk sac in the ostrich (struthio camelus) is an important aspect of its post-hatching development (bertram & burger 1981). there is limited information as to when yolk sac resorption in ostrich chicks occurs (keffen & jarvis 1984; deeming 1995). the latter author suggested a 10–14 day period for yolk utilization to be completed. this process has been more intensively studied in avian species other than the ratites (paganelli, oliszowka & ar 1974; moran & reinhart 1980; matshusita 1986; murakami, akiba & horiguchi 1992; reidy, atkinson & leeson 1998). in the ostrich, failure to establish when yolk sac is considered “retained” has often led to unnecessary surgical excision of the yolk sac (deutectomy). this surgical operation would be justified in conditions such as omphalitis among others (huchzermeyer 1994). the main purpose of this study was to investigate the process of ostrich yolk sac resorption, the possible pertinent factors involved in this phenomenon and the clinical implications. materials and methods using sterile disposable gloves a total of 1 036 eggs were collected in the ostrich laying season from june 2002 to february 2003. these eggs were placed in sponge-padded, well-ventilated boxes twice a day, on the same day they were laid, to prevent brooding by the hens. the eggs were buffed with dry steel wool to remove dried mud on the surface and transported on their sides. subsequently they were labelled to indicate source camp and date of collection. the eggs were weighed on an electronic balance (adam equipment co., uk) and the mass of each egg recorded on the shell. the eggs were fumigated with a mixture of 200 g potassium permanganate 300 ml 40 % formaldehyde in a special 247 onderstepoort journal of veterinary research, 71:247–249 (2004) research communication yolk sac utilization in ostrich (struthio camelus) chicks e.z. mushi1, m.g. binta2 and r.g. chabo1 abstract mushi, e.z., binta, m.g. & chabo, r.g. 2004. yolk sac utilization in ostrich (struthio camelus) chicks. onderstepoort journal of veterinary research, 71:247–249 the mass of residual yolk sac expressed as a percentage of initial mass of the egg from which the chick hatched decreased sharply in the first 2 days post-hatching. a gradual reduction occurred between 3 and 10 days after which a sharp decline was noted between 11 and 13 days post-hatching. the highest number of chicks with unabsorbed yolk sac was noted on day 5 post-hatching followed by days 6 and 7. chick mortality followed the same pattern. the dynamics, causes and clinical consequences of yolk sac utilization are discussed. keywords: ostrich, struthio camelus, yolk sac 1 botswana college of agriculture, private bag 0027, gaborone, botswana 2 national veterinary laboratory, private bag 0035, gaborone, botswana accepted for publication 15 march 2004—editor room and afterwards stored at 12–18 °c, relative humidity (rh) of 70–80 % in a cold room. prior to transfer to the incubator, the eggs were warmed at 25 °c for 8–12 h and then set on day 10. incubation was done at 36.2 °c and 42 % rh. eggs were frequently weighed to assess water loss by the embryo. they were turned 45 ° to each side of the vertical, six times a day. as hatching was expected to occur at day 42, the eggs were incubated for between 35–39 days, after which they were placed in a hatcher where the temperature was 0.5–1 °c lower. eggs were now candled every 3 h to assess whether the chicks had pipped internally. hatching chicks were not assisted, in order to prevent the establishing of a weak population of chicks. once hatched, the umbilical cord was treated with a tetracycline-containing wound spray. the chicks were left to dry off completely and to settle down in a clean rest room. prior to their removal from the drying room, the chicks were given either a commercial probiotic (enteroform) or yoghurt depending on their availability. post-mortem examinations were conducted on all dead ostrich chicks from day 2 to day 13 posthatching. the carcasses were refrigerated at 4 °c and autopsies were performed at most 2 days after death. the mass of the chick and of any residual yolk sac in the abdomen were recorded. swabs of the yolk sac and viscera were taken for bacterial and fungal culture. results there was variation in the initial mass of the eggs, the average mass being 1.5 ± 0.5 kg. the postmortem examinations revealed that the chicks died from a variety of causes including omphalitis, starvation, dehydration, septicaemia and enteritis. high chick mortality was noted in the chicks aged between 3 and 11, peaking on days 6 and 7. it gradually declined after day 13. the highest number of dead chicks with unabsorbed yolk sac was in the 3–11 day age group as depicted in the histogram in fig. 1. the yolk sac mass was expressed as a percentage of the initial mass of the egg from which the chick hatched. at the time of hatching, it was noted that the yolk sac contributed to about 19.5 % of the initial egg mass. a sharp reduction in the mass of the yolk sac expressed as a percentage of the initial egg mass was realized in the first 48 h post-hatch. thereafter a gradual decline occurred from day 3 to day 10. subsequently, within 2 days, a sharp temporary increase was noted which fell precipitously to insignificant levels within a day. the bacteriological culture of yolk sacs yielded various bacterial species, amongst others staphylococcus spp., escherichia coli, bacillus lichenformis, achromobacter spp. and acinebacter calcoaceticus. discussion the mass of the 1 036 ostrich eggs that were incubated ranged from 1.4–1.6 kg. these values were comparable to those obtained by keffen & jarvis (1984) and foggin (1992). the results obtained in this study suggest that the presence of a yolk sac in a chick beyond day 13 should be considered as retained. studies conducted by murakami et al. (1992) indicated that the residual yolk sac plays a 248 yolk sac utilization in ostrich chicks � � � � � �� � � � � � � � � � � � � � � � � � � � � �� �� �� �� ���� � � �� �� �� �� � � � � � � � �� �� �� �� ���� � � � �� � �� � �� �� � �� �� �� �� � � � �� � � � fig. 1 number of chicks with unabsorbed egg yolk fig. 2 relationship between age of a chick (n = ) and the mass of the residual yolk expressed as percentage of the initial egg mass from which the chick hatched crucial role in complementing the nutrients absorbed to assure rapid growth of the chick once it has hatched. the yolk sac is therefore necessary for early initiation of growth, since dietary fat is only effective at 10 days after hatching (chamblee, brake, schultz & thaxton 1992). it has been found that in the avian uptake of fructose increases after hatching, which allows the inherent yolk glucose resources or reserves to become a major source of energy, as the yolk sac lipids are rapidly depleted (sklan 2003). the presence of the enzyme sucrase in the yolk sac of the chick emphasizes the role played by the yolk sac in growth initiation during pre-hatch and post-hatch period (matsushita 1986). there was a precipitous fall in the mass of yolk sac within the first 48 h period post-hatch. in newlyhatched broiler chicks, absorption of the yolk sac was found to precede initiation of growth within 24 h (chamblee et al. 1992). loss of mass of the egg yolk in the chickens between the ages of 3 and 11 days post-hatch was variable. the gradual decline in mass may partly be attributed to the varied rate at which yolk constituents, mainly lipids and carbohydrates, are utilized by the growing chick (moran & reinhart, 1980; reidy et al. 1998). the rate of yolk sac utilization is known to be influenced by exogenous feeding (noy & sklan 2001). it is also possible that various fractions of lipids are metabolized at different rates in the chicks as described by reidy et al. (1998). yolk sac utilization has been shown to be more rapid in fed rather than fasted birds, suggesting that the transport of yolk through the intestine could be increased by the greater intestinal motility found in chicks receiving feed (noy & sklan 2001). this finding lends support to the recommendation that ostrich chicks should be fed on the day they hatch, which is contrary to the common belief that dietary intake of carbohydrates and lipids delays yolk sac absorption. the results of this study suggest that unless the yolk sac is infected, its early removal, before ostrich chicks are 10 days old, may be detrimental. deutectomy has been shown to delay growth by 2 days (murakami et al. 1992). systemic infections or lack of exercise may also reduce chick metabolism and slow down yolk sac utilization. references bertram, b.c.r. & burger, a.e. 1981. aspects of incubation in ostriches. ostrich, 52:36–43. chamblee, t.n., brake, j.d., schultz, c.d. & thaxton, j.b. 1992.yolk sac absorption and initiation of growth in broilers. poultry science, 71:1811–1816. deeming, d.c. 1995. possible effect of microbial infection on yolk utilization in ostrich chicks. the veterinary record, 136: 270–271. foggin, c.m. 1992. veterinary problems of ostriches, in the topaz introduction to practical ostrich farming, edited by m.g. hallam. harare: the ostrich producers association of zimbabwe. huchzermeyer, f.w. 1994. transmissible diseases, in ostrich diseases, edited by f.w. huchzermeyer, agricultural research council, onderstepoort veterinary institute, pretoria, south africa. keffen, r.h. & jarvis, m.j.f. 1984. some measurements relating to ostrich eggs. ostrich, 55:182–187. matsushita, s. 1986. presence of sucrase in the yolk sac of the chick. journal of experimental zoology, 237:337–346. moran, e.t. & reinhart, b.s. 1980. poultry yolk sac amount and composition upon placement: effect of breeder age, egg mass, sex and subsequent change with feeding and fasting. poultry science, 59:1521–1528. murakami, h., akiba, y. & horigychi, m. 1992. growth and utilization of nutrients in newly-hatched chick with or without removal of residual yolk. growth development aging, 56:75–84. noy, y. & sklan, d. 2001. yolk and exogenous utilization in the post-hatch chick. poultry science, 110:490–495. paganelli, c.v., oliszowka, a.j. & ar, a. 1974. the avian egg: surface area, volume and density. condor, 76:319– 325. reidy, j.r., atkinson, j.l. & leeson, s. 1998. size and components of poultry yolk sacs. poultry science, 77:639– 643. sklan, d. 2003. fat and carbohydrate use in post-hatch chicks. poultry science, 82:117–122. 249 e.z. mushi, m.g. binta & r.g. chabo article information authors: elisabeth g. scheffer1,4 gert j. venter2,3 christopher joone1 nikolaus osterrieder4 alan j. guthrie1 affiliations: 1equine research centre, university of pretoria, south africa 2parasites, vectors and vector-borne diseases, arc-onderstepoort veterinary institute, south africa 3department of veterinary tropical diseases, university of pretoria, south africa 4institut für virologie, freie universität berlin, germany correspondence to: alan guthrie email: alan.guthrie@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 21 may 2011 accepted: 27 july 2011 published: 11 nov. 2011 how to cite this article: scheffer, e.g., venter, g.j., joone, c., osterrieder, n. & guthrie, a.j., 2011, ‘use of real-time quantitative reverse transcription polymerase chain reaction for the detection of african horse sickness virus replication in culicoides imicola’, onderstepoort journal of veterinary research 78(1), art. #344, 4 pages. doi:10.4102/ojvr.v78i1.344 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) use of real-time quantitative reverse transcription polymerase chain reaction for the detection of african horse sickness virus replication in culicoides imicola in this original research... open access • abstract • introduction • research method and design    • materials and method • results • discussion • conclusion • acknowledgments    • authors’ contributions • references abstract (back to top) despite its important role as vector for african horse sickness virus (ahsv), very little information is available on the dissemination of this virus in culicoides (avaritia) imicola kieffer (diptera: ceratopogonidae). this study reports on the applicability of a real-time quantitative reverse transcription polymerase chain reaction (rt-qpcr) to detect ahsv in dissected midges. a total of 96 midges were fed on ahsv-infected blood, after which one test group was dissected into head/thorax and abdomen segments immediately after feeding and the other only after 10 days of incubation. the majority of the midges (96%) ingested the virussuccessfully and there was no significant difference between the virus concentration in the heads/thoraxes and the abdomens immediately after feeding. after incubation, virus was detected in 51% of the midges and it was confined to the abdomen in the majority of these. the fact that virus was detected only in the heads/ thoraxes of four culicoides midges after incubation suggests the presence of a mesenteronal escape barrier. replication in the salivary glands was not shown. anincrease of the mean virus concentration in the abdomen after incubation indicates localised viral replication. the real-time rt-qpcr is recommended for further studies investigating the replication and dissemination of ahsv in culicoides midges. introduction (back to top) small biting midges in the genus culicoides (diptera: ceratopogonidae) are involved in the epidemiology and transmission of a number of orbiviruses of veterinary importance, including african horse sickness virus (ahsv) with nine known serotypes (howell 1962). this virus causes an infectious, non-contagious disease,african horse sickness (ahs), which is endemic in sub-saharan africa and can have a mortality rate of up to 95% in susceptible horses. based on its confirmed vector status, wide geographical distribution, abundance and host preference for larger mammals, the afro-asiatic culicoides (avaritia) imicola kieffer is considered the principle vector of ahsv in south africa (meiswinkel, venter & nevill 2004; nevill, venter & edwardes 1992). this species is also the most important vector of orbiviruses across vast geographic regions in africa, the mediterranean and southern europe (mellor, boorman & baylis 2000). following ingestion by a susceptible midge, ahsv infects and replicates in cells of the mesenteron before entering the haemocoel and infecting secondary target organs suchas the fat body and salivary glands (mellor 2000; wittmann & baylis 2000). a number of barriers to arbovirus infection appear to exist in culicoides midges, including the mesenteronal infection and escape barriers and the dissemination barrier. a salivary gland barrier has not been shown to be present in culicoides species (fu et al. 1999; mellor 1990). studies involving the north american vector culicoides (monoculicoides) sonorensis wirth and jones and bluetongue virus (btv) indicate infection of the salivary glands to be an essential prerequisite for the transmission of virus (jennings & mellor 1987). no comparable studies have been performed for c. imicola and/or ahsv. a number of real-time reverse transcription polymerase chain reaction (rt-pcr) assays have been described for ahsv (fernández-pinero et al. 2009; quan et al. 2010; rodríguez-sanchez et al. 2008), all with high sensitivity and a detection limit of 0.001–0.15 tcid50 per reaction. a real-time quantitative rt-pcr (rt-qpcr) with a unique approach of using circulating field isolates of ahsv (quan et al. 2010) has recently been used to determine the infection prevalence of ahsv inculicoides midges. the use of pcr to investigate the replication and distribution of ahsv in culicoides midges has not been described. the objective of this study was to investigate the replication and dissemination of ahsv in field-collected c. imicola by feeding, incubating and dissecting individuals and performing real-time rt-qpcr on the abdomens and the heads/thoraxes. research method and design (back to top) materials and method culicoides biting midges were collected alive using 220 v onderstepoort downdraught suction light traps (venter et al. 1998) at various sites near cattle at the arc-onderstepoort veterinary institute, south africa (25°39’s, 28°11’e; 1 219 m above sea level). after an acclimatising period of 2–3 days at 23.5 ºc and a relative humidity of 50% – 70%, field-collected midges were fed on defibrinated sheep blood containing ahsv serotype 6 at a concentration of 106.1 tcid50/ml through a chicken skin membrane (venter et al. 1991). after a feeding period of 30–40 min the blood-engorged females were separated into two groups: one group was dissected within hours after blood feeding (d0), whilst the other group was dissected after 10 days’ incubation (d10). the blood-engorged females were maintained on a 5% (w/v) sucrose solution containing antibiotics (500 iu penicillin, 500 μg streptomycin and 1.25 μg fungizone per 1 ml sucrose solution) at 23.5 °c (venter & paweska 2007). midges were identified as c. imicola by examination of wing pattern. straight vanna’s microscissors (agar scientific, essex, uk) were used to separate the abdomen (containing the midgut) from the head/thorax (containing the salivary glands). midges that could not be dissected immediately after feeding or incubation were stored overnight in a refrigerator at 4 °c or in a freezer at –70 °c if stored for a longer period. the dissected midges were subjected to real-time rt-qpcr following an adaption of the protocol described by quan et al. (2010). culicoides parts were placed separately in magna lyser green beads (roche products, south africa), containing 300 μl lysis/binding solution (am8500) from the ambion total nucleic acid extraction kit (am1836), to which 2.1 μl β-mercaptoethanol was added, or in 300 μl phosphate-buffered saline. after homogenisation in a magna lyser (roche products, south africa), 100 μl of each sample was mixed with 1 μl carrier rna, 60 μl isopropanol and 20 μl bead mix, the latter consisting of lysis/binding enhancer and magnetic beads. rna extraction was performed using either the magmax express magnetic particle processor (life technologies, usa) or the kingfisher flex automated purification system (thermo fisher scientific, finland). purified water and blood from a clinical case of ahs were used as negative and positive controls, respectively. an aliquot of 5 μl of each extract was mixed with 5 μl primers and probe for part of segment 8, which codes for the structural protein vp7 (quan et al. 2010), to obtain final concentrations of 400 nm for each primer and 180 nm for the probe in the 25 μl reaction. the samples were centrifuged, denatured at 95 °c for 1 min using a pcr machine (geneamp 9700, life technologies, usa) and rapidly chilled at –20 °c for 5 min. a total volume of 15 μl master mix (12.5 μl 2x rt-pcr buffer, 1 μl 25x rt-pcr enzyme and 1.5 μl purified water) was added before the samples were centrifuged again. rt-qpcrwas performed using the stepone plus real time pcr system (life technologies, usa) according to the manufacturer’s instructions. analysis of variance (anova) was used to differentiate between mean cycle threshold (ct) values. statistical differences between experimental groups were analysed using fisher’s exact test and/or χ2 analysis. p-values < 0.05 were considered statistically significant. results (back to top) the results of the rt-qpcr assays on the abdomens and heads/thoraxes of 47 d0 and 49 d10 c. imicola, respectively, are provided in table 1. ahsv was detected in 45 (95.7%) d0 midges, three of which (6%) contained virus only in the head/thorax. there was a significant difference between the number of culicoides that tested pcr positive for ahsv in the abdomen (89.4%) and in the head/thorax (34%). ahsv was detected in 25 d10 midges (51%), with a significantly higher number beingpcr positive for ahsv in the abdomen (49%) than in the head/thorax (8.2%). there was a significant (p < 0.001) decrease in the number of midges in which ahsv was detected in either the head/thorax or the abdomen immediately after bloodfeeding (95.7%) than after 10 days’ incubation (51%). based on the ct values no significant difference was identified in the ahsv concentration between heads/ thoraxes and abdomens of dc. imicola (p > 0.05). only one of the four positive d heads/thoraxes (25%) had a c value below the d mean, whereas 18 of the 24 positive abdomens (75%) had ct values below the mean of d0. table 1: summary of real-time rt-qpcr results for body segments of culicoides imicola after feeding on ahsv-6 infected blood. discussion (back to top) with use of rt-qpcr, ahsv rna was detected in 95.7% of the culicoides midges assayed immediately after feeding on an ahsv-infected blood meal. in previous studies, where similar infection techniques were used, ahsv was isolated only in 44% – 64% of the midges tested immediately after feeding when using cell culture systems (venter & paweska 2007; venter, graham & hamblin 2000). in the present study, ahsv rna was detected in 51% of the midges assayed after incubation. previous oral susceptibility studies using identical incubation conditions reported markedly lower virus recovery. depending on the virus isolate used, results for c. imicola ranged from 4.3% to 26.8% (paweska & venter 2003; venter & paweska 2007; venter et al. 2000). in these studies ahs virions were detected using virus isolation on cell culture systems. rt-qpcr, however, detects viral rna. this technique has been shown to be substantially more sensitive than virus isolation (quanet al. 2010), which may explain the higher values reported in the present study. in most of the d0 midges in which ahsv was found in the head/thorax, virus was also detected in the abdomen. the three c. imicola that tested pcr positive only in the head/thorax were probably harvested and immobilised whilst still taking up the blood meal. the ahsv loads detected in the heads/thoraxes and abdomensof dc. imicola were similar (p > 0.05), implying that no virus replication had taken place yet. however, the mean c value for the abdomens was lower in d10 midges (32.52) than in d0 midges (34.67). a drop of 3.32 in ct values implies a 10-fold increase of double-stranded rna (quan et al. 2010); the observed decrease of 2.15 therefore reveals approximately five times more viral rna in the abdomens of d10 midges compared to d0 midges. the results are even more prominent if one looks at the lowest c value of the abdomens (31.49 in d and 26.55 in d midges, respectively). this difference of almost five c values indicates more than a 50-fold increase of virus load in the abdomens, which was probably due to virus replication in the midgut cells. it has been shown that culicoides midges express various barriers that limit virus replication and transmission. the present results clearly illustrate that not all midges in a population are susceptible to infection with ahsv and that some individuals are able to clear the virus to below detectable levels within 10 days after feeding on a virus-infected blood meal. the mesenteronal infection barrier may have played a role in the proportion of d10 midges (49%) that were able to eliminate ahsv within 10 days without becoming infected. culicoides midges that were pcr positive in the abdomen but exhibited a ct value below detectable limits in the head/ thorax probably expressed a mesenteronal escape barrier, not allowing the virus to escape from the midgut cells. this result relates to a previous study where 43.6% of c. sonorensis exhibited such a barrier to btv (jennings & mellor 1987). in the present study, only four (8.2%) of the d10 midges were pcr positive in the head/thorax, indicating that they expressed neither a mesenteronal escape barrier nor a dissemination barrier. virus that is present in the head/thorax is presumably located in the salivary glands. all four these midges had a higher ct value in the head/thorax than in the abdomen (i.e. less viral rna in the head/thorax), implying that no additional viral replication had taken place in the salivary glands. the salivary glands were not specifically dissected but remained part of the heads/thoraxes. however, this study does not indicate whether this could have influenced the results and secondary viral replication in the salivary glands remains unlikely. the mean ct value of the heads/thoraxes of the d10 midges was not significantly different from that of the d0 midges (p = 0.3847). however, the value was 4.22 units higher than for the abdomens in the former test group, which indicates a substantially lower viral load in their heads/thoraxes. the finding also supports the hypothesis that viral replication did not occur in the salivary glands. conclusion (back to top) the real-time rt-qpcr used in the present study was an adapted version of the protocol optimised for detection of ahsv in blood and organ samples (quan et al. 2010). this adapted assay has recently been used to quantify viral loads in culicoides midge pools and now it has been shown to be a very sensitive method for investigating ahsv viral load differences in different body parts of culicoides midges as well. future studies investigating ahsv replication in culicoides midges should include investigations of ahsv viral load in salivary glands and/or saliva. acknowledgments (back to top) we thank the veterinary genetics laboratory and the department of tropical diseases of the university of pretoria for providing the laboratory facilities. this research was supported by the equine research centre of the university of pretoria and the freie universität, berlin provided a stipend for the principal investigator by means of the nafoeg stipend. authors’ contributions a.j.g. and n.o. were the project leaders, whilst e.g.s., g.j.v. and a.j.g. were responsible for the experimental and project design. the experiments were performed by e.g.s. and c.j. and statistics were performed by g.j.v., e.g.s. wrote the manuscript with contributions from all authors. references (back to top) fernández-pinero, j., fernández-pacheco, p., rodríguez, b., sotelo, e., robles, a., arias, m. et al., 2009, ‘rapid and sensitive detection of african horse sickness virus by real-time pcr’, research in veterinary science 86, 353–358. doi:10.1016/j.rvsc.2008.07.015, pmid:18782637 fu, h., leake, c.j., mertens, p.p. & mellor, p.s., 1999, ‘the barriers to bluetongue virus infection, dissemination and transmission in the vector, culicoides variipennis (diptera: ceratopogonidae)’, archives of virology 144, 747–761. doi:10.1007/s007050050540, pmid:10365165 howell, p.g., 1962, ‘the isolation and identification of further antigenic types of african horsesickness virus’, onderstepoort journal of veterinary research 29, 139–149. jennings, d.m. & mellor, p.s., 1987, ‘variation in the responses of culicoides variipennis (diptera, ceratopogonidae) to oral infection with bluetongue virus’, archives of virology 95, 177–182. doi:10.1007/bf01310778, pmid:3038052 meiswinkel, r., venter, g.j. & nevill, e.m., 2004, ‘vectors: culicoides spp.’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, 2nd edn., pp. 93–136, oxford university press, oxford. mellor, p.s., 1990, ‘the replication of bluetongue virus in culicoides vectors’, current topics in microbiology & immunology 162, 143–161. mellor, p.s., 2000, ‘replication of arboviruses in insect vectors’, journal of comparative pathology 123, 231–247. doi:10.1053/jcpa.2000.0434, pmid:11041993 mellor, p.s., boorman, j. & baylis, m., 2000, ‘culicoides biting midges: their role as arbovirus vectors’, annual review of entomology 45, 307–340. doi:10.1146/annurev.ento.45.1.307, pmid:10761580 nevill, e.m., venter, g.j. & edwardes, m., 1992, ‘potential culicoides vectors of livestock orbiviruses in south africa’, in t.e. walton & b.i. osburn (eds.), bluetongue, african horse sickness, and related orbiviruses: proceedings of the second international symposium, pp. 306–313, crc press, boca raton, florida. paweska, j.t. & venter, g.j., 2003, ‘oral susceptibility of south african culicoides species to live-attenuated serotype-specific vaccine strains of african horse sickness virus (ahsv)’, medical and veterinary entomology 17, 436–447. doi:10.1111/j.1365-2915.2003.00467.x, pmid:14651659 quan, m., lourens, c.w., maclachlan, n.j., gardner, i.a. & guthrie, a.j., 2010, ‘development and optimisation of a duplex real-time reverse transcription quantitative pcr assay targeting the vp7 and ns2 genes of african horse sickness virus’, journal of virological methods 167, 45–52. doi:10.1016/j.jviromet.2010.03.009, pmid:20304015 rodríguez-sanchez, b., fernández-pinero, j., sailleau, c., zientara, s., belak, s., arias, m. et al., 2008, ‘novel gel-based and real-time pcr assays for the improved detection of african horse sickness virus’, journal of virological methods 151, 87–94. doi:10.1016/j.jviromet.2008.03.029, pmid:18501973 venter, g.j., graham, s.d. & hamblin, c., 2000, ‘african horse sickness epidemiology: vector competence of south african culicoides species for virus serotypes 3, 5 and 8’, medical and veterinary entomology 14, 245–250. doi:10.1046/j.1365-2915.2000.00245.x, pmid:11016430 venter, g.j., hill, e., pajor, i.t.p. & nevill, e.m., 1991, ‘the use of a membrane feeding technique to determine the infection rate of culicoides imicola (diptera: ceratopogonidae) for 2 bluetongue virus serotypes in south africa’, onderstepoort journal of veterinary research 58, 5–9. pmid:1646980 venter, g.j. & paweska, j.t., 2007, ‘virus recovery rates for wild-type and live-attenuated vaccine strains of african horse sickness virus serotype 7 in orally infected south african culicoides species’, medical and veterinary entomology 21, 377–383. doi:10.1111/j.1365-2915.2007.00706.x, pmid:18092976 venter, g.j., paweska, j.t., van dijk, a.a., mellor, p.s. & tabachnick, w.j., 1998, ‘vector competence of culicoides bolitinos and c. imicola for south african bluetongue virus serotypes 1, 3 and 4’, medical and veterinary entomology 12, 378–385. doi:10.1046/j.1365-2915.1998.00116.x, pmid:9824821 wittmann, e.j. & baylis, m., 2000, ‘climate change: effects on culicoides-transmitted viruses and implications for the uk’, veterinary journal 160, 107–117. doi:10.1053/tvjl.2000.0470, pmid:10985802 bragg_1-8.qxd introduction infectious coryza (ic), caused by the bacterium haemophilus paragallinarum, remains a serious problem to the poultry industries in many countries, despite the wide-spread use of commercial vaccines. it has been demonstrated that there are four different serovars of nad-dependent h. paragallinarum currently in south africa (kume, sawata, nakase & matsumoto 1983; blackall, eaves & rogers 1990; bragg, coetzee & verschoor 1996). during an investigation of the incidence of the four serovars of nad-dependent h. paragallinarum in south africa, bragg et al. (1996) discovered that there had been a significant change in the incidence of the different serovars over a thirty-year period. it was shown that the incidence of serogroup a isolates had all but disappeared in the 1990s, while the incidence of serovar c-3 had increases significantly during this period. this lead bragg et al. (1996) to postu1 onderstepoort journal of veterinary research, 71:1–8 (2004) limitation of the spread and impact of infectious coryza through the use of a continuous disinfection programme r.r. bragg department of microbial, biochemical and food biotechnology, university of the free state, p.o. box 339, bloemfontein, 9300 south africa. e-mail: braggrr@sci.uovs.ac.za abstract bragg, r.r. 2004. limitation of the spread and impact of infectious coryza through the use of a continuous disinfection programme. onderstepoort journal of veterinary research, 71:1–8 the effect of a continuous disinfection programme, using the non-toxic disinfectant virukill, in layers, on the spread and impact of infectious coryza, caused by haemophilus paragallinarum was evaluated. in this experiment, both unvaccinated layers and layers vaccinated against infectious coryza were used. duplicate smaller groups of vaccinated and unvaccinated chickens were challenged with different serovars of both nad-dependent as well as nad-independent isolates of haemophilus paragallinarum. one group of chickens challenged with each of the different bacterial serovars was treated with the continuous disinfection programme, while the other group remained as the untreated controls. the clinical signs of infectious coryza were evaluated over a period of 20 days in each group. the egg production over this period was also evaluated. it was found in all experimental challenges, that the severity of the symptoms was reduced in the birds receiving the continuous disinfection programme. the drop in egg production was also found to be less severe in the treated groups when compared to the untreated control groups. the duration of infection was found to be either unchanged, or shorter in the birds treated with the continuous disinfection programme. in none of the experimental challenges was the duration or expression of clinical signs of ic increased due to the continuous disinfection programme. keywords: continuous disinfection, haemophilus paragallinarum, infectious coryza, virukill accepted for publication 24 june 2003—editor late that these changes in the incidence of the different isolates was possibly a result of the use of vaccines in this country not containing the correct serovars for this country. as a result of these findings, bragg et al. (1996) and bragg, greyling & verschoor (1997) postulated that there is a need for vaccines containing local isolates in countries where unique serovars of h. paragallinarum occur. bragg (2002b) further demonstrated that the serovar c-3 strain of nad-dependent h. paragallinarum was highly virulent when compared to the other serogroups of nad-dependent h. paragallinarum. the high virulence of serovar c-3 isolates could also be a possible reason for the increase in incidence of this serovar in south africa reported on by bragg et al. (1996). further evidence of the failure of commercially available vaccines for the control of ic was obtained recently in zimbabwe where a severe outbreak, which was caused by the serovar c-3 strain, was reported (bragg 2002a). the ic situation in south africa was further complicated with the discovery of nad-independent variants of h. paragallinarum in the early 1990s when horner, bishop & haw (1992) reported on a bacterium that appeared to be h. paragallinarum, but did not require nad for growth. they suggested that these isolates could not be h. paragallinarum because of the nad independence. mouahid, bisgaard, morley, mutters & mannheim (1992), bragg, coetzee & verschoor (1993) and bragg et al. (1997) suggested that these isolates were nad-independent variants of h. paragallinarum based on dna/ dna hybridization (mouahid et al. 1992) and reactions with monoclonal antibodies specific for h. paragallinarum (bragg et al. 1993). horner, bishop, jarvis & coetzee (1995) later conceded that their isolates were nad-independent h. paragallinarum. the main emphasis on the control of ic should remain on the establishment of the most suitable vaccine to combat all of the strains in a particular country. the development, testing and registration of a new vaccine, however, many take some time. there is thus a need for a simple method for the limitation of the effects of ic in the layers. the disinfectant (virukill, ica international chemicals, p.o. box 2312 stellenbosch, south africa 7601) is a patented formulation of a quaternary ammonium compound, in which the efficacy of the active ingredient has been boosted without increasing the toxicity of the product. it consists of a modification based on a 12 % didecyl dimethyl ammonium chloride (ddac) solution. this product has been shown to have a very low toxicity (data not shown) and a high efficacy against poultry viruses and bacteria (data not shown). because of this low toxicity and high efficacy, the possibilities of using this product for continuous disinfection during poultry production was investigated. the continuous disinfection programme consists of disinfection of hard surfaces during cleanout, treatment of the drinking water and spraying of the birds during production. bragg & plumstead (2003) demonstrated that the full continuous disinfection programme reduced mortalities in broilers under experimental conditions as well as under field conditions in the face of a newcastle disease virus challenge. this raised the question of the possibilities of using the full continual disinfection programme that was developed for broilers to control the impact of ic in layers. in these experiments, the effects of treating chickens that have been experimentally infected with various serovars of both nad-dependent and nadindependent h. paragallinarum, using the continuous disinfection program with ddac was investigated. at the time of these experiments, this disinfectant was the only product available that was registered in south africa (through act 36 of 1947) for use in the drinking water of birds and to spray the birds with. methods and materials chickens a total of 100 unvaccinated commercial layer chickens were obtained from a supplier of point-of-lay chickens. these chickens were obtained at 11 weeks of age before they were vaccinated against ic and originated from a site with no previous history of ic. the chickens were housed in layer facilities until they were used in the experiments. a total of 100 vaccinated commercial point-of-lay chickens were obtained from the same flock from which the unvaccinated chickens were obtained. these chickens had remained on the commercial farm and were vaccinated with the onderstepoort biological products (obp) ic vaccine at 12 weeks with a re-vaccination at 16 weeks. the obp ic vaccine contains serogroup a and c strains as well as the tongaat strain. these chickens remained on the farm until they were 18 weeks of age. after this time, they were moved to the same facilities as the 2 limitation infectious coryza through continuous disinfection programme unvaccinated groups. both vaccinated and unvaccinated chickens remained in the facilities until they were 25 weeks of age and were producing eggs. a total of 20 vaccinated and 20 unvaccinated chickens were removed and placed into layer cages in isolation facilities were they were further sub-divided into two groups of ten vaccinated and two groups of ten unvaccinated chickens. they were placed into cages in such a way that ten chickens were placed into the top five cages (two chickens per cage) and ten chickens were placed into the bottom row of cages. the vaccinated chickens were placed on one side of the battery of cages, while the unvaccinated chickens were placed onto the other side. the drinking water of the bottom row of cages was treated with 100 ppm ddac daily. thereafter the birds were challenged with one of the four different serovars of nad-dependent h. paragallinarum isolates or an nad-independent isolate according to the methods below. challenge and treatment methods the chickens were challenged according to the methods described by bragg (2001b) without modification. basically one chicken in the middle cage was selected and challenged with intra-sinus injection of 0.1 ml of the challenge bacterium. the remaining birds in the group were then infected via natural challenge routes. the clinical signs in the birds were recorded and scored for a 20-day period. the severity of the clinical signs was given a numerical value according to the methods described by bragg (2002b) such that birds with no clinical signs receive a score of 0, birds with mild clinical signs in the naturally infected birds get a score of 2, moderately infected birds received a score of 4 and severely infected birds receive a score of 6. the scoring of the clinical signs in the directly challenge bird was half that of the naturally infected birds. isolates which were used for challenge were 49 (a-1), 58 (b-1), 7 (c-2) and 46 (c-3) of the naddependent isolates and isolate 1343 (c-3), which is an nad-independent variant. all of the isolates used have been previously been isolated and serotyped by the author. as soon as the first clinical signs of ic were seen, the chickens in the bottom row of cages (the same chickens which receive virukill in the drinking water) were fogged with a 1 % solution of virukill such that each chicken received approximately 4 ml of diluted disinfectant. the egg production in each of the groups was also determined by daily counts of the eggs produced. at the termination of the experiment, bacterial samples were collected from both the left and right sinuses of the chickens for bacterial isolation, using the methods described by bragg et al. (1993). results challenge with nad-dependent isolates the different groups of chickens were challenged with the different serovars of nad-dependent isolates of h. paragallinarum. the results of the scoring of the clinical signs were used to produce a graphic representation of the course of the disease. the disease profiles for treated and untreated birds challenged with the different nad-dependent serovars of h. paragallinarum can be seen in fig. 1, for serovar a-1, fig. 2 for serovar b-1, fig. 3 for serovar c-2 and fig. 4 for serovar c-3. a summary of the mean disease scores for each of the different experimental groups, as calculated from the disease profiles, can be seen in table 1. the maximum disease score obtained in each of the experimental groups can also be seen in table 1. finally, the duration of the infection, as calculated from fig. 1–4 as the last day on which clinical signs were seen, is presented in table 1. statistical analysis of the disease profiles obtained was performed using a t-test and these results are included in table 1. challenge with nad-independent serovar c-3 isolate the disease profiles for treated and untreated birds challenged with nad-independent serovar c-3 can be seen in fig. 5 for vaccinated and unvaccinated birds. a summary of the mean disease scores and statistical analysis can be seen in table 1, together with the maximum disease score for each of the groups of chickens. finally, the duration of the infection, as calculated from fig. 5 as the last day on which clinical signs can be seen, is also presented in table 1. egg production eggs were collected from the cages on a daily basis. the number of eggs from each group were carefully recorded and the total number of eggs produced and the number of eggs per bird per day for the different treatments and challenges can all be seen in table 2. 3 r.r. bragg bacterial re-isolation twenty days after challenge, all of the surviving chickens were killed and bacterial re-isolations performed on both the left and right sinuses. it was found that there was very little difference between the groups of chickens treated with ddac and the groups not treated. approximately 60–70 % of all birds were infected in both the left and right sinuses. there was also no difference between the bacterial re-isolations from the vaccinated and unvaccinated chickens. it must be noted that clinical signs of ic were noted in all groups of chickens challenged with the different serovars of h. paragallinarum in these experiments, irrespective of the vaccination status of the birds or the use of the full continual disinfection programme. for this reason, h. paragallinarum could be re-isolated from each group of birds. discussion the effects of a full continuous disinfection programme, using ddac, on the disease profile can 4 limitation infectious coryza through continuous disinfection programme ��� ��� ��� ��� ��� ��� ��� � � � � � �� �� � � � � �� �� �� ��� ��� �������� � � � � �� �� �� �� �� �� � � � �� �� � � �� � ������������������ ��� ��� ��� ��� ��� ��� ��� � � � � � �� �� � � � � �� �� � � � � �� �� �� �� �� �� � � � �� �� � � �� ���������������������� �� ��� ��� �������� ���������������� fig. 1 disease profiles of vaccinated (a) and unvaccinated (b) chickens challenged with a serovar a-1 isolate of h. paragallinarum that were either treated with the continuous disinfection programme (treated) using virukill or not treated (untreated) in any other way �� ��� �� � � �� � � � �� � �� �� ��� ��� �������� � � � � �� �� �� �� �� �� � � � �� �� � � �� �� ��� �� � � � � � �� �� � � � � �� �� � � � � �� �� �� �� �� �� � � � �� �� � � �� ���������������������� ���������������� �� ��� ��� �������� � ������������������ fig. 2 disease profiles of vaccinated (a) and unvaccinated (b) chickens challenged with a serovar b-1 isolate of h. paragallinarum which were either treated with the continuous disinfection programme using virukill (treated) or not treated in any other way (untreated) clearly be seen in fig. 1–5. the full continuous disinfection program, which consisted of drinking water treatment as well as a daily fogging with ddac not only reduced the severity of infections, but also, in many cases reduced the duration of infection. in the case of vaccinated chickens challenged with serovar a-1, treatment with ddac not only reduced the overall clinical signs (which was found to be statistically significant) but also reduced the duration of infection to just 3 days. in the control group, which was housed and infected at the same time as the test group, clinical signs were still recorded up to 20 days post challenge. similar reductions in time of clinical signs were seen when the chickens were challenged with the nad-independent serovar c-3 isolates (table 1) as well as in unvaccinated birds challenged with serovar b-1). in many of the other experimental challenges, the reduction in the period when clinical signs were seen were not as marked as for the above examples. it must be stressed at this point that the scale of the x-axes of the graphs in fig. 1–5 are not the same. this is due to the fact that a marked difference in 5 r.r. bragg � � � � � � � � � � �� �� � � � � �� �� � � � � �� �� �� �� �� �� � � � �� �� � � �� �� ��� ��� �������� ������������������ � �� ��� ��� �������� � � � � � � � � � � �� �� � � � � �� �� � � � � �� �� �� �� �� �� � � � �� �� � � �� ���������������� ���������������������� fig. 3 disease profiles of vaccinated (a) and unvaccinated (b) chickens challenged with a serovar c-2 isolate of h. paragallinarum which were either treated with the continuous disinfection programme using virukill (treated) or not treated in any other way (untreated) �� ��� ��� �������� ���������������� � � � � �� �� � � � � �� �� � � � � � � � � � � �� �� �� �� �� �� � � � �� �� � � �� ���������������������� � � � � � � � � � � �� �� � � � � �� �� � � � � �� �� �� �� �� �� � � � �� �� � � �� � ������������������ �� ��� ��� �������� fig. 4 disease profiles of vaccinated (a) and unvaccinated (b) chickens challenged with a serovar c-3 isolate of h. paragallinarum which were either treated with the continuous disinfection programme using virukill (treated) or not treated in any other way (untreated) 6 limitation infectious coryza through continuous disinfection programme table 1 summary of the disease scores and profiles obtained when vaccinated and unvaccinated chickens challenged with the different serovars of nad-dependent and nad-independent isolates of h. paragallinarum and either treated with a continuous disinfection program or not treated isolate vaccinated treated# mean disease significance highest score duration score (days)## a-1 (dep*) yes yes 0.03 0.0350 0.2 3 a-1 (dep) yes no 0.12 0.6 19 a-1 (dep) no yes 0.16 0.3128 0.8 19 a-1 (dep) no no 0.21 1.2 18 b-1 (dep) yes yes 0.01 0.2801 0.2 2 b-1 (dep) yes no 0.02 0.2 3 b-1 (dep) no yes 0.03 0.0780 0.4 5 b-1 (dep) no no 0.08 0.4 17 c-2 (dep) yes yes 0.15 0.0009 2.0 14 c-2 (dep) yes no 0.87 2.4 > 20 c-2 (dep) no yes 1.25 0.0648 3.2 > 20 c-2 (dep) no no 1.69 4.8 > 20 c-3 (dep) yes yes 0.30 0.0836 0.8 14 c-3 (dep) yes no 0.43 1.2 > 20 c-3 (dep) no yes 2.01 0.3200 5.2 19 c-3 (dep) no no 2.45 6.2 19 c-3 (indep**) yes yes 0 0.0001 0 0 c-3 (indep) yes no 0.41 1.2 15 c-3 (indep) no yes 0.04 0.0006 0.4 2 c-3 (indep) no no 0.41 1.3 19 # treated with virukill in the drinking water at 100 ppm daily and fogged with a 1 % virukill solution daily after observation of the first clinical signs ## calculated as the last day on which clinical signs were seen in the group of chickens * nad dependent ** nad independent table 2 daily egg production and the number of eggs per chicken per day for the experimental period challenged vaccinated treated# total production eggs per bird per day a-1 (dep*) yes yes 95 0.48 a-1 (dep) yes no 83 0.42 a-1 (dep) no yes 102 0.51 a-1 (dep) no no 67 0.34 b-1 (dep) yes yes 125 0.63 b-1 (dep) yes no 129 0.65 b-1 (dep) no yes 116 0.58 b-1 (dep) no no 113 0.56 c-2 (dep) yes yes 62 0.31 c-2 (dep) yes no 48 0.24 c-2 (dep) no yes 107 0.54 c-2 (dep) no no 70 0.35 c-3 (dep) yes yes 97 0.49 c-3 (dep) yes no 120 0.60 c-3 (dep) no yes 71 0.36 c-3 (dep) no no 55 0.28 c-3 (indep**) yes yes 152 0.76 c-3 (indep) yes no 125 0.63 c-3 (indep) no yes 118 0.59 c-3 (indep) no no 121 0.61 # treated with virukill in the drinking water at 100 ppm daily and fogged with a 1 % virukill solution daily after observation of the first clinical signs * nad dependent ** nad independent virulence was detected between the different serovars of the nad-dependent isolates. it was interesting to note that in this experiment, the serovar c-3 isolate was found to be the most virulent in unvaccinated chickens. this was followed by serovar c-2. serovar a-1 showed mild virulence, while serovar b-1 was the least virulent. these results correspond to the findings of bragg (2002b) where similar virulence patterns were seen. it is also interesting to note that the mean disease scores obtained in these experiments were similar to those obtained by bragg (2002b) despite the fact that these chickens were not challenged at the same time. this indicates that the mean disease score remains constant for the different serovars over time. it was also interesting to note that the nad-independent isolate used in this experiment was much less virulent than the nad-dependent serovar c-3 isolate. this is in agreement with the findings of bragg (2002c) who demonstrated that the nadindependent isolates were less virulent than the nad-dependent isolates of the same serovar. in table 2, the effects of the full continuous disinfection program treatment on egg production can be seen. in most cases, the chickens subjected to the full continuous disinfection program laid more eggs than those not receiving the treatment. in some cases there is a substantial difference between the numbers of eggs per chicken per day in the treated group. for example, when unvaccinated chickens were challenged with serovar a-1 (which appears to be of low virulence) the egg production in the treated group was 0.51 eggs per bird per day. in the untreated control group, this was 0.34 eggs bird day. over the 20 days of the experimental challenge, this difference resulted in 102 eggs being produced by the ten chickens forming the treated group, while the control group produced only 67 eggs. similar substantial differences in egg production were also seen when unvaccinated chickens were challenged with serovar c-2. in most other cases improved egg production was recorded. where no improved egg production was seen as a result of the treatment the chickens showed only very mild clinical signs (for example when the chickens were challenged with serovar b-1). a notable exception to this was when vaccinated chickens were challenged with the nad-independent serovar c isolate. in this case, higher egg production was recorded in the control group. statistical analysis was not performed on the egg production data of the different groups as only ten birds per group were used. the egg production in these experiments was recorded as an indication of the effects of the full continuous disinfection on the impact of ic on the birds. it must be noted that the birds were sprayed with the disinfectant only when the first clinical signs of ic were recorded. the disinfectant was used in the drinking water from when the birds where placed into the cages. this method was selected to simu7 r.r. bragg ��� ��� ��� ��� ��� ��� ��� � � � � � �� �� � � � � �� �� � � � � �� �� �� �� �� �� � � � �� �� � � �� ��� ��� ��� ��� ��� ��� ��� � � � � �� �� � � �� � � � � �� �� � � � � �� �� �� ��� ��� �������� � � � � �� �� �� �� �� �� ���������������� �� ��� ��� �������� ���������������� ����������������������� ������������������ fig. 5 disease profiles of vaccinated (a) and unvaccinated (b) chickens challenged with an nad-independent serovar c-3 isolate of h. paragallinarum which were either treated with the continuous disinfection programme using virukill (treated) or not treated in any other way (untreated) late what would happen on a production farm, were spraying with a disinfectant would only start once clinical signs were seen. in the full continuous disinfection programme in broilers (bragg & plumstead 2003) birds were routinely sprayed with ddac from date of placement. if this approach was followed in these experiments, the impact of h. paragallinarum on the birds might have been even further reduced. conclusions from these results it can be seen that treatment of chickens infected with ic with the non-toxic disinfectant ddac reduced the duration and severity of clinical signs of the disease. furthermore, in most cases, treatment with the disinfectant also resulted in higher egg production, in the face of challenge by h. paragallinarum. it must be stressed that these experiments were done on a small scale in experimental layer cages with only ten chickens per group. although there is a good indication from these results that treatment with ddac can reduce the severity of infection, these finds need to be substantiated on full-scale production facilities where detailed statistical analysis could be performed on a larger population. it is also extremely important to note that the treatment of chickens with ddac should not be seen as a replacement for effective vaccination. in all of the cases, there was a significant difference between the disease profiles of the vaccinated chickens compared to the unvaccinated ones. in none of the cases examined was the disease profile of the unvaccinated chickens which had been treated with ddac less then that disease profile of vaccinated untreated chickens. the effective vaccination of chickens remains the main defence against ic. acknowledgements i am grateful to ica international chemicals, stellenbosch, who funded this research. references blackall, p.j., eaves, l.e. & rogers, d.g. 1990. proposal of a new serovar and altered nomenclature for haemophilus paragallinarum in the kume hemaglutinin scheme. journal of clinical microbiology, 28:1185–1187. bragg, r.r. 2002a. isolation of serovar c-3 haemophilus paragallinarum from zimbabwe: a further indication of the need for local vaccines against infectious coryza. onderstepoort journal of veterinary research , 69:129–132. bragg, r.r. 2002b. virulence of south african isolates of haemophilus paragallinarum. part 1: nad-dependent field isolates. onderstepoort journal of veterinary research, 69: 163–169. bragg, r.r. 2002c. virulence of south african isolates of haemophilus paragallinarum. part 2: naturally occurring nad-independent field isolates. onderstepoort journal of veterinary research, 69:171–175. bragg, r.r. & plumstead, p. 2003. continuous disinfection as a means to control infectious diseases in poultry. evaluation of a continuous disinfection programme for broilers. onderstepoort journal of veterinary research, 70:219–229. bragg, r.r., coetzee, l. & verschoor, j.a. 1993. plasmid encoded nad independence in some south african isolates of haemophilus paragallinarum. onderstepoort journal of veterinary research, 60:147–152. bragg, r.r., coetzee, l. & verschoor, j.a. 1996. changes in the incidence of the different serovars of haemophilus paragallinarum in south africa: a possible explanation for vaccination failures. onderstepoort journal of veterinary research, 63:217–226. bragg, r.r., greyling, j.m. & verschoor, j.a. 1997. isolation and identification of nad-independent bacteria from chickens with symptoms of infectious coryza. avian pathology, 26:595–606. kume, k., sawata, a., nakase, y. & matsumoto, m. 1983. serological classification of haemophilus paragallinarum with a hemagglutinin scheme. journal of clinical microbiology, 17:985–964. horner, r.f., bishop, g.c. & haw, c. 1992. an upper respiratory disease of commercial chickens resembling infectious coryza, but caused by a v factor independent bacterium. avian pathology, 21:421–427. horner, r.f., bishop, g.c., jarvis, c.j. & coetzee, t.h.t. 1995. nad (v-factor)-independent and typical haemophilus paragallinarum infecting commercial chickens: a five year field study. avian pathology, 24:453–463. mouahid, m., bisgaard, m., morley, a.j., mutters, r. & mannheim, w. 1992. occurrence of v-factor (nad)independent strains of haemophilus paragallinarum. veterinary microbiology, 31:363–368. 8 limitation infectious coryza through continuous disinfection programme abstract introduction host range the sequential discovery of human coronaviruses diversity and evolution of coronaviruses inter-host transmission of coronaviruses: what is known human-to-human transmission of coronaviruses conclusion acknowledgements references about the author(s) elijah n. mulabbi department of veterinary medicine, faculty of veterinary medicine, animal resources and biosecurity, makerere university, kampala, uganda robert tweyongyere department of veterinary medicine, faculty of veterinary medicine, animal resources and biosecurity, makerere university, kampala, uganda denis k. byarugaba department of veterinary medicine, faculty of veterinary medicine, animal resources and biosecurity, makerere university, kampala, uganda citation mulabb, e.n., tweyongyere, r. & byarugaba, d.k., 2021, ‘the history of the emergence and transmission of human coronaviruses’, onderstepoort journal of veterinary research 88(1), a1872. https://doi.org/10.4102/ojvr.v88i1.1872 review article the history of the emergence and transmission of human coronaviruses elijah n. mulabbi, robert tweyongyere, denis k. byarugaba received: 16 apr. 2020; accepted: 25 sept. 2020; published: 10 feb. 2021 copyright: © 2021. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract human coronaviruses are known respiratory pathogens associated with a range of respiratory illnesses, and there are considerable morbidity and hospitalisation amongst immune-compromised individuals of all age groups. the emergence of a highly pathogenic human coronavirus in china in 2019 has confirmed the long-held opinion that these viruses are important emerging and re-emerging pathogens. in this review article, we trace the discovery and emergence of coronaviruses (covs) over time since they were first reported. the review article will enrich our understanding on the host range, diversity and evolution, transmission of human covs and the threat posed by these viruses circulating in animal populations but overtime have spilled over to humans because of the increased proximity between humans and animals. keywords: coronaviridae; host range; diversity and evolution; transmission dynamics; mutation; recombination. introduction coronaviruses (covs) are positive-sense, single-stranded ribonucleic acid (rna) viruses with a linear, non-segmented viral genome. amongst all the rna viruses, they have the largest genome of about 27–32 kb, packed in a helical nucleocapsid. they are named covs because of the crown-like appearance of the surface projections. coronaviruses cause mainly respiratory and enteric diseases in mammals and birds with varying severity (dijkman et al. 2013; sipulwa et al. 2016; weiss & navas-martin 2005). coronaviruses are the largest group of viruses belonging to order nidovirales, family coronaviridae, in the sub-family coronavirinae. the sub-family coronavirinae is divided into four genera: the alpha (human coronavirus 229e [hcov-229e] and human coronavirus nl63 [hcov-nl63]); beta (human coronavirus oc43 [hcov-oc43], human coronavirus hku1 [hcov-hku1], severe acute respiratory syndrome coronavirus [sars-cov] and middle east respiratory syndrome–related coronavirus [mers-cov]); gamma and deltacoronaviruses (zhang et al. 2018). the gamma and deltacoronaviruses infect birds. coronaviruses have a highly conserved genomic organisation with a single large 5’ open reading frame (orf), constituting two-thirds of the genome encoding a replicase polyprotein, rna-dependent rna polymerase (rdrp) (fehr & perlman 2015). this is then followed by several additional orfs that encode both structural and non-structural proteins. these structural proteins include the nucleocapsid (n) protein, matrix (m) protein, envelope (e) protein and the spike (s) protein (masters & perlman 2013). the spike protein is the major determinant of the virulence of covs because it mediates the binding of the virus to the specific host receptor. host range coronaviruses exhibit a broad host range, infecting many mammalian and avian species, where they cause a broad spectrum of diseases in the respective hosts. the specificity to the host is because of the presence of specific receptors in the host, which interact with the cov through the spike protein during attachment and entry into the host (lim et al. 2016). it is by this convenience that most covs are named based on their host or site in the host at which the pathology manifests. although this is so, there is a great possibility of cross infection of covs because of their ability to jump species barriers, which in result makes them have a broad host range (cavanagh 2005). some covs are known to be promiscuous being detected in more than one host, like some bovine coronavirus (bcov, hcov-oc43 and other related viruses), which have been detected in cows, humans, horses, dogs, camels, deer, antelopes and giraffes (alekseev et al. 2008; hasoksuz et al. 2007; jin et al. 2007; lim et al. 2013), whilst others like the most recently described deltacoronaviruses are host-specific and confined to a single host. bats have been known to be reservoir hosts of alpha and betacoronaviruses, which predominantly affect mammals, whilst birds are reservoir hosts of gamma and deltacoronaviruses, which affect avian species and some mammalian species (drexler, corman & drosten 2014; woo et al. 2012). specifically, the host of hcov-229e are bats, hcov-nl63 are bats and palm civets, hcov-oc43 are cattle, hcov-hku1 are mice, sars-cov are bats and palm civets, mers-cov are bats and camels, and for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) the hosts are yet to be established but suspected to be bats and an unknown mammal intermediate host (bolles, donaldson & baric 2011; corman et al. 2018; guo et al. 2020; reusken et al. 2016). the host receptors of human covs have been identified: aminopeptidase n for hcov-229e, angiotensin-converting enzyme 2 for sars-cov, sars-cov-2 and hcov-nl63, cd26 commonly called dipeptidyl peptidase 4 for mers-cov and 9-o-acetylated sialic acid for hcov-oc43 and hcov-hku1 (bonavia et al. 2003; hofmann et al. 2005; huang et al. 2015; lu, hu et al. 2013; van der hoek 2007; zou et al. 2020). having noted earlier that covs have the ability to break species barriers, this definitively points to the variations that occur in the spike protein and ultimately the evolution of covs. the sequential discovery of human coronaviruses coronaviruses have been described for close to a century as avian infectious bronchitis virus (ibv), which is the first cov to be isolated in 1937, followed by murine hepatitis virus (mhv), which was reported a decade later (bailey et al. 1949; cavanagh 2005; cheever et al. 1949). this shows that animal covs have been known since the 1930s, including those that are highly pathogenic to animals, such as transmissible gastroenteritis virus (tgev), bovine coronavirus (bcov) and feline infectious peritonitis virus (fipv) (saif 2004). the first human cov to be isolated was hcov-229e during a study of respiratory illness amongst medical students in the university of chicago and was named 229e because it was isolated from student specimen 229e (hamre & procknow 1966). subsequently, in 1965, viruses b814 and hcov-oc43 were isolated from patients with common cold. by inoculation onto organ cultures of the respiratory tract, these viruses together with hcov-229e were shown to have similar morphological characteristics with the ibv of chicken that had been isolated earlier (hamre & procknow 1966; mcintosh et al. 1967; tyrrell & bynoe 1966). since then, several human and animal covs have been studied and have been shown to have a characteristic virion structure by electron microscopy after negative staining. the first diagnostic assays were based on tissue cultures and serological surveillance by measuring the significant rise in antibody titer. hcov-229e and hcov-oc43 were the only strains followed up after 1965 up to 1990s because they were the only strains that could easily be cultured, the other strains (oc16, oc37, oc38, oc44 and oc48) including the first identified human cov, b814, were lost because of the inability to culture then; thus, no further investigations could be done on them (van der hoek 2007). with increasing research and discovery of new scientific techniques, the number and the importance of covs have grown exponentially; there has been discovery of many novel covs in animals and humans. before 2002, only hcov 229e and hcov oc43 were known to be circulating in human population causing common cold, and they were relatively considered as harmless because they caused mild illness. however, in 2002, a cov causing severe acute respiratory illness was discovered and was named severe acute respiratory syndrome coronavirus (sars-cov). severe acute respiratory syndrome coronavirus initially emerged in 2002–2003 in guangdong province in south china and caused severe respiratory infection with high morbidity and mortality that had not been experienced with covs before. the disease was more pronounced in the elderly, infants and those with underlying conditions that make them immunocompromised (tsang & zhong 2003). although sars-cov disappeared a year after its emergence (feng & gao 2007), new strains of covs have continuously been discovered, and indeed in 2003, two more covs were discovered – hcov-nl63 and hcov-hku1 (drexler et al. 2014; van der hoek et al. 2004; woo et al. 2005). hcov-nl63 was isolated in the netherlands in 2003 from a child suffering from bronchiolitis and conjunctivitis, and this was not an isolated case as the virus was identified in clinical specimens from seven additional individuals who included infants and adults (van der hoek et al. 2004). in 2004, hku1 was discovered in hong kong from a 71-year-old man with pneumonia who had just returned from shenzhen, china (woo et al. 2005). ten years after the discovery of sars-cov, another human cov that causes severe respiratory illness was discovered in 2012, initially named human coronavirus erasmus medical center (hcov-emc), but later the international committee on the taxonomy of viruses named it middle east respiratory syndrome (mers) cov (chan et al. 2015; de groot et al. 2013; zaki et al. 2012). mers-cov was first identified in saudi arabia from a 60-year-old man with acute pneumonia, developed respiratory distress, renal failure and finally succumbed to the disease (widagdo et al. 2017). mers-cov causes a series of highly pathogenic lower respiratory tract infections with a higher case fatality rate of 35% compared to 11% of sars-cov (chan-yeung & xu 2003; donnelly et al. 2019). however, unlike sars which disappeared a year after its discovery, mers has remained causing outbreaks in the middle east, notably in saudi arabia (2014–2016) and in south korea (2015) (berry, gamieldien & fielding 2015; fehr & perlman 2015). just 7 years after the discovery of mers-cov, in december 2019, a novel human cov that is similar to sars-cov was discovered and isolated from patients in the hubei province, china, suffering from pneumonia, but has since spread to many countries across all continents (zhu et al. 2020). this novel virus causes coronavirus disease (covid-19), which has been declared a pandemic by the world health organisation (summers et al. 2020). this novel cov, initially designated as 2019-ncov but now named sars-cov-2, forms the seventh cov that infects humans (zhu et al. 2020). diversity and evolution of coronaviruses in all the covs that have so far been reported, occurrence has been spontaneous and their source has been mysterious. schools of thought have suggested recombination and the low fidelity of the rdrp as the main contributing factors that drive the evolution and diversity of the different covs with their hosts. recombination involves the exchange of genetic information between two non-segmented rna genomes, contributing to the genetic stability of picoraviruses and covs where it has been demonstrated to occur (lai et al. 1985). homologous recombination occurs between two regions of high sequence similarity of a single strain of a cov. ribonucleic acid recombination in covs can occur almost anywhere on the genome, forming many recombinants with multiple crossover events. this shows that covs have a high frequency of recombination, which gives them an emergence and epidemiological advantage (lai 1992a). the frequency of recombination in covs is so high that even in the absence of selection pressure, recombinants can be formed and can become the most dominant population amongst the viruses (lai 1992b; simon-loriere & holmes 2011). non-homologous recombination occurs if a cell is infected by two strains of a given species of cov, and exchange between genetically different rna genomes can occur resulting in a progeny with the sequence derived from both the parent strains (cavanagh 2005; simon-loriere & holmes 2011). although the probability of this non-homologous recombination amongst distantly related strains is small because of the need of sequence similarity, it is quite common in covs. coronaviruses have a mutation frequency just like other rna viruses because of the error-prone rdrp, which lacks a proofreading and mismatch repair capability. mismatch repair processes are not possible for genomes of most rna viruses because they replicate and package single-stranded genomes. the only mechanism of correcting the mismatch would be a replicase-associated proofreading mechanism that the polymerase lacks (elena & sanjuán 2005; steinhauer, domingo & holland 1992). this makes covs have high mutation rates, which can result in potentially adaptively useful genetic variation (drake et al. 1998). all these events, together with re-assorting mutations, give covs a potential to generate novel viral phenotypes by expanding their repertoire of essential genes with many new genes (forni et al. 2017). this plasticity of the genome provides covs a selective advantage and fast adaptability to usual and unusual natural hosts propelling them along the evolution path and may yield virus strains of unexpected virulence. all human covs originate from bats; so, bats definitely play an important role as the gene source in the evolution of human covs. in the lineage of bat covs, they are known to have jumped to other bat species and other mammalian species including humans, with each interspecies jumping resulting in dichotomous evolution to give rise to alphacoronaviruses and betacoronaviruses (woo et al. 2012). this jumping phenomenon is quite common in the lineage of covs although the mechanism by which they jump and switch to new hosts is not clearly understood. the great diversity of covs circulating in the bats has been attributed to their rich species diversity, high population densities and the ability to fly over long distances. the frequency and diversity of covs in bats have been globally detected in all continents with appreciable genetic similarity to human covs (anthony et al. 2017; li et al. 2005; tong et al. 2009). inter-host transmission of coronaviruses: what is known as noted earlier that bats and birds are reservoir hosts of all the covs and act as the gene source in the evolution pathway of these viruses, transmission starts from them although the mechanism of zoonotic transmission from bats to humans is unclear. it has, however, been suggested that intermediate hosts such as carnivores or herbivores are involved in the transmission of these viruses (cotten et al. 2013; enserink 2013; graham & baric 2010). human coronavirus 229e (hcov-229e) and alpaca-cov have been shown to have a close relationship with the diverse covs that exist in hipposiderid bats in africa, which are the natural hosts of that lineage (corman et al. 2015). humans can come into contact with hipposiderid bats in their natural habitats, which can suggest direct transmission of hcov-229e from bats rather than from alpaca, which do not share habitats with these bats. however, further analysis shows that covs that have close genomic similarity with hcov-229e occur in camelids and suggests that hcov-229e evolved towards the human genotype in camelids thereby identifying camelids as the zoonotic source of human infection. the host switching of 229e virus from bats to humans must have been facilitated by deletions in the spike gene of the bat-associated 229e viruses (corman et al. 2018). human coronavirus oc43 (hcov-oc43) is a betacoronavirus. betacoronaviruses have strains circulating in many highly divergent mammalian hosts such as primates, lagomorphs, artiodactyls, perissodactyls, rodents and carnivores (alekseev et al. 2008; drexler et al. 2014; erles et al. 2003; guy et al. 2000; hasoksuz et al. 2007; lau et al. 2012; lim et al. 2013; majhdi, minocha & kapil 1997; tsunemitsu et al. 1995; woo et al. 2014). bovine coronavirus is the best studied representative of these animal covs; the great diversity of beta-covs in livestock suggests them to be the zoonotic sources of hcov-oc43. like other covs, mutations in the spike gene reflect the adaptation of hcov-oc43 to the human host. hcov-oc43, like other beta-covs, have no ancestral link to bats but rather to rodents because of their close similarity to mouse hepatitis virus (mhv), thus believed to have speciated in rodents (corman et al. 2018). the mechanism of transmission from rodents to bovine is not well documented but the close proximity between humans and bovine causes the spillover to humans. human coronavirus nl63 (hcov-nl63) has been found to be related to covs of bats in the families vespertillionidae and hipposideridae, which points to the ancestry origin of hcov-nl43 (drexler et al. 2010; gloza-rausch et al. 2008; pfefferle et al. 2009; tao et al. 2017). the mechanism of transmission of hcov-nl43 to humans is not documented with no zoonotic reservoir so far identified. there is no viral sequences relating hcov-hku1 to other animal species, save the relationship it has with rodent-associated viruses (wang et al. 2015; woo et al. 2005). just like hcov-nl63, the mechanism of transmission of hcov-hku1 to humans is not documented with no zoonotic reservoir so far identified. considering the recent epidemics in humans with the spread of sars-cov, mers-cov and sars-cov-2, new insights have occurred in the transmission patterns of covs. sars-cov was previously thought to have originated from wild animals, the civet cats, raccoon, dogs and ferret-badger (guan et al. 2003) but cumulative phylogenetic studies have pointed to a bat origin of sars-cov. this is because there is no evidence for the circulation of sars-cov like viruses in palm civets in both the wild and breeding facilities (wang et al. 2006). therefore, these animals are only incidental hosts of sars-cov, and the live markets were probably sites where the interspecies transfer of the animal virus to human occurred (weiss & navas-martin 2005). sars-cov has been reported to infect macaque monkeys and domestic cats but transmission from these domestic cats to man has not been demonstrated (fouchier et al. 2003; navas-martin & weiss 2003). however, its ability to infect other animals suggests that sars-cov could be having a natural wild reservoir from which future outbreaks can originate. the first search for the reservoir of mers-cov focused on bats because of the genetic close relatedness of mers-cov to tylonycteris bat cov hku4 and pipistrellus bat cov khu5. however, molecular clock analysis suggested that these bat covs are unlikely to be the direct ancestor of mers-cov (chan, lau & woo 2013). molecular and serological surveys in dromedary camels from oman, canary islands, qatar and saudi arabia give evidence that these animals are the reservoir of mers-cov (azhar et al. 2014; haagmans et al. 2014; raj et al. 2014; reusken et al. 2013). dromedary camels in saudi arabia harbour several viral genetic lineages including those that actually caused human outbreaks, and in addition, an infectious virus has been isolated from them pointing at their important role in the transmission of mers-cov (sabir et al. 2016). the closeness of dromedary camels to humans results in continuous zoonotic transmission of the mers-cov to humans, thus explaining the cause of new infection in humans compared to sars-cov, where no new infections have been reported in humans since january 2004 because there is limited human–bat or human–intermediate host interactions (baseler et al. 2016; wang, potter et al. 2005). severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the newly discovered human cov, has been linked to a zoonotic source with a spillover to humans at huanan seafood wholesale market in wuhan, china. there were a number of non-aquatic birds and rabbits that were on sale in that market before the outbreak (mackenzie & smith 2020). through next-generation sequencing, sars-cov has been showed to be closely related to two bat-derived sars-like covs, bat-slcovzc45 and bat-slcovzxc21, which have been previously characterised from bats in china (hu et al. 2018). this is consistent with the fact that bats are natural reservoirs of covs. just like other human covs that have intermediate hosts, it is likely that sars-cov-2 has an unidentified intermediate animal host sold in the seafood market from which the virus spread to humans (li, zai et al. 2020; liu et al. 2020). this is so because there is no proof that there was proximity between humans and bats that host the virus, because in december the bats are known to have been hibernating (lu et al. 2020). the genomic sequences of sars-cov 2 from different patients have a 99.9% identity, which suggested that the virus originated from one source within a very short period and spread quite rapidly. human-to-human transmission of coronaviruses once zoonotic viruses have succeeded in breaking the species barrier to infect humans, their success in the human population depends on its ability to acquire sustained human-to-human transmissibility. human-to-human transmission of covs occurs directly or indirectly through airborne route or contact route. direct transmission requires the physical contact between an infected person and a healthy person, followed by the transfer of the virus through touching, contact with body fluids and inhalation of respiratory secretions. indirect transmission requires contact between a susceptible person with a contaminated surface and resultant transfer of the virus into entry points into the body like the respiratory pathway. the four human covs that are endemic in human populations and cause self-limiting common cold (hcov 229e, hku1, nl63 and oc43) are droplet-transmitted; these virus-containing droplets are released to the environment when an infected person breathes, coughs, sneezes or talks. this efficient human-to-human transmission of these virus is sustainable because these viruses replicate mainly in the central and upper parts of the respiratory tract (perlman & netland 2009; richard et al. 2017; wege & ter meulen 1982). sustainable human-to-human transmission is also dependent on the stability of the viruses in the environment. large virus-containing droplets released from infected persons do not remain suspended in the air for long but rather move for a short distance and surfaces or mucosa of close contacts. smaller droplets can remain suspended in air for long period of time and carried further from the infected person. hcov-nl63 can survive for 7 days in aqueous solution and respiratory sections and remains infective at room temperature for long (abdul-rasool & fielding 2010; müller et al. 2008). hcov-229e can survive and be detectable after 6 days at 20 °c and 50% relative humidity; this survival can be enhanced further at lower temperature and higher humidity (ijaz et al. 1985). literature about the stability in the environment of hcov-ku1 and hcov-oc43 is scanty but human-to-human transmission is the documented mode of transmission. human-to-human transmission of sars-cov and mers-cov occurs through modes like droplets, direct human-to-human transmission and through fomites. however, long-term and sustained human-to-human transmission has not been established in these zoonotic viruses. mers-cov has a lower human-to-human transmission than that of sars-cov (lu, liu et al. 2013). human-to-human transmission of sars-cov occurs rapidly, which explained the rapid spread of sars across 29 countries within only 6 months (groneberg et al. 2003; peiris, yuen et al. 2003). three instances of laboratory-acquired sars-cov infection were reported in singapore, china and taiwan, but in each of these cases, there were no sustained human-to-human transmission to cause a threat of a recurrent global outbreak but rather highlighted the potential risk when biosafety procedures in the laboratory are not adhered to (enserink & du 2004; liang et al. 2004; lim et al. 2004). human-to-human transmissions amongst family members contributed only 13% – 21% of the mers cases and 22% – 39% sars cases. this shows that the nosocomial route is the main route of transmission of these viruses because substantial virus shedding occurs only after the onset of symptoms and that is when patients seek treatment in medical centres (anderson et al. 2004; baseler et al. 2016; cowling et al. 2015; peiris, lai et al. 2003). studies showed that 44% – 100% of the mers-cov-infected individuals during the outbreak were linked to hospitals where they got the infection, and a similar observation was made for some clusters of sars patients (chowell et al. 2015; hunter et al. 2016). mers-cov remains stable and viable for 48 h at 20 °c and 40% relative humidity, whilst sars-cov for 5 days at 22 °c – 25 °c and 40% – 50% relative humidity. however, both lose viability rapidly at a higher temperature and humidity (van doremalen, bushmaker & munster 2013). this stability enables these viruses to be transmitted as aerosols; because sars-cov is more stable, it has a more sustained transmission than mers-cov through aerosols. fomite transmission of these viruses is possible because of the stability of these viruses; they stay on inanimate surfaces long enough such that by the time humans touch the surfaces, they get infected (lee & wong 2015). this explains the infection of humans who were not in close proximity with index patients in the respective outbreaks. sars-cov was shown to be excreted in stool and remains infectious in sewerage for 2 days; therefore, broken sewerage systems can be a mechanism of spread of sars-cov (wang, li et al. 2005). the newly identified sars-cov-2 has been suggested to be transmitted through droplet transmission and human-to-human transmission as evidenced with clusters of infected families and medical workers who have not had any exposure to animal markets where the infection started (carlos et al. 2020; chang et al. 2020; li, guan et al. 2020; wang et al. 2020). just like other hcovs, nosocomial transmission has been implicated as an important mode of acquiring infection; actually one study suggested hospital transmission to be 41% amongst the patients (wang et al. 2020). the stability of sars-cov-2 on contaminated surfaces and remaining viable for hours in aerosols or days on surfaces allow virus transmission through aerosols and fomites (van doremalen et al. 2020). sars-cov-2 has been detected in stool, suggesting that viral shading in stool is a potential route of transmission when persons get in contact with contaminated sewerage and also points to the possibility of fecal-oral transmission of the virus (young et al. 2020). in comparison with sars-cov and mers-cov, where intestinal infections were observed at some later stages of infection, patients infected with sars-cov-2 may harbour the virus in the intestine in the early or later stages of infection. detection of sars-cov in oral swabs, anal swabs and blood point to body fluids as an alternative mode of transmission through body fluids (zhang et al. 2020). there are more studies being done, which will improve our understanding on the human-to-human transmission dynamics of sars-cov-2. conclusion human covs are increasingly becoming important emerging pathogens from the previously known mild infections to severe acute respiratory infection with high fatality rates. like other emerging pathogens, human covs pose a challenge to science and medicine because of the scantly information about them before they emerge from initially unknown zoonotic sources. with the increasing human population and climate change, people have increased proximity with animals, be either encroaching on their habitats or trade, and therefore zoonotic spillover of these viruses is continuously likely to occur. the biggest challenge here is that therapies and vaccination remedies have not been developed to match the emergence of these viruses, leaving treatment to be limited to non-specific supportive therapy. since bats have been known to be an important reservoir of human viruses for several years, there should be continuous efforts to characterise the covs circulating in bat populations. there is a need to study the mechanisms through which these viruses find their way to the intermediate hosts because these intermediate hosts play an important role in linking bats to human populations. the possibility of direct transmission from bats to humans can also be investigated. not only should these intermediate hosts be identified but also viral surveillance studies have to be expanded to wild and domestic animals such as bovine, rodents and carnivores in order to establish the important human covs that occur in these animals before they spill over to humans and cause pandemics. mutations and recombination that occur in rna viruses should be the contributory factor in the emergence of these viruses where they become able to utilise the human receptors that are required for successful infection and the progress of disease. therefore, there is continuous need to monitor the effect of these mutations that occur in these viruses over a given period of time such that their emergence does not take the population by surprise and also precautionary measures should be taken early enough. with lack of therapies, avenues of animal-to-human transmissions must be blocked. this will require stringent regulatory mechanisms to control the trading of wild species in markets, creating buffer zones around habitats of wild animals and also an overall change in the cultural practices of communities. these can be enriched with the other avenues that are popularly used such as case isolation, quarantine and limiting the overall population mobility in this era where there is rapid expansion of transport networks. the one health concept will always be a great approach in detecting, containing and eliminating public health risks from zoonotic pathogens. acknowledgements the authors wish to acknowledge the makerere univeristy walter reed project and the staff of the emerging infectious diseases laboratory. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions m.n.e. is the corresponding author, wrote the draft of the review article and r.t. and d.k.b. read and gave comments for improving the manuscript. ethical consideration the authors confirm that ethical clearance was not required for the study. funding information this review article received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. data availability statement data sharing is not applicable to this article as no new data were created whilst writing this review article. disclaimer the views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors. references abdul-rasool, s. & fielding, b.c., 2010, ‘understanding human coronavirus hcov-nl63’, the open virology journal 4, 76. pmid: 20700397. 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https://doi.org/10.1056/nejmoa2001017 zou, x., chen, k., zou, j., han, p., hao, j. & han, z., 2020, ‘single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection’, frontiers of medicine 14, 1–8. https://doi.org/10.1007/s11684-020-0754-0 abstract introduction methods and materials results discussion acknowledgements references appendix 1 about the author(s) tlou a. ngoepe department of immunology, agricultural research council-onderstepoort veterinary research, pretoria, south africa department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa department of immunology, faculty of health sciences, university of pretoria, pretoria, south africa alri pretorius department of immunology, agricultural research council-onderstepoort veterinary research, pretoria, south africa department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa helena c. steyn department of immunology, agricultural research council-onderstepoort veterinary research, pretoria, south africa mirinda van kleef department of immunology, agricultural research council-onderstepoort veterinary research, pretoria, south africa department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation ngoepe, t.a., pretorius, a., steyn, h.c. & van kleef, m., 2023, ‘th1 and th2 epitopes of cowdria polymorphic gene 1 of ehrlichia ruminantium’, onderstepoort journal of veterinary research 90(1), a2070. https://doi.org/10.4102/ojvr.v90i1.2070 original research th1 and th2 epitopes of cowdria polymorphic gene 1 of ehrlichia ruminantium tlou a. ngoepe, alri pretorius, helena c. steyn, mirinda van kleef received: 02 aug. 2022; accepted: 17 nov. 2022; published: 23 mar. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract cowdria polymorphic gene 1 (cpg1, erum2510, erum_rs01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (dna) prime combined with dna boost and dna prime combined with protein boost, respectively, against heartwater infection via needle challenge. to localise its antigenic regions for inclusion in a multi-epitope dna vaccine against heartwater, erum2510 was cleaved into five overlapping subfragments. these subfragments were expressed individually in an escherichia coli host expression system and evaluated for their ability to induce proliferative responses, th1 and th2 cytokines (interferon gamma [ifn-γ] and interleukin 4 [il-4]) via enzyme-linked immunospot (elispot), quantitative real time polymerase chain reaction (qrt-pcr) and flow cytometry. recombinant (r)proteins 3 and 4 were shown to induce immunodominant th1 and th2 immune responses characterised by the secretion of effector cytokines ifn-γ and il-4 in addition to differential messenger ribonucleic acid (mrna) expression of tumour necrosis factor (tnf), il-2, il-1, il-18, il-10, transforming growth factor (tgf), granulocyte-macrophage colony-stimulating factor (gm-csf) and inducible nitric oxide synthase (inos). thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. a peptide pool comprising p9 and p10 derived from rprotein 3 induced a th1-biased immune response. a peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed th1 and th2 immune response characterised by secretion of ifn-γ and differential mrna expression of il-1, il-2, il-10, il-12, inos, tgf, tnf and gm-csf. only one of the peptides (p29) induced secretion of il-4. phenotypic analysis showed significant activation of cluster of differentiation 8+ (cd8+), cluster of differentiation 4+ (cd4+) and b+ lymphocyte populations. findings suggest that erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater. contribution: this study will facilitate the design of an effective multi-epitope dna vaccine against heartwater that will contribute to control this economically important disease in sub-saharan africa and beyond. keywords: heartwater; vaccine epitopes; pbmc; lymphocyte proliferation assay; elisa; elispot; cytokine qrt-pcr; flow cytometry. introduction heartwater is an infectious, noncontagious and lethal tick-borne disease of domestic and some wild ruminants (dumler et al. 2001) that is characterised by high fever, nervous signs, oedema of the heart and lungs and eventually death. the causative agent of heartwater is a rickettsia, ehrlichia ruminantium, which is transmitted by ticks of the genus amblyomma (camus et al. 1996). it is regarded as an economically important disease that causes stock losses in sub-saharan africa, thereby threatening food security (uilenberg 1982). currently, control of the disease relies mainly on the use of acaricides, antibiotic treatment (peregrine 1994) and the commercial, infection and treatment blood vaccine (van der merwe 1987). the latter has several drawbacks, as it requires that the infective blood be kept on dry ice and thawed immediately before inoculation; it requires treatment with antibiotics; and it does not protect against all isolates (allsopp 2009). therefore, there is a need to develop a vaccine that is safe, affordable, effective and does not require transport on dry ice, as most vaccines do still require a cold chain. to date, a number of experimental heartwater vaccines – for example, inactivated, attenuated and deoxyribonucleic acid (dna) – have been developed, and all have demonstrated varying levels of inadequate protection against a natural field challenge (collins et al. 2003). when the humoral major antigenic protein (map1) of e. ruminantium was used as a dna prime, recombinant protein boost vaccine in mice, it induced variable protection (13% – 67%) against virulent needle challenge, characterised by a cellular and humoral immune response (nyika et al. 2002). however, there are no reports that this vaccine was tested in sheep, goats or cattle. the 1h12 cocktail dna vaccine containing four e. ruminantium genes provided 100% protection against a virulent needle challenge in sheep (collins et al. 2003) but only 20% protection in sheep to a natural tick challenge in the field (pretorius et al. 2008). thereafter, selected 1h12 epitopes and multiple-vaccine candidate e. ruminantium epitopes that induce varied cellular immune responses (thema et al. 2019a, 2019b) were combined in a multi-epitope dna vaccine. this vaccine induced 60% protective efficacy in sheep after a laboratory tick challenge (tshilwane et al. 2019). it was traditionally thought that protective immunity against e. ruminantium is not antibody related because antiserum from heartwater immune cattle was not able to passively transfer protective immunity (du plessis 1970). however, various serological tests and western blot analysis of serum from heartwater immune animals revealed the presence of e. ruminantium-specific antibodies (rossouw et al. 1990). mwangi and coworkers (1998) later showed that e. ruminantium immune cattle elicited a predominant cellular immune response characterised in vitro by cd4+, cd8+ and γδ+ t cells, and the cytokines interleukin 2 (il-2), interferon gamma (ifn-γ), tumour necrosis factor (tnf)-α and tnf-β. in contrast, a minor b cell response suggestive of an antibody response was also detected. the function of antibodies in protective immunity against heartwater is still unknown, and it may be involved in opsonisation, complement-mediated killing and/or antibody-dependent cell-mediated cytotoxicity (totté et al. 1999). because a protective immune response to e. ruminantium infection has both cellular and humoral components, perhaps a subunit vaccine that includes both will result in improved protection. thus, the effectiveness of the multi-epitope dna vaccine may be further improved by combining well-defined specific pathogen epitopes that are able to trigger both humoral and cellular immunity (hajissa et al. 2019). erum2510 (erum_rs01380) is a previously identified polymorphic gene of e. ruminantium (perez et al. 1997) and referred to as cowdria polymorphic gene 1 (cpg1). it was shown to induce 100% protective immunity in ruminants as a heterologous dna prime and protein boost vaccine against a needle challenge (pretorius et al. 2010). antigen-specific lymphocyte proliferation was detected, as well as antibodies directed to rerum2510 after immunisation. based on these results, it was hypothesised that erum2510 contains th1 and th2 epitopes that could confer optimal protective immunity when included in the multi-epitope dna vaccine against heartwater. therefore, this study reports the use of epitope mapping to identify erum2510 epitopes that induce protective immune responses characterised by cd8+, cd4+ and b+ cells and the expression of th1 and th2 cytokines. methods and materials cloning of erum2510 gene subfragment e. ruminantium (welgevonden) was grown in bovine endothelial cells (zweygarth & josemans 2001) and genomic dna prepared by percoll density gradient and dneasy blood and tissue kit (qiagen) as described previously (mahan et al. 1995). for ease of cloning an adenine (a) and thymine (t) rich gene sequence, erum2510 was cleaved into six overlapping subfragments, for which amplicon specific primers were designed (appendix 1). to facilitate directional cloning, a cacc nucleotide sequence was added to the 5′ end of the forward primer. the dna subfragments were amplified in a 25 μl reaction containing 31 ng e. ruminantium (welgevonden) genomic dna, 1 × q5 high-fidelity master mix (new england biolabs® inc.) and 0.4 μm forward and 0.4 μm reverse primers (inqaba biotec). the dna subfragments were each polymerase chain reaction (pcr) amplified using the following cycling parameters: initial denaturation for 2 min at 95 °c, followed by 30 cycles of denaturation at 95 °c for 30 s, annealing at 55 °c for 30 s, extension at 72 °c for 3 min and a final extension at 72 °c for 7 min, using a geneamp® pcr 9700 amplifier (applied biosystems). amplicons were cloned in the pet102 directional topo® vector (invitrogen) following the protocol of the manufacturer. clones containing inserts of the correct size were sequenced at inqaba biotec (pretoria, south africa) using the t7 reverse primer and trxfus forward primer (1 μmol) to ensure that the sequence was correct and that the his-tag was in frame. expression and purification of rerum2510 recombinant proteins were expressed in the bl21 (de3) strain of escherichia coli (e. coli) as thioredoxin, his-tagged fusion proteins, using the overnight express™ instant terrific broth media (gershoni et al. 2007) (novagen®), as described previously (thema et al. 2016). proteins were extracted using the bugbuster™ protein extraction reagent, following the protocol of the manufacturer (novagen®) and recombinant protein purified using protino® ni-ted 150 packed columns, following instructions of the manufacturer. recombinant proteins were analysed using sodium dodecyl sulphate – polyacrylamide gel electrophoresis (sds-page) and anti-his (roche) western blot analysis according to standard protocols. briefly, the purified rproteins were separated on a 12% criterion pgx™ precast sds-page gel (bio-rad) and transferred to a polyvinylidene fluoride (pvdf) membrane. the membrane was incubated with anti-his6 antibodies (1:6000 dilution) (roche), followed by a secondary antibody (goat anti-mouse immunoglobulin g [igg], 1 in 20 000 dilution), and the protein bands were visualised using bm blue peroxidase (pod) precipitating substrate (roche). peptide synthesis a total of thirty-seven 16-mer peptides (overlapping by 8 amino acids, figure 1-a1) were designed, spanning the length of erum2510 subfragments 3 (peptides 1–19) and 4 (peptides 20–37). they were synthesised by genscript usa inc., new jersey, united states (us). experimental animals immune animals two merino sheep (sm2 and s6823) were bought from a heartwater-free area in the free state province, south africa, where the amblyomma hebraeum tick is absent. they tested negative for e. ruminantium, using the pcs20 quantitative real-time polymerase chain reaction (qrt-pcr), before the experiment began (steyn et al. 2008). sheep were infected with heartwater (welgevonden strain)–infected ticks. for the primary infection, 10 adult heartwater-infected ticks were allowed to feed on the naïve sheep. sheep were monitored daily for disease symptoms (body temperature above 41.5 °c, loss of appetite, heavy breathing, depression, hanging head, stiff gait, exaggerated blinking, chewing movements, anorexia and signs of nervous symptoms). sheep were treated using terramycin®100 (1 ml/10 kg body weight) after onset of the febrile response. both animals then received an infected tick challenge, similar to the primary infection, 30 days after antibiotic treatment and were monitored daily for onset of clinical symptoms (thema et al. 2016). heartwater infection of sheep (approximately 12 days after primary infection) and ticks (before sheep infection) was confirmed by pcs20 qrt-pcr (steyn et al. 2008). the severity of infection was determined by scoring the clinical signs according to a reaction index (ri). these animals were scored according to their temperature reaction and symptoms displayed, as well as treatment received (pretorius et al. 2007). peripheral blood mononuclear cell isolation blood (50 ml per sheep) was collected in bd vacutainer® – ethylenediaminetetraacetic acid (edta) (beckton, dickson) tubes. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using ficollhistopaque®-1077 (sigma aldrich®), as instructed by the manufacturer. peripheral blood mononuclear cells were re-suspended in 1 ml complete roswell park memorial institute 1640 media (crpmi) containing 25 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes), 10% foetal calf serum (fcs), 2 mm l-glutamate, 5 × 10-5 m mercaptoethanol, penstrep (0.2 mg/ml, streptomycin sulphate, 0.1 mg/ml, sodium benzylpenicillin [invitrogen]) and cells counted using the tc1o automated cell counter (bio-rad). immunological assays lymphocyte proliferation assay the lymphocyte proliferation assay (lpa) was carried out in triplicate in a 96-well half-area plate in a total volume of 100 μl/well (costar), as described previously (sebatjane et al. 2010). peripheral blood mononuclear cells (4 × 106 cells/ml) were stimulated with recombinant proteins and/or synthetic peptides (10 μg/ml). crude welgevonden antigen prepared from cell culture (10 μg/ml), as described previously (van kleef et al. 2002), and concanavalin a (cona, 10 μg/ml) were used as positive controls and unstimulated pbmcs as negative control. cells were incubated at 37 °c, with 5% co2 for five days, and proliferation was determined by measuring the incorporation of 0.5 μci of [methyl-3h]thymidine (sigma-aldrich®) added during the final 18 h of the assay using a liquid scintillation & luminescence counter (trilux). results were expressed as the stimulation index (si) calculated by the counts per minute (cpm) of the test antigen (averaged from triplicate wells) divided by the mean cpm of the unstimulated pbmc. a response was considered positive if two times higher than the si of unstimulated pbmc with a significant p-value of ≤ 0.05. enzyme-linked immunospot assay (interferon gamma and interleukin 4) the expression of ifn-γ and interleukin-4 (il-4) was monitored using the bovine, ovine and equine ifn-γ or il-4 enzyme-linked immunospot (elispot™) kit (mabtech) according to instructions of the manufacturer and as described previously (liebenberg et al. 2012). briefly, the ifn-γ or il-4 monoclonal antibody–coated elispot plates (millipore) were incubated with pbmc (2 × 105 cells per well) in triplicate with erum2510 rproteins and/or synthetic peptides (10 μg/ml). welgevonden antigen (10 μg/ml) and cona (10 μg/ml) as positive controls and unstimulated pbmc as negative control. the plates were developed after 48 h incubation at 37 °c in a humidified 5% co2 incubator. results are represented as spot-forming cells (sfc) per million, and the number of sfc produced after stimulation with antigen was compared with the number of sfc with no stimulation. results were considered positive if the sfc of stimulated cells was 2× higher than sfc of unstimulated cells with a p ≤ 0.05. spot-forming cells were enumerated using an automated ks elispotax10 reader (zeiss). cytokine profiling using quantitative real time polymerase chain reaction cytokine profiling was carried out using qrt-pcr as described previously (liebenberg et al. 2012). peripheral blood mononuclear cells (4 × 106/ml) were stimulated with 10 μg/ml rprotein and/or synthetic peptides overnight (18 h) at 37 °c in a humidified incubator at 5% co2. stimulated pbmc were harvested and resuspended in 1 ml trizol reagent (sigma aldrich®). the total ribonucleic acid (rna) was isolated according to the tri®-reagent protocol, and genomic dna contamination was removed using the quantitect reverse transcription kit (qiagen) according to the instructions of the manufacturer. the rotor-gene sybr green qrt-pcr kit (qiagen) was used to quantify cytokine rna targets using the rotor-gene q (qiagen). ovine qrt-pcr primers for housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (gapdh) and β-actin, and cytokines il-4, il-1α, transforming growth factor (tgf)-β, granulocyte-macrophage colony-stimulating factor (gm-csf), il-10, il-12, il-18, tnf-α, il-2 and inducible nitric oxide synthase (inos) (table s1) were synthesised by inqaba biotec. the cycling conditions were set as follows: 5 min at 95 °c, 40 cycles of two-step cycling at 95 °c for 5 s and 60 °c for 10 s. glyceraldehyde 3-phosphate dehydrogenase and β-actin are common housekeeping genes that were used to normalise cytokine gene expression. gene expression was measured by relative quantitation using the 2-δδct method (livak & schmittgen 2001). one fold increase (fi) indicates that the cytokine messenger ribonucleic acid (mrna) concentration of the stimulated sample is twice that of nonstimulated samples, and the cytokines were considered to be upregulated if the mrna increase difference was fi > 1 compared with both housekeeping genes. phenotypic analysis by flow cytometry peripheral blood mononuclear cells were incubated in triplicate with all of the individual erum2510 rproteins (10 µg/ml) or peptides (10 µg/ml), cona (10 μg/ml, positive control) and unstimulated pbmc (negative control), for 72 h at 37 °c in a humidified incubator at 5% co2. the resulting cells were surface stained and intracellular cytokine stained, as described previously (thema et al. 2016). surface staining: cells were incubated for 15 min at room temperature with primary monoclonal antibodies recognising cd4 (immunoglobulin m [igm], cell line gc50a), cd8 (igg1, cell line cact80c), cd45ro (immunoglobulin g3 [igg3], cell line gc44a) or b cell (igm, cell line baq44a) markers (washington state university, pullman, united states) at a dilution of 1:100 in phosphate-buffered saline (pbs) containing 0.5% fcs, 0.09% sodium azide. after two washes, secondary goat antimouse antibodies specific to igm-apc (serotec), igg1-pe (serotec) and igg3-fitc (serotec) were diluted to 1:8, 1:30 and 1:10, respectively, and added to the cells. after 15 min incubation and two washes, the cells were fixed with 2% formaldehyde (except those for intracellular cytokine staining [ics]). flow cytometry data were collected with a beckman coulter fc500 flow cytometer and analysed using kaluza software version 1.2 (beckman coulter, brea, california, united states). intracellular cytokine staining: cells to be stained intracellularly for cytokines were treated with bd golgiplug™ protein transport inhibitor containing brefeldin a (bd biosciences™) 4 h before harvesting. after surface staining (ss), these cells were fixed and permeabilised using the bd cytifix and cytopermtm kit (bd biosciences) according to instructions of the manufacturer. thereafter, the cells were stained with mouse antibovine ifn-γ-alexafluor488 (1:20, celtic diagnostics) by incubation for 30 min. the cells were then washed twice and flow cytometry data collected with a beckman coulter fc500 flow cytometer and analysed using kaluza software version 1.2. statistical analysis the difference of significance between lpa, elispot assay and phenotypic analysis results were determined by student t-test; the difference was considered significant at a p-value of ≤ 0.05. ethical considerations all animal research was performed in accordance with the stipulations of the animal ethics committee at the arc onderstepoort veterinary research, the university of pretoria animal use and care committee (v096-13), and approved by the south african department of agriculture, forestry and fisheries (daff) section 20 of the animal diseases act of 1984 (act no 35 of 1984). results cloning of erum2510 gene subfragments erum2510 was divided into six overlapping subfragments of 450 bp each, which were amplified by pcr using gene-specific primers. four of these subfragments (fragments 3–6) were successfully cloned in the pet 102 directional topo® expression vector, and this was confirmed by pcr using vector-specific primers (t7 reverse and trxfus forward primer). numerous attempts to clone pcr products of erum2510 subfragment 1 and 2 individually failed, and as a result, these were cloned as one fragment of 900 bp encoding a predicted protein of 44.5 kda. expression and purification of erum2510 rproteins in e. coli the recombinant erum2510 expressed subfragments 3–6 had the predicted size of 30 kda each, with the exception of the two gene subfragments 1–2, which were cloned and expressed as one gene subfragment encoding a predicted protein of 45.5 kda (figure 2-a1). three of the rproteins (subfragments 1–2, 4 and 5) were purified from the soluble supernatant, whereas rproteins translated from the gene subfragments 3 and 6 were purified from inclusion bodies. the protein concentration of the proteins ranged from 356 µg/ml to 1355 µg/ml. lymphocyte proliferation assay with recombinant proteins two sheep (sm2 and s6823) were infected with e. ruminantium by tick feeding and treated when they developed heartwater symptoms of a febrile response. after the tick challenge, similar to the primary infection, both sheep were considered to be immune to e. ruminantium because they did not develop any heartwater disease symptoms. the ability of immune pbmc, collected from these sheep, to undergo clonal proliferation was evaluated with the lpa. peripheral blood mononuclear cells from both sheep had significant and positive si of 3 after stimulation with the crude welgevonden-strain antigen. rprotein 3 showed a significant positive si of 3 (sm2) and 10 (s6823), whereas rprotein 4 had a significant positive si of 4 (sm2) and 20 (s6823) (figure 3-a1). rprotein 6 induced significant proliferation only in pbmc from s6823 (si 4) (figure s3). enzyme-linked immunospot assays (interferon gamma and interleukin 4) with recombinant proteins rproteins 3 and 4 induced the highest secretion of ifn-γ and il-4 in pbmc from s6823 (figure 1a, c). however, the same erum2510 rproteins failed to induce secretion of il-4 in pbmc from sm2, although ifn-γ was secreted (figure 1b, d). figure 1: secretion of interferon gamma (ifn-γ) (a, b) and interleukin 4 (il-4) (c, d) by immune peripheral blood mononuclear cells (pbmc) from s6823 (a, c) and sm2 (b, d) stimulated with erum2510 rproteins and crude welgevonden-strain antigen (positive control) and unstimulated pbmc (negative control). data are presented as the average number of spot-forming cells per million. cytokine profiling with recombinant proteins erum2510 rproteins were screened for their ability to induce the expression of il-1, il-2, il-4, il-10, il-12, il-18, inos, tnf, tgf and gm-csf (table 1). the cytokine profile induced by the rproteins in pbmc from both sheep was characterised by: rprotein 3 (il‑1, il-2, il-10 and tnf), rprotein 4 (il-1, il-4, il-10, il-12, il-18, tnf, tgf and gm-csf), rprotein 5 (il-1, il-18 and tnf) and rprotein 6 (inos). table 1: relative fold change in cytokine mrna isolated from sm2 and s6823, stimulated with erum2510 rproteins and normalised to endogenous reference genes glyceraldehyde 3-phosphate dehydrogenase (gapdh) and β-actin, relative to an untreated negative control (unstimulated peripheral blood mononuclear cell). phenotypic analysis with recombinant proteins a statistically significant increase of the cd4+ifn-γ+ population in immune pbmc from s6823 was observed after incubation with erum2510 rprotein 4 (4.29%) and 6 (2.8%) (figure 2a). similar results were also observed in immune pbmc from sm2, rprotein 4 (2.3%) and rprotein 6 (1.5%) (figure 2b). in contrast, none of the erum2510 rproteins induced significant activation of cd8+ifn-γ+ population in immune pbmc from both sheep (figure 2a, b). rproteins 4 and 6 were also shown to induce statistically significant activation of memory cd4+ t lymphocytes in immune pbmc from s6823 (38.6%; 29.4%, respectively) while only rprotein 6 induced a similar response in sm2 (24.9%) (figure 4-a1). however, none of the erum2510 rproteins induced statistically significant activation of memory cd8+ t lymphocytes (figure 4-a1). only rprotein 4 induced a statistically significant increase of the b+ population in immune pbmc from s6823 (figure 3a). figure 2: average percentage of peripheral blood mononuclear cells (pbmc) from s6823 (a) and sm2 (b) that stained positive for cd4 and interferon gamma (ifn-γ) and cd8 and ifn-γ expression markers after stimulation with erum2510 rproteins 3, 4 and 6. unstimulated pbmc was included as negative control. peripheral blood mononuclear cells were stimulated with 10 μg/ml rprotein and incubated for 72 h, after which the percentage of cells was determined by flow cytometry. figure 3: average percentage of immune peripheral blood mononuclear cells (pbmc) from s6823 (a) and sm2 (b) that stained positive for b cell expression markers after stimulation with erum2510 rproteins 3, 4 and 6. unstimulated pbmc was included as negative control. peripheral blood mononuclear cells were stimulated with 10 μg/ml protein and incubated for 72 h, after which the percentage of cells was determined by flow cytometry. peptide synthesis data obtained from immunological assays carried out with five erum2510 rproteins led to the conclusion that rproteins 3 and 4 were the most immunogenic. rprotein 4 was shown to be immunodominant, with an ability to induce cytokines associated with both th1 and th2 responses. based on this, overlapping synthetic peptides were designed spanning the length of erum2510 rproteins 3 and 4 and evaluated for their ability to induce recall immune responses. lymphocyte proliferation assay with peptides to identify immunogenic peptides of rproteins 3 and 4 that are recognised by immune pbmc from s6823 and sm2, all 37 peptides synthesised were individually screened in a lpa assay. only four peptides from rprotein 3 (p9, p10, p17 and p18) and four from rprotein 4 (p24, p25, p28 and p29) induced statistically significant proliferation above the cut-off point in pbmc isolated from both sheep (figure 5-a1). the proliferative response induced in sm2 pbmc by peptides was similar to those obtained with the rproteins 3 and 4. however, the proliferative responses induced in s6823 by peptides was shown to be slightly lower than those obtained by rproteins 3 and 4. these eight peptides were selected for further immune analysis. enzyme-linked immunospot assay with peptides eight of the selected peptides from the lpa assay (10 μg/ml) were incubated in triplicate with immune pbmc isolated from sm2 and s6823 in an elispot assay. all eight peptides induced significant expression of ifn-γ in pbmc from both sheep, with the exception of peptide 29, which did not induce significant responses in s6823 (figure 4). only peptide 28 was shown to induce the expression of both il-4 and ifn-γ from sm2 (figure 4); however, no significant expression of il-4 was observed for s6823. for further analysis, the eight peptides were pooled into four groups: pool a, rprotein 3, p9 and p10; pool b, rprotein 3, p17 and p18; pool c, rprotein 4, p24 and p25; pool d, rprotein 4, p28 and p29. figure 4: secretion of interferon gamma (ifn-γ) (a, b) and interleukin 4 (il-4) (c, d) by immune peripheral blood mononuclear cells (pbmc) from sm2 and s6823 stimulated with erum2510 rproteins and peptides at a concentration of 10 μg/ml. only those peptides with positive results are shown with (a, c) rprotein 3 and corresponding peptides; (b, d) rprotein 4 and corresponding peptides. unstimulated pbmcs were included as negative control. data are presented as the average number of spot-forming cells per million. cytokine profiling with peptides the mrna cytokine fold expression profiles induced by the peptides were highly variable between sheep and are listed in table 2. although the values were generally higher when gapdh was used as housekeeping gene, they did correlate with β-actin housekeeping gene results. in s6823 but not sm2, il-4 and inos was induced when pbmc were stimulated with pools b, c and d. in sheep sm2 but not s6823, il-1, tnf and tgfb were produced by all pools tested. table 2: relative fold change in cytokine mrna in peripheral blood mononuclear cells from sm2 and s6823 stimulated with rerum2510 synthetic peptides and normalised to endogenous reference genes glyceraldehyde 3-phosphate dehydrogenase (gapdh) and β-actin, relative to an untreated negative control (unstimulated peripheral blood mononuclear cell). phenotypic analysis with peptides to determine the population of lymphocytes responding to peptides, flow cytometry was carried out. peptide pools a and c induced a significant increase of cd4+ifn-γ+ cells by pbmc from s6823 (figure 5a). peptide pools a, b and d induced a significant increase of cd4+ifn‑γ+ in sm2 (figure 5b). in contrast to the rprotein result, peptide pool d induced a significant increase of cd8+ifn-γ+ cells by pbmc from both sheep (figure 5). peptide pools a, b, c and d induced a significant activation of memory cd4+ t lymphocytes (figure 6-a1). in contrast to results obtained from erum2510 rproteins, all peptide pools (a, b, c, d) induced a significant increase of memory cd8+ t cells by immune pbmc from both sheep. peptide pool d induced a statistically significant increase of b cells in both sheep (figure 6). figure 5: average percentage of peripheral blood mononuclear cells (pbmc) from s6823 (a) and sm2 (b) that stained positive for cd4 and interferon gamma (ifn-γ), and cd8 and ifn-γ expression markers after stimulation with erum2510 peptide pools. peripheral blood mononuclear cells were stimulated with 10 μg/ml protein and incubated for 72 h, after which the percentage of cells was determined by flow cytometry. unstimulated pbmcs were included as negative control. figure 6: average percentage of peripheral blood mononuclear cells (pbmcs) from s6823 (a) and sm2 (b) that stained positive for b cell expression markers after stimulation with erum2510 peptide pools. peripheral blood mononuclear cells were stimulated with 10 μg/ml protein and incubated for 72 h, after which the percentage of cells was determined by flow cytometry. unstimulated pbmcs were included as negative control. discussion cowdria polymorphic gene 1 (cpg1, erum2510) was previously shown to induce lymphocyte proliferation, antibodies and protection against heartwater in sheep using a dna prime and rprotein boost immunisation strategy (pretorius et al. 2010). this implicates erum2510 as a potential vaccine candidate for inclusion in an epitope-based vaccine against heartwater. to identify its antigenic epitopes, erum2510 was divided into six overlapping subfragments. subfragments 1 and 2 were cloned as a single fragment and subfragments 3–6 were cloned individually to produce five rproteins. erum2510 rprotein 3 and 4 induced proliferation of pbmc obtained from both sm2 and s6823, with the strongest response observed in s6823. in contrast, all the erum2510 rprotein subfragments induced significant expression of ifn-γ in pbmc from both sheep quantified in the elispot assay. however, significant expression of il-4 was induced by rprotein 3 and 4, only in s6823 pbmc. the expression of additional cytokines associated with protection by immune pbmc in response to erum2510 rproteins was investigated by qrt-pcr. erum2510 rprotein 4 induced strong expression of il-2 and tnf-α (th1) mrna, accompanied by strong expression of il-10 and il-4 (th2) mrna. these results thus correlate with the elispot assay for sheep s6823. however, there were differences observed in this study between gene expression and protein levels detected in pbmc from sm2 in response to rprotein 4. the overproduction of il-4 mrna induced by this rprotein may have activated mrna post-transcriptional expression regulation, which includes mrna degradation mechanisms in order to avoid the detrimental effects caused by the overproduction of il-4 protein (junttila 2018; wierenga & messer 2000). although the mechanism of the observed regulation is not known, rna decay can be mediated by rna decay–promoting proteins, namely brf1, ksrp and auf1, which was not tested for in the current study (khabar 2007). this highlights the importance of comparison between mrna expression levels and protein cytokine secretion levels, as the detection of mrna does not necessarily mean that the protein is produced (shebi et al. 2010). thus, conclusions derived from cytokine mrna levels alone should be made with caution unless protein expression can be verified. erum2510 rprotein 4 induced significant proliferation of b+ lymphocytes, cd4+cd45r0+ t cells and cd4+ifn-γ+ t cells. none of the erum2510 rproteins assayed were shown to induce significant activation of cd8+ t cells in both sheep. results therefore indicate that in both sheep, the cytokine profile induced by erum2510 rprotein 3 and 4 is characteristic of a th1 and th2 mixed immune response. the expression of il-4 and ifn-γ mrna strongly suggest that rproteins 3 and 4 contain th1 and th2 (b cell) epitopes that could induce a cellular and humoral response against e. ruminantium. combined th1 and th2 responses induced by a rprotein have been described (daifalla, bayih & gedamu 2015; stachyra et al. 2019). for example, mice vaccinated with the recombinant saposin-like protein fhsap-2, from fasciola hepatica, induced high detectable levels of igg1, igg2 and ige, as well as high levels of il-10 and ifn-γ indicative of a mixed th1 and th2 response (espino et al. 2005). thus, the induction of th1 and th2 mixed responses to pathogen antigens is not uncommon and is beneficial to the immune response. the similar strong co-expression of th1 and th2 cytokine mrna observed suggests that erum2510 rproteins 3 and 4 contain both th1 and th2 epitopes, possibly working in concert to eliminate infectious agents and ensure control of the immune response. because erum2510 rprotein 3 and 4 were shown to be the most immunogenic, they were divided into 37 overlapping synthetic peptides to identify specific epitopes. the immune response induced by the synthetic peptides was firstly characterised by lpa assay. eight out of 37 peptides induced pbmc from both animals to proliferate. these eight peptides (rprotein 3: p9, p10, p17, p18; rprotein 4: p24, p25, p28, p29) were then screened with elispot, and they all induced statistically significant expression of ifn-γ in pbmc isolated from both sheep, as did their respective rproteins. only peptide 28, derived from rprotein 4, induced statistically significant expression of il-4 in sm2, whereas the rprotein did not. this may indicate that there are epitopes in the rprotein that suppress the immune response induced by the peptide, thus emphasising the importance of epitope mapping. furthermore, no peptide induced expression of il-4 in pbmc from s6823, while both rprotein 3 and 4 did. the ability of peptide 28 to induce expression of ifn-γ and il-4 suggests that it is a th1 and th2 epitope. previous studies have similarly reported on the ability of a peptide to induce th1 and th2 responses (chaturvedi et al. 1996; wong et al. 2004). selected peptides were then pooled for qrt-pcr and flow cytometry assays. peptide pool a (rprotein 3: p9, p10) induced a th1-biased response characterised by the expression of il-12, tnf and gm-csf. peptide pool d (rprotein 4: p28, p29) induced a th1 and th2 response characterised by the expression of ifn-γ, il-2, tgf and il-4 (p28). phenotypic analysis showed that peptide pool a (rprotein 3: p9, p10) and peptide pool c (rprotein 4: p24, p25) activated a significant percentage of cd4+ t cells expressing ifn-γ in s6823 pbmc. interestingly, all peptide pools were able to induce significant activation of cd8+cd45r0+ t cells and cd4+cd45r0+ t cells. a higher percentage of cd8+cd45r0+ t cells than cd4+cd45r0+ t cells was noted for peptide pool a (rprotein3: p9, p10) and peptide pool d (rprotein 4: p28, 29) in both sheep. this contrasts with the data obtained from erum2510 rproteins, wherein none of the rproteins were shown to activate cd8+cd45r0+ t cells. this may be because the rproteins were not processed correctly or that they contained other epitopes that interfered with the cd8+ t cell response (wang et al. 2005). the variability in response of the two sheep to the same antigen observed in the study can be attributed to the usage of outbred animals (genetic variability), which has been reported by other groups (esteves et al. 2004; thema et al. 2019a, 2019b). major histocompatibility complex (mhc) typing of sm2 (results not shown) and s6823 (thema et al. 2016) showed different ovar-drb1 alleles at exon 20, s6823: *0201, 0801 and sm2: *0323, 0333. although these preliminary findings indicated that the peptides produced a ‘weaker’ response in s6823 compared with sm2, they have the potential to induce an immune response associated with protection. therefore, they should be further investigated as vaccine candidates, using larger statistically significant groups of sheep, goats and cattle. in conclusion, multi-epitope vaccines containing both cellular and humoral epitopes induce strong and often enhanced immune responses, as shown in studies on plasmodium falciparum, malaria (shi et al. 2000), human immunodeficiency virus (hiv) (karpenko et al. 2007) and herpes simplex virus (wang et al. 2011), to name a few. the ability of erum2510 rprotein 4 and synthetic peptides (p28, p29) to induce statistically significant th1, th2 and b cell responses in this study, as well as antibodies directed to rerum2510 and protection of sheep in a previous study (pretorius et al. 2010), merits investigating them as vaccine candidates in a multi-epitope vaccine against heartwater. acknowledgements competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions t.a.n. performed all experimental work and wrote the article; a.p. was involved in idea conceptualisation, advising and editing the article; h.c.s. was involved in animal work and editing the article; m.v.k. obtained funding, advised and cowrote the article. funding information the authors acknowledge the financial support received from the economic competitiveness support programme and the agricultural research council – onderstepoort veterinary research (arc-ovr), south africa. data availability the data that support the findings of this study are available on request from the corresponding author. disclaimer the views expressed in this article are of the authors and not an official position of the institution or funder. references allsopp, b.a., 2009, ‘trends in the control of heartwater: tick-borne diseases’, onderstepoort journal of veterinary research 76(1), 81–88. https://doi.org/10.4102/ojvr.v76i1.69 camus, e., barré, n., martinez, d. & uilenberg, g., 1996, heartwater (cowdriosis). a review, pp. 136–138, office international des epizooties, paris. chaturvedi, p., yu, q., southwood, s., sette, a. & singh, b., 1996, ‘peptide analogs with different affinities for mhc alter the cytokine profile of t 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journal of veterinary research 68, 37–40. appendix 1 table 1-a1: rotor-gene q ovine quantitative real time polymerase primers used to quantify cytokine mrna targets in quantitative real time polymerase chain reaction assay. figure 1-a1: schematic representation of overlapping peptides spanning the length of erum2510 fragment 4 used for epitope mapping. overlapping peptides used in this study were designed in the same format. figure 2-a1: anti-his6 western blot analysis illustrating purified recombinant proteins (indicated with arrows). (kda: molecular weight marker with size indicated in kda; l1: sub-fragment 1-2, l2: sub-fragment 3, l3: sub-fragment 4, l4: sub-fragment 5, l5: sub-fragment 6). figure 3-a1: average stimulation index (si) indicating proliferative response of immune peripheral blood mononuclear cells isolated from sm2 (a) and s6823 (b) to five erum2510 sub-rproteins. crude welgevonden strain antigen was included as a positive control and unstimulated peripheral blood mononuclear cells as negative control. horizontal dashed line represents a stimulation index of 2, which is the cut-off point for a positive response. figure 4-a1: average percentage of peripheral blood mononuclear cells from s6823 (a) and sm2 (b) that stained positive for cd4 and cd45ro; and cd8 and cd45ro expression markers after stimulation with erum2510 rproteins 3, 4 and 6. unstimulated peripheral blood mononuclear cells was included as negative control. peripheral blood mononuclear cells were stimulated with 10 μg per ml protein and incubated for 72 h after which the percentage of cells was determined by flow cytometry. figure 5-a1: average stimulation index of immune peripheral blood mononuclear cells isolated from sm2 (a, b) and s6823 (c, d) after incubation with erum2510 rproteins and peptides. (a, c) rprotein 3 and corresponding peptides; (b, d) rprotein 4 and corresponding peptides. unstimulated peripheral blood mononuclear cells were included as negative control. the horizontal dashed line represents a stimulation index of 2, which is the cut-off point for a positive response. figure 6-a1: average percentage of peripheral blood mononuclear cells from s6823 (a) and sm2 (b) that stained positive for cd4 and cd45r0, and cd8 and cd45r0 expression markers after stimulation with erum2510 peptide pools. peripheral blood mononuclear cells were stimulated with 10 μg/ml protein and incubated for 72 h after which the percentage of cells was determined by flow cytometry. unstimulated peripheral blood mononuclear cells were included as negative control. article information authors: nicolaas j. pienaar1,2 peter n. thompson1 affiliations: 1department of production animal studies, university of pretoria, south africa2directorate animal health, department of agriculture, forestry and fisheries, south africa correspondence to: nicolaas pienaar postal address: private bag x04, onderstepoort 0110, south africa dates: received: 07 oct. 2011 accepted: 24 nov. 2012 published: 05 mar. 2013 how to cite this article: pienaar, n.j. & thompson, p.n., 2013, ‘temporal and spatial history of rift valley fever in south africa: 1950 to 2011’, onderstepoort journal of veterinary research 80(1), art. #384, 13 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.384 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. temporal and spatial history of rift valley fever in south africa: 1950 to 2011 in this original research... open access • abstract • introduction • materials and methods • results    • 1950–1951    • 1951–1952    • 1952–1953    • 1953–1954    • 1954–1955    • 1955–1956    • 1956–1957    • 1957–1969    • 1969–1970    • 1970–1973    • 1973–1974    • 1974–1975    • 1975–1976    • 1976–1977    • 1977–1978    • 1978–1979    • 1979–1980    • 1980–1981    • 1981–1982    • 1982–1983    • 1983–1984    • 1984–1985    • 1985–1986    • 1986–1987    • 1987–1988    • 1988–1989    • 1989–1990    • 1990–1991    • 1991–1998    • 1999    • 2000–2007    • 2008    • 2009    • 2010    • 2011 • cumulative temporal and spatial distribution of outbreaks • discussion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ several outbreaks of rift valley fever (rvf) have been documented in south africa since it first occurred in the country in 1950. however, there is no comprehensive account of the timing, location and extent of all known outbreaks. as part of a study investigating the epidemiology of rvf in south africa, a full history of outbreaks was compiled using references to the disease in south africa from scientific literature, annual reports, disease reports and animal disease databases. the geographic location and temporal occurrence of each outbreak were recorded as accurately as allowed by the available records. the result was a better and more complete picture than has hitherto been available of the spatial and temporal distribution of rvf in south africa for the period between 1950 and 2011. several smaller outbreaks which had not been described previously in literature were documented. extensive outbreaks occurred in the central interior of the country (free state, eastern cape and northern cape provinces), interspersed with smaller outbreaks or long intervening periods of absence, whilst smaller outbreaks occurred in the eastern part of the country (kwazulu-natal, mpumalanga and gauteng). introduction top ↑ rift valley fever (rvf) is a zoonotic viral disease of ruminants that is transmitted by mosquitoes of various genera. in animals rvf can be inapparent, or cause peracute or acute disease. the most noted effect is abortions, but it can also cause fever leading to mortalities in young animals, especially lambs. humans can contract rvf following contact with tissues of infected animals. the symptoms in humans are mostly those of a febrile, influenza-like illness, but in severe cases it can lead to haemorrhagic fever, encephalitis, retinal haemorrhage and even death (swanepoel & coetzer 2004). rvf typically occurs in south africa as outbreaks interspersed with long periods of absence. the mechanisms of virus survival during the interepidemic periods are unknown, but may include transovarial transmission in the vectors or low-level transmission between vectors and hosts (swanepoel & coetzer 2004). new outbreaks are triggered by an explosion in vector numbers as a result of the abundance of water due to increased rainfall, flooding or even human activity such as dam building. areas where rvf has occurred in the past are likely to experience re-occurrences (gerdes 2004). rvf was first diagnosed in the kenyan rift valley in 1931 (daubney, hudson & garnham 1931) and has since also been diagnosed in zimbabwe, zambia, mozambique, namibia, madagascar, tanzania, somalia, sudan, egypt, senegal, mauritania and on the arabian peninsula, amongst others (swanepoel & coetzer 2004). occurrences of rvf outbreaks in kenya have been detailed by linthicum et al. (1999) and by murithi et al. (2010). since the first recorded occurrence in south africa in 1950 (alexander 1951), a large number of outbreaks have been listed by swanepoel and coetzer (2004), but several of the smaller outbreaks have never been recorded in the literature. although information regarding these outbreaks is available in veterinary services disease reports and annual reports, no comprehensive record of rvf outbreaks in south africa exists. this information is important not only for understanding the epidemiology of the disease but also in guiding management attempts. this paper documents the occurrence of rvf in south africa on a temporal and spatial basis and attempts to document all the recorded instances of rvf outbreaks in south africa. materials and methods top ↑ to fully document the occurrence of rvf in south africa, a comprehensive search was conducted of both published and unpublished literature. annual reports of the national department of agriculture and the directorate of veterinary services/animal health from 1950 to 2000 were searched. since its first occurrence in south africa in 1950, rvf has been a notifiable or reportable disease and therefore the disease reports of the veterinary services were considered to be a good source of information on occurrence of the disease. annual reports of individual veterinary laboratories were not available. however, facilities for laboratory confirmation of rvf were already in place in 1950 and as the laboratory services were part of the veterinary services all occurrences of rvf mentioned in these reports were considered to have been laboratory confirmed, unless otherwise mentioned. if the disease had not been confirmed in a laboratory but was documented in a report, we mentioned it in our results but did not include it in our statistics. disease reports to the world organisation for animal health (oie) and the southern african regional commission for the conservation and utilisation of the soil (sarccus) were also included in the search. a literature search combining the keywords ‘rift valley fever’ and ‘south africa’ was performed on the electronic databases pubmed (u.s. national library of medicine, bethesda), sciencedirect (elsevier, amsterdam) and cabdirect (cab international, wallingford). all articles that indicated a year and location of an outbreak, including reports of rvf in humans, were used.disease reports from south african veterinary regions and provincial veterinary services between 1950 and 2011, stored in the archives of the directorate animal health, and the animal disease database of the directorate animal health (1992–2011) were major sources of reports on rvf in south africa. disease reports prior to 1994 referred to veterinary regions, which were demarcated according to the main animal production activities in each region. the veterinary services of south africa were also divided into these regions. figure 1 shows a map and a list of these regions, as well as the pre-1994 provincial boundaries of south africa. after 1994, veterinary services were provincialised according to the new provincial boundaries (figure 2). figure 1: veterinary regions and provinces of south africa before 1994. figure 2: south african provincial boundaries after 1994 and the kruger national park. all available information on rvf outbreaks was collated by season (july–june), covering a total of 61 seasons between 1950/1951 and 2010/2011. an outbreak was defined as the occurrence of laboratory-confirmed rvf in a specified location during a season. in this study, ‘epidemic’ is used to refer to an occurrence of multiple related outbreaks in the same season. for each outbreak, as much detail as possible was obtained with respect to the location (region, district or, if possible, exact location) and time (season or, if possible, month) of the event. south africa was divided into 462 blocks spanning a half degree latitude by a half degree longitude and each outbreak was assigned to one or more of these blocks. this was necessary to account for the temporal variation in the spatial resolution of the outbreak reports. however, in some cases the spatial resolution of the veterinary services disease reports was very low and only the veterinary region or district was mentioned. in such cases, all the blocks within that area were recorded as having experienced an outbreak, except where it was known or was concluded following further investigation that an outbreak had never occurred or was extremely unlikely to have occurred in a certain area. the cumulative number of seasons during which each block had experienced an outbreak was then calculated and plotted on a map of the country. where the month of occurrence was mentioned, outbreaks were also tabulated by season to show the temporal distribution of outbreaks within each season. a detailed analysis of the association between potential climatic and other risk factors and the occurrence of rvf in south africa will be reported on separately. results top ↑ during the 61-year study period (1950–2011) one or more rvf outbreaks were reported in 27 seasons. south africa experienced three major epidemics that affected an extensive area of the country (1950–1951, 1974–1976 and 2010–2011). the rest of the reports concerned smaller epidemics, individual outbreaks or isolated cases, some of which were not laboratory confirmed. all reported outbreaks are detailed by season and a summary of all the laboratory-confirmed outbreaks is given in table 1. maps show post-1994 provincial boundaries. however, some references are made to veterinary regions, the names and boundaries of which differ in many cases from those of both previous and present provinces of south africa (refer to figures 1 and 2).since separate laboratory reports could not be found, it was assumed that, unless otherwise stated, all rvf outbreaks reported in the annual reports of the veterinary services were laboratory confirmed. several reports stated that outbreaks were reported but could not be confirmed by the laboratory; very little information was given and these reports are mentioned in the following sections but not included in our statistics. 1950–1951 rvf was recorded in south africa in this period in the northern cape province, western orange free state and the southern transvaal (figure 3). this was the first time rvf had been diagnosed in south africa and no reports of any similar disease prior to this epidemic could be found. it started in the western free state in december 1950 and continued until april 1951. specific areas mentioned are the wolwespruit and dealesville areas of the boshof district, vaalhartz, the vaal river barrage, loskop dam, koffiefontein and standerton (alexander 1951). at first the disease was not recognised, but after a veterinarian performing a post-mortem inspection on a farm south of johannesburg fell ill, it was recognised as rvf (mundel & gear 1951) and the virus (lineage o) was isolated (grobbelaar et al. 2011). during this epidemic sheep mortalities were estimated at 100 000, together with an estimated 500 000 abortions (gerdes 2004). it was recognised that the epidemic was associated with the ‘panveld’ area of the orange free state, characterised by shallow grassland depressions that fill with water seasonally (alexander 1951). figure 3: districts and veterinary regions affected by rift valley fever in south africa, 1950–1951. 1951–1952 a single laboratory-confirmed diagnosis of rvf on 30 january 1952 was reported from the farm weltevreden in the wolvenspruit area of the boshof district (figure 4) (van der linde 1953). figure 4: rift valley fever in the orange free state in south africa between 1951 and 1953. 1952–1953 during april 1953 outbreaks were confirmed (positive mouse protection test) in the gannapan and legpan areas of the fauresmith (luckhoff) district, northwest of luckhoff in the orange free state (figure 4). these pans have a geographical reference of 29°40’s, 24°40’e. farms affected were eldorado, legpan, bossiespan a & b, wolvenkraal, nelsdam, overskot, koppiesdam, de rif, alpha and wolvenplaat. it was also noted that the outbreaks were associated with the flooding of the nearby pans (van der linde 1953). a team from the south african institute for medical research was sent to investigate. their report mentioned that several people contracted the disease after handling meat and also suggested that standing water in pans could have contributed to the outbreak (gear et al. 1955). 1953–1954 no outbreaks of rvf were found although several suspected outbreaks were investigated (alexander 1955a). no further details on the suspected outbreaks could be found. 1954–1955 a large number of samples from suspected rvf outbreaks were tested, but no outbreaks were confirmed (alexander 1955b). no further details were given on the suspected outbreaks. 1955–1956 a total of 28 outbreaks were reported from the orange free state (wesselsbron, odendaalsrus, soutpan, dealesville, boshof, hoopstad, ventersburg and bothaville districts), as shown in figure 5 (alexander 1956). virus isolation confirmed that the outbreak was caused by a lineage i rvf virus (grobbelaar et al. 2011). figure 5: districts affected by rift valley fever in south africa during the period 1955–1956. 1956–1957 the annual report for the period 1956–1957 mentions two outbreaks in the western orange free state, but does not provide further detail regarding the exact place and time (division of veterinary services 1957). 1957–1969 no outbreaks of rvf were reported (division of veterinary services 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969). neither two suspected outbreaks in the orange free state in the period 1965–1966 nor several suspected outbreaks in the period 1966–1967 could be confirmed by the laboratory (division of veterinary services 1966, 1967). 1969–1970 severe livestock losses occurred owing to outbreaks reported from the standerton, frankfort, kroonstad, koppies, odendaalsrus, vryburg and the lower umfolozi districts (figure 6) (division of veterinary services 1970). figure 6: districts affected by rift valley fever in south africa during 1969–1970. 1970–1973 no outbreaks were reported for this period (division of veterinary services 1971, 1972, 1973). 1973–1974 during this period south africa’s largest rvf epidemic began. it had the largest extent, lasted three years and likely affected more animals than any other epidemic, although no reliable estimates are available. a mortality rate of up to 95% was reported amongst young lambs, with widespread abortions and deaths in adult animals (division of veterinary services 1974). the outbreak was caused by a lineage l rvf virus (grobbelaar et al. 2011). all areas except the winter rainfall area, the eastern transvaal and transvaal reported widespread occurrence of the disease (figure 7). in natal the disease occurred only in the eshowe state veterinary area. the kimberley, bloemfontein and fauresmith areas and the eastern cape and karoo veterinary region suffered severe losses owing to the disease. heavy rains and large tracts of standing water created ideal conditions for the rvf virus vectors. the epidemic continued through the winter (which was warmer than average), spilling over into the next season (division of veterinary services 1974). figure 7: veterinary regions and one district in natal affected by the 1973–1974 rift valley fever epidemic in south africa. 1974–1975 the major epidemic of 1973–1974 continued during this season, being most severe in the north-western cape province, southern orange free state and the western parts of transvaal (figure 8). other areas specifically mentioned were the calvinia area in september 1974, areas along the orange river, the nyl river valley near potgietersrus, and east griqualand. a few cases were reported from natal (division of veterinary services 1975). areas specifically mentioned as being affected by this epidemic in an unpublished report of the western cape veterinary region (anonymous 1976) were steinkopf, springbok, brandvlei, sutherland, williston, nieuwoudtville, vredendal, lutzville, calvinia (february 1975), clanwilliam, vanrhynsdorp (october 1974), kenhardt (september 1974), pella (august 1974), graafwater (may 1975), lamberts bay and boesmanland (may 1975). during this epidemic human cases appeared to be more severe, with more complications and deaths than had previously been noted (mcintosh et al. 1980a). lineage l rvf virus was isolated from several human cases (grobbelaar et al. 2011). barnard and botha (1977) published a map indicating reported rvf outbreaks, which is included in figure 8. figure 8: veterinary regions affected by the rift valley fever epidemic during 1974 to 1975. 1975–1976 the epidemic that had begun in 1973 continued, but with fewer losses than during the previous seasons. the kenhardt district reported a severe outbreak towards the end of the season and a few cases were reported from the pietermaritzburg district. no cases were reported from the northern and eastern transvaal regions (division of veterinary services 1976a). an unpublished report of the western cape veterinary region (anonymous 1976) mentions outbreaks in the brandvlei area. 1976–1977 a few outbreaks were reported from the mtunzini and lower umfolozi districts of natal and the queenstown and middelburg areas of the eastern cape province (figure 9) (division of veterinary services 1977a). the monthly disease reports (division of veterinary services 1976b, 1977b) from the regional veterinary services reported outbreaks in gordonia in the free state region (august 1976), the vryburg district of the free state region (september 1976), the middelburg and welkom districts of the free state region (october 1976), the viljoenskroon district of the highveld region and vryburg district of the free state region (november 1976), the queenstown district of the eastern cape and karoo region (january 1977), the queenstown and middelburg districts of the eastern cape and karoo region (february 1977), a few cases from the eastern cape and karoo region and a few suspected cases from the free state region (march 1977). figure 9: districts affected by rift valley fever outbreaks in south africa during 1976–1977. 1977–1978 a few cases were reported from the free state and the eastern cape and karoo regions (figure 10) (division of veterinary services 1978a). the monthly disease reports from the various veterinary regions (division of veterinary services 1977b, 1978b) reported outbreaks in the koppies district of the free state region, where an animal owner also contracted rvf (november 1977). the middelburg veterinary laboratory confirmed one case in the eastern cape and karoo region (january 1978) and reported a suspected case from upington (may 1978). a private veterinarian reported a case from the welkom district of the free state region (february 1978). figure 10: veterinary regions and districts affected by rift valley fever outbreaks in south africa during 1977–1978. 1978–1979 the monthly disease reports of the veterinary regions (division of veterinary services 1978b, 1979) reported outbreaks in the middelburg and prieska districts of the eastern cape and karoo region (november 1978), the pietermaritzburg and mtonjaneni districts of the natal region (december 1978), the estcourt district of the natal region (february 1979) and the bethlehem district of the free state region (april 1979) (see figure 11). figure 11: districts affected by rift valley fever outbreaks in south africa during 1978–1979. 1979–1980 a single outbreak was reported in the mtunzini district of the natal region (directorate of animal health 1980). 1980–1981 several outbreaks of rvf were reported by private veterinarians from the highveld region, but none were laboratory confirmed (directorate of animal health 1981). 1981–1982 three outbreaks were reported from the hlabisa district in the natal region, one outbreak was reported from the eastern cape and karoo region, and another from the bethlehem and kroonstad areas (figure 12) (directorate of animal health 1982). a disease report for 1982 (division of veterinary services 1983a) reported only one rvf outbreak, with no geographical reference, during april 1982, so it is likely that most of the mentioned outbreaks occurred between july and december 1981. figure 12: veterinary region and districts affected by rift valley fever outbreaks in south africa during 1981–1982. 1982–1983 small outbreaks of rvf were reported from the natal region and the highveld region (division of veterinary services 1983b). a disease report by the division of veterinary services (1984a) reported rvf outbreaks during march, april and june 1983. 1983–1984 small outbreaks of rvf were reported from the natal region and the western cape region (figure 13) (division of veterinary services 1984b). however, the outbreak in the western cape region was likely to have occurred in the northern part of the region (now the western part of the northern cape province), and therefore not within the winter rainfall region. a disease report by the division of veterinary services (1984a) reported rvf outbreaks during october, november and december 1983, with 18 outbreaks in november 1983. the report by the division of veterinary services for the following year (directorate of veterinary services 1985c) reported one rvf outbreak in february and one in april 1984. figure 13: veterinary regions affected by rift valley fever outbreaks in south africa during 1983–1984. 1984–1985 small outbreaks of rvf were reported from the utrecht district of natal and the kroonstad and henneman districts in the highveld region. a foetus from the transvaal region tested positive for rvf (figure 14) (directorate of veterinary services 1985a). the report by the division of veterinary services for 1984 (directorate of veterinary services 1985c) reported no rvf outbreaks during the period july–december 1984. figure 14: veterinary regions and districts affected by rift valley fever outbreaks in south africa during 1984–1985. 1985–1986 in this period, 13 outbreaks of rvf were reported from the estcourt, utrecht and ubombo districts of the natal region (director of the natal region of veterinary services 1986b) and seven outbreaks from the port elizabeth, middelburg, beaufort west, hofmeyer and graaff reinett districts of the eastern cape and karoo region (figure 15) (director of the eastern cape and karoo region of veterinary services 1986; directorate of veterinary services 1986b). the 1985 annual report to the oie (directorate of veterinary services 1985b) reports one outbreak during august 1985, without giving a geographic location. however, the monthly report from natal for august 1985 indicated that an outbreak in the estcourt district occurred during this month. the 1986 annual report to the oie (directorate of veterinary services 1986a) reported two outbreaks during february 1986. the monthly reports from natal (director of the natal region of veterinary services 1986a) indicated outbreaks in ubombo during february 1986 and in hlabisa during march 1986. figure 15: districts affected by rift valley fever outbreaks in south africa during 1985–1986. 1986–1987 no cases of rvf were reported for this period (directorate of veterinary services 1987). the annual disease report to the oie for 1987 reported one case in march 1987, but did not give a geographic location (directorate of veterinary services 1989a); the year of last occurrence was given as 1986. 1987–1988 the annual disease report to the oie for 1987 mentioned one case in october 1987, but did not give a geographic location (directorate of veterinary services 1989b). 1988–1989 although the climatic conditions were seemingly favourable for the disease and the veterinary services had predicted outbreaks, none were reported (directorate of animal health 1989). 1989–1990 the annual report to the oie (directorate of veterinary services 1989b) indicated one outbreak between january and march 1989, but without any indication of the geographic location of the outbreak. 1990–1991 during february 1991 five outbreaks of rvf were reported from the estcourt district and two from the kliprivier district of natal (figure 16) (director of the natal region of veterinary services 1991). figure 16: districts affected by rift valley fever outbreaks in south africa during 1990–1991. 1991–1998 according to the directorate of animal health/veterinary services disease database (directorate of animal health 2012), no outbreaks of rvf were reported. 1999 during january 1999 rvf was diagnosed in captive african buffalo (syncerus caffer) in a boma (holding pen for wildlife) at skukuza in the kruger national park (figure 17) (state veterinarian skukuza 2000; directorate of animal health 2012). six buffalo cows aborted and positive virus isolation confirmed rvf. the virus was determined to belong to lineage c (grobbelaar et al. 2011). figure 17: rift valley fever outbreak in captive african buffalo at skukuza during january 1999. 2000–2007 no outbreaks of rvf were reported during this period (directorate of animal health 2012). 2008 between january and june 2008 rvf was reported from the nkomazi, mbombela and dr j.s. moroka local municipalities in mpumalanga, the bela-bela and ba-phalaborwa local municipalities in limpopo, the nokeng tsa taemane, tshwane and kungwini local municipalities in gauteng and the potchefstroom, moretele and madibeng local municipalities in the north west province (figure 18) (directorate of animal health 2012). a total of 15 outbreaks were reported, with 353 animal cases and 103 animal deaths. the species affected were cattle, goats, sheep and african buffalo (directorate of animal health 2012). the national institute for communicable diseases (nicd) (2008) reported 14 human cases during 2008 and lineage c rvf viruses were isolated from humans, cattle and african buffalo (grobbelaar et al. 2011). figure 18: rift valley fever outbreaks in south africa during 2008. 2009 during february to june 2009 several outbreaks of rvf occurred in the ingwe, kwa sani and umngeni local municipalities in kwazulu-natal and the matatiele local municipality in the eastern cape province, and one outbreak occurred in the mbombela local municipality in mpumalanga (figure 19) (directorate of animal health 2012). a total of 19 outbreaks were reported, with 210 animal cases and 66 animal deaths. the species affected were cattle and sheep (directorate of animal health 2012). a lineage c rvf virus was isolated from several human cases (grobbelaar et al. 2011). figure 19: rift valley fever outbreaks in kwazulu-natal and mpumalanga during february to june 2009 and in the northern cape during october to november 2009. during october to november 2009 the northern cape province experienced rvf outbreaks associated with irrigated lands along the lower orange river in the kakamas region (directorate of animal health 2012). a total of 19 outbreaks were reported, with 53 animal cases and 35 animal deaths. the species affected were cattle, sheep and goats (directorate of animal health 2012). the nicd (2009) reported seven human cases associated with these outbreaks and a rvf virus, this time from lineage h, was isolated (grobbelaar et al. 2011). 2010 a major epidemic of rvf occurred during the first half of 2010. it was first reported from the bultfontein and brandfort areas of the free state and ultimately spread to all provinces except kwazulu-natal. the free state, northern cape and eastern cape were the most severely affected (figure 20). the epidemic started in february 2010 and continued until june 2010. for the first time in the history of rvf in south africa several outbreaks occurred in the winter rainfall region of the western cape (directorate of animal health 2012). the virus responsible for the 2010 outbreaks belonged to lineage h (grobbelaar et al. 2011). figure 20: rift valley fever outbreaks in south africa in 2010. during this period a total of 484 outbreaks were reported, with 14 342 animal cases and 8877 animal deaths. the epidemic affected mostly sheep (13 117 cases reported) followed by cattle and goats. indigenous wildlife that were reported to have shown signs of rvf during 2010 were springbok (antidorcas marsupialis), blesbok (damaliscus dorcas dorcas), bontebok (d. dorcas phillipsi), waterbuck (kobus ellipsiprymnus), african buffalo, sable (hippotragus niger), kudu (tragelaphus strepsiceros), nyala (tragelaphus angasii) and gemsbok (oryx gazella). some exotic species that were affected were fallow deer (cervus dama), llama (lama glama), alpaca (lama pacos), asian buffalo (bubalus bubalis) and ibex (capra ibex). in most of these indigenous and exotic wildlife species these were the first documented cases of rvf (directorate of animal health 2012). 2011 during 2011 a smaller epidemic of rvf occurred, involving mostly the eastern cape and some outbreaks in the western and the northern cape provinces (figure 21). a total of 135 outbeaks were reported, involving 4139 animals. sheep were most affected (3491 animals), followed by goats (327 animals) and 195 cattle (directorate of animal health 2012). figure 21: rift valley fever outbreaks in south africa in 2011. wild animals affected were springbok, bontebok and buffalo. one fallow deer and a cape hare (lepus capensis) also showed clinical signs of rvf (directorate of animal health 2012).during 2012 no outbreaks of rvf were reported (directorate of animal health 2012). for easy reference, a summary of all the recorded laboratory-confirmed rvf outbreaks in south africa between 1950 and 2011 is given in table 1. table 1: summary of rift valley fever outbreaks in south africa, 1950–2011. cumulative temporal and spatial distribution of outbreaks top ↑ the temporal distribution and indication of the extent of the rvf outbreaks are shown in figure 22. prior to about 1990, bar heights overestimate the number of blocks that experienced outbreaks as some earlier reports mentioned only the veterinary region, in which case all the squares within that region were counted as having experienced an outbreak. however, the three major rvf epidemics (1950–1951, 1973–1976 and 2010–2011) can be identified. figure 22: the temporal distribution of reported rift valley fever outbreaks in south africa during the period 1950–2010. the geographic distribution of all the reported rvf outbreaks since 1950 and the cumulative number of seasons during which at least one outbreak was recorded for each half degree square block are shown in figure 23. for the same reason as in figure 22 the extent of earlier outbreaks, particularly in the central and western regions, is overestimated. in contrast, because outbreaks in the eastern parts of the country generally remained localised, the relative frequency with which they were initiated in that region is under-emphasised in this representation. nevertheless, the main region in which extensive outbreaks occurred is clearly visible in the central interior of the country. figure 23: the cumulative number of seasons during which rift valley fever outbreaks occurred in south africa during the period 1950–2011. for outbreaks where the actual month of occurrence was mentioned, table 2 gives a summary of the months during which rvf outbreaks occurred during each season. most outbreaks occurred from january onwards, but where outbreaks occurred before january, this often followed outbreaks late during the previous season. table 2: the temporal distribution of rift valley fever outbreaks in south africa (1950–2010) for each season where the month of the outbreak is known. discussion top ↑ the search for documented outbreaks of rvf in south africa yielded potentially useful results. for the first time the location and season of all the recorded rvf outbreaks in south africa have been documented. this will facilitate further research into the epidemiology of the disease in south africa and will promote a better understanding of the disease, leading to improved management, limiting the loss of livestock and human life. the poor spatial and, to a lesser extent, temporal precision in the reporting of earlier outbreaks undoubtedly reduced the accuracy of our calculated cumulative statistics prior to 1990 shown in figures 22 and 23. several other factors may have influenced the reporting rate of rvf, including awareness by farmers, veterinarians and animal health workers, farmer perception and compliance, veterinary coverage of remote areas, and possibly low clinical expression of the disease in small, localised outbreaks. nevertheless, it is likely that the status of rvf as a reportable or notifiable disease helped to ensure that the passive surveillance system captured most outbreaks of the disease.several outbreaks not previously recorded in the literature were documented during this study, for example the outbreaks during the 1978–1979 season and the small outbreaks between 1982 and 1985. the results indicate that the occurrence of rvf has been more frequent and more widespread than is generally believed, likely because the disease was often absent from an area for long periods and new farmers and veterinarians subsequently being unaware that an area had experienced outbreaks in the past. it is therefore imperative that farmers and veterinarians should continuously be made aware of the risk of rvf outbreaks and the need to vaccinate. the occurrence of a large epidemic in 2010 will increase awareness of rvf for several years to come, but the disease will likely largely disappear again and be forgotten in the future. this study can serve as motivation for vaccination programmes during interepidemic periods. the general pattern that emerged from this analysis is that large epidemics tended to occur in the central parts of the country (free state, eastern cape and northern cape), separated by long periods of absence or with small, localised intervening outbreaks. smaller outbreaks tended to occur in the eastern parts of the country (kwazulu-natal and gauteng). outbreaks often occurred in these areas during the same season, but several times the outbreaks in the eastern part of the country were apparently not associated with those in the central region, for example in 1991, 2008 and 2009. grobbelaar et al. (2011) showed that rvf virus isolates from across africa grouped into 15 lineages, of which eight have been detected in south africa, and that the viruses responsible for the outbreaks in the eastern parts of the country in 2008 and 2009 (lineage c) were different from those associated with outbreaks in the central regions in 2009 and 2010 (lineage h). apart from this, differences in the pattern of outbreaks between the regions could not be identified during this study, but it is possible that different vector species are responsible (jupp 2004). in addition, differences in climate, topography, vegetation and host populations are likely to have had a major influence, but fall beyond the scope of this paper and will be assessed in further analyses. the reservoir or mechanism for maintenance of the rvf virus during interepidemic periods in south africa has not been established; however, several possibilities exist. the first is that transovarial transmission takes place in certain aedes spp. mosquitoes, associated with shallow grassland depressions or ‘dambos’ (linthicum et al. 1985; pepin et al. 2010). an increase in these mosquitoes triggers an outbreak, which is propagated by other mosquito species (gerdes 2004). however, studies have not been able to demonstrate transovarial transmission in south africa (mcintosh et al. 1980b). each of the three major epidemics in the central interior of south africa was associated with a different rvf virus lineage (grobbelaar et al. 2011), indicating that long-term survival of virus in dormant mosquito eggs is unlikely to have been the main mechanism for virus survival between these epidemics. another possibility is low-level circulation of virus between animals and vectors, without resulting in clinical signs or severe outbreaks (pepin et al. 2010). serological evidence of low-level circulation of rvf virus in african buffalo has been found in the kruger national park (r.g. bengis, pers. comm., 27 october 2010) and antibodies have been found in several wildlife species in kenya (evans et al. 2008). davies et al. (1992) also showed low-level circulation amongst cattle in zambia. the possibility of an enzootic transmission cycle involving wildlife or domestic stock therefore requires further investigation. incursions of vectors from possible endemic areas, either within south africa or in neighbouring countries, is also a potential mechanism for initiation of outbreaks in south africa and may be related to meteorological conditions. it is also possible that the movement of infected livestock or other unknown carriers could occur and initiate outbreaks. closely related viruses have been isolated from geographically distant parts of africa (grobbelaar et al. 2011), suggesting that long-distance dispersal of either vectors or infected animals occurs, and phylogeographic analysis indicates a complex pattern of long-distance viral movement throughout africa (soumaré et al. 2012). further investigation of the epidemiology of rvf and modelling of its transmission in southern africa, taking into account virus phylogenetics and a variety of potential risk factors, is required to test these hypotheses. our analysis showed that most reported outbreaks occurred during mid to late summer, from january onwards. this coincided with the period of highest rainfall in most areas, which, coupled with warm temperatures, would have resulted in greater vector numbers. where outbreaks occurred before january, this usually followed outbreaks late during the previous season; in other words, the epidemic ‘survived’ the winter. this was well documented during the period from 1974 to 1976, when outbreaks occurred throughout the winter of 1975 and continued into the next season when a warm, wet winter was experienced. this presumably resulted in the vectors maintaining the infection through the winter. from the results (also see figure 22) it is clear that certain areas were more prone to extensive rvf outbreaks. this agrees with a kenyan study (murithi et al. 2010), which showed that districts where outbreaks had occurred in the past were five times more likely to experience a subsequent outbreak. the areas in south africa that experienced the most extensive outbreaks were in the central interior of the country, particularly the western free state, north-western part of the eastern cape and the central part of the eastern northern cape. the literature consulted for this study did not provide enough detail on the spread of rvf within a season, but the analysis of the 2010 epidemic suggests that the disease spreads from one area to the next once a large epidemic is triggered. a plausible explanation for this could be that enzootic vectors trigger the outbreak, which is then spread by epizootic vectors (swanepoel 2009). factors such as animal movement could also play an important role in the spread of the disease during an epidemic. from the results it also becomes evident that after a large epidemic, such as the ones experienced during 1950–1951 and 1973–1976, outbreaks continue to occur for several years after the epidemic. this would suggest that south africa may experience more outbreaks over the next few years in the wake of the 2010 epidemic. in addition, unless vaccination is continued, large epidemics are likely to recur once the proportion of immune animals in the population wanes and favourable environmental conditions occur. it is therefore necessary to raise awareness of this situation and to implement vaccination programmes that should be sustained through the interepidemic periods, particularly in the high-risk areas identified in this study. in addition, more research is required to investigate the mechanisms for rvf viral maintenance during interepidemic periods in southern africa to elucidate the risk factors for not only the initiation but also the perpetuation of outbreaks. findings will contribute to refine techniques for predicting future outbreaks. acknowledgements top ↑ the authors would like to thank dr grietjie de klerk, dr sunelle strydom and the late dr willie ungerer for their assistance and patience in compiling this report, the director of animal health for permission to conduct this study, and the provincial directors of veterinary services for permission to use their data. we also wish to thank all the national and provincial officials who assisted with the data collection. three reviewers offered constructive comments, which helped to improve the manuscript. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions n.j.p. (university of pretoria) performed the study and wrote the article. p.n.t. (university of pretoria) was the academic study leader for the msc degree of which this report formed part and assisted in study design, data analysis and writing of the article. references top ↑ alexander, r.a., 1951, ‘rift valley fever in the union’, journal of the south african veterinary medical association 22, 105–109. alexander, r.a., 1955a, ‘annual report for 1 july 1953 to 30 june 1954 of the directorate of veterinary services’, department of agriculture, pretoria. alexander, r.a., 1955b, ‘jaarverslag vir 1 julie 1954 tot 30 junie 1955 vir die afdeling veeartsenydiens [annual report for 1 july 1954 to 30 june 1955 of the section veterinary services]’, department of agriculture, pretoria. alexander, r.a., 1956, ‘jaarverslag vir 1 julie 1955 tot 30 junie 1956 vir die afdeling veeartsenydiens [annual report for 1 july 1955 to 30 june 1956 of the section veterinary services]’, department of agriculture, pretoria. anonymous, 1976, ‘report on rift valley fever from the western cape 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http://dx.doi.org/10.1051/vetres/2010033 soumaré, p.o.l., freire, c.c.m., faye, o., diallo, m., de oliveira, j.v.c., zanotto, p.m.a. et al., 2012, ‘phylogeography of rift valley fever virus in africa reveals multiple introductions in senegal and mauritania’, plos one 7, e35216. http://dx.doi.org/10.1371/journal.pone.0035216 state veterinarian skukuza, 2000, ‘annual report for january 1999 to december 1999’, directorate of veterinary services. swanepoel, r. & coetzer, j.a.w., 2004, ‘rift valley fever’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, 2nd edn., pp. 1037–1070, oxford university press, cape town. swanepoel, r., 2009, ‘re-emergence of rift valley fever in south africa: how can we better predict and respond?’, keynote address at the oie regional seminar on rift valley fever, bloemfontein, 16–18 february. van der linde, n.t., 1953, ‘a recent epidemic of rift valley fever in the orange free state’, journal of the south african veterinary medical association 24, 145–148. article information authors: katendi changula1 a.s. mweene1 affiliations: 1school of veterinary medicine, university of zambia, zambia correspondence to: katendi changula postal address: po box 32379, lusaka, zambia how to cite this abstract: changula, k. & mweene, a.s., 2012, ‘filoviral haemorrhagic fevers: a threat to zambia?’, onderstepoort journal of veterinary research79(2), art. #465, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.465 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. filoviral haemorrhagic fevers: a threat to zambia? in this abstract... open access filoviral haemorrhagic fevers (fvhf) are caused by agents belonging to filoviridae family, ebola and marburg viruses. they are amongst the most lethal pathogens known to infect humans. incidence of fvhf outbreaks are increasing, with affected number of patients on the rise. whilst there has been no report yet of fvhf in zambia, its proximity to angola and democratic republic of congo, which have recorded major outbreaks, as well as the open borders, increased trade and annual migration of bats between these countries, puts zambia at present and increased risk. previous studies have indicated bats as potential reservoir hosts for filoviruses. an increasing population with an increasing demand for resources has forced incursion into previously uninhabited land, potentially bringing them into contact with unknown pathogens, reservoir hosts and/or amplifying hosts. the recent discovery of a novel arenavirus, lujo, highlights the potential that every region, including zambia, has for being the epicentre or primary focus for emerging and re-emerging infections. it is therefore imperative that surveillance for potential emerging infections, such as viral haemorrhagic fevers be instituted. in order to accomplish this surveillance, rapid detection, identification and monitoring of agents in patients and potential reservoirs is needed. international co-operation is the strategy of choice for the surveillance and fight against emerging infections. due to the extensive area in which filoviral infections can occur, a regional approach to surveillance activities is required, with regional referral centres. there is a need to adopt shared policies for the prevention and control of infectious diseases. there is also need for optimisation of currently available tests and development of new diagnostic tests, in order to have robust, highly sensitive and specific diagnostic tests that can be used even where there are inadequate laboratories and diagnostic services. article information authors: dirk booyse1 burk a. dehority2 affiliations: 1department of anatomy and physiology, university of pretoria, south africa 2department of animal sciences, ohio state university, usa correspondence to: dirk booyse email: dbooyse@op.up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 16 nov. 2010 accepted: 16 feb. 2011 published: 21 july 2011 how to cite this article: booyse, d. & dehority, b.a., 2011, ‘rumen protozoa in south african sheep with a summary of the worldwide distribution of sheep protozoa’, onderstepoort journal of veterinary research 78(1), art. #307, 7 pages. doi:10.4102/ojvr.v78i1.307 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) rumen protozoa in south african sheep with a summary of the worldwide distribution of sheep protozoa in this original research... open access • abstract • introduction • materials and methods    • ethical considerations • results and discussion • conclusion • references abstract (back to top) protozoa species were identified in rumen contents of four domestic sheep (ovis aries) from south africa. all animals were fed a forage diet which consisted of 50% lucerne and 50% teff hay. ten new host records were identified, bringing the total number of species and forms observed in sheep in south africa to 30. the occurrence and geographic distribution of ciliate protozoa in both domestic and wild sheep from around the world are summarised. it was found that 15 genera and 131 species occur in domestic sheep globally. introduction (back to top) other than several early reports from south africa on rumen ciliate protozoa in domestic sheep (fantham 1920, 1921, 1926; van der wath & myburgh 1941), no additional research in this respect has been published. these previous studies identified a total of only 12 species and forms. studies from other areas of africa are also limited (see, e.g. selim et al. [1996] for egypt and selim et al. [1999] for libya). to date only 17 species have been observed in domestic sheep across africa. materials and methods (back to top) each sample was washed in a pitchford–visser filter, similar to the one used for trematode egg counts. the outer sieve had an aperture of 37 μm, whilst apertures in the inner sieve were 110 μm. this procedure retained all the coarse material in the inner sieve and the protozoa were captured in the outer sieve. the sample was recovered from the outer sieve by means of a drain tap. the washed sample was drained into a container, allowed to stand and settle for 15 min and then most of the supernatant was decanted. this allowed concentration of the sample to a final volume of 40 ml. a measured volume of alcohol was added to preserve the sample. a few drops of methyl blue stain were added to stain the protozoa and nucleus (booyse, boomker & dehority 2010). a 0.1-ml aliquot of stained sample was transferred onto a glass microscope slide using a ‘finnpipette’, commonly used in chemistry laboratories. the sample was covered with a glass cover slip and examined with a standard nikon microscope fitted with a panasonic digital camera. photographs were taken to aid identification. each sample was examined in triplicate. owing to the observed lack of entodinium spp. in the washed samples, additional samples of rumen contents from the four sheep were obtained. sub-samples (0.1 ml) of these samples were stained and examined under the microscope to verify the presence of entodinium spp., which had been lost through the 37-µm filter. ethical considerations housing and care of fistulated animals in the department of anatomy and physiology were approved on an ongoing basis by the university of pretoria animal care and use committee, as sheep are routinely used as rumen fluid donors, both for experimental and for teaching purposes. results and discussion (back to top) a total of 20 species and forms of protozoa were observed in the four sheep used in the present study. of these species, 10 were identified in south africa for the first time (table 1). this brings the total number of species and forms reported from sheep in south africa to 30. all entodinium species listed in table 1 were identified in whole rumen content (i.e. unfiltered samples). it is noteworthy that for these new host records in south africa at least three of the entodinium species are closely related or possibly similar to entodinium dubardi (dehority 1994). these are entodinium caudatum f. dubardi, entodinium parvum and entodinium simplex. entodinium nanellum would have been included in this group, but had been observed previously. two of the other entodinium species reported as a new host record, namely entodinium exiguum and entodinium longinucleatum, can occur on a variation line with e. dubardi. however, two rather distinctive features allow identification as a separate species: for e. exiguum, a straight oesophagus that does not curve towards the macronucleus, and for e. longinucleatum, a macronucleus extending the entire length of the cell. the number of species and forms observed for other geographical locations range from fewer than 10 to 49, as shown in table 2. the number found in the present study is about halfway between these extremes. in general, several reports where only a limited number of species were observed, appear not to be comprehensive studies; that is, only a few species were identified rather than a complete listing of all species present. göçmen et al. (1999) observed that the protozoan fauna of sheep in turkey and far eastern areas (e.g. china and japan) appeared to be more diverse than in european and american sheep. a number of the studies listed in table 2 were reported between 1920 and 1985, before the most widely used classification scheme of lee, hutner and bovee (1985) was published. therefore, the older species names in the subfamily diplodiniinae listed in the original reports have been corrected according to the more recent classification scheme. for example, eodinium spp. are now classified under diplodinium and eremoplastron spp. under eudiplodinium. diploplastron affine is now classified as metadinium affine. in more recent studies by göçmen (1999a, 1999b), classification of both epidinium spp. and ophryoscolex spp. have been redefined and are used in table 2. the total number of protozoa found in each of the localities indicated in table 2 is summarised and listed at the end of the table. table 3 lists species and forms of protozoa observed in wild bighorn and dall sheep in the usa. bighorn sheep are found in the rocky mountains from colorado to canada, whilst the dall sheep live further north, reaching into alaska. many of the protozoa found in these two species (10 in bighorn and 11 in dall sheep) are also present in domestic sheep. although not reported from domestic sheep, at least two species have been observed in turkish cattle, namely entodinium dalli, and a second form, entodinium dalli rudidorosospinatum (göçmen & őktem 1996). of particular interest is the presence of two new species of polyplastron, neither of which has been observed elsewhere. it is possible that as further in-depth studies are conducted, we will find a somewhat uniform distribution of protozoal species around the world. table 1: rumen ciliate protozoa observed in domestic sheep (ovis aries) in south africa. table 2: compilation of the protozoan fauna reported for domestic sheep (ovis aries) from numerous geographical locations around the world. the numbers in each locality refers to the references listed below the table. table 3: protozoa recorded in bighorn (ovis canadensis) and dall (ovis dalli) sheep. the numbers in each locality refers to the references listed below the table. conclusion (back to top) we identified 10 species of rumen ciliate protozoa that had not been previously observed in rumen contents of south african sheep. this brings the total of species observed in this country to 30. the collection of protozoal fauna in south african sheep was compared to previous data reported for sheep worldwide and appeared to be somewhat less diverse. references (back to top) banerjee, k., 1955, ‘studies on parasitic ciliates from indian ruminants’, proceedings of the zoological society 8, 87−101. booyse, d.g., boomker, e.a. & dehority, b.a., 2010, ‘protozoa in the digestive tract of wild herbivores in south africa, i: warthogs (phacochoerus aethiopicus)’, zootaxa 2492, 63−68. bush, m. & kofoid, 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& torun, s., 1997, ‘turkiye evcil koyun (ovis ammon aries)’larinin işkembe siliyat (protozoa: ciliophora) faunasi hakkinda bir ön calişma: ifamilya isotrichidae (trichostomatida) ve entodiniidae (entodiniomorphida)’, turkish journal of zoology 21, 475−502. selim, h.m., imai, s., yamato, o., el kabbany, a., kiroloss, f. & maede, y., 1996, ‘comparative study of rumen ciliates in buffalo, cattle and sheep in egypt’, journal of veterinary and medical science 58, 799−801. doi:10.1292/jvms.61.303 selim, h.m., imai, s., sheik, a.k., attia, h., okamoto, e., miyagawa, e. et al., 1999, ‘rumen ciliate protozoal fauna of native sheep, friesian cattle and dromedary camel in libya’, journal of veterinary and medical science 61, 303−305. van der wath, j.g. & myburgh, s.j., 1941, ‘studies on the alimentary tract of merino sheep in south africa. vi. the role of infusoria in ruminal digestion with some remarks on ruminal bacteria’, onderstepoort journal of veterinary science and animal industry 17, 61−85. wertheim, p., 1935, ‘infusorien aus dem widerkauermagen von gebiete jugoslawiens nebs einer ubersicht dieser tierchen von balkanhalbinsel bereich und ein kurzer bericht uber die pferdedarminfusorien, zugleich revision der familie ophryoscolecidae’, veterinarski archiv 5, 386−537. inpankaew_161-165.indd introduction cryptosporidium parvum is an obligate intracellular protozoan parasite that mainly infects the gastrointestinal tract of a wide range of vertebrates including livestock and humans (current & garcia 1991). infection of immunocompetent humans can induce acute, self-limiting diarrhoea. in contrast, infection of immunodeficient individuals frequently results in persistent, severe and life-threatening diarrhoea (gu errant 1997). cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in aids patients in thailand. moreover, manatsathit, tansupasawas dikul, wanachiwanawin, setawarin, su wanagool, pra kas vejakit, leelakusolwong, eampokalap & kach intorn (1996) reported that c. parvum is the most common enteric pathogen contrib161 onderstepoort journal of veterinary research, 76:161–165 (2009) seroprevalence of cryptosporidium parvum infection of dairy cows in three northern provinces of thailand determined by enzyme-linked immunosorbent assay using recombinant antigen cpp23 t. inpankaew1, 3, s. jittapalapong1, j. phasuk1, n. pinyopanuwut1, w. chimnoi1, c. kengradomkit1, c. sunanta2, g. zhang3, g.o. aboge3, y. nishikawa3, i. igarashi3 and x. xuan3* abstract inpankaew, t., jittapalapong, s., phasuk, j., pinyopanuwut, n., chimnoi, w., kengra domkit, c., sunanta, c., zhang, g., aboge, g.o., nishikawa, y., igarashi, i. & xuan, x. 2009. seroprevalence of cryptosporidium parvum infection of dairy cows in three northern provinces of thailand determined by enzyme-linked immunosorbent assay using recombinant antigen cpp23. onderstepoort journal of veterinary research, 76:161–165 cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in aids patients in thailand. cryptosporidiosis outbreaks in humans may be attributed to contamination of their drinking water from infected dairy pastures. a 23-kda glycoprotein of c. parvum (cpp23) is a sporozoite surface protein that is geographically conserved among c. parvum isolates. this glycoprotein is a potentially useful candidate antigen for the diagnosis of cryptosporidiosis by enzyme-linked immunosorbent assay. therefore, we investigated the seroprevalence of c. parvum infection in dairy cows in northern thailand using an elisa based on recombinant cpp23 antigen. sera were randomly collected from 642 dairy cows of 42 small-holder farmers, which had the top three highest number of the dairy cows’ population in northern thailand, that included chiang mai, chiang rai and lumpang provinces. the overall seroprevalence of the infection was 4.4 %, and the seropositive rates for the three provinces were 3.3 % in chiang mai, 5.1 % in chiang rai and 3 % in lumpang. these results suggest that cattle could play a role in zoonotic cryptosporidiosis in thailand. keywords: cryptosporidium parvum, cpp23, dairy cow, elisa, thailand * author to whom correspondence is to be directed. e-mail: gen@obihiro.ac.jp 1 department of parasitology, faculty of veterinary medicine, kasetsart university, bangkok, thailand 2 chiang rai provincial office, department of livestock develop ment, chiang rai, thailand 3 national research center for protozoan diseases, obihiro university of agriculture and veterinary medicine, inada-cho, obihiro, hokkaido 080-8555, japan accepted for publication 14 august 2008—editor 162 cryptosporidium parvum infection of dairy cows in thailand uting to 20 % of the causes of chronic diarrhoea in aids patients in thailand. in the developing world, cryptosporidium constitutes part of a complex group of parasitic, bacterial and viral diseases that leads to an inability of infected individuals to achieve their full potential. this complex group of infections classified by the world health organization (who) as “neglected diseases”, initiate common diseases associated with poverty (savioli, smith & thompson 2006). crypto sporidiosis outbreaks in humans are thought to be due to contamination of their drinking water from infected dairy pastures. in a recent survey in the nong pho dairy region of central thailand using cryptosporidium-specific antigen (csa) it was found that the seroprevalence rate of cryptosporidium infection in dairy cows was 9.4 % (jittapalapong, pinyopanuwat, chimnoi, siripanth & stich 2006). this finding suggested that the infection could also be endemic in other areas in thailand where the same study had not been done. hence, there was need for a further investigation into the distribution of cryptosporidiosis in cattle in these regions. furthermore, the diagnosis of c. parvum infection relies almost exclusively on the microscopic detection of oocysts in faeces, but this method is relatively time consuming, subjective and unreliable especially when oocysts shedding in the faeces has ceased. nevertheless, a serological test based on elisa is capable of partially fulfilling diagnostic requirements because serum antibodies against the parasite persist even after oocysts shedding in faeces has ceased. a 23-kda glycoprotein of c. parvum (cpp23) is a sporozoite surface protein that is geographically conserved among c. parvum isolates. this conserved glycoprotein is potentially a useful candidate antigen for the diagnosis of cryptosporidiosis by elisa since it is likely to detect c. parvum strains in various geographical regions (perryman, kapil, jones & hunt 1999). therefore, an elisa based on the recombinant cpp23 antigen was used in this study to investigate the prevalence of c. parvum infection in dairy cows in the northern part of thailand. materials and methods parasite cryptosporidium parvum isolate (hnj-1 strain) was used in this study (abe, kimata & iseki 2002). cloning of cpp23 gene purified c. parvum oocysts were lyzed in 0.1 m trishcl (ph 8.0) containing 1 % sodium dodecyl sulphate (sds), 0.1 m nacl, and 10 mm edta and then treated with proteinase k (100 μg/mℓ) at 55 °c for 2 h. the genomic dna pellets were extracted with phenol/chloroform followed by ethanol precipitation. the dna pellets were dissolved in a te buffer (10 mm tris-hcl, ph 8.0, and 1 mm edta) and used as a template dna for pcr. the truncated cpp23 gene without sequences encoding a hydrophobic signal peptide and a c-terminus was amplified by pcr using oligonucleotide primers, 5’-acggatccaaaaatgggttgtt-3’ and 5’-acggatcctaatttaggcatca-3’, both containing bamhi sites introduced to facilitate cloning. the pcr product was digested with bamhi and then cloned into the bamhi site of the bacterial expression vector, pgex-4t-3 (promega, usa). the resulting plasmid was designated as pgex/cpp23. expression of the cpp23 gene in escherichia coli the recombinant cpp23 gene was expressed as a glutathione s-transferase (gst)-fusion protein (gst-cpp23) in jm109 e. coli (promega, usa) as described by takashima, xuan, kimata, iseki, kodama, nagane, nagasawa, matsumoto, mikami & otsuka 2003 and then purified. elisa the elisa was performed as reported by bannai, nishigawa, seo, nakamura, zhang, kimata, ta kashima, li, igarashi & xuan 2006. briefly, the purified gst-cpp23 was diluted to an optimal concentration (5 μg/mℓ) in a 50 mm carbonate-bicarbonate buffer (ph 9.6), of which 50 μℓ were added separately to duplicate wells for each sample. coated plates were incubated at 4 °c overnight. after the unabsorbed antigen was discarded, the wells were blocked with pbs containing 3 % skim milk (blocking solution, 100 μℓ per well) at 37 °c for 1 h. the plates were then washed once with pbs containing 0.05 % tween 20 (pbs-t). fifty microlitres of serum diluted in the blocking solution (1:100) were added to each well and incubated at 37 °c for 1 h. after incubation, the wells were washed six times with pbs-t and subsequently incubated with 50 μℓ of goat anti-bovine igg-horseradish peroxidase conjugate (icn biochemical, usa) (1:4 000) at 37 °c for 1 h. after six washes, 100 μℓ of substrate solution [0.05 % 2,2’-azino-bis (3-ethylbenz-thiazoline-6-sul163 t. inpankaew et al. phonic), 0.2 m sodium phosphate, 0.1 m citric acid, 0.003 % h2o2] were added to each well. after 1 h reaction at room temperature, the optimal density (od) was read at 415 nm by using an mtp-120 elisa reader (corona electric, japan). the elisa titre was expressed as the reciprocal of the maximum dilution that showed an elisa value equal to or greater than 0.1, which is the difference in absorbance between that for the antigen (gst-cpp23) well and that of the control antigen (gst) well. sera blood samples (n = 642) were collected from the caudal or jugular vein of dairy cows belonging to 42 small-holder farmers of the top three highest number of dairy cow population in chiang mai (150 samples), chiang rai (392 samples) and lumpang provinces (100 samples) (fig. 1). sera were separated after sedimentation of blood cells and were stored at –20 °c until use. results and discussion the truncated cpp23 gene without sequences encoding a hydrophobic signal peptide and a c-terminus was inserted into the bacterial expression vector pgex-4t-3, and expressed as a gst fusion protein (gst-cpp23) in e. coli. the gst-cpp23 reacted strongly with sera from c. parvum-infected cattle but not with sera from uninfected cattle (data not shown). this result indicated that the gstcpp23 can be adopted as a useful antigen for serodiagnosis of c. parvum infection. the prevalence of c. parvum infection in cattle has been reported in many parts of the world, such as canada (40.6 %) (trotz williams, jarvie, martin, leslie & peregrine 2005), usa (8.7 %) (fay er, trout & graczyk 2000), spain (8.4 %) (cas tro-hermida, almeida, gonzález-warleta, correia da costa, rumbo-lorenzo & mezo 2007), australia (48 %) (becher, robertson, fraser, palmer & thompson 2004), japan (12 %) (sakai, tsushima, nagasawa, ducusin, tanabe, uzaka & sarashina 2003) and vietnam (33.5 %) (nguyen, nguyen, le, le hua, van nguyen, honma & nakai 2007). in thailand, most investigations have been carried out in humans, but less is known of the infection in animals, particularly in dairy cows, which might be the carrier of the parasite. the elisa with gst-cpp23 as antigen was used to investigate the seroprevalence of c. parvum infection in dairy cows in the northern part of thailand. the overall seroprevalence of c. parvum infection was 4.4 % (28/642). this is lower than the prevalence (9.4 %) in nong pho dairy areas (the central part of thailand) as has previously been reported (jittapalapong et al. 2006). the seropositive rates in three provinces were 3.3 % (5/150) in chiang mai, 5.1 % (20/392) in chiang rai and 3 % (3/100) in lumpang (table 1). chiang rai province was the highest endemic area for c. parvum infection in this investigation. the numbers of dairy farm harbouring infected cows were from 16 to 42 (38 %) and the farm infection prevalence was 37.5 % (3/8), 37.9 % (11/29) and 40 % (2/5) in chiang mai, chiang rai and lumpang respectively. a widespread infection rate in dairy cows due to c. parvum in the northern fig. 1 map of thailand showing the location of the three provinces from which blood samples were collected: chiang mai prov ince (cm), chiang rai province (cr) and lumpang province (lp) 164 cryptosporidium parvum infection of dairy cows in thailand region of thailand was therefore determined in this study. the seroprevalence of c. parvum infection rates varied in different age groups, ranging from 2.63 % to 14.29 % (table 2). the age of the animal is one of the most important risk factors associated with cryptosporidiosis. however, the occurrence of c. par vum infection in the current study was found in all age groups but no statistically significant differences between the age groups were determined. nevertheless, it has been shown that asymptomatic adult domestic ruminants, such as dairy cows, sheep and goats may act as carriers and may be a source of infection for younger animals (fayer et al. 2000; bomfim, huber, gomes & alves 2005). the current study demonstrated a low prevalence rate of c. parvum in dairy cows; however, asymptomatic cattle can serve as important natural reservoirs for this parasite. these data also indicate a potential risk of c. parvum transmission to the human population in thailand and the need for more attention to be paid to its control in dairy cattle because of its zoonotic nature. acknowledgements we thank the provincial veterinary officers in the department of livestock development in chiang rai, chiang mai and lumpang provinces for their help in collecting the blood samples from the dairy farms. we also thank all the diary cow owners cooperated in this study. this project was financially supported by the faculty of veterinary medicine, kaset sart university research development institute (kurdi), the japan international cooperation agency (jica) and the program of founding research center for emerging and re-emerging infectious diseases, mext japan. references abe, n., kimata, i. & iseki, m. 2002. identification of genotypes of cryptosporidium parvum isolated from a patient and dog in japan. journal of veterinary medical science, 64:65– 168. bannai, h., nishigawa, y., seo, j., nakamura, c., zhang, s., kimata, i., takashima, y., li, j., igarashi, i. & xuan, x. 2006. development and evaluation of an enzyme-linked immunosorbent assay using recombinant p23 for the detection of antibodies to cryptosporidium parvum in cattle. journal of protozoology research, 16:9–15. becher, k.a., robertson, i.d., fraser, d.m., palmer, d.g. & thompson, r.c. 2004. molecular epidemiology of giardia and cryptosporidium infections in dairy calves originating from three sources in western australia. veterinary para sitology, 123:1–9. bomfim, 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herpesvirus-1 expressing p23 protein of cryptosporidium parvum induces neutralizing antibodies in rabbits. journal of parasitology, 89:276–282. trotz williams, l.a., jarvie, b.d., martin, s.w., leslie, k.e. & peregrine, a.s. 2005. prevalence of crypto sporidium infection in southwestern ontario and its association with diarrhea in neonatal diary calves. canadian veterinary journal, 46:349–351. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: eltayb m. abuelzein1 mofeed a. al-khaliyfa2 ahmed a. gameel3 affiliations: 1king fahad medical research centre, king abdulaziz university, saudi arabia 2almarai company, riyadh, saudi arabia 3faculty of veterinary medicine, university of khartoum, sudan correspondence to: eltayb abuelzein email: eabuelzein@yahoo.com postal address: po box 80216, jeddah 21589, saudi arabia dates: received: 24 jan. 2011 accepted: 04 aug. 2011 published: 09 nov. 2011 how to cite this article: abuelzein, e.m., al-khaliyfa, m.j. & gameel, a.a., 2011, ‘natural in utero infection of neonatal calves with bovine viral diarrhoea virus on a large dairy farm in saudi arabia’, onderstepoort journal of veterinary research 78(1), art. #318, 4 pages. http://dx.doi.org/10.4102/ ojvr.v78i1.318 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) natural in utero infection of neonatal calves with bovine viral diarrhoea virus on a large dairy farm in saudi arabia in this original research... open access • abstract • introduction • materials and methods    • ethical consideration • results • discussion • conclusion • acknowledgements    • author contributions • references abstract (back to top) the dairy industry is a large and important business in saudi arabia. although farms are administered to high international standards, some reproduction problems, of uncertain aetiology, are encountered. the most frequently seen are conception failures, abortions, stillbirths and the birth of weak or malformed calves. these conditions are suggestive of bovine viral diarrhoea virus (bvdv) infection. unfortunately, very little published information is available regarding the impact of this disease on cattle populations in saudi arabia. as a consequence, the present study was carried out and is the first of its kind in saudi arabia and the gulfregion. the aim of the study was to elucidate the role of in utero bvdv infection leading to the birth of weak or malformed calves on a large dairy farm in saudi arabia. the study was divided into two parts. firstly, apparently healthy neonatal calves were sampled for the detection of pre-colostral serum antibodies to bvdv. the presence of these antibodies indicates exposure of the foetus to bvdv during the last two trimesters of gestation. secondly, tissue samples from malformed neonatal calves were examined for the presence of bvdv antigens. detection of such antigens confirms exposure of the foetus to the virus during the first trimester of gestation. the results of the investigation indicated that 36.1% of the neonatal calves were exposed to bvdv infection in utero. this is higher than what has been reported in the literature and suggests that dairy farmers in the arabian peninsula need to be made aware of the dangers of bvdv infections in their herds. the epidemiological significance of the results is discussed. introduction (back to top) bovine viral diarrhoea (bvd) (olafson, maccallum & fox 1946) is caused by bovine viral diarrhoea virus (bvdv), a pestivirus of the family flaviviridae. this disease has long been a scourge to dairy and other cattle populations in the world (world organization for animal health 2008). cattle of all ages are susceptible and infection can be acquired in utero. embryos and foetuses so infected may exhibit a range of clinical signs, including death, and infection can also lead to the birth of persistently infected (pi) calves (grooms 2004). therefore, bvdv infection can have major implications for the cattle industry (kampa 2006; lindberg 2002). massive economical losses are attributed to bvdv infection in the dairy industry worldwide (heur, healy & zerbini 2007). in developed countries, the impact of bvd on the cattle industry has been well documented (kampa 2006; lindberg 2002). unfortunately, in most developing countries, the economical losses due to bvd have not been studied, despite the presence of substantial dairy industries. this is also the situation in saudi arabia. the harsh and arid ecosystems in saudi arabia are generally unsuitable for raising cattle. however, being an affluent country, saudi arabia has succeeded in establishing big dairy farms that supply both local markets and other gulf states with milk and dairy products. cattle on these farms are mainly imported holstein friesians. however, some holstein friesians are bred locally by artificial insemination, using imported semen. many of these dairy farms experience reproduction problems of uncertain aetiology, including gynaecological problems such as conception failures, abortions, stillbirths and malformations of neonatal calves, which are suggestive of bvdv infection. despite this situation, little published information is available regarding bvd in the country. the present study is the first of its kind in the region. it was designed to investigate in utero exposure of foetuses to bvdv by means of detecting bvdv antigens in the tissues of malformed neonatal calves and pre-colostral antibodies to bvdv in the serum of apparently healthy calves. the presence of bvdv antigens in the tissues of malformed calves would indicate their exposure to the virus during the first trimester of gestation (kampa 2006), a time when their immune systems are incompetent and thus tolerant to the infection. the presence of pre-colostral serum antibodies to bvdv in neonates indicates foetal infection during the last two trimesters of gestation, by which time the immune system can produce specific antibodies in response to infection (graham 2001; moening & liess 1995). materials and methods (back to top) the farm studied was established at al-kharj oasis (150 km south of riyadh, saudi arabia) in 1977. it was developed according to international standards and is managed by qualified personnel. there are 15 000 holstein friesian milk cows on the farm, which have been imported as in-calf heifers. according to the farm authorities they were imported from bvdv-free herds and were tested prior to importation for the presence of bvdv. a few cows were bred locally following artificial insemination using imported semen, which, according to the farm administration, was also tested for the presence of bvdv. the cows are zero grazed and are completely isolated from other animals from which they can contract infection. they are fed with total mixed rations and milked four times a day at six-hourly intervals. the holstein friesian breed is adapted to temperate geographical zones. therefore, to harness their full genetic potential in the harsh and hot saudi arabian climate, special consideration was given to their housing on the farm. the cows are housed in a semi-closed system with some degree of free movement. cooling is maintained by large fans and cool water sprays that produce a fine mist. these cooling conditions greatly reduce the heat stress on the cows. each cow house on the farm can accommodate 239 milk cows. the dimensions are 160 m x 43 m x 6.5 m (length x width x height). each house comprises a shaded area for the day and a non-shaded area for the night and contains locking stanchions and drinkers. the feeders are in a line along the house. lights are switched on from 18:00 to 06:00. the floors are cleaned while the cows are being milked. the farm also has a veterinary clinic, isolation and quarantine units, a colostrum area and an incinerator. the bvd vaccination schedule used on the farm is such that heifer calves are vaccinated at the age of 56 days. maiden heifers are vaccinated at the age of 13 months and in-calf heifers and dry cows are vaccinated 21 days before calving. according to the farm administration only live bvdv vaccine is used on this farm. for the study of pre-colostral serum antibodies, individual blood samples were collected from 284 apparently healthy neonatal calves (133 bull calves; 151 heifer calves) before colostrum feeding, using vacutainers and following standard methods of serum separation (oie 2008). sera were collected over a period of four weeks. the sera were inactivated by heating at 56 °c for 30 min and then stored at –20 °c until use (2–3 weeks). an enzyme-linked immunosorbent assay (elisa) (bio–x diagnostics, jemelle, belgium) was used to detect antibodies in the pre-colostral sera using 96-well elisa plates. the plates provided by the manufacturers were presensitised by monoclonal antibodies to which the bvdv was reacted and were thus ready to receive the test sera. all assay procedures were as described by the manufacturer. a volume of 0.05 ml of each reactant was used per well. the incubation temperature was set at 37 °c, except for that of the substrate, which was at room temperature (24 °c – 26 °c). washing was carried out automatically by filling the wells and aspirating the washing buffer three times. briefly, the test procedure was as follows: • the test sera were diluted 1:100 in the dilution buffer and added in duplicate wells. positive and negative control sera were included in the test. the plates were then incubated for 1 h and washed. • a peroxidase-labelled antibovine igg monoclonal antibody was added to each well at the dilution recommended by the manufacturers. the plates were incubated for 1 h and washed. • the enzyme substrate (hydrogen peroxide) and chromogen tetramethylbenzidine were added to each well, followed by incubating the plates for 10 min in the dark. • the stop solution was added to each well and the reaction was read at 450 nm in an elisa plate reader and the optical density (od) readings were recorded. for interpretation of the results the mean od value of the negatives was subtracted from the mean of the positive ones. the signal read for each serum sample was divided by the signal of the positive serum and multiplied by 100 to express it as a percentage. the degree of positivity (from 1+ to 5+) for each test serum was determined using a quality control table provided with the kit. a total of 346 tissue samples were collected from 62 euthanized malformed neonatal calves for the detection of bvdv antigens. the samples included tissue from the brain, the spinal cord, the spleen, the liver, the lungs, mesenteric lymph nodes, the kidneys, the ovaries and the abomasums, as well as some cerebrospinal fluid. each sample was placed in a sterile container and put in an ice box for immediate transport to the laboratory. the samples were stored at –86 °c until tested with an antigen-capture elisa (usually within two weeks of sample collection). an antigen-capture elisa designed to detect bvdv antigens in tissues (idexx herschel, idexx laboratories, schiphol-rijk, the netherlands) was performed according to the manufacturer’s instructions. the procedure included preparing the tissues, performing the test, and collecting and interpreting the results. all reagents and plates were provided as part of the kit. reactant volumes, incubation times, the washing procedure and result collection were as described earlier. briefly, the test procedure was as follows: • ready-for-use microtitre elisa plates with immobilised monoclonal antibodies specific to bvdv were provided in the kit. • the tissue sample suspected to contain bvdv antigen was added to sensitised plates. following incubation and washing, the plates received the conjugate, which consisted of the specific antibody to bvdv conjugated to horseradish peroxidase. the plates were again incubated and washed. • the substrate solution was added. in the presence of the peroxidase enzyme the substrate is converted into a product which reacts with chromogen to generate a blue colour. interpretation of the results was performed according to the formula where odsample, odnc and od pc denote the optical density readings of the sample, the negative control and the positive control, respectively. samples for which s:p ≤ 0.20 were classified as negative, whilst those for which s:p ≤ 0.30 were classified as positive. ethical consideration all the ethical considerations regarding this manuscript were taken into account. results (back to top) table 1 shows the results of the elisa for antibodies in the pre-colostral sera. of the 284 serum samples tested, 76 (26.8%) contained antibodies against bvdv. positive reactions were seen in 27.8% of the samples from the bull calves and in 25.8% of the samples from the heifer calves. according to the manufacturer’s criteria, positive serum reactions can be graded from weak (+) to very strong (5+). a value of 3+ is regarded as indicative of a strong reaction. figure 1 shows the distribution of reaction strengths in pre-colostral sera. of the positive serum samples, 5.5% were weakly positive (+), 16.2% showed a slightly stronger reaction (2+), and 78.3% showed a strong reaction (43.2% scored 3+; 35.1% scored 4+). no samples showed a very strong reaction (5+), as defined by the manufacturer. bvdv antigen was detected in tissue samples of 49 (79%) malformed neonatal calves. table 2 summarises the corresponding elisa results. table 3 shows the total number of neonatal calves that were exposed to bvdv infection in utero (i.e. malformed calves that tested positive for bvdv antigen as well as calves displaying pre-colostral antibodies to bvdv). figure 1: distribution of reaction strengths obtained from positive pre-colostral sera. table 1: detection of pre-colostral antibodies against bovine viral diarrhoea virus in neonatal calves’ sera. table 2: positive antigen detections in tissues from malformed neonatal calves. table 3: summary of exposure of neonate calves to bovine viral diarrhoea virus infection in utero. discussion (back to top) results of the study showed that 26.8% of the calves examined had developed pre-colostral antibodies to bvdv and that both sexes were affected to almost the same extent. this indicates that these calves had been infected in utero during either the second or the last trimester of gestation. at that age the immune system of the foetus is capable of responding to the bvdv infection by producing antibodies. the results also indicated that 78.3% of the positive sera yielded strong reactions. compared to those of other published reports, the result of 26.8% positive reactions in pre-colostral serum suggests that infection in this particular herd is three to four times higher than elsewhere (see for example schefers et al. 2008 for us results). this is not surprising, as no strict control policy for bvd, other than vaccination, was implemented on this particular farm. vaccination alone seemed to have little beneficial effect and should therefore preferably be combined with other control measures, for example removal of pi animals. furthermore, the bvdv antigen was detected in all types of the tissue sampled, albeit at different rates. this indicates that the virus was pantropic in its distribution in the foetal tissues. this is not unexpected, as the malformed calves were immune tolerant at the age when they were exposed to the bvdv (during the first trimester of gestation). in addition, bvdv is known to show a high affinity towards rapidly dividing cells, which makes foetal tissues favoured sites for virus replication (cornish, van olphen & cavender 2005; duffell & harkness 1985). our overall results showed a high level of detection frequency (51.2%) of the bvdv antigen in tissues of the malformed calves. however, the incidence rate ranged from 8.3% (lungs) to 87.8% (kidneys). the highest incidences were seen in the kidneys (87.8%), livers (85%) and spleens (61.3%). the results may be attributed to the excretion of the virus and immune complexes via the kidneys and the liver. it is also the case that bvdv is often associated with macrophages and other immune-competent cells present in the spleen. moreover, the stationary macrophages, such as the mesangial cells in the kidney and the kupffer cells in the liver, can trap the bvdv. such trapping would be expected to add to the virus load in these organs. the observation of both malformed calves and calves presenting with pre-colostral antibodies to bvdv suggests that the dams were subjected to bvdv infectionthroughout their pregnancies. this indicates continuous circulation of the virus on the farm and probably the presence of pi animals (alkhaliyfa, abuelzein & gameel 2010). indeed, the dams themselves could have been pi animals (lindberg 2002). it is also possibile that the semen used for artificial insemination on this farm might have contained bvdv and it is recommended that this potential source be tested for the presence of the virus. published information suggests that the most important sources of bvdv infection within and between dairy cattle herds are pi animals (lindberg 2002). pi animals therefore constitute a continuous and major threat to dairy cattle. it is recommended that the identification of pi animals should be a pre-requisite to planning control measures against bvd in a particular geographical region, whether bvdv is endemic or not (duffell & harkness 1985; fredriksen, sandvik & loken 1999; ridpath, hessman & neill 2006). the overall results of the present investigation showed that 36.1% of the neonatal calves tested on this farm were infected by bvdv in utero. the malformed calves had to be euthanised. in view of this high incidence we recommend that an urgent assessment of the economic damage caused by these annual losses should be carried out. conclusion (back to top) we recommend that dairy farms in saudi arabia should adopt a policy of regular testing for the presence of animals persistently infected with bvdv and subsequentremoval of such animals. the low efficacy of the adopted vaccine regimen on this particular test farm warrants its re-evaluation. it is vital that farmers be made aware of the high financial costs associated with bvdv infection. acknowledgements (back to top) the authors gratefully acknowledge the kind assistance of staff members on the farm. the authors would also like to thank prof. p.s. mellor for his valuable comments. author contributions e.m.a. is the principal investigator who planned this work, supervised the research process (field and laboratory investigations) and wrote the manuscript.m.a.a. conducted the clinical work, collected the materials and was involved in the laboratory work. a.a.g. was involved in the identification of the different types of malformations in the neonate calves and supervised the sampling from the malformed calves. all the authors participated in brain storming discussions that involved the research work and the manuscript. references (back to top) alkhaliyfa, m.a., abuelzein, e.m.e. & gameel, a.a., 2010, ‘identification of cattle persistently infected with bvdv by ear-notch testing in saudi arabia’, veterinary record 167, 660–661. cornish, t.e., van olphen, l.a. & cavender, j.l., 2005, ‘comparison of ear notch immunohistochemistry, ear notch antigen-capture elisa, and buffy coat virus isolation for detection of calves persistently infected with bovine viral diarrhea virus’, journal of veterinary diagnostic investigation 17, 110–117. http://dx.doi.org/10.1177/104063870501700203, pmid:15825490 duffell, s.j. & harkness, j.w., 1985, ‘bovine virus diarrhoea-mucosal disease infection in cattle’, veterinary record 117, 240−245. http://dx.doi.org/10.1136/vr.117.10.240, pmid:4095876 fredriksen, b., sandvik, t. & loken, t., 1999, ‘level and duration of serum antibodies in cattle infected experimentally and naturally with bovine virus diarrhoea virus’, veterinaryrecord 144, 111−114. 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http://dx.doi.org/10.1177/104063870802000515, pmid:18776097 world organization for animal health, 2008, ‘bovine viral diarrhoea’, in manual of diagnostic tests & vaccines for terrestrial animals: mammals, birds & bees, 6th edn., vol.2, pp. 698–711, oie publications, paris. article information author: martyn h. jeggo1 affiliation: 1australian animal health laboratory, victoria, australia correspondence to: martyn jeggo postal address: australian animal health laboratory, csiro livestock industries, pmb 24, geelong vic 3220 how to cite this abstract: jeggo, m.h., 2012, ‘first international one health congress’, onderstepoort journal of veterinary research 79(2), art. #471, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.471 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. first international one health congress in this abstract... open access more than 650 people from around 60 countries attended the 1st international one health conference, held in melbourne from 14 to 16 february 2011. scientists, clinicians, government and community members from a range of disciplines came together to discuss the benefits of working together to promote a one health approach to human, animal and environmental health. one health embraces systems thinking and recognising the interdependence of people, animals and environment. the conference was hosted by the commonwealth scientific and industrial research organisation (csiro) and was supported by international agencies, the australian and canadian governments, and industry.the organising committee recognised from the outset, the need to provide a forum not just for scientific presentation, but for open discussion and dialogue around the policy and political issues, as well as the science that drives the one health agenda. the committee was also cognizant of the need to embrace a definition of one health that includes food security and food safety and included the social and economic pressures that shapes this area. the meeting was therefore organised under four themes with plenary sessions followed by breakout parallel sessions for each of these. the themes covered disease emergence, environmental drivers, trade, food security and food safety, and science policy and political action. the plenary session commenced with one or two keynote presentations by world leaders on the topic being covered, followed by panel discussions involving six to eight experts and involving all participants at the congress. each of the panel members spoke briefly on the topic covered by the keynote speaker and were asked to be as provocative as possible. the discussions that followed allowed debate and discussion on the keynote presentations and the panel members comments. this was followed by six to eight parallel breakout sessions involving in depth papers on the session’s topic. throughout the conference at various times, sponsored sessions dealt with particular areas of science or policy providing a further framework not only to learn current science but for debate and discussion. a full copy of all abstracts is available on the web at http://www.springerlink.com. in concluding the congress recognised the interdependence of, and seeks to improve human, animal and environmental health; recognised that communication, collaboration and trust between human and animal health practitioners is at the heart of the one health concept; agreed that a broad vision that includes other disciplines such as economics and social behaviour is essential to success. the congress stressed the need to promote the ‘do-able’ such as improving surveillance and response for emerging infectious diseases whilst developing the broader approach. it identified a need to emphasise community participation and development of community capacity, and especially, an open transparent dialogue with both a ‘ground up’ and ‘top down’ approach that would lead to an improved understanding of our ecosystems, including molecular ecobiology, are an essential part of one health. abstract introduction materials and methods statistical methods results discussion conclusion acknowledgements references about the author(s) rhulani makhuvele toxicology and ethnoveterinary medicine, agricultural research council-onderstepoort veterinary research, onderstepoort, south africa department of paraclinical sciences, faculty of veterinary science, university of pretoria, onderstepoort, south africa kenn foubert department of pharmaceutical sciences, university of antwerp, antwerp, belgium nina hermans department of pharmaceutical sciences, university of antwerp, antwerp, belgium luc pieters department of pharmaceutical sciences, university of antwerp, antwerp, belgium luc verschaeve department of risk and health impact assessment, sciensano, brussels, belgium department of biomedical sciences, university of antwerp, antwerp, belgium esam elgorashi toxicology and ethnoveterinary medicine, agricultural research council-onderstepoort veterinary research, onderstepoort, south africa department of paraclinical sciences, faculty of veterinary science, university of pretoria, onderstepoort, south africa citation makhuvele, r., foubert , k., hermans , n., pieters , l., verschaeve, l. & elgorashi, e., 2022, ‘protective effects of methanolic leaf extracts of monanthotaxis caffra against aflatoxin b1-induced hepatotoxicity in rats’, onderstepoort journal of veterinary research 89(1), a1968. https://doi.org/10.4102/ojvr.v89i1.1968 original research protective effects of methanolic leaf extracts of monanthotaxis caffra against aflatoxin b1-induced hepatotoxicity in rats rhulani makhuvele, kenn foubert, nina hermans, luc pieters, luc verschaeve, esam elgorashi received: 17 aug. 2021; accepted: 25 jan. 2022; published: 23 mar. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract aflatoxins are potent hepatotoxic and carcinogenic secondary metabolites produced by toxigenic fungi. the present study investigated the protective effect of methanolic leaf extracts of monanthotaxis caffra (mlemc) against aflatoxin b1-induced toxicity in male sprague-dawley rats. the rats were randomly divided into 6 groups of 8 animals each. five groups were administered orally for seven days with three different concentrations of mlemc (100 mg/kg, 200 mg/kg and 300 mg/kg), curcumin (10 mg/kg) or vehicle (25% propylene glycol). the following day, these groups were administered 1 mg/kg b.w. of aflatoxin b1 (afb1). the experiment was terminated three days after administration of afb1. group 6 represented untreated healthy control. serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatinine and liver histopathology were evaluated. methanolic leaf extracts of m. caffra decreased the levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and creatinine in the sera of rats as compared with the afb1 intoxicated group. co-administration of mlemc improved the histological characteristics of the hepatocytes in contrast to the afb1 treated group, which had mild to severe hepatocellular injuries including bile duct proliferation, bile duct hyperplasia, lymphoplasmacytic infiltrate and fibrosis. extracts of m. caffra were beneficial in mitigating the hepatotoxic effects of afb1 in rats by reducing the levels of liver enzymes and preventing hepatic injury. keywords: mycotoxins; aflatoxins; annonaceae; liver toxicity; liver enzymes; amelioration. introduction aflatoxins (afs) are group of difuranocoumarins toxic secondary metabolites produced by aspergillus flavus, aspergillus parasiticus and aspergillus nomius, which contaminate various foodstuff and feed. the most predominant major afs that contaminate food and feed commodities include afb1, afb2, afg1 and afg2 and their hydroxylated metabolites, namely afm1 (ráduly et al. 2020). aflatoxin b1 is the most potent hepatotoxin, mutagen, teratogen, immunosuppressive and carcinogen amongst other major groups of afs in nature (zarev et al. 2020). when ingested, it is bio-transformed in the liver primarily by cyp3a4 and cyp1a2 isoforms that are members of the cytochrome p450 (cyp) superfamily of drug metabolising enzymes and generate a highly reactive species, namely afb1-exo-epoxide and other metabolites (bedard & massey 2006; rushing & selim 2019). these reactive species bind to the macromolecules causing toxicity and mutations, which lead to lipid peroxidation, necrosis, cell damage, cell death, deoxyribonucleic acid (dna) lesions, carcinogenicity and other genetic diseases (bedard & massey 2006; zarev et al. 2020). aflatoxin b1 deleteriously affects global public health as it is the major contributor to the worldwide occurrence of hepatocellular carcinoma (hcc). the toxin also works synergistically with hepatitis b and c viruses to significantly increase the risk of hcc far above either factor individually (rushing & selim 2019). surgery and liver transplant offer limited treatment options as they are only useful in the treatment of early stages of hcc. other non-surgical options such as chemoand radio-therapies are only successful in patients with localised liver tumour (darvesh, aggarwal & bishayee 2012). therefore, research that focuses on the development of alternative preventative and therapeutic strategies may lead to valuable findings that may be effective in the control of hcc. although the principal toxic target of afb1 is the liver, several reports showed that exposure to afb1 has adverse renal effects as it increases the levels of creatinine and urea in animal experiments (el-mahalaway 2015; yilmaz et al. 2018). previous studies also revealed that oxidative stress resulting from afb1 consumption leads to myocardial cell membrane destruction manifested by a marked increase in lactate dehydrogenase (ldh) activity (mannaa et al. 2014). the biosynthesis and activation of afs can be attenuated using bioactive compounds present in herbal products. plant extracts have been studied for their antifungal growth inhibition and mycotoxin detoxification. several studies have been conducted on the protective effects of plant extracts and its bioactive compounds against afb1-induced hepatotoxicity (choi et al. 2010; soni et al. 1997; tang et al. 2007). curcumin and ellagic acid are examples of phytochemicals isolated from plants and they inhibited hepatocarcinogenicity in rat and chicken models (gowda et al. 2008; soni et al. 1997). these compounds are known for their potent free radical scavenging ability and exert their anticarcinogenic effect primarily through prevention of oxidative stress (darvesh et al. 2012). furthermore, phytochemicals such as lycopene and quercetin have been reported to inhibit biotransformation of afb1 toxicity by inducing detoxification enzymes (choi et al. 2010; tang et al. 2007). monanthotaxis caffra (annonaceae) is a shrub occurring in evergreen forests of eastern cape, kwazulu-natal and mpumalanga provinces of south africa (national research council 2008). this plant species has been used in traditional medicine against tumours, microbial and parasitic infections in veterinary and human health (mulholland et al. 2000; okhale et al. 2016). methanolic leaf extract of m. caffra has been also reported for its antigenotoxic properties against afb1-induced genotoxicity (makhuvele et al. 2018a). furthermore, crotepoxide, a known antitumour compound and other polyoxygenated cyclohexane derivative, 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene were isolated from m. caffra (makhuvele et al. 2018b; mulholland et al. 2000). the in vivo hepatoprotective effects of m. caffra against mycotoxin-induced toxicity has never been explored. therefore, this study aimed to investigate the protective effects of methanolic leaf extracts of m. caffra (mlemc) against afb1-induced hepatotoxicity on rats. materials and methods chemicals aflatoxin b1, dimethyl sulfoxide (dmso) and curcumin were purchased from sigma-aldrich (st. louis, united states). methanol and acetonitrile were bought from van waters & rogers inc. (radnor, usa) vwr and propylene glycol was from merck (darmstadt, germany). preparation of methanolic leaf extract of m. caffra leaves of m. caffra (annonaceae) were collected from lowveld national botanical gardens (south africa) in march 2015. the identities of the plants were confirmed by mrs. e. van wyk, university of pretoria, south africa. a voucher (number: pru 122761 for m. caffra) was deposited in the h.g.w.j. schweickerdt herbarium of the university of pretoria. the plant material was dried in an oven set at 45 °c and thereafter, ground to a fine powder. the powdered plant material was stored in airtight glass container in the dark at room temperature until use. the powdered leaf material of m. caffra (350 g) was added to 3500 ml of 80% methanol and extracted to exhaustion by maceration at room temperature. the plant extracts were filtered through whatman no. 1 filter paper and concentrated to dryness under reduced pressure using buchi rotary evaporator. in vivo evaluation of hepatoprotective effect of methanolic leaf extracts of m. caffra animals a total of 48 sprague-dawley male rats (7 weeks old), weighing between 150 g and 200 g, were obtained from south african vaccine producers (savp; johannesburg, south africa). the study was carried out using single gender as a way of reducing variation. during the experiment, the rats were kept in separate cages (2 per cage) under a controlled temperature of ±22 °c, and humidity at ±50% in a light and dark cycle of 12 h. the rats were fed with a conventional rodent diet and water, available ad libitum for the duration of the study. rats were provided with enrichment including wooden sticks for gnawing, tissues and egg containers. all enrichment items and cages, water bottles and bedding were sterilised before use. rats were acclimatised and closely monitored under laboratory conditions for five days prior to treatment. study design the sprague-dawley rats were randomly divided into six groups of eight animals each (n = 8): group a (healthy control, did not receive any treatment). group b (negative control, received 25% propylene glycol), group c (positive control, received curcumin dissolved in 25% propylene glycol (10 mg/kg body weight). the dose of curcumin was selected based upon previous studies where the dosage produced a marked hepatoprotective effects against aflatoxin b1 (poapolathep et al. 2015). group d, e and f rats were treated with 100 mg/kg, 200 mg/kg and 300 mg/kg body weight per day of mlemc, respectively. the doses of the plant extracts were in line with those reported in literature (sathya, kokilavani & ananta 2012). all treatments were administered once a day by oral gavage in the morning for 7 consecutive days. on day 8, all treated rats were administered 1 mg/kg b.w of afb1 (zarev et al. 2020), dissolved in reverse osmosis water by oral gavage, except the healthy control group. after three days, rats were sacrificed by using an isoflurane inhalation protocol for anaesthesia (roustan, perrin & courbiere 2012). biochemical analysis blood samples were collected from the lateral tail vein from each rat on day 0 and by cardiac puncture on day 10 following anaesthesia with isoflurane prior to sacrifice for determination of serum biochemistry. the blood samples were centrifuged at 1500 × g, at 4 °c for 15 min and the serum was collected, then evaluated for serum enzyme level. serum aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), ldh and creatinine were measured using cobas integra 400 kits from roche following manufacturer’s instructions. histopathological studies the liver was excised and then fixed in 10% buffered formalin for histological analysis. the organ was sliced and processed according to routine histology tissue processing in an automated tissue processor. after tissue processing, the sections were cut into 5 µm – 6 µm and the slides were prepared and stained in an automated haematoxylin and eosin tissue stainer before histology examination. the slides were examined with a bx 63 olympus electron microscope with olympus cellsens dimension version 1.12 software. statistical methods data are presented as mean ± standard deviation. differences between groups were determined using one-way analysis of variance (anova). the standardised residuals were tested for deviations from normality using dunnett’s, t-test and shapiro–wilk’s test. least significant difference (lsd) test was used to determine statistical significance between the means of treated groups and the controls. the data were considered significant at p < 0.05. results effect of treatment on animal general conditions and body weight neither mortality nor clinical signs of illness and abnormalities were reported in all rats administered with mlemc, negative (afb1) and curcumin positive control treatments in this study. the administration of mlemc did not affect the body weight, feed consumption and behavioural patterns of the rats at all tested concentration during 10 days of experiment. based on these results, mlemc can be considered safe for consumption by animals. effect of treatment on serum biochemistry effects of different concentrations of mlemc on serum levels in rats induced with afb1 are presented in figure 1. the levels of alt and ast significantly increased in afb1-treated rats (group b) in comparison to the healthy and curcumin positive control groups (p < 0.05). the levels of these enzymes markedly decreased in rats administered with afb1+mlemc at different concentrations and the positive control curcumin (p < 0.05). furthermore, there were no significant differences observed in alt and ast levels between the healthy and curcumin control group and the afb1+mlemc treated groups. no significant differences were observed in the levels of alp and creatinine between the healthy, afb1 intoxicating group and curcumin control group and those treated with different concentrations of mlemc (figure 1). the ldh levels of afb1-treated rats were significantly higher when compared to the ldh levels of healthy and curcumin treated group and the afb1+mlemc treated groups, which were not statistically different from each other (figure 2). moreover, a significant decrease in the ldh level in rats treated with curcumin was observed (p < 0.05). figure 1: effect of methanolic leaf extracts of m. caffra on the serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase activities and lactate dehydrogenase following aflatoxin b1-induced hepatoxicity in rats. figure 2: effect of methanolic leaf extracts of m. caffra on the levels of creatinine following aflatoxin b1-induced hepatotoxicity in rats. effect of treatment on liver histopathology the effects of administration of mlemc on liver histopathology are presented in figure 3. most rats in afb1-intoxicated group (negative control) showed mild to severe hepatocellular degeneration with bile duct proliferation, hydropic degeneration, mild to moderate portal fibrosis in some of the larger tracts and portal lymphoplasmacytic infiltrates with the occasional neutrophil was observed in the periportal areas further extending into the periportal zone and disrupting the limiting plates, thus causing piecemeal necrosis. scattered single cell hepatocellular necrosis was observed and the remaining hepatocytes had mildly granular and eosinophilic cytoplasm. the presence of phagocytic macrophages was also reported. these histological alterations were also observed in rats co-administered with afb1+curcumin and afb1+mlemc but the injuries were substantially reduced to slight signs of sublethal non-specific hepatocellular injuries. slight granular eosinophilic cytoplasms were also observed in all of the hepatocytes. inflammation or necrosis were not observed and the portal tracts were within normal limits. figure 3: effect of methanolic leaf extracts of m. caffra on histology of the hepatocyte. the liver section was stained with h&e (40 ×). (a) healthy control group; (b) negative group; showed mild to severe hepatocellular injury, the black arrow indicates bile duct proliferation, red arrow shows lymphoplasmacytic infiltrate, white arrow indicates fibrosis and orange arrow indicates normal bile duct; (c) afb1+curcumin group showing normal liver architect (d) afb1+ mlemc 100 mg/kg; (e) afb1+mlemc 200 mg/kg and (f) afb1+mlemc 300 mg/kg. discussion human and animals are exposed to different xenobiotic substance such as aflatoxins that cause deleterious effects on the biomolecules and cellular membrane by inducing oxidative damage (el-agamy 2010). the liver is an essential organ of human and animals, which is involved in metabolism of xenobiotics (colakoglu & donmez 2012). aflatoxin b1 is a well-known hepatotoxic agent. it induces cellular and tissue damage to the hepatocytes, thus resulting in aflatoxicosis, hcc, necrosis, cirrhosis, etc. (shan 2020; zarev et al. 2020). aflatoxin b1 mediates these various deleterious effects through the induction of oxidative stress (el-agamy 2010). methanolic leaf extracts of m. caffra ameliorated afb1-induced genotoxicity in in vitro antigenotoxic assays (makhuvele et al. 2018b). due to this antigenotoxic effect of mlemc, we hypothesised that mlemc will ameliorate afb1-induced hepatotoxicity in rats. therefore, we further investigated the protective effects of mlemc against afb1-induced hepatotoxicity in rats. in our study, afb1 intoxication significantly increased the serum levels of the liver enzymes ast and alt in comparison with untreated control group, which implies that afb1 caused cellular damage to the liver because of the release of these enzymes, which are normally located in the cytoplasm of the liver cells, in the blood stream. the higher standard deviation observed in the afb1 intoxicated group were because of the two rats which did not respond to afb1, their serum levels were not afffected by administration of afb1. in general, ast and alt are released into the blood stream when there is a liver damage or loss of cell membrane intergrity. these serum enzymes are considered as common biomarkers for the diagnosis of liver damage (amacher 1998). alanine aminotransferase and ast were normal in healthy control group whilst co-adminsteration of afb1+curcumin and afb1+mlemc (100 mg/kg, 200 mg/kg and 300 mg/kg) normalised the serum enzymes alt and ast. no significant difference was observed between the mlemc and curcumin-treated groups in comparison with the healthy control group. this is a clear indication that mlemc and curcumin ameliorated the toxic effect of the afb1, by inhibiting afb1-induced liver cell injury damage and consequently lowering the ast and alt levels in the blood. curcumin was used as a positive control in this study as it is renowned for its protective effects against afb1-induced hepatocarcinogenesis and hepatotoxicity in rodents, broilers and ducklings by lowering the levels of serum markers and lipid peroxidation (chuang et al. 2000; gowda et al. 2008; mathuria & verma 2008). methanolic leaf extracts of m. caffra demonstrated protective effect against afb1-induced toxicity at all tested concentrations. however, the highest concentration of mlemc showed high reduction comparably with the lower concentration, although no significant difference was observed. an increase in ldh levels in rats intoxicated with afb1 only was observed as compared with untreated control. increased ldh activity is one of the major events involved in liver damage after afb1 administration, as it is only released in the blood stream when the liver is injured (choi et al. 2010; nayak & sashidhar 2010). however, this increase in ldh activity was not observed in the healthy control and in rats treated with different concentrations of afb1+mlemc. a significant decrease in the ldh level in rats treated with curcumin was observed, which is in line with literature reports (nayak & sashidhar 2010). creatinine, a major marker of kidney function, is the final metabolite of creatine conversion. increased creatinine as an index of impaired kidney function because of chronic exposure to afb1 was reported in chickens and rats (el-mahalaway 2015; valchev et al. 2014). in this study, significant changes were not observed in the serum level of creatinine in rats treated with a single dose of afb1, which is in line with previous reports where nephrotoxicity was observed only in animals having chronic exposure to afb1 (el-mahalaway 2015; valchev et al. 2014). the result of biochemical analysis of the protective effects of mlemc was also confirmed by the histopathological investigations. aflatoxicosis is characterised by hydropic changes, vacuolar degeneration, bile duct proliferation and lymphoplasmacytic infiltration in exposed hepatocytes. however, the histopathological effects of afb1 are directly proportional to the concentration and exposure time to afb1 (do & choi 2007; yaman, yener & celik 2016). aflatoxin b1 intoxication caused mild to severe hepatocellular injury accompanied by bile duct proliferation, hydropic changes and lymphoplasmacytic infiltrate in the hepatocytes of exposed rats. this histological effect of afb1 on rat hepatocytes has been reported in literature (yaman et al. 2016). minimal degree of the above-mentioned histological characteristics in rats exposed to co-administered afb1+curcumin and afb1+mlemc were observed in this study, thus implying that curcumin and mlemc had recuperative effects against afb1-induced acute toxicity. curcumin has been reported in literature to possess ameliorative effects on the histology of the liver and other organs (el-agamy 2010; gowda et al. 2008). members of the genus monanthotaxis are known to contain essential oils (parmena, mgina & joseph 2012), alkaloids, flavonoids and cyclohexane epoxides (mulholland et al. 2000). chemical analysis of the 80% extract of m. caffra revealed it is rich in cyclohexane epoxide derivatives including crotepoxide and 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene and a mixture of two related benzoyloxy cyclohexidiene derivatives (makhuvele et al. 2018b). bioassay-guided fractionation of the extract using vitotox genotoxicity assay yielded the two antimutagenic compounds crotepoxide and 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene. in addition to their antimutagenic effects against aflatoxin b1-induced mutagenicity in ames and vitotox assays, the antitumour or anticarcinogenic properties of these polyoxygenated cyclohexane derivatives have been reported in literature. crotepoxide inhibited the expression of tumour necrosis factor (tnf) regulated gene products involved in anti-apoptosis such as bcl-2, bcl-xl, cyclin d1, cox-2, bax, bid, c-myc, mmp-9 and vefg, etc. furthermore, crotepoxide also inhibited the tumours by preventing the activation of genes that are involved in tumorigenesis at gene levels (alonso-amelot 2016). the compound also possessed antitumour properties against various carcinoma in rats and mice (parmena et al. 2012; shing & tam 1996; starks et al. 2012). the protective effects of these plant extracts against afb1 hepatotoxicity may be due to the presence of the above-mentioned compounds, which are found in annonaceae plant species. conclusion the present study showed that pre-treatment of rats with mlemc protected hepatocytes from afb1-induced hepatotoxicity as evidenced by the normalisation of the serum enzyme levels of ast, alt and ldh and the reduction of hepatocellular lesions. this effect was comparable to that produced by curcumin a known hepatoprotective agent against afb1-induced toxicity. this hepatoprotective effects results from the synergistic effects of the compounds present in the mlemc. our results suggest that mlemc can be considered as potential natural agent for prevention of afb1-induced hepatotoxicity. further studies on long time treatment of mlemc against afb1-induced hepatotoxicity in rats are recommended. acknowledgements the authors would like to thank the lowveld national botanical garden for permission to collect plant material for the study. they also thank mrs. e. van wyk for assisting with plant identification. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions e.e., l.p., l.v. and n.h. designed the study. r.m., k.f. and e.e. performed the experiments and analysed data. r.m. drafted the manuscript. e.e., n.h., l.p. and l.v. revised the manuscript. ethical considerations all animals received humane care in accordance with the guidelines of the university of pretoria animal ethical committee. the protocol (v073-15) was approved by the university of pretoria animal ethics committee. funding information the study was supported by the national research foundation (grant number cprr 87746 and nrf/fwo 87964), fonds wetenschappelijk onderzoek (fwo) grant number g001014n, flemish interuniversity council (vlir) under grant, zein2014z184, and agricultural research council. data availability the raw data that support the findings of this study are available on request from the corresponding author, e.e. disclaimer the views and opinions expressed in this article are those of the authors and do not reflect the official position or policy of the affiliation. references alonso-amelot, m.e., 2016, ‘multitargeted bioactive materials of plants in the curcuma genus and related compounds: recent advances’, in f.r. atta-ur-rahman (ed.), studies in natural products chemistry, vol. 47, pp. 111–200, elsevier, amsterdam. amacher, d.e., 1998, ‘serum transaminase elevations as indicators of hepatic injury following the administration of drugs’, regulatory toxicology and pharmacology 27(2), 119–130. https://doi.org/10.1006/rtph.1998.1201 bedard, l.l. & massey, t.e., 2006, ‘aflatoxin b1-induced dna damage and its repair’, cancer letters 241(2), 174–183. 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t., theunis, m., elgorashi, e., apers, s., ionkova, i. et al., 2020, ‘in vitro antigenotoxicity, in silico adme prediction and protective effects against aflatoxin b1 induced hepatotoxicity in rats of an erythrina latissima stem bark extract’, food & chemical toxicology 135, 110768. https://doi.org/10.1016/j.fct.2019.110768 article information authors: johan c.a. steyl1 leon prozesky1 wilhelm h. stoltsz2 john a. lawrence1 affiliations: 1department of paraclinical sciences, university of pretoria, south africa2department of veterinary tropical diseases, university of pretoria, south africa correspondence to: johan steyl postal address: private bag x04, onderstepoort 0110, south africa dates: received: 02 aug. 2011 accepted: 13 dec. 2011 published: 20 apr. 2012 how to cite this article: steyl, j.c.a., prozesky, l., stoltsz, w.h. & lawrence, j.a., 2012, ‘theileriosis (cytauxzoonosis) in roan antelope (hippotragus equinus): field exposure to infection and identification of potential vectors’, onderstepoort journal of veterinary research 79(1), art. #367, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.367 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. theileriosis (cytauxzoonosis) in roan antelope (hippotragus equinus): field exposure to infection and identification of potential vectors in this original research... open access • abstract • introduction • materials and methods    • experimental design    • hand-rearing of disease-free roan antelope calves    • selection of trial animals and preparation for field tick exposure    • monitoring of theileria infection    • treatment of clinical theileriosis    • tick feeding on theileria sp. (sable) carrier animals    • tick salivary glands dissection, staining and polymerase chain reaction-testing    • field ticks • results    • monitoring of theileria infection       • tick infestation       • rectal temperature       • habitus       • peripheral lymph node palpation       • lymph node aspiration       • peripheral blood smears       • mucous membrane examination       • blood samples for polymerase chain reaction detection of theileria sp. (sable)    • treatment of clinical theileriosis    • tick feeding on theileria sp. (sable) carrier animals    • salivary gland investigation of prefed ticks    • field ticks • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ four hand-reared, naïve roan antelope, 4 months of age, were exposed to naturally infected pasture on a game farm in mpumalanga province, south africa, where roan are known to die from theileriosis. various clinical parameters were recorded during this period. the predominant ticks parasitising these animals at the time (january to february), were rhipicephalus appendiculatus and rhipicephalus evertsi evertsi adults. after a period of 5 weeks the animals developed signs of clinical theileriosis and were treated with buparvaquone to prevent mortality. primary hyperplasia of the local draining lymph nodes (lnn. anorectales) near the feeding site of adult r. evertsi evertsi indicated possible transmission of theileria sp. (sable) by this tick species. after recovery from theileriosis, these animals were confirmed carriers of theileria sp. (sable) by pcr (polymerase chain reaction) and dna probe analysis. laboratory-bred larvae and nymphs of r. evertsi evertsi and r. appendiculatus respectively, were fed on the ears of these roan antelope. salivary glands from moulted and prefed adult ticks of each species were dissected and stained for theileria spp., and the pcr and dna probe applied to a representative batch of dissected glands. r. appendiculatus adults collected from grass in infected camps were also dissected after prefeeding them on rabbits. salivary glands of both tick species showed infected acini on staining and were also positive for theileria sp. (sable) only, on multiprotozoal pcr-screening analysis. there was no statistical significant difference between the infection rate and the intensity of infection between the two tick species. r. appendiculatus ticks collected from grass were also pcr-positive for theileria sp. (sable). introduction top ↑ historically, a theileria sp. (named theileria hippotragi at the time) have been known to occur in roan antelope, hippotragus equinus, in west africa since 1912 (todd & wolbach 1912) and in south africa since 1974 (wilson et al. 1974). a theileria sp. also infects sable antelope (hippotragus niger) in southern africa in regions where theileriosis occurs in roan (neitz 1957; wilson et al. 1974). gene sequencing and comparison of isolates from recent cases in western roan (hippotragus equinus koba) in malelane (a town in the eastern parts of mpumalanga province in south africa) confirmed, as had been suspected previously, that the theileria sp. involved in southern africa is the same in roan and sable (nijhof et al. 2005). it was originally isolated in lymphoblast cell culture from a sable that died of theileriosis in south africa and referred to as theileria sp. (sable) (stoltsz & dunsterville 1992). theileria sp. (sable) is certainly responsible for roan and sable mortalities in mpumalanga and elsewhere in south africa. this was supported by postmortal diagnosis of theileriosis in these species over a wide area of the northern and eastern parts of south africa. identification of the theileria sp. involved, and confirmation of theileria sp. (sable) as the pathogen, was performed by using a recently developed pcr (polymerase chain reaction) and dna probe on reverse line blot (rlb) assay (nijhof et al. 2005) for diagnostic purposes. currently, no serological test exists that can be utilised in screening for theileria sp. (sable) exposure or status. opportunistic, randomly collected blood samples from various other species in the malelane area tested positive for theileria sp. (sable), (personal experience). these included nyala (tragelaphus angasii), blue wildebeest (connochaetes taurinus), reedbuck (redunca arundinum), blesbok (damaliscus pygarus phillipsi) and bushbuck (tragelaphus scriptus). no mortality as a consequence of theileriosis has been described in these species.rhipicephalus appendiculatus (brown ear tick) is a well-known vector of theileria sp. in southern africa (norval, perry & young 1992) and is also one of the more dominant species and widely distributed throughout the northern and eastern parts of south africa. these factors have led to the suspicion that r. appendiculatus should be considered a strong contender as a vector for theileriosis in roan antelope. early field observations, however, during outbreaks of the disease in roan, also supported the possibility of rhipicephalus evertsi evertsi as a vector for theileria sp. (sable). the tick vector(s) for theileriosis in roan antelope has not been determined and therefore this study set out to establish the role of r. appendiculatus and r. evertsi evertsi as vectors for theileria sp. (sable). materials and methods top ↑ experimental design for this investigation, a farm in the summer rainfall region of south africa known to suffer regularly from roan mortalities because of theileriosis, was selected in the mpumalanga province of south africa. diagnosis of mortality as a consequence of theileriosis was confirmed by the section of pathology, faculty of veterinary science, university of pretoria. the farmer, a breeder of endangered antelope species, was keeping the roan-breeding stock grouped in camps ranging in size from 20 ha to 30 ha.hand-reared roan calves known to be pcr-negative for theileria sp. (sable) were exposed to ticks in a camp with a recent history of mortality as a result of theileriosis in roan antelope. constant monitoring of various clinical parameters indicative of theileria sp. infection was performed on these animals. after recovery from clinical theileriosis, these calves were confirmed carriers of theileria sp. (sable). laboratory-bred immature stages r. appendiculatus and r. evertsi evertsi originating from cattle in the area were allowed to feed on these calves. after they moulted to adults, salivary gland dissections, staining, and pcr-screening for theileria spp. and other blood parasites were performed. hand-rearing of disease-free roan antelope calves roan calves born during the 2002 calving season were hand-reared under tick-free conditions. calves were collected from the pasture 24 hours after birth to ensure passive colostral immunity transfer and to prevent theileria sp. infections from natural tick challenge (shaw 2002). on arrival at the hand-rearing facility all calves were thoroughly dipped with a contact pyrethroid dip (bayticol®) and clearly identified by using plastic ear tags with a sequential numbering system. table 1: list of haemoparasites screened for by polymerase chain reaction, reverse line blot method. there was no record of disease or mortality as a result of theileriosis during the period of hand-rearing of the roan antelope calves (44 calves in total). a few animals died from dietary and/or viral diarrhoea. the latter was mainly the result of rotavirus infection as diagnosed by lancet laboratories during the first 6 weeks of age. thereafter, there was no disease occurrence of any kind up to weaning at 7 months of age. selection of trial animals and preparation for field tick exposure four male roan calves (numbers 210, 218, 222 and 242), 4 months of age, were randomly selected. the animals were physically restrained and blood was collected in ethylenediaminetetraacetic acid (edta), stored at -20 °c, and submitted for pcr-testing as developed by nijhof et al. (2005). a wide range of dna-probes for various known theileria, babesia, anaplasma and ehrlichia species (table 1), including controls and theileria sp. (sable), was applied to the submitted samples (nijhof et al. 2005). the blood samples collected from the four selected roan bull calves were all negative on pcr (rlb)-method, that is, the reverse line blot method) for all the haemoparasites, including theileria sp. (sable).on receipt of the pcr results, the four roan males were chemically immobilised by using a drug cocktail consisting of 20 µg/kg – 35 µg/kg etorphine hcl and 0.3 mg/kg azaparone. they were translocated whilst under anaesthesia over a short distance to graze pastures in a camp on the farm that was known to be infected with theileriosis. mortality in roan as a result of theileriosis had occurred in this camp no longer than 6 months prior to this experiment, and it had since been accommodating a roan herd. this herd was transferred to an adjacent camp 2 weeks prior to the experiment. the four males were purposely left undipped in an attempt to expose them to ticks as soon as possible after arrival. this field trial was conducted during january and february 2003, coinciding with the tick activity season and tick numbers were close to a peak. monitoring of theileria infection the animals were still being bottle-fed and as a result it proved very easy to habituate them to handling, thus facilitating a wide spectrum of possible monitoring parameters. several parameters were monitored over a period of 6 weeks. the time to noticeable tick infestation, species present, attachment sites and subjective assessment of tick load were recorded. rectal temperature was measured twice daily in the morning and afternoon at feeding times with a calibrated digital thermometer. a rectal temperature higher than 40 °c, especially if the ambient temperature was below 28 °c, was considered a fever reaction. subjective scoring values of 1–4 were applied to assess feeding behaviour, with 4 indicating good general health and behaviour progressively deteriorating to 1, which implied clinical illness. the peripheral lymph nodes were palpated to assess swelling. scoring of lymph node size (0–4) with 4 being visibly enlarged without palpation, and 0 normal on palpation, was given. fine-needle aspiration, using a sterile 1½” 20g hypodermic needle, was simultaneously performed on the superficial cervical and other lymph nodes if they were visibly enlarged at times of blood collection. thin smears were prepared as described for peripheral blood smear staining and examination. peripheral blood smears were obtained every second day by pricking of the tail tip with a ¾” 21g needle, fixed and stained by using the diff quik® staining method. the sample was immediately examined with a standard light microscope for lymphoblastic transformation and theilerial schizont and/or piroplasm presence. blood smears were examined for other vector-borne haemoparasitic infections such as anaplasma spp. and babesia spp. blood was also collected in edta every 7 days during chemical immobilisation and kept at -20 °c freezer for later pcr-testing. clinical observation included examination of the ocular conjunctival mucosae and the colour was subjectively assessed at regular intervals. treatment of clinical theileriosis a clinical case of theileriosis was defined by using the following criteria: pyrexia above 40 °c shortly followed by, preceded or accompanied by, theilerial schizonts in circulation detectable by blood smear examination. sick animals received anti-theilerial therapy by using buparvaquone (butalex®) at 2.5 mg/kg intramuscularly once. if the fever did not abate 48 hours later, a second injection was administered. a staggered treatment regimen was applied to the different animals in an attempt to determine the efficacy of anti-theilerial therapy in relation to the first day of fever. the following regimen was applied: animal 218 was treated on the first day of pyrexia. animal 210 was treated one day, animal 222 two days, and animal 242 three days, post-pyrexia. tick feeding on theileria sp. (sable) carrier animals clean ticks for theileria pick-up experiments were obtained from engorged females of r. evertsi evertsi and r. appendiculatus, originating from cattle in the malelane area and transferred to the laboratory for egg laying by using standard operating procedures (heyne, elliott & bezuidenhout 1987). larvae of r. appendiculatus (3-host tick) were fed on rabbit ears and backs after hatching and left to engorge. these engorged larvae were collected and stored in batches of 250 in ventilated plastic vials to moult and mature to nymphae under controlled laboratory conditions. eggs from r. evertsi evertsi (2-host tick) were weighed and divided into batches that contained an estimated 500 eggs each. these eggs were left to hatch and mature before the onset of the tick feeding trial.upon pcr confirmation of carrier status for theileria sp. (sable), the four animals were relocated to tick-free boma conditions. they were prepared for tick feeding by applying linen ear-bags to their ears (heyne et al. 1987). two modifications were made to the described method. a head bandage was applied with adhesive tape (elastoplast®) and temporary nylon skin sutures were strategically placed to prevent animals from shaking loose the ear-bags. the animals were chemically immobilised to fit the ear-bags safely and to facilitate the placement of ticks. the four animals were divided into pairs. one pair received r. evertsi evertsi larvae and the other received nymphae of r. appendiculatus, which were applied randomly in batches of 500 per ear per animal. after 6 days, the animals infested with the r. appendiculatus nymphae were immobilised and the bags with engorged nymphae removed. it was necessary to scrape off engorged, but still attached, nymphae by using the shaft of a scalpel handle. the ear-bags of the animals containing r. evertsi evertsi larvae were removed after 14 days and engorged nymphae collected. tick salivary glands dissection, staining and polymerase chain reaction-testing the engorged nymphae of both tick species were kept under standard laboratory conditions until moulted to adults (heyne et al. 1987). the ensuing adult ticks were then fed on laboratory rabbits for a period of 4 days to allow salivary gland maturation to occur (morzaria et al. 1999; world organisation for animal health 2008). the rabbits were euthanised with sodium pentobarbitone (eutha-naze®) and ticks were removed immediately with curved forceps, taking care not to put pressure on the anterior region of the attached ticks. these ticks were placed in a tick-proof ventilated container and, within 4 hours, individually embedded with the scutum facing up in a petri dish containing paraffin wax. ticks, 100 of r. evertsi evertsi and 150 of r. appendiculatus, of equal numbers of male and female ticks, were then dissected in phosphate buffered saline (pbs) by using a standard stereomicroscope. salivary glands were removed and transferred to a glass microscope slide to dry. the slides were then processed by staining with methyl-green and pyronin (walker et al. 1979) and examined for theileria spp. infected salivary acini with a standard light microscope.a representative sample of about 50 ticks for each species was dissected, the salivary glands removed and suspended in 2 ml vials containing an isotonic saline solution for pcr detection of theileria sp. (sable) and possible other protozoal organisms. a screening profile of a wide range of vector-borne haemoparasites including theileria spp., anaplasma spp., ehrlichia spp., babesia spp., and others (table 1) was applied to these samples using the rlb method. field ticks approximately 160 adult r. appendiculatus ticks collected by hand from grass stalks during the february from various roan-grazing camps on the farm, were allowed to feed on rabbits for 4 days and were included as a separate sample. these camps experienced mortalities as a result of theileriosis about 6 months earlier, 2 months into the roan-calving season. a total of 100 ticks were dissected and stained as above. additional salivary glands from 30 ticks were submitted for pcr. results top ↑ monitoring of theileria infection tick infestation larval infestation occurred only after about 2 days and adult infestation 5 days after introduction of the roan into the camp. this was marked by increased scratching activity, especially of the ears. the adult stage of r. evertsi evertsi feeding in perianal and inguinal regions was first noted. their numbers never increased to more than 20 per animal. rhipicephalus evertsi evertsi immatures (larvae and nymphae) were present in low numbers in the outer ear canal. secondly, r. appendiculatus adults started feeding around the same time but occupied other regions involving the head, neck and outer surface of the ear pinna. their numbers were higher than r. evertsi evertsi, ranging from approximately 40 to 80 at a time per animal. immatures were also present in low numbers on the inner peripheral area of the ear pinna.occasionally other tick species could be found, either singly or in very low numbers of less than five. these included rhipicephalus (boophilus) species around the neck and shoulder, and amblyomma hebraeum in the inguinal and perianal region. figure 1: marked (a) anorectal and (b) superficial cervical lymphadenomegaly. rectal temperature rectal temperatures recorded in the afternoons were found on average to be higher than morning temperatures and as such were not considered. morning rectal temperatures were indicative of a true pyrexia (figures 2–5). the earliest pyrexic reaction (higher or equal to 40 °c) was observed in animal 218 at 34 days post-release into the infected camp. the days to first pyrexia for the other animals were as follows: animal 210 on day 36, animal 222 on day 37, and animal 242 on day 39. the fever reactions in the animals had specific temporal relationships with blood smear and lymph node size changes (figures 2–5). rectal temperatures remained high for a day or two after anti-theilerial therapy. figure 2: animal 218: blood smear findings, lymph node findings and day of treatment in relation to rectal temperature over time. figure 3: animal 210: blood smear findings, lymph node findings and day of treatment in relation to rectal temperature over time. figure 4: animal 222: blood smear findings, lymph node findings and day of treatment in relation to rectal temperature over time. figure 5: animal 242: blood smear findings, lymph node findings and day of treatment in relation to rectal temperature over time. habitus during the first 2 weeks of exposure the animals showed slight tick worry but soon became habituated. all animals were feeding well and continuously scored 4 until the day of pyrexia. although still drinking milk, enthusiasm decreased and all animals lost their appetite within 24 hours after the first pyrexia. all scored around 1 to 2 at this stage. the duration of poor habitus was longer for animals 222 and 242 and continued whilst their rectal temperatures remained high. within 24 h after the rectal temperatures dropped to within the normal range, habitus steadily improved over 3 days. grazing activity was poor for all animals during the first 10 days after treatment for theileriosis. approximately 2 weeks after treatment for theileriosis, all animals showed a marked loss in body condition, which relates to poor habitus during recovery from the infection. peripheral lymph node palpation the superficial cervical lymph node (sln) was the easiest to palpate and score. a gradual increase in size of all four animals’ sln could be detected, scoring around 2 from 2 weeks after field introduction. five days prior to the first animal (218) showing pyrexia, the anorectal lymph nodes (aln), bilateral to the tail base, were visibly enlarged (score 4), (figure 1a). at the time of first pyrexia, a marked general enlargement of peripheral lymph nodes could be detected, scoring between 3 and 4 (figure 1b). this temporal pattern was the same for all four animals. within 7 days after treatment for theileriosis, the lymph node size decreased considerably and reverted to a score of 2, with the anorectal nodes becoming visibly and palpably undetectable 10 days after treatment. lymph node aspiration at introduction of the roan to tick-infested veld, lymph node aspirates showed predominantly small lymphocytes, with few blast-transformed lymphocytes and an occasional macrophage. at first enlargement of the sln. (score 2) there was an increase in the blast-transformed lymphocyte complement and also occasional eosinophils. this finding continued until the first anorectal lymph node impression smear was performed in reaction to marked enlargement (score 4 on animal 218, 5 days prior to pyrexia). at this stage, no equivalent enlargement could be detected in the sln’s or in any other lympho-centres. initially, on cytology of aln aspiration, blast-transformed lymphocytes predominated, some of which were atypical and appeared neoplastic. the cytological features in these cells were large, round to ovoid, often indented to bean-shaped nuclei, containing one or two large nucleoli and increased amounts of amorphous basophilic cytoplasm. regular mitotic figures also featured. it sometimes proved difficult to differentiate between these ’neoplastic‘ lymphoblasts and immature monocytes. no typical theilerial schizonts (koch’s bodies) could be detected at this stage on the aln impression smears. at the time of pyrexia, all peripheral lymph nodes scored 4 and impression smears showed marked lymphocytic blast transformation, in addition to cytoplasmic protozoal inclusion bodies characteristic of theilerial schizonts. within 1 week of treatment for theileriosis, the impression smears were characterised by decreased lymphoblast numbers, large numbers of pyknotic and karyorrhexic lymphocytes and nuclear debris in the background. theilerial schizonts were difficult to find from 72 h post-buparvaquone therapy and when present, showed marked pyknosis.similar lymph node changes and cytological findings could be demonstrated in the other animals at the time of their respective reactions. peripheral blood smears in comparison to blood smears performed prior to field exposure, peripheral smears showed increased numbers of eosinophils from about 1 week into the trial that coincided with slight lymph node enlargement (score 2). from 3–5 days prior to pyrexia, a progressive atypical lymphoblastosis could be detected. this change continued, with the first cytoplasmic theilerial schizonts demonstrable in lymphoblasts 24 hours prior to the first fever reaction. the first intra-erythrocytic theilerial piroplasms could be seen from the day of fever, with numbers increasing as the disease progressed. as a result of antitheilerial therapy at the time of pyrexia, the numbers of theilerial schizonts and piroplasms decreased over the following 4–6 days until only the occasional piroplasm could be detected. examination for other microscopically detectable haemoparasites was negative. mucous membrane examination the mucous membrane colour was pink at the start of the trial and remained so until around 5 days after the first fever reaction. the mucous membrane colour at this stage varied from pale pink at first, progressively turning pale yellow around 2 days later. animal 242 showed more intensely pale yellow mucous membranes in comparison to the others. the normal pink mucous membrane colour returned within 2 weeks after treatment. blood samples for polymerase chain reaction detection of theileria sp. (sable) polymerase chain reaction results showed that all four animals were negative prior to field exposure. they became positive for theileria sp. (sable), coinciding with the first fever reaction and remained as such 6 weeks later. treatment of clinical theileriosis animals responded well to treatment with buparvaquone with clinical parameters improving and returning to normal within the expected time range for each (see above). animals 222 and 242, which received delayed treatments, 2 and 3 days respectively after the first pyrexia, required a second injection of buparvaquone because their rectal temperatures remained high for longer than 48 hours post-treatment. the other two animals showed a significant drop in rectal temperature within 48 hours, and did not require a second injection of buparvaquone. tick feeding on theileria sp. (sable) carrier animals tick yields from individual animals varied considerably depending on the species. from a total of 2000 r. evertsi evertsi larvae applied to ear-bags in two animals, only about 380 viable engorged nymphae could be recovered. in comparison, the number of r. appendiculatus nymphs, which dropped engorged, was about 590 in total (figures 6a and 6b). figure 6: (a) calico cloth ear-bags protecting rhipicephalus appendiculatus nymphae feeding on a roan antelope calf and (b) engorged rhipicephalus appendiculatus nymphae ready to be collected. salivary gland investigation of prefed ticks feeding ticks on rabbits was very successful, with more than 90% of placed ticks attaching after 12 hours, which resulted in high recovery rates of ticks after 4 days of feeding. methyl-green and pyronin tick salivary gland staining was typical, as described by walker et al. (1979), (figure 7). the quantitative results are illustrated (table 2). figure 7: methyl-green and pyronin stain. table 2: infection rate with theileria in ticks recovered from carrier roan. the pcr (rlb) results for 50 dissected salivary glands from r. evertsi evertsi and r. appendiculatus were positive for theileria sp. (sable) in both species. field ticks the infection rate in adult r. appendiculatus collected from grass, is shown (table 3). table 3: methyl-green and pyronin tick salivary gland staining and polymerase chain reaction. infection rate in adult rhipicephalus appendiculatus ticks collected from grass. discussion top ↑ there has been no attempt made previously to identify the vector of theileriosis in roan, but field observations that outbreaks usually coincided with heavy infestations with r. appendiculatus, led to the assumption that this species was a potential vector. rhipicephalus evertsi evertsi has been viewed as an apparently incidental parasite, but was included in this investigation as it is known to transmit other theileria sp. in ticks fed experimentally on carrier animals, no statistically significant difference in infection rate per infected tick or the number of ticks infected with theileria sp. (sable) could be demonstrated between r. evertsi evertsi and r. appendiculatus. from these and pcr findings, it can be concluded that both species become infected with theileria sp. (sable) and are potential vectors. primary anorectal hyperplastic or ‘neoplastic’ lymphadenomegaly was a very important finding in support of identifying the main vector for theileriosis in roan exposed to natural tick infection. it is well known from theilerial kinetic studies, that the local draining lymph node(s) is the first to enlarge during the initial stages of theileriosis in cattle (morrison et al. 1981).the drainage area for the anorectal lymph node includes the perianal region and tail. the only ticks feeding at this site at the time of lymphoid proliferation were r. evertsi evertsi adults, suggesting that r. evertsi evertsi was more likely to be the transmitting vector than r. appendiculatus in these cases. this, however, does not exclude r. appendiculatus as a vector, as pcr results on dissected salivary glands showed that it was also infected with theileria sp. (sable). as a consequence of the need for chemical restraint to perform lymph node aspirates, daily lymph node aspiration to determine the temporal relationship of primary nodal (ln. anorectales) theilerial schizont appearance, versus the first appearance in other lymph nodes and the peripheral blood, was not possible. during tick salivary gland dissections, one field-collected r. appendiculatus tick exhibited a very high infection with theileria. this infection could not be confirmed to be theileria sp. (sable), and comment on its significance would be speculative. if it is assumed that this infection was theileria sp. (sable), it would indicate that r. appendiculatus ticks are capable of acquiring heavy infection rates, supporting its role as a vector significantly. tick yields from ears were very low for r. evertsi evertsi in comparison with r. appendiculatus. this could be ascribed to prolonged housing in ear-bags and exposure to host-inflicted trauma from head-shaking and scratching. based on the rectal temperature results, it would be difficult at this stage to be sure about the incubation period of theileriosis in roan, as it was not possible to determine the day of infection by infected ticks under field conditions. at best, one can state that it appears to be less than 33 days (earliest temperature reaction) in most cases. an infection trial, possibly with an infected tick-derived stabilate or known infected live ticks, would be necessary to settle this issue. the use of buparvaquone as anti-theilerial chemotherapy is highly effective against theileriosis in roan antelope, but the staggered delay in treatment in relation to the first day of pyrexia resulted in animals 222 and 242 (treatment delay of 2 and 3 days respectively) requiring a second injection of buparvaquone. this finding revealed the importance of administering buparvaquone as close as possible to the initial fever reaction. a delay of anti-theilerial therapy may result in more serious complications and death, even if treated with a very effective anti-theilerial drug such as buparvaquone. no animal in this trial needed supportive therapy for opportunistic infections such as pneumonia that is known to be a complication in cattle recovering from east coast fever (muraguri, kiara & mchardy 1999; wagner et al. 1975). during the first 2 weeks of field tick exposure, a small increase in size of superficial cervical lymph nodes could be palpated and peripheral blood smear eosinophilia was detected.these changes were ascribed to expected hyperplasia [see sln fine-needle aspiration (fna) impressions smears] and inflammation as a result of ectoparasitic (mainly ticks) dermatitis. post-pyrexic changes in the mucous membrane colour of the animals were indicative of anaemia: (pale pink), later becoming complicated by icterus (pale yellow). these changes are consistent with signs occasionally observed in cattle suffering from east coast fever (theileria parva), (lawrence, de vos & irvin 1994), or theileria annulata infection (pipano & shkap 2004). loss of body condition during the post-therapeutic recovery phase was mostly likely a consequence of less time spent grazing with more time spent lying down and resting, although the animal would still drink milk reluctantly. anaemia and systemic inflammation also accounted for additional nutritional demands on the animal. it was the first time that theileriosis, which has been well studied in cattle, has been followed clinically in detail under field conditions in a wild antelope species. previously, limited observations were recorded with experimental and natural theileria taurotragi infection in eland antelope (grootenhuis et al. 1980). careful monitoring of various clinical parameters revealed many similarities in the clinical development and progression of theileriosis (especially east coast fever) in cattle and in roan antelope. conclusion top ↑ clinical theileriosis in roan antelope responds favourably to buparvaquone treatment, provided that it is administered as close as possible to the primary pyrexic phase. for more exact pathogenesis studies, parasite transmission and disease monitoring will have to be repeated in a controlled environment with known infection times and dose rates. valuable information gathered during field tick exposure demonstrated that r. evertsi evertsi and r. appendiculatus are two potential vectors of theileriosis in roan antelope. to determine the role of both species in the epidemiology of theileriosis outbreaks in roan, tick infection and transmission trials that involve carrier versus piroplasmaemic hosts should be compared separately for both tick species. acknowledgements top ↑ our sincere appreciation for the financial, infrastructural, and personnel support provided by mr j. hume, owner of mauricedale game ranch. a special acknowledgement of gratitude to mrs anita york, without whose dedication and love for these animals this project may never have reached its full potential. polymerase chain reaction tests were performed by the molecular laboratory in the department of veterinary tropical diseases, university of pretoria. the research team also acknowledges the significant funding contribution from the department of trade and industry of south africa through the technology and human resources for industry programme (thrip). competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions l.p. (university of pretoria) was the project leader. j.c.a.s. (university of pretoria) and j.a.l. (university of pretoria) were responsible for the experimental and project design. j.c.a.s. (university of pretoria) performed all the experiments. w.h.s. (university of pretoria) made conceptual contributions. j.c.a.s. (university of pretoria) wrote the manuscript and j.a.l. (university of pretoria) edited the language and grammar. blood sample testing was performed by the molecular biology laboratory in the department of veterinary tropical diseases, university of pretoria. references top ↑ grootenhuis, j.g., morrison, w.i., karstad, l., sayer, p.d., young, a.s., murray, m. et al., 1980, ‘fatal theileriosis in eland (taurotragus oryx): pathology of natural and experimental cases’, research in veterinary science 29, 219–229. pmid:7193340 heyne, h., elliott, e.g.r. & bezuidenhout, j.d., 1987, ‘rearing and infection techniques for amblyomma species to be used in heartwater transmission experiments’, onderstepoort journal of veterinary research 54, 461–471. pmid:3448576 lawrence, j.a., de vos, a.j. & irvin, a.d., 1994, ‘east coast fever’, in j.a.w. coetzer, g.r. thomson & r.c. tustin (eds.), infectious diseases of livestock with special reference to southern africa, pp. 309–325, oxord university press, cape town. morrison, w.i., buscher, g., murray, m., emery, d.l., masake, r.a., cook, r.h. et al., 1981, ‘theileria parva: kinetics of infection in the lymphoid system of cattle’, experimental parasitology 52, 248–260. http://dx.doi.org/10.1016/0014-4894(81)90080-1 morzaria, s.p., spooner, p., bishop, r. & mwaura, s., 1999, ‘the preparation of a composite stabilate for the immunisation against east coast fever’, in s. morzaria & s. williamson (eds.), proceedings of an fao/ ilri/oau-ibar workshop, nairobi, kenya, 10–12 march 1997, pp. 56–61. muraguri, g.r., kiara, h.k. & mchardy, n., 1999, ‘treatment of east coast fever: a comparison of parvaquone and buparvaquone’, veterinary parasitology 87, 25–37. http://dx.doi.org/10.1016/s0304-4017(99)00154-5 neitz, w.o., 1957, ‘theileriosis, gonderiosis and cytauxzoonoses’, onderstepoort journal of veterinary research 27, 275–429. nijhof, a.m., pillay, v., steyl, j., prozesky, l., stoltsz, w.h., lawrence, j.a. et al., 2005, ‘molecular characterization of theileria species associated with mortality in four species of african antelopes’, journal of clinical microbiology 43, 5907–5911. http://dx.doi.org/10.1128/jcm.43.12.5907-5911.2005, pmid:16333074 norval, r.a.i., perry, b.d. & young, a.s., 1992, the epidemiology of theileriosis in africa, academic press, london. pipano, e. & shkap, v., 2004, theileria annulata theileriosis, oxford university press, oxford. shaw, m.k., 2002, ‘theileria development and host cell invasion’, in d.a.e. dobbelaere & d.j. mckeever (eds.), world class parasites: theileria, pp. 1–22, kluwer academic publishers, norwell, massachusetts, usa. stoltsz, w.h. & dunsterville, m.t., 1992, ‘in vitro establishment and cultivation of a cytauxzoon sp. (theileria sp.) from a sable antelope (hippotragus niger, harris 1838)’, journal of the south african veterinary association 63, 182. todd, j.l. & wolbach, s.b., 1912, ‘parasitic protozoa from the gambia’, journal of medical research 26, 195–218. pmid:19972051 wagner, g.g., jessett, d.m., brown, c.g.d. & radley, d.e., 1975, ‘diminished antibody response to rinderpest vaccination in cattle undergoing experimental east coast fever’, research in veterinary science 19, 290–211. walker, a.r., mckellar, s.b., bell, l.j. & brown, c.g.d., 1979, ‘rapid quantitative assessment of theileria infection in ticks’, tropical animal health and production 11, 21–26. http://dx.doi.org/10.1007/bf02237760 world organisation for animal health, 2008, ‘theileriosis’, in manual of diagnostic tests and vaccines for terrestrial animals, 6th edn., 2.4.16, pp. 789–804, viewed 23 june 2011, from http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/ wilson, d.e., bartsch, r.c., bigalke, r.d. & thomas, s.e., 1974, ‘observations on mortality rates and disease in roan and sable antelope on nature reserves in the transvaal’, southern african wildlife management association journal 4, 203–206. 2195belozerov_pp325-330.qxd introduction in southern africa the cattle egret (bubulcus ibis) is a partial migrant, especially in the highveld and the drier northwest, that disperses during winter after breeding activities in spring and summer (martin 1997). they are highly colonial breeders, often breeding in large numbers in mixed-species heronries (maclean 1993; martin 1997; steyn 1996), and there is usually a high degree of breeding synchrony throughout the heronry (steyn 1996). heronries are usually abandoned after the breeding season, but the birds may use the same site for breeding year after year (brown, urban & newman 1982). the nest consists of a platform of sticks or reeds in tree or reedbed (maclean 1993), and it is not known to what extend nests of a previous season are reused. the tick argas (persicargas) arboreus kaiser, hoogstraal & kohls, 1964 is a common parasite of the cattle egret (bubulcus ibis), as well as other water birds, in their heronries all over the african continent (khalil, hoogstraal & oliver 1980). the feeding of ticks, egg-laying by adult females, and development of the larval and nymphal tick stages coincide with the birds’ nesting period during the spring-summer season. during autumn and winter dormant adult ticks and nymphs (both unfed and engorged) are mostly confined to suitable niches in the birds’ nesting trees, frequently under loose bark, and in cracks in the trees (active ticks also inhabit these niches during the day-time in the spring-summer period). the dormant state of the adult ticks is related to reproductive diapause controlled by photoperiod and 325 onderstepoort journal of veterinary research, 70:325–330 (2003) population structure of argas aarboreus (acari: argasidae) ticks associated with seasonally abandoned mixed heronries, dominated by cattle egrets (bubulcus iibis), in south africa v.n. belozerov1, d.j. van niekerk2 and h.j.b. butler2 abstract belozerov, v.n., van niekerk, d.j. & butler, h.j.b. 2003. population structure of argas arboreus (acari: argasidae) ticks associated with seasonally abandoned mixed heronries, dominated by cattle egrets (bubulcus ibis), in south africa. onderstepoort journal of veterinary research, 70:325–330 during winter populations of argas arboreus from heronries of the cattle egret, bubulcus ibis, in south africa are composed of adults, with some predominance of males, and ii–iv instar nymphs, in a state of diapause. the period of tick activity, including reproduction and development of eggs, larvae and n1 nymphs, is synchronized with the nesting and breeding season of their avian hosts. it begins during spring with the return of birds to the heronry, and ceases in autumn through induction of reproductive diapause in engorged females, and behavioural diapause in unfed nymphs and adult ticks. many ticks showed morphological anomalies and malformations, the study of which could possibly be used for monitoring of environmental pollution. keywords: argas arboreus, argasid ticks, birds, bubulcus ibis, heronries * author to whom correspondence is to be directed 1 biological research institute, st. petersburg state university, stary peterhof, 198504, russia e-mail: valentin.belozerov@paloma.spbu.ru 2 department of zoology and entomology, university of the free state, p.o. box 339, bloemfontein, 9300 south africa accepted for publication 24 june 2003—editor temperature (khalil 1974; 1976; khalil & shanbaky 1976). this life cycle appears to be similar in many regions of the tick’s distribution, including egypt (kaiser 1966; guirgis 1971; hafez, abdel-malek & guirgis 1971; 1972) and south africa (belozerov & kopij 1997). argas arboreus overwinters mainly as adults. during the winter in egypt, which lasts from october through march and during which birds are absent from the heronries, adult ticks represent 70–85 % of the total overwintering tick population, the rest consisting of n2–4 instars (guirgis 1971). in one study in south africa, adult ticks represented not less than 65 % of the overwintering tick population soon after their hosts begin breeding in the heronry (belozerov & kopij 1997). unfortunately, this study only included the nesting period of the birds, and data were not gathered when the birds were absent from the heronry. in this article additional data are presented on the population structure of overwintering a. arboreus ticks at some south african heronries, dominated by cattle egrets, immediately before and after birds return to the nesting sites. materials and methods the study was carried out in heronries consisting of several bird species, of which cattle egrets were the dominant species and the sacred ibis, threskiornis aethiopicus, additional inhabitants. in some colonies, reed cormorant, phalacrocorax africanus and the african spoonbill, platalea alba, and their nests, were also present. ticks were collected at heronries within the free state province, south africa, at the localities listed below. wolwekop farm (wf) this heronry is situated in trees on the retaining wall of an earth dam on the farm wolwekop (26°40’ e; 29°27’ s) in the dewetsdorp district. it is in the same locality where a. arboreus (belozerov & kopij 1997) and their main hosts, the cattle egret (kopij 1997) were studied previously. the dominant tree species used for nesting by the birds at this heronry were acacia karroo and rhus lancea. soetdoring nature reserve (snr) a heronry situated at the water-edge of the krugersdrift dam (25°57’ e; 28°51’ s) in the soetdoring nature reserve. the dominant tree species used for nesting were also a. karroo and r. lancea. willem pretorius game reserve (wpgr) this heronry was situated on a small island in the allemanskraal dam (27°09’ e; 28°18’ s) in the willem pretorius game reserve. eucalyptus sp. trees and rhus sp. shrubs were mainly used for nesting. all these heronries have been in existence for many years. ticks, including nymphs and eggs, were collected from under loose bark and in cracks in trees, and preserved in 70 % ethyl alcohol until they were examined in the laboratory. in the wf and snr heronries ticks were collected on 5 and 6 october 2000, before the birds returned. in the wpgr heronry, ticks were collected on 4 november 2000 when the cattle egrets had just returned for nesting (mainly in eucalyptus trees), but sacred ibises had young nestlings in nests which were constructed in rhus sp. shrubs only 30–50 m away. ticks were collected separately from the eucalyptus trees and rhus shrubs. results the population structure of the tick populations in the various heronries is graphically illustrated in fig. 1. wf heronry (fig. 1a) the bulk of the tick specimens (76.5 %; 237/310) consisted of adult ticks, with the sex ratio (1:1.2) in favour of males. the remaining ticks were n2 and n3–4 stage nymphs in about equal proportions. no eggs, larvae or 1st instar nymphs were found. snr heronry (fig. 1d) adult ticks comprised 79.2 % (251/317) of the overwintering population with the sex ratio (1:1.4) in favour of males. the rest of the population consisted mostly of n3–4 stage nymphs (18.0 %), while n2 nymphs contributed only 2.8 % of the population. neither larvae nor n1 nymphs were collected, and only a small batch of about 12 recently laid, viable eggs was found. wpgr heronry (fig. 1e) a total of 399 ticks were collected from the eucalyptus trees that housed cattle egret nests. the adults predominated (71.2 %), whilst the female to male ratio was 1:1.9. about a quarter of the sample consisted of n2–4 nymphs, and n1 nymphs were present. numerous batches of freshly laid eggs were observed under the bark of the trees. 326 argas arboreus (acari: argasidae) ticks in south africa �������� ��� ������ ������� � � ��� �� ������ � ������ �� ! ������ " �������� �#�$� ��������� ��� �� ��� �� �� ! �%� �� &�'����((� � �%�� ��)�*�������� ��� ������ ������!�� + ��� �� ��� �� �� ! ��� �� &�*,-.��((� / ��)�*���� ��01$2������� ��� ��������� &�*,-.��((� ��� �� ��� �� �� �%�� � ������ �� ! � ���� 3 4,-�,�.����! ��� ������ �������� ��� �� !���� � !%� �� �� ! ����� �� �%� �� 5 ��)�*���� ��01$2�� ��� ����� �� �� �� �� � !!���� �� ! ����� ��� �� � �� � !%� �� 327 v.n. belozerov, d.j. van niekerk & h.j.b. butler fig. 1 population structure of argas arboreus at wolwekop (a, b, c), soetdoring nature reserve (snr; d) and willem pretorius game reserve (wpgr; e, f) heronries, south africa. data of figures b and c from belozerov & kopij (1997) the 209 ticks collected from rhus sp. shrubs in which the sacred ibis nests with young nestlings were located were composed of n2–4 nymphs (71.8 %), and adult ticks were much less common (fig. 1f). the sex ratio of adults was close to parity (52 % males and 48 % females). n1 stage nymphs were present, but no larvae were observed in either of the two samples (fig. 1e and f). abnormalities of a. aarboreus many ticks in all samples had various abnormalities in their external morphology, the most common being damaged, stumpy and shortened legs, as well as body asymmetry and the appearance of spots with changed cuticular structure. the number of ticks with limb and body abnormalities were especially high at wf (29.7 %; 92/310 ticks), while the least malformations were observed in ticks from snr (7.9 %; 25/317 ticks). at wpgr 16.8 % (67/ 399) of ticks collected on eucalyptus sp. trees and 20.6 % (43/209) of ticks collected on rhus sp. shrubs had abnormalities. in the october sample collected at wf before the return of the birds, a progressive increase in malformations was observed from n1 nymphs (13.5 %) to adult ticks (33.4 %). in the november sample from wpgr, where birds had already returned to the heronry, the greatest proportion of malformations was evident in the n3–4 stage nymphs (21–24 %) with lower proportions in adult ticks (15–17 %) and n2 nymphs (11–18 %). discussion the proportion of adult and n2–4 nymphs is similar for the samples from wf (fig. 1a), snr (fig. 1d) as well as the eucalyptus tree sample from wpgr (fig. 1e). by contrast, the rhus sample collected only 30–50 m away from, and concurrently with the eucalyptus tree sample was completely different, with n1–4 nymphs dominating the sample (fig. 1f). since the rhus sample were collected at a heronry that was in a more advanced breeding cycle phase (nestlings) than the other samples, which were collected from heronries that were in the very early phases of the breeding cycle (i.e. just before or after the birds returned), it is possible that the breeding phase of the birds might have an influence on the ticks life cycle. an earlier study of the ticks at the wf heronry (belozerov & kopij 1997) did show changes in the population composition of the ticks (fig. 1b, c). just after the birds’ return to the heronry 65.0 % of the tick population consisted of adults, with n3–4 nymphs accounting for the balance (fig. 1b). many recently laid eggs were also found. two months later, when most birds had nestlings, n1–4 nymphs dominated the sample and adult ticks contributed only 24.7 % to the population (fig. 1c). it therefore seems likely that the life cycle of a. arboreus is indeed closely linked to that of their avian hosts. the data obtained confirm the strong seasonality of the argas ticks’ life cycle, which is characterized by a definite alternation between active and dormant periods that correspond to the seasonal rhythms in the activities of their hosts. tick feeding, development and reproduction is synchronized with the nesting and breeding periods of the birds during the spring-summer season both in egypt (guirgis 1971; hafez et al. 1971; 1972) and in south africa (belozerov & kopij 1997). the current study also confirms the results of previous studies that adult and n2–4 nymphal a. arboreus are the stages that overwinter (guirgis 1971; hafez et al. 1971; 1972; belozerov & kopij 1997). in both southern and northern populations of a. arboreus, adult ticks comprise 70–85 % of the overwintering reserve, with n2–4 nymphs making up the balance. eggs, larvae and n1 nymphs do not overwinter and their appearance and development coincide with the spring-summer period. this synchronization, undoubtedly, is made possible by a photoperiodically-controlled reproductive diapause in adult females (khalil 1974; 1976). the period of egg-laying, followed by the development of larvae and n1 nymphs, is temporally limited due to the reproductive diapause, and this results in the accumulation of an overwintering reserve of older nymphs and adult ticks. the data of guirgis (1971) suggest that behavioural diapause of unfed adults and nymphs are essential for the maintenance of the normal seasonal cycle of a. arboreus. this was shown by the behaviour of ticks during the winter of 1966–1967 in egypt when the seasonal rhythm of birds was disturbed and the ticks retained their dormant state, although the birds did not leave the heronries. this behavioural diapause ensures the dormant state of unfed adult and nymphs, and results in preservation of the population structure in the overwintering population of ticks until their hosts return to the heronries. returning birds are met by overwintered hungry ticks that are ready to attack their hosts, as soon as the birds begin nesting and breeding. this explains the earlier onset of tick development in places colonized by sacred ibises (fig. 1f), compared to those colonized at a later stage by cattle egrets (fig. 1e). the engorged females overwinter in a reproductive diapause state and are activated by increased daylength during spring. oviposition is initiated at the time their hosts return to the heronry after wintering elsewhere, or even earlier as evidenced by the eggs found before the birds had returned to the snr heronry. a decrease in the proportion of nymphs in the tick populations during autumn, both in egypt and south africa, after birds had left the heronries (belozerov & kopij 1997), indicates that engorged nymphs, in contrast to females, do not display an autumnal developmental arrest, but continue to moult. this results in an increased proportion of adults within the overwintering reserve of a. arboreus. this latter fact, however, requires verification under experimental conditions. our data on the population structure of a. arboreus confirm previous conclusions that the life cycle of this parasite of water birds that nest in colonies in africa is normally univoltine. there are, however, additional options for either biennial development or bivoltine development with seasonal synchronization by means of facultative diapause at the n2–4 and adult stages. the combination of the three types of development, with overlapping life cycles of different duration, results in an increased hardiness and stability of local populations of this tick. similar patterns in development are also characteristic of argasid ticks in more temperate climates. the duration of the life cycle of the pigeon tick argas (argas) reflexus fabr. in europe varies between 3 328 argas arboreus (acari: argasidae) ticks in south africa and 11 years, but development of generations is well synchronized by means of various seasonal adaptations, which are specific for every postembryonic stage. the overwintering reserve in this species is therefore more complex, consisting of unfed and engorged larvae, n1–4 and adult ticks in a state of diapause (dautel & knulle 1997). in the cosmopolitan fowl tick a. (persicargas) persicus oken the ability to overwinter is present in all stages of its life-cycle, including the egg stage (galuzo 1957), and this explains the very wide geographic distribution of this tick. similar adaptations in ixodes (ceratixodes) uriae white, a circumpolar parasite of colonial sea birds, enable the perfect coordination of its development with the breeding seasons of its hosts under the severe conditions of both the subarctic (karpovich 1973; eveleigh & threlfall 1974) and subantarctic regions (frenot, de oliveira, gauthier-clerc, deunnf, bellido & vernon 2001), in spite of the long duration of its life cycles there (3–7 and 2–4 years respectively). this is possible through overwintering of all postembryonic stages (in unfed state), as well as by an extended egg diapause. thus, the set of regulatory stages with an ability for diapause is quite vast, but rather variable in ticks parasitizing colonial birds. in cosmopolitan a. persicus and circumpolar i. uriae they include the egg and all postembryonic stages, in european a. reflexus all postembryonic stages, and in african a. arboreus only adult ticks and n2–4 instars. the off-host location of a. arboreus larvae is unknown. according to belozerov & kopij (1997) and guirgis (1971) very few or no larvae (either unfed or engorged) are found hiding under bark and in its crevices with nymphs and adult ticks. it is much easier to find larval ticks when they are feeding on fledged nestlings of cattle egrets. some data suggest that engorged larvae can be found in the litter under trees after detachment from nestlings (guirgis 1971). this requires further investigation. it would appear that malformations of the nymphs and adults of a. arboreus are frequent. many different forms of teratological abnormalities induced by regeneration and external factors have been described by buczek (1994) in adult and larval a. reflexus and a. persicus, and by campana-rouget (1959) in nymphs of ornithodoros parkeri and ornothodoros turicata. investigations conducted recently on ixodid ticks of the genus ixodes (alekseev & dubinina 1993; zharkov, dubinina, alekseev & jensen 2000) have revealed a wide distribution of morphological anomalies (in 10–48 % of ticks in different populations) and their dependence on pollution levels in the environment. the conclusion from these investigations concerning biomonitoring of the environment by investigating morphological anomalies in ticks, can be used for the same purpose in population studies of a. arboreus. acknowledgements we thank prof. l.j. fourie for his comments on this article. references alekseev, a.n. & dubinina, e.v. 1993. abnormalities in ixodes ticks (ixodoidea, ixodinae). acarina. russian journal of acarology, 1:73–85. belozerov, v.n. & kopij, g. 1997. changes in population stage structure of an argasid tick argas arboreus during nesting season of its host, the cattle egret bubulcus ibis, in south africa. acarina. russian journal of acarology, 5:63– 68. brown, l.h., urban, e.k. & newman, k. (eds.) 1982. the birds of africa. 1. london: academic press. buczek, a. 1994. teratologia kleszczi (acari, ixodida). katowice: slaska akademia medyczna (in polish). campana-rouget, y. 1959. la teratologie des tique. annales de parasitologie humaine et comparee, 34:209– 260, 354–431. dautel, h. & knulle, w. 1997. life cycle and seasonal development of postembryonic argas reflexus (acari: argasidae) at two thermally different locations in central europe. experimental and applied acarology, 21:697–712. eveleigh, e.s. & threlfall, w. 1974. the biology of ixodes (ceratixodes) uriae white, 1851 in newfoundland. acarologia, 16:621–635. frenot, y., de oliveira, e., gauthier-clerc, m., deunnf, j., bellido, a. & vernon, p. 2001. life cycle of the tick ixodes uriae in penguin colonies: relationships with host breeding activity. international journal for parasitology, 31:1040–1047. galuzo, i.g. 1957. argasid ticks and their epizootologic significance. alma-ata (in russian). guirgis, s.s. 1971. the subgenus persicargas (ixodoidea, argasidae, argas). 11. ecology and seasonal dynamics of a.(p.) arboreus in egypt. journal of medical entomology, 8: 407–414. hafez, m., abdel-malek, a.a. & guirgis, s.s. 1971. the subgenus persicargas (ixodoidea, argasidae, argas). 12. biological studies on the immature stages of a.(p.) arboreus in egypt. journal of medical entomology, 8:421–429. hafez, m., abdel-malek, a.a. & guirgis, s.s. 1972. the subgenus persicargas (ixodoidea, argasidae, argas). 14. biological studies on the adult stage of a.(p.) arboreus in egypt. journal of medical entomology, 9:19–29. kaiser, m.n. 1966. the subgenus persicargas (ixodoidea, argasidae, argas). 3. the life cycle of a.(p.) arboreus, and a standardized rearing method for argasid ticks. annals of the entomological society of america, 59:496–502. karpovich, v.n. 1973. the life cycle of ceratixodes putus under murman conditions. parazitologiya, 7:128–134 (in russian, english summary). 329 v.n. belozerov, d.j. van niekerk & h.j.b. butler khalil, g.m. 1974. the subgenus persicargas (ixodoidea, argasidae, argas). 19. preliminary studies on diapause in a.(p.) arboreus. journal of medical entomology, 11:363–366. khalil, g.m. 1976. the subgenus persicargas (ixodoidea, argasidae, argas). 26. argas (p.) arboreus: effect of photoperiod on diapause induction and termination. experimental parasitology, 40:232–237. khalil, g.m. & shanbaky, n.m. 1976. the subgenus persicargas (ixodoidea, argasidae, argas). 25. argas (p.) arboreus: effect of temperature on diapause termination. experimental parasitology, 39:431–437. khalil, g.m., hoogstraal, h. & oliver, j.h., jr. 1980. biological evaluation of the systematic validity of the african argas (persicargas) arboreus and the asian-australian a. (p.) robertsi (ixodoidea, argasidae). international journal for parasitology, 10:253–259. kopij, g. 1997. timing of colony occupation, clutch size and breeding success in the cattle egret bubulcus ibis related to nest location in a south african heronry. acta ornithologica, 32:169–174 . martin, a.p. 1997. cattle egret bubulcus ibis, in the atlas of southern african birds. 1: non-passerines, edited by j.a. harrison, d.g. allan, l.g. underhill, m. herremans, a.j. tree, v. parker & c.j. brown. johannesburg: birdlife south africa. maclean, g.l. 1993. roberts’ birds of southern africa. cape town: john voelcker bird book fund. steyn, p. 1996. nesting birds. the breeding habits of southern african birds. cape town: fernwood press. zharkov, s.d., dubinina, e.v., alekseev, a.n. & jensen, p.m. 2000. anthropogenic pressure and changes in ixodes tick populations in the baltic region of russia and denmark. acarina. russian journal of acarology, 8:137–142. 330 argas arboreus (acari: argasidae) ticks in south africa herold_203-210.qxd introduction after the outbreak of rinderpest at the turn of the 19th century the bulk of the african buffalo (syncerus caffer) population was restricted to the kruger national park, the hluhluwe and umfolozi nature reserves and the addo elephant national park. since the addo population was the only one free from foot-and-mouth disease (fmd) and corridor disease (cd) most of the animals outside the three areas mentioned derive from them (de vos 1987). african buffaloes are considered long-term carriers of the three sat types of the fmd virus (hedger & condy 1985; bengis, thomson & de vos 1987) as well as of theileria parva lawrencei, the causative agent of cd (potgieter, stoltsz, blouin & roos 1988). furthermore, they are considered a source of re-infection of domestic cattle for bovine tuberculosis (bengis, kriek, keet, raath, de vos & huchzermeyer 1996) and brucellosis (herr & marshall 1981). transporting african buffaloes in south africa is therefore subject to strict regulations. different approaches to satisfy the demand for buffaloes free of specific diseases (bfsd) among private game farmers, to increase the genetic variety outside the endemic areas and to reap the existing 203 onderstepoort journal of veterinary research, 71:203–210 (2004) comparison of three different media for freezing of epididymal sperm from the african buffalo (syncerus caffer) and influence of equilibration time on the post-thaw sperm quality f.c. herold1, k. de haas1, d. cooper2, b. colenbrander3, j.o. nöthling1, w. theunisen4, b. spillings4 and d. gerber1 abstract herold, f.c., de haas, k., cooper, d., colenbrander, b., nöthling, j.o., theunisen, w., spillings, b. & gerber, d. 2004. comparison of three different media for freezing of epididymal sperm from the african buffalo (syncerus caffer) and influence of equilibration time on the post-thaw sperm quality. onderstepoort journal of veterinary research, 71:203–210 assisted reproductive techniques might prove themselves useful tools in producing buffaloes free of specific diseases (bfsd), which are in demand in south africa. freezing protocols for african buffalo semen must not only result in good post-thaw qualities, but must also be practical. epididymal sperm from six mature african buffalo bulls was collected, diluted with three different semen extenders and frozen. pre-freezing equilibration times between 2 and 9 h were tested. total and progressive motility, longevity and acrosomal integrity were measured and compared. the use of triladyl™ proved to result in better post-thaw parameters than the other two diluents. equilibration times of between 4 and 9 h did not influence post-thaw sperm qualities significantly. for some of the treatments, exposure to semen extenders before freezing for less than 4 h resulted in inferior postthaw semen parameters. keywords: african buffalo, andromed®, epididymal, red ovine freezing buffer, sperm, syncerus caffer, triladyl™ 1 section of reproduction, department of production animal studies, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 kzn nature conservation service, private bag x01, st. lucia, 3936 south africa 3 department of farm animal health, section of reproduction, faculty of veterinary medicine, utrecht university, yalelaan 12, 3854 cm utrecht, the netherlands 4 wildlife biological resource centre, endangered wildlife trust, p.o. box 582, pretoria, 0001 south africa accepted for publication 9 march 2004—editor benefits of larger trophy sizes of animals from the kruger national park, have been adopted (gerber 2000). special breeding programmes are one and, in future, assisted reproductive techniques might become another. attempts to freeze epididymal sperm from african buffaloes have been made in the past (bartels, lambrechts, kidson & friedmann 1996; lambrechts, van niekerk, coetzer, cloete & van der horst 1999; kilian, lubbe, bartels, friedmann & denniston 2000; gerber, irons, arlotto & cooper 2001; gerber, irons, herold & cooper 2002; lubbe, bartels, kilian, friedman & mortimer, unpublished data 2000) and total post-thaw motility varied from 19 % to 45 %. semen diluents used in all previous trials contained egg yolk. the presence of hormones (hartmann, lacorn & steinhardt 1998) in them as well as problems arising from lack of hygiene may affect the fertilizing capacity of spermatozoa (müller-schlösser, aires, hinsch & hinsch 2001). since collection of sperm from the african buffalo is often done in the field, ideal conditions are not necessarily present. a useful freezing method must therefore not only result in high post-thaw qualities but must also be practical. the aim of this study was to evaluate the influence of equilibration time of different semen extenders on post-thaw sperm qualities such as motility, longevity and acrosomal integrity, parameters that are routinely used for semen evaluation (stewart & o’hagan 1972; correa & zavos 1995; garner, thomas & gravance 1999). the study was also designed to evaluate the suitability of an egg yolk-free semen diluent (andromed®, minitüb, germany) and to compare it to two egg yolkcontaining media (triladyl™, minitüb, germany and red ovine freezing buffer, imv, france). materials and methods epididymides were collected from six african buffalo bulls culled during a disease eradication programme in huhluwe/umfolozi national park in september 2001. this programme was performed under the responsibility of the kwazulu-natal nature conservation service. all animals were killed by means of rifle shot and only bulls which had three or more pairs of permanent lower incisors (about 3–3.5 years of age) were used in the trial. the testes and epididymides from each bull were collected through a scrotal incision within 10 min after culling. an effort was made to remove as much as possible of the vas deferens. the vas deferens, the tail of the epididymis and part of the body were dissected free from the testis. the epididymal duct was then dissected free and transected on the site in the tail of the epididymis where the tubular diameter becomes distinctly larger distally. a blunted 23 or 25 g hypodermic needle connected to a 10 ml syringe was then inserted into the lumen of the vas deferens and the sperm was flushed with air in a retrograde direction from the tail of the epididymis and collected in a sterile plastic tube. all epididymides were flushed within 30 min of death of the animal. the harvested sperm of both epididymides of the same bull were mixed and split into three aliquots, each of which was poured into a 15 ml plastic tube, containing 13 ml of one of the three different cryodiluents. the three semen extenders used were triladyl™, andromed® (both minitube, germany) and red ovine freezing buffer (imv, france). the glycerol concentrations of these media are 6.6 %, 6.8 % and 3.5 %, respectively. the concentration of the sperm/diluent mixture was determined with a haemocytometer and was between 100 and 150 x 106 sperm cells per ml. the sperm-diluent suspension was mixed and the three 15 ml tubes were placed in a 500 ml water bottle containing water at a temperature of 20 °c. the water bottle was placed into a styrofoam box containing water and ice cubes in such a relationship that the temperature of the water was 4 °c resulting in a cooling rate of 0.25 °c/min. the samples were then transported to a laboratory. evaluation of fresh semen before further processing the total and progressive sperm motility from each solution was evaluated by means of eyeball assessment using a phase contrast microscope. percentages of progressive and total motile sperm cells were recorded. freezing of semen after 2 h five 0.25 ml french straws were loaded from each of the three sperm dilutions, dried and placed 4 cm above liquid nitrogen for 10 min before being plunged into and stored in a nitrogen tank until thawing. the 15 ml tubes containing the remaining sperm dilutions were further kept in water bottles in a water bath at 4 °c. the procedure of packing and freezing straws was repeated eight times every hour. 204 freezing of epididymal sperm from african buffalo (syncerus caffer) thawing of semen after a storage time of between 4 and 37 days the straws were thawed in a water bath at a temperature of 37 °c for at least 30 s. the contents of two straws of the same treatment were emptied into two different pre-heated (37 °c) 3 ml perspex tubes and stored in a water-bath at 37 °c. evaluation of the post-thaw semen quality motility an amount of 25 µl of the thawed semen were further diluted with 100 µl of the corresponding diluent to determine the total and progressive sperm motility. the motility of the semen from both thawed straws was determined by eyeball assessment using a phase contrast microscope. in the rare event of remarkable differences this was attributed to handling, and the percentage of total and progressive motile sperm cells of the sample, which had a higher motility, was recorded. longevity the longevity of the sperm was assessed by evaluating the total and progressive motility of the equilibrated sperm immediately (0), 1 and 2 h after thawing. during this time the sperm samples were kept in a water bath at 37 °c. acrosomal integrity the acrosomal integrity was evaluated after a storage time of 5 months. the content of a thawed 0.25 ml straw was then emptied into an eppendorf tube. the semen diluent mixture was washed twice with 200 µl of pbs by centrifugation at 600 g for 5 min. the supernatant was removed and the pellet re-suspended with 200 µl of pbs. fifty microlitres of igepal ca-630 (sigma-aldrich ltd, atlasville, south africa, cat no: d1626) and 50 µl of ethd-1 (ethidium homodimer, laboratory specialist services ltd, clareich, south africa, cat no: e-1149) were added and the suspension incubated for 5 min at 37 °c. igepal ca-630 was added to permeabilize the sperm membrane and ethd-1 to stain the dna red. then 5 µl of 1 mg/ml ssdna (deoxyribonucleic acid sodium from salmon, sigma-aldrich ltd, atlasville, south africa, cat no: d1626) were added to bind the free ethd-1 and it was further incubated for 1–2 min. spermatozoa were fixed by addition of 50 µl of 4 % paraformaldehyde and 1 % glutaraldehyde in pbs and kept in the dark for 1 min before being washed once with 200 µl of pbs by centrifugation at 600 g for 5 min. the supernatant was removed and the pellet re-suspended with 50 µl of pbs. fifty microlitres of fitc-pna (lectin from arachis hypogaea*fluorescein, sigma-aldrich ltd, atlasville, south africa, cat no: l7381) were added and the suspension was incubated for 10 min at 37 °c. it was again washed once with 200 µl of pbs by centrifugation at 600 g for 5 min, the supernatant was removed, re-suspended with 25–100 µl of pbs and kept in the dark. equal amounts of 2 µl sperm suspension and of antifade (slowfade antifade kit, laboratory specialist services ltd, clareich, south africa, cat no: s-2828) were placed on a microscopic slide and covered with a cover slip. the edges of the coverslip were sealed with nail polish to prevent drying out. the samples were examined under an epifluorescence microscope (bh2-rfca; olympus, tokyo, japan) at a magnification of 400–1 000. spermatozoa were classified according to their appearance as acrosome intact, acrosome damaged or devoid of acrosome (fig. 1). one hundred spermatozoa of each sample were evaluated and the results expressed as percentages. statistical analysis anova a “repeated measure one way anova” was used to test for differences between the eight different equilibration times separately for the use of andromed®, trilady™ or red ovine freezing buffer. the same test was used to compare the different media. to identify different groups the tukey test was used. in the event that normality failed, the friedman repeated measures anova on ranks was used. total and progressive motility were compared separately at every measurement. longevity after it became evident that equilibration times between 4 and 9 h did not yield different results, the mean values for these equilibration times were used for each buffalo and motilities before freezing, immediately, 1 h and 2 h after thawing were compared with each other by means of a “repeated measure one way anova”. to identify different groups the tukey test was used. results total and progressive motilities of fresh sperm samples varied between the bulls and averaged 57 ± 205 f.c. herold et al. 206 freezing of epididymal sperm from african buffalo (syncerus caffer) table 1 comparison of total and progressive motilities (%) ± sd of all three media. each value represents the mean of equilibration times of 4–9 h. values with different superscripts within columns differ significantly. measurements were taken before freezing (fresh), immediately (0 h), 1 h and 2 h after thawing. rofb = red ovine freezing buffer medium fresh – fresh – 0 h – 0 h – 1 h – 1 h – 2 h – 2 h – total progessive total progressive total progressive total progressive andromed® 57 ± 17 31 ± 21 46 ± 13a 14 ± 14a 48 ± 12a 7 ± 11a 45 ± 12a 3 ± 6a triladyl™ 56 ± 13 19 ± 11 54 ± 13a,b 19 ± 12a 56 ± 13a,b 21 ± 15b 49 ± 12a 16 ± 11b rofb 58 ± 14 24 ± 15 57 ± 12b 29 ± 15b 58 ± 15b 10 ± 13a 51 ± 16a 2 ± 6a fig. 1 spermatozoa from the african buffalo after freezing and thawing, stained with fitc-pna 1 = sperm cell with intact acrosome, 2 = sperm cell with damaged or reacting acrosome and 3 = sperm cell with reacted or lost acrosome ������������ �� ������� ����� �� � � �� �� � � �� �� � � � � ���� �� � � �� �� �� �� �� �� �� � � � � � � � � � � � � � � fig. 2 comparison of total sperm motility (%) in respect of different pre-freezing equilibration times (2–9 h) and longevity for sperm frozen in the diluents andromed®, triladyl™ and red ovine freezing buffer (rofb) all values are means of the six bulls. a = andromed®, t = triladyl™ and r = red ovine freezing buffer. motilities were evaluated immediately (0 h), 1 h and 2 h after thawing � �� � � � � � �� � � � �� �� � � ���� �� � � ������������ �� ������� ����� �� � � �� �� �� �� �� �� �� � � � � � � � � � � � � fig. 3 comparison of progressive sperm motility (%) in respect of different equilibration times (2–9 h) and longevity for sperm frozen in the diluents andromed®, triladyl™ and red ovine freezing buffer (rofb) all values are means of the six bulls. a = andromed®, t = triladyl™ and r = red ovine freezing buffer. motilities were evaluated immediately (0 h), 1 h and 2 h after thawing ������������ ������������ ������� ������������� ������������� �������� ��� ���� ��������� � �� �� � � � � � � � �� �� �� �� �� �� �� �� �� �� � � �� �� �� �� �!� � � � � �� � � �� � �� � � �� �� � fig. 4 comparison of longevity for sperm frozen in the diluents andromed®, triladyl™ and red ovine freezing buffer (rofb) each value represents the mean of equilibration times between 4 and 9 h. measurements were taken immediately (0 h), 1 h and 2 h after thawing for total (tot) and progressive (prog) motility 19 % (mean ± sd, range 33–80 %) and 31 ± 23 % (range 10–60 %), respectively. post-thaw total motility (t = 0) of sperm frozen with andromed®, triladyl™ or red ovine freezing buffer was 43 ± 15 %, 52 ± 14 %, and 56 ± 13 %, respectively. equilibration time significantly affected post-thaw motility. short equilibration times (2 and 3 h) negatively affected post-thaw sperm motility, being 27 ± 19 % and 43 ± 12 %, 45 ± 10 % and 50 ± 20 % as well as 48 ± 10 % and 60 ± 20 % for andromed®, triladyl™ and red ovine freezing buffer, respectively (fig. 2). equilibration periods over 3 h resulted in significantly higher motility percentages, being 46 ± 13 %, 54 ± 13 % and 57 ± 12 % for andromed®, triladyl™ and red ovine freezing buffer, respectively. no differences were observed between the longer (4–9 h) incubation periods. total motility did not significantly decline within 2 h after thawing, apart for samples frozen with red ovine freezing buffer (table 1 and fig. 4). post-thaw progressive motility (t = 0) of sperm frozen with andromed®, triladyl™ or red ovine freezing buffer was 13 ± 13 %, 18 ± 12 % and 28 ± 16 %, respectively. equilibration time significantly affected post-thaw motility. short equilibration times (2 and 3 h) negatively effected post-thaw sperm motility being 6 ± 9 % and 12 ± 14 %, 7 ± 7 % and 20 ± 13 % as well as 17 ± 17 % and 35 ± 20 % for andromed®, triladyl™ and red ovine freezing buffer, respectively (fig. 3). equilibration periods over 3 h resulted in significantly higher motility percentages, being 14 ± 14 %, 19 ± 12 % and 29 ± 15 % for andromed®, triladyl™ and red ovine freezing buffer, respectively. no differences were observed between the longer (4–9 h) incubation periods. progressive motility did significantly decline within 2 h after thawing to almost 0 in samples frozen with andromed® and red ovine freezing buffer (3 ± 6 % and 2 ± 6 %, respectively) but did not decline in samples frozen with triladyl™ (fig. 4). when acrosomal integrity was evaluated andromed proved to be inferior to both other media. the percentage of intact acrosomes did not differ within diluents or equilibration times. the acrosomal integrity was therefore analysed again to test for differences between the diluents when all the equilibration times were pooled. triladyl™ resulted in 56 ± 7 % intact acrosomes, which is significantly higher than the results for andromed® (52 ± 7 %). rofb (55 ± 6 %) did not differ from the other two extenders. when the percentages of lost acrosomes were compared, an equilibration time of 2 h resulted in significantly lower percentages than when equilibrated for 8 h (3 % and 6 %, respectively) using red ovine freezing buffer. furthermore, the use of andromed® resulted in significantly higher percentages (6 %) of lost acrosomes than both other media (4 % and 3 % for triladyl™ and rofb, respectively), when equilibrated for 2 h. andromed also yielded significantly higher numbers of lost acrosomes compared to red ovine freezing buffer after an equilibration time of 5 h (8 % and 4 %, respectively) and compared to triladyl™ when equilibrated for 8 h (7 % and 3 %, respectively). discussion epididymal sperm from the african buffalo can successfully be frozen-thawed in triladyl™ extender when adequate equilibration times are applied. owing to the deleterious effects of tissue degeneration it is recommended that spermatozoa be recovered immediately post-mortem (hopkins, armstrong, hummel & junior 1988). therefore, we decided to flush epididymal sperm as soon after culling as possible (in our case this was 30 min) and keep the samples in semen diluents rather than transport the whole epididymides as such. many reports are available about the exposure time of semen to glycerol, but different results were achieved. in ejaculated bull semen a shorter equilibration time has been recommended because longer exposure to glycerol was suggested to be detrimental to sperm survival. exposure times compared were 0.5 and 18 h (o´dell & hurst 1956; berndtson & foote 1972). contrary to these reports are results that indicate no differences in the percentage of intact acrosomes and motile spermatozoa in bovine semen samples when equilibrated for 0.5 or 2 h (wiggin & almquist 1975a). wiggin & almquist (1975b) also reported that equilibration times of more than 4 h are detrimental to semen and recommended 2 h. in equids, no difference in the postthaw motility have been found after samples were exposed to pre-freeze equilibration times between 30 and 120 min (jimenez 1987). no reports are available for african buffalo semen equilibrated for different times using glycerol containing media before freezing. since laboratories are often some hours away from the site of collection, we compared equilibration times between 2 and 9 h rather then shorter ones as done in some of the above-mentioned studies. our results were similar to those in the study described by jimenez 207 f.c. herold et al. 1987). we also did not observe any differences between equilibration times longer than 3 h for any of the media used. although we used a similar concentration of glycerol we did not observe any detrimental effect owing to exposure times exceeding 4 h. we found equilibration times of 2 and 3 h to be inferior. these differences can be due either to the fact that we used epididymal sperm or to species differences. a post-thaw semen parameter which is rarely measured, is longevity and our results show that it might be useful to compare this more often. when post-thaw motilities were compared immediately after thawing the use of red ovine freezing buffer resulted in higher total motility than andromed® and in a higher progressive motility than for both andromed® and triladyl™. after incubation for 2 h in a 37 °c water bath the progressive motility for using red ovine freezing buffer decreased to 2 ± 6. this means that only 8 % of the spermatozoa that were progressively motile immediately after thawing were still intact 2 h after thawing. this figure was 18 % for andromed® and 82 % for triladyl™. the decrease of sperm motility when rofb and andromed® were used was only significant when compared to the values before freezing, but not when compared to the ones immediately after thawing. this fact was attributed to the low number of samples, as only six buffaloes were available. the trend seemed to be that progressive motility decreased less over the 2 h observation period when sperm is frozen with triladyl™. ordinary spermatozoa staining techniques such as eosin/nigrosin are not useful to evaluate acrosomal integrity. electron microscopy is an accurate method for assessing the acrosomal status, but it is expensive and very laborious. indirect immunofluorescence techniques employing monoclonal antibodies are not available for african buffaloes. lectins are widely used to bind to glyconconjugates of the outer acrosomal membrane and are made visible by markers such as fluorescein isothiocynate (fitc). staining spermatozoa with fitc-pna is comparatively simple and allows the easy assessment of the acrosomal status. fitc-pna has been used to stain spermatozoa of stallions (rathi, colenbrander, bevers & gadella 2001), boars (flesch, colenbrander, van golde & gadella 1999), bulls (garner et al. 1999), dogs (sirivaidyapong, bevers, gadella & colenbrander 2001) and humans (mortimer, curtis & miller 1987). the percentages of intact acrosomes in our study were low in comparison to previous work, where percentages of 67–77 % (gerber et al. 2001), 80–87 % (gerber et al. 2002) and 58–64 % (lambrechts et al. 1999) intact acrosomes were reported. since we used (at least when triladyl™ was used) very similar freezing protocols it is possible that the higher percentages of intact acrosomes found by these researchers can be attributed to the techniques used to evaluate the acrosomal integrity. it appears therefore that eosin/nigrosin staining is inferior to fitc-pna staining to selectively stain the acrosomal membrane for the evaluation of acrosomal integrity. since during routine inseminations many cows are not inseminated at the optimal time, it would not be surprising if longevity has a significant effect on non-return rates. whether an increased longevity, as it was found in the present study, has an influence on the non-return rate needs to be tested. an interesting observation of our study was that progressive motility was higher or as high immediately after thawing as it was before freezing when triladyl™ (19 % and 19 %, respectively) or red ovine freezing buffer (29 % and 24 %, respectively) were used. it is unlikely that sperm samples can be frozen without damaging any sperm. we therefore assume that some of the spermatozoa that were not moving when evaluated before thawing, were in a quiescent stage and not immotile because of damage. it is known that spermatozoa of bos taurus bulls (carr & acott 1984) as well as of rats, guinea pigs, hamsters and humans (turner & reich 1985) are kept in the epididymides in a quiescent state, even though they are fully capable of moving. the reason for this quiescence in bulls was suggested to be a ph-dependent factor present in cauda epididymal fluid (cef), which becomes inactivated as well as diluted during addition of seminal plasma to epididymal spermatozoa upon ejaculation (acott & carr 1984). the ability to immobilize spermatozoa temporarily has also been attributed to glycerolphosphocholin (gpc) and carnitin, which are both present in high concentrations in rat cef (turner, d’addario & howards 1978). in bulls the half-time of motility initiation upon dilution is given as being 2 to 5 min (acott & carr 1984). it is therefore possible that not all spermatozoa were released from their quiescent state when we first evaluated the motility. another possibility is that triladyl™ as well as red ovine freezing buffer contain substances that prolong the quiescent state of spermatozoa. in any case, it seems that some of the spermatozoa, which were recorded as immotile before freezing, were in fact in a quiescent state and only subsequently became motile owing to longer expo208 freezing of epididymal sperm from african buffalo (syncerus caffer) sure to the semen diluent as well as dilution of cef. since we assume it to be an advantage to freeze epididymal spermatozoa in the quiescent state because more energy is conserved by them, it is desirable to investigate this phenomenon further. it can be concluded from our results that equilibration times between 4 and 9 h do not influence postthaw semen qualities and no attention must therefore be paid to it. we further conclude that shorter equilibration times are detrimental. this knowledge is very valuable when freezing sperm of wild animal species in the field. furthermore, it was found that triladyl™ was superior to red ovine freezing buffer and andromed® when used as a freezing diluent for epididymal african buffalo sperm. although andromed® is a totally defined medium, it cannot be recommended, since the results obtained in this study concerning progressive motility, longevity and acrosomal integrity of the sperm were inferior to those when triladyl™ was used. acknowledgements the authors would like to thank imv france and minitüb germany for providing the buffers and extenders, respectively. references acott, t.s. & carr, d.w. 1984. inhibition of bovine spermatozoa by caudal epididymal fluid: ii. interaction of ph and a quiescence factor. biology of reproduction, 30:926–935. bartels, p., lambrechts, h., kidson, a. & friedmann, y. 1996. the potential of breeding disease-free african buffalo using assisted reproductive technology. proceedings of a symposium on the african buffalo as a game ranch animal, onderstepoort, south africa, 1996:75–78. bengis, r.g., kriek, n.p.j., keet, d.f., 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1956. the effect of glycerol equilibration time on the freezing of bovine spermatozoa in egg yolk sodium citrate and skim milk semen extenders. journal of dairy science, 39:1156–1160. 209 f.c. herold et al. potgieter, f.t., stoltsz, w.h., blouin, e.f. & roos, j.a. 1988. corridor disease in south africa: a review of the current status. journal of the south african veterinary association, 59:155–160. rathi, r., colenbrander, b., bevers, m.m. & gadella, b.m. 2001. evaluation of in vitro capacitation of stallion spermatozoa. biology of reproduction, 65:462–470. sirivaidyapong, s., bevers, m.m., gadella, b.m. & colenbrander, b. 2001. induction of the acrosome reaction in dog sperm cells is dependant on epididymal maturation: the generation of a functional progesterone receptor is involved. molecular reproduction and development, 58:451– 459. stewart, d.l. & o’hagan, c. 1972. the prediction of the fertility of bull semen from laboratory tests. seventh international congress on animal reproduction and artificial insemination, munich, 1972:1279–1283. turner, t.t., d’addario, d. & howards, s.s. 1978. further observations on the initiation of sperm motility. biology of reproduction, 19:1095–1101. turner, t.t. & reich, g.w. 1985. cauda epididymal sperm motility: a comparison among five species. biology of reproduction, 32:120–128. wiggin, h.b. & almquist, j.o. 1975a. effect of glycerol equilibration time and thawing rate upon acrosomal maintenance and motility of bull spermatozoa frozen in plastic straws. journal of animal science, 40:302–305. wiggin, h.b. & almquist, j.o. 1975b. combinations of glycerol percent, glycerol equilibration time, and thawing rate upon freezability of bull spermatozoa in plastic straws. journal of dairy science, 58:416–419. 210 freezing of epididymal sperm from african buffalo (syncerus caffer) abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) alaster samkange department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa department of production animal clinical studies, faculty of health sciences and veterinary medicine, university of namibia, windhoek, namibia simbarashe chitanga department of veterinary pre-clinical, faculty of health sciences and veterinary medicine, university of namibia, windhoek, namibia department of biomedical sciences, faculty of health sciences, university of zambia, lusaka, zambia school of life sciences, college of agriculture, engineering and sciences, university of kwazulu-natal, durban, south africa georgina n. tjipura-zaire directorate of veterinary services, windhoek, namibia vimanuka g. mutjavikua department of production animal clinical studies, faculty of health sciences and veterinary medicine, university of namibia, windhoek, namibia jan w. smith department of production animal clinical studies, faculty of health sciences and veterinary medicine, university of namibia, windhoek, namibia luis neves department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa centro de biotecnologia, universidade eduardo mondlane, maputo, mozambique tshepo matjila department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, pretoria, south africa citation samkange, a., chitanga, s., tjipura-zaire, g.n., mutjavikua, v.g., smith, j.w., neves, l. et al., 2023, ‘seroprevalence and associated risk factors of bovine neosporosis in the khomas region of namibia’, onderstepoort journal of veterinary research 90(1), a2077. https://doi.org/10.4102/ojvr.v90i1.2077 original research seroprevalence and associated risk factors of bovine neosporosis in the khomas region of namibia alaster samkange, simbarashe chitanga, georgina n. tjipura-zaire, vimanuka g. mutjavikua, jan w. smith, luis neves, tshepo matjila received: 19 sept. 2022; accepted: 23 jan. 2023; published: 05 apr. 2023 copyright: © 2023. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract neospora caninum is a coccidian parasite that occurs worldwide and is one of the most important causes of abortion, especially in cattle. however, no studies have been performed in namibia to determine the n. caninum status in livestock. therefore, this study aimed to determine the seroprevalence of n. caninum in cattle and the associated risk factors in the khomas region of namibia. a total of 736 sera were collected from cows in 32 farming establishments. these comprised 698 beef and 38 dairy cattle sera and were tested using a commercial enzyme-linked immunosorbent assay (elisa) kit. questionnaires were concurrently administered to determine possible risk factors associated with n. caninum seropositivity. a total of 42 sera were positive (all beef), giving an animal-level seroprevalence rate of 5.7%. eight of the 32 establishments had at least one positive animal, giving a herd-level seroprevalence of 25%. there was no significant association between seropositivity and the presence of dogs, jackals, history of abortions, farm size, number of cattle or average annual rainfall. the establishments with moderate to high numbers of feliformia were 9.8 times more likely to be seropositive to n. caninum than those with none to low levels of the former (p = 0.0245). the authors concluded that the seroprevalence level of n. caninum in the khomas region was relatively low compared with other parts of the world and that the role of feliformia in the epidemiology of bovine neosporosis needed to be further investigated. contribution: serological evidence of bovine neosporosis and the associated risk factors are reported in namibia for the first time. this study contributes to the scientific body of knowledge on n. caninum in africa, which is currently limited. keywords: seroprevalence; cows; risk factors; n. caninum; khomas; namibia. introduction neospora caninum is an obligate intracellular coccidian parasite of the apicomplexa phylum and toxoplasmatidae family, which occurs worldwide (dubey et al. 1988; gharekhani et al. 2021; goodswen, kennedy & ellis 2013). the parasite primarily infects dogs and cattle as well as all major domestic livestock species, companion animals, chickens, sparrows, wildlife and captive animals, including deer, rhinoceros, rodents, rabbits, coyotes, dingos, wolves and foxes (donahoe et al. 2015; fereig & nishikawa 2020; mcallister 2022). the canids are considered the definitive hosts, in which infection results in polyradiculoneuritis and polymyositis in young dogs, with dermatitis and neurological manifestations being characteristic in adult dogs (decôme et al. 2019; fereig & nishikawa 2020). neosporosis is of significant economic importance in livestock, particularly cattle, in which infection is characterised by abortions, stillbirths, the birth of weak neonates, congenital malformation, increased numbers of culled cows and decreased milk yield (gharekhani, yakhchali & berahmat 2020; kierbić et al. 2019), with associated annual losses running into hundreds of millions of united states dollars (demir, eşki & ütük 2020; reichel et al. 2013). infection in these intermediate hosts is through ingestion of food and water contaminated with sporulated oocyst (dubey, schares & ortega-mora 2007; mcallister 2022), with the subsequent vertical transmission in infected herds playing a more significant role (gharekhani et al. 2020; lefkaditis et al. 2020; sinnott et al. 2017). several risk factors have been identified to be associated with infection in livestock, and these include the presence and number of farm dogs (dubey & schares 2011), farm production systems and practices (bartels et al. 2006; dubey et al. 2007; ghalmi et al. 2012; otranto et al. 2003; pare et al. 1998), antibodies against other pathogens such as bovine viral diarrhoea (björkman et al. 2000), human population density (schares et al. 2004) and the region within countries (bartels et al. 2006). while n. caninum is presumed to occur worldwide (reichel, wahl & ellis 2020), there is wide variation in prevalence across countries and regions, with some countries not having any data. within the southern african region, evidence of bovine neosporosis has been reported in south africa and zimbabwe, with varied prevalence (adesiyun et al. 2020; jardine & last 1993, 1995; jardine & wells 1995; njiro et al. 2011). other hosts that have been reported to show evidence of infection in southern africa include dogs (jardine & dubey 1992), birds (lukášová et al. 2018) and wildlife (seltmann et al. 2020). despite the importance of the livestock sector in namibia and the possible impact that neospora infection can have on the productivity of this sector, there has been no study to determine the prevalence, distribution and potential risk factors associated with infection in commercial cattle production. therefore, the purpose of this study was to fill this knowledge gap by determining the seroprevalence of n. caninum infection in cows and the associated risk factors. materials and methods study population and setting the study area was namibia’s khomas region, located in the central part of the country (figure 1). namibia’s sub-tropical climate varies from arid to semi-arid, and it is the driest country in sub-saharan africa (mwazi & shamathe 2007). the country’s central highlands receive an annual rainfall of between 300 mm and 400 mm and have an altitude of up to 1900 m (kandiwa et al. 2017). the vegetation is predominantly shrub-veld and ambient temperatures range from 7 °c in winter to 33 °c in summer (kandiwa et al. 2019). the khomas region has about 556 farming establishments, with approximately 44 000 primarily commercial beef cattle (directorate of veterinary services 2018). in addition, there are a few resettlement farms and communal settlements. figure 1: map showing the 14 regions of namibia in relation to the khomas region, which lies in the central area of the country. the study animals were the cows in the khomas region of namibia. the females were targeted because vertical transmission is the most important in the epidemiology of n. caninum (de aquino diniz et al. 2019; dubey et al. 2007). in addition, wei and co-workers found that female cattle had a higher seroprevalence rate than males (wei et al. 2022). therefore, targeting females increased the probability of detecting positive animals. study design this was a cross-sectional study design in which 32 farming establishments in the khomas region of namibia were selected. these comprised 26 commercial beef farms, three communal beef herds, two dairy, and one resettlement farm. sampling and data collection sample size calculations were performed according to the methods by pfeiffer (2002). for farm selection, estimated herd-level and individual animal-level prevalence rates of 20% and 10%, respectively, were used (fereig et al. 2016; nasir et al. 2012). a multistage sampling strategy was used to select the farming establishments included in this study. the khomas region was first divided into clusters ranging from three to seven farming units. seven clusters were chosen using convenience sampling, especially targeting farms that had previously reported abortions. next, a stratified random sampling technique was used to select individual animals to be sampled at farm level, and a total of 736 cows were selected. a minimum of 10 cattle were sampled from each of the 32 farming establishments. a questionnaire was administered during the collection of serum samples to use that data to determine the possible risk factors of n. caninum in the khomas region of namibia. plain vacutainer® blood tubes and 20-gauge needles were used to collect blood from the coccygeal or jugular veins. sera were extracted by centrifugation at 6000 revolutions per minute (rpm) for 10 min, after which they were stored at −20 °c until testing. serological analysis indirect enzyme-linked immunosorbent assay (elisa) (idexx neospora x2®) (idexx laboratories, inc., maine 04092, united states [us]) was used to detect specific anti-n. caninum immunoglobulin g (igg) antibodies in the bovine sera according to the manufacturer’s instructions. the assays were duplicated, with absorbance values measured at 650 nm, with the sample to positive (s/p) ratio of 0.50 as the cut-off value (negative < 0.50 and positive > 0.50). data analysis the possible risk factors associated with n. caninum in sampled farming establishments were captured on questionnaires. the questionnaire and serology results were then captured in a microsoft excel 2013 spreadsheet. the captured data were analysed using descriptive statistics, chi-square test, odds ratios (ors) and multiple regression analysis at a 95% confidence level. regression analysis was used on quantitative data to analyse the relationship between the dependent variable (number of n. caninum seropositive cases per establishment) and the independent variables (the numbers of cattle and dogs per establishment, farm size and average annual rainfall). the chi-square test and ors were used to analyse the relationship between categorical data (the history of abortions, sightings of stray dogs, number of jackals, number of feliforma and rain scores over the previous three seasons) and the n. caninum status of each establishment. finally, descriptive statistics were used to calculate the seroprevalence rates. all the statistical analyses were carried out using microsoft excel 2013. results in this study, 736 cattle (698 beef and 38 dairy) were sampled from 32 farming establishments, 30 of which were beef herds, and the remaining two were dairy. eight of the 32 establishments had at least one positive animal, giving an overall herd-level prevalence of 25% (8/32), and they were widely distributed across the sampling sites (figure 2). an overall animal-level prevalence of 5.71% (42/736) was observed, which varied widely across the farming establishments. for example, in the eight seropositive establishments, animal-level seroprevalence rates ranged from 3.03% (1/33) to 80.95% (17/21) (table 1). the herd-level and animal-level seroprevalences for beef cattle only were 26.67% (8/30) and 6.02% (42/698), respectively. all the seropositive establishments were beef-producing commercial farms. the herd-level and animal-level seroprevalences for the two dairy farms sampled were 0% (0/2 & 0/38). in addition, all three communal establishments and one resettlement farm sampled (all beef) were seronegative. figure 2: google earth map showing the distribution of the cattle farming establishments sampled in the khomas region of namibia, which is delineated in yellow. the eight positive farms are marked red, and the negative ones in blue. table 1: a summary of the neospora caninum enzyme-linked immunosorbent assay test results for cattle sampled in the khomas region of namibia. the results of possible risk factors for n. caninum seropositivity in cattle investigated using a questionnaire are shown in table 2. the table summarises the results of the statistical analyses of the putative risk factors for seropositivity. table 2: a summary of the statistical analyses results performed on the possible risk factors associated with neospora caninum seropositivity. eighteen of the 32 establishments (56.25%) sampled had a history of abortions during the previous 5 years, ranging from low levels of less than 10% (score 1), moderate levels of 10% to 15% (score 2) to at least one overt abortion outbreak (score 3) in the same period (figure 3). eleven of the 18 farms that reported at least one incident of abortions in the previous 5 years reported at least one incident of moderate to high abortion levels (scores between 2 & 3). five of the eight farming establishments with at least one n. caninum seropositive animal had a history of previous abortions. however, this was not statistically significant on the chi-square test (p = 0.497). figure 3: distribution of abortion of scores among sampled establishments. the annual rainfall received per farming establishment averaged 344 mm per year and ranged from 224 mm to 600 mm annually. the average annual rainfall for 72% (23/32) of the establishments ranged between 301 mm and 400 mm per year, and 25% (8/32) of the establishments received 201 mm to 300 mm annually (figure 4). however, the average rainfall per establishment was not significantly associated with n. caninum seropositivity on multiple regression analysis (p = 0.143). figure 4: average annual rainfall distribution among the sampled farming establishments. the sighting of feliformia (primarily hyenas, cheetahs and leopards) at the farming establishments was significantly associated with n. caninum seropositivity among the cattle on the chi-square test and or analysis. the establishments with moderate to high numbers of feliformia on their properties were 9.8 times more likely (or = 9.8; 95% confidence interval [ci]: 0.061 to 4.504) to be seropositive to n. caninum than those with none to low levels (p = 0.0245) (figure 5). figure 5: feliformia score distribution among sampled establishments. except for one, all the other 31 establishments had at least one dog, ranging in number from 1 to 41, with the latter being at a resettlement farm with many livestock owners and households (figure 6). the average number of dogs per establishment sampled was six. the presence of dogs in the establishments was not significantly associated with seropositivity on multiple regression analysis (p = 0.433). on chi-square analysis, there was also no significant association between n. caninum seropositivity and sightings of stray dogs in sampled establishments (p = 0.838). figure 6: distribution of dogs in sampled farming establishments. all the farming establishments reported observing high numbers of jackals on their properties (score 3); this was, however, not significantly associated with n. caninum seropositivity on chi-square analysis (p = 0.854). the average number of cattle per sampled establishment was 301, ranging from 13 to 1205 animals. the modal range was between 201 and 400 cattle (figure 7). on multiple regression analysis, there was no significant association between cattle numbers per establishment and seropositivity (p = 0.946). there was also no significant association between farm size and seropositivity on multiple regression analysis (p = 0.713). figure 7: distribution of the numbers of cattle in sampled cattle herds. discussion this study reports an overall animal-level seroprevalence of 5.7%, and to the best of the authors’ knowledge, this is the first study on n. caninum seroprevalence in cattle reported from namibia. this observed prevalence is comparable to that reported within the region in south africa, where 5.17% seroprevalence was reported in beef cattle (chisi et al. 2013). however, unlike in the latter study, where all the dairy farms sampled were positive (chisi et al. 2013), the two small-scale dairy herds sampled in this study were both negative. these results could be because the dairy industry in namibia is still in its infancy, with an estimated herd of only 3000 cattle (bieldt 2005). in addition, the largest dairy farm in the country was not sampled because it fell outside of the study area. a similarly low-seroprevalence rate of 3.8% was also detected in one study in iran, where it was attributed to a combination of warm and dry climate followed by cold and dry conditions (noori et al. 2019). these climatic conditions were deemed unfavourable for the survival of n. caninum oocysts in the environment (noori et al. 2019). on the other hand, humid conditions coupled with mild to warm environmental temperatures are ideal for the sporulation and survival of oocysts (dubey et al. 2007) and have also been associated with higher incidences of n. caninum abortions (wouda, bartels & moen 1999). northwest china has a generally hot and dry climate with very little rainfall in the summer months compared with the other parts of the country, and the area has been found to have the lowest n. caninum seroprevalence rate of 9.4% in the country (wei et al. 2022). these scenarios perfectly mirror the semi-arid conditions of the khomas region of namibia, where this study was conducted. a study on cattle in northern tanzania (arusha region) determined a seroprevalence rate of 21.5% (semango et al. 2019), which is much higher than the 5.7% found in this study. given the semi-arid nature of namibia’s climate, it could affect the survival and sporulation of the n. caninum oocysts in the environment (dubey et al. 2007; noori et al. 2019) and therefore reduce transmission. on the other hand, northern tanzania is more humid and has higher average annual rainfall (kimaro, mor & toribio. 2018) compared with namibia, which provides a more conducive environment for the sporulation of n. caninum oocysts. the same argument could also explain the relatively low prevalence rate found in namibia compared with other countries such as the northern part of algeria (ghalmi et al. 2012), argentina (moore et al. 2003), china (qian et al. 2017), colombia (llano et al. 2018), north and central america, asia, europe and india (hebbar et al. 2022). the low animal-level n. caninum seroprevalence rate in this study could also be attributed to the fact that 95% (698/736) of the samples tested were from beef cattle compared with only 5% (38/736) from dairy cattle. beef cattle are less susceptible to n. caninum than dairy cattle, and therefore they tend to have lower seroprevalence rates (fort et al. 2015; gharekhani et al. 2020; haddad, dohoo & vanleeuwen 2005; quintanilla-gozalo et al. 1999; ribeiro et al. 2019). communal, dairy and resettlement establishments were all negative, possibly because the number of herds sampled from this category was very small; a larger sample size might have given a different result. furthermore, feliforms are less likely to be found in this category of establishments because of higher human population densities. however, the dog population is also expected to be higher with the increased human population, especially in communal setups (butler & bingham 2000). this study found no significant association between the number of dogs at the farming establishments and n. caninum seropositivity. similar findings were also made in studies in tanzania (mathew 2017; semango et al. 2019). however, as dogs are the definitive hosts of n. caninum (goodswen et al. 2013; mcallister et al. 1998), these findings are somewhat surprising. other studies have indeed confirmed that the presence of farm dogs increases the risk of n. caninum infection in cattle (abdeltif et al. 2022; dubey et al. 2007; fávero et al. 2017; robbe et al. 2016) and goats (rodrigues et al. 2020), most likely through faecal contamination of pastures and open water sources with oocysts. the lack of significant association with the number of dogs found in the present study might have been caused by the fact that most of the farm dogs were confined to the homesteads and therefore did not have carte blanche access to livestock pastures. this, in turn, meant that the risk of pasture contamination, regardless of the number of dogs at the farming establishment, was markedly reduced. in their study, guimaraes and co-workers found a positive correlation in n. caninum seroprevalence between cattle and dogs (guimaraes et al. 2004). therefore, a low seroprevalence found in cows in this study could reflect the same scenario in farm dogs. however, such an inference can only be speculative because our study did not test the farm dogs. the n. caninum positive status was significantly associated (p < 0.05) with the presence of feliformia (brown hyenas, leopards and cheetahs) but not black-backed jackals, despite most farmers reporting many sightings of jackals on their properties. domestic dogs (canis familiaris) and black-backed jackals (canis mesomelas) look physically similar; therefore, the possibility of mistaking these two species in places where the presence of feliformia was reported cannot be entirely disregarded. in a recent namibian study, brown hyenas (hyaena brunnea) and black-backed jackals (canis mesomelas) were found to be seropositive to n. caninum (seltmann et al. 2020); however, the significance of these findings in light of the results of this study need to be further investigated. furthermore, workers in tanzania have also suggested the possible involvement of wildlife in the epidemiology of n. caninum after finding no association been dog ownership and cattle seropositivity (semango et al. 2019). in one study, self-rearing of replacement heifers was associated with an increased risk of bovine neosporosis (otranto et al. 2003). however, this study found no such association, despite all the establishments indicating that they self-reared replacement heifers. this could be explained by the low n. caninum seroprevalence rate in the khomas region, which reduces the risk of vertical parasite transmission. this study found no significant association between abortion history and n. caninum seropositivity. one study in northeast algeria made similar findings, and the authors concluded that those cows were resistant to n. caninum abortions (abdeltif et al. 2022). however, further investigations would be needed, given the extreme biological importance of the latter findings. the same scenario could also be at play in the khomas region of namibia, especially considering that significant variations in seropositivity have been found between countries, within countries, regions in the same country, and breeds (dubey et al. 2007). another study in southeastern iran found no association between n. caninum seropositivity and a history of abortions (noori et al. 2019). however, other studies have demonstrated a significant association between n. caninum seropositivity and a history of abortions (ghalmi et al. 2012; llano et al. 2018; moore et al. 2009). as noticed earlier, this discrepancy could be because of variations between countries or regions. conclusion the findings in this study were generally in agreement with some studies in other parts of the world. however, the seroprevalence level of n. caninum in the khomas region is much lower than in most other parts of the world. furthermore, the role of feliformia in the epidemiology of bovine neosporosis needs to be further investigated. acknowledgements the authors would like to thank the meat board of namibia for funding the purchase of the elisa kits used in this study and the directorate of veterinary services in namibia for availing their laboratory facilities and technical staff. also, they would like to sincerely thank dr vonai charamba and dr alec bishi for guiding the statistical analysis. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.s., s.c., l.n. and t.m. were responsible for the conception and design of the study. a.s., g.n.t.-z., j.w.s. and v.g.m. did the sample collection and processing. a.s. did the data analysis and writing of the first draft of the manuscript. all authors participated in the revisions of the manuscript and read and approved the final version of the manuscript. ethical considerations this study was approved by the university of namibia ethics committee and the university of pretoria’s research ethics committees (nec0007 & rec087-21). funding information the meat board of namibia funded the purchase of the idexx elisa kits used in this 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caninum-associated abortion storms in dairy herds in the netherlands (1995 to1997)’, theriogenology 52(2), 233–245. https://doi.org/10.1016/s0093-691x(99)00125-9 salih_303-307.qxd introduction it has been shown that hyalomma anatolicum anatolicum is the principal vector of theileria annulata, the cause of tropical theileriosis in the sudan, although other hyalomma ticks are also known to be able to transmit the disease (um el hassan, jongejan & morzaria 1983). the natural infection rate in the salivary glands of hyalomma ticks is an important parameter in the study of the epidemiology of tropical theileriosis in cattle (sangwan, chaudhri, sangwan & gupta 1994). moreover, infection rates can be a useful indicator of transmission of theileria and have been used as an index in combination with routine serological and parasitological surveys in cattle (young 1981). different methods have been used to study infection rates of theileria species in salivary glands of ticks (walker, susan, meckellar, bell & brown 1979). in general, histological and histochemical methods for detecting the infection have the disadvantage that it is not possible to determine with certainty the parasite species infecting the tick (melrose, brown & sharma 1980). this has been overcome by using pcr targeting a t. annulata-specific gene such as the one encoding the 30-kda major merozoite surface antigen (tams-1) (d’oliveira, van der weide, jacquiet & jongejan 1997). in the sudan, walker, latif, morzaria & jongejan (1983) studied the natural infection rate for theileria in ticks from shambat (khartoum state) and nishishiba (central sudan). they found that 38 % and 303 onderstepoort journal of veterinary research, 72:303–307 (2005) natural infection rates and transmission of theileria annulata by hyalomma anatolicum anatolicum ticks in the sudan d.a. salih1*, o.e. sharieff1^, a.g. lazarus2, s.m. hassan3 and a.m. el hussein1 abstract salih, d.a., sharieff, o.e., lazarus, a.g., hassan, s.m. & el hussein, a.m. 2005. natural infection rates and transmission of theileria annulata by hyalomma anatolicum anatolicum ticks in the sudan. onderstepoort journal of veterinary research, 303–307 hyalomma anatolicum anatolicum nymphs were collected from two localities in the sudan: eddamer in northern sudan and wad-medani in central sudan. they were allowed to moult to adult ticks, which were assessed for theileria infection in their salivary glands using feulgen stain. at eddamer, 49.6 % of 123 ticks examined were infected with theileria and the mean intensity of infection was 1.3 (i.e. the number of infected acini/number of infected ticks). at wad-medani, 8.6 % of 162 ticks were infected and the mean intensity of infection was 7.9. the prevalence of infection was higher in female than in male ticks at both localities. when adult h. a. anatolicum were applied onto two susceptible calves, both animals developed the severe form of theileriosis. keywords: hyalomma anatolicum, infection rates, sudan, theileria annulata, transmission * author to whom correspondence is to be directed. e-mail: diaeldin2000@hotmail.com 1 central veterinary research laboratories, p.o. box 8067, al amarat, khartoum, sudan 2 wildlife conservation police, p. o. box 77, juba, sudan 3 faculty of veterinary medicine, university of khartoum, p.o. box 32, khartoum north ^ deceased accepted for publication 20 june 2005—editor 86 % of them were infected with theileria with a mean of 37 and 19.5 parasite masses per tick in shambat and nishishiba, respectively. similarly, jongejan, morzaria, mustafa & latif (1983) determined the infection rates of hyalomma marginatum rufipes ticks fed on calves experimentally infected with t. annulata and showed that the infection rate can reach 100 %. the purpose of this study was to assess the natural infection rates of hyalomma a. anatolicum with t. annulata at two localities in the sudan, and to attempt to transmit the infection experimentally to calves. materials and methods collection and maintenance of ticks fully engorged nymphs were collected at two localities, eddamer (17°58´n, 34°96´e), northern sudan and wad-medani (14°4´n, 33°51´e), central sudan. particular attention was paid to wall crevices, masses of cobwebs and dried cow-pats which were likely to provide sheltered moulting sites. the ticks were maintained in the laboratory in an incubator at 28 °c and 85 % relative humidity for moulting. after moulting, they were identified according to the descriptions provided by hoogstraal (1956), hoogstraal & kaiser (1959) and okello-onen, hassan & essuman (1999). only adult h. a. anatolicum were included in this study. the ticks were maintained in flat-bottomed plastic tubes, each of which was labelled with the date of collection and locality. feeding of ticks on rabbits and dissection in order to allow the ticks to harden their cuticles after moulting, they were left in the incubator for a further 10 days. thereafter, they were applied onto rabbits (crosses of new zealand x local) using ear bags according to the methods described by bailey (1960). the feeding on rabbits was carried out in order to stimulate the maturation of theileria sporozoites (cunningham, brown, burridge & purnell 1973). a total of 180 ticks from eddamer and 240 from wad-medani were placed on six rabbits. they comprised an equal number of male and female ticks. the day of application of ticks was designated as day 0 of feeding. on day 3, the attached ticks were gently removed using a pair of forceps and immediately dissected. dissection was carried out under a stereoscopic microscope. both salivary glands of each tick were gently freed from the posterior tracheas, placed on a clean microscope slide with a drop of normal saline. they were then fixed in absolute methanol for 10 min and air dried. the slides were labelled to indicate the locality of origin and sex of the tick. feulgen stain the feulgen stain used to stain the salivary gland smears was prepared as has been described previously (anonymous 1983). the smears were first hydrolyzed for 1 h in 5n hcl and subsequently stained in the feulgen stain for 1 h. thereafter, they were gently washed with distilled water, heat dried at 60 °c overnight, and then mounted with depex under a coverslip. examination of the slides the slides were examined under the x40 objective lens of a standard microscope. the parasitized acinar cells were distinguished by their large hyperplastic nuclei, which stained intensely red. acini in which there were infected cells containing densely stained sporozoites and sporoblasts that pushed the nuclei to one side were counted. the infection rate was expressed as follows: infection rate (%) = number of infected ticks total number of ticks dissected x 100 in addition, two other infection parameters were determined according to the method of sangwan et al. (1994). these concerned the mean intensity of infection (number of infected acini / number of infected ticks) and the mean abundance of infection (number of infected acini / number of ticks examined). transmission of t. annulata by h. a. anatolicum for this purpose, ticks collected as fully engorged nymphs from eddamer and wad-medani and from the same batch that had been subjected to the infection rate assessments as set out above were used. after moulting to adults, transmission of t. annulata was carried out by applying 14 females and 17 males from eddamer or wad-medani to both ears of two calves number 382 and number 387, respectively. sera from the calves were tested before application of the tick for t. annulata antibodies using the indirect fluorescent antibody test (ifa)(salih 2003). after the ticks had been applied both calves were monitored on a daily basis using clinical and parasitological parameters. 304 transmission of theileria annulata by hyalomma anatolicum anatolicum ticks in sudan data analysis the data on the infection rates were subjected to chi-square in order to test the variation between males and females, and the localities. results theileria infection rates in ticks the number of h. a. anatolicum examined for theileria parasites infections and the number of theileria parasite masses found in their salivary glands at the two localities are shown in table 1. a total of 123 and 162 h. a. anatolicum collected from eddamer and wad-medani, respectively, were dissected to assess the infection rate in their salivary glands. forty-two females (61.8 %) and 19 males (34.5 %) from eddamer were infected, whereas from wadmedani 13 females (15.8 %) and 1 male (1.25 %) were positive. the prevalence of infection was higher in female than male ticks at both localities (table 1). the mean number of infected acini was significantly higher in females than in males (p < 0.05). in addition, the intensity and abundance of infection were also significantly higher in females than in males (p < 0.01). transmission of t. annulata by h. a. anatolicum prior to tick application both calves were shown to be negative for t. annulata antibodies using the ifa test. theileria annulata was successfully transmitted to both calves (table 2). the period that elapsed for the onset of fever after transmission by ticks from wad-medani and eddamer was 8 and 16 days, respectively. calf 387 died of tropical theileriosis, whereas calf 382 recovered from the infection without treatment. macroschizonts and piroplasms appeared earlier in calf 387 than in calf 382 (table 2). discussion infection rates of theileria in ticks are considered a useful indicator for the level of theileria challenge in combination with data on theileria infection rates in cattle (young 1981). the infection rate in h. a. anatolicum (49.6 %) collected in eddamer indicated that cattle are subjected to a high challenge with t. annulata sporozoites and that ticks feeding as nymphs have a high chance of becoming infected with the parasite. in comparison, at wad-medani a 305 d.a. salih et al. table 1 infection rate, intensity and abundance of infection of theileria in salivary glands of male and female h. a. anatolicum ticks collected as nymphs from eddamer and wad-medani localities ticks no. no. positive mean no. of mean mean intensity examined ( %) infected acini abundance of of infection infection eddamer females 68 42 (61.8) 81.0 1.2 1.9 males 55 19 (34.5) 12.0 0.2 0.6 both sexes 123 61 (49.6) 46.5 0.7 1.3 wad-medani females 82 13 (15.8) 54.0 2.0 13.7 males 80 1 (1.25) 2.0 0.7 2.0 both sexes 162 14 (8.6) 28.0 1.4 7.9 table 2 transmission of theileria annulata to experimental calves by h. a. anatolicum adults collected as nymphs from the field prepatent periods animal no. locality challenge days to duration of by adult fever fever rectal schizont piroplasm days to ticks temp. death (days) (°c) (days) (days) 382 eddamer 14 females 16 3 40.8 18 19 recovered 17 males 387 wad14 females 8 11 40.4 11 16 23 medani 17 males much lower infection rate (8.6 %) was observed, which may indicate that cattle are subjected to a lower challenge with t. annulata in this area and that ticks feeding as nymphs have a lower chance of becoming infected. the fact that lower numbers of ticks were infected at wad-medani, but with higher numbers of parasites per tick may reflect an endemically unstable situation compared with eddamer where most ticks were infected but with lower numbers of parasites per tick. in a previous study in the sudan, h. a. anatolicum was shown to have high natural infection rates with theileria, ranging between 38 % and 86 % (walker et al. 1983). infection rates of h. a. anatolicum with t. annulata were subsequently estimated, using the same method as used in this study, to be 80 % in the ticks collected from the field (kuku area, khartoum) (el-imam 1999) and 96 % in ticks fed experimentally on infected calves (bakheit 1998). the high infection rate in ticks collected from eddamer may be influenced by the animal husbandry system used. this consist of small enclosures in which susceptible crossbred (zebu x friesian) cattle are maintained, and which creates an ideal microhabitat for h. a. anatolicum ticks. hence, opportunities for animals to become infected and develop immunity to t. annulata at a younger age are greater than at wadmedani where semi-intensive cattle raising is practiced. it is interesting, however, to note that earlier investigations by walker et al. (1983) indicated very high (86 %) infection rates in nishishiba (a village nearby wad-medani). at the time of that investigation, however, the drought which prevailed at the time of the current investigations had not yet started and there was no influx of animals carrying ticks from h. a. anatolicum infested areas for grazing in the el gezira agricultural scheme, which is situated in the wad-medani area. this might have resulted in the interbreeding of different tick populations and the development of more heterogenous and resistant offspring, in contrast to a more a stable population in the eddamer area. this hypothesis may be supported by the fact that h. a. anatolicum has now established in the blue nile province (south of wadmedani), an area where, in the early 1980s, this tick was not present (salih, hassan, el hussein & jongejan 2004). the finding that the intensity of infection was higher in female than in male ticks is in agreement with the fact that the total number of type iii acini, which are arranged distally to the main salivary gland ducts, is higher in females than in males (ochanda, young, welles, medley & perry 1996). male ticks detach irregularly to re-attach next to feeding females in order to mate, which may contribute to a lower number of sporozoites in the male ticks. female ticks may thus play a more important role in transmission than male ticks (ochanda et al. 1996). moreover, the development of theileria parasites in vector ticks depends on several extrinsic and intrinsic factors which remain to be elucidated as stated by sangwan et al. (1989, cited by sayin, karaer, dincer, cakmak, inca, yukari, eren, vatansever, nalbantoglu & melrose 2003). it is known that h. a. anatolicum can also transmit other theileria spp. such as theileria lestoquardi (hooshmand-rad & hawa 1973) and theileria equi (schein, rehbein, voigt & zweygarth 1981). the results of the transmission experiment show that h. a. anatolicum collected from the field are infected with t. annulata and caused tropical theileriosis in susceptible calves, confirming earlier observations made by um el hassan et al. (1983). since histological staining methods are insufficient to differentiate between theileria species, in our ongoing studies larger number of ticks from a wider survey area are being examined by the pcr-based methods of d’oliveira et al. (1997). in conclusion, this study confirms the importance of h. a. anatolicum ticks as vectors of t. annulata and the need to protect susceptible animals against tropical theileriosis possibly through vaccination with attenuated cell culture vaccines such as the one currently under development in the sudan. acknowledgements this research was partially supported by the international atomic energy agency (iaea) through technical co-operation project no. sud/05/027, entitled “control of ticks and tick-borne diseases”. we thank prof. f. jongejan, utrecht university for critically reading the manuscript and for his valuable suggestions and comments. this work is published by kind permission of the director-general, animal resources research corporation. references anonymous 1983. ticks and tick-borne diseases in eastern and southern africa. zimbabwe. studies on theileriosis and economics of tick control. manual of laboratory technique. field document no. 3. (gcp/zim/013/den). 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latif, a.a., morzaria, s.p. & jongejan, f. 1983. natural infection rates of hyalomma anatolicum anatolicum with theileria in sudan. research in veterinary science, 35:87–89. young, a.s. 1981. the epidemiology of theileriosis in east africa, in advances in the control of theileriosis, edited by a.d. irvin, m.p. cunningham & a.s. young. the hague: martinus nijhoff. 307 d.a. salih et al. article information author: kim a. kayunze1 affiliation: 1development studies institute, sokoine university of agriculture, tanzania correspondence to: kim kayunze postal address: po box 3024, morogoro, tanzania how to cite this poster: kayunze, k.a., 2012, ‘impact of hiv and aids on food security in rufiji district, tanzania’, onderstepoort journal of veterinary research 79(2), art. #482, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.482 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. impact of hiv and aids on food security in rufiji district, tanzania in this poster... open access relatively high prevalence of hiv and aids and food insecurity in rufiji district whilst the linkage between the two problems was not known was the basis of this study. data were collected amongst 225 households between november 2005 and october 2006 through participatory rural appraisal (pra), household income and expenditure survey (hies) and structured interviews. binary logistic regression was used for analysis in which case the dependent variable was food security in terms of food insecure (0) and food secure (1) based on kilocalories consumed per adult equivalent per day. the independent variables included having been affected by hiv and aids in terms of not affected (0) and affected (1). the results reveal that the odds for households affected by hiv and aids to be food secure were 0.705 times as high as the odds for households not affected by hiv and aids to be food secure. this means that households affected by hiv and aids were less likely to be food secure as opposed to those not affected by hiv and aids. the b statistic for having been affected by hiv and aids was negative (b = -0.350) meaning that being affected by hiv and aids had negative impact on food security. however, the wald statistic which shows the magnitude of impact was small (0.251) and not significant (p = 0.617). this shows that hiv and aids had little impact on food security. based on these findings, it is concluded that although being affected by hiv and aids has negative impact on food security, it does not automatically make households food insecure, especially in a short run, and that some non-hiv and aids factors like high dependency ratio and low ability to buy food have bigger negative impact than that of hiv and aids on food security. on the basis of the conclusion, it is recommended that efforts to improve food security amongst households affected by hiv and aids should consider both hiv and aids and non-hiv and aids factors. petney_313-318.qxd introduction several surveys on the ixodid ticks infesting a variety of rodent species have been conducted in south africa (rechav 1982; horak, sheppey, knight & beuthin 1986; howell, petney & horak 1989; horak, fourie, novellie & williams 1991; fourie, horak & van den heever 1992; braack, horak, jordaan, segerman & louw 1996; horak & boomker 1998; horak & cohen 2001). some of these rodents are significant, if not preferred, hosts of the immature stages of several tick species of which the adults are important parasites of domestic animals (norval & mason 1981; rechav 1982; norval 1984; braack et al. 1996). despite their small size, the potential of rodents for rapid population growth and for maintaining high population levels can contribute significantly to the dynamics of tick species that use them as hosts (norval 1979; horak, spickett & braack 2000). in order to assess the significance of tick/rodent host relationships in the epidemiology of tick-borne 313 onderstepoort journal of veterinary research, 71:313–318 (2004) striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks in the eastern cape province t.n. petney1, i.g. horak2*, d.j. howell3 and s. meyer2** abstract petney, t.n., horak, i.g., howell, d.j. & meyer, s. 2004. striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks in the eastern cape province. onderstepoort journal of veterinary research, 71:313–318 striped mice, rhabdomys pumilio, were trapped over a period of 17 months in the thomas baines nature reserve, and placed in cages, over water, until all the ticks they harboured had detached. the mice were then returned to the reserve. four ixodid tick species were recovered from the mice of which the larvae and nymphs of rhipicephalus follis and rhipicephalus simus were the most numerous. most larvae of r. follis detached from mice trapped from march to july, and most nymphs in march and from june to september. most larvae of r. simus detached from mice trapped from december to march, and most nymphs from january to march and during may and june. seven ixodid tick species were collected from striped mice, house rats, rattus rattus, vlei rats, otomys spp. and praomys sp. captured in the vicinity of human dwellings or animal holding facilities in the grahamstown district. the striped mice captured in the thomas baines reserve harboured considerably larger numbers of ticks than any of the rodent species in the more urbanized localities. keywords: eastern cape province, ixodid ticks, rhabdomys pumilio, rhipicephalus follis, rhipicephalus simus, rodents, seasonality, striped mice * author to whom correspondence is to be directed ** present address: university of pretoria biomedical research centre, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa 1 tick research unit, rhodes, unversity, grahamstown, 6140 south africa. present address: school of pharmaceutical, molecular and biomedical sciences, university of south australia, gpo box 2471, adelaide sa 5001, australia 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: ivan.horak@up.ac.za 3 department of agriculture and water supply, uitenhage, 6320 south africa accepted for publication 30 april 2004—editor diseases in south africa, it is essential to know which tick species infest which rodent species and what levels the intensity of infestation can reach. in a previous paper (howell et al. 1989), we suggested that striped mice, rhabdomys pumilio, were of no significance in the epidemiology of heartwater, caused by ehrlichia ruminantium, in domestic ruminants, because they did not represent significant hosts for the immature stages of amblyomma hebraeum, the principal vector of this organism. in this contribution we wish to present data on other tick species that infested the same striped mice, and on the tick burdens of striped mice and other murid rodents in more urbanized habitats. materials and methods the methods employed to collect ticks from the striped mice captured in the thomas baines nature reserve, eastern cape province (33°23’ s, 26°28’ e) have been described in detail by howell et al. (1989) and will be repeated only briefly here. except during september 1987, when no mice were trapped, striped mice were trapped each month from july 1987 to november 1988. these mice were kept in cages over water and once all their ticks had detached the mice were released in the vicinity of the sites at which they had been trapped. the ticks were collected from the water, dried and allowed to moult. when moulting was judged to be complete all the ticks that had moulted and those that had failed to moult were identified and counted. the counts of those that had moulted were converted to the previous life stage and added to those of the larvae or of the nymphs that had detached from the mice, but that had failed to moult. murid rodents were also trapped around the stables and dwellings at the field station of the tick research unit, rhodes university, around an animal shelter situated in a semi-rural environment close to the field station, and around the outbuildings and homestead of the small holding “faraway”, all within a 3 to 10 km radius of the centre of the city of grahamstown (33°19’ s, 26°32’ e), eastern cape province. these rodents were killed and processed for the recovery of ticks as described by horak et al. (1986). results and discussion four ixodid tick species were recovered from the mice trapped in the thomas baines nature reserve (table 1). of these the larvae and nymphs of rhipicephalus follis and rhipicephalus simus were the most numerous and the most prevalent. four of the five a. hebraeum larvae and the single nymph recovered from the mice failed to moult to nymphs and to an adult respectively. howell et al. (1989) have discussed the implications of this failure, coupled with the small number of ticks of this species collected from the mice, in relation to the epidemiology of heartwater, in detail. twenty-four of the 36 larvae, and 11 of the 13 haemaphysalis leachi nymphs recovered from the mice also failed to moult to the next life stage. most r. follis larvae detached from mice that were trapped from march to july, and most nymphs from those trapped in march and from june to september (fig. 1). of these 1 806 larvae and 512 nymphs failed to moult. most larvae of r. simus detached from mice trapped from december to march, and most nymphs from those trapped from january to march and during may and june (fig. 2). of these 803 larvae and 172 nymphs did not moult. the larvae and nymphs of r. follis and r. simus are very similar in appearance (walker, keirans & horak 2000) and although we attempted to separate them, it was particularly difficult to do so for the engorged larvae and nymphs that had died before moulting. accordingly we cannot vouch for the absolute accu314 striped mice as hosts for immature ixodid ticks in eastern cape province table 1 ixodid ticks in 172 collections from striped mice in the thomas baines nature reserve number of ticks recovered no. of tick species larvae nymphs total collections amblyomma hebraeum 5 1 6 6 haemaphysalis leachi 36 13 49 18 rhipicephalus follis 13 313 704 14 017 162 rhipicephalus simus 2 206 321 2 527 116 total 15 560 1 039 16 599 169 table 2 ixodid ticks collected from 14 rhabdomys pumilio in the grahamstown district total number of ticks no. of rodents tick species larvae nymphs total infested haemaphysalis leachi 42 11 53 6 ixodes sp. 6 0 6 3 rhipicephalus (boophilus) sp. 1 0 1 1 rhipicephalus follis 13 3 16 3 rhipicephalus simus 37 0 37 6 rhipicephalus simus group 8 0 8 1 total 107 14 121 10 racy of the numbers that we have recorded for these species. ten of the 14 r. pumilio examined in the grahamstown district were infested with ticks, and five species, of which h. leachi and r. simus were the most numerous and prevalent, were recovered from them (table 2). forty of the 73 otomys spp., five of the 14 praomys sp., and five of the 13 rattus rattus examined in the grahamstown district were infested and a total of six species were recovered, of which h. leachi was amongst the most numerous and prevalent (tables 3–5). general the importance of the availability of rodents for the immature stages of tick species preferring these small mammals as hosts, on the subsequent numbers of adults of the particular species, has been alluded to by norval (1979) and demonstrated by horak et al. (2000). the latter authors recorded a marked increase in the numbers of adult r. simus and rhipicephalus turanicus, both of which use rodents as preferred hosts for their immature stages, on the vegetation of a landscape zone in the kruger national park approximately 8 months after a rodent explosion. on the other hand, no such increase in the numbers of adult rhipicephalus appendiculatus, of which the immature stages feed on antelopes or hares, was noted. the most striking differences between the tick burdens of the mice in the reserve and those of the rodents around human structures in the present study were the intensity of infestation and its prevalence and the species composition of the tick populations. the mean burdens of the various rodent 315 t.n. petney et al. � � � � �� � �� � �� � � � � ��� ��� ��� ��� ��� �� � ����������� �� ��� �� �� �� � � � � � � � � �� � �� � �� � � � � � ������ �� ������ ���� ���� � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � �� � �� � � � � � �� �� �� �� �� �� �� �� �� � � � � � �� � �� � �� � � � � ������ ���� ���� � � � � � � � � � � � � � � � � � �� ������ ����������� � �� fig. 1 the seasonal occurrence of the immature stages of rhipicephalus follis on striped mice in the thomas baines nature reserve, eastern cape province fig. 2 the seasonal occurrence of the immature stages of rhipicephalus simus on striped mice in the thomas baines nature reserve, eastern cape province species examined close to grahamstown varied between four ticks on infested praomys sp. and 12 on infested r. pumilio, compared to 98 on infested mice in the thomas baines reserve. amblyomma hebraeum the significance of the six immature a. hebraeum recovered from the mice in the reserve compared to the abundance of these stages on the large bovids and the vegetation at the same locality has been discussed in an earlier publication (howell et al. 1989). the single larva and nymph collected from the otomys spp. and the praomys sp. respectively examined in the grahamstown district were not engorging and appeared to be dying, indicating an unsatisfactory host/parasite relationship, further emphasising the unsuitability of murid rodents as hosts for the immature stages of this tick. haemaphysalis leachi the immature stages of h. leachi, of which the adults are specific parasites of domestic dogs and cats and the larger wild felids (norval 1984; horak jacot guillarmod, moolman & de vos 1987; horak, braack, fourie & walker 2000), were proportionally most numerous on the rodents examined close to grahamstown. this is not surprising as workers at the field station and the owner of “faraway” kept domestic dogs as pets, and dogs also accounted for a large proportion of the animals housed at the animal shelter. in a survey of the ticks infesting dogs on “faraway” and on a neighbouring property con316 striped mice as hosts for immature ixodid ticks in eastern cape province table 3 ticks collected from 73 otomys spp. in the gahamstown district number of ticks recovered no. of rodents tick species larvae nymphs females total infested amblyomma hebraeum 1 0 0 1 1 haemaphysalis leachi 48 9 0 57 15 ixodes alluaudi 0 0 2 2 2 ixodes bakeri 0 2 4 6 4 ixodes spp. 11 0 0 11 6 rhipicephalus follis 51 8 1 60 12 rhipicephalus simus 33 18 0 51 18 rhipicephalus simus group 4 27 0 31 3 total 148 64 6 218 40 table 4 ticks collected from 14 praomys sp. in the gahamstown district total number of ticks no. of rodents tick species larvae nymphs total infested amblyomma hebraeum 0 1 1 1 haemaphysalis leachi 10 0 10 3 ixodes bakeri 0 1 1 1 ixodes sp. 1 0 1 1 total 11 2 13 5 table 5 ixodid ticks collected from 13 rattus rattus in the grahamstown district total number of ticks no. of rodents tick species larvae nymphs total infested haemaphysalis leachi 68 2 70 5 ixodes sp. 1 0 1 1 rhipicephalus follis 0 1 1 1 total 69 3 72 5 ducted between august 1983 and july 1986, a total of 6 146 adult h. leachi were collected and comprised 71.3 % of all ticks recovered from the dogs (horak et al. 1987). with this number of adult ticks on the dogs at “faraway” higher burdens could have been expected on the rodents examined there. however, the immature stages do not feed exclusively on rodents, but also on dogs (horak et al. 1987; jacobs, fourie & horak 2004), and this could be a reason for the smaller burdens. the h. leachi recovered from the striped mice in the reserve were probably the progeny of adult ticks feeding on wild carnivores there. ixodes spp. all stages of development of ixodes alluaudi and ixodes bakeri infest shrews and rodents, with the latter tick showing a preference for otomys spp. (walker 1991). the unidentified ixodes larvae collected from some of the rodents probably belonged to one or both of these species. rhipicephalus (boophilus) sp. the single larva collected from a striped mouse at the field station near grahamstown is a “ straggler” originating from ticks on cattle kept on the premises. rhipicephalus follis eland, taurotragus oryx, examined in the thomas baines nature reserve during 1982 and 1984, and an african buffalo, syncerus caffer, examined during 1985 harboured adult r. follis. it is the ticks on these hosts, and probably also on burchell’s zebras, equus burchelli, in the reserve, which gave rise to the large numbers of immature ticks recovered from the striped mice at this locality. large numbers of adult r. follis have also been recorded on eland in the mountain zebra national park near cradock in the eastern cape province (horak et al. 1991) and the greatest intensity of infestation appeared to occur from august to march. superimposing the seasonality of these adult ticks on that of the large number of larvae detaching from mice trapped in the thomas baines nature reserve from march to july, and of nymphs detaching during march and from june to september (fig. 1) indicates that this three-host tick probably only completes one life cycle annually. the immature stages of r. follis collected from the rodents examined close to grahamstown probably originated from adults infesting cattle at the field station, horses at the animal shelter and dogs on “faraway”. horses are also good hosts of r. follis, which at the time was identified as rhipicephalus sp. (near r. capensis) (horak, knight & de vos 1986), as was the single adult tick on a dog at “faraway” (horak et al. 1987). rhipicephalus simus adult ticks on the eland, african buffaloes, zebras and carnivores in the reserve were the most likely source of immature r. simus on the striped mice at this locality, and on dogs, horses and cattle the source of the immature stages on the rodents examined in the vicinity of grahamstown. in an earlier survey a total of 1 351 adult r. simus was collected from dogs on “faraway” and on a neighbouring property, and these ticks were most numerous from august to april (horak et al. 1987). superimposing this pattern of seasonality on that determined for the immature stages on the striped mice in the thomas baines reserve indicates that this tick also only completes one life cycle annually, with most adults present during spring and summer, most larvae from mid to late summer, and most nymphs in late summer, autumn and winter. a similar survey conducted on striped mice in the andries vosloo kudu reserve, approximately 30 km north-east of grahamstown, yielded most r. simus larvae from march to june and nymphs from june to september (rechav 1982). unfortunately rechav (1982) did not state whether or not he collected the immature stages of other tick species from the mice. acknowledgements the cape department of nature conservation and in particular mr brad fike are thanked for their support during the study in the thomas baines nature reserve. dr amy jacot guillarmod of “faraway” and dr jenny randles of the animal shelter gave us permission to collect rodents at these localities. mr eddy williams assisted with the collection of ticks from the rodents examined close to grahamstown. this research was supported by financial grants from the department of agriculture and from the foundation for research development (now the national research foundation). references braack, l.e.o., horak, i.g., jordaan, leonora c., segerman, joyce & louw, j.p. 1996. the comparative host status of red veld rats (aethomys chrysophilus) and bushveld gerbils (tatera leucogaster) for epifaunal arthro317 t.n. petney et al. pods in the southern kruger national park, south africa. onderstepoort journal of veterinary research, 63:149–158. fourie, l.j., horak, i.g. & van den heever, j.j. 1992. the relative host status of rock elephant shrews elephantulus myurus and namaqua rock mice aethomys namaquensis for economically important ticks. south african journal of zoology, 27:108–114. horak, i.g., potgieter, f.t., walker, jane b., de vos, v. & boomker, j. 1983. the ixodid tick burdens of various large ruminant species in south african nature reserves. onderstepoort journal of veterinary research, 50:221–228. horak, i.g., knight, m.m. & de vos, v. 1986. parasites of domestic and wild animals in south africa. xx. arthropod parasites of the cape mountain zebra (equus zebra zebra). onderstepoort journal of veterinary research, 53:127–132. horak, i.g., sheppey, k., knight, m.m. & beuthin, c.l. 1986. parasites of domestic and wild animals in south africa. xxi. arthropod parasites of vaal ribbok, bontebok and scrub hares in the western cape province. onderstepoort journal of veterinary research, 53:187–197. horak, i.g., jacot guillarmod, amy, moolman, l.c. & de vos, v. 1987. parasites of domestic and wild animals in south africa. xxii. ixodid ticks on domestic dogs and on wild carnivores. onderstepoort journal of veterinary research, 54:573–580. horak, i.g., fourie, l.j., novellie, p.a. & williams, e.j. 1991. parasites of domestic and wild animals in south africa. xxvi. the mosaic of ixodid tick infestations on birds and mammals in the mountain zebra national park. onderstepoort journal of veterinary research, 58:125–136. horak, i.g. & boomker, j. 1998. parasites of domestic and wild animals in south africa. xxxv. ixodid ticks and bot fly larvae in the bontebok national park. onderstepoort journal of veterinary research, 65:205–211. horak, i.g., braack, l.e.o., fourie, l.j. & walker, jane b. 2000. parasites of domestic and wild animals in south africa. xxxviii. ixodid ticks collected from 23 wild carnivore species. onderstepoort journal of veterinary research, 67:239–250. horak, i.g., spickett, a.m. & braack, l.e.o. 2000. fluctuations in the abundance of boophilus decoloratus and three rhipicephalus species on vegetation during eleven consecutive years. proceedings of the third international conference on ticks and tick-borne pathogens: into the 21st century. institute of zoology, slovak academy of sciences, bratislava, slovakia, edited by m. kazimírová, m. labuda & p.a. nuttall: 247–251. horak, i.g. & cohen, marlene. 2001. hosts of the immature stages of the rhinoceros tick, dermacentor rhinocerinus (acari, ixodidae). onderstepoort journal of veterinary research: 68, 75–77. howell, d.j., petney, t.n. & horak, i.g. 1989. the host status of the striped mouse, rhabdomys pumilio, in relation to the tick vectors of heartwater in south africa. onderstepoort journal of veterinary research, 56:289–291. jacobs, p.a.h., fourie, l.j. & horak, i.g. 2004. a laboratory comparison of the life cycles of the dog ticks haemaphysalis leachi and rhipicephalus sanguineus. onderstepoort journal of veterinary research, 71:15–28. norval, r.a.i. 1979. the limiting effect of host availability for the immature stages on population growth in economically important ixodid ticks. journal of parasitology, 62:285–287. norval, r.a.i. & mason, c.a. 1981. the ticks of zimbabwe. ii. the life cycle, distribution and hosts of rhipicephalus simus, koch 1844. zimbabwe veterinary journal, 12:2–9. norval, r.a.i. 1984. the ticks of zimbabwe. ix. haemaphysalis leachi and haemaphysalis spinulosa. zimbabwe veterinary journal, 15:9–17. rechav, y. 1982. dynamics of tick populations (acari: ixodidae) in the eastern cape province of south africa. journal of medical entomology, 19:679–700. walker, jane b. 1991. a review of the ixodid ticks (acari, ixodidae) occurring in southern africa. onderstepoort journal of veterinary research, 58:81–105. walker, jane b., keirans, j.e. & horak, i.g. 2000. the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world. cambridge: cambridge academic press. 318 striped mice as hosts for immature ixodid ticks in eastern cape province 2192ker_pp299-305.qxd introduction memory is a property common to a diverse range of tissues, such as the brain and immune system (herweg & chang 2001; rosen 2001), but is it possible for the heart to remember? indeed, this appears to be the case—cardiac memory has been demonstrated in the heart of the human, dog, rat and rabbit (rosenbaum & blanco 1982; goldberger & kadish 1999; herweg & chang 2001; rosen 2001, 2002). cardiac memory is manifested as peculiar changes of the t wave, seen on the electrocardiogram (rosenbaum & blanco 1982; goldberger & kadish 1999; rosen 2001, 2002). there are three waveforms on an electrocardiogram (ecg). the p wave reflects atrial activation, the qrs complex ventricular activation, and the t wave ventricular repolarization (rosen 2001). the t wave actually reflects both transmural and apico-basal gradients for ventricular repolarization. ultimately, t waves derive from a balance between the inward and outward ion currents that occur in individual ventricular myocytes (rosen 2001, 2002). the factors affecting t wave expression are regional differences in these ionic currents with the action potentials induced by them and the temporal sequence of ventricular activation (rosen 2001). the consequence is voltage gradients in the ventricle, both transmurally and apico-basally (rosen 2001). but how is cardiac memory manifested in the t wave and what type of 299 onderstepoort journal of veterinary research, 70:299–305 (2003) the heart remembers: observations of cardiac memory in the dorper sheep heart j. ker1, e.c. webb2, j.a. ker3 and p.a. bekker1 abstract j. ker, e.c. webb, j.a. ker & p.a. bekker. 2003. the heart remembers: observations of cardiac memory in the dorper sheep heart. onderstepoort journal of veterinary research, 70:299–305 memory is a property common to a diverse range of tissues. cardiac memory has been demonstrated in the human, dog, rat and rabbit. this is a peculiar phenomenon, reflected in the t wave of the electrocardiogram. the heart is able to remember periods of alterations in the sequence of ventricular activation and once there is a return to a normal sequence of ventricular activation the t waves may manifest memory. cardiac memory is noted when the t wave during normal ventricular activation retains the vector of the previous abnormal qrs complex, caused by a period of altered ventricular activation. possible mechanisms of memory in the heart are alterations of the transient outward potassium current (ito) in ventricular myocytes and new protein synthesis inside myocytes. these two mechanisms operate in shortand long-term cardiac memory respectively. currently, it is unknown whether memory may have adverse structural consequences in the heart. we were able to demonstrate memory in the hearts of dorper wethers and this is the first report of cardiac memory in dorper sheep. keywords: cardiac memory, dorper sheep, electrocardiograph 1 department of physiology, faculty of medicine, university of pretoria, p.o. box 24318, gezina, pretoria, 0031 south africa. e-mail: james.ker@med.up.ac.za 2 department of animal and wildlife sciences, faculty of agricultural sciences, university of pretoria, pretoria, 0002 south africa 3 department of internal medicine, faculty of medicine, university of pretoria, pretoria, 0002 south africa accepted for publication 11 june 2003—editor stimuli is considered worthy of remembrance by the heart? only one event is remembered by the heart and that is a period (or periods) of altered ventricular activation (rosenbaum & blanco 1982; goldberger & kadish 1999; rosen 2001). a variety of clinical scenarios are able to cause abnormal ventricular activation and these include: ventricular pacing, intermittent left bundle branch block, premature ventricular complexes and ventricular preexcitation (rosenbaum & blanco 1982; geller & rosen 1993; nirei & kasanuki 1997; geller & carlson 1999; goldberger & kadish 1999; sporton & holdright 2001). normally, a depolarization impulse is initiated in the atrium and reaches the ventricles by way of the specialized conduction system (van dam 1989). however, during one of the above situations of altered ventricular activation, ventricular activation is initiated in one of the ventricles itself and the depolarization front moves in a lateral direction to the other ventricle. it is these periods of altered (abnormal) ventricular activation that the heart may remember. when cardiac memory is noted, the direction (polarity) of the t wave is similar to the direction (polarity) of the qrs complexes noted during the period(s) of abnormal ventricular activation (goldberger & kadish 1999). rosenbaum & blanco (1982), in their original description of cardiac memory, noted a specific sequence in cardiac memory. periods of abnormal ventricular activation (an altered sequence of ventricular depolarization) may induce a change in the t wave, which will be noted after return to a normal sequence of ventricular activation. the t wave will retain the vector of the previous abnormal qrs complex—the polarity or direction of this t wave will be the same as that of the abnormal qrs complex(es). we wanted to know if the dorper sheep heart is able to manifest cardiac memory. materials and methods this study was performed with the approval of, and adherence to, the guidelines of the pretoria biomedical research centre`s animal use and care committee. clinically normal dorper wethers (n = 6), all between the ages of 9 and 12 months, and weighing between 35 and 40 kg were used in the study. they were each fed on lucerne hay ad libitum, supplemented with 300 g per day of pelleted concentrate (10 mj me/kg dm with 14 % crude protein) and had free access to water at all times. the wethers were placed into two groups. the wethers in one of the groups (n = 4) were sedated with ketamine hydrochloride (brevinaze) at a dose of 100 mg intramuscularly once only while those in the second group (n = 2) were sedated with midazolam (dormicum) at a dose of 30 mg intramuscularly once only. they were then placed in the right lateral decubitus position. two different sedatives were used to exclude the possibility that the choice of sedative may have electrocardiographic effects, either potentiating or inhibiting the occurrence of cardiac memory. after an interval of 10 min there were no spontaneous movements and baseline electrocardiographs (ecgs) were obtained. method of electrocardiography • einthoven’s triangle was moved from the frontal to the sagittal plane, as described before by schultz & pretorius (1972), by moving the standard and unipolar limb electrodes as follows: (a) avr moved from the right fore limb to the head between the ears; (b) avl moved from the left fore limb to the sacrum; (c) avf moved from the left hind limb to the sternal angle; and (d) the earth electrode was placed on the right hind leg, just above the hock. • the six precardial leads were placed as follows: (a) v1 placed 7 cm to the right of the sternal angle; (b) v2 placed 7 cm to the left of the sternal angle; (c) v3 placed 4.5 cm below and 1 cm to the left of v2; (d) v4 placed 4.5 cm below and 1 cm to the left of v3; (e) v5 placed 4.5 cm below and 1 cm to the left of v4; and (f) v6 placed 4.5 cm below and 1 cm to the left of v5. • meditrace 200, disposable ecg conductive, adhesive electrodes were used. the skin areas where ecg electrodes were placed were shaven and the electrodes were secured with super glue (bostik). • a 12 lead electrocardiogram was performed with a schiller at-2 plus six channel electrocardiograph. the paper speed was set at 25 mm/s. 300 the heart remembers: cardiac memory in dorper sheep we induced premature ventricular complexes (pvcs) to alter the normal sequence of ventricular activation by positioning a spring-wire guide on the endocardial aspect of the right ventricle via the left internal jugular vein, utilizing the seldinger technique. the position of the spring-wire guide was confirmed by x-ray and mechanical movement of the wire produced premature ventricular complexes. results cardiac memory was demonstrated in five of the six dorper wethers and memory t waves were seen in leads iii and v1 only. lead iii proved to be the most useful lead to demonstrate cardiac memory as memory t waves were seen in all the cases in this lead while only one wether demonstrated memory t waves in lead v1. 301 j. ker fig. 1 example 1—cardiac memory, demonstrated in lead iii 302 the heart remembers: cardiac memory in dorper sheep fig. 2 example 2—cardiac memory, demonstrated in lead vi 303 j. ker fig. 3 example 3—cardiac memory, demonstrated in lead iii fig. 1 demonstrates an example from an ecg of one of the dorper wethers. the first two beats are normal sinus beats. the t waves are biphasic (arrow). the third beat is a premature ventricular complex (broken arrow). note that the polarity of the qrs complex of the pvc differs from that of the normal complexes—the qrs complex of the normal beats are positive while that of the pvc complexes are negative. the fourth beat is the first normal beat after the pvc. the t wave of this beat is negative—the polarity of this t wave follows the polarity of the qrs complex of the pvc—the heart “remembers”. this memory t wave is indicated by the double arrow. in fig. 2 an example of cardiac memory in lead v1 is illustrated, and in fig. 3 an example of cardiac memory in lead iii is shown. the conventions regarding labeling and arrows are the same as for fig. 1. discussion cardiac memory t waves may develop after either short or long periods of altered ventricular activation and is called short-term and long-term cardiac memory respectively (goldberger & kadish 1999; rosen 2001). however, there is no consensus yet on the period of time required to separate shortfrom long-term cardiac memory (goldberger & kadish 1999). rosenbaum & blanco (1982) in the first cardiac memory experiments needed 15 min of right ventricular pacing to demonstrate memory t waves. goyal & syed (1998) were recently able to observe cardiac memory after only 1 min of right ventricular pacing in humans. t wave changes may occur under a variety of circumstances (rosen 2001). for many years it was thought that all t wave changes may be explained by wilson’s formulation (levine & lown 1952; rosenbaum & blanco 1982; rosen 2001). this classic formulation states that all t wave changes are either primary or secondary in nature (wilson & macleod 1931). a primary t wave change is any change in the polarity, amplitude or duration of a t wave, where the qrs complex preceding the altered t wave is normal (levine & lown 1952; rosenbaum & blanco 1982; rosen 2001). therefore, primary t wave changes are independent of the qrs complex and are derived from intrinsic ventricular ion channels and other electrical determinants of ventricular repolarization (rosen 2001). secondary t wave changes, on the other hand, derive from an altered sequence of ventricular activation—the preceding qrs complex must be abnormal (rosenbaum & blanco 1982; rosen 2001) and these t wave changes are seen during the period of abnormal ventricular activation. with the discovery of cardiac memory there came a third type of t wave change—the pseudoprimary t wave change (rosen 2001). t wave changes caused by memory are not secondary, because the preceding qrs complex is normal—memory is only seen during reversion to normal sinus rhythm (rosenbaum & blanco 1982; rosen 2001, 2002). they are also not primary, as they were preceded by a period of altered ventricular activation, and are therefore classified as “pseudoprimary”. the mechanisms of cardiac memory involve alterations in ventricular ion channels and the synthesis of new proteins (rosen 2001, 2002). in the induction of short-term cardiac memory, both the transient outward potassium channel (ito) and the hormone angiotensin ii play an integral role, because if the ito, or the receptors for angiotensin ii are blocked, short-term cardiac memory will not occur (rosen 2001, 2002). long-term cardiac memory is suppressed if the synthesis of new proteins is prevented (shvilkin & danilo 1998; rosen 2001, 2002). in the central nervous system, memory is a process of long-term potentiation, induced by repetitive exposure to a signal that results in new protein synthesis (shvilkin & danilo 1998). neuroscientists rely on cycloheximide, an inhibitor of new protein synthesis, to differentiate between these two mechanisms (shvilkin & danilo 1998). a significant delay in the development of long-term cardiac memory has been observed in the dog after the administration of cycloheximide (shvilkin & danilo 1998). currently, it is not known whether the development of cardiac memory may have any possible adverse structural consequences in the heart (rosen 2001, 2002). on a subcellular level cardiac memory is associated with a reduction in density of the gap junctional protein connexin 43 (cx43) and also with changes in the distribution of cx43, changing from concentration at the longitudinal poles of myocytes to a more uniform distribution across the lateral margins of the cell (patel & plotnikov 2001; rosen 2002). furthermore, these changes in cx43 are non-uniform—greater epicardially than endocardially (patel & plotnikov 2001). the possible adverse structural effects of cardiac memory on the heart deserves further study. we propose that our ovine model of cardiac memory in the dorper sheep heart is a valid model that may serve as the basis for further investigation of such possible cardiac structural alterations. 304 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in comprehensive electrocardiology, theory and practice in health and disease, edited by p.w. macfarlane & t.d. veitch lawrie. new york: pregamon press. wilson, f.n. & macleod, a.g. 1931. the t deflection of the electrocardiogram. transatlantic association of american physicians, 46:29–38. 305 j. ker article information authors: christopher j. kasanga1 r. sallu2 c.a.r. mpelumbe-ngeleja2 j. wadsworth3 n.p. ferris3 g.h. hutchings3 p.p. wambura1 m.g.s. yongolo2 n.j. knowles3 donald p. king3 m.m. rweyemamu1 affiliations: 1sokoine university of agriculture, tanzania 2central veterinary laboratory, tanzania 3wrlfmd, institute for animal health, united kingdom correspondence to: christopher kasanga postal address: po box 3019, sokoine university of agriculture, morogoro how to cite this abstract: kasanga, c.j., sallu, r., mpelumbe-ngeleja, c.a.r., wadsworth, j., ferris, n.p., hutchings, g.h. et al., ‘infectious diseases of economic importance: molecular biological characteristics of foot-and-mouth disease viruses collected in tanzania from 1967 to 2009’, onderstepoort journal of veterinary research 79(2), art. #474, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.474 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. infectious diseases of economic importance: molecular biological characteristics of foot-and-mouth disease viruses collected in tanzania from 1967 to 2009 in this abstract... open access foot-and-mouth disease (fmd) is endemic in tanzania. since the first reports in 1954, fmd has caused significant economic losses in the country due to mortality and morbidity of livestock and costs associated with controlling the disease. the aim of this study was to review the serotype and genetic relationships of the fmd virus (fmdv) recovered from outbreaks in tanzania, and compare them with viruses detected from elsewhere in the sub-saharan region. at the world reference laboratory for foot-and-mouth disease (wrlfmd), a total of 106 fmd viruses have been isolated from samples collected between 1967 and 2009 from northern, southern, eastern and central parts of tanzania. the presence of fmdv was determined by laboratory methods such as vi, cf, antigen elisa and rt-pcr. phylogenies of vp1 sequences were determined by the neighbour-joining method. foot-and-mouth disease virus sat1 was the most frequent serotype (46.2%; n = 49) isolated in tanzania followed by o (26.4%; n = 27), a (14.1%; n = 15) and sat 2 (11.3%; n = 13). genotyping showed that type o viruses fell into either the east africa 1 (ea-1) or ea-2 topotypes, type a’s into the africa topotype (genotype i), type sat 1’s into topotype i and type sat 2’s into topotype iv. this study reveals that serotypes a, o, sat1 and sat2 cause fmd outbreaks in tanzania. recent samples from outbreaks in 2008, 2009 and 2010 have been typed as serotypes a, o, sat1 and sat2. phylogenetic analysis of fmdv isolates from tanzania showed that they are genetically related to lineages and topotypes from west and east africa. in tanzania, lack of comprehensive animal movement records and inconsistent vaccination programs make it difficult to determine the exact source of fmd outbreaks or to trace the transmission of the disease over time. therefore, further collection and analysis of samples from domestic and wild animals, together with improved local epidemiological investigation of fmd outbreaks is required to elucidate the complex epidemiology of fmd in the sub-saharan region. article information authors: ahmed elghali1 shawgi m. hassan2 affiliations: 1veterinary research institute, khartoum, sudan2department of parasitology, university of khartoum, sudan correspondence to: ahmed elghali postal address: po box 8067, khartoum, sudan dates: received: 16 mar. 2012 accepted: 14 sept. 2012 published: 06 dec. 2012 how to cite this article: elghali, a. & hassan, s.m., 2012, ‘ticks infesting animals in the sudan and southern sudan: past and current status’, onderstepoort journal of veterinary research 79(1), art. #431, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.431 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ticks infesting animals in the sudan and southern sudan: past and current status in this research communication... open access • abstract • introduction • northern sudan • central sudan • western sudan • eastern sudan • republic of south sudan • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ in this review, we collate information about ticks identified in different parts of the sudan and south sudan since 1956 in order to identify gaps in tick prevalence and create a map of tick distribution. this will avail basic data for further research on ticks and policies for the control of tick-borne diseases. in this review, we discuss the situation in the republic of south sudan as well as sudan. for this purpose we have divided sudan into four regions, namely northern sudan (northern and river nile states), central sudan (khartoum, gazera, white nile, blue nile and sennar states), western sudan (north and south kordofan and north, south and west darfour states) and eastern sudan (red sea, kassala and gadarif states). introduction top ↑ ticks may have both direct and indirect adverse effects on their host animals. they feed on blood, leading to anaemia, and the injuries caused by their attachment damage hides and predispose animals to secondary bacterial infections that may lead to mastitis. these wounds may also be attractive to the myiasis-causing flies. ticks can cause paralysis and probably transmit a greater variety of organisms that cause diseases than any other arthropod (hoogstraal 1956). the economically most important tick-borne diseases are theileriosis, babesiosis, ehrlichiosis (heartwater) and anaplasmosis. the study of ticks that parasitise domestic animals is of considerable importance in that it assists in determining their host preference, their seasonality and geographic distribution, the diseases they may transmit and their control (king, getting & newson 1988). on-host ticks may be affected by factors such as species, breed and immune status, whilst free-living ticks are affected by climate, geographic region and seasonality. during the late 1940s and early 1950s hoogstraal (1954, 1956) collected ticks from more than 50 localities in southern sudan and 15 localities in northern sudan. he recorded 38 tick species in the southern sudan provinces, seven in other provinces and 18 that were found in both southern sudan and other provinces.in this review, we collate the findings on ticks identified in various regions of the sudan and in south sudan since hoogstraal’s survey (1956). the information should assist in filling some of the gaps in our knowledge about the ticks of the sudan and south sudan in order to compile a map illustrating tick distribution in both countries. the data could also provide a basis for further research on ticks and tick-borne diseases, which would be useful for developing control policies. for the purposes of the review, we have included data from the republic of south sudan as well as from the sudan, divided into northern sudan (northern and river nile states), central sudan (khartoum, gazera, white nile, blue nile and sennar states), western sudan (north and south kordofan and north, south and west darfour states) and eastern sudan (red sea, kassala and gadarif states). the localities are shown in figure 1. figure 1: states of the sudan and southern sudan. northern sudan top ↑ hoogstraal (1956) identified amblyomma exornatum, hyalomma dromedarii, hyalomma excavatum, hyalomma impeltatum, hyalomma impressum, hyalomma marginatum, hyalomma scupense (= hyalomma detritum), hyalomma rufipes, hyalomma truncatum, rhipicephalus evertsi evertsi, rhipicephalus praetextatus and rhipicephalus sanguineus in the northern provinces. later, salih et al. (2004) collected hyalomma anatolicum from cattle in atbara and eddamer, and hy. dromedarii from all localities sampled, as well as rhipicephalus (boophilus) decoloratus, hy. impeltatum, hy. truncatum, hy. rufipes, r. evertsi evertsi, r. praetextatus and ticks belonging to the r. sanguineus group from several localities. ahmed, elhussein and elkhidir (2005) reported that in the river nile state 74% of the ticks feeding on sheep were hy. anatolicum, 15% r. praetextatus, 9% r. sanguineus group, 2% r. evertsi evertsi and 0.5% hy. dromedarii. in the same area the species composition of ticks feeding on camels was hy. dromedarii (89%), hy. impeltatum (7.7%), hy. anatolicum (3.3%), hy. truncatum (0.29%), hy. rufipes (0.25%), r. praetextatus (0.30%) and r. sanguineus group (0.09%) (elghali & hassan 2009). the predominant tick species feeding on horses in atbara were hy. anatolicum (= hyalomma anatolicum anatolicum) (92%) together with hy. dromedarii, r. evertsi evertsi and r. sanguineus (salim 2008). central sudan top ↑ hoogstraal (1956) recorded a. exornatum, amblyomma lepidum, amblyomma variegatum and r. (b.) decoloratus across central sudan, except in khartoum. hy. scupense, hy. dromedarii, hy. excavatum, hy. impeltatum, hy. impressum, hy. marginatum and hy. rufipes were also recorded in central sudan, except in the blue nile region (hoogstraal 1956). other species recorded in all states by hoogstraal (1956) included r. evertsi evertsi, r. praetextatus and r. sanguineus. just more than 25 years later the dominant tick species north and south of wad medani was hy. anatolicum and a r. (boophilus) species, respectively (fao 1983). tatchell (1983) stated that hy. rufipes, r. evertsi evertsi and r. (b.) decoloratus were ubiquitous in their distribution along the blue nile, whilst the distribution of hy. anatolicum was restricted to north of wad medani. he further reported that a. lepidum was common only south of a line between wad medani and khartoum, whilst rhipicephalus (boophilus) annulatus was restricted to the area south of wad medani. ticks of the blue nile and white nile ecosystems were studied by jongejan et al. (1987), who found 24 tick species infesting livestock and wildlife in these areas. amongst these tick species rhipicephalus (boophilus) geigyi, rhipicephalus bergeoni and rhipicephalus camicasi were recorded for the first time in the sudan. they also reported that r. (b.) annulatus had extended its distribution further north into the blue nile, gezira and khartoum regions. during 1979 to 1982, the immature stages of a. lepidum, hy. anatolicum and r. evertsi evertsi were found feeding on cattle and sheep and immature a. variegatum specimens were collected from cattle (latif 1985). latif (1985) also found hy. impeltatum nymphs on camels and sheep and hy. dromedarii nymphs on camels for the first time. in the khartoum area, gad elrab (1986) reported hy. anatolicum (= hy. a. anatolicum), hy. dromedarii, hy. truncatum, r. evertsi evertsi, r. praetextatus and ticks of the r. sanguineus group on local sheep. similarly, osman (1999) collected hy. anatolicum, r. evertsi evertsi and ticks of the r. sanguineus group from sheep on the khartoum university farm, north of khartoum. abdoon, osman and el wasila (1992) collected 1344 ticks from horses, amongst which 97.4% were hy. anatolicum and the rest hy. dromedarii, hy. rufipes and r. evertsi evertsi. in the southern parts of khartoum, adjacent to gazera state, exotic cattle were found to be infested with hy. anatolicum, hy. rufipes, r. (b.) decoloratus, r. evertsi evertsi and ticks of the r. sanguineus group (mohamed, aziz & kheir 1998). according to mohammed (2002) a. lepidum was the predominant tick species in the blue nile state and infestation of host animals occurred throughout the year. according to lazarus (2002), who collected ticks from cattle in and around wad medani in central sudan from may to july 2002, hy. anatolicum was the most abundant species, followed by r. evertsi evertsi, hy. rufipes, r. sanguineus, a. lepidum and r. (b.) decoloratus. all his collections of a. lepidum were male specimens. elimam (2003) found that 11 tick species infested cattle in kosti (central sudan), amongst which a. variegatum, hy. anatolicum and hy. excavatum were reported for the first time in this area. mohammed, osman and elrabaa (2004) recorded a. lepidum, hy. anatolicum (70% and 83%, respectively) at soba and kuku and hy. dromedarii, hy. impeltatum, hy. rufipes, r. evertsi evertsi and ticks of the r. sanguineus group on cattle in khartoum. salih et al. (2004) collected a. lepidum from cattle along the blue nile, from wad medani to damazin and along the white nile, from eduaim to rabak. they also found a. variegatum in kosti. in addition, they collected hy. anatolicum in khartoum and in relatively high numbers in um benin (blue nile, south of wad medani), whilst hy. dromedarii was present in all localities sampled except kosti. other ticks recorded by salih et al. (2004) in other localities of central sudan were r. (b.) decoloratus, hy. impeltatum, hy. rufipes, hy. truncatum, r. evertsi evertsi, r. praetextatus and ticks of the r. sanguineus group. export sheep (hamari and watish) brought to the al kadaro slaughterhouse (khartoum state) from the white nile, blue nile, kordofan and gadarif states were found to be infested with a. lepidum, hy. anatolicum, hy. dromedarii, hy. impeltatum, hy. rufipes, hy. truncatum, r. (b.) decoloratus, r. camicasi, r. evertsi evertsi, rhipicephalus guilhoni and rhipicephalus muhsamae (elfaki 2005). mohammed and hassan (2007) studied the ticks that infested sheep in sennar state and recorded a. lepidum, hy. anatolicum (= hy. a. anatolicum), hy. truncatum, r. (b.) decoloratus, r. camicasi, r. evertsi evertsi, r. guilhoni and r. muhsamae. salim (2008) found that the dominant tick species infesting horses in kosti was hy. anatolicum (= hy. a. anatolicum) (92%), whilst hy. dromedarii, r. evertsi evertsi and r. sanguineus were also present. in the butana area, eltigani (2009) recorded a. lepidum, a. variegatum, hy. anatolicum, hy. dromedarii, hy. impeltatum, hy. rufipes, hy. truncatum, r. (b.) decoloratus, r. evertsi evertsi and r. sanguineus on camels. western sudan top ↑ hoogstraal (1956) recorded a. exornatum, a. lepidum, a. variegatum and r. (b.) decoloratus in western sudan and r. (b.) annulatus in kordofan. he also reported the presence of hy. dromedarii, hy. excavatum, hy. impeltatum, hy. impressum, hy. marginatum, hy. rufipes, hy. scupense, r evertsi evertsi, r. praetextatus and r. sanguineus. although osman et al. (1982) later recorded various species of amblyomma, rhipicephalus (boophilus) and hyalomma in kordofan, the dominant tick species was hy. impeltatum. osman (1997) also argued that the unusual distribution of a. lepidum and a. variegatum on sheep and goats in the nuba mountains required further study. in a subsequent study, cattle in kadogli and dilling in kordofan state were found to be infested with a. lepidum, a. variegatum, hy. rufipes, hy. truncatum, r. (b.) annulatus, r. (b.) decoloratus, r. evertsi evertsi, r. praetextatus and ticks of the r. sanguineus group (sowar 2002). in elobeid, the predominant tick species on horses were hy. anatolicum (92%), whilst hy. dromedarii, r. evertsi evertsi and r. sanguineus were also present (salim 2008). salih et al. (2004) recorded a. lepidum on cattle at several localities in western sudan, whilst a. variegatum was found in elobeid and nyala, and hy. dromedarii in all localities sampled. in darfour, osman (1978a) found that hy. rufipes, hy. truncatum and r. sanguineus were the dominant species. he also recorded hyalomma turanicum, r. (b.) annulatus, rhipicephalus cuspidatus and rhipicephalus sulcatus for the first time in darfour and r. guilhoni and rhipicephalus turanicus for the first time in the sudan. osman (1978b) recorded a. variegatum, hy. dromedarii, hy. rufipes, hy. truncatum, r. (b.) decoloratus, r. evertsi evertsi, r. praetextatus, r. sulcatus and r. turanicus on sheep at jebel marra (marra mountain). ticks belonging to the genera amblyomma, rhipicephalus (boophilus), hyalomma and rhipicephalus were reported from cattle, camels and horses in and around nyala town, with hy. anatolicum present on dairy farms in nyala (gaafar 2008). adam (2005) reported a single male hy. anatolicum in buram. abdalla (2007) recorded 15 tick species belonging to four genera in south darfour. these included a. lepidum, a. variegatum, hy. anatolicum (only in nyala town), hy. dromedarii, hy. impeltatum, hy. impressum, hy. rufipes, hy. truncatum, r. (b.) annulatus, r. (b.) decoloratus, r. evertsi evertsi, r. guilhoni, r. muhsamae, r. praetextatus and r. sanguineus. with the exception of r. guilhoni, r. muhsamae and r. praetextatus, the same tick species as well as hy. excavatum were found on dairy cattle in nyala (gaafar 2008). according to ibrahim (2009), the dominant tick species infesting cattle and sheep in north darfour was hy. impeltatum, whilst a. lepidum, a. variegatum (accidental introduction possibly by migrating birds), hy. dromedarii, hy. rufipes, hy. truncatum, r. (b.) decoloratus and r. evertsi evertsi were also present. eastern sudan top ↑ in eastern sudan, hoogstraal (1956) recorded a. exornatum, a. lepidum, hy. dromedarii, hy. excavatum, hy. impeltatum, hy. impressum, hy. marginatum, hy. rufipes, hy. scupense and r. (b.) decoloratus. he also reported hy. truncatum in kassala and r. evertsi evertsi, r. praetextatus and r. sanguineus in all provinces. subsequently karrar, kaiser and hoogstraal (1963) stated that a. lepidum numbers were high (15.5 ticks per host) in wooded savannah areas (river atbara), the cultivated gash delta and in the acacia seyal savannah of river gash in kassala. they also reported r. sanguineus on sheep, goats, cattle, camels and donkeys and that hy. dromedarii was the most abundant tick species on camels. the latter species presented together with a. lepidum, hy. excavatum, hy. impeltatum, hy. rufipes, hy. truncatum, r. praetextatus and r. sanguineus. in the same area, mohamed and yagoub (1990) identified r. evertsi evertsi and r. sanguineus on cattle, sheep and equines (horses and donkeys), hy. excavatum and hy. rufipes on cattle and equines, whilst hy. dromedarii, hy. marginatum and r. (b.) annulatus were collected only from cattle. also in kassala, imam (1995) collected 4844 ticks from sheep, including the species a. lepidum, hy. anatolicum, hy. impeltatum, r. (b.) decoloratus, r. evertsi evertsi and ticks of the r. sanguineus group. salih et al. (2004) collected a. lepidum from cattle in gadarif and port-sudan, whilst a single hy. anatolicum was collected from one animal in port-sudan and hy. dromedarii from all localities except kassala. in gadarif, the predominant species on horses was hy. anatolicum (92%), with hy. dromedarii, r. evertsi evertsi and r. sanguineus also being present (salim 2008). elghali and babikir (unpublished data) identified 291 ticks collected from 66 cattle in elgalabat (near the ethiopian boarder) during 2008, of which 60.6% were a. lepidum, followed by a. variegatum, hy. anatolicum, hy. rufipes, hy. truncatum, r. (b.) decoloratus, r. evertsi evertsi, r. sanguineus and r. turanicus. in the red sea state, the predominant cattle ticks have been found to be hy. anatolicum, hy. dromedarii and hy. impeltatum (khalid 2009). hy. anatolicum was also abundant in the toker arera. other species, including r. camicasi, r. (b.) decoloratus, r. evertsi evertsi and r. guilhoni have been collected in lesser numbers (khalid 2009). republic of south sudan top ↑ hoogstraal (1956) recorded amblyomma cohaerens, a. exornatum, amblyomma latum, a. lepidum, amblyomma marmoreum, amblyomma nuttalli, amblyomma pomposum, amblyomma rhinocerotis, a. variegatum and amblyomma tholloni in southern sudan, together with r. (b.) annulatus and r. (b.) decoloratus. he recorded dermacentor circumguttatus and dermacentor rhinocerinus in equatoria province and haemaphysalis aciculifer, haemaphysalis bequaerti, haemaphysalis hoodi, haemaphysalis houyi, haemaphysalis leachii muhsami (= haemaphysalis muhsamae), haemaphysalis parmata and hy. rufipes in all southern states. he also recorded ixodes cavipalpus, ixodes nairobiensis ixodes rasus, ixodes schillingsi, ixodes simplex and ixodes vespertilionis in equatoria and bahr el ghazal provinces. furthermore, he recorded margaropus reidi in bahr el ghazal province and rhipicephalus appendiculatus, rhipicephalus arnoldi, rhipicephalus bequaerti, rhipicephalus compositus, r. cuspidatus, rhipicephalus distinctus, r. evertsi evertsi, rhipicephalus kochi, rhipicephalus longicoxatus, rhipicephalus longus, rhipicephalus muehlensi, r. praetextatus, rhipicephalus pravus, r. sanguineus, rhipicephalus simpsoni, r. sulcatus, rhipicephalus supertritus and rhipicephalus tricuspis in the three provinces of south sudan. rhipicephalus appendiculatus was first reported at kajo kaji and yei in 1950 (hoogstraal 1956). later it was also reported in chukudum, river, nimuli, yambio, nagichot and juba (julla 2003). morzaria et al. (1981) reported a. lepidum, a. variegatum, hy. rufipes, r. appendiculatus, r. (b.) decoloratus, r. evertsi evertsi, r. praetextatus and r. pravus on cattle in the south. the dominant tick species in this region is a. variegatum (fao 1983). jongejan et al. (1987) reported that a. lepidum and a. variegatum had extended their distributions between latitude 5° n and 12° n, whilst when compared to r. (b.) decoloratus, r. (b.) annulatus occupied the forest and wetter areas. jongejan et al. (1987) also recorded r. (b.) geigyi from a single locality in south sudan. by 1997, rhipicephalus (boophilus) microplus had not yet been recorded in the sudan (latif & hassan 1997). in the pibor area in jonglei state, korok (2005) found that a. lepidum represented 56.6% of the tick population, r. sanguineus 20.5% and r. evertsi evertsi 16.2%. other tick species he recorded were a. variegatum, hy. rufipes, r. (b.) annulatus, r. (b.) decoloratus and r. praetextatus. marcellino (2008) recorded a. variegatum, hy. rufipes, r. appendiculatus, r. (b.) decoloratus, r. evertsi evertsi, r. praetextatus and ticks of the r. sanguineus group on cattle in central equatoria. he reported that a. variegatum was found in all the sampling localities. r. appendiculatus was abundant in the juba area, whilst only a single male r. appendiculatus was identified in mangalla and none in terekeka. discussion top ↑ in the sudan and south sudan there are different geographic and climatic situations, ranging from the desert zone in the north (with an annual rainfall of less than 100 mm) to wooded savannahs in the south (with annual rainfall of more than 1000 mm). this extraordinary climatic and ecological diversity, coupled with a wide range of domestic and wildlife hosts, serve to accommodate the biological requirements of a variety of tick species. hassan and salih (unpublished data) report that factors such as animal movement, habitat change, drought, desertification and global climatic changes may force ticks to extend their distribution ranges beyond their known geographic regions. they report that a. variegatum has extended its range north of 12° n, whilst hy. anatolicum has moved south of 14° n and r. (b.) annulatus was found in the semiarid zone. abdalla and hassan (2010) have recently reported on the distribution of a. variegatum, hy. anatolicum and r. (boophilus) species in various localities of darfour state. the distribution of some tick-borne diseases has been altered to some extent because of certain changes in the environment, the movement of animals to new pastures and the spread of certain tick species to new ecological zones. heartwater, for instance, was originally restricted to the eastern parts of the country where a recognised vector, a. lepidum, was abundant (abdel rahim & shommein 1984; karrar et al. 1963). according to osman and hassan (2003), a. lepidum is restricted to the eastern region, from torit to kapoeta in the south to kassala in the north. abdalla (2007) reported the presence of antibodies to ehrlichia ruminantium (heartwater causative agent) in umdafug and in reheid-arbirdi in southern darfour state, and concluded that these results were due to changes in the geographic distribution of ticks and hence the disease. furthermore, heartwater has recently become endemic in kordofan and darfour (mohammed, unpublished data). tropical theileriosis (theileria annulata infections) has been detected in new regions, to where the tick vector hy. anatolicum has extended its distribution range (abdalla 2007; gaafar 2008). salih et al. (2004) collected hy. anatolicum from cattle at umbenein, in the southern regions of the blue nile state, proving that the species has extended its distribution southwards, which may lead to the emergence of tropical theileriosis in these areas. adam (2005) detected t. annulata antibodies around nyala and in elradom, southern darfour. in the same state, abdalla (2007) reported t. annulata antibodies in cattle in eid-elfirsan and concluded that this finding is linked to geographic changes in the distribution of the vector tick. the recovery of hy. anatolicum in several localities in darfour state (abdalla & hassan 2010) confirm the observation. similarly, east coast fever (theileria parva infection) coincides with the distribution of r. appendiculatus in the southern part of south sudan. the first outbreak of east coast fever was reported in 1950 in the kajo kaji and yei districts on the western bank of the nile and extended up to juba (julla 1985, 1994). the disease was found to be more prevalent in areas that were used for grazing during the dry season, namely apuk toich, river lol and the aweil district (zessin & baumann 1982). in the south, scattered tick collections proved that r. appendiculatus has moved northwards up to bore (julla 2003) and may lead to the occurrence east coast fever in the southern regions of the north. conclusion top ↑ in conclusion, numerous tick species are distributed throughout the sudan and south sudan and these include the economically most important vectors of disease. several of the vector tick species have expanded their distribution beyond their previously recognised geographical zones, which may lead to the expansion of tick-borne diseases to new areas where outbreaks of disease are expected to be drastic. furthermore, ticks and tick-borne diseases in the sudan represent one of the most important obstacles to livestock production. hence it seems that without control of ticks and tick-borne diseases, it would be almost impossible to increase livestock production with foreign-breed animals, selection from indigenous breeds or through cross-breeding (osman 1976). acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this paper. authors’ contributions a.e. (veterinary research institute) suggested the concept of the review, collected most of the used articles and other references, designed the review, wrote the first draft of the review and submitted the review. s.m. h. (university of khartoum) added some references, improved the written paper and revised all information, particularly the recent tick names. references top ↑ abdalla, m.m., 2007, ‘studies on ticks and tick-borne diseases of cattle in south darfour state, sudan’, mvsc thesis, department of parasitology, university of khartoum.abdalla, m.m. & hassan, s.m., 2010, ‘current status of distribution of ticks (acari: ixodidae) infesting cattle in south darfour state, sudan’, university of khartoum journal of veterinary science and animal production 1(2), 76–97. abdel rahim, a.i & shommein, a.m., 1984, ‘heartwater in the sudan with emphasis on eastern region’, paper presented at the 11th veterinary conference of animal wealth, kassala, eastern sudan, 10–14th april, pp. 129–139. abdoon, a.m.o., osman, o.m. & el wasila, m., 1992, ‘the epidemiology of equine babesiosis in khartoum district, sudan. 1 tick prevalence and their parasite rate’, bulletin of animal health and production in africa 40, 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piroplasmosis of horses in the sudan’, mvsc thesis, department of parasitology, university of khartoum. sowar, a.e., 2002, ‘epidemiology and ecology of ticks and some tick-borne diseases in (kadogli and dilling) southern kordofan state, sudan’, mvsc thesis, department of preventive medicine, university of khartoum. tatchell, r.j., 1983, ‘tick and tick-borne diseases control. the sudan. tick ecology and tick control’, technical report, ag: gcp/sud 024/den, food and agriculture organization of united nations, rome. zessin, k.h. & baumann, m.p., 1982, ‘report on the livestock disease survey, bahr el ghazal province’, technical cooperation germany/sudan, gtz, berlin. article information authors: k. kistiah1,2 john frean1,2 j. winiecka-krusnell3 a. barragan3 affiliations: 1parasitology reference unit, national institute for communicable diseases, south africa 2university of the witwatersrand, johannesburg, south africa 3department of parasitology, mycology and environmental microbiology, swedish institute for infectious diseases, sweden correspondence to: john frean postal address: private bag x4, sandringham 2131, south africa how to cite this poster: kistiah, k., frean, j., winiecka-krusnell, j. & barragan, a., 2012, ‘unexpectedly low seroprevalence of toxoplasmosis in south africa’, onderstepoort journal of veterinary research 79(2), art. #486, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.486 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. unexpectedly low seroprevalence of toxoplasmosis in south africa in this poster... open access toxoplasmosis is an infection of warm-blooded vertebrates caused by the obligate intracellular protozoan parasite, toxoplasma gondii. it is one of the most common parasitic diseases of humans, infecting approximately one-third of the world’s population. in persons with advanced hiv, toxoplasmosis represents a major opportunistic infection of the central nervous system. approximately two-thirds of all people living with hiv live in sub-saharan africa. in areas such as this, toxoplasmosis could theoretically pose a huge threat. there is little known about t. gondii prevalence in humans in africa. geographically, prevalences vary widely on this continent, as observed in other parts of the world. there is limited historical information about the disease in south africa. more knowledge is needed at a regional level about the risk of toxoplasmosis, diagnostic issues, and measures to reduce the risk to susceptible persons. the seroprevalence of t. gondii in selected populations, namely hiv-positive and hiv-negative individuals, and a more general sample biased towards pregnant women, was therefore investigated and found to be 9.8% (37/376), 12.8% (48/376) and 6.4% (32/497) respectively. compared with historical data from south africa, the prevalence has decreased substantially; however, the incidence of clinical disease is unknown, despite the very high burden of hiv and aids cases (5.9 million and 0.7 million, respectively in 2009). this study provided information relating to the diagnosis and current seroprevalence of t. gondii in south africa. many questions still remain to be answered however, to fully understand the impact of this parasite on the country’s population. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) emmanuel m. seakamela bacteriology division, onderstepoort veterinary research, agricultural research council, pretoria, south africa department of life and consumer sciences, college of agriculture and environmental sciences, university of south africa, pretoria, south africa letlhogonolo diseko bacteriology division, onderstepoort veterinary research, agricultural research council, pretoria, south africa dikeledi malatji department of life and consumer sciences, college of agriculture and environmental sciences, university of south africa, pretoria, south africa lavhelesani makhado bacteriology division, onderstepoort veterinary research, agricultural research council, pretoria, south africa mmatau motau bacteriology division, onderstepoort veterinary research, agricultural research council, pretoria, south africa kudakwashe jambwa bacteriology division, onderstepoort veterinary research, agricultural research council, pretoria, south africa kudakwashe magwedere directorate of veterinary public health, department of agriculture, land reform and rural development, pretoria, south africa nombasa ntushelo department of biometry, infruitec, agricultural research council, cape town, south africa itumeleng matle bacteriology division, onderstepoort veterinary research, agricultural research council, pretoria, south africa citation seakamela, e.m., diseko, l., malatji, d., makhado, l., motau, m., jambwa, k. et al., 2022, ‘characterisation and antibiotic resistance of yersinia enterocolitica from various meat categories, south africa’, onderstepoort journal of veterinary research 89(1), a2006. https://doi.org/10.4102/ojvr.v89i1.2006 project research registration: project number: p10000156 note: additional supporting information may be found in the online version of this article as online appendix 1. original research characterisation and antibiotic resistance of yersinia enterocolitica from various meat categories, south africa emmanuel m. seakamela, letlhogonolo diseko, dikeledi malatji, lavhelesani makhado, mmatau motau, kudakwashe jambwa, kudakwashe magwedere, nombasa ntushelo, itumeleng matle received: 24 feb. 2022; accepted: 17 aug. 2022; published: 07 nov. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract yersinia enterocolitica infections impose a significant public health and socioeconomic burden on human population in many countries. the current study investigated the prevalence, antimicrobial resistance profile and molecular diversity of y. enterocolitica in meat and meat products across various retail outlets in selected provinces of south africa (sa). in a cross-sectional study, a total of 581 retail meat and meat products were collected from four cities across three provinces of sa. samples were from beef and pork products, which included 292 raw intact, 167 raw processed, and 122 ready-to-eat (rte) meats. samples were analysed using classical microbiological methods for isolation, identification and biotyping of y. enterocolitica. conventional polymerase chain reaction (pcr) was performed for confirmation, serotyping, screening of virulence (n = 11) and antimicrobial resistance (n = 18) genes. phenotypic antimicrobial resistance profiles were determined against 12 antibiotics discs, using disc diffusion method. the overall prevalence of 12% (70/581) was reported across all cities with contamination proportion reported in samples collected from raw intact 15% (43/292), followed by raw processed 11% (18/167) and rte meats 7% (9/122). all positive isolates were of biotype 1a with 7% (5/70) belonging to bioserotype 1a/o:8. most of the isolates harboured ymoa, ystb, fepd, ail, fepa, inva and myfa virulence genes. high antimicrobial resistance frequency was observed for ampicillin (94%), cephalothin (83%) and amoxicillin (41%), respectively. of the 18 tested antimicrobial resistance genes, blatem was the most predominant (40%) followed by cmla (21%). this study reveals the presence of antimicrobial resistant y. enterocolitica possessing virulent genes of public health importance in products of animal origin, therefore, health monitoring and surveillance of this pathogen is required. keywords: yersinia enterocolitica; meat; prevalence; biotypes; bioserotypes; serotypes; virulence genes; antimicrobial resistance genes. introduction yersinia enterocolitica is a foodborne pathogen with a widespread distribution in nature (bancerz-kisiel & szweda 2015). it is commonly isolated from different animals, food products and environmental sources (novoslavskij et al. 2013; platt-samoraj et al. 2015). as a zoonotic foodborne pathogen, y. enterocolitica can be transmitted to humans through consumption of contaminated food products especially pork and beef (syczyło et al. 2018). the contamination of meat and meat products can occur at any stage along the food supply chain because of poor hygienic practices, improper handling and cooking processes (indrawattana et al. 2011). of the various food products surveyed in countries with systems to monitor y. enterocolitica infections, meat and meat products are widely found as important vehicles for this pathogen and are implicated in several high-profile outbreaks of yersiniosis (sakai et al. 2005; zdolec & kiš 2021). y. enterocolitica is a heterogeneous pathogen that has been divided into six biotypes (1a, 1b, 2, 3, 4 and 5) and at least 70 different serotypes based on a combination of biochemical and serological tests (bottone 2018). biotypes are further categorised into three groups (1a, 1b, 2–5) based on their degree of pathogenicity. of the three groups, 1a is presumably non-pathogenic, followed by the moderate pathogenic (2–5) and the highly pathogenic 1b group (bancerz-kisiel et al. 2018). strains belonging to 1b/o:8, 2/o:5,27, 2/o:9, 3/o:3 and 4/o:3 serotypes are the most prevalent causative agents of human illness at an infectious dose of 104 to 108 bacterial cells or more orally (hancock, schaedler & macdonald 1986; momtaz, davood rahimian & safarpoor dehkordi 2013). the disease induced by pathogenic strains of y. enterocolitica is characterised by diverse symptoms ranging from a mild but self-limiting gastroenteritis to acute mesenteric lymphadenitis (bottone 1997). urinary and respiratory osteoarticular infection, as well as endocarditis, erythema nodosum, bacteremia and sepsis have been associated with human yersiniosis (krajinović, tambić andrašević & baršić 2007). pathogenicity of y. enterocolitica strains particularly biotypes 1b and 2–5 and some members of 1a are attributed to the presence of a chromosomal and 70-kb pyv plasmid genes that control the production and functions of proteins which promote the invasion, manipulation and survival in the host where they cause diseases (fàbrega & vila 2012; fredriksson-ahomaa 2017). furthermore, the pathogenicity of this bacterium is commonly associated with the presence of chromosomal ail (attachment-invasion locus) and ysta (yersinia stable toxin) genes (pierson & falkow 1993). other important plasmid-borne virulence genes in y. enterocolitica includes ystb, ystc, yada, yop, virf, inva, myfa and ymoa (bancerz-kisiel et al. 2018). y. enterocolitica has been reported to be highly susceptible to majority of antibiotics except β-lactams agents such as penicillin, ampicillin and the first-generation cephalosporins (abdel-haq et al. 2006; peng et al. 2018). however, the prevalence of multi drug-resistant y. enterocolitica strains isolated from food and environmental sources has been on the rise in the last decade, because of excessive use and overreliance on antibiotics in livestock production and antimicrobial resistance genes transmission among different species (sharma et al. 2018; wang et al. 2021). thus, foods of animal origin are recognised as important vehicles for the transmission of potential antimicrobial resistant y. enterocolitica in humans (durán & marshall 2005). prevalence of y. enterocolitica infections is carefully monitored in developed countries. in europe, human yersiniosis is the third most common bacterial enteric disease after campylobacteriosis and salmonellosis (chlebicz & śliżewska 2018). in 2016, human yersiniosis was estimated to have affected nearly 117 000 people in the united states, including 640 cases requiring hospitalisation and 35 deaths (https://www.cdc.gov/yersinia/index.html). on the contrary, no sufficient diagnostic data is currently available in many african countries and no exact number of cases of the disease is known for those countries (chlebicz & śliżewska 2018; nesbakken 2013). in south africa (sa), there is a dearth of qualitative data on the occurrence of y. enterocolitica in retail meat and meat products from different animal species. in a recent study, madoroba et al. (2021) isolated y. enterocolitica from 17% of meat and meat products; however, no indication of biotypes, serotypes and virulence properties of the strains was provided, making it difficult to determine whether these isolates corresponded to saprophytic or pathogenic species. therefore, the aim of this study was (1) to investigate the prevalence of y. enterocolitica in pork and beef meat products from retail outlets of sa, (2) to determine the association of y. enterocolitica with various chromosomal and plasmids virulence genes, and (3) to assess the antimicrobial susceptibility profiles and antimicrobial resistance genes of the isolated y. enterocolitica strains. materials and methods study design and sample collection a cross-sectional study was conducted to determine the prevalence and characteristics of y. enterocolitica in retail outlets from four cities (pretoria, rustenburg, bronkhospruit and emalahleni) across three provinces (gauteng, north west and mpumalanga) of sa. all formal retail outlets in the country selling groceries are mandated to affiliate with supermarket association of sa. these retail outlets were divided into four categories namely, chain, large, medium and small outlets (mokgophi et al. 2021). a total of 58 outlets were randomly selected from comprehensive list of active outlets obtained from the supermarket association of sa across four cities to participate in this study. the number of samples collected across various categories of retail outlets was determined as shown in online appendix 1, table s1. determination of sample size and sample collection the sample size was determined by predicting 50% expected prevalence with 95% confidence interval (ci) and 5% level of significance as cited by thrusfield (2007) in a formula: where pexp is the expected prevalence and d is the desired precision. a pexp value of 50% and a d value of 5%: therefore, the minimum sample size in this study was supposed to be 384 meat samples. a total of 581 meat samples were collected between september 2020 and february 2021 through sampling once per each retail outlet. the 581 meat and meat products were directly purchased from the selected outlets. location, animal species and sample category are summarised in online appendix 1, table s2. considering the diversity of samples collected in this study, samples were further divided into six sample types (online appendix 1, table s3). all collected samples were placed into sterile plastic bags and placed in cooler boxes with ice packs. transportation was done in such a way that the temperatures of the samples were maintained at less than 4 °c until the samples arrived at the feed and food analysis laboratory at agricultural research council, onderstepoort veterinary research (arc-ovr). the samples were subjected to microbiological analysis within 24 h upon collection. microbiological analysis isolation and identification of y. enterocolitica in the current study, the presence of y. enterocolitica in meat and meat products was detected in accordance with international organization for standardization (iso) 10273:2017 as described by madoroba et al. (2021), with minor modifications. in short, 25 g of meat samples were inoculated into 225 ml of peptone sorbitol bile broth (thermofisher scientific, johannesburg, sa), followed by homogenisation for 2 min and incubation at 30 °c for 24–48 h. in addition, homogenised samples were incubated at 8 °c for 10 days. following incubation, broth samples (50 μl) were inoculated onto cefsulodin-irgasan-novobiocin (cin) (thermofisher scientific, johannesburg, sa) and macconkey agar (onderstepoort biological product, pretoria, sa). the inoculated plates were incubated aerobically at approximately 30 °c for 24–48 h. colonies resembling ‘bull eye’ that appeared small with deep red centres and clear zones around on cin and small colourless on macconkey agar were considered presumptive and were subjected to biochemical tests. the biochemical tests were performed using rapid™ one system (thermofisher scientific, johannesburg, sa) for identification of enterobacteriaceae according to manufacturer’s instructions. confirmation of y. enterocolitica by pcr genomic deoxyribonucleic acid (dna) was extracted from the 24 h pure y. enterocolitica growth culture on blood agar, following the protocol of the high pure pcr template preparation kit (roche, germany). the microcentrifuge tube containing the extracted dna was stored at –80 °c for further analysis. the quantity and purity of the dna was assessed using qubit fluorometric quantitation (thermofischer scientific, waltham, ma, us). the confirmation of presumptive y. enterocolitica isolates was carried out by conventional pcr as previously described by novoslavskij et al. (2010). this pcr targets the y. enterocolitica specific 16srrna, primers (inqaba biotechnical industries, pretoria, sa) as well as pcr conditions are indicated in online appendix 1, table s4. y. enterocolitica atcc® 23715 and e. coli atcc® 25922 were used as positive and negative controls respectively. the pcr product underwent gel electrophoresis for 1 h at 120 volts using 1.5% agarose gel. the gels were viewed under ultraviolet (uv) using gel documentation system (vacutec, sa) (online appendix 1, figure s1). bio-group and serotype identification the bio-group identification was performed by biochemical tests (lipase activity, esculin, indole, acid from d-xylose trehalose and nitrate reduction) as previously outlined by pham, bell and lanzarone (1991). serotyping was performed using a multiplex pcr (mpcr-1) assay that targets the four genes: wbbu, per, wbca and wzt as described by garzetti et al. (2014). a whole genome sequenced y. enterocolitica (pew01) was used as a positive control. primers and pcr conditions are indicated in online appendix 1, table s4. the pcr products were electrophoresed on a 3% agarose gel for 3 h at 100 volts. the gels were viewed under uv using gel documentation system (vacutec, sa). the presence of virulence-associated genes conventional pcr assays were performed to screen for the presence of 11 virulence-related genes (mpcr-2, mpcr-3, duplex and single-plex) (online appendix 1, table s4). the primer sequences, pcr conditions and the references are summarised in online appendix 1, table s4. a whole genome sequenced y. enterocolitica (pew01) was used as a positive control. the pcr products underwent gel electrophoresis on a 3% agarose gel and results were viewed using a gel documentation system (vacutec, sa) (online appendix 1, figure s2 and figure s3). phenotypic antimicrobial resistance test agricultural research council, onderstepoort veterinary research (arc-ovr): general bacteriology laboratory (national reference laboratory), various veterinarians and antimicrobial resistance (amr) experts in the country were consulted pertaining to which antibiotics are commonly used in the livestock production. a total of 12 antimicrobial agents were selected for this study as outlined in table 2. all confirmed isolates of y. enterocolitica were subjected to antimicrobial susceptibility test (ast) using kirby bauer disk diffusion method as described by european committee on antimicrobial susceptibility testing (eucast) guidelines (2021). the overnight pure cultures of y. enterocolitica on blood agar (thermofisher, johannesburg, sa) were inoculated into sterile saline solution tubes and diluted to the equivalent concentration of 0.5 mcfarland standard. the bacterial suspension was then inoculated aseptically on mueller–hinton agar plates (thermofisher, johannesburg, sa). six antibiotic discs were placed per inoculated plate followed by incubation at 30 °c for 24 h. after incubation, the zone of inhibition around individual discs was determined and interpreted as resistant or susceptible using the eucast guidelines for enterobacteriaceae. y. enterocolitica attc® 23715 and escherichia coli atcc® 25922 reference strains were used as a positive and negative controls, respectively. pcr amplification of amr genes conventional pcr assays were performed to screen for the presence of 18 amr genes as listed in online appendix 1, table s5. briefly, all pcr reactions were performed in a total volume of 25.0 μl, which contained 12.5 μl 2× taq master mix, 4.5 μl nuclease free water (with exception to β-lactams; 3.5 μl and tetracycline 5.5 μl), 5 μl template dna (40 ng/μl 100 ng/μl) and 0.5 μl of each primer. the pcr cycling conditions consisted of an initial denaturation at 94 °c for 3 min, 30 cycles of denaturation at 94 °c for 30 s, various annealing temperatures for the respective genes (online appendix 1, table s5), and extension at 72 °c for 1 min followed by a final extension at 72 °c for 10 min. the pcr products underwent gel electrophoresis on a 3% agarose gel and results were captured using a gel documentation system (vacutec, sa). statistical analysis the prevalence of 11 virulence genes and five serogroups were tested on 581 meat samples. the 581 meat samples were classified by location, animal species, sample category, sample type, and retail category. the meat samples were then grouped into 23 different categories using only location, animal species and sample category as classifying variables, as shown in online appendix 1, table s2. the recorded data was binary with 1 indicating positive results and 0 indicating negative results. chi-square test of equal proportions performed on each classifying variable, which is location, animal species, sample category, sample type and retail category. chi-square tests of association were performed between classifying variables and the bacterium, y. enterocolitica. a multiple correspondence analysis was performed on meat sample data with 23 possible categories of meat samples tested for 11 virulence genes and five serogroups. a multiple correspondence analysis was performed to study associations between virulence genes and serogroups. a multiple correspondence analysis was performed using xlstat software (version 2020.5, addinsoft, new york, us). the binary data was subjected to a chi-square test using frequency procedure (proc freq) of sas statistical software version 9.4 (sas institute inc., cary, nc, us). ethical considerations the protocol for this study was approved by the ethics committee of the onderstepoort veterinary research (approval number: 20.10) and university of south africa prior to the start of the study (approval number: 2021/caes_hrec/081, 10 may 2021. results prevalence of yersinia enterocolitica in meat and meat products of the total number of collected meat samples, 12% (70/581) were positive for y. enterocolitica. sixty percent (42/70) of the positive samples were from beef products and the remaining 28/70 (40%) were from pork products as indicated in table 1. there was no statistical difference (p = 0.6065) in the distribution of y. enterocolitica among tested animal species. the distribution of the positive samples by location revealed that y. enterocolitica was significantly (p = 0.0004) found in samples collected from rustenburg (n = 30; 20%) followed by emalahleni (n = 10; 15%) and pretoria (n = 30; 10%). all samples from bronkhospruit tested negative for y. enterocolitica (table 1). the prevalence of y. enterocolitica according to retail outlets size was found to be higher in samples from chain outlets 15% (n = 33) as compared to 14% (n = 23), 7% (n = 9) and 6% (n = 5) in large, small and medium outlets, respectively. however, no statistical difference (p = 0.0631) was reported in the sample types. table 1: prevalence of y. enterocolitica in various meat products. regarding the meat types, it was found that raw-intact meat products (n = 43; 15%) were highly contaminated with y. enterocolitica compared to raw-processed meat (n = 18; 11%) and ready-to-eat (rte) meats (n = 9; 7%). however, there was no statistical difference (p = 0.0934) observed in the contamination of various meat types by y. enterocolitica. the prevalence of y. enterocolitica in various sample types was found to be high in tripe (n = 7; 27%) followed by bone or skeleton tissues (n = 6; 18%), organs (n = 9; 16%), muscles (n = 21; 12%) and processed meat (n = 25; 10%) samples, while biltong showed extremely lower contamination level (n = 2; 5%). no statistical difference (p = 0.0758) was reported in the sample types. biotyping and serotyping of y. enterocolitica biotyping analysis showed that all 70 positive isolates belonged to biotype 1a, while the pcr serogroups yielded that 7% (n = 5) belonged to serotype o:8 (bioserotype 1a/o:8). the remaining isolates 93% (n = 65) were non-typeable. occurrence of virulence genes among y. enterocolitica isolates figure 1 shows the overall presence of various virulence genes examined in the current study. the yada, virf and ysta genes were not detected in all tested isolates; however, ymoa gene was predominantly detected in 80% (n = 56) of the isolates followed by ystb gene 70% (n = 49). the fepd, fepa, fes, ail, inv and myfa genes were found in 59% (n = 41), 56% (n = 39), 31% (n = 22), 29% (n = 20), 19% (n = 13) and 14% (n = 10) of the isolates, respectively. figure 1: distribution of virulence genes in isolates of y. enterocolitica. prevalence of resistance to antimicrobial agents general antimicrobial resistance prevalence overall, the resistance of the 70 y. enterocolitica isolates to 12 antibiotics was determined and the results are presented in table 2. among the tested antibiotics, the highest resistance of y. enterocolitica isolates was observed in ampicillin 94% (n = 66), followed by cephalothin 83% (n = 58), amoxycillin 41% (n = 29) and tetracycline 19% (n = 13). low levels of resistance were recorded against a wide range of antibiotics including imipinem 3% (n = 2), gentamycin 3% (n = 2), piperacillin 1% (n = 1), amikacin 1% (n = 1), aztreonam 1% (n = 1) and ciprofloxacin 1% (n = 1). no resistant isolates were observed against trimethoprim-sulphamethoxazole. table 2: classes and concentrations of antimicrobial agents used in the study. frequency of antimicrobial resistant y. enterocolitica isolates by meat types among the tested antibiotics against isolates recovered from various meat types, the highest resistance to ampicillin range from 100% for raw processed meat to 93% (n = 40) for raw intact meat and 89% (n = 8) for rte meats. it was further found that 89% (n = 8) of isolates from both rte and raw processed meats were highly resistant towards cephalothin as compared to raw intact (n = 34; 79%) meat isolates. further resistance was observed in raw meat isolates (n = 20; 47%) towards amoxycillin while a less and even more lower resistance was observed in processed (n = 7; 39%) and rte (n = 2; 22%) isolates, respectively. no statistical significance was observed against various antibiotics and meat types (data was not shown in the table). frequency of resistant strains by serotype of y. enterocolitica five isolates belonging to bioserotype 1a/0:8 were also subjected to different antibiotics of which all (n = 5; 100%) were resistant towards ampicillin. it was further observed that 60% (n = 3) were resistant towards cephalothin while 40% (n = 2) were resistant towards amoxycillin and tetracycline, respectively. prevalence of resistance patterns and multi-resistant isolates of y. enterocolitica a total of 12 resistance patterns were detected in the y. enterocolitica isolates. the predominant resistance pattern of cephalothin-amoxycillin-ampicillin (kf-amc-amp) was reported in isolates at a frequency of 27% (n = 16) and 24% (n = 6) for beef and pork respectively (table 3). other predominant patterns included cephalothin-ampicillin (kf-amp) and tetracycline-ampicillin (te-amp). the overall, mar (multi antibiotic resistance) frequency was 13% (n = 9/70) with one isolate exhibiting resistance against 10 different antimicrobial agents (ipm-kf-te-ak-c-cip-cn-amc-tzp-amp). the occurrence of mar in beef and pork isolates was 17% (n = 7/42) and 7% (n = 2/28), respectively. the number of antibiotics observed in resistance patterns for mar isolates differed ranging from 3 to 10 (table 3). table 3: resistance patterns and multi-antibiotic resistance exhibited by y. enterocolitica isolates recovered from various meat types. antimicrobial resistance genes seventy isolates were screened against 18 antimicrobial resistance genes using pcr. of the 70 isolates, 40% (n = 28), 21% (n = 15), 9% (n = 6) and 7% (n = 5) harboured blatem, cmla, blashv (β-lactams) and tetb (tetracycline) genes respectively. the other genes were either detected in 4% (n = 3) or less as shown in table 4. all 1a/0:8 isolates showed various distribution patterns towards amr genes with blatem detected in 60% (n = 3) while sul3 and qnra were each detected in 40% (n = 2) of the isolates (table 4). table 4: distribution of antimicrobial resistance genes in y. enterocolitica. discussion this study investigated the prevalence and characteristics of y. enterocolitica in meat and meat products recovered from retail outlets. the overall prevalence of 12% was recorded which was lower than 18% recently reported by madoroba et al. (2021). the variation in prevalence between these studies was mainly because of differences in sample size, meat matrices tested, and geographical locations. compared to the current study, a lower prevalence has been reported in egypt at 6% (younis, elkenany & dowidar 2021), france at 5% (esnault et al. 2018), and india at 1% (latha et al. 2017). the high prevalence in the current study is very alarming and poses a potential serious public health hazard in the country. lack of surveillance programmes in meat products, inadequate hygiene and absence of one health policies may be the contributing factors. y. enterocolitica is a zoonotic pathogen that can be transmitted from animal reservoirs to humans, along the food supply chain. y. enterocolitica was arbitrarily present in beef (13%) samples than in pork (11%) which is inconsistent with previous studies that reported lower prevalence in beef and pork samples. for instance, yang et al. (2013) reported a prevalence of 2% and 1% from beef and pork samples respectively in korea, while latha et al. (2017) found y. enterocolitica in india at 1% for both beef and pork samples. a study by odoi et al. (2021) in japan reported high prevalence of y. enterocolitica at 28% for beef and 21% for pork samples. pork and pork-based products have been found to be contaminated with high proportion of y. enterocolitica as pigs can harbour this pathogen for long periods without showing any clinical signs (laukkanen-ninios, fredriksson-ahomaa & korkeala 2014; moreira et al. 2019). the results of our study suggest that the distribution of y. enterocolitica varied among tested samples with beef products having a higher potential of causing yersiniosis but more importantly highlight that it will be a strong incentive to monitor the occurrence of y. enterocolitica in various animal species and their products other than the common reservoirs (moreira et al. 2019). prevalence of y. enterocolitica varied significantly in different cities. it is common for bacteria to be unevenly distributed because of various factors including geographical and the intrinsic strain characteristics. the results of the present study provide a baseline information on geographical distribution and molecular characterisation of this pathogen in south africa. this information is important for future epidemiological studies and also informs on which cities will require more attention in terms of surveillance, control and prevention measures to avoid possible outbreaks. the current study revealed that the prevalence of y. enterocolitica was significant between various retail outlet categories with chain and large accounting for the highest contamination proportions compared to small and medium outlets. the variation from various retail outlets is largely influenced by hygiene standards of meat processing and handling in individual facility (indrawattana et al. 2011). the high contamination from large and chain retail outlets highlights the necessity for outlet management and workers to always be on guard to eliminate public health risks. the recovery of y. enterocolitica from retail meat and meat products does not necessarily mean that contamination took place in the retail environment (sauders et al. 2009). however, re-contamination from working areas, tools, machinery, operators and survival of strains under harsh conditions have been reported as the main source of y. enterocolitica in retail products. good animal husbandry, good hygiene and effective sanitation applications across meat value chain can minimise bacterial load in meat. however, in sa, some of the good hygiene practices were observed to be inadequate along meat value chain (matle et al. 2019) which could be the reason for high prevalence of y. enterocolitica in retail outlets. there was a disproportionally high contamination in raw intact meat (15%) as expected because similar results were reported elsewhere (fukushima et al. 1997; siriken 2004). contamination rate in raw processed (11%) and rte (7%) meats is a concern as majority of human yersiniosis cases have been linked to these products (grahek-ogden et al. 2007). the high prevalence of y. enterocolitica in rte meat products can pose serious health risk to consumers as these products are consumed without any further cooking or pathogen inactivation. the current results are inconsistent with those reported by madoroba et al. (2021). a study conducted in italy reported contamination at 1% and 3% in raw and processed meat respectively (bilge & leyla 2010), while younis, mady and awad (2019) reported contamination at 13% and 10% in raw and processed meat respectively in egypt. it has been widely documented that poor environmental conditions, contaminated working areas, tools, workers’ hands and storage conditions are the fuelling factors towards high contamination of meat and meat products along meat value chain (diyantoro & wardhana 2019; fasanmi et al. 2018; sabina et al. 2011). because of the wide spectrum of meat matrices analysed in the current study, samples were grouped into six sample types with the highest contamination rates reported in tripe, bone or skeleton tissue, organs and muscle samples. processed samples consisted of wide range of products such as minced meat, wors, cabanossi, russian, patties, burger, polony and vienna, which accounted for more than 55% of all meat products produced in the country (http://www.statssa.gov.za/publications/p03101/p031012019.pdf). the high presence of y. enterocolitica in tripe is of great concern. matle (2016) reported that 57% – 67% of sa population consume tripe as a staple food especially in the winter season. the presence of y. enterocolitica in processed meat and biltong samples is alarming as these products are consumed by many people including children; naidoo and lindsay (2010) have reported these products as the most commonly consumed rte meat product in sa. the results show a need for proper surveillance of y. enterocolitica in products such as biltong so that the extent of contamination can be determined in these products. this is important for public health and regulatory standpoints. the prevalence of y. enterocolitica in products such as tripe, has previously been reported by nortjé et al. (1999) in their assessment of the incidence of y. enterocolitica in offal from gauteng province which was lower compared to the results from this study. erickson et al. (2019) also evaluated the prevalence of y. enterocolitica from meat products in the us in which they reported the presence of 1% and 7% in tripe and organs, respectively. bio typing and serotyping of y. enterocolitica characterisation of y. enterocolitica into biotypes and serogroups is necessary for distinguishing virulent from avirulent strains. the differentiation is also useful for source tracking of the strains that are implicated in human outbreaks and provides epidemiological markers that are critical for disease investigations (nadon et al. 2001). the occurrence of biotype 1a which was identified in all the isolates from present study is in agreement with the general prevalence of this biotype found in meat products from retail outlets as reported in other studies (grant, bennett-wood & robins-browne 1999; kraushaar et al. 2011; pham et al. 1991). biotype 1a strains are widely distributed in the environment and have frequently been isolated from food samples of animal origin and symptomatic humans (stephan et al. 2013). certain strains of biotype 1a can cause sporadic extra-intestinal infections and have been implicated in gastrointestinal outbreaks (tennant, grant & robins-browne 2003). even after many years of clinical isolation of biotype 1a, the pathogenicity of this biotype is still poorly understood (sabina et al. 2011). the importance of the finding of our study is that it provided valuable information into y. enterocolitica strain diversity found in meat products consumed in the country which can be used for policy making decision. biotype 1a is the most heterogeneous of the six biotypes of y. enterocolitica and its most common serotypes are o:5, o:7, o:8, o:10 and non-typeable strains (paixão et al. 2013; sihvonen 2014). the current study reported that 7% of isolates belonged to bioserotype 1a/o:8 while 93% were non-typeable. bioserotype 1a/o:8 has seldom been reported in clinical cases (thong et al. 2018); however, its role in causing infection in human should not be underestimated. virulence genes the pathogenicity of y. enterocolitica is attributed to the expression of plasmid and chromosomal virulence genes. biotype 1a is traditionally regarded avirulent because it does not carry ail, inva, ysta, yada and virf genes which are known to be associated with pathogenicity in y. enterocolitica (bancerz-kisiel et al. 2018). inva, ail, ystb were detected in 19%, 29% and 70% of the isolates. other important gene in pathogenicity of y. enterocolitica is myfa which was found in 14% of our isolates. these genes (inv, ail and myfa genes) are involved in infection stages such as adhesion, invasion and enhancement of epithelial cell penetration as well as evasion of the immune system (drummond et al. 2012; uliczka et al. 2011; younis et al. 2019) while ystb is responsible for causing diarrhoea (bancerz-kisiel et al. 2018; sabina et al. 2011). batzilla et al. (2011) argued that biotype 1a represents a potential group of emerging y. enterocolitica strains that share known and putative virulence-associated features with the pathogenic bioserotypes which seems to be true in the current isolates. the detected biotype 1a strain reported in the current study is of concern as they carry genes that are important for initiating infections in human. wang et al. (2021) investigated the presence of virulence genes in 51 biotype 1a isolates in china and reported that ymoa, fepd and fes while ail, ysta, yada and virf were negative. ye et al. (2015) examined 11 virulence genes on 70 biotype 1a isolates from china, in which all strains lacked ysta, yada and virf. in switzerland (stephan et al. 2013), a study was carried out on 21 y. enterocolitica biotype 1a for the presence of yada, virf, ail, ysta, ystb and myfa of which all genes except ystb was not found. phenotypic antimicrobial resistance it is well known that most strains of y. enterocolitica exhibit resistance to β-lactam antibiotics, such as ampicillin and cephalothin (fàbrega, ballesté-delpierre & vila 2015). the high prevalence of resistance to ampicillin, amoxicillin and cephalothin in our study agrees with published reports of beef and pork-associated y. enterocolitica by others in egypt (younis et al. 2019), china (ye et al. 2015) and greece (gousia et al. 2011). the high prevalence of resistance to ampicillin, amoxicillin and cephalothin among y. enterocolitica detected in the current study was as expected. tetracycline which is one of the important antibiotics used for the treatment of human yersiniosis (fàbrega et al. 2015) is also among the extensively used antibiotics in sa for the treatment of bacterial infections in production animals (mokgophi et al. 2021). in the present study, 19% of the isolates were resistant to tetracycline which was higher than that reported by ye et al. (2015) and aghamohammad et al. (2015) of 6% and 13%, respectively. this extensive use may be propelled by the fact that tetracyclines are relatively cheap, easily accessible and their use in veterinary practice as stock remedies have been permitted by the south africa fertilizers, farm feed, agricultural and stock remedies act 24 of 1977 as over-counter medication. the 19% of resistance reported in our study against tetracycline might be attributed to widespread application of this antibiotic in the country (sabtu, enoch & brown 2015). less than 3% resistance rates were observed in imipinem, gentamycin, piperacillin, chloramphenicol, amikacin, aztreonam and ciprofloxacin, while all isolates were susceptible to trimethoprim-sulphamethoxazole. the low level of resistant y. enterocolitica against imipinem, gentamycin, piperacillin, chloramphenicol, amikacin, ciprofloxacin, aztreonam and trimethoprim-sulphamethoxazole could be because of their infrequent use in animal production (henton et al. 2011). similar prevalence of resistance to antimicrobial agents has been reported for y. enterocolitica strains recovered from food-producing animals in other countries (li et al. 2018; sharifi et al. 2011; ye et al. 2015). heterogeneity of the antimicrobial resistance pattern is thought to be depending on several factors including the bioserotypes and geographical distribution (fàbrega et al. 2015). in the present study, five y. enterocolitica 1a/0:8 isolates were resistant to ampicillin (100%), cephalothin (60%), amoxycillin (40%) and tetracycline (40%). these results were consistent with previous studies elsewhere in the world (bonardi et al. 2010; li et al. 2018). a study carried out in italy, tested nine y. enterocolitica 1a/o:8 isolates and reported 55% and 22% resistance rate in ampicillin and amoxycillin, respectively (bonardi et al. 2010), which was lower than that of our study. on the contrary, li et al. (2018) examined three y. enterocolitica 1a/o:8 isolates of which all were resistant to amoxycillin and cephalothin while 67% were resistant to ampicillin. multi antibiotic resistance patterns in bacteria have made treatment with common antimicrobials very difficult (frieri, kumar & boutin 2017). in this study, 12 resistant patterns were observed of which three most common patterns kf-amc-amp, kf-amp and te-amp were exhibited by 31%, 30% and 17% of isolates, respectively, while other patterns were carried by 9% or less isolates. the extensive and popular use of conventional antibiotics in production animals to prevent diseases and stimulate growth could be the contributing factors towards high occurrence of mar and resistance rates towards β-lactams and tetracyclines in sa which was observed in the current study. furthermore, easy and unauthorised accessibility to these antibiotics may also play a role in high resistance rates. the capabilities of antibiotics in controlling fatalities caused by important human and animal infections may be reduced because of high mdr occurrences thereby posing a public health risk (doyle, lennox & bell 2013) which may worsen in immunocompromised people because of their high susceptibility to infections (olaniran, nzimande & mkize 2015). this is highly concerning as over 30% of south africans are immunocompromised because of ageing, pregnancy and comorbidities such as hiv and tb (http://www.statssa.gov.za/publications/p03101/p031012019.pdf). genotypic antimicrobial resistance plasmid-borne genes (blatem, blacmy-2, blashv and blapse) coding for resistance to β-lactams and extended-spectrum cephalosporins (cephalothin) were detected at different frequencies in this study with 40% of the isolates harbouring blatem gene. the blatem gene is known to encode tem β-lactamase enzyme which is responsible for conferring resistance to penicillin family (bailey et al. 2011; ejaz et al. 2021). in accordance, a study in poland (kot & rainko 2009) reported the presence of this gene in y. enterocolitica isolates from food samples. moreover, phenotypic intrinsic resistance of y. enterocolitica to ampicillin and cephalothin observed in this study might be because of blatem gene. blacmy-2, blashv, blapse and tetb showed a relatively low frequency (> 9%) which is consistent with previous studies on gram negative bacterium (dougnon et al. 2021; santos et al. 2020). other significant antimicrobial gene found in this study was cmla which was detected in 21% of our isolates. the cmla gene is responsible for resistance in chloramphenicol via a non-enzymatic mechanism and membrane-linked efflux proteins. these are responsible for the selective pumping of antibiotics from the bacteria cytoplasmic matrix thereby reducing accessibility to cell organelles (møller et al. 2016; white et al. 2000). other genes that need to be highlighted are those that encode for phenicols, quinolones, trimethoprim and sulphonamides resistance. almost all y. enterocolitica strains in this study have displayed very low frequency (< 4%) or absence of those genes (table 4). absence or very low frequency for genes encoding resistance against those groups of antibiotics has been reported in majority of previous studies regardless of the source of isolation. these findings are not surprising as they correlate with low resistance level reported by phenotypic results. conclusion this study is arguably among the first in the country to investigate both the phenotypic and genotypic characteristics as well as antimicrobial profiling of y. enterocolitica isolated from retail beef and pork meat products. therefore, it can be concluded that retail beef, pork meat and meat products carry potentially pathogenic y. enterocolitica bioserotype 1a/o:8. in most cases, these bioserotypes harboured various virulence genes known to be associated with human yersiniosis, thus presenting a potential public health risk in the country. it can also be concluded that y. enterocolitica isolates in this study showed resistance to most clinically significant antibiotics. adherence to proper hygiene practices across the meat value chain can reduce y. enterocolitica contamination of beef and pork products. acknowledgements the authors acknowledge gauteng department of agriculture and rural development (gdard, republic of south africa) for partly funding this study under project p10000153. we also like to thank arc-ovr for providing all research facilities for this work. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions study conceptualisation and supervision: i.m and d.m.; methodology, e.m, l.d, k.j, l.m, m.m.; software and formal analysis: n.n; writing – original draft preparation, e.m and i.m.; writing – review and editing: l.d.; k.j. k.m and d.m.; funding acquisition, i.m. all authors have read and agreed to the 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https://doi.org/10.1590/1519-6984.229998 zdolec, n. & kiš, m., 2021, ‘meat safety from farm to slaughter – risk-based control of yersinia enterocolitica and toxoplasma gondii’, processes 9(5), 815. https://doi.org/10.3390/pr9050815 article information authors: thelma meiring1,2 leon prozesky2 eben r. du preez3 dirk j. verwoerd4 affiliations: 1idexx laboratories, onderstepoort, south africa 2department of paraclinical sciences, university of pretoria, south africa 3sd morris consulting services, johannesburg, south africa 4karan beef feedlot, heidelberg, south africa correspondence to: thelma meiring email: thelma@idexxsa.co.za postal address: po box 12731, onderstepoort 0110, south africa dates: received: 17 feb. 2011 accepted: 17 mar. 2011 published: 25 aug. 2011 how to cite this article: meiring, t., prozesky, l., du preez, e.r. & verwoerd, d.j., 2011, ‘the diagnosis and prevalence of persistent infection with bovine viral diarrhoea virus in south african feedlot cattle’, onderstepoort journal of veterinary research 78(1), art. #323, 8 pages. doi:10.4102/ojvr.v78i1.323 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: (online) the diagnosis and prevalence of persistent infection with bovine viral diarrhoea virus in south african feedlot cattle in this original research... open access • abstract • introduction • materials and methods    • collection of specimens    • immunohistochemistry staining    • enzyme-linked immunosorbent assays • results    • immunohistochemistry staining    • enzyme-linked immunosorbent assays • discussion • conclusion • acknowledgements • references abstract (back to top) bovine viral diarrhoea virus (bvdv) infection is an important viral infection affecting the cattle industry today. the prevalence of this infection in south african feedlots is unknown. ear notch biopsies were collected from chronic poor doers and animals that appeared unthrifty upon entering feedlots, as well as animals entering the hospital pen with respiratory disease for the first time. a total of 1690 samples were collected: 1074 from the former category and 616 from the latter. a routine immunohistochemistry staining protocol showed that 49 animals tested positive, of which 43 (4%) came from the feedlot entry group and six (1%) from the hospitalised group. the prevalence of persistently infected cattle from this selected, nonrandom sample entering six large south african feedlots was found to be 2.9%, which is higher than the international rule of thumb that 0.5% of all cattle entering feedlots are persistently infected. there was no clear correlation between persistent infection and respiratory disease. serum samples were also collected when possible and 10 positive cases were found. results from enzyme-linked immunosorbent assays for antigen and antibody performed on these sera correlated well with those from the immunohistochemistry staining method in six cases, but in four cases the animals tested falsely positive owing to nonspecific staining. immunohistochemistry staining on ear notch biopsies is thus a reliable diagnostic method to identify persistently infected animals with bvdv, but the pathologist should be aware of nonspecific positive staining. introduction (back to top) bovine viral diarrhoea virus (bvdv) is one of the most economically important pathogens in the cattle industry today (firat et al. 2002; hilbe et al. 2007; houe, lindberg & moennig 2006; radostits et al. 2000; van vuuren 2005). it causes a multitude of different diseases including subclinical benign diarrhoea (bovine virus diarrhoea), peracute highly fatal diarrhoea, haemorrhagic and thrombocytopaenic disease, reproductive failure, fatal mucosal disease of persistently infected (pi) animals infected early in utero, and abortions and malformations (firat et al. 2002; radostits et al. 2000). in addition, bvdv causes a general immunosuppression (peterhans, jungi & schweizer 2003) that is strongly associated with chronic nonresponsive respiratory disease in feedlots (haines et al. 2001). bvdv is a member of the genus pestivirus (family flaviviridae). there are two biotypes, designated as noncytopathic (ncp) and cytopathic (cp), depending on their effect on tissue culture cells. the ncp type is the most common and the most important and is also the one causing persistent infection in animals. there is considerable antigenic diversity and cross-reactivity amongst isolates of bvdv. the virus has been divided into genotypes i and ii, depending on antigenic and genetic differences (kampa et al. 2007). both ncp and cp isolates exist within each genotype. genotype i has recently been subdivided into two subgenotypes, bvdv1a and bvdv1b (potgieter 2004; radostits et al. 2000). additional subgenotypes have been proposed for strains that are unique to southern africa (potgieter 2004). the ncp type of bvdv is predominant in nature (firat et al. 2002; radostits et al. 2000). when the ncp virus infects the foetus between day 42 and day 125, the immune system develops a tolerance towards the virus and an immune response to that specific bvdv strain does not develop. (hilbe et al. 2007; kampa et al. 2007; luzzago et al. 2006; mcclurkin et al. 1984). the calves are born persistently viraemic and continue to shed virus every day for the rest of their lives. the neonatal mortality of pi calves is high and they are often born weak (moczygemba 2003). others are normal and healthy at birth. fatal mucosal disease may develop within 6–24 months of age if the animal is super-infected with a homologous strain of a cp virus or if the ncp virus in the body mutates to a homologous cp virus (potgieter 2004; radostits et al. 2000). the bvdv is of economic importance because of associated abortion, congenital defects, still births, increased neonatal mortality, prenatal and postnatal growth retardation, suboptimal reproductive performance, death from mucosal disease and premature disposal of pi animals (cornish et al. 2005; firat et al. 2002; hilbe et al. 2007; radostits et al. 2000). economic impact specifically associated with feedlots includes expenses incurred for treatment, additional labour (care and movement of animals), premature culling because of chronic disease (repeat treatments) and profit reduction because of reduced growth and performance during and after illness (hessman et al. 2009). pens containing a single pi animal experience a pull rate (animals removed to hospital pen because of apparent illness) that is 33% higher than pens without pi animals. pens adjacent to those with pi animals also experience a similar increase in morbidity rate or pull rate. loneragan et al. (2005) found a pull rate incidence as high as 43%. the rule of thumb in the united states feedlot industry is that 1% of calves born per year in an infected herd are pi animals. about half of these are expected to die before weaning, which sets the industry’s average intake rate of pi animals at 0.5% (ishmael 2003). feedlots are particularly at risk of receiving pi animals. although pi calves can present as stunted animals with an unthrifty coat, not all pi animals are in poor condition and it is therefore not possible to assess infection status exclusively from the physical appearance of an animal (potgieter 2004). the real danger associated with pi animals is that they are persistently viraemic and immunosuppressed and shed virus either constantly or intermittently, thus being the main source of infection for other animals (cornish et al. 2005; kampa et al. 2007; loneragan et al. 2005; luzzago et al. 2006; van vuuren 2005). both brock et al. (1998) and moczygemba (2003) suggest that the levels of viraemia in pi calves are cyclic and that higher levels of virus and shedding may occur under stressful situations, for example, transport and entry into a feedlot. niskanen, lindberg & traven (2002) infected 10 calves with bvdv and bovine coronavirus (bcv) and put them with noninfected calves. none of the latter calves contracted bvdv, but all became bcv positive. the finding suggests that acutely infected animals do not secrete bvdv effectively and appear not to be a major source of infection for other animals; rather, pi animals appear to be the major source of bvdv infection (potgieter 2004). bvdv infects the lymphoid system and is immuno-suppressive (potgieter 2004; ridpath et al. 2002) as it infects the dendritic or stromal macrophages that support and nurse lymphoid cells during their development. damage to these cells destroys the lymphoid population, which leads to lymphopaenia and immunosuppression (wren 2001). immunohistochemistry (ihc) staining of skin biopsies is a reliable, fast diagnostic tool to identify the presence of bvdv antigen (hilbe et al. 2007; houe et al. 2006; luzzago et al. 2006; thur, zlinszky & ehrensperger 1996). researchers have sampled skin from various locations on pi animals and found that results were identical, irrespective of the location from which the sample was collected (thur et al. 1996). the presence of bvdv antigen in keratinocytes in the stratum basale and stratum spinosum has been documented in clinically normal pi cattle (bielefeldt ohmann 1988; dubois et al. 2000; sandvik 1999; thur et al. 1996). according to njaa et al. (2000), bvdv has a tropism for lymphocytes, mononuclear phagocytes and epithelial cells. in their study of formalin-fixed skin biopsies, positive ihc staining for bvdv was found in the cytoplasm of keratinocytes, sebaceous epithelial cells, mononuclear cells in the dermis and vascular smooth muscle cells. the epidermal staining occurred diffusely and predominantly in the stratum basale and stratum spinosum. the most prominent staining was found in the isthmus and infundibulum of hair follicles. previous studies showed ihc staining to be as reliable as virus isolation (vi) to detect pi cattle (broderson, white & smith 1998; thur et al. 1996). the advantages of ihc staining are that tissues are fixed in formalin and the procedure is more rapid and economical than vi. there is little chance of identifying an acutely infected animal as falsely positive according to skin biopsy. njaa et al. (2000) found positive ihc staining for bvdv on four skin samples in animals acutely infected with a high dose of bvdv. the staining was, however, confined to a few small, discrete foci in the stratum spinosum, with little extension into follicular ostia, and was distinguishable from the more extensive staining in pi animals. libler-tenorio, ridpath and neill (2003) found that bvdv antigen in inoculated calves was present only in lymphoid tissue and the intestinal mucosa but not the skin. ridpath et al. (2002) infected animals with six different isolates and could not detect virus in any of the skin biopsies. hilbe et al. (2007) detected positive staining only in the skin of pi animals and no positive stains in acutely infected animals. based on these results pi animals are rarely expected to be confused with acutely infected animals when skin biopsies are used, which suggests that the technique can be used as a reliable, fast diagnostic tool to diagnose pi animals (luzzago et al. 2006). antigen-capture enzyme-linked immunosorbent assay (elisa) can also be used to test for the presence of bvdv antigen and several different elisas are commercially available (brinkhof, zimmer & westenbrink 1996; brock 1995; hilbe et al. 2007; sandvik 1999). elisa is a versatile diagnostic method to detect almost any immunoreactive molecule and can also be used to detect serum antibodies (hilbe et al. 2007). clinically healthy pi calves older than 2 months are nearly always elisa-negative for antibodies (sandvik 1999). exceptions occur when pi calves ingest high titres of maternal antibodies to a different strain and some of these persist for longer than 3 months, or when exposed to different field or vaccine strains, the pi calf mounts a (poor) humoural response. the objectives of this study were to: • determine, by means of ihc evaluation of skin biopsies, the prevalence of pi animals entering feedlots • determine whether pi animals are at higher risk of contracting respiratory disease in feedlots than noninfected animals • determine the reliability of ihc by investigating nonspecific staining or staining of few mononuclear cells with re-evaluation and/or restaining with another colour • determine the reliability of ihc staining for identifying pi animals in comparison with the antibody and antigen capture elisas. materials and methods (back to top) collection of specimens ear notch biopsies were collected from calves at the point of entry into feedlots, from calves presented to the hospital pen for the first time and from chronic poor doers from the following feedlots: karan beef, mollevel, taaiboschbult, gysbertshoek, hurland and sparta beef. the calves selected at point of entry were poor looking with a dull coat, unthrifty, thin, and potbellied and were suspected to be persistently bvdv infected. the chronic poor doers were animals that had received more than three treatments in the hospital pen and were placed on pasture because of poor performance in the feedlot. dead animals in this group were also sampled. a total of 1074 specimens were collected from first-time entrants and chronic poor doers between march 2005 and october 2005, and 616 from animals entering the hospital pen for the first time because of respiratory disease between may 2006 and august 2006. ear notch samples measuring 10 mm x 5 mm collected with an ear tag applicator were fixed in 10% buffered formalin and presented to the laboratory for examination, within 3–4 weeks after collection. ear notch samples were taken because of the ease of collection, but biopsy specimens could have been taken from any skin area, as bvdv antigen is evenly distributed in skin samples (thur et al. 1996). immunohistochemistry staining sections were cut and embedded in wax blocks by routine standard operating procedures at the ihc laboratory of the pathology section, faculty of veterinary science, onderstepoort. seven specimens were placed in a single wax block as demonstrated in figure 1. each animal could thus be identified individually and costs were minimised. figure 1: wax block demonstration. the abc vector elite immunoperoxidase staining procedure was used (vectastain universal elite abc kit from merck, vector laboratories, uk), shown in figure 2. figure 2: abc vector elite immunoperoxidase staining procedure. sections of 3–4 μm were cut from the wax blocks. the tissues were then mounted on pretreated superfrost plus glass slides for immunoperoxidase staining. the slides were dried overnight in an oven at 58 °c to enhance tissue section adhesion. routine dewaxing and rehydration was performed (10 min in xylol and 3 min each in 100%, 96% and 70% alcohol according to the modified version of the method suggested by bancroft and stevens [1982]). the abc vector elite immunoperoxidase staining procedure, as described earlier, was used (figure 2). after dewaxing and rehydration antigen was demasked according to the proteolytic enzyme digestion (pronase) method. a 3% hydrogen peroxide solution (in distilled water) was used for 5 min to block endogenous peroxidases. sections were rinsed twice: once in distilled water and once in phosphate buffered saline – bovine serum albumin (pbs–bsa) buffer for 5 min per rinse. the pronase (sigma protease type xiv) solution was prepared by adding 50 mg of the pronase powder to 100 ml of pbs–bsa buffer that had been heated to 37 °c. the sections were incubated in the pronase–buffer solution in the oven at 37 °c for 30 min. sections were then rinsed in distilled water and then in pbs–bsa buffer for 5 min per rinse. a drop of refrigerated normal rabbit serum was added to 10 drops pbs–bsa buffer, placed directly on sections and left for 20 min. the excess normal serum was gently shaken off the slides and slides were gently wiped clean around tissue sections. the primary monoclonal bvd antibody (cornell university, new york) was applied to all the appropriate sections at a dilution of 1:1000 and left to stain for 1.5 h on the bench at room temperature. negative controls were prepared using only pbs–bsa buffer or an irrelevant monoclonal antibody. the sections were rinsed twice; once in distilled water and once in pbs–bsa buffer for 5 min each. the secondary biotinylated antiserum (rabbit anti-mouse antibody, diluted at 1:500) was applied on sections for 30 min and left at room temperature. the sections were then rinsed twice; with distilled water followed by pbs–bsa buffer for 5 min each. peroxidase-conjugated avidin (vector laboratories, uk) was then applied (prepared according to the manufacturer’s instructions) for 30 min on the bench at room temperature. the slides were rinsed twice as described before. the sections were placed in a diaminobenzidine (dab) or novared substrate (using a droplet method) for approximately 1 min. as soon as clear positive staining was observed macroscopically on the positive control slide, all other slides were rinsed in distilled water. the sections were then counterstained with haematoxylin for 3–4 min. the sections were then rinsed under running tap water for 10 min to remove excess dab or novared substrate followed by routine dehydration through increased alcohol concentrations (70%, 96% and 100%) and xylol (vector laboratories, uk). sections were then mounted and cover with a cover slip. enzyme-linked immunosorbent assays both bvdv antibody elisas and bvdv antigen capture elisas (idexx laboratories bvdv antibody test kit, zul.no.bgvv-b233 version 06-44000-02 and idexx laboratories bvdv antigen test kit/serum plus, zul.no.bgvv-b230 version 06-43860-02, respectively) were performed according to standard operating procedures by golden vetlab (now idexx south africa), woodmead, south africa. results (back to top) immunohistochemistry staining in total, 1690 ear notch biopsies were received for processing and ihc staining (1074 from calves entering feedlots and chronic poor doers; 616 from first-time admissions to the hospital because of respiratory disease). of the total sample, 49 (2.9%) stained positive for bvdv antigen; 43 positive stains were found amongst the feedlot entrants and six positive stains were found amongst the samples from the hospitalised calves. cases that showed granular dab or novared-coloured staining in the keratinocytes of the epidermis, epithelial cells in hair follicles, smooth muscle cells and fibroblasts were regarded as positive (njaa et al. 2000). figures 3–6 demonstrate positive staining observed with dab or novared. figure 3: positive diaminobenzidine staining in the keratinocytes of the epidermis (top arrow) and hair follicle epithelium (middle and bottom arrow) of a persistently infected calf (magnification: 40x). figure 4: positive novared staining in the epidermis (top arrow) and hair follicle epithelium (bottom arrow) of a persistently infected calf (magnification: 40x). figure 5: positive novared-stained fibroblasts in the dermis (arrows) of a persistently infected calf (magnification: 40x). figure 6: positive novared staining of hair follicle epithelium (arrows) in a persistently infected calf (magnification: 40x). all samples were evaluated twice, because an abnormally high number of cases appeared to stain positively in one group at the first evaluation. in this group (from taaiboschbult) 75 of 154 cases apparently stained positive. this raised concern and the staining patterns were investigated. it was concluded that a high number of cases showed nonspecific positive staining (vide infra). after re-evaluation, as described below, only 12 positive cases were identified. nonspecific staining was defined as nongranular dab or novared staining visible mainly in spindle and round cells in the dermis (figure 7). in the samples investigatde during this study only spindle-shaped cells and round cells stained positive and no positive staining was evident in keratinocytes, hair follicle epithelial cells or smooth muscle cells. figure 7: nonspecific positive novared staining of mast cells in the dermis (arrow; magnification: 40x). positive staining of mast cell granules was suspected (s. clift, pers. comm.), which was confirmed after staining the sections with ziehl–neelsen (zn) stain (figure 8). most of the bovine mast cells in the skin were spindle shaped, which was confirmed with zn staining. mast cell granules stain blue (basophilic) with the zn stain (university of rochester 2007). figure 8: ziehl–neelsen basophilic granular staining of spindle-shaped mast cells in the dermis (arrows; magnification: 40x). mast cells stained positive more often with the dab colour marker than with novared. the majority of the nonspecific cases were stained with dab. another problem with using dab as colour marker was that the colour pigment was sometimes difficult to distinguish from melanin pigment in the skin (yellow to golden colour). novared was preferred as colour marker in this study as its unique brick-red colour precludes confusion with any other. enzyme-linked immunosorbent assays serum samples to test for bvdv antibodies and antigens were available from only 10 of the animals that originally tested positive during ihc staining. the results are presented in table 1. table 1: results of enzyme-linked immunosorbent assays for bovine viral diarrhoea virus. of the 10 animals that tested positive during ihc staining, only six tested positive for viral antigen with the elisa. pi animals will always be antigen positive, as they are consistently viraemic. this result indicated that animals 6, 7, 8 and 9 were not pi. these cases were re-evaluated, and as discussed later, they showed nonspecific positive staining in mast cells, leading to the false positive diagnosis. a pi animal will not develop antibodies against the strain of bvdv that infected it in utero (mcclurkin et al. 1984). of the six animals that tested positive for viral antigen, three did not have antibodies against bvdv, which confirms their pi status. however, if a pi animal is infected with a significantly different strain of bvdv after birth, it could develop antibodies to that strain (mcclurkin et al. 1984). this explains why three animals that tested positive for viral antigen during elisa and ihc staining, also tested positive for antibodies against bvdv. discussion (back to top) ihc staining on ear notch biopsies is considered a fast and accurate diagnostic tool to identify animals that are persistently bvdv infected (hilbe et al. 2007; houe et al. 2006; luzzago et al. 2006; thur et al. 1996). the overall prevalence of pi calves entering feedlots in this study was 2.9%, which is much higher than the 0.5% estimate of ishmael (2003). the estimate was based on the assumption that 1% pi animals are born into infected herds per year and that half of the animals are expected to die before weaning. less than 0.5% of such animals will thus be available to enter feedlots owing to the early deaths of pi animals. unpublished data from a south african study in july 2004 indicate a prevalence of pi animals of 0.56% in a single feedlot based on 2994 samples randomly collected from 20 000 calves entering the specific feedlot (w. schultheiss, pers. comm., 01 september 2010). the samples in the present study were, however, taken from a selected population of calves, which were visually suspect animals and chronic poor doers, and therefore results cannot be directly compared to those of other studies. feedlots receive large numbers of animals from a potentially large number of infected herds, increasing the possible number of pi animals that enter their feedlot population. loneragan et al. (2005) state that when one pi animal is identified in a herd, the herd likely contains others as well and it is thus likely that pi animals entering a feedlot are clustered by herd of origin. in addition, many truckloads of calves entering south african feedlots are sourced from auctions and speculators, thus representing a collection of comingled animals from a variety of herds. many of these high-risk calves are culled from breeding herds because of poor growth performance at weaning. a higher percentage of positive animals may thus be present in a feedlot compared to a single infected herd where, based on data from north america and europe, approximately 1–2% of calves born are infected persistently. the samples were collected mainly by feedlot personnel and distinctions were not made between animals entering the feedlot for the first time and chronic poor doers. it is thus not possible to determine how many suspected pi first-time entrants and how many chronic poor doers were sampled. it was, however, clear from the samples received that feedlot personnel preferred sampling chronic poor doers compared to first-time entrants. selection of first-time entrants was based on their physical appearance, including appearing pot-bellied, unthrifty and thin. the feedlots were specifically interested in the possible causes of chronic poor doers, as it is an important problem in the feedlot industry that results in financial losses. this suggests that the likelihood of a pi animal to become a chronic poor doer in a feedlot is increased, which may also have played a role in the higher prevalence (2.9%) observed in this study. loneragan et al. (2005) found that pi animals were more likely to become chronically ill or die. in the group of animals entering the hospital pen for the first time (collected separately at the end of the study), only 1% were infected persistently according to ihc staining. the hypothesis that persistent infection with bvdv would increase the likelihood of contracting a respiratory illness soon after admission owing to the immunosuppressive nature of the disease was not substantiated during the study. however, the seasonal nature of bovine respiratory disease, the varying profile of calf ages and different purchasing strategies by the participating feedlots, coupled with the relatively small sample of hospital cattle in this study, preclude any firm conclusions on the contributory role of bvdv to bovine respiratory disease in south african feedlots. in addition, a significant number of calves that die following persistent infection with bvdv show severe complicated respiratory pathology with or without salmonellosis (d.j. verwoerd, pers. comm., 03 february 2011). further studies on the role of bvdv in the health of south africa feedlot cattle are clearly indicated. other factors causing respiratory disease in cattle in feedlots include the time of year (more prevalent in winter), extremely dusty conditions, stressors such as over-stocking, social structure in the pen, rain and mud, wind, poor adaptation, nonvaccinated (not pretreated) animals and respiratory disease following acidosis. in the unpublished study by schultheiss mentioned earlier, no specific evidence of an increase in pneumonia in pi calves was found, but the presence of a pi animal in a pen increased the risk of the other calves in the pen contracting pneumonia threefold (w. schultheiss, pers. comm.). this also corresponds to other studies such as that by loneragan et al. (2005), who found an increase in respiratory disease in pi calves. the authors found the incidence of respiratory tract disease to be 43% greater in cattle with an opportunity for direct contact with a pi animal. these cattle also required more treatments for respiratory disease compared with cattle not exposed to a pi animal, thus incurring substantially higher medical costs. the reliability of ihc staining to diagnose pi animals was also tested in this study. one of the most important findings was that nonspecific staining may lead to diagnosing an animal incorrectly as being infected persistently with bvdv. this problem was investigated after an inappropriate number of positive cases were diagnosed. nonspecific positive staining was observed in round as well as spindle-shaped mast cells. as the pathologist becomes more proficient in evaluating these sections, it becomes clear when mast cells are staining positive because the granules have a distinctly different shape from the granular virus staining. all sections diagnosed as positive were re-evaluated to exclude any false positives from the final results. although nonspecific staining was observed with both stains, it was far more frequently seen with dab staining. it is not certain why mast cell granules stain positively with the bvdv antibody. this phenomenon is not seen in every negative case and mast cells granules do not always take up stain. the possibility of over-staining should also be considered, as many of the affected sections showed colour changes in the connective tissue and cartilage, which is indicative of over-staining. all positively diagnosed animals must have positive staining in keratinocytes of the epidermis (stratum basale and stratum spinosum), hair follicle epithelium, smooth muscle cells of blood vessels and spindle-shaped cells in the connective tissue of the dermis (fibroblasts) (njaa et al. 2000; also findings fromt his study). if these parameters are considered and cases are carefully examined, ihc staining can be considered to be a fast, reliable and cost-effective tool to identify pi animals. cornish et al. (2005) found that the ihc stain detected 100% of pi calves. the monoclonal antibody used in the ihc staining method received from cornell university was specifically developed for research purposes and is considered to be of the highest quality. all samples in this research project were stained with it. one of the limitations of this study was the inability to collect serum samples from all animals sampled. serum samples for testing were available for only 10 pi animals as diagnosed according to ihc staining. of these, six calves were positive for bvdv antigen (consistently viraemic) and three had antibodies to bvdv (table 1). the idexx herdcheck bvdv antibody test kit has high specificity (> 99.7%) and sensitivity (nearly 100%) and detects bvdv types i and ii antibodies (idexx laboratories n.d.). however, it cannot be used on its own to identify a pi animal, for the following reasons. a pi animal will not have antibodies against the bvdv strain that infected it in utero because the immune system recognises the virus as self. animals infected in utero after the immune system is competent can develop an antibody response, as can pi animals infected after birth with a different strain (mcclurkin et al. 1984). they will, however, be antibody negative towards the strain that caused the initial infection. in this study three of the six pi animals showed the presence of antibody, indicating infection by a different strain of virus after birth. a pi animal will have a positive result with the bvdv antigen capture elisa because it is persistently viraemic. an acutely infected animal will, however, also test positive, and this test is therefore not able to confirm pi status. the test is based on the robust ems (gp48) antigen, which is consistently present in large quantities in both serum and tissue, making it easy to detect with the test kit. this antigen is highly stable, yielding reliable results even after long storage. of the sampled animals, four were negative for antigen but positive for antibody, indicating that they were not infected persistently. re-evaluation of the immunoperoxidase sections indicated that all four of these were incorrectly diagnosed originally as positive owing to the presence of nonspecific staining. based on the information obtained in this study, ihc staining is a good and reliable tool to diagnose pi animals. the finding corroborates those of cornish et al. (2005), hilbe et al. (2007), houe et al. (2006), luzzago et al. (2006) and thur et al. (1996). however, the pathologist evaluating these sections should be able to differentiate clearly positive from nonspecific staining according to the advised parameters. too few skin samples were accompanied by serum samples to compare determinations by ihc staining and elisa antibody and antigen statistically. in general, ihc staining is more cost effective, as only one test needs to be performed compared with both elisas. currently the cost for one animal’s ihc stain test is approximately equal to the cost of one of the elisas. conclusion (back to top) the incidence of pi calves entering feedlots in south africa is estimated to be approximately 0.56%. in this study the incidence was 2.9% because the samples were collected from a selected population of calves that appeared visually suspect or were recognised as poor doers. it was concluded that the possibility of a pi animal to become a chronic poor doer increases in a feedlot situation compared to calves raised extensively. this study confirmed the reliability of the ihc staining method to identify pi animals, despite the possibility of wrong diagnosis owing to nonspecific staining. acknowledgements (back to top) we would like to express our appreciation to the following people: dr sarah clift for her support and help with interpretation of the ihc stains, and troubleshooting; ms vanessa prinsloo for cutting of sections; mrs marie smit for performing the ihc stains; prof. j.a. lawrence for his valuable inputs. references (back to top) bancroft, a.s. & stevens, a., 1982, theory and practice of histological techniques, 2nd edn., chruchill livingstone, edinburgh. bielefeldt ohmann, h., 1988, ‘bvd virus antigens in tissues of persistently viraemic, clinically normal cattle: implications for the pathogenesis of clinically fatal disease’, acta veterinaria scandinavica 29, 77−84. pmid:2849295 brinkhof, j., zimmer, g. & westenbrink, f., 1996, ‘comparative study of four enzyme-linked immunosorbent assays and a co-cultivation assay for the detection of antigens associated with bovine viral diarrhoea virus in persistently infected cattle’, veterinary microbiology 50, 1−6. doi:10.1016/0378-1135(95)00201-4 brock, k.v., 1995, ‘diagnosis of bovine viral diarrhoea virus infections’, veterinary clinics of north america: food animal practice 11, 549−561. pmid:8581862 brock, k.v., grooms, d.l., ridpath, j. & bolin, s.r., 1998, ‘changes in levels of viraemia in cattle persistently infected with bovine viral diarrhoea virus’, journal of veterinary diagnostic investigation 10, 22−26. pmid:9526856 broderson, b.w., white, a.k. & smith, d.r., 1998, ‘immunohistochemical test on skin biopsies as a method for detection of cattle persistently infected with bovine viral diarrhoea virus’, proceedings of american association of bovine practitioners 31, 246−252. cornish, t.e., van olphen, a.l., cavender, j.l., edwards, j.m., jaeger, p.t., vieyra, l.l. et al., 2005, ‘comparison of ear notch immunohistochemistry, ear 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samples and immunohistochemistry of skin sections for detection of acute bovine viral diarrhoea infections’, journal of veterinary diagnostic investigation 14, 303−307. pmid:12152809 sandvik, t., 1999, ‘laboratory diagnostic investigations for bovine viral diarrhoea infections in cattle’, veterinary microbiology 64, 123−134. doi:10.1016/s0378-1135(98)00264-8 thur, b., zlinszky, k. & ehrensperger, f., 1996, ‘immunohistochemical detection of bovine viral diarrhoea virus in skin biopsies: a reliable and fast diagnostic tool’, journal of veterinary medicine series b 43, 163−166. doi:10.1111/j.1439-0450.1996.tb00301.x university of rochester, 2007, microwave ziehl–neelsen method for acid-fast bacteria, viewed 29 may 2010, from http://www.urm.rochester.edu/path/zqu/stainsmanual/index.html van vuuren, m., 2005, ‘bovine viral diarrhoea virus infection in livestock in southern africa’, cab reviews: perspectives i n agriculture, veterinary science, nutrition and natural resources no. 009, 1−5wren, g., 2001, ‘bvdv vaccination’, bovine veterinarian, november–december, 4−12. wren, g., 2001, ‘bvdv vaccination’, bovine veterinarian, november–december, 4−12. article information authors: tiny m. hlokwe1 akinbowale o. jenkins2 elizabeth m. streicher3 estelle h. venter2 dave cooper4 jacques godfroid5 anita l. michel2 affiliations 1tuberculosis laboratory, arc–onderstepoort veterinary institute, south africa 2department of veterinary tropical diseases, university of pretoria, south africa 3department of biomedical sciences, stellenbosch university, south africa 4ezemvelo kzn wildlife, st lucia, south africa 5department of food safety and infection biology, norwegian school of veterinary science, norway correspondence to: tiny hlokwe email: hlokwet@arc.agric.za postal address: private bag x05, onderstepoort 0110, south africa dates: received: 20 sept. 2010 accepted: 19 jan. 2011 published: 20 june 2011 how to cite this article: hlokwe, t.m., jenkins, a.o., streicher, e.m., venter, e.h., cooper, d., godfroid, j. & michel, a.l., 2011, ‘molecular characterisation of mycobacterium bovis isolated from african buffaloes (syncerus caffer) in hluhluwe-imfolozi park in kwazulu-natal, south africa’, onderstepoort journal of veterinary research 78(1): art. #232, 6 pages. doi:10.4102/ojvr.v78i1.232 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) molecular characterisation of mycobacterium bovis isolated from african buffaloes (syncerus caffer) in hluhluwe-imfolozi park in kwazulu-natal, south africa in this original research... open access • abstract • introduction • methods    • sample collection       • bacterial isolation and molecular identification       • genomic dna extraction       • genotyping       • spoligotyping       • variable number of tandem repeats typing       • polymorphic g–c-rich sequence and is6110 typing • results    • mycobacterium bovis isolation and molecular identification    • spoligotyping    • analysis of variable number of tandem repeats    • polymorphic g–c-rich sequences       • is6110 typing • discussion • conclusion • acknowledgements    • author’s contributions • references abstract (back to top) bovine tuberculosis (btb), a chronic disease of mammals caused by mycobacterium bovis, is a threat to south african wildlife. it has been reported that african buffaloes (syncerus caffer) are reservoir hosts of btb in south african wildlife populations. this study reports on the molecular identification and typing of 31 m. bovis isolates collected between 1993 and 2008, mainly from buffaloes but also from two lions and a bush pig, in the hluhluwe-imfolozi park (hip) in kwazulu-natal. to study the dynamics of btb in the buffalo populations, 28 m. bovis isolates from the hip and epidemiologically related parks were characterised using regions of difference deletion analysis for species identification and spoligotyping, variable number of tandem repeats (vntr), polymorphic g–c-rich sequences and is6110 restriction fragment length polymorphism (rflp) genotyping methods. at least three distinct m. bovis genotypes were found amongst hip samples. the combination of vntr typing (using a 16-loci panel) and is6110 rflp revealed the presence of three additional genetic profiles in individual buffaloes, demonstrating that the highest level of discrimination was achieved by these typing methods. one of the observed spoligotypes (sb0130) was dominant and represented 75% of isolates from buffaloes. a novel m. bovis spoligotype (sb1474), which is reported for the first time in this study, was observed in 14.3% of isolates from buffaloes. based on the observed genetic relationships, the findings suggest independent introductions from at least three unrelated sources. these findings improve the knowledge regarding the diversity of circulating m. bovis strains in the hip. introduction (back to top) bovine tuberculosis (btb) is a chronic, contagious disease caused by mycobacterium bovis, a gram-positive acid-fast bacterium. this pathogen is particularly noted for its diverse host tropism, which includes humans, livestock and several wildlife species. the first report of btb in cattle in south africa was made by hutcheon (1880), whilst it was first diagnosed in african buffaloes (syncerus caffer) in the hluhluwe-imfolozi park (hip) in 1986 (jolles 2004). spillover of btb to other species has been documented (michel et al. 2006; michel et al. 2009). the hip is situated in kwazulu-natal, south africa and the area of 100 000 ha is almost entirely surrounded by communal farmland and livestock. it is the fourth largest game reserve in south africa and has a buffalo population of approximately 3500 heads. the molecular characterisation of m. bovis is essential in understanding disease transmission between species and the spatial distribution of this infection in wildlife populations. in a previous study, molecular characterisation of m. bovis isolated from wildlife in the hip was performed using a combination of three genetic tools, namely spoligotyping and restriction fragment length polymorphism (rflp), including is6110 and polymorphic g–c-rich sequence (pgrs) typing (michel et al. 2009). in that study, m. bovis isolates, the majority of which were derived from buffaloes, had been collected in the hip and two epidemiologically related parks between 1993 and 2000. with one exception (tb 328), which had a shift in one of the bands (michel et al. 2009), the isolates all displayed the same is6110 rflp banding pattern. in addition, spoligotyping yielded only one type identical to sb0130 in the international m. bovis database (www.mbovis.org). this report also showed that epidemiologically unrelated strains are in circulation in the hip and in the greater kruger national park (michel et al. 2009). previous studies showed that variable number of tandem repeats (vntr) typing is more discriminatory for m. bovis isolates than spoligotyping (allix et al. 2006). in a study by romero and co-workers (2008), both spoligotyping and vntr were found to display similar discriminatory capacity. on the basis of the above information, the aim of this study was to investigate the circulating strains of m. bovis in the hip further. regions-of-difference (rd) deletion analysis was used for the identification of the isolates and spoligotyping, vntr, pgrs and is6110 rflp techniques were used for genotyping. methods (back to top) sample collection tissue samples, collected from tuberculin skin tests and/or buffaloes that had tested positive for gamma interferon during btb surveys between 1993 and 2008, were transferred to the tuberculosis laboratory at the onderstepoort veterinary institute (ovi) for bacterial culture. a total of 28 buffaloes, from the hip (n = 20), munyawana game reserve (n = 4), st lucia wetland game parks (n = 3) and a private game reserve (n = 1), were included in the study. buffaloes from munyawana and st lucia wetland game parks (both in kwazulu-natal) were originally from the hip, but were moved to the new locations between 1997 and 2000. a buffalo from a private game reserve (situated in mpumalanga province) was born to a dam originating from the hip. additional samples included in this study were collected during ad-hoc post-mortem examinations from two lions (panthera leo) from, respectively, the hip and munyawana game reserve, and a bush pig (potamochoerus larvatus) from the hip. bacterial isolation and molecular identification the tissue samples were processed at the tuberculosis laboratory, ovi, according to standard procedures (alexander et al. 2002; bengis et al. 1996). the samples were inoculated onto löwenstein-jensen media supplemented with pyruvate and incubated at 37 °c for up to 10 weeks. acid-fast bacterial isolates were subjected to polymerase chain reaction (pcr) amplification using primers that target a sequence encoding the mpb 70 antigen to identify mycobacterium tuberculosis complex bacteria (alexander et al. 2002; bengis et al. 1996; michel et al. 2009). m. tuberculosis complex bacteria were identified following the presence of a 372-bp pcr product. deletion analysis was performed as described by warren et al. (2006) for species identification. the rd4 and rd9 genomic regions of difference were targeted using a multiplex pcr approach (warren et al. 2006). the pcr products were separated on a 2–3% agarose gel to allow for good definition of the bands. m. bovis isolates were confirmed by the presence of two specific bands of 268 bp and 108 bp for rd4 and rd9, respectively. genomic dna extraction a puregenetmdna extraction kit was used to extract genomic dna from isolates according to the manufacturer’s instructions (gentra systems, minneapolis), with minor modifications. cells treated overnight with glycine were heat killed at 94 °c for 10 minutes and allowed to cool. a volume of 500 μl extraction buffer (50 g/l monosodium glutamic acid; 6.06 g/l tris-hcl (ph 7.4); 9.3 g/l edta) was added to the culture and cells were suspended by using an inoculating loop. following centrifugation, 300 µl lysis solution and 5 µl rnase a solution (4 mg/ml) (roche diagnostics, randburg) were added and the sample was then incubated at 37 °c for 30 min. a volume of 150 µl protein precipitation solution was added and samples were centrifuged at maximum speed for 2 min, followed by precipitation of the aqueous phase with isopropanol (merck, halfway house). the resulting pellet was dissolved in 40 µl te buffer (10 mm tris-hcl; 1 mm edta). the extracted dna was stored at –20 °c until use. genotyping either pure genomic dna (extracted using the puregenetm dna extraction kit) or cell lysates (prepared by boiling the m. bovis cells for 25 min and storing at –20 °c until used) were used for the pcr-based typing methods (i.e. spoligotyping and vntr typing). for rflp typing, pure genomic dna was used. spoligotyping spoligotyping was done according to a standardised international method (kamerbeek et al. 1997) using a commercially available kit (isogen biosciences bv, maarsen). m. tuberculosis h37rv, m. bovis bcg and sterile distilled water were used as controls. the spoligotype patterns were compared to those stored in the m. bovis spoligotype database (www.mbovis.org). variable number of tandem repeats typing cell lysates or genomic dna was used for pcr amplification. initially, vntr analysis, also called multiple locus variable analysis, was carried out using primers to amplify the etr-a to etr-f loci (frothingham & meeker-o’connell 1998). additional vntr typing was performed on 10 loci (mtub 2, mtub 12, mtub 21, mtub 29, mtub 30, mtub 39, miru 2, miru 10, miru 16 and qub 11a) as described by le fleche et al. (2002). pcr was performed using an eppendorf ag 22331 hamburg thermocycler (merck eppendorf, hamburg). the pcr products were separated on a 2% agarose gel at 85 v for 3 h and visualised under 312 nm uv light (spectroline® model tc-312a transilluminator, westbury). a 100-bp ladder (inqaba biotechnology, pretoria) was included as a marker. the band sizes were determined using quantity one® 1-d analysis software installed in the gel doc system (bio-rad laboratories, hercules, ca), converted to copy numbers (for exact tandem repeats [etrs], see frothingham & meeker-o’connell 1998; for additional loci, see le fleche et al. 2002) and saved in a spreadsheet. the genotyping data were exported to bionumerics software (applied maths, sint-martens-latem). the categorical coefficient option was used for cluster analysis to create a dendogram. polymorphic g–c-rich sequence and is6110 typing pgrs and is6110 rflp typing were performed as previously reported (cousins et al. 1998; michel et al. 2008). the gelcompar ii system (applied maths, sint-martens-latem) was used for analysis of the is6110 rflp results, with the similarity coefficient of dice and the unweighted pair group method with arithmetic averages for clustering maximum tolerance at 1.2%. pgrs rflp typing patterns results were analysed manually. results (back to top) mycobacterium bovis isolation and molecular identification bacterial growth was observed on löwenstein-jensen culture slopes after 6–10 weeks of incubation. the presence of acid-fast bacilli was confirmed by means of ziehl–neelsen stains. isolates were classified as members of the m. tuberculosis complex following detection of a 372-bp fragment by pcr using primers targeting the sequence that codes for the mpb 70 antigen of the m. tuberculosis complex. deletion analysis using multiplex pcr targeting rd4 and rd9 (warren et al. 2006) further confirmed the isolates as m. bovis owing to the amplification of dna products with band sizes of 268 bp and 108 bp, respectively (results not shown). spoligotyping spoligotyping was performed on all 31 m. bovis isolates: 28 from buffaloes; two from lions and one from a bush pig. the results (figure 1 and figure 2) revealed four different spoligopatterns. the most prevalent spoligotype pattern was found in 24 of the 31 (77.4%) m. bovis isolates, including 21 isolates from buffaloes, two from lions and that of the bush pig. this spoligotype pattern was marked by the absence of spacers 3, 9, 11, 16 and 39–43, which is consistent with sb0130 (www.mbovis.org). the strain type was identified in isolates collected from the hip during the study period (1993–2008) and has been reported earlier (michel et al. 2009). a second spoligotype pattern was found in four buffaloes (tb 6452, tb 6453, tb 6459 and tb 6461) sampled in 2007. this m. bovis spoligopattern has not been described in the m. bovis database before and was allocated a new code, sb1474. it is characterised by the lack of spacers 3, 6, 8, 9, 10, 11, 12, 16, 22, 23 and 39–43. a third spoligotype pattern was found in two m. bovis isolates from buffaloes sampled in 2007 and 2008 (tb 6458 and tb 6696, respectively). the pattern, designated sb0121, is delineated by the absence of spacers 3, 9, 16, 21 and 39–43. this pattern is similar to that for m. bovis bcg except that spacer 21 is present in the latter. another spoligotype pattern was isolated in 2000 from a buffalo (tb 1714) that was translocated from the hip and was kept in a boma at munyawana game reserve. this particular pattern was characterised by the absence of spacers 3, 6, 8, 9, 10, 11, 12, 16 and 39–43. it is identical to sb0140 in the international m. bovis database. figure 1: results from different typing methods applied to mycobacterium bovis isolates from the hluhluwe-imfolozi park and epidemiologically related game reserves. figure 2: geographical distribution of spoligotype and is6110 patterns of mycobacterium bovis isolates from the hluhluwe-imfolozi park. analysis of variable number of tandem repeats results of the vntr analyses on all 31 isolates, using a total of 16 loci, are summarised in figure 1. comparison of the copy numbers of the vntr profile of each isolate revealed five profiles. the most prevalent vntr profile was found in 23 of the 31 m. bovis isolates from buffaloes, lions and a bush pig. this vntr profile was essentially shared by tb 328 (second profile) and tb 1714 (third profile), except for the copy numbers relating to locus mtub 12 (in tb 328) and mtub 29 (in tb 1714). the remaining two vntr profiles were found in four (tb 6452, tb 6453, tb 6459, tb 6461) and two m. bovis isolates from buffaloes (tb 6458 and tb 6696) collected in 2007 and 2008, respectively). the genetic diversity observed in these isolates were attributed to the use of a combination of the loci etr-a, etr-b, etr-c, etr-e, mtub 2, mtub 12, mtub 21, mtub 29 and qub 11a. polymorphic g–c-rich sequences pgrs typing was performed on five m. bovis isolates cultured from buffaloes sampled in 2007 (figure 1: tb 6452, tb 6453, tb 6459, tb 6461 and tb 6458). the pgrs profiles of 16 isolates have been analysed previously and were found to share a unique profile (michel et al. 2009). in the present study, three pgrs dna profiles were observed. one profile (hip-2) was present in three of the five m. bovis isolates analysed, whilst the two remaining isolates each exhibited a genetically different profile (hip-3 and e, respectively) as shown in figure 1. is6110 typing the same five m. bovis isolates as analysed by pgrs typing were subjected to is6110 typing. three is6110 patterns were identified, namely c17, c18 and c8v. m. bovis isolates with identical pgrs patterns also displayed identical is6110 patterns (figure 1). discussion (back to top) this study presents data obtained from m. bovis isolates from the hip and three epidemiologically related game reserves. pcr-based genotyping tools (i.e. spoligotyping and vntr typing) were primarily applied to characterise 31 m. bovis isolates, complemented with pgrs and is6110 typing on some of the samples for added discriminatory power. most m. bovis infections examined in this study were caused by a unique genotype, which corresponded to a spoligopattern that had previously been identified as sb0130 (michel et al. 2009). these results were in line with those from vntr typing, except for vntr typing singling out one isolate (tb 328) on the basis of a difference in locus mtub 2. this finding was supported by previous is6110 typing results (michel et al. 2009). based on the high degree of homology between tb 328 and the dominant vntr typing profile, it is unlikely that tb 328 resulted from a separate introduction of m. bovis into the hip; rather, it may be linked to the dominant strain through an evolutionary process. spoligotyping and vntr typing results were also found to correspond to a unique genotype isolated in buffaloes sampled in 2007 (tb 6452, tb 6253, tb 6459, tb 6461). the spoligopattern has not been described in the hip previously nor included in the international m. bovis database and was therefore designated a new code, sb1474. however, when is6110 and pgrs typing data were considered for this group of isolates, both markers were able to discriminate between two profiles (e.g. c17 and c18). the genetic diversity of these strains collected in 2007 suggests that they did not evolve from the dominant hip strain (sb0130) and therefore most likely represent an independent introduction of m. bovis into the hip. during the period prior to 2000 buffalo tb 1714 was translocated from the hip to munyawana game reserve where it was euthanised and sampled in 2000. spoligotyping revealed a strain, sb0140, which is related to the novel sb1474. the novel strain (sb1474) differs from sb0140 by deletion of spacers 22 and 23 in addition to the loss of spacers 3, 6, 8, 9, 10, 11, 12 and 16, which is descriptive of the bovis2 family (brudey et al. 2006). the origin of strain sb0140 remains elusive, despite identical pgrs and is6110 typing results with sb0130 as shown in figure 1. evidence of another independent source of m. bovis in the hip was found in two buffaloes (tb 6458 and tb 6696) sampled in 2007 and 2008, respectively, which both carried the spoligotype sb0121. this particular spoligotype pattern was previously isolated in cattle in gauteng, mpumalanga and the limpopo provinces of south africa (michel et al. 2008), as well as in buffaloes and other wildlife in the kruger national park (michel et al. 2009). additional is6110 and pgrs typing (only on tb 6458) confirmed that this genotype was a member of the c8 variant strain complex, which is the cause of the btb epidemic in the kruger national park (michel et al. 2009). it is unclear whether the strain could have been introduced more recently from infected cattle or wildlife translocated from areas neighbouring the kruger national park to areas neighbouring the hip. alternatively, the c8 strain as found in the kruger national park may have been more widely spread in the eastern parts of the country than previously known and undergone similar evolutionary processes as described for the kruger national park (michel et al. 2009). it is also possible that, at a particular time, strains present in buffaloes sampled in 2007 (western part of the hip) and 2008 (eastern part of the hip) may not have been present in buffaloes sampled before 2002 (eastern part of the hip). indeed, earlier behavioural studies indicate that hip buffaloes have relatively stable and small home ranges (compared to those in the kruger national park) owing to the abundant year-round food and water availability in the hip (dora 2004). very little mixing, if any, of breeding herds is known, with adult bulls implicated as the main source of infection among herds. the use of molecular typing methods in the present study proved to be useful in confirming the origins of m. bovis strains translocated to, for example, st lucia wetland game parks in kwazulu-natal and a private game reserve in mpumalanga. in all cases the m. bovis strain types were identical to the most prevalent type in the hip. the methods show that the prevalent strain has spilled over to wildlife species other than buffaloes, as evidenced by the cases of two lions and a bush pig in the present study, and by is6110 typing in a previous study by michel and co-workers (2009). the identification of previously undetected m. bovis strains in the hip is of great concern, since it indicates the possibility of multiple introductions in the past and a persistent risk for new m. bovis introductions into the park, as well as a risk of spillover to surrounding communal cattle farms. this presents a challenge for controlling the disease in livestock and has an impact on wildlife management and potentially also on public health (michel et al. 2006; romero et al. 2008). conclusion (back to top) the molecular typing methods described here have been useful in studying the epidemiology of btb in the hip and provide valuable information for future disease management strategies. this study further suggests that detection surveys for btb are needed in communal cattle herds around the hip to identify and efficiently manage sources of m. bovis infection. acknowledgements (back to top) we thank dr noel smith and staff at the veterinary laboratory agency for vntr analysis training and providing the vntr a, b, c, e and f primers, gilles vergnaud of the institute of genetics and microbiology, university of paris xi orsay, also for vntr analysis training, dr roy williams for creating the map used in figure 2, and dr claude sabeta for critically reviewing the manuscript. the work was funded jointly by the department of agriculture, the department of science and technology, the belgian directorate-general for development cooperation (research project 9740x) and the university of pretoria. author’s contributions t.m.h. and a.o.j. contributed equally to the design of the study and preparation of the manuscript. e.m.s. performed spoligotyping of isolates. d.c. provided information relating to animals involved and their locations. a.l.m., j.g. and e.h.v. were involved in drafting the manuscript and revising it critically. all authors read and approved the final manuscript. references (back to top) alexander, k.a., pleydell, e., williams, m.c., lane, e.p., nyange, j.f.c. & michel, a.l., 2002, ‘mycobacterium tuberculosis: an emerging disease of free-ranging wildlife’, emerging infectious disease 8, 592−595. allix, c., walravens, k., saegerman, c., godfroid, j., supply, p. & fauville-dufaux, m., 2006, ‘evaluation of the epidemiological relevance of variable-number tandem-repeat genotyping of mycobacterium bovis and comparison of the method with is6110 restriction fragment length polymorphism analysis and spoligotyping’, journal of clinical microbiology 44, 1951−1962.doi:10.1128/jcm.01775-05, pmid:16757584, pmid:1489394 bengis, r.g., kriek, n.p.j., keet, d.f., raath, j.p., de vos, v. & huchzermeyer, h.f.k.a., 1996, ‘an outbreak of bovine tuberculosis in a free-living buffalo population in the kruger national park’, ondestepoort journal of veterinary research 63, 15−18. pmid:8848298 brudey, k., driscoll, j.r., rigouts, l., prodinger, w.m., gori, a., al-hajoj, s.a. et al., 2006, ‘mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (spoldb4) for classification, population genetics and epidemiology’, bmc microbiology 6, 23. doi:10.1186/1471-2180-6-23, pmid:16519816, pmid:1468417 cousins, d., williams, s., liebana, e., aranaz, a., bunschoten, a., van embden, j. et al., 1998, ‘evaluation of four dna typing techniques in the epidemiological investigations of bovine tuberculosis’, journal of clinical microbiology 36, 168−178. pmid:9431942, pmid:124829 dora, c.a., 2004, ‘the influences of habitat structure and landscape heterogeneity on african buffalo (syncerus caffer) group size in hluhluwe-imfolozi park, south africa’, msc thesis, university of oregon. frothingham, r. & meeker-o’connell, w.a., 1998, ‘genetic diversity in the mycobacterium tuberculosis complex based on variable numbers of tandem dna repeats’, microbiology 144, 1189−1196.doi:10.1099/00221287-144-5-1189, pmid:9611793 hutcheon, d., 1880, ‘tering, consumption, tables mesenterica’, annual report, colonial veterinary surgeon, cape of good hope. jolles, a., 2004, ‘disease ecology of bovine tuberculosis in african buffalo’, phd thesis, department of ecology and evolutionary biology, princeton university. kamerbeek, j., schouls, l., kolk, a., van agterveld, m., van soolingen, d., kuijper, s. et al., 1997, ‘simultaneous detection and strain differentiation of mycobacterium tuberculosis for diagnosis and epidemiology’, journal of clinical microbiology 35, 907−914. pmid:9157152, pmid:229700 le fleche, p., fabre, m., denoeud, f., koeck, j.l. & vergnaud, g., 2002, ‘high resolution, on-line identification of strains from the mycobacterium tuberculosis complex based on tandem repeat typing’, bmc microbiology 2, 37. doi:10.1186/1471-2180-2-37, pmid:140014 michel, a.l., bengis, r.g., keet, d.f., hofmeyr, m., de klerk, l.m., cross, p.c. et al., 2006, ‘wildlife tuberculosis in south african conservation areas: implications and challenges’, veterinary microbiology 112, 91−100.doi:10.1016/j.vetmic.2005.11.035, pmid:16343819 michel, a.l., hlokwe, t.m., coetzee, m.l., mare, l., connoway, l., rutten, v.p. et al., 2008, ‘high mycobacterium bovis genetic diversity in a low prevalence setting’, veterinary microbiology 126, 151−159. doi:10.1016/j.vetmic.2007.07.015, pmid:17720336 michel, a.l., coetzee, m.l., keet, d.f., maré, l., warren, r., cooper, d. et al., 2009, ‘molecular epidemiology of mycobacterium bovis isolates from free-ranging wildlife in south african game reserves’, veterinary microbiology 133, 335−343. doi:10.1016/j.vetmic.2008.07.023, pmid:18786785 romero, b., aranaz, a., sandoval, a., alvarez, j., de juan, l., bezos, j. et al., 2008, ‘persistence and molecular evolution of mycobacterium bovis population from cattle and wildlife in donana national park revealed by genotyping variation’, veterinary microbiology 132, 87−95. doi:10.1016/j.vetmic.2008.04.032, pmid:18539410 warren, r.m., gey van pittius, n.c., barnard, m., hesseling, a., engelke, e., de kock, m. et al., 2006, ‘differentiation of mycobacterium tuberculosis complex by pcr amplification of genomic regions of difference’, international journal of tuberculosis lung disease 10, 818−822. pmid:16850559 article information author: joe brownlie1 affiliation: 1royal veterinary college, united kingdom, england correspondence to: joe brownlie postal address: royal veterinary college, hawkshead lane, north mymms, hatfield, herts, al9 7ta, united kingdom how to cite this proceeding: brownie, j., 2012, ‘a foresight vision for infectious diseases in africa’, onderstepoort journal of veterinary research79(2), art. #459, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.359 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a foresight vision for infectious diseases in africa in this proceeding... open access • introduction • the emerging and future risks from infectious diseases • international dimension of foresight and infectious diseases • the birth of southern african centre for infectious disease surveillance • the importance of southern african centre for infectious disease surveillance • references introduction top ↑ in 2005, the united kingdom (uk) through their foresight programme, within the government office for science (formerly the uk office for science and innovation) launched a project to consider the future risks from infectious diseases for humans, animals and plants (http://www.bis.gov.uk/foresight/our-work/projects/published-projects/infectious-diseases). whilst defining these risks, foresight also explores the very best science that may be brought to transform our ability to manage the future challenges. a report of the future threats was published in 2006 (brownlie et al. 2006) following the published report, we highlighted (king et al. 2006) that infectious diseases account for a quarter of all human mortality, and a similar fraction of morbidity (world health organisation 2005). infectious diseases of crops and livestock cost the global economy uncounted billions of euros every year. on top of this, sudden epidemics of infectious diseases can deliver humanitarian and economic shocks on a scale difficult to absorb. according to the world bank, the 2003 severe acute respiratory syndrome (sars) epidemic, which killed fewer than 1000 people, was responsible for an estimated 2% fall in gdp across east asia; an influenza pandemic could kill millions of people and cost 700 billion euros globally in a single year (european union 2005). in recent years, there have been numerous outbreaks of livestock and crop diseases costing individual countries billions of euros, for example foot-and-mouth disease (fmd) in taiwan and the uk; bovine spongiform encephalopathy (bse) in the uk; classical swine fever (csf) in netherlands; soybean rust in brazil; southern corn blight in the united states (us); and most recently, avian influenza in egypt. the un millennium development goals, as well as having explicit targets for reducing the burden of human diseases (particularly hiv and/or aids, tuberculosis and malaria), also have targets for reducing poverty and hunger, but these are compromised by crop and livestock diseases. in most developing regions, where the impacts of infectious disease are greatest, there is now little hope of meeting any of the millennium development goals by 2015 (united nations 2005). it was clearly evident that outbreaks of disease can move rapidly and spread across a country, across regions and, in some cases, become global. the best strategy was to stop the disease ‘in its tracks’ as early as possible. for this to happen, we needed to be able to undertake extremely rapid detection and accurate identification of the pathogens; this would facilitate the correct control measures to be put in place, whether antimicrobials, vaccination or a culling policy. in those early stages, and thereafter, understanding where the outbreak is and its progress through the region is critical for control; thus effective monitoring surveillance and directed epidemiology provides the authorities with key information to target appropriate control measures. the global nature and threat posed by many diseases is self-evident. for any foresightful look at their potential risk, account must be taken of their global prevalence and impact. with this in mind, the project enlisted over 300 experts in some 30 countries for consultation and used a variety of methods, including delphi studies (which use formal methods to generate forecasts from groups of experts), expert reviews, workshops, mathematical modelling and commissioned research. the emerging and future risks from infectious diseases top ↑ at the conclusion of the final analysis, eight future categories of infectious diseases were identified as high risk and where improved detection systems would make a difference over the next 10−25 years. • new and emerging diseases, such as sars and bse, and novel variants, such as h5n1 subtype influenza a, are anticipated to continue threaten nations. • infections becoming resistant to treatment, including antibiotic-resistant bacterial infections, such as tuberculosis and methicillin-resistant staphylococcus aureus (mrsa) and those where mutation overcomes present vaccine protection, such as influenza. • zoonoses, that is, infections transferring to humans from animals, which can be associated with livestock, pets and, in many cases, with wildlife, for example, sars, avian influenza, plague, lyme disease and anthrax. this category includes foodborne infections such as escherichia coli o157 or salmonella. • hiv and/or aids, tuberculosis and malaria, the ‘big three’ tropical diseases covered by un millennium development goal 6. • epidemic plant diseases, such as cassava mosaic virus, coffee wilt disease and banana blight, currently of concern in east africa. • acute respiratory infections, a category that covers pandemic influenza and a variety of other viral and bacterial infections. • sexually transmitted infections (stis), including but not only hiv and/or aids, which are increasing in incidence in many parts of the world. • transboundary animal diseases, such as fmd, csf and newcastle disease, which remain amongst the most important barriers to international trade in livestock and livestock products. international dimension of foresight and infectious diseases top ↑ it is obvious that global disease needs a global vision. to this end, the project looked at infectious disease risks not just within the uk but also across sub-saharan africa and in china. in sub-saharan africa, it was clear that the importance of infectious disease could not be greater (rweyemamu, otim-nape & serwadda 2006). it carries the heaviest burden of infectious disease of any region; the impact of hiv and/or aids, malaria and tuberculosis cannot be over-emphasised whilst the so-called ‘neglected diseases’ are ever-present and must not be forgotten. the seriousness of plant diseases is quickly seen on whole communities who solely rely on them for their own food and for nutrients for their livestock. the ultimate consequence of crop failure is starvation, rural migration, de-population of whole tracts of agricultural land but, at worse, it is death. for livestock, the situation in some areas and some times, is not much better. twelve of the 15 transboundary livestock diseases, formerly considered by the oie as the most contagious (and which, in many countries, are notifiable to their government), are to be found in africa. in the uk, they are all exotic. the situation in africa has worsened in recent years with the further spread of diseases such as contagious bovine pleuropneumonia (cbpp), of peste des petits ruminants virus (pprv) and rift valley fever virus (rvfv). this scourge of animal disease cuts deeper where there is poor governance and reduced infrastructure for the livestock extension services and limited diagnostic services. to compound the problem, these highly infectious diseases close all-important regional and global livestock markets that are so valuable to provide income back into the further development of agricultural communities. the recent advances in technology have promoted rapid pen-side tests that have the potential to help with the diagnosis of infectious diseases. for sub-saharan africa, these tests, to be effective, will need to operate across a number of different countries, diverse systems of culture and governance and be cost effective; the question of global equity then becomes a critical issue. for those of us involved with the sub-saharan africa foresight project, it became clear that to have any long-term value or sustainability, a new paradigm for an integrated approach to control infectious diseases was needed. this gave rise to the ‘pan-african vision and strategy for the management of infectious diseases’ (rweyemamu et al. 2006). the mission statement, at that time, for a pan-african vision and strategy for the management of infectious diseases was: a pan-african concerted effort, shared by au member governments reflecting the needs of the african society and supported by the international community, with the goal of society protecting from the ravages of dangerous infectious disease that compromise human, and animal and plant health, improved livelihoods, agriculture and economic development. the birth of southern african centre for infectious disease surveillance top ↑ the foresight ‘detection, identification and monitoring of infectious diseases (dim)’ project provided a number of findings which were taken up by a number of governments, agencies and organisations across the world. however, one of the outcomes that was pertinent only to sub-saharan africa, was the creation of a centre for infectious diseases. in time, it was hoped that this would provide the african continent with a capability for diagnosis and identification of any new and emerging diseases; a capability similar to that provided by the centres for disease control and prevention (cdc) in atlanta for the usa. such a centre would give greater ‘ownership of disease’ to african countries and thereby promote an african capability that would underpin the national and international control programmes, some of which were of only african interest. to create such a centre, we needed to start ‘small’ (in relative terms, for such an enormous continent), establish a local grouping of countries, find a vision and obtain some start up funds that would be independent of individual government sponsorship. the initial project was to join medical and veterinary institutions in tanzania in a ‘smart partnership’ with relevant uk institutions (together to focus on human and animal diseases in a virtual centre incorporating a small but selected number of institutes, organisations and universities from the two countries. the proposal (‘piloting african-uk partnership through ‘onemedicine’ surveillance and detection of infectious diseases in tanzania’) was submitted for funding to the wellcome trust in the uk in 2008 with the royal veterinary college (principal investigator = professor joe brownlie) as the uk institution and with the veterinary school at the university of sokoine, tanzania. this proposal failed to obtain funding but did start a series of actions both within the wellcome trust and within our tanzanian collaborators – most notably professor mark rweyemamu. the reasons for failure included the need for african leadership, wider country involvement and better ‘take-up’ by the medical sciences. the invitation from the wellcome trust to reconsider the proposal required a deeper and more extensive african involvement. at this point, an inspired african leader took the reins and rode the proposal through and over the difficult cross-country obstacle race to win the race and be awarded the invaluable prize of wellcome trust funding. that leader was professor mark rweyemamu and the ultimate prize was the creation of the southern african centre for infectious diseases and surveillance (sacids) with 5 southern african countries involved (republic of south africa, tanzania, zambia, botswana, mozambique, democratic republic of congo) and a number of institutions from london, uk (the royal veterinary college, the london school of hygiene and tropical medicine and the london international development centre). the proposal to wellcome trust was entitled sacids. the importance of southern african centre for infectious disease surveillance top ↑ the main thrust of sacids is create a virtual centre within southern african countries with the capacity to provide effective diagnostic capability and research of international standard on infectious diseases, both medical and veterinary, and to provide training to the next generation of young african scientists with the long term ambition to populate the centre and the relevant institutions within the southern african countries. this requires both new graduate courses in the molecular sciences and epidemiology to be designed, approved and initiated in sacids member universities and to rapidly incorporate the new technologies that would underpin national diagnosis and surveillance. returning to the foresight programme for a moment (king et al. 2006), we can see that ‘a wide range of technological advances from remote sensing to nanotechnology were reviewed and ultimately four were selected for detailed consideration. novel information technologies for the capture, analysis and modelling of data are already being developed: these will allow data to be collected electronically from hand-held devices, or from remote sensors, and analysed and modelled in real time. genomics and post-genomics approaches will allow the rapid characterisation of pathogens. mass screening of people, animals and plants in transit should become feasible through non-invasive detection systems for volatile organic chemicals or atypical electromagnetic profiles. portable devices will become available for diagnosing infections in individual patients, animals or plants, satisfying a growing demand for cheap, quick, easy-to-use, over-the-counter products, perhaps resembling today’s home pregnancy test kits. some of these technologies will be generic, such as ‘lab-on-a-chip’ screening for a range of infectious agents, non-specific diagnostics based on detecting activated immune responses, or simple tests to differentiate between viral and bacterial infections to aid the appropriate prescription of antibiotics. better disease detection capability is vital but will present challenges as well as opportunities. new technologies must be embedded within functional national or international surveillance systems. in practice, while the widespread use of self-administered tests, for example, could provide valuable data, it is not clear whether or how government agencies would gain access to it. other kinds of information e.g., records from mobile phones or traffic cameras to follow movements, are potentially valuable for disease control purposes, particularly in outbreak situations, but the public might not accept their use for this purpose. there is always the danger that disease data could be used to discriminate against individuals without providing any benefit or compensation. similarly, technologies deployed by developed countries could disadvantage developing countries by restricting travel and trade on the basis of the presence or even the suspicion of an infectious disease. finally, everywhere in the world, better disease data might raise public alarm and expectations of effective action, whether or not this is realistic. this requires that surveillance is operationally linked to an appropriate response. for example, the global plan to stop tb relies on the combination of case detection and directly observed therapy (dots) (world health organisation 2006) and plans to combat influenza involve surveillance, drug delivery and vaccine production (fouchier et al. 2005). southern african centre for infectious disease surveillance has the real opportunity to co-ordinate the most important and impressive programme across the southern african countries to combat infectious diseases in humans and livestock. it needs to foster interdisciplinary approaches to infectious disease research that transcend both national and also traditional intellectual boundaries, such as those between medicine and veterinary medicine or between virology, bacteriology, mycology and parasitology. a better understanding of patterns of infectious disease also needs input from disciplines as diverse as anthropology, economics and climatology. quantifying these relationships and understanding their dynamics requires inputs from statistics and mathematics to generate credible models. health systems research is needed to understand how new technologies can be used most effectively, and must include consideration of the needs, expectations, capabilities and sensitivities of end users and other stakeholders (king et al. 2006). sacids has already done what a couple of years ago would have been considered highly unlikely; with a little more effort over the next few years, it could achieve the impossible – a fully functional sacids that has morphed into a continental sub-saharan centre for infectious disease surveillance and control. references top ↑ brownlie, j., peckham, c., waage, j., woolhouse, m., lyall, c., meagher, l. et al., 2006, foresight. infectious diseases: preparing for the future future threats. office of science and innovation, london, p. 1–83. european union, 2005, impact assessment avian influenza, viewed n.d., from http://archive.defra.gov.uk/ fouchier, r., kuiken, t., rimmelzwaan, g. & osterhaus, a., 2005, ‘global task force for influenza’, nature 435, 419–420. http://dx.doi.org/10.1038/435419a, pmid:15917779 king, d.a., peckham, c., waage, j.k., brownlie, j. & woolhouse, m., 2006, ‘infectious diseases – preparing for the future’, science 303, 1392–1393. http://dx.doi.org/10.1126/science.1129134, pmid:16959992 rweyemamu, m., otim-nape, w. & serwadda, d., 2006, foresight. infectious diseases: preparing for the future, africa, office of science and innovation, london, p. 1–120. united nations, 2005, the millennium development goals report 2005, un, new york. world health organization, 2006, stop tb strategy to fight the global tuberculosis epidemic, viewed n.d., from http://www.who.int/tb/features_archive/stop_tb_strategy_launch/en/index.html regassa_333-336.qxd bovine babesiosis or redwater, caused by babesia bovis and babesia bigemina, results in serious economic losses worldwide (carson & phillips 1981; mccosker 1981). it is estimated that half a billion cattle in tropical and subtropical countries are at risk to babesiosis (ristic & levy 1981). both b. bigemina and b. bovis occur in many areas of south africa, coinciding with the distribution of their vectors (de vos 1979). babesia bigemina is transmitted by both rhipicephalus (boophilus) decoloratus and rhipicephalus (boophilus) microplus and has a much wider distribution than b. bovis, which is only transmitted by r. (b.) microplus (de vos 1979). rhipicephalus (b.) microplus is expanding its range in south africa (tønnesen, penzhorn, bryson, stoltsz & masibigiri 2004). bovine babesiosis is not an important cause of mortality in cattle in endemically stable situations (norval, fivaz, lawrence & dailecourt 1983). endemic stability to bovine babesiosis occurs when the inoculation rate of babesia by ticks into cattle is sufficiently high to infect virtually all calves while they are protected by colostral and innate immunity (mahoney & ross 1972). if the inoculation rate is low and calves are not infected during this period, then endemic instability and clinical disease 333 onderstepoort journal of veterinary research, 71:333–336 (2004) research communication progression towards endemic stability to bovine babesiosis in cattle introduced onto a game ranch assefa regassa, b.l. penzhorn* and n.r. bryson department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort 0110, south africa abstract regassa, assefa, penzhorn, b.l. & bryson, n.r. 2004. progression towards endemic stability to bovine babesiosis in cattle introduced onto a game ranch. onderstepoort journal of veterinary research, 71:333–336 an opportunity to study progression toward endemic stability to babesia bigemina arose when cattle were reintroduced onto a game ranch in 1999 after an absence of three years. the study was conducted between august 2000 and june 2001. the unvaccinated breeding cows were sampled only once. calves born during october 1999 were initially vaccinated against b. bigemina and babesia bovis at the age of 4 months and were then bled at 10, 17 and 20 months of age. calves born during 2000 were bled at 7 and 8 months of age. sera were collected from all the cattle sampled and later tested for antibodies against b. bigemina and b. bovis using the indirect fluorescent antibody (ifa) test. although endemic stability to b. bigemina had not been achieved at nooitgedacht 2 years after resumption of cattle ranching, the high seroprevalence in the unvaccinated 8month-old calves suggested that the situation was approaching stability and that calf vaccination against bovine babesiosis was not required. tick control should therefore be restricted to prevent excessive tick worry. only vaccinated cattle were positive to b. bovis and it was concluded that the parasite was absent from the ranch. keywords: babesia bigemina, babesia bovis, bovine babesiosis, endemic stability, immunization, redwater, south africa * author to whom correspondence is to be directed accepted for publication 7 april 2004—editor results. according to the definition of norval et al. (1983), an endemically stable situation occurs when 81–100 % of the herd are infected with a particular babesia species. an opportunity to study progression towards endemic stability in a newly introduced cattle population arose when cattle were reintroduced onto a 2 780ha game ranch in 1999 after an absence of 3 years —the ranch had been managed solely for wildlife in the interim. the ranch (nooitgedacht; 24° 33’ s; 28° 36’ e), in the mokopane (previously potgietersrus) district, limpopo province, south africa, is hilly, with an average altitude of 1 380 m above sea level. the vegetation was classified as sour bushveld (acocks 1988). the rainfall during the periods july 1999 to june 2000 and july 2000 to june 2001 was 1 000 mm and 670 mm, respectively. the antelope population on nooitgedacht comprised eland (taurotragus oryx) (n = 26), gemsbok (oryx gazella) (n = 21), red hartebeest (alcelaphus buselaphus) (n = 30), blue wildebeest (connochaetes taurinus) (n = 38), blesbok (damaliscus pygargus philipsi) (n = 180), impala (aepyceros melampus) (n = 200), greater kudu (tragelaphus strepsiceros) (n = 60), nyala (tragelaphus angasii) (n = 3), waterbuck (kobus ellipsiprymnus) (n = 30), reedbuck (redunca arundinum) (n = 28), mountain reedbuck (redunca fulvorufula) (n = 20), grey duiker (sylvicapra grimmia) (n = 40), steenbok (raphicerus campestris) (n = 10) and klipspringer (oreotragus oreotragus) (n = 10) (f.s.h. du preez, personal communication 2000). nooitgedacht ranch falls outside the known distribution of r. (b.) microplus. the r. (b.) decoloratus vector tick population on nooitgedacht would probably have remained at fairly high levels even in the absence of cattle, as impalas and greater kudus, which were present in relatively large numbers, are preferred hosts for this species (horak, boomker, spickett & de vos 1992; horak, gallivan, braack, boomker & de vos 2003). from 1999 onwards the management objectives at nooitgedacht were to produce brahman steers and to run a brahman stud. the founding cattle breeding stock were obtained from kareefontein ranch, 100 km south of nooitgedacht, an area where babesia bigemina was endemic. the cattle were not dipped or treated with antibabesial drugs before being moved. we were unable to ascertain what the level of infection in the herd had been at the time of introduction. outbreaks of clinical babesiosis had not been recorded on nooitgedacht ranch, however, and no clinical cases of the disease were reported during the study period (f.s.h. du preez, personal communication 2001). ticks on the cattle were controlled by hand-spraying with bayticol (2 % flumethrin, bayer), at irregular intervals, whenever the owner judged the tick burden to be excessive. our study commenced in august 2000, one year after cattle had been reintroduced to nooitgedacht. the first calf crop (n = 30), born in october 1999, had been vaccinated against b. bigemina and b. bovis at the age of 4 months. the main objective of our study was to assess whether endemic stability to b. bigemina had been achieved in the newly introduced cattle population, but we also report on the effects of vaccinating against b. bovis in the 1999 calf crop. the study involved breeding cows, as well as calves born during october 1999 and october 2000. cattle were selected for sampling by the simple random sampling technique (thrusfield 1995). the breeding cows (n = 50) were sampled once only, when sampling was commenced in august 2000. the calves born during october 1999 were sampled when they were 10 months old (n = 49), and then re-sampled at 17 (n = 39) and 20 months (n = 30). the october 2000 calf crop was sampled at 7 months (n = 47) and 8 months (n = 20) of age. the cattle were restrained with a neck-clamp and blood was collected aseptically from the caudal vein into 10 ml plain vacutainer tubes (sherwood medical) using 20-gauge needles (becton dickinson). at the laboratory, the tubes were centrifuged and the serum decanted. the sera were then frozen and stored at the department of veterinary tropical diseases until they were transferred to the onderstepoort veterinary institute, where serological testing was performed. all serum samples were tested for the detection of antibodies against babesia bigemina using standard indirect fluorescent antibody test (ifat) procedures (anon. 2000). data generated from the work were recorded and analyzed by means of the sas statistical package, version 8.1. comparative analyses were carried out using the chi-square test. the prevalence of antibodies to b. bigemina and b. bovis in vaccinated and unvaccinated cattle of various age groups is shown in fig. 1. seventeen percent of the 7-month-old calves were seropositive to b. bigemina. twenty-eight days later (at the age of 8 months), this had increased significantly to 70 % (χ2 = 10.1411, p = 0.0015). the prevalence of antibodies to b. bigemina in 10and 17-month-old cattle did not differ significantly (χ2 = 0.9594, p = 0.3273), while the 10-month-old 334 endemic stability to bovine babesiosis in cattle calves had significantly higher antibody prevalence to b. bovis than the 17-month-old cattle (χ2 = 5.8419, p = 0.0156). the prevalence of antibodies to b. bigemina in 17and 20-month-old cattle did not differ significantly (χ2 = 2.1473, p = 0.1428). this was also the case with prevalence of antibodies to b. bovis (χ2 = 0.3221, p = 0.5704). seventy-two percent of the breeding cows were seropositive to b. bigemina 1 year after being transferred to nooitgedacht ranch and their serological status was significantly higher than that of the 20month-old cattle, born on the ranch (χ2 = 12.5644, p = 0.0004). the breeding cows were all negative to b. bovis. in endemically stable scenarios, the age incidence of b. bigemina parasitaemia is generally lower in older animals when compared with the younger ones (mahoney 1969). although the serological status of the breeding cows at the time of transfer to nooitgedacht was unknown, this group retained a higher seroprevalence to b. bigemina than their calves. the breeding cows were probably the main source of b. bigemina infection to ticks after the reintroduction of cattle to the ranch. babesia bigemina infections rarely persist for longer than a year, and infected cattle normally only remain infective to ticks for 4–7 weeks (johnston et al. 1978; mahoney 1969). any loss of infection would contribute to a reduced parasite transmission rate, as fewer animals would serve as sources of infection to ticks. the antelope that remained on the ranch mingled freely with the cattle. as is the case with cattle, greater kudus and impalas (ca 260 on the ranch) are also preferred hosts of r. (b.) decoloratus. it seems likely, therefore, that a substantial number of larvae of this tick species attaching to cattle during the study period were the offspring of adults that had fed on antelope, and were therefore not infected with b. bigemina. the overall level of infection of the vector tick population would thus tend to be low. in the vaccinated 1999 calf crop, which was sampled at the age of 10, 17 and 20 months, the seroprevalence to b. bigemina decreased with age. this may have been due to the loss of ifa antibody titres as a result of a low number of superinfections. todorovic (1975) reported that cattle challenged with b. bigemina-infected blood reached peak levels of ifa reacting antibody titres 21 days post infection and the titres decreased gradually thereafter. de vos (1977, unpublished data cited by de vos 1979), also using the ifa test, found that 93 % of the cattle were positive to b. bigemina 2 months after vaccination, whilst only 60 % were still positive 19 months later. he observed that in the absence of adequate natural challenges, the titres of vaccinated cattle decreased and more cattle became seronegative. the percentage of animals positive to b. bovis at the age of 10, 17 and 20 months declined with increasing age, probably due to loss of ifat-reacting antibody titres in the absence of natural challenge. using the ifat, de vos (1977, unpublished data cited by de vos 1979) determined that seroprevalence in a vaccinated herd gradually decreased in the absence of adequate natural challenge: a herd that was 97 % positive to b. bovis 2 months post vaccination, was only 60 % positive 19 months later. the unvaccinated 2000 calf crop showed a sharp increase in prevalence of antibodies to b. bigemina at 8 months, a situation approaching endemic stability. a similar trend was also observed in calves of the same age group in a trial conducted concurrently at another ranch in limpopo province (regassa, penzhorn & bryson 2003). mahoney (1969) found that the age incidence of b. bigemina parasitaemia rose from zero at birth, attained a maximum between 6 months and 2 years of age and then declined sharply in the older animals. in summary, endemic stability to b. bigemina had not been achieved at nooitgedacht 2 years after resumption of cattle ranching. the high seroprevalence in the unvaccinated 8-month-old calves suggested that the situation was approaching stability and that calf vaccination was not required. tick control should therefore be restricted to prevention of excessive tick worry. 335 a. regassa, b.l. penzhorn & n.r. bryson �� �� �� �� � � � �� �� �� �� ��� � � �� � � �� � � � � � � � ��� �������� ������������ � � ������������� fig 1. prevalence of antibodies against babesia bigemina and babesia bovis, as determined by ifa test, in vaccinated (10-, 17and 20-month-old) and unvaccinated (7-, 8and 30–140-month-old) brahman cattle at nooitgedacht ranch acknowledgements this project (no. 36-5-471) was approved by the research committee and the animal use and care committee of the faculty of veterinary science, university of pretoria, and was funded by the ethiopian agricultural research organization as part of an m.sc. research project. mr f.s.h. du preez, the owner of nooitgedacht ranch, is gratefully acknowledged for permission to conduct the research on his property and provision of all required information. dr h. hansen introduced us to mr du preez. the ranch staff are thanked for their assistance when specimens were collected. data analysis was done in collaboration with mrs rina owen and mr sollie millard, department of statistics, university of pretoria. references acocks, j.p.h. 1988. veld types of south africa. pretoria: government printer (memoirs of the botanical survey of south africa, no. 57). anonymous. 2000. manual of standards for diagnostic tests and vaccines, 4th ed. paris: office international des epizooties. carson, c.a. & phillips, r.s. 1981. immunologic response of the vertebrate host to babesia, in babesiosis, edited by m. ristic & j.p. kreier. new york: academic press. de vos, a.j. 1979. epidemiology of bovine babesiosis in south africa. onderstepoort journal of veterinary research, 50: 357–362. horak, i.g., boomker, j., spickett, a.m. & de vos, v. 1992. parasites of domestic and wild animals in south africa. xxx. ectoparasites of kudus in the eastern transvaal lowveld and the eastern cape province. onderstepoort journal of veterinary research, 59:259–273. horak, i.g., gallivan, g.j., braack, l.e.o., boomker, j. & de vos, v. 2003. parasites of domestic and wild animals in south africa. xli. arthropod parasites of impalas, aepyceros melampus, in the kruger national park. onderstepoort journal of veterinary research, 70:131–163. johnston, l.a.y., leatch, g. & jones, p.n. 1978. the duration of latent infection and functional immunity of droughtmaster and hereford cattle following natural infection with babesia argentina and babesia bigemina. australian veterinary journal, 54:14–18. mahoney, d.f. 1969. bovine babesiosis: a study of factors concerned in transmission. annals of tropical medicine and parasitology, 63:1–4. mahoney, d.f. & ross, d.r. 1972. epizootiological factors in the control of bovine babesiosis. australian veterinary journal, 48:292–298. mccosker, p.j. 1981. the global importance of babesiosis, in babesiosis, edited by m. ristic & j.p. kreier. new york: academic press. norval, r.a.i., fivaz, b.h., lawrence, j.a. & dailecourt, t. 1983. epidemiology of tick-borne diseases of cattle in zimbabwe: i. babesiosis. tropical animal health and production, 15:87–94. regassa, a., penzhorn, b.l. & bryson, n.r. 2003. attainment of endemic stability to babesia bigemina in cattle on a south african ranch where non-intensive tick control was applied. veterinary parasitology, 116:267–274. ristic, m. & levy, m.g. 1981. a new era of research toward solution of bovine babesiosis, in babesiosis, edited by m. ristic & j.p. kreier. new york: academic press. thrusfield, m. 1995. veterinary epidemiology, 2nd ed. london: blackwell science. todorovic, r.a. 1975. serological diagnosis of babesiosis: a review. tropical animal health and production, 7:1–14. tønnesen, m.h., penzhorn, b.l., bryson, n.r., stoltsz, w.h. &. masibigiri, t. 2004. displacement of boophilus decoloratus by boophilus microplus in the soutpansberg region, limpopo province, south africa. experimental and applied acarology, 32:199–208. 336 endemic stability to bovine babesiosis in cattle article information authors: makhosazana motloang1 justin masumu2 barend mans1 peter van den bossche3,† abdalla latif1 affiliations: 1parasites, vectors & vector-borne diseases programme, arc-onderstepoort veterinary institute, onderstepoort, south africa2universite pedagogique nationale, kinshasa, democratic republic of the congo 3institute of tropical medicine, antwerpen, belgium correspondence to: makhosazana motloang postal address: private bag x05, onderstepoort 0110, south africa dates: received: 16 june 2011 accepted: 19 oct. 2011 published: 21 feb. 2012 how to cite this article: motloang, m., masumu, j., mans, b., van den bossche, p. & latif, a., 2012, ‘vector competence of glossina austeni and glossina brevipalpis for trypanosoma congolense in kwazulu-natal, south africa’, onderstepoort journal of veterinary research 79(1), art. #353, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.353 note: †, 1962–2010 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. vector competence of glossina austeni and glossina brevipalpis for trypanosoma congolense in kwazulu-natal, south africa in this original research... open access • abstract • introduction • materials and methods    • field collection of tsetse flies    • determination of infection rates in tsetse flies    • maintenance of tsetse flies    • experimental infection of colony flies using infected animals    • experimental infection of susceptible animals using field-collected flies    • statistical analyses    • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ tsetse-transmitted trypanosomosis (nagana) has been the cause of stock losses in the recent past and still presents a major problem to livestock owners in certain areas of kwazulu-natal, south africa. over 10 000 cattle mortalities were reported in the 1990 nagana outbreak. although information on the distribution and abundance of the tsetse flies glossina brevipalpis and glossina austeni in kwazulu-natal exists, data on their vector competence are lacking. this study aimed to determine the rate of natural trypanosoma congolense infection by field-collected as well as colony-reared flies of these species. a total of 442 field-collected g. brevipalpis and 40 g. austeni flies were dissected immediately after collection to determine their infection rates, whilst 699 g. brevipalpis and 49 g. austeni flies were fed on susceptible animals in 10 and four batches, respectively, for use in xenodiagnosis experiments. teneral colony flies were fed on infected animals and dissected 21 days post infection to confirm their infectivity testing. glossina austeni harboured 8% immature and mature infections. in g. brevipalpis, the infection with the immature stages was lower (1%) and no mature infections were observed. although all four batches of g. austeni transmitted t. congolense to four susceptible animals, no transmission resulted from 10 batches of g. brevipalpis fed on susceptible cattle. colony-derived g. austeni (534) and g. brevipalpis (882) were fed on four bovines infected with different t. congolense isolates. both g. austeni and g. brevipalpis acquired trypanosome infection from the bovines, with immature infection ranges of 20% – 33% and 1% – 4%, respectively. parasites, however, only matured in g. austeni (average = 4%). glossina austeni plays a larger role in the epidemiology of animal trypanosomosis in kwazulu-natal than g. brevipalpis and therefore more focus should be aimed at the former when control measures are implemented. introduction top ↑ the tsetse zone in south africa encompasses an area of 18 000 km2, representing the southernmost distributional limit of this genus in africa (kappmeier-green, potgieter & vreysen 2007). of the four tsetse species (diptera: glossinidae) historically found in south africa, only glossina austeni (savanna species) and glossina brevipalpis (fusca species) still exists in the north-eastern parts of kwazulu-natal. the fly habitat is confined to protected nature reserves, game parks, indigenous forests and river beds (kappmeier, nevill & bagnall 1998). the other two species, namely glossina morsitans morsitans and glossina pallidipes, which were considered to be the most efficient vectors of trypanosomes in general, had been eradicated in 1897 and 1954, respectively (du toit 1954). glossina austeni and g. brevipalpis were not considered to be important vectors of trypanosomosis in south africa (fuller 1923).despite the eradication of g. morsitans morsitans and g. pallidipes, clinical cases of trypanosomosis were still diagnosed in cattle, horses and dogs between 1955 and 1989 (bagnall 1993). in 1990, a serious outbreak of the disease in cattle, locally referred to as nagana, was reported at dip tanks in the vicinity of the hluhluwe–umfolozi game reserve and the infection was attributed to trypanosoma congolense and trypanosoma vivax. the treatment of cattle with trypanocidal drugs, combined with tsetse control efforts using deltamethrin ‘pour-on’ insecticide and odour-baited, insecticide-impregnated targets, was successful in controlling the outbreak (bagnall 1993), but the strategy was considered unsustainable because preventing tsetse fly reinvasion could not be guaranteed (kappmeier et al. 1998). the temporary nature of this approach was evidenced by the recurrence of trypanosomosis, which has reverted to the high levels seen in 1990, before these temporary control measures were instituted (van den bossche et al. 2006). the reoccurrence of outbreaks changed the previous perception that g. austeni and g. brevipalpis were of minor importance in trypanosome transmission and reaffirmed that they are responsible for the cyclical transmission of nagana (kappmeier & nevill 1999; kappmeier-green et al. 2007). subsequent tsetse surveys conducted from 1991 to 1999 revealed a relative higher prevalence and abundance of g. brevipalpis compared with that of g. austeni in the affected areas (bagnall 1993; kappmeier 2000). recently, a study conducted at the mvutshini dip tank found trypanosome infections in 61% of suspected sick cattle using the buffy coat procedure (van den bossche et al. 2006). the survey indicated that nagana was still prevalent in kwazulu-natal and that t. congolense was the dominant causative organism. of the two tsetse species, g. brevipalpis was found to be more abundant and therefore believed to be responsible for causing disease in cattle around the hluhluwe–umfolozi game reserve. the importance of a species as a vector is not determined only by its abundance, but also by its vector competence; that is, its ability to become infected and transmit pathogens (leak 1998). there are two key stages in the interaction of some trypanosomes (e.g. trypanosoma brucei or t. congolense) with tsetse flies, namely the initial establishment of infection in the midgut and the subsequent maturation of the trypanosomes to produce infective stages in the salivary glands or oesophagus (aksoy, gibson & lehane 2003). the current study was therefore conducted to identify the principle vector of trypanosomes in both field and controlled experimental conditions by comparing the vector competence of g. austeni and g. brevipalpis in transmitting t. congolense to cattle. materials and methods top ↑ field collection of tsetse flies glossina brevipalpis and g. austeni were collected between 2006 and 2008 from areas known to be endemic with nagana in north-eastern kwazulu-natal (figure 1). flies were collected using the h-trap designed for collecting these two species (kappmeier 2000). traps were baited with a mixture of 1-octen-3-ol and 4-methylphenol, dispensed from heat-sealed sachets in the presence of acetone. the traps were emptied daily and flies were transported to the agricultural research council (arc) field research station at kuleni in a cooler box. collected flies were divided into two groups, of which one was immediately used to determine the infection rate and the other group was used in xenodiagnosis experiments. flies were couriered to the arc-onderstepoort veterinary institute (ovi) laboratory, situated 600 km from the collection areas. figure 1: location of collection sites for glossina brevipalpis and glossinan austeni. determination of infection rates in tsetse flies a total of 442 g. brevipalpis and 40 g. austeni specimens were dissected immediately after collection and examined for trypanosome infections. an additional 195 g. brevipalpis specimens were dissected after transmission experiments to confirm the results of the infectivity testing in cattle. dissections were conducted according to the method of lloyd and johnson (1924) and as described by leak, ejigu and vreysen (2008). flies were dissected to expose immature infections (non-infective trypanosome forms) in the midgut and mature infections (infective trypanosome forms) in the proboscis (jordan 1976; van den bossche et al. 2004). the respective organs were placed onto microscope slides containing droplets of phosphate-buffered saline (with glucose) and covered with cover slips. infection rates were determined by direct observation of prepared slides under a compound microscope using a 10× eyepiece and a 25× objective. maintenance of tsetse flies at the arc-ovi, field-captured flies were maintained under the same conditions as the colony-reared flies. the arc-ovi houses well-established colonies of g. austeni and g. brevipalpis. these colonies were established in 2002 with seed materials of g. austeni obtained from the tsetse and trypanosomosis research institute in tanga, tanzania and g. brevipalpis supplied by the insect pest control sub-programme of the international atomic energy agency (iaea) laboratories in seibersdorf, austria. colony flies were maintained at 75% relative humidity and 24 °c and fed on artificial membranes using high-quality gamma-irradiated bovine blood according to the standard operating procedure of the food and agriculture organization/iaea (2006). experimental infection of colony flies using infected animals the artificial infection of colony flies was carried out to compare the results with the infection rate in field-collected flies. each of four susceptible cattle was infected intravenously with (1 × 106) organisms using four different t. congolense isolates. three of the isolates (i, ii and bmrngk2) were isolated from cattle at boomerang farm and the fourth (mvu10) from cattle at the mvutshini community dip tank. all isolates were maintained in mice. molecular characterisation of all four isolates, according to a method described by geysen, delespaux and geerts (2003), revealed that they belonged to t. congolense. the infected cattle were monitored daily for the development of trypanosome parasitaemia and fever, whilst packed cell volume (pcv) values were also determined. flies were fed on cattle when parasites were detected in the buffy coat preparations. experimental animals were clipped on their flanks and localised areas shaved with a scalpel blade to expose the skin (akol & murray 1983). flies were confined in cages in groups of not more than 20 and allowed to feed until fully engorged. the engorged flies were maintained on artificial feeding membranes (as described for the colony flies) for 21 days before dissection (masumu et al. 2006). experimental infection of susceptible animals using field-collected flies to demonstrate the infectivity of field-collected flies in cattle, 10 batches of g. brevipalpis (699 flies in total) and four batches of g. austeni (49 flies in total) were used in the transmission experiments. the batch sizes were determined by the number of flies captured in an area on a specific date. for each area the number of collections ranged from 16 to 180 and 6 to 40 for g. brevipalpis and g. austeni, respectively. flies were fed as mentioned earlier. where the number of flies per batch exceeded 20, more cages were used. each batch was allowed to feed on a susceptible nguni bovine, except for one batch of g. austeni, which was fed on a goat. the animals were monitored daily for the development of trypanosome parasitaemia, fever and changes in pcv over 30 days. animals considered anaemic (i.e. pcv = 18% for three consecutive days) received treatment with 3.5 mg/kg diminazene aceturate (berenil tm). statistical analyses variation in infection prevalence between isolates and tsetse flies was analysed using the statistical program genstat® (payne et al. 2007). testing was performed at the 5% significance level, with p < 0.05 used as the cut-off for statistical significance. ethical considerations ethical approval for the experiments was obtained from the animal ethics committee of the ovi (ref. 07/20/c174) and animal use and care committee of the university of pretoria, faculty of veterinary sciences (ref. vo56-09). results top ↑ a total of 637 g. brevipalpis and 40 g. austeni specimens collected from the field were dissected and their infection prevalence assessed (table 1). analysis revealed 8% immature and mature trypanosome infections the midgut and proboscis of g. austeni. on the other hand, only 1% of immature infections were detected in the midgut of g. brevipalpis. table 1: prevalence of trypanosome parasites in midgut and proboscis of field-collected glossina austeni and glossina brevipalpis. to confirm the infection rate results obtained for the field-collected tsetse flies, laboratory-controlled fly infectivity experiments were conducted. a total of 534 colony-reared g. austeni and 882 g. brevipalpis, fed on experimentally infected parasitaemic cattle, were dissected. dissection results indicated that all four isolates (boomerangi, boomerangii, bmrngk2 and mvu10) became established in the midgut of both g. austeni (20% – 33%) and g. brevipalpis (1% – 4%) (table 2). of the infected flies, 22% (n = 121) immature infections and 4% (n = 21) mature infections were found in g. austeni fed on four cattle infected with different t. congolense isolates. only 2% (n = 17) immature infections were seen in g. brevipalpis and no parasites were detected in the proboscis. table 2: trypanosome infection rate in midgut and proboscis of glossina austeni and glossina brevipalpis colony flies fed on infected cattle (calculated p-values). vector competence was also assessed to confirm the results on the infection rates with trypanosomes in g. brevipalpis and g. austeni collected from game parks and communal dip tanks in kwazulu-natal. the infectivity of g. brevipalpis fed on susceptible cattle under controlled conditions was not shown and no trypanosome transmission was observed from any of the flies (as many as 180 per animal). on the other hand, transmission with g. austeni was achieved with a small number of feeding flies (i.e. fewer than 10 per animal). there were no significant differences in the immature infection prevalence between the four isolates in g. austeni (p = 0.158). however, there was a significant difference (p = 0.025) in the immature infection prevalence of these isolates in g. brevipalpis. a higher number of midgut infections were observed to have resulted from boomerangi and ii (4.3% and 2.6%, respectively) than from mvu10 and bmrngk2 (0.36% and 0.50%, respectively), as shown in table 3. in contrast, the number of g. austeni with mature infections from the four isolates differed significantly (p = 0.007). flies infected with boomerangi and ii isolates had a higher infection prevalence (9.5% and 8.2%, respectively) compared with those infected with isolates mvu10 and bmrngk2 (2.6% and 1.6%, respectively) (table 3). mature infections were not detected in the proboscis of any g. brevipalpis flies. table 3: infection prevalence in the midgut and proboscis of infected colony glossina austeni and glossina brevipalpis flies. after the flies had fed on the animals, parasites were detected in blood samples within 14, 17 and 22 days for cattle and 15 days for the goat. all animals subsequently presented with clinical signs of anaemia (table 4). table 4: infectivity of glossina austeni and glossina brevipalpis collected from different field sites and fed on susceptible hosts under controlled conditions. discussion top ↑ in the present study, only 1% of t. congolense infections were found in the midgut of g. brevipalpis. in contrast, the infection rate in the midgut and the proboscis of g. austeni was significantly higher, with 8% detected in both organs. the age structure of the two glossina species had not been determined in the field-collected flies. it has been reported that older flies are more likely to be infected than younger flies (harley 1966; jordan 1976; woolhouse et al. 1994). the factors affecting the trypanosome infection rate in flies and contributing to their being refractory to infection have been detailed and discussed by several authors (jordan 1976; roditi & lehane 2008; welburn & maudlin 1999). factors such as age may influence the readiness with which flies can be infected. according to harley (1967), the longer the female of some fly species, such as g. brevipalpis, lives, the more likely she is to be infected with t. congolense. in contrast, species such as g. austeni can be readily infected when they are 1 day old and less successfully later (jordan 1976; ward 1968). in our study, both g. austeni and g. brevipalpis colony specimens were fed a day after emergence and were able to establish infections in the midgut. however, trypanosomes could not develop to maturity in g. brevipalpis. the results showing the poor efficiency of g. brevipalpis as a vector was unexpected based on their higher population densities in areas close to the hluhluwe–umfolozi game reserve where high infection prevalence in cattle had been reported (gillingwater, mamabolo & majiwa 2010; van den bossche et al. 2006). these results further suggest that g. austeni is the major vector of trypanosomes in the area, despite its relatively low population density as reported by esterhuizen et al. (2005) and hendrickx et al. (2003). the relatively low numbers of g. austeni collected in this study indicate either that the natural population density is low or that the h-trap is not effective for the collection of this species. similar observations were reported by gaturaga, maloo and loehr (1989) when they collected only 33 flies representing two glossina species over a period of one year, despite a high trypanosome infection rate (22%) amongst cattle. they attributed the low numbers of g. austeni collected, assumed to be the major vector, to the inefficiency of the biconical trap used. interestingly, flies infected with isolates boomerangi and ii exhibited more midgut infections in g. brevipalpis and more mature infections in g. austeni than those infected with isolates mvu10 and bmrngk2. both fly species seemed to be more susceptible to infection with boomerang i and ii isolates; however, g. brevipalpis is refractory to subsequent parasite maturation occurring in the proboscis. goossens et al. (2006) found a very low prevalence of t. congolense in cattle (0.8%) on mafia island, tanzania, where g. brevipalpis is the only tsetse fly species encountered and widely distributed. they attributed the low prevalence to a combination of factors, such as frequent use of prophylactic treatment of cattle with trypanocidal drugs, a low feeding frequency of g. brevipalpis on cattle and the low vectorial capacity of the fly. in contrast, wilson, dar and paris (1972) found the infection rate with t. congolense in field-collected g. brevipalpis to be about 2% in their study in uganda. the injection of the infected proboscis collected from these flies produced patent infection in mice and thus demonstrated the ability of t. congolense to mature in the proboscis. the present data do not support results from two recent studies on the trypanosomes infection rates in tsetse flies conducted in the same area using only polymerase chain reaction (pcr) analysis (gillingwater et al. 2010; mamabolo et al. 2009). mamabolo et al. (2009) detected trypanosome dna in 89% of the flies examined but the results did not specify the species origin. furthermore, this molecular test does not distinguish between mature and immature infections. in the study of mamabolo et al. (2009), the injection of a suspension of macerated fly proboscis did not produce any viable infections in mice that were monitored for the development of trypanosome parasitaemia. similarly, gillingwater et al. (2010) reported a higher percentage (20%) of flies testing positive for trypanosome dna in the midgut with only 1.6% of mature infections. again, the results were not separated according to species. the higher infection rates detected by the pcr analysis may be related to recent feeding of the flies on infected animals although these trypanosomes would not necessarily develop successfully in the midgut or the proboscis. conclusion top ↑ a wealth of entomological data have been collected over the years in kwazulu-natal, which were used by hendrickx et al. (2003) to produce distribution and prediction maps. however, parallel data on the epidemiology of the disease have not been generated to support the intention of the veterinary authorities to control or eradicate tsetse flies from south africa. the results from the current study support the findings of goossens et al. (2006) that g. brevipalpis is not the main vector of t. congolense in kwazulu-natal, despite its higher abundance, whereas g. austeni has been shown to have a higher vector competence. focus should, therefore, be directed towards the control of g. austeni in the province whilst more research is still needed to develop more efficient traps to monitor the population dynamics of this species before, during and after control operations. acknowledgements top ↑ the department of science and technology and the arc are thanked for financial support dr gert venter provided valuable comment on an earlier version of the manuscript. mr dannie de klerk assisted with field collections and feeding on susceptible animals. mr jerome ntshangase and mr gazu are thanked for deploying, and maintaining traps and assistance with field collections. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions m.m. (arc-onderstepoort veterinary institute) was the project leader and performed most of the experiments and wrote the manuscript. j.m. (universite pedagogique nationale) was a student mentor who assisted in the execution of some experiments and also edited the manuscript. a.l. (arc-onderstepoort veterinary institute) was the project supervisor and also contributed to revision and editing of the manuscript. b.m. (arc-onderstepoort veterinary institute) was the co-supervisor of the study and contributed to revision and editing of the manuscript. p.v.d.b. (institute of tropical medicine) was a co-supervisor to j.m. 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(eds.), 2008, collection of entomological baseline data for tsetse area-wide integrated pest management programmes, food and agricultural organization, rome. lloyd, l. & johnson, w.b., 1924, ‘the trypanosome infections of tsetse flies in nigeria and a new method of estimation’, bulletin of entomological research 14, 265–288. mamabolo, m.v., ntantiso, l., latif, a. & majiwa, p.a.o., 2009, ‘natural infection of cattle and tsetse flies in south africa with two genotypic groups of trypanosoma congolense’, parasitology 136, 425–431. http://dx.doi.org/10.1017/s0031182009005587, pmid:19250578 masumu, j., marcotty, t., geysen, d., geerts, s., vercruysse, j., dorny, p. et al., 2006, ‘comparison of the virulence of trypanosoma congolense strains isolated from cattle in a trypanosomosis endemic area of eastern zambia’, international journal for parasitology 36, 497–501. http://dx.doi.org/10.1016/j.ijpara.2006.01.003, pmid:16516215 payne, r.w., murray, d.a., harding, s.a., baird, d.b. & soutar, d.m., 2007, genstat for windows introduction, 10th edn., vsn international, hemel hempstead. roditi, i. & lehane, m.j., 2008, ‘interactions between trypanosomes and tsetse flies’, current opinion in microbiology 11, 345–351. http://dx.doi.org/10.1016/j.mib.2008.06.006, pmid:18621142 van den bossche, p., de deken, r., brandt, j., geerts, s., geysen, d. & berkvens, d., 2004, ‘the transmission of mixed trypanosoma brucei/t. congolense infections by tsetse (glossina morsitans morsitans)’, veterinary parasitology 119, 147–153. http://dx.doi.org/10.1016/j.vetpar.2003.11.008, pmid:14746974 van den bossche, p., esterhuizen, j., nkuna, r., matjila, t., penzhorn, b., geerts, s. et al., 2006, ‘an update of the bovine trypanosomosis situation at the edge of hluhiuwe-imfolozi park, kwazulu-natal province, south africa’, onderstepoort journal of veterinary research 73, 77–79. pmid:16715881 ward, r.a., 1968, ‘the susceptibility of glossina austeni into infection with trypanosoma brucei,’ transactions of the royal society of tropical medicine and hygiene 62, 672–768. welburn, s.c. & maudlin, i., 1999, ‘tsetse-trypanosome interaction: rites of passage’, parasitology today 15, 399–403. http://dx.doi.org/10.1016/s0169-4758(99)01512-4 wilson, a.j., dar, f.k. & paris, j., 1972, ‘a study on the transmission of salivarian trypanosomes isolated from wild tsetse flies’, tropical animal health and production 4, 14–22. http://dx.doi.org/10.1007/bf02357090, pmid:4671472 woolhouse, m.e.j., bealby, k., mcnamara, j.j. & silutongwe, j., 1994, ‘trypanosome infections of the tsetse fly glossina pallidipes in the luanwa valley, zambia’, international journal for parasitology 24, 987–993. http://dx.doi.org/10.1016/0020-7519(94)90164-3 article information authors: ivan g. horak1,4 gordon j. gallivan2 arthur m. spickett3 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa 2187 cluny street, ottawa, canada 3parasites, vectors & vector-borne diseases programme, onderstepoort veterinary institute, south africa 4department of zoology and entomology, university of the free state, south africa correspondence to: ivan horak email: ivan.horak@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 09 sept. 2010 accepted: 10 nov. 2010 published: 25 feb. 2011 how to cite this article: horak, i.g., gallivan, g.j. & spickett, a.m., 2011, ‘the dynamics of questing ticks collected for 164 consecutive months off the vegetation of two landscape zones in the kruger national park (1988–2002). i. total ticks, amblyomma hebraeum and rhipicephalus decoloratus’, onderstepoort journal of veterinary research 78(1): art. #32, 10 pages. doi:10.4102/ojvr.v78i1.32 copyright notice: © 2011. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) the dynamics of questing ticks collected for 164 consecutive months off the vegetation of two landscape zones in the kruger national park (1988–2002). part i. total ticks, amblyomma hebraeum and rhipicephalus decoloratus in this original research... open access • abstract • introduction • materials and methods    • study localities    • climate    • tick collection    • statistical methods • results and discussion    • total ticks    • amblyomma hebraeum    • rhipicephalus decoloratus • conclusion • acknowledgements • references abstract (back to top) despite a large number of studies on tick biology, there is limited information on long-term changes in tick populations. this study thus aimed to assess the long-term population dynamics of questing ixodid ticks in two landscape zones of the kruger national park (knp). questing ixodid ticks were collected in the knp from august 1988 to march 2002 by monthly dragging of the vegetation in three habitats (grassland, woodland and gully) at two sites (nhlowa road and skukuza). findings pertaining to total tick numbers and amblyomma hebraeum and rhipicephalus decoloratus specifically are presented here. fourteen tick species were collected, as well as four others that could be identified only to generic level. more ticks (211 569 vs 125 810) were collected at nhlowa road than at skukuza. larvae were the most commonly collected stage of all the major tick species. a. hebraeum was the most commonly collected tick (63.6%) at nhlowa road, whereas r. decoloratus accounted for 15.3% of the ticks collected there. at skukuza, 31.6% and 27.1% of the collected ticks were r. decoloratus and a. hebraeum respectively. most a. hebraeum larvae were collected in summer and the fewest in winter and early spring, mostly in woodland and least often in grassland habitats. most r. decoloratus larvae were collected in spring and the fewest in autumn and winter, and were more frequently collected in woodland and grassland than in gullies. the largest collections of most tick species were made during the early 1990s, while numbers were lowest in the mid-1990s after a drought during 1991 and 1992 and then increased towards the late 1990s, followed by a final decrease. the changes in tick numbers over time probably reflect differences in their host communities at the two sites and the effect of climatic conditions on both hosts and free-living ticks. the population dynamics of questing ticks reflect a complex interaction between ticks, their hosts and the environment. introduction (back to top) tick populations are influenced by a complex interaction between the ticks, their hosts and the environment. there have been many studies regarding the various factors affecting the tick–host–environment association. however, most have been of relatively short duration (typically 1–2 years) and there is little information on longer-term changes in tick populations. in south africa, horak and boomker (1998) collected ticks from bontebok, (damaliscus pygargus dorcas) and grey rhebok (pelea capreolus) in the bontebok national park over a 10-year period. while there were marked changes in the abundance of the major tick species over time, the changes are difficult to interpret because the ticks were collected only during february of each year. horak et al. (1993) and horak et al. (1995b) monitored the changes in tick populations on scrub hares (lepus saxatilis) at skukuza in the kruger national park (knp) monthly over a period of 6 years. most amblyomma hebraeum larvae were collected from the hares between november and march or april and an erratic decline in numbers was observed over the 6-year survey period. the numbers of larvae of the two-host tick hyalomma truncatum peaked on the hares between may and august every year and the nymphs between june and september. no noticeable decline in their numbers was evident during the 6 years of the survey. the knp, encompassing nearly 2 million hectare, is a large nature reserve in north-eastern south africa. acocks (1988) classified five vegetation types in the knp and at a more detailed level gertenbach (1983) described 35 landscape zones. numerous surveys of the ticks parasitising wildlife in the knp have been conducted (horak, de vos & brown 1983; horak, de vos & de klerk 1984; horak et al. 1987b; horak et al. 1988; horak et al. 1991; horak et al. 1992; horak et al. 1993; horak, de vos & braack 1995a; horak et al. 1995b; spickett et al. 1995; braack et al. 1996; horak et al. 2000a; horak et al. 2003). these studies have recorded the host associations of tick species, their patterns of seasonal abundance, and the effects of host age and gender and droughts on the intensity of infestation. there have also been various surveys of free-living ticks in the park (spickett et al. 1992; horak et al. 2006b). these studies have examined differences among landscape zones and the effects of droughts and burning on free-living tick populations. in the present communication we describe changes in the populations of questing ticks, and specifically of two major species, a. hebraeum and rhipicephalus decoloratus, collected from the vegetation in two landscape zones in the knp monthly over a period of 164 months. preliminary results on five tick species collected during either the first 11 or 12 years of the survey, and on one species collected throughout the survey, have previously been published (horak, spickett & braack 2000b; horak, emslie & spickett 2001; horak et al. 2006a). materials and methods (back to top) study localities the study was conducted at nhlowa road and skukuza in the knp. the nhlowa road site was located in the south of landscape zone 17, which has been described as a sclerocarya caffra–acacia nigrescens savanna (gertenbach 1983). this landscape zone extends from the south-eastern border of the knp to beyond the satara tourist rest camp, approximately 125 km to the north. it is an open, treed savanna with moderate shrub and dense grass cover (gertenbach 1983). the study site (approximately 25°15’s, 31°55’e; alt. approximately 200 m above sea level) was about 8 km long and 2 km wide and straddled the nhlowa gravel tourist road between the crocodile bridge tourist rest camp in the south and the lower sabie tourist rest camp in the north.the skukuza site was situated in the central north of landscape zone 4, the thickets of the sabie and crocodile rivers (gertenbach 1983). this landscape zone extends from the middle of the southern border of the knp to north of the skukuza tourist rest camp, a distance of approximately 50 km. the vegetation consists of thorny thickets characterised by a. nigrescens and combretum apiculatum, with a sparse grass layer (gertenbach 1983). the site (approximately 25°04’s, 31°32’e; alt. approximately 260 m above sea level) was about 7 km long by 2 km wide and straddled the gravel road serving as a firebreak along the banks of the seasonally flowing nwaswitshaka river. the site commenced approximately 4 km west of the skukuza tourist rest camp and continued west to the gravel tourist road that joins the tarred doispane and napi roads. climate the climate in the southern knp is described as tropical with summer rainfall. both sites were within the 600 mm – 650 mm isohyets (gertenbach 1980). annual rainfall (june–may) at the skukuza rest camp averaged 609 mm over the period of the study, with a range of 275 mm – 1122 mm (figure 1a). although the annual rainfall often differed between the two sites in a given year (figure 1a and 1b), average annual rainfall at the lower sabie rest camp (the closest climate station to the nhlowa road site) was similar to that at the skukuza rest camp. weather readings only commenced at the lower sabie rest camp between 1991 and 1992 and rainfall at the nhlowa site, which was located in arid lowveld, was probably lower than that at the lower sabie rest camp located in the lowveld (acocks 1988). based on the long-term rainfall patterns in the knp (gertenbach 1980; whyte & joubert 1988), the study commenced toward the end of a dry cycle that ended in the early 1990s, while the latter part of the study was conducted in a wet cycle.average temperatures were approximately 0.8 °c cooler at skukuza than at lower sabie, with maximum temperatures differing by 0.4 °c and minimum temperatures by 1.3 °c (figure 1c). average relative humidity at the skukuza rest camp was similar to that at the lower sabie rest camp at 08:00 but was generally lower at 14:00 (figure 1d). table 1: number of ticks collected by dragging at the skukuza and nhlowa road sites in the kruger national park from august 1988 to march 2002, organised according to species and life stage. tick collection questing ticks were collected by drag-sampling the vegetation with flannel strips (spickett et al. 1992). in addition to the flannel strips, the operator responsible for dragging the strips also wore flannel leggings. three drags, each approximately 250 m long and 1 m wide, were performed in each of three habitats at each site, namely grassland, woodland and gully. the three habitats were selected subjectively on their visual appearance. because of the limited number of gullies at each site, the same gullies were usually sampled every month, whereas the sampling locations in grassland and woodland usually varied from month to month. after each drag the ticks collected on the flannel strips and the leggings were removed using sharp-pointed forceps. ticks were stored in 70% ethyl alcohol in internally labelled, plastic-stoppered glass vials for subsequent identification and counting.sampling commenced in august 1988 and continued in every habitat at both localities until march 2002, except after heavy rainfall or during a bush fire, or upon sighting elephants (loxodonta africana), african buffaloes (syncerus caffer) or lions (panthera leo) in a habitat. figure 1 a) annual (june–may) rainfall, b) monthly rainfall, c) mean monthly maximum and minimum temperatures and d) mean monthly relative humidities at 08:00 (upper lines) and 14:00 (lower lines) at skukuza and lower sabie (the two rest camps closest to the study sites) during the study period. statistical methods the tick counts were log10 transformed prior to analysis to reduce the effects of over-dispersion (petney, van ark & spickett 1990). independent variables of interest in the analysis were site, year, season, habitat and rainfall. year was normally taken as the seasonal year (june–may) used to define the annual rainfall in the knp, unless the occurrence of a tick stage was restricted to a calendar year. when the numbers were sufficient, the seasonality of the total number of ticks and each stage of a species were tested for both sites using serial autocorrelation (spss inc. 1994). multifactor analyses of variance were used to assess the effect of site, year, season and habitat on the total number of ticks and each stage. when their occurrence was strongly seasonal, months with zero counts were usually excluded to improve the sensitivity of the analysis. a serial cross-correlation was used to assess the relationship between rainfall and tick numbers. results and discussion (back to top) the total number of ticks and the respective species collected at each site are summarised in table 1. fourteen species were identified, while a further four species could be identified only to generic level. nine of the species were collected in sufficient numbers to allow an evaluation of trends, while the other five species were collected only infrequently and in small numbers. total ticks more ticks were collected at nhlowa road than at skukuza (p < 0.0001). at nhlowa road, a. hebraeum accounted for 63.6% of the ticks collected and was the species most commonly collected in all years (figure 2a). r. appendiculatus (17.3%) and r. decoloratus (15.3%) were the other commonly collected ticks, with r. appendiculatus the second most commonly collected tick between the 1989 and 1994 collection periods and was subsequently replaced by r. decoloratus. at skukuza, r. zambeziensis (38.3%) was the most commonly collected tick, followed by r. decoloratus (31.6%) and a. hebraeum (27.1%). r. zambeziensis was the most commonly collected tick between 1989 and 1993 and again in 1998 (figure 2b). r. decoloratus was the most common species in other years, except in 2000 when a. hebraeum was most commonly collected. all of the commonly collected tick species have regularly been collected from large ungulates in the knp, mostly as larvae (horak et al. 1983; horak et al. 1984; horak et al. 1988; horak et al. 1992; horak et al. 2003; horak, golezardy & uys 2007). the differences in the most common tick species and the numbers of ticks between the two sites probably reflect the differences in the large herbivore assemblages. the open savanna of the nhlowa road site is used by white rhinoceroses (ceratotherium simum), giraffes (giraffa camelopardalis), african buffaloes, greater kudus (tragelaphus strepsiceros) burchell’s zebras (equus burchellii), blue wildebeest (connochaetes taurinus) and impalas (aepyceros melampus). rhinoceroses and giraffes are excellent hosts of adult a. hebraeum and african buffaloes are excellent hosts of adult a. hebraeum and r. appendiculatus (horak et al. 1987a; horak et al. 2007). zebras, kudus and impalas are good hosts of all stages of development of r. decoloratus (horak et al. 1984; horak et al. 1992; horak et al. 2003). the most common ungulates in the thickets of the skukuza site are impalas, kudus, bushbuck (tragelaphus scriptus) and giraffes, although black rhinoceroses (diceros bicornis), buffaloes and zebras are also present. from 1988 to 1994 the populations of several ungulate species in the knp, including kudus, warthogs (phacochoerus africanus) and buffaloes declined by more than 50%. declines of 20% – 30% were also observed in the populations of other species, such as blue wildebeest and impalas, while the populations of some species, such as zebras and giraffes, did not change significantly (ogutu & owen-smith 2005). for many of the ungulate species that declined, there were significant lags in the population response to changes in rainfall and the recovery of those populations was delayed after the annual rainfall increased, probably because of the increased adult mortality during the years with low rainfall (owen-smith & mason 2005; owen-smith, mason & ogutu 2005). the ungulate species that declined are important hosts of the major tick species and the delayed recovery of ungulate populations probably contributed to the low tick numbers throughout the latter half of the 1990s, even though the survival of larval ticks should have increased along with the higher rainfall. changes in the size of the host population have previously been suggested as an explanation for the differences in the number of ticks parasitising impalas in the droughts of 1982 and 1992 in the skukuza region of the knp (horak et al. 1995a; horak et al. 2003). the impala population was relatively large prior to the drought of 1982, during which ‘terminally affected’ impalas were more heavily infested with ticks than healthy impalas (horak et al. 2003). the infestations on healthy impalas in 1982 were similar to those of impalas in 1980, a non-drought year. the impala population declined since the late 1980s and in 1992 tick infestations on impalas were much lower than during the early 1980s. the difference between the trends for 1980 and 1992 were due to the lower numbers of a. hebraeum, r. decoloratus and r. zambeziensis, species for which impalas and kudus are important hosts. there was significant seasonality in total tick numbers(p < 0.001) at skukuza (figure 3a), with a marked decline in tick numbers from january to april and a sharp increase in may. this reflected the seasonal increase in the numbers of r. zambeziensis larvae. at nhlowa road, the seasonal change in tick numbers was not statistically significant (p = 0.2) because the winter decline in the numbers of a. hebraeum larvae was offset by the increases in r. appendiculatus larvae in the winter and r. decoloratus larvae in the spring (figure 3b). mean monthly tick collections (june–may) varied significantly between years (p < 0.001) and there was a significant year-by-site interaction (p < 0.001) because the year-to-year changes were not consistent between the two sites (figure 2a and 2b). however, the highest numbers at both sites occurred in the early 1990s, declined towards the mid-1990s and again increased towards the turn of the century. annual rainfall declined since 1988, resulting in the drought of 1991, after which it again increased and peaked by 1999. however, tick numbers and rainfall were only weakly correlated, which suggests that annual rainfall was not the primary determinant of the numbers of questing ticks and that changes in ungulate populations over time may also have influenced the observed changes. total tick collections were highest in woodland and lowest in grassland (p < 0.001). this pattern was consistent at both sites (table 2) and among months and years. the habitat distribution reflects the distribution of a. hebraeum at nhlowa road, and of a. hebraeum and r. zambeziensis at skukuza. table 2: mean monthly tick collections [log10(n + 1)] organised according to habitat. amblyomma hebraeum once stimulated, questing a. hebraeum larvae are extremely active and readily climb onto the flannel strips and leggings. large mammals, such as rhinoceroses, giraffes, buffaloes, elands (taurotragus oryx) and kudus are important hosts of all stages of this tick, as are warthogs. smaller antelope, such as impalas, bushbuck and grey duikers (sylvicapra grimmia), as well as scrub hares, helmeted guinea fowls (numida meleagris) and carnivores are good hosts of the immature stages (horak et al. 1987a; horak et al. 1988; horak et al. 2007).larvae and nymphs were more commonly collected at nhlowa road than at skukuza (p < 0.0001). larvae accounted for more than 99.9% of a. hebraeum collected at both sites. despite significant variation between years in the mean monthly collections of larvae at the two sites, the trends were similar, with higher numbers in the early 1990s, followed by a decline in the mid-1990s and an increase in the late 1990s (figure 2a and 2b). the numbers of a. hebraeum larvae were significantly correlated with annual rainfall during the preceding year (p = 0.02) at skukuza, but not at nhlowa road (p > 0.2). the numbers of collected a. hebraeum nymphs did not differ significantly between years (p = 0.4). the differences between the two sites’ tick populations may be explained by the differences in host species. the populations of smaller ungulates, such as impalas, which predominate at skukuza, appear to respond more rapidly to fluctuations in rainfall (ogutu & owen-smith 2005). the drought during 1991 and 1992 also appeared to have caused the emigration of large mammals from the skukuza area (horak et al. 1995a), which would have further exacerbated the impact of declining host populations on the numbers of questing a. hebraeum larvae in this area. after the mid-1990s there was a greater increase in the numbers of a. hebraeum larvae at skukuza than at nhlowa road, as the populations of warthogs and impalas increased after the drought. in contrast, the populations of the larger ungulate species, such as buffaloes and elands, which predominate at the nhlowa road site, recovered much slower than the populations of smaller ungulates predominating at skukuza. a. hebraeum larvae were seasonal at both sites (figure 3a and 3b; p < 0.001), with numbers peaking in the summer and then reaching a minimum in the winter and early spring. at skukuza, the lowest numbers were collected in july and did not increase until november, whereas at nhlowa road the numbers declined gradually to a nadir in september, then increased. however, the difference in population sizes between the sites was not statistically significant (p = 0.27). although only small numbers of nymphs were collected, a seasonal pattern was evident at nhlowa road (p < 0.05), with a peak in the late summer and early autumn. the peak activity of questing a. hebraeum larvae during the summer rainfall period is consistent with that observed in other summer rainfall areas in southern africa (petney et al. 1987). a similar pattern of seasonal activity was observed on scrub hares examined at monthly intervals around skukuza for the first 6 years of the present survey (horak et al. 1993; horak et al. 1995b) and warthogs examined in the southern knp from 1980 to 1981 (horak et al. 1988). the intensity of infestation of a. hebraeum larvae appeared to be non-seasonal on greater kudus (horak et al. 1992). however, on impalas, the seasonal peaks of infestation were in the autumn and spring (horak et al. 2003), while the peaks on helmeted guinea fowls occurred in the summer and winter (horak et al. 1991), and on zebras in the winter (horak et al. 1984). the differences between the seasonal activity of questing a. hebraeum larvae and their seasonal abundance on hosts may be related to a number of factors. the intensity of infestations may differ among individuals of a host species at a given time; thus, the apparent seasonal pattern may depend on the individuals sampled. also, many of the larger ungulates exhibit seasonal changes in distribution. even within restricted home ranges, both kudus and impalas appear to shift habitat use seasonally within the knp (young 1972; owen-smith 1979). these shifts in habitat use may alter the exposure to questing a. hebraeum larvae for mobile species, whereas the intensity of infestation on more sedentary species, such as scrub hares and warthogs, more accurately reflects the activity of questing ticks. according to petney, horak and rechav (1987), a. hebraeum larvae are restricted to well-drained, shaded habitats with grass cover. in the present study, larger numbers of a. hebraeum larvae were collected in the woodland than in the grassland (p < 0.001) at both sites (table 2), and this difference was consistent over years and months (figure 4a and 4b). two factors may account for such distribution. firstly, the large herbivore hosts of adult a. hebraeum are inclined to rest or shelter in the shade of trees during the heat of the day and hence more engorged female ticks may detach there. engorged female ticks may detach in grassland habitats, but here these extremely large, slow-moving creatures could easily be seen by birds and other predators. secondly, the higher relative humidity and lower saturation deficits in the woodland and gullies may have increased larval survival. nymphs were collected mostly in the gullies and least often in the grassland. figure 2: mean monthly collections of the total number of ticks and the three major tick species by year at a) nhlowa road and b) skukuza. figure 3: mean (± 95% confidence interval) of the total number of ticks and of the larvae of the three major tick species by month at a) skukuza and b) nhlowa road. rhipicephalus decoloratus this tick infests a wide range of ungulate hosts in the knp and accounted for the majority of ticks collected from blue wildebeest, burchell’s zebra, impalas and greater kudus (horak et al. 1983; horak et al. 1984; horak et al. 1992; horak et al. 2003). small to medium-sized intermediate feeders or browsers, such as impalas and greater kudus, appear to support much larger burdens than the grazers (zebras, blue wildebeest and buffaloes) or large browsers such as giraffes (horak et al. 1983; horak et al. 1984; horak et al. 1992; horak et al. 2003; horak et al. 2007). more r. decoloratus larvae were collected at skukuza than at nhlowa road (p = 0.004), which may be explained by the predominance of impalas and greater kudus at skukuza. there was a significant year-to-year variation in the numbers of larvae (p < 0.001) as well as a significant year-by-site interaction (p < 0.001). the numbers at skukuza were higher than the numbers at nhlowa road between 1988 and 1990 (figure 2a and 2b). the numbers at skukuza declined rapidly from 1990 to 1992 and remained lower than those at nhlowa road from 1992 to 1994. in contrast, the numbers at nhlowa road were relatively constant from 1988 to 1991, followed by a decline until 1994. the numbers at both sites increased between 1995 and 1998, followed again by a decline. however, the decline at skukuza was much less pronounced than at nhlowa road. the numbers of r. decoloratus larvae were not significantly correlated with annual rainfall. r. decoloratus was seasonal at both sites (p < 0.001), with the largest collections made in the spring and the smallest in autumn and winter (figure 3a and 3b). the seasonal pattern differed between the sites (p = 0.048). at skukuza the numbers remained low from february to july, and then increased sharply to peak during september–november before declining again. at nhlowa road, the changes from the nadir in april to the peak in november appeared to be more gradual. average autumn and winter temperatures at nhlowa road were typically 1 °c – 2 °c higher than at skukuza and the effect of winter temperatures may have been less pronounced than at skukuza. however, the increase in the number of questing larvae over the winter suggests that other factors, such as seasonal shifts in habitat use by host animals may also be important. the seasonal pattern of questing larvae corresponds to the seasonal patterns on ungulate hosts in the knp (horak et al. 1983; horak et al. 1984; horak et al. 1992; horak et al. 2003). although r. decoloratus can complete more than one life cycle annually and is present throughout the year, a number of factors may contribute to the seasonality of this tick. firstly, the development of the non-parasitic stages is temperature dependent, with prolonged pre-oviposition, oviposition and incubation periods at lower temperatures (londt 1974, 1977; robertson 1981; short et al. 1989; spickett & heyne 1990). the threshold for development is approximately 10 °c (short et al. 1989; spickett & heyne 1990). in the eastern cape and gauteng provinces, south africa, the majority of eggs are produced by female ticks towards the end of the summer and later, over-winter (robertson 1981; spickett & heyne 1990). as the temperature rises in spring these eggs hatch more or less synchronously (robertson 1981), giving rise to an increase in questing larvae on the vegetation at this time. the seasonal pattern of r. decoloratus larvae at skukuza is consistent with the above interpretation, although larvae were active throughout the winter, probably because the average temperature of 16 °c – 17 °c for june and july is well above the threshold temperature for development. a second factor that may impact on the seasonal pattern of r. decoloratus is host resistance. at skukuza and in the biyamiti region of the knp, the increase in the intensity of infestation of r. decoloratus on impalas from august (horak et al. 2003) was similar to the pattern observed for questing ticks at skukuza. however, the intensity of infestation on impalas declined more rapidly than the numbers of questing ticks. the initial increase in the intensity of infestation on impalas was caused by an increase in larvae numbers. this resulted in a rapid increase in the larvae: adult ratio in august and september (gallivan & horak, unpublished observations 2003) because the increase in the infestations of nymphs and adults lagged behind the increase in the infestations of larvae. the larvae: adult ratio declined in october, and in december the intensity of the total r. decoloratus infestation declined, possibly as a result of the development of resistance. the increase in the numbers of r. decoloratus on impalas from august occurred towards the end of the winter when diet quality is reduced (monro 1980; van rooyen 1992) and impalas are in poorer condition (gallivan, culverwell & girdwood 1995). resistance to tick infestation is reduced on a low plane of nutrition (o’kelly & seifert 1969; sutherst et al. 1983). thus, impalas were exposed to large numbers of questing r. decoloratus larvae at a time when they had reduced resistance to infestation. the decrease in the larvae: adult ratio in october corresponded to the beginning of the annual rainfall period, which is associated with an improvement in diet quality. sutherst et al. (1983) have shown that a smaller percentage of r. microplus larvae mature on cattle maintained on a higher plane of nutrition and that the percentage that matures varies seasonally, with a peak in the winter when forage quality is low and a rapid decline in the spring when forage quality improves. the decline in the percentage of r. decoloratus larvae maturing on impalas at skukuza from october onwards would reduce the number of females detaching and, subsequently, the number of questing larvae in the summer and early autumn. blue wildebeest and zebras are the predominant species in landscape zone 17, and buffaloes, kudus, and giraffes are also present in large numbers (gertenbach 1983). blue wildebeest and zebras migrate between the northern and southern parts of zone 17 annually, moving north in the summer and south in the winter (gertenbach 1983). blue wildebeest are poor hosts of ixodid ticks, but burchell’s zebras are relatively efficient hosts (horak et al. 1983; horak et al. 1984; gallivan & horak 1997) and may harbour burdens of r. decoloratus similar to the burdens on impalas (horak et al. 1984; horak et al. 2003). the movement of blue wildebeest and zebras into the nhlowa road site during the autumn would increase the number of detaching females and contribute to the increasing numbers of questing larvae during the winter and spring. the northward movement of these host species in the spring would limit the availability of hosts and hence, the numbers of questing larvae. at skukuza the numbers of questing r. decoloratus larvae declined rapidly following the 1991 drought, but subsequently increased to a peak in 1998. the higher summer temperatures and lower relative humidity during the drought of the early 1990s probably reduced larval survival. in addition, there was a marked reduction in the size of the impala population (horak et al. 1995a). the combination of reduced larval survival and low host density resulted in a considerable reduction in intensity of infestation on impalas during the 1991 drought (horak et al. 2003). the number of impalas at skukuza increased again in 1992 (horak et al. 1995a), but the numbers of questing r. decoloratus larvae remained low until 1994. the delay in the recovery of the population of questing r. decoloratus larvae suggests that even though the impala population recovered, the low intensity of infestation probably limited the number of detaching female ticks. consequently the population of questing r. decoloratus larvae did not recover until the intensity of infestation increased on impalas and other hosts. the decline in the numbers of questing r. decoloratus larvae at nhlowa road following the 1991 drought was more gradual than at skukuza and the nadir was not reached until 1994. the diversity of large host species at nhlowa road may have altered the response. while there were marked decreases in the populations of some hosts (e.g. kudus and buffaloes), the populations of others like giraffes and zebras did not decline (ogutu & owen-smith 2005). similar to the situation at skukuza, numbers of r. decoloratus larvae peaked in 1998 at nhlowa road. this increase in numbers was apparent from march to june 1998, declined to a nadir in august and then reached a new peak in october. the numbers declined dramatically after november and very few larvae were collected in 1999. the average daily temperatures in the autumn, winter and the early spring of 1998 were approximately 1 °c above the mean. however, while the development rates and fecundity of r. decoloratus are temperature dependent (londt 1977; short et al. 1989), a 1 °c temperature increase is unlikely to account for a fivefold increase in tick numbers. instead, a combination of increasing host populations and a dry year may have contributed to the marked increase in the numbers of questing r. decoloratus larvae. the recovery of ungulate populations following a population decline may take several years, depending on the fecundity of the species. in the knp, the recovery of the populations of mid-size ungulates, such as impalas and kudus, appears to require about 5 years (ogutu & owen-smith 2005) and increases in populations are associated with an increase in juvenile survival (owen-smith & mason 2005). juvenile impalas harbour higher burdens of r. decoloratus than adults (horak et al. 2003), while juvenile kudus harbour similar burdens to the adults (horak et al. 1992). the increase in ungulate populations following the 1991 drought would have coincided with a dry summer and winter during 1997 and 1998 and again during 1998 and 1999 (figure 1b). in 1998 rainfall at lower sabie was only 64 mm from february to august, whereas the mean from 1993 to 2001 was 290 mm. the dry weather from the latter half of the summer throughout the winter probably created significant nutritional stress, a factor which reduces resistance to infestation and which has a greater effect on juveniles than adult animals (gallivan et al. 1995). thus the increase in the ungulate populations during 1998 and 1999 with a higher proportion of juveniles, which may have higher intensities of infestation than adults and can be more susceptible to nutritional stress, resulted in a host population of which a large proportion had a reduced resistance to infestation. the combination of a large number of susceptible hosts and more rapid development of the free-living stages probably contributed to a rapid increase in the numbers of questing r. decoloratus larvae. interestingly, the numbers of questing larvae declined dramatically in december 1998, following more than twice the average rainfall being recorded than in october and november. more r. decoloratus larvae were collected in the woodland and grassland than in the gullies (p < 0.001). this difference was significant at nhlowa road, but not at skukuza (table 2). the differences among habitats were consistent over years and months (figure 5a and 5b). general: despite its lengthy time frame, this study provides information only on trends in tick populations, firstly because sampling by dragging flannel cloths collects mostly only ticks that quest for hosts from the vegetation. nymphs and adults of a. hebraeum hunt for hosts from the soil surface (bryson et al. 2000), while the larvae quest for hosts from the vegetation. the substantial difference between the number of collected larvae, and nymphs and adults (table 1) is an indication of the differences in their host-finding strategies. because r. decoloratus is a one-host tick, only its larvae seek for hosts, doing so by questing from the vegetation. the numbers of r. decoloratus larvae collected from flannel cloths are thus a better representation of the tick population seeking hosts than is the case with a. hebraeum. secondly, only a small proportion of the total area of each collection locality was sampled. the nine 250 m2 drag samples conducted each month are equivalent to a total of only 2 250 m2 at each site. the skukuza site covered 7 km x 2 km (14 000 000 m2) and the nhlowa road site covered 8 km x 2 km (16 000 000 m2). consequently sampling covered only 0.014% – 0.016% of the area at each site. extrapolating from the drags to the whole site yields changes in the magnitude of millions for the major tick species. from 1989 to 1994 the decline in numbers at skukuza was equivalent to 1200 ticks per month or 14 400 ticks per year. extrapolating to the whole site, this represents a decline of 90 million ticks of which only a miniscule fraction was collected by drag-sampling the vegetation. figure 4: mean (± 95% confidence interval) collections of amblomma hebraeum larvae by a) year and b) month for each habitat at nhlowa road and skukuza. figure 5: mean (± 95% confidence interval) collections of rhipicephalus decoloratus larvae by a) year and b) month for each habitat at nhlowa road and skukuza. conclusion (back to top) the objective of the study was to assess the long-term population dynamics of questing ixodid ticks in two landscape zones of the knp and to this end collections were made monthly from the vegetation over a period of 164 months. this paper dealt with changes in the populations of questing larvae and nymphs of a. hebraeum and larvae of r. decoloratus. changes in the populations of the two ticks were related to rainfall, season, host populations, and their own biology. the study indicated that meaningful results on the seasonality of tick species, of which one or more stages quest for hosts from the vegetation, can be obtained by drag-sampling. it also highlighted the advantages of long-term surveys as opposed to those of shorter duration. this study contributes to the current understanding of the complexity of the population dynamics of these tick species, both of which are of considerable economic importance. acknowledgements (back to top) we are indebted to the south african national parks (sanparks) for placing their staff and facilities in the knp at our disposal. we gratefully acknowledge the assistance of dr leo braack and mr andré potgieter with arranging the logistics for the collections. ms heloise heyne and ms andrea van niekerk assisted with many of the field collections, as did a number of nature conservation and veterinary students. our special thanks, however, are reserved for the sanparks staff at skukuza, who were our armed guards and assisted with the collection of the many thousands of ticks from the flannel strips and leggings. the research was funded by the faculty of veterinary science (university of pretoria) sanparks, bayer animal health, the university of the free state and the national research foundation. references (back to top) acocks, j.p.h., 1988, veld types of south africa with accompanying veld type map. 3rd edn., department of agriculture and water supply, pretoria braack, l.e.o., horak, i.g., jordaan, l.c., segerman, j. & louw, j.p., 1996, ‘the comparative host status of red 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1972, ‘observations on the movement patterns and daily home range size of impala, aepyceros melampus (lichtenstein), in the kruger national park’, zoologica africana 7, 187–195. tuppurainen_153-164.qxd introduction lumpy skin disease (lsd) in cattle is caused by a capripoxvirus. it is characterized by fever, enlarged lymph nodes, firm, circumscribed nodules in the skin and ulcerative lesions particularly of the mucous membrane of the mouth. it usually occurs at regular intervals in endemic areas or it may cause epidemics, which spread fairly rapidly throughout a region or country (davies 1991). field and experimental evidence have proved that lsd is not highly contagious. the morbidity rates in 153 onderstepoort journal of veterinary research, 72:153–164 (2005) the detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques e.s.m. tuppurainen, e.h. venter* and j.a.w. coetzer department of veterinary tropical diseases, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract tuppurainen, e.s.m., venter, e.h. & coetzer, j.a.w. 2005. the detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques. onderstepoort journal of veterinary research, 72:153–164 lumpy skin disease (lsd) is a disease of cattle, primarily in africa and madagascar and rarely in the middle east. it is caused by a capripoxvirus that belongs to the family poxviridae. the disease is of economic importance in endemic areas. effective control of lsd requires accurate and rapid laboratory techniques to confirm a tentative clinical diagnosis. comparative studies on different diagnostic tests used at different stages of the disease have not been done. the aim of this study was to compare several of these tests. six seronegative bulls, between 11 and 20 months of age, were infected intravenously and kept in an insect-free facility. the course of the infection was monitored. during a 3-month period blood samples and skin biopsies were collected for virus isolation and polymerase chain reaction (pcr). skin biopsies were also examined using transmission electron microscopy (tem). the incubation period in infected animals varied from 4–5 days. the length of the viraemic period did not correlate with the severity of clinical disease. viraemia was detected from 1–12 days using virus isolation and from 4–11 days using the pcr, which is longer than has previously been reported. virus was isolated from skin biopsies until day 39 post infection (p.i.) and pcr could demonstrate viral dna until day 92 p.i. transmission electron microscopy of negatively stained skin biopsies detected lsd virus only in one of the four bulls that developed skin lesions until day 33 p.i. the pcr was a fast and sensitive method to demonstrate viral dna in blood and skin samples. it could detect viral nucleic acid in skin lesions 53 days longer than virus isolation. virus isolation from blood and skin samples was sensitive and reliable, but as a single test it may be too time-consuming to use although this depends on how rapidly the diagnosis must be confirmed. in conclusion, this study showed the pcr to be superior in detecting lsd virus from blood and skin samples. however, virus isolation is still required when the infectivity of the lsd virus is to be determined. keywords: lumpy skin disease, polymerase chain reaction, virus isolation * author to whom correspondence is to be directed. e-mail: estelle.venter@up.ac.za accepted for publication 6 april 2005—editor natural outbreaks vary from 3–85 %. in experimentally produced infections only 40–50 % of inoculated animals have shown clinical signs. the mortality rate is usually low and less than 10 % (thomas & maré 1945). a presumptive diagnosis of the disease can be made based on clinical signs. however, mild and inapparent disease may be difficult to diagnose and rapid laboratory methods are needed to confirm the diagnosis. laboratory diagnosis of lsd can be performed either by identification of the agent using transmission electron microscopy (tem), by its isolation in cell cultures, by a direct fluorescent antibody test (fat) or by detection of antibody using conventional serological tests such as the serum virus neutralization test (snt), indirect fluorescent antibody test (ifat) and agar-gel immunodiffusion test (agid). routine histopathology and immunohistological staining provide a relatively inexpensive tool to diagnose the disease. indirect and antigen trapping elisas as well as the polymerase chain reaction (pcr) have been developed more recently for detecting lsd antibodies and antigen (carn, kitching, hammond & chand 1994; carn 1995; ireland & binepal 1998; heine, stevens, foord & boyle 1999). the disease is of economic importance because of decreased milk yield and mass loss, infertility and permanent damage to hides. it is endemic in most parts of sub-saharan africa and madagascar and rarely causes outbreaks in the middle east (oie’s computer data page, handistatus ii, multiannual animal disease status). vaccination is the only effective method to control the disease in endemic countries. in previously lsd-free countries, in the event of an outbreak, the rapid confirmation of a clinical diagnosis is essential so that eradication measures, such as quarantine, slaughter-out of affected and in-contact animals, and ring vaccinations can be implemented as soon as possible (carn 1993). the aim of this study was to monitor the clinical signs of bulls experimentally infected with lsd virus and to determine virus persistence in blood and skin lesions using virus isolation, pcr and tem. materials and methods experimental animals six bulls, between 11 and 20 months of age, from a herd where vaccination against lsd was not practised were used as experimental animals. they were thin-skinned, european breeds: two holsteinfriesians, two dexters and two dexter crosses (table 1). before they were purchased and again before the onset of the experiment the animals were tested to be seronegative using the snt. during the experiment the animals were kept in isolation in the insect proof facility of the university of pretoria’s biomedical research centre. before the onset of the experiment the bulls had a period of 2 weeks to acclimatize to the new environment. the experimental animals were numbered with ear tags (1–4 and 7–8). preparation of virus suspension and animal inoculation a virulent, south african field isolate strain v248/93 of lsd virus that had been passaged three times in cell cultures was used to infect the animals. bovine dermis cells prepared from a foetal calf’s ears were cultured in 75 cm² tissue culture flasks in a growth medium comprising minimum essential medium (mem) with l-glutamine (highveld biological), 0.2 % sodium bicarbonate (highveld biological), 5 % foetal calf serum (adcock ingram) and gentamycin 0,05 mg/ml (genta 50 phenix, 50 mg/ml). two hours after preparation of the cells 0.5 ml of the virus suspension was inoculated into the 75 cm² flasks containing bovine dermis cells in the growth medium. virus was harvested when 90–100 % of the cells in the monolayer were infected and showed the typical cytopathic effect (cpe) caused by lsd virus. culture flasks were briefly frozen at –70 °c and then thawed. the cell suspension was centrifuged for 3 min at 2 000 rpm, the supernatant removed and aliquoted in 1.8 ml or 3.5 ml volumes in cryotubes and stored as reference stock virus at –70 °c until used. the reference stock virus was titrated on bovine dermis cells growing in the wells of 96-well microtitre plates containing the growth medium, and the tissue culture infected dose 50 (tcid50) of the virus was calculated using the method of reed & muench (1938). the bulls were inoculated intravenously (iv) with 2 ml of the virus suspension at a titre of 5 log tcid50. clinical observations, sample collection and processing after infection the bulls were clinically examined daily and their rectal temperatures were measured twice a day for 30 days p.i. blood and affected skin 154 lumpy skin disease virus in experimentally infected cattle biopsy samples were collected at various intervals. after inoculation blood samples in edta and in heparin were collected daily for 30 days p.i. and then at 3-day intervals up to day 40. after collection of the blood samples they were aliquoted in 1.8 ml volumes in cryotubes. the heparinized blood was stored at –70 °c and the edta blood at –20 °c. biopsies of the nodular skin lesions comprising epidermis, dermis and subcutis were collected for virus isolation, pcr and tem. samples were collected at weekly intervals for more than 3 months. the skin area was anaesthetized locally using 2 % lignocaine® (lignocaine hcl, bayer ah) before the biopsies were taken. those animals showing signs of discomfort during the procedure were tranquillized by administration of domosedan® (detomidine, novartis/orion pharma) sedative. standard sterile techniques were used to take the samples and the resultant skin wounds were sutured with 2-0 ethicon vicryl (johnson & johnson intl). samples were divided into three pieces, which were placed in 1.8 ml cryotubes. these were stored either at –20 °c for pcr or at –70 °c for virus isolation. on the day of collection of the skin nodules, samples were submitted to the electron microscopy unit, department of anatomy and physiology, faculty of veterinary science, university of pretoria. the presence of virus was determined in the samples using the pcr, virus isolation and tem using several standard operating procedures (sops) of the department of veterinary tropical diseases and electron microscopy unit or using other published procedures as described below. diagnostic methods virus isolation heparanized blood samples bovine dermis cells at ± 50 % confluency growing in tubes containing the growth medium described above were infected with 0.5 ml heparinized blood. after 24 h the medium was removed and the cells were washed twice with phosphate buffered saline containing mg2+ and ca2+ (pbs+) and gentamycin (0.05 mg/ml). the medium was replaced with mem containing 5 % foetal calf serum, l-glutamine, 0.2 % sodium bicarbonate and gentamycin (0.05 mg/ml). the cell cultures were observed daily for cpe. after 14 days negative cultures were frozen briefly at –70 °c and thawed. a second passage was done and observed for 14 days. isolates were stored at –70 °c. skin biopsies for virus isolation, biopsy tissue was minced using sterile scissors and forceps and then using sterile techniques ground with a pestle in a mortar containing sterile sand. ten millilitres pbs+ containing gentamycin (0.1 mg/ml), ampicillin (0.05 mg/ml) (intramed ampicillin inj. 500 mg) and amphotericin b (5 µg/ml) (inj. fungizone, bristol-myers squibb) were added. the suspension was left to stand overnight at 4 °c. the samples were partially clarified by centrifugation at 2 000 rpm for 3–5 min to remove gross particles and 0.5 ml of supernatant was used to inoculate monolayers of bovine dermis cells growing in the medium described above. cultures were observed daily for 14 days for cpe. the growth medium was replaced if it appeared cloudy due to bacterial or fungal contamination. after 14 days negative cell cultures were blind-passaged onto a fresh cell culture monolayer and observed for another 14 days or until typical cpe changes occurred. a negative control for virus isolation consisted of a cell monolayer without any virus and a positive control consisted of a cell monolayer inoculated with 0.2–0.5 ml lsd virus (v 248/93) suspension at a titre of 4 log tcid50. polymerase chain reaction the extraction method used was a modification of the method described by schwartz, varde, nadelman, wormsel & fish (1997) and gubbels, de vos, van der weide, viseras, schouls, de vries & jongejan (1999). a volume of 200 µl blood in edta frozen at –70 °c was thawed. the samples were suspended in 100 µl lysis buffer containing 0.378 g kcl, 1 ml tris (1.0 m, ph 8), 0.5 ml tween 20 and 60 % guanidine thiocyanate (roche diagnostics) in 100 ml. protein bands were digested by adding 1 µl of proteinase k (10 mg/ml) (invitrogen, laboratory specialist services ltd) to each sample. the portions of the skin biopsies to be used in this test were first cut into fine pieces using sterile scissors and forceps and ground with a pestle in a mortar. the samples were suspended in 1 ml lysis buffer containing 60 % guanidine. a volume of 10 µl proteinase k (10 mg/ml) was added. all the samples were incubated at 56 °c overnight and then heated for 10 min at 100 °c to denature the enzyme. a phenol:chloroform:isoamylalcohol (25:24:1, v/v, invitrogen) solution was added to each sample at a 1:1 volume and the samples were mixed while incubating at room temperature for 10 min. after cen155 e.s.m. tuppurainen, e.h. venter & j.a.w. coetzer trifugation (13 000 rpm for 15 min) the upper, aqueous phase was collected and dna was precipitated using ice cold, 100 % ethanol. after centrifugation (13 000 rpm for 15 min) pellets were washed with 70–75 % ethanol and centrifuged again at 13 000 rpm for 1 min. pellets were dried thoroughly and resuspended in 30 µl of distilled water. the pcr primers were developed from the viral attachment protein encoding gene and have the following sequences (ireland & binepal 1998): forward primer 5’-d tttcctgatttttcttactat3’ reverse primer 5’-d aaattatatacgtaaataac 3’ the size of the amplicon was 192 bp (ireland & binepal 1998). a platinium® quantitative pcr supermix-udg (2x) reaction mixture (invitrogen, life technologies) was used for amplification of nucleic acid templates. dna amplification was carried out in a final volume of 25 µl containing 12.5 µl platinium® quantitative pcr supermix-udg, 1 µl 0.20 mm each primer, 9.5 µl distilled water and 1 µl dna sample. the reaction was carried out in a perklin-elmer gen amp 9600 pcr system. to remove any urasil residues from the pcr mixture the pcr started with one cycle of 42 °c for 2 min and 94 °c for 10 min. the initial cycle was 94 °c for 1 min, 50 °c for 30 s and 72 °c for 1 min. this was followed by 40 cycles of 94 °c for 1 min, 50 °c for 30 s, and 72 °c for 1 min, and a final elongation step of 72 °c for 1 min to complete the extension of the primers (ireland & binepal 1998). a water control and previously tested positive and negative control samples were always included when blood and skin specimens were tested using the pcr. amplified products were analysed using a 100 bp dna ladder (whitehead scientific ltd) as a molecular marker on 1.5 % agarose gels. gels were stained using ethidium bromide (1 µg/ml) in tris edta (te) and amplicons were visualized using an uv transilluminator at a wave length of 590 nm. positive reactions were confirmed according to size. transmission electron microscopy the preparation and negative staining of skin specimens for tem were made according to the sops of the electron microscopy unit. samples were ground in a mortar with a pestle and a small volume of distilled water was added. aqueous material was centrifuged for 15 min at 5 000 rpm. the supernatant was collected and centrifuged again for 45 min at 13 000 rpm. each pellet was rinsed carefully with distilled water and diluted with one droplet of water. pellets were stained with 3 % phosphotungstic acid (pta). grids were examined using a philips cm 10 transmission electron microscope in the electron microscopy unit. results clinical signs before the onset of the experiment all the bulls were healthy, eating well and had apparently adapted to their new environment. bulls 1 and 2 were the smallest but were in good condition. bulls 3 and 4 were in good physical condition with shiny hair coats. bulls 7 and 8 were very thin. the animals were divided into three groups according to the severity of the clinical signs after inoculation as shown in table 1. two of them developed severe, generalized disease (group 1), two manifested mild disease with fever and a few skin lesions on the neck and back (group 2), and two had only transient fever but no other signs (group 3). in this study the day of inoculation was counted as day 1 p.i. an increase in rectal temperature, which lasted for 3–14 days, was recorded in all animals from days 5 and 6 p.i. (fig. 1). group 1: in bulls 1 and 2 the fever persisted for 10–14 days; in bull 1, at the height of the reaction, it exceeded 41 °c (41.5 °c) for 3 days (fig. 1). after the main fever reaction bull 1 had a rise in temper156 lumpy skin disease virus in experimentally infected cattle table 1 grouping of animals according to the severity of clinical signs group no. clinical signs animal no. age (months) mass (kg) breed 1 severe 1 11 232 dexter 2 15 250 dexter 2 mild 7 17 236 friesian 8 20 310 friesian 3 inapparent 3 16 295 dexter cross 4 14 321 dexter cross ature (39.2 °c) for 1 day on day 22 p.i. bull 2 also had a rise in temperature (39.4 °c) on day 24 p.i. both animals developed ulcerative lesions on the muzzle and nostrils and numerous skin nodules over the entire body. in addition, they showed salivation, lachrymation, a mucopurulent nasal discharge and a slight enlargement of their superficial lymph nodes. bull 1 had subcutaneous oedema between the tarsal joint and fetlock of the right hind leg. because of respiratory distress, high fever and inappetence bull 1 was treated with 12 ml of sulfatrimetoprim (sulfatrim 240®, phenix, virbac) per day administrated either iv or im for 10 days and 4 ml of phenylbutazone (phenylarthrite®, 200 mg/ml, centaur, bayer) every second day, iv. these treatments were started on day 10 p.i. group 2: bulls 7 and 8 had elevated rectal temperatures for 5–7 days which did not exceed 40.5 °c. a few skin lesions developed mainly on the neck, back and perineal area of both animals but they showed no other clinical signs. both animals developed a ringworm infection 30–34 days p.i. group 3: in bulls 3 and 4, the fever lasted 3–6 days and only on 1 day did it exceed 40 °c in bull 4. a slight rise (39.3 °c) in rectal temperature was recorded for bull 3 on day 18 p.i. no other clinical signs were observed in either of the animals. virus isolation heparinized blood samples group 1: virus was isolated from bull 1 for 11 days from days 6–16 p.i. fever commenced on day 5 p.i. and virus was isolated from the blood sample collected on the following day (day 6 p.i.). the rectal temperature of bull 2 started to rise on day 6 p.i. and virus was isolated from its blood for 11 days between days 6 and 16 p.i. group 2: virus was isolated from bull 7 only once (on day 16 p.i.), 10 days after the fever had commenced (on day 6 p.i.). from bull 8 virus was isolated four times between days 5 and 16 p.i. (on days 5, 8, 10 and 16 p.i.), which was 1 day before the rectal temperature started to rise (on day 6 p.i.). group 3: from bull 3 virus was isolated three times between days 11 and 16 (on days 11, 12 and 16 p.i.). this was 5 days after the onset of fever (on day 6 p.i.). from bull 4 virus was isolated seven times between day 5 (i.e. on the same day that the fever commenced) and day 16 p.i. (on days 5, 6, 9, 10, 12, 14 and 16 p.i.). skin biopsies group 1: virus was isolated from bull 1 on day 10 157 e.s.m. tuppurainen, e.h. venter & j.a.w. coetzer �� �� �� �� �� �� � � � � � � � � � � � � � � � � � � � � � � � � ��������� �� �� �� �� � �� � �� �� �� �� �� �� �� �� fig. 1 temperature reaction in the bulls 158 lumpy skin disease virus in experimentally infected cattle ��������� �� �� �� �� � �� � �� �� �� �� �� �� �� �� � � � � � � � � � � � � � � � � � � � � � � fig. 2 virus isolations on bovine dermis cell cultures from blood samples collected in heparin � � � � � � � � � � � � � � � � � � � � � � � �� ��������� �� �� �� �� � �� � �� �� �� �� �� �� �� �� fig. 3 pcr results from edta blood samples p.i. when the first skin sample was collected and for the last time on day 39 p.i. from bull 2 virus was isolated once, 26 days after the first skin lesions appeared on day 33 p.i. group 2: no virus was isolated from skin biopsies collected from bull 7. from a skin lesion of bull 8 virus was isolated on day 10 p.i. group 3: the bulls of this group did not develop any skin lesions. polymerase chain reaction edta blood samples group 1: both bulls 1 and 2 gave positive pcr results on days 2 and 3 p.i. bull 1 was positive seven times between days 8 and 14 p.i. (on days 8–14). on day 24 p.i. it became positive again but only for 1 day. bull 2 gave positive pcr results seven times between days 5 and 15 p.i. (on days 5, 7, 9, 10, 12, 13 and 15). after the main viraemic period bull 2 showed positive pcr results again on days 19, 25, 26 and 28 p.i. group 2: bull 7 gave one positive pcr result on day 3 p.i. it had pcr positive samples on days 5, 11 and 13 p.i. after which it remained negative. bull 8 registered positive results on days 9, 11 and 12 p.i. and one positive result on day 26 p.i. group 3: bull 3 gave its first positive pcr result on day 4 p.i. between days 10 and 13 p.i. it was pcr positive three times on days 10, 12 and 13 p.i. after 8 negative days it became positive again on days 22, 25 and 27 p.i. bull 4 gave its first positive pcr result on day 3 p.i. between days 9 and 15 p.i. it gave four positive results on days 9, 12, 14 and 15 p.i. subsequently on days 19, 25 and 26 p.i. it was pcr positive again. skin biopsies group 1: the first skin lesions appeared in bull 1 on day 6 p.i. and in bull 2 on day 7 p.i. the pcr failed to detect viral dna from the skin biopsy of bull 1 collected on day 10 p.i. even though lsd virus was isolated in cell culture and it was demonstrated using tem. from bull 1, 17 samples were collected, mainly skin biopsies, but also several scabs. the first pcr-positive skin nodule was collected on day 18 p.i. and the skin biopsies of this bull remained positive until day 92 p.i. however, biopsies collected from this animal on days 25, 47, 75 and 81 p.i. were all negative. the first two skin samples collected on days 11 and 18 p.i. from bull 2 were negative, but positive pcr results were obtained in bioposies taken on day 25 p.i. and, apart from the one collected on day 81 p.i., it remained positive until day 85 p.i. group 2: bulls 7 and 8 developed very few skin lesions. these occurred mainly in the area of the neck and back but a small number were present in the perineal area. the first lesions in bull 7 developed on day 10 p.i. and skin biopsies from it were pcr-positive on days 12 and 25 p.i. the skin biopsy collected on day 18 p.i. was pcr negative. no skin lesions were observed in this animal after day 25 p.i. skin lesions appeared in bull 8 on day 8 p.i. the pcr demonstrated viral dna from skin biopsies only 2 days after the appearance of the first skin lesion on day 10 p.i. this animal remained positive until day 18 p.i. the last skin biopsy collected on day 25 p.i. was negative. group 3: these bulls did not develop skin lesions. transmission electron microscopy the results of the tem examinations are given in table 2. group 1: lumpy skin disease virus was detected in the first skin biopsy collected on day 10 p.i. from bull 1, which was 4 days after the first appearance of skin lesions. in biopsies collected on days 18 and 25 p.i. the presence of lsd virus was suspected but not confirmed. virus was again detected from a specimen collected on day 33 p.i. no virus was detected in ten biopsy specimens collected from bull 1 between days 39 and 106 p.i. or from the skin specimens of bull 2 collected between days 11 and 106 p.i. virus was detected in a skin nodule of bull 2 collected on day 33 p.i. group 2: bull 7 did not have any positive samples. in bull 8 virus was detected on day 25 p.i. discussion the effective control or eradication of lsd in endemic and non-endemic areas requires rapid and accurate diagnostic methods to confirm a presumptive diagnosis. recent research has focused mainly on transmission of the disease (kitching & taylor 1985; kitching & mellor 1986; carn & kitching 1995a; chihota, rennie, kitching & mellor 2001) and development of recombinant vaccines (ngichabe, wamwayi, barrett, ndungu, black & bostock 1997; aspden, van dijk, bingham, cox, passmore 159 e.s.m. tuppurainen, e.h. venter & j.a.w. coetzer & williamson 2002; ngichabe, wamwayi, ndungu, mirangi, bostock, black & barret 2002). no data on comparative studies of different diagnostic methods of lsd have been published. the use of the pcr for the detection of lsd virus nucleic acid from skin biopsies and tissue culture supernatant has been published (ireland & binepal 1998; markoulatos, mangana-vougiouka, koptopoulos, nomikou & papadopoulos 2000) but no studies have been undertaken on the persistence of the virus in blood and skin using the pcr. in the study described here, a virulent south african field isolate of lsd virus was used to infect the six bulls. the intravenous route of infection was selected because previous data indicate this route to be the most effective way to produce severe generalized disease in experimentally infected cattle (carn & kitching 1995a). the incubation period of 4–5 days recorded in the bulls in this study is in agreement with that reported in earlier publications, in which the incubation period after experimental infection was 4–14 days (haig 1957; capstick 1959; prozesky & barnard 1982; carn & kitching 1995b). all the bulls became infected and developed fever and viraemia. however, the severity of clinical manifestations varied in the bulls (table 1). this correlates well with earlier observations that less than half of the cattle infected experimentally with lsd virus or naturally exposed during an outbreak will develop generalized disease (weiss 1968; prozesky & barnard 1982; carn & kitching 1995b). in our study two bulls developed severe disease, two bulls showed mild disease and two bulls showed no clinical disease apart from fever (table 1). the fever reaction correlated well with the development of clinical signs. fever persisted for the longest period (12 days on average) in animals of group 1 that showed the most severe disease (table 3). the mean duration of fever in mildly affected animals (group 2) was 6 days. the two animals that did not develop clinical signs (group 3) had the shortest febrile reaction (4.5 days on average). no clear 160 lumpy skin disease virus in experimentally infected cattle table 2 transmission electron microscopy results of the skin biopsies of the bulls day p.i. bull 1 bull 2 bull 7 bull 8 10 pos nd nd neg 11 nd neg nd nd 12 nd nd neg nd 18 suspect neg neg neg 25 suspect neg neg suspect 33 pos suspect nd nd 39 neg neg nd nd 47 neg neg nd nd 54 neg nd nd nd 60 neg nd nd nd 67 neg nd nd nd 75 neg nd nd nd 81 neg nd nd nd 92 neg nd nd nd 99 neg neg nd nd 106 neg neg nd nd 113 neg nd nd nd nd = not done table 3 temperature reaction relative to the development of skin lesions group no. animal no. onset of fever duration of average duration of appearance of skin (day p.i.) fever (days) fever (days) lesions (day p.i.) 1 1 5 14 12 6 2 6 10 7 2 7 6 7 6 10 8 6 5 8 3 3 6 3 4.5 no skin lesions 4 5 6 no skin lesions biphasic fever response as has been reported in previous publications was detected (barnard, munz, dumbell & prozesky 1994). bulls 1, 2 and 8 developed skin lesions 1–2 days after the onset of fever. bull 7 developed skin lesions 4 days after the rectal temperature started to rise. this finding correlates well with the observation that skin lesions appear within 48 h of the first rise in temperature (weiss 1968) or within 4–7 days of subcutaneous inoculation of the virus (prozesky & barnard 1982). in this experiment bovine dermis cells (bdc) were chosen to be used in virus isolation because they are susceptible to lsd virus infection and show typical cpe. although bdc are primary cells, they live long enough to ensure the isolation of the virus. in the present study the appearance of cpe took sometimes only 4 days to develop but it usually became apparent between the 7th to the 10th day. on one occasion it took as long as 12 days to develop. negative cell cultures were blind-passaged once and sometimes twice to ensure that the results were negative. the severity of the clinical signs did not correlate with the length of viraemia in the infected bulls. virus was isolated for the longest period of time (12 days) from the blood of bull 4, which developed no clinical signs, and bull 8, which was only mildly affected. however, the viraemic period in these two bulls was only one day longer than that of bulls 1 and 2 that both showed severe disease (11 days). the mean length of viraemia detected by virus isolation in the animals of groups 2 and 3 was 6.5 and 9 days, respectively (table 4). the viraemia in the bulls that developed no clinical sings persisted for a longer period on average than that in the bulls that showed mild disease. no virus could be isolated from the blood samples of the bulls after day 16 p.i., which is similar to the observations of carn & kitching (1995b). the bulls remained viraemic for a longer period than has previously been determined. weiss (1968) demonstrated virus in the blood of experimentally infected cattle for a period of 4 days following the appearance of fever and generalized skin lesions. in a more recent study lsd virus was isolated in cultures of lamb testis cells from blood samples of experimentally infected cattle for a period of 9 days, from the 3rd to the 11th day p.i. (carn & kitching 1995b). in the present study lsd virus was isolated from the blood of all the bulls for 1–12 days between days 5 and 16 p.i. when seven cattle were infected with lsd virus using the intradermal and subcutaneous routes at multiple sites on the side of the neck, viraemia was detected in only two of the animals using virus isolation techniques (prozesky & barnard 1982). the total volume of the virus suspension used in their study to infect each animal was 2 ml and the titres varied between 4.5 and 6.0 log tcid50. in an experiment to determine the effect of the infecting dose and the route of infection, carn & kitching (1995b) used different titres of a virus suspension (2–6.5 log tcid50) to infect cattle by placing lsd virus on the conjunctiva or using the intranasal, intradermal and intravenous routes. eleven out of 25 animals infected intradermally developed only a local reaction at the inoculation site and a viraemia was detected in two of them. only five of all the 40 infected animals showed both severe generalized disease and a viraemia, which persisted for up to 9 days. of these five animals, three were infected via the intravenous route, one intranasally and one via the intradermal route. in the present study virus was isolated from blood samples of four of the bulls collected 1 day before (bull 8), the same day (bulls 2 and 4) or 1 day after (bull 1) the onset of fever. however, from bull 7, that showed mild disease, virus was isolated only 10 days after the onset of fever and from bull 3 that did not develop clinical signs apart from the fever, virus was isolated 5 days after its onset. depending on how rapidly the confirmation of the presumptive diagnosis is required, the virus isolation technique may be too time-consuming because members of the genus capripoxvirus grow very slowly in cell cultures and the development of cpe may take up to 14 days on primary isolation (alexander, plowright & haig 1957). several passages may also be required before the virus becomes adapted and will grow on cells. the results of this study indicate that in animals showing mild or inapparent disease it took 5–10 days after the onset of the febrile reaction before lsd virus could be isolated from blood samples (table 4), which will delay the confirmation of the diagnosis for even longer. a tentative diagnosis of lsd is usually based on clinical signs. the disease in animals that develop only a few skin lesions and/or transient fever may therefore be difficult to diagnose and may be confused with pseudo-lumpy skin disease (barnard et al. 1994). the present study indicates that virus isolation on bdc cultures is a useful method but it may be too time-consuming to be used as a first diag161 e.s.m. tuppurainen, e.h. venter & j.a.w. coetzer nostic method. it is nevertheless a valuable tool to detect the viability of the virus but should be used in combination with other tests. there are no published reports on the persistence of lsd virus in the blood of experimentally infected cattle using the pcr. in this study five of the bulls (1–4 and 7) were found to be pcr-positive on days 2–4 p.i. (fig. 3), the virus in the blood probably being the virus that was inoculated. the blood samples of all the animals were negative on day 6 p.i. the blood samples of all the experimental animals became pcr positive within 4 days after the onset of fever (mean 2 days) and skin lesions appeared approximately 1–4 days (mean 2 days) after the onset of the fever reaction (table 4). the results of this experiment indicate that the pcr was able to detect the nucleic acid of the lsd virus from the blood samples of the experimental bulls on the same day that the skin nodules appeared. the blood of bull 2 remained pcr positive for the longest period (11 days). the mean duration of the viraemic period in all the bulls determined using the pcr was 7 days and it was 8.8 days using virus isolation. the pcr could detect virus in the blood of animals that showed severe, mild or inapparent disease. the blood samples of all the animals were pcr negative after day 15 p.i., which correlates well with the virus isolation results (in which virus was isolated up to day 16 p.i.) (table 4). the blood samples of all the bulls except bull 7 were pcr positive for 1–4 days between days 19 and 28 p.i. as discussed above bulls 1–3 showed slightly elevated rectal temperatures between days 18 and 24. no virus could be isolated during this period from the blood samples of any of the bulls. during this time bulls 1 and 2 developed severe skin lesions over their entire bodies. bulls 7 and 8 showed very few lesions, but none were detected in bulls 3 and 4. virus was isolated from skin biopsies of bull 1 between days 10 and 39 p.i. and from those of bull 2 only on day 33 p.i. this finding indicates that the skin lesions contained viable virus between days 19 and 28 p.i. this study has revealed the pcr to be a fast and sensitive diagnostic technique to detect lsd virus nucleic acid in blood samples. the reason why lsd virus could not be isolated from the skin biopsies collected from bull 7, and why it was isolated only once from those collected from bulls 2 and 8, is not clear. the concentration of the virus in the skin lesions may have been too low, the size of the sample may have been too small or a failure in the techniques used in processing the samples may have occurred. 162 lumpy skin disease virus in experimentally infected cattle table 4 the temperature reaction and the appearance of skin lesions relative to virus isolation and pcr results of blood samples. the numbers in brackets indicate the number of positive test results during the time period group no. animal no. onset of appearance of positive virus viraemic positive viraemic fever skin lesions isolation period pcr period (day p.i.) (day p.i.) (days p.i.) (days) (days p.i.) (days) 1 1 5 6 6–16 (11x) 11 8–14 (7x) 7 2 6 7 6–16 (11x) 11 5–15 (7x) 11 2 7 6 10 16 (1x) 1 5–13 (3x) 9 8 6 8 5–16 (4x) 12 9–12 (3x) 4 3 3 6 no skin lesions 11–16 (3x) 6 10–13 (3x) 4 4 5 no skin lesions 5–16 (7x) 12 9–15 (4x) 7 table 5 detection of lsd virus / nucleic acid in skin lesions using tem, vi and pcr the numbers in brackets indicate the number of positive test results during the time period group no. animal no. appearance of positive tem results positive virus positive pcr skin lesions isolation (day p.i.) (days p.i.) (days p.i.) (days. p.i.) 1 1 6 10-33 (2x) 10–39 (4x) 18–92 (11x) 2 7 suspected on day 33 p.i. 33 (1x) 25–85 (8x) 2 7 10 none none 12–25 (2x) 8 8 suspected on day 25 p.i. 10 (1x) 10–18 (2x) 3 3 no skin lesions 4 no skin lesions virus was isolated from the skin lesions of bull 1 on days 10, 18, 33 and 39 p.i. in this animal lsd virus remained viable for 33 days after the appearance of skin nodules. from bull 2 virus was isolated once, on day 33 p.i., which was 26 days after the appearance of the skin lesions. these results are in agreement with the earlier observation of weiss (1968) that viable lsd virus particles remain for 33 days in the skin nodules after their first appearance. transmission electron microscopy demonstrated lsd virus in skin biopsies of bull 1 between days 10 and 33 p.i. this correlates well with the isolation of virus in cell cultures (days 10–39 p.i.). this finding also corresponds with the observations of davies, krauss, lund & taylor (1971) that virus could be detected by tem in skin specimens collected between days 9–21 p.i. why the virus was detected in the skin biopsies of only one experimental animal is unclear. incorrect procedures used in preparing the samples could be a reason or possibly more than one grid should have been prepared from each specimen as described by davies et al. (1971). the size of the sample may also have been too small, which resulted in the virus concentration in the specimen being too low. in bulls 7 and 8 the pcr was able to detect lsd virus nucleic acid from the skin biopsies already 2 days after the appearance of skin nodules. however, it was not able to demonstrate lsd virus dna from the first skin biopsy collected from bull 1 on day 10 p.i. even though the virus was isolated in cell culture and detected by tem. the negative result may indicate a failure in the dna extraction procedure or the presence of dna amplificator inhibitors. in experiments done with sheep pox skin lesions, a dilution of skin biopsy samples at 1:10 was needed to remove dna amplificatory inhibitors (markoulatos et al. 2000). the pcr could demonstrate viral dna in the skin biopsies of bull 1 (until day 92 p.i.) and in bull 2 (until day 85 p.i.) for up to 3 months. the skin biopsies of bull 7 remained positive until the last skin nodule was collected on day 25 p.i. and bull 8 until day 18 p.i. no viral dna was detected from biopsies collected from healthy-looking skin after all the skin lesions had disappeared. since the pcr only detects nucleic acid, this result does not necessarily imply that lsd virus remains infective for such a long time in skin lesions. these observations indicate that the pcr is a more sensitive test to detect lsd virus or viral nucleic acid in skin biopsies than virus isolation or tem examination. the results of tem, vi and pcr are compared in table 5. in previous studies the pcr has been shown to have a greater sensitivity to detect capripoxviruses from skin biopsies than antigen trapping elisa (ireland & binepal 1998). in conclusion, this study revealed that the pcr is a useful method for a rapid confirmation of the diagnosis of lsd. virus isolation is still needed to detect the infectivity of the virus. the pcr could demonstrate lsd virus nucleic acid in blood and skin samples. using both pcr and virus isolation techniques the persistence of the virus in blood and skin of experimentally infected animals was found to be for longer than had previously been shown. according to this study even though tem is usually considered to be a fast and reliable method, a negative result should be confirmed by using another method. acknowledgements we thank prof. p.g. howell for his valuable advice and constant support during this project, prof. r.c. tustin for his constructive editing of the article, ms a-m. bosman for assistance in molecular biology, ms d. nel and ms a. durand for assistance in virology and serology, and the personnel of the department of anatomy and physiology, electron microscopy unit for assistance in transmission electron microscopy examination of skin lesions. the financial support received from the following institutes is greatly appreciated: research and science foundation of farmos, finnish veterinary foundation, department of veterinary tropical diseases, faculty of veterinary 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bello university, nigeria correspondence to: ndudim ogo postal address: parasitology division, national veterinary research institute, pmb 01, vom, nigeria dates: received: 03 june 2011 accepted: 26 sept. 2011 published: 07 feb. 2012 how to cite this article: ogo, n.i., onovoh, e., okubanjo, o.o., galindo, r.c., de la lastra, j.p. & de la fuente, j., 2012, ‘molecular identification of cordylobia anthropophaga blanchard (diptera: calliphoridae) larvae collected from dogs (canis familiaris) in jos south, plateau state, nigeria’, onderstepoort journal of veterinary research 79(1), art. #349, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.349 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. molecular identification of cordylobia anthropophaga blanchard (diptera: calliphoridae) larvae collected from dogs (canis familiaris) in jos south, plateau state, nigeria in this original research... open access • abstract • introduction • materials and methods    • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ myiasis-causing larvae were extracted from dogs attending veterinary clinics in plateau state, nigeria and subjected to molecular analysis involving polymerase chain reaction amplification of the 28s rrna gene of blowflies, cloning and sequencing techniques. all larvae were confirmed as cordylobia anthropophaga blanchard (diptera: calliphoridae) after the initial morphological identification. this is the first molecular identification of any myiasis-causing fly species in nigeria and may serve as a reliable alternative to morphological identification where samples are not well preserved or difficult to identify to species level. introduction top ↑ the infestation of vertebrate animals with larvae of dipteran flies is refered to as myiasis. skin injuries on animals or the presence of excretory products (urine and/or faeces) in dry sand or clothing serve as an attractant to the flies. the flies subsequently lay their eggs in wounds or sleeping places of the animals, especially on straws, sand and sometimes on urine-smelling clothing (ockenhouse et al. 1990). hatching of eggs and the eventual penetration of larvae into the skin occurs through contact with the larvae from the environment or clothing; subsequently, the larva feeds and thrives on dead or living tissues and body fluid. growth and development of the larvae from the first to the third instar larvae and the eventual pupae occur within a set time (depending on the fly species involved in the myiasis), but is determined by temperature rather than humidity (stevens & wall 2001). non-migratory cutaneous furunculoid myiasis of animals and humans in the tropics (africa) and subtropics (south america) are usually associated with cordylobia species (cordylobia anthropophaga, cordylobia ruandae and cordylobia rodhaini) and dermatobia hominis (diptera: oestridae), respectively (adam et al. 2006; olumide 1994; tamir, haik & schwartz 2003). however, canine myiasis is mostly due to c. anthropophaga because dogs, humans and small rodents serve as reservoir hosts for the larvae (mcgraw & turiansky 2008). the prevalence of canine myiasis associated with c. anthropophaga in nigeria varies. ogo et al. (2009) reported a 100% prevalence, suggesting a preference for dogs, although many myiasis-causing flies are endemic to the country (olumide 1994; oluwatosin & fadahunsi 2003; uva & onyeka 1998). several methods are used for diagnosis of myiasis in nigeria (dipeolu 1975; dipeolu & olufarati 1975; idowu & olotu 1976). these include clinical presentation, morphological characterisation of the larvae (e.g. slits of the posterior spiracles located on the posterior spiracular plates) and occasional identification of the adult fly. the time-consuming method of definitively rearing larvae to adults under soil and temperature-dependent conditions is also used occasionally. advances in molecular techniques provide a new impetus to the diagnosis and identification of a broad range of organisms, including myiasis-causing flies (hall et al. 2009; stevens & wall 2001). molecular identification can be used on dead, live or preserved samples. the fact that it can be used effectively on all larval stages will eliminate the need to rear the larvae to adults for identification (ames, turner & daniel 2006). several genetic markers from mitochondrial dna [cytochrome oxidase gene (coi, coii, 12s mtdna)] and ribosomal dna (16s rrna, 28s rrna) have been used in identification, population and evolution studies of myiasis-causing flies of the families oestridae, calliphoridae and sarcophagidae (otranto & stevens 2002). this study reports on the use of polymerase chain reaction (pcr), cloning and sequencing of the 28s rrna gene of blowflies as an aid in the identification of c. anthropophaga larvae from dogs in nigeria. materials and methods top ↑ different stages of larvae (n = 200) were randomly collected from the dermal layer of the skin of 50 dogs visiting veterinary clinics in jos south (9°47´n, 8°51´e) in plateau state, nigeria. each extracted larva was preserved in 70% ethanol for molecular studies. morphological identification of the larvae was performed at the department of parasitology, national veterinary research institute, vom. identification of c. anthropophaga was based on morphological characteristics and the presence of three slits at the posterior spiracular plate of the larva, according to hall and smith (1993) and soulsby (1982). as a confirmation of such identification, we subjected larvae to molecular analysis at the instituto de investigacion en recursos cinegeticos irec (csic-uclm-jccm) in ciudad real, spain. the larvae were washed in distilled water for 1 hour before drying in a laminar flow cabinet under ultraviolet light. individual larvae were placed on a petri dish in 1 ml phosphate-buffered saline (ph 7.4) and dissected using a sterile scalpel blade. the internal organs were collected with a watchmaker’s forceps under a stereo microscope (nikon smz 1500, new york) and transferred into 1 ml trizol reagent® (sigma-aldrich, st. louis). dna was extracted according to the manufacturer’s instructions (sigma-aldrich) and stored at –20 °c after the dna concentration had been determined using a nanodrop® nd 1000 spectrophotometer (nanodrop products, wilmington). pcr amplification was initiated by using the forward and reverse primers d1f: 5’-ccc cct gaa ttt aag cat at-3’ and d2r: 5’gtt aga ctc ctt ggt ccg tg-3’, which amplify the 28s rrna d1–d2 regions of blowflies (stevens & wall 2001). the primers and other reagents in the reaction mixture included the dna template (50 ng/µl – 150 ng/µl), 10× high-fidelity pcr buffer with 15 mm mgcl2 (5 µl), deoxyribonucleotide triphosphate mix (2 mm of each base), 10 pmol of each forward and reverse primer and high-fidelity pcr enzyme mix (5 u/µl) made up to 50 µl with nuclease-free water. conditions for the pcr were an initial denaturation at 94 °c for 2 min, followed by 30 cycles of denaturation at 94 °c for 1 min, annealing at 55 °c for 2 min and extension at 72 °c for 1.5 min. the reaction terminated with a final extension step at 72 °c for 10 min. control reactions were performed without the addition of dna to the reaction so as to rule out contamination during pcr. electrophoresis of the amplicons from the pcr on 1.5% agarose gel was used to calculate the size of the amplified fragments compared to a standard molecular weight marker (1 kb massruler®, fermentas). the amplicons were purified with the purelink™ purification kit (invitrogen, carlsbad, ca) according to the manufacturer’s instructions, then ligated into the pgem®-t vector system i (promega, madison) and transformed into jm109 high-efficiency competent escherichia coli cells according to the manufacturer’s protocol. the transformed cells were subsequently plated on x-gal⁄iptg plates (iptg, isopropyl β-d-1-thiogalactopyranoside). the small colourless positive transformants were selected and cultured overnight in lesogeny broth-ampicillin medium at 37 °c. plasmid dna was purified with the qiaprep® spin miniprep kit (qiagen, valencia, ca) whilst a proteus vulgaris ii enzyme (pvu ii) kit (fermentas®, vilnius) was used to digest the plasmid dna for size evaluation on 1% agarose prior to sequencing. at least two clones from each purified plasmid were submitted for sequence confirmation in an automatic sequencer (3730 dna analyzer, applied biosystems®, carlsbad, ca). our sequences for c. anthropophaga were compared with sequences previously deposited in the gene bank using the basic local alignment search tool (blast) of the ncbi database (http://www.ncbi.nlm.nih.gov/blast). in the phylogenetic analysis, sequence alignment was obtained by using bioedit software (version 5.0.9) (hall 1999). the genbank accession numbers of the 28s rrna gene sequences used to construct a phylogenetic tree were c. anthropophaga ‘isolate yaounde’ (aj551432) and oestrus ovis (aj551428) as an out-group. the distance matrix was calculated by use of kimura-2 parameters, whilst 1000 bootstrap replicates were used to estimate the reliabilities of the nodes on the phylogenetic tree. a bootstrap value of 70% is considered significant evidence for phylogenetic grouping (hills & bull 1993). the output of the tree was constructed with the mega 4.1 program (tamura et al. 2007). ethical considerations animals were handled humanely during sample collection, under the supervision of veterinarians, and procedures complied with nigerian laws. results top ↑ all the intact secondand third-stage larvae examined were positively identified as c. anthropophaga and showed the typical morphological characteristics of being oval in shape, with an average size of 11 mm – 16 mm. dna amplification for the 28s rrna gene of blowflies, using an amplicon size of 651 base pairs, was obtained. a blast search for all the cloned and sequenced amplicons recorded across the ddbj/embl/genbank databases showed 98% – 100% similarity to the partial 28s rrna gene of c. anthropophaga ’isolate yaounde‘. the sequences from our samples were all identical, resulting in the submission of only one sequence to genbank under the accession number jf806640. in the phylogenetic tree shown in figure 1, our sequences were clustered with c. anthropophaga ’isolate yaounde‘ from cameroon with 62% as bootstrap value. this low value infers a close relationship between the two strains. figure 1: a neighbour-joining tree based on 630 base pair sequences of the 28s rrna genes of the blowfly (cordylobia anthropophaga), with 1000 bootstrap replicate values. discussion top ↑ cutaneous myiasis usually presents with numerous erythematous, furunculoid skin lesions on any part of the body and oozes serous fluid. pressure on such lesions lead to the expulsion of the larvae and liquefied tissue. in the tropics, myiasis may be caused by the larvae of different dipteran flies, attracted by the humidity and temperature of the microenvironment of the skin. broken or wounded skin is at a higher risk of infestation (farkas et al. 2009). in nigeria, cutaneous myiasis in both dogs and humans is usually attributed to c. anthropophaga, with the majority of the reports from the western and northern parts of the country (dipeolu & olufarati 1975; edungbola 1982; ogo et al. 2009). the fly is also present in the neighbouring cameroon (guillozet 1981). the close proximity between nigeria and cameroon may account for the similarity in identity and phylogenetic analysis of the sequences obtained during our study and those reported by stevens and wall (2001), thus suggesting that the same strain is circulating between the two countries. molecular identification as reported here can be used in addition to morphological identification based on the physical characteristics of the larvae and the specific posterior spiracular plates of c. anthropophaga. however, the reported method provides unambiguous results and could therefore serve as an alternative to morphological methods when preservation methods are not optimal or when there is doubt about similar or closely related fly species. this method can also be applied in animal forensics to determine the period of neglect or abuse and time of death of an animal by identifying the larvae and fly species present on carcasses (anderson & huitson 2004). conclusion top ↑ the use of molecular techniques in the identification of c. anthropophaga larvae in nigeria suggest that the technique may also be useful in studying other aspects of larval infestation, such as epidemiology, pathogenicity, population genetics and the mechanism of resistance to pharmaceutical compounds. acknowledgements top ↑ we thank dr. isabel fernández de mera for her advice during sample analysis and the head of research, animal health group at the instituto de investigacion en recursos cinegiticos, irec (csic-uclm-jccm), ciudad real, spain for providing the facilities to carry out this research. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions n.i.o. (national veterinary research institute), e.o. (national veterinary research institute) and o.o.o. (department of veterinary parasitology) conceived and designed the project and were all involved in sample collection and morphological identification of the larvae. n.i.o. (national veterinary research institute), r.c.g. (instituto de investigacion en recursos cinegiticos), j-m.p.d.l.l. (instituto de investigacion en recursos cinegiticos) and j.d.l.f. (instituto de investigacion en recursos cinegiticos) conducted the molecular studies. n.i.o. (national veterinary research institute) and o.o.o. (department of veterinary parasitology) wrote the manuscript. references top ↑ adam, a.a., rahman, a.o.a., nail, a.s.m. & imam, a.m.m., 2006, ´cutaneous myiasis due to dermatobia hominis: a case report from eastern sudan´, sudan journal of medical science 1, 147–148. ames, c., turner, b. & daniel, b., 2006, ´the use of cytochrome oxidase i gene (coi) to differentiate two uk blowfly speciescalliphora vicina and calliphora vomitoria´, forensic science international 164, 179–182. pmid:16504435 anderson, g.s. & huitson, n.r., 2004, ´myiasis in pet animals in british columbia: the potential of forensic entomology for determining duration of possible neglect´, canadian veterinary journal 45, 993–998. pmid:15646845, pmcid:554754 dipeolu, o.o., 1975, ´a survey of the ectoparasitic infestation of dogs in nigeria´, journal of small animal practice 16, 123–127. http://dx.doi.org/10.1111/j.1748-5827.1975.tb05725.x dipeolu, o.o. & olufarati, b., 1975, ´letter: furuncular myiasis caused by larva of 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lagelu l.g.a. of oyo state´, african journal of clinical and experimental microbiology 4, 44–47. otranto, d. & stevens, j., 2002, ´molecular approaches to the study of myiasis causing larvae´, international journal for parasitology 32, 1345–1360. http://dx.doi.org/10.1016/s0020-7519(02)00095-4 soulsby, e.j.l., 1982, helminths, arthropods and protozoa of domesticated animals, elbs and bailliere tindall, london. stevens, j.r. & wall, r., 2001, ´genetic relationship between blowflies (calliphoridae) of forensic importance´, forensic science international 120, 116–123. http://dx.doi.org/10.1016/s0379-0738(01)00417-0 tamir, j., haik, j. & schwartz, e., 2003, ´myiasis with lund’s fly (cordylobia rodhaini) in travelers´, journal of travel medicine 10, 293–295. http://dx.doi.org/10.2310/7060.2003.2732 tamura, k., dudley, j., nei, m. & kumar, s., 2007, ´mega4: molecular evolutionary genetics analysis (mega) software version 4.1.’, molecular biology and evolution 24, 1596–1599. http://dx.doi.org/10.1093/molbev/msm092, pmid:17488738 uva, c.u.t. & onyeka, j.o.a., 1998, ´preliminary studies on myiasis in domestic animals slaughtered in jos abattoir, plateau state, nigeria´, african journal of natural science 2, 111–112. article information authors: christopher j. kasanga1 raphael sallu2 fredrick kivaria3 mathias mkama2 joseph masambu2 mmeta yongolo2 chanasa mpelumbe-ngeleja2 philemon n. wambura1 donald p. king4 mark m. rweyemamu1 affiliations: 1faculty of veterinary medicine, sokoine university of agriculture, tanzania 2central veterinary laboratory, dar-es-salaam, tanzania 3national epidemiology unit, ministry of livestock development and fisheries, dar-es-salaam, tanzani 4institute for animal health, woking, united kingdom correspondence to: christopher kasanga postal address: po box 3019, sokoine university of agriculture, morogoro how to cite this proceeding: kasanga, c.j., sallu, r., kivaria, f., mkama, m., masambu, j., yongolo, m. et al., ‘foot-and-mouth disease virus serotypes detected in tanzania from 2003 to 2010: conjectured status and future prospects’, onderstepoort journal of veterinary research79(2), art. #462, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.462 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. foot-and-mouth disease virus serotypes detected in tanzania from 2003 to 2010: conjectured status and future prospects in this proceeding... open access • abstract • introduction • materials and methods    • sample collection    • laboratory analysis of samples    • data analysis • results • discussion • conclusion • acknowledgements • references abstract top ↑ this study was conducted to investigate the presence of foot-and-mouth disease virus (fmdv) in different geographic locations of tanzania. epithelial tissues and fluids (n = 364) were collected from cattle exhibiting oral and foot vesicular lesions suggestive of fmd and submitted for routine fmd diagnosis. the analysis of these samples collected during the period of 2002 and 2010 was performed by serotype-specific antigen capture elisa to determine the presence of fmdv. the results of this study indicated that 167 out of 364 (46.1%) of the samples contained fmdv antigen. of the 167 positive samples, 37 (28.4%) were type o, 7 (4.1%) type a, 45 (21.9%) sat 1 and 79 (45.6%) sat 2. two fmdv serotypes (o and sat 2) were widely distributed throughout tanzania whilst sat 1 and a types were only found in the eastern zone. our findings suggest that serotypes a, o, sat 1 and sat 2 prevail in tanzania and are associated with the recent fmd outbreaks. the lack of comprehensive animal movement records and inconsistent vaccination programmes make it difficult to determine the exact source of fmd outbreaks or to trace the transmission of the disease over time. therefore, further collection and analysis of samples from domestic and wild animals are being undertaken to investigate the genetic and antigenic characteristics of the circulating strains, so that a rational method to control fmd in tanzania and the neighbouring countries can be recommended. introduction top ↑ foot-and-mouth disease virus (fmdv; family picornaviridae, genus: aphthovirus) exists as seven serotypes (o, a, c, asia 1, southern african territories 1–3 [sat 1–3]) and causes a highly contagious disease of ruminants and swine. foot-and-mouth disease (fmd) is endemic in most of sub-saharan africa and is considered to be one of the most widely distributed transboundary animal diseases (tad) in the world (oie and fao reports 2003). in tanzania, fmd is the most important viral tad (swai et al. 2009). fmdv in endemic settings across the world have been categorised into six pools; each comprising a different geographic location with different predominant serotypes. the fmdv pools include pool 1 in east asia (o, a and asia 1), pool 2 in central asia (o, a and asia 1), pool 3 in europe and south asia (o, a and asia 1), pool 4 in southern, eastern and horn of africa (a, o, sat 1, 2 and 3), pool 5 in western africa (o, a, sat 1 and 2), and pool 6 in southern africa (sat 1, 2 and 3) (paton, sumption & charleston 2009). tanzania links east africa and southern africa in a region that overlaps between pools 4 and 6.since its first documentation in 1927 and first isolation of the virus in 1954, many fmd outbreaks have occurred across different areas of tanzania. unrestricted animal movements are an important mechanism by which fmd is spread within and across international borders (di nardo, knowles & paton 2011; kivaria 2003). in order to limit the spread and economic impact of the disease, the control measures implemented during outbreaks in tanzania typically consist of quarantine and restriction of animal movements particularly in areas with well-defined farming systems (kivaria 2003). however, the presence of multiple fmd serotypes, and the occurrence of subclinical forms of the disease renders fmd control very difficult, particularly in pastoral agricultural systems where resources are limited. establishing and quantifying the distribution of fmdv serotypes in different eco-climatic regions in the country will contribute to the understanding of fmd epidemiology, and provide knowledge to researchers, vaccine manufacturers and policy makers to more efficiently deploy resources to control fmd field outbreaks. of the seven known fmdv serotypes, four (o, a, sat 1 and sat 2) have been previously identified and reported in tanzania (mlangwa 1983; rweyemamu & loretu 1972; rweyemamu et al. 2008b; swai, mrosso & masambu 2009; vosloo et al. 2002). despite the fact that fmd is endemic in tanzania, only limited studies have been conducted to describe the spatial and temporal distribution of fmd outbreaks (picado et al. 2010) and fmdv serotypes in the country (rweyemamu & loretu 1972; swai et al. 2009). furthermore, no specific detailed studies have been undertaken to describe the molecular epidemiology of tanzanian fmdv serotypes. these approaches can define the distribution patterns of viruses and factors determining endemicity of fmd in tanzania, and data from such studies will provide important information that will underpin future efforts to control fmd. therefore, the aim of this current study was to use routine laboratory diagnostic methods to establish the spatial and temporal distribution of fmdv serotypes in different regions of tanzania. sample collection targeted fmd outbreaks that occurred in the country between 2003 and 2010. materials and methods top ↑ sample collection all samples were collected from confirmed fmd outbreaks (based on clinical signs) in cattle in different regions of tanzania. the samples were collected by staff from seven strategically placed veterinary investigation centres in the country. these samples were obtained from regions namely arusha and kilimanjaro (northern zone), mwanza and kagera (northern-lake zone), tabora, rukwa and kigoma (western zone), mtwara (southern-coastal zone), iringa and mbeya (southern zone), dodoma and singida (central zone) and dar-es-salaam, pwani and morogoro (eastern-coastal zone). epithelial tissues and fluid from oral and foot lesions were collected and immediately placed in virus transport media composed of equal amounts of sterile glycerol (50% v/v) and 0.04 m phosphate buffered saline at ph 7.2–ph 7.6, stored at +4 °c and transported to the central veterinary laboratory (cvl), dar-es-salaam. laboratory analysis of samples vesicular fluids and tissue epithelia were analysed for fmdv antigens, and the viral antigen typed into different serotypes by the antigen detection elisa (iah, pirbright) as previously described (hamblin, armstrong & hedger 1984; ferris & dawson 1988). data analysis the dependent variable tested was the seropositivity to fmdv antigen (positive or negative) and specific serotype, in this case types a, o, sat 1 and sat 2. the independent variables investigated were the geographic location of the origin of samples and/or fmd outbreaks. results top ↑ a total of 364 samples from different eco-climatic regions in tanzania were examined for fmdv antigen. of the tested samples, 167 (45.9%) were positive to at least one of the four serotypes of fmdv. of the 167 positive samples, 37 (22.0%) were serotype o, 7 (4.2%) serotype a, 45 (27%) serotype sat 1 and 78 (46.8%) serotype sat 2 (table 1). there were no samples that tested positive for more than one fmdv serotype. serotype sat 2 was identified throughout the northern, southern, western, eastern and central zones of tanzania (figure 1). serotypes o, sat 1 and sat 2 were identified at least from one geographic region every year from 2003 to 2010 (table 1). serotype a was exclusively found in the eastern coastal zone (figure 1), and was only detected more recently in 2009 (table 1). interestingly, serotype sat 1 was consistently detected only in the eastern coastal region throughout the eight years (2003–2010). serotype o was mainly found in the southern and northern highlands as well as the lake zones. animals from which all the tested samples were obtained had no documented history or evidence of vaccination against fmdv. table 1: summary of foot-and-mouth disease virus serotypes detected in tanzania from 2003 to 2010. figure 1: geographic distribution of foot-and-mouth disease virus serotypes detected in tanzania between 2003 and 2010. discussion top ↑ fmd is known to be endemic in tanzania. these findings show that fmdv is widely distributed in many parts of the country with at least four serotypes being found. the four serotypes that were detected during the period of from 2003 to 2010 were o, a, sat 1 and sat 2. this is broadly in agreement with findings from previous studies, which reported the detection of these serotypes in this country from late 1950s (rweyemamu & loretu 1972; vosloo et al. 2002). these observations suggest that serotypes o, a, sat 1 and sat 2 have been, and are also associated with the recent fmd outbreaks in different areas of tanzania. we have observed that the three serotypes (o, sat 1 and sat 2) were detected consistently every year from 2003 to 2010 (table 1). studies conducted by others have reported the existence of serotypes o and sat 2 in tanzania to be as far back as in 1950s with the detection of sat 1 for the first time in 1971 (rweyemamu & loretu 1972, 1973). together, these findings indicate that types o and sat 2 are old in tanzania and have consistently been observed in the country. these observations raise some questions such as, what factors could be involved in the successive existence of these serotypes? are the viruses detected in 1950s genetically and antigenically related to the most recent strains? are viruses from different geographic regions genetically related? all these call for a need of performing a detailed study on the molecular epidemiology of fmdv in tanzania. the current and previous studies indicate some enigma relating to the dynamics of serotype a in tanzania. during the period covered by this study (i.e. from 2003 to 2010) it was detected only in 2009 in the eastern coastal zone. rweyemamu and loretu (1972) reported regular detection of this sero-type in the northern, northern-lake and central zones of tanzania for up to 1971. swai et al. (2009), however, did not find serotype a in the samples collected between 1997 and 2004. the factors that may have led to the apparent disappearance of serotype a in tanzania for possibly up to 38 years (from 1971 up to 2009) have not been elucidated. they could include high fragility of the virus and low number of samples that were brought for analysis. however, the reappearance of the type a viruses could possibly be a consequence of cross-border spread of the virus from the neighbouring countries, implying that unlike serotypes o, sat 1 and sat 2, serotype a might not be truly endemic in tanzania. similar observations have also been made in iran that reported the emergence of serotype a sub-lineage ’a-iran-05’ (after long time of no periodic outbreaks of type a viruses), which has no closely related antecedents (knowles et al. 2009). together, these observations indicate the possibility of emergence of different serotype a strains in different geographic areas. therefore further studies are required to determine the genetic characteristics of the old and new serotype a isolates detected in tanzania. this will unravel the evolutionary characteristics of type a sub-lineages, which could be useful for the control of emerging type a variants in the region. as the government of the united republic of tanzania is planning to adopt a strategic control programme for fmd through vaccination and controlled animal movements, a wider knowledge and understanding of fmdv dynamics and epidemiology should be taken into consideration. this will require the identification of high-risk ’hotspots’ as well as potentially infected and fmd-free zones. this task will however need considerable political commitment and laboratory resources to define the spatial and temporal distribution of fmd outbreaks and serotypes in the country. the heterogeneity of fmdv serotypes observed in this study (table 1) and their spatial occurrence (figure 1) highlights the need for continuous surveillance of fmd so as to monitor the infection status and spread of fmdv serotypes in livestock and wildlife in tanzania. as risk factors and transmission characteristics differ in each region (swai et al. 2009; picado et al. 2010), a regional fmd surveillance system involving tanzania and the neighbouring countries will provide information on serotype spatiotemporal distribution for effective control of the disease. serotype sat 2 was detected almost throughout the country for the whole period of 2003–2010 (table 1), with the highest detection rate of 46.8%. additional information from veterinary investigation centres (vics) in tanzania (data not shown) indicates that sat 2 strains were obtained from regions with relatively higher number of livestock, especially cattle. these findings suggest that sat 2 could be incriminated as the cause of many fmd outbreaks in tanzania. efforts to isolate and characterise the viruses through genome sequencing are required so as to understand the genetic and antigenic features of field strains; information that is necessary for selection of appropriate vaccine candidate strains for use in tanzania and neighbouring countries. serotype o seems to be widespread in the northern, southern, western and eastern zones, and not in the central parts of tanzania (figure 1). this observation agrees with the previous reports in tanzania (mlangwa 1983; mowld 2003). in addition, serotypes a and sat 1 were also not detected from the central parts of tanzania. however, it is important to recognise that the absence of serotypes o, a and sat 1 from the central parts of tanzania could be due to low number of samples submitted to cvl for diagnosis that may be related to under reporting of outbreaks, inappropriate cold chain facilities and logistics rather than the actual situation of disease occurrence. the control of fmd, especially following outbreaks, requires timely identification and characterisation of circulating fmdv serotypes in a given geographic area. tanzania is found in the great lakes (also known as the east african community (eac) or southern-east africa fmd epidemiological cluster characterised by a substantial diversity of circulating strains or topotypes (rweyemamu et al. 2008a). five serotypes (a, o, c, sat 1 and sat 2) are known to be endemic in this cluster (sahle et al. 2007; vosloo et al. 2002), and a sixth serotype (sat 3) has only been isolated from african buffaloes in uganda in 1970 (hedger, forman & woodford 1973). in the current study, we detected serotypes a, o, sat 1 and sat 2 in cattle from different geographic locations in tanzania. so far serotypes c and sat 3 have never been detected in tanzania. whether these serotypes, that have been reported to prevail in the eac cluster, also exist in tanzania remain unclear and need further systematic investigation. the current spatiotemporal distribution of fmdv serotypes reported in this study indicates the absence of fmdv in southeastern areas of tanzania bordering mozambique (figure 1). this area has also been designated as low-density fmd area with no evidence of fmd outbreaks (picado et al. 2010). these findings suggest that the southeastern corner, precisely the mtwara corridor, could be considered as a potential fmd-free zone whereby strict surveillance and control programmes should be implemented for production of livestock meant for exportation. in this study we have observed an inconsistency serotype distribution in tanzania (figure 1). the heterogenic distribution of the different serotype could be ascribed to several factors such as presence of diverse susceptible wildlife reservoirs, inadequate diagnostic capacities, diverse farming systems, socio-economic factors, uncontrolled animal movements, genetic and antigenic variation of the pathogen as well as lack of clear disease control policies (rweyemamu 1984). however, the laboratory capacity in tanzania and neighbouring countries has been considered to be inadequate in terms of facilities, equipment and diagnostic kits. therefore, enhancement of laboratory capacity to undertake disease surveillance is of a paramount importance so as to keep pace with the accurate and timely identification of fmdv field strains, which is required for appropriate control strategies of fmd in the region. we did not test any samples from wildlife in this study as all samples were obtained from fmd outbreaks in cattle. however, the epidemiological role of wildlife such as the african buffalo (syncerus caffer) as carriers of particularly the sat serotypes has been widely discussed (hedger et al. 1973; vosloo et al. 1996). a number of regions from which samples were derived are known to be potentially livestock-wildlife interface areas with predominantly high population of buffalos. further studies are required to elucidate the epidemiological link of fmdv maintenance in wildlife, and transmission of the virus from livestock to wild animals or wild animals to livestock. furthermore, the temporal and spatial dynamics of infection need to be conducted with the analysis of host animal distributions and contact opportunities, sero-surveys to estimate the level of infection and use of modern available techniques to track fmdv incursions into disease free areas. conclusion top ↑ we have detected the four fmdv serotypes o, a, sat 1 and sat 2, which are cocirculating in tanzania. sero-type a seems to occur in waves with periods (years) of apparent absence between epidemics. the presence of multiple serotypes and the complex epidemiology of fmd complicate the control of the disease through vaccination and establishment of fmd-free zones. our findings also emphasize the importance of undertaking continuous surveillance to monitor the emergence and spread of fmdv strains. therefore, further studies in both domestic and wild animals are required to investigate the genetic and antigenic characteristics of the circulating strains so that a rational control method for fmd in tanzania and neighbouring countries can be recommended. acknowledgements top ↑ the authors wish to recognise the assistance provided by farmers and staff in veterinary investigation centres for sample collection during fmd outbreaks. we appreciate the contribution of staff at the central veterinary laboratory – temeke during laboratory analysis of samples. we are grateful to the world reference laboratory (wrl), pirbright, uk who provided elisa kit for the study. the ministry of livestock development and fisheries, united republic of tanzania funded this work. the authors also acknowledge the southern african centre for infectious disease surveillance for providing financial support (to cjk and rs) to present this paper at the first one health conference in africa, johannesburg, south africa, july 2011. references top ↑ di nardo, a., knowles, n.j. & paton, d.j., 2011, ‘combining livestock trade patterns with phylogenetics to help understand the spread of foot and mouth 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58, 44–52. http://dx.doi.org/10.1111/j.1865-1682.2010.01180.x rweyemamu, m.m. & loretu, k., 1972, ‘observations on foot-and-mouth disease in tanzania’, bulletin of epizootic diseases of africa 22, 101–104. rweyemamu, m.m. & loretu, k., 1973, ‘observations on the first epidemic of foot-andmouth disease type sat 1 in tanzania’, bulletin of epizootic diseases of africa 21, 393–400. rweyemamu, m.m., 1984, ‘antigenic variation in foot-and-mouth disease: studies based on the virus neutralization reaction’, journal of biological standardization 12, 323–37. http://dx.doi.org/10.1016/s0092-1157(84)80013-x rweyemamu, m., roeder, p., mackay, d., sumption, k., brownlie, j., leforban, y. et al., 2008a, ‘epidemiological patterns of foot-and-mouth disease worldwide’, transboundary and emerging diseases 55, 57–72. http://dx.doi.org/10.1111/j.1865-1682.2007.01013.x rweyemamu, m., roeder, p., mackay, d., sumption, k., brownlie, j. & leforban, y., 2008b, ‘planning for the progressive control of foot-and-mouth disease worldwide’, transboundary and emerging diseases 55, 73–87. http://dx.doi.org/10.1111/j.1865-1682.2007.01016.x sahle, m., dwarka, r.m., venter, e.h. & vosloo, w., 2007, ‘comparison of sat-1 foot-and mouth disease virus isolates obtained from east africa between 1971 and 2000 with viruses from the rest of sub-saharan africa’, archives of virology 152, 797–804. http://dx.doi.org/10.1007/s00705-006-0893-x swai, e.s., mrosso, a. & masambu, j.i.g., 2009, ‘occurrence of foot-and-mouth disease serotypes in tanzania: a retrospective study of tongue epithelial tissue samples’, tanzania veterinary journal 26, 7–12. http://dx.doi.org/10.4314/tvj.v26i1.49232 vosloo, w., bastos, a.d., kirkbride, e., esterhuysen, j.j., janse van rensburg d., bengis., r.g. et al., 1996, ‘persistent infection of african buffalo (syncerus caffer) with sat-type foot-and-mouth disease viruses: rate of fixation of mutations, antigenic change and interspecies transmission’, journal of general virology 77, 1457–1467. http://dx.doi.org/10.1099/0022-1317-77-7-1457 vosloo, w., bastos, a.d., sangare, o., hargreaves, s.k. & thomson, g.r., 2002, ‘review of the status and control of foot-and-mouth disease in sub-saharan africa’, revue scientifique et technique (oie) 21, 437–44. article information authors: yona sinkala1 d. pfeiffer2 c. kasanga3 j.b. muma4 m. simuunza4 a. mweene4 affiliations: 1ministry of livestock and fisheries development, lusaka, zambia 2royal veterinary college, united kingdom 3sokoine university of agriculture, tanzania 4school of veterinary medicine, university of zambia, zambia correspondence to: yona sinkala postal address: po box 30041, lusaka, zambia how to cite this abstract: sinkala, y., pfeiffer, d., kasanga, c., muma, j.b., simuunza, m. & mweene, a., 2012, ‘foot-and-mouth disease control in zambia: a review of the current situation’, onderstepoort journal of veterinary research 79(2), art. #472, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.472 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. foot-and-mouth disease control in zambia: a review of the current situation in this abstract... open access zambia has been experiencing low livestock productivity as well as trade restrictions owing to the occurrence of foot-and-mouth disease (fmd) and contagious bovine pleura pneumonia (cbpp). foot-and-mouth disease was first recorded in zambia in 1933 in the western province and since then the country has experienced repeated outbreaks. bearing in mind the pressure that may be existing on the many risk factors for fmd including climate change, there is need to review our knowledge on fmd control. we present the spatial distribution of the fmd outbreaks that have been recorded in zambia in the last twenty years, and the effect of the vaccinations and movement control that have been applied. we propose further strain characterisation of previous fmd outbreaks, including full sequence of vp1 gene and the 5’utr site. the data will be geo-coded and populated with risk factor attributes. we also present preliminary findings of the buffalo and cattle probang sampling that was conducted in lochnivar and kafue national park. we further probang sampled 25 buffalo at each interface area in sioma ngwezi, lukusuzi and lower zambezi national parks. villages in close proximity to the buffalo populations as well as those not in close proximity will be multistage cluster sampled for comparison. the data will be geo-coded and populated with risk factor and foot-and-mouth disease virus (fmdv) characterisation attributes. data collected using a pre-tested structured questionnaire will be geo-coded and populated with identified risk factors and stored in a database and will be spatially modelled to determine their effect on fmd occurrence and control measures. new outbreaks of fmd that may occur will be investigated to find out if there are new strains involved, species affected and predisposing risk factors. the authors conclude that impacts of fmd on livelihoods if appropriate control measures are not put in place are far more devastating especially at community level. presented with the current poverty levels failure to institute result oriented control measures will exacerbate the already life-threatening situation. article information authors: mohamed m. sirdar1,2 jackie picard3,4 shahn bisschop2 bruce gummow2,4 affiliations: 1agriculture and animal production project, poultry section, national cooperative corporation, khartoum, sudan 2department of production animal studies, university of pretoria, south africa 3department of veterinary tropical diseases, university of pretoria, south africa 4school of veterinary and biomedical science, james cook university, australia correspondence to: bruce gummow postal address: private bag x04, onderstepoort 0110, south africa dates: received: 14 july 2011 accepted: 13 dec. 2011 published: 21 may 2012 how to cite this article: sirdar, m.m., picard, j., bisschop, s., gummow, b., 2012, ‘a questionnaire survey of poultry layer farmers in khartoum state, sudan, to study their antimicrobial awareness and usage patterns’, onderstepoort journal of veterinary research 79(1), art. #361, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.361 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a questionnaire survey of poultry layer farmers in khartoum state, sudan, to study their antimicrobial awareness and usage patterns in this original research... open access • abstract • introduction • materials and methods    • census of layer farms in khartoum state    • questionnaire survey    • data analysis    • spatial analysis    • ethical considerations • results    • census of commercial layer farms in khartoum state, sudan    • questionnaire analysis • discussion    • census of layer farms in khartoum state, sudan    • questionnaire survey • conclusion • acknowledgements    • competing interests    • authors’ contributions • references • footnotes abstract top ↑ an initial census of layer farms in khartoum state, sudan, was carried out in late 2007 and early 2008 and found that there were 252 layer farms with a total population of 2 221 800 birds. this paper reports the findings of the census. based on this information, a structured questionnaire survey of 92 farms was then conducted in the state in april 2008 to collect data on antibiotic usage, demographic data and public health awareness. ninety-eight per cent of participating farms comprised open-sided houses. it was found that 49% of the farms surveyed were on antibiotic treatment when the survey was conducted, whilst 59% of the farms had used antibiotics within the last 3 months. the study found that farmers and producers had a lack of knowledge about antimicrobial residues, their withdrawal periods and the risk posed by the consumption of these residues. the study also concluded that traditional farming systems in sudan relied heavily on antimicrobial medication to control disease and almost half of the farms surveyed were treating their flocks with antimicrobials. in addition to this, there was a lack of disease control programmes which probably resulted in a massive use of antibiotics to control endemic diseases. this was further compounded by the absence of governmental supervision and control on the use of drugs. introduction top ↑ khartoum state is responsible for almost 90% of sudan’s poultry production (ministry of agriculture, animal resources and irrigation 2005). antimicrobials are used in layer hens in sudan, mainly to treat and prevent bacterial infections. these antimicrobials are similar to those used in human medicine, which include aminoglycosides, tetracyclines, beta-lactams, quinolones, macrolides, polypeptides, amphenicols and sulphonamides (stolker & brinkman 2005).a study that was conducted concurrently with this one showed that eggs from a high proportion of farms and layer houses contained antimicrobial residues (sirdar 2010). similarly, investigations in nigeria and tanzania showed that a high proportion of table eggs contained antimicrobial residues (kabir et al. 2004; nonga et al. 2010). this is very different to countries in western europe, australia and north america, where it would be unusual to detect any antibacterial substances in table eggs. there is little known as yet about farmers’ perceptions or other factors that play a role in the cause of this problem in some african countries. this study set out to attempt to gain a better understanding of what sudanese poultry farmers know and think about antimicrobial use. only through understanding these factors, the problem may eventually be resolved and lead to a healthier lifestyle for africans. in addition, little has been published about the demographics of the poultry layer industry in sudan, and so a secondary objective of this study was to carry out a census of the poultry layer industry in khartoum state, sudan. materials and methods top ↑ census of layer farms in khartoum state a census to determine the size and structure of commercial layer farms in khartoum state, sudan, was conducted between december 2007 and january 2008. the sampling frame consisted of all known layer farms in the three localities of khartoum state. the identification of farms was based on data from an internal publication by the state ministry of agriculture, animal resources and irrigation of 2005, from day-old chick suppliers and from poultry veterinarians. in addition, information from farm owners about other farms was used to identify farmers not already listed. the sampling unit at the time of the survey was a layer farm producing eggs or pullets, or layer farms not currently in production. for each area the following was recorded: the location of each farm, the number of farms in each area, the number of layer houses per farm, the capacity of each layer house, and the farming system used. questionnaire survey a structured questionnaire was designed to collect information on farm management procedures used on each layer farm, besides investigating local knowledge and understanding issues that surround antibiotic usage in food-producing animals.the sampling frame for the questionnaire was all known layer farms that were producing eggs at the time of the survey in khartoum state (figure 1). data were obtained on antibiotics recently used, antibiotics used in the last 3 months, reasons for using the antibiotics, diseases currently on the farm, diseases recorded in the last 3 months, withdrawal period, methods of storing antibiotics, quality control and policies of antibiotics usage in the poultry industry. perceptions of the public health risk of antibiotic residues in table eggs were also investigated. in addition to that, the farming system, chicken breed, breeding system, number of chickens per house, number of houses per farm and current age of the flock were recorded. to determine the antibiotics used at the time of the survey, labels and empty bottles of antibiotics were collected and the data were recorded. all elements of the questionnaire were categorical variables, structured as closed ended questions. the only continuous questions regarded the age of the flock, the number of chickens per house and the number of houses per farm; these also were coded later and recorded as categorical variables. the questionnaire was not subjected to pretesting or repeatability testing; it was designed in english and the contents were translated into arabic during the interview. the validity of the questionnaire was assessed by comparing the results of the questionnaire with reliable criteria, that is, the related questions in the same questionnaire form and known facts such as the absence of rules and regulations of antibiotic usage in sudan. the survey was carried out in april 2008, covering the whole state, and all information needed in the questionnaire form was captured through direct interview. the respondents were the owners or managers of the farms. thirty-four of the farms that participated in the questionnaire survey were correlated to results from a separate survey on their farms that investigated the presence of antimicrobial residues in eggs (sirdar 2010). in this survey three eggs from each house on a farm were tested for antimicrobial residues and if one or more eggs were found to contain antimicrobial residues, the farm was considered as positive for residues in eggs. these 34 respondents’ results were analysed further to determine whether there were any statistical associations between what farmers answered (table 1) and the known presence of antimicrobial residues on their farms. table 1: the association between factors associated with antimicrobial residues and the presence of antimicrobial residues in eggs. data analysis the information captured from the census was recorded and summarised (table 2). the questionnaire data derived from all known layer farms was captured into, and analysed by using, epiinfo™1 version 3.5.1. several descriptive statistics, including frequencies, means, medians and statistical associations between several factors were measured (chi-square and fisher exact test). strengths of associations were assessed by calculating odds ratios and data were stratified to look for any possible confounding effects. for the 34 selected farms, the association between questions answered (factors), and the presence or absence of antimicrobial residues in eggs on these farms in april 2008 (sirdar 2010), was assessed by using the two-tailed fisher exact test (epicalc 2000 software2). factors related to the presence or absence of antimicrobials on the farm (p < 0.05) were re-examined in a multivariate model by using multiple unconditional logistic regression to control confounding. models were built by using forward elimination with switching because of the small sample size: those with p > 0.05 on the wald test were removed one at a time until all factors left in the model were statistically significant at p < 0.05 (thrusfield 2005). logistic regression, therefore, was used to determine the best set of factors. whilst a value of p < 0.05 was considered significant; p-values between 0.06 and 0.1 were considered numerically reportable as potential trends. table 2: areas covered in the survey and the proportion of farms surveyed in each area in khartoum state, 2008. spatial analysis the mapping program, google earth version 4.3,3was used to trace the farms and areas of sampling and to record the coordinates of all the farms sampled in khartoum state. all coordinates were entered into the spreadsheet program, excel (microsoft corporation, usa 2003). data were converted from excel files for use in arcview 9.3 (esri redlands 2009). africa, sudan and khartoum state shape files were downloaded from www. maplibrary.org4. maps of khartoum state showing the sampling locations were created by using arcview 9.3. ethical considerations the project was an approved university of pretoria research project (v047-07), which included ethical approval by the animal use and care committee. no live animals were used in the study. results top ↑ census of commercial layer farms in khartoum state, sudan the census covered all three localities of the state: khartoum north (bahry), khartoum and omdurman. the census showed that there were 252 layer farms containing 764 commercial layer houses in the state, with a total capacity of 2 221 800 birds. the locations of layer farms sampled in the entire khartoum state are shown (figure 1). figure 1: location of farms surveyed in khartoum state, sudan, april 2008. questionnaire analysis the questionnaire survey was conducted in april 2008. ninety-two farms participated in the survey. the questions and the results of the questionnaire survey, have been summarised (table 3).layer farms in 17 different production areas were surveyed (table 2). eleven areas (65%) were in bahry locality and the remainder (35%) was in khartoum locality. there were no farms in production in omdurman locality whilst the survey was conducted. about 59% of the farms that were surveyed were in bahry locality and 41% were in khartoum locality. a high proportion of farms were surveyed in kalakla north (100%), kalakla and dikhenat (94%), and soba garb (82%) areas. in contrast, farms in tyba hassanab were the least surveyed (13%), because owners in this area were not willing to participate in the survey. only a few farms were included in el-samrab and el-shegailab, because the total number of farms in these areas was low and houses were found to be used as layer rearing houses at the time of the survey. table 3: topics covered by the questionnaire and the questionnaire survey results, khartoum state, 2008. only one out of twelve closed systems in khartoum state was prepared to take part in the questionnaire survey, which unfortunately biased the survey towards farms with traditional open houses. the majority of the farmers interviewed were small-scale producers who had a maximum of 1000 birds (95%) that were mainly distributed into one or two houses (83%).the most common breed in the survey was hisex (51%), as it is considered to be the most tolerant of the breeds to high ambient temperatures. in addition to that, hisex was introduced to sudan over 3 decades ago and the local supplier had established a good business-relationship with the farmers. nevertheless, lohman (13%) and hyline (5%), both only introduced to the sudanese market in the last 4 years by foreign companies, were starting to capture a good share of the market. bovan breed (a dutch breed introduced to sudan in the 1980s) shared 9% of the total breeds found in the state, whilst the unknown breeds (not including indigenous breeds) kept by the owners interviewed, were 14%. thirty-seven per cent of the farms surveyed had multiple ages on the same farm. when comparing this figure with the all-in all-out breeding system (63%), it gives an erroneous idea of age distribution because most of the farms surveyed had only one house on the farm that was used for egg production at a time. in 68% of the surveyed farms, the age of the flocks varied from 4 to 12 months. although there was one age group within each layer house, the distance between layer houses on a farm, or between farms, was less than 30 meters, indicating that even when the system used is all-in all-out, it could be affected also by other age groups on the farms and between farms. from the answers to the questionnaire (n = 92), it was found that the odds of antibiotic use at the time of the survey were 21 times greater on farms that had concurrent disease (7 < or < 36, 95% confidence interval), which implies that usage was more for treatment than for prophylaxis. this is supported further by the finding that farms that have used antibiotics in the 3 months prior to the survey had 18 times greater odds of concurrent disease during the same period (or = 18; 6 < or < 54, 95% confidence interval; chi-square = 32; p < 0.01). when the use of antibiotics were more closely examined, it was found that the association between prophylactic uses of antibiotics and the presence of diseases on farms was insignificant (p-value = 0.15), whilst the association between therapeutic and prophylactic use at the same time with the presence of disease on farms, was significant with a p-value = 0.02. the odds of antibiotics used for therapeutic purposes were 17 times greater on farms that had diseases than on those that did not have diseases. the fourth strata (purpose of antibiotic use unknown) showed an insignificant association with disease on farms. the summary odds ratio of the stratified analysis was 22; the adjusted odds ratio was 27, chi-square was 37, and p-value < 0.01, which suggested some confounding effect within ’purpose for use of antibiotics‘. when placed into a logistic regression model, there was still a strong association between therapeutic use and the presence of disease (or = 15), but the association was no longer significant (wald p > 0.05) and this was probably because of low numbers in some of the strata. farmers who indicated that they did not know drugs in eggs can affect human beings, were much more likely not to know that drugs pass from chicken body to eggs (or = 28; 95% confidence interval = 6 < or < 141); (chi-square = 23; p < 0.02). these comprised 83% of the farmers, and therefore suggest a widespread lack of knowledge about drug behaviour and its effects amongst sudanese farmers. information was available on 34 farms as to the antimicrobial status of their eggs, that is, whether residues had been detected in one or more eggs (sirdar 2010), and consequently the association between what famers answered, and the presence or absence of antimicrobial residues was examined. the factors considered for farms positive for antimicrobial residues in khartoum state have been listed (table 1). the fisher exact test showed no significant association between any of the factors examined in the questionnaire and the presence of antimicrobial residues on the farms, with the exception of whether they followed a withdrawal period or not. the majority (92%) of farmers on farms where antimicrobials were found indicated that they did not follow a withdrawal period. a number of regression models were created by using various combinations of factors. in none of the models were any of the factors shown to be statistically significant. the last two factors that were eliminated from the models were adherence to a withdrawal period, and whether farmers thought drugs passed from a chicken’s body to its eggs. discussion top ↑ census of layer farms in khartoum state, sudan the census conducted in this study was necessary because the unpublished census conducted in 2005 by the state ministry of agriculture, animal resources and irrigation, after the avian influenza (ai) outbreak, only recorded farms affected by ai and did not differentiate between broiler and layer farms (ministry of agriculture, animal resources and irrigation 2005). this census proved to be a challenge because most farms are not registered with the local authorities, the land ownership or occupancy was not always recorded, and the land use was fluid. the initial information on farm locations was dependent on the internal data of the state ministry of agriculture, animal resources and irrigation, which were not complete. in order to expand on this information, a snowball approach was adopted by using information gathered from the original field veterinarians and farm owners to locate other farms in the area. in addition, the day-old chicks’ suppliers provided useful data about layer farms in the state and further farms were located from unpublished reports. whilst the census was not ideal, the result was more extensive than previous censuses carried out in the layer industry of sudan, and thus contributed valuably to the update of available information on this production sector.the last census conducted by the state ministry of agriculture, animal resources and irrigation, in 2005, showed that there were 527 poultry (broiler and layer) farms in khartoum state, whilst this census revealed that there were 252 layer farms in the state, with 166 farms (66%) located in bahry, 78 farms (31%) in khartoum, and 8 farms (3%) in omdurman locality. most of the farms in bahri locality were smallholdings. the farms were generally clustered in groups of 10–20 farms along the nile, with most farms in the cluster neighbouring each other. for this reason, when conducting the spatial analysis, the main challenge was to record the coordinates of each farm sampled because there were small differences of seconds and even fractions of seconds between them. almost 50% of the farms recorded at the time of the census were not producing eggs for various reasons, which included: • that many farmers lacked the financial resources to restock their farms after the depopulation and condemnation of carcasses that resulted from the 2006 avian influenza (ai) outbreak. the sudanese government had only compensated the farmers with 60% of the direct cost (carcass price) divided into three payments. at the time of the census, many farmers had not yet received full compensation. this problem was compounded by the dramatic increase in animal feed prices during 2008. • a shortage of day-old chicks, because the suppliers were unable to cover the whole demand associated with the ban of day-old chicks and fertile egg imports after the ai outbreak. this lead to an increase in the price of day-old chicks, making it cost-ineffective for small-scale producers. • the lack of government protection of small-scale producers. this made them highly vulnerable to the effects of disease, market forces and the weather. furthermore, there were no state-run or industry-run disease control programmes; therefore the introduction of diseases such as newcastle disease and salmonellosis caused massive fatalities and chronic respiratory disease that resulted in severe production losses. • that farmers who use traditional housing for breeding can produce only during the cooler winter months (from late october until january). • that some farms were in the downtime period preparing for another cycle. • that the pullets were not yet in lay. • that farmers switched from layer to broiler production. usually in sudan, farmers raise day-old laying chicks until they start to lay, and then continue in the same house until the end of their production cycle. after the culling of the batch, the farmer may use the same farm for producing broilers or to start a new cycle of layers. the data provided by this census do not therefore cover the full production potential of the sudanese layer industry. it does, however, provide a baseline and guide for researchers and officials who wish to compile a more complete database concerning the poultry industry. in addition to that, it will serve as a primary source of data for all who are interested in the sudanese poultry industry. the government in sudan has subsequently established a census forum to create their own database of all livestock farms in the state and they will benefit from the data provided in this research. questionnaire survey ninety-two farms participated in the questionnaire survey conducted in april 2008. the participants of the questionnaire were 52% of the total number of farms (178 farms) in the areas surveyed. the main reason for the low participation was that some farms were not in production at the time of the questionnaire, as explained above. other reasons included the absence of the farm owner or manager, or a refusal to participate for personal reasons.it was clear from the survey that traditional farming systems relied heavily on antimicrobial medication to control disease; 49% of farms were treating their flocks with antimicrobials, whilst a further 9% had used antimicrobials within 3 months prior to this survey. the main purpose of the antibiotics was to treat (61%) a variety of diseases including salmonellosis (30%) and chronic respiratory disease (25%). this high level of disease is believed to be as a result of the type of housing, poor environmental sanitation, poor biosecurity, close grouping of farms and poor management. almost all the antibiotic classes were found in the sudanese market for purchase either as separate products or as products in combination with multivitamins and minerals. oxytetracycline, however, has the added advantage of a highly competitive price, a broad-spectrum coverage and is combined with multivitamins, so that it is the most commonly used antibiotic, with 25% in current use and 23% having used it in the last 3 months. these findings agree with babiker et al. (2009), who classified salmonellosis and respiratory disease as highly prevalent in layer flocks in khartoum state. oxytetracycline appears to be widely used on poultry farms in africa; mitema et al. (2001), kabir et al. (2004) and nonga et al. (2010) found that it was the most used antibiotic in kenya, nigeria and tanzania, respectively. other commonly used antibiotics were tylosin (19%) and the broad-spectrum enrofloxacin (14%) which is used to treat infectious coryza and mycoplasma infections in birds, and colistin (14%) which is used to treat diarrhoea (reinhardt et al. 2005). in all poultry production systems globally, the preferred method of treatment by 97% of the farms was by the mass medication of drinking water. feed was not used as route of administration because the feed mills used for food preparation tend not to fully homogenise small quantities of drugs in feed, which results in an uneven distribution of drugs in the feed. furthermore, sick birds will continue to drink, but will not eat. prophylactic antimicrobial therapy was less common and was not associated with disease (p < 0.15). this finding was expected because small-scale farmers may not be able to afford the cost of treating prophylactically as a result of their limited resources. the failure of the logistic regression models to show any significant associations between the known presence of antimicrobials in eggs on a farm, and certain answers given by farmers, was most likely because of the small sample size in the stratified data (table 1), which reduced the power of the tests. the forward selection process with replacement nevertheless still produced a model that was consistent with the fisher exact test results, indicating that the major factor associated with the presence of antimicrobials in eggs was a lack of compliance with withdrawal period and a lack of understanding that residues can pass from the chickens to eggs. it highlights the importance of risk communication and how ignorance of an african public about the behaviour of antimicrobials in chickens and humans is probably the single most important contributor to the fact that many african countries have antimicrobial residue problems. most sudanese farmers do not believe that drugs in eggs affect human beings, or that drugs can pass from the chicken body to its eggs. there was a significant association between those (85% of respondents) who believed that drugs do not pass from the hens’ bodies to their eggs, and those (89% of respondents) who do not believe that drugs in table eggs can affect human beings (p < 0.02). furthermore, 75% of the farmers apparently did not understand the concept of a drug withdrawal period in eggs. an overwhelming majority of respondents (95%) were not aware of any government regulations pertaining to the sale of eggs during the withdrawal period of antimicrobials. this is partially because there is an absence of any rules and regulations in sudan governing antimicrobial use in poultry production or animal production. it was, therefore, not surprising that 98% of the farmers questioned, continued to sell eggs whilst their hens were on antibiotic treatment. the lack of knowledge about the withdrawal periods is greater than for farmers from tanzania (nonga el al. 2010), where 80% knew about the withdrawal period, but still sold eggs during this period. in the same way as the sudanese poultry farmers, the tanzanian farmers were unaware that antimicrobials in eggs have any detrimental effect on human beings. the problem was compounded further by a lack of quality control measures applied to egg products, such as cracked eggs, grading of eggs, cleaning of dirty eggs or fumigation of eggs. in sudan, 95% of farmers do not apply any quality control measures to eggs. conclusion top ↑ the census conducted in this study concluded that there was a gap in information in the layer industry in sudan and that this study provided more reliable baseline information than was previously available. there is still a need for more efficient census data for the poultry industry in sudan.the main reason for the high prevalence of antimicrobial residues in the layer industry was probably the lack of knowledge of farmers and producers about antimicrobial residues, their withdrawal periods, and the risk posed by the consumption of these residues. the study also concluded that traditional farming systems in sudan relied heavily on antimicrobial medication to control disease and almost half of the farms surveyed were treating their flocks with antimicrobials. in addition there was a lack of disease control programmes which probably resulted in a massive use of antibiotics to control endemic diseases. the situation was compounded further by the absence of governmental supervision and control on the use of drugs. consequently, it was concluded that a solution to the residue problem would be intensive extension and educational programmes on responsible and appropriate antibiotic use. this would include avoidance of certain antibiotics and the following of withdrawal periods, coupled with the government formulating simple regulations for the use of antibiotics and their withdrawal. furthermore, farmers should be educated on alternative methods of infectious disease management, such as vaccination, environmental sanitation and disease containment, which would decrease the use of antibiotics. acknowledgements top ↑ the authors would like to acknowledge the national research foundation (south africa), the university of pretoria (production animal studies department & tropical diseases department) and the islamic zakaat fund (malawi), for funding this study. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions this work was conducted by m.m.s. (national cooperative corporation) as part of his msc in epidemiology at the university of pretoria. m.m.s. (national cooperative corporation) was involved in all aspects of the project and carried out the fieldwork for the project and some of the laboratory analysis. the principle supervisor for this project was b.g. (university of pretoria) who was responsible for the design and management of the project and the guidance of m.m.s. (national cooperative corporation) in all aspects of the project, but particularly in the epidemiology. j.p. (university of pretoria) acted as a co-supervisor and provided assistance and inputs principally into the microbiological components of the project. all three of these authors contributed extensively to the writing of this article. s.b. (university of pretoria) acted as a co-supervisor to m.m.s. (national cooperative corporation) and as a poultry consultant. references top ↑ al-ghamdi, m., al-mustafa, z., el-morsy, f., al-faky, a., haider, i. & essa, h., 2000, ‘residues of tetracycline compounds in poultry products in the eastern province of saudi arabia’, public health 114, 300–304. http://dx.doi.org/10.1016/s0033-3506(00)00350-4babiker, m.a., tawfeig, a., yahia, i.e. & noura, k., 2009, ‘mortality and disease status in layer chicken flocks reared in traditional farms in khartoum, sudan’, international journal of poultry science 8, 264–269. http://dx.doi.org/10.3923/ijps.2009.264.269 kabir, j., umoh, v.j., audu-okoh, e., umoh, j.u. & kwaga, j.k.p., 2004, ‘veterinary drug use in poultry farms and determination of antimicrobial drug residues in commercial eggs and slaughtered chicken in kaduna state, nigeria’, food control 15, 99–105. http://dx.doi.org/10.1016/s0956-7135(03)00020-3 ministry of agriculture, animal resources and irrigation, khartoum state, sudan, 2005. internal publication. mitema, e.s., kikuvi, g.m., wegener, h.c. & stohr, k., 2001, ‘an assessment of antimicrobial consumption in food producing animals in kenya’, journal of veterinary pharmacology & therapeutics 24, 385–390. http://dx.doi.org/10.1046/j.1365-2885.2001.00360.x nonga, h.e., simon, c., karimuribo, e.d. & mdegela, r.h., 2010, ‘assessment of antimicrobial usage and residues in commercial chicken eggs from small holder poultry keepers in morogoro municipality, tanzania’, zoonoses and public health 5, 339–344. reinhardt, a.k., gautier-bouchardon, a.v., gicquel-bruneau, m., kobisch, m. & kempf, i., 2005, ‘persistence of mycoplasma gallisepticum in chickens after treatment with enrofloxacin without development of resistance’, veterinary microbiology 106, 129–137. http://dx.doi.org/10.1016/j.vetmic.2004.11.016, pmid:15737482 sirdar, m.m., 2010, ‘antibiotic residues in commercial layer hens in khartoum sate, sudan, 2007–2008’, master thesis, dept. production animal studies, university of pretoria. stolker, a.a. & brinkman, u.a., 2005, ‘analytical strategies for residue analysis of veterinary drugs and growth-promoting agents in food-producing animals–a review’, journal of chromatography a 1067, 15–53. http://dx.doi.org/10.1016/j.chroma.2005.02.037, pmid:15844509 thrusfield, m., 2005, veterinary epidemiology, 3rd edn., blackwell science ltd., oxford, united kingdom. footnotes top ↑ 1.http://www.cdc.gov/epiinfo/epiinfo.htm 2.epicalc 2000, version 1.02, joo gilman & mark myatt 1998, brixton books. 3.http://earth.google.com/download-earth.html 4.www.maplibrary.org/stacks/africa/sudan/khartoum/index.php article information authors: gordon j. gallivan1 andrea spickett2 heloise heyne2 arthur m. spickett2 ivan g. horak3,4 affiliations: 1187 cluny street, ottawa, canada 2parasites, vectors & vector-borne diseases programme, onderstepoort veterinary institute, south africa 3department of veterinary tropical diseases, university of pretoria, south africa 4department of zoology and entomology, university of the free state, south africa correspondence to: ivan horak email: ivan.horak@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 09 sept. 2010 accepted: 02 feb. 2011 published: 19 apr. 2011 how to cite this article: gallivan, g.j., spickett, a., heyne, h., spickett, a.m. & horak, i.g., 2011, ‘the dynamics of questing ticks collected for 164 consecutive months off the vegetation of two landscape zones in the kruger national park (1988–2002). part iii. the less commonly collected species’, onderstepoort journal of veterinary research 78(1): art. #41, 9 pages. doi:10.4102/ojvr.v78i1.41 copyright notice: © 2011. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) the dynamics of questing ticks collected for 164 consecutive months off the vegetation of two landscape zones in the kruger national park (1988–2002). part iii. the less commonly collected species in this original research... open access • abstract • introduction • materials and methods • results and discussion    • amblyomma marmoreum    • dermacentor rhinocerinus    • haemaphysalis elliptica    • hyalomma truncatum    • rhipicephalus evertsi evertsi    • rhipicephalus simus    • rhipicephalus turanicus • conclusion • acknowledgements • references abstract (back to top) despite many studies regarding tick ecology, limited information on long-term changes in tick populations exist. this study assessed the long-term population dynamics of the less frequently collected questing ixodid ticks in the kruger national park (knp). from 1988 to 2002, monthly dragging of the vegetation was performed in three habitats (grassland, woodland and gully) at two sites in the knp (nhlowa road, landscape zone 17, and skukuza, landscape zone 4). amblyomma marmoreum and rhipicephalus evertsi evertsi were collected as larvae most commonly. most a. marmoreum larvae were collected at skukuza and numbers peaked from march to july. more r. evertsi evertsi larvae were collected at nhlowa road and numbers peaked in summer and in winter, while at skukuza there was a single peak in spring. haemaphysalis elliptica, rhipicephalus simus and rhipicephalus turanicus were collected as adults most commonly. more ha. elliptica and r. turanicus were collected at nhlowa road than at skukuza, while r. simus numbers from the two sites were approximately equal. ha. elliptica were collected most often between february and june, and r. simus and r. turanicus during february and march. all three species were collected more frequently in gullies than in grassland or woodland. their numbers increased in 1994/1995 following an eruption of rodents, the preferred hosts of the immature stages. the different host-seeking strategies of ticks largely determine the development stage at which they are likely to be collected during vegetation dragging and reflect a complex interaction between ticks, their hosts and the environment. introduction (back to top) many studies have examined the association between ticks, their hosts and the environment, but most have been of relatively short duration (typically 1–2 years) and there is little information on longer-term changes in tick populations. in earlier publications (horak, gallivan & spickett 2011; spickett, gallivan & horak 2011) we described changes in the numbers of questing ticks and the population dynamics of the four major tick species, amblyomma hebraeum, rhipicephalus appendiculatus, rhipicephalus decoloratus and rhipicephalus zambeziensis. the questing ticks were collected monthly by dragging flannel strips over the vegetation at two sites in the kruger national park (knp) over a period of 164 months. in the present publication we describe changes in the numbers of the less commonly collected species, namely amblyomma marmoreum, dermacentor rhinocerinus, haemaphysalis elliptica, hyalomma truncatum, rhipicephalus evertsi evertsi, rhipicephalus simus and rhipicephalus turanicus. materials and methods (back to top) the knp is a large nature reserve of nearly 2 million ha in north-eastern south africa. five vegetation types (acocks 1988) and 35 landscape zones (gertenbach 1983) have been identified in the reserve. the study sites at skukuza and nhlowa road and the associated climate, methods of tick collection and statistical analyses have been described in detail by horak et al. (2011). the climate in the southern knp is described as tropical with summer rainfall. annual rainfall is measured from june to may; consequently, we have chosen june to represent the commencement of each year. briefly, questing ticks were collected monthly by drag-sampling the vegetation with flannel strips (spickett et al. 1992) at two sites in the knp, namely nhlowa road and skukuza. the nhlowa road site was in landscape zone 17, which has been described as a sclerocarya caffra – acacia nigrescens savanna, and the skukuza site in landscape zone 4 (thickets of the sabie and crocodile rivers). three drags, approximately 250 m long and 1 m wide, were made at a normal walking speed in each of three visually selected habitats (grassland, woodland and gully) at each site. in addition to the flannel strips, the operator responsible for dragging the strips also wore flannel leggings. because of the limited number of gullies at each site, the same gullies were usually sampled every month, whereas the sampling locations in grassland and woodland usually varied from month to month. after each drag the ticks on the flannel strips and the leggings were removed using sharp-pointed forceps and stored in 70% ethyl alcohol in internally labelled, plastic-stoppered glass vials for later identification and quantification. sampling commenced in august 1988 and continued in every habitat at the two localities until march 2002, except after heavy rainfall, during a bush fire or upon sighting elephants (loxodonta africana), african buffaloes (syncerus caffer) or lions (panthera leo) in the vicinity. the identification of the immature stages of ticks, particularly of the lesser-known species, can be a difficult and time-consuming exercise. the larvae and nymphs of a. marmoreum have been described by theiler and salisbury (1959), those of d. rhinocerinus by keirans (1993), of ha. elliptica by apanaskevich, horak and camicas (2007), of hy. truncatum by apanaskevich and horak (2008), and of the various rhipicephalus species by walker, keirans and horak (2000). results and discussion (back to top) annual rainfall (june–may) at the skukuza rest camp averaged 609 mm over the period of the study, with a range of 275 mm – 1122 mm (figure 1a). average annual rainfall at the lower sabie rest camp, the closest climate station to the nhlowa road site, was similar to that at skukuza. mean monthly maximum temperatures fluctuated around 33 °c in mid-summer, while minimums rarely fell below 5 °c in winter (figure 1b). figure 1: (a) annual rainfall (june-may) and (b) mean monthly maximum and minimum temperatures at skukuza and lower sabie , during the study period. the numbers of all species collected from the vegetation are summarised in table 1. four taxa could be identified only to a generic level, including 188 amblyomma sp. larvae, 104 haemaphysalis sp. larvae, a single ixodes sp. nymph and a single rhipicephalus pravus group larva. counts of specimens of these four taxa and those of the four major tick species, namely a. hebraeum, r. appendiculatus, r. decoloratus and r. zambeziensis are listed collectively as ‘ other species’ in the table. population dynamics of the four major tick species have been discussed in earlier publications (horak et al. 2011; spickett et al. 2011). except for ha. elliptica, r. simus and r. turanicus, of which adults were the most commonly collected stage, larvae were the most commonly collected stage of all these tick species. table 1: numbers of the less common tick species collected by dragging at the skukuza and nhlowa road sites in the kruger national park (august 1988–march 2002). amblyomma marmoreum leopard tortoises (geochelone pardalis), the largest tortoise species in south africa (branch 1998), are the preferred hosts of the adults of a. marmoreum and hence it is colloquially known as the south african tortoise tick (horak et al. 2006a). adult ticks are rarely found on hosts other than reptiles, but the larvae infest a wide range of hosts, including carnivores, herbivores, hares, birds and reptiles (horak et al. 2006a). amblyomma marmoreum larvae were collected more frequently at skukuza than at nhlowa road (p < 0.001) and accounted for 98.8% of the a. marmoreum specimens collected (table 1). the riverine thickets at skukuza probably afforded greater protection for younger tortoises against predators and this may explain why larvae were collected more frequently at skukuza than at nhlowa road. the collections were seasonal (p < 0.001), with most larvae collected between march and july. there was a definite peak in may at skukuza but not at nhlowa road (figure 2a). this corresponds to the seasonality of a. marmoreum larvae on greater kudus (tragelaphus strepsiceros), impalas (aepyceros melampus), scrub hares (lepus saxatilis) and helmeted guineafowls (numida meleagris) examined in the knp (horak et al. 1991; horak et al. 1992; horak et al. 1993; horak et al. 1995; horak et al. 2003), and countrywide on leopard tortoises (horak et al. 2006a). figure 2: (a) mean collections of amblyomma marmoreum larvae (mean ± 95% confidence interval) per month and (b) mean monthly collections per year at nhlowa road and skukuza. although there was considerable year-to-year variability, the numbers of a. marmoreum larvae collected each year appeared to decline from 1990 to 2001 (figure 2b). the size of the collections was not significantly correlated with annual rainfall or rainfall over the activity period (february–september). the numbers did not differ significantly among habitats (p = 0.20), but there was a trend towards more frequent collections in the gullies than in grassland and woodland (table 2). the slightly more frequent collections of a. marmoreum larvae in gullies could imply that tortoises prefer to browse on the more succulent vegetation usually found there. table 2: mean monthly tick collections [log10 (n + 1)] by habitat. dermacentor rhinocerinus adult d. rhinocerinus are found almost exclusively on black and white rhinoceroses (diceros bicornis and ceratotherium simum) (keirans 1993), of which nearly all in the southern knp are infested and on which the tick may occur in large numbers (knapp et al. 1997). adult ticks quest for their preferred hosts at a height of 1 m or more on thick-stemmed, tall grass species (horak, personal observations 2000). they are seldom picked up on flannel strips and only two adults were collected (table 1). because adult ticks have a very strong affinity to rhinoceroses they have not been collected from any of the numerous host species we examined in the knp. it is this affinity that probably makes the operator dragging the flannel strips and the strips themselves unattractive to the ticks. the rodent hosts of the immature stages have been detected only recently (horak & cohen 2001). in mthethomusha nature reserve (adjacent to the knp and where there are also white rhinoceroses) three rodent species, namely bushveld gerbils (tatera leucogaster), red veld rats (aethomys chrysophilus) and natal multimammate mice (mastomys natalensis) were found infested with the immature stages of d. rhinocerinus (horak & cohen 2001). the small size of these animals implies that the immature stages of d. rhinocerinus quest for hosts from the soil surface rather than the vegetation, and only seven larvae were collected from the flannel strips (table 1). no bushveld gerbils or red veld rats examined in the knp were infested (braack et al. 1996). haemaphysalis elliptica haemaphysalis elliptica adults were more commonly collected at nhlowa road than at skukuza (p = 0.001). landscape zone 17 is at the centre of lion activity in the knp (gertenbach 1983), which may account for the greater number of adult ticks in this area. there was a significant difference between the numbers of adults collected over years (p < 0.001). some were collected from 1988 to 1990, but few were collected from 1991 to 1993. the numbers then increased, with a marked peak in 2000 (figure 3a). the patterns were similar at both sites, but there was a significant year-by-site interaction (p < 0.001), because more were collected at skukuza in 1988 and 1989 while more were collected at nhlowa road in the other years. the collections of adult ticks were correlated with annual rainfall of the preceding year at both sites (r > 0.67; p < 0.01). a significant correlation with rainfall 2 years before was also observed at skukuza (p = 0.64; p < 0.05), and a positive but non-significant correlation with rainfall observed at nhlowa road (p = 0.48; p < 0.2). the dramatic increase in numbers in 1999 and 2000 followed 2 years of above-average rainfall in 1998 and 1999. following a rodent explosion in 1993 (horak, spickett & braack 2000b), after above-average annual rainfall in 1992, numbers peaked slightly at nhlowa road in 1994 and 1995. the increase in numbers of ha. elliptica adults around the turn of the century was probably also associated with an increase in rodent populations during a period of above-average rainfall. according to the time series assessment, the seasonality of ha. elliptica adults was not statistically significant, probably because of the small numbers collected in most years. however, an anova showed a significant difference between months (p < 0.001), with the largest numbers collected in late summer and autumn (february–june) and the smallest numbers in spring (september–november) (figure 3b). a similar pattern was observed at both sites. there are no data regarding the seasonality of adult ha. elliptica on carnivores in the knp, but in north-eastern kwazulu-natal, south-east of the knp, adult ticks on dogs in rural communities were found mostly from january to march or april (horak, emslie & spickett 2001). haemaphysalis elliptica larvae were more commonly collected at skukuza than at nhlowa road. most larvae were collected in 1989 and 1990, and only sporadically thereafter. the largest numbers were collected in september and november, and only two of the 60 were collected between january and april. nymphs were collected sporadically in the early 1990s and again towards the turn of the century. eight of the 10 nymphs were collected between july and october. the september–november and july–october peaks for collections of ha. elliptica larvae and nymphs, respectively, correspond to the seasonal peaks in ha. elliptica/spinulosa larval and nymphal infestations on red veld rats in the southern knp (braack et al. 1996). figure 3: (a) mean monthly collections of haemaphysalis elliptica adults from each site per year, (b) mean collections (± 95% confidence interval) per month from each site and (c) mean collections (± 95% confidence interval) from each habitat per year. as shown in table 2, the largest numbers of ha. elliptica adults were collected in the gullies, while fewest were collected in woodland (p < 0.01). this pattern was consistent over months and at both sites. however, there was a significant difference between years (p = 0.02), with the largest numbers collected in the gullies until the late 1990s figure 3c) followed by more frequent collections in grassland and woodland. eight of the ten nymphs were collected in the gullies. tall grasslands appear to support a higher diversity of small mammal populations (monadjem 1997), but many of the rodent hosts of the immature stages of ha. elliptica prefer sheltered, rocky areas (skinner & smithers 1990). during periods of below-average rainfall, when grass swards were shorter and ground cover was reduced, gullies were probably the preferred habitat of many of the rodent hosts. around the turn of the century the majority of ha. elliptica adults were collected in grassland and woodland. denser ground cover and taller grass swards following a period of higher rainfall would have provided a more suitable habitat for many rodent species, and an increase in rodent populations would have led to an expansion of habitat use. thus, the population dynamics and habitat distribution of questing ha. elliptica are probably determined by the effects of rainfall on the populations of rodent hosts of the immature stages, rather than the carnivore hosts of the adults. hyalomma truncatum although only 24 larvae and five adult hy. truncatum were collected from the vegetation (table 1), these low numbers should not be regarded as a reflection of actual abundance of this tick in the knp. during the first 6 years of this survey, five scrub hares were examined monthly around skukuza, and the 360 examinations during this time yielded a total of 36 647 larvae and 9896 nymphs of this two-host tick (horak et al. 1993; horak et al. 1995). adult hy. truncatum have been collected from the large browsers and grazers in the knp, including giraffes (giraffa camelopardalis), burchell’s zebras (equus burchellii), elands, (taurotragus oryx), white rhinoceroses and kudus, as well as warthogs (phacochoerus africanus) (horak, de vos & de klerk 1984; horak et al. 1988; horak et al. 1992; horak, golezardy & uys 2007; knapp et al. 1997). the discrepancy between the numbers collected by dragging and the numbers on scrub hares strongly suggests that hy. truncatum larvae quest for hosts from the soil surface. the adults definitely quest for hosts from the soil surface and can readily be seen scuttling along the ground towards any large potential host. rhipicephalus evertsi evertsi rhipicephalus evertsi evertsi is a two-host tick that infests a wide range of host species (walker et al. 2000). in the knp, burchell’s zebras and giraffes appear to be preferred hosts for all stages (horak et al. 1984; horak et al. 2007), while impalas and scrub hares are important hosts for the immature stages (horak et al. 1993; horak et al. 1995; horak et al. 2003). larvae were most commonly collected during dragging (table 1) and, theoretically, only larvae and adults should quest for hosts. the single nymph that was collected had probably become dislodged just before or after moulting and was questing for a host. very few adults were collected, probably because they quest for hosts from the soil surface or, like the adults of many species, they do not attach to flannel strips. in previous drag-sampling studies in landscape zones 4 and 17 there was no evidence of seasonality in the numbers of questing r. evertsi evertsi larvae collected (spickett et al. 1992; horak et al. 2006b). the present study, however, showed significant periodicity at both sites (p < 0.001). at nhlowa road there were two annual peaks (november– february and june–july), whereas at skukuza, there was only a single peak (november–december) with a nadir between may and june (figure 4a). based on the development times seen in the laboratory, r. evertsi evertsi can complete more than one life cycle annually at the prevailing temperatures in the knp (horak et al. 2003). at nhlowa road the summer and winter peaks correspond to those observed on burchell’s zebras (horak et al. 1984), suggesting that the seasonal pattern was related to the pattern of zebra migration. at skukuza, the late spring peak and late autumn nadir differed not only from the pattern observed in other drag-sampling studies but also from that observed on impalas and scrub hares in this landscape zone (horak et al. 1993; horak et al. 2003). the reasons for the differences between the seasonal patterns observed in the present study and previous studies are unknown. they may be related to seasonal differences in habitat use by ungulates within landscape zone 4, or they may be because of the relatively short duration of the sampling periods (1–4 years) in the earlier studies. in the present study there was considerable variability among years and the general seasonal pattern was seen only over the longer time period. figure 4: mean collections (± 95% confidence interval) of rhipicephalus evertsi evertsi larvae (a) by month and (b) by year at the two sites. the numbers of r. evertsi evertsi larvae collected differed significantly among years (p < 0.001) and there was a significant year-by-site interaction (p = 0.028), with proportionally greater changes at skukuza than at nhlowa road. however, the trends were similar at both sites, with peaks in the early 1990s, a decline through the mid-1990s, and subsequent increases in the late 1990s. numbers declined in 2000 (figure 4b). the numbers were not significantly correlated with seasonal rainfall at either site. the apparent differences in the annual trends at the two sites may be related to differences in the host communities. the populations of the larger ungulates at nhlowa road, such as zebras and giraffes, appear to be less sensitive to changes in rainfall (ogutu & owen-smith 2005) than the populations of smaller ungulates, such as impalas, at skukuza. larvae were more commonly collected at nhlowa road than at skukuza (p < 0.001) and there was a significant habitat-by-site interaction (p = 0.002). at nhlowa road, larvae were least frequently collected in the gullies and most commonly in the grassland (table 2), probably because of the large numbers of zebras and other grazers at this locality. at skukuza, r. evertsi evertsi larvae were most commonly collected in the woodland, probably because of the presence of large numbers of impalas and other browsers, but the difference among habitats was not statistically significant (p > 0.1). the differences among habitats were not significant among months or years (p > 0.4). rhipicephalus simus adults were the most commonly collected stage of r. simus. the preferred hosts of adult r. simus are large ruminants, such as african buffaloes, and monogastric animals, such as large carnivores, warthogs, zebras and rhinoceroses (walker et al. 2000). large numbers of r. simus adults have been collected from lions, leopards, warthogs, burchell’s zebras and white rhinoceroses in the knp (horak et al. 1984; horak et al. 1988; horak et al. 2000a; knapp et al. 1997). the collections of r. simus were seasonal, with a peak between february and march and a nadir between august and september (figure 5a). the numbers did not differ significantly between the two sites (p = 0.2) and the seasonal patterns were similar. the peak in adult numbers corresponds with that on burchell’s zebras and warthogs in the knp (horak et al. 1984; horak et al. 1988) and on warthogs in swaziland in a sclerocarya caffra – acacia nigrescens savanna (gallivan & surgeoner 1995). figure 5: mean collections (± 95% confidence interval) of rhipicephalus simus adults (a) by month and (b) by year at nhlowa road and skukuza. there was a significant difference between the numbers of r. simus adults collected over the years (p < 0.001). numbers declined from 1988 to 1991, then increased again in 1994, followed by a peak in 2000 (figure 5b). the trends differed significantly between the two sites (p < 0.001). at skukuza, numbers tended to be higher between 1988 and 1989 than from 1997 to 2000. however, at nhlowa road the numbers of r. simus adults were higher from 1993 to 1995, with a marked peak in 1994. the numbers of adult r. simus collected at skukuza were significantly correlated with annual rainfall during the 2 previous years (p < 0.05). at nhlowa road, the numbers were significantly correlated with rainfall 2 years before (p = 0.05), but not with that of the preceding year. the decline in numbers in the early 1990s corresponded to a period of decreasing annual rainfall and to a marked decline in the warthog population in the knp (ogutu & owen-smith 2005). the decline in annual rainfall probably also caused a decline in the populations of rodents, which are the preferred hosts of the immature stages of this tick (braack et al. 1996; horak et al. 2005; norval & mason 1981; petney et al. 2004). following a period of above-average rainfall in 1992, there was an eruption of rodent populations in 1993 (horak et al. 2000b) that may have accounted for the large number of adult r. simus collected at nhlowa road during 1994. as with the adults of ha. elliptica, the increase in the numbers of r. simus adults around the turn of the century was probably associated with an increase in rodent populations during a period of above-average rainfall. as shown in table 2, adult r. simus were most commonly collected in the gullies and least frequently in the grassland (p < 0.001). there was a significant difference in the habitat distribution between the two sites (p = 0.03). at skukuza, 48.3% of the ticks were collected in the gullies and 16.4% in grassland, whereas at nhlowa road, 69.2% were collected in the gullies and only 7.6% in grassland. habitat distribution differed significantly among months (p = 0.025), with the most pronounced difference during february and march, the period of peak activity. there was also a marginally significant difference in habitat distribution among years (p = 0.1), with highest numbers in the gullies; however, in 2000 the numbers were marginally higher in woodland. as in the case of ha. elliptica, the habitat distribution probably corresponds to the pattern of habitat use by the hosts of the immature stages. rhipicephalus simus larvae were more frequently collected at skukuza than at nhlowa road. almost half (45.8%) were collected in 1989, while the second largest collection (14.6%) was made in 2001. other collections were sporadic. at skukuza, most r. simus larvae were collected from january to june, but at nhlowa road most were collected from june to november. at skukuza, more larvae were collected in the gullies than in grassland, whereas at nhlowa road, most larvae were collected in woodland. rhipicephalus turanicus adult r. turanicus have been collected from lions, leopards, cheetahs, african wild dogs, feral cats and scrub hares in the knp, but none of the animals were heavily infested (horak et al. 1993; horak et al. 1995; horak et al. 2000a). no immature stages have been collected from animal hosts within the knp or adjacent nature reserves. scrub hares examined on farms around hluhluwe in north-eastern kwazulu-natal were infested with all stages of r. turanicus (horak et al. 1995) and crested francolins (francolinus sephaena) on a farm in limpopo were infested with larvae (uys & horak 2005). adult ticks were also common on dogs in maputo province, mozambique, adjacent to the knp (neves, afonso & horak 2004). rhipicephalus turanicus was most commonly collected at nhlowa road (97.8%) and adults were the most commonly collected stage (table 1). as mentioned before, landscape zone 17 is at the centre of lion distribution within the knp (gertenbach 1983) and the herds of large grazers would support populations of other carnivores. collections were seasonal (p < 0.001), with a peak between february and march (figure 6a). there were significant differences between the numbers collected among years (p = 0.002), with peaks in 1991, 1994, 1995, 1999, 2001 and 2002 (figure 6b). the numbers of r. turanicus adults were not correlated with rainfall and, as shown in table 2, ticks of this stage were collected in the gullies more often than in woodland (p < 0.001). this pattern was consistent among years and months. figure 6: (a) mean collections (± 95% confidence interval) of rhipicephalus turanicus adults by month at the two sites and (b) total collections by year at nhlowa road. there was a strong correlation (r = 0.7; p = 0.009) between the numbers of adult r. simus and adult r. turanicus collected annually at nhlowa road. the year-to-year patterns of the two species were similar and both species were most commonly collected in the gullies. however, woodland yielded the fewest r. turanicus adults, whereas the grassland yielded the fewest r. simus adults. the distribution of adult r. turanicus among habitats and year-to-year collection patterns were also similar to those of adult ha. elliptica at the nhlowa road site. conclusion (back to top) in order to determine the population dynamics of free-living ixodid ticks, questing ticks were collected monthly for 164 months from the vegetation at two localities in the knp. of the collected species, 14 could be identified to specific level. larvae of a. marmoreum and r. evertsi evertsi, and adults of ha. elliptica, r. simus and r. turanicus were collected in sufficient numbers to conclude that they quest for hosts from the vegetation. the host-seeking strategies of their remaining developmental stages and those of the tick species collected only in small numbers still need to be investigated. the value of long term sampling at more than one locality and within various habitats at each locality yielded several answers and posed a number of questions. should more samples be taken within the predominant habitat at a locality, for example in the thickets and woodlands of a thicket locality and in the grasslands of a savanna locality, to obtain more accurate results pertaining to an entire locality? what are the reasons for markedly more numerous questing ticks in a treed savanna locality compared with a locality composed largely of thickets? what value can be added by simultaneously sampling the major host species within a study site for ticks and determining host densities and daily and seasonal movements? finally, logistical challenges to what can be achieved in the field in a large nature reserve need to be considered, for example (1) the dangers posed to field workers by large predators, african buffaloes, rhinoceroses and elephants, (2) the number of drag samples that can be accomplished in a day to ensure reasonable homogeneity, as the total length of the drags placed end to end covered a distance of 2250 m at each locality, and (3) the number of people, besides an armed ranger, needed to collect, within a reasonable time frame, the thousands of ticks sometimes collected in a single drag. acknowledgements (back to top) we are indebted to the south african national parks (sanparks) for placing their staff and facilities in the knp at our disposal. we gratefully acknowledge the assistance of dr leo braack and mr andré potgieter with arranging the logistics for the collections. a number of nature conservation and veterinary students assisted with many of the field collections. our special thanks, however, are reserved for the sanparks staff at skukuza, who were our armed guards and assisted with the collection of the many thousands of ticks from the flannel strips. the research was funded by the university of pretoria (faculty of veterinary science), sanparks, bayer animal health, the university of the free state and the national research foundation. references (back to top) acocks, j.p.h., 1988, veld types of south africa with accompanying veld type map, 3rd edn., department of agriculture and water supply, pretoria. apanaskevich, d.a., horak, i.g. & camicas, j-l, 2007, ‘redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haemaphysalis (rhipistoma) leachi (audouin, 1826) (acarina, ixodidae)’, onderstepoort journal of veterinary research 74, 181−208. pmid:17933361 apanaskevich, d.a. & horak, i.g., 2008, ‘the genus hyalomma. vi. systematics of h. 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three largest wild ruminant species in southern africa’, onderstepoort journal of veterinary research 74, 231−242. pmid:17933365 horak, i.g., gallivan, g.j. & spickett, a.m., 2011, ‘the dynamics of questing ticks collected for 164 consecutive months off the vegetation of two landscape zones in the kruger national park (1988–2002). i. total ticks, amblyomma hebraeum and rhipicephalus decoloratus’, onderstepoort journal of veterinary research 78(1), art. #32, 10 pages, doi:10.4102/ojvr.v78i1.32 keirans, j.e., 1993, ‘dermacentor rhinocerinus (denny, 1843) (acari, ixodida, ixodidae), redescription of the male, female and nymph and first description of the larva’, onderstepoort journal of veterinary research 60, 59−68. pmid:8332318 knapp, s.e., krecek, r.c., horak, i.g. & penzhorn, b.l., 1997, ‘helminths and arthropods of black and white rhinoceroses in southern africa’, journal of wildlife diseases 33, 492−502. pmid:9249695 monadjem, a., 1997, ‘habitat preferences and biomasses of small mammals in swaziland’, african journal of ecology 35, 64−72. doi:10.1111/j.1365-2028.1997.042-89042.x neves, l., afonso, s. & horak, i.g., 2004, ‘ixodid ticks on dogs in and around maputo and elsewhere in mozambique’, onderstepoort journal of veterinary research 71, 279−283. pmid:15732454 norval, r.a.i. & mason, c.a., 1981, ‘the ticks of zimbabwe. ii. the life cycle, distribution and hosts of rhipicephalus simus koch, 1844’, zimbabwe veterinary journal 12, 2−9. ogutu, j.o. & owen-smith, n., 2005, ‘oscillations in large mammal populations: are they related to predation or rainfall?’, african journal of ecology 43, 332−339. doi:10.1111/j.1365-2028.2005.00587.x petney, t.n., horak, i.g., howell, d.j. & meyer, s., 2004, ‘striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks’, onderstepoort journal of veterinary research 71, 313−318. pmid:15732458 skinner, j.d. & smithers, r.h.n., 1990, mammals of the southern african subregion, university of pretoria, pretoria. spickett, a.m., horak, i.g., van niekerk, a. & braack, l.e.o., 1992, ‘the effect of veld-burning on the seasonal abundance of free-living ixodid ticks as determined by drag-sampling’, onderstepoort journal of veterinary research 59, 285−292. spickett, a.m., gallivan, g.j. & horak, i.g., 2011, ‘the dynamics of questing ticks collected for 164 consecutive months off the vegetation of two landscape zones in the kruger national park (1988–2002). part ii. rhipicephalus appendiculatus and rhipicephalus zambeziensis’, onderstepoort journal of veterinary research 78(1): art. #233, 9 pages. doi:10.4102/ojvr.v78i1.233, pmid:1297958 theiler, g. & salisbury, l.e., 1959, ‘ticks in the south african zoological survey collection – part ix – “the amblyomma marmoreum group”’, onderstepoort journal of veterinary research 28, 47−124. uys, a.c. & horak, i.g., 2005, ‘ticks on crested francolins and vegetation in limpopo province, south africa’, onderstepoort journal of veterinary research 72, 349−343. walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae), a guide to the brown ticks of the world, cambridge university press, cambridge. introduction various authors have documented the ixodid tick species infesting domestic animals and wildlife in south africa (theiler 1962; walker 1991; walker, keirans & horak 2000). furthermore, theiler and her co-workers, in a series of papers published in the 1940s and 1950s, have plotted the specific localities at which many of these ticks were collected and howell, walker & nevill (1978) have converted the latter into maps. however, with the exception of the various rhipicephalus spp. mapped by walker et al. (2000), the published geographic distributions of many species that occur in south africa are based on surveys conducted approximately 60 years ago (theiler 1962). the ticks infesting domestic and wild animals in mozambique have also been well documented (theiler 1943, 1962; tendeiro 1954; dias 263 onderstepoort journal of veterinary research, 76:263–276 (2009) species composition and geographic distribution of ticks infesting cattle, goats and dogs in a temperate and in a subtropical region of south-east africa i.g. horak1*, n. nyangiwe2, c. de matos3 and l. neves4 abstract horak, i.g., nyangiwe, n., de matos, c. & neves, l. 2009. species composition and geographic distribution of ticks infesting cattle, goats and dogs in a temperate and in a subtropical region of south-east africa. onderstepoort journal of veterinary research, 76:263–276 the species and distribution of ticks infesting cattle, goats and dogs in the eastern region of the eastern cape province, south africa and maputo province, mozambique were determined from collections made from these animals at 72 localities in the former region and 30 in the latter. eleven ixodid and one argasid species were recovered in the eastern cape province and 15 ixodid species in maputo province. the most common ticks infesting cattle and goats in both provinces were am blyomma hebraeum, rhipicephalus (boophilus) microplus, rhipicephalus appendiculatus and rhipi cephalus evertsi evertsi. the dominant species on dogs were haemaphysalis elliptica and rhipi cephalus si­ mus. the geographic distributions of the major species and some of the minor species in both regions were plotted. the partial or complete displacement of the indigenous tick rhipicephalus (boophilus) decoloratus by the introduced species r. (b.) microplus was a major feature of both surveys. keywords: cattle, dogs, eastern cape province, geographic distribution, goats, ixodid ticks, maputo province, mozambique, south africa * author to whom correspondence is to be directed. e-mail: ivan.horak@up.ac.za 1 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa; and department of zoology and entomology, university of the free state, p.o. box 339, bloem fontein, 9300 south africa 2 döhne agricultural development institute, private bag x15, stutterheim, 4930 south africa, and department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 3 direcção de ciências animais, instituto de investigação agrária de mozambique, c.p. 1922, maputo, mozambique; and dep artment of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 4 faculdade de veterinária, universidade eduardo mondlane, c.p. 257, maputo, mozambique accepted for publication 19 february 2009—editor 264 ticks infesting cattle, goats and dogs in south-east africa 1993), but their geographic distributions are largely unknown. some of the more recent data for the eastern cape province is that of baker (1982), who summarized the results of acaricide resistance studies he and his co-workers had conducted, and plotted the localities in the eastern region of the eastern cape province at which resistant strains of various tick spe cies had been collected. latterly nyangiwe, horak & bryson (2006) collected ticks from dogs at 40 localities in this region and plotted the distributions of four of the eight species that they had recovered. in maputo province neves, afonso & horak (2004) and de matos, sitoe, neves, bryson & horak (2008) collected ten species of ticks from dogs and plotted the distribution of two of these. to our knowledge no other recent data are available for these regions. walker (1991) listed 77 species of ixodid ticks that occur in south africa, and two new species, rhip i­ cephalus oreotragi and rhipicephalus warburtoni have subsequently been added to the list (walker et al. 2000). thirty-seven of these ticks have been recorded on domestic animals. the names of two of these 37 species, namely haemaphysalis leachi and hyalomma marginatum turanicum, neither of which occur in south africa, have been reinstated as haemaphysalis elliptica and hyalomma glabrum, both of which are valid indigenous species (apanaskevich & horak 2006; apanaskevich, horak & camicas 2007). a third species, hyalomma margi­ na tum rufipes has recently been raised to species level, namely hyalomma rufipes (apanaskevich & horak 2008). dias (1993) recorded 59 ixodid tick species in mozambique, and listed 26 of these as parasitizing domestic animals. of the latter 26 species ambly­ omma theilerae has been synonymized with ambly­ omma hebraeum (camicas, hervy, adam & morel 1998), haemaphysalis humerosoides is considered a synonym of haemaphysalis leachi (camicas et al. 1998), and rhipicephalus reichenowi is considered a synonym of rhipicephalus planus (walker et al. 2000). we now also know that the tick dias identified as rhipicephalus capensis does not occur in mozambique and, although he listed rhipicephalus sanguineus from cattle, in our opinion the latter tick is more likely to be rhipicephalus turanicus. the recent surveys of neves et al. (2004) and de matos et al. (2008) have added h. elliptica, haemaphysalis spinulosa, ixodes cavipalpus, a rhipicephalus sp. (near r. pravus) and r. turanicus to the species listed by dias (1993). several of dias’ studies on the ticks of mozambique commenced more than 50 years ago and although he probably listed nearly all the species that occur there, he unfortunately did not always record the localities at which particular species were collected. furthermore, the civil war that ended during the 1990s and its consequences for humans, livestock and wildlife, plus the livestock restocking programme that followed, have probably all had an effect on the species composition and geographic distribution of ticks in mozambique. thus with the exception of the surveys conducted by neves et al. (1994) and de matos et al. (2008), there is little current information on the ticks that occur there. the present studies were undertaken in an attempt to rectify this situation for both the eastern region of the eastern cape province, south africa and for ma puto province, the most southerly of the ten provinces of mozambique. at the same time this provided the opportunity to compare the species composition of ticks infesting cattle, goats and dogs in a temperate region with that of ticks infesting the same host species in a subtropical region, both with long coastlines. materials and methods localities during 2004 and 2005 ticks were collected from cattle, goats, dogs and the vegetation at 72 localities in the eastern region of the eastern cape province, south africa. this region lies between 30°22’ and 32°39’ s, and 27°45’ and 29°55’ e, and the terrain rises from sea level in the south to approximately 2 000 m in the north-east in the drakensberg mountain range. its inland climate is classified as that of plateau slopes, and its approximately 250 km long indian ocean seaboard has a subtropical coastal climate. summer rainfall varies between 500 and 1 000 mm annually, and winter rainfall between 125 and 375 mm. midsummer mean daily temperatures vary between 15.0 °c and 25.0 °c and those of midwinter between 7.5 °c and 17.5 °c. the vegetation along the coast is described as coastal forest and thornveld. adjacent to the latter in the west lies a strip of vegetation described as eastern province thornveld and in the east a strip described as ngongoni veld. with the exception of ribbons of valley bushveld along rivers courses, the inland vegetation, including that in the mountainous north, is described as highland sourveld (acocks 1988). in 2004, 2005 and 2006 ticks were also collected at 30 localities spread throughout maputo province, 265 i.g. horak et al. mozambique, from the same host species as in the eastern cape province, and from the vegetation. the southern boundary of this province is approximately 500 km north-east of the northern limit of the eastern cape province survey region. maputo province is located between 24°15’ and 26°52’ s, and 31°48’ and 33°05’ e, and has a climate classified as subtropical lowveld. its coastline is approximately 320 km long, and the landscape rises from the indian ocean in the east to about 800 m above sea level in the lebombo mountain range in the west. annual summer rainfall varies between 500 and 750 mm and winter rainfall between 125 and 250 mm. midsummer mean daily temperatures vary between 25.0 °c and 27.5 °c and midwinter temperatures between 15.0 °c and 20.0 °c. the vegetation along the coast is described as shrubland with patches of wetland, mangrove and deciduous trees along riverbanks and dunes. inland it is dominated by woodland and associations of savannah and mopane (colophospermum mopane) in the north-west district of magude. the inland vegetation in the south is dominated by forest from the mountain district of namaacha to the southern mozambican border. animals whenever possible, ticks were collected from five approximately year-old cattle, five goats and five dogs at each of the sampling sites (nyangiwe et al. 2006; nyangiwe & horak 2007; de matos et al. 2008). however, the prescribed numbers of animals at the stipulated number of localities were not always realised. in addition, ticks were collected from 24 dogs at two villages in south-west maputo province during a rabies inoculation campaign, as well as from two dogs at two villages in the south-east of the province (de matos et al. 2008). the geographic coordinates of each site at which ticks were collected were recorded and used to plot the distributions of the various tick species. tick collections ticks were collected from the ears, bodies, bellies, feet, tails and peri-anal regions of the cattle and goats on the single occasion that each locality was visited. to minimize stress to the dogs as well as injuries to the researchers, they were often muzzled and thereafter restrained by their owners. the collections of ticks from the animals were not intended to be exhaustive, and focused mainly on adult ticks, but several nymphs of a. hebraeum and rhipiceph alus appendiculatus were also collected. in addition, quest ing ticks were collected by dragging flannel strips over the vegetation as described by horak, gallivan, spickett & potgieter (2006), for a distance of 100 m at three places close to each of the collection sites. the ticks recovered from the animals and the flannel strips were placed in 70 % ethyl alcohol in internally labelled vials and transported to laboratories in south africa or maputo, where they were identified and counted using stereoscopic microscopes. results and discussion a total of 11 ixodid tick species and one argasid spe cies were recovered in the eastern region of the eastern cape province (table 1), and 15 ixodid tick species in maputo province (table 2). amblyomma hebraeum, rhipicephalus (boophilus) microplus, r. appendiculatus and rhipicephalus evertsi evertsi were most prevalent on cattle, goats and the vegetation, and h. elliptica on dogs in both provinces (tables1 and 2). furthermore, a large number of cattle and dogs in maputo province were infested with rhipicephalus simus (table 2). amblyomma hebraeum this tick was present mainly in the coastal region of the eastern part of the eastern cape province, where the vegetation is described as coastal forest and thornveld interspersed with valley bushveld (acocks 1988). however, a considerable number of collections were also made further inland (fig. 1a). with the above mentioned exception, the distribution of a. hebraeum in the survey region in the eastern cape province is similar to that plotted for it by howell et al. (1978). estrada-peña, horak & petney (2008) noted that a trend towards more intense periods of drought in the highveld of zimbabwe favoured the expansion of a. hebraeum in this region of that country and that temperature did not have an effect on this expansion. a similar phenomenon could be responsible for the spread of this tick into the higher lying areas of the eastern cape province. amblyomma hebraeum was widespread throughout maputo province (fig. 1b). this widespread distribution complements its extensive distribution in south africa to the south and west of this province as illustrated by howell et al. (1978) and walker & olwage (1987). haemaphysalis elliptica when the surveys of neves et al. (2004) and nyangiwe et al. (2006) on the ticks of dogs in mozambique 266 ticks infesting cattle, goats and dogs in south-east africa t a b l e 1 s p e ci e s a n d t o ta l n u m b e r o f tic ks c o lle ct e d f ro m c a tt le , g o a ts , d o g s a n d t h e v e g e ta tio n in t h e e a st e rn r e g io n o f th e e a st e rn c a p e p ro vi n ce , s o u th a fr ic a t ic k sp ec ie s t o ta l n o . o f ti ck s co lle ct ed n o . o f ti ck s co lle ct ed f ro m c at tl e, g o at s, d o g s an d t h e ve g et at io n a n d n u m b er o f th es e in fe st ed c at tl e (n = 3 60 ) g o at s (n = 3 60 ) d o g s (n = 2 00 ) v eg et at io n ( n = 7 2) t ic ks n o . in fe st ed t ic ks n o . in fe st ed t ic ks n o . in fe st ed t ic ks l o ca lit ie s p o si ti ve a m bl yo m m a he br ae um 7 3 4 5 9 2 1 3 8 6 8 6 1 5 6 1 9 1 8 2 h ae m ap hy sa lis e lli pt ic a 2 0 1 – – – – 1 9 3 9 3 8 5 h ae m ap hy sa lis s pi nu lo sa 1 – – – – 1 1 – – h ya lo m m a ru fip es 3 2 3 0 2 1 2 2 – – – – ix od es p ilo su s g ro u p 3 – – 1 1 2 2 – – r hi pi ce ph al us ( b oo ph ilu s) d ec ol or at us 1 6 5 2 5 6 2 1 – – 1 3 8 2 5 r hi pi ce ph al us ( b oo ph ilu s) m ic ro pl us 2 2 8 9 9 6 8 2 4 2 2 3 6 1 1 3 – – 1 0 8 5 6 3 r hi pi ce ph al us a pp en di cu la tu s 4 8 1 7 2 1 5 9 2 7 1 1 9 2 4 2 9 6 2 9 1 9 7 0 5 4 3 r hi pi ce ph al us e ve rt si e ve rt si 5 3 4 2 1 9 5 7 3 1 6 2 2 5 1 3 3 4 2 2 1 1 3 2 6 6 r hi pi ce ph al us s an gu in eu s 2 5 – – – – 2 5 7 – – r hi pi ce ph al us s im us 5 4 – – – – 5 4 2 9 – – o to bi us m eg ni ni 4 4 2 – – – – – – t o ta l 13 6 67 5 73 5 – 4 48 4 – 36 2 – 3 08 6 – t a b l e 2 s p e ci e s a n d t o ta l n u m b e r o f tic ks c o lle ct e d f ro m c a tt le , g o a ts , d o g s a n d t h e v e g e ta tio n in m a p u to p ro vi n ce , m o za m b iq u e t ic k sp ec ie s t o ta l n o . o f ti ck s co lle ct ed n u m b er o f ti ck s co lle ct ed f ro m c at tl e, g o at s, d o g s an d t h e ve g et at io n a n d n u m b er o f th es e in fe st ed c at tl e (n = 1 45 ) g o at s (n = 1 29 ) d o g s (n = 1 32 ) v eg et at io n ( n = 2 3) t ic ks n o . in fe st ed t ic ks n o . in fe st ed t ic ks n o . in fe st ed t ic ks l o ca lit ie s p o si ti ve a m bl yo m m a he br ae um 3 1 2 8 1 5 8 8 1 3 6 1 1 3 0 1 1 8 2 1 0 3 3 2 0 0 1 0 h ae m ap hy sa lis e lli pt ic a 4 4 9 – – – – 4 4 7 6 2 2 1 h ae m ap hy sa lis s p . 7 – – – – 7 6 – – h ya lo m m a ru fip es 1 1 1 – – – – – – ix od es c av ip al pu s 1 1 1 1 7 4 3 1 – – r hi pi ce ph al us ( b oo ph ilu s) m ic ro pl us 7 4 8 6 6 7 9 0 1 2 9 3 5 1 4 3 – – 3 4 5 1 7 r hi pi ce ph al us a pp en di cu la tu s 1 9 8 9 1 2 2 2 9 5 5 1 4 4 7 8 6 2 4 5 1 3 r hi pi ce ph al us e ve rt si e ve rt si 1 1 2 9 5 9 3 1 0 3 5 1 3 8 3 4 3 1 9 9 r hi pi ce ph al us k oc hi 3 0 9 5 2 1 8 – – – – r hi pi ce ph al us lo ng us 4 1 1 3 1 – – – – r hi pi ce ph al us p ra vu s g ro u p 1 2 8 3 2 2 2 2 – – r hi pi ce ph al us s an gu in eu s 1 5 3 – – – – 1 5 3 1 7 – – r hi pi ce ph al us s im us 7 1 2 3 3 6 1 0 2 2 7 1 6 3 4 6 9 8 3 3 r hi pi ce ph al us tr ic us pi s 3 – – 3 1 – – – – r hi pi ce ph al us tu ra ni cu s 7 8 9 7 1 1 6 8 1 8 – – t o ta l 15 1 92 10 5 58 – 2 57 2 – 1 24 8 – 81 4 – 267 i.g. horak et al. fig. 1 the geographic distribution of ambly om­ ma hebraeum in (a) the eastern region of the eastern cape province, south af ri ca, and (b) maputo prov ince, mo zam bique and in the eastern cape province were published this tick was referred to as h. leachi. it has subsequently been re-established as an old taxon, namely h. elliptica, originally described by koch in 1844 from the cape of good hope (apanaskevich et al. 2007). nyangiwe et al. (2006) and de matos et al. (2008) have plotted the geographic distribution of h. ellip­ tica in the eastern region of the eastern cape province and in maputo province and these maps will not be repeated here. although collections were made at only 20 of the 40 localities at which dogs were examined in the eastern cape province, these were spread nearly throughout the survey region, leading us to believe that the tick is as widespread in this region as illustrated by howell et al. (1978). in maputo province it was collected at 26 of the 27 local ities at which dogs were examined. norval (1984) has suggested that wherever there are dogs and adequate numbers of rodents to act as hosts for its immature stages, h. leachi (h. elliptica) is likely to be present. the distribution map of howell et al. (1978) portrays a more extensive distribution for h. leachi (h. elliptica) in the east of south africa, adjacent to mozambique, than in the west of the former country. the widespread distribution of h. ellip tica in maputo province thus supplements the already extensive distribution of this tick in the southeast of the sub-continent. a n !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !(!( !( !( !( !(!(!( !( !( !( !(!( !( !( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede amblyomma hebraeum !( b n 268 ticks infesting cattle, goats and dogs in south-east africa hyalomma spp. hyalomma rufipes was present at 16 localities in the eastern cape province (fig. 2), and its distribution in this region is similar to that mapped for it by ho well et al. (1978). walker (1991) states that h. ru fipes is absent in many of the moister eastern regions of south africa. its presence in the eastern cape survey region may thus reflect a trend towards slightly drier conditions in this area. hyalomma rufipes was collected at only one locality in maputo province. this is not surprising, as its distribution as illustrated by howell et al. (1978), shows that it is not present in north-east kwazulu-natal or in the kruger national park, south africa, immediately to the south and to the west of maputo prov ince. furthermore, dias (1993) states that the distribution of h. rufipes is restricted to the drier regions of some of the more northerly provinces of mozam bique, namely inhambane, sofala, tete and niassa. no hyalomma truncatum were collected in either of the survey regions. the north-eastern limits of this tick’s distribution in south africa appear to coincide with the lebombo mountain range that lies between the kruger national park in the west and maputo province in the east (howell et al. 1978). during the cooler months of the year nearly every scrub hare, lepus saxatilis, in the kruger national park is infested with the immature stages of h. truncatum (horak, spickett, braack & penzhorn 1993), and most giraffes, giraffa camelopardalis, with adults throughout the year (horak, golezardy & uys 2007). ixodes spp. three ticks belonging to the ixodes pilosus group of species were recovered at three sampling sites in the eastern cape province, one from a goat and one each from two dogs. these collection localities lie within the distribution range mapped for this group of ticks by howell et al. (1978). eleven ixodes cavipalpus were collected from a calf, four goats and a dog at a single locality in maputo province. although dias (1993) listed five species of ixodes that occur in mozambique, he did not include i. ca­ vipalpus amongst these. according to walker (1991) the latter tick is possibly the most widely distributed ixodes sp. in the afrotropical region, and has been collected from a wide variety of hosts (theiler 1962). rhipicephalus (boophilus) spp. rhipicephalus (b.) decoloratus was collected at 29 of the 72 survey localities in the eastern cape province (fig. 3a), compared to not one of the 30 localities in maputo province. conversely r. (b.) micro­ plus was present at nearly every sampling locality in both provinces (fig. 3b, c). howell et al. (1978) mapped the distributions of both these ticks in south africa. at that time r. (b.) decoloratus was widespread in the eastern region of the eastern cape province, whereas the distribution of r. (b.) microplus was restricted to coastal pockets. using their own more recently obtained data, baker, jordaan & robertson (1981) and baker (1982) illustrated a considerably more extensive fig. 2 the geographic distribution of hy a lom ma rufipes in the eastern region of the eastern cape province, south africa n 269 i.g. horak et al. fig. 3 the geographic distribution of rhipi cephalus (bo oph i lus) decolor atus in (a) the eastern region of the eastern cape province, south africa, and of rhipicephalus (bo oph ilus mi­ cro plus in (b) the eastern region of the eastern cape province and (c) maputo prov ince !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !(!(!( !( !( !(!( !( !( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede rhipicephalus (boophilus) microplus !( c a n b n n 270 ticks infesting cattle, goats and dogs in south-east africa distribution for the latter tick in this region. the results of the present survey indicate that r. (b.) mi­ croplus has further extended its range, and that the 1978 distribution pattern of the two ticks has now been reversed. in the present study a single nymph and 26 adult r. (b.) decoloratus were collected from six cattle and a goat at four localities in the eastern cape province, and questing larvae from the vegetation at 25 localities. in contrast 16 nymphs and 1 288 adult r. (b.) microplus were collected from 242 cattle and 113 goats at 69 of the 72 localities surveyed, and 1 085 larvae from the vegetation at 62 localities in this province. at the time dias (1993) compiled his lists of the ticks of mozambique he stated that r. (b.) decoloratus was common wherever there were cattle, in all regions of the country, and that it was also present on several wild mammals. in support of this observation greater kudus, tragelaphus strepsiceros, and impalas, aepyceros melampus, in the kruger na tional park, south africa, to the west of maputo prov ince, harbour very large numbers of r. (b.) decoloratus and no r. (b.) microplus (horak, boom ker, spickett & de vos 1992; horak, gallivan, braack, boomker & de vos 2003). this implies that in earlier times wildlife and probably also domestic cattle in maputo province were very likely to have been infested with r. (b.) decoloratus. this is no longer the case for domestic stock, as it appears as if the latter tick has now been displaced by r. (b.) microplus. dis placement has also been reported in zimbabwe (mason & norval 1980), the eastern province of zambia (berkvens, geysen, chaka, madder & brandt 1998) and tanzania (lynen, zeman, bakuname, di giulio, mtui, sanka & jongejan 2008). the reasons for this displacement are numerous. the total length of the life cycle of r. (b.) microplus is slightly shorter than that of r. (b.) decoloratus (arthur & londt 1973; londt & arthur 1975), and female r. (b.) microplus produce slightly more eggs than those of r. (b.) decoloratus (spickett & malan 1978). its spread may thus be enhanced by its slightly shorter life cycle and slightly higher egg production. moreover, cross mating between the two spe cies results in sterile eggs being produced (spickett & malan 1978), and although male ticks prefer to mate with conspecific females (norval & sutherst 1986), they will also mate with females of the other species (spickett & malan 1978). the males of r. (b.) microplus are sexually mature a few days sooner than those of r. (b.) decoloratus (londt & arthur 1975), and thus in mixed infestations they have a greater chance of mating with females of their own species. furthermore, if the sex ratio of male to female r. (b.) microplus is similar to that of r. (b.) decoloratus on naturally infested hosts, viz. approximately 2:1 (horak et al. 1992, 2003), the excess numbers of r. (b.) microplus males could mate with r. (b.) decoloratus females, rather than the converse happening. as females apparently mate only once, the cross-mated females would produce sterile eggs and r. (b.) microplus would consequently constitute an ever-increasing proportion of future mixed populations of the two ticks. fig. 4 the geographic distribution of rhip i cephalus append iculatus in (a) the eastern region of the eastern cape province, south africa and (b) maputo province, mo zambique, and of rhipi cephalus evertsi evertsi in (c) the east ern region of the eastern cape province and (d) maputo province a n 271 i.g. horak et al. fig. 4 (cont.) !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede rhipicephalus appendiculatus !( b c !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !( !(!(!( !( !( !(!( !( !( !( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede rhipicephalus evertsi evertsi !( d n n n 272 ticks infesting cattle, goats and dogs in south-east africa in southern mozambique this displacement has been augmented by the decimation of livestock and wildlife during the war of independence and the civil strife that followed. this alone possibly led to the near extinction of r. (b.) decoloratus in this region. when restocking with cattle commenced after the war, r. (b.) microplus was probably introduced or reintroduced on these animals, whereas few of the larger wildlife species, and with them r. (b.) dec ol­ oratus, have returned to maputo province. rhipicephalus appendiculatus this tick was collected at 70 of the 72 collection localities in the eastern region of the eastern cape province (fig. 4a) and, with the exception of the most southerly region of maputo province, where it was collected at only one locality, it was also widespread throughout maputo province (fig. 4b). its distribution in south africa, as illustrated by walker et al. (2000), indicates that it is absent, or only sparsely present, in north-east kwazulu-natal, where this province abuts on the southern border of maputo province. the tick’s scant distribution in north-east south africa thus supports its sparseness or absence in the south of maputo province. its widespread presence in the remainder of maputo province adds this region to its already widespread distribution in south-east africa. rhipicephalus evertsi evertsi rhipicephalus evertsi evertsi is widespread throughout both survey regions (fig. 4c, d), confirming its position as one of the most widely distributed rhipi­ cephalus species in sub-saharan africa (walker et al. 2000). although domestic and wild equids are probably the preferred hosts of all stages of development of this tick (norval 1981), cattle, goats and sheep are also good hosts, while dogs are seldom infested (walker et al. 2000). this pattern of host infestation is also apparent in the present surveys (tables 1, 2). the tick distribution maps published by walker et al. (2000) for rhipicephalus spp. in sub-saharan afri ca, illustrate the widespread distribution of r. e. evertsi in the eastern half of south africa, but do not include a single site in southern mozambique. this we are sure is due to a lack of reportage and not actual absence. its presently recorded widespread distribution in maputo province (fig. 4d), complements its distribution in the adjacent regions of south africa (walker et al. 2000). rhipicephalus kochi this tick has a very limited distribution in south africa. it is present only in the far north-east regions of kwazulu-natal and limpopo provinces where these border on mozambique (walker et al. 2000), and it is thus usually only encountered on wild animals in south africa. it was collected from cattle and goats in maputo province within the distribution range recorded for it in this region by walker et al. (2000). rhipicephalus pravus group this group of ticks comprises several apparently closely related species. all stages of development of some of these have been illustrated and described (walker et al. 2000), whereas others remain innominate and undescribed. one of the latter ticks has been designated rhipi­ cephalus sp. (near pravus) and its distribution stretches across southern africa from northern namibia in the west, through botswana and northern south africa to southern mozambique in the east (walker et al. 2000), while the distribution of another, designated rhipicephalus sp. (near punctatus) extends from south-western angola in the west, through zambia and zimbabwe to northern mozambique in the east (walker et al. 2000). the tick collected in maputo province resembles the rhipicephalus sp. (near pravus) entity, and its distribution corresponds with that of the latter tick as illustrated by walker et al. (2000). adult ticks of this species infest a variety of domestic and wild hosts, including hares, and the immature stages infest elephant shrews and hares (walker et al. 2000). in the present survey three cattle, two goats and two dogs were infested with adult ticks (table 2). rhipicephalus sanguineus all stages of development of r. sanguineus are nearly exclusively parasites of domestic dogs (walker et al. 2000), and adult ticks were collected only from dogs in both survey regions, at two localities in the eastern cape province and at eight in maputo province (fig. 5a). the free-living stages of this tick’s life cycle have adapted to the environment in and around the resting places of dogs in man-made structures such as sheds, dog kennels and human dwellings, whence they quest for their hosts (horak 1982; walker et al. 2000). its presence on dogs at ten of the rural sampling localities in the present surveys suggests that these animals were, at least for some 273 i.g. horak et al. fig. 5 geographic distribution of (a) rhipi cephalus sanguineus, (b) rhipiceph alus simus, and (c) rhipi ceph alus tura nicus in maputo province, mo zam bique !( !( !( !(!( !( !( !(!( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede rhipicephalus sanguineus !( a n !( !( !( !( !( !( !( !( !( !( !( !( !( !( !(!( !( !( !( !(!(!( !( !( !( !(!( !( !( !( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede rhipicephalus simus !( b n !( !( !( !( !( !( !(!(!( !( !( !( !( !( !( !( !( !( !( !( mozambique south africa swaziland matola maputo bella vista moamba sede magude sede naamacha sede marracuene sede rhipicephalus turanicus !( c n 274 ticks infesting cattle, goats and dogs in south-east africa part of the day, chained, or caged at the homes of their owners, or that they had a preferred resting place in or next to these homes. rhipicephalus simus nyangiwe et al. (2006) and de matos et al. (2008) have mapped the geographic distribution of r. si­ mus in the eastern region of the eastern cape province and in maputo province based on collections from dogs. although r. simus is widespread in the moister regions of southern africa, adult ticks are seldom collected in large numbers (walker et al. 2000). in the eastern region of the eastern cape province it was collected only from dogs, and then only from 14 of the 53 localities at which dogs were examined. its absence on cattle in the eastern cape at the same localities at which infested dogs were present is difficult to explain. in contrast slightly more cattle than dogs were infested in maputo prov ince, where r. simus was widespread and present at 24 of the 30 localities sampled (fig. 5b). the immature stages of this tick are reliant on rodents as hosts (petney, horak, howell & meyer 2004), and horak, spickett & braack (2000) have recorded a significant increase in the numbers of questing adult r. simus on the vegetation in the southern kruger national park in the year following a rodent explosion at the same locality. the small number of hosts infested with adult r. simus in the eastern cape province compared with the large number in maputo province could possibly reflect differences in both the overall abundance and species richness of the rodent fauna in the two regions. rhipicephalus turanicus the inability to distinguish this tick from r. san­ guineus has created many problems as to their ident ities, host preferences, life cycles and geographic distributions. the studies of pegram, clif ford, walker & keirans, (1987a) and pegram, keirans, clifford & walker (1987b) have addressed this problem, but have, as yet, not solved it. this is probably mainly due to the widespread distributions of both of these ticks and with these the inevitable geographic variability in their morphology. in her review of the ixodid ticks of southern africa walker (1991) reported that r. turanicus was present in scattered localities in transvaal province, south africa (a province that has now been divided into the four northern provinces of this country) and from bergville in kwazulunatal province. the newfound ability to identify this tick with greater certainty resulted in walker et al. (2000) recording it at 11 localities in south africa 9 years later. it is now frequently collected in south africa from dogs, hares and larger birds and also questing from the vegetation, but has as yet not been found in the eastern cape province. a total of 78 adult r. turanicus was collected in maputo province, mainly from dogs, but cattle and a goat were also infested (table 2). it was particularly prevalent in the central region of maputo province, but collections were also made in the far south and north of the province (fig. 5c). questing adult ticks have been collected from the vegetation in the southern kruger national park, south africa, to the west of maputo province, where they are most abundant during the late summer and autumn (horak et al. 2000). as with r. simus the immature stages of this tick are reliant on rodents as hosts (walker et al. 2000), and horak et al. (2000) recorded an increase in the numbers of questing adult ticks on the vegetation in the kruger national park in the year after a rodent explosion. we assume that dias (1993) did not recognize r. turani­ cus as a separate species and included it with r. sanguineus in his list of ticks for mozambique. rhipicephalus spp. a number of unconfirmed locality records for rhip i­ ceph alus longus exist in central and northern mozam bique (walker et al. 2000), and the present collection is the furthest south to date. rhipicephalus tricuspis has been reported in the northern provinces of south africa and in central and northern mozambique, with a single record just north of maputo province (walker et al. 2000). three ticks were collected from a single goat in maputo province in the present survey (table 2). otobius megnini a single calf, at each of two localities in the eastern region of the eastern cape province, was infested with the nymphs of the argasid tick, otobius megnini (table 1). no argasid ticks were collected in maputo province. acknowledgements we thank the various cattle, goat and dog owners for permission to collect ticks from their animals and for their assistance with these collections. our sincere thanks go to messrs k. kogina, b. zenani and z.m. nyangiwe, who assisted with the tick collec275 i.g. horak et al. tions in the eastern cape province, and mr c. sitoe for his assistance with the collections in maputo province. we are most grateful to the directorates of agriculture, eastern cape province and of animal science, mozambique, for permitting n. nyangiwe and c. de matos to conduct the eastern cape and mozambican segments of the study as part of their official duties. dr roy williams, of the arc-onderste poort veterinary institute, mapped the distributions of the ticks. the south african element of the project was financed by funds provided by bayer healthcare south africa and by the technology and human resources for industry programme (thrip) of the department of trade and industry, and the national research foundation. the finances for the mozambican side came mainly from a sida/sarecjoint ineve-eduardo mondlane uni versity, veter inary faculty, research project, no. # 27. references acocks, j.p.h. 1988. veld types of south africa with accompany­ ing veld type map, 3rd ed. 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1936. systematic para­ sitology, 10:3–26. pegram, r.g., keirans, j.e., clifford, c.m. & walker, jane b. 1987b. clarification of the rhipicephalus sanguineus group (acari, ixodoidea, ixodidae). ii. r. sanguineus (latreille, 1806) and related species. systematic parasitology, 10:27–44. petney, t.n., horak, i.g., howell, d.j. & meyer, s. 2004. striped mice, rhabdomys pumilio, and other murid rodents as hosts for immature ixodid ticks. onderstepoort journal of veterinary research, 71:313–318. spickett, a.m. & malan, j.r. 1978. genetic incompatibility between boophilus decoloratus (koch, 1844) and boophilus microplus (canestrini, 1888) and hybrid sterility of australian and south african boophilus microplus (acarina: ixodidae). onderstepoort journal of veterinary research, 45:149–153. tendeiro, j. 1954. an ixodological survey of mozambique. garcia de orta, 3:231–236. theiler, gertrud. 1943. notes on the ticks off domestic stock from portuguese east africa. estação anti-malárica de lourenço marques: imprensa nacional de moçambique. theiler, gertrud. 1962. the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region). project s 9958. report to the director of veterinary services, onderstepoort. mimeographed. walker, jane b. & olwage, a. 1987. the tick vectors of cowdria ruminantium (ixodoidea, ixodidae, genus amblyom­ ma) and their distribution. onderstepoort journal of veteri n­ ary research, 54:353–379. walker, jane b. 1991. a review of the ixodid ticks (acari, ixodidae) occurring in southern africa. onderstepoort jour­ nal of veterinary research, 58:81–105. walker, jane b., keirans, j.e. & horak, i.g. 2000. the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world. cambridge: cambridge academic press. article information authors: donald p. king1 m. madi1 v. mioulet1 j. wadsworth1 c.f. wright1 b. valdazo-gonzález1 n.p. ferris1 n.j. knowles1 j. hammond1 affiliations: 1institute for animal health, pirbright, united kingdom correspondence to: donald king postal address: po box 3019, sokoine university of agriculture, morogoro how to cite this proceeding: king, d.p., madi, m., mioulet, v., wadsworth, j., wright, c.f., valdazo-gonzález, b. et al., 2012, ‘new technologies to diagnose and monitor infectious diseases of livestock: challenges for sub-saharan africa’, onderstepoort journal of veterinary research 79(2), art. #456, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.456 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. new technologies to diagnose and monitor infectious diseases of livestock: challenges for sub-saharan africa in this proceeding... open access • abstract • foot-and-mouth disease • polymerase chain reaction technologies for foot-and-mouth disease virus detection • isothermal amplification • using genome sequence data to define trans-boundary movements of foot-and-mouth disease virus • foot-and-mouth disease viral evolutionary dynamics • diagnostic challenges for sub-saharan africa • conclusions • acknowledgements • references abstract top ↑ using foot-and-mouth disease (fmd) as an example, this review describes new tools that can be used to detect and characterise livestock diseases. in recent years, molecular tests that can detect and characterise pathogens in a diverse range of sample types have revolutionised laboratory diagnostics. in addition to use in centralised laboratories, there are opportunities to locate diagnostic technologies close to the animals with suspected clinical signs. work in this area has developed simple-to-use lateral-flow devices for the detection of fmd virus (fmdv), as well as new hardware platforms to allow molecular testing to be deployed into the field for use by non-specialists. once fmdv has been detected, nucleotide sequencing is used to compare field strains with reference viruses. transboundary movements of fmdv are routinely monitored using vp1 sequence data, while higher resolution transmission trees (at the farm-to-farm level) can be reconstructed using full-genome sequencing approaches. new technologies such as next-generation sequencing technologies are now being applied to dissect the viral sequence populations that exist within single samples. the driving force for the use of these technologies has largely been influenced by the priorities of developed countries with fmd-free (without vaccination) status. however, it is important to recognise that these approaches also show considerable promise for use in countries where fmd is endemic, although further modifications (such as sample archiving and strain and serotype characterisation) may be required to tailor these tests for use in these regions. access to these new diagnostic and sequencing technologies in sub-saharan africa have the potential to provide novel insights into fmd epidemiology and will impact upon improved strategies for disease control.effective control of infectious diseases is reliant upon accurate diagnosis of clinical cases using laboratory tests, together with an understanding of factors that impact upon the epidemiology of the infectious agent. a wide range of new diagnostic tools and nucleotide sequencing methods are used by international reference laboratories to detect and characterise the agents causing outbreaks of infectious diseases. in the past, high costs (initial capital expenses, as well as day-to-day maintenance and running costs) and complexity of the protocols used to perform some of these tests have limited the use of these methods in smaller laboratories. however, simpler and more cost-effective formats are now being developed that offer the prospect that these technologies will be even more widely deployed into laboratories particularly those in developing regions of the world such as sub-saharan africa. foot-and-mouth disease top ↑ this short review focuses on foot-and-mouth disease (fmd) and highlights new diagnostic approaches that can be used to detect and characterise pathogens causing livestock diseases. foot-and-mouth disease is a trans-boundary viral disease that is endemic in sub-saharan africa, much of south asia (middle east, indian sub-continent and southeast asia) and parts of south america. the causative agent, fmd virus (fmdv), is a picornavirus (genus: aphthovirus) with a positive-sense rna genome of approximately 8300 nucleotides in length. foot-and-mouth disease virus exists as seven genetically discrete serotypes, five of which are currently endemic in sub-saharan africa. this virus causes an acute disease in cloven-hoofed animals (i.e. cattle, sheep, goats, pigs and buffalo) that is associated with the development of vesicles on epithelial surfaces of the mouth and feet. foot-and-mouth disease virus infection also generates a transient viraemia in infected animals that typically lasts for approximately five days (figure 1; alexandersen et al. 2003). tests that exploit these clinical windows in an infected animal form the basis of laboratory approaches currently used to diagnose fmd. these assays aim to detect fmdv in epithelium and fluid from vesicles, as well as in blood and swabs from mucosal surfaces (oral and nasal swabs). in addition, fmdv-specific antibody responses in exposed animals can be detected using serological assays; however these methods are not discussed in this review. in general terms, the virological assays utilise three different strategies: propagation of fmdv, detection of viral antigenic proteins, or use of molecular assays to amplify specific rna sequences. the earliest versions of these tests exploited approaches that were largely used to propagate fmdv for vaccine production (brown 2003). although initially developed during the 1920s and used up until the 1950s, the obvious disadvantages of these systems placed limitations on their use for wide scale fmd diagnostics. indeed, these systems were superseded by improved highly sensitive in-vitro cell cultures used to propagate fmdv, such as primary bovine thyroid cells (snowdon 1966), permanent pig kidney cultures (ib-rs-2; de castro 1964) and more recently a goat tongue cell line (brehm et al. 2009). virus isolation approaches can be highly sensitive (depending upon the cell culture system used) although it can be slow taking between one and four days to generate a result. propagation of fmdv in cell cultures is still widely considered to be the ‘gold-standard’ test for fmd diagnosis. the development and establishment of cell culture systems has been paralleled by improvements to immunological (enzyme-linked immunosorbent assays: elisa) assays designed to detect fmdv antigen using polyclonal antisera (ferris et al. 1988) or characterised monoclonal antibody reagents (ferris et al. 2011). capture elisas are more rapid than virus isolation, but they have lower analytical sensitivity and are also inappropriate for use with certain sample types such as blood, milk and swabs. more recently, lateral-flow devices (lfds, also referred to as immuno-chromatographic strip tests) have been developed for the detection of fmd viral antigen. these simple-to-use and rapid tests utilise fmdv specific antibody reagents (normally monoclonal antibodies) in a format similar to the sandwich capture elisa used for laboratory diagnosis. positive test signal is generated by the diffusion of coloured, antibody-coated latex beads or colloidal gold particles through a membrane towards an immobilising band of trapping antibody. an lfd has been developed for the detection of all seven fmdv serotypes which uses a pan-serotypic monoclonal antibody (ferris et al. 2009). in addition, sample preparation in field conditions can be achieved using simple disposable tissue homogenizers for preparing epithelial suspensions. in terms of diagnostic sensitivity and specificity, the overall performance of this lfd is similar to laboratory-based antigen elisa, although the diagnostic sensitivity of the current test is lower for sat 2 field strains (ferris et al. 2009). this lfd and a simple homogenising kit is now commercially available (via boehringer ingelheim vetmedica svanova, uppsala, sweden). data from the field illustrates the potential for the lfd to be used in locations close to animals to provide rapid support to veterinarians in their clinical assessment of suspected fmd cases. the simplicity and stability of the lfd may be important features for fmd diagnosis in sub-saharan african countries. in particular, ability to rapidly recognise fmdv in clinical material may improve the selection of diagnostic samples that are shipped to reference laboratories for subsequent isolation and/or strain characterisation. in addition, recent results have also indicated that it is possible to recover rna from fmdv positive lfds that can be used in rrt-pcr assays and sequencing studies. further lfd assays are now in development and future formats, such as the sat 2 device that has recently been evaluated (ferris et al. 2010), may also allow rapid serotyping of field strains. figure 1: diagnostic windows for fmd showing temporal distribution of fmdv in different biological samples ([1-black] vesicular epithelium; [2-red] blood and [3-orange] mucosal swabs) and (4-blue) fmdv-specific antibodies in sera. the dotted axis-line represents incubation period that is dependent upon infectious dose, virus strain, species and other host determinants. this figure was generated using representative ‘in contact’ experimental cattle data from previous studies (alexandersen et al. 2003) and unpublished data from iah. polymerase chain reaction technologies for foot-and-mouth disease virus detection top ↑ amplification of specific nucleic acid sequences using reverse transcription polymerase chain reaction (rt-pcr) is now widely used for the laboratory detection of fmdv. these molecular assays are suitable for the diverse range of different samples that might be submitted for laboratory investigation (tissues, blood, swabs, oesophageal or pharyngeal (op) scrapings, faecal samples and milk). over the past 15 years, improvements have been made to rt-pcr protocols used for the detection of fmdv. initially, assays that targeted conserved regions of the genome (3d: meyer et al. 1991; rodrıguez et al. 1994 and 5’ untranslated region [5’ utr], reid et al. 2000) utilised agarose gel electrophoresis for the detection of amplified products. however, these labour intensive procedures have a high risk of generating false positives due to carry-over of chain reaction (pcr) amplicons and are therefore not generally considered ideal for routine testing of large numbers of samples. real-time rt-pcr (rrt-pcr) assays have now largely replaced agarose gel based assay formats. these more rapid fluorescence-based approaches are highly sensitive enabling simultaneous amplification and quantification of fmdv specific nucleic acid sequences. in addition to enhanced sensitivity, the benefits of these closed-tube rrt-pcr assays over conventional endpoint detection methods include a reduced risk of cross-contamination, their large dynamic range, an ability to be scaled up for high-throughput applications and the potential for accurate target quantification. several assays have been developed to detect fmdv that use 5’-nuclease assay (taqman®) system to detect pcr amplicons (callahan et al. 2002; oem et al. 2005; reid et al. 2002). other formats exploited for fmdv-specific rrt-pcr assays include the use of modified minor groove binder (mgb) probes (mckillen et al. 2011; moniwa et al. 2007), hybridisation probes (moonen et al. 2003), primer-probe energy transfer (priproet: rasmussen et al. 2003) and rt-linear-after-the-exponential pcr (late pcr: reid et al. 2010). in order to minimise human operator errors and increase assay throughput, these assays can be automated using robots for nucleic acid extraction (reid et al.; moonen et al. 2003). together with the implementation of quality control systems, these improvements have increased the acceptance of the rrt-pcr assays for routine diagnostic purposes. in addition to use in centralised laboratories, there are opportunities to deploy rrt-pcr technologies close to the animals with suspect clinical signs. these test formats may be particularly suitable for use in fmd-endemic areas such as countries within sub-saharan africa where the time taken to collect and dispatch samples to a laboratory for disease investigation can be protracted. work in this area has explored the use of new hardware platforms to allow pcr testing to be deployed into the field for use by non-specialists (callahan et al. 2002; hearps, zhang & alexandersen 2002; king et al. 2008). the focus of current work is the development of hardware platforms (madi et al. 2011) incorporating a simple-to-use and robust template extraction process such that all the steps of the assay can be performed without user intervention. these steps include (1) nucleic acid extraction, (2) pcr set-up, (3) amplification and (4) unambiguous calling of results. the use of homogeneous systems has previously been recognised an important aspect for the implementation of molecular methods for field detection of fmdv (hearps et al. 2002). currently, there is only limited access to these technologies outside of europe and north america. in addition to the performance of these equipment and assays, the availability and cost of consumables, as well as mechanisms to locally service the machines (in the event of equipment failure) will be important factors for the routine use of these tests in countries within sub-saharan africa. isothermal amplification top ↑ the recent development of portable equipment for pcr has made molecular diagnosis of fmd in the field an achievable goal. however, this approach relies on precision thermocycling requiring instrumentation which can be fragile, prohibitively expensive and that will require decontamination when transferred from one site to another. as an alternative to pcr, isothermal (single temperature) amplification methods for the detection of fmdv have been developed. since the specific amplification step for both nucleic acid sequence based amplification (nasba) and reverse-transcription loop-mediated amplification (rt-lamp) formats occurs at a constant temperature, there is less reliance upon expensive equipment and there is obvious potential to use of these assays as the basis of an inexpensive (or even disposable) molecular test. two different isothermal strategies: nasba and rt-lamp are discussed in this review. nucleic acid sequence based amplification technology is a continuous, isothermal and enzyme-based method to amplify single stranded rna and is therefore particularly suited to the detection of viruses with rna genomes such as fmdv (compton 1991). assays employ three enzymes: t7 rna polymerase, reverse-transcriptase and ribonuclease-h, a set of target-specific forward and reverse oligonucleotide primers and two types of detection probes. the forward primer has a 5’ extension containing the promoter sequence for t7 bacteriophage dna-dependent rna polymerase, while the reverse primer has a 5’ extension containing a complementary binding sequence for a dna oligonucleotide detection probe. using an fmdv specific nasba assay (collins et al. 2002), concordant results between nasba and rrt-pcr were generated for 87% of fmd samples (lau et al. 2008). simple enzyme-linked oligonucleotide capture nasba formats using elisa plate readers have also been evaluated and these show promise for use in laboratories where more expensive equipment may not be available (lau et al. 2008). reverse-transcription loop-mediated amplification is an isothermal autocyling strand-displacement dna synthesis technique which utilises four specific primers to recognise six regions of the target genome (notomi et al. 2000). formation of loop structures enables explosive polymerase-based enzymatic amplification, which generates double-stranded, multi-sized amplicons. pan-serotypic rt-lamp assays have been designed for fmdv (dukes, king & alexandersen 2006; li et al. 2009). validation data indicates that rt-lamp has equivalent analytical sensitivity to rrt-pcr and may be less sensitive to inhibition by problematic sample matrices such as op fluids and faecal samples. reverse-transcription loop-mediated amplification products are generated in abundance and can be detected using equipment to monitor turbidity, agarose gels or real-time pcr machines. furthermore, it is also possible to visualise dual-labelled lamp amplicons using novel lateral flow devices (james et al. 2010). in addition, changes in free-cation (mg2+) concentration in positive rt-lamp reactions can be visualised using a colour change of a dye indicator such as hydroxynaphthol blue (bearinger et al. 2011). together with simple methods to prepare template rna, these simple readouts for rt-lamp may prove useful in field settings. using genome sequence data to define trans-boundary movements of foot-and-mouth disease virus top ↑ once fmdv has been detected, nucleotide sequencing is a useful tool used to compare field strains with reference viruses and allows important phenotypic characteristics to be elucidated, such as antigenic determinants present on the viral capsid. genetic characterisation of foot-and-mouth disease virus routinely uses vp1 sequence data generated by rt-pcr. this region of the fmdv genome is approximately 630 nucleotides in length and encodes a protein (1d) that comprises an important component of the fmd viral capsid. these sequences are used to categorise field strains into discrete sub-groups (or topotypes) which frequently show geographical clustering based on the historical distribution of the virus (di nardo, knowles & paton 2011). these analyses provide evidence for the transboundary movements of fmdv and provide critical support to regional and country-level programmes to control fmd. within sub-saharan africa, three main epidemiological pools of fmdv have been recognised (paton, sumption & charleston 2009): west africa comprising serotypes o, a, sat 1 and sat 2; east africa comprising serotypes o, a, sat 1, sat 2 and sat 3 and southern africa with serotypes sat 1, sat 2 and sat 3. recent fmd outbreaks in the united kingdom (during 2001 and 2007) have highlighted the limitation to which vp1 sequence data can be used to discriminate sequences from field cases of disease. vp1 is relatively short (~8% of the genome length) and as a consequence, phylogenetic trees generated from viral sequences recovered within outbreaks clusters are typically flat with poor resolution. furthermore, limiting sequence analysis to only vp1 reduces the ability to identify broad-scale recombination events that may drive step-changes in the generation of new genetic and antigenic variants. identifying the sources of fmd outbreaks can play an important role in disease control: however, this can be confounded by incomplete epidemiological evidence and the numerous routes by which the virus can spread (via aerosols, movements of infected animals or their products, and spread of fomites on contaminated persons and objects). recent advances in laboratory methodologies allow rapid sequencing of complete fmdv genomes (abdul-hamid et al. 2011; cottam et al. 2009a). for the first time, this opens up the potential for using genome sequencing to reconstruct virus transmission trees with extremely high resolution and to reveal and identify the origin of unresolved transmission events within discrete infection clusters quickly. using full fmdv genome sequences determined from field samples collected from the 2001 fmd outbreak in the uk, it was shown that transmission events at the level of farm-to-farm spread could be reconstructed (cottam et al. 2006, 2008b). known patterns of spread of the virus were be reproduced by statistical parsimony-based analyses of this data. however, in some cases, these genetic data supported transmission histories different from those suggested by conventional contact tracing studies (cottam et al. 2006). during the 2007 fmd outbreak in the uk, full genome sequencing was used in real-time to support epidemiological investigations (cottam et al. 2008a). analyses of these data have revealed the most likely chain of transmission events, and predicted undisclosed infected premises prior to their discovery by serological surveillance. thus, for the first time, results indicate that full-genome sequencing can be used for fine-scale epidemiology to reveal and identify the origin of fmdv causing outbreaks. foot-and-mouth disease viral evolutionary dynamics top ↑ foot-and-mouth disease evolves rapidly due to its large population size, high replication rate and poor proof-reading ability of its rna-dependent rna polymerase. within cells, fmdv exists as heterogeneous populations comprising similar but non-identical genomes. consensus (sanger) sequencing identifies the predominant sequence present in a sample, but does not provide any information regarding the structure of minority variants that are present. next-generation sequencing (ngs) techniques offer an unprecedented ‘step-change’ increase in the amount of sequence data that can be generated from a sample to reveal nucleotide substitutions present in only a small fraction of the population. using ngs performed on a genome analyzer platform (illumina), the viral populations within bovine epithelial samples (foot lesions) have been determined (wright et al. 2011). this approach revealed the fine polymorphic sub-structure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. some of the higher frequency polymorphisms identified encoded changes in the heparan sulphate binding site revealing intermediate stages in the evolution of a tissue-culture adapted viral genome upon replication within a mammalian host. conventional sanger sequencing and ngs methods can provide powerful datasets that have the potential to revolutionise our understanding of the patterns of viral evolution and factors that impact upon fmdv circulation in endemic countries. capital equipment and running cost to undertake sequencing studies are decreasing all the time. regional and national centres of molecular and sequencing expertise are now becoming established in some african countries to provide support for research into the epidemiology of livestock diseases such as fmd. although currently limited to use in specialised molecular facilities, there is a prospect that new more affordable formats of ngs technologies will become available soon. diagnostic challenges for sub-saharan africa top ↑ this short review describes new tools that have recently been developed to support the diagnosis and epidemiology of fmd. the driving force for these improvements has largely been influenced by the priorities of developed countries in europe and north america with fmd-free (without vaccination) status. however, it is important to recognise that these technologies also show considerable promise for use in fmd-endemic countries, although further modifications may be required to tailor these tests for use in these regions. the presence of multiple serotypes of fmd in the majority of sub-saharan african countries can influence local disease diagnostic priorities. rather than pan-serotypic fmdv assays that are developed in fmd-free countries, a particular priority for endemic countries is the development of serotype-specific (and lineage-specific) typing assays that can be used rapidly to monitor incursions of exotic and emerging fmdv lineages into new geographical regions. the high genetic diversity of circulating fmdv strains (particularly sat serotypes) provides a constant challenge for diagnostic laboratories in fmd endemic countries in africa, and it is important that on-going validation is undertaken to ensure tests remain fit for purpose and are able to detect new viruses as they continue to evolve. furthermore, it is critical that robust protocols for sample collection and archiving in the field are adopted (belsham et al. 2011). our understanding of fmd epidemiology in africa is patchy and currently suffers from a lack of good quality laboratory and sequence data. the global control of fmd strategy currently being developed jointly by oie and fao is expected to rely heavily on the use of these next generation tools for more accurate and rapid diagnosis of fmd at the local level. however, without improvements to the quality and coverage of samples collected from field outbreaks, it is unlikely that the epidemiological picture of the disease in the region will become clear enough to make a significant impact upon disease control. conclusions top ↑ these new diagnostic tools can play a critical role in our ability to detect and monitor the spread of fmd in endemic regions of the world. however, it is also important to recognise that effective monitoring and control of fmd is reliant upon adequate resources, these are principally financial but also include availability of trained field personnel and a strong supporting laboratory infrastructure. in addition, international cooperation, transparency between different countries, sharing of epidemiological data and ownership of disease are also key factors in the control of important trans-boundary disease such as fmd. acknowledgements top ↑ the authors of this article are supported by grant funding from uk department of environment, food and rural affairs (defra se1124, se1126, se2938 and se2937), eu noe epizone (contract no. food-ct-2006-016236) and the combating infectious diseases of livestock for international development (cidlid) project funded by dfid/bbsrc/scottish government (towards the strategic control of foot-and-mouth disease in africa: new techniques for a neglected problem). the authors also acknowledge southern african centre for infectious disease surveillance (sacids) for providing travel support (to dk) to present this paper at the 1st one health conference in africa, johannesburg, july 2011. references top ↑ abdul-hamid, n.f., fırat-saraç, m., radford, a.d., knowles, n.j. & king, d.p., 2011, ‘comparative sequence analysis of representative foot-and-mouth disease virus genomes from southeast asia’, virus genes 43, 41–45. http://dx.doi.org/10.1007/s11262-011-0599-3, pmid:21479678 alexandersen, s., quan, m., murphy, c., knight, j. & 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virology 85, 2266–2275. http://dx.doi.org/10.1128/jvi.01396-10, pmid:21159860 article information authors: george dautu1 calvin sindato2 aaron s. mweene1 kenny l. samui1 polly roy3 robert noad4 janusz paweska5 phelix a.o. majiwa6 antony j. musoke6 affiliations: 1department of disease control, university of zambia, zambia 2national institute for medical research, tabora, tanzania 3london school of hygiene and tropical medicine, london, united kingdom 4royal veterinary college, hatfield, united kingdom 5national institute for communicable diseases, johannesburg, south africa 6onderstepoort veterinary institute, onderstepoort, south africa correspondence to: george dautu postal address: po box 32379, lusaka, zambia how to cite this abstract: dautu, g., sindato, c., mweene, a.s., samui, k.l., roy, p., noad, r. et al., 2012, ‘rift valley fever: real or perceived threat for zambia?’, onderstepoort journal of veterinary research 79(2), art. #466, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.466 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. rift valley fever: real or perceived threat for zambia? in this abstract... open access • abstract • introduction • aetiology • epidemiology • risk factors associated with rift valley fever • conclusion • acknowledgements    • competing interests • references abstract top ↑ rift valley fever (rvf) in zambia was first reported in 1974 during an epizootic of cattle and sheep that occurred in parts of central, southern and copperbelt provinces. in 1990, the disease was documented in nine districts of the provinces of zambia. in the last two decades, there have been no reports of rvf. this long period without reported clinical disease raises questions as to whether rvf is a current or just a perceived threat. to address this question, world organisation for animal health (oie) disease occurrence data on rvf for the period 2005−2010 in the southern africa development community (sadc) was analysed. from the analysis, it was evident that most countries that share a common border with zambia had reported at least one occurrence of the disease during the period under review. due to the absence of natural physical barriers between zambia and most of her neighbours, informal livestock trade and movements is a ubiquitous reality. analysis of the rainfall patterns also showed that zambia received rains sufficient to support a mosquito population large enough for high risk of rvf transmission. the evidence of disease occurrence in nearby countries coupled with animal movement, and environmental risk suggests that rvf is a serious threat to zambia. in conclusion, the current occurrence of rvf in zambia is unclear, but there are sufficient indications that the magnitude of the circulating infection is such that capacity building in disease surveillance and courses on recognition of the disease for field staff is recommended. given the zoonotic potential of rvf, these measures are also a prerequisite for accurate assessment of the disease burden in humans. introduction top ↑ rift valley fever (rvf) is an economically important, emerging arthropod-borne viral disease of both livestock and man. the disease was first identified in 1931 following sudden death of lambs and ewes on a single farm along the shores of lake naivasha in the greater rift valley of kenya (daubney et al. 1931, reviewed in pepin et al. 2010). the importance of the disease lies in its public health impact and the economic losses resulting from the cessation of trade in livestock and livestock related products. this has been shown by the prolonged import bans from countries in the horn of africa where rvf has been registered, causing great hardship to the livestock trade based communities. in zambia, rvf was first reported in 1974 during an epizootic of cattle and sheep that occurred in chisamba (central province) and mazabuka (southern province) districts and some parts of copperbelt province (hussuein et al. 1987). human death due to rvf disease in chisamba was also previously reported (watts et al. 1984). several other epizootics have been reported in the same areas (department of veterinary and tsetse control services annual reports 1975−1989). for a long time the disease was known to be confined to the same outbreak areas. however, a sero-epidemiological study carried out between january 1990 and march 1991 showed that the disease could have a country wide distribution (samui et al. 1997). although rvf is considered endemic in zambia, the clinical disease has not been reported in the last two decades. this long period without reported cases raises questions as to whether rvf is a current, or just a perceived threat. this article reviews some of the reasons as to why rvf has not been reported in zambia in the recent past through focusing on the aetiology, epidemiology and risk factors associated with the disease. furthermore, the oie disease occurrence data on rvf for the period 2005−2010 in the southern africa development community (sadc) will be analysed. aetiology top ↑ rift valley fever is an arthropod-borne viral disease caused by a rift valley fever virus (rvfv) of the family bunyaviridae and genus phlebovirus. the rvfv genome is made up of three segments namely l, m and s which are packaged together in the virions in the form of ribonucleoprotein (rnp). the l and m segments present in the virus particle are of negative polarity. the l segment encodes a single protein which is the viral rna-dependent rna polymerase (muller et al. 1994; pepin et al. 2010), and for the precursor to the glycoproteins. the m segment encodes four proteins, nsm1, nsm2 and two glycoproteins, gc and gn (collett et al. 1985; collett et al. 1986; schmaljohn & hooper 2001). the s segment utilises an ambisense strategy to code for two proteins, the nucleoprotein n, in the negative polarity, and a non-structural protein, nss, in the positive polarity (giorgi et al. 1991). the virus is resistant to heat and could stay active for four months at 21 °c and 3 hours at 56 °c (flick et al. 2005). however, it can be inactivated by strong calcium or sodium hypochlorite, especially when treated for three hours at 56 °c (sossah 2009). there is only one serotype of rvfv known to date (martin et al. 2008). in zambia, there is no evidence regarding the physical isolation of rvf virus from the field. however, there is enough serological evidence to suggest the presence of the virus (davies et al. 1992). clinical manifestations of the disease in ruminant livestock, especially sheep and cattle, are characterised by high mortality (100% in neonatal animals and 10% − 20% amongst adult animals) and high abortion rates particularly in infected pregnant animals (coetzer 1977, 1982; swanepoel 1994). in humans the disease is self-limiting, although complications of hemorrhagic fever, retinitis, blindness, and encephalitis may occur in 1% − 2% of affected individuals with a case fatality of approximately 10% − 20% (madani et al. 2003). epidemiology top ↑ rift valley fever disease is an important endemic problem in sub-sahara africa which includes zambia. the disease outbreaks in africa occur at irregular intervals of 5−15 years in the savannah grasslands and 25−35 years in the semi-arid regions. rift valley fever virus has demonstrated a real capacity to emerge in virgin areas as shown by the outbreaks in egypt (1977, north of the sahara desert), madagascar (1979), saudi arabia and yemen (2000), outside the continent of africa (centre for disease control and prevention 2000; morvan et al. 1992; shoemaker et al. 2002). rift valley fever virus has two transmission cycles, namely the enzootic and epizootic cycles. enzootic cycle occurs during periods of normal amounts of rainfall. in the enzootic cycle, rvf virus is maintained by low-level activity within the mosquito vector population involving transovarial transmission with occasional infection and amplification of virus in wildlife such as african buffaloes (syncerus caffer) or susceptible livestock. epizootic or epidemic cycles occur following extended periods of exceptionally plentiful rainfall and subsequent flooding of dambos, which results in the emergence of abundant numbers of floodwater aedes mosquitoes. these transovarially infected mosquitoes feed on susceptible livestock (e.g. sheep and cattle) that rapidly develop high-titer viremias and signs of clinical disease. the infected livestock in turn infect secondary bridge mosquito vectors such as the culex or anopheles spp. (coetzer 1977, 1982; turell et al. 1984) and thereafter, human infections develop as a result of bites from infected mosquitoes (aedes, culex or anopheles spp.), exposure to infectious aerosols, handling of aborted fetal materials, or percutaneous injury during slaughtering or necropsy of viremic animals (meegan 1981; van velden et al. 1977). it is unclear whether humans have any important biological role as amplification hosts in the rvf virus epizootic or epidemic life cycle. the past distribution of rvf outbreak in zambia is well documented (department of veterinary and tsetse control services annual reports, 1975−1989; davies et al. 1992; hussein et al. 1987; samui et al. 1997). the high risk areas have been identified where several rfv epizootics had occurred. these areas include ndola in the copperbelt province, chisamba in the central province, and lusaka and mazabuka in southern province (hussein et al. 1987). rift valley fever clinical signs were limited to susceptible bos taurus cattle and imported sheep. however, a rvf sero-epidemiological study carried out in 5 traditionally managed herds that graze in the kafue flats (flood plain grasslands [figure 1]) showed that rvf was not only a threat to the commercial exotic breeds but also to the indigenous local breeds. for instance, a study carried out by ghirotti et al. (1991) in the kafue flats showed that 14% of the indigenous cattle tested seroconverted to rvf. the 14% rvf sero-prevalence rate was attributed to high concentration of wild and domestic ruminants grazing together in the flood plains during the dry season. it is worthwhile to mention here that no studies have been done to determine the role of wildlife in the maintenance of rvfv in zambia. furthermore, the sero-epidemiological study carried out between january 1990 and march 1991 in at least one district of each of the nine provinces showed the existence of rvf in the respective districts studied (samui et al. 1997). the high positive rates were also observed in areas where cattle grazed in dambos or flood plains (table 1 and figure 2). the results of this study suggest that rvf was not only endemic in the commercial farms of chisamba, lusaka and mazabuka but could be endemic throughout most of the cattle producing parts of the country. the implication of these results are that the traditional farmers who graze their cattle in the flood plains or dambos together with all those involved in livestock production are particularly at risk of contracting rvf if it is still circulating at high prevalence in cattle, sheep and goats, and if the local environment is favourable for transmission of the virus. figure 1: map of zambia showing the location of the kafue flats. table 1: distribution of rift valley fever amongst cattle in zambia. figure 2: map of zambia showing sampling location denoted with square dots. in zambia the disease has not been reported for the last two decades. this period without detected disease does not necessary mean that rvf is not a threat to zambia. this is so because from past rvf research, a low level of rvf virus transmission has been detected in livestock and humans during inter-epizootic periods (iep). for example, a study carried out in animals born before the 1997−1998 and after the 2006−2007 outbreaks in kenya showed a low igg prevalence against rvf, indicating that virus transmission continued in kenya during an iep (rostal et al. 2010). similarly another study carried out in senegal during an iep in sheep and goats indicated a 2.9% seroprevalence (chevalier et al. 2003). in zambia, a study carried out during 1982−1986 on a sentinel herd using indigenous breeds at lutale in mumbwa showed a low level of seasonal rvf virus activity of 3% − 8% (davies et al. 1992). the studies carried out in zambia and other parts of africa clearly support the existence of low degree of rvfv transmission during the iep and that this low level of seasonal virus activity could generate epizootics as witnessed by the 1985−1986 epizootics in zambia (hussein et al. 1987). more interestingly, evidence of interepidemic human transmission of rvfv has been reported. in kenya, research done on children born after the documented rvf outbreak of 1997−1998 showed that low-level interepidemic transmission to humans continued to occur (labeaud et al. 2008). although there are no studies done on interepidemic human transmission of rvfv in zambia, the results of the previous studies done in animals and humans during iep clearly shows that rvf is a serious threat to zambia.although a low level virus activity has been demonstrated during iep in studies carried out in kenya and zambia (davies et al. 1992; rostal et al. 2010), no rvf associated abortion or death was observed. this implies that the infected livestock developed no clinical signs or developed mild febrile illness with no obvious clinical disease. the lack of specific rvf signs during iep implies that the presence of rvf could only be detected through specific, well-focused, active surveillance. therefore countries like zambia with limited resources to carry out this type of surveillance during iep could have problems in detecting the threat of rvf early and subsequently fail to report the disease. analysis of the oie disease occurrence data on rvf for the period 2005−2010 in the sadc region showed that most countries that share a common border with zambia had reported at least one occurrence of the disease during the period under review. since conditions which predispose to rvf activities tend to occur on a regional level (davies et al. 1992), the failure to detect the disease could be linked to the weak national surveillance system. inability of the field veterinary staff to recognise the clinical, pathological and epidemiological features of the disease is yet another challenge as far as reporting of rvf occurrence is concerned. for example, when confronted with a disease that involves abortion during iep, rvf is not included on the list of differential diagnosis. it is worthwhile to mention here that in zimbabwe, rvf-associated abortions were found in cattle over a period of 7 inter-epizootic years (1971−1977) and the temporal pattern suggested a possible annual emergence of infected mosquitoes (swanepoel 1981). this report shows clearly why it important to include rvf in the list of differential diagnosis especially when specimens are collected from cattle that have aborted. however, the diagnosis of rvf during iep is further undermined by a shortage of rvf reagents which comes as a result of lack of planning or funding. it is worth mentioning here that during iep, awareness and preparedness tend to decrease drastically as limited resources required for surveillance activities are redirected to other areas. risk factors associated with rift valley fever top ↑ there are several factors associated with the occurrence of rvf. these includes climatic conditions (rainfall, temperature, cloud cover), geographical features (dambos, flood plains), vegetation cover, livestock trade (both local and international) and human activities (such as building of dams, irrigation schemes). rainfall is one of the determinants of rfv outbreaks and this has been analysed in relation to the rvf epizootics in kenya (anyamba et al. 2009, 2010; davies et al. 1985; richards et al. 2010). zambia receives a good amount of rainfall annually and the rainfall pattern is divided into three agro-ecological zones namely region i, ii and iii (figure 3). region i, the driest, is most prone to drought and receives less than 800 mm of rain annually. this region includes the zambezi and luangwa valleys. region ii covers the central part of zambia extending from the east through to the west. it receives rainfall of between 800 mm and 1000 mm. region iii covers the northern part of the country and receives more than 1000 mm of rainfall in a season. region ii and iii are more prone to flooding and have high incidences of malaria due to high vector activities. therefore, the amount of rain tend to increase towards the north and decrease towards the south. the rainfall is considered to influence the onset of disease by producing a rising water table, to the point where seasonal flooding occurs, particularly in certain geomorphic formations known as ‘dambos’. figure 3: map of zambia showing the three agro-ecological zones. flooding of the dambos results in the emergence of abundant numbers of floodwater aedes sp., in particular aedes mcintoshi (linthicum et al. 1984). these transovarially infected mosquitoes are responsible for initiating epizootics of rvf, which then recruit other vectors for its propagation (linthicum et al. 1985). it should be noted that the flooded dambos are the most favoured breeding sites for a variety of mosquito species that are capable of transmitting rvf (davies & highton 1980). above all the humid conditions and cloud cover that exist during prolonged rainy periods allow a greater proportion of the adult aedes population to survive through more feeding-oviposition cycles than in the hot, dry conditions usually prevailing in these areas (davies et al. 1985) vegetation changes, due to a change in climatic conditions, has an effect on mosquito habitats. for instance, in the rainy season the proliferation of vegetation and increase in vegetation biomass favours the increase in population of mosquito species that are capable of transmitting diseases to livestock and humans. the dry season does not favour vegetation proliferation and hence there are fewer mosquito-borne diseases. in zambia, a sentinel herd study was carried out in 1982−1986 to determine whether annual rvf virus activity occurred and was associated with seasonal rains. the results showed that a low level rvf virus activity of 3% − 8% did occur in each of the years. however, in 1985−1986, more than 20% of the animals seroconverted and this greater activity was associated with vegetation changes (davies et al. 1992). the vegetation change was detected by remote sensing satellite imagery. livestock trade has previously been associated with the introduction of rvf in news areas (centre for disease control and prevention 2000; madani et al. 2003). there is a lot of livestock trade between zambia and her neighbours, which means that the introduction and spread of new diseases from neighbouring countries is high. oie disease occurrence data on rvf for the period 2005−2010 in the sadc region showed that most countries that share a common border with zambia had reported at least one occurrence of the disease during the period under review (figure 4 oie, world animal health information database [wahid] interface). due to the absence of natural physical barriers between zambia and most of her neighbours and given that the conditions which predispose to rvf activity do occur on a regional level, there is a high probability that rvfv could be circulating in zambia. figure 4: map showing the occurrence of rift valley fever in the southern africa development community region in the period 2005–2010. conclusion top ↑ this review demonstrates that rvfv is a threat to zambia as the environmental risk factors conducive for its propagation are widely distributed in most livestock producing areas. despite the threat posed by rvf in zambia, little research has so far been done. most studies documented so far were limited to the high risk areas and only conducted during rvf outbreaks. little is known about rvf virus activities during iep both in the high risk and low risk areas. currently, there is no information regarding the different types of rvf virus strains found in zambia. nothing is known of their virulence, pathogenicity or distribution in the different ecological zones of the country. the current prevailing hypothesis is that rvf virus is maintained in the eggs of aedes mosquitoes which are seasonal floodwater breeders (davies et al. 1985). however, different aedes spp. have been implicated in the transmission of rvfv in different regions of africa. for example, ae. ochraceus, ae. vexans arabiensis and ae. dalzieli are known vectors of rvfv in west africa (fontenille et al. 1998) where as ae. mcintoshi/ circumluteolus are known vector of rvfv in east africa (huang 1985). in zambia, the potential mosquito vector species that might be involved in the enzootic or epizootic cycles has never been documented. baseline data regarding their distribution and ecology is missing. the role of these mosquitoes in the maintanance of rvfv is not well understood. human infection through direct contact with aborted foetuses, meat and other animal byproducts during rvf outbreaks (labeaud et al. 2008), the specifics of what types of animal exposure are most risky, have not yet been elucidated. although human death due rvf was reported in the endemic areas of zambia (watts et al. 1984), no studies have been done to determine whether rvf transmission to human occurs during iep. lastly, the current rvf early warning system needs to be improved by including spatial and population parameters so as to achieve higher precision and confidence. therefore, in order to control rvf in the endemic and non-endemic areas of zambia, future research should aim at addressing the above mentioned gaps. the data generated from such research will help veterinary, health policy makers, planners and other stakeholders in prioritising, designing and implementing cost effective and sustainable rvf control programs. acknowledgements top ↑ this work has been supported by wellcome trust grant wt087546ma to the southern african centre for infectious disease surveillance (www.sacids.org) and its collaborating uk partner institutions, namely the london school of hygiene and tropical medicine (http://www.lshtm.ac.uk), the royal veterinary college (http://www.rvc.ac.uk) and the london international development centre (http://www.lidc.org.uk). competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. references top ↑ anyamba, a., linthicum, k.j., small, j., britch, s.c., pak, e., de la rocque, s. et al., n.d., ‘prediction, assessment of the rift valley fever activity in east 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(eds.), fields virology, 4th edn., pp. 1581–1602, lippincott, williams & wilkins, philadelphia, pa. shoemaker, t., boulianne, c., vincent, m.j., pezzanite, l., al-qahtani, m.m., al-mazrou, y. et al., 2002, ‘genetic analysis of viruses associated with emergence of rift valley fever in saudi arabia and yemen, 2000−2001’, emerging infectious diseases 8, 1415−1420. pmid:12498657 sossah, c., 2009, ‘oie regional seminar on re-emergence of rvf in southern africa’, how to better predict and respond, sossah, south africa. swanepoel, r.c.j., 1994, ‘rift valley fever’, in j.a.w. coetzer, g.r. thompson, r.d. tustin (eds.), infectious diseases of livestock with special reference to southern africa, pp. 688−717, oxford university press, cape town. turell, m.j., bailey, c.l. & rossi, c.a., 1984, ‘increased mosquito feeding on rift valley fever virus-infected lambs’, american journal of tropical medicine and hygiene 33, 1232−1238. pmid:6150656 van velden, d.j., meyer, j.d., olivier, j., gear, j.h. & mcintosh, b., 1977, ‘rift valley fever affecting humans in south africa a clinicopathological study’, south african medical journal 51, 867−871. pmid:561445 watts, t.e., tembo, g. & ngandu, n.h., 1984, ‘rift valley fever in chisamba, zambia’, medical journal of zambia 18, 10−11. article information author: georges tshilenge1 affiliation: 1central veterinary laboratory, kinshasa 1, democratic republic of congo correspondence to: georges tshilenge postal address: po box 8842, kinshasa 1, democratic republic of congo how to cite this poster: tshilenge, g., 2012, ‘immunogeno: protective mechanism for rift valley fever in the democratic republic of congo’, onderstepoort journal of veterinary research 79(2), art. #483, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.483 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. immunogeno: protective mechanism for rift valley fever in the democratic republic of congo in this poster... open access in democratic republic of congo (drc), this pathology is not well documented. no epidemic of the rvf has not been reported but sera samples collected in six provinces surveyed from 2005 to 2006 revealed 14% of apparent prevalence and, high apparent prevalence (20%) of antibodies against rvf virus was reported in katanga province during the same survey; this serological evidence was associated with abortions cases in cattle (mulumba et al. 2009). livestock immunisation is important for control of rift valley fever virus (rvfv) epidemics; however immunisation of susceptible domestic animals in endemic countries does not protect animals against the clinical disease but prevents the propagation of virus to human population through reduction of the amplification degree in host animals. the humoral immunity is sufficient for protection for animals as well as for humans. the infection caused by rvfv leads to neutralisation of the immunity of the animal (barnard 1979). various immunological studies have been made on the characterisation of immune response during rvfv epidemics but, until now several studies have been concentrated on the response of the innate immune particularly based on signal interferon system than the response of the adaptive immune and cell mediated humoral immune. the available information on the immune response related to rvfv does not seem to provide enough information on various mechanisms of the response immune system. the aim of the study is based on mechanism of immune response system including protective effect of immunisation against rvfv. in addition, epidemiological and molecular studies will be assessed. as a matter of fact, following studies will be conducted: • evaluation of the immunological protection against rift valley fever in vaccinated and non-vaccinated cattle using igg and igm elisas in katanga province • assessment of cellular response to rift valley fever disease in vaccinated and naturally infected cattle • molecular characterisation of rvfv strains circulating in vaccinated and non vaccinated cattle • assessment of protective effect related to vaccinal strains in cattle, using a longitudinal survey. the studies will be carried out northern katanga province within two areas, one with historyof circulation of rvfv and other without history rvfv circulation. whole blood, spleen, liver, lymph node will be collected as target tissues from cattle carcasses. in addition, goats and sheeps samples will be collected alongside from each area in order to clarify the disease situation. serological tests based on the detection of ig m and ig g will be used. diva tests, lamp, and ihc techniques will be used. within previously vaccinated areas in the above mentioned areas and those that are not vaccinated, the collected samples will be analysed using rt-pcr/rt-lamp. in vitro experimental studies systems will be carried out using animal pmbcs that will be infected with wild type of rvf virus as well as with vaccinal strains, such as clones 13 and mp12 to characterise various cell types such as cd4 t cells, cd8 t cells, b-cells, nk cells and, macrophages will be studied with regard to activation and apoptosis signals on various post – infection days, using flow cytometry. a pool of animals will be vaccinated with the clone 13 and another with the mp12 to determine the traceability. the monitoring of the immune response will be done through the measurement of immunoglobulin g (ig g) and immunoglobulin m (ig m). rt-pcr, spectrophotometer or facs methods will be used for the dosage of cells t cd4 + and cell t cd8+. meintjes_pp39-53.qxd introduction farmers in the kuruman district of the northern cape province, republic of south africa have recently experienced mortalities in their free-ranging cattle herds. the mortalities, approximately 200 head of cattle, were ascribed to nephrotoxicity induced by a previously unknown poisonous plant (du plessis, joubert, prozesky, naudé, herman & van der westhuizen 2004). clinically, weakness, malaise, and perineal and brisket oedema were observed. necropsy findings included severe anasarca, ascites, perirenal oedema and nephrosis, and mild hepatosis. microscopical examination revealed extensive degeneration and necrosis of tubular epithelial cells in the renal cortex, accompanied by large numbers of protein and, sometimes, cellular casts in the tubular lumens. dosing trials conducted by the division of toxicology, onderstepoort veterinary institute confirmed nolletia gariepina (dc) mattf. as the causative plant (du plessis 2004). the primary aim of the current trial was to elucidate the pathophysiological changes in renal function in sheep induced by n. gariepina. a secondary objective was to evaluate the toxic effects and pathological findings following administration of this nephrotoxic plant to sheep. 39 onderstepoort journal of veterinary research, 72:39–53 (2005) toxicity, pathophysiology and pathology in sheep following dosing of the nephrotoxic plant nolletia gariepina (dc) mattf. r.a. meintjes1, c.j. botha2 and l. prozesky2 abstract meintjes, r.a., botha, c.j. & prozesky, l. 2005. toxicity, pathophysiology and pathology in sheep following dosing of the nephrotoxic plant nolletia gariepina (dc) mattf. onderstepoort journal of veterinary research, 72:39–53 ingestion of the plant nolletia gariepina was confirmed as the cause of acute mortalities in cattle in the kuruman area of the northern cape province of south africa. the aim of this trial was to investigate the toxic effects of this plant with respect to clinical signs, pathophysiology and pathology using the sheep as a model. at dosages of 1.5 g dried, milled plant material/kg body mass there were no detectable abnormal findings, while at dosages of 2.8–3.0 g/kg most of the animals died acutely. in subacutely affected sheep, depression, inappetance, teeth grinding, tachycardia, weak ruminal movements and recumbency were noticed. the most prominent pathophysiological changes observed, included a sharp rise in non-protein nitrogen substances in the plasma, remarkable decline in glomerular filtration rate, increase in sodium and potassium excretion, and a rise in urine gamma glutamyltransferase activity. macroscopically a severe nephrosis was present in all the animals. the most important findings detected histologically were necrosis of the proximal convoluted tubular epithelium and large numbers of protein casts in the lumens. keywords: kidney, nephrotoxicity, nolletia gariepina, plant poisoning, sheep 1 department of anatomy and physiology, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: roy.meintjes@up.ac.za 2 department of paraclinical sciences, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa. e-mail: christo.botha@up.ac.za and leon.prozesky@up.ac.za accepted for publication 27 july 2004—editor materials and methods nolletia gariepina was collected on the farm tom brown, situated in the quadrant 2226 cb of the kuruman district, northern cape. the plant material was air dried, finely milled and frozen (at –10 °c) before being used. nine mutton merino wethers, 9–18 months of age, were individually placed in steel metabolic crates in an experimental animal housing facility. the room temperature was set and maintained at 22 °c. lucerne hay and drinking water were available ad lib. to facilitate the collection of unsoiled urine the sheep were fitted with faecal collection bags. the total volume of urine voided per day was collected in refrigerated containers located under each crate. the sheep were placed in the metabolic crates for an adjustment period of 5 days prior to a 5-day control phase during which baseline values for the different parameters were established as each animal served as its own control. immediately following the control period the sheep were deprived of feed for 24 h and then dosed with dried, milled plant material (1.5–3 g/kg body mass) by stomach tube as indicated in table 1. daily clinical examinations (habitus, rectal temperature, pulse, respiration and rumen motility) were conducted to evaluate the animals’ health. feed and water intake and urine volume were recorded daily. an aliquot of urine was collected for conventional urinalysis (combur urinary dip stick [merck]). urinary creatinine, sodium (na) and potassium (k) concentrations, gamma glutamyltransferase (ggt) activity and osmolarity were also determined. the ggt:creatinine ratios served as early detection of renal tubular damage. blood samples were collected for determination of haematocrit, osmolarity and plasma protein, na, k, creatinine and urea concentrations. in addition, ggt and aspartate aminotransferase (ast) activities were determined to establish if any hepatic damage had occurred. from the above variables the following were derived: 1. glomerular filtration rate (gfr) the plasma clearance of endogenous creatinine was equated to gfr on the assumption that creatinine is neither secreted nor absorbed by the renal tubule in sheep (nawaz & shah 1984; bastl, rudnick & nairns 1985). thus, gfr = uvol.u[creatinine] / pl[creatinine] (l/d) where uvol = volume of urine passed per day (l/d) u[creatinine] = creatinine concentration in urine (mmolar) pl[creatinine] = creatinine concentration in plasma (mmolar) 2. fractional excretion of sodium (fena) (bastl et al. 1985) this represents the amount of filtered sodium excreted in the urine expressed as a percentage. fena = uvol.u[na].100 / gfr.pl[na] (%) where pl[na] = sodium concentration in plasma (mmolar) u[na] = sodium concentration in urine (mmolar) the carcasses of sheep that died or were euthanased (overdose of pentobarbitone sodium [euthanaze, centaur] administered intravenously) were submitted for necropsy. tissue specimens (approximately 10 mm3) from several organs were collected and fixed by immersion in 10 % neutral buffered formalin for at least 5–7 days. specimens were routinely processed for light microscopy and stained with haematoxylin and eosin (he). renal tissue specimens were collected from two cases (no. 13 and 42) within minutes of death, cut into 1 mm2 blocks and fixed in labelled glass vials containing 2.5 % gluteraldehyde in 4 % 0.1 m millonig’s buffer. samples were routinely processed for electron microscopy using a transmission electron microscope and photographed. results toxicity the clinical signs observed and pathological findings are summarized in table 1. four of seven animals that were dosed with 3 g/kg n. gariepina plant material died acutely within 24 h. one sheep, which was depressed, recumbent and exhibited respiratory distress (hyperventilation, groaning, frothing at the mouth) was euthanased 8 h after being dosed with 3 g/kg milled plant material. only two animals, which had received 3 g/kg plant material, survived 40 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. 41 r.a. meintjes, c.j. botha & l. prozesky t a b l e 1 d o si n g r e g im e n o f d ri e d , m ill e d n o lle tia g a ri e p in a p la n t m a te ri a l a d m in is te re d o ra lly t o s h e e p , a n d c lin ic a l s ig n s a n d p a th o lo g ic a l f in d in g s s h e e p d o si n g r e g im e n n o . a g e b o d y d a y d o se c lin ic a l s ig n s* m a cr o sc o p ic le si o n s m ic ro sc o p ic le si o n s (m o n th s) m a ss (g /k g ) (k g ) a 9 1 8 3 7 0 3 w e a k ru m in a l m o ve m e n ts , m o d e ra te t o s e ve re n e p h ro si s s e ve re s u b a cu te n e p h ro si s w ith t u b u la r u n co m fo rt a b le , ta ch yc a rd ia ( d 0 ); d ila ta tio n in a p p e ta n ce , re cu m b e n cy , co n m o d e ra te h e p a to si s g e st e d m u co u s m e m b ra n e s (d 1 ); m ild h e p a to si s d e p re ss e d ( d 2 ); h a b itu s im p ro ve d , m ild a sc ite s a n d h yd ro p e ri ca rd iu m te e th g ri n d in g ( d 3 ); s tr o n g r u m e n m ild m o n o n u cl e a r in te rs tit ia l p n e u m o n ia m o ve m e n ts ( d 6 ); e u th a n a si a ( d 7 ), b o d y m a ss 3 2 kg d ys p la si a o f sm a ll in te st in e m u co sa a 5 1 8 3 0 0 3 d e p re ss e d , in a p p e ta n ce , w e a k s e ve re n e p h ro si s w ith p e ri re n a l o e d e m a s e ve re s u b a cu te n e p h ro si s w ith t u b u la r ru m in a l m o ve m e n ts , ta ch yc a rd ia , a n d h a e m o rr h a g e s d ila ta tio n co n g e st e d m u co u s m e m b ra n e s, te e th g ri n d in g ( d 1 ); r e cu m b e n cy m o d e ra te h e p a to si s w ith w id e sp re a d t u b u la r re g e n e ra tio n (d 2 ); d e h yd ra tio n ( d 4 ); e u th a n a se d h a e m o rr h a g e s in s e ro sa o f g a ll b la d d e r (d 5 ), b o d y m a ss 2 6 kg m ild h e p a to si s g e n e ra lis e d s e ro sa l h a e m o rr h a g e s o f fo re s to m a ch s m ild m o n o n u cl e a r in te rs tit ia l p n e u m o n ia d ys p la si a o f sm a ll in te st in e m u co sa d ys p la si a o f sm a ll in te st in e m u co sa a 1 3 b 1 4 2 8 0 3 s lig h t fr o th in g a t m o u th , u n co m fo rt m o d e ra te n e p h ro si s a n d h e p a to si s d iff ic u lt to e va lu a te t h e k id n e y a n d li ve r a b le , d ie d d u ri n g n ig h t (d 0 ) b e ca u se o f co e xi st e n t a u to ly si s s e ve re p u lm o n a ry c o n g e st io n a n d o e d e m a p u lm o n a ry c o n g e st io n w ith w id e sp re a d h a e m o rr h a g e s g e n e ra lis e d h a e m o rr h a g e s a 1 4 r 1 4 3 7 0 3 d ie d d u ri n g n ig h t (d 0 ) g e n e ra lis e d c o n g e st io n a n d d iff ic u lt to e va lu a te t h e k id n e y a n d li ve r h a e m o rr h a g e s b e ca u se o f co e xi st e n t a u to ly si s s e ve re n e p h ro si s p u lm o n a ry c o n g e st io n w ith w id e sp re a d h a e m o rr h a g e s s e ve re p u lm o n a ry c o n g e st io n a n d o e d e m a * c lin ic a l s ig n s in o rd e r o f a p p e a ra n ce 42 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. t a b l e 1 (c o n tin u e d ) s h e e p d o si n g r e g im e n n o . a g e b o d y d a y d o se c lin ic a l s ig n s* m a cr o sc o p ic le si o n s m ic ro sc o p ic le si o n s (m o n th s) m a ss (g /k g ) (k g ) s 5 1 2 3 0 0 3 r e cu m b e n cy , d ie d d u ri n g n ig h t (d 0 ) a 4 1 8 3 2 0 3 r e cu m b e n cy , h yp e rv e n til a tio n , g ro a n in g , d is tr e ss e d , sl ig h t fr o th in g a t m o u th , h e a d b e n t to w a rd s sh o u ld e r, e u th a n a si a ( d 0 ) a 1 3 r 1 8 4 1 0 3 r e cu m b e n cy , d ie d d u ri n g n ig h t (d 0 ) 4 2 9 2 3 0 1 .5 n /a ( d 0 – 6 ) m o d e ra te n e p h ro si s s e ve re d iff u se n e p h ro si s 7 2 .2 5 w e a k ru m in a l m o ve m e n ts ( d 8 ); a cu te p n e u m o n ia o f a p ic a l a n d c a rd ia c m o d e ra te t o s e ve re h e p a to si s st ro n g e r ru m in a l m o ve m e n ts ( d 1 1 ); lo b e s re co ve re d ( d 1 3 ) p u ru le n t b ro n ch o p n e u m o n ia 1 1 9 2 .8 d e p re ss e d , in a p p e ta n ce ( d 1 1 9 ); re cu m b e n cy , ta ch yc a rd ia , ru m in a l st a si s, t e e th g ri n d in g , g ro a n in g , e u th a n a se d in e xt re m is (d 1 2 0 ) 1 3 9 2 1 .5 0 1 .5 n /a ( d 0 – 6 ) m o d e ra te t o s e ve re n e p h ro si s p ro m in e n t d ila ta tio n o f tu b u le s co n ta in in g g ra n u la r o r p ro te in c a st s 7 2 .2 5 n /a ( d 7 – 1 3 ) m ild , w a te ry a sc ite s l in in g e p ith e liu m o f tu b u le s fla tt e n e d 1 1 9 2 .8 s lig h tly d e p re ss e d , ta ch yc a rd ia , w ith n o s ig n s o f re g e n e ra tio n d e cr e a se d a p p e tit e ( d 1 2 0 ); w e a k ru m in a l m o ve m e n ts ( d 1 2 1 ); s tr o n g e r s o m e g lo m e ru li w e re d ila te d ru m in a l m o ve m e n ts ( d 1 2 5 ); t e e th g ri n d in g , ru m in a tin g ( d 1 2 6 ); m ild , m u lti fo ca l, ch ro n ic in te rs tit ia l e u th a n a si a ( d 1 2 8 ) n e p h ri tis m ild s w e lli n g o f h e p a to cy te s m ild , d iff u se m o n o n u cl e a r in te rs tit ia l p n e u m o n ia * c lin ic a l s ig n s in o rd e r o f a p p e a ra n ce 43 r.a. meintjes, c.j. botha & l. prozesky ��� � � �� � � �� � � � � � �� �� �� �� �� �� � �� � �� �� � � � � �� ��� ������������ ��� ������������ ��� �������������� ��� �������������� � �� ��� � � �� � � �� � �� � �� � � � � � �� ����� ��� �� ��� ��� � �� � �� �� � � � � �� ��� ������������ ��� ������������ ��� �������������� ��� �������������� fig. 1 serum urea concentration in four sub-acutely affected sheep following administration of dried, milled nolletia gariepina plant material fig. 2 serum creatinine concentration in four sub-acutely affected sheep following administration of dried, milled nolletia gariepina plant material for longer periods, but were depressed, anorexic and had weak rumen movements and tachycardia. administration of half the dose (1.5 g/kg) to two wethers elicited no clinical effects. following a washout period of 7 days the same two sheep were dosed with 2.25 g/kg n. gariepina plant material. only one animal was mildly affected clinically (decreased appetite and weak ruminal movements), but was judged fully recovered after 6 days. more than 3 months later the same two wethers were dosed again with 2.8 g/kg and at this dose, moderate to severe clinical signs developed (table 1). pathophysiology non-protein nitrogen (npn) substances in serum the most noticeable aberrations in blood chemistry were the remarkable increases in serum urea and creatinine concentrations in sheep that survived for 24 h or longer (fig. 1 and 2). serum ast and ggt activities a moderate increase in ast activity (96–157 u/l) above the reference range (5–60 u/l) occurred in some sheep. there was no noticeable increase in ggt activity. daily urine volumes following dosage of the sheep with n. gariepina, even at doses of 1.5 g/kg, urine production in general increased. in some cases, however, e.g. sheep a5 and sheep a9 dosed with 3 g/kg, the increase was preceded by virtual anuria on the day immediately following dosage (table 2). urinalysis semi-quantitative urinalysis (urinary dip stick) revealed mild proteinuria and presence of haemoglobin in the urine after dosing. glomerular filtration rate (gfr) dramatic decreases in gfr were calculated in sheep, which survived at least one day post-dosing, when the plant material was dosed in excess of 1.5 g/kg (table 3). urinary sodium excretion during the control phase no sodium could be detected in the urine, but within 24 h of dosing the nolletia plant material at doses of 2.8 g/kg and above, urinary sodium concentrations rose. the fractional excretion of sodium is given in table 4. urinary potassium excretion the fractional excretion of potassium increased, even after receiving relatively low doses (1.5 g/kg) of nolletia plant material. as potassium is excreted by the nephron tubule, values in excess of 100 % are encountered, sometimes even under control conditions. 44 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. table 2 urine volumes (l) of sheep-control values and values following dosing with nolletia gariepina dose rate sheep control day 1 day 2 day 3 day 4 day 5 day 6 day 7 no. mean (sd) post post post post post post post dosing dosing dosing dosing dosing dosing dosing 1.5 g/kg 42 0.21 (0.06) 0.64 0.66 0.71 13 0.275 (0.069) 0.35 0.33 0.27 2.25 g/kg 42 0.21 (0.06) 0.64 1.42 3.047 1.7 2.129 2.042 13 0.275 (0.69) 0.71 0.37 0.445 0.44 0.52 0.56 2.8 g/kg 42 0.33 (0.11) died 13 0.161 (0.044) 0.59 0.28 0.64 0.71 0.6 0.6 1 3.0 g/kg a9 0.874 (0.25) 0.099 1.37 1.46 1.068 1.305 1.46 2.67 a5 0.643 (0.165) 0 0.86 3.12 1.21 0.66 euth* a4 0.868 (0.207) euth.* s5 0.32 (0.15) died a14r 0.573 (0.14) died a13b 0.378 (0.15) died a13r 0.754 (0.1) died * euth. = euthanasia 45 r.a. meintjes, c.j. botha & l. prozesky table 3 glomerular filtration rates (l/day) of sheep—control values and values following dosing with nolletia gariepina dose rate sheep control day 1 day 2 day 3 day 4 day 5 day 6 day 7 no. mean (sd) post post post post post post post dosing dosing dosing dosing dosing dosing dosing 1.5 g/kg 42 37.29 (4.06) 43.05 44.43 13 49.07 (8.25) 46.83 38.33 2.25g/kg 42 37.29 (4.06) 4.8 6.56 1.93 3.07 13 49.07 (8.25) 15.42 26.38 23.85 37.44 2.8 g/kg 42 37.73 (8.09) died 13 33.11 (9.48) 16.53 28.06 10.71 1.62 2.18 4.01 3.0 g/kg a9 87.79 (11.95) 0.49 5.27 2.76 1.15 1.59 1.89 4.73 a5 62.03 (15.15) –a 8.74 15.99 4.65 1.95 euth* a4 41.89 (2.06) euth.* s5 75.32 (1.96) died a14r 68.14 (1.55) died a13b 33.22 (10.55) died a13r 103.41 (12.19) died * euth. = euthanasia a = no urine produced table 4 fractional excretion of sodium (%) and potassium (%) in sheep—control values and values following dosing with nolletia gariepina dose rate sheep no. control day 1 day 2 day 3 day 4 day 5 day 6 day 7 mean post post post post post post post (sd) dosing dosing dosing dosing dosing dosing dosing 1.5 g/kg 42 na 0 0 0 k 82 (4.2) 101 195 13 na 0 0 0 k 101 (11) 101 150 2.25 g/kg 42 na 0 0 0 0 0 k 82 (4.2) 444 695 2 533 1 989 13 na 0 0 0 0 0 k 101 (11) 180 178 276 229 2.8 g/kg 42 na 0 died k 51 (18) 13 na 0 1 0.1 5.4 16 7.8 9.0 k 37 (14) 33 45 166 614 573 378 3.0 g/kg a9 na 0 5.7 1.8 11.8 13.9 11.9 16.5 k 97 (53) 315 845 1 028 1 225 1 084 796 a5 na 0 –a 0 5.9 10.1 5.25 euth* k 89 (15) –a 45 533 673 648 a4 na 0 euth.* k 137 (39) s5 na 0 died k 89 (38) a14r na 0 died k 102 (20) a13b na 0 died k 124 (46) a13r na 0 died k 68 (8) * euth. = euthanasia a = no urine produced urine osmolarity in most of the animals, which did not die within 24 h of dosing the plant material, urine osmolarity decreased markedly (table 5). urinary ggt activity although no change in serum ggt activity was noticed following dosing with nolletia plant material, urine ggt activity tended to rise and then fall within a week (table 6). pathology macroscopic pathology a severe nephrosis was present in all the animals. the kidneys exhibited various degrees of swelling, bulged slightly on cut surface and ranged in colour 46 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. table 5 urine osmolarity (mosm/l) of sheep—control values and values following dosing with nolletia gariepina dose rate sheep control day 1 day 2 day 3 day 4 day 5 day 6 day 7 no. mean (sd) post post post post post post post dosing dosing dosing dosing dosing dosing dosing 1.5 g/kg 42 2 516 (357) 1 267 1 721 13 2 647 (79) 2 312 2 900 2.25 g/kg 42 2 516 (357) 624 566 496 566 634 13 2 647 (79) 813 1 877 2 182 2 237 2 440 2.8 g/kg 42 1 452 (222) died 13 2 355 (163) 1 311 990 658 567 659 635 3.0 g/kg a9 1 375 (364) 582 508 520 550 613 584 444 a5 1 470 (597) –a 493 255 539 567 euth.* a4 1 071 (145) euth.* s5 1 948 (456) died a14r 1 875 (406) died a13b 1 947 (366) died a13r 1 498 (243) died * euth. = euthanasia a = no urine produced table 6 urine gamma glutamyltransferase (ggt) concentrations (units/l) in sheep—control values and values following dosing with nolletia gariepina dose rate sheep control day 1 day 2 day 3 day 4 day 5 day 6 day 7 no. mean (sd) post post post post post post post dosing dosing dosing dosing dosing dosing dosing 1.5 g/kg 42 25 (13) 10 10 13 26 (8) 15 15 2.25 g/kg 42 25 (13) 36 75 30 18 13 26 (8) 9 36 24 9 2.8 g/kg 42 14 (7) died 13 35 (23) 9 48 117 69 30 39 21 3.0 g/kg a9 10.5 (1) 66 165 90 42 36 21 10 a5 13 (3) –a 96 71 71 58 euth.* a4 18 (15) euth.* s5 16 (7) died a14r 19 (6) died a13b 12 (3) died a13r 16 (2) died * euth. = euthanasia a = no urine produced 47 r.a. meintjes, c.j. botha & l. prozesky from light brown to greyish-white (fig. 3 and 4). widespread serosal haemorrhages; mild accumulation of fluid in the body cavities; moderate hepatosis characterized by mild swelling of the liver with rounded edges, slightly pale colour and a taut capsule; and lung congestion and oedema were often seen (table 1). light microscopy kidney a severe nephrosis was the most striking lesion present. the epithelium of the proximal convoluted tubules (pct) in the cortex and straight tubules in the cortico-medullary junction was the most severefig. 3 and 4 severe nephrosis in a sheep dosed with nolletia gariepina plant material 3 4 ly affected. epithelial cells showed karyolysis, karyorrhexis or pyknosis of nuclei with clumping and dissociation of the brightly eosinophilic cytoplasm (necrosis), or increased eosinophilia and vacuolated appearance (hydropic degeneration) of the cytoplasm. the tubules contained large numbers of protein casts in the lumens (fig. 5 and 6). single neutrophils were present in the interstitial tissue and a mild neutrophilia was evident in the blood vessels. in one animal (no. 13) a few tubules were dilated and single tubules exhibited moderately basophilic epithelial cells with large, vesicular nuclei indicative of early regeneration. no specific lesions were visible in the glomeruli. 48 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. fig. 5 and 6 widespread necrosis of renal tubular epithelium, particularly in the proximal convoluted tubules. the tubules contain large numbers of protein casts in the lumens 5 6 liver mild hepatocellular swelling with accumulation of multiple small vacuoles in the cytoplasm of hepatocytes (indicating hydropic degeneration) was the most striking lesion in most animals. scattered hepatocytes throughout the parenchyma were necrotic, evidenced by increased eosinophilia of the cytoplasm and karyorrhexis, karyolysis or pyknosis of the nuclei. no specific lesions were visible in the portal tracts and bile ducts. 49 r.a. meintjes, c.j. botha & l. prozesky fig. 7 proximal convoluted tubule epithelial cell. note the swollen mitochondria with loss of internal structure ( ). parallel orientation of mitochondria to the cell axis is lost fig. 8 proximal convoluted tubule epithelial cell. note the increase in intracellular electrondense bodies interpreted as primary lysosomes ( ) 7 8 transmission electron microscopy kidney the most striking changes occurred in the pct of both animals (fig. 7–10). individual cells lining the tubules could not be identified because of loss of the cell membranes. affected tubules were demarcated by the intact basement membrane. the usually conspicuous brush border and apical canaliculi of the epithelial cells had disappeared. the cytoplasmic organelles and nuclei were disorganized and dispersed as a loose mass within the tubular lumen. 50 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. fig. 9 and 10 proximal convoluted tubule epithelium. note the loss of cell membranes ( ) with chromatin margination (c). cytoplasmic organelles are disorganized and dispersed as a loose mass within the tubular lumen( ). the basal membrane is intact ( ) 9 10 c c nuclear changes ranged from chromatin margination and karyolysis to pyknosis. chromatin margination, the most common change exhibited, was evident as condensation of the chromatin in irregular clumps (karyorrhexis), or as a thin continuous rim along the inner membrane of the nuclear envelope, with disappearance of the chromatin from other areas of the nucleus and loss of nucleolus. some affected nuclei were interpreted as being in an intermediate stage between margination and lysis. pyknotic nuclei were recognized by the shrunken nucleus with diffuse condensation of the chromatin. the most conspicuous change in the proximal tubular epithelial cell cytoplasm was increased numbers of electron-dense, single membrane-bound bodies interpreted as primary lysosomes. also present were variably sized, single membrane-bound vacuolar bodies which contained medium electron–dense, finely granular material that could not be identified because of the advanced stage of breakdown and degradation and most probably represented autolysosomes. scattered between the necrotic cellular organelles were residual bodies loaded with undigested electron-dense residues of various kinds. the normal basal distribution of the mitochondria, with parallel orientation to the cell axis, was lost. mitochondria had a rounded form and some were severely swollen with a loss of internal structure, whereas others were electron-dense and could not be distinguished unequivocally, by morphological features alone, from other vesicular structures in the cell including primary lysosomes. discussion following ingestion of n. gariepina there appears to be a threshold before toxicity will be induced. when 3 g/kg of dried, milled plant material was dosed orally, five of the seven sheep died acutely or were euthanased for humane reasons. on the other hand, administration of 1.5 g/kg elicited no clinical signs and 2.25 g/kg only induced mild clinical signs. during the reported field outbreaks, the cattle were deprived of feed for 24–48 h and, following their release, they presumably consumed large quantities of the plant material (du plessis 2004; du plessis et al. 2004), thus most probably exceeding the threshold level. although this plant is highly toxic it has, except for the outbreak in cattle, never before been associated with poisoning of livestock (du plessis 2004). the acute mortalities observed in this study could be ascribed to other toxins, besides the nephrotoxin(s), contained by the plant. analysis of the plant material for cyanogenic potential gave negative results. the nephrotic syndrome corroborated by the gross and histological findings was only reproduced in sheep that survived for longer than 24 h. the concentrations of non-protein nitrogen substances in the plasma generally increase when gfr decreases by more than 75 % (more than 75 % kidney damage has occurred). this is because of the large “reserve capacity” of the kidneys (rose & renkke 1994). in the animals, which did not die acutely, kidney damage was so severe, enough to induce marked increases in the plasma concentrations of creatinine and urea as well as a low gfr. as no specific damage to the glomerulus was described on histopathology and there is no reason to believe that the azotaemia was pre-renal (the animals were not dehydrated, neither was any cardiac pathology detected), the drastic fall in gfr was due to blockage of proximal tubules by cellular debris and protein casts (as was obvious on the histopathological findings). the reverse pressure across the filtration membrane due to such blockage would effectively stop the filtration process. the appearance of sodium in the urine and the rise of potassium concentration in the urine, as well as the increase in urine volume, despite the marked fall in gfr, could be ascribed to two factors. the flow of filtrate in non-damaged nephrons may have been so rapid that there was little chance for the reabsorption of sodium, potassium and water. more likely, however, in damaged nephrons (proximal tubular epithelial damage with loss of brush border), which still permitted filtrate flow, sodium, potassium and water reabsorption would be compromised. most of the sodium, potassium and water reabsorption occurs in the pct and damage to the renal epithelium in this region would account for losses of large amounts of electrolytes and water (by osmotic diuresis) (knepper & burg 1983). the reduction in urine osmolarity indicates, that, in spite of the natriuresis and kaliuresis, more water relative to electrolytes was lost. this may be a phenomenon associated purely with proximal tubular damage or possibly the distal part of the nephron duct becomes refractory to the effects of antidiuretic hormone (as in chronic nephritis). gamma glutamyltransferase is present in the brush border of the proximal convoluted tubule where it facilitates transfer of glutamate into the tubular cells (welbourne & mathews 1999). a rise in urine ggt activities when there is proximal tubular damage, is 51 r.a. meintjes, c.j. botha & l. prozesky therefore in accordance with the pathological findings in this section of the nephron tubule, as was seen in this trial. the pathological lesions in the experimental sheep correspond to a large extent with the lesions described in the experimentally produced cases in cattle, but were much less pronounced compared to the lesions in the field outbreak in cattle (du plessis 2004). in both species under experimental conditions gross lesions included mild to moderate accumulation of fluid in body cavities, severe nephrosis and a moderate hepatosis. contrary to the situation in the experimentally produced cases in cattle, perirenal oedema was not a striking feature in sheep, whereas widespread serosal haemorrhages were present in the latter. nephrosis was characterized by degeneration and necrosis of particularly the pct and affected tubules contained large numbers of protein casts in the lumens. the mild neutrophyllic infiltration in the interstitial tissue was most likely in response to the tubular necrosis. scattered tubules were dilated and in one animal early tubular epithelial regeneration was displayed. transmission electron microscopy confirmed that the basement membrane of affected tubules was still intact and a significant increase of autolysosomes was displayed in the affected lining epithelial cells of the pct. no structural visible lesions were evident in the glomeruli. most animals revealed mild hepatocellular swelling, with accumulation of multiple small vacuoles in the cytoplasm of hepatocytes indicating hydropic degeneration. the pct epithelium is the most susceptible to both toxic and hypoxic injury because of its high metabolic rate (jamison, myers & neild 1997; confer & panciera 2001). degeneration and necrosis of the tubular epithelium in the kidneys (nephrosis), especially the pct, is a non-specific lesion. it can be hypoxic or nephrotoxic in origin. the former follows on a period of hypotension causing marked renal ischaemia. complete ischaemia of more than 2 h duration causes irreversible tubular necrosis, especially of the pct (confer & panciera 2001). nephrotoxic acute tubular necrosis (atn) can be distinguished from ischaemic atn by two histological features viz. extensive necrosis of predominantly pct epithelium and intact tubular basement membranes. the opposite is true for ischaemic atn, where necrosis is patchy and mostly affects the pct with disruption of the basement membrane (maxie 1993; jamison et al. 1997; confer & panciera 2001). toxic damage to tubular epithelium has a better prognosis if the animal survives the initial toxic insult, as the basement membranes that are necessary for epithelial regeneration are intact (maxie 1993), and some nephrotoxic drugs can cause ischaemia rather than direct toxic damage to the renal tubules (jamison et al. 1997). in conclusion, dosing of n. gariepina plant material to sheep in doses exceeding 2.8 g/kg resulted in acute death of most animals and marked changes in the clinical chemistry of the survivors characterized by a sudden, marked decrease in gfr, azotaemia, elevated fractional excretion of na and k, and increased urinary ggt activity. sheep, which died or were euthanased, exhibited severe nephrosis that was histologically characterized by necrosis of predominantly lining epithelial cells of the pct and the presence of protein and cellular casts. acknowledgements we thank ms h. engelbrecht and ms h. rossouw, section of physiology; ms e. myburgh, section of clinical pathology; ms e. van wilpe and ms d. meyer, electron microscopy unit, faculty of veterinary science, university of pretoria; and ms l. labuschagne, division of toxicology, onderstepoort veterinary institute for their valuable assistance. the authors also express their gratitude to the national research foundation (nrf) of south africa and mr w. du plessis, the farmer, for funding this trial. the research reported here emanates from project 36.5.520, approved by the animal use and care and research committees of the faculty of veterinary science, university of pretoria. references bastl, c.p., rudnick, m.r. & nairns, r.g. 1985. assessment of renal function: characteristics of the functional and organic forms of acute renal failure, in the kidney: physiology and pathophysiology, edited by d.w. seldin & g. giebisch. new york: new york press. confer, a.w. & panciera, r.j. 2001. the urinary system, in thomson’s special veterinary pathology, 3rd ed., edited by m.d. mcgavin, w.w. carlton & j.f. zachary. missouri: mosby inc. du plessis, e.c. 2004. pathological investigation of the nephrotoxic effects of the shrub nolletia gariepina (dc) mattf. in cattle. mmedvet (path) dissertation, university of pretoria. du plessis, e.c., joubert, j.p.j., prozesky, l., naudé t.w., herman, p. & van der westhuizen, g.b.m. 2004. nephrotic syndrome in cattle caused by the shrub nolletia gariepina in the kalahari sandveld of southern africa, in poisonous plants and related toxins, edited by t. 52 toxicity, pathophysiology and pathology of the nephrotoxic plant nolletia gariepina (dc) mattf. acamovic, c.s. stewart & t.w. pennycott. wallingford: cabi publishing. jamison, r.l., myers, b.d. & neild, g. 1997. acute renal failure, in nephrology, edited by r.l. jamison & r. wilkinson. london: chapman & hall medical. knepper, m. & burg, m. 1983. organisation of nephron function. american journal of physiology, 244, f579–f589. maxie, m.g. 1993. the urinary system, in pathology of domestic animals, 4th ed., edited by k.v.f. jubb, p.c. kennedy & n. palmer. california: academic press inc. nawaz, m. & shah, b.h. 1984. renal clearance of endogenous creatinine and urea in sheep during summer and winter. research in veterinary science, 36:220–224. rose, b.d. & renkke, h.g. 1994. review of renal physiology, in renal pathophysiology—the essentials. baltimore: williams & wilkins. welbourne, t.z. & mathews, j.c. 1999. glutamate transport and renal function. american journal of physiology, 277 (4 pt2), f501–f505. 53 r.a. meintjes, c.j. botha & l. prozesky article information authors: anita l. michel1 roy g. bengis2 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa 2directorate veterinary services, skukuza, south africa correspondence to: anita michel postal address: private bag x04, onderstepoort 0110, south africa how to cite this proceeding: michel, a.l. & bengis, r.g., 2012, ‘the african buffalo: a villain for inter-species spread of infectious diseases in southern africa’, onderstepoort journal of veterinary research 79(2), art. #453, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.453 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the african buffalo: a villain for inter-species spread of infectious diseases in southern africa in this proceeding... open access • abstract • introduction    • status of african buffaloes    • infectious diseases transmitted by african buffaloes    • factors related to african buffaloes in disease transmission    • factors related to the pathogen    • factors related to the environment    • impact of infectious diseases on african buffaloes and their environment    • diagnosis and control of infectious diseases in african buffaloes • conclusion • references abstract top ↑ the african buffalo (syncerus caffer) is a large wild bovid which until recently ranged across all but the driest parts of sub-saharan africa, and their local range being limited to about 20 km from surface water. they are of high ecological value due to their important role as bulk feeders in the grazing hierarchy. they also have high economic value, because they are one of the sought after ‘big five’ in the eco-tourism industry. in africa, buffaloes have been recognised for some time as an important role player in the maintenance and transmission of a variety of economically important livestock diseases at the wildlife and/or livestock interface. these include african strains of foot-and-mouth disease (fmd), corridor disease (theileriosis), bovine tuberculosis and bovine brucellosis. for a number of other diseases of veterinary importance, african buffaloes may also serve as amplifier or incidental host, whereby infection with the causative pathogens may cause severe clinical signs such as death or abortion as in the case of anthrax and rift valley fever, or remain mild or subclinical for example heartwater. the long term health implications of most of those infections on the buffalo at a population level is usually limited, and they do not pose a threat on the population’s survival. because of their ability to harbour and transmit important diseases to livestock, their sustainable future in ecotourism, trade and transfrontier conservation projects become complex and costly and reliable diagnostic tools are required to monitor these infections in buffalo populations. introduction top ↑ the transmission and spread of infectious diseases amongst and between domestic and wild animals can occur directly through contact (foot-and-mouth disease [fmd], bovine tuberculosis and brucellosis), or indirectly through the agency of haematophagous arthropod vectors such as mosquitoes, tsetse flies and ixodid ticks (kock 2005). in addition, environmental contamination with infected ticks (theileriosis) or fomites (anthrax) can infect livestock even when the habitat is not shared at the same time. some of these diseases can affect a variety of wildlife hosts, but only very few species play a decisive role in disease maintenance and transmission at the wildlife and/or livestock interface. the increasing competition for land available for livestock – based agriculture or wildlife ranching and conservation has highlighted the need for an integrated approach to sustainable livestock and wildlife health, use and management. in this debate the african buffalo features as a villain to those who wish to protect livestock populations from devastating diseases, as they undermine national and international disease eradication schemes, which have been implemented and executed with significant success, and at great cost in the past (munag’andu et al. 2006). on the other hand, conservationists and wildlife ranchers take on a distinctly different attitude towards controlling livestock diseases transmitted by wildlife (bengis, kock & fischer 2002). in this overview the role of the african buffalo, both as a victim and asymptomatic carrier in the transmission of the most important livestock diseases at the wildlife and/or livestock interface, is reviewed. links between disease ecology and buffaloes’ ecology are examined in an attempt to reveal why buffaloes are successful maintenance hosts for several of those diseases. status of african buffaloes since the early days of european settlement in southern and eastern africa, african buffaloes have attracted the admiration of hunters and have consequently been a highly sought after trophy animal for the past three centuries (prins 1996). in addition, they gained extraordinary value for other ecotourism related purposes during the past decades (van der merwe, saayman & krugell 2004). the african buffalo (syncerus caffer) is one of the most ubiquitous large herbivores which can tolerate a wide range of climatic conditions and habitats as long as it is provided with access to an abundant supply of water (grimsdell 1969; sinclair 1977). ecologically, the african buffalo is a bulk grazer and occupies an important niche through opening up habitats that are preferred by short-grass grazers (de vos & bengis 1994). currently the conservation status of the african buffalo is satisfactory with no immediate threat of extinction (friedmann & daly 2004). infectious diseases transmitted by african buffaloes african buffaloes have been recognised for some time for various roles in disease transmission amongst wildlife species and at the interface with livestock. amongst the best known diseases are foot-and-mouth disease, caused by the african strains sat 1, 2 and 3 (fmd viruses) and corridor disease (theileriosis, caused by theileria parva), owing to the disastrous, large scale clinical outbreaks they may cause in livestock (anonymous 2007; norval, perry & young 1992). however, african buffaloes have been implicated in a number of other epidemiological roles involving both indigenous and alien livestock diseases. diseases which originated in africa and co-evolved with african wildlife species including african buffaloes are defined as indigenous. they generally do not pose a threat on the survival of their hosts’ populations because of the evolutionary development of unique coping mechanisms. well known indigenous diseases maintained by african buffaloes include fmd caused by the sat strain viruses (thomson 1995), corridor disease and african trypanosomosis (anonymous 2007, 2009; de vos & bengis 1994; norval et al. 1992). in contrast, foreign, so-called alien livestock diseases, such as bovine tuberculosis (mycobacterium bovis) and bovine brucellosis (brucella abortus), have been introduced and successfully established themselves in buffaloes which serve as wildlife maintenance host (de vos et al. 2001). rinderpest falls within the same grouping of alien diseases but its extreme pathogenicity and high mortality precluded the development of a wildlife reservoir (de vos & bengis 1994). the devastating effect of rinderpest on many cloven hoofed african ungulate species and african buffaloes (as an epidemic end host) in particular at a local or regional population level was more important ecologically and epidemiologically than their potential role in transmission of the disease to livestock. in 2011, rinderpest was officially eradicated from the world (morens et al. 2011) and therefore its significance for interspecies transmission no longer exists. due to their wide geographical range and distribution, african buffaloes have been furthermore implicated as amplifiers or incidental hosts in the epidemiology of a number of other indigenous infectious agents, including those confined to specific localised geographic areas, for example trypanosomes is limited to certain savannah and forested areas endemic for tsetse flies, and buffalo are one of the preferred hosts for these flies and develop a symptomatic carrier state for this parasite (molooa et al. 1999). anthrax, caused by brucella anthracis is limited to certain anthrax endemic areas of the african continent (anonymous 2009). for rift valley fever little is known about the precise mechanisms of virus maintenance and transmission. however, buffaloes have been associated with a possible sentinel role during epidemics and a possible maintenance role during interepidemic periods, respectively (bengis et al. 2010; labeaud et al. 2011). factors related to african buffaloes in disease transmission the epidemiology of infectious diseases is determined by factors related to the host, the environment and the causative pathogen. in the case of african buffaloes a number of intrinsic behavioural characteristics are instrumental to its role in hosting and transmitting livestock diseases. they are a highly social species living in large herds of up to 1000 animals, whose gregarious behaviour provides ideal conditions for direct pathogen transmission via aerosol and body secretions (grimsdell 1969; michel et al. 2006). individual or small groups of buffaloes have also been shown to migrate over large distances within short periods of time, either in response to drought or as a result of the dispersal strategies for heifers or bachelor bulls. these events, like fission and fusion events driven by changes in the seasonal availability of grazing, take place on a regular basis and constitute a powerful vehicle for pathogen dispersal across herds (cross, lloyd-smith & getz 2005; halley, vandewalle & taolo 2002). factors related to the pathogen as regards the chronic, slow alien livestock diseases, such as bovine tuberculosis and bovine brucellosis, the lack of coping mechanisms in naïve african buffaloes may result in deleterious long term effect on certain buffalo populations. some pathogens exhibit a very strict and narrow host range, for example t. parva, whilst m. bovis is capable of causing disease in a very broad spectrum of domestic and wild animal species. amongst its wildlife hosts, maintenance host potential has been confirmed for african buffaloes and is suspected for greater kudu and possibly others (michel et al. 2006). indirect transmission of pathogens via environmental contamination is more effective if the organism can survive for significant periods of time outside of its host(s). whilst the survival of most viruses outside the host is very short, m. bovis has been shown to survive for between five days and four weeks in the environment (tanner & michel 1999) and the spore forming bacillus anthracis bacterium is ideally equipped for long term (years) survival outside of a host (de vos 2004). factors related to the environment african buffaloes occur in many different habitats including woodlands, grasslands, swamps, floodplains and thickets, at a range of altitudes, provided they have access to an abundant supply of water and good quality grazing. this species’ ability to survive in all but the more arid habitats, allows it to host a variety of pathogens under diverse environmental conditions. for example, the abundance of the aedine mosquitoes, the endemic vector of the rift valley fever virus, is influenced by wet climatic shifts which are frequently associated with increased virus transmission to domestic and wild ruminants (bengis et al. 2010). in addition, african buffaloes spend a considerable percentage of their time in and near water (ryan, knechtel & getz 2006) which further increases their risk of exposure to vectors. in addition, african buffaloes are a preferred host for certain ixodid tick species, the vectors of various protozoal and rickettsial parasites of bovids. the modern wildlife industry grows at an average annual rate of 5.6% in terms of area exempted for game ranching and is mainly based on ecotourism, hunting and live game trade (cloete, taljaard & grove 2007). this rapid growth rate in conjunction with the historical iconic role of the african buffalo as a member of the ‘big five’ for viewing, photographing and hunting has lead to an increase in the numbers and distribution of buffaloes on private land. in certain parts of south africa the number of game farms registered for keeping buffaloes has equalled or exceeded that of livestock, resulting in an expansion of the wildlife and/or live stock interface and hence a risk in bi-directional disease transmission. high population densities and frequent translocation of african buffaloes between private game properties for commercial gain are contributing to cumulative risk of african buffalo to contract or transmit livestock diseases. for this reason, in south africa buffaloes and cattle may not be kept together on shared rangeland. however, pathogens do not generally respect fences. against the background of the establishment of large wildlife conservation areas and transfrontier parks in southern africa, the potential role of buffaloes in the transmission of infectious diseases at the wildlife and/or livestock interface cannot be ignored. high expectations have been linked to the creation of large conservation areas in terms of improved and sustainable livelihoods for the communities in and around those wildlife areas. however, increased numbers of livestock and buffaloes, in the absence of game deterrent fences and often a lack of effective livestock vaccines, will inevitably increase the risk of disease transmission, and make disease eradication from livestock virtually impossible (kock 2005). impact of infectious diseases on african buffaloes and their environment amongst the infectious diseases, fmd and corridor disease and african trypanosomosis are truly asymptomatic in african buffaloes, which act as biological reservoirs of infection in an endemic cycle. the fmd infection occasionally escapes into other sympatric wild cloven-hoofed species, and where livestock are in contact with african buffaloes, they may become infected resulting in devastating outbreaks with a high socio-economic impact (vosloo et al. 2005). rift valley fever, is a seasonal vector born disease and viremia and abortions have been reported in african buffaloes (evans et al. 2008). their exact role in the epidemiology of rift valley fever is however, still unknown but results of serological investigations in kenya and south africa indicated that african buffaloes become infected with the virus (evans et al. 2008; labeaud et al. 2011). bovine tuberculosis in african buffaloes has been well documented and can be described as a chronic, progressively debilitating condition (de vos et al. 2001). it has spread throughout the buffalo population of the kruger national park and spilled into more than a dozen other wildlife species. there is a continuous risk for spillback into livestock and an associated zoonotic risk for animal owners, which is undetermined (berg et al. 2011; michel, müller & van helden 2010). anthrax is a sporadic, usually fatal disease affecting buffaloes in numerous endemic areas in sub-saharan africa (de vos 1990; mohan & gotts 1970). herbivores are known to be more susceptible to anthrax than omnivores and carnivores, and losses in african buffaloes, greater kudu, nyala and waterbuck have been significant in outbreaks of this disease in southern and eastern africa (de vos 2004). when buffaloes are affected by anthrax, they serve as highly effective multipliers of b. anthracis, which can contaminate the soil and run-off water, and carcasses become amplifiers for blowflies. anthrax may be transmitted to neighbouring livestock farms and initiate an outbreak amongst cattle, and the converse has also been experienced when an outbreak in cattle spilled over into wildlife in an adjoining conservation area (malilangwe – zimbabwe) (de vos 1990). it has been speculated that african buffaloes are the species with the highest susceptibility to rinderpest, which decimated the buffalo populations all over africa during the rinderpest epidemic between 1888 and 1899 (de vos & bengis 1994). diagnosis and control of infectious diseases in african buffaloes availability of diagnostic tests for wildlife species is very often the biggest limitation in diagnosing infectious diseases in target wildlife species which are taxonomically far removed from the closest livestock counterparts. the african buffalo falls into the subfamily bovinae together with domestic cattle, which is helpful and provides a basis for adapting and developing diagnostic tests for relevant infections in buffaloes. however, it needs to be emphasised that all tests applied in buffaloes still require validation to prove fitness for purpose as stated by the world organisation for animal health (oie) (oie 2009). the question whether infectious diseases in buffaloes require controlling is a contentious one and is best discussed per disease grouping. since the causative agents of certain indigenous diseases, namely fmd viruses, trypanosomes and theilerias, are widely maintained by african buffaloes and generally do not pose a threat to these population’s existence, diagnostic testing is usually applied for monitoring and surveillance activities at population level. for movement purposes and international trade, the same tests (including direct pathogen detection for corridor disease and trypanosomosis) are applied at individual animal level in conjunction with herd testing records. it is accepted that fmd can at best be contained in wildlife areas that have buffaloes, but eradication will not be achievable (vosloo et al. 2005). the situation is essentially the same for indigenous diseases detected less frequently in african buffaloes, such as rift valley fever and anthrax, where outbreaks are monitored strictly for epidemiological information and analyses. a very different scenario is presented in the cases of bovine tuberculosis and bovine brucellosis, which are both alien diseases introduced into the buffalo population most likely through contact with infected cattle. following their successful establishment in african buffaloes as their wildlife reservoir or maintenance host, the state veterinary and conservation authorities have been faced with a set of previously unknown challenges. apart from the need to prevent spillover of the diseases to neighbouring livestock, these diseases may spread uncontrolled within infected conservation areas. although our current knowledge on the impact of bovine tuberculosis and bovine brucellosis does not indicate an immediate threat on buffalo survival at the population level, it would be premature to make any prediction about the long term impact at this point in time. as no effective vaccine exists for bovine tuberculosis in animals and given the extremely broad host spectrum of the causative organism, the real threat goes beyond buffalo conservation but concerns severely affected sympatric species such as lions and kudus (michel et al. 2009). it is fair to speculate that african buffaloes will not remain the only maintenance host in an endemically infected ecosystem such as the kruger national park and disease management and control strategies are urgently called for. two effective vaccines are registered for the control of bovine brucellosis in cattle, but their efficacy and safety in african buffaloes has not been tested, as the disease is currently considered to have little effect on african buffaloes and the ecosystem (godfroid 2004). pre-movement testing of buffaloes for both bovine tuberculosis and brucellosis is an absolute requirement to minimise the risk of disease transmission to uninfected populations. in the case of bovine tuberculosis serological tests are not useful to detect infection and the intradermal tuberculin test (skin test) is applied in conjunction with the interferon gamma assay (michel et al. 2011; michel & simoes 2009). conclusion top ↑ it is clear that disease transmission at the interface is bi-directional and african buffaloes are culprits or villains in respect of some diseases, whilst they have fallen victim to alien diseases transmitted from cattle. as the wildlife and/or livestock and/or human interface is rapidly expanding and gaining in intensity and complexity, there should be no room for a blaming game, but it must be appreciated that african buffaloes form an integral part of the indigenous fauna of africa and are inseparable from their indigenous pathogens. a strictly separate approach is needed for the alien diseases which may directly affect the health of buffaloes or indirectly the conservation of wildlife by turning infected areas into ‘conservation islands’ and making them unavailable for conservation in the bigger sense. to enable ecologists, veterinary researchers and 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africa’, revue scientifique et technique 14, 503–520. pmid:8593386 van der merwe, p., saayman, m. & krugell, w., 2004, ‘factors that determine the price of game’, koedoe 47, 105–113. vosloo, w., bastos, a.d.s., sahle, m., sangare, o. & dwarka, r.m., 2005, ‘virus topotypes and the role of wildlife in foot and mouth disease in africa’, southern and east african experts panel on designing successful conservation and development interventions at the wildlife/livestock interface: implications for wildlife, livestock, and human health. ahead animal health for the environment and development, forum. iucn/ssc veterinary specialist group, southern africa sustainable use specialist group et al., iucn, gland, switzerland and cambridge, uk, 14–15 september 2003. article information authors: justin masumu1,2,3 g. tshilenge1,2 v. mbao4 affiliations: 1southern african centre for infectious disease surveillance, chuo kiikuu, morogoro, tanzania 2laboratoire vétérinaire central, kinshasa, democratic republic of the congo 3institut national de recherche biomédicale, avenue de la democratie, kinshasa i/rdc 4centre for ticks and tick-borne diseases, lilongwe, malawi correspondence to: justin masumu postal address: po box 3297, chuo kiikuu, morogoro, tanzania how to cite this abstract: masumu, j., tshilenge, g. & mbao, v., 2012, ‘epidemiological aspects of bovine trypanosomosis in an endemic focus of eastern zambia: the role of trypanosome strain variability in disease pattern’, onderstepoort journal of veterinary research 79(2), art. #468, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.468 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. epidemiological aspects of bovine trypanosomosis in an endemic focus of eastern zambia: the role of trypanosome strain variability in disease pattern in this abstract... open access • abstract • bovine trypanosomosis in eastern zambia: background • biological characteristics of trypanosome strains circulating in eastern province • involvement of the trypanosome strain variability in the geographic distribution of bovine trypanosomosis in eastern province • involvement of the trypanosome strain variability in the expression of bovine trypanosomosis in eastern province • trypanosome strain variability and the development of drug resistance in eastern province • from the past to the future • trypanosome-related factors and disease control strategies • conclusion • acknowledgements    • competing interest • references abstract top ↑ bovine trypanosomosis displays various epidemiological aspects in various areas. in some instances the disease has a high prevalence in animals with high impact on production whereas in other cases the disease has a low impact on production despite a high level of infection in animals. in addition epidemiological changes are frequently observed in various areas and are related to many factors including the vectors, the host, the parasites, the environment as well as the livestock management. however the implication of these factors in these changes is not fully elucidated. in eastern zambia, factors predicting the establishment of severe infection in cattle are all present. however trypanosomosis occurring in cattle in this area has a low impact on livestock production. several studies on the characterisation of trypanosome strains circulating in domestic and wild animals have been conducted in order to clarify the epidemiology of this disease in this area. these studies aimed at evaluating genetic and biological characteristics of these strains including their virulence profiles, their transmissibility by tsetse flies, their resistance to drugs and interference between different strains. in this review these findings are analysed in order to elucidate the implication of trypanosome strain variability in the distribution and the expression of this disease in the study area. the evolutionary trends of the situation occurring in this study area are also explained. use of these findings is the context of disease control in the study area is further discussed. bovine trypanosomosis in eastern zambia: background top ↑ bovine trypanosomosis is endemic in eastern province of zambia. previous epidemiological studies using parasitological, serological or molecular techniques showed that the prevalence of the disease varies in different districts (machila, sinyangwe & mubanga 2001; sinyangwe, delespaux & brandt 2004; simukoko, marcotty & phiri 2007). the same variation is observed within a particular district where a high prevalence of the disease is observed in some villages compared to other. in some cases no infection has been detected in cattle kept in some villages. in 1996, trypanosome infections were found in cattle in only 74% of sampling sites with a prevalence of infection varying between villages from 0% to 64% (sinyangwe et al. 2004). such variations are related to many factors including the habitat fragmentation of tsetse flies that affects tsetse density in various areas (ducheyne, mweempwa & de pus 2009). for example the decrease prevalence of the infection in cattle from 1998 to 2003 in petauke where tsetse habitat has widely been fragmented supports this observation. the temporal trends of the infection in cattle revealed no (sinyangwe et al. 2004) or little (simukoko et al. 2007) variations between seasons although higher prevalence seems to be observed during the rainy season (simukoko et al. 2007) when tsetse flies are more abundant (van den bossche & de deken 2002). such variations influence drug management and, although farmers treat their animals throughout the year, most drugs are selectively administered during this period (van den bossche, doran & connor 2000). in this area, the same selection approach is used by farmers in administrating trypanocides drugs in animals. in most cases oxen and cows are more treated (van den bossche et al. 2000) since these animals categories, especially oxen are more infected with trypanosomes (simukoko et al. 2007). although this selective treatment resulted in the reduction (1.5 treatments per year) of drugs administered to animals (van den bossche et al. 2000) it could not limit the development and spread of drug resistance in the area. from 1996 (sinyangwe et al. 2004) to 2003 (delespaux, dinka & masumu 2008), drug resistance to diminazene aceturate increased fivefold although drug use didn’t change during this period of time (van den bossche et al. 2000). despite a high proportion of resistant strains in the area farmers continue using trypanocides which effect needs to be evaluated. trypanosoma congolense remains the most prevalent trypanosome species affecting cattle in this area with more than 95% of the infections (sinyangwe et al. 2004; simukoko et al. 2007). in eastern province only the savannah type has been previously characterised in livestock (masumu, marcotty & geysen 2006a). this type was previously shown to be highly virulent in cattle (bengaly, sidibe & ganaba 2002). furthermore it is efficiently transmitted by glossina morsitans morsitans, the main vector of trypanosomes in this area. bloodmeal analyses revealed that cattle provide 75% of meals to g. m. morsitans in this area (van den bossche & staak 1997) and thus are highly exposed to infections with these highly virulent t. congolense savannah strains circulating in this area. however the cattle breed (angoni) reared on the plateau of eastern province are not trypanotolerant. taken together, these factors, including a high level of resistance to trypanocides in trypanosome strains circulating in cattle and the restricted number of treatment per year, favour for the development of an epidemic situation in cattle on the plateau of eastern province. however economical surveys revealed that the disease has a low impact on livestock production (doran 2000). the isolation and characterisation of trypanosome isolates from cattle in this endemic area revealed sound features that explain the epidemiology of bovine trypanosomosis in this particular area. in this review trypanosome characteristics observed in this area will be analysed for their implication in the epidemiology of bovine trypanosomosis on the plateau of eastern province in zambia. biological characteristics of trypanosome strains circulating in eastern province top ↑ trypanosomes can only influence the distribution and expression of the disease in various areas and different hosts only if they are genetically and phenotypically different. in order to test this, t. congolense circulating in the domestic and wild animals in eastern province were characterised at genetic level (masumu, geysen & vansnick 2006b; masumu, geysen & van den bossche 2009a) as well as for biological characters including the virulence profile (masumu et al. 2006a; van den bossche, chitanga & masumu 2011), the transmissibility by tsetse flies (masumu, marcotty & ndeledje 2006c; masumu, akoda & van den bossche 2010) and the possible existence of cross-protection between different strains (masumu et al., 2009). it appears that many strains of t. congolense circulate in the eastern province (masumu et al. 2006b; 2009a). very few isolates co-circulate in different areas indicating a more restricted distribution. in trypanosomes circulating in domestic animals most of the strains were of low virulence (masumu et al. 2006a). only 20% of them were highly virulent. on the other hand most of the trypanosomes circulating in wild animals were highly virulent (van den bossche et al. 2011) highlighting the trypanotolerant trait of these animals. the presence of high prevalence of virulent strains in wild animals is also a clear indication of these animals being reservoirs of these strains in eastern province. in domestic cycle where trypanosomes circulate only in domestic animals, geographical distribution of these strains indicated that high virulent trypanosome strains are present in limited villages (4 out of 11) and different virulent profiles were observed in various areas suggesting an uneven distribution of those virulent trypanosome strains (masumu et al. 2006a). whether the circulation of low virulent trypanosome strains in domestic animals could protect them from the adverse affect of the high virulent strains was also assessed (masumu et al. 2009b). indeed there is a clear indication of the existence of cross-protection between trypanosomes of high and low virulence but the degree of protection varies in different combinations of strains. this finding suggests that the effect of virulent trypanosome strains may vary depending on the combination of the high and low virulent strains in a particular area. only in areas where most virulent strains do not cross-react with the majority of low virulent strains will the disease have high impact on animal health and thus livestock production. in areas where high level of cross-protection is observed between strains of high and low virulence the effects of such virulent strains will be minimal. the epidemiological importance of the virulent strains was further stress by the fact they were efficiently transmitted by tsetse flies compared to the strains of low virulence (masumu et al. 2006c). involvement of the trypanosome strain variability in the geographic distribution of bovine trypanosomosis in eastern province top ↑ the geographical distribution of trypanosomosis depend on multiple factors including the abundance and infection rate of tsetse flies, the livestock management, the level of drug resistance and drug use in a particular area and the degree of livestock to resist to tsetse fly bites as well as to trypanosome strains circulating in the area. in the context of eastern province, significant variations are observed in the distribution of tsetse in various areas. in most cases high prevalence of the disease is observed in areas highly infested by tsetse flies. within the same geographic area such variations are related to the abundance of tsetse flies throughout the year. in eastern province it appears that tsetse flies are more abundant during the rainy season (van den bossche & de deken, 2002) explaining the high prevalence of the disease previously observed during this period (simukoko et al. 2007). such variations may also be explained by the variations observed in the strains of t. congolense circulating in these areas with a high prevalence of the disease likely being observed in areas where most trypanosome strains are efficiently transmitted by tsetse flies. although highly virulent strains are efficiently transmitted that low virulent ones, further analyses revealed that during the chronic phase of infection the transmission of low virulent strains increases (masumu et al. 2010). this indicates that high and low virulent strains are all transmitted by tsetse flies in this area since most low virulent strains induced chronic infections in cattle. the distribution of the disease can also be influenced by the level of drug resistance to trypanocides in strains circulating in various areas. it can be assumed that high prevalence of infection will be observed in areas where the level of drug resistance is also high. this can partially be explained by the persistence of infection after treatment with trypanocides in animals infected with resistant strains. in addition although there is not evident correlation between resistance and transmission to tsetse flies (personal observation) the level of drug resistance in individual trypanosome strains may influence the infection rate in individual tsetse flies in case of persistence of drugs in tsetse after feeding on treated animals prior infection through an infected bloodmeal. in eastern province high prevalence was observed in areas where the level of drug resistance was also high (sinyangwe et al. 2004). involvement of the trypanosome strain variability in the expression of bovine trypanosomosis in eastern province top ↑ the expression of trypanosomosis in various areas varies considerably (van den bossche 2001). in most cases severe infections are observed when animals are kept near game areas whereas a rather mild infection occurs in areas where wild animals are rather absent. in eastern province bovine trypanosomosis is rather mild on the plateau but when livestock are kept near the luangwa valley where wild animals are abundant they suffer from severe infections. this was previously suggested to result from the presence of heterologous challenge in wild areas as a consequence of the circulation of diverse strains in these areas (van den bossche 2001). our findings revealed that even in areas where wild animals are absent several trypanosome strains circulate in domestic animals suggesting that the number of trypanosome strains is not important for the expression of the disease in a given area (masumu et al. 2009a). instead from the findings on virulence and cross-protection obtained in eastern province it appears that the expression of the disease in various areas is largely dependent on the virulence of trypanosome strains circulating in each particular area with severe disease being observed in areas where virulent strains are more prevalent. our findings on high prevalence of virulent trypanosome strains in the wild environment compared to the plateau where wild animals are absent and where most strains are of low virulence support this observation (van den bossche 2001). however as indicated previously high level of interference between high and low virulent strains may attenuate the effects of the disease in animals previously infected with low virulent strains even after challenge with virulent strains (masumu, marcotty & geerts 2009b). the low impact the disease has on the plateau of eastern province may be explained by the circulation in animals of a high prevalence of low virulent trypanosome strains and the existence of a high level of cross-protection between the few virulent strains and the high prevalent low virulent strains. on the other hand the high prevalence of virulent strains in the wild environment explains the severity of the disease observed in these areas. trypanosome strain variability and the development of drug resistance in eastern province top ↑ the rapid development of drug resistance in eastern province arise many questions. on the plateau of eastern province a fivefold increase of drug resistance was observed from 1996 (sinyangwe et al. 2004) to 2003 (delespaux et al. 2008). in the absence of factors underlying the development of drug resistance in the area (van den bossche et al. 2000), such increase may be explained by the circulation of resistant strains between different areas. however our findings on genetic characterisation on the plateau indicate that the circulation of trypanosome strains is very limited (masumu et al. 2009a). even in geographically close villages similar strains were rarely observed. indeed the circulation of trypanosome strains in various areas can be facilitated by the movement of the infected host or infected vectors. in this area livestock movement is very restricted. in addition tsetse flies do not move freely in various areas due to the fragmentation of their habitats. variations in the distribution of tsetse flies are more linked to the distribution of cattle (van den bossche & staak 1997). these two factors are in favour of a more restriction of trypanosome strains circulating in various areas. such restriction cannot facilitate the circulation of resistant trypanosome strains. instead the development of drug resistance seems to be induced locally. although genetic exchange was only reported in trypanosoma brucei (jenni, marti & schweizer 1986) not t. congolense, it may be assumed that the spread of drug resistance in eastern province is related to genetic exchange (delespaux, dinka & masumu 2008) between resistant and susceptible trypanosome strains. indeed resistant and susceptible trypanosome strains do circulate in animals belonging to the same village (masumu et al. 2006a). since two different strains can infect an individual animal, and tsetse flies can be infected more than once, the presence of two different strains in the same fly will favour the exchange of genetic material. further studies are needed to clarify the role of genetic exchange in the spread of drug resistance. from the past to the future top ↑ the high prevalence of low virulent trypanosome strains and the presence of high level of interference between strains of high and low virulence in eastern province, an area where tsetse flies take their blood meal principally from a susceptible animal i.e. cattle (van den bossche & staak 1997), is a perfect illustration of an endemic situation where the vector (tsetse flies), the parasite (trypanosomes) and the host (cattle) live in harmony. the current situation results from the fragmentation of tsetse habitat and the elimination of wild animals. indeed many decades from now wild animals were prevalent in this area. consequently the virulence profile of trypanosome strains was similar to that occurring in luangwa valley where the trypanotolerant trait of wild animals selects against trypanosomes of low virulence. however the elimination of wild animals on this plateau led to a situation where tsetse depends on livestock for their survival (van den bossche & staak 1997) and trypanosome circulate only in susceptible animals. since the presence of virulent strains induces severe infections in these susceptible animals, the later are either treated or die from their infection. this results in the elimination of these virulent strains and the subsequently high prevalence of strains that affect animal health to a lesser degree. only virulent strains that can interfere with strains of low virulence can persist in the susceptible host animal thus explaining the persistence of 20% of the virulent strains in this area (masumu et al. 2006a). the situation prevailing on the plateau of eastern province indicates clearly that bovine trypanosomosis is endemic in this area. although the prevalence of the disease varies largely in different areas (machila et al. 2001; sinyangwe et al. 2004; simukoko et al. 2007), further changes are still to occur in the future in various areas. for example, petauke district that had a high prevalence of the disease and a high prevalence of drug resistance in 1990s (sinyangwe et al. 2004) is no longer the major focus of bovine trypanosomosis in eastern province. the high level of fragmentation resulting from the increased surface of cultivation of crops induced a spectacular reduction of tsetse fly abundance and subsequently a low prevalence in animals (simukoko et al. 2007). on the other hand katete district remains an area of concern since the level of infection in animals is still high (simukoko et al. 2007) and more important the increase of drug resistance is alarming (delespaux et al. 2008). it’s obvious that this situation is far from being stable in the near future. indeed the presence of a wild environment in luangwa valley where about 14% of wild animals are infected with trypanosomes (anderson, mubanga & fevre 2011) constitutes a serious threat to livestock production in eastern province. although habitat fragmentation renders difficult to tsetse flies to move towards districts like katete, petauke, other district like msoro that is located near the game is constantly at high risk of trypanosomosis. in addition this district may constitute a buffer zone between the high risk area of the valley and the plateau of eastern province. in this district the high challenge of tsetse flies and the tranfer of highly virulent trypanosome strains from the wild environment will result in a high infection rate in livestock including pigs and small ruminants that are rather resistant to trypanosomes. this was demonstrated in a survey where all livestock species (cattle, pigs, sheep and goats) kept in msoro district were highly infected (> 10%) with trypanosomes contrarily to the situation occurring on the plateau where most infections are restricted to cattle (simukoko et al. 2007). indeed in the absence of wild animals, the presence of such infections in livestock other than cattle is epidemiologically very important. first, in such areas of high risk of trypanosomosis farmers prefer exploiting small ruminants and pigs for their relative tolerance to t. congolense. their relative number will exceed that of cattle. further since these animals are trypanotolerant and rarely treated with trypanocides they will maintain trypanosomial infection for a long period of time and thus increase the risk of tsetse flies being infected with trypanosomes even virulent to cattle. such risk will increase the severity of the disease in cattle in those particular areas explaining the habitual low production observed in cattle in such areas. second this encroachment of people and livestock to the game area constitutes a new epidemiological situation where virulent trypanosome strains circulating in wild animals (van den bossche et al. 2011) will gradually be introduced into the livestock management area and finally reach the plateau of eastern zambia. whilst such movement of virulent trypanosome strains from the wild to the interface will be favoured by tsetse flies movement, the transfer of these strains from the interface to the plateau of eastern province will probably be occasioned by livestock movement. since cattle movement is very restricted small ruminants and pigs will be the major factor for the circulation of trypanosomes between areas and the introduction of virulent trypanosome strains in various areas including the plateau where the situation is currently more endemic and rather stable. this will be facilitated for example by the commercial and social exchanges between families in the area that increases the movement of animals from one area to another. when infected with trypanosomes, pigs and small ruminants will favour the circulation of these virulent strains even in areas further away from the game areas. it would be advisable to conduct a follow up of trypanosome strains circulating on the plateau e.i. in katete district where trypanosomes are more resistant but rather low virulent and at the interface e.i. in msoro district where a high prevalence of virulent strains may be present whilst being less or no resistant to trypanocides. trypanosome-related factors and disease control strategies top ↑ several control strategies have been used in eastern province for bovine trypanosomosis. the extent of drug resistance to either diminazene aceturate or isometamidium chloride indicates clearly that caution should be made in using these drugs to control trypansomosis in cattle. it is not well known how cattle withstand the infection in this particular area where drug use is minimal (van den bossche et al. 2000) and a high proportion of trypanosome strains are resistant to either or both drugs (delespaux et al. 2008; sinyangwe et al. 2004). amongst the reasons is the circulation of a high prevalence of low virulent strains in livestock. indeed infections induced by these strains have minimal effect on livestock production. in most cases low virulent strain can affect reproductive parameters but do not necessarily cause animal death. like in theileriosis the presence of such infection can be beneficial in protecting animals against adverse effects of virulent strains. in this area, farmers use production-oriented strategy where only sick animals are treated. although this strategy could not reduce the development of drug resistance it reduces animal mortality (van den bossche et al. 2000). our findings and the presence of high prevalence of resistant strains are in favour of such control strategy. not only treating sick animals also is economically acceptable by resource-limited farmers, it favours the establishment of premonition state in animals that are subsequently protected against adverse effects of virulent strains. it’s possible that this strategy will remain the strategic option used by farmers in the future although it does not boost the reproduction of infected animals (van den bossche et al. 2000). such control strategy cannot be applied in areas displaying a different epidemiological situation like in msoro or the game areas of luangwa valley where the trypanosome profiles are rather threatening for livestock. conclusion top ↑ many factors can influence the epidemiology of bovine trypanosomosis in various areas. very few studies have so far been deeply conducted for the understanding of the involvement of the parasite in the epidemiology of this disease. indeed this can only be possible if sound information is yielded at genetic and biological levels. this implies the development of appropriate tools for genotypic and phenotypic characterisation. in the situation prevailing on the plateau of eastern province of zambia where wild animals are not present and tsetse flies depends largely on a susceptible host, the outcome of the disease on livestock health and production was found to be correlated with the profiles of trypanosome strains circulating in animals. disease control through parasite management leading to the establishment of premonition state in animals is thus encouraged and further supports the control strategy so far adopted by farmers. similar features 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variations in the distribution and abundance of the tsetse fly, glossina morsitans morsitans in eastern zambia’, medical and veterinary entomology 16, 170–176. http://dx.doi.org/10.1046/j.1365-2915.2002.00360.x van den bossche, p., 2001, ‘some general aspects of the distribution and epidemiology of bovine trypanosomosis in southern africa’, international journal for parasitology 31, 592−598. http://dx.doi.org/10.1016/s0020-7519(01)00146-1 van den bossche, p., doran, m. & connor, r.j., 2000, ‘an analysis of trypanocidal drug use in the eastern province of zambia’, acta tropica 75, 247−258. http://dx.doi.org/10.1016/s0001-706x(00)00059-0 van den bossche, p. & staak, c., 1997, ‘the importance of cattle as a food source for glossina morsitans morsitans westwood (diptera: glossinidae) in katete district, eastern province, zambia’, acta tropica 65, 105−109. http://dx.doi.org/10.1016/s0001-706x(97)00658-x olivier_187-199.indd introduction the utilization of freshwater fish to supplement protein demand by humans, especially in rural areas, is a topic widely researched. nevertheless, south africa has always lagged behind in this effort when compared to densely populated countries such as china, taiwan and israel. many reasons for this state of affairs can be given, like the customary supply of marine fish from south africa’s long shoreline with its rich, but lately diminishing, fish community. south africa’s inland and rural communities have also not to a large extent developed an established appetite for freshwater fish. this may be due to the relatively effortless and uncomplicated accessibility to competitively priced alternative sources of protein, such as chicken, small ruminants, pigs and cattle. the position may, however, change drastically. declining tendencies in marine fisheries output have been reported as long ago as the 1970s (noble & hemens 1978; cram 1980; allanson & jackson 1983). since then more documented evidence of this phenomenon is available. the imbalance between the development of land protein resources on the one hand and population growth linked to rising costs of refrigeration and transport of marine protein products on the other, may, however, force the pendulum to move towards utilization of aquaculture products as supplementary food sources. this, together with an increased interest in the development of aquaculture practices, has spurred the initiation of research efforts in aspects such as intensive freshwater fish husbandry, harvesting of fish stocks from natural freshwater habitats (roode 1978; saay187 onderstepoort journal of veterinary research, 76:187–199 (2009) report on some monogenean and clinostomid infestations of freshwater fish and waterbird hosts in middle letaba dam, limpopo province, south africa p.a.s. olivier1*, wilmien j. luus-powell1 and j.e. saayman1, 2 abstract olivier, p.a.s., luus-powell, wilmien j. & saayman, j.e. 2009. report on some monogenean and clinostomid infestations of freshwater fish and waterbird hosts in middle letaba dam, limpopo province, south africa. onderstepoort journal of veterinary research, 76:187–199 this report deals with the results of a parasitological study done as part of a post-impoundment ecological study of middle letaba dam, limpopo province, south africa. it involved a seasonal survey protocol with particular attention to the diversity and prevalence of the parasitic fauna of the indigenous fish community of the dam and the role of selected fish-eating birds in the life cycle and distribution of fish helminths. the potential species composition of fish of the dam is provided. monogenean and clinostomatid parasites encountered are listed and infestation statistics of fish and fish-eating bird hosts are presented. the results of this study also provide information on new distribution and host records of the encountered monogeneans. keywords: clinostomidae, middle letaba dam, monogenea, south africa * author to whom correspondence is to be directed: olivierp@ ul.ac.za 1 department of biodiversity, university of limpopo (turfloop campus), private bag x1106, sovenga, 0727 south africa 2 present address: 7 protea street, albertinia, 6695 south africa accepted for publication 1 september 2008—editor 188 monogenean and clinostomid infestations of freshwater fi sh and waterbird hosts in limpopo, south africa man 1984; hecht 1985; hecht, uys & britz 1988) and the enhancement of guidelines, aims and objectives for aquaculture-supportive research in south africa (safriel & bruton 1984). prioritizing freshwater fish as a supplementary alternative supply of protein for humans indisputably accentuates the importance of fish health in general. in this regard, the significant role of scientific knowledge with regards to indigenous as well as introduced fish parasites can hardly be over-emphasized. over the last decade the importance of studying fish parasites, including aspects such as their diversity, distribution, infestation rates and pathology have been realized by many south african researchers. this report deals with the results of parasitological studies done as part of a multidisciplinary research programme on the post-impoundment ecology of the middle letaba dam. the study was commissioned in 1987 by the (then) department of devel opment aid with the government service of the (then) gazankulu government as beneficiary. the study involved a seasonal survey protocol and particular attention was given to the diversity and prevalence of the parasitic fauna of the indigenous fish community of the dam. attention also focused on the role of selected waterbirds in the life cycle and distribution of some fish helminths (with special reference to members of the clinostomidae) using piscivorous birds as final hosts. materials and methods study area middle letaba dam is situated at 30°24’ s, 23°16’ e, about 50 km west of the city of giyani, in the limpopo province of south africa. the catchment area of the dam includes two river systems: the sen wabathweni river with a catchment area of 757 km2 and a mean annual rainfall of 757 mm, and the middle letaba river with a catchment area of 1 042 km2 and a mean annual rainfall of 708 mm. the most important tributaries of these systems are the koedoes, brandboontjies and houtbos rivers. at full water level, middle letaba dam has a surface area of 1 943 ha and a mean depth of 9.5 m (maximum of 34 m). water temperatures (day and night temperatures) and ph values were frequently measured at various positions in the dam. precipitation data were obtained from the weather bureau station in the vicinity of the dam. water temperatures did not fluctuate much seasonally, with average seasonal day temperatures being 27.5 °c (autumn), 17.5 °c (winter), 22 °c (spring) and 27 °c (summer) and average night temperatures being 26 °c (autumn), 16,5 °c (winter), 20 °c (spring) and 25.5 °c (summer). reasons for these relatively small differences may be due to the fact that the middle letaba dam is situated in a subtropical environment and that at the time of the survey the dam was only about 30 % full. as a result, there was no clear delineation between the epilimnion and hypolimnion resulting in a thorough mixing of the entire water column. for the period of the survey ph values of the impoundment were relatively constant, with average seasonal values ranging between 7.9 (autumn), 8.3 (winter), 7.7 (spring) and 8.1 (summer). procuring and transport of host specimens parasitological surveys were done seasonally from january 1984 to january 1989. the majority of fish hosts were captured using gill nets of various stretched mess sizes, ranging from 30–180 mm but beach seine nets were used in shallower water. where the physical conditions did not allow the use of gill nets fish hosts were collected by means of electro-fishing. the captured fish were transported live to the field laboratory where they were kept in containers with well aerated water. avian hosts were shot and their beaks were immediately sealed with elastic bands to avoid the escape of any clinostomid worms lodged in the buccal cavity and oesophagus. the birds were transported in sealed plastic bags to the field laboratory for immediate autopsy and parasitic infestation analysis of the complete alimentary tract. examination of hosts for parasites immediately prior to examination, the fish were killed by severing the spinal cord just posterior to the cranium. monogenean parasites were collected from the gills of the fish with the aid of a stereo microscope and smears were made from the skin and mucosa of the stomach. sampled monogeneans were fixed in hot (70 °c) alcohol-formalin-acetic acid (afa) and stored in 4 % buffered formalin. some of the monogeneans were mounted on microscope slides in glycerine jelly under slight cover slip pressure, and the cover slip sealed with clear nail varnish. clinostomids were carefully removed from the buccal cavity and oesophagus of the birds, fixed in hot (70 °c) afa, and preserved in 80 % ethanol. the clinostomids that were collected were mounted on microscope slides for microscopic investigations or stored in 80 % ethanol for further studies. 189 p.a.s. olivier, w.j. luus-powell & j.e. saayman prevalence of infestation (%) was calculated as the number of infested hosts/number of examined hosts x 100. mean intensity of infestation was calculated as the total number of parasites/number of infested hosts. standard deviation was not calculated due to meaningless values as a result of too small sample sizes (rózsa, reiczigel & majoros 2000). for consistency, name changes of hosts since the beginning of this study are incorporated, following that of skelton (2001) for fish hosts and hockey, dean & ryan (2005) for bird hosts. results and discussion fish species composition of middle letaba dam a pilot-survey (done prior to the parasitological survey) of the catchment area of the proposed middle letaba dam was carried out to determine the possible species composition of fish which could eventually inhabit the dam (olivier, kruger, van der waal, viljoen & viljoen 1986). the latter investigation complements a previous study by gaigher & mcpott (1973), and mainly correlates with their findings, except for barbus radiatus peters and synodontis zam bezensis peters which the latter authors encountered in the middle letaba river and glossogobius giuris (hamilton-buchanan) which they collected at the confluence of the middle letaba and klein letaba rivers. based on the results of these two surveys, the potential fish species diversity of the dam is relatively low, limited to about 28 species with clarias gariepinus burchell and oreochromis mossambicus (peters) the numerically dominant species (table 1). of the species mentioned in table 1, mesobola brevianalis (boulenger), opsaridium zambezense (gilchrist et thompson), barbus lineomaculatus boulenger, b. radiatus, cyprinus carpio l., micralestes acutidens (peters), amphilius uranoscopus (pfef fer), schilbe intermedius rüppell, chiloglanis pretoriae van der horst, s. zambezensis, micropterus salmoides (lacepède), tilapia rendalli (boulenger) and g. giuris were not collected from the dam during the present parasitological survey. some of these may table 1 potential species composition of fish of middle letaba dam and its catchment area gaigher & mcpott 1973 n olivier et al. 1986 sampled during this study anguilla mossambica peters mesobola brevianalis (boulenger) opsaridium peringueyi (gilchrist & thompson) barbus lineomaculatus boulenger barbus unitaeniatus günther barbus bifrenatus fowler barbus viviparus weber barbus toppini boulenger barbus radiatus peters barbus trimaculatus peters barbus paludinosus peters labeobarbus marequensis (a. smith) labeo rosae steindachner labeo ruddi boulenger labeo cylindricus peters labeo molybdinus du plessis cyprinus carpio linnaeus micralestes acutidens (peters) amphilius uranoscopus (pfeffer) schilbe intermedius (rüppell) clarias gariepinus (burchell) chiloglanis pretoriae van der horst synodontis zambezensis peters micropterus salmoides (lacepède) pseudocrenilabrus philander (weber) tilapia rendalli (boulenger) oreochromis mossambicus (peters) glossogobius giuris (hamilton-buchanan) 1 – – 82 13 46 68 – 89 83 32 24 64 68 4 – – – – 111 – – – 69 – 177 – + + + + + + + + x + + + + + + + + + + + + + x + + + + x + x x x + + + + x + + + + + + + x x x x + x x x + x + x n = number of fish sampled for parasitological analyses + = species present x = species not collected 190 monogenean and clinostomid infestations of freshwater fi sh and waterbird hosts in limpopo, south africa still successfully colonize middle letaba dam. however, c. pretoriae and o. zambezense are considered to prefer rivers as their favoured habitat and will, in all probability, not establish themselves in the dam (polling, mokgalong & saayman 1983). synodontis zambezensis and g. giuris might also not colonize the dam as they naturally occur only in downstream habitats (gaigher 1973). the unique construction of the overflow of the dam, forming an impenetrable barrier to upstream migration, will prevent these two species from reaching the dam. of the 28 species of fish listed in table 1, only 15 were collected during the parasitological survey and, therefore, could be considered as possible parasite hosts (only a single specimen of anguilla mossambica peters was collected during the summer survey of 1984, but revealed no parasites). monogenean parasites to date, very few records of monogenea of south african freshwater fish appeared in the published literature (e.g. paperna 1980; mashego 1983; khalil & polling 1997; mashego 2000; luus-powell, mashego & khalil 2003; christison, shinn & van as 2005). during this study monogenean parasites were collected from a total of 831 hosts specimens of as many as 14 different fish species (table 2). table 2 host/parasite checklist of monogenean parasites from middle letaba dam with an indication of host specificity for each species of parasite host n parasite host range barbus unitaeniatus günther 73 dactylogyrus sp. 1 3 barbus bifrenatus fowler 13 dactylogyrus sp. 4 3 barbus viviparous weber 46 dactylogyrus sp. 4 3 barbus toppini boulenger 68 dactylogyrus sp. 4 3 barbus trimaculatus peters 70 dactylogyrus afrolongicornis paperna dactylogyrus afrolongicornis alberti paperna dactylogyrus afrosclerovaginatus paperna dactylogyrus allolongionchus paperna dactylogyrus meyersi price, mcclellan, druckenmiller & jacobs dactylogyrus sp. 1 dactylogyrus sp. 2 dactylogyrus sp. 3 1 1 1 1 1 3 2 1 barbus paludinosus peters 83 dactylogyrus dominici mashego dactylogyrus teresae mashego 1 1 labeobarbus mareqensis (a. smith) 32 dactylogyrus spinicirrus (paperna & thurston) dactylogyrus sp. 1 dactylogyrus sp. 2 1 3 2 labeo rosae steindacher 24 dactylogyrus sp. 7 dactylogyrus sp. 8 dactylogyrus sp. 9 dactylogyrus sp. 10 dogielius sp. 1 2 2 2 2 1 labeo ruddi boulenger 64 dactylogyrus sp. 7 dactylogyrus sp. 9 dactylogyrus sp. 9 dactylogyrus sp. j dactylogyrus sp. 10 dactylogyrus sp. 12 dogielius sp. 2 dogielius sp. 3 dogielius sp. 4 2 2 2 2 1 1 1 1 1 labeo cylindricus peters 68 dactylogyrus sp. 5 2 labeo molybdinus du plessis 4 dactylogyrus sp. 5 dactylogyrus sp. 6 2 1 191 p.a.s. olivier, w.j. luus-powell & j.e. saayman host n parasite host range clarias gariepinus (burchell) 111 quadriacanthus allobychowskiella paperna quadriacanthus aegypticus el-naggar & serag quadriacanthus clariadis paperna quadriacanthus sp. 1 quadriacanthus sp. 2 quadriacanthus sp. 3 quadriacanthus sp. 4 quadriacanthus sp. 5 quadriacanthus sp. 6 quadriacanthus sp. 7 quadriacanthus sp. 8 quadriacanthus sp. 9 quadriacanthus sp. 10 gyrodactylus rysavyi ergens macrogyrodactylus clarrii gussev macrogyrodactylus karibae douellou & chishawa 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 pseudocrenilabrus philander (weber) 69 cichlidogyrus tilapiae enterogyrus cichlidarium 2 2 oreochromis mossambicus (peters) 105 cichlidogyrus halli (price & kirk) cichlidogyrus sclerosus paperna & thurston cichlidogyrus tilapiae paperna cichlidogyrus sp. 1 cichlidogyrus sp. 2 enterogyrus cichlidarum paperna scutogyrus gravivaginus (paperna & thurston) 1 1 2 1 1 2 1 n = number of hosts examined 1 = one-host parasite 2 = two-host parasite 3 = three-host parasite table 2 (cont.) although some of the parasites encountered are of uncertain specific status, their generic status and host distribution were positively verified. the dactylogyrid species were obtained from the gills of their respective hosts. gyrodactylid parasites were found to be present on either the skin or the gills of the host. species of chichlidogyrus paperna and scutogyrus pariselle & euzet are harboured on the gills of their hosts, while enterogyrus chichlidarum paperna occurs in the mucosa of the stomach of both its hosts (pseudocrenilabrus philander (weber) and o. mossambicus). during the current study, members of three monogenean families, represented by eight genera and 47 species (19 known species and 28 unidentified species), were encountered (table 2). species of these families are known to be variably host specific, with host ranges restricted to one or only a few closely related species. this trend is supported by the results of this study (table 2). in general, members of the genus dogielius bikhovski were found only on two of the four labeo hosts and species of quadriacanthus paperna, gyrodactylus paperna and macrogyrodactylus malberg were found on c. gariepinus only. species of cichlidogyrus were restricted to p. philander and o. mossambicus, while scutogyrus gravivaginus (paperna & thurston) was collected from o. mossambicus only. species of the genus dactylogyrus diesing are parasites of barbus and labeo species. more specifically, table 2 clearly illustrates the phenomenon of host specificity with no less than 37 of encountered species found on a single host, eight parasitized two-host species and only two were harboured by three different hosts. this corresponds with findings of mashego (1983) who reported on eight of the parasites collected during the present study parasitizing the same host species, although from different geographical regions in south africa. these are dactylogyrus afrolongicornis afrolongicornis paperna, dactylogyrus afrolongicornis alberti paperna, dactylogyrus afrosclerovaginatus paperna, dactylogyrus allolongionchus paperna and dactylogyrus meyersi price, mclellan, druckenmiller & jacobs from barbus trimaculatus peters, dactylogyrus dominici mashego 192 monogenean and clinostomid infestations of freshwater fi sh and waterbird hosts in limpopo, south africa and dactylogyrus teresae mashego from barbus pa ludinosus peters, and dactylogyrus spinicirrus (paperna & thurston) from labeobarbus marequensis (smith, 1841). in general, the intensity of monogenean infestations was found to be very low, on average five parasites per infested host. however, the diversity of monogeneans on c. gariepinus (with no less than 16 species), with a more profound and variable intensity of infestation, is noteworthy. immediately after the closure of the dam in 1984, very low intensities were observed (mean intensity = 1.0) followed by a tremendous increase, reaching a peak in the summer of 1986/1987 (mean intensity = 700.0). this was followed by a steady decline to as low as a mean intensity of less than 1.0 in january 1988. the latter could be ascribed to the greater volume of water as a result of good rains in december/january of 1987/ 1988 (more than 250 mm; mean annual for the catchment area is 732 mm) resulting in a decline in host density. gyrodactylids have no free swimming larval stages and infestation between individual hosts is probably by direct contact (paperna 1980). during the period of hyperinfestation, infested specimens of c. gariepinus all developed a thick layer of greyish-white mucus over the entire skin, in all probability caused by the severe irritation caused by the parasites. other pathological signs observed included proliferation of epithelial cells at the point of attachment, erosion of the skin and fading of skin colour, all of which are in accord with similar observations reported by paperna (1980). monogeneans encountered from middle letaba dam revealed some species representing either first parasite records, or new host records from south africa. this can be summarized as follows: 1. gyrodactylus von nordman (gyrodactylidae). christison et al. (2005) provides a summary of all the gyrodactylids that have been reported from southern africa. these include one known, gyrodactylus transvaalensis prudhoe & hussey, and three unidentified species as records from south africa. none of the latter was collected during this survey. however, the authors agree with christison et al. (2005) that the records of the three unidentified species referred to by bragg (1991) and lombard (1968), should not be considered as part of the south african fauna due to their association with alien fish hosts (rainbow trout and large mouth black bass, respectively). however, gyrodactylus rysavyi ergens, described and reported to date only from egypt (ergens 1973), was encountered during the present study from c. gariepinus, and represents a new distribution record from south africa. 2. macrogyrodactylus malberg (gyrodactylidae). during this study two macrogyrodactylid species (macrogyrodactylus clarii gussev and macrogyrodactylus karibae douëllou & chishawa) were encountered from c. gariepinus and have previously been reported on by khalil & mashego (1998). the latter authors also reported on macrogyrodactylus congolensis (prudhoe) collected from c. gariepinus at mokgoma-matlala dam, south africa. 3. dactylogyrus diesing (dactylogyridae). eleven dactylogyrid species were previously reported from south africa (khalil & polling 1997): d. afro longicornis paperna, d. afrolongicornis alberti, d. afrosclerovaginus, d. allolongionchus, d. dominici, dactylogyrus enidae mashego, dac tyl ogyrus jubbstrema price, korach & mcpott, d. myersi, dactylogyrus pienaari price, korach & mcpott and d. spinicirrus d. teresae mashego. of these, only three species, d. enidae, d. jubbstrema and d. pienaari, were not encountered during this study. of the remaining seven species collected, it is worthwhile to note that d. afrosclerovaginus was collected from barbus trimaculatus peters (previously reported from barbus paludinosus peters), representing a new host record. additional to the above, 12 unidentified dactylogyrid species were found from six hosts (table 2). 4. dogielius bikhovski (dactylogyridae). none of the more than 20 described species from africa (khalil & polling 1997) was either sampled during the present study or is previously known from south africa. however, four unidentified species, one from labeo rosae steindachner and three from labeo ruddi boulenger, were collected. 5. quadriacanthus paperna (dactylogyridae). three formerly described species (quadriacanthus aegypticus el-naggar & serag, quadriacanthus allobychowskilla paperna and quadriacanthus clariadis paperna) and ten unidentified species, all sampled from c. gariepinus and all being first records for the genus from south africa, were collected. 6. chichlidogyrus paperna (ancyrocephalidae). only one species, chichlidogyrus papernastrema price, peebles & bamford is previously known 193 p.a.s. olivier, w.j. luus-powell & j.e. saayman from south africa. this species was not collected during the present survey, possibly due to the absence of the type host, tilapia sparrmanii boulenger. however, five chichlidogyrid species were encountered and are new records for south africa: chichlidogyrus halli (price & kirk), chi chlidogyrus sclerosus paperna & thurston, chichlidogyrus tilapiae paperna and two unidentified species. 7. enterogyrus paperna (ancyrocephalidae). en tero gyrus cichlidarum paperna was the only species collected and represents a first record of the genus from south africa. 8. scutogyrus pariselle & euzet (ancyrocephalidae). scutogyrus gravivaginus (paperna & thurston) was the only species collected and it is a first record of the genus from south africa. digenean species of the family clinostomidae positive species designation of trematodan metacercariae is usually difficult due to their undifferentiated genitalia, therefore adult stages from the definitive host are frequently the only way to achieve positive identification. however, the fact that most trematodes, including their larval stages, are highly host specific (paperna 1980; grobler & mokgalong 2002) can be applied as a useful tool for identification. furthermore, diagnosis during this survey was somewhat easier as trematode infestations (especially that of clinostomids) of resident piscivorous birds acting as definitive hosts, were simultaneously investigated (mokgalong 1996). in the past, many problems were experienced with the specific diagnostic designations of members of the family clinostomidae (grobler, mokgalong & saayman 1999). feizullaev & mirzoeva (1983) provided some clarity on solving this problem when they synonomized 35 species as one, clinostomum complanatum (rudolphi). these included clino stomum vanderhorsti ortlepp and clinostomum tilapiae ukoli, both of which prompted much discussion, confusion and misidentification. recent studies using molecular techniques clearly revealed that material previously identified as c. tilapiae, found in the gill chambers of o. mossambicus, is in fact neutraclinostomum intermedialis (lamont) (grobler et al. 1999; grobler & mokgalong 2002). the demonstration of high host specificity (o. mossambicus as intermediate fish host and the darter, anhinga rufa (daudin), as definitive avian host) for n. intermedialis (mokgalong 1996) further supports the view of considering c. tilapiae sampled from gill chambers of o. mossambicus, as a junior synonym of n. intermedialis. this approach is followed for the present report. during the present survey three species of the family clinostomidae were sampled from six different fish hosts (table 3). the results concerning these species are discussed separately below and, where applicable, additional data of similar studies from different habitats in south africa are presented as comparisons. neutraclinostomum intermedialis (lamont) this parasite was exclusively procured from the branchial chambers of o. mossambicus and, during this study, a total of 379 specimens were collected. a total of 177 fish hosts were examined and showed a prevalence of 48 % and a mean intensity of 4.4 (table 3). the largest number of hosts were encountered during summer (n = 87), with almost 50 % (n = 39) represented by smaller fish (total length < 10 cm). fish longer than 30 cm were virtually absent from this survey, but the data of a single large specimen is nevertheless included in table 4. table 3 species of the family clinostomidae collected from infested intermediate hosts from middle letaba dam parasite host n prev. mi. neutraclinostomum intermedialis (lamont) oreochromis mossambicus (peters) 177 48 4.4 clinostomum complanatum (rudolphi) barbus unitaeniatus günther barbus trimaculatus peters labeobarbus marequensis (a. smith) pseudocrenilabrus philander (weber) 82 89 13 47 2.4 1.1 7.7 6.4 1 2 1 1 euclinostomum heterostomum (rudolphi) oreochromis mossambicus (peters) clarias gariepinus (burchell) 177 21 24.9 4.7 2.2 9 n = number of hosts examined prev. = prevalence of infestation (%) mi. = mean intensity of infestation 194 monogenean and clinostomid infestations of freshwater fi sh and waterbird hosts in limpopo, south africa the highest prevalence (57.1 % for all seasons) was found within the < 10 cm length group with a 63.6 % registered for the winter survey within the same length group (the autumn survey was not taken into account due the fact that only two hosts were examined). the lowest prevalence was found for hosts of 20.1–30 cm in length suggesting smaller fish to be more susceptible to infestation. the highest mean intensity (11.3), however, was recorded during the summer survey for the 20.1–30 cm length group with as many as 20 parasites per host. this length group in fact recorded the highest mean intensity values for all seasonal surveys, except during spring (table 4). neutraclinostomum intermedialis does indicate some, yet not pronounced, seasonal variations in prevalence and intensity of infestation. this relatively small seasonal variation in infestation rates is to be expected in a young, subtropical impoundment where seasonal climatic conditions are not sharply demarcated. furthermore, comparison of infestation rates amongst different sexes of the host failed to illustrate any noteworthy differences. mokgalong (1996) clearly illustrated a. rufa to be the definitive host for n. intermedialis, with 75 % of the examined hosts from middle letaba dam infested (table 5). he further reported high infestation values of n. intermedialis for this bird (intensity = 7–72 and mean intensity = 35). these values correspond well (prevalence of 50–100 %) if compared with similar data for other water bodies in the limpopo province (mokgalong 1996). the high infestation values of a. rufa in middle letaba dam, and the fact that it is one of the most abundant resitable 4 seasonal analysis, per length group (tl. cm) of oreochromis mossambicus (peters), for neutraclinostomum intermedialis (lamont) infestations in middle letaba dam season < 10 cm 10.1–20 cm 20.1–30 cm > 30 cm all size groups summer number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 39 53.9 1–21 4 83 24 45.8 1–7 4.3 47 23 13.1 5–20 11.3 34 1 100 4 4 4 87 41.4 1–21 4.7 168 autumn number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 2 100 3–4 3.5 7 10 80 2–16 5.6 45 3 33.3 – 8 8 – – – – – 15 73.3 2–16 5.5 60 winter number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 11 63.6 1–5 2.4 17 4 75 4–5 4.3 13 25 40 1–17 3.7 37 – – – – – 40 50 1–17 3.4 67 spring number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 18 55.6 1–10 5.5 55 14 50 1–9 4 28 3 33.3 – 1 1 – – – – – 35 51.4 1–10 4.7 84 all seasons number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 70 57.1 1–21 4.1 16 52 55.8 1–16 4.6 133 54 27.8 1–20 5.6 80 1 100 4 4 4 177 48 1–21 4.5 379 195 p.a.s. olivier, w.j. luus-powell & j.e. saayman dent fish-eating birds (olivier, saayman & polling 1991), tend to indicate that intensity of infestation in the intermediate host (o. mossambicus) may well increase drastically in the near future. saayman (1986) investigated n. intermedialis infestations in o. mossambicus in various other water bodies of the limpopo and olifants rivers drainage systems in the limpopo province (table 6). only three of these water bodies (nile flood pans, nwanedzi river and tompi seleka fish station) exhibit infestation values comparable to that of middle letaba dam. values obtained from tompi seleka are to be expected as it represents a situation where fish are kept at high densities in production ponds. these ponds were also densely populated with snails, the invertebrate intermediate host for n. intermedialis, (no data on the species of the snails are available) and aquatic macrophytes. the ratio of shore to water area in production ponds is far greater than that for impoundments, resulting in enhanced opportunities for released cercariae to locate suitable hosts. physical conditions in the nile flood pans and pools in the nwanedzi river are almost similar to the situation at tompi seleka and this may explain the high levels of infestation recorded for these two localities. clinostomum complanatum (rudolphi) in middle letaba dam only four of the potential fish host species (table 1) were infested with metacertable 5 parasite/host checklist of definitive hosts (piscivorous birds) at middle letaba dam infested with adult members of clinostomidae parasite host n prev. in. mi. neutraclinostomum intermedialis (lamont) anhinga rufa (daudin) 44 75 7–72 35 clinostomum complanatum (rudolphi) phalacrocorax lucidus (lichtenstein) phalacrocorax africanus (gmelin) anhinga rufa (daudin) ardea cinerea linnaeus nycticorax nycticorax (linnaeus) 11 30 44 3 3 63 60 96 66 66 1–37 1–7 3–190 1–3 2–4 11 4 48 2 3 euclinostomum heterostomum (rudolphi) phalacrocorax luidus (lichtenstein) phalacrocorax africanus anhinga rufa (daudin) ardea cinerea linnaeus ardea melanocephala anon ardea purpurea linnaeus 11 30 44 3 5 2 88 30 80 100 100 50 1–21 1–10 1–11 4–6 1–8 1 7 6 5 5 5 1 n = number of hosts examined prev. = prevalence of infestation (%) in. = intensity of infestation mi. = mean intensity of infestation table 6 comparison of infestation values of neutraclinostomum intermedialis (lamont) in oreochromis mossambicus (peters) in different water bodies of the limpopo and olifants rivers drainage systems—data from saayman (1986) waterbody n prev. in. mi. middle letaba dam glen alpine dam luphephe-nwanedzi dams nwanedzi river nzhelele dam seshego dam nile flood pans piet gouws dam lepellane dam tompi seleka fish station 177 89 157 66 15 35 58 59 114 25 48 14.6 10.7 54.6 7 38 62.1 35.6 28.9 64 1–21 1–6 1–5 1–8 1 1–5 1–13 1–14 1–6 1–2 4.5 2 1.4 3.2 1 3 3.5 2.8 2.1 1.5 n = number of hosts examined prev. = prevalence of infestation (%) in. = intensity of infestation mi. = mean intensity of infestation 196 monogenean and clinostomid infestations of freshwater fi sh and waterbird hosts in limpopo, south africa cariae of c. complanatum (table 3). in all of the infested hosts the parasites were always found encysted on the peritoneum of visceral organs with an explicit preference for the peritoneum of the ventral surface of the swim bladder. extremely low prevalence and intensity values were recorded (table 3). saayman (1986) investigated several indigenous fish species from various localities in the olifants and limpopo drainage systems for c. complanatum infestations. from the numerous hosts investigated, only five species (including marcusenius macrolepidotus (peters) which was not encountered in middle letaba dam) were infested (table 7). from the infested hosts, a total of 1985 parasites were procured with prevalence and intensity values, as given in table 7, profoundly higher than those recorded for middle letaba dam. seven piscivorous birds at middle letaba dam were identified by mokgalong (1996) as regular final hosts for c. complanatum (table 5). although relatively low numbers of birds were examined, the data of table 5 clearly indicate a. rufa as the predominant definitive host for this parasite. reed cormorants, phalacrocorax africanus (gmelin), grey herons, ardea cinerea linnaeus. and black-crowned night herons, nycticorax nycticorax linnaeus, seem to act as subsidiary final hosts. if the results from the present study and that of saayman (1986) are evaluated, the following important observation needs to be elucidated: s. intermedius and m. macrolepidotus seem to be the major intermediate hosts for c. complanatum (table 7). both were, however, absent from middle letaba dam at the time of the survey, but s. intermedius does occur in the catchment area of the dam (table 1). it can, therefore, be expected that the latter will colonize the dam at some stage. as the biological requirements for the completion of the life cycle of the parasite are established in the dam, it could well be expected that s. intermedius would immediately become infested after colonization. euclinostomum heterostomum (rudolphi) during this study, e. heterostomum was recorded regularly, encysted in muscle tissue of the host. the major intermediate host was identified as o. mossambicus from which a total of 96 parasites were collected (table 8). a single male c. gariepinus (totable 7 fish intermediate hosts from various localities in the olifants (o) and limpopo (l) rivers drainage systems infested with clinostomum complanatum (rudolphi)—data from saayman (1986) host n prev. in. mi. locality and drainage system schilbe intermedius (rüppell) 399 28.7 1–21 3.4 luphephe-nwanedzi dams (l) nwanedzi river (l) glen alpine dam (l) magalakwena river (l) nzhelele dam (l) nile flood pans (l) ga-selati river (o) olifants river (o) letaba estates (o) marcusenius macrolepidotus (peters) 524 54.7 1–122 6.7 luphephe-nwanedzi dams (l) nwanedzi river (l) glen alpine dam (l) nzhelele dam (l) nile flood pans (l) mohlapitse river (o) letaba estates (o) barbus unitaeniatus günther 2 50 1 1.0 letaba estates (o) barbus toppini weber 1 100 8 8.0 letaba estates (o) chiloglanis pretoriae van der horst 115 5.2 1–4 2.2 luvhuvhu river (l) nwanedzi river (l) mohlapitse river (o) n = number of hosts examined prev. = prevalence of infestation (%) in. = intensity of infestation mi. = mean intensity of infestation 197 p.a.s. olivier, w.j. luus-powell & j.e. saayman tal length = 66,3 cm), from a sample of 21 specimens examined, collected during the winter survey, was found harbouring nine specimens of e. heterostomum. this may suggest that c. gariepinus, in extreme cases, can act as a reservoir host. the data of table 8 reflect that all length groups of o. mossambicus may become infested with the parasite although it appears as if larger fish (total length longer than 20 cm) may be more vulnerable to infestation. no significant seasonal variations in infestation, nor any prevalence amongst different sexes, were observed. in middle letaba dam it was established that members of the ardeidae, phalacrocoracidae and anhingidae act as final hosts for the adults of e. heterostomum (table 5), the latter firmly attached to the lining of the buccal cavity and oesophagus. except for p. africanus with a prevalence of 30 %, all the other hosts demonstrated higher prevalence values ranging from 50–100 % with intensity values ranging from 1–21. white-breasted cormorants, phalacrocorax lucidus (lichtenstein), and a. rufa seem to be the major definitive hosts, but, a. cinerea, black-headed herons, ardea melanocephala (anon), and purple herons, ardea purpurea linnaeus, may well prove to be equally or even more important, pending the examination of larger numbers of hosts. although clinostomid metacercarial cysts attain a relatively large size, and notwithstanding the relatively high infestation values, it does not appear as if these parasites cause any deleterious affects to semi-adult and/or adult hosts. this observation is supported by paperna (1980). in fingerlings and table 8 seasonal analysis per length group (tl, cm) of oreochromis mossambicus (peters) for euclinostomum heterostomum (rudolphi) infestations in middle letaba dam season < 10 cm 10.1–20 cm 20.1–30 cm > 30 cm all size groups summer number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 39 12.8 1 1 5 24 33.3 1–9 2.6 21 23 56.5 1–8 2.2 29 1 100 5 5 5 87 31 1–9 2.2 60 autumn number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 2 – – – – 10 20 1–2 1.5 3 3 – – – – – – – – – 15 13.3 1–4 1.5 3 winter number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 11 9.1 1 1 1 4 – – – – 25 36 1–4 2.1 19 – – – – – 40 25 1–4 2 20 spring number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 18 5.6 1 1 1 14 28.6 1–6 3 12 3 – – – – – – – – – 35 14.3 1–6 2.6 13 all seasons number of hosts examined prevalence of infestation intensity of infestation mean intensity of infestation number of parasites collected 70 10 1 1 7 52 26.9 1–9 2.6 36 54 43.1 1–8 2.2 48 1 25 1 1 5 177 24.9 1–9 2.2 96 198 monogenean and clinostomid infestations of freshwater fi sh and waterbird hosts in limpopo, south africa smaller fish species, however, severe infestations are likely to be detrimental. paperna (1980) reported mortalities amongst very young (40–60 mm) o. mossambicus, even with intensity values of as low as 3–5 worms. during the present survey a number of juvenile specimens of o. mossambicus infested with e. heterostomum showed definite signs of locomotory impairment. severe infestation with n. intermedialis is also likely to cause respiratory inhibition. britz, saayman & van as (1984) and britz, van as & saayman (1984) reported considerable physical and histological damage to the oesophageal mucosa of avian hosts infested with clinostomid worms. this supports earlier reports from baugh & pandey (1969) and ukoli (1970). the latter author observed that if worms are removed from the oesophageal region of the host, part of the host tissue is torn away, leaving a deep lesion which bleeds freely. the study at middle letaba dam confirmed these observations in almost all infested avian hosts. acknowledgements the authors thank the department of development aid, premier food industries ltd. and the research development and administration of the university of limpopo for financial assistance. we also thank the (then) gazankulu government for their involvement and support and the department of biodiversity, university of limpopo for infrastructure and technical support. references allanson, r.b. & jackson, p.b.n. 1983. limnology and fisheries potential of lake pk le roux. pretoria: csir (south african 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice course doi:10.4102/ojvr.v79i2.477http://www.ojvr.org page 1 of 2 how to cite this course: simuunza, m.c., ‘msc one health analytical epidemiology’, onderstepoort journal of veterinary research 79(2), art. #477, 2 pages. http://dx.doi. org/10.4102/ojvr.v79i2.477 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright: © 2012. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. c ou rs e de liv er y w ill m ai nl y be d on e by e xp er ts f ro m t he sc ho ol s of v et er in ar y m ed ic in e an d m ed ic in e of th e u ni ve rs ity of z am bi a. e xp er ts fr om ot he r so ut he rn a fr ic an ac ad em ic an d re se ar ch in st itu tio ns w ith in th e sa c id s co ns or tiu m a s w el l as s ta ff f ro m c ol la bo ra tin g re se ar ch i ns tit ut io ns i n in du st ri al iz ed c ou nt ri es , es pe ci al ly th e r oy al v et er in ar y c ol le ge (r v c ) an d th e l on do n sc ho ol o f h yg ie ne a nd t ro pi ca l m ed ic in e (l sh t m ) w ill al so p ar tic ip at e in c ou rs e de liv er y. t hi s w ill c re at e a bl en d of p ro fe ss io na l kn ow le dg e an d sk ill s in e pi de m io lo gy a nd o ne h ea lth th at w ill pr ov id e a un iq ue le ar ni ng en vi ro nm en t. sa c id s is a o ne h ea lth c on so rt iu m o f so ut he rn a fr ic an ac ad em ic a nd r es ea rc h in st itu tio ns in vo lv ed w ith in fe ct io us di se as es of hu m an s an d an im al s in th e d em oc ra tic r ep ub lic o f c on go ( d r c ), m oz am bi qu e, s ou th a fr ic a, z am bi a an d t an za ni a. s ta ff f ro m t he se i ns tit ut io ns a re w or ki ng in sm ar t pa rt ne rs hi p w ith co lle ag ue s fr om l sh t m a nd r v c – t w o u ni ve rs ity o f l on do n c ol le ge s w hi ch pa rt ic ip at e in th e in te rd is ci pl in ar y l on do n in te rn at io na l d ev el op m en t c en tr e (l id c ). sa c id s al so co lla bo ra te s w ith th e in te rn at io na l l iv es to ck r es ea rc h in st itu te ( il r i) a nd c on tin ue s to f or ge l in ka ge s w ith o th er in st itu tio ns fr om in du st ri al is ed c ou nt ri es . t he sa c id s he ad qu ar te rs is lo ca te d at th e so ko in e u ni ve rs ity of a gr ic ul tu re . t hi s ex ci tin g ne w c ou rs e fo cu se s on p ro vi si on o f kn ow le dg e in a na ly tic al e pi de m io lo gy a nd o n ho w to a pp ly th is k no w le dg e in th e co nt ro l o f di se as es in bo th h um an a nd a ni m al p op ul at io ns ( o ne h ea lth c on ce pt ). t he c ou rs e ha s be en d es ig ne d to t ra in a ne w g en er at io n of h um an c ap ita l w hi ch w ill p ro vi de th e ne ce ss ar y le ad er sh ip i n so lv in g va ri ou s he al th ch al le ng es u si ng e pi de m io lo gi ca l to ol s to i m pr ov e th e he al th a nd w el fa re o f o ur c om m un iti es . a pp lic an ts f or t he c ou rs e sh ou ld h av e a de gr ee i n m ed ic in e, v et er in ar y m ed ic in e, o r a ba si c de gr ee i n bi ol og ic al s ci en ce s. g ra du at es in h ea lth r el at ed fi el d of st ud y su ch as st at is tic s, de m og ra ph y, fo od sc ie nc e an d pu bl ic h ea lth m ay a pp ly p ro vi de d th ey ha ve in te re st in he al th an d ha ve ex pe ri en ce of ha vi ng w or ke d in a n in st itu tio n de al in g w ith h ea lth is su es . t he re a re l im it ed s ch ol ar sh ip s av ai la bl e fo r th is c ou rs e pr ov id ed b y th e w el lc om e t ru st th ro ug h sa c id s. t he m sc i n o ne h ea lth a na ly tic al e pi de m io lo gy w ill be of fe re d by th e sc ho ol of v et er in ar y m ed ic in e in co lla bo ra tio n w ith th e sc ho ol of m ed ic in e at th e u ni ve rs ity of z am bi a. a ll th e st ud en ts w ill be re gi st er ed by th e sc ho ol of v et er in ar y m ed ic in e. t he sc ho ol of v et er in ar y m ed ic in e w as e st ab lis he d in 1 98 4 an d ha s so m e of th e be st l ea rn in g fa ci lit ie s in z am bi a. t he s ch oo l ha s lo ng st an di ng ex pe ri en ce in co nd uc tin g po st gr ad ua te t ra in in g an d re se ar ch i n di se as es o f ve te ri na ry a nd p ub lic h ea lth im po rt an ce . t he sc ho ol is lo ca te d w ith in th e u ni ve rs ity of z am bi a m ai n ca m pu s. c ou rs e c oo rd in at or : d r m ar tin c . s im uu nz a e m ai l: m ar tin .s im uu nz a@ un za .z m ; m si m uu nz a@ ly co s. co m ph on e: + 26 0 21 1 29 37 27 m ob ile : + 26 0 96 88 08 51 1 fa x: + 26 0 21 1 29 37 27 w eb si te : w w w .u nz a. zm a dd re ss : d ep ar tm en t o f d is ea se c on tr ol sc ho ol o f v et er in ar y m ed ic in e u ni ve rs ity o f z am bi a p. o . b ox 3 23 79 l us ak a, z am bi a. source: author’s original material as presented at the conference course continues on the next page → msc one health analytical epidemiology m.c. simuunza http://dx.doi.org/10.4102/ojvr.v79i2.462 http://dx.doi.org/10.4102/ojvr.v79i2.462 course doi:10.4102/ojvr.v79i2.477http://www.ojvr.org page 2 of 2 a ck no w le dg em en ts t he o ne h ea lth a na ly tic al e pi de m io lo gy m sc p ro gr am m e is su pp or te d by s a c id s th ro ug h a gr an t f ro m th e w el lc om e t ru st (w w w .w el lc om e. ac .u k) u nd er th e a fr ic an in st itu tio n in iti at iv e. t he c ou rs e is a tw o y ea r fu lltim e m sc p ro gr am m e of th e u ni ve rs ity o f z am bi a ca le nd ar o f se m es te rs an d ac ad em ic y ea rs c om pr is in g of a t au gh t co m po ne nt i n th e fi rs t ye ar a nd a r es ea rc h pr oj ec t cu lm in at in g in to th e su bm is si on o f d is se rt at io n in th e se co nd y ea r. st ud en ts w ill u nd er ta ke th e fo llo w in g co re c ou rs es : � r es ea rc h m et ho do lo gy a nd c om pu te r a pp lic at io ns � pr in ci pl es o f e pi de m io lo gy a nd b io st at is tic s � d is ea se s ur ve ill an ce a nd r is k a na ly si s � e m er gi ng a nd r eem er gi ng d is ea se s � in fe ct io us d is ea se m od el in g an d g eo gr ap hi ca l i nf or m at io n sy st em � a dv an ce d st at is tic al m et ho ds in e pi de m io lo gy � o ne h ea lth m ed ic in e an d g lo ba liz at io n in a dd iti on s tu de nt s w ill b e ab le to c ho os e on e el ec tiv e co ur se fr om th e fo llo w in g: � h ea lth e co no m ic s, p ol ic y, m on ito ri ng a nd e va lu at io n � m ol ec ul ar e pi de m io lo gy a nd b io in fo rm at ic s � e nv ir on m en ta l e pi de m io lo gy y ea r tw o sh al l co m pr is e su pe rv is ed r es ea rc h w or k cu lm in at in g in to s ub m is si on o f a di ss er ta tio n an d sh al l b e un de rt ak en o ve r a pe ri od o f tw o se m es te rs o f fu lltim e st ud y. t he to pi c (r es ea rc h pr op os al ) of th e di ss er ta tio n sh al l be g ui de d by t he o ne h ea lth t he m e an d sh al l be a pp ro ve d by t he s ch oo l po st gr ad ua te c om m itt ee in th e la st s ev en w ee ks o f t he s ec on d se m es te r o f p ar t i . “ ... be tw ee n an im al a nd h um an m ed ic in e th er e ar e no d iv id in g lin es -no r s ho ul d th er e be ." r ud ol f v ir ch ow (1 82 119 02 ). t hi s co ur se a im s to e qu ip s ci en tis ts w ith s ki lls in a na ly tic al e pi de m io lo gy fo r t he ef fi ci en t p re ve nt io n an d co nt ro l o f h um an , a ni m al a nd z oo no tic d is ea se s. o ne he al th is a s ci en tif ic c on ce pt w ho se m is si on is to fo st er c lo se r p ro fe ss io na l in te ra ct io n, c ol la bo ra tio n an d ed uc at io na l o pp or tu ni tie s ac ro ss th e ve te ri na ry , m ed ic al a nd a lli ed s ci en ce s fo r t he p ur po se o f i m pr ov in g hu m an a nd a ni m al h ea lth . u po n co m pl et io n of th e m sc in o ne h ea lth a na ly tic al e pi de m io lo gy , g ra du at es w ill b e ab le to : � d em on st ra te k no w le dg e of th e co nc ep t of “ o ne h ea lth ” an d its a pp lic at io n in th e de ve lo pm en t of h ea lth p ol ic y an d th e co nt ro l an d pr ev en tio n of in fe ct io us d is ea se s. � d em on st ra te kn ow le dg e on ho w in te ra ct io ns be tw ee n hu m an an d an im al p op ul at io ns a nd e nv ir on m en ta l ch an ge s ca n le ad to e m er ge nc y an d re em er gi ng o f i nf ec tio us d is ea se s. � pl an , u nd er ta ke a nd a na ly ze d at a fr om a re se ar ch p ro je ct c on ce rn in g hu m an , an im al a nd z oo no tic d is ea se s an d be ab le t o m on ito r an d ev al ua te a ct iv iti es fo r po lic y an d pr og ra m m e de ve lo pm en t. � a pp ly a s ci en tif ic s ty le o f w ri tin g in th e pr es en ta tio n of re se ar ch . � a pp ly e co no m ic a nd s oc io -e co no m ic co nc ep ts a nd m et ho ds i n th e de si gn , im pl em en ta tio n an d ev al ua tio n of he al th d el iv er y se rv ic es . � d et er m in e th e fa ct or s af fe ct in g th e oc cu rr en ce an d sp re ad of di se as e th ro ug h hu m an a nd a ni m al p op ul at io ns an d be a bl e to p re ve nt o r co nt ro l su ch sp re ad . source: authors original material as presented at the conference msc one health analytical epidemiology m.c. simuunza (continues...) article information authors: e. lum1,2 h.k. kimbi1,2 j. mbuh1 j. ndamukong-nyanga1,2 a. l. njunda3 j. lello4 affiliations: 1department of plant and animal sciences, university of buea, cameroon 2research foundation for tropical diseases and the environment, buea, cameroon 3department of medical laboratory science, university of buea, cameroon 4school of biosciences, organism and environment group, cardiff university, cardiff correspondence to: antony musoke how to cite this poster: lum, e., kimbi, h.k., mbuh, j., ndamukong-nyanga, j., njunda, a.l. & lello, j., 2012, ‘co-infections of malaria and soil-transmitted helminths in localities with different levels of urbanization in the mount cameroon region’, onderstepoort journal of veterinary research 79(2), art. #487, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.487 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. co-infections of malaria and soil-transmitted helminths in localities with different levels of urbanisation in the mount cameroon region in this poster... open access malaria co-exists with intestinal helminths and they have different effects on infected individuals. a total of 235 and 208 children from ekona and great soppo respectively of both sexes aged 4–14 years were enrolled into a cross-sectional study.capillary blood was collected for detection and determination of malaria parasitaemia as well as pcv. stool samples were collected for quantitative determination of helminth ova by kato-katz technique. the prevalence of malaria and helminths was higher in ekona than great soppo. in great soppo, trichuris was the most prevalent helminth than great soppo and an association was found between these co-infections. more children were co-infected in ekona and co-infecting species were ascaris and plasmodium falciparum. the prevalence of malaria and intestinal helminths as well as co-infection was lower in great soppo than in ekona, probably due to increased urbanization in great soppo than ekona. article information authors: qiong liu1 yuheng gong1 yuqin cao1 xintian wen1 xiaobo huang1 qigui yan1 yong huang1 sanjie cao1 affiliations: 1college of veterinary medicine and institute of preventive veterinary medicine, sichuan agricultural university, china correspondence to: sanjie cao postal address: 46 xinkang road, yucheng district, yaan city, sichuan province, china dates: received: 09 sept. 2012 accepted: 03 dec. 2012 published: 06 mar. 2013 how to cite this article: liu, q., gong, y., cao, y., wen, x., huang, x., yan, q. & et al., 2013, ‘construction and immunogenicity of a ∆dapxic/ompp2 mutant of actinobacillus pleuropneumoniae and haemophilus parasuis’, onderstepoort journal of veterinary research 80(1), art. #519, 6 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.519 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. construction and immunogenicity of a ∆apxic/ompp2 mutant of actinobacillus pleuropneumoniae and haemophilus parasuis in this original research... open access • abstract • introduction • materials and methods    • bacteria isolates and growth conditions    • construction of vector for homologous recombination    • electroporation of actinobacillus pleuropneumoniae and selection of recombinants    • identification    • growth and genetic stability of the mutant strain    • haemolytic activity and nad dependency test    • safety and vaccination efficacy of the mutant strain in mice    • serological measurement • results    • identification of actinobacillus pleuropneumoniae apxic mutant lacking antibiotic resistance markers    • growth and genetic stability of the mutant strain    • haemolytic activity test    • safety of the mutant strain in mice    • serological measurement • discussion • conclusion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ the apxic genes of the actinobacillus pleuropneumoniae serovar 5 (sc-1), encoding the apxi-activating proteins, was deleted by a method involving sucrose counter-selection. in this study, a mutant strain of a. pleuropneumoniae (sc-1) was constructed and named dapxic/ompp2. the mutant strain contained foreign dna in the deletion site of ompp2 gene of haemophilus parasuis. it showed no haemolytic activity and lower virulence of cytotoxicity in mice compared with the parent strain, and its safety and immunogenicity were also evaluated in mice. the ld50 data shown that the mutant strain was attenuated 30-fold, compared with the parent strain (ld50 of the mutant strain and parent strain in mice were determined to be 1.0 × 107 cfu and 3.5 × 105 cfu respectively). the mutant strain that was attenuated could secrete inactivated apxia rtx toxins with complete antigenicity and could be used as a candidate live vaccine strain against infections of a. pleuropneumoniae and h. parasuis. introduction top ↑ actinobacillus pleuropneumoniae (ap) is the causative agent of porcine pleuropneumoniae, a highly contagious disease contributing to fatalities in pig production worldwide (sebunya & saunders 1983). the pathogen is transmitted by air or direct contact with infected pigs (chiers et al. 2002). haemophilus parasuis is a commensal bacterium in the upper respiratory tract of conventional pigs, but under appropriate conditions, can invade and cause severe systemic disease (glasser`s disease) (hoefling 1991). both bacteria are members of the family of pasteurellaceae; their growth requires a v factor (nad), but no x factor (hemin). in recent years, the porcine industry has suffered a significant increase in the incidence, morbidity and mortality associated with glasser`s disease (oliveira & pijoan 2004) an infection often combined with ap, which has caused serious economic loss world-wide.the apx is characterised as one of the major virulence determinants of a. pleuropneumoniae (frey & nicolet 1990). up till now, four different apx toxins (apxi to apxiv) have been described and varying combinations of apx toxins are produced in different a. pleuropneumoniae serovars. for instance, serovars 1, 5, 9 and 11 generate apxi and apxii (tascon et al. 1993). amongst the apx toxins, apxi, which is secreted by the most virulent serovars such as serovars 1, 5, 9, 10 and 11, has shown strong haemolytic activity and cytotoxic activity (kamp et al. 1991). meanwhile, apxi, apxii and apxiii are strongly immunogenic and involved in the induction of protective immunity. the toxin apxi is encoded on polycistronic operons apxicabd (frey et al. 1994). in these operons, the a gene encodes the protoxin, whilst the c gene encodes a cyltransferase, which is involved in the post-translational activation of the protoxin in the cytoplasm (issartel, koronakis & hughes 1991). the b and d genes respectively encode an atpase and a protein adaptator to facilitate apx toxin secretion (thanabalu et al. 1998). the inactivated whole-bacteria vaccine has been demonstrated to elicit protection against infection from the same serovar of a. pleuropneumoniae (hensel et al. 2000) and subunit vaccines have been tested to provide some immunity with reduction in clinical symptoms and pulmonary lesions (goethe et al. 2000). however, in terms of inactivated whole-bacteria vaccine, it was not able to generate cross protection to infection from heterologous serovars or to provide long-lasting immunity. secondly, in terms of subunit vaccines, although the subunit vaccines contained the mixed outer membrane proteins and although proteins secreted from several serovars or recombinant toxins have been shown to protect animals from clinical infection (maas et al. 2006), the relatively higher production cost of mixing different antigens would undoubtedly limit their application. in contrast, pigs surviving infection with one serovar are at least partially protected against infection by other serovars. consequently, developing a live vaccine by attenuation of a. pleuropneumoniae has become a dominant research field. the outer membrane protein p2 (ompp2) of h. parasuis is encoded by the ompp2 gene and is an immunodominant porin with considerable antigenic heterogeneity amongst different serovars of h. parasuis (mullins et al. 2009). the ompp2 has been targeted as a potential vaccine candidate. as a porin, ompp2 plays a role in colonisation and has been shown binding to specific components of human mucin (reddy et al. 1996). it was demonstrated that ompp2 could be expressed in h. parasuis and that it shows good antigenicity. inactivated bacterial vaccines and subunit vaccines are currently the main ways of preventing porcine pleuropneumoniae. however, the multitude of serovars (15 serovars) and the limited cross-protection conferred by bacterins or apx toxin-based subunit vaccines (jolie, mulks & thacker 1995) makes treatment of a. pleuropneumoniae infection difficult. a live vaccine is considered to offer the best prospects of obtaining cross-protection against this bacterium. in the current study, an attenuated mutant a. pleuropneumoniae strain with the ompp2 gene of h. parasuis serovar 5 (nagasaki) (takahashi et al. 2001) was developed as a potential vaccine candidate without introducing an antibiotic resistance marker by using a sucrose counter-selection strategy (oswald et al. 1999). actinobacillus pleuropneumoniae serovar 5 (sc-1), is one of the predominant serovars in china, and produces both apxi and apxii toxins. the apxic region was targeted for disruption with a gene of h. parasuis to produce a mutant strain that secretes an inactive toxin with full antigenic properties. here, we describe the construction of the homologous recombination vector, the characterisation of the resultant mutant strain and the testing of its virulence and protective efficacy against a. pleuropneumoniae and h. parasuis infection in mice. materials and methods top ↑ bacteria isolates and growth conditionsc haemophilus parasuis serovar 5 (nagasaki) and a. pleuropneumoniae serovar 5 (sc-1) were selected for this study. the bacterial strains were both grown on tryptic soy agar (tsa, difcotm, bd) and in tryptic soy broth (tsb, difcotm, bd) supplemented with 1% (wt/vol) nad. all bacterial strains were grown at 37 °c.escherichia coli dh5α was routinely maintained in luria–bertani medium (lb) or maintained in lb supplemented with 100 mg/ml of ampicillin or 50 mg/ml of kanamycin when containing the relevant vector. the vector pboskdic, constructed by dr guobin wang, was used for constructing the vector of the homologous recombination. construction of vector for homologous recombination the first step was to transform the restriction site on the right homologous arm of homologous recombination vector pboskdic with spei and mlui. the primers used in this study are listed in table 1. a pcr of the right homologous arm was conducted by initial denaturing at 94 °c for 5 min, followed by 30 cycles of denaturing at 94 °c for 45 sec, annealing at 55 °c for 30 sec and extension at 72 °c for 1.5 min, with a final extension at 72 °c for 10 min. the reconstructive vector was called pboskdic-1. the ompp2 gene of h. parasuis was then amplified by pcr with the same pcr conditions as described above, the amplicons of ompp2 were checked in agarose gel electrophoresis, purified and cloned in pmd-simple-19t vector (takara biotechnology [dalian] co., ltd), followed by subcloning between spei and mlui sites of the reconstructive vector pboskdic-1. the vector was use for homologous recombination and called pboskdapxic/ompp2 (figure 1). table 1: sequences of primers used for construction of the transfer vector. figure 1: schematic representation of the construction of the actinobacillus pleuropneumoniae mutant strain. electroporation of actinobacillus pleuropneumoniae and selection of recombinants the transfer vector pboskdapxic/ompp2 was extracted by tm vector mini kit ii (omega). the competent cells of a. pleuropneumoniae serovar 5 (sc-1) were prepared according to the general optimisation protocol for the transformation of bacteria provided by the electroporation system (gene pulser xcelltm, bio-rad). pulse conditions were as follows: cuvette 0.2 cm-diameter; voltage 2.5 kv; capacitance 25 uf; resistance 800 ω; time constant 5 ms. after electroporation, the cells were transferred into 1 ml tsb supplemented with nad and incubated immediately at 37 °c, at 180 r/min for 3 h. the recovered cells were plated onto the tsa plates, supplemented with 1% (wt/vol) nad and 50 ug/ml of kanamycin, incubated overnight at 37 °c.kanamycin resistant colonies were selected and inoculated onto tsa-sucrose plates, which were incubated at 37 °c to confirm that the sucrose sensitivity was conferred by transfer of the vector (figure 1). identification the genomic dna of the parent and mutant strains was extracted individually. primer pairs ic-f and ic-r were used to amplify the relevant fragments from the genomic dna of the parent and mutant strains. the dna fragments of these strains were separated on a 1% agarose gel. growth and genetic stability of the mutant strain the mutant strain was inoculated into tsb supplemented with 1% (wt/vol) nad, the bacterial concentration was determined at od600 every hour, and the value recorded. the growth curves were drawn to compare features of growth cycles. the genetic stability of the inserted ompp2 gene in the genome of the mutant strain was tested. this was done by propagating the mutant strain for 10 passages serially in tsb supplemented with 1% (wt/vol) nad, and then amplifying the relevant fragments by pcr from the colony of each passage. haemolytic activity and nad dependency test the haemolytic activity and nad dependency of the parent and mutant strains were examined. briefly, they were inoculated onto sheep blood agar plates overnight at 37 °c. escherichia coli dh5α was used as negative control. the haemolytic activity of the strains was recorded. at the same time, the growth was observed of the mutant strain inoculated in the tsb, supplemented with 1% (wt/vol) nad and incubated overnight at 37 °c. safety and vaccination efficacy of the mutant strain in mice cultures of a. pleuropneumoniae serovar 5 were grown overnight at 37 °c in tsb supplemented with nad, followed by dilution to 1:1000. the diluted culture was incubated again until the od600 reached 0.8. at this point, the viable count of a. pleuropneumoniae dapxic/ompp2 was found to three concentrations (table 2). for the virulence test, three groups of eight 6-week-old female balb/c mice (sichuan province huaxi experimental animal centre, chengdu, china) were injected intraperitoneally (i.p.) with 200 µl tsb medium containing various bacterial counts of a. pleuropneumoniae or h. parasuis. survival of the mice was recorded after 72 h (table 2). the lethal dose of 50% (ld50) value of the mutant strain dapxic/ompp2 was calculated by karber’s method. table 2: virulence of the mutant strain and the parent strain of actinobacillus pleuropneumoniae and haemophilus parasuis in mice. serological measurement serum was isolated from whole blood collected from mice at four points in time: before vaccination (day 0), before the secondary vaccination (day 14), before the third vaccination (day 28) and before challenge (day 42). inactivated a. pleuropneumoniae and h. parasuis were used as a somatic antigen by elisa. briefly, the 96-well enzyme-linked immunosorbent assay (elisa) microplates were coated overnight at 4 °c with 0.08 mg/ml of inactivated a. pleuropneumoniae and h. parasuis diluted in 100 ml of 50 mm sodium carbonate buffer, ph 9.6. plates were washed three times with washing buffer (0.05% tween 20 in pbs, pbst) and blocked for 1 h at 37 °c with blocking buffer (1% bsa in pbst). after three washes, serum samples were diluted to 1:100 in blocking buffer, added to each well (100 µl per well) in duplicate and incubated for 1 h at 37 °c. after three washes, 100 µl of hrp-conjugated goat antis mice igg (sba, america), diluted to 1:5000 in blocking buffer, was added to each well and incubated at 37 °c for 1 h. after three washes, 50 µl tmb was added to each well at room temperature in dark reaction for 15 min the reaction was then stopped by adding 100 µl of 2 m h2so4 per well. the absorbance was read at 450 aa with an elisa micoplate reader (model 680, bio-rad). results top ↑ identification of actinobacillus pleuropneumoniae apxic mutant lacking antibiotic resistance markers after electroporation of a. pleuropneumoniae competent cells with the vector pboskdapxic/ompp2, some transformants were screened from growth on tsa plates containing kanamycin and sucrose, respectively. three transformants with the phenotype of kanr and sucs were chosen for propagation in tsb medium without kanamycin or sucrose to promote the second order recombination event and excised the kanamycin resistance and sacb genes. finally, the cells were further grown overnight in tsb-nad in the absence of kanamycin, then grown onto tsa-sucrose plates. the sucr colonies were replica plated onto tsa-sucrose and tsa-sucrose-kanr plates to choose the colonies with the kans and sucr phenotype.the apxic gene of a. pleuropneumoniae was identified by pcr (figure 2), the genomic dna of the parental strain had an amplicon of about 800 bp and the mutant stain an amplicon about 1400 bp. the amplification of the ompp2 gene of h. parasuis yielded a band of 1100 bp. this suggests that the ompp2 gene of h. parasuis was inserted into the apxic gene of a. pleuropneumoniae at the deletion site. figure 2: confirmation of apxic gene disruption in the genome of the mutant strain by polymerase chain reaction amplification using primer pair ic-f and ic-r. growth and genetic stability of the mutant strain no obvious difference was observed in vitro growth curves of the parental strain and mutant strains, indicating that the ompp2 gene of h. parasuis that took the place of the apxic gene had no significant influence on the growth of a. pleuropneumoniae (figure 3). figure 3: growth curves of the parent and mutant strains. both strains showed an identical growth response. a pcr fragment with a size of 1400 bp was amplified from all ten serial passages in tsb supplemented with 1% (wt/vol) nad, indicating that the ompp2 gene of h. parasuis was stably inserted into the genome of the mutant strain, and the apxic gene of a. pleuropneumoniae had been deleted (figure 4). figure 4: stability of the inserted ompp2 gene in the genome of the mutant strain over 10 passages by polymerase chain reaction. haemolytic activity test the functional activity of the apxi toxin produced by dapxic/ompp2 was examined in the haemolytic assay. fully active apxi toxin had haemolytic activity, as demonstrated by clear zones surrounding the colonies of a. pleuropneumoniae on sheep blood agar (figure 5, section p). the apxic gene of a. pleuropneumoniae was deleted, and the ompp2 gene of h. parasuis showed no haemolytic activity, thus the mutant strain was lacking haemolytic activity. this was evident from the absence of a clear zone surrounding the colonies of the a. pleuropneumoniae mutant on sheep-blood agar (figure 5, section m). figure 5: identification of haemolysis of mutant strain. safety of the mutant strain in mice to test the virulence of the mutant strain, various concentrations of a. pleuropneumoniae dapxic/ompp2 and serovar 5 of h. parasuis and a. pleuropneumoniae parent strain were prepared and administrated to mice via the intraperitoneal route. no deaths were observed in mice that received 2.0 × 106 cfu of h. parasuis and 8.0 × 106 cfu of dapxic/ompp2, whilst 100% mortality was recorded in mice that received the parent strain at doses of 2.0 × 106 cfu. therefore, compared to the parent strain and serovar 5 of h. parasuis, virulence of the mutant was attenuated at least 30-fold (table 2). serological measurement serum was isolated from whole blood collected from mice at four points in time: before vaccination (day 0), before the secondary vaccination (day 14), before the third vaccination (day 28) and before challenge (day 42). antibodies against the somatic antigen of inactivated a. pleuropneumoniae and h. parasuis were examined using elisa (see figure 6). the elisa titres were defined as the highest serum dilution resulting in od450 twice and the negative control serum at a dilution of 1:100. figure 6: indirect elisa analysis of the igg antibody in murine serum after immunisation. six-week-old balb/c mice were subcutaneous injected three times every two weeks with the mutant strain at a dosage of 3.0 x 106 cfu (group a and b) 200 μl per mice. the mice of group b and d were vaccinated with 7.0 x 105 cfu of the parent strain (serovar 5 of actinobacillus pleuropneumoniae), using inactivated bacteria as a coating antigen. absorbance was measured at 450 nm in an automated plate reader. results are expressed as means of the od450 ± s.d. (n = 3). numbers on the x-axis indicate week post-primary immunisation. discussion top ↑ it is known that apxi toxin is a very important member of the rtx family and common structural features are present in the rtx toxins. when used as a subunit vaccine, it could offer a good level of protection in mice against a. pleuropneumoniae infection (seah, frey & kwang 2002). the apxic contains 13 repeated nonapeptides and plays an important role during the interaction between apxi toxins (issartel et al. 1991). in earlier studies, expression of the apxc gene was generally inactivated by insertion of foreign marker genes or by deletion of the gene (liu et al. 2007). these mutant strains have the potential to reverse virulence by releasing the inserted genes and transmitting the marked antibiotic resistance gene to the animal’s normal bacterial flora. live vaccines are considered to offer the best prospect for obtaining cross-protection against a. pleuropneumoniae serovar 5 and h. parasuis serovar 5. in 1999, oswald constructed an a. pleuropneumoniae ureac mutant strain using a new method, the sucrose counter-selectable system, but its application in a. pleuropneumoniae has been limited so far (oswald et al. 1999). the method demonstrated that it is possible to modify the a. pleuropneumoniae genome without inserting an antibiotic resistance marker.based on these previous studies, we constructed a live attenuated apxi inactivation mutant strain by deleting the apxic toxin, and inserting the ompp2 gene of h. parasuis. the a. pleuropneumoniae serovar 5 (sc-1) was selected as the parent strain for the mutant construction, because it is one of the most prevalent serovars in china, and is capable of secreting apxi and apxii. the results indicate that the mutant strain dapxic/ompp2 is capable of secreting apxi, apxii and ompp2 of h. parasuis after deletion of apxic as the dapxic/ompp2 still contains apxib and apxid genes, which encode proteins that are essential for the secretion of the toxin (welch 1991). compared with the parent strain, virulence of the mutant strain (dapxic/ompp2) was attenuated 30-fold, providing more evidence that the apxi of a. pleuropneumoniae serovar 5 is necessary for full virulence (goethe et al. 2000). in terms of haemolytic activity, the mutant strain had lower visible haemolytic activity on agar plates. it was proved that apxic gene was absence from the genome of a. pleuropneumoniae (sc-1). the serological measurement of mice serum show that the mutant strain provided lower protection against h. parasuis than a. pleuropneumoniae. this is possibly because a. pleuropneumoniae is the parent strain, whilst the expression of ompp2 gene in the mutant strain is not enough to protect against invasion of h. parasuis. another aspect is that the ompp2 gene is not the most important antigen of h. parasuis, but the mutant strain could still protect mice against h. parasuis and a. pleuropneumoniae. in summary, the mutant strain dapxic/ompp2 had greatly reduced virulence. it needs to be pointed out that the mouse model is only a preliminary assay and full studies in pigs are needed to test this mutant in future. conclusion top ↑ in this study, we successfully constructed a mutant strain of a. pleuropneumoniae (sc-1) named dapxic/ompp2, whilch can provide the protection against h. parasuis and a. pleuropneumoniae. acknowledgements top ↑ this work was supported by the science expertise of public interest career – agriculture (no.201303034). competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions s.c. (sichuan agricultural university) was the project leader, q.l. (sichuan agricultural university) and y.g. (sichuan agricultural university) were responsible for experimental and project design and y.c. (sichuan agricultural university), x.w. (sichuan agricultural university) , x.h. (sichuan agricultural university), y.h. (sichuan agricultural university) and q.y. (sichuan agricultural university) performed some of the experiments. references top ↑ chiers, k., donne, e., van overbeke, i., ducatelle, r. & haesebrouck, f., 2002, ‘actinobacillus pleuropneumoniae infections in closed swine herds: infection patterns and serological profiles’, veterinary microbiology 85, 343–352. http://dx.doi.org/10.1016/s0378-1135(01)00518-1frey, j., haldimann, a., nicolet, j., boffini, a. & prentki, p., 1994, ‘sequence analysis and transcription of the apxi operon (hemolysin i) from actinobacillus pleuropneumoniae’, gene 142, 97–102. http://dx.doi.org/10.1016/0378-1119(94)90361-1 frey, j. & nicolet, j., 1990, ‘haemolysin patterns of actinobacillus pleuropneumoniae’, journal of clinical microbiology 28, 232–236. goethe, r., gonzales, o.f., lindner, t. & gerlach, g.f., 2000, ‘a novel strategy for protective actinobacillus pleuropneumoniae subunit vaccines: detergent extraction of cultures induced by iron restriction’, vaccine 19, 966–975. 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pore’, embo journal 17, 6487–6496. http://dx.doi.org/10.1093/emboj/17.22.6487 welch, r.a., 1991, ‘pore-forming cytolysins of gram-negative bacteria’, molecular microbiology 5, 521–528. http://dx.doi.org/10.1111/j.1365-2958.1991.tb00723.x introduction the gross morphological features of the avian tongue have been described in numerous species (see mclelland 1979 for a review of the earlier lit­ erature) and the structural adaptations of this organ linked to diet and mode of feeding (gardner 1926, 1927). many of these studies, particularly the earlier works, presented comparative information on the macroscopic features of the tongue with a view to providing taxonomic data (lucas 1896, 1897; gard­ ner 1926, 1927; harrison 1964). this information was subsequently used to classify the tongue of birds into various categories. gardner (1926, 1927), for example, recognised eight categories based on the function and adaptations of this organ. harrison (1964), on the other hand, proposed the classifica­ tion of avian tongues into five functional groups, namely tongues specialized for collecting food, eat­ ing, swallowing, taste and touch, and nest building. echoing the suggestion by gardner (1926, 1927) that microscopic data would enhance the under­ standing of macroscopic features, recent studies have generally supplied more comprehensive infor­ mation on the structure of the avian tongue by using both light and electron microscopy in addition to macroscopic descriptions (kobayashi, kumakura, 335 onderstepoort journal of veterinary research, 76:335–345 (2009) morphology of the tongue of the emu (dromaius novaehollandiae). i. gross anatomical features and topography m.r. crole* and j.t. soley department of anatomy and physiology, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract crole, m.r. & soley, j.t. 2009. morphology of the tongue of the emu (dromaius novaehollan­ diae). i. gross anatomical features and topography. onderstepoort journal of veterinary research, 76: 335–345 despite numerous papers addressing the topic, the gross morphology of the ratite tongue and more specifically that of the emu, has been superficially or poorly described. this paper presents the first definitive macroscopic description of the emu tongue and reviews, consolidates and compares the scattered information on the gross morphology of the ratite tongue available in the literature. twenty­ three heads obtained from birds at slaughter were used for this study. specimens were fixed in 10 % neutral buffered formalin, rinsed and the gross anatomy described. the emu tongue is divided into a body and a root. the body is triangular, dorsoventrally flattened, pigmented and displays caudally directed lingual papillae on both the lateral and caudal margins. the root, a more conspicuous struc­ ture in comparison to other ratites, is triangular, with a raised bulbous component folding over the rostral part of the laryngeal fissure. following the general trend in ratites, the emu tongue is greatly reduced in comparison to the bill length and is specifically adapted for swallowing during the cranioin­ ertial method of feeding employed by palaeognaths. this study revealed that it is not only the shape of the tongue that differs between ratites, as previously reported, but also its colour, appearance of its margins and root, and its length in comparison to the bill, and the shape of the paraglossum. keywords: dromaius novaehollandiae, emu, gross morphology, ratite, tongue * author to whom correspondence is to be addressed: e­mail: martina.crole@up.ac.za accepted for publication 4 march 2009—editor 336 morphology of tongue of emu (dromaius novaehollandiae). i. gross anatomical features and topography yoshimura, inatomi & asami 1998; jackowiak & godynicki 2005; jackowiak & ludwig 2008; tivane 2008). due to their commercial importance, the tongue and associated hyobranchial apparatus of domestic poul­ try have been described in detail (see calhoun 1954 for a review of the earlier literature; hodges 1974; mclelland 1975; nickel, schummer & seiferle 1977; homberger & meyers 1989). during the past 180 years numerous publications on the ratite tongue have appeared in the form of sketches, descriptions and comparisons (meckel 1829; cuvier 1836; gadow 1879; owen 1879; py­ craft 1900; göppert 1903; duerden 1912; farag gi­ ana 1933; roach 1952; feder 1972; mccann 1973; cho, brown & anderson 1984; fowler 1991; bonga tomlinson 2000; gussekloo & bout 2005; porches­ cu 2007; crole & soley 2008; jackowiak & ludwig 2008; tivane 2008). many of these studies, how­ ever, provide incomplete and sometimes mislead­ ing information on the macroscopic features of this organ. this situation is exacerbated by the fact that some descriptions are based on limited numbers of specimens ranging from embryos to fully mature birds, resulting in conflicting information that is diffi­ cult to interpret. the most comprehensive studies of a ratite tongue are those of jackowiak & ludwig (2008) and tivane (2008) on the ostrich, although the former authors neglected to reference any of the earlier literature on this topic. to date there have only been four reports on the gross morphology of the emu tongue. the most complete gross morphological description is that of faraggiana (1933) who studied a single excised specimen of the tongue and laryngeal mound. crole & soley (2008) described the basic features of the emu tongue. in a study of feeding in palaeognathous birds, bonga tomlinson (2000) depicts the outline of the emu tongue in relation to the hyobranchial apparatus and surrounding mandibular rami, and briefly describes the presence of lingual papillae. cho et al. (1984) simply note that “the emu tongue has a serrated edge”. this paper presents the first definitive macroscopic description of the emu tongue and reviews, consoli­ dates and compares the scattered information on the gross morphology of the ratite tongue available in the literature. this study not only contributes to a better understanding of the upper digestive tract of the emu but also provides data that can be used for more meaningful future comparative studies of the ratite tongue. materials and methods specimen collection the heads of 23 adult (14–15 months) emus of ei­ ther sex were obtained from a local abattoir (oryx abattoir, krugersdorp, gauteng province, south africa) immediately after slaughter of the birds. the heads were rinsed in running tap water to remove traces of blood and then immersed in plastic buck­ ets containing 10 % neutral buffered formalin. they were allowed to fix for approximately 4 h while being trans ported to the laboratory, after which they were immersed in fresh fixative for a minimum period of 48 h. care was taken to exclude air from the oro­ pharynx by wedging a small block of wood in the beak. specimen preparation and recording the specimens were rinsed in running tap water and each preserved head was used to provide infor­ mation on the gross anatomical features of the tongue and its topographical relationships within the oropharyngeal cavity. this was achieved by incising the right commissure of the beak, disarticulating the quadratomandibular joint and reflecting the mandi­ ble laterally to openly display the roof and floor of the oropharynx (fig. 1). the length (from the apex to the caudal edge of the caudal papillae) and width (between the tips of the last lateral papillae) (fig. 2) of 16 tongues were measured and the lateral and caudal lingual papillae counted. the bill length was measured on the mandibular rhamphotheca from the commissure to the rostral bill tip. relevant ana­ tomical features were described and recorded using a canon 5d digital camera with a 28–135 mm lens and a canon macro 100 mm lens for higher magni­ fication photographs. three tongues were removed from the heads by lift­ ing the organ from the floor of the oropharynx and cutting through the frenulum as well as the paired ceratobranchiale and urohyale of the hyobranchial apparatus. the mucosa was stripped from the tongues to expose the intraglossal elements (fig. 7 and 8) of the hyobranchial apparatus. all specimens were studied and described at the department of anatomy and physiology, faculty of veterinary sci­ ence, university of pretoria, south africa. this study was approved by the research committee of the fac ulty of veterinary science (protocol no. v040/08) and complied with the regulations regarding the ethical use of animals. the terminology used in this study was that of nomina anatomica avium (baumel, king, breazile, evans & vanden berge 1993). 337 m.r. crole & j.t. soley results topography the tongue of the emu consists of a rostral pigment­ ed body and a caudal, variably pigmented root, both of which lie within the confines of the non­pigment­ ed regions of the roof and floor of the oropharynx (fig. 1). the tongue body occupies the middle third of the floor of the oropharynx and is a triangular structure with the apex pointing rostrally. the tongue root (fig. 1 and 4) extends from the caudal lingual papillae to the glottis and is flanked by, but does not extend to, the paired ceratobranchiale of the hyo­ branchial apparatus. in the closed gape, the caudal margin of the tongue body lies beneath and in con­ tact with the rostral border of the choana, whereas the triangular tongue root fits snugly into the rostral aspect of the choana. in some tongues the apex is observed, in the closed gape, to make contact with the base of the median palatine ridge which origi­ nates at the border of the pigmented and non­pig­ mented regions of the palate. tongue body (corpus linguae) the tongue body is dorsoventrally flattened (fig. 5) with the dorsum being slightly raised in the centre and sloping towards the margins. it varies in length between 21–27 mm (average of 23.6 mm), and in width between 20–29 mm (average of 25.9 mm) (fig. 2). the apex (apex linguae) is rudimentary and varies in shape from a sharp point (fig. 1), to a blunt or rounded tip. in some instances the apex is invaginated by a shallow groove forming two small­ er points (fig. 2). the dorsal surface (dorsum ling­ uae) is pigmented giving it an ash­grey/brown col­ our in formalin­fixed specimens (fig. 1 and 2). the ventral surface (ventrum linguae) (fig. 6) is lighter in colour than the dorsal surface with the epithelium appearing glass­like (transparent). the rostromedi­ al region of the ventrum is slightly concave. a con­ spicuous, light­coloured, finger­like projection ex­ tends along the midline from the tip of the frenulum to end bluntly caudal to the apex (fig. 6). this line represents the rostral projection of the basihyale (see below) (fig. 8). from the rostrolateral surfaces of the frenulum two raised bands (crura) (fig. 6), are directed and taper towards the apex. numerous pale doughnut­shaped structures with a darker cen­ tre are clearly visible beneath both the dorsal and ventral surfaces of the tongue body (fig. 2 and 3). light microscopy confirmed that each of these struc­ tures constitutes a glandular unit with a central lu­ men/duct opening onto the lingual surface (crole & soley 2008), which grouped together form the lin­ tb cp w l* * a 2 fig. 2 dorsal view of the tongue body (tb) showing the apex (a), lateral lingual papillae (*) and caudal lingual papillae (cp). tongue body length (l) was measured from the apex to the caudal papillae. the width (w) was measured between the tips of the last lateral papillae. bar = 5 mm fig. 1 emu head opened along the right commissure to reveal the positioning of the tongue within the oropharynx. the body of the tongue (t) lies within the non­pigmented re­ gion of both the roof (nr) and floor (nf) of the oropharynx, and the small tongue root (*) extends from the base of the tongue body to the rostral tip of the glottis (arrow­ heads). the apex (a) of the tongue lies close to the bor­ der of the pigmented and non­pigmented regions. other noticeable features of the oropharynx include the broad mandibular rhamphotheca (mr), the interramal region of the non­pigmented floor with its numerous folds (arrows), the laryngeal mound (lm), the median palatine ridge (pr), the choana (c), infundibular cleft (ic), pharyngeal folds (pf) and proximal oesophagus (o). bar = 5 mm pr mr mr nr nf t lm pf c ic o * 1 a 338 morphology of tongue of emu (dromaius novaehollandiae). i. gross anatomical features and topography gual glands (gll. linguales) (mclelland 1993). in some tongues, these structures are obscured due to a darker colouration of the dorsum and only the openings, resembling pits, are visible (fig. 4). margins (margo linguae) the three margins of the tongue body display two sets of lingual papillae (fig. 1 and 2), the left and right lateral lingual papillae (papillae linguae late­ rales) and the caudal lingual papillae (papillae ling­ uae caudales). the first lateral papillae originate on either side of and just caudal to the apex. these are the smallest of the lateral papillae and are directed laterally or caudolaterally. the rest of the papillae progressive­ ly point more caudolaterally and become longer and more slender. the last papillae are the longest and most caudally directed, and in some specimens ex­ hibit a pale tip. in some instances individual papillae emanate from the base of adjacent papillae (fig. 2) and not directly from the lingual margin. the number of papillae present on the lateral lingual margins is variable and not necessarily equal on both sides. although the left and right lateral margins demon­ strate a similar range of papillae (three to eight on the left side and five to eight on the right side), there appears to be a consistently higher number of papil­ lae on the right margin than compared to the left. the average number of lateral papillae on the tongues studied is 11.2. the doughnut­shaped struc­ tures seen below the surface (fig. 3) end abruptly just beyond the root of the lingual papillae, although in the last lateral and caudal papillae they extend to the papillae tips. the caudal lingual papillae (fig. 1, 2 and 4) are ru­ dimentary and poorly defined compared to the lat­ eral papillae and demarcate the caudal boundary of the tongue body. in some instances (n = 4) the cau­ dal papillae appear as a fused, centrally positioned structure with variable incisures and small projec­ tions (fig. 4). in other specimens (n = 4) the fused component is flanked on either side by a single, more typical papilla. in a number of tongues (n = 8) the fused component displays a shallow median groove resulting in the formation of two median pa­ pillae which are accompanied by a variable number (zero to two) of adjacent papillae (fig. 2). the cau­ dal papillae vary in number between one to four (av­ erage two and a half). in one specimen, a structure similar in appearance to a lingual papilla was ob­ served to project dorsally from the mucosa covering the left ceratobranchiale, just caudal to the last lat­ eral papilla. tongue root (radix linguae) the tongue root (fig. 1 and 4) is a fleshy triangular structure, which in most specimens, is non­pigment­ ed. the caudal extremity of the root ends as a rounded, raised bulbous structure (pigmented in some specimens) that extends into the rostral as­ pect of the laryngeal fissure (glottis). the mucosa of the tongue root is continuous with the rest of the cb tb cb le le * 4 fig. 4 dorsal view of the triangular tongue root, showing the caudal extremity of the tongue root (*) folding over the laryngeal entrance (le). in this specimen, the caudal lin­ gual papillae (arrows) of the tongue body (tb) appear fused with variable incisures and small projections being apparent. the rostral parts of the paired ceratobranchi­ ale (cb) are seen bordering the tongue root. note the pitted surface of the tongue body, representing the open­ ings of the large underlying glands. bar = 1 mm d lp 3 fig. 3 ventral view of the lateral lingual papillae showing the abrupt transition (arrows) between the presence of dough­ nut­shaped structures (d) and the unelaborated surface of the papillae (lp). bar = 1 mm 339 m.r. crole & j.t. soley mucosa covering the oropharyngeal floor and forms a shallow groove where it abutts the paired cerato­ branchiale and the raised margins of the laryngeal fissure (fig. 4). the surface of the root displays the same doughnut­shaped structures seen on the tongue body, particularly in the midline. a shallow retrolingual recess exists between the ventral as­ pect of the caudal lingual papillae and the tongue root. frenulum (frenulum linguae) the frenulum (fig. 5 and 6) is a fleshy non­pigment­ ed structure which attaches the caudal half of the tongue body to the oropharyngeal floor. it is triangu­ lar in shape, with the rostral attachment to the ven­ trum of the tongue forming the point of the triangle. the mucosa along the lateral edges is thrown into longitudinal folds. these folds are obliterated when 5 d tb fr lf c v fig. 5 the dorsoventrally flattened tongue body (tb) shown in lateral profile. the folds of the frenulum (fr) are not visi­ ble as the tongue body is in the raised position. dorsum (d), ventrum (v), tongue root tip (arrows), laryngeal fis­ sure (lf), choana (c). bar = 5 mm 6 bb cbcb u fr cc fig. 6 the tongue body and frenulum in ventral view. note the extent of the rostral projection of the basihyale (double­ headed arrow). the position of the body of the basihyale (bb), rostral parts of the paired ceratobranchiale (cb) and the urohyale (u) are indicated and occur in triangular formation running within the frenulum (fr). the dough­ nut­shaped structures can be clearly seen below the sur­ face. crura (c). bar = 5 mm br bb pgpg u cbcb 7 8 cb cb br bb pgpg u fig. 7 and 8 the lingual skeleton shown in dorsal (7) and ventral (8) view the broad paraglossum (pg) lies dorsal to the rostral projection of the basihyale (br) within the tongue body. the body of the basihyale (bb), the rostral parts of the paired ceratobranchiale (cb) and the urohyale (u) are all imbedded within the frenulum (see fig. 6). bar = 5 mm 340 morphology of tongue of emu (dromaius novaehollandiae). i. gross anatomical features and topography the tongue body is lifted dorsally from the oropha­ ryngeal floor (fig. 5). the rostral point of the frenu­ lum houses the body of the basihyale while the two lateral edges enclose the rostral parts of the paired ceratobranchiale which merge rostrally with the body of the basihyale (fig. 6). extending caudally from the body of the basihyale, along the midline, is the urohyale, also housed within the frenulum (fig. 6) (see also fig. 8). lingual skeleton the lingual skeleton consists of the paraglossum and the rostral projection of the basihyale (fig. 7 and 8), both of which are imbedded in the tongue body. the paraglossum is a broad, thin, teardrop­ shaped cartilaginous plate imbedded within the lin­ gual parenchyma. the rostral tip is pointed while the base varies from gently rounded, to scalloped. the paraglossum is situated dorsal to the rostral projection of the basihyale, to which it is attached by loose connective tissue. the basihyale runs almost the full length of the paraglossum, ending near its rostral tip. the edges of the paraglossum do not ex­ tend to the apex or lingual margins, nor into any of the lingual papillae. discussion there is no definitive information in the literature on the topography of the emu tongue within the oro­ pharynx. the sketch by faraggiana (1933) shows the tongue in relation only to the laryngeal mound whereas bonga tomlinson (2000) simply depicts the outline of the emu tongue body in relation to the hyobranchial apparatus and mandibular rami. from the specimens examined in the current study it was observed that the apex of the tongue did not extend further than half the distance from the commissure to the rostral bill tip. this contrasts with the position­ ing of the tongue body indicated by bonga tomlinson (2000), which shows it to occupy a far more rostral position relative to the surrounding structures. how­ ever, despite differences in the appearance of the various ratite tongues, the topographical relation­ ships of this organ in the emu are generally similar to those illustrated in the ostrich (göppert 1903; farag­ giana 1933; bonga tomlinson 2000; jacko wi ak & ludwig 2008; tivane 2008), greater rhea (gadow 1879; pycraft 1900; faraggiana 1933; gussekloo & bout 2005), cassowary (p. johnston, personal com­ munication 2008) and kiwi (mccann 1973). the general shape of the tongue in birds usually mimics that of the bill (bradley 1915; harrison 1964; koch 1973; hodges 1974; nickel et al. 1977) or the palate (mclelland 1979). however, in comparison to other bird families, the ratite tongue is greatly re­ duced in length relative to the bill (faraggiana 1933; ziswiler & farner 1972; mclelland 1979; bailey, men sah­brown, samour, naldo, lawrence & gar ner 1997; bonga tomlinson 2000; gussekloo & bout 2005; jackowiak & godynicki 2005; jackowiak & ludwig 2008), a feature also noted in the emu (see table 1). tongue structure in birds is highly variable and closely related to feeding (mclelland 1979), with the ratite tongue being described as a rudimen­ tary or vestigial organ adapted for rapid swallowing of large food items (gadow 1879; pycraft 1900; mclelland 1979; bonga tomlinson 2000). two spe­ cific adaptations of the avian tongue for swallowing have been recognized, namely, the occurrence of caudally directed lingual papillae (harrison 1964; mclelland 1979; king & mclelland 1984) and/or a reduction in tongue size (mclelland 1979). the emu tongue body displays both of the above mentioned adaptations, as does that of the cassowary (p. john­ ston, personal communication 2008). two reasons for tongue reduction in ratites can be advanced. in birds that swallow food whole (harrison 1964; mclel­ land 1979) the tongue is unnecessary and therefore rudimentary (harrison 1964; king & mclelland 1984) as well as non­protrusable (king & mclelland 1984). it is also suggested that because of the cranioiner­ tial feeding method employed by ratites, a longer tongue extending to the bill tip would be injured due to the rapid bill closure involved in this feeding tech­ nique (bonga tomlinson 2000). there are surprisingly few accounts documenting the general appearance of the emu tongue, with both fowler (1991) and sales (2006, 2007) simply quoting the observation of cho et al. (1984) that “the tongue of the emu has a serrated edge”. the fringed appearance of the emu tongue body is also illustrated by bonga tomlinson (2000). the most comprehensive description of the general shape of the emu tongue is that of faraggiana (1933) who described the basic features noted in this study. how ever, as this author was limited to a single spec­ i men, some differences were apparent. in addition to the rounded apex described by faraggiana (1933), pointed or split apices were observed in the present study, whereas the tongue body appeared broader than that depicted in the earlier work. it is clear from previous studies that the shape of the tongue body differs between ratites (cho et al. 1984). these differences in tongue shape are compared in table 1 and indicate that the tongues of the emu and cassowary (p. johnston, personal communica­ 341 m.r. crole & j.t. soley t a b l e 1 c o m p a ra tiv e f e a tu re s o f th e r a tit e t o n g u e s p ec ie s b o d y sh ap e r o o t sh ap e p ig m en ta ti o n b o d y m ar g in s t o n g u e le n g th co m p ar ed t o lo w er b ill le n g th ( % )+ e m u (d ro m ai us n ov ae ho lla nd ia e ) t ri a n g u la r 1 5 , 2 0 t ri a n g u la r 1 5 , 2 0 b o d y: y e s 1 5 , 2 0 r o o t: v a ri a b le 2 0 s e rr a te d 9 , 1 3 , 1 4 , 1 5 , 2 0 l a te ra l 9 , 1 4 , 1 5 , 2 0 a n d c a u d a l p a p ill a e 9 , 1 5 , 2 0 2 0 .8 # – 2 3 .8 # o st ri ch (s tr ut hi o ca m el us ) t ri a n g u la r o r ∩ ­s h a p e d 4 , 6 , 1 3 , 1 4 , 1 7 , 1 8 s h o rt a n d /o r b lu n t 3 , 4 , 6 , 8 , 1 3 , 1 4 , 1 7 , 1 8 c a u d a l “ lin g u a l p o ck e t” 1 , 2 , 9 , 1 4 , 1 6 , 1 7 , 1 8 f la t 1 7 , 1 8 , 2 1 b o d y: n o 1 8 r o o t: n o 1 8 , 2 1 s m o o th 1 8 t w o c a u d o la te ra l p ro je ct io n s (l in g u a l h o rn s) 1 , 2 , 7 , 9 , 1 7 , 1 8 2 0 9 – 2 1 .4 # 2 5 1 7 g re a te r rh e a (r he a am er ic an a) t ri a n g u la r w ith r o u n d e d a p e x 9 , 2 1 f la t 2 1 b o d y: y e s 9 , 1 1 l in g u a l h o rn s n o t 9 , 2 1 r o o t: n o 2 1 s m o o th 9 , 1 4 t w o g lo b o se , b ila te ra l ca u d o la te ra l p a p ill a e 1 4 t w o c a u d a l l in g u a l h o rn s/ p ro je ct io n s 9 , 2 1 1 9 # – 2 0 .9 # d a rw in ’s r h e a (p te ro cn em ia p en na ta ) v ­s h a p e d w ith p o in te d a p e x 1 3 – – s m o o th 1 3 – c a ss o w a ry (c as ua riu s ca su ar iu s) t ri a n g u la r, lo n g e r th a n w id e 4 r o st ra l r o u n d e d a p e x fr e e o f p a p ill a e , n o ca u d a l p a p ill a e 1 9 f la t 1 9 b o d y: n o 1 9 r o o t: n o 1 9 b a ck w a rd p o in tin g t ip s 4 d e n tic u la te 6 s im ila r to t h e e m u b u t a d iff e re n t p a tt e rn 1 9 1 3 1 9 k iw i (a pt er yx a us tr al is m an te lli ) t ri a n g u la r lo n g ­p yr ifo rm ; tip o b tu se , re tu se o r tr u n ca te 1 2 n o 5 , 1 2 s m o o th 5 , 1 2 (a pt er yx h aa st i) o b lo n g , co n st ri ct io n b e lo w t ra n sv e rs e m id lin e ; a p e x tr u n ca te o r re tu se 1 2 (d e p ic te d , b u t n o t la b e le d 1 2 ) n o 1 2 b lu n t 1 2 9 .5 *– 1 4 .2 * (a pt er yx o w en i) s im ila r to a . h aa st i, w ith la rg e r co n st ri ct io n 1 2 n o 1 2 f o ld e d 1 2 + t h e se a re a p p ro xi m a te m e a su re m e n ts * e xt ra p o la te d f ro m t h e m e a su re m e n ts ( sp e ci e s n o t m e n tio n e d ) in r o a ch ( 1 9 5 2 ) # o w n m e a su re m e n ts u n d e rl in e d n a m e s in d ic a te a s ke tc h is s u p p lie d , b o ld in d ic a te s p h o to g ra p h s: 1 m e ck e l ( 1 8 2 9 ) 2 c u vi e r (1 8 3 6 ), 3 m a ca lis te r (1 8 6 4 ), 4 g a d o w ( 1 8 7 9 ), 5 o w e n ( 1 8 7 9 ), 6 p yc ra ft ( 1 9 0 0 ), 7 g ö p p e rt ( 1 9 0 3 ), 8 d u e rd e n ( 1 9 1 2 ), 9 f a ra g g ia n a ( 1 9 3 3 ), 1 0 r o a ch ( 1 9 5 2 ), 1 1 f ed er (1 9 7 2 ), 1 2 m cc a n n ( 1 9 7 3 ), 1 3 c h o e t al . (1 9 8 4 ), 1 4 b o n g a t o m lin so n ( 2 0 0 0 ), 1 5 c ro le & s o le y (2 0 0 8 ), 1 6 p o rc h es cu ( 2 0 0 7 ), 1 7 ja ck o w ia k & l u d w ig ( 2 0 0 8 ), 1 8 t iv an e (2 0 0 8 ), 1 9 jo h n st o n (p e rs o n a l c o m m u n ic a tio n ), 2 0 p re se n t st u d y, 2 1 p e rs o n a l o b se rv a tio n 342 morphology of tongue of emu (dromaius novaehollandiae). i. gross anatomical features and topography tion 2008) share similar gross morphological fea­ tures. it should be noted, however, that it is not only tongue shape that differs between ratites. the ap­ pearance of the tongue body margins, tongue root, the prevalence of pigmentation, tongue size relative to the length of the bill, the occurrence of special features (for example, the lingual pocket in the os­ trich), and the shape and composition of the para­ glossum all define differences in ratite tongue struc­ ture and appearance (see table 1). it is also noteworthy that in birds with an omnivo­ rous diet the tongue conforms to a generalised pat­ tern described as triangular with a pointed apex, with the chief adaptive feature being that of caudally pointing spines (papillae) on the caudal margin (gardner 1927). this statement would certainly be true for the emu, which also enjoys a varied diet (davies 1978). lingual papillae (dorsal, lateral and caudal) are a common feature of the avian tongue and have been described in numerous species (gardner 1926, 1927; mclelland 1979; king & mclelland 1984; bai­ ley et al. 1997; kobayashi et al. 1998; mclelland 1990) including domestic poultry (calhoun 1954; zis­ wiler & farner 1972; mclelland 1975; nickel et al. 1977; king & mclelland 1984; mclelland 1990). how ever, it would appear that lingual papillae are not a common or well­developed feature in ratites (ta ble 1), a characteristic also noted by bonga tomlinson (2000). apart from the lateral papillae of the emu (table 1) and cassowary (gadow 1879; pycraft 1900), the rest of the ratites documented display smooth lateral tongue margins. in the little spotted kiwi (mccann 1973) the lateral tongue mar­ gins are narrowly infolded, but show no papillae. the lateral lingual papillae of the emu tongue show a lack of bilateral symmetry which involves differ­ ences in both number and shape, with a greater number of papillae usually being observed on the right margin. faraggiana (1933) also noted that the number of papillae was not the same on each side of the tongue body whereas bonga tomlinson (2000) provides a definitive number of five lingual papillae on the lateral margins. in contrast, as noted in this study, the number of papillae displays a nor­ mal variation between specimens of 3–8 on the left and 5–8 on the right margins. the caudal lingual papillae of the emu tongue are ru­ dimentary compared to other bird species and even though identifiable, are often not well­developed. the sketch by bonga tomlinson (2000) neglects to depict the caudal lingual papillae in this species. in comparison to the other ratites, the emu appears to be the only member which possesses structures recognisable as caudal lingual papillae (table 1). however, in the ostrich and greater rhea (table 1) the caudolateral aspect of the tongue body displays papillae­like extensions. whether these structures represent true caudal lingual papillae remains un­ determined. the function of the lingual papillae is reportedly to assist in the aboral transport of food (mclelland 1979; king & mclelland 1984). in the emu the lin­ gual papillae may be instrumental in removing smaller food particles from the roof of the orophar­ ynx in a similar fashion to that proposed by bonga tomlinson (2000) for palaeognathous birds (see below). some confusion exists in the literature regarding the naming of the caudal extremity of the tongue body (the tongue base) and the tongue root (moore & elliott 1946) with both terms being used inter­ changeably (mclelland 1975). in domestic poultry the tongue is clearly defined into a free rostral tip (apex), a body and a caudal root (mclelland 1993). descriptions of the tongue using this terminology exist for a number of species (see, for example, faraggiana 1933; bailey et al. 1997; jackowiak & godynicki 2005; jackowiak & ludwig 2008). based on the work of lillie (1908) and bradley (1915) it is generally accepted that the border between the tongue body and root is the row of caudal lingual papillae (botezat 1910; moore & elliott 1946; gentle 1971; nickel et al. 1977; bailey et al. 1997). this border coincides with the boundary between the oral and pharyngeal cavities as described for anas by zweers et al. 1977 (cited by mclelland 1993). some authors appear to use the term ‘tongue base’ synonymously with ‘tongue root’ (nickel et al. 1977; gussekloo & bout 2005). in some studies the cau­ dal aspect of the tongue body has been termed the tongue base (warner, mcfarland & wilson 1967; mclelland 1975; bhattacharyya 1980; bonga tom­ linson 2000) or even the tongue root (koch 1973; mclelland 1979; mclelland 1990; kobayashi et al. 1998) whereas in other publications the term tongue base is used but not defined (bacha & bacha 2000; calhoun 1954). alternative terminology used for the tongue root includes the posterior part of the tongue (gentle 1971), the sensory area (bhattacharyya 1980) and the preglottal part of the tongue (hom­ berger & meyers 1989; liman, bayram & koçak 2001). the importance of clarity in correctly identifying and naming the various components of the tongue has 343 m.r. crole & j.t. soley been pointed out by moore & elliott (1946), particu­ larly in regard to the location of taste buds. failure to recognise the caudal aspect of the tongue (the tongue root) as part of the tongue could lead to invalid conclusions about the presence of taste buds in this organ, as they are reportedly concentrated in this region (moore & elliott 1946; gentle 1971; nick el et al. 1977; bacha & bacha 2000; al­mansour & jarrar 2004). a clearly defined triangular structure represents the tongue root in the emu and is positioned between the caudal margin of the tongue body and the laryn­ geal entrance. this structure seems to be unique to the emu as in other ratites the tongue root is repre­ sented by a featureless stretch of mucosa (table 1). the structure of the tongue root in kiwi species is unclear (mccann 1973). the extension of the tongue root into the rostral aspect of the laryngeal entrance (faraggiana 1933; present study) represents an in­ teresting modification not observed or illustrated in other ratites (ostrich and greater rhea) (göppert 1903; faraggiana 1933; gussekloo & bout 2005; por chescu 2007; jackowiak & ludwig 2008; tivane 2008). the positioning of the tongue root would also appear to assist in sealing the rostral part of the larynx when the glottis is closed, almost assuming the role of an epiglottis, which is not present in birds (kaupp 1918; calhoun 1954; king & mclelland 1984; nickel et al. 1977). this argument regarding the role of the tongue root functioning as an epiglot­ tis in the emu has been proposed by gadow (1879) but disputed by faraggiana (1933). the tongue root of the emu also appears to play a special role in as­ sisting to close off of the rostral aspect of the choa­ na in the closed gape. the choana of most birds is divided into a rostral slit­like part (pars rostralis) and a caudal triangular part (pars caudalis) (king 1993) with the tongue commonly closing off the rostral part of the choana (mclelland 1975, 1979). in the emu, the triangular choana (fig. 1) is not divided into ros­ tral and caudal parts and therefore the tongue body plays no part in closing off the choana in the closed gape. instead, the tongue root partially closes off the rostral aspect of the choana in this species. little mention is made in the literature of the frenu­ lum in birds. a possible reason for this may be its general lack of remarkable features, serving simply to attach the tongue to the oropharyngeal floor (mclelland 1979). in the emu, the frenulum is a rel­ atively large structure which houses part of the hyo­ branchial apparatus. the lateral margins are longi­ tudinally folded which would seem to indicate that the tongue is capable of a certain degree of move­ ment. this observation lends further support to the role played by the tongue of palaeognaths in cranio­ inertial feeding and in drinking. during swallowing in palaeognaths the tongue is lifted and contacts the palate before moving caudally, thereby scraping any food caudal to the tongue into the proximal oesophagus (bonga tomlinson 2000). palaeognaths transport food from their bill tips to the oesophageal entrance via the cranioinertial feeding method (bonga tomlinson 2000), also described as the ‘catch and throw’ method by gussekloo & bout (2005). the transport of food into or close to the oesophageal entrance is facilitated by a large gape and marked depression of the tongue. tongue de­ pression enlarges the ‘buccal cavity’ (oropharyn­ geal cavity), which assists in moving food to the caudal oropharynx, while retraction of the tongue assists in the final transport of fluid to the oesoph a­ gus during drinking (gussekloo & bout 2005). there­ fore, despite the emu tongue showing such relatively reduced dimensions and rigidity, it possess a surpris­ ingly large range of movements in both the rostro­ caudal (though unable to protrude) and dorsoventral planes by virtue of the relatively large, folded frenu­ lum and the association of the hyobranchial appara­ tus with the tongue body and frenulum. the lingual skeleton of the emu is formed by the me­ dian, unpaired paraglossum and the rostral projec­ tion of the basihyale of the hyobranchial apparatus. the paraglossum is related dorsally to the rostral projection of the basihyale as also described by bonga tomlinson (2000) in the emu and the greater rhea. however, the findings of this study contrasted with those of bonga tomlinson (2000) in that the rostral projection of the basihyale extended further rostrally, ventral to the paraglossum, than that de­ picted by the author. the paraglossum of the emu was teardrop­shaped with a pointed rostral tip and a rounded base al­ though it is depicted by parker (1866) in dromaius irroratus as inverted heart­shaped and by bonga tomlinson (2000) in dromaius novaehollandiae as arrowhead­shaped. in ratites the paraglossum re­ mains cartilaginous and does not ossify in older birds (bonga tomlinson 2000), a situation also ap­ parent in the emu. the shape of the paraglossum differs be tween the ratites. the paraglossum of the emu (dromaius irroratus and novaehollandiae), rhea (rhea americana) and cassowary (casuarius ben­ netii) are all basically arrowhead­shaped, although individual differences are apparent, particularly re­ garding the form of the base (parker 1866; bonga tomlinson 2000; present study). the paraglossum 344 morphology of tongue of emu (dromaius novaehollandiae). i. gross anatomical features and topography of the kiwi (apteryx australis) (parker 1891) is also a single structure but is much narrower than that of the emu, rhea and cassowary and has a split, elon­ gated base. the ostrich paraglossum is divided into two narrow paraglossalia which flank the rostral pro­ jection of the basihyale and are located ventrola­ teral to it (bonga tomlinson 2000; tivane 2008). this arrangement differs radically from that of the emu, where the rostral projection of the basihyale lies ventral to the paraglossum, and the other ratites and has lead to some authors not recognising or misinterpreting the narrow, paired structure (meckel 1829; parker 1866; webb 1957; jackowiak & lud­ wig 2008) present in the ostrich tongue. the tongue of birds is a rigid organ due to the pres­ ence of the paraglossum (koch 1973) and, except in parrots, the absence of intrinsic musculature (zis­ wiler & farner 1972; koch 1973; nickel et al. 1977; mclelland 1990). the rigidity afforded by the para­ glossum in palaeognathous birds is needed for the swallowing phase in order to push the food into the oesophagus. the rostral projection and body of the basihyale, situated ventrally in the tongue body, con­ nects the hyobranchial apparatus with the tongue, and due to its close association, retracts the tongue during swallowing. the great mobility of the hyobran­ chial apparatus in birds, attributed to the fact that it does not articulate with the skull (mcleod 1939), is the main contributor to the movement of the tongue (king & mclelland 1984; bonga tomlinson 2000). acknowledgements the authors thank mr peter duncan for providing the emu heads; dr catarina tivane for collection of the specimens; mrs charmaine vermeulen for the photography; ms marté smit for preparing the fig­ ures; the support staff of the department of anatomy and physiology, faculty of veterinary science, uni­ versity of pretoria; carole long, secretary of the otanewainuku kiwi trust, avi holzapfel (leader of the kiwi recovery group) and susan cunningham from new zealand for their assistance in acquiring literature on the kiwi; peter johnston from the live transplant unit and department of anatomy, uni­ versity of auckland, new zealand for his information on the cassowary; and the university of pretoria for financial support. references al­mansour, m.i. & jarrar, b.m. 2004. structure and se­ cretions of the lingual salivary glands of the white­cheeked bulbul, pycnonotus leucogenys (pycnontidae). saudi jour­ nal of biological sciences, 11:119–126. bacha, w.j. & bacha, l.m. 2000. digestive system, in color atlas of veterinary histology, edited by d. balado. phila del­ phia: lippincott williams & wilkins. bailey, t.a., mensah­brown, e.p., samour, j.h., nal­ do, j., lawrence, p. & garner, a. 1997. comparative morphology of the alimentary tract and its glandular deriva­ tives of captive bustards. journal of anatomy, 191:387–398. baumel, j.j., king, a.s., breazile, 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digestive system, in birds – their structure and function, 2nd ed. london: bailliere tindall. kobayashi, k., kumakura, m., yoshimura, k., inatomi, m. & asami, t. 1998. fine structure of the tongue and lin­ gual papillae of the penguin. archivum histologicum cytolo­ gicum, 61:37–46. koch, t. 1973. splanchnology, in anatomy of the chicken and domestic birds, edited by b.h. skold & l. devries. ames, iowa: the iowa state university press. lillie, f.r. 1908. the development of the chick. new york: hen­ ry holt and co. liman, n., bayram, g. & koçak, m. 2001. histological and histochemical studies on the lingual, preglottal and laryngeal salivary glands of the japanese quail (coturnix coturnix ja­ ponica) at the post­hatching period. anatomia, 30:367–373. lucas, f.a. 1896. the taxonomic value of the tongue in birds. auk, 13:109–115. lucas, f.a. 1897. the tongues of birds. reports of the united states national museum, 1895:1003–1020. macalister, a. 1864. on the anatomy of the ostrich (struthio 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buchhandlung. moore, d.a. & elliott, r. 1946. numerical and regional dis­ tribution of taste buds on the tongue of the bird. journal of comparative neurology, 84:119–131. nickel, r., schummer, a. & seiferle, e. 1977. digestive system, in anatomy of the domestic birds. berlin: verlag paul parey. owen, r. 1879. memoirs on the extinct and wingless birds of new zealand; with an appendix of those of england, austra­ lia, newfoundland, mauritius and rodriguez. vol. 1. london: john van voorst. parker, t.j. 1891. observations on the anatomy and develop­ ment of apteryx. philosophical transactions of the royal so­ ciety of london, b., 182:25–134. parker, w.k. 1866. on the structure and development of the skull in the ostrich tribe. philosophical transactions of the royal society of london, 156:113–183. porchescu, g. 2007. comparative morphology of the diges­ tive tract of the black african ostrich, hen and turkey. ph.d. thesis, agrarian state university of moldova. pycraft, w.p. 1900. on the morphology and phylogeny of the palaeognathae (ratitae and crypturi) and neognathae (cari­ natae). transactions of the zoological society of london, 15: 149–290. roach, r.w. 1952. notes on the new zealand kiwis (1). new zealand veterinary journal, 1:38–39. sales, j. 2006. digestive physiology and nutrition of ratites. avian and poultry biology reviews, 17:41–55. sales, j. 2007. the emu (dromaius novaehollandiae): a review of its biology and commercial products. avian and poultry bi ology reviews, 18:1–20. tivane, c. 2008. a morphological study of the oropharynx and oesophagus of the ostrich (struthio camelus). m.sc. thesis, university of pretoria. warner, r.l., mcfarland, l.z. & wilson, w.o. 1967. microanatomy of the upper digestive tract of the japanese quail. american journal of veterinary research, 28:1537– 1548. webb, m. 1957. the ontogeny of the cranial bones, cranial pe­ ripheral and cranial parasympathetic nerves, together with a study of the visceral muscles of struthio. acta zoologica, 38: 81–202. ziswiler, v. & farner, d.s. 1972. digestion and the diges­ tive system, in avian biology, edited by d.s. farner, j.r. king & k.c. parkes. new york: academic press. introduction trypanosomes remain a constant threat to the lives of humans, cattle and other domesticated animals, throughout large regions of africa and south amer­ ica. in africa, the disease in domestic animals is mainly caused by t. congolense and t. vivax, with t. congolense being the most pathogenic species in east africa and t. vivax in west africa (kaaya, win­ qvist & johnson 1977). methods of screening ani­ mals for trypanosomosis for epidemiological studies include direct microscopic observation of parasites in the buffy coats following concentration of blood using the microhaematocrit centrifugation technique (mhct), or serological tests that include the com­ plement fixation test and the card agglutination test (claes, ilgekbayeya, verloo, saidouldin, geerts, buscher & goddeeris 2005). however, recently a variety of polymerase chain reaction (pcr)­based techniques have been proposed. unlike the sero­ logical tests, pcr methods have largely overcome difficulties of sensitivity and specificity associated with direct parasite examination. a number of pcr primers have been developed for detection of species and subspecies of t. congo­ lense (savannah) (masiga, smyth, hayes, bromidge & gibson 1992); t. congolense (kilifi) (masiga et al. 1992) and t. vivax (masake, majiwa, moloo, makau, njuguna, maina, kabata, jole­moiyoi, & nantulya 1994). since these are species­specific tests, sev­ 285 onderstepoort journal of veterinary research, 76:285–289 (2009) detection of natural trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of its1 rdna s. biryomumaisho1*, s.e. melville2, e. katunguka­rwakishaya1 and g.w. lubega3 abstract biryomumaisho, s., melville, s.e., katunguka­rwakishaya, e. & lubega, g.w. 2009. detection of natural trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of its1 rdna. onderstepoort journal of veterinary research, 76:285–289 different species of trypanosomes may infect their mammalian hosts both singly or in combination. this study was undertaken to determine the trypanosome species that may be afflicting pigs in uganda. blood was collected from pigs of all ages and sexes from two districts, kasese in western and jinja in central uganda. of the 133 pig blood samples from kasese that were tested for trypano­ somes using the microhaematocrit centrifugation technique (mhct), none was found to be infected. however, of the 253 pigs from jinja district, nine were infected with trypanosomes of which three had t. vivax as determined by mhct. however, application of the its1 rdna pcr test revealed that eight pigs had t. vivax in mixed infections and one pig had t. vivax monolithic infection. these observa­ tions show that under certain circumstances, pigs may be important reservoirs for, as well as hosts to, t. vivax, contrary to earlier reports. keywords: its­pcr, pigs, trypanosoma vivax, uganda * author to whom correspondence is to be directed. e­mail: biryomumaisho@vetmed.mak.ac.ug 1 department of veterinary medicine, makerere university, p.o. box 7062, kampala, uganda 2 department of pathology, university of cambridge, cam­ bridge, cb2 1qp, uk. present address: hughes hall, uni ver­ sity of cambridge, cambridge, cb1 2ew, uk 3 department of veterinary parasitology and microbiology, makerere university, p.o. box 7062, kampala, uganda accepted for publication 19 february 2009—editor 286 trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of its1 rdna eral primer sets would be required to test each sam­ ple for all species of trypanosomes. attempts have been made to develop a single molecular marker to accurately distinguish between species in a single pcr test using the internal transcribed spacers (its) located within the ribosomal rna genes, both for trypanosomes and a number of other organisms (schlotter, hauser, von haeseler & tautz 1994) and samuel (1998). the internal transcribed spacer (its) region of ribo­ somal dna (rdna) is a popular target for universal and specific trypanosome identification because of its highly conserved flanking regions, and size vari­ ability among trypanosome species and subgroups (hernandez, martin­parras, martinez­robles & schvartz man 1993). a suitable its primer set for try panosomes (kin 1 and kin 2) was developed by mclaughlin, ssenyonga, nanteza, rubaire, wafula, hansen, vodkin, novak, gordon, montenegro­ james, mdachi, sharipo, chang & kakoma (1996) and subsequently was evaluated by desquesnes, mclaughlin, zoungrana & davila (2001) but the detection of t. vivax with these primers was less sensitive. subsequently, njiru, constantine, guya, crow ther, kiragu, thomson & davila (2005) evalu­ ated new primers (cf and br) for its 1 rdna; these primers showed 100 % homology with t. vivax rdna sequence in genbank, accession number u22316. it has been proposed previously that the principal hosts of t. vivax are cattle, sheep, goats, horses and camels, but not dogs and pigs (losos 1986). stephen (1986) suggested that pigs are refractory to t. vivax infection since when he challenged pigs with wild­caught tsetse flies infected with t. vivax and t. congolense in a ratio 2:1, no t. vivax para­ sites were subsequently detected in the blood of these animals. however, a recent study by ng’ayo, ngiru, eucharia, muluvi, osir & masiga (2005) in western kenya showed that pigs can, indeed, be infected with t. vivax. in their study intended to de­ termine the animal reservoirs of human sleeping sickness, of the ten pigs infected with trypano­ somes, three carried t. brucei and five had t. vivax. similarly, in a study of trypanosome molecular dif­ ferential diagnosis in livestock in south western uganda, balyeidhusa, enyaru, matovu, nerima, akol & sebikali (2006) detected three t. vivax infec­ tions in 22 pigs. the present study, therefore, was designed to con­ duct further investigations of the trypanosome spe­ cies that affect pigs reared in two distinct agroeco­ logical zones in uganda. materials and methods collection and handling of pig blood samples blood was collected from pigs of all ages and both sexes on smallholder farms in the districts of west­ ern (kasese) and central (jinja) uganda. fewer pigs than cattle were kept by farmers in the two districts; therefore, all pigs that were reared in villages where the cattle and goats were bled, were also bled. a total of 133 and 253 pigs were bled from kasese and jinja districts, respectively. in larger pigs (weigh­ ing more than 50 kg) blood was obtained from the marginal ear vein and in smaller pigs, from the ante­ rior vena cava or the cutaneous abdominal vein. two to five millilitres of blood were dispensed into edta­coated collection tubes. of this blood, 20 µℓ was spotted onto whatman fta cards. the remain­ ing blood was used for preparation of microhaema­ tocrit tubes for subsequent centrifugation and exam­ ination for trypanosomes as described by mur ray, mur ray & mcintyre (1977). speciation of trypanosomes with microhaematocrit centrifugation/dark ground microscopy technique (mhct) capillary tubes were centrifuged at 1 020 g for 5 min and the presence of trypanosomes detected by ob­ servation of parasite motion just above the buffy coat. in a sample where trypanosomes were detect­ ed, the microhaematocrit tube was cut just below the buffy coat to include 1 mm of the erythrocyte layer, and the contents including about 1 cm of plas­ ma were transferred to a glass slide and covered with a cover slip. the wet smear was observed for trypanosomes under the microscope with reduced illumination and classified into species basing on their movements whereby t. congolense vibrates without progressing, t. brucei swims and t. vivax moves fast across the field (radostatis, gay, blood & hinchcliff 2000). extraction of dna from blood spotted on fta cards in this study, dna was extracted from trypanosome­ positive pigs because we intended to identify sam­ ples for further molecular studies (results not pre­ sented in this paper). dna was extracted from dry blood spotted on fta cards using the qiaamp® mini kit (2003) protocol and subsequently used for speciation of infecting trypanosomes by pcr ampli­ fication of its1 rdna. briefly, three or four 2 mm­ diameter circles of whole dried blood spot were punched out of the fta cards and placed in a 1.5 mℓ 287 s. biryomumaisho et al. microfuge tube, covered with cell lysis buffer and incubated at 85 °c for 10 min. genomic dna was extracted by adding proteinase k and incubating the extract at 56 °c for 1 h. the dna was precipi­ tated with ethanol and subsequently eluted in 110 µℓ of buffer ae, aliquoted and stored at –20 °c until re­ quired for use as template dna for testing individual dna extracts for infecting trypanosome species by its1 rdna pcr. amplification of the rdna intergenic sequence by the polymerase chain reaction (its1-pcr) the 25 µℓ total volume pcr mix contained 2.5 µℓ of 10x pcr buffer (15 mm mgcl2, 0.25 µℓ of each10 mm dntp solution), 0.5 µℓ containing 10 mm (10x mix­ ture) of the forward and reverse its1 rdna primers, 0.125 µℓ of hotstar taq dna polymerase and 5 µℓ of the dna extracted from fta blood spots. the its1 rdna primers used were: • forward (cf) 5’ccggaagttcaccgatattg • reverse (br), 5’ttgctgcgttcttcaacgaa reactions were performed in a ptc­100 peltier thermal cycler which was programmed as follows: lid preheating at 96 °c for 15 min, followed by 38 cycles each consisting of denaturation at 94 °c for 30 s, annealing at 56 °c for 1 min and extension at 72 °c for 90 s. the mixture was incubated at 72 °c for 10 min for the final extension step. the products were left at 4 °c in the thermocycler pending electro­ phoresis in a 1.5 % agarose gel containing 0.5 µg/mℓ ethidium bromide and flooded with 1x tbe buffer. electrophoresis was performed at 55 v for 30 min. the positive control trypanosome dna the t. brucei positive control dna was extracted from the procyclic form of strain 927 (t. brucei treu 927/4 pal/ke/70/eatro 1534). trypano soma con­ golense and t. vivax dna were extracted from strains il 3000 and il 2569, respectively, which were kindly provided by dr phelix majiwa. results identification of infecting trypanosomes using mhct and its1 rdna pcr the results of mhct trypanosome speciation from pig blood samples (table 1) based on movement characteristics of trypanosomes showed three spe­ cies, namely, t. brucei, t. congolense and t. vivax. the movement characteristics of trypanosomes from pig no. 128 were not characteristic of any of the three species; hence the infecting trypanosomes species were not assigned. all infections were confirmed by re­testing with its1 rdna pcr. species identification using the its1 rdna pcr anal ysis detected more species of trypanosomes in individual samples compared to the mhct except for pig number 164. trypanosoma vivax was detect­ ed by both techniques; t. brucei was the most com­ mon trypanosome and was detected in eight of the nine samples by its1 rdna pcr, mainly in mixed infections. the mhct detected only three mixed in­ fections compared to eight with the molecular tech­ nique. determination of the trypanosome species by pcr analysis was achieved by comparing individual pcr amplicons with the corresponding band size of the positive control (tb, tc and tv) bands (fig. 1). pres­ ence of more than one band denoted presence of mixed trypanosome infections while single bands denoted monolithic infections. of the nine pig sam­ ples, three showed banding patterns corresponding table 1 speciation of trypanosomes isolated from pigs in jinja district pig identity trypanosome species by mhct by pcr of its1 rdna 123* 124 128 132 146 147 148 162 164 t. brucei t. congolense not classified t. brucei, t. congolense t. brucei t. vivax t. brucei t. vivax, t. brucei t. brucei, t. vivax t. brucei, t. congolense, t. vivax t. brucei, t. congolense, t. vivax t. brucei, t. vivax t. brucei, t. congolense, t. vivax t. brucei, t. congolense t. brucei, t. vivax t. brucei, t. vivax t. brucei, t. vivax t. vivax * the numbers identifying pigs were assigned in the order pigs were bled. the figures do not re ­ present identification tags of pigs 288 trypanosoma vivax infections in pigs with microhaematocrit centrifugation and amplification of its1 rdna to mixed trypanosome infections comprising of t. vivax, t. congolense and t. vivax; four had t. vivax and t. brucei; one t.brucei and t. congolense while one is t. vivax monolithic infection. the band sizes for its1 rdna pcr amplicons from the control samples of trypanosome dna and also trypanosome dna extracted from the animal hosts correspond approximately to those given by njiru et al. (2005). they reported that members of subge­ nus trypanozoon (t. brucei brucei; t. evansi, t. b. rho desiense and t. b. gambiense) gave a constant product of approximately 480 base pair (bp); t. con­ golense (savannah) 700 bp, t. congolense (kilifi) 620 bp; t. congolense (forest) 710 bp and t. vivax 250 bp. although agarose gels as used here cannot unequi­ vocally resolve band sizes that vary by less than 100 bp (to distinguish between the t. congolense subgroups), fig. 1 clearly shows that each band ob­ tained from the ugandan isolates corresponded in size with one of the positive control samples. discussion the lack of sensitive and suitable diagnostic tests hinders the collection of accurate epidemiological data and institution of control programmes for live­ stock diseases. this study focused on accurate de­ tection of parasites and how various species of trypanosomes could simultaneously be detected from blood samples of porcine hosts. notable was the detection of t. vivax in pigs that have hitherto been reported to be refractory to the infection. there have been previous reports of t. vivax not being a parasite of pigs (losos 1986; stephen 1986; desquesnes & davilla 2002), but rather of cattle as the principal hosts. recent advances in diagnostics, especially pcr­based techniques, have yielded dif­ ferent results showing that t. vivax can infect pigs, and suggesting it could be a causal organism of dis­ ease in pigs. the detection of natural t. vivax infec­ tions in pigs by both parasitological (mhct) and molecular (its1 rdna­pcr) techniques in the present study indicates that pigs do serve as hosts to t. vivax. the unique conditions under which in­ fection and transmission occur are yet to be estab­ lished. this finding is in agreement with ng’ayo et al. (2005) who detected more significant t. vivax in­ fections in goats and pigs than in sheep. sub se­ quently, balyeidhusa et al. (2006) reported three t. vivax infections in pigs by its­pcr. while the pres­ ence of parasites in animal hosts may not necessar­ ily lead to serious disease, it is important to deter­ mine whether pigs are significant hosts to t. vivax since control of livestock trypanosomosis usually targets cattle. to date, pigs have always been excluded from trypanosomosis control exercises (personal obser­ vations), but these data suggest it may be neces­ sary to treat pigs in areas where prevalence of cat­ tle trypanosomosis is relatively high (exceeding 10 % prevalence). fig. 1 agarose gel (1.5 % w/v) banding pattern after amplification of its1 rdna of trypano­ somes from pigs bled in jinja district, central uganda. m = lambda ladder size marker (100 base pairs); tb = t. brucei; tc = t. congolense; tv = t. vivax; ­ve = negative con­ trol (i.e. containing no dna). the numbers assigned to each amplified dna sample on the gel reflect the order in which pigs were bled m tb tc tv 123 124 128 132 146 147 148 162 164 -ve 289 s. biryomumaisho et al. at present we do not know to what extent infection with t. vivax causes symptomatic disease in pigs. it is possible that pathology is less severe but that it plays a more important role as a reservoir for infec­ tion of tsetse and onward transmission to more sus­ ceptible species, thus aiding the maintenance of this parasite in the ecosystem. future clinical pa­ thology studies could be helpful in defining the im­ portance of infection of pigs. acknowledgements the authors thank the district veterinary officers of kasese and jinja districts for allowing us to utilize the district veterinary laboratories to process the blood samples. we also thank innovations at ma ke­ rere (i@mak.com) for sponsoring the field study and the association of commonwealth universities for financial support to savino biryomumaisho while at the department of pathology in the university of cambridge. references balyeidhusa, a.s.p., enyaru, j.c.k., matovu, e., neri­ ma, b., akol, m. & sebikali, c. 2006. molecular differen­ tial diagnosis of african trypanosomosis in domestic animals in north west uganda, in proceedings of the 11th international congress of parasitology, glasgow, medimond, 2006: 7–12. claes f., ilgekbayeya, g.d., verloo, d., saidouldin, t.s., geerts, s., buscher, p. & goddeeris, b.m. 2005. comparison of serological tests for equine trypano­ somosis in naturally infected horses from kazakhstan. vet­ erinary parasitology, 131:221–225. desquesnes, m. & dávila, a.m.r. 2002. applications of pcr­based tools for detection and identification of animal trypanosomes: a review and perspectives. veterinary para­ sitology, 109:213–231. desquesnes, m., mclaughlin, g., zoungrana, a. & dá­ vila, a.m.r. 2001. detection and identification of trypan o­ soma of african livestock through a single pcr based on internal transcribed spacer 1 of rdna. international journal of parasitology, 31:610–614. kaaya, g.p., winqvist, g. & johnson, l.w. 1977. clin ico­ pathological aspects of trypanosoma congolense infection in goats. bulletin of animal health and production in africa, 25:397–408. hernandez, p., martin­parras, l., martinez­robles, m.l. & schvartzman, j.b. 1993. conserved features in the mode of replication of eukaryotic ribosomal rna genes. embo journal, 12:1475–1485. losos, g.j. 1986. infectious tropical diseases of domestic ani­ mals. protozoal diseases. avon, uk: the bath press. masake, r.a., majiwa p.a., moloo, s.k., makau, j.m., njuguna j.t., maina, m., kabata jole­moiyoi, o.k. & nantulya, v.m. 1994. sensitive and specific detection of trypanosoma vivax using the polymerase chain reaction. experimental parasitology, 85:193–205. masiga, d.k., smyth, a.j., hayes, p., bromidge, t.j. & gibson, w.c. 1992. sensitive detection of trypanosomes in tsetse flies by dna amplification. international journal of parasitology, 64:207–218. mclaughlin, g., ssenyonga, s., nanteza, a., rubaire, a., wafula, o., hansen, r.d., vodkin, m., novak, r.j., gordon, v.r., montenegro­james, s., mdachi, m.r., shapiro, s., chang, k.p. & kakoma, i. 1996. pcr­ based detection and typing of parasites, in parasitology for the 21st century, edited by m. azcel & m.z. alkan. walling­ ford, oxon: cab international. murray, m., murray, p.k. & mcintyre, w.i.m. 1977. an improved parasitological technique for the diagnosis of afri­ can trypanosomosis. transactions of the royal society of tropical medicine and hygiene, 71:325–326. ng’ayo, m.o., njiru, z.k., eucharia, u.k., muluvi, g.m., osir, e.o. & masiga, d.k. 2005. detection of trypano­ somes in small ruminants and pigs in western kenya: impor­ tant reservoirs in the epidemiology of sleeping sickness. kinetoplastic biology and disease, 4:5. njiru, z.k., constantine, c.c., guya, s., crowther, j., kiragu, j.m., thompson, r.c.a. & davila, a.m.r. 2005. the use of its1 rdna pcr in detecting pathogenic african trypanosomes. parasitology research, 95:186–192. radostatis, o.m., gay, c., blood, d.c. & hinchcliff, k.w. 2000. diseases caused by trypanosomes. veterinary medicine, 9th ed. london: w.b. saunders company limited. samuel, r.e.a. 1998. its sequences from nuclear rrna sug­ gest unexpected phylogenetic relationships between euro­ mediterranean, east asiatic and north atlantic taxa of quercus (gagaceae). plant systematics and evolution, 211: 129–139. schlotterer, c., hauser, m.t., von haeseler, a. & tautz, d. 1994. comparative evolutionary analysis of rdna its regions in drosophila. molecular biology and evolution, 11:513–522. stephen, l.e. 1986. trypanosoma vivax. trypanosomiasis: a veterinary perspective, 1st ed. oxford: pergamon press. tolosa_323-326.indd introduction taeniosis and hydatidosis are parasitic zoonoses that present major public health problems in lower income countries (taylor, coop & wall 2007). bovine cysticercosis, caused by the metacestodes of taenia saginata is cosmopolitan and occurs in developing as well as in industrialized countries (dorny, phiri, gabriel, speybroeck & vercruysse 2002). the prevalence is high in some african countries (over, jansen, van olm 1992). in ethiopia, the prevalence of bovine cysticercosis ranges from 3.1 % in the central part to 26.25 % in the southern part of the country (tembo 2001; dawit 2004; nigatu 2004; hailu 2005; abunna, tilahun, megersa, regassa & kumsa 2008). the prevalence of human taeniosis, caused by t. saginata, ranges from 10 % to 70 %. the tradition of eating raw beef is a major risk factor contributing to this high prevalence (mamo 1988). hydatidosis, caused by the meta cestodes of echinococcus granulosus, is endemic in ethiopia and studies from various parts of the country indicate that the infection ranges from 25.7 % to 63 % and 4.4 % to 18.8 % in cattle and sheep, respec323 onderstepoort journal of veterinary research, 76:323–326 (2009) prevalence of bovine cysticercosis and hydatidosis in jimma municipal abattoir, south west ethiopia t. tolosa1, w. tigre1, g. teka1 and p. dorny2, 3* abstract tolosa, t., tigre, w., teka, g. & dorny, p. 2009. prevalence of bovine cysticercosis and hydatidosis in jimma municipal abattoir, south west ethiopia. onderstepoort journal of veterinary research, 76:323–326 a cross-sectional study to determine the prevalence of bovine cysticercosis and hydatidosis was conducted from october 2007 to march 2008 in cattle slaughtered at the jimma municipal abattoir. cyst distribution and viability of bovine cysticercosis and hydatidosis were also determined. a total of 512 carcasses were inspected of which 15 (2.93 %) and 161 (31.44 %) were infected with taenia saginata metacestodes and hydatid cysts, respectively. from a total of 109 cysticerci collected from infected carcasses, 47 (43.12 %) were viable. the anatomical distribution of the cysticerci was, shoulder muscle (39.5 %), heart (33.9 %), neck muscle (13.8 %), tongue (10.1 %), masseter muscles (1.8 %) and diaphragm (0.9 %). of the 1171 hydatid cysts collected 223 (19.0 %) were fertile, 505 (43.1 %) sterile, 49 (29.8 %) calcified and 94 (8.0 %) contained pus. a greater proportion of fertile cysts were found in the lungs than in other organs. it was concluded that these zoonotic cestodes deserve due attention to safeguard public health, and that further studies are needed on genotyping, epidemiology and public health importance of echinococcus granulosus in the study area. keywords: cattle, cysticercosis, echinococcus granulosus, ethiopia, hydatidosis, prevalence, taenia saginata * author to whom correspondence is to be directed. e-mail: pdorny@itg.be 1 jimma university, college of agriculture and veterinary medicine, school of veterinary medicine, p.o. box, 307, jimma, ethiopia 2 laboratory of parasitology, faculty of veterinary medicine, ghent university, salisburylaan 133, 9820 merelbeke, belgium 3 department of animal health, institute of tropical medicine, nationalestraat 155, 2000 antwerpen, belgium accepted for publication 16 march 2009—editor 324 bovine cysticercosis and hydatidosis in jimma municipal abattoir, south west ethiopia tively (tamene 1986; abduljewad 1988; mohammed 1988; woubet 1988; yilkal 1989; fikre 1994; mussie 1995). in jimma town, in the southwestern part of ethiopia, the hygienic conditions are poor and raw beef consumption is common. backyard slaughtering of domestic animals, particularly, sheep and goats; and feeding stray dogs with condemned organs are common practices (abduljewad 1988). these habits promote the transmission of both cestode infections. hence, this study was conducted with the objective of determining the prevalence of bovine cysticercosis and hydatidosis, slaughtered in the municipal abattoir of jimma. materials and methods the study was conducted in the municipal abattoir of jimma in the southwestern part of ethiopia, 352 km southwest (sw) of addis ababa. mixed crop and livestock, in an extensive system, are the main production systems practised in the area. several species of livestock are reared in the zone, but cattle are the most predominant. they are used as assets, for draft power, meat, milk and hides. animals are sold only when they become less productive in terms of milk production or draft power. the most common con straints to livestock production are related to communal grazing and watering, poor shelter and underfeeding. a cross sectional study on bovine cysticercosis and hydatidosis was conducted from october 2007 to march 2008 on randomly selected animals slaughtered at the jimma municipal abattoir. the study animals originated from different districts of the jimma region. sex, age, breed and origin of each animal were recorded. during meat inspection, carcasses and their respective organs were carefully examined in accordance to the procedures of the ethiopian ministry of agriculture meat inspection regulation (1972) for the detection of t. saginata cysticercosis and hydatidosis. visual inspection and palpation, followed by multiple incisions in the liver, kidney, lung, kidney, heart, diaphragm, shoulder, tongue and masseter muscle, were made to detect the presence of cysticerci and hydatid cysts. cysts were carefully removed from the carcass and organs and transported to the parasitology laboratory of the school of veterinary medicine, jimma uni versity, for further examination. the viability of t. saginata cysticerci was assessed by incubating the cysts in a normal saline solution containing 30 % ox bile at 37 °c for 2 h. the cysts were regarded as viable if the scolex evaginated during the incubation period. the evaginated cysticerci were examined microscopically for species identification (gracey, collins & huey 1999). hydatid cysts were carefully incised and designated as fertile if protoscolices were found. fertile cysts were subjected to a viability test (macpherson, zeyhle & roving 1985) and infertile hydatid cysts were classified as sterile or calcified (soulsby 1982). table 1 taenia saginata cysticercosis in cattle slaughtered at jimma municipal abattoir (n = 512): infection levels in different organs organs range of number of cysticerci recovered total/prevalence shoulder muscle heart tongue masseter muscle neck muscle diaphragm 1–10 1–10 2–3 1–2 2–5 1 43 (39.45) 37 (33.94) 11 (10.10) 2 (1.83) 15 (13.76) 1 (0.92) total 109 (100) table 2 viability of hydatid cysts according to the affected organs from cattle slaughtered at the jimma municipal abattoir (n = 512) organ inspected fertile sterile calcified pus formation total lung liver spleen heart kidney 174 (21.16) 38 (12.46) 5 (26.32) 4 (22.22) 2 (28.57) 366 (44.52) 109 (35.74) 14 (73.68) 11 (61.11) 5 (71.43) 219 (26.64) 127 (41.64) 0 (0) 3 (16.67) 0 (0) 63 (7.66) 31 (10.16) 0 (0) 0 (0) 0 (0) 822 305 19 18 7 total 223 (19.04) 505 (43.13) 349 (29.80) 94 (8.03) 1 171 325 t. tolosa et al. results all 512 cattle inspected were adult males and from similar agro-ecological sites and husbandry systems. of those, 15 (2.93 %) were infected with cysticerci. a total of 109 cysticerci were collected and occurred, in decreasing order, in the shoulder muscle (43), heart (37), neck muscle (15), tongue (11), masseter muscle (2) and diaphragm (1) (table 1). forty-seven (43.12 %) were viable and 62 (56.88 %) were degenerated or calcified. viable cysticerci were observed in the shoulder (21), cardiac (11), neck (8), tongue (6) and diaphragm (1) muscles, in descending order. the degenerated or calcified cysts were recovered from heart (26), shoulder (22), neck (7), tongue (5), and masseter (2) muscles, in decreasing order. out of a total of 512 cattle carcasses, 161 (31.44 %) were infected with hydatid cysts, a total of 1 171 hydatid cysts being collected from the infected animals. of these cysts, 223 (19.04 %) were fertile, 505 (43.13 %) sterile, 349 (29.80 %) calcified and 94 (8.03 %) contained pus (table 2). cysts were found in the lungs (57 %), liver (34 %), spleen (5 %), heart (3 %) and kidneys (1 %). discussion in this study the prevalence of cysticercosis was 2.93 %, which is comparable to reports on the subject emanating from central ethiopia (3.11 %) (tembo 2001), but lower than that reported in other parts of the country (dawit 2004; nigatu 2004; hailu 2005; abunna et al. 2008). the different prevalences reported in these studies might be due to several factors of which husbandry systems, hygiene differences and eating habits are among the most important. in addition, diagnosis of bovine cysticercosis by meat inspection underestimates the true prevalence, especially when infection is light (dorny & praet 2007). the higher prevalence of cysticercosis in developing countries is associated with poor sanitary infrastructure, low awareness and improper disposal of sewage, which also pertains to ethiopia, where the widespread habit of eating raw meat is an additional important risk factor. the high prevalence of hydatidosis of 31.45 % is in accord with findings in other parts of the country. most of the slaughtered animals were adults, which were most likely culled due to inefficiency for draught purpose. old animals are likely to have a higher possibility of acquiring infection due to their longer exposure to infection and to lower immunity to combat infection. in this study, the lungs were the most frequently affected organ (57 %), followed by the liver (34 %), which is in agreement with other studies in cattle in ethiopia (mohammed 1988; abdul juwad 1988). also, a much higher proportion of fertile cysts was observed in the lungs than in any other organ. the fertility rate of hydatid cysts may increase with advancing age of the hosts due to reduced immunological compatibility of animals (himo nas 1987). the prevalence of hydatidosis is likely to be high in ethiopia owing to several factors favouring transmission, among which are, keeping of dogs in close association with ruminants, low public awareness on the role of offal in transmitting hydatid cysts, feeding of dogs with hydatid infected organs and backyard slaughtering practices. in addition, jimma town has a large population of stray dogs that visit the abattoir grounds to be fed with condemned organs (personal observations). to reduce infections of these cestodes to cattle and prevent transmission to humans attention should be given to such considerations aspersonal hygiene, meat hygiene, control of stray dog populations and cestodicidal treatment of dogs in the study area. while taeniosis in humans has generally little clinical significance, hydatid disease is a serious condition that warrants control measures to be taken. in order to assess the zoonotic potential of e. granulosus, genotyping of parasites isolated from cattle, sheep and goats should be undertaken. acknowledgements this work was supported by the flemish inter university council (vlir-jimma university iuc programme ethiopia-belgium). the cooperation of jimma municipal abattoir is appreciated. references abduljewad, a. 1988. hydatidosis prevalence at jimma abattoir. d.vm. thesis, faculty of veterinary medicine, addis ababa university. abunna, f., tilahun, g., megersa, b.a., regassa, a. & kumsa, b. 2008. bovine cysticercosis in cattle slaughtered at awassa municipal abattoir, ethiopia: prevalence, cyst viability, distribution and its public health implication. zoonoses and public health, 55:82–88. dawit, s. 2004. epidemiology of t. saginata taeniasis and cysticercosis in north gondor zone. dvm thesis, faculty of veter inary medicine, addis ababa university. dorny, p., phiri, i., gabriel, s., speybroeck, n. & vercruysse, j. 2002. a sero-epidemiological study of bovine cysticercosis in zambia. veterinary parasitology, 104: 211– 215. 326 bovine cysticercosis and hydatidosis in jimma municipal abattoir, south west ethiopia dorny, p. & praet, n. 2007. taenia saginata in europe. veterinary parasitology, 149:22–24. fikre, l. 1994. echinococcosis/hydatidosis in konso (southern ethiopia): an assessment trial of its prevalence, economic and public health importance. d.vm. thesis, faculty of veterinary medicine, addis ababa university. gracey, j.f., collins, d.s. & huey, r.j. 1999. meat hygiene, 10th ed. london: harcourt brace & company. hailu, d. 2005. prevalence and risk factors for taenia saginata cysticercosis in three selected areas of eastern shoa. m.sc. thesis, faculty of veterinary medicine, addis ababa university. himonas, c. 1987. the fertility of hydatid cysts in food animals in greece. helminth zoonoses. amsterdam, the nether lands: martinus nijhoff publishers. macpherson, n.l., zeyhle, e. & roving, t. 1985. an echinococcosis pilot control programme for north west turkana, kenya. annals of tropical medicine and parasitology, 78: 188–192. mamo, e. 1988. some common zoonotic helminths, in the ecology of health, edited by a.z. zein & h. kool, ministry of health, addis ababa. ministry of agriculture 1972. meat inspection regulations. legal notice no. 428 negarite gazexa, addis ababa. mohammed, a. 1988. study on prevalence and economic significance of bovine hydatidosis in gamo-goffa region. d.vm. thesis, faculty of veterinary medicine, addis ababa university. mussie, h. 1995. bovine hydatid disease: an assessment trial of its prevalence and economic importance at bahir dar slaughterhouse. d.vm. thesis, faculty of veterinary medicine, addis ababa university. nigatu, k. 2004. cysticercus bovis: development and evaluation of serological tests and prevalence at addis ababa abattoir. m.sc. thesis, faculty of veterinary medicine, addis ababa university. over, h.j., jansen, j. & van olm, p.w. 1992. distribution and impact of helminth diseases of livestock in developing countries. rome, italy: fao (animal production and health paper, 96). soulsby, e.j.l. 1982. helminths, arthropods and protozoa of domesticated animals, 7th ed. london: bailliere tindall. tamene, m. 1986. a preliminary study on echinococosis/hydatidosis of livestock (cattle, sheep and goats) in gondar administrative region. d.vm. thesis, faculty of veterinary medicine, addis ababa university. taylor, m.a., coop, r.l. & wall, r.l. 2007. veterinary parasitology, 3rd ed. oxford: blackwell publishing. tembo, a. 2001. epidemiology of taenia saginata taeniasis and cysticercosis in three selected agroclimatic zones in central ethiopia. s.sc. thesis, faculty of veterinary medicine, addis ababa university & free university of berlin. woubet, s. 1988. prevalence of cattle hydatidosis and its economic significance in robe municipality abattoir. d.vm. thesis, faculty of veterinary medicine, addis ababa university. yilkal, a. 1989. hydatidosis in cattle, sheep, pigs; cysticercus tenuicollis in sheep around dessie and the efficacy of hagenia abyssinica (kosso) on taenia hydatigena. d.vm. thesis, faculty of veterinary medicine, addis ababa uni ver sity. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice mtshali_67-75.qxd introduction animal diseases in general and tick and tick borne diseases (ttbd) in particular are among the many factors which directly and indirectly hamper the growth of the livestock sector. livestock is important because of the roles it play as a source of food, in generating employment, delivery of energy (dung, biogas), fertilizer, weed control, use of marginal lands, investment and savings, and transport (sansoucy 1995). tick-borne diseases of major economic importance in southern africa which affect cattle are heartwater caused by ehrlichia ruminantium, babesiosis caused by babesia bigemina and babesia bovis, anaplasmosis caused by anaplasma marginale, and theileriosis caused by theileria parva. of much lesser importance in cattle are the generally non-pathogenic mild theilerioses caused by theileria mutans, theileria velifera and theileria taurotragi, spirochaetosis (borreliosis) caused by borrelia theileria, benign babesiosis caused by babesia occultans, and bovine ehrlichiosis caused by ehrlichia bovis (horak 1982). in africa tick-borne diseases are important and they are considered as one of the greatest animal dis67 onderstepoort journal of veterinary research, 71:67–75 (2004) a sero-epidemiological survey of blood parasites in cattle in the north-eastern free state, south africa m.s. mtshali1*, d.t. de waal2 and p.a. mbati1 abstract mtshali, m.s., de waal, d.t. & mbati, p.a. 2004. a sero-epidemiological survey of blood parasites in cattle in the north-eastern free state, south africa. onderstepoort journal of veterinary research, 70:67–75 a survey to determine the incidence of parasites in cattle (n = 386) was conducted in the north eastern free state between august 1999 and july 2000. giemsa-stained blood smears were negative for blood parasites. a total of 94 % of the cattle were sero-positive for babesia bigemina by indirect fluorescent antibody test while 87 % were sero-positive for anaplasma by enzyme-linked immunosorbent assay. the observation of negative blood smears but high incidence of positive serological results for anaplasma and babesia for the same group of cattle indicates that this area is endemic for these diseases but with a stable disease situation. all the animals were sero-negative for b. bovis and this is probably because the tick vector (boophilus microplus) which transmits the disease is not present in the free state province. two tick species belonging to the family ixodidae were found on cattle, namely boophilus decoloratus and rhipicephalus evertsi evertsi. in the present study significant differences in seasonal burdens of b. decoloratus occurred, with the highest infestations recorded from february to june. the presence of r. evertsi evertsi throughout the year without any or with small fluctuations in winter months was observed, with a peak from february to may keywords: anaplasma, babesia, cattle, elisa, epidemiology, ifat * author to whom correspondence is to be directed. e-mail: mtshalims@qwa.uovs.ac.za 1 parasitology research programme, qwa-qwa campus, university of the free state, private bag x 13, phuthaditjhaba, 9866 south africa 2 department of veterinary microbiology and parasitology, faculty of veterinary medicine, university college dublin, ireland accepted for publication 23 october 2004—editor ease problem. it is accepted that theileriosis caused by t. parva, dermatophilosis and heartwater are the major tick-borne or tick-associated diseases of grazing cattle. babesiosis and anaplasmosis may be important in certain regions and may cause problems in grazing and zero-grazing situations. the complexity of determining the direct and indirect economic impact of tick-borne diseases and their control is reflected in the fact that only rough estimates are available for the cost of some of the components (de castro 1997). materials and methods study area the north-eastern free state was divided into three study sites that included harrismith (29°5’e, 28°18’s), kestell (28°38’e, 28°20’s) and qwa-qwa (28°50’e, 28°35’s) which are three established study sites of uniqwa’s parasitology research programme. they are located at 42, 28 and 5 km respectively away from the qwa-qwa campus of the university of the free state. experimental animals a total of 10–16 animals were randomly selected and examined monthly at each of the three study sites over a continuous period of 12 months from august 1999 to july 2000. they were kept under small-scale farming systems. during the day they graze on communal pastures. the usual grazing time is 7–9 h per day, the duration being determined by the time of the year. at night, cattle are herded, milked and kept in kraals with a manure floor. parasitological diagnosis cattle were individually restrained in a mobile facility consisting of a collecting kraal, race, crush and neck clamp. thin and thick blood smears were made using standard techniques, fixed in absolute methanol and stained with a 10 % neutral buffered giemsa stain. both thin and thick smears were examined using a 100 x oil immersion objective lens of a nikon se light microscope. serological diagnosis blood samples were collected aseptically from the tail vein into 10 ml vacuum tubes (becton dickinson vacutainer systems europe, uk) without anticoagulant. they were centrifuged for 30 min at 2 000 rpm and the sera were harvested into cryovials, and kept at –35 °c until used for serological analysis. an indirect fluorescent antibody test described by morzaria, brocklesby & harradine (1977) was used to detect b. bigemina and b. bovis antibodies. a competition inhibition enzyme-linked immunosorbent assay (ci-elisa) described by visser, mcguire, palmer, davis, shkap, pipanoe & knowles (1992) was employed to detect a. marginale antibodies, and the monoclonal antibody was obtained from the onderstepoort veterinary institute, pretoria, south africa. determination of packed cell volume values in the cattle peripheral blood for packed cell volume was taken from the tip of the tail of the cattle by pricking the skin with the point of sharp scissors. the drop of blood that formed was collected into micro-haematocrit tubes. the blood was centrifuged in a microhaematocrit centrifuge (gemmy industrial corp.) for 10 min. a micro-haematocrit reader scale was used to determine the pcv readings of the studied animals. collection and identification of tick species ticks were collected on a monthly basis from august 1999 to july 2000 from the same groups of cattle from which blood samples had been collected. only adult male and female ticks in all stages of engorgement were collected from the right-hand sides of the animals. the method of counting ticks on one body side only is generally used to estimate the overall level of tick infestation on animals (hermans, dwinger, buening & herrero 1994; dreyer, fourie & kok 1998b).the right sides of animals were divided into 13 different regions and ticks from each region were collected separately (dreyer et al. 1998b). these regions did not necessarily correspond to recognized anatomical regions. the position of attached adult ticks was first checked by visual examination and then palpating the hair coat of the animals. the ticks were removed using entomological forceps from each of the defined body areas and placed in labelled containers filled with 70 % alcohol. the ticks were identified to species level and counted under a standard stereoscopic microscope (kyowa, tokyo). identification was done with the aid of the descriptions of hoogstraal (1956) and walker (1961). the number of ticks collected was multiplied by two to give an indication of total tick burden of each host. 68 blood parasites in cattle in north-eastern free state, south africa data presentation and analysis the data sets were analyzed statistically using analysis of variance techniques. the software programme used was the statistical analysis system (sas) for all the serological data. another software programme used was statistica 1998 edition for windows (statsoft, inc.). a one-way analysis of variance (anova) test (barnard, gilbert & mcgregor 1993) was employed to determine significant differences in burdens of boophilus decoloratus and rhipicephalus evertsi evertsi during winter (may to july), spring (august-october), summer (november to january) and autumn (february to april). this was followed by the least significant difference (lsd) test, to indicate the seasons causing the variance (zar 1974). a one-way anova-test followed by a lsd test was used to determine if any significant variability existed in seasonality of the tick species in the three different localities (barnard et al. 1993). results parasitological screening for haemoparasites no blood parasite was detected in the thick and thin blood smears prepared from a total of 386 cattle examined in the three study sites. the majority of cattle in the area are of mixed-breed origin which are predominantly friesian-crosses. all the animals sampled appeared healthy and did not show any clinical signs of disease. serological diagnosis of anaplasma and babesia in the cattle the average seroprevalence of the cattle (n = 139) in the harrismith area (χ2 test, statistical value = 79.317, df = 1, prob = 0.001) for a. marginale was 88 % by elisa while 92 % of the animals were sero-positive (χ2 test, statistical value = 98.482, df = 1, prob = 0.001) for b. bigemina by ifat. the sera from the harrismith area were negative (100 %) for b. bovis (table 1). the average seroprevalence of the cattle (n = 127) in the kestell area (χ2 test, statistical value = 59.598, df = 1, prob = 0.001) for a. marginale was 84 % by elisa while 91 % of the animals were sero-positive (chi-square test, statistical value = 86.811, df = 1, prob = 0.001) for b. bigemina by ifat. the sera from the kestell area were negative (100 %) for b. bovis (table 2). the average seroprevalence for the cattle (n = 120) in qwa-qwa (χ2 test, statistical value = 73.633, df = 1, prob = 0.001) for a. marginale was 89 % by elisa. all the animals were sero-positive (100 %) for b. bigemina by ifat. the sera from the qwaqwa area were negative (100%) for b. bovis (table 3). means of od values of tested sera of the cattle as determined by elisa the mean monthly value and standard deviation (sd) of optical density (od) values of the tested sera from the cattle (n = 386) in the three areas were determined. the mean monthly od values of the cattle in all the three sites ranged between 0.236–0.302 as shown in table 4. means of pcv values in the cattle the mean monthly pcv values of the cattle in all the three sites ranged between 26–31 % as shown in table 5. 69 m.s. mtshali, d.t. de waal & p.a. mbati table 1 the prevalence of anaplasma and babesia in cattle (n = 139) in harrismith as determined by elisa and ifat respectively anaplasma marginale frequency percent cumulative cumulative frequency percent n 17 12 17 12 p 122 88 139 100 babesia bigemina n 11 8 11 8 p 128 92 139 100 babesia bovis n 139 100 139 100 p 0 0 0 0 n = the number of screened animals that were negative p = the number of screened animals that were positive 70 blood parasites in cattle in north-eastern free state, south africa table 2 the prevalence of anaplasma and babesia in cattle (n = 127) in kestell as determined by elisa and ifat respectively anaplasma marginale frequency percent cumulative cumulative frequency percent n 20 16 20 16 p 107 84 127 100 babesia bigemina n 11 9 11 9 p 116 91 127 100 babesia bovis n 127 100 127 100 p 0 0 0 0 n = the number of screened animals that were negative p = the number of screened animals that were positive table 3 the prevalence of anaplasma and babesia in cattle (n = 120) in qwa-qwa as determined by elisa and ifat respectively anaplasma marginale frequency percent cumulative cumulative frequency percent n 13 11 13 11 p 107 89 120 100 babesia bigemina n 0 0 0 0 p 120 100 120 100 babesia bovis n 120 100 120 100 p 0 0 0 0 n = the number of screened animals that were negative p = the number of screened animals that were positive table 4 mean monthly od values of tested sera from cattle (n = 386) in harrismith, kestell and qwa-qwa as determined by elisa month n mean sd august (1999) 33 0.247 0.083 september 31 0.236 0.089 october 32 0.246 0.066 november 32 0.256 0.114 december 30 0.302 0.121 january (2000) 31 0.275 0.096 february 30 0.251 0.091 march 33 0.235 0.082 april 39 0.245 0.077 may 33 0.290 0.090 june 32 0.251 0.081 july 30 0.249 0.010 n = the number of sampled animals sd = standard deviation from the mean table 5 mean monthly pcv values of cattle (n = 386) in harrismith, kestell and qwa-qwa month n mean sd august (1999) 33 29 6.33 september 31 29 6.29 october 32 28 4.94 november 32 31 4.58 december 30 28 6.86 january (2000) 31 29 6.78 february 30 30 3.88 march 33 30 5.05 april 39 28 4.92 may 33 29 5.53 june 32 26 6.11 july 30 26 6.90 n = the number of sampled animals sd = standard deviation from the mean tick species infesting the cattle and their numbers a total of 5 930 adult ticks belonging to two species were collected from the cattle (n = 386) during the period under review. these are summarized in table 6. high tick infestations were observed on the animals from qwa-qwa. boophilus decoloratus was generally the dominant species when comparing all the three study sites, whereas rhipicephalus evertsi evertsi was only dominant in qwa-qwa. significant differences (p = 0.641, at 5 % significance level) in the seasonal burdens of b. decoloratus occurred, with the highest infestations recorded from february to june (fig. 1). peaks were observed in may to june and december to february. rhipicephalus evertsi evertsi was present throughout the year with some small fluctuations in the winter months, but with a peak in october (fig. 2). increased numbers of r. evertsi evertsi were present on the animals from february to may, with a peak occurring in may, dropping off to low levels in june and july in kestell. a peak of r. evertsi evertsi was observed from february to may in both harrismith, and qwa-qwa. seasonal burdens and variations of the two tick species were compared for the three localities, harrismith, kestell and qwa-qwa as is shown in fig. 1 and 2. seasonal variations among the three localities for the two tick species were small and mostly insignificant, and data from the three localities were thus combined for a discussion of the seasonal dynamics. discussion serological and parasitological diagnosis of parasites in the cattle it has been shown that, based on the information gained from a serological study, the immune status of cattle in an area can be classified into an endemically stable (81–100 % positive sera) situation, a situation approaching stability (61–80 %), an unstable (21–60 %) situation, a minimal disease situation (1–20 %), and a disease-free situation (0 % positive sera) (norval, lawrence & daillecourt 1983). where the inoculation rate of babesia and anaplasma is adequate to ensure that all calves are infected while they are protected by innate and/or colostral immunity, clinical disease is minimal and endemic stability is achieved. the disruption of an existing situation of endemic stability is usually associated with drought 71 m.s. mtshali, d.t. de waal & p.a. mbati table 6 tick species of the studied animals (n = 386) in harrismith, kestell and qwa-qwa site n boophilus rhipicephalus decoloratus evertsi evertsi harrismith 139 934 492 kestell 127 960 418 qwa-qwa 120 1 348 1 778 total 386 3 242 2 688 n = the number of sampled animals fig.1 mean monthly b. decoloratus burdens in cattle sampled in harrismith, kestell and qwa-qwa during a 12month study period (august 1999 to july 2000) fig. 2 mean monthly r. evertsi evertsi burdens in cattle sampled in harrismith, kestell and qwa-qwa during a 12month study period (august 1999 to july 2000) �� �� �� �� �� �� � � � � � � � � � � � � � � �� �� � � � � � ���������������� ���� ���� ������� ���������� ������� ���������������� ������� ���������� ������� � � � � � � � � � ���� ���� �� �� �� �� �� �� �� �� �� � � � � � �� �� � � � � � conditions (de vos 1979), or increased tick control (norval et al. 1983). endemic instability, on the other hand, describes the situation in a herd where some animals fail to become infected within nine months of birth. when such susceptible individuals encounter infected ticks, clinical disease develops (dreyer, fourie & kok 1998a). with the ifat a total of 94 % of the studied animals were positive for b. bigemina indicating a significantly large number of animals exposed to this parasite. when comparing the three study sites, all the animals in qwa-qwa were positive (100 %) for b. bigemina, 92 % of the animals in harrismith were positive and 91% of the animals in kestell were positive for b. bigemina by ifat. based on the finding that 94 % of cattle were seropositive for b. bigemina, it is evident that cattle in the north-eastern free state fit into the group of infection rates that is indicative of an endemically stable situation. no clinical cases of babesiosis due to this organism were observed during the 12-month study period. although there were differences in seroprevalence between the cattle in harrismith, kestell and qwa-qwa areas, these differences were small and not statistically significant. a similar study by dreyer et al. (1998a) investigating tick-borne diseases of cattle in botshabelo and thaba nchu indicated a situation approaching endemic stability with 62.42 % of the studied animals having b. bigemina antibodies. all the sera tested were negative for b. bovis in all the three study sites. as far as it is known, b. bovis is only transmitted by boophilus microplus (de vos 1979; de vos & jorgensen 1992) which does not occur in the free state province (howell, walker & nevill 1978). dreyer et al. (1998a) found an average seroprevalence of 19.47 % against b. bovis in cattle sampled in the botshabelo-thaba nchu area. these low seroprevalence indices indicated a situation of minimal disease, and the risk of clinical disease outbreak due to this organism was thus small. the occurrence of b. bovis antibodies in cattle of botshabelo-thaba nchu area (dreyer et al. 1998a), is difficult to explain, because no b. microplus ticks were found in this region (dreyer 1997). endemic instability defines the situation in which some animals in the herd fail to become infected for a considerable period after birth. this could be due to a host-parasite imbalance resulting from infrequent transmission. disease is then seen when susceptible animals in a herd encounter infected ticks. the creation of unstable situations in south africa is to a large extent dependent on two factors: unfavourable climatic conditions and the injudicious control of ticks. one or both of these factors may influence the stability of either of the babesia spp. in any given area (de vos 1979). the average seroprevalence of cattle for a. marginale was 87 % in the three study sites. a seroprevalence above 81 % is indicative of a situation of endemic stability. when comparing the three study sites, the animals of qwa-qwa had the highest positive percentage of 89 %. this was followed by harrismith where 88 % of the animals being positive for a. marginale by elisa. in kestell 84 % of the animals were positive for a. marginale by elisa. endemic areas of anaplasmosis in south africa appear to have high vector activity and, for the maintenance of a stable situation, the obvious source of infection would be infected cattle. it is still believed that adequate vector challenge will ensure early exposure of calves and result in the development of protective immunity (potgieter 1979). natural epidemic areas occur where none of the young calves are exposed to early infection because of the effect on vector survival resulting from geographical and climatic restrictions. drastic fluctuations of these restrictions may result in the temporary settlement or elimination of vectors in these areas, with obvious consequences. human-induced epidemics may result within endemic areas, if excessive tick control is practiced. if vector ticks are eradicated, a farmer will eventually breed susceptible stock. under such conditions a lapse in the dipping programme, the possible introduction of carrier animals, the neglect by neighbours to dip their cattle, and the presence of certain wild ruminants and migrating cattle could lead to serious disease outbreaks (potgieter 1979). there was no significant difference (p = 0.006, at 1 % significance level) in the incidence of either anaplasmosis or babesiosis between the seasons. further there was no significant (p = 0.008, at 1 % significance level) association on the occurrence of anaplasma and babesia infections when using the likelihood ratio of chi-square indicating that the two infections are independent. the observation of negative blood smears but high prevalence of positive serological results for anaplasma and babesia for the same group of animals indicates that the north eastern free state is endemic for these diseases but with a stable disease situation. it is known that indigenous breeds 72 blood parasites in cattle in north-eastern free state, south africa can become very resistant to ticks (bonsma 1981) and acquire immunity to tick-borne diseases if exposed at an early age (ross & lohr 1968). elisa od values of tested sera and pcv readings of the cattle this study has shown that the animals had pcv ranges that fall within the normal pcv range of “healthy” cattle. this means that the animals studied are healthy carriers of anaplasmosis and babesiosis. although anaemia can be caused by factors other than tick-borne diseases, it remains one of the most reliable indicators of tick transmitted diseases. the pcv profile and average pcv of cattle is affected by the number of ticks infecting animals and the prevalence of anaplasma and babesia. this observation can be used as an additional indicator for tick-borne diseases even when anaplasma and babesia cannot be detected by parasitological diagnostic tests. the general linear models procedure showed no significant effect (p = 0.922, at 5 % significance level) of site, month, breed, b. bigemina or a. marginale on od values. there was no significant effect (p = 0.0160, at 5 % significance level) of site, month, breed, b. bigemina or a. marginale on pcv when using the general linear models procedure, and there was no significant association (p = 0.7129, at 5 % significance level) between pcv and od values when using correlation analysis (pearson correlation coefficients). tick species infecting the cattle a total of 5 930 adult ticks belonging to two species were collected during 12-month period from august 1999 to july 2000 in the harrismith, kestell and qwa-qwa areas. boophilus decoloratus the “blue tick” was generally a dominant species, but is inactive in winter and spring. in the present study significant differences in seasonal burdens of this onehost tick occurred, with the highest infestations recorded from february to june. this is similar to results obtained by robertson (1981) in the eastern cape province and dreyer et al. (1998b) in the free state province, where b. decoloratus was abundant from may to june but sparse from july to january. in a study done by rechav (1982) in the eastern cape, no definite pattern of seasonal occurrence was observed but the lowest numbers on hosts were also seen in early spring, with peaks in summer and autumn (february to june). the various peaks in adult infestation resulting in population “waves”, indicate the completion of several generations in one year (punyua, latif, nokoe & capstick 1991). peaks were observed in the present study, namely, in may to june and december to january. boophilus decoloratus is responsible for the transmission of two tick-borne diseases, namely anaplasmosis and babesiosis. this tick is usually most evident from about september until the end of june, and may breed throughout the year in warmer areas. in colder climates, however, it is inactive in winter and early spring (howell et al. 1978). the observation in our study is similar to that of howell et al. (1978) in that a small rise in tick numbers was noted during september, and a peak in june when the blue ticks were mostly evident was obtained. the positive correlation (r2 = 0.364, p < 0.05) between burdens of tick species and warm or cold seasons was expected as ticks are ectotherms. such a correlation was also reported by fourie, kok & heyne (1996) following their study in southwestern free state. according to rechav (1982), environmental temperature is probably the main regulating factor in the seasonal patterns of the blue tick. the presence of r. evertsi evertsi the “red-legged tick” throughout the year with only small fluctuations in the winter months was also observed by punyua et al. (1991), fivaz & de waal (1993), and dreyer et al. (1998b). in this study, the peak observed from march to may resembled the january to may peak on cattle in a kwazulu-natal study (baker & ducasse 1967), the april to may peak in a study by rechav (1982) in the eastern cape, the march to may peak in a study on sheep in the free state (horak, williams & van schalkwyk 1991), and the march to may peak on cattle in botshabelo and thaba nchu (dreyer et al. 1998b). results obtained in this study indicated that temperature, and specifically the winter temperature, was probably the major factor regulating the seasonal activity of the red-legged tick. the continuous presence of adult ticks throughout the year indicates that more than one life cycle can be completed annually, as was suggested by matson & norval (1977). rhipicephalus evertsi evertsi is one of the most important vectors of theileria equi in horses in south africa (howell et al. 1978), and transmits b. bigemina (de vos & jorgensen 1992) and a. marginale (potgieter 1979) in bovines. certain adult r. evertsi evertsi strains can produce a toxin resulting in “spring lamb paralysis” in lambs, kids and calves. 73 m.s. mtshali, d.t. de waal & p.a. mbati high ear infestations with the immature stages of this species can result in ear irritation with secondary bacterial infections of ear canals (howell et al. 1978). red-legged ticks are most active in summer, though some specimens can be found all through the year. in kwazulu-natal and the eastern cape the numbers of immature red-legged ticks start to increase early in november, are at their peak from january to april and then slowly decrease again. adult numbers are highest from january to the end of may (howell et al. 1978). the tick is inactive in winter and spring. rhipicephalus evertsi evertsi was the second most abundant tick species in harrismith and kestell. this is probably due to the availability of its other preferred hosts, such as sheep and goats, which generally graze together with cattle. seasonal fluctuations of both tick species in the three localities were very similar but their burdens in cattle in qwa-qwa were significantly highest compared to the other two localities. a first possible reason for higher tick infestations in qwa-qwa was that the body condition of cattle in this locality was always poorer compared to that in the other two localities. this can result in a lowered tick resistance resulting in higher tick burdens (de castro & newson 1993). a possible reason for the poor body conditions could have been the severe overgrazing of the veld in this specific locality. farmers in this area appeared unmotivated and unwilling to herd their cattle to better grazing further away. a second possible reason for the higher tick burdens could have been the higher stocking densities, resulting in more contact with hosts (hlatshwayo 2000). another possible reason for higher tick burdens in qwa-qwa is that farmers in this area do not control ticks or tick-borne diseases. a possible reason these farmers not to control ticks is that most of them are resource-poor farmers, and they do not have the necessary resources and knowledge to control ticks (hlatshwayo 2000). acknowledegments we are grateful to the farmers in the north eastern free state for their cooperation. we sincerely thank dr laura lopez, mr olivier matthee, ms andrea spickett and mr jonas komape of the onderstepoort veterinary institute, pretoria, south africa for helping us with the sero-diagnostic techniques. we also thank mr mothea tsosane, mr thobelani lose and the late ms zolelwa mxhebe for their assistance with data collecting. we would also like to acknowledge dr peter njuho of the university of natal, pietermaritzburg for helping us with statistical analysis. this study was funded by the national research foundation and the university of the north, qwa-qwa campus. references baker, m.k. & ducasse, f.b.w. 1967. tick infestation of livestock in natal. the predilection sites and seasonal variations of cattle ticks. journal of south african veterinary medical association, 38:447–453. barnard, c.j., gilbert, f.s. & mcgregor, p.k. 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potgieter, f.t. 1979. epizootiology and control of anaplasmosis in south africa. journal of the south african veterinary association, 50:367–432. punyua, d.k., latif, a.a., nokoe, s. & capstick, p.b. 1991. tick (acari: ixodidae) infestations on zebu cattle in western kenya: seasonal dynamics of four species of ticks on traditionally managed cattle. journal of medical entomology, 28:630–636. rechav, y. 1982. dynamics of tick populations (acari: ixodidae) in the eastern cape province of south africa. journal of medical entomology, 19:679–700. robertson, w.d. 1981. a four year study of the seasonal fluctuations in the occurrence of the blue tick boophilus decoloratus (koch) in the coastal regions of the eastern cape, in tick biology and control, edited by g.b. whitehead, & j.d. gibson. proceedings of an international conference held at the tick research unit, rhodes university, grahamstown. 27–29 january 1981. ross, j.p.j. & lohr, k.f. 1968. serological diagnosis of babesia bigemina infection in cattle by the indirect fluorescent antibody test. research in veterinary science, kabete kenya, 9:557–562. sansoucy, r. 1995. livestock—a driving force for food security and sustainable development. world animal review, 84/85:5–17. visser, e.s., mcguire, t.c., palmer, g.h., davis, w.c., shkap, v., pipanoe. & knowles, d.p. 1992. the anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized anaplasma species. infection and immunity, 60:5139–5144. walker, j.b. 1961. some observations on the classification and biology of ticks belonging to the genus rhipicephalus with special reference to the immature stage. east african medical journal, 38:232–246. zar, j.h. 1974. biostatistical analysis. englewood cliffs, new jersey: prentice-hall inc. 75 m.s. mtshali, d.t. de waal & p.a. mbati article information authors: bronwyn c. cloete1 andré bester2 affiliations: 1western cape provincial veterinary laboratory, histology section, south africa2department of industrial and systems engineering, cape peninsula university of technology, bellville campus, south africa correspondence to: bronwyn cloete postal address: private bag 5020, stellenbosch 7599, south africa dates: received: 27 jan. 2012 accepted: 12 aug. 2012 published: 02 nov. 2012 how to cite this article: cloete, b.c. & bester, a., 2012, ‘a lean six sigma approach to the improvement of the selenium analysis method’, onderstepoort journal of veterinary research 79(1), art. #407, 13 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.407 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a lean six sigma approach to the improvement of the selenium analysis method in this original research... open access • abstract • introduction    • setting       • key focus       • background       • objectives       • contribution to field    • literature review • materials and methods    • materials    • setting    • procedure    • ethical considerations • results • trustworthiness    • reliability of analytical data    • validity of analytical data • discussion    • outline of the results    • practical implications    • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ reliable results represent the pinnacle assessment of quality of an analytical laboratory, and therefore variability is considered to be a critical quality problem associated with the selenium analysis method executed at western cape provincial veterinary laboratory (wcpvl). the elimination and control of variability is undoubtedly of significant importance because of the narrow margin of safety between toxic and deficient doses of the trace element for good animal health. a quality methodology known as lean six sigma was believed to present the most feasible solution for overcoming the adverse effect of variation, through steps towards analytical process improvement. lean six sigma represents a form of scientific method type, which is empirical, inductive and deductive, and systematic, which relies on data, and is fact-based. the lean six sigma methodology comprises five macro-phases, namely define, measure, analyse, improve and control (dmaic). both qualitative and quantitative laboratory data were collected in terms of these phases. qualitative data were collected by using quality-tools, namely an ishikawa diagram, a pareto chart, kaizen analysis and a failure mode effect analysis tool. quantitative laboratory data, based on the analytical chemistry test method, were collected through a controlled experiment. the controlled experiment entailed 13 replicated runs of the selenium test method, whereby 11 samples were repetitively analysed, whilst certified reference material (crm) was also included in 6 of the runs. laboratory results obtained from the controlled experiment was analysed by using statistical methods, commonly associated with quality validation of chemistry procedures. analysis of both sets of data yielded an improved selenium analysis method, believed to provide greater reliability of results, in addition to a greatly reduced cycle time and superior control features. lean six sigma may therefore be regarded as a valuable tool in any laboratory, and represents both a management discipline, and a standardised approach to problem solving and process optimisation. introduction top ↑ setting the function of the biochemistry laboratory at western cape provincial veterinary laboratory (wcpvl) is to render accurate and precise analytical data to pathologists so that correct diagnoses can be made. focus on quality in analytical testing laboratories, including the wcpvl biochemistry laboratory, has enjoyed increasing importance as sans 17025 (2005) became more prominent in laboratories country-wide. this quality evolution is similar to that experienced by manufacturing and other service sectors of the world economy. quality is defined as a spectrum of activities and processes that shape the characteristics of a product or service. thus, an undisputed benefit to a laboratory that desires reliability as a characteristic of their results output will follow from adopting a quality approach, which can address the root causes of sources of variability in laboratory processes (nevalainen 1999). lean six sigma is one such approach.lean six sigma is a combination of certain tools and techniques that provides laboratory quality practitioners with a means to improve processes and reduce cycle times. this approach incorporates the use of six sigma methodology, which inherently focuses on gathering data, analysing the collected data, and thereafter improving the process yield, as well as the lean methodology which identifies key areas of variation (khalil, khan & mahmood 2006). key focus from a quality-engineering perspective, the selenium analysis test method may be considered to be analytical laboratory service process. according to cawley (2000), statistical quality control (sqc) techniques are being employed with significant success to improve analytical confidence and the capability of laboratory processes, and they benefit continuous process improvement efforts. furthermore, laboratory managers need to understand that there is an underlying process in generating analytical results, the process can be managed, and the process must evolve through a programme of continuous improvement.an approach to improve the selenium analysis process at wcpvl led to the statement of the following research hypothesis, ‘h0: variation in process, time and control procedures has a direct impact on the disparity in selenium testing results’. in this hypothesis, variation in process, time and control serve as the dependent variable, whilst disparity in selenium testing results serves as the independent variable. background selenium is one of the rarest chemical elements and is considered an essential trace element (tarin 2006). selenium analysis processes have, however, traditionally posed several challenges because of the complex chemical properties of selenium. the main difficulty with the selenium analysis process is sample mineralisation. organic forms of selenium (dimethylselenide and dimethyldiselenide) volatilise from a sample at a temperature exceeding 70 °c; therefore, a loss of selenium yield can occur in an open digestion system and could, as a result, return inaccurate results (kurkova, skrypnik & zaeckiene 2008). furthermore, for selenium analysis to occur, total decomposition of organic material is essential, because selenium values cannot be detected analytically and measured without the release of selenium from the prevalent protein form (selenomethionine and selecysteine) found in biological samples when digested in an acid matrix. as a result, an adequate and total digestion process must be followed. in addition, certain selenium detection techniques such as fluorimetry, hydride generation atomic absorption spectrophotometry (hg-aas), and hydride generation inductively coupled plasma mass spectrometry (hg-icp-ms), require chelate or hydride formation, which are convenient only in total digestion (tarin 2006). objectives the primary objective of this paper was ‘quality improvement on the selenium analysis process by following a lean six sigma model’. the define, measure, analyse, improve and control (dmaic) phases of six sigma are based on deming’s plan-do-check-act (pdca) cycle. according to moen and norman (2011), the pdca cycle is firmly rooted in the traditional phases of the scientific method. secondary research objectives were ‘to determine whether a modified microwave sample digestion procedure was capable of reducing risks associated with the digestion procedure’. in addition, the objective was ‘to optimise the yield of the selenium and ultimately to translate it into an improvement in quality in terms of reliability of results’. contribution to field the approach followed by the researcher is regarded as a model for improvement, which is as a rule employed by any biochemistry or analytical chemistry laboratory. this approach incorporates standard chemistry laboratory validation techniques, and serves as an example of how a quality-methodology can be used to assure and improve analytical quality. literature review in support of an analytical test method being described as a service process, the american association for clinical chemistry (aacc) (2011) describes an analytical method as ‘... a science professionally conducted with rigorous statistical analysis, quality controls, and extensive oversight’, whilst wang (2008) deliberates that the quality improvement process starts with a diagnostic journey of the process, where problems as well as symptoms are identified. thereafter a hypothesis is formulated and tested, and root causes are identified. finally, remedial action is taken and the process is then continuously monitored.scott (2007) contends that a plethora of process improvement approaches exist and is of the opinion that ‘continuous process improvement methods include define, measure, analyse, improve and control, plan-do-study-act, six sigma and total quality management’. (foster 2007), however, points out that there are seven basic tools, known also as the seven basic tools, typically used for process improvement. whilst the basic tools are commonly used in a logical order, they can, however, be used in any order. these quality-tools are ishikawa diagrams (root cause and effect diagrams), pareto charts, process maps (flowcharts), check sheets, histograms, scatter plots and control charts. berte’s (2007) opinion is that a means of improving laboratory test processes at bench level are accomplished by quality improvement programmes. programmes include the use of statistical quality-tools, which provide a visual means to understand quality-control data, so that timely action can be taken when method problems are detected. the merger between six sigma and lean results in a powerful lean six sigma tool, because the strengths from each individual method are secured (table 1). table 1: comparison between lean and six sigma. statistical process control (spc) is defined as ‘the application of statistical methods to the measurement and analysis of variation in a process’ (gryna, chua & defeo 2007). woodall (2000:341) describes spc as ‘a sub-area of statistical quality control (sqc), consisting of methods for understanding, monitoring and improving process performance over time’. woodall (2000) and hare (2001) shared the opinion that understanding process variation is key to spc. these authors maintain that there are at least two basic kinds of variation, which affect a process, namely common cause variation and special cause variation. park and anthony (2008:5) state that ’... variation is the main enemy of quality’, whilst jiang, murphy and tsui (2006) believes that the objective of statistical process control is to identify and remove special cause variation as quickly as possible.wu, pearn and kotz (2009:339) consider process capability studies to be ‘an important and well-defined tool in applications of statistical process control (spc) to continuous improvement of quality and productivity’. the authors state that the relationship between specification limits and tolerance limits of a process, and the actual process performance, may be quantified by using suitable process capability indices. foster (2007) maintains that a process can be considered capable if individual results consistently meet specification; however, it is important to note that a process is considered stable only if special cause variation is not present in the process. as a result process capability studies are arguably seen to be the most fundamental toolset available for quality-improvement purposes on a process, as summarised by marquis (2011), stating: the clinical chemist must check whether uncertainty of all his analytical methods is compatible with the tolerance required by medical needs. it is the aim of the capability index. if the uncertainty interval of a method is greater than the tolerance interval, the analytical method must be discarded or improved. (n.p.) for process capability studies to be conducted on an analytical chemistry process, it is important to determine and obtain the appropriate result measures (also known as r-criteria) from the process being studied. r-criteria represent the true process results, and as a result r-criteria are data, which are collected of overall process performance (process management international 2009). r-criteria closely track how well customer requirements are being met. this is executed by the determination of accuracy and precision of the analytical chemistry process. a controlled experiment is therefore designed and conducted in order to obtain quantitative result measures (r-criteria) or process results. a further measure known as process measures (p-criteria), represents process characteristics that are expected to affect the ‘process results’ (dahlgaard, kristensen & gopal 2002). from a lean six sigma perspective, r-criteria are considered to be quality-control points, whilst p-criteria are quality checkpoints. p-criteria are data measured or obtained from practical process steps. the state of the process may therefore be measured by a quality checkpoint or p-criteria. failure modes and effects analysis (fmea) is described as a reliability analysis tool, or a methodology to make process designs more reliable. failure modes and effects analysis is the systematic consideration of each component in a system, and involves the identification, analysis and documentation of the possible failure modes within a system, as well as the effects of each potential failure in the system (foster 2007). some of the benefits of fmea are listed below: • improvement of safety, quality and reliability • improvement of a company’s image and its competitiveness • increased satisfaction from a user standpoint • reduction in development cost • records of actions taken to reduce risk. van leeuwen et al. (2009) argue that, as a quality-tool, fmea has high improvement potential when applied to clinical laboratory processes. failure modes and effects analysis is a highly structured systematic assessment, which generates a comprehensive review to safeguard against system performance problems. failure modes and effects analysis is able to assure process and quality features, which are designed into the modified process (fmea information centre 2011). blumberg, cooper and schindler (2011:13) are of the opinion that ‘good research follows the structure of the scientific method’, from which the analogy can be drawn that the structured lean six sigma approach, which is inherently scientific and comprises distinct phases, provides a suitable platform to conduct research in a scientific laboratory environment. allen (2006) regards six sigma as: ... an organised and systematic problem-solving method for strategic system improvement that relies on statistical methods and the scientific method to make dramatic reductions in customer defined defect rates and/or improvements in key outputs variables. (p. 8) following the lean six sigma process map makes it possible for value to be identified within a process. process efficiency of both current state and future state can be determined. this drives improvement actions and tasks to a future state process. the lean six sigma process map also addresses process waste through kaizen analysis, and identifies muda (non-value activities), muri (volatile work-in-progress) and mura (un-standardised working practice during sample processing) present in a process. process standardisation is regarded as a form of process improvement (american society for quality 2011; anvari, ismail & hojjati 2011; byrne, lubowe & blitz 2008; hubbard 2010). as a result, the analogy can be drawn that lean six sigma provides a dominant and innovative tool, developed on the basis provided by the scientific method and capable of process improvement and concurrent reduction of process variation on an analytical chemistry process. many techniques have been developed for selenium analysis, including fluorimetry, electrothermal atomic absorption (et-aas), hg-aas, neutron activation analysis (naa), and different mass spectrophotometry methods. these methods include gas chromatography spectrophotometry (gc-ms), thermal ionisation mass spectrophotometry and, more recently, inductively coupled plasma mass spectrophometry (icp-ms). all of these methods (except naa and et-aas for some sample types) require a prior sample digestion to decompose organic matter before detection can occur (ducros et al. 1994). the technique of choice depends on the sample matrix, sample concentration and the type of information required (e.g. isotope or selenium species). the selenium analysis process thus predominantly involves two main sequential steps, namely, digestion of samples and thereafter, detection of the selenium in samples. any changes to improve any one part of the process cannot be made without consideration for the other, because of the complex chemical nature of the element selenium (tarin 2006). as a result of the very narrow margin between deficient and toxic levels of selenium in healthy organisms, it is crucial that any laboratory rendering the service of selenium analysis takes steps to ensure the accuracy of their diagnostic service. thus, during the development of an improved selenium analysis technique, it is important to take into consideration the specific process requirements, namely, total decomposition of organic material (chemically releasing selenium from the selenomethionine and selenocysteine form in biological samples), and the preservation of selenium yield (as selenium volatilises from samples at a temperature exceeding 70 °c). sample digestion in a closed system, such as a microwave digestion system, was believed to be capable of overcoming this challenge. for fluorometric detection of selenium, however, the oxidation state of selenium, as well as interference from nitrate ions and analyte ph which occurs as a result of microwave digestion, severely inhibits accurate selenium detection (kurkova et al. 2008). microwave digestion, as a means to overcome challenges associated with open heat-block digestion, is therefore only possible with an alternative detection method such as hydride generation or hg-aas. materials and methods top ↑ materials this research was focussed on the improvement of an analytical method, (referred to as an analytical process, in quality terms), and therefore the primary subject of research is considered to be the trace element, namely selenium, being analysed in the process under scrutiny. setting the focus of this research vested with investigating potential improvements of the digestion procedure as a means to mitigate quality concerns pertaining to the selenium analysis process employed by wcpvl. research has highlighted that microwave digestion was a suitable alternative to open-heat-block sample digestion, as microwave digestion would lead to augmented digestion, in terms of selenium yield and recovery. furthermore, under microwave conditions, higher digestion temperatures can be reached, increasing the rate of digestion. as a result, it reduces digestion time from hours to minutes. oxidising conditions are maintained stably throughout the digestion procedure, and complete digestion can be achieved with one acid, eliminating the need to mix acids and consequently reducing the risk of contamination (tarin 2006).laboratory trials conducted, returned that successful modification of the digestion method could not be accomplished without the additional modification of the detection technique. it was found that fluorometric detection of selenium was impossible in samples which had not undergone the required chemical redox reaction. the open-block digestion process enabled chemical redox reaction but microwave digestion did not return the same results; therefore, further processing of microwave digested samples was required. furthermore, fluorometric selenium detection was also inhibited by a low sample ph and excess nitric acid, which remained in samples after microwave digestion. as a result, a standard operating procedure (sop), which employed the use of both microwave digestion and hg-aas detection, was required. procedure the research method followed the lean six sigma five phases, commonly known as dmaic, representing the acronym for ‘define’, ‘measure’, ‘analyse’, ‘improve’ and ‘control’; therefore, the following research design was followed:• define(d) the problem or selenium analysis improvement opportunity phase • measure(d) the process criteria phase • analyse(d) the result criteria phase • improve(d) and optimise(d) the selenium analysis process phase • control(led) the selenium analysis process. systematic data analysis thus took place in the order which is specified by the lean six sigma process map. during the define phase of lean six sigma, the purpose of the process was established. furthermore, the extent to which the process met the requirements (customer) was defined during this phase, as well as how the process was monitored. the influence and extent of variation present in the process was also determined during this phase. research design operational implications of the define phase involved: • the identification of process steps from a process map • evaluation of a root cause analysis diagram • assignable causes of process problems were listed • pareto analysis was conducted to highlight the most critical problem(s) in the process • a detailed current state value map was drafted • the improvement opportunity to be addressed was selected. during the measure phase both types of measures which were required, namely process measures (p-criteria) and result measures (r-criteria), were collected. measures were analysed to provide an assessment of the current performance of the process, and to obtain information of changes to the process. signals of potential problems in the process were also identified through the evaluation of p-criteria. a controlled experiment was conducted to obtain r-criteria. the controlled experiment involved the processing of 10 non-probability, purposively selected liver samples from an archived population of all the previously processed samples processed by the laboratory. in addition to these, an in-house control sample was added to the sample group. all samples in this sample group were processed with the current state selenium analysis method, and replicated 13 times. furthermore, during the six trial runs, an international certified reference material (crm) sample was included to determine the deviation from trueness from the true accurate value (concentration). the confidence intervals of the crm, and the quality of the standard curve fit, served as a guideline for the determination of analytical accuracy. a blank sample, as well as a set of five standard calibration samples, was included with each process trial run. the experiment was designed so that measurement data obtained from the calibration standards served the purpose of assessing the analytical precision of the process. during the analyse phase of the lean six sigma process, the primary tools used to analyse data are inherently six sigma tools, namely statistical tools such as spc, including analysis of variation (anova) and confidence intervals, as well as regression analysis. this phase is referred to as ’listening to the voice of the process‘ (process management international 2009). the analysis and interpretation of r-criteria was thus conducted in the following order: • total regression uncertainty analysis. during the total regression uncertainty analysis, quantitative method characteristics of the standard calibration curve of the current state fluorimetry selenium analysis process were identified through the analysis of raw data r-criteria. • certified reference material analysis. the accuracy and precision of crm measurements were analysed also with respect to average linearity and characteristics established for total regression analysis of the average process standard calibration curve. • precision, analytical bias and horrat (horwitz ratio) analysis. the precision or closeness with which results of the replicated analyses of a sample agree, the bias or consistent deviation of analytical results from true value’, as well as the horrat ratio or method’s performance with regard to precision, of the current state selenium analysis process were determined. • analysis of individual standard curves of process runs. data collected from each individual process run, were analysed with statistical tools by plotting the data into scatter plots and histograms and with anova hypothesis testing. • systematic error analysis. a cochran variant of the t-hypothesis test was conducted as a means to evaluate systematic error, related to accuracy in the process. this determined whether systematic error present could be considered as statistically significant by assessing whether or not the average crm measurement obtained from process runs was different from the value stated by the reputed crm supplier. • process capability analysis. this involved statistical analysis that determined whether the analytical test process was capable of performing within the tolerance level as specified by the internationally certified crm supplier. divergent from process capability studies conducted by manufacturing quality practitioners, in the specialist area of clinical chemistry, an important factor known as total analytical uncertainty, has to be taken into account. as a result, based on literature review, process capability indices were adapted accordingly to make provision for the important laboratory consideration. during the improve phase, focus shifted to the evaluation of p-criteria once again. an identifiable characteristic of this phase involved improvement solutions being developed to address root causes of problems identified. as a result, alternative remedies were evaluated to secure a quality improvement on the process. a quality-tool known as kaizen analysis, taken from the lean bouquet of quality-tools, was the primary tool used in this phase. the improve phase provided improvement direction, and led to the redesign of the selenium analysis process. the optimise process sub-phase as employed to secure the best possible process improvement solution and this sub-phase relied on r-criteria data, in addition to p-criteria. the p-criteria were used to develop improvement remedies as part of the research conducted to improve the selenium analysis process, and r-criteria obtained from the improved designs were used to evaluate the effectiveness of the solutions developed. the following r-criteria analysis and interpretation formed part of the optimise sub-phase: • process variance analysis. statistical analysis determined whether the variance observed in the two analytical processes differed with statistical significance from each other. an independent t-test was used to compare the variance around the means of the two laboratory methods. • non-parametric difference in mean analysis. this type of analysis was performed to determine the extent of the quality improvement, as a result of the lean six sigma process modification, whereby the r-criteria process means of the two analytical processes were statistically compared. the variable of interest in this type of analysis was the difference between the values of replicate observations, rather than the values of the observations themselves. the control phase of lean six sigma utilised a feedback loop, whereby the standard performance of the improvement process was compared to actual performance, and action was taken to maintain the gains of improvements secured. in this phase, the quality-tool, fmea, was used as the final step in the approach to the improvement of the selenium analysis process of the western cape provincial veterinary laboratory. the steps of this phase were, (1) design controls and document the improved process, (2) validate the measurement system, (3) determine the final process capability and (4) implement and monitor controls. the dmaic method emphasises data analysis and fact-based decision making. the essence of the dmaic method is to reduce variation in a process in order to achieve high conformance to customer requirements. as a result, the lean six sigma methodology provides an exceptional research structure and design, as used for the purpose of improving an analytical process. ethical considerations no human or animal participants were used during research conducted for this paper, and as a result, no ethical considerations were applicable. results top ↑ the lean six sigma approach to the quality improvement of the selenium analysis process was regarded as a technique, which provided quality practitioners with a mechanism to reduce process time, whilst minimising variation and reducing process waste simultaneously. ultimate process improvement was initially directed during the initial stages as a result of analogies being drawn from the literature review, and in the latter stages, by analogies drawn from the analysis and interpretation of data obtained through the dmaic phases of lean six sigma.a schematic of results obtained during the define phase is seen in the form of an ishikawa chart (figure 1), a pareto chart (figure 2, table 2 and 3) and a current state value stream (figure 3 and table 4). the phase entailed a detailed evaluation of the current state selenium analysis process from data obtained from the process steps. the use of basic quality-tools enabled the analysis of the primary process data, or p-criteria. figure 1: ishikawa root cause analysis diagram. figure 2: pareto chart – pareto analysis: selenium analysis process. table 2: table of assignable causes of quality problems in selenium analysis process. table 3: effects of problems in selenium analysis process. figure 3: current state value stream map. table 4: results of p-criteria analysis. results obtained during the measure phase, which primarily involved the analysis of the future state value stream map has been depicted (figure 4). during this phase, additional r-criteria were collected for analysis by means of a controlled experiment. figure 4: future state value stream map. the analyse phase involved intensive statistical analysis of quantitative data, whereby the r-criteria obtained from the process were analysed. the results of total regression analysis on standard calibration curves (table 5) and crm analysis (table 6) yielded result measures known as r-criteria for further analysis. table 5: total regression analysis. table 6: certified reference material analysis. further analysis of this data included the determination of precision, bias and horrat of the process, systematic error analysis, process capability analysis, anova hypothesis testing, the analysis of control chart data, and led to the following analogies:• in the current state selenium analysis process, result quality was variable • in the current state process, accuracy and process precision could be improved • the overall current state process performance was poor • systematic error was present in the current state selenium analysis process • the current state selenium analysis process was experiencing a disparate loss in selenium yield from process run to process run. the statistical analysis of research data centred on the determination of the following: the calculation of the tstat, known as the calculated t statistic, also known as tcalc. in statistics, the t distribution is used to estimate the population mean when ơ is unknown. the tcrit, or critical value was obtained from the t-distribution table. the calculated f-statistic, or fcalc, also known as fstat was calculated. in statistics, the f-distribution is used for testing the equality between the variances of populations. the fcrit, or critical value was obtained from the f-distribution table. analysis of variation, a statistical methodology, was used to compare the means of groups. the regression ss, also known as regression sum of squares (ssr), which is equal to the sum of the squared differences between the predicted value of y and the mean of y was determined, whereas the residual ss, also known as the error sum of squares (sse), which is equal to the sum of the squared differences between the observed value of y and predicted value of y, was also determined. significant linearity exists where ssr > sse. furthermore, the regression ms was determined. the regression ms is also known as regression mean square (msr) = , where ssr is regression sum of squares or explained variation and residual ms, which is where k = number of independent or explanatory variables in the regression model. furthermore, the sy/x was calculated. the standard error of regression, also known as standard error of the estimate, or residual standard deviation or random calibration uncertainty, is a measure of the amount of error accrued in predicting a y-value for each given x-value. this statistic measures the variability of the actual y-values from the predicted y-values. it is a measure of variation around the fitted line of regression and is measured in units of the dependent variable y, and was determined. the sb, also known as standard error of slope or sb1, is a statistic that determines the sensitivity of the calibration function or the rate at which the signal changes with concentration. the sa, standard error of the intercept or sa1, is the error associated with the actual intercept point of the line. the intercept of the regression line has implications for the smallest detectable signal (measured response) and the corresponding lowest concentration. these were also calculated during statistical analysis of the research data. confidence levels (cl), is the probability that the interval estimate will include the population parameter. b, also known as the corresponding confidence level for b associated with sb (interval of the slope), was calculated by using the t-statistic for (n-2) degrees of freedom. tsb, calculated as the product of tcrit and sb, whilst a, or the corresponding confidence level for a, associated with sa (y-intercept), was calculated in the same way as that of the slope. tsa, calculated by using the tcrit and sa; the limit of detection (lod) were determined as xlod or the concentration representing lod, and ylod or the response representing the lod. the limit of quantitation (loq) was calculated where yloq, known as the signal representing the loq, was calculated. the improve phase involved the kaizen analysis of p-criteria, in addition to further r-criteria analysis of data obtained from the improved analytical method process (table 7). table 7: kaizen approach to improvement to selenium analysis process. analogies drawn from the analysis of p-criteria, which highlighted the root causes of quality problems and identified the improvement opportunities, were:• sample digestion was the root cause of problems associated with the process of selenium analysis • a value stream of activities was identifiable in the current state process • muda were identified as a form of process waste to be removed through modification • muri was identified as a form of process waste to be removed through modification • mura was identified as a form of process waste to be removed through modification • heijunka (process improvement) was possible in the form of process modification • the design of process control features assures the quality output of the modified process design. analysis that took place in the control phase of lean six sigma entailed fmea, a quality-tool, whereby p-criteria, or information on potential failure points of the improved process, were analysed (table 8). table 8: failure modes and effects analysis recommendations. in summary the key research findings were that the selenium analysis process at wcpvl was problematic in terms of delivering quality of results. the open heat-block sample digestion procedure was identified as the major root cause of quality problems; however, a modification to the digestion procedure could not be accomplished successfully without a modification in selenium detection, from fluorometric detection to hydride generation detection. the current state selenium analysis process operated at 37.5% process efficiency; however, a value stream was identifiable in the process. optimisation of the value stream to a modified process resulted in 90% potential efficiency.the current state selenium analysis process was found to be reasonably accurate and reasonably precise, but a definite systematic error is present in the process. systematic error is a known cause of poor accuracy and precision. unstandardised process steps resulted in a systematic error in the process. this process performance is unacceptable and the process requires heavy control and inspection. with a process capability index of 1.02 the process is, however, potentially capable. process variation was out of statistical control, and data analysis found evidence of disparate loss in selenium yield, as process results varied quantitatively from weekly process runs. process redesign offered a remedy in the form of a modified selenium process, assumed to produce superior results in terms of accuracy and precision. modified process design enabled control features to be built into the design. trustworthiness top ↑ reliability of analytical data in order to ensure reliability of analytical data during the research process, scientific measurements are not reported as single values, but rather as ranges or averages with estimates of error surrounding a value after repeated measurements of the value. the statistical standard deviation of the range of measurements obtained from 13 repeated trial runs was tabulated and used to compute a confidence interval around research results. confidence intervals provide an estimate of the probability that a similar result will be found if the study is repeated. validity of analytical data according to capri and egger (2003) ’uncertainty is inherent in scientific research‘. an important requirement of scientific research is, therefore, to identify and quantify uncertainty and to assess the reliability of scientific data. research data obtained and used for the purpose of this study were consequently statistically analysed, and uncertainty was determined according to a technique known a total regression analysis as proposed by fraser (2011).data error was categorised as random error or systematic error. random error is derived from unpredictable variation, and therefore cannot be controlled. reducing uncertainty in the system, however, helps to correct systematic error as well as to minimise random error. increasing the amount of observations of a measurement is considered to be one avenue of reducing uncertainty, although uncertainty may never be reduced to zero (carpi & egger 2003). as a consequence of error, scientific measurements were not reported as single values, but rather as ranges, or averages with estimates of the error surrounding the value, after repeated measurement of the value. all analytical data for the research conducted were reported with confidence levels to account for error, as well as the associated confidence limits, to reflect the precision of statistical estimates computed during research. furthermore, the inclusion of crm analysis conducted as part of the research process also supports the validity of research data results. discussion top ↑ outline of the results as a means to prove or disprove the research hypothesis, which reads, ‘variation in process, time and control procedures have a direct impact on the disparity in selenium testing results’, the study focussed on the stated research objectives. in alignment with the primary research objective, which was set ‘to establish an alternative more accurate and safer digestion procedure within the selenium analysis process in order to attain quality improvement of the process’; suitable quality-tools were selected and used in a very precise manner to realise this objective. this practical demonstration of the successful use of quality-tools in a lean six sigma framework, in a clinical chemistry research environment, enabled process improvement through the identification of significant quality risks to the process being studied. the outcome of this approach was a modified sop, which inserts functional practical benefit to the operational system in the biochemistry laboratory.a secondary objective ‘to determine if a modification of the existing hot plate digestion to microwave digestion is capable of reducing the risks associated with the digestion procedure’, was stated. the result of the process capability studies during lean six sigma highlighted that the elimination of quality risks, such as process variation, results in an improved analytical process. the literature review (desert research institute 2004; university of alberta, department of environmental health and safety 2011) highlighted that the use of perchloric acid is hazardous to the process operator and to the environment. as a result, a modification of the selenium analysis method away from the use of perchloric acid may be regarded empirically as a form of process improvement. furthermore, the use of the fmea quality-tool culminated in further functional practical value to the biochemistry operational system by ensuring that risks associated with the modified selenium analysis method, were minimal. key research findings which focussed on the secondary objective stated as ‘determining if a modification of the existing digestion method will result in the optimisation of selenium yield of the process’, was generated from result data, which served as evidence of the disparate loss of selenium yield during the current state selenium analysis process. this selenium loss was attributed to process variation, and a standardised modification of the selenium analysis process overcomes this quality problem. foster (2007) contends that the improve, or kaizen, phase of lean six sigma involves off-line experimentation, whereby the factors affecting process performance are analysed. kurkova et al. (2008) stated that, at temperatures exceeding 70 °c, organic forms of selenium volatilise from samples. optimisation of the process to microwave digestion and hg-aas detection was therefore regarded as a quality improvement of the process. the final research objective stated, namely ‘to determine whether a modification of existing digestion procedure will ultimately translate into an improvement in terms of reliability of results’, had clear tangent planes to the independent variable of the research hypothesis, that is, quality improvement and laboratory results. key findings, based on the analysis of research data, were that both accuracy and precision of the current state selenium process would be improved by a modified selenium analysis process. both process capability studies and process performance studies conducted supported this finding, which further emphasised the importance of this practical improvement to biochemistry laboratory processes. reliability of an analytical method is the ability of the method to be both accurate and precise. therefore, an improvement in both accuracy and precision with the modified selenium analysis process, would ultimately improve the reliability of the analytical process, and thereby the quality of the process. practical implications the significant benefit gained from this research, has been the practical value secured by the improvement of an analytical biochemistry process in a very demanding diagnostic environment. an improvement in terms of process efficiency and reliability renders valuable resources available for other activities required in the section, such as laboratory technologist time, and equipment resources. furthermore, by reducing cycle time, results can be produced by the section in a reduced time period, ensuring reliability of diagnoses. limitations of the study results from this research were reflective of normal operating conditions of wcpvl and is not a reflection of process or system operation during emergency situations that may occur, such as an outbreak of a controlled disease. recommendations the following recommendations were made as a result of this research study:• the current state selenium analysis process must be modified in order to assure the reliability of process results • process modification is necessary to address process variability that resulted from process steps, time and control procedures in the process, which were found to be responsible for disparate results • process modification from open heat-block to microwave digestion is recommended • process modification from fluorometric selenium detection to hydride generation detection is recommended • it is recommended that the modified process be monitored, and that continuous evaluation take place according to the lean six sigma cycle to maintain a focus on continuous improvement of the selenium analysis process. conclusion top ↑ measurable benefit can be attained from research that has been adequately constructed and designed, with a clear and consistent focus on the desired research objectives. conclusions were drawn from key research findings in response to each of the research objectives. process standardisation in terms of time and stricter temperature control in a closed digestion system is regarded as capable of overcoming process variation. from this statement, it follows that a modified process design, focussed on both sample digestion and detection, is capable of overcoming unacceptable systematic error process variation and provide the process with better control features. furthermore, a modified selenium analysis process, which includes a modified sample digestion procedure, will result in the reduction of biohazard and other risks associated with the selenium analysis process, and the assumption is made that a modification from open heat-block digestion will optimise the selenium yield of the analytical laboratory process.in addition, a modified process design would ultimately translate into an improvement in quality in terms of the reliability of process results. on the basis of conclusions drawn from key research findings, it can be stated with confidence that: there is significant evidence to accept the stated null-hypothesis, and it was found that variability of the dependant variable of the research hypothesis, associated with the process, had a significant and detrimental impact on the independent variable of the research hypothesis, namely, the result quality of the selenium analysis process. (cloete 2011, n.p.) as a result, the research hypothesis was accepted as true. acknowledgements top ↑ the research was funded by the department of agriculture of the western cape. tremendous thanks are extended to the management and staff at wcpvl for their support and assistance during the research process. the authors would like to thank both prof. dr j. andré watkins and mr andré bester for their mentorship and supervision. competing interests the authors declare that there are no competing interests that may have inappropriately influenced the presentation of information in this paper. authors’ contributions b.c.c. (western cape provincial veterinary laboratory) is a m. tech (quality) student at the cape peninsula university of technology and conducted the research. a.b. 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ahmadu bello university, nigeria correspondence to: minka salka postal address: private bag 2134, d.a.c., ahmadu bello university, mando-kaduna, nigeria dates: received: 09 dec. 2011 accepted: 07 mar. 2012 published: 24 may 2012 how to cite this article: salka, m.n. & olusegun, a.j., 2012, ‘modulatory effect of ascorbic acid on physiological responses of transported ostrich chicks’, onderstepoort journal of veterinary research 79(1), art. #388, 7 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.388 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. modulatory effect of ascorbic acid on physiological responses of transported ostrich chicks in this original research... open access • abstract • introduction • materials and methods    • thermal environmental and rectal temperature data    • experimental birds and measurements    • vehicle loading and transportation procedures    • statistical analysis    • ethical considerations       • potential benefits and hazards • results    • thermal conditions and rectal temperature during transportation    • aberrant behaviours    • heterophil to lymphocyte ratio • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the aims of this study were to determine the modulating role of ascorbic acid (aa) on rectal temperature (rt), heterophil to lymphocyte (h to l) ratio and aberrant behaviours of ostrich chicks transported by road for 4 h during hot-dry conditions. twenty ostrich chicks aged 2.5 months, of both sexes and belonging to the red neck breed, served as subjects of the study. the chicks were assigned randomly to aa-treated and control groups, consisting of 10 chicks each. the aa-treated group was administered orally with 100 mg/kg body weight of aa dissolved in 5 ml of sterile water 30 min before transportation, whilst the control group was given the equivalent of sterile water only. the thermal load (tl) experienced in the vehicle during transportation fluctuated between 31 °c and 89 °c, as calculated from the ambient temperature and relative humidity. transportation induced hyperthermia, lymphopenia, heterophilia and aberrant behaviours of pecking, wing fluffing and panting, which were ameliorated by aa administration. the relationships between the tl, journey duration and physiological variables of rt, h to l ratio and aberrant behaviours recorded during transportation were significantly and positively correlated in the control group. in aa-treated group the relationships were not significantly correlated. in conclusion, the results showed for the first time that aa ameliorated the adverse effects of stress caused by road transportation on the aberrant behaviours, rt and h to l ratio of ostrich chicks during the hot-dry season. introduction top ↑ transportation of animals is known to induce both psychological and physiological stress and stress-induced behavioural changes may result in accompanying major physiological disturbances such as aberrant behaviours, immune-suppression, weight loss and disruption of general homeostasis (ayo et al. 2009; minka & ayo 2008, 2010; stewart 1994). it has been established that changes in rectal temperature (rt), heterophil to lymphocyte ratio (h to l) and behavioural activities of animals, including birds, are reliable indices of stress and are often used in the development of management strategies aimed at increasing productivity. these changes are important for on-the-spot evaluation of the health status and adaptability of animals to various stress factors, including transportation stress (ayo, oladele & fayomi 2002; minka & ayo 2007a, 2007b, 2008; warriss et al. 1993). very often, behavioural changes are the main signs of distress and the first signs of disease in animals (ayo et al. 2002).the high levels of stress suffered by ostriches during road transportation has been sharply criticised by human rights and animal welfare activists because this transportation poses a great risk to both the driver and the birds (foggin 1992; hoffman & lambrechts 2011; minka & ayo 2008; wotton & hewitt 1999). the lack of adequate recommendations relating solely to the welfare of ostriches during road transportation has aggravated the problem (ministry for agriculture, fisheries and food 1998; minka & ayo 2008; piccione et al. 2005). for example, in nigeria, ostriches are mostly purchased and transported by road from the northern guinea savannah zone, where they are predominantly reared, to new farms, markets, zoos and game reserves in other parts of the country (ayo & minka 2004; minka 2003; minka & ayo 2007c, 2008). this road transportation covers distances ranging from 200 km to over 1000 km and uses vehicles specifically not meant for transportation of ostriches, which demonstrates little consideration for the ostriches’ welfare. in spite of the management procedures toward alleviation of transportation stress in ostriches, which centred solely on vehicle modification and design, reports indicate that the birds still suffer considerable stress during transportation. in addition, the use of neuroleptics and other central nervous depressants resulted in many ostriches developing rhabdomyopathy. besides, the necessity of using such drugs in transported animals is an indication that those animals’ welfare is already poor and compromised (pfitzer & lambrechts 2001). it has, therefore, become necessary to search for safer agents that may alleviate the stresses ostriches suffer during their transportation by road without contravening their welfare and productivity. several studies have successfully employed ascorbic acid (aa), otherwise known as vitamin c, as an ameliorating agent against road transportation stress in livestock, including poultry, because of its antioxidant and inhibitory properties on free radicals. the effects of stress are also apparently reduced, as evidenced by lower cortisol levels (ayo et al. 2009; minka & ayo 2007b, 2010, 2011). however, such studies in ostriches are currently lacking in the available literature. therefore, the aims of the present study were to evaluate the effects of a 4 h road transportation on thermal load (tl), behaviour, rt and immunosuppression of ostrich chicks and to suggest aa as an ameliorating agent in ostrich chicks transported by road during hot-dry conditions. materials and methods top ↑ thermal environmental and rectal temperature data the present study was conducted at the college of agriculture and animal science, kaduna, which is located at 11°10′ n, 07°38′ e in the northern guinea savannah zone of nigeria. the ambient temperature (at), relative humidity (rh) and rt were recorded at the sites of the study for seven days before and after transportation, at 07:00, 14:00 and 18:00 each day. these parameters were recorded hourly during the journey. the at and rh were recorded using a wet-bulb and dry-bulb thermometer (thermometers direct ltd, aldershot, united kingdom). the rh was obtained by calculating the depression point from the at and wet-bulb temperature values. the temperature humidity index (thi), a measure of tl, recorded during the transportation period was obtained from the at and rh using a thi calculator. the rt was recorded with a digital clinical thermometer (cocet, kangfu medical equipment co., zhejiang, china), inserted about 5 cm into the rectum via the cloaca for 3 min until an alarm was heard, indicating the end of the reading. experimental birds and measurements twenty apparently healthy, 2.5-month-old farmed ostrich chicks, belonging to the red neck breed and of both sexes, served as subjects. the birds weighed 20.7 kg on average and were obtained from a standard commercial farm, located in kaduna in the northern guinea savannah zone of nigeria. the chicks were fed ostrich growers’ mash (feed master ltd, kaduna, nigeria) with a nutritive value of 2465.0 kcal/kg metabolised energy, 22% protein and 12% fibre. they were given access to water ad libitum in both the farms from which and to which they were transported. one week before transportation, the birds were assigned randomly into aa-treated and control groups, consisting of 10 chicks each. blood samples were obtained from the ostriches two days prior to transportation in order to obtain base-line (pre-transportation) values. on transportation day and 30 min before loading, the aa-treated group was given 100.0 mg/kg body weight of aa orally, dissolved in 5 ml of sterile water, whilst control group was given the equivalent of sterile water only. the 30 min time lapse from aa administration to loading was allowed for the absorption of the aa before the commencement of the journey. the rt values of the ostriches were recorded 30 min before and after loading and subsequently every hour of the transportation. during transportation, the behavioural events of individual chicks were recorded continuously, as described by das, srivastava and das (2001) and minka and ayo (2010). the behaviour was recorded by four observers inside the vehicle, with each observer recording the on-going behaviour of five chicks in a similar pattern. the number of ostriches that exhibited behavioural events such as panting (hyperventilation), wing fluffing and pecking were recorded whenever they were observed for each hour of the transportation period. blood samples were obtained immediately on arrival and on the third day after transportation. at each period of blood sampling 5.0 ml of blood was taken from a wing vein into a heparinised test tube. monolayer blood films were made from each sample, fixed and stained for differential leucocyte counts (dacie & lewis 1975). results for heterophils (h) and lymphocytes (l) are presented. the h to l ratio was calculated by dividing the value of h by that of l. vehicle loading and transportation procedures the ostriches were transported in a modified bedford van. the floor of the van, which had no holes or gaps, was covered with chopped wood shavings, on top of which was placed a non-slip rubber mat. the windows were covered with grills that permitted the regulation of ventilation. the van was partitioned into 10 compartments with detachable and perforated plastic materials measuring 0.70 m high. each compartment housed two ostrich chicks comfortably and with enough space for them to lie down. although recommendations on stocking density for ostrich chicks during transportation are lacking (wotton & hewitt 1999), in the present study the chicks were stocked at a density of 0.35 m2 per chick. each partition was fixed in place after loading each ostrich into the vehicle. similarly, the partitions were detached separately during unloading, starting from the rear of the van and moving to the front. transportation was undertaken during the hot-dry period of april. the vehicle travelled for 4 h – from 08:00 to 12:00 – covering a distance of 220 km on an asphalt road, with an average speed of 50 km/h. food and water were withdrawn completely 6 h before and during the journey period. on arrival, the ostriches were immediately unloaded individually into a pen, where they were fed the same ration given to them before the transportation. statistical analysis the mean and standard error of different behavioural events were calculated. data were subjected to the student’s t-test, whilst pearson’s correlation analysis was employed in calculating the relationships between the journey periods, at and behavioural activities. values of p < 0.05 were considered significant. ethical considerations the handling, loading and transportation of the ostrich chicks were in accordance with national and international guidelines on humane handling and transportation of animals and birds (farm animal welfare council 2003; federal republic of nigeria 1988; maff 1998) and as described by sales and smith (1995) and wotton and hewitt (1999). the college of agriculture and animal science at the ahmadu bello university in kaduna, nigeria approved all of the protocols required for the study period, under the permit number vcn-002519. potential benefits and hazards both physical and psychological trauma during transportation were anticipated because of the bi-pedal nature of the ostriches, which often leads to traumatic injury, fracture and rabdomyolopathy. thus, the birds were transported as recommended by the national and international guidelines cited above. to ensure the safety of the birds, the primary author, a veterinary surgeon registered with the veterinary council of nigeria, participated fully in the transportation of the birds. results top ↑ thermal conditions and rectal temperature during transportation the at, rh and tl inside the vehicle rose with each hour of the journey, attaining their peak at the fourth hour when the transportation was completed (table 1). the rt recorded in aa-treated ostrich chicks 30 min after aa administration and just before loading rose (p < 0.05) from the mean base-line value of 38.5 °c ± 0.2 °c to 39.6 °c ± 0.1 °c. thereafter, the rt of the aa-treated birds declined and fluctuated between the values of 38.5 °c and 39.0 °c during the post-loading and transportation periods and did not differ (p > 0.05) from the base-line values. in the control group, the rt values recorded after loading and during each hour of the transportation period fluctuated between 38.3 °c and 40.1 °c, significantly higher than the base-line values and corresponding values obtained in the aa-treated ostriches (figure 1a). table 1: fluctuations in thermal microenvironment variables inside the vehicle during a four-hour road transportation of ostriches. figure 1: the effects of loading and transportation on, (a) rectal temperature, (b) the exhibition of piloerection behaviour, (c) the exhibition of panting behaviour and (d) the exhibition of pecking behaviour of control and ascorbic acid (aa)-treated ostriches. aberrant behaviours the aberrant behaviours of piloerection (figure 1b), panting (figure 1c) and pecking (figure 1d), recorded in the control ostriches increased (p < 0.05) with the journey duration and tl; these behaviours manifested apparently during the last hours of the journey when the thermal environment variables were at their peak. in aa-treated ostriches, panting was the only aberrant behaviour that significantly (p < 0.05) manifested at the last hour of the journey. heterophil to lymphocyte ratio the h to l ratio of 0.67 ± 0.30 recorded in the control chicks immediately after transportation was higher (p < 0.05) than the base-line value of 0.33 ± 0.10 and the corresponding value of 0.34 ± 0.10 recorded in aa-treated chicks after transportation. the h to l ratio recorded in the aa-treated group immediately and three days after transportation was not different from the base-line ratio. however, the control chicks had still not returned to the base-line h to l ratio three days after transportation (table 2). table 2: responses of heterophils and lymphocytes of ascorbic acid-treated (n = 10) and control (n = 10) ostriches to road transportation stress. the relationships between the tl, journey duration and physiological variables of rt, h to l ratio and aberrant behaviour recorded during the transportation were significantly and positively (p < 0.001; r = 0.814) correlated in the control group. in aa-treated group the relationships were not significantly correlated (table 3). table 3: relationships between the hour of the journey, thermal load, rectal temperature and aberrant behaviours in ascorbic acid-treated (n = 10) and control (n = 10) ostriches during the road transportation period. discussion top ↑ the results obtained in the present study showed that the transportation of ostrich chicks during the hot-dry season is very stressful and poses a risk to the health of the birds, findings which agreed with previous reports (foggin 1992; minka & ayo 2007c, 2008; mitchell et al. 1996). the fact that the ostrich is known to withstand very high environmental temperatures without any consequential adverse effects on its homeostasis (stewart 1994) suggests that the mean at value of 35 °c – recorded during transportation in the present study, and which was far below the temperature (50 °c) at which ostriches begin to experience adverse effects to their homoeostasis – may not be a major cause of transportation stress in the ostriches. this may be true because, unlike mammals and other birds, ostriches have excellent ability to thermoregulate under high at. however, during transportation, they suffer considerable stress when the at values are either above or below 20 °c (payne 1993; piccione et al. 2005), which is the recommended thermoneutral zone for ostriches (skadhauge 1981).the mean tl value of 51.0 °c ± 7.8 °c and at value of 33.6 °c ± 1.1 °c recorded during the journey were similar to the tl and at values of 44.0 °c and 34.0 °c, respectively, obtained during transportation of pullets, and which were reported to induce heat stress in pullets (minka & ayo 2010). the fact that the tl increased with the hour of the day and journey duration suggests that as the journey progressed the effect of tl on the birds also rose. in addition, panting behaviour of the birds increased the rh in the vehicle, which in turn increased tl. this apparently exacerbated the mechanism of thermoregulation by increasing evaporative heat loss and inducing hyperthermia in the ostriches. other studies have shown that even at an at of 18.0 °c and rh of 89% ostriches still suffer transportation stress (payne 1993). it appears therefore that other transportation factors such as handling, loading, vehicle vibration, motion, noise and novelty of environment may be the major stress factors affecting the ostrich during transportation, rather than high at and rh. this requires further investigation. the base-line values of rt recorded in both treated and control ostriches before transportation did not differ from each other and from the normal range rt values of 38.3 °c to 39.3 °c established for ostrich chicks (skadhauge 1981; stewart 1994), although the rts were at their lower limit values. the results were similar to those obtained by stewart (1994), who showed that ostriches, in contrast to other birds, have an rt below 40.0 °c and in the range of the eutherian mammals. the increase in the rt value recorded in aa-treated group 30 min after the administration of aa, but before loading, was within the normal range for the ostrich, but was higher than that of the control, which demonstrated that administration of aa induced rt elevation in the ostriches. this finding supports earlier results that aa exerts a biphasic effect on rt. firstly, it increases the rt whenever the value is at a lower limit threshold as observed in aa-treated ostriches in the present study. secondly, it reduces rt values that are at the maximum threshold (ayo, minka & fayomi 2005; ayo, owoyele & dzenda 2007). the increase in rt values recorded in the control group 30 min after handling and loading, but before the transportation, showed that these procedures were stressful to the ostriches. similar observations were made during the handling, loading and transportation of birds (minka & ayo 2007b, 2011). the increase in rt value at this stage of transportation may not be associated with an increase in tl, but could be explained as a result of physical activity; and also by stress-induced hyperthermia, which is the regulated shift of the thermoregulatory set-point mediated by prostaglandin e and interleukin 1 and 6 (kent & ewbank 1983; piccione et al. 2007). stress-induced hyperthermia is associated with cortisol release and is known to attain peak level within 10 min of administration of a stress factor (piccione et al. 2007). the fact that a non-significant (p < 0.05) increase in rt value was recorded post-loading in the aa-treated ostriches suggested, for the first time, that aa alleviated the effects of handling and loading stress on rt of the ostriches. the mechanism of action of aa on the handling and loading stress factors may be caused by the fact that aa is an inhibitory vitaminergic neurotransmitter in the hypothalamus, which plays an important role in thermoregulation by inhibiting cortisol, the chief hormone of stress (balz 2003; kannan et al. 2002). this is an important finding because handling and loading of ostriches are the most critical procedures and the major cause of poor welfare (minka & ayo 2007c, 2008). the progressive increase in rt value during the transportation, especially in the control birds, suggested that transportation itself, apart from the handling and loading procedures, further increased the release of cortisol, which apparently continued to alter rt. the present finding agrees with those of knowles et al. (1996), hartung (2003) and minka and ayo (2008), who established that the period of transportation of an animal is one of the most traumatic in its life. this is particularly true of ostriches, as they are very social birds and relatively minor changes in their social status may cause severe stress (stewart 1994). the effects of stress caused by road transportation acting concomitantly with high at and rh on the ostriches in the vehicle further aggravated the stressful conditions of the birds. the insignificant (p > 0.05) change in rt value recorded in the aa-treated ostriches, compared to those obtained in the control, further indicated that aa did not only ameliorate the stress imposed by handling and loading, but also the stresses encountered during the actual process of road transportation. such stresses included vehicle motion, noise, vibration and speed, high at and rh, long duration of journey, deprivation of food and water, novelty of environment, and confinement. the findings of the present study demonstrated that aa reduced significantly the stress on rt induced by road transportation. the results were similar to those obtained during transportation of ostriches and poultry by road (minka & ayo 2008, 2010). similar ameliorating effects of aa on the rt of pullets have been documented (minka & ayo 2010, 2011). the behavioural responses recorded during the third hour of transportation revealed some unusual and aberrant behaviours such as pecking (directed to the ground or to objects), wing fluffing (piloerection) and panting (polypnoea), especially in the control ostriches. the manifestations of these behaviours suggest that the ostriches were stressed and the level of stress increased with the duration of the journey and tl. although ostriches are known to have a wide range of thermoregulatory set points, it has been reported that ostrich chicks are affected severely by hot weather (minka & ayo 2007c; reiner, seitz & dzapo 1996; stewart 1994), as confirmed by the present study. the aberrant behaviours of polypnoea and piloerection were responses of the birds to increased demand in oxygen and heat dissipation brought about by the high at and rh recorded in the vehicle during the transportation. because ostriches, like other birds, have no sweat glands, the only means available for cooling is evaporation via polypnoea and piloerection by exposing the bare skin on their thorax and upper legs to enhanced convection and radiation losses. similar observations on the behaviours of ostriches exposed to different thermal conditions have also been reported (samson 1996). although the vehicle cabin was well ventilated, the air entering the vehicle was very hot and did not exert any cooling effect on the birds. this is characteristic of the hot-dry season in the zone of study. the present result showed that ostriches began to pant when the tl value was 40 °c. the result obtained in aa-treated birds demonstrated that aa has reduced the effect of tl on the birds and facilitated the physiological mechanism of thermoregulation. it has been established that aa decreases heat load through reduction in heat production or increasing heat loss by enhancing thermal exchange between the body and the environment (chang et al. 1993; minka & ayo 2010; tauler et al. 2003). similarly, aa enhanced the adaptability of poultry and animals to environmental stress (mckee & harrison 1995; minka & ayo 2010). the mechanism of action of aa in reducing heat stress may also be through the detoxification of reactive oxygen species known to be in abundance in the body during stressful conditions (minka & ayo 2007b; tauler et al. 2003). the intense pecking behaviour exhibited by the ostriches in the control group may be associated with frustration, hunger and metabolic stress caused by transportation procedures, which are tasking and completely novel to the birds (foggin 1992). the fact that pecking behaviour was less manifested in aa-treated birds suggested that aa reduced the bird’s frustration. aa is known to be the first vitaminergetic neurotransmitter and it plays a significant role in inhibiting cortisol, the chief hormone of stress, and the limbic region involved in the elicitation of fear and frustration (balz 2003; karanth et al. 2000). the significant (p < 0.05) increase in h to l ratio recorded in the control ostriches post-transportation indicated that the immune system of the birds was compromised as a result of transportation up to the third day of the post-transportation period. the h to l ratio has been used as the most common reliable index of stress measurement from blood analysis (kannan et al. 2002; minka & ayo 2007b, 2011). a similar effect of transportation stress on h to l ratio was reported in poultry, including ostriches (minka & ayo 2007b, 2008, 2011) and the increase in h to l ratio has been reported to reflect the effects of elevated corticosteroids in the circulation induced by stress (kannan et al. 2002; schalm, jain & carroll 1975). the insignificant change in h to l ratio obtained in aa-treated ostriches post-loading was an indication of reduced physiological stress. the result showed, for the first time, that aa alleviated the stresses induced by both the transportation proper and the post-transportation procedures on the lymphocytes and heterophils of the ostriches. aa has been reported to be a chain-breaking antioxidant, involved in the prevention and restriction of free-radical chain formation and propagation. consequently, aa enhances the immune status of the ostriches by protecting the blood cells, including heterophils and lymphocytes, from oxidative damage induced by free radicals and cortisol (alok et al. 2003; elsayed, farghaly & abu-taleb 2008; tauler et al. 2003). although there are no recommended doses for aa as an antioxidant in ostriches, it has been shown that it ameliorates heat stress in ostriches in supplementation, at a dose of 250 mg/kg of feed (elsayed et al. 2008). conclusion top ↑ the results of the present study have demonstrated for the first time that aa administration prior to the commencement of road transportation was beneficial to ostrich chicks and may be used to improve the bird’s welfare and make their transportation more economically viable. for example, aa ameliorated the adverse effects of stress caused by road transportation on the behaviour, rt and h to l ratio of ostrich chicks. it also enhanced the homeostatic mechanisms of behavioural, thermoregulatory and cellular immune responses of ostrich chicks to the stressful conditions imposed by road transportation during the hot-dry season. aa is readily available, cheap, safe and easy to administer in ostrich chicks, making it an ideal supplement for use during transportation. acknowledgements top ↑ the authors are grateful to all laboratory staff of the college of agriculture and animal science at the ahmadu bello university, kaduna for their technical assistance. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions m.n.s. 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219–227. reiner, g., seitz, k. & dzapo, v., 1996, ‘a survey of farming environment and ostrich behaviour’, in d.c. deeming (ed.), improving our understanding of ratites in farming environment, ratite conference proceedings, 27–29 march, university of manchester, uk, pp. 52–56. sales, j. & smith, w.a., 1995, incubation and management of ostrich and ostrich products, university printers, stellenbosch. samson, j., 1996, ‘behavioural problems in farmed ostriches in canada’, canadian veterinary journal 37, 412–414. pmid:8809393, pmcid:1576451 schalm, o.w., jain, n.c. & carroll, e.j., 1975, veterinary haematology, 3rd edn., lea and febiger, philadelphia. skadhauge, e., 1981, osmoregulation in birds, springer-verlag, berlin. stewart, j.s., 1994, ‘ostrich behaviour and behavioural problems’, proceedings of the annual conference of avian veterinarians, 28–30 september, reno, usa, pp. 104–109, association of avian veterinarians, denver. tauler, p., aguilo, a., gimeno, i., fuentespina, e., tur, j.a. & pons, a., 2003, ‘influence of vitamin c diet supplementation on endogenous antioxidant defense during exhaustive exercise’, european journal of physiology 446, 658–664. warriss, p.d., kestin, s.c., brown, s.n., knowles, t.g., wilkins, l.j., edwards, j.e. et al., 1993, ‘the depletion of glycogen stress and indices of dehydration in transported broilers’, veterinary record 149, 391–398. wotton, s.b. & hewitt, l., 1999, ‘transportaton of ostriches – a review’, veterinary record 145, 725–731. pmid:10972110 article information authors: christopher j. kasanga1 t. yamaguchi2 h.m. munang’andu3 p.n. wambura1 k. ohya4 h. fukushi4 affiliations: 1department of veterinary microbiology and parasitology, sokoine university of agriculture, tanzania 2faculty of agriculture, tottori university, japan 3department of paraclinical sciences, university of zambia, zambia 4faculty of applied biological sciences, gifu university, japan correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this abstract: kasanga, c.j., yamaguchi, t., munang’andu, h.m., wambura, p.n., ohya, k. & fukushi, h., 2012, ‘genomic sequence of infectious bursal disease virus from zambia suggests evidence for genome re-assortment in nature’, onderstepoort journal of veterinary research 79(2), art. #473, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.473 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. genomic sequence of infectious bursal disease virus from zambia suggests evidence for genome re-assortment in nature in this abstract... open access infectious bursal disease virus (ibdv) is a bi-segmented rna virus, which belongs to the genus avibirnavirus of the family birnaviridae. two serotypes, 1 and 2, exist in ibdv. the serotype 1 ibdvs are the causative agents of infectious bursal disease (ibd) in chickens worldwide and lead to immunosuppression in young birds. genome re-assortment has been speculated to occur and contribute to the emergence of new ibdv strains. however, evidence was lacking until recently when two re-assortant viruses were detected in china. in this study, we determined the complete nucleotide sequence of an ibdv, designated kzc-104, from a confirmed natural ibd outbreak in lusaka, zambia in 2004. the genome consisted of 3074 and 2651 nucleotides in the coding regions of segments a and b, respectively. alignment of both nucleotide and deduced amino acid sequences, and phylogenetic analysis revealed that the genome segment a of kzc-104 was derived from a very virulent strain, whereas its segment b was derived from a classical attenuated strain. on blast search, the full-length segments a and b sequences showed 98% closest nucleotide homology to the very virulent strain d6948 and 99.8% closest nucleotide homology to the classical attenuated strain d78, respectively. this is a unique ibdv reassortant strain, which has emerged in nature involving segment b of a live attenuated vaccine. this observation provides direct evidence for the involvement of vaccine strains in the emergence of reassortant ibdv in the field. taken together, these findings suggest an additional risk of using live ibdv vaccines, which may act as genetic donors for genome re-assortment. further studies are required to investigate the epidemiology and biological characteristics of reassortant strains so that the appropriate and safe ibdv vaccines can be recommended. article information authors: maryke l. ferreira1 annemariè avenant-oldewage1 affiliations: 1department of zoology, university of johannesburg, south africa correspondence to: annemariè avenant-oldewage postal address: po box 524, auckland park 2006, south africa dates: received: 29 oct. 2012 accepted: 06 feb. 2013 published: 28 mar. 2013 how to cite this article: ferreira, m.l. & avenant-oldewage, a., 2013, ‘notes on the occurrence of trypanosoma sp. (kinetoplastida: trypanosomatidae) in freshwater fishes from south africa’, onderstepoort journal of veterinary research 80(1), art. #529, 4 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.529 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. notes on the occurrence of trypanosoma sp. (kinetoplastida: trypanosomatidae) in freshwater fishes from south africa in this research communication... open access • abstract • communication    • ethical considerations • acknowledgements    • competing interests    • author’s contributions • references abstract top ↑ a total of 257 fishes from four families, clariidae, cichlidae, cyprinidae and schilbeidae were collected from three localities: the sand river dam, swaziland; the nylsvlei nature reserve, south africa and the vaal dam and vaal river barrage, south africa. only fishes (n = 154) from clariidae and cichlidae were found to be infected with trypanosomes. a total of 221 clarias gariepinus (burchell 1822) were collected from the vaal dam and vaal barrage area, south africa. of these, 74% (89/121) were infected with trypanosomes from the vaal dam and 63% (63/100) from the vaal river barrage, with no seasonal infection pattern. a prevalence of 25% (1/4) was found in c. gariepinus from the sand river dam, swaziland, and a 50% (1/2) prevalence was found in tilapia sparrmanii from the nylsvlei nature reserve, south africa. standard measurements conformed closely to the morphometric and morphological descriptions of trypanosoma mukasai. this article provides new locality records for t. mukasai from the vaal dam, vaal river barrage and nylsvlei nature reserve (south africa) and the sand river dam (swaziland). tilapia sparrmanii collected in the sand river dam in swaziland is also noted as a new host record. communication top ↑ since the first piscine trypanosome was discovered from the blood of salmo fario by valentin in 1841, more than 190 trypanosome species have been recorded from around the world (woo 1998). several authors have recorded piscine trypanosomes from many of the major freshwater systems of africa (baker 1960, 1961), however, only three known species are noted from africa: trypanosoma toddi bouet, 1909; trypanosoma mukasai hoare, 1932 and trypanosoma tobeyi dias, 1952. baker (1960) noted that nuclear position might be an important factor in separating the three african freshwater fish trypanosome species. it was noted that small morphological differences separate t. toddi from t. tobeyi and that t. mukasai and t. tobeyi might prove to be a synonym of the same trypanosome (baker 1960). in 1962, pienaar reported trypanosoma clariense from clarias gariepinus (burchell, 1822) in the north west province (pienaar 1962). this species was first described by fantham (1919) from the same host. it was suggested by smit, van as and davies (2004) that t. clariense might be a synonym of t. mukasai, owing to the apparent common staining properties and morphometrics. trypanosoma mukasai was originally described from four species of cichlids (hoare 1932) and has since been known to infect a broad range of fish hosts across africa (negm-eldin 1997, 1998). some of the most recent records are those of davies and van as (2000), smit, van as and davies (2004), smit, moll and van der bank (2008) and hussein et al. (2010). according to smit, van as and davies (2004), morphometrically variable haemoflagellates were found within a single host from the okavango delta, botswana. however, despite these findings, they concluded that all okavango trypanosomes examined might be t. mukasai due to their nuclear indices ni ˃ 1. since these findings, davies et al. (2005) reported that two genotypic groups of trypanosomes (even though morphologically similar) are present in the fish from the okavango. a great deal of molecular research has been done in recent years regarding the phylogenetic relationships of trypanosomes (davies et al. 2005; gibson et al. 2005; gu et al. 2007, 2010; jakes, o’donoghue & adlard 2001; karlsbakk, haugen & nylund 2006; martin et al. 2002; seghal, jones & smith 2001; votypka et al. 2002; votypka, lukes & obornik 2004). the aim of this article is to increase the distribution and host data of a species of trypanosome preliminarily identified as t. mukasai.fishes were collected using fishing rods from the sand river dam (25° 59′ 16″ s, 31° 42′ 16″ e) (channelled from the inkomati river), swaziland (february 2012) and with gills nets from the nylsvlei nature reserve (24° 39′ 17.28″ s, 28° 41′ 27.6″ e), limpopo province, south africa (may 2012); vaal dam (26° 53′ 40.99″ s, 28° 8′ 43.98″ e) and vaal river barrage (26° 45’ 53.28” s, 27° 41’ 30.12” e) from the vaal river system, south africa (november 1998 to february 2000 and february 2012). fish were identified (see table 1) and blood films were prepared, air dried, fixed with methanol and stained with phosphate-buffered giemsa solution prior to screening (davies et al. 2005). microscopic images of the blood protozoans were captured and the organisms were measured with a 100x oil immersion objective on a zeiss axioplan 2 photomicroscope. standard measurements of the haemoflagellates were taken according to the method of karlsbakk (2006). no possible leech vectors were observed. of the 257 fishes collected, positive infections were observed in two families, clariidae and cichlidae. fish numbers and prevalence are recorded in table 1. no seasonal pattern was notable. out of the clarias gariepinus collected from the vaal dam, 74% (89/121) were infected and from the vaal barrage 63% (63/100) were infected (table 1). a total of 427 parasites were counted in the vaal dam over four months, with 262 parasites counted in the vaal river barrage. parasite intensities were not calculated due to the variation in blood volume used for blood film preparation. there was no seasonal pattern observed with regard to prevalence. all 60 labeo capensis, 57 labeo umbratus and 35 cyprinus carpio collected during the same sample trip were uninfected. of the 26 fishes collected from the four different piscine families in the sand river dam, swaziland, 25% (1/4) of c. gariepinus were infected with trypanosomes (table 1). ten fish from two families were collected at nylsvlei nature reserve, south africa. a 50% (1/2) prevalence was found in tilapia sparrmanii, with two parasites present (table 1). trypomastigote sizes varied between the three sites (see table 2). the cytoplasm is stained dark blue with a pale section anterior to the kinetoplast, the nucleus is stained light pink and the flagellum (undulating membrane) originating near the kinetoplast is trailing anteriorly beyond the body as a free flagellum. the tail is short and almost absent (see figure 1a–c). nuclear indices (ni values) (becker & overstreet 1979) for trypanosomes measured at all three sites were ni ˃ 1, indicating an anteriorly positioned nucleus (table 2). table 1: fish species collected, total number of fish, prevalence and total number of fish hosts infected with trypanosomes from the sand river dam, swaziland, nylsvlei nature reserve, south africa, vaal dam and vaal barrage, south africa. figure 1: light micrographs of giemsa stained trypanosoma sp. (a) trypomastigote stage in clarias gariepinus burchell, 1822 blood film from the vaal dam, south africa. (b) trypomastigote stage in clarias gariepinus blood film from the sand river dam, swaziland. (c) trypomastigote stage in tilapia sparrmanii smith, 1840 blood film from the nylsvlei nature reserve, south africa. table 2: morphometrics of trypanosomes collected at three sampling sites (sand river dam, swaziland, nylsvlei nature reserve, south africa and vaal dam/vaal barrage, south africa). as reported by hoare 1932, t. mukasai measured 40 µm – 55 µm in length, with the ‘nucleus near the middle of the body and the kinetoplast at the posterior end. the undulating membrane was strongly developed and a short free flagellum is present’ (see hoare 1932). trypomastigotes measured in the current study have similar body lengths, a nucleus just in front of the midline, posterior kinetoplast and prominent undulating membranes. from this, it can be concluded that the trypanosoma sp. in the current study resembles and possibly is t. mukasai, which can be confirmed by molecular sequencing. future research should include molecular assessment of trypanosomes from all three sampling sites. it is evident that a phylogenetic analysis of all freshwater fish trypanosomes of africa is long overdue, especially to resolve the confusion regarding the synonymy of species. sand river dam, swaziland, nylsvlei nature reserve, south africa, vaal dam and vaal river barrage, south africa serve as new locality records for t. mukasai, and t. sparmanii as a new host record. ethical considerations top ↑ the parasites were obtained from fish sampled for the approved projects by mr beric gilbert and mr ebi hussain. ethical clearance was obtained from the university of johannesburg faculty ethics committee in 2011 for both these projects. fish were sampled according to the guidelines of a permit obtained from the gauteng nature conservation for annemarièavenant-oldewageand from the limpopo nature conservation for r. greenfield. the fish from swaziland were caught during a fishing competition. potential health and safety hazards were disclosed to the ethics committee. staff and students were covered by the universitity’s 3rd party insurance. all researchers participated voluntarily. acknowledgements top ↑ the university of johannesburg is thanked for providing the funding for the project. dr. r. greenfield and prof h. van der bank of the university of johannesburg are thanked for providing blood films from swaziland and nylsvlei nature reserve. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. author’s contributions m.l.f. (university of johannesburg) studied slides from all localities, stained slides from all localities apart from the vaal river and provided the first draft of the manuscript. a.a.o. (university of johannesburg), provided all of the vaal river data and slides, and collaborated on various drafts of the manuscript. references top ↑ baker, j.r., 1960, ‘trypanosomes and dactylosomes from the blood of freshwater fishes in east africa’, parasitology 51, 515–526. http://dx.doi.org/10.1017/s0031182000025592baker, j.r., 1961, ‘trypanosomes of african freshwater fish: an addendum’, parasitology 51, 263. http://dx.doi.org/10.1017/s0031182000068657, pmid:13686112 davies, a.j., gibson, w., ferris, v., basson, l. & smit n.j., 2005, ‘two genotypic groups of morphologically similar fish trypanosomes from the okavango delta, botswana’, diseases of aquatic organisms 66, 215–220. http://dx.doi.org/10.3354/dao066215, pmid:16261936 fantham, h.b., 1919, ‘some parasitic protozoa found in south africa. vi’, south african journal of sciences 20, 493–500. gibson, w.c., lom, j., peckova, h., ferris, v.r. & hamilton, p.b., 2005, ‘phylogenetic analysis of freshwater fish trypanosomes from europe using ssu rrna sequences and random amplification of polymorphic dna’, parasitology 130, 405–412. http://dx.doi.org/10.1017/s0031182004006778, pmid:15830814 gu, z.m., wang, j.g., li, m., zhang, j.y., ke, x.l. & gong, x.n., 2007, ‘morphological and genetic differences of trypanosoma in some chinese freshwater fishes: difficulties of species identification’, parasitology research 101, 723–730. http://dx.doi.org/10.1007/s00436-007-0536-5, pmid:17558522 gu, z., wang, j., ke, x., lui, y., lui, x., gong, x. et al., 2010, ‘phylogenetic position of the freshwater fish trypanosome, trypanosoma ophiocephali (kinetoplastida) inferred from the complete small subunit ribosomal rna gene sequence’, parasitology research 106(5), 1039–1042. http://dx.doi.org/10.1007/s00436-010-1759-4, pmid:20177907, pmcid:2848340 hoare, c.a., 1932, ‘on protozoal blood parasites collected in uganda’, parasitology 24, 210–224. http://dx.doi.org/10.1017/s0031182000020564 hussein, a.n.a., rabie, s.a., mohammed, n.e. & hussein, n.m., 2010, ‘light and scanning electron microscopic studies of trypanosome parasites infecting freshwater fishes in quena governorate, egypt’, egyptian academic journal of biological sciences 2, 17–31. jakes, k.a., o’donoghue, p.j. & adlard, r.d., 2001, ‘phylogenetic relationships of trypanosoma chelodina and t. binneyi from australian tortoises and platypuses inferred from small subunit rrna analyses’, parasitology 123, 483–487. http://dx.doi.org/10.1017/s0031182001008721, pmid:11719959 karlsbakk, e., 2006, ‘aspects of the morphology and ecology of some north atlantic marine fish trypanosomes’, ph.d. dissertation, university of bergen, bergen, norway. karlsbakk, e., haugen, e. & nylund, a., 2006, ‘morphology and aspects of growth of a trypanosome transmitted by the marine leech johanssonia arctica (piscicolidae) from northern norway’, folia parasitologica 52, 209–215. martin, d.s., wright, a.d.g., barta, j.r. & desser, s.s., 2002, ‘phylogenetic position of the giant anuran trypanosomes trypanosoma chattoni, t. fallisi, t. mega, t. neveulemairei, and t. ranarum inferred from 18s rrna gene sequences’, journal of parasitology 88, 566–571. pmid:12099428 negm-eldin, m.m., 1997, ‘trypanosoma mukasai (hoare, 1932) in its biological vector batracobdelloides tricarinata (blanchard, 1897) and their life cycles’, deutsche tierärztliche wochenschrift 104, 215–219. pmid:9239952 negm-eldin, m.m., 1998, ‘further studies on trypanosoma mukasai hoare, 1932 and its biological vector batracobdelloides tricarinata (blanchard, 1897)’, deutsche tierärztliche wochenschrift 105, 175–181. pmid:9646550 pienaar, u. de v., 1962, haematology of some south african reptiles, witwatersrand university press, johannesburg. sehgal, r.n.m., jones, h.i. & smith, t.b., 2001, ‘host specificity and incidence of trypanosoma in some african rainforest birds: a molecular approach’, molecular ecology 10, 2319–2327. http://dx.doi.org/10.1046/j.1365-294x.2001.01339.x, pmid:11555273 smit, n.j., davies, a.j. & van as, j.g., 2000, ‘a trypanosome from the silver catfish (schilbe intermedius) in the okavango delta, botswana’, bulletin of the european association of fish pathologists 20, 116–119. smit, n.j., van as, j.g. & davies, a.j., 2004, ‘fish trypanosomes from the okavango delta, botswana’, folia parasitologica 51, 299–303. smit, n.j., moll, j. & van der bank, f.h., 2008, ‘the first record of trypanosomes from zambezi grunters and spotted squeakers in the zambezi river, namibia and their retention in captivity’, suid-afrikaanse tydskrif vir natuurwetenskap en tegnologie 4, 16–22. votypka, j., obornik, m., volf, p., svobodova, m. & lukes, j., 2002, ‘trypanosoma avium of raptors (falconiformes): phylogeny and identification of vectors’, parasitology 125, 253–263. http://dx.doi.org/10.1017/s0031182002002093, pmid:12358422 votypka, j., lukes, j. & obornik, m., 2004, ‘phylogenetic relationship of trypanosoma corvi with other avian trypanosomes’, acta protozoologica 43, 225–231. woo, p.t.k., 1998, ‘diplomonadida, kinetoplastida and amoebida (phylum sarcomastigophora)’, in p.t.k. woo (ed.), fish diseases and disorders: noninfectious disorders, vol. 2, pp. 69, cabi, oxfordshire. article information authors: wani l. marcellino1 ibrahim i. julla2,† diaeldin a. salih3 abdel r.m. el hussein3 affiliations: 1ministry of animal resources and fisheries, juba, republic of south sudan 2college of veterinary science, university of bahr el ghazal, sudan 3central veterinary research laboratories, al amarat, sudan correspondence to: wani marcellino email: wanimarcellino@yahoo.co.uk postal address: p.o. box 126, juba, south sudan dates: received: 11 may 2011 accepted: 07 sept. 2011 published: 15 nov. 2011 how to cite this article: marcellino, w.l., julla, i.i., salih, d.a. & el hussein, a.r.m., 2011, ‘ticks infesting cattle in central equatoria region of south sudan’, onderstepoort journal of veterinary research 78(1), art. #336, 5 pages. http://dx.doi.org/10.4102/ ojvr.v78i1.336 note: †, 1953–2011 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) ticks infesting cattle in the central equatoria region of south sudan in this original research... open access • abstract • introduction • materials and methods • results • discussion • conclusion • acknowledgments    • competing interests    • authors’ contributions • references abstract (back to top) ticks infesting cattle represent a serious problem for improvement of cattle productivity in south sudan. there has been limited information on ticks and tick-borne diseases in southern sudan. this study was initiated to update the current distribution of ticks infesting cattle in the central equatoria region of south sudan. the surveys for the present study were conducted at various cattle camps in juba, mangalla and terekeka between december 2004 and june 2005. a total of 2322 ticks were collected from the bodies of 88 randomly selected cattle. ticks were preserved in 70% ethanol for later identification. seven ixodid tick species were found to infest cattle in juba whilst six species were recorded in mangalla and only four species in terekeka. amblyomma variegatum was the most common and widely distributed species found on cattle across all the study locations. amblyomma lepidum was not found during this study. based on these findings, it would be advisable to preempt the situation and institute containment procedures before possible east coast fever outbreaks occur. introduction (back to top) in sudan, approximately 68 ixodid tick species that infest a variety of animal species, including birds and reptiles, have been identified (hoogstraal 1956; jongejanet al. 1987; osman 1978). in the equatoria region of sudan, hoogstraal (1956) identified 46 species and subspecies from nine genera. in another study, julla (1994) identified 13 species and subspecies from five genera infesting livestock, namely amblyomma lepidum, amblyomma variegatum, rhipicephalus (boophilus) annulatus, rhipicephalus (boophilus) decoloratus, haemophysalis leachi leachi, hyalomma rufipes, hyalomma truncatum, rhipicephalus appendiculatus, rhipicephalus evertsi evertsi, rhipicephalus pravus, rhipicephalus sanguineus group and rhipicephalus simus. korok (2005) identified some of these species in the pibor area of jonglei state, but r. appendiculatus was not found in the area. there has been limited information on ticks and tick-borne diseases in southern sudan. the most detailed study is that of hoogstraal (1956), which confirmed the presence of some of the most economically important tick species, including r. appendiculatus. morzaria et al. (1981) reported the occurrence of r. appendiculatus in chukudum, aswa river, palataka, nimule and juba. julla (1994) reported that r. appendiculatus was abundant in and around juba, while malak et al. (2006) encountered the presence of r. appendiculatus during a participatory epidemiology study in the kajo kaji and yei river counties. more recently, marcellino (2008) encountered the presence of r. appendiculatus in and around juba. the total annual rainfall and topography determine the natural vegetation that covers the region. the ecological zones vary from savanna woodland, green belt and thick forest in the south to tropical rain forest in the north, which covers most of the region. multiple species of evergreen trees and tall grass, with a south-to-north transition of forest, are present. the bahr-el jebel river crosses the region from south to north and divides it into two almost equal parts. there are many seasonal streams that flow from the central and south-western hills towards the main streams. the region is rich in wildlife, with many small and large game species occurring mostly in the open savanna grassland. their seasonal movement, commencing with the start of the rains, is towards ethiopia, kenya and uganda. a variety of wildlife, including the african buffalo (syncerus caffer), are found in three established national parks and six game reserves in south sudan. african buffalo are known reservoir hosts of theileria parva. this study was initiated to update the current distribution of ticks infesting cattle in the central equatoria region of south sudan. materials and methods (back to top) the surveys for the present study were conducted at various cattle camps in juba, mangalla and terekeka between december 2004 and june 2005. ticks were collected from cattle at kansuk, logo west, tokiman (both eastern and western divisions), gumbo, kamiro, kworijic and nyambulung in the juba district. inmangalla samples were collected from cattle in the vicinity of the towns mangalla and malang, whilst in terekeka samples were collected from cattle camps in the villages terekeka, lujura, tukoro and yari (figure 1). all the sites are stocked with the indigenous nilotic zebu cattle breed, which is kept under traditional management systems. this involves all cattle being tethered at night, which aided in the sampling. all visible adult ticks were collected from the bodies of 88 randomly selected cattle using a pair of blunt forceps. each animal was cast and restrained and particular attention was paid to predilection sites specific to different tick species, such as the ears, briskets, dewlap, perineum, udder, hump, inner hind legs, eyes and tail. collected ticks were preserved in 70% ethanol in labelled vials for transport to the laboratory. sampling records included a reference number for each tick and the date and locality of sampling. ticks were identified according to the methods of hoogstraal (1956), okello-onen, hassan and essuman (1999) and walker et al. (2003). figure 1: the central equatoria region of southern sudan, indicating sampling localities. results (back to top) a total of 2322 ticks were collected from 88 animals at 14 sites in juba, mangalla and terekeka (table 1). seven tick species from four ixodid genera were identified. these included a. variegatum, hyalomma rufipes, r. (b.) decoloratus, r. appendiculatus, r. evertsi evertsi, r. sanguineous group, and r. simus. amblyomma variegatum was found across the study area. rhipicephalus appendiculatus, however, was abundant only in the juba area; only one male specimen was identified from mangalla andnone was found in terekeka. table 1 summarises the number of adult ticks collected from the study area. the highest tick counts were recorded at mangalla, naying and surai, and the lowestat kansuk, kabu, malang, logo west and tokiman west. the most abundant tick species collected in this survey was a. variegatum (57.7%), followed by h. rufipes (10.6%), r. evertsi evertsi (10.4%), r. (b.) decoloratus (10.1%) and r. appendiculatus (7.8%). less frequently collected species were the r. sanguineous group (2.7%) and r. simus (0.7%). of the collected ticks, 1340 (65.3%) were male and 794 (34.7%) were female. male specimens outnumbered female ticks in all the study areas except kansuk, where 66.7% and 33.3% female and male adult ticks, respectively, were collected. the geographic distribution and prevalence of tick species in the 14 locations are summarised in table 2. tick loads were highest in cattle camps at terekeka (66.3%) and nyambulung (59.0%) while the lowest tick loads were recorded at malang (13.5%) and board yard (17.8%). table 1: numbers of ticks collected on cattle at selected locations in the central equatoria region, sudan (december 2004 and january–june 2005). table 2: tick loads at selected locations in the central equatoria region, sudan (december 2004; january–june 2005). discussion (back to top) ticks and tick-borne diseases are widespread (food and agriculture organization 1983), but are most prevalent and exert their greatest impact in the tropical andsubtropical regions of the world (bram 1983). in many countries they present a major health impediment to the development and improvement of livestock. in addition to being efficient disease vectors, ticks may cause direct economic losses to livestock owing to damage caused by blood loss, hide, skin and teat damage, production and weight loss, and increased control costs. approximately 68 ixodid tick species have been recorded in sudan (fao 1983; hoogstraal 1956; jongejan et al. 1987; karrar, kaiser & hoogstraal 1963; osman 1978). the seven species found during this study have also previously been identified by hoogstraal (1956), morzaria et al. (1981) and julla (1994) in chukudum and malak et al. (2006) in the kajo kaji and yei counties. however, in the current survey, no specimens of r. appendiculatus were found in terekeka during any of the twocollections (may 2005 and june 2005) and only one male specimen was recorded in mangalla. amblyomma variegatum, the tropical bont tick, was the most common and widely distributed species found on cattle across all the study locations. previously, this species was reported to infest cattle, sheep, goats and buffaloes. its adult stage is known to be present throughout the year, with heavier infestations in the rainy season (walker et. al. 2003). at juba, the most abundant tick species were a. variegatum and r. appendiculatus. rhipicephalus sanguineus group and r. simus were found infesting only cattle at board yard and gumbo cattle camps although they are known to infest domestic dogs. rhipicephalus simus occurs in savanna regions and has never been encountered in very large numbers (walker et al. 2003). seven ixodid tick species were found to infest cattle in juba whilst six species were recorded in mangalla and only four species in terekeka. amblyomma variegatum, h. rufipes, r (b.) decoloratus and r. evertsi evertsi were prevalent in the whole study area. amblyomma lepidum was not found during this study, but was reported in previous studies to be rare in the area and mainly found on cattle at slaughterhouses (julla 1994). although only one male r. appendiculatus specimen was found, the high prevalence of antibodies against t. parva in cattle in mangalla (marcellino 2008) suggests that the distributional area of east coast fever may extend northwards to bor. this may be related to dinka bor cattle having moved through the juba–mangalla area, where the disease is endemic, on their way back home from the west equatoria region following the signing of comprehensive peace agreements in january 2005.the prevalence of t. parva antibodies and its vector, r. appendiculatus, in this area is contrary to the prediction of morzaria et al. (1981) that r. appendiculatus occurs in a cool climate with high rainfall and generally does not tolerate daily maximum temperatures higher than 30 °c or a dry season longer than four months. the climate of juba does not appear to be conducive to its establishment and maintenance. in fact, further north of juba conditions became more arid. the spread of the tick vector is further supported by studies of lessard et al. (1990), in which the computer model climex predicted that most of southern sudan is suitable for the establishment and maintenance of r. appendiculatus. however, the northern limit of its distribution is still not very clearly delineated; the present study extended only 110 km north of juba. based on these findings, it would be advisable to preempt the situation and institute containment procedures before possible east coast fever outbreaksoccur. in addition, with regard to the epidemiology of theileriosis in general, and t. parva in particular, antibodies to t. parva in cattle from the bahr el ghazal region have been demonstrated by zessein and bauman (1982). detailed epidemiological studies are needed to confirm the absence or presence of r. appendiculatus in the area. the absence of r. appendiculatus in terekeka during the present survey may be attributed to overstocking and subsequent overgrazing, resulting in reducedvegetation cover. hornby (1935) found that grass cover correlated positively with numbers of r. appendiculatus in tanzania, where overgrazing and overstocking had resulted in reduced numbers of ticks due to a lack of grass cover. these observations have also been endorsed by studies of yeoman and walker (1967), norval (1977) and short et al. (1989). a wider distribution of r. appendiculatus is expected to have a serious impact on the future of cattle improvement programmes in areasendemic to theileriosis. a systematic survey involving monthly collection of ticks throughout the year is required in terekeka to gain epidemiological data relevant to theileriosis in the area. conclusion (back to top) seven ixodid tick species were found to infest cattle in juba whilst six species were recorded in mangalla and only four species in terekeka. amblyomma variegatum, the tropical bont tick, was the most common and widely distributed species found on cattle across all the study locations. amblyomma lepidum was not found during this study. at juba, the most abundant tick species were a. variegatum and r. appendiculatus. rhipicephalus sanguineus group and r. simus were found infesting only cattle at board yard and gumbo cattle camps although they are known to infest domestic dogs. based on these findings, it would be advisable to preempt the situation and institute containment procedures before possible east coast fever outbreaks occur. acknowledgments (back to top) the authors would like to thank the animal resources research corporation in khartoum, sudan for providing funding for the study. we also thank our colleagues, mr. john gubek, mr. patrick lodu and mr. joel jada of the juba veterinary research laboratory for their assistance with fieldwork. we would like to thank dr arthur m. spickett of the arc-ovi, south africa for critically reading the manuscript. this work is published by permission of the undersecretary of the ministry of animal resources and fisheries, republic of south sudan. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions w.l.m. was the principle investigator. he collected the samples, identified tick species, performed the statistical analysis and drafted the manuscript. i.i.j. contributed to tick identification and manuscript writing. d.a.s. contributed to statistical analysis and preparation of the manuscript. a.r.m.e.h. was the project leader and contributed to the final preparation of the manuscript. references (back to top) bram, r.a., 1983, ‘tick-borne livestock diseases and their vectors: the global problem’, world animal review [fao animal production and health, paper no. 36], pp. 54–59, food and agriculture organization, rome. fao. see food and agriculture organization. food and agriculture organization, 1983, ‘studies on important tick-borne diseases of cattle’, technical report, no. 2 ag:gcp/sud /024/den, food and agriculture organization, rome. hoogstraal, h., 1956, african ixodoidea. i. ticks of sudan (with special reference to equatoria provine and with preliminary reviews of genera boophilus, margaropus, and hyalomma), us department of navy (bureau of medicine and surgery), washington, d.c. hornby, h.e., 1935, ‘east coast fever: a warning’, veterinary record 15, 476. jongejan, f., zivkovic, d., pegram, r.g., tatchell, r.j., fison, t., latif, a.a. et al., 1987, ‘ticks (acari: ixodidae) of the blue and white nile ecosystem in the sudan with particular reference to the rhipicephalus sanguineous group’, experimental and applied acarology3, 331–346. http://dx.doi.org/10.1007/bf01193169 julla, i.i., 1994, ‘studies on the epidemiology of theileriosis in equatoria of sudan with emphasis on east coast fever‘, phd thesis, dept. of preventive veterinary medicine, university of khartoum. karrar, g., kaiser, m.n. & hoogstraal, h., 1963, ‘ecology and host-relationships of ticks (ixodidae) infesting domestic animals in kassala province, sudan, with special reference to amblyomma lepidum donitz’, bulletin of entomological research 54, 509–522. korok, m.j., 2005, ‘ecological and epidemiological studies on ticks (acari: ixodidae) in pibor area, jonglei state’, mvsc thesis, dept. of preventive veterinary medicine, university of khartoum. lessard, p.l., eplattenier, r., norval, r.a.i., kundert, k., dolan, t.t., croze, h. et al., 1990, ‘geographical information systems for studying the epidemiology of cattle diseases caused by theileria parva’, veterinary record 126, 255–262. pmid:2327044 malak, a., kiara, h., mpoke, l., muriuki, s., tereka, t. & ounga, t., 2006, ‘an epidemiological study of east coast fever and other livestock diseases in kajo kaji and yei counties of southern sudan’, ilri kenya. p. 58. marcellino, w.l., 2008, ‘prevalence and economic impact of east coast fever in central equatoria state’, msc thesis, animal resource research council, sudan academy of sciences. morzaria, s.p., tatchell, r.j., minor, r., pederson, v., julla, i.i., rahim, a. et al., 1981, ‘preliminary studies on the epidemiology of theileriosis in eastern equatoria province of the sudan’, in a.d. irvin, m.p. cunningham & a.s. young (eds.), advances in the control of theileriosis, pp. 83–85, marthinus njihoff publishers, the haque. norval, r.a.i., 1977, ‘tick problems in relation to land utilization in rhodesia’, veterinary journal 8, 33–38. okello-onen, j., hassan, s.m. & essuman, s., 1999, taxonomy of african ticks: an identification manual, icipe science press, nairobi. osman, o.m., 1978, ‘preliminary notes on the distribution of ticks (acarina: ixodidae) in darfur province, sudan’, bulletin of animal health and production in africa 26, 329–333. short, n.j., floyd, r.b., norval, r.a.i. & sutherst, r.w., 1989, ‘development rates fecundity and survival of developmental stages of ticks rhipicephalus appendiculatus, boophilus decoloratus and b. microplus under field conditions in zimbabwe’, experimental and applied acarology6, 123–141. http://dx.doi.org/10.1007/bf01193981 walker, a.r., bouattour, a., camicas, j.i., horak, i.g., latif, a.a., pegram, r.g. et al., 2003, ticks of domestic animals in africa: a guide to identification of species, bioscience reports, edinburgh. yeoman, g.h. & walker, j.b., 1967, the ixodidae ticks of tanzania: a study of the zoogeography of the ixodidae of an east african country, commonwealth institute of entomology, london. zessein, k.h. & bauman, m.p.o., 1982, report on livestock disease survey: bahr el ghazal province, sudan, gesellschaft für technische zusammenarbeit (gtz), berlin. elghali_177-185.indd introduction little publish information on ticks infesting camels is available. camels in egypt were found to be mainly infested by hyalomma dromedarii (95 %) together with hyalomma marginatum subspecies, hyalomma anatolicum excavatum and hyalomma species nymphs (van straten & jongejan 1993). in the same area and on the same animal species diab, el kady & shouky (2001) reported that h. dromedarii, hy alomma impeltatum, h. a. excavatum and h. a. anatolicum represented 96 % of the tick population with a higher infestation in march to november and a mean monthly total of 22–78 ticks per animal. about 62 % of adult ticks were collected from the tail, anus, brisket and udder, and 91 % of nymphs were found infesting humps, neck, ears and sides. in saudi arabia, a total of 1 045 adult h. dromedarii and 174 h. a. excavatum together with 110 ticks that included h. a. anatolicum, h. impeltatum, hyalomma schulzei, hyalomma truncatum, hyalomma mar gina tum rufipes and hyalomma marginatum turanicum as well as 143 hyalomma spp. nymphs were collected from 10 camels (el khalifa, al asgah & diab 1985). in yemen, arab republic, the most abundant livestock ticks were hyalomma spp., particularly on camels, but with a very low burden (macar tan, hunter, pegram & bourne 1987). in the sudan, karrar, kaiser & hoogstraal (1963) reported that h. dromedarii was the main tick species of camels together with amblyomma lepidum, h. impeltatum, rhipi177 onderstepoort journal of veterinary research, 76:177–185 (2009) ticks (acari: ixodidae) infesting camels (camelus dromedarius) in northern sudan a. elghali1* and s.m. hassan2 abstract elghali, a. & hassan, s.m. 2009. ticks (acari: ixodidae) infesting camels (camelus dromedarius) in northern sudan. onderstepoort journal of veterinary research, 76:177–185 monthly total body tick collections from 13-20 camels were conducted for 2 consecutive years (2000– 2001) in northern sudan. tick populations were correlated with locality, season, predeliction site, sex and coat colour. hyalomma dromedarii was found to be the predominant (89 %) tick species infesting the camels. other tick species found in very low numbers were hyalomma impeltatum (7.7 %), hy alomma anatolicum anatolicum (3.3 %), hyalomma truncatum (0.29 %), hyalomma marginatum rufipes (0.25 %), rhipicephalus praetextatus (0.30 %) and rhipicephalus sanguineus group (0.09 %). nymphs of the genus hyalomma were collected in significant numbers. adult ticks significantly preferred to attach to the lower parts of the camel’s body for feeding while the nymphs preferred the back of the animal. female camels harboured more ticks than males while higher infestations were recorded on camels with a grey coat colour compared to those with a brown coat colour. ticks were found on camels throughout the year and increased in numbers during march to october with a peak in september. keywords: camels, hyalomma spp., rhipicephalus spp., sudan, ticks * author to whom correspondence is to be directed. e-mail: elghaliaahmed@hotmail.com 1 atbara veterinary research laboratory, p.o. box 121, atbara, sudan 2 department of parasitology faculty of veterinary medicine uni versity of khartoum, sudan accepted for publication 1 september 2008—editor 178 ticks (acari: ixodidae) infesting camels (camelus dromedarius) in northern sudan atbara sudan tiyat abu ho sam wadi abu rigaiba ed damer camel grazing area gangari river al basli nikhilas r iv e r 17°45’ 17°50’ 34°00’ scale 1 250 000 34°15’ w mukabrab al drou sa l halag al humer al u shera gersi 34°00’ n il e muuran alrouk al gezen al shaet n fig. 1 map of the study area in nothern sudan showing the localities from where ticks were colected (●) and atbara research laboratory (■) 179 a. elghali & s.m. hassan cephalus sanguineus sanguineus, rhipicephalus simus, h. a. excavatum, h. truncatum and h. m. ru fipes. dolan, wilson, schwart, newson & field (1983) collected h. dromedarii, h. m. rufipes and rhipicephalus pulchellus from camels in kenya. the objective of this study was to conduct population studies of ticks infesting camels in northern sudan. materials and methods the study area extends along the western banks of river atbara starting from ed damer in northern su dan (fig. 1). this area is in the arid (desert) zone (annual rainfall ranges between 0–100 mm) with very poor vegetation cover which mainly consists of scattered bushes and small trees, i.e. prosopis chilen sis and capparis decidua. large acacias such as acacia nilotica nilotica, acacia seyal, acacia tortilis tortilis, acacia tortilis raddiana, ziziphus spina christa and hyphaena thebaica are abundant (el amin 1989). small-scale farms are cultivated along the river banks where camels graze. they graze on grass and post-harvest agricultural residues and browse on bushes and trees. during the wet seasons they move to the butana area, approximately 300 km to the south-east, where the grazing is better. tick collection and identification total body collections of ticks from 13–20 adult camels were carried out on a monthly basis bar for 2 consecutive years (2000–2001). ticks from each predilection site were put into separate vials containing 70 % ethanol. the vials were labeled to indicate date, time, locality, animal number, sex, host coat colour and predilection site. the predilection sites included head (head and neck), back (hump region and flank), tail (tail shaft and perineum), lower parts (brisket, belly, udder or scrotum and inner sides of thigh) and feet (knee to pad). the ticks were identified according to hoogstraal (1956), hoogstraal & kaiser (1959) and walker et al. (2003), and results were recorded according to table 1 means (± se) of ticks collected from camels along the river atbara and ed damer in 2000–2001 year tick species river atbara (123) ed damer (62) 2000 h. dromedarii (m) h. dromedarii (f) h. dromedarii (t) h. impeltatum (m) h. impeltatum (f) h. impeltatum (t) h. a. anatolicum (m) h. a. anatolicum (f) h. a. anatolicum (t) hyalomma nymphs 125.0 ± 12.2a 40.4 ± 5.1a 165.4 ± 16.3a 12.4 ± 1.1a 5.7 ± 0.6a 18.2 ± 1.4a 3.8 ± 0.5a 7.3 ± 0.8a 11.1 ± 1.1a 5.0 ± 0.8a 49.5 ± 5.9b 13.6 ± 1.9b 63.1 ± 7.2b 3.3 ± 0.7b 0.45 ± 0.2b 3.8 ± 0.7b 1.1 ± 0.3b 0.2 ± 0.1b 1.2 ± 0.3b 2.5 ± 0.9b mean total ticks 194.7 ± 17.3a 68.1 ± 7.8b ratio (male:female) 3.1 ± 0.21b (105) 7.1 ± 1.2a (56) 2001 h. dromedarii (m) h. dromedarii (f) h. dromedarii (t) h. impeltatum (m) h. impeltatum (f) h. impeltatum (t) h. a. anatolicum (m) h. a. anatolicum (f) h. a. anatolicum (t) hyalomma nymphs 111.4 ± 9.5a 43.4 ± 4.1a 154.9 ± 12.9a 10.8 ± 1.1a 0.37 ± 0.1a 11.2 ± 1.1a 1.02 ± 0.6a 0.88 ± 0.2a 1.9 ± 0.8a 7.9 ± 1.0a 40.2 ± 4.0b 13.7 ± 1.6b 53.9 ± 5.1b 2.8 ± 0.4b 0.02 ± 0.02b 2.9 ± 0.38b 0.02 ± 0.02b 0.07 ± 0.03b 0.1 ± 0.04a 4.9 ± 0.9b mean total ticks 168.0 ± 13.7a 56.8 ± 5.3b ratio (male:female) 3.6 ± 0.24a 4.6 ± 0.6a means ( ± se) followed by the same letter in each row are not significantly different at 5 % level, based on ryan’s q test (regwq) ( ) = no. of camels used m = male f = female t = m + f 180 ticks (acari: ixodidae) infesting camels (camelus dromedarius) in northern sudan table 2 total tick burdens on camels in river nile state, northern sudan, 2000 and 2001 year location h. dromedarii h. impeltatum h. a. anatolicum hyalomma nymphs 2000 river atbara ed damer 20 295 3 906 2 214 236 1 353 74 615 155 total no. of ticks 24 201 2 450 1 427 770 % of adults 86.2 8.7 5.1 – ratio (nymphs:adults) 1:36.5 2001 river atbara ed damer 16 275 3 024 1 176 162 200 6 830 274 total no. of ticks 19 299 1 338 206 1 104 % of adults 92.6 6.4 1.0 – ratio (nymphs:adults) 1:18.9 total 43 500 3 788 1 633 1 874 % of grand total abundant adults 88.9 7.7 3.3 – ratio (nymphs:adults) 1:26.1 table 3 total numbers of tick species collected from camels in very low numbers during 2000 and 2001 in river nile state, northern sudan tick species males females total % of total adult ticks h. truncatum h. marginatum rufipes r. praetextatus r. sanguineus group 127 78 84 26 16 46 65 16 143 124 149 42 0.29 0.25 0.30 0.09 table 4 means ( ± se) of ticks collected from male and female camels in river nile state, northern sudan, 2000–2001 year tick species n male camels n female camels 2000 h. dromedarii h. impeltatum h. a. anatolicum 63 63 63 82.2 ± 12.3b 8.4 ± 1.1b 5.7 ± 1.2b 122 122 122 156.4 ± 16.0a 15.9 ± 1.4a 8.9 ± 1.1a hyalomma nymphs 63 3.0 ± 1.0a 122 4.8 ± 0.8a total ticks 63 96.3 ± 3.4b 122 181.2 ± 17.3a ratio (male:female) 63 5.5 ± 1.0a 122 3.8 ± 0.4b 2001 h. dromedarii h. impeltatum h. a. anatolicum 33 33 33 89.8 ± 13.2b 6.8 ± 2.1a 1.1 ± 0.4a 128 128 128 127.5 ± 11.2a 8.7 ± 0.9a 1.3 ± 0.6a hyalomma nymphs 33 7.9 ± 1.9a 128 6.5 ± 0.8a total ticks 33 97.7 ± 14.5b 128 137.5 ± 12.0a ratio (male:female) 33 3.4 ± 0.3a 128 4.1 ± 0.3a means ( ± se) followed by the same letter in each row are not significantly different at 5 % level, based on ryan’s q test (regwq) n = no. of animals used 181 a. elghali & s.m. hassan t a b l e 5 m e a n ( ± s e ) n u m b e rs o f tic ks c o lle ct e d f ro m c a m e ls in r iv e r n ile s ta te in 2 0 0 0 a n d 2 0 0 1 m o n th n h . d ro m e d a ri i h . im p e lt a tu m h . a . a n a to li c u m h y a lo m m a n y m p h s t o ta l ti c k s r a ti o ( m a le :f e m a le ) 2 0 0 0 ja n u a ry f e b ru a ry m a rc h m a y ju n e ju ly a u g u st s e p te m b e r o ct o b e r n o ve m b e r d e ce m b e r 2 0 2 0 1 7 1 6 1 9 1 6 1 6 1 4 1 7 1 5 1 5 7 0 .9 ± 8 .4 b 7 0 .4 ± 1 0 .1 b 8 2 .2 ± 1 7 .3 b 8 4 .6 ± 1 7 .6 b 1 0 4 .7 ± 2 2 .4 b 1 2 4 .2 ± 2 4 .9 b 1 3 6 .3 ± 4 2 .1 a b 2 5 6 .6 ± 7 3 .0 a 2 5 2 .5 ± 6 3 .7 a 1 7 1 .1 ± 5 1 .5 a b 1 3 7 .9 ± 3 3 .2 a b 1 7 .3 ± 3 .4 a 1 0 .9 ± 2 .1 a 1 1 .4 ± 3 .2 a 1 4 .1 ± 3 .6 a 8 .6 ± 1 .9 a 9 .9 ± 2 .6 a 1 1 .1 ± 3 .4 a 1 8 .1 ± 5 .3 a 1 7 .4 ± 4 .7 a 1 2 .7 ± 3 .8 a 1 6 .6 ± 4 .5 a 1 5 .5 ± 3 .5 a 9 .5 ± 3 .0 a b 7 .0 ± 1 .7 a b 1 4 .0 ± 5 .1 a 6 .9 ± 2 .0 a b 9 .4 ± 2 .9 a b 4 .8 ± 1 .3 a b 1 .8 ± 0 .7 b 2 .4 ± 0 .7 b 6 .7 ± 1 .8 a b 4 .7 ± 2 .0 a b 1 .8 ± 0 .6 b 0 .4 ± 0 .2 b 3 .4 ± 2 .3 a b 3 .6 ± 1 .2 a b 3 .7 ± 1 .7 a b 2 .4 ± 1 .0 a b 9 .3 ± 4 .0 a b 3 .6 ± 2 .0 a b 2 .9 ± 1 .5 a b 1 1 .3 ± 3 .6 a 5 .9 ± 2 .4 a b 1 0 3 .7 ± 1 1 .8 b 9 0 .8 ± 1 4 .1 b 1 0 0 .6 ± 1 .9 b 1 1 3 .1 ± 2 4 .1 b 1 2 0 .2 ± 2 5 .7 b 1 4 3 .5 ± 2 9 .4 a b 1 5 2 .2 ± 4 5 .9 a b 2 7 6 .5 ± 7 8 .2 a 2 7 2 .4 ± 6 8 .2 a 1 9 0 .6 ± 5 5 .7 a b 1 5 9 .3 ± 3 9 .9 a b 2 .9 ± 0 .3 b 5 .8 ± 1 .4 a b 6 .6 ± 1 .6 a b 2 .1 ± 0 .2 b 4 .6 ± 1 .1 a b 8 .1 ± 3 .0 a 3 .8 ± 0 .8 b 6 .5 ± 1 .7 a b 3 .0 ± 0 .3 b 2 .4 ± 0 .4 b 2 .7 ± 0 .3 b 2 0 0 1 ja n u a ry f e b ru a ry m a rc h a p ri l m a y ju n e ju ly o ct o b e r n o ve m b e r d e ce m b e r 1 3 1 4 1 6 1 5 1 6 1 9 1 7 1 6 1 8 1 7 1 5 4 .8 ± 4 2 .6 a b 8 9 .4 ± 2 6 .4 a b 1 7 0 .8 ± 4 7 .6 a 1 5 8 .5 ± 4 9 .2 a b 1 1 5 .3 ± 2 1 .5 a b 1 2 1 .2 ± 2 .9 a b 1 5 8 .6 ± 2 7 .0 a b 9 3 .5 ± 8 .3 a b 6 0 .8 ± 1 0 .9 b 8 6 .5 ± 1 6 .1 a b 1 3 .1 ± 3 .4 a b 3 .7 ± 2 .7 a b 1 5 .8 ± 5 .1 a 8 .4 ± 3 .1 a b 7 .1 ± 2 .2 a b 5 .6 ± 1 .2 b 4 .5 ± 0 .9 b 7 .3 ± 1 .7 a b 8 .4 ± 1 .1 a b 6 .0 ± 1 .4 a b 2 .8 ± 1 .0 a 1 .3 ± 0 .5 a 2 .1 ± 0 .6 a 0 .7 ± 0 .3 a 0 .6 ± 0 .3 a 0 .2 ± 0 .1 a 0 .4 ± 0 .3 a 0 .0 ± 0 .0 a 4 .4 ± 4 .4 a 0 .4 ± 0 .2 a 9 .0 ± 2 .2 a b 9 .5 ± 3 .0 a b 5 .7 ± 1 .8 a b 6 .0 ± 1 .8 a b 4 .3 ± 1 .8 a b 8 .0 ± 2 .6 a b 1 2 .4 ± 4 .0 a 3 .2 ± 0 .8 b 3 .6 ± 1 .6 b 6 .6 ± 1 .7 a b 1 7 0 .7 ± 4 6 .0 a 9 9 .4 ± 2 9 .2 a 1 8 8 .6 ± 5 1 .9 a 1 6 7 .8 ± 5 2 .4 a 1 2 3 .0 ± 2 3 .4 a 1 2 6 .9 ± 2 3 .0 a 1 6 3 .5 ± 2 7 .7 a 1 0 0 .8 ± 8 .9 a 7 3 .6 ± 1 1 .8 a 9 2 .9 ± 1 6 .7 a 2 .4 ± 0 .3 3 .8 ± 0 .6 2 .8 ± 0 .3 3 .6 ± 1 .3 3 .4 ± 0 .5 4 .1 ± 0 .7 5 .8 ± 0 .8 4 .3 ± 0 .9 5 .2 ± 0 .7 3 .4 ± 0 .9 m e a n s (± s e ) fo llo w e d b y th e s a m e le tt e r in e a ch c o lu m n a re n o t si g n ifi ca n tly d iff e re n t a t 5 % le ve l, b a se d o n r ya n ’s q t e st ( r e w g q ) n = n o . o f a n im a ls u se d t a b l e 6 m e a n ( ± s e ) n u m b e r o f tic ks c o lle ct e d f ro m d iff e re n t si te s o n c a m e ls t ic k s p e c ie s h e a d b a c k t a il a n d u n d e r ta il l o w e r p a rt s f e e t h . d ro m e d a ri i h . im p e lta tu m h . a . a n a to lic u m 2 2 .7 ± 1 .7 b 1 .7 ± 0 .1 6 b 0 .0 6 ± 0 .0 2 b 1 .7 ± 0 .4 2 c 0 .0 9 ± 0 .0 2 c 0 .0 3 ± 0 .0 2 b 2 3 .8 ± 1 .6 b 1 .9 ± 0 .2 b 0 .2 4 ± 0 .0 4 b 7 6 .9 ± 7 .4 a 5 .4 ± 0 .6 a 1 .2 ± 0 .2 a 1 8 .6 ± 1 .6 b 1 .2 ± 0 .1 b 0 .2 5 ± 0 .0 6 b h ya lo m m a n ym p h s 1 .0 4 ± 0 .2 6 b 4 .5 ± 0 .5 a 0 .3 ± 0 .1 b 0 .9 ± 0 .2 b 0 .2 4 ± 0 .1 5 b r a tio ( m a le :f e m a le ) 4 .1 ± 0 .3 a b 2 .8 ± 1 .1 b 3 .4 ± 0 .2 a b 3 .2 ± 0 .2 b 4 .6 ± 0 .9 a m e a n s (± s e ) fo llo w e d b y th e s a m e le tt e r in e a ch r o w a re n o t si g n ifi ca n tly d iff e re n t a t 5 % le ve l, b a se d o n r ya n ’s q t e st ( r e w g q ) n o . o f a n im a ls = 2 4 9 182 ticks (acari: ixodidae) infesting camels (camelus dromedarius) in northern sudan table 7 mean (± se) number of ticks collected from male and female camels of different coat colour in river nile state in 2000 and 2001 camel gender tick species grey brown female h. dromedarii (m) h. dromedarii (f) h. dromedarii (t) h. impeltatum (m) h. impeltatum (f) h. impeltatum (t) h. a. anatolicum (m) h. a. anatolicum (f) h. a. anatolicum (t) 140.3 ± 14.6a 54.8 ± 6.2a 195.0 ± 19.7a 13.7 ± 1.4a 1.1 ± 0.3a 14.8 ± 1.5a 1.3 ± 0.6a 2.0 ± .3a 3.3 ± 0.8a 71.4 ± 6.0b 26.7 ± 3.1b 98.2 ± 8.3b 5.6 ± 0.6b 0.54 ± 0.4b 6.2 ± 0.9b 0.2 ± 0.1b 0.6 ± 0.3b 0.8 ± 0.3b hyalomma nymphs 7.8 ± 1.1a 6.0 ± 0.8a total 213.2 ± 21.1a 105.1 ± 8.9b ratio adult 3.8 ± 0.3a 4.0 ± 0.4a male h. dromedarii (m) h. dromedarii (f) h. dromedarii (t) h. impeltatum (m) h. impeltatum (f) h. impeltatum (t) h. a. anatolicum (m) h. a. anatolicum (f) h. a. anatolicum (t) 79.0 ± 13.1a 27.2 ± 3.8a 106.2 ± 16.0a 6.5 ± 1.3a 0.9 ± 0.2a 7.4 ± 1.3a 0.9 ± 0.2a 1.1 ± 0.3a 1.9 ± 0.4a 42.9 ± 7.9a 12.5 ± 3.2b 55.4 ± 10.8a 6.4 ± 3.6a 0.4 ± 0.4a 6.8 ± 3.5a 0.2 ± 0.2b 0.7 ± 0.5a 0.9 ± 0.7a hyalomma nymphs 7.7 ± 1.7a 2.9 ± 0.8a total 115.5 ± 17.3a 63.1 ± 13.4a ratio (male:female) 4.0 ± 0.6a 4.4 ± 0.7a means (± se) followed by the same letter in each row are not significantly different at 5% level, based on ryan’s q test (rewgq females : grey 108, brown 74 males: grey 50, brown 17 m = male, f = female and t = m + f table 8 correlation analysis between ticks collected from camels with monthly meteorological values in 2000–2001 in river nile state year met. data h. nymphs h. d. h. i. h. a. a. total ticks 2000 min. (185) max. (168) rh9 (185) rh3 (185) rain (185) 0.02 0.06 –0.003 –0.13 0.24*** 0.16* 0.12 0.01 –0.05 0.08 –0.02 –0.06 0.1 0.11 –0.01 –0.02 –0.1 0.001 0.08 –0.03 0.15* 0.1 0.01 –0.04 0.07 2001 min. (172) max. (172) rh9 (172) rh3 (172) rain (172) 0.02 –0.01 0.09 0.02 0.11 0.02 0.04 –0.11 –0.16* –0.1 –0.17* –0.16* –0.003 –0.01 –0.07 –0.1 –0.11 0.08 0.08 –0.07 –0.005 0.2 –0.09 –0.14 –0.1 * p < 0.05, ** p < 0.01 and *** p < 0.001 h. d. = h. dromedarii h. i. = h. impeltatum h. a. a. = h. a. anatolicum min. = minimum temperature max. = maximum temperature rh9 = relative humidity at 900 h rh3 = relative humidity at 1 500 h ( ) = no. of observations 183 a. elghali & s.m. hassan the body site and genus, species, gender and developmental stage of each tick collected. the parameters studied included tick species infesting camels, seasonality, effects of camel gender and coat colour and preferred attachment sites of each tick species. the number of ticks was correlated with the mean monthly minimum ambient temperatures, mean monthly maximum ambient temperatures, and mean ambient relative humidity and total monthly rainfall. results in the 2 study years 2000 and 2001, h. dromedarii represented 86.2 % and 92.6 %, respectively, of the ticks infesting the camels (table 1), with a mean of 88.9 % (table 2). mean tick numbers per camel were 165.4 ± 16.3 and 154.9 ± 12.9 in 2000 and 2001, respectively. other ticks collected in some numbers were h. impeltatum (7.7 %) and h. a. anatolicum (3.3 %) (table 2). tick species found in very low numbers were h. truncatum, h. marginatum rufipes, rhipicephalus praetextatus and r. sanguineus group (table 3). the ratio of male to female ticks ranged between 3.1 ± 0.21 and 7.1 ± 1.2 for the 2 years, respectively. the total ratio of hyalomma nymphs to adults of all hyalomma species was 1:26.1. female camels were found to carry more ticks than males. this result was highly significant for h. dromedarii (table 4). hyalomma dromedarii numbers increased from april and reached a peak in september and october in 2000 (table 5). in 2001, the highest infestation was recorded in march although there was no significant difference between months. hyalomma impeltatum showed no significant differences between months in 2000 but the least mean tick numbers were collected in july and august. peak numbers of this species were collected in march 2001 (table 5) and the least in february, june and july. more h. a. anatolicum were collected in january 2000 and in november 2001 with no significant differences between months. hyalomma nymphs were prevalent throughout 2000 and 2001 with peaks in november 2000 and july 2001 (table 5). the total numbers of adult ticks collected were significantly higher in 2000 during september and october and from january to june but in 2001 there were no significant differences in the total numbers ticks collected per month. during the collection period, male ticks outnumbered females with the exception of h. a. anatolicum (table 5). the udder, scrotum, belly and brisket were the preferred sites for feeding of h. dromedarii (53.5 %), h. impeltatum (52.5 %), and h. a. anatolicum (65.4 %) (table 6). the hump and sides was much less preferred (1.2 %, 0.9 % and 1.7 % of h. dromedarii, h. impeltatum and h. a. anatolicum, respectively) while the nymphs preferred the back (64.5 %) (table 6). female camels with a grey coat colour harboured significantly more ticks than females with a brown coat colour (table 7). this was shown clearly by individual tick species, their gender and also with mean total ticks collected. however, there were no significant differences in the numbers of nymphs harboured by grey and brown female camels. male grey coloured camels harboured more ticks than brown coloured males but the difference was only significant for female h. dromedarii and for male h. a. anatolicum. hyalomma dromedarii infestation was positively correlated in 2000 with mean minimum and maximum ambient temperatures, relative humidity and rainfall (table 8) while in 2001, this was positively correlated only with mean minimum and mean maximum ambient temperatures. hyalomma impeltatum in 2000 and h. a. anatolicum in both years were found to increase with increased humidity and decreased temperature. generally, tick infestation was not strongly correlated with meteorological values. it was, however, observed that hyalomma nymphs significantly increased in number with rainfall during 2000. discussion this study was conducted in a semi-desert zone where rainfall is very low and of short duration (july to september). the river atbara is a factor in that it creates suitable microhabitats for ticks with acacia trees, bushes, few grasses and cultivated areas and plays a role with the movement of camels to butana from july to november in impacting on the seasonal population changes of ticks. in the present study, h. dromedarii represented the main tick species infesting camels (88.9 %) followed by h. impeltatum (7.7 %) and h. a. anatolicum (3.3 %). it was also found that h. dromedarii females were the main tick species found engorged on the camels, while females of the other species (h. impeltatum, h. a. anatolicum, h. m. rufipes and rhipicephalus spp.) were not engorged or only partially engorged. this could be due the fact that cam184 ticks (acari: ixodidae) infesting camels (camelus dromedarius) in northern sudan els are not the preferred hosts of the latter tick species. other tick species found in this study in small numbers were h. truncatum, h. m. rufipes, r. sanguineus group and r. praetextatus. in egypt, the main tick species infesting camels is h. dromedarii representing up to 95 % of ticks (van straten & jongejan, 1993; diab et al. 2001). in eastern sudan, karrar et al. (1963) reported that h. dromedarii is the main tick species feeding on camels. other tick species they found on camels were r. sanguineus, r. simus, h. a. excavatum and amblyomma lepidum. in the current study, a. lepidum was not found despite the fact that some camels are taken each year to the butana area where this tick species was previously found (karrar et al. 1963). ahmed (1999) studied tick infestations on sheep in the study area and found that 74 % of the ticks were h. a. anatolicum, 15 % r. sanguineus group, 2 % r. simus, 9 % rhipicephalus evertsi evertsi and 0.5 % h. dromedarii. this strongly implies that sheep are not preferred hosts for h. dromedarii, while camels are not preferred hosts for h. a. anatolicum. he also reported that sheep were not infested with h. impeltatum, h. truncatum and h. m. rufipes. in the current study r. e. evertsi was not found feeding on the camels. these two studies in the same ecological zone where camels and sheep co-exist imply that there is a strong host preference of these ticks. in this study most ticks were collected from the lower parts of the camels. unlike other animal species (hoogstraal 1956), the tail, despite the fact that it is short in camels, was found to be an important feeding site of ticks particularly the edges. this could be due the fact that the tail of camels has a less fat component or that due to the physiological status of camels in that the body temperature fluctuates during the day and that of the tail is possibly the least affected. the high percentage (64.5 %) of nymphs found on the back particularly the hump also differs from other animal species. this is could be due to the fact that the nymphs ( which attached as larvae) seek shelter within the hairy hump area and that it provides more humidity, or it may be that h. dromedarii nymphs prefer the back of their hosts. for instance, hassan (1997) working on cattle, found that nymphs of rhipicephalus appendiculatus and amblyomma variegatum preferred the head region and the underside of the hosts, respectively. the high number of ticks collected from under the tail in this study is in agreement with the findings of diab et al. (2001). hylomma m. rufipes was found mainly under the tail particularly around the anus. the predilection site of this tick species is not different from that of cattle (hoogstraal 1956; hassan 1997). seasonality of ticks infesting camels was observed by diab et al. (2001) who reported that high tick infestations in egypt occurred during march to november. in the present study, there was no clear pattern of seasonality. ticks were found on camels throughout the year but it was observed that the highest infestation occurred from march to october. this could be attributed to the fact that the non-parasitic flat stages could survive well during winter which was reflected as infestations during the following summer. on the other hand, it was observed that ticks do not go into diapause during winter when ambient temperature drops to about 10 °c. hylomma a. anatolicum feeding on sheep in the same area were also not found to go into diapause, according to ahmed (1999) who found high infestations in winter. it is also possible that h. dromedarii unlike r. appendiculatus (pegram & banda 1990) does not go into diapause. this study revealed that female camels carried significantly more ticks than the males. this was true for all adult tick species. hassan (1997) reported similar findings for cows with higher loads of r. appendiculatus and a. variegatum than oxen. pregnancy and lactation stress may lower the resistance of females to tick infestation (ali 2004). however, this was not true for nymphs as both genders of camels carried loads the differences of which were not significant. this could be due to differences in resistance of male and female camels against larvae that feed and moult on the host, but not against nymphs. camels with a grey coat colour carried significantly more ticks than those with a brown coat colour. this was true for all tick species particularly those which were found feeding on female camels. working with cattle, hassan (1997) found that hosts with a black coat colour were infested with fewer r. append iculatus and a. variegatum compared to cattle of a brown and white coat colour. he attributed this phenomenon to the fact that a dark coat colour absorbs more heat, changing the microhabitat of the ticks which leads to a high mortality rate or the ticks dropping off without feeding. camels in this area are of two types, namely brown and grey, and the difference in tick load may indicate a difference in their innate resistance to tick infestation. the current study revealed that male ticks of all species except h. a. anatolicum outnumbered females. similar results were reported by hoogstraal (1956), 185 a. elghali & s.m. hassan kaiser, sutherst, bourne, gorissen & floyd (1988) and hassan (1997). this is due to the fact that females detach from the hosts after a few days of feeding to oviposit while males remain for several weeks before dropping (hoogstraal 1956). however, h. a. anatolicum females were found to outnumber males. this could be due to the fact that camels are probably not their preferred hosts and hence males either do not attach or drop off without attaching. very few engorged h. a. anatolicum females were collected indicating that few males were available for mating with females to complete engorgement. references ahmed, b.m. 1999. studies on epizootiology of theileria (lestoquardi nomen novum) hirci (apicomplexa: theileriidae) in river nile state. m.sc. thesis, nile valley university. ali, a. 2004. studies on immune response of rabbits to hylomma anatolicum anatolicum and hylomma dromedarii (acari: ixodidae). ph.d. thesis, university of khartoum. diab, f.m., el kady, g.a. & shouky, a. 2001. bionomics of ticks collected from sinai. 2. abundance, attachment sites and density of ticks infesting arabian camels. journal of egyptian society of parasitology, 31:479–489. dolan, r., wilson, a.j., schwart, h.j., newson, r.m. & field, e.r. 1983. camel production in kenya and its constraints. tick infestations. animal health, 15:179–183. el amin, h.m. 1989. trees and shrubs of the sudan. exeter: ithaca press. el khalifa, m.s., al asgah, n.a. & diab, f.m. 1985. ticks (acari: ixodidae) infesting common domestic animals in al gasim province, saudi arabia. journal of medical ento mology, 21:114–115. hassan, s.m. 1997. ecological studies on rhipicephalus appen diculatus and amblyomma variegatum (acari: ixo di dae): drop-off rhythms, development, survival and seasonal population dynamics. ph.d. thesis, kenyatta university. hoogstraal, h. 1956. african ixodoidea. 1. ticks of the sudan with special reference to equatoria province and with preliminary reviews of the genera boophilus margaropus and hyalomma. washington d.c.: us navy. hoogstraal, h. & kaiser, m.n. 1959. observations on egyptian hyalomma ticks (ixodoidea: ixodidae ). 5. biological notes and differences in identity of hyalomma anatolicum and its subspecies anatolicum koch and excavatum koch among russian and other workers identity of hyalomma lusitanicum koch. entomological society of america, 52:243– 261. kaiser, m.n., sutherst, r.w., bourne, a.s., gorissen, l. & floyd, r.b. 1988. population dynamics of ticks on ankole cattle in five ecological zones in burundi and strategies for their control. preventive veterinary medicine, 6:199– 222. karrar, g., kaiser, m.n. & hoogstraal, h. 1963. ecol ogy and host-relationship of ticks (ixodoidea) infesting domestic animals in kassala province, sudan with special reference to amblyomma lepidum donitz. bulletin of entomological research, 54:509–522. macartan, b.m., hunter, a.g., pegram, r.g. & bourne, a.s. 1987. tick infestation on livestock in yemen arab republic and potential as vectors of livestock diseases. tropical animal health and production, 19:21–31 pegram, r.g. & banda, d.s. 1990. ecology and phenology of cattle ticks in zambia: development and survival of free living stages. experimental and applied. acarololgy, 8:291–301. van straten, m. & jonjegan, f. 1993. ticks (acari: ixodidae) infesting the arabian camel (camelus dromedarius) in sinai, egypt with a note on acaricidal efficacy of ivermectin. experimental and applied acarololgy, 17:605–612. walker, a.r., bouatour, a., camicas, j.l., estrada peña, a., horak, i.g., latif, a.a., pegram, r.g. & preston, p.m. 2003. ticks of domestic animals in africa. a guide to identification of species. bioscience report, 42 comiston drive, edinburgh eh10 5 qr scotland, uk. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: maryke l. ferreira1 annemariè avenant-oldewage1 affiliations: 1department of zoology, university of johannesburg, south africa correspondence to: annemariè avenant-oldewage postal address: po box 524, auckland park 2006, south africa dates: received: 04 feb. 2013 accepted: 04 may 2013 published: 13 june 2013 how to cite this article: ferreira, m.l. & avenant-oldewage, a., 2013, ‘selected haematological changes in clarias gariepinus (burchell, 1822) infected with a trypansosoma sp. from the vaal dam, south africa, onderstepoort journal of veterinary research 80(1), art. #572, 3 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.572 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. selected haematological changes in clarias gariepinus (burchell, 1822) infected with a trypansosoma sp. from the vaal dam, south africa in this research communication... open access • abstract • introduction • materials and methods • results • discussion • conclusion • acknowledgements    • competing interests    • author's contributions • references abstract top ↑ the use of haematological techniques to assess fish health is generally accepted. the aim of the current study was to determine selected haematological changes that occur in clarias gariepinus (burchell, 1822). infected with trypanosomes. blood films were prepared according to standard techniques to confirm trypanosome infections and whole blood was collected, the serum and plasma separated, and prepared for albumin and total protein concentration analysis. plasma albumin levels were significantly higher in infected wild caught fish than in uninfected wild caught fish and uninfected breeding stock. serum albumin levels were significantly lower in infected wild caught fish when compared to uninfected breeding stock. the total plasma and serum protein levels were within the normal range for c. gariepinus, that is, 3 g – 6 g/100 ml. the total plasma protein levels varied significantly between the three groups. however, the total serum protein levels were only significantly different between uninfected breeding stock and uninfected wild caught fish, as well as uninfected breeding stock and infected wild caught fish. the total protein levels were significantly higher in infected wild caught fish than in the other groups, a possible indication of an infection or inflammatory host response. introduction top ↑ to date, approximately 200 species of piscine trypanosomes are known (gupta & gupta 2012). in nature, trypanosome infections are common, especially where the leech vector is abundant. the effect of a trypanosome infection depends solely upon the host’s energy resources, and the impact ranges from physiological (tandon & chandra 1977), metabolic, pathological (lom & dykova 1984), biochemical (gupta & gupta 1986) and asymptomatic, to behavioural anomalies and syndromes (barber, hoare & krause 2000). haematological techniques are important for the assessment of the impact of trypanosome infections on fish health (shah & altindağ 2004). osman, fadel and ali (2009) reported that clarias gariepinus (burchell, 1822) infected with trypanosoma mukasai hoare, 1932 showed significant decreases in total serum protein, albumin, globulin levels, albumin/globulin ratios and cholesterol concentrations. these authors also noted an increase in serum aspartate aminotransferase and alanine aminotransferase enzyme activities. however, no significant effect was noted for serum urea, creatinine and uric acid concentrations (see osman et al. 2009). gupta and gupta (2012) recorded erythropenia due to piscine trypanosomiasis in various fish hosts. symptoms of piscine trypanosomiasis range from anaemia (woo 1981; islam & woo 1991) to leucocytosis, hypoglycaemia and hypocholesterolemia (gupta & jairajpuri 1983; gupta & gupta 1986). several authors have reported on the effect of trypansome infection on albumin (osman et al. 2009; kharat & kothawade 2012; kovyrshina & rudneva 2012) and total protein levels (mlay et al. 2007; osman et al. 2009; kharat & kothavade 2012). according to gupta and gupta (2012), increased erythroblasts, haemoblasts and macrophages can also occur as clinical manifestations. the aim of the current study is to determine whether any haematological changes occur in the african sharptooth catfish, c. gariepinus infected with trypanosomes. materials and methods top ↑ twenty-one c. gariepinus were collected from the vaal dam (26°52.249′s 28°10.249′e), south africa, using gill nets. the fish were kept in fully prepared aquaria. non-infected c. gariepinus breeding stock were maintained at the university of johannesburg aquarium, south africa. blood films were prepared from all fish. infections were confirmed by observing haemoflagellates in methanol fixed and giemsa stained films prepared according to the method described by davies et al. (2005). blood samples were collected from both trypanosoma infected (wild caught) and non-infected (wild caught and breeding stock) fish and centrifuged to concentrate cellular components. serum was obtained by allowing blood to clot for 30 min – 45 min (yielding serum without fibrinogen) and then centrifuged at 3000 rpm for 15 min. in a duplicate set, the plasma was separated by centrifuging unclotted blood at 3000 rpm for 10 min. plasma and serum were respectively transferred to separate eppendorf tubes and stored in liquid nitrogen until further examination. samples were thawed at room temperature. bcg albumin (diag-250) and total protein (qcpr-500) values were measured following standard operating procedures supplied by biocom biotech. data are presented as mean ± standard error. a two-way analysis of variance (anova) was done to calculate any significant differences between infected and uninfected groups using graph pad prism 6, version 6.01, significance was set at 95%. results top ↑ the trypanosomes in c. gariepinus blood films resemble t. mukasai. morphological characteristics include a nucleus anterior to the mid-line, similar to the descriptions of baker (1960), negm-eldin (1997), smit, davies and van as (2000) and ferreira and avenant-oldewage (2013). of the 21 c. gariepinus collected from the vaal dam, only 19% (4/21) were infected. parameters between plasma and serum were similar (table 1). significant differences (p < 0.05) were noted between albumin and total plasma protein levels. the two-way anova indicated significant differences between plasma albumin levels between uninfected breeding stock and infected wild caught fish, as well as between uninfected wild caught fish and infected wild caught fish (table 1). serum albumin levels differed significantly between uninfected breeding stock and uninfected wild caught fish, as well as between uninfected breeding stock and infected wild caught fish (table 1). significant differences in the total plasma protein were seen between all three groups of fish, although total serum protein differed significantly only between uninfected breeding stock and uninfected wild caught fish, uninfected breeding stock and infected wild caught fish, and uninfected wild caught and infected wild caught fish. total protein in uninfected wild caught fish was significantly higher than in uninfected wild caught fish or uninfected breeding stock fish serum and plasma. table 1: clarias gariepinus (burchell, 1822) collected from the vaal dam, south africa haematological parameters tested. it was also noted that some of the fish collected from the vaal dam and vaal barrage were infected with proteocephalus glanduligerus (janicki, 1928), tetracampos ciliotheca (wedl, 1861) and unidentified nematodes, and the parasitemia in these fish was very low. the presence of cestodes was previously recorded from this locality (madanire-moyo & avenant-oldewage 2013). discussion top ↑ blood parameters are patho-physiological indicators of the whole body and are thus important in diagnosing the health status of fish (maheswaran et al. 2008). the albumin concentration in fish plasma and serum reflects the health of the animal, liver function, metabolic status and stress conditions (kovyrshina & rudneva 2012). plasma albumin levels were significantly higher (table 1) in trypanosome infected wild caught fish than in uninfected wild caught and uninfected breeding stock. the lowest levels were observed in uninfected fish reared as breeding stock. in a study done by kharat and kothawade (2012) on the haematology of clarias batrachus (linnaeus, 1758) with trypanosomosis, higher levels of albumin were reported in infected fish. however, these levels were not significantly different from those of uninfected fish. osman et al. (2009) reported low serum albumin levels in trypanosome-infected c. gariepinus, which resulted in generalised oedema and haemodilution. in the current study, serum albumin levels were significantly lower for infected wild caught fish when compared to uninfected breeding stock, indicating haemodilution caused by the trypanosome infection. parasite infection is most likely not the only factor responsible for variation in albumin levels, since it has been noted that diet composition (chukwuma, obioma & christopher 2010; mcqueen et al. 2011; kovyrshina & rudneva 2012), fish species, age, life cycle stage, sexual maturity, health and environmental factors (kovyrshina & rudneva 2012) also affect albumin levels. thus the high albumin levels observed in uninfected breeding stock may also be attributed to their diet and/or their age (hasnain et al. 2004). low plasma protein levels are often linked to starvation and depletion of energy stores (lockhart & metner 1984; cunjak 1988). plasma proteins include globulins, fibrinogens and albumins, and are vital in transporting materials via the circulation system (inyang, daka & ogamba 2010). adams, brown and goede (1993) recorded the normal range for total protein to be between 3 g – 6 g/100 ml and adeyemo et al. (2003) recorded the reference value to be 3.8 mg ± 0.11 mg/100 ml. in the current study, total protein values for all groups were within the range proposed by adams, brown and goede (1993) (table 1). uninfected breeding stock levels (plasma and serum) were the lowest, indicting depletion of energy stores (table 1). fish infected with trypanosomes had a higher total protein plasma and/or serum levels. according to khan (1977, 1980), a decrease in plasma protein is associated with trypanosome infections. this was also noted by smirnova (1970) for serum protein levels of burbot, lota lota (linnaeus, 1758), infected with trypanosoma lotae (smirnova, 1970) and similarly by tandon & joshi (1973) for fish infected with trypanosoma vittati (tandon & joshi, 1973) and trypanosoma maguri (tandon & joshi, 1973). conclusion top ↑ wild fishes are frequently infected with a variety of pathogens and are regularly subjected to various stress factors, thus it is difficult to assign abnormalities to any one particular cause (woo 2006). future studies should include assessment of other parasites (internal and external) to exclude their effect on blood parameters. hence the present study should be seen as preliminary. acknowledgements top ↑ the university of johannesburg is thanked for providing infrastructure and funding to annemariè avenant-oldewage. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. author’s contributions m.l.f. (university of johannesburg) was a post-doctorate student and performed analysis on blood and provided the first draft of the manuscript. a.a-o. 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publishing, wallingford. article information authors: tingting cui1 jun xiong1 yongzhi wang2 xintian wen1 xiaobo huang1 yong huang1 xiaoping ma1 zhongkai hu1 qin zhao3 sanjie cao1 affiliations: 1college of veterinary medicine and institute of preventive veterinary medicine, sichuan agricultural university, china2sichuan rural science and technology development centre, chengdu, china 3college of food science, sichuan agricultural university, china correspondence to: sanjie cao postal address: 46 xinkang road, ya’an 625014, china dates: received: 27 june 2012 accepted: 31 oct. 2012 published: 04 mar. 2013 how to cite this article: cui, t., xiong, j., wang, y., wen, x., huang, x., huang, y. et al., 2013, ‘construction of an artificial recombinant bicistronic plasmid dna vaccine against porcine rotavirus’, onderstepoort journal of veterinary research 80(1), art. #498, 8 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.498 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. construction of an artificial recombinant bicistronic plasmid dna vaccine against porcine rotavirus in this original research... open access • abstract • introduction • materials and method    • bacterial strains, plasmid, virus, cell lines and experiment mice    • construction and reforming of the eukaryotic bicistronic expression vector pvaxd-asd    • construction of vaccine plasmid and its transformation into attenuated salmonella typhimurium    • transfection of cos-7 cells and transient expression    • detection of the transcription of nsp4 and vp7 genes in vivo    • stability and safety of recombinant attenuated salmonella typhimurium    • inoculation of mice    • collection and analysis of samples    • measurement of interferon-γ production    • ethical considerations • results    • reforming eukaryotic expression vector pvax1-asd and pvaxd-asd    • construction of the transient expression of vaccine plasmid pvax1-asd-vp7, pvax1-asd-nsp4, pvaxd-asd-nsp4-vp7    • transient expression of recombinant plasmids in cos-7 cells    • transcription of recombinant plasmids in vivo using attenuated salmonella typhimurium as transgenic vehicle    • stability and safety of recombinant attenuated salmonella typhimurium    • production of serum anti-rotavirus igg antibodies    • production of intestinal lavage anti-rotavirus iga antibodies    • detection of interferon-γ secretion by splenocytes • trustworthiness • discussion • conclusion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ the attenuated salmonella typhimurium χ4550 strain was used to harbour a reconstructed bicistronic dna vaccine against porcine rotavirus, which carried the rotavirus nonstructural protein 4 (nsp4) and vp7 genes simultaneously. using a balanced lethal system, the kanamycin resistance gene of expressing eukaryotic plasmids pvax1 and pvaxd were replaced by the aspartate β-semialdehyde dehydrogenase (asd) gene. the nsp4 cleavage product (259–525) of rotavirus osu strain and vp7 full-length genes were amplified by reverse transcription polymerase chain reaction and then inserted into the eukaryotic single-expression plasmid, pvax1-asd, and the eukaryotic dual-expression plasmid, pvaxd-asd, respectively. the recombinant plasmids pvax1-asd-nsp4, pvax1-asd-vp7 and pvaxd-asd-nsp4-vp7 were transformed into the attenuated s. typhimurium χ4550 strain by electrotransformation. an indirect immunofluorescence assay of the expressed cos-7 cell suggested that the recombinant s. typhimurium χ4550 strain was constructed successfully. the recombinant s. typhimurium χ4550 strain was orally administered to balb/c mice. the group immunised with dual-expression plasmids produced a significantly higher level of serum immunoglobulin g (igg) and intestinal immunoglobulin a (iga) than the group immunised with single-expression plasmids. these results indicated that eukaryotic bicistronic plasmid dna vaccines could be successfully constructed to enhance humoural, mucosal and cellular immune response against rotavirus infection. introduction top ↑ rotavirus is a highly infectious, triple-layered icosahedral, non-enveloped virus particle with a genome of 11 segments of double-stranded ribonucleic acid (rna) (petrie, estes & graham 1983). it is recognised as the most important cause of acute gastroenteritis and dehydration in young children and animals (estes 2001). although highly effective, currently available reassortant tetravalent rhesus rotavirus vaccines were found to cause a higher occurrence of intussusception in young children (glass et al. 2006). moreover, the safe rotavirus-like particle (vlp) vaccines induce only partial protection and intestinal antibody secreting cell responses when used in conjunction with live attenuated human rotavirus (gonzalez et al. 2004; yuan et al. 2001) and 2/6-vlp vaccines do not protect suckling mouse pups from rotavirus diarrhoea (coste et al. 2000). thus, effective protective rotavirus vaccines that are unlikely to cause intussusception would be highly desirable. the use of dna vaccines is a new approach to protect animals and children against rotavirus. vlp and dna vaccines may constitute a third generation of rotavirus vaccines (chen et al. 1998; o’neal et al. 1997). it was previously reported that parenteral immunisation with rotavirus vp4, vp6 and vp7 dna vaccines induce high levels of serum rotavirus immunoglobulin g (igg) but fail to protect mice against viral challenge (chen et al. 1997; herrmann et al. 1999; yang et al. 2001). it is therefore necessary to develop an efficient method to increase the immunogenicity of dna vaccines.the rotavirus nonstructural protein 4 (nsp4) is the first described viral enterotoxin and may inducing doseand age-dependent diarrhoea in neonatal mice without causing histological alterations (ball et al. 1996). when mice were infected with rotavirus, nsp4-specific immunoglobulin a (iga) antibodies were generated in response to mucosal immunisation and transcytosed from the basal lamina into the mucosal epithelium (gebert 1997). further, orally immunised mice produced maternal antibodies against nsp4, generating passive protection in newborn mouse pups against rotavirus challenge (ward, rich & besser 1996). thus, nsp4 is thought to be a promising candidate for a rotavirus dna vaccine that induces a significant and effective immune response (ball et al. 2005). vp7 is a structural protein of the outer capsid of a rotavirus and is encoded in the seventh, eighth or ninth gene segment of the genome (depending on the viral strain) (song & hao 2009). vp7 may be the dominant immunogen for the production of neutralising antibodies, which are thought to have an important role in the protection against rotavirus-induced diarrhoea (ward et al. 1990, 1993) and associate resistance to protect the host against diarrhoea upon challenge with the virulent parental rotaviruses (hoshino et al. 1988). matsui et al. (1989) demonstrated that vp7 monoclonal antibodies (mabs) have two distinct epitopes, with the specific epitopes defined by vp7-specific mab 57-8 mediating protection against at least two rotavirus serotypes. from this we concluded that vp7 plays an important role in resisting rotavirus challenge. in this study, a bicistronic plasmid was formed as a co-expression vector carrying the c-terminal of the porcine rotavirus (prv) nsp4 and vp7 genes, and then inserted into the salmonella typhimurium χ4550 strain as a dna vaccine that was orally administered to balb/c mice. the s. typhimurium χ4550 strain consists of attenuated bacteria of which the asd gene has been deleted. we evaluated the mucosal and systemic antibody responses. the results showed that the co-expression dna vaccine mediated by a bicistronic plasmid could significantly enhance humoural and mucosal immune response. materials and method top ↑ bacterial strains, plasmid, virus, cell lines and experiment mice the attenuated s. typhimurium χ6212, χ3730 and χ4550 strains and plasmid pya3342 were kindly provided by professor kai schulze of the helmholtz centre for infection research, braunschweig, germany. the eukaryotic expression vector pvax1 was purchased from invitrogen, carlsbad. it ‘contains cytomegalovirus (cmv) immediate-early promoter for efficient expression and bovine growth hormone (bgh) poly a signal for mrna stability’ (yang et al. 2009). the osu rotavirus strain was used to generate cdna and produce virus antigen stock, which was applied as the antigen in the enzyme-linked immunosorbent assays (elisas). ma104 cells and cos-7 cells were purchased from the china center for type culture collection (wuhan, china). six-week-old inbred balb/c female mice were obtained from the chengdu institute of biological products (chengdu, china). construction and reforming of the eukaryotic bicistronic expression vector pvaxd-asd the vectors pvaxa and pvaxb were constructed by reforming the multiple cloning site of pvax1. the product of pvaxa polymerase chain reaction (pcr) (-pcmv-mcs-bghpolya-) was linked with pvaxb to form vector pvaxd with two different multiple cloning sites. dual-enzyme digestion identification showed that the bicistronic vector pvaxd was successfully constructed. the aspartate β-semialdehyde dehydrogenase (asd) gene was amplified by pcr and digested by restriction enzymes bamhi and xbai from plasmid pya3342. we then used the klenow fragment enzyme character to fill the sticky end of dna fragments derived by enzyme restriction digestion to a flat end, and linked it with pvax1 and pvaxd vectors that were removed from the 5’ terminal phosphate group by calf intestinal alkaline phosphatase. the reformed plasmids, pvax1-asd and pvaxd-asd, were reconstructed successfully and plasmid pvaxd-asd was sequenced by invitrogen, shanghai. construction of vaccine plasmid and its transformation into attenuated salmonella typhimurium viral rna was extracted from ma104 cells infected with the osu rotavirus strain by using the rnasimple total rna kit (tiangen, beijing). total cdna was obtained from the viral total rna by a reverse transcriptase reaction procedure. the nsp4 segment was amplified from the cdna using the sense primer s1 5’-ctgcagccgccatggaacaggttactac-3’ (forward) and the antisense primer 5’-ctcgagctcacata gacgcagttacttccgac-3’ (reverse). underlined areas indicate the psti and xhoi sites, respectively. the vp7 segment was amplified from the cdna using the sense primer s2 5’-gctagccgccatgtatggtattgaat-3’ (forward) and the antisense primer 5’-ggtaccatctagactcggt aataaaaggcag-3’ (reverse). underlined areas indicate the nhei and kpni sites, respectively. the amplified dna fragments of the nsp4 and vp7 genes were cloned into the pmd19-t vector (takara) and sequenced. the nsp4 fragment was released from the pmd19-t plasmid by psti and xhoi digestion and ligated into the reformed pvax1-asd plasmid. pvaxd-asd vectors were cut by the same enzymes. the recombinant plasmids were named pvax1-asd-nsp4 and pvaxd-asd-nsp4. the vp7 fragment was subsequently released from the pmd19-t plasmid by nhei and kpni digestion and ligated into the pvax1-asd and pvaxd-asd-nsp4 vectors, cut by the same enzymes. the recombinant plasmids were named pvax1-asd-vp7 and pvaxd-asd-nsp4-vp7. these plasmids, including pvax1-asd-vp7, pvax1-asd-nsp4, pvaxd-asd-nsp4-vp7 and pvax1-asd, were electroporated into s. typhimurium χ3730 with a cuvette (1.8 kv, 200 ω, 25 μf).positive transformants were electroporated into s. typhimurium χ4550 using the same method as described above. lastly, the positive transformants were verified by pcr amplification and digested with restriction enzymes. the resulting strains were named χ4550 (pvax1-asd-vp7), χ4550 (pvax1-asd-nsp4), χ4550 (pvaxd-asd-nsp4-vp7) and χ4550 (pvax1-asd). transfection of cos-7 cells and transient expression cos-7 cells were seeded in six wells of tissue culture plates (costar, shanghai). cells were transfected with the recombinant plasmids pvax1-asd-vp7 in the first well, pvax1-asd-nsp4 in the second well, pvaxd-asd-nsp4-vp7 in the third and fourth wells, and pvax1-asd in the fifth and sixth wells according to the instructed protocol provided by the supplier (invitrogen life technologies, carlsbad). cells were washed with phosphate buffered saline (pbs) after 36 hours, then fixed with an ice-cold methanol and acetone mixture (1:1) at 4 °c for 30 min and washed again with pbs. two kinds of diluted primary antibody were collected from rabbits immunised by prokaryotic express protein vp7 and nsp4, respectively, and incubated at 37 °c for 1 h. diluted secondary antibodies (fitc-conjugated goat anti-rabbit igg) were then added to all groups and plates were incubated at 37 °c for 1 h. detection of the transcription of nsp4 and vp7 genes in vivo six-week-old mice were orally inoculated with 1 × 109 cfu of χ4550 (pvaxd-asd-nsp4-vp7). three days post immunisation, payer’s patches were removed from three mice and pooled. cellular rna was isolated from homogenised payer’s patches with trizol (takara, dalian) according to the manufacturer’s instructions. the transcripts of prv nsp4 and vp7 genes in payer’s patches were analysed by means of reverse transcription (rt) pcr using specific primers s1 and s2 described earlier (yang et al. 2009). stability and safety of recombinant attenuated salmonella typhimurium a single bacterial colony of the selected recombinant χ4550 (pvaxd-asd-nsp4-vp7) strain was inoculated into antibiotic-free luria-bertani (lb) liquid medium and incubated at 37 °c, 200 r/min until the optical density of the culture reached approximately 0.6 at 600 nm. we then inoculated the bacterium (1:50) into antibiotic-free lb liquid medium and incubated samples at 37 °c, 200 r/min. over the next 18 hours, at three-hour intervals, the bacterium was transferred from the lb liquid medium onto lb agar medium and identified by rt-pcr with specific primer s2. a gavage needle was used to inoculate six-week-old balb/c mice (groups of three) intragastrically with χ4550 (pvaxd-asd-nsp4-vp7) at a dosage of 1×108 cfu, 1×109 cfu and 5×109 cfu. they were boosted with the same dosages two weeks later. at the same time, the control group was given pbs at the same dosages. the health of the mice was observed every day for 30 days. inoculation of mice eight-week-old balb/c mice were randomly assigned to six groups of 15 each: • group a: pbs (ph 7.2) • group b: χ4550 (pvax1-asd) • group c: χ4550 (pvax1-asd-nsp4) • group d: χ4550 (pvax1-asd-vp7) • group e: χ4550 (pvaxd-asd-nsp4-vp7) • group f: χ4550 (pvax1-asd-ns4)+ χ4550 (pvax1-asd-vp7). on day 1 mice of groups b, c, d and e were orally immunised with 0.2 ml salmonella suspension (1 × 109 cfu) and boosted with the same dosage twice at two-week intervals. the co-immunised group (f) received the same dosage of two equally mixed salmonella suspensions (1 × 109 cfu) on days 1, 14 and 28. the control group (a) received 0.2 ml pbs on days 1, 14 and 28. collection and analysis of samples three mice from each group were sacrificed for sera and intestinal lavage on days 1, 14 and 28. blood was collected from the submaxillary sinuses of the mice and sera were prepared and stored at –20 °c until use. the intestinal fluid of each mouse was prepared by dicing the entire small intestine, excluding fat and the pancreas, and triturating it in 5 ml pbs containing 1 mmol/l phenylmethylsulfonyl fluoride (pmsf). to remove cellular debris, samples were centrifuged at 12 000 × g for 30 min. the supernatant was collected as resultant intestinal lavage and stored at –20 °c until use.for measurement of rotavirus serum igg and intestinal iga levels in immunised mice, an indirect elisa was used as described previously (yang et al. 2009). data of serum igg and intestinal iga titres were analysed using one-way analysis of variance. significant differences were found amongst all immunisation groups (p ≤ 0.05). measurement of interferon-γ production spleens of three mice from group e were removed aseptically and pooled into 10 ml rpmi1640 medium 14 days after the third immunisation. splenocytes were isolated by using a cell constrainer and centrifuged at 1000 rpm for 10 min at room temperature. the cell pellet was re-suspended in 10 ml of the mentioned medium and the concentration of cells was adjusted to 5 × 106 cells/ml. an aliquote of 2 ml of this cell suspension and 20 mg of stimulant protein nsp4 and vp7 equivalent admixture were added to each well of a 24-well pate. plates were incubated in co2 at 37 °c for 72 h. the splenocyte culture supernatants were collected for interferon-γ (ifn-γ) analysis using an ifn-γ elisa kit according to the manufacturer’s instructions (fangcheng biotech, beijing). ethical considerations animals were handled humanely during sample collection under the supervision of veterinarians and according to procedures complying with chinese laws. results top ↑ reforming eukaryotic expression vector pvax1-asd and pvaxd-asd the asd segments with flat ends were ligated into plasmid pvax1 and pvaxd and then heat-shock transformated into χ6212 competent cells. fragments of 1400 base pairs (bp) were amplified by rt-pcr from a single bacterial colony, indicating that the vectors pvax1-asd and pvaxd-asd were reformed successfully (figure 1). figure 1: identification electrophoresis of recombinant pvax1-asd and pvaxd-asd with coenobium polymerase chain reaction. construction of the transient expression of vaccine plasmid pvax1-asd-vp7, pvax1-asd-nsp4, pvaxd-asd-nsp4-vp7 a fragment of 300 bp was amplified by rt-pcr from the prv osu strain. sequence analysis showed that the amplified sequence was 267 bp in length and encoded 89 amino acids. the result of sequence alignment showed that the predicted amino acid sequence exhibited 97.8% homology with the sequence of the nsp4 gene fragment (87–175) of the prv osu strain. the nsp4 gene fragment was inserted into the pvax1-asd and pvaxd-asd vectors. another 1000-bp fragment was amplified by rt-pcr from the prv osu strain. sequence analysis showed that the amplified sequence was 987 bp in length and encoded 326 amino acids. the result of sequence alignment showed that the predicted amino acid sequence exhibited 99.4% homology with the sequence of the vp7 gene fragment of the prv osu strain. the vp7 gene fragment was inserted into the pvax1-asd and pvaxd-asd-nsp4 vectors. the recombinant plasmids pvax1-asd-nsp4, pvax1-asd-vp7 and pvaxd-asd-nsp4-vp7 were electroporated into s. typhimurium χ4550 strain. they were subsequently detected by enzyme restriction analysis (figures 2, 3 and 4). figure 2: identification electrophoresis of recombinant pvax1-asd-nsp4 with restriction enzyme. figure 3: identification electrophoresis of recombinant pvax1-asd-vp7 with restriction enzyme. figure 4: identification electrophoresis of recombinant pvaxd-asd-nsp4-vp7 with restriction enzyme. transient expression of recombinant plasmids in cos-7 cells the expression of recombinant plasmids pvax1-asd-vp7, pvax1-asd-vp7 and pvaxd-asd-nsp4-vp7 was demonstrated by an indirect immunofluorescence assay. cytoplasmic fluorescence was observed in cos-7 cells transfected with all the recombinant plasmids, but not with the parental vector pvax1 (yang et al. 2009) (figure 5). figure 5: indirect immunofluorescence detection of the expression of recombinant plasmids in cos-7 cells. (a) indirect immunofluorescence result of cos-7 cells transfected with pvax1-asd-vp7; (b) indirect immunofluorescence result of cos-7 cells transfected with pvax1-asd-nsp4; (c) indirect immunofluorescence result of cos-7 cells transfected with pvaxd-asd-nsp4-vp7, by using anti-nsp4 serum; (d) indirect immunofluorescence result of cos-7 cells transfected with pvaxd-asd-nsp4-vp7, by using anti-vp7 serum and (e) indirect immunofluorescence result of cos-7 cells transfected with pvax1-asd, by using anti-vp7 serum. transcription of recombinant plasmids in vivo using attenuated salmonella typhimurium as transgenic vehicle three days after intragastrically inoculating mice with attenuated s. typhimurium χ4550 (pvaxd-asd-nsp4-vp7), payer’s patches were removed from three mice and used for rna extraction. payer’s patches are not only the main colonisation site of attenuated s. typhimurium but also an important immunologically relevant site in the context of mucosal responsiveness. two fragments of 300 bp and 1000 bp, respectively, were amplified by rt-pcr (figures 6 and 7). figure 6: detection of the transcription of the vp7 gene in vivo after mice were immunised orally with recombinant salmonella typhimurium (reverse transcription polymerase chain reaction). figure 7: detection of the transcription of the nsp4 gene in vivo after mice were immunised orally with recombinant salmonella typhimurium (reverse transcription polymerase chain reaction). stability and safety of recombinant attenuated salmonella typhimurium selected single bacterial colonies of recombinant χ4550 (pvaxd-asd-nsp4-vp7) were inoculated into antibiotic-free lb medium. the recombinant plasmids were subsequently identified by rt-pcr for 18 hours at three-hour intervals. the positive ratio reached 100%, showing that the recombinant plasmid was of high stability. balb/c mice that had been inoculated orally with χ4550 (pvaxd-asd-nsp4-vp7) at different dosages were used for safety analysis. no adverse reactions were observed during the observation period; the diet, stools and health of the mice were all normal. production of serum anti-rotavirus igg antibodies following oral immunisation with all recombinant s. typhimurium vaccines, all mice developed detectable serum igg titres against rotavirus. serum anti-rotavirus igg was measured by indirect elisa using a 1:100 dilution of immunised mouse serum. as shown in figure 8, the anti-rotavirus igg was produced fast and appeared 28 days post inoculation. in group b and c, we could infer that the antibody level of vp7 expression was significantly higher than nsp4 expression (p < 0.05). in addition, the co-expression of recombinant s. typhimurium χ4550 (pvaxd-asd-nsp4-vp7) produced the highest antibody level in group e, which was somewhat similar to that of group d and group f. no specific anti-rotavirus antibodies were observed in serum samples of group a (pbs) or group b (χ4550 [pvax-asd]). the experiment demonstrated that the co-expression s. typhimurium vaccine may considerably enhance the humoural immune response. figure 8: indirect enzyme-linked immunosorbent assay analysis of the anti-rotavirus igg antibody in murine serum after immunisation with different vaccines. production of intestinal lavage anti-rotavirus iga antibodies the ability of the recombinant s. typhimurium vaccine to induce a mucosal immune response was determined by detecting the level of iga antibodies to rotavirus in intestinal lavage samples. as shown in figure 9, the anti-rotavirus iga was produced fast and first appeared after 14 days. a one-way analysis of variance indicated that there was not much difference amongst groups c, d, e and f, which received χ4550 (pvax1-asd-nsp4), χ4550 (pvaxd-asd -vp7), χ4550 (pvaxd-asd-nsp4-vp7), χ4550 (pvax1-asd-nsp4) and χ4550 (pvaxd-asd -vp7), respectively. up to day 28 post inoculation, there were significant differences amongst each group. the igg antibody level of co-expression group e was the highest, that of group b and c was similarly low and for co-immunised group f the antibody level was notably higher than that of group c and d, but somewhat lower than for group e. no specific anti-rotavirus antibodies were observed in the intestinal lavage sample of group a (pbs) or group b (χ4550 [pvax-asd]). figure 9: indirect enzyme-linked immunosorbent assay analysis of anti-rotavirus iga antibody in murine serum after immunisation with different vaccines. detection of interferon-γ secretion by splenocytes the secretion of ifn-γ by splenocytes in the supernatant of the cultures was detected by elisa. a total of 1097.693 pg/ml ifn-γ was secreted by group e, which was much higher than that of the control group (group b) at 781.7125 pg/ml. this indicated that the immunity of mice was considerably increased post inoculation with the χ4550 (pvaxd-asd-nsp4-vp7) vaccine. trustworthiness top ↑ we declare that the experiments were conducted in a reliable, authentic and valid manner. discussion top ↑ this experiment demonstrated that a bicistronic dna vaccine that encodes the prv vp7 protein and the c-terminal of the nsp4 protein simultaneously and is delivered by an attenuated s. typhimurium χ4550 strain, was a potent vaccine, which induced a significantly higher level of serum igg, mucosal iga and splenocyte ifn-γ than in the mice immunised with a single-expression vaccine.dna immunisation is a considerable vaccination strategy, which has many desirable characteristics for an ideal vaccine, including induction of broad immune responses and long-lasting immunity against diseases. in addition, oral delivery of plasmid dna vaccines with bacteria as carriers is more efficacious than traditional dna vaccines. compared ‘to immunization with naked plasmid dna, no further plasmid amplification and purification steps are needed, thereby reducing cost and labour extensively’ (schoen et al. 2004). oral immunisation with a rotavirus dna vaccine was first demonstrated by chen et al. (1997). yang et al. (2001) reported that intramuscular immunisation of mice with vp6 dna vaccines induced high levels of vp6-specific serum igg and iga antibodies but not fecal iga antibodies. our study provided evidence that attenuated s. typhimurium strain χ4550 could be used as an oral delivery vector for prv dna vaccines. the target gene could be expressed both in vitro and in vivo to develop a specific humoural, mucosal and cellular immune response against prv. live bacteria as dna vaccine vectors offer many potential advantages in contrast to inactivated or attenuated vaccines. they are able to deliver multiple antigens safely with easy oral immunisation. bacteria naturally contain immunostimulatory molecules such as lipopolysaccharides, which can function as an adjuvant to provoke immune responses (pasetti, levine & sztein 2003). besides, salmonella carrier strains have been shown to escape from the primary vacuole of the phagosome and transfer plasmids effectively to mammalian host cells in vivo (schoen et al. 2004). live salmonella vectors have also been shown to induce a systemic immune response, including humoural, cellular and mucosal immunity against pathogen infection (lintermans & greve 1995). safety is a prerequisite for plasmid dna vaccines with live bacteria as vectors. in this study, we reconstructed the new plasmid by removing the kanamycin resistance gene and replaced it with the asd gene using a balanced lethal system. rt-pcr was used to ensure that the plasmids, pvax1-asd and pvaxd-asd, were reconstructed successfully. the asd gene is a diaminopimelic acid (dap) biosynthetic gene, which is an important component of the cell wall of gram-negative bacteria. as bacterial amino acids are not found in eukaryotes, no salmonella could survive without dap. an s. typhimurium χ4550 strain without an asd gene was chosen as the plasmid dna carrier as it was the only strain in which reconstructed plasmids could survive. during the experiment, the diet, stools and health of mice were all normal and no adverse reactions were observed. all the results suggested that the reconstructed s. typhimurium χ4550 strain vaccine was safe. the structural protein vp7 of a rotavirus constitutes the outer shell of the virion and elicits the production of distinct neutralising antibodies in the host. it is thought to play an important role in protecting rotavirus-induced diarrhoea. in previous research a murine model was constructed to express the rotavirus vp7 protein with a transmissible gastroenteritis virus vector to immunise and provide protection against infections (ribes et al. 2011). ‘rotavirus-specific antibodies were found only after immunization by the intraperitoneal route. partial protection against rotavirus-induced diarrhoea was achieved in suckling balb/c mice born to dams immunized with the recombinant virus’ (ribes et al. 2011) when they were orally challenged with the homotypic rotavirus. according to this study, improving the immunity of vp7 was stringently demanded. kavanagh et al. (2010) also demonstrated that the nsp4 protein of a rotavirus has mucosal adjuvant properties, because it is a multifunctional protein that functions in rotavirus morphogenesis and pathogenesis and was the first viral enterotoxin to be described. this discovery correlates with the observation that inclusion of specific adjuvants in vaccines can modify the presentation modality of antigens to the immune system and improve the induction of the immune response over that induced by the same antigen given alone (buonaguro, tornesello & buonaguro 2009 in kavanagh et al. 2010). for these reasons, we reconstructed a bicistronic dna vaccine harbouring the vp7 and nsp4 proteins synchronously. both serum igg and mucosal iga antibody response against the rotavirus were detected post inoculation with the dna vaccine. the vaccine expressing only the nsp4 protein elicited a weak anti-rotavirus igg and iga antibody response. however, the vaccine expressing only the vp7 protein induced a significantly stronger response, similar to that induced by co-immunised nsp4 and vp7 proteins. the bicistronic dna vaccine co-expressing the vp7 and nsp4 proteins induced the highest igg and iga antibody levels. thus, we concluded that the immunogenicity of a rotavirus dna vaccine could be improved by co-expressing the vp7 and nsp4 proteins. this study was the first to construct a eukaryotic bicistronic plasmid dna vaccine harboured by the prokaryotic expressing s. typhimurium χ4550 strain and elicited desirable humoural and mucosal immune response against rotavirus. further experiments are needed to elucidate the immunologic mechanism of this recombinant dna vaccine. conclusion top ↑ we described the construction of a bicistronic plasmid dna vaccine against porcine rotavirus, which carries the nsp4 and vp7 genes simultaneously. balb/c mice were orally immunised with an attenuated s. typhimurium χ4550 strain expressing the vaccine. significant levels of serum igg, intestinal iga and ifn-γ were induced by the reconstructed bicistronic dna vaccine, suggesting that it may be a promising vaccine candidate against porcine rotavirus. acknowledgements top ↑ we thank the science and technology support programme, sichuan province for integration and demonstration of the key technology research on modern pig industry. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions s.c. 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http://dx.doi.org/10.1016/s0264-410x(00)00543-0 yuan, l., iosef, c., azevedo, m.s.p., kim, y., qian, y., geyer, a. et al., 2001, ‘protective immunity and antibody-secreting cell responses elicited by combined oral attenuated wa human rotavirus and intranasal wa 2/6-vlps with mutant escherichia coli heat-labile toxin in gnotobiotic pigs’, journal of virology 75, 9229–9238. http://dx.doi.org/10.1128/jvi.75.19.9229-9238.2001, pmid:11533185 article information authors: stephen b. hughes1 melvyn quan2 alan guthrie3 martin schulman1 affiliations: 1department of production animal science, university of pretoria, onderstepoort, south africa2department of veterinary tropical diseases, university of pretoria, onderstepoort, south africa 3equine research centre, university of pretoria, onderstepoort, south africa correspondence to: stephen hughes postal address: section of reproduction, department of production animal science, faculty of veterinary science, private bag x04, university of pretoria, onderstepoort 0110, south africa. dates: received: 03 jan. 2012 accepted: 18 june 2013 published: 30 aug. 2013 how to cite this article: stephen b., hughes, s.b., quan, m., guthrie, a. & schulman, m., 2013, ‘development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor and insulin receptor expression in equine tissue’, onderstepoort journal of veterinary research 80(1), art. #402, 7 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.402 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. development of real-time reverse transcription polymerase chain reaction assays to quantify insulin-like growth factor and insulin receptor expression in equine tissue in this original research... open access • abstract • introduction • materials and methods    • sample collection    • rna extraction    • design of primers for amplification and sequencing of equine receptor mrna    • igf1r and insr mrna amplification and sequencing    • quantitative two-step real-time rt-pcr    • generation of igf1r and insr standard curves • results    • amplification and sequencing of equine igf1r and insr mrna    • quantitative two-step real-time rt-pcr    • igf1r and insr standard curves    • application of quantitative real-time rt-pcr assays • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the insulin-like growth factor system (insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor and six insulin-like growth factor-binding proteins) and insulin are essential to muscle metabolism and most aspects of male and female reproduction. insulin-like growth factor and insulin play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. in order to better understand the local factors that regulate equine physiology, such as muscle metabolism and reproduction (e.g., germ cell development and fertilisation), real-time reverse transcription polymerase chain reaction assays for quantification of equine insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were developed. the assays were sensitive: 192 copies/µl and 891 copies/µl for insulin-like growth factor 1 receptor, messenger ribonucleic acid and insulin receptor respectively (95% limit of detection), and efficient: 1.01 for the insulin-like growth factor 1 receptor assay and 0.95 for the insulin receptor assay. the assays had a broad linear range of detection (seven logs for insulin-like growth factor 1 receptor and six logs for insulin receptor). this allowed for analysis of very small amounts of messenger ribonucleic acid. low concentrations of both insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples, this was most likely due to the high level of glucose metabolism and glucose utilisation by these tissues. the assays developed for insulin-like growth factor 1 receptor and insulin receptor messenger ribonucleic acid expression have been shown to work on equine tissue and will contribute to the understanding of insulin and insulin-like growth factor 1 receptor physiology in the horse. introduction top ↑ gene expression analysis is of fundamental importance in biomedical research (bustin 2002). the various methods used include: northern blotting, in situ hybridisation, ribonuclease (rnase) protection assays, complementary deoxyribonucleic acid (cdna) arrays and real-time reverse transcription polymerase chain reaction (rt-pcr) (giulietti et al. 2001). polymerase chain reaction based assays are the most commonly used method for characterising gene expression and comparing messenger ribonucleic acid (mrna) levels in different samples (bustin 2002). the polypeptide hormones, insulin-like growth factor 1 (igf1) and insulin (ins) are similar in structure (rinderknecht & humbel 1978; torres et al. 1995). they elicit similar biological responses, regulate cell proliferation and mediate metabolic signals, but with differing potencies (morgan, jarnagin & roth 1986). these ligands cross-react competitively with igf1r receptor (igf1r) and ins receptor (insr) (torres et al. 1995). igf1r and insr are closely related members of the tyrosine kinase receptor super-family (garrett et al. 1998). these receptors are expressed in almost all mammalian cell types and are large trans-membrane tetrameric glycoproteins, which consist of two alpha-subunits and two beta-subunits (α2β2) and are linked by disulfide bonds (garrett et al. 1998). each alpha-subunit contains the extra-cellular ligand-binding site and is about 125 kilo-dalton (kda) – 140 kda in size. each beta-subunit has a trans-membrane domain and the catalytic intra-cellular tyrosine kinase domain is about 95 kda – 97 kda. tyrosine kinases are enzymes that phosphorylate the tyrosine portion of proteins (abbott, bueno, pedrini & murray et al. 1992). these receptors, like their ligands, are encoded by distinct genetic loci, which are believed to have evolved from a common ancestral gene (kim & accili 2002). they share > 50% overall amino acid sequence identity and 84% identity with tyrosine kinase domains (jones & clemmons 1995; pandini et al. 2002). in domestic animals, igf1 and ins play essential roles in reproduction (velazquez, spicer & wathes 2008). to understand the roles igf1 and ins play in the local regulation of the formation and competence of equine oocytes and spermatozoa, real-time rt-pcr assays were developed to evaluate igf1r and insr gene expression in equine tissues. materials and methods top ↑ sample collection to develop the assays, testes were harvested from each of three healthy friesian, thoroughbred, and warmblood stallions that had been admitted for routine orchidectomies at the onderstepoort veterinary academic hospital (ovah). to evaluate the assays, tissue samples from four mixed-breed adult horse mares were collected immediately after slaughter at a horse abattoir. the samples collected were obtained from the liver, spleen, kidney, lung, heart, skeletal muscle and endometrium.all tissue samples were collected using sterile forceps and a surgical blade and the blocks (5 mm × 5 mm × 5 mm) of tissue were placed into plastic 2.0 ml cryotubes (nunc, usa), containing approximately 10 volumes of rnalater® (ambion, usa). the samples were kept at 4 °c for 24 hours and then stored at -80 °c until analysis. rna extraction to extract rna, a sample of tissue was thawed at room temperature and approximately 30 mg of tissue was placed into a magna lyser® green beads tube (roche, germany) containing 900 μl rlt buffer (qiagen, usa) and 9 μl 14.3 m β-mercaptoethanol (β-me) (sigma). the samples were homogenised with a magna lyser® instrument (roche, germany) and two runs of 7000 revolutions per minute (rpm) for 45 seconds were performed; they were cooled on ice for 2 minutes between runs. samples were kept at room temperature for 30 minutes, then centrifuged at 14 000 rpm for 3 minutes. the supernatant was removed (approximately 600 μl) and transferred to 1.5 ml micro-centrifuge tubes. rna was extracted from the samples using the rneasy® mini kit (qiagen, usa) according to the manufacturer’s instructions and eluted in 50 μl nuclease-free water. rna concentration and purity was determined from the a260/a280 ratio, as measured by a nanodrop nd-1000 spectrophotometer (thermo fisher scientific, usa). design of primers for amplification and sequencing of equine receptor mrna the complete equus caballus genome had not been published at the time of this study. only a trace-file archive of the genome was available publically on genbank (ncbi) (n.d.). the human mrna sequences for igf1r (nm 000875) and insr (nm 000208) were assumed to be closely related to the equine equivalents and were used to perform a discontinuous megablast (altschul et al. 1990) with the equine trace-file archive to identify equine igf1r and insr trace file sequences. trace files were assembled using the staden package (staden 1996; staden, beal & bonfield 2000). a local alignment between each equine genomic dna contig and the respective human mrna sequence was performed using the water program in emboss (rice, longden & bleasby 2000). equine exons were identified as regions with a high degree of alignment between the human mrna and the equine dna sequences. the non-exon sequences were deleted from the trace file and the position of the exon-intron junctions noted. the exons were reassembled to yield a putative equine mrna sequence. the positions of the exon-exon junctions corresponded with the human exon-exon junctions. the putative equine mrna sequences were used to design primers with the aid of fastpcr® software v3.6.89 (kalender 2007). igf1r and insr mrna amplification and sequencing a one-step reverse transcription polymerase chain reaction (rt-pcr) for igf1r and insr mrna was performed with the geneamp® gold rna pcr core kit (lifetech, usa) according to the manufacturer’s instructions. two 25 μl reactions were performed per rna sample (2 µl) for each primer pair. cycling conditions for the rt-pcr were 42 °c for 12 minutes, 95 °c for 10 minutes and 40 cycles at 94 °c for 20 seconds, 57 °c for 30 seconds and 72 °c for 60 seconds. this was followed by a final extension step at 72 °c for 7 minutes and a hold at 4 °c on a geneamp® pcr system 9700 (lifetech, usa). amplification of the target dna was confirmed by running five μl of pcr product on an ethidium bromide-stained 1.5% agarose gel, electrophoresed at 110 volts and the dna was visualised by uv trans-illumination. the two rt-pcr reactions per sample were combined and purified using a qiaquick® pcr purification kit (qiagen, usa), according to the manufacturer’s instructions. the purified dna was eluted in 50 µl elution buffer (eb) (qiagen, usa).the same primers used for amplification were used for direct sequencing. a bigdye® terminator v3.1 cycle sequencing kit (lifetech, usa) was used according to the manufacturer’s instructions. phred (ewing & green 1998; ewing et al. 1998) and the staden package (staden 1996; staden, beal & bonfield 2000) were used for base calling and sequence assembly. the assembled igf1r and insr sequence for each of the three breeds of stallion was aligned with the equivalent human mrna sequence using clustalw software (embl-european bioinformatics institute n.d.). quantitative two-step real-time rt-pcr the sequences generated were used with primer express® software (lifetech, usa) to design equine igf1r and insr mrna primers and taqman® tamra™ probes (lifetech, usa). the primers were designed to amplify 68 and 74 nucleotide regions of the respective igf1r and insr genes. the probes were designed to span exon-exon junctions to ensure detection of mrna amplification over genomic dna. the probes were labeled with different fluorescent dyes (fam and vic) to allow differentiation between igf1r and insr. taqman® reverse transcription reagents (lifetech, usa) were used as described in the supplied protocol. reverse transcription was performed with random hexamers. a volume of 7.7 μl rna was added to the reagents up to a total volume of 20 μl/reaction. cdna from the tissue samples (liver, spleen, heart, lung, kidney, muscle and endometrium) collected from four horses was placed in a 9700 thermal cycler (lifetech, usa) and the following conditions were used: 25 °c for 10 minutes, 42 °c for 30 minutes, 95 °c for 5 minutes and a hold at 4 °c. a lightcycler® taqman® master kit (roche, germany) was used as described in the supplied protocol for the real-time pcr. five μl of cdna, a final concentration of 0.5 μm primers and a final concentration of 0.1 μm probe were used per reaction. cycling conditions in a lightcycler 2.0 (roche, germany) were as described in the protocol, with an annealing temperature of 60 °c. with each sample tested, a no-rt control was run as a control to screen for genomic dna contamination. rna copies per milligram of tissue were calculated using the following formulae: the volume (μl) of rna extracted (indicated with a) using the rneasy spin column (qiagen, usa), b indicated 7.7 μl of rna that were converted into 20 μl of cdna and 5 μl of cdna used or reaction and c indicates the 30 mg of tissue was homogenised in 900 μl rlt buffer (qiagen, usa) and 600 μl used for rna extraction. generation of igf1r and insr standard curves the real-time rt-pcr products were purified with the qiaquick pcr purification kit (qiagen, usa) and quantified using a nanodrop nd-1000 spectrophotometer (thermo fisher scientific, usa). the amplicons were cloned using the pgem® t easy vector system (promega, usa) according to the manufacturer’s instructions. the plasmids were linearised with sal 1 restriction enzyme. plasmids were sequenced using t7 and sp6 primers to ensure the absence of mutations in the inserts. the linearised plasmid was transcribed using the megashortscript™ kit (ambion, usa) according to the manufacturer's instructions.rna was resuspended in 20 μl te buffer (ambion, usa) and quantified using a nanodrop nd-1000 spectrophotometer, using an extinction coefficient of 33 for small rna < 200 nucleotides (megashortscript™ protocol). the rna concentrations and the molecular weight of the transcripts were used to calculate the rna copy numbers. aliquots of ten-fold dilution series (1 × 100 to 1 × 109 copies/μl) were prepared and stored at -80 °c until needed. the control step for the presence of plasmid dna in the serial rna dilutions was checked by running real-time pcr on the rna, without first performing a reverse transcription reaction (no-rt control). real-time rt-pcr of the standard dilution series, using 7.7 μl of rna per reaction, was repeated in triplicate on two separate days. all the data were used to calculate a linear regression equation in microsoft excel® (microsoft, usa) of cycle threshold (ct) against log copy number. the regression equation was used to calculate mrna copy numbers per reaction from the ct. efficiency of the assay was calculated using the following equation: efficiency = 10-1/slope – 1. [eqn 4] the limit of detection was defined as the copy number concentration where 95% of the rt-pcr’s would yield a positive result (ct ≤ 40) and was calculated using the karber equation (karber 1931). results top ↑ amplification and sequencing of equine igf1r and insr mrna two pairs of primers were designed to amplify an approximate 1000 base pair (bp) region of the putative equine igf1r mrna in two overlapping segments: forward_1 (gct aat tgt gaa gtg gaa ccc ac), reverse_1 (gta caa agt gaa tgg ccg gag), forward_2 (aca atg tca cag acc cag agg ag), reverse_2 (aga aca cag gat cag tcc acg ac); and one pair to amplify a 488 bp region of the putative insr mrna: forward_3 (tga tgt gta ccc cgt gcc tgg) and reverse_3 (tgg tct tca ggg cga tgt cgt).a region of both equine igf1r and insr mrna that spanned exon-exon junctions was amplified by pcr and products of the expected size were obtained (figure 1). these rt-pcr products were visualised on a gel; for igf1r the expected sizes were 520 bp and 453 bp, and for insr 488 bp. figure 1: bromide-stained 1.5% agarose gel of igf1r (520 and 453 bp) and insr (488 bp) pcr products, with positive (300 bp) and negative rt-pcr controls and dna ladders in first and last wells. the pcr products were sequenced and the sequences obtained for igf1r and insr mrna were identical for each breed of horse. basic local alignment search tool (blast) (altschul et al. 1990) showed a 92% and 93% identity of the equine igf1r and insr mrna sequences with the human homologs (table 1). table 1: results of the discontiguous megablast of the real-time rt-pcr product sequence showing the five most similar igf1r and insr mrna sequences. quantitative two-step real-time rt-pcr the equine mrna primers and taqman® tamra™ (inqaba biotechnical industries, south africa) probes that were designed were: igf1r – forward primer (cag tgc ctc caa ctt cgt ctt), reverse primer (ccg gcc cag gaa tgt ca) and probe (vic-caa gaa cca tgc ctg cag aag gag ca), and the product size amplified was 67 nucleotides. insr – forward primer (tcc gga agt tac gcc taa ttc), reverse primer (ctg gtt gtc caa ggc gta ga) and probe (fam-tga gta gtt ccc gat ttc caa ggt ctc t), and the product size amplified was 73 nucleotides (figure 2). figure 2: location and sequence of primers (arrows) and taqman® tamra™ probes (blocks) designed for quantification, (a) igf1r and (b) insr by real-time rt-pcr. igf1r and insr standard curves clones with the real-time rt-pcr product insert were sequenced to confirm the presence and sequence of the insert. no mutations in the clones were detected.transcription of the plasmid yielded 2.40 μg rna/μl and 4.51 μg rna/μl for igf1r and insr respectively. plasmid dna contamination was evaluated by running a no-rt control and a no template control in the real-time pcr. plasmid dna was detected, but for igf1r, the dna concentration was 9800, and for insr 76 000 times less than the rna; this was therefore considered to not be significant (table 2). table 2: comparison between rt and no-rt controls, using cycle threshold (ct) values of an igf1r and insr dilution series. the calculated regression equation for the igf1r dilution series was: y = -3.301x + 45.59 [eqn 5] the calculated regression equation for the insr dilution series was: y = -3.444x + 47.42 (figure 3). [eqn 6] figure 3: linear regression of six replicates of (a) igf1r and (b) insr standard curves (from ct rt-pcr results). the efficiency and sensitivity of the igf1r assay are 1.01 and 192 copies/μl and for the insr assay 0.95 and 891 copies/μl respectively. application of quantitative real-time rt-pcr assays the log transformed mean igf1r mrna concentration of equine (n = 4) tissue samples (liver, spleen, heart, lung, kidney, muscle and endometrium) was 104.91 ± 101.04 standard deviations (sd) copies per mg tissue with a range of 103.66 (spleen) to 106.19 (heart) (figure 4). using rna as the unit of measurement, the mean igf1r mrna concentration was 104.78 ± 101.63 sd per μg rna with a range of 102.92 (spleen) to 106.83 (muscle). in all cases, with the exception of heart and muscle, the igf1r mrna concentrations per mg tissue were higher than the concentration per μg rna. figure 4: mean log, (a) igf1r (b) insr mrna copies per mg tissue (downward diagonal) and ìg rna (black) ± standard error. discussion top ↑ this study describes the development of real-time rt-pcr assays for the preliminary characterisation of igf1r and insr mrna expression in selected equine tissues. in order to develop these real-time rt-pcr assays, equine igf1r and insr trace files were identified from a megablast with human igf1r and insr mrna. this initial step was necessary as the only public horse genome data that were available at the time of this study was a dna trace-file archive, accessible through genbank (ncbi) (n.d.). this increased the complexity of the study. there is more equine genome data available now, but much of the mrna sequence data are predicted, whereas the equine mrna data used in this study were sequenced directly. the assays were designed to span exon-exon junctions to preferentially amplify mrna over genomic dna. exons were identified as areas with a high degree of identity between the equine dna trace-files and the human mrna sequences. large regions of non-similar sequences separated the exons in the equine trace files. the exon-exon junctions of the equine mrna were identical to that of the human, suggesting a common homology. both assays were efficient, sensitive and had a broad linear range of detection (seven logs for igf1r and six logs for insr). the assays worked well in our hands and were sensitive and specific for the detection of equine igf1r and insr mrna. igf1r and insr were present on the surface of almost all cell types (desoye et al. 1997; leroith et al. 1995; seino, seino & bell 1990). however, the expression of these receptors in different tissue types may vary depending on the metabolic and mitogenic characteristics of the cells within these tissues and there may be variation between individual animals due to age (georgieva et al. 2003; hess & roser 2001; lackey, gray & henricks 2000), nutritional state (balage et al. 1990; leroith et al. 1995) and stage of reproductive cycle (desoye et al. 1997; ginther et al. 2003; lackey et al. 2000; shimizu et al. 2008; silva, figueiredo & van den hurk 2009). in our study the igf1r and insr profiles in various equine tissues were similar. low concentrations of both igf1r and insr mrna were detected in endometrium, lung and spleen samples, whilst high concentrations were detected in heart, muscle and kidney samples. the high level of insr expression in the heart, muscle and kidney is probably associated with the high level of glucose metabolism and utilisation by these tissues. for all tissues, except the endometrium, the insr mrna concentrations were higher than the igf1r mrna concentrations. the mean insr:igf1r mrna ratio was highest in the liver tissue (22:1) and lowest in the endometrial tissue (1:2). the liver manufactures most of the circulating igf1 (jones & clemmons 1995; navarro et al. 1999) and has the highest levels of igf1 mrna and peptide expression, but the lowest expression of igf1r mrna (jones & clemmons 1995). it is highly probable that an organ manufacturing a hormone with an endocrine function will have a low concentration of receptors for that hormone. this would ensure that the hormone enters the general circulation and exerts its endocrine effect elsewhere in the body. conclusion top ↑ the sensitivities of the assays at 95% using the karber equation are 192 copies/μl and 891 copies/μl for igf1r mrna and insr respectively. this allows analysis of very small amounts of mrna. however, for these assays to be reliable they need extensive and accurate optimisation. hence, a study to describe the steps used to optimise and validate the assays for real-time rt-pcr of equine igf1r and insr mrna is needed. such a study should attempt to avoid co-amplification of genomic dna, reduce inter-assay variability and be normalised to a relevant housekeeping gene. measuring gene expression at the mrna level requires the inclusion of a reliable housekeeper gene for accurate data interpretation (housekeeper genes are evenly expressed amongst different tissues of an organism, at different stages of development, between normal and diseased states and should not be affected by experimental treatment itself (e.g., beta-actin, glyceraldehyde-3-phosphate dehydrogenase and 18s ribosomal rna). the assays may then be used routinely and incorporated into gene expression research in the field of equine medicine, such as joint cartilage injury and repair, as well as in the field of equine reproduction, such as stallion fertility. quantifying the amount of mrna using real-time pcr enabled the measurement of differential gene expression, but the level of mrna transcription may not be necessarily proportional to the level of mrna translation due to rna-binding translational activators and repressors. hence, real-time pcr should be coupled to techniques for measuring or quantifying protein levels (taqman® protein assays, western blotting, elisa or immuno-precipitation immuno-fluorescence). acknowledgements top ↑ competing interests the authors would like to thank the following groups at the faculty of veterinary science, university of pretoria, for their financial support and use of their laboratories: equine research centre, veterinary genetics laboratory and the department veterinary tropical diseases. authors’ contributions m.s. 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pmid:10547834 torres, a.m., forbes, b.e., aplin, s.e., wallace, j.c., francis, j.l. & norton, r.s., 1995, ‘solution structure of human insulin-like growth factor ii. relationship to receptor and binding protein interactions’, journal of molecular biology 248, 385–401. http://dx.doi.org/10.1016/s0022-2836(95)80058-1 velazquez, m.a., spicer, l.j. & wathes, d.c., 2008, ‘the role of endocrine insulin-like growth factor-i (igf-i) in female bovine reproduction’, domestic animal endocrinology 35, 325–342. http://dx.doi.org/10.1016/j.domaniend.2008.07.002, pmid:18703307 article information authors: j.b. muma1 martin simuunza1 k. mwachalimba1 m. munyeme1 b. namangala2 c. hankanga3 g. sijumbila4 r. likwa ndonyo4 a. mwanza3 a. simanyengwe mweene1 affiliations: 1department of disease control, university of zambia, zambia 2department of paraclinical studies, university of zambia, zambia 3department of clinical studies, university of zambia, zambia 4school of medicine, university of zambia, zambia 5department of veterinary and livestock development, ministry of livestock and fisheries development, zambia correspondence to: yona sinkala postal address: po box 30041, lusaka, zambia how to cite this course: muma, j.b., simuunza, m., mwachalimba, k., munyeme, m., namangala, b., hankanga, c. et al., 2012, ‘development of a curriculum for training in o ne health analytical epidemiology at the university of zambia’, onderstepoort journal of veterinary research 79(2), art. #475, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.475 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. development of a curriculum for training in one health analytical epidemiology at the university of zambia in this course... open access • abstract • introduction • development of the course curriculum • structure of the drafted curriculum • teaching strategies • career opportunities for graduates • admission criteria • conclusion • acknowledgement • references abstract top ↑ recently, the world has witnessed emergence of novel diseases such as avian influenza, hiv and aids, west nile virus and ebola. the evolution of these pathogens has been facilitated mainly by a constantly evolving animal-human interface. whilst infectious disease control was previously conceptualised as either public health or animal health related issues, the distinction between disciplinary foci have been blurred by multiple causal factors that clearly traverse traditional disciplinary divides. these multiple evolutionary pressures have included changes in land use, ecosystems, human-livestock-wildlife interactions and antibiotic use, representing novel routes for pathogen emergence. with the growing realisation that pathogens do not respect traditional epistemological divides, the ‘one health’ initiative has emerged to advocate for closer collaboration across the health disciplines and has provided a new agenda for health education. against this background, the one health analytical epidemiology course was developed under the auspices of the southern african centre for infectious diseases surveillance by staff from the university of zambia with collaborators from the london school of hygiene and tropical medicine and the royal veterinary college in london. the course is aimed at equipping scientists with multidisciplinary skill sets to match the contemporary challenges of human, animal and zoonotic disease prevention and control. epidemiology is an important discipline for both public and animal health. therefore, this two-year programme has been developed to generate a cadre of epidemiologists with a broad understanding of disease control and prevention and will be able to conceptualise and design holistic programs for informing health and disease control policy decisions. introduction top ↑ in recent years, there has been increased discourse in the global health community on the subject of one health (meisser, schelling & zinsstag 2011; zinsstag et al. 2011). one health is a systems approach to health in social-ecological systems incorporating the health of humans, animals and the environment. this has been prompted by the unprecedented emergency of novel zoonotic pathogens such as severe acute respiratory syndrome (sars), avian influenza, west nile virus, rift valley fever and ebola, amongst others. furthermore, it has been recognised by the world health organisation (who) that some of the classical zoonoses such as rabies, cysticercosis, trypanosomosis and bovine tuberculosis have not been given the appropriate attention (briggs & hanlon 2007; cleaveland, haydon & taylor 2007; fooks 2005; who 2005). taylor, latham and woolhouse (2001) observed that out of 1415 human pathogens, 61% are zoonotic. the increased risks of pathogen transmission from animals to humans have been attributed to a number of anthropogenic factors such as complex patterns of global change; the inextricable interconnection of humans through trade and travel; increased intensification of animal husbandry; the development of peri-urban systems for livestock production, increased domestication of wildlife in game ranches and encroachment of people and their livestock into wildlife areas (zinsstag et al. 2011). the steady growth in the world population has resulted in limited space for human habitation, leading to continuous human encroachment on wildlife sanctuaries and this has amplified the risk of occurrence of new emerging diseases (marcotty et al. 2009). a joint who, food and agriculture organisation (fao) and world organisation for animal health (oie) report on emerging zoonoses (2004), argued that zoonotic diseases are on the increase owing to shortfalls in public health infrastructure and policy, a paucity of public health scientific studies to answer questions and build expertise, and a lack of integrated human and animal health surveillance (who 2005). the avian influenza pandemic demonstrated that no single profession can fight these disease threats single-handedly. therefore, all players from human, veterinary and environmental health are indispensible in addressing the threats to health confronting our world. all these and many other factors have drawn professions from different disciplines to engage in joint efforts in finding solutions to the threat that infectious diseases pose to human and animal survival (powdrill, nipp & rinderknecht 2010). dedomon (2010) noted that, one health is a paradigm that serves the needs of all and encourages the spirit of collaboration and scientific inquiry for the common good. not only has the need for interdisciplinary participation been acknowledged in projects involving control of zoonoses (marcotty et al. 2009; roth et al. 2003; zinsstag et al. 2007) but there has been a new awakening to change the way health professions are educated (marcotty et al. 2009; zinsstag et al. 2011). consequently, we have seen increased advocacy for joint training in public health which has resulted in more learning institutions taking up the challenge of breaking traditional educational boundaries in health training. a good example of integrated training where the one health concept has be embraced principally and structurally is the college of veterinary medicine, nursing and allied health (cvmnah) at tuskegee university in the united states of america (habtemariam 2011). the college is composed of the school of veterinary medicine (animal health) and the school of nursing and allied health (human health) and is said: to be the only one of its kind in the united states where animal health (veterinary medicine) and human health (nursing & allied health) are merged under one college within the framework of one health-one medicine. (habtemariam 2011) further, in 2003, kansas state university started to offer a new master’s program in one health public health, with collaboration from agriculture, arts and sciences, human, ecology and veterinary medicine (michael 2010). more recently, sokoine university of agriculture introduced a course in one health molecular biology aimed at increasing collaboration in diagnosis of infectious diseases, between the medical and veterinary sectors across sub-saharan africa and it envision to improvement of the quality of laboratory diagnosis and the standardisation of diagnostic methods across human and veterinary diagnostic laboratories, which is essential in facilitating international trade as advocated for by the sanitary and phytosanitary (sps) agreement under the world trade organisation (wto). this paper discusses the development of the one health analytical epidemiology at the university of zambia, whose aim is to equip scientists with skills in analytical epidemiology for the efficient prevention and control of human, animal and zoonotic diseases. development of the course curriculum top ↑ the curriculum was developed by staff from the university of zambia’s schools of medicine and veterinary medicine. after the initial document was developed by the local staff, consultations were held with collaborators from the london school of hygiene and tropical medicine (lshtm), the royal veterinary college (rvc) in london. further consultations were held with staff from the sokoine university of agriculture in tanzania. curriculum development started with the formation of a joint working group comprising representatives from the medical and veterinary schools of the university of zambia (unza). the working group drew on expertise from epidemiology, public health and demography. the first committee meeting was held in september 2009 and involved brain storming on the modus operadi regarding the process of curriculum development. a work plan was formulated, where targets were set and responsibilities assigned to each member of the committee, which included the review of reference materials collected from collaborating institutions. subsequently, key courses were identified and a draft course outline proposed (table 1). each member of the working group was assigned to develop a structure for the course they were comfortable with. after a series of meetings, a zero-draft was developed and circulated amongst members of the group for review and comments. finally, a four day workshop was organised for in-depth review of the zero draft. the output of the workshop was a working document that was ready for external review. external consultations were in two forms. firstly, there was soliciting for course materials of related programmes from collaborating institutions and personal discussions with the expert in the identified fields. materials were collected from the lshtm and the rvc in london, and muhimbili university of health and allied sciences (muhas) in tanzania. the curriculum for msc in public health currently running at the school of medicine at unza was also included in the reference material. secondly, a visit was made to the uk in april 2011, with the main purpose of presenting the working document to collaborators in the north and receiving comments. a series of meetings were held of small groups of specialists in epidemiology and economics at both lshtm and the rvc. the visits presented us with an opportunity to discuss the curriculum with leading experts and improve curriculum design. during this final visit, the outline of the draft curriculum and its contents were thoroughly revised and modified. two of the proposed courses were re-conceptualised and renamed and another two were merged in order to conform to the one health concept. the visit also presented an opportunity to learn and share knowledge on the techniques of developing the curriculum and somehow strengthened the interactions with various professionals of different expertise in the field of medicine and veterinary medicine. upon returning from the uk, a visit was made to the sokoine university of agriculture in tanzania where further consultations with regional expertise were held. after this external review, the curriculum was finalised and presented to the joint school of veterinary medicine and school of medicine board of studies and then subsequently to the university of zambia senate for final approval in june 2011. structure of the drafted curriculum top ↑ the output of the discussions, workshops and inter-institutional consultations resulted into the curriculum for the master of science in one heath analytical epidemiology that will be offered by the university of zambia, starting from september 2011. the programme is structured such that by the end of the training, students of msc one health analytical epidemiology should be able to do the following: • demonstrate knowledge of the concept of ‘one health’ and its application in the development of health policy and the control and prevention of infectious diseases • demonstrate knowledge on how interactions between human and animal populations and environmental changes can lead to emergency and re-emerging of infectious diseases • plan, undertake and analyse data from a research project concerning human, animal and zoonotic diseases and be able to monitor and evaluate activities for policy and programme development • apply a scientific style of writing in the presentation of research • apply economic and socio-economic concepts and methods in the design, implementation and evaluation of health delivery services • determine the factors affecting the spread of disease through human and animal populations and be able to prevent or control such spread. the courses that the students will have to undertake in order for the above stated learning outcomes to be achieved are shown in table 1. it is hoped that at the end of their training students will have acquired skills in analytical epidemiology for efficient prevention and control of human, animal and zoonotic diseases. table 1: programme structure for the one health analytical epidemiology and distribution of courses by semester. the programme shall comprise two parts, a taught component executed by coursework in the first year (part i) and a research project that will culminate in the submission of a dissertation in the second year (part ii). the two parts shall be undertaken consecutively. the first part (part i) of the programme shall be undertaken over a period of two semesters (semesters 1 and 2) of fulltime study and shall comprise:• course work as required by the school board of graduates studies (http://www.unza.zm) • lectures, practicals and tutorials as well as participation in field trips and seminars • continuous assessment which will include written tests, assignments, laboratory or field reports and seminar presentations • a written examination at the end of each semester • submission of a research proposal for part ii of the programme for approval. the second part (part ii) of the program shall be undertaken over a period of two semesters (semesters 3 and 4) of full-time study and shall comprise supervised research work culminating in the submission of a dissertation. the topic (research proposal) of the dissertation shall be guided by the one health theme and shall be approved by the school postgraduate committee in the last seven weeks of the second semester of part i. for successful implementation of the course, no student will be permitted to proceed to part ii of the degree program unless he or she has satisfied the requirement of part i. teaching strategies top ↑ course delivery will mainly be done by experts from the schools of medicine and veterinary at the university of zambia. experts from other southern african academic and research institutions within the southern african centre for infectious disease surveillance (sacids) consortium as well as staff from collaborating research institutions in industrialised countries, especially the rvc and lshtm will also participate in course delivery. this will create a blend of professional knowledge and skills in epidemiology and one health that will provide a unique learning environment. career opportunities for graduates top ↑ students graduating with an msc in one health analytical epidemiology would be professionals with international competence to work in the local and regional markets in training and research institutions, government public health system and international bodies tasked with management of human and animal health such as who, oie, fao et cetera. admission criteria top ↑ all applications will be considered through the directorate of research and graduate studies of the university of zambia. the following shall be eligible to apply for the masters degree in analytical epidemiology at the university of zambia: • graduates from any recognised university in the field of biological sciences, medicine, veterinary medicine/science and any other related field of study. • the minimum requirement for medical and veterinary candidates is a first degree with at least b grade for epidemiology, statistics or community medicine. applicants must also have obtained a minimum grade of b in a level mathematics. • medical and veterinary graduates with at least one year of related medical or veterinary experience will have an added advantage. • graduates from biological sciences must have at least a merit grade and those with at least two years of post-qualifying experience will have an added advantage. • candidates wishing to pursue any specific course(s) under this programme may be considered, and will be awarded a certificate upon successful completion. such students shall not be eligible for the award of the msc. in one health analytical epidemiology. • in general, rules and regulations prescribed by the directorate of research and graduate studies at the university of zambia shall apply. (http://www.unza.zm) we have developed a master of science programme in one health analytical epidemiology which is aimed at furthering the holistic approach in combating the threat of infectious diseases using the combined resources of human and veterinary professionals and allied sciences. this has come out of the recognition that the control of diseases in the southern african development community (sadc) region requires acquisition and strengthening of human knowledge and skills by training professionals with competence in one health and epidemiology. thus, graduates of this training programme will not only be epidemiologists, but will also have an understanding of the concept of one health. this curriculum is tailored towards epidemiological understanding of disease dynamics and thus does not claim to offer all that is needed to be learnt on the subject of ‘one health’. however, it is acknowledged that epidemiology is a key discipline in effective management of human, animal and environmental health. in order to make the programme more applicable, other vital subjects such as global health and economics have been fused in so as to supplement what is ordinarily known as classical epidemiology. collectively, these tools are meant to help in understanding the close association that exists between human and animal health as evidenced in the recent occurrence of the h1n1 avian influenza pandemic. the programme will therefore emphasise the need for inter-sectoral collaboration in disease surveillance, epidemic disease preparedness and response as well as development of enabling policy platforms, across the human, animal and eco-health sectors. it has become apparent that the responsibility to ensure public health, especially as it pertains to the risk of emerging and re-emerging zoonoses, is a shared responsibility of professionals in the human and veterinary medicine and other allied sciences such as ecology (michael 2010). marcotty et al. (2009) strongly encouraged collaboration between the veterinary and the medical sectors, in the diagnosis, monitoring and control of zoonotic brucellosis and tuberculosis. these authors further, added that useful collaborations could be nurtured at undergraduateand postgraduate-training levels, through, for example, common public-health modules or courses. this programme has been designed to train a new generation of world class scientists who will serve to address the heavy burden and threat of emerging and re-emerging infectious diseases in africa that greatly hamper public health and animal health and thus socio-economic development of developing countries (who 2005). the successful implementation of the programme strongly depends on support from other sacids consortium universities in southern africa as well as the lshtm, rvc and other internationally reputable institutions. conclusion top ↑ it is hoped that the training programme will create increased collaboration between professions in medical, veterinary and allied sciences and create a platform for sharing their knowledge and resources to better promote human, animal and environmental health. the medical profession would, benefit greatly from an improved knowledge and understanding of the epidemiology of zoonotic diseases whilst the veterinary sector would, have a better appreciation of what is expected from them, in terms of controlling the zoonoses in order to assure public health. acknowledgement top ↑ the wellcome trust is thanked for funding the development of the curriculum through sacids. the curriculum development was part of wider sacids consortium projects under the framework of the one heath concept. we are further grateful for the support we received from the london international development centre, london school of hygiene and tropical medicine, the royal veterinary college, sokoine school of agriculture, muhimbili university of health and allied sciences and the university of zambia. we would also like to thank professor kenny samui for his contribution to the successful development of this curriculum. references top ↑ briggs, d. & hanlon, c.a., 2007, ‘world rabies day: focusing attention on a neglected disease’, veterinary record 161, 288–289. http://dx.doi.org/10.1136/vr.161.9.288, pmid:17766802 cleaveland, s., haydon, d.t. & taylor, l., 2007, ‘overviews of pathogen emergence: which pathogens emerge, when and why?’, current topics in microbiology and immunology 315, 85–111. http://dx.doi.org/10.1007/978-3-540-70962-6_5 cleaveland, s., laurenson, m.k. & taylor, l.h., 2001, ‘diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence’, philosophical transactions of the royal society b: biological sciences 356, 991–999. http://dx.doi.org/10.1098/rstb.2001.0889, pmid:11516377 dedomon, e.r., 2010, ‘the one health initiative through a 50-year lens’, one health news letter fall, 3–5. fooks, a.r., 2005, ‘rabies remains a “neglected disease”’, eurosurveillance 10, 211–212. pmid:16972345 habtemariam, 2011, the college of veterinary medicine, nursing and allied health, viewed 31 august 2011 from, http://www.tuskegee.edu/academics/colleges/cvmnah/office_of_the_dean_-_cvmnah.aspx marcotty, t., matthys, f., godfroid, j., rigouts, l., ameni, g., gey van pittius, n. et al., 2009, ‘zoonotic tuberculosis and brucellosis in africa: neglected zoonoses or minor public-health issues? the outcomes of a multi-disciplinary workshop’, annals of tropical medicine and parasitology 103, 401–411. http://dx.doi.org/10.1179/136485909x451771, pmid:19583911 meisser, a., schelling, e. & zinsstag, j., 2011, ‘one health in switzerland: a visionary concept at a crossroads?’, swiss medical weekly 141, 13201. michael, b., 2010, ‘an innovative approach to graduate public health education’, one health newsletter fall, 1–2. powdrill, t.f., nipp, t.l. & rinderknecht, j.l., 2010, ‘one health approach to influenza: assessment of critical issues and options’, emerging infectious diseases 16, e1. http://dx.doi.org/10.3201/eid1608.100673, pmid:20678310 roth, f., zinsstag, j., orkhon, d., chimed-ochir, g., hutton, g., cosivi, o. et al., 2003, ‘human health benefits from livestock vaccination for brucellosis: case study’, bulletin of world health organisation 81, 867–876. pmid:14997239 taylor, l.h., latham, s.m. & woolhouse, m.e., 2001, ‘risk factors for human disease emergence’, philosophical transactions of the royal society b: biological sciences 356, 983–989. http://dx.doi.org/10.1098/rstb.2001.0888 who, 2005, the control of neglected zoonotic diseases: a route to poverty alleviation, geneva, switzerland. zinsstag, j., schelling, e., roth, f., bonfoh, b., de savigny, d. & tanner, m., 2007, ‘human benefits of animal interventions for zoonosis control’, emerging infectious diseases 13, 527–531. http://dx.doi.org/10.3201/eid1304.060381, pmid:17553265 zinsstag, j., schelling, e., waltner-toews, d. & tanner, m., 2011, ‘from “one medicine” to “one health” and systemic approaches to health and well-being’, preventive veterinary medicine 101, 148–156. http://dx.doi.org/10.1016/j.prevetmed.2010.07.003, pmid:20832879 article information authors: esmey b.e. moema1 pieter h. king1 chantelle baker2 affiliations: 1department of biology, university of limpopo, medunsa campus, south africa2electron microscope unit, university of limpopo, medunsa campus, south africa correspondence to: esmey moema postal address: po box 139, medunsa 0204, south africa dates: received: 06 feb. 2012 accepted: 18 apr. 2012 published: 14 sept. 2012 how to cite this article: moema, e.b.e., king, p.h. & baker, c., 2012, ‘descriptions of strigea cercariae from the gauteng and north west provinces, south africa’, onderstepoort journal of veterinary research 79(1), art. #410, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.410 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. descriptions of strigea cercariae from the gauteng and north west provinces, south africa in this original research... open access • abstract • introduction • materials and methods    • ethical considerations • results    • strigea cercaria a    • strigea cercaria b    • strigea cercaria c • trustworthiness    • reliability    • validity • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis, amphistomosis and fasciolosis. two freshwater snail species, lymnaea natalensis, krauss 1848 and bulinus tropicus, krauss 1848 were sampled from five localities in gauteng and one locality in the north west province from 2007 to 2010. these snails were collected in order to study their cercarial sheddings. they were found to be infected with three different types of strigea cercariae, of which the morphology was studied using standard light and scanning electron microscopy techniques. introduction top ↑ strigea cercariae are characterised by a tail with long furcae (longifurcate), a pharynx (pharyngeal) and the presence of both oral and ventral suckers (distome). these cercariae are known to encyst in snails, tadpoles, reptiles and fish. adults, as intestinal parasites of birds and mammals, are usually classified in either the family strigeidae or diplostomatidae (frandsen & christensen 1984).the most comprehensive work on cercarial types, distribution and morphology in southern africa was published by porter (1938). fain (1953) studied the cercariae from snails in lake albert (on the border of the democratic republic of the congo and uganda) and vercammen-grandjean (1960) from lake kivu (on the border of the democratic republic of the congo and rwanda). later, combes (1980) presented the ‘world atlas of cercariae’, whereafter king and van as (1997, 2001) described the cercariae shed by bulinus tropicus, krauss 1848 in the free state. jansen van rensburg (2001) described the cercariae from snail species in the okavango delta, botswana and nadasan and appleton (2003) investigated the cercariae in the vicinity of durban, kwazulu-natal. surveys on cercarial sheddings in the tshwane area, gauteng province and one locality in the north west province revealed two freshwater snail species to shed three different types of strigea cercariae. lifecycle studies on these parasites frequently failed, mainly because of their extremely high host specificity with regard to their first, second and definitive hosts. the main aim of this project was to gather morphological information on these cercarial types, which, in the future, may assist in establishing possible linkages with strigea metacercarial stages in local fish populations from these areas. materials and methods top ↑ the freshwater snails, lymnaea natalensis, krauss 1848 and b. tropicus were collected over a period of four years (2007–2010) from four localities, namely, supersand dam (25º35′02.42′′ s – 28º10′38.98′′ e, altitude 1198 m a.s.l.) situated ± 2 km north of the bon accord dam in the onderstepoort area, gauteng; the boekenhoutskloof farm dam (25º32′45.18′′ s – 28º26′12.47′′ e, altitude 1214 m a.s.l.), approximately 15 km north of the roodeplaat dam nature reserve, gauteng; metsi-pepa (26º11′52.63′′ s – 27º09′51.92′′ e, altitude 1473 m a.s.l.) along the n14 krugersdorp–ventersdorp main road and ± 35 km from ventersdorp, north west province; and northern farm (25º55′49.18′′ s – 27º58′19.11′′ e, altitude 1375 m a.s.l.) situated north of johannesburg, gauteng, bordering the diepsloot informal settlement. the snails were collected using metal scoops, as described by van eeden (1960), or collected manually from the ventral surface of water lily leaves. in the laboratory they were kept in plastic containers and fed fish flakes. they were exposed to indirect daylight to stimulate the natural shedding of cercariae. the cercariae were stained with nile blue sulphate or neutral red vital stains and studied alive using a nikon compound microscope (nikon, tokyo, japan). one or two stained cercariae were placed in a drop of water on a microscope slide, covered with a glass cover slip and moved once or twice over a bunsen burner to slow down cercarial movement. drawings were made by means of a drawing tube. in all cases, 20 specimens were measured, with all measurements given in micrometres (µm) and with minimum and maximum values indicated along with the mean value and standard deviation in parenthesis. owing to the low infections in the snails, no other intra-molluscan stages were studied. for scanning electron microscopy (sem), specimens were fixed overnight in 2.5% glutaraldehyde, washed in millonig’s phosphate buffer (ph = 7.2) and dehydrated through a graded ethanol series for 30 s – 60 s in each concentration. thereafter, they were critically point dried (polaron, watford, uk), mounted on aluminium stubs, sputter coated with gold (emscope; quorum technologies, ashford, uk) and examined using a leica stereoscan 420 sem (leica electron optics, uk) at the electron microscope unit of the university of limpopo (medunsa campus). ethical considerations this research project met the requirements of the research ethics committee (bp 05/2005) and the animal ethics committee (aec 02/05) of the university of limpopo. snails were kept under controlled laboratory conditions with sufficient light, air and food according to acceptable standards. they were individually placed in containers and cercariae collected after natural shedding. results top ↑ the freshwater snails, l. natalensis and b. tropicus were found to host three types of strigea cercariae. table 1 accounts for the total collection of these two snail species from the four localities over a period of four years from 2007 to 2010. the prevalence of each infection is also indicated. table 1: prevalence of infections of two freshwater snail species collected from four different localities across the gauteng and north west provinces, south africa. strigea cercaria a snail host: l. natalensis. locality: supersand dam, gauteng province.the cercaria (figure 1a; figures 2a and 2b) consists of an elongated and cylindrical body, measuring 164 µm – 185 µm (180 µm ± 7 µm) long × 49 µm – 60 µm (52 µm ± 4 µm) wide and a tail stem ending in two caudal rami (figure 1b). the tail stem is shorter than the length of the body, measuring 198 µm – 206 µm (201 µm ± 3 µm) × 24 µm – 29 µm (26 µm ± 2 µm), with the caudal rami shorter than the tail stem, at 169 µm – 174 µm (172 µm ± 2 µm) × 16 µm – 18 µm (17 µm ± 1 µm). figure 1: light microscope projection drawings of three types of strigea cercariae shed by lymnaea natalensis and bulinus tropicus, (a) head and (b) forked-tail of strigea cercaria a, (c) head and (d) forked tail of strigea cercaria b and (e) head and (f) forked tail of strigea cercaria c. a pear-shaped oral sucker, 52 µm – 69 µm (59 µm ± 4 µm) × 18 µm – 31 µm (27 µm ± 4 µm) is situated at the anterior end of the body. sensory receptors with short cilia were observed alongside the oral sucker (figure 2c). a pre-pharynx, 4 µm – 7 µm (6.5 µm ± 1.0 µm) × 3 µm – 4 µm (3.5 µm ± 1.0 µm) connects the oral sucker to the pharynx, 12 µm – 19 µm (14 µm ± 2 µm) × 10 µm – 21 µm (13 µm ± 3 µm) , followed by an oesophagus, 7 µm – 12 µm (8 µm ± 1 µm) × 4 µm – 6 µm (5 µm ± 1 µm) that extends to just anterior of the acetabulum. the oesophagus divides into two intestinal caeca, 82 µm – 85 µm (83 µm ± 1 µm) × 5 µm – 6 µm (5.5 µm ± 1.0 µm) that extend posteriorly and terminates close to the posterior end of the body. figure 2: strigea cercaria a, depicted using light micrographs of (a) whole mount and (b) head of cercariae with penetration glands (pg) posterior to acetabulum, as well as scanning electron micrographs of, (c) oral sucker (os), (d) acetabulum (ac), (e) joint between body and tail (*) and (f) sensory receptors with long cilia (lcr) on the tail stem. the acetabulum (figure 2d), 35 µm – 39 µm (36 µm ± 1 µm) × 33 µm – 37 µm (35 µm ± 2 µm) is aspinose and located 28 µm – 38 µm (33 µm ± 4 µm) from the oral sucker. it is notably smaller than the oral sucker. four penetration glands posterior to the acetabulum (figure 1a; figure 2b) are arranged as follows: two penetration glands, 7 µm – 15 µm (10 µm ± 2 µm) × 9 µm – 16 µm (13 µm ± 2 µm) flanking each other, followed by two large unpaired penetration glands lying tandem and posterior to the first pair. the first unpaired penetration glands measure 6 µm – 10 µm (8 µm ± 2 µm) × 16 µm – 24 µm (19 µm ± 2 µm) and the second pair, 6 µm – 10 µm (8 µm ± 1 µm) × 15 µm – 27 µm (19 µm ± 3 µm).a small bipartite excretory bladder, 7 µm – 10 µm (8 µm ± 1 µm) × 7 µm – 11 µm (9 µm ± 3 µm) is situated at the posterior end of the body (figure 1a). two main collecting tubes arise from the bladder, extend anteriorly and divide into anterior and posterior collecting tubules anterior to the acetabulum. fourteen pairs of flame cells were found in the body, of which six pairs are located in the anterior half and the remaining eight pairs are located in the posterior half of the body. the flame cell formula is: 2([2 + 2 + 2] + [1 + 2 + 2 + 1 + 2]) = 28. the excretory pores open sub-terminally on either side of the caudal rami (figure 1b). posteriorly directed minute spines that are denser in the anterior part of the organism cover the body tegument (figure 2c), whereas fewer and more widely separated spines cover the tail stem (figure 2e), with sparse sensory receptors with long cilia on its surface (figure 2f). six hair-like structures (figure 1b), three anteriorly and three posteriorly, were observed on each side of the tail stem. a series of caudal bodies were also observed in the tail stem (figure 1b). the caudal rami are covered with spines that are visually fewer when compared to the spines found on the tail stem. strigea cercaria b snail host: b. tropicus. locality: northern farm dam, gauteng province.strigea cercaria b (figures 1c and 1d; figures 3a and 3b) comprises a cylindrical body, 88 µm – 155 µm (109 µm ± 21 µm) × 45 µm – 60 µm (50 µm ± 4 µm) and a fork tail. the tail stem, 75 µm – 127 µm (105 µm ± 16 µm) × 28 µm – 36 µm (33 µm ± 3 µm) is shorter than the body length, with the caudal rami measuring 123 µm – 161 µm (149 µm ± 11 µm) × 10 µm – 21 µm (16 µm ± 3 µm). the oral sucker (figure 3c), 16 µm – 40 µm (30 µm ± 7 µm) × 25 µm – 33 µm (30 µm ± 3 µm) is covered by fairly large and sharp backwardly pointing spines (figures 3c and 3d). the oral sucker opens directly into a pharynx, 7 µm – 10 µm (9 µm ± 1 µm) × 7 µm – 12 µm (10 µm ± 2 µm), followed by a short oesophagus, 6 µm – 30 µm (13 µm ± 7 µm) × 3 µm – 6 µm (4 µm ± 1 µm) (figure 1d). figure 3: strigea cercaria b, depicted using light micrographs of (a) whole mount and (b) head of cercaria, as well as scanning electron micrographs of, (c) oral sucker (os), (d) spines (sp) around the oral sucker, (e) acetabulum (ac) and (f) tail tegument (tt). the oesophagus bifurcates into two intestinal caeca, 31 µm – 49 µm (42 µm ± 5 µm) × 4 µm – 9 µm (6 µm ± 1 µm), encircling the acetabulum and terminate posteriorly to the acetabulum. the acetabulum (figure 3e), 18 µm – 30 µm (25 µm ± 4 µm) × 18 µm – 25 µm (22 µm ± 2 µm) is smaller than the oral sucker and situated 43 µm – 90 µm (58 µm ± 15 µm) mid-ventrally from the anterior end of the body. two non-pigmented eyespots are situated 46 µm – 48 µm (47 µm ± 1 µm) from the anterior end of the body and measure 7 µm – 10 µm (9 µm ± 1 µm) × 4 µm – 9 µm (6 µm ± 2 µm) (figure 1d).four small penetration glands (figure 1d), 4 µm – 7 µm (6 µm ± 1 µm) in diameter, are situated at the terminal end of each intestinal caecum. a small bipartite excretory bladder, 3 µm – 9 µm (7 µm ± 2 µm) × 7 µm – 15 µm (10 µm ± 3 µm), is located at the posterior end of the body. two main collecting tubes arise from the bladder, extend anteriorly and divide into anterior and posterior collecting tubules anterior to the acetabulum giving rise to capillaries that terminate in flame cells (figure 1d). the flame cell formula is: 2([2 + 1] + [2 + 2]) = 14. the excretory pore opens sub-terminally on the lateral side of each caudal ramus (figure 1c). the body surface is covered with many posteriorly facing spines that are concentrated more at the anterior end of the body. these spines are large and concentrated around the oral sucker (figure 3c), and become smaller towards the posterior. a row of large spines surrounds the acetabulum wall (figure 3e). four hair-like structures (figure 1d) were observed: three on the anterior half and one posterior on both sides of the body. the tail stem (figure 3f) is covered with only a few short spines that are randomly positioned. the caudal rami are covered with fewer posteriorly directed spines. both the tail stem and the caudal rami have hair-like structures, eight were found on both sides of the tail stem and two on the postero-lateral side of each caudal ramus (figure 1c). strigea cercaria c snail host: l. natalensis. locality: metsi-pepa, north west province.strigea cercaria c is elongated with a large oval-shaped body, 151 µm – 184 µm (171 µm ± 10 µm) × 45 µm – 52 µm (50 µm ± 3 µm), and a fork tail that is almost twice as long as the body (figures 1e and 1f; figures 4a and 4b). the tail stem measures 413 µm – 508 µm (479 µm ± 31 µm) × 66 µm – 90 µm (72 µm ± 9 µm). the caudal rami, at 407 µm – 497 µm (477 µm ± 29 µm) × 30 µm – 60 µm (38 µm ± 8 µm), are as long as the tail stem and taper posteriorly into sharp-ending points (figure 1f). the body (figure 1e; figure 4b) displays a pear-shaped oral sucker, 48 µm – 64 µm (57 µm ± 5 µm) × 22 µm – 33 µm (28 µm ± 4 µm), that is longer than wide and very protrusible. sensory receptors with short cilia dominate the area around the mouth opening (figures 4d and 4e). posteriorly directed spines (figures 4d and 4e) are concentrated at the anterior end of the body, especially around the oral sucker, but are fewer in number at the posterior end of the body. sensory receptors with long cilia were observed dorsally on the body surface (figure 4f). figure 4: strigea cercaria c, depicted using light micrographs of (a) whole mount, (b) head of cercaria and (c) caudal bodies (cb) in the tail stem, as well as scanning electron micrographs of, (d) oral sucker (os), (e) sensory receptors with short cilia (scr) surrounding the oral sucker and (f) sensory receptors with long cilia (lcr) anteroventrally on the body. a small oral opening (figure 4d) situated in the middle of the oral sucker opens into a pre-pharynx, 9 µm – 15 µm (12 µm ± 2 µm) × 4 µm –7 µm (6 µm ± 1 µm). it continues into a muscular pharynx, 10 µm – 15 µm (11 µm ± 2 µm) × 9 µm – 15 µm (11 µm ± 2 µm), leading to a short oesophagus, 7 µm – 12 µm (9 µm ± 2 µm) × 3 µm – 6 µm (4 µm ± 1 µm). the oesophagus divides at the level of the acetabulum into two very large intestinal caeca, 67 µm – 97 µm (80 µm ± 8 µm) × 10 µm – 16 µm (13 µm ± 2 µm) that terminate just above the excretory bladder at the posterior end of the body.an acetabulum, 12 µm – 39 µm (33 µm ± 8 µm) × 30 µm – 39 µm (34 µm ± 3 µm), with spines on the luminal wall is situated 37 µm – 40 µm (39 µm ± 1 µm) posterior to the elongated oral sucker. three finely granular penetration glands occur in tandem on either side of the body, just posterior to the acetabulum. the first pair measures, 9 µm – 15 µm (13 µm ± 2 µm) × 10 µm – 13 µm (11 µm ± 1 µm), the second pair, 7 µm – 15 µm (13 µm ± 2 µm) × 10 µm – 13 µm (11 µm ± 1 µm) and the third pair, 10 µm – 16 µm (14 µm ± 2 µm) × 10 µm – 13 µm (12 µm ± 1 µm). the excretory system consists of an oval-shaped bladder, 7 µm – 10 µm (8 µm ± 1 µm) × 6 µm – 10 µm (8.0 µm ± 1.9 µm) from where the main excretory ducts on each side of the bladder run anteriorly towards the middle of the body. these ducts then divide at the level of the acetabulum into anterior and posterior collecting tubules which branch into smaller capillaries bearing flame cells. the flame cell formula is as follows: 2 (3 + 3) = 12. two hair-like structures were observed bilaterally on the anterior end and seven pairs on the posterior half of the tail stem (figure 1f). caudal bodies in the tail stem are irregular in size and shape, and vary in number (figure 1f; figure 4c). trustworthiness top ↑ reliability repeated measurement (n = 20) of morphological characteristics by both line drawings and comparative information from sem micrographs, corroborate our findings and thus improves reliability. validity all measurements included are of internationally accepted and well-recognised taxonomic descriptors in this discipline. discussion top ↑ all three of the cercariae were identified as strigea cercariae according to the identification keys provided by frandsen and christensen (1984), classifying them as longifurcate-pharyngeal distome cercariae of the superfamily strigeoidea. porter (1938) described three fork-tailed cercariae shed by l. natalensis, namely cercaria scheerpoortia and cercaria magaliesia both from hartebeestpoort dam (north west province) and cercaria maritzburgensis from pietermaritzburg (kwazulu-natal province). fain (1953) described nine strigea cercariae, which included one secreted by l. natalensis. furthermore, vercammen-grandjean (1960) described six strigea cercariae which included five secreted by biomphalaria species and one by l. natalensis from lake kivu. more recently, king and van as (2001) described a fork-tailed cercaria secreted by b. tropicus from the free state province, whilst jansen van rensburg (2001) described a fork-tailed cercaria from l. natalensis from the okavango delta, botswana and nadasan and appleton (2003) described a pharyngeal longifurcate distome cercaria shed by b. tropicus from durban, kwazulu-natal. the present study adds to the descriptions of strigeid cercariae by the abovementioned authors from various regions in africa. with its unique grouping of penetration glands, strigea cercaria a is closely related to the strigea cercaria described by fain (1953) but has penetration glands that are located at the post-acetabular region, whereas the one previously described has penetration glands situated at the pre-acetabular region. other differences were size and shape of the body, as well as the flame cell formula. with its cluster penetration glands, strigea cercaria b is similar to the strigea cercaria described by king and van as (2001) secreted by b. tropicus in the free state province. the present cercaria is, however, smaller and has fewer penetration glands and flame cells. strigea cercaria b has seven pairs of flame cells, three pairs anteriorly and four pairs posteriorly to the acetabulum; whereas, the one described by king and van as (2001) has two pairs anteriorly and two pairs posterior to the acetabulum. although the internal morphology of these two cercariae is similar, sem revealed external differences; for example, the surface spines and features of the acetabulum. the cercariae described by porter (1938) and jansen van rensburg (2001) were all shed by l. natalensis. all of the abovementioned cercariae differ from the description of the present cercaria based on the position and shape of the penetration glands, body size and caudal bodies in the tail. interestingly, strigea cercaria b was collected only once from l. natalensis from boekenhoutskloof farm dam. it is too soon to comment whether or not this was an accidental infection or whether the parasite is adapting to a new first intermediate host. with regard to the position and size of the penetration glands, strigea cercaria c is clearly different in most morphological aspects to that described by king and van as (2001) and jansen van rensburg (2001). this cercaria, however, resembles most of the characteristic features of c. scheerpoortia, as described by porter (1938), especially the three pairs of penetration glands on each side of the body posterior to the acetabulum. strigea cercaria c has three pairs of flame cells anteriorly and three pairs posteriorly, unlike c. scheerpoortia, which has three pairs of flame cells occurring anteriorly, two pairs occurring posteriorly and one pair occurring in the tail stem. the cercaria described here also has hair-like structures found on either side of the body and the tail, which differs from the strigea cercaria described by porter (1938). other differences are the size of the intestinal caeca, tail stem morphology and pharynx size and shape. four types of metacercariae were obtained from local freshwater fish from the same localities. two types of strigea metacercariae were found to encyst in the muscle tissue of tilapia sparrmanii, smith 1840 and pseudocrenilabrus philander, weber 1897, one was found in the cranial cavity of clarias gariepinus, burchell 1822 and a fourth in the vitreous chamber of t. sparrmanii. according to olsen (1974) members of the superfamily strigeoidea belong to the families diplostomatidae and strigeidae, which both include intestinal parasites of fish-eating and frog-eating birds and mammals. gibson, jones and bray (2001) mentioned that members of the strigeidae are mainly specific to birds but are also found in reptiles. only one genus, duboisiella baer, 1938 occurs in mammals. the lifecycles of these strigea cercariae are presently still unknown, but they may develop into adults in either of the families mentioned above. with these parasites infecting a wide spectrum of freshwater fish, it is important to gather much more information on their larval stages first in order to attempt lifecycle studies in the future. conclusion top ↑ in this study, l. natalensis was found to host two different strigea cercariae. the first is characterised by two penetration glands flanking each other, followed by two unpaired penetration glands lying tandem and posterior to the first pair. the second cercaria is characterised by three pairs of granular penetration glands found in tandem posterior to the acetabulum. bulinus tropicus secreted one strigea cercaria characterised by two non-pigmented eyespots and a cluster of four penetration glands situated at the terminal end of each intestinal caecum.the three strigea cercariae described in the present study were unrelated to any previous investigations described in the text. this investigation thus adds important findings with regard to morphological characterisation of these parasites. our prelimenary studies have shown that many freshwater fish species are infected with various strigea metacercariae which were collected from the same waterbodies that are presently utilised by the neighbouring communities. it is therefore imperative to trace the origin of these cysts in local fish species that may serve as the main source of protein. acknowledgements top ↑ scanning electron microscopy equipment utilised during this investigation was supported by the department of science and technology in partnership with the national research foundation of south africa. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions e.b.e.m. (university of limpopo) was the principal investigator, as this study formed part of her doctoral thesis. p.h.k. (university of limpopo) was the postgraduate supervisor of this study and c.b. (university of limpopo) was a co-worker involved with all aspects of micrography and technical finishing of photographic plates. references top ↑ combes, c., 1980, ‘atlas mondial des cercaires [world atlas of the cercariae]’, mémoires du muséum national d’histoire naturelle, paris 115, 1–236.fain, a., 1953, contribution à l’étude des formes larvaires des trematodes au congo belge et spécialment de la larvae de schistosoma mansoni [contribution to the study of the larval forms of the trematodes in belgian congo and especially of the larvae of schistosoma mansoni]. mémoires institut royal colonial belge section de sciences naturalles et médicales 22, 1–312. frandsen, f. & christensen, n.o., 1984, ‘an introductory guide to the identification of cercariae from african freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance’, acta tropica 41, 181–202. pmid:6206702 gibson, d.i., jones, a. & bray, r.a., 2001, keys to the trematoda, vol. 1, cabi publishing, london. jansen van rensburg, c.j., 2001, ‘snail borne larval trematodes of the okavango delta, botswana’, msc dissertation, department of zoology, university of the free state. king, p.h. & van as, j.g., 1997, ‘morphology and scanning electron microscopy of cercariae shed by bulinus tropicus (krauss, 1848) in the free state, south africa’, journal of african zoology 111, 301–312. king, p.h. & van as, j.g, 2001, ‘cercariae shed by bulinus tropicus (krauss, 1848) in the free state, south africa’, journal of african zoology 36, 95–105. nadasan, d.s. & appleton, c.c., 2003, ‘studies on larval trematode infections in freshwater snails in durban, south africa. i. morphology and surface topography of cercariae’, proceedings of a workshop on african freshwater malacology, kampala, uganda, 133–166. olsen, o.w., 1974, animal parasites – their life cycles and ecology, 3rd edn., university park press, baltimore. porter, a., 1938, ‘the larval trematoda found in certain south african mollusca with special reference to schistosomiasis (bilharziasis)’, south african journal of medical research institute viii(xlii), 471. van eeden, j.a., 1960, ‘key to the genera of south african freshwater and estuarine gastropods (mollusca)’, annals of transvaal museum 24, 1–17. vercammen-grandjean, p.h., 1960, les trematodes du lac kivu sud. musée royal de l’afrique centrale, tervuren [the trematodes of lake kivu south. royal museum of central africa, tervuren]. belgique annales novelle serie in 4˚ sciences zoologiques 5, 1–171. article information authors: ilana c. van wyk1 joop boomker1 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa correspondence to: ilana van wyk email: ilana@conradie.net postal address: private bag x04, onderstepoort 0110, south africa dates: received: 20 nov. 2010 accepted: 29 aug. 2011 published: 11 nov. 2011 how to cite this article: van wyk, i.c. & boomker, j., 2011, ‘parasites of south african wildlife. xix. the prevalence of helminths in some common antelopes, warthogs and a bushpig in the limpopo province, south africa’, onderstepoort journal of veterinary research 78(1), art. #308, 11 pages. http://dx.doi.org/10.4102/ ojvr.v78i1.308 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2495 (print) issn: 2219-0635 (online) parasites of south african wildlife. xix. the prevalence of helminths in some common antelopes, warthogs and a bushpig in the limpopo province, south africa in this original research... open access • abstract • introduction • materials and methods    • materials    • survey areas    • study animals    • collection and preservation of parasites    • parasite counts and identification    • data analysis    • ethical considerations • results and discussion    • animals       • impalas       • kudus       • bushbuck       • nyala       • blue wildebeest       • black wildebeest       • gemsbok       • waterbuck       • warthogs       • bushpig    • helminths       • agriostomum       • cooperia       • cooperioides       • elaeophora       • haemonchus       • impalaia       • murshidia       • oesophagostomum       • setaria       • trichostrongylus       • trichuris       • avitellina       • stilesia       • echinococcus       • taenia spp.       • fasciola • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract (back to top) little work has been conducted on the helminth parasites of artiodactylids in the northern and western parts of the limpopo province, which is considerably drier than the rest of the province. the aim of this study was to determine the kinds and numbers of helminth that occur in different wildlife hosts in the area as well aswhether any zoonotic helminths were present. ten impalas (aepyceros melampus), eight kudus (tragelaphus strepsiceros), four blue wildebeest (connochaetes taurinus), two black wildebeest (connochaetes gnou), three gemsbok (oryx gazella), one nyala (tragelaphus angasii), one bushbuck (tragelaphus scriptus), one waterbuck (kobus ellipsiprymnus), six warthogs (phacochoerus aethiopicus) and a single bushpig (potamochoerus porcus) were sampled from various localities in the semi-arid northern and western areas of the limpopo province. new host–parasite associations included trichostrongylus deflexus from blue wildebeest, agriostomum gorgonis from black wildebeest, stilesia globipunctata from the waterbuck and fasciola hepatica in a kudu. the mean helminth burden, including extra-gastrointestinal helminths, was 592 in impalas, 407 in kudus and blue wildebeest, 588 in black wildebeest, 184 in gemsbok, and 2150 in the waterbuck. excluding probstmayria vivipara, the mean helminth burden in warthogs was 2228 and the total nematode burden in the bushpig was 80. the total burdens and species richness of the helminths in this study were consistently low when compared with similar studies on the same species in areas with higher rainfall. this has practical implications when animals are translocated to areas with higher rainfall and higher prevalence of helminths. introduction (back to top) game ranching constitutes a significant part of the land use practices in the limpopo province, south africa. parasites play an important role in regulating host populations in a natural environment; therefore, knowledge about the helminth infections of wildife in an area is essential. farming with game creates an unnatural system and disrupts the balance between parasite and host. in this stressful environment animals become diseased or can even die from parasite loads that they would have survived under natural conditions. trophy hunting and the production of venison are increasing in the province and condemnation of carcasses due to macroscopic pathology caused by parasites can lead to financial losses. little work has been conducted on the helminth parasites of artiodactylids in the northern and western parts of the province. the aims of this study were to determine the species and numbers of helminth that occur in different wildlife hosts in the area, and to determine whether any zoonotic helminths were present. tothis end, a total of 37 animals from various localities in the limpopo province were examined and their parasites identified and counted. materials and methods (back to top) materials the animal species, number of collections and the collection dates and localities (with their global positioning system coordinates) are summarised in table 1. table 1: the collection data for the various animals examined. survey areas the survey was performed in the northern parts of the limpopo province near the soutpansberg and the town musina, which lies on the limpopo river, and the western parts near the town lephalale. the vegetation in the soutpansberg region is mixed bushveld with sections falling within the mist belt on the escarpment. the musina area is semi-arid and the vegetation type varies between thornveld and mopani veld (acocks 1988). the rainfall recorded between july 2006 and june 2007 for the soutpansberg area and musina was 300 mm and 500 mm, respectively (south african weather service 2007). rainfall is strictly confined to the summer. the climate is hot during summer and cool during winter, with occasional frost. the western parts of the province are semi-arid and the vegetation type varies between sweet bushveld and mixed bushveld (acocks 1988). rainfall from july 2006 to july 2007 in the lephalale area was 178 mm (south african weather service 2007). the summers are hot and the winters cool. study animals with the exception of one gemsbok (oryx gazella) and the nyala (tragelaphus angasii), the study material was obtained from animals that had been culled or hunted during the hunting season (may–september) of 2006 and 2007. a few organ samples from one of the gemsbok and the carcass of the nyala were submitted to the louis trichardt veterinary laboratory for post-mortem examination. in addition to the warthogs (phacochoerus aethiopicus) that had been culled, the carcasses of several warthogs from a meat processing plant were examined fortrichinella spp. as only the eviscerated carcasses were available, only samples from the diaphragms and the intercostal muscles could be collected for examination. collection and preservation of parasites the carcasses of the culled animals were first inspected visually for the presence of taenia or echinococcus metacestodes on the cut surfaces and those of the pericardium, heart, diaphragm and liver. when permitted, the tongue was freed and examined visually and palpated. only single metacestodes were encountered and collected, which were subsequently preserved in 70% alcohol. carcasses that were destined for export were not skinned until they reached the abattoir. no incisions were made into the muscles, except for the masseter and the diaphragm. the heads and capes of animals hunted for trophies were preserved; therefore, no incisions of the masseter muscles or tongues could be made. the intestinal helminths as well as those in the heart, lungs and liver of the antelopes were collected as described by boomker, horak and de vos (1989) but digests of abomasal and intestinal mucosae were not performed. depending on the volume of ingesta of the different parts of the gastrointestinal tract, aliquots of a quarter, a tenth or a 25th were made and examined. the entire abomasal content of the bushbuck (tragelaphus scriptus) was examined. the helminths of warthogs and the bushpig (potamochoerus porcus) were recovered as described by boomker et al. (1991a). in addition, muscle samples from the diaphragm and intercostal and masseter muscles were crushed (between glass slides) and examined under a stereoscopic microscope for the presence of trichinella spp. parasite counts and identification aliquots of ingesta and organ washings were fixed and preserved in 70% ethyl alcohol. these were transported to the laboratory, where they were examined under a stereoscopic microscope and all helminths were removed. the nematodes were cleared in lactophenol, identified under a standard microscope and counted. malenematodes were identified to species level, whereas female nematodes, especially where two or more related species occurred in a single host, were identified to the generic level only. fourth-stage larvae were identified only to the generic level. live adult cestodes were relaxed in water in petri dishes. once all movement had ceased they were fixed in 5% formalin for 24 h and further preserved in 70% ethyl alcohol. scoleces were dissected out of the metacestodes and mounted en face in berlese’s fluid under slight pressure from a cover slip. adult cestodes were stained in carmalum or mayer’s acid haemalum, cleared in xylene and mounted on glass slides in canada balsam (gibbons, jones & kahlil 1996). data analysis sampling was not truly random and depended on circumstances to some extent. therefore, the significance of the results could not be compared statistically. however, descriptive statistics were used as applicable. ethical considerations the samples in this study were opportunistically collected from animals that had been either hunted or culled for reasons other than this study. results and discussion (back to top) animals impalas twelve helminth species, ten nematodes and two cestodes were recovered from impalas (aepyceros melampus) in this study. the helminth burdens are presented in table 2. cooperia hungi was most prevalent, followed by impalaia tuberculata, oesophagostomum columbianum and trichostrongylus deflexus. haemonchus krugeri had the highest mean intensity (375 worms), followed by c. hungi (280 worms) and i. tuberculata (140 worms). impala and their parasites have been studied by several authors and some significant contributions have been made by anderson (1992), heinichen (1973), horak (1978, 1980, 1981) and mönnig (1933) in south africa and gallivan et al. (1989) in swaziland. because of their feeding ecology, impala are exposed to the helminths of both browsers and grazers. impala also share pastures with domestic livestock on many game ranches in south africa and considerable overlap between thehelminth fauna of sheep, cattle and impalas occurs (horak 1978). cooperia hungi is considered a definitive parasite of impalas and the other nine nematode species all occurred in five or fewer animals, which classifies them as eitheroccasional or accidental parasites (horak 1980). in horak’s (1978) study on impala at nylsvley, more than 90% of the worms were retarded in the fourth stage at different times of the year. as digests were notcarried out in this study, the recovery rate of larval stages was low, which may account for the low total helminth counts. the harsh environment in the northern and western parts of the limpopo province would necessitate helminth parasites to employ strategies to protect the vulnerable, free-living larval stages, not only during winter when temperatures and rainfall are low, but also during summer, when temperatures are high and rainfall often low. owing to the higher rainfall and milderclimate at nylsvley it is doubtful whether the same levels of larval retardation exist for worms in the current study area. the relatively few helminth species recovered could also be due to a smaller diversity of antelope species in the sampling areas. where a large number of antelopespecies are present, cross-transmission can take place more readily (boomker, anthonissen & horak 1988), which increases the diversity of parasite species. although two impalas from nwanedi in the soutpansberg had eosinophilic granulomata in the lungs, no parasites were recovered from these lesions. the mostcommon gross lesions associated with the lungworm pneumostrongylus calcaratus in impalas in swaziland were firm nodules along the lateral borders of the caudal lung lobes (gallivan et al. 1989). those authors recorded an 85% prevalence of this nematode in impalas in mlawula nature reserve, swaziland. pneumostrongylus calcaratus has been found to be prevalent in the eastern transvaal lowveld (now limpopo and mpumalanga) as indicated by ortlepp (1962) and young and wagener(1968), and in the northern part of kwazulu-natal (anderson 1992). young and wagener (1968) found a high prevalence in impala in the southern kruger national park, whereas horak (1978, 1980) did not recover lungworms from impalas at nylsvley nature reserve or from pafuri in the northern kruger national park. horak (1981) ascribed this difference in prevalence to climate. he suggested that pneumostrongylus is restricted to warm, moist regions, where the mollusk intermediate host(urocyclus [elisolimax] flavescens) occurs. table 2: total helminth burdens of examined impala. kudus five nematode species and one trematode were recovered from kudus (tragelaphus strepsiceros) in this study. the total worm burdens are summarised in table 3. the kudu with the highest burden had 2690 worms, of which 2425 were haemonchus contortus. excluding this kudu, the mean burden for the remaining seven kuduswas 81 worms. this is a classical example of overdispersion of the parasites. the lowest burden was six worms, comprising the two species elaeophora sagitta and fasciola hepatica, which both use intermediate hosts. the mean helminth burden, including e. sagitta and f. hepatica, was 407 worms. haemonchus contortus was the most prevalent worm and was recovered from six of the eight kudus, followed by e. sagitta recovered from three kudus. according to the criteria of horak (1980), haemonchus vegliai, cooperia neitzi, cooperia acutispiculum, e. sagitta and t. deflexus are definitive parasites of kudus in thekruger national park (boomker et al. 1989) and in the eastern cape (boomker, horak & knight 1991b). the amphistomes, schistosoma mattheei, moniezia benedeni, taenia larvae, i. tuberculata, agriostomum gorgonis and strongyloides papillosus are considered occasional parasites in kudu (boomker et al. 1991b). kudus are known to harbour parasites from other species of antelopes. boomker et al. (1991b) found that kudus in the addo elephant national park harbourednematodirus helvetianus and dictyocaulus spp., thought to originate from cattle in the area. the h. contortus recovered in the current study probably originated from goats. cooperioides hamiltoni, a parasite described from impalas, was recovered from kudus in this study and is considered an occasional parasite of kudus (boomkeret al. 1988). except for one kudu from the soutpansberg area, the helminth burdens were relatively low but the range was similar for that of kudu sampled in the etosha gamereserve (boomker et al. 1988) and the eastern cape (boomker et al. 1991b). this is in contrast to kudus sampled in the kruger national park (boomker et al. 1989), which showed much higher burdens. the kudus were mainly from the southern region of the park, which has an annual rainfall of 600 mm – 700 mm. the dry climate of the localities in the present study area is more similar to that in etosha and the eastern cape and is too harsh for the survival of the free-living infectivestages of helminths (boomker et al. 1989). of the helminths considered definitive parasites of kudu in the kruger national park (boomker et al. 1989) only c. neitzi and e. sagitta were recovered from the kudus in this study. however, c. neitzi was recovered only in one kudu and in small numbers. elaeophora sagitta was recovered from three kudus from two of the localities in this study and also not in significant numbers. their presence is an indication of the abundance of their intermediate host (boomker 2007). because kudus are browsers and roam considerably they are subject to reinfection with their own parasites only to a limited extent (boomker, horak & de vos 1986). however, animals that roam are more likely to become infected with accidental parasites (horak 1980). agriostomum gorgonis and oesophagostomum spp. are considered accidental parasites as they occurred only in one kudu each and in low numbers. table 3: total helminth burdens of examined kudu. bushbuck no parasites were recovered from the single animal recovered in this survey. this may be a result of underdispersion or the low stocking densities of antelopes on the farm of origin, which, in turn, will also limit the exposure of animals to infection or cross-infection with helminths. nyala the carcass of the nyala bull was submitted to the louis trichardt veterinary laboratory for a post-mortem examination in october 2007. clostridium novyi was diagnosed as the cause of death. the animal was in poor condition and died during the rutting season. it was expected that the animal would have harboured some parasites under these conditions, yet none was recovered. the nyala originated from a farm where cattle and antelopes shared the same pasture. deworming of cattlecould be a possible reason for the absence of helminths as the parasite contamination on the pasture would be reduced. also, the nyala died at the end of the dry season, before the first spring rains, when larvae were still in hypobiosis or absent from the pasture. blue wildebeest five nematode species were recovered from blue wildebeest (connochaetus taurinus) in this study and their burdens are summarised in table 4. the highest total burden in an animal was 550 worms. the mean helminth burden of the four blue wildebeest was 407 worms. the species with the highest intensity was h. contortus (500 per individual), followed by haemonchus bedfordi (375 per individual). this parasite appears to be common in blue wildebeest and has been recovered from animals from the kruger national park (horak, de vos & brown 1983b) as well as from the kalahari gemsbok national park (now kgalagadi transfrontier park) (boomker et al. 1986). cooperia connochaeti was recovered from three of the four blue wildebeest in this study. it is considered a definitive parasite for this species. the seasonal patterns of several parasites of wildebeest from the kruger national park have been attributed to a combination of climatic factors and migratory patterns of the wildebeest in the park (horak et al. 1983b). on small game farms animals generally have limited migration patterns and therefore the risk of becoming reinfected from the contaminated pastures increase. table 4: total helminth burdens of examined blue wildebeest. black wildebeest one of the two black wildebeest (connochaetes gnou) sampled harboured 100 h. bedfordi only. the other presented with a total of 1075 worms, comprising 50 a. gorgonis, 875 h. bedfordi and 150 o. columbianum. the helminths of black wildebeest have received little attention (boomker et al. 2000). wildebeest appear to be fairly resistant to parasitic infections, as animals fromthe golden gate highlands national park and rietvlei nature reserve harboured relatively low burdens of only a small number of helminth species when compared with blue wildebeest from the kruger national park (horak et al. 1983b). in an earlier study, boomker et al. (2000) found that a black wildebeest from the mountain zebra national park harboured a single taenia sp. larva, 26 haemonchus spp. females and one haemonchus spp. larva. neither of two black wildebeest from the karoonational park sampled during that study harboured any worms (boomker et al. 2000). gemsbok two nematode species and the larval stages of two cestode species were recovered from the gemsbok sampled in the present study. one of the two gemsbok from musina had 350 c. hungi, one setaria hornbyi and seven taenia hydatigena larvae, whilst the other had no nematodes and only six t. hydatigena and three taenia hyaenae larvae. the gemsbok from kerneels young trust harboured three calcified setaria spp. and two t. hydatigena larvae. the larval stages of t. hydatigena were found attached to the mesenterium and liver of all three gemsbok. the t. hyaenae larvae occurred in the triceps and heartmuscles. the metacestodes were identified according to the number and size of their rostellar hooks (verster 1969). although gemsbok are relatively common in the limpopo province, they are historically not endemic to this region. the translocation of antelopes to climatic orvegetation regions where they did not occur historically can be hazardous to their health (boomker et al. 2000). earlier studies found the total helminth burden of gemsbok from the west coast national park, where the antelopes do not naturally occur, to be 28 681 (boomker et al. 2000), compared with 5877 for gemsbok from the kalahari gemsbok national park (now kgalagadi trasfrontier park) (boomker et al. 1986). the high burden for gemsbok in the west coast national park isattributed to the climatic conditions in the area, as the climate appeared to favour parasites but caused stress in the gemsbok (boomker et al. 2000). in the limpopo province the climate is rather similar to that of the kalahari, which may be the reason for the small numbers of helminths recovered from gemsbok in the present study. waterbuck five helminth species were recovered from the waterbuck (kobus ellipsiprymnus), namely cooperia curticei, trichuris spp., o. columbianum, avitellina spp. and stilesia globipunctata. the helminth with the highest intensity was c. curticei (2075), followed by trichuris spp. (50) and o. columbianum (25). this is approximately half of what was recovered from a single waterbuck from pretoriuskop in the kruger national park, which harboured 4082 helminths (boomker et al. 1986) and included h. bedfordi (813), cooperia fuelleborni (1524), c. hungi (363), cooperia yoshidai (1088), i. tuberculata (225) and o. columbianum (25) (boomker et al. 1986). warthogs ten helminth species were recovered from the warthogs in this study. the total burdens of each parasite are presented in table 5. two nematode genera occurred in all the warthogs, namely oesophagostomum mwanzae and probstmayria vivipara. murshidia hamata was recovered from four of the six warthogs and oesophagostomum mocambiquei from three. murshidia pugnicaudata was recovered from only two warthogs. hydatid cysts of echinococcus granulosus were recovered from the lungs of one of the warthogs. the helminth with the highest intensity was p. vivipara, with burdens ranging between 276 000 and 825 000 per host. the helminth with the second highest intensity was o. mwanzae, with a total burden of 3700 recovered from one warthog. the highest total helminth burden that included p. vivipara, was 825 940 including for warthog bwh1. however, warthog bwh3 had the highest helminth burden (5920) that excluded p. vivipara. no trichinella were found in any of the carcasses. four of the warthogs originated from bushman’s safaris, two of which were examined in august 2006 and two in august 2007. the two warthogs from the 2006 sample harboured murshidia spp. but no o. mocambiquei, but the converse was true for the warthogs examined in 2007. the two warthogs sampled in 2006 also had lower total helminth burdens (excluding p. vivipara), namely 162 and 702, respectively. the warthogs sampled in 2007 had total burdens (excluding p. vivipara) of 5920 and 4810, respectively. it is possible that although lephalale had below-average rainfall during the 2006/2007 season, rain during the dry season in 2007 could have affected the abundance of parasites in the region. the helminths of warthogs in southern africa have been described by several authors, including boomker et al. (1991a), horak et al. (1983a, 1988) and ortlepp (1964). the helminth parasites of warthogs as listed by round (1968) include parasites from across africa. however, many helminths occur only in certain regions as is evident from the work of ortlepp (1964), horak et al. (1983a, 1988) and boomker et al. (1991a). table 5: total helminth burdens of examined warthogs. bushpig a total of 80 worms representing two nematode species, namely physocephalus sexalatus (10) and globocephalus versteri (70), were recovered from the single bushpig. the total worm burden of the bushpig was low compared with that of warthogs. ortlepp (1964) made no note of the burdens of the worms he recovered from bushpigs. because only one animal was examined, a low burden due to underdispersion could not be ruled out. little work has been conducted on the helminth parasites of bushpigs. ortlepp (1964) examined material from mozambique and the northern transvaal (now limpopo), which has been included in the list of helminth parasites of round (1968). bushpigs are difficult to sample because of their largely nocturnal activity andtheir relatively limited distribution in forested areas. helminths agriostomum agriostomum gorgonis was recovered only from one kudu and one black wildebeest, and in small numbers from both animals. it is considered an occasional parasite of kudus in the kruger national park (boomker et al. 1989). no record could be found of the worm in black wildebeest, but it has been listed as a parasite of bluewildebeest (horak et al. 1983b; round 1968). the free-living stages of hookworms require moist climatic conditions for survival and the low rainfall in the present study area would have been limiting to the survival of the larvae of these parasites. cooperia cooperia hungi was recovered from only one warthog in this study. it has previously been recovered from warthogs at hoedspruit in the eastern transvaal (now eastern part of the limpopo province) (boomker et al. 1991a). it is primarily a parasite of impala (horak 1978) and is considered an accidental parasite in warthogs(boomker et al. 1991a). cooperioides cooperioides hepaticae is the only extra-intestinal trichostrongyloid of impalas and is considered a common parasite of impalas in the kruger national park (pletcher et al. 1988; young & wagener 1968). pletcher et al. (1988) suggest that smaller burdens in adult impalas compared with yearlings may be due to acquired partial immunity in adults due to previous exposure. cooperioides hepaticae is generally considered to be of minor pathological significance unless it is present in largenumbers in combination with other trichostrongyles, or when burdens are associated with poor nutritional conditions (pletcher et al. 1988). in the present study, c. hepaticae was recovered from only two impalas and both had very small burdens. one reason for the low prevalence and intensity could bethat the animals sampled were all adults and thus would have had an acquired immunity. also, the animals were all sampled before the start of the rainy season and therefore the peak burdens of this parasite might have been missed. the dry climate of the study area will affect the survival of free-living stages of the parasites. elaeophora elaeophora sagitta is a definitive parasite of the tragelaphine antelopes (boomker et al. 1988). this worm was recovered only from three kudus (i.e. none were recovered from the bushbuck or nyala) during the current study. this can be because neither the parasite nor its vector, thought to be a tabanid fly (boomker et al. 1986), is common in the study area. haemonchus haemonchus contortus was the most prevalent worm in kudus in this study. it was recovered from six kudus from three different localities in the study area. except for one kudu, the individual burdens of the worm was small. at langgedachte, h. contortus was collected from three blue wildebeest and three kudus. at this game farm, domestic livestock and game are kept together.haemonchus contortus is considered a parasite of domestic animals and the high prevalence is possibly due to cross-transmission from domestic animals and the high stocking densities on the farm. impalaia impalaia tuberculata is a parasite of several antelope species, in particular impalas and blesbok, (damaliscus dorcas phillipsi) (boomker 1977; horak 1978). it was recovered from only one warthog in this study. warthogs are not considered to be definitive hosts of these parasites (boomker et al. 1991a; horak et al. 1988) and infection was probably accidental. murshidia ortlepp (1964) recovered m. hamata from pilgrim’s rest and zululand, but m. pugnicaudata only from pilgrim’s rest. no murshidia spp. have been recovered from warthogs in namibia (horak et al. 1983a). both species have been recovered from warthogs from hoedspruit (boomker et al. 1991a) and the kruger national park(horak et al. 1988). only m. hamata has been recovered from warthogs in the north-western transvaal (now the limpopo province) (boomker et al. 1991a). between 50 and 250 m. hamata were recovered from four of the warthogs in this study. murshidia pugnicaudata, however, was recovered in small numbers from only two animals: one from the musina area in the north and the other from the lephalale area in the west. both species therefore occur in the north, east and west of the province, but m. pugnicaudata possibly not in the north-west. however, it is not stated from where the warthogs in the north-western transvaal originated (boomker et al. 1991a). it should be noted that m. pugnicaudata was recovered only from warthogs examined during 2006. it is possible that climatic factors played a role in the prevalence of the parasites. oesophagostomum oesophagostomum columbianum is a common parasite of many antelope species (boomker 2007). it was the most prevalent helminth in this study and was recoveredfrom four hosts, namely the waterbuck, blue and black wildebeest, and impalas. in none of these animals did this worm occur in large numbers. the typical lesions in the large intestines of sheep due to o. columbianum infestation were not found in any of the examined animals. horak (1981) considers o. columbianum as a definitive parasite of impala. both o. mwanzae and o. mocambiquei have been recovered from warthogs from hoedspruit (boomker et al. 1991a), the kruger national park (horak et al. 1988) and pilgrim’s rest (ortlepp 1964). both species were also recovered in this study from animals that originated from the musina and lephalale areas. it would appear thatthese are the only two oesophagostomum species that occur in warthogs in the limpopo and mpumalanga provinces. although o. mwanzae is the dominant of the two species, both can be considered definitive parasites of warthogs in the area. setaria setaria spp. are filarid worms and the adults are usually found in the abdominal cavity of their hosts. mosquitoes are the intermediate hosts (reinecke 1983). thesetaria spp. are considered non-pathogenic and are usually found only at necropsy. trichostrongylus trichostrongylus deflexus has been described as a separate species only relatively recently (boomker & reinecke 1989). mönnig (1933) described specimens oftrichostrongylus instabilis from impala with extreme variation in the spicule shape. similarly, horak (1980) found the same extreme variation in spicule shape fromtrichostrongylus males that he recovered from sheep, calves and goats that had been artificially infected from faecal cultures of impalas. he referred to these worms as trichostrongylus colubriformis. boomker and reinecke (1989) re-evaluated some of these specimens and proposed that these worms represent a definitive species,which they named t. deflexus. horak et al. (1983b) reported t. colubriformis from blue wildebeest from the kruger national park. they made no mention of the spicule morphology. after examining the material collected by horak et al. (1983b) from the wildebeest, boomker and reinecke (1989) considered t. colubriformis from this material as synonymous with t. deflexus. trichostrongylus deflexus was recovered from blue wildebeest and impalas in the current study. trichuris ortlepp (1937) described trichuris barbertonensis from an ox. the spicules and sheath measured between 6.83 mm and 7.3 mm and did not end in a bulb or swelling as is described for trichuris globulosa or trichuris ovis. two individual adult trichuris worms were recovered from the large intestine of the waterbuck in the current study. the spicules of the male measured 5.8 mm in length and did not end in a bulb or swelling either. it is possible that the specimen collected from the waterbuck was t. barbertonensis. many trichuris spp., of which t. globulosa is the most widespread, have been reported to occur in antelopes in southern africa (ortlepp 1937). trichuris spp. are of greater concern in enclosures and zoos than in open spaces owing to their monoxenous life cycle and thick-shelled eggs (boomker 2007). environmental build-up ofthe parasite is not likely to happen in a free-range system. avitellina avitellina spp. is common in semi-arid areas (reinecke 1983) but was recovered only from the waterbuck. stilesia stilesia globipunctata fragments were recovered from the waterbuck. no previous record of this parasite in waterbuck could be found. both stilesia and avitellina require an invertebrate intermediate host, such as oribatid soil mites. adult worms are therefore more likely to be found in animals that feed close to the ground (boomker et al. 1986). in this study stilesia hepatica was recovered from only one impala. gallivan et al. (1996) reported no difference between seasons or sexes, but in their study adult animals had larger burdens, possibly indicating that it is an accumulative infection. echinococcus hydatid cysts were recovered from the lungs of one warthog. echinococcus granulosus has remarkably low intermediate host specificity (macpherson 1985). hydatidcysts have been recovered from many species, including impalas, warthogs and zebras (equus burchellii) (young 1975). possible definitive hosts of echinococcus spp. on the farm in lephalale are spotted hyenas (crocuta crocuta), black-backed jackals (canis mesomelas), african wild cat (felis lybica) (verster & collins 1966), and domestic dogs. taenia spp. the larval stage of t. hydatigena is much larger than that of other species and is thus more easily identified (verster 1969). the intermediate hosts are ruminants andpigs, whilst the final hosts are canines and other wild carnivores. in sheep the migratory tracts through the liver, commonly known as hepatitis cysticercosa, can cause the death of an animal (reinecke 1983). no macroscopic liver pathology was noted in any of the gemsbok affected. taenia hyaenae utilises many ruminant species as intermediate hosts. the definitive hosts for this species are hyaenas and african wild dogs (lycaon pictus) (loos-frank 2000). spotted hyaenas, brown hyaenas (hyaena brunnea), african wild dogs, black-backed jackals and leopards (panthera pardus) are all known to frequent the farm from which these gemsbok came. cysticerci that resemble this species in the number and shape of the rostellar hooks have previously been recovered from impalasand sable antelopes (hippotragus niger) (verster 1969). macroscopically it cannot be distinguished from the cysticerci of other taenia spp., including taenia saginata. only microscopically can t. saginata be identified by the absence of rostellar hooks, an important consideration during meat inspection. humans are the definitive hosts for the latter cestode, which has been recovered from various game species. larval t. saginata has been found in several hosts in africa; for example graber(1959) found the species in dorcas gazelles (gazella dorcas), red-fronted gazelles (gazella rufifrons) and gazella spp. in chad, le roux (1937) observed it in oribis (ourebia ourebi) in zambia, sousa dias (1950) found it in cape buffaloes (syncerus caffer) in angola and nelson, pester and rickman (1965) in bushbuck in kenya. also, the metacestodes of taenia solium have been found in warthogs in africa (bain 2001) and in wild boars (sus scrofa) in europe (eslami & farsad-hamdi 1992). itis evident that there is a potential public health risk associated with the consumption of venison infected with metacestodes. hydatid material of human origin collected in sub-saharan africa all conform to the sheep strain of e. granulosus (macpherson & wachira 1997). however, this doesnot prove non-infectivity of other e. granulosus strains, species or subspecies to humans. because of the seriousness of echinococcus spp. infection, meat inspection should be thorough and the necessary precautions should be taken when handling carnivores (young 1975). fasciola two specimens of f. hepatica were recovered from a kudu from the nwanedi game reserve. fasciola hepatica has not been described from kudus previously. as browsers, kudus are not normally exposed to aquatic vegetation and thus few get infected with trematodes (condy 1972). fasciola gigantica has been reported ingreater kudu on a game ranch in zambia (zieger et al. 1998) as well as in rhodesia (now zimbabwe), where kudu deaths were attributed to this parasite (condy 1972; hammond 1972). kudus appear to be very susceptible to infection (condy 1972). the nwanedi nature reserve, where the infected kudu was examined, has a large dam that is fed by the nwanedzi river. this creates a favourable environment for the freshwater snail that serves as intermediate host to the flukes. domestic stock have previously had access to the reserve and are currently kept upstream from the dam, which could be a possible source of contamination. wildlife that have been introduced to the reserve are other possible sources. fasciola gigantica is the most common liver fluke in domestic ruminants in africa and accounts for most liver fluke infections in wild animals (hammond 1972). conclusion (back to top) the aim of the study was to describe the helminth parasites of the common wildlife species in the northern and western parts of the limpopo province. the climate of these areas is harsh and dry, with a large region that is considered to be semi-arid. this has an impact on the prevalence of the parasites that occur in these areas. in total 36 animals were examined and their helminths recorded. new host records for species include a. gorgonis in black wildebeest, s. globipunctata in waterbuck and f. hepatica in kudu. for the study area as a whole, comparisons with other studies provided valuable information. the total burdens and species variation of especially the adult helminths in this study were consistently lower compared with studies in areas with higher rainfall. the only trematode recovered was f. hepatica from a kudu at nwanedi. the dry climate is not conducive to the survival of freshwater snails that act as intermediatehosts. these parasites typically occur in areas with wetter or more humid conditions. there are practical implications for the low prevalence and species variation of helminths. when animals are translocated from the drier parts of the limpopoprovince to areas with higher rainfall, they will be relatively naive to the higher parasite loads in those areas and/or completely naive to other parasites, such as lungworms. on the other hand, animals originating from areas with higher rainfall are likely to harbour higher parasite loads. they should be dewormed prior to translocation to dry areas, because the stress of the dry climate and change in vegetation can cause them to succumb to the parasite loads they already harbour. at the same time, new parasites could be introduced, with variable results to the wildlife already present. game is kept together with domestic stock on many farms in the province. helminths that typically occur in domestic stock were recovered from some antelope species. haemonchus contortus was recovered from all the kudus and blue wildebeest from langgedachte. high stocking densities of animals on this farm likely contributed to the frequency of cross-transmission of parasites between species. with parasites that have a high fecundity, such as haemonchus or oesophagostomum, environmental build-up may become considerable under certain conditions or specific times of the year. this will impact on worm control programmes for domestic animals, but may also cause morbidity in game at times. some parasites are more problematic in intensive situations, for example when animals are kept in a boma or very small camps. this is especially true for parasites with a monoxenous life cycle such as trichuris spp. animals that are kept intensively should be dewormed to prevent environmental build-up of the parasite. under extensive conditions, these parasites are unlikely to cause any problems. the only known zoonotic helminth recovered was e. granulosus from the lungs of a warthog. the infective stage for humans is the egg that is deposited by the final host – dogs – which are often fed uncooked offal. one should be aware that the parasite can become established in the local human population through this route, especially in communal areas where people live in close association with their animals. cysticerci of t. hyaenae and t. hydatigena were recovered from the gemsbok. although these tapeworms are not known to be zoonotic, the cysticerci are notmacroscopically distinguishable from the zoonotic cysticeri of t. solium or t. saginata. this has public health implications because the cysticerci render the meat aesthetically unacceptable and, in addition, a lack of proof of non-infectivity does not prove that these cestodes are not in fact infective to humans. the host species included in the study are some of the more common ones that occur on game farms in the limpopo province. the parasites of many wildlife species, including relatively common species such as giraffes (giraffa camelopardalis), common reedbuck (redunca arundinum) (boomker et al. 1984) and grey rhebuck (pelea capreolus) (see boomker et al. 2000) but also scarcer game such as red duikers (cephalophus natalensis) (see boomker et al. 1984), have not been studied extensively, particularly in the limpopo province. because these animals all share the same, mostly confined environment, scientific game farming practices require more knowledge on the parasites, including the helminths, of these species. acknowledgements (back to top) the project was funded partly by a study bursary from the department of agriculture, limpopo. the late mr. ryno watermeyer from the helminthology section at the department of veterinary tropical diseases and ms. i. venter of the louis trichardt state veterinary laboratory are thanked for their technical assistance. thanks also go to mr. r. van wyk of the musina research station, mr. s. joubert of kerneels young trust, mr. m. vermaas of langgedachte, mr. n. van zyl of bushman’s safaris and mr. c. van heerden, for making material available. competing interests the authors declare that there were no competing interests that may have inappropriately influenced the presentation of information in this paper. authors’ contributions i.c.v.w. conducted the study in partial fulfillment of an msc degree. j.b. acted as supervisor for the thesis and made conceptual and editorial contributions. references (back to top) acocks, j.p.h., 1988, veld types of south africa. botanical research institute, pretoria. 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journal of the south african veterinary medical association 39, 81–86. zieger, u., boomker, j., cauldwell, a.e. & horak, i.g., 1998, ‘helminths and botfly larvae of wild ungulates on a game ranch in central province, zambia’, onderstepoort journal of veterinary research 65, 137–141. pmid:9741058 article information authors: p. dorny1 c. kabwe1 k. kirezi1 k. lukanu1 p. lutumba1 v. maketa1 p. matondo1 k. polman1 n. praet1 n. speybroeck1 j. sumbu1 affiliations: 1university of kinshasa, kinshasa, democratic republic of congo correspondence to: antony musoke how to cite this poster: dorny, p., kabwe, c., kirezi, k., lukanu, k., lutumba, p., maketa, v. et al., 2012, ‘cysticercosis in the democratic republic of congo’, onderstepoort journal of veterinary research 79(2), art. #485, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.485 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cysticercosis in the democratic republic of congo in this poster... open access cysticercosis, caused by taenia solium eggs, is a zoonotic disease whose consequences can be severe especially in the cerebral localisation (neuorcysticercosis). indeed, neurocysticercosis is the first cause of epilepsy amongst the infectious etiology group. following the increase of epilepsy cases in kinshasa and bas-congo, it was important to assess the fraction attributable to neurocysticercosis especially as data on cysticercosis in democratic republic of congo (drc) dating from 1970. a joint study between veterinary and human doctors was conducted in the provinces of bas-congo and kinshasa between 2008 and 2010. blood samples were collected from the general population, patients with epilepsy and pigs. these samples were analysed using elisa antigen in the laboratory of the institute of tropical medicine in antwerp. patients positive to elisa antigen took the ct scan exam for the confirmation of neurocysticercosis. in the province of kinshasa, of 530 epileptic patients, 6.3% were identified as neurocysticercosis cases. out of a total of 498 pigs, 38.9% were positive for cysticercosis. in the province of bas-congo, of 943 inhabitants from malanga village, 21.6% were positive with predominance in males (26.4% versus 17.5%). a total of 145 pigs from 5 villages were examined and 41.2% found positive. we can conclude that cysticercosis in the drc has been neglected for a long time and cysticercosis could be a real major public health problem. prospective studies addressing the consequences of cysticercosis in communities are needed in order to prevent epilepsy due to neurocysticercosis. article information authors: reza kheirandish1 masoud sami2 shahrzad azizi3 mohammad mirzaei1 affiliations: 1department of pathobiology, shahid bahonar university of kerman, iran2department of food hygiene and public health, shahid bahonar university of kerman, iran 3department of pathobiology, islamic azad university, shahrekord branch, iran correspondence to: reza kheirandish postal address: department of pathobiology, shahid bahonar university, kerman, iran dates: received: 30 mar. 2012 accepted: 14 sept. 2012 published: 29 nov. 2012 how to cite this article: kheirandish, r., sami, m., azizi, s. & mirzaei, m., 2012, ‘prevalence, predilection sites and pathological findings of taenia multiceps coenuri in slaughtered goats from south-east iran’, onderstepoort journal of veterinary research 79(1), art. #436, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.436 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence, predilection sites and pathological findings of taenia multiceps coenuri in slaughtered goats from south-east iran in this original research... open access • abstract • introduction • materials and methods    • study area    • sample collection    • pathological examination    • ethical considerations • results    • prevalence rate and predilection sites    • macroscopic and histopathologic findings • trustworthiness • discussion • conclusion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ coenurosis is a zoonotic disease in a variety of ruminants caused by the metacestode of taenia multiceps. the coenuri in the brain and spinal cord of sheep and goats have been identified as coenurus cerebralis whilst those reported in other tissues have been named coenurus gaigeri. this study was conducted during the spring and summer of 2011. out of 25 739 goats inspected in slaughterhouses, 23 carcasses (0.09%) revealed one or multiple visible swellings on the different muscles and visceral organs. the coenuri, of variable sizes, were found mainly in the muscles of the thigh, shoulder and neck, and were less common in the abdominal muscles and subcutaneous tissues. coenuri were also found in the diaphragm, tongue, intercostal muscles, lung, parotid area and tunica adventitia of the aorta in a goat with severe infection. the brains of slaughtered goats that had coenuri in their skeletal muscles were examined and coenuri were found in two specimens (8.69%). the coenuri were located in the occipital lobe, the anterior part of the right cerebrum and the parietal lobe of the left cerebrum. histopathologically, coenuri in the brain caused pressure atrophy and liquefactive necrosis in the surrounding tissues, hyperaemia, perivascular cuffing, neuronal degeneration, neuronophagia, satellitosis, diffuse microgliosis and astrocytosis. coenuri in the skeletal muscles caused degenerative and necrotic changes, hyalinisation and myositis. in the lung, tissues around the coenurus revealed atelectasis and focal interstitial fibrosis. in the present study, concurrent occurrence of coenuri in the central nervous system and skeletal muscles supports the hypothesis that c. cerebralis and c. gaigeri are different names for the metacestodes of the same species of tapeworm. introduction top ↑ coenurosis (gid or sturdy) is a zoonotic disease caused by the metacestodes of taenia multiceps in various livestock species. coenurosis is an economically important disease as it causes serious problems in especially the sheep industry. taenia multiceps inhabits the small intestine of the definitive hosts, which include dogs and wild carnivores. carnivores are mainly infected by eating the uncooked offal of domestic animals, in which the larval stages of the tapeworms such as t. multiceps and echinococcus spp. occur. when vegetation contaminated with eggs is ingested by the intermediate hosts – in this case ruminants – the oncospheres hatch in the small intestine and pass through the intestinal wall to reach the brain via the bloodstream, where they develop further (soulsby 1982; verster 1965). in addition to infecting the brain, the larval stage also develops in the subcutis and muscles, and in the body cavities (schuster, sivakumar & wieckowsky 2010). intramuscular and subcutaneous coenuri are referred to as taenia gaigeri by some authors (boch & supperer 1983) whilst others regard it as synonymous with t. multiceps (verster 1969; verster & bezuidenhout 1972). the intermediate host apparently also plays a role in determining the kind of coenurus (schuster et al. 2010; soulsby 1982). coenurus gaigeri, the metacestode of t. gaigeri has been reported in the subcutaneous tissues of sheep and goats. the morphological features of the coenuri occurring in the brain and other tissues have been reported to be similar (soulsby 1982). the metacestode, especially in goats, have been documented with an alternative name, c. gaigeri (bhalla & negi 1962; el sinnari, tageldin & sumri 1999; sharma et al. 1995; sharma & chauhan 2006). this study was aimed at describing the pathological lesions caused by t. multiceps, the site of predilection, and the size and number of coenuri in different tissues of naturally infected goats. materials and methods top ↑ study area this study was performed in kerman province, iran, during the spring and summer of 2011. the province of kerman is in the south-east of iran and covers an area of 181 714 km2. the geographical latitude and longitude of this area is 30°15´n and 57°01´e, respectively. kerman city is located in an arid desert area with a semi-moderate and dry climate. the maximum and minimum temperatures reach 39.6 °c and –7 °c, respectively. rainfall is irregular and scarce. the density of livestock animals in this area is 33 animals per square kilometre. sample collection the carcasses of 25 739 goats were inspected for coenuri (13 882 in the spring and 11 857 in the summer) at the kerman slaughterhouse in south-east iran. the tissue locations, numbers and sizes of coenuri from each goat were recorded. the macroscopic and microscopic lesions of the coenuri were evaluated. the heads of the infected goats were separated from the rest of the carcasses, and after removing the skin, a cross-section was cut through the area just caudal to the frontal bone using a handsaw. this was followed by two parallel cuts on the parietal bone. the bone was removed using a chisel and hammer and the meninges were incised with a scalpel blade to expose the brain. the brain was examined for the presence of coenuri. the spinal cord was examined after the removal of vertebral arches. pathological examination the coenuri and the surrounding tissues were fixed in 10% neutral buffered formalin for histopathological examination. the samples were dehydrated in graded ethanol and embedded in paraffin wax. sections were cut at 5 μm thickness, mounted on glass slides and routinely stained with hematoxylin and eosin. each section was examined by light microscopy. ethical considerations the study was approved by the local ethics committee of our faculty, in accordance with the ethics standards of the ‘principles of laboratory animal care’. results top ↑ prevalence rate and predilection sites with regard to the prevalence rate and predilection sites of coenuri, 23 out of 25 739 goats inspected in the slaughterhouse (0.09%) revealed one or more visible swellings on the different skeletal muscles and visceral organs. of the affected carcasses, 13 were found in spring and 10 in summer. the swellings were soft and pitted on pressure and varied from 2.1 cm × 3.4 cm to 8.5 cm × 10.2 cm. clear watery fluid and whitish clusters of scolices were visible through the thin and transparent wall of the coenuri (figure 1). as shown in figures 2 and 3, the coenuri were found mainly in the muscles of the thigh (15) and neck and shoulder region (10), whilst less frequently in the subcutaneous tissues and perineal fat (5) and abdominal muscles (3). although the coenuri were usually observed at the superficial surfaces of affected muscles, they sometimes extended very deep into the muscles, especially in the neck region. figure 1: multiple coenurui embedded superficially and deep between the muscles of the neck and shoulder (arrowheads). figure 2: a coenurus at the base of the tongue. figure 3: microscopic features of coenurosis in the brain. figure 4: inflammatory cells including giant cells (arrowheads) and neutrophils (astrisk) around the coenurus. a total of 68 coenuri were recorded in a single carcass, with most located in the skeletal muscles. single coenuri were found in the diaphragm, the intercostal muscles, the right lung, the pelvic cavity and at the base of the tongue (figure 2). single coenuri were also located in the tunica adventitia of the aorta above the base of the heart and close to the parotid gland. the brain was infected with three coenuri, of which two occurred in the occipital lobe of the right hemisphere and the other in the anterior part of the cerebrum near the olfactory lobe. cerebral coenuri were detected in two of the 23 infected goats (n = 2/23, 8.69%). in another case, the parietal lobe of the left cerebrum was infected with a single coenurus . none occurred in the spinal cord. macroscopic and histopathologic findings coenuri in the brain caused damage to surrounding tissues, including thinning of the cerebral grey and white matter owing to focal pressure atrophy and liquefactive necrosis. the meninges were hyperaemic and oedematous, and microscopically there were degenerative and necrotic lesions in the brain. within the brain, sections of coenuri of various shapes were surrounded by marked eosinophilic necrotic tissues, degenerated neutrophils and mononuclear cells. a zone of chronic cellular reaction consisting of langhans giant cells and mononuclear cells was present on the outer layer and a capsule of fibrous connective tissue enclosed the whole coenurus (figures 3 and 4). cerebral tissues around the coenuri revealed hyperaemia, perivascular cuffing predominantly comprising mononuclear cells, neuronal degeneration, demyelination, neuronophagia, satellitosis, diffuse microgliosis and astrocytosis. in the skeletal muscles, the metacestodes caused mechanical destruction with associated degenerative and necrotic changes, hyalinisation, and focal to diffuse inflammatory cell infiltration that included lymphocytes and eosinophils. in the lung, the coenurus caused atelectasis and focal interstitial fibrosis in the pulmonary parenchyma around the coenurus. trustworthiness top ↑ the investigators who undertook the gross observations and histopathological studies in the present study were unaware of the experimental design and grouping details. the histopathological studies performed blind by three different pathologists. discussion top ↑ infection by the larval stage of the tapeworm t. multiceps in small ruminants is common worldwide (oryan et al. 2010; shivapraksh & thimma reddy 2009; uslu & guclu 2007), but rare in horses (ed. fraser 1991), cattle (avcioglu et al. 2011) and humans (webbe 1994). reports from asian countries note the central nervous system and skeletal muscles as the main predilection sites in the intermediate hosts (alim et al. 2002; gosh et al. 2005; islam et al. 2006; moghaddar 2007; oryan et al. 2010; sharma & chauhan 2006). these sites have not been reported as often from countries outside asia, for example in sudan (hago & abu-samra 1980; ramadan, magzoub & adam 1973), oman (el sinnari et al. 1999) and namibia (bohrmann 1990).cerebral coenurosis occurs principally in sheep whilst extra-cerebral coenurosis is more common in goats. the clinical signs of the cerebral form include ataxia, hypermetria, blindness, head deviation, stumbling, paralysis, and head pressing and circling, and may be confused with other nervous diseases. the mortality rate may reach 100%. in most cases, animals with muscle infections remain without clinical symptoms and are diagnosed only at slaughter (soulsby 1982). in rare cases, treatment is administered (verster & tustin 1990). praziquantel and albendazole have been reported to be effective, with cure rates of 60% – 85% in humans (verster & tustin 1982). coenuri in the skeletal muscles may cause muscular pain or impairment of movement and eventually result in recumbency (sharma & chauhan 2006). locomotory disorders in the hind limbs may be related to pressure on the sciatic nerve caused by coenuri (ramadan et al. 1973). the present study has investigated the prevalence and pathologic characteristics of coenurosis in slaughtered goats in kerman province, iran. the prevalence of coenurosis was recorded as 0.09% (n = 25 739). the main tissues infected by coenuri were the muscles of the thigh, shoulder and neck, and less frequently the abdominal muscles, subcutaneous tissues and cerebrum. single coenuri were found in the diaphragm, tongue, intercostal muscles, lung, parotid area and tunica adventitia of the aorta in a severely infected goat. the occurrence of coenuri in the tongue, parotid area and tunica adventitia of the aorta is reported here for the first time, although gharagozlou, mobedi and akhavan (2003) described pulmonary coenurosis previously in goats from iran. in addition, their phylogenetic analysis of mitochondrial deoxyribonucleic acid (co1 and nd1) showed that c. gaigeri and coenurus cerebralis were not different species. in the present study, the location of coenururi in the central nervous system and other extra-cranial tissues (e.g. the skeletal muscles and subcutaneous and visceral organs) was observed. this finding supports the hypothesis that c. cerebralis and c. gaigeri are not different species. genetic characterisation and phylogenetic position is a diagnostic tool for identification of various species of metacestodes (huttner et al. 2008). godara et al. (2011) reported simultaneous coenuri in the cerebrum and liver of goats but found no coenuri in the skeletal muscles or subcutaneous tissues. they also concluded that c. cerebralis and c. gaigeri are not different species. in other studies with regard to goats, coenuri were reported only from subcutaneous and skeletal muscles. patro et al. (1997) found coenuri only in the subcutaneous tissues of goats. shivapraksh and thimma reddy (2009) reported an outbreak of multiple subcutaneous coenuri in a herd of goats. they identified the subcutaneous coenuri as c. gaigeri because of their extra-cranial locations (i.e. in the neck, prescapular region, abdomen and limbs). jain and shah (1982) described that the subcutaneous location is an uncommon site for coenuri in sheep when compared to the brain and spinal cord. in our study, histopathological findings in the skeletal muscles included degenerative and necrotic changes, hyalinisation and myositis, which are in agreement with a previous report (oryan et al. 2010). in addition, the coenuri in the cerebrum caused perivascular cuffing, neuronal degeneration, demyelination, neuronophagia, satellitosis, diffuse microgliosis and astrocytosis. similar findings were described in previous studies (godara et al. 2011; nourani & pirali kheirabadi 2009; oryan, moghaddar & gaur 1994). in this study, no significant difference was observed between the prevalence of coenurosis during the spring and summer (10 infected goats in spring and nine in summer). the observation is in agreement with that of sharma, singh and tiwari (1998), who described c. cerebralis infections in sheep not to be associated with seasonal variation, but rather to occur throughout the year. cancedda et al. (2002) showed that coenurosis incidence in sardinia was highest during spring and the autumn. in jordan, abo-shehada et al. (2002) reported 11 of 12 infected heads during spring and autumn. the survival of the t. multiceps eggs in pasture, distribution of the final host and grazing behaviour of the intermediate hosts are factors that influence the rate of infection (desouky, badawy & refaat 2011). the difference with regard to incidence in the different agro-climatic zones may be attributed to diverse geographical, sociological and ecological factors (sharma & chauhan 2006). conclusion top ↑ the present study has indicated various predilection sites in coenurosis, including the brain, the skeletal muscles and other organs and tissues. the finding points to c. cerebralis and c. gaigeri being synonyms for the larval stage of t. multiceps in various animal species. further studies are necessary to clarify the tendency of coenuri to occur in the subcutaneous tissues, skeletal muscles and visceral organs of goats. acknowledgement top ↑ this research was financially supported by the research council of shahid bahonar university, kerman. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions r.k. (shahid bahonar university) was the project leader and pathologist, m.s. (shahid bahonar university) was responsible for the project design, sample collection and performing some experiments, s.a. (islamic azad university) performed pathology and m.m. 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africa’, onderstepoort journal of veterinary research 32, 7–118. pmid:5335506 verster, a., 1969, ‘a taxonomic revision of the genus taenia linnaeus, 1758 s. str.’, onderstepoort journal of veterinary research 36, 3–58. pmid:5407584 verster, a. & bezuidenhout, j.d., 1972, ‘taenia multiceps larva from a gemsbok’, onderstepoort journal of veterinary research 39, 123–124. pmid:4664320 verster, a. & tustin, r.c., 1982, ‘treatment of the larval stage of taenia multiceps with praziquantel’, journal of the south african veterinary association 53, 107–108. pmid:7120267 verster, a. & tustin, r.c., 1990, ‘treatment of cerebral coenuriosis in sheep with praziquantel’, journal of the south african veterinary association 61, 24–26. pmid:2269985 webbe, g., 1994, ‘human cysticercosis: parasitology, pathology, clinical manifestations and available treatment’, pharmacology and therapeutics 64, 175–200. http://dx.doi.org/10.1016/0163-7258(94)90038-8 article information authors: 1peter masoko 1jacobus n. eloff 2jackie picard affiliations: 1department of paraclinical sciences, phytomedicine programme, university of pretoria, south africa 2department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, south africa correspondence to: kobus eloff email: kobus.eloff@up.ac.za postal address: department of paraclinical sciences, phytomedicine programme, university of pretoria, private bag x04, onderstepoort, 0110 south africa keywords combretum; crust formation; erythema; exudates; terminalia; wistar rats; wound healing dates: received: 04 nov.2009 accepted: 28 aug.2010 published: 01 oct. 2010 how to cite this article: masoko, p., eloff, j.n., picard, j., 2010, the use of a rat model to evaluate the in vivo toxicity and wound healing activity of selected combretum and terminalia (combretaceae) species extracts,onderstepoort journal of veterinary research 77(1):art. #2, 7 pages. doi: 10.4102/ojvr.v77i1.2 copyright notice: © 2010. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 1025-9848 (print) issn: 2071-9736 (online) the use of a ratmodel to evaluate the in vivo toxicity and wound healing activity of selected combretum and terminalia (combretaceae) species extracts in this original research... open access • abstract • introduction • materials and methods    • plant collection    • plant storage    • extraction procedure    • preparation of extract    • selection of rats    • housing and feeding conditions    • preparation of animals    • wound creation    • topical application of aqueous creams    • observations • results • discussion • conclusion • acknowledgements • references abstract (back to top) wound healing is a fundamental response to tissue injury and several natural products have been shown to accelerate the healing process. the present study was undertaken to determine the safety and efficacy of the topical treatment of acetone leaf extracts of combretum imberbe, combretum nelsonii, combretum albopuntactum and terminalia sericea based on their in vitro antimicrobial activity. four circular full-thickness skin wounds were made on the backs of eight anaesthetised wistar rats using aseptic techniques. the treatments were administrated topically using 10% and 20% concentrations of each extract in aqueous cream in separate treatments. indications of erythema, exudate, crust formation, swelling and ulceration were used to determine the wound healing process. all of the wounds closed completely within 17 days. throughout the experiment, a subcutaneous probe was used to determine that the body temperature and body weight of the rats were within the normal range. c. imberbe and c. nelsonii extracts accelerated wound healing, but there was no significant difference in wound contraction using 10% and 20% concentrations of the extracts in cream. the results also showed the potential usefulness of this model to measure accelerating wound healing. the extracts could perhaps overcome defects associated with healing failure in chronic wounds and prevent secondary bacterial and fungal infections. introduction (back to top) the wound healing process involves a highly coordinated cascade of cellular and immunological responses over a period of time. the first phase (also known as the coagulation phase) occurs immediately after the traumatic injury and serves to seal the wound with a fibrin and platelet plug and to initiate the inflammatory process. cytokines produced by the platelets cause leukocytes from the blood circulation to amass at the site of injury. the predominant cell type in the first three days after injury is neutrophils, which phagocytose microbes and foreign debris. thereafter, activated macrophages at the wound site assist in wound repair by the release of proteases for wound debridement and by the phagocytosis of microbes, cell debris and damaged neutrophils. together with other cells, macrophages in the area produce various cytokines, chemokines and growth factors, which, in turn, cause the influx and activation of more macrophages, as well as the stimulation of the adaptive immune response (waldorf & fewkes 1995). granulation tissue forms 2–3 days after the injury and consists predominantly of fibroblasts. immediately following the injury and during granulation, keratinocytes migrate into the matrix and proliferate under the influence of growth factors produced by macrophages, keratinocytes and fibroblasts; while, at the same time, neovasculisation occurs. in the second week after the injury, the wound contracts as a result of the activity of adapted fibroblasts known as myofibroblasts. collagen production and remodelling continues with scar formation (tsirogianni, moutsopoulous & moutsoupoulos 2006). injuries are common in humans, especially in environments where there is a high prevalence of violent acts and accidents. owing to poor or delayed wound management, these wounds often become infected with either commensal or environmental microorganisms, or both. infected wounds heal more slowly, re-epithelialisation is more prolonged and there is a risk of systemic infection (priya et al. 2002). people in rural areas in south africa often use medicinal plants for the initial treatment of wounds as they are easily accessible and cheap. in these areas, the most common way to treat wounds with such plants is by using a decoction of one or more of the plants as a wash. this is followed by the application of a powder made from the plants directly onto the wound, after which the wound is covered by a bandage. the plant material is sometimes carbonised before pounding into a powder. an emulsion of a powder prepared from various parts of the plant and oil from the seeds of lannea microcarpa (anacardiaceae), or butter of butyrospermum parkii (sapotaceae) can also be used, as well as juice obtained by squeezing or heating the plant leaves or roots. these treatments are usually repeated every day until the wounds are healed. bandages most often consist of whole, fresh leaves, or a strip of cotton cloth (inngjerdingen et al. 2004). the process of wound healing is promoted by several plant products (suguma, chandrakasan & joseph 1999), which contain active principles such as triterpenes, alkaloids, flavonoids (sharma et al. 1990) and biomolecules (chithra, sajithlal & chandraksan 1995). these agents usually influence one or more phases of the healing processes. to date, the wound healing properties of two tropical plants, centella asiatica (suguma, sivakumar & chandrakasan 1996) and terminalia chebula (suguma, surjeet & chandrakasan 2002), have been demonstrated in rat models. many species of the combretaceae family that are widely distributed in africa are used in traditional medicine for treating a myriad of conditions including, pneumonia, syphilis, abdominal pains, conjunctivitis, diarrhoea, leprosy, scorpion stings and mumps (hutchings et al. 1996). some species of this plant family are also used for wound healing, for example, hutchings et al. (1996) have reported that the vhavenda people of northern south africa use the leaves of terminalia sericea for the treatment of wounds and menorrhagia, while the bark of this species is used to treat wounds and the roots for diarrhoea, infertility and venereal diseases (mabogo 1990). mabogo (1990) has reported that the roots of combretum molle are used by the vhavenda for wound healing, while, in ghana the leaves of this species are used for wound dressing. other plant species within the combretaceae family that are used for medicinal purposes include combretum kraussii, the leaves of which have been used to treat wounds (hutchings et al. 1996) and combretum micranthum, the leaves and fruits of which can be used for the topical treatment of wounds (iwu 1993; oliver-bever 1986). van wyk et al. (1997) have also reported that t. sericea and c. kraussii are used for the topical treatment of wounds. acetone extracts of combretum imberbe, combretum nelsonii combretum albopuntactum and t. sericea have antibacterial properties that work against staphylococcus aureus, pseudomonas aeruginosa, enterococcus faecalis and escherichia coli (eloff 1999). in earlier studies, we found that combretum and terminalia extracts demonstrated remarkable activity against candida albicans, cryptococcus neoformans, microsporum canis, sporothrix schenckii and aspergillus fumigatus, having a minimum inhibitory concentration (mic) as low as 0.02 mg/ml mosmann 1983). this study was conducted by applying the extracts of c. imberbe, c. nelsonii, c. albopuntactum and t. sericea to wounds created in wistar rats to determine if these extracts induced any skin irritation, damage or other negative effects. materials and methods (back to top) this research was approved by the research and animal use and care committee of the university of pretoria, south africa (vi 010/05 approval number). plant collection leaves were collected in the summer of 2003 from four species of trees of the combretaceae family in the lowveld national botanical gardens in nelspruit, south africa. voucher specimens in the garden herbarium and tree labels verified the identity of the plants. the selection of the species was based on their high antifungal activity which had been demonstrated in previous in vitro studies (masoko et al. 2005, 2007) and low in vitro toxicity (masoko, 2006). leaves were collected from the four species belonging to different sections of the genus (carr 1988), c. imberbe wawra (hypocrateropsis engl. & diels), c. nelsonii duemmer (angustimarginata engl. & diels), c. albopunctactum suesseng (ciliatipetala engl. & diels) and t. sericea burch ex dc (psidioides). plant storage leaves were separated from the stems and dried at room temperature. most scientists have tended to use dried material because there are fewer problems associated with the large-scale extraction of dried plants rather than fresh plant material (eloff 1998a). the dried plants were milled to a fine powder in a macsalab mill (model 200 lab; eriez, bramley, south africa) and stored at room temperature in closed containers in the dark until used. extraction procedure the dried leaves of each species were individually extracted by treating 500 g of finely ground plant material with 5 l of acetone (technical grade; merck, johannesburg, south africa) in big glass containers (eloff 1998b). the containers and contents were vigorously shaken for 3 h – 5 h in a labotec model 20.2 shaking machine at high speed. the particulate matter was allowed to sediment and the supernatant was filtered and evaporated using a rotavaporator (r-114; büchi, new castle, usa) and decanted into pre-weighed labelled beakers. the process was repeated three times to exhaustively extract the plant material, after which the extracts were combined. the solvent was removed under a stream of air in a fume cupboard at room temperature. the extract was then weighed to quantify the extraction efficiency. preparation of extract each dried extract was ground to a fine powder with a pestle in a mortar and mixed with an aqueous cream – obtained from reitzer pharmaceuticals (pty) ltd (sandton, south africa) – consisting of distilled water, white petroleum jelly, mineral oil, emulsifying wax and phenoxyethanol to a concentration of either 10% (1 g extract per 10 g cream) or 20% (2 g extract per 10 g cream) and kept at 4 °c until used. selection of rats ten healthy male wistar rats weighing 150 g – 200 g were used. the test was conducted using a single gender in order to reducing variability and to minimise the numbers required (organisation for economic co-operation and development (oecd), 2000). at the commencement of the study, each rat was 8–12 weeks old and the weight variation of animals used did not exceed ± 20% of the mean weight of all previously dosed animals (anon. 2001). housing and feeding conditions the rats were kept at the university of pretoria biomedical research centre and housed in separate cages at a temperature of 22 °c (± 2 °c) and relative humidity (50% – 60%) in a light–dark cycle of 12 hours. they were fed a conventional rodent diet obtained from epol (pty) ltd (randburg, south africa) and had an unlimited supply of drinking water. environmental enrichment, for example, bedding (wood wool), was provided to keep the rats occupied. previous work suggests that the provision of enrichment items, such as wood wool, gives laboratory rats the opportunity to perform exploratory and gnawing activities. this can be used to improve their well-being and to distract them from tampering with dressings (zhu et al. 2006). preparation of animals the cages of the rats were labelled from one to eight for identification purposes. the rats were kept in their cages for at least five days prior to commencement of the experiment to allow for acclimatisation to the laboratory conditions (spielmann et al. 1999) and they were handled daily during this period. they were not immunosuppressed. wound creation the hair on the back of each of the rats was removed with electrical clippers and bare areas were disinfected using a solution of 0.5% chlorhexidine in 70% alcohol, which was allowed to dry after application. the rats were anaesthetised with isoflurane (0.01 μg/kg – 0.05 μg/kg). four evenly spaced wounds were made in the skin within the bare area using a punch to obtain biopsies with a diameter of 6 mm (simosen, petersen & groth 2002). the whole process was carried out in a biosafety class ii cabinet to limit infection. a temperature probe (microchip; identipet, johannesburg, south africa) was injected subcutaneously into the rump of each rat, so that the body temperature could be routinely monitored during the course of the experiment without unduly disturbing the animal. topical application of aqueous creams the treatments were applied on separate lesions (a, b, c and d) three times a week as follows: a, no treatment, b, cream only, c and d were 10% and 20% of crude extract, respectively. these lesions were randomly selected and two rats were selected for each plant (i.e. eight rats in total were used). to avoid interference by the rat of mixing of treatment a dressing was applied. observations a blind study was done and the observations for each rat were recorded separately. every monday, wednesday and friday, at 09:00, each rat was taken out of its cage, anaesthetised with isoflurane, its wound dressing removed and various parameters were measured, including their weight and body temperature; the latter was read electronically via an electronic reader. the lesions on each rat were rated using the following parameters, (1) the presence and type of exudate, (2) erythema, (3) swelling, (4) ulceration and (5) crust formation. the scoring criteria are shown in table 1. each lesion diameter was also measured in millimeters in the vertical and horizontal planes using the electronic digital calipers. the wounds were then cleaned using clean cotton wool and the treatments were reapplied, after which the wounds were re-dressed and the rat allowed to recover and placed in his cage.the rats were observed individually at least once during the first 30 minutes after treatment and then periodically during the next 24 hours, with special attention being given during the first 4 hours of this period. all the rats were observed daily for any indication of illness and interference with the wound dressing. clinical signs of disease, included changes in the skin and fur, diarrhoea, lethargy, sleep, weight loss and coma were also checked. provision was made for the early termination of those rats that were either ill or interfered excessively with the wound dressings. all the rats were euthanised by carbon dioxide inhalation when their wound had fully contracted and upon evidence of scar formation. at necropsy, a pathologist examined the liver, heart, lungs, intestines, lymph nodes and kidneys of the rats for gross abnormalities. results (back to top) with the exception of rat 1, no rat in this study lost weight. rat 1 was observed to have lost weight on day 3, but by day 5 its weight was once again within the normal range (figure 1). the rats’ temperatures were also within the expected values of 35 °c – 37 to percentages of the original size. since the results of the two rats which were treated the same were practically identical, the averages of these measurements were used. to find out if treatment accelerated wound healing, the wound sizes were compared to that of the untreated wound. combretum imberbe extracts (figure 3a) healed the wound faster than their control, followed by c. nelsonii (figure 3b). the controls healed faster than the 10% concentration of c. albopunctactum, but slower than the 20% extract of this species (figure 3c). the 10% concentration and the 20% concentration of the t. sericea extracts, in cream, produced almost similar results and were slightly better than the untreated wound (figure 3d). the resultant healing was also quantified on the basis of erythema (figure 4), exudate (figure 5) and crust formation (figure 6). figure 1: weights of rats for 3 weeks figure 2: body temperatures of rats measured for 3 weeks figure 3: lesion sizes of wounds treated with (a) combretum imberbe, (b) combretum nelsonii, (c) combretum albopunctactum, (d) terminalia sericea for 3 weeks figure 4: the influence of the crude extracts on the wound erythema of rats figure 5: the influence of the crude extracts on the exudate formed on rats figure 6: the influence of the crude extracts on the crust formed on rats discussion (back to top) herbal products are usually perceived as ‘safe’ and are often applied to wounds without any in vitro or in vivo toxicity tests being done. this, however, cannot be assumed in all cases. for example, jatropha curcas l (euphorbiaceae) was found to have esterases and lipases (staubmann et al. 1999) and it also has disinfectant, antiparasitic (fagbenro-beyioku, oyibo & anuforom 1998), antiviral (matsuse et al. 1999) and molluscicidal activities (liu et al. 1997). jathropa curcas was, however, found to be toxic (makkar, becker & schmook 1998). this study was designed using simple in vivo method to determine any adverse effects, as well as to compare the relative effectiveness of acetone leaf extracts of c. nelsonii, c. imberbe, c. albopunctactum and t. sericea on wound healing activity. the in vitro cytotoxicity of acetone extracts of these four species was evaluated on artemia salina nauplii and vero monkey kidney cells. the vero cells were a more sensitive indicator of toxicity (masoko 2006). at a concentration of 25%, the acetone had no adverse effects on the fungi tested, or on a. salina nauplii and vero kidney cells. the results on brine shrimps indicated that the four leaf extracts had lc50 values above 20 μg/ml, which is the recommended cut-off point for detecting cytotoxic activity (geran et al. 1972). the podophyllotoxin toxin standard had lc50 of 7 µg/ml. in the mtt assay, c. imberbe extract had the highest lc50 of 168.6 µg/ml and c. nelsonii had the lowest (75.7 µg/ml) the lc50 for c. albopunctactum and t. sericea were 102.9 µg/ml and 121.7 µg/ml, respectively (masoko 2006). an ld50 of 30 µg/ml was considered to be non-toxic according to the criteria of the american national cancer institute (suffness & pezzuto 1990). although, in practice, local treatments are usually repeated every day until the wounds are healed, it was decided to treat the rats every second day to minimise effects associated with handling and the continuous use of anaesthetics. it was also thought that the plant extracts would retain their antimicrobial activity over that period. wound contamination was unlikely as the wounds were covered with an occlusive dressing. we found that the rats neither showed any sign of irritation to any of the treatments, including the aqueous cream control, nor did the wounds swell or exhibit noticeable pus formation or ulceration. the wounds were completely healed after 17 days and remained so when the rats were euthanised on day 21. the created wounds, as well as the contraction and healing of them are depicted in figure 7. with the exception of rat 1 and rat 5, which had transient weight loss and an elevated body temperature, none of the rats showed any signs of clinical disease. it was also not certain whether the illness in these two rats was due to the wound manipulations or a clinical manifestation of interstitial pneumonia that was endemic in these two rats and which was observed at necropsy. we found that the extracts of c. imberbe and c. nelsonii were superior in their wound healing abilities. the wound treated with the c. imberbe extracts (figure 3a) closed more rapidly than that of the cream and untreated control. in fact, wound closure was slowest in the untreated control. on day 12, an apparent increase in wound size of the controls was noted. this was due to the fact that there was a marked crust formation, making it difficult to measure the wound edges. all the wounds were healed by day 17. the same healing pattern occurred with c. nelsonii (figure 3b), with the exception that the wound size increased on day 8, rather than day 5. in the cases of c. albopunctactum (figure 3c) and t. sericea (figure 3d), there was little difference in the contraction of the wounds in comparison to the controls, indicating that these two plants had little or no effect on wound contraction. the use of the 20% concentration extracts is recommended because these have a better antifungal effect (masoko et al. 2005) and the 10% extract of t. sericea was found to have a slightly negative effect on wound healing on day 5. all the extracts, especially c. imberbe (data not shown) were slightly better in reducing the erythema (figure 4) of the wounds, in comparison to the controls. the exudates were slightly more prominent with the extracts (figure 5). this could be as a result of the difficulty to distinguish between exudates and extract remnants. there was essentially no noticeable difference in crust formation between the different treatments (figure 6). the most prominent group of biologically active compounds isolated from c. imberbe are the triterpenes (angeh 2005). asiaticoside is the most abundant triterpene glycoside, which is effective in wound healing and apparently acts by enhancing the induction of antioxidant levels at an early stage of wound healing (shukla et al. 1999). in other studies, we isolated three biologically active triterpenes: terminolic, asiatic and arjunolic acids from c. nelsonii (masoko 2006). wound healing activity may be attributable, in part, to triterpene-rich fractions within the plant extracts we used. however, extracts from the whole plant may have better wound healing properties than a single active ingredient, possibly due to other unidentified active compounds which act synergistically (williamson 2001). wound contraction begins almost concurrently with collagen synthesis. the rate of contraction depends on the degree of tissue laxity and shape of the wound, with loose skin wounds and square wounds healing the fastest. thus, rat skin, being loose, tends to heal faster than human skin, with wound contraction significantly contributing to wound closure (stipcevic, piljac & piljac 2006). consequently, wound contraction, which is usually more rapid than epithelisation, causes a decrease in the overall healing time of rat wounds (cross et al. 2005). although rats are not ideal for studying the efficacy of therapy on wound healing in humans due to the differences in the skin structure, they can still be used as a model for wound healing because laboratory rats are inbred and thus there is little variation in their wound healing due to genetic differences, allowing only few animals to be used (reed et al. 1996). laboratory rats are also relatively cheap and easy to handle and their use usually attracts less ethical objections than other animals such as pigs etc. there are also advantages in the use of rats as a research model, such as the availability of a broad knowledge based on rat wound healing and they are well established as wound models (kimuro et al. 2005; nayak et al. 2005; sumitra, manikandan & suguna 2005). products which have been found to improve wound healing in rats have also done so in humans. victor-vega et al. (2002) compared wound healing on rats and humans and found, through their experiments, that the adenosine a2a receptor antagonist significantly accelerated wound closure when applied topically. this effect was even more remarkable when compared to the recombinant human platelet-derived growth factor administered locally. although a pig’s skin structure closely resembles that of humans, pigs were not used for practical reasons. the housing, feed and care of pigs is more expensive than other models and the psychological well-being of the pigs must be addressed by providing them with conspecific visual interaction, various toys and hand-fed treats, under professional supervision. these forms of enrichment serve to lower the distress that may otherwise be experienced and potentially confound the experimental results (fries et al. 2005), but would be too time-consuming. figure 7: photographic representation of created wound (a) and 100% healed wound after treatment administered (b) table 1: evaluation of erythema and exudate conclusion (back to top) our data indicate that the tested plant extracts had no adverse effects on wound healing in rats and, in fact, the acetone extracts of c. imberbe and c. nelsonii appeared to promote wound healing. this is remarkable as there was no evidence of wound infection during the trials. although the mechanism of wound healing is unclear, our data indicate that further investigation of the combretaceae family may be useful in this regard. taken in conjunction with the data collected in this study and the in vitro antifungal activity of selected species, we suggest that the four selected species will also have an antifungal activity on infected wounds. we are currently assessing the effect of four selected species on rats infected with candida albicans, cryptococcus neoformans, microsporum canis and sporothrix schenckii. acknowledgements (back to top) we would like to acknowledge the national research foundation for their provision of funding, 'mr rudi kotze and mr johan hurter, who allowed us to collect plant material from the lowveld national botanical garden, and mr n.p. selahle, who helped with the care of the rats and the experiment. references (back to top) angeh, j., 2005, ‘isolation and characterization of antibacterial compounds present in members of combretum section, hypocrateropsis’, phd thesis, department of paraclinical sciences university of pretoria. anon., 2001, guidance document on using in vitro data to estimate in vivo starting doses for acute toxicity. nih publication number 01–4500. national institute of environmental health sciences, research triangle park. carr, j.d., 1988, combretaceae in southern africa, tree society of southern africa, johannesburg. chithra, p., sajithlal g.b. & chandraksan, g., 1998, ‘influence of aloe vera cross, s.e., naylor, i.l., coleman, r.a. & teo, t., 1995, ‘an experimental model to investigate the dynamics of wound contraction’, british journal of plastic surgery 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10-20. introduction in nile crocodiles (crocodylus niloticus), crocodile poxvirus (crv) infection causes small, superficial skin lesions covered by dark brownish crusts or scabs. microscopically the lesions are characterised by the presence of masses of intracytoplasmic inclusions in proliferating epidermis cells (horner 1988; huchzermeyer, huchzermeyer & putterill 1991). the morphology of crv particles in negatively stained preparations was described by gerdes (1991) and in 2006, afonso, tulman, delhon, lu, vil joen, wallace, kutish & rock published the genomic sequence of crv from c. niloticus. minute superficial lesions, below which were deeply penetrating holes found in skins of american alligators (alligator mississippiensis) were dubbed “pix” (dickson, cardeilhac & ashley 2002). they were believed to be a manifestation of a lymphohistiocytic proliferative syndrome associated with west nile virus infection (nevarez, mitchell, johnson & kinler 2007). similar pin prick-like lesions were found in nile crocodile skins and dubbed “pit holes” (huchzermeyer & putterill 2004). affected skins are downgraded which has severe financial implications for the farmer. this paper describes the histopathology of these lesions, the morphological 311 onderstepoort journal of veterinary research, 76:311–316 (2009) identification and partial sequencing of a crocodile poxvirus associated with deeply penetrating skin lesions in farmed nile crocodiles, crocodylus niloticus f.w. huchzermeyer1*, d.b. wallace2, 3, j.f. putterill2 and g.h. gerdes2 abstract huchzermeyer, f.w., wallace, d.b., putterill, j.f. & gerdes, g.h. 2009. identification and partial sequencing of a crocodile poxvirus associated with deeply penetrating skin lesions in farmed nile crocodiles, crocodylus niloticus. onderstepoort journal of veterinary research, 76:311– 316 when large numbers of crocodile skins were downgraded because of the presence of small pin pricklike holes, collapsed epidermal cysts were found deep in the dermis of juvenile crocodiles while forming cysts were observed in hatchlings. histopathology of these forming cysts showed the presence of intracytoplasmic inclusions in proliferating and ballooning epidermal cells. pox virions were seen in electron microscope preparations made from the scabs of such early lesions. the partial sequencing of virus material from scrapings of these lesions and comparison of it with the published sequence of crocodile poxvirus showed the virus associated with the deep lesions to be closely related, but different. to differentiate between the two forms of crocodile pox infection it is suggested that the previously known form should be called “classical crocodile pox” and the newly discovered form “atypical crocodile pox”. the application of strict hygiene measures brought about a decline in the percentage of downgraded skins. keywords: crocodylus niloticus, dermatitis, electron microscopy, genome sequencing, poxvirus * author to whom correspondence is to be directed. e-mail: crocvet@mweb.co.za 1 p.o. box 12499, onderstepoort, 0110 south africa 2 onderstepoort veterinary institute, private bag x05, onderste poort, 0110 south africa 3 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa accepted for publication 19 february 2009—editor 312 crocodile poxvirus associated with skin lesions in farmed nile crocodiles, crocodylus niloticus identification of a poxvirus from these lesions and its comparison with crv using partial sequence analysis. materials and methods three tanned crocodile skins were made available for gross examination of the skin lesions (“pit holes”) and photography. one affected crocodile from each of four age groups, three-, two-, one-year and hatchlings were selected by examination of their ventral skin. these animals were killed by an approved slaughter technique. they were skinned and samples of skin with visible lesions were fixed in 10 % phosphate buffered formalin. an additional affected hatchling was selected in a similar manner to that mentioned above and scrapings were taken from some of the visible skin lesions. in order to obtain additional material for study purposes, skin samples were excised from affected crocodiles during subsequent routine post mortem examinations. light microscopy the formalin-fixed skin samples were routinely processed for light microscopy. sections were cut at ca 6 µm thickness, stained with haematoxylin and eosin, and examined using a leica dm-6000b compound light microscope (leica microsystems, ir, gmbh). electron microscopy unfixed scrapings from the crust of a recently formed lesion in the skin of a hatchling crocodile were homogenised using an ultra-turrax homogeniser (jan ke & kunkel, ika-werk, gmbh & co kg, staufen) in double distilled water and routinely prepared for negative staining. low speed centrifugation was used to remove coarse detritus. after ultra-centrifugation of the supernatant, the resultant pellet was resuspended in 3 % phospho-tungstic acid (pta) at ph 6.4, applied to formvar coated copper grids and examined using a jeol jem-1200ex mk-i transmission electron microscope (jeol, tokyo, japan) at 80 kv. partial sequencing of the virion gene dna was extracted from virions in the scrapings according to an adapted method of esposito, condit & obijeski (1981). lesion scrapings were added to phosphate-buffered saline (pbs) and ground with diatomaceous earth using a mortar and pestle. the emulsified solution was cleared by low speed centrifugation in a beckman avanti (beckman, usa) bench-top centrifuge (2 000 rpm [450 g] for 2 min). the supernatant fraction was diluted 1:1 in viral-lysis buffer consisting of 1 % sds (sigma, usa), 20 mm b-mercaptoethanol (merck, germany), and 20 mm edta. proteinase-k (roche, germany) (at a final concentration of 100 µg/mℓ) was added and the mixture was incubated at 56 °c for 2 h in order to release viral dna. calf liver trna (0.5 µℓ of a 10 mg/ mℓ stock) (roche, germany) and 1/10th volume 3 m sodium acetate (ph 5.3) was added and the dna extracted with phenol/chloroform (sigma, usa), and precipitated with 2.5 volumes 96 % ethanol. after air-drying, the dna was resuspended in 50 µℓ sterile, ultra-pure hplc-quality water (sigma, usa) and stored at –20 °c for pcr analysis. pcr analysis of viral dna for the design of primers, a unique region of crocodile poxvirus (crv) open reading frame (orf) 019 sequence was chosen (afonso et al. 2006). two oligonucleotide primers were designed (zcp1f – cta gac gaa gaa ctg gta c, zcp1r – gga gat ctt ggt gcg gta) covering a 577 base pair (bp) region of orf019. pcr amplification was carried out using these primers and a geneamp 2400 thermal cycler (perkin-elmer, usa). a 50 µℓ reaction volume was prepared consisting of 5 µℓ 10x pcr buffer (containing 20 mm mgcl2) (takara bio medicals, japan), 4 µℓ 2.5 mm dntps (takara bio medicals, japan), 0.5 u taq dna polymerase (takara ex taqtm, takara biomedicals, japan), 1 µℓ of each primer (20 pmoles of each), 1 µℓ of template dna (~ 0.1 ng) and 38 µℓ sterile ultra-pure hplc-quality water (sigma, usa). template dna was denatured for 60 s at 95 °c, followed by 35 cycles of denaturation at 95 °c for 15 s, primer annealing at 56 °c for 45 s, and strand extension at 72 °c for 60 s. sequencing and analysis of pcr-amplified viral dna for sequence analysis, primer zcp1f was chosen as sequencing primer. pcr-amplified dna was purified using a qiaquick spin column (qiagen, germany) and 10 ng was added to 3.2 pmoles primer in a 12 µℓ reaction volume. sequencing was performed using an abi prism 3100 genetic analyser (hitachi, japan) and bigdyetm chemistry from a bigdyetm terminator cycle sequencing kit (applied biosystems, usa). sequence data was then aligned and compared with the published crv sequence (afonso et al. 2006). 313 f.w. huchzermeyer et al. results macroscopic in tanned crocodile skins, small deep pin prick-like holes were found randomly distributed in some of the smooth scales of the ventral skin (fig. 1). the pit holes were distinct from the sensory pores which are found usually one to each ventral scale situated close to its caudal edge (fig. 1) and which are not deep holes. fig. 2 collapsed and healed cyst in the dense dermis. its epidermal lining signifies healing, 10x fig. 3 part of a newly forming cyst in hatchling skin. note the finger-like epidermal projections (columns) penetrating further into the dermis (black arrows). several inclusion bodies are indicated by the white arrows, 5x fig. 4 intracytoplasmic inclusions in the proliferating epidermal cells in a newly formed cyst, indicated by the arrows, 10x fig. 5 an early stage of a newly forming cyst with intracytoplasmic inclusion bodies. f is a fold artefact induced by sectioning, 10x fig. 1 pit holes (encircled area and white arrows) in ventral scales of tanned nile crocodile skin and showing sensory pores close to the caudal margin of the scales (black arrows), 0.75x 2 4 3 5 314 crocodile poxvirus associated with skin lesions in farmed nile crocodiles, crocodylus niloticus histopathology the histopathology of the skin lesions of the threeyear-old and two-year-old crocodiles consisted of collapsed cysts with a folded surface that was lined entirely by a complete epidermis without any trace of an inflammatory reaction (fig. 2). the skin of the one-year-old crocodile showed similar collapsed cysts, each with a healed surface. however, under the epidermis there were small perivascular infiltrations of lymphocytes. in the skin of the hatchling croc odiles there were debris-filled cysts, partially lined by an apparently proliferating epidermis. this epidermis appeared to form extending columns of cells deep into the dermis (fig. 3) and, in one region, the proliferating epidermal cells were ballooned and contained intracytoplasmic inclusion bodies (fig. 4). in a subsequent routine post mortem case, newly formed cysts were found to be packed entirely with ballooning cells containing intracytoplasmic inclusions (fig. 5). electron microscopy electron microscopic examination of the scrapings from the edge of these lesions revealed the presence of pox virions with cross striations, typical of those found with crv. the accompanying micrograph (fig. 6) is of the slightly larger c-form where stain has partially penetrated the particle obscuring detail of the surface thread. the double membrane or outer coat of the particle is, however, visible. viral dna dna isolated from the scrapings was pcr-amplified and analysed using agarose gel electrophoresis. only the predicted amplification product of 577 bp was obtained (data not shown). the amplified dna was purified, sequenced and the resulting sequence (533 bp) aligned with crv (fig. 7). as expected, the sequence aligned within orf019 of crv (afonso et al. 2006). at the nucleotide level (fig. 7a) there are seven differences, relating to five at the amino acid level (fig. 7b). discussion the epidermis of the crocodilian skin consists of straight layers of squamous cells without epidermal ridges embracing dermal papillae. the epidermis is covered by tough rigid scale-keratin over the scales and by a more flexible interscale keratin in the folds between the scales (richardson, webb & manolis 2002). in previously described cases of crocodile poxvirus infections in nile crocodiles, there were circumscribed lesions with severe proliferation of the epidermis, causing a depression into the underlying loose dermis with occasional ulceration. in the proliferating and ballooning epidermal cells, large numbers of intracytoplasmic inclusions, comprising greater numbers of bollinger bodies and fewer borrel bodies were found (horner 1988; huchzermeyer et al. 1991; buoro 1992; pandey, inoue, ohshima, okada, chihaya & fujimoto 1990). these lesions do not cause permanent scars in the tanned skin. in contrast, the lesions found in the present investigation penetrated deeply through the loose dermis and into the dense dermis causing cysts to develop that collapsed at a later stage but did not close or heal completely. this penetration was accompanied by extended columns of epidermal cells. even after the healing of the ulcers, the deeply sited cysts collapsed, but remained as permanent lesions comprising the “holes” seen macroscopically in the tanned skins. in the first cases examined, only a few ballooning epidermal cells at the entrance of the cysts appeared to contain bollinger and borrel bodies. for this reason it was deemed necessary to examine crust-scrapings utilising electron microscopy. however, in a subsequent case that was examined, cysts completely filled with ballooning epidermis cells containing intracytoplasmic inclusions were encountered. the intimate association between the mani festations of crocodile poxvirus infection and the deep cysts leaves little doubt regarding the causative role of the poxvirus, particularly in the absence of the pathogenic west nile virus in southern africa. fig. 6 crocodile poxvirus particle attached to a collagen fibre from the crust covering a newly formed cyst. cf – collagen fibre; vp – virus particle, 40 000x 315 f.w. huchzermeyer et al. in the united states of america, pitting of alligator skins was a cause of concern (haire 1997). this was before the first outbreak of west nile virus infection in alligators was reported in that country (miller, mauel, baldwin, burtle, ingram, hines & frazier 2003). this indicates the possibility that two different agents may cause similar permanent lesions. members of the poxvirus family cannot be assigned to different genera using electron microscopy and viral particles are generally compared morphologically to those of the well-described vaccinia virus which is the type species of the orthopox genus (medzon & bauer 1970). the mature virion is brick to ovoid-shaped and measures 235–330 nm x 170– 285 nm. two forms are recognized, the m or mulberry form and the c or capsule form (doane & anderson 1987). commonly, the outer envelope is absent, revealing the complex structure of the particle coat or membrane with irregularly arranged surface threads or double ridges outlined by the pta stain (gerdes 1991). the new virus seen in this investigation in its c-form did not differ morphologically from the known crocodile poxvirus. fig. 7 sequence alignment of a region of orf 019 of crv with the “pit” poxvirus (amplified using pcr) at the nucleotide level (a) and amino acid level (b). differences in nucleotides (*) and amino acids (grey shading) are shown a crv ctggtacgtcgcggtcggctcctcttccgggtcgcgcgcggaggcggtgaaggcgttgat 60 pit ctggtacgtcgcggtcggctcctcttccgggtcgcgcgcggaggcggtgaaggcgttgat 60 crv cgcgtcgatgccggcggccgaaaaaatgtacgcgcagggatctggcgcgtcggggctcga 120 pit cgcgtcgatgccggcggccgaaaaaatgtacgcgcagggatcgggcgcgtcggggctcga 120 * crv gcacgccatcatatccatggcggcgtagcgcgagaagtactcccgggacagcacgatttg 180 pit gcacgccatcatgtccatggcggcgtagcgcgagaagtactcccgggacagcacgatttg 180 * crv gtgctcgtacgcgaagggctcctccagcgccgagacggggatcacgcgcatgatggtctc 240 pit gtgctcgtacgcgaagggctcctccagcgccgagacggggatcacgcgcatgatggtctc 240 crv gaccgcgccgagcgcgttcaggaagcgaaggtacagcgtctcgcgcgtctcggggcccag 300 pit gaccgcgccgagcgcgttcagaaagcggaggtacagcgtttcgcgcgtctcggggctcag 300 * * * * crv cgtgatggagacgcacagcaagtcgtgccggatcccgctcagcggcggaaaccggtcgta 360 pit cgtgatggagacgcacagcaggtcgtgccggatcccgctcagcggcggaaaccggtcgta 360 * crv ctcgacgatggagtgctcgaacgggtcggccgcgccgtcctcgtccgggtcgctgccgtc 420 pit ctcgacgatggagtgctcgaacgggtcggccgcgccgtcctcgtccgggtcgctgccgtc 420 crv gtccacgtcgtcgccgtcggtgaagtacccggcgtcgccgcgctcggcctccagcatccc 480 pit gtccacgtcgtcgccgtcggtgaagtacccggcgtcgccgcgctcggcctccagcatccc 480 crv gtcgcgcacgcccgcgcgtcgataggcagggcgtcgtttttgatcgagaacgt 533 pit gtcgcgcacgcccgcgcgtcgataggcagggcgtcgtttttgatcgagaacgt 533 b crv lvrrgrllfrvargggegvdrvdaggrknvragiwrvgararhhihggvarevlpgqhdl 60 pit lvrrgrllfrvargggegvdrvdaggrknvragigrvgararhhvhggvarevlpgqhdl 60 crv vlvregllqrrdgdhahdgldraervqeakvqrlarlgaqrdgdaqqvvpdpaqrrkpvv 120 pit vlvregllqrrdgdhahdgldraervqkaevqrfarlgaqrdgdaqqvvpdpaqrrkpvv 120 crv lddgvlervgravlvrvaavvhvvavgevpgvaalglqhpvaharasigrasflien 177 pit lddgvlervgravlvrvaavvhvvavgevpgvaalglqhpvaharasigrasflien 177 316 crocodile poxvirus associated with skin lesions in farmed nile crocodiles, crocodylus niloticus the limited sequence data generated for this virus indicates that it is closely related to, but distinct from, the crocodile poxvirus, crv. in view of the fact that there are two closely related crocodile poxviruses causing entirely different lesion morphologies, we propose the disease described in this paper to be called “atypical” crocodile pox and the previously described disease to be called “classical” crocodile pox. whereas the “classical” crocodile pox infection is characterised by superficial lesions without permanent scars, the “atypical” crocodile pox infection causes deeply penetrating lesions and permanent scars or “pit holes”. the introduction of strict hygiene measures in the rearing houses on the affected farm, particularly thorough cleaning and disinfection of the pens resulted in a reduction in the percentage of downgraded skins. this has however, not resulted in the elimination of the infection from the farm. acknowledgements staff of the pathology section, department of paraclinical sciences, faculty of veterinary science, univ ersity of pretoria are thanked for preparing and staining the skin sections. references afonso, c.l., tulman, e.r., delhon, g., lu, z., viljoen, g.j., wallace, d.b., kutish, g.f. & rock, d.l. 2006. genome of crocodile pox virus. journal of virology, 80:4978– 4991. buoro, i.b.j. 1992. pox-like virus particles in skin lesions of five nile crocodiles in kenya. discovery and innovation, 4: 117–118. dickson, h., cardeilhac, p. & ashley, j.d. 2002. pix skin disease in the american alligator (alligator mississippiensis), in crocodiles. proceedings of the 16th working meeting of the crocodile specialist group, iucn – the world con ser­ vation union, gland, switzerland and cambridge, uk: 187. doane, f.w. & anderson, n. 1987. poxviridae, in electron microscopy in diagnostic virology. a practical guide and atla s. cambridge: cambridge university press. esposito, j., condit, r. & obijeski, j. 1981. the preparation of orthopoxvirus dna. journal of virological methods, 2:175–179. gerdes, g.h. 1991. morphology of poxviruses from reptiles. veterinary record, 128:452. haire, d. 1997. concern over american alligator skin pitting. crocodile specialist group newsletter, 16:22–23. horner, r.f. 1988. poxvirus in farmed nile crocodiles. veter­ inary record, 122:459–462. huchzermeyer, f.w., huchzermeyer, k.d.a. & putterill, j.f. 1991. observations on a field outbreak of pox virus infection in young nile crocodiles (crocodylus niloti­ cus). journal of the south african veterinary association, 62: 27–29. huchzermeyer, f.w. & putterill, j.f. 2004. pitholes in nile crocodile skins, in crocodiles. proceedings of the 17th working meeting of the crocodile specialist group, iucn – the world conservation union, gland, switzerland and cam bridge, uk: 492–493. medzon, e.l. & bauer, h. 1970. structural features of vaccinia virus revealed by negative staining, sectioning and freeze-etching. virology, 40:860–867. miller, d.l., mauel, m.j., baldwin, c., burtle, g., ingram, d., hines, m.e. & frazier, k.s. 2003. west nile virus in farmed alligators. emerging infectious diseases, 9: 794–799. nevarez, j.g., mitchell, m.a., johnson, a.j. & kinler, n. 2007. establishing an association between west nile virus exposure and the development of lymphohistiocytic proliferative syndrome in american alligators, alligator missis­ sippiensis. journal of herpetological medicine and surgery, 17:4–7. pandey, g.s., inoue, n., oshima, k., okada, k., chihaya, y. & fujimoto, y. 1990. poxvirus infection in nile crocodiles (crocodylus niloticus). research in veterinary science, 49:171–176. richardson, k.c., webb, g.j.w. & manolis, s.c. 2002. crocodiles: inside out. chipping norton: surrey beatty and sons. article information how to cite this item: how to cite this item: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011, onderstepoort journal of veterinary research 79(2), art. #488, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.488 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. acknowledgement in this acknowledgement... open access the work in the papers and abstracts was supported by wellcome trust grant wt087546ma to the southern african centre for infectious disease surveillance (www.sacids.org) and its collaborating uk partner institutions, namely the london school of hygiene and tropical medicine (http://www.lshtm.ac.uk/), the royal veterinary college (www.rvc.ac.uk) and the london international development centre (www.lidc.org.uk).the work presented by esron karimuribo et al. was supported by rockefeller foundation. article information authors: adrienne saif1,2 john frean1,2 jenny rossouw2 anastasia n. trataris1,2 affiliations: 1university of the witwatersrand, south africa 2special bacterial pathogens reference unit, national institute for communicable diseases, national health laboratory services, south africa correspondence to: adrienne saif postal address: private bag x4, sandringham 2131, south africa how to cite this poster: saif, a., frean, j., rossouw, j. & trataris, a.n., 2012, ‘leptospirosis in south africa’, onderstepoort journal of veterinary research 79(2), art. #478, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.478 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. leptospirosis in south africa in this poster... open access leptospirosis is a common zoonosis worldwide. it has a ubiquitous distribution and causes a wide spectrum of disease. leptospirosis therefore has a broad reservoir host range, and many infected species of animals excrete leptospires in their urine, which leads to contamination of soil and water. typical descriptions of the disease include a biphasic (anicteric form) and fulminant disease in the icterohaemorrhagic form. only a few local case reports of human leptospirosis have been published, the most recent one being in 1974. a rodent-related zoonosis study (ratzooman) was conducted from 2003 until 2006 in three provinces (limpopo, kwazulu-natal and the eastern cape). of the people sampled in cato crest (durban, kwazulu-natal province), 43/217 (19.8%) were seropositive for leptospirosis. of the clinical samples sent to the special bacterial pathogens reference unit from all over the country for testing in 2009, 16/176 (9%) were igm positive; in 2010 and january 2011 to may 2011, 14/215 (6.5%) and 12/96 (12.5%), respectively, were igm positive. the apparent incidence of leptospirosis in the south african population is moderately high based on the detected positives in suspected cases; it is thought that the circulating infection rate may be even higher when looking at the ratzooman results. this may be due to underreporting and undiagnosed cases. communities in informal settlements in urban areas are especially at risk as infected rodent populations are a continuous source of transmission. article information authors: joseph n. kuria1 stephen m. gathogo2 affiliations: 1department of veterinary pathology, microbiology and parasitology, university of nairobi, kenya2department of veterinary services, ministry of livestock development, kenya correspondence to: joseph kuria postal address: po box 29053, nairobi 00625, kenya dates: received: 01 mar. 2013 accepted: 20 may 2013 published: 31 july 2013 how to cite this article: kuria, j.k.n. & gathogo, s.m., 2013, ‘concomitant fungal and mycobacterium bovis infections in beef cattle in kenya’, onderstepoort journal of veterinary research 80(1), art. #585, 4 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.585 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. concomitant fungal and mycobacterium bovis infections in beef cattle in kenya in this research communication... open access • abstract • introduction • materials and methods • results and discussion • conclusion • acknowledgments    • competing interests    • authors' contributions • references abstract top ↑ bovine tuberculosis is an important zoonosis and accurate diagnosis is important for its surveillance. post-mortem diagnosis may, however, be compromised by lesions caused by other pathogens. in an investigation on its prevalence in slaughter cattle in kenya, mycobacterium bovis and dimorphic fungi were inadvertently identified separately or concurrently in tuberculous lesions. beef carcasses were inspected for lesions in two abattoirs in nairobi. tissues with lesions were collected and transported to the laboratory. smears of lesions were stained by acid-fast procedure and examined microscopically. lesions were cultured in löwenstein-jensen (lj) and in bbltm mycobacterium growth indicator tubes (mgit) media. mycobacteria isolates in lj medium were identified by dna typing. smears of bbltm mgit cultures were acid-fast stained and examined microscopically. tissue sections were stained with periodic acid-schiff reagent before examination. of the 929 carcasses examined, 176 had granulomatous lesions. dimorphic fungi were detected as acid-fast negative cells in 58 (32.9; 33.5%) of the lesion smears, either alone (29.0; 16.4%) or concurrently with acid-fast bacilli (29.0; 16.4%). the fungi were also detected in some bbltm mgit-culture smears and lesioned tissue sections. the fungi were identified, by means of cellular morphology, as paracoccidioides brasiliensis and blastomyces dermatitidis. a total of 64 isolates of mycobacteria were recovered in lj medium, 19 of which were identified as m. bovis. the present report documents native p. brasiliensis infections outside the presumed endemic region and b. dermatitidis infections in a livestock animal. the findings further indicate the importance of dimorphic fungi as a differential diagnosis of bovine tuberculosis in the region. introduction top ↑ bovine tuberculosis is caused by mycobacterium bovis and manifests in cattle as granulomatous lesions mainly in the lungs and lymph nodes of the thorax (liebana et al. 2008; office international des epizooties [oie] 2009). although post-mortem examination and culture are effective procedures for the diagnosis of bovine tuberculosis (oie 2009), the sensitivity of the procedure is affected by the presence of non-tuberculous parasitic, bacterial or mycotic granulomas and bacterial abscesses, which may be indistinguishable macroscopically from tuberculous granuloma (liebana et al. 2008; oie 2009). a study in mali cattle revealed that 72% of tuberculous lesions detected during standard meat inspections were due to pathogens other than m. bovis (muller et al. 2009). dimorphic fungi, which include blastomyces dermatitidis and paracoccidioides brasiliensis, are known causative agents of endemic systemic mycoses. although b. dermatitidis has a worldwide distribution, p. brasiliensis is geographically restricted to south and central america (chakrabarti & shivaprakash 2005; mcewen et al. 1995; restrepo 1985). paracoccidioides brasiliensis is mainly a human pathogen, causing chronic granulomatous pulmonary or disseminated infection (borges-walmsley et al. 2002). however, infections have been reported in domestic and captive wild animals (bagagli et al. 2003; costa & fara-netto 1978; costa et al. 1995; ricci et al. 2004). human paracoccidioidomycosis cases reported outside the endemic region are considered imported (onda et al. 2011; van damme et al. 2006). blastomyces dermatitidis primarily affects humans and dogs, causing a chronic suppurative or granulomatous respiratory infection that can disseminate to other tissues and organs, especially the skin (chapman et al. 1997). however, infections have also been reported in cats and horses (schmiedt et al. 2006). this report documents p. brasiliensis and b. dermatitidis infections in beef cattle in kenya in association with granulomas grossly indistinguishable from those caused by m. bovis. materials and methods top ↑ beef cattle carcasses were inspected for tuberculosis lesions in two abattoirs in nairobi, kenya, between july 2009 and november 2009. the animals slaughtered originated mainly from the nomadic pastoral communities in the arid and semi-arid northern areas of the country. carcasses were randomly selected and inspected according to procedures established by legislation. the lungs, the pleura, abdominal organs and the lymph nodes of the head region, thoracic cavity and the mesentery were palpated, incised and examined. samples of affected tissues were collected individually into sterile plastic bags, transported to the laboratory and preserved at -20 °c . the samples were then processed to be examined for acid-fast bacilli (afb) and for mycobacterium cultures according to standard procedures (oie 2009; world health organization [who] 1998a, 1998b). tissue samples were homogenised using griffith tubes, decontaminated with 4% sodium hydroxide and neutralised with phosphate buffered solution. smears were then prepared from the homogenates, stained according to the ziehl-neelsen (zn) method and examined microscopically. samples of the homogenates were cultured in löwenstein-jensen (lj) medium and bbltm mgittm medium (mycobacterium growth indicator tubes, becton, dickinson and co, usa). smears were prepared from the bbltm mgittm culture tubes and stained with the zn stain. mycobacteria isolated in lj medium were identified by molecular, analysis using the genotype mtbc assay kit (hain lifescience, nehren, germany), as described by gathogo, kuria and ombui (2012). briefly, a loopful of bacteria was collected from each afb-positive lj slant, suspended in 300 μl of purified water in a 1.5-ml microcentrifuge tube and incubated in boiling water for 20 min to inactivate the mycobacteria. this was followed by incubation in an ultrasonic bath to break the mycobacterial cell walls. dna was extracted by centrifugation and amplified in a thermocycler. the amplification conditions consisted of 5 min of initial denaturation at 95 °c, 10 cycles of 30 s each at 95 °c and 2 min each at 58 °c, 20 cycles of 25 s each at 95 °c, 40 s at 53 °c and 40 s at 70 °c, and a final extension at 70 °c for 8 min. hybridisation and detection were carried out using a semi-automated washing-and-shaking device (twincubator®, hain lifescience, nehren, germany). labelled hybridisation membrane strips were added into wells of the plastic reaction tray containing the amplicons and hybridisation reagents, after which plates were incubated. colourimetric detection of hybridised amplicons was performed by addition of streptavidin-conjugated alkaline phosphatase and the appropriate substrate. after the final washing step, the strips were air dried, fixed on a data sheet and examined visually.subsequent to detection of fungi in the zn preparations, histological examination of the lesions was carried out to confirm the presence of fungi. formalin-fixed samples of the lesions were routinely processed for histopathology and embedded in paraffin wax. they were sectioned at 5 μm thickness, stained with the periodic acid-schiff reagent and examined microscopically. results and discussion top ↑ of the 929 carcasses examined in total, 176 (19%) presented with tuberculous lesions. the majority of the lesions were localised (173/176; 98.29%) and were observed in bronchial lymph nodes (94), posterior mediastinal lymph nodes (94), lung parenchyma (14), liver parenchyma or portal lymph nodes (3), and in other lymph tissues (57). fungi were detected as acid-fast negative cells in smears of 58 cases (32.9%), either alone (29; 16.4%) or concurrent with afb (29; 16.4%). mycobacterium spp. were detected from only 35 cases (19.8%). of the 58 cases showing the presence of fungi, four presented with lesions in the lungs, whilst lesions in lymph nodes and other tissues and organs were seen in the remaining cases (table 1). the fungi comprised two types: blastospores with multiple buds attached to the mother cell by a narrow neck, resembling a mariner’s wheel and typical of p. brasiliensis, or large thick-walled cells with a single broad-based bud, identified as b. dermatitidis (figure 1a and 1b). these fungi were also detected in some bbltm mgit cultures (figure 1c and 1d) and b. dermatitidis cells were detected in some tissue sections (figure 1e and 1f). mycobacterium spp. were isolated from all 64 lesions that were afb positive, with 19 isolates being identified as m. bovis. table 1: cause of granulomatous lesions recovered from slaughter cattle in nairobi, kenya, as detected according to direct acid-fast staining, culture and dna typing. figure 1: photomicrographs of dimorphic fungi observed in tuberculous lesions in beef cattle in kenya, (a) paracoccidioides brasiliensis and (b) blastomyces dermatitidis fungal cells in lymph node lesion smears, (zn x 1000); (c) p. brasiliensis and (d) b. dermatitidis cells in bbl cultures, (zn x 1000). note the multiple buds, shown at b, on the flattened p. brasiliensis cell; (e) p. brasiliensis and (f) b. dermatitidis yeast cells in lymph node sections, (pas x 1000). the results of the investigation indicated that granulomatous lesions in the beef cattle carcasses examined were caused either separately by mycobacterium spp. or dimorphic fungi, or concurrently by both bacterial and fungal infections. bovine tuberculosis is endemic in many african countries (ayele et al. 2004). in tropical and subtropical countries the disease is of particular importance as a large proportion of the population depends on livestock for their livelihood. lack of control or eradication of the disease affects trade in animals and animal products (biet et al. 2005). the zoonotic nature of bovine tuberculosis further puts the human populations at risk. accurate diagnosis is therefore important in design and implementation of control programmes. in the present report dimorphic fungi were identified in tuberculous-like lesions as acid-fast negative cells. p. brasiliensis and b. dermatitidis are known to cause granulomatous lesions grossly similar to those caused by tuberculosis (baily et al. 1991; borges-walmsley et al. 2002; frean, blumberg & maureen 1993). in the presumed endemic region, paracoccidioidomycosis has also been found to occur concurrently with tuberculosis in 10% of human cases (borges-walmsley et al. 2002). in the present report, the fungi occurred concurrently with tuberculosis in 16.4% of the cases and separately in 17% of the cases. clinically, dimorphic fungi mimic tuberculosis (borges-walmsley et al. 2002; parvin et al. 2010) and chronic systemic mycoses caused by dimorphic fungi may therefore be confused with tuberculosis during ante-mortem examination of animals. examination of acid-fast stained smears of post-mortem specimens can, however, provide a preliminary diagnosis of mycobacterial disease (oie 2009).previous reports indicate that beyond the presumed endemic geographical region, paracoccidioidomycosis occurs only in patients who have previously resided in south or central america (miyaji & kamei 2003; shelbume & hamil 2002). detection of p. brasiliensis in cattle in the present report was an indication that this fungal infection does, however, occur outside the endemic area. it is noteworthy that climatic conditions similar to those in the presumed endemic regions of south and central america occur in northern kenya. although b. dermatitidis infections are most common in dogs and humans in north america, some isolated cases have been reported in africa, israel, india and bangladesh (carman et al. 1989; parvin et al. 2010; rouhou et al. 2008). in africa, and especially south africa, infections have previously been reported only in humans. conclusion top ↑ the present report documents native p. brasiliensis infections outside the reported endemic region and b. dermatitidis infection in a livestock animal. dimorphic fungi should therefore be considered as an important differential diagnosis for tuberculosis during meat inspection in the region. in the present investigation, 16.4% of the cases were found to be caused exclusively by the fungi. bovine tuberculosis is an important zoonosis and its diagnosis at slaughter requires condemnation of parts of or the whole carcass. dimorphic fungi are not zoonotic and a diagnosis would therefore avert condemnation. however, observation of infection in cattle in the present report may also indicate infection in other animals, including humans, in the region. acknowledgments top ↑ we acknowledge the facilitation of the management of kenya meat commission and njiru abattoirs in sample collection, the national tuberculosis reference laboratory for analytical facilities and the department of veterinary services for partial funding of the study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.n.k. 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gibson s. kibik7 julius d. keyyu5 peter godfrey-faussett8 paul van helden9 mecky i. matee1 affiliations: 1department of microbiology and immunology, muhimbili university of health and allied sciences, tanzania 2department of biochemistry, muhimbili university of health and allied sciences, tanzania 3school of public health and social sciences, muhimbili university of health and allied sciences, tanzania 4development studies institute, sokoine university of agriculture, morogoro 5tanzania wildlife research institute, arusha, tanzania 6the royal veterinary college, london, united kingdom 7kilimanjaro clinical research institute, tumaini university, tanzania 8london school of hygiene and tropical medicine, london, united kingdom 9dst/nrf centre of excellence for biomedical tuberculosis research/mrc centre of molecular and cellular biology, division of molecular biology and human genetics, faculty of health sciences, university of stellenbosch, south africa correspondence to: erasto mbugi postal address: po box 65001, dar es salaam, tanzania how to cite this proceeding: how to cite this proceeding: mbugi, e.v., kayunze, k.a., katale, b.z., kendall, s., good, l., kibik, g.s. et al. 2012, ‘”one health” infectious diseases surveillance in tanzania: are we all on board the same flight?’, onderstepoort journal of veterinary research79(2), art. #500, 7 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.500 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. this article was republished with the author’s full affiliation. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ‘one health’ infectious diseases surveillance in tanzania: are we all on board the same flight? in this proceeding... open access • abstract • introduction • health policy systems in tanzania • zoonoses with reference to previous veterinary studies • climatic change and one health • drug resistance • effective change • one health and bureaucratic organisation • conclusion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ infectious diseases account for nearly 40% of the burden of human mortality and morbidity in low-income countries, of which 7% is attributable to zoonoses and 13% to recently emerging diseases from animals. one of the strategic approaches for effective surveillance, monitoring and control of infectious diseases compromising health in both humans and animals could be through a combination of multiple disciplines. the approach can be achieved through a joint effort from stakeholders comprising health professionals (medical and veterinary), social, economic, agricultural, environmental and other interested parties. with resource scarcity in terms of number of staff, skills and facility in low-income countries, participatory multi-sectoral and multidisciplinary approaches in limiting the burden of zoonotic diseases could be worthwhile. we review challenging issues that may limit the ‘one health’ approach for infectious diseases surveillance in tanzania with a focus on health policy and how best the human and animal health systems could be complemented or linked to suit the community in need for disease control under the theme’s context. introduction top ↑ the concept of ‘one health’, if deployed in resource poor countries with proper management may be the best approach in sustainable control of emerging and re-emerging zoonoses in these countries. a recent report (jones et al. 2011) suggests that infectious diseases account for nearly 40% of the human disease burden with respect to morbidity and mortality in low-income countries. one-fifth of this is attributable to zoonoses or diseases which are classified as recently emerged from animals (7% and 13% respectively). the 1st ‘one health’ international congress was held in melbourne (from 14 february to 16 february 2011) with substantial representation from the southern african centre for infectious diseases surveillance (sacids), senior and young researchers from around the world as well as senior representatives from united nations (un), food and agriculture organisation of the united nations (fao), world health organisation (who) and office international des epizooties (oie). the meeting addressed, amongst many issues, the ‘one health’ concept of infection and immunity and the concept was illustrated by discussion about influenza virus which was first isolated from pigs in 1931 as a probable wildlife reservoir (doherty 2011). the connection and suspicion of connection to an avian and porcine reservoir of recent influenza outbreaks, originating usually in asia, is widely known (taubenberger & morens 2006) and it is likely that the 1918 ‘flu epidemic had a similar origin (morse 2004). one of the major issues in control of infections transmissible to man and animals is the lack of or limited joint approach to ameliorate the situation. thus the need for joint action with a combination of technologies and conceptual frameworks from both medical and veterinary professionals, social scientists and ecologists to launch approaches to contain the emerging and re-emerging infectious diseases. again, a reference to the asian situation is useful: in this scenario, we may expect regular emergence of influenza epidemics, which cycle between poultry and pigs kept in close contact, and where the virus gains virulence in this cycling, enabling it to become highly virulent to humans in due course. the need for effective integration between human and animal surveillance has been repeatedly identified as a key to successful surveillance of emerging and re-emerging infectious diseases (cleaveland, laurenson & taylor 2001; kahn 2006; kuiken et al. 2005; morse 1995; murphy 1998; shears 2000; woolhouse 2002; woolhouse & gowtage-sequeria 2005; world bank 2010).nevertheless, there are challenges to the ‘one health’ concept, particularly regarding its operationalisation. early detection and surveillance of the warning signs of infectious diseases is one major key towards successful control of diseases, in both humans and animals, but the lack of participatory health policy that focuses on multi-sectoral contributions towards one health is a major hurdle, particularly in resource-poor countries. this means that having an interdisciplinary consensus commitment and collective networks with leading consortia such as the sacids, one health central and east africa (ohcea) and united states agency for international development (usaid) organisation for prevention of disease outbreaks in central africa (respond) as prominent networks could initiate ‘one health’ action by breaking down barriers and creating networks. these could contribute to alleviating cross-transmission of infections at the human/animal/ecosystem interface which is a critical step to limit interspecies disease transmission. the approach could exploit skills from various disciplines, to prevent emerging and re-emerging infectious diseases from becoming prevalent, probably with minimal costs in terms of human and other resources. in this context, the major focus could be on social and cultural determinants of health that necessitate a holistic, integrative and interdisciplinary approach to alleviate health problems. the animal health for the environment and development (ahead) grouping in south africa, which is also affiliated with the wildlife conservation society (wcs) is an example of organisations applying one health approaches in disease surveillance and control. zoonotic infections from both livestock and wildlife in some countries contribute to infections in man for example, 60% zoonoses of which 70% originate from wildlife (atlas 2011; who 2011). it is clear that the risk of disease outbreaks is associated with climate, land use, scarce resources, innate ecosystem characteristics as well as poverty (daszak 2011). there is growing demand for meat and animal products, which is likely to lead to increasingly intensive farming and in turn and increased risk for disease. international livestock research institute (ilri) (kenya) reports that ‘a new infectious disease emerges every four months, and 75% of them originate in animals’, according to a recent report in nature. gilbert (2011) stated: such emergent diseases can have severe socio-economic, health and environmental impacts. whereas rich nations are controlling livestock diseases effectively, developing countries, including many in africa and asia, lag dangerously behind. (gilbert 2011) cook et al. (1996) reported an association between human tubercle bacillus (tb) cases and tuberculin positive cattle and clearly indicated the need for both medical and veterinary authorities’ joint action to arrest infection and subsequent disease. yet there have been few studies on risk factors for transmission from wild or domestic animals to people and between wildlife and domestic livestock in the literature (jones et al. 2011). therefore, the effect of one health action could be more than just an attempt to limit zoonotic infectious diseases, since it will extend to food security. it has been predicted that lack of food security is an increasing and serious risk to life, the lack of which is likely to result in more deaths in some areas than the combination of malaria and hiv and aids (nabarro 2011). it has been reported that up to 40% of household income in developing countries can be livestock dependent (mcdermott, director of ilri, and his ilri colleague delia grace at a conference in new delhi [leveraging agriculture for improving nutrition and health]). therefore, all these health related issues need attention and by addressing them together and holistically is likely to be worthwhile. the only question is whether all disciplines, including the broad definitions of natural and social sciences, are ready to be integrated. environment, for example, can play role in disease transmission to both humans and animals. evidence exists from some parts of the world for contamination of meat with clostridium difficile (riley 2011) raising concern on human and animal health through cross contamination. for example, preventing the spread of some infections from wildlife (e.g. ebola fever), can be achieved by effecting behavioural changes in hunters, but this is perhaps best done by professionals other than medical or veterinary workers, since it requires a good understanding of educational approaches at the appropriate level to hunters concerning wildlife health. an associated benefit of such intervention would enable the hunters to participate in wildlife health monitoring systems (reed & cameron 2011; ondzie et al. 2011). the issue of social mobilisation is vital in one health so there needs to be a strong emphasis on the need for national, regional and international collaboration in disease control (odugleh-kolev, who offices, geneva, switzerland). diseases have extensive social impact, and high economic costs, thus the need for a participatory and a well-coordinated approach, which requires the full multidisciplinary approach, including economists and politicians. there are several drivers of disease emergence and re-emergence, and we know that evolution of microorganisms involve a dynamic relationship with their hosts and environment (morens, folkers & fausi 2004). therefore, smart surveillance, using for example phylogenetics, can reveal the source of disease and direction of flow. such information can provide critical information to enable the problems to be tackled by the appropriate professionals with the correct tools in the practice of the ‘one health’ approach. health policy systems in tanzania top ↑ despite advocating ‘one health’ approaches in infectious disease surveillance in developing countries such as tanzania, the concept may be challenged and compromised by the existing health policies in the country. the current health policy and the tanzanian veterinary act (the tool that provides guidelines for veterinary practice activities) may not have a common point of intersection. this could be a result of parallel working organs that do not interact or a habitually conservative notion that ‘a vet should be a vet’ and ‘a medic should be a medic’. this concept could also be applicable to other professionals, whose roles could be critical if the ‘one health’ concept should be attempted. the concept of ‘one health’ can be misunderstood and needs to be introduced carefully; for example, one could think that with the concept, a vet can work in a medical hospital, and similarly a medical practitioner in a vet clinic. this is not the idea under ‘one health’, since the goal is to attempt to utilise professional skills from both veterinarians and medical practitioners as well as other professionals in arresting zoonotic diseases. the differential diagnosis of zoonotic diseases in humans may require the skills of both health professionals, followed up with preparation of intervention plans enabling the optimal use of veterinary intervention as a means to reduce the burden of human disease from an animal source (michel, müller & van helden 2010). the idea is to create preparedness in disease control through early disease surveillance, which is a critical initial step in restraining spread of diseases in developing countries. a study by karimuribo et al. (2011) points out lack of formal routine collaboration between sectors in disease surveillance with cross-sector collaboration being triggered only following disease emergencies. this has resulted in poor success in controlling disease outbreaks because of the lack of joint preparation for epidemics. examples include diseases such as avian influenza, rift valley fever (rvf) and african swine fever. the zoonotic potential of many diseases is not even recognised by many animal care practitioners, let alone medical practitioners (swai, schoonman & darbon 2010). the emergence of novel diseases across the animal and human domains is said to result from the rapid increase of both human and domestic animal populations. this increase has consequently increased the contact rate (jones et al. 2011; mazet et al. 2011; michel et al. 2006). this fact necessitates the need for a change in strategy for disease control, shifting from a single to a multidisciplinary approach. country-specific health systems can largely contribute to limiting efforts for ‘one health’ despite any good will for successful strategic disease control. available reports (department for international development 2011) have in principle indicated the public health burden of zoonotic tuberculosis in tanzania. for example, this report identifies mycobacterium bovis, mycobacterium tuberculosis and atypical mycobacterial species isolated from biopsy samples from human patients to be the main causes of tuberculous adenitis in humans. therefore the report highlights the need for a veterinary-medical collaborative effort to control tuberculosis in tanzania. collaborative efforts between these health professional partners have been improving over time but are largely dependent on the respective policies that govern the two bodies independently. not only the policies, but also a change in mindset of the individual practitioners from the two but closely related fields to a holistic approach in the control of zoonoses is needed for adoption of one health. clearly, a major limiting factor for both professionals is the extremely limited resource base, shortage of qualified personnel leading to a lack of time to deal with routine daily problems and the huge burden of disease. there is thus insufficient time or inducement to reflect and plan to move to a common ground. the tanzanian health policy currently emphasises the need for planning health development since resources and technology are limited (ministry of health of tanzania 2003). the policy also recognises the role of traditional and alternative health care to tanzanians, which is a significant component of the health care since about 60% of the population use traditional and alternative care systems for their day-to-day health care. however, it does not indicate the need for joint efforts between the veterinary, medical and other professionals as players which would provide for the establishment of ‘one health’. the tanzanian ministry of health and social welfare (ministry of health and social welfare of tanzania 2009) stresses the value of partnership for delivering the millennium development goals (mdgs). in its report, it recognises the need for preparedness for emerging and re-emerging diseases due to intensive cross-border contacts and globalisation. it is amongst the strategic plans for the ministry of health and social welfare of tanzania (mohsw) to strengthen surveillance, prevention, diagnosis and treatment of neglected infectious tropical diseases. however, the strategic plan does not clearly indicate the role of other professionals who could be instrumental for early diagnosis and prevention of infections particularly zoonoses. the concept of effective integration of the medical and veterinary disciplines has been suggested to be particularly necessary in the low income countries where facilities are limited (shears 2000). perhaps the time has come to address this in formulating a new mohsw plan in collaboration with the other relevant ministries, for example, ministries of: agriculture, food security and co-operatives; livestock and fisheries development; water and irrigation and natural resources and tourism. zoonoses with reference to previous veterinary studies top ↑ as far as veterinary and medical practices are concerned, zoonoses are not part of routine differential diagnosis in tanzania. this has, in addition, not been part of advisory packages to livestock keepers (john, kazwala & mfinanga 2008; swai et al. 2010). this lack of attention to otherwise preventable diseases has consequently resulted in under-reporting and lack of proper treatment to patients and livestock owners in need. a study by swai et al. (2010) on the knowledge and attitude towards zoonoses in both animal health workers and livestock keepers proposed, based on their findings, the need for public health promotion and education using an inter-disciplinary one-health collaboration between vets, public health practitioners and policy makers. it was further suggested that there should be joint action amongst these stakeholders and that they are the key to a more efficient and effective approach to the diagnosis and control of zoonoses in tanzania. the current inadequate communication between veterinary and human health care professionals in approaching these issues plus the limited data on zoonoses in tanzania (cripps 2000) is a factor constraining timely diagnosis and control of these diseases. climatic change and one health top ↑ climate change influences the pathogen flow, disease transmission and spread to various hosts. in a similar context, climate change is predicted to affect the geographical distribution and population densities of wildlife, which consequently will affect disease dynamics (jones et al. 2011). strong evidence suggests pathogen flow between people, livestock and non-human primates; and predicts that flow will increase concomitant with forest fragmentation and edge effects (jones et al. 2011). on the other hand, agro-ecosystem change has been said to decrease disease risk and flow from wildlife to domestic animals whilst also selecting for decreased pathogenicity (jones et al. 2011). in its strategic plans, the tanzanian ministry of health highlights those threats that may rapidly prove to be destructive, for example avian flu, severe acute respiratory syndrome (sars) and other new threats due to climate change (ministry of health and social welfare of tanzania 2009). the one health approach should therefore acknowledge and attempt to incorporate complex socioecological dynamics that influence the health of humans and animals (both livestock and wildlife). clearly, this will not be possible without the inclusion of social and natural scientists in the one health plan. the ohcea, which is another network for infectious disease surveillance and control, has objectives that mainly focus on emerging diseases for sustainable health and the need for a transformational change that allows continuous improvement of health and well-being of humans, livestock as well as wildlife ecosystems. the main goal, like other ‘one health’ based networks, focuses on a holistic approach to improve public and animal health. linked ‘one health’ networks will make information sharing within and between vicinities easier in terms of skills and approach. in a similar context, the success for a ‘one health’ approach in disease surveillance and control rests on acceptance and active participation of both national and international multidisciplinary key partners. available evidence suggests that socio-economic determinants of health are more powerful determinants of risk than biological factors (jones et al. 2011). therefore, the need for biocultural and economic approaches for ‘one health’ operationalisation is necessary. the major challenge in these health approach issues is the inequalities within and between human populations that produce different levels of disease burden in human populations. attention to food safety and security, particularly livestock products from sub-saharan africa and a comprehensive, focused result-based collaborative approach whilst taking responsibility towards ‘one health’ is a key towards success. therefore, people should address the ‘one health’ concept at national and international levels with special focus on possible identification of points of intervention, feasibility and acceptability of the approach by the community in need. this is possible through a joint approach amongst disciplines in disease control strategies. drug resistance top ↑ antimicrobial drug resistances as well as possible cross-resistances are other major challenges to the one health approach in surveillance and control of diseases. in human tuberculosis, for instance, drug resistance as well as multiple drug and extensively multi-drug resistance (mdr and xdr-tb) pose a challenge to the treatment of human tuberculosis. it is the strategy of the tanzanian ministry of health and social welfare to introduce and implement multi-drug and extensively multi-drug resistance tb management (ministry of health and social welfare of tanzania 2009). however, the strategic plan does not cite specific key role players, nor highlight the risk of treating m. bovis infection in humans with standard first line drugs without careful follow-up, given that m. bovis is innately resistant to pyrazinamide. given this, there is a very high risk that multi-drug resistant m. bovis could become a reality and may flow back to an animal reservoir over time. however, if veterinarians and other professionals are given opportunities where appropriate, the outcome should be more favourable. the advantage is to arrest the situation early and at relatively low cost whilst sharing the available scarce resources in order to avoid high or unaffordable costs later. successful operationalisation of the concept of ‘one health’ can be achieved however, it needs a careful assessment of areas of weakness as far as health policies are concerned (delegates at the 1st one health conference, melbourne, february 2011). amongst these we need to highlight the possible economic impacts of emerging infectious diseases, thus the need for political and policy actions to rectify the situation. these all are achievable through improved sentinel surveillance systems and better laboratory and multidisciplinary epidemiological research. the ‘one health’ concept targets early detection of disease at the animal or human and ecosystem interface to fulfill its mandate. this is not a straightforward approach, as it touches on multi-sectoral policies, consensus and community education. in most communities, animals are deeply interwoven with the social system and are used as ‘savings’ amongst the rural people (michel et al. 2004), making it difficult to enforce culling even when animals are clearly infected with a disease. this is particularly problematic if there is little or no compensation as is the case in tanzania. therefore, communities as stakeholders need to be willing participants to the processes. education to the community will definitely aid in avoiding misunderstanding of the concept of ‘one health’ amongst people of different calibre. a concern regarding the ‘one health’ approach to disease surveillance is the possibility of misunderstanding the concept itself. it should be understood that the aim of the concept is to increase preparedness for tackling infectious diseases, special attention being given to the emerging unexpected and novel events with avian flu, influenza, rvf and sars as few examples. effective change top ↑ awareness is the key to eventual change from one system to another. dedicated efforts are needed to ensure education of stakeholders in ‘one health’, particularly increasing awareness targeting specific groups such as farmers and lay people who might have no knowledge or awareness of the concept. a broader perspective on zoonoses should be deployed, particularly to change the local knowledge in both traditional and smallholder livestock keeping systems which might be limited to a restricted number of zoonoses such as anthrax, rabies and tuberculosis (swai et al. 2010). attention to large scale commercial farming is also important. effective positive changes under the theme ‘one health’ require involvement and engagement at the higher hierarchal level of leadership, using compelling arguments regarding environment, sustainability, lives at risk, economic impact, security as well as instability. this will hopefully lead to appropriate action at policy level, which will include the setting of balanced and reasonable priorities. the idea is to have a cross-agency and cross-disciplinary collaboration in science and humanities and bring together the key policy players whose role in ‘one health’ is crucial. the greatest challenge to this approach is the presence of outdated policies in different government sectors for disease control that might require transformation in terms of thinking amongst these key players into practical and action-oriented plans. a clear definition of ‘one health’ has to be spelled out. the provisionally suggested definition is that ‘one health is the prevention of human disease through control of animal infections and diseases in all ecosystems (stakeholders at the 1st one health conference, melbourne, australia, february 2011). to achieve this goal, having a cadre of dedicated and integrated medical and veterinary professionals as well as other key players including sociologists, policy makers and the politicians should be a priority. this holistic approach in control of zoonoses could be rewarding particularly in third world countries where the cost of disease (not only in economic terms) is high and there are limited human and other resources. the barriers and bridges for an inter-professional participatory approach in zoonotic disease control have been identified and critically analysed (world health organisation/department for international development animal health program, food and agriculture organisation & office international des epizooties 2006). these factors have been clustered into bridges and barriers (table 1), with barriers outnumbering bridges and have been proposed to be useful checklists to guide planning, organisation and management of zoonosis research in future. the idea is to allow veterinary and medical authorities to identify and tackle some of the overarching problems such as the lack of emphasis on zoonotic diseases in the training of both medics and vets. this is critical if we want to control zoonotic diseases under a ‘one health’ approach, as it may be influenced by a bureaucratic modus operandi in decision-making, resulting from the lack of well-defined policies for inter-sectoral linkage as a disease control strategy. table 1: bridges and barriers in controlling zoonotic diseases. one health and bureaucratic organisation top ↑ the ‘one health’ approach provides the opportunity for joint global health initiatives with the opportunity to use resources optimally compared to use in one sector only. with this approach it is a good idea to identify cross organisational boundaries with the aim of reducing redundancy, whilst increasing efficiency and improving outcomes for people, animals, the environment and society (comments from joseph anelli, a stakeholder in ‘one health’ at the 1st one health conference, melbourne, february 2011). various pandemics have in principle indicated the impact that emerging infectious diseases can have economically, socially, medically, and environmentally, suggesting benefits of using a ‘one health’ approach in education to address global health and sustainability challenges (barret et al. 2010). this approach could provide added value through combined efforts, rather than integration of the veterinary profession into the medical profession for example, which is neither a desirable outcome nor the intention. it is quite clear that the idea of ‘one health’ cannot be overemphasised; it is ‘predict’ and ‘prevent’ rather than ‘cure’ and ‘treat’ (simon easteal, stakeholder in ‘one health’ at the 1st one health conference, melbourne, february 2011). this means that for a successful one health approach, greater understanding of biological systems including the molecular mechanisms is needed. in addition, we need a better understanding of the epidemiology of infectious diseases at the human and animal (livestock and wildlife) intersection. this is achievable through education and training to achieve knowledge and skills gain amongst different health professionals. in addition, it would be important to raise awareness by facilitating communication and inter-disciplinary collaboration on research, information sharing amongst veterinary, public health, agricultural personnel and policy makers (coulibaly & yameogo 2000). the need to understand the dynamics of infectious diseases in their complex multi-host communities, their threats to public health, livestock economies and wildlife has been spelled out earlier (cleavaland et al. 2001). the simplest way to achieve this at ground level at minimal cost is to organise joint meetings and symposia with different professionals to address a common disease problem, for example, avian influenza. using such an example, one can have professionals such as vets, clinicians, conservation specialists in birds and bird movement and animal and laboratory technicians. it is the systematic quantification of these cross-species transmitted pathogens that need a joint approach amongst stakeholders to restrain their impacts that could be viewed in public health, conservation and economic perspectives. these all efforts will be successful with policies that allow multi-sectoral approach in disease control, particularly those of interest to both animals and humans. conclusion top ↑ zoonotic diseases are arguably more important in low-income countries than in high-income resource-rich countries. the emergence and re-emergence of zoonotic diseases in low-income countries is due to a number of factors including deficient policies, population growth, environment and ecosystem change and a misconception of the ‘one health’ concept amongst professionals. in addition, these factors are compounded by a low level of preparedness. finally, there is the need to work towards acceptance by communities and authorities in need for a change in strategies to control zoonotic infections. this necessitates the need for effective understanding of ‘one health’ and associated networks to communicate and generate a shared database of ‘one health’ perspectives for effective control of both commonly known and emerging zoonoses. perhaps the most relevant route to identify the optimal points of intervention should be through national and international collaboration and coordination using available resources in terms of personal skills and technology for disease surveillance, diagnosis, treatment and control. the change in policy to favour a holistic approach and interaction in disease surveillance and control could be a remarkable boost to environmental preservation, conservation strategies, as well as human and animal health and economic well-being. acknowledgement top ↑ the sacids is acknowledged for providing the opportunity for a postdoctoral research fellowship to em. financial support was received from the wellcome trust sub-awardees grant. prof. mark rweyemamu, the director sacids is acknowledged for stimulating the idea and advice. the london international development centre in the united kingdom and its staff is acknowledged for collaboration and providing the opportunity for preparation of this manuscript. prof. jeff waage is acknowledged for his dedicated efforts to provide complementary material support and encouragement during preparation of this manuscript. muhimbili university of health and allied science, particularly, the school of medicine and the department of biochemistry are acknowledged for granting time for preparation of this work. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions e.v.m. (muhimbili university of health and allied sciences) participated in reviewing the literature, conceptualising, drafting and writing the manuscript. b.z.k. (tanzania wildlife research institute) participated in initial drafting of the manuscript and critical manuscript review. s.k. (the royal veterinary college) participated in critical manuscript review. l.g. (the royal veterinary college) assisted in critical manuscript review and focusing of the manuscript. g.s.k. (kilimanjaro clinical research institute) critically reviewed the manuscript. j.d.k. (tanzania wildlife research institute) conceived the idea, assisted in drafting and critical review of the manuscript. p.v.h. (university of stellenbosch) assisted in critical review of the manuscript. p.g.f. (london school of hygiene and tropical medicine) assisted in drafting the manuscript and critically reviewed the manuscript. m.i.m. (muhimbili university of health and allied sciences) directed the whole process of drafting and critical review of manuscript. k.a.k. 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http://siteresources.worldbank.org/intard/resources/ppp_web.pdf world health organisation, 2011, strong intersectoral partnerships in health: managing zoonotic public health risks at the human-animal-ecosystem interface, department of food safety and zoonoses (fos), world health organization, geneva. world health organisation/department for international developmentanimal health program, 2006, ‘the control of neglected zoonotic diseases: a route to poverty alleviation’, report of a joint who/dfid-ahp meeting with the participation of fao and oie, geneva, 20 and 21 september 2005. woolhouse, m.e.j., 2002, ‘population biology of emerging and re-emerging pathogens’, trends in microbiology 10, s3–s7. woolhouse, m.e.j. & gowtage-sequeria, s., 2005, ‘host range and emerging and reemerging pathogens’, emerging infectious diseases 11, 1842–1847. http://dx.doi.org/10.3201/eid1112.050997, pmid:16485468 article information authors: calvin sindato1 esron karimuribo2 e.g. mboera3 affiliations: 1national institute for medical research, tabora, tanzania 2sokoine university of agriculture, tanzania 3national institute for medical research, dar es salaam, tanzania correspondence to: calvin sindato postal address: po box 482, tabora, tanzania how to cite this abstract: sindato, c., karimuribo, e. & mboera, e.g., 2012, ‘the epidemiology and socio-economic impact of rift valley fever epidemics in tanzania: a review’, onderstepoort journal of veterinary research 79(2), art. #467, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.467 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the epidemiology and socio-economic impact of rift valley fever epidemics in tanzania: a review in this abstract... open access a review was conducted to provide comprehensive update on rift valley fever (rvf) in tanzania, with particular attention devoted to trend of occurrence, epidemiological factors, socio-economic impact and measures which were applied to its control. information presented in this paper was obtained through extensive literature review. rift valley fever was documented for the first time in tanzania in 1977. this was followed by epidemics in 1997 and 2007. contrary to the latest epidemic in 2007 sporadic cases of rvf during the previous epidemics were confined to mainly livestock and mostly affecting northern parts of tanzania. the latest disease epidemic expanded to cover wider areas (mostly northern and central zones) of the country involving both human and domestic ruminants. during the latest disease outbreak 52.4% (n = 21) of regions in tanzania mainland were affected and majority (72.7, n = 11) of the regions had concurrent infections in human and animals. phylogenetic comparison of nucleotide and amimo acid sequences revealed different virus strains between kenya and tanzania. epidemiological factors that were considered responsible for the previous rvf epidemics in tanzania included farming systems, climatic factors, vector activities and presence of large population of ruminant species, animal movements and food consumption habits. majority of the rvf positive cases in the latest epidemic were livestock under pastoral and agro-pastoral farming systems. the disease caused serious effects on rural people’s food security and household nutrition and on direct and indirect losses to livestock producers in the country. psycho-social distress that communities went through was enormous, which involved the thinking about the loss of their family members and/or relatives, their livestock and crop production. socially, the status of most livestock producers was eroded in their communities. cessation of lucrative trade in ruminants resulted in serious economic losses to the populations who were totally dependent upon this income. livestock internal market flows drastically dropped by 37% during latest epidemic. rift valley fever epidemics had dramatic impact of rvf outbreak on the international animal trade in which there was a 54% decline in exports equivalent to loss of $352 750.00. the estimate of loss as a result of deaths for cattle was $4 243 250.00 whereas that of goats and sheep was $2 202 467.00. steps taken to combat epidemics included restriction of animal movements, ban of the slaughter of cattle and vaccination of livestock and health education. from past epidemics we have learnt that each subsequent outbreak had expanded to cover wider areas of the country. the disease had dramatic socio-economic impacts both at community and nation at large. the main challenges related to the control of rvf outbreaks included lack of preparedness plan for rvf, poor coordination and information transmission, limited facilities and manpower for rvf outbreak intervention. control of the 2007 rvf epidemic was largely the result of animal and human health agencies working in an integrated manner. article information authors: benjamin mogoye1 colin n. menezes2 martin p. grobusch2,3,4 kerstin wahlers5 john frean1 affiliations: 1national institute for communicable diseases, national health laboratory service, south africa 2department of internal medicine, chris hani baragwanath hospital and university of the witwatersrand, south africa 3institute of tropical medicine, university of tübingen, germany 4department of internal medicine, university amsterdam, the netherlands 5comprehensive infectious diseases center, university hospitals, germany correspondence to: martin grobusch postal address: department of internal medicine, chris hani baragwanath hospital and the university of the witwatersrand, faculty of health sciences, 7 york road, johannesburg 2193, south africa how to cite this abstract: mogoye, b., menezes, c.n., grobusch, m.p., wahlers, k. & frean, j., 2012, ‘human cystic chinococcosis in south africa’, onderstepoort journal of veterinary research 79(2), art. #469, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.469 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. human cystic echinococcosis in south africa in this abstract... open access cystic echinococcosis (ce) is caused by the tapeworm, echinococcus granulosus. the tapeworms resides in the small intestines of canids and the lifecycle involves both intermediate and definitive hosts. humans are accidental intermediate hosts. cystic echinococcosis is an economically important infection constituting a threat to public health, and is considered an emerging disease around the world. there are at least 10 echinococcus strain types (g1 – g10), each exhibiting diversity of morphology, development and host range. the epidemiology of ce is poorly understood in south africa. a retrospective data analysis of the national health laboratory service (nhls) laboratory information system on echinococcosis serology, microscopy and histopathology results in eight provinces (excluding kwazula-natal) showed an overall positivity rate in submitted diagnostic samples of 17.0% (1056/6211), with the eastern cape (30.4%), north west (19.0%) and northern cape (18.0%) provinces showing highest rates. the data showed considerable variability between provinces. the review also showed that most proven cases were negative on serology, implying that the actual number of patients could be underestimated. to our knowledge, no data exist about the prevalent strains of e. granulosus and this prospective study will attempt to fill that gap. the aim is to genotype strains causing the disease in south africa. two different polymerase chain reaction (pcr) methods will be used to respectively target the 12s rrna and nad 1 genes. to date, three samples have been genotyped as g1, g5 and g6; suggesting diversity of strains prevalent in the country, but more data is needed for a clearer picture. article information authors: kim a. kayunze1 angwara d. kiwara2 eligius lyamuya3 dominic m. kambarage4 jonathan rushton5 richard coker6 richard kock7 mark m. rweyemamu4,8 affiliations: 1development studies institute, sokoine university of agriculture, tanzania 2institute of development studies, muhimbili university of health and allied sciences, tanzania 3muhimbili university of health and allied sciences, tanzania 4sokoine university of agriculture,tanzania 5royal veterinary college, hawkshead lane, united kingdom 6mahidol university, thailand 7royal veterinary college, united kingdom 8royal veterinary college, university of london, united kingdom correspondence to: kim kayunze postal address: po box 3024, morogoro, tanzania how to cite this proceeding: kayunze, k.a., kiwara, a.d., lyamuya, e., kambarage, d.m., rushton, j., coker, r. et al., 2012, ‘a socio-economic approach to one health policy research in southern africa’, onderstepoort journal of veterinary research 79(2), art. #460, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.460 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a socio-economic approach to one health policy research in southern africa in this proceeding... open access • abstract • introduction • materials and methods • findings from literature    • wild animals-livestock-humans interactions and disease occurrence       • burden of disease in humans and animals    • measurement of burden of disease in humans       • burden of disease in animals       • linkage between burden of disease and other factors       • linkage between burden of disease and health policies       • linkage between burden of disease and the practice of health care services       • linkage between burden of disease and psycho-social theories of health care       • linkage between burden of disease and some socioeconomic factors       • burden of disease and poverty    • conclusion and recommendations • acknowledgements • references abstract top ↑ one-health approaches have started being applied to health systems in some countries in controlling infectious diseases in order to reduce the burden of disease in humans, livestock and wild animals collaboratively. however, one wonders whether the problem of lingering and emerging zoonoses is more affected by health policies, low application of one-health approaches, or other factors. as part of efforts to answer this question, the southern african centre for infectious disease surveillance (sacids) smart partnership of human health, animal health and socio-economic experts published, in april 2011, a conceptual framework to support one health research for policy on emerging zoonoses. the main objective of this paper was to identify which factors really affect the burden of disease and how the burden could affect socio-economic well-being. amongst other issues, the review of literature shows that the occurrence of infectious diseases in humans and animals is driven by many factors, the most important ones being the causative agents (viruses, bacteria, parasites, etc.) and the mediator conditions (social, cultural, economic or climatic) which facilitate the infection to occur and hold. literature also shows that in many countries there is little collaboration between medical and veterinary services despite the shared underlying science and the increasing infectious disease threat. in view of these findings, a research to inform health policy must walk on two legs: a natural sciences leg and a social sciences one. introduction top ↑ infectious diseases which occur in an epidemic or explosive form attract national, regional or international attention because of their propensity for causing high morbidity and rapid transboundary spread across national borders or even across continents and because of their potential for causing high mortality in affected populations and national and/or international socio-economic impacts. those that occur in endemic form or cause chronic disease tend to attract less public attention, although locally they might even be of a higher socio-economic impact (maudlin, eisler & welburn 2009). this latter category includes many of what have been referred to as ‘neglected’ or ‘lingering’ zoonoses. in general most of the newly recognised emerging or re-emerging infectious diseases of humans or animals have tended to be of the epidemic or transboundary type (world bank 2010). an emerging disease may be defined as one ‘that is newly recognized or newly evolved, or that has occurred previously but shows an increase in incidence or expansion in a geographical, host or vector range’ (fao/oie/who 2004). the majority (i.e. about 60%) of all infectious diseases of humans and most (i.e. about 75%) emerging infectious diseases of humans have been shown to have an animal origin, and thereby of zoonotic nature (jones et al. 2008; taylor, latham & woolhouse 2001; otte et al. 2007; woolhouse & gowtage-sequeria 2005, cited by shaw 2009). examples of such emerging diseases include ebola, avian influenza, pandemic influenza, human immunodeficiency virus (hiv) and aids, bovine spongiform encephalitis (bse) and the nipah virus. examples of the so-called neglected or lingering zoonoses include anthrax, bovine tuberculosis (tb), brucellosis, cysticercosis and neurocysticercosis, cystic echinococcosis or hydatid disease, rabies, zoonotic sleeping sickness or human african trypanosomosis (hat), and food-borne zoonoses, including salmonella (salmonellosis), campylobacter (campylobacteriosis), and escherichia coli (colibacillosis) infections of animal origin affecting millions of people annually. in order to address the infectious disease burden effectively – especially in developing countries – there is, therefore, an increasing body of opinion that advocates not only an enhanced collaboration between the human and veterinary medical sectors (i.e. the so-called one medicine, schwabe 1969) but also inter-sectoral collaboration across the public, animal and environmental health sectors, involving both the natural and social sciences (coker et al. 2011; zinsstag et al. 2011). in this regard current health policies, whether for human or animal health, need some re-examination as to their fitness-for-purpose for the one health approaches. as part of an effort to generate evidence to inform policy development for re-examining how existing health systems are structured, resourced, and managed to create synergies between animal and human health, and in the process to reduce the effect of zoonotic disease burdens, the sacids smart partnership of human health, animal health and socio-economic experts published, in april 2011, a conceptual framework to support one-health research for policy on emerging zoonoses (coker et al. 2011). the present paper builds on this framework to examine factors that have been reported to affect the burden of disease and how such a burden affects socio-economic well-being so that research projects, especially in sub-saharan africa, could be geared towards analysing some of the linkages documented in literature. the specific objectives were to, (1) analyse how interactions amongst wildlife, livestock, and humans lead to occurrence of infectious diseases, (2) explore the application of one health approaches to controlling infectious diseases, (3) investigate how one health approaches, theoretical factors, health policies, and socio-economic factors explain burden of disease and (4) illustrate the impact of burden of disease on poverty. the findings were intended to generate empirical information on which advocacy might be based for more collaboration, and how to realise it, for more effective control of lingering and emerging infectious diseases. materials and methods top ↑ the starting point for this paper was the sacids conceptual framework described by coker et al. (2011), presented in figure 1. the main assumption for the framework is that wild animals are reservoirs of pathogens and that the pathogens can spread to humans directly or indirectly. as seen in figure 1, biological interventions can be applied at the following three levels: prevention of pathogens against crossing from wild animals to livestock; prevention of pathogens which are in livestock from crossing to humans and once the pathogens are in humans, controlling them to reduce morbidity and mortality in humans. these interventions can result in biological improvements at three levels of: figure 1: a framework for research to inform one-health policy. • a change in incidence of pathogens in livestock derived from wildlife • a change in antibiotic resistance in organisms in livestock • reduced incidences of disease and improved survival following infection. success in those interventions is expected to contribute to attainment of the highest level of success, which is having healthy animal and human populations, economic security, and social stability. however, that highest level of success can hardly be attained without active participation of other stakeholders. that is why in the sacids framework such stakeholders have been identified, as seen under the titles ‘context’ and ‘mechanism’. the stakeholders are other natural scientists (besides veterinary and medical personnel), ecologists and agricultural scientists to address environmental and agricultural issues; socio-economists to address societal issues including human behaviour and economic issues and policy-makers to address health service organisation issues, patterns and provision and access, fiscal systems, regulation and governance, information and planning aspects. findings from literature top ↑ wild animals-livestock-humans interactions and disease occurrence infectious diseases can spread directly or indirectly from one person to another, one animal to another, or from animals to persons and vice versa. wild animals are known to be reservoirs of pathogens some of which may not affect them due to their genetic make-up and adaptation to wild conditions, albeit some of the pathogens can cause disease in livestock and in humans. but infectious diseases can also cross from either humans to wild animals (e.g. human tb) or from livestock to wildlife (e.g. bovine tb). a major transboundary animal disease of cattle that used to cause heavy mortality in wild ungulates, whose elimination from the maasai eco-system of tanzania and kenya resulted in a progressive increase in the population of wildlife, notably the wildebeest in the serengeti (kock 2003) and whose global eradication depended on concerted action only in the cattle population, was rinderpest (fao & oie 2011).interactions amongst wildlife, livestock and humans can favour the spread of the pathogens either directly to people through contact with wild animals harbouring the pathogens, contact with contaminated wild products, or consuming wildlife products, including bush-meat. these interactions are best explained with the aid of a diagram like the one in figure 2 by institutes of medicine (2009, in who 2010). a major inference from figure 2 is that interactions amongst the members of the natural ecosystems (e.g. human encroachment and land use, etc), food and agriculture systems (e.g. expanding agricultural production, etc.), and human living environments (including increasing population density and growth, etc.) can lead to disease occurrence or infection spread. in their analysis of interaction trends amongst wildlife, livestock and humans, jones et al. (2008) concluded that the majority of emerging infectious diseases of humans (71.8%) originate from wildlife. figure 2: interactions that may lead to disease occurrence. the interactions in figure 2 are also explained by two theories: • the island biogeography theory • the parasite-stress theory of human sociality. the former states that pathogens that lead to disease occurrence are identified at three levels of: • interactions within species sources of pathogens • interactions between recipient host species and species sources of pathogens • interactions within recipient host species (macarthur & wilson 1967). the latter states that humans’ ontogenetic experiences with infectious diseases as well as their evolutionary historical interactions with these diseases exert causal influences on human psychology and social behaviour (thornhill 2010). this theory emphasises the causal role of non-zoonotic parasites, which are characteristic of disease transmission from one person to another one, rather than zoonotic parasites which transmit diseases from vertebrate animals to humans. burden of disease in humans and animals burden of disease is a measure of financial cost, mortality, morbidity, or other indicators for humans and animals at the individual, community, herd or flock, farm, national, or global level due to diseases. it is normally measured in terms of disability-adjusted life years (dalys). a daly is equivalent to the loss of one year of ‘healthy life’ and allows the burden of disease in a population to be measured as the gap between current health and an ideal situation where everyone lives to old age, free of disease and disability (mathers et al. 2001). this definition is from a medical point of view; it is in human medicine that burden of disease started being measured, and the measurement is described below. measurement of burden of disease in humans burden of disease in humans is measured in various ways using various indicators, which are presented in table 1. table 1: indicators for computing burden of disease in humans. although who publishes annual tables showing how many dalys a year different diseases are estimated to cost, the zoonotic component of infectious diseases is largely missing from the league of tables (coleman 2002, in shaw 2009). in some works, total burden of disease is calculated; where this is done, conventionally, direct burden of disease in people is measured in dalys, and all the other components (direct losses in animals and costs of prevention and treatment in people and animals) are measured in monetary terms (brazier et al. 2007; drummond et al. 2005; shaw 2009). burden of disease in animals unlike in humans where assigning monetary values to people’s losses of life complicates calculation of burden of disease, in animals the calculation is straightforward because most direct losses due to illness and due to death have objective monetary values (shaw 2009). however, the calculation is complicated by the presence of many animal species which have various roles in the human society. in spite of the differences and complexities in calculating burdens of disease in humans and animals, table 2 gives the ways of how to do the calculations. table 2: items for calculation of burden of disease in terms of disability-adjusted life years. linkage between burden of disease and other factors collaborative efforts of many disciplines and experts in those disciplines to deal with infections diseases to reduce the burden of diseases in humans and animals is one thing; there are other factors which can enhance or constrain the pace towards controlling the diseases. in this paper, the other factors considered are health policies, the practice of health care services on the ground, theoretical factors, and socio-economic factors, which are discussed below. linkage between burden of disease and health policies health policy means different things to different people, but its compressive definition which is widely acceptable is given by walt (1994) as a set of statements stipulating courses of action that affect the set of institutions, organisations, services, and funding arrangements of the health care system and goes beyond health services by including actions or intended actions by public, private and voluntary organisations that have an impact on health. she adds that health policy is concerned with environmental and socio-economic effects on health as well as with health care provision. however, many books on health policy focus narrowly on the health care system only. therefore, some scholars, for example nancy (1987, in walt 1994), prefer talking about health public policy in order to differentiate the broader definition from the narrow one.health policies are affected by related international polices and by other policies which have nothing to do with health care or services for example, (1) environmental pollution, (2) insecurity and instability (whether caused by employment or violence), (3) economic regulation and deregulation and (4) contaminated water and poor sanitation, all of which increase morbidity and mortality. in view of this, the implementation of health policies may be constrained or enhanced by these other policies. besides these, also cost-sharing affects health policies. for example, in africa, it is widely known that the policy of cost sharing in both animal health and human health since the 1980s has complicated access to the services. this situation is well explained by rushton and leonard (2009) as follows: before the 1980s, particularly from the late 1940s, animal health had been regarded as a predominantly public service and thus was predominantly provided by governments. but since the 1980s the provision of the services has been increasingly opened to market institutions. however, animal health, like human health, is subject to market failures, and there remains a role for the state in their correction, through the provision of selected goods and services, the setting and monitoring of regulations and taxes and subsidies. in the human health sector cost-sharing has been characterised by people contributing for health services but ending up not getting the services, for example prescription for medicines to buy instead of being given the medicines. this connotes high burden of disease, especially in rural areas where people cannot afford paying for health services in private health facilities, which have proliferated concomitantly with the rise in cost-sharing in public health facilities. linkage between burden of disease and the practice of health care services with respect to the practice of health care services, delivery of health services is indicated by a number of variables, which should be applicable to individuals, households, communities, populations, nations, regions, and globally so that comparisons in the levels of the services can be possible. for human diseases, who (2010) gives a number of indicators, which are divided in the following categories: (1) health service coverage, (2) risk factors, (3) health workforce, infrastructure and essential medicines, (4) health expenditure ratios and per capita health expenditures and (5) health inequities. under each of those categories there are a number of indicators. the indicators are not reproduced here for saving space, but they are readily available on the interment. better health services result into less burden of disease, and vice versa.about health service practice for animals, as in human health, health indicators are very important in order to prevent, control and treat animal diseases effectively. according to european commission (2007), simple and reliable performance indicators help to measure progress towards animal health, guide policy, inform priorities, and target resources. the indicators can be divided into hard indicators of animal health (e.g. disease prevalence, number of animals eliminated) and softer indicators tracking the confidence, expectations and perceptions of citizens. in rural areas of developing countries, like for human health, animal health statuses and services are poor vis-à-vis urban areas, mainly due to fewer animal health facilities and fewer animal health personnel. as a result, according to who (2006), zoonoses typically affect isolated rural livestock keeping communities and those living in urban slums. linkage between burden of disease and psycho-social theories of health care psycho-social theories of health care seeking behaviour are amongst various factors that explain burden of disease. the theories explain the determinants of behaviour that lead people to accessing and utilising health services. some of the determinants are common in seeking health care services for humans and for animals, and these include local people’s experiences with diseases, availability of traditional versus modern treatment, knowledge and beliefs about diseases, decision process for seeking health services, and parochial versus cosmopolitan outlook of diseases. the behavioural aspects that are practised in turn determine the extent to which health services are accessed. some of the prominent theories are parsons’ sick role, mechanic’s general theory of help seeking, suchman’s stages of illness and medical care, theory of reasoned action (tra), and theory of planned behaviour (tpb). besides the theories, there are models for health care seeking behaviour. the difference between the theories and the models is that the former consider decision points or stages of health care seeking, but the latter can be regarded as containing sets of interacting variables (rebhan 2009). one of the prominent models is the health belief model (hbm). in view of this model, if individuals do not perceive the illness as serious, they will not seek treatment or preventive measures for themselves, for their household members or for their livestock (rosenstock, strecher & becker 1994; sheeran & abraham 1995, in hausmamm-muela, ribera & nyamongo 2003). in the interest of saving space, only some of the theories and models are described here.the tra and the tpb are closely associated as the latter is an extension of the former. by tra, fishbein and ajzen (1975) argue that attitude and subjective norm are the primary determinants of behaviour. however, no behaviour is specified in both of the theories. therefore, various researchers in medical and agricultural systems (including livestock production) have applied the two theories in various situations and found them applicable to explaining correlations between certain factors (drivers of behaviour) and behaviour (good or bad) that people express. for example, both tra and tpb have been used to study farmers’ conservative behaviours, and tra has been used to study attitude towards buying feeds for livestock, and adoption of olive oil in british kitchens (jackson et al. 2006). if the behaviour is assumed to be the way how people act in choosing sources of health care services that people prefer for themselves, for their household members, or for their livestock, the two theories can be used in the same way as the health care utilisation model (hcum), which was developed by andersen (1968) looking at three categories of determinants of choosing where to seek health services, (1) predisposing characteristics (demographics, position within the social structure, and beliefs of health services benefits), (2) enabling characteristics (resources found within the family and the community) and (3) need-based characteristics, including the perception of need for health services, whether individual, social, or clinically evaluated perceptions of need (wolinsky 1988, in rebhan 2009). the model has undergone modifications a number of times, but its current form centres specifically on treatment selection – whether people go for traditional healers, modern healers, drug sellers, self-treatment, or no treatment. linkage between burden of disease and some socio-economic factors it is widely known that the most basic social services are education, health, water supply, and communication services. health services, which are amongst other social services, are affected by the other services. some sophisticated social services in view of current technologies like mobile phones and the internet influence access to health services in various ways. the linkage between social aspects and health are detailed in the report of the who’s commission on social determinants of health (who 2008). the report gives three overarching recommendations for improving health:• improving daily living conditions • tackling the inequitable distribution of power, money, and resources • measuring and understanding the problem of health inequity and assessing its impact of action. burden of disease and poverty poverty is pronounced deprivation in well-being (world bank 2001). it is a multidimensional phenomenon whose comprehensive definition would have to include all of its indicators, which are innumerable. accordingly, no attempt is made to present its definition here, but it is well known that its indicators include deficiencies in basic needs, especially food, shelter, and clothes; and in social services like education, healthcare, and water supply. it is also indicated by vulnerability, exposure to risk, voicelessness, powerlessness, and capabilities that a person has (sen 1999; world bank 2001). there are various ways of measuring poverty, but they are avoided in this short paper. however, at least it is worth mentioning that income is a poor indicator of well being since it is volatile and some people having much income may not use it to obtain important needs. this view is supported by sen (1999) who argues that resources are imperfect indicators of well-being and alkire, qizilbash and comim (2010) who contend that income is a fuzzy measure of poverty. therefore, non-monetary indicators are preferable to monetary ones, either to supplement the latter or alone. the preference for using non-monetary indicators grew in the 1990s after sen (1999) came up with the capability approach to poverty measurement, which is linked with the human development perspective and is now fashionable in measuring poverty. empirical information shows that healthier people are more productive and that wealthier people can obtain things that make them healthier. for example, studies which were conducted in colombia, peru, and nicaragua in the mid-1990s showed that reduced exposure to disease is associated positively with the health of adults and also with greater individual income-generating capacity (savedoff & schultz 2000). with respect to animal health, the burden of disease affects not only livestock keepers but also consumers of livestock products like meat, milk, and eggs. for livestock keepers, there may be losses of income. it is known that about 42% of the poor worldwide are dependent on livestock as their livelihoods but that imperfect or missing markets often trap them in low income equilibriums, preventing them from benefiting from the increased demand for animal protein (otte & pica-ciamarra 2009). besides the problem of market imperfections, diseases affect much the ability of livestock keepers through low productivity and mortality of their livestock. accordingly, it is obvious that amongst livestock keepers infectious diseases contribute to impoverishing them. in view of this, the same authors (otte & pica-ciamarra 2009) contend that if poverty alleviation is a policy goal, policy makers should identify, design and implement public actions that allow poor livestock producers to take advantage of the increasing demand for meat, milk and eggs. conclusion and recommendations we have seen in the literature reviewed that the occurrence of infectious diseases is driven by causative agents (viruses, bacteria, parasites, etc) and mediator conditions (social, cultural, economic or climatic) which facilitate the infection to occur and aid spread or transmission of the infection. the causative agents are best understood through the natural sciences whilst the mediator conditions are best understood through the social sciences. accordingly, the research framework that recognises the contribution that socio-economists can play in collaboration with biological scientists to harness innovation in science and technology in order to improve the capacity to detect, identify and monitor infectious diseases of humans and animals and their interactions in order to better manage the risk posed by them is one that is likely to provide the type of evidence-based policy impact.the practice of one health approaches is long overdue. adopting them should not be debatable, but the modalities of how to adopt and practise them should be the discussion, in view of the factors that enhance and those that constrain them. accordingly, factors that constrain the adoption and practice of one health should be curbed and those that promote it should be fostered. using social research methods (e.g. questionnaire-based interviews, key informant interviews, life histories, pra, fgd, ethnography, grounded theory, probing, and prompting), and tools (e.g. questionnaires, checklists of items for discussion, likert-type scales, index scales, and differential semantic scales) can add value to explanation of disease emergence, re-emergence, and persistence as well as burden of disease; the tools are good at studying attitudinal and behavioural aspects, which cannot be studied biologically. moreover, economics of controlling diseases facilitate quantification of burden of disease, for example by determining burden of disease in monetary terms and using the separable costs method of cost-effective analysis to determine equitable sharing of costs amongst various sectors working collaboratively to control diseases. the one health driven policy research framework to examine the extent to which one health approach can help to streamline health policies in the public, animal, and wildlife health sectors in such a way that they facilitate more collaboration between natural and social scientists so as to increase the effectiveness of interventions to stem infectious diseases for better socio-economic well-being is more focused on health policies. however, there may be other factors apart from health policies constraining the collaboration, which may include health service provision on the ground, psycho-social theories of health care seeking behaviour, and socio-economic factors. accordingly, such a framework should include an assessment of such factors as determinants of collaboration amongst various stakeholders, to find which of them are more associated with enhancing or deterring the collaboration and reduction of the burden of disease. infectious diseases increase the burden of disease in humans and animals, and the burden in turn aggravates poverty, especially amongst rural and sub-urban people whose economy mainly depends on livestock. therefore, there should be equitable control of human and animal diseases in rural and urban areas. literature shows that the majority of infections, new or old, either do or have the propensity to move across species (human and animals) or may have originated from animals before assuming the human-to-human transmissibility. one health approaches seem to be the logical strategy. but one should not under-estimate the challenges by the current organisational systems; even for the shared problem of zoonoses between animal and human health; fao (2006) has observed the challenges. acknowledgements top ↑ this work has been supported by wellcome trust grant wt087546ma to the southern african centre for infectious disease surveillance (www.sacids.org) and its collaborating uk partner institutions, namely the london school of hygiene and tropical medicine (www.lshtm.ac.uk), the royal 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johannesburg july, 14 2011, viewed 30 august 2011, from http://www.sacids.org/kms/resources/onehealth_johannesburg_zinsstagetal_2011%20%282%29.pdf gous_419-441.indd introduction tuberculosis, caused by the bacterium myco bac terium tuberculosis, also referred to as human tuberculosis (htb), is an uncommon disease in species other than humans. it is a primary human path ogen but may infect domestic or wildlife species that are in close or prolonged contact with humans (fourie 1983; michalak, austin, diesel, ba con, zimmerman & maslow 1998; michel & huch zermeyer 1998; montali, mikota & cheng 2001). although m. tuberculosis is the most common infection in humans, mycobacterium bovis is responsible for an increasing proportion of human tuberculosis cases (o’reilly & daborn 1995; cosivi, grange, daborn, raviglione, fujikura, cousins, robinson, huchzermeyer, de kantor & meslin 1998). much attention has been given to the zoonotic potential of tuberculosis in wildlife populations and the threat they present to human health (fredrickson, barton, ragan & roberts 1971; thoen 1988; stetter, mikota, gutter, monterosso, dalovisio, degraw & farley 419 onderstepoort journal of veterinary research, 76:419–441 (2009) the pathology of tuberculosis caused by mycobacterium tuberculosis in a herd of semi-free-ranging springbok (antidorcas marsupialis) t.a. gous* and m.c. williams section of pathology, department of paraclinical sciences, faculty of veterinary science university of pretoria, private bag x04, onderstepoort, 0110 south africa abstract gous, t.a. & williams, m.c. 2009. the pathology of tuberculosis caused by mycobacterium tuberculosis in a herd of semi-free-ranging springbok (antidorcas marsupialis). onderstepoort journal of veterinary research, 76:419–441 the first detailed description of the pathology of tuberculosis, caused by mycobacterium tuberculosis, in springbok is reported. the springbok were part of a semi-free-ranging herd kept on the grounds of ithemba laboratory for accelerator based science (labs) in the kuils river district of the western cape province, south africa. mycobacterium tuberculosis was isolated from three animals out of a total of 33 sampled, with two animals showing tuberculosis lesions. the index case was an adult ewe that showed advanced miliary tuberculosis with marked macroscopic and microscopic lesions in the lungs, pleura and respiratory lymph nodes, and numerous acid-fast bacilli. six healthy rams were sampled nine months later and a pilot study indicated miliary tuberculosis lesions in one ram, which again were macroscopically most prominent in the lungs, pleura and respiratory lymph nodes. macroscopic lesions were also noted in the sternal, iliac, prefemoral and retropharyngeal lymph nodes. microscopy in this animal revealed lesions in the macroscopically affected organs as well as numerous other lymph nodes, and suspected lesions occurred in the testicle and colon. acid-fast bacilli were scarce to moderate in affected organs. because of the miliary nature of the lesions in both affected animals, the route of infection could not be established conclusively. the lesions in most affected organs of both animals resembled classical tuberculous granulomas. a main study conducted on healthy animals 19 months after the pilot study failed to find any animal with tuberculosis lesions in the group of 25 sampled, and all were negative for mycobacteria via mycobacterial culture. keywords: mycobacterium tuberculosis, pathology, semi-free-ranging, springbok * author to whom correspondence is to be directed. present address: western cape provincial veterinary laboratory, private bag x5020, stellenbosch, 7599 south africa accepted for publication 16 april 2009—editor 420 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) 1995; cosivi et al. 1998; michalak et al. 1998; de lisle, mackintosh & bengis 2001). the reverse situation, however, has received little attention—the threat tuberculous humans present to wildlife. almost all reports published on htb in indigenous wild animals in south africa refer only to wild animals in captivity, notably non-human primates such as chacma baboons (papio ursinus) and vervet monkeys (cercopithecus aethiops) (fourie 1983; michel, venter, espie & coetzee 2003). trans mission to semi-free-ranging and free-ranging wildlife is an extremely rare occurrence and only one report describes htb in free-ranging indigenous wildlife, viz. banded mongooses (mungos mungo) in botswana and suricates (suricata suricatta) in south africa (alexander, pleydell, williams, lane, nyange & michel 2002). in the rest of the world htb in wild animals has been recorded exclusively in captive wildlife, and does not appear to occur naturally in free-living animals. the range of species infected includes nonhuman primates, elephants, ungulates, carnivores, marine mammals and psittacine birds (thoen, richards & jarnagin 1977; forshaw & phelps 1991; montali et al. 2001). the first case of tuberculosis in a springbok (antidorcas marsupialis) was recorded by robinson in 1951 at the pretoria zoological gardens (robinson 1953). the animal was killed after a positive reaction to the tuberculin test, and post mortem examination revealed extensive tuberculous lesions in the lungs. acid-fast bacilli were numerous in smears made from the lung lesions. although pure cultures of m. tuberculosis were obtained, subsequent cultures from guinea pigs that were inoculated with affected lung material were considered to be of the bovine type. a pure culture of m. tuberculosis was obtained from an in-contact springbok that died shortly afterwards, but inoculation of guinea pigs was never attempted. the identity of the causative mycobacterium species in both these cases is there fore, at best, equivocal. no description of the pathology was given in either of the animals other than “extensive tuberculosis lesions in the lungs”. the paper by hofmeyr (1956) mentions one case of tuberculosis in a springbok at the pretoria zoological gardens, but gives no additional information regarding the causative mycobacterium species or pathology. in december, 1999 htb was diagnosed in a springbok ewe that originated from a herd of semi-freeranging springbok that are kept on the grounds of the ithemba laboratory for accelerator based science (labs) near faure in the kuils river district of the western cape province, south africa. buildings on the 42 ha site are interspersed with artificially maintained kikuyu (pennisetum clandestinum) and kweek (cynodon dactylon) lawns, with groves of exotic alien vegetation consisting almost exclusively of port jackson willow (acacia saligna). apart from springbok, other semi-free-ranging game species on the premises include burchell’s zebras (equus burchelli), bontebok (damaliscus dorcas dor cas), grysbok (raphicerus melanotis) and steenbok (raphi cerus campestris). when the initial case of tuberculosis was diagnosed in a springbok there were 48 springbok, six bontebok, four zebras and a few grysbok and steenbok present. this study documents the pathology of htb in the springbok of ithemba labs. after the index case was diagnosed, an opportunity to examine more springbok presented itself on 26 september 2000 when the ithemba labs staff environmental club decided to cull six animals due to unsustainable numbers. one animal showed advanced generalized (miliary) tuberculosis, and m. tuberculosis was subsequently isolated from a pool of four organ specimens. this prompted the main study during which 25 animals were culled from april 2002 to july 2002 for detailed pathological and bacteriological investigation. materials and methods part one: index case animals on 20 december 1999 a prostrate and weakened adult springbok ewe was presented for necropsy at the western cape provincial veterinary laboratory (wcpvl) in stellenbosch to establish the cause of the clinical signs. the ewe was euthanased by means of an overdose of intravenous barbiturates and a full necropsy performed. necropsy revealed the possibility of tuberculosis and various specimens to confirm the diagnosis were collected as indicated below. on 29 june 2000 an old springbok ram was found dead at ithemba labs after it had appeared “tired” for at least a week prior to death. the carcass was presented for necropsy at the wcpvl but advanced decomposition made detailed examination impossible. however, lesions suggestive of tuberculosis were present in the lungs, and specimens were collected as indicated below. 421 t.a. gous & m.c. williams macroand histopathology all macroscopic lesions found at necropsy of the index case were recorded. the following tissue specimens were collected from the index case for histopathological examination and fixed in 10 % buffered formalin: heart (multiple specimens), lung (multiple specimens), mediastinal lymph node, bronchial lymph node, liver, spleen, kidney, adrenal glands and alimentary tract (oesophagus, abomasum, small intestine, large intestine and rectum). specimens from the ram consisted of lung tissue and pulmonary lymph nodes. the formalin-fixed tissues were embedded in paraffin wax and sections were cut at 5 μm and routinely stained for histopathological study with haematoxylin and eosin (h&e). all sections were also stained with ziehl-neelsen (zn) stain to detect acid-fast bacilli. microscopic lesions were recorded after light microscopic examination. bacteriology a fresh unpreserved mediastinal lymph node that showed lesions suggestive of tuberculosis was aseptically collected from the index case. two fresh unpreserved lung specimens were aseptically collected from the ram. these specimens were all frozen (–20 °c) and dispatched in a frozen state, within 5 days of collection, to the tuberculosis laboratory at the onderstepoort veterinary institute (ovi) for mycobacterial culture. the specimens were cultured for mycobacterium spp. according to the standard isolation and identification methods for mycobacteria used by the ovi. these methods have been described (anon. 1983; bengis, kriek, keet, raath, de vos & huchzermeyer 1996; keet, kriek, penrith, michel & huchzermeyer 1996). part two: pilot study animals on 26 september 2000 the ithemba labs staff environmental club decided to cull six springbok due to unsustainable numbers. at this stage, htb was known to be present within the herd and a pilot study was designed to evaluate these animals for the presence of tuberculosis. the animals were randomly selected by selecting every second animal encountered, and consisted of clinically healthy rams comprising three sub-adult and three adult animals. the animals were stunned before death by professional hunters using the intracranial gunshot method with .22 calibre rifles. as soon as each animal fell to the ground the throat was cut to cause exsanguination and death. animals were identified numerically and chronologically as they were euthanased, and then transported to the wcpvl for necropsy and sampling. macroand histopathology the main aim of the pilot study was to detect animals with macroscopic and microscopic tuberculosis lesions, and to describe the lesions in detail. the hallmark tuberculosis lesion is the granuloma, or tubercle (dungworth 1993), and any macroscopic lesion resembling granulomatous inflammation was described. these were lesions showing any of the following characteristics: pinpoint to larger size, multiple to single, distinctly outlined or poorly demarcated, flat to elevated above the organ surface, pale to pigmented, with or without necrosis, calcification, liquefaction, abcessation or encapsulation. tissue specimens were collected for histo path ological examination and all, except the lymph nodes and specimens for mycobacterial culture (see below), were fixed in 10 % buffered formalin. these included the brain, eyes, heart (multiple specimens), lung (multiple specimens), liver, kidneys, spleen, adrenal glands, testes, thymus (if present) and alimentary tract (tongue, oesophagus, abomasum, duodenum, jejunum, ileum and colon). specimens from other organs and tissues were only collected if macroscopic abnormalities were noted. fresh unpreserved lymph nodes were collected aseptically and each lymph node placed in a separate sterile container for processing within 24 h of collection which entailed aseptic sectioning of each lymph node into thin (1–2 mm) slices for macroscopic evaluation. specimens from each lymph node were then placed in 10 % buffered formalin, and the remainder dispatched for mycobacterial culture. lymph nodes sampled included the tonsils, parotid (left and right), retropharyngeal (left and right), mandibular (left and right), bronchial, mediastinal, sternal, hepatic, splenic, ileocaecal, mesenteric, iliac, inguinal, prescapular (left and right), prefemoral (left and right) and popliteal (left and right) lymph nodes. the formalin-fixed tissues were embedded in paraffin wax and sections were cut at 5 μm and stained with h&e and zn. microscopic lesions were recorded after light microscopic examination. the suspect macroscopic granulomatous lesions were confirmed and described microscopically. additional granulomatous inflammatory lesions not 422 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) detected macroscopically, were given microscopic descriptions. for the purpose of this study, microscopic granulomatous inflammation was defined as a lesion showing any combination of the following: central caseous or coagulative necrosis, with or with out calcification, surrounded by macrophages, epithelioid cells and multinucleated giant cells, and more peripherally lymphocytes, plasma cells, macrophages and varying degrees of fibroplasia, with or without encapsulation. the reaction may be modified by haemorrhage, accumulation of fibrin and neutrophil infiltration. the presence or absence, location and relative numbers of acid-fast bacilli were noted after zn staining. bacteriology specimens for mycobacterial culture comprised fresh unpreserved tissue samples from the lung (multiple specimens), liver, spleen, kidneys, ileum and all the lymph nodes listed above. these specimens were placed in individual sterile containers and immediately refrigerated at 2–4 °c. the lymph nodes were processed as indicated above. spec imens were then frozen (–20 °c) and dispatched in a frozen state, within five days of collection, to the tuberculosis laboratory at the ovi for mycobacterial culture, where they were pooled into the following four groups per animal: lungs, other organs (liver, spleen, kidneys and ileum), organ lymph nodes (bronchial, mediastinal, hepatic, splenic, ileocaecal and mesenteric) and carcass lymph nodes [tonsils, parotid (left and right), retropharyngeal (left and right), mandibular (left and right), sternal, iliac, inguinal, prescapular (left and right), prefemoral (left and right) and popliteal (left and right)]. these tissue pools were each cultured for mycobacterium spp. according to the same protocol mentioned above. part three: main study animals in 2002 the springbok numbers had increased to 62 animals and the ithemba labs staff environmental club decided to cull several of them, which afforded the opportunity to sample more animals for tuberculosis investigations. professional hunters were employed to cull 25 animals, consisting of 15 adult ewes and ten adult rams, from april 2002 to july 2002. one ewe showed right hind leg lameness, while the others all appeared clinically healthy and were randomly selected. random selection was achieved by shooting every second animal encountered. the animals were shot in batches of between five and ten animals per day of sampling. euthanasia was performed as described above. animals were identified numerically and chronologically as they were euthanased, and transported to the wcpvl for necropsy and sampling. macroand histopathology similar to that of the pilot study, the aim of the main study was to detect animals with macroscopic and microscopic tuberculosis lesions, and to describe the lesions in detail. the same macroscopic and microscopic criteria for evaluation were therefore applied. tissue specimens were collected for histopatho logical examination and all, except the lymph nodes and specimens for mycobacterial culture, were fixed in 10 % buffered formalin. these included specimens from the brain, eyes, heart (multiple specimens), lung (multiple specimens), liver, kidneys, spleen, adrenal glands, testes, ovaries, uterus, mammary gland, urinary bladder, pancreas and alimentary tract (tongue, oesophagus, rumen, aboma sum, duodenum, jejunum, ileum and colon). spe c i mens from other organs were only collected if macroscopic abnormalities were noted. fresh unpreserved lymph nodes were collected, handled and processed as described above. lymph nodes sampled included the tonsils, parotid (left and right), retropharyngeal (left and right), mandibular (left and right), bronchial, mediastinal, ileocaecal, mesenteric, iliac, inguinal, prescapular (left and right), prefemoral (left and right) and popliteal (left and right) lymph nodes. the formalin-fixed tissues were processed, stained and examined for granulomatous inflammation in a similar fashion to those of the pilot study. bacteriology specimens for mycobacterial culture comprised fresh unpreserved lung (multiple specimens), all the lymph nodes listed above and any other organ that showed suspected tuberculosis lesions. these spec imens were placed in individual sterile containers, immediately refrigerated at 2–4 °c and the lymph nodes processed as for the pilot study. after macroscopic examination of the lymph nodes, they were pooled into four groups as follows: head (tonsils, parotid, retropharyngeal and mandibular), thoracic (bronchial and mediastinal), abdominal (mesenteric and ileocaecal), peripheral (prescapular, prefemoral, popliteal and inguinal). specimens were then frozen (–20 °c) and dispatched in a frozen state, 423 t.a. gous & m.c. williams within five days of collection, to the tuberculosis laboratory at the ovi for mycobacterial culture. at the ovi the lymph node pools, lungs, and other organ specimens were each cultured individually for mycobacterium spp. according to the protocol outlined above. results part one: index case the index case was an adult ewe (a1) and a second possible case an adult ram (a2) macroscopic pathology a1 showed marked emaciation. hard and dry faecal pellets were present in the rectum, resulting in constipation and rectal prolapse. there was depletion of peri-renal and mesenteric fat reserves, and the fat in the coronary groove of the heart and bone marrow cavity was soft, translucent and jelly-like (serous atrophy of fat). these changes reflected marked cachexia. the carcass of a2 showed advanced post mortem autolysis and putrefaction with evidence of post mortem predation. significant lesions were found in the following organs: lung the lungs of a1 showed moderate numbers of multifocal, firm, round to oval, greyish white, nodular granulomatous lesions throughout the lungs that were more numerous in the caudal lobes. the nodules measured 1–10 mm in diameter. some of the granulomatous nodules displayed irregular, multifocal, central, yellowish, caseous necrotic foci measuring 2–5 mm in diameter, with moderate calcification, and were moderately encapsulated. the entire visceral and parietal pleura of a1 was covered in moderate numbers of multifocal, round to ovoid, non-encapsulated, translucent to greyish white plaques and nodules measuring 1–10 mm in diameter. these nodules resembled “tb-grapes” and consisted mostly of fibrous connective tissue showing no to few, multifocal, irregularly shaped, pale yellowish, central caseous necrosis measuring 1–5 mm in diameter, and often prominent calcification. the right lung lobe of a2 was consolidated, and both lobes showed a few round to oval, whitish, firm, nodular areas, measuring 1–10 mm in diameter, which were considered to be granulomas. these lesions, which were suspected to be tuberculosis were more numerous in the cranial lobes. there were moderate (a2) to large (a1) numbers of adult bronchonema magna nematodes present in the lumen of the trachea at the tracheal bifurcation. lymph node the bronchial and mediastinal lymph nodes of a1 were enlarged and measured 30 x 10 mm, and 50 x 10 mm, respectively. both lymph nodes showed multifocal to confluent irregular areas, measuring 1–10 mm in diameter, of pale yellowish caseous necrosis with prominent calcification. the necrotic areas were moderately encapsulated. spleen the spleen of a1 was moderately enlarged and had an increased (“meaty”) consistency. the red pulp was prominent and the white pulp indistinct. liver the liver of a1 showed moderate hepatomegaly with focally disseminated necrosis, the latter being represented by numerous pinpoint, pale white foci distributed throughout the parenchyma. kidney the kidneys of a1 were pale brown and moderately swollen. adrenal gland both adrenal glands of a1 displayed marked enlargement of the cortex as a result of hyperplasia. histopathology the microscopic appearance of the lesions in the organs mentioned above, as well as significant lesions detected in other organs, are described below. the multinucleated giant cells encountered were always a mixed cell population consisting of both the langhans’ and foreign-body types, except when otherwise stated. no acid-fast bacilli were encountered in the organs that did not show significant lesions. lung there were numerous multifocal to confluent necrogranulomas visible throughout the lung parenchyma of a1. these consisted of a central area of caseous necrosis that was usually calcified, surrounded by a 424 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) rim of moderate numbers of macrophages and epithelioid cells, and low numbers of multinucleated giant cells. there was an outer layer of lymphocytes and plasma cells, within a moderately developed fibrous capsule. numerous multifocal smaller, and often indistinctly outlined granulomas, without necro sis and calcification, consisting of macrophages, epithelioid cells, lymphocytes and plasma cells were also visible. these smaller granulomas were often infiltrated by low to moderate numbers of neutrophils. the remainder of the lungs showed multifocal areas of alveolar neutrophil infiltration admixed with moderate to large numbers of macrophages. low to moderate numbers of macrophages were also present in alveolar walls. a few bronchioles in the lung of a1 contained sections of numerous nematode parasites. lesions that were ascribed to this nematode infection consisted of moderate to marked hyperplasia and hypertrophy of bronchiolar epithelium and smooth muscle. the lamina propria of the bronchiolar epithelium was infiltrated by moderate numbers of lymphocytes and plasma cells, and small numbers of eosinophils. moderate numbers to numerous globular leukocytes, with moderate to abundant, variably-sized, round, eosinophilic, cytoplasmic globules, occurred in the intercellular areas of the bronchiolar epithelium. the bronchiolarand perivascular-associated lymphoid tissue was moderately hyperplastic. some bronchioles, notably those parasitized by adult nem atodes, were filled with a moderate amount of exudate consisting of fibrin, neutrophils, eosinophils and small numbers of macrophages. ziehl-neelsen staining of the lungs of a1 revealed numerous medium-sized, slender, acid-fast bacilli in the cytoplasm of macrophages, epithelioid cells and multinucleated giant cells in necrogranulomas along the edge of the necrotic centre. bacilli were also present in moderate numbers in macrophages and epithelioid cells of the smaller granulomas. freelying acid-fast bacilli in brionchioles were admixed with cellular debris, and there were also bacilli in the cytoplasm of intra-brionchiolar macrophages. the lungs of a2 were markedly autolysed and putrefied, with the presence of numerous bacteria many of which morphologically resembled clostridium spp. there were multifocal areas of what appeared to be coagulative and caseous necrosis, separated from the adjacent lung tissue by a prominent reaction zone consisting of haemorrhage, fibrin exudation and possible neutrophils. large numbers of gram-negative cocco-bacilli were present in the necrotic areas and reaction zone. the remainder of the lungs of this animal showed marked haemorrhage, protein-rich alveolar oedema, fibrin exudation and widespread alveolar macrophage infiltration. all sections stained negatively for the presence of acid-fast bacilli with zn staining. the lung lesions in a2 probably reflected necrotic bacterial pneumonia with possible early abscessation. lymph node the mediastinal lymph nodes of a1 displayed large multifocal to confluent necrogranulomas, involving almost the entire lymph node, with only a small rim of relatively normal tissue remaining. these necrogranulomas consisted of a central caseous necrotic area that was usually moderately calcified, with a surrounding layer of low to moderate numbers of macrophages, epithelioid cells and multinucleated giant cells. a few extremely large giant cells, containing numerous centrally located nuclei, were visible. the outer layer consisted of a well-developed fibrous capsule that was infiltrated by moderate numbers of lymphocytes and plasma cells, and low to moderate numbers of haemosiderin-containing macrophages. a few smaller granulomas, consisting of macrophages and epithelioid cells, with peripheral lymphocyte and plasma cell infiltration, were also visible. the relatively normal lymph node tissue showed numerous multifocal nests, consisting of macrophages, epithelioid cells and multinucleated giant cells. the bronchial lymph nodes of a1 showed less extensive but otherwise similar lesions to the above. additionally, there were mutifocal areas of coagulative necrosis in the relatively normal lymph node tissue that were infiltrated by moderate to large numbers of neutrophils. the medullas were infiltrated by large numbers of macrophages, many of which showed erythrophagocytosis. in the mediastinal and bronchial lymph nodes of a1 large numbers of medium-sized, slender, acid-fast bacilli were present along the edges of the necrotic caseous exudate. the bacilli often formed thick clumps. acid-fast bacilli were also present in large numbers in the cytoplasm of macrophages, epithelioid cells and multinucleated giant cells surrounding the necrotic centre, as well as in the multifocal nests formed by these cells. the respiratory lymph nodes of a2 showed similar advanced post mortem changes to the lungs. there was a focal area of suspected coagulative necrosis in the cortex, with accumulation of fibrin and necrotic cellular debris. ziehl-neelsen staining was negative for acid-fast bacilli in this animal. 425 t.a. gous & m.c. williams spleen the spleen of a1 showed marked multifocal to confluent necrotic to necrogranulomatous splenitis. this was represented by numerous foci of coagulative to caseous necrosis of macrophage aggregates in the red pulp. the necrotic areas were infiltrated by moderate numbers of neutrophils. moderate numbers of macrophages, and low numbers of multinucleated giant cells, were also present throughout the red pulp. there was moderate extramedullary haematopoiesis. medium-sized, slender, acid-fast bacilli were numerous in the cytoplasm of most macrophages. liver the liver of a1 displayed marked focally disseminated coagulative necrosis of hepatocytes, with mild to moderate neutrophil and macrophage infiltration. the sinusoids and portal tracts were infiltrated by low to moderate numbers of macrophages, and low numbers of multinucleated giant cells, that often formed small microgranulomas. moderate numbers of macrophages occurred in the portal venules and lymphatics, as well as in the central veins. there was a focal area of capsular fibroplasia and mature fibrosis. in sections stained with zn there were numerous medium-sized, slender, acid-fast bacilli in necrot ic areas, and low to moderate numbers in the cytoplasm of macrophages of both the macrophages occurring throughout the liver, as well as those inside portal venules and lymphatics, and central veins. kidney numerous multifocal necrogranulomas occurred in the cortex and cortico-medullary junction in the kidney of a1. these microgranulomas consisted of low to moderate numbers of macrophages in their centres, some of the cells being necrotic and surrounded by moderate to large numbers of lymphocytes and plasma cells. in zn-stained sections large numbers of medium-sized, slender, acid-fast bacilli were found in the cytoplasm of macrophages in the microgranulomas, but were also noted in scattered individual macrophages. a few such bacilli were also present in the cytoplasm of mesangial cells in a few renal corpuscles. adrenal gland the adrenal gland of a1 showed marked hyperplasia of the zona fasciculata with marked accumulation of clear, single to multiple, variably-sized, round vacuoles in the cytoplasm of the cells, representing lipid droplets. scattered cells in this zone were necrotic. there was scanty macrophage infiltration in the sinusoids of all zones of the cortex. zn staining revealed moderate numbers of medium-sized, slender, acid-fast bacilli, often forming clumps, in the cytoplasm of macrophages and scattered parenchymal cells of all zones of the cortex. gastro-intestinal tract the small intestine, colon and rectum of a1 showed scanty to low numbers of multifocal nests of macrophages in the lamina propria of the mucosa. these organs, as well as the abomasum, also had small numbers of widely scattered individual macrophages in the same location. the lamina propria of the mucosa of the small intestine was also infiltrated by moderate numbers of lymphocytes and plasma cells. scanty nematode larvae were present in gastric glands of the mucosa of the abomasum, without an inflammatory reaction. ziehl-neelsen staining of the abomasum, small intestine, colon and rectum revealed low to moderate numbers of mediumsized, slender, acid-fast bacilli in the cytoplasm of macrophages located in the mucosa. bacteriology mycobacterium tuberculosis was isolated from a mediastinal lymph node of a1. the lung specimens from a2 were negative for mycobacterium spp. after 10 weeks’ incubation. part two: pilot study three adult (b3, b5 and b6) and three sub-adult (b1, b2 and b4) rams were sampled. macroscopic pathology all the animals were in good condition, except for b4 which showed moderate emaciation. b2 and b6 did not show any noteworthy macroscopic lesions. significant lesions were found in the following organs of the other animals: lung the lungs of b4 showed lesions highly suggestive of tuberculosis. there were four large and fairly distinctly demarcated, multifocal, granulomatous, nodular lesions. one which was situated in the cranial edge of the left cranial lobe, was irregularly ovoid and 20 x 10 mm in size, yellowish, granulomatous and non-encapsulated. two granulomas that occurred in the dorso-caudal area of the left and right 426 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) caudal lobe respectively, were similar in appearance. they were round and 20 mm in diameter, and had an indistinct arborescent central area of yellowish caseous necrosis containing numerous individual necrotic foci measuring 1–2 mm in diameter. the individual necrotic foci and entire nodules were surrounded by mildly developed fibrous connective tissue. the fourth granuloma occurred in the craniomedial aspect of the right caudal lobe and was round, 30 mm in diameter with a central, pale yellow, caseous necrotic area surrounded by a welldeveloped fibrous capsule. apart from these large nodular granulomas, there were numerous non-encapsulated, multifocal to confluent, translucent to greyish white, round to irregular granulomas measuring 1–5 mm in diameter distributed throughout the lung parenchyma. the entire visceral and parietal pleura was cover ed in numerous, multifocal to confluent, round to ovoid, translucent to pale whitish, non-encapsulated plaques and nodules, measuring 1–10 mm in diameter. these nodules resembled “tb-grapes” and consisted mostly of fibrous connective tissue showing no or mild multifocal areas of pale yellowish caseous necrosis measuring 1–5 mm in diameter on cut section. b5 displayed moderate numbers of adult b. magna nematodes in the lumen of the trachea and both bronchi, especially at the tracheal bifurcation. liver the livers of b1, b3 and b4 showed scanty to low numbers of multifocal, round, distinctly outlined, pinpoint to 3 mm diameter, pale white foci randomly distributed throughout the parenchyma. lymph nodes the following lymph nodes of b4 showed macroscopic lesions highly suggestive of tuberculosis: • bronchial: multifocal to coalescing, non-encapsulated, round to ovoid, pale yellowish, caseous necrotic to granulomatous areas measuring 1– 15 mm in diameter, with mild to moderate calcification. • mediastinal: multifocal to coalescing, non-encapsulated, ovoid to irregular, granulomatous areas measuring 2–18 mm in diameter. there was a small focal, round, 1 mm-diameter area of dull yellowish necrosis. • sternal: multifocal, non-encapsulated, round, white to slightly yellow, necrotic areas of up to 1 mm in diameter, and a focal, round, non-encapsulated area of granulomatous inflammation measuring 4 mm in diameter. • iliac: multifocal, encapsulated, round to oval, pale yellowish, caseous necrotic foci measuring 3– 7 mm in diameter, with moderate calcification. • right prefemoral: focal, non-encapsulated, round area of yellowish caseous necrosis measuring 3 mm in diameter, with moderate calcification. • right retropharyngeal: focal, non-encapsulated, round area of slightly yellow-tinged necrosis measuring 1 mm in diameter. histopathology the microscopical lesions in the organs mentioned above, as well as significant lesions detected in other organs, are described below. the multinucleated giant cells encountered were always a mixed cell population consisting of both the langhans’ and foreign-body types, except when otherwise stated. all lesions in all organs were negative for acid-fast bacilli, except where otherwise stated. lung the lungs of b4 showed numerous multifocal to confluent necrogranulomas throughout the parenchyma. the necrogranulomas consisted of a central area of caseous necrosis that was often moderately calcified, surrounded by large numbers of macrophages and epithelioid cells, and low to moderate numbers of multinucleated giant cells. enveloping this layer was an outer layer consisting of low to moderate numbers of lymphocytes and plasma cells, within a moderately developed fibrous capsule. some granulomas did not show central necrosis. in a few necrogranulomas there was infiltration of moderate numbers of neutrophils into the necrotic centre with occasional pus formation. the surrounding bronchioles were plugged with necrotic cellular debris consisting of neutrophils, macrophages and respiratory epithelial cells. the pleura showed a proliferative response characterized by prominent fibroplasia with moderate numbers of multifocal to confluent granulomas in some of which were areas of coagulative to caseous necrosis in their centres. the granulomas consisted of large numbers of macrophages and epithelioid cells, and moderate numbers of lymphocytes and plasma cells. scanty to low numbers of neutrophils infiltrated the granulomas. the mesothelium showed moderate hypertrophy and hyperplasia. 427 t.a. gous & m.c. williams ziehl-neelsen staining of the lungs and pleura revealed small numbers of medium-sized, slender acid-fast bacilli in the cytoplasm of macrophages, epithelioid cells and multinucleated giant cells of the granulomas. moderate numbers of acid-fast bacilli were present along the peripheral edge of the necrotic exudate in some necrogranulomas. the lungs of b5 displayed small numbers of sections of adult nematodes in some bronchioles. there was mild to moderate epithelial and smooth muscle hypertrophy and hyperplasia of bronchioles, with infiltration of small numbers of eosinophils, lymphocytes and plasma cells in the lamina propria of the bronchiolar epithelium, and occasionally perivascularly. small numbers of globular leukocytes, with moderate to abundant, variably-sized, round, eosinophilic, cytoplasmic globules, occurred in the intercellular areas of the bronchiolar epithelium. liver the multifocal pale white foci in the livers of b1, b3 and b4 seen macroscopically consisted of nodular portal aggregations of moderate to large numbers of lymphocytes and plasma cells; scanty eosinophils and macrophages were found in them only in b4. the liver of b5 showed a focal lesion similar to those of b4 but with only scanty eosinophil infiltration but no macrophages. a small focal granuloma occurred in the parenchyma of b5, which consisted of central caseous necrosis surrounded by small numbers of macrophages, epithelioid cells and eosinophils. all animals showed infiltration of scanty to low numbers of lymphocytes, plasma cells and eosinophils in the portal tracts, without the formation of distinct nodules. the liver lesions were possibly a response to a previous parasitic larval migration. lymph nodes the lymph nodes of b4 that had macroscopic lesions showed the following changes: • bronchial: there were multifocal to confluent necrogranulomas consisting of a central caseous necrotic area that was moderately calcified, with a surrounding layer of moderate numbers of macrophages, epithelioid cells and scanty multinucleated giant cells, admixed with moderate numbers of lymph o cytes and plasma cells. there was a poorly to moderately developed outer fibrous capsule. numerous small multifocal granulomas, that did not show central necrosis and calcification, occurred in the remainder of the lymph node. these granulomas consisted of small numbers of macrophages, epi thelioid cells and numerous (most ly langhans’) giant cells. ziehl-neelsen staining revealed small numbers of medium-sized, slender, acid-fast bacilli in the peripheral edge of the necrotic centre of ne c rogranulomas, and also in the cytoplasm of macrophages surrounding the necrotic centre. • mediastinal: multifocal to coalescing granulomatous lymphadenitis, represented by numerous small granulomas composed of small numbers of macrophages and epithelioid cells, and numerous, mostly langhans’, giant cells. one granuloma showed mild central calcification. very rare medium-sized, slender, acid-fast bacilli were present in the cytoplasm of epithelioid cells and multinucleated giant cells following zn staining. • sternal: there was a focal granuloma that displayed central caseous necrosis with moderate calcification. this was surrounded by moderate numbers of macrophages and epithelioid cells, and scanty multinucleated giant cells, admixed with moderate numbers of lymphocytes and plasma cells. there was a poorly-developed fibrous capsule. numerous small multifocal granulomas, similar in appearance to the small granulomas in the bronchial lymph node, occurred in the remainder of the lymph node. ziehl-neelsen staining revealed rare medium-sized, slender, acid-fast bacilli in the peripheral edge of the necrotic centre of the necrogranuloma, and also in the cytoplasm of macrophages that occurred in the small granulomas. • iliac: there were multifocal necrogranulomas, similar in appearance to the necrogranulomas in the bronchial lymph node, but with more numerous multinucleated giant cells and a well-developed fibrous capsule. a few small granulomas, consisting of low numbers of macrophages and epithelioid cells, occurred in the remainder of the lymph node. the pattern of zn staining was similar to that in the bronchial lymph node. • right prefemoral: there were multifocal necrogranulomas, similar in appearance to the necrogranulomas in the bronchial lymph node, but with large numbers of multinucleated giant cells. the necrogranulomas were surrounded by a few satellite granulomas, some showing central caseous necrosis, composed of moderate numbers of macrophages, epithelioid cells, multinucleated giant cells, lymphocytes and plasma cells. most showed a moderately developed outer fibrous capsule. the pattern of zn staining was similar to that in the bronchial lymph node. 428 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) • right retropharyngeal: there was a focal granuloma that consisted of central caseous necrosis with early calcification, surrounded by numerous macrophages and epithelioid cells, admixed with small numbers of lymphocytes, plasma cells and neutrophils. the macrophages and epithelioid cells exhibited occasional round, variably-sized, single to multiple, clear cytoplasmic vacuoles resembling fat gobules. the granuloma had an outer layer of poorly developed fibrous connective tissue. the remainder of the lymph node showed numerous scattered macrophages, epithelioid cells and multinucleated giant cells that were occasionally arranged in granulomas. ziehl-neelsen staining revealed very rare medium-sized, slender, acid-fast bacilli in the peripheral edge of the necrotic centre of necrogranulomas, and also in the cytoplasm of macrophages surrounding the necrotic centre. the microscopic lesions in lymph nodes of b4 that did not exhibit macroscopic abnormalities were as follows: • left retropharyngeal: there was one poorly-developed granuloma in the cortex that consisted of small numbers of macrophages and epithelioid cells, and scanty multinucleated giant cells. scanty individual langhans’ giant cells occurred in the cortex and paracortex. • left mandibular, right mandibular and left prescapular: there was moderate sinus histiocytosis with a few nests composed of small to moderate numbers of macrophages in the cortex. • left prefemoral: a focal granuloma occurred in the cortex and consisted of a central necrotic area that was completely calcified, surrounded by moderate numbers of macrophages and epithelioid cells, numerous multinucleated giant cells and small numbers of lymphocytes, plasma cells, neutrophils and eosinophils. there was a poorly-developed fibrous capsule. very scanty mediumsized, slender, acid-fast bacilli were present in the cytoplasm of macrophages after zn staining. • mesenteric: there were moderate numbers of small granulomas consisting of moderate numbers of langhans’ giant cells, and smaller numbers of macrophages and epithelioid cells. scanty individual multinucleated giant cells were also visible. • ileocaecal: a few small granulomas consisting almost exclusively of multinucleated giant cells occurred in the cortex. one multinucleated giant cell displayed characteristics of both langhans’ and foreign-body giant cells, i.e. with nuclei arranged both in a row along the periphery of the cell, and as a group centrally. • inguinal: there was a single small granuloma in the cortex which consisted of a few macrophages. two large individual multinucleated giant cells (one each of the langhans and foreign-body type) occurred in the cortex. there was a nest of short, acid-fast bacilli in the cytoplasm of a macrophage in the medulla of a mesenteric lymph node of b2, unaccompanied by an inflammatory reaction. the lymph node also showed small multifocal areas of calcification in the cortex and medulla, and marked sinus histiocytosis. lesions in the mesenteric lymph nodes of b1 and b5, and the ileocaecal lymph node of b1 reflected nematode larval migration. b1 showed two granulomas in the ileocaecal lymph node, one each in the cortex and capsule, and one cortical granuloma in the mesenteric lymph node. the cortical ileocacecal granuloma displayed remnants of a nematode larva that was surrounded by numerous multinucleated giant cells, and small numbers of macrophages and epithelioid cells. the capsular granuloma displayed a similar reaction, but without nematode larval remnants visible. the granuloma in the mesenteric lymph node of b1 showed mild central coagulative necrosis, surrounded by small numbers of macrophages and epithelioid cells. b5 showed a focal granuloma in the cortex of a mesenteric lymph node that resembled the cortical ileocaecal granuloma of b1. diaphragm the parietal pleura covering the diaphragm of b4 displayed lesions similar to those in the lung pleura, but with the presence of mild central calcification of necrogranulomas. ziehl-neelsen staining was positive for scanty medium-sized, slender, acid-fast bacilli in the cytoplasm of a few epithelioid cells in necrogranulomas. testes the testes of b4 showed infiltration of moderate numbers of lymphocytes and plasma cells, and scanty to small numbers of macrophages in the interstitium. there were scanty microgranulomas, consisting of macrophage nests surrounded by small numbers of lymphocytes and plasma cells. gastro-intestinal tract the colon of b4 displayed four granulomas, one in the submucosa, two in the muscle layer and one in 429 t.a. gous & m.c. williams the serosa. the submucosal granuloma showed a central nematode larva within necrotic tissue that was infiltrated by scanty eosinophils, and surrounded by numerous multinucleated giant cells and smaller numbers of macrophages, epithelioid cells, lymphocytes and plasma cells. the other three granulomas were similar in appearance, but without the central nematode larva, necrosis and eosinophils, and only scanty multinucleated giant cells. there was a focal granuloma in the submucosa of b5 that consisted of a few central multinucleated giant cells surrounded by moderate numbers of macrophages, epithelioid cells, lymphocytes, plasma cells and eosinophils. the colon of all six animals showed scanty to small numbers of coccidia, in various stages of their lifecycle, in the epithelium of the mucosa of the colon, without an inflammatory reaction. there was a single nematode larva visible in a mucosal gastric gland of the abomasum in b4, without a noticeable inflammatory reaction. bacteriology mycobacterium tuberculosis was isolated from all organ pools of b4, and the organ lymph node pool of b6. mycobacteria other than tuberculosis (mott) were isolated from the lungs, organ lymph nodes and carcass lymph nodes of b2, and organ lymph nodes and organs other than the lungs of b3. part three: main study fifteen adult ewes (c1, c2, c3, c4, c5, c6, c7, c8, c10, c11, c12, c13, c14, c19 and c22) and 10 adult rams (c9, c15, c16, c17, c18, c20, c21, c23, c24 and c25) were sampled. macroscopic pathology all the animals were in good condition and only c1 showed clinical signs consisting of right hind leg lameness before euthanasia. ewes c3, c11, c12, c13 and c22 were in the early stages of pregnancy, while ewes c2 and c6 were in an advanced stage of pregnancy. c4, c7 and c8 did not show any noteworthy macroscopic lesions. significant lesions were found in the following organs of the other animals: kidney in the right kidney of c1 was a lesion highly suggestive for tuberculosis. in the cortex there was a focal, round, fairly distinctly demarcated, 8 mm diameter, dull-white, nodular, granulomatous lesion that bulged slightly from the surface. the nodule showed central caseation and was encapsulated by a fairly well-developed fibrous capsule. part of this nodule was pooled with specimens from the uterus of c1 (see below) and submitted for mycobacterial culture. liver there were scanty to small numbers of round, distinctly outlined, pinpoint to 3 mm diameter, pale white foci randomly distributed throughout the parenchyma in the livers of c3, c9, c10, c11, c12, c13, c14, c15, c16, c17, c18, c19, c20, c21, c22, c23, c24 and c25. lymph node both prescapular lymph nodes and the right mandibular lymph node of c1 were enlarged and showed significant proliferative changes consisting of multifocal to confluent, indistinctly outlined, oval to round nodules measuring 4 mm (right prescapular lymph node) and 15 mm (left prescapular and right mandibular lymph nodes) in diameter throughout the node. these nodules were moderately firmer in consistency than the rest of the lymph node. the right parotid and right retropharyngeal lymph nodes of c9 were enlarged and both showed marked focal, proliferative, indistinctly outlined, oval nodules measuring 23 x 10 mm (right parotid lymph node) and 15 x 10 mm (right retropharyngeal lymph node) in the cortex. these nodules were firmer in consistency than the rest of the lymph node. the right parotid lymph node showed poor distinction between the medulla and cortex. all of the above lymph node changes probably reflected marked reactive follicular hyperplasia, but early tuberculous granulomatous inflammation could not be ruled out macroscopically. skeletal muscle the gastrocnemius muscles of c20 showed bilaterally symmetrical, focal, 5 x 20 mm accumulations of a gritty, granular, pale white material, resembling calcified tissue, underneath the outer muscle sheath. uterus the endometrium of c1 showed multifocal petechiae. there were well-developed caruncules that showed multifocal petechiae and ecchymoses, and multifocal dull yellow-white areas of necrosis meas430 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) uring from pinpoint to 3 mm in diameter. the caruncules were covered with a moderate amount of purulent exudate. there were multifocal petechiae and ecchymoses measuring 3–5 mm in diameter in the cervical mucosa. several specimens of affected tissue from the uterus and cervix were pooled with a specimen from the kidney (see above) of c1 and submitted for mycobacterial culture. the uterus of c5 was congested and displayed similar haemorrhagic lesions to those seen in c1. however, no areas of necrosis or purulent inflammation were visible. lung the following animals had moderate numbers of adult b. magna nematodes present in the lumen of the trachea and both bronchi, notably at the tracheal bifurcation: c2, c5, c10, c24 and c25. abdominal cavity several mature unidentified cestode larvae that mor phologically resembled taenia hydatigena cysticerci were visible in the abdominal cavity of c3 and c1. they were attached to the liver (c3) and abomasum (c1), or were lying loose and un attached (c1). three cestode larvae of c1 that were attached to the abomasum, and one that was lying loose, were firm, round to irregularly-shaped nodules consisting of a calcified and inspissated centre, surrounded by a fairly well-developed fibrous capsule. the three nodules attached to the abomasum measured 15 mm in diameter while the loose nodule measured 30 mm in diameter. eyelid in the right ventral eyelid of c9 was a focal purulent sinus tract that extended from the surface for 20 mm into the eyelid and was 5 mm wide. there was extensive purulent inflammation of the eyelid and surrounding subcutaneous tissues. this lesion may have been a traumatic injury and probably resulted in the hyperplastic lesions observed in the right parotid and right retropharyngeal lymph nodes (see above) of this animal. histopathology the microscopical lesions of the organs mentioned above, except for the eyelid, as well as significant lesions detected in other organs are described below. the multinucleated giant cells encountered were always a mixed cell population consisting of both the langhans’ and foreign-body types. all lesions in all organs were negative for acid-fast bacilli with zn staining. kidney the focal lesion in the right kidney of c1 consisted of a central area of caseous necrosis that contained necrotic cellular debris, mostly necrotic neutrophils, and showed early calcification. this was surrounded by moderate numbers of macrophages and epithelioid cells, and numerous lymphocytes and plasma cells. there was a fairly well-developed outer layer consisting of fibrous connective tissue. liver the macroscopic multifocal pale white foci in the liver consisted of nodular aggregations of a mixture of moderate to large numbers of lymphocytes, plasma cells and eosinophils in portal tracts, and occasionally in the parenchyma. c25 had a small focal granuloma in a portal tract consisting of a central area of coagulative necrosis surrounded by small numbers of macrophages and epithelioid cells. in one animal (c1) there was a small focal empty cyst in the parenchyma that was surrounded by a thin layer of degenerative and necrotic hepatocytes, with haemorrhage and infiltration of moderate numbers of neutrophils and eosinophils. the cyst was probably parasitic in nature and had a thin outer wall, with an inner adherent single row of spindleloid cells projecting into the cyst cavity. all animals, except c9 and c20, showed infiltration of scanty to moderate numbers of lymphocytes, plasma cells and eosinophils in the portal tracts, without the formation of distinct nodules. the lesions present in the liver were probably in response to previous parasitic larval migration. the liver of c4 displayed multifocal areas, consisting of one to multiple open fusiform slits or clefts throughout the parenchyma, and also involving portal tracts. the cleft aggregates were surrounded by low to moderate numbers of macrophages, epithelioid cells and fibroblasts, with occasional multinucleated giant cells and small numbers of lymph ocytes, plasma cells and eosinophils. the cause of the clefts could not be established but they are unlikely to have been related to tuberculosis. lymph node the prescapular lymph nodes and right mandibular lymph node of c1, and the right parotid and right retropharyngeal lymph nodes of c9 displayed typical reactive hyperplastic changes, without evidence 431 t.a. gous & m.c. williams of possible tuberculosis. these changes were marked and consisted of numerous cortical secondary reactive follicles with prominent germinal centres, necrosis of individual lymphocytes in follicles with increased tingible-body macrophage activity, a prominent hyperplastic paracortical zone and sinus histiocytosis. the following lymph nodes showed focal to multifocal microscopic granulomatous lymphadenitis: mesenteric (c1, c2, c9, c10, c11, c12, c13, c14, c16, c17, c18, c20, c21, c22, c23, c24 and c25), ileocaecal (c1, c8, c12, c16, c19, c24 and c25), bronchial (c1 and c7) and inguinal (c20). the lesions were characterized by variable numbers (usually single) of centrally-located nematode larvae, or their necrotic remnants, in the following animals: mesenteric lymph nodes of c1, c2, c9, c11, c12, c17 and c25; ileocaecal lymph nodes of c1, c8, c12, c19 and c24; bronchial lymph nodes of c1 and c7; and the inguinal lymph nodes of c20. the nematode larvae were occasionally surrounded by low to moderate numbers of eosinophils that often displayed degenerative and necrotic changes. granulomas without central nematode larvae occasionally showed central caseous necrosis. all animals showed infiltration of variable numbers of a mixture of macrophages, epithelioid cells, multinucleated giant cells, lymphocytes, plasma cells and eosinophils around the central parasite or necrotic area. the bronchial and mediastinal lymph nodes of c22 showed accumulations of moderate to large numbers of macrophages, containing cytoplasmic foreign particulate matter, in the cortex and medulla. the macrophages were often arranged in nests. tonsils one of the tonsils of c2 displayed a focal granuloma that consisted of a central area of caseous necrosis surrounded by moderate numbers of macrophages. moderate numbers of small plant fibres were present in the crypts and it is likely that the granuloma was a foreign-body reaction. skeletal muscle c20 displayed multifocal, well-defined and variablysized lakes of calcification in muscle and the surrounding connective tissue that were rimmed and separated form each other by moderetaly-developed fibrous connective tissue. the connective tissue was infiltrated by moderate to large numbers of lymphocytes and plasma cells, and small to moderate numbers of macrophages. the lesion resembled calcinosis circumscripta. uterus the lesions in the uterus of c1 were marked and consisted of multifocal haemorrhages and coagulative necrosis of the endometrium. the necro-haemorrhagic exudate contained large numbers of bacterial rods morphologically resembling clostridium spp. the remainder of the endometrium showed infiltration of large numbers of neutrophils, and there was moderate early granulation tissue formation consisting of angiogenesis, macrophage infiltration and fibroplasia. a few endometrial blood vessels contained fibrin thrombi. the lesions probably represented post-partum involution with bacterial endometritis. the uterus of c5 showed marked vascular congestion. the endometrium was infiltrated by numerous macrophages that contained abundant cytoplasmic haemosiderin pigment. there were a few multinucleated giant cells and early granulation tissue formation similar to c1. small numbers of lymphocytes and plasma cells were also present. the lesions probably reflected post-partum involution. lung the following animals showed moderate, multifocal, parasitic, eosinophilic and granulomatous bronchopneumonia with moderate numbers of adult nematodes visible in brionchioles, as well as moderate numbers of larvae present in alveoli: c1, c2, c6 and c8. the lesions consisted of moderate to marked hyperplasia and hypertrophy of bronchiolar epithelium and smooth muscle. there was occasional mild bronchiolar epithelial squamous metaplasia in bronchioles plugged with adult parasites. a few bronchioles were ruptured, resulting in free-lying adult nematodes in the parenchyma that were surrounded by moderate to large numbers of macrophages and multinucleated giant cells, and moderate numbers of lymphocytes, plasma cells and eosinophils. there was widespread infiltration of small to moderate numbers of lymphocytes, plasma cells and eosinophils in the lamina propria of the bronchiolar epithelium, and often perivascularly. moderate numbers to numerous globular leukocytes, with moderate to abundant, variably-sized, round, eosinophilic, cytoplasmic globules occurred in the intercellular areas of the bronchiolar epithelium. the reaction to the larvae in the alveoli consisted of infiltration of moderate to large numbers of eosinophils, macrophages, multinucleated giant 432 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) cells, lymphocytes and plasma cells, with mild to moderate epithelialization of pneumocytes and mild fibrosis. similar lesions were present in c5, c7, c9, c16 and c17, with c22 and c25 showing very mild changes, but no nematode larvae could be found in the alveoli of any of these seven animals. the following animals showed mild lesions suggestive for nematode infestation, but no parasites were visible: c3, c4, c10, c11, c12, c13, c14, c15, c18, c19, c20, c21, c23 and c24. the lesions in these animals were generally confined to the bronchioles, and consisted of mild to moderate epithelial and smooth muscle hypertrophy and hyperplasia, with infiltration of small numbers of eosinophils, lymphocytes and plasma cells in the lamina propria of the bronchiolar epithelium, and occasionally perivascularly. low to moderate numbers of globular leukocytes, with moderate to abundant, variablysized, round, eosinophilic, cytoplasmic globules often occurred in the intercellular areas of the bron chiolar epithelium. abdominal cavity the parasitic nodules found in the abdominal cavity of c1 had a central cavity that was filled with caseous necrotic material that was markedly calcified. the calcified necrotic centre was surrounded by a thin layer of macrophages, multinucleated giant cells, lymphocytes and plasma cells. there was a well-developed fibrous connective tissue capsule. gastro-intestinal tract apart from the nematodes causing lesions in the liver, lymph nodes and lungs, several gastro-intestinal tract organs were also infested with parasites. the following animals had scanty to small numbers of nematode larvae present within gastric glands in the abomasum, usually with a negligible to mild lymphocyte, plasma cell and eosinophil inflammatory reaction: c1, c2, c5, c6, c7, c10 and c25. the ileum of c13 and the jejunum of c25 each had a focal parasitic granuloma in the submucosa and muscle layer, respectively. both granulomas consisted of a central nematode larva surrounded by small to moderate numbers of macrophages, epithelioid cells, multinucleated giant cells (only c13), lympho cytes and plasma cells. a single nematode parasite was found in the lumen of the jejunum of c24. the following animals showed scanty to small numbers of coccidia, in various stages of their lifecycle, in the epithelium of the mucosa of the colon, with a minimal inflammatory reaction: c4, c7, c9, c15, c16, c17, c18, c20, c21 and c24. bacteriology mycobacterial culture was performed on the organ pools mentioned above, as well as on an additional pool for c1 that consisted of specimens from the kidney and uterus. all pools of all animals were negative for mycobacterium spp. after 10 weeks’ incubation. summary of results table 1 is a summary of the macroscopical, microscopical and mycobacterial culture results of all 33 springbok. mycobacterium tuberculosis was confirmed via culture in only three springbok (a1, b4 and b6), indicating a prevalence of 9 %. two (a1 and b4) of the three culture-positive animals displayed tuberculosis lesions, while b6 did not show any significant macroscopic or microscopic pathology. mycobacteria other than tuberculosis (mott) were isolated from two animals (b2 and b3). b2 was positive for one nest of acid-fast bacilli in one macrophage of a mesenteric lymph node, but no lesions were present. table 2 indicates the presence of macroscopic and microscopic lesions, presence of acid-fast bacilli, and organs that did not show lesions for all organs sampled in both animals with tuberculosis lesions. b4 had prominent macroscopic tuberculosis lesions in the lungs, pleura and bronchial, mediastinal, sternal and iliac lymph nodes. the right prefemoral and right retropharyngeal lymph nodes showed small macroscopic lesions. microscopic tuberculosis lesions with acid-fast bacilli were present in all the above organs, as well as in the left prefemoral lymph node. the following organs of b4 did show microscopic but not macroscopic lesions that were typical or highly suggestive for tuberculosis, although no acid-fast bacilli were encountered in them: testis, colon and the left retropharyngeal, mesenteric, ileocaecal, inguinal, left mandubular, right mandibular and left prescapular lymph nodes. in the other affected springbok (a1) marked macroscopic tuberculosis lesions in the lungs, pleura, liver and bronchialand mediastinal lymph nodes were present. because this animal was presented for a routine diagnostic necropsy, only a limited set of organs were sampled for microscopy. the only lymph nodes sampled for microscopy in this animal were the bronchial and mediastinal lymph nodes. microscopic tuberculosis lesions with acid-fast bacilli were present in all the organs of a1 that showed macro433 t.a. gous & m.c. williams scopic lesions, as well as the following organs: kidney, adrenal gland, abomasum, small intestine, colon and rectum. the heart and oesophagus were also examined, but no macroscopic or microscopic tuberculosis lesions were noted. macroscopic lesions encountered ranged from encapsulated (lungs of both animals, pulmonary lymph nodes of a1 and an iliac lymph node of b4) to nonencapsulated (pleura of both and mediastinal lymph node of b4). macroscopic granulomas with or without obvious caseous necrosis were prominent in the lungs of both, while the iliac lymph node of b4 showed only necrotic foci. the lesions in the pleura of both animals were nodular, plaque-like and proliferative to almost sarcomatous. the liver of a1 showed marked focally disseminate necrosis, which proved to be small necrogranulomas microscopically. the degree of calcification varied from absent (liver of a1) to moderate (lungs and bronchial lymph table 1 summary of results springbok macroscopy (granulomatous lesions) microscopy (granulomatous lesions positive for acid-fast bacilli) mycobacterial culture a1 +a + + a2 sb –c – b1 – – – b2 – – mottd b3 – – mott b4 + + + b5 – – – b6 – – + c1 s – – c2 – – – c3 – – – c4 – – – c5 – – – c6 – – – c7 – – – c8 – – – c9 s – – c10 – – – c11 – – – c12 – – – c13 – – – c14 – – – c15 – – – c16 – – – c17 – – – c18 – – – c19 – – – c20 – – – c21 – – – c22 – – – c23 – – – c24 – – – c25 – – – a positive b suspect c negative d mycobacteria other than tuberculosis 434 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) nodes of both). the size of lesions varied from single pinpoint lesions (right retropharyngeal of b4), to miliary (lungs of both), to multifocal (sternal lymph node of b4 and lungs of both). the microscopic lesions varied in terms of severity, relative numbers of inflammatory cells, degree and type of necrosis, and abundance of acid-fast bacilli. lesions were marked in organs such as the lungs, table 2 comparison of organs showing macroscopic and microscopic tuberculosis lesions, acid-fast bacilli and no lesions in the two m. tuberculosis culture-positive springbok (a1 and b4) organ macroscopic lesions microscopic lesions acid-fast bacilli no lesions a1 b4 a1 b4 a1 b4 a1 b4 lung +a + + + + + –b – pleura + + + + ndc + – – bronchial ln. + + + + + + – – mediastinal ln. + + + + + + – – sternal ln. nsd + ns + ns + ns – iliac ln. ns + ns + ns + ns – r-prefemoral ln. ns + ns + ns + ns – l-prefemoral ln. ns – ns + ns + ns – r-retropharyngeal ln. ns + ns + ns + ns – l-retropharyngeal ln. ns – ns + ns – ns – r-mandibular ln. ns – ns + ns – ns – l-mandibular ln. ns – ns + ns – ns – l-prescapular ln. ns – ns + ns – ns – mesenteric ln. ns – ns + ns – ns – ileocaecal ln. ns – ns + ns – ns – inguinal ln. ns – ns + ns – ns – spleen – – + – + – – + liver + – + – + – – + kidney – – + – + – – + adrenal gland – – + – + – – + testicle ns – ns + ns – ns – abomasum – – + – + – – + small intestine – – + – + – – + colon – – + + + – – – rectum – ns + ns + ns – ns oesophagus – – – – – – + + heart – – – – – – + + brain ns – ns – ns – ns + eyes ns – ns – ns – ns + thymus ns – ns – ns – ns + tongue ns – ns – ns – ns + tonsils ns – ns – ns – ns + r-parotid ln. ns – ns – ns – ns + l-parotid ln. ns – ns – ns – ns + hepatic ln. ns – ns – ns – ns + splenic ln. ns – ns – ns – ns + r-prescapular ns – ns – ns – ns + r-popliteal ns – ns – ns – ns + l-popliteal ns – ns – ns – ns + a present b absent c no microscopy done d not sampled 435 t.a. gous & m.c. williams pleura and bronchial lymph nodes of both, and liver, spleen and kidney of a1. mild lesions consisting of individual macrophages or only a few nests of macrophages were present in the adrenal gland, abomasum, small intestine, colon and rectum of a1, and the testes, left and right mandibular lymph nodes, and left prescapular lymph node of b4. marked granulomatous inflammation, with large numbers macrophages, epithelioid cells and multinucleated giant cells, was present in the lungs and bronchial lymph nodes of both animals, the mediastinal lymph node of a1, and the sternal, iliac, right prefemoral, left prefemoral and right retropharyngeal lymph nodes of b4. the pleura of b4 displayed prominent fibroplasia. giant cells were of both the langhans’ and foreign-body type, but langhans’ giant cells predominanted in the bronchial and mediastinal lymph nodes of b4. some granulomas showed central caseous necrosis (lungs and bronchial lymph nodes of both), while necrosis was absent in others, often in the same organ (lungs and bronchial lymph nodes of both, left retropharyngeal and mesenteric lymph nodes of b4). the liver, spleen and kidney of a1 showed prominent multifocal coagulative necro sis of granulomas that were composed of moderate num bers of macrophages, and small numbers epithelioid cells and multinucleated giant cells. the mesenteric and ileocaecal lymph nodes of b4 did not show macroscopic lesions, but prominent microscopic granulomas consisting of large numbers of multinucleated giant cells were present. the giant cells were mostly of the langhans’ type in the case of the mesenteric lymph node. one multinucleated giant cell in the ileocaecal lymph node showed characteristics of both foreign-body and langhans’ giant cells. multi nu cle ated giant cells in lesions comprised both types, with langhans’ giant cells usually outnumbering the foreign-body type. necrogranulomas in the lungs of b4, and bronchial lymph node, spleen and liver of a1 were infiltrated by moderate numbers of neutrophils, but macroscopically visible pus formation was not a feature of the lesions. small numbers of neutrophils infiltrated the lesions in the lung of a1, and the pleura, left prefemoral and right retropharyngeal lymph nodes of b4. all the organs of a1 sampled for microscopy, except the oesophagus and heart, showed lesions and the presence of acid-fast bacilli. for b4, the presence of acid-fast bacilli was generally related to the severity of the lesions. they were visible in almost all organs showing macroscopic pathology, while not visible in the organs that only showed microscopic pathology (except for the left prefemoral lymph node). acidfast bacilli were encountered in b4 in the lungs, pleura and bronchial, mediastinal, sternal, iliac, right prefemoral, left prefemoral and right retropharyngeal lymph nodes. acid-fast bacilli were numerous (lung, spleen, liver, kidney and the bronchial and mediastinal lymph nodes of a1), moderate in number (adrenal gland of a1 and lung of b4), to small in number (abomasum, small intestine, colon and rectum of a1, and pleura and the following lymph nodes of b4: bronchial, sternal, iliac and right prefemoral). very rare acid-fast bacilli were visible in the mediastinal, left prefemoral and right retropharyngeal lymph nodes of b4. the tuberculosis and suspected tuberculosis lesions seen in a1 and b4 are summarized in table 3. the granulomatous lesions seen in the testes, colon and following lymph nodes: left retropharyngeal, left and right mandibular, left prescapular, mesenteric, ileocaecal and inguinal of b4 are included in the table as tuberculosis lesions, even though no acid-fast bacilli were detected. the multifocal granulomas in the colon of b4 were probably caused by migrating nematode larvae, but sections of a nematode larva were encountered in only one of the four granulomas. discussion this study represents the first confirmed cases of m. tuberculosis in springbok. previous reports of tuberculosis in springbok did not conclusively establish the mycobacterium species involved, and no detailed description of the pathology was given (robinson 1953; hofmeyr 1956). mycobacterium tuberculosis was cultured from three springbok (a1, b4 and b6), indicating a prevalence of 9% (3/33), but only two (a1 and b4) showed tuberculosis lesions. this emphasizes the importance of mycobacterial culture in diagnostic surveys for tuberculosis. there is no documented information on the prevalence of m. tuberculosis in infected semi-free-ranging and free-ranging wildlife. this is not surprising, as htb is exceptionally rare in wildlife populations not kept in captivity, although it may be an important emerging disease in certain freeranging wildlife species in some regions of the world (alexander et al. 2002). in captive wildlife, m. tuberculosis was found to be the third most frequently isolated mycobacterium (11 %) among a total of 263 mycobacterial isolates obtained from zoos and wildlife parks in the united states of america in the mid1970s (thoen et al. 1977). between 1991 and 2001 436 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) t a b l e 3 s u m m a ry o f tu b e rc u lo si s le si o n s in t h e t w o m . tu b e rc u lo si s cu ltu re -p o si tiv e s p ri n g b o k (a 1 a n d b 4 ) o rg a n n e c ro s is c a lc if ic a ti o n m a c ro p h a g e s e p it h e li o id c e ll s g ia n t c e ll s n e u tr o p h il in fi lt ra ti o n f ib ro u s e n c a p s u la ti o n a c id -f a s t b a c il li a 1 b 4 a 1 b 4 a 1 b 4 a 1 b 4 a 1 b 4 a 1 b 4 a 1 b 4 a 1 b 4 l u n g + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + p le u ra a + + + + + n d b + + + n d + + + n d + /– n d + – – n d + b ro n ch ia l l n . + + + + + + + + + + + + + + + + + + + + + + + + + + + + + – + + + + + + + m e d ia st in a l l n . + + + + /– + + + /– + + + + + + + + + + + + + – – + + – + + + + /– s te rn a l l n . n s c + n s + n s + + + n s + + + n s + + + n s – n s + /– n s + ili a c ln . n s + + + n s + + n s + + + n s + + + n s + + + n s – n s + + n s + r -p re fe m o ra l l n . n s + + + n s + + n s + + + n s + + + n s + + + n s – n s + n s + l -p re fe m o ra l l n . n s + n s + n s + + n s + + n s + + + n s + n s + n s + /– r -r e tr o p h a ry n g e a l l n . n s + n s + n s + + + n s + + + n s + + + n s + n s + /– n s + /– l -r e tr o p h a ry n g e a l l n . n s – n s – n s + n s + n s + /– n s – n s – n s – r -m a n d ib u la r ln . n s – n s – n s + n s – n s – n s – n s – n s – l -m a n d ib u la r ln . n s – n s – n s + n s – n s – n s – n s – n s – l -p re sc a p u la r ln . n s – n s – n s + n s – n s – n s – n s – n s – m e se n te ri c ln . n s – n s – n s + n s + n s + + n s – n s – n s – ile o ca e ca l l n . n s – n s – n s + /– n s + /– n s + + + n s – n s – n s – in g u in a l l n . n s – n s – n s + n s – n s + /– n s – n s – n s – s p le e n + + + n a d – n a + + n a + n a + n a + + n a – n a + + + – l iv e r + + + n a – n a + + n a + n a + n a + + n a – n a + + + – k id n e y + + n a – n a + + n a + /– n a + /– n a – n a – n a + + + – a d re n a l g la n d + /– n a – n a + n a – n a – n a – n a – n a + + – t e st ic le n s – n s – n s + n s – n s – n s – n s – n s – a b o m a su m – n a – n a + /– n a – n a – n a – n a – n a + – s m a ll in te st in e – n a – n a + n a – n a – n a – n a – n a + – c o lo n – – – – + + + – + + – – – – – – + – r e ct u m – n s – n s + n s – n s – n s – n s – n s + n s f re q u e n cy o f d e te ct io n o r se ve ri ty : – = a b se n t a o n ly m a cr o sc o p y d o n e f o r a 1 + /– = r a re ly f o u n d b n o m ic ro sc o p y d o n e + = l o w c n o t sa m p le d + + = m e d iu m d n o t a ff e ct e d + + + = h ig h 437 t.a. gous & m.c. williams a total of 12 cases of m. tuberculosis infection in eight different species were recorded at the national zoological gardens of south africa in pretoria (michel et al. 2003). the generalized nature of the lesions in both affected springbok makes it difficult to establish the route of infection, although the oral route appears most likely. the lung lesions in both animals involved the interstitium and alveolar walls rather than the bronchioles which strongly suggests the oral route of infection. however, the pulmonary lymph nodes in both animals showed marked macroscopic lesions, while the mesenteric and ileocaecal lymph nodes only showed moderate lesions, suggesting infection via inhalation. the behavioral pattern of springbok would have facilitated exposure to human excretions in the environment, supporting the oral route of infection. investigations into the possible source of infection revealed that two of the groundskeepers were under treatment for confirmed m. tuberculosis infection (h. kettner, personal communication 2000). the springbok were semifree-ranging and never handled, so it is highly un likely that aerosol transmission occurred from the infected humans to the springbok. the lung lesions in the springbok were not markedly encapsulated and the possibility of aerosol shedding from open lung lesions, and subsequent intraspecific transmission after the initial infection should also be considered. cattle infected with m. tuberculosis usually do not develop progressive disease and typically only show small granulomas in the pharyngeal, thoracic and mesenteric lymph nodes (dungworth 1993; o’reilly & daborn 1995; cousins, huchzermeyer, griffin, brückner, van rensburg & kriek 2004). captive and free-ranging wildlife, however, often develop extensive lesions and progressive pulmonary disease after infection with m. tuberculosis (lomme, thoen, himes, vinson & king 1976; montali et al. 2001; alexander et al. 2002). generalized or miliary tuberculosis occurs when there is haematogenous dissemination of both free and macrophage-associated mycobacteria from the primary site of infection throughout the body. this is most frequently the result of breakdown of a blood vessel by an expanding caseating granuloma or cavitating lesion (dungworth 1993; lópez 2001). susceptible hosts also tend to develop rapidly progressing generalized lesions more readily than resistant animals (de lisle, bengis, schmitt & o’brien 2002), indicating that springbok should be regarded as highly susceptible. lesions were found in numerous organs (table 3), and it is likely that a1 would have shown lesions in many more organs if sampling had been done more extensively. the lesions observed in the two affected springbok were dissimilar in appearance in the various organs. the lesions varied in appearance in terms of extent and macroand microscopic appearance, as indicated in table 3. the variability in lesion appearance is probably a reflection of the progression of the infection as determined by the host’s cell-mediated immune response, and ranged from early small granulomatous lesions to chronic encapsulated necrogranulomas and proliferative nodules (dungworth 1993; neill, pollock, bryson & hanna 1994; cousins et al. 2004). this variability in the appearance of tuberculosis lesions has also been noted for african buffalo infected with m. bovis in the kruger national park of south africa (kriek, de vos, bengis, huchzermeyer, keet & raath 1992; kriek 1994). this pronounced variation should be considered when assessing the macroscopic lesions for diagnostic purposes in single animals. the macroscopic and microscopic appearance of the tuberculosis lesions in the springbok mostly conformed to the classical tuberculous granulomatous inflammatory lesions as described for humans, nonhuman primates and various domestic animals and wildlife. more specifically the lesions resembled lesions found in herbivores and omnivores (francis 1958; thoen & himes 1981; dungworth 1993). there were indistinct to discrete granulomas with or without central caseous necrosis, surrounded by variable, but usually numerous, macrophages, epithe lioid cells, multinucleated giant cells, lymphocytes and plasma cells. fibrous encapsulation was absent to moderate. calcification was generally mild to moderate and microscopic neutrophil infiltration was only occasionally observed. the pleural nodular lesions were more proliferative, with prominent fibroplasia. multinucleated giant cells consisted of both lang hans’ and foreign-body types, with langhans’ giant cells occasionally predominating. this is in accordance with thomson (1984) who states that the distinction between langhans’ and foreign-body giant cells is not valid, as both types are found in the same lesions and there is no relationship between the type and aetiological agent. acid-fast bacilli were scanty to numerous, with large clumps visible in the necrotic exudate of the pulmonary lymph nodes of one animal. some organs showed typical or highly suggestive tuberculous granulomatous lesions but acid-fast bacilli could not be detected. this marked variation in numbers of acid-fast bacilli, and even absence, in typical lesions is commonly found in cattle (cousins et al. 2004). 438 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) some animals, notably certain wildlife species, show obvious differences regarding the appearance and distribution of lesions when compared to those observed in cattle infected with m. bovis (de lisle et al. 2002). the lesions in the springbok differed from those in cattle mainly in the degree of calcification. the springbok showed minimal to moderate calcification while cattle often show marked calcification of caseous necrogranulomas (dungworth 1993). renal lesions in cattle are limited to the cortex (dungworth 1993). the renal lesions in a1 occurred in the cortex but were confined to the cortico-medullary junction. acid-fast bacilli were numerous in the renal granulomas but there were also rare acid-fast bacilli in the mesangial cells of renal corpuscles. abscessation and fistulation of the parotid lymph nodes are common findings in greater kudu infected with m. bovis (bengis & keet 1998; keet, kriek, bengis & mi chel 2001), but absent in most other species, including springbok. in contrast to herbivores and omni vores, certain carnivore species such as lions show lesions that are more proliferative in nature without caseation, calcification or multinucleated giant cells, but acid-fast bacilli are often numerous (keet et al. 1996; keet, kriek, penrith & michel 1998; de lisle et al. 2002). the proliferative nodular lesions in the pleura of the springbok resembled the pleural lesions of cattle (dungworth 1993), and showed, in contrast to those in lions, caseation, calcification and acid-fast bacilli. scanty multinucleated giant cells were found in the pleural lesions of the springbok. there are few detailed reports on the pathology of m. tuberculosis in wildlife. most reports only superficially mention or describe the pathology, and concentrate mainly on the epidemiology, mycobacteriology or zoonotic aspects of the disease (thoen et al. 1977; michalak et al. 1998; oh, granich, scott, sun, joseph, stringfield, thisdell, staley, workmanmalcolm, borenstein, lehnkering, ryan, soukup, nitta & flood 2002; michel et al. 2003). there is only one report that gives some indication of the pathology of probable m. tuberculosis infection in springbok. in this report, two springbok from the pretoria zoological gardens showed extensive tuberculosis lesions of the lungs, with numerous acid-fast bacilli in smears from the lung lesions. one of the springbok displayed pus in the lesions that was of a soft creamy consistency (robinson 1953). the two springbok with tuberculosis lesions in the present study also showed extensive lung lesions with moderate to large numbers of acid-fast bacilli, but no macroscopically visible pus. two east african oryxes (oryx gazella beisa) from the same pen at the jackson zoological park in missouri, usa, had tuberculosis as a result of m. tuberculosis infection. one animal died from tuberculosis while the other was euthanased as a result of a positive idt test. both showed generalized disease with extensive necrogranulomatous lesions in the lungs, liver, mediastinal and mesenteric lymph nodes. one animal also had necrogranulomas in the uterus. acid-fast bacilli were visible in the affected organs (lomme et al. 1976). the lesions were generally similar in the springbok, but neither a1 or b4 had macroscopically visible lesions in the mesenteric lymph nodes. springbok a1 was a ewe but the uterus was not sampled for microscopic analysis, and therefore the presence of lesions could not be confirmed, although there were no macroscopic lesions. mycobacterium tuberculosis infection in captive asian elephants (elephas maximus) results in necrogranulomas primarily in the lungs and thoracic lymph nodes. in animals suffering from extensive involvement of both lungs, severe calcified necrogranulomas and cavitating lesions with pulmonary abscesses develop that often cause the death of the animal. typical microscopic findings include granulomas with giant cells in the lymph nodes and lungs initially, that progress to extensive necroand pyogranulomatous pneumonia in advanced cases. acid-fast bacilli are sparsely distributed but more easily found in central areas of caseation in the lungs, rather than in the lymph nodes where they are usually rare (montali et al. 2001). springbok a1 was markedly weakened and recumbent when it was euthanased for necropsy. this animal would have probably died naturally from tuberculosis, indicating that tuberculosis can be fatal, similar to the east african oryx and asian elephant. springbok b4, however, had tuberculosis as an incidental finding. although the springbok showed marked lesions in the lungs, there was never cavitation or abscessation, and only moderate calcification. the numbers of acid-fast bacilli in the springbok were low to numerous, with large numbers visible in the necrotic exudate of the lungs, and bronchial and mediastinal lymph nodes of a1. humans and non-human primates infected with m. tuberculosis typically show pulmonary disease with involvement of pulmonary lymph nodes, and occasionally systemic dissemination. macroscopic granulomas are characterized by caseation and cavitation. microscopically the lesions display classical tuberculous granulomatous inflammation with calci439 t.a. gous & m.c. williams fication, fibrous encapsulation and variable numbers of acid-fast bacilli (thoen & himes 1981; kumar, cotran & robbins 1997; montali et al. 2001). the springbok in the current study had similar lesions but cavitation was not a macroscopic feature. the main differential diagnosis for the tuberculosis lesions in the springbok was granulomatous inflammation caused by migrating nematode larvae. adult b. magna lungworms in the airways of the lungs did not cause granulomatous inflammation, except where bronchioles had ruptured. granulomas occurred in the lungs, various lymph nodes, but mostly the mesenteric lymph node, tonsil, liver, kidney and gastro-intestinal tract. inspissated and calcified parasitic larval nodules occurred in the abdominal cavity attached to the liver and abomasum of two animals. the lesions were differentiated from tuberculous granulomas by the presence of larvae in cross-section of most granulomas, the absence of acid-fast bacilli, and infiltration of eosinophils. many animals had multifocal nodular aggregations of lymphocytes and plasma cells with variable numbers of eosinophils in the liver, particulary the portal tracts. these aggregations probably represent lymphoid follicles in response to parasitic migration and were all negative for acid-fast bacilli. the focal granuloma in the kidney of c1 resembled a typical tuberculous granuloma both macroscopically and microscopically, but no acid-fast bacilli were visible. the uterus of c1 and c5 showed necro-haemorrhagic bacterial endometritis and gran ulomatous endometritis, respectively. these lesions probably reflect post-partum involution, with bacterial infection in the case of c1. the lesions did not show acid-fast bacilli, and mycobacterial culture of pooled kidney and uterus specimens from c1 was negative. the gastrocnemius muscle of c20 displayed bilaterally symmetrical areas of calcification macroscopically, which was determined to be calcinosis circumscripta microscopically. the liver of c20 showed multifocal granulomatous inflammation that surrounded groups of open fusiform clefts. the cause of the clefts could not be established but they probably contained an immunogenic substance which elicited a foreign-body reaction. acid-fast bacilli could not be found in the lesions, and the animal was culture-negative for mycobacteria. mycobacterial culture is regarded as the gold standard for diagnosing tuberculosis (de lisle et al. 2002). mycobacterium tuberculosis was cultured from three springbok (a1, b4 and b6) in this study, but only two had displayed lesions of tuberculosis. the historical literature is often ambiguous as to the identity of mycobacteria isolated from animals with tuberculosis. the paper by robinson (1953) on tuberculosis of springbok indicates that pure cultures of m. tuberculosis were obtained from one animal. however, subsequent cultures from guinea pigs that were inoculated with affected lung material were considered to be of the bovine type. an incontact springbok that died shortly afterwards yielded a pure culture m. tuberculosis, but inoculation of guinea pigs was never attempted. the identity of the causative mycobacterium species in both these cases should be regarded as equivocal, although m. bovis may have been involved. the case of tuberculosis mentioned by hofmeyr (1956) gives no additional information regarding the causative myco bacterium species. springbok b6 did not show any macroscopical or microscopical tuberculosis lesions, and the isolation was made from the organ lymph node pool. this pool comprised the bronchial, mediastinal, hepatic, splenic, ileocaecal and mesenteric lymph nodes. this animal could therefore have been infected via either the oral or inhalation route, with bacterial spread to lymph nodes draining either the lungs or gastro-intestinal tract. springbok b6 may have been sampled during the early infection phase before there was enough time for detectable lesions to develop. another possible reason for the absence of lesions is localized containment of the infection as a result of an effective immune response. the isolation of mott from b2 and b3 indicates that these animals were exposed and infected by environmental mycobacteria, without the development of tuberculosis lesions. springbok b2 showed a single cluster of acid-fast bacilli in the cytoplasm of a macrophage in a mesenteric lymph node, without an inflammatory reaction. these acid-fast bacilli were short, straight and thick-set rods, in contrast to the m. tuberculosis bacilli in the infected springbok which were all medium to fairly long, slender and often curved. although the microscopic morphology of acid-fast bacilli cannot be used to identify the causative organism, the morphological difference between the mott and m. tuberculosis was striking. the morphology of m. bovis appears similar to m. tuberculosis (cousins et al. 2004). this difference may therefore be a useful microscopical indicator of the mycobacterium species involved, although mycobacterial culture will always be needed to confirm the identity. although the source of infection could not be established conclusively, it is likely to be one or both of 440 pathology of tuberculosis caused by mycobacterium tuberculosis in semi-free-ranging springbok (antidorcas marsupialis) the m. tuberculosis positive groundskeepers. unfortunately, both groundskeepers terminated their employment several months before the index case, and it was not possible to obtain m. tuberculosis isolates from them for genomic typing to confirm transmission. although the initial infection of the springbok at ithemba labs is likely to be from humans (zooanthroponosis), subsequent intraspecific transmission cannot be ruled out. tuberculosis was not recorded in any of the other wildlife species on the property during the study period, and only time will tell if they are also infected. the wildlife at ithemba labs are kept in a semi-free-ranging state on the grounds, which is securely fenced, and it is unlikely that they will act as a source of infection for humans. the site is also closed to the general public. ithemba labs is situated in the cape town metropole, which has one of the highest reported incidence rates of tuberculosis in humans in the world (anon. 2001; anon. 2004). with this high incidence rate in humans, the zooanthroponotic risk of tuberculosis is far greater than the zoonotic potential of tuberculosis in this area. acknowledgements we express our gratitude to drs gideon brückner and james kitching of the directorate veterinary services, western cape department of agriculture for funding of the mycobacterial cultures, and allowing us the use of their necropsy facilities; to messrs paul slingers and chrisjan jantjes for assisting with the necropsies; to dr horst kettner of the ithemba labs staff environmental club for organizing and assisting with the culling of the springbok; to dr anita michel and her staff at the tuberculosis laboratory of the arc onderstepoort veterinary institute for conducting the mycobacterial examinations; to messrs errol van kerwel and andré meyer, and ms gwen du plessis at pathcare for the processing, sectioning and staining of all the histopathological sections, and to prof. joop boomker of the department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria for identification of the nematodes. references alexander, k.a., pleydell, e., williams, m.c., lane, e.p., nyange, j.f.c. & michel, a.l. 2002. mycobacterium tuberculosis: an emerging disease of free-ranging wildlife. emerging infectious diseases, 8:598–601. anon. 1983. laboratory manual. tuberculosis laboratory, section of bacteriology. onderstepoort veterinary institute, pret oria, south africa. anon. 2001. south african national tuberculosis association. latest tuberculosis statistics. http://www.santa.org.za/main5. html. ano n. 2004. cape town tb control. a partnership between the provincial 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animals. journal of the american veterinary medical association, 170:987–990. thomson, r.g. 1984. general veterinary pathology. phila delphia: w.b. saunders company. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice austin_227-234.indd introduction in the genus lamproglena von nordmann, 1832 only the adult females are gill parasites of fishes. the majority of species occur in freshwater and the only exception, lamproglena lichiae von nordmann, 1832 was collected from the red sea (fryer 1968) on the doublespotted queenfish, scomberoides lysan. six of the 39 species of this genus recorded are from southern africa (moll & avenant-oldewage 2008) and include lamproglena monodi capart, 1944; lamproglena clariae fryer, 1956; lamproglena barbicola fryer, 1961; lamproglena cornuta fryer, 1964; lamproglena hoi dippenaar, luus227 onderstepoort journal of veterinary research, 76:227–234 (2009) ecological parameters of lamproglena hoi (copepoda: lernaeidae) infection on the bushveld smallscale yellowfish, labeobarbus polylepis (boulenger, 1907) a. austin and a. avenant-oldewage* department of zoology, university of johannesburg, kingsway campus, p.o. box 524, auckland park, johannesburg, 2006 south africa * e-mail address: aoldewage@uj.ac.za accepted for publication 12 november 2008—editor abstract austin, a. & avenant-oldewage, a. 2009. ecological parameters of lamproglena hoi (copepoda: lernaeidae) infection on the bushveld smallscale yellowfish, labeobarbus polylepis (boulenger, 1907). onderstepoort journal of veterinary research,76:227–234 this study describes the distribution and aspects of the ecology of lamproglena hoi. bushveld smallscale yellowfish, labeobarbus polylepis (boulenger, 1907) were collected during june 2006 from the phongolo and assegaai rivers, march 2005 and october 2006 from the elands river, and january 2007 and june 2008 from the komati river in mpumalanga, south africa and examined for the presence of parasites. lamproglena hoi specimens were collected from the gill filaments of the host. specimens were fixed with warm afa (alcohol-formaldehyde-acetic acid) and preserved in 70 % ethanol. the identification of parasites took place in the laboratories of the university of johannesburg. twenty-five copepods (prevalence 21 %, mean intensity = 4.17, abundance = 0.86) were collected on 29 fish in the phongolo river and 46 copepods (prevalence 40 %, mean intensity = 3.83, abundance = 1.53) were collected on 30 fish in the assegaai river. one hundred and sixty eight copepods (prevalence 52 %, mean intensity = 12.92, abundance = 6.72) were collected on 25 fish in 2005, and 527 copepods (prevalence 95 %, mean intensity = 27.74, abundance = 26.35) were collected on 20 fish in the elands river. one hundred and sixteen copepods (prevalence 75 %, mean intensity = 7.73, abundance = 5.80) were collected on 20 fish in 2007, and 273 copepods (prevalence 63 %, mean intensity = 16.06, abundance = 10.11) were collected on 27 fish in 2008 in the komati river. labe obarbus polylepis from these four rivers was found to have a relatively high l. hoi infection. inseminated l. hoi females (immature) attach to the host in winter and their ovaries become conspicuous (mature). in spring fertilized eggs are stored in egg sacs hanging from the body (gravid), indicating that fertilized eggs start to hatch in spring and continued hatching into summer. parasites prefer the median part of the second gill arch for attachment. no correlation exists between the number of parasites recorded on the gills and the sizes (total lengths) of yellowfish sampled. keywords: copepoda, crustacea, ecology, labeobarbus polylepis, lamproglena hoi, yellowfish 228 lamproglena hoi infection on bushveld smallscale yellowfi sh, labeobarbus polylepis (boulenger, 1907) powell & roux, 2001; lamproglena hepsetii van as & van as, 2006. lamproglena hoi was described from specimens collected in the spekboom river, mpumalanga (dip penaar, luus-powell & roux 2001). it is an ectoparasite on the gills of the lowveld largescale yellowfish, labeobarbus marequensis and the bush veld smallscale yellowfish, labeobarbus polylepis. labeobarbus polylepis is an indigenous species endemic in the southern tributaries of the limpopo river system, as well as the inkomati and phongolo system, above altitudes of 600 m and prefers permanent rivers with deep pools, riffles and runs (skelton 2001). the only publication that exists on l. hoi was by dippenaar et al. (2001) and described the morphological features of this species by means of scanning electron micrographs and drawings. the aim of this study is to contribute to the understanding of the ecology of this parasite on l. polylepis. materials and methods sampling sites and hosts specimens of four labeobarbus polylepis (boulenger, 1907) populations were collected from in komati and phongolo river systems within the natural geographical distribution range of the species. twenty nine specimens were collected from the phon golo river (27°22’17.93” s, 30°35’24.50” e) and 30 specimens from the assegaai river (27°4’48.22” s, 30°49’15.09” e) during june 2006. in the inkomati system, 25 specimens were sampled from the elands river (25°36’56.09” s, 30°30’55.29” e) in march 2005 and 20 in october 2006. twenty specimens were collected from the komati river (25°53’40.94” s, 30°17’1.19” e) in january 2007 and 27 in june 2008 (fig. 1). collection of fish and parasites fish specimens were captured by means of gill nets and killed by severing the spinal cord behind the head. they were weighed and their sizes were determined in the field by measuring their total length. the fish were dissected by cutting from the anus towards the head and the sex was determined. the gills were removed, dissected apart, placed in water and examined for parasites with the aid of a dissecting microscope. the gill arches were divided into three areas: anterior, median and posterior, and the positions of the parasites on each gill arch were noted. females of l. hoi were collected and fixed in warm alcohol-formaldehyde-acetic acid (afa) and preserved in 70 % ethanol. fig. 1 map indicating the geographic distribution of labeobarbus polylepis sampled in four rivers (elands, phongolo, assegaai and komati rivers) in mpumalanga, south africa. dots indicate the position of the sampling sites south africa south africa nooitgedacht dam kwena dam elands river ngodwana dam ngodwana river vygeboom dam crocodile river komati river phongolopoort dam swaziland mozambique phongolo river heyshope dam assegaai river usutu river n 229 a. austin & a. avenant-oldewage identification of parasites the identification of the parasites took place in the laboratory of the university of johannesburg. parasites were identified as l. hoi based on their morphology (dippenaar et al. 2001). three developmental stages were observed in l. hoi females. inseminated females were categorized as immature when attaching to the gill arches of the host. when the ovaries became conspicuous the females were considered mature. subsequently, fertilized eggs are stored in egg sacs hanging from the body and females were classified as gravid. data presentation lamproglena hoi specimens were counted and the data obtained were graphically represented. a comparison between the infections in the four l. polylepis populations was done. geographical locality, seasonality and gender specificity of l. hoi were determined. the location of the parasites on the gills was recorded. the correlations between the total length of the host and the number of parasites were determined. infection levels were expressed as prevalence, mean intensity and abundance according to the definitions of bush, lafferty, lotz & shostak (1997). results the combined surface area of the second and third gills of l. polylepis is larger than that of the first and fourth gills (fig. 2) and the fact that the former two gills are situated centrally in the gill chamber allows for maximum water flow over this area. lamproglena hoi congregates on the outside of the gill filaments and attaches close to the gill arches with the posterior end facing away from gill arches (fig. 3a and 3b). in young females the gut content appears green (fig. 3b) and no proliferation of gill tissue is apparent. in mature females the gut is filled with a red substance that appears to be blood (fig. 3a) and proliferation of gill tissue is observed (fig. 3b). infection statistics of lamproglena hoi prevalence, abundance (relative density) and mean intensity sampling in the phongolo and assegaai rivers was done in winter, while the specimens in the elands river were collected in autumn and spring and in the komati river in summer and winter. the prevalence, mean intensity and abundance of the four rivers show seasonal variance (fig. 7). the prevalence of l. hoi on the gills of l. polylepis varied between the different rivers. the prevalence in l. fig. 2 gill arches of labeobarbus polylepis in the gill chamber. numbers indicate the gills’ position in succession from anterior to posterior fig. 3 (a) photograph of a gill arch of labeobarbus polylepis to indicate attachment sites of lamproglena hoi parasites. gill is divided into three sites (anterior (a), median (m) and posterior (p)), and (b) an enlargement to show attachment and proliferation at attachment site. arrows indicate proliferation a b 230 lamproglena hoi infection on bushveld smallscale yellowfi sh, labeobarbus polylepis (boulenger, 1907) polylepis in the phongolo river (21 %) and assegaai river (40 %) were relatively low, compared to the considerable higher prevalence in the elands river (52 % and 95 %) and komati river (63 % and 75 %). the highest mean intensity value recorded for l. hoi was in the elands river and increased from 12.92 in autumn to 27.74 in spring (fig. 4). the lowest values noted were 3.83 in the assegaai river and 4.17 elands river autumn 2005 elands river spring 2006 0 10 20 30 40 50 60 70 80 90 100 v a lu e prevalance (%) mean intensity (no.) abundance (no.) fig. 4 graph depicting the prevalence, mean intensity and abundance of lamproglena hoi on the gills of labeobarbus polylepis in autumn 2005 and spring 2006 in the elands river in mpumalanga, south africa v a lu e prevalance (%) mean intensity (no.) abundance (no.) phongolo river winter 2006 assegaai river winter 2006 0 5 10 15 20 25 30 35 40 45 fig. 5 graph depicting the prevalence, mean intensity and abundance of lamproglena hoi on the gills of labeobarbus polylepis in winter 2006 in the phongolo and as se gaai rivers in mpumalanga, south africa v a lu e prevalance (%) mean intensity (no.) abundance (no.) komati river summer 2007 komati river winter 2008 0 10 20 30 40 50 65 70 80 fig. 6 graph depicting the prevalence, mean intensity and abundance of lamproglena hoi on the gills of labeobarbus polylepis in summer 2007 and winter 2008 in the komati river in mpu ma langa, south africa 231 a. austin & a. avenant-oldewage in the phongolo river during winter (fig. 5). the rest of the values ranged between 7.73 (summer) and 10.11 (winter) in the komati river (fig. 6). for this fish species, the abundance values of l. hoi ranged from 6.72–26.35 (autumn and spring) in the elands river to 0.86–1.53 (winter) in the phongolo and assegaai rivers and then increased to 5.80– 16.06 (summer and winter) in the komati river (fig. 4, 5 and 6). ecological parameters seasonality when the data sets from the various surveys are combined a pattern becomes apparent. the highest number of l. hoi was collected in spring. the highest prevalence, mean intensity and abundance of l. hoi were also recorded in spring. all parameters decreased toward summer and the lowest numbers were observed during winter surveys (fig. 7). in autumn 58 % of females were mature; this percentage increased in winter to 95 % and 96 %, respectively with 4 % and 5 % immature, indicating that eggs are not released during winter and that young females are present in this season. in spring 84 % of females were gravid and egg sacs were hanging from the body. in summer 55 % females were gravid, indicating that fertilized eggs start to hatch in spring and continued hatching into the summer season, as gravid females decreased in number from spring to summer (fig. 8) and non parasitic nauplii were observed in close proximity to the egg sacks. v a lu e autumn 2005 summer 2007 0 10 20 30 40 50 65 70 100 90 80 winter 2006 winter 2006 spring 2006 winter 2008 seasonal variance prevalance (%) mean intensity (no.) abundance (no.) p e rc e n ta g e ( % ) mature immature gravid 0 10 20 30 40 50 60 70 80 90 100 elands river autumn 2005 elands river spring 2006 phongolo river winter 2006 assegaai river winter 2006 komati river summer 2007 fig. 7 graph illustrating the seasonal variance of prevalence, mean in tensity and abundance of lamproglena hoi on the gills of labeobarbus polylepis found in four rivers in mpumalanga, south africa fig. 8 graph showing composition of immature, mature and gravid lamproglena hoi females percentages in autumn, winter, spring and summer 232 lamproglena hoi infection on bushveld smallscale yellowfi sh, labeobarbus polylepis (boulenger, 1907) location of parasites on the gills the gill arches were divided into three areas: anterior, median and posterior and the positions of the parasites on each gill arch were noted. from a sub sample, a total of 729 parasites were collected on the gills of l. polylepis, 49 % were found on the left gills and 51 % on the right gills. on both the left and right side, 79 % of the parasites were attached to the median part of the gill arch. the gill arch’s anterior gill area had 14 % of parasites and the posterior gill area 8 %. the second gill on both the left and right sides had the highest numbers of parasites (fig. 9). the position of l. hoi on the hosts collected from the elands river in autumn 2005 was excluded from the sub sample. gender specificity in the sub sample (excluding eland river data 2005) of 126 infected fish, the number of infected males (n = 56 or 81 % of infected fish) was much higher than that of the females (n = 13 or 19 %). however, mainly male fish were collected in the komati river, hence the large infection on male hosts (n = 31). host size preference no correlation existed between the number of parasites and the size of the host (fig. 10). discussion the highest number of l. hoi females was observed during spring and it decreased from summer to autumn. the lowest number of parasites occurred in winter. abundance and mean intensity values differ, the numbers in both cases were lower in spring time (komati river) than in autumn (elands river). labeobarbus polylepis in the elands river demonstrated extremely high numbers of l. hoi infections during autumn and spring. the hosts in the komati 0 20 40 60 80 100 120 location of parasites on gills 11 37 5 19 103 11 13 72 2 6 73 4 10 49 3 15 87 6 20 83 10 7 69 14 a m p a m p a m p a m p a m p a m p a m p a m p l2 l3 l4 r1 r2 r3 r4 n u m b e r o f l a m p ro g le n a h o i 0 10 20 30 40 50 65 70 100 90 80 length of fish (mm) y = 0.0011x + 13.777 r2 = 2–05 0 100 200 300 400 500 600 n u m b e r o f l a m p ro g le n a h o i fig. 9 graph showing the position (anterior (a), median (m) or posterior (p)) of the lamproglena hoi on the left (l) and right gill (r) arches of labeobarbus polylepis fig. 10 graph showing the number of lamproglena hoi compared to the total size of labeobarbus poly lepis hosts 233 a. austin & a. avenant-oldewage river showed exceptionally high numbers of parasites in winter compared to those in the phongolo and assegaai rivers. khan & thulin (1991) suggested that since ectoparasites are directly exposed to the river water, they are in contact with pollutants in it, which could reduce continued occurrence or reproductive tempo. according to marx & avenantoldewage (1996) and avenant-oldewage (2003) higher numbers of ectoparasites occur in less contaminated waters, while more polluted water has the reverse effect. the elands river is geographically isolated from the komati river by two waterfalls, but the sampling localities in both rivers are not highly impacted (ferreira, wepener & van vuren 2008). the komati river drainage area is mainly covered by forests and the river is therefore not impacted by anthropogenic activities. the less polluted elands and komati rivers have higher numbers of copepod ecto par a sites than those fish in the phongolo and assegaai rivers. this finding corroborates the results of avenantoldewage (2003) for l. clariae in the oli fants river and that of tsotetsi, avenant-oldewage & mashego (2004) on l. clariae in the vaal river. only three developmental stages of the life cycle of l. hoi females were observed. the immature stage occurred in winter and the highest percentage of mature females was also observed during this season. in spring, gravid females dominated in numbers on the gills, and this phenomenon continues into summer. it appears that fertilized eggs hatch in spring and summer. the females die after egg production as is evident from the decline in the number of gravid females as spring goes into summer (84 % compared to 55 %). there were no significant differences in the parasite load between the left and right gill, which indicates that l. hoi do not have a gill side preference. rhode (1993) speculated that the preference for a particular gill side is due to the asymmetrical body shape of many parasites. lam proglena hoi is bilaterally symmetrical which may possibly explain why there is no gill side preference. rhode (1993) furthermore suggested that the distribution of parasites on different gills differs because of the preference or size of the gills. marx & avenant-oldewage (1996) found that l. clariae concentrated near the ends of the gill arches which are completely different from l. hoi which congregates all over the gill surface. furthermore, tsotetsi et al. (2004) reported that l. clariae specimens attach midway along the gill filament so that the genital segment is in line with the apex of the filament. in l. hoi, the parasite attaches close to the bony part of the gill arch as well as along the length of the filament towards the tip. seventy-nine percent of the l. hoi were attached to the median part of the gill arch. this differs from the findings of tsotetsi et al. (2004), who determined that 52 % l. clariae parasites attach to the median part of gill arch. according to tsotetsi et al. (2004) the anterior part of the clarias gariepinus gill arch also harboured 14 % of parasites, similar to the finding in this study. the posterior gill arches of l. poly le pis harboured fewer l. hoi parasites (8 %) compared to l. clariae (34 %) were situated on the pos terior gill arches of c. gariepinus (tsotetsi et al. 2004). more l. hoi parasites preferred the median position on the gill arches. the second gill arch on both sides had more l. hoi parasites than any other gills. the increased gill surface and water flow through the gill chamber might explain why there are more l. hoi parasites in these positions. it is suggested that water flow in these areas provides the ideal opportunity for attachment. this will furthermore provide an advantage for distribution of offspring. on the other hand, tsotetsi et al. (2004) found a higher occurrence of l. clariae on the fourth gill on both sides. the fourth gill arch of c. gariepinus is shorter than the others and consists of fewer, thinner gill filaments. tsotetsi et al. (2004) suggested that the protection or diminished turbulence in the part of the gill chamber may offer an explanation for attachment location. in 2004 tsotetsi et al. found that l. clariae had no preference regarding the sex of the host. mainly male fish were collected during this study in the komati river which makes this sample survey biased and a conclusion could not be made on gender preference. data of the present study showed no correlation between the number of parasites and the host size. this is in agreement with the results of tsotetsi et al. (2004) on l. clariae on c. gariepinus and those of sproston, yin & hu (1950) on ophiocephalus argus and number of lamproglena chinensis. similar results for other copepod studies have been reported by marcogliese (1991) for caligus műller, 1758 and lo, morand & galzin (1998) for lernaea cyprinacea linnaeus, 1758. the increase in the size of the host will lead to an increase in gill surface and volume water flow through the gill chamber. however, these advantages have no affect on the number of parasites. 234 lamproglena hoi infection on bushveld smallscale yellowfi sh, labeobarbus polylepis (boulenger, 1907) conclusion the numbers of l. hoi specimens on the gills of l. polylepis from the four rivers indicate a relatively high prevalence, mean intensity and abundance, and the occurrence of a seasonal variance. these observations corroborate those of tsotetsi et al. (2004) for l. clariae. the median part of the gill arch on the second gill on both the left and right sides was the preferred attachment site. no correlation was found between the number of parasites and the host size or sex. acknowledgements the authors acknowledge the financial assistance provided by the water research commission and the university of johannesburg. we express our gratitude to gordon o’brien and quinton tam for their assistance in the field and laboratory. gina walsh is thanked for fig. 2 and quinton tam for fig. 3. references avenant-oldewage, a. 2003. lamproglena and lernaea (copepoda) as possible bio-indicators of environmental deterioration in the olifants river. journal of south african veterinary association, 72:96. bush, a.o., lafferty, k.d., lotz, j.m. & shostak, a.w. 1997. parasitology meets ecology on its own terms: margolis et al. revisited. journal of parasitology, 83:575–583. dippenaar, s.m., luus-powell, w.j. & roux, f. 2001. lam proglena hoi n. sp. (copepoda: lernaeidae) from two yellowfish hosts, barbus marequensis and barbus polylepis, caught in a river in mpumalanga, south africa. onderstepoort journal of veterinary research, 68:209–215. ferreira, m., wepener, v. & van vuren, j.h.j. 2008. die invloed van papierpulpmeule-aktiwiteite op die visgemeenskapstruktuur van die elandsrivier, mpumalanga. tydskrif vir natuurwetenskap en tegnologie, 27:83–94. fryer, g. 1968. the parasitic crustacea of african freshwater fishes; their biology and distribution. journal of zoology, london, 156:45–95. khan, r.a. & thulin, j. 1991. influence of pollution on parasites of aquatic animals. advanced parasitology, 30:201–238. lo, c.m., morand, s. & galzin, r. 1998. parasite diversity/ host age and size relationship in three coral-reef fishes from french polynesia. international journal for parasitology, 28: 1695–1708. marcogliese, d.j. 1991. seasonal occurrences of lernaea cyprinacea on fishes in belews lake, north carolina. journal of parasitology 77:326-327. marx, h.m. & avenant-oldewage, a. 1996. redescription of lamproglena clariae fryer, 1956 (copepoda, lernaeidae), with notes on its occurrence and distribution. crustaceana, 69:503–523. moll, j. & avenant-oldewage, a. 2008. ’n oorsig oor die verspreiding, taksonomie en aspekte van die ekologie van die genus lamproglena (copepoda), ’n ektoparasiet op die kieue van visse. tydskrif vir natuurwetenskap en tegnologie, 27:107–124. rohde, k. 1993. ecology of marine parasites. wallingford, uk: cab international. skelton, p. 2001. a complete guide to the freshwater fishes of southern africa, 2nd ed. halfway house: southern book publishers. sproston, n.g., yin, w.y. & hu, t. 1950. the genus lamproglena (copepoda parasitica): the discovery of the life-histories and males of two chinese species from food fishes, revealing their relationship with lernaea, and of both to the cyclopoidea. sinensia, n.s. 1:51–54. tsotetsi, a.m., avenant-oldewage, a. & mashego, s.n. 2004. aspects of the ecology of lamproglena clariae (copepoda: lernaeidae) from the vaal river system, south africa. journal of crustacean biology, 24:529–536. von nordmann, a. 1832. mikrographische beiträge zur natur geschichte der wirbellosen tiere. zweites heft. berlin: g. reimer. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: souaibou farougou1 mariama gagara1 guy a. mensah2 affiliations: 1polytechnic school of abomey-calavi, university of abomey-calavi, republic of benin2agricultural research centre of agonkanmey, national institute of agricultural research of benin, republic of benin correspondence to: souaibou farougou postal address: po box 2009, main recipe, cotonou 01, republic of benin dates: received: 05 dec. 2012 accepted: 03 july 2013 published: 09 sept. 2013 how to cite this article: farougou, s., gagara, m. & mensah, g.a., 2013, ‘prevalence of peste des petits ruminants in the arid zone in the republic of niger’, onderstepoort journal of veterinary research 80(1), art. #544, 6 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.544 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence of peste des petits ruminants in the arid zone in the republic of niger in this original research... open access • abstract • introduction • materials and methods    • study areas    • blood sample collection    • laboratory analysis of blood samples    • statistical analyses • results    • overall prevalence of peste des petits ruminants    • prevalence of peste des petits ruminants according to location    • prevalence of peste des petits ruminants according to age    • prevalence of peste des petits ruminants according to sex    • polymerase chain reaction analysis • discussion • conclusion • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ the study aimed to determine the prevalence of peste des petits ruminants in the arid zone (niamey, tillabéry and tahoua) of the republic of niger. a serological survey was conducted and 519 serum samples were collected from 253 unvaccinated sheep and 266 unvaccinated goats. the sample included 340 female animals (168 sheep and 172 goats) and 160 kids and lambs (78 lambs and 82 kids). a competitive enzyme-linked immunosorbent assay yielded an overall seroprevalence of 45.0%. the prevalence in sheep was 42.0% compared with 47.9% in goats. the seroprevalence observed amongst small ruminants in tahoua (49.8%) and tillabéry (46.6%) was significantly higher (p = 0.001) than that observed in animals from niamey (25.1%). it was also higher (p = 0.04) in sheep younger than two years (51.8%) than in adults (37.6%). conversely, the seroprevalence showed no significant difference between male animals (35.8% in sheep; 50.1% in goats) and female animals (45.1% in sheep; 46.4% in goats). the prevalence of the disease observed amongst the sheep and goat populations confirms the continued danger of this disease in the areas studied. it is therefore necessary to develop strategies such as improving livestock services, providing effective vaccines and implementing a vaccination programme for an effective control of the disease in sub-saharan africa. introduction top ↑ small ruminants in the republic of niger contribute significantly to the gross domestic product of the country. of the total of 80 403 tons of meat produced in the country, small ruminants contribute 29 709 tons, about 37.0% (countrystat 2012). compared with the studies carried out on cattle, current knowledge of small ruminant diseases is fragmentary and incomplete. the persistence of, amongst others, peste des petits ruminants (ppr) is explained by the essentially transhumant mode of livestock management and low immunisation coverage. only 16.4% of the sheep and 14.2% of the goat population are vaccinated against ppr (countrystat 2012). ppr is an infectious and highly contagious viral disease of wild and domesticated small ruminants. it is characterised by digestive disorders, erosive stomatitis and necrotising pneumonia (jones et al. 1993) and can be controlled by vaccination, quarantine and disinfection (chen et al. 2010; diallo et al. 2007; jones et al. 1993). the geographical distribution of ppr includes sub-saharan africa, the middle east (abu-elzein et al. 1990; lefevre et al. 1991; taylor 1984; taylor, al busaidy & barrett 1990) and the indian subcontinent (kwiatek et al. 2007; shaila et al. 1996; taylor et al. 2002). ppr is caused by a virus belonging to the genus morbillivirus (gibbs et al. 1979). often manifesting in epizootic form, it is of vital economic importance because of its associated high mortality rate. in spite of the direct and indirect losses caused by ppr in animal husbandry in the republic of niger, few studies have been conducted to determine its epidemiological parameters such as animal movements, management issues (bloch & diallo 1991) and introduction of animals that are important in ppr transmission and its persistence. the purpose of this study was to assess the seroprevalence of ppr in the arid zone in niamey, tahoua and tillabéry in the republic of niger. materials and methods top ↑ study areas out of the eight regions that make up the republic of niger, niamey, tillabéry and tahoua (figure 1) were selected because of reports of alleged cases of ppr. in tahoua and tillabéry, five districts were randomly selected for the purpose of this study, whilst in niamey, three districts were selected. figure 1: location of farms surveyed in the republic of niger. the three regions combined contain 37.0% of the sheep and 32.0% of the goat populations. local livestock are kept under semi-intensive conditions and graze pastures during the rainy season (june to september; rainfall = 200 mm – 900 mm). hay is provided during the dry season (october to may). the diet is supplemented with agro-industrial and food byproducts such as corn bran, rice bran, millet bran, sorghum bran and brewer’s spent grain, as well as vitamin and mineral supplements in the form of salt licks. animals get water ad libitum from the rivers and have access to open shelters all year round, except for those meant for fattening and pregnant or lactating female animals, which are kept in covered shelters. average weather data for the sample collection period are provided in table 1. table 1: weather data for the study area. blood sample collection the samples were collected randomly from 15–20 small ruminants per breeding unit in the districts of tahoua and tillabéry and from 8–10 small ruminants in the district of niamey, where fewer small ruminants occur.blood samples of sheep and goats were collected over four months (november to february) during the dry season, when a resurgence of various animal diseases, including ppr, is generally reported by veterinary service workers. we collected a total of 519 blood samples from unvaccinated sheep and goats (according to the veterinary office) in selected districts of niamey (niamey i, iii and v), tahoua (birni-n’konni, illela, abalak, madoua, bambay) and tillabéry (n’dounga, fakara, bani bangou, kokoro, tabala) (table 2). table 2: distribution of samples by region. samples were organised according to age (animals younger than two years vs animals older than two years). information gathered from farmers as well as the dentition helped to determine the ages of the animals.blood samples were collected in plain vacutainer tubes. after centrifugation the resulting serum was transferred to mictotubes and frozen at -20 °c until analysis. each tube was marked by codes that represented information of the location of the breeder and the age and sex of the animals. during the collection of blood samples, special attention was given to animals showing signs of suspected ppr. in such cases, a nasal swab was taken for further analysis by polymerase chain reaction (pcr) to confirm ppr. a total of 26 such blood samples were collected from animals in the four regions surveyed. laboratory analysis of blood samples serum samples were analysed for ppr antibodies at the department of niger veterinary laboratories, using a competitive enzyme-linked immunosorbent assay (elisa). competitive elisa kits of cirad (emvt, france) and the pirbright institute (united kingdom) were used in this study. the analyses were performed in accordance with the procedure described by libeau et al. (1995), choi et al. (2005) and wang et al. (2007). optical densities were read in a spectrophotometer at 492 nm. the mean inhibition percentage of a serum sample was calculated from two wells. samples with an inhibition percentage higher than 50.0% were considered positive (aslam et al. 2009; zahur et al. 2011). the competitive elisa kit used for the diagnosis of ppr had a sensitivity and specificity of 92.2% and 98.4%, respectively, compared with the test virus neutralisation standard (gopilo 2005). the detection of viral rna by reverse transcriptase pcr (rt-pcr) was performed in accordance with the method described by bao et al. (2008) and couacy-hymann et al. (2002). viral rna was extracted from nasal swabs using the rneasy mini kit (qiagen, courtaboeuf, france) according to the manufacturer’s instructions. for pcr, a pair of primers amplifying a fragment of 351 bp (np3: 5’-cgg tct aga aaa tcg ctc cac ctg-3’ and np4: 5’-cct ctc ctc cct ggt cca gaa tct-3’) was used (couacy-hymann et al. 2002). an aliquot of 25 µl of the amplification mixture contained 1 µl of each primer (5 pmol/l), 5 µl of cdna, 1 µl of deoxyribonucleotide triphosphate (dntp), 5 µl of 5 × buffer, 1 µl of taq polymerase (1.25 u/l) and 12 µl of sterile distilled water. after an initial denaturation at 95 °c for 5 min, amplification proceeded with 29 cycles of denaturation at 94 °c for 30 s, 30 s of annealing at 55 °c and an extension phase at 72 °c for 1 min, followed by a final extension of 5 min at 72 °c. the pcr products were visualised after electrophoresis on 1.5% agarose gel. the samples that had been analysed by pcr were considered positive if a dna fragment of 351 bp was obtained. statistical analyses collected data were entered into a spreadsheet according to sex and age for each locality. the apparent prevalence of ppr was calculated by dividing the number of positive samples by the total number of samples tested. the true prevalence was estimated using the method of rogan and gladen (1978). the z bilateral test included in the statistica software package (statistica 2003) was used to compare the prevalences. differences were considered significant at p < 0.05. results top ↑ overall prevalence of peste des petits ruminants of the 519 blood samples processed by serology, 231 tested positive for ppr, representing a prevalence of 45.0%. the prevalence in goats was 5.9% higher than in sheep, although this was not a significant difference (p = 0.11) (table 3). table 3: prevalence of peste des petits ruminants in sheep and goats according to location. prevalence of peste des petits ruminants according to location the prevalence of ppr in sheep and goats varied between sampling areas (table 3). except for the tahoua region, the true prevalence in goats was higher than in sheep. the prevalence in the tahoua region was 24.7% higher (p < 0.001) than for the niamey region. although the prevalence was 3.2% higher than for the tillabéry region, the difference was not statistically significant. however, the prevalence of ppr determined in the tillabéry region was 21.5% higher than that found for the niamey region (p < 0.001). prevalence of peste des petits ruminants according to age in all regions, the prevalence of ppr in sheep younger than two years was higher than in both sheep and goats older than two years (table 4). in the tillabéry and niamey regions, positive cases and prevalences in goats younger than two years were higher than in goats older than two years (table 4). the ppr prevalence amongst young small ruminants (52.4%; n = 359) was significantly higher than in adults (40.9%; n = 160). table 4: prevalence of peste des petits ruminants in sheep and goats according to age. prevalence of peste des petits ruminants according to sex except for the niamey region, the prevalence of ppr in female sheep was higher (although not significantly) than in male sheep in all regions (table 5). however, the particularity of the niamey results can be explained by the small sample size collected in this region. the prevalence in male goats was higher, but not significantly, than in female goats in all regions, except for the niamey region (table 5). the overall prevalence found amongst female animals was 45.8% (n = 179), which was not significantly different from that in male animals (43.5%; n = 340). table 5: prevalence of peste des petits ruminants in sheep and goats according to sex. polymerase chain reaction analysis the pcr analysis of samples from nasal swabs confirmed the presence of the ppr virus (figure 2). of the 26 samples analysed, eight were positive. the pcr results in these animals confirmed those of serological analyses. figure 2: agarose gel (1.5% w/v) of the polymerase chain reaction amplification products performed on nasal swabs. discussion top ↑ the prevalence of ppr (45.0% for all sera tested) was investigated in north-western niger (tahoua, niamey and tillabéry) and is close to that observed in other regions in africa. in mali, tounkara et al. (1996) and sangare et al. (2007) found prevalences of 32.0% and 37.0%, respectively, whilst awa et al. (2002) found a prevalence of 30.0% in cameroon. these results could be accounted for by the fact that in arid and semi-arid areas where ppr develops in an enzootic manner, cases generally appear only when other factors contribute to a weakening in the animals. in the republic of niger, cold weather conditions from november to february and low temperatures predispose animals to lung diseases. during this period, pastures are virtually non-existent and the resulting shortage of food lessens the resistance of animals, thus making them susceptible to various diseases. the seroprevalence in other regions is lower than found in our study. in fact, prevalences below 30.0% have been noted in many regions of the world. for example, a seroprevalence of 28.9% was observed in northern burkina faso (sow et al. 2008), 24.0% in oman (taylor et al. 1990), 22.4% in turkey (özkul et al. 2002), 19.0% in côte d’ivoire (couacy-hymann et al. 2002) and 10.0% in ethiopia (abraham et al. 2005). the present study showed that the ppr seroprevalence was slightly higher in goats (47.9%) than in sheep (42.0%) although the difference was not significant. this trend is consistent with that observed in cameroon (awa et al. 2002), where the seroprevalence in goats (44.0%) was higher than that observed in sheep (29.0%). the same trend is observed in pakistan, where khan et al. (2007) found a higher seropositivity in goats (51.2%) than in sheep (39.0%). in north-western mali, sangare et al. (2007) noted a seroprevalence of 44.0% in goats and 34.0% in sheep. however, these results contradict those noted by sow et al. (2008) in northern burkina faso, where the seroprevalence was 33.1% in sheep and 23.0% in goats. in turkey, seroprevalences of 29.2% and 20.0% were found in sheep and goats, respectively (özkul et al. 2002). in ethiopia, abraham et al. (2005) found a prevalence of 13.0% in sheep and 9.0% in goats. these varying results show that the sensitivity to ppr is not necessarily linked to the given species of small ruminants, but rather to rearing conditions and other individual factors. furthermore, it appears from the study that the seroprevalence of ppr varies according to area. the prevalence noted amongst sheep and goats was much higher in tahoua (49.8%) and tillabéry (46.6%) than in niamey (25.1%). the relatively high prevalence in tahoua and tillabéry could be due to a higher number of ppr-affected animals from which blood samples were collected. the overall seroprevalence found amongst female animals was 45.8%, which was not significantly different from that found in male animals. the ppr seroprevalence amongst young animals (51.8%) was much higher than amongst adults (42.0%). this can be accounted for by the fact that the susceptibility of animals to infection by the ppr virus is linked to age (lefevre 1991). young animals (between 2 and 18 months old) seem more prone to ppr than adults. these results are not consistent with those by sow et al. (2008), who noted a prevalence of 33.4% in adults compared with 28.0% in young animals. tounkara et al. (1996) further noted that the ppr seroprevalence is higher in older small ruminants because in an enzootic area they are more prone to contamination by the virus. pcr analyses confirmed the presence of the ppr virus in surveyed farms. similar results were obtained by balamurugan et al. (2006). conclusion top ↑ this study showed that the seroprevalence of ppr is relatively high (45.0%) amongst sheep and goats in the north-western region of the republic of niger. precarious breeding conditions, malnutrition and nutritional deficiencies due to an uncertain rainy season and harsh climates (intense cold and dust) are, to a large extent, the predisposing factors.in a country where farming contributes greatly to the gross domestic product, it is important to protect livestock against diseases associated with serious economic impact, such as ppr. however, the republic of niger is currently confronted with conflicting issues, marked by rapid population growth and an increasing need for animal protein without the resources allocated to the health improvement of livestock being adjusted accordingly. these elements make research and development choices complex and difficult. priority themes and strategies should be adapted, taking into account the prevailing disease conditions and means of intervention. given the economic imperatives, sound decisions should be made to allocate funds to activities that would have an optimal positive impact on livestock productivity in a short period. the fight against ppr plays a significant role in this regard. further work should investigate the role of other pathogens and environmental risk factors in the spread of the disease to improve the understanding of the emergence of the disease and its socio-economic impact. acknowledgments top ↑ the authors would like to thank dr. issoufou maikano, his team of the directorate of veterinary laboratory and the karkara ngos agents for their contribution to the realisation of this study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions s.f. 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2190kabongo_pp273-279.qxd introduction bovine viral diarrhoea virus (bvdv) is a pathogen of cattle with a worldwide distribution. it causes a variety of prenatal and postnatal clinical syndromes. together with the viruses of border disease (bd) and hog cholera (hc) it forms the genus pestivirus within the family flaviviridae (horzinek 1991; collett 1992). the genome of bvdv is a single-stranded, positivesense, non-polyadenilated rna of approximately 12.5 kb in length. it has two non-coding regions at the 5’ end (5’ntr) and at the 3’ end (3’ntr) of the genome. translation occurs in a cap-independent manner from a single large open reading frame (orf) that encodes a polyprotein of about 4 000 amino acids. the polyprotein is coand post-translationally processed by viral and host cell proteases 273 onderstepoort journal of veterinary research, 70:273–279 (2003) molecular analysis of bovine viral diarrhoea virus isolates from south africa n. kabongo1,2, c. baule3 and m. van vuuren1 abstract kabongo, n., baule, c. & van vuuren, m. 2003. molecular analysis of bovine viral diarrhoea virus isolates from south africa. onderstepoort journal of veterinary research, 70:273–279 the presence of bovine viral diarrhoea virus in south africa has been confirmed by several serological surveys. however, little is known about its biological properties. twenty five isolates obtained by isolation in tissue culture and detected by means of the antigen capture elisa from clinically sick cattle and from foetal calf serum in south africa were characterized on the basis of analysis of the 5’ non-translated (ntr) region of the genome. a reverse-transcription polymerase chain reaction (rt-pcr) was used to amplify specific sequences from the 5’ntr of the genome. the oligonucleotide primers corresponding to positions 105–125 and 399–378, respectively, in the sequence of bvdv strain nadl were used to generate the pcr products. both strands were sequenced directly with these primers and fluorescence-labelled dideoxynucleotides in an automated nucleic acid sequencer. reference strains of pestiviruses [(bvdv type i, bvdv type ii, border disease virus (bdv) and hog cholera virus (hcv)] and isolates from a previous investigation on bvdv in southern africa were included for comparative purposes. all the bvdv strains obtained during this study belong to subgroups of bvdv genotype i. no association could be demonstrated between the geographic origin of the isolates. a number of isolates formed another branch separate from the existing branches ia, ib and ic. these findings suggest that extensive genetic diversity can be found within bvdv type i isolates from southern africa. isolates that group with the classical bvdv type i strains, particularly of american origin, coexist with variants that appear to represent a local genetic pool and or variants evolving from the classical strains. keywords: bvdv 5’ntr, phylogeny, reverse-transcription polymerase chain reaction, sequencing, south africa 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 bacteriology division, onderstepoort veterinary institute, agricultural research council, private bag x05, onderstepoort, 0110 south africa 3 swedish university of agricultural sciences, veterinary faculty, department of veterinary microbiology, section of virology, biomedical center, box 585, s-751 23 uppsala, sweden accepted for publication 12 may 2003—editor into mature structural and non-structural proteins. the bvdv genome encodes four structural proteins: capsid protein (c), envelope protein with intrinsic rnase activity (erns), transmembrane glycoprotein (e1) and envelope glycoprotein (e2). at least seven non-structural proteins (n pro, p7, ns2-ns3, ns4 a, ns4 b, ns5 a and ns5 b) are found (collett 1996; thiel, plagemann & moennig 1996). the 5’ntr of the genome is highly conserved among pestiviruses and for this reason it has been used as a target for molecular detection of pestiviruses and for genetic discrimination among and within pestivirus genotypes (pellerin, vandenhurk, lecomte & tijssen 1994; lowings, ibata, needham & paton 1996; baule, van vuuren, lowings & belák 1997; becher, orlich, shannon, horner, konig & thiel 1997; vilcek, alenius, paton, mittelholzer & belák 1999). two genotypes of bvdv have been described on the basis of the 5’ntr analysis, namely bvdv type i, which is subdivided into subgroup ia and represented by the reference strain nadl, and subgroup ib, represented by the reference strain osloss. bvdv type ii has strain 890 as reference and comprises especially isolates associated with a new form of acute infection in cattle, the haemorrhagic syndrome, originally described in north america (pellerin et al. 1994). bvdv type ii has also been shown to include ovine isolates (vilcek, nettleton, paton & belák 1997; ridpath, neil, frey, landgraft & thiel 2000); and, recently, to be present outside north america (canal, strasser, hertig, masuda & peterhans 1998; dean & leyh 1999; flores, weiblen, gil, tobias, lima, garcez & botton 2000). the natural transmission of pestiviruses which are not highly host specific between hostspecies has prompted a new classification of the members of the pestivirus genus. the suggested classification takes into account the antigenic and genomic relationship rather than the species of origin (becher et al. 1997; sullivan, chang & akkina 1997; vilcek et al. 1997). accordingly, the genus pestivirus is divided into four genotypes: genotype 1 (pestivirus type 1) that includes the present bvdv type i strains; genotype 2 (pestivirus type 2) represents isolates of hcv; genotype 3 (pestivirus type 3) includes sheep and pig isolates defined as “true bd” viruses and genotype 4 (pestivirus 4) includes isolates of cattle and sheep currently defined as bvdv type ii. the presence of bvdv in southern africa has been confirmed by several serological surveys (theodoridis, boshoff & botha 1973; theodoridis & boshoff 1974; barnard 1977; depner, hubschle & liess 1991; giangaspero, alders, baer, blondel & morgan 1991; van vuuren 1991; baule & banze 1994; muvavarirwa, mudenge, moyo & javangwe 1995; ferreira, lourens & van vuuren 2000). however, little is known about its biological properties. the aim of this paper was to expand knowledge on the genetic characteristics of local isolates and those from the previous study by baule et al. (1997). materials and methods specimens specimens of blood, organs and lymphoid tissues from sick and dead cattle were obtained from feedlots, commercial beef farms and dairy farms or were submitted by private practitioners and feedlot consultants. other specimens comprised cell lines (n = 3) submitted for testing for the presence of adventitious viruses, and pooled serum obtained from foetuses (n = 7) at an abattoir. some specimens were tested in duplicate, which accounts for the total number of 117 (table 1). tissue filtrates of the original specimens or sera were inoculated either on madin darby bovine kidney (mdbk) line cells or primary and secondary cells calf foetal kidney cells (cfk). the viruses that were isolated, were identified by means of specific fluorescein-conjugated antisera and antigen capture elisa tests. rt-p c r of the 5’ntr of the bvdv genome total rna was extracted from supernatants of infected cells, tissue homogenates and serum specimens, using trizol (gibco, life technologies), according to the manufacturer’s instructions. cdna was synthesized by random priming with pdn6 (amersham-pharmacia, uppsala, sweden) using moloney murine leukaemia virus reverse transcriptase (m-mlv rt) (gibco, life technologies), as follows: 5 µl of total rna were mixed with 0.02 u of pdn6 and 3 µl of ddh2o and denatured at 65 °c for 10 min, then quickly chilled on ice. a reaction mix containing 4 µl of 5x 1st strand buffer, 2 µl of 0.1 m dtt, 0.5 µl of each dntp (10 mm each), 24 u of rnase inhibitor (rna guard, amersham-pharmacia) and 200 u of m-mlv rt was added. synthesis was carried out at 37 °c for 90 min, followed by the inactivation of the enzyme at 95 °c for 5 min. a polymerase chain reaction (pcr) was used to amplify specific sequences from the 5’ntr of the genome. the oligonucleotide primers used were as 274 molecular analysis of bovine viral diarrhoea virus isolates from south africa follows (corresponding to positions 105–125 and 399–378, respectively, in the sequence of bvdv strain nadl): forward – 5’-agccatgcccttagtaggact-3’ reverse – 5’-actccatgtgccatgtaca-3’ amplification was carried out in a total volume of 50 µl containing 10 mm tris-hcl, ph 9.0, 50 mm kcl, 1 µg/µl of bsa, 0.2 mm of each deoxynucleotide, 15 pmol of each primer, 2.5 mm mgcl2, 2.5% formamide, 1 u of taq dna polymerase (perkin elmercetus, norwalk, ca, usa), and 5 µl of cdna. the reaction mixes were overlaid with two drops of mineral oil. pcr cycles were as follows: 5 cycles of denaturation at 94°c for 45 s, annealing at 55°c for 45 s and extension at 72 °c for 1 min, followed by 30 cycles of denaturation at 94 °c for 45 s, annealing at 50°c for 45 s and extension at 72°c for 1 min. a final extension step at 72 °c for 7 min was included. precautions to avoid contamination as described by belák & ballagi-pordány (1993) were followed throughout the rt-pcr. the pcr products were visualized by ethidium bromide staining, after electrophoresis on 2 % agarose gels. sequencing and sequence analysis the amplicons were purified using the qiaquick dna purification kit (qiagen), according to the manufacturer’s instructions and spectrophotometrically quantified. both strands were sequenced directly with the same primers used to generate the pcr products and fluorescence-labelled dideoxynucleotides in an automated nucleic acid sequencer (abi prism 377). the primers were selected based on alignments of sequences of various pestiviruses (bvdv type i, csfv and bdv). highly conserved parts of the 5’ntr were used for the selection of primers. these primers have also been evaluated for the amplification of bvdv type ii as well and were therefore suitable for the detection of all known pestiviruses. nucleotide sequence editing, analysis and alignments were done using multiple programmes from the dnastar package (dnastar inc., madison, wi.). the phylogenetic analysis presented was completed following alignment of nucleotide sequences using the megalign. reference strains of pestiviruses, nadl-bvdv type i, subgroup ia (american type), osloss-bvdv type i, subgroup ib (european type), 890-bvdv type ii, bdv and hcv and isolates ic from a previous investigation on bvdv in southern africa (baule et al. 1997) were included for comparative purposes. the criteria for assignment of genotype were based on sequence similarity as shown in the phylogenetic tree. strains branching with or similarly to nadl are considered subgroup ia, with osloss subgroup ib and so forth. the embl/genbank/ddbj for the nucleotide sequences corresponding accession numbers are: af041040, m31182, m96751, m96687, l32885, l32888 and sequences selected from u97409-u97481. the phylogenetic tree was edited with the deneba canvas (5.0) graphic programme. results a total of 117 specimens were subjected to molecular characterization of which 25 were confirmed positive with pcr (table 1). eighteen isolates obtained by isolation in tissue culture and seven isolates detected in foetal calf sera by means of the antigen capture elisa were confirmed as bvdv with pcr. eight other specimens that included two sera, three buffy coats, two spleens and one lung gave inconclusive readings with the fa test in cell cultures. two were confirmed negative and six yielded a weak band with pcr. they were not molecularly analyzed and were not included in the phylogenetic tree (fig. 1). all of the strains were identified as bvdv i, either subgroups bvdv ia (nadl-like) or bvdv ib (osloss-like) or bvdv i*. table 2 shows isolates that were confirmed by pcr and the predominant clinical signs associated with them. seven isolates obtained from 156 pooled serum specimens and three cells lines of unknown history were included under the heading “others”, since no clinical syndrome could be ascribed to them. the phylogenetic assignment of these isolates, compared to reference strains of pestiviruses and to sequences from a previous investigation with bvdv isolates from southern africa is shown in fig. 1. the phylogenetic tree was generated based on a comparison of 245 nucleotide long sequences in the 5’ntr. the distances were calculated using the neighbor-joining method. the bvdv isolates listed in fig. 1 were determined to be bvdv type i. the 25 isolates analyzed were phylogenetically discriminated as follows: two (st22f/99, st2g/99), segregated clearly as subgroup ia; none was found under subgroup ib; three (st25g/99, st23f/99, st24g/99) were included in a cluster provisionally termed ic (baule et al. 1997), whilst the remaining isolates formed a separate cluster named i*. 275 n. kabongo, c. baule & m. van vuuren 276 molecular analysis of bovine viral diarrhoea virus isolates from south africa �������� �������� �� � ��� �������� ���� ��� ����� ��� �� ���� ������� ����� ��� ��� � ��� ������� ������� ����� ��� �������� ��� ����� �������� �������� ������� ������� ����� � ����� � �������� �������� ����� ��� ������ ��� ������� �������� ����� ��� ������� ������� ������ ������� �������� � ����� ������� ������� ������ ��� ���� �������� �� ��� ������ � ���� ����� �� ������ ������ ������ ������ ���� � � ������ �������� ������ ������ ������ ������ �������� ������ ������� ��� ��� � ����� ������� ������ �� ���� � ����� �������� ��� ���� ��� ���� ������ ����� � �� ����� ����� ��� ��������� ������� � ����� ��������� ������� �������� ������� �������� ��������� ������ �������� ����� �� �������� �������� � � ����� ����� ������������ ��� ��� ����������� ����� ��� ��� � ��� ��� �� ��� �� � �� ! � � � " fig. 1 phylogenetic analysis of bvdv isolates and sequences from genbank reference strains of bvdv type i (nadl, osloss), bvdv type ii (890), bdv, hcv and isolates from a previous investigation on bvdv are included. isolates obtained during the present investigation are indicated in bold. the phylogeny is based on analysis of 5'ntr of the genome. the isolates (see table 2) cluster into subgroup ia, subgroup ic and subgroup i* discussion there was no relationship between the geographic origin, the nature of the clinical signs and the typing of the bvdv isolates. animals from the north-west (nw); free state (f); g (gauteng) and eastern cape (ec) provinces were infected with the same strain. this may inter alia be the result of the free movement of animals, the absence of closed herds or vaccination. throughout south africa, there is a diversity of farming systems from extensive to intensive, including closed herds where artificial insemination (ai) is used. isolates were obtained from samples collected in feedlots, dairy herds and commercial beef farms in all provinces, indicating the ubiquitousness of bvdv in south africa. the reverse-transcription pcr based on the 5’ntr of the virus genome and further sequencing enabled differentiation of bvdv genotypes and subgroups; this is of epidemiological importance and might be of value in control programmes. it has been reported that direct detection of the virus in serum or homogenized tissue specimens clinical samples by rt-pcr is often unsuccessful (elkholy, bolin, ridpath, arab, abou-zeid, hamman & platt 1998). this might be due to either the presence of certain elements in the clinical specimens that are inhibitory to reverse transcriptase or taq polymerase enzymes or to masking of the target template by proteins coagulated during extraction of nucleic acids in the clinical specimens. 277 n. kabongo, c. baule & m. van vuuren table 1 bvdv isolates obtained by virus isolation, elisa and confirmed with pcr specimens number elisa positive virus isolation positive pcr positive serum 54 7 4 11 whole blood 32 3 3 3 spleen 10 nt 3 3 lung 8 nt 2 2 lymph nodes 10 nt 3 3 cell lines 3 nt 3 3 total 117 7 18 25 nt: not tested * three buffy coats that tested positive on both tests table 2 predominant clinical syndrome associated with bvdv isolates and their origin respiratory respiratory diarrhoea diarrhoea others +pyrexia +pyrexia (n = 7) (n = 3) (n = 3) (n = 2) (n = 10) ncpst5f/98 ncpst11nw/98 ncpst1g1/98 ncpst3g/98 ncpst14ec/99 ncpst6g/98 ncpst12nw/98 ncpst2g2/98 ncpst17g/99 ncpst15f/99 ncpst7f/98 ncpst13nw/98 ncpst4g/98 ncpst16f/99 ncpst8f/98 ncpst18f/99 ncpst21g/98 ncpst19f/99 ncpst9nw/98 ncpst20nw/98 ncpst10nw/98 ncpst22f/99 ncpst23f/99 ncpst24g/99 ncpst25g/99 ncp: noncytopathogenic * this classification is based on the predominant clinical signs observed by the clinician in naturally infected animals. the enteric syndrome manifests as acute or chronic diarrhoea; and the respiratory syndrome as nasal discharge, respiratory distress, sneezing and coughing, while “others” include those of unknown history and those in which no clinical syndrome ascribed to the case ** identification of isolates: ncp: noncytopathic biotype, followed by s for south africa, t for tropical diseases, the isolate id and the area where it came from, the number after province of origin, where applicable, represents number of samples from the same sender in order of submission, which is followed by year of isolation. the letters that represent the province of origin: nw: north-west; f: free state; ec: eastern cape; g: gauteng six clinical samples from which virus had not been isolated showed a weak band with rt-pcr although it was situated at the correct molecular weight position. these six specimens were not molecularly analyzed nor were they included in the phylogenetic tree. the results obtained with pcr were in agreement with those obtained by virus isolation in all the negative cases except in seven out of 156 pooled sera that were negative for virus isolation after one passage but tested positive on antigen capture elisa. this confirms the need for more than one passage before virus becomes detectable with the fa test. all the bvdv strains obtained during this study were ncp bvdv i (bvdv ia (nadl-like), bvdv ic subgroups or bvdv i* although theodoris isolated cp bvdv in 1974. no association could be demonstrated between the geographic origin of the isolates and branch discrimination. the three groupings formed by the south african isolates (subgroup ia, cluster ic and cluster provisionally called i*) included bvd viruses from different regions: f, g, nw and ec. it is worth noting their similarity to isolates of the bvdv cluster provisionally termed ic in a previous investigation (baule et al. 1997) which did not segregate with either the ia or the ib subgroups. the presence of isolates of this cluster in south africa may reflect a local genetic subgroup that is spreading in the region since genotype i shows an intragenotyping diversity. this might have occurred because of cattle movement or the use of biological such as cell culture-derived vaccines. no type bvdv type ii were found, however the vaccine appears to be protective against both types i and ii. a number of isolates i* (n = 20) formed another branch separate from ia, ib or ic. this branch was, however; distinct from the one defining a cluster preliminary termed id by baule et al. (1997) and was found to comprise isolates particularly distinct from the ia and ib subgroups. these findings suggest that an extensive genetic diversity can be found within bvdv type i isolate from southern africa. isolates that group with the classical bvdv type i strains, particularly of american origin, coexist with variants that appear to represent a local genetic pool and/ or variants evolving from the classical strains. a clustering of isolates with regards to farms of origin was not observed with the isolates investigated, as has been reported by others (paton 1995; vilcek et al. 1999). differences in farming practices, i.e. extensive farming versus intensive farming may contribute to this difference in virus ecology. closed herds and restricted contact among cattle may be a determinant factor to establish bvdv in a herdspecific manner. most herds from which the samples originated were managed extensively. references barnard, b.j.h. 1977. virus diseases in feedlot cattle in 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van vuuren reviewer acknowledgement open accesshttp://www.ojvr.org page 1 of 1 onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of onderstepoort journal of veterinary research: alan guthrie alan walker arthur spickett augustine estrada-pena bruce gummow cattoli giovanni dmitry apanaskevich elizabeth boomker eva de clerq flip van schalkwyk francis vercammen francois malan fred potgieter graeme cumming ivan horak jackie picard jim mclachlan johan vorster leon prozesky we appreciate the time taken to perform your review successfully. manuel fondevilla marthie ehlers maryke henton maxime madder moritz jansen van vuuren morne de la rey nick kriek otto koekemoer quentin wessels rick last rudolph bigalke shahn bisschop tertius gous trevor petney truuske gerdes ulrike seitzer victor cortese yasuhiko suzuki in an effort to facilitate the selection of appropriate peer reviewers for onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on www.ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ojvr.org 2. in your ‘user home’ [http://www.ojvr.org/ index.php/sajhrm/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to onderstepoort journal of veterinary research. please do not hesitate to contact me if you require assistance in performing this task. margo martens submissions@ojvr.org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 journal of veterinary research onderstepoort foreword_iii.indd in 1908 a pan-african veterinary conference formed part of the inauguration ceremony of the onderste poort veterinary laboratory. attended by 18 delegates from 12 countries in southern africa, including the four colonies and three protectorates forming british south africa, rhodesia, german south west africa, portuguese east africa, mada gascar and the belgian congo, discussions focussed on the animal diseases of the region with the emphasis on trypanosomosis (nagana) and east coast fever. the successful meeting was followed by a series of similar conferences held in different african countries during the first half of the 20th century. it was therefore fitting that the centenary celebrations of the onderstepoort complex in 2008 should again be accompanied by a pan-african meeting of those involved in combating animal diseases on the continent. the intention was to celebrate accomplishments of the past but also to look into the future and plan the way forward. the focus was on research achievements and needs but due consideration was also given to the important contributions of veterinary training, veterinary field services and vaccine production. the invited contributions of a number of experts in the field of african animal diseases are recorded for posterity in these proceedings. scientific and peer review committee: d.w. verwoerd (chair) a.j. musoke b.l. penzhorn m. van vuuren w. vosloo w.j.s. botha g.r. thomson pretoria march 2009 iii foreword proceedings: onderstepoort centenary pan-african veterinary conference onderstepoort south africa, 7–9 october 2008 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice markotter_257-262.indd introduction rabies is caused by all members of the lyssavirus genus in the family rhabdoviridae of the order mononegavirales, a group of single stranded negative sense rna viruses currently consisting of seven genotypes (gts). classical rabies viruses (rabv) are grouped in the lyssavirus genotype 1 (gt 1) and occur worldwide (tordo, benmansour, calisher et al. 2004). two biotypes of rabv occur in africa; the mongoose biotype and the canid biotype (von teichman, thomson, meredith & nel 1995; nel, sabeta, von teichman, jaftha, rupprecht & bingham 2005). in addition, gt 2 (lagos bat virus (lbv)), gt 3 (mokola virus (mokv)) and gt 4 (duvenhage virus (duvv)) have been reported exclusively from the african continent (nel & markotter 2007). there is little surveillance for these gts but all have been reported from south africa and other african locations in recent years (paweska, blumberg, liebenberg, hewlett, grobbelaar, leman, croft, nel, nutt & swanepoel 2006; markotter, kuzmin, rupprecht, randles, sabeta, wandeler & nel 2006a; markotter, randles, rupprecht, sabeta, wandeler, taylor & nel 2006b; sabeta, markotter, mohale, shumba, wandeler & nel 2007; kuzmin, niezgoda, franka, agwanda, markotter, beagley, urazova, breiman & rupprecht 2008). the gold standard for lyssavirus diagnostics approved by both the world organization for animal health (oie) and the world health organization (who) (who 2005), the fluorescent antibody test 257 onderstepoort journal of veterinary research, 76:257–262 (2009) evaluation of a rapid immunodiagnostic test kit for detection of african lyssaviruses from brain material w. markotter1*, d. york2, c.t. sabeta3, w. shumba3, g. zulu3, k. le roux4 and l.h. nel1 abstract markotter, w., york, d., sabeta, c.t., shumba, w., zulu, g., le roux, k. & nel, l.h. 2009. evaluation of a rapid immunodiagnostic test kit for detection of african lyssaviruses from brain material. onderstepoort journal of veterinary research, 76:257–262 a rapid immunodiagnostic test kit was evaluated against a selection of isolates of lyssavirus genotypes occurring in africa. the test was carried out in parallel comparison with the fluorescent antibody test (fat) and isolates representing previously established phylogenetic groups from each genotype were included. the specificity of the rapid immunodiagnostic test compared favourably with the fat and was found to detect all representatives of genotypes 1, 2, 3 and 4 in brain samples of either field cases or suckling mouse brain inoculates. keywords: africa, diagnostics, lyssavirus, rabies, rapid test * author to whom correspondence is to be directed. e-mail: wanda.markotter@up.ac.za 1 department of microbiology and plant pathology, university of pretoria, pretoria, 0002 south africa 2 molecular diagnostic services, private bag x20, westville, dur ban, kwazulu natal, 3630 south africa 3 oie rabies reference laboratory, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 4 allerton veterinary laboratory, private bag x2, cascades, pieter maritzburg, 3202 south africa accepted for publication 1 july 2009—editor 258 evaluation of immunodiagnostic test kit for detection of african lyssaviruses from brain material (fat) (dean, abelseth & atanasiu 1996), is performed on brain tissue. these preparations are stained with anti-lyssavirus serum (conjugate) labelled with fluorescein isothiocyanate (fitc) and viewed under a fluorescence microscope. this test therefore cannot be performed under field conditions since specialized equipment is needed. it is important that rabies diagnosis be quick and reliable in order to ensure correct post exposure prophylaxis (pep) decisions and prevent fatal infections in cases of human exposures. diagnostics in epidemiological studies aimed at prevention and control of the disease in the relevant reservoir species should also be as practical as possible. routine diagnostic tests should focus on brain tissues (brainstem is the optimal region). diagnosis of lyssaviruses in humans is fundamentally different from diagnosis in animals due to the fact that the former most often requires ante-mortem diagnosis while in animals it is usually performed post-mortem (trimarchi & smith 2002). methods involved in antemor tem diagnosis depend on a high level of technical expertise and a well equipped laboratory. in africa and other developing regions of the world, lyssavirus diagnostics and surveillance are seriously hampered due to the lack of facilities and logistical support for reliable execution of the fat. fur thermore, in some instances where a diagnostic facility does exist and is operational, the need to efficiently transport samples to a central facility can often not be met. these obstacles in obtaining a diagnostic result from field specimens have led to serious underreporting of the disease and have ultimately resulted in a lack of commitment to control the disease. a rapid immunodiagnostic test kit (ridt) for rabv, that could offer advantages towards overcoming some of the difficulties mentioned above, has been developed (kang, oh, lee, park, park, hong, lee, cho & song 2007). this lateral flow test uses gold conjugated detector antibodies, including a monoclonal antibody directed against the lyssavirus nucleoprotein. according to kang et al. (2007), the test is rapid and simple and does not require any specialized equipment or technical expertise—but their study included only gt 1 viruses of which none were of african origin. since gt 1–4 lyssaviruses occur in africa, the test should be able to identify all these gts in order to be of any potential use in africa. it was therefore our aim to evaluate this test for its ability to detect the most diverse isolates within those african lyssavirus gts and variants known to us. materials and methods samples a total of 25 samples that included 21 diverse representatives of all the african lyssavirus genotypes and four negative controls (table 1), were tested. samples were selected based on previous phylogenetic studies to be representative of different phylogenetic groups within each genotype (nel et al. 2005; sabeta et al. 2007; cohen, sartorius, sabeta, zulu, paweska, mogoswane, sutton, nel, swanepoel, leman, grobbelaar, dayson & blumberg 2007; markotter, kuzmin, rupprecht & nel 2008). fluorescent antibody test (fat) results were compared with those obtained with the standard fluorescent antibody test (fat) (dean et al. 1996) using a polyclonal fluorescein isothiocyanate conjugated immunoglobulin (onderstepoort veterinary institute, oie rabies reference labor atory). rapid immunodiagnostic test (ridt) the ridt test was performed as described by the manufacturer (animal genetics, inc). briefly, a 20 % brain suspension of each isolate was prepared in pbs. a swab supplied with the test kit was dipped into the brain homogenate after which it was transferred to the assay diluent for extraction. after 1 min, four drops of the sample were added to the sample well, using the supplied dropper. final results were read 5 min after application of the sample. all samples were tested in duplicate. in each case, the appearance of two lines was considered a positive result (one in the test zone and one in the control zone), while the formation of only one line in the control zone was considered negative. purified monoclonal antibody against the rabies virus nucleoprotein was attached to the test zone and purified goat anti-mouse igg was attached to the control zone (kang et al. 2007). results all 21 samples that tested positive with the fat also tested positive with the ridt and all four negative samples tested negative (table 1 and fig. 1). although the intensity of the test lines was found to vary between different virus samples, all the tests were clearly readable and there were no cases of doubtful interpretation. most samples reacted after about 2 min and none of the samples indicated any 259 w. markotter et al. table 1 list of lyssavirus-positive samples included in the evaluation of the specificity of the rapid immunodiagnostic test (ridt) against african lyssaviruses* sample number genotype host sample laboratory identification number year of isolation geographical origin reference 1 gt 1 (canine biotype) canine original canine brain 272/06 2006 south africa, limpopo cohen et al. 2007 2 gt 1 (canine biotype) canine original canine brain 567/04 2004 south africa, kwazulu natal cohen et al. 2007 3 gt 1 (canine biotype) canine original canine brain 479/96 1996 south africa, limpopo cohen et al. 2007 4 gt 1 (canine biotype) black-backed jackal original jackal brain 819/05 2005 south africa, north west province cohen et al. 2007 5 gt 1 (canine biotype) bat-eared fox original bat-eared fox brain 31/05 2005 south africa, eastern cape cohen et al. 2007 6 gt 1 (mongoose biotype) galerella sanguinea original mongoose brain 22107 1994 zimbabwe nel et al. 2005 7 gt 1 (mongoose biotype) cynictis penicillata original mongoose brain 669/90 1990 south africa, mpumalanga nel et al. 2005 8 gt 1 (mongoose biotype) cynictis penicillata original mongoose brain 767/95 1995 south africa, free state nel et al. 2005 9 gt 1 (mongoose biotype) cynictis penicillata original mongoose brain 364/96 1996 south africa, eastern cape nel et al. 2005 10 gt 1 (mongoose biotype) atilax paludinossus original mongoose brain 113/91 1991 south africa, western cape nel et al. 2005 11 gt 2 (lineage a) bat (epomophorus wahlbergi) original bat brain lbvsa2008 2008 south africa unpublished 12 gt 2 (lineage a) bat (rousettus aegyptiacus) suckling mouse brain lbvafr1999 1999 senegal markotter et al. 2008 13 gt 2 (lineage b) bat (eidolon helvum) suckling mouse brain lbvnig1956 1956 nigeria markotter et al. 2008 14 gt 2 (lineage c) mongoose (atilax paludinossus) suckling mouse brain lbvmongoose 2004 2004 south africa markotter et al. 2008 15 gt 3 feline suckling mouse brain 12341 1981 zimbabwe sabeta et al. 2007 16 gt 3 feline suckling mouse brain 543/95 1995 south africa, east london sabeta et al. 2007 260 evaluation of immunodiagnostic test kit for detection of african lyssaviruses from brain material sample number genotype host sample laboratory identification number year of isolation geographical origin reference 17 gt 3 feline suckling mouse brain 97/252 1997 south africa, pietermaritzburg sabeta et al. 2007 18 gt 3 canine suckling mouse brain 173/06 2006 south africa sabeta et al. 2007 19 gt 4 human suckling mouse brain duvvsa2006 2006 south africa paweska et al. 2007 20 gt 4 bat (unknown) suckling mouse brain duvvsa1981 1981 south africa paweska et al. 2007 21 gt 4 human suckling mouse brain duvvsa1970 1970 south africa paweska et al. 2007 * as negative controls, phosphate buffered saline (pbs), uninfected mouse, uninfected canine and uninfected bat brain were used fig. 1 results of the rapid immunodiagnostic test (ridt) after testing negative control samples as well as representatives of african lyssaviruses (table 1). samples 1–21 indicate a positive result with different representatives of african lyssaviruses. negative control samples (pbs, canine and bat uninfected brain) indicated a neg ative result. c = control line and t= test line 261 w. markotter et al. non-specific reactions. no difference in results between field samples and isolates that were passaged in suckling mouse brains was observed. discussion rabies is endemic in most developing countries. for most countries in africa no rigorous epidemiological data for lyssaviruses exist—largely due to the lack of operational rabies diagnostic facilities. the fat requires a fluorescent microscope, which is not only expensive but needs to be well maintained and is sensitive to power surges and blackouts that are common throughout africa. many afri can governments simply cannot comply with the requirements that would ensure fully competent fat diagnostics. in other cases, where a diagnostic facility may exist, its usefulness is restricted to specific geo graphic locations and samples from remote areas seldomly or never reach these laboratories. under these circumstances, the ridt may be a useful tool—in our hands it has demonstrated a high specificity (100 %) against representatives of all the known african lyssavirus genotypes when compared to the fat. this is a very simple test that can be performed in less than 10 min without any specialized equipment, infrastructure, or high level of training. there are no critical points to field use such as cold storage, since the test kit contains everything required for the diagnosis and is stable at ambient temperatures. as indicated in another evaluation of the test, it is capable of detecting low amounts of virus – at an excellent level of sensitivity (slightly less sensitive than a well executed fat) (kang et al. 2007). in our hands, several field isolates were examined, including a gt 2 infected bat brain, and all field isolates were detected as positive using the ridt. from these results, the ridt could be a very useful additional tool in field surveillance for lyssavirus infections in those many areas where no other diagnostic method is available or where samples simply cannot easily reach a diagnostic laboratory due to logistical and infrastructural limitations. in these latter scenarios, suspect rabies cases are simply not tested at all (personal observations at several locations in africa). the ridt could specifically assist in better understanding of the epidemiology of lyssavirus infections in wildlife if application in an on-site manner is considered. just one example applies to kudu (tragelaphus strepsiceros) antelope, in which rabies is, in fact, a significant, but still fairly unqualified problem in namibia and potentially elsewhere. areas of wildlife reserve are often very remote, and it is rarely possible for conservationists, game rangers or farmers to duly collect and send samples to a diagnostic laboratory for testing. brain material is required for the ridt and where no adequate necropsy facilities are available, other methods for removal of brain material without the risk of opening the scull, could be applied—including, for example, a trucut needle biopsy through superior orbital fissure (tong, leung, lee & lam 1999). it must be stressed that a reliable rabies diagnosis can only be made post-mortem using brain material and that saliva must not be recommended. the simplicity of the ridt kit also urges us to point out a further area of caution: all personnel performing the test should be vaccinated against rabies and trained to follow the safety regulations and procedures for working with lyssaviruses, and in cases of clear potential human exposures, positive diagnosis should be considered as a matter of course while additional testing by fat should be sought as a matter of urgency. in conclusion, we do not argue that the ridt should be used as replacement for the well accepted gold standard fat. in fact, the preferential use of the fat should be encouraged and implemented wherever possible. we do, however, consider that the ridt could be a useful onsite test under field conditions and in developing countries with limited diagnostic resources. the technique has the potential of enhancing epidemiological surveillance of lyssaviruses under such conditions and in remote areas where lyssaviruses infections otherwise go unnoticed or unquantified. after the diagnosis samples can also be stored in phosphate-buffered 50 % glycerol saline and stored at 4 °c, or can be kept at room temperature for up to 4 months for future characterization, if needed. in this regard the ridt, together with a renewed global focus on rabies as one of the most seriously neglected zoonoses, may assist in obtaining at least some rabies epidemiological information from those many parts of africa that report rabies haphazardly or not at all. acknowledgements we thank animal genetics, inc who supplied the rapid immunodiagnostic kits for this study. references cohen, c., sartorius, b., sabeta, c., zulu, g., paw eska, j., mogoswane, m., sutton, c., nel, l., swanepoel, r., leman, p.a., grobbelaar, a.a., dyason, e. & blumberg, l. 2007. epidemiology and viral molecular virus characterization of reemerging rabies, south africa. emerging infetcious diseases, 3:1879–1886. 262 evaluation of immunodiagnostic test kit for detection of african lyssaviruses from brain material dean, d.j., abelseth, m.k. & atanasiu, p. 1996. the fluorescent antibody test, in laboratory techniques in rabies, edited by f.x. meslin, m.m. kaplan & h. koprowski. geneva: world health organization. kang, b., oh, j., lee, c., park, b., park, y., hong, k., lee, k., cho, b. & song, d. 2007. evaluation of a rapid immunodiagnostic test kit for rabies virus. journal of virological methods, 145:30–36. kuzmin, i.v., niezgoda, m., franka, r., agwanda, b., mar kotter, w., beagley, j.c., urazova, o.y., breiman, r.f. & rupprecht, c.e. 2008. lagos bat virus in kenya. journal of clinical microbiology, 46:1451–1461. markotter, w., kuzmin, i., rupprecht, c.e., randles, j., sabeta, c.t., wandeler, a.i. & nel, l.h. 2006a. isolation of lagos bat virus from water mongoose. emerging infectious diseases, 12:1913–1918. markotter, w., kuzmin, i., rupprecht, c.e. & nel, l.h. 2008. phylogeny of lagos bat virus: challenges for lyssavirus taxonomy.virus research, 135:10–21. markotter, w., randles, j., rupprecht, c.e., sabeta, c.t., wandeler, a.i., taylor, p.j. & nel, l.h. 2006b. recent lagos bat virus isolations from bats (suborder megachiroptera) in south africa. emerging infectious diseases, 12:504–506. nel, l.h. & markotter, w. 2007. lyssaviruses. critical reviews in microbiology, 33: 301–324. nel, l.h., sabeta, c.t., von teichman, b., jaftha, j.b., rup precht, c.e. & bingham, j. 2005. mongoose rabies in southern africa: a re-evaluation based on molecular epidemiology. virus research, 109:165–73. paweska, j.t., blumberg, l., liebenberg, c., hewlett, r.h., grobbelaar, a.a., leman, p.a., croft, j.e., nel, l.h., nutt, l. & swanepoel, r. 2006. fatal human infection with rabies-related duvenhage virus, south africa. emerging infectious diseases, 12:1965–1967. sabeta, c.t., markotter, w., mohale, d.k., shumba, w., wandeler, a.i. & nel, l.h. 2007. recent mokola virus isolations from domestic mammals in south africa. emerg ing infectious diseases, 13:1371–1373. tong, r., leung, k.m., lee, k.c. & lam, a.w. 1999. trucut needle biopsy through superior orbital fissures for rabies diagnosis. the lancet, 354:2137–2138. tordo, n., benmansour, a. & calisher, c. et al. 2004. virus taxonomy: the classification and nomenclature of viruses, in the eight report of the international committee on taxonomy of viruses, edited by c.m. fauquet, m.a. mayo, j. maniloff, u. desselberger & l.a. ball. london: elsevier/academic press. trimarchi, c.v. & smith, j.s. 2002. diagnostic evaluation in rabies, edited by a.c. jackson & w.h. wunner. london: else vier science. von teichman, b.f., thomson, g.r., meredith, c.d. & nel, l.h. 1995. molecular epidemiology of rabies virus in south africa: evidence for two distinct virus groups. journal of general vorology, 76:73–82. who 2005. expert consultation on rabies. geneva: world health organization (technical report 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice ndhlovu_235-248.indd introduction tick infestation and tick-borne diseases (tbds) are important conditions affecting livestock health and productivity in zimbabwe (norval 1979; lawrence, fog gin & norval 1980) and in large areas of subsaharan africa (norval, donachie, meltzer, deem & mahan 1995). examples of major tbds are anaplasmosis, babesiosis, heartwater and theileriosis. ticks are responsible for direct damage to livestock through their feeding habits. the damage is manifested as hide damage, damage to udders, teats and scrotum, myiasis due to infestation of damaged sites by maggots and secondary microbial infections (norval 1983a; meltzer & norval 1993; dreyer, fourie & kok 1998a). there are also productivity losses associated with the various tick species that can occur (norval, sutherst, kurki, gibson & kerr 1988; norval, sutherst, jorgensen, gibson & kerr 1989; stachurski, musonge, achu-kwi & saliki 1993; meltzer, norval & donachie 1995; jonsson 2006). these are not usually obvious to the communal or smallholder farmer but are important if raising of cattle is done at a commercial level. related to productivity losses, concepts such as economic damage threshold and economic threshold (meltzer & 235 onderstepoort journal of veterinary research, 76:235–248 (2009) tick infestation, and udder and teat damage in selected cattle herds of matabeleland south, zimbabwe d.n. ndhlovu1, p.v. makaya2 and b.l. penzhorn1* abstract ndhlovu, d.n., makaya, p.v. & penzhorn, b.l. 2009. tick infestation, and udder and teat damage in selected cattle herds of matabeleland south, zimbabwe. onderstepoort journal of veterinary research, 76:235–248 a cross-sectional survey was conducted to determine tick infestation, and udder and teat damage in 286 lactating cows and heifers at six properties in the smallholder and commercial sectors in gwanda district of matabeleland south province, zimbabwe. eight tick species were identified: amblyomma hebraeum, hyalomma truncatum, hyalomma marginatum rufipes, rhipicephalus evertsi evertsi, rhipicephalus (boophilus) decoloratus, rhipicephalus appendiculatus, rhipicephalus zambeziensis and rhipicephalus simus. overall, 81.5 % of the cattle were tick infested; prevalence of tick-infested cattle was significantly higher on communal land (93.8 %) and recently claimed land (85.1 %) than on commercial farms. the mean tick load on infested cattle on communal land was significantly higher than in the other two sectors. although 53 % of the sampled cattle had some degree of udder and teat damage, very few farmers (2.6 %) treated their cattle for these conditions. udder damage was ca. two times and three times, respectively, more likely to occur in cattle on communal land compared to cattle on recently claimed land and commercial farms. the occurrence of r. appendiculatus and r. zambeziensis indicate that the cattle population in the study area is at high risk of a theileriosis outbreak, a tick-borne disease that has not been reported from this area. keywords: teat damage, tick infestation, udder damage, zimbabwe * author to whom correspondence is to be directed. e-mail: banie.penzhorn@up.ac.za department of veterinary tropical diseases, faculty of vet erinary science, private bag x04, onderstepoort, 0110 south africa 2 division of veterinary services, central veterinary laboratory, box cy 551 causeway harare, zimbabwe accepted for publication 17 november 2008—editor 236 tick infestation and udder and teat damage in cattle of matabeleland south, zimbabwe norval 1993; young & haantuba 1997) need to be considered in tick and tbd control. meltzer & norval (1993) defined the economic damage threshold for amblyomma hebraeum -infested cattle as the minimum average weekly standard female tick burden sufficient to cause damage, equal in dollar value to the cost of applying tick control. this figure can be changed due to damage to udders and teats, infestation of cattle by screwworm chrysomya bezziana and the nutritional condition of the animal. according to young & haantuba (1997), the economic threshold is the lowest pest population that causes a reduction in profit, or equivalently the pest population where the benefits of control equal the costs of eliminating the pest. young & haantuba (1997) reported that the economic threshold for rhipicephalus appen diculatus differed between infected and noninfected ticks. when infectious ticks were considered, then the economic threshold was reached even when only a single tick was present. compulsory short-interval dipping of cattle for the control of ticks and tbds has been enforced in zimbabwe since the early part of the previous century (norval 1979; norval 1983a; norval, perry & hargreaves 1992). initially, intensive dipping was introduced for the control of east coast fever following its introduction from east africa in 1901/1902 during the restocking that followed the rinderpest pandemic of 1896 and the anglo-boer war of 1899–1902 (norval 1979; lawrence et al. 1980; norval 1983a; norval et al. 1992). as the ox-wagon was the main mode of transportation during this period, the pandemic had disastrous consequences. in recent years, the division of veterinary services has faced budgetary constraints and has instituted strategic dipping to reduce costs. strategic dipping entails weekly dipping of cattle during periods of high tick activity in the months of november to april and either fortnightly or monthly dipping during the other months. plunge dipping with amitraz 25.50 % m/m (“tickbuster® wp”, zimbabwe phosphate industries) is practised in the communal areas. in the commercial sector farmers are responsible for the control of ticks and tbds, and they usually practise intensive dipping regimens that are characterized by weekly dipping to ensure that cattle are tick-free. at the same time, vaccines are available for the control of anaplasmosis, babesiosis, zimbabwean theileriosis and heartwater. these vaccines can be procured from the central veterinary laboratory in harare. the agrarian land reform that started in full force in 2000 has seen movement of livestock into former commercial farms and the breakdown of fences between these commercial farms and communal areas. the above scenarios have implications as regards tick species dynamics, their impacts and their distribution in matabeleland south province, zimbabwe. the objective of this cross-sectional study was to characterize tick species, their distribution and their impact on cattle with special emphasis on udder and teat damage at selected dip-tanks and farms of matabeleland south province, zimbabwe. results from the study will add to existing knowledge, guide animal health managers and lay a foundation for further research on ticks and tbds in matabeleland south province. materials and methods study area and experimental animals the study was conducted in gwanda district (fig. 1) of matabeleland south province during the months of january, march and april 2007. a communalarea dip-tank, switsha (21°10’ s, 29°07’ e), was used as a pilot survey site to assess the completeness of the data-capture forms and to assess the ability to identify ticks by officers involved in the study. the other study properties were timber farm (21°01’ s, 29°09’ e) and double cross farm (21°10’ s, 29°09’ e), these two properties being a2 farms. new farmers who were formerly communal farmers own these two farms (a2 sector). the two commercial farms were judds farm (20°53’ s, 29°39’ e) and blanket farm (20°05’ s, 28°27’ e), which are owned and managed by the original farmers from before the land reform. in addition to switsha dip tank, cattle from two communal dip-tanks were sampled: sivume (20°36’ s, 28°31’ e) and nyandeni (20°40’ s, 28°55’ e). the study properties were all located in natural regions 4 and 5 of zimbabwe (ta wonezvi 2005; international crops research insti tute for the semi-arid tropics [icrisat] 2007). this region is characterized by low and erratic rainfall of below 450 mm in region 5 and of between 450 mm and 650 mm in region 4 (icrisat 2007). generally, in these regions, the year can be divided into three seasons: a warm, wet season from mid-november until april, a cool dry season from april to august and a hot dry season from september up to mid-november (colborne, norval & spickett 1981). according to icrisat (2007), the common vegetation type in gwanda district is comprised of colo phospermum mopane, combretum spp., acacia spp., boscia and grewia spp. woodlands. the 237 d.n. ndhlovu, p.v. makaya & b.l. penzhorn common grass species are aristida and panicum spp. a total of 286 female cattle (lactating cows and heifers) were sampled. this does not include the 17 cattle that were sampled at switsha dip tank during the pilot survey. the number of cattle sampled from each property was: blanket farm n = 34, double cross farm n = 46, judds farm n = 39, timber farm n = 21, nyandeni dip-tank n = 55 and sivume dip-tank n = 91. the properties were chosen through convenient sampling, while the sample animals were sampled systematically (thrusfield 1995) at the dip-tanks and farms. as absolute ages of individual animals were unknown, parity (i.e. number of calves borne) was used as an indicator of age. tick counting, collection and identification cattle were restrained in a cattle race for tick counts and tick collections. counting of ticks was done on one side of the animal while all ticks on the udder were counted. the counting was done according to the method of londt, horak & de villiers (1979). the method targeted counting of ticks at their predilection sites on the live animal (baker & ducasse 1967). no attempt was made to identify immature stages or to record gender of the ticks. ticks were counted from six sites: both surfaces of the pinna, the neck region including the dewlap, the fore-leg including the axilla and hoof, upper perineum, the tail brush and the lower perineum which included the whole udder. at least two ticks of each species were collected by hand from the six predilection sites, of three visibly infested cattle after initial identification in situ, and preserved in 70 % alcohol for confirmation of their identity under a stereo-microscope at the central veterinary laboratory in harare. ticks from the same site on the animal and same dip-tank or farm were put in the same bottle, which was labelled with the collection date, site of collec n s w o matobo bulilimamangwe beitbridge matebeleland south province umzingwane gwanda insiza switsha ................... timber farm ........... double cross farm . nyandeni ................ sivume .................... blanket farm .......... judds farm ............. key zimbabwe fig. 1 maps of zimbabwe and mattabeleland south province showing study areas in gwanda district 238 tick infestation and udder and teat damage in cattle of matabeleland south, zimbabwe tion on the animal and the name of the property. tick infestation was defined according to the animal health act (cattle cleansing) regulations, 1993 (gov ernment of zimbabwe 1993). udder and teat damage udder damage in this study was defined as conditions that affected the udder such as induration of one or more quarters, abscessation and open wounds. udder damage was assessed by visual inspection, palpation to check for indurations and from the clinical history given by the owner of the animal. further, it was also determined whether one or more quarters were affected. farmers were verbally questioned on the methods they used to manage conditions affecting teats and udders; these questions were mainly directed to those farmers whose cattle had the condition of interest. the degree of udder damage was further graded as severe, mild, slight, none and other. teat damage was defined to include blocked teats, lost teats and teats from which milk containing pus could be expressed. the damage to teats was assessed by visual inspection, manual palpation and milking all quarters to check for blockages. teat damage was graded as very severe, severe, mild, slight and none. the information on tick numbers, tick species, udder damage, teat damage, treatment or non-treatment and parity of the animal (number of calves borne) were entered into two data-capture forms. one of the forms captured information on udder damage and teat damage, treatment/non-treatment and tick counts at the various predilection sites. the other form captured information on tick species and their numbers. after field collection, the data were entered into excel spreadsheets (microsoft 2000). these data were also transformed into a database in microsoft access 2000. the overall number of tick-infested cows and heifers was calculated from the total number of animals sampled and expressed as a percentage. tick infestation was evaluated in relation to the farming sector, predilection site and parity of the animal. udder and teat damage was assessed in relation to farming sector, parity and tick infestation status of the animal. farming sector, predilection site and parity of the animal categories were generated as follows: three for farming sector (communal, a2 and commercial), six for predilection site (pinna, neck, leg, tail, upper and lower perineum) and three for parity (1–3, 4–6 and > 7). the two-sample proportion-test and two-sample t-test for independent groups in statistix (analytical software 1996, version 1.0) was used to measure differences in proportions between categories and to evaluate the differences in mean number of tick counts for the different categories, respectively. values of p < 0.05 were considered as significant. association between tick infestation and the potential putative risk factors and that between udder and teat damage and the potential putative risk factors was evaluated using epi-info version 6. for tick infestation, the analysis focused on two risk factors, namely farming sector and parity of animal while that for udder and teat damage focused on farming sector, parity and tick infestation status of the animals. the association was assessed by calculating the chi-square (2) test for association, the relative risk (rr) and 95 % confidence intervals (thrusfield 1995). an rr greater than 1 indicates a positive statistical association between factor and outcome, while that less than 1 indicates a negative statistical association and an rr of 1 suggests no association (thrusfield 1995). results tick infestation according to farming sector, predilection site and parity of animals of the 286 cows and heifers sampled, 233 (81.5 %) were infested with ticks (table 1). the communal (93.8 %) and a2 (85.1 %) sectors recorded a significantly higher (p < 0.05) prevalence compared to the commercial farming sector (53.4 %); there was no significant difference between the former two farming sectors. occurrence of tick infestation was significantly associated with the communal sector (2 = 47.8, p < 0.001) and the a2 sector (2 = 14.8, p < 0.001) compared to the commercial sector. tick infestation was found to be approximately two times more likely to occur on the communal sector (rr = 1.8, 1.4 < rr < 2.2) and the a2 sector (rr = 1.6, 1.3 < rr < 2.02) compared to the commercial farming sector. of the 8 792 ticks counted in situ, the following species and groups were identified, in descending order of prevalence (table 2): amblyomma hebraeum (36 %), hyalomma truncatum/hyalomma mariginatum rufipes (29 %), rhipicephalus evertsi evertsi (17.7 %), rhipicephalus (boophilus) decoloratus (6.5 %), rhipicephalus simus (5.4 %) and rhipicephalus zambeziensis/appendiculatus (5 %). of the 8 792 ticks counted, the communal sector (70 %) contributed the highest percentage with the 239 d.n. ndhlovu, p.v. makaya & b.l. penzhorn a2 and commercial sectors together contributing 30 % (table 3). the tick index varied among the farming sectors with the communal sector (42.2) recording the highest index followed by the a2 (19.4) and the commercial sector recording the lowest (table 3). similarly, the mean tick counts varied significantly (p < 0.05) among the farming sectors (table 3); irrespective of the tick species, the communal sector recorded a significantly overall higher mean tick count compared to the a2 and commercial sectors. however, there was no significant overall mean tick count difference between the a2 and commercial sectors. significantly (p < 0.05) higher mean counts of a. hebraeum, h. truncatum and r. e. evertsi were recorded for the communal sector compared to the a2 and commercial farming sectors (table 3). however, a. hebraeum mean tick count was significantly higher for the a2 sector compared to the commercial sector while that for r. e. evertsi was significantly higher for the commercial sector compared to the a2 sector. there was no significant difference in the mean tick count of h. trun catum between the a2 and commercial sectors. there was no significant difference in the mean tick count table 1 number of cattle infested with ticks and the prevalence of tick infestation according to farming sector farming sector no. of cattle sampled no. of cattle infested prevalence (%) communal a2 commercial 146 67 73 137 57 39 93.8a 85.1a 53.4c total 286 233 81.5 figures with a different superscript in a column are significantly different at p < 0.05 table 2 total tick counts according to tick species identified by visual inspection tick species total tick count % a. hebraeum h. truncatum* r. e. evertsi r. (b). decoloratus r. simus r. zambeziensis** 3 165 2 575 1 559 574 477 442 36.0 29.3 17.7 6.5 5.4 5.0 total 8 792 * includes h. m. rufipes ** includes r. appendiculatus table 3 total tick counts, mean tick counts and tick index according to farming sector and tick species identified by visual inspection tick species farming sector communal a2 commercial *ttc mean  s.e. *ttc mean  s.e. *ttc mean  s.e. a. hebraeum h. truncatum r. e. evertsi r. (b). decoloratus r. simus r. zambeziensis 2 198 1 938 1 169 511 227 119 16a  1.2 15a  0.9 11a  0.8 8a  1.2 6a  0.8 7a  1.6 621 297 84 4 41 252 9.9b  0.8 5b  0.5 3.7b  0.8 2a  0.9 2b  0.3 6a  0.7 346 340 306 59 209 71 5.9c  0.7 7b  0.9 5.8c  0.3 4a  0.9 7a  1.1 3b  0.5 overall 6 162 12.5a  0.5 1 299 6.4b  0.4 1 331 5.8b  0.3 total cattle sampled 146 67 73 tick index 42.2 19.4 18.2 mean tick counts with a different superscript in a row for each tick species are significantly different at p < 0.05 * ttc = total tick count 240 tick infestation and udder and teat damage in cattle of matabeleland south, zimbabwe of r. (b). decoloratus among all the farming sectors. rhipicephalus simus recorded a significantly lower mean tick count for the a2 sector compared to the commercial and communal sectors while that for r. zambeziensis was significantly lower for the commercial sector compared to the a2 and communal sectors. the upper perineum accounted for the highest percentage (34.3 %) of the ticks counted in situ, followed by the lower perineum (26.9 %) with the leg (6.2 %) accounting for the lowest percentage (table 4). mean tick counts varied significantly (p < 0.05) according to the predilection site (table 4); irrespective of the tick species, the upper and lower perineum recorded significantly higher mean tick counts compared to the other sites. the tail and the pinna also recorded significantly higher mean tick counts compared to the neck and the leg. tick samples that were sent to the central veterinary laboratory (table 5) revealed that r. zambeziensis was present at all sampled sites, while r. appen diculatus was only present at three sites. other tick species, which were confirmed at the laboratory, were h. m. rufipes, which was identified from all the sites but sivume dip tank and h. truncatum, which was identified at four sites, but not at judds farm. the highest percentage of a. hebraeum and r. appendiculatus was recorded on the lower perineum while that of r. e. evertsi and h. m. rufipes was on the upper perineum, that for r. (b). decoloratus, r. simus and h. truncatum was on the tail and that for r. zambeziensis was on the pinna (table 6). there was no significant difference in the prevalence of tick infestation according to parity (table 7). udder and teat damage according to farming sector and parity of animals the communal sector recorded a significantly higher (p < 0.05) prevalence of udder and teat damage compared to the a2 and commercial farming sectors and there was also a significant difference between the latter two sectors (table 8). occurrence of udder damage was significantly associated with the communal sector compared to the a2 (2 = 21, p < 0.001) and commercial sectors (2 = 49, p < 0.001). similarly, teat damage was significantly associated with the communal sector compared to the a2 (2 = 22.3, p < 0.001) and commercial sectors (2 = 64.8, p < 0.001). teat damage was also significantly associated with the a2 sector (2 = 8.6, p = 0.003) compared to the commercial sector. table 4 total, percent and mean tick counts of ticks identified visually according to predilection site on sampled animals predilection site total tick count % of total mean  s.e upper perineum lower perineum tail pinna neck leg 3 016 2 358 1 252 952 669 545 34.3 26.9 14.2 10.8 7.6 6.2 11.8a  0.6 10.1a  0.6 7.6b  0.6 6.7b 0.5 4.8c  0.5 4.2c  0.3 total 8 792 mean tick counts with a different superscript in a column are significantly different at p < 0.05 table 5 specimens identified by the central veterinary laboratory tick species diptank/farm total switsha double cross judds nyandeni sivume a. hebraeum r. evertsi evertsi r. appendiculatus r. zambeziensis r. (b.) decoloratus r. simus h. truncatum h. marginatum rufipes 26 * 0 11 0 1 6 6 4 2 0 20 0 11 5 3 2 1 1 3 18 5 0 2 * * 1 5 0 3 5 5 1 3 11 2 17 6 11 0 33 6 13 41 35 26 27 16 total ticks counted 50 45 32 19 51 197 * not submitted 2 4 1 d .n . n d h l o v u , p .v . m a k a y a & b .l . p e n z h o r n table 6 predilection site of laboratory-identified tick species predilection site tick species total a. hebraeum r. e. evertsi r. zambezi ensis r. append i culatus r. (b). decol oratus r. simus h. trun ca tum h. rufipes (%) (%) (%) (%) (%) (%) (%) (%) (%) upper perineum lower perineum tail pinna neck leg 3 (9.1) 18 (54.5) 5 (15.2) 0 ( 1 (3) 6 (18.2) 5 (83.3) 0 1 (16.7) 0 0 0 2 (4.9) 1 (2.4) 4 (9.8) 21 (51.2) 13 (31.7) 0 ( 0 9 (69.2) 1 (7.7) 1 (7.7) 2 (15.4) 0 ( 6 (17.1) 3 (8.6) 11 (31.4) 1 (2.9) 6 (17.1) 8 (22.9) 0 ( 1 (3.8) 23 (88.5) 0 ( 0 ( 2 (7.7) 3 (11.1) 4 (14.8) 14 (51.9) 0 ( 2 (7.4) 4 (14.8) 14 (87.5) 0 ( 2 (12.5) 0 ( 0 ( 0 ( 33 (16.8) 36 (18.3) 61 (31.0) 23 (11.7) 24 (12.2) 20 (10.2) total 33 ( 6 ( 41 ( 13 ( 35 ( 26 ( 27 ( 16 ( 197 ( 242 tick infestation and udder and teat damage in cattle of matabeleland south, zimbabwe udder damage was found to be approximately two times and three times more likely to occur on the communal sector (rr = 1.8, 1.4 < rr < 2.5; rr = 3.2, 2.1 < rr < 4.9) compared to the a2 and commercial farming sectors, respectively. similarly, teat damage was found to be approximately two times and four times more likely to occur on the communal sector (rr = 1.8, 1.4 < rr < 2.5; rr = 4.3, 2.6 < rr < 7.1) compared to the a2 and commercial farming sectors, respectively. teat damage was also found table 7 number of cattle infested with ticks and the prevalence of tick infestation according to parity parity total sample number infested prevalence (%) 1–3 4–6 > 7 199 80 7 162 65 6 81.4a 81.3a 85.7a 286 233 figures with a different superscript in a column are significantly different at p < 0.05 table 8 number and proportion of cows and heifers with udder and teat damage according to farming sector farming sector total sampled udder damage (%) teat damage (%) communal a2 commercial 146 67 73 108 (74.0a) 27 (40.3b) 17 (23.3c) 111 (76.0a) 28 (41.8b) 13 (17.8c) total 286 152 (53.1) 152 (53.1) figures with a different superscript in a column are significantly different at p < 0.05 table 9 number and proportion of cows and heifers with udder and teat damage according to parity parity total sampled udder damage (%) teat damage (%) 1–3 4–6 > 7 199 80 7 115 (57.8a) 33 (41.3b) 4 (57.1ab) 117 (58.8a) 29 (36.3b) 4 (57.1ab) total 286 152 (53.1) 150 (52.4) figures with a different superscript in a column are significantly different at p < 0.05 table 10 tick infestation and udder damage situation of sampled animals tick infestation and udder damage status number of animals (total animals tick infested/not tick infested) % tick infested animals with udder damage tick infested animals with no udder damage not tick infested animals with udder damage not tick infested animals with no udder damage 133 (233) 100 (233) 19 (53) 34 (53) 57.1 42.9 35.8 64.2 table 11 tick infestation and teat damage situation of sampled animals tick infestation and teat damage status number of animals (total animals tick infested/not tick infested) % tick infested animals with teat damage tick infested animals with no teat damage not tick infested animals with teat damage not tick infested animals with no teat damage 135 (233) 98 (233) 17 (53) 36 (53) 57.9 42.1 32.1 67.9 243 d.n. ndhlovu, p.v. makaya & b.l. penzhorn to be approximately two times more likely to occur on the a2 sector (rr = 2.4, 1.3 < rr < 4.1) compared to the commercial sector. animals of parity 1–3 recorded a significantly higher (p < 0.05) prevalence of udder and teat damage compared to those of parity 4–6, but there was no significant difference between those of parity 1–3 and parity > 7 and those of parity 4–6 and parity ≥ 7 (table 9). occurrence of udder (2 = 5.6, p = 0.02) and teat (2 = 10.7, p < 0.001) damage was significantly associated with parity 1–3 compared to parity 4–6. udder (rr = 1.4, 1.1 < rr < 1.9) and teat (rr = 1.6, 1.2 < rr < 2.2) damage was found to be 1.4 times and approximately two times more likely to occur on animals of parity 1–3 compared to those of parity 4–6, respectively. association between tick infestation and udder and teat damage the percentage of tick-infested animals with udder damage was higher than those with tick infestation but with no udder damage (table 10). similarly, the percentage of tick-infested animals with teat damage was higher than those with tick infestation but with no teat damage (table 11). occurrence of udder (2 = 5.5, p = 0.02) and teat (2 = 10.6, p < 0.001) damage was significantly associated with tick infestation compared to no tick infestation. udder (rr = 1.6, 1.04 < rr < 2.2) and teat (rr = 1.8, 1.2 < rr < 2.7) damage was found to be approximately two times more likely to occur on tickinfested animals compared to those with no tick infestation. treatment for udder and or teat damage only four animals were treated out of a total of 152 cattle with udder and teat damage. the properties where treatment was given were blanket farm and double cross farm. discussion tick species and infestation eight species of ticks were found to be infesting cattle at the different study sites. the ticks belonged to three genera, namely amblyomma, rhipiceph alus (including the subgenus boophilus) and hya lom ma. according to punyua, latif, nokoe & cap stick (1991), cited by hlatshwayo, mbati & dipeolu (2002), it is rare to find more than six species infesting their hosts in any one given ecological zone. in this case, eight species of ticks were found infesting cattle, which indicated that the study area probably comprised of two or more ecological zones. on the other hand, this could be one of those rare instances where more than six tick species are found in one ecological zone, as was evident at judds farm, sivume dip tank and nyandeni dip tank, where seven tick species were identified at the same study sites. related species such as r. appendiculatus and r. zam beziensis, as well as h. truncatum and h. marginatum rufipes, coexisted at these sites. in descending order of abundance, the tick species identified were a. hebraeum, hyalomma sp., r. e. evertsi, r. (b.) decoloratus, r. simus and the r. appendiculatus/zambeziensis group. these findings are partly in agreement with those of bryson, tice, horak, stewart & du plessis (2002) who found that in some parts of south africa, particularly in the north west province, the commonest ticks infesting cattle were a. hebraeum, r. appendiculatus and r. e. evertsi while in mpumalanga province the commonest tick was r. (b.) decoloratus. these findings are in contrast to those of several workers (dreyer, fourie & kok 1998a; mekonnen, hussein & bedane 2001; hlatshwayo et al. 2002) who found that the commonest tick species infesting cattle was r. (b.) decoloratus in some parts of the free state province of south africa and in central ethiopia. regassa (2001) found the commonest tick in the borana province of ethiopia to be rhipicephalus pulchellus. amblyomma hebraeum the occurrence of a. hebraeum in this part of the country and the absence of amblyomma variegatum is in agreement with the findings of peter, perry, o’callaghan, medley, shumba, madzima, burridge & mahan (1998a). according to these authors, the northern limit of a. hebraeum is latitude 17° south, while above this northern limit it is replaced by a. variegatum. peter, perry, o’callaghan, medley, shumba, madzima, burridge & mahan (1998b) also reported that a. hebraeum was predominant in the lowveld and had encroached into some parts of the highveld of zimbabwe. the absence of the related species, a. variegatum, was expected as its distribution is limited to the zambezi valley and the surrounding dry lowveld areas in the north-western parts of zimbabwe (norval, perry, meltzer, kruska & booth 1994). in the east, it is found in the burma valley, which is at the eastern border of zimbabwe (norval 1983b). amblyomma hebraeum and a. variegatum in zimbabwe have an area of overlap (peter 244 tick infestation and udder and teat damage in cattle of matabeleland south, zimbabwe et al.1998a). in an area of overlap there is inter-specific mating with the production of sterile hybrids. it is also reported that a. variegatum completely replaces a. hebraeum over a period of three years (norval et al. 1994). the survival of a. hebraeum is favoured by the presence of alternative wildlife hosts for the adult stage while this is limited to african buffalo (syncerus caffer) for a. variegatum (norval et al. 1994). petney, horak & rechav (1987) stated that the main host species of a. hebraeum vary from place to place, with hosts of more than 100 kg in mass being regularly infested, while hosts below this mass are not usually infested. the immature stages infest birds and small mammals (horak, mac ivor, petney & de vos 1987). amblyomma hebraeum has also been found to infest the leopard tortoise (geochelone pardalis) and the angulate tortoise (chersina angulata) (dower, petney & horak 1988). amblyomma spp. ticks are important vectors of ehrlichia ruminantium, the cause of heartwater (peter et al. 1998b). hyalomma species two species, h. truncatum and h. marginatum rufipes, were collected in this study, while chatikobo, kusina, hamudikuwanda & nyoni (2001) collected only h. m. rufipes from cattle in the sanyati smallholder farming area in the north-western part of zim babwe. horak, swanepoel & gummow (2001) and van niekerk, fourie & horak (2006) reported the presence of h. truncatum, h. m. rufipes, h. glabrum and h. marginatum turanicum in south africa. hyalomma glabrum and h. m. turanicum were not recovered during the current study. hyalomma truncatum and h. m. rufipes occurred together at 80 % of the study sites, which is in agreement with van niekerk et al. (2006) who stated that there was considerable overlap in the distributions of h. truncatum and h. m. rufipes. the upper perineum was the attachment site of 87.5 % of h. m. ru fipes, while 12.5 % were collected from the tail. this tick did not infest the other parts of the animal. this study confirmed the predilection site for h. m. rufipes to be the upper perineum, in agreement with baker & ducasse (1967) and londt et al. (1979). the related h. truncatum was collected at 80 % of the study sites. the majority of the ticks, ca. 52 %, were collected from the tail, while all attachment sites on the animal, except for the pinna, yielded ticks. these findings differ slightly from those of londt et al. (1979) who collected the tick from all six predilection sites, with most of the ticks being collected from the upper perineum. rhipicephalus evertsi evertsi the occurrence r. e. evertsi at all sites was consistent with the findings of norval (1981) who reported that this tick species occurred in all parts of zimbabwe except in the zambezi valley. this tick is able to tolerate a wide range of climatic conditions (hamel & gothe 1978) and some strains can cause paralysis in sheep. rhipicephalus (boophilus) decoloratus with the exception of double cross farm, rhipicephalus (boophilus) decoloratus was found at all study sites. the failure to find r. (b.) decoloratus at double cross farm could have been because cattle had been dipped three days previously and as such this tick could have been removed by the acaricide. cattle are the main hosts of r. (b.) decoloratus (londt et al. 1979), with horses and donkeys being less important hosts. this tick can also be found on a large number of wild ungulates, including impalas (aepyceros melampus), bushbuck (tragelaphus scrip tus) and kudus (tragelaphus strepsiceros) (horak, potgieter, walker, de vos & boomker 1983). the species is distributed throughout most of the wetter regions of south africa and is present in the eastern parts of zimbabwe (norval et al. 1992; walker, bouattour, camicas, estrada-peña, horak, latif, pegram & preston 2003). the related r. (b.) microplus was not found in this lowveld area. the absence of r. (b.) microplus was expected, as it has a limited distribution in zimbabwe (katsande, mazhowu, turton & munodzana 1996); distribution of this tick is in the eastern region (chimanimani, chipinge, mutare, rusape and wedza), northern, north-eastern and the north-western region of zimbabwe (katsande et al. 1996). rhipicephalus simus this tick species was collected from all study sites. as expected, the bulk of the collections (ca. 88 %) were from the tail switch. these findings are similar to those of londt et al. (1979), where 100 % of the ticks were collected from the tail switch. interestingly, r. simus was found in large numbers on some properties during this study; in contrast, bryson et al. (2002) indicated that while this tick was widely distributed, it was seldom found in large numbers. the large numbers identified at some study sites would indicate that there was an abundance of murid rodents in the area, which are the hosts for the immature stages of this tick species (walker et al. 2003; van niekerk et al. 2006). the other reason for the 245 d.n. ndhlovu, p.v. makaya & b.l. penzhorn abundance would be the dipping method: if farmers do not thoroughly wet the tail switch or ignore it completely, then this tick would thrive. rhipicephalus appendiculatus and rhipicephalus zambeziensis these two species co-existed at 60 % of the study sites. in zimbabwe r. appendiculatus is confined to the moister regions, which includes the highveld. the related r. zambeziensis replaces r. appendiculatus in the hot, dry river systems in the lowveld of zimbabwe (norval, walker & colborne 1982; cited by mooring & mazhowu 1995). madder, speybroeck, bilounga, helleputte & berkvens (2005) stated that survival of r. zambeziensis was better than that of r. appendiculatus under more extreme conditions of humidity and temperature. as such, r. zambeziensis was more adapted to hotter and drier areas while r. appendiculatus was more adapted to cooler and wetter conditions. in zimbabwe and other countries within the region, the different instars of r. appendiculatus exhibit a strict pattern of seasonal occurrence (latif, hove, kanhai, masaka & pegram 2001), with one generation occurring each year. adults are mostly active and abundant in the rainy season (mid-december to may), larvae in the cool season (march to october) and nymphs in the dry months of june to october (latif et al. 2001). competitive displacement between the two species possibly occurs in those areas where their distributions overlap (mooring & mazhowu 1995). there is an apparent displacement of r. appendiculatus by r. zambeziensis as one proceeds southward in the study area. of the r. appendiculatus specimens collected, 84 % were from sivume diptank, the north ern-most study site, as compared to 4.8 % of r. zambeziensis collected at the same study site. in contrast, ca. 49 % of r. zambeziensis specimens were collected at double cross farm, the southernmost study site, but no r. appendiculatus were collected at that location. although r. zambeziensis was collected from all sites on the animal, except the leg, 83 % of the specimens were collected from the neck and pinna. in contrast, 69 % of r. ap pen diculatus was collected from the lower perineum. the location at extreme ends of the host, of these two related ticks could be due to chemicals emitted by either tick species, which serve to repel the other related species. according to the animal health act (cattle cleansing) regulations (government of zimbabwe 1993), tick infestation arises when at least 10 % of the cattle have ≥ 10 live ticks on them. in this study, 81 % of the cattle were regarded as tick infested. five of the six study sites (ca. 83 %) were regarded as tick infested properties as more than 10 % of the cattle had ≥ 10 ticks. generally, such properties would be placed under quarantine until the animals were tick free. cattle can become tick infested if they are not regularly dipped or if ticks have developed resistance to the acaricide being used. the low level of tick infestation (6 %) at blanket farm could have been due to dipping that had taken place 2 days previously. interestingly, at the other sites such as double cross farm and timber farm where dipping had taken place 3 days previously, the herds were deemed to be tick infested as more than 10 % of the cattle had ≥10 live ticks. at these sites one would have expected the residual effect of the acaricide to kill or repel the ticks, but this was not the case. an abundance of live ticks on cattle even after recent dipping can occur for various reasons, amongst others, development of resistance to the particular acaricide being used (meltzer & norval 1993), or poor mixing of the acaricide. the other factor that can contribute to increased tick abundance on cattle, even after regular and recent dipping, is the abundance of the free-living stages in the veld, which could be due to favourable environmental conditions. as this study was conducted during summer, it would be expected that free-living stages would be in abundance in the veld and that the pick-up rate of ticks from the veld would be correspondingly high. udder and teat damage farmers view tick infestations as causes of teat damage (masika, sonandi & van averbeke 1997; hlatshwayo & mbati 2005). ticks also cause udder damage, predispose to mastitis, abscessation and hide damage (meltzer & norval 1993; dreyer et al. 1998a; dreyer et al. 1998b; tibary & anouassi 2000). of the 286 milking cows and heifers sampled in this study, 53 % had some degree of udder and/or teat damage. this is in agreement with results from kenya (latif, punyna & capstick 1987, cited by meltzer & norval 1993), where 52 % of the milking cattle surveyed had lost one or more teats. while no teats were reported lost in the current study, teat and udder damage was encountered in the form of blocked teats, pus discharges and indurations of quarters. the reason teats not being lost is that farmers at most of the properties surveyed practised some form of regular tick control. if a breakdown in tick 246 tick infestation and udder and teat damage in cattle of matabeleland south, zimbabwe control, for whatever reason, were to occur for a prolonged period, then the prevalence of lost teats would emerge as an important issue. cows of lower to middle parity were the ones most affected by conditions of udder and teat damage. this differed from findings in mozambique, where it was found that animals > 4 years old were more prone to damage (asselbergs & lopes pereira 1989, cited by meltzer & norval 1993). the reason for the apparent difference could be due to the method of estimating age of the animals. in this study, animals were aged according to parity rather than actual years. as such, the lower and middle parity groups included cows > 4 years old, making comparison with the kenyan study impossible. of concern, was the fact that farmers treated only four (2.6 %) of the 152 animals with udder/teat damage. this low rate of treatment was due to lack of remedies and the fact that farmers were content as long as there was at least one functional quarter and teat from which a calf could suckle. in conclusion, tick infestation as defined by the animal health act of zimbabwe (government of zimbabwe 1993) was prevalent in the milking cattle sampled, especially in communal herds of gwanda. such a situation is ideal for the establishment of endemic stability to the various tbds. on the other hand, higher tick infestations can lead to damaged udders and teats, which was not deemed important by the farmers, since only a small proportion of them treated their affected animals. this attitude would change if communal farmers became commercially oriented and took into consideration potential productivity losses associated with certain levels of tick infestation and the effect on the growth rates of calves suckling from two or fewer teats and quarters. tick species such as r. appendiculatus and r. zambeziensis were found in considerable numbers, but the disease that they transmit, theileriosis, has not been reported from the study area. nevertheless, these cattle are at risk of contracting the disease should a theileria parva-infected animal be introduced into the area. currently these ticks are important in the study area for the direct damage that they cause. acknowledgements we thank the director of the division of veterinary field services, dr msongelwa mangena, the provincial veterinary officer for matabeleland south, who availed all the resources needed to successfully conduct the study, and the central veterinary laboratory staff who assisted in tick 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information author: david nabarro1 affiliation: 1special representative of the united nations secretary general for food security and nutrition correspondence to: david nabarro postal address: 2 un plaza room 2613, new york 10017, usa how to cite this preface: nabarro, d., 2012, ‘one health: towards safeguarding the health, food security and economic welfare of communities’, onderstepoort journal of veterinary research 79(2), art. #450, 3 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.450 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. one health: towards safeguarding the health, food security and economic welfare of communities in this preface... open access • one health thinking • a serious global food security situation • drivers for animal disease • better food security and food safety through one health principles • putting one health thinking into practice it is a privilege for me to be invited to join this conference organised by young african scientists who are exploring the opportunities for integrated approaches to animal and human health. thank you for inviting me.in my early professional assignments i worked with communities in southeast asia and east africa. i sensed that their food systems, patterns of agriculture, nutrition and health were inextricably linked. their leaders often told me that dealing with a range of different professionals was intensely frustrating and what they needed was multi-skilled professionals who really understood the challenges people face. they need to appreciate how rural life and livelihoods vary from season to season, or change over the years as climatic conditions, access to infrastructure or new initiatives by government or civil society alter the environment in which people live and work. in my presentation today i shall focus on ways in which the ‘one health’ approach is contributing to food security and economic well-being of communities as well as helping to safeguard their personal health. i appreciate that you have been discussing these issues in depth in the last two days: i hope that you will share your observations with me. one health thinking top ↑ one health is a way of thinking that reflects the reality of people’s lives and livelihoods. that thinking encourages a focus on new kinds of outcomes, policies, actions and research agendas − different from those which will be pursued if we stay within our normal professional disciplines. one health thinking, outcomes, policies, action and research are important for: • increasing the number of people who enjoy food security − who have stable access to nutritious foods for their needs • ensuring bio-secure and ecologically sustainable production of safe food for people to eat • encouraging accessible local markets and fair international trade in agricultural products. the risk of disease emergence at the interface between animals, humans and ecosystems is at the current core of one health thinking. it can include illnesses that have an indirect effect on human health through their impact on people’s livelihoods … and the drivers that increase the risk of emerging diseases (climate change, resource depletion, land degradation, demand, lack of bio-security, contamination). we should not try to define one health thinking and action too tightly. please do not see one health as a scientific discipline or international programme. instead let us look at how different stakeholders − in government, amongst farmers and food processors, within consumer groups and civil society − use the concepts in practice. a serious global food security situation top ↑ we face a global situation where an estimated 925 million people go hungry. the effects of recent food price increases are likely to deepen the vulnerability of those who spend between 50% and 80% of their family budget on food, mostly basic staples. efforts to respond to food insecurity, food safety and food trade challenges have addressed all four dimensions of food security namely, availability, access, utilisation and stability. progress has been made at several levels such as: • countries affected by food insecurity • investors in countries’ efforts to improve food security • the organisations that support both countries and investors and • the system for governance of international arrangements to assist affected countries (including the revitalised committee on food security). rising prices present a great opportunity for farmers to respond to growing demand. but farmers need inputs and cash to do this. they need help to manage the risks associated with trying to produce more. and they need the infrastructure to market their food. well-functioning food markets and trade have huge potential to increase small farmers’ integration into value chains so as to increase the benefits they can capture from the trade in their products. drivers for animal disease top ↑ farmers seeking to increase their livestock holdings have particular challenges. they can find themselves facing outbreaks of disease in their animals: these might contribute to the emergence of infectious disease emergence at the human-animal-ecosystem interface. the livestock sector is an area in which one health thinking and action can make a difference to lives and livelihoods. estimates from the world bank on the projection for the increase in meat production over the next 40 years indicate that most of this will occur in the developing world. improving livestock production in a sustainable and humane manner that balances both carbon and water footprints is a major challenge facing the one health community. for the 75% of the world’s poor that are rural and dependent on agriculture, disease outbreaks in livestock not only put at risk their immediate food source, but it also puts at risk their livelihoods and resilience capacity − and that affects their long-term food security. disease outbreaks which reduce the availability of live animals and livestock products can reduce household income, undermine the diets of household members, impair nutritional status and increase risks to health, especially of women and children. outbreaks can also impair the wider market availability for those products. chronic food insecurity also drives risky behaviours related to animals: no one who is well-fed would consider consuming the carcass of an animal that has died of disease. effects of animal disease extend to people who work in production and processing − including livestock and agri-food workers, transporters and sellers. one health thinking helps us find ways to limit these risks and encourage resilient livelihoods. agricultural intensification and lack of biosecurity can also result in food borne disease: the who (world health organisation) estimates that in 1997, food contamination cost up to $35 billion in the united states alone due to medical costs and lost productivity. fear of losing business can also result in further disease spread in a clandestine way. the potential negative economic impacts of restricting the movement and sale of diseased animals or animal products (a common response to disease emergence) may unfortunately drive some linked to the industry to try to work around bans on sales and trade in order to maintain their income − and in the process, potentially extends a disease outbreak and makes it harder to control. the world bank has calculated that: the emergence of bse, sars, h5n1, and influenza a(h1n1) have caused over us$20 billion in direct economic losses over the last decade and much more than us$200 billion in indirect losses. better food security and food safety through one health principles top ↑ in the un (united nations) system we seek coordinated responses to food insecurity and unsafe food through movements of multiple actors. these encourage a broad range of stakeholders to work in synergy as they pursue immediate and longer term food security outcomes. the work goes best if undertaken under the leadership of national authorities. we focus on multi-disciplinary and multi-stakeholder movements, anchored to institutions but not controlled by them. we also encourage multi-stakeholder platforms where different groups work together and coordinate support particularly for smallholder-based food security initiatives and nutrition-sensitive agricultural development. we have developed a comprehensive framework for action (cfa) for food security and a road map for scaling up nutrition (sun) to guide the emergence of these movements. what have we learnt over the past few years in applying one health thinking to food and nutrition security, food safety, markets and trade? • firstly, the need for country leadership: effective action to ensure food security and safe food for all happens if led from within countries. • secondly, the need for clarity on results: in our work the emphasis has been on equitable and sustained improvements in people’s lives, long-term health and resilient livelihoods. • thirdly, the need for joint working: by institutions which tend to function separately. these include government services − and their ministries − with separate responsibilities for animal and human health, the environment, as well as for agriculture and trade. • fourthly, the value of being inclusive: working with and responding to stakeholders outside government − particularly organisations of smallholder farmers and those who work in the livestock val ue chain, businesses, agriculture unions, consumers’ organisations, local food producers. putting one health thinking into practice top ↑ government ministers expect us bureaucrats and professionals to advise them on how best to avoid risk associated with disease at the animal-human-ecosystem interface. we have made real progress in the area of h5n1 highly pathogenic avian influenza, and in relation to sars and spongiform encephalopathy. but we have much more to do with regard to sustainable livelihoods of pastoral communities, rabies, or what some refer to as the forgotten zoonoses. i know that you have been discussing some of these in your meeting. i am keen to see one health thinking become a central feature for responsible national and international policy making. this means putting the lessons we have learnt into practice: • firstly, never forgetting that our primary clients are those most at risk of food insecurity or most likely to be affected by unsafe food or market failure. • secondly, working together for sensible and realistic policies to improve food and nutrition security, sustainable and bio-secure production, safe livestock products, functioning markets and fair trade. • thirdly, overcoming our tendency to work in our professional niches and bureaucratic silos, and instead sharing data and analyses, developing joint policies, doing research together, implementing joint investigations and being accountable for delivering results. it helps if we undergo training together, too. • fourthly, focusing on outcomes that have meaning to the business community, to human health, animal welfare and wildlife advocates, to politicians and to the media and then working hard to demonstrate and communicate our results. within the un system our secretary general advocates for comprehensive, and increasingly integrated responses to the current challenges we face in the health, food and agriculture, climate change and trade interface. he encourages broad-based partnerships that focus on results. he requires our different organisations to work together and to link up with governments, regional organisations, private companies, civil society and − most importantly − farmers’ organisations. he wants us to explain the virtues of working in a joined up way and of breaking down professional silos. only then can we contribute to safeguarding the health, food security and economic prospects of poor communities. we have seen real progress in the african union − especially with pastoral communities and in the work being done by fao (food and agriculture organisation), who and the oie (office of international education). we are delighted to see the emergence of multi-disciplinary networks of professionals dedicated to supporting equity, reducing poverty and encouraging resilient livelihoods. we are pleased that young professionals like you are taking responsibility for working effectively at interfaces in ways that respond to people’s realities. you listen to the people you serve, to each other, to politicians and other stakeholders and to your own instincts as you take this important work forward. you are helping to build trust and respect between people and professionals, between different professional disciplines and between departments and organisations. you are impatient for results so you try to work in synergy rather than in competition. you appreciate what matters to people rather than spending time telling them what they should do − or feel. much more needs to be done. here are my requests to you today: • please continue to work in ways that reflect one health thinking and action. • please look for opportunities to advocate for one health – in national parliaments, regional assemblies and globally; with farmers’ organisations, labour unions and civil society groups; with businesses and entrepreneurs. • please do what you can to inspire wider and stronger networks of people who are excited by the potential of effective one-health working that empowers communities to act in ways that improve health and livelihoods. • please contribute to the growing movement for one health that works in the service both of poor communities and of the global good, brings together women and men, farmers and consumers as well as social and physical scientists, and engages all nations. article information authors: epaphras a. muse1 esron d. karimuribo2 george c. gitao3 gerald misinzo1 lesakit s.b. mellau2 peter l.m. msoffe2 emmanuel s. swai4 mbyuzi o. albano4 affiliations: 1department of veterinary microbiology and parasitology, sokoine university of agriculture, tanzania 2department of veterinary medicine and public health, sokoine university of agriculture, tanzania 3department of veterinary pathology, microbiology and pathology, university of nairobi, kenya 4veterinary investigation centre arusha, tanzania correspondence to: epaphras muse postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this proceeding: muse, e.a., karimuribo, e.d., gitao, g.c., misinzo, g., mellau, l.s.b., msoffe, p.l.m. et al., 2012, ‘epidemiological investigation into the introduction and factors for spread of peste des petits ruminants, southern tanzania’, onderstepoort journal of veterinary research 79(2), art. #457, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.457 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. epidemiological investigation into the introduction and factors for spread of peste des petits ruminants, southern tanzania in this proceeding... open access • abstract • introduction • material and methods    • study area    • study design and data collection    • questionnaire survey    • samples collection for competitive enzyme linked immunosorbent assay analysis    • samples collection for reverse transcriptase polymerase chain reaction    • statistical data analysis • results    • disease prevalence    • management practices    • introduction of peste des petits ruminants in southern tanzania    • observed clinical signs    • economic losses    • disease awareness and vaccination • discussion • conclusion • aknowledgement • references abstract top ↑ a study was carried out to confirm and identify sources and elucidate factors associated with the introduction of peste des petits ruminants (ppr) in southern tanzania. this study was conducted in tandahimba and newala districts of mtwara region following suspected outbreak of ppr in the area. qualitative data were collected using semi-structured questionnaires and in-depth interviews of key informants who included goat and sheep owners with suspected cases of ppr and animal health service providers as well as local administrative authority. additionally, 216 serum samples and 28 swabs were collected for serological and virological laboratory disease confirmation. the results show that ppr was first introduced in likuna village of newala district in february 2009 through newly purchased goats from the pugu livestock market located about 700 km in the outskirts of dar es salaam city. factors which contributed to spread of ppr included communal grazing and the cheap prices of sick animals bought by livestock keepers for slaughtering in other villages. laboratory findings confirmed presence of ppr in the area by rt-pcr and serological analysis revealed that seroprevalence was 31%. these findings have confirmed, for the first time, introduction of ppr in southern tanzania. the presence of ppr poses high risk of southward spread of the disease to other southern african countries in the sadc region thus calling for concerted and collaborative efforts in prevention and control of the disease to avoid losses. further elaborate studies on the spread, prevalence and risk factors associated with the disease should urgently be investigated. introduction top ↑ peste des petits ruminants is an acute, highly contagious infectious disease of small domestic ruminants and small wild ruminants, such as antelopes, impala and gazelles (abu elzein et al. 2004; nussieba et al. 2009). the disease is caused by peste des petits ruminants (ppr) virus (pprv). pprv belongs to a morbillivirus genus of paramixyoviridae family. pprv is a single serotype that is differentiated into four (i-iv) lineages (forsyth & barrett 1995; couacy-hymann et al. 2002). the geographical distribution of ppr lineages varies as lineage i and ii have been commonly reported in west africa (ashley et al. 2010), lineage iii has been reported eastern africa except for sudan which has been found to harbour lineage iv in addition to lineage iii (khalafalla et al. 2010). on the other hand, lineage iv has been reported in central and north africa, asia and china (ozkul et al. 2002; wang et al. 2009; awa et al. 2000; ayari-fakhfakh et al. 2010; balamurugan et al. 2010; khalafalla et al. 2010).the disease is transmitted by direct contact involving secretions or excretions from infected animals to healthy animals in close proximity. clinically, ppr is characterised by sudden onset of depression, fever, lacrimation, sores in the mouth, dyspnoea and coughing, foul smelling diarrhoea and death. post-mortem findings, normally restricted to the alimentary tract, consist of extensive erosive stomatitis and heamorrhagic gastro-enteritis, and often include streaks of congestion along the folds of the mucosa resulting in the characteristic ’zebra-striped‘ appearance (chauhan et al. 2009). secondary bronchopneumonia is common. the disease is characterised by high morbidity and mortality (50% – 80%) in naive sheep and goats populations, impacting negatively on the livelihoods, food security and socio-economic activities of livestock keepers in affected areas. the disease also impacts negatively on the local and international livestock trade markets. in tanzania the disease limits the efforts of farmers and government in attaining the millennium development goal for eradicating extreme poverty and hunger. in tanzania, ppr was first confirmed in 2008 in the northern areas (kivaria et al. 2009; swai et al. 2009), where it was confined until recently when it was suspected to have been introduced in southern areas in 2010 (fao 2010). the presence of ppr in southern tanzania would pose a high risk for spread to the whole of the southern african development community (sadc) threatening to devastate the livelihoods and food security of millions of small herders and agro-pastoralists (fao 2010). until the current study was designed, no official confirmation of ppr in southern parts of the country had been carried out. the objectives of this study were: • to confirm the presence of ppr in goats and sheep in newala and tandahimba districts, mtwara region • to identify sources and factors that contributed to the introduction and spread of the ppr in southern tanzania • to describe the epidemiological factors and losses in the affected villages. the hypotheses were to confirm if suspected cases seen in goats and sheep in southern tanzania were caused by pprv and to find out which factors facilitate the spread of the disease in this zone. material and methods top ↑ study area the study was carried out in eight selected villages in two districts of mtwara region, newala and tandahimba districts, in southern tanzania between march and may 2011 (figure 1). the estimated census for humans, goats and sheep in these districts are shown in table 1. the study area was purposively targeted following reports of suspected ppr outbreak that decimated small ruminants in the districts. this area borders mueda district of the cabo delgado province of mozambique with a small ruminant population that is naive to ppr. the study area lies between 2° 11′ and 6° 14′ s, and 35° 11′ and 38° 26′ e at an elevation of 100 m – 800 m above sea level. the rainy season starts in november and/or december to april and/or may with an average annual precipitation of 893 mm – 1001 mm. the mean monthly temperature varies from 23 °c to 27 °c and relative humidity varies from 79% to 87%. figure 1: showing sampling points and introduction of peste des petits ruminants in newala (star). table 1: the proportions of households, morbidity, mortality, chemotherapy, vaccination and statistics for goats, sheep and people. study design and data collection the current study employed a cross-sectional study design where selected villages were visited once between march 2011 and may 2011. purposive sampling of animals was carried out in villages with suspected ppr cases based on opinion from local veterinary officials and leaders. upon identification of household with suspected cases, the researcher (eam) conducted detailed interviews with owners of goats and sheep. this was complemented with random selection of five animals, either goats or sheep, which were subjected to detailed clinical examination as well as sample collection. questionnaire survey a semi-structured questionnaire was developed and field tested on a few households keeping goats and sheep in the two districts. the interviews focused on collection of information on flock size, species, age and sex, health and vaccination status, and management. age was approximated and classified as kids or lambs (≤ 3 months), weaners (> 3 to ≤ 9 months) and adults (> 9 months). health status data was collected by recording history of disease outbreak or occurrence, its clinical signs, overall number of sick animals (used to compute morbidity) as well as overall and specific deaths associated with observed clinical cases (used to compute crude and case fatality of ppr). flock management data included access to animal health and extension services (presence, type and frequency of services), action taken after the outbreak, live animal market visiting frequency and addition of new animals, and suspected source of the infection. in-depth interviews of key informants were conducted to obtain opinion from local livestock field officers (lfos) as well as from local government officials in affected villages and wards. the district veterinary officer (dvo) of newala district and veterinary officers at mtwara veterinary investigation centre (vic) were also interviewed to ascertain where and when ppr was first introduced into southern tanzania and which disease control measures were instituted before the current study. samples collection for competitive enzyme linked immunosorbent assay analysis a total of 216 serum samples were collected from goats and sheep in newala and tandahimba districts. initially, blood samples were collected from the jugular vein of each animal using plain vacutainer tubes. the samples were labelled accordingly to allow identification of each animal and flock sampled and kept in a slanted position overnight to allow serum separation from clotted blood samples. serum was decanted and aliquoted into 1.5 ml cryovials before being transported and stored temporarily at the vic, mtwara. finally the serum samples were shipped in a cool box chilled on ice packs to the laboratory at the vic, arusha where serological analysis was carried out. a monoclonal antibody (mab) based competitive enzyme linked immunosorbent assay (celisa) (diallo et al. 1995) was used for the detection of antibodies in sera to pprv using approved competitive elisa kit as described by singh et al. (2004a) and swai et al. (2009). according to the manufacturer, the sensitivity and specificity (for both animal and flock levels) of this celisa are 99.4% and 94.5%, respectively. briefly, the elisa plates were coated with ppr antigen; the unbound antigen was washed away using buffer then samples were added; rabbit antimouse-horseradish peroxidase (hrpo) conjugate was added and incubated with constant agitation in each stage. substrate solution (o-phenylenediamine dihydrochloride containing h2o2) was added allowing for a colour reaction to develop which was halted with the addition of an equal volume of 1 m h2so4. the elisa micro plates were read with an immunoskan reader (flow laboratories, uk) with an inference filter of 492 nm and connected to a computer loaded with elisa data information (edi) software for automated reading and calculation of the percentage inhibition (pi) values. samples collection for reverse transcriptase polymerase chain reaction samples (n = 28) from nasal and eye discharges as well as saliva from oral ulcers were collected from clinically sick sheep and/or goats using sterile swabs which were placed in a viral transport media containing antibiotics and antifungals. the samples were transported on ice to sokoine university microbiology laboratory for further analysis. virological analysis employed reverse transcriptase polymerase chain reaction (rt-pcr) to confirm involvement of ppr in suspected cases. briefly, the test was carried out as follows: the rna extraction from samples was done using commercial rna extraction kit (qiagen®); the rna was converted to cdna using a reverse transcriptase enzyme (superscript iii platinum one-step quantitative, invitrogen®). the cdna was amplified using pprv specific np3 and np4 primers as previously described by couacy-hymann et al. (2002). pcr products were analysed by electrophoresis and visualised in a uv transilluminator. statistical data analysis villages, farms and individual animal data were stored in microsoft excel 2007 (version 12). descriptive statistics for the animal and flock level explanatory variables examined in the study were computed using microsoft excel. proportions were calculated for seroprevalence and factors that included animal species, sex and age, location of the flock, health status, management and veterinary services provision. the statistical significance for the proportions was compared using chi-square test in epi info software version 5 (centre for disease control and prevention). a confidence limit of less than 5% was used to indicate a significant level. separate statistical analyses were performed for the data from the two species because previous studies have indicated that virus infection rate and epidemiology may be very different in the two species (waret-szkuta et al. 2008). study village maps were created using quantum gis (qgis 1.4.0 version) enceladus (open source geospatial foundation). results top ↑ disease prevalence the presence of the ppr infection in southern tanzania was confirmed by the rt-pcr test whereby 53.6% of the samples were positive from both sheep and goats.the overall ppr seroprevalence was 31% (95% ci = 24.9% – 37.6%) in the two districts (figure 2). tandahimba district recorded higher seroprevalence (55.5%) compared to the seroprevalence in 5.7% in newala district, the difference which was also statistically significant (p < 0.001). mnyawa village recorded highest seroprevalence followed by nannala, ngunja, and mchichira villages of tandahimba district (figure 2). mnima research farm had high seroprevalence followed by chiwonga, mapili and mnauya village in that order in newala district. there was no statistical difference in the seroprevalence recorded in goats (35.3%) compared to that in sheep (30.7%). similarly, there was no statistical difference in the ppr seroprevalence in female (36.8%) compared to that recorded in male (29.8%) animals sampled in this study. age-wise, 32.1% of the adults and 22.7% of the kids or lambs were seropositive, however, there was no statistical difference. figure 2: seroprevalence by villages or district. figure 3: proportions (%) for seroprevalence by sex (left) and species (right). the ppr prevalence based on reported cases observed by interviewees is shown in table 1. overall, 57% of 79 households visited had experienced suspected ppr cases in their flocks. again, a significantly higher proportion of households in tandahimba (81.3%) had ppr cases than those in newala district (19.4%; p < 0.001). a similar trend was also observed with respect to morbidity of ppr when tandanimba recorded higher morbidity (73.1%) compared to that in newala district (4.8%; p < 0.001). clinically, a significantly higher proportion of goats (90.9%) were reported to be sick compared to only 9.1% sheep which were sick (p < 0.001). a similar trend in mortality was recorded when the proportion of goats and sheep which died was 93.4% and 6.6%, respectively (p = 0.059). a good number of small ruminant owners also reported to treat their animals using antibiotics as shown in table 1. management practices it was observed that goats and sheep in the two districts are managed in two different ways. the first group keeps animals in small group in small houses on a raised floor at night and graze in a community land during day time. the second group manage animals collectively by keeping them together in groups in relatively large animal houses on ground floor and utilise communal grazing. goats and sheep are kept for meat and for sale in order to generate income for the household. there were more male respondents interviewed (90.0%) in comparison to female respondents (10.0%; table 2). only a few respondents in newala district tethered their animals at night (10.1%) and most (94.9%) farmers kept their animals in a small raised house at night. all (100%) of the respondents utilised communal grazing areas (table 2). introduction of peste des petits ruminants in southern tanzania the in-depth interview revealed that ppr was introduced in the southern tanzania for the first time in goats and sheep in february 2009. the current study managed to trace a village where sick animals started which was likuna village of newala district. it was observed that ppr introduction was through newly purchased goats from the pugu livestock market located about 679 km in the outskirts of dar es salaam city. these animals were brought in about one week prior to the disease outbreak. other villages in close proximity of this village that reported outbreak of ppr in the same month were kikuyu, makote, namiyonga, lidumbe and mkunya. it was confirmed that these five villages had also received some of these goats from pugu livestock market. the disease spread to neighbouring tandahimba district through buying sick and cheap animals from newala district with the intention of selling them at tandahimba live animal markets and butcheries. observed clinical signs different clinical signs were reported by livestock keepers in suspected ppr cases. the signs included diarrhoea, lacrimation, nasal discharges, respiratory distress, oral ulcers and skin nodules (figure 4). figure 4: clinical signs presented by peste des petits ruminants disease. economic losses the economic loss due to mortality in two districts was 25.7% and case fatality rate was 52.6% (table 1). case fatality rate was higher in newala district (92.9%) compared to (51.2%) in tandahimba district (p < 0.000). disease awareness and vaccination over 60.8% of respondents in tandahimba were aware of the disease outbreak and 49.4% had their own animals affected (table 2). very few livestock keepers (2.9%) knew what disease it was and only (6.5%) though that they had seen it in the past. during the outbreak, animal owners (35.4%) from the two districts treated their animals using chemotherapy whereas 22.6% were from newala and 43.8% were from tandahimba (table 1). more than half (58.9%) had veterinary extension workers in their villages but half (52.9%) of the farmers reported regular health inspection for their animals, although the frequency of inspection varied (table 2). all (100%) respondents had not vaccinated their animals (table 1). the source of the infection and spread was suspected by farmers to be communal grazing and change of weather conditions, with more animals being affected during the rainy season. table 2: proportions of respondents’ sex, animal management, awareness of the disease, source and spreading of the infection and veterinary extension services. discussion top ↑ this study has confirmed the aetiology of the disease outbreak that occurred in the districts of tandahimba and newala in southern tanzania to be ppr in march 2011. it has also identified the village where the first introduction and consequently, the source of ppr spread in southern tanzania happened. although it took only one year from first official confirmation of the disease in northern tanzania to introduction in southern tanzania, it has taken about two years to confirm this disease through efforts of the current study. this is consistent with the findings by karimuribo et al. (2011) of delayed confirmation following introduction of ppr in the country. these findings point to the importance of having efficient disease surveillance and diagnostic capacity especially for emerging and re-emerging fatal diseases affecting animal populations. in this study it was found out that the source of the disease was through the introduction of new animals purchased from live animal market. similar sources of the disease have been implicated before (singh et al. 2004a; muhammad et al. 2009). the proportions of sero-converted animals examined in the area under study was low (31.0%) compared to the reports from northern tanzania (45.5%) (swai et al. 2009) but at the flock level the prevalence was high (48.9%). ozkul et al. (2002) in turkey found comparable findings whereby the overall prevalence was low based on antibody test and higher based on clinical signs. low ppr seroprevalence have also been reported in tunisia (ayari-fakhfakh et al. 2010). although the seroprevalence was low in this study it has been shown elsewhere that seroprevalence can be as high as 45.5%, 78% and 92.5%, as reported in cameroon, nigeria and sudan respectively (ekue et al. 1992; obidike et al. 2006; osman et al. 2008) using neutralisation and haemagglutination tests. when the competitive elisa method was used the seroprevalence was reported to be 51% and 50% (khan et al. 2008; misbah et al. 2009) in pakistan. the inconsistency in the seroprevalence of antibodies to pprv in different areas is attributed to variations in a number of factors including the management system, levels of immunity, diagnostic test, sampling procedures used and technical know-how of the researchers (singh et al. 2004b; waret-szkuta et al. 2008). in this study, goats were reported to show an acute form of the disease whilst sheep showed a sub-acute or chronic form; this finding is supported by other studies (obi, rowe & taylor 1984; swai et al. 2009). although, the differences observed between males and females was not significant swai et al. (2009) observed sex differences in sheep whereby males were more affected compared to females. with respect to age category, the highest prevalence of ppr was observed in adults compared to other age category. this result is in agreement with other finds observed in ethiopia and india (singh et al. 2004a; waret-szkuta et al. 2008) where they reported high prevalence in adults. association with seasonal (weather) changes observed by farmers has been reported elsewhere (singh et al. 2004a; muhammad et al. 2009). even though the ppr was introduced in newala, it was more prevalent in tandahimba district. the reasons are thought to be first selling of sick animals from newala to tandahimba animal markets and butcheries and secondly the livestock keepers in tandahimba district managed their animals by utilising more (60.8%) communal grazing compared to newala district (39.2%) (table 2). this study revealed that more than half of the farmers have little or no access to veterinary services. lack of appropriate veterinary services and inadequate infrastructure especially in the local live animal markets in the country, may facilitate disease transmission. extra attention from the government to upgrade the handling and penning facilities of these market places could result in reduced level of disease transmission. efforts should be make to increase livestock and public awareness with respect to this new disease in the area. the current ongoing government efforts to perform ppr vaccination of goats and sheep in unaffected villages should be encouraged. conclusion top ↑ this study has confirmed, for the first time, the presence of the ppr in southern tanzania. in addition, the study has identified the source of introduction of ppr to be newly purchased animals from pugu livestock market. given that no vaccinations had been carried out against ppr, our result confirms natural transmission of ppr virus under field conditions in the southern tanzania. the spread of this disease to southern tanzania poses a high risk of the disease spread to southern countries (sadc countries including mozambique, zambia and malawi) with naïve goat and sheep populations. further studies on virus isolation, disease status in wildlife and temporal trends events are required to define the epidemiology of ppr in large area of the southern tanzania. national, regional and international collaborative efforts are required to contain and control the disease. aknowledgement top ↑ we would like to thank the project of ruforum (ru 2009 grg 17tads) for supporting this work. we also extend our appreciations to the staff at mtwara veterinary investigation centre, field officers and farmers for their invaluable contributions in terms of their time and cooperation. in addition, we value vic arusha sua technician for laboratory technical assistance. references top ↑ abu elzein, e.m.e., housawi, f.m.t., bashareek, y., gameel, a.a., alafaleq, a.i. & anderson, e.c., 2004, ‘severe ppr infection in 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haemophilus paragallinarum is a gram-negative, polar staining, non-motile bacterium. in 24-h cultures it appears as short rods, or coccobacilli, 1–3 µm in length and 0.4–0.8 µm in width, with a tendency for filament formation. this organism causes an acute respiratory disease of chickens known as infectious coryza (ic), a disease first recognised as a distinct entity in the late 1920s. since the disease proved to be infectious and primarily affected the nasal passages, the name “infectious coryza” was adopted (blackall 1989). the major economic effect of the disease is an increased culling rate in meat chickens and a reduction in egg production (10– 40 %) in laying and breeding hens. the disease is limited primarily to chickens and has no public health significance (yamamoto 1991). all the commercially available bacterins against ic consist of inactivated broth cultures of a combination of two or three different serotypes. although vaccines against ic have been used in south africa since 1975, it became apparent in the 1980s that the vaccines were becoming less effective in controlling the disease (bragg, coetzee & verschoor 1996). this may be due to the emergence of previously unknown serovars, serogroups or changes in the population dynamics. vaccine efficiency is therefore a problem and an alternative to available vaccines is needed. 139 onderstepoort journal of veterinary research, 71:139–152 (2004) genetic organisation of the capsule transport gene region from haemophilus pparagallinarum o. de smidt, j. albertyn*, r.r. bragg and e. van heerden department of microbial, biochemical and food biotechnology, university of the free state p.o. box 339, bloemfontein, 9300 south africa abstract de smidt, o., albertyn, j., bragg, r.r. & van heerden, e. 2004. genetic organisation of the capsule transport gene region from haemophilus paragallinarum onderstepoort journal of veterinary research, 71:139–152 the region involved in export of the capsule polysaccharides to the cell surface of haemophilus paragallinarum was cloned and the genetic organisation determined. degenerate primers designed from sequence alignment of the capsule transport genes of haemophilus influenzae, pasteurella multocida and actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the h. paragallinarum capsule transport gene locus. this fragment was used as a digoxigenin labelled probe to isolate the complete h. paragallinarum capsule transport gene locus from genomic dna. the sequence of the cloned dna was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. the four genes were designated hcta, b, c and d (for h. paragallinarum capsule transport genes) and the predicted products of these genes likely encode an atp-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated abc (atp-binding cassette) transporters. keywords: capsular transport genes, haemophilus paragallinarum, infectious coryza * author to whom correspondence is to be directed. e-mail: albertynj.sci@mail.uovs.ac.za accepted for publication 11 november 2003—editor capsules are found on the surface of a wide range of bacteria and are often important for virulence. these polysaccharide structures have been the subject of intensive investigation because of their usefulness as vaccines for prevention of bacterial infections (lee 1987; boulnois & roberts 1990). many researchers sought to understand the role of the capsule in virulence by identifying the genes involved in capsular polysaccharide export and biosynthesis. the genetic organization of the group ii capsule gene loci of haemophilus influenzae type b (kroll, zamze, loynd & moxon 1989; kroll 1992), escherichia coli k1 and k5 (boulnois, roberts, hodge, hardy, jann & timmis 1987; jann & jann 1990), pasteurella multocida m1404 (b:2) (boyce, chung & adler 2000) and actinobacillus pleuropneumoniae serotype 5a (ward & inzana 1997) have been determined and are very similar. in each of these species, a central dna segment necessary for capsular polysaccharide biosynthesis is flanked by dna encoding proteins for capsule export. substantial homology exists in the genes required for capsular polysaccharide export among these species, suggesting a common evolutionary origin (frosch, edwards, bousset, kraube & weisgerber 1991). genetically defined acapsular mutants have been shown to have reduced virulence in a number of organisms (boyce et al. 2000). a mutant defective in the export of the p. multocida capsule was constructed by allelic exchange and virulence assays showed the acapsular p. multocida to be 106 fold less virulent than their encapsulated counterparts (boyce & adler 2000). similar studies have been conducted on the bexa gene of h. influenzae (kroll, hopkins & moxon 1988). a frame shift mutation engineered at a restriction site within the open reading frame resulted, when introduced into the cap locus in the chromosome, in the expression of a mutant phenotype. the noncapsulated mutants of a. pleuropneumoniae reported by inzana, todd & veit (1993) showed extreme stability and induced a protective immune response without any symptoms of disease. this not only proves the capsule’s involvement in virulence but also offers the opportunity to investigate the possibility of producing live vaccines. in an attempt to understand the genetic organization of the capsular genes of h. paragallinarum degenerate pcr primers, based on the capsule loci of h. influenzae, a. pleuropneumoniae and p. multocida, were used to amplify a section of the capsule transport genes of h. paragallinarum. this section was employed as a probe to clone the full-length transport region. materials and methods bacterial strains haemophilus paragallinarum strain 1742, obtained from the department of poultry health, university of pretoria, south africa, was grown in tm/sn medium (1 % biosate peptone, 1 % nacl, 0.5 % glucose, 0.1 % starch and 0.0005 % thiamine solution, oleic acid-albumin complex and chicken serum as supplements) as described by blackall & yamamoto (1990), in which 1.5 % agar was used to solidify the medium if required. in liquid culture the organisms were grown without aeration and on solid media in a candle jar at 37 °c. escherichia coli strain sure2 (stratagene) was grown with aeration in luria-bertani (lb) broth (sambrook, fritsch & maniatis 1989) under selective pressure with 60 µg/ml ampicillin in liquid and solid media when required. preparation and analysis of genomic and plasmid dna genomic dna was prepared from 20, 5 ml liquid cultures of h. paragallinarum grown for 16 h (towner 1991). the cells were harvested by centrifugation at 3 000 g for 10 min at 4 °c and the mass of the pellet was determined. the pellet was washed in tebuffer (10 mm tris-hcl, 1 mm edta) ph 8 and centrifuged again at 3 000 g for 5 min at 4 °c. the pellet was re-suspended in 40 ml/0.5 g cells buffer (50 mm tris-hcl, ph 8, 0.7 mm sucrose) and lysozyme (20 mg/ml) was added before the suspension was incubated on ice for 5 min. six hundred microlitres edta (0.5 m, ph 8) and 500 µl 10 % sds were added for each 0.5 g cells, gently mixed and placed on ice for 5 min. after the addition of 10 ml/0.5 g cells digestion buffer (1 % sds, 50 mm tris-hcl ph 8, 0.1 m edta, 0.2 m nacl, 0.5 mg/ml proteinase k), the suspension was incubated at 55 °c for 3–16 h with mild shaking. one time the volume of ph calibrated phenol (ph 7.8) was added to the lysate and incubated a further 3 h at 25 °c with constant inversion. cell debris was removed by centrifugation at 4 000 g for 10 min and the supernatant mixed with 0.1x the volume 5 m nacl and placed on ice for 5 min. genomic dna was precipitated with 10 ml 100 % ethanol, spooled and washed in 1 ml 70 % ethanol. after drying, the pellet was suspended in 500 µl/ 0.5 g cells te-buffer and incubated at 50 °c for 1 h or kept at 4 °c overnight before use. 140 genetic organisation of capsule transport gene region from haemophilus paragallinarum plasmid dna was isolated by a rapid alkaline lysis method described by sambrook et al. (1989) and suspended in te-buffer containing 10 µg/ml rnase. genomic and plasmid dna were analysed by restriction enzyme digestion. plasmid dna was digested with ecori or hindiii for 1 h, while genomic dna was digested using bamhi, ecori, hindiii, psti or xbai for 3–16 h. all the enzymes used in these digestions were obtained from roche molecular biochemicals. pcr analysis and cloning techniques pcr analysis was performed in a perkin-elmer geneamp 2400 thermocycler. haemophilus paragallinarum genomic dna (60 ng) was used as template and pcr reactions were carried out in 50 µl volumes. the reaction mixtures consisted of a 10x dilution of reaction buffer (100 mm tris-hcl, 15 mm mgcl2, 500 mm kcl, ph 8.3), 2 pmol of each degenerate primer (table 1) in different combinations, 0.2 mm dntp mixture and 5 u of taq polymerase (roche). the reaction conditions consisted of an initial denaturation cycle of 94 °c for 5 min followed by 25 cycles of 94 °c for 30 s, 45 °c for 30 s, 72 °c for 2 min and a final elongation cycle of 72 °c for 5 min. the same reaction constituents and conditions were used for amplification of the partial h. paragallinarum capsule transport gene locus and for production of a dna probe for screening. pcr products were purified and dna fragments were recovered from agarose gels with the gfxäpcr dna and gel band purification kit (amersham pharmacia biotech). purified fragments were cloned into either pgem-t easy or pgem-3z (promega). escherichia coli strain sure2 was grown to early log phase at 18 °c in sob-media as described by hanahan (1983). competent e. coli cells were prepared according to the method of inoue, nojima & okayama (1990). blotting techniques southern hybridisation was used as a method to identify fragments in digested genomic dna that encode the capsule transport gene locus and colony hybridisation to identify positive clones containing the recombinant plasmids. genomic dna was digested with bamhi, ecori, hindiii, psti or xbai and the fragments separated by agarose gel electrophoresis. the dna was transferred to a magnacharge nylon membrane (micron separations, inc.) by 1 h downward capillary transfer as described by chomczynski (1992). dna was linked to the membrane with the gs gene linker™ (bio-rad) prior to hybridisation. colony blotting to screen for the presence of clones containing the transport gene locus was performed on transformants grown for 16 h on lb plates containing ampicilin. blotting proceeded as described by the dig system users’ guide for filter hybridisation (roche molecular biochemicals). colonies were lifted from the growth media and fixed on a magnacharge nylon membrane. the membrane was subjected to lysis in 10 % sds and denaturation solution (0.5 m naoh, 1.5 m nacl) followed by neutralisation (1 m tris-hcl ph 7.5, 1.5 m nacl) and washing twice in ssc until all the cell debris was removed. hybridisation and colorimetric detection were performed as described in the dig nucleic acid detection kit (roche molecular biochemicals). probe labelling and screening methods the 2.6 kb fragment used as a hybridisation probe was amplified from h. paragallinarum genomic dna with primers hctd-1f and hcta-1r (table 1). this fragment was prepared as the hct-probe by random prime labelling with digoxigenin using the dig labelling and detection kit. 141 o. de smidt et al. table 1 degenerate and sequence specific oligonucleotides used for the amplification of the capsule transport gene locus from h. paragallinarum degenerate primers hctd-1f 5’gat aaa gat wtw gth tat gtr tcr aat gca cc -3’ hctc-1f 5’gcb tcy gat att tat rtt tcd saa tcd ag -3’ hctc-1r 5’cya aat ama rtt gyt ggc gat c -3’ hctb-1f 5’atg atg tgg cgh aat gcd tc -3’ hctb-1r 5’aac att tcy gwr ccr tga atc aty gg -3’ hcta-1r 5’aty ttr gtt tcw cat agc ccg wvt -3’ g sequence specific primers hctd-1r 5’ggt gca ttc gac aca tat ac -3’ hcta-1f 5’att tta gtt tct cat agt cca acc g -3’ the amount of labelled dna was determined by comparison of the intensity of the spots of a serial dilution of the hct-probe to that of a labelled control (supplied by the manufacturer). sequencing and analysis plasmid construct phcta-d was used as a template for sequencing. sequencing reactions were performed with the abi prism big dye terminator v3.0 cycle sequencing ready reaction kit and data collected on an abi prism 377 dna sequencer (perkin-elmer biosystems). data was analysed using sequencing analysis v3.3. sequences were reverse complemented and compared by using sequence navigator v 1.0.1 and assembled using auto-assembler v1.4.0 and dnassist v2.0. sequence submission sequence of the transport gene locus was submitted to genbank, accession number ay116594. results partial amplification of the h. pparagallinarum capsule transport gene region genomic dna was isolated from h. paragallinarum and used as a template for pcr amplification of the h. paragallinarum capsule transport genes. the capsule transport gene sequences of h. influenzae (bexa-d genes), a. pleuropneumoniae (cpxa-d genes) and p. multocida (hexa-d genes) were obtained from genbank (accession no. x54987, u36397 & af067175) and submitted to a multiple sequence alignment using dnassist v 1.0.2. six degenerate primers were designed (table 1) from areas in these aligned gene sequences where the sequence was highly conserved. the pcr performed with different oligonucleotide combinations (fig. 1a), showed amplification of fragments of expected sizes in lanes 1 (~2.6 kb), 2 (~2.3 kb), 3 (~1.9 kb), 4 (~1.6 kb) and 6 (~1.1 kb). the relative position of each of these fragments in the proposed h. paragallinarum transport gene region is indicated in fig. 1b. lanes 5 and 7 showed either non-specific or no amplification. more than one band was visible in some lanes due to non-specific priming and a low annealing temperature of 45 °c. the estimated ~2.6 kb fragment amplified by the oligonucleotides hctd-1f and hcta-1r (fig. 1a, lane 1), was cloned into pgem-t easy and designated phct. the nucleotide sequence of this fragment was determined and analysis revealed considerable homology with the capsule transport genes of related organisms (h. influenzae, a. pleuropneumoniae and p. multocida). this high degree of homology among the four species indicated that the sequenced 2638 bp insert in phct represented part of the capsule transport gene region of h. paragallinarum. by comparison with the capsular transport genes of p. multocida, this fragment contained homologues of hexc and hexb as well as small regions of the 3’ end of hexd and the 5’ region of hexa. construction of a mini-library to isolate the entire capsule transport gene region to facilitate the cloning of the full-length capsular transport region, the phct insert was used as a probe (designated hct) in southern blotting followed by colony hybridisation. genomic dna of h. paragallinarum was digested with five different restriction enzymes, transferred to a nylon membrane and hybridised with a digoxigenin labelled hct-probe under stringent conditions. southern blotting and hybridisation indicated that a hindiii fragment of ~6.15 kb (fig. 2, lane 3) hybridised to the hctprobe. hybridisation products visible in lanes 1, 2, 4 and 5 at a position of ~21 kb correspond to the relative position of undigested genomic dna or unresolved large restriction fragments when using restriction enzymes bamhi, ecori, psti and xbai. the hindiii fragments resolved between 6 kb and 6.5 kb were excised from the gel, purified and cloned into vector pgem-3z to construct a minilibrary. colony hybridisation was used as a screening method to identify positive clones containing the transport genes. a total of 93 colonies were visible within 1 day of transformation and two colonies showed hybridisation with the hct-probe after screening under stringent conditions. plasmid dna, extracted from the above-mentioned colonies and digested with hindiii, revealed the presence of a ~6.15 kb insert. to confirm that these plasmid constructs did contain the capsule transport region, the 5’ and 3’ terminal regions were sequenced. sequencing confirmed that both clones were identical and also gave an indication of the orientation in which the ~6.15 kb fragment was ligated into the vector. sequence alignment to known capsular genes, using the above-mentioned sequences, indicated that the ~6.15 kb fragment did in fact contain the relevant capsule region. pcr reactions were performed to determine which part or parts of the hct-probe features in the 142 genetic organisation of capsule transport gene region from haemophilus paragallinarum 143 o. de smidt et al. m 1 2 3 4 5 6 7 5.1 2.0 1.6 1.4 21 kb 0.83 2 600 bp 2 300 bp 1 900 bp 1 600 bp 1 100 bp 860 bp 1 kb hctd-1f hctc-1f hctb-1f hcta-1rhctc-1r hctb-1r hcta hctb hctc hctd 1 100 bp a b fig. 1 a amplification of segments of the h. paragallinarum capsule transport gene region the different degenerate oligonucleotides were used in the following combinations: hctd-1f & hcta-1r (lane 1), hctc-1f & hcta-1r (lane 2), hctd-1f & hctb-1r (lane 3), hctc-1f & hctb-1r (lane 4), hctd-1f & hctc-1r (lane 5), hctb-1f & hcta-1r (lane 6) and hctc-1f & hctc-1r (lane 7) b schematic representation of the proposed h. paragallinarum transport gene region indicating the relative positions of the degenerate oligonucleotides used in fig. 1a the pcr fragments expected were as follows: 2 600 bp (lane 1, fig. 1a), 2 300 bp (lane 2, fig. 1a), 1 900 bp (lane 3, fig. 1a), 1 600 bp (lane 4, fig. 1a), 1 100 bp (lane 5, fig. 1a), 1 100 bp (lane 6, fig. 1a), 860 bp (lane 7, fig. 1a) ~6.15 kb fragment. these pcr reactions were performed using sequence specific oligonucleotides designed according to the sequence obtained from the 2.6 kb phct fragment. sequence specific oligonucleotide hctd-1r was used in combination with t7 (fig. 3, lane 1) and hcta-1f in combination with sp6 (fig. 3, lane 2) (sp6 and t7 have binding sites on opposite sides of the multiple cloning site of pgem-3z). amplification of two bands were visible, a ~1.5 kb band in lane 1 representing the segment upstream and a ~2.1 kb band in lane 2 indicating the segment downstream from the hct-probe sequence. these results and the high degree of sequence homology with the transport genes of related organisms, verified that the ~6.15 kb fragment represents the entire h. paragallinarum capsule transport region and was designated phcta-d. the nucleotide sequence of the full-length capsular transport region was determined through primer walking using the ~6.15 kb hindiii restriction fragment of phcta-d. analysis of the complete sequence revealed that the h. paragallinarum capsule transport gene region is 3 792 bp in length with a gc content of 37 %, comprising four open reading frames representing the four capsule transport genes designated hctdcba (fig. 4 and 5). hctd contains 1 188 nucleotides and terminates at a tga stop codon, encoding a putative protein of 395 amino acids. the next open reading frame, hctc, starts at the third base of the hctd stop codon and encodes a putative protein of 387 amino acids. the third base of the stop codon at the 3’end of hctc is the first base of the atg at the start of hctb, 798 nucleotides in length and coding for a putative protein of 265 amino 144 genetic organisation of capsule transport gene region from haemophilus paragallinarum ~6.15 kb m 1 2 3 4 5 5.1 2.0 1.6 1.4 21 kb ~2.1 kb ~1.5 kb m 1 2 5.1 2.0 1.6 1.4 21 kb fig. 2 southern blot analysis of digested genomic dna hybridised with the hct-probe under stringent conditions. genomic dna was digested with bamhi (lane 1), ecori (lane 2), hindiii (lane 3), psti (lane 4) and xbai (lane 5) for 3 h. arrow indicates positive hybridisation with a fragment ~6.15 kb in size in the hindiii digestion fig. 3 amplification of the regions upand downstream from the hct-probe sequence within the ~6.15 kb clone. a ~1.5 kb fragment was amplified when oligonucleotide hctd-1r was used in combination with t7 (lane 1). a ~2.1 kb fragment was amplified by oligonucleotides hcta-1f and sp6 (lane 2) 0 3.79 kb 2 kb 3 kb1 kb hctd 1 188 bp hctb 798 bp hcta 648 bp hctc 1 164 bp hct-probe fig. 4 genetic map of the capsule transport gene region of h. paragallinarum. the locations and directions of transcription of the four open reading frames hctdcba are indicated. the 2.6 kb fragment (hct) used as the dna probe in fig. 2 is also indicated fig. 5 nucleotide sequence of the capsule transport region. three thousand nine hundred and twenty three nucleotides of the sequence are shown, from arbitrary points 167 bp upstream of hctd to 131 bp downstream of hcta. the four open reading frames are indicated as hctd, hctc, hctb and hcta, in each case from the first atg, with the translated peptide sequence beneath. the underlined regions are referred to in the text 145 o. de smidt et al. –167 …ga taa gtg ttg ata taa ata aaa ttt ccc gag tct tta –130 –129 aaa aat tgg aat tat ttt tat aaa aaa gtt ttc tac agg aaa ttg –85 –84 agc aaa aat taa taa tta tct atg ata att act cac ttt taa tag –40 hctd –39 aaa aat cat gat caa aaa caa aat aat taa ggt aaa act atg cgt 6 met arg 2 7 aaa tcg ctg att gca gta agt tac tgc tta tta tta atg tct tgg 51 3 lys ser leu ile ala val ser tyr cys leu leu leu met ser trp 17 52 tct tat ttg cca aat tca gga ccg agc aaa ggc aat att gag gta 96 18 ser tyr leu pro asn ser gly pro ser lys gly asn ile glu val 32 97 gtc aat aaa cag aaa tcc aat gag gat ttg ctt gca gta cag ttg 141 33 val asn lys gln lys ser asn glu asp leu leu ala val gln leu 47 142 atc gag gtg aat aat aaa gtt gcg gaa agt atg ttt aat caa caa 186 48 ile glu val asn asn lys val ala glu ser met phe asn gln gln 62 187 cac cct caa tca ttt ttg cag ttt cct tca tca aaa gca cat tat 231 63 his pro gln ser phe leu gln phe pro ser ser lys ala his tyr 77 232 cat ggg gta gtt aaa tgc tgg tgt tta ctt gat att act ctc tgg 276 78 his gly val val lys cys trp cys leu leu asp ile thr leu trp 92 277 gaa gca ccc gcc agc aac ttt gtt tgg cag tgt gtt gaa tca agc 321 93 glu ala pro ala ser asn phe val trp gln cys val glu ser ser 107 322 cgg tgt gtc ggg cgg aca aag cac tca ctt acc gga aca ggt ggt 366 108 arg cys val gly arg thr lys his ser leu thr gly thr gly gly 122 367 tat agc aat gga aga ata acc att cct ttt gtt ggt gca tta aaa 411 123 tyr ser asn gly arg ile thr ile pro phe val gly ala leu lys 137 412 gta gca ggg aaa aca ccg gag cag atc caa tct gaa att gtt gga 456 138 val ala gly lys thr pro glu gln ile gln ser glu ile val gly 152 457 cgt tta caa gca att gcc aat caa cca caa gca gtg gtg cga att 501 153 arg leu gln ala ile ala asn gln pro gln ala val val arg ile 167 502 gtg aag aat aat tct gct aat gtg acg gtt tta act aaa tcg act 546 168 val lys asn asn ser ala asn val thr val leu thr lys ser thr 182 547 act att cga atg gct tta act gct tac ggt gaa cga agt gtt aga 591 183 thr ile arg met ala leu thr ala tyr gly glu arg ser val arg 197 592 tgc tat tgc ggc agc agg tgg agc cgg tgg tat gtg caa aga tgt 636 198 cys tyr cys gly ser arg trp ser arg trp tyr val gln tyr cys 212 637 ttc agt gcg act gac tcg tgg gaa atc agg gtg caa acg att tct 681 213 phe ser ala thr asp ser trp glu ile arg val gln thr ile ser 227 682 tta gcc agg att aac gga ggg agc cac agg caa aat atc cta tta 726 228 leu ala arg ile asn gly gly ser his arg gln asn ile leu leu 242 727 cgt tcc ggc gat gta gta acg tta tta aat aat cca ctt tct ttc 771 243 arg ser gly asp val val thr leu leu asn asn pro leu ser phe 257 772 act gca atg ggt gcg gta gga aat agt aaa gaa att cgt ttt tcg 816 258 thr ala met gly ala val gly asn ser lys glu ile arg phe ser 272 817 gca gaa ggt tta act tta gca gaa gca atc ggt cgt tta ggt gga 861 273 ala glu gly leu thr leu ala glu ala ile gly arg leu gly gly 287 fig. 5 (continued) 862 ttg aat gat gat cgt gca gat cca aga gga gta ttt atc ttt cgt 906 288 leu asn asp asp arg ala asp pro arg gly val phe ile phe arg 302 907 tat gtt cca ttt gaa gaa atg ccc tta agt aaa caa aat gaa tgg 951 303 tyr val pro phe glu glu met pro leu ser lys gln asn glu trp 317 952 caa gcc aag ggg tat cac aac gga atg aaa att cca aca gta tat 996 318 gln ala lys gly tyr his asn gly met lys ile pro thr val tyr 332 997 caa gcg aat tta ctt gaa cct caa tca atg ttt tgg att caa caa 1041 333 gln ala asn leu leu glu pro gln ser met phe trp ile gln gln 347 1042 ttt cca att aaa gat aaa gat att gtt tat gta tct aat gca cca 1086 348 phe pro ile lys asp lys asp ile val tyr val ser asn ala pro 362 1087 ttg gct gaa tac caa att tat tcg tat gat tta cgc cac cgt tgc 1131 363 leu ala glu tyr gln ile tyr ser tyr asp leu arg his arg cys 377 1132 aac tac acc gcc ggt ttc aac tgt aaa caa gtg tta ata atc tgt 1176 378 asn tyr thr ala gly phe asn cys lys gln val leu ile ile cys 392 hctc 1177 agg ggg aga tga tg gaa caa aat gta gta gtt caa tcg aaa gaa 1220 393 arg gly arg *** met glu gln asn val val val gln ser lys glu 406 1221 caa ctg aga aag tta aaa cag tgg ttg cga aaa att aat ctg tta 1265 407 gln leu arg lys leu lys gln trp leu arg lys ile asn leu leu 421 1266 ttt tta ctg acg gtg att att ccg act ttt tgt tcg tta ttt tat 1310 422 phe leu leu thr val ile ile pro thr phe cys ser leu phe tyr 436 region a1 (421–442) 1311 ttt tct att tgg gct tcc gat gtt tat att tcg gag tcc agt ttt 1355 437 phe ser ile trp ala ser asp val tyr ile ser glu ser ser phe 451 1356 att gtg cgt tct tct cgt gct cag gca tcg ctc gga ggt atg ggg 1400 452 ile val arg ser ser arg ala gln ala ser leu gly gly met gly 466 region a2 (458–475) 1401 gct tta ttg cag agt atc ggt ttt gct cgt tcg caa gat gat act 1445 467 ala leu leu gln ser ile gly phe ala arg ser gln asp asp thr 481 1446 ttt acg gtg caa gaa ttt atg cgt tcg cgt aat gcg ttg aca aca 1490 482 phe thr val gln glu phe met arg ser arg asn ala leu thr thr 496 1491 ttg gaa agt gag tta ccg gtg aga aaa ttt tat gaa gat gaa ggg 1535 497 leu glu ser glu leu pro val arg lys phe tyr glu asp glu gly 511 1536 gat ttt ttc agc ccg ttt aat ccg tta ggt ttt ttt aat gaa cag 1580 512 asp phe phe ser pro phe asn pro leu gly phe phe asn glu gln 526 1581 gaa ttg ttt tat caa tat ttt cgt aaa cat ttg atg att aat atc 1625 527 glu leu phe tyr gln tyr phe arg lys his leu met ile asn ile 541 1626 gat tct tta tct ggg tat tgc tac ttt aca ggt tcc gtg ggt tta 1670 542 asp ser leu ser gly tyr cys tyr phe thr gly ser val gly leu 556 1671 atg gct gac ctc cgg cac caa caa gaa tta aat gga agc cat tat 1715 557 met ala asp leu arg his gln gln glu leu asn gly ser his tyr 571 1716 tgc cat ttt ggc ggg aaa cca ttt agt gga ata aac tca atg atc 1760 572 cys his phe gly gly lys pro phe ser gly ile asn ser met ile 586 1761 gtg cac gta aag ata caa tta ctt tgc gga aca atc ggt aat gaa 1805 587 val his val lys ile gln leu leu cys gly thr ile gly asn glu 601 1806 gca gaa aaa tat ttg tct gaa acc tcg aca gcc tta agc caa tat 1850 602 ala glu lys tyr leu ser glu thr ser thr ala leu ser gln tyr 616 146 genetic organisation of capsule transport gene region from haemophilus paragallinarum fig. 5 (continued) 1851 cgt gta aaa aat ggg ata ttt gat att ggg gca caa tct gaa tcg 1895 617 arg val lys asn gly ile phe asp ile gly ala gln ser glu ser 631 1896 att tta act tta gtg cag aag ttg cag gat gaa ctg att gcc att 1940 632 ile leu thr leu val gln lys leu gln asp glu leu ile ala ile 646 1941 cag acg caa ctt gat cag gtg agg ggc gtt atc tcc gga tac cct 1985 647 gln thr gln leu asp gln val arg gly val ile ser gly tyr pro 661 1986 cag gtt aaa gtg tta aag gca agg caa ttt gaa agt att cgt gaa 2030 662 gln val lys val leu lys ala arg gln phe glu ser ile arg glu 676 2031 aga agt ggc aca aca att gaa tcc ggg gtt ttt gag ggg aaa cca 2075 677 arg ser gly thr thr ile glu ser gly val phe glu gly lys pro 691 2076 ttc ttt aac aac aca atc agc aga gta cca gcc gtt aat tta gat 2120 692 phe phe asn asn thr ile ser arg val pro ala val asn leu asp 706 2121 gaa acc ttg gca aaa cag caa tta aca gct gca atg tct tgc gtt 2165 707 glu thr leu ala lys gln gln leu thr ala ala met ser cys val 721 2166 aca agt ggc aaa gaa gaa gct gga aga caa cag ctt tat ctg gaa 2210 722 thr ser gly lys glu glu ala gly arg gln gln leu tyr leu glu 731 2211 att att gct aaa cct agc cat cca gat tta gca ttg gaa ccg cac 2255 737 ile ile ala lys pro ser his pro asp leu ala leu glu pro his 751 2256 cgt ttg tac aat att ttg gca act ttg att ctt gga tta gtt att 2300 752 arg leu tyr asn ile leu ala thr leu ile leu gly leu val ile 766 region a3 (753–777) 2301 tat ggc gtt tca act tta tta tta gcc ggt gtg aga gag cat aag 2345 767 tyr gly val ser thr leu leu leu ala gly val arg glu his lys 781 hctb 2346 aac tga tg cag tat ggt gaa caa act tcg tta aaa gat tca ttt 2389 782 asn *** met gln tyr gly glu gln thr ser leu lys asp ser phe 795 2390 act atc caa gga cgg gtg ttg aaa gcg ttg ttg ttg cgt gaa att 2434 796 thr ile gln gly arg val leu lys ala leu leu leu arg glu ile 810 2435 atc act cgt tat ggt cgt aaa aat tta ggc ttt ttg tgg gtt gtt 2479 811 ile thr arg tyr gly arg lys asn leu gly phe leu trp val val 825 2480 cgt gag cca ttt ttg atg agc cta gtt att gtg gta atg tgg cat 2524 826 arg glu pro phe leu met ser leu val ile val val met trp his 840 2525 ttt ttt cgt gct gat cgc ttt tca aca tta aac att gtt gct ttt 2569 841 phe phe arg ala asp arg phe ser thr leu asn ile val ala phe 855 2570 gca atg acg gtt atc cat tat tat gga tgt ggc gta atg ctt cta 2614 856 ala met thr val ile his tyr tyr gly cys gly val met leu leu 870 2615 acc gtg caa tta gcg gga atg gat tcc aat atc cca tta ctt tta 2659 871 thr val gln leu ala gly met asp ser asn ile pro leu leu leu 885 2660 tca cgt aat gta cgt cct ctt gat acg ctt ttt tct cgt atg att 2704 886 ser arg asn val arg pro leu asp thr leu phe ser arg met ile 900 2705 ttg gag att gct ggt gcg act gta gca caa att gtg atg tta gtg 2749 901 leu glu ile ala gly ala thr val ala gln ile val met leu val 915 2750 att tta att gct att gat tgg atc ggc ttg cca aat gat gtg ttg 2794 916 ile leu ile ala ile asp trp ile gly leu pro asn asp val leu 930 2795 tat atg ctt ttt gct tgg ttc tta atg gca ctg ttt gcc att ggt 2839 931 tyr met leu phe ala trp phe leu met ala leu phe ala ile gly 945 2840 tta ggt tta att att tgt gct att tct tat tat tta gag ttt ttc 2884 946 leu gly leu ile ile cys ala ile ser tyr tyr leu glu phe phe 960 147 o. de smidt et al. fig. 5 (continued) 2885 ggt aaa att tgg gga aca tta tct ttt gtg atg ttt cct att tcc 2929 961 gly lys ile trp gly thr leu ser phe val met phe pro ile ser 975 2930 ggt gca ttc ttt tta gtg aat agt ttg cca aac aat ctg caa tct 2974 976 gly ala phe phe leu val asn ser leu pro asn asn leu gln ser 990 2975 att ttg ctt tgg ttt cca atg gtt cac ggt acg gaa atg ttt cgt 3019 991 ile leu leu trp phe pro met val his gly thr glu met phe arg 1005 region b (990–1009) 3020 cac ggt tat ttt ggt tct tca gtt att aca atg gaa tca ccg agt 3064 1006 his gly tyr phe gly ser ser val ile thr met glu ser pro ser 1020 3065 tat tta ttt att tgt gat ttg gtg atg tta tta atc ggt cta ctg 3109 1021 tyr leu phe ile cys asp leu val met leu leu ile gly leu leu 1035 hcta 3110 atg gtg ggt agt ttt agt aat agg att aat gca aga tg att agt 3153 1036 met val gly ser phe ser asn arg ile asn ala arg *** met ile ser 1050 3154 gta gac cac gtt tat aaa aaa tat caa aca cgg aca ggt tcg gta 3198 1051 val asp his val tyr lys lys tyr gln thr arg thr gly ser val 1065 3299 ccc gta tta aat gat att aat ttt agc ctt acc aaa gaa gaa aaa 3243 1066 pro val leu asn asp ile asn phe ser leu thr lys glu glu lys 1080 3244 att ggt att tta ggt cgc aac gga gca gga aaa tca cca tta att 3288 1081 ile gly ile leu gly arg asn gly ala gly lys ser pro leu ile 1095 region c1 (1080–1092) 3289 cgt tta atg agt ggt gtt gaa gct cca act tca gga ata att cga 3333 1096 arg leu met ser gly val glu ala pro thr ser gly ile ile arg 1110 3334 cga gaa atg agc att tct tgg cca tta gcc ttt agc ggt gca ttc 3378 1111 arg glu met ser ile ser trp pro leu ala phe ser gly ala phe 1125 3379 caa ggt agc tta acg gga atg gat aat tta cgc ttc att tgt cgt 3423 1126 gln gly ser leu thr gly met asp asn leu arg phe ile cys arg 1140 region c2 (1131–1138) 3424 att tat aat gct gat att aat tat gtt act gaa ttt acg gaa tcc 3468 1141 ile tyr asn ala asp ile asn tyr val thr glu phe thr glu ser 1155 3469 ttt tcc gaa ttg ggc aat tat tta tat gag cct gta aaa aat tat 3513 1156 phe ser glu leu gly asn tyr leu tyr glu pro val lys asn tyr 1170 3514 tct tca gga atg aaa gca cgc tta gct ttt gca ttg tcg tta tcc 3558 1171 ser ser gly met lys ala arg leu ala phe ala leu ser leu ser 1185 3559 gtt gag ttt gat tgc tat ctc att gat gaa gtg att gcc gtt gga 3603 1186 val glu phe asp cys tyr leu ile asp glu val ile ala val gly 1200 3604 gat tct cgt ttt agt gat aaa tgt cgc tat gaa ctt ttt gaa aaa 3648 1201 asp ser arg phe ser asp lys cys arg tyr glu leu phe glu lys 1215 3649 cgc aaa gat cgt tcc att att tta gtt tct cat agt cca acc gct 3693 1216 arg lys asp arg ser ile ile leu val ser his ser pro thr ala 1230 3694 att aga caa tat tgt gat aat gca aaa gta tta gat aaa gga aaa 3738 1229 ile arg gln tyr cys asp asn ala lys val leu asp lys gly lys 1245 3739 ttg tta gat ttc tct tct att gat gag gct tat caa tat tat aat 3783 1246 leu leu asp phe ser ser ile asp glu ala tyr gln tyr tyr asn 1260 3784 cag aca tag agg tta gat ttt aaa ata aaa taa cgt tac ttt ctt 3883 1261 gln thr *** 1260 3829 gct tta tca taa att tca atg gct ata gtt aag ttc gaa ata aat 3873 3874 caa ggt aac aag ctg aat aca gtg aaa aat agc act ttt tat gcc 3918 3919 aag gt… 148 genetic organisation of capsule transport gene region from haemophilus paragallinarum acids. hctb terminates with a tga stop codon where it overlaps with the hcta start codon. hcta contains 648 nucleotides, encodes a putative protein of 215 amino acids and terminates at a tag stop codon. downstream of hcta all reading frames in both directions are closed with multiple stop codons. part of an open reading frame is present upstream of hctd, which showed considerable homology with the p. multocida hyaa biosynthesis gene. the overlapping stop and start codons in the hct genes (fig. 4 and 5) indicate that these four genes are probably transcriptionally coupled. discussion analysis of the h. paragallinarum hctdcba gene cluster revealed a clear bias toward codons rich in nucleotides a and t (37 % gc content) consistent with the 39 % gc content of the h. influenzae capsule gene cluster (kroll, loynds, brophy & moxon 1990) and 37 % gc content of the h. influenzae genome overall (roy & smith 1973). it also correlates with the calculated gc contents of a. pleuropneumoniae (40 %) and p. multocida (37 %). the gene lengths and region size correlate well with those of related organisms, all belonging to the family pasteurellaceae (table 2). blast searches of the combined, non-redundant nucleotide and protein databases at the national centre for biotechnology information (ncbi) indicated that h. paragallinarum hctdcba were highly homologous at both the nucleotide and amino acid levels to h. influenzae bexdcba (kroll et al. 1990), a. pleuropneumoniae cpxdcba (ward & inzana 1997), p. multocida hexdcba (chung, zhang & adler 1998) and neis149 o. de smidt et al. hctb (207) s i l l w f p m v h g t e m f r h g y f bexb (208) s i a l w f p m i h g t e m f r h g y f oppb (209) r t a r a k g l p m r r i i f r h a l k fig. 6 alignment of the relatively hydrophilic portions of hctb, bexb and oppb. the number in brackets is the position of the first amino acid in each sequence. identical amino acids in all three genes are boxed, and the matches of the oppb sequence to the dassa/hofnung consensus are underlined table 2 comparison of capsular transport gene and protein sizes and % identity and similarity between proteins from h. paragallinarum 1742 with those of related bacterial species h. paragallinarum related bacterial species comparison on protein level orfa proteinb orfa accession no.c % identity % similarity hcta (648) hcta (215) bexa (654) (h. influenzae) p10640 77.2 85.1 cpxa (615) (a. pleuropneumoniae) u36397 78.6 85.6 ctrd (651) (n. meningitidis) m57677 78.6 87.4 hexa (660) (p. multocida) af067175 75.3 85.6 hctb (798) hctb (265) bexb (798) (h. influenzae) p19391 57.7 86.0 cpxb (798) (a. pleuropneumoniae u36397 57.7 84.9 ctrc (798) (n. meningitidis) m57677 58.1 86.7 hexb (798) (p. multocida) af067175 58.5 82.3 hctc (1164) hctc (387) bexc (1134) (h. influenzae) p22930 43.7 67.2 cpxc (1167) (a. pleuropneumoniae) u36397 43.9 65.6 ctrb (1164) (n. meningitidis) m57677 38.7 58.1 hexc (1137) (p. multocida) af067175 49.9 71.8 hctd (1188) hctd (395) bexd (1182) (h. influenzae) p22236 42.5 63.0 cpxd (1212) (a. pleuropneumoniae) u36397 42.5 65.0 ctra (1176) (n. meningitidis) m57677 41.5 62.5 hexd (1182) (p. multocida) af067175 43.0 68.1 a open reading frame of each capsule transport gene and corresponding nucleotide size in base pairs b predicted proteins for each capsule transport gene and protein size in amino acids c genbank accession numbers of capsular transport sequences from related bacterial species seria meningitidis ctrabcd (frosch, muller, bousset & muller 1992) (table 2). the predicted amino acid sequences of the hct genes showed significant identity with the capsule transport genes of related organisms. the predicted hcta protein showed on average 77 % identity and 85.4 % similarity with the a proteins of h. influenzae, a. pleuropneumoniae and p. multocida. hcta contains the atp-binding domains a (gxlgrxgxgks) and b (xxdnlrfi) (walker, sarste, runswick & gay 1982) at amino acids 1080–1092 and 1131– 1138 respectively (fig. 5, regions c1 and c2), which are conserved in the bexa and cpxa homologues (kroll et al. 1990; fath & kolter 1993; ward & inzana 1997). the nucleotide homology as well as the high degree of similarity between homologous proteins, supports the speculation that hcta might encode an atp-binding protein component of a polysaccharide export apparatus. hctb protein showed an average of 58 % identity and 84.4 % similarity with its corresponding homologues and is predicted to be a hydrophobic protein over most of its length, containing at least six potential membrane-spanning a-helical domains (kyte & doolittle 1982; kroll et al. 1990 ). a short relatively hydrophilic region starting at amino acid 990 (fig. 5, region b) aligned with a similar region in oppb of salmonella typhimurium (hiles, gallagher, jamieson & higgins 1987) and bexb from h. influenzae (kroll et al. 1990). furthermore, each showed a marginal sequence similarity to a consensus thought to be involved in intermolecular interactions in the oligopeptide transporter (dassa & hofnung 1985). fig. 5 (region b) shows the position of this sequence on hctb and fig. 6 shows an alignment of the relatively hydrophilic portions of hctb, bexb and oppb. hctb is therefore a candidate for an integral innermembrane component of the putative polysaccharide exporter. the multiple protein sequence alignment of hctc with the respective c proteins of h. influenzae, a. pleuropneumoniae and p. multocida showed a lower homology (average of 45.8 % identity and 68.2 % similarity) in comparison to hcta and b with their corresponding homologues. transposon mutagenesis of bexc (kroll et al. 1990) suggested that this gene might be a periplasmic protein. prediction of protein subcellular localisation of the hexc protein performed with psort (nakai & kahehisa 1991), suggested an inner membrane protein, possibly with a periplasmic domain, concurring with the transposon mutagenesis data on bexc (chung et al. 1998). the n-terminus of bexc containing phosphatase activity suggests that the protein is either excreted into the periplasm with cleavage of an n-terminal leader peptide or anchored in the bacterial inner membrane by an uncleaved n-terminal domain to protrude into the periplasm. it is therefore a candidate for a periplasmically orientated component of a capsular polysaccharide exporter. ward & inzana (1997) predicted the cpxc protein of a. pleuropneumoniae to be relatively hydrophilic with hydrophobic domains near the n and c-termini that may serve as membrane anchors. three long hydrophobic stretches of amino acid sequence with membrane-spanning potential allowing the possibility of anchoring at more than one site have been identified in bexc (kroll et al. 1990). similar stretches of sequence are present in hctc at amino acids 421– 442, 458–475 (fig. 5, regions a1 and a2) at the proposed n-terminal and 753–777 at the c-terminal (fig. 5, region a3). considering this information and the facts known about the hctc homologues, it is proposed that this protein serves as the second component of a protein complex involved in polysaccharide export across the cytoplasmic membrane (reizer, reizer & saver 1992). hctd showed an average of 42.6 % identity and 65.3 % similarity with the predicted d proteins of h. influenzae, a. pleuropneumoniae and p. multocida. hctd showed similarity of 63 % with bexd and 62.5 % with crta from h. influenzae and n. meningitidis respectively. ctra from neisseria meningitidis is believed to be an outer membrane protein with porin properties (frosch et al. 1992). in addition, bexd and its homologues is believed to be outer membrane associated (kroll et al. 1990; rosenow, esumah, roberts & jann 1995), mutations in the bexd gene coding for this corresponding protein accumulated polysaccharides in the periplasmic space (bronner, clarke & whitfield 1994). based on these similarities with ctra and bexd, hctd it is probably an outer membrane protein involved in capsular polysaccharide transport across the outer membrane, possibly with porin properties. these data are therefore consistent with the hypothesis that the hctabcd gene cluster encodes proteins that form an export complex for capsule polysaccharides. the findings will greatly facilitate the investigation at molecular level of the role of the h. paragallinarum capsule in pathogenesis. however, confirmation of the importance of each gene product and elucidation of the function of each protein will require characterization of the phenotypic impact of in-frame deletions or other mutations in the respective genes. in-frame deletions might lead 150 genetic organisation of capsule transport gene region from haemophilus paragallinarum 151 to reduced virulence with the possible use as a live vaccine. acknowledgements this study was supported financially by the national research foundation (nrf) as a student scholarship to o. de smidt and core funding to j. albertyn. financial contributions from the department of microbial, biochemical and food biotechnology, university of the free state are gratefully acknowledged. references blackall, p.j. 1989. the avian haemophili. clinical microbiology reviews, 2:270–277. blackall, p.j. & yamamoto, r. 1990. infectious coryza, in a laboratory manual for the isolation and identification of avian pathogens (3rd ed.), edited by h.g. prachase, l.h. arp, c.h. domermuth & j.e. pearson. ames, iowa: american association of avian pathologists. boulnois, g.j., & roberts, i.s. 1990. genetics of capsular polysaccharide production in bacteria. current topics in 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sequences in the aand b-subunits of atp synthase, myosin, kinases and other atp-requiring enzymes and a common nucleotide binding fold. embo journal, 1:945–951. ward, c.k. & inzana, t.j. 1997. identification and characterization of a dna region involved in the export of capsular polysaccharides by actinobacillus pleuropneumoniae serotype 5a. american society of microbiology, 65:2491–2496. yamamoto, r. 1991. infectious coryza, in diseases of poultry (9th ed.), edited by m.s. hofstad, h.j. barnes, b.w. calnek, w.m. reid & h.e. yonder jr. iowa state university press. 152 genetic organisation of capsule transport gene region from haemophilus paragallinarum article information authors: bersissa e. kumsa1,2 shewit mekonnen1 affiliations: 1department of parasitology, addis ababa university, ethiopia 2department of experimental sciences, university of padova, italy correspondence to: bersissa kumsa email: bersissak@yahoo.com postal address: po box 34, bishoftu, ethiopia dates: received: 25 feb. 2011 accepted: 19 apr. 2011 published: 04 oct. 2011 how to cite this article: kumsa, b.e. & mekonnen, s., 2011, ‘ixodid ticks, fleas and lice infesting dogs and cats in hawassa, southern ethiopia’, onderstepoort journal of veterinary research 78(1), art. #326,4 pages. doi:10.4102/ojvr.v78i1.326 copyright notice: © 2011. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. issn: 0030-2465 (print) issn: 2219-0635 (online) ixodid ticks, fleas and lice infesting dogs and cats in hawassa, southern ethiopia in this original research... open access • abstract • introduction • materials and methods    • study area    • study animals    • study methodology    • ectoparasite collection and identification    • data analysis • results • discussion • conclusion • references abstract (back to top) this study investigated the prevalence, risk factors and species composition of ticks, fleas and lice infesting dogs and cats in and around hawassa in southern ethiopia. in total, 200 dogs and 100 cats were examined from november 2008 to april 2009. of the dogs and cats examined, 99.5% and 91.5%, respectively, wereinfested with one or more species of ticks, fleas or lice. the overall prevalence was higher in dogs than in cats. a total of six different species of ectoparasites were collected and identified from dogs, but only three species from cats. ctenocephalides felis was the predominant species amongst the animals, with a prevalence of 82.9% on dogs and 67% on cats. other prevalent species on dogs included ctenocephalides canis (73.8%), heterodoxus spiniger (4%), nymphs of amblyomma spp. (3.5%),pulex irritans (2.5%) and haemaphysalis leachi (0.5%). c. canis (18%) and p. irritans (6%) were also found on cats. more female than male fleas and lice were observed. the study revealed that the prevalence of fleas, ticks and lice on dogs was not significantly different between male and female animals or between young and adult dogs. however, the prevalence of these ectoparasites was significantly higher in female than in male and in adult than in young cats. the study showed that theprevalence of ectoparasites on both dogs and cats was significantly higher on animals with flea allergy dermatitis (fad) than those without fad, and on animals with lesions on their skin compared with those without lesions. introduction (back to top) dogs and cats are the most commonly owned companion pets worldwide. for instance, in the usa 60% – 70% of households own at least one companion animal (moriello 2003). in ethiopia, many families own dogs and cats. dogs are kept for guarding, hunting or as pets, especially in larger towns, whilst cats are mainly keptfor hunting house rodents. despite the presence of a large population of dogs and cats in ethiopia there is paucity of information regarding the number, breeds, management systems and health status of these animals (yacob, ataklty & kumsa 2008). ectoparasites are one of the major constraints to the health and performance of domestic animals worldwide. ectoparasites are common on dogs and cats, presenting an important cause of pruritic as well as nonpruritic skin disorders. they transmit a variety of diseases and cause hypersensitivity. ticks, fleas and lice are the predominant ectoparasites encountered on domestic dogs and cats in several countries around the world. their feeding or movement patterns may haveseveral direct and indirect effects on their hosts (alcaino, gorman & alcaino 2002; rinaldi et al. 2007). common ixodid tick species that infest dogs and cats belong to the genera amblyomma, rhipicephalus and haemaphysalis. ticks cause direct damage owing to theirblood-feeding behaviour, act as vectors for bacterial, rickettsial, viral and protozoal diseases and may also cause tick paralysis owing to poisonous injections (marchiondo et al. 2007; wall & shearer 1997). many species of fleas have been recorded from different animal species. echidinophaga gallinacea (the sticktight poultry flea), pulex irritans (human flea), leptosylla segnis (rat flea), and ctenocephalides felis and ctenocephalides canis are the most commonly reported species of fleas fromdogs and cats. the infestation of an animal with fleas generally reflects poor management by the owners. the effects of flea bites include severe pruritus, self-inflicted trauma and flea allergy dermatitis (fad), a specific skin disease of dogs and cats (soulsby 1982). fleas harboured by pets can serve as intermediate hosts for the nematode, acanthocheilonema reconditum, and the dog tapeworm, dipylidium caninum, both of which can parasitise humans (soulsby 1982). xenosylla cheopis is the chief vector of the human pathogen yersinia pestis, causing human plague (marchiondo et al. 2007; wall & shearer 1997). in addition, fleas have been implicated in the transmission of the etiologic agent of cat-scratch zoonosis (comer, paddock & childs 2001). despite the large numbers of dogs and cats in ethiopia, very few studies have been conducted on the ectoparasites of these animals. as a result there is paucity of information on the distribution, species composition, public awareness and zoonotic importance of ectoparasites of dogs and cats in ethiopia (melkamu 2008; yonas 2008). thus, the current study was aimed at determining the species composition and prevalence of ticks, lice and fleas on domestic dogs and cats in hawassa in thesouthern parts of ethiopia. materials and methods study area (back to top) study area the study was conducted in hawassa, which is capital of the southern nations and nationalities peoples regional state (snnpr), located 275 km south of addis ababa. geographically the area lies between the latitudes 4°27´n and 8°30´n and between the longitudes 34°21´e and 39°1´e. the annual mean temperature of thearea is about 25 °c, whereas the annual mean rainfall varies from 801 mm to 1000 mm. study animals ectoparasites were sampled during a house-to-house survey of dogs and cats in hawassa and regular weekly visits to a veterinary clinic. the animals included in the study were randomly selected for examination. study methodology a cross-sectional study was performed from november 2008 to april 2009 based on the house-to-house survey and regular examination of animals presenting at the selassie veterinary clinic in hawassa. ectoparasite collection and identification both dogs and cats were thoroughly examined for the presence of ectoparasites during regualar examinations lasting 10 min – 15 min (soulsby 1982). the host’s skin was also brushed with a fine comb after being rubbed with a piece of cotton soaked in ether. the use of a plastic comb and white paper allowed for thorough examination for the presence of ectoparasites. live ticks were removed from the animals manually by using forceps, whilst fleas and lice were brushed off the dogs’ fur onto white sheets of paper using a brush and plastic comb. all collections were preserved in a solution of 70% ethanol and identified to species level undera stereomicroscope, according to the standard morphological identification keys as described by soulsby (1982), urquhart et al. (1996) and walker et al. (2003). clinical symptoms suggestive of fad were mainly itching and associated lesions (e.g. erythema, crusted papules, excoriations, alopecia, scales, desquamation and hyperkeratosis) were assessed during physical examination of the animals (durden et al. 2005; koutinas et al. 1995; rinaldi et al. 2007). data analysis all collected data were entered into spreadsheets and analysed with statistical software (spss, version 15). data were summarised as descriptive statistics (meanand percentages) and displayed as tables. the confidence level for the analyses was set at 95%, with significance level assessed at p < 0.05. results (back to top) as shown in table 1, 199 of the examined dogs (99.5%) were infested with one or more species of ectoparasites. c. felis was the most prevalent species (82.9%), whereas c. canis was the second most common species (73.8%). nymphs of amblyomma spp. were the most prevalent ticks on dogs, with a prevalence of 3.5%, followed by haemaphysalis leachi (0.5%). of the total of 100 cats examined, 91 (91%) were infested by one or more species of ectoparasites (table 2). as for the dogs, c. felis was the most common species found on cats (67%), followed by c. canis (18%). no ticks were found on cats during this study. the results showed that the overall prevalence of fleas, ticks and lice on dogs was not significantly different (p > 0.05) between male and female, or between young and adult dogs (tables 3 and 4). however, the overall prevalence of these ectoparasites was significantly higher (p < 0.05) amongst female compare with male cats, and also amongst adult compared with young cats (tables 3 and 4). a total of 1542 ectoparasites were collected from dogs during this study. species included c. felis (female = 803; male = 175), c. canis (female = 303; male = 84), p. irritans (female = 19; male = 4), heterodoxus spiniger (nymphs = 13; female = 78; male = 25), nymphs of amblyomma spp. (n = 19) and h. leachi (female = 4; male = 5). a total of 493 ectoparasites were collected from cats during the study period. species included c. felis (female = 300; male = 72), c. canis (female = 83; male = 20), and p. irritans (female = 15; male = 3). amongst fleas and lice, the number of female individuals was higher than that of male individuals. results showed that, for both cats and dogs, the prevalence of ectoparasites was significantly higher (p < 0.05) on animals presenting with fad than on those without fad (table 5). the prevalence of ectoparasites was significantly higher on animals with skin lesions than on those without lesions (table 6). table 1: overall prevalence of ectoparasites on examined dogs (n = 200) in hawassa. table 2: overall prevalence of ectoparasites on examined cats (n = 100) in hawassa. discussion (back to top) in this study a very high overall prevalence of ectoparasites was recorded amongst both dogs (99.5%) and cats (91%) in hawassa. this prevalence suggests that these ectoparasites are very common and present major problems with regard to the health, use and performances of these important animals in the study area. this observation supports findings from debre zeit (yonas 2008) and from nekempte in western oromia in ethiopia (melkamu 2008). it also agrees with reports fromelsewhere in the world (alcaino et al. 2002; aldemir 2007; beck et al. 2006; canón-franco & pérez-bedoya 2010; rinaldi et al. 2007). the higher prevalence of ectoparasites recorded on dogs (99.5%) than on cats (91%) in this study may reflect more efficient grooming behaviour of cats, as has beensuggested by eckstein and hart (2000). ctenocephalides felis was identified as the most common ectoparasite amongst both animal groups. this finding is in line with earlier reports from ethiopia (melkamu2008; yonas 2008) as well as elsewhere in the world (alcaino et al. 2002; aldemir 2007; beck et al. 2006; canón-franco & pérez-bedoya 2010; gonzalez, castro & gonzalez 2004; gracia et al. 2008; rinaldi et al. 2007). c. canis was identified as the second most common ectoparasite species amongst both host groups, which also agrees with the earlier mentioned reports. the current study was the first to examine the prevalence of ectoparasites amongst dogs and cats in hawassa. studies from various parts of the world indicated that c. felis, c. canis and p. irritans are the most common flea species that infest dogs and cats, which are regarded as the primary hosts for these three parasitic species. it was reported that c. felis was the most dominant species on dogs in chile (alcaino et al. 2002), georgia, usa(durden et al. 2005), southern italy (rinaldi et al. 2007), and several areas in germany (beck et al. 2006), spain (gracia et al. 2008) and colombia (canón-franco & pérez-bedoya 2010). however, aldemir (2007) and xhaxhiu et al. (2009) reported that c. canis was the predominant species of ectoparasites on dogs. ctenocephalides felis was the most prevalent species amongst domestic cats examined in this study, which agrees with the findings reported by beck et al. (2006) in several areas of germany and xhaxhiu et al. (2009) in albania. another species of flea collected from the study animals was p. irritans (2.5% and 6% from dogs and cats, respectively), which is in line with findings by koutinas et al. (1995), alcaino et al. (2002), durden et al. (2005), gracia et al. (2008) and canón-franco and pérez-bedoya (2010). yonas (2008) also reported c. felis as the most dominant ectoparasite species on dogs and cats in central oromia, ethiopia, followed by c. canis. nymphs of amblyomma spp. represented the most prevalent tick species on dogs (3.5%), followed by h. leachi (0.5%). no ticks were recovered from cats duringsample collection. the prevalence of h. spiniger was 4%, which is lower than what was previously reported by yonas (2008) in debre zeit. this may be attributed to several factors, forexample, variations in agroecology, time of study and animal management in various study areas. a significant difference (p < 0.05) was observed in the prevalence of ectoparasite infestation between young and adult cats (table 4), which may be due to young cats being confined to houses and therefore having greater exposure to ectoparasites infestation like fleas, as well as less efficient grooming behaviour than adult cats (eckstein & hart 2000). similar to most other studies of ectoparasite prevalence amongst animals, more female than male fleas and lice were found on animals in this study. the most probable reason for this is that female individuals usually have a longer lifespan than the male individuals. male individuals also spend more time off the host and are therefore more prone to predation or starvation than female individuals (durden et al. 2005). table 3: comparison of the overall prevalence of ectoparasites on male and female study animals. table 4: comparison of the overall prevalence of ectoparasites on young and adult study animals. table 5: overall prevalence of ectoparasites on dogs and cats with and without flea allergy dermatitis. table 6: association between overall prevalence of ectoparasites and skin lesions amongst animals. conclusion (back to top) in view of the findings it is possible to conclude that different species of fleas, ticks and lice represent common health and welfare problems of dogs and cats inhawassa. further studies regarding the role of these ectoparasites in transmission of diseases to dogs and cats, zoonotic importance, comparative prevalence and load, and the importance of dogs and cats as alternative hosts in different agroecology, urban and rural areas in ethiopia are recommended so as to design an applicable control programme in the country. references (back to top) alcaino, h.a., gorman, t.r. & alcaino, r., 2002, ‘flea species from dogs in three cities of chile’, veterinary parasitology105, 261−265. doi:10.1016/s0304-4017(01)00626-4 aldemir, o.s., 2007, ‘epidemiological study of ectoparasites in dogs from eruzurum region in turkey’, revue de medecine veterinaire 158(03), 148−151. beck, w., boch, k.k., mackensen, h., wiegand, b. & pfister, k., 2006, ‘qualitative and quantitative observations on the flea population dynamics of dogs and cats in several areas of germany’, veterinary parasitology 137, 130−136. doi:10.1016/j.vetpar.2005.12.021, pmid:16442233 canón-franco, w.a. & 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production in africa. a decline in reports of outbreaks of cbpp in affected countries does not augur well for the implementation of internationally coordinated control actions due to absence of science based evidence for disease prevalence which underpins control and preventive actions. according to provost, perreau, breard, le goff, martell & cottew (1987), the first description of cbpp was in 1550 by gallo. in the early 1800s, the disease was widespread over most of europe. in 1843, it was introduced into the united states via a dairy cow that was purchased off a ship from eng land. by 1884, cbpp had become so widespread and destructive in the united states that the federal government established the bureau of animal in dus try to combat the disease. in1887, the first intensive campaign to control an animal disease by quarantine and slaughter began in the united states. the mmmsc bacteria was first isolated and propagated in artificial media in france by nocard & roux in 1898. in 1854, cbpp was introduced into south 13 onderstepoort journal of veterinary research, 76:13–17 (2009) contagious bovine pleuropneumonia (lungsickness) in africa w. amanfu fao ectad unit, regional animal health centre/eastern africa au offices, museum hill-westlands road, nairobi, kenya abstract amanfu, w. 2009. contagious bovine pleuropneumonia (lungsickness) in africa. onderstepoort jour nal of veterinary research, 76:13–17 contagious bovine pleuropneumonia (cbpp) or lung sickness, is an insidious pneumonic disease of cattle caused by mycoplasma mycoides subspecies mycoides small colony variant (mmmsc) and it is one of the major diseases affecting cattle in africa. with the imminent eradication of rinderpest from africa (somali ecosystem) cbpp has become the disease of prime concern in terms of epizootics that affect cattle on the continent. the control and/or eradication of the disease have suffered from unsustained control actions due to lack of operational funds to support such actions and deterioration in the quality of veterinary services in many countries affected by the disease. stamping out procedures which were adopted by botswana to control the disease (1995–1997) cannot be carried out by many countries currently affected by cbpp due to the high financial cost, the widespread nature of disease, animal welfare considerations and the potential loss of a valuable genetic resource base. the current scenario of cbpp disease epidemiology in sub-saharan africa requires that proactive measures are taken to safeguard countries in southern africa which are currently free from cbpp from being contaminated by the disease thus affecting the beef industry and people’s livelihoods; and to progressively control the disease in endemic zones of western and central africa. this presentation discusses the epidemiology of cbpp in africa, diagnosis of the disease, regional strategies that could be deployed to prevent and control the spread of the disease on the continent and research thrusts on cbpp. 14 contagious bovine pleuropneumonia (lungsickness) in africa africa by a friesian bull imported from holland. the disease spread rapidly in africa and within 2 years, caused the death of over 100 000 cattle in south africa (thiaucourt, van der lugt & provost 2004). the disease was eradicated from south africa in 1924, zimbabwe in 1904, botswana in 1939 and australia in 1972. after virtual elimination of cbpp from europe in the 19th century, the disease reappeared in portugal and spain in 1951 and 1957, respectively. outbreaks have been reported in southern france on a few occasions, the latest being in 1984. in italy the disease reappeared in 1990 but was eliminated by 1993. epidemiological considerations of cbpp in africa cbpp represents a major constraint to cattle production in africa and is regarded as the most serious infectious animal disease affecting cattle now that rinderpest is almost eradicated from the continent. in the 1970s, the disease situation seemed to be under control. as such, the interest of veterinary authorities, regional and international animal health organisations shifted towards priorities other than cbpp. however, after almost 20 years of respite, cbpp made a dramatic come back on two major fronts–one in the east of the continent and the other in the south. almost at the same time, there was a resurgence of the disease in previously known endemic areas of the continent (west africa) probably as a result of cessation of rinderpest vaccination and with it, the waning of the state of immunity in cattle population to cbpp that used to be conferred through the use of the combined rinderpest/cbpp vaccine. the chronology of events on cbpp outbreaks in africa shows two distinct epidemiological trends: • epidemic outbreaks of disease in previously disease-free areas • increased incidence of disease in endemically infected areas. in the early 1960s and 1970s, sustained research in africa, notably muguga (kenya), farcha (chad), vom (nigeria) and elsewhere in africa on cbpp, coupled with a massive international campaign code named joint project 16, resulted in the disappearance of clinical disease from most parts of africa. however, as a result of economic decline, poorly resourced veterinary services and civil conflicts, the disease made a spectacular come back in the late 1980s and early 1990s. outbreaks of cbpp in previously disease-free countries or parts of countries over more than a decade, outbreaks of cbpp in previously uninfected areas have occurred in tanzania (1990, 1992 and 1994), democratic republic of congo (1991), rwanda (1994), botswana (1995), north-western zambia (1997 and still continuing) and burundi (1997). the underlying common factor to these outbreaks in previously free areas were uncontrolled or illegal movement of cattle from known infected cattle populations and represent a failure of surveillance systems, emergency preparedness and early reaction to these outbreaks. the botswana out break occurred after over half a century of freedom from the disease (amanfu, masupu, adom, raborokgwe & bashiruddin 1998a). this outbreak was eradicated by the slaughter of about 320 000 infected and in-contact cattle and restocking with about 70 000 cattle (amanfu, sediadie, masupu, benkirane, geiger & thiaucourt 1998b). international cooperation in the eradication of cbpp in botswana was fostered between south africa, namibia and others. the technical expertise of onderstepoort vet erinary institute, coupled with technical assistance from the food and agriculture organization of the united nations (fao) and the deep commitment of the government of botswana to eradicate the disease, ensured that the eradication of cbpp from botswana was a success. in zambia, cbpp appears to be confined to the western province but the influx of cbpp infected cattle from angola has contributed to the maintenance of this infected zone with spread to the rest of the country and further southwards to sesheke. elsewhere in east and central africa, the outbreaks were not effectively controlled with the result that cbpp is now endemic in rwanda, burundi, kenya, central african republic and tanzania from where the disease threatens malawi, mozambique and northern zambia. increased incidence in endemically infected countries most countries within the previously infected zones of west, central and east africa have over the years reported an increase in the incidence of the disease. the reasons behind this increase are decreased surveillance and control due to a variety of factors among which are: • changing ecological/environmental factors such as availability of water and grazing areas leading to increased movement of cattle over long dis15 w. amanfu tances in search of water and feed. stress factors may contribute to exacerbation of the disease. • cattle movement control which was easily enforceable during the colonial era appears unenforceable in recent times. the deterioration and decrease in mobility of field veterinary staff that are expected to enforce cattle movement regulations, has also been a contributory factor. in most countries the implementation of structural adjustment programmes (withdrawal of most gov ernment services, vehicles, sudden and poorly implemented shift of responsibilities from public to private, etc.), and changes in chain of command within veterinary services, led to rapid decline in the activities of veterinary departments and with that, disease control in general. • lack or insufficient resources allocated to cbpp control activities in particular reduced funding for vaccination, possibly associated with the ces sation of externally funded vaccination campaigns against rinderpest whereby vaccination against cbpp was carried out simultaneously. • cost recovery for cbpp vaccination and farmers’ unwillingness to pay because of association of vac cines with tissue reactions after administration of vaccines in some cases. • potency and field performance of cbpp vaccines (t1-44 and t1-sr). • reduced disease surveillance in the field, abattoir and the laboratory. • recently, over-concentration on the prevention and control of highly pathogenic avian influenza has led to decreased efforts in cbpp surveillance and reporting. cbpp diagnosis an overview of the current state of techniques available for the diagnosis of cbpp clearly demonstrates that recent advances in the study of immunology and molecular biology have and will continue to open up avenues for improved cbpp diagnosis. the tools currently available for cbpp diagnosis include clinical signs (fig. 1), pathologic lesions (pleurisy, lung hepatisation and interlobular thickening) (fig. 2), isolation and identification of the causative agent, immunoblotting, serology and pcr techniques. details of standard diagnostic tools for cbpp have been well documented by thiaucourt et al. (2004). recent advances in molecular biology have allowed accurate characterization of mmmsc strains at a molecular level and insertion sequence analysis has permitted the differentiation of cbpp agents of african, european or australian origin. a pan-african laboratory network for cbpp one of the major developments in the past was the establishment of the fao/iaea coordinated research programme through which national veterinary laboratories were provided with tools to diagnose cbpp, including further validation of cbpp c-elisa fig. 1 acute clinical form of cbpp. note the extended neck and abducted fore limbs indicative of respiratory dis tress source: m. niang; fao project (2008)-lcv, bamakomali fig. 2 acute gross lung lesion of cbpp. note the pronounced interlobular thickening with marbled appearance source: m. niang; fao project (2008)-lcv, bamakomali 16 contagious bovine pleuropneumonia (lungsickness) in africa and a pen-side test in the field. the advantages of having a cbpp network included easier management of information through electronic messaging systems and the provision of an ideal forum for research and development of regional strategies for the progressive control of cbpp. in addition, this facilitated standardization of reagents, protocols, data analysis and interpretation of results of cbpp tests. a network arrangement offered opportunities to facilitate dissemination of knowledge and technologies in a uniform manner. the network system needs to be revived and the international support to improvements in functional capacity of laboratories for avian influenza diagnostics could be adapted to support cbpp diagnostics as well. control options, containment and eradication of cbpp in africa the epidemiological nature of cbpp, where there is persistence of infection and transmission of the disease (often over long distances) through sub-acute and chronic cases, dictates that control and eradication programmes must be both comprehensive and consistently applied over about 15–20 years to be successful. on the other hand, a ‘piece-meal’ approach to cbpp control and eradication is almost certainly doomed to failure. it will predispose countries to cbpp endemicity, discourage both animal health officials and farmers and will make control/ eradication both difficult and costly. in many areas where cbpp currently occurs or which are at high risk of the disease, the potential natural epidemiological range of the disease extends over territory which may encompass more than any one country and may indeed include several countries. this may occur where there are traditional cattle trading, herding, nomadic or transhumance patterns that cover a large region. examples of this are to be found in the well-recognized ecological zones for cbpp in west/ central africa, east africa and northern parts of southern africa. the best method to control cbpp is by stamping out, but this is beyond the means of most african countries affected by the disease. significant progress towards cbpp control in these eco logical zones will only be possible if there is a high degree of cooperation between neighbouring countries in the development and implementation of regionally coordinated cbpp prevention, pre paredness and control/eradication programmes. risk of infection can be minimised by the prevention of contact between infected and healthy herds together with active disease surveillance. emergency preparedness, including early warning and early reaction based on contingency planning and establishment of the necessary infrastructure must therefore be the cornerstone of cbpp management in africa. community involvement based on education and training is essential to ensure early warning and early reaction. a regional programme must have the capacity for innovation and flexibility. cbpp control, like many transboundary animal diseases, must be regarded as a public good. control strategies for cbpp in different ecological and epidemiological zones have been discussed and published in joint fao/iaea/au-ibar/oie con sultative group meetings (fao 1998, fao 2001, fao 2004 and fao 2007). musisi, bamhare, belemu, chisembele, chitate, kabilika, kimera, kitalyi, munsimbwe & njau (2007) described the evolution of cbpp in sadc countries following the acknowledgement by the chief veterinary officers (cvos) of sadc countries at their workshop in pretoria 2003 of the threat cbpp poses to the cattle industry in the region and counter epizootic measures that have to be instituted following the recognition by the cvos to tackle cbpp control in the region with all seriousness. this was done in phases, namely emergency and recovery phases. control actions in sadc countries have been implemented by the fao supported by the government of south africa. success is limited, but in tanzania, zanzibar still remains free of cbpp. the spread of cbpp southwards has been arrested such that to date there has been no spread of cbpp disease to malawi, as indi cated through sustained surveillance in that country. research thrust the fao/oie/au-ibar/iaea joint consultative group meeting on cbpp has afforded these organizations and the international scientific community an excellent technical platform for exchange of ideas and synthesis of strategic initiatives aimed at the progressive control and prevention of cbpp. earlier meetings of the cbpp consultative group have identified the need for development and validation of new strategies, vaccines, diagnostic tests and the potential use of antibiotics for control of cbpp in africa (fao 2001, 2004 and 2007). in view of the epidemiological situation of cbpp in africa, most countries use vaccination, usually carried out by official veterinary services, as a form of control. the t1-44 vaccine, an attenuated live mmmsc vaccine strain, has been in use since 1956. safety and efficacy of the vaccine has been questioned at different fora by rweyemamu, litamoi, palya & sylla (1995), 17 w. amanfu thiaucourt, yaya, wesonga, huebschle, tulasne & provost (2000) and march (2004). the live attenuated vaccines currently in use are a compromise between virulence, immunogenicity and safety. it has been argued in certain quarters that improving the quality of existing cbpp vaccines is more likely to deliver significant beneficial effects than developing a new generation of vaccines, which will be an expensive and time consuming process. with the support of the wellcome trust, the moredun institute, university of edinburgh, uk has proposed or is currently working on the following research themes on vaccines and diagnostics in collaboration with various partners (schnier & mckeever 2007): • comparison of safety and efficacy of buffered cbpp vaccine with conventional t1-44 vaccine • estimation of the social and economic impact of vaccination • evaluation of different vaccine distribution systems • evaluation of different diagnostic tests, e.g. latex agglutination test (lat antigen) lppq-elisa, taqm-pcr • development of a new generation vaccine • evaluation of immunological responses to vaccination. research on molecular epidemiology and improvements in the diagnostics of cbpp amongst other initiatives by the cirad group in montpellier, france in collaboration with partners, is ongoing. the future the control of cbpp by affected countries appears uncertain, partly owing to missing gaps in the pathogenesis of the disease, lack of plausible epidemiological data from which sustained control actions could be based, weak veterinary services and poor economic standing of most countries affected by the disease in africa. the feasibility of the use of currently available tools for diagnosis and control coupled with reinforcement of awareness creation, disease search using community based animal health workers must be strengthened to bring hope to cattle farmers in africa. the control of cbpp in angola is pivotal to the mitigation of risk of cbpp spread to sadc countries. a template for prevention and control was presented by windsor (2000). references amanfu, w., masupu, k.v., adom, k., raborokgwe, m.v. & bashiruddin, j.b. 1998a. an outbreak of contagious bovine pleuropneumonia in ngamiland district of northwestern botswana. the veterinary record, 143:46–48. amanfu, w., sediadie, s., masupu, k.v., benkirane, a., geiger, r. & thiaucourt, f. 1998b. field evaluation of a competitive elisa for the detection of contagious bovine pleuropneumonia in botswana. revue d’elevage et de medicine veterinaire des pays tropicaux, 51:189–193. fao 2001. summary of presentations and discussions. report of the second meeting of the fao-oie-oau-iaea con sultative group on contagious bovine pleuropneumonia (cbpp), rome, italy, 24–26 october 2000. rome: fao: 3–9. fao 2004. summary of recommendations. report of the third meeting of the fao-oie-au/ibar-iaea consultative group on contagious bovine pleuropneumonia (cbpp), rome, italy, 12–14 november 2003. rome: fao: 5–6. fao 2007. summary of recommendations. report of the fourth meeting of the fao-oie-au/ibar-iaea consultative group on contagious bovine pleuropneumonia (cbpp), rome, italy, 6–8 november 2006. rome: fao: 13–15. march, j.b. 2004. improved formulations for existing cbpp vaccines—recommendations for change. vaccine, 22:4358– 4364. musisi, f.l., bamhare, c., belemu, j., chisembele, c., chi tate, f., kabilika, s., kimera, s., kitalyi, j., munsimbwe, l. & njau, p. 2007. the evolution of cbpp in southern africa: 2004–2006. proceedings; fao-oie-au/ ibar-iaea consultative group meeting on contagious bovine pleuropneumonia (cbpp), rome, italy, 6–8 november 2006. rome: fao: 89-101. provost, a., perreau, p., breard, a., le goff, c., martel, j.l. & cottew, g.s. 1987. contagious bovine pleuropneumonia. revue scientifique et technique (oie), 6:625– 679. rweyemamu, m.m., litamoi, j., palya, v. & sylla, d. 1995. contagious bovine pleuropneumonia vaccines: the need for improvements. revue scientifique et technique (oie), 14:593–601. schnier, c. & mckeever, d. 2007. development and validation of new technologies for control of cbpp in east africa. proceedings: fao-oie-au/ibar-iaea consultative group meeting on contagious bovine pleuropneumonia (cbpp). rome, italy, 6–8 november 2006. rome: fao: 73–78. thiaucourt, f., yaya, a., wesonga, h., huebschle, o.j.b., tulasne, j.j. & provost, a. 2000. contagious bovine pleuropneumonia-a reassessment of the efficacy of vaccines used in africa. annals of the new york academy of science, 916:71–80. thiaucourt, f., van der lugt, j.j. & provost, a. 2004. contagious bovine pleuropneumonia, in infectious diseases of livestock. vol. 3, 2nd ed., edited by j.a.w. coetzer & r.c. tustin. cape town: oxford university press. windsor, r.s. 2000: the eradication of contagious bovine pleuropneumonia from south western africa: a plan for action. annals of the new york academy of science, 916:326– 332. << /ascii85encodepages false /allowtransparency 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: anastasia n. trataris1,2 lorraine arntzen1 jennifer rossouw1 john frean1,2 allan karstaedt3 affiliations: 1national institute for communicable diseases, national health laboratory service, south africa 2university of the witwatersrand, south africa 3chris hani baragwanth hospital, bertsham, south africa correspondence to: anastasia trataris postal address: centre for emergingand zoonotic diseases special bacterial pathogens reference laboratory how to cite this poster: trataris, a.n., arntzen, l., rossouw, j., frean, j. & karstaedt, a., 2012, ‘bartonella henselae and bartonella quintana seroprevalence in hiv-positive, hiv-negative and clinically healthy volunteers in gauteng, south africa’, onderstepoort journal of veterinary research79(2), art. #479, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.479 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. bartonella henselae and bartonella quintana seroprevalence in hiv-positive, hiv-negative and clinically healthy volunteers in gauteng, south africa in this poster... open access bartonella is a genus of opportunistic, gram-negative bacilli transmitted from animals to human hosts. bartonellae are newly emerging pathogens that can cause a variety of clinical manifestations in both immunocompromised and healthy persons.the aims were to determine the igg and igm seroprevalences of bartonella henselae and bartonella quintana in immunocompromised and immunocompetent individuals using an immunofluorescence assay (ifa). a total of 382 hiv-positive outpatients of the chris hani baragwanth hiv-clinic, 382 retrospective residual samples from hiv-negative antenatal patients, and 42 clinically healthy volunteers were tested using a commercially available ifa kit to determine the prevalence of igg and igm antibodies to b. henselae and b. quintana. the igm and igg seroprevalences for the hiv-positive patients were 14% (53/382) and 32% (121/382), respectively, compared to 18% for both igm (62/342) and igg (63/342) in the hiv-negative antenatal patients. similarly, the prevalence for igm was 17% (7/42) and igg was 19% (8/42) for the clinically healthy volunteers. hiv-positivity appears to be a significant risk factor for bartonella infection, compared with healthy subjects. although ifas have a high sensitivity for bartonella antibody detection, they have various limitations including cross-reactivity with other closely-related human pathogens. course doi:10.4102/ojvr.v79i2.476http://www.ojvr.org page 1 of 1 how to cite this course: wambura, p., ‘msc: in one health molecular biology’, onderstepoort journal of veterinary research 79(2), art. #476, 1 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.476 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright: © 2012. the authors. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. source: author’s original material as presented at the conference course structure year 1  research methods  statistics and data management  advanced molecular biology  immunology of infectious diseases  one health medicine  comparative genomics and bioinformatics  techniques in molecular and cellular biology  pathogen evolution and emerging infectious diseases during the second semester students will receive mentoring to develop a research proposal, which must be presented to an academic panel for approval. the research proposals will be related, as far as possible, to one health themes identified in the main sacids framework, and will address:  pathogen biology  host pathogen interactions  detection, identification and surveillance of disease  biodiversity or environmental determinants of one health year 2 during the second year, the learning process will be pro-active and will include a literature review, intensive laboratory work, statistical analysis and writing a dissertation partnerships the curriculum has been developed in partnership with the uk’s london school of hygiene and tropical medicine, the royal veterinary college and others, which offer world-class postgraduate programmes in health and related disciplines. course modules are taught by academicians from sua and muhimbili university for health and allied sciences (muhas) supported by internationally recognised staff from sacids partner institutions and others. experienced scientists from sacids institutions will combine strong capacity in molecular biology and one health to provide a unique blend of professional knowledge and skills for the graduates of this innovative msc programme. while the sacids financial support targets students from the sadc region, students from other parts of africa or elsewhere who are able to meet their own cost are also eligible. acknowledgement the one health molecular biology msc course is supported by sacids through a grant from the wellcome trust under its african institutions initiative ( www.wellcome.ac.uk ) msc in one health molecular biology the msc in one health molecular biology course is offered at the faculty of veterinary medicine of sokoine university of agriculture. the course focuses on practical skills and a thorough understanding of how to apply molecular biology to one health – an emerging discipline which promotes collaboration between the veterinary and medical professions. upon completion, researchers will be highly-skilled professionals able to:  demonstrate knowledge and understanding of molecular biological techniques and their applications in the detection, identification and monitoring of infectious diseases of humans and animals  demonstrate specialist knowledge for understanding how prophylactic or curative treatments or therapies for diseases may be developed through molecular research of host and pathogen genomes  devise molecular biological approaches to find newer ways that will improve the health of humans, animals and our environment  join international phd programmes or secure employment in academic and research institutions course director: professor philemon wambura email: info@sacids.org faculty of veterinary medicine sokoine university of agriculture p. o. box 3019 morogoro, tanzania sokoine university of agriculture msc: in one health molecular biology p. wambura http://dx.doi.org/10.4102/ojvr.v79i2.462 http://dx.doi.org/10.4102/ojvr.v79i2.462 burridge_41-51.qxd introduction wildlife are important hosts for ticks in many regions of the world (hoogstraal & aeschlimann 1982). in an attempt to develop a practical and stress-free method for control of ticks on wild animals, a selfmedicating applicator was designed and tested successfully on deer (sonenshine, allan, norval & burridge 1996). it did not, however, lend itself to use with large numbers of animals. consequently, an improved self-medicating applicator was developed, which could be retrofitted to existing devices that attract wild animals, such as troughs containing feed or water (burridge, simmons & simmons 2000, 2003; simmons, burridge & simmons 2001). this improved applicator was designed to transfer oilbased acaricides passively to wild animals, but its versatile design was such that it had the potential to be used to treat animals of any size with any oilbased compound. consequently preliminary trials with this applicator were formulated to test whether the concept of delivery of oil-based treatments to both domestic and wild animals through the device was practical using various animal species naturally infected or infested with endoor ecto-parasites. initial studies were conducted to determine the efficacy of the applicator as a method for delivery of treatments to control gastrointestinal nematodes in cattle and deer in the united states, flies on cattle in the united states, and ticks on cattle in south africa. the results of these trials are described in this report. 41 onderstepoort journal of veterinary research, 71:41–51 (2004) development of a novel self-medicating applicator for control of internal and external parasites of wild and domestic animals m.j. burridge1, l.a. simmons1, e.h. ahrens2, s.a.j. naudé3 and f.s. malan3 abstract burridge, m.j., simmons, l.a., ahrens, e.h., naudé, s.a.j. & malan, f.s. 2004. development of a novel self-medicating applicator for control of internal and external parasites of wild and domestic animals. onderstepoort journal of veterinary research, 71:41–51 four trials, three in the united states and one in south africa, were conducted to evaluate the potential value of a novel self-medicating applicator in the passive control of gastrointestinal nematodes in cattle and deer, and of flies and ticks on cattle using oil-based treatments. the results of the trials demonstrated that this applicator is an effective and practical device for the passive treatment of both deer and cattle for trichostrongyle infections using the endectocide, moxidectin (cydectin®, fort dodge animal health, usa), of cattle for horn fly (haemotobia irritans) infestations using the insecticide, cyfluthrin (cylence®, bayer ag, germany) and of cattle for tick infestations (in particular amblyomma hebraeum and rhipicephalus appendiculatus) using the acaricides deltamethrin and amitraz (delete all®, intervet, south africa). keywords: amitraz, cattle, cyfluthrin, deer, deltamethrin, fly control, moxidectin, nematode control, self-medicating applicator, tick control 1 department of pathobiology, college of veterinary medicine, university of florida, p.o. box 110880, gainesville, florida 32611-0880, usa 2 3551 zenner-ahrens road, kerrville, tx 78028, usa 3 intervet research unit, p.o. box 124, malelane, 1320 south africa accepted for publication 23 september 2003—editor materials and methods self-medicating applicator the applicator, given the trademark of the appligator™ (university of florida), is a semicircular device composed of a solid outer pipe made of rigid polyvinyl chloride containing a rigid porous internal pipe made of high-density polyethylene, with an upper portion of the outer pipe removed to allow animals to contact the internal porous material (fig. 1). it was attached to a feed trough using plastic ties or nuts and bolts, and primed with a predetermined volume of oil-based compound sufficient to saturate the porous pipe, after which an additional measured treatment dose is added to the porous pipe. both the priming and treatment doses were added by syringe through treatment fill ports in the exposed porous pipe. when cattle or deer feed from the trough the applicator deposits the compound on the neck region, thus treating them in a stress-free manner without the need for handling equipment. treatments anthelmintic the anthelmintic used was cydectin® (fort dodge animal health, fort dodge, iowa, usa) containing 0.5 % of the second-generation endectocide moxidectin. moxidectin was selected because it is highly efficacious against gastrointestinal parasites of cattle and deer (craig 1999), it is of low toxicity in terms of tissue residues and ecological safety (herd 1995), it has been used successfully for internal parasite control in farmed deer in new zealand (audigé, wilson & morris 1998; waldrup, mackintosh, duffy, labes, johnstone, taylor & murphy 1998), it has a persistent effect against target nematodes (eysker & eilers 1995; hubert, kerboeuf, cardinaud & blond 1995; rendell & callinan 1996), and it is formulated as an oil-based pour-on that is absorbed through the skin. insecticide the insecticide used is cylence® (bayer ag, leverkusen, germany) containing 1% cyfluthrin. cyfluthrin was selected because it was commercially marketed for use in fly control on cattle in the usa, it is available as an oil-based pour-on formulation, and it is an effective insecticide (sulaiman, pawanchee, othman, jamal, wahab, sohadi, rahman & pandak 1998; vale, mutika & lovemore 1999). acaricide the acaricide used was delete all® (intervet, isando, south africa) containing 0.5 % deltamethrin, 2 % amitraz and 0.5 % piperonyl butoxide. this acaricidal combination was selected because it is formulated as an oil-based pour-on and because deltamethrin and amitraz are effective for the control of ticks on cattle in africa (haigh & gichang 1980; luguru 1991; kagaruki 1996; mekonnen 2001). experimental designs anthelmintic trial nineteen male fallow deer (dama dama), resident on a private wildlife ranch in putnam county, florida, usa, were selected for the cervid anthelmintic trial. they were 2–5 years of age and were naturally infected with trichostrongyles. the deer were randomly assigned to one of two fenced pastures on the ranch. ten of them were kept in a pasture containing an applicator attached to a feed trough (the applicator group) and the remaining nine on the other pasture with no attachments to the feed trough (the control group). eight brahman, angus and hereford cattle from a privately owned farm in hendry county, florida, were selected for the bovine anthelmintic trial. they were 1–4 years of age and consisted of four heifers, two steers and two bulls, all naturally infected with trichostrongyles. the cattle were assigned randomly by sex to one of two fenced pastures so that each group contained two heifers, one steer and one bull. they were divided into an applicator group and a control group as for the deer. the 19 deer and eight cattle were individually restrained in squeeze chutes, and faecal material was removed manually from the rectum of each animal for examination for trichostrongyle eggs. the animals were then returned to their respective pastures on either the wildlife ranch or the cattle farm. the applicators were primed with enough moxidectin to saturate the porous columns, then additional moxidectin (80 ml for the ten deer and 84 ml for the four cattle) was added to the devices to form reservoirs for treatment, based on a dosage of 0.5 mg moxidectin per kg body mass. commercial deer or cattle feed was placed in the feed trough in each of the pastures, and the animals were allowed to feed. while feeding, the ten deer and the four cattle in the applicator groups received a total of 80 ml and 84 ml of moxidectin respectively, until all 42 self-medicating applicator for control of parasites of wild and domestic animals available moxidectin in the reservoirs had been transferred to the necks of the animals. the deer and cattle were restrained individually again on days 6 and 12 and on days 7, 14, and 21 respectively, after initiation of the trials to obtain a series of post-treatment faecal samples. each faecal sample was placed in a plastic bag immediately after collection, and the bag was sealed and stored on ice for transport to the laboratory. the sample was quantitatively examined for trichostrongyle eggs using a modification of the mcmaster egg-counting method (whitlock 1948). a 4 g amount of faeces was mixed with 26 ml of fecal float (phoenix pharmaceuticals inc., st. joseph, missouri, usa), poured through a layer of cheese cloth, and distributed to a two-chambered mcmaster slide. after a 5-min interval the mcmaster slide egg-counting grid was examined microscopically at 100x magnification for the presence of trichostrongyle eggs. insecticidal trial sixteen 2 to 5-year-old brahman-cross cows from a privately owned ranch in starr county, texas, usa, were selected for the insecticidal trial. they were randomly assigned in equal numbers to one of two fenced pastures at the ranch, and were divided into an applicator group and a control group as in the anthelmintic trial. the trial commenced in march when horn flies (haematobia irritans) were naturally abundant on the cattle on the ranch. counts of horn flies were made visually on each of the 16 cattle in the trial on the day prior to onset of the trial. the applicator was primed with enough cyfluthrin to saturate the porous column, then 96 ml 43 m.j. burridge et al. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � � � � � � � � � � � � � � � �� � � � � � � � � � � � � � � � � � � � ��������� ������ � ��� ����� ��� ���� � ����� ����� �� ��� ��� � �� ������� � ��� ��������� � �������� ��� �� � ��� � � � � fig. 1 schematic drawing of cross-section of an appligator™ self-medicating applicator mounted on the edge of a feed trough of additional cyfluthrin were added to the device to form a reservoir for treatment of the cattle, based on a dosage of 12 ml for animals exceeding 363 kg body mass. commercial cattle feed was placed in the trough in each pasture, and the animals were allowed to feed. counts of horn flies were made on day 1 and then weekly from days 7–42 to obtain a series of post-treatment fly counts. acaricidal trial fifteen beefmaster-cross cattle on a farm belonging to the intervet research unit in malelane, mpumalanga province, south africa were selected for the acaricidal trial. the cattle were 1-year-old animals of mixed sexes and were randomly assigned in equal numbers to one of three fenced tick-infested pastures at the farm. five cattle were kept on a pasture containing an applicator attached to a feed trough (the applicator group) and five each in two other pastures with no attachments to the feed troughs (the positive control and negative control groups). the trial commenced in january when ticks (including amblyomma hebraeum, rhipicephalus appendiculatus, rhipicephalus simus and boophilus spp.) were naturally abundant on the pastures of the farm. counts of adult ticks were made by species on each of the 15 cattle on the day prior to onset of the trial. the ticks were counted macroscopically in situ while the animals were restrained on a cement floor in a crush pen with both a head and a body clamp. two people performed the tick count on each animal, and a third person recorded the data. disposable latex gloves were worn during tick counting, with new sets of gloves worn for each experimental group. the applicator was primed with enough deltamethrin/amitraz to saturate the porous column, then 95.5 ml of additional deltamethrin/amitraz was added to the device to provide a reservoir for treatment of the cattle, based on a dosage of 0.1 ml per kg body mass. cattle on the pasture containing the positive control group were treated by application of deltamethrin/amitraz pour-on along the back line using a dosage of 0.1 ml of pour-on per kg body mass. cattle in the pasture containing the negative control group were treated only for ethical reasons when their tick burdens became too heavy, and in those instances the acaricide used was triatix cattle spray® (intervet, isando, south africa) containing 12.5 % amitraz at a rate of 5 l per animal. commercial cattle feed was added to the trough in each pasture, and the animals were allowed to feed. cattle in the applicator and positive control groups were treated with deltamethrin/amitraz weekly from days 0 to 77 using an applicator and pour-on, respectively. after tick counts had been made, cattle in the negative control group were treated with amitraz for ethical reasons on days 0, 14, 28, 35, 49, 63 and 84 due to the heavy tick challenge. counts of adult ticks were made on day 7 after treatment and then weekly to day 84 to obtain a series of post-treatment tick counts by species. statistical analyses the data were analyzed for statistical differences in trichostrongyle egg counts, fly counts and tick counts between the test groups using the two-tailed t test. for egg and fly counts, differences between treated and untreated animals were analyzed for each day on which counts were made. for tick counts, differences between treated animals (the applicator and positive control groups) and untreated animals (the negative control group) were analyzed only for those days when the negative control cattle were treated for ethical reasons, with the tick counts on negative control cattle preceding treatment. these analyses were selected to minimize 44 self-medicating applicator for control of parasites of wild and domestic animals table 1 effect of moxidectin on trichostrongyle infections in fallow deer when applied passively using a self-medicating applicator animal no. no. of trichostrongyle eggs per by group gram of faeces by day of trial day 0 day 6 day 12 applicator group 362 75 0 a 367 25 0 0 374 300 0 0 400 100 a 0 441 75 0 0 444 25 0 0 489 75 0 0 523 100 0 0 544 125 a 0 561 75 0 0 control group 307 50 50 50 364 50 75 75 372 125 125 125 410 75 50 50 467 125 125 150 490 25 25 a 533 100 125 100 568 150 100 175 586 75 100 75 a = no sample collected due to empty rectum the effect of treatment of the negative control cattle. ideally from the scientific point-of-view, the negative controls should not have been treated, increasing the number of ticks on each animal, but ethically these cattle had to be treated periodically to minimize the impact of the heavy burdens on their health and well-being. results anthelmintic trials the ten deer in the applicator group had a mean burden of 97.5 trichostrongyle eggs per gram (epg) of faeces before treatment with moxidectin from an applicator. the egg counts in all deer in the group dropped to zero by day 6 post-treatment and remained at zero by day 12 post-treatment (table 1). the trichostrongyle egg counts were significantly less (p < 0.001) in the deer treated using the applicator than were those in the untreated deer on both days 6 and 12 of the trial. the four cattle in the applicator group had a mean trichostrongyle egg count of 300 epg of faeces before treatment with moxidectin. after treatment, the egg counts dropped to zero in three of the four cattle, the exception being steer no. 109 in which the count dropped to 50 epg (table 2). this steer did not feed from the trough during the first day of the trial and thus had no contact with the applicator 45 m.j. burridge et al. table 2 effect of moxidectin on trichostrongyle infections in cattle when applied passively using a self-medicating applicator no. of trichostrongyle eggs per gram of faeces by day of trial animal no. by group day 0 day 7 day 14 day 21 applicator group 102 250 0 0 0 105 475 0 0 0 109a 200 50 50 50 111 275 0 0 0 control group 101 225 175 175 200 107 175 200 225 300 110 400 475 500 550 113 200 175 175 175 a steer no. 109 did not make contact with the applicator during the first day of trial table 3 effect of cyfluthrin on horn fly infestations on cattle when applied passively using a self-medicating applicator no. of horn flies on animal by day of trial animal no. by group day -1 day 1 day 7 day 14 day 21 day 28 day 35 day 42 applicator group 34 150 0 0 4 18 70 220 470 39 300 0 0 4 20 50 200 500 41 200 0 0 2 26 110 240 500 42 200 0 0 10 24 100 240 480 43 250 0 0 4 14 68 180 500 44 400 0 0 12 18 76 300 450 45 300 0 0 6 16 60 200 600 47 550 0 0 14 22 80 250 450 control group 38 180 200 470 400 400 400 450 600 48 250 250 500 380 420 380 470 460 49 300 400 550 400 370 300 440 500 50 500 450 600 470 410 500 450 450 55 280 300 400 360 350 360 400 500 61 200 250 450 350 380 400 380 450 73 550 600 560 500 450 500 500 480 93 300 400 600 480 400 450 500 550 46 self-medicating applicator for control of parasites of wild and domestic animals t a b l e 4 e ff e ct o f d e lta m e th ri n /a m itr a z o n t ic k in fe st a tio n s o n c a tt le w h e n a p p lie d p a ss iv e ly u si n g a s e lfm e d ic a tin g a p p lic a to r n o . o f a d u lt tic ks a o n a n im a l b y d a y o f tr ia l a n im a l n u m b e r b y g ro u p d a y -1 d a y 7 d a y 1 4 d a y 2 1 d a y 2 8 d a y 3 5 d a y 4 2 d a y 4 9 d a y 5 6 d a y 6 3 d a y 7 0 d a y 7 7 d a y 8 4 a p p lic a to r g ro u p b 0 1 /0 1 4 8 5 7 8 1 7 1 7 1 1 2 7 1 4 9 1 8 5 1 6 2 0 7 0 1 /0 2 5 4 2 4 5 1 1 2 2 2 3 3 4 1 3 2 0 1 7 1 5 4 3 1 8 0 1 /1 1 4 4 8 4 0 1 3 9 1 2 1 9 1 2 1 1 1 3 4 1 6 3 0 1 /2 2 4 2 3 2 9 4 4 0 7 1 2 6 6 4 5 6 3 6 3 9 1 9 1 5 2 3 1 9 0 2 /0 4 3 8 5 6 2 4 6 3 9 1 8 7 1 4 2 2 6 7 3 2 0 5 2 2 2 1 1 p o si tiv e c o n tr o l g ro u p b 0 1 /1 3 3 8 2 1 9 2 1 0 2 7 7 1 8 2 2 7 6 5 7 7 2 7 1 9 5 6 0 1 /1 4 4 5 1 3 2 0 2 0 3 1 3 5 2 2 4 4 8 9 9 7 7 1 1 6 3 6 2 5 1 1 0 1 /2 4 4 9 2 4 3 6 1 4 0 1 0 0 1 6 0 3 2 5 3 1 9 9 3 2 2 2 1 2 1 4 0 1 /3 2 4 5 7 8 7 8 7 8 6 1 6 9 2 4 5 8 2 2 8 1 7 1 0 9 5 0 2 /0 8 2 9 9 6 9 7 7 3 1 1 0 6 8 3 6 1 2 3 1 7 1 4 4 6 n e g a tiv e c o n tr o l g ro u p c 0 1 /0 3 3 5 5 2 9 4 5 4 6 1 1 6 6 5 2 1 5 1 1 5 4 3 2 2 4 7 2 0 2 1 8 9 4 1 1 7 0 1 /0 6 3 5 6 3 1 2 6 5 1 1 5 8 8 3 6 1 7 4 1 5 2 3 1 7 8 3 3 1 6 3 0 1 0 0 1 3 3 0 1 /1 2 3 8 3 3 7 3 5 4 6 1 8 5 6 3 6 2 1 6 1 8 3 4 1 0 1 2 4 4 1 8 3 4 1 0 9 1 2 8 0 1 /2 6 3 9 4 3 7 3 6 7 1 1 6 3 7 2 6 1 7 2 1 3 1 5 0 1 4 0 4 3 1 2 8 1 0 7 1 2 7 0 2 /0 7 5 0 0 2 4 1 6 3 5 4 7 9 1 2 1 5 4 1 7 3 7 1 3 4 1 4 8 3 2 6 6 6 1 4 6 a a m bl yo m m a he br ae um , r hi pi ce ph al us a pp en di cu la tu s, r hi pi ce ph al us s im us a n d b oo ph ilu s sp p . tic ks b e a ch a n im a l t re a te d w ith d e lta m e th ri n /a m itr a z a t w e e kl y in te rv a ls f ro m d a y 0 t h ro u g h d a y 7 7 c e a ch a n im a l t re a te d w ith a m itr a z o n d a ys 0 , 1 4 , 2 8 , 3 5 , 4 9 , 6 3 a n d 8 4 f o r e th ic a l r e a so n s d u e t o h e a vy t ic k ch a lle n g e 47 m.j. burridge et al. t a b l e 5 e ff e ct o f d e lta m e th ri n /a m itr a z o n a m bl yo m m a he br ae um in fe st a tio n s o n c a tt le w h e n a p p lie d p a ss iv e ly u si n g a s e lfm e d ic a tin g a p p lic a to r m e a n n o . o f a d u lt tic ks o n a n im a l b y d a y o f tr ia l t ri a l g ro u p a d a y -1 d a y 7 d a y 1 4 d a y 2 1 d a y 2 8 d a y 3 5 d a y 4 2 d a y 4 9 d a y 5 6 d a y 6 3 d a y 7 0 d a y 7 7 d a y 8 4 a p p lic a to r 7 3 .4 2 4 1 6 .2 9 5 .4 6 .8 7 .6 3 .2 3 .8 2 4 .2 1 .6 2 .2 p o si tiv e c o n tr o l 7 4 .6 1 2 .6 6 .2 4 3 .6 1 .4 1 .4 0 .8 0 .2 6 .8 6 .6 4 .6 3 .4 n e g a tiv e c o n tr o l 8 6 .8 1 4 .4 5 3 .2 4 .8 5 9 .4 8 .4 1 .6 3 7 .8 2 .2 4 3 .2 2 8 1 4 .6 a a p p lic a to r g ro u p a n d p o si tiv e c o n tr o l g ro u p t re a te d w ith d e lta m e th ri n /a m itr a z a t w e e kl y in te rv a ls f ro m d a y 0 t h ro u g h d a y 7 7 ; n e g a tiv e c o n tr o l g ro u p t re a te d w ith a m itr a z o n d a ys 0 , 1 4 , 2 8 , 3 5 , 4 9 , 6 3 a n d 8 4 f o r e th ic a l r e a so n s d u e t o h e a vy t ic k ch a lle n g e t a b l e 6 e ff e ct o f d e lta m e th ri n /a m itr a z o n r hi pi ce ph al us a pp en di cu la tu s in fe st a tio n s o n c a tt le w h e n a p p lie d p a ss iv e ly u si n g a s e lfm e d ic a tin g a p p lic a to r t ri a l g ro u p a m e a n n o . o f a d u lt tic ks o n a n im a l b y d a y o f tr ia l d a y -1 d a y 7 d a y 1 4 d a y 2 1 d a y 2 8 d a y 3 5 d a y 4 2 d a y 4 9 d a y 5 6 d a y 6 3 d a y 7 0 d a y 7 7 d a y 8 4 a p p lic a to r 3 6 3 .8 4 .8 4 .4 1 3 4 .2 2 7 .6 1 4 .4 7 .2 9 .8 0 .4 0 .2 0 .2 0 .6 p o si tiv e c o n tr o l 3 2 0 .6 1 1 1 .8 8 4 .2 6 5 .2 1 4 0 .6 2 2 .8 5 4 .6 7 .2 3 .8 3 6 1 1 .2 4 .4 3 .8 n e g a tiv e c o n tr o l 2 8 5 .6 2 4 8 .4 5 2 2 1 0 9 5 3 2 .4 1 4 8 .6 1 4 6 .6 3 7 5 .2 6 0 3 0 1 .2 2 1 .2 7 9 .4 1 0 6 .8 a a p p lic a to r g ro u p a n d p o si tiv e c o n tr o l g ro u p t re a te d w ith d e lta m e th ri n /a m itr a z a t w e e kl y in te rv a ls f ro m d a y 0 t h ro u g h d a y 7 7 ; n e g a tiv e c o n tr o l g ro u p t re a te d w ith a m itr a z o n d a ys 0 , 1 4 , 2 8 , 3 5 , 4 9 , 6 3 a n d 8 4 f o r e th ic a l r e a so n s d u e t o h e a vy t ic k ch a lle n g e until day 1 post-treatment. the trichostrongyle egg counts were significantly less (p < 0.02) in the cattle treated using the applicator than were those in the untreated cattle on days 7, 14 and 21 of the trial. insecticidal trial the eight cattle in the applicator group had a mean infestation of 293.8 horn flies before treatment with cyfluthrin from an applicator. after treatment, the fly counts on these cattle dropped to zero by day 1 post-treatment, remained at zero through day 7, and gradually increased from a mean of 7.0–493.8 during days 14–42 post-treatment (table 3). the fly counts were significantly less (p < 0.001) on the cattle treated using the applicator than on the untreated cattle on days 1 through 35 of the trial. acaricidal trial control of all tick species the five cattle in the applicator group had a mean infestation of 456.6 adult ticks before treatment with deltamethrin/amitraz from an applicator, after which their mean adult tick count fell with weekly treatments to 11.6 adult ticks by day 84 of the trial (table 4). similarly, the five cattle in the positive control group had a mean infestation of 416.2 adult ticks before treatment with deltamethrin/amitraz pour-on, after which their mean adult tick count fell with weekly treatments to 8.4 adult ticks by day 84 of the trial (table 4). in contrast, the tick challenge in the negative control group was so heavy that the cattle in this group had to be treated on days 14, 28, 35, 49, 63 and 84 of the trial for ethical reasons. the adult tick counts were significantly less (p < 0.001) on both the cattle treated using the applicator and those treated by pour-on than were those for the cattle in the negative control group on days 14, 28, 35, 49, 63 and 84 of the trial. furthermore, adult tick counts were significantly less (p < 0.05) on the cattle treated using the applicator than on those treated by pour-on on days 14, 21 and 42 of the trial. control of amblyomma hebraeum the five cattle in the applicator group had a mean infestation of 73.4 adult a. hebraeum (range 35– 114) before treatment with deltamethrin/amitraz from an applicator, after which the mean adult a. hebraeum count fell with weekly treatments to 2.2 adult ticks (range 0–5) by day 84 of the trial (table 5). similarly, the five cattle in the positive control group had a mean infestation of 74.6 adult a. hebraeum (range 47–100) before treatment with deltamethrin/amitraz pour-on after which the mean adult a. hebraeum count fell with weekly treatments to 3.4 adult ticks (range 2–5) by day 84 of the trial (table 5). in contrast, the mean adult a. hebraeum count in the negative control group remained high, necessitating treatment with amitraz on days 14, 28, 35, 49, 63 and 84 of the trial for ethical reasons. the adult a. hebraeum counts were significantly less (p < 0.01) on both the cattle treated using the applicator and those treated by pour-on than were those for the cattle in the negative control group on days 14, 28, 49, 63 and 84 of the trial. control of rhipicephalus appendiculatus the five cattle in the applicator group had a mean infestation of 363.8 adult r. appendiculatus (range 295–430) before treatment with deltamethrin/amitraz from an applicator, after which the mean adult r. appendiculatus count fell dramatically with weekly treatments to 0.6 adult ticks (range 0–2) by day 84 of the trial (table 6). similarly, the five cattle in the positive control group had a mean infestation of 320.6 adult r. appendiculatus (range 229– 362) before treatment with deltamethrin/amitraz pour-on, after which the mean adult r. appendiculatus count fell with weekly treatments to 3.8 adult ticks (range 0–8) by day 84 of the trial (table 6). in contrast, tick challenge with r. appendiculatus in the negative control group was so heavy that the cattle in the group had to be treated on days 14, 28, 35, 49, 63 and 84 of the trial for ethical reasons. the adult r. appendiculatus counts were significantly less (p < 0.001) on both the cattle treated using the applicator and those treated by pour-on than on the cattle in the negative control group on days 14, 28, 35, 49, 63 and 84 of the trial. furthermore, adult r. appendiculatus counts were significantly less (p < 0.05) on the cattle treated using the applicator than were those for those treated by pour-on on days 7, 14, 21, 28, 42 and 70 of the trial. discussion since the advent of deer farming in the early part of the last century, it has become apparent that nematode parasites can cause severe disease and death in deer and, when infections are subclinical, they can lead to reduced productivity (fletcher 1982; mackintosh, mason, manley, baker & littlejohn 1985). control of nematodes has relied on 48 self-medicating applicator for control of parasites of wild and domestic animals treatment of deer with ivermectin administered by injection, as an oral drench or by topical application (mackintosh et al. 1985; rehbein & visser 1997). more recently moxidectin (waldrup et al. 1998) has been the anthelmintic of choice. the administration of anthelmintics to deer requires the use of handling facilities, and it induces stress in the animals and incurs labour costs. these constraints increase the costs of parasite control, result in losses of deer due to stress and/or handling accidents, and limit the frequency of treatments. use of self-medicating applicators is an alternative delivery method for anthelmintics to deer, and has the advantages of eliminating stress in the deer and of minimizing costs in labour and facilities. there are numerous reports summarizing the adverse impact of nematodes on the productivity of cattle (williams 1983; gibbs & herd 1986; craig 1988; hawkins 1993; reinemeyer 1994; clymer 2001; vercruysse & claerebout 2001). however, nematode control programmes are often difficult or impossible to implement economically on cattle farms which lack animal handling facilities (herd 1988). herd (1988) reviewed new anthelmintic delivery systems, such as medicated feed blocks and water dispensers, designed to simplify worm control in bovines by eliminating the necessity to handle animals or to put them in a crush to deliver the anthelmintic. he pointed out that, if these new delivery systems were to be used, profitable worm control strategies could be introduced to farms where lack of handling facilities had previously prevented any type of control programme. he argued that farmers want fast, simple and easy deworming programmes that involve minimal handling of cattle. self-medicating applicators could provide such an anthelmintic delivery system for cattle producers. it is apparent from the cattle trial in florida, that animals must feed readily from the trough to which the applicator is attached in order to receive an appropriate dose of anthelmintic. steer no. 109 was unaccustomed to supplemental feeding and did not feed from the trough during the first day of the trial and, consequently, was only partially treated for trichostrongyles. it is recommended, therefore, that animals to be treated using a self-medicating applicator be allowed to acclimatize to feeding from a trough (or whatever receptacle to which the applicator is attached) before treatment commences. the results of the trial on the cattle ranch in texas demonstrated that self-medicating applicators can be effective devices for the passive treatment of cattle for fly infestations. other self-medicating devices have been developed for fly control on cattle. they include dust bags, cable back-rubbers and oilers. dust bags typically consist of two burlap sacks one inside the other which contain insecticidal dust (adkins & seawright 1967). dust bags are suspended in a place which cattle frequent such as over gate openings, which force the animals to brush against the bags, dispensing the insecticidal dust from the burlap sacks onto their heads and backs. dust bags require shelter and may be unsatisfactory in humid climates (foil & hogsette 1994). cable back-rubbers consist of a chain or barbed wire suspended between two posts, with the chain or wire wrapped with burlap sacks which are soaked with an insecticidal solution (rogoff & moxon 1952) which is typically an insecticide diluted with diesel or mineral oil (dobson & peterson 1963). cattle passing under and contacting the back-rubbers are treated. the oiler consists of a tank containing insecticide which is attached to a post and from which is suspended a rubbing element such as a rope or a mop-like device (barlow & surgeoner 1979). when cattle rub the element, small quantities of insecticide are delivered to it, some of which is passed on to the cattle. the results of the trial on the research farm in south africa demonstrated that self-medicating applicators can be effective devices for the passive treatment of cattle for tick infestations. other self-medicating devices have been developed for tick control on animals. they include the duncan applicator and the ‘4-poster’ device. the duncan applicator consists of a drum-like base incorporating a feed bin, with an acaricide container on top of a treatment column rising from the centre of the bin (duncan & monks 1992). the duncan applicator has been used to control ticks on eland (taurotragus oryx), african buffaloes (syncerus caffer) and cattle using the acaricide flumethrin (duncan & monks 1992; duncan 1992). the ‘4-poster’ device consists of a central feed bin with a feeding/application station on each side of the bin, each station consisting of one bait port and two vertical pesticide-impregnated application rollers (pound, miller, george & lemeilleur 2000). the ‘4-poster’ device has been tested experimentally in the united states as a passive method for control of ticks on white-tailed deer (odocoileus virginianus) using the acaricide amitraz (pound, miller & george 2000). the results of this and other studies have demonstrated that the concept of passive delivery of treatments to animals using self-medicating applicators has practical potential in the control of gastroin49 m.j. burridge et al. testinal nematodes, flies and ticks. these devices should be of value for parasite control particularly in situations where animals are difficult to handle, such as on game ranches, and where handling facilities are not available due to lack of economic resources. furthermore, self-medicating applicators provide a method for the control of vectors of diseases of public health importance where the primary hosts of the vectors are wildlife, such as with control of lyme disease through control of its tick vectors on wild deer. acknowledgements we thank dr b. clymer of fort dodge animal health and mr r. jooste of bayer zimbabwe for providing the cydectin® and cylence® for the anthelmintic and insecticidal trials respectively. we also thank the farmers in the united states for the use of their facilities in some of the trials. references adkins, t.r. & seawright, j.a. 1967. a simplified dusting station to control face flies and horn flies on cattle. journal of economic entomology, 60:864–868. audigé, l.j.m., wilson, p.r. & morris, r.s. 1998. a survey of internal parasites and parasite control on north island deer farms. new zealand veterinary journal, 46:203–215. barlow, l.a. & surgeoner, g.a. 1979. the efficacy of self-applicating devices for control of the face fly, musca autumnalis (diptera: muscidae) and the horn fly, haematobia 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egg-counting technique and apparatus. journal of the council for scientific and industrial research, 21:177– 180. williams, j.c. 1983. ecology and control of gastrointestinal nematodes of beef cattle. veterinary clinics of north america, large animal practice, 5:183-205. 51 m.j. burridge et al. article information authors: stanly fon tebug1,6 gilson r. njunga2 mizeck g.g. chagunda3 jacob p. mapemba4 julius awah-ndukum5 steffi wiedemann6 affiliations: 1international livestock research institute, kenya2central veterinary laboratory, malawi 3future farming systems group, scottish agricultural college, scotland 4world university service of canada 5school of biomedical and biological sciences, university of plymouth, devon 6institute of animal breeding and husbandry, university of kiel, germany correspondence to: steffi wiedemann postal address: institute of animal breeding and husbandry, christian-albrechts-university kiel, olshausenstrasse 40, 24098 kiel, germany dates: received: 20 march 2013 accepted: 31 oct. 2013 published: 28 feb. 2014 how to cite this article: tebug, s.f., njunga, g.r., chagunda, m.g.g., mapemba, j.p., awah-ndukum, j. & wiedemann, s., 2014, ‘risk, knowledge and preventive measures of smallholder dairy farmers in northern malawi with regard to zoonotic brucellosis and bovine tuberculosis’, onderstepoort journal of veterinary research 81(1), art. #594, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.594 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. risk, knowledge and preventive measures of smallholder dairy farmers in northern malawi with regard to zoonotic brucellosis and bovine tuberculosis in this original research... open access • abstract • introduction    • materials and methods       • study area, population and sampling strategy       • knowledge and practices of dairy farmers with regard to zoonoses       • brucella antibody and tuberculin skin test       • statistical analysis • results    • knowledge and practices of dairy farmers with regard to zoonoses    • brucella antibody and tuberculin skin test • discussion • conclusion • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ milk production using local cattle breed-types is an age-old practice in malawi. although dairy farming is becoming more common as a result of the increasing population and demand for milk and milk products, there is limited knowledge of the farmers’ awareness of zoonotic disease risks, their preventative practices and the disease burden in animals. this study determined dairy farmers’ general knowledge of zoonoses, assessed their risks for infection with zoonotic bovine tuberculosis (btb) and brucellosis, and evaluated farm practices to prevent disease transmission. a questionnaire was drawn up and administered by the authors. it was used to collect information about the knowledge and preventive practices of 140 out of 684 registered dairy farmers at mzuzu agricultural development division, northern malawi. during a second visit to 60 out of the 140 farms, a total of 156 and 95 cattle were tested for brucellosis and tuberculosis, respectively. most farmers (77.1%) knew or had heard of zoonotic diseases, whilst 75.0% correctly named at least one zoonotic disease. more survey participants named tuberculosis as a zoonotic disease compared to brucellosis (74.3% versus 2.9%). the most commonly named means of transmission were milk (67.0%) and meat (56.0%). almost all survey participants (96.4%) practised at least one farm activity that could lead to potential transmission of brucellosis or btb, including sale (67.0%) and consumption (34.0%) of unpasteurised milk. antibodies against brucellosis were found in 12 cattle (7.7%), whilst one animal (1.1%) reacted to the tuberculin skin test. general knowledge about possible transmission of diseases between humans and animals was high, although most farmers practised risk behaviours that could potentially expose the public to milk-borne zoonotic diseases such as brucellosis and btb. furthermore, some animals had positive results for brucellosis and tuberculosis tests. therefore, improvement of zoonotic disease prevention programmes, as well as further investigation into the prevalence and risk factors for zoonoses, is recommended. introduction top ↑ dairy farming is gaining importance in malawi. this is a result of the increasing population and demand for milk and milk products in the country’s major cities. most dairy farmers are registered and organised into about 50 milk bulking groups (mbgs) around the country’s major cities: blantyre (southern region), lilongwe (central region) and mzuzu (northern region)(banda et al. 2011). the mbgs are mostly run and managed by farmers who collect milk from members within a radius of eight km. following milk testing on specific gravity and alcohol testing for acidity, milk is bulked in a cooling tank. milk is bought in bulk by the processors and a bonus is paid to the participating farmers for higher-bulk quantities. the dairy cattle breeds are predominantly holstein-friesians, jerseys and their crosses with the indigenous malawi zebu (banda et al. 2011; tebug et al. 2012a). these animals are mainly stall-fed or grazed on communal pastureland near human dwellings. although smallholder dairy farming plays a salient role in meeting the increasing demand for milk as well as milk products, and serves as an important source of employment in malawi, these animals may also transmit diseases like brucellosis and tuberculosis to humans. brucellosis and tuberculosis are considered to be the most important and widespread zoonotic diseases (who/fao/oie 2004). in sub-saharan africa, prevalence rates of brucellosis in humans as high as 13.0% have been reported in some communities (kunda et al. 2007; schelling et al. 2003; swai & schoonman 2009). malawi is described as non-endemic, because no case of brucellosis has been documented in humans (pappas et al. 2006). however, pyrexia of unknown origin is not uncommon (walsh et al. 2000). in contrast, tuberculosis in humans is relatively well documented and continues to be a major public health problem in malawi (bowie 2006; nyirenda 2006). according to the who global tb control report for 2012 there was an estimated prevalence rate of 164 per 100 000 population for tuberculosis (all forms) in the year 2011 (oie 2012). though no case of human tuberculosis due to mycobacterium bovis (m. bovis) (bovine tuberculosis, btb) has been reported in the country, isolation of m. bovis from milk and human tissues samples elsewhere is not uncommon (kazwala et al. 1998, 2001). in cattle, recent reports show that the prevalence rates of btb are as high as 13.2% in the neighbouring countries of tanzania and zambia (swai et al. 2005; swai & schoonman 2012). similarly, prevalence rates of brucellosis ranging from 1.2% to 14.0% have been reported in cattle reared in different livestock systems in tanzania and zambia (chimana et al. 2010; karimuribo et al. 2007). the most recent study carried out on malawian cattle in 1986 revealed a prevalence of 3.8% and 0.3% for btb and brucellosis, respectively (bedard, martin & chinombo 1993). both brucellosis and tuberculosis are considered to be occupational hazards; consumption of unpasteurised milk and physical contact with infected animals have been identified as the two most common routes for animal-to-human transmission (fetene, kebede & alem 2011; makita et al. 2008; schelling et al. 2003). livestock farming is believed to be an at-risk occupation, since close contact with animals is inevitable during routine farm activities (shitaye, tsegaye & pavlik 2007; swai & schoonman 2009, 2010). furthermore, cultural habits such as the consumption of fresh or soured milk may hamper preventive measures. low levels of formal education may further render knowledge dissemination and control programmes to be difficult (ayele et al. 2004; shitaye et al. 2007). the risk of transmission of btb and brucellosis has been reported to be influenced by livestock-keeping systems and environmental factors such as closeness to stock routes, access to surface drinking water, location of farms, age of animals and farmers’ knowledge of preventive measures (kazwala et al. 2001; swai & schoonman 2010). disease control schemes, including compensation to farmers for infected animals that have to be culled, are not really feasible – this is mainly due to limited resources in most developing countries, including malawi. despite this, like in most parts of africa, little official information about occurrence of btb and brucellosis is available for malawi (mcdermott & arimi 2002). therefore, information about livestock owners’ awareness, disease identification skills and preventive farm practices, which have received more recent attention, could be used to optimise disease control (john, kazwala & mfinanga 2008; mosalagae, pfukenyi & matope 2011; munyeme et al. 2010). the present study in the northern region of malawi was designed to determine dairy cattle farmers’ general knowledge about zoonotic diseases and preventive farm practices, as well as risk of transmission from dairy cattle to farmers with regard to btb and brucellosis. materials and methods study area, population and sampling strategy the present study was carried out in two districts (mzimba and nkhata bay) of the northern region of malawi. over 90.0% of the dairy cattle population in the region is found in these districts (banda et al. 2012; mzuzu agricultural development division 2009). the study population comprised 684 farmers, who were organised into 12 mbgs. each mbg had an average of 42 (range: 4–65) member farms, and each member farm had an average of 2.2 (range: 1–9) animals, including 1.2 cows per farm (banda et al. 2012; tebug et al. 2012a, 2012b). mzuzu agricultural development division provided a list of mbgs and dairy farmers. the data used in this study were collected in two phases. the first phase was conducted between february 2011 and june 2011. seven mbgs were randomly selected using a table of random numbers, from which 30.0% (140/472) of the farmers were selected and included in the survey. the second phase was carried out during the month of april 2011. one hundred and fifty-six animals from 138 farms in all 12 mbgs (146 cows and 10 bulls) and 95 cows from 74 farms were randomly selected and tested for brucellosis and btb, respectively.< knowledge and practices of dairy farmers with regard to zoonoses a questionnaire was developed to assess farmers’ general knowledge about zoonotic diseases, as well as preventive farm practices with regard to btb and brucellosis. farmers were interviewed by the same team, which was comprised of the first author (sft) and a veterinary assistant. this questionnaire was pre-tested for clarity, and to avoid confounding questions, on a pilot group of 15 farmers; in the case of inconsistent questions, it was modified accordingly. information contained in the first section included: (1) the location of the farm; (2) age and sex of the farm owner; (3) duration that the farmer had been dairy farming; (4) herd size; and (5) origin of the animals. in the second section, knowledge of zoonotic diseases was assessed. farmers were asked: (1) if they knew or had heard of a disease that is naturally transmitted between animals and man; (2) to name or describe known zoonotic disease(s); and (3) to state known route(s) of transmission. in the last section, preventive measures against zoonoses (such as btb and brucellosis) and milk-handling practices were assessed. in this section, farmers were asked: (1) if they or any family member had ever undergone medical examination for zoonotic diseases; (2) if their animals had ever been tested by a veterinarian for any zoonotic diseases; (3) whether or not milk was processed or boiled before consumption; and (4) where the milk was sold. brucella antibody and tuberculin skin test blood samples (about 7.5 ml) were collected from the jugular vein of the cattle. after coagulation and centrifugation (1500 x 15 min) serum was extracted and stored at 2 °c – 4 °c for 3–21 days until analysis was carried out. a competitive enzyme linked immunosorbent assay kit (brucella-ab c-elisa, svanova biotech ab, uppsala, sweden) was used for serological analysis (centre veterinary laboratory, lilongwe, malawi). this test distinguishes between brucella infected animals, brucella strain 19 vaccinated animals and animals infected with cross-reacting gram-negative bacteria. samples were tested in singles following the manufacturer’s recommendations (svanova biotech ab) as described elsewhere (bayemi et al. 2009).for intradermal tests for btb, bovine and avian purified protein derivatives (ppd) (supplied by id-lelystad, netherlands, and veterinary laboratories agency, weybridge, uk, respectively) were used. intradermal injections of 0.1 ml of bovine ppd and avian ppd were administered on shaved sites of the mid-neck region. the injection sites were examined 72 ± 6 h later and any swelling was measured with a pair of callipers. interpretation of the results was based on the world organisation for animal health (oie) recommendations (oie 2009). briefly, animals with a difference in skin thicknesses after bovine tuberculin and avian tuberculin injections in the subcutaneous immunotherapy test (scit) (by subtracting the increase in avian site from the increase in the bovine site) of > 4 mm, > 2 mm but < 4 mm and < 2 mm were considered positive, inconclusive and negative, respectively (oie 2009). statistical analysis the data obtained were entered in microsoft excel® (microsoft, usa) and transferred to minitab® 16 statistical software (minitab, inc., state college, pennsylvania, usa). descriptive statistics were generated and the association between different variables (dairy farmer and farm characteristics) and knowledge or farm practices with regard to zoonoses were assessed by chi-square (χ2) test. odds ratios (or) and confidence intervals (ci) were calculated to assess potential risk indicators associated with brucellosis seroprevalence in a univariate logistic regression model. potential risk indicators included in the models were identified based on previously reported risk factors (shitaye et al. 2007; swai & schoonman 2009, 2010) and on availability of data, under practical conditions in malawi, such as area, grazing system, origin of cows, breeding practice and age of animals. unadjusted risk indicators associated with brucellosis in this study in the univariate model (p ≤ 0.25) were included in a multivariate logistic model. the age of animals and duration in dairy farming were grouped into two categories each (above respective median value, below respective median value). for all χ2 tests, as well as for logistic univariate and multivariate models, p-values < 0.05 were considered to be significant. results top ↑ knowledge and practices of dairy farmers with regard to zoonoses one hundred and eight (108/140) (77.1%) survey participants had heard or believed that there are diseases that are naturally transmitted between animals and man, 14.0% (20/140) were not sure, whilst 9.0% (12/140) were certain that they had never heard of such diseases (table 1). three-quarters (105/140) of the survey participants correctly named at least one zoonotic disease. about two-thirds (95/140) of all the survey participants correctly named one route of zoonotic disease transmission. bovine tuberculosis was the most commonly named zoonotic disease and milk was the most frequently mentioned potential means of zoonotic disease transmission. sale of unpasteurised milk was known by most of the farmers to be a potential risk factor for disease transmission. all survey participants consumed home-produced milk. almost all survey participants (96.0%) practised at least one activity that could lead to milk-borne transmission of btb or brucellosis, such as no or irregular testing of animals for those diseases (26/140), as well as consumption (48/140) or sale (71/140) of unpasteurised milk (table 2). a significantly higher proportion (p < 0.05) of farmers from nkhata bay district (51.1%) consumed unpasteurised milk compared to those in mzimba district (25.0%). a higher percentage of women (57.1%) compared to men (37.5%) (p < 0.02) named at least one mode of transmission of zoonotic diseases. table 1: named zoonotic diseases, possible means of transmission and preventative measures in smallholder dairy farms. table 2: association of some smallholder dairy farmer characteristics, milk-borne disease awareness and milk consumption habits. table 3: animal-level univariate logistic analysis of risk factors associated with brucellosis seroprevalence. brucella antibody and tuberculin skin test of the 156 animals tested, 12 (7.7%) had antibodies against brucella species. in the univariate logistic regression models, a higher age and free or partial grazing were associated with occurrence of antibodies against brucellosis (p < 0.05) (table 3). in the final multivariate logistic regression model, only animals older than or equal to five years were more likely (or = 6.97; 95.0% ci = 1.41–34.36) to have experienced a brucella infection than those younger than five years. one out of 95 (1.1%) cattle was positive for btb. discussion top ↑ knowledge of zoonoses amongst dairy farmers was high; more farmers reported btb (74.3%) as a zoonotic disease than brucellosis (15.0%). a similar observation was made in a study of animal handlers in cameroon, where 68.0% knew btb as zoonotic (awah ndukum et al. 2010). the percentage of farmers who named btb as a zoonotic disease was higher than the 39.6% and 16.1% of cattle owners and smallholder dairy farmers in zambia and zimbabwe, respectively (mosalagae et al. 2011; munyeme et al. 2010). in contrast to both of these other studies, in which the majority (88.0% and 74.8%, respectively) of the survey participants were male, most (60.0%) of the survey participants in the current study were women. significantly, in the present study, more women than men named one mode of transmission (p < 0.05) and knew of milk-borne diseases (p = 0.05), which may explain the overall high-level of awareness observed. the relatively high level of awareness of btb may also be due to tuberculosis in humans, which is closely associated to hiv and aids; this is a leading cause of death (estimated at 35.8% of all deaths) in malawi (bowie 2006; nyirenda 2006).unlike tuberculosis, it was found that little is known about brucellosis, which does not reflect the apparent disease burden in animals. poor knowledge of brucellosis is thought to significantly impede people who are infected with brucellosis from seeking medical services; this is thought to have contributed to under-diagnosis and under-reporting of zoonoses in neighbouring tanzania (john et al. 2008; kunda et al. 2007). given the relatively high number of cattle with antibodies against brucellosis, low level of awareness, low formal education, as well as the unpasteurised milk-consumption habit observed in the present study, this is likely to be the case in northern malawi (table 2). unpasteurised milk is either consumed as fresh milk or as fermented curdled sour milk (chambiko). higher risk of infection with btb and brucellosis has been found to be associated with non-heated milk consumption (fetene et al. 2011; kochar et al. 2007; makita et al. 2008). fermentation of unpasteurised milk to ph values below ph 4.0 has been shown to not inhibit the growth of brucella strains (zuniga estrada et al. 2005). this investigation demonstrated that animals are not checked for zoonotic diseases on a regular basis. few farms (19.0%) reported that a veterinarian had tested their animals at least once for a zoonotic disease (mainly tuberculosis) due to cost reasons and lack of knowledge. as previously noted (mcdermott & arimi 2002), information on zoonotic disease burden in most african countries remains scarce. although the prevalence recorded in this study (1.1%) may not be a true reflection of the real situation (because of the small sample size used), reaction to the tuberculin skin test and the presence of antibodies against brucella infection in dairy cattle demonstrate that these diseases occur in the area. this is also not surprising given that both diseases had been reported previously in malawian cattle (bedard et al. 1993). in addition, about 76.0% of the dairy cattle population in the study area are of exotic breeds (tebug et al. 2012a), most of which were imported from countries like zambia and south africa where brucellosis has also been reported in cattle (chimana et al. 2010; hesterberg et al. 2008; swai & schoonman 2010). brucellosis has a wide clinical spectrum in humans (kochar et al. 2007) and symptoms might be misdiagnosed for other febrile diseases such as malaria. the results of this study indicate that zoonotic diseases could be transmitted from dairy cattle to humans. therefore, further investigations, as well as concerted veterinary and medical efforts in the control of zoonotic diseases in malawi, would be beneficial. conclusion top ↑ evidence of both btb and brucellosis was present in cattle on smallholder dairy farms in northern malawi. most dairy farmers knew btb to be a zoonotic disease, yet farm practices that constitute a high potential public health risk, such as consumption of unpasteurised milk, were still common. despite the relatively high number of cattle with antibodies against brucellosis, little was known about this zoonotic disease. seroprevalence of brucellosis was higher in cows older than five years. therefore, efforts by both veterinary and medical personnel should focus on effective ways of improving: farmers’ knowledge of zoonotic btb and brucellosis transmission, the development of improved herd disease management plans, and the establishment of food safety systems. acknowledgments top ↑ the authors acknowledge the full collaboration of smallholder dairy farmers around mzuzu, the staff of the department of animal health and livestock production (mzuzu), the staff of mpoto dairy farmers association and the administration of world university service of canada, malawi program, for supporting this study. we are thankful to the management of the centre veterinary laboratory, lilongwe (malawi), mr h. banda, mr p. dzimbiri and mr r.b. nyirongo for their support. we would also like to thank dr b. chimera for his helpful suggestions and for facilitating the procurement of reagents used in this study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions s.f.t. 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1school of veterinary medicine, university of zambia, zambia 2muhimbili university of health and allied sciences, tanzania 3hokkaido university research center for zoonosis control, japan 4namwala district medical office, namwala district, zambia 5sokoine university of agriculture, tanzania 6london school of hygiene and tropical medicine, united kingdom correspondence to: b. hang’ombe postal address: po box 30972, lusaka, zambia how to cite this article: hang’ombe, b.m., ziwa, m., haule, m., nakamura, i., samui, k.l., kaile, d. et al., 2014, ‘surveillance and diagnosis of plague and anthrax in tanzania and zambia’, onderstepoort journal of veterinary research 81(2), art. #722, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.722 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. surveillance and diagnosis of plague and anthrax in tanzania and zambia in this abstracts... open access • abstract • acknowledgements abstract top ↑ yersinia pestis and bacillus anthracis are diseases that rarely occur, with devastating consequences. in africa, despite the diseases being rare, they are reported on a yearly basis in remote areas of the continent due to lack of proper surveillance and detection systems. in tanzania and zambia, studies have been ongoing to understand these pathogens in endemic areas.the studies apply the well established polymerase chain reaction (pcr) for detecting yersinia pestis dna in suspected human specimens, rodents and their fleas by dna extraction and primers targeting the plasminogen activator gene. in tanzania, 516 rodent and nine human samples were analysed for the presence of yersinia pestis dna. of these samples, four rodent samples belonging to mastomys and tatera species and two human specimens were found positive. as for zambia, 810 rodent samples were collected and analysed. yersinia pestis dna was detected in 33 samples, belonging to the tatera, rattus and mastomys species. as for fleas, the xenopsylla species from rodents were positive for yersinia pestis dna. the isolated bacteria were subjected to antimicrobial sensitivity tests, with results indicating a response pattern as recommended by the world health organization in the treatment of plague. in case of anthrax, suspected samples from hippopotamuses, soil and humans were screened through bacteria culture and confirmation by pcr targeting the pxo1 (protective antigen gene) and pxo2 (capsule) virulence plasmids. anthrax was detected in hippopotamuses, soil and humans, with the epidemiological link being confirmed through variable number of tandem repeats analysis. the presence of yersinia pestis dna in rodents and fleas may represent evidence that infected rodents and fleas are being maintained in plague endemic areas, consistent with the hypothesised enzootic maintenance of plague elsewhere, whilst bacillus anthracis is amplified by animals from soil into the human population. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). article information authors: grace n. madanire-moyo1 wilmien j. luus-powell1 pieter a. olivier1 affiliations: 1department of biodiversity, university of limpopo, south africa correspondence to: wilmien luus-powell postal address: private bag x1106, limpopo 0727, south africa dates: received: 28 july 2011 accepted: 12 oct. 2011 published: 16 feb. 2012 how to cite this article: madanire-moyo, g.n., luus-powell, w.j. & olivier, p.a., 2012, ‘diversity of metazoan parasites of the mozambique tilapia, oreochromis mossambicus (peters, 1852), as indicators of pollution in the limpopo and olifants river systems’, onderstepoort journal of veterinary research 79(1), art. #362, 9 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.362 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. diversity of metazoan parasites of the mozambique tilapia, oreochromis mossambicus (peters, 1852), as indicators of pollution in the limpopo and olifants river systems in this ... open access • abstract • introduction • materials and methods    • water quality parameters    • data analyses    • ethical considerations • results    • water quality    • parasite species composition, diversity and distribution • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ aquatic systems are affected by a variety of anthropogenic activities that decrease water quality through the introduction of organic and inorganic pollutants. to investigate the relationship between fish parasite communities and water quality, metazoan parasites were examined in 140 specimens of the mozambique tilapia (oreochromis mossambicus) sampled in three lakes in the limpopo province, namely the luphephe–nwanedi dams (regarded as unpolluted), the flag boshielo dam (regarded as moderately polluted) and a return water dam on a mine site (regarded as polluted). the monogenean parasites cichlidogyrus halli, digenean larval stages of clinostomum and diplostomum spp. and a gryporynchid cestode were found in or on o. mossambicus in all the sampled sites. the distribution of monogeneans (cichlidogyrus sclerosus, cichlidogyrus dossoui, cichlidogyrus tilapiae, scutogyrus longicornis and three enterogyrus spp.), metacercarial stages of two digeneans (neascus and acanthostomum spp.) and nematodes (an unidentified nematode, contracaecum sp., paracamallanus cyathopharynx and procamallanus laevionchus) was limited to the unpolluted and moderately polluted lakes. larval stages of diplostomum sp. were present in o. mossambicus collected from the unpolluted and polluted sites. the variability of the calculated infection indices (prevalence, mean abundance and mean intensity) and the parameters of species richness and diversity suggest that the structure of parasite communities are affected by the pollution levels of the water. the unpolluted reference site had the highest species richness and the highest overall parasite abundance values. introduction top ↑ in recent years there has been an increasing awareness that parasitism should be investigated in view of undesirable environmental conditions such as organic and inorganic pollution arising from anthropogenic activities (marcogliese 2005). environmental conditions are important for a host’s survival and well-being (sures 2008). a number of studies showed that a close and highly susceptible link exists between environmental conditions and parasitism (khan & billiard 2007; madanire-moyo & barson 2010; marcogliese 2005; marcogliese et al. 2006; oros & hanzelová 2009; schmidt et al. 2003; thilakaratne, mclaughlin & marcogliese 2007). for heteroxenous (indirect life cycle) metazoan parasites, environmental conditions must be favourable for all host levels (i.e. intermediate and final hosts) and for free-living stages of the parasites (dzikowski, paperna & diamant 2003). monoxenous (direct life cycle) metazoans, which are normally ectoparasites, are in constant contact with water, suggesting that poor water quality may adversely affect their diversity to a greater extent (avenant-oldewage 2001; pietrock & marcogliese 2003). as a result, populations of both heteroxenous and monoxenous parasites are expected to be affected by changing environmental conditions. some approaches for the use of parasites as bioindicators of environmental degradation are identical to those applied for free-living invertebrates (kennedy 1997) and fishes (fausch et al. 1990): • community data, especially diversity indices, are used to assess environmental health. the underlying hypothesis is that parasite diversity is highest in unpolluted waters, whereas pollution stress leads to a loss of species, change in dominance and reduction in diversity (blanar et al. 2009). in calculating diversity indices species richness and the abundance of each species are taken into account. the advantage of this approach is that knowledge on the identity of a species, its biology or susceptibility to pollutants is not required. a change in the index can therefore indicate a change in water quality, but it cannot provide any information on the nature of the change or on the identity of the pollutant (fausch et al. 1990). • another method is the use of multivariate analyses to assess pollution. this technique incorporates a number of factors, but has the disadvantage of requiring more expertise in calculation, and especially in interpretation. biotic indices are favoured for their low cost and simplicity (kennedy 1997). the present study was carried out to ascertain the relationship between metazoan parasite species composition and pollution levels. the mozambique tilapia, oreochromis mossambicus (peters, 1852), was chosen as the model fish because it is a hardy species with a remarkable tolerance for organic (noorjahan, dawood & nausheen 2003) as well as inorganic pollution (somanath 2003). materials and methods top ↑ fish were caught in gill nets of mesh sizes 30 mm – 110 mm over four seasonal samplings: april 2009 (autumn), july 2009 (winter), october 2009 (spring) and january 2010 (summer). fish were collected from one reference site and two polluted lakes of the limpopo and olifants river systems. the reference site, the luphephe–nwanedi dams (22°39.492’s, 30°25.342’e), are twin lakes within a 10 170 ha nature reserve and are connected by a channel (2.5 m deep). the lakes are relatively unpolluted owing to the absence of intensive agricultural, industrial and mining activities close to the lakes (oberholster, ashton & macmillan 2009). the moderately polluted site, the flag boshielo dam (24°49.057’s, 29°24.509’e), is situated in the middle region of the olifants river, the biggest tributary of the limpopo river. the olifants river has been gradually impaired because of an increase in agricultural and mining activities, industrial development and urbanisation (de villiers & mkwelo 2009). the polluted locality was a return water dam (23°59.622`s, 29°24.509`e) located within mining premises in the mogalakwena river subcatchment of the limpopo river. the mine uses sewage effluent from mokopane’s wastewater treatment plant as processing water in its mining operations. the water from the mine’s processing plants is pumped to the tailings dam, from where it overflows into the return water dam. the water quality at this locality is affected by industrial, sewage and agricultural activity upstream and mining effluents from the plant. examination of sacrificed fish for parasites, fixing, preservation, whole mount preparation and identification of parasites followed standard procedures used by authors such as douëllou (1993) for monogeneans, barson et al. (2008) and chibwana and nkwengulila (2010) for digeneans, anderson (1992) for nematodes, khalil, jones and bray (1994) for cestodes and avenant, van as and loots (1989) for dolops ranarum. water quality parameters surface water temperature, dissolved oxygen (do) content, salinity, ph, conductivity and total dissolved solids (tds) were determined in situ using a handheld ysi multi-parameter instrument. subsurface water samples were collected in acid-treated sampling bottles at all sampling sites. samples were frozen immediately and taken to an accredited laboratory for analyses of selected water parameters (iso 17025, sanas, laboratory no. t0391). data analyses levels of parasite infections were analysed according to bush et al. (1997). shannon-wiener and margalef (diversity), as well as evenness (equal distribution) biotic indices were calculated to compare the three communities of metazoan parasites. increasing values of the shannon-wiener and margalef indices indicate an increase in diversity. values of evenness can range from 0 to 1, with 0 indicating a completely uneven distribution of parasites in a sample and 1 indicating a totally even distribution. all indices were calculated according to magurran (1988).all infection data were normalised with √(χ) transformations to guarantee normality of distribution. all physical variables were standardised to zero mean and unit variance to make them dimensionless. principal components analysis (pca) was performed to determine the pattern of distribution of parasite species amongst the sites. results were considered significant at the 95% level (p < 0.05). ordination was achieved using canoco version 4 (ter braak & smilauer 1998). ethical considerations the protocol was approved by the animal ethics committee of the university of limpopo. fish were killed by severing the spinal cord whilst covering the eyes with a damp cloth. handling and treatment of animals were in accordance with the guidelines of the south african council on animal care (south african bureau of standards 2004). results top ↑ water quality conductivity, total alkalinity, and concentrations of nitrate, sulphate, chloride, potassium, calcium and magnesium were higher in the moderately polluted site and the polluted site, typical of areas receiving sewage input and agricultural, industrial and mining effluents (table 1). at both sites, levels of heavy metals such as aluminum, iron and lead surpassed the south african target water quality range (twqr) for aquatic life (department of water affairs and forestry 1996). furthermore, the return water dam had the highest concentrations of nutrients (ammonium, nitrate, nitrite and orthophosphate) amongst all sites and were consistently above the levels set by the south african twqr (table 1). table 1: summary of environmental variables measured in three lakes of the limpopo and olifants river systems. the results of the pca analysis of environmental variables in the three lakes are presented in figure 1. the first two axes explained 32.7% of total variation of the environmental characteristics. the flag boshielo dam was positively associated with do content and iron level but negatively associated with arsenic level (figure 1). the return water dam was significantly associated with increasing gradients of nutrients (nitrite, nitrate, sulphate, orthophosphate and ammonium) and inorganic constituents (magnesium, calcium, potassium, chloride, sodium, tds, conductivity and turbidity). this lake was negatively associated with do content (figure 1). figure 1: a principal components analysis ordination biplot showing limnological differences amongst the three sampling sites. parasite species composition, diversity and distribution a total of 140 specimens of o. mossambicus were collected and examined from the three localities (table 2). the metazoan parasites encountered included 20 species, comprising eight monogeneans [cichlidogyrus halli (price and kirk, 1967), cichlidogyrus sclerosus paperna and thurston, 1969, cichlidogyrus dossoui douëllou, 1993, scutogyrus longicornis (paperna and thurston, 1969), cichlidogyrus tilapiae paperna, 1960 and three species of enterogyrus paperna, 1963], five larval digeneans (neascus von nordmann, 1832, tylodelphys diesing, 1850, diplostomum nordmann, 1842, tetracotyle diesing, 1858 and clinostomum leidy, 1856), four nematodes [contracaecum raillet & henry, 1912, procamallanus laevionchus (wedl, 1862) and paracamallanus cyathopharynx (baylis, 1923) and an unidentified nematode larva], a gryporynchid cestode larva, a branchiuran, dolops ranarum (stuhlmann, 1891), and a copepod, ergasilus von nordmann, 1832 (table 3). table 2: some parasitological parameters for metazoan parasites of oreochromis mossambicus sampled from three lakes of the limpopo and olifants river systems. table 3: number and dominance of parasite taxa of oreochromis mossambicus sampled from three lakes of the limpopo and olifants river systems. mean parasite species richness and diversity indices (shannon-wiener, margalef and evenness of shannon-wiener) decreased from the reference site to the polluted site. the heteroxenous parasite species corroborated the general pattern seen for diversity indices. the reference site and the moderately polluted site had an equal number of monoxenous parasite species (table 2). the dominant species were metacercariae of tetracotyle and neascus spp. and a gryporynchid cestode at the luphephe–nwanedi dams, the flag boshielo dam and the return water dam, respectively (table 2). prevalence values for the monogeneans were greater at the flag boshielo dam than at the luphephe–nwanedi dams, except for enterogyrus sp. 1 and enterogyrus sp. 3 (figure 2a). prevalence values for metacercariae of a tylodelphys sp. were relatively similar in fish collected from the luphephe–nwanedi dams and the flag boshielo dam and the same trend was noted for diplostomum sp. larvae in fish collected from the luphephe–nwanedi dams and the return water dam (figure 2b). prevalence values for all nematodes encountered were higher in fish sampled from the luphephe–nwanedi dams than in fish sampled from the flag boshielo dam (figure 2c). the prevalence values for the larval stage of the gryporynchid cestode were relatively similar in fish collected from the luphephe–nwanedi dams and the flag boshielo dam, but highest in fish collected from the return water dam (figure 2d). figure 2: prevalence of (a) monogeneans, (b) digeneans, (c) nematodes and (d) gryporynchid cestode larvae, dolops ranarum and ergasilus sp., of fish sampled from the limpopo and olifants river systems. the mean abundance values for all monogeneans encountered were higher in the luphephe–nwanedi dams than in the flag boshielo dam (figure 3a). the mean abundance values for metacercariae of clinostomum sp. were relatively low in fish collected from all localities (figure 3b). larvae of the unidentified nematode and p. cyathopharynx were more abundant in the flag boshielo dam than in the luphephe–nwanedi dams (figure 3c). the gryporynchid cestode larvae were most abundant in fish from the return water dam (figure 3d). the mean intensity values for most metazoan parasites were highest at the luphephe–nwanedi dams, except for species such as c. halli, c. sclerosus, enterogyrus sp. 2, p. cyathopharynx and the larvae of the gryporynchid cestode (figures 4a-d). figure 3: mean abundance of (a) monogeneans, (b) digeneans, (c) nematodes and (d) gryporynchid cestode larvae (dolops ranarum and ergasilus sp.) of fish sampled from three lakes of the limpopo and olifants river systems. figure 4: mean intensity of (a) monogeneans, (b) digeneans, (c) nematodes and (d) gryporynchid cestode larvae (dolops ranarum and ergasilus sp.) of fish sampled from three lakes of the limpopo and olifants river systems. the eight most dominant parasite species from o. mossambicus comprised 80.7% of the total individual parasite specimens encountered (table 3). the proportional abundance of the six main taxonomic groups collected during the study revealed that metacercariae of digeneans and larval cestodes were more cosmopolitan than monogeneans (figure 5). figure 5: relative abundance of parasite groups in oreochromis mossambicus sampled from three lakes of the limpopo and olifants river systems. the first two factors in the pca accounted for 36.2% of the observed variance, which was based on the relative abundance of the parasite species (figure 6). all monogeneans, metacercariae of the digeneans (clinostomum and neascus spp.) and the nematodes were closely associated with o. mossambicus sampled from the luphephe–nwanedi dams and the flag boshielo dam. d. ranarum, metacercariae of tylodelphys and tetracotyle spp. were unique to the luphephe-nwanedi dams whilst larvae of diplostomum sp. were common in the luphephe–nwanedi dams and the return water dam. the gryporynchid cestode larvae were, to a greater extent, strongly associated with the return water dam (figure 6). figure 6: a principal components analysis ordination biplot showing parasite species distribution based on abundance amongst sites. discussion top ↑ the spatial distribution of water quality variables showed the pollution gradient differed amongst the sampling sites (return water dam < flag boshielo dam < luphephe–nwanedi dams), especially for trophic status variables (ammonia, nitrates, nitrites, orthophosphates and sulphates), turbidity, conductivity, tds and metals (lead and aluminium). the luphephe and nwanedi tributaries flow through areas of natural grassland, interspersed with areas of subsistence cultivation. there is very little urbanisation; as a result, the waters are relatively uncontaminated. results from this study were consistent with previous work describing the luphephe–nwanedi dams as essentially unpolluted (oberholster et al. 2009) and the flag boshielo dam as impacted by a combination of mining and agricultural effluents (ashton 2010; de villiers & mkwelo 2009). the results further demonstrate that the return water dam is a polluted site, with high levels of nutrients and metals. parasite communities at the polluted and reference sites showed some trends consistent with predicted parasite diversity in stressed environments (blanar et al. 2009). the reference site had the highest species richness and the highest overall parasite abundances. similar to the polluted site, the moderately polluted site had lower mean abundance and mean intensity values for most parasite species than the reference site. in addition, these two sites revealed a total absence of the branchiuran d. ranarum and metacercariae of tylodelphys and tetracotyle spp. fish from the polluted site had the lowest total parasite numbers, species richness and parasite diversity as reflected by the indices used in the present study. decreases in species richness and diversity in the helminth parasite communities of fishes can indeed result from aquatic pollution (marcogliese 2005). this naturally occurs through the loss of ectoparasitic monoxenous species that are in direct contact with organic and inorganic pollutants (pietrock & marcogliese 2003) or through the loss of heteroxenous parasites that use intermediate hosts sensitive to pollution (marcogliese 2005; pietrock, meinelt & marcogliese 2008). for example, the ectoparasites c. halli, c. sclerosus, c. dossoui, c. tilapiae, s. longicornis, d. ranarum and ergasilus sp. seem to be sensitive to pollution as they were not found in the polluted site. although the three enterogyrus spp. are endoparasitic, they were also absent from the polluted site, demonstrating their sensitivity to pollution. metacercariae stages of the digeneans (neascus, tylodelphys and tetracotyle spp.) and the nematodes (unidentified nematode, contracaecum sp., p. cyathopharynx and p. laevionchus) were absent at the polluted site, indicating their possible intolerance to pollution. these patterns in parasite communities are consistent with several similar studies of substantially disturbed environments (dzikowski et al. 2003; khan & billiard 2007; madanire-moyo & barson 2010; marcogliese 2005; marcogliese et al. 2006; oros & hanzelová 2009; schmidt et al. 2003; thilakaratne et al. 2007). for example, indirect life cycle helminth parasites that were present in a nearby reference estuary were missing from the parasite communities of the grey mullet (liza aurata and liza ramada) collected from a nearby polluted estuary (dzikowski et al. 2003). exposure to urban effluent was reflected in lower species richness of macroparasite communities of the flounder fish (platichthys flesus) in german blight in the north sea (schmidt et al. 2003). marcogliese et al. (2006) established that fish downstream of sewage outfall had lower total parasite abundances than fish collected from reference localities in lake st. louis. thilakaratne et al. (2007) reported a lower diversity of helminths at sewage effluent and metal-polluted sites than at a reference site in lake st. louis. winter flounder (pleuronectes americanus) in the vicinity of a pulp and paper mill had significantly lower abundances of intestinal helminths than those at a nearby reference locality (khan & billiard 2007). a fish community in a river in southern slovakia, polluted with cyanide and heavy metals, had half the diversity of gastrointestinal helminth parasites as a comparable community in a nearby reference river owing to a loss of species that exploit sensitive invertebrate species as obligatory intermediate hosts (oros & hanzelová 2009). component species richness and diversity of parasites of the sharptooth catfish (clarias gariepinus), decreased with organic pollution in the manyame catchment of zimbabwe (madanire-moyo & barson 2010). the gryporynchid cestode larvae were evidently associated with the polluted site. the first intermediate hosts of most cestodes are oligochaetes and these worms can tolerate organic pollution because they feed on organic particles (sibley, dixon & barton 2000). kostarev (1980) found high numbers of caryophyllaeus laticeps and attributed this to increased oligochaete populations where household sewage was discharged directly into reservoirs. thus, the increased abundance of these intermediate hosts in this study may be due to the high organic enrichment of the sediments that are suitable for the oligochaete intermediate hosts (sibley et al. 2000). in addition, a few endoparasitic cestodes are able to accumulate and tolerate higher levels of metal contaminants than their fish hosts (retief, avenant-oldewage & du preez 2007). there were no substantial differences between the moderately polluted site and the reference site with regard to parasite component community species richness and diversity. prevalence values of some parasites (e.g. most monogeneans) were comparatively lower in fish from the reference site than in those from the moderately contaminated site, although no significant trends were detected. a large percentage of the differences between these two sites was due to higher mean abundances of most parasite species at the reference site. thus, it appears that although moderate levels of contamination may affect parasite communities, the responses of parasites may be subtle or absent if a disturbance or pollution level is moderate (marcogliese et al. 2006). it may be difficult to detect in part because the responses of all parasite species are integrated in community analyses (kennedy 1997; marcogliese 2005). conclusion top ↑ the results suggest that parasite species composition and richness of freshwater fish parasites are influenced by environmental factors. however, the responses of parasites may be subtle or absent if a disturbance or pollution level is moderate. heteroxenous parasite species are transmitted through invertebrate and vertebrate hosts acting as intermediate or definitive hosts. changes in the structure of a parasite community mirror differences in the composition of the aquatic species, such as macro-invertebrate fauna, commonly used as indicators of water quality. these findings also suggest that monoxenous parasite species decrease in numbers in disturbed environments, as they are directly exposed to the effects of water quality. thus, parasite communities can be used as an early warning system for monitoring ecosystem degradation. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions w.j.l-p. (university of limpopo) was the project leader, p.a.o. (university of limpopo) the project co-leader and g.n.m-m. (university of limpopo) was responsible for experimental design and performed most of the experiments and calculations. references top ↑ anderson, r.c., 1992, nematode parasites of vertebrates: their development and transmission, cab international, wallingford. ashton, p.j., 2010, ‘the demise of the nile crocodile (crocodylus niloticus) as a keystone species for aquatic ecosystem conservation in south africa: the case of the olifants river’, aquatic conservation: marine and freshwater ecosystems 20, 489–493. http://dx.doi.org/10.1002/aqc.1132 avenant-oldewage, a., 2001, ‘protocol for the assessment of fish health based on the health index: report and a manual for training of field workers to the rand water board (report no. 2001/03/31)’, report, rand water, vereeniging. avenant, a., van as, j.g. & loots, g.c., 1989, ‘on the hatching and morphology of dolops ranarum larvae (crustacea: branchiura)’, journal of zoology (london) 217, 511–519. http://dx.doi.org/10.1111/j.1469-7998.1989.tb02506.x barson, m., bray, r.a., ollevier, f. & huyse, t., 2008, ‘taxonomy and faunistics of the helminth parasites of clarias gariepinus (burchell, 1822), and oreochromis mossambicus (peters, 1852) from temporary pans and pools of the save-runde river floodplain, zimbabwe’, comparative parasitology 75, 228–240. http://dx.doi.org/10.1654/4337.1 blanar, c.a., munkittrick, k.r., houlahan, j., maclatchy, d.l. & marcogliese, d.j., 2009, ‘pollution and parasitism in aquatic animals: a meta-analysis of effect size’, aquatic toxicology 93, 18–28. http://dx.doi.org/10.1016/j.aquatox.2009.03.002, pmid:19349083 bush, a.o., lafferty, k.d., lotz, j.m. & shostak, a.w., 1997, ‘parasitology meets ecology on its own terms: margolis et al. revisited’, journal of parasitology 83, 575–583. http://dx.doi.org/10.2307/3284227, pmid:9267395 chibwana, f.d. & nkwengulila, g., 2010, ‘variation in the morphometrics of diplostomid metacercariae (digenea: trematoda) infecting the catfish, clarias gariepinus in tanzania’, journal of helminthology 84, 61–70. http://dx.doi.org/10.1017/s0022149x09990083, pmid:19619367 de villiers, s. & mkwelo, s.t., 2009, ‘has monitoring failed the olifants river?’, water sa 35, 671–676. department of water affairs and forestry, 1996, south african water quality guidelines: aquatic ecosystems, 2nd edn., department of water affairs and forestry, pretoria. douëllou, l., 1993, ‘monogeneans of the genus cichlidogyrus paperna, 1960 (dactylogyridae: ancyrocephalinae) from cichlid fishes of lake kariba (zimbabwe) with descriptions of five new species’, systematic parasitology 25, 159–186. http://dx.doi.org/10.1007/bf00007007 dzikowski, r., paperna, i. & diamant, a., 2003, ‘use of fish parasite species richness indices in analyzing anthropogenically impacted coastal marine ecosystems’, helgoland marine research 57, 220–227. http://dx.doi.org/10.1007/s10152-003-0138-2 fausch, k.d., lyons, j., karr, j.r. & angermeier, p.l., 1990, ‘fish communities as indicators of environmental degradation’, american fisheries society symposium 8, 123–144. kempster, p.l., hattingh, w.h.j. & van vliet, h.r., 1980, ‘summarized water quality criteria’, technical report (tr 108), directorate of water affairs, pretoria. kennedy, c.r., 1997, ‘freshwater fish parasites and environmental quality: an overview and caution’, parassitologia 39, 249–254. khalil, l.f., jones, a. & bray, r.a. (eds.), 1994, keys to the cestode parasites of vertebrates, cab international, wallingford. khan, r.a. & billiard, s.m., 2007, ‘parasites of winter flounder (pleuronectes americanus) as an additional bioindicator of stress-related exposure to untreated pulp and paper mill effluent: a 5-year field study’, archives of environmental contamination and toxicology 52, 243–250. http://dx.doi.org/10.1007/s00244-006-0082-7, pmid:17160488 kostarev, g.f., 1980, ‘the effects of pollution on the fish parasite fauna in the kama reservoirs’, in biologicheskie resursy vodoemov zapadnogo urala, mezhyuzovskii sborrnik nauchnykh trudov [allow phonetic typing biological resources of the waters west of the urals], pp. 147–153, perskii gosudarstvennyi universitet, perskii. madanire-moyo, g. & barson, m., 2010, ‘diversity of metazoan parasites of the african catfish clarias gariepinus as indicators of pollution in a subtropical african river system’, journal of helminthology 84, 216–227. http://dx.doi.org/10.1017/s0022149x09990563, pmid:19761628 magurran, a.e., 1988, ecological diversity and its measurement, princeton university press, princeton. marcogliese, d.j., 2005, ‘parasites of the superorganism: are they indicators of ecosystem health?’, international journal for parasitology 35, 705–716. http://dx.doi.org/10.1016/j.ijpara.2005.01.015, pmid:15925594 marcogliese, d.j., gendron, a.d., plante, c., fournier, m. & cyr, d., 2006, ‘parasites of spottail shiners (notropis hudsonius) in the st. lawrence river: effects of municipal effluents and habitat’, canadian journal of zoology 84, 1461–1481. http://dx.doi.org/10.1139/z06-088 noorjahan, c.m., dawood, s.s. & nausheen, d., 2003, ‘impact of dairy effluent on biochemical constituents of fish oreochromis mossambica’, journal of ecotoxicology & environmental monitoring 13, 227–231. oberholster, p.j., ashton, p.j. & macmillan, p.m., 2009, ‘a water quality reconnaissance investigation of the nwanedzi dam, limpopo province’, report (gwdms 175044), csir (natural resources and the environment), pretoria. oros, m. & hanzelová, v., 2009, ‘re-establishment of the fish parasite fauna in the tisa river system (slovakia) after a catastrophic pollution event’, parasitology research 104, 1497–1506. http://dx.doi.org/10.1007/s00436-009-1356-6, pmid:19214574 pietrock, m. & marcogliese, d.j., 2003, ‘free-living endohelminth stages: at the mercy of environmental conditions’, trends in parasitology 19, 293–299. http://dx.doi.org/10.1016/s1471-4922(03)00117-x pietrock, m., meinelt, t. & marcogliese, d.j., 2008, ‘effects of cadmium exposure on embryogenesis of stagnicola elodes (mollusca, gastropoda): potential consequences for parasite transmission’, archives of environmental contamination and toxicology 55, 43–48. http://dx.doi.org/10.1007/s00244-007-9083-4, pmid:18166987 retief, n.r., avenant-oldewage, a. & du preez, h., 2007, ‘ecological aspects of the occurrence of asian tapeworm, bothriocephalus acheilognathi yamaguti, 1934 infection in the largemouth yellowfish, labeobarbus kimberleyensis (gilchrist and thompson, 1913) in the vaal dam, south africa’, physics and chemistry of the earth 32, 1384–1390. schmidt, v., zander, s., koerting, w. & steinhagen, d., 2003, ‘parasites of the flounder platichthys flesus (l.) from the german bight, north sea, and their potential use in ecosystem monitoring’, helgoland marine research 57, 236–251. http://dx.doi.org/10.1007/s10152-003-0159-x sibley, p.k., dixon, f.g. & barton, d.r., 2000, ‘impact of bleached kraft pulp mill effluent on benthic community structure in relation to environmental factors’, journal of aquatic stress and recovery 7, 229–246. http://dx.doi.org/10.1023/a:1009987123319 somanath, v., 2003, ‘impact of tannery effluent on bioenergetics of fishes’, journal of ecotoxicology & environmental monitoring 13, 161–173. south african bureau of standards, 2004, draft 2: the welfare of animals in laboratories, (scs140.38d sans 10386/3), south african bureau of standards, pretoria. sures, b., 2008, ‘host-parasite interactions in polluted environments’, journal of fish biology 73, 2133–2142. http://dx.doi.org/10.1111/j.1095-8649.2008.02057.x ter braak, c.j.f. & smilauer, p., 1998, canoco reference manual and user’s guide to canoco for windows: software for canonical community ordination (version 4.5), microcomputer power, ithaca. thilakaratne, i.d.s.i.p., mclaughlin, j.d. & marcogliese, d.j., 2007, ‘effects of pollution and parasites on biomarkers of fish health in spottail shiners, notropis hudsonius (clinton)’, journal of fish biology 71, 519–538. http://dx.doi.org/10.1111/j.1095-8649.2007.01511.x erasmus_97.indd 97 onderstepoort journal of veterinary research, 76:97 (2009) african horsesickness b.j. erasmus deltamune (pty) ltd., p.o. box 14167, lyttelton, 0140 south africa abstract erasmus, b.j. 2009. african horsesickness onderstepoort journal of veterinary research, 76:97 african horsesickness (ahs) played a major role in the history and development of southern africa, both in times of peace and of war. frequent epidemics of ahs resulted in major transportation impediments. the viral (filterable) nature of the aetiological agent was proven in 1900 by sir john m’fadyean in london, making it one of the first animal diseases for which this had been demonstrated. similarly, the cultivation of ahs virus in the brain of mice and its subsequent attenuation were also amongst the first such feats for animal viruses. interestingly, both milestones followed the initial work of max theiler (son of sir arnold) with yellow fever virus. the role of culicoides midges as vectors of bluetongue and ahs viruses by rene du toit in 1944 was another first, adding a neglected group of insects to the list of insect viral vectors. despite the pioneering work of many workers at onderstepoort, several challenges exist (such as an absolutely safe and efficacious vaccine). it is trusted that a combination of resolute researchers 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information author: eric beda1 affiliation: 1southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania correspondence to: eric beda postal address: po box 3297, morogoro, tanzania how to cite this poster: beda, e., 2012, ‘towards one health knowledge networks: a southern african centre of infectious disease surveillance case study’, onderstepoort journal of veterinary research 79(2), art. #480, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.480 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. towards one health knowledge networks: a southern african centre of infectious disease surveillance case study in this poster... open access • footnote the dynamic nature of new information and/or knowledge is a big challenge for information systems. early knowledge management systems focused entirely on technologies for storing, searching and retrieving data; these systems have proved a failure. juirsica and mylopoulos1 suggested that in order to build effective technologies for knowledge management, we need to further our understanding of how individuals, groups and organisations use knowledge. as the focus on knowledge management for organisations and consortia alike is moving towards a keen appreciation of how deeply knowledge is embedded in people’s experiences, there is a general realisation that knowledge cannot be stored or captured digitally. this puts more emphasis in creating enabling environments for interactions that stimulate knowledge sharing. our work aims at developing an un-obtrusive intelligent system that glues together effective contemporary and traditional technologies to aid these interactions and manage the information captured. in addition this system will include tools to aid propagating a repository of scientific information relevant to surveillance of infectious diseases to complement knowledge shared and/or acts as a point of reference. this work is ongoing and based on experiences in developing a knowledge network management system for the southern african centre of infectious disease surveillance (sacids), a one health consortium of southern african academic and research institutions involved with infectious diseases of humans and animals in partnership with world-renowned centres of research in industrialised countries. footnote top ↑ 1. using ontologies for knowledge management: an information systems perspective, igor jurisica, john mylopoulos, eric yu, university of toronto, toronto, ontario, canada. article information authors: ivan g. horak1,2 heloise heyne3 edward f. donkin4 affiliations: 1department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, south africa 2department of zoology and entomology, university of the free state, south africa 3arc-onderstepoort veterinary institute, onderstepoort, south africa 4department of production animal studies, faculty of veterinary science, university of pretoria, south africa correspondence to: ivan horak email: ivan.horak@up.ac.za postal address: faculty of veterinary science, private bag x04, onderstepoort 0110, south africa keywords domestic cats; ixodid ticks; 19 species; southern africa; wild felids dates: received: 19 april 2010 accepted: 10 aug. 2010 published: 24 nov. 2010 how to cite this article: horak, i.g., heyne, h. & donkin, e.f., 2010, parasites of domestic and wild animals in south africa. xlviii. ticks (acari: ixodidae) infesting domestic cats and wild felids in southern africa, onderstepoort journal of veterinary research 77(1): art. #3, 7 pages. doi: 10.4102/ojvr.v77i1.3 copyright notice: © 2010. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 1025-9848 (print) issn: 2071-9736 (online) parasites of domestic and wild animals in south africa. xlviii. ticks (acari: ixodidae) infesting domestic cats and wild felids in southern africa in this original research... open access • abstract • opsomming • materials and methods • results • discussion    • amblyomma hebraeum    • amblyomma marmoreum    • haemaphysalis colesbergensis    • haemaphysalis elliptica    • haemaphysalis zumpti    • hyalomma truncatum    • ixodes corwini    • ixodes pilosus group    • ixodes rubicundus    • other ixodes species    • rhipicentor bicornis    • rhipicentor nuttalli    • rhipicephalus capensis    • rhipicephalus gertrudae    • rhipicephalus sanguineus    • rhipicephalus simus    • rhipicephalus turanicus    • other rhipicephalus species    • otobius megnini • conclusion • acknowledgements • references abstract (back to top) ticks collected from domestic cats (felis catus), cheetahs (acinonyx jubatus), caracals (caracal caracal), african wild cats (felis lybica), black-footed cats (felis nigripes), a serval (leptailurus serval), lions (panthera leo), and leopards (panthera pardus) were identified and counted. thirteen species of ixodid ticks and one argasid tick were identified from domestic cats and 17 species of ixodid ticks from wild felids. the domestic cats and wild felids harboured 11 ixodid species in common. the adults of haemaphysalis elliptica, the most abundant tick species infesting cats and wild felids, were most numerous on a domestic cat in late winter and in mid-summer, during 2 consecutive years. the recorded geographic distribution of the recently described haemaphysalis colesbergensis, a parasite of cats and caracals, was extended by 2 new locality records in the northern cape province, south africa. introduction (back to top) although several surveys on the ticks infesting domestic dogs in southern africa have been conducted, little has been done about collecting ticks from domestic cats (felis catus). the only reported surveys of ticks on domestic cats are those of horak & matthee (2003), who identified ticks in 20 collections from cats presented at a private veterinary practice in stellenbosch, western cape province, apanaskevich & horak (2008), who identified ticks collected from 4 cats at colesberg in the eastern cape province and a single cat at prince albert in the western cape province, and horak et al. (2000), who collected ticks from 7 feral cats in the kruger national park. horak & matthee (2003) recorded 5 ixodid tick species from cats at stellenbosch, apanaskevich & horak (2008) described a new species, haemapysalis colesbergensis, from the collections which they had examined, and horak et al. (2000) recovered 7 ixodid tick species from feral cats in the kruger national park. considerably more time has been devoted to the ticks infesting wild felids than to those on domestic cats. horak et al. (1987c; 2000) examined collections from 6 wild felid species, of which caracals (caracal caracal) and lions (panthera leo) accounted for the majority. they recorded 22 ixodid tick species from the 90 felids that were examined. since the late 1970s and early 1980s, horak and heyne have identified ticks in chance collections made by themselves and others from domestic cats and from wild felids. furthermore, donkin has collected ticks regularly from his family cat over a period of 26 consecutive months. this paper documents our findings on the identities and numbers of ticks collected from domestic cats and from wild felids in southern africa. materials and methods (back to top) ticks were collected from 130 domestic cats (felis catus), 23 cheetahs (acinonyx jubatus), 21 caracals, 3 african wild cats (felis lybica), 5 black-footed cats (felis nigripes), a single serval, leptailurus serval, 9 lions and 8 leopards (panthera pardus). the collections from domestic cats, caracals, african wild cats, black-footed cats and the serval were all made in south africa. those from cheetahs were undertaken in south africa and namibia, while the collections from lions and leopards were carried out in south africa, namibia and botswana. the ticks were gathered from domestic cats by their owners or by us, and we have construed the 26 consecutive monthly collections from donkin’s cat as though they were taken from 26 separate cats. ticks were taken from wild felids by veterinarians, animal health technicians, or researchers, while the animals were immobilised for translocation or other purposes; however, complete collections were rarely possible. the species of animal which were host to the ticks, the date and the locality of the collection were recorded. if ticks were not immediately stored in 70% ethanol in glass or plastic vials until assessment, they were transferred to this medium as soon as they had been examined. the donkin family cat appeared to be a dominant male that roamed freely around the surrounding properties in the ‘die wilgers’ suburb of eastern pretoria, gauteng province, south africa. from june 2004 to july 2006, all visible ticks were taken from this cat at weekly intervals or less. the ticks collected during each month were pooled and separately preserved as described, until they were identified and counted, and their numbers used to determine the seasonal abundance of adult haemaphysalis elliptica. results (back to top) the numbers and species of ticks collected from domestic cats, the number of cats infested with each species and the province in which a particular tick species was collected are summarised in table 1. a total of 13 ixodid tick species and 1 argasid species were recovered from the domestic cats, and of these, h. elliptica, followed by h. colesbergensis were the most predominant. the ticks taken from various wild felids, and the province or country in which they were collected, are summarised in tables 2−6. of the 17 ixodid tick species recovered from wild felids, h. elliptica was again the most numerous. eleven ixodid tick species were common to the domestic cats and wild felids. table 1: ticks collected from 130 domestic cats in five of south africa’s nine provinces table 2: ticks collected from 23 cheetahs in south africa and namibia table 3: ticks collected from 21 caracals in the eastern cape, western cape and northern cape provinces, south africa table 4: ticks collected from 3 african wild cats, 5 black-footed cats and a serval in four of south africa’s nine provinces discussion (back to top) amblyomma hebraeum adult amblyomma hebraeum are parasites of very large herbivores such as domestic cattle, african buffaloes (syncerus caffer), elands (taurotragus oryx), giraffes (giraffa camelopardalis), and white and black rhinoceroses (ceratotherium simum and diceros bicornis) (norval 1983; horak et al. 1987b; walker 1991; horak, golezardy & uys 2007). the immature stages, particularly the larvae, attach to the same hosts as the adult ticks, as well as small domestic ruminants, a large variety of smaller wild ruminants, domestic and wild carnivores, scrub hares (lepus saxatilis), ground-frequenting birds and leopard tortoises (geochelone pardalis) (horak et al. 1987b, 2000, 2007; dower, petney & horak 1988; horak, emslie & spickett 2001). it is thus not surprising that two of the cats and a leopard were infested with immature ticks, while a single cheetah and a lion harboured a few adult ticks. amblyomma marmoreum the adults and nymphs of amblyomma marmoreum are parasites particularly of leopard tortoises (horak et al. 2006), whereas the larvae will infest leopard tortoises and the same hosts as those listed above for the larvae of a. hebraeum (horak et al. 1987b, 2006). wild felids feature prominently amongst these host species, with collections recorded from feral cats, cheetahs, caracals, lions and leopards (horak et al. 1987c, 2000). table 5: ticks collected from 9 lions in south africa, botswana and namibia table 6: ticks collected from 8 leopards in south africa, botswana and namibia haemaphysalis colesbergensis this tick has been described only recently in collections made from domestic cats in the northern cape and western cape provinces (apanaskevich & horak 2008). it had already been recorded on caracals in the eastern cape province during 1984 and 1985, but at the time was incorrectly identified as haemaphysalis leachi (horak et al. 1987c). upon re-examination, the species proved to be h. colesbergensis.the preference of h. colesbergensis for domestic cats and caracals as hosts is now confirmed by the collections reported here. this tick species harvested from domestic cats at fraserburg and calvinia, in the northern cape province, indicates new locality records. taken in conjunction with previous records (apanaskevich & horak 2008), the species is present in the northern cape, eastern cape and western cape provinces in regions with a semi-arid to arid climate. haemaphysalis elliptica in south africa and parts of southern africa, this species was known as h. leachi, with which it had previously been synonymised. however, apanaskevich, horak & camicas (2007) resurrected the name h. elliptica, and proved that it was a valid old southern african taxon. furthermore, they showed that h. leachi sensu stricto is a separate taxon, which is present further north in africa, and after re-examination of many of the specimens from earlier surveys in south africa, came to the conclusion that all of those identified as h. leachi were, in fact, h. elliptica.h. elliptica is a three-host species of which the larvae and nymphs use murid rodents as hosts (norval 1984; matthee et al. 2007) and are only exceptionally encountered on the same carnivore hosts as the adults (apanaskevich et al. 2007). it is one of the major species that was collected from domestic dogs in two surveys conducted in mozambique (neves, afonso & horak 2004; de matos, et al. 2008), and in several surveys conducted on dogs in south africa (horak et al. 1987c, 2001; horak 1995; jacobs et al. 2001; horak & matthee 2003; nyangiwe, horak & bryson 2006). it was also the most abundant and prevalent tick on domestic cats in a study conducted in the western cape province (horak & matthee 2003). furthermore, large numbers have been collected from the three largest wild cats in south africa, namely cheetahs, lions and leopards (horak et al. 1987c, 2000). with the exception of the survey carried out by de matos et al. (2008), in which the name h. elliptica was used for this tick, all of these surveys refer to this species as h. leachi. the relatively large number of domestic cats now recorded as infested, and the large numbers of ticks regularly collected from the donkin family cat, establish h. elliptica as a major parasite of these animals. it is the vector of babesia rossi in dogs (lewis et al. 1996 [then referred to as h. leachi]), but whether it also transmits babesia felis to cats remains to be demonstrated. seasonal fluctuations in the monthly numbers of adult h. elliptica harvested from the family cat are illustrated graphically in figure 1. although deducing the seasonality of a tick species from collections taken from a single host animal is decidedly risky, the fact that the collections were made over a period of 2 years and resulted in a reasonably similar annual pattern, somewhat underpins the results obtained by this approach. the number of ticks recovered monthly from the cat increased erratically from the commencement of the survey in june 2004, reaching a peak during january 2006. within this increasing pattern of infestation, tick numbers reached minor peaks in august 2004 and 2005 (late winter), with major peaks during the mid-summer months of january and february 2005, and december 2005 and january 2006. despite the smaller numbers of adult ticks present in late summer and winter, their presence during these seasons indicates that the life cycle of h. elliptica continues throughout the year in pretoria. this pattern of seasonality is similar to that on domestic dogs belonging to people in rural communities in north-eastern kwazulu-natal province, south africa (horak et al. 2001). only 13 nymphs were recovered along with the total of 1785 adult h. elliptica collected from the cats, and 12 nymphs from the wild felids, indicating that these animals alone would not be able to maintain this species. it also implies that there must have been an abundance of murid rodents in the vicinity to serve as hosts for the immature stages of the tick (braack et al. 1996). figure 1: total numbers of adult haemaphysalis elliptica collected each month, from june 2004 to july 2006, from a male domestic cat in the eastern suburbs of pretoria, gauteng province, south africa haemaphysalis zumpti according to walker (1991), the adults of haemaphysalis zumpti are parasites of small carnivores. the presence of h. zumpti on a single domestic cat, 5 black-footed cats and 6 caracals, while none were collected from cheetahs, lions and leopards, confirms this observation. hyalomma truncatum adults of hyalomma truncatum prefer domestic cattle and large, wild bovids as hosts (norval 1982; horak et al. 2007), but a single tick has been collected from an african wild cat and 4 from a lion in zimbabwe (norval 1982), while in south africa single ticks have also been taken from a cheetah and two lions (horak et al. 2000). the 3 infested lions were all examined within the core distribution range of this tick in southern africa (theiler 1962; walker 1991). ixodes corwini the original description of ixodes corwini by keirans, clifford, & walker (1982) is based on a tick collected from a cape clawless otter (aonyx capensis) in the western cape province, and it was also recorded by them as present on mongooses and genets. horak et al. (1987c) recovered i. corwini from dogs on smallholdings near grahamstown in the eastern cape province, and we now report it as present on domestic cats in the same region, as well as from east london in the same province, and from knysna in the western cape province. ixodes pilosus group there appear to be 3 possible species within this group (mckay 1994). although ticks in this complex have a very wide host range, including domestic dogs, caracals, various antelopes and scrub hares (horak et al. 1987c; horak & boomker 1998), large numbers of adult ticks are seldom taken from a single host. the collection of ticks from 2 cats and 2 caracals in this study and from 7 cats in a survey by horak & matthee (2003), confirms a partiality of this group for felids. horak et al. (1987c) recorded adult ticks mainly from january to may on 22 caracals examined in the eastern cape province. ixodes rubicundus although stampa (1959), theiler (1962), horak, moolman & fourie (1987a) and walker (1991) all record caracals amongst a variety of other mammals as hosts of adult ixodes rubicundus, none of them has mentioned its presence on domestic cats or dogs. jacobs et al. (2001) report the occurrence of 7 ticks of this species on dogs and horak & matthee (2003), who harvested ticks from dogs and cats, record the presence of 9 such ticks on dogs, but none on cats. the presence of i. rubicundus ticks on 2 domestic cats in our study suggests that they may be good hosts of this species, but that their usually owner-restricted home ranges preclude the likelihood of their becoming infested. ticks of this species were collected from 2 of the 3 african wild cats examined and 8 of the 21 caracals. stampa (1959) recovered ticks from both these felid species. the immature stages of i. rubicundus infest elephant shrews (elephantulus spp.), and smith’s red rock rabbits (pronolagus rupestris), and the adults infest sheep, goats and cattle, as well as various wild bovids (stampa 1959; horak et al. 1987a; fourie, kok & heyne 1996; fourie, horak & woodall 2005). careful examination of caracals has, however, indicated that they are infested by all stages of development of this species (horak et al. 1987a, 1987c), and that they can therefore most likely support the life cycle of the tick in the absence of all other host species. other ixodes species a single ixodes myotomus female was collected from a domestic cat. this species normally infests bush karoo rats (otomys unisulcatus) (walker 1991), and the cat may have become naturally infested, or it could have acquired infestation by eating a rat on which an unattached or loosely attached female tick was present. however, the geographic distribution of o. unisulcatus does not include the pretoria district, where the tick was recovered, and we must therefore assume that other murid rodent species can also harbour i. myotomus. a relatively large number of ixodes spp. nymphs, of which the largest proportion probably belonged to the ixodes pilosus group, were recovered from domestic cats, and two were taken from caracals. a cat also harboured a single ixodes sp. male, and cats and caracals ixodes spp. females, which we were unable to identify. rhipicentor bicornis the genus rhipicentor contains only two species, and the distributions of both are restricted to africa (theiler 1961, 1962; walker 1991). the hosts of the immature stages of rhipicentor bicornis are unknown, but as in the case of rhipicentor nuttalli, are likely to be rock elephant shrews (elephantulus myurus). theiler (1962) records adult r. bicornis on jackals, genets and several wild felids. in this study, 10 cheetahs, a lion and a leopard in namibia, as well as a single caracal in the northern cape province, south africa, were infested with adult ticks. rhipicentor nuttalli fourie et al. (2002) have demonstrated that rock elephant shrews are the preferred hosts of the immature stages of this three-host tick. the adults target domestic dogs, hyaenas, lions, leopards, south african hedgehogs (atelerix frontalis) and porcupines (hystrix africaeaustralis) (theiler 1962; norval & colborne 1985; walker 1991; horak et al. 2000). in this study, a single domestic cat, 5 cheetahs, 2 caracals and a leopard were infested with adult ticks. theiler (1962) mentions that the adults are common on domestic dogs in the clanwilliam district of the western cape province, south africa, and in the omaruru district, namibia, during late summer. infestation with adult ticks can lead to paralysis in dogs (perchman 1976; norval & colborne 1985). rhipicephalus capensis the distribution of rhipicephalus capensis is restricted to south africa in a narrow zone along the western coast of the western and northern cape provinces, with a single occurrence recorded in the eastern cape province (walker, keirans & horak 2000). we collected a single female tick from a leopard in the western cape province in this study. rhipicephalus gertrudae rhipicephalus gertrudae is present only in south africa and namibia (walker et al. 2000). it is a three-host tick and its adults feed on cattle, sheep, wild bovids and domestic and wild carnivores, while its immature stages prefer murid rodents (walker et al. 2000; matthee et al. 2007). until 2000, only three adult r. gertrudae had been recorded on domestic dogs and none from cats (walker et al. 2000). however, as soon as surveys were conducted within its distribution range, this situation changed. jacobs et al. (2001) collected r. gertrudae from 7 dogs examined in central free state province, while horak & matthee (2003) found 83 instances on dogs and 2 on domestic cats in the western cape province. we now add a further 5 instances from cats, and 3 from caracals to the existing 3 records for caracals, as well as a cheetah in namibia, as a new host record. in the southern and north-western winter rainfall regions of south africa, r. gertrudae is found predominantly on sheep and dogs during may to october (horak & fourie 1992; horak & matthee 2003). in namibia and in the semi-arid regions of the western central summer rainfall region of south africa, it is most abundant on cattle from september or october, to february or march (biggs & langenhoven 1984; fourie, kok & heyne 1996). rhipicephalus sanguineus it may appear illogical to include a discussion on a tick species that was not even collected in this study, but the mere fact of its absence is significant. rhipicephalus sanguineus and h. elliptica have been the most abundant tick species found on domestic dogs during the various surveys conducted on these animals in southern africa (goldsmid 1963; horak 1995; bryson et al. 2000; jacobs et al. 2001; horak & matthee, 2003; neves et al. 2004; de matos et al. 2008). however, not a single r. sanguineus was recovered from domestic cats or wild felids in our survey, or in that conducted on 23 species of wild carnivores by horak et al. (2000), or that by horak & matthee (2003) on domestic cats in the western cape province. these findings serve as additional confirmation of the near-strict host preference of r. sanguineus for domestic dogs. rhipicephalus simus rhipicephalus simus replaces r. gertrudae in the more moist eastern regions of south africa, and its immature and adult stages have the same host preferences as r. gertrudae (walker et al. 2000). although walker et al. (2000) list only a single collection of r. simus from a domestic cat, they record several from wild felids. horak et al. (2000) added 2 instances of r. simus on cheetahs, 17 on lions and 2 on leopards to this list, while this study adds 4 from domestic cats and 1 each from a cheetah and a lion. rhipicephalus turanicus the identities of rhipicephalus turanicus and r. sanguineus have been confused so frequently that numerous incorrect host records for r. sanguineus have been published. both ticks occur on domestic dogs, sometimes even simultaneously (de matos et al. 2008), but with extremely rare exceptions, it is r. turanicus, and not r. sanguineus that is recovered from hosts other than domestic dogs (walker et al. 2000). the adults of r. turanicus have a very wide host range, including nearly all domestic animals, as well as wild canids, felids, suids, equids, bovids, leporids and even some of the larger species of birds (walker et al. 2000). walker et al. (2000) record 4 occurrences of r. turanicus on domestic cats and 35 on wild felids, to which we now add 2 from cats and 2 from lions. other rhipicephalus species the collection of a rhipicephalus evertsi evertsi nymph from a caracal and its adults from a leopard should be viewed as accidental or opportunistic infestations, as this tick does not normally target felids (walker et al. 2000). the stable cat from which an adult rhipicephalus zambeziensis was recovered is likely to have acquired infestation from a stray tick during a study involving this tick species. r. zambezienis adults have been collected previously from cheetahs, lions and leopards (horak et al. 2000; walker et al. 2000). otobius megnini the spinose ear tick, as this argasid tick species is known colloquially, is dependent for its survival on man-made structures and the micro-environment in its immediate vicinity. hence its presence in the ears of cattle that are confined at night in stone kraals, calves reared in unplastered calf-pens constructed from bricks (howell, walker & nevill 1978; fourie & horak 1990), stabled horses, people that ride or that are associated with horses (naudé et al. 2001; huchzermeyer 2002), or dogs and cats kept in the vicinity of infested premises. some of the cats examined in this study were stable cats, and one of them harboured 40 otobius megnini nymphs in its ears. conclusion (back to top) nineteen ixodid tick species were recovered from domestic cats and the seven species of wild felids that inhabit south africa. the cats and wild felids are good hosts of only one tick species of veterinary importance, which is h. elliptica, the vector of b. rossi to domestic dogs. acknowledgements (back to top) the authors acknowledge with gratitude the collections of ticks made from domestic cats by drs maureen baker, jenny randles, and the late amy jacot guillarmod, from black-footed cats by dr nadine lamberski, and from cheetahs and a leopard by prof. h.j. bertschinger. the study was funded by the university of pretoria, the university of the free state and the national research foundation. references (back to top) apanaskevich, d.a., horak, i.g. & camicas, j.-l., 2007, ‘redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haemaphysalis (rhipistoma) leachi (audouin, 1826) (ixodida, ixodidae)’, onderstepoort journal of veterinary research 74, 181−207. apanaskevich, d.a. & horak, i.g., 2008, ‘two new species of african haemaphysalis ticks (acari: ixodidae), carnivore parasites of the h. (rhipistoma) leachi group’, journal of parasitology 94, 594−607. biggs, h.c. & langenhoven, j.w., 1984, ‘seasonal prevalence of ixodid ticks on cattle in the windhoek district of south west africa/namibia’, onderstepoort journal of veterinary research 51, 175−182. braack, l.e.o., horak, i.g., jordaan, l.c., segerman, j. & louw, j.p., 1996, ‘the comparative host status of red veld rats (aethomys chrysophilus) and bushveld gerbils (tatera leucogaster) for epifaunal arthropods in the southern kruger national park, south africa’, onderstepoort journal of veterinary research 63, 149−158. bryson, n.r., horak, i.g., höhn, e.w. & louw, j.p., 2000, ‘ectoparasites of dogs belonging to people in resource-poor communities in north west province, south africa’, journal of the south african veterinary association 71, 175−179. de matos, c., sitoe, c., neves, l., bryson, n.r. & horak, i.g., 2008, ‘ixodid ticks on dogs belonging to people in rural communities and villages in maputo province, 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research 72, 293−301. goldsmid, j.m., 1963, ‘ticks infesting dogs in the salisbury area of southern rhodesia’, journal of the south african veterinary medical association 34, 609−610. horak, i.g., 1995, ‘ixodid ticks collected at the faculty of veterinary science, onderstepoort, from dogs diagnosed with babesia canis infection’, journal of the south african veterinary association 66, 170−171. horak, i.g., moolman, l.c. & fourie, l.j., 1987a, ‘some wild hosts of the karoo paralysis tick, ixodes rubicundus neumann, 1904 (acari: ixodidae)’, onderstepoort journal of veterinary research 54, 49−51. horak, i.g., macivor, k.m. de f., petney, t.n. & de vos, v., 1987b, ‘some avian and mammalian hosts of amblyomma hebraeum and amblyomma marmoreum (acari: ixodidae)’, onderstepoort journal of veterinary research 54, 397−403. horak, i.g., jacot guillarmod, a., moolman, l.c. & de vos, v., 1987c, ‘parasites of domestic and wild animals in south africa. xxii. ixodid ticks on domestic dogs and on wild 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south african veterinary association 72:2. jacobs, p.a.h., fourie, l.j., kok, d.j. & horak, i.g., 2001, ‘diversity, seasonality and sites of attachment of adult ixodid ticks on dogs in the central region of the free state province, south africa’, onderstepoort journal of veterinary research 68, 281−290. keirans, j.e., clifford, c.m. & walker, j.b., 1982, ‘the ixodes (afrixodes) oldi group (acari: ixodidae) from sub-saharan africa with descriptions of five new species’, journal of medical entomology 19, 309−329. lewis, b.d., penzhorn, b.l., lopez-rebollar, l.m. & de waal, d.t., 1996, ‘isolation of a south african vector-specific strain of babesia canis’, veterinary parasitology 63, 9−16. matthee, s., horak, i.g., beaucournu, j-c., durden, l.a., ueckermann, e.a. & mcgeoch, m.a., 2007, ‘epifaunistic arthropod parasites of the four-striped mouse, rhabdomys pumilio, in the western cape province, south africa’, journal of parasitology 93, 47−59. mckay, i.j., 1994, ‘two new species of ixodid ticks closely related to ixodes pilosus (acari: ixodidae)’, journal of the south african veterinary association 65, 158. naudé, t.w., heyne, h., van der merwe, i.r. & benic, m.j., 2001, ‘spinose ear tick, otobius megnini (dugès, 1884) as the cause of an incident of painful otitis externa in humans’, journal of the south african veterinary association 72, 118−119. neves, l., afonso, s. & horak, i.g., 2004, ‘ixodid ticks on dogs in and around maputo and elsewhere in mozambique’, onderstepoort journal of veterinary research 71, 279−283. norval, r.a.i., 1982, ‘the ticks of zimbabwe. iv. the genus hyalomma’, zimbabwe veterinary journal 13, 2–10. norval, r.a.i., 1983, ‘the ticks of zimbabwe. vii. the genus amblyomma’ zimbabwe veterinary journal 14, 3–18. norval, r.a.i., 1984, ‘the ticks of zimbabwe. ix. haemaphysalis leachi and haemaphysalis spinulosa’, zimbabwe veterinary journal, 15, 9−17. norval, r.a.i. & colborne, j., 1985, ‘the ticks of zimbabwe. x. the genera dermacentor and rhipicentor’, zimbabwe veterinary journal 16, 1−4. nyangiwe, n., horak, i.g. & bryson, n.r., 2006, ‘ixodid ticks on dogs in the eastern region of the eastern cape province, south africa’, onderstepoort journal of veterinary research 73, 305−309. perchman, g.e., 1976, ‘rhipicentor infestation in the dog: a case report’, rhodesian veterinary journal 7, 15−16. stampa, s., 1959, ‘tick paralysis in the karoo areas of south africa’, onderstepoort journal of veterinary research 28, 169−227 + 1 map. theiler, g., 1961, ‘a contribution to the knowledge of african ixodidae. the genus rhipicentor’, revue de zoologie et de botanique africaines 66, 297−308. theiler, g., 1962, ‘the ixodoidea parasites of vertebrates in africa south of the sahara (ethiopian region)’, project s 9958, report to the director of veterinary services, onderstepoort. mimeographed, 260 pp. walker, j.b., 1991, ‘a review of the ixodid ticks (acari, ixodidae) occurring in southern africa’, onderstepoort journal of veterinary research 58, 81−105. walker, j.b., keirans, j.e. & horak, i.g., 2000, ‘the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world’, cambridge university press, cambridge. article information authors: jan a. van wyk1 estelle mayhew2 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa 2department for telematic education innovation, university of pretoria, south africa correspondence to: jan van wyk postal address: private bag x04, onderstepoort 0110, south africa dates: received: 19 nov. 2012 accepted: 20 dec. 2012 published: 13 mar. 2013 how to cite this article: van wyk, j.a. & mayhew, e., 2013, ‘morphological identification of parasitic nematode infective larvae of small ruminants and cattle: a practical lab guide’, onderstepoort journal of veterinary research 80(1), art. #539, 14 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.539 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. morphological identification of parasitic nematode infective larvae of small ruminants and cattle: a practical lab guide in this original research... open access • abstract • introduction • materials and methods    • faecal cultures and harvesting of infective larvae    • larva preservation    • larva preparation for identification    • larva identification       • length of sheath tail extension       • proportion of sheath tail extension comprising a filament • differential larval count    • identification procedure • ethical considerations • results and discussion    • differentiation of larvae    • salient features of l3 of the common nematode genera and/or species       • trichostrongylus spp.       • ostertagia and teladorsagia spp.       • haemonchus spp.       • cooperia spp.       • the hookworms, bunostomum spp. and gaigeria pachyscelis       • oesophagostomum spp. and chabertia ovina       • nematodirus spp.       • strongyloides papillosus       • dictyocaulus spp.       • protostrongylid lungworm larvae       • free-living nematodes • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ in 2004, a new concept was introduced for simplifying identification of larvae of the common nematodes of cattle, sheep and goats that comprises estimates of the lengths of the sheath tail extensions of infective third-stage larvae (l3) of each genus and/or species to that of trichostrongylus spp., instead of having to be dependent only on measurements in micrometre. for example, if the mean length of the sheath tail extension (the extension of the sheath caudad, beyond the caudal tip of the larva) of trichostrongylus colubriformis and trichostrongylus axei is assumed to be ‘x’, then that of haemonchus contortus is 2.0–2.7 ‘x’ – a difference that is not difficult to estimate. an additional new approach suggested now, particularly for l3 of species and/or genera difficult to differentiate (such as chabertia ovina and oesophagostomum columbianum), is to estimate the proportion of the larval sheath tail extension comprising a terminal thin, whip-like filament. for the experienced person, it is seldom necessary to measure more than one or two sheath tail extensions of l3 in a mixed culture, because the identity of most of the remaining l3 can thereafter be estimated in relation to those measured, without having to take further measurements. the aim of this article was to present the novel approach in the form of a working guide for routine use in the laboratory. to facilitate identification, figures and a separate organogram for each of small ruminants and cattle have been added to illustrate the distinguishing features of the common l3. introduction top ↑ based on a review by van wyk, cabaret and michael (2004) and helminthological literature over nine decades, the present article is aimed, through a novel approach and illustrations drawn to scale, at facilitating the morphological identification of infective nematode larvae (l3) of the common nematodes of small ruminants and cattle. diagnosis of parasitic nematode infections of ruminants, both qualitative and quantitative, is largely still dependent on relatively inaccurate methods such as faecal worm egg counts and accompanying larva identification, without which no indication can be obtained of the identities of most of the common worm genera, excepting for those genera with morphologically distinct ova, for example strongyloides papillosus, nematodirus spp. and trichuris spp. in contrast, ova of ostertagia, teladorsagia, trichostrongylus, oesophagostomum, chabertia spp. and, to some extent, cooperia, bunostomum and gaigeria spp. are either difficult or impossible to differentiate without measurements and computations that are, as yet, impractical for routine use. although some progress has been made with computerised identification (christie & jackson 1982), this has not been developed for general use. large differences in the pathogenicity of the common worm genera make it essential to know which nematode genera are responsible for cases of morbidity of animals. at present, the only practical method available for routine laboratory estimation of the proportions of the worm genera present in the living animal, is to identify the larvae that are found in fresh faeces (mostly lungworm larvae) or those that develop in faecal cultures (gastrointestinal nematodes). however, it is often only the experienced person who can identify the larvae with a high degree of accuracy and few such persons remain for training the inexperienced. for many of the nematode genera, distinguishing features such as the shape of the cranial extremity (the head) of the larva are practically indistinguishable to all but the practised eye. measuring first-stage larvae (l1) of protostrongylids and the l3 of strongyles, or the length of the sheath tail extension (ste) (from the caudal tip of the larva to the tip of the ste – ‘c’ in figure 1) can aid identification, but, being too time-consuming, it is not practical to measure each larva during routine differential diagnosis. in this article, a simplified, better-structured method is described for differentiating the l1 of various genera of protostrongylids and the l3 of strongyles from one another. whilst some conventional characteristics are still used for identification, the novel approach principally comprises a comparison of the lengths of the stes of l3 of the unknown identity to those of trichostrongylus axei and trichostrongylus colubriformis and estimating the proportion (if any) which comprises a whip-like filament (‘d’ in figure 1). materials and methods top ↑ only some methods which are not more or less universal laboratory proceedings are included below. faecal cultures and harvesting of infective larvae there are numerous different methods for preparing faecal cultures. however, in this laboratory they are prepared as follows by a modification of the technique of roberts and o’sullivan (1950), as described by reinecke (1973) for all but the various lungworm and nematodirus species. in short, sheep faecal pellets are thoroughly crumbled before being mixed with sufficient vermiculite chips to yield a crumbly mixture which is lightly compacted, using non-porous stampers, to a depth of about 5 cm in wide-mouthed glass jars of approximately 1 l capacity. a hole is left in the centre of the culture by holding a stamper vertically in the centre of the jar whilst the mixture is lightly compacted around it. the culture is moistened sufficiently to ensure that it does not dry out whilst being incubated, but without it becoming water-logged. thereafter, the jar is incubated in the dark at 26 °c – 28 °c for 5–7 days, during which time it is checked periodically and moistened if necessary. note that, as discussed by van wyk et al. (2004), the length of both the larva and its ste may vary in relation to the amount of moisture in the culture medium (rossanigo & gruner 1996), so this needs to be standardised for consistent results.after the 7-day period of incubation of the culture, the inside of the jar is sprayed lightly with water from a wash bottle before being placed in bright light that stimulates the l3 to migrate up the inner surfaces of the vessel’s walls. the culture is harvested repeatedly over several days by holding the jar at a slant with the mouth pointing downwards and then spraying the inner walls with a wash bottle and allowing the larval suspension to drain into suitable containers. as gaigeria pachyscelis and bunostomum phlebotomum do not migrate readily up the walls of the culture jars, they are harvested by filling the culture jar with water, allowing it to stand for a few minutes to allow the air to escape from the culture, adding water to the jar until the water meniscus protrudes above the lip of the jar, placing an overturned petri dish over the mouth of the jar and keeping the petri dish in position whilst the jar is inverted (borgsteede & hendriks 1974; eckert 1960). water is then added to the petri dish and the rim of the jar is lifted slightly from the bottom of the dish on one side by slipping two glass microscope slides under it. the preparation is left for a few hours for l3 to migrate into the water and to settle, before the water in the petri dish is removed with a pipette for larval identification and counting. because nematodirus spp. ova need up to 14 days to hatch, fungal overgrowth commonly makes the above culture method unsuitable for members of this genus. thus the ova are initially concentrated in relatively little faecal suspension by sieving through a combination of sieves with apertures of different sizes. so-called visser sieves (a set consisting of three tubular sieves fitting one into the other) (malan & visser 1993) are ideal, with sieve openings of about 200 mm in the inner tube, 150 mm in the middle and 38 mm – 60 mm apertures in the outer tube sieve. the latter sieve is capped at the bottom to accumulate the contents in about 20 ml of water, whilst a tap is provided in the cap for drainage. the faecal sample is placed in the inner tube sieve before being thoroughly washed with a rosette of spray using an adjustable garden spray on a hose, with the effluent passing through all three tubes. the inner tube sieve is then removed and the contents of the middle tube washed similarly before it is removed in turn and the process repeated for the outer tube. nematodirus spp. and marshallagia marshalli ova are retained in suspension in the inner tube and are cleaned further by progressive removal of the faecal particles through alternate differential sedimentation and flotation, respectively in water and 40% sucrose solution, ending with a watery suspension (reinecke 1973). for final ‘cleaning’ of the ova, the suspension of ova and faecal particles is poured into a flat-bottomed glass crystallising dish to a depth of about 3 cm and left to stand for about a minute before the contents of the dish are carefully poured at a slow and even pace into a second dish of the same sort. the ova, having a higher relative density than most of the faecal particles, sink relatively rapidly to the bottom and many of them adhere to the dish, whilst the majority of the faecal particles remain in the suspension that is poured off. the ova are harvested by washing into a beaker, with the use of a wash bottle. the process is repeated until few ova, appearing to the naked eye as a whitish, granular layer on the bottom of the dish, are visible after decantation. the cleansed nematodirus spp. ova are next cultured in water containing a pinch of potassium dichromate (k2cr2o7) to control fungal growth until the l3 have emerged. continual aeration of the suspension seems to be beneficial for stimulating hatching. for nematodirus spathiger, the yield of l3 can be improved considerably by storing the ova at about 4 °c for a week before incubation (viljoen 1972). in the case of nematodirus battus, formalin is added weekly to the culture over the 5–6 weeks of incubation to a final concentration of 1%, incubating the ova for an hour and then washing them on a 53 μm sieve before continued incubation (f. jackson pers. comm., 1998 – anonymous, ‘moredun research institute parasitology division standard operating procedures’). nematodirus spp. ova can also be ‘hatched’ artificially (f. jackson pers. comm., 1998 – moredun sops): well cleansed, fully embryonated ova (i.e. on the point of hatching) are concentrated to obtain about 1 ml of solid material (eggs) per 10 ml of water. about 0.25 ml of egg suspension is pipetted in a line on a thick glass base plate measuring 300 mm × 200 mm × 8 mm, covered with a similar ‘crushing’ glass plate and firm pressure is applied on the top plate until the ova are felt to crack. stereoscopic examination should reveal that almost all the eggs have cracked, with larvae emerging. the larvae emerge fully when the preparation is re-suspended in water. larva preservation most l3 that die from natural causes or are killed (e.g. with heat) become granular and less translucent in appearance and tend to curl up, such as when killed with formalin. however, if alive, they can be relaxed and preserved practically unchanged in appearance for training and reference purposes. formalin is added to a final concentration of about 1% – 2% to the suspension of larvae, which are then killed by heating the suspension to 55 °c – 57 °c for about a minute (note that most of the common infective larvae can survive for even some hours in much higher concentrations of formalin than the amount described above). larvae killed and preserved in this way do not curl up, largely retain their brightly translucent appearance and their internal structures are more clearly discernable than those of l3 killed by heating alone (van wyk et al. 2004). larva preparation for identification a drop of larval suspension is deposited on a glass microscope slide and the larvae killed with lugol’s iodine solution that is pre-diluted to a level where it takes a few minutes before the l3 become darkly stained. the reason for this is that it is more difficult to observe internal structures of larvae (such as the shape of the oesophagus that is important in some cases) and also to differentiate free-living nematodes from l3 of bunostomum and gaigeria spp. (which rapidly stain almost uniformly brown over their entire length) from the majority of the others, in which the cranial part of the larva initially stains considerably less intensively than the rest. larva identification note that, in this article, the tip of the cranial extremity of a larva is referred to as its ‘head’ and the caudal tip as its ‘tail’ (figure 1) and the free sheath beyond the tail tip as the ste.morphological identification of l3 of most parasitic nematodes is based principally on examination of the caudal and cranial extremities, although other features such as the length or shape of the oesophagus or cranial refractile spots are important in some genera. note, however, that once exsheathed, l3 of relatively few genera can be differentiated. even when a space has formed between the cranial tip and the sheath of an aging larva, the characteristic shape of the head appears distorted and more squared than usual, thus increasing the chances of incorrect identification. length of sheath tail extension the length of the ste is a very important criterion for identification and, to facilitate its application in larval differentiation, the ste of every larva being evaluated is related to that of tr. colubriformis and tr. axei. to this end, the length of the ste (‘c’ in figure 1) of tr. colubriformis and tr. axei (± 33 mm) is represented by ‘x’, to which that of each l3 encountered in a culture is related as follows: length of ste of l3 being identified (‘c’ in figure 1) = ste / ‘x’. proportion of sheath tail extension comprising a filament if the ste ends in a narrow, thin filament (‘d’ in figure 1), the proportion of the total length of the ste that this comprises is calculated, although with practice it can usually be estimated without the need for measurement. note, however, that there is no exactly definable point of transition from the sheath filament to the cranial portion of the ste per genus or species; it is invariably a more or less gradual process, with no precise point of inflexion. on the other hand, because of relatively large differences in filament proportion between those species or genera such as chabertia ovina and oesophagostomum spp. that are difficult to differentiate (table 1), this fact does not detract from its usefulness as a criterion for identification. individual values in tables 1 and 2 were derived as described by van wyk et al. (2004). in some cases, the lengths of the stes were not listed in the papers reviewed by them, but could be calculated from the tables, estimated from photographs and/or figures drawn to scale, or could be calculated from histograms (keith 1953). differential larval count top ↑ identification procedure firstly, a stage micrometer is used to determine, for each microscope objective lens, the number of divisions of the graticule in the ocular lens that span 33 mm; that is, the mean length of the stes of tr. axei and tr. colubriformis, defined as ‘x’ for the purposes of the present system of identification (table 1). then the stes of the l3 encountered in the diagnostic samples are compared, in turn, to the number of divisions (usually four divisions at 100× magnification) recorded for tr. colubriformis and tr. axei. whilst the experienced person will use 10× ocular and objective microscope lenses (i.e. about 100× total magnification) or even somewhat less for the largest part of each differential count, about double this magnification will be necessary for the exceptional larval specimens that are difficult to identify, as well as for routine differentiation by the novice.conventionally, the first 100–200 l3 encountered per count are identified for estimating the proportion of a given sample which each genus comprises. whilst l3 can generally be identified only to genus, this is not absolute. because the l3 of trichostrongylus spp. of small ruminants are difficult to differentiate from those of teladorsagia spp., they are commonly grouped during the count. however, when exsheathed, l3 of nematodirus spp. and intestinal trichostrongylus spp. can be differentiated from those of tr. axei and teladorsagia spp. in having digitate appendages on the tail, whilst the others have none (figure 2 [mcmurtry et al. 2000] and figure 3). table 1: measurements of third-stage larvae of small ruminants, including sheath tail extensiona, ‘x’-values and the proportion of the sheath tail extension comprising a filament. table 2: measurements of third-stage larvae of cattle, including sheath tail extensiona, ‘x’-values and the proportion of the sheath tail extension comprising a filament. whilst exsheathing of intestinal trichostrongylus spp. aids in their identification, larvae that are exsheathed are often difficult to differentiate, as mentioned above. this impasse of having to differentiate some l3 after they have been exsheathed whilst others must perforce be ensheathed can be overcome to some extent by conducting differential counts in two steps. initially, ensheathed trichostrongylus, teladorsagia species are grouped, whilst the rest are identified to the level of genus or species. thereafter, as a second step, the l3 are exsheathed and only those with specific digitate terminal caudal appendages (figure 2, intestinal trichostrongylus spp.) are differentiated from the rest of the first 100–200 encountered. subsequently, the proportion of teladorsagia spp. can be computed as follows: • step 1: do differential count of ensheathed l3 and let % teladorsagia spp. + trichostrongylus spp. = x. • step 2: exsheathe the l3 and repeat differential count. let % intestinal trichostrongylus spp. (mcmurtry et al. 2000) = y. then % teladorsagia spp. + tr. axei = x y. unfortunately, with the above approach it is not possible to differentiate tr. axei from teladorsagia spp., but if it is important to do so, the method of lancaster and hong (1987) can be employed as follows during the first step for a rough estimate, whilst keeping in mind that cabaret (pers. comm., 2003) experienced the lancaster and hong (1987) technique to have a wide margin of error (see discussion below): • step 1: do differential count of ensheathed l3 and let % teladorsagia spp. (lancaster & hong 1987) = x; and let % trichostrongylus spp. (lancaster & hong 1987) = y. • step 2: repeat differential count after having exsheathed the l3 and let % intestinal trichostrongylus spp. (mcmurtry et al. 2000) = z. then % t. axei = y z. figure 1: diagram of a nematode infective larva, depicting (a) total length, (b) tip of larva tail, (c) sheath tail extension and (d) filament. figure 2: identification to species level of trichostrongylus spp., based on differences in the morphology of the tips of the larva tails: (a, b) trichostrongylus colubriformis, (c, d) trichostrongylus vitrinus, (e) trichostrongylus axei and (f) teladorsagia circumcincta. ethical considerations top ↑ this article is based on research conducted in the 1970s and early 1980s. whilst it was carried out strictly in accordance with ethical considerations overseen by the management of the onderstepoort veterinary research institute, at that stage there were no national or international guidelines for work of this nature of which the authors were aware. in 2004, a novel method of larvae evaluation was identified and described from the work of van wyk et al. (2004) and the present study illustrates this in terms of the differences between the larvae, whilst, at the same time, accurately drawing the ste filaments (this having been developed only recently, but without the need for further research in animals). results and discussion top ↑ differentiation of larvae most importantly, as emphasised by mönnig (1931), is the selection of only those points of comparison between l3 of the various genera and species that will enable swift identification with the minimum number of measurements. it is also necessary to acquaint yourself with the variation in the appearance of the various features under different depths of focus of the microscope.it is exceedingly difficult to photograph larval stes such that both the tip of the ste and the rest of the caudal extremity of a given larva is in focus simultaneously. hence the excellent photographs in the papers of corticelli and lai (1964) and henriksen (1972), and to some extent also of keith (1953), deserve particular mention, as these workers succeeded particularly well, and their photos can be put to very good use when training inexperienced persons to identify l3. on the other hand, some of the modern electronic photomicroscopes are able to integrate a series of photos at different depths of focus into a composite picture with the entire sheath tail in focus and this could be used fruitfully to this end. tables 1 and 2 contain summaries of the mean measurements and corresponding ‘x’-values of the different common worm genera and/or species (see van wyk et al. 2004, tables 1–7, for details gleaned from the literature). l3 of parasitic nematode genera are illustrated in this article in figures 2–13. as discussed by van wyk et al. (2004), in most cases where drawings were published previously, the shapes of the heads (cranial extremities) of the larvae and the proportional lengths of the filamentous portions of the stes were not drawn accurately to scale. many of the differences between worm genera are very small, making it difficult to observe and even more difficult to draw accurately. there are notable exceptions, such as the drawings of borgsteede and hendriks (1974), which are relatively accurate regarding the morphology of both the heads and stes of the l3; yet, identification remained so difficult, especially for the inexperienced, that another morphological feature was sought that could simplify the process. the sheath tail filament presented this possibility. figure 3: terminal appendages of nematodirus spp. exsheathed third-stage larvae: (a) nematodirus spathiger, lateral view, (b) nematodirus spathiger, dorso-lateral view, (c) nematodirus filicollis, lateral view, (d) nematodirus filicollis, ventro-lateral view, (e) nematodirus battus, lateral view and (f) nematodirus abnormalis, lateral view. figure 4: third-stage larvae of common nematodes of small ruminants, measured in micrometres (ìm). figure 5: cranial and caudal extremities of third-stage larvae of common nematodes of small ruminants, measured in micrometres (ìm). figure 6: third-stage larvae of common nematodes of cattle, measured in micrometres (ìm). figure 7: cranial and caudal extremities of third-stage larvae of common nematodes of cattle, measured in micrometres (ìm). figure 8: cranial and caudal extremities of ensheathed third-stage larvae of nematodirus spp., measured in micrometres (ìm). figure 9: differentiation of third-stage larvae of trichostrongylus spp. (left) and ostertagia spp. (right), depicting (a) initial point of inflexion craniad and (b) ‘shoulder’ of ostertagia spp. figure 10: exsheathed third-stage larva of oesophagostomum columbianum with a total of 20 or 21 intestinal cells (larva thawed after having been frozen in liquid nitrogen). the potential of the ste filament for differentiation of l3 was not recognised previously. most earlier workers either did not take it into consideration as a distinguishing feature, or it was mentioned in general without investigating its potential for identifying l3 to the level of genus or species (corticelli & lai 1964; gibbons et al. 2012). this omission could perhaps be ascribed to the fact that, as mentioned, the transition between the filament and the rest of the ste is not sharp and thus cannot be pinpointed with certainty, especially because it is also affected by the depth of focus of the microscope.with few exceptions, such as some nematodirus spp. and intestinal trichostrongylus spp., the infective larvae of the common nematodes of domestic ruminants can be identified only to genus level. however, as differences between species of a given genus in characteristics such as pathogenicity and, to some extent, susceptibility to anthelmintics are generally small, this limitation is seldom a serious disadvantage. morphological differences between some genera (e.g. trichostrongylus spp. and teladorsagia spp.) are so small that the ‘x’ system is ineffective for differentiation and the stes have no filaments. however, these are the exceptions, as the differences are mostly large enough to make it possible after a few measurements (in divisions on the graticule) of l3 in a mixed culture to judge the various ‘x’-values without the necessity for many (if any) further measurements. experience has shown that even relatively small differences can be recognised without recourse to measurement. for instance, whilst this system was in the early stages of development we became alerted, without having to resort to measurements, to a pure culture of trichostrongylus falculatus (the sheath tail of which differs from the common trichostrongylus spp. by only 0.5 ‘x’) when faecal samples were submitted after a field outbreak of deaths in sheep in the free state province. on the other hand, variations within species or genera (tables 1 and 2) must be kept in mind and it remains essential to take note of and to use other morphological features, such as the shape of the head (figures 5–8, 14 and 15) and the refractile bodies in the heads of cooperia spp. (below), in addition to the ‘x’ system. it is also advisable to have l3 of at least tr. colubriformis and/or tr. axei, but preferably of more genera, in pure culture available in each laboratory where either diagnostic or research differential larval counting is performed. only small numbers of these l3 are required at a time, with the result that a single batch killed and preserved as described above can suffice for years. figures 14 and 15 are organograms that can function as illustrated, interactive keys for the stepwise morphological identification of common nematode l3 of small ruminants and cattle, respectively. they were compiled largely for the purpose of training of the inexperienced and are intended for use together with the various figures depicting prominent distinguishing features of the morphology of the l3 concerned. salient features of l3 of the common nematode genera and/or species trichostrongylus spp. whilst very similar, there are some distinguishing features between different trichostrongylus species of domestic ruminants, for instance in length of ste (e.g. ‘x’ and no filament in tr. colubriformis and tr. axei and 1.7 ‘x’ in tr. falculatus and trichostrongylus rugatus) (o’callaghan 2004; van wyk et al. 2004) and the presence or absence of digitate appendages on the caudal extremity of the larvae (figure 2) (mcmurtry et al. 2000). common to all the trichostrongylus species included in this article is that the ste is without a filament and tapers so sharply that it resembles the point of a sharpened wooden pencil. the very short ste of tr. colubriformis and tr. axei is used as the basis of the classification system (van wyk et al. 2004). when exsheathed, the l3 of tr. axei resemble those of teladorsagia spp. in that the tip of the tail is smooth, in contrast to the irregular protuberances mentioned for intestinal trichostrongylus spp. however, the differences can be discerned only at high magnification and are usually visible only in larvae that are exsheathed. hence, as mentioned above, this feature requires a second step in the differential larval count procedure. ostertagia and teladorsagia spp. small ruminants: the ste of the teladorsagia spp. of small ruminants overlaps considerably in length with that of trichostrongylus spp. and is very similar in appearance, resembling a sharpened pencil point, and both are without a terminal filament (table 1). added to this is that, as mentioned, the tip of the tail of the teladorsagia spp. l3 is smooth, similar to that of tr. axei. according to lancaster and hong (1987), the head of ovine teladorsagia species has a slight ‘shoulder’ close to its cranial tip (illustrated in figure 9), whilst that of trichostrongylus spp. does not. this is fully described and depicted in lancaster and hong (1987) and van wyk et al. (2004). note, however, that j. cabaret (pers. comm., 2003) reported an average of 30% incorrect identifications with use of this method for differentiating tr. colubriformis and teladorsagia circumcincta in mixed culture.whilst tr. axei and te. circumcincta do differ markedly in total length (with respective means of 720 mm and 820 mm), it is time-consuming to do such measurements. furthermore, the lengths of the l3 of trichostrongylus spp. and teladorsagia spp. overlap to a considerable extent (o’callaghan 2004), thus making it a relatively impractical for accurate discrimination. figure 11: exsheathed third-stage larva of chabertia ovina, with a total of 32 intestinal cells (larva thawed after having been frozen in liquid nitrogen). figure 12: tail morphology of lungworm larvae of five genera: (a) muellerius capillaris, (b) protostrongylus rufescens, (c) cystocaulus ocreatus, (d) dictyocaulus filaria and (e) neostrongylus linearis, with (i) and (ii) illustrating the different types of appendices to the larva tails. cattle: in this host species, in contrast to the situation in small ruminants, there is small chance of confusing the stes of ostertagia ostertagi and trichostrongylus spp. l3. at slightly more than 2 ‘x’, the ste of l3 of os. ostertagi is twice as long as that of the common trichostrongylus spp. and the presence of a short filament serves further to differentiate them from the l3 of both te. circumcincta and trichostrongylus spp. (tables 1 and 2). another useful distinguishing feature is that the ste of os. ostertagi ends in a blunter tip than that of haemonchus placei (borgsteede & hendriks 1974) (figures 6 and 7). haemonchus spp. as discussed by van wyk et al. (2004), there has been confusion in the past concerning the validity of h. placei as a separate species from haemonchus contortus, despite clear morphological and/or biological distinguishing features between the two both in the l3 and adult worms. the confusion was compounded by cross-infectivity of both species to sheep and cattle. the adults of the two species are indeed relatively difficult to differentiate morphologically, but with use of the novel approach to differentiation presented now, this should not apply to the l3.whilst the infective larva of h. contortus of small ruminants has an ste with a length of 2.2–2.7 ‘x’ and a filament comprising 10% – 15% thereof, the corresponding values of h. placei of cattle are 2.7–4.0 ‘x’ and ± 20% (figures 4–7 and tables 1 and 2). on the other hand, borgsteede and hendriks (1974) discuss the possibility of confusion between the l3 of h. placei and of os. ostertagi in cattle and point out the respective bullet-shaped and flatter heads and much finer and blunter ste tips of os. ostertagi and h. placei (see figures 6 and 7 in this article, as well as subsection 3.1.3. of van wyk et al. 2004 and, particularly, figure 3 of borgsteede & hendriks 1974). added to this is that the filament comprises a larger proportion of the ste of h. placei than it does in os. ostertagi (table 2) (van wyk et al. 2004) and, as discussed by van wyk et al. (2004), the intestine of haemonchus spp. is reported to end caudally in two terminal cells, compared to only one in ostertagia spp. cooperia spp. most striking is the presence of two unique refractile bodies in the head of the l3 of cooperia spp. (figures 4–7), constituting an important distinguishing feature from those of other genera. on the other hand, the stes of cooperia pectinata and cooperia punctata of cattle somewhat resemble those of os. ostertagi and h. placei, all having a filament of about 20% (table 2). however, in addition to the refractile bodies of cooperia spp., the head of the l3 of os. ostertagi is considerably more squared than that of cooperia spp. or h. placei. in contrast, the ste of cooperia oncophora in cattle is ± 3 ‘x’, more closely resembling the l3 of h. placei than that of os. ostertagi. another distinguishing feature of c. oncophora is that the caudal tip of the sheath of c. oncophora is clearly perceptible, whereas that of c. punctata ‘appears to vanish into nothingness’ (borgsteede & hendriks 1974), or is ‘refractile in appearance’ (anonymous 1977). borgsteede and hendriks (1974) also describe the head of the l3 of c. oncophora to be somewhat broader than that of c. punctata, although we have found this difference to be difficult to visualise. figure 13: morphology of the cranial extremities (heads) of free-living nematodes: (a) tylenchida, (b) dorylaimida and (c) rhabditida, depicting, (i) stoma, (ii) stylet, (iii) oesophagus, (iv) valve and (v) nerve ring. figure 14: organogram for identifying gastrointestinal nematode and lungworm larvae of small ruminants, including a general description of free-living nematodes. figure 15: organogram for identifying gastrointestinal nematode and lungworm larvae of cattle. notably, as reviewed by van wyk et al. (2004), the mean lengths of the stes of c. oncophora l3 cultured from sheep differ significantly from those from cattle. the respective lengths are about 73 mm (2.4 ’x‘, table 1) and 94 mm (3.1 ’x‘, table 2), with corresponding ranges of 62 mm – 82 mm and 65 mm – 116 mm, hence with little overlapping in the lengths of their stes, as confirmed by the fact that hansen and shivnani (1956) recorded only one measurement of less than 79 mm for c. oncophora from cattle and dikmans and andrews (1933) recorded a maximum of 82 mm for this species from sheep. the cooperia curticei l3 of sheep has an ste more closely resembling that of c. pectinata and c. punctata than that of c. oncophora, but it is important to note that, as mentioned, the ste of the latter is reported to be shorter in small ruminants than in cattle (see above). the hookworms, bunostomum spp. and gaigeria pachyscelis hookworm l3 (b. phlebotomum in cattle, and bunostomum trigonocephalum and g. pachyscelis in sheep and goats) are characteristically small in size, being almost 100 mm less in total length than those of h. contortus, the second shortest of the common species or genera. they stain uniformly dark with iodine, in contrast to the l3 of other nematodes that stain light brown cranially and dark brown caudally shortly after addition of the iodine. the stes vary from 2.4 ’x’ for b. phlebotomum l3 to 4.4 ’x‘ for g. pachyscelis, each with a filament comprising about 50% of the ste. the oesophagus has a prominent bulb caudally that is most easily observed in the live, active l3, but difficult to visualise after any but very short periods of staining with dilute iodine solution. the l3 of the following three genera are set apart from the rest by having conspicuously long stes. oesophagostomum spp. and chabertia ovina helminths of these two genera occur widely disseminated in the world, but ch. ovina occurs much less commonly than the other. whilst in small ruminants oesophagostomum venulosum occurs in mediterranean-type climate and oesophagostomum columbianum under warmer subtropical and tropical conditions, in cattle oesophagostomum radiatum is practically universally disseminated. a characteristic of the infective larvae of both genera is that they have relatively long stes (table 1 and 2). however, note that the filament of l3 of oesophagostomum spp. has been observed occasionally to break off, resulting in a much different appearance (mönnig 1931; j. van wyk pers. obs., 1996). whilst apparently not as yet reported for other species, presumably this is also a possibility and should be kept in mind for both ch. ovina and some others, such as l3 of nematodirus spp. with long ste filaments. the l3 of oesophagostomum and chabertia spp. resemble one another so closely that they are described as practically indistinguishable by some authors. however, much of the confusion appears to have resulted from misidentification in the first place, because the numbers and appearance of the intestinal cells, as well as distinguishing features of the stes present ready methods of differentiation. number of intestinal cells: whilst, in some instances, both oesophagostomum and chabertia genera are described and/or depicted as having 32 rectangular intestinal cells or as being indistinguishable (anonymous 1977; eckert 1960; gibbons et al. 2012), in others where only oesophagostomum spp. are described, they are listed as having or perhaps having up to 32 cells (mönnig 1931). in contrast, dikmans and andrews (1933) and borgsteede and hendriks (1974) correctly describe ch. ovina to have about 32 and oesophagostomum spp. about 20 intestinal cells, although the latter authors were uncertain whether some l3 of oesophagostomum could have up to 32 cells. unfortunately it is usually only in the very newly developed infective larvae that the shapes of the intestinal cells are clearly discernable, but even if not, it is sometimes possible to count the cells by examining the granular cell content under relatively high magnification. in addition, a further method has been found to illustrate the number of cells per genus very effectively; when exsheathed l3 of oe. columbianum and ch. ovina are frozen in liquid nitrogen and subsequently thawed, a bubble can generally be seen in each intestinal cell, very effectively differentiating the two genera (figures 10 and 11, re-photographed from van wyk 1977). the photographs clearly illustrate the difference in numbers of cells, being 18–22 in oe. columbianum and 28–32 in ch. ovina. intestinal cell shape: the intestinal cells of the l3 of the two genera under discussion also differ dramatically in shape, being triangular in oesophagostomum spp. and rectangular brick-shaped in ch. ovina (figures 4, 10 and 11). however, as discussed, the outlines and thus shapes of the cells are frequently not discernable in any but newly developed larvae. sheath tail extension filament: it is in the ste that the most consistent means of differentiation of oe. columbianum and ch. ovina l3 lies, not in its length, but mainly in the proportion of the length comprising the filament, being about 50% – 60% in the former and 25% in the latter (table 1). hence, despite it being difficult to pinpoint the exact point of inflexion between the filament and the cranial, non-filamentous portion of the ste, the margin of error resulting therefrom is small enough to allow accurate discrimination. in summary, oesophagostomum spp. and ch. ovina l3 respectively have ± 18–22 and 28–32 triangular intestinal cells, the lengths of the ste are ± 5 ‘x‘ and 4 ‘x’ and the filaments constitute from 40% to 70% of the ste by species in the former and 25% in the latter (figures 5 and 7; tables 1 and 2). nematodirus spp. the commonly encountered species are n. spathiger, nematodirus filicollis and nematodirus abnormalis of small ruminants, nematodirus helvetianus of cattle and n. battus of lambs and also calves.when sheathed, nematodirus spp. l3 are conspicuously longer than those of other nematode genera. they have only eight large intestinal cells, a considerably longer ste (except for n. battus) and prominent digital appendages on the tails of the larvae (figure 3). it is seldom necessary to identify l3 of nematodirus spp. in routine faecal cultures because the ova of the genus require at least two weeks to hatch, whilst faecal cultures are usually harvested after 5–7 days and require pre-exposure to low temperatures to be able to hatch (viljoen 1972). however, because nematodirus ova are much larger than, and hence easily distinguished from, those of most other parasitic nematodes, they can be recorded separately in routine faecal egg counts and any nematodirus l3 which may be encountered in cultures, are ignored and not included in the differential larval counts. it is important to note, however, that nematodirus ova can be confused in general appearance with those of m. marshalli, the ova of which are similarly considerably larger than those of most of the parasitic gastrointestinal nematodes (soulsby 1982). also, whilst the eggs of the other common nematodirus species are more or less oval in shape, that of n. battus is considerably smaller and much more oblong, resembling ‘oversize’ eggs of the other common gastrointestinal strongyles. strongyloides papillosus the l3 of this species are exceptionally thin, the oesophagus comprises about 40% of the total length, the tip of the tail is bifid and it has no covering second sheath as do the l3 of the other genera, hence also no ste. at the lower magnifications usually used for routine larva identification, it is not possible to see that the tip of the tail is bifid, but it does have the appearance of an ste, of which the tip of the filament has broken off. misidentification may occur if only the tail of this larva is examined instead of the entire larva, because the disproportionately long oesophagus will be missed and it sometimes seems – mistakenly so – as if it does have an ste. dictyocaulus spp. both dictyocaulus filaria of small ruminants and dictyocaulus viviparus of cattle are ovo-viviparous. hence, their ova hatch in their respective hosts and the l1 are passed in the faeces. for diagnosis, the l1 are recovered by baermannisation, otherwise a few faecal pellets or a blob of faeces can be placed in water in a petri dish and the surrounding water observed under a stereo microscope for larvae migrating out of the faeces. dictyocaulus spp. larvae are small in size and very lethargic, the tail ends caudally in a smoothly rounded tip (‘d’ in figure 12), no ste is discernable and the intestinal cells are indistinct and usually brown in colour. a striking difference between l1 of d. filaria and d. viviparus is a conspicuous bulbous thickening of the sheath on the head of the former that is absent from d. viviparus. similar to the infective larvae of nematodirus spp., larvae of dictyocaulus spp. are seldom encountered in representative numbers in routine larval cultures, but for a different reason; they migrate poorly out of such cultures unless collected as described for the recovery of hookworm larvae. striking photos of d. filaria and d. viviparus larvae can be seen on the website of gibbons et al. (2012). protostrongylid lungworm larvae muellerius, cystocaulus, neostrongylus and protostrongylus spp. are commonly present in sheep faeces in europe (cabaret 1986), whilst varestrongylus pneumonicus, which is found in small ruminants in asia, is recorded only in roe deer in temperate europe. the l1 of the protostrongylids are recovered from faeces using a baermann-derived technique (cabaret, dakkak & bahaida 1980; also various textbooks, e.g. hansen & perry 1994). the morphology of the tails of some of the larvae is shown in figure 12, but persons who encounter these l1 more commonly should study the more detailed descriptions in van wyk et al. (2004) and gibbons et al. (2012). free-living nematodes cultures of faeces picked up from the ground are often contaminated with free-living nematodes, which then tend to dominate faecal cultures to the extent that it is almost impossible to do a differential parasitic nematode larval count. thus it is preferable to collect faeces from the rectum for cultures, or from bags hung on the animal, although contamination has also been described despite the use of faecal collecting bags (van wyk et al. 2004), in which case it was overcome by shearing and thoroughly washing the buttocks of the animals.it is important to note that, even though they often resemble the l3 of parasitic nematodes, most of the free-living nematodes encountered in faecal cultures are adults and not larvae. in contrast with the majority of the parasitic larvae, the commonly encountered free-living nematodes stain uniformly, extremely dark brown with iodine, are relatively thick and cigar-shaped, have a rhabditiform oesophagus (i.e. with two conspicuous bulbs caudally) and a long tail with no covering sheath. however, the genera differ markedly from one another (as briefly indicated by van wyk et al. 2004) and these nematodes differ to such an extent from the parasitic larvae that there should seldom be confusion. conclusion top ↑ there are dwindling numbers of persons trained in ‘conventional’ helminthological techniques, such as larva identification, which is relatively difficult to learn even when persons with the required skill are available as mentors to the inexperienced. in addition, whilst the full range of pure cultures (and thus larvae) of the common gastrointestinal nematodes of both sheep and cattle were generally available previously, few laboratories maintain these any longer. thus, it is essential that every possible aid, such as the newly described use of the proportion of the ste in identification, be available to those who need to learn to differentiate infective larvae obtained by faecal culture. acknowledgements top ↑ this work was supported by the eu ‘parasol’ (eu-fp6) and ‘risc-net’ projects, the latter under the ‘cidlid’ call for applications, funded by a partnership between the biotechnology and biological sciences research council, the department for international development and the scottish government. hennie gerber (now deceased) maintained pure nematode cultures and lynne michael and regina alves were instrumental in preparations. jacques cabaret was the source of information on lungworm larvae, which are described in more detail in van wyk et al. (2004). ian carmichael went to much trouble to locate original material from trials of beveridge, martin and pullman (1985). michael o’callaghan drew the ‘f’ diagram in figure 3. hervé mauleon helped with descriptions of free-living nematodes. ron kaminsky, frank jackson, jacques cabaret, jacques cortet, eric morgan and flip van schalkwyk supplied l3 of a variety of species. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions j.a.v.w. (university of pretoria) was responsible for developing the novel approach to morphological identification of infective larvae of the common gastrointestinal nematode genera of small ruminants and cattle and wrote the manuscript. e.m. 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identification of nematodes of small ruminants and cattle simplified’, veterinary parasitology 119, 277–306. http://dx.doi.org/10.1016/j.vetpar.2003.11.012, pmid:15154594 viljoen, j.h., 1972, ‘morphology of the free-living stages of nematodirus species with some observations on their development under laboratory conditions’, journal of the south african veterinary association 43, 87–94. pmid:4680366 article information authors: stuart gordon1 charlotte bolwell1 chris rogers1 alan guthrie2 forgivemore magunda3 petronella hove3 affiliations: 1institute of veterinary, animal and biomedical sciences, massey university, new zealand2faculty of veterinary science, university of pretoria, south africa 3faculty of veterinary science, university of zimbabwe, zimbabwe correspondence to: stuart gordon postal address: private bag 11 222, palmerston north 4442, new zealand dates: received: 19 feb. 2013 accepted: 11 apr. 2013 published: 24 may 2013 how to cite this article: gordon, s., bolwell, c., rogers, c., guthrie, a., magunda, f. & hove, p., 2013, ‘descriptive epidemiology of african horse sickness in zimbabwe’, onderstepoort journal of veterinary research 80(1), art. #578, 5 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.578 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. descriptive epidemiology of african horse sickness in zimbabwe in this original research... open access • abstract • introduction • materials and methods    • selection of veterinary surgeries, data collection and analysis • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a study of the prevalence of african horse sickness in horses was conducted, using records from two private equine practices in harare for the period 1998–2004. results indicated a higher prevalence of the disease in horses in zimbabwe in the late rainy season (march – may). age of the horse was found to be a significant risk factor, with foals or yearlings appearing to be 1.80 times more likely to contract the disease compared with horses older than two years. the case fatality rate in foals or yearlings was also higher than in older age groups, but this difference was not significant. the vaccination status was an important risk factor, with vaccinated horses 0.12 times less likely to die from the disease compared with unvaccinated horses. young, unvaccinated horses therefore seem to be the most susceptible to the disease and have greater chances of fatality. this study highlights the importance of adequately protecting horses against african horse sickness by providing immunisation through vaccination and discusses the need to review current vaccination strategies being practiced in zimbabwe. introduction top ↑ african horse sickness (ahs) is an infectious and non-contagious disease, affecting all species of equidae, caused by a double-stranded rna orbivirus (calisher & mertens 1998). ahs was first recognised in southern africa, with the first major outbreak recorded in 1719, when more than 1700 animals died (theiler 1921 in mellor & hamblin 2004). although endemic in equidae in sub-saharan africa, outbreaks have also been recorded in north africa, the middle and near east and southern europe (maclachlan & guthrie 2010). both ahs and bluetongue viruses are transmitted by the biting midges culicoides. culicoides imicola is the field vector of ahs virus although culicoides bolitinos has been shown to play an important role in the transmission of ahs virus in the cooler upper highlands of south africa (meiswinkel & paweska 2003). the highest incidence of the disease usually occurs in the late summer and early autumn in years when the climatic conditions favour an abundance of culicoides midges (coetzer & guthrie 2004). the distribution of ahs virus is governed by a number of factors, including the efficiency of control measures, the presence of long-term vertebrate reservoirs and the occurrence of the culicoides vectors (mellor & hamblin 2004). it was shown by wellby et al. (1996) and mellor and hamblin (2004) that the virogenesis and infection rates of the culicoides vectors were directly proportional to an increase in temperature, but that the survival rates of culicoides were inversely related to temperature. this suggests that transmission should be possible only at certain times of the year, giving the disease the same seasonality as the vector species. previous studies conducted in zimbabwe have shown the disease is usually diagnosed in late summer and early autumn, with most cases occurring towards the end of the rainy season (march – may) (blackburn & swanepoel 1988a). in contrast, henning (1956) found the disease occurred slightly earlier in south africa, starting in february and declining sharply with the first frosts in may. the relationship between the fatality rate in horses due to ahs compared with the age of the affected horse and the immunity status induced by vaccination was studied in zimbabwe by blackburn and swanepoel (1988a). the authors reported 207 cases of clinically diagnosed ahs, with 107 deaths, between 1975 and 1980. furthermore, 56 of these cases, with 50 deaths, were recorded in foals. erasmus (1978) claimed, however, that the age, sex and body condition of the host animal have no influence on the susceptibility to ahs in naïve horses. ahs cases have been recorded in horses that were vaccinated, suggesting that vaccination does not provide adequate protection (blackburn & swanepoel 1988a). however, these findings were reported when the vaccine used in zimbabwe contained only ahs virus serotypes one to six, unlike the freeze-dried, polyvalent, modified live, attenuated ahs virus vaccine (onderstepoort biological products®) used today. the current vaccine contains serotypes one to four and serotypes six to eight. experimental studies have shown that antibody decline in foals with maternally derived immunity occurs between two and four months after birth (blackburn & swanepoel 1988b). more recently, crafford et al. (2013) reported that maternal antibodies to ahs virus acquired passively by foals born to vaccinated mares varied markedly, with further variation noted in the duration of passive immunity to individual ahs virus serotypes. this raises concern over the general belief amongst horse owners and equine veterinarians in zimbabwe that foals with maternally derived immunity should only be vaccinated when between six and nine months of age. reliable data on ahs in horses are available readily from private practitioners, although, since 1998, reporting of the occurrence of this notifiable disease by veterinarians to government veterinary officers has been erratic (o. kakono pers. comm., 09 november 2006). this has resulted in a gap in the knowledge about the disease status in the horse population across the country. the objectives of this study were to determine the practice prevalence of ahs in the harare region and the distribution of ahs cases by year, season, age, sex, and vaccination status. the effect of age and vaccination status on case fatality was also investigated. materials and methods top ↑ selection of veterinary surgeries, data collection and analysis a convenience sample of two private veterinary surgeries in harare was selected, based on reliable record keeping and a large equine caseload covering a wide practice area. records of ahs cases available at the two private veterinary surgeries, for the period 1998–2004, were used for this study. the population of horses at risk was 16 987, as determined from the annual horse census of the central veterinary laboratories of zimbabwe and from data provided by the horse society of zimbabwe, the mashonaland turf club, the thoroughbred association of zimbabwe, the polo association of zimbabwe and the polo x association of zimbabwe. horses included in this population were from the districts of marondera, harare and goromonzi, which represented the practice area of the two veterinary practices.diagnosis of ahs was based on clinical signs and post-mortem lesions recognised by the veterinarian or during laboratory diagnosis. the laboratory diagnosis was based on the antigen-capture enzyme-linked immunosorbent assay (elisa) as described previously by hamblin et al. (1991). no repeat cases of ahs in individual horses were recorded in any year. a total of 8529 horses, representing 50.2% of the 16 987 horses in the study area, were attended to by the two private practices during the study period. of the 8529 horses attended to, 980 (11.5%) were foals or yearlings (< two years of age). data were extracted from each case record, including the date of diagnosis, age, sex, vaccination status and disease outcome of each affected horse. the diagnosis date was used to determine the annual and monthly cases and the seasonality of the disease. the following seasonal categories were generated: early rainy season (december – february), late rainy season (march – may), cold dry season (june – august), and the warm dry season (september – november). age of the case horses was categorised into three groups: foals or yearlings (< two years of age), subadults (two to five years of age) and adults (> five years of age). the annual practice prevalence and annual case fatality rates for ahs were calculated for the years 1998–2004. the practice prevalence and case fatality rates for ahs, stratified by season, year and age, were calculated over the whole study period. the effect of age on the odds of contracting ahs was evaluated by calculating the odds ratio (or) and 95% confidence intervals (ci). the or was also calculated for the effect of age and the effect of vaccination status on the odds of fatality due to ahs. results top ↑ the practice prevalence of ahs during the study period was 1.96% (167/8529). the average annual number of ahs cases recorded was 24, with the highest practice prevalence recorded in 2003 and the lowest in 1998 (table 1). table 1: the annual practice prevalence of african horse sickness in horses recorded by two harare equine practices for the period 1998–2004. across all years, the highest numbers of ahs cases were recorded from march (16.2%; 27/167) to june (18.0%; 30/167), with peak numbers reached in april (21.0%; 35/167) and may (31.7%; 53/167). from july onwards, the number of cases decreased dramatically, with no cases recorded in august and november. the late rainy season recorded the highest number of cases (68.9%; 115/167), whilst the lowest number of cases (3.6%; 6/167) was recorded in the warm dry season (table 1). male horses accounted for 48.5% (81/167) of the ahs cases recorded, whilst female horses accounted for 36.5% of the cases (61/167). no sex identification was recorded in the medical records of 25 horses. the highest numbers of ahs cases were recorded in adult horses and subadults, with these groups accounting for 40.7% (68/167) of the total ahs cases recorded. the lowest number of cases (18.6%; 31/167) was recorded in foals or yearlings. foals or yearlings recorded a higher prevalence of ahs compared with horses older than two years (table 1). the odds of ahs developing were 1.8 times greater in foals or yearlings (95% ci: 1.3–2.9) compared with horses older than two years. the majority of the horses with ahs had previously been vaccinated against ahs (85%; 142/167). the average annual number of fatalities due to ahs was 9.6, with the highest case fatality rates recorded in 1998 and 1999 and the lowest in 2004 (table 1). foals or yearlings recorded the highest overall case fatality rate, followed by adults and subadults. the odds of foals or yearlings dying from ahs were 1.8 times greater (95% ci: 0.90–4.30) compared with horses older than two years, although the odds were not significantly different. unvaccinated horses recorded a higher overall case fatality rate compared with vaccinated horses (table 1). higher case fatality rates were recorded for unvaccinated foals or yearlings, subadults and adult horses compared with their respective vaccinated age categories (table 2). unvaccinated subadults had the highest case fatality rates, followed by unvaccinated adults and unvaccinated foals (table 2). the odds of vaccinated horses dying from ahs were 0.12 times less likely (95% ci: 0.044–0.350) compared with unvaccinated horses. table 2: case fatality rates for african horse sickness in vaccinated and unvaccinated horses according to age group. discussion top ↑ the prevalence of ahs in zimbabwe recorded during the present study was lower than in previous reports (blackburn 1982; blackburn & swanepoel 1988a; musuka 1999). blackburn and swanepoel (1988a) recorded 207 cases of ahs from 1654 horses studied in zimbabwe in 1980, representing a 12% prevalence compared to a 1.96% prevalence found in this study. this could possibly be attributed to improved vaccination and control measures practiced in the country during this current study period. it is also important to note that the prevalence of ahs recorded in this study may be an underestimation of true values, as results are based on a convenience sample taken from two veterinary surgeries only. not all cases of ahs in the horse population, within the catchment area under study, may have been presented to these two surgeries.the prevalence of ahs was higher in years that experienced early rains followed by a dry spell (rainfall data supplied by the dept. of meteorological services, harare) and there was a definite seasonal distribution, with most cases being diagnosed between late summer and autumn (february – june). these findings concurred with observations from earlier studies in zimbabwe (blackburn & swanepoel 1988a). the seasonality of the disease is also affected by vector patterns. the infection rates of the vector, culicoides, and the rate of virogenesis increase with increasing environmental temperature although the rate of adult fly mortality may also increase (mellor & hamblin 2004; wellby et al. 1996). culicoides imicola, however, breed only in wet or damp areas, which may explain why ahs cases are low in the warm, dry months despite high environmental temperatures favouring increased rates of virogenesis and infection (baylis, mellor & meiswinkel 1999). it has been proposed that a strong link exsits between major outbreaks of ahs in southern africa and the warm phase of the el niño–southern oscillation (enso) (baylis et al. 1999). it has been suggested that this link is due to the combination of heavy rain and drought that the enso brings to southern africa, creating breeding conditions that allow the culicoides vector to increase more than 200-fold (baylis et al. 1999; meiswinkel, nevill & venter 1994; mellor & hamblin 2004). the results of the present study support this suggestion, as the highest practice prevalence (3.67%) was recorded in 2003 when a large enso was reported. the lowest practice prevalence (0.57%) was recorded in 1998 when the enso was weak and a moderate la niña–southern oscillation was reported (national institute of water and atmospheric research 2013). furthermore, it has been predicted by wittmann and baylis (2000) that the distribution of c. imicola will become more widespread with increasing global temperatures, bringing the ahs virus into the range of other culicoides species that are known to be competent vectors of this disease. the results of the present study showed that the age of the horse was associated with the occurrence of ahs in this population, with foals or yearlings having greater odds of developing the disease than older horses. these findings concur with earlier observations in zimbabwe (blackburn & swanepoel 1988a), although erasmus (1978) claimed that age had no influence on susceptibility to the disease. as supported by the findings of this study, age susceptibility and survival may be linked to the immune status of a horse due to vaccination. the response to vaccination is therefore important, as the immune status of the horse modifies the form of the disease and thus the associated mortality. however, despite high levels of horse vaccination in zimbabwe, a high prevalence of ahs was recorded. a high prevalence of ahs in vaccinated horses in the country was also reported previously (blackburn & swanepoel 1988a). the polyvalent vaccine in use at the time of that study (1980) included attenuated strains of serotypes one to six only (blackburn & swanepoel 1988a). in the present study, however, cases still occurred in vaccinated horses, although the polyvalent vaccine used now also includes serotypes seven and eight. furthermore, weyer et al. (2013) showed that horses immunised against ahs virus can be infected both clinically and subclinically with ahs virus following natural infection in field conditions, with 16% of immunised horses being infected over a two-year period. the higher case fatality rates recorded in foals or yearlings may be due to incomplete protection against the ahs virus after the initial vaccination, which has been attributed to interference between the virus serotypes (coetzer & guthrie 2004). some strains of vaccine may also be immunogenically weak and several courses of vaccination may be required to achieve full immunity to the ahs virus, thus making foals or yearlings that have not yet received multiple vaccinations more susceptible (laegreid 1996). experimental studies conducted by blackburn and swanepoel (1988a) showed that multiple vaccinations with attenuated strains of ahs serotypes one to six led to a broader response to various serotypes and to a higher individual serotype titre. however, it has been highlighted that although protective responses to all the virus serotypes in the polyvalent vaccine may be achieved only after repeated immunisation, excessive administration of vaccine over many years appears to lead to a state of immunological unresponsiveness or hypersensitivity (erasmus 1978). this could explain why there is often an increase in cases of ahs in older horses. improper storage of the vaccine or incorrect administration of the vaccine may also contribute to vaccine failure. crafford et al. (2013) reported a mean half-life of only 20.5 days for passively acquired maternal neutralising antibody to ahs virus recorded in six foals in an endemic area in the western cape province of south africa. blackburn and swanepoel (1988b) showed that passively acquired antibodies in foals against individual ahs serotypes declined to undetectable levels between two and four months of age. furthermore, the vaccination of foals between three and four months of age resulted in a weak antibody response, which did not adversely affect pre-existing low levels of maternal antibody (blackburn & swanepoel 1988b). thus, foals from vaccinated dams may succumb to ahs as early as at three months of age and immunisation at an earlier age could therefore help to control foal susceptibility to and mortality from ahs. the effectiveness of the current vaccination strategies against ahs used in zimbabwe may, therefore, require revision. the vaccination of younger foals with maternally derived antibody against some serotypes of ahs could be considered instead of the current practice of vaccinating foals only older than six months in zimbabwe. the sex of the horse was not adequately investigated as a possible risk factor as the sex composition of the original population was unknown, thus necessitating the need for further studies in this area. conclusion top ↑ the age of the horse was found to be significantly associated with ahs, with foals or yearlings more likely to contract the disease compared with horses older than two years. the ahs case fatality rate in foals or yearlings was also found to be higher than in older age groups. furthermore, the vaccination status was found to be an important risk factor, with vaccinated horses less likely to die from ahs compared with unvaccinated horses. it appears, therefore, that young, unvaccinated horses seem to be the most susceptible to ahs and that they suffer greater chances of fatality. this reinforces the importance of adequately protecting horses against ahs through vaccination, highlighting the need to review current vaccination strategies being practised in zimbabwe. acknowledgements top ↑ the authors would like to acknowledge the cooperation and support of the two private practices in harare used for data collection in this study. the authors are also grateful for the information provided by the central veterinary laboratories of zimbabwe, the horse society of zimbabwe, the mashonaland turf club, the thoroughbred association of zimbabwe, the polo association of zimbabwe and the polo x association of zimbabwe. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions xxs.g. 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gholamreza razmi1 saeed yaghfoori1 affiliations: 1department of pathobiology, ferdowsi university of mashhad, iran correspondence to: gholamreza razmi postal address: po box 91775-1793, mashhad, khorasan razavi, iran dates: received: 29 june 2013 accepted: 07 aug. 2013 published: 02 oct. 2013 how to cite this article: razmi, g. & yaghfoori, s., 2013, ‘molecular surveillance of theileria ovis, theileria lestoquardi and theileria annulata infection in sheep and ixodid ticks in iran’, onderstepoort journal of veterinary research 80(1), art. #635, 5 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.635 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. molecular surveillance of theileria ovis, theileria lestoquardi and theileria annulata infection in sheep and ixodid ticks in iran in this original research... open access • abstract • introduction • material and methods    • field study    • blood samples    • microscopy of blood smears    • tick examination    • semi-nested pcr    • polymerase chain reaction-restriction fragment length polymorphism    • sequencing • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a molecular study was undertaken to detect theileria ovis, theileria lestoquardi and theileria annulata in sheep and tick vectors. investigation was conducted from 2010 to 2011 in the south of khorasan razavi province, iran. a total of 150 blood samples were collected from 30 different sheep flocks. in addition, ixodid ticks were sampled from the same flocks. the stained blood smears were microscopically examined for the presence of piroplasms and a semi-nested polymerase chain reaction-restriction (pcr) was used for subsequent molecular speciation. salivary glands were isolated from the ticks and subsequently analysed by semi-nested pcr. polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) was used to differentiate between t. lestoquardi and t. annulata from pcr-positive samples. theileria species infection was microscopically detected in 18.6% of blood smears. the presence of t. ovis and t. lestoquardi or t. annulata was detected by semi-nested pcr in 58.6% and 6.6% of blood samples respectively. in total, 169 ixodid ticks were collected from different areas of the province. the most prevalent ticks were rhipicephalus turanicus (n = 155; 91.7% of the total), followed by hyalomma anatolicum anatolicum (n = 8; 4.7%) and hyalomma marginatum turanicum (n = 6; 3.5%). from an organ pooling of 33 ticks, three pools of salivary glands from r. turanicus were positive for theileria species by semi-nested pcr. of the three r. turanicus samples testing positive for theileria species, two (6.1%) were positive for t. ovis and one (3.0%) for t. lestoquardi or t.annulata. amongst the 11 pcr-positive samples for t. lestoquardi or t. annulata, 10 were positive for t. lestoquardi and one sample was positive for both t. lestoquardi and t. annulata using pcr-rflp. the results also demonstrated that pcr-rflp could be used for the detection of t. ovis. based on the results, it can be concluded that t. ovis has a higher prevalence than t. lestoquardi, and that r. turanicus could be a possible vector for t. ovis and t. lestoquardi. finally, the pcr-rflp based on msp1 restriction enzyme is a simple method for differentiation of theileria species in sheep and ixodid ticks. introduction top ↑ at least six theileria species have been found to infect small ruminants. of these, theileria lestoquardi, theileria luwenshuni and theileria uilenbergi are considered to be highly pathogenic in sheep and goats. the other three species, theileria separata, theileria ovis and theileria recondita, are generally considered to be non-pathogenic or mildly pathogenic (perston 2001; schnittger et al. 2000; uilenberg 1995, 1997). theileria annulata, which is a causal agent of malignant theileriosis in cattle, can also infect sheep (leemans et al. 1999). theileria species have been successfully identified in sheep by using different molecular methods based on the hyper-variable v4 region of 18s rrna genes. the available primers based on 18s rrna genes have also cross-reacted with t. annulata and t. lestoquardi (gubbels et al. 1999; schnittger et al. 2004). it is necessary to differentiate between t. lestoquardi and t. annulata because both pathogens are transmitted by hyalomma anatolicum anatolicum and both can be found in small ruminants (spitalska et al. 2005). different molecular methods such as polymerase chain reaction (pcr) (kirvar et al. 1998), polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) (heidarpour et al. 2009; spitalska et al. 2005) and lockwood analytical method for prediction (lamp) (liu et al. 2008; salih et al. 2008) have been developed to differentiate the two theileria species. iran is an endemic region for t. ovis, t. lestoquardi and t. annulata infection in sheep and cattle. hyalomma anatolicum anatolicum is a common tick vector for t. lestoquardi and t. annulata. to date, t. annulata infection has also been reported in sheep from iran (zaeemi et al. 2011). the climate in the southern part of the khorasan razavi province is similar to other areas in iran where ovine theileriosis is present. in addition, many suspected cases of ovine theileriosis have been reported by local veterinary services in this area. the aim of the study is the molecular identification and differentiation of theileria species in sheep and tick vectors from these areas. material and methods top ↑ field study the study was conducted in the southern areas of the khorasan razavi province, which is located in the east of iran (figure 1). an approximate population of 1 493 838 sheep is distributed throughout this area. sheep husbandry is one of the most economically important occupations in this area. sheep and goat flocks are kept together and the average herd size is between 200–1000. the sheep outnumber goats and the ratio is reported to be 50–900 sheep and 30–200 goats in each flock. the climate is semi-arid, with cold winters and warm summers, and there is an average annual rainfall of 167 mm. the mean annual temperature ranges from 5.5 °c – 23.0 °c. figure 1: locality where the fieldwork was carried out in khorasan razavi province, iran. blood samples flocks with a clinical history of theileriosis in five different areas in the south of the khorasan razvai province during 2010–2011 were selected for this study. five sheep from each of these flocks were then randomly selected. blood was drawn from the jugular vein and collected in edta tubes. simultaneously, the body of each animal was inspected, and if ticks were found, they were placed into appropriately labelled vials. the blood and tick specimens were refrigerated during transport to the laboratory. microscopy of blood smears blood smears were air dried, fixed in methanol, and stained with 10% giemsa solution in phosphate-buffered saline (pbs), ph 7.2. the slides were microscopically examined using an oil immersion lens at 1000× magnification. tick examination the ticks collected from each infested sheep were counted and identified by microscopy using appropriate keys (estrada-pena et al. 2004; hoogstraal 1956; walker et al. 2003). the ticks were grouped into pools of five, according to their species and sex. the salivary glands from each tick pool were dissected and placed in 0.85% saline. the ticks and isolated organs were all stored at -20 °c. semi-nested pcr total genomic dna was extracted from edta blood and tick samples using a dna isolation kit (cinna gene, tehran, iran). semi-nested pcr was performed according to the method of shayan and rahbari (2005). in the first round of amplification, a forward primer (p1), 5′-cacagggaggtagtgacaag-3′, and a reverse primer (p2), 5′-aagaatttcacctatgacag-3′, were used to yield an amplification product, which allowed differentiation between theileria and babesia. amplification was conducted in 20 μl reaction volumes (accupower pcr premix kit, bioneer, seoul, south korea) with a final concentration of 250 μm of each dntp in 10 mm tris-hcl, ph 9.0, 30 mm kcl, 1.5 mm mgcl2, 1 u taq dna polymerase, and 10 pmol of each pcr primer (takapouzist co., tehran, iran). one microlitre of dna template was added to each reaction mixture and water was added up to a total volume of 20 μl. the reactions were subjected to the following cycling conditions using the bio-rad thermocycler: 95 °c for 5 min, 36 cycles with denaturing at 94 °c for 45 s, primer annealing at 54 °c – 58 °c for 45 s, and an extension step at 72 °c for 45 s, followed by final extension at 72 °c for 10 min. the products were refrigerated at -4 °c. the pcr products were separated by electrophoresis on a 1.5% agarose gel with tbe buffer and visualised using ethidium bromide and a uv-illuminator (vilber lourmat, germany). visible bands at 426 base pair (bp) – 430 bp for theileria species were produced in the first round of pcr. in the second round, amplification by internal primers was used to specifically detect t. ovis (p2/p4) and t. lestoquardi/t. annulata (p2/p3) as shown in table 1. the pcr products were also separated using electrophoresis on a 1.5% agarose gel to determine the presence of specific bands for t. ovis and t. lestoquardi/t. annulata. for positive controls, t. annulata and t. lestoquardi gdna (provided by the razvi vaccine and serum research, karaj branch, karaj, iran), and t. ovis gdna (provided by dr parviz shayan, parasitology dept., faculty of veterinary medicine, tehran university, tehran, iran) were used. a sample containing sterile water was used as a negative control and simultaneously as a control for contamination in each pcr amplification. table 1: the sequences of primers used in semi-nested polymerase chain reaction. polymerase chain reaction-restriction fragment length polymorphism theileria. annulata and t. lestoquardi were differentiated by polymerase chain reaction-restriction (pcr-rflp) analysis of the first round of semi-nested pcr products with msp1 enzyme (mbi fermentas, lithuania) according to the method of spitalska et al. (2004). this involved mixing 10 μl pcr products (0.5 μg – 1 μg), 18 μl nuclease-free water, 2 μl 10× buffer tango, 1 μl msp1 in 31 μl total volume. the reaction was incubated at 37 °c for 6 h. five microlitres of restricted samples were transferred to a 1.5% agarose gel with a 100 bp dna ladder (mbi fermentas, lithuania) for fragment size determination. dna fragments were thereafter separated by horizontal electrophoresis in 1 × tae buffer at 80 v for one hour. the gel was stained using commercial ethidium bromide. the restriction analysis patterns for theileria species are shown in table 2. the msp1 restriction enzyme was able to cut amplified products from t. ovis to distinguish it from theileria species. table 2: results of restriction enzyme on nucleotide sequences of different theileria species in sheep. sequencing selected positive products were sequenced in the facilities of bioneer inc. (seoul, south korea). sequences were analysed by using ncbi blast, national institute of health, usa ( http://www.ncbi.nlm.nih.gov). results top ↑ theileria species infection was microscopically detected in 18.6% of blood smears with low parasitic infections (0.2% – 0.01%), whilst semi-nested pcr findings revealed that 58.6% (n = 88) of blood samples were positive for t. ovis and 6.6% (n = 10) for t. lestoquardi or t. annulata (table 3). the results also indicated that all positive samples from blood smears were positive when using semi-nested pcr, whereas no piroplasms were seen by microscopy in 70 pcr-positive animals. in total, 169 ixodid ticks were collected from different areas of the province. the most common tick was r. turanicus (n = 155; 91.7% of the total). other ticks included h. a. anatolicum (n = 8; 4.7%) and hyalomma marginatum turanicum (n = 6; 3.5%). from a total of 33 tick organ pools, three pools of salivary glands from r. turanicus were positive for theileria species by semi-nested pcr. of these positive samples, two (6.1%) were positive for t. ovis dna and one (3%) for t. lestoquardi or t. annulata dna (table 4). pcr amplification using the first primer pair produced a major product between 426 and 430 bp for theileria dna. the amplified product of samples positive for t. lestoquardi or t. annulta in the first round of semi-nested pcr was further analysed by pcr-rflp. mspi digestion gave very distinct profiles that differentiated t. annulata and t. lestoquardi. the results of enzymatic digestion (n = 10, 90.9%) by mspi showed t. lestoquardi and mixed infection (n = 1, 9.1%) with t. annulata and t. lestoquardi (figure 2). this method also showed that the enzyme modified is able to distinguish t. ovis (figure 3). the sequencing results also confirmed the pcr-rflp results. the sequenced pcr products were found to be 100% identical to the t. lestoquardi 18s rrna genbank references (jq917458.1, af081135.1, kc778786.1, kc778785.1) by blast analysis. table 3: molecular and microscopic detection of theileria species in blood samples of sheep in the south of the khorasan razavi province. table 4: frequency of tick infestation with the results of molecular methods for detection theileria species in salivary glands of ixodid ticks. figure 2: restriction digests of theileria species amplification products. figure 3: comparison of restriction digests on theileria species amplification products. discussion top ↑ the ovine piroplasm prevalence of 18.6% detected in this study population was higher than that reported in a previous study in the southern areas of the khorasan razavi province of iran (razmi, eshrati & rashtibaf 2006). however, in contrast to previous studies, the current strategy to collect blood samples was focused on flocks with a known history of disease. in general, the infections detected in the blood smears were low and the morphological differentiation between different theileria species was not possible. molecular tools provide complementary diagnostic information, and accordingly, semi-nested pcr was used to achieve a simultaneous differentiation between different theileria species in sheep (shayan & rahbari 2005). with this molecular approach, 58.6% and 6.6% of blood samples were dna-positive for t. ovis and t. lestoquardi /t. annulata, respectively. the results demonstrated a much higher diagnostic sensitivity using pcr when compared to the conventional microscopic examination of blood smears. our results also indicated that many of the sheep might serve as a reservoir for infection, with a very low parasitaemia that cannot be detected by microscopy. in a previous study, spitalska et al. (2005) detected t. lestoquardi infection in 76.0% of sheep with appropriate clinical signs in the fars province (in southern iran) using pcr and dna sequencing techniques. heidarpour et al. (2009) reported that 12.5% and 87.5% of sheep examined in the eastern half of iran were positive for t. ovis or t. lestoquardi, respectively. zaeemi et al. (2011) also detected t. ovis in 40.2% and t. lestoquardi in 54.8% of sheep in the western half of iran. a comparison of these observations and this study’s results indicate that the frequency of t. ovis infection in the khorasan razavi province is higher than that in any other areas of iran, and that the t. lestoquardi infection is lower. in the present study, climatic factors may have influenced the differences in the frequency of piroplasm infection in the north when compared to the south of the province. a high prevalence of t. ovis has also been reported in some areas of turkey (aktas, altay & dumanli 2007) and china (ge et al. 2012). during this study, the infected sheep did not show any clinical signs, whereas highly fatal disease due to t. lestoquardi has been reported in sheep in the south and southeast of iran (hashemi-fesharaki 1997). the low incidence of clinical cases seen in the current study may be due to the low levels of infection with t. lestoquardi. three tick species were identified in sheep in the south khorasan razavi province. rhipicephalus turanicus was the dominant tick species in this area; it has also been reported with high prevalence in sheep from the mountainous areas such as the khorasan province (rahbari, nabian & shayan 2007; razmi, najarnejad & rashtibaf 2011). based on the semi-nested pcr results, t. ovis and t. lestoquradi infections were only detected in a few salivary glands in r. turanicus. molecular analysis revealed that rhipicephalus bursa and rhipicephalus sanguineus could be a possible vector for t. ovis (aktas, altay & dumanli 2006; telmadarraiy et al. 2012). hyalomma anatolicum has been shown to be an important vector for t. lestoquardi in iran and other countries (hooshmand-rad & hawa 1973; kirvar et al.1998; taha & elhussein 2010; uilenberg 1997). in our study, few samples of h. anatolicum were collected and theileria species were not detected by pcr. the low frequency of h. anatolicum has previously been reported for sheep in the khorasan province (razmi et al. 2003; razmi, najarnejad & rashtibaf 2011). the primer for semi-nested pcr is derived from the hypervariable region v4 of 18s rrna, and it detects and differentiates simultaneously theileria and babesia in dna samples from sheep (shayan & rahbari 2005). however, this technique cannot differentiate between t. annulata and t. lestoquardi. the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) can easily discriminate between t. lestoquardi and t. annulata by a single restriction digest (spitalska et al. 2004). furthermore, 91% of positive samples for t. lestoquardi/ t. annulata were differentiated as t. lestoquardi and 9% were found to have a mixed t. annulata and t. lestoquardi infection. this technique also shows that the method is applicable for t. ovis differentiation. recently, different theileria species in sheep were detected by nested pcr-rlfp (heidarpour et al. 2009; zaeemi et al. 2011). naturally occurring t. annulata infection has also been reported in sheep in iran and sudan (taha et al. 2013; zaeemi et al. 2011). conclusion top ↑ in summary, our study indicated that microscopic examination of stained blood smears has limited value for differentiation between theileria species, especially with low parasitaemia levels in sheep. the results of molecular examination indicated that t. ovis has a high prevalence when compared to t. lestoquardi in the southern khorasan razavi province. the dominant tick was found to be r. turanicus and it may act as an important vector for t. ovis and t. lestoqaurdi. finally, pcr-rflp based on msp1 restriction enzyme digestion is an applicable method for differentiation between t. lestoquardi, t. annulata and t. ovis species in sheep and tick vectors. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions g.r. 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ticks’, parasitology research 107, 1009–1012. http://dx.doi.org/10.1007/s00436-010-1968-x, pmid:20607288 taha, k.m., salih, d.a., ali, a.m, omer, r.a. & el hussein, a.m., 2013, ‘naturally occurring infections of cattle with theileria lestoquardi and sheep with theileria annulata in the sudan’, veterinary parasitology 191, 143–145. http://dx.doi.org/10.1016/j.vetpar.2012.08.003, pmid:22951302 telmadarraiy, z., oshaghi, m.a., hosseini-vasoukolaei, n., yaghoobi-ershadi, m.r., babamahmoudi, f. & mohtarami, f., 2012, ‘first molecular detection of theileria ovis in rhipicephalus sanguineus ticks in iran’, asian pacific journal of tropical medicine 5, 29–32. http://dx.doi.org/10.1016/s1995-7645(11)60240-x uilenberg, g., 1995, ‘international collaborative research: significance of tick-borne hemoparasitic diseases on world animal health’, veterinary parasitology 57, 19–41. http://dx.doi.org/10.1016/0304-4017(94)03107-8 uilenberg, g., 1997, ‘general review of tick-borne diseases of sheep and goats worldwide’, parasitologia 39, 161–165. walker, a.r., bouattour, a., camicas, j.l., estradapeña, a., horak, i.g., latif, a. et al., 2003, ‘ticks of domestic animals in africa: a guide to identification of species’, bioscience reports, edinburgh, scotland, u.k. zaeemi, m., haddadzadeh, h.r., khazraiinia, p., kazemi, b. & bandehpour. m., 2011, ‘identification of different theileria species (theileria lestoquardi, theileria ovis, and theileria annulata) in naturally infected sheep using nested pcr-rflp’, parasitology research 108, 837–843. http://dx.doi.org/10.1007/s00436-010-2119-0, pmid:20978792 tait_33.indd 33 onderstepoort journal of veterinary research, 76:33 (2009) the genetics of african trypanosomes a. tait wellcome trust centre for molecular parasitology, glasgow university, uk abstract tait, a. 2009. the genetics of african trypanosomes. onderstepoort journal of veterinary research, 76:33 the occurrence of genetic exchange is not only a fundamental property of an organism but its occurrence provides us with methods of analysis that can be used to dissect the basis of important traits. this paper will review the current evidence for a sexual cycle in trypanosoma brucei and illustrate how genetic analysis can be used as a tool to identify genes of relevance to the disease, its treatment and transmission. the role of this process in the generation of diversity in the field will be illustrated by considering populations of the three sub-species of t. brucei and how the availability of genome sequence data has been exploited to study whether genetic exchange occurs in the trypanosomes infecting livestock. finally, the use of population genetics as a methodology to identify genes under selection will be discussed and this will be illustrated as an approach to validating markers for drug resistance with examples from other parasitic protozoa. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information author: christopher j. kasanga affiliation: 1southern african centre for infectious disease surveillance, sokoine university of agriculture, tanzania correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: kasanga, c.j., 2014, ‘the changing landscape of the molecular epidemiology of foot-and-mouth disease virus in southern africa north of limpopo and east africa’, onderstepoort journal of veterinary resear ch 81(2) art. #730, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.730 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. licensee: aosis openjournals. this work is licensed under the creative commons attribution license. the changing landscape of the molecular epidemiology of foot-and-mouth disease virus in southern africa north of limpopo and east africa in this abstracts... open access • abstracts • acknowledgements abstracts top ↑ foot-and-mouth disease (fmd) is endemic in most countries of southern and eastern africa. it affects cloven-hoofed animals that include livestock and wildlife. fmd is caused by fmd virus (fmdv), the single stranded positive sense rna virus, with a high rate of genetic mutation(s). southern and eastern africa relies profoundly on livestock production as a source of economic growth and livelihoods. despite the importance of fmd in africa, the epidemiology of fmdv and factors contributing to the endemicity of fmd in susceptible animal populations are not clearly known in the region. in this study, molecular characteristics of fmd virus detected in southern africa north of limpopo and eastern africa were determined using methods such as vi, antigen-elisa, rt-lamp, real-time rt-pcr, sequencing of the vp1 and phylogenetic analysis. the findings of this study indicate that serotypes a, o, sat 1, sat 2 and sat 3 predominate in both the african buffalo (syncerus caffer) and cattle in the region. furthermore, the performance of rt-lamp discloses its superiority to real time rt-pcr with high potential for the specific detection and surveillance of infectious diseases of humans and animals in the region. in-depth phylogenetic analysis of vp1 sequences reveals the existence unassigned topotype(s) among serotype sat 1 viruses detected from buffalo in mozambique. whole genome sequencing of the virus within the unassigned topotype unravels close genetic relationship to viruses detected in southern africa with 94% closest nucleotide homology to sat1rhod5/66 strain isolated from cattle in zimbabwe in 1966. the presence of multiple serotypes and topotypes, uncontrolled animal movements and the presence of several risk factors for disease occurrence complicate control of fmd in the region. the rt-pcr and whole genome sequencing strategy established in the current study could be deployed to study the evolutionary characteristics of viruses sampled from cattle and buffalo at different locations in time and space. it is suggested that in-depth genome analysis should be conducted to uncover sequence-based evidence for fmdv endemicity in the region. this information is necessary for fmd control strategies in most countries in africa. the genome sequencing approach allows for targeted and cost-effective fmd control strategies in the endemic settings of southern and eastern africa rather than application of blanket and expensive interventions – an important consideration for resource constrained countries in the region. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). article information authors: christopher j. kasanga1 rahana dwarka2 gaothlele thobokwe3 jemma wadsworth4 nick j. knowles4 misheck mulumba5 ezekia ranga6 jimis deve11 cornelius mundia10 patrick chikungwa8 laureta joao9 raphael sallu7 mmeta yongolo7 philemon n. wambura1 mark m. rweyemamu1 donald p. king4 affiliation: 1southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania2onderstepoort veterinary institute, university of pretoria, south africa 3botswana vaccine institute, botswana 4wrlfmd, the pirbright institute, united kingdom 5southern african development community secretariat, botswana 6ministry of livestock development and fisheries, tanzania 7tanzania veterinary laboratory agency, tanzania 8southern african development community, transboundary animal diseases section, malawi 9southern african development community, transboundary animal diseases section, angola 10southern african development community, transboundary animal diseases section, zambia 11southern african development community, transboundary animal diseases section, mozambique correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: kasanga, c.j., dwarka, r., thobokwe, g., wadsworth, j., knowles, n.j., mulumba, m. et al., 2014, ‘molecular biological characteristics of foot-and-mouth disease virus in the african buffalo in southern africa’, onderstepoort journal of veterinary research 81(2), art. #728, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.728 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. molecular biological characteristics of foot-and-mouth disease virus in the african buffalo in southern africa in this abstracts... open access • abstract • acknowledgements abstract top ↑ foot-and-mouth disease (fmd) is endemic in most countries in southern africa. african buffaloes (syncerus caffer) are known to play a significant role in the transmission and dynamics of fmd virus (fmdv) in wildlife-livestock interface areas. the aim of this study was to investigate the serotype and determine the genetic relationships of fmdv recovered from animals in tanzania, zambia and mozambique, and compare them with viruses detected from elsewhere in the sub-saharan region. a total of 150 oesophageal-pharyngeal (probang) samples collected in 2010 from cattle and buffaloes in marromeu (mozambique), katavi (tanzania) and lochninvar (zambia) national parks were used in this study. the presence of fmdv was determined by laboratory methods such as vi, antigen elisa and real-time rt-pcr. phylogenies of vp1 sequences were determined by the neighbour-joining method. the overall fmdv genome detection rate was 6.7% (n = 10), with sat 1 being the most frequent serotype (60%; n = 6) isolated in cattle and buffaloes in mozambique, tanzania and zambia followed by sat 3 (30%; n = 3) and sat 2 (10%; n = 1). genotyping showed that type sat 1 viruses fell into either the topotype 1 (nwz) or unassigned topotypes, type sat 2 into the africa topotype i and type sat 3s into topotype iv (sez). this study reveals that serotypes sat 1–3 are maintained in cattle and buffaloes in livestock-wildlife interface areas in marromeu, katavi, and lochinvar national parks. phylogenetic analysis of fmdv isolates from tanzania, zambia and mozambique showed that they are genetically related to lineages and topotypes from africa. this information contributes to the understanding of the epidemiology of fmd in southern africa. in mozambique, tanzania and zambia, lack of consistent surveillance systems and animal movement controls make it difficult to determine the exact source of fmd and transmission dynamics of fmdv. further studies are needed to elucidate the complex epidemiology of fmd in africa. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). 2193myburgh_cmyk_pp307-316.qxd introduction blackflies were not considered significant pests in south africa but developed pest status along many rivers following the building of dams, canals, irrigation schemes or hydro-electrical plants along those rivers (nevill 1988). this is because impoundments promote the development of suspended organic material and a more constant seasonal flow of water, which in turn creates ideal conditions for immature blackflies (howell & holmes 1969; car 1983; nevill 1988) to increase in numbers in rapids downstream of such structures (chutter 1963; palmer 1991). moreover, these structures change many of the rivers from seasonal to perennial and thus create ideal and continuous breeding conditions for simuliids (myburgh 1999a; b). at present, blackflies are common and significant pests along the orange, vaal, great fish, sundays and gamtoos rivers. periodic outbreaks are also experienced along the eerste, olifants and berg rivers (edwardes & palmer 1994; palmer 1997) (fig. 1). it is expected that blackflies may acquire pest status along the liebenbergsvlei river follow307 onderstepoort journal of veterinary research, 70:307–317 (2003) review of blackfly (diptera: simuliidae) control in south africa e. myburgh1 and e.m. nevill2 abstract myburgh, e. & nevill, e.m. 2003. review of blackfly (diptera: simuliidae) control in south africa. onderstepoort journal of veterinary research, 70:307–317 the medical, veterinary and economic importance of blackflies in south africa, and the historical development of blackfly control programmes in various south african rivers, are reviewed in this paper. in 1996 it was estimated that blackflies can cause more than r 88 million damages per annum along the middle and lower orange river where simulium chutteri is considered the main pest species. a clear link between the construction of dams and the spread of the blackfly problem was shown. four phases characterize the development of blackfly control in south africa: (1) during the 1960s blackflies in the vaal river were controlled with ddt; (2), during the 1970s and into the 1980s blackflies were controlled using water-flow manipulation; (3) when used at strategic times, water-flow manipulation could be used to enhance the effect of natural predator populations; and (4) during the 1990s the organophosphate temephos and toxins produced by the bacterium bacillus thuringiensis var. israelensis were tested for their efficacy against blackflies. the larvicides temephos and b. thuringiensis proved to be effective and are still used in several control programmes. the latest research focuses on the factors that influence adult blackfly survival and annoyance, as well as the development of methods that can be used to protect sheep from blackfly attacks. keywords: bacillus thuringiensis var. israelensis, blackfly, economic importance, orange river, simulium, temephos, water manipulation 1 arc-onderstepoort veterinary institute, blackfly field station, upington, south africa. present address: p.o. box 835, duiwelskloof, 0835 south africa. e-mail: myburg@lantic.net 2 arc onderstepoort veterinary institute, private bag x5, onderstepoort, 0110 south africa accepted for publication 12 june 2003—editor ing the completion of the lesotho highlands water scheme, although so far this has not happened (fig. 1). currently 39 blackfly species are known to occur in southern africa (palmer 1997; palmer & de moor 1998). these include five members of the genus paracnephia and 34 simulium species (palmer 1997). the latter genus includes the mammalian pests simulium chutteri and simulium damnosum sensu latu and the avian pest species simulium adersi and simulium nigritarse sensu latu. the arc-onderstepoort veterinary institute (arcovi) has been involved in the development and implementation of blackfly control programmes for more than three decades. in this paper, the advances made towards the control of blackflies in south africa are reviewed, the medical, veterinary and economic importance of the pest is outlined and current and future blackfly research by the arc-ovi is discussed. medical, veterinary and economic importance of blackflies in addition to feeding on sugar, which is used as a fuel for flight (hocking 1953; davies, peterson & wood 1962; hunter 1977; sutcliffe 1986), anautogenous adult female blackflies also require a blood meal (welton, bass, ladle & merritt 1987; palmer 1997; gibson & torr 1999) for ovarian development (davies & peterson 1956; peterson 1959; crosskey 1990). because of their blood-feeding activity they are considered ideal disease vectors (crosskey 1990) and are probably best known for transmitting the filarial nematode onchocerca volvulus to humans (nelson 1991; davies 1994; hougard, yaméogo, sékétéli, boatin & dadzie 1997; gibson & torr 1999). the resulting disease known as onchocercosis or “river blindness” has left more than 20 million people infected and millions blind in west africa and south america (rodriquez-perez, 308 review of blackfly (diptera: simuliidae) control in south africa fig. 1 known blackfly problem areas in south africa ���������� ���� � �� �� ������� ���� ��� ��� ��� ������� ����������� �������� �� �� ��� �� �� ������ �� �� ���� ���� ������� �� �� ��!"������ ������ #������ �� �� #���� ��! �� �� ������� �� �� ���� ���$�%��! ���� &����� ���� �" ��! ������ '�� ��� ����� ��� (������� ���� � �� �� ������� �� �� ���%����� ������������ #����� ��� ���%�� �����������$%�� �� ��� �� �� )�� � �� �� '��� �� �� �������� *�!�����%�� '��� ����� � ����� �����!�� ��� '���!���� )��� &��%��%�� ��� �� �� �����+� '��� ��� #��!������� ���+���� ���%����� �� �� , ��� ������ �) � reyes-villanueva & rodriquez 1995; samba 1995; hougard et al. 1997; molyneux & davies, 1997). furthermore, in humans, the bites of some blackfly species can cause allergic reactions known as “blackfly fever” or simuliotoxicosis (crosskey 1990; palmer 1997). this condition is characterized by swelling, itching, haemorrhage and oedema (de villiers 1987) which, in severe cases, requires medical attention (mason & schemanschuk 1990). in animals, blackflies have been implicated in the spread of leucocytozoonosis (anderson & voskuil 1963; crosskey 1993), bovine onchocercosis, (crosskey 1990; hadi & takaoka 1995), the cytoplasmic polyhedrosis virus, the iridescent virus, vesicular stomatitis virus (bernardo & cupp 1986; bridges, mccluskey, salman, hurd & dick 1997; maré 1998), avian trypanosomes (crosskey 1993), myxomatosis (williams & williams 1966; kettle 1984) and dirofilaria species (simmons, edman & bennett 1989). it has also been shown that allergic reactions to blackfly bites, similar to that described in humans, can lead to the death of cattle (mason & schemanschuk 1990). in south africa, simuliids have been implicated in the spread of two pathogens to animals namely a chlamydophila (previously chlamydia) sp., that causes blindness in sheep and abortion in cattle (de moor 1982a), and rift valley fever virus. mechanical transmission of this virus, by s. chutteri, may have contributed to the major rift valley fever outbreak between prieska and groblershoop in 1975 (mcintosh, jupp, dos santos & barnard 1980). blackflies readily attack exposed parts of the bodies of livestock, such as the eyes, ears and teats (anderson & voskuil 1963), and the resulting wounds are prone to secondary infections which sometimes lead to the death of animals (palmer 1997). blackflies also cause considerable irritation (annoyance) to livestock (anderson & voskuil 1963; crosskey 1990; kok, fourie & oberem 1994). in south africa, sheep under attack from blackflies will bundle together and hide their heads underneath each other. during these periods the sheep do not feed or mate, and this results in loss of mass and a reduction in lambing percentage (palmer 1997). in southern new zealand and canada the irritation value alone of the pest is high enough to have it classified as the most significant insect pest in these areas (gibson & torr 1999). according to edman & simmons (1985) the annoyance of haematophagous simulium species can be severe enough to warrant large-scale control operations. blackfly annoyance also leads to economic losses through reduced efficiency of agricultural and industrial workers, interference in recreation, and reduced real estate values (mason & shemanschuk 1990). although there are numerous reports of blackfly epidemics in south africa, only steenkamp (1972) has made a detailed study of the economic impact of the pest. he reported physical destruction of the teats of some cows and a reduction in milk production of up to 35 kg milk per week per cow (30–50 % reduction). in poultry he found a 10–15 % reduction in egg production. other reports confirm that cows can loose their udders and sheep their ears as a result of secondary infections that develop because of blackfly wounds (de moor 1986). during a more recent, though smaller-scale survey along the vaal river, farmers reported that blackflies killed lambs, caused losses of 60% in total farm stock production and reduced milk production by as much as 55 l per cow per week (o’keeffe 1985). in 1996 the northern cape agricultural union estimated that blackflies, if left uncontrolled, can cause potential losses of more than r88 million per annum to the stock industry along the orange river (palmer 1997). this figure is based on a 25 % reduction in lamb production and excludes other figures such as land depreciation and tax losses to the state. today s. chutteri is considered to be the most important blackfly pest species in south africa. this species occurs along the orange, vaal and great fish rivers, and, to a lesser extent, the sundays river, but it is most abundant and causes the largest economic problems along the middle and lower orange river (palmer 1997). simulium chutteri is a large-river species endemic to southern africa which, under favourable conditions, can become the most abundant blackfly species in this region with larval densities exceeding 500 000/m2 (palmer & de moor 1998). it is a multivoltine species with 11–13 generations per annum (palmer, edwardes & nevill 1996). simulium chutteri occurs throughout the year although an increase in biting activity is usually experienced in spring and early summer (august to november) and autumn (april to may) (jordaan & van ark 1990; palmer, edwardes & nevill 1995b) suggesting that it is adapted to moderate weather conditions. history of blackfly control in south africa attempts to control blackflies in south africa started with the use of ddt in 1965. this was followed by water manipulation in the 1970s and 1980s, strategically timed to maximize the effect of inver309 e. myburgh & e.m. nevill tebrate predators. since the late 1980s the use of larvicides has been tested and developed into planned control programmes. these phases are described in more detail below: phase 1: ddt applications periodic blackfly outbreaks, especially following floods, were experienced along the vaal river prior to 1940 (howell & holmes 1969), but after the completion of the vaal barrage in 1923, vaalharts diversion weir in 1936 and vaal dam in 1938, blackfly numbers steadily increased (de moor 1986). since 1950 frequent blackfly outbreaks have been reported along the vaal river (howell & holmes 1969; nevill 1988). four species have been incriminated in the outbreaks: s. chutteri (chutter 1968; howell & holmes 1969), s. damnosum s.l., s. nigritarse (steenkamp 1972) and s. adersi (begemann 1980). after a severe blackfly outbreak during 1963 in the warrenton district (chutter 1968; howell & holmes 1969) the first extensive studies were undertaken on the ecological requirements of local blackfly species (chutter 1968). these were followed in 1965 by the first attempts to control the pest using ddt which, at that time was described as “the perfect weapon for the perfect target” (brown 1962). between 1965 and 1967 ddt was applied several times to the vaal river from structures suspended above sluice gates or by fixed-wing aircraft (howell & holmes 1969). the ddt applications resulted in the growth of benthic algae on rocks, which was attributed to the eradication of most invertebrates as a result of the low target specificity of ddt (car & de moor 1984). nevill (1988) noted that the algal mats had the benefit that they did not allow new generations of blackfly larvae to attach to affected rocks. although high larval mortalities were obtained with ddt, rapid reinfestation was recorded following the disappearance of the algal mats (howell & holmes 1969). the control programme was suspended in 1967 because of the environmental damage caused by ddt. after major floods in 1974, begemann (1980) found blackfly larvae in great numbers in the vaal river, indicating that the blackfly problem had not been solved. phase 2: water-flow manipulation the 1970s saw the spread of the blackfly problem along the vaal river after the completion of the bloemhof dam in 1970 (car 1983). moreover, during the period 1972 to 1978 the gariep and van der kloof dams were completed in the orange river and this allowed s. chutteri to also develop to pest proportions in this river system (nevill 1988; jordaan & van ark 1990). in 1975 the orange fish tunnel, linking the gariep dam to the great fish river (fig. 1), was completed and reports indicated that s. chutteri also developed pest status in the great fish river during the years following the completion of the tunnel (coetzee 1982; o’keeffe 1985). these problems led to the second phase in the battle against blackflies, namely the use of water-flow manipulation. water-flow manipulation is the process by which the water levels of rivers are artificially fluctuated to expose and desiccate the sessile blackfly pupae as well as forcing the larvae to move to undesirable sites where they are prone to starvation and predation (howell, begemann, muir & louw 1981). howell et al. (1981) started flowfluctuation trials in 1977 at the vaalhartz diversion weir and found a drop in the numbers of immature blackflies for up to 30 km downstream of the weir. they followed this with trials in the orange river during 1978 at the van der kloof dam where water flow was interrupted for approximately 66 h. here they reported similar successes. the authors recommended that cut-offs in water-flow be implemented twice annually, during may and august. they furthermore claimed that s. chutteri lost its pest status in sections of the river where regular water-flow fluctuations were implemented. separate trials by car (1983) confirmed that a reduction in the water level of the orange river reduced the number of immature blackflies in the river. the greatest effect on larvae could be found during winter and he recommended a cut-off in water-flow during july/august when the majority of the population is in the larval stage. phase 3: integrated water flow manipulation during the same period de moor (1982a; b), working along the vaal river, proposed a third method of blackfly control. this method involved an integrated approach where data on the life-cycle, population dynamics and microhabitat preferences of the six most abundant simulium species, and their natural aquatic invertebrate predators, were used to determine the best time to carry out a series of river-flow cessations. water-flow regulation was then applied to halt the build-up of populations and maintain s. chutteri at levels at which they could be controlled by natural predators. although integrated water-flow manipulation can be regarded as the most cost-efficient and ecologically least disruptive of the available methods, de moor & car (1986) 310 review of blackfly (diptera: simuliidae) control in south africa noted that the method is limited by the availability of impoundments upstream of simulium breeding sites and is therefore impractical. furthermore, from the start of the 1980s there were major agricultural developments along the orange river and the traditional crops were expanded to include winter crops such as wheat and peas. nevill (1988) concluded that the need for additional irrigation water, as well as the release of water from the gariep and van der kloof dams, for the generation of hydroelectricity, made the further use of strategically-timed water-flow manipulation impractical along the orange river. researchers also realized that the long distances over which water flow had to be manipulated made the sustainable use of this method unrealistic (jordaan & van ark 1990). waterflow manipulation was therefore discarded as a control method along the orange river. phase 4: bti and temephos applications bti is an acronym for bacillus thuringiensis berliner var. israelensis de barjac (serotype h-14) a naturally occurring bacterium that, on sporulation, produces protein crystals with larvicidal activity against filterfeeding diptera such as mosquitoes and blackflies. the protein crystals are classified as a delta-endotoxin and must first be activated by the digestive proteases of the target organism to become a toxic substance. once ingested the alkaline conditions of the stomach dissolve the crystal and release the delta-endotoxin. the toxin has an affinity for the stomach wall lining causing the cells to first swell then rupture. when enough stomach cells burst, the alkaline fluid of the midgut can enter the blood. this increases the alkalinity of the blood and results in a general paralysis and death. blackfly larvae need to filter out sufficient spores for bti to be effective. this larvicide therefore works best in clear water where there is no dilution of spores by silt particles or algae. also because bti is a biological product produced by a fermentation process, whereafter it must be formulated for use in water, it has so far been impossible to produce a very highly concentrated product. during the 1980s bti gained ground as a biological agent for the control of simuliids after studies by undeen, takaoka & hansen (1981) and lacey, escaffre, philippon, sékétéli & guillet (1982) indicated that this blackfly larvicide was effective in other parts of the world. following these reports, car & de moor (1984) conducted trials during 1982 in the vaal river and reported high larval blackfly mortalities. laboratory and field trials by car (1984) also showed that bti was effective in controlling blackflies, but that its toxicity was considerably reduced in polluted rivers with a high sewage level and high chloride concentration. subsequent trials in the orange river during 1983 confirmed the efficacy of bti against blackflies and its low toxicity to non-target organisms (de moor & car 1986). the alternative to bti was the organophosphate temephos [“abate”—sa cyanamid (pty) ltd], specially formulated for blackfly control as a 20 % suspension concentrate. this larvicide had proven to be very effective and environmentally acceptable in west african rivers during the onchocercosis control programme. temephos can and is best used in rivers that have silt particles in suspension. the larvicide adsorbs to the silt particles that, in turn, are filtered out and concentrated in the midgut of the blackfly larvae, causing them to poison themselves. due to its mode of action and the low dosage rate used, less temephos formulation is needed to produce the same effect as bti. also temephos can be applied to silt-laden rivers, which is often the case at high flow rates. large-scale trials to control blackflies breeding in the orange river, using bti and temephos larvicides, started in 1991 after a severe blackfly outbreak followed a flood in 1988 (palmer 1997). fortunately, new, improved and more practical bti formulations [“teknar hp-d” (registered trademark of thermo trilogy corporation registered and marketed in south africa by wefco marketing) and “vectobac” (valent biosciences (pty) ltd)] had become available. both bti and temephos proved to be effective against s. chutteri larvae (palmer 1995) and led to the establishment of an annual blackfly control programme along the orange river (palmer, edwardes & nevill 1996). however, since each of the two larvicides has a different mode of action and different formulations, the river conditions under which they can be used also differ. the application of presently available formulations of bti is restricted to rivers with relatively low flow rates less than 100 cubic metres per second (< 100 cumecs). application is usually by helicopter, the bti being sprayed 50 m to 100 m above rapids, in a zig-zag path across the river to allow dispersion before the bti reaches the larvae attached to rocks in the rapids. in this way the larvae have a greater chance of filtering out a lethal dose of bti spores. temephos is the only larvicide which can be applied to the orange river when flow rates are 300 cumecs and higher. under these circumstances it is effective for up to 50 km 311 e. myburgh & e.m. nevill downstream of the point of application. at such high flow rates it is applied from a vehicle moving slowly across a bridge or from a boat (palmer, edwardes & nevill 1995a, b; 1996). various studies on the impact of these two larvicides on non-target organisms showed that they were safe for use in the orange river (palmer 1993; palmer & palmer 1995). at the same time trials were done to assess the downstream carry of these two larvicides (palmer et al. 1995a) and on the timing of larvicide applications (palmer et al. 1995b). furthermore, methods were developed for rapidly assessing larval and pupal abundance before and after larvicide applications (palmer 1994). these trials lead to the development of larvicidal programmes in the vaal and orange rivers. however, due to financial and logistical constraints, the vaal river larvicidal programme was discontinued. currently the national department of agriculture implements the orange river blackfly control programme (orbcp). between two and 19 temephos and bti applications are needed annually to control the pest (palmer 1997; myburgh 1999b). ideally the two larvicides should be used alternately to inhibit selection of resistant individuals, especially to temephos. however, in practice this is not always possible as at high flows, i.e. greater than 100 cumecs, bti cannot be used due to the high dosage and costs involved. for a detailed review of the orbcp see palmer et al. (1996), palmer (1997; 1998) and myburgh (1999b). blackfly control programmes along the vaal, great fish, sundays and gamtoos rivers vaal river simulium chutteri is the predominant species although s. damnosum can also be a problem. following the discontinuation of larviciding sections of the vaal river, weekly water-flow interruptions were introduced in 1997 at bloemhof dam and vaalharts weir. this approach was very successful (myburgh, 1997; 1998a, b; 1999a). for various reasons it has been difficult to develop an effective routine water-flow interruption programme at the vaal barrage, but this may still be possible. a major shortcoming of this approach is that water-flow cannot be interrupted when dams are full. an integrated water-flow/larvicide programme is needed for the vaal river. great fish river the main pest species is s. chutteri, although s. damnosum, simulium medusaeforme, s. nigritarse and simulium mcmahoni have also been recorded. blackfly breeding is mainly restricted to 33 rapids over a distance of 90 km. this river has a median flow of 3.5 to 4.6 cumecs. it is therefore feasible for local farmers, using hand-held sprayers, to apply larvicide across the entire width of the river. first control attempts started in 1984 using an early formulation of bti, but were discontinued (palmer 1997). following the success of the orbcp, the latest formulation of bti was used in a renewed fish river control programme. this got underway in 1993. in order to overcome the negative effect that the high silt load in this river has on the effect of the larvicide, the bti is now applied at double the recommended dosage rate. the larvicide is supplied by the national department of agriculture (myburgh 1997; 1998a, b; 1999a). sundays river the blackfly problem along the sundays river also started when an additional and regular supply of water was received from the orange river after 1977, via the fish-sundays tunnel and canal (palmer 1997). the main species are s. adersi (lower river) and s. damnosum (upper river), while s. chutteri numbers are low. this is a small river with a median flow of only 2–3 cumecs. the main breeding is restricted to 26 rapids over 41 km, but in-stream vegetation creates additional breeding surfaces (palmer 1997). it is therefore possible for persons, in the affected sections, to monitor and treat the river themselves, if trained and supplied with bti. initial control efforts were made in 1995 by sunlands irrigation board. in late 1997, sundays river citrus cooperative took over the responsibility, the bti being supplied by the national department of agriculture (myburgh 1997; 1998a, b; 1999a). gamtoos river this is a small river with a median flow varying between 1.5 and 2.0 cumecs. no s. chutteri occur here, the main species being s. adersi and s. medusaeforme (palmer 1997). in 1995 the river was surveyed from the air for rapids which are the main breeding sites. fifty-five rapids over a distance of 66 km were recorded but in-stream trailing vegetation increased the available surfaces for blackfly larvae to attach. gamtoos irrigation board 312 review of blackfly (diptera: simuliidae) control in south africa employees have been trained, and this board has taken on the responsibility of controlling blackfly larvae in the river and in canals. the bti is supplied by the national department of agriculture (myburgh 1997; 1998a, b; 1999a). present and future blackfly research although an effective blackfly control programme is in place along the middle and lower orange river, major outbreaks still occur (palmer 1997; myburgh 1999a). some of these outbreaks can be attributed to human error (including unavailability of the helicopter and late ordering of larvicides), but a lack of information on several blackfly population dynamic factors makes planning of control actions difficult and inaccurate (myburgh 2002). for example, in the current planning of control actions, fundamental fitness traits such as fecundity, dispersal, feeding, survival and longevity are poorly understood, although studies elsewhere have shown that these are all important considerations in the planning of control actions (colbo & porter 1979; de moor 1982b). due to the significance of these factors, the arc-ovi, under contract to the water research commission from 2000 to 2002, conducted a study to address some of the aspects believed to influence adult blackfly survival. these studies showed that various physiological and ecological variables, such as body size, mass, lipid and glycogen levels, longevity, climatological factors (myburgh 2002; 2003) and nectar availability (myburgh, bezuidenhout & nevill 2002) affect the survival of adult blackflies. the results of this study can be used, to some extent, to explain the typical seasonal variation found in the annoyance levels exhibited by adult blackflies along the lower orange river and therefore, outbreaks can be predicted with more accuracy and blackfly control programmes can be adjusted accordingly. current research focuses on the development of methods to protect livestock from the attacks of adult blackflies. these studies include the use of insecticides and shelters. another aspect requiring attention is that blackflies along the orange river may have developed resistance to temephos so that larval control programmes are no longer as effective when this larvicide is used. if this is the case, a strategy to overcome the resistance problem should be developed and/or a new larvicide tested and registered for use in south africa. moreover, the reason why periodic outbreaks during the summer months occur along the lower orange river, below onseepkans (fig.1), is not known and this problem requires investigation. in addition, the development of an effective control programme for the vaal river blackfly problem needs to be developed as the control programme, developed for s. chutteri along the orange river, is not entirely suitable for use along the vaal river as the two river systems differ in certain respects. some of the ways in which the vaal river differs from the orange river, are the following: • dams and other impoundments are more frequent along its course. • it is not used for irrigation as extensively as the orange river, and is then mostly via canals fed from weirs, e.g. vaalharts scheme—therefore, the use of waterflow manipulation as a blackfly control option would be less disruptive to agriculture. • it flows through a comparatively densely populated part of south africa so there is a greater recreational demand on or near it. • because of its proximity to large cities the blackflies, which breed along its length, impact directly on associated human activities, e.g. conference venues or, indirectly, e.g. racing stables— human activities with a high commercial value. • because the area through which it flows is less arid than that of the lower orange river, livestock production is more intensive and cattle are more important, especially dairy cattle—the immediate negative effect of blackfly attacks will be more apparent. • although s. chutteri is the dominant blackfly species, as it is along the orange river, a second species, s. damnosum, may also occur in pest proportions. as its name implies s. damnosum also feeds on humans—therefore exacerbating the nuisance effect of blackflies. • simulium damnosum does not scatter eggs on the water surface, as does s. chutteri, but attaches them to surfaces below the water level. this suggests that methods to control each of the two blackfly species could differ. • in recent years, the lesotho highlands water scheme has resulted in the regular supply of water, via the liebenbergvlei river to the vaal dam, ensuring a more constant high flow of water in the vaal river. this could favour the production of constantly high levels of blackflies 313 e. myburgh & e.m. nevill and may create new breeding sites upstream of the vaal dam. from the foregoing it is 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the control of blackfly in the orange, vaal, gamtoos, sundays and fish rivers for the contract year 1997/ 1998. report no: ovi 1998/001/upt for the national department of agriculture, directorate of agricultural resource conservation, pretoria, june 1998. myburgh, e. 1998b. blackfly control in south africa. interim report on the control of blackfly in the orange, vaal, gamtoos, sundays and fish rivers for the contract year 1998/ 1999. report no: ovi 1998/006/upt for the national department of agriculture, directorate of agricultural resource conservation, pretoria, december 1998. myburgh, e. 1999a. control of blackflies in south africa. final report on the control of blackfly in the orange, vaal, gamtoos, sundays and fish rivers for the contract year 1998/ 1999. report no: ovi 1999/006/upt for the national department of agriculture, directorate of agricultural resource conservation, pretoria, june 1999. myburgh, e. 1999b. control of the pest blackfly, simulium chutteri, in the orange river, south africa. journal of the south african veterinary association, 70:46. myburgh, e. 2002. the influence of developmental temperature on the adult survival of simulium chutteri (diptera: simuliidae). msc thesis, university of pretoria. myburgh, e. 2003. factors that influence adult blackfly (diptera: simuliidae) survival along the lower orange river, south africa. water research commission report no. 1019/1/03, pretoria, south africa. myburgh, e., bezuidenhout, h.b. & nevill, e.m. 2002. the role of flowering plant species in the survival of blackflies (diptera: simuliidae) along the lower orange river. koedoe, 44:63–70. nelson, g.s. 1991. human onchocerciasis: notes on the history, the parasite and life cycle. annals of tropical medicine and parasitology, 85:83–95. nevill, e.m. 1988. the creation of permanent blackfly problems by the construction of dams, in long-term data series relating to south africa’s renewable natural resources, edited by i.a.w. macdonald & r.j.m. crawford. south african national scientific programmes report no.157, csir, pretoria,south africa: 353–355. o’keeffe, j.h. 1985. the blackfly problem in the great fish river. the naturalist, 29:3–8. palmer, r.w. 1991. ecological effects of impoundments in the buffalo river, eastern cape, with particular reference to the distribution of blackflies (diptera: simuliidae), ph.d. thesis, rhodes university. palmer, r.w. 1993. short-term impacts of formulations of bacillus thuringiensis var. israelensis de barjac and the organophosphate temephos, used in blackfly (diptera: simuliidae) control on rheophilic benthic macroinvertebrates in the middle orange river, south africa. south african journal of aquatic science, 19:14–33. palmer, r.w. 1994. a rapid method of estimating the abundance of immature blackflies (diptera: simuliidae). onderstepoort journal of veterinary research, 61:117–126. palmer, r.w. 1995. biological and chemical control of blackflies (diptera: simuliidae) in the orange river. water research commission report no. 343/1/95, pretoria, south africa. palmer, r.w. & palmer, a.r. 1995. impacts of repeated applications of bacillus thuringiensis var. israelensis de barjac and temephos used in blackfly (diptera: simuliidae) control, on macroinvertebrates in the middle orange river, south africa. south african journal of aquatic science, 21: 35–55. palmer, r.w., edwardes, m. & nevill, e.m. 1995a. downstream carry of larvicides used in the control of pest black flies (diptera: simuliidae) in the orange river, south africa. journal of vector ecology, 21:37–47. palmer, r.w., edwardes, m. & nevill, e.m. 1995b.timing of larvicide treatments for the control of pest blackflies (diptera: simuliidae) in a semi-arid environment in south africa. journal of vector ecology, 21:48–59. palmer, r.w., edwardes, m. & nevill, e.m. 1996. control of pest blackflies (diptera: simuliidae) along the orange river, south africa: 1990–1995. onderstepoort journal of veterinary research, 63:289–304. palmer, r.w. 1997. principles of integrated control of blackflies (diptera: simuliidae) in south africa. water research commission report no. 650/1/97, pretoria, south africa. palmer, r.w. 1998. an overview of black fly (diptera: simuliidae) control in the orange river, south africa. israel journal of entomology, 32:99–110. palmer, r.w. & de moor, f.c. 1998. annotated records of blackfly (diptera: simuliidae) distribution in southern africa. african entomology, 2:223–251. peterson, b.v. 1959. observations on mating, feeding and oviposition of some utah species of black flies (diptera: simuliidae). the canadian entomologist, 91:147–155. rodriquez-perez, m.a., reyes-villanueva, f. & rodriquez, m.h. 1995. estimating the gonotrophic cycle and survivorship of simulium ochraceum (diptera: simuliidae) 315 e. myburgh & e.m. nevill during routine vector surveillance in southern mexico. journal of the american mosquito control association, 11:360– 362. samba, e.m. 1995. ten years of onchocerciasis control. report of the world health organization, geneva, for 1995, no. ocp/gva/85.1b: 43–57. simmons, k.r., edman, j.d. & bennett, s.r. 1989. collection of blood-engorged black flies (diptera: simuliidae) and identification of their source of blood. journal of the american mosquito control association, 5:541–546. steenkamp, j.a. 1972. ‘n ondersoek na die wisselwerking tussen sommige ekologiese faktore en die bevolkings van simulium damnosum theobald en s. nigritarse coquillett (simuliidae: diptera) in die vaalrivier by parys. d.sc. thesis, potchefstroom university. sutcliffe, j.f. 1986. black fly host location: a review. canadian journal of zoology, 64:1041–1053. undeen, a.h., takaoka, h. & hansen, k. 1981. a test of bacillus thuringiensis var. israelensis de barjac as a larvicide for simulium ochraceum, the central american vector of onchocerciasis. mosquito news, 41:37–40. welton, j.s., bass, j.a.b., ladle, m. & merritt, w.j. 1987. distribution of oviposition sites and characteristics of the egg development in the ”blandford fly” simulium posticatum (diptera: simuliidae). journal of applied ecology, 24: 865–879. williams, e.h. & williams, p.h. 1966. a note on an apparent similarity in distribution of onchocerciasis, femoral hernia and kaposi’s sarcoma in the west nile district of uganda. east african medical journal, 43:208–209. 316 review of blackfly (diptera: simuliidae) control in south africa cameron_11-12.indd introduction when contemplating the history of botulism (lamsiekte) in south africa, one of the first realizations is that the initial observations were not done by scientists or veterinarians but by travellers such as le vaillant in 1881 (le vaillant 1896). this was followed by the visits and reports by lichtenstein (1812), a medical doctor, james bleckhouse, an evangelist and professor ferdinand strauss from stuttgart, who all noted peculiar grazing behaviour (gutsche 1979). they must have been shocked by the revelation of the hot, dusty and dry climate as well as the prevailing poverty in the north-western parts of south africa. the important concept to learn and remember from this phase is that all research starts by observation astute and deductive—and this led the government of the time to realise that lamsiekte was not only a disease or aberration but a serious community problem. this in turn led to a more formal approach by the cape government who obtained the services of brentford and hollier-little and appointed duncan hutcheon at port elizabeth in 1880. in cooperation with gammack and jotello soga, working in kimberley, it was established that lamsiekte and stiffsickness were the same condition; both associated with malnutrition. early research further research was subsequently done by spreull at koopmansfontein and borthwick in grahamstown. progress was, however, impeded by the disruption caused by the anglo-boer war (1899–1902). the complexity of the condition was nevertheless realized. frei was of the opinion that the condition was caused by an infectious agent whereas burtt-davey believed that a toxic plant was involved, a theory that was initially shared by theiler. in the meantime the disease continued to cause cattle losses in the vryburg area and bechuanaland (now botswana). a turning point came in 1911 that was instigated by charles butler. he revived the bechuanaland farm11 onderstepoort journal of veterinary research, 76:11–12 (2009) a brief history of botulism in south africa c.m. cameron south african veterinary association, p.o. box 25033, monument park, 0105 south africa abstract cameron, c.m. 2009. a brief history of botulism in south africa. onderstepoort journal of veterinary research, 76:11–12 when looking back into the history of botulism and contemplating the final understanding of the syndrome and the ultimate solutions, there are four facets that stand out clearly. the first is that much of the solution was guided by astute observations, curious travellers, committed veterinarians and particularly farmers themselves who were able to relate the occurrence of the condition to climatic and grazing conditions. secondly, there was the identification of the osteophagia and pica syndrome which led to the feeding of bone-meal as a successful mitigating measure as well as the establishment that botulism was not due to a plant poisoning. thirdly, the solution of the problem depended on the integration of experience and knowledge from diverse disciplines such as soil science, animal behaviour and husbandry, nutrition, botany and ultimately advanced bacteriology and the science of immunology. finally it required the technical advancement to produce toxoids in large quantities and formulate effective aluminium hydroxide precipitated and oil emulsion vaccines. 12 history of botulism in south africa ers association and persuaded the authorities to take action. a meeting was held at vryburg on 8 june 1911 where j. burtt-davey (government botanist) gave a lecture, arnold theiler addressed the farmers and they in turn made the farm “armoedsvlakte” as well as cattle available for feeding experiments. this new initiative gave rise to further intensive research inter alia by the swiss botanist marguerite henrici in 1914. p.r. viljoen, previously from the allerton laboratory in natal, was also transferred to “armoedsvlakte” in 1914. but once again, progress was impeded by human conflict—both collectively and individually. the major element was the outbreak of the first world war and the reticence of government to purchase “armoedsvlakte”. this situation was compounded by the resignation of arnold theiler but he fortunately reassumed duty after negotiations and was assigned to “armoedsvlakte” in 1919. concomitantly ph de kock became president of the farmers union and actively propagated the employment of a chemist and botanist. the final solution within days of being posted to “armoedsvlakte” thei ler, through astute and intensive observations, conceived his theory which he recorded as follows: “soil and vegetation deficient in phosphorus (remediable with phosphatic manuring) produced pica or osteophagia in cattle compelling them to find phosphorous in putrid bones and carcase material (remediable by feeding bone meal and other phosphor-rich material). the position in carrion of all sorts (dead meerkats, spring-hares, ostriches, tortoises, household refuse, etc.)—a toxiogenic saprophyte (later identified as the cause of a form of botulism) could be eliminated by clearing pasturage of all such material” (gutsche 1979)”. based on this perception, initial solutions were developed of which feeding of bone-meal and phosphate was the most important. the transfer of robinson to onderstepoort from allerton would herald the research that ultimately established the relationship between consumption of carrion and intoxication by the clostridium botulinum exotoxin. in his 12th report, theiler highlights the contributions by p.r. viljoen, h.h. green, p.j. du toit and e.m. robinson (theiler 1927). the first isolation of clostridium botulinum was accomplished by du toit and rob inson in 1928 and the final identification was achieved by robinson in 1930. an effective vaccine was produced by mason (mason, steyn & bisschop 1938) and refined by jansen (1971a, b) and jansen, knoetze & visser (1976). a fundamental truth emanating from this review is that a final solution required integration of indigenous knowledge, experience and observations of farmers with disciplines such as epidemiology, botany, toxicology, pathology and bacteriology. this observation is ultimately in harmony with the current philosophical perspective of: “one world, one medicine, one health”. references gutsche, t. 1979. there was a man. the life and times of sir arnold theiler k.c.m.g. of onderstepoort. cape town: howard timmins. jansen, b.c. 1971a. the toxic antigenic factors produced by clostridium botulinum types c & d. onderstepoort journal of veterinary research, 38:93–98. jansen, b.c. 1971b. the quantitative determination of the toxic factors produced by clostridium botulinum (van ermengen, 1886) types c & d. onderstepoort journal of veterinary research, 38:177–180. jansen, b.c., knoetze, p.c. & visser, f. 1976. the antibody response of cattle to clostridium botulinum types c and d toxoids onderstepoort journal of veterinary research, 43: 165–174. le vaillant, f. 1896. travels into the interior parts of africa. vol ii. earliest reports. lichtenstein, m.h.k. 1812. trav els in southern africa in the years 1803–1808 (english edition). van riebeeck society. mason, j.h., steyn, h.p. & bisschop, j.h.r. 1938. the immunization of bovines against lamsiekte. journal of the south african veterinary medical association, 9:65–70. robinson, e.m. 1930. the bacteria of the clostridium botulinum c and d types. sixteenth report of the director of vet erinary services. union of south africa. theiler, a. 1927. lamsiekte (parabotulism) in cattle in south africa. eleven and twelfth reports of the director of veterinary education and research, part ii: 821–1361. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: esron d. karimuribo1 kuya sayalel2,3 eric beda1 nick short4 philemon wambura1 leonard g. mboera5 lughano j.m. kusiluka3 mark m. rweyemamu1 affiliations: 1southern african centre for infectious disease surveillance, morogoro, tanzania2ngorongoro conservation area authority, ngorongoro arusha, tanzania 3the open university of tanzania, dar es salaam, tanzania 4royal veterinary college, london, united kingdom 5national institute for medical research, dar es salaam, tanzania correspondence to: esron karimuribo postal address: po box 3297, sua morogoro, tanzania how to cite this proceeding: karimuribo, e.d., sayalel, k., beda, e., short, n., wambura, p., mboera, l.g. et al., 2012, ‘towards one health disease surveillance:the southern african centre for infectious disease surveillance approach’, onderstepoort journal of veterinary research 79(2), art. #454, 7 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.454 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. towards one health disease surveillance: the southern african centre for infectious disease surveillance approach in this proceeding... open access • abstract • introduction • materials and methods    • one health surveillance    • study design    • baseline study on assessment of surveillance systems    • design of southern african centre for infectious disease surveillance one health surveillance strategy in southern africa    • study sites    • data analysis • results    • baseline study on the performance of surveillance system in the animal and human health sector in tanzania       • disease surveillance structure       • performance of surveillance systems    • one health surveillance strategy in southern africa • discussion • conclusion • acknowledgements • references abstract top ↑ africa has the highest burden of infectious diseases in the world and yet the least capacity for its risk management. it has therefore become increasingly important to search for ‘fit-for-purpose’ approaches to infectious disease surveillance and thereby targeted disease control. the fact that the majority of human infectious diseases are originally of animal origin means we have to consider one health (oh) approaches which require inter-sectoral collaboration for custom-made infectious disease surveillance in the endemic settings of africa. a baseline survey was conducted to assess the current status and performance of human and animal health surveillance systems and subsequently a strategy towards oh surveillance system was developed. the strategy focused on assessing the combination of participatory epidemiological approaches and the deployment of mobile technologies to enhance the effectiveness of disease alerts and surveillance at the point of occurrence, which often lies in remote areas. we selected three study sites, namely the ngorongoro, kagera river basin and zambezi river basin ecosystems. we have piloted and introduced the next-generation android mobile phones running the epicollect application developed by imperial college to aid geo-spatial and clinical data capture and transmission of this data from the field to the remote information technology (it) servers at the research hubs for storage, analysis, feedback and reporting. we expect that the combination of participatory epidemiology and technology will significantly improve oh disease surveillance in southern africa. introduction top ↑ although there have been some recent advances in the diagnosis and management of human infectious diseases, they still are a significant impact and burden on global economies and public health (jones et al. 2008). infectious diseases are responsible for a quarter of all human deaths worldwide (king et al. 2006). most of these are as a result of emerging infectious diseases (eids), defined as infections that have newly appeared in a population or have existed but are rapidly increasing in incidence or geographic range (morse 1995). there are various drivers for the occurrence of eids including socio-economic, environmental and ecological factors. analysis of origins of eids for longer than six decades concluded that over 60% are zoonotic of which about 72% originate in wild animals (jones et al. 1978). it has also been observed that when considering spatial distribution of origins of infectious diseases, the majority are prevalent in and affect developing countries in the tropics, particularly in africa and south-east asia. effective disease surveillance is required to ensure freedom from eids or else timely intervention in order to reduce risks and impact on animal and human populations. disease surveillance is commonly defined as an ongoing systematic collection, analysis and interpretation of data essential to the planning, implementation and evaluation of disease management practice, closely integrated with the timely dissemination of these data to those who need to know (mboera, rumisha & kitua 2001; thacker & berkelman 1988). although the purpose and objectives of disease surveillance may differ between different health sectors, it is generally agreed that surveillance is useful for rapid detection of new and/or foreign diseases, provides evidence of freedom from diseases within a defined geographic area or population, accurately delineates the distribution and occurrence of diseases relevant to disease control and provides evidence required to assess progress and success of disease control or rededication (fao 2004). most developing countries have limited disease surveillance capacity and so need to ensure optimal use of available resources. however, previous studies indicated that both animal and human health sectors are poorly resourced in terms of both clinical and public health service provision (james & muchiri 2006; jones 2011; karimuribo et al. 2011). considering that most emerging infectious disease conditions in public health sectors, especially in tropical countries, are of animal (wildlife and domestic) origin (jones et al. 2008; wolfe, dunavan & diamond 2007), it is important to encourage collaboration between the animal and human health sectors in order to minimise risks associated with such infections. the purpose of this work is to share findings based on our experience in southern africa focusing on the oh surveillance strategy developed by the southern african centre for infectious disease surveillance (sacids). materials and methods top ↑ one health surveillance in this paper, one health (oh) surveillance is defined as collaborative efforts between the human and animal (wildlife and domestic) health sectors to conduct systematic collection of disease event data, analysis of this data and a timely dissemination of surveillance results to guide interventions aimed to prevent or control disease in human and animal populations. study design this study involved two phases. phase 1 involved conducting a baseline survey to assess the performance of current disease surveillance in animal and human health sectors. lessons learnt from these exercises were used to improve the design of the oh surveillance system. the second phase involved designing an oh surveillance system suitable for the southern african region. baseline study on assessment of surveillance systems a rapid situation analysis was carried out in may 2010 to understand the structure and requirement of surveillance systems in the animal and human health sectors in tanzania. this was followed by assessing the performance of the disease surveillance systems in the two health sectors by visiting animal and human health facilities in ngorongoro district, carried out in november 2010. during field visits, individuals responsible for detection and reporting of disease events were interviewed using a structured questionnaire. a total number of 14 wards of ngorongoro district were visited where all resident ward livestock field officers (lfo) were interviewed. the study also collected data from 13 health facilities, representing approximately 62% of all health facilities in ngorongoro district. the exclusion of an individual health facility was based on lack of access to key personnel or disease surveillance records during the field visit. respondents were asked to specify frequency of collection and submission of disease surveillance reports, diseases reported and constraints and challenges faced during disease surveillance in animal and human health sectors. field data collection was complemented by visiting the district veterinary office (dvo) and district medical office (dmo) at the ngorongoro district council headquarters where disease surveillance reports submitted for the period from 2005 to 2010 were retrieved and analysed. the assessment of disease surveillance in both animal and human health sectors was based on measuring the completeness and accuracy of surveillance reports focusing on a period of five years (2005–2010) before the study. other factors that might have influenced the timely submission of disease reports such as the distance from a health facility to the district headquarters were also recorded. design of southern african centre for infectious disease surveillance one health surveillance strategy in southern africa a participatory approach was used at an initial project inception workshop organised to agree on study sites and surveillance approaches. during the workshop, held in january 2010, it was agreed to develop and pilot oh surveillance in three ecosystems namely ngorongoro, kagera river basin and zambezi river basin. the workshop recommended initial pilot activities to be carried out in one ecosystem (ngorongoro) and then up-scaling the approach to the other two project sites. implementation of the project activities in ngorongoro ecosystems required a collaborative approach coordinated by sacids and national centre for infectious disease surveillance (natcids). other key collaborators in the development of the oh surveillance strategy in tanzania were: the ministry of health and social welfare (epidemiology unit), the ministry of livestock and fisheries development (epidemiology section) and two academic institutions responsible for veterinary (sokoine university of agriculture) and medical (muhimbili university of health and allied sciences) training in the country. the project also partnered with collaborators from uk, namely the royal veterinary college (rvc) and imperial college london, who assisted in sharing with the southern colleagues their experiences in infectious disease surveillance and the use of appropriate technologies and tools to support oh disease surveillance. recommendations of the uk foresight report on infectious diseases (brownlie et al. 2006) were also considered whilst devising oh surveillance strategy in southern africa. study sites the current project on oh disease surveillance adopted three study sites namely ngorongoro, kagera river basin and zambezi river basin ecosystems. the first ecosystem represents an area of maximum human-wildlife-domestic animal interactions. the ecosystem is located in a remote area in the arusha region which borders kenya in the north and the tanzanian serengeti ecosystem in the west. the ecosystem is predominantly inhabited by the maasai pastoral communities who keep cattle, goats and sheep and are in close proximity with wild animals in the wildlife protected areas of ngorongoro conservation area (nca). the other two sites (kagera river basin and zambezi river basins) represent cross-border ecosystems where oh surveillance could be potentially effective in diagnosing and managing infectious diseases across borders. kagera river basin is an ecosystem located in the great lake region of eastern africa which links uganda, rwanda, burundi and tanzania. the ecosystem has a relatively high incidence of communicable diseases with cross-border spread and is considered to be a high risk potential entry-point for haemorrhagic fever (especially ebola and marburg) which occur in the democratic republic of congo (drc). zambezi river basin is located in the southern africa and is the fourth largest basin in africa after the congo, nile and niger river basins. the zambezi river has its source in western province of zambia and flows through eastern angola, namibia (eastern caprivi strip), northern botswana and through victoria falls (shared between zambia and zimbabwe) before entering lake kariba. the project site focused on two districts of zambia (kazungula and sesheke) which share international borders with zimbabwe, botswana, namibia and angola in the zambezi river basin ecosystem. data analysis data collected during baseline study on assessment of the performance of disease surveillance systems were summarised and where applicable, descriptive statistics computed. data trends by ward and health facilities were demonstrated using graphical presentation of the results. the names of health facilities were coded to ensure a blind contribution in disease reporting to comply with ethical clearance conditions. results top ↑ baseline study on the performance of surveillance system in the animal and human health sector in tanzania disease surveillance structure the surveillance structure between the animal and human health sectors in tanzania was found to be similar (table 1). the initial detection of disease events in both health sectors starts in the communities where sick individuals are detected by community-based reporters. the current systems use official cadres who are the lfos (in animal health sectors) and the health facility in-charge or integrated disease surveillance and response (idsr) focal person (in human health sectors) to prepare and submit disease surveillance reports to the higher levels. the central coordinating level for disease surveillance and response is at the district level (dvo and dmo). the two offices are responsible for transmitting reports to higher authorities through the intermediate (zonal vics and rmo) or sometimes directly to the central level in the ministry responsible for animal health and human health, respectively. the similarities in surveillance structures of the two health sectors offer opportunities for increased collaborations between veterinary and medical professionals with regards to disease surveillance and response. with respect to frequency of reporting, the animal health sector demands submission of disease reports on a monthly basis using field and abattoir surveillance reports. in the case of notifiable diseases, the officials are required to report disease events immediately. under the idsr system, officials are required to report diseases under surveillance on weekly (epidemic-prone conditions) and monthly (epidemic-prone, diseases of public health importance and those targeted for eradication) basis. performance of surveillance systems a total number of 13 human health facilities (dispensaries, health centres and hospitals) were visited and participated in this study. the facilities were located between 1 km and 237 km from the ngorongoro district council headquarters (table 2). more than 69% of these facilities are owned by the local government authority, the ngorongoro district council. it was also found that organisations such as the roman catholic and the evangelical lutheran churches of tanzania run some health facilities that, in addition to offering health services, are involved in the capture and reporting of disease events in the study area.the performance of disease surveillance in animal and human health sectors, defined by the number of surveillance reports received every month is shown in figure 1. overall, the reporting was better in the human health sector than in the animal health sector. there were some years such as 2008 and 2009 when the dvo did not receive a single disease report. similarly the dmo did not receive monthly reports from human health facilities throughout 2005. generally, some wards and health facilities performed better in submitting disease surveillance reports compared to others (figures 2a and figure 2b). it was clear that those wards and facilities close to the district council headquarters submitted reports more regularly than the distant wards and facilities. one health surveillance strategy in southern africa the oh surveillance strategy was developed as a result of a participatory and consultative process summarised in table 3. before project implementation, applications were submitted to the appropriate bodies responsible for ethical clearance in the national institute for medical research (nimr) and the tanzania wildlife research institute (tawiri). holding an inception workshop for key stakeholders interested in oh surveillance was helpful in selecting study sites as well as identification of appropriate mobile technologies and tools to assist surveillance. the subsequent meetings of the natcids and the joint technical committee (jtc) in august and september 2010 respectively, defined the oh surveillance strategy to be adopted in ngorongoro and later in two other project sites. the meetings agreed that the strategy should consist of two complementing systems namely:• community-based active surveillance (cas) system which was designed to actively capture disease events in animal and human populations using simple case definitions of symptoms and syndromes occurring in communities. it was also agreed the cas system would use community-based health reporters who would actively screen for the occurrence of disease events in human, wildlife and domestic animal populations. data on these events would be recorded and transmitted through android mobile phones using the epicollect data capture application in near to real time. • district-based passive surveillance (dps) system uses existing surveillance strategies in animal and human (idsr) health sectors with enhanced performance through application of mobile technologies in transmission of near to real time data in the two health sectors. collaborating with other institutions in the united kingdom (royal veterinary college and imperial college london) as well as those in south-east asia (biophics, ministry of public health thailand, mbds and instedd, cambodia) assisted in the improvement of the oh surveillance system developed by sacids. the two systems (cas and dps) are linked together at the data analysis point. data collected through cas and dps systems from pilot sites located in tanzania are stored centrally on a server located at sacids headquarters. southern african centre for infectious disease surveillance acts as a custodian and stores data on behalf of the ministry of livestock and fisheries development and the ministry of health and social welfare who own the data. at sacids, data are analysed and summarised as reports that are shared with the two ministries and field-based disease management units at district headquarters. a similar model is proposed for dealing with handling data collected in zambezi river basin when data storage and analysis is expected to be the role of the university of zambia veterinary school (unza vet) on behalf of respective ministries responsible for animal and human health. there is still on-going discussion on an appropriate model to adopt for collecting, storage and analysis of data from the kagera river basin which is under the auspices of the east african community (eac). table 1: similarities between disease surveillance structure in animal and human health sectors in tanzania. discussion top ↑ similarities in the current disease surveillance structure in animal and human health sectors provide opportunities for collaboration between the two sectors. for instance, under the current idsr strategy, emphasis on disease management is placed in hands of district health facility levels (franco, setzer & banke 2006). similarly, the molfd demands the dvo to be in-charge of managing disease epidemics in animal populations. as both the dmo and dvo work under the umbrella of the district executive officer, it is logical to work together in the management of disease epidemics in animal and human populations in their respective districts. this has been happened in some instances during rift valley fever and anthrax outbreaks in ngorongoro district between 2006 and 2009 (b.m. miran, pers. comm., 2010). it was also found that sometimes animal and human health officials in ngorongoro district do share vaccine storage facilities during surge demand of resources. this usually happens during disease vaccination campaigns when teams of vaccinators camping in remote areas require storage facilities for proper handling of vaccines. this experience is not new to resource-challenged remote areas as reported in other countries where sharing resources such as transport logistics and equipment reduces costs (schelling et al. 2007). the current oh strategy designed by sacids where one person (community-based health reporter) actively searches for occurrence of disease events is another good example of optimising the use of limited resources. findings of the baseline survey agree with previous findings of poor performance of disease surveillance in animal and human health sectors in tanzania (allport et al. 2005; mboera et al. 2001). the situation is made worse with the delayed reporting of sick individuals at health facilities where disease events are normally captured. a study by shayo et al. (2003) indicated that the majority of rural-based individuals stay at home or consult traditional healers before visiting health facilities to seek medical services. similar findings have been reported in the animal health sectors where sick animals are usually managed by farmers or community-based animal health workers in remote areas before intervention of veterinarians (karimuribo & swai 2006). the baseline study also reported a significant variation in completeness of surveillance report in both animal and human health sectors. although some wards and health facilities seem to do better than others, the overall picture confirms poor surveillance coverage. in-depth interviews of district officials responsible for animal and human health confirmed that the lack of human resources to supervise and manage surveillance systems may seriously affect the performance of the system. this is confirmed, for example, by at the fact that there was no dvo between 2006 and 2009 in ngorongoro when there was sharp decline in the number of surveillance reports submitted to the district headquarters. this situation was reversed in 2010 after recruiting a veterinarian to head the veterinary section in the district. other examples were noted where poor reporting was associated with times when key idsr staff responsible for submitting weekly or monthly reports were away from their work stations. this experience had also been reported previously by rumisha et al. (2007) when poor disease reporting under idsr was attributed to staff being on annual leave. although quantitative data on timeliness was not collected in the current study, interview with officials responsible for disease surveillance in the animal and human health sectors indicated that there is always delayed reporting, a problem which is more critical in the animal than in the human health sector. for instance, monthly reports in the animal health sector can be delayed by six to nine months before being received by the epidemiology section (f. kivaria, pers. comm., 2010). in the human health sector, the timely submission of weekly and monthly reports have been reported to be only 8% and 24%, respectively (rumisha et al. 2007). the problem of poor timely reporting is mainly attributed to the paper-based transmission of data coupled with challenging infrastructure and communication networks especially in rural areas. given the challenges of surveillance in the animal and human health sectors, the sacids designed a ‘fit-for-purpose’ oh surveillance strategy. the strategy is considered appropriate for southern africa as it has taken into consideration the situations and challenges prevailing on the ground. key considerations include the relatively higher proportion of patients receiving treatment at home from traditional healers before visiting health facilities, lack of proper diagnostic facilities at community and village levels, limited human diagnostic and mobility resources in remote areas as well as poor infrastructure for efficient communication between rural communities and district or ministry headquarters (strasser 2003). the concept of collaborative efforts in managing infectious diseases in tanzania and other southern african countries is not new. the emergence of innovative and appropriate technologies, approaches and tools for participatory epidemiology and disease surveillance (hussain et al. 2005; jost et al. 2007), such as the use of mobile technologies (aanensen et al. 2009; despont-gros et al. 2005) is likely to mean that infectious disease surveillance can be improved in future. this will contribute to better public health and economic and social stability in africa. it is also anticipated that the oh surveillance will foster stronger collaborative links between the animal and human health professionals and consequently improve management and control of infectious diseases in animal and human health sectors. figure 1: efficiency of monthly disease reporting in the animal and human health sectors in ngorongoro district from 2005 to 2010. figure 2: completeness of disease reporting in the (a) animal and (b) human health sector. table 2: health facilities and wards visited during assessment of surveillance systems in ngorongoro district. table 3: timeline of events that contributed to the development of southern african centre for infectious disease surveillance one health surveillance strategy in southern africa. conclusion top ↑ the approach designed by sacids on oh surveillance is considered suitable for detecting and containing infectious diseases in animal and human populations in countries with limited resources such as those in southern africa. adoption of mobile technologies and appropriate surveillance approaches will improve timely and complete capture of events that would have been otherwise have been missed using routine surveillance systems in the animal and human health sectors. it is concluded that the oh surveillance strategy is timely and relevant to sub-saharan africa. acknowledgements top ↑ we would like to acknowledge support and cooperation received from various individuals in the tanzanian ministry of livestock and fisheries development, ministry of health and social welfare, officials at ngorongoro district council working with the dvo and dmo offices who assisted retrieval of disease surveillance reports. we are grateful to clinical officers, in-charge of different health facilities and wards visited in ngorongoro district as well as community members who participated in this study. we thank our colleagues at imperial college london particularly dr david aanensen and mr chris powell for their valuable support in capacity building related to setting up, field-testing and use of smartphones running the epicollect application in southern africa. this study was funded by the rockefeller foundation through a research support grant 2009 dsn 305. references top ↑ aanensen, d.m., huntley, d.m., feil, e.j., al-own, f. & spratt, b.g., 2009, ‘epicollect: linking smartphones to web applications for epidemiology, ecology and community data collection’, plos one 4, e6968. http://dx.doi.org/10.1371/journal.pone.0006968, pmid:19756138 allport, r., mosha, r., bahari, m., swai, e. & catley, a., 2005, ‘the use of community-based animal health workers to strengthen disease surveillance systems in tanzania’, revue scientifique et technique (oie) 24, 921–932. pmid:16642762 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164–190. wickramage, k. & nellapalli, p., 2008, ‘community participatory methods in disease surveillance and public health in war-affected camps, and its potential contribution to peace building’, international electronic journal of health education 11, 95–108. wolfe, n.d., dunavan, c.p. & diamond, j., 2007, ‘origins of major human infectious diseases’, nature 447, 279–283. http://dx.doi.org/10.1038/nature05775, pmid:17507975 article information authors: adwoa asante-poku1 kwame g. aning2 bashiru boi-kikimoto3 dorothy yeboah-manu1 affiliations: 1department of bacteriology: noguchi memorial institute for medical research, ghana2school of veterinary medicine, college of agriculture and consumer sciences, university of ghana, ghana 3department of bacteriology: veterinary division, ministry of food and agriculture, ghana correspondence to: dorothy yeboah-manu postal address: po box lg581, university of ghana, ghana how to cite this article: asante-poku, a., aning, k.g., boi-kikimoto, b. & yeboah-manu, d., 2014, ‘prevalence of bovine tuberculosis in a dairy cattle farm and a research farm in ghana’, onderstepoort journal of veterinary research 81(2) art. #716, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.716 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/kms/ frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence of bovine tuberculosis in a dairy cattle farm and a research farm in ghana in this proceedings... open access • abstract • introduction • materials and methods    • study area and cattle population    • comparative tuberculin skin test    • slaughter and sample collection    • cultivation of mycobacterial species    • ziehl-neelsen microscopy    • biochemical assay    • dna extraction    • spoligotyping    • 16srrna gene sequencing    • taqman single nucleotide polymorphim typing assay    • data analysis • results    • tuberculin reactivity in cattle    • direct microscopy    • identification and characterisation of mycobacterium species • discussion • acknowlegements    • competing interests    • authors’ contributions • references abstract top ↑ the aim of the study was to estimate the prevalence of bovine tuberculosis (btb) and to identify the mycobacterial species causing btb in a dairy farm and research farm. six hundred and eighty-five cattle were screened for btb by using the comparative intradermal tuberculin test (ctt). positive reactors were slaughtered and carcasses were taken for isolation of mycobacterial species. this was followed by speciation of isolates using both standard conventional and molecular assays. seventeen of the cattle were positive by ctt, giving a crude btb prevalence of 2.48% among cattle from the two farms. six of the 17 samples (35.30%) yielded positive acid-fast bacilli cultures and three of the isolates were identified as mycobacterium tuberculosis complex (mtbc), which were sub-divided into two mycobacterium tuberculosis sensu scrito (mtb) and one mycobacterium africanum; the remaining three were mycobacterium other than tuberculoisis (mott). spoligotyping further characterised the two mtb isolates as ghana (spoligotype data base 4 number 53) and latin american mediterranean (lam), whilst spoligotyping and single nucleotide polymorphism (snp) analysis typed the m. africanum as west african 1. microseq 500 analysis identified two of the mott as mycobacterium flavescens and mycobacterium moriokaense respectively, whilst the remaining one could not be identified. this study observed the prevalence of bovine tb among cattle from two farms in ghana as 2.48% and confirms the public health importance of m. africanum as a pathogen in ghana. introduction top ↑ bovine tuberculosis (btb) is a major public health problem that has been neglected over the years, especially in sub saharan africa. it is considered to be among the seven highly neglected zoonotic diseases of the world and has a major impact on international trade of animal products (sahraoui et al. 2009). despite its importance, very little has been done regarding the creation of awareness of the disease. bovine tuberculosis has been controlled in the developed countries due to the successful implementation of the test and slaughter (ts) policy of all infected cattle and compensation of affected farmers by governments. sub saharan africa, which is home to more than half of the world’s cattle population, has been the hardest hit for various reasons. firstly, in most african countries cattle are used to show economic status in the society and secondly they serve as the main source of income for many farmers (otte & chilonda 2002). moreover, countries in africa are yet to fully implement the ts policy; this is mainly because of the lack of financial commitment on the part of governments to compensate farmers with infected animals.tuberculosis is caused by a group of gram-positive bacterial species that are genetically close; they are referred to as mycobacterium tuberculosis complex (mtbc). even though they are genetically similar, they appear to differ in host specificity. whilst mycobacterium tuberculosis (m. tuberculosis) and mycobacterium africanum (m. africanum) are said to be the main pathogens of human tb, mycobacterium bovis (m. bovis) is thought to be the main causative agent of btb. however, some findings from recent studies seem to have challenged this notion. whilst some studies implicated m. bovis in humans (grange & yates 1996; de kantor & ritacco 1994), others showed the involvement of m. tuberculosis and m. africanum in cattle (romero et al. 2011; cadmus et al. 2010). bovine tuberculosis is diagnosed in the field by the detection of a delayed hypersensitive response to purified protein derivative (ppd) antigen in live cattle; this is perceived to be the first point of care diagnostic tool. however, this test lacks specificity due to antigenic similarity among various members of the mycobacterium tuberculosis complex (mtbc) and also with other mycobacterial species. microscopic detection of acid-fast bacilli in impression smears is simple and performed in the laboratory, but lacks specificity and sensitivity. microbiological isolation and specie identification are the final proof methods for diagnosis of btb (proaño-pérez et al. 2011). yet, microscopy is still widely used in sub saharan african because it is very cheap. ghana is one of the countries where it is believed that btb is still an epizootic disease (veterinary services division 2008), yet data about the disease is very scanty. in ghana, veterinary officers usually diagnose btb based on post mortem detection of tb lesions in animal carcasses during inspection of slaughtered animals at various abattoirs. however, macroscopic occurrence of lesions usually indicates the advanced stages of bovine tb (corner 1994; shitaye et al. 2006). the present study was designed to estimate the prevalence of btb among cattle from a dairy and research farm using the comparative intra-dermal tuberculin test, followed by laboratory analysis for isolation and speciation of the infecting bacteria. materials and methods top ↑ study area and cattle population the study was conducted in two government-owned farms (farm a and farm b) located 16.09 km apart in the greater accra region of ghana (figure 1). these two farms were selected based on the availability of demographic data on the cattle. farm a, is situated on hilly land with easy access to fodder. the farm stocks mainly cattle, a few horses and goats, which are all for breeding purposes. it has a herd size of 400 (28 bulls, two breeding bulls and 370 cows or heifers) made up of sanga cattle from burkina faso and friesan-sanga. grazing is within the confines of the farm and the cattle are kept close to human housing. it is the main source of pasteurised milk and other milk products, such as yoghurt, within the community and surrounding areas. farm b, which was set up mainly for research and teaching, is situated on low-lying land with rich fodder. the farm stocks various animals including sheep, goats, cattle, ducks, chickens and donkeys. it has a herd size of 285 comprising sanga and white fulani breeds. although its main purpose is research and teaching, the farm undertakes some commercial activities to complement the research activities. ante-mortem examination (including sex, breed and body condition) of all the cattle was performed by two veterinary personnel and trained staff. figure 1: map of greater-accra region of ghana indicating the adenta district where the present study’s farms were located. comparative tuberculin skin test comparative tuberculin skin test was performed according to the previously outlined protocol (ó reilly & daborn 1995). briefly: two injection sites were located at the middle side of the neck, one above the other, separated by at least 12 cm. the hair was shaved around the sites to a radius of about 2 cm. the skin folds at both sites were measured with a calliper and the measurements were recorded. an aliquot of 0.1 ml bovine purified protein derative (ppd-b) was subsequently injected intradermally into the lower injection site, and similarly, an avian purified protein derative (ppd-a) was injected at the upper site. after 72 h, the thickness of the skin folds at both sites was measured and recorded. the tuberculin test results were interpreted based on the world organisation for animal health (oie) recommended cut-off of greater than 4 mm skin-fold thickness (world organisation for animal diagnostic manual 2004). a bovine reactor was defined as an animal in which the relative increase in skin thickness at the injection site for ppd-b was at least 4 mm greater than the increase in skin thickness at the injection site for ppd-a. a negative reactor was identified when there was no reaction to the bovine antigen or when the difference of the skin thickness at the injection sites did not exceed 2 mm. an inconclusive reaction was recorded if reaction to both ppd-b and ppd-a exceeded 2 mm, but the difference between the bovine and avian reaction was < 4 mm. slaughter and sample collection detailed veterinary inspection was carried out on all m. bovis tuberculin-positive cattle. the lungs, liver, spleen, kidney and mammary gland were palpated carefully and inspected both externally and internally. mandibular, retropharyngeal, tracheobronchial, mediastinal, hepatic, mesenteric and supramammary lymph nodes were sliced into thin sections and inspected in situ for detection of visible lesions. about 2 cm3 of the suspected tissue specimens were taken from infected organs using a sterile knife, the sample was then kept in phosphate buffered saline (pbs) ph 7.2 in sterile 50 ml centrifuge tubes and transported on ice to the noguchi memorial institute for medical research for microbiological analysis. no ethical clearance was required for this study because it was performed on slaughtered animals and organ confiscation is part of routine monitoring in ghana. cultivation of mycobacterial species sample processing was carried out using aseptic techniques in a biosafety cabinet to avoid cross-contamination between samples. specimens were processed according to standard methods (thoen & steele 1995). in brief: 1 cm3 of suspected lesion was manually homogenised in a sterile mortar containing 2 ml phosphate buffered saline. the resulting suspension was transferred into a sterile screw cap tube and decontaminated using the petroff method. after neutralisation, the homogenate was concentrated by centrifuging for 20 min at 3000 rpm and left to stand for 5 min before opening; this was to allow the generated aerosols to settle. decontaminated specimens were inoculated on four lowenstein-jensen slopes; two were supplemented with 0.4% sodium pyruvate to enhance the isolation of m. bovis and m. africanum, the remaining two were supplemented with glycerol for isolation of m. tuberculosis. the cultures were incubated at 37 °c and were read weekly for macroscopic growth until 12 weeks. ziehl-neelsen microscopy smears prepared from decontaminated specimen and bacterial isolates were stained by ziehl-neelsen and graded according to the international union against tuberculosis and lung diseases (iuatld) guidelines. biochemical assay isolates that were confirmed as acid-fast bacilli (afb) were further characterised by susceptibility to p-nitro benzoic acid (pnb), pyrazinamidase activity (pza), nitrate reduction and niacin production using standard procedures (world health organization 1998). dna extraction dna of confirmed afb-positive isolates was extracted using a previously described protocol (kaser et al. 2009). about 5 μl loop full of harvested bacteria was heat killed in 300 μl of extraction buffer (50 mm tris–hcl, 25 mm ethylenediaminetetraacetic acid (edta) and 5% monosodium glutamate). after cooling, 100 μl of a 50 mg/ml lysozyme solution was added and incubated with shaking for two hours at 37 °c. sixty micro litres of 20 mg/ml proteinase k solution in a 10 x buffer [100 mm tris–hcl, 50 mm edta, 5% sodium dodecyl sulphate (ph 7.8)] were then added and incubated at 45 °c overnight. the bacterial cell wall was fully disrupted by adding 200 μl of 0.1 mm-diameter zirconia beads (biospec products) to each sample and was vortexed at full speed for four minutes. beads and undigested tissue fragments were removed by centrifugation at 14 000 rpm for three minutes and the supernatants were transferred to fresh tubes for phenol-chloroform (fluka) extraction. the dna contained in the upper phase was precipitated with ethanol and re-suspended in 100 μl of water. spoligotyping commercially prepared amino-linked spacer oligonucleotides were diluted to the indicated concentration with 0.5 mm nahco3. membranes were then prepared following previously published procedures (goyal et al. 1997). in brief: the membrane (pall biosupport, michigan) was activated by incubation in 16% (weight or volume) 1-ethyl-2-(3-dimethylaminopropyl)carbodiimide (sigma chemical, st. louis, missouri) for ten minutes at 25 °c. following a brief wash with deionised water, 150 μl of each diluted oligonucleotide (spacer) was applied in a line by using a miniblotter system (mn45; immunetics, cambridge, massachusetts). after incubation at room temperature for five minutes, excess non-bound oligonucleotide solutions were removed from the membrane by aspiration. the membrane was inactivated by incubation in 100 mm naoh for nine minutes at room temperature, followed by a brief wash with 2 × sspe (0.36 m nacl, 20 mm nah2po4, 2 mm edta, ph 7.7) (gibco-brl, grand island, newyork) and a five-minute incubation in 2 × sspe–0.1% sodium dodecyl sulphate (sds) at 58 °c. the membrane was incubated in 20 mm edta for 20 min at room temperature and stored at 4 °c until use.spoligotyping assay was then carried out as previously outlined (kamerbeek et al. 1997). 16srrna gene sequencing non-tuberculous isolates identified by biochemical analysis were sequenced for specie identification using microseq 500, as previously published. sequences obtained were used in blast searches of databases at ncbi. taqman single nucleotide polymorphim typing assay taqman real time pcr was performed according to standard procedures using probes designed by stucki et al. (2012). briefly: two microlitres of dna was added to a 10-μl-reaction mix containing 0.21 um each reverse (3’ggcctgtgacccgttcaac 5’) and the forward (3’tccagcaggtgaccatcgt 5’) primers. in addition, 0.83 um each of probe a for ancestral allele (vic-cgtggacctcatg-mgbnfq) and probe b for mutant allele (6fam-cgtggacctgatgcmgbnfq) and 5 μl taqman universal mastermix ii (applied biosystem, carlsbad, usa) were added to the reaction mix. the reaction was then carried out using applied biosystem, carlsbad, usa thermal cycler under the following conditions: 60 °c for 30 s, 95 °c for 10 min, 95 °c for 15 s and 60 °c for 1 min for 40 cycles, then 60 °c for 30 s. the fluorescence intensity in the vic and fam (6-fluorescein amidite) channels was measured at the end of each cycle. data analysis tuberculin skin test positivity was calculated using oie interpretations and the crude prevalence rates were calculated by dividing the positives by the total cattle population. the significance of observed difference between the two farms was calculated using the chi square test; a p-value of < 0.05 indicated significance. the obtained spoligotyping pattern was compared with those available in the international spoligotype database. species, lineages and clades were assigned according to signatures described in the database. results top ↑ tuberculin reactivity in cattle seventeen of the 685 (2.48%) cattle screened had a positive reaction to mycobacterium bovis ppd. thirteen of the positive reactors were from farm b, giving a farm-specific prevalence of 4.56% (13/285), which was higher than that from farm a (p = 0.003), which had four positives, giving a prevalence rate of 1.0% (4/400). the average middle neck size induration before ppd injection was six mm on both farms. the average post injection skin induration recorded for farm a animals showed a slightly higher skin thickness of 13.25, whilst farm b recorded 12.77 mm skin thickness (table 1). table 1: distribution of tuberculin test positive cattle between the two farms. direct microscopy direct microscopy after decontamination had two of the 17 (11.8%) samples yielding positive afb, and direct microscopy from impression smears had all samples negative by afb. identification and characterisation of mycobacterium species six out of the seventeen cultured samples 6/17 (35.29%) yielded growth that was confirmed as acid-fast bacilli. nine (52.94%) showed no growth after 12 weeks of incubation, whilst two (11.77%) got contaminated with massive fungal growth. three (50.00%) of the afb isolates were confirmed by susceptibility to para nitro benzoic acid as belonging to mtbc and the remaining three were classified as mycobacteria other than tuberculosis (mott) by showing resistance to pyrazinamide. spoligotyping analysis classified two as m. tuberculosis sub lineage latin american mediterranean (lam) and ghana, respectively, the remaining one as m. africanum sub lineage west african 1; this was confirmed by snp analysis.comparing our obtained isolate patterns with the spold4 database, 1/3 (33.3%) of the mtbc isolates that had previously defined shared spoliogotype number, whilst the remaining two had undefined patterns. microseq 500 analysis identified two of the mott as mycobacterium flavescen and mycobacterium moriokaense respectively; the remaining one could not be identified (table 2). table 2a: identity of isolates obtained from the study using both biochemical and molecular assays. table 2b: identity of isolates obtained from the study using both biochemical and molecular assays. discussion top ↑ this study analysed the prevalence of btb in two dairy farms. the overall crude prevalence rate among cattle was 2.48%. this figure is far lower than the findings of a previous study that analysed prevalence of btb in the dangbe-west district (figure 1) of the same administrative region, which had a prevalence rate of 13.80% (bonsu, laing & akanmori 2000). the risk of btb is influenced by a number of factors including: age of animals; breed; herd size; farming practices in terms of stock densities, pasture systems and contact between animals. the main difference in the animals used for this study and another study by bonsu et al. (2000) was the pasture systems used: the present study used animals from a dairy farm, the other study used animals from free grazing farms that were dotted in rural communities. studies conducted in both developed and african countries have shown that dairy cows experience more stress during gathering for milking, which increases the risk of transmission and susceptibility to btb (humblet, boschiroli & saegerman 2009). nevertheless, the practices in rural ghanaian communities, where animals from different farms congregate around the same stream for watering (bonsu et al. 2000), facilitate contact with both humans and animals from different environs. this increases the risk of transmission and susceptibility to btb and could account for the observed differences. in addition, the cattle populations in the current study are from farms where veterinary officers frequently screen animals for infections (dr kikimoto, pers. comm. n.d.). thus, our observation confirms the importance of a test and slaughter control strategy for btb.the distribution of positive animals was not uniform between the farms studied; farm b, which is situated on low land, had a higher farm-specific prevalence than farm a, which is on a hill. local african breeds have been proven over the years to have a high level of resistance to btb due to the high endemicity in africa. exotic breeds, which are mostly from developed counties where the disease has been controlled, are more susceptible to btb (müller et al. 2011). this could explain the observed difference, as farm a stocks mostly local breeds (only two exotic bulls), whereas farm b has few local breeds (10) and mainly exotic animals. in addition, cattle that graze on hilly terrains tend to spread out, which leads to less contact, whilst those that feed on low and wet lands tend to congregate around the areas with rich fodder, which leads to more contact (hall et al. 1978; ameni et al. 2007). thus, the difference in the landscape of the two farms might have contributed to this difference. six afb-positive isolates were obtained; of these, three were mycobacteria other than tuberculosis (mott). the isolation of mott from carcasses of btb-positive reactor cattle not only signifies the importance of these bacteria in pastoral communities but also gives an indication of the mycobacterial status of the environment and water sources, as well as their potential to cause infection. these bacteria are commonly found in environmental sources such as water and soil and have a high survival rate. the farm sites used in the study have created pools within the farms from which the animals drink. these pools were found to be contaminated with animal waste, which is well known to enhance the growth of environmental mycobacterial species. two of the most commonly found mott (m. flavescens and m. moriokaense) were identified in this study. these two, in addition to several others such as mycobacterium terrae, mycobacterium avium, mycobacterium chelonae, mycobacterium gordonae, mycobacterium fortuitum, mycobacterium flavescens and mycobacterium smegmatias, have been implicated as possible causes of btb (cleaveland et al. 2007). the identification of m. tuberculosis and m. africanum as causative agents of btb is not surprising. members of mtbc are generally known to be genetically similar, but differ in host specificity. however, in regions like sub saharan africa that have a high burden of human tb coupled with close animal-human contact, there are occasional cases where cross-host specie infections occur (grange & yates 1996; de kantor & ritacco 1994; romero et al. 2011; cadmus et al. 2010). whilst most studies give the prevalence of m. tuberculosis infection in cattle as ≤1.00%, there are a few exceptions, like algeria and sudan with 6.20% and 7.40% prevalence, respectively (ocepak et al. 2005; boulahbal, benelmouffok & brahimi 1978; sulieman 2002). mycobacterium tuberculosis was isolated from two out of 685 (0.22%) cattle tested with the tuberculin skin test, which put the result in the range of < 1.00%. present knowledge shows that m. tuberculosis does not appear to have an indigenous animal host or reservoir and that the animals which become infected represent most probably accidental hosts acquiring it from humans. in this study, though not directly proven, it can be speculated that animal attendants with active pulmonary tb could be the source of m. tuberculosis infection, transmitting it via sputum or urine. the isolation of m. africanum from cattle in ghana is not surprising. m. africanum has been identified as an important tb-causing pathogen unique to west africa. country-specific prevalence among pulmonary tb patients can be as high as 50%; it causes about 20% of all tb cases in ghana (yeboah-manu et al. 2011; addo et al. 2007). a study conducted in nigeria that analysed 180 mycobacterial isolates from cattle also identified m. africanum in cattle. equally, coscolla et al. (2013) isolated an mtbc from a wild chimpanzee in côte d’ivoire that was shown by comparative genomic and phylogenomic analyses to belong to a new lineage of mtbc that is closer to m. africanum. all of these findings confirm the importance of m. africanum as a pathogen of public health importance to west africa. more resources and efforts need to be put in for understanding both the epidemiology and biology of this pathogen for effective tb control. acknowlegements top ↑ the authors wish to express their profound gratitude to the technical staff, veterinary division, ministry of food and agriculture and to all farms workers of the two government farms included in this study. their immense cooperation contributed towards the success of this work. funding was obtained from the unicef/undp/world bank/who special program for research and training in tropical diseases for dym. competing interests the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. authors’ contributions d.y.m. (university of ghana) conceived and designed the project; a.a.p. (university of ghana) and b.b.k (university of ghana) were responsible for performing the experiment; a.a.p., d.y.m. and k.g.a. 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article information authors: charlotte e. ellis1 vuyokazi e. mareledwane1 roy williams1 david b. wallace2 phelix a.o. majiwa1 affiliations: 1molecular epidemiology and diagnostics programme, agricultural research council-onderstepoort veterinary institute, south africa 2new generation vaccine programme, agricultural research council-onderstepoort veterinary institute, south africa correspondence to: charlotte ellis postal address: private bag x05, onderstepoort 0110, south africa dates: received: 27 aug. 2013 accepted: 11 feb. 2014 published: 27 may 2014 how to cite this article: ellis, c.e., mareledwane, v.e., williams, r., wallace, d.b. & majiwa, p.a.o., 2014, ‘validation of an elisa for the concurrent detection of total antibodies (igm and igg) to rift valley fever virus’, onderstepoort journal of veterinary research 81(1), art. #675, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.675 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. validation of an elisa for the concurrent detection of total antibodies (igm and igg) to rift valley fever virus in this original research... open access • abstract • introduction • research method and design    • recombinant nucleoprotein preparation    • pre-coating and pre-blocking of plates    • double-antigen elisa procedure       • igm capture elisa       • rift valley fever competition elisa    • panel of sera included in the validation of the double-antigen elisa • results    • titration of the recombinant nuceloprotein horseradish peroxidase conjugate    • repeatability of the double-antigen elisa    • analytical specificity    • analytical sensitivity    • diagnostic accuracy    • diagnostic sensitivity and specificity • ethical considerations • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ rift valley fever virus (rvfv) infects humans and livestock, causing haemorrhaging and abortions in animals. three major rvf epizootics have occurred in south africa since the 1950s and the outbreak in 2010 had a mortality rate of 10.7% in humans. accurate and early detection is therefore essential for management of this zoonotic disease. enzyme-linked immunosorbent assays (elisas) have been developed for the detection of either igm or igg antibodies to rvfv in animal sera. in this study, data are presented on the validation of a double-antigen elisa for the simultaneous detection of both classes of antibodies to rvfv in a single test. elisa plates were coated with a recombinant nucleoprotein. the nucleoprotein, conjugated to horseradish peroxidase, was used as the detecting reagent. a total of 534 sera from sheep and cattle were used in the validation. the sheep sera were collected during a rvf pathogenesis study at the agricultural research council (arc) – onderstepoort veterinary institute and the cattle sera were collected during an outbreak of rvf in 2008 at the arc – animal production institute in irene, pretoria. the elisa had a diagnostic sensitivity of 98.4% and a specificity of 100% when compared to a commercial celisa. this convenient and fast assay is suitable for use in serological surveys or monitoring immune responses in vaccinated animals. introduction top ↑ rift valley fever virus (rvfv) belongs to the genus phlebovirus, family bunyaviridae (bishop et al. 1980) and infects humans and livestock, primarily causing haemorrhaging and abortions in animals. the virus is mainly transmitted by mosquitoes, is endemic throughout much of africa and, in recent years, has also spread to saudi arabia and yemen. three major rvf epizootics have occurred in south africa, in 1950–1951, 1973–1975 and, more recently, 2008–2011 (métras et al. 2012). during the outbreak of 2010, 242 human cases were confirmed with a mortality rate of 10.7% (national institute for communicable diseases 2011). during the 2011 outbreak, 32 human cases were confirmed, with no fatalities. in non-endemic countries, rvfv is a concern as a potential agent for bioterrorism. accurate and reliable diagnosis of rvf and efficient surveillance of the disease are therefore essential. rift valley fever virus is an enveloped bunyavirus with a tri-segmented, negative-sense rna genome. the large (l) segment encodes the viral rna-dependent rna polymerase, whilst the medium (m) segment encodes the external glycoproteins (gn and gc) and the non-structural protein (nsm). the small (s) segment is ambisense, coding for the nucleoprotein (n) in the antigenomic sense and the non-structural protein (nss) in the genomic direction (pepin et al. 2010). within the virion, the gn and gc are expressed as a precursor polypeptide. these glycoproteins, being exposed on the outer surface of the virus during infection, are recognised by the host immune system and induce the production of neutralising antibodies. the n protein, gn and gc all elicit the production, firstly, of igm and then igg rvfv-specific antibodies from day 4 – day 8 after infection (jansen van vuren et al. 2007; williams et al. 2011). various techniques are used for the diagnosis of rvf, including virus isolation, virus neutralisation, antigen detection, polymerase chain reaction (pcr) and detection of specific antibodies. virus isolation and virus neutralisation tests take days to perform and present a health hazard to laboratory staff. for this reason, there is a trend to use recombinant antigens in an enzyme-linked immunosorbent assay (elisa) format. most of the elisas employ the recombinant n-protein (fafetine et al. 2007; jansen van vuren et al. 2007; williams et al. 2011). recently, a new elisa was described using the gn protein expressed in an insoluble form in escherichia coli (jäckel et al. 2013). previously, we reported the development and validation of an igg and igm elisa that uses a recombinant n-protein of rvfv (williams et al. 2011). the disadvantage of these elisas is that two separate tests have to be performed, one to detect igm and another to detect igg in sera. the development and validation of a double-antigen elisa (dag elisa) for rvf, which detects both igm and igg in one test, are described in this study. the assay uses a recombinant n-protein (rn) expressed in e. coli as the capture antigen and a rn-horseradish peroxidase (hrp) conjugate for detection – hence the term dag elisa. the main advantage of the dag elisa is that it is non-species specific, as is the case with the previously validated igg and igm elisa. the commercial celisa (idvet innovative diagnostics, grabels, france) has the same advantages, but is very expensive, making it impractical for routine use (comtet et al. 2010). although the dag elisa is useful for detecting the presence of rvfv-specific antibodies, it cannot differentiate between igm and igg classes of antibody. however, this limitation can be overcome by performing follow-up testing using the igm capture elisa described previously (williams et al. 2011). research method and design top ↑ recombinant nucleoprotein preparation a cdna encoding of the n protein gene of rvfv was inserted into a pstaby expression vector (delphi genetics inc., charleroi, belgium) with a protein tag to facilitate purification of the expressed rn. se1 e. coli cells (delphi genetics inc., charleroi, belgium) were transformed with this plasmid and glycerol stocks of the transformants prepared. for expression of rn, an overnight inoculum was prepared in 20 ml luria broth (lb) and the next day upscaled to 1000 ml – 2000 ml lb culture. after induction with 1 mm iptg overnight at 18 °c, the cells were harvested and lysed with bugbuster, containing benzonase and reclysozyme (all from merck kgaa, darmstadt, germany). the cleared cell lysate was used to purify the rn on a metal chelate affinity chromatography column (williams et al. 2011). the rn-hrp was prepared as described by williams et al. (2011). pre-coating and pre-blocking of plates the optimal dilution of the antigen was predetermined using a checker-board titration (crowther 1995). nunc polysorp plates (nunc, roskilde, denmark) were coated with diluted rn in pbs at 100 µl per well. the plates were incubated overnight at room temperature. the following day, they were washed three times with 300 µl tst washing solution (50 mm tris/150 mm nacl/0.1% tween 20, ph 8.0) per well and then blocked with 100 µl of 1 × stabilcoat buffer (surmodics inc., eden prairie, minnesota, usa) per well. after 1 h at room temperature, the solution was discarded without washing and the plates tapped gently on paper towelling to remove remaining solution. the plates were then dried for 2 h in a laminar flow cabinet, vacuum sealed and stored at 4 °c – 8 °c until required. double-antigen elisa procedure the pre-coated, pre-blocked elisa plates were equilibrated to room temperature and 50 µl of dilution buffer (3% non-fat milk powder in tst buffer) was added to each well. thereafter, 50 µl of control or serum samples were added (the dilution buffer only served as the conjugate control). the plates were incubated for 45 min at room temperature and then washed three times with tst buffer. the rn-hrp conjugate was diluted in dilution buffer and 100 µl added per well. the plates were then incubated for 30 min at room temperature and washed as before, after which 100 µl of tmb ready-to-use substrate (invitrogen corporation, frederick, maryland, usa) was added to each well and incubation was performed in the dark for 10 min. then, 100 µl of stop solution (2 n h2so4) was added per well and the absorbance was read at a450 nm. the raw absorbance (a) values were expressed as a percentage positive (pp) of the positive control, using the following formula: igm capture elisa the rvfv-specific igm capture elisa was used as part of this study to confirm outlier results (williams et al. 2011). rift valley fever competition elisa a commercial, indirect competition elisa (celisa) kit (id screen rift valley fever multi-species elisa; idvet innovative diagnostics, grabels, france) was used according to the manufacturer’s instructions for detecting rvfv-specific antibodies. given that it has a validated diagnostic sensitivity and specificity of 100%, the kit was used as the standard reference for evaluating the dag elisa. the results were calculated as competition percentage, using the following formula: a suspect or negative (s/n) value of ≤ 40% was considered to be positive. panel of sera included in the validation of the double-antigen elisa an rvf pathogenesis study was performed in sheep using a dose-titration approach at the agricultural research council – onderstepoort veterinary institute (arc–ovi) as part of the development and evaluation of a recombinant rvfv vaccine. this study provided an opportunity to collect a large panel of sera for the validation of the dag elisa. briefly, 30 6-month-old merino sheep, confirmed sero-negative for rvfv, were divided randomly into five equal groups and infected intravenously with a virulent south african rvfv strain (m35/74), using titres ranging from 102 to 106 plaque-forming units (pfu) per animal per group. blood samples were collected daily for the first 10 days post infection (pi) and then every fourth day until the end of the trial on day 27 pi. all samples were tested using both the dag elisa and a commercial celisa. for the purposes of this validation, the results of the dag elisa were compared with those of the celisa. the celisa detects antibodies to rvfv n protein and, according to the manufacturer, has a 100% diagnostic sensitivity and specificity when compared to the serum neutralisation test. a total of 412 sera from the rvf pathogenesis study were tested and compared in the dag elisa and the celisa, of which 216 were positive and 196 negative. in addition, 121 bovine sera from a natural rvf outbreak in 2008 on the farm at the arc – animal production institute (irene, pretoria) were included, of which 26 were positive and 95 negative. the results of all sera tested were included in the receiver operating characteristic (roc) analysis to determine the cut-off value for the dag elisa. results top ↑ all samples were tested using both the dag elisa and a commercial celisa. for the purposes of this validation, the results of the dag elisa were compared with those of the celisa. titration of the recombinant nuceloprotein horseradish peroxidase conjugate the rn-hrp conjugate was titrated on a pre-coated, pre-blocked rvfv igg elisa plate to determine the optimal dilution of the conjugate to be used in the validation of the dag elisa. this was determined to be 1:640, where the absorbance (a450) of the positive control was ≥ 1.00 and that of the negative control was 0.02 (figure 1). figure 1: two-fold serial dilution of the recombinant nucleoprotein horseradish peroxidise conjugate on pre-coated elisa plates to determine the optimal dilution for use in the double-antigen elisa. repeatability of the double-antigen elisa the stability of the rn antigen-coated plates was established previously as part of the validation of the rvfv igg elisa (fafetine et al. 2007). each control was tested in quadruplicate wells on each plate and served as internal quality controls. the repeatability of the positive control, negative control and the conjugate control was demonstrated with the average and the standard deviation for 15 individual plates tested on 15 different days (figure 2). the a450 nm of the positive serum ranged between 0.70 and 1.80, of which the negative control was < 0.10 and the conjugate control < 0.03. (a450 nm of < 0.10 for the negative control is acceptable for an elisa validation). the percent coefficient of variance (% cv) within plates varied between 1.30% (lowest) to 12.50% (highest). the % cv of the positive control between the 15 plates was 20.49%. figure 2: repeatability of the double-antigen elisa when tested with the control sera for (a) the positive control, (b) negative control and (c) conjugate control. analytical specificity although the possibility of cross-reacting antibodies being detected by the dag elisa was not addressed here, previous antigenic cross-reactivity studies in sheep (swanepoel et al. 1986) and field studies in cattle (davies 1975; swanepoel 1976) did not find any cross-reactivity of sera from rvfv-infected convalescing animals with any other african phleboviruses. analytical sensitivity to determine the detection limit of the dag elisa and the celisa, serial two-fold dilutions of the positive control were made and tested in both elisas. the results are presented for the titrations of the positive control (figure 3). in both elisas, the undiluted positive control was used to calculate the pp values of the dilutions. figure 3: analytical sensitivity of the double-antigen elisa compared to the celisa for the detection of the positive control and dilutions thereof. diagnostic accuracy all samples were tested with the dag elisa and commercial celisa. for the purposes of this validation, the results obtained using the dag elisa were compared with those obtained for the celisa. the average antibody titres of the sera from infected sheep were used to plot the mean of the dag elisa and celisa results (figure 4). the igm and igg serological immune responses of the sheep after infection with rvfv strain m35/74 are presented in figure 4. in addition, the separate responses for igm and igg were also plotted on this graph, as mentioned, using the individual igg and igm elisas. the first detectable antibodies occurred at day 5 pi, with the first peak in levels between day 6 pi and day 9 pi, as measured using the celisa and dag elisa, which correlated with the igm response as supported by the igm capture elisa. both the celisa and dag elisa then showed a decrease in antibody levels, corresponding to a drop in igm levels. from day 9 pi, igg antibodies were detectable (igg indirect elisa), rising sharply until day 14 pi, then remaining relatively constant, but with a slight rise, until day 27 pi, the last day of sampling of the animals. however, with the dag elisa, a steady downward trend was observed from day 10 pi until day 14 pi, then continuing in line with the igg response of the indirect igg elisa until day 27 pi. the pp values for the sera from the negative and positive reference groups are plotted in figure 5. of the 291 negative sera tested, 286 had pp values of ≤ 20%, whilst only five had pp values of ≥ 20% but ≤ 40% – and of the 243 positive sera tested, only two sera had pp values of ≥ 20% but < 40%, whilst the remainder had pp values of ≥ 45%. a selected cut-off value (positive–negative threshold) should differentiate optimally between subpopulations of infected and uninfected animals. a roc curve analysis (graphpad prism 5.0 2010) was performed on the 291 negative and 243 positive sera using a 95% confidence interval (table 1). the roc curve of the dag elisa sera data used in this study had an area under the curve equal to 0.998. the sensitivity and specificity of the dag elisa was plotted against the entire range of possible pp values (figure 6). the cross-over point of the two lines indicates the pp value for optimum sensitivity and specificity, in this case a pp of > 30.0%, where the likelihood range is the largest, namely 288.6, with the sensitivity calculated at 99.2% and the specificity at 99.7%. by including a suspect range of values between 30.0% and 40.0%, the sensitivity was calculated at 98.4% and the specificity at 99.7% (table 2). table 1: receiver operating characteristic curve analysis of the double-antigen elisa. table 2: cut-off value ranges for the double-antigen elisa. figure 4: average anti-rift valley fever virus nucleoprotein-specific antibody titres in sheep sera collected after infection with virulent rift valley fever virus. figure 5: average anti-rift valley fever virus nucleoprotein-specific antibody titres in sheep sera collected after infection with virulent rift valley fever virus. figure 6: percentage positive values for the negative (n = 291) and positive (n = 243) sera. diagnostic sensitivity and specificity the selection of a cut-off value allowed test results to be divided into positive or negative categories. the dag elisa results of the sera were classified as true positive or true negative if they were in agreement with the celisa results or, alternatively, as false positive or false negative if they differed from the celisa results. in some instances, the results of the igm capture elisa were taken into consideration. if the cut-off value of ≥ 40% was used as positive, the sera were classified accordingly (table 3). some outliers are included in table 4. with the idvet celisa, a number of the sera were classifiable as negative or suspect, but were positive in the igm elisa. these sera were obtained on day 4 pi or day 5 pi when the igm antibodies were first measurable. the cut-off point for a positive result in the igm elisa was calculated at pp values of > 7%.a number of the sera tested negative or suspect using the celisa (sera #231, #234, #192), but tested positive in the igm and the dag elisas. serum #216 and serum #191 were positive in the igm elisa, but suspect in the dag elisa and negative, or suspect, in the celisa. none of these sera had detectable levels of igg. the igg level only started to appear from day 9 to 10 after inoculation. table 3: summary of diagnostic sensitivity and specificity. table 4: outliers as tested using the three elisas. ethical considerations top ↑ ethical approval for all procedures involving the rvf pathogenesis trial in sheep was granted by the arc-ovi animal ethics committee, pretoria, south africa. discussion top ↑ the rvfv outer surface glycoproteins, gn and gc, induce the production of neutralising antibodies, which afford protection in infected and vaccinated animals (collett et al. 1985). however, the n protein is the most immunodominant antigen when compared to the glycoproteins, as it elicits higher levels of antibodies when analysed on western blots containing the viral proteins (data not shown) and is highly specific for rvfv (jansen van vuuren et al. 2007; williams et al. 2011). when expressed in e. coli, the recombinant n-protein is soluble and has excellent stability at 4 °c (fafetine et al. 2007). pre-coated elisa plates have a shelf life of at least two years when sealed and stored at 4 °c (data not shown). production of the recombinant n-protein is very convenient when compared to the risks involved in working with live rvfv and the processes required for viral inactivation. the soluble nature of the antigen allows for conjugation to hrp, making it ideal for use in diagnostic elisas.in this study we developed and compared a dag elisa using recombinant rvfv n-protein. the major advantage of the dag elisa and the commercial celisa with which it was compared is that they can simultaneously detect igm and igg classes of antibody and neither is species-specific. the dag elisa has a diagnostic sensitivity and specificity of 98.4% and 100%, respectively, whilst its analytical sensitivity compares favourably with the celisa. however, the main reason for developing and using the dag elisa in-house is that it is affordable and therefore accessible to diagnostic veterinary laboratories of many developing countries where rvf is endemic. conclusion top ↑ the data presented in this study show that the dag elisa can be used as a safe, reliable, fast and highly accurate diagnostic tool, with good potential for use in serological surveys or for monitoring immune responses in vaccinated animals. acknowledgements top ↑ this research project was funded in part by galvmed. the authors wish to thank the arc-ovi transboundary animal diseases programme team for animal care, sample collection and processing during the rvfv pathogenesis study. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions the elisa validation was performed by v.e.m. (arc-onderstepoort veterinary institute) the rn production, purification and rn-hrp conjugate preparation was undertaken by c.e.e. (arc-onderstepoort veterinary institute). the manuscript was written and submitted by c.e.e., with editing by r.w. (arc-onderstepoort veterinary institute), d.b.w. (arc-onderstepoort veterinary institute) and p.a.o.m. 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feng1 hui-ying fan1 jing-yi li1 ji-dang chen1 bin zhang1 affiliations: 1the key laboratory of animal disease control and prevention of the ministry of agriculture, south china agricultural university, china2department of biotechnology research, veterinary research institute of guangdong academy of agricultural sciences, china correspondence to: cheng-gang xu postal address: 483 wushan road, tianhe district, guangzhou city, guangdong province, china dates: received: 05 oct. 2011 accepted: 13 dec. 2011 published: 24 apr. 2012 how to cite this article: zhang, j., shen, h., liao, m., ren, t., guo, l., xu, c. et al., ‘detection of haemophilus parasuis isolates from south china by loop-mediated isothermal amplification and isolate characterisation’, onderstepoort journal of veterinary research 79(1), art. #383, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.383 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. detection of haemophilus parasuis isolates from south china by loop-mediated isothermal amplification and isolate characterisation in this original research... open access • abstract • introduction • materials and methods    • bacterial strains    • clinical samples    • bacterial isolation    • dna extraction    • development of the loop-mediated isothermal amplification and polymerase chain reaction methods    • detection of haemophilus parasuis using loop-mediated isothermal amplification and polymerase chain reaction    • serotyping of haemophilus parasuis isolates    • genotyping of haemophilus parasuis by the pulsed-field gel electrophoresis method    • ethical considerations • results    • rapid, sensitive and specific detection of haemophilus parasuis using loop-mediated isothermal amplification    • detection and isolation of haemophilus parasuis    • serotyping and genotyping of haemophilus parasuis isolates • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ haemophilus parasuis is the etiological agent of glässer’s disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. in this study, a loop-mediated isothermal amplification (lamp) test was developed to improve the specificity, facility and speed of diagnosis of h. parasuis isolates. the lamp assay rapidly amplified the target gene within 50 min incubation at 63 °c in a laboratory water bath. the lamp amplicon could be visualised directly in the reaction tubes following the addition of sybr green i dye. the detection limit of this lamp method was 10 cfu/ml, which was 10 times more sensitive than the earlier 16s rrna polymerase chain reaction (pcr) test conducted by oliveira, galina and pijoan (2001), and no cross-reactivity was observed from other non-h. parasuis strains. this lamp test was evaluated further on 187 clinical specimens from pigs suspected of being infected with h. parasuis. forty-three were found positive by bacterial isolation of h. parasuis, as well as by the 16s rrna pcr and lamp tests. the 43 h. parasuis isolates were classified into 9 serovars and had 37 genetic patterns when analysed by pulsed-field gel electrophoresis (pfge). this displayed that various h. parasuis serovars and genotypes were widely distributed in south china. therefore, the speed, specificity and sensitivity of the lamp test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of h. parasuis in favour of controlling glässer’s disease. introduction top ↑ haemophilus parasuis is the causative agent of glässer’s disease in swine, a disease characterised by fibrinous polyserositis, meningitis and polyarthritis (li et al. 2009; olvera, segalés & aragón 2007). in recent years, diseases in swine caused by h. parasuis have attracted worldwide attention (baumann & bilkei 2002). it is therefore necessary to carry out timely and effective monitoring of h. parasuis to identify strains circulating between farm animals as a means of defining the epidemiology and types of h. parasuis strains present in respective areas in order to control and prevent outbreaks.haemophilus parasuis is a slow-growing, fastidious organism with specific nutritional requirements, making its isolation difficult (oliveira, galina & pijoan 2001). moreover, detection of h. parasuis using conventional methods, including culture, biochemical or immunological assays is not effective and requires several days to complete (angen et al. 2007). the polymerase chain reaction (pcr) provided a major advance in the diagnosis of h. parasuis infections (oliveira et al. 2001), including nested pcr (jung et al. 2004) and real-time pcr assays (turni, pyke & blackall 2009). however, pcr assays are limited by the equipment required to carry out the reactions and the need for skilled personnel. fortunately, a promising candidate for a new detection method is loop-mediated isothermal amplification (lamp), which amplifies dna with high specificity, efficiency and rapidity under isothermal conditions. it is faster, more specific and easier to perform than conventional pcr (nagamine, hase & notomi 2002; notomi et al. 2000). the development of lamp procedures has been reported for many different clinical applications: the diagnosis of viral and bacterial infections (iwamoto, sonobe & hayashi 2003; minami et al. 2006; song et al. 2005; wang et al. 2009), for diagnosis of protozoal diseases (kuboki et al. 2003; thekisoe et al. 2005) and for canine and equine piroplasmosis (alhassan et al. 2007; ikadai et al. 2004). the infb gene of h. parasuis has been proven to be useful as a genetic marker for phylogenetic studies (turni et al. 2009), making it a better alternate gene to the 16s rrna gene in establishing the lamp method. to date, 15 h. parasuis serovars have been recognised using immunodiffusion tests with heat-stable antigens. however, about 25% of isolates are non-typable using this methodology (vanier et al. 2006). in view of the fact that serotyping does not provide enough discrimination of h. parasuis isolates, an optimised and standardised pulsed-field gel electrophoresis (pfge) method established by our research team was used to characterise the h. parasuis isolates. this method provides a higher discriminatory power, allowing for the typing of previously undefined isolates (zhang et al. 2011). in the present study, 15 serovar reference strains were used to develop the lamp assay of h. parasuis. in addition, the h. parasuis strains isolated from clinical specimens collected from south china, were serotyped and characterised by pfge patterns. materials and methods top ↑ bacterial strains the test was optimised using the 15 reference strains for the different serovars of h. parasuis and the field isolates of actinobacillus pleuropneumoniae (serovar 5), a. pleuropneumoniae (serovar 9), two strains of bordetella bronchiseptica, pasteurella multocida, streptococcus suis, salmonella typhimurium and escherichia coli. clinical samples from 2008 to 2010, 187 specimens, including lung, nasal swab, synovia and heart from pigs suspected of being infected with h. parasuis were collected in south china. bacterial isolation all 187 specimens were cultured on tryptic soy agar (oxoid inc., basingstoke, uk) containing 10 mg/ml nicotinamide adenine dinucleotide (nad, sigma-aldrich, st. louis, usa) and 5% bovine serum (gibco®, invitrogen, carlsbad, usa) for isolation of h. parasuis strains. plates were incubated at 37 °c for 24 h – 72 h and presumptive small and translucent bacterial colonies were selected for further characterisation by pcr, lamp and biochemical testing. dna extraction template dna used for lamp and 16s rrna pcr were prepared as follows: bacterial cells of each strain from colonies on trypticase soy agar (tsa) were re-suspended in double-distilled water (ddh2o) to achieve a concentration of approximately 106 cfu/ml. in order to examine the detection limit for lamp and 16s rrna pcr, a 10-fold dilution series of h. parasuis of serovar 5 with ddh2o was made. the cells were heat-treated in a boiling water bath for 10 min and were centrifuged at 12 000 rpm/min for 10 min. the resulting supernatant was used as the template for the lamp and 16s rrna pcr. development of the loop-mediated isothermal amplification and polymerase chain reaction methods four primers corresponding to fip, bip, f3 and b3 (table 1) were used for the lamp assay and designed based on the conserved regions of the infb gene sequence (genebank accession numbers: ef424388), according to criteria established previously (notomi et al. 2000; tomita et al. 2008). the reaction mixture consisted of 0.8 μm of each fip and bip primers, 0.2 μm of each f3 and b3 outer primers, 400 μm of each dntp (takara bio inc., shiga, japan), 1 m betaine (sigma-aldrich, st. louis, usa), 1x thermopol buffer (new england biolabs inc., ipswich, usa), 2 mm mgso4, 8 u of bst dna polymerase large fragment (new england biolabs inc., ipswich, usa) and 2 μl of genomic dna. the reaction mixture was incubated at 61 °c, 62 °c, 63 °c, 64 °c or 65 °c using a heat block for 20 min, 30 min, 40 min, 50 min or 60 min, respectively. reactions were terminated by increasing the temperature to 80 °c for 4 min. to determine the sensitivity of the lamp assay, different dilutions of h. parasuis from 1 × 106 cfu/ml to 1 × 101 cfu/ml (i.e. a series of 10-fold dilutions) was prepared and compared with pcr by using the same templates at identical concentrations. the details of the primers (table 1) and conditions for the pcr assay for the detection of h. parasuis were carried out as described previously (oliveira et al. 2001). cross-reactivity between the primers used in lamp pcr with dna isolated from a. pleuropneumoniae, b. bronchiseptica, p. multocida, s. suis, s. typhimurium and e. coli was also carried out. genomic dna extracted from the 15 h. parasuis reference strains were used as positive controls and distilled water was used as a negative control. table 1: loop-mediated isothermal amplification (lamp) primers designed for detection of the haemophilus parasuis infb gene. detection of haemophilus parasuis using loop-mediated isothermal amplification and polymerase chain reaction detection of the lamp reaction products was performed using 2% agarose gel electrophoresis prepared in 0.5x tris-borate-edta buffer stained with 0.5 g/ml ethidium bromide. banding patterns were examined visually under natural or uv light following the addition of 1 μl sybr green i dye to the reaction tube. pcr products were subjected to electrophoresis on a 2% agarose gel and visualised as above. serotyping of haemophilus parasuis isolates serotyping of h. parasuis isolates using gel diffusion (gd) and indirect hemagglutination assays (iha) were performed as described previously (cai et al. 2005; tadjine et al. 2004) and all isolates were tested using gd first. if a definitive serotype could not be determined, respective isolates were then examined by iha testing. isolates that could not be identified by either test were defined as non-typable (serovar nt). genotyping of haemophilus parasuis by the pulsed-field gel electrophoresis method haemophilus parasuis field isolates were genotyped using the pfge method established by our research team to determine the number of different genotypes (zhang et al. 2011). a dendrogram illustrating the genetic relatedness between identified h. parasuis isolates was generated using bionumerics 4.0 software (applied maths, inc., austin, usa), with a 1.5% tolerance for fragment shifts. ethical considerations all animal experiments conducted for the purposes of this study conform to the chinese national laws regarding the use of animals in biomedical research. results top ↑ rapid, sensitive and specific detection of haemophilus parasuis using loop-mediated isothermal amplification the lamp method described in this study was performed in a simple water bath at 63 °c for 50 min. all positive lamp reactions produced a characteristic ladder of multiple bands and the results could also be seen after staining with sybr green i (figure 1). the detection limit of lamp was 10 cfu/ml, which was 10 times more sensitive than 16s rrna pcr (figure 1). lamp accurately detected all 15 h. parasuis reference strains and no amplification products were detected when either dna from the other swine bacterial pathogens, or the negative (no template) reaction control was used (figure 2). figure 1: detection of (a) infb loop-mediated isothermal amplification (lamp) and (b) polymerase chain reaction products, as well as visual detection of infb-lamp products under (c) daylight and (d) ultraviolet light. figure 2: specificity tests of loop-mediated isothermal amplification in agarose gel under ultraviolet light for haemophilus parasuis detection. detection and isolation of haemophilus parasuis a total of 187 clinical specimens were obtained from pigs in south china that were suspected of being infected with h. parasuis. eighty-nine (47.6%) and 64 (34.2%) samples tested positive for h. parasuis by lamp and pcr, respectively. forty-three samples (23.0%) were positive for h. parasuis by bacterial isolation and all the 43 samples tested positive for h. parasuis by pcr and lamp (table 2). table 2: comparative analysis of haemophilus parasuis detection by either loop-mediated isothermal amplification, polymerase chain reaction or bacterial isolation. serotyping and genotyping of haemophilus parasuis isolates nine of the fifteen recognised serovars were identified amongst the isolates collected from south china. serovar 5 was the most prevalent (23.3%), followed by serovar 4 (16.3%), whilst 25.6% of strains were non-typable by either gd or iha. serovars 2, 6, 7, 9, 12, 13 and 15 were represented by a small number of strains only. all 43 h. parasuis isolates were typed by pfge and great genetic diversity was observed. a total of 37 different pfge patterns were observed from the 43 h. parasuis isolates (figure 3 and table 3). in addition, strains that could not be typed by gd or iha tests were genotyped by pfge and displayed unique pfge patterns. genotyping analysis demonstrated that the h. parasuis population was heterogeneous and no dominant clones predominated. figure 3: dendrogram of pulsed-.eld gel electrophoresis subtype patterns of all 43 haemophilus parasuis field strains identified. table 3: characterisation of 43 haemophilus parasuis field isolates from south china. discussion top ↑ haemophilus parasuis is a commensal of the upper respiratory tract in domestic pigs and is also the etiological agent of glässer’s disease (oliveira et al. 2001). however, the bacterium can cause acute disease when introduced into immunologically naive herds (little 1970). laboratory diagnosis of h. parasuis using conventional methods such as culture, biochemical and immunological assays is time-consuming and laborious. haemophilus parasuis is a fastidious bacterium and its isolation in pure culture from diseased animals is usually difficult; therefore, rapid and accurate identification of h. parasuis isolates is essential for the diagnosis of glässer’s disease and a key element for controlling the disease (aarestrup, seyfarth & angen 2004; oliveira & pijoan 2004). a fast and accurate lamp test was developed to improve the diagnosis of h. parasuis infections. this lamp test can be used to detect h. parasuis in both pure culture and clinical samples. the lamp method can amplify dna with high specificity, efficiency and speed under isothermal conditions (nagamine et al. 2002; tomita et al. 2008). the lamp assay also can be performed on-site, using a simple and inexpensive experimental set-up, such as a water bath or heat block that provides a constant temperature. in addition, the lamp reaction products can be evaluated by a visual inspection of the colour change of the mixture when sybr green i dye is added. after isolation of h. parasuis from the clinical specimens, the lamp can be performed as a substitute for 16s rrna pcr tests for bacterial identification, eliminating the need for time-consuming electrophoresis and costly specialised equipment, as well as reducing the time for the diagnosis of h. parasuis infection considerably. it has been reported that the 16s rrna gene is not species-specific to enable h. parasuis to be distinguished from other closely related species when the short amplification sequence of real-time pcr was used (turni et al. 2009). recently, researchers have reported that the infb gene is useful as a genetic marker for phylogenetic studies. this observation is also true for h. parasuis, in that the infb gene be used to distinguish h. parasuis from all other closely related species, such as actinobacillus suis and a. pleuropneumoniae (hedegaard et al. 2000; turni et al. 2009). in this study, we evaluated the applicability and efficacy of infb-lamp for the identification of h. parasuis from 187 different specimens. analysis of these samples by infb-lamp resulted in the positive identification of 89 h. parasuis samples, compared to only 64 positive samples identified by pcr. the results of this study also indicate that h. parasuis is widely distributed in south china. moreover, the infb-lamp technique has the potential to be applied in clinical settings in which 16s rrna-pcr is too sophisticated to be implemented. in addition, it is a suitable tool for investigating the epidemiology of h. parasuis in a clinic. detection and isolation of h. parasuis is important in understanding the epidemiology of h. parasuis infections and can provide critical information used in the design of vaccines specific to strains endemic to certain geographic locations. in this study, the h. parasuis serovars present in south china were determined by gd and iha. these results showed that 32 (74.4%) h. parasuis isolates could be assigned accurately to a specific serovar, whilst nine (20.9%) h. parasuis serovars were identified and 11 (25.6%) isolates are typically non-typable. so, the pfge method was used to analyse the genetic diversity of h. parasuis isolates collected from south china and a computer analysis program was used to determine similarities between isolates, thereby providing a more objective assessment of clonality (in conjunction with epidemiologic data) that can be used to establish h. parasuis as the etiologic agent of animal disease in this region. conclusion top ↑ in this study, our results indicate that infb-lamp can be used in the diagnosis of h. parasuis infections, which is essential in defining the exact prevalence of glässer’s disease in south china. combining this assay with traditional serotyping methods and a highly discriminatory pfge genotyping technique will provide comprehensive information regarding the epidemiology of h. parasuis in south china. knowledge of the h. parasuis strains present in this region can be used in the rational design of vaccines against these isolates, in addition to providing information useful to epidemiologic research. acknowledgements top ↑ we would like to thank professor huanchun chen (college of veterinary medicine, huazhong agricultural university) for providing 15 h. parasuis reference strains. this work was supported by grants from the program for new century excellent talents in university (grant no. ncet-06-0752) and guangdong technology planning committee (grant no. 2006b0152, 2009a0201006, 2009b030803050 and 2011b020306010). competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions c.x. (south china agricultural university) was the project leader, h.s. (south china agricultural university) and j.z. (south china agricultural university) were responsible for experimental and project design and m.l. (south china agricultural university), s.f. (south china agricultural university) and b.z. (south china agricultural university) were in charge of clinical samples collection. l.g. (south china agricultural university) and j.l. (south china agricultural university) performed most of the bacterial isolation and pcr detection, whilst h.f. (south china agricultural university), j.c. (south china agricultural university) and t.r. 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lacouture, s., jacques, m. & gottschalk, m., 2006, ‘haemophilus parasuis invades porcine brain microvascular endothelial cells’, microbiology 152, 135–142. http://dx.doi.org/10.1099/mic.0.28312-0, pmid:16385123 wang, y., chen, p., guo, h.b., chen, y., liu, h. & he, q.g., 2009, ‘loop-mediated is othermal amplification targeting the apxiva gene for detection of actinobacillus pleuropneumoniae’, fems microbiology letters 300, 83–89. zhang, j., xu, c., guo, l., ke, b., ke, c., zhang, b. et al., 2011, ‘a rapid pulsed-field gel electrophoresis method of genotyping haemophilus parasuis isolates’, letters in applied microbiology 52, 589–595. article information authors: edward k. kariuki1,2 barend l. penzhorn2 ivan g. horak2 affiliations: 1department of veterinary and capture services, kenya wildlife service, kenya 2department of veterinary tropical diseases, university of pretoria, south africa correspondence to: ivan horak postal address: private bag x04, onderstepoort 0110, south africa dates: received: 10 apr. 2012 accepted: 22 aug. 2012 published: 21 nov. 2012 how to cite this article: kariuki, e.k., penzhorn, b.l. & horak, i.g., 2012, ‘ticks (acari: ixodidae) infesting cattle and african buffaloes in the tsavo conservation area, kenya’, onderstepoort journal of veterinary research 79(1), art. #437, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.437 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ticks (acari: ixodidae) infesting cattle and african buffaloes in the tsavo conservation area, kenya in this research communication... open access • abstract • communication • conclusion • acknowledgements    • competing interests    • author’s contributions • references abstract top ↑ several ixodid tick species are shared between domestic cattle and african buffaloes (syncerus caffer). so too, are a number of tick-borne diseases. the aim of the study was to compare the species composition of ticks that infest cattle and buffaloes utilising the same habitat within the tsavo conservation area, kenya. to this end, 25 cattle and 62 buffaloes were each opportunistically sampled for ticks on a single occasion in february 2010. eight species, namely amblyomma gemma, amblyomma lepidum, hyalomma albiparmatum, hyalomma rufipes, hyalomma truncatum, rhipicephalus evertsi evertsi, rhipicephalus pravus and rhipicephalus pulchellus infested both cattle and buffaloes. three species, rhipicephalus (boophilus) sp., rhipicephalus kochi, and rhipicephalus muehlensi were collected only from cattle, and three species, hyalomma impeltatum, rhipicephalus humeralis and rhipicephalus praetextatus were present only on buffaloes. the attachment sites of the various tick species were also recorded. new locality records for h. impeltatum and h. truncatum and the first confirmed locality record for rhipicephalus praetextatus sensu stricto in kenya were documented. communication top ↑ the adults of a large number of ixodid tick species infest both domestic cattle and african buffaloes (syncerus caffer) in kenya as well as elsewhere in africa (horak 1999; horak, golezardy & uys 2007; londt, horak & de villiers 1979; walker 1974). several of these tick species are vectors of disease, but whereas buffaloes are seldom affected by tick-borne diseases, they can have devastating effects on cattle. the two most noteworthy of these diseases are heartwater, caused by ehrlichia ruminantium and transmitted by certain amblyomma species and east coast fever and corridor disease, caused by theileria parva and transmitted by rhipicephalus appendiculatus (allsopp, bezuidenhout & prozesky 2004; lawrence, perry & williamson 2004). although the tick species that infest cattle and african buffaloes in kenya and in the eastern cape province of south africa have been meticulously recorded (horak 1999; horak et al. 2007; walker 1974), to our knowledge no surveys have been done on the ticks that occur on these animals in a common habitat. moreover, the ticks infesting cattle and wildlife in kenya and their geographic distributions were last reviewed in the 1960s (walker 1974). since then, vegetation cover and farming practices have undergone several changes, and tick species diversity and distribution have probably also changed in response to these events. the objectives of the present study were to identify and compare the species of ticks that infest cattle and african buffaloes sharing the same habitat within the greater tsavo conservation area in south-eastern kenya. the predilection attachment sites of adult ticks on the two host species were also recorded. the survey was conducted in the tsavo east and tsavo west national parks and the privately owned taita ranches and human settlement areas which form a partial corridor between the parks in the south-east. since the parks are not fenced, most cattle utilise the park’s pastures during the dry seasons, with their owners finding themselves in the parks without intent, or deliberately entering them in search of fresh pastures. on the other hand, the ranches, numbering approximately 20, constitute a vital dispersal area for wildlife. the parks and ranches comprise the greater tsavo conservation area, which lies between 04.20° s, 37.65° e and 01.92° s, 39.36° e in the south-east of kenya and borders on tanzania in the south. the vegetation of the parks comprises open plains alternating from grasslands and bushlands to semi-arid acacia shrub and woodlands (tolvanen 2004). during february 2010 a single opportunistic collection of ticks was made from individual cattle at a community cattle dip-tank at bachuma (an area adjacent to the tsavo east national park), and from a small herd of cattle held at the maktau gate of tsavo west national park because of illegal entry into the park, as well as from individual animals at other randomly distributed localities. a total of 25 cattle were sampled in this way. all these animals had been subjected to weekly spraying with an acaricide. in the same month, ticks were also collected opportunistically from each of 62 immobilised african buffaloes during a rinderpest surveillance campaign within the tsavo conservation area. tick collection was done by hand or with forceps from six sites on each animal (one ear, one side of the neck, one leg including the foot, the whole tail including the tail brush, the whole upper perineum, and one half of the lower perineum). ticks from each body region of each animal were first placed separately in bijou bottles containing boardman’s solution (80% water, 3% di-ethyl ether and 17% alcohol) in order to kill them. they were removed from the solution after 24 hours and placed in vials containing a long-term preservative (80% alcohol, 14% water and 5% glycerol to which 1% chloroform was added to preserve their colour). identification labels were written in pencil and placed in the sample bottles with the ticks in order to ensure correct identification of the samples. tick identification and counting were done using a stereoscopic microscope. taxonomic descriptions and illustrations were used as aids towards identification (walker 1960; walker & olwage 1987; walker, keirans & horak 2000). ticks whose identity could not be determined, were sent to the department of veterinary tropical diseases, university of pretoria, for identification. the species and numbers of ticks collected from cattle and buffaloes in the tsavo conservation area are summarised in tables 1 and 2. a total of 14 ixodid tick species were collected from the cattle and buffaloes and 8 of these species infested both hosts. rhipicephalus (boophilus) sp., rhipicephalus kochi and rhipicephalus muehlensi were present only on cattle, and hyalomma impeltatum, rhipicephalus humeralis and rhipicephalus praetextatus were collected only from buffaloes. the results indicate that a large number of tick species, of which the adults may infest cattle and/or buffaloes, are present in the tsavo conservation area. walker (1974) recorded 14 such species in this region. three of the species mentioned by walker (1974), namely amblyomma variegatum, r. appendiculatus and rhipicephalus maculatus were not collected in this survey. the absence of a. variegatum, a major vector of e. ruminantium and r. appendiculatus, the vector of t. parva, on the 87 animals currently sampled, begs further investigation in the light of walker’s earlier survey. in the present survey the proportions of cattle and buffaloes infested with amblyomma gemma were similar, as were the proportions infested with amblyomma lepidum. both ticks are potential vectors of e. ruminatium, but are not regarded as playing a significant role in its transmission in the field (allsopp et al. 2004; walker & olwage 1987). amblyomma gemma was collected from the neck of cattle and from the leg, upper and lower perineum and tail of both cattle and buffaloes, whilst a. lepidum was collected from the neck, leg and lower perineum. these attachment sites largely agree with those of yeoman and walker (1967) and walker (1974), who recorded the ventral surface of the body from the dewlap and axilla to the escutcheon as preferred attachment sites for these ticks. four hyalomma species were collected in this study. two of the 25 cattle examined were infested with hyalomma albiparmatum and six of the 62 buffaloes. walker (1974) recorded 30 collections of this tick from cattle and none from buffaloes. ticks were collected from the tail and tailbrush of cattle, and from the tail and lower perineum of buffaloes, the same sites as those reported by walker (1974). few collections of h. impeltatum have been reported from kenya and none from the region of the tsavo conservation area (walker 1974). the collection of three ticks from the lower perineum of a single buffalo at this locality thus constitutes a new locality record. no ticks belonging to this species were collected from cattle. walker (1974) lists 25 confirmed records of collections of hyalomma rufipes from cattle in kenya and five from buffaloes. in this survey, six cattle and 31 buffaloes were infested and ticks were recovered from the upper perineum and tail of cattle and from the ears, legs, lower perineum and the tail of buffaloes. these attachment sites agree with those recorded by londt et al. (1979). walker (1974) reports 31 confirmed records of hyalomma truncatum in 892 collections from cattle and 10 in 119 collections from buffaloes. in the present study, two cattle and two buffaloes were infested. h. truncatum was present on the upper perineum and tail of cattle and on the legs and tail of buffaloes. in addition to these attachment sites, londt et al. (1979) recorded it from the lower perineum. the collection of h. truncatum in the tsavo conservancy constitutes a new locality record for this species. the distance between this site and the closest locality recorded by walker (1974) is approximately 250 km. table 1: ticks collected from cattle in the tsavo conservation area, kenya. table 2: ticks collected from african buffaloes in the tsavo conservation area, kenya. the single female rhipicephalus (boophilus) sp. tick, collected from a cow, could not be identified to species level because of damage to its mouthparts. walker (1974) reports two records of rhipicephalus (boophilus) sp. in the tsavo region. okelo-onen et al. (2001) collected ticks from cattle in the kwale district, approximately 150 km to the southeast of the tsavo conservation area, and identified rhipicephalus microplus on cattle in kenya for the first time. they speculated that it may have been present along the kenyan coast for some time but that it had not been detected there, or that it may have spread from the northern coastal region of tanzania where it was known to exist (yeoman & walker 1967). rhipicephalus microplus is a cattle tick, but was not collected in the present study, indicating that it may not have spread inland.collections of rhipicephalus evertsi evertsi from cattle and buffaloes were common. these findings support earlier data, which show that the preferred hosts of this tick are large animals such as cattle, elands (taurotragus oryx) and zebras (horak et al. 1991; londt et al. 1979). ticks collected from the cattle were attached in the upper perineum, whilst those from buffaloes were present on the upper and lower perineum and the tail, confirming the observations of londt et al. (1979). collections of r. humeralis were made only from buffaloes and none from cattle. this differs from the records of walker (1974), who reported 16 of 892 collections from cattle and only one from 119 buffaloes. ticks were collected from the ears, neck, legs, upper and lower perineum. judging by walker’s findings, this tick seems to prefer african elephants, loxodonta africana, as hosts, of which she found 28 of 78 to be infested (walker 1974). a single specimen of r. kochi was collected from the cattle. though not recovered from buffaloes during this study, it has previously been collected from these animals (walker et al. 2000). rhipicephalus muehlensi was collected from cattle and none from buffaloes, and the ears were the preferred site of attachment, confirming previous observations on nyalas (tragelaphus angasii) by horak, boomker and flamand (1995). although no r. muehlensi was collected from buffaloes in this survey, or in the survey conducted by walker (1974), it has been collected from these animals in other studies (horak et al. 1983). rhipicaphalus praetextatus was collected only from buffaloes and none were found on cattle. when this tick does occur on cattle, infestations are usually light to moderate, whereas buffaloes are good hosts (walker et al. 2000). rhipicaphalus praetextatus was collected from the ear, neck, lower perineum and tail, with the greatest number of ticks collected from the tail. because of confusion between the identification of this tick and rhipicaphalus simus, walker et al. (2000) indicated that all locality records for r. praetextatus prior to theirs in kenya should be regarded as presumed. the present collections now confirm the presence of rhipicaphalus praetextatus sensu stricto within the tsavo conservation area. walker (1974) recorded a few collections of rhipicaphalus pravus sensu stricto from cattle in kenya, and none from buffaloes. the single female tick collected from a buffalo in this study was attached to an ear, whilst ticks were attached to the ears, legs and lower perineum on the 13 infested cattle. this attachment pattern confirms earlier observations that rhipicaphalus pravus may attach at several sites on the animal’s body, including the dorsal surface of the ear and its base, the horn base, escutcheon, perineum, groin and heels (yeoman & walker 1967). of all the tick species collected, rhipicaphalus pulchellus was the most numerous on both cattle and buffaloes. r. pulchellus was present in all collection sites on cattle, with the highest numbers recovered from the ears, followed by the upper perineum and the tail. similarly, ticks were collected from all sites on buffaloes, but on these animals most were present on the upper perineum followed by the tail, ears and the lower perineum. similar records of r. pulchellus attaching in multiple sites have been noted for cattle, sheep and camels (walker et al. 2000). conclusion top ↑ the aim of the study was to determine the ixodid tick species infesting cattle and buffaloes sharing the same habitat in the tsavo conservation area, kenya. ticks were collected opportunistically from both host species and a total of 87 animals were sampled and 14 ixodid tick species recovered. eight of these species, namely a. gemma, a. lepidum, h. albiparmatum, h. rufipes, h. truncatum, r. evertsi evertsi, r. pravus and r. pulchellus were collected from cattle and buffaloes. this finding indicates that not only ticks but also some of the diseases transmitted by ticks could be shared by these hosts, and that there is a need to establish the disease status of cattle and buffaloes in the area. in addition new locality records for h. impeltatum and h. truncatum were determined and the first locality record for r. praetextatus sensu stricto in kenya is reported. acknowledgements top ↑ kenya wildlife service veterinary department is thanked for administrative support and the staff of the department for their cooperation. we are grateful to dr maxime madder for assistance with tick identification, mayuko koga, george paul and kingori edward for their assistance in the field and to paul macharia for being a caring driver and massai language translator in the field. this research was supported by grants from the national research foundation to blp and igh. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. author’s contributions e.k.k. (kenya wildlife service) collected and identified the ticks and wrote the first draft of the manuscript. b.l.p. (university of pretoria) was responsible for planning the project and assisted with writing the manuscript. i.g.h. (university of pretoria) assisted e.k.k. (kenya wildlife service) with the identification of the more problematic tick species and assisted with the compilation of the manuscript. references top ↑ allsopp, b.a., bezuidenhout j.d. & prozesky, l., 2004, ‘heartwater’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, pp. 507–535, oxford university press southern africa, cape town.horak, i.g., potgieter, f.t., walker, j.b., de vos, v. & boomker, j., 1983, ‘the ixodid tick burdens of various large ruminant species in south african nature reserves’, onderstepoort journal of veterinary research 50, 221–228. pmid:6646664 horak, i.g., fourie, l.j., novellie, p.a. & williams, e.j., 1991, ‘parasites of domestic and wild animals in south africa. xxvi. the mosaic of ixodid tick infestations on birds and mammals in the mountain zebra national park’, onderstepoort journal of veterinary research 58, 125–136. pmid:1923374 horak, i.g., boomker, j. & flamand, j.r.b., 1995, ‘parasites of domestic and wild animals in south africa. xxxiv. arthropod parasites of nyalas in north-eastern kwazulu-natal’, onderstepoort journal of veterinary research 62, 171–179. pmid:8628570 horak, i.g., 1999, ‘parasites of domestic and wild animals in south africa. xxxvii. ixodid ticks on cattle on kikuyu grass pastures and in valley bushveld in the eastern cape province’, onderstepoort journal of veterinary research 66, 175–184. pmid:10631707 horak, i.g., golezardy, h. & uys, a.c., 2007, ‘ticks associated with the three largest wild ruminant species in southern africa’, onderstepoort journal of veterinary research 74, 231–242. pmid:17933365 lawrence, j.a., perry, b.d. & williamson, s.m., 2004, ‘corridor disease’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, pp. 468–471, oxford university press southern africa, cape town. londt, j.g.h., horak, i.g. & de villiers, i.l., 1979, ‘parasites of domestic and wild animals in south africa. xiii. the seasonal incidence of adult ticks (acarina: ixodidae) on cattle in the northern transvaal’, onderstepoort journal of veterinary research 46, 31–39. pmid:460821 okello-onen, j., hassan, s.m., essuman, s., kariuki, d.p. & mbogo, s.k., 2001, ‘boophilus microplus (acari: ixodida) reported for the first time in kenya’, bulletin of animal health and production in africa 49, 146–149. tolvanen, r., 2004, ‘nature conservation areas in kenya – tsavo east and west national parks’, in p. pellikka, j. ylhaisi & b. clarks (eds.), taita hills and kenya. seminar, reports and journal of a field excursion to kenya, pp. 59–63, expedition reports of the department of geography, university of helsinki. walker, j.b., 1960, notes on the common tick species of east africa, cooper mcdougal & robertson, nairobi. walker, j.b., 1974, the ixodid ticks of kenya: a review of present knowledge of their hosts and distribution, commonwealth institute of entomology, london and reading. walker, j.b. & olwage, a., 1987, ‘the tick vectors of cowdria ruminatum (ixodoidea: ixodidae, genus amblyomma) and their distribution’, onderstepoort journal of veterinary research 54, 353–379. pmid:3329325 walker, j.b., keirans, j.e. & horak, i.g., 2000, the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world, cambridge university press, cambridge. http://dx.doi.org/10.1017/cbo9780511661754 yeoman, g.h. & walker, j.b., 1967, ixodid ticks of tanzania. a study of the zoogeography of the ixodidae of an east african country, commonwealth institute of entomology, london. article information authors: calvin gomo1,2 shuvai musari2 michel de garine-wichatitsky3,4 alexandre caron3,4 davies m. pfukenyi5 henriette van heerden1 affiliations: 1department of veterinary tropical diseases, university of pretoria, south africa 2central veterinary laboratory, harare, zimbabwe 3cirad, upr agir, department environment and societies, harare, zimbabwe 4cirad, upr agir, department environment and societies, montpellier, france 5faculty of veterinary science, university of zimbabwe, zimbabwe correspondence to: henriette van heerden postal address: private bag x04, onderstepoort 0110, south africa dates: received: 17 feb. 2012 accepted: 03 oct. 2012 published: 07 dec. 2012 how to cite this article: gomo, c., musari, s., de garine-wichatitsky, m., caron, a., pfukenyi, d.m. & van heerden, h., 2012, ‘detection of brucella abortus in chiredzi district in zimbabwe’, onderstepoort journal of veterinary research 79(1), art. #417, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.417 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. detection of brucella abortus in chiredzi district in zimbabwe in this original research... open access • abstract • introduction • materials and methods    • study area and sample collection • cultures    • polymerase chain reaction    • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ brucellosis is an endemic disease in zimbabwe caused by the genus brucella. brucella seroprevalence was recently reported to be high in the wildlife-livestock interface in the chiredzi district and the neighbouring gonarezhou national park (gnp) in zimbabwe, and higher amongst communal cattle with an abortion history and access to grazing in gnp than amongst communal cattle with no abortion history or access to grazing in gnp. the aim of this study was to investigate brucella species in brucellosis seropositive cattle in the chiredzi district with access to gnp using isolation and identification. isolation of brucella species from whole blood (n = 18) and milk samples (n = 10) from seropositive animals with an abortion history was based on the rose bengal test (rbt) and enzyme-linked immunoassays (enzyme-linked immunosorbent assay [elisa]; indirect elisa and complement elisa), using microbiology and polymerase chain reaction (pcr) methods. brucella abortus was cultured and identified from blood and milk collected from seropositive cows in both communal areas. the brucella-specific 16-23s intergenic spacer (its) pcr and multiplex amos-pcr assays verified the identification of the cultures. our results confirmed that b. abortus is present in cattle on communal farms in the chiredzi district in zimbabwe and might cause cattle abortions. the need for implementing control measures and raising public awareness on zoonotic transmission of brucellosis are recommended. introduction top ↑ bovine brucellosis is a bacterial disease caused by brucella abortus. in addition to its zoonotic importance, it also affects animal health and production (godfroid et al. 2005; pappas et al. 2005). consumption of contaminated foods or occupational exposure remains the major source of infection in humans. brucellosis is primarily recognised as an occupational hazard for veterinarians, farmers, laboratory technicians, slaughterhouse workers, and others who work with animals and their products. the main source of infection for the public is through ingestion of unpasteurised dairy products. the bacteria can also be transmitted through raw or undercooked meat from infected animals. the brucella species generally considered pathogenic for humans, in decreasing order of virulence, are brucella melitensis, brucella suis and b. abortus (baldwin & goenka 2006). identification of brucella spp. is important in surveillance and eradication efforts. currently, mainly serological screening of potential hosts and to a lesser extent isolation and identification of the pathogen from potential hosts are used for the diagnosis of brucellosis. culturing (isolation and identification) of brucella spp. is recognised as the ‘gold standard’, but is time consuming and complex, and positive animals sometimes yield negative culture results (alton et al. 1988; oie 2008; whatmore 2009). most brucella cultures have been isolated from aborted foetuses, milk, hygroma fluid, or lymph nodes from infected animals. madsen (1989) and mohan et al. (1996) identified b. abortus biovar 1 cultures from aborted foetuses. the same biovar (bv) was also isolated from an aborted foetus of a waterbuck in wankie (hwange) national park (condy & vickers 1969) and from eland hygroma fluid on a game ranch in zimbabwe (condy & vickers 1972). matope et al. (2009) isolated primarily b. abortus bv 1 and to a lesser extent b. abortus bv 2 from aborted foetuses and milk samples from infected herds in zimbabwe. these authors also isolated b. melitensis bv 1 from an aborted foetus of a goat in zimbabwe (matope et al. 2009). it is difficult to obtain positive brucella cultures from blood and positive cultures are only obtained from 10% – 70% of infected human infections since successful isolation depends on the duration, the localisation of the infection and the type of brucella species (al-attas et al. 2000). various polymerase chain reaction (pcr) assays are available for differentiating brucella at the genus, species and/or biovar level. genus-specific pcr assays like 16-23s rrna intergenic spacer (its) region (keid et al. 2007) detect only brucella, whereas multiplex pcr assays differentiate brucella at the species level (bricker & halling 1994, 1995; garcia-yoldi et al. 2006; halling, tatum & bricker 1993). the automated multiplex oligonucleotide synthesizer (amos) multiplex pcr assay distinguishes b. abortus (bv 1, 2 and 4), vaccine strains b. abortus rb51 and s19, b. melitensis (bv 1, 2 and 3), vaccine b. melitensis rev1, brucella ovis, and b. suis (bv 1) (bricker & halling 1994, 1995; halling et al. 1993). brucellosis is endemic in sub-saharan african countries. in zimbabwe it was first diagnosed from aborted cattle in 1913 (bevan 1931). various studies in the country showed a higher brucella infection in commercial than communal areas (madsen 1989; matope et al. 2010; swanepoel, blackburn & lander 1976). a recent study in the wildlife-livestock interface (malipati and pesvi) and non-interface (chomupani and pfumare) communal areas in the south-east lowveld of chiredzi district (figure 1) showed a significantly higher brucella seroprevalence in cows with an abortion history and in cattle grazing in parks (gonarezhou national park [gnp] and kruger national park [knp]) (gomo et al. 2012). although bovine brucellosis was demonstrated through serology (gomo et al. 2012), no isolation or characterisation of the bacteria was done. due to the potential health risk to community members, the objective of the present study was to further characterise the brucellosis species from infected herds in the malipati and pesvi communal areas in chiredzi district, which were found seropositive using the rose bengal test (rbt) and competitive enzyme-linked immunoabsorbent assay (celisa) in the study by gomo et al. (2012) and rbt and indirect enzyme-linked immunoabsorbent assay (ielisa) in this study. brucella-specific pcr and amos-pcr assays were used to confirm the identity of the brucella isolates. materials and methods top ↑ study area and sample collection the study was conducted in the chiredzi district in the south-east lowveld of zimbabwe as described earlier by gomo et al. (2012). the malipati and pesvi communal areas in the chiredzi district share boundaries with the gnp in zimbabwe and the unfenced region of the northern knp (separated by the limpopo river), respectively (figure 1). the two communal areas were selected based on high brucella seroprevalence, reports of abortion and no history of vaccination (chiredzi veterinary services, pers. comm., 2009; gomo et al. 2012). samples were collected from cattle at the malipati and pesvi dip tanks during 2008 and 2009. the malipati dip tank is located about 1 km from the unfenced gnp and pesvi dip tank lies adjacent to the unfenced knp across the limpopo river (dip tank 3 km from northern boundary of knp). whole blood (n = 18) as well as milk (n = 10) samples (table 1) were collected from herds with an abortion history and that tested seropositive using rbt and celisa by gomo et al. (2012). the ielisa was done on samples from malipati and pesvi communal cattle (700 serum samples of 1038 tested cattle) that were part of the study of gomo et al. (2012) to confirm their seropositive status. cultures top ↑ only milk (n = 10) and blood samples (n = 18) collected from animals which had a history of abortion and had tested positive for brucella antibodies using serological tests were cultured for b. abortus isolation at the central veterinary laboratory (cvl) in harare, zimbabwe (table 1). milk samples were centrifuged at 6000 g – 7000 g for 15 min; skim milk was discarded and the cream and sediment were mixed and spread on brucella selective medium and blood agar (ba) (quinn et al. 1994). the inoculated brucella selective media and ba plates were placed in a jar with gas (6% [oxygen] o2, 10% [carbon dioxide] co2 and 84% [nitrogen] n2) at 37 °c and examined for 10 days. plates that did not show any growth after 10 days were discarded as negative. suspected brucella colonies were transferred to ba, on which brucella appeared small (1 mm diameter), round, grey and non-haemolytic. suspected colonies were gram and modified ziehl neelsen (stamp‘s) stained and the reactions to oxidase and catalase were observed (quinn et al. 1994). speciation of brucella colonies was done using microbiology tests (excluding the phage tests) as indicated by alton et al. (1988) and oie (2008).for blood culturing, each 5 ml blood sample was added to biphasic medium (trypticase soy solid and liquid phase; ruiz 1961) and incubated at 37 °c with 5% co2 atmosphere for 10 days (ruiz et al. 1997). the solid phase was prepared with 12 ml of trypticase soy agar and the liquid phase consisted of 30 ml trypticase soy broth. inoculated solid and liquid phase bottles were checked every 24 hours to evaluate haemolysis and turbidity. once the bacterial growth was detected by turbidity and haemolysis, the colony was sub-cultured and gram stain was performed to confirm the presence of gram-negative rods in the broth and on the agar slant. colonies were stained with gram and modified ziehl neelsen (stamp’s) stains and the reactions to oxidase and catalase were observed (quinn et al. 1994). brucella speciation was done as described for the milk cultures. figure 1: the location of the two communal areas (malipati and pesvi) that were surveyed in the chiredzi district in zimbabwe that borders the gonarezhou national park and the kruger national park. table 1: information of rose bengal test seropositive bovines from which whole blood and milk samples were collected in the pesvi and malipati communal regions in zimbabwe. polymerase chain reaction dna was extracted from isolates obtained from blood and milk cultures using the qiagen dna mini kit (qiagen) according to the manufacturer‘s instructions. each 16-23s its pcr (keid et al. 2007) amplification reaction was prepared in a total volume of 25 µl containing 50 mm potassium chloride (kcl), 10 mm tris(hydroxymethyl)aminomethane hydrochloride (tris–hcl) (ph 9.0), 2.0 mm magnesium chloride (mgcl2), 200 µm of each deoxynucleotide triphosphates (dntp), 0.4 µm of each primer, 2.5 µl dna template and 1.5 u gotaq® hot start polymerase (promega). polymerase chain reaction conditions included an initial denaturation at 95 °c for 5 min followed by 35 cycles consisting of 30 s of denaturation at 95 °c, 30 s of annealing at 56 °c, and 30 s of elongation at 72 °c, with a final elongation at 72 °c for 5 min. the amos-pcr condition was used as previously described by bricker and halling (1994, 1995). the pcr reaction consisted of 1.5 mm mgcl2, 1x pcr buffer (promega), 250 μm dntps, 5’ primer cocktail consisting of b. abortus, b. melitensis, b. ovis and b. suis specific primers each (0.2 μm) and 1 μm is711-specific primer, 1 u gotaq® hot start polymerase (promega) and 2.5 μl dna per 25 μl reaction. all the pcr samples were analysed by electrophoresis in a 2% agarose gel, stained with ethidium bromide (0.5 mg/ml), and the dna bands were visualised under ultraviolet (uv) light (uvp transilluminator model tm-20). the dna of brucella reference strains obtained from the brucella culture collection, france (bccn), namely: b. abortus bv 1 (544 = bccn r4), b. abortus bv 2 (86/8/59 = bccn r5), b. abortus bv 4 (292 = bccn r7), b. melitensis (16m = bccn r1), b. suis bv 1 (1330 = bccn r12), b. ovis (63/290 = bccn r17) and brucella canis (rm6/66 = bccn r18) were included as positive controls. ethical considerations research involving animals have been approved by the animal use and care committee of the university of pretoria and were done according to the national code of welfare standards for each animal species. results top ↑ the seroprevalence was 8.3% of the 700 cattle samples from the malipati and pesvi in the chiredzi district based on rbt and ielisa. the prevalence of the individual communal areas were 9% (n = 490) and 6.7% (n = 210) in malipati and pesvi, respectively. table 1 indicates the rbt, celisa and ielisa serological results of bovine sampled for culturing. brucella abortus was isolated from two seropositive cows in the malipati and pesvi regions that were seropositive for rbt, ielisa and celisa and both had an abortion history (table 1). the isolates had microscopic and bacteriological characteristics typical of the brucella genus, namely gram-negative coccobacilli, non-motile, positive for modified ziehl-neelsen staining with oxidase and catalase production. both the brucella cultures could only be identified to species level, namely b. abortus, and due to unavailability of phage tests at cvl the biovar(s) could not be determined (alton et al. 1988).dna extracted from isolates from blood, milk and brucella reference strains produced a 214 bp product that is specific to brucella using the its66 and its279 primers for the 16-23s rdna its region (keid et al. 2007). the two isolates from blood and milk identified as b. abortus produced the unique 498 bp fragment specific to b. abortus bv 1, 2 and 4 using the multiplex amos-pcr (bricker & halling 1994, 1995; figure 2). discussion top ↑ brucella abortus was isolated and confirmed with amos-pcr assay as b. abortus bv 1, 2 or 4 strains (bricker & halling 1994, 1995) from seropositive cows with an abortion history in the malipati and pesvi interface regions in the chiredzi district in zimbabwe. these isolates were established from brucella infected cattle samples from the malipati and pesvi regions with a seroprevalence of 10.3% (gomo et al. 2012). the brucella seroprevalence rate reported in this study (8.3% of n = 700) using rbt and ielisa was relatively similar to the seroprevalence of 10.3% (n = 1038) using rbt and celisa reported by gomo et al. (2012). since none of the sampled cattle had been vaccinated against brucellosis, the detected antibodies were most likely due to a natural infection by brucella species, which was confirmed by celisa results (gomo et al. 2012) and amos-pcr assay as wild-type b. abortus. the celisa and amos-pcr differentiate between natural infections and vaccine strains (bricker & halling 1994, 1995; nielsen et al. 1989). figure 2: identification and differentiation of brucella abortus isolated from seropositive cows in the chiredzi district using automated multiplex oligonucleotide synthesizer multiplex polymerase chain reaction. due to the unavailability of phage tests that identify biovars, the b. abortus isolates could only be identified to species level using microbiology tests (alton et al. 1988). the amos-pcr confirmed the two isolates as b. abortus bv 1, 2 or 4 since the multiplex pcr cannot distinguish b. abortus bv 1, 2 and 4 from one another (figure 2; bricker & halling 1994, 1995). previous studies have demonstrated the presence of b. abortus bv 1 and 2 in cattle in different parts of zimbabwe (madsen 1989; matope et al. 2009; mohan et al. 1996). the majority of the b. abortus isolates were found to be biovar 1 (84.6%, 11/13) with the remaining ones being biovar 2 (matope et al. 2009). brucella abortus bv 1 appears to be the predominant cause of brucellosis in cattle in zimbabwe (matope et al. 2009). similarly, in neighbouring south africa, biovar 1 has been shown to contribute about 90% whilst biovar 2 accounted for 10% of all the b. abortus isolates (bishop, bosman & herr 1994). only two cultures were obtained from milk (n = 10) and blood (n = 18) samples from seropositive cows with an abortion history (7%). sensitivity of culturing brucella species from blood varies from 10% – 70% of suspected human infections (al-attas et al. 2000; pappas et al. 2005; ruiz et al. 1997) depending on the growth conditions (ruiz et al. 1997), duration, localisation of the infection and type of brucella species (al-attas et al. 2000; ruiz et al. 1997). the biphasic method was used to isolate brucella from whole blood samples as described by ruiz (1961) and incubated for 10 days since ruiz et al. (1997) obtained 100% cultures over a maximum time of 216 hours (9 days). we obtained a low percentage of cultures (7%) from known brucella seropositive samples with unknown stage of brucellosis. the low sensitivity of culturing brucella in this study clearly indicates that other culturing techniques should be investigated. the lysis centrifugation (lc) technique has been reported by espinosa et al. (2009) to be the preferred technique for brucella culturing at all stages of brucellosis, since it yields 25% more positive results and provided results 10 days earlier than the biphasic method. the lc technique is a yield-optimisation method that uses lysis of erythrocytes in a citrate solution followed by isolation of brucella bacilli by centrifugation that concentrated the bacilli and assist growth (espinosa et al. 2009). the use of the lc technique for brucella culturing with a longer incubation period (40 days [espinosa et al. 2009] compared to 10 days in our study) should rather be used for brucella culturing from blood and milk in future. evidence of brucella infections in cattle in the study area has been serologically demonstrated previously (gomo et al. 2012). the isolation of b. abortus from brucella seropositive animals confirms the presence of brucellosis and indicates that b. abortus might causes abortions in the studied areas since both the cows from which b. abortus were isolated had an abortion history. the purchase of unknown brucella-status cattle from the commercial to the communal sector for the purposes of restocking herds and genetic improvements and an increased uncontrolled movement of cattle due to agrarian reforms in the country are reported as the likely source of spread of brucellosis into the communal sector (matope 2008; matope et al. 2010). in addition, sharing of grazing land and watering points between cattle and wildlife at the studied interface is also likely to be a source of transmission of the disease in both directions as b. abortus has been isolated from cattle (madsen 1989; matope et al. 2009; mohan et al. 1996; this study) and wildlife (condy & vickers 1969, 1972). the identification of b. abortus known to occur in cattle and wildlife in zimbabwe is significant since it is one of the species generally considered pathogenic for humans (baldwin & goenka 2006). the tradition of consuming unpasteurised milk in rural areas, low awareness of the zoonotic importance of brucellosis, close intimacy with livestock and provision of assistance during parturition may increase the risk of human exposure to b. abortus infections in the study area. despite the prevalence of brucellosis in the study area, no published information is available with regard to human brucellosis. however, public awareness in the chiredzi communities should be increased to reduce the risk of human exposure to b. abortus infection. conclusion top ↑ brucella abortus was isolated from blood and milk collected from seropositive cows in the chiredzi district and therefore the community members in the chiredzi regions like pesvi and malipati should be informed of the risk of human exposure to brucella infection. the isolation of b. abortus from seropositive cows confirms that this species could be associated with cattle abortions in the chiredzi district in zimbabwe. however, further studies are recommended to determine the distribution of b. abortus biovars and human brucellosis prevalence in the area. the need for implementing control measures and raising public awareness on zoonotic transmission of brucellosis is recommended. serially, serological testing for brucellosis before translocation, culling of seropositive animals, increased controlled livestock movement and calfhood vaccinations should be instituted for the control of the disease. in addition, simple, user-friendly extension material to make cattle owners aware of this disease and its control should be produced and disseminated to them and the extension staff. acknowledgements top ↑ this work was supported financially by the institute of tropical medicine (itm) in antwerp, belgium and the national research foundation in south africa. we would also like to acknowledge the research conducted within the framework of the research platform production and conservation in partnership (rp-pcp grant/project ahe#1 2007 to 2009) that established insight into the seroprevalence of brucellosis in the chiredzi region, zimbabwe. we would like to thank staff from ovi-arc bacteriology section and virology staff from cvl, harare for helping in the microbiology and serology tests. we would also like to thank dr laure guerrini for providing the map of the chiredzi district. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions c.g. (university of pretoria) and h.v.h. (university of pretoria) designed the project and wrote the manuscript. c.g. (university of pretoria) and s.m. (central veterinary laboratory) were involved in morphology identification of the culture and serological testing of the sera. c.g. (university of pretoria) conducted the molecular studies. m.d.g.-w. (cirad), a.c. (cirad) and d.m.p. 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brucellosis in cattle in rhodesia’, rhodesia veterinary journal 7, 24–31. whatmore, a.m., 2009, ‘current understanding of the genetic diversity of brucella, an expanding genus of zoonotic pathogens’, infection, genetics and evolution 9, 1168–1184. http://dx.doi.org/10.1016/j.meegid.2009.07.001 abstract introduction methods results discussion conclusion acknowledgements references about the author(s) sitira williams department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom isabella endacott department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom abel b. ekiri department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom mirende kichuki college of veterinary medicine and biomedical sciences, sokoine university of agriculture, morogoro, tanzania mariana dineva department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom erika galipo department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom vadim alexeenko department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom ruth alafiatayo department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom erik mijten zoetis, zaventem, belgium gabriel varga zoetis, zaventem, belgium alasdair j.c. cook department of veterinary epidemiology and public health, faculty of health and medical sciences, university of surrey, guildford, united kingdom citation williams, s., endacott, i., ekiri, a.b., kichuki, m., dineva, m., galipo, e. et al., 2022, ‘barriers to vaccine use in small ruminants and poultry in tanzania’, onderstepoort journal of veterinary research 89(1), a2007. https://doi.org/10.4102/ojvr.v89i1.2007 note: additional supporting information may be found in the online version of this article as online appendix 1, 2, 3 and 4. original research barriers to vaccine use in small ruminants and poultry in tanzania sitira williams, isabella endacott, abel b. ekiri, mirende kichuki, mariana dineva, erika galipo, vadim alexeenko, ruth alafiatayo, erik mijten, gabriel varga, alasdair j.c. cook received: 25 feb. 2022; accepted: 01 june 2022; published: 04 aug. 2022 copyright: © 2022. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract vaccination is an important disease prevention and control measure; however, vaccine adoption by livestock farmers in tanzania is still low. this cross-sectional study examined the challenges to vaccine use faced by livestock owners and animal health professionals (ahps) in tanzania. a questionnaire was administered to 216 households that kept small ruminants and poultry and 19 ahps’ data were collected electronically via the survey platform qualtrics, and descriptive statistics were performed. households with poultry reported vaccinating mostly against newcastle disease (91.7%), fowl pox (48.1%) and gumboro disease (37.0%), whilst households with small ruminants reported contagious caprine pleuropneumonia (62.2%), sheep and goat pox (17.1%), foot-and-mouth disease (7.3%) and peste des petits ruminants (7.3%). the households’ decision to vaccinate was mostly influenced by knowledge of diseases (82.4%), disease history on the farm (69.4%) and vaccine price (63.4%). most households (54.6%) experienced challenges when purchasing vaccines, including high vaccine cost (78.0%), long distance from vaccine source (61.0%) and vaccine unavailability (21.2%). the findings suggest that improving the knowledge of livestock owners regarding the priority diseases and the benefits of vaccination, establishing more vaccine suppliers, improving vaccine distribution and access and training ahps and households on appropriate vaccine storage and handling are necessary to improve vaccine adoption and ensure vaccine quality and effectiveness. keyword: vaccine; small ruminant; goat; sheep; poultry; challenges; tanzania; africa. introduction in tanzania, as in the rest of sub-saharan africa, endemic livestock diseases are an obstacle to livestock production because of direct losses in livestock mortality, morbidity and the impact on the livelihoods of livestock keepers. newcastle disease (nd) in poultry, contagious caprine pleuropneumonia (ccpp) in small ruminants (goats and sheep), contagious bovine pleuropneumonia (cbpp) and east coast fever (ecf) in cattle are the diseases most often reported by farmers in tanzania (covarrubias, nsiima & zezza 2012). to counter the impact of such endemic diseases, vaccines are used in livestock to maintain animal health and improve overall production (roth & sandbulte 2021). in small ruminants (goats and sheep), there is an economic benefit to controlling and potentially eradicating key endemic diseases, such as peste des petits ruminants virus (ppr), which causes high morbidity and mortality in goats and sheep (jones et al. 2016). beyond disease control, vaccination has the potential to increase animal-source food consumption, improve household income and reduce food insecurity (knueppel et al. 2010). although vaccines are known to prevent livestock production losses and reduce the incidence of disease, vaccine adoption in small-holder livestock production in tanzania is still low. small-holder livestock producers face several barriers to vaccine use, and there are many interrelated factors that have been suggested to either promote or constrain the use of vaccines, including access to veterinary services, access to distributors and retailers, geographical location (urban vs. rural) (covarrubias et al. 2012), cost of vaccine and need for refrigeration in the field (babiuk & wallace 2018). another important factor reflected at the household level is gender imbalances (female-headed compared to male-headed households) (babiuk & wallace 2018; covarrubias et al. 2012). the barriers to vaccine use are likely to differ by livestock species. in small ruminants, ccpp and ppr cause high morbidity and mortality rates and therefore are of high concern to small-ruminant holders (mbyuzi et al. 2015). however, high concern has not necessarily equated to high vaccine use. for instance, although vaccination is known to be the most effective form of prevention against major livestock diseases, including foot-and-mouth disease (fmd), an endemic disease across many regions in tanzania, only 5% of livestock households reported vaccinating against fmd (casey-bryars et al. 2018). the unavailability of appropriate vaccines, lack of effective policies on vaccine quality, quality control and beliefs on vaccine efficacy have been cited as blocks to vaccine usage (casey-bryars et al. 2018; railey et al. 2018). although previous literature suggests there are a multitude of factors that may constrain the uptake of vaccines in ruminants, there is still little known about the constraints to vaccine uptake in small ruminants, specifically. in poultry, nd is amongst the most prevalent diseases, with a mortality rate of up to 90% – 100%, and it is reported to particularly affect rural and remote areas (hugo et al. 2017). despite this, vaccination for nd was reported to be low in a previous study that investigated the barriers underpinning the use of the nd vaccine in tanzania (campbell et al. 2018). the reported key factors related to nd vaccination in tanzania have included flock size, knowing someone who vaccinated, use of traditional medicine (campbell et al. 2018), local support with vaccination, knowledge about nd signs, previous vaccine use and gender (campbell et al. 2018, 2019). although previous literature provides useful knowledge about the challenges to nd vaccine use, it is not clear or known if the same challenges apply to other prevalent key poultry diseases that can negatively impact poultry health and production, such as infectious bronchitis, gumboro disease, marek’s disease, escherichia coli and salmonellosis. the role of gender is also important to consider when investigating barriers to vaccine use in livestock. gender imbalance in vaccine access has been suggested; for example, access to livestock vaccines and animal health information is limited in north-eastern uganda (yusuf 2013). women are disproportionately affected by challenges in accessing veterinary services, disease information and veterinary pharmaceuticals (galiè et al. 2017). regarding livestock ownership, women are more likely to own poultry and small ruminants, whilst cattle ownership is substantially more male-orientated (njuki & sanginga 2013). livestock ownership can provide a sustainable income, which may be particularly important for improving the livelihoods of rural women (njuki & sanginga 2013). it is therefore important to consider gender dynamics in livestock production when identifying ways to reduce gender imbalances and to help increase productivity and income generation for female-headed livestock businesses. the aim of this study was to explore the challenges to the use of livestock vaccines experienced by small-ruminant and poultry farmers, as well as the challenges in providing veterinary services faced by animal health professionals (specifically animal health or livestock extension officers) in tanzania. the role of gender in small-ruminant and poultry production with respect to vaccine use was also investigated. exploring the barriers to the use of livestock vaccines in tanzania will help inform the development of strategies to increase vaccine adoption by small-ruminant and poultry farmers. methods study population and setting the target study population comprised household heads (referred to as households) of livestock holdings that kept either poultry and/or small ruminants, as well as animal health or livestock extension officers (here after referred to as animal health professionals) in selected regions of tanzania. for the purposes of this study and in the context of tanzania, an animal health or livestock extension officer is defined as a person who holds either a diploma or a certificate in an animal health-related field, works closely with the community and reports livestock diseases within their local community areas to the local government. study design in this cross-sectional study, five study regions across tanzania were targeted and purposively selected based on the following criteria: (1) areas must have a reasonably large number of small-ruminant and poultry holdings based on the knowledge of key stakeholders and (2) areas must be geographically dispersed sufficiently to capture locational differences regarding the barriers to vaccine use. the following five regions met the above criteria and were thus included in the study: mwanza (north), arusha (north-east), kilimanjaro (north-east), morogoro (central) and pwani and dar es salaam (east) (figure 1). within each study region, three districts were randomly selected, and within each district, three wards were randomly selected, resulting in a total of nine wards per region and an overall total of 45 wards in the five regions. figure 1: map of the study regions in tanzania. initially, this study was designed to involve focus group interviews targeting a total of 480 study participants, including 450 households (optimally 30 households per district) and 30 animal health professionals selected from the 45 wards. however, because of the coronavirus disease 2019 (covid-19) international and local travel restrictions and public health guidelines at the time of implementation in june 2020, the study approach was revised to telephone interviews. after revision of the study approach to telephone interviews, the breakdown of the overall total of 480 participants was as follows: (1) one-on-one telephone interviews with households (approximately 450 participants, 10 participants per ward who kept poultry and small ruminants); and (2) one-on-one telephone interviews with animal health professionals (approximately 30 participants from the 45 wards). to generate contact information for potential participating households, initially, district veterinary officers were contacted and asked to provide contact information for animal health professionals based in each of the selected study wards located in their districts. the animal health professionals were then asked to provide a list of households in their ward, including five households with small ruminants and a minimum of five animals per herd and/or a list of five households with poultry and a minimum of 10 chickens per flock. the determination of the cut-off number of small ruminants or chickens per household was subjective; local animal health professionals were consulted to determine the number of animals kept by a typical household. for the purposes of this study, the households were classified as primarily small-ruminant households or poultry households, based on the number of animals owned; households that owned at least five sheep or goats were classified as small-ruminant households and those that owned at least 10 local chickens or 50 chickens on commercial flock were classified as poultry households. data collection and analysis two questionnaires were designed to collect data from household heads of the livestock holdings (appendix 1 material s1) and from animal health professionals (appendix 2 material s2). the questionnaire tools collected data on the following parameters: demographics, livestock holdings, vaccine use, diseases of most concern, prioritised vaccines needed, factors that influence decision to vaccinate, vaccine source, challenges experienced when obtaining or purchasing vaccines and when using vaccines on animals, vaccination campaigns, traditional treatment methods used and the role of gender of small-ruminant and poultry household heads in vaccine use. the questionnaires were translated to kiswahili and interviews were conducted in kiswahili, the most common language in tanzania, as well as in english. to ensure interpretation was not changed following translation, individuals who were local and fluent in kiswahili were requested to review the translated questionnaire tools and revisions were made before they were finalised and administered to the study participants. prior to survey administration, pretesting of the surveys was completed with five households and five animal health professionals, and the questions were revised further for clarity. a field team of five interviewers was created, which included interpreters knowledgeable in the other local languages. the study team was based and conducted the telephone interviews from a central location in morogoro. interview time slots with the participants were scheduled by the field team prior to the interview at a time most convenient for each respondent. each participant received two reminder calls regarding the date and time of interviews (a week and a day before the interview) with the aims of reducing time-consuming constraints and avoiding delays and rescheduling of the interviews. one-on-one telephone interviews were conducted with the target participants and responses were entered on tablets using an online survey on the qualtrics platform. verbal consent was obtained before the start of each interview. data were collected over a period of 16 days, from 09 november 2020 to 24 november 2020. the data were downloaded from the qualtrics platform and analysed using descriptive statistics in microsoft excel and r (version 1.3.1093). ethical considerations ethical review and approval were granted by the research and publication committee of the college of veterinary medicine and biomedical sciences, sokoine university of agriculture (approval date: 14 january 2020). results there was difficulty in reaching the initially targeted 480 study participants after revision of the study approach to telephone interviews. in the end, a total of 309 households were successfully reached via telephone based on the contact information provided by animal health professionals, and 223 households agreed to participate in the study and were interviewed, providing a response rate of 72.2% (223 out of 309). after excluding seven incomplete interviews, 216 (96.9%) households were included in the analysis. of the 30 animal health professionals initially contacted, three were not reachable at the time of the study; six of the 27 animal health professionals did not respond to the survey; and of the remaining 21, 19 (90.5%) had complete interviews and were included in the analysis. results from household surveys respondent characteristics the largest proportion of the household respondents were from the pwani and dar es salaam region (61 out of 216, 28.2%), followed by mwanza (48 out of 216, 22.2%), kilimanjaro (43 out of 216, 19.9%), morogoro (34 out of 216, 15.7%) and arusha (30 out of 216, 13.9%). most of the respondents were men (133 out of 216, 61.6%); the owners of the herd or flock and those in charge of managing the herd or flock were also predominantly men (62.0% and 63.0%, respectively) (table 1). table 1: characteristics of the participating household heads (households) (n = 216). almost all respondents (213 out of 216, 98.6%) were also the actual owners of the livestock holding (table 1). nearly half of the respondents had between one and five years of experience in managing their herd or flock (98 out of 216, 45.4%). most of the telephone interviews with households were conducted in kiswahili (145 out of 216, 67.1%) (table 1). characteristics of the livestock holdings poultry households accounted for most respondents (117 out of 216, 54.2%), followed by small-ruminant households (99 out of 216, 45.8%) (appendix 3 table s1). chickens (174 out of 216, 80.6%), goats (115 out of 216, 53.2%) and cattle (99 out of 216, 45.8%) were the three most common animals kept by the households. chickens (104 out of 216, 48.1%) were reported to provide the most income for households (appendix 3 table s1). for poultry households (n = 117), the most reported average flock size was between 10 and 20 birds (41 out of 117, 35.0%) (appendix 3 table s1). for small-ruminant households (n = 99), the most reported average herd size was between 10 and 50 goats or sheep (44 out of 99, 44.4%) (appendix 3 table s1). vaccine use most of the respondents (190 out of 216, 88.0%) had vaccinated their flock or herd in the last 12 months (appendix 3 table s2): 108 poultry households and 82 small-ruminant households. within those who reported vaccinating, the most common diseases vaccinated against in poultry were nd (99 out of 108, 91.7%), fowl pox (52 out of 108, 48.1%) and gumboro disease (40 out of 108, 37.0%) (figure 2), and in small-ruminants they included ccpp (homa ya mapafu in kiswahili) (51 out of 82, 62.2%), sheep and goat pox (14 out of 82, 17.1%), fmd (6 out of 82, 7.3%) and ppr (6 out of 82, 7.3%) (figure 2; appendix 3 table s2). figure 2: diseases households vaccinated against in small ruminants and poultry. diseases of most concern to households and prioritised vaccines needed over one-third of respondents (83 out of 216, 38.4%) reported that they were concerned about diseases they could not source a vaccine for. the diseases of most concern to small-ruminant households included ‘circling’ disease (actual disease and causative name is not known) (21 out of 40, 52.5%), ccpp (11 out of 40, 27.5%), fmd (4 out of 40, 10.0%) and east coast fever (4 out of 40, 10.0%) (figure 3). poultry households reported fowl pox (16 out of 28, 57.1%), nd (8 out of 28, 28.6%) and coccidiosis (4 out of 28, 14.3%) as the diseases of most concern (figure 3; appendix 3 table s3). figure 3: diseases of most concern to small-ruminant and poultry households. when households (n = 216) were asked about vaccines considered to be of high priority but to which they did not have access, small-ruminant households reported ‘circling’ disease vaccine (17 out of 99, 17.2%) and ccpp vaccine (8 out of 99, 8.1%), whilst poultry households reported fowl pox vaccine (18 out of 117, 15.4%) and nd vaccine (11 out of 117, 9.4%) (appendix 3 table s3). factors that influence households’ decision to vaccinate when respondents were asked about which factors influenced their decision to vaccinate their animals against a certain disease, the most frequently reported factors were the knowledge of diseases (178 out of 216, 82.4%), the history of disease on the farm (150 out of 216, 69.4%) and the price of vaccines (137 out of 216, 63.4%) (figure 4; appendix 3 table s4). when asked to rank the top two most influential factors in their decision-making, the most frequently reported factors were the price of vaccine (97 out of 216, 44.9%), the knowledge of diseases (54 out of 216, 25.0%) and the distance to vaccine source or supplier (42 out of 216, 19.4%) (appendix 3 table s4). when households were asked about potential consumer-associated drivers and socio-economic factors that would most influence their decision to vaccinate their herd or flock, the three most frequently reported factors were government support with access to expensive vaccines (80 out of 216, 37.0%), information and education regarding administering vaccines safely (58 out of 216, 26.9%) and funding and investment opportunities in agriculture (32 out of 216, 14.8%) (appendix 3 table s4). figure 4: factors influencing the households’ decision-making to vaccinate their animals. vaccine source many of the respondents sourced their vaccines from a veterinary or agricultural drug shop (140 out of 216, 64.8%) or directly from a veterinarian or an animal health officer (92 out of 216, 42.6%) (appendix 3 table s2). most of the respondents travelled between 30 min and 1 h (79 out of 216, 36.6%), followed by under 30 min (71 out of 216, 32.9%) to the source of vaccine purchase (appendix 3 table s2). the animal health officer (115/216, 53.2%) was the most common primary provider of veterinary care and support for the herd or flock, and households predominantly sought advice on vaccine use and vaccination protocols for their herd or flock from a veterinarian or an animal health officer (157 out of 216, 72.7%) (appendix 3 table s2). challenges experienced by households when obtaining or purchasing vaccines and when using vaccines on animals over half of the respondents (118 out of 216, 54.6%) experienced challenges when obtaining or purchasing vaccines for their animals (appendix 3 table s3). overall, the most reported challenges included the high cost of vaccines (92 out of 118, 78.0%), the long distance from vaccine supplier or source (72 out of 118, 61.0%) and the unavailability of vaccines and/or vaccines not being in stock (25 out of 118, 21.2%) (appendix 4 figure s1; appendix 3 table s3). the most reported challenges when obtaining or purchasing vaccines did not differ by species. amongst the small-ruminant households (52 out of 99, 52.5%), the three most reported challenges were the high vaccine cost (39 out of 52; 75.0%), the long distance to vaccine supplier or source (28 out of 52; 53.8%) and the vaccines being unavailable or out of stock (13 out of 52, 25.0%). amongst the poultry households (56.4% (66 out of 117, 56.4%), the three most reported challenges included the high cost of vaccines (53 out of 66; 80.3%), the distance to vaccine supplier or source (44 out of 66; 66.7%) and the unsuitable vaccine package size (14 out of 66; 21.2%). almost half of the respondents (100 out of 216, 46.3%) experienced challenges with regard to the use of vaccines on the farm (appendix 3 table s3). overall, the most reported challenges included problems related to vaccine storage (50 out of 100, 50.0%), ineffectiveness of vaccines against disease (42 out of 100, 42.0%) and vaccine side effects (39 out of 100, 39.0%) (appendix 4 figure s2; appendix 3 table s3). the most reported challenges with regard to the use of vaccines did not differ by species. in small-ruminant households (39 out of 99, 39.4%), the most reported challenges were problems with vaccine storage (15 out of 39, 38.5%), ineffectiveness of vaccine (13 out of 39, 33.3%) and vaccine side effects (13 out of 39, 33.3%). in poultry households (61 out of 117, 52.1%), the three most reported challenges included vaccine storage problems (35 out of 61, 57.4%), the ineffectiveness of vaccines (29 out of 61, 47.5%) and vaccine side effects (26 out of 61, 42.6%). vaccination campaigns about one-third of respondents (64 out of 216, 29.6%) reported that their local government had conducted a vaccination campaign in their area in the last 5 years (appendix 3 table s5). those who reported government vaccination campaigns in their area (n = 64) specified that ccpp vaccine (10 out of 64, 15.6%) and rabies vaccine (8 out of 64, 12.5%) were promoted in the vaccination campaigns (appendix 3 table s5). regarding actual vaccination during the campaign, ccpp (8 out of 64, 12.5%) and anthrax (7 out of 64, 10.9%) were the top diseases vaccinated against in the campaigns (appendix 3 table s5). traditional treatment methods used by households to determine what other methods were used by households to treat their herd or flock, the respondents were asked if they had ever used traditional methods to treat animals against diseases or poor health. in total, 44.4% (96 out of 216) of respondents reported that they had used traditional treatment methods for their animals (appendix 3 table s5). of these, most reported using aloe vera leaves (41 out of 96, 42.7%) or neem leaves (11 out of 96, 11.5%); papaya leaves, moringa and ash were also used by a small proportion of households (appendix 3 table s5). the role of gender of small-ruminant and poultry household heads in vaccine use to gain further understanding regarding the role of gender in livestock and poultry production across different regions in tanzania, the respondents were asked whether they considered gender (i.e. being male or female) to be a barrier to vaccine access; only four respondents (4 out of 216, 1.9%), which included two men and two women, agreed with this statement (appendix 3 table s5). when these households were further asked about the ways gender acted as a barrier to vaccine access, the reported reasons included financial opportunities to buy expensive treatment or vaccines (1 out of 4, 25.0%) and that the administration and/or application method of vaccines on their animals was difficult for women (1 out of 4, 25.0%) (appendix 3 table s5). results from survey of animal health professionals respondent characteristics the animal health professionals included in this study worked mainly in the arusha (5 out of 19, 26.3%) and pwani regions (5 out of 19, 26.3%); the remaining respondents worked in morogoro (4 out of 19, 21.1%), mwanza (3 out of 19, 15.8%) and kilimanjaro (2 out of 19, 10.5%). animal health professionals were predominantly men (14 out of 19, 73.7%) and the highest level of education attained by most of the respondents was a diploma (13 out of 19, 68.4%) (appendix 3 table s6). most of the telephone interviews with these respondents were conducted in both english and kiswahili (7 out of 19, 36.8%) (appendix 3 table s6). employment characteristics when the respondents were asked to list the average number of animal health professionals (e.g. animal health officers, veterinarians, para-veterinarians, etc.) who work in each of the ward(s) that they also worked in, the most frequently reported number of animal health professionals per ward was 2 (12 out of 19, 63.2%) (appendix 3 table s6). most of the respondents reported providing veterinary services to over 20 households on average (12 out of 19, 63.2%) (appendix 3 table s6). regarding their current role, most respondents were animal health officers (12 out of 19, 63.2%), and others were livestock extension officers (7 out of 19, 36.8%) (appendix 3 table s6). most respondents had worked as animal health professionals for 5–15 years (11 out of 19, 57.9%), and the most frequently reported activities they participated in were treating animals (18 out of 19, 94.7%) and advising households on the health and management of their animals (16 out of 19, 84.2%) (appendix 3 table s6). the three most common animal species for which the respondents provided veterinary services (e.g. advice and treatment) in their wards were cattle (18 out of 19, 94.7%), goats (18 out of 19, 94.7%) and sheep (16 out of 19, 84.2%) (table 2). table 2: vaccine use, vaccine source and average travel time to vaccine source for households, as reported by animal health professionals (n = 19). vaccine use most animal health professionals reported that poultry households in their ward typically vaccinated their poultry flocks (17 out of 19, 89.5%), and the average poultry flock size vaccinated was mostly less than 50 birds (5 out of 17, 29.4%) or over 500 birds (5 out of 17, 29.4%) (table 2). overall, the most common poultry diseases vaccinated against were nd (14 out of 17, 82.4%), fowl pox (13 out of 17, 76.5%) and gumboro disease (8 out of 17, 47.1%) (table 2). most animal health professionals reported that small-ruminant households in their ward vaccinated their herds (15 out of 19, 78.9%), and the average herd size vaccinated was mostly over 50 animals (8 out of 15, 53.3%) (table 2). the most common diseases vaccinated against by small-ruminant households were ccpp (homa ya mapafu) (13 out of 15, 86.7%), sheep and goat pox (7 out of 15, 46.7%) and fmd (4 out of 15, 26.7%) (table 2). diseases of most concern to households and prioritised vaccines needed in ward(s) about half of animal health professionals (9 out of 19, 47.4%) reported that there were small-ruminant diseases of concern to households and for which they were currently unable to source a vaccine (appendix 3 table s7). the respondents reported that ccpp (5 out of 9, 55.6%), ppr (5 out of 9, 55.6%), fmd (2 out of 9, 22.2%) and circling disease (2 out of 9, 22.2%) were the main small-ruminant diseases households were most concerned about (appendix 4 figure s3; appendix 3 table s7). when animal health professionals (n = 9) were asked to report at most three priority vaccines that small-ruminant households in their ward(s) could not access at the time, ppr (4 out of 9, 44.4%) and ccpp (3 out of 9, 33.3%) were most frequently reported (appendix 4 figure s3). similarly, about half of animal health professionals (9 out of 19, 47.4%) reported that there were poultry diseases of concern to households and for which they were currently not able to source a vaccine (appendix 3 table s7). coccidiosis (2 out of 9, 22.2%), marek’s disease (2 out of 9, 22.2%), typhoid (2 out of 9, 22.2%) and nd (2 out of 9, 22.2%) were reported as the top poultry diseases of most concern to households (appendix 4 figure s4; appendix 3 table s7). gumboro (2 out of 9, 22.2%), nd (2 out of 9, 22.2%) and typhoid (2 out of 9, 22.2%) were the most reported vaccines when respondents were asked to list the top three priority vaccines that the poultry households could not access at the time (appendix 4 figure s4). factors reported by animal health professionals that influence households’ decision to vaccinate given the experience of the animal health professionals in veterinary diagnostic services, they were asked to give their opinion on the factors that influenced the decisions of poultry and small-ruminant households in their ward(s) to vaccinate their animals against a certain disease. the most frequently reported factors were knowledge of the disease (17 out of 19, 89.5%), history of the disease on farm (16 out of 19, 84.2%) and the occurrence of the disease on other local farms in the area (15 out of 19, 78.9%) (appendix 3 table s8). when asked to select the two main factors, the price of vaccines (8 out of 19, 42.1%), the availability of vaccines (4 out of 19, 21.1%) and the distance to the vaccine source or supplier (4 out of 19, 21.1%) were most frequently reported (appendix 3 table s8). most respondents stated that animal health officers (7 out of 19, 36.8%) and livestock extension officers (5 out of 19, 26.3%) were most used by poultry or small-ruminant households for veterinary support (appendix 3 table s8). vaccine source most respondents reported that poultry or small-ruminant households sourced vaccines from veterinary or agricultural drug shops (14 out of 19, 73.7%) or directly from a veterinarian or an animal health officer (11 out of 19, 57.9%) (table 2). the majority of respondents reported that poultry or small-ruminant households in their ward(s) travelled between 30 min and 1 h (6 out of 19, 31.6%), followed by under 2 h (6 out of 19, 31.6%), on average, to the source where they could purchase their vaccines (table 2). challenges experienced by households when obtaining or purchasing vaccines or when using vaccines on animals, as reported by animal health professionals most animal health professionals (17 out of 19, 89.5%) reported that poultry and small-ruminant households experienced challenges when obtaining or purchasing vaccines for their animals, and the most frequently reported challenges included the distance from vaccine supplier or source (10 out of 17, 58.8%), the high cost of vaccines (8 out of 17, 47.1%) and vaccines not being available or not in stock (7 out of 17, 41.2%) (appendix 4 figure s5; appendix 3 table s9). most animal health professionals (13 out of 19, 68.4%) also reported that poultry and small-ruminant households experienced challenges in relation to the use of vaccines on their animals, and the most frequently reported challenges included vaccine side effects (9 out of 13, 69.2%), problems related to vaccine storage (6 out of 13, 46.2%) and the ineffectiveness of vaccine against disease (5 out of 13, 38.5%) (appendix 4 figure s6; appendix 3 table s9). additional challenges faced by households and animal health professionals the animal health professionals were asked about other challenges or constraints experienced by poultry or small-ruminant households in relation to the health and production of their animals (i.e. other than vaccine-related challenges). the responses were categorised into themes to allow for meaningful interpretation. the most common responses from the animal health professionals were ‘lack of service providers’ (8 out of 19, 42.1%), ‘lack of education and knowledge’ (5 out of 19, 26.3%) and ‘lack of equipment and facilities’ (5 out of 19, 26.3%) (appendix 3 table s10). the animal health professionals were also asked about the challenges they experienced in the provision of veterinary services to small-ruminant or poultry households, and the most reported challenges were the lack of tools and equipment for use in clinical work (14 out of 19, 73.7%), insufficient number of animal health professionals in their ward(s) (13 out of 19, 68.4%) and poor road and transport access to households (13 out of 19, 68.4%) (appendix 3 table s10). vaccination campaigns most animal health professionals reported that their local government had conducted a vaccination campaign in their wards in the last five years (11 out of 19, 57.9%) (appendix 3 table s11). when these respondents (n = 11) were asked to list the vaccines promoted in the campaigns, the most mentioned vaccines were rabies (5 out of 11, 45.5%), ccpp (4 out of 11, 36.4%), anthrax (2 out of 11, 18.2%) and nd (2 out of 11, 18.2%) (appendix 4 figure s7; appendix 3 table s11). additionally, based on the responses of the animal health professionals (n = 11), the most mentioned diseases vaccinated against in the campaigns were rabies (5 out of 11, 45.5%), ccpp (4 out of 11, 36.4%) and contagious bovine pleuropneumonia (3 out of 11, 27.3%) (appendix 4 figure s7). traditional treatment methods used on the ward(s) most animal health professionals (12 out of 19, 63.2%) reported that poultry or small-ruminant households in their ward(s) used traditional methods to treat against diseases or poor health of their herd or flock; the most frequently reported traditional treatment methods used included aloe vera leaves (used for chickens) (7 out of 12, 58.3%) and neem leaf (6 out of 12, 50.0%) (appendix 3 table s11). the role of gender in vaccine adoption on the ward(s) when animal health professionals were asked whether they considered gender to be a barrier to vaccine access on their wards, only one respondent (1 out of 19, 5.3%) reported that this was the case, and the reported reason for this was that ‘males tend to leave all farmer managerial activities for the females’ (appendix 3 table s11). discussion vaccine use most poultry and small-ruminant households reported to have vaccinated their livestock against at least one disease in the last 12 months, and this information was corroborated by the animal health professionals. although most respondents reported vaccinating against at least one disease in the last 12 months, the proportion that vaccinated against diseases considered of most concern to small-ruminant and poultry households was extremely low. this finding is significant because vaccination is considered one of the most important preventive measures in disease prevention and control. the results suggest that there is a need for improvement in vaccination adoption through strategies such as increasing awareness of vaccines and related benefits to livestock owners. in addition, it is important to address the reported challenges faced by households related to access and availability of good-quality vaccines, because vaccines considered to be of high priority could not be accessed by households. the involvement of local leaders in efforts to improve vaccine use may also contribute to the success of vaccination drives, as it fosters the feeling of local ownership of the programme and may prevent conflicts with other development activities within the community villages (msoffe et al. 2010). factors that influence the decision of households to vaccinate their animals the current study suggests that knowledge is an important component of a strategy aimed at improving vaccine adoption. the factors reported to influence the decision of households to vaccinate animals included knowledge of the disease, history of the disease on farm and vaccine price. knowledge of the disease and price of vaccine were also ranked amongst the top two most influential factors. in addition, in the current study, about one-third of the respondents reported that their local government had conducted a vaccination campaign in their area within the last five years. this practice may have contributed positively to vaccine adoption in that community, as indicated by the high number of households that reported vaccinating their livestock against at least one disease in the last 12 months. two previous studies conducted in tanzania also suggested that knowledge was relevant in vaccination adoption. a previous study reported that knowing someone who vaccinated increased the odds of a household vaccinating, suggesting that provision of relevant knowledge to the community may be a useful strategy to increase vaccine adoption (campbell et al. 2018). another study reported that previous vaccine use, gender and support in the village were important factors and recommended that strategies to improve vaccine uptake needed to go beyond simply making vaccines available (lindahl et al. 2019). improving awareness of the diseases of concern, the relevant target priority vaccines and the related benefits of vaccinating livestock against disease amongst small-ruminant and poultry farmers may help increase vaccine uptake. challenges related to vaccine access and use experienced by households high cost of vaccines, the long distance to vaccine suppliers or sources and vaccines not being available and/or not being in stock were the most common challenges faced by households when obtaining or purchasing vaccines. not surprisingly, the high cost of vaccines and the long distance to vaccine suppliers or sources were also ranked amongst the top factors that influenced the decision of households to vaccinate animals. the reported challenge of the high cost of vaccines highlights the importance of household income as a key driver of vaccine use. cost of vaccines and distances to vaccination points were also reported as barriers to vaccine uptake in a study in ruminants in neighbouring uganda and kenya (mutua et al. 2019). a previous study in tanzania that examined poultry farmers’ preferences and ‘willingness to pay’ reported that farmers’ ‘willingness to pay’ was positively influenced by observation of the benefits of vaccination from previous vaccination events (campbell et al. 2019). improving awareness of the benefits of vaccinating against disease through vaccination awareness campaigns may help improve a farmer’s ‘willingness to pay’ and consequently vaccine uptake. the challenges of long distances to suppliers or sources combined with the unavailability of vaccines suggest that there are few vaccine sources, and distant geographic location from the vaccine sources might constrain vaccine access and consequently vaccine use. the establishment of more vaccine suppliers, including retailers and distributors which are more geographically dispersed, may help shorten the travelling distances and thus improve vaccine distribution and access, as well as vaccine adoption. the most common challenges to vaccine use on the farm reported by households and animal health officers were vaccine storage, ineffectiveness of vaccines and vaccine side effects. although the reasons for the ineffectiveness of vaccines were not investigated in this study, these findings suggest that there is a need to train both animal health officers and households on appropriate vaccine storage and handling to ensure that the quality and the effectiveness are not compromised before administration to the animals. awareness of the potential vaccine side effects is equally important for minimising misinformation and resentment of vaccine uptake. the most common animal health and production-related challenges faced by households were the lack of service providers, lack of knowledge and the lack of equipment and facilities, whilst those faced by animal health officers were the lack of tools and equipment to use in clinical work, an insufficient number of animal-health professionals in their ward(s) and poor road and transport access to farmers. these findings highlight potential weaknesses in veterinary care infrastructure and service delivery. addressing these challenges requires input from both the public and private sectors including the government and animal industry. the study findings suggest that traditional methods are considered an important option in the treatment of animal diseases. over two-thirds of household respondents reported using traditional treatment methods for their animals, and of these, most reported using aloe vera leaves or neem leaves. this finding was corroborated by animal health officers, who reported that households used traditional methods to treat their animals. the use of traditional medicine alone and/or in conjunction with western medicine to manage livestock diseases or conditions is well known. a previous study amongst poultry farmers in tanzania reported that the use of traditional medicine was associated with a decrease in the likelihood of awareness of nd vaccines and of previous vaccination (campbell et al. 2018); this suggests that traditional medicine may have a competitive relationship with vaccination. it is important to note that a link (treatment effect) between traditional methods and disease or condition treated was not investigated in this study. further investigations are required to determine the impact of the use of traditional methods on vaccine uptake, to identify and link the specific traditional method or type of plants and the conditions or diseases targeted and to explore the potential effectiveness of such treatments. role of gender in small-ruminant and poultry production findings from the current study indicate that women were less involved in activities and decision-making related to small-ruminant and poultry production. when the few respondents who considered gender a barrier to vaccine access (four households and one animal health professional) were asked about the reasons for this, households reported that the purchase of treatments and vaccines was expensive and the vaccine administration and/or application method was difficult for women. the animal health professionals reported that men tended to leave all farmer managerial activities for the women. these results corroborate the findings of a previous study that also reported a gender disparity in animal health and production roles in tanzania (njuki & sanginga 2013). gender disparity amongst men and women in livestock production is reported to be highly associated with ownership of land; women in tanzania have less access and control of land and resources in comparison to their male counterparts (ndiyo & urassa 2003; acosta et al 2022). in addition, women’s involvement in livestock production activities in tanzania is limited, and women are more likely to be involved in management and administration responsibilities (campbell et al. 2018). the reported gender imbalance in ownership of livestock can potentially affect food security, as well as women’s access to veterinary services, disease information and veterinary pharmaceuticals (galiè et al. 2017). although there are laws already in place for women’s rights to attain land, training and campaigning around equal ownership of land and access to livestock production services and resources are still vital to help women grow, develop and increase their livestock (idris 2018; lyimo-macha & mdoe 2002). limitations the change in study approach from focus groups and ethnographic interviews to telephone interviews because of the covid-19 pandemic disruptions may have impacted the quality of the study findings, as in-depth interviews could not be performed. we could not observe the commonalities and differences between the respondents and explore the responses given, which would have been possible through conducting focus groups. in addition, it was more time-consuming to organise and conduct the interviews; however, given the challenges, this approach was more appropriate for reaching a broader target audience, considering that the respondents were geographically dispersed. the number of animal health professionals recruited for the study was low; this could be related to the lack of service providers or animal health professionals in the targeted wards, as reported by the animal health professionals who participated in the current study. because of the small sample size of animal health professionals, the findings from the survey in this study group cannot be generalised to all animal health professionals in tanzania. the meaning of the survey questions following translation into kiswahili may have affected respondents’ interpretations of the questions. for example, the findings might have been affected if questions were misunderstood and answered incorrectly. to minimise this risk and ensure interpretations were not changed, individuals who were local and fluent in kiswahili were requested to review the translated questionnaire tools, and revisions were made before the tools were finalised and administered to study participants. telephone network issues during data collection might have also affected the quality and completeness of the collected data. for instance, some responses may have been rushed, as a handful of farmers were using their own mobile data to participate in the survey and were concerned about the length of time taken to complete the survey. additionally, some interviews were delayed, paused or rescheduled when network issues were experienced. conclusion although most households vaccinated their livestock against at least one disease in the last 12 months, the results suggest that there is a need for improvement in vaccination adoption. as in previous studies, the current study suggests that awareness of the diseases of concern and knowledge of the target priority vaccines and the related benefits of vaccinating livestock against disease should be considered together with other relevant strategies aimed at improving vaccine adoption. there is a need to address the challenge of the high cost of vaccines. improving awareness of the benefits of vaccinating through vaccine awareness campaigns may help improve farmer’s ‘willingness to pay’ and consequently vaccine uptake. the challenges of long distance and unavailability of vaccines may be addressed through the establishment of more vaccine suppliers to shorten the travelling distances and improve distribution and access to vaccines. such efforts will likely require key inputs from the animal industry, particularly the pharmaceutical industry. training of both animal health professionals and households on appropriate vaccine storage and handling is needed. this would ensure that the vaccine quality and effectiveness are not compromised before administration to the animals. improving access and availability of good-quality vaccines and addressing the weaknesses in the veterinary care infrastructure and service delivery are necessary. efforts to improve vaccine adoption and use will require a multisectoral collaborative approach that involves several key players, including poultry, sheep and goat owners; veterinary professionals; the pharmaceutical industry; and the local and national governments. finally, the findings indicated a gender disparity in activities and decision-making for smallruminant and poultry production; women were less involved and male-headed households predominated. more work is needed to promote and support women to get involved in livestock production as owners or workers, such as through promotion of animal health information and services at the community level. acknowledgements the authors would like to thank the following individuals for their support: they thank the household heads and animal health officers or livestock extension officers who spared their time to complete the data collection instruments. they also thank the tanzania study interview team, mwokozi mwanzalila, asha makweta, ashura mhidini, baraka edson mwamundela and filbert ewald mdee, without whose hard work and commitment this study would not have been possible. they thank tetiana miroshnychenko (zoetis-alpha initiative zaventem, belgium) and dr isaac odeyemi (zoetis outcomes research team, dublin, ireland) for guidance on the alpha initiative activities. they are also thankful to maryam manjenje (tanzania) for logistical assistance during the study implementation in the country. finally, they are grateful to adam trish (university of surrey, vhive team) for administrative and project management assistance. competing interests this study was supported by the africa livestock productivity and health advancement (alpha) initiative, co-funded by zoetis and the bill & melinda gates foundation. funding from zoetis was an unrestricted grant. two of the co-authors (e.m. and g.v.) are employed by zoetis. authors’ contributions i.e. and a.b.e. were responsible for the conceptualisation of the study; i.e., a.b.e. and a.j.c.c. contributed towards methodology; i.e., s.w., m.k., e.g., r.a and a.b.e. investigated data; s.w., i.e., v.a. and a.b.e. performed analysis and interpretation; s.w., i.e., m.d. and a.b.e. were responsible for writing (original draft preparation); s.w., i.e., e.g., r.a., m.d., v.a., e.m., g.v. and a.b.e. were responsible for writing (review and editing); a.b.e. and m.k. supervised; a.b.e, m.k., r.a. contributed to project administration; a.j.c.c. and g.v. were responsible for funding acquisition. all authors have read and agreed to the final version of the manuscript. funding information this research was funded by the african livestock productivity and health advancement (alpha) initiative, co-funded by the bill & melinda gates foundation (bmgf) (grant no. opp1165393) and zoetis. funding from zoetis was an unrestricted grant. data availability the data that support the 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district, tanzania’, tanzania journal of institutes of development studies 1, 25–34. njuki, j. & sanginga, p., 2013, women, livestock ownership and markets: bridging the gender gap in eastern and southern africa, routledge, london. railey, a.f., lembo, t., palmer, g.h., shirima, g.m. & marsh, t.l., 2018, ‘spatial and temporal risk as drivers for adoption of foot and mouth disease vaccination’, vaccine 36(33), 5077–5083. https://doi.org/10.1016/j.vaccine.2018.06.069 roth, j.a. & sandbulte, m.r., 2021, ‘the role of veterinary vaccines in livestock production, animal health, and public health’, in s. metwally, a. el idrissi & g. viljoen (eds.), veterinary vaccines: principles and applications, pp. 1–10, john wiley & sons, ltd, iowa state university, ames, iowa, usa. article information authors: alessandra scagliarini1 silvia piovesana1 filippo turrini1 federica savini1 fortune sithole2 cheryl m. mccrindle3 affiliations: 1dipartimento di scienze mediche veterinarie, university of bologna, italy2department of paraclinical sciences, university of pretoria, south africa 3school of health systems and public health, university of pretoria, south africa correspondence to: alessandra scagliarini postal address: dipartimento di scienze mediche veterinarie, university of bologna, via tolara di sopra 50, 40064 ozzano emilia, bologna, italy dates: received: 27 june 2012 accepted: 17 oct. 2012 published: 07 dec. 2012 how to cite this article: scagliarini, a., piovesana, s., turrini, f., savini, f., sithole, f. & mccrindle, c.m., 2012, ‘orf in south africa: endemic but neglected’, onderstepoort journal of veterinary research 79(1), art. #499, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.499 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. orf in south africa: endemic but neglected in this original research... open access • abstract • introduction • materials and methods    • questionnaire data analysis    • laboratory assays • results    • field observations and analysis of questionnaires    • spatial and temporal distribution of outbreaks    • diagnosis and genomic characterisation • discussion • conclusion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ a survey amongst sheep and goat producers and veterinarians was undertaken to collect epidemiological data on orf in south africa. previous epidemiological studies on the presence of the disease in the country have not been documented and this report is the first descriptive epidemiological study of orf in south africa. a seven-month investigation, realised by direct and indirect interviews and field observation, enabled us to outline incidence and risk factors of this disease and to better understand how the local farmers in rural areas relate to it. the results may contribute to better management of the disease in rural areas. by means of molecular analyses the phylogenetic relationships between field isolates from different areas have been identified. the findings gave a first important contribution to the general assessment of the economic impact of orf virus infections and the extent of the risk to human health. introduction top ↑ orf virus (ov) is the type species within the genus parapoxvirus (ppv) in the family poxviridae, subfamily chordopoxvirinae and it is the causal agent of a papular dermatitis affecting sheep, goats and occasionally other artiodactyla species (inoshima, morooka & sentsui 2000; kummeneje & krogsrud 1979). typical ov lesions progress from erythema to macules, papules, vesicles, pustules and scabs, which are usually located around the mouth or nares of lambs and kids, on the teats of nursing ewes or around the coronets. the shed scabs contain high titres of stable virus, which, if kept dry, can remain infectious long enough to sustain annual outbreaks of the disease. severe outbreaks of orf can occur in naïve flocks or herds, in which lesions are extensive and proliferative and do not spontaneously regress. the disease can be particularly severe in nursing lambs and kids where death from starvation and thirst can occur within days from lesions developing. during outbreaks, morbidity can approach 100%, whilst mortality is usually less than 1%. however, mortality can increase by 20% – 50% as a result of secondary complications such as stress, immunosuppression or concomitant disease and can exceed 90% in the case of ‘malignant orf’ (hosamani et al. 2009; kitching 2004; mazur & machado 1989; scagliarini et al. 2006). the diagnosis is generally based on clinical signs, which are typical, but can be confused with those shown in other vesicular diseases of sheep and goats. these include foot and mouth disease (watson 2004), bluetongue (baipoledi, nyange & hyera 2002), peste des petits ruminants (rossiter & taylor 1994) and sheep and goat pox (kitching 2004; zheng et al. 2007), which are all listed by the world organisation for animal health (oie) as high-impact diseases. other diseases that resemble orf include papillomatosis (smith et al. 2002) and bacterial diseases such as staphylococcal dermatitis and dermatophilosis (bath et al. 2011; kitching 2004; smith et al. 2002). rapid and accurate differential diagnosis is therefore essential (mcelroy & bassett 2007; watson 2004), as early recognition of orf not only allows for institution of proper control measures and specific management strategies for unweaned animals, but also prevents zoonotic transfer. orf represents a zoonotic disease and an occupational hazard (lederman et al. 2007; steinhart 2005). in the united kingdom it has been identified as the most frequent occupational zoonosis (buchan 1996) and is also described as common in australasia (crumbie 1998). in addition to occupational exposure in veterinarians and dairy farm workers, it has been associated with religious slaughter (ghislain, dinet & delescluse 2001; uzel et al. 2005). in humans, ov infections can cause painful lesions on the hands, fingers and face, which respond poorly to treatment and may take months to regress. reports exist of atypical proliferating forms of giant orf in immunocompromised individuals, which can require hospitalisation (rogers et al. 1989). further complications such as erythema multiform, bullous pemphigoid, swan neck deformity, paresthesia and autoimmune blistering disorders have also been reported (ara et al. 2008; lederman et al. 2007; white et al. 2008). although orf is often described as endemic, the actual prevalence of infections amongst livestock herds is greatly underestimated (hosamani et al. 2009). the danger to consumers from unpasteurised milk is unknown; however, according to the food and agriculture organization of the united nations (fao 2011) milk from diseased animals has to be discarded. this may have a negative impact on the profitability of dairy farms, particularly since the lesions remain for a long time and can cause mastitis. studies on the economic implications of the disease have not been published despite it being regarded as one of the most important viral infections of sheep and goats in both developing (perry et al. 2002) and developed countries (haig & mcinnes 2002). according to the world organisation for animal health, south africa has more than 26 billion sheep and 5 billion goats (oie 2010). sheep are bred for wool as well as meat production. angora goats are farmed for mohair and several indigenous breeds of goats for meat. there are also a number of goat breeds that are used for milk production in both commercial and informal sectors (schoeman, cloete & olivier 2010). orf in sheep and goats is recognised in south africa (kitching 2004), but the epidemiology is not well documented. the aim of this investigation was to identify the risk factors and study the molecular epidemiology of orf in sheep and goats in south africa. materials and methods top ↑ the study area comprised the whole sheep and goat farming sector in south africa. this sector is highly dualistic and can typically be divided into communal farming and commercial farming. in south africa, the majority of sheep are farmed commercially, whilst goats are more hardy and, except for angora and dairy breeds, are typically encountered in small-scale farming and subsistence systems. commercial sheep and goat farms are mainly found in the eastern and northern cape, whilst in the rest of south africa the majority of animals are owned by small-scale and communal farmers (schoeman et al. 2010). an extensive outbreak investigation was conducted over a period of seven months, from september 2009 to march 2010. the sampling methodology was based on two types of questionnaires, of which the first one was sent out by e-mail to all veterinarian members of both the south african veterinary association and the south african society for veterinary epidemiology and preventive medicine. this questionnaire focused on awareness of the disease, the period of the year with the highest incidence, the species involved, and the age and the percentage of affected animals. an open-ended question was included at the end of the questionnaire to allow respondents to add more information on the disease. the second questionnaire was addressed to local farmers and administered using structured interviews during field visits to rural areas where veterinarians had identified outbreaks. the statements were translated into the local languages with the assistance of animal health technicians working for the state veterinary services. pictures of lesions in sheep and goats were shown to make the topics more understandable. the questionnaire focused on the awareness of the disease, time of the year with the highest incidence, age and percentage of animals affected, duration of the disease, localisation of the lesions, treatments and previous experience of transmission to humans. an accurate observation of the environment, animals, and breeding and management systems were undertaken in the areas where farmers were interviewed. questionnaire data analysis data obtained from the analysis of the questionnaires were recorded in a spreadsheet and subjected to qualitative statistical analyses using the statistical package for the social sciences (spss). laboratory assays when suspected cases of orf were observed, scab samples from affected animals were collected using forceps and stored separately at –20 °c in plastic bags or tubes until laboratory processing. in addition, scab samples were collected from cattle showing skin and mucosal lesions. the total dna was extracted from the sampled scabs using the commercially available nucleospin tissue® kit (machery and nagel, düren, germany) according to the manufacturer’s instruction. to assess the presence of ov, the dna was used as template for a qualitative polymerase chain reaction (pcr) able to amplify a sequence of 594 nucleotides internal of the parapoxvirus-conserved gene b2l. primers and cycling conditions were described previously (inoshima et al. 2000). the ov vaccine strain (orf freeze-dried vaccine reg. no g1110 act 36/1947, onderstepoort biological products ltd., onderstepoort, south africa) was used as a positive control and thus subjected to dna extraction and pcr. pcr products representative of outbreak macro-areas as well as the orf vaccine produced by the onderstepoort laboratory were sequenced and compared. sequence analyses were performed by nucleotide and amino acid alignment according to the clustalw method as implemented in the software megalign (dnastar suite, lasergene inc., madison). a phylogenetic tree was generated after clustalw alignment with published sequences of parapoxviruses retrieved from genbank (table 1). table 1: parapoxvirus sequences used in the phylogenetic analysis of orf virus. results top ↑ field observations and analysis of questionnaires a total of 300 questionnaires were sent by email to members of the south african veterinary association and the south african society for veterinary epidemiology and preventive medicine. preliminary analysis showed that private veterinarians working exclusively with companion animals had never experienced cases of orf. the analysis thus focused on 80 questionnaires, from across the country, answered by 29 private and 51 state veterinarians. amongst these, 84.3% of the state veterinarians and 58.6% of the private veterinarians confirmed to have experienced orf outbreaks. of the veterinarians who had observed orf, 50% (n = 30) stated that the disease mostly affected goats, 20% (n = 12) had seen it mainly in sheep, whilst 26.7% (n = 16) had observed it in both species. regarding the time of the year in which outbreaks mainly occur, 50% of the veterinarians indicated the rainy season (from october to march), 13.3% (n = 8) the dry season and 13.3% (n = 8) reported the disease throughout the year. the majority of the veterinarians (48.4%; n = 29) indicated a morbidity rate ranging from 1% to 20%, whilst a minority (8.3%; n = 5) estimated the morbidity rate to be higher than 40%. during the survey period, only one case of transmission to humans was reported. this was the result of a goat bite to a veterinarian, who developed a blister on her fingers and recovered three weeks after the onset of the lesion (figure 1). figure 1: human case of orf: blistering lesion on a finger. a total of 161 farms were visited, of which 106 were classified as ‘rural’ and 55 as ‘commercial’. the data from structured interviews indicated that 75% (n = 121) of farmers had prior knowledge about orf and that 90% (n = 110) of them had experienced outbreaks within the last two years. farmers reported that the disease lasts 2–3 weeks (33.6%; n = 37), 1 month (43.6%; n = 48) and 2 months (6.4%; n = 7), whilst 16.4% (n = 18) were unable to reply to this question. the majority of the farmers (79.3%; n = 96) reported outbreaks during the rainy season, whilst 9.9% (n = 12) observed a higher incidence of the disease during the dry season. the remaining 10.8% (n = 13) of respondents were unable to reply to this question. farmers were also interviewed about the age of affected animals: 80.2% (n = 97) identified young animals (kids or lambs) as being more affected by orf virus, whilst 13.2% (n = 16) stated that the disease could occur in either young or adult animals. only 3.3% of respondents (n = 4) observed the disease in adult animals only. respondents consequently pointed out a correlation between disease occurrence and the lambing period, which was mostly concurrent. distribution of the lesions was also recorded and the face was indicated as the most frequent site (61.9%; n = 75). this localisation was often coupled with other sites, such as the ears (14.9%; n = 18), the legs (10.7%; n = 13) and the udder (6.7%; n = 8). an association between the occurrence of the disease amongst flocks and the presence of thorny acacia species, which grow and are browsed during the rainy season, was also observed. with regard to traditional methods used by farmers to treat the disease, it was found that 61.8% (n = 68) of the respondents treated sick animals during outbreaks, whilst 38.2% (n = 42) did not. the most common recorded treatment consisted of application of vaseline® oil (a registered skin product made of petroleum jelly) or machine oil. copper sulphate and zinc preparations were also described. farmers in rural areas scratch open the lesions by hand or using stones before treating them with oil when the disease is severe. only a minority of farmers use antibiotics and seek veterinary assistance. in some rural communities in the north west province the disease is treated using the root of a plant called morototshwetshwe or sebete, which has been identified as cassia italica (table 2). table 2: treatments or remedies for orf reported by farmers. less than 1% of the interviewed farmers used a commercial vaccine to prevent the disease, whilst the application of so-called auto-vaccination (using ground scabs applied to scarified skin) was reported by farmers from the eastern and northern cape provinces and confirmed by local veterinarians. spatial and temporal distribution of outbreaks during the period of investigation, 54 outbreaks of orf-like diseases amongst sheep and goats were reported. of these, 44 occurred in rural areas and the rest on commercial farms. a total of 39 outbreaks of suspected parapoxvirus infections were visited and scab samples were collected for subsequent analysis. in addition, state veterinarians reported a further 15 outbreaks via email or phone, but these were neither visited nor sampled owing to logistical constraints. the majority of the outbreaks occurred in goat flocks and orf incidence appeared to be directly related to the sheep and goat populations of the provinces (figure 2). in the investigated outbreaks, different degrees of severity of the disease were recorded, although it appeared that clinical signs seen in sheep were milder than in goats (figure 3a and 3b). figure 2: map of the outbreaks (september 2009 – march 2010) and animal species involved. figure 3: (a) typical clinical case of orf virus infection in a sheep (sample a52); (b) proliferative orf lesions in a boer goat (sample a93). diagnosis and genomic characterisation the diagnostic pcr performed on the dna extracted from the scabs collected from diseased animals confirmed parapoxvirus infection through the amplification of a specific 594 base pair fragment of the b2l gene. the nucleotide sequences of the amplified products confirmed, in all cases, ov as the causative agent in sheep and goats. the residue identity of b2l sequences ranged from 96.9% to 100% and from 97.5% to 100% at the nucleotide and amino acid level, respectively. a 100% identity has been shown between field strains sa52, isolated from sheep in the north west province, a90 collected from sheep in the western cape, sa142 isolated from goats in mpumalanga and the vaccine strain used in south africa. a complete identity was also found between strains sa88 and sa106, isolated from goats in limpopo and the western cape, respectively, and strains isolated from goats in kwazulu-natal (sa89), gauteng (sa87), limpopo (sa118), the eastern cape (sa93) and the northern cape (sa126). the pcr and subsequent sequencing performed on the dna extracted from the scabs collected from cattle in two mixed herds in limpopo allowed us to demonstrate pseudocowpox virus (pcpv). the two identified pcpv strains (sa85 and sa86) showed an identity of 99.3% and 99.4% at nucleotide and amino acid level, respectively, leading us to conclude that the disease in cattle was not caused by a unique virus. discussion top ↑ although it is generally accepted that orf is endemic in south africa, the extent of the problem is unknown. the fact that 54 outbreaks occurred during the observation period in the whole study area indicated that the prevalence and the economic impact of the disease are probably underestimated by the veterinary profession. this can be due to the fact that only a limited number of farmers report outbreaks or seek veterinary assistance (table 2). it is postulated that the underestimation of the importance of orf can be attributed to the fact that it is not a controlled or notifiable disease. as a result there is little or no data recorded by the veterinary authorities and no surveillance programmes on ov are running in south africa. however, vaccine is produced and animal health technicians are consulted to collect samples when farmers suspect the presence of a more serious disorder such as foot and mouth disease or bluetongue. commercial breeders are often assisted by private veterinarians for specific cases only and so do not form part of routine surveillance. consequently, private veterinarians report that the highest incidence is in sheep, which are farmed for wool and meat on commercial farms. in contrast, goats, which are known for their adaptability and fertility and browse in thornbush areas (malan 2000), are mainly found in the informal rural sector. thus, according to state veterinarians, who work in rural areas, goats are mainly involved in outbreaks. according to the answers from the questionnaires, the morbidity of the disease observed by veterinarians (1% – 20%), was far lower than that observed during the field outbreaks we have inspected, where between 90% and 100% of animals in a flock showed clinical signs. our observations lead us to confirm that goats rather than sheep are mainly affected, which is in agreement with reports by veterinarians. the largest number of affected animals were boer goats. according to existing reports (e.g. de la concha-bermejillo et al. 2003; guo et al. 2003; ndikuwera 1992) as well as our own observations, boer goats appear to be more prone to developing severe ov infection (figure 3b), which seriously impacts animal welfare and consequently the farming economy. the majority of veterinarians, as well as the farmers, agreed that ov infections have a higher incidence during the rainy season (summer). this indication substantially differs from the epidemiological data observed in other countries, where the highest incidence has been reported in the dry season, when the consumption of dried, fibrous grass contributes to oral lesions and virus penetration (lewis 1996; rodríguez et al. 2011). environmental observation indicated that many plant species belonging to the acacia genus were associated with the incidence of orf. these species are the dominant woody plants in the bushveld areas of south africa. they produce spiked or hooked thorns and tender, succulent leaves early in the rainy season, which probably cause micro-lesions around the lips and faces of browsing goats. the virus enters through damaged mucosa and the scabs of an infected animal remain on the thorns, which can affect another animal. sheep are grazers rather than browsers, although they also eat smaller thorn bushes. considering the seasonal and self-limiting nature of orf, the apparent low mortality (according to farmers) and short immunity conferred, it was not surprising that less than 1% of the farmers reported using commercial vaccines to prevent the diseases; instead they use homemade vaccines produced from scabs of sick animals. this procedure constitutes a further risk for transmission, as the scabs produced after vaccination contain live virus, which can survive in the environment for long periods (nettleton et al. 1996). furthermore, scratching the scabs off infected animals to produce the homemade vaccine, as well as treating them with oil, is considered a risk factor for zoonotic transmission to farmers. during our investigation, we were able to collect ethnoveterinary information such as traditional remedies to cure orf. in particular, we found that some farmers treated animals with a decoction of the roots of c. italica, both topically and orally and it was claimed that the remedy resulted in recovery from the disease within a few days. previous reports have demonstrated the antimicrobial, anti-inflammatory, antipyretic, analgaesic, purgative and antitumor activity of c. italica (assane et al. 1994; jain et al. 1997; kazmi et al. 1994; masoko et al. 2010). at present there is no information about the antiviral properties of c. italica extracts and further studies will be needed to confirm this property. orf shares clinical manifestations with other notifiable vesicular diseases of ruminants, such as bluetongue and foot and mouth disease. it was hypothesised that veterinarians operating in the provinces with a higher risk of foot and mouth disease might have a better knowledge of ov owing to regular surveillance and monitoring in flocks and herds. this hypothesis was, however, not confirmed, as our data showed no significant difference in the knowledge level with respect to province (p = 0.076). during the study period, outbreaks of both bluetongue and foot and mouth disease occurred in south africa, underlining the importance of a rapid differential diagnosis. pcr is currently the most affordable and rapid method for the direct diagnosis of orf and also represents the preliminary step for genomic characterisation. the sequence of the ppv b2l gene, a homologue of the major envelope antigen, p37k, of the vaccinia virus, has been widely used as a molecular target for virus detection and diagnosis of infection and for identification of species within the genus parapoxvirus (inoshima et al. 2010). the molecular characterisation based on the partial nucleotide and amino acid sequences of representative strains of all the identified outbreaks (table 2), showed a high level of conservation of the b2l gene. previous reports observed mutations within the b2l gene sequence of ov strains originating from the same outbreak (abrahao et al. 2009; chan et al. 2007; guo et al. 2004; hosamani et al. 2006; klein & tryland 2005). our results lead us to conclude that diseased sheep and goats were all affected with ov, whilst samples collected from cattle were shown to be positive for pcpv, leading us to promptly exclude other look-alike vesicular diseases in the investigated flocks. pseudocowpox was already reported in south africa in poorly managed farms, where it causes economic losses because of reduced milk production (munz & dumbell 2004). our analyses allowed us to exclude a phylogenetic clustering linked to the geographical origin. in particular, we showed that strains sa52, sa90 and sa142 collected from the north west, western cape and mpumalanga provinces clustered together. these strains were also found to be identical to the ov strain contained in the locally produced vaccine (figure 4). vaccination against ov results in scabby lesions as in naturally infected animals. scabs that are formed are rich in virus, which, when shed, contribute to the environmental pool of the virus and can be the cause of further outbreaks. however, since the vaccine had never been used on the farms where samples were collected, the vaccine strain could be excluded as the causative agent of the outbreaks. on the contrary, the complete identity of several field strains with the vaccine strain seems to indicate that these viruses have been circulating over a long period of time. the phylogenetic analysis inferred from b2l amino acid alignment confirmed the presence of a limited number of ov variants circulating in the country (figure 4). this hypothesis seems to be further supported by the phylogenetic analyses inferred from the data of the concatenated alignment of three ov protein sequences, which allowed the identification of two clusters corresponding to the two main variants circulating in the country (data not shown). the limited movements of breeding animals within south africa and a high level of adaptation of the ov to its host may represent a possible explanation for our observation. figure 4: phylogenetic tree generated using the neighbour-joining method (saitou & nei 1987) after clustalw alignment of the amino acid sequences of the major envelope protein encoded by the b2l gene segment. the effects of zoonotic infections on human health are usually greatest on livestock keepers who live in close proximity to their animals. approximately 46% of south africans still live in rural areas, coming regularly into close contact with farm animals. for this reason it is remarkable that, in spite of a widespread presence of the disease in farmed animals, neither farmers nor veterinarians reported cases of orf infection in humans. ov infections in humans are consequent to the handling of infected animals or animal by-products or vaccination. for this reason, farmers, abattoir workers, veterinarians and shearers are at greatest risk of infection. during our investigation only one human case was confirmed (in a veterinarian) in september 2010. she was working in kwazulu-natal and became infected during foot and mouth disease surveillance whilst inspecting a goat with vesicular lesions in its mouth. the demonstration that pcpv is circulating amongst cattle suggests that there are, indeed, further sources for parapoxvirus transmission to humans in south africa. although pcpv and ov have already been identified as a cause of zoonotic diseases in south african veterinarians (gummow 2003), parapoxvirus infections can be classified as a neglected zoonosis, as they are likely to be under-recognised by medical practitioners. ov and pcpv normally cause painful lesions on the hands, fingers and face, and in several cases lymphadenopathy is recorded, which is difficult to differentiate from other distal upper extremity entities such as sporothricosis, mycobacterium marinum and leishmaniosis (schmidt et al. 2006). it also shares risk factors and clinical features with other life-threatening diseases such as localised orthopox virus infections, tularaemia and anthrax (lewis-jones 2004); missed or mistaken diagnosis constitutes ground for reflection on the sometimes incapacitating consequences of the disease on certain categories of patients, such as children and immunocompromised individuals (lederman et al. 2007). considering that south africa has one of the highest incidences of hiv and aids in the world, with an estimated 5.6 million people infected in 2009, it is possible that cases of giant ulcerative and recurrent forms may be misdiagnosed and not recognised, whilst a correct diagnosis would allow physicians to formulate appropriate therapy and management. conclusion top ↑ in conclusion, the study design of this work was based on an investigation performed on natural outbreaks of orf in sheep and goats in south africa. in line with classic epidemiology, disease determinants linked to the host, agent and environment and their interactions were described, providing the first descriptive epidemiological data on orf outbreaks in south africa. questionnaire responses from veterinarians and farmers have led to a better understanding of the host range, seasonal distribution of cases and environmental risk factors. a comparison between the actual number of outbreaks observed and the opinions of veterinarians on the likely incidence of orf suggests that the incidence of the disease is underestimated in south africa. our investigation led us to identify pcpv strains circulating in the cattle population, suggesting the necessity to collect more epidemiological data on parapoxvirus infections resembling vesicular diseases, also in bovines.although only one human case was reported during the observation period, the risk of zoonotic transmission, especially in low-income rural areas, is likely to be far higher, if based on the diagnosed human cases in other countries with similar levels of orf in livestock. acknowledgement top ↑ the authors wish to thank prof. kobus eloff for his support in identifying the medicinal plant used to treat orf in the field and dr laura gallina for her technical support in genomic sequencing. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this paper.< authors’ contributions a.s. (university of bologna) was responsible for the project design. s.p. (university of bologna) performed the field survey and collected samples. f.t. (university of bologna) performed most of the laboratory experiments. f.s. (university of bologna) contributed to the manuscript drafting. f.s. (university of pretoria) made conceptual contributions and was responsible for critical review of the manuscript. c.m.m. 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virology, national veterinary research institute, mozambique 2department of veterinary tropical diseases, university of pretoria, south africa 3avimune, pretoria, south africa 4department of production animal studies, university of pretoria, south africa 5agricultural research council, onderstepoort veterinary institute, south africa correspondence to: estelle venter postal address: private bag x04, onderstepoort 0110, south africa dates: received: 02 feb. 2012 accepted: 18 apr. 2012 published: 31 aug. 2012 how to cite this article: fringe, r., bosman, a.-m., ebersohn, k., bisschop, s., abolnik, c. & venter, e., 2012, ‘molecular characterisation of newcastle disease virus isolates from different geographical regions in mozambique in 2005’, onderstepoort journal of veterinary research 79(1), art. #409, 7 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.409 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. molecular characterisation of newcastle disease virus isolates from different geographical regions in mozambique in 2005 in this original research... open access • abstract • introduction • materials and methods    • sampling    • virus isolation    • virus characterisation    • ethical considerations • results    • confirmation of virus isolation    • characterisation of the newcastle disease virus • trustworthiness • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ newcastle disease (nd) is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. viral determinants for newcastle disease virus (ndv) virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. the prevalence of newcastle disease in village poultry in mozambique is not well documented and strains of ndv involved in yearly outbreaks are unknown. the fusion (f) protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. newcastle disease viruses from various geographical regions of mozambique were sequenced and compared genetically to published sequences obtained from genbank. samples were collected in three different areas of mozambique and ndv was isolated by infection of embryonated chicken eggs. sequence analysis of the f-protein encoding gene was used to classify 28 isolates from mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in genbank. the isolates obtained from mozambique grouped mainly into two clades. in the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from mozambique that were previously described. in the second clade, 16 isolates group together with sequences obtained from genbank originating from australia, china, south africa and the usa. eleven of these isolates showed a high similarity with sequences from south africa. the number of samples sequenced (n = 28), as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in mozambique in 2005. viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. from this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in mozambique. introduction top ↑ newcastle disease (nd) is regarded as one of the most serious diseases in the poultry industry (alexander 1991). the causative agent, newcastle disease virus (ndv), is transmitted by ingestion or inhalation and produces a disease with varied clinical manifestations, varying from severe acute to subclinical infection, depending on the virulence of the virus strain (millar, chambers & emmerson 1988). the virus is classified in the family paramyxoviridae, genus avulavirus which encompasses a diverse group of single-stranded, negative-sense, non-segmented rna viruses. ten distinct avian paramyxovirus (apmv-1 – apmv-10) groups are recognised and have been defined using standard serological tests (miller et al. 2011). avian paramyxovirus-1 and ndv are synonymous, although the term pigeon paramyxovirus type 1 (ppmv-1) has been used to distinguish the antigenic variant of apmv-1 virus responsible for the continuing panzootic in racing pigeons (alexander 1998; office internationale des epizooties [oie] 2004). both paramyxovirus (pmv)-2 and pmv-3 viruses have also been isolated from domestic poultry (alexander et al. 1997). the first reported outbreaks of nd occurred in 1926 on the island of java, indonesia. subsequently, an outbreak of the disease was reported in the same year near newcastle-on-tyne, which led to its description the following year (bruce et al. 2002; oie 2004; seal et al. 2002). from 1926 to 1940, severe cases of the disease were reported near the sea ports of the indian ocean. the virus first infected poultry in the tropical rain forests of south-east asia. once established in poultry, the worldwide spread of nd was probably the result of trade in live infected poultry. presently, nd has a worldwide distribution, although, in some countries such as australia, only viruses of low virulence for chickens have been reported (alexander et al. 1997). newcastle disease has been endemic in southern africa causing outbreaks of the disease in poultry (pfitzer et al. 2000) and entry through the port of durban was first recorded during 1944 (kaschula et al. 1945). in kenya, the first recorded entry of the disease was through the port of mombassa during 1934 (valadão 1950). although the virus was first isolated in 1946, the disease had already been clinically diagnosed in mozambique in 1938 (valadão 1950). the disease is one of the major constraints of rural poultry production which could otherwise play an important role in poverty alleviation. today it is responsible for high mortality in chickens in both traditional and commercial sectors throughout the country (fringe & dias 1991; valadão 1950). the paramyxovirus genome consists of a negative-sense, single-stranded ribonucleic acid (rna) that encodes for six genes: the nucleoprotein, phosphoprotein, matrix protein, fusion protein (f), haemagglutinin-neuramidase (hn) protein and polymerase protein, respectively. the hn protein mediates attachment of the virion to sialic acid-containing receptors, whilst the f protein mediates fusion of the viral envelope with cellular membranes (lamb & kolakofsky 2001; pantua et al. 2005; scheid & choppin 1974). analysis of the nucleotide sequence data from the f gene, encoding for the fusion protein of ndv, has allowed differentiation of closely related viruses resulting in epidemiological evidence of the virus origin (alexander et al. 1997). newcastle disease viruses were previously classified into nine genetic groups (i–ix), with genetic groups vi and vii divided into several subtypes (ballargy-pordany et al. 1996; tsai et al. 2004). more recent studies of the f and l (rna-dependent, rna polymerase gene) genes revealed two distinct classes: i and ii within nd serotype 1. phylogenetic analysis revealed that class i and ii viruses each consist of at least nine genotypes, designated 1–9 and i–ix, respectively (lui et al. 2003). only class ii viruses are responsible for fatal infections in poultry and they have been responsible for major outbreaks worldwide. newcastle disease viruses have been subsequently re-classified broadly into six lineages (1–6), which correspond to the existing groups (i–ix), and these lineages are divided into 13 sub-lineages (aldous et al. 2003). avirulent viruses are grouped in lineages 1 and 6, whilst virulent viruses are grouped in lineages 3, 4 and 5; lineage 2 contains both virulent and avirulent viruses. it seems that some lineages tend to group together geographically whilst others circulate worldwide (aldous et al. 2003). for example, isolates obtained from south africa and the middle east region group together in lineage 5 (genotype vii) and those from the far east and western europe group in lineage 3d (genotype viii) and 5b (viib) (lee et al. 2004). two novel genetic lineages, namely 5b (viib) and 3d (viii) were described by herczeg et al. (1999). they claimed that these groups were responsible for the 1990 and 1995 outbreaks in southern africa, including mozambique. this classification system (ballargy-pordany et al. 1996) made it possible to detect the origin of the virus and evidence exists that the nd strains circulating in southern africa have a common ancestor in the far east and southern europe (abolnik et al. 2004; liang et al. 2002). the prevalence of nd in village poultry in mozambique is not documented and only one report revealed the molecular characterisation of ndv isolates circulating in mozambique between 1990 and 1995 (herczeg et al. 1999). in the present study, the aim was to characterise genetically ndv strains occurring during 2005 in various geographical regions of mozambique using nucleic acid sequences and to compare these results with ndv sequencing results that have been described in south africa, as well as other published sequencing data from ndv elsewhere in africa. this will lead to a better understanding of the epidemiology of the disease and will generate useful data that can be applied to adapt or adjust control programmes in mozambique. materials and methods top ↑ sampling samples from suspected ndv infected and dead chickens were collected from various villages in the southern (72 samples), central (18 samples) and northern (30 samples) regions of mozambique (figure 1). cloacal and tracheal swabs, as well as faeces, were collected from live chickens and samples from the trachea, lung, spleen, intestine, liver, heart, kidney, proventriculus and brain were collected from dead chickens. swabs were treated separately according to the procedure described by the oie manual (2004), whilst other organ samples were placed in isotonic phosphate buffered saline (pbs), ph 7.0 – ph 7.4, until processing. virus isolation virus isolation was performed at the virology department of the national veterinary research institute (nvri), in maputo, mozambique. suspensions of organs and/or swabs were first centrifuged in a bench-top centrifuge at 4000 rpm for 5 min. antibiotics (penicillin [2000 units/ml], streptomycin [0.01 ml/1 ml], gentamycin [50 µg/ml] and mycostatin [1000 units/ml]) were added to the supernatants and incubated for 1 h – 2 h at room temperature (oie 2004). a volume of 0.2 ml of the supernatant was then inoculated into the allantoic cavity of five 9–11-day-old, specific pathogen free (spf) embryonated chicken eggs according to the procedure described in the oie manual (2004). eggs were then incubated at 37 °c for 4–7 days. allantoic fluid was harvested and tested for the presence of a haemagglutinating virus by the haemagglutination (ha) test. samples that tested positive with the ha test were tested for the presence of ndv using polyclonal anti-ndv serum in a haemagglutination-inhibition (hi) test. aliquots of ndv-positive allantoic fluid were prepared and stored at -70 °c for polymerase chain reaction (pcr) and sequencing analysis (alexander 1991, 1998; oie 2004). virus characterisation nucleic acid was extracted from allantoic fluid positive for ndv (n = 44/120) using trizol ls (invitrogen; life technologies™, carlsbad, usa) and/or using a qiaamp® viral rna mini kit, according to the manufacturer’s instructions (qiagen gmbh, hilden, germany). the geneamp®gold rna pcr reagent kit (applied biosystems, foster city, usa) was used to generate first strand complementary deoxyribonucleic acid (cdna). the following reaction was compiled: 2 µl of ± 75 ng of extracted rna was added to the rt mixture (5 × reaction buffer, 2.5 mm mg2+, 250 µm of each dntp, 10 units per 20 µl rnase inhibitor, 10 mm dtt, 1.25 µm random hexamer, 15 units per 20 ml multiscribe reverse transcriptase) in a final volume of 20 µl rnase-free water. the cdna product (10 µl) was amplified at 25 °c for 10 min, followed by 42 °c for 15 min. polymerase chain reaction was then conducted to amplify a 1180 base pair fragment spanning the regions between nucleotides 581 of the f and nucleotides 610 of the matrix protein (m), using previously published primers: m610-5’ctg tac aat ctt gcg ctc att gtc-3’ and reverse primer ndvf581-5’ctg cca ctg cta gtt gtg ata atc c-3’ (abolnik et al. 2004). the pcr amplification mixture consisted of 2 µl cdna, 1 × rt-pcr buffer, 1.75 mm mg2+, 200 µm of each primer, 2.5 units per 25 µl amplitaq gold deoxyribonucleic acid (dna) polymerase, in a total volume of 25 µl. the pcr was carried out using the same cycling conditions previously described by abolnik et al. (2004). the pcr products were separated by electrophoresis using a 1.5% agarose gel stained by ethidium bromide (0.2 mg/ml). the results were visualised under uv-illumination and documented using a kodak edac gel documentation system (kodak, rochester, usa). amplification products were submitted to inqaba biotechnical industries (pty) ltd, pretoria, south africa for sequencing. the same set of primers was used for sequencing as described for the pcr. sequencing data of the partial f gene was assembled using gap4 of the staden package (bonfield, smith & staden 1995; staden 1996; staden, beal & bonfield 2000). sequence data for the f0 cleavage site was assembled and edited to a total length of 382 base pairs using gap 4 of the staden package (version 1.6.0 for windows; 2006) (bonfield et al. 1995). sequences were deposited in genbank under accession numbers, as summarised in table 1. to compare sequences obtained in this study with previously published sequences, a basic local alignment search tool (blast) search was performed using the blastn algorithm. the assembled sequences were aligned with related sequences obtained from genbank using clustalx (version 1.81 for windows; 2000). the alignment was truncated manually to the size of the smallest sequence. similarity matrices were constructed using the two-parameter model of kimura (kimura 1980) and the jukes and cantor (1969) correction model for multiple base changes. phylogenetic trees were constructed using neighbour-joining (saitou & nei 1987) and the maximum parsimony methods by using the mega 3.0 software package (kumar, tamura & nei 2004). to assign confidence values to topologies, bootstrapping (felsenstein 1985) was applied: 1000 replicates per tree for distance methods. analysis of the deduced amino acid sequences of the collected samples and data obtained from genbank was performed by construction of a phylogenetic tree using the maximum parsimony tree. mega 4 software was used and subjected to 1000 replicates of bootstrap. figure 1: map of the geographical areas in mozambique where samples were collected during the study period. ethical considerations samples were sent mainly to the national veterinary research institute in mozambique for diagnostic purposes. other samples were collected by a veterinarian (dr raul fringe) under the authority of the national veterinary research institute, mozambique. results top ↑ confirmation of virus isolation samples (n = 120) were collected from the northern, central and southern regions of mozambique. newcastle disease virus isolates (n = 39) from embryonated chicken eggs were confirmed by ha and hi testing. table 1: samples included in the phylogenetic analysis. characterisation of the newcastle disease virus twenty-eight pcr amplicons were used for sequencing (table 1). sequences from these samples, together with sequencing data obtained from genbank, were aligned and phylogenetic analysis was performed. nucleic acid sequences were analysed by constructing a neighbour-joining tree (figure 2) (saitou & nei 1987). similar results were obtained by the maximum parsimony method. nucleotide sequences were translated into amino acids and also analysed by the maximum parsimony method (results not shown) (tamura et al. 2007). the samples obtained from mozambique in 2005 grouped mainly into two clades (figure 2). in the first clade, 12 isolates (ndv 7, 10, 21, 25, 26, 28, 30, 31, 35, 36, 39 and 41) grouped together with sequences of isolates representing genotypes vii, viib, viic and viid (abolnik et al. 2004; herczeg et al. 1999). taking the high bootstrap values in account, these samples clearly can be assigned to genotype viib. four of these isolates (ndv 7, 10, 25 and 39) cluster in close relationship with sequences derived from isolates from mozambique (af136780, af136778, af136777 and af136779) previously described by herczeg et al. (1999). the four isolates used in this study were collected in the southern region, one (ndv 10) from the magude district in maputo province (figure 1) and two from hamoine district in inhambane province (ndv 25 and 39), whilst one sample (ndv 7) was collected from the central region (sofala province, peri-urban zone). the other eight isolates (ndv 21, 26, 28, 30, 31, 35, 36 and 41) group together and share a node to the previously mentioned group. these samples were collected from the hamoine district in inhambane province (central–southern region) (ndv 31 and 41), the monape district in nampula province (northern region) (ndv 21) and the chibuto district in gaza province (southern region) (ndv 28 and 30), as well as from the peri-urban zone of manica province (central region) (ndv 26) and the sabie location of maputo province (ndv 35 and 36) (figure 1 and figure 2). in the second clade, 16 isolates grouped together with sequences obtained from genbank originating from australia and south africa and, to a lesser extent, to isolates from china and the usa (figure 2). eleven of these isolates (ndv 14, 16, 17, 18, 19, 20, 23, 32, 34, 43 and 44) showed a high similarity with sequences from south africa (af532741 and af532742, both from kwazulu-natal province, and af532743, from gauteng province) and an isolate from queensland, australia (m24693) described by toyoda et al. (1989) (figure 2). two isolates (ndv 40 and 42) that share the same node branched together with two isolates from australia (ay935491 and ay935495). these isolates originated from new south wales and were published by kattenbelt, stevens and gould (2006). there is no indication that these isolates are vaccine strains. three isolates (ndv 3, 12 and 22) branched separately but in a close similarity to isolate m24692 from australia described by toyoda et al. (1989), as well as the grouping of samples in this study mentioned above (figure 2). analysis of the deduced amino acid sequences of the collected samples and data obtained from genbank did not change the phylogenetic grouping of samples (results not shown). figure 2: results of the neighbour-joining analysis of the sequences of the partial fusion protein encoding gene, showing the phylogenetic relationship of newcastle disease virus isolates collected in mozambique in 2005 with other known newcastle disease virus sequences described in south africa and other countries worldwide. different lineages are indicated on the right. trustworthiness top ↑ validated standard operating procedures were used in the laboratory and all the relevant controls were used in all laboratory tests performed. results obtained can therefore be declared trustworthy. discussion top ↑ outbreaks of nd in mozambican poultry and the devastation of the infection has been reported for decades, yet only one previous study (herczeg et al. 1999) focused on characterising the causative strains, with samples collected from 1990 to 1995. they concluded that outbreaks in mozambique and southern africa were to the result of strains of lineage 5b (viib) and 3d (viii) that were most likely endemic to the region, or were recently introduced. for the present study, 120 samples (mainly swabs) were collected in 2005 from various villages in different geographical areas in mozambique. we aimed to determine the strains that circulated in the country, 10 years after the first description of lineage 5b in mozambique. of the 28 strains genetically analysed in the present study, 11 were classified as lineage 5b (viib) and were phylogenetically closely related to strains described in mozambique in 1994–1995 (heczeg et al. 1999), as well as from south africa (1993–1999) and botswana (1998) (abolnik et al. 2004). these virulent strains were distributed across all regions sampled. lineage 5d (viia), that was first detected in south africa in 1999 and continues to cause major outbreaks in the region in the past, was not detected in the samples tested here, neither were pigeon paramyxovirus strains (lineage 4) detected in the poultry sampled. the majority of the viruses we analysed were classified in lineage 1. lineage 1 not only contains strains that are frequently isolated from wild birds, but also strains that have been adapted for use as vaccines; for example, the queensland/v-4 (‘v-4‘), ulster 2c/67 and i-2 (derived from v-4) that have been used as live vaccines in many countries (aldous et al. 2003). the avinew-61tm vaccine, a vg/ga derivative is a lineage 1 strain that applied widely in south africa, and strains that were assumed to be derivates of this live vaccine were isolated frequently in south africa in a previous study (abolnik et al. 2004). the v-4 vaccine was used in the macie district, gaza province in the 1990s in mozambique and the i-2 vaccine was introduced in 1998, first in laboratory trials at the national veterinary research institute and then in villages of the manhiça district, maputo province in the southern region of mozambique. in the following years, the vaccine was used to vaccinate village chickens in five provinces, viz. gaza, inhambane, tete, zambezia and nampula. thus, it is likely that the lineage 1 strains detected in this study are derivates of the v-4 and i-2 vaccines that are widely used in mozambique. conclusion top ↑ in this study, we present a ’snapshot‘ of the nd situation in mozambique in 2005, 10 years after a previous study identified lineage 5b that also affected south africa. but, the latter was affected subsequently by an outbreak of lineage 5d at the time of sampling, and which then completely replaced lineage 5b. this highly virulent strain did not appear to have spilled over to mozambique by 2005. lineage 5b therefore persisted in mozambique from at least 1994 until 2005; however, the current (2012) status remains unknown. acknowledgements top ↑ the research project was funded by the swedish agency for research cooperation. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions e.v. 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microbiology 104, 19–30. http://dx.doi.org/10.1016/j.vetmic.2004.09.005, pmid:15530736 valadão, f.g., 1950, ‘newcastle disease in province of mozambique’, veterinary journal 12, 47–73. article information authors: massimo scacchia1 andrea di provvido1 carla ippoliti1 uqbazghi kefle2 tesfaalem t. sebhatu3 annarita d’angelo1 fabrizio de massis1 affiliations: 1istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, teramo, italy2veterinary services, asmara, eritrea 3national animal and plant health laboratory, asmara, eritrea correspondence to: fabrizio de massis postal address: istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’, campo boario, 64100 teramo, italy dates: received: 08 june 2012 accepted: 27 mar. 2013 published: 23 apr. 2013 how to cite this article: scacchia, m., di provvido, a., ippoliti, c., kefle, u., sebhatu, t.t., d’angelo, a. & de massis, f., 2013, ‘prevalence of brucellosis in dairy cattle from the main dairy farming regions of eritrea’, onderstepoort journal of veterinary research 80(1), 4 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.448 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence of brucellosis in dairy cattle from the main dairy farming regions of eritrea in this original research... open access • abstract • introduction • material and methods    • study areas    • animals    • sample collection    • sample testing    • statistical analysis • results    • brucellosis at sub-regional level • discussion • conclusions • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ in order to get a reliable estimate of brucellosis prevalence in eritrean dairy cattle, a cross-sectional study was carried out in 2009. the survey considered the sub-population of dairy cattle reared in modern smalland medium-sized farms. samples were screened with the rose bengal test (rbt) and positive cases were confirmed with the complement fixation test (cft). a total of 2.77% (417/15 049; credibility interval ci: 2.52% – 3.05%) of the animals tested in this study were positive for antibodies to brucella species, with a variable and generally low distribution of positive animals at regional level. the highest seroprevalence was found in the maekel region (5.15%; ci: 4.58% – 5.80%), followed by the debub (1.99%; ci: 1.59% – 2.50%) and gash-barka (1.71%; ci: 1.34% – 2.20%) regions. seroprevalence at sub-regional levels was also generally low, except for two sub-regions of debub and the sub-region haicota from the gash-barka region. seroprevalence was high and more uniformly distributed in the maekel region, namely in the asmara, berik and serejeka sub-regions. considering the overall low brucellosis prevalence in the country, as identified by the present study, a brucellosis eradication programme for dairy farms using a test-and-slaughter policy would be possible. however, to encourage the voluntary participation of farmers to the programme and to raise their awareness of the risks related to the disease for animals and humans, an extensive public awareness campaign should be carefully considered, as well as strict and mandatory dairy movement control. introduction top ↑ brucellosis is one of the most common zoonotic diseases in the world. the geographical distribution of brucellosis constantly changes as new foci emerge or re-emerge. the disease occurs worldwide in both animals and humans, except in those countries where bovine brucellosis has been eradicated. the worldwide economic losses due to brucellosis are extensive, not only in terms of animal production but also in terms of human health. however, when the incidence of brucellosis is controlled in the animal reservoirs, there is a corresponding and significant decline in the incidence in humans (seleem, boyle & sriranganathan 2010). in eritrea, brucellosis is an important disease in both animals and humans; most human cases appear to be related to the consumption of unpasteurised milk (kebedom et al. 2008). literature shows that there have been some seroprevalence studies carried out in domestic animals (omer et al. 2000a, 2000b) and humans (omer et al. 2002). however, they were limited both in terms of number of animals tested and their geographical extent. knowledge about brucellosis prevalence in a country is of the utmost importance in order to set up a strategy to control and eradicate the disease. therefore, in 2009, the ministry of agriculture carried out a new serological survey for brucellosis in eritrea. the objective was to estimate the seroprevalence of brucellosis in dairy cattle in eritrea, in order to identify strategies for control and eradication of the disease in the country. material and methods top ↑ study areas eritrea is divided into six administrative regions: maekel, debub, anseba, gash-barka, northern red sea and southern red sea. the study was carried out in the first five regions, where dairy farming is practiced. southern red sea was excluded because there are limited dairy activities due to the dry climate. the population of dairy cattle reared in modern small and medium-sized farms were included in the study. these farms are mainly located in the urban and peri-urban areas in all of the five regions selected for the study. animals the dairy cattle population in eritrea is estimated to be around 28 000 and is comprised of three types of breeds: holstein friesian (90%); indigenous breeds, mainly barka (approximately 8% – 9%); and crossbreeds of holstein friesian and barka (approximately 1% – 2%). the three main cattle husbandry systems are: the intensive urban and peri-urban system (mainly devoted to dairy cattle); the extensive mixed crop-livestock system used in the southern highlands; and the extensive (traditional) pastoral husbandry system used in the western and eastern lowlands. the serological study included all sexually mature dairy cattle (both male and female) present in individual dairy farms in urban and peri-urban areas and was planned and coordinated by the ministry of agriculture of eritrea during 2009. there is no vaccination against animal brucellosis in eritrea, and thus all samples were from unvaccinated animals. sample collection sera were collected in 10 ml sterile tubes by jugular venipuncture. sera were stored in refrigerators (0°c – 4°c) and transported in iceboxes to the central veterinary laboratory in asmara. regional veterinary teams established for this purpose collected the samples. sample testing all the tests were carried out at the central veterinary laboratory in asmara. serum samples were tested for brucella antibodies using the rose bengal test (rbt) as a screening test and all positive reactors were confirmed with the complement fixation test (cft). both tests were performed according to the provisions of the world organization for animal health (oie) manual of diagnostic tests and vaccines for terrestrial animals (world organization for animal health (oie) 2008). to increase the specificity of test results, only samples that tested positive on both rbt and cft were considered to be positive for brucella. statistical analysis results obtained in this study were estimated and compared using a bayesian approach (sivia 1996). this is an application of the bayes theorem (bayes 1763), which allows the investigator to integrate any previous knowledge (expressed as a prior probability distribution), with the likelihood of obtaining a certain result if the animals are infected (likelihood functions), with the results obtained by the application of the tests to a given population (collected data). the final results are probability distributions of the proportion of infected animals in the population (posterior probability). probabilities of the various possible prevalence values were estimated using a binomial likelihood function and an uninformed uniform (0, 1) prior distribution. as existing knowledge on the prevalence of infected animals was considered to be virtually zero, an uninformed uniform (0, 1) prior distribution was used. the uniform (0, 1) distribution states that prior to the collection of data, all true probability values are considered possible within the range defined for the number of infected animals (prevalence calculation at animal level). the upper (ucl) and lower (lcl) 95% credibility levels (limits of the 95% credibility interval [ci]) were calculated using a beta probability distribution (vose 2000). the primary advantage of using a bayesian method over a traditional statistical method is that uncertainty in each parameter is modelled as a probability distribution, thus, the posterior distributions that are obtained in the analysis can be used to calculate the 95% ci for prevalence. in contrast to confidence intervals, credibility intervals have a direct probabilistic interpretation (wagner et al. 2003). calculations were performed using ms-excel® for windows®, version 2003 (ms excel 2003). results top ↑ the total number of animals sampled from the different regions was 15 049. a total of 2.77% (ci: 2.52% – 3.05%) tested positive to antibodies to brucella species (table 1). table 1: test results for brucellosis in dairy cattle at regional level in eritrea, 2009. the geographical distribution of test results at regional level is shown in figure 1. the distribution of positive animals at regional level is variable and generally low. the highest seroprevalence was found in the maekel region (5.15%; ci: 4.58% – 5.80%), followed by the debub (1.99%; ci: 1.59% – 2.50%) and gash-barka (1.71%; ci: 1.34% – 2.20%) regions. in the anseba and northern red sea regions, seroprevalences were 0.70% (ci: 0.43% – 1.15%) and 1.47% (ci: 0.81% – 2.69%) respectively (see table 1 and figure 1). as far as the maekel region is concerned, the lcl of the percentage of positive animals is higher than the ucl of the percentage of positive animals identified in the other regions. therefore, the seroprevalence recorded in the maekel region is significantly higher than all of the other regions investigated. as far as the anseba region is concerned, the ucl of the percentage of positive animals is lower than the lcl of the percentage of positive animals identified in the maekel, debub and gash-barka regions. therefore, the seroprevalence recorded in the anseba region is significantly lower than the ones recorded in the maekel, debub and gash-barka regions. figure 1: geographical distribution of test results for brucellosis in dairy cattle in eritrea at regional level in 2009. brucellosis at sub-regional level seroprevalence at sub-regional levels (figure 2) is generally low, except for three sub-regions in the maekel region (asmara, berik and serejeka), two sub-regions in the debub region (adikeih and adiquala) and the sub-region of haikota in the gash-barka region. however, the number of infected animals in the sub-regions of adikeih, adiquala and haicota is small (22, 17 and 10 respectively). figure 2: geographical distribution of test results for brucellosis in dairy cattle in eritrea at sub-regional level in 2009. discussion top ↑ the results of this study show that the percentage of dairy cattle positive for brucella antibodies in rbt and cft in eritrea is generally low. this excludes the maekel region (figure 1), which shows an overall percentage slightly above 5% (table 1). the seroprevalence recorded in the maekel region is significantly higher than the other regions. this is in contrast to the seroprevalence recorded in the anseba region, which is significantly lower than those recorded in the maekel, debub and gash-barka regions. this suggests that further investigation is needed to assess the possible causes of the observed variation of brucellosis prevalence in the different regions. this is confirmed by the fact that at sub-regional level the seroprevalence is generally low (figure 2), (except for two sub-regions in debub and the sub-region haicota from the gash-barka region) and the number of infected animals in these sub-regions is small (only 22, 17 and 10, in adikeih, adiquala and haicota sub-regions, respectively).studies investigating brucellosis in dairy cattle have been scarce in the area concerned. omer et al. (2000) found an individual seroprevalence of 8.2% in dairy herds kept under the intensive husbandry systems of the farms around the capital, asmara. higher percentages of brucellosis-positive dairy cattle were obtained in a study carried out by omer et al. (2007) in the kassala area, eastern sudan, near to the eritrean border. this study was conducted during 2004 – 2006 and identified a mean of 10.9% of cattle testing positive. however, this figure could have been overestimated due to the low number of cattle tested during the period (n = 694). losses due to brucellosis are extensive, not only in terms of animal production, but also in human health. however, when the incidence of brucellosis is controlled in the animal reservoirs, there is a corresponding and significant decline in the incidence in humans de massis et al. (2005). it is hoped that the results of the present study will encourage the start of a brucellosis-eradication programme using the test-and-slaughter policy in eritrea. the number of seropositive animals is low and costs of slaughtering could be handled either by the owners themselves or through partial or total compensation from the dairy associations or the government. the latter strategy would greatly help the owners in restocking animals and affording the indirect economic losses related to the presence of the disease in herds. however, one of the keys to a successful eradication programme is the voluntary participation of the animal owners. to reach this objective it is important to increase the public awareness about the risks related to the disease in animals and human beings. this requires an increase in the knowledge of disease control and eradication by the veterinary services, which would increase the trust of farmers in the system in order to guarantee their complete and voluntary participation in the eradication campaign. moreover, to complement and support the eradication programme, the participation of the farmers should be encouraged through establishing some market-reward system (i.e. easy market accesses to farmers with brucellosis-free herds and restrictions to access the milk markets for owners with seropositive herds). conclusions top ↑ the percentage of dairy cattle positive for brucella antibodies in rbt and cft in eritrea is generally low, except for the maekel region (see figure 1), which shows an overall percentage slightly above 5%. considering the overall low brucellosis prevalence in eritrea, a brucellosis-eradication programme for dairy cattle herds through a test-and-slaughter policy could be considered. however, to encourage voluntary farmer participation and to raise awareness about the risks related to the disease in animals and human beings, an extensive public awareness campaign is vital, as well as strict and mandatory dairy movement control. acknowledgements top ↑ activities were carried out in the framework of the oie twinning project on brucellosis between the national veterinary laboratory of eritrea and the istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’ in teramo, italy. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.s. (istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’) was the project leader. m.s. and a.d.p. (istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’) were both responsible for the project design. c.i. (istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’) assisted in data calculations and map production. u.k. (national animal and plant health laboratory) made conceptual contributions and carried out the literature review. t.t.s. (national animal and plant health laboratory) and a.d’a. (istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’) made conceptual contributions. f.d.m.. (istituto zooprofilattico sperimentale dell’abruzzo e del molise ‘g. caporale’) carried out data analysis and wrote the manuscript. references top ↑ bayes, t., 1763, ‘an essay towards solving a problem in the doctrine of chances’, philosophical transactions of the royal society of london 53, 370–418 viewed 01 december 2011, from www.stat.ucla.edu/history/essay.pdfde massis, f., di girolamo, a., petrini, a., pizzigallo, e., & giovannini, a., 2005, correlation between animal and human brucellosis in italy during the period 1997-2002. clinical microbiology and infection 11, 632–636. http://dx.doi.org/10.1111/j.1469-0691.2005.01204.x, pmid:16008615 kebedom, a., teclebirhan, t., tekest, t., zekarias, b., mufunda, j. & gebremichael, a., 2008, ‘recent lessons learnt from the outbreak of brucellosis in dekemhare, anseba, eritrea’, journal of the eritrean medical association 3, 19–21, viewed 01 december 2011, from http://www.ajol.info/index.php/jema/article/view/49622 ms excel, 2003, computer software, microsoft windows, redmond, washington. omer, m.k., assefaw, t., skjerve, e., tekleghiorghis, t. & woldehiwet, z., 2002, ‘prevalence of antibodies to brucella spp. and risk factors related to high-risk occupational groups in eritrea’, epidemiology and infection 129, 85–91. http://dx.doi.org/10.1017/s0950268802007215, pmid:12211600, pmcid:2869878 omer, m.k., skjerve, e., holstad, g., woldehiwet, z. & macmillan, a.p., 2000a, ‘prevalence of antibodies to brucella spp. in cattle, sheep, goats, horses and camels in the state of eritrea; influence of husbandry systems’, epidemiology and infection 125, 447–453. http://dx.doi.org/10.1017/s0950268899004501, pmid:11117970, pmcid:2869619 omer, m.k., skjerve, e., woldehiwet, z. & holstad g., 2000b, ‘risk factors for brucella spp. infection in dairy cattle farms in asmara, state of eritrea’, preventive veterinary medicine 46, 257–265. http://dx.doi.org/10.1016/s0167-5877(00)00152-5 omer, m.m., abdelaziz, a.a., abusalab, s.m.a. & ahmed, a.m., 2007, ‘survey of brucellosis among sheep, goats, camels and cattle in kassala area, eastern sudan’, journal of animal and veterinary advances 6, 635–637. seleem, m.n., boyle, s.m. & sriranganathan, n., 2010, ‘brucellosis: a re-emerging zoonosis’, veterinary microbiology 140, 392–398. http://dx.doi.org/10.1016/j.vetmic.2009.06.021, pmid:19604656 sivia, d.s., 1996, data analysis. a bayesian tutorial, clarendon, oxford. vose, d., 2000, risk analysis: a quantitative guide, 2nd edn., john wiley & sons, chichester. wagner, b., gardner, i., cameron, a., doherr, m.g., 2003, ‘statistical analysis of data from surveys, monitoring, and surveillance systems’, in m.d. salman (ed.), animal disease surveillance and survey systems: methods and applications, blackwell publishing, ames, iowa. http://dx.doi.org/10.1002/9780470344866.ch5 world organization for animal health (oie), 2008, manual of diagnostic tests and vaccines for terrestrial animals, viewed 01 december 2011, from http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/ article information authors: joyce siwila1 kabemba e. mwape1,2 affiliations: 1department of clinical studies, university of zambia, zambia2department of veterinary tropical diseases, university of pretoria, south africa correspondence to: joyce siwila postal address: po box 32379, lusaka 10101, zambia dates: received: 10 jan. 2012 accepted: 18 apr. 2012 published: 23 aug. 2012 how to cite this article: siwila, j. & mwape, k.e., 2012, ‘prevalence of cryptosporidium spp. and giardia duodenalis in pigs in lusaka, zambia’, onderstepoort journal of veterinary research 79(1), art. #404, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.404 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence of cryptosporidium spp. and giardia duodenalis in pigs in lusaka, zambia in this original research... open access • abstract • introduction • materials and methods    • study area, sample collection and analysis    • statistical analysis    • ethical considerations • results    • overall prevalence    • prevalence by farm and pig class    • prevalence by sex    • association between infection and faecal consistency • trustworthiness • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ this study was aimed at determining the prevalence of cryptosporidium spp. and giardia duodenalis in pigs which were being raised in intensive management systems. faecal samples were collected from pigs of all age groups from three different piggery units. samples were collected directly from the rectum for piglets and weaners and from the floor within 2 min – 5 min of excretion for sows and boars. at the time of collection, faecal consistency was noted as being normal, pasty or diarrhoeic. samples were analysed further using the merifluor® cryptosporidium/giardia immunofluorescence assay. all piggeries had at least one pig infected with either parasite. from a total 217 samples collected, 96 (44.2%; confidence interval [ci] = 37.6% – 50.9%) were positive for cryptosporidium spp., whilst 26 (12%; ci = 7.6% – 16.3%) had g. duodenalis parasites. of all the pigs, 6.9% (15/217) harboured both parasites. with regard to cryptosporidium spp. infection, statistically significant differences were observed amongst the three units (p = 0.001), whereas no significant differences were observed for g. duodenalis infection (p = 0.13). prevalence was higher in weaners as compared to other pig classes for both parasites, with significant differences being observed for g. duodenalis infection (p = 0.013). there was, however, no difference in infection between male and female pigs for both parasites. furthermore, most infections were asymptomatic. from the study results it was clear that cryptosporidium spp. and g. duodenalis infections were prevalent amongst pigs in the piggeries evaluated and, as such, may act as a source of infection for persons who come into contact with them. introduction top ↑ cryptosporidium spp. and giardia duodenalis are protozoan parasites infecting a wide range of domestic animals including livestock, dogs and cats (fayer 2004; thompson 2004; xiao & fayer 2008). both cryptosporidiosis and giardiosis, the respective diseases which these parasites cause, are reported to be zoonoses, although the status of giardiosis as a zoonosis is still debatable (monis & thompson 2003; smith et al. 2006). each genus comprises a complex of species and genotypes, some of which are pathogenic and some specific to particular hosts (caccio et al. 2005; smith et al. 2006; xiao et al. 2004). of the eight known assemblages, only two assemblages of g. duodenalis, a and b, are considered to be zoonotic (feng & xiao 2011).transmission occurs directly or indirectly by the ingestion of either oocysts or cysts from infected individuals, via contaminated water, food and pasture (castro-hermida et al. 2005; graczyk, fayer & cranfield 1997; preiser, preiser & madeo 2003; xiao, herd & bowman 1994; xiao, herd & rings 1993). with cryptosporidiosis, clinical manifestations vary and depend on the age and health status of the host, the infective dose and the genetic background of the parasite (xiao & fayer 2008), with stressed and immunologically compromised animals being more susceptible (enemark et al. 2002; ramirez, ward & sreevatsan 2004). in young livestock, the disease can be debilitating and cause severe illness and/or death (de graaf et al. 1999). in calves, cryptosporidiosis is often characterised by profuse watery diarrhoea of acute onset, anorexia, dehydration and weight loss (de graaf et al. 1999; fayer et al. 1998). for naturally infected pigs, the infection is typically asymptomatic even in young animals (maddox-hyttel et al. 2006; ramirez et al. 2004). however, symptomatic infections do occur in young piglets less than three weeks of age, with inappetance, depression, vomiting, diarrhoea and mortality being the most common signs (enemark et al. 2002; enemark et al. 2003; rotkiewicz et al. 2001). on the other hand, g. duodenalis infections in domestic animals are often asymptomatic, although clinical disease does occur in young animals (geurden et al. 2006; o’handley et al. 1999; robertson et al. 2000). in calves, g. duodenalis mostly affects those between five and ten weeks of age (thompson 2000), but infections may also occur in calves as young as four days (xiao & herd 1994) and in adult cows (maddox-hyttel et al. 2006). infection may result in numerous episodes of diarrhoea, which, in turn, adversely affects production and results in economic loses for producers (xiao 1994). other clinical signs may include chronic pasty diarrhoea, lethargy, weight loss and poor condition (thompson 2000). the occurrence of g. duodenalis in pigs has been reported in all age groups, from piglets to sows and boars, from europe and north america (langkjaer et al. 2007; maddox-hyttel et al. 2006; olson et al. 1997; xiao et al. 1994), with age specific prevalence ranging from 7% – 84%. little is known about the prevalence of cryptosporidium spp. and g. duodenalis in pigs in africa and in particular zambia. the aim of the present study was to determine the prevalence of cryptosporidium spp. and g. duodenalis in pigs of all age groups from three piggeries in zambia. materials and methods top ↑ study area, sample collection and analysis the university of zambia, located in the country’s capital, lusaka, has three piggery units that are run by three different departments of the university: the schools of veterinary medicine (ve) and agricultural sciences (ag), located within the campus premises, and on liempe farm (li), located in a neighbouring district about 20 km away from the main campus. the piggery units are used for teaching and commercial purposes. the present study was carried out in these units to determine the infection status of the pigs with cryptosporidium spp. and g. duodenalis. management is intensive and similar on all the three units. weaned piglets were housed in groups of 10–20 depending on the size of the pen, whilst sows and boars were housed individually. piglets were housed together with their dam. all pig houses had solid floors and all the pigs received piped water.the study was carried out from march to june 2011. visits were made to each unit and a single faecal sample was collected from each pig. the faecal samples were collected rectally using a gloved hand from piglets and weaners. efforts were made to collect the faecal samples from the rectum of sows and boars and, where not possible, the samples were picked from the floor within 2 min – 5 min of being dropped. these latter samples were taken from the upper area of the droppings to ensure that the sample had not been potentially contaminated through contact with the floor. faecal consistence was noted and categorised as either normal (hard, formed stool), pasty (pasty, unformed) or diarrhoeic (soft, unformed) at the time of collection. the samples were transported immediately to the laboratory in cool containers packed with ice packs, where they were placed in clean stool specimen containers containing 10% formalin. one part of stool was mixed with three parts of 10% formalin and fixed at room temperature for at least 1 h before further analysis. a commercial immunofluorescence assay (merifluor® cryptosporidium/giardia ifa) (meridian diagnostics inc., cincinnati, usa) for the detection of cryptosporidium spp. and g. duodenalis was used to identify cryptosporidium spp. oocysts and g. duodenalis cysts according to manufacturer’s instructions. (oo)cysts were visualised using an immunofluorescence microscope. a sample was identified as positive if at least one (oo)cyst was identified under the microscope. statistical analysis data were entered in an excel spreadsheet and verified for accuracy before being pasted into stata data editor. statistical analysis was carried out using computer software stata version 10.1 (2007). proportions, differences in infections amongst the piggeries, age groups and gender were determined by chi-square test and fisher’s exact test, where appropriate. data were reported as absolute values or as percentages. all results were considered significant at p < 0.05. ethical considerations permission to conduct the study in the piggery units was sought from the respective piggery managers. faecal samples were collected by trained veterinarians adhering to the zambian regulations and guidelines on animal husbandry. results top ↑ overall prevalence a total of 217 faecal samples were collected from the three piggery units, comprising samples from suckling piglets aged 2–5 weeks (n = 32), weaned piglets aged 7 weeks – 6 months (n = 163), gilts or sows (n = 15) and boars (n = 7) both aged more than 6 months. of the total samples collected, 112 were from female pigs and 105 from male pigs. the overall prevalence of cryptosporidium spp. was 44.2% (96/217; confidence interval [ci] = 37.6% – 50.9%), whilst that of g. duodenalis was 12.0% (26/217; ci = 7.6% – 16.3%). of all the pigs, 6.9% (15/217) harboured both parasites. all farms had at least one pig infected with either parasite. prevalence by farm and pig class prevalence amongst the three farms or locations in the different age groups and pig classes is indicated in table 1. on all the farms, the number tested was higher for weaners and so was the number of positive animals. with regard to cryptosporidium spp. infection, statistically significant differences were observed amongst the three farms (p = 0.0010). between farm differences were also observed for li and ve (p = 0.0110) and between ag and ve (p < 0.0001) but not for li and ag (p = 0.0770). on the other hand, no significant differences were observed amongst the farms for g. duodenalis infection (p = 0.1300), as was the case for between farms; li and ag (p = 0.3100), li and ve (p = 0.2600). however, differences were observed between ve and ag (p = 0.0430).the overall prevalence of cryptosporidium spp. in the different pig classes was 31.3% (10/32) in piglets, 46.6% (76/163) in weaners, 53.3% (8/15) in sows and 28.6% (2/7) in boars. on the other hand, the overall prevalence of g. duodenalis in the pig classes was 6.3% (2/32) in piglets, 10.4% (17/163) in weaners, 40.0% (6/15) in sows and 14.3% (1/7) in boars. significant differences were further observed amongst the pig classes for g. duodenalis (p = 0.0130), whilst no difference was observed for cryptosporidium spp. infection (p = 0.3000). table 1: prevalence of cryptosporidium spp. and giardia duodenalis arranged by pig class, sampled from three piggeries in lusaka, zambia over the study period from march to june 2011. prevalence by sex the prevalence was similar in both male pigs (41.9%; 44/105) and female pigs (46.4%; 52/112) for cryptosporidium spp. (p = 0.50). this was also the case for g. duodenalis, with prevalences of 9.5% (10/105) and 14.3% (16/112) in male and female pigs, respectively (p = 0.28). association between infection and faecal consistency most of the faecal samples collected were formed and had a normal consistency. only four (1.8%) of the samples were diarrhoeic and nine (4.2%) were pasty. half of the diarrhoeic faeces contained cryptosporidium spp. oocysts, but the parasites were absent in the pasty samples. almost half (46.1%; 94/204) of the normally formed faeces had cryptosporidium spp. parasites. the difference amongst the three categories was significant (p = 0.024). trustworthiness top ↑ the test used in the present study (merifluor® cryptosporidium/giardia) is reliable and has been used in many reported studies. parasite identification, however, requires an experienced technician or researcher. the samples in the present study were analysed by an experienced researcher and verified independently by another. the study was conducted on a small scale as a result of limited funds. a larger sample size with more piggeries included would have helped to make more generalised conclusions. discussion top ↑ in the present study, the prevalence of cryptosporidium spp. and g. duodenalis was determined for three university farms in zambia. this is the first report of the two protozoan parasites in zambian pigs. the overall prevalence of cryptosporidium spp. was 44.2%, a result that is significantly higher than what has been reported elsewhere. for example, olson et al. (1997) reported a prevalence of 11.0% from 236 canadian pigs, whilst fiuza et al. (2011) reported a prevalence of 2.2% from 91 pigs in rio de janeiro, brazil. in addition, kváč et al. (2009) reported a prevalence of 21.1% from 413 samples in the czech republic, whilst in spain, quílez et al. (1996) reported a prevalence of 21.9% from 620 pigs. comparable results for cryptosporidium spp. have, however, been reported in pig manure from different farms in canada in which 44.3% of the samples were positive for cryptosporidium spp. and 50.8% had g. duodenalis parasites (farzan et al. 2011); the g. duodenalis percentage being much higher than what we report in the present study. the overall prevalence of g. duodenalis in the present study was 12%. our results are in agreement with those reported by olson et al. (1997) in which they found a prevalence of 9% in pigs from canadian farms and that by xiao et al. (1994) (7%) from pigs in ohio, usa. nevertheless, higher prevalence rates (44%) have been reported elsewhere (koudela et al. 1991). the differences in the prevalence of both cryptosporidium spp. and g. duodenalis in the present study and that from other studies highlighted above can be attributed to differences in sample sizes, as well as sample processing and detection methods. the results in the present study are reported with caution. the prevalences for both cryptosporidium spp. and g. duodenalis are probably underestimated because only one sample was collected and tested. it has been shown that parasite excretion can be intermittent (buret et al. 1990) and therefore it is suggested that three samples be collected consecutively and tested (van gool et al. 2003). the triple sample collection was not conducted in the present study because of limited resources. in all the three piggeries, prevalence of cryptosporidium spp. was higher than that of g. duodenalis for all pig classes. the prevalence was higher in weaners than in any other pig class for both parasites, but significant differences amongst the three units was only observed for g. duodenalis. this finding of higher infection rates in weaners corroborates reports by other authors (maddox-hyttel et al. 2006; quílez et al. 1996) in which they found pigs aged one to six months to be infected with cryptosporidium spp., an indication that infection is more common in younger animals. establishment of infection is reported to be delayed until weaning (quílez et al. 1996). however, olson et al. (1997) reported higher prevalence in animals older than six months compared to younger animals. of the infected pigs, 98% were asymptomatic, whilst only two were symptomatic. this is in agreement with previous reports that indicated largely asymptomatic infections in most pigs (quílez et al. 1996; ramirez et al. 2004) for both cryptosporidium spp. and g. duodenalis. conclusion top ↑ the present study has demonstrated that cryptosporidium spp. and g. duodenalis are prevalent in pigs and most infections are asymptomatic. these pigs may act as a source of infection for humans through direct contact with faeces and faecal contaminated materials. it is therefore important that control measures be considered and put in place to prevent such infections. further studies are warranted to determine the infection status of other pigs in other areas of the country, including free range pigs which are common in rural areas. acknowledgements top ↑ the authors are grateful to the piggery managers for agreeing to participate in the study. mr bright chulu is also thanked for his technical support. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions j.s. (university of zambia) and k.e.m. (university of zambia) were responsible for the design of the study and the sample collection. j.s. (university of zambia) performed the laboratory analysis of the samples and the statistical analysis of data. j.s. (university of zambia) wrote the first draft of the manuscript. j.s. (university of zambia) and k.e.m. 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3southern african development community secretariat, botswana 4ministry of livestock development and fisheries, tanzania correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: peter, e., kasanga, c.j., sallu, r., mathias, m., yongolo, m., mulumba, m. et al., 2014, ‘screening for foot-and-mouth disease virus in livestock-wildlife interface areas of tanzania’, onderstepoort journal of veterinary research 81(2), art. #723, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.723 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. screening for foot-and-mouth disease virus in livestock-wildlife interface areas of tanzania in this abstracts... open access • abstract • acknowledgements abstract top ↑ rapid and accurate diagnosis is paramount in understanding the infection status of foot-and-mouth disease (fmd) virus (fmdv) in animals. in this study, the singleplex real-time rt-pcr (qrt-pcr) assay employing the callahan 3df-2, 3df-r primers and callahan 3dp-1 probe was used in screening for fmdv genome on oesophageal-pharyngeal (op) fluids. the op samples were collected from cattle and african buffaloes in livestock-wildlife interface areas of mikumi, mkomazi and ruaha national parks in tanzania in 2011, which included national parks and the surrounding areas. the detection rates of fmdv genome were 5.88% (n = 3), 19.44% (n = 7) and 41.18% (n = 21) in mkomazi, ruaha and mikumi national parks, respectively. fmdv detection rates in ruaha and mikumi were significantly higher in the african buffaloes (p < 0.05) compared to those in cattle. there was no correlation of fmdv detection with either age or sex of the animals in the three national parks. these findings indicate that cattle and buffaloes in mikumi, ruaha and mkomazi were naturally infected with fmdv. furthermore, the higher fmdv detection rates in buffaloes suggest that buffaloes could potentially act as reservoirs for fmdv and possibly play a significant role in transmission of the virus to other in-contact susceptible animals. further studies including serotyping, virus isolation, experimental infection and sequencing of the viruses, are required to elucidate the complex epidemiology of fmd in cattle and buffaloes in the livestock-wildlife interface areas in tanzania. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). article information authors bugwesa z. katale1,2 e.v. mbugi1 s. kendal3 r.d. fyumagwa2 g.s. kibiki4 p. godfrey-faussett5 j.d. keyyu2 p. van helden6 m.i. matee1 affiliations 1department of microbiology and immunology, muhimbili university of health and allied sciences, tanzania 2tanzania wildlife research institute, njiro, arusha, tanzania 3centre for emerging, endemic and exotic disease, royal veterinary college, united kingdom 4kilimanjaro christian medical college, kilimanjaro clinical research institute, tumaini university, moshi, tanzania 5department of infectious and tropical diseases, london school of hygiene and tropical medicine, united kingdom 6dst/nrf centre of excellence for biomedical tuberculosis research/mrc centre of molecular and cellular biology, division of molecular biology and human genetics, faculty of health sciences, university of stellenbosch, correspondence to: bugwesa katale postal address: po box 95001, dar es salaam, tanzania how to cite this proceeding: katale, b.z., mbugi, e.v., kendal, s., fyumagwa, r.d., kibik, g.s., godfrey-faussett, p. et al., 2012, ‘bovine tuberculosis at the human-livestock-wildlife interface: is it a public health problem in tanzania? a review’, onderstepoort journal of veterinary research 79(2), art. #463, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.463 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. bovine tuberculosis at the human-livestock-wildlife interface: is it a public health problem in tanzania? in this proceeding... open access • abstract • introduction • distribution of bovine tuberculosis in animals in tanzania • countrywide survey of mycobacterium bovis infection in humans • risk factors for mycobacterium bovis infection and disease in humans • role of husbandry practice in transmission of bovine tuberculosis • challenges for control of bovine tuberculosis in cattle in tanzania • conclusion • acknowledgement    • competing interest    • authors’ contributions • references abstract top ↑ despite the apparent public health concern about bovine tuberculosis (btb) in tanzania, little has been done regarding the zoonotic importance of the disease and raising awareness of the community to prevent the disease. bovine tuberculosis is a potential zoonotic disease that can infect a variety of hosts, including humans. the presence of multiple hosts including wild animals, inefficient diagnostic techniques, absence of defined national controls and eradication programs could impede the control of bovine tb. in tanzania, the diagnosis of mycobacterium bovis in animals is mostly carried out by tuberculin skin testing, meat inspection in abattoirs and only rarely using bacteriological techniques. the estimated prevalence of btb in animals in tanzania varies and ranges across regions from 0.2% to 13.3%, which is likely to be an underestimate if not confirmed by bacteriology or molecular techniques. mycobacterium bovis has been detected and isolated from different animal species and has been recovered in 10% of apparently healthy wildebeest that did not show lesions at post-mortem. the transmission of the disease from animals to humans can occur directly through the aerosol route and indirectly by consumption of raw milk. this poses an emerging disease threat in the current era of hiv confection in tanzania and elsewhere. mycobacterium bovis is one of the causative agents of human extra pulmonary tuberculosis. in tanzania there was a significant increase (116.6%) of extrapulmonary cases reported between 1995 and 2009, suggesting the possibility of widespread m. bovis and mycobacterium tuberculosis infection due to general rise of human immunodeficiency virus (hiv). this paper aims to review the potential health and economic impact of bovine tuberculosis and challenges to its control in order to safeguard human and animal population in tanzania. introduction top ↑ mycobacterium tuberculosis, mycobacterium bovis, mycobacterium bovis bcg, mycobacterium canettii, mycobacterium africanum, mycobacterium pinnipedii, mycobacterium microti, mycobacterium caprae, the dassie and the oryx bacillus, and the recently discovered mycobacterium mungi are closely related species that form the m. tuberculosis complex (mtbc). mycobacterium tuberculosis and m. bovis are the most important species in the complex which commonly cause human and animal tuberculosis (tb), with concomitant negative consequences for human and animal health and economic costs. the probability of m. bovis transmission is more likely to occur between animals, particularly those in close contact such as herd animals (grange & collins 1987). humans can also be infected by m. bovis from contact with infected animals or animal products and the likelihood of infection and disease, as with human forms of tb are exacerbated by crowding and stress (figueroa-munoz & ramon-pardo 2008). the transmission of m. bovis between humans or from humans to animals is very rare. although the occurrence of m. bovis in humans is relatively minor compared to the burden from m. tuberculosis as far as we know, there is concern that the hiv and aids pandemic may have magnified this risk. transmission of m. bovis at the livestock-wildlife or human-animal interface occurs essentially because of overlap in their territories (aranaz et al. 2004). encroachment of wildlife sanctuaries by humans in tanzania has increased the likelihood of this interaction and infection. specifically, boundary regions areas in protected areas are used increasingly for grazing of livestock and agriculture and this corresponds with areas where the remaining population of wildlife has been concentrated by this land-use pressure (etter et al. 2006). factors as source of infection and transmissions of m. bovis include persistence of m. bovis in infected animals after death and survival of m. bovis in the environment (aranaz et al. 2004). it is not only domestic animals that can experience pathology from m. bovis, but a wide range of wild animal species in africa, including lion (panthera leo), buffalo (syncerus caffer), wildebeest (connochaetes taurinus), kudu (tragelaphus strepsiceros), bushbuck (tragelaphus scriptus), topi (damaliscus lunatus) and a number of others (cleveland et al. 2005). these animals can be a source of infection for livestock and humans (aranaz et al. 2004) and in tanzania, a classic example is the illegal hunting of resident and migratory herbivores in protected areas (loibooki et al. 2002; magige et al. 2008; sinclair & arcese 1995). the transmission of bovine tuberculosis from wildlife to humans in such cases is by direct contact between infected animals and hunters, either via aerosol contamination when the carcass is opened, through entry of organisms via cuts in the skin or through the alimentary system (fanning & edwards 1991; georghiou et al. 1989; robinson et al. 1988). tuberculosis in man is generally characterised by loss of weight, weakness, poor appetite, http://www.emedicinehealth.com/script/main/art.asp?articlekey=58790 cough, and night sweats. mycobacterium tuberculosis is the most common cause of human tb, however an unknown proportion of cases occur due to m. bovis (acha & szyfres 1987) at least partly because it is impossible to distinguish tuberculosis infection caused by m. bovis from m. tuberculosis from clinical signs alone. bovine tuberculosis (btb) is often subclinical; when present, clinical signs are not specifically distinctive and are characterised by weakness, anorexia, emaciation, dyspnoea, enlargement of lymph nodes, and cough, particularly with advanced tuberculosis (oie 2009). pathologically, btb is characterised by the formation of granulomas (tubercles) that are usually yellowish and either caseous, caseo-calcareous or calcified and are sometimes encapsulated (de lesle et al. 2002). however, in disseminated cases, multiple small granulomas may be found in numerous organs such as female genitalia (http://www.cfsph.iastate.edu). mycobacterium bovis may present as extra pulmonary tuberculosis often as cervical lymphadenitis (kleeberg et al. 198`4; mfinanga et al. 2004). based on this premise, m. bovis may present either as pulmonary tb (47%) or extrapulmonary cases (53%), whereas m. tuberculosis presents as 82% pulmonary and 18% extrapulmonary cases (owendidactic.org, n.d.). currently, m. bovis accounts for only 1% of all human tb in developed countries as compared to 10% in the developing world (etchechoury et al. 2010). according to a who report (2010), there was a significant increase (116.6%) of extrapulmonary cases of tb reported in tanzania between 1995 and 2009, which is suggestive of an emerging m. bovis epidemic, however, information on the contributions of m. bovis infection to extra pulmonary cases is very limited and it is possible that these extra cases may also be hiv-related (amanfu 2006). the survey conducted by national tuberculosis and leprosy program, tanzania (ntlp) in 2009 reported that 20.994 (37.2%) out of 64.417 tb patients in tanzania were co-infected with hiv. according to the world bank (2010) report on development indicators, the prevalence of hiv (% of population aged 15−49) in tanzania is 5.6%. tanzania has the third largest domestic stock population in africa (after ethiopia and sudan). according to the ministry of livestock development and fisheries (mldf) national census conducted in 2007/2008, the total numbers were 18.5 million cattle, 13.1 million goats, 3.6 million sheep and 53 million poultry. the livestock production system is a pastoral and agro-pastoral system, where movement of animals searching for pasture and water is unrestricted. the presence of large numbers of livestock in traditional settings and where animals are kept in close contact with little veterinary service contributes to spread of disease. previous studies conducted in pastoral communities in the arusha region found that a history of tb in the family, drinking raw milk, eating raw animal products, poor ventilation and having poor knowledge concerning transmission of tuberculosis were risk factors for m. bovis infection and disease (mfinanga et al. 2004). the diagnosis of tb in cattle in tanzania is done by tuberculin skin testing (tst), meat inspection in abattoirs and rarely by bacteriological or molecular techniques the commonly used diagnostic test for human tb in primary health centres and hospitals is microscopic examination and culture, and speciation are not done routinely (mfinanga et al. 2004). although meat is recommended to be abbatoir-inspected before entering markets, proper meat inspection is not effectively carried out due to the inadequacy of the veterinary service sector as a result of the withdrawal of public veterinary services. in addition, the recommended test and slaughter policy, a disease control program based on slaughter of positive reactors animals, is not properly implemented (see figure 1) despite our knowledge that this policy has successfully reduced the prevalence of bovine tuberculosis (michel et al. 2009). it is only algeria, burkina faso, cameroon, morocco, namibia and south africa out of 48 countries in africa that apply a test-and slaughter policy as a control measure and consider bovine tuberculosis as a notifiable disease (cosivi 1998). the lack of public finances are obstacles in the control of bovine tuberculosis in many countries. in tanzania, lack of clear policies on how bovine tuberculosis can be controlled and the failure of health authorities to recognise m. bovis as cause of tuberculosis hinder the control of the disease (kazwala et al. 2006). few studies have confirmed m. bovis in humans in tanzania (kazwala et al. 2001; mfinanga et al. 2004), however, there is a body of evidence for m. bovis infection in man and a description of the relationships between m. bovis isolates found in humans and cattle. in their study, kazwala et al. (2001) found that m. bovis isolates from man had a 70% – 80% genetic relatedness to those found in cattle, arguably suggesting an infection and evolutionary relationship between them. mycobacterium bovis infection in man has also been reported in other countries in africa including nigeria, zaire and egypt (cosivi 1998; idrisu et al. 1977; idigbe et al. 1986; nafeh et al. 1992). this paper aims to review the current situation of m. bovis infection in animals and discuss the zoonotic importance of m. bovis in tanzania. the paper also highlights the burden of tuberculosis, risk factors for infection, communities’ knowledge on prevention of the disease and challenges to its control in order to safeguard the human and animal population in tanzania. distribution of bovine tuberculosis in animals in tanzania top ↑ mycobacterium bovis was demonstrated in tanzania for the first time in 1952 (markham 1952). thereafter, it has been isolated from livestock, wildlife and humans. the prevalence of m. bovis in cattle varies between districts, with more infection in older cattle than yearlings and calves (kazwala et al. 2001). variation in m. bovis infection in different geographical areas of the country suggests that there are m. bovis infection foci (or hotspots). shirima et al. (2003) suggested that many factors could contribute to m. bovis foci, including the presence of mycobacteria in the environment, management practices where animals are extensively grazed and overcrowded at watering points and auction markets. according to cleveland et al. (2007), flooding has also been suggested as a propagating factor of m. bovis in the environment. reports from several studies that have been conducted in various districts have reported m. bovis infection in livestock as well as wildlife. the prevalence of presumed m. bovis infection determined by using a single intradermal comparative tuberculin test (sictt) was 1.7% (n = 181) and 0.4% (n = 259) in kibaha and morogoro respectively (mdegela et al. 2004) (table 1). durnez et al. (2009) reported a prevalence of 2.4% (n = 728) m. bovis infection in cattle from 49 herds belonging to extensive and intensive management systems. the prevalence of m. bovis infection reported by this study was in same range as demonstrated previously in the same region by shirima et al. (2003). according to kazwala et al. (1996), the highest prevalence of 13.3% m. bovis infection was reported in the southern highlands and larger herds of cattle had a higher rate of bovine tuberculosis. the prevalence of m. bovis infection in cattle in other parts of the country are as follows: shinyanga, mwanza, bukoba: 0.2% in intensively managed farms (jiwa et al. 1997), rift valley districts (babati, hanang, mbulu and karatu) 0.93% (kazwala et al. 2001), manyara region 0.9% (cleveland et al. 2007). the prevalence of m. bovis in cattle has been reported to be higher in intensive systems than in pastoral production systems (shirima et al. 2003). however, in contrast, durnez et al. (2009) reported a higher prevalence of bovine tuberculosis in the extensive than in an intensive system. husbandry practices in the country could contribute to the difference in prevalence of m. bovis infection in extensive and intensive systems. free movement of animals, overcrowding in communal grazing areas and watering points might contribute to its spread. a study by kazwala and colleagues (2006) reported the similarity of m. bovis isolates from different geographical locations, which was attributed to migration of cattle as well as sale to local communities. uncontrolled movement of cattle together with a decline in service of public sector in the provision of veterinary services impeded disease control programs in tanzania. the withdrawal of a public veterinary service forced livestock keepers to take the responsibility of treating their own livestock in order to fill this vacuum (ftp.fao.org/docrep/fao). in tanzania, m. bovis infections have been confirmed in a number of wildlife species including buffalo (syncerus caffer), african civet (civettictis civetta, n = 1), lion (panthera leo), wildebeest (connochaetes taurinus), topi (damaliscus lunatus) and lesser kudu (tragelaphus imberbis) (table 2). of particular note, m. bovis has been recovered from apparently healthy wildebeest that did not show lesions at post-mortem (cleveland et al. 2005). these reports are not comprehensive surveys and little information is available on the disease status in wildlife. analysis of serum samples by using enzyme immunoassay (eia) detected m. bovis antibodies in 4% of serengeti lions, 6% (n = 17) buffalo (syncerus caffer) in tarangire and 2% (n = 41) wildebeest in the serengeti (cleveland et al. 2005). it is important to note that the who recently issued a statement on the unreliability of serology to diagnose tb and these results are thus at best likely to be a significant underestimate and quantitatively inaccurate. countrywide survey of mycobacterium bovis infection in humans top ↑ tuberculosis accounts for approximately 6% of all deaths and 8% of all diseases in humans (ntlp 2007). however, the contribution of m. bovis to human tuberculosis in tanzania is unknown, owing to the absence of efforts in most laboratories in hospitals and health centres to differentiate between the species of the m. tuberculosis complex. despite the lack of data, according to kazwala et al. (2001), m. bovis infection is considered as a pathogen of concern to people living in rural areas. in many developed countries, human tb caused by m. bovis accounts for around 1% of all tb cases, and sporadic cases occur either in elderly people by reactivation of ancient infections or in immigrants from countries where bovine tb has not been eradicated (de la rua-domenech 2006; etchechoury et al. 2010). in the developing world the contribution of m. bovis to human tuberculosis is higher and account for an estimated 10% of all tb cases (cousins et al. 1999; etchochoury et al. 2010). shitaye et al. (2007) reported that m. bovis infection in man depends on the prevalence of the disease in cattle, socioeconomic conditions, consumer habits, food hygiene practices and medical prophylaxis measures. human tuberculosis due to m. bovis is mostly the result of transmission from cattle to man and in many cases results into extrapulmonary manifestation (cosivi 1998; daborn et al., 1997; kazwala et al. 2001; mfinanga et al. 2004; amanfu et al. 2006; munyeme & munang’andu 2011). it has been suggested that m. bovis infection in man increases proportionately to the total number of tb cases and that hiv is a major factor for development of active tb disease (cosivi 1998). in developing countries m. bovis infection in humans is also increasing due to the lack of control and diagnostic measures, and pasteurisation of milk (etter et al. 2006). thus there is every reason to be seriously concerned that the hiv pandemic will result in an increase of human tuberculosis due to m. bovis, and a greater degree of transmission of infection to other humans and to animals could well occur. information on cross transmission of m. bovis infection between livestock, wildlife and man in tanzania is limited. this situation is similar to that of other developing countries where m. bovis infection in man is almost certainly underreported (cosivi et al. 1998; munyeme & munang’andu 2011) due to the lack of diagnostic facilities to distinguish tuberculosis caused by m. bovis and m. tuberculosis. in africa, consumption of raw fresh milk and improperly cooked or raw meat, and the attitude of some communities which regard bushmeat (poached or hunted wildlife) as a cheap source of protein, represents one of the major risk factors for humans with respect to infection with m. bovis (aranaz et al. 2004; etter et al. 2006). kazwala et al (1998) found that out of 805 milk samples that were collected, 31 (3.9%) were positive for mycobacteria. in this study, atypical mycobacteria represented with 87% of the positive samples, however, 6.5% contained m. bovis. whilst these samples represent a minority of the positives, the results show that raw milk is a threat to public health. moreover, durnez et al. (2009) reported a high prevalence of m. bovis and recovered atypical mycobacteria isolates from milk samples in and around morogoro, tanzania, and concluded that the populace, especially cattle owners in an extensive system, should be educated concerning bovine tuberculosis. the high level presence of atypical mycobacteria in milk also poses a danger to immunocompromised individuals, especially hiv and aids patients. mfinanga et al. (2004), in their study in the arusha region, northern tanzania, investigated 457 biopsy specimens, of which 65 (14.2%) were positive on culture for mycobacteria. in this study, the proportion of atypical mycobacteria was 31 (47.7%) compared to 7 (10.8%) m. bovis, and 27 (41.5%) m. tuberculosis. they concluded that atypical mycobacteria were more common than m. tuberculosis and therefore hiv and raw milk are major risk factors identified for m. bovis and non-tuberculous mycobacterial adenitis. the finding that atypical mycobacteria are common was confirmed in a study by durnez et al. (2011), in morogoro, tanzania, where 7.3% of 645 terrestrial small mammals sampled in cattle farms were positive for atypical mycobacteria. a high proportion of the atypical mycobacteria were recovered in insectivores as opposed to rodents. insectivores feed on insects that spend most of their time in the ground. the recovery of atypical mycobacterium from this source is not surprising perhaps, since mycobacteria are well known environmental and soil dwelling microbes. what is important in this work is that durnez et al. (2011) established a direct correlation between the proportion of atypical mycobacterium in reacting and non-reacting tuberculin farms, complicating the interpretation of tuberculin skin testing (tst) results. mycobacterium bovis is one of the well-known causative agents of human extra-pulmonary tuberculosis. this situation prevails in tanzania, where an early study (daborn et al. 1997), showed that seven out of nineteen lymph node biopsies from suspected extra-pulmonary tuberculosis patients were infected with m. tuberculosis and four with m. bovis. in most developing countries, the extent of human tuberculosis due to m. bovis and the frequency of m. bovis extra-pulmonary tuberculosis is not known (chen et al. 2009; cosivi 1998). however, between 1995 and 2009, the number of reported extra-pulmonary tb cases increased from 6195 to 13 417 in tanzania (who 2010, see table 3), which is in all likelihood an underestimate (kazwala et al. 2001). the available literature shows that m. bovis infections are correlated with people who keep large numbers of cattle (kazwala 1996) and most cases of extra-pulmonary tb were found in regions with a high proportion of cattle to humans (kazwala et al. 1993). a who (2006) zoonotic survey reported the following extr-apulmonary cases in several regions with a high population of cattle in tanzania; arusha (30%), mbeya (28.1%), iringa (27.3%), shinyanga (19.8%), mara (19.7%), dodoma (19.4%) and mwanza region (10.8%). a study conducted by mfinanga et al. (2004) found that a disproportionately high number of mycobacterial adenitis was found in subsistence farmers and livestock keepers in arusha and mbeya region which is suggestive of cross transmission of m. bovis. a high proportion of cattle are kept in traditional settings in rural areas where knowledge regarding m. bovis infection is generally minimal (mfinanga et al. 2004). according to pušić et al. (2008), the persistence of bovine tuberculosis is mostly linked to the traditional extensive breeding system and free-ranging cattle. mycobacterium bovis is resistant to pyrazinamide, one of the four first line tb antibiotics and prognosis is often poor (who 2010). given this scenario, it is not surprising that multidrug-resistant strains (mdr) of m. bovis have been detected in the usa (bouvet et al. 1993) and spain (guerrero et al. 1997; rivero et al. 2001). a study conducted in tanzania reported genetic relatedness of m. bovis isolates in man to those found in cattle (kazwala et al. 2006). these authors found one strain of m. bovis from a human patient in arusha region that had the same genotype as m. bovis from cattle within the same geographical area. risk factors for mycobacterium bovis infection and disease in humans top ↑ the risk factors for bovine tuberculosis are similar in different geographical areas. according to a study conducted in pastoral communities in the northern part of tanzania, the risk factors for bovine tuberculosis in man were found to be traditional practices such as sleeping in the same house as animals, lack of knowledge regarding the disease and its risks, hiv and aids, raw milk consumption and poor ventilation of houses (mfinanga et al. 2004). in sub-saharan africa, active competition between large-scale commercial food enterprises and smaller, less regulated farmers who frequently ignore safety standards for hygiene and product quality, increases the risk of zoonotic tuberculosis (etter et al. 2006). consumption of raw fresh milk is also a risk factor for bovine tuberculosis. mdegella et al (2004) and colleagues in their study in morogoro and kibaha districts concluded that despite the low prevalence of tuberculosis in milk in the study herds, milk consumers are at high risk of being infected with the disease and insisted that farmers should be educated about the risk of bovine tuberculosis and associated health risks. mfinanga et al. (2003) in their study on the role of livestock keeping in tuberculosis trends in pastoral communities in babati, hanang, mbulu and karatu districts in the arusha region, found that all ethnic groups possessed habits and beliefs that increased the risk of being infected with both bovine and human tuberculosis. in their study, mdegella et al. (2004) found that 14% of milk samples (n = 109) were positive for atypical mycobacteria. in addition, mfinanga et al. (2004) found that several activities, including handling animals and animal products, specifically milking, herding cattle and goats, hunting, slaughtering, handling skins and hides, moving cow dung and plastering walls with dung or mud, might increase risk of zoonotic tuberculosis. role of husbandry practice in transmission of bovine tuberculosis top ↑ husbandry practices in tanzania are divided into three categories, namely, extensive, intensive and semi-intensive systems. the extensive system is traditional and the most popular husbandry practice, and is the main source of milk and meat for tanzania but receives very little attention from veterinary services. the extensive farming system is practised mostly in rural areas where animals share grazing land and watering points. most of the cattle kept in tanzania are zebu (bos indicus) which are relatively resistant to diseases (frankel & soule 1981; wambura et al. 1998). the intensive systems are usually dairy and pig farms which are located in peri-urban areas and are intended for milk and pork production. in these systems, animals are frequently kept indoors and fed complete rations, but in some cases they are grazed outside to supplement feeding. a single comparative intradermal tuberculin test (scitt) survey conducted in different farming systems in the eastern zone of tanzania found that bovine tuberculosis occurred both in intensive and pastoral farming systems, with significantly higher prevalence in the intensive system than in pastoral systems (shirima et al. 2003). this could be attributed to husbandry practices in especially dairy cattle that are confined indoors, where close contact between animals and lack of ventilation increase chances of disease transmission. however, this is contrary to results presented by durnez et al. (2009) in a more recent study, who showed that m. bovis infection in extensive farming systems was higher than intensive systems. the contradiction of infection rate in different farming systems could be explained by considering the management practices of each farming practice, or that systems have changed over time. in the extensive system, free movement of cattle which share grazing land and watering points facilitate disease transmission. poor animal housing and drainage systems designs in intensive farm systems and water supply are key elements that could play a big role in diseases transmission (pool 1945). challenges for control of bovine tuberculosis in cattle in tanzania top ↑ in tanzania, m. bovis is considered as a neglected disease and it has not been assigned as a notifiable disease (kazwala et al. 2006). when it comes to disease control, most resources are directed to notifiable diseases such as contagious bovine pleuropneumonia, african swine fever, rinderpest, contagious caprine pleuropneumonia and rift valley fever. extensive husbandry practices are widespread and cattle move from one place to another searching for grazing and watering points. this situation is exacerbated during drought, when nomadic tribes move and establish temporary settlements in areas where grazing land and water are available. bovine tuberculosis could be eradicated at the national level if attention is given at policy level. according to collins (2006), the success of a national eradication programme, include a clear identification of the goals, of the policies that guide actions and of the sequences of actions that are required within the programme to accomplish these goals. eradication is possible if movement of cattle is controlled, if there is compulsory testing of all cattle within specified intervals, if positive reactors are removed (slaughtered in a controlled manner), if compensation is provided to farmers for all positive reactors, if compulsory identification is done, and if there is establishment and maintenance of disease free areas, and sufficient funds and manpower to fulfil the task are provided (http://www.pathobiologics.org/ivphc/ref/mccrindle_shangai_2006.pdf). however, this has been impossible for most developing countries because of cost implications. in the netherlands and australia, eradication of bovine tuberculosis was successfully achieved due to the practical involvement of farmers as stakeholders (collins 2006). however, the success of bovine tuberculosis eradication programmes in developed countries was achieved at a time when herds were smaller, and the intensity and demands of production were lower (collins 2006). the presence of maintenance hosts in wildlife populations also impede bovine tuberculosis eradication programs (etter et al. 2006). white et al. (2008) reported that the presence of multiple hosts for bovine tuberculosis complicate control measures not only because of resistance variation between the different host species but also because of ecological and behavioural differences. the african buffalo is a known maintenance host of bovine tuberculosis. aerosol transmission of m. bovis within buffalo herds is favoured by their social behaviour (michel et al. 2006) and can be transferred to domestic cattle by intermingling. globally, the presence of wildlife maintenance hosts threatens m. bovis eradication programs (etter et al. 2006). in tanzania, the national policy on control of wildlife diseases in protected areas is to leave nature to take its own course. there are very few circumstances where treatment or intervention occurs or is allowed. wildlife immunisation is not allowed in national parks, game reserves and game controlled areas. in circumstances where bovine tuberculosis control in wildlife is not practised and communities around protected area conduct illegal bush hunting in wildlife areas, the risk for cross-transmission of diseases to livestock and humans remain very high. according to cross and gertz (2006) vaccination could potentially control bovine tuberculosis, but combining vaccination and culling of infected animals is a more attractive management option. this is perhaps of little importance for tb control at this stage, since there is no evidence that vaccination against tb would be successful, even if attempted. furthermore, vaccination of cattle against bovine tuberculosis or improvement in tuberculosis testing procedures will have no effect on wildlife tuberculosis prevalence (kao et al. 1997). in britain, culling of a maintenance host of bovine tuberculosis, the badgers (meles meles), increased the prevalence of bovine tuberculosis in the cattle population because of ecological and social disturbances of the badger populations (white et al. 2008). thus, the reduction of transmission risk between species is not a simple matter. however, a reduction of or minimising contact between wildlife and livestock could serve as a priority for future management of the disease in tanzania. collins and grange (1983) reported that ‘it is axiomatic that no control measures against transmissible diseases can be totally effective unless all reservoirs of the causative agent can be eliminated’. as in other parts of the world, the challenge facing the control of tuberculosis is the lack of an effective vaccine. the current tb vaccine, m. bovis bacille calmette-guérin (bcg) provides little or no protection against pulmonary tuberculosis in cattle and man (hogartha et al. 2005). nevertheless, bovine tuberculosis could be controlled if there are sound control measures such as regular skin testing and removal of reactors, meat inspection in abattoirs, restriction of cattle movements (pušić et al. 2008). conclusion top ↑ there is a remarkable paucity of information available on the zoonotic importance of bovine tuberculosis in humans, particularly in developing countries such as tanzania. the lack of diagnostic facilities to distinguish between m. bovis and m. tuberculosis is a challenge. moreover, high proportions of atypical mycobacteria in clinical specimens indicate a widespread environmental occurrence of these organisms, which further complicates accurate diagnosis. lack of clear policies and implementation regarding control of bovine tuberculosis in cattle impedes control of the disease. widespread evidence of m. bovis infection in animals and humans should be an alarm sign for medical and veterinary health professionals and government bodies. this illustrates the importance of the ‘one health concept’ that can bring together medical and veterinary practitioners as an important tool to fight diseases of public health and economic importance. acknowledgement top ↑ the authors would like to thank the southern african centre for infectious diseases surveillance (sacids) funded by the welcome trust for support. prof mark rweyemamu, the director sacids is acknowledged for his ideas and encouragement in writing of the manuscript. the sacids collaborative institutions in the united kingdom (royal veterinary college and london school of hygiene and tropical medicine) through london international development centre (lidc) are also acknowledged for their support during a stay in the uk. competing interest the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions b.z.k. (muhimbili university of health and allied sciences) reviewed the literature, drafted and wrote the manuscript. e.v.m. (muhimbili university of health and allied sciences) contributed to the drafting and critical review of the manuscript. s.k. (royal veterinary college) made conceptual contribution and critical review of the manuscript. r.d.f. (tanzania wildlife research institute) made conceptual contribution and significant review of the manuscript. g.s.k. (kilimanjaro clinical research institute) assisted in critical review of the manuscript and made contribution on preparation of the manuscript. p.g.f. (london school of hygiene and tropical medicine) made conceptual contribution and critical review of the manuscript. j.d.k. (tanzania wildlife research institute) contributed to the drafting, conceptualization and critical review of the manuscript. p.v.h. (university of stellenbosch) made conceptual contribution and critical review of the manuscript. m.i.m. 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serengeti ii, ‘dynamics, management, and conservation of an ecosystem’, the university of chicago press, ltd., london. wambura, p.n., gwakisa, p.s., silayo, r.s. & rugaimukamu, e.a., 1998, ‘breed-associated resistance to tick infestation in bos indicus and their crosses with bos taurus’, veterinary parasitology 77, 63−70. http:// dx.doi.org/10.1016/s0304-4017(97)00229-x white, p.c.l., bohm, m., marion, g. & hutchings, m.r., 2008, ‘control of bovine tuberculosis in british livestock − there is no “silver bullet”, trends in microbiology 16, 420−427. http://dx.doi.org/10.1016/j.tim.2008.06.005 world bank report, 2010, development indicators, viewed on 15 july 2011, from  http://data.worldbank.org/news/world-development-indicators-2010-released world health organization (who), 2006, ‘the control of neglected zoonotic diseases, a route to poverty alleviation’, report of a joint who/dfid-ahp meeting with the participation of fao and oie, geneva, 20 and 21 september 2005, from  http://www.int/zoonoses/report_sept06.pdf. world health organization (who), 2010, global tuberculosis control, global, region and country-specific for key indicators, viewed 25 june 2011, from http://www.who.int/tb/data article information authors: tingiya sikombe1,3 christopher j. kasanga2 yona sinkala3 martin simuunza3 john muma3 rahana dwarka4 misheck mulumba5 cornelius mundia6 aaron mweene3 affiliations: 1central veterinary research institute, zambia2southern african centre for infectious disease surveillance, sokoine university of agriculture, tanzania 3school of veterinary medicine, university of zambia, zambia 4onderstepoort veterinary institute, university of pretoria, south africa 5southern african development community secretariat, botswana 6southern african development community, transboundary animal diseases section, zambia correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: sikombe, t., kasanga, c.j., sinkala, y., simuunza, m., muma, j., dwarka, r. et al., 2014, ‘typing and serological surveillance of fmdv in the african buffaloes in zambia’, onderstepoort journal of veterinary research 81(2) art. #742, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.742 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. typing and serological surveillance of fmdv in the african buffaloes in zambia in this abstract... open access • abstract • acknowledgements abstract top ↑ foot-and-mouth disease (fmd) is endemic in zambia. little is known of the epidemiology of fmd virus (fmdv) in the country and this has led to the continuous occurrence of fmd in southern zambia. fmd severely impacts pastoral and agro-pastoral communities who are most reliant on livestock products for economy and food security. southern zambia is densely populated with livestock and wildlife, which are usually in contact almost throughout the year. the control of fmd in zambia is mainly done strategically through vaccination, but this is complicated by the presence of buffaloes and traditional cattle farmers’ practice of transhumance in areas harbouring wild animals. the current research aims at determining the infection status and fmd virus (fmdv) serotype(s) circulating in domesticated and wild animals in southern zambia. a targeted cross-sectional study will be employed in this study. sera and oesophageal-pharyngeal (op) fluids will be obtained from cattle and buffaloes in selected areas of zambia. epidemiological data such as age, sex, health, and vaccination status of animals will be taken during sampling. infection status will be determined by nsp-elisa targeting the 3abc region of fmdv genome. fmdv serotypes will be examined by lpb-elisa and/or antigen elisa on op samples. this research has the potential to unravel the infection status and serotype(s) of circulating fmdv strains in zambia. this information will be useful in designing a rational strategy of control of fmd in zambia and the neighbouring countries. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). article information authors: itumeleng i. setshedi1,2 gerda fouche1 john dewar2 vinesh maharaj1 martin s. myer1,2 affiliations: 1csir bio-prospecting, pretoria, south africa 2department of life and consumer sciences, university of south africa, south africa correspondence to: itumeleng setshedi postal address: po box 3024, morogoro, tanzania how to cite this poster: setshedi, i.i., fouche, g., dewar, j., maharaj, v. & myer, m.s., 2012, ‘phytochemical isolation of compounds from sceletium tortuosum and activity testing against plasmodium falciparum’, onderstepoort journal of veterinary research 79(2), art. #481, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.481 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. phytochemical isolation of compounds from sceletium tortuosum and activity testing against plasmodium falciparum in this poster... open access malaria is a major health care problem in tropical regions due to the increasing resistance of plasmodium falciparum against widely available antimalarial drugs. traditional societies relied on medicinal plants to treat parasitic infections. as a result, drugs like quinine and artemisinin were isolated from herbs and barks (varughese et al. 2010). sceletium tortuosum has been used as medicine for social and spiritual purposes by san hunter gatherers and khoi pastoralists. sceletium tortuosum is rich in alkaloids, one of the important classes of natural product producing treatment for parasitic infections (kayser et al. 2002). laboratory preparation of extracts of fresh s. tortuosum plant material was conducted mimicking traditional methods of preparation using organic solvents. mesembrine was isolated from a methanol extract using conventional column chromatography. sixteen extracts and mesembrine were evaluated for antiplasmodium activity using a plasmodium lactate dehydrogenase culture sensitivity assay with chloroquine as reference drug. of the sixteen extracts, four showed activity against p. falciparum with ic50 ranging between 1.47 µg/ml and 7.32 µg/ml. extracts prepared from stored material at -20 °c showed no antiplasmodium activity. the four originally active extracts were re-screened six months later, but the antimalarial activity could not be reproduced. to determine discrepancy in biological results, chemical profiling of the extracts was done using high performance liquid chromatography technique. differences were observed in the profiles of the active extracts when compared to those of stored plant material. the instability of plant constituents observed could be a result of plant storage suggesting that the plant is best used when fresh. article information authors: johan l. bekker1 louw c. hoffman2 piet j. jooste3 affiliations: 1department of environmental health, tshwane university of technology, south africa 2department of animal sciences, stellenbosch university, south africa 3department of biotechnology and food technology, tshwane university of technology, south africa correspondence to: johan bekker postal address: private bag x680, pretoria 0001, south africa dates: received: 29 feb. 2012 accepted: 20 aug. 2012 published: 05 dec. 2012 how to cite this article: bekker, j.l., hoffman, l.c. & jooste, p.j., 2012, ‘wildlife-associated zoonotic diseases in some southern african countries in relation to game meat safety: a review’, onderstepoort journal of veterinary research 79(1), art. #422, 12 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.422 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. wildlife-associated zoonotic diseases in some southern african countries in relation to game meat safety: a review in this review article... open access • abstract • introduction    • background    • ’office internationale des epizooties‘ controlled diseases    • disease control in angola, botswana, mozambique, namibia, south africa, zimbabwe • disease occurrence in game in angola, botswana, namibia, south africa and zimbabwe • overview of some zoonotic diseases associated with game • emerging and re-emerging zoonotic diseases • disease surveillance and management • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ with on-going changes in land use practices from conventional livestock farming to commercial, wildlife-based activities, the interface or interaction between livestock and wildlife is increasing. as part of the wildlife-based activities of ecotourism, breeding and hunting, game farmers are also exploring the utilisation of meat from hunted or harvested game. the expanding interface or increased interaction between livestock and wildlife increases the risk of disease incidence and the emergence of new diseases or the re-emergence of previously diagnosed diseases. the risk is not only related to domestic and wild animal health, but also to the occupational hazards that it poses to animal handlers and the consumers of game meat. this review endeavours to highlight the role that game plays in the spreading of zoonotic diseases to other animals and humans. examples of zoonotic diseases that have occurred in wild animals in the past, their relevance and risk have been summarised and should function as a quick reference guide for wildlife veterinarians, ecologists, farmers, hunters, slaughter staff, processors and public health professionals. introduction top ↑ the progressive expansion of game ranching as an extensive form of game farming on developed (fenced) land in southern africa has resulted in ranching with endangered and/or rare species which can to a greater or lesser extent, be found in free community with livestock. in many parts of the world, farmers are changing from conventional livestock farming to commercial, wildlife-based activities such as ecotourism, which usually requires a wide diversity of frequently translocated species and adequate populations for tourist viewing (bengis, kock & fisher 2002). with game becoming more valuable and therefore stocked at higher densities on smaller properties, disease prevention has become a very important aspect of game ranch management (bester & penzhorn 2002). a survey amongst south african game farmers revealed that 34.3% of these farmers are farming with both domestic and game animals (bekker 2011). skinner (1970) expressed concern regarding the dangers that game poses to domestic livestock by acting as reservoir hosts of pathogens of livestock which can cause epizootic disease and also that the handling and management of wild animals in relation to disease control is difficult. in support of this, bengis et al. (2004) were of opinion that the abovementioned situations will probably enhance and intensify the wildlife-livestock interface and that the potential for ’cross-over‘ of diseases will increase. the cross-over of epizootic outbreaks represents a serious threat both to wildlife and, via reverse spill-over (‘spill-back‘), to sympatric populations of susceptible domesticated animals (daszak, cunningham & hyatt 2000). bengis et al. (2002) also indicated that foreign animal diseases cycling in livestock may cross the interface and infect wildlife. wildlife therefore plays an important role in the spreading of transmissible animal diseases. in the united states, the presence of brucellosis amongst elk and bison in the yellowstone national park is considered a potential threat to domestic animals grazing in the park (daszak et al. 2000). godfroid (2002) was of the opinion that the development of the game farming industry has contributed to the re-emergence of brucellosis. diseases such as malignant catarrhal fever (mcf) in wild herbivore species often show few clinical signs, but the disease is often deadly to cattle. according to li et al. (1996), the viruses responsible for mcf are an important obstacle to the propagation of endangered ruminant species in the wild, in captivity, or on game farms. although the office internationale des epizooties (oie) has declared that rinderpest has now been eradicated from the surface of the earth and that all 198 countries and territories in the world with rinderpest-susceptible animals are free of the disease (food and agricultural organization [fao] 2011), gortázar et al. (2007) have pointed out that true multihost diseases that are regarded as eradicated are the worst, because a single spill-over from wildlife to livestock may have severe consequences for health and the economy. rinderpest, for example, was eradicated in nigeria in 1974 after the jp15 campaign, but was reintroduced between 1980 and 1983 into two states of nigeria from infected cattle to wildlife, and this resulted in the loss of an estimated one million cattle and numbers of wildlife, of which 207 buffalo, 20 warthog, eight waterbuck and two bushbuck carcasses were recovered (shanthikumar & atilola 1990). other examples of multihost diseases include bovine tb (michel et al. 2006; phillips et al. 2003), foot and mouth disease (scoones et al. 2010) and avian influenza (alexander 2000; gauthier-clerc, lebarbenchon & thomas 2007).regarding zoonotic diseases, cleaveland, laurenson and taylor (2001) then already indicated that 61% (868) of the 1415 species of infectious organisms known to be pathogenic to humans were of zoonotic nature. some of these diseases can be transmitted through the consumption of animal-infected material or through the handling of such material (occupational disease). for the purpose of this review, human tuberculosis as a result of mycobacterium bovis is briefly discussed. several publications have already appeared on the prevalence of bovine tuberculosis in african wildlife and elsewhere in the world (e.g. ayele et al. 2004; michel 2002; michel et al. 2006; parra et al. 2006; zieger et al. 1998). it was estimated by the world health organization (who) that m. bovis is the causative bacterium in 3% of all tuberculosis cases (who 1994a). although high priority is placed on the meat inspection function at formally registered abattoirs to identify and remove carcasses with lesions of m. bovis from the food supply chain, asymptomatic animals or localised lesions could pass through the inspection system unnoticed (van der merwe et al. 2009). poor and rural communities, furthermore, often obtain meat from uncontrolled and unregistered slaughter facilities where there is no meat inspection. regarding game abattoirs, bekker (2011) showed that in south africa there are only a few registered game abattoirs, which are mostly used for export purposes, and that the real number and location of game slaughter facilities located on game farms are unknown to the relevant authorities and that almost no meat inspection is done on game farms. according to cosivi et al. (1998), the intake of uninspected game meat may increase the risk of contracting tuberculosis. a number of authors such as cosivi et al. (1995), cosivi et al. (1998), de la rua-domenech (2006) and thoen, lobue and de kantor (2006) have reported on human tuberculosis due to m. bovis globally. some other examples of zoonotic diseases contracted by humans include a cutaneous anthrax outbreak that occurred in rural paraguay through touching the meat of a sick cow (harrison et al. 1989); an anthrax occurrence in mashonaland east province of zimbabwe due to handling, eating and drinking products of an infected animal (mwenye, siziya & peterson 1996); brucellosis contracted by workers in meat packing plants (taylor & perdue 1989; white et al. 1974) and eating of caribou meat (chan, baxter & wenman 1989); human trichinellosis which occurred in northern italy after eating raw horse meat (pozio et al. 1988); and the 2010 outbreak of rift valley fever (rvf) in south africa where 172 cases and 15 human deaths after direct contact with rvf-infected livestock were reported by the national health department in south africa (who 2010). pavlin, schloegel and daszak (2009) indicated that the simplest way to minimise the risk of zoonotic disease is the reduction of opportunities for transmission of diseases from wildlife to humans. although not clear for the neighbouring countries of south africa, the survey amongst south african game farmers by bekker (2011) indicated, however, that 41% of farmers with mixed farming systems have no control measures to prevent animal interaction in order to prevent disease spreading between game and domestic animals. furthermore, specific gaps have been identified in game farming practices insofar as the establishment of written health plans, routine health inspections, screening for zoonotic diseases, provision of quarantine camps and record keeping of health and withdrawal periods as suggested by the globalgap integrated farm assurance standard (globalgap 2007). furthermore, liddell and baily (2001) were of the opinion that the lack of records of animal health treatment undermines the ability to track the inputs used during this animal production stage of the supply chain. this desktop study provides a review of disease control mechanisms by the oie, angola, botswana, namibia, south africa and zimbabwe as well as a summary of the diseases associated with game occurring in the aforementioned countries and the relevance as well as the risk of cross-infections related to some zoonotic diseases that have occurred with game. this review should therefore function as a quick reference guide for wildlife veterinarians, ecologists, farmers, hunters, slaughter staff, processors and public health professionals. background animal hosts, vectors and the environment in a natural ecosystem are in a dynamic balance, with occasional pathogen emergence and impact, but disease is not frequent when compared to artificial or domesticated animal farming systems. the success of preventing disease on a farm revolves around the abovementioned balance. pressures on the environment such as the expected increase in human population (1 billion in africa from 2009 to 2050), rapid urbanisation in developing countries and the global demand for livestock products (requiring higher stock rates) cause multiple animal health problems (thornton et al. 2009). as an example, both scenarios of overstocking and introducing animals into habitats that they are not adapted to (e.g. lechwe in the free state province of south africa and gemsbok in high rainfall areas) are becoming more common in south africa. these drivers may cause disease vectors to increase with host species not suited for their habitat (and vice versa) to be affected first by a disease (bester & penzhorn 2002). ‘office internationale des epizooties’ controlled diseases internationally, the oie is the intergovernmental organisation responsible for improving animal health worldwide and is recognised as a reference organisation by the world trade organization (wto). with the consideration of international spread, zoonotic potential, significant spread in native populations and emerging diseases as criteria, the oie keeps a list of so-called ’oie listed‘ diseases for listing. all the countries relevant to this review (angola, botswana, mozambique, namibia, south africa, zimbabwe) are member countries of the oie. it is required that all member countries will report to the oie on incidences of any of the oie listed diseases (oie 2011a). disease control in angola, botswana, mozambique, namibia, south africa, zimbabwe policies and legislation normally form part of governmental animal health and food and meat control systems with the aim to prevent or control the spreading of disease to animals and humans. regarding southern africa, thomson and penrith (2011) reported comprehensively on the animal health policy, legislation and trade in beef in the five participating states of the kavango-zambezi transfrontier conservation area (kaza tfca) which include angola, botswana, namibia, zambia and zimbabwe and of which, botswana, namibia and zimbabwe are neighboring countries of south africa. thomson and penrith (2011) also list some trade related protocols of the southern african development community (sadc) dealing with rules of origin for products to be traded between the member states of the sadc, sanitary and phytosanitary (sps) rules, and technical barriers to trade (tbt). the following two tables provide non-exhaustive summaries of existing legislation in angola, botswana, namibia, south africa and zimbabwe on the control of animal diseases and zoonosis (table 1) and trade in meat and meat products (table 2). disease occurrence in game in angola, botswana, namibia, south africa and zimbabwe top ↑ wildlife diseases have become more important in recent years and have led to an explosion of related knowledge (gortázar et al. 2007). a non-exhaustive summary of zoonotic diseases that have occurred in wildlife animals in angola, botswana, namibia, south africa and zimbabwe is therefore presented (table 3). table 1: summary of some legislation governing animal diseases in angola, botswana, namibia, south africa and zimbabwe. table 2: summary of some legislation governing trade in meat and meat products in angola, botswana, namibia, south africa and zimbabwe. overview of some zoonotic diseases associated with game top ↑ several worldwide outbreaks of animal related diseases such as bovine spongiform encephalopathy (bse), avian influenza, rvf, et cetera and the associated media declarations have alerted consumers about the safety of meat in general. because of these disease outbreaks, consumers are more informed and concerned about the safety and quality of meat products (hoffman et al. 2005; radder & le roux 2005). the industry has a moral responsibility towards the consumer in this regard. gregory (2000) pointed out that the public puts their faith in the food authorities and the food industry to provide them with safe products. to achieve this, gortázar et al. (2007) suggest that there should be close cooperation between multidisciplinary professionals such as wildlife ecologists, veterinarians and public health professionals. regarding game meat production, certainly the game farmer, game hunting associations, processors (including those on farms), wholesale and retail markets and consumer interest groups should join in this multidisciplinary team. according to bekker (2011), treatment of sick game animals is mostly done by veterinarians (59.5%) followed by trained farm staff (31.4%) and untrained farm staff (9.1%). from a veterinary health perspective, blaha (1999) indicated that food animal practitioners play an important part in animal health management in general. in addition to veterinarians and other animal practitioners, veterinary public health practitioners can also play a role to ensure animal health and the provision of safe meat derived from slaughter animals to the consumer. resulting from a desk top study, a non-exhaustive summary of the relevance as well as the risk of cross infections related to some of the zoonotic diseases that have occurred in the wild animals listed in table 3 is also provided (table 4). emerging and re-emerging zoonotic diseases top ↑ sleeman (2006) raised a concern that the handling and eating of game meat by humans will expose them to new micro-organisms and contaminants and will result in the emergence of new diseases. over the past years, emerging diseases have frequently hit the headlines, of which most have a zoonotic foundation. emerging and re-emerging zoonotic diseases are those that are newly identified, newly evolved or have occurred previously, but have more recently shown an increase in incidence or expansion into a new geographical area, host or vector range (bengis et al. 2004; paulsen & smulders 2004). though not zoonotic, an outbreak of african swine fever (asf) has for example recently occurred in the eastern parts of the gauteng province and the western parts of the mpumalanga province of south africa. both these areas fall outside south africa’s asf control area (gdard 2012). although the sources of these outbreaks are still unknown, the southward distribution and expansion of warthogs and tampans associated with the species could also contribute to the spreading of asf beyond the borders of the asf control areas (rmrdsa 2010). although daszak, cunningham and hyatt (2000) in their description of the relationship between wildlife, domestic animal and humans indicated that most emerging diseases impact on almost all groups, pavlin, schloegel and daszak (2009) were of the opinion that most emerging infectious diseases are associated with wild animal zoonotic diseases. infectious pathogens in wild animals as possible reservoirs have become increasingly important due to their substantial impacts on human health, domestic animal production, wildlife-based economies, wildlife conservation and global biodiversity (bengis et al. 2004; daszak et al. 2000; paulsen & smulders 2004). according to the fao (2010), ± 60% of emerging infectious diseases of humans are zoonotic, of which 72% originate from wildlife. in addition, christensen (1996) indicated that food of animal origin holds a threat to human health due to the diseases that it may transfer to humans and other animals.risk factors influencing the occurrence of diseases in wild animals are based on (1) the movements or translocations of wild or domestic animals and animal products, (2) the result of wildlife surplus, (3) the changing agricultural practices to farming with wild animals that support the transmission of pathogens between wild and domestic animals, (4) host and vector expansion, (5) the association with ’spill-over‘ from domestic animals to wildlife populations living in the same proximity, (6) the relation to human intervention, (7) diseases with no overt human or animal involvement, (8) the increase in human population and their relation to local and international travel and contact with wildlife populations and their products, (9) adaptations of pathogens themselves to certain situations, (10) changes in the environment and ecosystems that increase the transmission of infectious agents through open-air breeding and (11) enhancement of diagnostic and epidemiological techniques which assist in early detection of emerging or re-emerging infective agents (bengis et al. 2004; daszak et al. 2000; gortázar et al. 2007). bengis et al. (2004) warned that the geographical expansion of pathogens and/or their vectors as a result of global warming and other associated climatic changes and the continued collision between human populations and wildlife in the human endeavour to advance into new habitats and ecosystems will increase the risk of new emerging diseases in years to come. they also pointed out that the most likely populations to be affected are those in less developed countries and poorer communities. several opportunities exist for diseases to spread from one animal to another as they move around and associate with each other. diseased animals, for example, die and are left to scavengers to clean, which in turn are infected and they spread the disease further. this process implies, however, that there may be some extent of dilution which will make the organisms responsible for disease less abundant and less likely to persist. dilution occurs when other hosts in a community are also exposed instead of only being present in one highly competent reservoir host species alone (begon 2008). ogden and tsao (2009) indicated that this method of dilution will result in fewer new infections and thereby reduce the basic reproductive number (r0) of the pathogen. in their study on lyme disease, however, they also indicated that the extent of amplification or dilution of the disease is dependent on the mechanisms of completion, host contact rates and host resistance. bengis et al. (2002) indicated that the most probable transmission mechanisms include (1) aerosols contaminating feed, water and range and (2) flightless vectors such as ticks, and winged vectors such as mosquitoes, and that one of the most important factors in disease transmission between livestock and wildlife is the creation of new interfaces. in addition to this, the increase in the distribution of game meat may also now be seen as a possible risk in the spreading of diseases. table 3: summary of some zoonotic diseases reported to have occurred in game in angola, botswana, namibia, south africa and zimbabwe. disease surveillance and management top ↑ from a study by bekker (2011), there is evidence of minimal disease surveillance and management amongst game farmers. according to paskin (1999) the early detection of disease increases the chance to arrest the disease prior to it causing damage. detection requires that a surveillance system must be in place as required by the globalgap integrated farm assurance standard (globalgap 2007). surveillance activities can be defined as:all regular activities aimed at ascertaining the health status of a given population with the aim of early detection and control of animal diseases of importance to national economies, food security and trade. (paskin 1999) disease surveillance is not a haphazard activity and therefore requires proper planning and techniques. a summary of aspects that bengis et al. (2002) have provided as guidance in this regard is presented here (table 5). from an economic perspective, it also makes sense to have proper prevention measures in place. the oie (2006b) indicated in a report that dealt with an economical analysis of the costs of prevention versus the costs of outbreak, that the benefits accrued from improved prevention and control measures outweigh the costs of prevention and control investment. as an example, tambi et al. (1999) proved that rinderpest control in africa through the implementation of the pan-african rinderpest campaign (parc) in 1986 has been a wise public investment decision and that both producers and consumers gained from it economically. conclusion top ↑ this review shows that game plays an important role in animal disease spreading to other domesticated and wild animals and provides summaries of the occurrence of some zoonotic diseases in game in angola, botswana, namibia, south africa and zimbabwe, current disease and meat (food) control legislation in these countries and the relevance as well as the risk of cross-infections related to some zoonotic diseases that have occurred with game. of particular concern to game meat safety are the zoonotic diseases that can be transmitted through the consumption of meat and meat products. the consumer has to be safeguarded from possible infection (zoonosis) and therefore meat inspection of game carcasses is essential (skinner 1970). however, as previously indicated, several of the zoonotic diseases are also an occupational hazard as farmers, farm workers, hunters, slaughter staff, veterinarians, processors, et cetera , can become infected whilst handling the animals or meat thereof. table 4: summary of relevance as well as the risk of cross infections related to some zoonotic diseases that have occurred in wild animals. table 5: factors to consider with disease detection and management. pavlin et al. (2009) indicated that the simplest way to minimise the risk of zoonotic disease is the reduction of opportunities for transmission of diseases from wildlife to humans. to achieve this, gortázar et al. (2007) suggested a multidisciplinary approach of wildlife ecologists, veterinarians and public health professionals. in addition, oberem and oberem (2011) pointed out the importance of disease-causing investigations carried out on dead animals by experts who will know how to prevent further spreading of the diseases, especially zoonotic diseases. in this regard, the animal disease investigation laboratories also play an important role as they can assist with the carrying out of routine investigations and surveillance programmes (especially notifiable, controlled and zoonotic diseases and research on animal disease and related issues) (geering, roeder & obi 1999). the implementation of a comprehensive surveillance plan should be of critical importance to veterinary authorities (gortázar et al. 2007) and will require innovative measures to improve vigilance (bengis et al. 2004). in this regard, the south african department of agriculture, forestry and fisheries (daff), for example, have an epidemiology division whose mandate is contained in the animal diseases act (act no. 35 of 1984) (as amended) (south african government 1984) and its main functions include (ungerer 2008):• developing, analysing and auditing policies for the surveillance of animal diseases and diseases that can be transmitted from animals to humans • developing, analysing and auditing policies on disease reporting in the different provinces of south africa • conducting risk assessments at a national level • acting as contact point for communicating risk and the occurrence of animal diseases in south africa to foreign governments and international bodies • managing animal disease information and reporting thereof to the world organization for animal health (oie), southern african development community (sadc) and the african union (au) • managing of animal disease information by using a geographic information system • managing of animal disease early warning systems (sadc, nationally and internationally) • ensuring that there are adequate government veterinary laboratory services at provincial level and elsewhere, for example in the kruger national park, and by working in collaboration with the onderstepoort veterinary institute of the agricultural research council • enforcement of quality systems in diagnostic veterinary laboratories • auditing the enforcement of policy for reference laboratories. however, these measures will only be effective if wild game ranchers follow the correct procedures in contacting the relevant authorities. bekker (2011), however, has illustrated negligence in certain farming practices and a lack of knowledge of farmers that may impact negatively on a country’s surveillance plan. to prevent a situation where farmers and other stakeholders may not place a high value on participating in achieving the main objectives of a surveillance plan, it is recommended that policy makers and law enforcers at all levels of government in collaboration with other wildlife industry stakeholders such as wildlife veterinarians, ecologists, farmers, hunters, slaughter staff, processors and public health professionals establish strategies to ensure that all stakeholders are adequately trained in the role that they play in the control of zoonotic diseases in game. industry organisations such as farmer and hunting associations as ’bridging organisations‘ have a role to play with the provision of information or knowledge by (1) encouraging dialogue between governmental policy makers and industry, (2) acting as channels for policy makers to direct and apply new strategies at the industry-level, (3) assisting with the bottom-up flow of information at industry level and (4) providing platforms for information exchange (berkes 2009; tarnoczi & berkes 2010). in addition, bridging organisations can provide a platform for trust building, sense making, learning, vertical and horizontal collaboration, and conflict resolution (hahn et al. 2006). meaningful knowledge is most likely to result in concept development, attitudinal change and positive behaviour. acknowledgements top ↑ we would like to thank the library and information services of the tshwane university of technology library for their support in obtaining information used in this review. this study was also partly funded by stellenbosch university overarching strategic plan food security initiative. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions this manuscript forms part of a phd study where the student j.l.b. (tshwane university of technology) was mainly responsible for the compilation of the review published on zoonotic diseases. l.c.h. (stellenbosch university) was the first study promoter and p.j.j. 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world. it is situated in pretoria, a small town near onderstepoort.” this was reputedly said by an american visitor in the 1910s. it is possibly anecdotal but i heard it for the first time more than 60 years ago when i was at school contemplating studying veterinary science. i am using it to illustrate that onderstepoort is not only nationally famous. and onderstepoort and theiler are ‘synonymous’. i shall try to explain why this is so. i shall also try to capture the so-called ‘spirit of onderstepoort, referred to by theiler on more than one occasion to motivate his staff when their morale, or perhaps even his own, was low. in 1936 p.j. du toit & cecil jackson, in their lengthy obituary of theiler, defined it as: “that ideal of service, that conscientiousness of endeavour, that pride in a task, that confidence of success in the face of difficulties”. what they clearly had in mind was the legacy of the example set by theiler that became the unwritten code of ethics of the institute. theiler was therefore that ‘spirit’. there were pre-onderstepoort and onderstepoort phases in theiler’s career, both lasting ± 2 decades. all that it really involved as far as my theme is concerned was a change in address—from the lower town to the upper town—and i shall therefore consider them in the same breath. early career theiler suffered an appalling physical mishap a few weeks after his arrival in this country from switzerland in 1891. virtually penniless, he took on a job as a farmhand on the farm of the businessman and kruger-concessionary, nellmapius, situated where irene is now. in an unguarded moment, while cutting fodder in a steam-driven chaff cutter, his left hand was accidentally severed at the wrist. he barely survived, psychologically broken, his career 3 onderstepoort journal of veterinary research, 76:3–7 (2009) introductory keynote address theiler and the ‘spirit of onderstepoort’ r.d. bigalke 231 charles street, brooklyn, pretoria, 7405 south africa abstract bigalke, r.d. 2009. theiler and the ‘spirit of onderstepoort’. onderstepoort journal of veterinary research, 76:3–7 although theiler became internationally known for his pioneering veterinary research a distant century ago, there are probably few veterinary researchers today who have not heard of him. onderstepoort, the research institute he created, is equally well, if not even better, known. moreover, the name onderstepoort is not only associated with his institute but also with the only south african faculty of veterinary science, another theiler ‘baby’. the purpose of this presentation is to determine why this was so, and to what extent the fame was justified. was it due to the ‘spirit of onderstepoort’ sometimes referred to by theiler in the early as well as later stages of his career, or was there perhaps more to it, and what was theiler’s share in the development of that spirit, that fame? 4 keynote adress: spirit of onderstepoort in tatters. it also shaped his destiny. he slowly recovered and had a wooden prosthesis made in germany which was subsequently always gloved. theiler soon developed two major career objectives: • an ‘own’ research institute • an ‘own’ faculty both objectives were amazingly achieved in due course by this dynamic man. already famous and having befriended powerful senior officials and some politicians of the zuidafrikaansche republiek (zar), he used persistent coercion to have a tin shanty building, which he converted into a laboratory, allocated to him in 1898. theiler was conscripted into the boer forces when the anglo-boer war broke out in 1899, serving as the only veterinarian on the zar side compared to dozens in the british army. he saw service mostly on the natal front but returned to his laboratory after the conventional part of the war was over. he was appointed as government veterinary bacteriologist by the milner regime in 1903. but theiler aimed much higher than that. he wanted a proper bacteriological institute like the ones he had seen overseas. again he sought the assistance of senior officials and powerful politicians. in this case lord selborne, who replaced lord milner as high commissioner in 1905, was particularly supportive. he was a horse breeder and lover and held theiler in high esteem, particularly because of his research on african horsesickness. theiler had also managed to catch the eye of general louis botha, prime minister of transvaal and minister of agri culture. the massive budget for acquiring the land and erecting the laboratory was therefore readily passed by the legislature. the product was the ‘extravagant palace of science’, as it was dubbed by the politicians, which cost a staggering £60 000. it was built on the farm de onderstepoort in 1908 and its official name was the veterinary bacteriological laboratories of transvaal, but soon became known just as onderstepoort. a visionary scientist true to his mission-orientated vision, theiler had provided state of the art facilities for: • diagnostic work to identify the host of economically important diseases of livestock • research aimed at finding a solution for these diseases • vaccine production (four viral vaccines—one of which was small pox that he had been contracted to produce because his vaccine was better than anyone else’s in the country, two bacteriological vaccines and two diagnostic agents) • a lecture room and hostel for students, although there were none and would not be any for the next 15 years. he had clearly pulled the wool over somebody’s eyes. theiler’s dream of an ‘own’ faculty on his own campus was only realised in 1920. he once again played a pivotal role in influencing the decisions made by the bureaucrats. he gave evidence to two commissions of enquiry, providing the latter with a comprehensive business plan in which he outlined what was already available at onderstepoort because he wanted the faculty on his own campus. of course he held the trump cards in the form of an existing institute and appropriately trained scientists who could be used as teachers on a part-time basis. he also had the support of the prime minister, general j.c. smuts. in 1920 he was appointed in the newly created, exalted position of dean and director of veterinary education and research at the age of 51. the existing facilities were used, only three new buildings being required. his first students graduated in 1924. research achievements research aimed at providing solutions for the many serious diseases of livestock in southern africa (r&d) was theiler’s other obsession. he had many successes. he was a multi-disciplinarian and scored amazing breakthroughs in many of the subjects. i have selected five highlights that had major implications, both locally and internationally, on animal health and production. rinderpest remember that when rinderpest (rp) hit the zar and cape colony in 1896, the pandemic had already swept unchecked through the entire africa. theiler and hutcheon’s attempts (the latter in the cape colony) at stamping out, which had been used with fair success in europe, were a failure. there was no vaccine, although an international prize had been offered for its development. theiler had been appointed as government veterinarian by the zar government specifically to combat rp. he and watkins-pitchford of the natal colony opted for a serum-virus type vaccine, for which 5 r.d. bigalke models certainly existed. in a tin shanty laboratory in the marico bushveld, close to christmas in 1896, they developed a successful vaccine in less than 6 weeks. the ‘publish or perish’ concept was as yet foreign to them and the great and experienced robert koch, who had been hired by the cape col ony, pipped them at the post with his primitive, often fatal, bile ‘vaccine’. theiler never again made that mistake. east coast fever theiler’s second research triumph occurred in 1903 with east coast fever when he concluded that rhodesian redwater was not a form of redwater at all but an entirely different disease. he based his findings on the work done by lounsbury in 1902, in which he established that the three-host tick rhipicephalus appendiculatus was an excellent vector, and on his own observations on the morphology of the parasite and the pathology of the disease. koch came to the same conclusion in 1903 but still clung to his boophilus decoloratus transmission hypothesis. this time theiler got the accolade, the parasite being named theileria in due course. it was watkins-pitchford, however, who discovered the most effective early control method for east coast fever, i.e. very short interval dipping. anaplasmosis anaplasmosis was theiler’s next conquest. the great theobald smith, working on texas fever in the usa in the late 1890s, was happy to regard the marginal points that he observed in anaemic cattle as a sequel to redwater. however, by 1908 theiler had enough evidence to describe the points as micro-organisms in their own right. he used the generic name anaplasma because he could not see any cytoplasm—marginale for the highly pathogenic one and centrale for the more benign one. the latter he immediately turned into a very useful vaccine that is still being used in this country today. the strain was also given to several countries where anaplasmosis is also a problem, such as australia, israel and some south american countries. lamsiekte two things have always intrigued me about lamsiekte: • why it took theiler so long to unravel the riddle of its aetiology. • whether viljoen’s life-long grudge against theiler for receiving insufficient credit for his research contribution was justified. when i read gutsche’s biography on theiler, i was inclined to agree that viljoen had been bamboozled by theiler, which did not surprise me. however, on studying the original literature i completely changed my mind. i say it took a long time, because by 1912 (remember that he only had the ‘flash of lightning’ in 1919) theiler knew that: • hutcheon had advocated the use of bonemeal as a prophylactic in the early 1880s. • hutcheon’s colleague, borthwick, had shown, in a well-controlled field experiment in 1895, that lamsiekte did not occur in cattle fed a liberal allow ance of bonemeal. • bone-craving had been identified by hutcheon and several farmers as being associated with the occurrence of the disease (a ‘premonitory’ sign, he called it). • hutcheon regarded an aphosphorosis of the grazing as the primary cause of the disease. • walker (1909) and mitchell (1911), the latter a bit more convincingly, had produced a few cases of lamsiekte by feeding cattle on rotten bones. the only explanation i can offer is that theiler got stuck on his pet ‘grass toxin’ theory and that he misinterpreted hutcheon’s views on aphosphorosis. when he walked with the cattle on the farm armoedsvlakte in the autumn of 1919—at this stage i want to go back a year to p.h. de kock, president of the vryburg farmers association, who said, to which theiler took great umbrage, that ‘it would do no harm if some experts became cattle herds’ to find the cause of the disease—and saw osteophagia (bone-craving) with his own eyes, the penny immediately dropped. viljoen (1918) also latched onto the grass toxin theory. he stated categorically in his 1918 publication that he had not observed osteophagia on armoedsvlakte during the 4 years that he had been there and that he did not believe that it was a ‘premonitory’ sign. but he had personally seen and studied almost 200 cases of lamsiekte on the farm during this period. therefore, osteophagia must have occurred frequently while he was there. how is it possible that he had not observed it? the only possible explanation is that he had never walked with the cattle! he did not even attempt to produce lamsiekte by feeding rotten bones. he did produce two cases with blowfly pupae from a rotten carcass but failed to recognise the condition as lamsiekte. i don’t think he deserved more credit than he got. 6 keynote adress: spirit of onderstepoort theiler had actually done nothing more than confirm a hypothesis that was about 30 years old—even ‘spirits’ can sometimes miss the boat. but he confirmed it with a vengeance. within a year he had chemical proof that the grazing was definitely phosphorus-deficient and had followed the phosphorus cycle over the seasons; studied bone-craving in all its manifestations; produced lamsiekte by feeding cattle on rotten bones; developed a prophylactic dosage programme for lamsiekte with bonemeal; and produced the bacterial toxin concerned in culture—at this stage it was a mixed culture of anaerobes, but he could produce lamsiekte by injecting the supernatant fluid into cattle. perhaps even more important was theiler’s concurrent discovery of the crucial importance of phosphorus in the growth and production of cattle. a photograph published shows some animals of the same age but two of these animals that did not receive bonemeal have obviously lost a year’s growth. what was theiler like as a person? he knew that he had become indispensable to the zar and post-anglo-boer war governments and used this knowledge to befriend powerful people, especially politicians such as lord selborne, general louis botha and later even general smuts. if he did not get what he thought he needed badly or was criticised in parliament, as happened a few times with lamsiekte, his strategy was to threaten to resign. botha fell for this ‘blackmail’ time and again. this was perhaps not so strange. at that stage theiler was almost synonymous with the department of agriculture, botha having little else to brag about in parliament than the achievements of this incredibly successful scientist. he just could not afford to lose the services of such a dynamic person. theiler obtained all sorts of concessions like lengthy sabbatical leave on three occasions to study in switzerland. how he managed to be so productive despite all these visits is a miracle. when other divisions had their budgets cut, theiler got what he had asked for or even more. he sometimes even used this tactic to improve his own employment position. theiler also used his fame to network with the top veterinary scientists overseas, who soon recognised him as one of their peers, the expert on african tropical diseases. he attended three international veterinary conferences in succession, the first in badenbaden in 1898, only 2 years after his appointment as government veterinarian in the zar. people like lav eran (france), bang (denmark), hutyra (hungary), nocard (france), peroncitto (italy), dschunkowsky (russia) and theobald smith (usa) and others became his close colleagues. theiler was almost ‘obsessed’ with continuing education for himself, especially at swiss universities. why? the most likely explanation is that, as a multidisciplinarian who personally directed every research project at his institute, he thought it was essential to know as much of the discipline as the scientist(s) concerned did. when he later appointed non-veterinary scientists, like the excellent biochemist harry green, this need became even greater. theiler was in awe of the basic or ‘mother’ sciences such as botany, chemistry, biochemistry and zoology: ‘foster by all means the pure sciences. they are, in the hands of experts, the medium of solving the many economic problems of south africa”, he said. but he ensured that his handpicked successor, p.j. du toit (ph.d.), also obtained a veterinary degree before he appointed him! theiler may have been a spirit, but he was no angel. his leadership style was very autocratic and he consequently lost some excellent researchers, foremost being his compatriot k.f. meyer who left in 1910 and became a very eminent scientist in the usa. another was watkins-pitchford. theiler had more than 200 publications to his credit. however, there is not a single one of which he is not the senior author, if not the sole author. i strongly doubt whether he always gave sufficient credit to his co-workers. on the other hand, he seemed to have no qualms when his researchers published under their own names, as long as he had vetted their articles. theiler detested bureaucracy. his open lines of communication with botha and smuts functioned well and he won his ‘battles’ by ‘blackmailing’ them. but when general kemp appeared on the scene as minister of agriculture in 1924, in the new government of general hertzog, ‘blackmail’ backfired. kemp wanted to get rid of him—probably theiler’s 1914 knighthood and the sir arnold title rankled with this ex boer general. had it not been for the undoubted intercession of his deputy, p.j. du toit, theiler would have been pensioned off at the age of 57. theiler then withdrew from the fray leaving p.j. du toit in charge of the administration of onderstepoort while he concentrated on his research, his students and his famous annual reports. theiler’s ‘bitterness burst its bounds’, according to his biographer gutsche, when he was retired at 60 in 1927 ‘on the grounds of superannuation’ without even being offered the emeritus appointment he had expected. he left south africa on a world trip, 7 r.d. bigalke was feted wherever he went and offered some tempting appointments, for example in australia, and at various universities, which he refused, settling down in switzerland with his bones and other pathological specimens to do research and study at universities, and going on the personal holidays he had never taken while at onderstepoort. he returned to south africa in 1934, when denys reitz replaced kemp as minister of agriculture, to work (unpaid) at onderstepoort. he went to london in 1936 to attend a conference at which his son max—a medical doctor employed in the usa—was due to speak on his research on the development of a vaccine against yellow fever, for which max was later awarded a nobel prize, but died from a heart attack shortly before the conference was due to begin. is the ‘spirit’ still alive today? to the elements of theiler’s legacy i would add: • r&d aimed at serving farmers, large as well as small • a devotion to research • workaholism (if there is such a word). when i was at onderstepoort (1956–1988) all these elements were still very much in place. being paid by the government to work at a research institute where idealism prevailed over materialism was a rare privilege. my contact with onderstepoort is limited these days. however, when i have the opportunity to wander on the beloved, peaceful old campus, particularly late in the day, if not at night, i get a distinct feeling that that ‘spirit’ is still around. acknowledgements my sincere gratitude is due to ms h. heyne of the onderstepoort veterinary institute for assistance with the preparation of a powerpoint presentation. i also wish to acknowledge the use of material from the book there was a man. the life and times of sir arnold theiler k.c.m.g. of onderstepoort, published by howard timmins, cape town and from the archives of the onderstepoort veterinary insti tute. references bigalke, r.d. & verwoerd, d.w. 2008. a century of research achievements by the onderstepoort veterinary insti tute. trans actions of the royal society of south africa, 63: 31–51. du toit, p.j. & jackson, c. 1936. the life and work of sir arnold theiler. journal of the south african veterinary medical association, 7:134–186. gutsche, t. 1979. there was a man. the life and times of sir arnold theiler k.c.m.g. of onderstepoort. cape town: howard timmins. henning, m.w. 1956. animal diseases in south africa (3rd ed.). south africa: central news agency. jansen, b.c. 1977. the growth of veterinary research in south africa, in a history of scientific endeavour in south africa, edited by a.c. brown. cape town: the royal society of s.a. neitz, w.o. 1957. theileriosis, gonderiosis and cytauxzoonosis: a review. onderstepoort journal of veterinary research, 27:275–430. theiler, a. 1903. rhodesian tick fever. transvaal agricultural journal, 1:93–110. theiler, a. 1909. a contribution to our knowledge of gall-sickness. transvaal agricultural journal, 8:423–435. theiler, a. 1911. further investigations into anaplasmosis of south african cattle. first report of the director of veterinary research: 7–46. theiler, a., viljoen, p.r., du toit, p.j., meier, h. & robinson, e.m. 1926. lamsiekte (parabotulism) in cattle in south africa. eleventh and twelfth reports of the director of veterinary education and research: 822–1361. viljoen, p.r. 1918. investigations into lamsiekte in cattle. fifth and sixth reports of the director of veterinary research: 255–320. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: simbarashe katsande1 gift matope1 masimba ndengu2 davies m. pfukenyi2 affiliations: 1department of paraclinical veterinary studies, university of zimbabwe, zimbabwe 2department of clinical veterinary studies, university of zimbabwe, zimbabwe correspondence to: davies pfukenyi postal address: po box mp 167, mount pleasant, harare, zimbabwe dates: received: 27 sept. 2012 accepted: 28 jan. 2013 published: 28 mar. 2013 how to cite this article: katsande, s., matope, g., ndengu, m. & pfukenyi, d.m., 2013, ‘prevalence of mastitis in dairy cows from smallholder farms in zimbabwe’, onderstepoort journal of veterinary research 80(1), art. #523, 7 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.523 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence of mastitis in dairy cows from smallholder farms in zimbabwe in this original research... open access • abstract • introduction • materials and methods    • study sites and selection of study sites    • study animals    • assessment of milking practices    • milk sample collection and clinical examination    • laboratory tests    • data analysis    • ethical considerations • results    • descriptive statistics    • individual animal logistic regression model    • farm level logistic regression model • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a cross-sectional study was conducted to determine the prevalence of sub-clinical and clinical mastitis and the associated factors in cows from selected smallholder dairy farms in zimbabwe. physical examinations were conducted on all lactating cows for evidence of signs of clinical mastitis. composite milk samples were collected from all lactating cows for bacterial culture and somatic cell counting. cows were categorised as clinical if they exhibited clinical features of mastitis, or sub-clinical if no apparent signs were present but they had a positive bacterial isolation and a somatic cell count of at least 300 x 103 cells/ml. farm-level factors were obtained through a structured questionnaire. the association of mastitis and animaland herd-level factors were analysed using logistic regression. a total of 584 animals from 73 farms were tested. overall, 21.1% (123/584) had mastitis, 16.3% (95/584) had sub-clinical mastitis and 4.8% (28/584) had clinical mastitis. herd-level prevalence was 49.3%. coagulase-negative staphylococci (27.6%), escherichia coli (25.2%), staphylococcus aureus (16.3%), klebsiella spp. (15.5%) and streptococcus spp. (1.6%) were the most common isolates. in individual cows, pure dairy herds (or = 6.3) and dairy crosses (or = 3.1) were more likely to have mastitis compared to mashona cows. farms that used pre-milking teat dipping were associated with reduced mastitis prevalence. further research is needed on the prevalence of mastitis and a comparison of data for both smallholder and commercial dairy farms in all regions of zimbabwe should be undertaken. introduction top ↑ in zimbabwe, smallholder dairy farming developmental programmes were initiated in 1983 (ngongoni et al. 2006; muchenje et al. 2007). their contribution to national-marketed milk increased gradually from 2% in 1998 and to an estimated 5% in 2002 (ngongoni et al. 2006). smallholder dairy farming has the potential to increase the milk production base of the country, improve household nutrition and empower rural communities with income-generating ventures. it can assist farmers to diversify, lessen farming risks and create an opportunity for use of idle forage (ngongoni et al. 2006). shortage of and poor quality fodder have been reported to be two of the major limiting factors to smallholder dairy farming in the country (ngongoni et al. 2007). mastitis is another major limiting factor; it is a disease of dairy cattle that causes low milk production and high economic losses (ericsson-unnerstad et al. 2009; tesfaye, regasa & kelay 2009). mastitis is an inflammation of the mammary gland that is mostly caused by infectious agents, especially bacteria, but it can also be due to physical or chemical agents. somatic cell counts (scc) in milk are commonly used as indicatorsof mastitis, on the basis that an increase reflects an immune responseto the presence of infection in the mammary gland (green et al. 2008). mastitis is associated with high scc; this is mostly due to increased leukocyte levels, as they are involved in the removal of the infectious agents, and also exfoliated epithelial cells (bagnicka et al. 2011). leukocytes are responsible for destroying bacteria, and the enzymes left behind during the phagocytic process significantly reduce the shelf life of milk and milk products (ingalls 2009). however, animals with low scc can also have the mastitis pathogen present. demands by consumers and processors for safe, high-quality dairy products are major factors in motivating the need for additional improvements in mastitis control in dairy herds. mastitis could be reduced by improving milking procedures and hygiene in the herd (karimuribo et al. 2008; kivaria, noordhuizen & msami 2007). dry cow treatment, milking technique,post-milking teat dipping and antimicrobial treatment of clinicalmastitis are examples of management factors that have a significanteffect on the reduction of mastitis cases and bulk tank milk scc (lievaart, barkema, hogeveen & kremer 2009; lievaart et al. 2009). in zimbabwe, there is little current information about the prevalence of mastitis, factors associated with mastitis and the bacteria mainly responsible for intra-mammary infections in the smallholder dairy sector. the only data that is available is from studies that were conducted more than a decade ago (perry et al 1987; makaya, aarestrup & olsen 1996; kudinha & simango 2002). therefore, the objectives of this study were to determine (1) the individual animaland herd-level prevalence and factors associated with mastitis and (2) the most common bacteria associated with sub-clinical and clinical mastitis in cows from smallholder dairy farms in zimbabwe. materials and methods top ↑ study sites and selection of study sites a convenient sample of smallholder dairy farms (n = 73) in zimbabwe was selected. selection was based on the presence of farms that were producing and selling milk to milk-collecting centres when the study commenced, and on agro-ecological regions and locations of the areas. the four selected smallholder dairy centers were: dowa (18º32’s, 32º07’e) in agro-ecological region iia; guruve (16º20’s, 30º35’e) and marirangwe (18º06’s, 30º48’e) both in agro-ecological region iib; and nharira (19º15’s, 31º15’e) in agro-ecological region iii. study animals animal ownership by the smallholder dairy farmers and the demographics of cows sampled are presented in table 1. all smallholder dairy farms in the respective study areas with at least a single lactating cow were recruited to the study. a total of 73 smallholder farmers were still operational when the study commenced. the average herd size per farm ranged from 10 to 16, with a median of 13 animals. the breeds kept by these farmers included the local indigenous sanga type (bos indicus) commonly known as ‘mashona’, exotic dairy breeds (bos taurus), such as red dane and jersey, dairy crosses, such as friesian, jersey and red dane crosses, and beef crosses, such as brahman, hereford and mashona crosses. all of the lactating cows (n = 584) on the selected farms were eligible for milk sample collection. a total of 584 lactating cows were sampled with most being the local indigenous mashona breed (44%) and dairy crosses (43.7%), whilst a few were pure dairy (2.4%) and beef crosses (9.9%). due to the unavailability of records, the exact ages and milk production levels of the sampled cows was not determined. however, the parity of the cow was recorded. assessment of milking practices a pre-tested structured questionnaire with close-ended questions was used to collect data on milking practices and other farm management variables thought to influence the prevalence of mastitis in smallholder dairy cows. these factors included milking procedures, milking place and frequency of milking, cleaning and drying methods of teats, source of water (open or closed source), pre-and post-milking teat dipping and milking practices for suspected mastitic cows. the questionnaire was administered through a face-to-face interview. the pre-testing of the questionnaire was carried out on a few selected farmers who farmed in one of the study areas; clarity of questions and its user friendliness was checked and later revised. the survey was carried out between november 2009 and march 2010. milk sample collection and clinical examination the collection of milk samples for bacterial culture and somatic cell counting was carried out during the same period as the questionnaire survey. teats were washed using clean running water, wiped dry with disposable paper towels and disinfected using 70% ethyl alcohol. to minimise contamination with bacteria from the skin around the teat canal, the first streams of milk were discarded. composite quarter milk samples were collected aseptically from all lactating cows and were transported to the laboratory in cooler boxes on ice (approximately 4 ºc) where milk samples were plated out and preparations for microscopic cell count commenced on the day of sampling. a thorough physical examination for evidence of clinical mastitis was conducted on all lactating cows that were sampled in this study. clinical findings such as secretions, abnormalities of size and shape of the udder, its consistency and temperature were assessed by visual inspection and palpation. a cow was considered to have clinical mastitis if it fulfilled at least two of the clinical findings, (1) pain reaction upon palpation, (2) changes in colour and consistency of milk (blood tinged milk, watery secretions, clots, pus) and (3) change in consistency of the udder (lakew, tolosa & tigre 2009). cows that did not have clinical mastitis were tested further for sub-clinical mastitis based on culture and somatic cell counting. a cow showing no apparent clinical signs of mastitis and whose milk was macroscopically normal, but had a positive bacterial culture and scc of at least 300 x 103 cells/ml (international dairy federation 1987), was regarded as positive for sub-clinical mastitis. laboratory tests all the tests for bacterial culture and isolation were performed at the central veterinary laboratory in harare. milk samples were cultured for bacteria as described by haltia et al. (2006). all samples were plated on blood agar (oxoid, basingstoke, hampshire, uk), macconkey agar (oxoid) and edward’s agar (oxoid) to detect most aerobic pathogens, enteric bacteria and streptococci respectively. the plates were incubated aerobically at 37 ºc for up to 48 h. a milk sample was considered positive for mastitis pathogen(s) if at least a single colony of a potential pathogen was detected and positively identified by biochemical tests. plates showing mixed and confluent growths, with no evidence of single discernible colonies, were not investigated further. primary identification of staphylococci was based on colony morphology, catalase test, gram-staining morphology and differentiated from micrococci on the basis of the oxidative-fermentative (of) test carried out on semi-solid of medium (difco, becton, dickinson and company, franklin lakes, nj, usa). the staphylococci were also tested for production of coagulase enzyme by the tube method as described by quinn et al. (1994). isolates that produced gram-positive cocci in clusters, and were catalase positive, glucose-fermentative, resistant to bacitracin and did not produce coagulase were identified as coagulase-negative staphylococci (cns). staphylococcus aureus isolates were differentiated from other coagulase-positive staphylococci (staphylococcus intermedius and some strains of staphylococcus hyicus) on the basis of mannitol fermentation on mannitol salt agar (oxoid), susceptibility to 5 µg novobiocin and 300 units of polymixin b as reported by kudinha and simango (2002). the enteric bacteria were identified using colony morphology, oxidase test, lactose fermentation on macconkey agar (oxoid), indole production test, citrate utilisation, and other standard biochemical tests as described by quinn et al. (1994).somatic cell counts (scc) were carried out in the bacteriology laboratory of the central veterinary laboratory in harare. the scc were determined by using the breed’s direct smear method; spreading 0.1 ml of milk to cover about 1 cm2 of a glass slide, drying and staining with methylene blue (quinn et al. 1994). the somatic cells were counted microscopically over approximately 50 fields under oil immersion for each sample, and the total number of cells was recorded using a counter. the counts were converted to the total number of somatic cells per ml using the formula suggested by quinn et al. (1994). table 1: smallholder farmers’ cattle ownership and demographics of sampled cows. data analysis the proportion of mastitis-positive cows was calculated against the total number of animals investigated; this was based on the clinical features of mastitis (clinical mastitis), the pathogen isolated and a scc of at least 300 x 103/ml (sub-clinical mastitis). the prevalence of mastitis in individual cows was calculated according to the study area, breed, parity and history of mastitis. a herd was denoted as positive for mastitis if at least a single animal with mastitis (either clinical or sub-clinical) was detected. the farm-level prevalence of mastitis was calculated according to the study area and milking management practices, such as the use of preand post-milking teat dipping, milking frequency, cleaning of teats prior to milking, use of disposable paper towels, and source of water (open or closed source) for milking. the association between mastitis (0 = negative and 1 = positive) and categorical animaland farm-level variables was assessed in univariable analyses using fisher’s exact x2 test. all the variables having p < 0.25 were further investigated using multivariable logistic regression analysis (tables 3 and 4) for an association with mastitis. due to the low numbers of mastitis-positive animals per farm (low clustering effect), an adjustment for clustering per herd was not considered critical as the logistic regression was unlikely to result in artificially low coefficients (matope et al. 2011). two multivariable logistic regression models were built; one for individual animals (table 3) and another for farm level variables (table 4) using stata version se 10.0 (stata corp., texas, usa). in both models, the outcome variable was the mastitis status (0 = negative and 1 = positive). the models were manually constructed by forward-selection, applying the maximum likelihood estimation procedure and statistical significance contribution of individual predictors (or group of predictors) using the likelihood ratio test (matope et al. 2010; dohoo, martin & stryhn 2003). the presence of interaction between variables was checked by constructing a two-product term and forcing it into the model and examining the changes in the coefficients and p-values of the main effects (matope et al. 2010). evidence of confounding was checked by dropping one of the variables and assessing changes of the coefficients (matope et al. 2010). goodness-of-fit of the logistic regression models was assessed using the hosmer-lemeshow test, whilst predictive ability was determined using the receiver operating characteristic (roc) curve (dohoo et al. 2003; matope et al. 2010). ethical considerations the ethical and the higher degrees committees of the faculty of veterinary science and the department of animal science, faculty of agriculture, approved the use of animals and all protocols or procedures in this study. the objectives of this study were well explained to all participating smallholder dairy farmers who all expressed their consent to participating in the study. questionnaire responses were given on a voluntary basis and respondents were permitted to withdraw their consent up to seven days before data submission. standard operating procedures were used for the collection of milk samples and the dairy cows were supplied with adequate food and clean drinking water. results top ↑ descriptive statistics a total of 584 animals from 73 farms were tested. of these, 21.1% (123/584) had mastitis, with 16.3% (95/584) and 4.8% (28/584) having sub-clinical and clinical mastitis respectively. only one cow (0.8%) with signs of clinical mastitis was negative on bacterial culture, but had a scc > 300 x 103 cells/ml. the farm-level prevalence of mastitis was 49.3% (36/73). from the mastitis-positive cows, coagulase-negative staphylococci (cns) (27.7%), escherichia coli (25.2%), s. aureus (16.3%), klebsiella spp. (15.4%) and streptococcus spp. (1.6%) were the most predominant isolates obtained. it was found that 3.2% had mixed pathogens (various combinations of pairs of e. coli, klebsiella spp., cns and streptococcus spp.) and 9.0% had contaminated growths (table 2). cows with clinical mastitis were associated with e. coli (32.1%), s. aureus (21.4%), cns (14.3%), klebsiella spp. (10.7%), mixed e. coli and klebsiella spp. (3.6%), contaminated growths (unidentified pathogens) (14.3%) and no bacterial isolates (3.6%). individual animal logistic regression model on univariable analysis, study area, breed, parity and history of mastitis were found to have significant associations with mastitis. however, the final logistic regression model revealed that study area and breed (mashona, pure dairy, dairy crosses and beef crosses) were independently associated with mastitis in individual cows (table 3). farm level logistic regression model cleaning of the teats was not included in the regression model since all farmers practiced it. on univariable analysis; post-milking teat dipping, frequency of milking, fore-milk testing, cloth use and water source were found not to be significantly associated with mastitis in herds. the final logistic regression model revealed that study areas and pre-milking teat dipping were independently associated with mastitis in herds (table 4). table 2: summary of the bacterial isolates from smallholder dairy cows with mastitis. table 3: summary of univariable and multivariable logistic regression analysis of associations between mastitis and potential risk factors for individual animals. table 4: summary of univariable and multivariable logistic regression analysis of associations between mastitis and potential herd-level risk factors. discussion top ↑ the results of the present study show that utilisation of recognised pure exotic dairy breeds, such as the holstein, jersey or red dane, in the selected smallholder dairy farms is uncommon. instead, farmers predominantly use crosses of these dairy cattle and beef breeds (mainly mashona) for their dairy farming. this may be reflective of either the absence of a sound breeding policy for pure exotic dairy breeds by resource-limited smallholder farmers, or preference of the local beef breeds, which are better adapted to the harsh environments and offer more resistance to diseases prevalent in these areas. in individual cows, the prevalence of sub-clinical mastitis recorded during the present study is comparable to that reported from other smallholder farming sectors (harouna et al. 2009; karimuribo et al. 2008; kivaria, noordhuizen & kapaga 2004). compared to individual milk samples, composite milk samples may be less effective for the isolation of micro-organisms due to the dilution factor from uninfected quarters. the prevalence of clinical mastitis in our study (4.8%) is within the range of other reports from smallholder dairy farms in some countries in sub-saharan africa (abera et al. 2011; getahun et al. 2008; kivaria et al. 2004). this provides further support of other studies in the region, which have concluded that sub-clinical mastitis is more prevalent than clinical mastitis (almaw, zerihun & asfaw 2008; lakew, tolosa & tigre 2009). this is likely to be partly influenced by virulence of the circulating bacterial strains and the levels of immunity of the cows to these pathogens. the relatively high prevalence of sub-clinical mastitis recorded in this study could possibly indicate a substantial financial loss through decreased milk production due to progressive destruction of the alveolar epithelial cells in the mammary gland (zhao & lacasse 2008). mungube et al. (2005) reported a substantial loss due to subclinical mastitis; the overall financial loss for each cow per lactation was estimated to be us $78.65 (tesfaye et al. 2009). however, most smallholder farmers are not well informed about the invisible losses from sub-clinical mastitis and do not normally recognise the condition (mungube et al. 2004). all of the smallholder farmers clean the teats of dairy cows with running water prior to milking, but, as observed earlier (millogo et al. 2008), the implementation of mastitis preventive measures such as using separate drying cloths for each cow, and practicing preand post-milking teat dipping are noted to be infrequent amongst the farmers. a lower prevalence of both clinical and sub-clinical mastitis has been reported on farms where separate towels for teat cleaning and post-milking teat dipping were routinely practiced (kivaria et al. 2007). despite the fact that some of these risk factors are not found to be statistically significant in the final logistic regression model, presumably due to low sample sizes, the need to implement mastitis preventive measures such as the use of pre-milking and post-milking teat dipping cannot be overemphasised. the isolation of cns, s. aureus, e. coli, klebsiella spp. and streptococcus spp. from mastitic cows in this study supports findings from previous studies conducted in both commercial and smallholder dairy farms in zimbabwe (perry et al. 1987; makaya et al. 1996; kudinha & simango 2002), south africa (swartz, jooste & novello 1984) and smallholder dairy farms in other regions of africa (getahun et al. 2008; lakew et al. 2009). as observed in other studies in zimbabwe and elsewhere (makaya et al.1996; kudinha & simango 2002; petzer et al. 2009), sub-clinical mastitis is most commonly associated with cns. this highlights the importance of the role of cns in sub-clinical mastitis and that some of them are more pathogenic than is generally assumed. therefore, the general mastitis-control programmes, such as dry cow-therapy, that take these pathogens into consideration will be more beneficial. in this study, environmental bacteria e. coli and klebsiella spp. predominate over s. aureus in both sub-clinical and clinical mastitis; this is contrary to other reports where s. aureus was the most common isolate (swartz et al. 1984; tesfaye et al. 2009). inadequate hygiene in the dairies in our study, especially during the rainy season when cow udders are mainly soiled due to animals lying in muddy overnight cow pens, is likely to increase the risk to infection with environmental pathogens. pure exotic dairy cows (or = 6.3), cows of dairy crosses (or = 3.1) and beef crosses (or = 2.6) are more likely to be positive for mastitis compared to the local indigenous mashona breed. the difference in mastitis prevalence might reflect a number of general resistance characteristic attributes of the breeds, such as udder and teat morphology. since cows in the same study area are all under similar conditions, management factors such as grazing, kraaling, milking procedures, et cetera are unlikely to influence this breed difference. hence, it is likely that the indigenous breeds have over the years become better adapted to regional stressors than the exotic breeds. similarly, as in previous studies (green et al. 2008; harouna et al. 2009), indigenous breeds (in this instance mashona) are found to have significantly lower scc than exotic dairy breeds and their crosses (data not shown). therefore, due to these breed differences in levels of scc, the diagnosis of sub-clinical mastitis, especially in indigenous cows like mashona, may be improved by lowering the cut-off points for scc rather than adopting the universal criteria that were designed for high-milk producing cows. in support of previous observations (byarugaba et al. 2008; getahun et al. 2008), the present study shows a progressive increase in the prevalence of mastitis with increased parity. this apparent increase in the risk of mastitis in older cows, especially after the fourth parity, could be attributed to chronic sub-clinical mastitis especially caused by host-adapted pathogens such as s. aureus (bradley 2002). however, studies in other regions have reported higher prevalence in heifers compared to older cows; this could possibly be due to differences in management practices (mcdougall et al. 2009). although, based on the available data, the differences in farm-level prevalence of mastitis in this study cannot be explained fully, it is likely that this can be attributed to animal husbandry practices. for instance, marirangwe farms that keep the highest proportions of dairy crosses are associated with a higher prevalence compared to dowa, which keep very few dairy crosses and predominantly the mashona breed. since the selection of the study areas was influenced by the availability of operational farms, the inclusion of more study areas across the country could have revealed differences in prevalence of mastitis according to the different agro-ecological regions. nevertheless, considering that sub-clinical mastitis is quite prevalent and that common mastitis pathogens such as cns, e. coli, klebsiella spp. and s. aureus were isolated from all the study areas, the wide-spread of mastitis in smallholder dairy farms in zimbabwe cannot be ruled out. in conclusion, this study established an overall mean cow-level mastitis prevalence of 21.1%, where subclinical (16.3%) is more prevalent than clinical (4.8%) mastitis. mastitis is found to be prevalent in all of the study areas, with a mean of 49.3%. pure exotic dairy cows, cows of dairy crosses and beef crosses have greater odds of mastitis compared to the mashona breed. the bacteria most commonly associated with mastitis include cns, e. coli and klebsiella spp. and s. aureus. at farm-level, the differences in mastitis prevalence can be related to management factors like the use of pre-milking teat dipping. further research is needed on mastitis prevalence in all regions of zimbabwe in both smallholder and commercial dairy farms. a comparison of data between these distinct dairy farming practices is an area of further research. acknowledgements top ↑ this project was sponsored by the southern african development community/implementation and coordination of agricultural research and training (sadc/icart) programme for regional training. the authors are grateful to the smallholder farmers and veterinary laboratories who participated in the study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions d.m.p. (university of zimbabwe) was the project leader responsible for study design and data analysis, g.m. (university of zimbabwe) and m.n. (university of zimbabwe) were co-leaders responsible for conceptual contributions and writing of the manuscript and s.k. 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42(5), 925–931. http://dx.doi.org/10.1007/s11250-009-9509-2 zhao, x. & lacasse, p., 2008, ‘mammary tissue damage during bovine mastitis: causes and control’, journal of animal science 86, 57–65. http://dx.doi.org/10.2527/jas.2007-0302 article information authors: jonathan rushton1 barbara häsler2 nicoline de haan3 ruth rushton4 affiliations: 1animal health economics, rvc, united kingdom 2agrihealth, rvc and lcirah, united kingdom 3food and agriculture organisation of the united nations, rome, italy 4forensic psychologist, independent consultant, united kingdom correspondence to: jonathan rushton postal address: royal veterinary college, hawkshead lane, north mymms, hatfield, herts, al9 7ta how to cite this proceeding: rushton, j., häsler, b., de haan, n. & rushton, r., 2012, ‘economic benefits or drivers of a “one health” approach: why should anyone invest?’, onderstepoort journal of veterinary research 79(2), art. #461, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.461 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. economic benefits or drivers of a ‘one health’ approach: why should anyone invest? in this proceeding... open access • abstract • introduction    • background       • economic logic for investment in one health       • looking at resources in the health system       • responses to specific diseases and health problems       • where resources are scarce       • where resources are underutilised • discussion and conclusions • acknowledgements • references abstract top ↑ one health concepts and ideas are some of the oldest in the health discipline, yet they have not become main stream. recent discussions of the need for one health approaches require some reflection on how to present a case for greater investments. the paper approaches this problem from the perspective of the control and management of resources for health in general. it poses the following questions, (1) where do we need extra resources for one health, (2) where can we save resources through a one health approach and (3) who has control of the resources that do exist for one health? in answering these questions three broad areas are explored, (1) the management and resources allocated for diseases, (2) the isolation of parts of the society that require human and animal health services and (3) the use of resources and skills that are easily transferable between human and animal health.the paper concludes that one health approaches are applicable in many scenarios. however, the costs of getting people from different disciplines to work together in order to achieve a true one health approach can be large. to generate tangible benefits requires careful management of specialist skills, knowledge and equipment, which can only be achieved by a greater openness of the human and animal health disciplines. without this openness, policy makers will continue to doubt the real value of one health. in summary the future success of one health is about people working in the research, education and provision of health systems around the world embracing and managing change more effectively. introduction top ↑ since 2008 a number meetings have taken place and documents produced (food and agriculture organisation/world animal health organisation/world health organisation 2008; chatham house 2010; canadian public health agency 2009; world bank 2010) that have raised the need for a more holistic approach to problems that affect the health of humans, animals and the general environment. such an approach is not new, but the need for it has been given a much sharper focus with the increasing incidence of diseases that have the potential of creating large economic impacts, human deaths and losses of environmental diversity. the articulation of the need to adopt a one health approach was accepted at a meeting of governments (imcapi) in hanoi in april 2010. this was followed by a meeting in stone mountain, georgia, usa to discuss how one health can be operationalised. one of the recommendations of that meeting was the need for a document that clearly presents an investment strategy for one health. whilst the authors acknowledge the ongoing work in this area, the general feeling is that the one health approach is still some way from being main stream with human and animal health policy making. the questions that come to mind are: • why is one health not main stream? • if one health is important, how can the case for a major paradigm shift be more persuasively presented? the paper explores these questions from the perspective of resource allocation namely by looking at, (1) where extra resources are needed for one health, (2) where resources can be saved with a one health approach, and (3) who has control of the resources that do exist for one health at the moment. background the environments generating health problems are dynamic. the human population continues to grow with expansions greatest in the last fifty years occurring in the developing world (figure 1). figure 1: world population from 1850 to 1999. in addition to the growth in populations there are constant changes in the movement of people and settlement patterns. some the most dramatic have been the movements of people from central america and mexico to north america in the last 15 years and in general the movement of people to urban environments. to keep pace with these changes livestock systems and their associated value chains have evolved. delgado et al. (1999) described this as a livestock revolution; in fact it was a second revolution, as the first occurred in the 1800s in europe and the associated colonies (rushton 2009). this first revolution was largely based on ruminant production, and the second and most recent revolution being based largely on intensive monogastric systems and to some extent from a growth in milk production. the world has seen greater specialisation and intensification of livestock systems leading to increased output per animal and per unit of labour. there has also been massive expansion of livestock populations particularly poultry and pigs and a concentration and clustering of livestock populations. in general there has been an increase in the sophistication and globalisation of livestock product value chains.originally these changes in the livestock sector were celebrated although there were early concerns of poorer livestock producers being left behind (de haan et al. 2001; heffernan 2002; food and agriculture organisation 2005; owen et al. 2005) and negative impacts on the environment (de haan, steinfeld & blackburn 1997; steinfeld et al. 2006). what was not anticipated were the growing problems with the control of transboundary animal diseases and more specifically the resurgence of zoonotic diseases (greger 2007). one of the issues that has been raised it is that as domestic livestock populations increase there has been greater contact with wildlife. also as human populations have pushed into new areas there has been increased contact between human populations and wildlife. therefore potentially two different sources of diseases either through direct contact with wild animals or through domestic species possible acting as liaison hosts. in addition to these contacts with wildlife the emerging food chains have generated greater levels of moral hazard (asymmetry of information) where people consuming livestock products are unlikely to know how animals are raised and fed, and how the product was handled and stored before it arrives on the plate. an extreme example of this comes from the uk where only 339000 people work in agricultural holdings (only 0.6% of the population) yet they produce food and therefore can affect the wellbeing of 60 million people (see figure 2). figure 2: health contributions to the food chain. responses to these existing and emerging challenges have been strong with greater control of many transboundary animal diseases, and success stores such as the global eradication of rinderpest and the regional eradication of diseases such as foot and mouth disease and classical swine fever. however, there have been major setbacks such as bovine spongiform encephalopathy, sars and highly pathogenic avian influenza h5n1. the emergence of these problems and the apparent increase in the frequency at which such pathogens emerge (woolhouse & gowtage-sequeria 2005; woolhouse 2008; jones et al. 2009) indicate a need to re-assess how the world deals with change and manages health.as the livestock sector and human society changes there is a need to monitor with different intensities and manage health risks in different ways. where risks can be quantified there has been a tendency towards allocating resources for monitoring and management in a targeted manner. yet the predictions of the emergence and re-emergence of disease problems have not been strong with obvious examples being bse, h5n1, h1n1. there are two possibilities from this scenario: what we are observing are not predictable events they are random and cannot be identified through commonly used statistical methods or that our current risk models are inadequate at simulating reality. either could be true, the more immediate challenge is to redirect resources so that problems as they emerge are addressed proportionately to the impact they cause and that response are not based on fears that are held. to achieve this there is a need for information on how resources are currently allocated and whether this use of use resources could be improved. economic logic for investment in one health investments in one health need to recognise two different aspects:• disease impact • costs of disease in terms of losses in production of livestock • costs of controlling the disease • human health impacts and costs • avoidable losses – the costs of disease that can be avoided by implementing a disease control programme. the disease impact gives some idea of the economic importance of a problem and whether there is a need to dedicate further resources in terms of education and research. the decision on surveillance and intervention needs to assess if their costs are less than the avoidable losses generated. the following sections will explore areas in one health where this could be the case. looking at resources in the health system the section is divided into three different areas, (1) resources dedicated to specific disease problems across humans and animals, (2) human populations that have poor access to resources for health and (3) resources can be moved between human and animal health issues easily. responses to specific diseases and health problems diseases can be relatively easily split into those that are problematic in humans, those that are problematic in animals and those can cause problems in both human and animal populations. the authors would suggest that the most appropriate approaches to diseases that cause problems in humans or animal populations need specialised approaches and in general this is how human health and animal health systems have evolved. however, the diseases that cause problems in both humans and animals – the zoonoses – require generalised approaches (figure 3). figure 3: disease groupings and the appropriate responses. some of the zoonotic diseases cause significant impacts in specific locations (knobel et al. 2005 for rabies), and some that are classified as zoonotic cause huge disruptions (otte et al. 2010 for hpai h5n1). however, the zoonotic diseases as a whole do not tend to have a large enough impact in human and animal populations at the same time to have warranted the creation of generalised health service that approaches the problems from a combined human and animal population perspective (maudlin, eisler & welburn 2009). for example, tuberculosis is one of world’s major human disease problems, but the causative and self maintaining pathogen of this disease is not mycobacterium bovis, the pathogen in cattle, it is mycobacterium tuberculosis. therefore whilst the disease complex as a whole could be considered zoonotic, the pathogens tend to be species specific with some spillover into other species populations. similarly, brucellosis pathogens are specific to livestock species with spillover into humans where the pathogen can remain but not spread between people. probably the most challenging pathogens in terms of affecting both human and animal populations are the parasitic problems such as cysticercosis and cystic echinococcosis.this is not to argue that zoonotic diseases are not important, but that as a group they do not seem to cause sufficient impact across both human and animal populations for societies to have a cadre of people who work across species or organisations that follow the disease across animal and human populations. the problem actually lies in where resources are spent on diseases that circulate in animals and cause spillover impacts in the human population. for example brucellosis causes a significant economic impact, but the costs of controlling the disease in animals are greater than the benefits generated in the animal population, it is only when benefits from the prevention of disease in humans is taken into account that the costs are exceeded (roth et al. 2003). the implications for such disease problems is the need for one health thinking at a much higher level of budgetary and resource allocation, so that control campaigns in animals are sufficiently well financed to lead to benefits in humans. this requires a proactive and preventative approach to disease management, a recognition that disease can be managed further upstream, which requires significant shifts in resource allocation. it does not necessarily mean closer working mechanisms in the field. there are examples where there has been a need and a successful implementation of one health in the eradication of disease with strong field level coordination such as the control of cystic echinococcosis and echinococcus in places such as new zealand and the ongoing attempts to remove cysticercosis from the northern area of peru (gonzalez 2011). it is also argued that the non-communicable diseases is a problem that deserves more thought on how best to harness one health approaches. food chains process and refine food for both animals and humans and this has important implications on food intake nutritional health and resulting diseases. these aspects are rarely treated as one health issues and are invariably observed and worried about rather than thinking of the underlying causes. they would require a more general rather than disease specific approach. in common with all the disease groups there is the need for an understanding of the role of human behaviour in terms bringing host and pathogen together. there is also a need to understand and use how we react when a disease is present which could be in a positive manner in terms of controlling disease and also in negative manner leading to the maintenance of disease. the latter could be due to ignorance and/or economic gain. finally human behaviour plays a role in consumption and therefore the emergence of non-communicable diseases. to attract and retain resources that are applied to specialised activities in terms of dealing with diseases there would be a need for an agreement of the human and animal health leaders on who leads, who implements and who gets the resources. in some cases there will be strong arguments that integrated field level approaches are not necessary, but that these require one health thinking at a budgetary allocation point. other diseases do require much more integrated approaches such as specific parasitic diseases and the non-communicable diseases. where resources are scarce many people live in geographical isolation in areas where they are reliant on livestock. making resources available in these areas for either human or animal health is difficult due to the limited availability of trained resources and the lack of demand for such services. the need for one health approaches would make sense in terms of matching overall demand for animal and human health services and the potential to supply adequate services. strong arguments for generalised services and these have been well documented (schelling et al. 2007; zinsstag et al. 2007).some lessons from these generalised approaches would benefit from examining the literature on the integrated rural development programmes (roling & wagemarkers 1998; morton, matthewmaan & barton 1997; van veldhuizen et al. 1997). indeed, one health services probably need to incorporate aspects of animal production and genetics, water and sanitation and potentially plant health in such regions. where resources are underutilised many human health and animal health facilities are built that replicate capacity and in some cases have relatively low throughput. in addition there is human capacity building in data collection, storage and analysis skills in the two health areas. in the case of human capacity this can often be in too few numbers and/or with a low demand of their skills in their specific health field. low throughput and low demand often leads to poor calibration of standards and variable output of results. small numbers of trained people limit interchange and advancement in knowledge. there are strong arguments that certain aspects of human and veterinary diagnostics, data collection and analysis need to be combined to create synergies which will improve resource use. where resources, skills and institutions could provide a service to both human and animal health delivery and budget constraints limit how much redundancy can be allowed – laboratories are an obvious target, and more creative use could be made of epidemiology and socio-economics skills. better linkages of human and animal health surveillance data may potentially be useful for emerging diseases. discussion and conclusions top ↑ the environment that leads to the emergence and re-emergence of health problems is dynamic and constantly changing. these changes have led to responses in terms of strengthening disease surveillance, internationally through who, oie, fao and nationally through multi and bi lateral programmes plus regional agreements. these have generated benefits in terms of: • improved understanding of health problem emergence and re-emergence in order to respond in a proportionate and timely manner. • generalised systems of health delivery where resources are scarce – very specific situations. • combined use of infrastructure and skillsets to improve the use of underutilised resources and create synergies. the benefits are not constant as the environment is constantly changing. these changing benefits have changing costs that can only be estimated with better monitoring systems of: • livestock systems • value chains • people working within and using these chains. yet we have weak systems to monitor the working and behaviour of livestock systems and their associated chains implying that one health agenda should be expanded to include environmental concerns and human behaviour. political reality of adopting a one health agenda also requires thought and needs to be realistic with the development of evidence of added value from one health approaches through systematic data collection and analysis. the current lack of evidence reflects a lack of funding, collaboration, management and support, and future work needs to pose the following questions: • how do we improve the monitoring of facilitating environment so we can in real time: • estimate health problem impact with more accuracy • estimate the costs (direct costs and institutional costs) of monitoring and control • estimate benefits from mitigation activities. we need to search for proportionate and rational responses that involve individuals, communities, non-governmental organisations (ngos), private and public sectors, and to recognise that no one mechanism will suit all situations – it requires a systems and people centred approach with strong technical leadership. acknowledgements top ↑ madelon meijer, oxfam, the netherlands; malla hovi, ahvla, uk; wantanee kalpravidh, david castellan – fao, bangkok; nick taylor university of reading, uk; liz redmond – fsa, uk; katharina stärk, dirk pfeiffer, javier guitian – rvc, uk; mark rweyemamu and the sacids team; jeff waage – leverhulme centre for integrative research on agriculture and health, lidc, uk. references top ↑ canadian public health agency, 2009, ‘report of the expert consultation on one health’, winnapeg, march 2009, canadian government, ottawa, canada. chatham house, 2010, ‘shifting from emergency response to prevention of pandemic disease threats at source’, chatham house, london, uk. de haan, c., schillhorn van veen, t., brandenburg, b., gauthier, j., le gall, f., mearns, r. et al., 2001, ‘livestock development: implications for rural poverty, the environment and global food security’, the world bank, washington dc, usa. de haan, c., steinfeld, h. & blackburn, h., 1997, livestock and the environment: finding a balance, wrenmedia, suffolk, uk. delgado, c., rosegrant, m., steinfeld, h., ehui, s. & courbois, c., 1999, livestock to 2020. the next food revolution, food, agriculture and the environment discussion paper 28. ifpri, washington dc, usa. food and agriculture organisation, 2005, ‘livestock policies and poverty reduction in africa, asia and latin america’, pro-poor livestock policy initiative policy brief, fao, rome italy. food and agriculture organisation/world animal health organisation/world health organisation, 2008, ‘contributing to one world, one health a strategic framework for reducing risks of infectious diseases at the animal–human–ecosystems interface’, fao, rome, italy. gonzalez, a., 2011, email, 29 november, agonzale@jhsph.edu. greger, m., 2007, ‘the human/animal interface: emergence and resurgence of zoonotic infectious diseases’, reviews in microbiology 33, 243–299. heffernan, c., 2002, ‘livestock and the poor: issues in poverty focused development’, in e. owen, t. smith, m.a. steele, s. anderson, a.j. duncan, m. herrero et al., (eds.), responding to the livestock revolution: the role of globalisation and implications for poverty alleviation bsas publication 33, 229–245. jones, k.e., patel, n.g., levy, m.a., storeygard, a., balk, d., gittlemman, j.l. et al., 2008, ‘global trends in emerging infectious diseases’, nature (london) 451, 990–993. http://dx.doi.org/10.1038/nature06536, pmid:18288193 knobel, d.l., cleaveland, s., coleman, p.g., fevre, e.m., meltzer, m.i., miranda, m.e.g. et al., 2005, ‘re-evaluating the burden of rabies in africa and asia’, bulletin of the world health organization 83, 360–368. pmid:15976877, pmcid:2626230 maudlin, i., eisler, m.c. & welburn, s.c., 2009, ‘neglected and endemic zoonoses’, philosophical transactions of the royal society of london. series b, biological sciences 364, 2777–2787. morton, j., matthewman, d. & barton, d., 1997, livestock production extension: issues, case studies and policy options. nri socio-economic series 12. chatham, uk: natural resources institute. owen, e., best, j., devendra, c., ku-vera, j., mtenga, l., richards, w. et al., 2005, ‘introduction the need to change the “mind-set”’, in e. owen, a. kitalyi, n. jayasuriya & t. smith (eds.), livestock and wealth creation. improving the husbandry of animals kept by resource-poor people in developing countries, nottingham university press, nottingham, uk, pp 1–11. otte, m.j., hinrichs, j., rushton, j., roland-holst, d. & zilberman, d., 2008, ‘impacts of avian influenza virus on animal production in developing countries’, perspectives in agriculture, veterinary science, nutrition and natural resources, cabi, 3 no. 080. roling, n.g. & wagemakers, m.a.e. (eds.), 1998, facilitating sustainable agriculture. participatory learning and adaptive management in times of environmental uncertainty, cambridge university press, cambridge. roth, f., zinsstag, j., orkhon, d., chimed-ochir, g., hutton, g., cosivi, o. et al., 2003, ‘human health benefits from livestock vaccination for brucellosis: case study’, bulletin of the world health organization 81, 867–876. rushton, j., 2009, the economics of animal health & production, cab international, wallingford (uk) & cambridge (usa). schelling, e., bechir, m., ahmed, m.a., wyss, k., randolph, t.f. & zinsstag, j., 2007, ‘human and animal vaccination delivery to remote nomadic families, chad.’, emerging infectious diseases 13, 373–379. http://dx.doi.org/10.3201/eid1303.060391, pmid:17552089, pmcid:2725911 steinfeld, h., gerber, p., wassenaar, t., castel, v., rosales, m. & de haan, c., 2006, livestock’s long shadow environmental issues and options, fao, rome, italy. van veldhuizen, l., waters-bayer, a., ramirez, r., johnson, d. & thompson, j., 1997, farmers’ research in practice – lessons from the field, intermediate technology publications, london. woolhouse, m.e., 2008, ‘epidemiology: emerging diseases go global’, nature (london) 451, 898–899. http://dx.doi.org/10.1038/451898a, pmid:18288175 woolhouse, m.e.j. & gowtage-sequeria, s., 2005, ‘host range and emerging and reemerging pathogens’, emerging infectious diseases 11, 1842–1847. http://dx.doi.org/10.3201/eid1112.050997, pmid:16485468, pmcid:3367654 world bank, 2010, people, pathogens and our planet. volume 1: towards a one health approach for controlling zoonotic diseases, the world bank, washington, usa. zinsstag, j., schelling, e., roth, f., bonfoh, b., savigny, d.d. & tanner, m., 2007, ‘human benefits of animal interventions for zoonosis control’, emerging infectious diseases, 13, 527–531. http://dx.doi.org/10.3201/eid1304.060381, pmid:17553265, pmcid:2725951 bragg_93-98.qxd introduction the bacterium, haemophilus paragallinarum (bibberstein & white 1969) causes infectious coryza (ic), which is an upper respiratory tract infection in chickens, both layers and breeders. the naddependent strains of h. paragallinarum are very fastidious organisms requiring two supplements to the medium which are essential for their growth. these are nad at 1.56–25 µg/ml of medium (page 1962) and between 1.0–1.5 % nacl (rimler, davies & page 1977). chicken serum has also been found to be required by most pathogenic strains (sawata, kume & nakase 1980), while other isolates have been reported as showing better growth in the presence of 1 % chicken serum ( blackall & reid 1982; hinz 1973). it has been demonstrated that there are three serogroups of h. paragallinarum (page 1962; kume, sawata, nakase & matsumoto 1983; blackall, eavers & rogers 1990a ) consisting of at least nine different serovars (blackall, eavers & aus 1990b) and that the protection afforded by a vaccine is determined by the serovars of the bacterium present in the vaccine (rimler et al. 1977; kume, sawata & nakase 1980) the development of a pcr test which is specific for h. paragallinarum (chen, miflin, zhang & blackall 93 onderstepoort journal of veterinary research, 71:93–98 (2004) the testing and modification of a commercially available transport medium for the transportation of pure cultures of haemophilus pparagallinarum for serotyping r.r. bragg1, p. jansen van rensburg, e. van heerden and j. albertyn department of microbial, biochemical and food biotechnology, university of the free state p.o. box 339, bloemfontein, 9300 south africa abstract bragg, r.r., jansen van rensburg, p., van heerden, e. & albertyn, j. 2004. the testing and modification of a commercially available transport medium for the transportation of pure cultures of haemophilus paragallinarum for serotyping. onderstepoort journal of veterinary research, 71:93–98 haemophilus paragallinarum, the causative agent of infectious coryza in poultry, is an extremely fastidious organism requiring specific growth conditions for isolation. for complete control of the disease in regions where more that one of the serovars of the different serogroups occurs, it is essential that the bacterium causing the problem be isolated and serotyped. this work describes the modification and testing of transport media, which will ensure the survival of the causative agents in suspected infectious coryza cases for transport to a laboratory where the bacterium can be isolated and serotyped. the various transport media used are based on commercially available amies transport medium supplemented with the different supplements used for the growth of h. paragallinarum. it was established that the bacterium remains viable for up to 18 days in amies transport medium containing all the supplements when stored at 4 °c or 37 °c. at room temperature or 25 °c, there was no difference in the survival of h. paragallinarum in commercial amies transport medium (without charcoal) and amies transport medium with supplements. keywords: infectious coryza, haemophilus paragallinarum, transport medium 1 e-mail: braggrr.sci@mail.uovs.ac.za accepted for publication 23 october 2003—editor 1996) has greatly assisted in the diagnosis of ic, particularly in regions where the poultry production facilities are far removed from diagnostic laboratories. miflin, chen, bragg, welgemoed, greyling, horner & blackall (1999) demonstrated that the pcr test established by chen et al. (1996) was specific for both the nad-dependent and nadindependent isolates. they also demonstrated that it could differentiate between h. paragallinarum and other closely related organisms which can be isolated from the sinuses of chickens. unfortunately this test is not serogroup or serovar specific. serotyping of isolates from regions where there have been vaccination failures remains essential and in order to achieve this, the causative bacterium must be isolated and serotyped. infectious coryza remains a serious problem in many developing countries where technical facilities for the serotyping of isolates are often not available and in order to achieve this they must be transported to betterequipped laboratories. in addition, many of the laboratories that lack the facilities to perform serotyping also lack those to freeze-dry isolates. this makes the transport of isolates from developing countries very difficult. currently, there is no suitable transport medium for the long distance transport of h. paragallinarum. transport media for neisseria gonorrhoeae, which is also an extremely fastidious organism are, however available. this organism can be transported successfully in amies transport medium with or without the addition of charcoal. there are many similarities in the growth requirements of n. gonorrhoeae and those of h. paragallinarum, such as their micro-aerophilic nature and the fact that both organisms show improved growth in the presence of serum. in the light of these similarities, attempts were made to establish a transport medium for h. paragallinarum, based on that for n. gonorrhoeae. materials and methods a sample of the modesto strain of h. paragallinarum, which is a serogroup c isolate (serovar c-2) was obtained from the faculty of veterinary science, university of pretoria as a freeze-dried culture. this bacterium was reconstituted on tmb broth (reid & blackall 1984) and was stored by freeze-drying or in microbanktm at –70 °c. the identity of the bacterium as h. paragallinarum was tested using the pcr test developed by chen et al. (1996) without any modifications. the purity of the culture was established by plating the bacterium onto blood tryptose agar (bta) plates together with a feeder culture of staphylococcus aureus, and observation of typical satellitism (de blieck 1932). these tests were also used to confirm the identity and purity of all samples stored on the different transport media developed and tested in these experiments. eight different variations on amies transport medium were evaluated for the transport of h. paragallinarum (table 1). these variations include commercially available amies transport swabs or media (tubes) with or without charcoal; (abbreviated as a/ts, a/wc and a/-c respectively). in addition, four different supplements were added to amies transport medium to determine if any of these supplements has as effect on the viability of h. paragallinarum. amies transport medium with charcoal (a/wc) and without charcoal (a/-c) were prepared in sterile petri dishes. the plates were inoculated with pure cultures of h. paragallinarum and incubated at 37 °c for 4 days, after which viability of each culture was tested by taking samples from which were plated onto bta plates with a feeder culture of staphylococcus aureus. the identity of each sample showing positive growth was confirmed through pcr. amies transport swabs (a/ts) without charcoal were tested for their suitability for transporting pure cultures of h. paragallinarum at four different temperatures. sixty four swabs were made from 18 hold cultures of h. paragallinarum growing on bta plates, containing a feeder culture. sixteen of these swabs were stored at 4 °c, 16 were kept at room temperature (which ranged from between 21 °c to 28 °c during the course of these experiments), 16 were incubated at 25 °c and the remaining 16 were incubated at 37 °c. two of the swabs were selected each day from the 4th day of storage from each of the four groups and were plated out onto bta plates with a feeder culture of s. aureus. five of the colonies were randomly selected for confirmation of identify of the isolated bacterium by the pcr test. one hundred and twenty eight samples of h. paragallinarum growing on bta plates were collected on sterile swabs which were then placed into tubes containing different versions of the transport medium (a/nad, a/t, a/a-o, a/serum and a/supp; table 1). the tubes were divided into four groups of 32 samples each and each group was maintained at 94 transportation of pure cultures of h. paragallinarum for serotyping 95 4 °c, 25 °c, 37 °c or at room temperature. two samples were removed daily from the 4th day of incubation for a total of 19 days and the swabs were processed to determine survival of the sample. twelve swabs were used to collect samples from the sinuses of broiler breeders suffering from ic on a farm in zimbabwe. after collection the swabs were placed into tubes containing amies transport medium. another four swabs were collected from the drinking water in sheds where clinically infected broiler breeders were housed. all of these swabs were transported to johannesburg international airport from harare, zimbabwe as hand luggage, followed by further transportation by car to bloemfontein where they were processed by plating them out onto bta plates with a feeder culture. identity of the bacterial colonies was confirmed as h. paragallinarum by pcr. the total time elapsed from collection of the samples to processing in the laboratory was 5 days. samples of pure cultures of h. paragallinarum were collected from bta plates on sterile swabs which were placed into tubes containing one of the following modified transport media: swabs, a/nad, a/t, a/a-o, a/serum and a/supp (table 1). these samples were sent by airmail post to cape town, after which they were returned to the laboratory in bloemfontein and processed as described above. the total time spent in transit was 6 days. results and discussion the results obtained when samples of h. paragallinarum were stored at different temperatures in amies transport medium with (a/wc) and without charcoal (a/-c) are in table 2. from the results obtained it was established that h. paragallinarum survived better on amies transport medium without charcoal. all further experiments were therefore conducted in the absence of charcoal. the survival of h. paragallinarum in amies transport swabs without charcoal at different temperatures for various days was tested (table 3). viable bacteria could be isolated after 9 days in amies transport medium kept at 4 °c and 37 °c. at 25 °c and room temperature viable bacteria could be isolated for up to 8 days of storage. to explore the possibility of further enhancing the viability of isolates during transport, amies transport medium without charcoal was supplemented with different combinations of the supplements required by h. paragallinarum for growth (a/nad, a/t, a/a-o, a serum and a/supp; table 1). these media were tested through inoculation with h. paragallinarum and subsequent storage at four different temperatures. a comparison of the maximum survival time can be seen in table 4. with storage at 4 °c, the best results were obtained in the medium that received the full compliment of supplements. it was found that viable bacteria could be recovered after a total of 18 days. viable bacteria could be found in all of the media tested for at least 15 days at 4 °c. with storage at 25 °c the best results were obtained in the medium with the full group of supplements. however, the duration of survival was greatly reduced with viable bacteria only isolated from samples stored for up to 7 days. this is in sharp contrast to the survival of the bacteria for 18 days in the same medium at 4 °c (table 4). the survival pattern for h. paragallinarum stored in the different versions of the transport medium held at room temperature were very similar to the results obtained when the samples were stored at 25 °c. as in the previous cases (i.e. storage at 4 °c and 25 °c) the best results were obtained with the medium containing all of the supplements. the results obtained when h. paragallinarum was stored at 37 °c on the different versions of the medium were more similar to the results obtained when the samples were stored at 4 °c. as in all of the cases the best results were obtained with medium containing all of the supplements. these results were substantially different from the results obtained when the different media were stored at room temperature and at 25 °c. it can be seen from this data that the best survival was obtained in the medium with all of the supplements and viable bacteria were still detected in this medium after 18 days at 37 °c. to further test the effectiveness of commercial available amies transport swabs, samples obtained from zimbabwe were tested for viability. these isolates were serotyped and found to be serovar c-3. (bragg 2002). during transport of samples it is unlikely that a constant temperature can be attained. a trial experiment was therefore conducted with samples transported in different media from bloemfontein to cape town and returned back to bloemfontein during a 6-day period. all isolates transported in the different media where found to be viable after this 6-day period (table 5). r.r. bragg et al. table 4 comparison of the maximum survival time in days of h. paragallinarum in the different transport media. the figure in brackets is the percentage of isolates surviving on the last day when growth was detected temperature media 4 °c 25 °c room temperature 37 °c a/ts 9 (100 %) 8 (50 %) 8 (100 %) 9 (100 %) a/nad 15 (100 %) 6 (50 %) 7 (50 %) 15 (100 %) a/t 16 (50 %) 6 (50 %) 6 (100 %) 16 (50 %) a/o-a 15 (100 %) 5 (100 %) 6 (100 %) 16 (100 %) a/serum 15 (100 %) 6 (100 %) 7 (50 %) 15 (100 %) a/supp. 18 (100 %) 7 (100 %) 8 (100 %) 18 (100 %) table 2 growth after revival of h. paragallinarum streaked onto plates of amies transport medium with (a/wc) and without (a/-c) the addition of charcoal after 4 days’ incubation at 37 °c media number tested number showing growth confirmation by pcr a/wc 3 0 n/a* a/-c 3 1 positive * n/a = not applicable and there were no surviving colonies 96 transportation of pure cultures of h. paragallinarum for serotyping table 1 abbreviations of the different transport media evaluated for the transport of h. paragallinarum abbreviation tube or with with supplements swab charcoal supplements a/wc tube yes no none a/-c tube no no none a/ts swab no no none a/nad tube no yes 2.5 ml/l of a 1 % nad a/t tube no yes 10 ml/ thiamine hydrochloride a/o-a tube no yes 50 ml/l oleic-albumin complex (o-a) a/serum tube no yes 10 ml/l heat inactivated chicken serum a/supp tube no yes 2.5 ml/l of a 1 % nad 10 ml/l thiamine hydrochloride 10 ml/l heat inactivated chicken serum 50 ml/l oleic-albumin complex (o-a) table 3 results obtained when commercially available amies transport swabs without charcoal (a/ts) was used to store h. paragallinarum at different temperatures storage temperature days 4 °c 25 °c room temperature 37 °c 4 2/2 2/2 2/2 2/2 5 2/2 2/2 2/2 2/2 6 2/2 2/2 2/2 2/2 7 2/2 2/2 2/2 2/2 8 2/2 1/2 2/2 2/2 9 2/2 – – 2/2 10 – – – – 11 – – – – 97 r.r. bragg et al. one of the main objectives of these experiments was to establish a procedure to collect samples from clinically infected chickens from remote areas and the transport of these samples to a laboratory where h. paragallinarum can be isolated and serotyped. this was achieved during a transport simulation where samples were sent by postal service from bloemfontein to cape town and returned to bloemfontein during a 6-day period. viable samples were obtained from all transport media tested. more importantly, similar results were obtained with samples collected in zimbabwe and transported to bloemfontein for further processing; in this case viable h. paragallinarum bacteria were isolated, serotyped and found to belong to serovar c-3 (bragg 2002). the addition of supplements required for the growth of h. paragallinarum into amies transport medium without charcoal increased the storage time of h. paragallinarum in media containing full supplementation at 4 °c and 37 °c. the storage time was not increased when the supplements were added, and the tubes stored at 25 °c and at room temperature. it is not always possible to ensure that the environmental temperature of the samples can be maintained at 4 °c for 18 days during transportation, particularly under third world conditions. as h. paragallinarum was isolated in commercially available amies transport media without charcoal for up to 8 days with storage at either 25 °c or at room temperature, it is suggested that this commercially available product be used to transport samples of h. paragallinarum. it was successfully used when samples were transported from zimbabwe. when temperatures of 4 °c can be guaranteed, amies transport medium with the full range of supplements will increase the time from 9–18 days that samples can be successfully transported. references biberstein, e.l. & white, d.c. 1969 a proposal for the establishment of two new haemophilus species. journal of medical microbiology, 2:75–78. blackall, p.j. & reid, g.g. 1982. further characterization of haemophilus paragallinarum and haemophilus avium. veterinary microbiology, 7:359–367. blackall, p.j., eavers, e.l. & rogers, d.g. 1990a. proposal of a new serovar and altered nomenclature of haemophilus paragallinarum in the kume haemagglutinin scheme. journal of clinical microbiology, 135:469–474. blackall, p.j., eavers, e.l. & aus, g. 1990b. serotyping of haemophilus paragallinarum by the page scheme: comparison of the use of agglutination and hemagglutinationinhibition tests. avian diseases, 34:643–645. bragg, r.r. 2002. isolation of serovar c-3 haemophilus paragallinarum from zimbabwe: a further indication of the need for local vaccines against infectious coryza. onderstepoort journal of veterinary research, 69:129–132. chen, x., miflin, j.k., zhang, p. & blackall, p.j. 1996. development and application of dna probes and pcr tests for haemophilus paragallinarum. avian diseases, 40:398– 407. de blieck, l. 1932. a haemoglobinophilic bacterium as the cause of contagious catarrh of the fowl. veterinary journal, 88:9–13. hinz, k-h., 1973. differentiation of haemophilus species from fowls. 1. cultural and biochemical studies. avian pathology, 2:211–229. kume, k., sawata, a. & nakase, y. 1980. immunological relationship between page’s and sawata’s serotype strains of haemophilus paragallinarum. the veterinary record, 41: 757–760. kume, k., sawata, a., nakase, y. & matsumoto, m. 1983. serologicalclassification of haemophilus paragallinarum with a haemagglutinin system. journal of clinical microbiology, 17:958–964. miflin, j.k., chen, x., bragg, r.r., welgemoed, j.m., greyling, jm., horner, r.f. & blackall, p.j. 1999. confirmation that pcr can be used to identify nad-dependent and nad-independent haemophilus paragallinarum isolates. onderstepoort journal of veterinary research, 66: 55–57. page, l.a. 1962. haemophilus infection in chickens. 1. characteristics of 12 haemophilus isolates recovered from diseases chickens. american journal of veterinary research, 23:85–95. reid, g.g. & blackall, p.j. 1984. pathogenicity of australian isolates of haemophilus paragallinarum and haemophilus avium in chickens. veterinary microbiology, 9:77–82. table 5 isolation of h. paragallinarum from different transport media sent to cape town and returned to bloemfontein in different transport media over a period of 6 days media number tested number positive transport swabs 3 3 a/nad 3 3 a/t 3 3 a/a-o 3 3 a/serum 3 3 a/supp 3 3 rimler, r.b., davies, r.b. & page, r.k. 1977. infectious coryza: cross protection studies, using seven strains of haemophilus paragallinarum. american journal of veterinary research, 38:1587–1589. sawata, a., kume, k. & nakase, y. 1980. biological and serological relationships between page’s and sawata’s serotyping of haemophilus paragallinarum. american journal of veterinary research, 41:1901–1904. 98 transportation of pure cultures of h. paragallinarum for serotyping article information authors: michael p. combrink1 graham carr2 ben j. mans1 frances marais1 affiliations: 1parasites, vectors and vector-borne diseases programme, arc-onderstepoort veterinary institute, south africa 2howick veterinary clinic, howick, south africa correspondence to: michael combrink postal address: private bag x05, onderstepoort 0110, south africa dates: received: 18 june 2012 accepted: 17 sept. 2012 published: 06 dec. 2012 how to cite this article: combrink, m.p., carr, g., mans, b.j. & marais, f., 2012, ‘blocking babesia bovis vaccine reactions of dairy cattle in milk’, onderstepoort journal of veterinary research 79(1), art. #491, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.491 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. blocking babesia bovis vaccine reactions of dairy cattle in milk in this original research... open access • abstract • introduction • materials and methods    • animals    • vaccination    • block treating vaccine reactions    • monitoring reactions to babesia infection    • molecular genotyping of the babesia bovis vaccine strain    • ethical considerations • results • trustworthiness • discussion • conclusion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ the use of 1.16 mg/kg (one third) of the recommended dose of diminazene aceturate, administered indiscriminately to cattle on day seven of the unfrozen babesia bovis and babesia bigemina bivalent live blood vaccine reaction, was an infection and block treatment method of immunisation used successfully with no known adverse effect on the parasites or the development of protective immunity. continuing with this practice after replacement of the unfrozen vaccine with deep-frozen monovalent b. bovis and b. bigemina live blood vaccines resulted in reports of vaccine failure. laboratory investigation indicated the harmful effect of block treatment in preventing the development of durable immunity against b. bigemina as opposed to the much lesser effect it had on b. bovis. consequently the practice was no longer recommended. a b. bovis vaccination attempt aimed at controlling the disease of dairy cows in milk (n = 30) resulted in 20% fatalities during the expected vaccine reaction period. the practice of block treating b. bovis was therefore reinvestigated, this time in a field trial using dairy cattle in milk (n = 11). using 0.88 mg/kg (one quarter) of the recommended dose of diminazene administered on day 12 of the b. bovis vaccine reaction resulted in only two animals (n = 5) testing ≥ 1/80 positive with the indirect fluorescent antibody test (ifat) although parasites could be demonstrated in three. in the untreated control group, by contrast, five of the vaccinated animals (n = 6) tested ≥ 1/80 positive with ifat and parasites could be demonstrated in all. the unsatisfactory outcome obtained in this study, combined with that of the earlier investigation, indicated that there are more factors that influence successful vaccination than previously considered. it is therefore concluded that block treatment of the live frozen south african cattle babesiosis vaccines reactions is not recommended. introduction top ↑ babesia bovis is an arthropod-transmitted pathogen in cattle, rated as being the tick-borne disease with the biggest economic impact on cattle farming in the tropical and subtropical areas of africa, asia, australia and south america (bock et al. 2004). the prevalence of the disease is closely related to the tick vectors’ distribution, which is confined by humidity and temperature (gothe 1967; yeoman & walker 1967). in south africa, the disease is transmitted by the one-host tick, rhipicephalus (boophilus) microplus, naturally inhabiting the savanna climatic regions of wooded grasslands that are also used as cattle pasture (walker et al. 2003). disease control by tick eradication is not a solution in those areas where the vector is already well established (de vos 1979). an alternative, more acceptable practice is to limit the degree of tick control, thereby allowing natural endemic stability to develop. however, relying on this as a realistic approach is made impossible by the multiple tick-borne disease ecosystems of southern africa (bezuidenhout 1985; perry et al. 1985). the control of multi-host ticks invariably also affects the one-host ticks and ultimately has a negative effect on the epidemiology of bovine babesiosis through reduction of the tick population to the extent that endemic stability cannot be maintained (de waal 1996). the advisable approach to managing the disease would therefore be to integrate the strategic use of acaricides and the application of the appropriate vaccine, which should also prove the most cost-efficient method of control (de waal & combrink 2006). for many years, the only available babesiosis vaccine was a chilled bivalent live blood vaccine containing parasites of both b. bovis and babesia bigemina. as calves up to nine months of age usually show highest resistance to the disease and seldom develop serious symptoms, it is generally recommended that vaccination be restricted to this age group (trueman & blight 1978). despite this recommendation, a large proportion of vaccines are administered to older, more susceptible animals. although the vaccine strains used were attenuated, the shortcoming of the chilled vaccine was the fatal reactions it caused in older cattle if left untreated. for this reason a method of infection and block treatment was investigated, whereby one third (1.16 mg/kg) of the recommended dose of diminazene aceturate is administered indiscriminately to cattle on day seven after inoculation of the vaccine, which proved to be quite successful with no known adverse effects to the parasites or to the development of protective immunity (de waal 1996). table 1: babesia bovis vaccine reactions of dairy cattle in milk. in april 1998, the bivalent chilled vaccine was replaced by deep-frozen monovalent live blood vaccines of b. bovis and b. bigemina. the block treating method was continued until reports of vaccine failures associated with the use of this practice prompted a reinvestigation of this approach. it was found that treatment of the frozen b. bigemina vaccine reactions with diminazene on day seven at dose levels as low as 0.35 mg/kg (one tenth the recommended dose) will still kill all parasites (combrink & troskie 2004) and thus prevent the development of a durable immunity. although treatment at 1.16 mg/kg (one third the recommended dose) was not as harmful to the b. bovis parasites, it was found that the prepatent period of the vaccine reaction was extended for a longer period. due to the detrimental effect of diminazene on the vaccine parasites as well as various other factors, such as the degree of natural resistance of different cattle breeds and individual animals to babesia parasites, the infectivity of frozen vaccine being less predictable than that of unfrozen vaccine (due to demise of parasites during freezing and thawing), the non-conformance of diminazene preparations to manufacturer’s label claims (tettey et al. 2002) and the accuracy of the drug dose administered, all influencing successful immunisation, the block treating of b. bovis and b. bigemina vaccine reactions was no longer recommended. in renewed efforts aimed at specifically controlling b. bovis, veterinarians have again started to vaccinate older cattle and have found that, although not completely safe, the risk involved in losing a few animals due to vaccination outweighs the risk of losing large numbers of animals due to natural disease outbreaks. vaccination of 2134 nutritionally challenged adult beef cattle and 30 normally rationed adult dairy cows in milk with the b. bovis vaccine resulted respectively in 16 (0.75%) and six (20%) fatalities during the two to four weeks following inoculation (nick fischer pers. comm., february 2011). it has been suggested (kuttler, zaugg & johnson 1984) that environmental and other stressors could compromise natural resistance to disease, but no published information could be found of this being of significance under field conditions. however, it is known that the effect of stressors is even more pronounced in the case of intensive lactating dairy cattle (johnson & vanjonack 1976), which may explain the higher fatality rate experienced in this group. having thus observed a specific need for use of the b. bovis vaccine in milk-producing animals, and considering that laboratory block treating of this vaccine’s reaction was found not as completely harmful to the parasites, we decided to reinvestigate the old practice. materials and methods top ↑ animals eleven non-pregnant friesian dairy cows, four to nine years old, between 54 and 83 days in milk, with body condition scores of 1.5 to 2.5 out of five, and which had no antibody titres to b. bovis antigen in the ifa test (gray & de vos 1981; joyner et al. 1972), were used in the field trial to determine the effect of block treating b. bovis vaccine. animals were randomly divided into two groups of which one consisted of six and the other of five cows each (table 1). they were dipped for ticks before and for the duration of the experiment. frost and cold weather experienced during this period restricted tick activity to a minimum. vaccination the commercial frozen live-blood vaccine (de waal 1996; de waal & combrink 2006) used in this study was the b. bovis ‘s’ strain (de vos 1978; callow, mellors & mcgregor 1979). the vaccine was dispatched from onderstepoort on dry ice and directly before use thawed in water at 37 °c. all 11 cows were each vaccinated with 1 ml of the vaccine intramuscularly and were then returned to the farm’s normal dairy herd management system. block treating vaccine reactions previously, vaccine reactions were block treated on day seven of the vaccine reaction, using one third (1.16 mg/kg) of the recommended dose of diminazene. this was done to correspond with the five to seven day pre-patent reaction period of the b. bigemina parasite used in the old bivalent, unfrozen vaccine. the practice was retained even after replacement of the bivalent, unfrozen vaccine with deep-frozen, monovalent babesiosis vaccines, in the event that both are used concomitantly. considering that the pre-patent period of the b. bovis vaccine applied in the field ordinarily varies between 9 to 19 days (combrink personal observation) the parameter for block treatment in this study was moved from day seven to 12 after inoculation. the rationale for treating later was to increase the potential of parasite survival, but still allow for the treatment to be effective in case of an early well-advanced reaction.very few farmers have access to animal weighing facilities and it has been observed by author graham carr that the weight estimation of clients in his practice is on average, an overestimate rather than an underestimate. therefore, in the absence of determining a specific normal error in weight estimation for this study, and which may not be objectively applicable to all farmers, it was decided to use one quarter (0.88 mg/kg) of the recommended dose of diminazene, found effective in controlling the vaccine parasite reaction (combrink & troskie 2004). the rationale for using a one quarter dose was that this would help restrict the total dose administered to less than one third, should the weight of the animal be greatly overestimated by 32%. for the sake of uniformity, a commercially obtainable weight measuring tape (rondo™) was used. monitoring reactions to babesia infection rectal temperatures, blood smears and packed cell volumes (pcv) of experimentally infected cattle were monitored daily. antibody titres for babesia were determined in sera collected before, and at 17, 24 and 30 days after vaccination, using the indirect fluorescent antibody test (ifat) (gray & de vos 1981). the ifat was until recently listed in the world organization for animal health manual of diagnostic tests and vaccines for terrestrial animals as being suitable for the diagnosis of disease within a local setting, but has now been largely replaced by the enzyme-linked immunosorbent assay (elisa) as the test of choice for babesia spp., mainly because of objectivity in interpretation of results. however, the elisa has not yet been validated for use at the onderstepoort veterinary institute and the ifat is still used for research and serological surveys in south africa, where only titres of dilutions 1/80 or higher are considered as positive (bessenger & schoeman 1983). the infectivity of the vaccine strain in cattle was determined by demonstrating the babesia parasites in stained blood smears or positive seroconversion following vaccination. molecular genotyping of the babesia bovis vaccine strain total genomic dna was extracted from 200 µl of whole blood collected in ethylene-diaminetetraacetic acid (edta) tubes using automated magnapure protocols according to established diagnostic procedures and eluted in 100 µl of elution buffer (mans et al. 2011). genotypic analysis of b. bovis samples were conducted using the 1bf (tgtgttaatgtaactcagcccg) and 2br (aaagcctgttagttgatggacc) primers for the bv80 gene as previously described (lew et al. 1997a, 1997b). general polymerase chain reaction (pcr) reaction used 2.5 µl of genomic dna, 25 µl greentaq (fermentas), 20.5 µl pcr-grade water and 1 µl of each primer at 10 pmoles final concentration. the pcr protocol consisted of denaturation at 95 °c for 2 min, 45 cycles of denaturation at 95 °c (30 s), annealing at 58 °c (30 s) and extension at 72 °c (1.5 min), followed by a final extension at 72 °c (7 min). the amplified products were analysed by electrophoresis on a 1.2% agarose gel using standard conditions. blood from the b. bovis vaccine production stock material (s22) was used as positive control and blood from a bovine kept under quarantined tick-free conditions as well as blood from the b. bigemina vaccine production stock material were used as negative controls. the positive control gave a single amplified pcr product of ~700 bp, whilst both negative controls yielded no pcr products. blood collected from the animals before vaccination tested negative. ethical considerations ethical approval for the study was obtained from the animal ethics committee of the arc-ovi (ref. ov14/02/p001). animal experimental and welfare work was performed by a veterinarian (dr graham carr) registered with the savc. results top ↑ all six animals in group one that only received the vaccine (table 1) were found b. bovis positive on blood smear examination. five tested ≥ 1/80 positive on ifat. two experienced febrile reactions in excess of 40 °c, occurring on days 16 and 17 post vaccination for the one animal and between days 15 to 18 for the other, who also required treatment on day 18 due to an increasing parasitaemia and severe anaemia (pcv 14). molecular genotyping of both animals experiencing febrile reactions confirmed the presence of the b. bovis vaccine strain, whilst no other genotypes were detected. three of the five animals in group two that received the vaccine and 0.88 mg/kg diminazene administered on day 12 post inoculation (table 1) were found b. bovis positive on blood smear examination. two tested ≥ 1/80 positive on ifat. one experienced a very mild febrile reaction peaking at 39.5 °c on day 18, and b. bovis molecular genotyping of this animal indicated only the vaccine parasite being present. one of the blood smear positive animals also had a coincidental anaplasma marginale relapse infection, which left untreated was instrumental in the anaemia (pcv 18) experienced. the parasite prepatent period average of 15.5 ± 5.9 days obtained for group one (n = 6) was not dissimilar to the 17.3 ± 8.0 days of group two (n = 3). trustworthiness top ↑ laboratory testing of samples was performed in compliance with sanas iso/iec 17025:2005 accreditation requirements for a molecular and serology diagnostic laboratory (ref. v0017) and daff 001/daff 002 molecular (pcr) and serology section requirements for an approved veterinary laboratory (ref. daff-30). discussion top ↑ field vaccination of 30 cows in milk with b. bovis resulted in six (20%) fatalities occurring during the expected vaccine reaction period. blood smear examination at the time revealed the cause as b. bovis but it was not confirmed whether it was from the vaccine strain or a field isolate (nick fischer pers. comm., february 2011). in another b. bovis vaccination attempt by fischer, blood was then collected from a sick cow, which when tested with pcr showed the only parasite genotype present to be that of the b. bovis vaccine. in this study, although only six animals were used, vaccination of the dairy cows resulted in one (17%) of the six animals developing clinical disease that required treatment, which also showed that unmonitored vaccination of dairy cattle in milk is not advisable.in the present study, six of the 11 cows used tested ≥ 1/40 for b. bigemina on ifat prior to vaccination with b. bovis and during the course of the study circulating parasites for b. bigemina could be demonstrated microscopically on blood smears in three of these animals as well as in two of the five that tested completely negative. in 2010 combrink et al. found that only 53% field cattle (n = 260) and 58% laboratory cattle (n = 12), which had previously been infected with a b. bigemina field isolate, seroconverted (≥ 1/80) after b. bovis vaccination, yet b. bovis parasites could be demonstrated microscopically in all of the laboratory cattle and when challenged with a b. bovis field isolate this group did not show clinical reactions compared with an unvaccinated control group (n = 6). therefore, considering this effect that b. bigemina can have on b. bovis vaccine serology and in the present study having found only five of the six animals in group one positive on ifat, although all were blood smear positive, it becomes clear that the microscopic demonstration of the parasite was the only reliable way of validating exposure of animals to the b. bovis vaccine organism. conclusion top ↑ in this study, then, failing to demonstrate b. bovis parasites on blood smears in two of the five cows of group two is indicative of the harmful effect that block treatment can have on the b. bovis vaccine organism. comparing the unsatisfactory results from this study with those of combrink and troskie (2004), it is clear that there are more factors that influence successful vaccination than what was then considered by the authors. probably the most obvious factors to be added is the effect that existing b. bigemina infection in an animal has on subsequent infection with b. bovis (legg 1935; wright et al. 1987) and the differences between animal husbandry practised during laboratory and field experimentation. consequently, it can only be endorsed that block treatment of the live frozen south african cattle babesiosis vaccines reactions is not recommended. acknowledgement top ↑ we thank bruce allwood for making his farm and cattle available for this study. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions m.p.c. (arc-onderstepoort veterinary institute) was the project leader. m.p.c. (arc-onderstepoort veterinary institute) and g.c. (howick veterinary clinic) were equally responsible for experimental and project design. m.p.c. (arc-onderstepoort veterinary institute) performed laboratory microscopy and experimental data analysis. g.c. (howick veterinary clinic) performed all animal experimental and welfare work (collection and preparation of samples, monitoring and treatment of animals). b.j.m. (arc-onderstepoort veterinary institute) performed pcr work. f.m. (arc-onderstepoort veterinary institute) performed serology diagnostic work. m.p.c. (arc-onderstepoort veterinary institute) wrote the manuscript and b.j.m. 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challenge with virulent babesia bovis’, infection and immunity 55, 364–368. pmid:3542832 yeoman, g.h. & walker, j.b., 1967, ‘the ixodid ticks of tanzania’, commonwealth institute of entomology, london. article information authors: mujeeb r. fazili1 hiranya k. bhattacharyya1 manzoor u.r. mir2 abdul hafiz1 noore a. tufani1 affiliations: 1teaching veterinary clinical complex, faculty of veterinary sciences and animal husbandry, sher-e-kashmir university of agricultural sciences and technology of kashmir, india2division of veterinary biochemistry, faculty of veterinary sciences and animal husbandry, sher-e-kashmir university of agricultural sciences and technology of kashmir, india correspondence to: mujeeb fazili postal address: 78-hig (lane 6, green park) colony, bemina, srinagar, kashmir 190018, india dates: received: 11 nov. 2013 accepted: 25 june 2014 published: 12 nov. 2014 how to cite this article: fazili, m.r., bhattacharyya, h.k., mir, m.u.r., hafiz, a. & tufani, n.a., 2014, ‘prevalence and effect of oxytetracycline on congenital fetlock knuckling in neonatal dairy calves’, onderstepoort journal of veterinary research 81(1), art. #710, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.710 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prevalence and effect of oxytetracycline on congenital fetlock knuckling in neonatal dairy calves in this original research... open access • abstract • introduction • material and methods • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ musculoskeletal system deformities were observed in 24 (34.3%) of 70 neonatal dairy calves that presented with different congenital abnormalities. among them, 19 calves (27.1%), the majority of which were crossbred jersey calves of either gender with mean (± s.e.) body weight 22.00 kg ± 1.17 kg and aged 7.11 ± 1.16 days, were presented for treatment of congenital knuckling. five of the knuckling calves had additional concurrent congenital conditions and were excluded from the present study. all of the remaining 14 calves showing moderate, bilateral fetlock knuckling had a wooden or polyvinyl chloride (pvc) splint applied to the palmar or plantar aspect of the affected limbs. all of the animals received a dose of the analgesic tolfenamic acid intramuscularly, and were randomly allocated to two equal groups. calves of group i additionally received oxytetracycline (20 mg/kg intravenous daily for 3 days). the condition resolved satisfactorily in 83.3% and 80.0% calves from the two groups, respectively. the left and right fetlock angle (mean ± se) reduced significantly (p ≤ 0.01) from 50.57° ± 4.20° to 4.00° ± 2.27° and 48.71° ± 2.37° to 5.33° ± 3.03°, respectively in animals of group i. in group ii calves, the angles showed reduction from 50.86° ± 2.94° to 4.20° ± 2.75° and from 48.71° ± 3.14° to 6.80° ± 3.34°, respectively. from the present study, it was concluded that bilateral moderate fetlock knuckling in the neonatal dairy calves can be managed satisfactorily with early application of splints. supplementary use of oxytetracycline at repeated doses of low toxicity had only a marginally beneficial effect. introduction top ↑ congenital contracted flexor tendon, observed most frequently as flexion of the metacarpophalangeal or metatarsophalangeal joint within 1 or 2 weeks of birth, is a common defect in numerous breeds of cattle (leipold, hiraga & dennis 1993; simon et al. 2010). aetiologic origins include inherited factors, in utero nutrition, malposition and the foetus being too large relative to the dam (anderson, desrochers & st jean 2008; ferguson 1997). the diagnosis can easily be made by the abnormally flexed position of the affected limb. the deformity may be mild (if the calf is able to walk on the feet but the heels do not contact the ground), moderate (if the animal is able to remain standing on the tip of the toe with the dorsal aspect of the hoof lying perpendicular to the ground but walks on the dorsal aspect of the pastern, fetlock or carpus) or severe (if the affected animal stands and walks on the dorsal aspect of the pastern, fetlock or carpus). severe flexural deformities are also accompanied by arthrogryposis, involvement of multiple limbs and the head and neck, and severe carpal deformities. the animals with flexural deformities may be unable to nurse, so failure to acquire passive immunity transfer may be a complicating factor (weaver, st jean & steiner 2005). chronic deformity may lead to skin ulceration on the dorsum of the fetlock and, subsequently, to septic arthritis.successful treatment of flexural deformities depends on the site and severity of the deformity and on the appropriate use of medical, physical and, possibly, surgical therapy. moderate cases are routinely treated by using a bandage, splint or cast and by providing analgesia (anderson et al. 2008). splints can be very useful for restoring the limb to normal alignment and orientation. in a new-born foal, a single high dose of oxytetracycline administered intravenously (iv) at 44 mg/kg together with the application of the splints has been reported to result in a moderate decrease in the metacarpophalangeal joint angle (madison et al. 1994). although oxytetracycline (3.0 g in 250 ml physiological saline) iv has been given in calves, its routine use at such a high single dose is not recommended because of its high nephrotoxicity (ducharme 2004). anderson et al. (2008) reported some success with lower doses of oxytetracycline but the details of dose and frequency of administration were not provided. the present study was planned to determine the prevalence of congenital fetlock knuckling and to ascertain the benefit of using a low-toxicity dose of oxytetracycline continuously for 3 days supplementary to the application of splints in neonatal calves with this type of moderate bilateral deformity. material and methods top ↑ seventy newborn calves with different congenital external deformities were presented to the teaching veterinary clinical complex, faculty of veterinary sciences, sher-e-kashmir university of agricultural sciences & technology of kashmir, shuhama, srinagar, kashmir, india by rural owners for treatment over a period of 3 years (2006–2008). knuckling of the distal limb joints was detected in 19 of these calves. fourteen (14) of the calves with moderate bilateral fetlock knuckling and without any additional deformity were included in this treatment trial. forelimbs (figure 1) were involved in 12 calves, and in two calves, hindlimbs (figure 2) were affected. the physiological parameters recorded included respiration rate (rr), heart rate (hr) and rectal temperature (t). the blood samples were assessed for packed cell volume (pcv), haemoglobin (hb) and total plasma protein (tpp). figure 1: photograph of a neonatal calf with moderate fetlock knuckling of the forelimb. figure 2: photograph of a neonatal calf with moderate fetlock knuckling of the hindlimb. for assessment of the angle at the affected fetlock joints, the calves were restrained without any premedication in lateral recumbency. the foot of the limb placed on the upper side was extended slightly and, using a protractor, the angle formed between a central longitudinal arbitrary line on the lateral aspect of the metacarpal or metatarsal bone with that of the central line of the corresponding foot was recorded (sirin et al. 2014). the calves were then restrained on the contralateral side and the fetlock angle recorded in the same manner. after injecting xylazine (inj. xylaxin, indian immunological ltd.) 0.05 mg/kg intramuscularly (im) or diazepam (inj. calmpose, ranbaxy laboratories ltd. india), 0.1 mg/kg iv, the animals were restrained in lateral recumbency. a splint made from an appropriately sized well-padded wooden or polyvinyl chloride (pvc) pipe cut into a one-third or half semi-tubular shape was applied to the palmar/plantar aspect of the affected limbs of all the calves from the foot, excluding the claw, up to and including the knee/hock joint. all the calves were given a single dose of 2.0 mg/kg im tolfenamic acid (inj. maxtol, intas pharmaceuticals limited, india) as an analgesic. the animals were then allotted randomly to one of the two equal groups (n = 7), group i and group ii. the animals of group i were given oxytetracycline (inj. terramycin, pfizer india ltd.) at 20.0 mg/kg. the calculated dose of oxytetracycline was added to 250 ml of normal saline and infused iv daily for three consecutive days. the first dose was given at the hospital and the calf discharged. the remaining two doses were administered by the field veterinarian at the owner’s premises. the animals of group ii received no additional medication. the owners were advised to assist the calves in standing and suckling. they were also directed to monitor the proper position of the splint, and to apply three to four additional layers of bandage after carefully stretching the limb every alternate day. the owners were also instructed to remove the splints on day 10 and to encourage the animals to stand and walk for two more days before presentation at the hospital for further evaluation. the proportion of the calves with knuckling among the neonates showing musculoskeletal congenital defects was calculated as a percentage. the mean ± s.e. values of the fetlock angles were compared between the groups before treatment and within groups after 12 days or 20 days following institution of the treatment. the significance was calculated by t-test at p ≤ 0.01. results top ↑ musculoskeletal system deformities accounted for 34.3% (n = 24) of the 70 neonatal dairy calves that were presented with different congenital abnormalities (table 1). nineteen calves, ten male and nine female showed knuckling; they contsituted 27.1% of the total number of calves presented with congenital deformities. the majority (16) of these calves were crossbred jersey calves. two crossbred holstein friesian calves and one local non-descript calf also exhibited the condition. the body weight (mean ± s.e.) and the age of the knuckling calves was 22.00 kg ± 1.17 kg (range: 14 kg – 35 kg) and 7.11 ± 1.16 days (range: 1–25 days), respectively. all the calves except one were born unassisted. the dams of these calves had a mean ± s.e. age of 6 ± 0.62 years (range: 3–12 years) and their parity was 2.53 ± 1.17 (range: 1–5). the owners reported that such abnormalities were not encountered in the earlier calves of these dams. table 1: prevalence of congenital fetlock knuckling in neonatal dairy calves. forelimbs alone were affected in the greatest number (left = 16, right = 14) of the calves, in which bilateral knuckling (n = 14) outnumbered unilateral (n = 2). bilateral hindlimb knuckling was present in two calves. all four limbs were affected in one 3-day-old 16.0 kg crossbred jersey female calf. the remaining five calves suffered from additional congenital conditions. four had single additional defects (arthrogryposis = 2, atresia ani = 1 and brachygnathism = 1) but one had multiple (arthrogryposis, kinked neck and tail) additional defects. only fetlock joints were involved in most (n = 16) of the calves (14 forelimb and 2 hindlimb). in two of these animals the carpal joints were also simultaneously involved. carpal joints only were affected in another calf. the mean ± s.e. values of all the physiological and haemato-biochemical parameters were within the normal range for the neonatal calves; however, two animals in each group showed mild tachycardia (>150 beats/min). whilst the animals were comfortably restrained in lateral recumbency, no difficulty was encountered in recording the fetlock joint angle. in calves of group i, the mean ± s.e. angle in the left and the right fetlock joints was 50.57° ± 4.20° and 48.71° ± 2.37° , respectively. in group ii calves, the deflection angle was 50.86° ± 2.94° and 48.71° ± 3.14°, respectively. the difference in the angles of the corresponding joints in calves of the two groups was not significant. all the calves from both groups except one from group i maintained the splints properly. one of the calves from group ii was lost to follow-up. another owner had removed a wooden splint from the left leg of his calf on day 7 due to the development of pressure sores. these three calves were excluded from the study. the outcome of the treatment in animals of both the groups is presented in table 2. in animals of group i, 9 out of 12 limbs of the affected six calves had resolved completely when examined 12 days after institution of the treatment. in these calves, the left and the right fetlock angles were 4.00° ± 2.27° and 5.33° ± 3.03°, respectively (table 3). reduction in the angles, when compared to the pre-treatment values, showed high statistical significance (p ≤ 0.01). the affected feet showed full weight-bearing and no lameness was detected on progression. deformity in one of the legs in three calves had reduced to 20°, 24° and 35°, respectively. in two of these calves, regular reinforcement of the splints had not been undertaken. splints were reapplied for a further week. one of them recovered fully (increasing the success to 10/12; 83.3%) but the remaining two animals still had 15° and 20° angle deformity on removal of the splints. they subsequently continued to show mild bending of the fetlock and lameness despite weight-bearing. table 2: outcome of treatment in neonatal dairy calves with moderate fetlock knuckling. table 3: mean ± se values of fetlock joints (degrees) in knuckling calves. in animals of group ii, 7 out of 10 limbs of the six calves healed completely. in these calves, the left and the right fetlock angles were 4.20° ± 2.75°and 6.80° ± 3.34°, respectively. in these calves, the reduction in the angles from pre-treatment values also showed high statistical significance (p ≤ 0.01). one of the remaining calves with mild deflection (15°) of both the limbs showed complete cure in one limb without subsequent reapplication of the splints. the other calf with a 20° angle deviation of one limb did not respond favourably during the next week. at the end of the study period, the deformity had fully resolved in 8/10 (80.0%) limbs in the six calves. discussion top ↑ congenital anomalies have been estimated to occur in 0.2% to 5.0% of calves (distl & bahr 2005; priester, glass & waggoner 1970; saperstein 2002). a prevalence of 6.37% among a total of 361 cattle presented for treatment of different congenital and acquired ailments was previously recorded from kashmir (fazili & syed 2003). from kashmir it has been reported that 6.37% of the total 361 cattle presented for treatment comprised of congenital abnormalities (fazili & syed 2003). the musculoskeletal system was involved in 34.3% of the calves presented for treatment of external congenital defects. such defects were previously observed in only 24.0% of the total number of calves with congenital abnormalities (leipold, huston & dennis 1983; samad 2002). malformations of the distal limbs are among the most frequent congenital anomalies found in humans and animals (fazili & syed 2003; leipold & dennis 1987; talamillo et al. 2005). most of the calves in this study were crossbred jerseys, which are currently the most predominant cattle breed in kashmir.the previous siblings of all the presented affected calves were reportedly normal at birth. all of the affected calves were single births. the aetiologic origins of contracted flexor tendons include inherited factors, in utero nutrition, malposition, and overcrowding caused by the size of the foetus relative to the dam (anderson et al. 2008; ferguson 1997). five of the 19 calves with knuckling showed arthrogryposis, atresia ani, brachygnathism, kinked neck and kinked tail as additional defects. according to one earlier report, contracted tendons may occur together with other congenital abnormalities such as cleft palate, dwarfism and arthrogryposis (anderson et al. 2008). the number of male calves with contracted flexors in the present study was slightly higher (53.2%) than the females (47.4%). in contrast to this finding, more females than males have been reported in a previous study involving only six calves (simon et al. 2010). the mean values of the various physiological parameters recorded in the present study’s calves with knuckling of the fetlock were all in normal range (piccione et al. 2010). mild tachycardia recorded in two calves of both groups could be attributed to the distress experienced by these calves whilst trying to get up and move. most of the calves with contracted flexors were presented within the first week of their life and had not developed any complications. forelimbs were involved in the majority of the calves with contraction of the flexor tendons. this finding is in accordance with earlier reports (ferguson 1997; krishnamurthy 1993; verschooten et al. 1969). most of the calves showed bilateral defects, as also reported by weaver et al. (2005). goniometry is a simple, inexpensive, reliable, objective and non-invasive method (govoni et al. 2012). for the first time, the fetlock joint angle in calves with contracted flexors has been recorded. the goniometric measurements of limb joints only proximal to the fetlock joints in healthy, awake holstein friesian calves restrained in lateral recumbency (sirin et al. 2014) and standing sheep (govoni et al. 2012) were recently reported. in the calves of the present study, the angle of the fetlock deflection varied from 30° to 65°. according to verschooten et al. (1969), the degree of contracture varies greatly. the mean angle of the corresponding sides in calves of both the groups was similar, thus making the comparison of the outcome valid. the application of the splints was easy whilst the animals were properly sedated. use of diazepam or xylazine with some muscle relaxant effect (thurmon, tranquilli & benson 1996) was helpful in stretching the contracted tendons. the purpose of splints is to align the limb so that the animal’s body weight can stretch the tight tendons and ligaments; pvc is more frequently used than wooden splints. good quality pvc piping is not only light but also has a concave inner surface that fits well to the contour of the limb. according to ferguson (1997), they are simple and effective in calves. wooden splints, although light in weight, may get dislodged or lead to pressure sores due to their flat contact surface. the toes are left un-bandaged to support some weight and to further stretch the tendons. it has been advised that the splints should be changed on a daily basis and applied with enough tension to produce a non-contracted limb (ferguson 1997). in the present study, the owners were not directed to change the splints frequently due to fear of improper reapplication by them, but the need for repeated reinforcement and careful monitoring was stressed. one of the calves of group ii developed pressure sores under the splint. scrupulous management of splints in neonates ensures avoidance of skin necrosis at potential pressure points (weaver et al. 2005). non-steroidal anti-inflammatory drugs (nsaids) provide analgesia to the calf and are useful for decreasing the pain associated with stretching of the contracted soft tissue caused by weight-bearing, passive stretching exercises, splints or casts (anderson et al. 2008). only a single dose of an nsaid (tolfenamic acid) was used to avoid accentuation of the nephrotoxic effects of oxytetracycline. the mechanism whereby oxytetracycline exerts its effect is unknown but it is most likely associated with a muscle relaxant effect (madison et al. 1994). the safe dose of oxytetracycline in cattle is 6 mg/kg – 11 mg/kg body weight (papich & rivière 2009). according to lairmore et al. (1984), tetracyclines should only be administered to cattle at recommended doses, to avoid problems of nephrotoxicosis. oxytetracycline and tetracycline given to young animals can cause bones and teeth to become yellow, brown or grey, and high doses and long-term administration may delay bone growth and healing (trent & redic-kill 1997). in the present study, a high single dose of oxytetracycline was not used, to avoid toxicity. following administration of oxytetracycline, kidney function tests could not be conducted. it was not possible to ascertain the discolouration of teeth and deleterious effects on bone growth and healing, as they require a longer post-administration period to develop. most flexural limb deformities resolve with persistent nonsurgical management (anderson et al. 2008). the results of the present study indicated that moderate fetlock knuckling resolved completely in most (80.0%) of the calves by proper application of splints. only a marginal increase (3.3%) in the positive outcome was noted with oxytetracycline administration at a low-toxicity dose. considering the nephrotoxic potential of high doses of oxytetracycline in neonatal calves, the benefit of its use in knuckling calves needs to be confirmed. proper application and maintenance of the splints for 10 days was sufficient for managing most of the neonatal calves presented with moderate fetlock knuckling. according to anderson et al. (2008), treatment of congenital flexural deformities should be initiated soon after recognition of the problem. as the animal gets older the contracted tissues become less responsive. conclusion top ↑ from this study it was concluded that:• forelimb bilateral knuckling is one of the most prevalent congenital musculoskeletal abnormalities affecting neonatal dairy calves. • most neonatal calves with moderate fetlock knuckling that present early for treatment can be managed satisfactorily with the proper application of the splints. • use of oxytetracycline at a low-toxicity dose (20 mg/kg iv) daily for three consecutive days has a small additional beneficial effect in managing moderate fetlock knuckling. additional studies to ascertain the real benefit of using potentially toxic doses of oxytetracycline in neonatal calves with congenital knuckling are needed before continuing its routine use. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.r.f. (sher-e-kashmir university of agricultural sciences and technology of kashmir) conceived the idea, undertook clinical management and assessment of the outcome of the cases, wrote and revised the manuscript. h.k.b. (sher-e-kashmir university of agricultural sciences and technology of kashmir) assisted m.r.f. in clinical management and evaluation of the outcome of the calves with fetlock knuckling. m.u.r.m. (sher-e-kashmir university of agricultural sciences and technology of kashmir) conducted biochemical evaluations of the animals. n.a.t. (sher-e-kashmir university of agricultural sciences and technology of kashmir) recorded anamnesis, conducted haematological evaluation of the animals and statistical analysis of data. a.h. 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(ed.), large animal internal medicine, 3rd edn., pp. 1465–1515, mosby, st. louis. simon, s., william, m.b.j., rao, g.d., sivashanker, r. & kumar, r.s., 2010, ‘congenital malformations in ruminants and its surgical management’, veterinary world 3, 118–119. sirin, o.s., çelik, m.t., özmen, a. & avki, s., 2014, ‘measurements of normal joint angles by goniometry in calves’, veterinary and comparative orthopaedics and traumatology 27, 120–123. http://dx.doi.org/10.3415/vcot-13-04-0046 talamillo, a., bastida, m.f., fernandez-teran, m. & ros, m.a., 2005, ‘the developing limb and the control of the number of digits’, clinical genetics 67, 143–153. http://dx.doi.org/10.1111/j.1399-0004.2005.00404.x thurmon, j.c., tranquilli, w.j. & benson, g.j., 1996, ‘preanesthetics and anesthetic adjuncts’, in lumb & jones’ veterinary anesthesia, 3rd edn., pp. 183–209, 186–194, williams & williams, baltimore. trent, a.m. & redic-kill, k.a., 1997, ‘clinical pharmacology’, in p.r. greenough (ed.), lameness in cattle, 3rd edn., pp. 56–70, w.b. saunders company, philadelphia. verschooten, f., de moor, a., desmet, p., watte, r. & gunst, o., 1969, ‘surgical treatment of congenital arthrogryposis of the carpal joint associated with contraction of the flexor tendons in calves’, veterinary record 85, 140–171. http://dx.doi.org/10.1136/vr.85.6.140 weaver, a.d., st jean, g. & steiner, a., 2005, bovine surgery and lameness , 2nd edn., blackwell publishing ltd, oxford. http://dx.doi.org/10.1002/9780470751138 reviewer acknowledgement open accesshttp://www.ojvr.org 189 page 1 of 1 onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of onderstepoort journal of veterinary research: abdalla latif alan walker amanda lochner andrew mercer andrew potts anita michel annemarie avenant-oldewage annemarie labuscagne arthur spickett banie penzhorn basil allsopp bernard faye braam bezuidenhoud david gray dionne crafford dirk berkvens dmitry apanaskevich estelle venter evelyn madoroba flip van schalkwyk francois malan fred potgieter fredrick kivaria fritz huchzermeyer geoffrey fosgate gert venter gift matope heloise heyne henriette van heerden henrik christensen in an effort to facilitate the selection of appropriate peer reviewers for onderstepoort journal of veterinary research, we ask that you take a moment to update your electronic portfolio on www.ojvr.org for our files, allowing us better access to your areas of interest and expertise, in order to match reviewers with submitted manuscripts. if you would like to become a reviewer, please visit the journal website and register as a reviewer. to access your details on the website, you will need to follow these steps: 1. log into the online journal at http://www. ojvr.org 2. in your ‘user home’ [http://www.ojvr.org/ index.php/ojvr/user] select ‘edit my profile’ under the heading ‘my account’ and insert all relevant details, bio statement and reviewing interest. 3. it is good practice as a reviewer to update your personal details regularly to ensure contact with you throughout your professional term as reviewer to onderstepoort journal of veterinary research. please do not hesitate to contact me if you require assistance in performing this task. margo martens submissions@ojvr.org tel: +27 (0)21 975 2602 fax: +27 (0)21 975 4635 journal of veterinary research onderstepoort ivan horak james ker johan vorster jannie crafford joseph van heerden kerstin junker kgomotso sibeko lieza odendaal marco romito marna sinclair maryke henton mervyn mansel michael reichel moritz jansen van vuuren ned donkin neil duncan patricia forbes patrick masika paul van helden piet king richard burroughs richard kock roy meintjes samson mukaratirwa sonja matthee tanguy marcotty theo de waal wayne jorgensen werner giesecke wilmien luus-powell we appreciate the time taken to perform your review successfully. palmer_75-79.indd introduction arnold theiler’s identification of anaplasma marginale as the microbial agent of a specific disease, which he designated anaplasmosis, was only one of his remarkable achievements carried out in south africa from the late 19th century through the early decades of the 20th. equally as impressive was his subsequent recognition of a less virulent strain, which he termed a. marginale variety centrale, and its implementation as a vaccine—which continues in use today. [theiler’s original taxonomy of this less virulent strain has not been subsequently amended and, according to modern nomenclature, is referred to as a. marginale subspecies centrale.] on this occasion of the 100th anniversary of both theiler’s original identification of a. marginale and the founding of the onderstepoort veterinary institute, i am honoured to have been invited to present this lecture and daunted by the challenge of reviewing thei ler’s work from a centennial perspective. to man age this challenge, i will focus on only a few of the prescient observations of theiler’s 1908–1909 report, “anaplasma marginale (gen. and spec. nov.): the marginal points in the blood of cattle suffering from a specific disease”. discussion in this landmark paper, theiler (1910) identified a. marginale as the etiologic agent of a specific disease characterized by fever and anemia resulting in lethargy, anorexia, and a significant case fatality rate. this connection was especially notable in that the punctuate basophilic intraerythrocytic inclusions that characterize a. marginale in stained blood smears had been reported by smith & kilborne (1893) some 15 years earlier as stages of the protozoan piroplasma bigeminum (now babesia bigemina)—a reasonable conclusion given the presence of multiple distinct stages of protozoan parasites. theiler’s initial suspicion that there were two different etiologic agents involved in what was termed 75 onderstepoort journal of veterinary research, 76:75–79 (2009) sir arnold theiler and the discovery of anaplasmosis: a centennial perspective g.h. palmer department of veterinary microbiology and pathology and school for global animal health washington state university, pullman, washington, usa 99164-7040 abstract palmer, g.h. 2009. sir arnold theiler and the discovery of anaplasmosis: a centennial perspective. onderstepoort journal of veterinary research, 76:75–79 sir arnold theiler’s research in 1908/09 led to the discovery of the first rickettsial pathogen, anaplasma marginale, and set the stage for his development and implementation of an effective live vaccine based on a less virulent strain, a. marginale ss. centrale. his 1910 report, describing a. marginale, is among the classic monographs in infectious disease research, presenting not only observations in exacting detail but also highlighting the deductive reasoning leading to association of a new pathogen with a specific disease. with a centennial perspective and both conceptual frameworks and molecular tools unimaginable in theiler’s time, the significance of several observations in the original report— cyclic bacteremia, strain superinfection, and taxonomic position—is now clear and highlight the broad applicability of key principles of pathogen biology. 76 sir arnold theiler and the discovery of anaplasmosis redwater of cattle in south africa, was based on his view that animals recovered from infection with b. bigemina should be immune to further challenge with this pathogen. therefore, when animals deliberately inoculated with b. bigemina in england and subsequently imported into south africa underwent a clinically similar disease in the transvaal, he addressed two alternative hypotheses: (i) the disease was due to a different strain of b. bigemina against which the first strain was not protective; and (ii) the disease was a result of infection with a distinct pathogen. while identification of antibody and t-lymphocytes as primary mediators of protective immunity were still decades in the future, his clear understanding of the specificity of adaptive immunity was evident and key in his discovery of a. marginale as the etiologic agent of the disease. much of this first report on a. marginale describes his methodical approach to differentiation of a. marginale from b. bi gemina as separable etiologic agents, each responsible for a specific disease, previously lumped together as redwater or texas fever. most impressively theiler used a combination of approaches including epidemiologic analysis to identify regions where only one of the two diseases were present or where both occurred but at different times of the year, detailed association of microscopic observations with the clinical course of dual and single pathogen infections, and linkage of specific clinical signs and post-mortem lesions to the diseases caused by each pathogen. although theiler’s detailed observations, so characteristic of that era’s leading microbiologists, illuminate the first report, his critical review of the existing literature and observations of other scientists, including smith & kilborne in the united states, knuth & lignieres in argentina, and kossel and his co-workers in europe, provides evidence of his ability to integrate disparate observations into a unifying conclusion. a final characteristic that stands out from theiler’s initial work on anaplas mosis is the accord paid to the field observations of the farmers themselves that there were often two bouts of disease in cattle occurring at dif ferent times of the year—observations that strengthened theiler’s initial belief that two distinct pathogens were respons ible and allowed him to correlate microscopic observations with each clinical syndrome. this respect for the insight of farmers is very reminiscent of that which led smith & kilborne (1893) to the then unthought-of discovery that arthropods could transmit microbial pathogens. from a centennial perspective, with 100 years of investigation generating a conceptual framework of pathogenic microbiology and immunology unavailable to theiler as well as the then unimaginable tools of electron, fluorescence and confocal microscopy, pcr and whole genome sequencing, and high-throughput proteomics, it is remarkable how well theiler’s conclusions have stood the test of time. furthermore, buried in the 1910 manuscript are several observations which can now be understood—a true complement to theiler in that his observations were so clearly presented. the first of these is his observation that there were cyclic peaks of a. marginale bacteremia. theiler noted: “marginal points on 32nd day…62nd day with re-appearance of marginal points.” unlike african trypanosomes, for which ross & thomson (1910) reported cyclic waves of parasitemia, light microscopy has insufficient sensitivity to reliably track the fluctuations in a. marginale bacteremia, except in the acute and very early post-acute periods observed by theiler. although theiler clearly understood that a. marginale persisted in infected animals and that the pathogen levels were much lower during this phase, the basis for this persistence would remain poorly understood until more sensitive detection methods were developed. initially, southern hybridization (kieser, eriks & palmer 1990) and then pcr (french, mcelwain, mcguire & palmer 1998) allowed definitive detection of the cyclic waves of bacteremia between 102–107 organisms per mℓ of blood during persistent infection. these observations, in turn, led to the hypothesis that persistence was mediated by outer membrane protein antigenic variation and subsequent discovery of the immunodominant and antigenically-variable major surface protein (msp)-2 (reviewed in palmer, brown & rurangirwa 2000). research over the past decade has elucidated how the small (1.2 mb) a. marginale genome generates the tremendous number of variants needed to evade immune clearance for long-term persistence. this mechanism, termed segmental gene conversion (palmer & brayton 2007), uses a combination of recombination of complete donor alleles and oligonucleotide segments of these alleles into a single expression site to generate 103–104 unique msp-2 variants. most interesting is that there is compelling evidence that variants generated by recombination of complete donor alleles have a strong in vivo fitness advantage (palmer, futse, leverich, knowles, rurangirwa & brayton 2007). these variants arise preferentially in acute infection (during which bacteremia reaches ≥ 108 a. marginale per mℓ) and in the initial bacteremic peak in the post-acute period (futse, brayton, knowles & palmer 2005). this in vivo fitness results in bacteremia levels during the 77 g.h. palmer first post-acute peak that exceed 107 a. marginale per mℓ and thus can be observed microscopically. in contrast, subsequent bacteremic cycles during persistent infection are increasingly composed of organisms with less fit variants generated from multiple donor alleles and peak at levels just below reliable microscopic detection (futse et al. 2005; palmer et al. 2007). this understanding is consistent with both the timing and levels observed by theiler (1910)—detection of the acute and first postacute bacteremia followed by long-term persistence with retention of infectivity but below levels of microscopic detection. this understanding of the interplay between antigenic variation and pathogen fitness is relevant not only to a. marginale but numerous persistent microbial pathogens, including the human immunodeficiency virus (hiv) (goulder & watkins 2004; kent, fernandez, dale & davenport 2005). a second observation made by theiler that can now be explained is the occurrence of strain superinfection. at several places in his original 1910 monograph, he notes that re-infection with a milder course of disease occurs and raises the question as to whether this may be attributable to a. marginale strain differences: “an animal may be successfully inoculated more than once, although the second inoculation will only cause a slight reaction. this second reaction might be due to a difference in the strain of the anaplasma, the primary reaction gives sufficient ground immunity to protect an animal against severe lesions and death from a subsequent infection.” this observation formed the basis for his subsequent development of a less virulent strain, a. marginale ss. centrale, as a vaccine to prevent severe morbidity and death upon challenge with highly virulent a. marginale (theiler 1911; 1912). however, the basis for this strain superinfection remained unexplored until genome sequencing of multiple a. marginale strains (brayton, kappmeyer, herndon, dark, tibbals, palmer, mcguire & knowles 2005; rodriguez, palmer, knowles & brayton 2005) began to provide clues as to how a second strain could evade the immunity induced against an already established primary strain infection. during infection with the primary strain, immunity is sequentially developed against the msp2 variants expressed by recombination of complete donor alleles and subsequently by segmental recombination. consequently, a second strain must evade this immunity and does so only by encoding at least one distinct variant allele in its genome (futse, brayton, dark, knowles & palmer 2008). among highly similar strains with shared variant alleles strain superinfection either does not occur or is rare—the latter perhaps occurring in a narrow window following infection with the initial strain, but before broad immunity to the initial variant repertoire has been induced. this same mechanism appears to underlie the use of the a. marginale ss. centrale vaccine as the repertoire of this vaccine strain (shkap, molad, brayton, brown & palmer 2002) is distinctly different from the senso stricto a. marginale strains examined to date. understanding the basis for the strain superinfection first observed by theiler has a broader impact for infectious diseases. the pressures for genomic divergence among microbial pathogens where host immunity against a dominant strain limits new transmission, are not unique to a. marginale—similar pressures appear to manifest in pathogens as taxonomically distinct as hiv, hepatitis c virus and trypanosoma brucei (blackard & sherman 2007; hutchinson, picozzi, jones, mott, sharma, welburn & carrington 2007; piantadosi, chohan, chohan, mcclelland & overbaugh 2007). the strain structure of a. marginale is remarkably diverse and serves as an illustrative model as to how pathogens emerge under conditions of natural transmission and specifically how diversification occurs in, unlike the rna viruses, a relatively genomically stable organism. a final observation now viewed through the prism of time is theiler’s discussion of the taxonomy of a. marginale. theiler’s clear statement that this was a specific disease agent, apart from b. bigemina, was highly significant as it led to development of specific vaccines and, later, specific treatments for these diseases—similar in several clinical features but with very different etiologies and epidemiology. theiler’s choice of the genus name anaplasma reflected his observations that the inclusion lacked any observable cytoplasm, distinct from the other intra-erythrocytic protozoa in which the basophilic nucleus and eosinophilic cytoplasm could be distinguished microscopically: “they differ from any known blood parasite by the absence of a protoplasmic body and consist only of chromatin substance, thus resembling to a certain extent, the bacteria. they represent, in my opinion, a new genus of protozoa which i propose to call anaplasma and the species under consideration anaplasma marginale.” although a. marginale would be variously classified as a virus or protozoa, the initial observations by theiler are consistent with its correct classification as a novel bacterial genus. anaplasma marginale was the first rickettsial pathogen to be identified, although it was not correctly placed in the order rickettsiales until the 7th edition of bergey’s manual in 1957. the genus name has remained and, based on precedent and the guiding principles of bacterial nomenclature, now also gives 78 sir arnold theiler and the discovery of anaplasmosis its name to one of the two families that compose the order rickettsiales, anaplasmataceae (the other be ing the rickettsiaceae). interestingly, cowdry (1925) working in south africa would identify the first pathogen representative of a second genus, ehrlichia, in the family anaplasmataceae with his discovery of e. ruminantium as the cause of african heartwater. [cowdry placed the newly discovered organism in the genus rickettsia. this was later placed in a novel genus, cowdria, honouring his discovery. recent taxonomic re-organization (dumler et al. 2001) has unified highly related organisms in the genus ehrlichia.] the genera anaplasma and ehrlichia include the tick-transmitted pathogens with in the family anaplasmataceae and it is notable that pioneering work in south africa established the fundamentals of transmission, pathogenesis and immunity that have now been applied to a greatly ex panded number of animal and human pathogens in these genera. dumler’s highly cited paper (2001), revising the taxonomy of the family anaplasma taceae, has stood the test of complete genome sequencing, confirming the relationships among the members, and has led to better understanding of the biology of these organisms and their consequent diseases. thus today, research initiated by theiler has a widespread impact on understanding pathogen biology far beyond that envisioned a century ago. anaplasma marginale still holds many challenges for investigation and for improvement of disease control. among these, three stand out: (i) development of safe, standardized and effective vaccines; (ii) understanding the basis for age and breed innate resistance; and (iii) understanding transmission dynamics and virulence leading to severe outbreaks. despite theiler’s development of the a. marginale ss. centrale as a live vaccine in 1910/11, disease control remains suboptimal and, along with other tick-borne infections, exacts a disproportionate burden on small-holder farmers in resource poor countries (minjauw & mcleod 2003). addressing these challenges is incumbent on the research community to both continue the theiler tradition of disease investigation leading to improvement of animal health and to meet our global responsibilities to enhance human development and well-being. references blackard, j.t. & sherman, k.e. 2007. hepatitis c virus coinfection and superinfection. journal of infectious diseases, 195:519–524. brayton, k.a., kappmeyer, l.s., herndon, d.r., dark, m.j., tibbals, d.l., palmer, g.h., mcguire, t.c. & knowles, d.p. 2005. complete genome sequencing of ana plasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. proceedings of the national academy of sciences usa, 102:844–849 cowdry, e.v. 1925. etiology of heartwater: observation of a rickettsia, rickettsia ruminantium, in tissues of infected animals. journal of experimental medicine, 42:231–252. dumler, j.s., barbet, a.f., bekker, c.p., dasch, g.a., palmer, g.h., ray, s.c., rikihisa, y. & rurangirwa, f.r. 2001. reorganization of genera in the families rickettsi aceae and anaplasmataceae in the order rickettsiales: unification of some species of ehrlichia with anaplasma, cowdria with ehrlichia and ehrlichia with neorickettsia, descriptions of six new species combinations and designation of ehrlichia equi and ‘he agent’ as subjective synonyms of ehrlichia phagocytophila. international journal of systematic and evolutionary microbiology, 51:2145–2165. french, d.m., mcelwain, t.f., mcguire, t.c. & palmer, g.h. 1998. expression of anaplasma marginale major surface protein 2 variants during persistent cyclic rickettsemia. infection and immunity, 66:1200–1207. futse, j.e., brayton, k.a., knowles, d.p. & palmer, g.h. 2005. structural basis for segmental gene conversion in generation of anaplasma marginale outer membrane protein variants. molecular microbiology, 57:212–221. futse, j.e., brayton, k.a., dark, m.j., knowles, d.p. & palmer, g.h. 2008. superinfection as a driver of genomic diversification in antigenically variant pathogens. proceedings of the national academy of sciences usa, 105:2123–2127. goulder, p.j.r. & watkins, d.i. 2004. hiv and siv ctl escape: implications for vaccine design. nature reviews immun ology, 4:630–640. hutchinson, o.c., picozzi, k., jones, n.g., mott, h., sharma, r., welburn, 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: davies m. pfukenyi1 samson mukaratirwa1 affiliations: 1biodiversity/evolutionary biology group, school of life sciences, university of kwazulu-natal, westville campus, south africa correspondence to: samson mukaratirwa postal address: school of life sciences, university of kwazulu-natal, private bag x54001, durban 4001, south afri dates: received: 29 apr. 2013 accepted: 26 june 2013 published: 19 aug. 2013 how to cite this article: pfukenyi, d.m. & mukaratirwa, s., 2013, ‘a review of the epidemiology and control of gastrointestinal nematode infections in cattle in zimbabwe’, onderstepoort journal of veterinary research 80(1), art. #612, 12 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.612 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a review of the epidemiology and control of gastrointestinal nematode infections in cattle in zimbabwe in this review article... open access • abstract • introduction    • species of nematodes infecting cattle in zimbabwe    • cooperia    • haemonchus    • trichostrongylus    • trichuris    • other genera    • survival of free-living stages in the environment    • factors influencing gastrointestinal nematode infection in cattle       • season and host-age       • production and husbandry practices       • impact on production    • control       • chemical treatment    • pasture management, biological control and nutrient supplementation    • host resistance    • vaccination • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ in this review, the main gastrointestinal nematodes infecting cattle in zimbabwe and the epidemiological factors influencing their occurrence are reviewed and discussed. nineteen gastrointestinal nematode species that belong to seven families have been found to occur in cattle in zimbabwe. the main genera reported to date are cooperia, haemonchus, trichostrongylus and oesophagostomum and the dominant species are cooperia pectinata, cooperia punctata, haemonchus placei and trichostrongylus axei. the mixed infection by several species from the genera is the cause of parasitic gastroenteritis in cattle in zimbabwe. production and husbandry practices, season, host age and environment are considered to be the main factors that influence gastrointestinal nematode infection in cattle. the geographical distribution of the gastrointestinal nematodes is also reviewed in relation to the climatic conditions of the country. various control options are discussed and how they are applicable to the zimbabwean situation. based on reports and existing data on the epidemiological features of the gastrointestinal nematode infection in cattle, practical control measures are critically reviewed and recommendations are made for a national control programme. introduction top ↑ gastrointestinal (gi) nematode infections of cattle are constraints on the efficient raising of cattle on pasture throughout the world (gasbarre, leighton & sonstergard 2001). these parasites have two main features of epidemiological importance: there is no multiplication of their pre-parasitic stages and their infective stages, whether acquired directly by the host or indirectly via intermediate hosts, always give rise to a single adult male or female parasite in the definitive host (chiejina 1994). therefore, the number of infective stages present in the host environment at any given period is related to the number of worm eggs passed by the host and this largely determines the number of parasites potentially capable of being established in a susceptible host. however, characteristics such as breed, age and nutritional status of the host as well as characteristics of the environment also have a considerable influence on the parasites and their capacity to infect and inflict damage to the host (rivera et al. 1983). most studies on gi nematode ecology in cattle have concluded that climatic conditions play an important role in the survival and transmission of parasite eggs and larvae (rivera et al. 1983). therefore, to be effective, control measures depend on a sound understanding of the epidemiology of the disease in both the host and the environment. in this review, the information on the epidemiology of gi nematodes of cattle reported to date in zimbabwe is presented and the different options recommended for their control are discussed. species of nematodes infecting cattle in zimbabwe gastrointestinal nematodes of cattle have different pathogenic effects (charlier et al. 2009) and it is important to establish which broad groups are present in a herd, area, country or region. furthermore, some of these nematodes have different development times and stages outside and inside the definitive host; knowledge of which is important for effective control measures. gastrointestinal nematode infections in cattle in zimbabwe have been reported by various authors (bryant & norval 1985; pandey 1989; eysker, jansen & pandey 1990; pandey, nyanzunda & chitate 1990a; pandey et al. 1990b; duncan & forbes 1992; obwolo et al. 1992; pandey, chitate & nyanzunda 1993; vassilev 1993, 1994, 1999; obwolo et al. 1995; moyo et al. 1996; moyo et al. 1997; moyo et al. 2003; magaya et al. 2000; pfukenyi et al. 2007) and unpublished records from the state central veterinary research laboratory, harare, zimbabwe. parasitic gastroenteritis (pge) in cattle is caused by mixed infections of several nematode species (table 1). nineteen gi nematode species belonging to seven families have been reported to infect cattle in zimbawe (table 1). the most important and widely prevalent nematodes belong to the family trichostrongylidae, which is composed of the genera cooperia, haemonchus, trichostrongylus and ostertagia and to the family chabertiidae, which is composed of the genus oesophagostomum. the genera bunostomum, nematodirus, strongyloides, toxocara and trichuris are of lesser frequency (pandey et al. 1993; vassilev 1994, 1999; moyo et al. 1996; pfukenyi et al. 2007). table 1: checklist of gastrointestinal nematode species reported in cattle in zimbabwe. cooperia five cooperia species have been reported to infect cattle in zimbabwe (table 1). cooperia pectinata, cooperia punctata and cooperia spatulata are the most common species in cattle in the tropics (horak 1981) and as expected they are present in cattle in zimbabwe (buckley 1933; moyo et al. 1996; magaya et al. 2000). cooperia pectinata and c. punctata are the most prevalent species and they have been found in both the highveld and lowveld regions of zimbabwe (vassilev 1993; moyo et al. 1996). cooperia oncophora is mainly found in temperate climates (shaw et al. 1998) and has been reported only once in zimbabwe (buckley 1933). this was most likely a case of misidentification (jooste 1989) as there have been no other reports within the country since then. however, the parasite was recorded in cattle in the eastern cape province of south africa (horak, evans & purnell 2004), which experiences low temperatures similar to the temperate regions of europe the importation of cattle from europe could probably explain the presence of the parasite in zimbabwe. although it is normally a species of wild ruminants (jooste 1989; madzingira & mukaratirwa 2002; madzingira et al. 2002a), cooperia hungi has also been reported in cattle (jooste 1989). this could have been caused by cross-transmission from wild ruminants in cattle-antelope mixed farming systems (madzingira & mukaratirwa 2002). haemonchus three haemonchus species have been recorded (table 1). haemonchus placei is the most prevalent species and has been found in both the highveld and lowveld regions of zimbabwe (vassilev 1993; obwolo et al. 1995; moyo et al. 1996, 2003; magaya et al. 2000; moyo 2006). in contrast, haemonchus similis and haemonchus contortus have been found mainly in the highveld region (madzingira et al. 2002a). both h. placei and h. similis are more adapted to cattle, whilst h. contortus is more adapted to sheep and goats (amarante et al. 1997). thus, cross-infection of the haemonchus species between sheep, goats and cattle and of h. contortus between cattle and some antelopes (madzingira et al. 2002a) has seemed to occur in zimbabwe and this should be taken into consideration when formulating control measures. trichostrongylus two trichostrongylus species have been reported (table 1). the most frequent species is trichostrongylus axei; there are a few reports of trichostrongylus colubriformis (moyo et al. 1996; moyo 2006). few reports of t. colubriformis in cattle in south africa are available (horak 1981; horak et al. 2004). trichostrongylus colubriformis is a parasite of sheep and its presence in cattle is likely to be due to cross-infection (horak et al. 2004). however, with the exception of t. axei, parasite species belonging to the trichostrongylus genus are not considered to be important in cattle (horak et al. 2004). trichuris the prevalence of infection with the trichuris species in cattle is generally low, with trichuris globulosa being the most frequently encountered (vassilev 1999). trichuris barbertonensis (jooste 1989) and trichuris ovis (buckley 1933) have been reported once in cattle, and according to jooste (1989) the presence of t. ovis in cattle was a misidentification. trichuris barbertonensis was also documented in goats and other wild ruminants such as impala (aepyceros melampus), steenbuck (raphicerus campestris) and buffalo (syncerus caffer) (table 2). table 2: checklist of cattle gastrointestinal nematode species reported in other domestic and wild ruminants in zimbabwe. other genera the other minor parasite species in terms of severity and prevalence of infection are from the genera bunostomum, nematodirus, oesophagostomum, ostertagia, toxocara and strongyloides (table 1). except in calves in the high-rainfall highveld region, bunostomum phlebotomum has been found in low numbers (moyo et al. 1996). similarly, the prevalence of oesophagostomum radiatum and toxocara vitulorum has been found to be low (moyo et al. 1996; pfukenyi et al. 2007). as expected, infection with strongyloides papillosus is confined to calves less than eight months of age due to their immature immune status (pandey et al. 1993; vassilev 1999; moyo et al. 1996; pfukenyi et al. 2007). ostertagia ostertagi (moyo et al. 1996) and nematodirus species (obwolo et al. 1992) have been reported on an irrigated farm and a communal farming area in the highveld region, respectively. jooste (1989) also reported nematodirus species in cattle, but the origin of the animals was not given. nematodirus filicolis (lawrence 1928) and ostertagia circumcincta (christe 1957) were also documented once in sheep. however, jooste (1989) indicated that the reports could have been misidentification. survival of free-living stages in the environment cooperia pectinata and c. punctata and to a lesser extent cooperia spatulata, h. placei and t. axei are the dominant species infecting cattle in zimbabwe with the cooperia species being the most prevalent (pandey et al. 1993; moyo et al. 1996; vassilev 1999; pfukenyi et al. 2007). free-living stages of nematode parasites of grazing animals differ in their responses to environmental conditions – notably temperature and moisture – these differences exist within and between nematode species (chiejina 1994). the larvae of c. pectinata are well adapted to extremes of heat, cold and to dry conditions (reinecke 1960; durie 1962; lima 1998). they survive and persist longer and migrate better than the other nematodes (reinecke 1960). studies in zimbabwe (moyo et al. 1996; pfukenyi et al. 2007) have also indicated that cooperia third-stage larvae (l3) are more resistant to adverse conditions like high temperature and dryness, as high numbers of larvae were observed during both the wet and dry seasons. these findings probably explain the predominance of the cooperia species. the lower prevalence of h. placei when compared to the cooperia species could probably be attributed to the l3 being more sensitive to hot, arid conditions and shorter survival periods (reinecke 1960). in support of this, a significantly higher prevalence of haemonchus species was observed during the wet months of the year than during the dry season and in the high-rainfall highveld regions than the drier lowveld regions (vassilev 1994; pfukenyi et al. 2007). however, the lower prevalence during the dry season could be attributed to hypobiosis, which is shown to occur in the highveld region during the same period each year (moyo et al. 1996; magaya et al. 2000). the relatively low to moderate prevalence of trichostrongylus species when compared to cooperia and haemonchus species could probably be attributed to the fact that lower temperatures are required for the development of its free-living stages (levine 1963; williams & mayhew 1967; lima 1998). higher monthly percent frequency of infective larvae of the trichostrongylus species was observed during the cold dry months (may to august) in zimbabwe (pfukenyi et al. 2007). the survival rate and migration of o. radiatum is lower in comparison to the cooperia species and h. placei (reinecke 1960). its low prevalence is attributed to its relatively long life cycle and low resistance to desiccation during its pre-infective stages (rivera et al. 1983). immunity may also play an important role in the differences noted; o. radiatum is extremely effective in eliciting strong, protective immune responses. a primary exposure of previously naïve animals to infection or even to parasite antigens results in a very significant reduction in the number of parasites that can become established after a subsequent infection (gasbarre & canals 1989). as a result, these parasites remain largely a problem for only young animals in the herd (gasbarre & canals 1989), a finding that is consistent with observations in zimbabwe (moyo et al. 1996; pfukenyi et al. 2007). however, other parasites such as the cooperia species and h. placei require a longer period of exposure before this level of protective immunity is seen (gasbarre & canals 1989). the survival of b. phlebotomum is extremely low compared to other nematode species and is only present in large numbers when well-distributed summer rainfall provides adequate moisture for larval development and survival (reinecke 1960). in addition, the possibility of infection, especially under extensive range-type operations with low animal density, is greatly reduced since the larvae do not migrate from the faeces, but penetrate the host’s skin (reinecke 1960; rivera et al. 1983). its occurrence in zimbabwe is sporadic and tends to be localised, favouring soils with high moisture content; this can be a problem in kalahari sandveld areas of the country (jooste 1987). toxocara vitulorum is an infrequent parasite under extensive farming conditions (bianchin & honer 1987). its prevalence is low in communal and on commercial farms where cattle are kept under extensive farming conditions (moyo et al. 1996; pfukenyi et al. 2007). however, its prevalence in calves on irrigated farms in the highveld region has been found to be high (pandey et al. 1990b; moyo et al. 1996). according to pandey et al. (1990b), under natural conditions t. vitulorum infection is low and the high prevalence that has been observed on irrigated farms is due to the unique system of farm management resulting in persistent moist or wet pastures. however, lactogenic transmission is an important source of infection to calves and the larvae are present in milk for up to three to four weeks after parturition (pandey et al. 1990b). in cows, resumption of larval development in late pregnancy allows further lactogenic transmission. tropical climates do not offer favourable conditions for the transmission and survival of ostertagia and nematodirus species (levine 1963), so both parasites are of little significance in zimbabwe. however, nematodirus helvetianus occurs in calves in the moist, temperate climates of the coastal provinces (eastern cape, western cape and kwazulu-natal) of south africa (reinecke 1983; horak et al. 2004). similarly, o. ostertagi has been reported in cattle on coastal pastures in the eastern cape province of south africa. however, the parasite species also occurs in cattle in the non-coastal areas of the eastern highveld of mpumalanga province (louw 1999) and the northeastern highveld of the free state province (tsotetsi & mbati 2003) in south africa. the presence of these parasites in zimbabwe could probably be attributed to importation of cattle from south africa. table 2 shows gi nematodes reported in cattle that were also documented in other domestic and wild ruminants in zimbabwe. the data show that c. pectinata, c. punctata, h. similis, s. papillosus, t. axei, t. globulosa and t. barbertonensis were found in at least two other domestic or wild ruminants, whilst the others were reported in only one other domestic and/or wild ruminant. these findings are important when considering different options for the control of these parasites in cattle. factors influencing gastrointestinal nematode infection in cattle season and host-age when cattle graze on natural pastures, climate plays an important role in the transmission of gi nematodes (gatongi, gathuma & munyua 1987). the climate in zimbabwe is characterised by a warm wet season from november and december to march and april, followed by a cool dry season (may to august) and later a hot dry period (september to october and november). the general trend of gi nematode faecal egg counts (fecs) is related to the rainfall pattern (obwolo et al. 1992; eysker et al. 1990; pandey et al. 1993; vassilev, 1994, 1999; moyo et al. 1996; pfukenyi et al. 2007). faecal egg counts (fecs) rise from november and december to reach a peak during march and april, followed by a decline to low levels during the dry period from may and june to october and november (pandey et al. 1993; vassilev, 1994, 1999; moyo et al. 1996; pfukenyi et al. 2007). similarly, the population of infective larvae on pastures increases to a maximum during the rainy season, which coincides with the peak in fecs (pandey 1989; eysker & pandey 1991; vassilev 1993; moyo et al. 1996, moyo et al. 1997). pasture larval counts are generally low during the dry season, with the population of infective larvae on pastures being negligible between june and october. however, an important observation is that although pasture infectivity decreases during the dry season, some larvae can be found on pasture throughout the dry season, particularly on farms with irrigated pastures (moyo et al. 1996).thus, during the rainy season (december to march), the rainfall and temperature are favourable for the development, survival and translation of infective larvae on herbage. these conditions result in increased availability of infective larvae on pastures, so the chances of cattle picking up infective stages of the parasites whilst grazing are high, leading to a buildup of high worm burdens in the host. this leads to an increase in fecs with peaks between february and march and april and may. during late autumn to early spring (may to october and november) the decline in fecs is due to lack of rainfall and to some extent lower and higher temperatures; these are conditions that are unfavourable for the development of eggs into infective larvae. hence, the wet months carry the highest risk of occurrence of parasitic gastroenteritis (pge) in cattle; therefore control measures should take this factor into consideration. however, it is also important to note that the most pathogenic gi nematode parasites, haemonchus species, survive the dry season as inhibited early fourth-stage larvae (hypobiosis), whereas the other species from the genera cooperia and trichostrongylus survive the dry season mainly as adults (moyo et al. 1996; magaya et al. 2000). the prevalence of gi nematodes as observed through faecal egg counts is higher in animals aged less than 12 months as opposed to adult cattle (pfukenyi et al. 2007). hence, adult cattle act as reservoirs of infection and constant sources of infection for the more susceptible young animals. a higher prevalence and egg count of gi nematodes was demonstrated in lactating and pregnant cows when compared to dry cows, bulls and oxen; this confirms the periparturient egg-rise phenomenon (pfukenyi et al. 2007) and thus serves as a source of pasture contamination. production and husbandry practices just as meteorological and other environmental factors largely determine the overall number of infective stages of nematode parasites at any given time, livestock husbandry systems and management practices have a major influence on the transmission of that infection to a susceptible host population (chiejina 1994). the methods of cattle management in zimbabwe are varied and are inextricably linked with systems of agricultural production as well as with local traditions and beliefs. however, the main husbandry systems are communal or rural, small-scale holder and commercial. mixed farming systems of cattle and game, particularly antelopes, were introduced on cattle ranches in the nineties and have become an important agricultural activity in zimbabwe (knottenbelt 1990; madzingira et al. 2002b). there are contradictory results on gi nematode infections in cattle on communal land. earlier studies reported low levels of infection leading to the conclusion that gi nematodes were of no importance and do not contribute significantly to the poor body condition and low productivity recorded in cattle (bryant & norval 1985; pandey et al. 1990a; obwolo et al. 1992). however, later studies (pandey et al. 1993; vassilev 1994; 1999; moyo et al. 1996; pfukenyi et al. 2007) showed high levels of gi nematode infections in communal cattle. the levels of infection, particularly during the rainy season, could vary depending on farming systems (moyo et al. 1996). faecal egg, pasture larval and worm counts were highest in communally-grazed cattle, intermediate in cattle on irrigated commercial farms and lowest in cattle on conventional commercial farms (moyo et al. 1996). these differences could be due to different management systems and different microclimatic conditions. communal cattle are kept under extensive free-range poor communal pastures with very little input of feed supplements. anthelmintic (ah) treatment of communal cattle is either non-existent or very occasional and irregular and, if given at all, is limited to a few sickly animals (pandey et al. 1993). in addition, the stocking rate is high, being estimated to be 0.37 livestock units per hectare; this may be increased more than two-fold during the rainy season as 50% – 60% of the land is used for growing crops during that period (pandey et al. 1993). thus, a combination of poor nutrition, overgrazing, high stocking rate and lack of ah medication results in heavy worm burdens in communally grazed cattle (pandey et al. 1993) compared to others. in contrast, cattle on conventional commercial farms are kept under modern extensive or intensive conditions where stocking rates are lower, particularly in the hot and dry lowveld region (moyo et al. 1996). in addition, commercial farmers tend to treat all animals with an ah at the beginning of the dry season (april to may) and/or the beginning of the rainy season (november to december) (moyo et al. 1996). hence, worm burdens in this category of cattle are lower than communally grazed cattle. the obvious reason for higher levels of infection and pasture contamination on irrigated commercial farms when compared to conventional commercial farms is the higher stocking rate that is allowed by the irrigation and furthermore, irrigation implies an increase of the length of infectivity of pasture as shown by higher pasture larval counts (moyo et al. 1996). in mixed cattle-game farming systems, cattle and game species, particularly antelopes, are reared together. mixed farming with cattle and antelope is attributed to bring about the possibility of cross-infection of helminth parasites (jooste 1987; knottenbelt 1989; madzingira et al. 2002a). a high stocking density of antelope and domestic ruminants is known to favour such cross-infection and the random translocation of game may broaden the endemic and host ranges of internal parasites (horak 1981). earlier studies in zimbabwe have shown cross-infections of h. contortus, t. axei, t. colubriformis, dictyocaulus viviparus, c. pectinata and c. punctata from domestic ruminants, including cattle, to antelopes (condy 1972; grobler 1981; jooste 1984, 1987; knottenbelt 1989, 1990). later, madzingira et al. (2002a) reported evidence of cross-infections of o. radiatum and s. papillosus from cattle to antelope species. no common helminths of antelope were recovered from cattle, implying that antelopes may act as reservoir hosts of infection for cattle. it appears that antelope are better hosts for helminths of cattle than cattle are for those of antelopes (horak 1980; boomker, du plessis & boomker 1983; boomker et al. 1984; madzingira et al. 2002a). thus, antelopes are more likely to suffer in a mixed farming system with cattle. however, the degree of cross-infection was low and the parasites involved occurred in low numbers, which may have little effect on the health of the antelopes (madzingira et al. 2002b). impact on production infections with gi nematodes in cattle do not necessarily result in disease. however, heavy infections result in clinical pge and this consequently leads to inappetence, impaired gastrointestinal function and changes in protein, energy, mineral metabolism and water balance (fox 1993). clinical pge is characterised by watery diarrhoea, weight loss or reduced weight gain, dull hair coat, anorexia and a general loss of condition (anderson et al. 1965). data on reduced feed intake due to gi nematode infections in cattle are scarce; however, forbes et al. (2000) observed that cattle treated with the ivermectin sustained-release bolus (ivomec® sr bolus, merial, usa), on average grazed 105 minutes longer per day than those with evidence of patent nematode infections. subclinical infections without obvious clinical signs constitute the majority of infections and cause economic losses due to sub-optimal performance (vercruysse & claerebout 2001).gastrointestinal nematodes are considered to be primarily important in first season grazing calves in summer (charlier et al. 2009). increased weight gain is the most often observed and best-documented benefit of gi nematode control in first season grazing calves (charlier et al. 2009). this effect on weight gain was studied in relation to anthelmintic treatment in western europe (shaw et al. 1998; dimander et al. 2000, dimander 2003; larsson et al. 2006). similar studies were conducted in zimbabwe (duncan & forbes 1992; vassilev 1993; magaya et al. 2000; moyo et al. 2003), which showed a significant effect of anthelmintic treatment on the enhancement of productivity and protection from the effects of infection with nematode parasites in cattle. in the last decade, substantial generated evidence showed that gi nematodes could also have negative impacts on the performance of adult cattle (charlier et al. 2009). the milk yield response to anthelmintic treatment in pastured dairy cows was observed to be around 1 kg/cow per day (charlier et al. 2009). in anthelmintic-treated beef cows, increased conception and calving rates, and reductions in calf mortality and calving-to-breeding intervals were shown (hawkins 1993; gross, ryan & ploeger 1999). similar studies in dairy cows showed shorter calving-to-conception intervals in treated cows when compared to untreated controls (walsh, younis & morton 1995; sanchez et al. 2002). however, no such studies have been conducted in zimbabwe and the impact of gi nematodes on production should be investigated in different categories of cattle. control the available epidemiological information on gi nematodes of cattle in zimbabwe was used in this review to design appropriate control measures. methods available for the control of gi nematode infections are mainly based on chemical treatment, non-chemical management practices, immune modulation and biological control. chemical treatment the use of anthelmintics is still the mainstay for nematode control. vercruysse and claerebout (2001) defined a number of threshold conditions for choosing to treat cattle with an ah drug. a therapeutic threshold is intended to identify animals with parasitic levels that necessitate immediate treatment and is basically the diagnosis of clinical disease. the phenomenon of parasite over-dispersion could be put to good use if those animals suffering from sufficient levels of infection to cause considerable production loss or health effects could be identified and treated individually (malan, van wyk & wessels 2001). in the northern hemisphere, cattle pge is caused by o. ostertagi together with cooperia and trichostrongylus species and it occurs mainly in first season grazing calves in summer. similarly, in zimbabwe and other southern africa countries, pge occurs mainly in young cattle in summer, but is mainly due to haemonchus together with cooperia and trichostrongylus species. the diagnosis of pge can be based on clinical signs confirmed by fecs or by determination of parasite specific serum antibody, gastrin or pepsinogen levels (berghen et al. 1993). in new zealand, the fec strategy achieved a reduction in the frequency of ah use (leathwick et al. 2006). however, the main drawback is that this is impractical in zimbabwe due to the limitations of cost and facilities and lack of farmers’ training on diagnosis of cattle pge based on clinical signs. the measurement of serum pepsinogen and gastrin levels is a specific tool in the diagnosis of ostertagiosis, which is not important in zimbabwe and other southern africa countries since the disease is not common in the region. similarly, an enzyme-linked-immunoabsorbent-assay (elisa) specific for the temperate c. oncophora has been developed (poot et al. 1997), but might not be useful for the tropical cooperia species. however, since the animals that are most susceptible to parasites and/or their effects, and hence require repeated ah treatments, are commonly the same within a season or between seasons (malan, van wyk & wessels 2001), the therapeutic or targeted (selective) animal treatment remains a beneficial approach. hence, for the time being, to improve the therapeutic approach in zimbabwe, simple user-friendly extension material on pge should be produced and disseminated to farmers and extension staff. for exploitation of the over-dispersion concept, research focused on production of convenient, inexpensive and accurate on-farm methods (e.g., faffa malan chart (famacha©) in small ruminants) for identifying animals for targeted selective treatment should be prioritised. the threshold for production-based treatment is essentially based on the identification of the so-called ‘sub-clinical’ infections that are characterised by no clinical signs of pge in untreated animals. however, the level at which gi nematode infection in cattle results in sub-clinical parasitism and any associated production losses has not been defined (vercruysse & claerebout 2001). useful tools to define subclinical parasitism such as pepsinogen and gastrin levels, serum and milk antibody levels and the copro-antigen detection test were evaluated in western europe for temperate gi nematodes (hilderson et al. 1992; kloosterman et al. 1996; guitian et al. 2000; agneessens, vercruysse & claerebout 2001). however, these tools have not been assessed for tropical gi nematodes. gi nematode infections should be prevented rather than cured, with the preventive measures being applied at herd level (vercruysse & claerebout 2001). based on the current increased knowledge about the epidemiology of gi nematodes in zimbabwe, preventive treatment strategies of pge in cattle can be formulated. gi nematode infections in cattle are high during the rainy season and low during the dry season. a strategic treatment should be carried out when pasture infectivity levels are low during the dry season and a tactical treatment at the peak of pasture infectivity levels during the rainy season (figure 1). the basis of the strategic treatment is to achieve a long-lasting effect on worm burdens so that re-infection during the dry season will be minimal and additionally the negative impacts of a combination of poor nutrition with nematode infections will be prevented (moyo et al. 2003). the rationale of the tactical treatment is to prevent clinical disease during the peak period of infection. figure 1: seasonal occurrence of gastrointestinal nematode infections in cattle in zimbabwe based on epidemiological reports and the recommended treatment options. the broad spectrum ah that are available in zimbabwe include: the benzimidazoles (bz) (albendazole, fenbendazole and oxfendazole); imidazothiazoles (levamisole); macrocyclic lactones (ml) (ivermectin and doramectin); with benzimidazoles being the most commonly used generic drugs. a broad-spectrum ah administered tactically in january and march (figure 1) would control immature and adult forms of h. placei, o. radiatum, cooperia species and trichostrongylus species. however, the timing of tactical treatments depends on several factors and hence no hard and fast rules can be applied for their implementation (van wyk 1990). for instance, rainfall over a number of days is more dangerous than a similar amount of rain on a single day (van wyk 1990). van wyk (1990) recommended that in general, it is advisable to treat tactically after good rains have fallen over a period of 15 to 30 days and this should commence about three to six weeks after the start of the good rains. hence, if infection, particularly with adult h. placei is severe from january to april, treatments with narrow-spectrum anthelmintics (closantel) that are effective against this species should be done. a strategic treatment with a broad-spectrum ah that is effective against arrested h. placei larvae and that has an extended effect against adult cooperia species and trichostrongylus species should be administered in july (figure 1). such tactical and strategic ah treatments are cost-effective parasite control in zimbabwe (duncan & forbes 1992; vassilev 1993; magaya et al. 2000; moyo et al. 2003). however, in the communal areas it is imperative that the tactical and strategic ah treatments are village-based as cattle are communally grazed and there is no benefit for only a few to carry out the recommended control measures. by utilising the dipping facilities, the ah treatments should be organised and preferably done at the same time within a village. one of the major constraints with tactical or strategic single-dose treatments is the labour involved in gathering and handling cattle, particularly in communal areas where animal-handling facilities are limited. the use of products with persistent action, particularly the mls, should be encouraged to reduce the frequency of ah treatments. for ease of use and convenience, pour-on products are now widespread and pour-on ml products are now dominant in the cattle ah market. furthermore, one of the other developments is the introduction of controlled release devices (boluses), which deliver anthelmintics either continuously or at targeted times. this offers the possibility of a single-dose treatment without re-course to further treatment and should be used in communal areas during dipping sessions. despite the acceptable performance of grazing cattle after ah treatment, populations of nematodes that are resistant to the most commonly used ah increasingly threaten this control (sutherland & leathwick 2011). although ah resistance of cattle nematodes has appeared to develop more slowly than those of small ruminants, literature suggests a rapidly escalating problem with 145 documented cases of ah resistance (sutherland & leathwick 2011). a review by sutherland and leathwick (2011) showed reports of ah resistance of cattle nematodes in argentina, australia, brazil, belgium, uk, germany, india, new zealand and usa. this involves all three of the broad-spectrum ah classes and at least ten species of nematodes. there is no literature about ah resistance of cattle nematodes in zimbabwe. however, bz-resistant cooperia species and h. contortus have been reported in sheep (boersema & pandey 1997; mukaratirwa, charakupa & hove 1997). given the rapid increase of reports in the literature about ah resistance of cattle nematodes, it is important that preventive ah treatments are integrated in the grazing management system to prevent under-treatment or over-treatment (vercruysse & claerebout 2001). there is also a pressing requirement to determine the extent of the problem in zimbabwe and other regional countries, as early detection of ah resistance is an important factor for sustainable chemical control. pasture management, biological control and nutrient supplementation pasture spelling and rotational grazing are both means of producing safe pastures and reducing the infectivity of the pasture, especially for more susceptible groups of animals (morley & donald 1980). survival times of infective larvae of up to nine months in temperate countries are often much longer than the generally accepted limits of up to eight weeks with pasture spelling (brundson 1980). however, relatively shorter survival times of 12 to 16 weeks for l3 bovine strongylids tend to occur in tropical countries (fabiyi, copeman & hutchinson 1988; waruiru et al. 1998). factors that favour worm control by pasture spelling under extensive conditions include low and very erratic rainfall leading to slow pasture growth and sparser plant cover (van wyk 1990). hence, the rapid decline in larval populations that have been noted in tropical climates could make rotational control, based on changing pasture, practicable. this is applicable in the more extensive commercial farming systems of the lowveld region of zimbabwe, where rainfall is low and very erratic and temperatures are high. under these conditions, the free-living stages of the worms are more exposed to adverse climatic conditions and can be expected to succumb sooner than when under more intensive conditions (van wyk 1990). it is, however, important to note that rotational grazing systems may return animals to a much higher rather than a reduced level of pasture infection (brundson 1980). under such circumstances, tactical treatment of the animals is recommended. however, in communal grazing systems, cattle are communally grazed and therefore practices such as rotational grazing and provision of clean pastures would not be feasible. practically no cross-transmission between sheep and cattle occur for oesophagostomum and bunostomum species (porter 1953; rose 1968; barger & southcott 1975; southcott & barger 1975). the two genera are important in zimbabwe and hence, the alteration of cattle and sheep could be an effective method for controlling these parasites in the country. however, species of cooperia and intestinal trichostrongylus can reproduce in the alternate host (porter 1953; rose 1968). cross-transmission of t. axei, equally pathogenic to sheep and cattle, is sufficiently effective that alternate grazing of these hosts at intervals of several months has nothing to offer in its control (kates & turner 1960). furthermore, alternate grazing of the most susceptible hosts (young sheep and young cattle) is not reliable to aid control of haemonchus species in either host (morley & donald 1980). hence, cross-transmission between sheep and cattle appears not to be sufficiently restricted, so that alternate grazing would offer substantial benefits in gi nematode control in zimbabwe, where the dominant parasites are h. contortus and trichostrongylus species in sheep, and cooperia species., h. placei and t. axei in cattle. older animals are generally more resistant to gi nematode infections than younger animals. an alternate grazing system through rotational grazing of paddocks by calves ahead of cows or heifers could be employed (morley & donald 1980). two further refinements are the so-called ‘forwards creep grazing’ whereby calves are grazed ahead of their dams, which follow them directly on the same pasture and ‘sideways creep grazing’, in which young animals have access to adjacent pasture that is not accessible to their dams (van wyk 1990). all forms can be used to control cattle gi nematode infections in the intensive and extensive commercial farming systems of the highveld and lowveld regions of zimbabwe. however, the system is managerially complex and therefore relatively difficult to apply (van wyk 1990) and is not feasible in the communal grazing areas of the country. in addition, one consequence of mixed or alternate grazing of different age groups of cattle is the exposure of older animals to higher levels of larval intake than they might otherwise experience (morley & donald 1980). except for o. ostertagi where it could lead to the accumulation of very large numbers of arrested larvae in heifers or cows leading to type ii ostertagiosis (morley & donald 1980), this may not be important for most genera in zimbabwe. crop aftermaths (stubble lands) can also be used as safe grazing during the dry season in all the cattle farming systems in zimbabwe and such aftermaths are practical for worm control (morley & donald 1980; van wyk 1990). they are seldom sufficient to accommodate all the susceptible animals and this also requires careful planning to have pasture available in appropriate quantity and quality (van wyk 1990). biological control includes the use of predatory fungi to kill a variety of nematode species and substantially reduce the intensity of infection (larsen 1999). with regard to the effect of nematophagous fungi against cattle parasitic nematodes, it is possible to reduce the number of infective larvae on herbage and subsequently to reduce the number of nematodes establishing in the grazing animals (wolstrup et al. 1994; larsen et al. 1995; nansen et al. 1995). challenges to fungal control are a requirement for daily administering of fungi to the host and achieving the required fungal density inside the dung. however, a nematode-killing fungus, duddingtonia flagrans, discovered in new zealand (skipp et al. 2002), showed a trapping efficiency rate of 78% and activity for up to 90 days on pasture, providing a viable alternative to reduce animal mortality from nematode infections (waghorn et al. 2003). surveys in neighbouring south africa have shown the presence of local strains of d. flagrans (durand et al. 2005). given the limited genetic variation between isolates from different parts of the world (faedo 2001), a survey of zimbabwean local strains would address any concerns regarding importing a foreign fungus. when this control approach becomes available for general application, it might be feasible in the small-scale commercial sector, but impractical in the communal set-up. plants or crops with natural anti-parasitic properties may play a role in future grazing systems for livestock (waller & thamsborg 2004). several tannin-containing forages, in particular those with condensed tannins (ct), showed ah activity against gi nematodes of sheep and goats (hoste et al. 2006). extracts of three tannin-containing plants, onobrychis viciifolia, lotus pedunculatus and lotus corniculatus, were shown to have in vitro ah activity against the cattle nematodes o. ostertagi and c. oncophora, with l. pedunculatus tannins being more active than those of o. viciifolia and l. corniculatus (novobilsky, mueller-harvey & thamsborg 2011). in zimbabwe, acacia karoo was shown to have a negative influence on the establishment and fecundity of h. contortus infection in boar goats in comparison to goats fed acacia nilotica and a basal diet (kahiya, mukaratirwa & thamsborg 2003). this was attributed to the high content of ct in a. karoo. further studies are required to determine the ah effects of tanniniferous plants on cattle gi nematodes and their use in helminth control in order to address the wide problem of ah resistance. well-nourished animals are generally more resistant to the effects of parasite infection. therefore, nutritional supplementation may reduce the requirement for ah control (knox, torres-acosta & aguilar-caballero 2006; torres-acosta et al. 2004). additional dietary protein (torres-acosta et al. 2004), selenium (au yeung et al. 2005; smith et al. 2005), as well as minerals (islam et al. 2006; koski & scott 2003) may each play a role in countering infections presumably through mechanisms such as enhancing host immunity or maintaining digestive tract integrity. host resistance increasingly attractive alternatives for the control of gi nematodes in cattle are the identification of the host genes that influence the acquired or innate resistance to the parasites and the use of the vast potential of the host genome to reduce parasite transmission in cattle populations (gasbarre et al. 2001). studies in the usa demonstrated that the number of nematode eggs per gram (epg) in faeces of pastured cattle is strongly influenced by host genetics (leighton, murrell & gasbarre 1989) and that the heritability of this trait is approximately 0.30 (gasbarre, leighton & davies 1990). however, this value is slightly higher than that reported in africa (zinsstag et al. 2000). in addition, epg values are not normally distributed and a small percentage of a herd is responsible for the majority of parasite transmission (gasbarre et al. 1990). in this over-dispersed distribution, most individuals have relatively low faecal epg values. a small percentage of animals, estimated to be between 15% and 25% of the total population (anderson & may 1985), exhibit high epg values. furthermore, the odds of certain bulls producing susceptible (high epg) calves was shown to be 20 times greater than in other bulls (gasbarre, leighton & bryant 1995). this suggests that genetic management of a small percentage of the herd could considerably reduce overall parasite transmission. hence, initiation of a selective-breeding programme has revealed that calves can be separated into three types (gasbarre et al. 2001). type i or innately immune calves, which never showed high epg values; type ii or acquired immune calves, which showed rises in epg values through the first two months on pasture and then fell and remained at levels associated with type i calves; type iii or immunologically non-responsive calves, which maintained high epg levels. the approximate percentage ratio of these calves is 25%; 50% and 25%, respectively. hence, according to gasbarre et al. (2001), accurate identification of the genes that control resistance will offer the producer several alternatives for disease control. the small percentage of susceptible animals can be targeted for drug administration and this approach would reduce both the cost of the anthelmintics that are used and the odds for selection of drug-resistant mutants. a second option would be to target susceptible animals for immunotherapy, which would involve vaccines of immunomodulation. a final option would be genetic selection to remove susceptible animals from the herd. thus, studies on cattle genetics for resistance are required in the country. vaccination vaccinations would prevent rather than cure infections and represent a more sustainable and environmentally friendly form of parasitic control compared to drugs. however, with the exception of the vaccine against d. viviparus, no vaccines are currently available commercially for the control of helminth infections in ruminants (knox et al. 2001). few of the experimental vaccines have proceeded to or successfully passed the next phase of development, which is the large-scale, cost-effective production of the vaccine (geldhof et al. 2007; redmond & knox 2006). hence, vaccines against parasites are unlikely to become available for the control of worms on farms within the next decade. however, a vaccine for the control of haemonchus species in sheep and cattle may be the exception (bassetto et al. 2010). dairy calves vaccinated three times with a gut membrane from h. contortus immunogen and then challenged with h. contortus and h. placei showed a significant reduction of egg counts for h. contortus (100.0%) and reduction in worm count for h. placei (68.4%) (bassetto et al. 2010). conclusion top ↑ nineteen gi nematode species that belong to seven families have been found to occur in cattle in zimbabwe. cooperia pectinata, c. punctata, h. placei and t. axei are the dominant species. production and husbandry practices, season, host-age and environment are considered to be the main factors that influence gi nematode infection in cattle in this country. based on current epidemiological information, well-organised national preventive treatment strategies should be employed to control gi nematode infections in cattle. a broad-spectrum ah administered tactically in january and march would control immature and adult forms of h. placei, o. radiatum, cooperia species and trichostrongylus species. it is recommended that a strategic treatment with a broad-spectrum ah that is effective against arrested h. placei larvae and that also has an extended effect against adult cooperia species and trichostrongylus species should be administered in july. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions d.m.p. 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1–17. vercruysse, j. & claerebout, e., 2001, ‘treatment vs. non-treatment of helminth infections in cattle: defining the thresholds’, veterinary parasitology 98, 195–214. waghorn, t.s., leathwick, d.m., chen, l.y. & skipp, r.a., 2003, ‘efficacy of the nematode-trapping fungus duddingtonia flagrans against three species of gastro-intestinal nematodes in laboratory faecal cultures from sheep and goats’, veterinary parasitology 118, 227–234. http://dx.doi.org/10.1016/j.vetpar.2003.09.018, pmid:14729170 waller, p.j. & thamsborg, s.m., 2004, ‘nematode control in ‘green’ ruminant production systems’, trends in parasitology 20, 493–497. http://dx.doi.org/10.1016/j.pt.2004.07.012, pmid:15363444 walsh, a., younis, p.j. & morton, j.m., 1995, ‘the effect of ivermectin treatment of late pregnant dairy cows in south-west victoria on subsequent milk production and reproductive performance’, australian veterinary journal 72, 201–207. http://dx.doi.org/10.1111/j.1751-0813.1995.tb03520.x, pmid:8526811 waruiru, r.m., munyua, w.k., thamsborg, s.m., nansen, p., bøgh, h.o. & gathuma, j.m., 1998, ‘development and survival of infective larvae of gastrointestinal nematodes of cattle on pasture in central kenya’, veterinary research communications 22, 315–323. http://dx.doi.org/10.1023/a:1006112802459, pmid:9778777 williams, j.c. & mayhew, r.l., 1967, ‘survival of infective larvae of the cattle nematodes cooperia punctata, trichostrongylus axei and oesophagostomum radiatum’, american journal of veterinary research 28, 629. pmid:6068246 wolstrup, j., gronvold, j., henriksen, s.a., nansen, p., larsen, m., bogh, h.o. et al. 1994, ‘an attempt to implement the nematode-trapping fungus duddingtonia flagrans in biological control of free-living stages of trichostrongyles in first year grazing calves’, journal of helminthology 68, 175–180. http://dx.doi.org/10.1017/s0022149x00013729, pmid:7930461 zinsstag, j., ankers, p.h., nije, m., smith, t., pandey, v.s., pfister, k. et al. 2000, ‘heritability of gastrointestinal nematode faecal egg counts in west africa village n’dama cattle and its relation to age’, veterinary parasitology 89, 71–78. http://dx.doi.org/10.1016/s0304-4017(99)00226-5 article information authors: william a. taylor1,2 john d. skinner1,† joop boomker3,4 affiliations: 1centre for veterinary wildlife studies, faculty of veterinary science, university of pretoria, south africa 2department of zoology and physiology, university of wyoming, united states of america3department of veterinary tropical diseases, university of pretoria, south africa 4department of zoology, university of johannesburg, south africa correspondence to: william taylor postal address: po box 71664, bryanston 2021, south africa dates: received: 08 jan. 2013 accepted: 18 apr. 2013 published: 16 may 2013 how to cite this article: taylor, w.a., skinner, j.d. & boomker, j., 2013, ‘nematodes of the small intestine of african buffaloes, syncerus caffer, in the kruger national park, south africa’, onderstepoort journal of veterinary research 80(1), art. #562, 4 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.562 note: †prof. john d. skinner passed away on 28 august 2011 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. nematodes of the small intestine of african buffaloes, syncerus caffer, in the kruger national park, south africa in this research communication... open access • abstract • communication • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the abundance and distribution of parasitic helminths in populations of african buffaloes, syncerus caffer, have not been well documented. a total of 28 buffaloes of different ages and sexes were sampled in the kruger national park, south africa, for nematodes of the small intestine. three nematode species were identified, namely cooperia fuelleborni, cooperia hungi and trichostrongylus deflexus, with c. hungi being a new country record for african buffalo in south africa. the overall prevalence was 71% and the average number of worms was 2346 (range: 0–15 980). this is a small burden for such a large mammal. sex, age and body condition of the buffaloes had no significant effect on worm occurrence. communication top ↑ helminths of african buffaloes, syncerus caffer, have not been well documented compared with many southern african ungulates (boomker et al. 1996). although at least 25 nematode species have been recorded in the gastro-intestinal tract (git) of buffaloes across africa (with 13 of these recorded in south africa), quantitative studies to date have used small sample sizes, such as four from the kruger national park (knp), south africa, and four from the hluhluwe-imfolozi game reserve (himgr), south africa (boomker et al. 1996). the average burdens from these two collections were 1086 and 2096 worms, respectively. such numbers appear low for an animal the size of a buffalo and the general impression is that buffaloes do not harbour significant intestinal nematode burdens (boomker et al. 1996). basson et al. (1970) examined the carcasses of 100 buffaloes in the knp for parasitic and other diseases and recorded nine helminth species in the gits, but they did not document numbers or how the worms were distributed within the host population. other large-scale studies of helminths in african buffaloes have been based on faecal egg counts (e.g. caron, cross & du toit 2003; penzhorn 2000), which do not identify nematodes at the species level and which cannot be considered reliable for assessing the size of nematode burdens (penzhorn 2000; reinecke 1983). overall, therefore, we know little about the abundance and distribution of nematode communities in buffalo populations. in 2005, south african national parks (sanparks) performed a large-scale cull of buffaloes in the southern section of the knp (25° s, 31° e) to assess the prevalence of bovine tuberculosis. the culling method involved trained personnel shooting buffaloes from a helicopter, with up to 20 individuals being taken from a single herd in one day. each herd was sampled once and culled animals approximately represented a random cross section of the population. immediately after shooting, a large professional sanparks crew were available to locate all dead animals, which were quickly eviscerated in the field. we obtained the small intestines (si) of 28 buffaloes from four herds over four days during the cull (04–05 july 2005 and 18–19 july 2005). time and personnel constraints prevented the processing of more samples. a cross section of buffalo genders and ages was taken, which included 16 male animals (seven of which were under two years of age) and 12 female animals (three of which were under two years of age) (table 1). the field evisceration allowed quick access to the git of selected animals and we ligated their sis within 1.5 h after death. samples were then transported to a wet laboratory in the knp for processing, where the parasites were collected based on procedures described by horak (1978). hearts, lungs and livers were not collected because they were required for tuberculosis analysis, whilst abomasa and large intestines were not examined owing to logistical constraints. aliquots, each representing a twentieth of the volume of the si contents, were collected for microscopic examination and stored in 10% formalin for later identification and counting. permission for collecting this biological material was obtained from the scientific division of sanparks, whilst the ethics committee of the faculty of veterinary science, university of pretoria approved of our methods. knp veterinary staff kindly provided data on the sex, age and body condition score of each animal. these variables were included in statistical models as potential predictors of worm burdens. table 1: individual buffalo statistics and nematode infection results for 28 african buffaloes culled in the kruger national park, july 2005. the degree of aggregation of the nematodes was estimated using the corrected moment estimate for aggregation (k), where k = (m2 – s2/n)/(s2 – m) and m denotes mean parasite burden, s2 denotes variance and n denotes sample size (wilson et al. 2002). a value of k < 1 indicates over-dispersion. using the statistical software programme r (r development core team 2008), the effects of buffalo sex, age and body condition on nematode presence or absence were tested with logistic regression using the ‘glm’ function and binomial family. buffalo sex (two categories) and body condition (five categories) were entered as factors, whilst age was entered as a continuous variable. to check the fit of the model, the difference between the residual deviance of the overall model (the model with three predictors) and the residual deviance of the null model (intercept only) was calculated, and this was used in a χ2 test with degrees of freedom (df) equal to the number of predictor variables. goodness-of-fit of the model was verified with a separate χ2 test, also based on the residual deviance and degrees of freedom. the effects of the predictor variables on the nematode counts were tested with a generalised linear model using the negative binomial distribution (glm function, negative binomial family), because nematodes were over-dispersed (see results). the only model that converged was a reduced model with one predictor (age). model fit and goodness-of-fit tests were based on residual deviance and degrees of freedom.three species of nematodes were identified, namely cooperia fuelleborni, cooperia hungi and trichostrongylus deflexus, with an overall prevalence of 71% (table 1). adult nematode counts ranged from zero (n = 8) to 15 980 (n = 1), with an average count of 2346 worms. the frequency distributions for the nematodes were highly aggregated (k = 0.35), indicating over-dispersion (figure 1). nematode larvae, which occurred in small numbers, were not counted. figure 1: observed frequency distributions of (a) all nematodes (k = 0.35) and (b) cooperia fuelleborni (k = 0.34) in 28 african buffaloes. for nematode presence or absence, the overall model did not fit the data significantly better than the null model (null deviance – residual deviance = 4.85; null df – residual df = 5.00; p = 0.43), indicating that none of the independent variables were good predictors of nematode infection. the goodness-of-fit test indicated that the logistic model fit the data (p = 0.16). for nematode counts, the overall negative binomial model did not converge, so a reduced model with a single predictor variable (age) was used. the reduced model did not fit the data significantly better than the null model (null deviance – residual deviance = 0.05; null df – residual df = 1.00; p = 0.82), indicating that buffalo age was not a good predictor of nematode burden. the goodness-of-fit test indicated that the negative binomial model fit the data (p = 0.21).out of 13 nematode species so far known to occur in the git of african buffaloes in south africa, six (agriostomum gorgonis, c. fuelleborni, cooperia pectinata, cooperia punctata, trichostrongylus axei and t. deflexus) had been found in the si (boomker et al. 1996). in the present study, c. hungi is a new country record, but not a new host record because it had previously been recorded in african buffaloes outside south africa (boomker et al. 1996). many of the nematode species recorded previously had been collected incidentally (boomker et al. 1996), so it is difficult to compare the number of species found in the present study (three species) with other studies. basson et al. (1970) found four si species in 100 buffaloes, but it is not clear how thoroughly these animals were examined. worm burdens found in the present study are slightly higher than found previously, where four animals in the knp had a mean burden of 1086 worms (range: 148–2017) and four animals in the himgr had a mean burden of 2096 worms (range: 0–8383) (boomker et al. 1996). these earlier studies examined the entire git, whilst the present study included worms only from the si. the high count of worms in the present study (15 980) is twice the largest burden found in previous studies (8383, in himgr), but this is probably the result of the larger sample size. when compared with the average nematode burdens found in the si of other south african grazing ungulate species, the average burden in buffaloes falls at the high end of the range. for example, mountain reedbuck, redunca fulvorufula, in the free state had an average si burden of 2047 worms in 41 animals (taylor et al. 2005), whilst bontebok, damaliscus dorcus, in the bontebok national park had an average si burden of 334 worms in 16 animals (boomker & horak 1992). given that african buffaloes are approximately 18 times larger than mountain reedbuck and 9 times larger than bontebok, these worm burdens are not high for their body size. to put these burdens in perspective: it has been estimated that 300 000 infective larvae of cooperia species need to be ingested to produce clinical signs in cattle (reinecke 1983). this finding concurs with the statement of boomker et al. (1996) that, taking the nematode species diversity into account, burdens are not significant and should not inconvenience buffaloes. the nematodes were highly aggregated, as is common for parasite distributions (wilson et al. 2002), with most buffaloes harbouring burdens less than the average of 2346 worms. with regard to the nematode occurrence in the present study, none of the independent variables included were good predictors in the buffalo. it is recognised, however, that the number of buffaloes collected (n = 28) represents a small sample, so the outcome of the generalised linear models, particularly the negative binomial model for nematode counts, should be treated with caution. some studies have shown that male animals are more prone to parasitic infection than female animals (wilson et al. 2002). one proposed explanation for such an observation is that higher testosterone levels suppress the immune system, but such a sex-dependent effect on nematode presence or absence was not apparent in this case. in fact, the average burden in female buffaloes (3783 worms) was higher than in male buffaloes (1189 worms), but the lack of convergence in the generalised linear model prevented statistical comparison. acknowledgements top ↑ our thanks go to the late ryno watermeyer for assistance with nematode identification, brian kuhn and ian bredin for assistance during nematode collection in the knp, and louis van schalkwyk, peter buss, roy bengis, at dekker and the knp scientific services for logistic assistance and providing permits. two anonymous referees helped improve the manuscript. the study was funded by the university of pretoria. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions w.a.t. (university of pretoria and university of wyoming) collected and identified the nematodes and wrote the manuscript. j.d.s. (university of pretoria) funded the fieldwork and assisted with project design and scientific feedback. j.b. (university of pretoria and university of johannesburg) assisted with the project design and equipment, scientific feedback, nematode identification and manuscript comments. references top ↑ basson, p.a., mccully, r.m., kruger, s.p., van niekerk, j.w., young, e. & de vos, v., 1970, ‘parasitic and other diseases of the african buffalo in the kruger national park’, onderstepoort journal of veterinary research 37, 11–28. pmid:5535831 boomker, j. & horak, i.g., 1992, ‘parasites of south african wildlife. xiii. helminths of grey rhebuck, pelea capreouls, and of bontebok, damaliscus dorcus dorcus, in the bontebok national park’, onderstepoort journal of veterinary research 59, 175–182. pmid:1437019 boomker, j., horak, i.g., penzhorn, b.l. & keet, d.f., 1996, ‘parasites of african buffaloes: a documentation’, in b.l. penzhorn (ed.), proceedings of a symposium on the african buffalo as a game ranch animal, pp. 90–103, onderstepoort, pretoria. caron, a., cross, p.c. & du toit, j.t., 2003, ‘ecological implications of bovine tuberculosis in african buffalo herds’, ecological applications 13, 1338–1345. http://dx.doi.org/10.1890/02-5266 horak, i.g., 1978, ‘parasites of domestic and wild animals in south africa. x. helminths in impala’, onderstepoort journal of veterinary research 45, 221–228. pmid:572950 penzhorn, b.l., 2000, ‘coccidian oocyst and nematode egg counts of free-ranging african buffalo (syncerus caffer) in the kruger national park, south africa’, journal of the south african veterinary association 71, 106–108. http://dx.doi.org/10.4102/jsava.v71i2.689, pmid:11030361 r development core team, 2008, r: a language and environment for statistical computing, r foundation for statistical computing, vienna. reinecke, r.k., 1983, veterinary helminthology, butterworths, pretoria. taylor, w.a., boomker, j., krecek, r.c., skinner, j.d. & watermeyer, r., 2005, ‘helminths in sympatric populations of mountain reedbuck (redunca fulvorufula) and gray rhebok (pelea capreolus) in south africa’, journal of parasitology 91, 863–870. http://dx.doi.org/10.1645/ge-436r.1, pmid:17089756 wilson, k., bjornstad, o.n., dobson, a.p., merler, s., poglayen, g., randolph, s.e., read, a.f. & skorping, a., 2002, ‘heterogeneities in macroparasitic infections: patterns and processes’, in p.j. hudson, a. rizzoli, b.t. grenfell, j.a.p. heesterbeek (eds.), the ecology of wildlife diseases, pp. 6–44, oxford university press, oxford. pmid:15997619 article information author: delia grace1 affiliation: 1international livestock research institute, nairobi, kenya correspondence to: delia grace postal address: po box 30709, nairobi, kenya how to cite this article: grace, d., 2014, ‘the business case for one health’, onderstepoort journal of veterinary research 81(2), art. #725, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.725 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the business case for one health in this proceedings... open access • abstract • introduction • background and definitions for one health • framework for categorising one health problems and interventions    • key areas       • joining up health resources: sharing health resources between sectors       • controlling zoonoses in animal reservoirs       • early outbreak detection       • pandemic prevention       • adding value to health research and development • discussion • conclusion • acknowledgements    • competing interests • references abstract top ↑ this article outlines a pathway to develop the business case for one health. it describes the origin and development of one health and then identifies five potential areas where one health can add value and reduce costs. these are: (1) sharing health resources between the medical and veterinary sectors; (2) controlling zoonoses in animal reservoirs; (3) early detection and response to emerging diseases; (4) prevention of pandemics; and (5) generating insights and adding value to health research and development. examples are given for each category along with preliminary estimates of the potential savings from adopting the one health approach. the literature reviewed suggests that one dollar invested in one health can generate five dollars worth of benefits and a global investment of us$25 billion over 10 years could generate benefits worth at least us$125bn. conservation implications: the time has come to make the bigger case for massive investment in one health in order to transform the management of neglected and emerging zoonoses and to save the lives of millions of people and hundreds of millions of animals whose production supports and nourishes billions of impoverished people per annum. introduction top ↑ this article is based on an invited keynote presentation given at the southern african centre for infectious disease surveillance (sacids) one health (oh) conference held in arusha, tanzania in april 2013 (grace 2013). it draws on the experiences of the international livestock research institute (ilri) that were gathered through a number of oh and ecohealth (eh) projects over the last decade and incorporates findings from a literature review. oh is a broad movement that recognises the fact that human, animal and ecosystem health are interdependent and that multidisciplinary collaborations are often necessary in order to attain optimum health solutions. the article was motivated by a growing consensus that although oh is well understood and appreciated it has yet to gain large-scale traction in the medical and donor communities. as of 2013, oh is endorsed by global standard makers, namely, the world organisation for animal health (oie), the world health organization (who) and the food and agriculture organization of the united nations (fao), as well as being supported by the world bank. the last decade has seen a large number of conferences and a series of international inter-ministerial meetings with a focus on avian and pandemic influenza. there are currently around 20 universities offering oh graduate degree courses, including institutes in america, africa, europe and asia. the oh initiative acts as a clearinghouse for information on oh (http://www.onehealthinitiative.com), the one health global network operates a web-portal (http://www.onehealthglobal.net/) to facilitate communication and the one health commission also shares information ( https://www.onehealthcommission.org/). the ecohealth journal publishes on eh and articles on both oh and eh have appeared in other major epidemiology and infection journals. in addition, 2012 saw a major new publication on eh (charron 2012), updating the classic by lebel (2003). however, despite this large and growing body of evidence supporting the usefulness of oh, the great majority of medical education, clinical practice, ancillary services, development programmes and research continue to operate within disciplinary boundaries. this lack of uptake of oh was attributed initially to there being insufficient evidence to convince practitioners and decision makers. however, notwithstanding the growing evidence for oh, the organisation of health by sectors still persists to a large extent. at least partly in response, attention has turned to the economic justification of oh, with an important article by the world bank setting out the potential savings from investing in oh in order to prevent pandemics (world bank 2012). yet others argue that promotion of oh requires an understanding of how major shifts in health policy and practice occur, as well as how the ability to influence opinion shifts. many models exist for both understanding and influencing policy change and most of these involve, either implicitly or explicitly, a ‘theory of change’. these models recognise that evidence is only one part of policy influence and that positive influence of policy is highly dependent on context (jones 2011). this article discusses the process of building a compelling business case for oh in the context of a theory of change for adoption thereof. we first discuss various definitions of oh. next, we present a ‘big five’ framework for categorising oh problems and related interventions. we then provide estimates from the literature on the costs of the oh problem as well as the likely costs and benefits of oh interventions, drawing on a small number of key articles and reports. finally, we provide recommendations for building a convincing business case for one health. background and definitions for one health top ↑ that human, animal and environmental health is related has been recognised throughout the historical development of medicine on all continents (zinsstag et al. 2011). in most indigenous and historical medical systems, animal and human diseases are conceptualised as having similar causes, manifestations and treatments. but as medicine developed into a profession, a separation grew between those who treated humans and those who treated animals, with human doctors given higher status. (that being said, entry to veterinary schools in several countries in recent decades has been more competitive than entry to medical schools.) in the 19th century, european medicine became the dominant paradigm and entrenched the largely sectoral approach wherein human and animal health was separate disciplines and there were only weak connections between ecology and health. the 20th century saw three major movements, all of which contributed largely to current thinking on oh. the first was the concept of ‘one medicine’ which arose out of the work of calvin schwabe with the dinka in sudan (zinsstag et al. 2011). the second movement was ‘ecosystem health’ or ‘ecohealth’. this adapted thinking from ecology and environmental management to the improvement of human health and wellbeing. important early work on mercury contamination in the amazon basin was supported by the canadian international development research centre, which has continued to support research and programmes and to develop the approach (charron 2012). the third movement, which took the title of oh, arose because of increasing concern of disease emergence at the interface between animals, humans and ecosystems (nabarro 2012). this was triggered by a series of disease emergences of global importance in the 1990s, including severe acute respiratory syndrome (sars), avian influenza and west nile virus and had strong participation from veterinary and, to a lesser extent, human public health. one health can be defined as the collaborative effort of multiple disciplines to attain optimal health for people, animals, and our environment. ecohealth has been defined as systemic, participatory approaches to understanding and promoting both health and well-being in the context of social and ecological interactions. both definitions emphasise multidisciplinarity and the importance of agriculture and ecosystem-based interventions in order to attain health goals. the first decade of the 21st century saw an increasing convergence of one health and ecohealth, leading to the development of a broader discipline which incorporates infectious and non-infectious disease, epidemiological and ecological methods and both disease control and development (zinsstag 2011). the boundaries of oh and eh are fuzzy, with interpretations differing between groups. for example, the stone mountain working group felt that oh initiatives should address human, animal and environmental health simultaneously. on the other hand, the ecohealth journal (http://www.ecohealth.net) publishes not only on zoonoses but also on diseases that affect only wildlife (such as bat white-nose syndrome). the disease theme of the consultative group on international agricultural research (cgiar) research program on agriculture for nutrition and health focuses on food-borne diseases and zoonoses but also goes beyond zoonoses and occupational hazards to consider such things as obesity and anthroponotic but agriculture-related diseases as being oh issues (e.g. malaria, which is linked to irrigation but is not a zoonosis). this article considers as oh both those interventions and actions which address diseases that are either actually or potentially common to both humans and animals. this includes toxicosis due to aflatoxins, heavy metals or other injurious substances; resistance to antibiotics, parasiticides or other drugs used to treat both humans and animals; classical zoonoses such as brucellosis and tuberculosis; and emerging diseases such as avian influenza which are either zoonotic or potentially zoonotic. framework for categorising one health problems and interventions top ↑ several reviews suggest areas where oh is most likely to make a difference (rushton et al. 2012; world bank 2012). this article identifies a ‘big five’ of key areas. for each, the review provides a brief description of the area for collaboration, cites examples and calculates estimates on costs averted and benefits obtained by an oh approach. key areas joining up health resources: sharing health resources between sectors sharing health resources across human and veterinary health sectors would appear to be an easy win. this is especially the case for laboratory facilities as the majority of pathogens and chemical hazards are common to both humans and animals. joint laboratory facilities are particularly important in developing countries where scarcity of human and financial resources challenge the sustained operation of laboratory resources. a second area is shared education resources; much of the pre-clinical curriculum is common to both human and veterinary medicine and there are similar overlaps in many paraveterinary and paramedical curricula. disciplines which work at a scale much higher than the individual patient (e.g. epidemiology) or much l ower (e.g. molecular biology) use essentially the same methods for both humans and animals and can eas ily combine forces. there are already some courses which target both veterinarians and medical doctor s, for example joint masters degrees and the integrated courses delivered by the south african field epidemiology and laboratory training programme (safeltp). surveillance is a third area where there are obvious advantages to systems which are at least joined up and possibly integrated. several o f the most important surveillance systems are ‘one health’; these include promed (http://www.promedmail.org), healthmap (http://healthmap.org/en/) and the global early warning system (glews, http://www.glews.net). a fourth possible area for joining forces is disease control, especially in remote, livestock-keeping communities. linking livestock vaccination with vaccination of children has been piloted in chad in communities where the majority of livestock, but no children, were vaccinated (schelling et al. 2007).estimating the benefits of collaboration across medical and veterinary services is challenging. the operation of a joint laboratory in winnipeg is estimated to have reduced overall costs by 26% (world bank 2012). the world bank (2012) estimates a 10.0% – 27.5% saving across a range of joint services for avian influenza control. the campaign in chad suggested that combining both human and livestock vaccination reduced overall costs by 15% (schelling et al. 2007). we assume that both of these are on an annual basis. human health expenditure in developing countries was estimated at us$521 billion in 2012 (ihme 2012). estimations for veterinary health expenditure are less solid, but combining data from a number of studies suggests an expenditure of us$1bn – us$2bn on public animal health services in developing countries (bonnet et al. 2011; gallacher 2007; oie 2009). based on the studies cited above, it can be assumed that joint operations can save around 10% of the combined medical and veterinary budget devoted to those functions which are amenable to sharing. best available evidence suggests that laboratories, education and management of zoonoses are services which can be shared and that these constitute 5% of the human health budget and 40% of the veterinary health budget (eurostat 2012). this implies that the total savings of joined-up services could be us$2.68bn per year. given estimates of the cost of collaboration, net savings of around us$3bn imply gross savings of around us$4bn per year. controlling zoonoses in animal reservoirs historically, most major zoonoses that have been controlled successfully have concentrated on the animal reservoir. this includes diseases such as brucellosis, tuberculosis, rabies, salmonellosis, cysticercosis, trichinellosis and others which have been controlled successfully in many countries. a large number of economic analyses have been conducted in order to compare the costs and benefits of control and/or localised elimination of zoonoses. table 1 summarises some representative studies. economic assessments conducted after a control campaign is finished give results comparable to ex ante assessments, which suggests that ex ante assessments are credible. (however, this may be less true when animal health services are weak.) whilst a wide range of benefit-to-cost ratios are reported, all the literature reviewed showed a positive ratio. the median ratio of benefits to costs was around four to one with human health benefits at least equal to animal health benefits and often greater. this approximate, but relatively robust, estimate allows an assessment of the costs and benefits of controlling zoonoses in developing countries. a recent study estimates that around one in seven (14%) livestock in developing countries each year are currently or recently infected with one or more zoonoses and that each infection reduces their productivity by around 10% (grace 2013). according to the fao, the value of livestock production in developing countries was us$639bn per year at the time of their study (faostat, 2012), suggesting that the productivity losses related to zoonoses is around us$9.26bn per year (assuming that current losses are 1.4% and that, without these, production would be us$648bn). in addition to morbidity, mortality is an important cause of loss for livestock. numerous studies on developing country livestock indicate that annual mortality is high. otte and chilonda (2002) provide the most thorough review (although only covering africa) and this implies a mortality rate of 21% weighted by age and species. assuming that half of livestock mortality is because of disease and that half the disease is a result of zoonoses (world bank 2011), then developing countries currently lose approximately 68 million tropical livestock units (tlu) because of zoonoses (this represents 25% livestock mortality as a result of zoonoses, namely, 50% of 50% of disease-related livestock mortality). to extrapolate, assuming that the value of a tlu is us$366, this costs developing countries us$25bn per year. on the other hand, official reports of the oie suggest that only around 400 000 livestock units are lost each year in developing countries – a mortality rate of 0.03% (world bank 2011). this implausibly low estimate is an artefact of under-reporting and under-estimation of losses by between two and three orders of magnitude (grace et al. 2012). the human health costs of zoonoses are typically equal to or greater than the livestock sector losses, a trend which is becoming more pronounced with time (world bank 2012). the aforementioned study suggested 2.2 million human deaths and 2.4 billion human illnesses a year from zoonoses (grace et al. 2012). using standard and conservative costs of human illness, we may assume losses of at least us$50 billion in 2013. this implies that the annual costs of zoonoses may be us$9bn in lost productivity, us$25bn in livestock mortality and us$50bn from human health – rounded up to us$85bn in all per year. based on the studies cited in table 1, the costs of control are typically one fourth the benefits and a control programme may extend for five to 10 years, so the us$85bn in annual losses could be averted by a expenditure of us$21bn over this period. (please note that, for simplicity, we do not consider discounting in this or other estimates). table 1: a selection of studies showing the costs and benefits of control of zoonoses. early outbreak detection in the case of highly contagious diseases, rapid response is key to reducing the cost of disease outbreaks. it is difficult to estimate the costs of a counterfactual, that is, the costs that would have been incurred had response to outbreaks been more timely. however, an approximate quantification of the savings obtainable by early and efficient management can be obtained by comparing the costs of epidemics caused by the same disease in different contexts. for example, control of the bovine spongiform encephalitis (bse) outbreak in britain is considered widely to have been suboptimal because of a range of factors (including the novelty of the disease; lack of understanding of impacts; over-estimation of the effectiveness of control; and the wide establishment of the disease). on the other hand, when the disease was introduced to other countries, response was more rapid and effective. canada can then act as a counterfactual for the uk and, by comparing the costs of the outbreaks in the two countries, we can estimate that if control had been as timely and effective in the uk as in canada, 88% of the costs could have been averted (table 2).contexts that affect the timeliness of control include: whether the disease is a surprise or anticipated; whether there is effective surveillance in place or not; and whether public services are well funded and well functioning or have suboptimal performance. by comparing effectively-controlled epidemics with poorly-controlled epidemics we estimate that well-functioning surveillance systems and timely responses may reduce the cost of outbreaks by 95%. the world bank estimates that outbreaks have cost on average us$6.7bn from 1997–2009 (world bank 2011). they estimate that a us$3.4bn investment in animal health systems per annum would support these systems so that they could function effectively and efficiently, enabling them to avert the losses incurred through delayed or inadequate response. a 95% reduction in costs amounts to us$6bn saved per year. table 2: comparing worst case and better case disease surveillance and response incidents. pandemic prevention in addition to the ongoing losses from disease outbreaks, which have become the ‘new normal’, there is considerable concern over the possibility of a civilisation-altering pandemic or plague. these have occurred regularly but infrequently throughout history and pre-history, with the most recent example being the hiv pandemic. in a landmark study, the world bank considers the possible impacts and costs of averting high impact but low probability pandemics (burns, van der mensbrugghe & timmer 2008). we draw attention to their key message: a severe pandemic costing us$3 trillion may occur, on average, once in a hundred years. if the investments in one health systems are made and such a pandemic is prevented, the global expected benefits are us$30 billion per year. every year, an investment of us$3.4 billion would produce an expected benefit of us$30 billion for the international community. (burns et al. 2008) this expenditure of us$3.4bn on strengthening veterinary services would hence deliver two streams of benefits: averting major pandemics with an expected benefit of us$30bn and improving the timeliness of response to outbreaks with an annual expected benefit of us$6bn a year. adding value to health research and development oh and eh lead to better research and disease control programmes as well as ecosystems better able to provide health as a regulatory service. evidence for the value of oh and eh has been presented at major conferences. the most recent include: addis (2011), arusha (2013), bangkok (2013), davos (2012), johannesburg (2011), kunming (2012), london (2010) and melbourne (2011). important meetings have also been held where global health leaders endorsed the approach: these include gatherings in new york (2004), winnipeg (2009), bellagio (2010), london (2009), and stone mountain (2010). a recent review identified 41 major oh initiatives (rockefeller foundation 2011), another review showcased 31 oh projects (vsf 2010) and a recent book sets out eh theory and practice (charron 2012). whilst a large and growing body of evidence supports the hypothesis that adopting both oh and eh improves the effectiveness and efficiency of health research and delivery, the costs and benefits of adopting oh and eh as an approach to research and development are difficult to quantify. it is generally accepted that approaches which are highly participatory and multidisciplinary may have additional costs. on the other hand, participatory approaches can decrease some costs and increase sustainability. we consider this to be an important area for future research, but are unable to provide a monetary estimate. discussion top ↑ we reviewed the literature in order to develop a framework for assessing the benefits of one health approaches and interventions and to summarise information on the values thereof. there is an enormous lack of information on the burden of zoonoses and much of the evidence that exists is not readily available; hence, the estimates in this review should not be considered as definitive, but rather as examples of how important benefits from oh and eh could be quantified. with this caveat, this preliminary review (presented at sacids 2013 [grace 2013]) has developed initial estimates of the possible costs of zoonoses, the investments needed to control them and the benefits derived therefrom. dollar estimates are summarised in table 3: a us$25bn annual investment over 10 years would generate annual benefits worth at least us$125bn (excluding discounting). additional benefits include saved dalys (disability adjusted life years) which reflect the disutility of illness, as well as conserved ecosystem health regulation through reduction of zoonoses spill-over to wildlife.developing a comprehensive and credible ex ante assessment of the business case for oh and eh requires investment. a first step is to develop and evaluate metrics that capture the impact of zoonoses and emerging disease on human health, the livestock sector, the broader economy and ecosystem health regulation. given the deficiencies of current official reporting systems, estimates of disease prevalence and impact are best obtained by literature review and ‘ground-truthing’ studies. however, long-term solutions need to involve the upgrading of reporting systems to a standard that ensures quality, transparency and reliability. this will require an appropriate incentive system to be in place in order to elicit such institutional changes amongst the key stakeholders and actors involved. appropriate policies can help to institutionalise these processes and functions at different levels of the reporting system hierarchy. the business case also needs to consider the options for controlling disease and their likely efficiency, effectiveness and acceptability. developing a detailed business case covering the economic case, options, risks and priorities for one health investments would require a multidisciplinary team with skills in epidemiology, economics and an understanding of developing country livestock sectors. we estimate that a fiveto 10-person team of experts, supported by research assistants and information technology, could build the business case in one year, whilst 30 people would take just months. we believe that a credible body of evidence with regard to the costs, benefits and feasibility of control of zoonoses would stimulate investments by donors and national governments, as well as by the non-profit and private sector. table 3: preliminary estimate of costs and benefits of one health investments over a 10-year period with benefits and costs per year. conclusion top ↑ emerging and neglected zoonoses have often been managed sectorally, but recent decades have shown, case after case, the benefits of one health management. the growing body of evidence suggests the time has come to make the bigger case for massive investment in one health in order to transform the management of neglected and emerging zoonoses and to save the lives of millions of people and hundreds of millions of animals whose production supports and nourishes billions of impoverished people per annum. acknowledgements top ↑ this work was funded in part by support from the ecosystem services for poverty alleviation programme (espa). the espa programme is funded by the department for international development (dfid), the economic and social research council (esrc) and the natural environment research council (nerc). i also thank lucy lapar and olga jonas for their review of a draft version of the article. competing interests the author declares that she has no financial or personal relationship(s) that may have inappropriately influenced her in writing this article. references top ↑ bonnet, p., lancelot, r., seegers, h. & martinez, d., 2011, contribution of veterinary activities to global food security for food derived from terrestrial and aquatic animals, article presented at the 29th general assembly, world organisation for animal health, paris, may 22–27, 79 sg/9.budke, c.m., jiamin, q., qian, w. & torgerson, p.r., 2005, ‘economic effects of echinococcosis in a disease-endemic region of the tibetan plateau’, american journal of tropical medicine and hygiene 73(1), 2–10. burns, a., van der mensbrugghe, d. & timmer, h., 2008, evaluating the economic consequences of avian influenza, world bank, viewed 11 december 2013, from http://documents.worldbank.org/curated/en/2006/06/10247442/evaluating-economic-consequences-avian-influenza charron, d.f. (ed.), 2012, ecohealth research in practice: innovative applications of an ecosystem approach to health, insight and innovation in international development, vol. 1, international development research centre/springer, canada. http://dx.doi.org/10.1007/978-1-4614-0517-7_1 department for environment, food and rural affairs (defra), 2011, bovine tb and badger control: consultation on guidance to natural england on the implementation and enforcement of a badger control policy, defra, uk. eurostat, 2012, healthcare statistics, viewed 04 august 2013, from http://epp.eurostat.ec.europa.eu/statistics_explained/index.php/healthcare_statistics food and agriculture organization of the united nations (faostat), 2012, what’s new in faostat?, viewed 04 august 2013, from http://faostat3.fao.org/home/index.html gallacher, m., 2007, economics of official veterinary services: the case of latin america, v.04 (fourth draft), viewed 04 august 2013, from http://www.oie.int/fileadmin/home/eng/support_to_oie_members/docs/pdf/cema_-_latin_am_case__en_.pdf grace, d., mutua, f., ochungo, p., kruska, r., jones, k., brierley, l. et al., 2012, mapping of poverty and likely zoonoses hotspots, zoonoses project 4, report to the uk department for international development, nairobi, kenya, ilri, p. 119. grace, d., 2013, one health approach makes sense in the changing socio-economic landscape of developing countries, article presented at the 27th annual joint scientific conference and 2nd one health conference in africa, 16–19 april, snow crest hotel, arusha, tanzania. grey,d.j., williams, g.m., li, y. & mcmanus, d.p., 2008, ‘transmission dynamics of schistosoma japonicum in the lakes and marshlands of china’, plos one 3(12):e4058. ihme (institute for health metrics and evaluation), 2012, financing global health 2012: the end of the golden age?, ihme, seattle. jones, h., 2011, a guide to monitoring and evaluating policy influence, odi background note, viewed 09 december 2013, from http://www.odi.org.uk/sites/odi.org.uk/files/odi-assets/publications-opinion-files/6453.pdf lebel, j., 2003, health: an ecosystem approach (in_focus), international development research centre, ottawa, canada. maijala, r. & peltola, j., 2002, finnish salmonella control program – efficiency and viability in food safety promotion, article presented at the 10th eaae congress, zaragoza, spain, august 28–31. mcdermott, j., grace, d. & zinsstag, j., 2013, ‘economics of brucellosis impact and control in low-income countries’, revue scientifique et technique (international office of epizootics) 32(1), 249–261. nabarro, d., 2012, ‘one health: towards safeguarding the health, food security and economic welfare of communities’, onderstepoort journal of veterinary research 79(2), art. #450, 3 pages. http://dx.doi.org/10.4102/ojvr.v79i2.450 oie (the world organisation for animal health), 2009, cost of national prevention systems for animal diseases and zoonoses in developing and transition countries, viewed 09 december 2013, from http://www.oie.int/fileadmin/home/eng/conferences_events/sites/oie-wb_conference_1007/oie-costs%20of%20national%20prevention%20systems-final%20report.pdf otte, m.j. & chilonda, p., 2002, cattle and small ruminant production systems in sub-saharan africa – a systematic review, food and agriculture organization of the united nations, rome. rockefeller foundation, 2011, portfolio of one health activities and case studies, rockefeller foundation and global initative for food systems leadership, university of minnesota. rushton, j., häsler, b., de haan, n. & rushton, r., 2012, ‘ economic benefits or drivers of a “one health” approach: why should anyone invest?’, onderstepoort journal of veterinary research 79(2), art. #461, 5 pages. http://dx.doi.org/10.4102/ojvr.v79i2.461 schelling, e., béchir, m., ahmed, m.a., wyss, k., randolph, t.f. & zinsstag, j., 2007, ‘human and animal vaccination delivery to remote nomadic families, chad’, emerging infectious diseases 13(3), 373–379. http://dx.doi.org/10.3201/eid1303.060391 shwiff, s.a., kirkpatrick, k.n. & sterner, r.t., 2008, ‘economic evaluation of an oral rabies vaccination program for control of a domestic dog-coyote rabies epizootic: 1995–2006’, journal of the american veterinary medical association 233(11), 1736–1741. http://dx.doi.org/10.2460/javma.233.11.1736 vsf (veterinarians without borders/vétérinaires sans frontières), 2010, one health for one world: a compendium of case studies, veterinarians without borders/vétérinaires sans frontières, canada. world bank, 2011, world livestock disease atlas: a quantitative analysis of global animal health data (2006–2009), world bank, washington, d.c., viewed 09 december 2013, from http://documents.worldbank.org/curated/en/2011/11/15812714/world-livestock-disease-atlas-quantitative-analysis-global-animal-health-data-2006-2009 world bank, 2012, people, pathogens and our planet: the economics of one health, world bank, washington, d.c., viewed 09 december 2013, from http://documents.worldbank.org/curated/en/2012/06/16360943/people-pathogens-planet-economics-one-health zinsstag, j., schelling, e., waltner-toews, d. & tanner, m., 2011, ‘ from one medicine” to “one health” and systemic approaches to health and well-being’, preventive veterinary medicine 101(3–4), 148–156. http://dx.doi.org/10.1016/j.prevetmed.2010.07.003 perry_49.indd 49 onderstepoort journal of veterinary research, 76:49 (2009) economic impacts of tick-borne diseases in africa b.d. perry ilri, nairobi, kenya abstract perry, b.d. 2009. economic impacts of tick-borne diseases in africa. onderstepoort journal of veterinary research,76:49 as we join onderstepoort in celebrating its centenary, it is worth reflecting that tick-borne infections of wildlife and livestock have been part of everyday life in africa for many, many generations. while much has changed since watkins-pitchford started developing short-interval dipping trials with arsenicals to contain the new epidemic of east coast fever in southern africa 100 years ago, when it comes to the impacts of tick-borne infections, many of the same challenges remain. these impacts are characterised at one extreme by the high cost and environmental inappropriateness of chemical acaricides used to prevent losses from the cluster of diseases, each with its particular host and vector association, and on the other by the lost potential of indigenous breeds in settings where they enjoy the ecological climax of endemic stability. between these extremes are the variable degrees of morbidity and mortality associated with endemic instability. this keynote paper will review the diversity of these impacts, and discuss the value of broader consideration of societal impacts of the diseases and their control. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice marwick_123-125.indd roots private practice became the major vocation of local veterinarians from about 1945. soga was probably the first qualified south african veterinarian to practice privately on a limited scale in the 1800s. jack boswell was the first locally qualified (in 1934) veterinarian to go directly into practice without first working for the government and is regarded as the father of private practice in south africa. boet bekker worked for the government before setting up a successful practice in 1935. predicting the future every day, millions of people read their horoscope in the hope that it might offer guidance for the future of their lives. but lacking the use of tea leaves or a crystal ball, all that i can say is if one reads the press and popular publications and takes yourself out of the box, one begins to see and understand the bigger picture of veterinary science moving on to the 21st century. the biggest impact that causes an industry to change usually comes from outside the industry and such changes are also going to impact on veterinary science in the future. therefore, one should constantly look over one’s shoulder to see who might be creating something new that could change veterinary practice, and then stay one step ahead to survive. i believe that veterinarians have without question the most honorable and must wonderful profession that exists on the planet. the range and diversity of the profession is such that a veterinarian can branch out into many sectors of veterinary science and there will always be a lot of willing takers of these services should there be an economic advantage if one should apply them. i am sure the future on into the 21st century is so bright for veterinarians that they are going to have to wear sun glasses. challenges facing us today before we look too far into the future of veterinary science, we should look at the biggest challenge that faces us as veterinarians, which is for a permanent solution to the veterinary workforce shortfall in south africa. the starting point to succeed can be found in four areas: recruitment, selection, education and retention. the veterinary workforce shortfall is a multi-faceted problem and i don’t claim to have all the answers. i think onderstepoort realizes the magnitude of the problem and is trying to do something about it. there are, however, some things that each and every veterinarian can do to help. become a gatherer, recruiter and mentor for young people interested in what you are doing as a veterinarian. whether you are employed in state or provincial services, private practice, industry, academy or research, you can impact on someone’s life dramatically. i had a very good veterinary education but learnt all of bovine medicine, reproduction and nutrition from mentors, all of whom excelled in their own fields. there is just so much that veterinary education can do to prepare students for their career path—the rest is up to the student and the mentor. i therefore see that we as veterinarians will have to play a vital role in education and early mentorship in the future if we wish to stem the tide of ever decreasing numbers of rural practitioners and intend to set the demographic representation of the veterinary workforce on a new course. 123 onderstepoort journal of veterinary research, 76:123–125 (2009) the future of private veterinary practice in south africa c. marwick p.o. box 248, clocolan, 9735 south africa 124 future of private veterinary practice in south africa if veterinarians wish to pass the life boat test, the faculty needs to make graduating veterinarians more relevant to society in the 21st century, as the needs of the veterinary profession is severely imbalanced with more than 75 % of qualifying veterinarians ending up in companion animal practice. this makes the present day delivery of veterinary services, though essential for companion animals, a luxury item that loses its support base in a strained society that is ever increasingly monitoring animal welfare and food security. i don’t think one can overplay the mystique of james herriot and we should remind ourselves that is why we do what we do and solutions to the veterinary workforce must start with us, the roll players in veterinary science. remember, ‘all politics are local’. future challenges the fundamentals of veterinary science are changing and veterinarians should prepare themselves for change. there is a shift from a national market that is commodity-focused to a global market which is much more value-added and niche-market focused. because most of the world’s population growth will take place in developing countries, the global demand for meat is projected to increase by 60 % by 2020. the second change is from emphasis on production increases to one on social acceptance of meat products. consumers have concerns about animal welfare and environmental issues, and they are becoming more interested in organically produced foods. veterinarians should be mindful that animal agriculturists, who have had a privileged status in the past, now have a lesser status caused by a public increasingly out of touch with agriculture (milk out of the bottle, etc.). a further shift is envisioning food as a health promoter, and not just for sustenance. scientific advances in the field of transgenic animals will usher in a new area in which animal products may be used exclusively as medicines and for preventative treatments. these products will involve intense efforts to educate the public as to their merit. finally, rural vet erinarians will be perceived as public advocates. veterinarians should be seen as protectors against zoonotic outbreaks as well as agri-terrorism attacks. all these changes will require the reinventing of the 21st century veterinarian. ‘when you are on a dead horse, the best strategy is to dismount.’ the solution is not to continue on the treadmill of producing meat faster and faster. step back and reassess your personal and professional pathway into the future. veterinarians can add value for their clients and expand professional capabilities by acquiring new skills and becoming involved in new activities. these include, to name a few, becoming an animal welfare advisor, food safety expert, bio-security planner, population health expert, participating in the management of the ecosystem for sustainable use, in the maintenance of wildlife health and a greater involvement in biomedical research, and acting as a public health advocate. veterinarians need to focus on improving communications skills, partnering with government agencies and departments, conducting environmental scans of their entire commodity chain and training and teaching producers. i am not here to give all the answers, but will suggest that the future is catching up with us veterinarians and there is an urgency to address the future and keep looking over your shoulder, for the future we will live in tomorrow is created by actions we take today. in the socio-political environment private veterinarians will have to play a vital roll in addressing the unique issues within south africa. i envisage that private veterinarians will be pivotal in delivering a service on behalf of the government with the aid of animal health technicians and primary health workers. the days of private veterinarians working in isolation are rapidly coming to an end and they need to build coalitions with state veterinary services, other professions as well as with the food animal industry as a whole. as we move on into the new millennium, we are truly at the crossroad of the private veterinarian’s history. primary health care, new economic models, political pressure to become more relevant to agri-economic environment, economic viability of the private practitioner and technological challenges have brought both opportunities and despair to the private practitioner. studies have shown that the continuing convergence of animal heath, human health and eco-system health is the new reality, and that the concept of one medicine should be embraced with the veterinarian playing a pivotal role, as almost every human disease can be caused, modified or altered by environmental agents. this has been clearly shown in the case of tb, hiv, monkey pox, avian influenza, etc. seventy-five percent of diseases that have 125 c. marwick emerged in the past 25 years are zoonotic in nature. the concept of one world, one health, one medicine, should be embraced by the veterinary profession in the future. the future is what we make of it and we need to adapt or die. by embracing new technology and keep ing ahead of the pack, the private practitioner in south 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice roeder_9.indd 9 onderstepoort journal of veterinary research, 76:9 (2009) rinderpest: an historical overview p. roeder global rinderpest eradication programme and hpai, fao, rome abstract roeder, p. 2009. rinderpest: an historical overview. onderstepoort journal of veterinary research, 76:9 rinderpest is one of the few diseases which have changed the course of world history. originally an asian disease, for centuries it had a devastating impact in europe when introduced by returning and marauding armies accompanied by cattle as well as by cattle trade. nowhere was its impact more dramatically expressed than in africa where the sequel to its introduction into the horn of africa was a devastating panzootic throughout sub-saharan africa during the last decade of the 19th century extending into the 20th century. massive deaths of livestock, wild animals and the people dependent on them led to widespread human misery and changed the face of the african continent forever. into the middle of the 20th century the story in africa and asia was one of repeated epizootics against a background of enzootic disease affecting both livestock and wildlife. colonial veterinary services were engrossed in rinderpest control and the resources needed for its control were often overwhelmed. yet, internationally-coordinated rinderpest programmes, of which the last is the global rinderpest eradication programme, have led to a point where there is growing confidence that the disease and rinderpest infection in the wild have been eradicated, having last been detected in pakistan in 2000 and kenya and sudan in 2001. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information author: hazel m. dockrell1 affiliation: 1department of immunology and infection, london school of hygiene & tropical medicine, united kingdom correspondence to: hazel dockrell postal address: department of immunology and infection, faculty of infectious and tropical diseases, london school of hygiene & tropical medicine, keppel street, london wc1e 7ht, uk how to cite this proceeding: dockrell, h.m., 2012, ‘one health or “joining the dots”’, onderstepoort journal of veterinary research 79(2), art. #458, 3 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.458 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. one health or ‘joining the dots’ in this proceeding... open access • discussion • acknowledgments • references discussion top ↑ to achieve the goals of the one health agenda, multiand interdisciplinary approaches are essential. it is now common to bring people from different backgrounds together to work on a single disease problem, with a large number of multi-partner consortia currently funded by the european commission, the european and developing countries clinical trials partnership (edctp), the bill and melinda gates foundation and others. within institutions, mechanisms to increase interactions across disciplines include common seminars, retreats, and the formation of themed virtual centres. despite the willingness of most scientists to cooperate in research towards a common goal, the existence of different professional cultures has been identified as a barrier in achieving one health objectives. cooperation, complementarities and convergence are required to achieve our goals. more effective communication, better teaching to increase awareness of one health issues and more meetings that bring both animal and human health experts together are needed in order to join the dots across the disciplines in one health.a ’one health’ approach to improving human and animal health worldwide is a priority, as about two-thirds of human infectious diseases and about three-quarters of emerging infectious diseases are thought to be zoonoses. in addition, changes to farming methods and to more intensive farming in some parts of the world, together with greater travel and contact with wildlife, all increase the risk of infectious spread from animals to humans. a pertinent example of what can happen when human and animal health agencies do not communicate well is illustrated by the outbreak of q-fever in the netherlands (enserink 2010). q-fever is caused by coxiella burnetti, an organism that can infect many species but it causes abortions and stillbirths in pregnant goats. in 2007, 182 human cases were diagnosed in the netherlands, however this increased to 1000 cases in 2008 and 2361 cases in 2009. due to fears of potential loss of income, the presence of infections in goats had not been communicated to human clinicians. in addition to the burden of human disease that resulted, there were 6 deaths in 2009, and the spread of the infection within intensively farmed goat herds resulted in the decision to cull all pregnant goats in order to control the epidemic. although it is still unclear whether the epidemic resulted from a particularly virulent strain of c. burnetti, or from the high intensity farming of goats in a highly populated country, it is clear that there are both human health and economic risks from such outbreaks. multiand interdisciplinary approaches are required to deal with such zoonotic threats to human health. environmental factors include the production system in use, host abundance, host species diversity, interactions both within and between species, and selective pressures such as the use of antibiotics (coker et al. 2011). biological pressures will include both the diversity of the pathogenic species, and its mode and dynamics of transmission. the health impact will also be affected by the transmissibility of the infection, and the risk of disease emergence and spread. finally there will be economic and social factors that include not just the economic impact of infection but the cultural, environmental and socioeconomic context. thus coker and colleagues have advocated that in order to achieve one health, many disciplines will need to collaborate and work together (coker et al. 2011). people working on the same disease, or with a common purpose, are usually more than willing to collaborate. a successful example of interdisciplinary collaboration from the london school of hygiene and tropical medicine is that of the gates malaria partnership (n.d.) and its successor, the malaria capacity development consortium (n.d.), funded by both the gates foundation and the wellcome trust. the gates malaria partnership (2001–2009) was able to fund research projects for phd students and postdoctoral scientists in 14 african countries as well as china, bolivia and pakistan, in disciplines ranging from laboratory science to entomology, intervention studies and health economics – yet all these projects became part of a network. the malaria capacity development consortium (2008–2013) is now continuing the same joined-up approach to research and training in malaria. there are other examples of successful consortia in the tuberculosis field. the european commission fp6-funded tbvac consortium had 32 partners in 9 european and 4 african countries, all united by the common goal of a new vaccine against tuberculosis (tuberculosis vaccine initiative n.d.a). this consortium included the discovery of new antigens, testing in animal models, work on delivery systems and adjuvants, on correlates of protection, and the early steps of testing the new vaccines – thus bringing together scientists with a range of disciplinary backgrounds. again, successful progress has led to a new consortium, aptly titled newtbvac (tuberculosis vaccine initiative n.d.b), with european commission fp7 funding, and the establishment of a foundation to facilitate european efforts towards the global development of new tb vaccines, the tuberculosis vaccine initiative (tbvi) (tuberculosis vaccine initiative, n.d.c). many other research consortia carry out large multicentre studies, such as the gates grand challenge funded biomarkers for tb in africa (biomarkers for tb n.d.), where cohorts of subjects at 7 african sites have been recruited and followed longitudinally for the development of disease, in order to identify biomarkers that predict the development of, or protection from, disease. working across different countries can bring additional insights; for example, when bcg vaccinated infants were compared for the immune responses induced three months post bcg vaccination, in the united kingdom (uk) and malawi, uk infants were found to have stronger th1 cytokine responses than malawian infants (lalor et al. 2009). however, the use of multiplex bead array assays revealed that the malawian infants were not merely poor responders – instead they made stronger responses in terms of other th2 and down-regulatory cytokines, thus illustrating the impact of environment on such immunity (lalor et al. 2011). sometimes more than one infection can be studied in a consortium, as in the idea consortium which is studying the effect of helminth co-infection on immunity against tb, hiv and malaria (idea n.d.). on occasion the spectrum of disciplines and backgrounds in such consortia becomes even wider, such as in the innovative vector consortium which combines scientists from academia and from industry (innovative vector consortium n.d.). to be successful, such projects need sufficient funding, good leadership, and regular meetings (preferably at a remote location where all those present have to focus on the business of the meeting). partnerships across countries and diseases are also a feature of current capacity building efforts. these include the european and developing countries clinical trials network (edctp n.d.) with its regional nodes of excellence, and the 7 wellcome trust-funded african institutions initiative capacity building consortia (wellcome trust n.d.). again, despite different backgrounds and levels of research expertise, such consortia can work together well, sharing experience and identifying new opportunities for collaborative research. institutions such as the london school of hygiene & tropical medicine (lshtm) also have their challenges in maximising the benefit of the breadth of disciplines and infections present within the institution. one approach taken by the lshtm has been to set up cross-faculty virtual centres, that can be disease specific (such as the malaria centre, or the newer tuberculosis centre), or topic specific such as the march centre that works on maternal, reproductive and child health, linking over 100 researchers who have research interests in some aspect of this topic (london school of hygiene & tropical medicine n.d.). within the bloomsbury area of london, a major cross-institution centre has been established, to break down barriers and allow innovative approaches to research and teaching in the area of international development. the london international development centre (london international development centre n.d.) is a partnership of six colleges situated in the bloomsbury area – the royal veterinary college, birkbeck college, the school of pharmacy, the institute of education, the school of african and oriental studies and the lshtm. lidc facilitates the cooperation between lshtm and the royal veterinary college on one health research and teaching, including the uk support for the southern african centre for infectious disease surveillance (sacids n.d.). the outcome of all these linkages and consortia is that scientists, far from working in isolated silos, are becoming so well linked to others in their field that there is a danger that individual research innovation may suffer at the expense of harmonised multicentre studies. partnerships and consortia are essential if we want to achieve our one health goals, but these need time, effort and adequate funding, and good management and planning are essential. together, the partners can make greater progress than they would on their own, but there still needs to be a place for original research ideas (dockrell 2010). if consortia and centres that are by definition cross disciplinary can work well, and if institutions can link researchers from different disciplines to work together, this raises the question why a recent publication by meisser and colleagues (meisser, schelling & zinsstag 2011) identified different professional cultures as the most important barrier to delivering on one health. jeff waage of the london international development centre has proposed that in order to make an inter-disciplinary approach to one health work, cooperation – where working together is necessary to achieve a particular goal, complementarity – where one party benefits from the other’s strengths or resources, and convergence – where societal change leads to common tools and agendas, are all needed (j. waage, pers. comm., n.d.). for example, cooperation is needed on cooperative projects on zoonotic diseases which link veterinary and public health surveillance and management activity. the increasing threat of anti-microbial resistance is one subject that is a clear public health priority and that requires a co-operative approach. complementarity may sometimes be less obvious, but can usually be found. for example, public health can make use of veterinary health’s superior rural delivery systems, whilst public health systems can have superior financing sources and mechanisms that could be exploited by those working in veterinary health. new technologies and tools can also drive convergence, such as with the cassette based point of care devices and genome sequencing tools that show promise as diagnostic tools for both human and animal infections. so the challenge for one health is to identify and overcome the barriers that prevent animal and human health professionals from working together. to forge cooperation, teams need to be built and the barriers that prevent interdisciplinary work broken down. complementarity could be achieved by educating medical students and veterinary students about each others’ activities so that both groups can ’ borrow’ good ideas. new teaching courses in one health would facilitate this. and to drive convergence, we need to identify drivers of change and to be proactive, thinking beyond zoonoses. the recent review by coker et. al. (2011) proposes a framework in which research to inform one health policy can be conducted. what is certain is that more effective communication, better teaching and more meetings that bring both animal and human health experts together are needed in order to join the dots across the disciplines in one health. acknowledgments top ↑ the author would like to thank richard coker and jeff waage for providing material for this review. references top ↑ biomarkers for tb, n.d., the gc#6-74 consortium, viewed 09 may 2012, from http://www.biomarkers-for-tb.net/consortium/the-consortium coker, r., rushton, j., mounier-jack, s., karimuribo, e., lutumba, p., kambarage, d. et al., 2011, ‘towards a conceptual framework to support one-health research for policy on emerging zoonoses’, lancet infectious diseases 11, 326–331. dockrell, h.m., 2010, ‘presidential address: the role of research networks in tackling major challenges in international health’, international health 2, 181–185. enserink, m., 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calmette-guerin vaccination in infancy’, journal of infectious diseases 199, 795–800. http://dx.doi.org/10.1086/597069, pmid:19434928 lalor, m.k., floyd, s., gorak-stolinska, p., ben-smith, a., weir, r.e., smith, s.g. et al., 2011, ‘bcg vaccination induces different cytokine profiles following infant bcg vaccination in the uk and malawi’, journal of infectious diseases 204, 1075–1085. http://dx.doi.org/10.1093/infdis/jir515, pmid:21881123 london international development centre, n.d., new approaches towards international development, viewed 09 may 2012, from http://www.lidc.org.uk/ london school of hygiene & tropical medicine, n.d., research centres and groups, viewed 09 may 2012, from http://www.lshtm.ac.uk/research/centres-groups/ malaria capacity development consortium, n.d., malaria capacity development consortium, viewed 09 may 2012, from http://www.mcdconsortium.org/ meisser, a., schelling, e. & zinsstag, j., 2011, ‘one health in switzerland: a visionary concept at a crossroads?’, swiss medical weekly 141, w13201. southern africa centre for infectious disease surveillance (sacids), n.d., southern africa centre for infectious disease surveillance, viewed 09 may 2012, from http://www.sacids.org/kms/frontend/index.php?m=3 tubeberculosis vaccine initiative (tbvi), n.d.a, tbvac, viewed 09 may 2012, from http://www.tbvi.eu/projects/tbvac.html tubeberculosis vaccine initiative (tbvi), n.d.b, newtbvac, viewed 09 may 2012, from http://www.tbvi.eu/projects/newtbvac.html tuberculosis vaccine initiative, n.d.c, tuberculosis vaccine initiative (tbvi), viewed 09 may 2012, from http://www.tbvi.eu/home.html wellcome trust, n.d., african institutions initiative, viewed 09 may 2012, from http://www.wellcome.ac.uk/funding/international/wtx055734.htm article information authors: christopher j. kasanga1 begoña valdazo-gonzález2 rahana dwarka3 jemma wadsworth2 nick j. knowles2 philemon n. wambura1 mark m. rweyemamu1 misheck mulumba4 jimis deve5 donald p. king2 affiliations: 1southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania2wrlfmd, the pirbright institute, united kingdom 3onderstepoort veterinary institute, university of pretoria, south africa 4southern african development community secretariat, botswana 5ministry of agriculture and livestock, mozambique correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: kasanga, c.j., valdazo-gonzález, b., dwarka, r., wadsworth, j., knowles, n.j., wambura, p.n.et al., 2014, ‘full genome sequencing to study the evolutionary characteristics of foot-and-mouth disease virus in southern africa’, onderstepoort journal of veterinary research 81(2), art. #729, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.729 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. full genome sequencing to study the evolutionary characteristics of foot-and-mouth disease virus in southern africa in this abstracts... open access • abstract • acknowledgements abstract top ↑ foot-and-mouth disease (fmd) is endemic in most countries of southern africa where it affects cloven-hoofed animals that include livestock and wildlife. southern africa relies profoundly on livestock production as a source of economic growth and livelihoods. despite the importance of fmd in southern africa, the epidemiology of fmd virus (fmdv) and factors contributing to the endemicity of fmd infection in susceptible animal populations are not clearly known in this region. in this study, we designed and optimised an rt-pcr strategy for amplification of the complete genome of fmdv sat 1, and sequenced the whole genome of one isolate, designated sat1/moz/buf-b16/2010, which was isolated from an african buffalo (syncerus caffer) in mozambique in 2010. preliminary analyses showed that the genome polyprotein of sat1/moz/buf-b16/2010 consisted of 7626 nucleotides. alignment of nucleotides and deduced amino acid sequences and phylogenetic analysis indicated that the virus is closely related to sat 1 strains from the southern african region. using a fasta search, the polyprotein showed closest nucleotide identity (94%) to sat1/rho/5/66 isolated from cattle in devuli ranch in southeast zimbabwe in 1966. this rt-pcr and whole genome sequencing strategy could be deployed to study the evolutionary characteristics of viruses sampled from cattle and buffalo at different locations in time and space. a full repertoire of protocols for all of the circulating fmdv lineages is now required to conduct an in-depth genome analysis to understand the factors that impact upon fmdv endemicity in the region. this information is necessary for strategies for the control of fmd in most countries in africa. the genome sequencing approach allows for targeted and cost-effective fmd control strategies in the endemic settings of africa rather than application of blanket and expensive interventions – an important consideration for resource constrained countries. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). article information authors: bernard m. hang’ombe1 i. nakamura2 d. kaile3 a.s. mweene1 k.l. samui1 b.s. kilonzo4 h. sawa2 c. sugimoto2 b. wren5 affiliations: 1school of veterinary medicine, university of zambia, zambia 2research center for zoonosis control, hokkaido university, japan 3namwala district medical officer, namwala district, zambia 4sokoine university of agriculture, united republic of tanzania 5school of hygiene and tropical medicine, london, united kingdom correspondence to: bernard hang’ombe postal address: po box 32379, lusaka, zambia how to cite this abstract: hang’ombe, b.m., nakamura, i., kaile, d., mweene, a.s., samui, k.l., kilonzo, b.s. et al., 2012, ‘identification of the plague reservoir in an endemic area of zambia’, onderstepoort journal of veterinary research 79(2), art. #464, 1 page. http://dx.doi.org/10.4102/ ojvr.v79i2.464 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. identification of the plague reservoir in an endemic area of zambia in this abstract... open access yersinia pestis, the bacterial agent of plague, is primarily a parasite of wild rodents that persists in permanent, discrete enzootic foci throughout the world. the disease is transmitted in humans by bites from fleas of wildlife rodent species. therefore surveillance is the ultimate public health solution through plague detection in domestic dogs, other carnivores and wild rodents. the investigations of die-offs amongst plague-susceptible colonial rodents are also significant to determine the presence of y. pestis in a susceptible population.this study details the identification of the plague reservoir in a suspected endemic area of zambia. the study was undertaken through rodent investigation for the presence of y. pestis. a total of 105 rodents were sampled routinely and during a suspected plague period. on dissection 4 (3.81%, 95% ci: 1.23−10.0) rodents sampled during an outbreak showed signs of spleen enlargement. the blood, liver, lymph nodes and spleen of each rodent were subjected to culture on 6% sheep blood agar and macconkey agar. colonies obtained were identified as y. pestis by colony morphologic features, biochemical profiles, mouse inoculation assay and polymerase chain reaction (pcr). the pcr primers used targeted the y. pestis plasminogen activator gene, chromosomal ferric iron uptake regulation gene and the outer membrane protein b gene. the isolates were also subjected to antibiotic sensitivity tests using the disk diffusion method on mueller-hinton agar with sensitivity being observed with ampicillin, amoxicillin, chloramphenicol, gentamycin, streptomycin, tetracycline and trimethoprim-sulfamethoxazole. the findings, identifies a natural reservoir of y. pestis in zambia providing the public health officials with a definite host for the control strategy. article information authors: tom g. wahl1 aleksey v. burdakov1 andrey o. oukharov1 azamat k. zhilokov1 affiliations: 1black and veatch, mosalarko plaza one, moscow, russia correspondence to: tom wahl postal address: black and veatch, mosalarko plaza one, 16 marksistskaya st., moscow 109147, russia how to cite this proceeding: wahl, t.g., burdakov, a.v., oukharov, a.o. & zhilokov, a.k., 2012, ‘electronic integrated disease surveillance system and pathogen asset control system’, onderstepoort journal of veterinary research 79(2), art. #455, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.455 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. electronic integrated disease surveillance system and pathogen asset control system in this proceeding... open access • abstract • problem statement • methods and approach • method 1: electronic surveillance using electronic integrated disease surveillance system    • methods and features    • results • method 2: biological pathogens tracking using pathogen asset control system    • results • acknowledgements • references abstract top ↑ electronic integrated disease surveillance system (eidss) has been used to strengthen and support monitoring and prevention of dangerous diseases within one health concept by integrating veterinary and human surveillance, passive and active approaches, case-based records including disease-specific clinical data based on standardised case definitions and aggregated data, laboratory data including sample tracking linked to each case and event with test results and epidemiological investigations. information was collected and shared in secure way by different means: through the distributed nodes which are continuously synchronised amongst each other, through the web service, through the handheld devices. electronic integrated disease surveillance system provided near real time information flow that has been then disseminated to the appropriate organisations in a timely manner. it has been used for comprehensive analysis and visualisation capabilities including real time mapping of case events as these unfold enhancing decision making. electronic integrated disease surveillance system facilitated countries to comply with the ihr 2005 requirements through a data transfer module reporting diseases electronically to the world health organisation (who) data center as well as establish authorised data exchange with other electronic system using open architecture approach. pathogen asset control system (pacs) has been used for accounting, management and control of biological agent stocks. information on samples and strains of any kind throughout their entire lifecycle has been tracked in a comprehensive and flexible solution pacs. both systems have been used in a combination and individually. electronic integrated disease surveillance system and pacs are currently deployed in the republics of kazakhstan, georgia and azerbaijan as a part of the cooperative biological engagement program (cbep) sponsored by the us defense threat reduction agency (dtra). problem statement top ↑ infectious diseases in the twenty-first century continue to cause economic and social disruptions becoming more severe due to the emerging diseases often of a zoonotic nature, active travel increasing potential of regional and international epidemics and pandemics, and potential bioterrorist threats.to adequately address these growing threats countries have to strengthen and improve their capability to perform early detection and rapid reporting of infectious disease situation and outbreaks, to timely and accurately verify presence or absence of high-consequence pathogens, and to comprehensively and rapidly respond to care for infected patients and reduce exposure of the wider population, and the accidental and/or deliberate release of high-consequence pathogens. there have been a number of attempts to improve detection, reporting and respond capabilities with the modern information and communications technology. however, most of the advances in this field tend to either concentrate on isolated vertical segments (e.g. hiv, tb or malaria), separately address human or veterinary areas, and be isolated from diagnostics (laboratory) and clinical data sources, be disintegrated with the international information systems (world health organisation [who] and world organisation for animal health [oie]), or be tailored only for certain environment and disease surveillance priorities without an ability to adjust. thus there is a need for a comprehensive information and communications technology solution, which would overcome the challenges of existing solutions and facilitate in early detection, rapid reporting and response. the creation of this comprehensive solution would require significant effort from the epidemiology and diagnostics experts society as well as investment in development and implementation of the solution. methods and approach top ↑ for the 10 years the united states defense threat reduction agency (dtra) has been actively implementing the cooperative biological engagement program (cbep), which goals closely match infectious disease threats most of the countries face: • combat bioterrorism and prevent the proliferation of biological weapons-related technology, pathogens and expertise • enhance host governments’ disease surveillance systems to detect and report bioterrorism attacks, epidemics and potential pandemics. to address these goals two information and communications technology solutions were created, (1) electronic integrated disease surveillance system (eidss) and (2) pathogen asset control system (pacs). electronic integrated disease surveillance system facilitates in early detection, rapid reporting and response to disease outbreaks, whilst pacs improves security of dangerous pathogens stored in the laboratories. method 1: electronic surveillance using electronic integrated disease surveillance system top ↑ electronic integrated disease surveillance system is an electronic system intended to facilitate in collecting, notifying, sharing and analysing surveillance data. it consists of several modules listed below together with a specification of tracked data: • human module » demographic data » disease-specific clinical data based on standard case definitions » epidemiological investigations » sample and laboratory tests tracking linked to a specific case » aggregate cases. • veterinary module » avian and livestock cases » farm or owner information » disease-specific clinical data based on standard case definitions » epidemiological investigations » sample and laboratory tests tracking linked to a specific case » penside tests » aggregate cases » active surveillance data. • laboratory » samples, tests assignment, tests results and batch tests » aliquots and derivatives » transfer in or out operations » links to clinical case data. • analysis » customised and predefined reports » statistic analysis » different ways of data representation: grids, charts, maps. figure 1: typical electronic integrated disease surveillance system architecture. information can be collected and shared through these modules in several ways providing a near real-time information flow that can be then disseminated to the appropriate organisations in a timely manner. figure 1 represents typical eidss hierarchy covering different administrative levels: district, region, country. the first way to collect information is through a number of distributed nodes which are continuously synchronised amongst each other. let us say a veterinary case is registered in a district. once this information is entered to the system, replication starts and transfers the case to the region level site, which in turn send it to the central data repository (cdr), then it goes other sites which are supposed to get such information. bi-directional regular data exchange amongst sites guaranties that all participants get actual and up-to-date information. if a node gets off-line it keeps working in disconnected mode and will be synchronised with other nodes once it gets on-line. the eidss web server is the other way to get information into the system (figure 2). data entered through the web site is distributed to other nodes of the system and available for review and editing according to users’ permissions. access to eidss from handheld devices is the third entry point (figure 2). web portal designed for handheld devices provides simplified interface and support a variety of devices. any combination of these methods can be used depending on infrastructure, organisational and other issues. in a combination with using of commercial ‘off-the-shelf’ readily available generic hardware it makes eidss suitable for environments with different challenges. figure 2: web and/or handheld access to electronic integrated disease surveillance system: data analysis. figure 3: pathogen asset control system conceptual schema. figure 4: repository inventory audit. figure 5: material classification. figure 6: freezer content report. methods and features following one health approach eidss integrates human and veterinary surveillance data along with a laboratory information. for example, it is possible to create an outbreak record in the system which ties together several human and veterinary cases. one can also link several veterinary cases together. adding a laboratory piece gives the opportunity diagnose a case with a most recent laboratory data. for instance, case entered in the veterinary service is transferred to the laboratory and available through the eidss lab module. as soon as test results are registered for this case in the laboratory they will be transferred to the veterinary service site and can be used for further investigations.flexibility and scalability is one of the fundamental principles of the eidss. one example of such flexibility is that cases can be tracked with different levels of specification: case-based tracking with a specific case definitions and investigation information, when every case is registered as individual record, can be used for especially dangerous diseases, case-based tracking with just an emergency notification information can be used for other important diseases, aggregate reporting which is supposed to track just summary data from district or regions for a certain period of time. electronic integrated disease surveillance system also allows to switch between different approaches depending on the current situation. case definitions used in the system also can be adjusted according to the specific requirements. electronic integrated disease surveillance system supports different types of surveillance: passive surveillance (case-based and aggregate) is available for human and veterinary diseases, active surveillance is supported for veterinary disease, vector surveillance is planned to be released in the next version. comprehensive analysis modules give users various capabilities to investigate and present collected data. using either predefined reports which duplicates officially-approved paper forms or customisable reports where one can build a report as needed, users are able to get access to any variables in the database and explore human, veterinary and lab. the gis component of the system allows attaching geo-coordinates to cases and map data in addition to charts and grid representations (figure 2). electronic integrated disease surveillance system provides means for integration with other local or international systems: the who module can transfer approved reports to computerised information system for infectious diseases (cisid) assisting in international health regulations (ihr) compliance, open architecture approach supported by eidss allows to establish communications with existing local electronic systems. regional cooperation between neighbouring countries is also possible on the base of eidss. full localisation of the system which includes electronic forms, reports and manuals, unified design and staff training make it easy to operate for non-experienced users. turn-key deployment and support contribute to the sustainability of the solution. results electronic integrated disease surveillance system is being developed since 2005 in collaboration with different institutions including the centers for disease control and prevention (cdc), us, the walter reed army institute for research (wrair), us, ministries of health and veterinary departments of kazakhstan, uzbekistan, azerbaijan and georgia, ukraine and armenia and numerous international medical and veterinary experts. following iterative approach eidss went through the number of expertise (more than 75 000 hours) and revisions.currently version 3 is deployed in azerbaijan (90 installations), kazakhstan (more than 150 installations) and georgia (more than 120 installations). the initial phase of deployment in armenia and ukraine. electronic integrated disease surveillance system has been officially recognised in azerbaijan in 2010 and recently in kazakhstan and georgia. electronic integrated disease surveillance system is a solution that improves the capacity to detect, diagnose and report bioterrorism attacks and potential pandemics, and supports bioresearch. it strengthens both regional and global disease surveillance with plans for expansion into african and asian regions and globally. method 2: biological pathogens tracking using pathogen asset control system top ↑ pathogen asset control system helps to track and control pathogens that are collected, investigated and stored in biological laboratories. it allows monitoring of the whole life-cycle of the pathogens with necessary levels of detail and appropriate security. pathogen asset control system can be implemented within different facilities, from small repositories with one centralised storage area to big multi-discipline institutions with distributed structure and numerous laboratories. server-client architecture allows immediate access to all necessary information for every user who has appropriate rights, which includes principal investigators, facility management, it administrators and other laboratory personnel. pathogen asset control system utilises barcode technology and radio-frequency identification (rfid) to uniquely track all individual vials with highly pathogenic materials and allows error-free fast data input and retrieval. handling dangerous biological materials and processing information about them requires certain level of safety and security, and pacs provides invaluable assistance in this area. separated access control to data, detailed audit trails, inventory audit function and other security features of the application help the owner to comply with international and local regulations in biosafety and biosecurity. considering that different organisations might have different requirements on what information must be tracked on biological materials and how, pacs allows several options for system customisation and adoption to local requirements. that includes designing barcode labels, managing repository configuration, creating different data entry forms depending on material type customise data entry forms and output reports. being flexible and adoptable to the owner’s requirements pacs employs all common operations with biological materials, such as registration, transfer, destruction, splitting and reculturing. all operations are supplied with printable reports and acts. pathogen asset control system provides wide opportunities to utilise information entered into the system for biological investigation support. a variety of different user defined fields and information in them, supplemented with the capability of creating custom reports provides an opportunity to output data in ways so it can be used for analysis of different parameters. results pathogen asset control system is an important part of the overall biosecurity solution and can be very efficient in every biological laboratory where implemented. it allows the necessary levels of tracking and control over dangerous pathogens and complies with strengthening or enforcing regulations in biosafety.being implemented in numerous laboratories in 6 different countries pacs proved its reliability and effectiveness. pathogen asset control system is currently undergoing certification and accreditation (c&a) process according to united states department of defense standards and should be offered to many government agencies as a system for tracking biological agents once c&a is completed. pathogen asset control system is a robust electronic system that competently helps to solve the important task of managing dangerous materials and will be in demand by biological laboratories. acknowledgements top ↑ mr tom g. wahl is a project manager for eidss and pacs product. he prepared the overview of the eidss and pacs and introduction. dr aleksey v. burdakov made contribution to all sections of the article, including problem statement. mr andrey o. oukharov wrote eidss section of the article. mr azamat k. zhilokov wrote pacs section. references top ↑ burdakov, a.v., 2005, ‘implementation of the communications and information technology part of the international program on treat agent detection and response in the republic of kazakhstan’, environment and population health 1, 37. wahl, t. & burdakov, a.v., 2007, ‘electronic integrated disease surveillance system (eidss)’, advances in disease surveillance 2, 171 article information authors: mathias mkama1 christopher j. kasanga2 raphael sallu1 ezekia ranga3 mmeta yongolo1 misheck mulumba4 mark rweyemamu2 philemon wambura2 affiliations: 1tanzania veterinary laboratory agency, tanzania2southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania 3ministry of livestock development and fisheries, tanzania 4southern african development community secretariat, botswana correspondence to: mathias mkama postal address: po box 9254, dar es salaam, tanzania how to cite this article: mkama, m., kasanga, c.j., sallu, r., ranga, e., yongolo, m., mulumba, m. et al., 2014, ‘serosurveillance of foot-and-mouth disease virus in selected livestock-wildlife interface areas of tanzania’, onderstepoort journal of veterinary research 81(2), art. #718, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.718 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. serosurveillance of foot-and-mouth disease virus in selected livestock-wildlife interface areas of tanzania in this proceedings... open access • abstract • introduction • materials and methods    • study area and blood samples obtained    • sample handling    • laboratory analysis • results • discussion and conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ foot-and-mouth disease (fmd) is caused by a virus of the genus aphthorvirus of the family picornaviridae. there is great scientific need for determining the transmission dynamics of fmd virus (fmdv) by drawing more attention to the livestock-wildlife interface areas. a variety of literature suggests that buffalo could serve as reservoir of fmdv in wildlife and cattle. however, many fmdv research studies conducted on experimentally infected cattle as carriers and groups of animal highly susceptible to fmdv (i.e. bovine calves) have shown lower chances of transmission of the virus between carriers and the susceptible groups. these findings underscore the importance of continued research on the role played by carrier animals on fmdv transmission dynamics under natural conditions. the aim of this research study was to determine fmdv infection status among buffalo and cattle herds in selected livestock-wildlife interface areas. the sampled areas included mikumi, mkomazi and ruaha national parks, where a total of 330 buffalo and bovine sera samples were collected. laboratory analysis of the samples was done through the nsp elisa technique using the priocheck® fmdv ns kit for detection of antibodies directed against 3abc non-structural proteins and confirming natural infections. results showed that 76.3% of tested sera samples were positive for fmdv. however, serotyping of nsp elisa seroreactors with lpbe is yet to be done. this information is important for further epidemiological studies towards developing effective fmd control strategies introduction top ↑ foot-and-mouth disease (fmd) is a highly contagious disease of cloven-hoofed (even-toed) animals, for example cattle, swine, buffalo, sheep and goats (alexandersen, zhang & donaldson 2002). it is a viral disease caused by the fmd virus (fmdv). the latter is said to be the first animal pathogen identified as a virus by loeffler and frosch in 1898 (brown 2003). fmdv belongs to the genus aphthorvirus of the family picornaviridae. characteristically fmdv is a single-stranded positive sense rna genome surrounded by an icosahedral capsid composed of 60 copies each of four structural proteins (vp1-4). there are seven serotypes of fmdv named o, a, c, sat1, sat2, sat3 and asian1 (donaldson, kitching & barnett 1996). however, infection with any one serotype does not confer immunity against another and within serotypes many strains can be identified by biochemical and immunological tests (office international des epizooties [oie] 2009). when fmdv infection occurs, antibodies directed against structural and non-structural proteins are produced (lubroth & brown 1995). conventional serological tests can only detect antibodies directed at structural fmdv proteins. this is a major challenge, especially in scenarios where both vaccinated and infected animals co-exist, because antibodies directed at the capsid proteins of fmdv are induced by both inactivated viruses (from vaccines) and live viruses (from infected or carrier animals) (frank et al. 2010). therefore it is not possible to differentiate the origin of the antibodies using the conventional elisa tests like lpbe and spce; instead elisa tests for detection of antibodies directed at non-structural proteins (nsps) were developed that can differentiate between vaccinated and infected animals (mackay et al. 1997). tanzania covers an area of about 945 000 km2 and two-thirds of the country’s land resources constitute rangelands, suitable for livestock-keeping activities (swai, mrosso & masambu 2009, cited in nyamurunda et al. 2007). tanzania ranks third in africa, after sudan and ethiopia, in terms of livestock population (moldf 2010). despite the large number of livestock it has been difficult to benefit from this valuable sector due to some limiting barriers, one of which is fmd. fmd is endemic to tanzania. it was first documented in 1927 and its first virus typed isolation was made in 1954 (rweyemamu & loretu 1972). of the six known serotypes of fmdv in africa, four have been isolated and identified in tanzania; these are type a, o, sat 1 and sat 2 (rweyemamu et al. 2008). tanzania’s fmdv status has a major impact on the country’s livestock sector as it currently does not export live animals and animal products to lucrative european markets. the purpose of this research study was to determine the fmdv infection status among buffalo and cattle herds in selected livestock-wildlife interface areas of tanzania. materials and methods top ↑ study area and blood samples obtained the blood samples from buffalo and cattle utilised in this study were obtained from four interface areas in tanzania that have been reported to have numerous incidents of fmd outbreaks. tanzanian national parks are not fenced like the southern african national parks; hence animals intermingle freely across their borders. the blood samples from buffalo and cattle were obtained from mikumi, katavi, ruaha and mkomazi national parks and surrounding cattle herds respectively. the blood samples were drawn from jugular veins using plain 4.5 ml vacutainer® tubes plunged in vacutainer® holders with an 18 g needle. sample handling the vacutainer® tubes filled three-quarters with blood were kept upright in racks contained in a cool box with ice packs and serum was extracted within 12 h of sampling. the aliquots of 2.0 ml serum samples were also kept on ice packs during shipping and at -20 °c for long-time storage in the laboratory awaiting analysis. laboratory analysis the fmd nsp laboratory analysis was done at the centre for infectious diseases and biotechnology (cidb) laboratory using a priocheck® fmdv ns elisa kit (prionics lelystad b.v., netherlands), batch number 7610440, manufactured in may 2012. the frozen sera samples were sorted, arranged in racks and allowed to thaw. that was followed by aliquoting 100 μl from each sample into u-bottomed microplates using a 20 μl – 200 μl capacity single-channel micropipette (eppendorf, germany). the aliquoting process followed the same plate layout as for test microplates indicated in the manufacturer’s instruction manual supplied with the test kit. the working room temperature was maintained at 25 °c.priocheck® fmdv ns is a blocking elisa that detects antibodies against the non-structural 3abc proteins of all seven fmdv serotypes and it may be used to detect infection in vaccinated animals (sorensen et al. 2005). after dispensing 20 μl of test sample into test plates for incubation, the rest of the procedures were done as illustrated in the manufacturer’s instruction manual. the different colour changes on every microwell reflected different levels of antigen-antibody reactions. the values of colour changes (optical density – od) were measured by using a multiskan ex elisa reader with filter set at wavelength of 450 nm using ascenttm software version 2.6. the results were expressed in terms of percentage inhibition (pi), which was derived from the equation pi = 100 – (od450 test sample/od max) x 100. pi < 50% meant that the sample was negative and pi ≥ 50% meant that the sample was positive. figure 1: map of tanzania showing sampling areas. figure 2: fmdv infection status in buffalos from four selected tanzanian national parks. figure 3: fmdv infection status in cattle herds surrounding four tanzanian national parks. results top ↑ a total of 330 sera samples were analysed by nsp elisa using the priocheck® fmdv ns kit for detection of antibodies directed against 3abc nsp; 248 sera samples, equivalent to 76.3%, tested positive. generally, the buffalo serum samples tested demonstrated higher percentages of positive samples than the cattle serum samples from the same area; however, the opposite is true for samples obtained from the mkomazi area (see figures 2 and figure 3). for buffalo serum samples tested (see figure 2) katavi (100%) had the highest fmdv infection rate, followed by ruaha (93.5%), mikumi (93.3%) and mkomazi (22.6%). for cattle serum samples tested (see figure 3) there was only a slight difference in fmdv infection status between mikumi (82.9%), ruaha (79.3%) and katavi (80.3%). mikumi (82.9%) had the highest percentage of positive samples and mkomazi the lowest (58.3%). discussion and conclusion top ↑ in this research study a total of 330 buffalo and cattle sera samples were analysed by nsp elisa using the priocheck® fmdv ns kit for detection of antibodies directed against 3abc non-structural proteins. a total of 248 out of 330 serum samples (76.30%) tested positive (see table 1). the results obtained showed different fmdv infection status in terms of pi value. the highest pi values of cattle serum samples from katavi, ruaha, mikumi and mkomazi were 95.48%, 93.56%, 94.24% and 94.56% respectively (see table 1). katavi presented the overall ceiling pi value (95.48%). katavi also presented the overall ceiling pi value (95.10%) for buffalo serum samples analysed. the highest pi values of buffalo serum samples from ruaha, mikumi and mkomazi were 94.95%, 94.82% and 91.65% respectively (see table 1). the high pi values could be due to two factors, namely vaccination or repeated vaccination with fmdv-specific (purified or unpurified) vaccines and natural infection with fmdv. the sampled herds have not been involved in any vaccination campaign against fmd for more than seven years. the same applies to buffalo herds, as vaccination for buffalos had never been practiced in tanzanian national parks. based on the field history and nsp elisa results the seroreactors observed during the nsp elisa test could be due to natural fmdv infection (oie 2010). the pi values obtained (see table 2) were categorised into two groups, namely 50–69 and 70–100, based on validation criteria for pi values of weak positive and positive controls indicated in the manufacturer’s instruction manual. the proportion of positive samples with pi values in the range 70–100 appeared to be higher than those with pi values in the range 50–69 (see table 2). this was indicative of recent fmdv infection with possible active fmdv circulation in the field. the sampled interface areas had a good number of buffalo and cattle herds, except for mkomazi national park, which had a considerable number of buffalo and cattle herds. the number and size of buffalo and cattle herds could be contributing factors in the demonstrated variation in fmdv infection status (pi value variations). in addition these areas vary in terms of pasture and water availability, which may cause the movement of pastoralists and buffalo herds. however, the epidemiology of fmd in tanzania remains complicated, as is the case in other sub-saharan countries (vosloo et al. 2002). the contributing factors include the large number of serotypes, the genetic and antigenic variation occurring in different regions, uncontrolled animal movement and the presence of multiple species of wildlife and domestic animals that can harbour the fmdv for variable time intervals. table 1: results for nsp elisa test using priocheck® fmdv ns kit. table 2: grouped pi value for buffalo and cattle samples from interface areas. acknowledgements top ↑ this research study was conducted at the tanzania veterinary laboratory agency – centre for infectious diseases and biotechnology (tvla-cidb) laboratory with support from sacids, sadc/tads, sua and the ministry of livestock and fisheries development. we would like to thank bvi for their support in terms of technical laboratory skills and in particular mr gladson joshua, laboratory technician. this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions m.m. (tanzania veterinary laboratory agency) was the first author and participated in study development, field sample collection, laboratory analysis and manuscript writing. c.j.k. and p.w. (both sokoine university of agriculture) provided technical support in developing this study. r.s. and m.y. (tanzania veterinary laboratory agency) participated in the laboratory analysis of the obtained samples. e.r. (ministry of livestock and fisheries development tanzania) and m.m. (sadc tads) participated in field sample collection. m.r. (sokoine university of agriculture) was the project leader. references top ↑ alexandersen, s., zhang, z. & donaldson, a., 2002, ‘aspects of the persistence of foot-and-mouth disease virus in animals – the carrier problem’, microbes and infection 4, 1099–1110. http://dx.doi.org/10.1016/s1286-4579(02)01634-9 brown, f., 2003, ‘the history of research in foot and mouth disease’, virus research 91, 3–7. http://dx.doi.org/10.1016/s0168-1702(02)00268-x donaldson, a.i., kitching, r.p. & barnett, p.v., 1996, ‘foot and mouth disease’, in oie standards commission (eds.), manual of standards for diagnostic tests and vaccines, pp. 47–56, office international des epizooties, paris, france. frank, n.m., chrisostom, a., alexandersen, s., willium, o., okurut, a. & kristen, t., 2010, ‘seroepidemiological investigation of foot and mouth disease virus serotypes in cattle around lake mburo national park in south-western uganda’, journal of veterinary medicine and animal health 2(4), 46–54. lubroth, j. & brown, f., 1995, ‘identification of native foot and mouth disease virus non-structural protein 2c as a serological indicator to differentiate infected from vaccinated livestock’, research in veterinary science 59, 70–78. http://dx.doi.org/10.1016/0034-5288(95)90034-9 mackay, d., forsyth, m., davies p., berlinzani, a., belsham g.j., flint, m. et al., 1997, ‘differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in elisa’, vaccine 16, 446–459. http://dx.doi.org/10.1016/s0264-410x(97)00227-2 ministry of livestock and fisheries development (molfd), 2010, ‘tanzania is rich in livestock resources in terms of number and variety’, viewed 07 april 2013, from http://www.mifugo.go.tz/livestock_resources/ nyamurunda, c., melewas, j., sendalo, d., mtenga, l. & rwezaula, d., 2007, ‘sustaining livestock productivity in challenging tropical environments as a contribution towards achieving millennium development goals’, proceedings of the 2nd joint tanzania veterinary association and tanzania society of animal production, 29 november to 1 december. office international des epizooties (oie), 2009, ‘foot and mouth disease’, viewed 14 april 2013, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.01.05_fmd.pdf office international des epizooties (oie), 2010, ‘terrestrial animal health code: article 8.5.48: foot and mouth disease’, viewed 17 march 2013, from http://web.oie.int/eng/normes/mcode/en_chapitre_1.8.5.htm rweyemamu, m. & loretu, k., 1972, ‘observation of foot and mouth disease in tanzania’, bulletin of epizootic diseases of africa 20, 101. rweyemamu, m., roeder, p., mackay, d., sumption, k., brownlie, j., leforban, y. et al., 2008, ‘epidemiological patterns of foot and mouth disease worldwide’, transboundary and emerging disease 55(1), 57–72. http://dx.doi.org/10.1111/j.1865-1682.2007.01013.x sorensen, k., de stricker, k., dyrting, k., grazioli, s. & haas, b., 2005, ‘differentiation of foot and mouth disease virus infected animals from expressed fmdv 3abc antigen and a 3abc monoclonal antibody’, archives of virology 150, 805–814. http://dx.doi.org/10.1007/s00705-004-0455-z vosloo, w., bastos, a., sangare, o., hargreaves, s. & thomson, g., 2002, ‘review of the status and control of foot and mouth disease in sub-saharan africa’, revue scientifique et technique de l’office international des epizooties 21(3), 437–449. article information authors: erasto v. mbugi1,2 bugwesa z. katale1,3 sharon kendall4 liam good4 gibson s. kibiki5 julius d. keyyu3 peter godfrey-faussett6 paul van helden7 mecky i. matee1 affiliations: 1departments of microbiology and immunology, muhimbili university of health and allied sciences, tanzania2department of biochemistry, muhimbili university of health and allied sciences, tanzania 3tanzania wildlife research institute, arusha, tanzania 4the royal veterinary college, london, united kingdom 5kilimanjaro christian medical college, tumaini university, tanzania 6london school of hygiene and tropical medicine, london, united kingdom 7dst/nrf centre of excellence for biomedical tuberculosis research/mrc centre of molecular and cellular biology, division of molecular biology and human genetics, faculty of health sciences, university of stellenbosch, south africa correspondence to: erasto mbugi postal address: po box 65001, dar es salaam, tanzania how to cite this proceeding: mbugi, e.v., katale, b.z., kendall, s., good, l., kibik, g.s., keyyu, j.d. et al., 2012, ‘tuberculosis cross-species transmission in tanzania: towards a one-health concept’, onderstepoort journal of veterinary research 79(2), art. #501, 6 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.501 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. this article was republished with the author’s full affiliation copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. tuberculosis cross-species transmission in tanzania: towards a one-health concept in this proceeding... open access • abstract • introduction • transmission at the human-livestock-wildlife interface • possible contributing factors to increased transmission at the interface • molecular epidemiological – diagnostic tools and their application in developing countries • one health as a disease control strategy • conclusions • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ for centuries, tuberculosis, which is a chronic infection caused by the bacillus mycobacterium tuberculosis has remained a global health problem. the global burden of tuberculosis has increased, particularly in the southern african region, mainly due to hiv, and inadequate health systems which has in turn given rise to emergent drug resistant tuberculosis (tb) strains. bovine tuberculosis (btb) has also emerged as a significant disease with the tendency for inter-species spread. the extent of interspecies btb transmission both in urban and rural communities has not been adequately assessed. the phenomenon is of particular importance in rural communities where people share habitats with livestock and wildlife (particularly in areas near national parks and game reserves). aerosol and oral intake are the major routes of transmission from diseased to healthy individuals, with health care workers often contracting infection nosocomially. although tb control has increasingly been achieved in high-income countries, the disease, like other poverty-related infections, has continued to be a disaster in countries with low income economies. transmission of infections occurs not only amongst humans but also between animals and humans (and occasionally vice versa) necessitating assessment of the extent of transmission at their interface. this review explores tuberculosis as a disease of humans which can cross-transmit between humans, livestock and wildlife. the review also addresses issues underlying the use of molecular biology, genetic sequencing and bioinformatics as t tools to understand the extent of inter-species cross-transmission of tb in a ‘one health’ context. introduction top ↑ humans and animals have had close interactions as long as we have historical knowledge of man. this interaction largely contributes to the evolution and ongoing transmission of shared infectious diseases. the need for a systematic global effort to monitor for emerging human is important since most major human infectious diseases are thought to have animal origins (wolfe, dunavan & diamond 2007), examples being helminths, tuberculosis (tb), brucellosis, foot-and-mouth disease, leptospirosis, anthrax and rift valley fever (rvf). studies on 1415 pathogenic diseases in human and animals (cleaveland, laurenson & taylor 2001) have revealed 61.6% as multi-host pathogens, suggesting the need for monitoring these important infectious diseases. available data indicate that more than 80% of human and cattle populations of africa live in an area with partial or no control of bovine tb (cosivi et al. 1998; who/dfid 2006; who 2004). there is ample evidence that such lack of control and close contact of the two species contributes to tb transmission, mostly mycobacterium bovis from cattle to humans. perhaps the best-studied example and illustration of the effectiveness of control measures was the situation in western europe (germany as a good example) prior to the 1950s, where m. bovis infection in cattle was common, and m. bovis was present in perhaps 15% of human cases. after strict control measures were introduced, m. bovis prevalence dropped effectively to zero in both species, with only the occasional reactivation case seen in the elderly, or in new immigrants coming from areas where m. bovis is not controlled. the extent of m. bovis infection and disease is not properly known in almost every environment. many tests, for example, tuberculin skin test (tst) and assays based on gamma interferon response may indicate exposure, but neither indicates disease nor an indication of whether infection is from mycobacterium tuberculosis, m. bovis, or possibly other mycobacteria, particularly those of the m. tuberculosis complex. the usual assumption is that if the test is done in a bovid, then the presence of or exposure to m. bovis is indicated, whereas if done in humans, then m. tuberculosis is indicated. however, this may not be true, since cross–reactivity does occur. at this stage, the gold standard for identification remains microbiological, whether culture or done by molecular techniques. tuberculosis can present in many ways, and in humans, a frequent manifestation of bovine tb is extra-pulmonary tuberculosis (eptb). unfortunately, m. tuberculosis can also present in extra-pulmonary form, although less frequently. extra-pulmonary tuberculosis driven by m. tuberculosis can also occur more frequently in children and individuals who are immunocompromised, for example, hiv positive (connolly et al. 2004; heymann 2011). therefore, although eptb can be used as an indicator (or rather, index of suspicion) of m. bovis infection in humans, this should only be considered seriously in areas or individuals where exposure to bovine tb is likely or suspected. nevertheless, eptb prevalence can be used as an indicator of m. bovis in humans. the control of this disease clearly requires significant efforts on the part of both veterinary and human health care practitioners and this article will address this cross-species issue in particular. transmission at the human-livestock-wildlife interface top ↑ transmission of bovine tuberculosis from animals to humans can occur most likely via aerosol exposure or through consumption of un-pasteurised milk and other affected animal products. reports from tanzania covering the period from 2008 to 2009 suggest that eptb accounted for over 20% of the cases of tb and showed a marginal annual increment (national tb control program report 2011, in preparation). this high level prevalence of eptb plus other data (cook et al. 1996) suggests the presence of mycobacteria other than m. tuberculosis occur extensively in the tanzanian population. we note that more extra-pulmonary cases are contributed by m. tuberculosis than m. bovis by total case load; however, such a high prevalence rate is unlikely to be due entirely to m. tuberculosis. a study by cook et al. (1996) in monze district of zambia, reported for cattle in larger herds, in households with previously report of human tuberculosis cases (preceding 12 months) to be more likely tuberculin test positive (six times) than those without previous reported human tb case. this could reflect tb cross-transmission between species. in the same year, a variety of different mycobacteria, including m. tuberculosis, were isolated from dairy cattle and milk in tanzania (kazwala et al. 1998). studies in the netherlands (majoor et al. 2011) indicate mainly (58.9%) extra-pulmonary m. bovis infection in humans. although individuals may develop the pulmonary form of m. bovis disease, person-to-person transmission is said to be unlikely, based on data from genotype analysis (majoor et al. 2011). there are reports on elephant-to-human transmission of tuberculosis due to indirect exposure to aerosolised m. tuberculosis (murphree et al. 2011). the infection has been exacerbated by delayed or inadequate disease control practices. other reports have indicated m. tuberculosis as an emerging disease in asian elephants in zoological collections (mikota et al. 2001). finally, there are unpublished reports that m. tuberculosis has been detected in a number of ethiopian cattle, transferred from the human owners directly to the animals (paul van helden from d young pers. comm., n.d.). the implication, impact and challenges underlying zoonotic tuberculosis to both humans and animals has been repeatedly emphasised (cataldi & romano 2007; cleaveland et al. 2005; kazwala et al. 1998, 2001a; michel 2002b; michel et al. 2006; michel, müller & van helden 2010; woodroffe et al. 2009). yet diagnosis by acid fast smear is still widely used and often the only non-clinical diagnostic since it is affordable. but this test, despite being the keystone of tuberculosis diagnosis in africa, does not permit differentiation between m. tuberculosis and m. bovis, or any other mycobacterial species. thus, it is essential that better diagnostics are applied in order that we may know the extent and nature of the problem at hand. in addition, the conventional means of control are difficulty to apply once the infection enters into wildlife species which are common maintenance hosts in most cases (michel 2002a; palmer 2007). animals are regarded to be the main source of zoonotic tuberculosis infection in humans. however, as outlined above, reports also indicate that humans are not the only victims from cross-species infection by tuberculosis, but are also potential sources of infection, even to free-living wildlife at their interface (alexander et al. 2002). this creates room for researchers to look at the disease from both sides of the coin. aerosol and oral transmission are the major routes of transmission in patients, with health care workers contracting infection nosocomially. in wildlife, similar routes are proposed with carnivores and omnivores said to contract infections via scavenging of bovine tuberculosis infected carcasses and killing and consumption of infected prey such as antelope or wild bovids (michel et al. 2006). this is evident from proven bovine tuberculosis in predators (keet et al. 2001). intensification of animal-human contact enhances the likelihood of cross-over and an increased risk of transmission for both m. tuberculosis as well as m. bovis at the interface. cross-transmission in infectious disease is not a newly emerging view but linked to the co-evolution of humans, the animals with which they co-exist and evolve and the microorganisms in their environment (diamond 2002; wirth et al. 2008). once a pathogen has crossed over into a new species of host, for example from animal to human, it may transform into a specialised pathogen of humans wolfe et al. (2007). a number of studies have been done reflecting the impact bovine tuberculosis may pose to wildlife (alexander et al. 2002; cleaveland et al. 2005; de garine-wichatitsky et al. 2010; michel et al. 2006, 2007). others have focused on livestock alone (aranaz et al. 2003; cousins 2001; durnez et al. 2009, 2011; shirima, kazwala & kambarage 2003; kazwala et al., 2001b) and others in humans alone (kazwala et al. 1998; mfinanga et al. 2004; ngowi et al. 2008), mostly using conventional diagnostic and evaluation means of the disease. a review published recently (michel et al. 2010) pinpointed bovine tuberculosis in particular, to be a disease of low-income countries with a multifaceted impact affecting the health of livestock, humans and the ecosystems. in the same review, it is also speculated that the disease is likely to increase in situations where hiv and aids occurs. recent work has characterised m. bovis isolates and reported that regional clonal complexes predominating in specific areas occur, for example af1 m. bovis complex in west-central africa (muller et al. 2009), af2 prevalent in east africa (berg et al. 2011) as well as european 1 with european origin (smith et al. 2011). thus we do not yet know for certain whether m. bovis is an endemic or alien disease to africa, or whether it is endemic to some areas (e.g. some parts of west and east africa) but alien to others. certainly, there is no evidence to suggest that any form of m. bovis is endemic or indigenous to south africa. better unpacking of this information is needed, since it may impact on how we view this disease and respond to it. we do not know the relative frequency of the african and european type m. bovis in any area and it may be that the european variety dominates as an alien invasive organism. it may be that indigenous african cattle and wildlife in some areas are relatively resistant to bovine tb, which we may take advantage of in our responses to the disease. several studies and reviews surveying the zoonotic tb status in animals and humans have been published (cataldi & romano 2007). different study designs, methods of analysis and various study-dependent objectives largely influence the outcome and interpretation of these studies. differences in outcomes amongst these different studies nevertheless reflect the diversity of disease dynamics in different communities and methodological shortcomings have been revealed (heldal et al. 2003; murray & alland 2002). a new concern is that cross-species transmission may play a role in emergent drug resistant tuberculosis. mycobacterium bovis is inherently resistant to pyrazinamide, one of the four first line antibiotics, and thus inadequate and/or inappropriate treatment of apparent m. tuberculosis in a human can easily result in acquisition of further resistance in m. bovis. drug-resistant m. bovis in human disease has already been reported in a number of countries (blazquez et al. 1997; hughes et al. 2003; sechi et al. 2001). this is essentially risky for african populations due to a close association of tb with hiv and often poor patient adherence and problems with implementing a good dots programme as advocated by word health organisation (who). likewise, transmission of drug resistant m. tuberculosis to animals and back to humans is not impossible, although likely to be very rare. in rural communities, milk pasteurisation is not common and m. bovis survival in cow’s milk is effective. therefore, despite reports that humans are less susceptible to bovine tb than m. tuberculosis (de la rua-domenech 2006) we cannot ignore these known problems and complications. possible contributing factors to increased transmission at the interface top ↑ the main problem with identifying or quantifying the transmission of zoonotic tuberculosis at the human-animal interface is the failure to adopt appropriate diagnostic tools that can discriminate between m. bovis and m. tuberculosis, or other species of mycobacteria at routine diagnostic settings, particularly in low-income countries. this is not surprising, given the huge burden of disease, particularly of m. tuberculosis and the low per capita income of those countries. a further reason for this problem is that many clinicians show no interest in differentiating the causative agent, since initial treatment is the same. as a result the contribution of mycobacteria such as m. bovis to the global human tb burden is almost certainly underestimated. despite these problem and long before such discriminatory tests became available, the test-and-slaughter scheme has proven to be an efficient control method for bovine tuberculosis in developed countries, although it is considered expensive (michel et al. 2010) and is not yet accepted by all farmers (bennett 2009; torgerson & torgerson 2009). consequently, the joint impact of these important pathogens to national and global economies is still unknown. different and inflexible policies guiding the practice of veterinary and medical practitioners are also potential barriers to what may be an immediate positive impact on zoonotic tb control strategies in most developing countries. the prevalence of human tb and livestock tb has been reported to correlate (cosivi et al. 1998) despite contradicting reports on animal-human active transmission of zoonotic tb (cousins, williams & dawson 1999; dankner & davis 2000; dankner et al. 1993; jalava et al. 2007; romano et al. 1995, 1996; zumarraga et al. 1999). the absence of genetic relatedness amongst m. bovis isolates leading to these controversies is in all likelihood simply a matter of incomplete sampling and reactivation disease (mignard, pichat & carret 2006). this is supported by a report from baker et al. (2006) who found many genotypes in human tb identical to patterns from farmed and wild animals. with the current advancement in molecular techniques for evaluation of infectious agents, exploiting these skills and using them for both epidemiological and evolutionary analysis of mycobacterial populations infecting humans and animals (baker et al. 2004) and assessment of their cross-transmission amongst these species is possible and important. pathogen flow and disease dynamics of m. bovis has been broadly reported in south africa’s wildlife and livestock (michel 2002b; michel et al. 2006, 2010) showing that the african buffalo is the major reservoir of infection in wildlife in that ecosystem. thus, although originally a naïve host and infected by cross-over from cattle initially, the buffalo have become a source of infection for many other species, through dissemination of bacilli in the environment or predation (keet et al. 1996; michel 2002a, 2002b; michel et al. 2004, 2006, 2007, 2010). although fairly the extensive studies have been done in the south african national parks, little has been done in the large national parks of tanzania, such as serengeti, ruaha and mikumi, where a large number of buffaloes are found. unlike the south african parks, where there is little or no contact with domestic stock, in tanzania infected buffalo could be reservoirs of bovine tb contributing to the increasing prevalence of tuberculosis in both humans and livestock, through mechanisms described below. some legal and controlled hunting is allowed in open areas, game controlled areas (gcas) and game reserves for game-meat in tanzania but there is no established meat inspection to monitor tb or other infections in these game products. in addition, people around game reserves and national parks practice illegal or bush meat hunting and hidden sale of game meat in villages and therefore difficult to control or conduct meat inspection. the situation is worsened during extensive droughts when food becomes scarce, resulting in increased poaching activities. in such circumstances, the contact between species at their interface and probability of infected meat entering the human food chain may increase with consequent spillover and cross-transmission of infection amongst populations (osofsky et al. 2005). in addition, the life expectancy of livestock held by small scale farmers is generally higher than in commercial livestock keeping and bovine tuberculosis as a chronic and progressive disease manifests itself more often in older animals, under nutritional or productive stress (michel et al. 2006). this socio-cultural behaviour when combined with close proximity with wildlife is detrimental to either ecosystem in terms of tuberculosis cross-transmission. molecular epidemiological diagnostic tools and their application in developing countries top ↑ the development of molecular tools for identification of m. bovis, the known zoonotic tb causing mycobacterium, and differentiation from other members of the m. tuberculosis complex have allowed the discovery of more cases in retrospective studies and have suggested new forms of transmission (cataldi & romano 2007). in addition, the usefulness of molecular tools in normal practices of prevention and control of tuberculosis has been addressed (mcnabb, braden & navin 2002). genotyping of mycobacterial isolates in the mycobacterium tuberculosis complex has the ability to distinguish amongst clonal populations and provide key information on disease dynamics. however, this approach has been insufficiently applied in developing countries due to lack of facilities, resources and scarce skilled personnel. molecular techniques such as pcr are powerful tools to identify mycobacteria and can potentially be used on cultured and original biological samples. application of more than one molecular technique can complement each other in differentiating various strains or isolates and shed light on the phylogenetics and ultimately the transmission chains, thereby allowing for improved control or policy development (michel et al. 2010).such actions require specific and dedicated support and government commitment for wide use and sustainability. very few molecular epidemiological studies on tuberculosis have been done in tanzania and even those few have been made possible mainly through collaborations. these studies have focused on the epidemiology of the disease in livestock or in wildlife, or have dealt with the disease in humans. as yet, tracking transmission across species using molecular tools such as rflp (michel 2002b) in tanzania has been limited or almost non-existent. one health as a disease control strategy top ↑ management of diseases in wildlife is complex because the current wildlife policy in tanzania does not generally advocate treatment or vaccination to control infection and disease. treatment or vaccination is only allowed if a disease outbreak occurs in endangered wildlife species or the outbreak poses a threat to the species population. of course it must also be acknowledged that vaccination is not effective in all diseases, tb being one such example. moreover, the logistics and funding required for treatment or vaccination in wildlife also make it largely impractical because it is almost impossible to get all animals, except perhaps very rare species (osofsky et al. 2005). in most of africa, small scale farmer’s livestock and their products are largely excluded from veterinary and veterinary public health control measures (michel et al. 2004). therefore, a change in policy and the political will that is necessary to support such recommendations will be necessary to reduce infection and disease. we need a two-pronged approach to address both technical and political issues to formulate a way forward. a policy that is focused on control of zoonotic tb and which addresses the need for a ‘one health’ approach, through which veterinary and medical professions as well as social scientists and ecologists can work together to limit disease would be a new venture with a chance of success. conclusions top ↑ due to differences in national policies which guide different sectors of the tanzanian economy (health, livestock and wildlife), the immediate suggested approach would be to prevent contact between wildlife and livestock and to control the disease (tb) in livestock through introduction of a compulsory test and slaughter practice, a proven recipe in other countries. this will reduce the transmission of disease not only between livestock and humans, but also limit transmission of infection from wildlife and vice versa. the implementation should include a policy that strictly prohibits livestock introgression to national parks and game reserves by implementing a boundary, beyond which animal keeping is unlawful. this however, has a problem in that even if livestock are kept out of this zone, wildlife will not understand that zone and will move into it, such that the ‘zone’ will then move outwards to contact livestock again posing a big challenge on the strategy. the other remaining challenge is to convince policy makers and creating awareness in livestock keepers concerning the magnitude of the problem its consequences and the importance of disease control even impacts across international borders. the serengeti national park, for example, spans two countries and animal movement to the other side of the park into the kenyan maasai mara might be contributing to an increased transmission risk not only to the wildlife in that country but also the livestock and humans in the neighbouring country. this is also applicable from kenya to tanzania, of course, as it would be for any transfrontier park. although some work has been done we still do not have any accurate estimate of the extent of human-animal cross-species transmission in tanzania, possibly elsewhere, with regard to tb. much of this is because of the technical complexity and cost of cultures and speciation. in addition, additional problems include bias in previous studies owing to sampling from urban areas and pulmonary samples as well as challenge of detecting infection rather than disease and attributing it to transmission, together with the assumption that it is a particular species of mycobacterium. studies are currently underway in and around the serengeti national park under the umbrella of the southern centre for infectious disease surveillance (sacids), to try to establish the clear trans-boundary interaction between human-livestock-wildlife and tb transmission. once one has a clear understanding of these factors, it may be possible to suggest an appropriate ‘one health’ approach to arrest pathogen flow and interrupt prevailing disease dynamics across species. in our opinion, research aiming to identify and characterise pathogens (not only mycobacterial species) of animals with potential to be infective to humans (wolfe et al. 2007) should be prioritised in order to enable us know the potential source of diseases to become proactive and hopefully prevent or limit outbreaks. to successfully implement this strategy, a sustainable and efficient collaborative approach involving multiple stakeholders from various sectors and working groups is needed. acknowledgement top ↑ the sacids is acknowledged for providing a postdoctoral research fellowship to em. financial support was received from the wellcome trust. prof. mark rweyemamu, the executive director of sacids is acknowledged for stimulating the idea and advice. the london international development centre in the uk and staff are acknowledged for collaboration. prof. jeff waage is particularly acknowledged for his dedicated efforts to provide complementary material support and encouragement during preparation of this manuscript. sian brown and sue van rensburg are acknowledged for the illustrative pictures used in this manuscript. muhimbili university of health and allied science, particularly, the school of medicine and the department of biochemistry are acknowledged for providing time for preparation of this work. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this paper. authors’ contributions e.v.m. (muhimbili university of health and allied sciences)participated in reviewing the literature, conceptualising, drafting and writing the manuscript. b.z.k. (muhimbili university of health and allied sciences) participated in initial drafting of the manuscript and critical manuscript review. s.k. (the royal veterinary college) participated in critical manuscript review. l.g. (the royal veterinary college) assisted in critical manuscript review and focusing of the manuscript. g.s.k. (tumaini university) critically reviewed the manuscript. j.d.k. (tanzania wildlife research institute) conceived the idea, assisted in drafting and critical review of the manuscript. p.v.h. (university of stellenbosch) assisted in critical review of the manuscript. p.g.f. (london school of hygiene and tropical medicine) assisted in drafting the manuscript and critically reviewed the manuscript. m.i.m. 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intransigent of these are infections that induce a ‘carrier’ state, where the animal remains infected, is still able to transmit organisms to the vector, but may show disease symptoms intermittently or not at all. indeed, it may be difficult to detect the infection by classical means. some examples are those infections caused by african trypanosomes, babesia parasites, anaplasma or borrelia bacteria (allred & alkhedery 2004; horn 2004; palmer, futse et al. 2006; bankhead & chaconas 2007). in many cases, it is now evident through the use of sensitive molecular diagnostic methods that a typical infection course consists of numerous relapsing peaks of organisms separated by time periods when they are not detected (burgdorfer & mavros 1970; kieser, eriks et al. 1990; calder, reddy et al. 1996; zheng, sentsui et al. 1997; granquist, stuen et al. 2008). this has been known for many years with trypanosome infections but only more recently for some of the other infectious agents. nevertheless, some molecular characteristics of these infections resemble those of trypanosomes, but with their own particular special features. i consider here our current understanding of some of these molecular characteristics. discussion taking african trypanosomes as the original, moststudied paradigm, we know that the variable surface glycoprotein (vsg) that covers the cytoplasmic 53 onderstepoort journal of veterinary research, 76:53–58 (2009) persistence mechanisms in tick-borne diseases a.f. barbet department of infectious diseases & pathology, college of veterinary medicine university of florida, gainesville, florida, usa abstract barbet, a.f. 2009. persistence mechanisms in tick-borne diseases. onderstepoort journal of veterinary research, 76:53–58 the use of new, highly sensitive diagnostic methods has revealed persistent infections to be a common feature of different tick-borne diseases, such as babesiosis, anaplasmosis and heartwater. antigenic variation can contribute to disease persistence through the continual elaboration of new surface structures, and we know in several instances how this is achieved. known or suspected mechanisms of persistence in babesial parasites include cytoadhesion and rapid variation of the adhesive ligand in babesia bovis and genetic diversity in several merozoite stage proteins of different babesia spp. in anaplasma, extensive variation in the pfam01617 gene family accompanies cycling of organism levels in chronic infection. one result from the pioneering research at onderstepoort is the definition of a related polymorphic gene family that is likely involved in immunity against heartwater disease. we are beginning to understand the sizes of the antigenic repertoires and full definition is close, with the possibility of applying simultaneous high-throughput sequencing to the order of 1 000 small genomes. we also, for the first time, can consider modifying these genomes and looking at effects on persistence and virulence. however, important biological questions remain unanswered; for example, why we are seeing a new emerging anaplasma infection of humans and is infection of endothelial cells by anaplasma significant to persistence in vivo. 54 persistence mechanisms in tick-borne diseases membrane is responsible for the ensuing battle with the host immune system and we have extensive information about its structure, variation and mechanisms of expression (barry & mcculloch 2001; vanhamme, pays et al. 2001; borst 2002; mcculloch 2004; pays, vanhamme et al. 2004). despite data showing large differences in vsg structure as the trypanosome undergoes antigenic variation during infection, we know that vsg expression is not totally random and vsg repertoires are not entirely distinct in different places (hutchinson, picozzi et al. 2007). in fact, there are examples of shared variants between vsgs in east and west africa and south america (vervoort, barbet et al. 1981; magnus, vervoort et al. 1982). nevertheless, the repertoires are large (with > 1 500 genes in each trypanosome encoding vsgs) and largely divergent in different places and at different times (barry, crowe et al. 1983; barry, marcello et al. 2005; berriman, ghedin et al. 2005; hutchinson, picozzi et al. 2007). moreover, the use of different segments of these >1 500 genes to create novel trypanosome identities through recombination extends the repertoire size enormously (roth, bringaud et al. 1989; kamper & barbet 1992; barbet & kamper 1993; marcello & barry 2007) . this concept of antigenic repertoire size is useful in analyzing the epidemiology of many relapsing infectious diseases and the possibilities for control. so, what do we know about tick-borne diseases important to animal production? babesia bovis expresses a novel surface antigen, known as the variant erythrocyte surface antigen (vesa) 1, on the surface of infected erythrocytes (o’connor, lane et al. 1997). it is thought to mediate binding of parasitized erythrocytes to endothelial cells, thereby causing parasite sequestration in small capillaries (o’connor, long et al. 1999). this helps the parasite to avoid passage through the spleen and consequent removal. sequestration in the microvasculature is an important feature of babesiosis and is often found in the dangerous cerebral form of the disease. vesa1 is strongly recognized during bovine infection, with variant-specific antibodies detected shortly after the respective parasitemic peaks containing the homologous organisms. in a recent study (al-khedery & allred 2006), a genomic expression site for vesa1 was described which had acquired at least three different short-coding dna segments from elsewhere in the genome, suggesting an analogous mechanism to amplify the vesa1 repertoire in babesia parasites to that in trypanosomes. the vesa1 gene repertoire size in a single babesia organism was estimated at > 350 (for one of the two vesa1 subunits) (al-khedery & allred 2006), although genome sequence data put the repertoire size at closer to 150 copies (brayton, lau et al. 2007), without considering the effects of repertoire amplification by recombination. therefore, in both trypanosomes and babesia, which are protozoans with genome sizes of 35 mb and 8.2 mb, respectively, there are large, amplifiable repertoires of variant surface antigens. one might expect the situation to be less complex in anaplasma because of the smaller genome size of prokaryotes (1.2 mb for anaplasma marginale). in fact, this appears to be the case, with two variant outer membrane proteins (omps) identified, msp2 and msp3 (barbet, lundgren et al. 2000; brayton, meeus et al. 2003; meeus, brayton et al. 2003). mes senger rna encoding these molecules is transcribed from single genomic expression sites to which approximately seven msp2 pseudogenes and seven msp3 pseudogenes contribute sequence by recombination (brayton, knowles et al. 2001). as with trypanosomes and babesia, the variant repertoire is amplifiable in a. marginale, because of the use of different pseudogenes and pseudogene segments to create mosaic expression site sequences. although this extends the repertoire of variant msp2 and msp3 enormously, the ultimate size is limited by the relatively small pool of donor pseudogenes. an important unknown question is how much this pool of donor sequences varies with time and location. we do know that many pseudogenes are shared by strains from diverse locations and that epidemic spread of the organism appears associated with the introduction of organisms carrying different msp2 pseudogenes (rodriguez, palmer et al. 2005). anaplasma phagocytophilum is a related species to a. marginale which, although known for > 200 years as a pathogen of ruminants (cases in more than 300 000 norwegian lambs annually) (stuen 2007), is now causing severe infections in dogs and humans (dumler, choi et al. 2005; lester, breitschwerdt et al. 2005; poitout, shinozaki et al. 2005; dumler, barat et al. 2007; jensen, simon et al. 2007; beall, chandrashekar et al. 2008; kohn, galke et al. 2008). interestingly, the a. phagocytophilum genome contains > 100 genes and pseudogenes homologous to msp2 of a. marginale and likewise a single expression site to which they donate variant sequences (barbet, meeus et al. 2003; wang, rikihisa et al. 2004; lin & rikihisa 2005; dunning hotopp, lin et al. 2006). as with a. marginale, the a. phagocytophilum ortholog (msp2/p44) is immunoprotective against homologous variants (palmer, oberle et al. 55 a.f. barbet 1988; wang, kikuchi et al. 2006). although the potential variant repertoire appears larger than in a. marginale, it is known that many similar variants are expressed in the multiple different animal species infected by a. phagocytophilum in the usa, possibly indicating a more recent introduction to the continent and less evolutionary diversification of the donor gene repertoire than in european strains (barbet, lundgren et al. 2006). of great significance to africa is the tick-borne disease of heartwater and, here too, we now know much more of the potential variant omp repertoire. this is, in large part, due to the pioneering research of the onderstepoort veterinary institute which developed culture methods to grow ehrlichia (formerly cowdria) ruminantium (bezuidenhout, paterson et al. 1985) and then to obtain a complete genome sequence (collins, liebenberg et al. 2005). we are fortunate in this case to have more than one genome sequence, from both an african and a caribbean strain (frutos, viari et al. 2006) of e. ruminantium. although the ehrlichias express a related group of variable but immunoprotective (nyika, mahan et al. 1998; nyika, barbet et al. 2002; yager, bitsaktsis et al. 2005) omps to the msp2s of the anaplasmas, they do not appear to have a similar system of antigenic variation that involves extensive recombination into a single genomic expression site. this is not to say that there are no antigenic differences in omps between strains and, in fact, breaks in protection against different strains have been a continual problem for development of vaccines against heart water disease (jongejan, thielemans et al. 1991; jongejan, vogel et al. 1993; mahan, allsopp et al. 1999). the msp2-related omps of e. ruminantium are encoded by a tandem group of 16 genes (van heerden, collins et al. 2004) one of which appears to encode the major omp expressed in mammals and a second the major omp in ticks (postigo, taoufik et al. 2008). this leaves a question concerning the role of the remaining paralogs. in ehr lichia chaffeensis, which has a similar genomic struc ture of msp2-related paralogs to e. ruminantium, sensitive proteomic methods have detected expression of most paralogs despite the dominant expression of certain paralogs in ticks or mammals (ge & rikihisa 2007; seo, cheng et al. 2008). analysis of rna transcripts has suggested that the same may be true for e. ruminantium (bekker, postigo et al. 2005). it is also clear that both major and minor expressed paralogs are variant in different strains of e. ruminantium throughout africa and the caribbean (reddy, sulsona et al. 1996; barbet, byrom and mahan, unpublished). therefore, all paralogs could be involved in expression of the total antigenic identity of e. ruminantium, as they appear to be in anaplasma. in a. marginale, an interesting hypothesis has been proposed that the ability of strains to superinfect (and ultimately to cause epidemic spread and influence cross-protection by vaccines) depends on whether or not a superinfecting strain has a different donor pseudogene (for omp expression) repertoire (futse, brayton et al. 2008). if it does, then the superinfecting strain can overcome pre-existing immunity developed over a long time period of infection in carrier animals. this is supported by data from the msp2 pseudogene repertoire and, currently, the involvement of the other known variant omp, msp3, in this process is not clear. there are opportunities to test this theory in other systems. for example, despite the > 100 genes and pseudogenes potentially contributing to msp2/p44 variants in a. phagocytophilum, there are two us strains, mrk and hz, which differ in (presence or absence of) only two pseudogenes (dunning hotopp, lin et al. 2006). one might predict heavy usage of these two pseudogenes in a superinfection of mrk-infected (longterm carrier) animals by hz. if true for other tickborne diseases, knowledge of the total repertoire of contributing variant genes to omp diversity becomes of paramount importance to understand disease epidemiology and for vaccine development. con ceivably, this might be achieved for the smaller genome organisms using second and third generation genome-sequencing capable of obtaining, e.g. 1 000 genome sequences of diverse e. ruminantium strains (margulies, egholm et al. 2005; holt & jones 2008). complementary to the new power of genome sequencing technology to evaluate variant repertoires in tick-borne diseases is the ability to introduce foreign dna and investigate phenotypic effects. this has been achieved with a. phagocytophilum by transposon mutagenesis (felsheim, herron et al. 2006), but directed methods of gene knock-out in a. marginale, babesia or ehrlichia are still lacking, although standard in trypanosomes and other parasites and bacteria for many years. potentially, these methods offer the ability to verify definitively the importance of omp variation in persistent infections, as well as to use other approaches towards vaccines through deliberate attenuation. despite the extensive work over the last 100 years, we are still discovering new and unforeseen aspects of these organisms, such as the ability to continuously culture a. marginale, thought to infect only erythrocytes, in endothelial cells (munderloh, lynch et al. 2004; 56 persistence mechanisms in tick-borne diseases wamsley & barbet 2008). therefore, although much is now known about these 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: etheresia pretorius1 ursula b. windberger2 hester m. oberholzer1 roland e. j. auer3 affiliations: 1department of anatomy, school of medicine, faculty of health sciences, university of pretoria, south africa 2center for biomedical research, medical university vienna, austria 3university of pretoria biomedical research centre, university of pretoria, south africa correspondence to: etheresia pretorius email: resia.pretorius@up.ac.za postal address: po box 2034, university of pretoria, pretoria 0001, south africa keywords dog; fibrin networks; ischemic stroke; platelets; scanning electron microscopy dates: received: 27 june 2010 accepted: 21 aug. 2010 published: 05 nov. 2010 how to cite this article: pretorius, e., windberger, u.b., oberholzer, h.m. & auer, r.e.j., 2010, ‘comparative ultrastructure of fibrin networks of a dog after thrombotic ischaemic stroke’, onderstepoort journal of veterinary research 77(1): art. #4, 4 pages. doi: 10.4102/ojvr.v77i1.4 copyright notice: © 2010. the authors. licensee: openjournals publishing. this work is licensed under the creative commons attribution license. issn: 1025-9848 (print) issn: 2071-9736 (online) comparative ultrastructure of fibrin networks of a dog after thrombotic ischaemic stroke in this original research... open access • abstract • introduction • materials and methods    • patient    • streptokinase    • samples    • blood analysis    • preparation of the blood sample to obtain a fibrin clot    • preparation of the washed fibrin clot for scanning electron microscopy • results    • blood analysis    • ultrastructural analysis • discussion • conclusion • conflict oof interest statement • acknowledgements • references abstract (back to top) a cerebrovascular accident or stroke is a rare condition in dogs, but previous studies suggest that it is now increasingly being recognised. platelets and fibrin networks are involved in haemostasis, which is disrupted during a thrombotic event. in this study we investigate the ultrastructure of the fibrin networks of a dog that had suffered ischaemic stroke, following suspected thrombo-embolism from clots that became dislodged during catheter maintenance (flushing with heparinised saline) 2 days after carotid artery catheter insertion. fibrin networks of blood samples that were collected immediately after the stroke, 15 min after treatment with streptokinase and 24 h after treatment, were studied. the results were compared to those of two control dogs. during a stroke, fibrin morphology changes to form a thick, matted layer. post-treatment ultrastructure shows that the fibrin morphology returns to that comparable to controls. our results show that during thrombotic risk, fibrin network morphology changes visibly and reduces the fibrinolytic activity of the coagulation system. introduction (back to top) a cerebrovascular accident or stroke is defined as the sudden onset of non-progressive, focal brain dysfunction as a result of ischaemic infarction or haemorrhage (garosi & mcconnell 2005). focal ischaemic stroke is caused by an interruption of the arterial blood flow to a dependent area of the brain parenchyma by a thrombus or an embolus. although research based on the human condition is much more prominent in current literature, it seems as if this rare condition is increasingly being recognised in dogs (wessmann, chandler & garosi 2008). the choice of diagnostic tool is typically magnetic resonance imaging (mri), particularly using diffusion-weighted images and magnetic resonance angiography for ischaemic stroke and gradient echo sequences for haemorrhagic stroke (wessmann et al. 2008). wessmann and co-workers (2008) mentioned that the underlying cause is not always identified in either humans or dogs. underlying conditions that may be associated with canine stroke include hypothyroidism, neoplasia, sepsis, hypertension, parasites, vascular malformation and coagulopathy. although research has been done to show which coagulation factors are involved in ischaemic stroke, there is little available on the morphology of the fibrin networks that might be involved in coagulopathy as the cause of ischaemic stroke. in this study, we investigated the ultrastructure of the fibrin networks of a dog that had suffered a chance ischaemic stroke following suspected thrombo-embolism from clots that became dislodged during catheter maintenance (flushing with heparinised saline) 2 days after carotid artery catheter insertion. fibrin networks of blood samples collected immediately after the stroke, 20 min after treatment with streptokinase and 24 h after treatment, were studied. the results were compared to that of two control dogs. materials and methods (back to top) patient the patient was a 9-year-old female, sterilised beagle dog, approximately 11.5 kg in body weight, and clinically healthy prior to the adverse event. streptokinase the streptokinase used was streptase (750 000 iu, zlb, behring gmbh, marburg, germany). the enzyme was reconstituted with 5 ml of 5% dextrose (adcock ingram, south africa), of which 2.5 ml was injected into a 200-ml dextrose infusion bag (adcock ingram, south africa). samples samples consisted of blood from two control dogs (beagles) and samples from a beagle dog directly after a stroke, 15 min after streptokinase treatment and 24 h after treatment. blood samples were collected by venipuncture from the cephalic vein as part of the diagnostic efforts in the treatment of the condition of the dog. blood analysis blood was collected in edta-, citrateand heparine-coated vacutainer® tubes (bd belliver, united kingdom) as well as plain vacutainer® serum tubes (bd belliver, united kingdom) for haematology, serum chemistry and clotting parameters. the analysis were performed by the clinical pathology laboratory of the onderstepoort veterinary academic hospital (see results section for a list of tests performed). preparation of the blood sample to obtain a fibrin clot all blood was collected in citrate tubes, (bd belliver, united kingdom) stored at 4 °c and processed shortly after collection. freshly prepared platelet-rich plasma (fprp) was prepared by centrifuging blood at 1000 rpm (using a hettich universal centrifuge) for 2 min. from the fprp, 20 µl was mixed with 20 µl thrombin (south african national blood services) on a 0.2 µm millipore membrane (microsep, south africa) to form the coagulum (fibrin clot). the filter was placed in a petri dish, on filter paper dampened with phosphate buffered saline (pbs) to create a humid environment and placed at 37 °c for 10 min. this was followed by a washing process where the millipore membranes containing coagulate were placed in pbs and magnetically stirred for 2 h. blood proteins trapped within the fibrin network were removed in this way. preparation of the washed fibrin clot for scanning electron microscopy washed fibrin clots were fixed in 2.5% glutaraldehyde (lasec, south africa), in dulbecco’s phosphate buffered saline (dpbs) buffer (lasec, south africa), ph 7.4, for 1 h. each fibrin clot was rinsed 3 times in phosphate buffer (lasec, south africa), for 5 min before being fixed for 1 h with 1% osmium tetra-oxide (oso4) (lasec, south africa) the samples were rinsed 3 times with distilled water for 5 min each rinse and dehydrated serially in 30%, 50%, 70% and 90% ethanol, and 3 times with 100% ethanol. the scanning electron microscopy (sem) procedures were completed by critical point drying of the material, mounting and examination of the tissue using a jeol 6000f fegsem. results (back to top) blood analysis table 1 represents the haematology of the ischaemic stroke dog. analysis of haemoglobin, red blood cell counts and white blood cell counts showed no abnormalities compared to controls. platelet and coagulation related variables were also not significantly different compared to reference ranges. ultrastructural analysis sem images of fibrin networks from the control animal are shown in figure 1 (two control animals were used and their clot morphology is similar). fibrin networks consist of major, thick fibres (a) forming the largest part of the network, with thread-like thin, minor fibres (b), distributed between the major fibres. this type of distribution is typical of animal fibrin networks (pretorius et al. 2009). figure 2 illustrates the ultrastructure of the fibrin networks of the dog after the ischaemic stroke. the fibrin networks show a dense, mat-like appearance (c), without the typical distinguishable separate major and minor fibrin layers. figure 3 shows the fibrin network, 15 min post-treatment. here, the thick, mat-like layer appears a little less pronounced and individual thick, major fibres (d) are more prominent than in figure 2. the thick, black arrow shows areas of thick, matted fibrin. figure 4 indicates the fibrin networks 24 h post-treatment. here, major, thick fibres (e) as well as the minor, thin fibres (f) can now be distinguished. this morphology appears comparable to that of the control morphology (figure 1). figure 1,2,3 & 4: 1. scanning electron microscope image of control dog fibrin network with thick, major fibres as well as thin, minor fibres 2. scanning electron microscope image of fibrin network from dog suffering from ischaemic stroke (pre-treatment) 3. scanning electron microscope image of fibrin network from dog suffering from ischaemic stroke, 15 minutes after treatment 4. scanning electron microscope image of fibrin network from dog suffering from ischaemic stroke, 24 hours after treatment discussion (back to top) in this study, a first-generation thrombolytic product, streptokinase, was used to stabilise a dog that had suffered ischaemic stroke following suspected thrombo-embolism from clots that became dislodged during catheter maintenance. streptokinase has no fibrin-binding capabilities and causes systemic plasminogen activation (through cleavage, to produce plasmin), with concomitant destruction of haemostatic proteins (longstaff, williams & thelwell 2008) and is a protein produced by beta-haemolytic streptococcus. it is used as an effective and inexpensive clot-dissolving product and is therefore a thrombolytic agent with a half-life of approximately 20 min. during thrombolytic activity, plasmin is produced in the blood to break down fibrin, which is arranged in fibrin networks composed of major, thick fibres and minor, thin fibres. under normal conditions, plasmin therefore dissolves fibrin networks, after it has been produced, to stop bleeding. in the case of a thrombotic event causing abnormal clotting, as was found in this study, streptokinase causes an increase in the production of plasmin, followed by breaking down of the unwanted blood clots. during this process, streptokinase forms a complex in the plasma with plasminogen to form an activator complex and this complex then forms plasmin from unbound plasminogen. streptokinase will therefore break down abnormal fibrin networks that caused the thrombotic event. table 1 shows the haematology of the stroke dog, compared to reference values and no significant differences were noted. however, ultrastructural analysis of the fibrin networks revealed key changes in morphology. the ultrastructures of the fibrin networks and platelets of the controls are typical and have also previously been seen in animals like the mouse (mus musculus), horse (equus caballus), vervet monkey (chlorocebus aethiops, previously cercopithecus aethiops), oryx (oryx gazella), sheep (ovis aries), penguin (spheniscus demersus), rabbit (oryctolagus cuniculus) and sea turtle (caretta caretta) (pretorius et al. 2009). typically, a fibrin network will consist of major, thick fibres, forming the main structure of the clot and is covered with a minor, fine fibrin network that is sparsely distributed inbetween the major fibres (figure 1). analysis of the fibrin clot, performed shortly after the ischaemic stroke had occurred, showed that the fibrin network appeared matted, forming a dense, thick layer. individual major fibres were fused and fibres were woven into a tight, nearly solid layer (figure 2). however, 20 min after treatment with streptokinase was completed, individual major fibres were visible, although the matted appearance was still present (figure 3, thick black arrow). no minor, thin fibres were detected. it is known that streptokinase acts as rapidly as 20 min after administration. at 24 h after treatment, major fibres were more prevalent and comparable to that of the control animal, and minor, thin fibres were also detected at this stage (figure 4. f). this suggests that the streptokinase administered to the dog stabilised the fibrin network nearly to the degree found in a control animal, thus restoring the normal conditions and reducing thrombotic risk and the occurrence of another ischaemic stroke. results from two previous studies by humphries et al. in 2008 and pretorius et al. in 2009 showed comparable morphological changes during rabbit pregnancy and human pregnancy (humphries, smit & pretorius 2008; pretorius et al. 2009). the studies established that in human and rabbit pregnancy, minor, thin fibres form a thin net on top of the major, thick fibres. however, this minor fibre net covering was formed over and inbetween the major fibres, with the major fibres still detectable in the fibrin clot. in pregnancy, it is known that there is an increased thrombotic risk. humphries et al. 2008 and pretorius et al. 2009 concluded that as a result of the more dense appearance of fibrin networks during pregnancy, clots might take longer to be broken down by normal fibrinolytic activity. in this case study of an ischaemic stroke in a dog, an even tighter, dense, mat-like fibrin network is seen in the dog prior to treatment (figure 2). table 1: blood analysis of dog with ischaemic stroke conclusion (back to the top) we conclude that during thrombotic risk, fibrin network morphology changed visibly and reduced the fibrinolytic activity of the coagulation system, which led to the ischaemic stroke observed. it is further suggested that in future, sem analysis might be included in the screening of both animals and, possibly, humans where thrombotic risk is suspected. conflict of interest statement (back to the top) the authors of this paper have no financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. acknowledgements (back to the top) we thank the national research foundation of south africa (nrf) for funding e. pretorius (indigenous knowledge systems [fa200403310004]) and we acknowledge the south african national blood service for providing the thrombostim preparation, which included the lprp and the human thrombin. references (back to the top) garosi, l.s. & mcconnell, j.f., 2005, ‘ischaemic stroke in dogs and humans: a comparative review’, journal of small animal practice 46(11), 521−529. humphries, p., smit, e. & pretorius, e., 2008, ‘ultrastructural morphology of platelets and fibrin networks of lactating and non-pregnant rabbits’, anatomia, histologia, embryologia 37, 285−288. longstaff, c., williams, s. & thelwell, c., 2008, ‘fibrin binding and the regulation of plasminogen activators during thrombolytic therapy’, cardiovascular and hematological agents in medicinal chemistry 6(3) 212−223. pretorius, e., bronkhorst, p., briedenhann, s., smit, e. & franz, r.c., 2009, ‘comparisons of the fibrin networks during pregnancy, non-pregnancy and pregnancy during dysfibrinogenaemia using the scanning electron microscope’, blood, coagulation and fibrinolysis 20(1) 12−16. pretorius, e., vieira, w.a., oberholzer, h.m. & auer, r.e.j., 2009, ‘comparative scanning electron microscopy of platelets and fibrin networks of humans and different animals’, international journal of morphology 27(1) 69−76. wessmann, a., chandler, k. & garosi, l., 2009, ‘ischaemic and haemorrhagic stroke in the dog’, the veterinary journal 180(3) 290−303. article information authors: angwara kiwara1 ave-maria semakafu1 gasto frumence1 affiliations: 1school of public health and social sciences, muhimbili university of health and allied sciences, tanzania correspondence to: angwara kiwara postal address: po box 35147, dar-es-salaam, tanzania how to cite this article: kiwara, a., semakafu, a-m., frumence, g., 2014., ‘the quest for one health: human resource training aspects’, onderstepoort journal of veterinary research 81(2) art. #720, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.720 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the quest for one health: human resource training aspects in this proceedings... open access • abstract • introduction    • objectives • methodology and marterials • results • discussion    • appropriately trained human resources for health – a critical input in one health    • why consideration of social determinants of health matter – lessons for one health    • production of human resource for health–appropriate training models    • the germ theory model    • the race for the germ theory    • the beginning of a new era    • antibiotics    • the epidemiological transition and unlearned lessons    • a proposed new model for the training of veterinarians and medical doctors    • what is the essence of this model?    • health workers count • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ appropriately trained human resources for health (hrh) are key inputs into one health. ‘… more than 50% of all infectious diseases of humans originate from animals and that, of the emerging diseases about 75% could be traced back to animal origin’ (rweyemamu et al. 2006). a comprehensive understanding of the social determinants of health, through an appropriate training model for hrh, is a key input. this study aimed to explore if human and veterinary medical schools were using such a model or providing time for this model in their curricula. specific objectives were to: determine the time that human and veterinary medical schools’ curricula provide for subjects or courses related to the social determinants of health; analyse the curricula contents to establish how they relate to the social determinants of health; and explore how a bio-medical model may influence the graduates’ understanding and practice of one health. a review of human and veterinary graduate-level medical schools’ curricula in east africa was performed in april 2013 and may 2013. the findings were: in the curricula, sdh contents for knowledge enhancement about one health are minimal and that teaching is germ theory model-driven and partisan. out of the total training time for physicians and veterinarians, less than 10% was provided for the social determinants of health-related courses. in conclusion, the curricula and training times provided are inadequate for graduates to fully understand the social determinants of health and their role in one health. furthermore, the germ theory model that has been adopted addresses secondary causes and is inappropriate. there is a need for more in-depth model. this article suggests that a vicious cycle of ill-health model must be taught. introduction top ↑ international public health experts are now focusing their attention on innovative ways of realising better health in all countries through an improved framework; this is because previous approaches have not delivered the expected results (solar et al. 2010; who 2010). despite sustained efforts by health experts and policy makers, ill health still exists for both animals and humans (who 2006). the one health approach has overwhelmed most of the debates (coker et al. 2011). these debates are justified given that ‘it has been established that more than 50% of all infectious diseases of humans originate from animals and that of the emerging diseases about 75% could be traced back to animal origin,’ (rweyemamu et al. 2006). it is further argued, and plausibly so, that the social and environmental ecology drives the social determinants of health, which researches have proven over time to be of primary importance in both animal and human health (who 2006; world bank 2010). objectives this article sought to contribute to an important aspect of enriching a one health approach, with a specific focus on analysing the key aspects of the training of health workers. it set out to specifically: • determine the time that human and veterinary medical schools’ curricula provide for subjects or courses related to the social determinants of health (sdh). • analyse curricula contents and how they relate to the sdh. • examine how the curricula contents may influence the graduates’ understanding and practice of one health. methodology and marterials top ↑ the curricula of five university-level human and veterinary medical schools in east africa were reviewed. the aim was to establish how much time was allocated to courses related to the sdh taught at the medical schools. these courses included: nutrition, community health sociology and development studies. other courses included the classical subjects at medical and veterinary schools such as: parasitology; microbiology; virology; pathology; pharmacology; and management of diseases. current curricula were obtained from the respective universities and schools in the region and analysed by desk review. the total times provided for the sdh-related subjects (nutrition, community health sociology and development studies) were compiled. likewise, the total times provided for the classical subjects were compiled. a thorough examination of the classical subjects was undertaken to decipher and categorise the contents as to whether they were related in any way with the sdh. finally, ten group discussions were held with final-year veterinary and human medicine students in conveniently selected schools.for the focus-group discussions, three questions guided the discussions. these questions were: • how do you understand one health? • would you work as one team after graduation? • would you facilitate joint training if you had the means to do so? results top ↑ it was established that students studying to become physicians or veterinarians spend on average ten semesters at university. this totals 6382 hours; out of this time, only 639 hours or 10% is spent on subjects that are directly related to the sdh. the rest of the time is spent on the classical subjects. compartmentalisation and isolated, specific moulding takes place very early on before the graduate goes out to practice. it was also established that the contents of the classical subjects present microorganisms to be the primary causes of ill health in animals and human beings. students are taught that these microorganisms exist and act in their own right, independent of the social and environmental ecology, to cause ill health. they are taught that the direct management of these microorganisms can overcome the problem of ill health in animals with disregard to the social and environmental ecology. given the established compartmentalisation during training and the disregard for the social and environmental ecology, one health is thus seriously threatened. the curricula of the veterinary and medical schools put the germ theory model as the lead model in the training of veterinarians and human medical doctors. this centralisation is not in favour of a one health approach. it also creates and reinforces the fatal culture among medical and veterinary students of despising social sciences as a tool in one health. furthermore, it has come to acquire unquestionable legitimacy in dealing with diseases in both human beings and animals for reasons to be further raised below. during the focus group discussions, responses were transcribed and themes grouped together in a qualitative analysis. it was established that the final-year human and animal health students at the institutions studied minimally understand one health. all of the ten focus group discussions had similar conclusions. in an attempt to search deeper, written explanations about what one health meant were given to all groups. regarding working as one team for one health, the responses were mixed. these future animal and human health experts were of the view that such collective work should be done during epidemic outbreaks affecting human beings and animals. otherwise they see themselves as holding specific authorities, each in their own discipline and area of work. in relation to joint training, where certain core courses like anatomy and physiology are provided on one campus and in one school with provision for separate training in senior years, the views were very clear. the position was that no joint training can or should be done. it seems that stereotypes have already caught up with the future animal and human health experts. discussion top ↑ appropriately trained human resources for health – a critical input in one health sub-saharan african countries are locked up in a fatal dilemma. they have a double-hump epidemiological transition (who 2006). unlike the developed world, they have the increasing problem of non-communicable diseases whilst still suffering from high levels of infectious diseases. the high burden of infectious diseases is more than 50% of animal origin (rweyemamu et al. 2006). this is why the region’s hrh need to be trained with this fact born in mind. that is why, more than in any other region of the world, one health matters most in sub-saharan africa and needs to be strengthened (who 2008). what are the issues? infectious diseases: the future report (2006) noted that critical issues in sub-saharan africa include an overwhelming presence of infectious diseases in the region, which poses a serious threat to the lives of both animals and humans (rweyemamu et al. 2006). these diseases attack humans and animals, causing morbidity, mortality and death from starvation. the implications of this, in the form of economic losses, social instability and a resultant downward spiral in livelihood, are immediate (brazier et al. 2007). ultimately, these countries are locked in a vicious cycle of poverty that is hard to escape from. this has serious economic and health implications given the fact that livestock agriculture represents 25% of the region’s gross domestic product (rweyemamu et al. 2006). both the economic and political reforms that have taken place in the region have worsened the spread of contagious diseases affecting both animals and humans. for example, the structural adjustment programmes have intensified movement of both humans and animals in the region, making it easier for infections to spread faster and further. larger-scale farming activities have been extended to new areas, increasing contact with wild stock and the possibility of new and fatal infections (world bank 2010). documented examples include ebola and a variety of hemorrhagic fevers. the direct effects of zoonoses and multiplicity of other diseases among animals and humans, who are continuously interacting, has lead to the economic and social decline. understanding the importance of these factors by health workers, among others, is necessary in the quest for one health in the region (winding 2007). civil wars and refugee crises have been driven by poor governance and have displaced large numbers of animals and humans into remote areas. at such times, control of movement is not possible. this facilitates spread of infectious diseases from one area to another and from animals to humans. under such conditions, infectious diseases of animal origin easily spread among the human population because of poor hygiene, over congestion, poor nutrition and lack of proper accommodation (drummond et al. 2005). a deepened understanding of the direct effects of hrh practices is necessary in both animal and human health. such practices include irrational prescription of antibiotics. this leads to emergence of resistant microorganisms. the many unregistered pharmaceutical outlets in the region and the use of incomplete doses enhance this practice (rweyemamu 2006). due to liberalisation of the economy, uncontrolled importation and sale of drugs is widespread. these drugs are often ineffective because they lack the active ingredients that are necessary. others have expired, but they are sold for use to uninformed consumers. in both instances, emergence of resistant organisms is a consequence. the resulting prolonged morbidity has a negative impact on the economy. many systemic factors have contributed substantially to a decline of both animal and human health, thus minimising the chances of realising one health. they are: • weak health care systems, which cannot perform proper diagnosis and disease management. • weak rural economies, which tend to push people to migrate to urban areas where life without employment and income is a recipe for behaviours that may lead to drug use and sexually transmitted infections, including hiv and aids. • inadequate government budgets, which are often dependent on development partner support. this may have unfavourable conditionality or may not be received as expected. • weak government-level veterinary services, which are mainly available in urban areas where demand is higher. • frequent policy and structural changes. • uncontrolled animal movement within the country or from neighbouring countries. for the east african region this last factor is a major threat. each year, wild animals move across the national boundaries in large numbers. likewise, pastoralists migrate across the region looking for better pastures for their livestock. these movements facilitate movement of old and new pathogens (rweyemamu et al. 2006). why consideration of social determinants of health matter – lessons for one health the training of hrh for one health must facilitate an objective understanding of one health and health in general as a multisectoral or multifactorial product of the interplay of socio-economic factors. such understanding will enable the trained hrh to objectively appreciate that establishment of one health is driven by human behaviour, social policies, economics, culture and more. the best tool that indicates the role of sdh in health is the human development index. the human development index (hdi) is a tool developed by the united nations to measure and rank countries’ levels of social and economic development (who 2010). the direct link between socio-economic factors and health is clearly indicated when life expectancy and maternal mortality rates are examined in countries ranked by hdi (table 1). the results of such ranking are indicated in the tables below.the analysis of life expectancy and maternal mortality rate in different countries has shown that the higher the hdi (favourable socio-economic conditions), the lower the maternal mortality rate (table 2). likewise, the higher it is, the higher the life expectancy. on the other hand, the lower the hdi (unfavourable socioeconomic conditions), the higher the maternal mortality rate and the lower the life expectancy. table 1: maternal mortality in relation to social determinants. table 2: life expectancy in relation to social determinants of health. production of human resource for health-appropriate training models cocker et al. (2011) and solar et al. (2010) concurred with the findings of this research that an appropriate model for the training of human resources for one health is necessary and must draw its contents from a multisectoral framework. this premise is a key factor, which determines how effective the produced human resources will be after training as a one health core team. a major prerequisite, however, is the presence of political will – this will enable the training centres, particularly those at the higher levels, to get enough funds to guide development of appropriate training models that they can implement. the models developed should be able to respond to the needs of one health in specific situations. an effective hrh must be prepared to understand the primary causes rendering the concept and practice of one health to be marginalised. as the curricula analysis above showed, only 10% of the training time is related to social determinants of disease. an attempt will now be made to unravel this puzzle by revisiting the origins of the germ theory of disease. the germ theory model for centuries, this model has dominated conceptualisation of disease, approach to curing disease and, particularly, what is taught at medical and veterinary schools. it is interesting to note the span of years over which the germ theory has remained a leading model in the training of health care professionals despite its lack of objectivity and dynamism as a major shortcoming. this shortcoming is proven by the epidemiological transition, which has manifested itself in the developed world where infectious diseases have been controlled. this phenomenon deserves urgent scientific enquiry. training through this model focuses on managing isolated episodes of animal or human illness. the view that any particular episode is not independent of the social or environmental ecology is not considered. treatment of cholera, for example, is by administering antibiotics and other drugs. a primary solution, like improvement of hygiene through better water supplies, which is a lasting solution, is emphasised the least by most clinical physicians and often it is not even included in the management plan. likewise, the treatment of trypanosomiasis invariably does not consider the environmental ecology. the germ theory, however, has left the developing countries with a legacy of the ‘magic bullets’ (antibiotics and other drugs) as a ‘panacea’ to diseases that are purely of social origin. the problem is not only the legacy but also the production of professionals who cannot conceptualise illness within its social context and so, in turn, empower the affected population to become healthier through appropriate advocacy of social approaches. the germ theory has deep roots: briefly, it was founded in two epochs: the race for the germ theory and what has been termed as the beginning of a new era, which is briefly revisited below. the race for the germ theory before james henle’s 1840 essay on ‘miasmata and contagion’, in which he formulated the modern germ theory, understanding of ill health or disease was speculative and predominantly secular (barry 2005). at the beginning of 500 bc, medicine became largely secular. it was believed that disease was an outcome of some imbalance in the body and that balance could be restored by intervention (warner 1992). this speculative era was replaced as society became more inquisitive. the beginning of a new era by 1500, methods of medicine were challenged. parcelius declared investigation of nature ‘not by following that which the old taught, but by our own observation of nature, confirmed by experiment and by reasoning thereon’ (barry 2005). later, vaselius dissected human corpses and concluded that galen’s findings had come from animals and were flawed (barry 2005). fracastorius hypothesised that diseases had specific causes and that ‘... contagion passes from one thing to another and is originally caused by infection of the imperceptible particle’. in 1628, came edward jenner’s work with cowpox on vaccination and immunisations. these were major landmarks in human and veterinary medicine (barry 2005). in 1860, pasteur proved that living things, not a chemical reaction, caused fermentation (warner 1992). the proof of pasteur’s work was by joseph lister who applied these findings to surgery. subsequently, robert koch’s ground-breaking work established the complete life circle of the anthrax bacillus, which showed that it formed spores that could lie dormant in the soil for years. in 1882, koch’s discovery of the tubercle bacillus – the cause of tuberculosis in animals and human beings – further confirmed the germ theory. later on in early 1900, while working at ocean road hospital in tanzania, koch discovered the malaria parasite for the first time (richard et al. 1964). these events and breakthroughs gave the germ theory authenticity and unprecedented power. what was theory became ‘fact’, the social origin of disease paradigm lost face and it suffered a major convulsion. the discovery of antibiotics incarcerated almost irreversibly the idea that social and environmental ecology were the primary origins of ill health in animals and humans. antibiotics with the discovery of antibiotics in the 1940s and their effect on infections, the germ theory attained an unprecedented authority and power. the dramatic effects of antibiotic prescriptions (penicillin and sulpha drugs) on syphilis, yaws, anthrax and other infections gave the theory fame and outright acceptance as the answer to ‘all diseases’. unquestionably, it assumed a central position in the health care professional’s training. the ‘magic’ power of antibiotics cast darkness on the sanitary movement, the epidemiological transition and emerging lessons. the epidemiological transition and unlearned lessons as the germ theory evolved, great lessons of public health importance accumulated. the sanitary movement matured in england through its impact in reducing morbidity and mortality caused by the black death and cholera epidemics (basch 1999). virchow publicly advocated that medicine (in animals and humans) was a social science and that medical professionals and veterinarians needed to have social science knowledge (basch 1999). chadwick was literally prosecuted for standing behind the sanitary revolution in england. mckeon wrote prolifically about how the decline in morbidity and mortality preceded the advent of antibiotics and other curative work (mckeown et al. 1974). it is a small wonder how the training of health professionals has managed to marginalise these lessons and focus almost exclusively on the germ theory as a major tool in training. the germ theory is ill informed and is turned upside down by the realities of the epidemiological transition. in this transition many societies have overcome infectious diseases (bacterial, viral or parasitic) through socio-economic measures. they are now confronted by non-communicable diseases, which the germ theory cannot understand. given these developments, the germ theory should have suffered its first fatal convulsion, however, it did not. what is the way out of this impasse? a proposal is presented below. a proposed new model for the training of veterinarians and medical doctors the most frequent human health problems, in both the developed and developing countries, have social determinants as their primary cause. typically, the top five disease categories in the developing countries include: infectious diseases (bacterial, parasitic, viral or fungal); nutrition-related diseases; drugs; alcoholism; and degenerative disorders (diabetes, cancers or cardiovascular diseases). all of these have social determinants as their primary cause. in the developed world, the major categories of diseases in order of importance are: degenerative disorders (cancers, work-related health problems, heart diseases and diabetes); road traffic accidents; alcoholism; and suicide. these also have social determinants as their primary causes. likewise, major veterinary challenges in the developing countries, such as foot-and-mouth disease, rinderpest (controlled, but with recurrences), tick fever, trypanosomiasis, rabies, rift valley fever, anthrax and bovine tuberculosis have policy-related, social and environmental ecology as their primary factors. if these are not understood one health will be a mirage. as such, the proposed training model must clearly show that social determinants of health are the primary cause of health or ill health. at the same time, the curricula in medical and veterinary schools need to build capacity for all health workers to understand health and ill health in that context and that micro organisms, as presented by the germ theory, are secondary causes. such a model must show a dynamic link between social determinants and health or ill health. it must also show, within the dynamic process, the role of the germ theory model-produced professionals and its outcomes, such as the vicious cycle of ill-health, chronic ill-health and death. what is the essence of this model? the model, as shown in figure 1, shows that those who succumb to ill health often adopt a wait-and-see approach or self medicate. this happens for both livestock and human illnesses. eventually when it does not work they consult health care experts. the health care experts under their germ theory training provide prescriptions for medications like antibiotics. some of the animals or patients recover, but some will acquire a chronic status and others will die. those animals that recover often survive on low scale morbidity and are a liability to farmers because their productivity (milk, meat, eggs) is low and are not good for nutrition or the market. these intensify the downward spiral of poverty. for humans, those who respond to medication are discharged out of the management regime and resume life in the society where they caught the previous ill-health problem. because the social determinants of health, which caused the previous ill-health problem, are still unaddressed, those who were cured by the antimalarials or antibiotics, for example, and even many more new ones, will become sick again and get locked up in the vicious cycle of ill health. the diagram below illustrates this movement in a cycle, as determined by exposure to social determinants of health for both animals and humans.this suggested training model will strongly underline that analysis and understanding of health and disease by all health professionals not only public health professionals, at all levels of training, is essential. it must begin with a detailed understanding of the social determinants of health (sdh) – society, social relations, economic well-being – and the development trends, as the primary causes of disease or ill health. bacteria, parasites, viruses and more are only secondary causes of ill health. the struggles focused at vertical elimination of bacteria, viruses and parasites through use of drugs alone have not succeeded anywhere and no country in the world has done this to date or should ever do it in future. figure 1: social determinants of health and the vicious cycle of health for animals and humans. health workers count due to the immense power bestowed on them by society and their acceptability, health care professionals in human medicine and veterinarians can be significant advocates of social change. these professionals, through the model above, can be trained to understand that the majority of ill-health is due to sdh and that it is possible to change society so that ill-health becomes history. a number of countries have controlled rinderpest or malaria because they have been able to nurture political will and intervene through social determinants, and made it possible for universal accessibility so that such diseases have been controlled. a voice coming from medical doctors and veterinarians will reach many individuals in the consultation room, households and communities within this context and break the vicious cycle of ill health through favourable sdh (figure 2). by intervening at the level of social determinants of health, as the cycle in the diagram below shows, more and more will remain healthy and ill health will be minimised. figure 2: social determinants of health and the broken vicious cycle of ill health for animals and humans. conclusion top ↑ for a long time it has been assumed that the graduate hrh would productively take the role in realisation of one health. however, the nature of their knowledge on the link between sdh and animal or human health and how the medical schools shape this knowledge has been a grey area and given minimum attention.the curriculum review referred to above has shown that the medical and veterinary schools use the germ theory model in training. this model, however, does not facilitate a deep understanding of the importance of sdh in healthcare, and the courses related to sdh are provided by only 10% of the graduates’ training time. the level of impact of hrh on one health will be determined by how veterinarians and human medicine professionals comprehensively understand the social determinants of health as the primary causes of ill health or health. the model used in training affects this understanding. the germ theory presents microorganisms as the primary causes of ill health. however, the microorganisms are outcomes of the social and environmental ecology. these outcomes are rife in the developing world and can only be fully understood by hrh who have been trained on the vicious cycle of ill health. such health workers will be the agents for change, as they will be able to objectively empower individuals and communities to actively participate in pursuit and defence of their own health and that of animals. this study recommends the vicious cycle of ill health as a model with which to enrich training of hrh in order to enable better performance in one health. acknowledgements top ↑ the authors thank the swedish international development agency for providing the required support to undertake the fieldwork, which has led to this article. they also thank the muhimbili university of health and social sciences for giving them time from their regular activities to undertake this review. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions a.w., a-m.s. and g.f. 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health, nutrition and population, washington dc, viewed 27 june 2013, from http://siteresources.worldbank.org/intard/resources/ppp_web.pdf article information authors: adrian d. knoetze1,2 niloshni moodley1 celia abolnik2 affiliations: 1rainbow veterinary laboratory, rainbow chicken farms, south africa2department of production animal studies, university of pretoria, south africa correspondence to: adrian knoetze postal address: po box 42, hammarsdale 3700, south africa dates: received: 10 feb. 2014 accepted: 03 june 2014 published: 09 sept. 2014 how to cite this article: knoetze, a.d., moodley, n. & abolnik, c., 2014, ‘two genotypes of infectious bronchitis virus are responsible for serological variation in kwazulu-natal poultry flocks prior to 2012’, onderstepoort journal of veterinary research 81(1), art. #769, 10 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.769 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. two genotypes of infectious bronchitis virus are responsible for serological variation in kwazulu-natal poultry flocks prior to 2012 in this original research... open access • abstract • introduction • materials and methods    • sample collection       • virus isolation       • virus neutralisation tests       • rna extraction and reverse transcription polymerase chain reaction • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ this study describes the isolation, serotyping and genotyping of 54 infectious bronchitis virus (ibv) cases predominantly in kwazulu-natal and compared to several isolates from other south african provinces between 2011 and 2012 and several historic isolates. the results indicate the division of isolates into two different genotypes of ibv within the province, massachusetts (mass)-like and qx-like. the ibv mass-like genotype was the most prevalent and was detected in 79% of the full spike protein s1 gene sequences. variation up to 22.3% was detected within local mass-type strains, supporting the hypothesis that multiple ibv serotypes may co-circulate in the same region simultaneously. additionally, more conservation was observed amongst mass serotypes versus qx-like serotypes, implying that vaccine use can influence the variability within the ibv population; this is deduced from the fact that the only live vaccine registered for use in south africa at the time of the study was of mass origin and no qx-like vaccines were available for use. this study offers the first published consolidation of ibv isolates from an area of south africa and identifies variation within the ibv population of the broiler flock within the study area over a 2-year period. introduction top ↑ infectious bronchitis virus (ibv) is a highly contagious coronavirus of poultry that causes disease characterised by upper respiratory or urogenital lesions. ibv is a listed disease according to the world organisation for animal health (oie) and can result in large losses due to mortality or loss of production. the disease has a worldwide distribution. the virus is shed both through the upper respiratory system and in faeces and may be detected within the bird’s gastrointestinal system for several weeks or months (cavanaugh & naqi 1997). clinical signs and lesions include respiratory symptoms, effects on egg production and egg shell quality and kidney pathology. mortality rates vary between 14% and 82% depending on the ibv serotype and coinfections with other pathogens, particularly escherichia coli and mycoplasma spp. (cavanaugh & naqi 1997; cook 1983; cook, huggins & ellis 1991; dhinakar raj & jones 1997).coronaviruses are enveloped single-stranded positive-sense rna viruses. the ibv genome is 27.569 kbp in length and contains four coronavirus structural protein genes, spike (s), envelope (e), membrane (m) and nucleocapsid (n), plus non-structural protein genes 3 and 5 and its viral replicase gene (rep). the order of these genes is 5′ rep-s-3-e-m-5-n 3′ (boursnell et al. 1987; spaan, cavanaugh & horzinek 1988). ibv s protein undergoes post-translational cleavage to s1 and s2 subproteins. the s1 protein is responsible for attachment to the host cell membrane and also has epitopes to which neutralising and haemagglutination-inhibiting antibodies bind (kant et al. 1992; koch et al. 1990). two hypervariable regions (hvrs) have been demonstrated within the 50–159 residue regions of the s1 gene. hvr1 lies within region 38–51 whilst hvr 2 is located between amino acids 99 and 115 (cavanaugh, davis & mockett 1988; cavanaugh et al. 1992; wang, xu & collisson 1997). a third hvr between amino acids 274 and 387 has also been described (lee & jackwood 2001). the s1, s2 and m proteins have all been shown to induce cell-mediated immunity with the n gene proposed as carrying the predominant t cell epitope (ignjatovic & galli 1994). the high degree of variability within the s1 gene has been attributed to the lack of proofreading by the viral rna-dependent rna polymerase and to recombination events during replication (toro, van santen & jackwood 2012). the emergence of variation within the ibv population has been widely described since 1951, when jungherr, chomiak and luginbuhl (1956) demonstrated that the connecticut isolate neither cross-neutralised nor cross-protected against the traditional h120 massachusetts (mass) isolate of the 1940s. the first published reports of ibv from south africa were by morley and thompson in 1984. this was confirmed as an unknown variant by cook et al. in 1999. these viruses were associated with swollen-head syndrome observed in infected flocks throughout southern africa. novel ibv has been described in nigeria (de wit, cook & van der heijden 2011) as well as zimbabwe, where a qx-like ibv strain has been reported (jackwood 2012). the south african poultry industry is the single largest agricultural subsector, accounting for an annual turnover of r31.7 billion per annum (south african poultry association 2012) with flock sizes ranging from several hundred to large complexes of 600 000 birds. perdue and seal (2000) listed the estimated losses per flock from an ibv infection as 10% to 20% of market value. this can devastate a poultry farm, where margins are relatively small. the economic impact of this disease is thus of great importance. currently available research findings do not describe variation over a large collection of south african ibv isolates and thus do not adequately describe the degree of variation within the south african broiler flock. the core question revolves around the extent of variation present in the south african ibv population. objectives of this study included identifying variation within ibv isolates, describing the correlation between serotyping and genotyping, as a method of variation identification and describing variation within the kwazulu-natal ibv isolates over time. materials and methods top ↑ sample collection over 100 ibv isolates were collected during the 18-month period; 46 of these were selected for the present study based on clinical history and pathology caused by the strain, geographic distribution and serum neutralisation test results. in addition, eight prior isolates were also included in the study. these isolates included six rainbow veterinary laboratory ibv reference strains and two historical isolates (table 1). seventy-five percent of isolates selected for the study originated from kwazulu-natal (kzn), 11% from the north west province, 3.5% each from the eastern cape and gauteng and 6% from the western cape. the weighting towards kzn isolates demonstrated the higher isolation rate from this province in addition to the higher suspected infection rate of this subpopulation within the study population. virus isolation samples for isolation were collected from postmortem samples on suspicion of ibv infection. organs collected included either tracheal samples or pooled tracheal and kidney samples. samples were collected from various postmortem facilities around south africa but all virus isolations were performed at the kzn laboratory.the standard virus isolation procedures are based on the published laboratory manual for the isolation, identification and characterisation of avian pathogens in 9 to 10-day-old embryonated chicken eggs via the allantoic sac inoculation route (gelb 1989). dead embryos were opened after candling; diagnosis of the cause of death was conducted and in the case of evidence that the embryo was infected with ibv, the allantoic fluid was collected. typical signs of ibv infection in the embryo, namely stunting, urate deposits in the mesonephros and clubbing of the down feathers, were noted. in the cases where no pathological signs were seen in the embryo, the allantoic fluid was still collected and reinoculated for an additional passage. a total of three passages were performed until the sample was confirmed negative. inoculations with supporting ibv clinical signs were confirmed for the presence of ibv through a chloroform inactivation test as well as diagnostic polymerase chain reaction (pcr) assay. the chloroform inactivation test differentiates between enveloped and non-enveloped viruses, and in combination with embryo pathology and haemagglutination tests a diagnosis of ibv can be made. pcr-confirmed ibv-positive allantoic fluid isolates were then stored at -70 °c until further processing. virus neutralisation tests viral antigen-containing allantoic fluid was treated in serial dilution with antisera collected against each reference isolate (gelb 1989). these antisera were produced through the hyperimmunisation of specific-pathogen-free leghorn chickens. rna extraction and reverse transcription polymerase chain reaction rna was extracted using trisure reagent (bioline) according to the procedure recommended by the manufacturer. a two-step pcr procedure was followed: viral cdna was generated by combining 5 µg of extracted rna, 4 µl 5x buffer (roche), 1 µl random hexamers (roche), 2 µl deoxynucleotides (dntp) (bioline), 0.3 µl ribonuclease (rnaase) inhibitor (affymetrix usb, 40u/µl), 0.5 µl molony murine leucosis virus (m-mulv) reverse transcriptase (roche) and diethylpyrocarbonate (depc) water (h2o) to a final reaction volume of 20 µl. the reaction was then incubated at 37 °c for 60 min.complementary dna (cdna) reactions were diluted by adding 20 µl pcr-grade water (roche). pcr was conducted with 10 µl 2x thermo scientific phusion flash high-fidelity pcr master mix (inqaba biotech, pretoria), 2 µl onderstepoort (op) ibv s1 forward primer, 2 µl op ibv s1 reverse primer, 1 µl pcr-grade h2o and 5 µl cdna. the target region was amplified in a veriti thermal cycler (life technologies) using a cycling profile of 98 °c for 10 s, 30 cycles of 98 °c for 5 s, 50 °c for 15 s and an elongation phase of 72 °c for 2 min. a final elongation step of 72 °c for 4 min was added. op ibv s1 for: 5′-gaacaaaasacngacttag-3′, op ibv s1 rev: 5′-ccataactaacataaggrcaa-3′. pcr products were electrophoretically separated on a 1% agarose gel stained with ethidium bromide. a kapa™ universal ladder was included to identify the 1700 bp s1 gene amplicon. bands of the correct size were excised and dna purified using qiaquick pcr purification kit (qiagen) following the recommended procedure. purified 1.7 kb pcr amplicons were submitted to inqaba biotech (pretoria) for sanger sequencing. results were edited using chromas lite and then aligned using bioedit version 7.1.11 (hall 1999). the basic local alignment search tool (blast) server was used to retrieve similar sequences selected from genbank for comparative analysis, together with important reference viruses. pairwise similarity was calculated using bioedit (hall 1999) and phylogenetic trees were constructed in mega 5.2 (tamura et al. 2011). full s1 gene sequences were deposited in genbank under the accession numbers kj200273–kj200291. partial sequences are available from the corresponding author on request. phylogenetic relationships were inferred using the neighbour-joining tree inference method with a jukes-cantor model of sequence evolution using mega 5.2 (tamura et al. 2011). one thousand bootstrap samplings were performed to assign confidence values to branching orders. ethical considerations top ↑ this study was approved by the university of pretoria’s animal ethics committee under reference number v006/12. results top ↑ a total of 54 isolates confirmed as ibv positive by serum neutralisation and diagnostic pcr were analysed by reverse transcription polymerase chain reaction (rt-pcr). the isolates were named in accordance with the system proposed by cavanaugh (2001) (table 1).forty-one samples produced a band of approximately 1700 base pairs (data not shown), of which full s1 gene sequences could be assembled for 19 isolates, with the remainder generating either partial forward or reverse sequences. despite numerous attempts on the service provider’s part to troubleshoot sequencing reactions, they were unable to generate the complementary forward or reverse reactions for 30 of the amplicons submitted. the reasons for this are unknown, since the exact same forward and reverse primers were used for sequencing as for rt-pcr, and numerous re-submissions of fresh amplified rt-pcr product in sufficient molar quantities were made. phylogenetic analysis of the full s1 gene sequences are presented in figure 1. partial sequence trees are not shown, but were used to genotypically classify the isolates (results are presented in table 1). isolates were classified as either qx-like (n = 4) or mass-type (n = 16). table 1: infectious bronchitis viruses characterised in this study. s1 gene nucleotide sequence comparisons (data not shown) revealed variability in qx-like isolates ck/za/3665/11, ck/za/4916/11, ck/za/4871/12 and ck/za/6720/12 when compared to a standard h120 mass serotype with a high number of base changes observed at positions 95–119, 142–148,165–172 and 695–708. this was evident in the translated amino acid sequences (figure 2), and notably between residues 58 and 62 of these isolates where isses is replaced with stnht. figure 2: multiple amino acid sequence alignment for full s1 proteins. hypervariable regions are indicated in grey. the first sequence is used as a reference and identities are plotted as ‘.’. figure 2 continues: multiple amino acid sequence alignment for full s1 proteins. hypervariable regions are indicated in grey. the first sequence is used as a reference and identities are plotted as ‘.’. in this qx-like group, residues 77–85 contained the motif kdvynqsva compared to the mass grouping with a motif of hggrvvna. a large insertion was also detected between residues 122 and 129 with a motif of ssgsgscp, which is not present in any of the mass-type isolates. two point mutations within ck/za/3665/11 resulted in the replacement of asparagine with histidine at position 24 and then aspartic acid with tyrosine at position 248. variation was observed between the two 2011 and the 2012 isolates within the qx-like group; the protein sequence at position 18–22 of the 2012 isolates was falwk and calcs as compared to the 2011 isolates at the same position. the most variable portion of the amino acid sequence for this group occurred at residue 400, where two of four isolates had consensus and variation between asparagine, histidine and tyrosine was seen. the remaining isolates were closely related to reference sequence h120 (mass serotype). single-point mutations were observed at various points across the isolates. no variation in nucleotide sequence is seen after base pair 792, demonstrating greater conservation in the second half of the gene. pairwise similarities were calculated between the study isolates (table 2) and selected serotype sequences obtained from genbank (table 3). pairwise comparisons against reference sequences support the description of two distinct groups of isolates. qx-like isolates shared 97.2% – 98.4% nucleotide sequence identities, corresponding to 97.6% – 98.8% identity at the amino acid level. these qx-like isolates only shared 77.7% – 79% nucleotide sequence and 85.9% – 88.6% amino acid sequence identities with the mass-like isolates. sequence variation within the mass-like group was less, at 98.9% – 100% and 99% – 100% nucleotide and amino acid similarities respectively (table 2). four isolates (ck/za/0890/81, ck/za/6689/12, ck/za/5315/11 and ck/za/2287/11) were very closely related. these sequences differed by 0.3% or four point mutations at positions 294, 302, 374 and 483. table 2: percentage similarity of study isolates with selected reference strains. table 2 continues: percentage similarity of study isolates with selected reference strains. figure 1: phylogenetic tree of full s1 gene nucleotide sequences (1665 bp) with isolates sequenced in this study highlighted. the remainder of the mass-type south african isolates shared high degrees of similarity with two point mutations common to all isolates when compared to the reference strain. the replacement of thymine with cysteine at residue 11 and thymine with adenine at residue 138 was found amongst all mass isolates. the highest frequency of variation in this case was found between base pairs 706 and 818, with 10-point mutations found in this region. these two distinct groupings were also evident when local strains were compared to international reference strains (table 3). ck/za/3665/11, ck/za/4916/11, ck/za/4871/12 and ck/za/6270/12s shared the highest nucleotide and amino acid homology with ibv qx-like chinese tc07-1 and uk 3355/09 strains. the south african mass-type viruses shared 99.2% – 99.7% nucleotide sequence identity and 96.5% – 96.7% amino acid similarity with isolate mass avail. table 3: percentage similarity of study isolates with selected reference strains. discussion top ↑ phylogenetic analysis of the s1 genes revealed two distinct groupings of ibv within the study isolates. the larger group contained 15 of the initial 19 full sequences and shared 99.0% – 99.7% amino acid sequence similarity with the h120 serotype. within this group there was 98.8% – 100% similarity at the nucleotide sequence level. reported s1 gene mutation rates under vaccine selective pressure are 2.5% (lee & jackwood 2001) and reports suggest that in the absence of a selective vaccine, mutation rates are twice as high than when under selective pressure (mckinley et al. 2011).when compared against the year or area of isolation it was found that the highly similar mass grouping originated from kzn between april 2011 and september 2011 and then again in october 2012. one isolate from the north west province from july 2011 and two from the western cape collected in october 2011 also fell within this group. strong correlations were observed between the isolates that originated outside kzn. despite the geographic areas being far apart there was movement of eggs and embryos between provinces during the study period. the variability of 0.4% observed between ck/za/6743a/11 and ck/za/6743b/11 is interesting, since these samples were collected from the same farm on the same day but originated from separate chicken houses or units. the existence of an insertion at 673 and point mutations at 4, 6, 296 and 879 demonstrate the existence of variability and selection of subpopulations on a single farm. the selective criteria for these subpopulations are unknown, but variables such as maternally derived antibody levels and timing of vaccination could apply greater selective pressure for the emergence of variants of the epitopes found on vaccine-derived viruses (de herdt et al. 2001; meulemans et al. 2001; mondal & naqi 2001). an alternative hypothesis is that the individual house conditions with regard to air quality and the degree of damage to the chicken’s tracheal epithelium and cilia affect the selection of s1 protein subpopulations. the use of especially the live day-old h120 vaccine and the close similarity of mass-type isolates to h120 must not be overlooked as the most likely influence on the prevalence of the h120 group. this implies that the most likely contributors to ibv serotypes within these flocks are mass-type live vaccines. however, there is the possibility that either through genetic drift or quasi-species selection, field-adapted virulent mass-type ibv was able to proliferate in suitable hosts. the basis of this statement is that all the samples collected were from clinical cases of respiratory disease and the presence of suggestive pathology on postmortem examination. recombination and reversion to virulence within mass-type ibv vaccines have been described and field experience over the years of use has found that poor vaccination technique, concurrent viral or bacterial infections as well as on-farm management conditions can lead to ibv pathology in light of vaccine usage (cavanaugh, davis & cook 1992). variability within the qx-like genotype was greater at 97.7% – 98.5% than that seen in the mass grouping at 99.0% – 99.7%. a temporal trend in genetic drift was observed. this observation is in contrast to evolutionary patterns within the mass group and supports the existence of genetic drift within this ibv subpopulation. the greatest similarity was noted when comparing it to the qx-like strains of european origin (97%) as opposed to the asian qx-like strains (94%), which provides circumstantial evidence of the spread of qx-like ibv from asia into europe and then downward into africa. the results of the kzn isolates would support the high host adaptability of the qx-like variant and identify it as the leading ‘variant’ within kzn poultry flocks. shaw, britton and cavanaugh (1996) investigated belgian nephrotropic ibv strains and reported that the s1 gene differed by 21% – 45% from vaccine strains whilst the s2 gene only had 10% – 12% differentiation. the 793/b strain was shown to display between 21% and 25% variation amongst field isolates, whilst still retaining the same serotype (adzhar & gough 1997). the ck/ch/ldl/971 strain of ibv was shown to have < 80% homology between the vaccine and pathogenic strain as well as between itself and other ibv types (liu et al. 2009). host-driven selection has been postulated and can occur as demonstrated during egg passage of several 793/b isolates; although this selection did not alter the pathogenicity, it does illustrate the quasi-species theorem regarding ibv populations (cavanaugh et al. 2005). jackwood, hilt and callison (2003) utilised real-time pcr techniques and melting curves to visualise the existence of quasi-species within the ibv beaudette strain. re-isolated virus has been demonstrated to contain subpopulations, again supporting the quasi-species theorem (mckinley, hilt & jackwood 2008). the correlation between qx-like serotypes and the actual sequence was inconsistent, as only 4 of the 11 serotype qx-like isolates were classified within the qx-like genotype. all seven remaining mass serotypes genotyped into the same group. by comparison, when examining partial s1 gene sequence data, none of the qx-like partial sequences were serotyped as qx-like. seven serotype qx-like isolates genotyped as mass, whilst the two isolates that did not fall into a known serotype at the time were split between the mass and qx-like genotype. the above results cast doubt on the correlation between serotype and genotype results. it has been described that small sequence variations may alter the virus neutralising epitope and thereby the serotype of the virus (cavanaugh et al. 1992). an alternative hypothesis could be that coinfection with multiple strains of ibv is responsible for clinical disease, and between the culture or serum neutralisation test and the subsequent successive passages and rt-pcr selection for host-specific strains there is selection for an alternate dominant strain (liu et al. 2009; xu et al. 2007). comparing these protein sequences to the isolates that both genotyped and serotyped as massachusetts, there is no obvious genetic marker that serotyped as qx-like in contrast to the group that serotyped as mass-like. conclusion top ↑ this study describes the isolation, identification and serotyping of a representative selection of ibv, predominantly in kwazulu-natal, between 2011 and 2012. the massachusetts (h120) serotype was the most prevalent serotype within the kzn poultry flock, according to the samples analysed in this study. the evidence suggested that the most commonly isolated mass-types are primarily derived from variation or mutation of vaccine strains administered to flocks. the qx-like serotype is the only non-vaccine serotype identified in the sample pool and had a greater degree of variation within the group than the mass-type isolates. the presumption therefore is that greater genotypic variation is seen amongst ibv serotypes that are not exposed to vaccine selective pressure. acknowledgements top ↑ we thank ms annemarie bosman for advice, dr nokuthula ntseki and rainbow chicken farms for the use of historical isolates and financial support of the study and drs jaco goosen and andrew van wijk for the submission of additional isolates. we would like to thank the institute for tropical medicine, antwerp, belgium for the funding of this study. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions a.d.k. 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http://dx.doi.org/10.1016/j.vetmic.2007.01.006 article information authors: mpoki mwabukusi1 esron d. karimuribo1,2 mark m. rweyemamu1 eric beda1 affiliations: 1southern african centre for infectious disease surveillance, tanzania2sokoine university of agriculture, department of veterinary medicine and public health, tanzania correspondence to: mpoki mwabukusi postal address: po box 3297, morogoro, tanzania how to cite this article: mwabukusi, m., karimuribo, e.d., rweyemamu, m.m. & beda, e., 2014, ‘mobile technologies for disease surveillance in humans and animals’, onderstepoort journal of veterinary research 81(2), art. #737, 5 pages. http://doi:10.4102/ ojvr.v81i2.737 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mobile technologies for disease surveillance in humans and animals in this proceedings... open access • abstract • introduction • research method and design    • case study sites    • implementation    • digital forms framework       • tools used to implement the digital forms    • sms forms framework       • tools used to implement the sms forms • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a paper-based disease reporting system has been associated with a number of challenges. these include difficulties to submit hard copies of the disease surveillance forms because of poor road infrastructure, weather conditions or challenging terrain, particularly in the developing countries. the system demands re-entry of the data at data processing and analysis points, thus making it prone to introduction of errors during this process. all these challenges contribute to delayed acquisition, processing and response to disease events occurring in remote hard to reach areas. our study piloted the use of mobile phones in order to transmit near to real-time data from remote districts in tanzania (ngorongoro and ngara), burundi (muyinga) and zambia (kazungula and sesheke). two technologies namely, digital and short messaging services were used to capture and transmit disease event data in the animal and human health sectors in the study areas based on a server–client model. smart phones running the android operating system (minimum required version: android 1.6), and which supported open source application, epicollect, as well as the open data kit application, were used in the study. these phones allowed collection of geo-tagged data, with the opportunity of including static and moving images related to disease events. the project supported routine disease surveillance systems in the ministries responsible for animal and human health in burundi, tanzania and zambia, as well as data collection for researchers at the sokoine university of agriculture, tanzania. during the project implementation period between 2011 and 2013, a total number of 1651 diseases event-related forms were submitted, which allowed reporters to include gps coordinates and photographs related to the events captured. it was concluded that the new technology-based surveillance system is useful in providing near to real-time data, with potential for enhancing timely response in rural remote areas of africa. we recommended adoption of the proven technologies to improve disease surveillance, particularly in the developing countries. introduction top ↑ the use of mobile and wireless technologies to support the achievement of health objectives (i.e. mhealth) has the potential to transform the face of health service delivery across the globe. a powerful combination of factors is driving this change. these include rapid advances in mobile technologies and applications, a rise in new opportunities for the integration of mobile health into existing ehealth services and the continued growth in coverage of mobile cellular networks (vital wave consulting 2009).the ability to collect data is the key to the success of many organisations operating in the developing world. given the weaknesses of current tools and the surge in mobile phone growth, there is an opportunity for mobile and cloud technologies to enable timely and efficient data collection and thus change how healthcare is delivered to millions of people (anokwa et al. 2009). smart phones offer pc-like functionality and web connectivity far superior to traditional mobile phones. built-in global positioning system (gps) receivers provide the detailed location of the phone, accelerometers can recognise changes in movement and cameras provide the ability to record static images as well as video. data networks allow built-in software to access the internet, providing access to web browsing, email, mapping (such as google maps) and the viewing and editing of office documents using touch screen keyboards (or hardware keyboards) for textual input (aanensen et al. 2009). the aim of this paper is to discuss and share the implementation and outcomes of a mobile technologies project introduced by the southern african centre for infectious disease surveillance (sacids), with the goal of improving disease surveillance in animal and human health sectors. research method and design top ↑ case study sites the study was conducted in different ecosystems characterised by interaction between humans and domestic and wild animals (ngorongoro and kibaha districts in tanzania), or cross-border districts shared by neighbouring countries such as ngara (tanzania) and muyinga (burundi) and the zambezi river basin ecosystem districts in zambia (sesheke and kazungula), bordering zimbabwe, namibia and botswana. in this kind of environment, the chance of contracting and spreading infectious diseases is very high. implementation selection of suitable technology (over a paper-based system of reporting – figure 1) and designing processes were carried out after a rapid situation analysis to assess existing surveillance systems in animal and human health in may 2010, in order to understand the structure and requirements of surveillance systems in the animal and human health sectors in tanzania (karimuribo et al. 2012). figure 1: a paper-based system of reporting, which may take three months to reach a ministry level. the two chosen technologies (digital forms and short messaging services [sms] – figure 2) were based on a server–client model (figure 3), wherein a mobile phone or personal computer operates as a client, communicating directly to the server via an internet connection, or sms via the global system for mobile communications (gsm) network. the server then stores all collected data from the field using mobile phones or personal computers and provides users access to the data via an integrated web-interface (sms and digital forms data can be accessed via the same interface). figure 2: a proposed system in which digital and mobile technology is used to speed up the reporting process. figure 3: a proposed system in which digital and mobile technology is used to speed up the reporting process. the server–client model can be described with three functional components: (1) data collection – referred as hardware devices and software combinations that enable surveillance data to be submitted to a central database, (2) data aggregation – a central server for hosting collected and sent or synchronised data. for each data collection technology (sms and digital form) there is an intermediary tool for receiving and placing data in the common database and (3) data visualisation and management – a web application that acts as interface between the database and user so as to simplify data management and analysis with the following capabilities: inputting data the web, providing map representation of geo-tagged data, viewing images and downloading data in excel format for further analysis one of the factors we considered before implementation was to use a cost-effective technology. open source technology was chosen, not only because it is cost-effective, but also because it has a wide community of developers, which gives us a wide area for improving the system in the future and the capability of deploying this in different languages for the end user. open source technology is defined as the production and development philosophy of allowing end users and developers to not only see the source code of software, but modify it as well. (garger & lamar 2010) digital forms framework first of all, the forms or questionnaires are prepared by officials related to the field (animal or human health) in hard copy format before being transferred to a digital format. following the digitisation process, they are uploaded onto mobile phones and the server. users can then download new forms, collect and synchronise data to the server. finally, through a web interface, data can be viewed and analysed (figure 4). figure 4: the digital forms framework, which moves through, (a) officials (animal and human sectors) designing a hard copy form, (b) digitising forms and uploading onto phones and the server, (c) users downloading forms, collecting and synchronising data to the server and (d) viewing and analysing surveillance data through the web interface. only authorised users provided with an account (username and password) can access the data via the web interface in different levels. on top of that, the web interface has the capability of viewing images and maps, which can result in a quick response in case of outbreak. on the left pane of the web interface (figure 5) there are four sections. firstly is ‘administration’, which will only be visible to administrators. secondly is ‘account’, where the users’ password and profile can be edited. thirdly is ‘forms’, where a form can be chosen to be filled out using the web interface through the ‘new’ button, listing submitted data through ‘list’ button and downloading data in an excel format through the ‘download’ button for further analysis. lastly there is ‘maps’, on which, through the ‘show’ button, you will be able to view a map that represents the geo-tagged data submitted. the middle pane lists a few details of the data submitted and by clicking on the ‘view’ button, a full list will be shown on the right pane, as seen in figure 5. data can also be printed using a ‘print’ button. figure 5: digital form data submitted by flora patta, a livestock field officer in endulen village in ngorongoro district, tanzania. tools used to implement the digital forms the primary tools used were smart phones (android version 2.3.5 – minimum required version is 1.6), a data server with ubuntu version 10.0.4.1 and the data collection applications, epicollect and open data kit. the two data applications follow the three model components mentioned above (data collection, data aggregate and data visualisation and management). the project adopted the data collection aspect without any changes, whereby applications are installed in smart phones and digitised forms uploaded into applications. the data aggregation, visualisation and management aspects were customised by the sacids information communication technology (ict) team from the source code provided by the two applications. the results of customisation were two applications using the same database and data accessible via a single web interface. sms forms framework kannel, an open source wireless application protocol and sms gateway, was implemented to build the sms application, whereby any phone with sms capability can be used. questions are designed to be answered in a code format (figure 6 and figure 7) and, at the back end, will be mapped to their respective answers in clear and simple language for easy readability via the web interface (figure 8). in figure 6, in the ‘question map’ column, q1–q6 represents question numbers and questions were formulated using swahili language. for multiple choice questions, the selections were also coded, for example, from the ‘code map’ column, 0 represents the word hapana, which means ‘no’, 1 represent ndio, which means ‘yes’, c1–c5 represents signs observed and d1–d5 represents districts names. in the field, data reporters will be given a sheet of questions together with codes and an example of how sms data is to be written. in the ‘shorthand’ column in figure 6 is an example that demonstrates the format: start with disease or form name, use a colon to separate one answer code from another and, for the multiple choice, use a comma to separate the selection codes. figure 7 shows an example of sms codes as they would appear when written and sent from a mobile phone. figure 6: the code and question map for mastitis, in addition to the shorthand on how the sms should be sent to the server. figure 7: the short messaging service codes as they are appear on mobile phones, written in swahili. figure 8: short messaging service codes submitted by community reporters recruited by talib suleiman, a phd candidate at sokoine university of agriculture researching mastitis in zanzibar, tanzania, mapped into question map and answers, respectively, in the swahili language. this sms application was officially rolled out at the end of 2013 for use by a phd student, talib suleiman, from the sokoine university of agriculture in tanzania for collecting mastitis data in zanzibar and rift valley fever data in ngorongoro. tools used to implement the sms forms the primary tools used were any phone capable of sending sms, the same data server used for the digital forms and the kannel application, which was then customised by sacids ict team using the provided source code by kannel community to build an application termed smscollect. we also used a modem that acted as an sms receiver. this modem connected to the server through software configurations in the server. sms’s sent by phones to the modem number are transferred directly from the modem to the server database to be converted into an easily readable format via the web interface. results top ↑ table 1 summarises the data collected via digital forms technology from january 2011 to may 2013 for the ngorongoro and kibaha districts and from september 2012 to may 2013 for the kagera river basin (ngara and muyinga districts). a total of four forms were used: animal disease surveillance, community health report for human and animals, and monthly and weekly reports for humans. table 1 indicates a good number of data reported compared with the few mobile phones that were distributed in study areas: kagera river basin (27 phones), ngorongoro (27 phones) and kibaha (7 phones). table 1: the total number of forms submitted for the kagera river basin between september 2012 and may 2013, for the ngorongoro and kibaha districts between january 2011 and may 2013 and for zambezi river basin between july 2012 and may 2013. discussion top ↑ through the use of this new digital and sms forms technology, officials from human and animal health have been able to conduct follow ups on disease cases more easily than before. as all data are sent in a digital format directly to the database, they can be accessed, downloaded in excel format and analysed within a very short time. the ability of downloading data in excel for further analysis has been useful for the district veterinary and medical officers in writing disease cases reports. figure 9 shows a pie chart that represents animal disease surveillance data submitted by kagera river basin livestock field officers’ via digital forms between september 2012 and may 2013. as data were submitted digitally, the stage in which data were entered manually into the database for analysis was skipped, potentially reducing the chance of human error affecting data capturing. using a map generated from gps coordinates submitted directly from the field, areas where disease cases occurred can be easily located. for example, figure 10 shows coordinates of data submitted by livestock field officers in the kagera river basin. the map shows that many cases occurred on the northern part of ngara, tanzania, where three countries – tanzania, burundi and rwanda – meet. here, livestock are being moved across the borders; there is also a livestock auction located around the area and a game reserve. with the combination of the abovementioned factors, the concentration of disease cases in this area is high. figure 9: the percentage of animal disease cases for the kagera river basin collected over a period of eight months, september 2012 – may 2013. conclusion top ↑ this new reporting system has been able to help provide a quick response to some of the cases and has therefore been deemed a successful tool that should be implemented on a wider scale in the field. one example of its success was seen in early january 2013, when a ngara district veterinary officer, dr richard ngowi, managed to conduct a quick follow up after a number of reported foot-and-mouth disease (fmd) cases (figure 9 shows a 10% prevalence of fmd over other diseases). as a result, on 04 february 2013, dr ngowi declared the district to be infected by fmd and under quarantine. the quarantine notice was subsequently posted to the sacids website (http://www.sacids.org/kms/frontend/?m=38). figure 10: map showing areas where animal disease cases were reported by livestock field officers between september 2012 and may 2013 in the kagera river basin. acknowledgements top ↑ the authors would like to acknowledge the support and cooperation we received from the rockefeller foundation (2009 dsn 305 grant), the ministries of health and social welfare in tanzania, burundi and zambia, the ministries of livestock in tanzania, burundi and zambia, the sokoine university of agriculture, as the hosting institute, the university of zambia as collaborating institute, as well as officials from ngorongoro, kibaha, ngara and muyinga districts, and the east african integrated disease surveillance network, imperial college london (for providing initial implementation support). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article.  authors’ contributions m.m. (southern african centre for infectious disease surveillance) was responsible in technology implementation and writing the manuscript. e.d.k. (sokoine university of agriculture) was the project leader and responsible for project design in epidemiology. m.m.r (southern african centre for infectious disease surveillance) was the project principal investigator. e.b. (southern african centre for infectious disease surveillance) was the project leader for the technological aspect, made contributions to the conceptual design of the overall information technology (it) infrastructure framework and provided guidance in its implementation. references top ↑ aanensen, d.m., huntley, d.m., feil, e.j., al-own, f. & spratt, b.g., 2009, ‘epicollect: linking smartphones to web applications for epidemiology, ecology and community data collection’, plos one 4(9), e6968. http://dx.doi.org/10.1371/journal.pone.0006968anokwa, y., hartung, c., brunette, w., borriello, g. & lerer, a., 2009, ‘open source data collection in the developing world’, computer 42(10), 97–99. http://dx.doi.org/10.1109/mc.2009.328 garger, j. & lamar, s., 2010, an introduction to open source technology, viewed 03 may 2013, from http://www.brighthub.com/computing/linux/articles/62137.aspx karimuribo, e.d., sayalel, k., beda, e., short, n., wambura, p., mboera, l.g. et al., 2012, ‘towards one health disease surveillance: the southern african centre for infectious disease surveillance approach’, onderstepoort journal of veterinary research 79(2), art. #454, 7 pages. http://dx.doi.org/10.4102/ojvr.v79i2.454 vital wave consulting, 2009, mhealth for development: the opportunity of mobile technology for healthcare in the developing world, un foundation – vodafone foundation partnership, washington, dc. article information authors: mark m. rweyemamu1 janusz paweska1 dominic kambarage1 filomena namuba1 affiliations: 1southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania correspondence to: mark rweyemamu postal address: po box 3297, chuo kikuu morogoro, tanzania how to cite this preface: rweyemamu, m.m., paweska, j., kambarage, d. & namuba, f., 2012, ‘towards one africa, one health: the southern african centre for infectious disease surveillance one health focus on infectious diseases’, onderstepoort journal of veterinary research 79(2), art. #449, 2 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.449 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. towards one africa, one health: the southern african centre for infectious disease surveillance one health focus on infectious diseases in this preface... open access • acknowledgements the genesis of the southern african centre for infectious disease surveillance (sacids) is rooted in the realisation that infectious diseases are and will continue to be a formidable challenge to human welfare and economic development in africa over the horizon of 30 years from 2005, and thereby impede africa’s ability to meet the targets of the un millennium development goals (mdg). several recent studies indicate that africa probably has the highest burden of infectious diseases of humans and animals and yet the least capacity for their detection, identification, monitoring and risk management. whilst climate change, changing in habitation and farming systems, globalisation of travel and trade are shared drivers for infectious diseases globally, certain other drivers have special relevance to africa. these include the high human-livestock-wildlife interaction, land use and socio-economic settings. the realisation of opportunities offered by new technologies for risk management of the conventional and emerging communicable diseases in humans and animals will require the integration of research across sectors (human, animal, environment) and disciplines (natural and social science) plus surveillance and disease control strategies that take account of cultural and governance settings. such approaches will, increasingly, have to be based on ecological systems, which often transcend administrative or national boundaries. it is against this background that african experts in infectious diseases of humans, animals and plants advocated in 2005 a pan-african vision for infectious disease management as: a pan-african concerted effort, shared by au member governments, reflecting the needs of african society and supported by the international community, with the goal of a society protected from the ravages of dangerous infectious diseases that compromise either human health or livelihoods and agriculture and economic development. they also recommended that such a vision was best developed through national and regional clusters. the above considerations propelled academic and research institutions involved with infectious diseases of humans and animals (domesticated or wild, terrestrial or aquatic) in southern africa to form the sacids (http:// www.sacids.org), with the vision of: a southern african society protected from devastating infectious diseases affecting the health of humans, animals, i.e. both terrestrial and aquatic, and plants, i.e. crop, forest and ornamental, thereby promoting livelihoods, socio-economic development including market access and the environment. the sacids mission is: to harness innovation in science and technology in order to improve southern africa’s capacity (including human, financial and physical) to detect, identify and monitor infectious diseases of humans, animals, plants and their interactions in order to better manage the risk posed by them. from the standpoint of such a vision and mission, the sacids ‘one health’ focus is to address infectious diseases in the endemic settings of sub-saharan africa, with a particular attention to southern, central and east africa through a collaborative effort between natural and social sciences to advance the understanding of interactions between humans, animals and the environment to improve public and animal health. thus the sacids model is one of a virtual centre that links the core institutions in southern africa with centres of excellence in the ‘north’, bound by a common mission and focus on one health and whose operation seeks to focus on intra-african as well as south-south-north collaboration. our research strategy is reflected in the themes of this conference, namely: 1. climate dependent, vector-borne diseases. 2. diseases with potential inter-species concern or spread between wildlife, livestock and humans. 3. diseases of economic importance. 4. bacterial rare diseases. 5. dangerous emerging diseases. 6. systems for disease surveillance and preparedness analysis. 7. socio-economic approaches to one health policy research. the sacids philosophy is to work towards: one africa, one health. accordingly, we seek to collaborate with other programmes, such as afrique one, eaidsnet, (east african integrated disease surveillance network) ohcea (one health central and eastern africa), afenet (african field epidemiology network), ruforum (the regional universities forum for capacity building in agriculture) and others that share this philosophy through a one health approach, irrespective of their specific strand of emphasis on ‘one health’. the new strategic framework of sacids to 2020 is: a sub-saharan african society protected from devastating infectious diseases affecting the health of humans, animals, i.e. both terrestrial and aquatic, and ecosystems, thereby promoting livelihoods, socio-economic development including market access and the environment our convening of the first one health conference in africa at the national institute for communicable diseases, johannesburg is testimony to our commitment to this goal. over the coming months and years we intend to collaborate with others of like mind to focus our attention on this vision through the study and application of one health approaches. we trust that the readers of this special issue of the onderstepoort journal of veterinary research will appreciate the diversity of disciplines and expertise that is already beginning to collaborate effectively towards the goal of: one africa, one health through smart partnerships. acknowledgements top ↑ southern african centre for infectious disease surveillance is funded primarily by grants from the wellcome trust, rockefeller foundation and google foundation. we are also grateful to the support of sokoine university of agriculture, and those sponsors, such as fao (food and agriculture organisation) of the united nations, who have funded some targeted activities. article information authors: kim a. kayunze1,2 angwara kiwara3 eligius lyamuya4 dominic m. kambarage5 jonathan rushton6 richard coker7 richard kock8 affiliations: 1rural development, sokoine university of agriculture, tanzania2postdoctoral research fellow in one health policy, southern african centre for infectious disease surveillance, sokoine university of agriculture, tanzania 3development studies, muhimbili university of health and allied sciences (muhas), institute of development studies, tanzania 4microbiology and deputy vice chancellor (academics, research and consultancy), muhimbili university college of health and allied sciences (muhas), tanzania 5veterinary medicine, sokoine university of agriculture, tanzania 6animal health economics, royal veterinary college, united kingdom 7public health, london school of hygiene & tropical medicine, united kingdom 8wildlife health and emerging diseases, ceeed, royal veterinary college, united kingdom correspondence to: kim kayunze postal address: po box 3024, morogoro, tanzania how to cite this article: kayunze, k.a., kiwara, a., lyamuya, e., kambarage, d.m., rushton, j., coker, r. et al., 2014, ‘practice of one health approaches: bridges and barriers in tanzania’, onderstepoort journal of veterinary research 81(2) art. #733, 8 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.733 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. practice of one health approaches: bridges and barriers in tanzania in this proceedings... open access • abstracts • introduction    • one health practice: historical background    • one health practice: current situation • materials and methods • results • discussion    • respondents’ involvement in collaborative work with other experts    • extent to which bridges enhance one health practice    • extent to which barriers impede one health practice       • actual factors that enhanced and impeded collaboration       • actual factors that enhanced collaboration       • actual factors that impeded collaboration • conclusions and recommendations • acknowledgements    • competing interests    • authors’ contributions • references abstracts top ↑ the practice of one health approaches in human and animal health programmes is influenced by type and scope of bridges for and barriers to partnerships. it was thus essential to evaluate the nature and scope of collaborative arrangements among human, animal and wildlife health experts in dealing with health challenges which demand intersectoral partnership. the nature of collaborative arrangement was assessed, and the respective bridges and barriers over a period of 12 months (july 2011 – june 2012) were identified. the specific objectives were to: (1) determine the proportions of health experts who had collaborated with other experts of disciplines different from theirs, (2) rank the general bridges for and barriers to collaboration according to the views of the health experts, and (3) find the actual bridges for and barriers to collaboration among the health experts interviewed. it was found that 27.0% of animal health officers interviewed had collaborated with medical officers while 12.4% of medical officers interviewed had collaborated with animal health experts. only 6.7% of the wildlife officers had collaborated with animal health experts. the main bridges for collaboration were instruction by upper level leaders, zoonotic diseases of serious impact and availability of funding. the main barriers to collaboration were lack of knowledge about animal or human health issues, lack of networks for collaboration and lack of plans to collaborate. this situation calls for the need to curb barriers in order to enhance intersectoral collaboration for more effective management of risks attributable to infectious diseases of humans and animals. introduction top ↑ one health approaches in human and animal health programmes are much desired, and although their implementation is enhanced by some factors (bridges), it is impeded by other factors (barriers). the one health initiative (2011) defines these approaches as collaborative efforts of multiple disciplines working locally, nationally and globally to attain optimal health for people, animals, plants and our environment. the approaches have a long history which can be traced to the 1700s, when variolation was used by medical and non-medical people collaboratively to confer specific immunity to smallpox using cowpox (riedel 2005). one health approaches have been being practised by various individuals and organisations. for example, in tanzania, such collaboration exists between medical and veterinary officers who collaborate in prevention, diagnosis, control and treatment of rabies in people and in livestock. other examples include sharing facilities such as refrigerators for storage of vaccines for humans and for livestock, and vehicles to carry out campaigns against zoonotic diseases. there is some empirical information on such collaborations; karimuribo et al. (2012) found that sometimes animal and health officers in ngorongoro district, tanzania, were sharing vaccine storage facilities, especially during times of disease vaccination campaigns when teams of vaccinators were camping in remote rural areas. although such collaborative activities are undertaken, up-scaling the same and undertaking more others is impeded by various barriers. general barriers to as well as bridges for such collaboration are well documented, for example by the who (2006); however, the extent to which they enhance or impede the practice of one health approaches are not known because no research has been done to generate such empirical information. therefore, the research from which this article emanated was done with the specific objectives to: (1) determine the proportions of health experts who had collaborated with other experts of disciplines different from theirs, (2) rank the general bridges for and barriers to collaboration according to the views of the health experts, and (3) find the actual bridges for and barriers to collaboration among the health experts interviewed. one health practice: historical background one health is not new; its practice can be traced as far back as to the mid-1700s when edward jenner (1749–1823), honoured as the father of immunology, contributed innovatively to immunisation and the ultimate eradication of smallpox using cowpox to confer specific immunity to smallpox (riedel 2005). the history of one health is also associated with rudolf ludwig karl virchow (1821–1902), honoured as the father of pathology. his statement that ‘politics is nothing else but medicine on a large scale’ can be interpreted to mean that helping people on a one-to-one basis is called medicine and doing the same on a large scale is called politics. he also argued that ‘between animal and human medicine there is no dividing line – nor should there be’. it is also he who coined the term ‘zoonosis’ (kahn et al. 2007, cited by monath, kahn & kaplan 2010). he practised one health by working in various disciplines, including human medicine, anthropology and public health. another person who has contributed prominently to one health is calvin w. schwabe (1927–2006) who, in 1964, coined the term ‘one medicine’ (schwabe 1969), now commonly called ‘one health’, and proposed a unified human and veterinary approach to zoonotic diseases in his seminal textbook veterinary medicine and human health (1984). he promoted one health by pioneering the use of human disease tracking techniques in the study of animal illnesses in the 1960s, and is honoured as the founder of veterinary epidemiology (world bank 2010). one health practice: current situation the need for one health practice is gaining popularity. many feel that it should be practised to contain new diseases like ebola haemorrhagic fever and avian influenza, which affect people and animals and quickly become pandemic. the increasing need for one health has resulted in the formation of a global organ, one world – one health (owoh), in september 2004. owoh has formulated twelve principles, known as the manhattan principles – which serve as recommendations for establishing a more holistic approach to preventing epidemic or epizootic diseases and for maintaining ecosystem integrity for the benefit of humans, their domesticated animals and the foundational biodiversity that supports humans (world bank 2010). new diseases such as ebola and avian flu remind us to be alert at all times as more hazardous diseases may appear at any time. thus we can avoid the post-world war ii complacency based on landmark medical attainments of the 1940s which were characterised by effective antimicrobial agents, along with the establishment of the principles and practice of immunisation. these attainments led george marshall, united states secretary of state at the time, to proclaim that the conquest of all infectious diseases was imminent – which was not true since the relationships between people, microbes and environment are complex and ever changing (coker, atun & mckee 2008). other organisations also promote one health. the wildlife conservation society (wcs), a non-governmental organisation based in the usa, fosters multidisciplinary research focusing on the interactions among human health, the environment, food and animal production. the federation of veterinarians of europe (fve), which was founded in 1975 (fve n.d.), practises one health with the aim to unite the european veterinary profession for the benefit of animal health, animal welfare and public health. in africa, an organisation called animal health for the environment and development (ahead) practises one health by focusing on several themes of critical importance to the future of animal agriculture, human health and livelihoods, and wildlife health and conservation (ahead n.d.). apart from these organisations, research projects in various parts of the world have focused on one health. mazet et al. (2009), for example, undertook a project in which they assessed the impact of interactions between water and disease in the ruaha ecosystem by simultaneously investigating medical, ecological, socioeconomic and policy issues driving the system. however, some projects implementing one health approaches have been short-lived and lacking capacity building components, which does not promote one health on a sustainable basis. unlike such projects, however, the southern african centre for infectious disease surveillance (sacids) uses approaches that promote one health on a long-term basis, including capacity building of current one health workers through training and research (sacids n.d.). although one health is advocated and is beginning to be adopted, it is practised by few organisations and people. therefore, there have been appeals for more organisations and people to practise it. for example, the who (2006) argues that if it was adopted it would greatly facilitate detecting and dealing with zoonoses, while at the same time ensuring better access to health inputs for both poor people and their livestock. the who (2006) also proposes a framework for collaborative action to stem zoonoses, which has four components: (1) one health: medical and veterinary, (2) research: epidemiology and new tools, (3) advocacy and information, and (4) diagnostics and surveillance. a strength of this framework is that it promotes the application of one health principles. however, it overlooks the social ecosystem and the roles of socioeconomic and cultural aspects. an example is people’s behaviour with regard to hunting and consuming bush-meat or close interaction with domestic animals, popular in sub-saharan africa and generally in the developing world, which may be a source of pathogens that may cause zoonotic diseases. the who (2006) also suggests that neglected zoonotic diseases could be eradicated through diagnosis and surveillance by: using effective surveillance systems for each of the ‘neglected zoonoses’; research on zoonoses with linkage to local public health systems; changing from single disease or vertical approach control measures to more integrated health promotion; and data sharing, monitoring and training, among other things. neglected (or lingering) zoonoses are ancient diseases affecting both humans and animals (domestic and wild) which keep re-emerging as public problems for a number of reasons. the who (2006) lists the following lingering zoonoses and is concerned about the fact that they seem to attract less public awareness: anthrax, bovine tuberculosis, brucellosis, cysticercosis and neurocysticercosis, cystic echinococcosis or hydatid disease, rabies, zoonotic sleeping sickness or human african trypanosomiasis (hat), and food-borne zoonoses including salmonella (salmonellosis), campylobacter (campylobacteriosis), and escherichia coli infections of animal origin affecting millions of people annually. apart from various organisations promoting one health, some individuals have advocated the adoption of one health. mahr (2006, cited by winding 2007) says, for example: animal health is truly at a crossroads; its convergence with human and ecosystem health dictates that the ‘one world, one health, one medicine’ concept must be embraced. we need our colleagues in human medicine, public health, and the environmental health sciences. together, we can accomplish more in improving global health than we can alone, and we have the responsibility to do so. (n.p.) although one health is advocated and some organisations are applying its approaches, its practice is impeded by various barriers, some of which are well documented (see e.g. who 2006). the barriers and bridges are not listed here, but their relevance was evaluated in the research, and the extent to which they apply are reported on. overcoming the barriers so as to increase collaboration by sharing responsibilities and coordinating global activities to address health risks, especially at the animal-human-ecosystems interface, could be on national agendas globally, and this can be facilitated internationally through the fao, oie and who. these organisations are committed to working more closely together to align activities related to the animal-human-ecosystem interface in order to support member countries (fao, oie & who 2010). materials and methods top ↑ this article is based on research conducted in ngorongoro and kibaha districts of tanzania in july 2012 and august 2012, covering the period from july 2011 to june 2012, whereby data were collected from 91 medical, veterinary and wildlife officers (51 medical officers, 30 veterinary officers and 10 wildlife officers). the officers were interviewed using a self-administered questionnaire, which they filled out in the presence of the researchers. the role of the latter was to clarify any items of the questionnaire in case the respondents needed clarification. the questionnaire was formulated with the aim to capture information on the respondents’ collaboration among themselves, factors which enhanced collaboration and those which impeded collaboration. the respondents were selected purposively depending on their availability and them working in areas where households which were interviewed in the same study were receiving human and animal health services. most of the respondents held diplomas, while a few held a bachelor’s degree and one held a master’s degree.the two districts, ngorongoro and kibaha, were selected purposively because both have pastoralists who interact intimately with their livestock, and whose livestock interact with ecosystems and wildlife. these interactions are likely to enhance transmission of zoonotic diseases such as rabies, brucellosis, tuberculosis, and anthrax from wildlife to livestock and from livestock to people. for effective detection, prevention, control and treatment of such zoonoses, collaboration of medical, veterinary and wildlife officers would be important. the pastoralists of kibaha district have originated from northern tanzania, including ngorongoro district. permission to conduct the study was obtained from sokoine university of agriculture and endorsed by ngorongoro and kibaha district executive directors. ethical consideration was observed as all participants were informed, before being interviewed, of the purpose of the study, what participation involved, confidentiality in the research, risks of participation, and their right to ask questions and withdraw from participation. the data collected from the respondents were analysed using the statistical package for social sciences (spss) programme. descriptive analysis was the main method of data analysis. frequencies, percentages, averages and minimum and maximum values of individual variables were computed. the extent to which general factors enhance collaboration (bridges) and others constrain collaboration (barriers) among medical, veterinary and wildlife officers were determined using pair-wise ranking to compare 14 general factors enhancing collaboration and 14 general factors constraining collaboration. in each of the cases, factors enhancing or constraining collaboration were compared, two at a time, until every factor was compared with every other factor. each time respondents were asked to give their view on which of the two was a more important factor. a dummy table (see figure 1) is provided to illustrate how the comparison was done. figure 1: tool used for pair-wise ranking of barriers and bridges. the numbers in figure 1 represent the 14 factors enhancing or constraining collaboration; each of the statements has to be written twice. the shaded cells that are placed diagonally represent areas where an answer would be written if comparing a statement with itself would make sense; those cells as well as those below them are not used. using the area below the shaded cells would result in duplication of information (of cells above them). the winning item of the two items that are compared is written in the cells above the shaded cells. each of the fourteen factors in each case had the possibility of being mentioned at most 13 times, in other words, the first statement could win 13 times throughout the second row to the right of number 1; the last statement could win throughout the last column under number 14; and any other statement could win in its respective rows and columns a total number of 13 times. for example, statement 5 could win four times in column 6 (below 5) and 9 times in row 6 to the right after the shaded cell in the row starting with 5. the average number of times each factor mentioned by all the respondents is expressed as a percentage of 13. the higher the percentage the more the important the factors are. results top ↑ the empirical information obtained from the respondents is reported in table 1 with respect to whether they had been involved in any collaborative work with other experts in disease diagnosis, surveillance, treatment or control or conducting campaigns against specific diseases. table 1: proportions of health experts who had collaborated with other experts. using the procedure of pair-wise ranking described above, the results of the respondents’ views on the extent to which general factors enhanced collaboration among medical, veterinary and wildlife officers are presented in table 2. table 2: views on extent to which general factors enhance collaboration. by using pair-wise ranking, respondents were also asked to share their views on the extent to which general factors constrain collaboration (see table 3). table 3: views on extent to which general factors constrain collaboration. besides the general bridges and barriers stated above, which had been compiled before data collection, the respondents were asked about the actual bridges and barriers that impacted on their possible collaboration with health experts in other fields. the responses are summarised in table 4. table 4: actual bridges and barriers. discussion top ↑ respondents’ involvement in collaborative work with other experts the responses summarised in table 1 regarding whether respondents had collaborated with experts from disciplines different from their own in the period 01 july 2011 to 30 june 2012 show that veterinary officers had collaborated with experts of other disciplines more than the other groups of experts, followed by medical officers. it is worth noting that collaboration took place even though there were no guidelines for collaboration. one could assume that if there were guidelines for collaboration, the level of collaboration would have been higher. the proportion of health experts who collaborated with health experts from other fields (as seen in table 1) were 19.1%, 63.0% and 17.9% for human health experts, animal health experts and wildlife experts respectively. this indicates a situation which can serve as a good starting point for up-scaling the practice of one health approaches. extent to which bridges enhance one health practice the results presented in table 2 show that the leading factors for enhancing collaboration were adequate transport facilities for medical, veterinary and wildlife officers (66.7%), common training in zoonotic diseases for both veterinary and medical doctors and fieldworkers (59.0%), one health policy formulation guiding applied research in health (57.4%), and advocacy for control of neglected zoonotic diseases (55.9%). these leading factors are very realistic; for instance, the issue of one health policy formulation or at least guidelines for mainstreaming one health practices in human health and animal health systems can form a solid foundation on which to base plans for allocation of resources for one health practice. such plans and resource allocation are recommended by various researchers and academics, including coker et al. (2008), brazier et al. (2007) and rushton et al. (2012). on the same issues, coker et al. (2008) argue that lack of resources for health is mainly due to low resources allocation for strengthening public health capacity and recommend that the allocation of such resources should be understood as a cost-effective investment to support preparedness and resilience. on the other hand, brazier et al. (2007) urge governments to allocate resources by policy making and by planning, especially once the judgement has been made that health care resources allocation cannot be left to unregulated markets. the judgement has to be based on empirical evidence; this is accentuated by rushton et al. (2012) who conclude that:political reality of adopting a one health agenda also requires thought and needs to be realistic with the evidence of added value from one health approaches through systematic data collection and analysis. (n.p.) on the same issue of judgement, zinsstag et al. (2012) argue that claiming a one health approach requires the demonstration of added value to what human and animal health working alone can achieve. the above list of factors enhancing collaboration had been compiled before the survey. in addition, respondents were asked to mention other factors which they thought enhance or have the potential to enhance collaboration among various health experts. sixty-two (62) of the respondents replied, giving the following responses: close relationship among experts (29.0%), common training in zoonoses (21.0%), information sharing (17.8%), more funds availability for zoonotic diseases (11.3%), implementing the concept of one health (8.1%), multisectoral collaboration (4.8%), and raising awareness among policy makers of the one health concept (3.2%). moreover, each of the following three factors was mentioned by 1.6% of the 62 respondents as other factors enhancing collaboration among various health experts: all health experts being under one ministry, strong policy and constitution, and having magazines or books for medical and veterinary officers. some salient points were made regarding these additional factors, such as the importance of raising awareness among policy makers of the one health concept and strong policy and constitution. if these were done, they would contribute to the enhancement of one health policy implementation, for example through budgeting for one health activities at the ministerial and district levels. this idea is in line with that of rushton et al. (2012), who argue that for diseases like brucellosis – which cause significant economic impact while the cost of controlling them in animals are greater than the benefits generated in the animal population, but the costs are exceeded when benefits from the prevention of the diseases in humans are taken into account – there is a need for one health thinking at a much higher level of budgetary and resource allocation, so that control campaigns in animals are sufficiently financed to lead to benefits in humans. extent to which barriers impede one health practice the results presented in table 3 show that the factors that mostly constrained collaboration were lack of consensus on priority-setting (65.0%), inadequate resources for dissemination of results and raising public awareness (64.8%), inadequate transport facilities for medical, veterinary and wildlife officers (62.5%), and applied research being not recognised or rewarded as important as basic research (55.5%). these four issues are related to the issues of planning and resource allocation discussed in the previous section. the respondents were also asked to mention other factors which they thought constrained collaboration. these other factors were mentioned by 79 of the respondents: lack of team work among health experts (17.7%), lack of relevant knowledge or skills needed for collaboration (11.4%), poor communication among health experts (10.1%), experts being based in different departments (10.1%), and insufficient budget (7.6%). the other factors were: selfishness of health experts not considering public benefits during collaboration (6.3%), inconvenient time for the implementation of activities due to lack of resources (5.1%), different health policies in human health sector and animal health sector (5.1%), lack of collaborative budgeting for human and animal health (3.8%), lack of common research on zoonotic diseases (3.8%), weak facilitation (3.8%), difference in knowledge and work ethics (2.5%), different management of zoonotic diseases (2.5%), and health experts not sharing their reports (2.4%). moreover, each of the following seven factors was mentioned by 1.3% of the respondents: shortage of personnel, applied research being not recognised or rewarded as basic research, confidentiality of medical data to be shared by veterinary and wildlife officers, authorities’ lack of appreciation for experts, bureaucracy in implementing animal health and human health programmes, and health experts being not interested in the one health concept. the respondents were also asked to give their views on whether the collaboration existing among medical, veterinary and wildlife experts was enough for effective prevention, control and treatment of zoonotic diseases; about two-thirds (66.3%) of them responded that it was not enough. they were then asked to give their views on how it should be improved. they made the following suggestions: making health experts of various disciplines work together (22.5%), emphasising training to both experts (18.3%), collaborative sharing of information from both sides (11.3%), formulating policies for collaboration between human health and animal health sectors (11.3%), conducting training to both experts on zoonotic diseases (9.9%), sufficient budget to enable collaboration (9.9%), conducting sharing experience meetings between human health experts and animal health experts (4.2%), involving communities (2.8%), and experts reaching consensus on improving collaboration among them (2.8%). moreover, each of the following suggestions were made by 1.4% of the respondents: transparency in implementation of one health practice activities, availability of qualified staff at district level, finding a mechanism for each ministry to address zoonotic diseases depending on the source, both ministries allocating budgets for prevention of zoonotic diseases, recognition of the work done by experts, and each district conducting monthly meetings to implement the one health concept. the suggestions given by the respondents for improving collaboration among health experts were generally consistent with their views on the extent to which general factors enhance collaboration. for example, common training in zoonotic diseases for both veterinary and medical doctors and fieldworkers ranked second among general factors enhancing collaboration, and the same issue of training ranked second among the suggestions for improvement of collaboration among health experts. actual factors that enhanced and impeded collaboration the factors enhancing and those impeding collaboration among medical, veterinary and wildlife officers were evaluated further by asking the respondents about the actual factors that were applicable to their situation; those who had collaborated listed the factors that had enabled them to collaborate, and those who had not collaborated listed the factors that had prevented them from collaborating. these factors are discussed below. actual factors that enhanced collaboration as seen in table 4, the leading factors that enhanced collaboration among various health experts were the disease(s) being a zoonosis or zoonoses which needed assistance from veterinarians (26.4%), the disease(s) being a zoonosis or zoonoses which needed assistance from medical doctors (25.5%), being instructed by upper-level leaders to cooperate with other health experts (22.7%), and the availability of funding for the collaboration (19.8%). the first two of the above-mentioned factors relate to the issue of common training. this issue is being addressed by organisations promoting one health on a long-term basis, such as sacids, which has established postgraduate training in one health, starting with an m.sc. (one health molecular biology) at the sokoine university of agriculture in tanzania and an m.sc. (one health analytical epidemiology) at the university of zambia. related training in one health has also been initiated by several other universities, for example the university of calgary in canada (zinsstag et al. 2012). the other two factors relate to the issues of planning and budgeting discussed in the previous section.further analysis of the results focused on the ways in which respondents had collaborated. the leading ways of collaboration were: disease treatment or control (17.1%), campaigns against specific diseases (15.2%), disease diagnosis (14.6%), disease surveillance (14.6%), and treatment or control of rabies (10.4%). the other ways in which respondents had collaborated were: conducting campaigns against rabies (9.8%), surveillance of rabies (7.9%), diagnosing rabies (7.3%), research (2.4%), and sending weekly and monthly reports to relevant authorities (0.6%). with regard to other diseases, collaboration pertained to only some aspects, and not to all aspects as with regard to rabies. other diseases that elicited collaboration were: anthrax diagnosis, surveillance, treatment and campaigns; rift valley fever (rvf) diagnosis, surveillance, treatment and campaigns; east coast fever (ecf) diagnosis, surveillance, treatment and campaigns; contagious bovine pleuropneumonia (cbpp) diagnosis, surveillance, treatment and campaigns; ppr diagnosis; tuberculosis diagnosis, surveillance, treatment and campaigns; brucellosis diagnosis, surveillance, treatment and campaigns; and trypanosomiasis surveillance. the financial supporters of the collaborative activities were parent ministries, especially the ministry of health and welfare, the ministry of livestock and fisheries and the ministry of natural resources and tourism, and various national and international organisations, including fao, who, tasaf, tawiri, ncaa, ndc, saids, tanapa, the bill and melinda gates foundation, local government authorities, sua and muhas. respondents were also asked about the outcomes of their collaborations; however, only 27 of them responded to this question. they said that disease outbreaks were effectively controlled (48.1%) and that more people became aware of disease control and treatment (14.9%). others (3.7%) said that the diseases were diagnosed and controlled; they succeeded to collect data about rinderpest; dogs were vaccinated against rabies; awareness about rabies was increased among community members; and community members were given more knowledge on how to prepare meat before consuming it. actual factors that impeded collaboration the leading factors that impeded collaboration among various health experts (see table 4) were the following: lack of policy statements to enforce collaboration among health experts (42.8%), lack of networking partners (25.0%), lack of knowledge of animal health expertise (14.3%), and lack of plans for collaboration (7.1%). some solutions to these salient issues have been discussed above, with the exception of networking. networking would be promoted through the formulation of guidelines and memoranda of understanding for collaboration among various health experts. conclusions and recommendations top ↑ the proportion of medical, veterinary and wildlife officers collaborating with experts in fields different from theirs are encouraging in view of the fact that there are no guidelines for collaboration. the assumption is that if there were guidelines for collaboration, the level of collaboration would be higher. in view of this, it is recommended that efforts be made to increase the proportion of medical, veterinary and wildlife officers collaborating in order to control lingering (neglected) zoonoses and relatively new diseases more effectively.the results of this study showed that respondents were able to rank general bridges for and barriers to collaboration and even add others. this implies that they are familiar with the different factors that enhance or impede collaboration among health experts. on the basis of this, it is recommended that health ministries work together to address impediments to collaboration among them and formulate regulations and memoranda of understanding for mainstreaming one health approaches in human and animal health systems, so as to increase collaboration. in view of the argument presented in the introduction that the fao, oie and who could facilitate national efforts to increase sectoral collaboration in order to address health risks, especially at the animal-human-ecosystems interface, country-specific factors and fao, oie and who initiatives and support should be considered when mainstreaming one health approaches in human and animal health systems. based on the findings of this study, the general bridges for and barriers to collaboration documented in literature hold true in practice. the following bridges were more relevant in this study: some diseases are zoonoses which need assistance from health experts from different fields, existence of funding for the collaboration, disease outbreaks, and availability of transport facilities. the following barriers to collaboration were prominent: lack of policy statement to enforce collaboration among experts, lack of networking partners, lack of knowledge about animal health expertise, and lack of plans for collaboration. therefore, efforts to bring about collaboration should give priority to these factors. acknowledgements top ↑ the authors thank the wellcome trust grant wt087546ma to the southern african centre for infectious disease surveillance (http://www.sacids.org) and its collaborating uk partner institutions, namely the london school of hygiene and tropical medicine (http://www.lshtm.ac.uk), the royal veterinary college (http://www.rvc.ac.uk) and the london international development centre (http://www.lidc.org.uk), for supporting this work technically and financially. also, the authors thank the respondents for giving of their precious time to fill in the questionnaire that was used for data collection. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions k.a.k. (sokoine university of agriculture) designed and conducted the research and drafted the article. a.d.k. (muhimbili university college of health and allied sciences) and d.m.k. (sokoine university of agriculture) were the primary mentors or supervisors; they provided input to improve the research process and the article. e.l. (muhimbili university college of health and allied sciences), r.c. (london school of hygiene and tropical medicine) and j.r. (royal veterinary college) were mentors or supervisors; they provided critical comments from time to time. r.k. 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controlling zoonotic diseases. report no. 50833-glb, world bank, agriculture and rural development health, nutrition and population, washington dc. world health organization (who), 2006, ‘the control of neglected zoonotic diseases: a route to poverty alleviation’, in report of a joint who/dfid-ahp meeting with the participation of fao and oie, geneva, 20–21 september, viewed 31 may 2011, from http://whqlibdoc.who.int/publications/2006/9789241594301_eng.pdf zinsstag, j., meisser, a., schelling, e., bonfoh, b. & tanner, m., 2012, ‘ from ‘two medicines’ to ‘one health’ and beyond’, onderstepoort journal of veterinary research 79(2), art. #492, viewed 07 june 2013, from http://dx.doi.org/10.4102/ojvr.v79i2.492 swan_105-114.indd introduction veterinary education is shaped by demographic, environmental, disease, technological, political, and economical influences and is required to serve the needs of a country in relation to its environment, animal population, and society in which it operates. the number of veterinary schools present within a country will affect the scope of veterinary training and responsibility of each school. a veterinary training programme and curriculum need constant review and adaptation to keep abreast of trends in both national and international developments and requirements. currently a curriculum would normally be revised every 5 years, thus allowing a new programme to be fully implemented every ten years, whilst small and non-critical modifications could be introduced during the interim (fernandes & lobovilela 2002). curricular changes are not necessarily linear extensions of existing programmes but they need to take cognisance of future scenarios affecting the profession over time spans of 10–20 years. the faculty of veterinary medicine, cairo university appears to be the oldest faculty in africa. it was established as a school of veterinary medicine in 1827 in rasheed in the north west of the egyptian delta (www.cu.edu.eg). in 1950 the school became the 105 onderstepoort journal of veterinary research, 76:105–114 (2009) veterinary education in africa: current and future perspectives g.e. swan and n.p.j. kriek faculty of veterinary science, university of pretoria, south africa abstract swan, g.e. & kriek, n.p.j. 2009. veterinary education in africa: current and future perspectives. onderstepoort journal of veterinary research, 76:105–114 veterinary education commenced in south africa in 1920 at the onderstepoort veterinary institute in south africa in association with the transvaal university college, now the university of pretoria. sir arnold theiler, director of veterinary research and education, was the first dean. today there are 46 veterinary training institutions in africa of which 21 are in sub-saharan africa. veterinary services are indispensable to the sustained health and wellbeing of animals and humans, and agricultural economies of countries worldwide. veterinary education, postgraduate training, and research, and adequate numbers of veterinarians, are essential to satisfy the millennium development goals, the objectives of nepad and the african union, and the agreements regulating international trade. the relevance of the veterinary profession internationally is currently subject to profound scrutiny. its contributions are assessed against major environmental, demographic, political, disease, technological and economic needs. the scope of veterinary training in future will have to emphasise veterinary public health, food safety, emerging diseases, international trade, bioterrorism, and biomedical research, within the context of a one-health system focusing on the interface between wildlife, domesticated animals, humans, and their environment. within the context of time available, it would mean reducing the time allocated to training in the field of companion animals. a brief history and scope of veterinary education; current international trends in veterinary education and provisioning; and some perspectives on future veterinary training and initiatives applicable to africa are provided. 106 veterinary education in africa faculty of veterinary medicine of the cairo uni versity. there are presently 46 veterinary schools on the african continent of which 21 are in sub-saharan africa (hailat 2005; mayen 2006). with the exception of the faculties in cairo, egypt (1827), university of pretoria (1920) and khartoum, sudan (1938) all other african veterinary schools were established after 1960. the earliest reference to veterinary education in south africa dates back to 1901/02 when the transvaal department of agriculture was formed and it was suggested that a ‘veterinary college for south africa’ be established. an initial attempt was made in 1911 to create a chair of veterinary science for sir arnold theiler. formal proposals that contained the details of a proposed training programme were thereafter lodged with the state commission on higher education in 1914. it was, however, only in 1919 that a formal invitation was extended to sir arnold theiler to create a faculty of veterinary science. sir arnold theiler was appointed on 1 april 1920 as the first dean of the newly established faculty of vet erinary science through the intervention of general jan smuts. the first seven students were registered in the faculty of agriculture of the transvaal university college (tuc) in 1920 and were later joined by an eighth student. all eight students qualified in 1924 as the first locally trained veterinarians. the faculty remained under the management of the onderstepoort veterinary institute but for academic purposes was part of the then transvaal univer sity college (tuc) under aegis of the university of south africa, which later became the university of pretoria. all lecturing staff remained in the employ of the department of agriculture. full responsibility for the faculty of veterinary science was transferred to the university of pretoria only in 1973. the faculty of veterinary science at the university of pretoria remained the only facility responsible for veterinary training in south africa until 1980 when a second faculty was established within the medical university of southern africa (medunsa) with the purpose to increase the ethnic diversity in veterinary training. this faculty admitted its first students in 1982 and produced its first graduates in 1987. in 1999, due to the excessive cost of veterinary training, the medunsa veterinary faculty was amalgamated with the original faculty at the university of pretoria to form the current new national faculty. the amalgamated faculty continues to utilise the facilities at the onderstepoort campus of the university of pretoria. current scope of veterinary training in africa many parts of africa have large populations of indigenous livestock of low productivity and with animal health problems as major constraints to productivity (kisauzi, tsutsumi & mlangwa 1993). veterinary training in africa for many years was focussed on producing veterinarians to address the needs of the livestock sector, and most veterinarians were employed by the state. the original veterinary faculties in africa were established as a result of prevailing devastating livestock diseases such as rinderpest, east coast fever, african horsesickness, and the urgent need for their con trol. the curricula were at that time, and in many cases are still, primarily focussed on training veterinarians able to recognise and initiate efficient animal disease control, apply effective treatment of diseased animals, enhance animal welfare and safe guarding human health. most curricula of veterinary faculties in east and southern africa extend over 5–6 years, are primarily discipline-based and cover the traditional preclinical, paraclinical and clinical subjects in producing a “generalist veterinarian” (fao workshop on harmonising the curricula of veterinary faculties in east and southern africa 2004). there is very little to no provision for electives in these curricula. in a review undertaken on the adjustment of curricula in veterinary faculties in sub-saharan africa, de deken, obwolo, thys & geerts (2004) reported that due to socio-economic changes there has been an increased privatisation of veterinary services. this has led to the increasing entrance of veterinarians into private practice in the region and has necessitated increased clinical training and the introduction of new practice and business management programmes. however, private veterinary practice, other than in south africa, is very limited and underdeveloped, and there is still very little need for clinical veterinary specialization in these countries. in contrast, in south africa, more than 70 % of veterinarians registered with the south african veterinary council are in private practice. this situation is reflected in its veterinary training programme in which there is extensive emphasis on training in the field of companion animal medicine. the survey by de deken et al. (2004) also examined how the veterinary curricula adjusted for crucial developments in the veterinary field, such as decentralisation and globalisation, increasing the risk of the spread of transboundary diseases. in addition to adjustments made to provide for privatisation of vet107 g.e. swan & n.p.j. kriek erinary delivery systems and private practice, most faculties have also included additional training in food quality control and in wildlife production and management. the importance of wildlife in ecotourism and its increasing relevance in mixed livestock and wildlife ranching in certain countries necessitated its introduction in undergraduate veterinary curricula. factors affecting the future of veterinary education political veterinary education at its highest quality provides training that meets the needs of a particular society to adapt to changing national, regional and international conditions. the recommendations of the millennium development goals (mdg) africa steering group 2008 (www.mdgafrica.com), as well as the comprehensive africa agricultural development programme (caadp) as developed by the new partnership for africa’s development (nepad) and the african union, provide a framework for the needs in africa for the immediate future and beyond. eradi cation of extreme poverty and hunger (mdg1), combating hiv/aids, malaria and other diseases (mdg6), ensuring environmental sustainability (mdg7) and developing a global partnership for development (mdg8) are the main millennium goals applicable to veterinary education. these goals are fully aligned with the priorities as set by nepad and the strategies of the african union. agriculture has been identified as central to reducing poverty, hunger and accelerating growth in africa. the majority of africa’s people live in rural areas associated with agrarian systems that are generally weak and unproductive (nepad 2001). the mdg recommendations place a particular focus on smallholder farmers and on women that account for 80 % of the region’s farmers. cooperation with the pharmaceutical industry in providing access to affordable essential drugs, and collaborating with the private sector in making available new technologies are regarded as important targets in mdg8. infectious diseases seriously affect four of the mdgs, which relate to human health, food supply and economic development (anon. 2006). high levels of political instability (www.times-publication.com/risk-assesment/risk-assesment.html) and civil wars (www.africasunnews.com/wars.html) will remain to seriously impact on the delivery of veterinary education in the countries in africa affected by these scourges. transboundary, emerging and re-emerging diseases infectious diseases, including viral, bacterial, fungal and parasitic diseases, account for more than 20 % of human deaths and 25 % of morbidity, disproportionately affecting the poor and reducing life expectancy in many sub-saharan countries (anon. 2006.) disease during outbreaks can spread rapidly, causing enormous economic losses, and impact markedly on human health and livelihoods. these effects are most severe in africa given that in sub-saharan africa livestock farming contributes up to 25 % of gross national product across the region, and that 12 of the world’s 15 major epidemic diseases of animals occur endemically in africa. currently, new diseases emerge about every eight months and the likelihood is that about 75 % of these are new zoonotic diseases (willis, monroe, pot worow ski, halbert, evans, smith, andrews, spring & bradbrook 2007). in the past 25 years, 38 new pathogens have emerged of which 75 % have caused disease in animals. about 60 % of all human pathogens are known to have crossed the species barrier. the emergence and re-emergence of diseases are contributed to by global weather changes, and are driven by diseases such as hiv/aids as is the case with tuberculosis in humans, the increasing emergence of drug resistance, and when natural ecosystems are stressed (anon. 2006.) while there is great uncertainty about the future it is assumed that many of the current major human and animal diseases will remain important during the course of the next 1025 years wildlife serves as reservoirs of the pathogens causing infectious diseases and is an important and continuing source of disease for both livestock and humans. incursions into their natural habitat and trade in meat (bush meat) and in exotic animals for the pet trade are major drivers of the transmission of these pathogens to humans and domesticated animals (anon. 2006.) most emerging diseases have originated from animal reservoirs. transboundary diseases have been classified as one of the eight classes of diseases that are considered particularly important during the next 10–25 years. global animal and public heath the delivery of veterinary services is increasingly being accepted as a global public good mainly because of the initiatives of the world organisation for animal health (oie) (brückner 2008). this not only places the veterinary profession under public 108 veterinary education in africa scrutiny but has created awareness regarding its role in promoting animal health and its linkages to safeguarding human health. this changing paradigm also requires, by operating in a world of shared risks and common opportunities, acknowledgement of the realities of mutual dependence and growing interconnection (international task force of global public goods, 2006 meeting global challenges: international cooperation in the national interest. final report, stockholm, sweden). in this context, the rate at which diseases now may spread across the globe, the potential risk of bioor agri-terrorism using zoonotic pathogens, the extensive illegal trade in animals, changes in spread of animal disease due to changes in human demographics and behaviour, and patterns of land-use are noteworthy (brückner 2008). as a result, food safety and export regulations for animal products are becoming increasingly strict and, additionally, they may be designed as non-tariff trade barriers to protect internal markets of individual countries or blocks of countries. the international community expects the profession to assume different approaches when dealing with the disease environment such as predicting disease outbreaks and spread by focussing on prevailing climatic conditions, appreciating the impact of disease due to observed events, and to consider potential epidemiological causal links (brückner 2008). there is an expectation that all health export declarations for live animals and animal products should be scientifically founded. veterinary education in africa cannot ignore these global animal health and public health issues. in this context, the oie is currently preparing a special issue of the scientific and technical review on expanding education of global health and public health within the veterinary curriculum worldwide. convergence of animal, human and environmental health the high prevalence of poverty and starvation, excessive human population growth, presence of devastating human diseases such as hiv/aids, tuberculosis and malaria, and the presence of large underdeveloped communities will continue to influence the future direction of veterinary education in africa. veterinarians, being trained in comparative medicine, should be well equipped to function at the interface between wildlife, domestic animals and humans. large overlap exists in the preclinical training of health care professionals and similar standards in public health services exist for humans and animals. recurring threats of zoonotic disease have created the need for convergence of animal and human health training and provisioning. it can be expected that public health agencies in future be staffed with professionals in both human and animal health. veterinary education in africa should therefore promote the collaboration of animal and human health to more effectively and competently address public health needs of disease, particularly related to transboundary, and emerging and re-emerging zoonotic diseases. during the next 20 years environmental degradation is likely to continue due to pollution, overproduction, habitat fragmentation, climate change and invasion of alien species. according to the foresight report on infectious diseases (anon. 2006), the greatest impact of new diseases would be to undermine global water and geochemical cycles by disrupting key plant and microbial systems that support them. natural ecosystems will continue to be a major source of wildlife diseases that may threaten agricultural systems and human health. technology there have been major advances in technology leading to widespread use of computer-linked sensors to carry out diagnostics, administer therapies and to perform tele-surgery over great distances. future technological tools offer powerful capacity for the detection, identification and monitoring (dim) of infectious diseases (anon. 2006). advanced data collection and processing ability could enable emerging diseases to be quickly spotted, providing valuable time to develop vaccines and to prevent its spread. new techniques for identifying and analysing new pathogens assist in the rapid understanding of the effect of newly detected diseases on populations and how they can be controlled before inflicting damage. faster, smaller and cheaper diagnostic services will open up new possibilities for use by professionals and individuals. africa has the greatest burden of disease and probably the lowest level of disease management resources and skills. new devices and dim approaches could reduce costs and make trade easier by enabling disease-free status of animals and measures to counter terrorism. veterinary education should capitalise on new technology to provide distance education and virtual simulations in education. this will facilitate increasing the number of students in certain modules, and 109 g.e. swan & n.p.j. kriek will support continuing education opportunities. education should promote the integration of technological advances in the delivery of professional services in remote areas, including the provision of diagnostics, therapeutics, bio-sensing, data collection and interpretation, as well as in national and international cooperation. global trends in veterinary education veterinary science and practice have evolved from the initial focus on horses in the 1800s to livestock through to the 1950s, whereafter emphasis of the profession shifted towards companion animal medicine in many parts of the world (willis et al. 2007). over the past decade, the emphasis of veterinary practice has shifted from the traditional role of treatment and care of individual animals and the protection of human health to mass therapies, the implementation of plans for eradication of animal diseases, the development of animal nutrition, and the shift from pathological control of abattoir animals to the concept of the abattoir as an epidemiological observatory where the results of farming activities may be verified (marabelli 2003). food safety has been placed at the forefront through the tools of hazard analysis and critical control point (haccp). the relevance of the veterinary profession has become the subject of profound public scrutiny. several publications have appeared over the past number of years that envision the future and describe the new challenges facing the profession and which have necessitated adjustment of veterinary education (pritchard 1988; willis et al. 2007). the initial response to the broadening of the scope and specialisation of veterinary practice was the introduction of various forms of core-elective curricula. the core is completed by all students leading to the development of essential generic veterinary, or dayone, competencies. tracking (or areas of emphasis within the curricula) was implemented to allow students to acquire greater knowledge and skills in selected areas (species-based). in addition, there has been an adaptation of technology and pedagogic processes that have fostered greater emphasis on problem-solving skills and self-learning abilities of students (prasse 2007). future educational requirements are driven by major environmental, demographic, political, disease, technological and economic needs (willis et al. 2007). emphasis will increasingly be placed on the contributions made by the profession in the fields of globalisation of animal and public health, international trade in animals and animal products, food safety, emerging diseases, changes in the relationship between veterinary medicine and the environment, bioterrorism, biomedical research, and changes in the relationship between animals and humans within the context of a one-health approach. a responsive and flexible veterinary education system has been proposed to meet the challenges of the future (willis et al. 2007). leadership, interpersonal skills,and the ability to participate in teams have become core competencies required by veterinarians working in this new environment. future educational perspectives in africa although local events and needs will inevitably continue to shape the scope, type and format of training required, veterinary education in africa cannot be divorced from global animal and human health events and educational trends. unique conditions and circumstances that influence and that will potentially determine the future direction and provision of veterinary education in africa, with a focus on sub-saharan africa, are described under the headings below. scope the challenge for veterinary education is to balance the societal needs of the various sectors requiring veterinary services nationally, particularly those of the public and private sectors, as well as taking cognisance of global animal and human health matters. this becomes more difficult when there is only one veterinary faculty, as is often the case in africa, in a country that has the responsibility for all its veterinary training requirements. it hampers the ability of a faculty to focus its training in specific, specialised areas in an attempt to utilize its capital and human resources more optimally. ethiopia, nigeria and sudan are the only countries in sub-saharan africa with more than one faculty. to address these needs, more attention in future needs to be given to recognising or developing regional centres of competence in specific fields within veterinary schools in the region, similar to those in sadc. it is foreseen that such centres of competence will be limited to fields such as wildlife, exotic species, poultry health and production, pig health and production, and aquaculture and fisheries. these are fields that are very important to most countries but require smaller numbers of veterinarians to be 110 veterinary education in africa trained per annum. students in the region choosing these fields could then be sent to the recognised competent facility for training, thus providing critical mass and concentration of effort. alternatively, faculty staff competent in these fields could be shared by the different faculties. this will provide the necessary recognition of faculties in the region and provide an opportunity to focus. scant information exists about the expected current and future needs of the different sectors of veterinary services in south africa, and presumably also in other parts of africa. such information is essential for the future educational planning of veterinary faculties. in south africa, a study has recently been undertaken by kmpg under the auspices of the department of agriculture, but the results and recommendations are still forthcoming. community engagement has recently become a core academic output of the university of pretoria. in this regard, the faculty is in the process of establishing two community clinics—one located in an urban developing community, including squatter dwel lings, and the other in a rural developing community located in an area at the interface between wildlife, domestic animals and humans. both clinics are intended to provide the full scope of veterinary services, including animal and human health in these communities and to prepare students for an impending one-year compulsory veterinary community service following graduation. involvement in the community allows opportunities for applied research projects with the aim of uplifting of the community. curricular structure and requirements it is assumed that a similar (as in most parts of the world) standardized, but locally adapted and relevant core programme should in future form the basis of training of all veterinarians throughout subsaharan africa. this programme should provide each veterinarian with general professional skills and attributes, underpinning veterinary scientific knowledge and understanding, as well as prerequisite clinical competencies and skills. professional attributes such as leadership and interpersonal skills, together with the development of adequate communication abilities, teamwork, an inculcation of ethical responsibilities, and a thorough knowledge of the national legal and statutory requirements and obligations affecting the profession, should form part of the future standard training of all veterinarians. comparative veterinary medicine, a feature that distinguishes the veterinary profession from all other health professions, must be retained as an essential outcome of the underpinning veterinary scientific knowledge and prerequisite clinical competencies of all veterinarians. clinical competencies and skills should focus on those required for general practice and concentrate on preventative medicine as applied to populations of animals rather than the skills of therapeutic medicine. all veterinarians in future should also be skilled in essential laboratory diagnostics, quantitative epidemiology and disease surveillance, and monitoring methodologies. more advanced training in companion animal clinical studies, animal health and production, regulatory veterinary medicine, wildlife, exotic species and any other required specialist area should form part of elective programmes. unique differences in elective programmes will exist between countries in the subregion, depending on socio-economic factors and societal needs. students either could be placed or selected into a particular track of advanced study, dictated largely by national needs, recruitment strategy and admission policy. educational innovation the existing and ever increasing volume of new veterinary information makes it imperative that future veterinary students are skilled in the use of information and in problem-solving. students will need to take more responsibility for their own development by providing sufficient opportunity for self-learning in the curriculum and the use of new innovative stimulatory teaching methods and approaches. the use of information communication technology (ict) and e-learning should become an integral part of the pedagogic mode of educational delivery. virtual training opportunities within the region should be developed, not only for continuing education purposes but also for distance education, through application of ict and the use of virtual simulations. this will allow the best use of unique competencies, not only within the region but also on an international level. training of staff and students in the effective use of ict and e-learning opportunities and the development of adequate electronic and telematic facilities would be needed to make this possible in sub-saharan africa. accreditation and harmonisation according to edwards (2004), there is societal expectation that all veterinary qualifications are of similar basic competencies and standard. in reality though there is an apparent disparity in the level of veterinary training received, particularly between 111 g.e. swan & n.p.j. kriek developed and developing countries, with higher standards employed by the major accrediting bodies (i.e. those in developed countries). the challenge is to develop and implement a global standard to enable recognition of veterinary competencies from around the world. it is envisaged that in future a country whose veterinary profession (and education) is not recognised internationally and who cannot meet the criteria of the sps agreement (of the world trade organisation), may be excluded from trade. some regional blocks of accreditation currently operate around the world, encompassing the major accrediting bodies based in the usa, europe, united kingdom and australasia. in 2002, a meeting of representatives of these bodies examined the possibility of a system of global accreditation. this was followed up with further global veterinary accreditation meetings in 2004 in london, uk and in 2007 in melbourne, australia. the south african veterinary council (savc), responsible for administering the registration of veterinarians and para-veterinarians and for setting the minimum requirements for veterinary training in south africa, was invited for the first time to send representatives to participate in the meeting in melbourne. an international accreditors working group (iawg) made up of representatives of all major accrediting bodies, as well as representatives of the savc, nominated at the meeting in melbourne, met in chicago in october 2007 to formulate recommendations for a proposed joint site visit at murdoch university, division of veterinary and biomedical sciences in 2009. a member of the savc will also, for the first time, form part of the visitation team, as an observer. wider participation in the global accreditation initiatives within africa should occur. the formation of a regional accreditation body in future, that could take this responsibility for faculties in southern and eastern africa or possibly even for sub-saharan africa, is foreseen. in the meantime, the representative of the savc participating in the current initiatives could serve this purpose. the existing experience of the savc in the licensure of veterinarians and para-veterinarians, setting of minimum standards for undergraduate and postgraduate veterinary specialist training, accrediting and monitoring the quality of veterinary training in south africa, should be recognised and used as an opportunity by other veterinary faculties in sub-saharan africa. sharing of this experience can serve as an interim arrangement with the purpose of instituting the proposed regional accreditation body within the next 5 years. this accreditation body will then take the responsibility of setting minimum standards of veterinary training and for the accreditation of veterinary faculties in the region. the licensure of veterinarians and ongoing monitoring of the quality of education will remain the responsibility of the local regulatory council or authority (such as the savc) of each of the countries. it is unrealistic and unlikely that all faculties in africa will be able to meet the full scope of envisaged global accreditation requirements, due to mainly socioeconomic constraints and political priorities. the high level and extent of veterinary clinical training in companion animals which is common in the developed world is also not required in most parts of africa. however, a level of agreement on core veterinary training, as well as on veterinary activities that affect global animal and human health and that are required to ensure free trade in animal and animal products, could be considered. this will allow veterinary faculties in parts of africa to focus on the relevant veterinary needs of the country rather than being required to comply with the full scope of veterinary practice to be recognised internationally. future recognition of different levels or fields of veterinary training and possibly area-specific licensure of veterinarians would need to be considered by the international and national authorities, respectively. regional recognition as well as recognition by international bodies such as the oie and fao, of faculties in the region with specific competence in veterinary fields that are not adequately covered in the veterinary curricula of some faculties could provide an opportunity for students to acquire the necessary broader training required at an international level. the excellent clinical facilities and veterinary academic hospital of international standard that exist in south africa could, for example, be recognised as a regional centre for clinical training, particularly in companion animals. an fao project to investigate harmonising the curricula of veterinary faculties in east and southern afri ca was undertaken in 2004/05. veterinary faculties from ethiopia, kenya, mozambique, south africa, tanzania, uganda and zimbabwe participated in the project. the project in our opinion indicated that full harmonisation of curricula between the different countries was unlikely due to socio-political differences and differences in the scope of veterinary training required between countries. however, it is proposed that standardisation or harmonisation of at least the core programme required for all veterinarians could be considered for the region. this 112 veterinary education in africa could be facilitated, accredited and monitored through the appointment of a regional accreditation body. postgraduate training veterinary education cannot be conducted effectively in the absence of a strong postgraduate and veterinary research programme. this is also a requirement of all the major accrediting bodies. a very limited capacity for postgraduate training exists in most african schools due to inadequate financial resources, facilities, and difficulty in recruiting and retaining academic staff. stimulation and development of postgraduate training and research in the region is therefore imperative. a regional msc programme that had been planned between sadc veterinary faculties (mozambique, south africa, tanzania, zambia and zimbabwe) (de deken et al. 2004) was finally not implemented, mainly due to unsustainable financial resources by these universities. to strengthen postgraduate training, regional centres of competence in postgraduate training need to be developed and recognised by the region and by international organisations, e.g. the oie and fao. it is foreseen that postgraduate programmes or modules be developed and funded through international collaboration and partnerships. to ensure success and sustainability of such programmes it would be imperative that they are academically eval uated and quality-assured at an international standard. an extensive range of postgraduate programmes is currently offered at the faculty of veterinary science in south africa (table 1). many students from africa already register in a variety of these programmes. the mmedvet programme, the only type of its kind in africa, could serve as a regional resource for specialist clinical training in the region. a primarily web-based msc (veterinary tropical diseases) degree programme offered by the department of veterinary tropical diseases in the faculty of veterinary science in south africa is an example of a programme that has been developed through regional and international collaboration and partnerships and that has been subject to international academic evaluation. the programme is structured in such a way that the learner can achieve a qualification in a specific field of study (career path) by selecting appropriate elective modules. this degree programme deals with all the high impact diseases listed by the oie and other important infectious and parasitic diseases of livestock and wildlife of global importance. it is therefore of great value not only to learners from the region but also to those from the developed world. in the developed world, many of the high-impact diseases have been eradicated, often at great cost, but still pose a distinct threat because of increased international trade in animals and their products. table 1 postgraduate programmes degree programmes duration (years) bvsc (hons) combination of 3–4 modules 2–3 mmedvet* anaesthesiology; bovine medicine; cattle herd health; clinical laboratory diagnostics; diagnostic imaging; equine medicine; equine surgery; laboratory animal science; ophthalmology; pathology; pharmacology; pig herd health; poultry diseases; small animal medicine; small animal surgery; small stock herd health; reproduction; toxicology; veterinary ethology; veterinary public health; wildlife diseases 4–6 taught masters** veterinary industrial pharmacology; veterinary tropical diseases 1–2 research masters*** anatomy and physiology; companion animal studies; paraclinical sciences; production animal studies; veterinary tropical diseases 1–2 phd anatomy and physiology; companion animal studies; paraclinical sciences; production animal studies; veterinary tropical diseases 3–5 * allows automatic registration as a specialist in south africa ** consists of coursework and a dissertation. the programme in veterinary tropical diseases is primarily web-based and consists of a number of career paths (laboratory-orientated career path, veterinary field services and general practice) *** consists of a dissertation only 113 g.e. swan & n.p.j. kriek a masters and phd degree programme in phytomedicine offered by the department of paraclinical science, faculty of veterinary science in south africa is a very extensive programme that also attracts large numbers of students from all over africa. the programme deals with identifying, isolation, chemical elucidation, and clinical testing of extracts from plants used for ethnoveterinary medicine. it would be a very worthy regional programme. one other postgraduate research programme that also warrants regional recognition is a nufu-funded phd programme on environmental toxicology and zoonotic diseases. it is a south-south-north collaborative programme between veterinary faculties in mozambique, tanzania, south africa, zambia and zimbabwe, representing the south-south partners, the norwegian school of veterinary science and the norwegian veterinary institute in oslo, norway as the northern partners. this programme was initiated in 2002 and has registered ten phd students over this period with three of these successfully completing their studies. the nufu funding for this programme runs out in 2009 after which it will have to become self-sustainable. such programmes are urgently required and should be promoted within africa. those created should be retained as far as possible and not be allowed to disintegrate. conclusion it is now a crucial time for the veterinary profession and for veterinary education. failure to respond to a changing world and its needs will have dire consequences for the future of the profession and its position in, and acceptance by, society at large. africa should and cannot isolate itself from international trends in veterinary education prompted by changing international expectations pertaining to the contribution made to society by the veterinary profession. there is a need for the development of a vision for future veterinary needs in africa to guide veterinary training in sub-saharan africa (through a similar foresight study as was undertaken by aavmc) (willis et al. 2007). collaboration in veterinary training within sub-saharan africa must be sought for both underand postgraduate training programmes. it is imperative that the african faculties participate in global accreditation initiatives. developing a curriculum within the context of time and resource limitations, given the broad scope and complexity of veterinary activities, is going to be a major challenge and may be impossible within the context of the current constructs. this is an even greater challenge given the general consensus that it is impossible to train veterinarians currently to address the complex needs of the various sectors of society that need veterinary inputs. cognizance should be taken of the following: • to remain relevant education must prepare veterinarians for future needs • it is important that training must be relevant to societal and political needs • there is a changing emphasis on global animal and public health issues. it is expected that firm recommendations in this regard will be forthcoming from the oie workshop of deans scheduled for august 2009 • a firm decision to broaden the scope and context of veterinary science education is fundamental in preparing the profession to navigate the future transition and to enable it to strengthen its position in society recommendations • africa should not dissociate itself from global trends as they relate to veterinary services • veterinary training must be relevant to local societal needs, political expectation and environmental changes • adequate training in sub-saharan africa can only succeed if there are regional collaboration and partnerships • participation in global accreditation initiatives is imperative for the veterinary profession in africa • a foresight study to envision the future of veterinary education in africa should be implemented as soon as it is possible • we will have to demonstrate leadership beyond that which is usual and conventional to achieve this goal not just in south africa but throughout africa • it is our responsibility to resurrect the ‘spirit of onderstepoort’ of sir arnold theiler defined as: “…that ideal of service, that conscientiousness of endeavour, that pride in a task, that confidence of success in the face of difficulties…” references anonymous 2006. foresight. infectious diseases: preparing for the future. executive summary. london: office of science and innovation. 114 veterinary education in africa brückner, g. 2008. new challenges for the veterinary profession in global animal disease control and the trade in animals and animal products. proceedings of faculty day, fac ulty of veterinary science, university of pretoria. de deken, r., obwolo, m.j., thys, e., geerts, s. 2004. the adjustment of curricula in veterinary faculties in sub-saharan africa. revue scientifique et technique, office international des epizooties, 23:383–90. einarsson, s., isaksson, n.i., kindahl, h., lindberg, r. 1994. veterinary education and research in ethiopia and tanzania. report from a fact-finding mission, swedish university of agricultural sciences, international rural devel opment centre, box 7005, s-750 07, uppsala, sweden. working paper—international rural development centre, swedish university of agricultural sciences. edwards, j. 2004. global perspectives of veterinary education: reflections from the 27th world veterinary congress. journal of veterinary medical education 31:9–12. fernandes, t., lobo-vilela, c. 2002. preamble. vet 2020 prospects for veterinarian profiles: requirements by the profession and the society. development of european educational strategies: design of veterinarian profiles identified by market needs for the year 2020. socrates thematic network project. hailat, n. 2005. present status and vision of veterinary higher education in the arab world. journal of veterinary medical education 32:151–154. kisauzi, d.n., tsutsumi, y., mlangwa, j.e. 1993. school of veterinary medicine, university of zambia, lusaka. revue scientifique et technique, office international des epizooties, 12:695–705. marabelli, r. 2003. the role of official veterinary services dealing with new social challenges: animal health and protection, food safety, and the environment. revue scientifique et technique, office international des epizooties, 22:363–371. mayen, f. 2006. a status report of veterinary education in ethiopia: perceived needs, past history, recent changes, and current and future concerns. journal of veterinary medical edu cation, 33:244–247. prasse k.w. 2007. foreword to the foresight project final report: envisioning the future of veterinary medical education. journal of veterinary medical education, 34:1–2. pritchard, w.r. 1988. veterinary education in africa: past, present and future. journal of veterinary medical education, 15:13–16. willis, n.g., monroe, f.a., potworowski, j.a., halbert, g., evans, b.r., smith, j.e., andrews, k.j., spring, l., bradbrook, a. 2007 envisioning the future of veterinary medical education: the association of american veterinary medical colleges foresight project, final report. journal of veterinary medical education, 34:5–15. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice ilemobade_35-40.indd introduction at this momentous occasion when we are celebrating 100 years of the establishment of the onderstepoort veterinary institute (ovi), it is worth recalling that it was here in south africa, on the ubombo hills of zululand that dr david bruce and his colleagues who, between 1894 and 1899, carried out experiments that led to the identification of the causal agent of ‘tsetse fly disease’ or ‘nagana’ (bruce 1897). the centenary marking that epoch finding took place in africa and europe some 8 years ago. just about the same time, the rinderpest epidemic broke on africa in 1896 and led to the massive destruction of bovines and indigenous antelopes, one of the major reasons for the apparent disappearance of tsetse fly in the confined areas of transvaal and zululand where the fly was prevalent. by 1905, however, with the containment of rinderpest and recovery of the wild antelopes and bovines, the flies were back and nagana became prevalent again. it was left for du toit and his colleagues (du toit 1954; du toit, kluge & fiedler 1954) to devise means of eradicating the major fly involved in the transmission of trypanosomosis, glossina pallidi pes, from the fly belts in zululand using insecticide (bhc) by helicopter application. the other two glossina species, glossina austeni and glossina brevipalpis, considered at the time to be relatively unimportant in the transmission of pathogenic trypanosomes in the country, were not much affected and has remained low-level vectors of try panosomosis in kwazulu-natal at this time. 35 onderstepoort journal of veterinary research, 76:35–40 (2009) tsetse and trypanosomosis in africa: the challenges, the opportunities a.a. ilemobade upline resources foundation, p.o. box 1308, akure, ondo state 34001, nigeria abstract ilemobade, a.a. 2009. tsetse and trypanosomosis in africa: the challenges, the opportunities. onderstepoort journal of veterinary research, 76:35–40 tsetse-fly and the disease it transmits, trypanosomosis, remain an enormous disease challenge in the 37 countries of sub-saharan africa where the impact continues to be manifest in disease burden, increased level of poverty and decreased agricultural productivity. the impact also extends over an estimated 10 million km2 (a third of the african continent) of land area, a third of which contains some well-watered part of the continent, thus denying humans and livestock of potentially rich arable and pastureland. the disease is a threat to an estimated 50 million people and 48 million cattle with estimated annual losses in cattle production alone of 1–1.2 billion us$. these losses are due to stock mortality and depressed productivity, which may be of meat, milk, reproduction or traction. beyond its direct effects on humans and livestock is its impact on african agriculture and the livelihood of the rural population in the affected countries: the fly and the disease influence where people decide to live, how they manage their livestock, and the intensity and the mix of crop agriculture. the combined effects result in changes in land use and environment which may, in turn, affect human welfare and increase the vulnerability of agricultural activity. trypanosomosis is, therefore, both a public health and an agricultural development constraint. the challenges that the elimination or control of tsetse fly and trypanosomosis pose as well as the opportunities to develop appropriate intervention technologies are discussed in this presentation. 36 tsetse and trypanosomosis in africa returning to the issue of my remit, it is enough to remind us that african trypanosomosis is a disease complex unique to sub-saharan africa, affecting both humans (sleeping sickness) and animals (nagana or african animal trypanosomosis [aat]). it is caused by a protozoan blood parasite, the trypanosome, and is transmitted by the tsetse fly (glossina) which is confined to sub-saharan africa. the impact of the disease extends over 37 countries and an estimated 10 million km2 (a third of the continent) of land area. of this 10 million km2, some three million are covered by equatorial rain forest while the remaining area contains some well-watered parts of the continent, thus denying humans and livestock potentially rich arable land and pastures. the disease is a threat to an estimated 50 million people and 48 million cattle with estimated annual losses in cattle production alone in the range of 1–1.2 billion us$ due to stock mortality, depressed productivity, which may be of meat, milk, reproduction or traction. but beyond its effects on humans and livestock is its impact on african agriculture and the livelihood of the rural population in the affected countries: the disease influences where people decide to live, how they manage their livestock and the intensity and the mix of crop agriculture. the combined effects result in changes in land use and environment, which affect human welfare and increase the vulnerability of agricultural activity. trypanosomosis is therefore both a public health and an agricultural development constraint. aat contributes to food insecurity and poverty over vast tracts of sub-saharan africa. apart from being a threat to livestock, it undermines environmentally sound and economically viable animal and agricultural production and rural development. eighty-five percent of the african poor are located in rural areas and more than 80 % of the population depends on agricultural production for their livelihood. tsetse and trypanosomosis (t & t) is one of the transboundary insect pest and disease problems that constitute a key bottleneck to sustainable agriculture and rural development (sard) in sub-saharan africa, including south africa. to respond to the prob lem, the food and agriculture organization (fao) conference in 1997 endorsed the establishment of the programme against african trypano somosis (paat). this programme seeks through the fao, international atomic energy agency (iaea), african union (au) and world health organization (who) to ensure a coordinated and sustainable approach through the development and implementation of integrated interventions against t & t. paat is concerned with providing affected countries and the donor community with the information needed both to improve understanding and to facilitate policy, technical, financial and decision-making with respect to interventions, including the underlying pest management principles. paat’s strength lies in its multidisciplinary and inter-sectoral approach to a problem that has traditionally been tackled through unilateral veterinary or entomological interventions. because tsetse fly does not respect international boundaries, effective control or elimination of the fly depends on concerted action by the tsetse-infested contiguous countries. the finding of dr david bruce and his colleagues that the causative agent of the scourge that afflicted humans and cattle in southern africa was a protozoan parasite belonging to the genus trypanosoma, apart from its great scientific significance, has helped to shape field control of the disease and its vector. the centenary celebrations both in europe and africa of david bruce’s finding was, however, confronted with many questions, a major one being the failure to rid the african continent of this scourge during the 100 years that had elapsed since the epoch discovery despite the huge investments in the treatment, control and elimination of the disease and its vectors, and the comparatively low reproductive capacity of the vector! the celebrations were also marked by the recognition by african heads of state and governments at their meeting in lomé in 2000, of: “…the seriousness of the problem of one of africa’s greatest constraints to socio-economic development, severely affecting human and livestock health, limiting land use, causing poverty and perpetuating underdevelopment on the continent…” following which they declared the: “…year 2001 as the year of control of the tsetse-fly, to mark the beginning of renewed efforts in the campaign for the eradication of tsetse flies…” this campaign, named pan african tsetse and trypanosomosis eradication campaign (pattec), is at the vanguard of mobilizing political and funding support for the programme to eradicate the fly and therefore, the disease, from the african continent. the challenges the demographic and food security challenge aat is a dynamic disease, both in space and time. the most important factor affecting the disease to37 a.a. ilemobade day is the rapidly expanding human population complicated by differing local situations. according to mattioli, feldmann, hendrickx, wint, jannin & slingenbergh (2004) about 620 million people live in tsetse-infested countries (cf. europe population: 730 mil lion) with rural populations accounting for 68 % of this figure, i.e. 420 million, with a total labour force of 255 million. of the latter figure, the agricultural labour force accounts for 180 million (> 70 %). with the population growth averaging 2.5 %, from where will the additional food to feed such rapidly expanding populations come? removal of the constraints imposed by tsetse fly and trypanosomosis on vast tracts of land denied to agriculture will therefore open up opportunities to utilize the land for food cultivation, reduce food deficit and rural to urban drift, and improve the livelihood of rural dwellers in affected countries. because tsetse is vulnerable to agricultural cultivation pressure, tsetse eradication will be facilitated and reinvasion of cleared areas will be checked. the challenge of controlling or eradicating the fly allsopp (2001) reviewed the currently available options for vector control against trypanosomosis in africa. the options include ground spraying using non-residual insecticides, sequential aerial application of low-dosage, non-residual aerosols (sat), the bait technology and the sterile insect technique (sit). different species of tsetse require different approaches to their control. generally however, the effectiveness of each approach depends on the ecological situation existing in a given area, including distribution patterns, environmental requirements, be havioural characteristics, densities, high resilience and farming systems. there are 30 species or subspecies of the tsetse fly, of which 22 are recognized as of economic importance because of their role in the transmission of pathogenic trypanosomes. there is a fairly well defined limit to their spread, the northern and southern limits. generally, distribution of the different species of tsetse is determined principally by climate and influenced by altitude, vegetation and the presence of suitable host animals. the distribution varies markedly—some are widespread all over sub-saharan africa, while others have relatively limited distribution. many of the affected countries harbour two to three species of tsetse flies. thus, the presence of multispecies and their widespread distribution is a major challenge. furthermore, when tsetse belts expand from one country to the other it creates a major problem in control or eradication unless the affected countries act in concert. an example is the recent reclamation work done in the okavango delta of bot swana which has been quite successful but did not include the neighboring countries of namibia and zambia where extensions of the tsetse belts to these countries are found. pattec has been encouraging the three countries to collaborate to ensure that no reinvasion of tsetse occurs in cleared areas; otherwise it will be difficult and costly to maintain a barrier. the presence of tsetse in difficult terrains, e.g. in gorges, lowlands, deltas and high forests as occurs in most of central africa where access is a major problem and where the only livestock kept are the trypanotolerant breeds is a major challenge to control or eradication. given the foregoing, it is believed that the technical means of either controlling or even eradicating tsetse are available and the problem lies in successfully implementing control measures in a sustainable man ner. tsetse elimination requires that once the process is started, it should be consistently pursued until the goal of either control or eradication is achieved. otherwise, because the fly has intrinsic ability to recover fast, the remnant may recover rapidly, and sometimes to levels above those before con trol was applied. the creation of artificial barriers to secure areas cleared of tsetse from reinvasion can be very expensive and may not be sustainable. this partly explains why it has proved difficult over more than 100 years to rid the continent of t & t. the challenge of selecting technologies and the sequence in which they are applied field observation has confirmed that selection of technologies appropriate to a given area and the sequence of their application is crucial to the success or otherwise of any intervention. area-wide integrated pest management (aw-ipm) of t & t, as a phased approach has been advocated, involving the creation and expansion of tsetse free zones and integrating various technologies that are appropriate and environmentally acceptable (pattec 2001; feldmann & hendrichs 2001). the selection of technology and the sequence in which they should be applied, however, would depend on prevailing conditions. some of these conditions include, for example, the size of the area, tsetse distribution patterns and abundance, tsetse vulnerability, livestock systems, agricultural practices, livestock breeds, availability of infrastructure and capacity; availability of and access to intervention technologies and market demands and trade opportunities. 38 tsetse and trypanosomosis in africa unless appropriate combinations of technologies are applied, depending on the different environmental and social situations, t & t interventions may fail to achieve the desired objectives. the challenge of a coordinated approach the sustainable control or eradication of t & t requires a coordinated and concerted approach. regrettably this has, for a long time, been lacking, especially in large-scale interventions. to address this issue in a realistic manner, a workshop was convened in may 2002, at the fao headquarters in rome under the umbrella of paat, where an agreement was reached by fao, au/pattec, iaea and who on a harmonized approach to the problem of t & t, and in particular the concept of aw-ipm and the criteria or guidelines for selecting priority areas for joint international intervention in the context of sustainable agriculture and rural development (sard) (fao 2002). these criteria are: • severity of the impact of t & t • opportunity to support poverty reduction, increase food security and maximize the economic returns through: – expansion and intensification of mixed farming – improved subsistence farming and/or production of cash crops – proper land use and tenure as components of sustainability – sustainable and environmentally appropriate utilization of natural resources. • factors contributing to increased feasibility of project activities and sustainable outcomes: – presence of natural barriers – possibility for artificial confinement of tsetse population – commitment and involvement of local authorities and communities – existence of technical and logistic support. the challenge of drug control livestock farmers have depended principally on the use of trypanocides to control aat in the face of years of breakdown of extension services in affected african countries. there is, however, a growing concern on the effectiveness of the currently available trypanocides due to reported widespread drug resistance. the existing trypanocides—isometamidium, diminazine and homidium salts—have been in use for over 40 years and it is estimated that annually, 35 million doses, amounting to about 35 million us$, are administered. with such a low level of use, major drug companies are unwilling to invest in new trypanocide research and development, with the result that no new drugs have come into the market for over 40 years, the last one being diminazine. there is therefore the imperative of ensuring that the existing trypanocides are used judiciously in order to further extend their life-span of effective usage and ensuring that livestock farmers and pastoralists are not frustrated. geerts & holmes (1998) have reviewed how drugs can be managed and suggested the way forward. while the use of try pan ocides by livestock farmers and pastoralists is popular because it is a private good, supply chains often hamper availability. the challenge of sustainable agriculture, rural development and natural endowments (sard) the issue of sard should be at the heart of any enduring project, especially t & t interventions, having regard to the painful history of failed projects and the importance of not allowing history to be repeated. the concept of sard has been around since the 1970s and was incorporated as one of the goals of agenda 21. sard has been described as a process that meets the following criteria: • basic nutritional requirements of present and future generations, qualitatively and quantitatively, are met while providing a number of other agricultural products. • provision of durable employment, sufficient income and decent living and working conditions for all those engaged in agricultural production. • maintains and, where possible, enhances the productive capacity of the natural resource base as a whole and the regenerative capacity of renewable resources, without disrupting the functioning of basic ecological cycles and natural balances, destroying the socio-cultural attributes of rural communities or causing contamination of the environment. • reduces the vulnerability of the agricultural sector to adverse natural and socio-economic factors and other risks, and strengthens self-reliance. many donor-supported large-scale t & t interventions in the past have failed to achieve their goals partly because sustainability was not built into such projects upfront and where this was done, it has not been followed through. 39 a.a. ilemobade the opportunities having briefly outlined the challenges that t & t pose, we must now turn to the opportunities deriving from their control or eradication. the first, of course, is that of opening up of the extensive land hitherto denied to livestock raising and crop agriculture. the success story of the elimination or eradication of tsetse from the island zanzibar using a combination of suppression followed by the use of sit and the recently reported case of successful elimination of tsetse using the sat from the okavango delta in botswana where tourism has reportedly started flourishing with cattle grazing along the delta, has encouraged the african development bank (afdb) to become involved in t & t intervention in six african countries. the six countries involved, namely, burkina-faso, ethiopia, ghana, kenya and mali have been granted about 70 million us$ in loans and grants to eliminate tsetse over a period of 5 years under the pattec initiative. work has already started with collection of baseline data. negotiation is also in progress in six additional countries to benefit from the afdb investment. it is however, important that this bold initiative succeed in order to encourage further investment in tsetse and trypanosomosis. it is hoped that the present crisis in the global economy will not have a serious adverse effect on further investment and thus hinder the achievement of the millennium development goals (mdg). other opportunities can be categorized along the following headings: the opportunity for improved technology the refinement of methodologies has led to the greater success of intervention programmes using improved technologies. aerial spraying using sequential aerosol technology, involving ultra-low level of synthetic pyrethroid insecticide, has led to the success reported in botswana where tsetse has, to all intent and purposes, been exterminated. demographic pressure the need to reduce the food insecurity by opening up land denied to agriculture and livestock-raising by t & t in africa is a critical and urgent one. in many affected countries, the presence of t & t forces rural people and their livestock to live in areas of less risk, with the attendant problem of unbalanced exploitation of natural resources leading to increased vulnerability of the environment and natural resources and expansion of rural activities into a fragile environment. an example is the ethiopian experience where the presence of t & t in the western and southwestern lowlands had forced people to move to the highlands to avoid the tsetse-infested lowlands, which are fertile and relatively underused for agriculture. the result is that the highlands are overpopulated and the land acutely degraded, while animal traction power needed for rural agriculture is severely limited. removal of the t & t constraints in such places, therefore, will open up the land for expanded and improved livestock raising and agricultural production, reduce the demographic pressure, ensure balanced utilization of land and resources— thereby providing opportunities for improvement and diversification of livestock-agricultural productions. further, opening up of land will create opportunities for meeting part of the needs of rapidly expanding population growth in africa. opportunities for a significant increase in livestock-crop agricultural production it has been shown that much of the primary cultivation in sub-saharan africa (about 90 %) is carried out by hand (fao 1987), thus limiting the size of cultivable areas for arable and cash crops. fao (1994a, b) stated that crops are a major commodity in 28 tsetse-infested countries and in 22 of these indigenous cattle meat is a major commodity. livestock alone contributes approximately 35 % of agricultural gdp in sub-saharan africa and agricultural produce worth 4.75 billion us$ is lost annually. if the constraint of t & t is removed, therefore, the following would be achieved: • significant opportunities to reduce livestock mortality and morbidity, enhance human and animal health and increase livestock productivity of meat and milk to satisfy demands, potential or actual, and to support expansion, intensification and harmonization of crops-livestock production. • increased livestock production and productivity. increased productivity in the context of sard does not necessarily mean the increase in livestock numbers as environmentalists are quick to point out. admittedly, whatever method is used to control or eradicate aat can result in overstocking with livestock and consequent degradation of the land, if an effective stocking level is not maintained. this has led to the concern expressed by environmentalists that control of t & t is detrimental to the environment by reason of soil degradation and overexploitation of natural resources in reclaimed areas. in other words, t & t is the protector of environment against exploitation. however, when the debate is considered in a proper perspective, that is, in the light 40 tsetse and trypanosomosis in africa of sard, the control or eradication of t & t should be seen as essential component of activities directed at achieving sustainable rural development. • the use of draught animals for ploughing and other farm activities, for example, transportation of produce and other materials, especially in rural areas, will be enhanced and expanded, leading to increased crop production, greater opportunities for diversification of agricultural activities and improved nutrient cycle. it has been shown that in mixed crop-livestock farming systems, farmers who use animal traction generate 25–45 % more income per unit of land and 140 % more income per unit of labour than farmers who used hoes. budd (1999) suggested that the use of draught animals alone has the potential to move families from subsistence existence into the cash economy, leading to a better standard of living. • expansion of area of cultivation would translate into more food and therefore, increased income for the farmer; provide additional employment opportunities and enhance food security generally. • increased contribution to agricultural gdp. it has been estimated by fao (1994b) that if the impact of t & t is removed, agricultural gdp will increase between 5 and 10 %. the annual losses in terms of agricultural production, estimated at 4.75 billion us$ annually, will be saved and will improve rural income and encourage cash economy, thus reducing rural-urban drift. the savings that will be realized from drug control and the relief from drug-resistant trypanosomes will encourage more farmers including crop farmers to invest in livestock agriculture in the affected countries. conclusion the challenges of controlling or eradicating a disease that has remained intractable for more than 100 years would appear insurmountable given the history of past failures, yet they provide opportunities to correct past failures, devise environmentallyfriendly technologies both for large-scale and farmer-based interventions and reassess the need for approaches that ensure that any intervention has a built-in mechanism for achieving sard. acknowledgement the author acknowledges that some of the information used in this presentation came from paat publications. appreciation is extended to dr raffaele mattioli, the focal person, paat secretariat fao, rome, for his support and for making available some of the information used in this paper. i am grateful to the organizers of the centenary celebrations for their invitation to participate in this exciting conference and for their financial support to attend. references allsopp, regg. 2001. options for vector control against trypanosomiasis in africa. trends in parasitology, 17:15–19. bruce, d. 1897. further report on the tsetse fly disease or nagana in zululand. london: harrison & sons. budd, l. 1999. dfid-funded tsetse and trypanosome research and development since 1980, vol. 2. economic analysis. ayles ford, u.k. dfid london. du toit, r. 1954. trypanosomosis in zululand and the control of tsetse flies by chemical means. onderstepoort journal of veterinary research. 26:317–331. du toit, r., kluge, e.b. & fiedler, d.g.h. 1954. the eradication of g. pallidipes from zululand by chemical means. proceedings of the international scientific council for trypano somosis research, 1954: 141. fao 1987. the state of food and agriculture. rome: fao. fao 1994a. the state of food and agriculture. rome: fao. fao 1994b. opening address of y. cheneau, in a systematic approach to tsetse and trypanosomosis control. proceedings of the fao panels of experts. rome, 1–3 december 1993. fao 2002. report of a workshop on paat-pattec harmonization. rome, 2–3 may 2002. feldmann, u. & hendrichs, j. 2001. integrating the sterile insect technique as a key component of area-wide tsetse and trypanosomosis intervention (paat technical and scientific series, no. 3). geerts, s. & holmes, p.m. 1998. drug management and parasite resistance in bovine trypanosomosis in africa (paat technical and scientific series, no. 1). rome: fao. mattioli , r.c., feldmann, u., hendrickx, g., wint, w., jannin, j. & slingenbergh, j. 2004. tsetse and try panosomosis intervention policies supporting sustainable animal agricultural development. food, agriculture & environment, 2:310–314. pattec 2001. pan african tsetse and trypanosomiasis eradication campaign plan of action. addis 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: louis j. la grange1,2 björn reininghaus1 samson mukaratirwa2 affiliations: 1department of agriculture, rural development, land and environmental affairs, mpumalanga, south africa 2school of life sciences, university of kwazulu-natal, westville campus, correspondence to: louis la grange postal address: private bag x11309, nelspruit 1200, south africa dates: received: 25 june 2014 accepted: 05 sept. 2014 published: 20 nov. 2014 how to cite this article: la grange, l.j., reininghaus, b. & mukaratirwa, s., 2014, ‘first report of a mixed infection of trichinella nelsoni and trichinella t8 in a leopard (panthera pardus) from the greater kruger national park, south africa’, onderstepoort journal of veterinary research 81(1), art. #836, 3 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.836 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. first report of a mixed infection of trichinella nelsoni and trichinella t8 in a leopard (panthera pardus) from the greater kruger national park, south africa in this original research... open access • abstract • introduction • materials and methods • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ at least three trichinella species, namely trichinella nelsoni, trichinella britovi and trichinella zimbabwensis, and one genotype (trichinella t8), have been isolated from sylvatic carnivores on the african continent. with the exception of t. britovi, the other species are known to circulate in wildlife of the kruger national park (knp), south africa, and knp neighbouring game reserves (collectively known as the greater knp area). lions (panthera leo) and spotted hyenas (crocuta crocuta) appear to be the most important reservoirs of t. nelsoni and trichinella t8 in the knp and surrounding areas. interspecies predation between lions and hyenas has been implicated as a primary mode of maintaining the life cycles of these two trichinella species. this is the first report of a mixed natural infection of t. nelsoni and trichinella t8 in a leopard (panthera pardus) from south africa. trichinella muscle larvae were identified to species level by multiplex polymerase chain reaction (pcr). probable sources of infection, based on the known dietary preference and prey species’ range of leopards, are also discussed. the described occurrence of trichinella species in a leopard from the greater knp area raises the question of possible sources of infection for this predator species. introduction top ↑ the genus trichinella is comprised of the species trichinella spiralis, trichinella nativa, trichinella britovi, trichinella pseudospiralis, trichinella murrelli, trichinella nelsoni, trichinella papuae, trichinella zimbabwensis and three genotypes (trichinella t6, t8, t9) (pozio & zarlenga 2005; pozio et al. 2009), and a newly described species, trichinella patagoniensis from patagonia, argentina (krivokapich et al. 2008, 2012). of these, at least three species (t. nelsoni, t. britovi and t. zimbabwensis) and one genotype (trichinella t8) have been isolated from sylvatic carnivores in africa (pozio et al. 1994, 2002, 2005, 2007). with the exception of t. britovi, these species have been reported in wild carnivores from the kruger national park (knp) and neighbouring game reserves of south africa (la grange, marucci & pozio 2010; la grange et al. 2013; marucci, la grange & pozio 2009). lions (panthera leo) and spotted hyenas (crocuta crocuta) appear to be important reservoirs of t. nelsoni and trichinella t8 in the greater knp area of south africa. interspecies predation between lions and hyenas has been hypothesised to play a role in the maintenance of the life cycles of these two parasite species (mukaratirwa et al. 2013). trichinella spp. infections in humans have been reported due to the consumption of undercooked meat derived from bush pigs (potamochoerus larvatus) in kenya (forrester 1964; forrester, nelson & sander 1961; hutcheon & pamba 1972; nelson 1970; okelo & bhatt 1987) and warthogs (phacocoerus africanus) in tanzania (bura & willett 1977). results from previous studies suggest that these species of sylvatic suidae may play an additional, yet less pronounced, role in the epidemiology of t. nelsoni and trichinella t8, and have shown that warthogs and bush pigs represent only a small percentage of naturally infected animal reservoirs in africa (mukaratirwa et al. 2013). limited passive surveillance based on convenient samples from 17 warthogs culled in the knp and adjacent nature reserves have, to date, not yielded any positive samples (la grange & mukaratirwa unpublished data; marucci et al. 2009). trichinella nelsoni infection in leopards (panthera pardus) has been reported from tanzania (pozio et al. 1997) and kenya (pozio 2007), whereas t. britovi has been described in a leopard from iran (mowlavi et al. 2009). in addition to one leopard tested in 1974 in knp (marucci et al. 2009), three more, originating from the nature reserves adjacent to the western knp, have been tested since 2012, with negative results (la grange & mukaratirwa unpublished data). leopards are solitary animals; they avoid contact with lions and hyenas and do not normally prey on them (hayward et al. 2006). this suggests the existence of alternate sylvatic species serving as sources of infection, such as small and medium-sized game species, including warthogs, bush pigs and baboons (papio spp.), all of which could potentially serve as sources of trichinella spp. infection. although natural infection with trichinella spp. has not been documented in free-ranging baboons, monkeys and bush pigs in south africa, they could possibly serve as hosts for trichinella spp. in southern africa because experimental infection of t. zimbabwensis in baboons, monkeys (cercopithecus aethiopis) (mukaratirwa et al. 2008) and domestic pigs (matenga et al. 2006; mukaratirwa & foggin 1996) has previously been successful. furthermore, they are known to hunt and feed on other vertebrates in the wild (butynski 1982). between 1966 and 1974, five baboons from the greater knp area have been tested, with negative results (marucci et al. 2009); an additional two have been tested since 2012 (la grange & mukaratirwa unpublished data). as in the case with bush pigs, the number of samples tested is limited and this precludes any assumptions that these species do not play a role in the epidemiology of the trichinella spp. in southern africa. experimental infections by mukaratirwa et al. (2008) have shown that baboons are highly susceptible to the parasite, with fatal cases, and this might be the case in the wild. in the present study, a mixed natural infection of t. nelsoni and trichinella t8 was confirmed in a leopard from a nature reserve in the west of the greater knp area of south africa. materials and methods top ↑ in july 2012, a fresh carcass of an adult male leopard (about 5 years old) was found in a riverine stretch in a nature reserve in the west of the greater knp area (24º47’57.1’’s, 31º29’24.0’’e), and this was reported to the local state veterinary services. on post-mortem examination the animal showed signs of severe and generalised haemorrhages, including widespread distinct suggillations in the large muscle groups of the forelimbs and hindlimbs. secondary rodenticide poisoning was identified as the most likely reason for the observed findings. as part of the post-mortem examination and sampling procedures, muscle specimens were collected for routine surveillance for trichinella infection and submitted for testing to the parasitology laboratory, school of life sciences, university of kwazulu-natal, westville campus, durban. muscle specimens that were collected consisted of sections of about 40 g, and were collected bilaterally from the musculus masseter, musculus tricipitis brachii, flexor muscle group of the antebrachium, musculus intercostales, musculus diaphragmaticus, musculus quadriceps femoris and the flexor muscle group of the lower hindlimbs, which were pooled as one source sample. muscle samples were artificially digested as previously described (nöckler & kapel 2007) and examined under a stereo microscope for the detection of trichinella larvae. larvae observed in the digest were washed and used to infect sprague-dawley rats and the remaining muscle tissues were preserved in 2% sodium azide solution and submitted to the international trichinella reference centre (itrc) in rome, italy, for harvesting of additional larvae for species identification. a sample of the larvae was deposited in the itrc in rome, italy (isolate code iss 4413). results top ↑ initial testing by artificial digestion showed an infection intensity of three larvae per gram (lpg) in pooled muscle samples. in subsequent experimental infection of three sprague-dawley rats, only one rat established infection with an establishment of 702 lpg of muscle tissues. three larvae were recovered from the muscle tissue submitted to itrc and identified by a multiplex polymerase chain reaction (pcr) (pozio & la rosa 2003). one larva was identified as t. nelsoni, whilst the two remaining larvae were identified as trichinella t8. discussion top ↑ according to hayward et al. (2006), wild pigs and baboons are not frequently preyed upon by leopards, whilst other carnivorous species such as jackals, cheetahs, civets and genets are preyed on more frequently. prey species composition of leopards reported by le roux and skinner (1989) in the western greater knp area included the common warthog, making up 4%, the chacma baboon (papio ursinus) 1%, vervet monkeys 6%, small mammalian carnivores (african civets and genets) 2% and reptiles (leopard tortoise, mozambique spitting cobra and rock monitor) 4%. other authors have found various carnivorous animals of mammalian and reptile origin to represent 12% of all kills by leopards in north-eastern namibia (stander 1997), and large carnivores to represent 4% in the knp (bailey 1993). the predation of other carnivorous species by leopards, like the jackals that scavenge on the carcasses of other wild carnivores, suggests a possibly important source of trichinella spp. infection. the confirmation of a mixed infection in a leopard from south africa may suggest that wild pigs play a lesser role in the natural epidemiology of trichinella spp. in southern africa, when compared to lions and hyenas. other mammals that are more frequently preyed upon by leopards may act as better reservoirs for these trichinella taxa. the absence of human infections from consumption of undercooked meat from wild pigs or products derived from wild pigs in southern africa may support this. the report of a mixed infection of two species of trichinella in a leopard confirms the importance of the greater knp area as a habitat of reservoirs for trichinella species circulating in sylvatic carnivores of this region (la grange et al. 2010). the confirmation of a mixed infection of t. nelsoni and trichinella t8 in a leopard further confirms the sympatric status of these parasites, and a mixed infection of the same species has been reported in a lion in the knp area (marucci et al. 2009). despite the low number of wild pigs tested from the knp and adjacent reserves, studies have revealed a 2.5% infection rate in bush pigs from kenya (nelson et al. 1963), 4.0% from warthogs in senegal (gretillat & chevallier 1970) and 4.3% in warthogs from kenya, south africa, senegal and tanzania (sachs 1970). these studies all preceded the elucidation of the trichinella taxon (pozio et al. 1997) but later studies have implicated t. britovi and t. nelsoni in 23.0% and 7.0% of all infections of swine, respectively (pozio 2005). infections of swine from kenya and tanzania have most probably involved t. nelsoni, whilst those reported from senegal are considered to be t. britovi (mukaratirwa et al. 2013; pozio 2007). in south africa, however, warthogs and bush pigs may very well act as reservoirs for both t. nelsoni and trichinella t8, since both have been previously found in other mammals from this region (marucci et al. 2009; mukaratirwa et al. 2013), despite the fact that the latter has never been isolated from wild pigs. the preliminary prevalence amongst leopards in knp and surrounding areas, as documented in the present report, is much higher at 25% and this possibly suggests alternative or additional sources of infection for these predators, although the number of animals tested is limited. the fact that leopards have a tendency to avoid preying on wild pigs (hayward et al. 2006) may support the aforementioned theory, but should be interpreted with caution since the limited data on trichinella spp. infection in leopards and wild pigs in southern africa do not provide conclusive evidence. furthermore, the fact that leopards prefer to avoid wild pigs does not imply that leopards never prey on these species at all. future research should include screening of other smaller carnivorous animals frequently preyed upon by leopards, and surveillance efforts should be intensified amongst wild suidae to elucidate the role of each in the natural epidemiology of the various trichinella species circulating in the knp area. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions l.j.l.g. (department of agriculture, rural development, land and environmental affairs) conducted the initial acquisition and testing of samples, and is the main author of the manuscript. b.r. (department of agriculture, rural development, land and environmental affairs) performed the post-mortem examination, collected the muscle samples and is co-author of the manuscript. s.m. (university of kwazulu-natal) conducted the experimental infection, dissection and testing of muscle samples of rats, submitted the sample to the itrc and is the secondary author of the manuscript. references top ↑ bailey, t.n., 1993, the african leopard: ecology and behaviour of a solitary felid, columbia university press, new york. bura, m.w. & willett, w.c., 1977, ‘an outbreak of trichinosis in tanzania’, east african medical journal 54, 185–193. butynski, t.m., 1982, ‘vertebrate predation by primates: a review of hunting patterns and prey’, journal of human evolution 11, 421–430. http://dx.doi.org/10.1016/s0047-2484(82)80095-x forrester, a.t.t., 1964, ‘human trichinellosis in kenya’, in a. corradetti (ed.), proceedings of the 1st international conference of parasitology, vol. ii, pp. 669–671, pergamon, oxford, rome. forrester, a.t., nelson, g.s. & sander, g., 1961, ‘the first record of an outbreak of trichinosis in africa south of the sahara’, transactions of the royal society of tropical medicine and hygiene 55, 503–513. http://dx.doi.org/10.1016/0035-9203(61)90072-4 gretillat, s. & chevallier, j.l., 1970, ‘preliminary note on the epidemiology of trichinosis in wild animals in western africa’, bulletin of the world health organization 43, 749–757 (in french). hayward, m.w., henschel, p., o’brien, j., hofmeyr, m., balme, g. & kerley, g.i.h., 2006, ‘prey preferences of the leopard (panthera pardus)’, journal of zoology 270, 298–313. hutcheon, r.a. & pamba, h.o., 1972, ‘report of a family outbreak of trichinosis in kajiado district – kenya’, east african medical journal 49, 663–666. krivokapich, s.j., cinthia, l., gonzàlez, p., graciana, g.m. & viviana, c., 2008, ‘molecular evidence for a novel encapsulated genotype of trichinella from patagonia, argentina’, veterinary parasitology 156, 234–240. http://dx.doi.org/10.1016/j.vetpar.2008.06.003 krivokapich, s.j., pozio, e., gatti, g.m., prous, c.l., ribicich, m., marucci, g. et al., 2012, ‘trichinella patagoniensis n. sp. 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http://dx.doi.org/10.1016/j.ijpara.2005.03.013 pozio, e., verster, a., braack, l., de meneghi, d. & la rosa, g., 1994, ‘trichinellosis south of the sahara’, in c.w. campbell, e. pozio & f. bruschi (eds.), trichinellosis, pp. 527–532, istituto superiore di sanità press, rome. pozio, e. & zarlenga, d.s., 2005, ‘recent advances on the taxonomy, systematics and epidemiology of trichinella’, international journal for parasitology 35, 1191–1204. http://dx.doi.org/10.1016/j.ijpara.2005.07.012 sachs, r., 1970, ‘zur epidemiologie der trichinellose in afrika [the epidemiology of trichinellosis in africa]’, zeitschrift für tropenmedizin und parasitologie 20, 117–126. stander, p.e., 1997, ‘the ecology of lions and conflict with people in north-eastern namibia’, in j. van heerden (ed.), proceedings of a symposium on lions and leopards as game ranch animals, onderstepoort, oct 1997, pp. 10–17. article information authors: shahrzad azizi1 reza kheirandish1 elham rahimi2 affiliations: 1department of pathology, shahid bahonar university of kerman, iran2faculty of veterinary medicine, shahid bahonar university of kerman, iran correspondence to: shahrzad azizi postal address: po box 761691411, kerman 761, iran dates: received: 03 june 2014 accepted: 18 aug. 2014 published: 12 nov. 2014 how to cite this article: azizi, s., kheirandish, r. & rahimi, e., 2014, ‘comparison of polymerase chain reaction and warthin-starry techniques to detect leptospira spp. in kidneys of slaughtered cattle’, onderstepoort journal of veterinary research 81(1), art. #821, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.821 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. comparison of polymerase chain reaction and warthin-starry techniques to detect leptospira spp. in kidneys of slaughtered cattle in this original research... open access • abstract • introduction • materials and methods    • sample collection    • polymerase chain reaction procedure    • microscopic investigations    • haematoxylin-eosin staining    • warthin-starry staining method    • statistical analysis • results    • gross and histopathological findings    • warthin-starry detection • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ leptospirosis is a worldwide zoonotic disease that is caused by gram-negative spirochaetes, leptospira species. affected animals excrete the organism in the urine into the environment and act as a source of infection. cattle are maintenance hosts for some serovars of leptospirosis and are important in the transmission of the infection to humans. at post mortem examination, affected cattle show white spots in their kidneys but these are not specific for leptospirosis. sometimes it is necessary that leptospirosis be diagnosed in the carcass. different direct methods, including polymerase chain reaction (pcr), warthin-starry silver stain (ws), immunofluorescence (if) and immunohistochemistry (ihc) can be used in order to diagnose leptospirosis in the affected tissues, such as kidney. the main advantage of the ws technique is direct visualisation of the bacteria in the tissue samples. silver staining is useful for retrospective studies on formalin-fixed and paraffin-embedded samples but little information is available on the sensitivity and specificity of the technique. the present study aimed to find a simple and inexpensive method that can be used in any laboratory and that also, if clinical samples are not available, can detect leptospira in tissue samples post mortem. this study was performed on 19 paraffin-embedded kidneys of slaughtered cows that grossly had focal to multifocal white spots. leptospirosis was confirmed in these samples with pcr based on the lipl32 gene. out of 19 pcr positive kidneys, leptospira was identified in 13 stained samples by ws. the kidneys revealed different grades of interstitial nephritis. no relationship was found between severity of lesions and presence of leptospires in the kidneys. the pcr results on the urine and blood were consistent with matching ws stained kidneys. out of 13 kidneys that were positive with silver staining, 7 matching blood and 10 matching urine samples were confirmed positive for leptospirosis with pcr. in this study, the ws technique provided fewer positive results than pcr. this may be as a result of a low burden of leptospira in the kidney, but the sensitivity of ws staining needs more investigation. introduction top ↑ leptospirosis is a zoonotic disease occurring in domestic and wild animals worldwide that is caused by gram-negative spirochaetes of the genus leptospira (adler & de la peña-moctezuma 2010; hazıroğlu & milli 2001). this disease is significant for public health. in livestock, leptospirosis leads to septicaemia, hepatitis, nephritis, mastitis, decreased production of meat and milk, abortion and stillbirth (hazıroğlu & milli 2001). leptospires enter through cuts or abrasions of the skin and mucus membranes such as the conjunctiva and urogenital system (lilenbaum et al. 2008; monahan, callanan & nally 2009). the leptospiraemic phase lasts for approximately 10 days until the specific immunoglobulins appear in the blood (adler & de la peña-moctezuma 2010). after the immune response, leptospires localise in the kidneys of animal hosts (monahan et al. 2009; scanziani, sironi & mandelli 1989; yang, wu & pan 2001) and contaminate the environment by excretion in the urine and act as a source of infection (bharti et al. 2003; faine et al. 1999).cattle are maintenance hosts for some serovars of leptospira and may transmit infection to humans (levett 2001). in necropsy, focal to multifocal white spots are seen in the kidney, but these lesions are not pathognomonic for leptospirosis. access to a proper diagnostic test in clinical cases or after death is essential for treatment and prevention of leptospirosis. diagnostic techniques should be easy to use, available and inexpensive, with high specificity and sensitivity. the routine and reference test for leptospirosis is the microscopic agglutination test (mat) but this technique is expensive and needs a specialised laboratory and living leptospira serovars (céspedes et al. 2007; hernández-rodríguez et al. 2011). molecular techniques have many advantages (levett 2001). polymerase chain reaction (pcr) is a rapid, sensitive, inexpensive assay and can identify low doses of bacteria (céspedes et al. 2007). this technique is useful for identification of fastidious and slow-growing organisms, and can be used easily even in non-specialised laboratories in comparison to mat (céspedes et al. 2007; hernándezrodríguez et al. 2011). the presence of leptospira in the tissue can be detected by direct methods including culture, special staining of tissue and immunohistochemistry (ihc) (ellis et al. 1983; thiermann 1977). microbiological culture is expensive and time consuming and may take more than 8 weeks for results (obregón et al. 2004; rahim, gorbanpour & haidari 2005). for ihc, special antibodies need to be provided. warthin-starry (ws) silver staining is used for detection of spirochaetes and other bacteria in tissues and smears. it is a simple method and may be performed in any laboratory, but information on detection of leptospires in tissue by silver impregnation is scarce (fornazari et al. 2012; ortega-pacheco et al. 2008). in this study, paraffin-embedded kidneys were used for ws staining. all kidneys grossly had white spots on their surfaces and leptospirosis was confirmed by pcr. the agreement between pcr and ws was evaluated in this study. materials and methods top ↑ sample collection this retrospective study was performed on 19 paraffin-embedded kidneys of slaughtered cattle. these kidneys macroscopically had focal to multifocal white spots on their surfaces and leptospirosis was confirmed with pcr based on the lipl32 gene in a previous study (azizi et al. 2012). in addition, data on serum and urine of these samples examined by pcr for leptospirosis are available. polymerase chain reaction procedure deoxyribonucleic acid (dna) was extracted from frozen kidneys, buffy coat and urine samples with a high-yield dna purification kit (cinnagen inc., pn811sc, iran), according to the manufacturer’s instructions. polymerase chain reaction based on the lipl32 gene was performed using the primers 5’atctccgttgcactctttgc3’ and 5’accatcatcatcatcgtcca3’ as previously described by tansuphasiri et al. (2006) to distinguish between pathogenic and saprophytic leptospira species (also see azizi et al. 2012). polymerase chain reaction amplification was performed using the following programme: an initial cycle of denaturation at 94 °c for 3 min, 30 cycles of denaturation at 94 °c for 1 min, annealing at 60 °c for 90 s, extension at 72 °c for 20 min, a final extension at 72 °c for 10 min and holding at 4 °c. the amplified products were analysed by electrophoresis on ethidium bromide-stained 2% agarose gels and the results were observed using ultraviolet (uv) light. a sample was considered positive when the 474 bp dna band was obtained (vital-brazil et al. 2010; also see azizi et al. 2012). microscopic investigations paraffin-embedded kidneys were sectioned at 5 μm thickness, stained with haematoxylin-eosin (he) and ws silver and examined for interstitial nephritis. interstitial nephritis was graded according to degree of inflammation as follows: (-) negative; (+) mild, < 4 foci; (++) moderate, 4–6 foci and (+++) severe, > 6 foci in each section (rossetti et al. 2004). haematoxylin-eosin staining for he staining, the slides were deparaffinised in two jars containing xylene (10 min per step) and then hydrated with distilled water. the nucleus was stained with haematoxylin for 5 min, after which the slides were rinsed in running tap water for 7 min to remove the haematoxylin. then eosin was applied for 1 min. finally, the slides were dehydrated in graded alcohol, cleared in xylene and mounted. warthin-starry staining method the sections of pcr positive kidney were deparaffinised, rehydrated with triple-distilled water, and immersed in 1% silver nitrate solution at 43 °c for 30 min. developer solution was prepared during this time. the slides were flooded in developer solution until they became light brown to yellow and then washed rapidly in hot tap water (56 °c). after that the slides were rinsed in distilled water and then dehydrated, cleared in xylene and mounted (luna 1968). investigation of tissue sections was carried out using an optical microscope with a magnification of ×1000. in addition, five kidneys that were negative for leptospirosis on pcr were used as negative control. statistical analysis the association between the results of silver impregnation and pcr data for urine and blood was determined by the kappa test (k) (thrusfield 2005). in this statistical test, complete agreement between data corresponds to k = 1, and lack of agreement corresponds to k = 0. results top ↑ gross and histopathological findings grossly, focal to multifocal white spots, 1 mm – 5 mm in diameter, were randomly distributed on the surface of the kidneys (figure 1). histopathological examination revealed mononuclear cells, especially lymphocytes and a few plasma cells, aggregated focally in the interstitial tissues (figure 2). the lesions were confined to the renal cortex. mononuclear cells were also infiltrated around the glomeruli and blood vessels. the degree of focal interstitial nephritis in 19 kidneys included 11 samples with mild lesions (+), five with moderate (++) and three with severe lesions (+++). figure 1: numerous white spots related to leptospirosis distributed on the surface of a bovine kidney. figure 2: severe infiltration of mononuclear cells (arrows) in the interstitial tissues of the renal cortex. figure 3: accumulation of inflammatory mononuclear cells around the glomeruli (asterisk). warthin-starry detection leptospira was identified in 13 (68.4%) out of 19 pcr positive kidneys by ws. urine (n = 11/19) and blood (n = 7/19) samples matching these kidneys were pcr positive in our previous study (azizi et al. 2012). leptospira was detected by silver staining in all seven kidneys with matching pcr positive blood samples. out of 13 ws positive kidneys, 11 matching urine samples had been positive with pcr. organisms were detected by ws in two kidneys of which the matching urine samples were negative with pcr (table 1). no relationship was established between degree of histopathological lesions and the presence of the organism. spirochaetes were observed as spiral or filiform on the apical surface of epithelial cells (figure 3) and in the lumens of cortical tubules (figure 4). low numbers of leptospira, from one to a maximum of five organisms, were found more frequently in the tubules of the superficial than the deep cortex, and none in the medullary region. the results of ws staining and pcr had moderate agreement with kappa (0.55, p < 0.01) for urine and (0.42, p > 0.02) for blood respectively. table 1: comparative results of polymerase chain reaction and warthin-starry staining for detection of leptospira in tissue samples. figure 4: warthin-starry staining of kidney. presence of filiform, dark brown leptospira (arrows) on the apical surface of epithelial cells in the cortex. figure 5: warthin-starry staining of kidney. presence of spiral shaped leptospira (arrow) in the lumen of renal tubule. discussion top ↑ leptospirosis is a spirochaetal infection that threatens both animal and human health. humans can be affected during occupational activities and contact with infected animals, for example meat inspectors and abattoir staff (orrego et al. 2003).abattoir studies offer an opportunity for tissue sampling and diagnosis of diseases. cattle act as maintenance hosts for leptospirosis (levett 2001). affected cattle may show multiple macroscopic white foci in the renal parenchyma, referred to as ‘white-spotted kidney’ (baker et al. 1989; hunter et al. 1987; jones et al. 1987). microscopically, these spots reveal interstitial nephritis, which is a common finding in leptospirosis but not pathognomonic for this disease and may occur with other pathogens. different technical approaches including pcr, ws, immunofluorescence (if) and ihc can be used in order to perform diagnosis of leptospirosis in the suspected tissues, such as kidneys (ahmad, shah & ahmad 2005). the main advantage of ws and if techniques is direct visualisation of the bacteria in the tissue samples. silver staining is useful for retrospective studies on formalin-fixed and paraffin-embedded samples, but little information is available on the sensitivity and specificity of the test (szeredi & haake 2006). the objective of the present study was to find a simple and inexpensive method that can be performed in any laboratory and can detect leptospira in affected cases post mortem if samples from living animals are not available. therefore, the original specimens were taken from a slaughterhouse. the findings of this study agreed with the results of previous studies indicating that leptospires were localised on the apical surface of epithelium and in the lumens of proximal convoluted tubules of kidneys in carrier animals (marshall 1974; sterling & thiermann 1981). seibold, keech and bokelman (1961) reported the presence of leptospires in the lumen of tubules at the corticomedullary junction of carrier cattle with an extensive inflammatory cortical reaction. they suggested that leptospires migrated to the corticomedullary tubules because of damage to the cortical tubules, but in this study no leptospira was identified in the medullary tubules. silver staining is not commonly used as a diagnostic method for leptospirosis. in the present study, ws staining was applied to evaluate it as a rapid, easy, available technique that can be performed on formalin-fixed tissues. some studies have described its efficacy for detecting leptospires in animals and humans (chappel et al. 1992; de brito et al. 1996; léon et al. 2006; ortega-pacheco et al. 2008; sebastian et al. 2005). a few studies have reported a low frequency of positive results using this method (chappel et al. 1992; hunter et al. 1987). in this study, ws technique showed fewer positive results than pcr. this may be because of a low burden of leptospira in the kidney. there are some difficulties of interpretation in this method because reticulin fibres stain with ws and leptospiral fragments are not recognisable, especially if few organisms are present (szeredi & haake 2006). pcr was found in this study to be a more sensitive technique that could detect low numbers or deformed organisms in the tissue. in this study, leptospira was detected in the kidneys of all seropositive animals by silver staining. similarly, ws positivity in the kidney is reported more frequently in seropositive animals in other studies. jansen et al. (2007) investigated leptospirosis in wild boars in germany. they found antibodies to leptospires in 25 (18%) out of 141 wild boars. a total of 29 kidney specimens (17 from seropositive and 12 from seronegative boars) were examined with he and ws. moderate to severe chronic lymphoplasmacytic interstitial nephritis was observed in 15 (88%) kidney samples from the 17 seropositive boars and five out of 12 seronegative boars. leptospires were detected by silver staining in three (30%) out of 10 specimens from seropositive wild boars with chronic interstitial nephritis and were confirmed by pcr targeting lipl32 in two of the leptospira-positive samples. these researchers stated that wild boars could be a potential source of human leptospirosis in urban environments. hodgin, miller and lozano (1989) easily found spirochetes in the cortical and medullary interstitium of the kidney as well as the liver and placenta of aborted foals by ws in the acute stage of disease. in the present investigation, the histopathological and ws findings were similar to results described by rossetti et al. (2004). they detected leptospires in the kidneys of 41 wild house mice (mus musculus) with three diagnostic techniques, namely bacteriology, ws and ihc. leptospira belonging to the ballum serogroup was isolated from 16 (39%) out of 41 samples by culture and identified in 18 (44%) and 19 (46%) kidney specimens by ws and ihc respectively. with ws staining, leptospires were identified in high numbers on the apical surface of epithelial cells and in the lumen of cortical tubules. ihc detected the presence of the agent in one sample that was negative with ws staining. these researchers suggested that low numbers of leptospires in that sample was the reason for ws negativity. histopathologically, 13 out of 19 infected mice showed interstitial nephritis caused by mononuclear cell infiltration. these researchers observed that these three techniques had a high level of agreement and no significant differences between them were present. in agreement with the present study, no relationship was found between severity of lesions and presence of leptospires. the higher sensitivity of pcr technique as opposed to ws has been reported in previous studies. fornazari et al. (2012) compared different techniques for diagnosing leptospirosis in kidney, liver and blood samples of 465 slaughtered sheep. the sera were analysed by mat. kidney and liver samples of seropositive animals were examined using four techniques, including bacterial culture, ws, conventional pcr (cpcr) and quantitative pcr (qpcr). with mat, 21 animals (4.5%) were positive. none were positive by bacteriological culture. ws and cpcr techniques detected four and six positive kidney samples respectively. eleven animals were positive by qpcr (eight kidney samples and three livers). their results showed that qpcr had the highest sensitivity amongst the techniques used to detect leptospira spp. in tissue samples, followed by cpcr, ws and bacterial culture. however, some studies have reported high sensitivity for ws. wild et al. (2002) examined formalin-fixed kidneys of 12 dogs with chronic interstitial nephritis for leptospirosis. leptospira was identified in six samples with silver staining. leptospiral antigens were detected by ihc in all ws positive samples. sections that were negative by silver staining were also negative by immunostaining. the association of interstitial nephritis with positive pcr results and demonstration of leptospires in the kidney specimens indicate that cows act as a maintenance host for leptospira spp. in this area (hernández-rodríguez et al. 2011). the differences between two techniques may be related to lack of experience with the ws technique (orrego et al. 2003). however, it is accepted that if silver staining is performed carefully, it is a highly sensitive and reliable method (thiermann 1977). further studies will be carried out to determine the bacterial burdens in various tissues, especially kidney, in different hosts. also, localisation of leptospira in the kidney should be investigated in the acute and chronic stages of disease to determine in which stage the bacterial burdens are greater and can be detected by the ws technique. acknowledgements top ↑ the authors would like to thank the vice chancellor of research at the shahid bahonar university of kerman for their financial support and cooperation. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions s.a. 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terefe1 feysel redwan1 endrias zewdu2 affiliations: 1college of veterinary medicine, haramaya university, ethiopia2department of veterinary laboratory technology, ambo university, ethiopia correspondence to: yitagele terefe postal address: po box 281, dire dawa, ethiopia dates: received: 28 aug. 2013 accepted: 27 nov. 2013 published: 19 feb. 2014 how to cite this article: terefe, y., redwan, f. & zewdu, e., 2014, ‘bovine cysticercosis and its food safety implications in harari people’s national regional state, eastern ethiopia’, onderstepoort journal of veterinary research 81(1), art. #676, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.676 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. bovine cysticercosis and its food safety implications in harari people’s national regional state, eastern ethiopia in this original research... open access • abstract • introduction • materials and methods    • description of the study area    • harar municipal abattoir    • origin and management of study animals    • study design    • study methodology       • abattoir survey       • cyst distribution and characterisation       • questionnaire survey    • data handling, management and analysis • results    • prevalence of bovine cysticercosis and associated risk factors    • anatomical distribution and characterisation of cysts    • status of taenia saginata taeniosis as a meatbornezoonosis in harar town • discussion • conclusion • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ taenia saginata cysticercosis is one of the zoonotic diseases that threaten food safety and food security, particularly in developing countries. a cross-sectional study was conducted to estimate the prevalence and cyst distribution in infected cattle, and food safety implications of taenia saginata cysticercosis in harari people’s national regional state, eastern ethiopia. post-mortem inspection of carcasses and organs of slaughtered cattle in harar municipal abattoir, cyst viability tests and interviews with randomly selected meat consumers were undertaken. the post-mortem inspection showed that of the 898 local zebu cattle slaughtered for human consumption and examined for the presence of cysticerci of t. saginata, 19.7% (177/898; 95% ci = 17.2–22.5) harboured at least one cyst in the muscles or organs inspected. of the edible anatomical sites with cysticerci, shoulder muscle, liver and heart together represented 65.4%, 66.0% and 65.4% respectively of relative prevalence, total cyst count and cyst viability. these edible sites are preferred above others by local people for preparation and consumption of raw or inadequately cooked meat dishes that are locally served as kurt, kitffo and dullet. the interviews revealed that among the 300 study participants, 182 (60.7%) had been infected by taeniosis at least once during the previous year and of these 99.0% had eaten raw or undercooked beef, the majority (88.3%) obtained from butchers assumed to provide officially inspected meat that was fit for consumption. this indicated that existing meat inspection processes were inadequate to prevent carcasses infected with t. saginata cysticerci from reaching consumers. the high prevalence of viable cysts in the edible parts of beef together with the widespread consumption of raw or undercooked beef indicated the importance of t. saginata cysticercosis as a food safety problem in eastern ethiopia. the promotion of policies to upgrade existing meat inspection procedures and public education to ensure effective prevention of t. saginata taeniosis in humans were recommended. introduction top ↑ foods of animal origin are often the preferred source of protein. however, if not properly prepared or handled, they can lead to food-borne infections. cysticercosis caused by taenia saginata is among the diseases that affect food safety (gajadhar, scandrett & forbes 2006). taenia saginata cysticercosis or bovine cysticercosis, caused by the metacestodes of t. saginata, is a cosmopolitan disease occurring in industrialised as well as developing countries (dorny et al. 2009). the european parliament and council in its directive 2003/99ec identified t. saginata cysticercus as one of the major food-borne pathogens that should be monitored to protect human health (abuseir et al. 2006). the prevalence is considered to be higher in developing countries because of poor sanitation, traditional cattle husbandry systems and inadequate meat inspection facilities (cabaret et al. 2002; dorny & praet 2007). as a result, the quality of human life, the aesthetic value of meat and the trading of meat and offal are compromised (alum, rubino & ijaz 2010; dorny & praet 2007; gajadhar et al. 2006).taeniosis or bovine cysticercosis has been recognised as a common problem in ethiopia for many years (webb 1957). previous studies have indicated that taeniosis or bovine cysticercosis is an important meat-borne zoonosis that affects the safety of food presented for human consumption in different areas of the country. the reported prevalence of t. saginata taeniosis in the human population in ethiopia has been found to range from 31.0% to 89.4% (abunna et al. 2008; bedu et al. 2011; endris & negussie 2011; megersa et al. 2010; tembo 2001). the reported prevalence of bovine cysticercosis in cattle populations across various regions of ethiopia was reported to range between 2.2% to 26.3% (kumar & tadesse 2011). beef production in eastern ethiopia is growing steadily to meet the ever-increasing demand for ‘harar senga’, in other words, beef cattle that provide high quality meat that is consumed raw or in the kurt tradition. in ethiopia there is a deep-rooted culture of consuming raw or inadequately cooked beef (kumar & tadesse 2011; tembo 2001). formulating a strategy for controlling the associated disease prevalence is a first step in the management of disease, but the epidemiological data for t. saginata cysticercosis in ethiopia are inadequate to develop effective recommendations. the objectives of this study were to estimate the prevalence and distribution of cysts in infected cattle and to assess the food safety implications of t. saginata cysticercosis in cattle in harari people’s national regional state, eastern ethiopia. materials and methods top ↑ description of the study area the study was conducted in harar town in harari people’s national regional state, located at 9°18’43”n latitude and 42°7’23”e longitude, 515 km east of addis ababa. the elevation of the town varies from 1600 m to 1920 m above sea level. the regional state has 52 245 cattle, 5631 sheep, 45 256 goats and 53 277 poultry (central statistical agency 2011); the human population consists of 99 321 urban and 84 023 rural residents. the health facilities in harar town comprise four hospitals, 32 clinics, nine pharmacies and 16 drug shops owned by the ministry of health and private entrepreneurs (central statistical agency 2008). harar municipal abattoir the abattoir is owned by the harari people’s national regional administrative office, which aims to provide officially inspected and safe meat (beef, goat meat, mutton and camel meat) for consumers. the abattoir has separate compartments to slaughter animals for christian and muslim residents. on average 90 cattle for christians and 70 cattle for muslims are slaughtered per week. the routine meat inspection service is provided by two animal health assistant veterinarians employed by the animal health department of the regional agricultural office. origin and management of study animals cattle used for this study were local zebu (bos indicus), which originated from the surrounding areas of the east hararghe zone of oromia regional state, specifically the areas fedis, kombolcha, haramaya, kulbi, chelenko, babile and gursum. as in many other parts of ethiopia, these areas use an extensive cattle management system where animals graze freely on available natural pasture, including bushes where some members of the community defecate in the open. animals also often drink water from wells or streams that may be contaminated by effluent from open sewers. study design a cross-sectional study was carried out from october 2009 to september 2010 on cattle slaughtered at harar municipal abattoir. by following a systematic random sampling method, a total of 898 cattle were randomly sampled and routinely inspected for the presence of t. saginata metacestodes in edible parts of the slaughtered cattle. the required sample size of the study was determined as described by thrusfield (2007) with a 95% confidence interval and 5% desired precision. in the absence of published evidence of disease presence in the study area, a value of 50% was assumed; this corresponded to a required minimum sample size of 384. study methodology abattoir survey ante-mortem and post-mortem examinations were conducted by visiting the abattoir three days a week. for the ante-mortem examination cattle were randomly selected by lottery. they were tagged with an identification number and sex, age and origin were recorded. age estimation was done based on owner information and dentition. for the post-mortem examination the heart, liver, lung, tongue, masseter muscle, shoulder muscle, neck muscle, intercostal muscle and diaphragm were inspected for the presence of cysticerci by applying a two-stage meat inspection procedure stipulated by ethiopian ministry of agriculture meat inspection regulations (ministry of agriculture 1972). during the first stage, visualisation and palpation of organs, muscles and carcass were carried out; during the second stage, further incisions were done in each case where one or more cysticerci were found (gracey, collins & huey 1999). cyst distribution and characterisation anatomical distribution of the cysts by organ or muscle affected and their status as active (fluid-filled) or calcified were determined, as described by gracey et al. (1999). all suspected active cysts found during post-mortem examination were removed with the surrounding tissue, enclosed in labelled bottles and taken to the veterinary parasitology laboratory at haramaya university. the viability of the cysts was tested in the laboratory by incubating them at 37 °c in 30% ox bile dissolved in normal saline for about one to two hours. a cyst was regarded as viable if the scolex evaginated during incubation. taenia saginata metacestodes were identified by the cysticercus size, presence of a rostellum and absence of hooks on the rostellum of the evaginated cyst (gracey et al. 1999). questionnaire survey a questionnaire survey was conducted to assess the food safety significance of t. saginata cysticercosis. it relied on the participant’s self-reported experiences to diagnose t. saginata taeniosis. the structured questionnaire comprised questions to obtain background information about the participants as well as 11 multiple choice questions, which had been pre-tested on 50 randomly selected individuals not included in the survey. the questionnaire was designed to obtain information on each participant’s opinion on the status of t. saginata taeniosis; this included their awareness of taeniosis as a meat-borne zoonosis, presence of proglottids in their faeces, in their underwear or on their body during the previous 12 months (between 2008/2009 and 2009/2010), the source and type of meat consumed, whether it was consumed raw or undercooked and if so in which form, namely kurt or raw beef; kitffo, traditional ethiopian food made from raw, finely chopped red lean beef mixed with butter and spices (pepper, sitchini); or dullet, an ethiopian traditional food prepared from raw or undercooked finely chopped offal (kidney, liver, heart, rumen) mixed with spices and butter or oil. three hundred randomly selected volunteer participants from the urban population in harar town from whom consent was obtained were interviewed in one of their own local languages, namely amharic, oromiffa or harari. following detailed discussion about the objectives of the study with each participant, the interview was conducted face-to-face. data handling, management and analysis abattoir and questionnaire data were entered into microsoft excel, coded and analysed using the statistical package spss version 15.0. descriptive statistics were used to summarise the data collected. the prevalence rate of bovine cysticercosis and t. saginata taeniosis was calculated as the number of cattle found positive for at least one cyst (active or calcified) during meat inspection divided by 898 and the number of participants replying that they had noticed proglottids divided by 300, respectively. non-collinear variables with p-value ≤ 0.05 in the univariate logistic regression were used to fit the multivariable logistic regression model. odds ratio was used to assess the associations between different risk factors and prevalence. a confidence level of 95% was used. a p-value ≤ 0.05 was considered statistically significant. results top ↑ prevalence of bovine cysticercosis and associated risk factors as shown in table 1, the abattoir survey showed that 19.7% (95% ci = 17.2–22.5) of the cattle examined were positive for the presence of at least one cyst in the organs or muscles inspected. the prevalence of bovine cysticercosis did not show any significant difference between male and female, different age groups or origin of cattle (see table 2). table 1: prevalence of taenia saginata cysts in cattle slaughtered in harar municipal abattoir. table 2: occurrence of taenia saginata cysts with regard to sex, age and origin of cattle slaughtered. anatomical distribution and characterisation of cysts the liver, shoulder, tongue, heart and masseter harboured at least one cyst. shoulder muscles were the most frequently infected (30.5%), followed by the liver (22.6%) and tongue (18.5%) (see table 3). among the 177 cattle harbouring metacestodes of t. saginata (see table 1), 131 (74.0%) had at least one cyst in a single organ or muscle whereas the remaining 46 (26.0%) had cysticerci in more than one organ or muscle.the number of cysts found in a given organ or muscle ranged from one to nine; the highest number was recorded in the shoulder muscle. among the different parts affected, shoulder muscles, liver and heart together accounted for 65.4%, 66.0% and 65.4% relative prevalence, cyst count and relative viability respectively (see table 3 and table 4). out of 661 cysts examined, 185 (28.0%), 284 (43.0%) and 192 (29.0%) were found to be viable, non-viable and calcified, respectively. the highest relative viability was recorded in the masseter muscle (31.3%), then the liver (30.1%) and shoulder muscles (27.2%) (see table 4). table 3: distribution of cysticerci of taenia saginata in edible parts of infected cattle. table 4: characterisation of cysticerci of taenia saginata collected from edible parts of infected cattle. status of taenia saginata taeniosis as a meat-borne zoonosis in harar town among the 300 people involved in the questionnaire survey, 182 (60.7%) reported t. saginata infection. the positive respondents confirmed that they had experienced t. saginata taeniosis by observing the white segments or proglottids expelled either in their faeces, underwear or on the body at least once in the previous year (between 2008/2009 and 2009/2010). it was found that 65% of respondents, who had sufficient knowledge of the risk of consumption of raw or undercooked meat and on taeniosis as a meat-borne zoonosis, reported t. saginata taeniosis. it was shown that 88% of the people involved in this study purchased beef from butcher shops that provide officially inspected beef for consumption. however, 62% of them acquired t. saginata taeniosis (see table 5). the prevalence of t. saginata taeniosis had a statistically significant association (p < 0.05) with the habit of eating raw or undercooked beef, sex, age, religion, marital status and income of the respondents (see table 6). table 5: participant perception, knowledge, source and habit of beef consumption on taenia saginata taeniosis. table 6: logistic regression analysis of potential risk factors associated with taenia saginata infection in harar town. discussion top ↑ meat inspection revealed that the prevalence of t. saginata cysticercosis in the harari people’s national regional state was about 20.0%. this is higher than previously recorded for kombolcha in north-eastern ethiopia (6.7%) (endris & negussie 2011), jimma in south-western ethiopia (2.9% and 4.4%) (megersa et al. 2010; tolosa et al. 2009), wolita sodo (11.3%) (regassa et al. 2009) and ziway (3.0%) (bedu et al. 2011) in southern ethiopia. the present result was lower than found in hawassa in southern ethiopia (26.3%) (abunna et al. 2008) and similar to that found in north-western ethiopia (18.5%) (kebede 2008). however, large differences can also be found within a region. in two studies conducted in and around addis ababa in central ethiopia, prevalence varied between 7.5% (kebede, tilahun & hailu 2009) and 89.4% (tembo 2001). such differences in prevalence may be associated with the number of cattle examined, the sensitivity of the meat inspection procedures, which can be affected by the site and method of incision, abattoir facilities and management, the motivation and competency of the meat inspectors and the willingness of the owner to cooperate (abunna et al. 2008; dorny & praet 2007; wanzala et al. 2003). the geographical differences in the habit of raw meat consumption, environmental and personal hygiene, animal husbandry practices, proximity to waste water and accessibility of taenicides for treating animals might also contribute to differences in prevalence (cabaret et al. 2002; dorny & praet 2007; kumar & tadesse 2011; wanzala et al. 2003). to obtain the best estimate of the prevalence of t. saginata cysticercosis and increased confidence in the results of this study, the researchers negotiated with butchers so that multiple incisions could be made during inspection; they also performed a detailed examination of all muscles and organs. the post-mortem examination identified that among the edible parts investigated the liver, shoulder, tongue, heart and masseter muscle require careful inspection to identify the cyst. shoulder and masseter muscles (abunna et al. 2008), shoulder, heart and tongue (megersa et al. 2010), and heart, masseter, tongue, triceps and thigh muscles (kebede 2008) previously have been reported as major sites to be inspected. shoulder muscles, liver and heart, which together constituted 65.4%, 66.0% and 65.4% relative prevalence, total cyst count and relative viability of the cysts respectively, are also the main components of the locally prepared traditional dishes of raw or undercooked meat called kurt, kitffo and dullet. the participants in this study indicated their strong preference for these dishes, highlighting their potential for strong links to t. saginata cysticercosis (abunna et al. 2008; endris & negussie 2011; kebede 2008; megersa et al. 2010). to safeguard consumer health, it is therefore essential to increase the area and number of edible sites examined during meat inspection (wanzala et al. 2003). as incising the meat at a large number of different sites to allow a more detailed examination reduces the market value of the carcass, these inspections will require the full support of butchers. the high prevalence (60.7%) of taeniosis recorded in harar town was similar to that found in hawassa (64.2%) (abunna et al. 2008), ziway (56.7%) (bedu et al. 2011) and jimma (56.7%) (megersa et al. 2010). it was higher than the report from kombolcha (31%) (endris & negussie 2011). the difference in prevalence of t. saginata taeniosis in different areas may be associated with the difference in occurrence of cysticercosis in cattle, the level of raw meat consumption and the meat inspection procedures practised. reports have indicated that the prevalence of t. saginata taeniosis may also vary in relation to age, sex, religion, marital status, educational status and income of individual (abunna et al. 2008; bedu et al. 2011; endris & negussie 2011; megersa et al. 2010). previous reports from ethiopia indicated that consumption of raw or inadequately cooked beef was strongly associated with t. saginata infection (abunna et al. 2008; bedu et al. 2011; endris & negussie 2011; megersa et al. 2010). of the 300 respondents interviewed, 54.7% preferred beef and 45.3% goat; 97.0% of the former and only 17.0% of the latter acquired t. saginata taeniosis. even though no respondents preferred mutton or pork, they stated that they may eat mutton on festive or social occasions but never eat pork. of the 62.0% who ate raw or inadequately cooked beef in any of the three forms, more than 97.0% acquired t. saginata taeniosis, confirming that kurt, kitffo and dullet are all sources of viable cysts of the parasite, as found in previous studies (abunna et al. 2008; bedu et al. 2011; endris & negussie 2011; megersa et al. 2010). it is therefore wise to focus strongly on those edible sites of the carcass used for the preparation of these traditional dishes during meat inspection and public education. among the 300 study participants, 265 (88.3%) used meat from butcher shops presumed to provide officially inspected meat, and of the 182 (60.7%) people who acquired t. saginata taeniosis, 164 (90.1%) reportedly purchased their meat from these butcher shops. the high prevalence of t. saginata taeniosis recorded in the survey suggests that meat inspections fail to detect the presence of the cysts and as a result infected meat is commonly passed for human consumption (wanzala et al. 2003). poor working conditions and high workload in the abattoir, as observed by the researchers, might have contributed to this apparent low efficiency of meat inspection. dorny and praet (2007) demonstrated an association between capacity to undertake proper and sensitive meat inspections and motivation of the meat inspectors; thus proper facilities form a crucial part of providing safe food for consumers. the researchers recognise the deep-rooted culture of consumption of raw or undercooked beef in ethiopia. over 90% of respondents were aware of the risk of consumption of raw or undercooked beef; however, 65% of them nevertheless reported being infected with t. saginata taeniosis. this finding is consistent with other reports that showed the overriding influence of culture on the consumption of raw or undercooked beef, irrespective of age, religion, marital status and income of the individuals (abunna et al. 2008; girma et al. 2012; megersa et al. 2010). conclusion top ↑ there was a high prevalence of t. saginata cysts in edible anatomical sites of cattle from harari people’s national regional state in eastern ethiopia. a high prevalence of t. saginata taeniosis was strongly associated with the consumption of raw or inadequately cooked beef. thus t. saginata cysticercosis is an economically important disease in cattle in this region, requiring effective interventions to ensure food safety. the study highlights the need to strengthen meat inspection services through multisectoral collaboration involving the human health, veterinary public health and educational sectors to better control the source and expression of the disease. it is recommended that continuous public education programmes are provided particularly to school children, who might serve as a foundation for bringing about cultural change in the long term that leads to avoidance of raw or inadequately cooked meat consumption. acknowledgments top ↑ the authors would like to thank the haramaya university for the financial support to conduct this study. we also thank the institute of tropical medicine (itm), antwerp, belgium who funded the first author to attend the scientific research paper writing workshop that facilitated the writing of this paper. our sincere appreciation also goes to dr chris beadle of csiro, australia for critically reviewing and editing this article. finally, we are very grateful to the harar municipal abattoir meat inspectors, owners of butcher shops and study participants for their collaboration, enthusiasm and willingness to share their experiences during this study. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions y.t. (haramaya university) designed the study, conducted the questionnaire survey, analysed the data and prepared the manuscript. f.r. (haramaya university) conducted the abattoir survey and the writing of the manuscript, and e.z. 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kyule, n.m., baumann, m.p. et al. 2003, ‘control of taenia saginata by post-mortem examination of carcasses’, african health science 3, 68–76. webb, a.h., 1957, ‘intestinal parasitism in beghemdir province, ethiopia’, journal of the national medical association 49, 310–314. article information authors: christo j. botha1 mariëtte truter2 adriaana jacobs2 affiliations: 1department of paraclinical sciences, university of pretoria, south africa 2biosystematics division, agricultural research council-plant protection research institute, south africa correspondence to: christo botha postal address: private bag x04, onderstepoort 0110, south africa dates: received: 10 april 2014 accepted: 11 july 2014 published: 20 nov. 2014 how to cite this article: botha, c.j., truter, m. & jacobs, a., 2014, ‘fusarium species isolated from pennisetum clandestinum collected during outbreaks of kikuyu poisoning in cattle in south africa’, onderstepoort journal of veterinary research 81(1), art. #803, 8 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.803 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. fusarium species isolated from pennisetum clandestinum collected during outbreaks of kikuyu poisoning in cattle in south africa in this research communication... open access • abstract • introduction • methods    • isolations    • dna extraction and amplification    • dna sequencing and sequence comparisons • results    • isolations    • dna extraction and amplification    • dna sequencing and sequence comparisons • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ kikuyu poisoning occurs sporadically in south africa. it is of major economic importance, as valuable dairy cows are often poisoned by it, and once affected, the mortality rate is high. pennisetum clandestinum samples were collected during eight outbreaks of kikuyu poisoning in cattle in the eastern cape province of south africa from 2008 to 2010. the kikuyu grass samples were submitted specifically for the isolation and molecular identification of fusarium species, as it was recently suggested that mycotoxins synthesised by fusarium torulosum could be the cause of this intoxication. ninety-four fusarium isolates were retrieved from the grass samples, of which 72 were members of the fusarium incarnatum/fusarium equiseti species complex based on morphology and phylogenetic analyses of the translation elongation factor 1α sequence data. the south african isolates from kikuyu identified as members of the f. incarnatum/f. equiseti species complex grouped together in six separate clades. the other isolates were fusarium culmorum (n = 3), fusarium redolens (n = 4) and fusarium oxysporum (n = 15). although f. torulosum could not be isolated from p. clandestinum collected during kikuyu poisoning outbreaks in south africa, the mycotoxicosis theory is still highly plausible. introduction top ↑ kikuyu poisoning, an intoxication of ruminants, is described as a ruminitis syndrome resulting in, among other manifestations, forestomach necrosis and dehydration (bourke 2007; kellerman et al. 2005). although kikuyu grass (pennisetum clandestinum hochst. ex chiov.) is usually grazed without deleterious effects, it sporadically becomes toxic (bourke 2007; kellerman et al. 2005). this usually happens when the kikuyu grass pasture has been subjected to severe stress in some form (like drought or invasion by army worm caterpillars) followed by a growth spurt during warm weather (bryson 1982; kellerman et al. 2005; newsholme et al. 1983). poisoning has been reported from new zealand, australia and eastern and southern africa (bourke 2007; bryson 1982; martinovich, mortimer & di menna 1972). although kikuyu poisoning occurs periodically in south africa, it is of major economic importance, as valuable dairy cows are often poisoned and, once affected, the mortality rate is high (bryson 1982). after moving ruminants, especially cattle, to kikuyu pastures there is usually a latent period of 24 h or longer before clinical signs, mainly gastrointestinal and neuromuscular, are observed. gastrointestinal signs are usually present; these include ruminal atony, distension and colic (kicking at the abdomen and grunting). ruminal irritation and malabsorption result in the accumulation of excessive fluid in the rumen, leading to sloppy ruminal contents, which may even gush from the mouth and nose at death. severe dehydration with sunken eyes and an unpliable skin is observed. the neuromuscular signs are distinguished by bulbar paralysis, resulting in an inability to swallow that is characterised by ‘sham-drinking’ and salivation. tremors and incoordination have also been described (bryson 1982; kellerman et al. 2005; newsholme et al. 1983). at necropsy, excessive and sloppy, watery ruminal contents (often bright green) are frequently observed. hyperaemia of the submucosa and necrosis and ulceration of the forestomach mucosa may be seen macroscopically. severe dehydration is also noticed. on microscopic examination, superficial epithelial necrosis of the rumen, reticulum and omasum are present. characteristically there is detachment or absence of the stratum corneum, the necrosis only involves the stratum spinosum and stratum granulosum, but with preservation of the stratum basale (newsholme et al. 1983). the possibility that kikuyu poisoning is a mycotoxicosis has been mooted in the scientific literature since the first reported outbreaks (martinovich et al.1972). although various fungi that are potentially toxic to animals (such as myrothecium spp.) have been cultured from kikuyu grass collected during outbreaks, they were not consistently present and there was little evidence of heavy fungal infestation of the pastures (bourke 2007; kellerman et al. 2005; newsholme et al. 1983). thus, the mycotoxicosis theory was placed on hold. however, more recently, australian researchers reported that an endophytic fungus, fusarium torulosum, was consistently isolated from kikuyu grass collected during an outbreak (bourke 2007; ryley et al. 2007). during the summers and late summers of 2008–2010 various reports were received of large numbers of cattle dying as a result of kikuyu poisoning in the eastern cape province of south africa (f. van niekerk, humansdorp, personal comm., 2009). sods of kikuyu grass (roots and base of plant) were collected during eight outbreaks; in five outbreaks, the clinical diagnosis was confirmed by the characteristic histopathology of lesions in the forestomach. the kikuyu grass was submitted particularly for the isolation and molecular identification of fusarium species. the specific objective was to verify if f. torulosum was also consistently present in kikuyu grass during outbreaks of intoxication in south africa. if this endophytic fungus is always present in poisonous kikuyu grass on two different continents, it could direct future research to identify an aetiological agent. methods top ↑ isolations from 2008 to 2010, sods of kikuyu grass were collected from various localities in the eastern cape province during eight outbreaks of kikuyu poisoning in cattle. isolations were made from leaf and stem material. small pieces (4 mm) of plant tissue were surface disinfected with 1% sodium hypochlorite, rinsed twice with sterile water, blotted dry and plated onto fusarium selective medium (20.0 g agar, 15.0 g peptone, 1.0 g kh2po4, 0.5 g mgso4.7h2o, 1 g pcnb, 20 ml streptomycin sulphate in 1 l water) in petri dishes (nelson, tousson & marasas 1983). petri dishes were incubated for 7–10 days at 25 °c under cool-white fluorescent light. the plates were checked regularly and all colonies with typical fusarium morphology were transferred to half-strength potato dextrose agar (pda) (merck, germany). single-spore isolates were preserved and retained in the culture collection of the national collections of fungi, biosystematics division, plant protection research institute, agricultural research council (arc-ppri), pretoria, south africa. all the single-spored isolates from p. clandestinum were used in the morphological comparison. isolates were grown on pda and synthetic low-nutrient agar (sna) (nirenberg 1976). colony colour and morphology were compared with that stipulated by leslie and summerell (2006). morphological characters were described from structures produced on sna, except the macroconidia morphology. ten measurements per isolate of the macroconidia and microconidia were also taken and the averages computed. dna extraction and amplification isolates were grown on half-strength pda at 25 °c for 7 days. dna was isolated using the dneasy plant mini extraction kit (qiagen, valencia, ca) and following the manufacturer’s protocol after the mycelium was placed in eppendorf tubes and ground with approximately 10 µg sterile, chemically treated sand. extracted dna was used as the template in polymerase chain reactions (pcr). part of the translation elongation factor (tef) 1-α gene was amplified using the primer set ef1 (5’-cgaatctttgaacgcacattg-3’) and ef2 (5’-ccgtgtttcaagacggg-3’) (o’donnell, cigelnik & nirenberg 1998). the pcr consisted of 1 x supertherm taq reaction buffer with mgcl2, dntps (250 µm each), primers (0.2 µm each), template dna (25 ng) and supertherm taq polymerase (0.5 u) (southern cross, south africa). the pcr conditions for the tef gene region were amplified by initial denaturation at 94 °c for 2 min. this was followed by 30 cycles of denaturation at 94 °c for 1 min, annealing at 52 °c for 1 min and elongation at 72 °c for 1 min, with a final elongation step at 72 °c for 5 min (jacobs et al. 2010). the resulting pcr amplicons were visualised on a 1% agarose gel under ultraviolet (uv) light and purified using a qiaquick pcr purification kit (qiagen, hilden, germany). dna sequencing and sequence comparisons dna sequences were determined from pcr amplicons using the abi prismtm dye terminator cycle sequencing ready reaction kit with amplitaq dna polymerase (applied biosystems, warrington, united kingdom) using the primers ef1 and ef2. sequences generated in the present study have been deposited in genbank (table 1). table 1: fusarium isolates obtained from pennisetum clandestinum collected during kikuyu poisoning outbreaks in the eastern cape province of south africa. table 1 (continues): fusarium isolates obtained from pennisetum clandestinum collected during kikuyu poisoning outbreaks in the eastern cape province of south africa. the partial sequence data for tef were compared against both the fusarium multilocus sequence typing (mlst) database and the fusarium database (geiser et al. 2004). complete dataset and alignments for previously published phylogenetic relationships within the fusarium incarnatum/fusarium equiseti species complex (fiesc) were used (o’donnell et al. 2009). gaps were treated as missing data in the subsequent analysis. phylogenetic analysis was based on parsimony using paup 4.0* (swofford 2002). heuristic searches were done with random addition of sequences (100 replicates), tree bisection-reconnection (tbr) branch swapping, and mulpar effective and maxtrees set to auto-increase. phylogenetic signal in the data sets (g1) was assessed by evaluating tree length distributions over 100 randomly generated trees (hillis & huelsenbeck 1992). the consistency (ci) and retention (ri) indices were determined for the tef data set. phylogenetic trees were rooted with fusarium concolor as the monophyletic sister outgroup to the rest of the taxa. bootstrap analyses were performed to determine branching point confidence intervals (1000 replicates) for the most parsimonious trees generated for the tef data set (also see jacobs & van heerden 2012). results top ↑ isolations ninety-four fusarium isolates were retrieved from grass samples collected from camps with reported kikuyu poisoning during eight outbreaks from 2008 to 2010. the fusarium isolates consisted of fusarium culmorum (n = 3), fusarium redolens (n = 4), fusarium oxysporum (n = 15) and fusarium species within the fiesc (n = 72) based on morphology (table 1). the f. equiseti isolates were characterised by the absence of microconidia and macroconidia with elongated apical cells. chlamydospores were present in all isolates. the colony colour ranged from white to brownish with age. isolates identified as f. incarnatum were characterised by morphology typical of fusarium semitectum. these included macroconidia with a curved apical cell and pyriform to obovate microcondia borne on monophialides and polyphialides. no chlamydospores were observed in these cultures. the f. oxysporum isolates were characterised by purple colonies, with chlamydospores forming after 4 weeks. the macroconidia were straight to slightly curved, whilst the microconidia were oval to elliptical, borne on short monophialides. fusarium redolens isolates formed oval to cylindrical microconidia on monophialides, whilst the macroconidia were thick-walled with hooked apical cells. isolates of f. culmorum were characterised by the absence of microconidia and robust, short, thick-walled macroconidia. dna extraction and amplification amplicons of the tef gene region were 640 bp in size. dna sequencing and sequence comparisons parsimony analysis of the tef gene region was performed to determine the phylogenetic placement of the kikuyu isolates within the fiesc. alignment of the tef data set by inserting gaps resulted in a total of 587 characters used in the comparison of the different species. all parsimony-uninformative and constant characters were excluded, resulting in 164 parsimony-informative characters. heuristic searches on the data set generated one most parsimonious tree. in the tef data set (figure 1), the south african isolates from kikuyu identified as members of the fiesc grouped together in six separate clades (a–f), of which four were supported by high bootstrap values (> 85%). the kikuyu isolates clustered with five of the 28 phylogenetic lineages in the fiesc, namely fiesc mlst 5b, 5e, 6a, 10a and 12a (o’donnell et al. 2009). isolates from these mlsts originated from cereals, human and mammalian samples. two unique clusters not associated with any of the included phylogenetic lineages in the fiesc were also observed amongst the south african isolates from kikuyu. based on the basic local alignment search tool (blast) results (table 2) from the two selected databases, the south african isolates clustered with seven of the 28 phylogenetic lineages in the fiesc, namely: fiesc mlst 1a, 5c, 5f, 6a, 10a, 12a and 22a (o’donnell et al. 2009); again, these samples are associated with cereal, human and mammalian origin. the analyses have fiesc mlst 6a, 10a and 12a in common. figure 1: phylogenetic tree produced using parsimony of the translation elongation factor gene with fusarium concolor as outgroup. table 2: molecular identification of fusarium isolates based on translation elongation factor sequence data. table 2 (continues): molecular identification of fusarium isolates based on translation elongation factor sequence data. discussion top ↑ contrary to the findings in australia, f. torulosum was not isolated from the p. clandestinum collected during eight outbreaks of kikuyu poisoning in south africa. nonetheless, a number of other fusarium species were isolated from kikuyu grass collected from toxic pastures during 2008 to 2010. the genus fusarium is recognised for the taxonomic difficulties associated with it and, based on the classification system used, the total number of species in this genus has ranged from 9–1000 (summerell & leslie 2011). fusarium isolates from toxic kikuyu grass pastures in south africa mainly represent species in the fiesc. the dna sequence comparisons based on the tef gene formed the most important basis for distinguishing these fusarium species. the tef 1 α gene was also the most informative of the four gene regions used by o’donnell et al. (2009) to distinguish the 28 phylogenetic species in the fiesc. the grouping of the south african isolates from p. clandestinum with five of these species, based on sequence data for the tef gene, confirms that the majority of the fusarium isolates obtained from p. clandestinum associated with kikuyu poisoning in cattle form part of the fiesc. historically, the genus fusarium has been known as a plant pathogen, but since the 1960s has also been reported to be associated with secondary metabolites responsible for fusarium-related mycotoxicoses in humans and animals (summerell & leslie 2011). in many of the fusarium-associated intoxications, the mycotoxin(s) are unknown but the toxins of veterinary importance synthesised by some of the strains include fumonisins, zearalenone and trichothecenes such as diacetoxyscirpenol, deoxynivalenol and t2-toxin (desjardins 2006; kellerman et al. 2005; marasas, nelson & tousson 1984). other lesser-known fusarium mycotoxins include beauvericin, enniatins and fusarochromanone (altomare et al. 1995; bryden et al. 2004; logrieco et al. 1998). the mycotoxicosis theory is thus still very plausible and is supported by the periodic occurrence of kikuyu poisoning usually during warm, humid weather when fungal growth in a pasture is likely to be optimal (martinovich et al.1972). fusarium torulosum is known to produce mycotoxins such as wortmannin and butenolide (bourke 2007; ryley et al. 2007). oral administration of wortmannin to rats is toxic at a dose as low as 4 mg/kg, causing gastric and myocardial haemorrhage (gunther, abbas & mirocha 1989). butenolide is reported to induce acute inflammation in the forestomachs of cattle (tookey & grove 1972). in addition, brazilian researchers described a ruminitis syndrome very similar to kikuyu poisoning (riet-correa et al. 2009). unlike kikuyu poisoning, this intoxication is caused by members of the asteraceae family, baccharis coridifolia and baccharis megapotamica, but the toxins in the plants are a range of trichothecenes synthesised by soil fungi (myrothecium spp.) and absorbed by the roots of the plant (riet-correa et al. 2009). if kikuyu poisoning is a mycotoxicosis, assays could be developed to be able to forecast risk and to warn farmers that intoxication may occur. conclusion top ↑ although f. torulosum could not be isolated from p. clandestinum when collected during eight outbreaks of kikuyu poisoning in south africa, various other fusarium isolates (n = 94) were retrieved from the toxic kikuyu grass. these isolates were predominantly members of the fiesc; however, the possibility that kikuyu poisoning is a mycotoxicosis should be further investigated and toxic grass samples could be subjected to analytical screening for fungal metabolites. acknowledgements top ↑ the authors would like to thank the private veterinary practitioners who collected and submitted the kikuyu grass samples, and dr kerry o’donnell for his willingness to make the original dataset available and for valuable advice. this work is based upon research supported by the national research foundation of south africa. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.j.b. (university of pretoria) was the principal investigator and study coordinator. m.t. (agricultural research council-plant protection research institute) and a.j. (agricultural research council-plant protection research institute) performed the mycological culturing and identification. a.j. was responsible for the phylogenetic analyses. all authors compiled, read and approved the final manuscript. references top ↑ altomare, c., logrieco, a., bottalico, a., mulé, g., moretti, a. & evidente, a., 1995, ‘production of type a trichothecenes and enniatin b by fusarium sambucinum fuckel sensu lato’, mycopathologia 129, 177–181. http://dx.doi.org/10.1007/bf01103344 bourke, c.a., 2007, ‘a review of kikuyu grass (pennisetum clandestinum) poisoning in cattle’, australian veterinary journal 85, 261–267. http://dx.doi.org/10.1111/j.1751-0813.2007.00168.x bryden, w.l., lowe, m., amba, t.m. & abbas, h.k., 2004, ‘fusarochromanone and wortmannin: novel fusarium toxins’, in t. acamovic, c.s. stewart & t.w. pennycott (eds.), poisonous plants and related toxins: 6th international symposium on poisonous plants proceedings, glasgow, scotland, 2001, pp. 63–69, cabi 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disease notes 2, 133–135. http://dx.doi.org/10.1071/dn07053 summerell, b.a. & leslie, j.f., 2011, ‘fifty years of fusarium: how could nine species have ever been enough?’, fungal diversity 50, 135–144. http://dx.doi.org/10.1007/s13225-011-0132-y swofford, d.l., 2002, paup*: phylogenetic analysis using parsimony (*and other methods), version 4, sinauer associates, sunderland. tookey, h.l. & grove, m.d., 1972, ‘toxic effects of a butenolide mycotoxin and of fusarium tricinctum cultures in cattle’, journal of the american veterinary medical association 160, 1522–1526. article information authors: j.j. muyembe-tamfum1,2 s. mulangu3,4 justin masumu1,3 j.m. kayembe2 a. kemp5 janusz t. paweska5 affiliations: 1institut national de recherche biomédicale, kinshasa i, democratic republic of the congo 2université de kinshasa, kinshasa xi, democratic republic of the congo 3southern african centre for infectious disease surveillance, chuo kiikuu, tanzania 4national institute of allergy and infectious diseases, national institutes of health, united states 5special pathogens unit, national institute for communicable diseases of the national health laboratory service, south africa correspondence to: justin masumu postal address: po box 3297, chuo kikuu morogoro, tanzania how to cite this proceeding: muyembe-tamfum, j.j., mulangu, s., masumu, j., kayembe, j.m., kemp, a. & paweska, j.t., 2012, ‘ebola virus outbreaks in africa: past and present’, onderstepoort journal of veterinary research 79(2), art. #451, 8 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.451 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ebola virus outbreaks in africa: past and present in this proceeding... open access • abstract • introduction • documented human and non-human primate outbreaks in africa • etiology • transmission • ecology • clinical features • diagnosis • treatment • control measures • conclusion • references abstract top ↑ ebola haemorrhagic fever (ehf) is a zoonosis affecting both human and non-human primates (nhp). outbreaks in africa occur mainly in the congo and nile basins. the first outbreaks of ehf occurred nearly simultaneously in 1976 in the democratic republic of the congo (drc, former zaire) and sudan with very high case fatality rates of 88% and 53%, respectively. the two outbreaks were caused by two distinct species of ebola virus named zaire ebolavirus (zebov) and sudan ebolavirus (sebov). the source of transmission remains unknown. after a long period of silence (1980–1993), ehf outbreaks in africa caused by the two species erupted with increased frequency and new species were discovered, namely côte d’ivoire ebolavirus (ciebov) in 1994 in the ivory coast and bundibugyo ebolavirus (bebov) in 2007 in uganda. the re-emergence of ehf outbreaks in gabon and republic of the congo were concomitant with an increase in mortality amongst gorillas and chimpanzees infected with zebov. the human outbreaks were related to multiple, unrelated index cases who had contact with dead gorillas or chimpanzees. however, in areas where nhp were rare or absent, as in kikwit (drc) in 1995, mweka (drc) in 2007, gulu (uganda) in 2000 and yambio (sudan) in 2004, the hunting and eating of fruit bats may have resulted in the primary transmission of ebola virus to humans. human-to-human transmission is associated with direct contact with body fluids or tissues from an infected subject or contaminated objects. despite several, often heroic field studies, the epidemiology and ecology of ebola virus, including identification of its natural reservoir hosts, remains a formidable challenge for public health and scientific communities. introduction top ↑ the first southern african centre for infectious disease surveillance (sacids) conference on ‘one africa, one health’ served as inspiration for this review to illustrate the concept through a typical emerging infection. ebola haemorrhagic fever (ehf) is caused by any of five genetically distinct members of the filoviridae family: zaire ebolavirus (zebov), sudan ebolavirus (sebov), côte d’ivoire ebolavirus (cebov), bundibugyo ebolavirus (bebov) and reston ebolavirus (rebov). côte d’ivoire ebolavirus has been associated with only one human case (le guenno et al. 1995). reston ebolavirus has only caused disease in non-human primates (nhp) and was found in swine suffering from porcine reproductive and respiratory disease syndrome (barrette et al. 2009). zaire, sudan and bundibugyo ebola viruses are responsible for most of the ehf outbreaks (feldmann et al. 2005; groseth, feldmann & strong 2007; towner et al. 2008) but zebov constitutes a particularly serious threat to both human and nhps in sub-saharan africa. ebola haemorrhagic fever has been associated with large human outbreaks, with case fatality rates for zebov as high as 90%. the case fatality rate of ebov in nhp is unknown but some ecological data suggest that ebov has contributed to declines of up to 98% of local great ape populations in gabon and the republic of congo (walsh et al. 2003).currently there are no approved antiviral drugs or vaccines against filoviruses. the prevention of ehf requires improving our understanding of the epidemiology of the disease, especially the role of wildlife, including bats, in the transmission of ebola virus to humans. in their exhaustive review on ebola virus, feldmann and geisbert (2011) tackled different fundamental aspects of ehf outbreaks. leroy, gonzalez and baize (2011) reviewed the major scientific advances in our understanding of the ecology, host interactions, and control of the ebola and marburg viruses. in the present review we report important features related to ebola outbreaks in africa based on previous findings and own observations during major outbreaks that occurred on the continent. documented human and non-human primate outbreaks in africa top ↑ ebola viruses constitute a serious threat to both human and wildlife health in the congo and nile basins. the first documented outbreaks were generally regarded as causing a mysterious disease, so dramatic in its effect that it inspired novelists and film producers. in most of the cases, the disease has appeared suddenly out of the elusive natural environment and dissipated slowly during the outbreak. the first outbreaks of ehf occurred almost simultaneously in 1976 in southern sudan (june) and northwestern zaire (now democratic republic of the congo, drc) (september). initially it was thought that the drc outbreak was due to dissemination of the sudan outbreak but, in fact, the outbreaks were caused by two antigenically and biologically distinct species named sebov and zebov. the index case in sudan was a worker in a cotton factory in nzara who subsequently was the source of nosocomial transmission in maridi hospital. the mortality rate amongst the 284 notified cases was 53% (who 1978a). the index case in the zaire or drc outbreak was a 44 year-old male instructor at the yambuku catholic mission school who fell ill after extensive travels in northern equateur province. he bought fresh and smoked antelope and monkey meat on his way back to yambuku. he was treated for presumptive malaria at the yambuku hospital, where the outbreak emerged subsequently. in total 318 cases were recorded, with a case fatality rate of 88%. close contact with an acute ebola case and receiving an injection with a reused, unsterilised syringe at the hospital were the major risk factors for virus transmission in humans (who 1978b). in 1979, nzara and maridi in sudan were again hit by a small outbreak, with 34 cases and 22 deaths (baron, mccormick & zubeir 1983), whereas a single case was described in a child at tandala hospital in drc (heymann et al. 1980). apparently, none of the cases had contact with wild animals. after an absence from 1980 to 1993, several independent foci of ebola virus transmission were recorded. most of them were caused by zebov and sebov but some were caused by the newly discovered species, namely ciebov and bebov: • ebola haemorrhagic fever in côte d’ivoire (1994): a large ebola virus outbreak occurred amongst chimpanzees living in the taï national park in côte d’ivoire. an ethologist was infected whilst performing an autopsy on a dead chimpanzee. the patient was treated as a presumptive malaria case in abidjan hospital, without success. there were no secondary cases. this was the first documented outbreak of ebola virus amongst nhp in nature and the first in west africa. the outbreak led to the discovery of a new species of ebola virus, namely ciebov (le guenno et al. 1995). • ebola haemorrhagic fever in gabon (1994, 1996, 1997 and 2001–2002): several viral haemorrhagic fever outbreaks, caused by zebov, were associated with the hunting of nhp. the 1994 outbreak involved gold-diggers in the minkebé forest who had killed a sick gorilla for food; the illness was initially confused with yellow fever (amblard et al. 1997; georges-courbot et al. 1997a, 1997b; leroy et al. 2002). • ebola haemorrhagic fever in the republic of the congo (2001–2002, 2003 and 2005): the first recorded outbreak of ebola occurred in 2001–2002. in 2003, zebov re-emerged, affecting 143 individuals in mbomo (17 cases) and kellé (126 cases), and 128 deaths were recorded (formenty et al. 2006). three independent index cases were identified in relation to hunting episodes and contact with gorillas. during this outbreak, intra-familial transmission was more important than nosocomial transmission. however, three health care workers were infected. in the same year, another small ebola outbreak, involving 35 cases and 29 deaths, had occurred. the last documented ebola outbreak in the country was reported in 2005, with 11 cases and nine deaths (nkoghe et al. 2011). • ebola haemorrhagic fever in the drc (1995): zebov reemerged in 1995 in the city of kikwit, with 400 000 inhabitants, 1000 km south of the location of the 1976 outbreak. in total, 315 cases and 250 deaths were recorded. a 31-year-old female ebola patient traveled during the early stage of her disease to kinshasa, where she was isolated in a private clinic. no secondary transmission occurred (khan et al. 1999). the main occupation of the index case was farming and preparing charcoal in one of the remnant forest areas near kikwit but the exact cause of infection or exposure is not known. there were no great apes but a lot of bats and rodents were present in the region. the risk factors for secondary human-to-human infection were mainly working in kikwit general hospital or preparing corpses for burial. almost 20% of the 250 victims were health care workers (guimard et al. 1999). • ebola haemorrhagic fever in the drc (2007–2008, 2008–2009): a further outbreak occurred in 2007, in the mweka health zone, west kasai province, involving 264 cases and 187 deaths with a case fatality rate (cfr) of 71%. kampungu city was the epicenter of the outbreak with 47% of cases, followed by the city of kaluamba (42% of cases). the index case was the chief of the village and a hunter. the outbreak was apparently associated with a massive fruit bat migration through this region (leroy et al. 2007). during this outbreak, the fatalities amongst health care workers were fewer. however, several human-to-human transmissions had occurred in churches where patients had been taken for prayers and nursing (doctors without borders, unpublished data). • in the 2008 ebola outbreak, kaluamba was affected again, with 32 cases and 14 deaths (cfr of 43.8%). the index case was believed to be an 18 year old girl who had died from a post-abortion haemorrhage. however, the source of her exposure remains unknown. this outbreak was reported to the national and provincial health authorities 21 days after the disease onset, compared to a period of four months in the 2007 epidemic. the observed low cfr in kaluamba outbreak is considered to be due to the early recognition of the disease and the prompt response of the national team. • ebola haemorrhagic fever in uganda (2000, 2007, 2011): an outbreak of sebov occurred in gulu in 2000 and spread to the cities of mbarara and masindi, with a total of 425 cases and 224 deaths (cfr of 52%) (lamunu et al. 2004). this was the largest epidemic caused by sebov. the outbreak was recognised from a cluster of human cases and was amplified by nosocomial transmission. uganda was again affected in 2007 when a new ebola species, bebov, killed 30 people out of 116 cases (cfr of 26%) (towner et al. 2008). an isolated case of ehf caused by sebov was reported from uganda in 2011 (who 2011). • ebola haemorrhagic fever in sudan 2004: the epicenter of this small outbreak of 17 cases and seven deaths (cfr of 41.2%) was the town of yambio, near to the two previous ebola sites (nzara and maridi) (onyango et al. 2007). the outbreak started with the admission of a 27-year-old man to yambio hospital with fever and haemorrhagic manifestations. the onset of symptoms started on 15 april 2004. the outbreak was contained rapidly with the establishment of infection control measures, thanks to the early recognition and confirmation of the outbreak by the kenya medical research institute (kemri). etiology top ↑ ebola virus belongs to the family filoviridae, in the order mononegavirales which includes rhabdoviridae and paramyxoviridae. the virion is pleomorphic, producing ‘u’-shaped, ‘6’-shaped, or circular forms but the predominant forms of the virion most frequently seen by electron microscope are long tubular structures. it contains one molecule of linear, single-stranded, negative-sense rna of 4.2x106 da. the virus was first recognised in 1976 when two unrelated ehf outbreaks occurred 800 km apart in northern zaire (yambuku) and southern sudan (nzara or maridi) (who 1978a, 1978b). it was given the name ‘ebola’ after the small river near the catholic mission of yambuku, the epicenter of the 1976 ehf outbreak. ebola virus is not restricted to africa. a new species, rebov, was described in cynomolgus monkeys (macaca fascicularis) imported from the philippines (manila) to a quarantine facility in reston, usa in 1989. subsequently, rebov has been re-isolated from cynomolgus monkeys and domestic pigs in the philippines (barrette et al. 2009). transmission top ↑ in most outbreaks, ebola virus is introduced into human populations via the handling of infected animal carcasses. in these cases, the first source of transmission is an animal found dead or hunted in the forest, followed by person-to-person transmission from index case to family members or health-care staff. animal-to-human transmission occurs when people come into contact with tissues and bodily fluids of infected animals, especially with infected nonhuman primates (leroy et al. 2004). transmission has been reported in côte d’ivoire where an ethologist was infected through handling an infected, dead chimpanzee in the taï forest (le guenno et al. 1995). it was confirmed that the deaths of chimpanzees were indeed due to ebola virus. in gabon and the republic of the congo, outbreaks in humans were associated with extensive deaths of chimpanzees and gorillas (rouquet et al. 2005). in contrast, the animal source of infection during the drc, uganda and sudan outbreaks has never been detected. however, when analysing the risk factors for primary transmission of ebov from a broad anthropological point of view, it is noticeable that the increase in ebola outbreak since 1994 is frequently associated with drastic changes in forest ecosystems in tropical africa. the perturbation of these ecosystems due to extensive deforestation and human activities in the depth of the forests may have promoted direct or indirect contact between humans and a natural reservoir of the virus. ebov infection has therefore been related to human economic activities like hunting (young hunters infected by a chimpanzee in the forest near mayibout, gabon in 1996), farming (the charcoal maker in the forest near kikwit, drc in 1995) and gold digging (in minkebé forest, gabon in 1994). in some cases, as in mweka (drc), ehf outbreaks seemed to be linked to the hunting of bats for bush meat (leroy et al. 2009). rarely, scientific activities have resulted in primary ebov infection, for example, in the case of the ethologist who was involved in wildlife studies in the taï forest in 1994. these examples show clearly that certain economic activities, which many populations depend on for their survival, are risk factors for ebov infection. this finding should be taken into account when public health measures need to be implemented in ebov-endemic areas. regarding human-to-human viral transmission, infection occurs in community and hospital settings through direct contact with infected fluids (blood, secretions and excretions) or tissues of an acute patient or through direct contact with contaminated materials. any clustering of deaths in the same family pointed to ehf during the larger outbreaks. in the community setting, new infections were related to the ministration of funeral rites, which involve ritual cleansing of the cadaver and removal of hair, finger nails, toe nails and clothing before burial. people visiting or taking care of infected persons in their homes or in hospitals also risk being exposed to ebola infections. however, we have observed major differences in ebov transmission cycles between community-based outbreaks and hospital-based outbreaks. when ebola virus is introduced into a village, the outbreak seems to end spontaneously with limited generations of cases. as shown for the drc epidemics in table 1, ebola virus was introduced into 55 villages around yambuku (1976) and 25 villages around kikwit (1995). the majority of the affected villages reported less than ten cases each. similarly, the chain of transmission of ehf in the village of ekata in gabon was very short after the exposure of four brothers to dead animals in 2001. in contrast, a hospital setting with low standards of hygiene and sanitation rapidly becomes a source of epidemic amplification, especially if barrier-nursing techniques and universal hygiene measures are not observed by health workers. as a consequence, these nosocomial outbreaks are characterised by a relatively high proportion of deaths amongst health care workers. ironically, it was the excessive fatalities amongst health care workers in kikwit hospital in 1995 that brought the outbreak to the attention of the public health authorities. from this observation, it is possible that ehf infections are not as rare as generally thought. isolated cases may frequently happen in the community without being reported. in support of this hypothesis, several epidemiological sero-surveys reported high prevalence of ebola antibodies in communities in the absence of reports of ebola outbreaks (becquart et al. 2010; busico et al. 1999; gonzalez et al. 2000). because igg antibodies are known to cross-react amongst ebola species (macneil, reed & rollin 2011), this high seroprevalence may be the outcome of exposure to yet unknown, less pathogenic or non-pathogenic variants of ebola virus. table 1: distribution of ebola cases in the affected villages in democratic republic of the congo. sexual transmission has been suggested in humans since filoviruses can be found in semen (bausch et al. 2007). aerosol infection is questioned since people sharing the same space with infected persons do not contract the infection even though aerosol infection of nhp has been demonstrated in the laboratory (leffel & reed 2004). ecology top ↑ tropical rain forests in africa constitute a common ecosystem for ebola virus emergence (i.e. the western congo swamp forests near yambuku, taï forest in côte d’ivoire and minkebé forest in gabon), providing rich animal biodiversity, and epidemics appear to be seasonal. documented human and non-human ehf outbreaks occurred mainly during wet seasons, marked by fruit abundance. the index case of the 1995 ehf outbreak in kikwit fell ill in january and the 1994 ehf outbreak amongst chimpanzees in the tai forest occurred in november, at the end of the wet season. the natural reservoir of infection remains unknown, but the virus clearly has a zoonotic origin. in some outbreaks where information is available, the human index cases have invariably had direct contact with gorillas, chimpanzees, antelope or bats. the search for a reservoir of ebov has been very aggressive. although great apes are generally involved in ehf outbreaks, nhps are not thought to be natural reservoirs but, rather, susceptible hosts (table 2) based on the sudden sharp decline in populations of the great apes in gabon and the republic of congo which coincided with ebov outbreaks in humans (pourrut et al. 2005). several other animal and plant species have been investigated for susceptibility and to determine a natural reservoir of ebov. during the 1976–1979 ehf outbreaks, several ecological studies were conducted in order to identify the reservoir of the virus in nature. studies were conducted on plants eaten by guinea pigs (tandala, drc), on monkeys (yambuku, drc) and on bats (nzara, sudan) but without success. further ecological investigations using modern technologies, were carried out during the subsequent episodes of ehf outbreaks (1994–2010) especially in kikwit, where thousands of rodents, insects and birds were screened. these investigations have not been successful for various reasons, one being that they are usually implemented retrospectively, several weeks or months after the index case has been infected by a putative reservoir. it is possible that by that time the putative reservoirs may have moved to another site. a surveillance system capable of early detection of ebola cases could allow animal reservoir studies in ‘real time’, which is not always easy in remote places in african forests. table 2: documented outbreaks of ebola virus amongst non-human primates and swine (1980–2005) in africa. experimental studies provide a more convenient, alternative method to identify candidate animal reservoirs and need not rely on an actual outbreak. studies on 33 varieties of 24 species of plants and on 19 species of vertebrates and invertebrates experimentally infected with ebola virus gave the first evidence that both insectivorous and frugivorous bats can support the replication and circulation of ebov (swanepoel et al. 1996). this evidence along with reports of bat exposures for some of the ebola index cases directed the research toward the bats as potential reservoirs. indeed, an ecological survey revealed the presence of zebov-specific antibodies in six bat species caught in the field (epomops franqueti, hypsignathus monstrosus, myonycteris torquata, micropteropus pusillus, mops condylurus and rousettus aegyptiacus) (pourrut et al. 2005). viral nucleic acid sequences of ebola virus was also found in three species of fruit bat during the 2001–2003 outbreaks in gabon and republic of the congo (leroy et al. 2005). these studies were pre-dated by the ecological investigation of the 1998–2000 marburg heamorrhagic fever outbreak in durba village in northeastern drc, which consisted of repeated occurrences of short transmission chains arising in workers in goroumbwa mine where large numbers of bats roosted (swanepoel et al. 2007). a hypothesis of recurrent introductions of infection into humans from a natural source was supported by the finding that multiple genetic lineages of virus circulated during the outbreak. diverse genetic lineages of marburg virus were detected in egyptian fruit bats, r. aegyptiacus, and two species of insectivorous bat in the mine (rhinolophus eloquens, the eloquent horse-shoe bat, and miniopterus inflatus, the greater long-fingered bat) and, furthermore, these lineages corresponded to the ones isolated from the humans in durba. due to the complexity of laboratory testing of specimens collected from animals potentially infected with filoviruses and the necessity to conduct the testing under biosafety level four (bsl-4) conditions, the results of the study were only published seven years later but in the meantime had been presented at numerous international scientific meetings and significantly assisted in focusing filovirus ecology studies on bats as putative reservoir species.in this context, an ebola ecology expedition was organised in may 2011 (figures 1 and figure 2) to undertake a preliminary study in luebo, drc where recent outbreaks of ebola haemorrhagic fever occurred, and where cases of the disease were linked to catching bats for human consumption (leroy et al. 2009). the expedition aimed at testing the techniques and materials to be used during a larger international expedition planned to take place early the following year. forty-four specimens of different bat species were sampled, including those which are strongly implicated as potential reservoirs of filoviruses (e.g. h. monstrosus). blood and tissues from various organs were collected and brought to the nicd or nhls for laboratory testing in the recently commissioned maximum security laboratory (bsl-4), which had undergone extensive and protracted upgrading and refurbishment. this maximum security laboratory constitutes a strategically important research resource for preparedness training and response to outbreaks of dangerous pathogens, but will also greatly support efforts by african scientists to unravel the elusive nature of filovirus transmission cycles (figure 3). experimental inoculation studies have recently been conducted at this facility with filoviruses and egyptian fruit bats. figure 1: the ebola ecology expedition team arriving in luebo, democratic republic of the congo, may 2011. figure 2: dissection of wild-caught bats. field laboratory deployed during ebola ecology study in luebo, democratic republic of the congo, may 2011. figure 3: processing and testing of blood and tissues from bats collected during the 2011 ebola ecology expedition to luebo, democratic republic of the congo in biosafety level four facility in sandringham-johannesburg, south africa. despite the finding of ebola virus nucleic acid, antigen or antibodies in bats, ebov has never been isolated from them or any putative animal reservoir so far. however, the isolation of replicative marburg virus, another member of the filovirus family, from wild fruit bats (towner et al. 2009), reinforces the assumption that bats are strong ebov reservoir candidates. based on this hypothesis, a model of virus dissemination has been proposed: bats can transmit the virus either directly to humans and nhps or through an unknown vector as illustrated in figure 4. figure 4: the potential chains of transmission of ebola virus may be described as involving 3 stages, from primates or bats to humans (especially hunters) in the wild (index case), from index case to secondary cases (introduction into the domestic environment) and from patient to healthcare personnel in the clinical setting. whilst primates and fruit bats are known to be sources of ebola virus in nature, the reservoir has not yet been identified with any certainty. clinical features top ↑ the onset of the disease is abrupt after an incubation period of two to 21 days. the clinical features can be divided into four main phases as follows, (1) phase a. influenza–like syndrome: the onset is abrupt with non-specific symptoms or signs such as high fever, headache, arthralgia, myalgia, sore throat, and malaise with nausea. (2) phase b. acute (day 1–6): persistent fever not responding to antimalaria drugs or to antibiotics, headache, intense fatigue, followed by diarrhea and abdominal pain, anorexia and vomiting. (3) phase c. pseudo-remission (day 7–8): during this phase the patient feels better and seeks food. the health situation presents with some improvement. some patients may recover during this phase and survive from the disease and (4) phase d. aggravation (day 9): in many if not most cases, the health status gets worse. the following symptoms may be observed: • respiratory disorders: dyspnea, throat and chest pain, cough, hiccups • symptoms of haemorrhagic diathesis: bloody diarrhoea, haematemesis, conjunctival injection, gingival bleeding, nosebleeds and bleeding at the site of injection consistent with disseminated intravascular coagulation (figure 5) • skin manifestations: petaechiae (not so obvious on black skin), purpura (morbiliform skin rash) • neuro-psychiatric manifestations: prostration, delirium, confusion, coma • cardio-vascular distress and hypovolaemic shock (death). from these clinical manifestations it is obvious that ehf may mimic many other tropical diseases like malaria, typhoid fever or yellow fever at the start of the disease. in most outbreaks, recognition of the disease is delayed because physicians are not accustomed to this new illness and the symptoms are generally non-specific. outside the epidemic context, it appears quite impossible to recognise the first ebola case in an outbreak on clinical grounds only. suspicion of ehf is only possible later during the aggravation phase. figure 5: ebola patient with haemorrhagic diathesis at transfusion and injection sites. diagnosis top ↑ early laboratory confirmation of suspected clinical haemorrhagic fever cases is essential to implement appropriate control measures. definitive diagnosis of suspected cases of ehf is usually made by pcr detection and virus isolation on vero cells. as a class-4 pathogen, ebola virus culture requires a maximum containment facility. additional laboratory diagnostic tests include elisas for the detection of ebola iggand igm-specific antibodies and virus antigens; more specialised molecular testing is also available but is not readily available in the usual clinical setting. in africa, laboratory confirmation of ebola cases has been challenging and early recognition of the first outbreaks were severely hampered as a result. because the disease was poorly known or rare, laboratory investigations were oriented towards the more common, endemic pathogens in the area. initially, salmonella typhi and yellow fever virus were suspected to be responsible for the 1976 yambuku outbreak. blood samples collected for cultures (that remained negative), widal tests and liver specimens presented for pathological examination showed inconsistent results: some specimens gave evidence of yellow fever whilst others were compatible with liver congestion. the yambuku outbreak was finally confirmed by viral culture at the institute of tropical medicine in antwerp thanks to blood samples collected from a belgian nun who fell ill in yambuku and was transferred to ngaliema hospital in kinshasa. for the kikwit cases in 1995, a nosocomial bloody diarrhea outbreak caused by shigella dysenteriae or s. typhi was initially considered, as these diseases were endemic to kikwit. preliminary bacteriological investigations were conducted on 97 stool samples and nine blood cultures. only four stool specimens tested positive for shigella species and all blood cultures remained negative. the index case of this nosocomial outbreak was a 36-year-old laboratory technician who underwent a laparotomy in the kikwit general referral hospital for suspected perforated bowel after a protracted febrile syndrome with headache, myalgia and asthaenia. contact with his body fluids appeared to be the main mode of transmission for the subsequent secondary cases, suggesting that the disease was a viral haemorrhagic fever. to confirm this hypothesis, 14 blood specimens from acutely ill persons were collected and sent to the institute of tropical medicine in antwerp, belgium. the evidence of acute ebola infection was obtained in all specimens either by viral isolation, antigen-detection elisa or rt-pcr. since 1994, the incidence of ebola outbreaks increased and, as a consequence, the awareness of the disease has improved and facilities capable of diagnosing ehv were established in africa. national public health laboratories in endemic countries like uganda (uvri), kenya (kemri) and gabon (cirmf) have already developed capacities to diagnose ehf by elisa and rt-pcr. south africa is the only african country with a maximum containment, enclosed suit laboratory where all class-4 viral pathogens can be handled safely. after the last ebola outbreak in kaluamba, drc (2008–2009), the ebola diagnostic technologies of elisas for the detection of antigens and igm antibody, and rt-pcr have been transferred to the inrb in kinshasa. treatment top ↑ managing ebola patients in the african setting was a major challenge because there was no effective antiviral drug and no specific vaccine available. only supportive care could be administered, to sustain cardiac and renal functions with prudent use of perfusion. oral rehydration was recommended but sometimes not realistic because of throat pain, vomiting and intense fatigue. the main objective was to provide optimal care to the patient with maximum protection of the medical and nursing staff. for that purpose, medical and nursing staff had been trained in donning and removing personal protective equipment (ppe) and applying barrier–nursing procedures. in a clinical experiment conducted late in the 1995 ebola outbreak in kikwit, human convalescent blood was used for passive immunisation to treat patients that had been infected naturally with zebov; seven out of eight patients who received blood transfusion from convalescent ebola patients survived (mupapa et al. 1999). such experiments, unfortunately, have not been repeated in further outbreaks because in vitro studies showed that antibodies against ebola had no neutralising activities. in addition, although monoclonal antibodies to the glycoprotein of ebola virus showed protective and therapeutic properties in mice, they failed to protect nhp (gupta et al. 2001; oswald et al. 2007). since ebola virus is generally considered as a potential biological weapon, it is urgent to develop effective antiviral drugs and vaccines. the ideal is to develop a candidate vaccine able to confer interspecies cross-protection against zebov, sebov, bebov and unknown ebola virus species. control measures top ↑ the corner-stone for controlling an outbreak of ehf is to interrupt the viral transmission chain. in order to reduce transmission, several strict public health measures need to be implemented as quickly as possible, including isolation of patients, barrier precautions and identification and tracking of all contacts. most of the time, outbreaks are managed by a core structure called the international committee on scientific and technical coordination, under the aegis of the world health organisation (who). this committee is in charge of implementing control measure activities on a daily basis and has the following working subgroups: • co-ordination committee, which is responsible for all epidemic response activities, chair daily meetings and write reports for public health authorities and health partners. • the patient management team is involved in the isolation of clinical cases in a quarantine ward, training of medical and relief personnel on the proper use of protective equipment (gloves, gowns, masks etc.), and providing medical care based on symptomatic therapy to maintain the vital respiratory, cardio-vascular and renal functions. the non-governmental organisation, doctors without borders (msf), has developed expertise in this field from involvement in outbreak response. • the hygiene and sanitation team is in charge of disinfection and burial of all ebola and non-ebola dead bodies under safe conditions. local red cross volunteers usually perform these activities. • the epidemiological surveillance team is in charge of active and passive case finding, contact tracing and rumor-verification of suspect cases or deaths in the community. • social mobilisation and health education are critical for controlling an ebola outbreak since resistance from the community to freely provide information on patients, deaths and contacts are commonplace. ebola haemorrhagic fever outbreaks have many socio-cultural aspects that need to be studied deeply as communities can reject the anti-epidemic control measures imposed by the international scientific and technical committee. the existence of rumours and legends related to the outbreaks could obscure the viral nature of the disease. sometimes the anti-epidemic control measures needed to be adapted to the local culture, for example, funeral practices as in the 2003 ebola outbreak in republic of the congo (hewlett et al. 2005). the members of this team should include medical anthropologists, local red cross volunteers and opinion leaders such as teachers, religious groups, et cetera, for public sensitisation, education and information. • the logistic support team is in charge of providing any administrative, logistic and technical support to the other teams, such as coordination of secretariat, transport and communication. • the laboratory and research team is in charge of collecting, storing and shipping of clinical samples for diagnostic confirmation. this team is also responsible for ecological studies to determine the origins of an outbreak. • psychosocial support for the affected family or families has been neglected during previous outbreaks, but this issue has become more and more important due to stigmatisation of survivors and their families by the community. conclusion top ↑ formerly sporadic, with high case fatality rates (up to 90%), the deadly ebola haemorrhagic fever outbreaks are becoming more and more frequent in africa, mostly in relation to increasing contact with infected wildlife. previous epidemics were detected after a long delay, especially because of the remoteness of the epidemic focus, the lack of laboratory facilities and the poor knowledge of the disease by doctors and nurses, who confused ebola disease with malaria or typhoid fever. the more recent epidemics in yambio (2004) and kaluamba (2008) resulted in low cfrs of 41.2% and 43.8%, respectively. this is mainly related to the early detection of the outbreaks followed by a prompt and vigorous response from public health authorities and their partners. ebola haemorrhagic fever epidemics constitute a significant public health concern in africa and an effective vaccine is needed urgently. such a vaccine would primarily benefit doctors, nurses and field epidemiologists working in endemic countries. the second target group would be the scientists working with ebola virus as well as veterinarians and those involved in wildlife conservation in endemic 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fisheries development, tanzania2cirad, campus international de baillarguet, france 3central veterinary laboratory, tanzania 4veterinary investigation centre, arusha, tanzania 5veterinary investigation centre, mtwara, tanzania correspondence to: fredrick kivaria postal address: po box 9152, dar es salaam, tanzania dates: received: 15 mar. 2013 accepted: 25 aug. 2013 published: 30 oct. 2013 how to cite this article: kivaria, f.m., kwiatek, o., kapaga, a.o., swai, e.s., libeau, g., moshy, w., et al., 2013, ‘the incursion, persistence and spread of peste des petits ruminants in tanzania: epidemiological patterns and predictions’, onderstepoort journal of veterinary research 80(1), art #593, 10 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.593 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the incursion, persistence and spread of peste des petits ruminants in tanzania: epidemiological patterns and predictions in this original research... open access • abstract • introduction • materials and methods    • the historical perspectives of peste des petits ruminants incursion into tanzania    • field investigation of the incursion of peste des petits ruminants    • clinical and pathological investigation • laboratory techniques    • competitive enzyme-linked immunosorbent assay for peste des petits ruminants    • real-time polymerase chain reaction analysis and lineage definition    • investigation on the persistence of peste de petits ruminants    • investigation on the spread of peste des petits ruminants in tanzania • results    • field investigations on the incursion of peste des petits ruminants       • clinical signs: macroand microscopical lesions       • cross-sectional study results       • serological results and risk indicators for positive serological status against peste des petits ruminants       • characterisation of the strain and determination of its geographical lineage       • persistence and spread of peste des petits ruminants • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references • footnote abstract top ↑ peste des petits ruminants virus, which causes a severe disease in sheep and goats, has only recently been officially declared to be present in tanzania. an epidemiological study was carried out between september 2008 and october 2010 to investigate the incursion, persistence and spread of the virus in tanzania. the investigation involved serosurveillance, outbreak investigation and computation of epidemiological indices such as the effective reproductive number, persistence and the threshold level for vaccination. field and molecular epidemiological techniques were applied to isolate, characterise and trace the origin of the virus in tanzania. a total of 2182 serum samples from goats and 1296 from sheep from 79 villages across 12 districts were investigated. village-level prevalence of infection was variable (0.00% – 88.00%) and was higher in pastoral than in agro-pastoral villages. the overall antibody response to the virus was 22.10% (ci95% = 20.72% – 23.48%). about 68.00% and 73.00% of seropositive goats and sheep, respectively, did not show clinical signs. the proportion of seropositive animals differed significantly (p ≤ 0.001) between age groups, sex and farming practices. real-time polymerase chain reaction results showed that the isolated strains belong to lineage iii, whose origin is in east africa and the middle east. this indicates that one of the northern neighbouring countries is most likely the source of infection. the computed overall effective reproductive number, the threshold level of vaccination necessary to eradicate the disease and persistence were 4.75% and 98.00%, respectively. these estimates indicate that achieving elimination of the peste des petits ruminants virus from pastoral flocks will require significant effort and development of highly effective intervention tools. introduction top ↑ in tanzania, sheep and goats are important livestock species as they are able to survive unfavourable climatic conditions of arid and semiarid environments; hence, they are species of choice of pastoralists. these animals are widely distributed across different agro-ecological zones and production systems and form an integral part of the livelihood of rural households. however, the contribution of sheep and goats to food security in tanzania has recently been threatened by outbreaks of peste des petits ruminants (ppr). the first outbreak of ppr in sheep and goats in tanzania was confirmed in december 2008. since then, sporadic outbreaks have been reported in many parts of the country. the three fundamental steps of exotic pathogen emergence are incursion, persistence and spread in a geographical context. in this article, incursion refers to the first confirmed arrival of ppr virus (pprv) in the country, persistence refers to the obstinate survival of pprv in sheep and goat populations in spite of vaccination and other biosecurity measures undertaken to eliminate the virus, and spread refers to the movement of pprv between different geographical localities. we use data on the first occurrence and the subsequent events of ppr incursion and spread in tanzania to illustrate the epidemiological factors that drive these sequential events amongst the pastoral sheep and goat population. the epidemiological data, clinical and pathological features of this outbreak and recovery and identification of pprv are also reported. materials and methods top ↑ the historical perspectives of peste des petits ruminants incursion into tanzania incursion of ppr into tanzania was suspected after a confirmed ppr outbreak in kenya in august 2006. the possible presence of the disease in tanzania was reported at the tanzania veterinary association annual scientific conference by the ngorongoro district veterinary officer in december 2007. in march 2008, a high mortality rate was observed amongst sheep and goats in the ngorongoro district and a technical team was dispatched to investigate the cause of the increased deaths. a total of 112 sheep and goats were subjected to clinical, pathological and serological investigations. both clinical and pathological investigations yielded inconclusive results, whilst serological investigation yielded negative results for ppr. however, reports of increased deaths in sheep and goats continued in the ngorongoro and mara districts. in june 2008, a second team was dispatched to the ngorongoro and mara districts, where, respectively, 404 and 84 serum samples were collected. of the 404 samples collected in the ngorongoro district, 129 (31.93%) tested positive for ppr during serological screening. none of the samples from the mara district tested positive. based on these rapid epidemiological assessment (rea) results, the director of veterinary services designed a cross-sectional study that was aimed at gauging the magnitude and extent of ppr spread in the country. field investigation of the incursion of peste des petits ruminants a cross-sectional study was carried out in 12 districts across the regions of arusha, kilimanjaro, manyara and tanga in the north of the country between september 2008 and july 2009. the aim of the study was to determine the magnitude of ppr in the study area and the extent of the southward spread. approximately 4 362 838 goats and 2 079 151 sheep were estimated to be at risk in the study, representing about 57.75% and 32.08% of the national goat and sheep populations, respectively. all sheep and goat flocks in the sampling area formed the population from which study flocks were randomly selected. multistage sampling (dohoo, martin & stryhn 2003), with four hierarchical stages, was used as sampling strategy. the first level of selection was the region; the arusha, kilimanjaro, manyara and tanga regions were purposely selected. within each of the selected regions, specific districts (karatu, longido, mbulu, monduli, ngorongoro, siha, simanjiro, korogwe, lushoto, mkinga, muheza and tanga-municipal) were also purposely selected. villages were randomly selected. within each of the selected villages, 40 animals (either sheep or goats) were randomly selected. the study regions and districts were purposely selected based on perceived risk due to either their bordering kenya or the presence of farming activities characterised by extensive livestock movement. owing to the characteristics of the pastoral and agro-pastoral livestock production systems, it is reasonable to consider all the sheep and goats in a village as one flock, with very little within-flock variation. a village was therefore considered to constitute a flock. the sample size was computed as suggested by dohoo et al. (2003): where z = 1.96, p = 0.32 (the prior prevalence from the rea study described earlier) and l = 0.05 (the desired level of precision or accuracy). the required sample size was accordingly calculated as n = 334.37. however, ppr is a highly contagious disease. to obtain a similar accuracy to that with simple random sampling, the sample size had to be recalculated. the aim was to sample at least 40 animals from each village, and from the rea study the average rate of ppr homogeneity (p) was estimated to be 0.156. the sample size was subsequently recalculated as: where n = 334.37, ρ = 0.156 and m = 40, representing the average number of animals to be sampled from each village (dohoo et al. 2003). therefore, the new sample size was equal to 2360.816 animals and 59 villages were to be included in this study. the design effect (d) was calculated as 7.084 according to: the standard error (s) was calculated as 0.026 according to: indicating that with this design the certainty associated with the true prevalence of sheep and goats with antibodies to ppr will be p ± 1.960 × 0.026 of the estimate. the prevalence was determined by dividing the total number of positive samples by the total number of samples (dohoo et al. 2003). the sampling frame from which the 59 study villages were randomly selected was obtained from the district agriculture office. clinical and pathological investigation in all villages that were visited, discussions were held with livestock owners and animals were inspected for evidence of classic ppr symptoms (severe mucopurulent nasal and ocular discharges, necrotic stomititis and respiratory distress). dead animals were necropsied for evidence of ppr lesions and organ collection. pieces of the lung, spleen, kidney, liver and mesenteric lymph nodes from one dead goat were collected for laboratory confirmation. in addition, two goats from the ngorongoro district, which presented with clinical signs of ppr, were purchased and sacrificed for collection of microbiological samples and pathological lesions. laboratory techniques top ↑ competitive enzyme-linked immunosorbent assay for peste des petits ruminants a nucleotide monoclonal antibody-based competitive enzyme-linked immunosorbent assay (celisa) was used for the detection of antibodies to pprv (fao/iaea 1993; oie 2000). this test was developed using a virus-neutralising monoclonal antibody directed against an epitope of the haemagglutinin protein specific to pprv. all serum samples tested in the present study were processed in duplicate according to the standard protocol for the detection of rinderpest antibodies (fao/iaea 1993; oie 2000). the results of the celisa were interpreted using software from the economic development institute developed by the food and agriculture organization/international atomic energy agency for the detection of rinderpest antibody (fao/iaea 1993). results were interpreted as previously described by libeau et al. (1995). samples that showed colour inhibition (inhibition of the enzymatic colour reaction) ≥ 50% when compared to wells containing the control (no serum), were considered positive. a confidence interval at the 95% level (ci95%) for the prevalence of antibodies to pprv in the population studied was calculated using standard statistical methods (snedecor & cochran 1989). real-time polymerase chain reaction analysis and lineage definition a total of six sheep samples from the ngorongoro district in the arusha region were analysed for viral identification and lineage definition. one sample consisted of ovine tissues (liver, spleen, heart and mediastinal lymph nodes) and five of heparinised whole blood. total rna was extracted and cdna was amplified as previously described (kwiatek et al. 2007). the polymerase chain reaction (pcr) used the pan-morbillivirus primers located in the middle of the gene (nad1: ccaaggattgcagaaatgatc, nad2: gctcctgcactaaacttgttc), which allow the amplification of the n-gene of all morbilliviruses to give a product of 230 bases. the pcr conditions were as follows: an activation of 10 min at 95 °c, 35 cycles of amplification (30 s at 94 °c, 30 s at 55 °c and 30 s at 72 °c) and a final extension step at 72 °c for 7 min. the pcr was performed in a 2720 thermal cycler (applied biosystems). the resulting pcr products were analysed by electrophoresis on an ethidium bromide-stained 1.5% agar gel.the nucleic acid sequences obtained from pcr products amplified with the nad1–nad2 primer were aligned with sequences from pprv strains that were maintained in our database. phylogenetic analysis was performed on the 224 nucleotides located on the nucleoprotein gene of the virus. the known pprv sequences were derived from strains isolated over a period of 20 years. they consisted of ‘historical’ strains, such as the nigeria 75/1, from which the ppr vaccine was developed, and also the dorcas strain isolated from a dorcas gazelle in a zoological collection in the united arab emirates, (furley, taylor & obi 1987). strains isolated between 1988 and 2000 originated from cameroon, india, senegal, turkey, ghana, guinea bissau, oman and the ivory coast. the alignment of nucleotide sequences was created by means of the clustal w program (vector nti, informax inc., rockville). phylogenetic analysis was carried out as described by kwiatek et al. (2007). bootstrap confidence values were calculated on 1000 replicates. the rbok vaccine strain of rinderpest virus was considered as an out-group. investigation on the persistence of peste de petits ruminants a fundamental parameter determining the success (persistence and spread) of any infectious organism in a host population is the basic reproductive number, r0, which measures the potential for spread of infection. it is defined as the average number of new infections caused when an infection enters an entirely susceptible population (anderson & may 1991). two things are worth noting from this definition. first, it is an average value that can change from population to population or over time, depending on the patterns of contact or biological influences at play. second, the subscripted identifier (zero) refers to the time at the start; that is, when invasion occurs. as the infection spreads, the population is no longer entirely susceptible. contacts are therefore wasted on infected or immune individuals and the reproductive number is reduced to an effective reproductive number, rt. the effective reproductive number has a threshold value of 1 to ensure persistence and can be used to estimate the level of immunisation or managerial change required to control an epidemic. in this study, rt was estimated from the average age at seroconversion by the method of dietz (1993). the deterministic formula used was: where ls is the expected average farm gate lifespan of sheep and goats and as is the average age at seroconversion. an estimate of rt was also determined as described by heffernan and wahl (2005), using reported morbidity and case fatality risk associated with ppr according to the formula: where rt is the reported ppr morbidity and at is the average ppr case fatality risk. the following assumptions were applied: • from an epidemiological perspective, the small ruminant (sheep and goat) population is assumed to be a single, homogeneous, free-mixing host population (referred to as ‘the flock’) and contacts between individuals were random. • the kidding and lambing rates were high enough to sustain an input of susceptible hosts into the population, thereby maintaining a constant chain of infection in the population. • screening of 250 sheep and 600 goat samples, randomly selected from the 2006–2008 sera that were collected from the study area during rinderpest elimination campaigns, yielded negative results for pprv antibodies. it was therefore logical to assume that the study animals were seronegative during the 12 months before sampling, and therefore fully susceptible to the invading pprv infection. • the duration of the infectious period is not related to age. • the deterministic model did not consider the probability of re-invasion of the recovered flock. the probability of persistence or spread, ppers, over the next generation was computed as ppers = 1 – (1/rt)n, where n is the number of infectious foci (the number of villages that reported ppr cases) and rt equals r0 when there is no immunity or previous infection within the population. the threshold level of vaccination (vt) necessary to eradicate the disease was calculated according to vt = 1 – rt–1, as described in anderson and may (1991). investigation on the spread of peste des petits ruminants in tanzania in december 2009, an outbreak of a disease characterised by the sudden onset of yellowish diarrhoea, mucoid nasal and ocular discharges with matting of the eyelids and erosive lesions of the gums followed by death was reported from likuna, a village in the newala district of the mtwara region in southern tanzania. participatory epidemiological investigations showed that a livestock trader, and a resident of the village, had brought 70 goats from the pugu market in the dar es salaam region to be sold during the approaching seasonal festivals (christmas and new year). the trade goats started to die after three days, without noticeable clinical signs. to avoid losses, the trader sold the whole consignment cheaply to the nearby villages of makote, mnazi mmoja, lidumbe and mambika. the disease affected the resident stock with high morbidity and mortality rates, estimated at 90% and 73% respectively. clinical examination was carried out in 30 randomly selected animals and necropsies were performed on 10 dead animals. ocular and nasal swabs and sera were collected for laboratory confirmation from 217 randomly selected animals. the epidemic curve was plotted to see the pattern of the outbreak, using the daily records made by the newala district veterinary officer. competitive elisa showed that antibodies against pprv were present in 59% of the 217 serum samples and pprv protein was detected in 34% of the screened ocular and nasal swabs. by march 2010 the disease had spread to the nearby districts of tandahimba and masasi, where, respectively, 84% of the 180 and 3% of the 125 samples screened by celisa yielded positive results for antibodies against pprv. in april 2010 about 400 small ruminants were reported to have died because of an unknown disease in two divisions of malinyi and mtimbira in the ulanga district of the morogoro region. epidemiological investigations suggested that the disease was ppr, which invaded the region through returning migratory pastoralists who had moved northwards from the mtwara and lindi regions. serological investigations revealed the presence of antibodies against pprv in 152 (76%) of the 200 serum samples tested by means of celisa. in 2011, antibodies to pprv were demonstrated in trade cattle sharing common grazing lands with 225 small ruminants in the mukuranga district of the coast region. although not susceptible to the disease, cattle are able to seroconvert in case of a high prevalence of ppr in small ruminants (abubakar et al. 2011). in october 2010 the director of veterinary services conducted risk-based serosurveillance in all districts that border mozambique, malawi and zambia. samples were collected from animals at randomly selected high-risk areas, such as border crossings, auctions, slaughter slabs and grazing and watering points, and other areas where animals from a number of villages congregate. a total of 720 serum samples were collected under the vaccine for control of neglected animal diseases in africa (vacnada) programme (funded by the european union) and screened for antibodies against pprv at the panvac laboratories in addis ababa, ethiopia. of the 720 samples, 266 (36.9%) tested positive for antibodies against pprv. clinical disease was detected in the mahuta ward of the tandahimba district, mtwara and according to rt-pcr, pprv protein was detected in 33.0% of the tissue samples taken from 28 goats here. the chronology and probable movement of ppr in tanzania is depicted in figure 1. figure 1: chronology and spread of peste des petits ruminants in tanzania. results top ↑ field investigations on the incursion of peste des petits ruminants clinical signs: macroand microscopical lesions clinical signs of ppr were observed in 90 goats and 60 sheep, independent of age or sex, in the ngorongoro, monduli, longido and siha districts. the clinical signs included diarrhoea, especially in young animals, elevated body temperatures (> 40 °c), serous rhinorrhoea and serous mucopurulent nasal discharges. peri-nasal areas were often found encrusted with mucopurulent discharges. however, such clinical signs were not observed in the flocks in the other study districts. two necropsied goats showed inflammation of the apical lung lobes, with multiple abscesses evident in one of the goats. the mesenteric lymph nodes in both carcasses were swollen and the small intestines were hyperaemic. microscopical lesions in the lungs were associated with bronchopneumonia and focal necrotic areas surrounded by inflammatory zones were also found. cross-sectional study results a total of 3478 serum samples (2182 from goats; 1296 from sheep) were obtained from 79 villages1 between september 2008 and july 2009. the distribution of the study variables was such that 72.70% of the study animals were female, whilst 27.30% were male. of the 3478 animals, 77.80% were classified as adults and 22.20% were classified as kids or lambs. the majority of the study animals (2162/3478; 62.16%) were from the agro-pastoral farming system, whilst 37.84% (1316/3478) came from the pastoral farming system. > serological results and risk indicators for positive serological status against peste des petits ruminants of the tested animals, 22.10% (ci95%: 20.72% – 23.48%) were classified as seropositive for pprv antibodies. up to 88.00% of the study animals tested positive for pprv, with a greater proportion of positive samples found in the arusha, manyara and kilimanjaro regions (in descending order) than in the tanga region. the percentage of pprv-positive samples are categorised according to region, district, farming practice and animal species in table 1. it was estimated that 68.00% of the 483 goats and 73.00% of the 286 sheep that tested positive for antibodies against pprv had not displayed clinical signs. the proportion of seropositive animals differed significantly according to age, sex and farming practice, but there was no significant association between animal species and seropositivity. results of the pairwise comparison are summarised in table 2. despite an overall frequency of 22.10% (ci95%: 20.72% – 23.48%), the seroprevalence of ppr appeared to be variable across the study districts; pastoral districts appeared generally to have suffered more from infection (z = 15.38; p ≤ 0.0001) than the agro-pastoral districts, and a seroprevalence of 0.00% was reported in two districts (table 2). table 1: seroprevalence of peste des petits ruminants in sheep and goats according to sampling area. table 2: risk for peste des petits ruminants seroconversion according to animal age, sex, species and production system. characterisation of the strain and determination of its geographical lineage two samples (one tissue sample and one from blood) were found positive for pprv by means of rt-pcr. to obtain epidemiological information and analyse the genetic relation of the 2008 tanzania strain (from the ngorongoro district) with strains of different geographical origins, the nucleic acid sequences obtained from pcr products amplified with the nad1–nad2 primer were aligned with sequences from pprv strains retrieved from genbank or maintained in our database. phylogenetic analysis was performed on the 224 nucleotides located on the nucleoprotein gene of the virus. the program mrbayes (version 3.1.2) was used to perform bayesian analysis. tree manipulations were compiled using the figtree program (version 1.3.1) ( http://tree.bio.ed.ac.uk/software/figtree/). the rbok wild strain of rpv and a measles virus strain were considered as out-groups. for support, phylogenetic analysis was also carried out as described in kwiatek et al. (2007). as expected, the 2008 tanzania strain of pprv fell into lineage iii, as is the case for east african strains, and it was closely related to the historical strains ibri and dorcas isolated since 1987 in the sultanate of oman (furley et al. 1987). the lineage is distinct from the one circulating in asia from south india to turkey (özkul et al. 2002). figure 2 shows the bayesian inference of pprv strains. the phylogenetic reconstruction was made using mrbayes software as mentioned earlier (huelsenbeck & ronquist 2001). the sequence evolution model proposed by jobb (2011) was hky. consequently, the number of substitution types was set at six and the bayesian reconstruction was run for 6 000 000 iterations. results were analysed when the standard deviation between the monte carlo markov chains fell below 0.01. trees were sampled every 1000 generations; thus the consensus tree was built up with a burn-in phase of 25% and posterior probabilities were displayed as node labels. the tree was rooted by two out-groups, namely measles and rinderpest morbilliviruses collected from genbank (fj416068 and x98291 respectively), and the four pprv lineages are highlighted in black brackets. tanzania_2009 analysed in this study is underlined in lineage iii. figure 2: phylogenetic tree based on the nucleoprotein gene of the peste des petits ruminants virus, positioning the tanzania strain in lineage iii. persistence and spread of peste des petits ruminants estimates of rt derived through deterministic and morbidity or mortality techniques for each of the investigated districts, as well as those based on pooled data for the districts, were similar. these estimates, together with their respective vaccination threshold (vt) estimates, are summarised in table 3a and table 3b. the overall persistence was estimated at 0.98. as shown in figure 3 the epidemic curve resembles a typical propagated outbreak, with peaks of primary, secondary and tertiary generations of cases spaced by an incubation period. the most probable period of exposure of the susceptible animals to pprv was calculated as 3–5 days before the index case was reported. it was of epidemiological interest to observe that clinical signs were not present in 68% of the 483 goats and 73% of the 286 sheep that tested seropositive for antibodies against pprv. the presence of these ‘silent spreaders’, coupled with uncontrolled extensive livestock movement within the country and across the international borders, may explain the detection of antibodies against pprv without clinical disease in the south-western parts of the country (tanzania–zambia and tanzania–malawi borders), as shown in figure 1. figure 3: epidemic curve of peste des petits ruminants outbreak in the newala district of the mtwara region in southern tanzania. table 3a: estimation of effective reproductive number for peste des petits ruminants virus infection of pastoral sheep and goat flocks in tanzania, using average lifespan and age indices. table 3b: estimation of effective reproductive number for peste des petits ruminants virus infection of pastoral sheep and goat flocks in tanzania, using morbidity and case fatality risk indices. discussion top ↑ our results are in agreement with observations made by swai et al. (2009), who confirmed the presence of ppr in tanzania. as ppr has not been previously reported in tanzania, it can be considered an emerging disease in tanzania. absence of ppr in tanzania was demonstrated serologically in 2000 (wambura 2000). the identification of the pprv strain, as well as the serological, virological and clinical findings of this study confirmed that the disease was ppr. clinical and post-mortem examination may be sufficient for the diagnosis of ppr in endemic areas, but molecular methods of characterisation are compulsory in areas where the outbreaks occur for the first time (scott 1990). genotypic classification based on the n-protein gene appeared to be an effective method for discriminating between geographically distinct lineages (kwiatek et al. 2007). pastoral districts appeared generally to have suffered more from infection (z = 15.38; p ≤ 0.0001) than the agro-pastoral districts (table 2). reasons for this may be related to extensive livestock movements and exchanges resulting in a high rate of contact between animals, which increases the potential for transmission of pprv. pastoral animals will often travel long distances during the dry season in search of fodder and water. consequently, a considerable proportion of the population becomes infected during this period, giving rise to the establishment of disease endemicity and continued, year-round circulation of the virus. farmers are often not aware of the pitfalls of mixing sick and healthy animal for grazing. for example, the study team saw diseased small ruminants and in-contact animals being sold at the market despite the ongoing outbreak. the purchase of new stock from the market and the return of unsold animals will often precipitate in severe outbreaks. the movement of animals, therefore, plays an important role in the spread and maintenance of pprv in flocks, as does the purchase of potentially infected animals and their subsequent introduction into naïve flocks. age appears to be a risk indicator (p ≤ 0.001) for seropositive ppr status, as suggested by the observed age profile according to which about 43% of the flock consists of young animals. although goats are considered to be more susceptible to pprv than sheep (abubakar et al. 2011), there was no association (p = 1.000) between animal species and seropositivity in the current study. this could be due to the fact that sheep and goats are equally exposed to the ppr risk factors such as movement and nutritional stresses, or it could be that the circulating field strain has overcome the natural resistance of sheep; different types of breed might affect the outcome of pprv infection. the significant relationship (p = ≤ 0.001) between sex and seropositive ppr status could be due to the uneven distribution of the two sexes in the study flocks. the origin of pprv in tanzania is not well established, but it is believed that it might have been introduced to tanzania through the movement of live, infected animals. this hypothesis is supported by the fact that the border districts such as longido and ngorongoro had the highest prevalence (table 1) of pprv antibodies; moreover, pprv was isolated from tissue samples that originated from the ngorongoro district. the results of molecular epidemiological techniques suggest cross-border transmission of actively circulating pprv into tanzania from neighbouring northern countries. the virus we isolated is pprv lineage iii, which includes viruses whose origins are in east africa. an outbreak in ethiopia in 1996 as well as one in the middle east was of this type (abubakar et al. 2011). thus, one of the northern neighbouring countries in the east africa region is most likely the source of infection. the evolving east african situation regarding ppr for the period 2004–2009 can be traced in the world organisation for animal health’s wahid database ( http://www.oie.int/wahis/public.php?page=disease_timelines) (oie 2009), where the different waves of incursion of ppr can be seen. the first is observed in kenya in march 2006, a second in uganda in 2007 and the last one in tanzania in december 2008. the majority of north-eastern countries such as ethiopia and sudan have been reporting the disease for more than 10 years (lefèvre et al. 1991). for pprv transmission, infected and healthy animals need to be in contact (lefèvre et al. 1991), which serves as an additional indication that the virus came into tanzania through progressive introduction of live, infected animals from these countries. trade, as well as the nomadic lifestyle of the people in this region, is to a great extent responsible for the phenomenon. moreover, the general extension of ppr emergence in africa is certainly linked to the expansion of trade in animals and subsequent movement of these animals into previously disease-free regions. this phenomenon is further advanced by the presence of a significant proportion of ‘silent spreaders’. the epidemic curve revealed that during the outbreak cases occurred over multiple incubation periods. the shape and other features of an epidemic curve (figure 3) can suggest hypotheses about the time and source of exposure, the mode of transmission, the causative agent, incubation period and the efficacy of control measures (dicker & gathany 1992). the outbreak had a successive series of peaks, reflecting increasing and decreasing numbers of cases in each generation. the epidemic waned after five generations, probably because either the number of susceptible animals fell below some critical level or the zoosanitary measures taken to control the disease became effective. the estimates of rt for pprv infection (table 3a) indicate that, on average, four secondary ppr infectious cases resulted from each primary infection in pastoral flocks. the variation in rt estimates observed (table 3a) is probably due to farming system and environmental variation, but it may also reflect the stochastic nature of pprv. at this estimated rt value (4), it might be expected that minor outbreaks of ppr infection would occur in which far fewer individuals seroconvert. this expectation is consistent with the number of reported sporadic ppr episodes. however, the generally poor reporting level suggests that an rt value of 4 may be an underestimate of pprv infection in the pastoral population. furthermore, the morbilliviruses are known to be highly contagious, with a basic r0 between 15 and 20 (anderson & may 1991), which increases the chances of an infection spreading or persisting within the population. however, given that the serological survey was conducted when the epidemic was still evolving, the host–pathogen relationship may not have reached equilibrium. furthermore, it is possible that the assumption that animals were seronegative during the previous 12 months might not be valid, as the epidemic had been evolving for some time before the animals were sampled. the value of rt derived in this study should therefore be cautiously considered a minimum estimate of the true population values. there are limitations to generalisation of this estimated rt for pprv infections in other sheep and goat populations. in the current study, the animals were assumed to constitute a free-mixing population, approximating a homogeneously mixed flock. whilst representative of the small ruminant farming system found in tanzania, this production environment is quite different from that found in highland areas, where grazing land is scarce. for example, the effective contact rate is likely to differ between tethered and free-ranging animals, which would affect estimates of rt. other factors, such as pprv strain, genetics of the flock, pre-incursion flock immunity, climate, population density and frequency of contact between flocks, may also affect rt. thus, rt for pprv may vary from the present estimates in other sheep and goat populations and environments. further refinement of the estimates, which should include variables such as seasonality, environmental contamination, and contribution of the wild life population, as well as estimation of rt in other sheep and goat production settings will be required to determine the general applicability of these estimates. an rt value of 4 predicts that 63.8% – 84.6% of small ruminants in a population would have to be vaccinated with a 100.0% efficient vaccine to reduce rt to less than 1 (table 3a). if, as stated earlier, rt is underestimated, the percentage vaccine coverage would also be underestimated and would need to be increased for effective control of pprv. for an imperfect vaccine or other interventions that are unlikely to be completely efficient (e.g. quarantine, movement restriction and closure of primary and secondary livestock auctions), effective coverage would probably have to approach 100.0%. the situation is further complicated by the possibility of undetected disease in areas where stock populations are sparse, the possibility that the disease may establish in the wildlife population and the possibility of delayed detection and recognition, which would provide a potential source for re-infection of sheep and goats. thus, whilst some interventions may reduce the number of infected sheep and goats, eliminating pprv from pastoral flocks will require significant effort and development of highly effective intervention tools, as for rinderpest campaigns. the control of transnational livestock movements across borders is difficult, especially in the northern districts where maasai pastoralists are found on either side of the tanzania–kenya border. the resulting transborder movements of cattle, goats and sheep for breeding, trade and other socio-economic functions could be the main predictor for introduction, transmission and maintenance of pprv in the country. without proper control mechanisms, these animal movements could contribute to and accelerate the spread of the virus regionally. indeed, the pprv-positive samples from the tanzania–mozambique, tanzania–malawi and tanzania–zambia borders (figure 1) indicate a southward movement of the disease. therefore, the importance of ppr as a threat to small ruminants should be considered together with other economically important diseases and measures should be taken to prevent subsequent spread of such diseases into the region of the southern african development community (sadc). conclusion top ↑ the objective of this work was to investigate the incursion, persistence and spread of pprv in tanzania. our results contribute to the epidemiological understanding that pprv is established in tanzania. extensive livestock movement, failure to observe basic zoosanitary measures, delayed reporting and lack of proper control strategies at the local and national levels are the main factors contributing to the persistence of pprv in tanzania. the establishment of pprv in tanzania serves as a reservoir for the rest of the sadc region through livestock movement and trade of infected animals. acknowledgements top ↑ we are grateful to the maasai herdsmen who gave their time and allowed us to sample their animals. the help and participation of staff at the department of veterinary services, ministry of livestock and fisheries development of tanzania, is highly appreciated. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions f.m.k. (ministry of livestock and fisheries development) was the group leader and responsible for the study design and data analysis. o.k. (cirad) and g.l. (cirad) were responsible for the laboratory and molecular epidemiology work. a.m.k. (central veterinary laboratory), j.g. (central veterinary laboratory), e.s.s. (veterinary investigation centre, arusha), w.m. (veterinary investigation centre, arusha) and a.o.m (veterinary investigation centre, mtwara) were responsible for fieldwork. references top ↑ abubakar, m., khan, h.a., arshed, j.m., hussain, m. & qurban, a., 2011, ‘peste des petits ruminants (ppr): disease appraisal with global and pakistan perspective’, small ruminant research 96, 1–10.anderson, r.m. & may, r.m., 1991, infectious diseases of humans: dynamics and control, oxford university press, london. dicker, r. & gathany, n.c., 1992, principles of epidemiology, 2nd edn., u.s. department of health and human services, public health service, centre for disease control and prevention (cdc), atlanta. dietz, k., 1993, ‘the estimation of the basic reproduction number for infectious diseases’, statistical methods in medical research 2, 23–41. dohoo, i., martin, w. & stryhn, h., 2003, veterinary epidemiologic research, atlantic veterinary college, charlottetown. food and agriculture organization/international atomic energy agency (fao/iaea), 1993, ‘peste des petits ruminants elisa kit. competitive enzyme immunoassay for detection of antibody to ppr virus. bench protocol’, version ppr 1.0, january 1993. joint fao/ieae programme, animal production and health. pirbright, united kingdom: world reference laboratory for rinderpest. furley, c.w., taylor, w.p. & obi, t.u., 1987, ‘an outbreak of peste des petits ruminants in a zoological collection’, veterinary record 121, 443–447. heffernan, j.m. & wahl, l.m., 2005, ‘monte carlo estimates of natural variation in hiv infection’, journal of theoretical biology 236, 137–153. huelsenbeck, j.p. & ronquist, f., 2001, ‘mrbayes: bayesian inference of phylogenetic trees ’, bioinformatics 17, 754–755. ( http://mrbayes.sourceforge.net/links.php) jobb, g., 2011, treefinder, computer software, viewed 17 august 2011 from http://www..treefinder.de kwiatek, o., minet, c., grillet, c., hurard, c., carlsson, e. & karimov, b., 2007, ‘peste des petits ruminants (ppr) outbreak in tajikistan’, journal of comparative pathology 136(2–3),111–119. lefèvre, p.c., diallo, a., schenkel, s., hussein, s. & staak, g., 1991, ‘serological evidence of peste des petits ruminants in jordan’, veterinary record 128, 110. libeau, g., diallo, a., calvez, d. & lefèvre, p.c., 1995, ‘a competitive elisa using anti-n monoclonal antibodies for specific detection of rinderpest in cattle and small ruminants’, veterinary microbiology 31, 147–160. office international des epizooties (oie), 2000, oie manual of standards for diagnostic tests and vaccines. list a and b diseases of mammals, birds and bees, oie, paris. özkul, a., akca, y., alkan, f., barrett, t., karaoglu., t., dagalp, s.b. et al., 2002, ‘prevalence, distribution, and host range of peste des petits ruminants virus, turkey’, emerging infectious diseases 8, 708–712. scott, g.r., 1990, ‘rinderpest and peste des petits ruminants’, in e.p.j. gibbs (ed.), virus diseases of food animals, vol. 2, disease monographs, academic press, new york. snedecor, g.w. & cochran, w.g., 1989, statistical methods, 8th edn., iowa state university press, ames, iowa. swai, e.s., kapaga, a., kivaria, f., tinuga, d., joshua, g. & sanka, p., 2009, ‘prevalence and distribution of peste des petits ruminants virus antibodies in various districts of tanzania’, veterinary research communications 33, 927–936. http://dx/doi.org/10.1007/s11259-009-9311-7 wambura, p., 2000, ‘serological evidence of the absence of peste des petits ruminants in tanzania’, veterinary record 146, 473–474. footnote top ↑ 1. an additional 20 villages were sampled under the vacnada project. article information authors: folorunso o. fasina1 japhta m. mokoele1,2 b. tom spencer1 leo a.m.l. van leengoed1,3 yvette bevis4 ingrid booysen4 affiliations: 1department of production animal studies, university of pretoria, south africa 2limpopo department of agriculture, groblersdal, south africa 3department of farm animal medicine, utrecht university, the netherlands 4centre for geoinformation science, department of geography, geoinformatics and meteorology, university of pretoria, south africa correspondence to: folorunso fasina email: dayo.fasina@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 24 june 2014 accepted: 28 aug. 2015 published: 27 nov. 2015 how to cite this article: fasina, f.o., mokoele, j.m., spencer, b.t., van leengoed, l.a.m.l., bevis, y. & booysen, i., 2015, ‘spatio-temporal patterns and movement analysis of pigs from smallholder farms and implications for african swine fever spread, limpopo province, south africa’, onderstepoort journal of veterinary research 82(1), art. #795, 11 pages. http://dx.doi.org/10.4102/ojvr.v82i1.795 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. spatio-temporal patterns and movement analysis of pigs from smallholder farms and implications for african swine fever spread, limpopo province, south africa in this original research... open access • abstract • introduction    • historical perspective on the disease in south africa with emphasis on limpopo • materials and methods    • data collection    • data analyses       • cartography, point mapping and one-way linkages       • social network analyses • results    • farm characteristics and trade relationship    • social network analyses • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ infectious and zoonotic disease outbreaks have been linked to increasing volumes of legal and illegal trade. spatio-temporal and trade network analyses have been used to evaluate the risks associated with these challenges elsewhere, but few details are available for the pig sector in south africa. regarding pig diseases, limpopo province is important as the greater part of the province falls within the african swine fever control area. emerging small-scale pig farmers in limpopo perceived pig production as an important means of improving their livelihood and an alternative investment. they engage in trading and marketing their products with a potential risk to animal health, because the preferred markets often facilitate potential long-distance spread and disease dispersal over broad geographic areas. in this study, we explored the interconnectedness of smallholder pig farmers in limpopo, determined the weaknesses and critical control points, and projected interventions that policy makers can implement to reduce the risks to pig health. the geo-coordinates of surveyed farms were used to draw maps, links and networks. predictive risks to pigs were determined through the analyses of trade networks, and the relationship to previous outbreaks of african swine fever was postulated. auction points were identified as high-risk areas for the spread of animal diseases. veterinary authorities should prioritise focused surveillance and diagnostic efforts in limpopo. early disease detection and prompt eradication should be targeted and messages promoting enhanced biosecurity to smallholder farmers are advocated. the system may also benefit from the restructuring of marketing and auction networks. since geographic factors and networks can rapidly facilitate pig disease dispersal over large areas, a multi-disciplinary approach to understanding the complexities that exist around the animal disease epidemiology becomes mandatory. introduction top ↑ infectious and zoonotic diseases outbreaks have intensified in past decades through more intense interconnectedness, rapid transport, the opening-up of borders and increasing volumes of legal and illegal trades (jones et al. 2013; perry, grace & sones 2013). spatio-temporal analyses have been used in veterinary medicine and other fields in recent times (jiang, ediger & bader 2009; paul & dasgupta 2012; rivas et al. 2012). specifically, geographic factors such as roads, water bodies, distances from other outbreaks and markets, among other factors, have been found to play important roles in disease transmission (food and agriculture organization of the united nations [fao] 2013; jori et al. 2009; korennoy et al. 2014; pastrana et al. 2014; rivas et al. 2010rivas et al. 2012; sánchez-vizcaíno, mur & mártinez-lópez 2012). in addition, trade practices have played major roles in the spread of infectious diseases among livestock (fournié et al. 2013; mccarron et al. 2015). limpopo province is very important in the epidemiology of some transboundary animal diseases in south africa. in particular, the province has been designated as a control zone for african swine fever together with certain areas of the north west and mpumalanga provinces (department of agriculture, fisheries and forestry [daff] n.d.; penrith 2013). furthermore, the province provides an active domestic animal–wildlife–human interface, making it an ideal location for one health studies involving zoonoses or disease interactions between wildlife and domestic animals. pig production systems and the particular contributions of the emerging small-scale pig farms (esspf) in the limpopo have been described recently (mokoele et al. 2014). however, the presence of a deeprooted dual market structure remains a major challenge facing small-scale pig producers in the province (antwi & seahlodi 2011). while the commercial pig farmers use the formal markets (standard abattoirs, processing plants and supermarkets), the esspf farmers can access only the informal markets (local auctions, backyard slaughter, pension sale points and local abattoirs/slaughter slabs). the most popular auction point and abattoir used by the esspfs from limpopo are belfast (mpumalanga) and bronkhorstspruit (gauteng) respectively. these locations, which are outside the province, present the esspfs with better marketing opportunities and higher incomes for their products. while the commercial interests of the esspfs are being secured through these more affluent markets, breach of biosecurity remains evident and the risk of introducing infectious pathogens to non-endemic areas remains high, with imminent threats to the pig industry nationally. martínez-lópez, pérez and sánchez-vizcaíno (2009) and lindström et al. (2009) have previously identified such risks elsewhere. asf is a severe, highly transmissible viral infection of domestic pigs that manifests itself as a haemorrhagic fever, which can cause mortality of up to 100% in domestic pigs with consequent devastating effects on the livelihoods of farmers who depend on pig production (bastos, fasina & king 2014; de glanville et al. 2014; fasina et al. 2012a; penrith 2013). to date, three types of epidemiological cycle have been described for asf in southern africa (penrith, thomson & bastos 2004). historical perspective on the disease in south africa with emphasis on limpopo the first documented outbreak of asf in south africa was recorded in 1928 and this was related to contact between wild pigs and domestic pigs (penrith 2013; penrith & vosloo 2009). between 1933 and 1939, historical outbreaks of swine fever were documented (de kock, robinson & keppel 1940); cases may have been classical or african swine fever, as no clear distinction was established between the two diseases in earlier years (penrith 2013). since 1939, south africa has experienced only sporadic outbreaks that have been limited to the asf-controlled zones of limpopo and the kruger national park (boshoff et al. 2007). although parts of the north west province are designated within the control zones, asf had not been reported there (daff 2014, table 1). recent events have, however, proved that the virus, although contained, has the capacity to spread rapidly from the controlled areas to new locations within south africa (table 1). table 1: reported outbreaks of asf, 1993–2012 in south africa in south africa between 1993 and 2012, 1309 cases of asf were documented between 1993 and 2012 from 71 outbreaks in south africa (table 1; daff 2014). limpopo has accounted for 76.1% of all outbreaks to date and the majority of outbreaks from other provinces have links with limpopo (table 1). in september–october 2011, mpumalanga and gauteng experienced the first outbreak of asf to occur outside the control area in recent times. briefly, some pigs died in a small piggery within the asf control area in lephalale (ellisras), limpopo with the subsequent sale of surviving pigs at an auction in sundra, mpumalanga, and their transportation to rietpoort abattoir in gauteng. by january 2012, a total of 172 farms including some 10 374 pigs have had primary or secondary contacts with the index farm and only the prompt and co-ordinated intervention of the meat inspectors, private and government veterinarians, the industry and the laboratory (transboundary animal diseases programme (tadp)) curtailed the outbreaks and eradicated the infections (evans 2012; spencer 2012; spencer & penrith 2014). in this study, we used the spatio-temporal data of locations, possible interactions and marketing structures practised by the emerging small-scale farming communities in limpopo to draw a network of esspf, conduct some spatio-temporal analyses and integrate the outcomes with historical and recent reports of asf outbreaks in south africa to map the probable risk, spread and consequences of asf. in addition, because limpopo has played a significant role in the outbreaks and dissemination of asf in south africa and the province has been declared an asf-endemic location, we used asf as a model for a rapidly spreading transboundary animal disease amongst pig populations. materials and methods top ↑ data collection as part of the questionnaire survey conducted for a study on the production systems and dynamics of the esspf in limpopo (mokoele et al. 2014), the geo-coordinates of all the surveyed esspf were obtained using the garmin nuvi® or the nokia lumia 635® and entered into a microsoft excel® spreadsheet. briefly, limpopo consists of five districts, namely sekhukhune, capricorn, waterberg, vhembe and mopani. a purposive sampling method was targeted at all the available esspf farmers (n = 185) in these districts. an original list of 85 small-scale pig farmers was produced by the limpopo department of agriculture but an additional 100 farms that fall within this category but were not listed were recruited into the survey because the preliminary evidence from the field suggested that there were many unlisted esspf in the province. the inclusion criterion for the present study was pig farms with ≤ 50 sows, located within the five districts of limpopo that have been active in pig production for at least one year (mokoele et al. 2014). a participatory research model approach was used to collect information from the farmers (raman, sanghi & chambers 1989). of the 185 esspfs sent questionnaires, a total of 164 (88.65%) participated fully and filled in the questionnaires completely. all the data were entered into microsoft excel® and checked for consistency, correctness and validity. a preliminary map was drawn to check that all the places surveyed fell within the correct locations within limpopo, based on the data obtained. data were formatted to meet the need for cartography software in esri's arcgis/arcview, the r and the nodexl software and exported appropriately. extracts of the reports on the asf outbreaks to date in south africa were integrated into the final social network maps in order to predict the risk of asf and advise on intervention strategies. data analyses cartography, point mapping and one-way linkages esri's arcgis 10.1 software was used to add into the gis all the specified geographic co-ordinates of the esspf farmers as xy co-ordinate data. the resulting event layer was then displayed as a point symbol (red dot) portraying the locations of the small-scale pig farmers on an administrative map of limpopo (figure 1, census 2011). additional fields were added to the initial attribute table of the feature to enable the selection of farms by their designated abattoir. thereafter the ‘xy to line’ feature tool was used to construct geodetic lines or linkages (figure 2a and 2b) representing the shortest distance between the farm and the destination point (the abattoir or auction point). pig farmers who used local slaughtering points within their localities were represented by symbols (red dots) on these two maps. in addition, layers of the primary and secondary road networks within limpopo and contiguous provinces were made on the map previously generated (figure 3). figure 1: map of surveyed limpopo province and locations for emerging small-scale pig farmers in limpopo, 2012. figure 2: (a) unidirectional links between esspf and destinations of final products. (b) unidirectional links between esspf and preferred auction points. figure 3: distribution of locations of surveyed farms based on preferred slaughter location/auction markets and the national and secondary road networks. social network analyses unidirectional networks: firstly, filtered data were imported from the open microsoft excel® workbook into the nodexl environment and manipulated appropriately for analyses based on the software manufacturer's instructions (nodexl version 1.0.1.326, connected action). unidirectional graphs with 28 vertices were produced. the graph's vertices were grouped by cluster using the clauset-newman-moore cluster algorithm and laid out using the harel-koren fast multiscale layout algorithm. the edge colours, widths and opacity were based on edge weight values. the vertex sizes were based on betweenness centrality (bc) values. for emphasis, a graph or network is a set of vertices and edges connected together [g = (v, e)], edge [e] is an association linking two vertices, and vertices [v] are points or locations joined by edges (jiang et al. 2009). multidirectional graphs: in the second instance, data were exported into the r software environment and mapped for in-going (in-degrees) and out-going (out-degrees) contacts using the modified method of nøremark and widgren (2015). multidirectional graphs were produced separately for in-degree and out-degree links. in addition, the k-cores (strength of contribution of each vertex in the total network) were produced based on the number of links that influence each vertex. results top ↑ farm characteristics and trade relationship a total of 164 esspf were categorised based on the number of sow units (su) as follows: 1–10 su (n = 124; median = 4, ci95% = 4–5); 11–20 su (n = 17; median = 15, ci95% = 13–16); 21–30 su (n = 7; median = 26, ci95% = 23–29); 31–40 su (n = 2; median = 38, ci95% = 6–69); 41–50 su (n = 1; median = not applicable, ci95% = not applicable). an abattoir was the preferred slaughter point for the majority of respondents (105/164; 64%) and the abattoir of choice for the esspf from limpopo was bronkhorstspruit in gauteng (55.9%) and phalaborwa (26.2%) and thabazimbi (10.4%), both located in limpopo. a total of 97.6% of the surveyed esspf preferably chose the belfast auction in mpumalanga to source their pigs or sell whole animals (figure 2a and 2b). importantly, some esspfs (34.1%), especially those from capricorn district prefer to slaughter pigs within the communities or at pension points (figure 2a and 2b). up to 306 pigs were marketed monthly (mean/month = 283) by the surveyed farmers and a total of 3396 pigs were sold in the year 2011 preceding the survey. the esspfs were randomly dispersed throughout limpopo, with a tendency for greater farm concentrations around capricorn, mopani and greater sekhukhune districts (figure 1). however, no specific pattern exists for the farm distributions or choice of slaughter or sale facility and for the distances from the closest national or secondary road(s) to the farms (table 2; figure 3). table 2: preferred point of sale for pigs and pig products and the distances to the nearest national or secondary roads. the average distances between each of the slaughter or sale locations and the nearest national or secondary road networks are shown in table 2. in addition, the distance covered to travel with the animals to the belfast auction may be as short as 50 km up to a distance of about 400 km. the distances travelled to get to local abattoirs or slaughter points were within 5 km for local slaughter points, 150 km for phalaborwa abattoir, 200 km for thabazimbi abattoir and up to 400 km for bronkhorstspruit abattoir (figure 2a and 2b; mokoele et al. 2014). the preferred means of transportation includes own vehicle (34.5%), hired vehicle (5.4%), shared vehicle (1.4%) and other means (58.8%, n = 148). social network analyses using the in-going (in-degree) and out-going (out-degree) movement to and from the vertices, the most important nodes for disease spread are locations (phalaborwa, tzaneen, belfast auction, bronkhorstspruit abattoir, elias motsoaledi, molemole), pension points or local markets, tubatse, local slaughter points and polokwane in that order (figure 2a and 2b, 4–7). phalaborwa also has the highest degree of centrality whereas the top ten vertices ranked by bc were the local slaughter points, phalaborwa, thabazimbi, bronkhorstspruit, molemole, ephraim mogale, letaba, tzaneen, makhado and blouberg (see figure 4 and online appendix 2). figure 4: network map of connected locations for emerging small-scale pig farmers in limpopo. overall, the unidirectional graph has 28 vertices and 2 unique edges with a total of 161 edges. there were 4 connected components with an average geodesic distance (shortest path between points) of 1.48. the graph density (a function of the edges and vertices that make up a network) had a value of 0.06 and an average clustering coefficient (a measure of the degree to which nodes cluster together within the network) of 0.000 (online appendix 2). discussion top ↑ this study revealed that informal pig movements and trade networks in limpopo exist. while we did not investigate the specific role of each farmer and the middlemen, we confirmed that trade-associated short and long-distance movements of pigs and pig products exist within the province (5 km – 400 km). monthly, up to 306 pigs may be transported individually or collectively in shared facilities over a short or long distance to major abattoirs or auction points and these transports may facilitate long-distance risk of disease dispersal. in a case of a rapidly spreading disease outbreak, trade by these farmers could facilitate the rapid spread of disease across broad geographic areas as was the case of the lephalale-sundra-rietpoort (limpopo-mpumalanga-gauteng) african swine fever outbreak of 2011–2012. it will be necessary to target the esspfs and associated downstream sector traders in this sector for animal health and biosecurity training. in addition, animal health intervention and prevention strategies should target these esspf (mccarron et al. 2015). previous reports have confirmed that the majority of the past outbreaks of asf have involved esspf from limpopo (daff 2014). figure 5: multidirectional network map of in-degree connectedness of locations of emerging small-scale pig farmers in limpopo, 2012. several important nodes (towns and cities) with high levels of interactions and the potential to contribute to or rapidly spread pig pathogens inadvertently were identified. these nodes acted as dispersal and sometimes collection points. phalaborwa, thabazimbi, bronkhorstspruit and belfast are among such important points and there is a need to create surveillance points and inspection nodes around these locations. the first two locations are within limpopo but the last two are located in the adjacent gauteng and mpumalanga, where higher market values are the main drivers for the esspfs travelling these distances. it will be important to improve pig marketing locally and add value to pork products in limpopo to discourage long-distance travel, because such trips can pose a risk of spread of infection outside the control area. although a movement control policy is implemented in south africa, which includes a prohibition of the movement of pigs out of the asf control area without a veterinary permit only issued under specified conditions, emergency situations and economic imperatives often challenge such policies. there is therefore a need for enhanced vigilance on the part of the authorities, including active surveillance and participatory epidemiology among the esspfs. in addition, there is a need to motivate esspf to apply good farming practice with improved biosecurity, which will be rewarded by access to a scaled-up pricing system for pork certified by local veterinarians through a government support system (fasina et al. 2010, fasina et al. 2012b; logar 2014). since local slaughter and pension point sales were also revealed as important trade nodes, these activities may conceal the existence of subclinical infections and prevent early detection of rapidly spreading transboundary animal diseases. limpopo is of major interest because the great majority of asf outbreaks over decades have occurred or originated there. it is an important disease node not only for asf but potentially for other pig diseases. while the movement of pigs between farms was not evaluated in this study, we are aware that such movements exist (mokoele et al. 2014). social networks associated with pig farms are important factors in the transmission of infectious diseases (leslie et al. 2015). our model has identified local slaughter points and other abattoirs as potentially having the greatest influence on disease dispersal. strict anticipatory planning should be implemented in these locations and controlled slaughter should be encouraged (kao et al. 2007; lindström et al. 2010). our evaluation and analyses are based on certain assumptions. first, we assumed that the records are complete and the data used in the evaluations are accurate; it is important for recording officials to pay particular attention to data entry in the future, as critical analysis based on available data may be warranted. we have made every effort to verify the data and confirm certain facts where there are doubts; secondly, we did not discriminate between respondents, partial respondents and non-respondents as we eliminated all partially filled-in questionnaires. this resulted in differing denominators and may sometimes hide some important answers. some degree of recall bias may have occurred with regard to certain questions that made reference to the past years. moreover, since only the surveyed farmers were included in the networks, it is possible that some other smallholder pig farmers exist whose inclusion may significantly influence and change the strengths and directions of the networks. thirdly, the social network analytic method used in this study has certain limitations including the following: an assumption that all disease algorithms often exhibit ‘small world’ properties in a situation of close interconnectedness in temporal and spatial locality (jiang et al. 2009); in the real world this may not always be true. an assumption that an analysis kernel of bc can resolve all complexities of interconnectedness by measuring the shortest distances passing through vertices (freeman 1977). the networks can sometimes identify false positive edges and false negatives especially where background noises are high in the dataset (ufimtsev & bhowmick 2013). false positives may arise when two vertices have low to medium bc scores, which can combine to produce one with a high bc score. we have reduced this by creating four different networks for each identified node. finally, the threshold in a given group testing may be affected by the false positive bc values. we have corrected for this influence in the course of the modelling. figure 6: multidirectional network map of out-degree connectedness of locations of emerging small-scale pig farmers in limpopo, 2012. in conclusion, we have produced evidence that geographic factors and trade-movement networks played roles in asf dynamics and dispersal in south africa and that managing these in limpopo province is critical for pig disease control. the degree of interconnectedness of the esspf in the province may facilitate disease spread by people, trade, animal movements and also possible sharing of transport and farm equipment as has been describe elsewhere (kao et al. 2007; leslie et al. 2015). while we have made effort to integrate the dynamics of recent and historical outbreaks of asf in south africa geo-temporally, it will be worthwhile to re-evaluate the specifics of the lephalale-sundra-rietpoort outbreak of 2011–2012 and determine quantitatively the potential implications of ‘along-the-road networks’, the effect of delayed response due to travel time to auction time and treatment using antibiotics. these may reveal important hidden evidence of disease dynamics in the pig industry in south africa figure 7: structural cohesion (k-core) of network map of connected locations of emerging small-scale pig farmers in limpopo, 2012. acknowledgements top ↑ we acknowledge the limpopo department of agriculture as the provider of funds for this survey as part of a postgraduate programme through the human resource development section, the director of veterinary services in limpopo province, dr mampane, and his team of managers in the districts and all the veterinary officials for their help with the collection of data in their respective districts. the department of production animal studies and the faculty of veterinary science, onderstepoort partially supported this work with funding and resources. we thank marc a. smith and the team at connected action consulting group (http://www.connectedaction.net/) for assistance with sna mapping. we gratefully acknowledge the input of assoc. prof. eric etter (university of pretoria) for assistance with multidirectional sna. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions f.o.f. (university of pretoria) and j.m.m. (university of pretoria) designed the study. j.m.m. carried out the field survey. f.o.f. carried out the statistical analyses. b.t.s (university of pretoria) and l.a.m.l.v.l. (university of pretoria) supervised the project. y.b. (university of pretoria) and i.b. 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vertices in complex networks’, eleventh workshop on mining and learning with graphs, chicago, il, viewed 03 june 2014, from http://snap.stanford.edu/mlg2013/submissions/mlg2013_submission_3.pdf mariner_135-140.indd context the world trend is towards increasing demand for livestock products as consumers’ incomes increase (delgado, rosegrant, steinfeld, ehui & courbois 1999). however, this change is taking place in the context of globalization, climate change and increasing requirements for quality and safety. producers in africa are now competing in the middle east export markets with those from mongolia, brazil and the ukraine (mariner 2006). will producers in developing countries be able to benefit from increasing demand in local, regional or international markets? or will other players capture this opportunity? beyond trade, there are other important considerations. in developing countries, the majority of farmers are small-scale producers. in addition to contributions to the national wealth, livestock production has the important function of providing livelihoods and social foundation for the bulk of the population (grace, jost, mcgregor-skinner & mariner 2008). eighty-five percent of all farmers are small-scale farmers and most of these are in developing countries (nagayets 2005). disease control policies need to consider the economic, social and political impacts of both the disease itself and the actions taken to control disease on diverse groups of stakeholders (mcdermott, mariner, rich & omore 2007). within the past few years, there has been renewed recognition that the emergence of new infectious diseases is an on-going natural process (jones, patel, levy, storeygard, balk, gittleman & daszak 2008). there is evidence that the changing interactions of man, livestock and the environment further accelerate the emergence of new pathogens. considerable resources have been made available for technical interventions to reduce the perceived threat of emerging diseases, but mostly with shortterm objectives. there have been calls by some to 135 onderstepoort journal of veterinary research, 76:135–140 (2009) more appropriate disease control policies for the developing world j.c. mariner international livestock research institute, p.o. box 30709, nairobi, 00100 kenya abstract mariner, j.c. 2009. more appropriate disease control policies for the developing world. onderstepoort journal of veterinary research, 76:135–140 investment in disease control should be targeted to critical points that provide the greatest benefit to the livelihoods of livestock-dependent stakeholders. risk-based targeting should balance the impacts of diseases against the feasibility of their control. this requires sensitive and specific surveillance systems that provide representative overviews of the animal health situation for accurate assessment of disease impact and transmission patterns. assessment of impact should include household and market effects. the key in surveillance is involving livestock owners using active methods that ensure their disease priorities are addressed. epidemiological targeting of interventions to critical points in disease transmission cycles should be done to obtain maximal disease reduction. interventions should be delivered in full partnership with both private and community-based stakeholders to assure high uptake and sustainability. in developing countries, approaches such as participatory disease surveillance and community-based animal health programs have been effective and comply with international animal health standards. 136 disease control policies reshape livestock production systems to reduce the threat of the emergence of new agents, but in general these proposals have neglected to address the socio-economic benefits of and incentives for existing production systems. as a result, proposals to date for restructuring have had limited impact. the real opportunity is to use the current interest in emerging disease and one health (american veterinary medical association 2008) to drive sustainable change in health institutions through careful analysis of the political and socio-economic forces shaping production and disease control institutions. objectives of disease control the principal objectives of animal disease control are to: • encourage increased production to support economic growth. • enhance access to markets and add value to livestock products. • enhance the livelihoods of producers and reduce vulnerability to adverse shocks. • protect the health of the public through the reduction in zoonotic and food-borne illness. components of appropriate disease control policies it is important to recognize at the outset that each country will have its own unique socio-economic context and that there is the risk of being overly prescriptive in describing appropriate animal health policy. in a general analysis, it is best to describe characteristics of appropriate policies rather than propose specific policies. disease control policies and health objectives are very much intertwined. policies often fail because the objectives that they set out to accomplish were not achievable within the existing or attainable resource base and social context. the first test of a policy should be a balanced assessment of resources and feasibility. policies that capture market forces and are driven by natural incentive systems are the most likely to succeed. in such cases stakeholders will be rewarded for adhering to policy. if the strategy requires individuals to take significant actions that are counter to their self-interest, failure is probable. negative incentives to enforce compliance with unpopular regulations are problematic at best and unlikely to succeed. strategies that mobilize civil society organizations such as veterinary associations, producer associations, community institutions and non-governmental organizations to work towards shared disease control goals are highly advantageous. to accomplish this, disease control authorities must be willing to share both the ownership and decision-making in the disease control programs with civil society. this does not mean that national veterinary authorities cede any of their regulatory and certification responsibilities as defined by the terrestrial animal health code (oie 2006). it does mean that authorities work with civil society to define priorities and select methods from among options of demonstrable efficacy. uncertain legal environments discourage investment in animal health institutions because of the increased economic risks associated with the lack of a predictable investment environment. in order for civil society to contribute effectively, the rule of law in the health sector must be solidly established. this means that appropriate legislation and subsidiary regulations must be in place and enforced. the objective is to provide potential health investors with a sense of security. institutions control disease. institutions are the organizations, individuals, rules, practices, technologies, values and customs that all work together to achieve an end. often animal health professionals focus on specific technical interventions, such as the type of vaccine and vaccination schedule to be applied, rather than design an effective institutional model for the selection of vaccines and their delivery. more appropriate disease control policies will involve a wider range of actors and take a holistic view of problems. the concept of one health (american veterinary medical association 2008), which recognizes that human, animal and environmental health are intertwined, is a synergistic approach that offers considerable advantage in terms of enhancing the efficacy of disease surveillance, prevention and control. the professions involved have significant technical capacities that complement each other. however, information-sharing, joint coordination of activities and integrated mitigations are limited. overcoming constraints to the implementation of one health strategies will primarily require new institutional models developed in the context of national professional culture and infrastructure. 137 j.c. mariner a recurring theme in successful animal disease control programs is participation and empowerment of a diverse set of actors under the overall facilitation and regulation of the national veterinary authority. existing policies should be assessed to establish the extent to which they adhere to these principles. technical principles for more effective disease control policies at the technical level, disease control programs must be based on strong epidemiological principles and risk-based approaches. this was a key lesson of the rinderpest eradication program. programs should seek to target critical control points where interventions will have maximum impact to reduce disease risk either by reductions in disease circulation or exposure (may & anderson 1984). this includes targeting of surveillance inputs to high-risk populations within the national system. disease transmission analysis may suggest key populations that play pivotal roles in the maintenance or amplification of pathological agents. on the other hand, a step in the disease cycle or a particular exposure may offer an entry point to control of the disease or elimination of its impact. the risk-based approaches engendered in the sanitary and phytosanitary (sps) agreement (wto 2008) and expanded upon in the terrestrial animal health codes (oie 2006) are leading to new strategies such as compartmentalization and commoditybased trade. these strategies shift the focus away from mass control designed to change the disease status of a region or country to smart approaches that channel resources to mitigate disease risk at the spot where they can directly add value to products. an important concept of the sps agreement is the focus on results in terms of an achievement of acceptable level of risk rather than on the prescription of specific sanitary methods. this opens the door to experimentation and the search for more effective methods adapted to local constraints and opportunities. many believe that key technical innovations can solve disease control problems by themselves. sometimes they do. more often, technological innovation requires institutional innovation that allows the benefit of the new technology to be captured. an important area is action research to develop, demonstrate and document methods to effect institutional change and the benefits of institutional change. two examples of the power of technological solutions coupled to institutional change are communitybased vaccination programs that utilize thermostable vaccines (mariner, akabwai, toyang, zoyem & ngangnou 1994) or participatory surveillance systems that use rapid diagnostic test technology (mariner, hussein, roeder & catley 2003). the history of the adoption of these technological innovations highlights the institutional challenges to effective exploitation of new technologies. examples a success story: rinderpest rinderpest eradication is an apparent success story with many lessons. initially, the rinderpest eradication strategy focused on standard technical interventions such as mass vaccination delivered through classical disease control infrastructure. this approach achieved some important initial gains in terms of disease suppression and the eradication of rinderpest from west africa, but was insufficient to eradicate the disease from more challenging remote and unstable areas of east africa. institutional innovations were also targeted such as international control of vaccine quality, promotion of the private sector and strengthening of legislation. at the technical level, thermostable rinderpest vaccine created new options for the delivery of vaccine to remote and unstable areas. this lead to successful experimentation with community-based approaches to vaccination in remote areas that ultimately made eradication possible in the most challenging areas of east africa (for example south sudan, karamoja in uganda and the afar region of ethiopia) (mariner et al. 1994). this was a major shift in stakeholder roles within animal health institutions. livestock owners were becoming service providers as well as the principal customers of the system. existing veterinary service providers felt threatened and were the focus of resistance to change. as community animal health worker networks became established in rinderpest endemic areas, epidemiologists gained access to traditional information networks that proved to be highly efficient mechanisms for gathering epidemiological intelligence on the occurrence of rinderpest. the learning processes that evolved to tap this valuable resource became formalized as participatory disease surveillance. this methodology was key to identifying the final foci of rinderpest for eradication and is a methodological legacy. 138 disease control policies beyond the obvious benefits of the elimination of a major animal health scourge, rinderpest eradication left behind more capable institutions that were better adapted to local conditions and able to control disease. a disease that re-emerged: contagious bovine pleuropneumonia contagious bovine pleuropneumonia (cbpp) is an example of a disease threat that was once largely controlled but has re-emerged as a major problem due to the evolving political economic context. in the colonial period and the era of early independence, cbpp was controlled through a combination of vaccination with a vaccine of limited efficacy, strict movement control and culling of apparently infected animals (hammond & branagan 1965). in recent times, control programs based on this strategy have broken down as stringent movement control and culling is often no longer politically feasible. veterinary services have continued to target cbpp as a disease of public interest and attempted to retain a monopoly on the mandate for control interventions based on a policy of free vaccination, despite inadequate resources to actually implement mandates. cbpp is a chronic, insidious disease that is able to persist indefinitely in relatively small populations. the prospects for elimination from even small populations with existing vaccines in an era when stringent movement control is no longer achievable are poor (mariner, mcdermott, heesterbeek, thomson, roeder & martin 2006). this suggests that policies should shift towards sustainable control where recurrent costs to government are minimized until more effective control tools become available. in this environment, farmers and field veterinarians began to experiment with antibiotics as the only available control option. official conventional wisdom held that treatment of clinical cases with antibiotics gave rise to persistent infections and their use in the treatment of cbpp was banned in most countries. despite the ban their use in the treatment of cbpp became widespread. the popularity of antibiotic treatment and the public perception of positive results with antibiotics led to research trials that demonstrated suppression of transmission in treated herds and an official reassessment of the role of antibiotics in cbpp control (ayling, baker, nicholas, peek & simon 2000; huebschle, godinho, rowan & nicholas 2004; huebschle, ayling, godinho, lukhele, tjipura-zaire, rowan & nicholas 2006). this is an example of innovation in civil society leading to the development of an evidence base for an institutional shift in policy. the willingness of livestock owners to invest in cbpp treatment combined with statements of their willingness to purchase vaccination, if it were available for purchase, suggests that livestock owners are sufficiently motivated to invest in integrated cbpp control options such as treatment of cases and vaccination of in-contact animals. policies should be lib er alized to allow private farmers to purchase comprehensive control from their private veterinarian that includes an appropriate vaccination schedule. static animal health policies in a changing political economic environment hindered rather than facilitated control and probably contributed to the resurgence of the disease. historic control policies were not achievable in the present political economic context due to the limited public resources available for animal health and the low potential for effective movement control. as a result: • technical targeting of policies became inappropriate. • policies did not harness prevailing financial incentives to control the disease. • strategies constrained rather than sought to actively mobilize civil society partners. • unrealistic regulatory environments lead to a breakdown of official coordination and control. • market forces ultimately lead to innovation and the prospect of more effective control strategies. a recurring challenge: rift valley fever in arid and semi-arid areas, explosive rift valley fever (rvf) epidemics are associated with high rainfall and flooding. the disease is transmitted by mosquitoes and conditions that favour the rapid expansion of mosquito populations lead to outbreaks. once predisposing weather conditions are in place, the sequence of events leading up to an outbreak is relatively predictable. however, the short time courses of outbreaks make timely response to unfolding epidemics very difficult. historically, most interventions have been applied after the peak of the outbreak and have had little if any impact on the course of the disease (ilri 2008; jost, nzietchueng, kihu, bett, njogu, swai & mariner, in press). to predict the 2006/07 rvf outbreak in east africa early warning models took advantage of the normalized difference vegetation index (ndvi), collected by remote sensing, in addition to other indicators linked 139 j.c. mariner to regional rainfall (fao 2006). as ndvi is based on vegetative change, the ‘early warnings’ were issued subsequent to actual onset of conditions on the ground that was favourable for mosquito population explosions. retrospective assessments of the outbreak using participatory epi dem iological techniques indicated that livestock owners were well aware of clinical presentation of rvf, which somali pastoralists referred to as sandik (jost et al., in press). construction of event timelines for kenya provided evidence that the livestock outbreak had begun prior to the early warnings and that communities recognized the first human cases followed an average 17.5 days after the first livestock cases. essentially no response by either human or animal health authorities was undertaken prior to the change in official disease status on 4 december 2006 caused by the first diagnosis of rvf on a hospitalized human patient. this was 50 days after the first clinical livestock cases described by livestock producers. this experience shows that rather than an all-ornone approach to decision-making based on confirmed diagnoses, responses to rvf need to be undertaken using a risk-based approach to decision-making. effective responses require true early warnings based on escalating risk of predisposing climatic events. in the case of east africa, sea surface temperature changes are an appropriate early indicator that should trigger the first action steps. the warnings produced in 2008 indicate that this lesson has been taken on board (fao 2008). decision-making should be phased and appropriate actions taken in steps calibrated to match the evolving level of risk. in the case of the 2006/07 outbreak, the following lessons should be considered: • a risk-targeted, one health approach should be taken to surveillance that tracks environmental, veterinary and human indicators of rvf. • early warning forecasts should begin with seasonal weather forecasts and communicate evolutions in risk profiles. • decision-making for disease prevention and control should be risk-based and matched to evolving conditions. • responses should be timely, phased and epidemiologically-targeted to critical control points that can affect the evolution of the outbreak. conclusion veterinary, health and environmental services will need to link with civil society partners to form strong, innovative disease control and quality assurance institutions that fully mobilize human and economic resources to respond rapidly to changing health needs. references american veterinary medical association 2008. one health: a new professional imperative. final report of the one health initiative task force. http://www.avma.org/ onehealth. ayling, r.d., baker, s.e., nicholas, r.a., peek, m.l. & simon, a.j. 2000. comparison of in vitro activity of danofloxacin, florfenicol, oxytetracycline, spectinomycin and tilmicosin against mycoplasma mycoides subspecies mycoides small colony type. veterinary record, 146:243–246. delgado, c., rosegrant, m., steinfeld, h., ehui, s. & courbois, c. 1999. livestock to 2020: the next food revolution. washington dc: international food policy research institute, food and agriculture organization of the united nations and international livestock research institute (food, agriculture and the environment discussion paper, 28). fao 2006. empres watch: possible rift valley fever activity in the horn of africa. rome: food and agriculture organization of the united nations. fao 2008. empres watch: climate models predict increased risk of precipitations in the horn of africa for the end of 2008. rome: food and agriculture organization of the united nations. grace, d., jost, c., mcgregor-skinner, g. & mariner, j.c. 2008. small livestock farmers: participation in national animal health programs. technical item, no. 1. report of the 76th general session of the office international des epizooties. paris: office international des epizooties. hammond, j.a. & branagan, d. 1965. contagious bovine pleuropneumonia in tanganyika. bulletin of epizootic diseases in africa, 13:121–147. huebschle, o., godinho, k., rowan, t. & nicholas, r. 2004. danofloxacin treatment of cattle affected by cbpp. veterinary record, 155:403. huebschle, o.j., ayling, r.d., godinho, k., lukhele, o., tjipura-zaire, g., rowan, t.g. & nicholas, r.a.j. 2006. danofloxacin (advocin) reduces the spread of contagious bovine pleuropneumonia to healthy in-contact cattle. research in veterinary science, 81:304–309. ilri 2008. learning the lessons of rift valley fever: improved detection and mitigation of outbreaks: participatory assessment of rift valley fever surveillance and rapid response activities. nairobi: international livestock research institute. jones, k.e., patel, n.g., levy, m.a., storeygard, a., balk, d., gittleman, j.l. & daszak, p. 2008. global trends in emerging infectious disease. nature, 451:990– 993. jost, c.c., nzietchueng, s., kihu, s., bett, b., njogu, g., swai, e. & mariner, j.c. in press. participatory epidemiological assessment of the rift valley fever outbreak in kenya and tanzania in 2006 and 2007. american journal of tropical medicine and hygiene. mariner, j.c., akabwai, d.m.o., toyang, j., zoyem, n. & ngangnou, a. 1994. community-based vaccination with thermostable vero cell-adapted rinderpest vaccine (thermovax). proceedings of the 7th international symposium on veterinary epidemiology and economics, nairobi: 507–509. 140 disease control policies mariner, j.c., hussain, m., roeder, p.l. & catley, a. 2003. the use of participatory disease searching as a form of active disease searching in pakistan for rinderpest and more. proceedings of the 10th international symposium on veterinary epidemiology and economics, vina del mar, chile. mariner, j. 2006. assessment of livestock and livestock product markets in selected countries in the middle east. nairobi: international livestock research institute. mariner, j.c., mcdermott, j., heesterbeek, j.a.p., thomson, g., roeder, p.l. & martin, s.w. 2006. a heterogeneous model of contagious bovine pleuropneumonia transmission and control in pastoral communities in east africa. preventive veterinary medicine, 73:75–91. mcdermott, j., mariner, j., rich, k. & omore, a. 2007. combating avian flu in developing countries: the role of applied research in improving rural livelihoods. bulletin of animal health and production in africa, special edition on avian influenza: 47–54. may, r.m. & anderson, r.m. 1984. spatial heterogeneity and the design of immunization programmes. math bioscience, 72:83–111. nagayets, o. 2005. small farms: current status and key trends. future of small farms research workshop, wye college. oie 2006. terrestrial animal health code. paris: office international des epizooties. wto 2008. the wto agreement on the application of sanitary and phytosanitary measures (sps agreement). http://www. wto.int/english/tratop_e/sps_e/spsagr_e.htm << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information author: sohail h. khan1 affiliation: 1poultry research institute, pakistan correspondence to: sohail khan postal address: poultry research institute, murree road, shamsabad, rawalpindi 46000, pakistan dates: received: 04 nov. 2013 accepted: 12 feb. 2014 published: 15 apr. 2014 how to cite this article: khan, s.h., 2014, ‘the use of green tea (camellia sinensis) as a phytogenic substance in poultry diets’, onderstepoort journal of veterinary research 81(1), art. #706, 8 pages http://dx.doi.org/10.4102/ ojvr.v81i2.706 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the use of green tea (camellia sinensis) as a phytogenic substance in poultry diets in this review article... open access • abstract • introduction    • chemical components       • amino acids       • carbohydrates       • minerals • anticoccidial and antimicrobial effects of green tea    • anticoccidial effects    • antiviral effects    • effects on intestinal flora • effect of green tea on broilers' performance • effect of green tea on layers' performance • effect of green tea on blood and egg yolk constituents • conclusion • acknowledgement    • competing interests • references abstract top ↑ this review examined the use of green tea (camellia sinensis) in the diets of poultry. research findings were obtained from various recent studies, where much attention was focused on the role of green tea in the promotion of both animal and human health. the review involved some of the currently available information about green tea, pertaining to its chemical composition, anticoccidial and antimicrobial effect, effect on broiler and layer performance and on blood and egg yolk constituents. to the author’s knowledge this is the first review paper on this topic. it will be helpful for poultry nutritionists and the poultry industry, although more detailed studies are still needed to elucidate the effects of green tea in poultry nutrition under various circumstances. introduction top ↑ as a result of the ban on the use of antibiotic growth promoters in animal diets in the european union as from january 2006, and the growing pressure on livestock producers in other parts of the world, alternative substances and strategies for animal growth promotion are being investigated. in this context, phytogenic and herbal products have received increased attention, because as natural additives they have a high acceptability amongst consumers (toghyani et al. 2010). phytogenic feed additives (also called phytobiotics or botanicals) are commonly defined as plant-derived compounds. they are incorporated into diets to improve the productivity of livestock through amelioration of feed properties, promotion of the animals’ production performance, and the quality of food derived from those animals (windisch et al. 2007). phytogenic feed additives comprise a wide variety of herbs (flowering, non-woody and non-persistent plants), spices (herbs with an intense smell or taste commonly added to human food) and essential oils (volatile lipophilic compounds derived by cold expression or by steam or alcohol distillation), or oleoresins (extracts derived by nonaqueous solvents). this class of feed additives has recently enjoyed increasing interest, especially for use in swine and poultry, as can be seen from a significant increase in the number of scientific publications from 2000 onwards. green tea (camellia sinensis) is also included in the list of phytogenic substances.next to water, tea is one of the world’s most highly consumed beverages and it has attracted much attention in recent years due to its numerous health benefits (perumalla & hettiarachchy 2011). tea was accidentally discovered by shen nung, a chinese emperor, in 2737 bc. when some leaves fell into boiling water, producing a distinct taste and fragrance, it was pronounced to be heavensent by the emperor (wheeler & wheeler 2004). tea (camellia sinensis l.) is a perennial, evergreen and cross-pollinated plant bearing white flowers and green fruits with two to three seeds. the difference in the flavour, colour and name of the tea depends on how the leaves are processed. there are four basic types of tea: black tea (tea leaves that are exposed to the air and allowed to fully oxidise or ferment, changing the leaves from green to black); oolong tea (it falls between black and green tea); green tea (less processed and not fermented); and white tea (the least processed of all teas). the polyphenolic compounds of green tea have been shown to improve body weight gain and feed efficiency in pigs (hossain, seok & chul 2012), cattle (sarker et al. 2010b) and broilers (biswas & wakita 2001a). ensiled or oven-dried green tea by-products have also been evaluated in goats for their nutritive potential as protein feedstuffs (kondo, kazumi & hiro-omi 2007). these compounds also maintain microflora balance and exhibit antimicrobial effects against pathogenic bacteria (guray et al. 2011; hara-kudo et al. 2005). it has been reported that green tea polyphenols have strong anti-oxidative properties (nishida et al. 2006); this is demonstrated by a reduction in thiobarbituric acid reactive substances (tbars) values and by maintaining the oxidative stability of broiler meat (yang et al. 2003) and egg yolk (uuganbayar et al. 2005). the green tea leaves, their by-products, and tea polyphenols can be offered as an ingredient or as a supplement to broiler feed for reducing mortality in diseased birds (cao, karasawa & guo 2005) and to hens for improving laying performance (uuganbayar et al. 2005, 2006), and for reducing the cholesterol content of eggs. yang et al. (2003) also reported that cholesterol levels were decreased and fatty acids of plasma and meat were improved when the animals were fed different levels of green tea by-products. in poultry diets, green tea and its derivatives like green tea extract, green tea leaves, green tea by-products, green tea polyphenols and green tea flowers are supplemented for improving performance. the aim of this article was to review the most recent studies on the beneficial effects of green tea and to evaluate its potential interest as a component of poultry diets. chemical components the chemical composition and other properties of green tea are complex. abdo et al. (2010) found that air-dried green tea leaves contained 7.80% moisture, 92.20% dry matter, 82.40% organic matter, 18.15% crude protein, 8.72% ether extract, 19.32% crude fibre, 9.80% ash, 36.21% nitrogen free extract and 3002 kcal/kg calculated metabolisable energy (me). green tea has over 200 bioactive compounds and contains over 300 different substances (labdar 2010). the chemical composition of tea is multifaceted, consisting of polyphenols (catechins and flavanoids), alkaloids (caffeine, threobromine, theophylline), volatile oils, polysaccharides, amino acids, lipids, vitamin c, minerals and other uncharacterised compounds (karori et al. 2007). amino acids green tea contains many amino acids, but l-theanine, specific to the tea plant, is the most abundant, accounting for 50.00% of the total amino acids. this form of theanine acts as an antioxidant, protecting cells from free radical damage, and also helping to induce relaxation and prevent anxiety by increasing serotonin and dopamine levels in nerve cells. amino acid degradation is involved in the biogenesis of the tea aroma (balentine 1997). abdo et al. (2010) reported that green tea leaves contained amino acids as 1.35% aspartic, 0.64% therionine, 0.67% serine, 1.98% glutamic, 0.68% proline, 0.76% glycine, 0.78% alanine, 0.78% valine, 0.55% isoleucine, 1.17% leucine, 0.57% tyrosine, 0.78% phenylalanine, 0.32% histidine, 0.85% lysine, 0.74% argnine, 0.37% methionine and 0.14% cystine. more than 600 different molecules of volatile fractions of tea have been isolated. these include terpenoids and degradation products of amino acids, carotenoids and linoleic acid (hara et al. 1995). carbohydrates amongst the bio-active compounds found in green tea, the largest component present in green tea leaves is carbohydrates (cellulosic fibre). the simplest compounds are catechins, a group of flavanoids called flavan-3-ols (yilmaz 2006). these catechins are synthesised in tea leaves through malonic acid and shikimic acid metabolic pathways with gallic acid as an intermediate derivative (naidu 2000). catechins are colourless, water-soluble compounds that impart bitterness and astringency to green tea infusions (wang et al. 2000). catechins constitute 15.00% to 30.00% of dry weight of green tea leaves, as opposed to 8.00% to 20.00% of oolong and 3.00% to 10.00% of black tea (amidor 2009). green tea extract contains six primary catechins, namely epicatechin (ec), epicatechin gallate (ecg), epigallocatechin (egc) and epigallocatechin-3-gallate (egcg) (kajiya et al. 2004). epigallocatechin-3-gallate is the most important and well-studied tea catechin owing to its high content (50.00%) in tea. it also has the most potent physiological properties in comparison to other components (taylor et al. 2005). ester-type catechins ecg and egcg are more bitter and astringent than ec and egc and these flavanoids have a greater synergistic action than individual tea components (fujiki 1999). uuganbayar et al. (2006) reported that total catechin contents were 15.73% for korean green tea, 15.60% for japanese green tea and 14.04% for chinese green tea. of the total catechin components, egc was predominant, accounting for 67.50% (korean green tea), 67.90% (japanese green tea) and 63.80% (chinese green tea) of the total catechin contents. in another study, green tea leaves contained active constituents of 1.01% total phenols, 105 mg/kg caffeine, 50 mg/kg catechin, 35 mg/kg ec, 185 mg/kg ecg and 17.5 mg/kg ascorbic acid (abdo et al. 2010). the catechin content showed considerable variability in one study. the levels of egcg ranged from 117 mg/l – 442 mg/l, egc from 203 mg/l – 471 mg/l, ecg from 16.9 mg/l – 150.0 mg/l, ec from 25 mg/l – 81 mg/l and catechin from 9.03 mg/l – 115.00 mg/l. moreover, caffeine contents in the green tea infusions studied were between 141 mg/l and 338 mg/l (reto et al. 2007). minerals minerals comprise about 4.00% – 9.00% of the inorganic matter of tea (chaturvedula & prakash 2011). abdo et al. (2010) assayed the mineral content profile in green tea leaves as 4.66% calcium, 1.62% total phosphorus, 865.1 mg/kg manganese, 146.3 mg/kg zinc and 858.1 mg/kg selenium. similarly, reto et al. (2007) evaluated some minerals in green tea samples and reported that potassium was found in larger amounts (92 mg/l – 151 mg/l), whereas the content of sodium, calcium, fluoride, aluminium, manganese and iron were 35 mg/l – 69 mg/l, 1.9 mg/l – 3.5 mg/l, 0.8 mg/l – 2.0 mg/l, 1.0 mg/l – 2.2 mg/l, 0.52 mg/l – 1.90 mg/l, 0.020 mg/l – 0.128 mg/l, respectively. costa, gouveia and nobrega (2002) observed large variations in the mineral content (aluminium, calcium, magnesium and manganese) in green tea from different origins. shu et al. (2003) observed marked variations amongst different tea varieties in accumulating fluoride and aluminium. xu et al. (2003) reported that the content of selenium in green teas was greatly increased by foliar application of selenium-enriched fertilisers; moreover, the selenium-enriched green tea exhibited significantly higher antioxidant activity than regular green tea. anticoccidial and antimicrobial effects of green tea top ↑ anticoccidial effects positive effects of green tea have been demonstrated in poultry diseases including coccidiosis (jang et al. 2007) and avian influenza (deryabin et al. 2008; song et al. 2007). seven species of eimeria (an intracellular protozoan) belonging to the phylum apicomplexa cause avian coccidiosis, which is an economically important disease in the poultry industry. eimeria infection causes extensive destruction of the intestinal epithelium that results in reduced feed efficiency, body weight gain and a temporary reduction in egg production (dalloul & lillehoj 2005).to date, only one study demonstrating an anticoccidial effect of green tea on eimeria parasites has been published (jang et al. 2007). the results showed that green tea (0.5% and 2.0%) fed to five-week-old chickens for two weeks prior to infection with eimeria maxima (e. maxima) (10 000 sporulated oocysts per bird) significantly (p < 0.05) reduced shedding of oocysts in faeces by 38.5% and 51.5%, respectively. the green tea-based diet, however, did not improve body weight loss due to infection with e. maxima. in general, the higher concentration of green tea supplementation showed a greater protective effect and reduction in faecal oocyst shedding. in this regard, green tea components have shown anti-parasitic activities in vitro by inhibiting egg hatching and larval development and inactivating the infective larvae of teladorsagia circumcincta and trichostrongylus colubriformis (molan et al. 2003, 2004). in view of the fact that hosts are infected by contaminated litter in the chicken houses, significant reduction of faecal oocyst shedding by green tea in the diet would lead to less environmental contamination by coccidia parasites. the fundamental mechanism responsible for green tea-mediated protection against coccidiosis is as yet unknown. however, many cytokines are known to elicit a protective cell-mediated immune response against intracellular pathogens including coccidia. an increased level of ifn-γ, in particular, has been associated with protective immunity against avian coccidiosis (yun et al. 2000). this subject needs to be further investigated in the other species of coccidia that infect chickens. antiviral effects numerous outbreaks of avian influenza virus infection (a/h5n1) have occurred recently, infecting domestic birds, chicken and ducks. the possibility of the emergence of a new strain of influenza virus capable of causing a pandemic in humans is potentially high and no vaccine effective against such a strain currently exists. song et al. (2007) synthesised catechin derivatives with different alkyl chain lengths and aromatic ring substitutions at the 3-hydroxyl group from egc and catechin and their anti-influenza-viral activity was evaluated in vitro and in ovo. marked antiviral activity was observed for derivatives carrying moderate chain lengths (7–9 carbons) compared to those with aromatic rings, whereas the 5′-hydroxyl group of the trihydroxy-benzyl moiety did not significantly contribute to antiviral activity. the derivatives exerted inhibitory effects for all six influenza subtypes tested, including three major types of currently circulating human influenza viruses (a/h1n1, a/h3n2 and b type), h2n2 and h9n2 avian influenza virus. the compounds strongly inhibited adsorption of the viruses on red blood cells. they also restricted the growth of avian influenza virus in ovo, with a minimum inhibition concentration of 5 μm to 10 μm; this far exceeds that of the neuraminidase inhibitor oseltamivir or m2 proton channel inhibitor amantadine. the antiviral activity appears to be mediated by interaction with haemagglutinin (ha) or viral membrane, rendering ha less fusogenic at the initial stage of infection. the broad spectrum activity against various subtypes of influenza viruses may complement the limitations of current antivirals and contribute to managing potentially emerging influenza pandemics. the structure-activity data of catechin derivatives may usefully guide future research endeavours for applying green tea catechins as alternative anti-viral agents. subsequently deryabin et al. (2008) investigated the potential anti-viral activity of a unique nutrient mixture (nm) (containing green tea extract, lysine, proline, ascorbic acid, n-acetyl cysteine and selenium, amongst other micronutrients) and its components on a/h5n1 at viral dosages of 1.0, 0.1 and 0.01 tcid50. antiviral activity was studied in cultured cell lines pk, bhk-21, and vero-e6. virus lysing activity was determined by co-incubation of virus a/h5n1 with nm for 0 minutes to 60 minutes, followed by residual virulence titration in cultured spev or bhk-21 cells. the nm demonstrated high antiviral activity evident even at prolonged periods after infection. its antiviral properties were comparable to those of conventional drugs (amantadine and oseltamivir); however, nm had the advantage of affecting viral replication at the late stages of the infection process. effects on intestinal flora the caecum is one of the areas of greatest microbial activity in the gastrointestinal tract of chickens. intestinal microflora plays an important role in the health status of host animals. therefore, a common approach to maintain host gut health is to increase the number of desirable bacteria in order to inhibit colonisation by invading pathogens (guo et al. 2004). terada et al. (1993) studied the effects of green tea polyphenols (gtp) on the caecal flora in eight 24-day-old and eight 56-day-old chickens that had consumed a basal diet or a diet supplemented with tea polyphenols (2 g/kg diet) for periods of up to 56 days. on day 24 of the experiment, the number of total bacteria and bacteroidaceae significantly decreased, but staphylococci increased. the frequency of the occurrences of pseudomonads and yeasts significantly increased, but that of moulds decreased. this may be associated with the fact that the caecal flora of the chicken is not established until four weeks of age, as reported by barnes et al. (1972). on day 56, lactobacilli increased to some extent, but enterobacteriaceae, including the detection rate of proteus, showed a significant decrease. it seems that the activities of the intestinal flora may lead to lower ph and a decrease in caecal putrefactive bacteria such as proteus. cao et al. (2005) observed the effects of gtp supplement on counts of caecal microflora in female broiler chickens fed on semi-purified diets from 28 days to 42 days of age. they found that the counts of bifidobacteria, bacteroidaceae, peptococcaceae, lactobacilli, eubacteria and lecithinase-positive bacteria (clostridia, streptococci, staphylococci and bacilli) in the gtp group were lower than those of the control group. they reported that green tea and its chemical components show antibiotic-like effects of non-selectively decreasing total counts of all microflora. murali et al. (2012) evaluated the effect of marinating chicken breast fillets with extracts of lemon, green tea and turmeric against campylobacter jejuni (c. jejuni) and salmonella enteritidis. this combination was found to be most effective against c. jejuni and salmonella enteritidis. all the bacteria were killed within 12 hours of incubation, there was a five-log reduction in growth within one hour of incubation, and it proved to be more effective than any of the extracts used alone. furthermore, combination of both lemon and green tea killed all c. jejuni isolates, but not salmonella enteritidis, within 24 hours of incubation. polyphenols extracted from green tea have been shown to have inhibitory effects on gram-positive bacteria as well as gram-negative bacteria (gadang et al. 2008). the efficacy of green tea extract against various bacterial strains can be related to differences in cell membranes (ikigai et al. 1993). yoda et al. (2004) investigated the antibacterial activities of egcg on various strains of staphylococcus (gram positive cocci) and gram negative rods including escherichia coli, klebsiella pneumoniae and salmonella. they found that 50 μg/ml – 100 μg/ml was required to inhibit growth of staphylococcus and concentrations higher than 800 μg/ml were required to inhibit gram-negative rods. hara-kudo et al. (2005) studied the antibacterial effects of catechins (major gtp) using clostridium and bacillus spores. incubation with crude catechins decreased the number of clostridium botulinum (c. botulinum) and clostridium butyricum (c. butyricum) spores, but not bacillus cereus (b. cereus) spores. furthermore, the effects of six catechin derivatives on spores were investigated. the epicatechin gallate, egc, egc and gallocatechin gallate (gc) were more effective in decreasing c. botulinum and c. butyricum spore numbers than catechin and ec. the vegetative growth of c. botulinum and b. cereus was inhibited by crude extracts of the catechins. specifically, purified gc and egc inhibited the vegetative growth of c. botulinum and b. cereus. the inhibitory effect of ec on b. cereus was similar to that of gc. however, toxin-production by b. cereus was not inhibited by catechin. damage to the membrane of c. butyricum spores by catechin derivatives was shown using fluorescent microscopy. this study showed that low concentrations of catechins, although requiring a long exposure time, inhibited the growth of bacterial spores. however, the effects of the purified derivatives of the catechins were not the same and gc and egc were found to be the most potent. spores that are generally resistant to many disinfectants were sensitive to catechins. in another study, amongst the catechins present in green tea extract, egcg and ecg were the most potent in exhibiting antimicrobial activity due to the galloyl moiety present in their structures (shimamura, zhao & hu 2007). effect of green tea on broilers’ performance top ↑ in this review, it was found that green tea inclusion in broiler diets had positive effects on growth performance and lean meat production of the broilers. biswas and wakita (2001a) added four levels of green tea powder (0.50%, 0.75%, 1.00% and 1.50%) to broiler starter and finisher diets. supplemental green tea powder tended to decrease feed intake and body weight gain at a higher dose, but tended to improve fcr. dressing percentage was not affected by green tea, although proportions of some parts of the carcass were influenced. the proportion of thigh meat was increased by the 1.50% level feed while that of wing meat was decreased in all treatment groups. the quantity and percentage of abdominal fat were decreased significantly with supplementation. similar findings, namely that when the green tea by-product level was increased the percentage of abdominal fat decreased in broilers, were reported by yang et al. (2003) and guray et al. (2011). kaneko et al. (2001) reported that 1.00%, 2.50% and 5.00% of green tea in broiler diets linearly reduced body weight gain of the chicks. similarly, uuganbayar (2004) also reported that 1.00% to 1.50% green tea supplement in broiler diet had the effect of reducing body weight gain of the chicks. yang et al. (2003) determined the optimum level of green tea by-product (0.50%, 1.00% and 2.00%) in diets without antibiotics and evaluated its effect on broiler performances. they observed non-significant differences in feed intake and feed efficiency amongst treatments. similar results were reported by cao et al. (2005) indicating that body weight gain, feed intake and feed efficiency from 28 days to 42 days of age were not improved; however, mortality was significantly reduced by supplementation with green tea by-products. recently, shomali, najmeh and saeed (2012) investigated the effects of high levels of green tea powder (1.0%, 2.0%, or 4.0%) on broiler growth performance for two weeks. differences in body weight, feed intake and fcr were insignificant as well. in contrast to the above studies, sarker, kim and yang (2010a) observed significantly increased weight gain (1210.61 g/bird) in broilers during the finishing period at the 0.5% level compared to the 1.0% (1033.36 g/bird) level of green tea. guray et al. (2011) supplemented a liquid hydroalcoholic extract of fresh green tea (0.1 g/kg or 0.2 g/kg) in broiler diets. the dietary green tea extract increased the body weight, feed efficiency, carcass weight and dressing percentage. the broilers in green tea supplemented groups consumed more feed than the control birds throughout the entire experimental period. the relative gut length of broilers in the high level of green tea group tended to be lower than those in the control group. the dietary green tea extract increased redness (1) and yellowness (2) values of the breast meat. thus, the green tea extract appeared to have a measurable impact on cie colour values of the breast meat in broilers. the authors concluded that the improved production results in the broilers with added green tea extract are directly connected with physiological mechanisms such as the regulation of the caecal microflora. the production sources of green tea used in all of the above studies were different, for example: japanese green tea powder (biswas & wakita 2001a; kaneko et al. 2001); japanese and chinese green tea or their polyphenols (cao et al. 2005); korean green tea powder (sarker et al. 2010a; yang et al. 2003); and eastern black sea coast of turkey tea powder (guray et al. 2011). all of these green tea sources had different compositions. the inconsistency amongst the studies may be explained by the differences in total catechin content and its major components such as epicatechin, epigallocatechin, epicatechin gallette, epigallocatechin gallette, of the green tea and green tea extract used in these studies. effect of green tea on layers’ performance top ↑ yamane et al. (1999) reported that 0.67% japanese green tea extracts mixed with drinking water slightly reduced the egg weight of the hens. similarly, biswas and wakita (2001b) also reported that 0.30% green tea powder lowered average egg weight. in contrast to these results, uuganbayar et al. (2005) observed that up to 2.00% green tea powder in layer diets had no adverse effect on egg production rate and egg weight compared with the control. sadao and yuko (2008) found that there was no significant difference in egg weight, rate of egg production or egg mass between the control group and the group of laying hens fed on diets supplemented with 1.00% green tea, while 5.00% and 10.00% resulted in the lowest values. similar results were obtained by biswas et al. (2000), who reported that the addition of 0.60% japanese green tea to the layer diet had no effect on egg production rate of hens in a long-term feeding experiment. recently, ariana et al. (2011) observed that supplementation of layer diets with 0.50% extract of green tea and 1.50% powder of green tea had no significant effects on feed intake, egg production and egg weight.in contrast to above studies, yang et al. (2003) reported that hens fed diets containing 4.00% and 6.00% green tea by-product showed a significantly increased egg production rate. al-harthi (2004) reported that the addition of 0.20% green tea yielded significantly better egg production and egg mass than the control group. abdel-azeem (2005) reported that the addition of powdered green tea flowers at 0.25%, 0.50% and 0.75% to a japanese quail grower diet improved feed conversion, especially at the 0.75 % level. he attributed the effect on feed conversion to the biological role of flavonoids present in green tea in terms of antimicrobial, antifungal, antiseptic and anti-inflammatory activities or the role of green tea as an antioxidant. uuganbayar et al. (2006) compared the effects of two levels (1.00% or 2.00%) each of korean, japanese and chinese green tea on laying performance. the results showed that egg production rates of the layers that were fed diets containing 1.00% or 2.00% green tea powders were significantly increased compared to that of the control. the feed intake was significantly decreased in korean green tea and chinese green tea groups at 2.00% inclusion levels. they concluded that incorporation of 1.00% or 2.00% korean, japanese and chinese green tea into layer diets regardless of origin had favourable effects on laying performance. sahin et al. (2010) found that 200 mg or 400 mg of egcg exerted antioxidant effects and linearly improved feed intake from 29.6 g/day to 30.9 g/day and egg production from 84.30%/day to 90.10%/day in heat-stressed quails. abdo et al. (2010) investigated the effect of adding green tea leaves (1.00% to 5.00%) and its aqueous extract (0.5 l/100 kg to 2.5 l/100 kg of ration) to laying-hen diets. the results revealed that the improvements in egg production, egg mass and feed conversion values due to 1.00% green tea leaves compared to the control were 5.59%, 6.79% and 7.84%, respectively. the corresponding level (0.5 l/100 kg diet) of hot water green tea extract resulted in improvements of 6.78%, 7.46% and 8.65%, respectively. egg quality also improved with supplementation of green tea in layer diets. biswas and wakita (2001a) found that a low level of green tea powder (0.3%) improved the haugh unit, and albumen percentage was higher, but yolk percentage was lower in the green tea powder group. uuganbayar et al. (2005) found that eggshell thickness was reduced significantly in the layer group fed the diets containing green tea powder regardless of dietary levels (0.5%, 1.0%, 1.5% and 2.0%). the yolk colour score (yellowness of egg yolk) was increased in the layers fed the 2.0% green tea diet compared with that of the control diet. later, uuganbayar et al. (2006) reported that the eggshell thickness and shape index were significantly reduced in layers fed 1.0% or 2.0% japanese green tea diets compared to that of the control. however, there were no significant differences in eggshell thickness of layers fed diets containing korean and chinese green tea inclusions compared to that of the control. sensory evaluation in terms of juiciness, texture, flavour and acceptability of boiled eggs did not vary between the green tea-supplemented treatments and control treatments. the albumen index, yolk index and haugh unit of the eggs were no different between green tea and control treatments. however, yamane et al. (1999) and biswas et al. (2000) reported that japanese green tea inclusion in the layer diet improved the haugh unit score of the eggs. they indicated that the improvement of the haugh unit score with green tea feeding was accompanied by a greater albumen height and physical stability of egg albumen. bravo (1998) showed that albumen tended to be thicker in groups fed green tea powder and attributed this effect to the possible transfer of green tea powder polyphenols into β-ovomucin. the β-ovomucin increases albumen durability by forming complexes with proteins and polysaccharides. on other hand, sadao and yuko (2008) reported that there were no significant differences in the yolk colour fan score or strength of the eggshell for four treatments (green tea powder added 0.00%, 1.00%, 5.00% and 10.00% of the hen diets). however, they observed that eggshell strength, thickness of the eggshell and haugh unit values decreased with increasing green tea powder intake, especially in the 10.00% group. abdo et al. (2010) reported that more than 1.00% green tea leaves (3.00% and 5.00%) and more than 0.5 l/100 kg diet green tea extract (1.5 l/100 kg and 2.5 l/100 kg) were required to improve both external and internal egg quality parameters significantly. the improvement in shell thickness values due to 3.00% green tea leaves or the corresponding level (1.5 l/100 kg diet) of aqueous extract compared to the control was 6.88%. yolk colour score was not influenced significantly by any of the treatments. however, there was a steady increase in yolk colour score with increasing levels of green tea leaves. regarding yolk colour, the results revealed that there was a 13.29% increase of β-carotene in animals fed green tea leaves and according to naber (1979), β-carotene is an important precursor for vitamin a, which upon colourisation is categorised as yellow. however, the amount found in egg yolks is only one tenth of the amount of xanthophylls, so its influence on colour is reported to be insignificant. recently, ariana et al. (2011) found that egg quality such as yolk colour, albumen height, haugh units, eggshell breaking strength and shell thickness were unaffected by 0.50% extract of green tea and 1.50% powder of green tea. however, yolk weight and yolk index were significantly improved by the green tea treatments. effect of green tea on blood and egg yolk constituents top ↑ el-deek and al-harthi (2004) showed that the addition of green tea at 5 g/kg of broiler feed had no significant effect on the chemical composition of plasma total lipids, cholesterol, plasma aspartate aminotransferase and alanine aminotransferase activities. abdel-azeem (2005) found that the addition of powdered green tea flowers at 0.25%, 0.50% and 0.75% to growing japanese quail diets improved antibody titre production. they also found that raising the level of green tea from 0.25% to 0.75% significantly decreased blood lipid fractions and increased high-density lipoprotein (hdl). yang et al. (2003) reported that the addition of green tea by-product to diets tended to decrease blood low-density lipoprotein (ldl) cholesterol content compared to the control group, although there were no significant differences amongst treatments. the addition of green tea by-product increased docosahexaenoic acid (dha) in blood plasma and tended to decrease the cholesterol content in chicken meat, but a significant difference was not observed. an optimum level of dha, belonging to the omega3 fatty acids group, may play an important role in the control of blood pressure and blood cholesterol content. abdo et al. (2010) reported that the addition of 3.00% and 5.00% green tea leaves, or between 0.5 l/100 kg and 2.5 l/100 kg green tea extract to hens’ diets significantly dicreased both total blood plasma cholesterol and total lipids. they also found that 1.5 l/100 kg green tea extract was required to increase the beneficial blood plasma hdl compared with the control. the reduction in blood plasma cholesterol and the increase in blood plasma hdl due to 1.5 l/100 kg diet of hot water green tea extract, compared to the control, were 4.66% and 7.14%, respectively. in another study, it was observed that adding green tea extracts to the layers’ diets significantly reduced serum cholesterol (130.8 g/dl vs 145.9 g/dl) and triglycerides (1291.1 g/dl vs 1393.0 g/dl) compared to the control. the ratios of hdl to cholesterol and hdl to ldl were significantly greater in treatment groups than those of the control group (ariana et al. 2011).green tea supplementation of layer diets reduced the cholesterol content of the egg yolk (biswass & wakita 2000). yamane et al. (1999) demonstrated that the fat content of egg yolks significantly decreased. however, unganbayar et al. (2005) reported no significant differences in the cholesterol contents of egg yolks between layers that were fed diets containing 0.5%, 1.0% and 1.5% green tea powder and control diets. they did find that administration of 2.0% green tea suppressed egg yolk cholesterol significantly compared to that of the positive control treatment. uuganbayar et al. (2006) reported that the total cholesterol content of egg yolks was slightly reduced in the layers fed 1.0% or 2.0% korean and japanese green tea diets, but without significant differences. amongst green tea treatments, chinese green tea treatments at both 1.0% and 2.0% inclusion levels significantly lowered egg yolk cholesterol content compared to that of the control. ariana et al. (2011) also found that egg yolk cholesterol and triglyceride contents were reduced by green tea supplementation. the caffeine and catechin content of green tea may have an inhibitory effect on intestinal absorption of lipids (koo & sang 2007). therefore, a green tea diet for birds may prevent an excessive accumulation of lipids in the liver and other tissues, including the egg yolk. the exact mechanism of how green tea by-product decreases yolk cholesterol is as yet unknown. cholesterol is mainly biosynthesised in the liver of laying hens and integrated into vitellogenin and very lowdensity lipoprotein particles, which are secreted into the bloodstream and then taken up by growing oocytes through receptor-mediated endocytosis (elkin 2006). therefore, it has been suggested that the decrease in the egg yolk cholesterol is dependent on diminution in cholesterol synthesised in the liver. hence, the decrease in total lipid and cholesterol may be due to the effect of green tea by-products on hepatic 3-hydroxy-3-methylglutaryl coenzyme a reductase that is required for cholesterol synthesis in the liver (ariana et al. 2011). the conversion of cholesterol to bile acids occurs solely in the liver and represents the main pathway for the removal of cholesterol from the body. this may also explain the reduced in cholesterol levels. liver weight and liver cholesterol content may also be explained by the disorderly effect of tea catechins on micelle formation. bile acids can be reabsorbed from the small intestine only in the form of micelles. green tea catechins prevent the reabsorption of bile acids by disturbing micelle formation and thus increasing bile acid excretion. in order to replenish the loss in bile acids, the conversion of cholesterol to bile acids in the liver will be enhanced and this further decreases the content of liver cholesterol (myant & mitropoulos 1977), which might affect the total cholesterol content of the egg yolk. conclusion top ↑ based on the results of 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journal of infection and chemotherapy 10, 55−58. http://dx.doi.org/10.1007/s10156-003-0284-0 yun, c.h., lillehoj, h.s., zhu, j., min, w., 2000, ‘kinetic differences in intestinal and systemic interferon-gamma and antigen-specific antibodies in chickens experimentally infected with eimeria maxima’, avian diseases 44, 305–312. http://dx.doi.org/10.2307/1592544 article information authors: joe kgaladi1 louis h. nel1 wanda markotter1 affiliation: 1department of microbiology and plant pathology, university of pretoria, south africa correspondence to: wanda markotter postal address: department of microbiology and plant pathology, university of pretoria, pretoria 0002, south africa dates: received: 27 aug. 2012 accepted: 24 nov. 2012 published: 08 mar. 2013 how to cite this article: kgaladi, j., nel, l.h. & markotter, w., 2013, ‘comparison of pathogenic domains of rabies and african rabies-related lyssaviruses and pathogenicity observed in mice’, onderstepoort journal of veterinary research 80(1), art. #511, 13 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.511 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. comparison of pathogenic domains of rabies and african rabies-related lyssaviruses and pathogenicity observed in mice in this original research... open access • abstract • introduction • materials and methods    • virus isolates    • sequencing and comparison of the ucleoprotein, phosphoprotein, matrix protein and glycoprotein coding regions of several lyssavirus isolates    • virus amplification and titration    • experimental infections    • collection of brain, salivary glands and blood    • fluorescent antibody test and taqman real-time polymerase chain reaction to determine the presence and quantity of viral antigen and viral rna    • rapid fluorescent focus inhibition test    • ethical considerations • results    • comparison of pathogenic domains       • the nucleoprotein       • the phosphoprotein       • the matrix protein       • the glycoprotein    • pathogenicity of representative african lyssaviruses in mice       • mortality of mice inoculated intramuscular with a dose of 105 tcid50       • effect of inoculation dose on mortality of mice       • comparison of the pathogenicity of representative phylogroup ii lyssaviruses       • comparison of the pathogenicity of cell culture and original bat brain material of lbv (lbvsa2008)    • viral rna concentration in organs and blood    • correlation between pathogenic domains and mortality of mice    • serological response of mice after inoculation with different lyssavirus isolates • discussion    • pathogenicity of different african lyssavirus isolates inoculated intramuscular in mice    • viral rna in the blood of mice infected with african lyssaviruses    • viral rna load in the brains and salivary glands of mice infected with african lyssaviruses    • serological response of mice to infection with african lyssaviruses    • relationship between pathogenic domains and pathogenicity profiles observed in mice       • nucleoprotein and phosphoprotein       • matrix protein       • glycoprotein       • combined effects of pathogenic domains on mortality • conclusion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ several lyssavirus species occur in africa (rabies virus, lagos bat virus, mokola virus, duvenhage virus, shimoni bat virus and ikoma lyssavirus), displaying a high sequence diversity between isolates belonging to the same species. there is limited information about comparative pathogenesis of these african lyssaviruses and this precludes authoritative opinion on the potential public and veterinary health impact. in this study, an analysis of representative african lyssaviruses attempted to correlate viral genomic sequence similarities and differences with the corresponding pathogenic profiles observed in mice. the study demonstrated that the virus isolates evaluated could be lethal to mice when introduced intramuscularly and that different isolates of the same lyssavirus species differ in their virulence. using real-time polymerase chain reaction (pcr), viral rna was detected in brain tissue, but no viral rna was detected in the salivary glands or blood of mice that succumbed to infection. comparison of known pathogenic domains indicated that pathogenicity is likely to be dependent on multiple domains. cumulatively, our results re-emphasised the realisation that the pathogenicity of a lyssavirus species cannot be deduced based on studies of only a single isolate of the species or a single pathogenic domain. introduction top ↑ rabies is a fatal disease of mammals, including humans, and is caused by viruses of the genus lyssavirus in the family rhabdoviridae, order mononegavirales. currently, the lyssavirus genus consists of twelve classified species (ictv official taxonomy: updates since the 8th report), of which five (rabies virus [rabv], lagos bat virus [lbv], mokola virus [mokv] and duvenhage virus [duvv]) have been isolated in africa. two putative lyssavirus species, shimoni bat virus and ikoma lyssavirus, have recently been isolated from a bat (hipposideros commersoni) in kenya and an african civet (civettictis civetta) in tanzania, respectively (kuzmin et al. 2010; marston et al. 2012). lbv, mokv and duvv probably occur exclusively in africa, as encounters with these viruses have not been reported from elsewhere. the african rabies-related lyssaviruses are characterised by the diversity of the limited number of isolates available within each of the virus species. in southern africa, rabv is present in the form of two distinct variants, adapted to canines (canid variant) and to herpestids (mongoose variant) (nel et al. 2005). despite availability of vaccines and other rabies control measures, rabv is estimated to cause over 55 000 human deaths each year – mostly in developing countries of africa and asia (knobel et al. 2005). there are five different clusters of mongoose variant viruses in southern africa (nel et al. 2005). the clusters are defined by the geographic regions in which the viruses were isolated. isolates from more than one different mongoose species were found within a cluster. cohen et al. (2007) reported six different clusters of canid variant viruses in southern africa. the clusters were defined by geographic location and included virus isolates from four provinces of south africa. four lineages (a-d) have been described for lbv (kuzmin et al. 2010; markotter et al. 2008). it was indicated that lineage a only shares 79.1% nucleoprotein gene nucleotide identity with lineage b and c (markotter et al. 2008) whilst lineage d only shares from 79.5% to 80.9% identity to all other lbv lineages (kuzmin et al. 2010). duvv isolates show very close sequence homology in southern africa but duvv isolated in kenya in 2006 constitutes a new lineage (van eeden, markotter & nel 2011). for mokv several different phylogenetic groupings were observed based on geographical location (sabeta et al. 2010).pathogenesis studies of rabv (canid variant) as well as other lyssavirus species such as european bat lyssavirus-1 (eblv-1), european bat lyssavirus-2 (eblv-2) and australian bat lyssavirus (ablv) in animal models are reported regularly (dietzschold, faber & schnell 2008). however, studies on the pathogenesis of lyssaviruses known to occur in africa are limited. although rabv is associated with most of the human and animal rabies cases reported each year, in recent years, increases in lbv, duvv and mokv infections have been reported (markotter et al. 2008; sabeta et al. 2010; van eeden et al. 2011). improved surveillance correlates with an increase in the number of cases. surveillance in the kwazulu-natal province of south africa between 2003 and 2006 resulted in a number of lbv isolates in fruit bats (epomophorus wahlbergi) (markotter et al. 2008). in experimental models, lyssaviruses are lethal when inoculated intracranially but other routes of infection such as intramuscular (i.m.), which is representative of natural infection through a bite, depend on the virus isolate as well as the dose of inoculum (badrane et al. 2001; markotter et al. 2009). badrane et al. (2001) showed that lyssaviruses can be divided into two phylogroups, namely phylogroup i (rabv, duvv, elbv-1, eblv-2 and ablv) and phylogroup ii (lbv and mokv). phylogroup i was reported to be lethal through the intracranial (i.c.) and i.m. routes whilst initially phylogroup ii was lethal only through the i.c. route in mice. one isolate as a representative of lbv and mokv administered at a dose of 3 x 105 ld50 and 3 x 107 ld50, respectively, was used. this led to the assumption that these viruses are less lethal and less of a public and veterinary health threat. however, kemp et al. (1973) reported that shrews succumb to mokv when inoculated via the i.m., subcutaneous and oral route of inoculation. recently, mice were reported to succumb to lbv infection, with lbv isolates administered i.m. in doses of 103, 106 and 104.9 ld50 (kuzmin et al. 2008; markotter et al. 2009). the same was observed for one isolate of mokv administered at a dose of 106 ld50 (markotter et al. 2009). some of the lbv isolates and the mokv isolate were not lethal at a lower dose of 103 ld50. it has been shown in experimental infections that inoculation with a high viral dose results in a higher percentage mortality than inoculation with low viral doses (kemp et al. 1973; markotter et al. 2009; niezgoda et al. 1997). lyssaviruses are commonly transmitted by a bite from a rabid animal, after which the virus is transported from the site of entry to the central nervous system where it multiplies to high amounts once in the brain. during the late stage of disease, centrifugal spread of the virus to non-neuronal tissues occurs (warrell & warrell 2004). rabv, lbv and mokv have been reported to be present in non-neuronal tissues of rabid animals: kidney, salivary glands, muscle, tongue, oral swab, ovary, vaginal swabs, adrenals, pancreas, liver, spleen and heart (jackson et al. 1999; kemp et al. 1973; kuzmin et al. 2008). viremia is considered not to occur in lyssavirus infected animals. however, viral rna was detected in the blood of clinically ill rabv infected mice and this should be further investigated (lodmell, dimcheff & ewalf 2006). virus neutralising antibodies (vna) have been shown to be important in clearance of rabv infection (hooper et al. 1998), but there are also other mechanisms that play a role in virus clearance. hooper et al. (1998) demonstrated that in addition to vna, inflammatory mechanisms also contribute to clearance of virus infection. coe and bell (1977) showed the presence of vna in syrian hamsters as early as day 5 post inoculation with rabv. however, clinical signs were observed from day 8 to 9 whilst death followed after 2–5 days of onset of symptoms. mice inoculated with rabv (silver haired bat variant) were shown to succumb to infection irrespective of production of high vna (roy et al. 2007). this was attributed to the inability of vna and other immune factors to cross the blood-brain barrier. not all animals that survive experimental infection develop vna (niezgoda et al. 1997; niezgoda et al. 1998). it has been suggested that the specific virus isolate as well as the dose of inoculation could influence production of vnas (niezgoda et al. 1997; niezgoda et al. 1998). the lyssavirus genome codes for five proteins, that is the nucleoprotein (n), phosphoprotein (p), matrix protein (m), glycoprotein (g) and rna polymerase (l). a number of domains on these proteins have been implicated in the pathogencity of these viruses. for example, the amino acids phe 273 and tyr 394 on the n protein of a pathogenic rabv strain have been shown to play a role in inhibition of interferon (ifn) system and chemokines (masatania et al. 2011). inhibition of ifn system and chemokines prevents expression of antiviral proteins at the site of infection, allowing the virus to be transported to the central nervous system. a k/rxtqt motif on the p protein was shown to be important for binding to the lc8 dynein light chain, therefore important for retrograde intracellular transport of the virus to the central nervous system (cns) (poisson et al. 2001). however, some studies showed that transport of rabv to the cns also occurred in mutants where this motif was changed (tan et al. 2007). mutation of the ppey motif on the m protein reduced pathogenicity of rabv when inoculated intranasally (i.n.) in mice (gholami et al. 2008). although domains important in pathogenicity have been indicated in several lyssavirus proteins, the g protein remains the most important. mutation of arg/lys 333 on the g protein was reported to result in non-pathogenic strains when inoculated i.m. (badrane et al. 2001; dietzschold et al. 1983). this position was thought to be the most important in determining pathogenicity. however, this assumption was challenged when the lethality of a rabv isolate could be reversed by an amino acid change elsewhere (asn 194 to lys) regardless of the amino acid present at position 333 in the g protein (faber et al. 2005). another rabv virus, the rc-hl virus strain, was shown not to be lethal in mice via i.c. and i.m. route irrespective of conservation of arg 333 (takayama-ito et al. 2006). however, this strain reverted to a lethal strain via the i.c. route when leu 268 was replaced by ile 268 (takayama-ito et al. 2006). different strains of mice as well as different passages of the virus strains were used in determining the importance of these pathogenic domains. mutations such as arg 333 on the g protein were shown to be important in pathogenicity using different passages and different strains of mice (coulon et al. 1998; dietzschold et al. 1983; seif et al. 1985). these results demonstrated the importance of comparing a number of pathogenic domains when studying the pathogenicity of specific lyssavirus isolates. in this study, the full coding regions of the n, p, m and g gene were sequenced for a total of 23 representative lyssavirus isolates, predominantly from southern africa. the pathogenicity of 11 of the 23 isolates was determined by i.m. inoculation of mice and pathogenic domains between isolates were compared. materials and methods top ↑ virus isolates a total of 23 lyssavirus isolates comprising of the four african lyssavirus species (rabv [mongoose and canid variants], lbv, mokv and duvv) were selected for this study (table 1) to represent, where possible the genetic diversity especially for southern africa, based on previous phylogenetic studies (cohen et al. 2007; markotter et al. 2008; nel et al. 2005). all isolates were passaged mouse brain tissue or passaged cell culture material (passage number unknown) except for lbvsa2008 which was obtained as original bat brain suspension. five-week-old national institute of health (nih) balb/c mice (south african vaccine producers, sandringham, south africa) were used for experimental infection. table 1: lyssavirus isolates used in this study for amino acid comparison of pathogenic domains and experimental infection of mice. sequencing and comparison of the ucleoprotein, phosphoprotein, matrix protein and glycoprotein coding regions of several lyssavirus isolates lyssavirus sequences were either determined by previous studies (markotter et al. 2008) or determined in this study using several primer sets and cycling conditions (available on request) (table 1). the pcr amplicons were purified using the wizard® pcr preps dna purification system (promega, madison, usa). purified pcr products were sequenced with the bigdyetm termination cycle sequencing ready reaction kit 3.1 (applied biosystems, california, usa) according to the manufacturer’s protocol and analysed on an abi 377 dna sequencer (applied biosystems, california, usa). pcr products were sequenced with both the forward and the reverse primers. the complete n, p, m and g genes were sequenced. the nucleotide sequences were converted to amino acids and aligned using bioedit sequence alignment editor v.7.0, clustalw subroutine (hall 1999). an alignment was made that encompasses domains on the n, p, m and g gene indicated to be important for pathogenicity. sequences of other rabv isolates were also included (table 1). virus amplification and titration ten per cent mouse brain suspensions were prepared in dulbecco’s modified eagle’s medium (dmem-10) (lonza, walkersville, usa) supplemented with 10% fetal calf serum (lonza, walkersville, usa). the suspensions were centrifuged at 3200 g for 15 min and 0.5 ml of the supernatant was used to infect 2 x 106 mouse neuroblastoma (mna) cells in 25 ml flasks (corning incorporated, new york, usa) followed by incubation at an atmosphere of 37 ºc and 5% co2 for 1 h. thereafter, 0.3 ml of the infected cells was added to labtek chamber slides (nalge nunc international, new york, usa) whilst the remaining was added to t25 flask together with 5 ml of dmem-10 and incubated for 72 h. after 48 h, the presence of lyssavirus antigen on labtek chamber slides was determined by fluorescence antibody test (fat) (dean, abelseth & atanasiu 1996) using anti-rabies polyclonal fluorescence isothiocyanate conjugate (fitc) (rabies unit, onderstepoort veterinary institute, agricultural research council, south africa). to determine virus yield from the t25 flask, monolayers of mna cells (2 x 106) in labtek chamber slides were infected with serial 10-fold virus dilutions. the slides were incubated for 48 h. thereafter, the slides were fixed in cold acetone (merck, modderfontein, sa), stained with anti-rabies polyclonal fitc and the tcid50 determined (reed & muench 1983). experimental infections mice were put in individually labelled cages. only 11 (table 1, indicated by †) of the 23 isolates were chosen for inoculation of mice after pathogenic domains were compared between the 23 representative african lyssavirus isolates. the number of isolates for each lyssavirus species was then reduced based on amino acid similarities in pathogenic domains. groups of 3 to 5 mice were inoculated i.m. with 0.05 ml of 105 tcid50 of rabv (canid variant) (31/05 and 819/05), rabv (mongoose variant) (113/91), lbv (lbvsa2008 and lbvnig1956), mokv (12341) and duvv (duvvsa2006). in addition to inoculation at a dose of 105 tcid50, one lbv isolate from lineage a (lbvafr1999) and another from lineage c (lbvmong2004) were inoculated at a higher dose of 108 tcid50 and lbv (lbvsa2004) and mokv (252/97) at a dose of 107 tcid50. furthermore, lbv (lbvsa2008) was inoculated at a lower dose (103 tcid50) also using 10% suspension of the original bat brain material. mice were monitored for 60 days. deaths and clinical signs/observations were recorded daily. the mice were euthanised with ketamine (anaket-v) (35 mg/kg body mass) upon development of clinical signs or on day 60 in cases where clinical signs were not observed. collection of brain, salivary glands and blood brain and salivary glands were collected after the mice were euthanised or died. necropsies were performed in class ii biosafety cabinets using sterile implements. the material was stored at −70 ºc until use. blood was collected weekly in bd microcontainertm (becton dickson, new jersey, usa) by retro-orbital route using 80 µl capillaries (superior marienfeld laboratory glassware, germany). the blood was centrifuged at 13 400 g for 10 min and serum was collected in 1.5 ml microcentrifuge tubes (quality scientific plastics, california, usa) and stored at −20 ºc until use. blood clots were also stored at −20 ºc. fluorescent antibody test and taqman real-time polymerase chain reaction to determine the presence and quantity of viral antigen and viral rna all brain samples were tested for the presence of lyssavirus antigen by fat using polyclonal fluorescein isothiocyanate anti-lyssavirus conjugate (dean et al. 1996). brain and salivary gland samples of mice that succumbed to lyssavirus infection were further analysed for the presence and concentration of viral rna by taqman real-time pcr as described in coertse et al. (2010). only the blood clots of mice taken just before the mice died or were euthanised after developing clinical signs were analysed for the presence of lyssavirus rna by taqman real-time pcr (coertse et al. 2010). integrity of the isolated rna was also tested using real-time pcr (nakahata et al. 2006). rapid fluorescent focus inhibition test virus neutralising antibodies (vna) were determined by a modification of the rapid fluorescent focus inhibition test (rffit) (kuzmin et al. 2008). the following challenge viruses were used: challenge virus standard (cvs) strain for both rabv (canid and mongoose variants), lbvsa2006 for all isolates of lbv except lbvafr1999, lbvafr1999 for lbvafr1999, mokv252/97 for all mokv isolates and duvvsa2006 for duvvsa2006. the 50% end point neutralising titres were calculated according to the method of reed and muench (1983). ethical considerations ethical approval (15/04/p001) for all procedures involving the use of mice was granted by the onderstepoort veterinary institute ethical committee, pretoria, south africa. results top ↑ comparison of pathogenic domains the nucleoprotein an alignment of proposed pathogenic domains on the n gene is shown in figure 1. phe 273 and tyr 394 were shown to be important for evasion of retinoic acid-inducible gene i mediated innate immunity and pathogenicity (masatania et al. 2011). it was found that phe 273 was conserved in all isolates analyzed in this study. tyr 394 was only conserved for rabv isolates with the exception of one representative rabv isolate (rc-hl) which had his in this position. all isolates of lbv, mokv and duvv had tyr substituted by phe. figure 1: multiple alignments of the partial nucleoprotein amino acids sequences of representative lyssavirus isolates. these regions were previously implicated in evasion of innate immunity and pathogenicity (aa273 and 394). the phosphoprotein an alignment of the partial p protein (aa140–aa150) indicating the region previously implicated in retrograde intracellular transport of the virus to the cns is indicated in figure 2. the four residues on position 144-148 (k/rxtqt, with x representing any amino acid) important for p protein binding to the lc8 dynein light chain were conserved for all rabv (both variants), lbv and duvv isolates compared in this study. the rc-hl isolate of rabv had a substitution of thr 148 to ile. mokv isolates had substitutions at positions 144 and 146 of the lc8 dynein light chain binding motif; arg/lys 144 and thr 146 were both replaced by ile with the exception of isolate mokv252/97 where val instead of ile substituted the arg/lys 144. figure 2: multiple alignments of partial phosphoprotein amino acids sequences of representative lyssavirus isolates. the box indicates the region important for binding to the lc8 dynein light chain with amino acids important for binding indicated by *. the matrix protein a partial alignment that encompasses domains on the m gene previously implicated in pathogenicity is shown in figure 3. figure 3: multiple alignments of partial matrix protein amino acids sequences (aa1–aa100) of representative lyssavirus isolates. the boxes indicate regions implicated in pathogenicity. firstly, the asap motif at position 22-25 was reported to be important for the pathogenicity of a related rhabdovirus, vesicular stomatitis virus (irie et al. 2007). the motif was conserved for hep-flury, rc-hl, mokv isolates and lbvafr1999, whilst for other rabv isolates and duvv ala was substituted by val and by pro in lbv isolates. secondly, a motif at position 35-38 (ppey) was shown to be necessary for efficient virion release and pathogenicity (wirblich et al. 2008). the ppey motif was conserved in all the lyssavirus isolates. the vpl motif (amino acid 39-41) which overlaps the ppey, ppeyvpl, was indicated to be important in rabv budding, though not as important as the ppey (wirblich et al. 2008). the vpl motif was also conserved except for the shbrv-18 isolate of rabv which had a substitution of val to ile in position 39. thirdly, lys 77 and asp 81 were shown to be important in disruption of the mitochondrion and induction of apoptosis (gholami et al. 2008). all rabv (canid and mongoose variants) isolates had arg 77 and glu 81. the era isolate of rabv, all isolates of lbv and mokv had arg 77 to lys substitution. lbv lbvsa2008, lbvsa2006, lbvmong2004 and lbvsa2004 and mokv 252/97 had a ser at position 81 whilst the other isolates of lbv lbvafr1999 and lbvnig1956 and mokv 12341, 543/95 and 173/06 had an asn at this position. duvv isolates had arg 77 as with rabv isolates, but the glu 81 was replaced by gly. lastly, mita et al. (2008) showed that mutation of val to ala at position 95 results in increased apoptosis. val 95 was conserved amongst all the lyssavirus isolates compared except for the rabv mongoose variant; 22107 and lbvafr1999 isolates, which had a substitution to ile. the glycoprotein an alignment of the partial g proteins of representative lyssavirus isolates is shown in figure 4, with domains previously implicated in pathogenicity indicated. most of the amino acids on the g protein that have been indicated to be important for pathogenicity were conserved for rabv (canid and mongoose variants) isolates in this study. figure 4: multiple alignments of the partial glycoprotein amino acids sequences (aa130–aa352) of representative lyssavirus isolates. the boxes indicate amino acids previously shown to be important for pathogenicity. firstly, the peptide fragment between amino acid 189 and 214 has been indicated to be important for rabv binding to the nicotinic acetylcholine receptor (lentz et al. 1984). within this fragment, substitution of asn 194 with lys was reported to result in increased pathogenicity (faber et al. 2005) whilst mutation of arg/lys 198 resulted in reduced pathogenicity (lentz et al. 1984). asn 194 was conserved for all rabv (both variants) except for the hep-flury isolate, which had a substitution of asn 194 to his. arg/lys 198 was conserved for all isolates in this study. substitutions were observed on this nicotinic acetylcholine receptor fragment for rabv (canid and mongoose variants) but there were more variations within lbv, mokv and duvv isolates than in rabv isolates.secondly, ala 242, asp 255 and ile 268 were reported to be important for the pathogenicity of the nishigahara strain with ile 268 being the most important residue (takayama-ito et al. 2006). the rabv isolates shbrv-18 and rc-hl had a substitution of ala 242 to ser. asp 255 from rabv isolates pv, lep-flury and hep-flury was substituted by gly and by asn in rc-hl. the rc-hl strain also had ile 268 replaced by leu. ala 242 was substituted by ser in lbv, mokv and duvv whilst asp 255 was substituted by asn in mokv and all lbv isolates except for lbvafr1999, which had this amino acid conserved, as with rabv and duvv. all lbv, mokv and duvv isolates had ile 268 conserved. this was with the exception of lbvnig1956, which had val in that position. thirdly, mutation of arg/lys 333 on the g protein was reported to result in non-pathogenic strains when inoculated i.m. (badrane et al. 2001; dietzschold et al. 1983). unlike all rabv species analysed in this study, hep-flury had gln at position 333 instead of arg/lys. all lbv isolates except for lbvafr1999 had arg 330 substituted by leu. lbvafr1999, mokv and duvv had lys 330 conserved. arg/lys 333 was substituted by asp in all lbv and mokv isolates. lastly, phe 318 and his 352 on the rabv g protein have been shown to be important for binding to the p75 neurotrophin receptor (p75ntr) (langevin et al. 2002). all lbv and duvv had phe 318 substituted with ile; this was with the exception of lbv isolates lbvnig1956 and lbvafr1999 which had leu as with all mokv isolates. his 352 was substituted with val in all lbv isolates except for lbvafr1999, which had met, as with all mokv isolates, whilst duvv had tyr on this position. a mokv isolate (mokzim) reported by badrane et al. (2001) to be non-pathogenic when inoculated i.m. had identical pathogenic domains on the g protein, as with mokv (12341), and differed from other mokv isolates with a single substitution of lys 198 to arg. pathogenicity of representative african lyssaviruses in mice mortality of mice inoculated intramuscular with a dose of 105 tcid50 all isolates investigated in this study, except for lbv (lbvnig1956) were lethal to mice through the i.m. route. all mice that died exhibited symptoms typical of lyssavirus infection. this included walking in circles, hind leg paralysis, loss of weight and ruffled fur. inoculation with different isolates did not result in differences in symptoms. all brains collected from mice that died or were euthanised during development of symptoms tested positive with fat. the control group and those that survived (euthanised at the end of the experiment) tested negative. mortality ranged from 0% to 75% with an incubation period from 7 to 29 days whilst the mean incubation period ranged from 9.25 to 26 days (figure 5 and table 2). lbv (lbvsa2008) had the highest mean incubation period compared to other lyssavirus isolates analysed in this study. mice inoculated with mokv had the highest percentage mortality (75%), followed by rabv (mongoose variant) (67%), lbv and duvv (50%), and then rabv (canid variant) (33%). figure 5: percentage mortality and mean incubation period of mice after intramuscular inoculation with a 105 tcid50 of different lyssavirus isolates. table 2: summary of experimental infections of mice with lyssavirus isolates. effect of inoculation dose on mortality of mice inoculation at a higher dose (108 tcid50) with lineage a (lbvafr1999) and c (lbvmong2004) lbv isolates showed a difference in mortality between these lineages. lineage a had higher percentage mortality and lower mean incubation period than lineage c (table 2). comparison of the pathogenicity of representative phylogroup ii lyssaviruses comparison of mokv and lbv pathogenicity at a higher dose (107 tcid50) indicated mokv (252/97) to be more lethal than lbv (lbvsa2004). the trend was the same at inoculation of these species at 105 tcid50 (table 2). comparison of the pathogenicity of cell culture and original bat brain material of lbv (lbvsa2008) inoculation with 10% suspension of the original bat brain material resulted in an increase in percentage mortality and a decrease in mean incubation period as compared to inoculation with a dose of 105 tcid50. however, no mortalities were observed when a dose of 103 tcid50 was used (table 2). viral rna concentration in organs and blood a taqman real-time pcr (coertse et al. 2010) was used to determine the presence and viral rna concentration from the brains, salivary glands and blood of mice that succumbed to african lyssavirus infection. salivary glands and blood of all mice tested were negative. the rna in these negative samples was intact based on amplification of the 18s rrna (data not shown). amplification of the 18s rrna from the salivary glands to comparable levels as the positive brain samples rules out the possibility of false negatives from the salivary glands. there was no viral rna detected in the brains of the control group. there was variation in viral rna concentration in the brains of mice inoculated with different lyssavirus species as well as those inoculated with isolates of the same species. the standard deviations were generally large, ranging from 43.71 mean copy number/ng of rna for lbv to 113 698 mean copy number/ng of rna for rabv (mongoose variant) when mice where inoculated at 105 tcid50, indicating that there were large differences in viral rna concentration in the brains of mice inoculated with the same virus isolate (table 2). since a small portion of the mixed whole brain was used for rna isolation, the difference in viral rna concentration from mice inoculated with the same dose of the same virus strain could be due to the differences in virus distribution during mixing of the brain material. correlation between pathogenic domains and mortality of mice the relationships between the integrity of pathogenic domains and incubation time and mortality in mice are shown in table 3. it can be assumed that the sequences used to identify differences in pathogenic domains were the same as the sequences of the inocula used for experimental infection of mice, since only a few passages were carried out from the sequenced material to obtain the titre used for experimental infection. comparison of pathogenic domains on the nucleoprotein, phosphoprotein, matrix and glycoprotein showed that substitutions of particular amino acids associated with such domains are not consistent between isolates of the same species. this was with the exception of isolates that were used as representatives of rabv (canid and mongoose variants) and duvv isolates which had the same amino acids on pathogenic domains. the conservation of most of the known pathogenic domains for phylogroup i (rabv [canid and mongoose variants] and duvv) as compared to phylogroup ii lyssaviruses (lbv and mokv) did not correlate with increased overall pathogenicity observed in mice. mokv demonstrated high percentage mortality compared to rabv and duvv when inoculated at the same viral dose despite having most of the known pathogenic domains mutated. generally there was no clear correlation between substitutions on pathogenic domains and mortality. table 3: comparison of amino acids of pathogenic domains on lyssavirus genomes. the mean incubation time and percentage mortality observed in mice are also indicated. serological response of mice after inoculation with different lyssavirus isolates the vna titre was not converted to international units as there are no available reference sera for lbv, mokv or duvv and therefore results were only reported as the titre at which neutralisation occurred. there were detectable vnas in all mice on day 7, with the exception of three mice that were inoculated with rabv (mongoose variant) (table 4). there was no clear difference in vna titre between mice that survived and those that died (day 7 and 14). vna titre increased on day 14 and a peak was reached on day 28, followed by a drop on days 42, 50 and 60. however, the titre was still above 200. all the mice inoculated with rabv (mongoose variant) maintained low vna titres throughout the experiment and one animal in this group survived until the end of the experiment. since only 3 mice were inoculated by the rabv (mongoose variant) of which 2 died at day 13 and 14 it could not be determined whether the vna would be comparable with other lyssavirus species at a later stage. the low blood volumes collected by ocular bleeds did not allow the neutralisation tests to be repeated using a rabv (mongoose variant) as a challenge virus to determine the possibility that the challenge virus was the reason for the low vna titre observed. table 4: virus neutralising antibodies titre in mice inoculated with different lyssavirus isolates as determined by the rapid fluorescent focus inhibition assay. discussion top ↑ pathogenicity of different african lyssavirus isolates inoculated intramuscular in mice lyssavirus isolates in this study represent a larger diversity of african lyssavirus isolates than in previous studies (badrane et al. 2001; kemp et al. 1973; kuzmin et al. 2008; markotter et al. 2009). all lyssavirus isolates except one lbv (lbvnig1956) isolate were lethal to mice through the i.m. route of infection. differences in pathogenicity within lyssavirus species were observed in lbv and rabv (canid and mongoose variants) isolates. this was in agreement with our previous recognition of this phenomenon between lbv isolates (markotter et al. 2009). the difference in pathogenicity between isolates of the same species indicated the need to compare a number of isolates within a species when evaluating the pathogenicity. the pathogenicity of phylogroup ii lyssaviruses were found to be in agreement with results from other recent studies (kuzmin et al. 2008; markotter et al. 2009) and those from one early study (kemp et al. 1973), all of which indicated that these lyssaviruses can be lethal (at relatively low doses) through the i.m. route. this finding was in contradiction to conclusions from badrane et al. (2001). however, the only lbv isolate that was not lethal in the present study, was the isolate used by badrane et al. (2001), lbvnig1956. mokv has been isolated from domestic mammals such as canines and cats (sabeta et al. 2010), whilst lbv has been isolated from a mongoose, bats and canines (markotter et al. 2006a; markotter et al. 2006b). if phylogroup ii lyssavirus isolates were only lethal via the i.c. route, there would be less isolation of these viruses in nature, since a bite by a rabid animal would have to penetrate the skull. therefore, the epidemiology of these viruses further supports the observation that lbv and mokv can be lethal when inoculated i.m. in animals. in this study, mokv isolates were more pathogenic than isolates used as representatives of phylogroup i lyssaviruses (rabv [canid and mongoose variants] and duvv). this does not necessarily mean that mokv isolates studied here are the more pathogenic lyssaviruses but it does demonstrate that the pathogenicity of mokv was previously underestimated. it also showed that different isolates within a species exhibit different pathogenicity profiles. when inoculation dose was increased, isolates that had high percentage mortality also had high mean viral rna concentration. there was also a decrease in mean incubation period in the high mortality group when comparing lbv isolates. this could indicate that inoculation with a high dose results in the virus reaching the cns faster, multiplying to high titre and therefore causing death more quickly. the virus titre of the 10% suspension of the original bat brain material (lbvsa2008) was not determined; therefore an increase in percentage mortality and a decrease in mean incubation period when mice were infected with this inoculum could indicate that the titre of the virus was higher than 105 tcid50 of the tissue culture material. another possible reason for the difference in pathogenicity of original bat brain and tissue culture material could be that mutations may have been introduced during passage in culture which influenced pathogenicity. this could not be confirmed since no original brain material was available for sequencing. lbv (lbvsa2008) inoculation at 103 tcid50 did not cause death as observed with our previous study, which showed some lbv isolates to be non-lethal when inoculated i.m. at 103 ld50 (markotter et al. 2009). viral rna in the blood of mice infected with african lyssaviruses the occurrence of rabv in the blood of infected animals is conflicting. the virus is considered to be absent in the blood of lyssavirus infected animals (borodina 1958; schindler 1961). however, there are a few studies which have reported contradictory results to what is generally known about viremia in lyssavirus infection. viremia was reported in rabv infected dogs (burne 1970). lodmell et al. (2006) also reported the presence of viral rna in the blood of rabv infected mice. the presence of viral rna in clinically ill mice in the study could be due to injury to organs including the blood-brain barrier, resulting in escape of the virus from the infected organs into the bloodstream. in this study, no viral rna was detected in the blood of mice after inoculation with different african lyssavirus isolates. viral rna load in the brains and salivary glands of mice infected with african lyssaviruses there were differences in mean viral rna concentration in the brains of mice inoculated with different virus isolates. differences were also observed in viral rna concentration in the brains of mice inoculated with the same isolate. this indicated that the pathogenicity of lyssaviruses does not necessarily depend on the amount of the virus in the brains of inoculated animals. no viral rna was detected in the salivary glands of any of the infected animals, regardless of the lyssavirus species or the outcome of the infection. a number of studies reported the presence of virus in the salivary glands of rabid animals (hughes et al. 2006; kemp et al. 1973; kuzmin et al. 2008; lima et al. 2005; shankar et al. 2004). the animal model in all these studies was different from this study, except that of lima et al. (2005). lima et al. (2005) reported viral rna in the salivary glands of mice that succumbed to rabies on days 10 to 21 using hemi-nested pcr. however, there was no viral rna in the salivary glands of mice that died on days 25 and 30. in that study, a pool of salivary glands from two to three mice was used for rna isolation. this finding supports the studies that showed that mice are not an ideal model for pathogenesis studies of lyssaviruses (smith 1996) and that pathogenesis studies should involve true reservoir species. however, mice are nevertheless a practical and useful first line (primary) animal model for lyssavirus research. serological response of mice to infection with african lyssaviruses it was found that the natural production of vna during infection did not correlate with survival, since there was no clear difference in the vna titres of mice that died and those that survived. although vna has been shown to be important in clearance of rabv infection (hooper et al. 1998), there are other mechanisms that also play a role in virus clearance (hooper et al. 1998). therefore, the presence of vna after challenge with a virus does not necessarily guarantee survival; this was also observed by coe and bell (1977) and roy et al. (2007). relationship between pathogenic domains and pathogenicity profiles observed in mice nucleoprotein and phosphoprotein lbv, mokv and duvv isolates were found to have a common mutation in one of the two pathogenic domains described by masatania et al. (2011). however, the mutation is not to the same amino acid as that of the non-pathogenic strain shown by masatania et al. (2011) and it is therefore not clear if this mutation influenced the pathogenicity of these species. the motif k/rxtqt (position 144-148) on the p protein was reported to be important for binding to the lc8 dynein light chain and subsequently transport to the central nervous system (cns) (poisson et al. 2001). mokv isolates in this study were lethal to adult mice. fat and real-time pcr detected antigen and viral rna respectively in the brains of mice that succumbed, indicating that the virus reached the cns. this is irrespective of substitution on two of the four amino acids. mokv also had short mean incubation period compared to rabv (mongoose variant), duvv and lbv (lbvsa2008) which had the motif conserved. tan et al. (2007) showed transport of rabv to the cns with the lc8 binding motif mutated. however, disruption of lc8 binding motif resulted in non-pathogenic strains due to reduced transcription and replication and not failure to travel to the cns (tan et al. 2007). in this study, when 105 tcid50 was used, mokv isolate (12341) had high mean viral rna concentration in the brain as compared to lbv (lbvsa2008) and duvv (duvvsa06) isolates in which the lc8 binding motif was conserved. matrix protein lbv (lbvafr1999 and lbvnig1956) and mokv (12341, 543/95 and 173/06) isolates had both residues (lys 77 and asn 81) on the m protein which were reported to increase apoptosis (gholami et al. 2008). these mutations seemed to have little or no influence in pathogenicity in this study. lbv (lbvafr1999) and mokv (12341) caused 80% and 75% mortality to mice respectively despite having these amino acids, whilst lbv (lbvnig1956) was not lethal. glycoprotein lys/arg 333 on the glycoprotein has been reported by a number of researchers to be important in lyssavirus pathogenicity (badrane et al. 2001; dietzschold et al. 1983; takayama-ito et al. 2006). all phylogroup ii lyssavirus isolates had arg/lys 333 substitution to asp. badrane et al. (2001) reported these isolates to be non-pathogenic i.m. in mice. however, the badrane et al. (2001) study was limited to one isolate of lbv and mokv. in this study, all phylogroup ii lyssaviruses (with the exception of one lbv isolate, lbvnig1956) were lethal i.m., as reported by previous studies (kemp et al. 1973; kuzmin et al. 2008; markotter et al. 2009). all lbv isolates except lbvafr1999 also had lys 330 substituted to leu whilst mokv isolates had this amino acid conserved. this did not result in reduced pathogenicity of the lbv isolates. in addition to these substitutions, mokv and lbv isolates had mutations in two of the amino acids (ala 242 and asp 255) important in the pathogenicity of the nishigahara strain whilst the third amino acid (ile 268) was conserved. this was with the exception of lbv (lbvnig1956), which had all the three amino acids substituted. takayama-ito et al. (2006) showed the rc-hl strain with mutations on the three amino acids to be non-lethal to mice via the i.c. route, but the strain was lethal when leu 268 was substituted by ile as with the nishigahara strain. this could explain why lbvnig1956 was found to be non-pathogenic whilst the other phylogroup ii lyssaviruses were found to be pathogenic. mokzim isolate reported by badrane et al. (2001) to be non-pathogenic and the mokv isolate, 12341, in this study had the same amino acids on all currently known pathogenic domains on the g protein. this shows that in addition to pathogenic domains, there are other factors that affect the pathogenicity of lyssaviruses. it could also indicate that there are other yet unidentified domains or regions that play a role in pathogenicity. rabv had less variation in the peptide fragment for acetylcholine receptor binding than lbv, mokv or duvv. the amino acids important in binding to the p75 neurotropin receptor were only conserved for rabv. pathogenicity of lbv, mokv and duvv despite these substitutions emphasised the realisation that lyssaviruses bind to a variety of receptors. combined effects of pathogenic domains on mortality within lbv, lbvafr1999 had most of the pathogenic domains conserved whilst lbvnig1956 had fewer than rabv. lbvafr1999 had a higher percentage mortality and shorter incubation period than lbvmong2004 when inoculated at the same dose. this suggests some cooperativity between pathogenic domains within lbv. however, this cooperative effect was not observed when comparing the different species. rabv (canid and mongoose variants) had most of the known pathogenic domains conserved, followed by duvv, as compared to lbv and mokv. when inoculated at the same dose, rabv (canid variant) had the lowest percentage mortality whilst rabv (mongoose variant) and duvv had the same percentage mortality as lbv but lower than that of mokv. the vaccine strain, hep-flury had arg 333 substitution to qln, whilst most other pathogenic amino acids were conserved compared to lbv, mokv and duvv. however, this strain was reported to be non-pathogenic via both the i.c. and i.m. routes (takayama-ito et al. 2006). this supports our previous observation that in addition to known pathogenic domains, there are other domains or factors that contribute to the pathogenicity of lyssaviruses. this nullifies the view that a specific amino acid is important for the pathogenicity of the genus lyssavirus based on a few isolates, especially of a single species. conclusion top ↑ the study supports previous studies indicating that all african lyssavirus species can be lethal when inoculated via the i.m. route and that the pathogenicity of a lyssavirus species should not be concluded based on a single isolate. furthermore, until all pathogenic domains have been mapped and the co-operation between these domains and other cellular factors are understood, the pathogenicity of an isolate or species cannot be determined by the analysis of the amino acids encoded in domains associated with pathogenicity without inoculation in an animal model. acknowledgements top ↑ we wish to thank the rabies unit, onderstepoort veterinary institute, pretoria, south africa, for providing the isolates, with few exceptions. duvvsa2006 isolate was kindly provided by the centre for emerging and zoonotic diseases, national institute of communicable disease (national heath laboratory services), sandringham, south africa. lbvafr1999 was kindly provided by dr cliquet from agene francaise de sécurité sanitaire des aliments, france and lbvnig1956 by the centers for disease control and prevention, atlanta, usa. we also thank the national research foundation, the international society for infectious diseases, the international foundation for science and the poliomyelitis research foundation for financial support. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions w.m. 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matrix protein is essential for efficient virion release and rv pathogenicity’, journal of virology 82, 9730–9738. http://dx.doi.org/10.1128/jvi.00889-08, pmid:18667490 article information          authors: pilar foronda1 josué plata-luis1 borja del castillo-figueruelo1 ángela fernández-álvarez1 aarón martín-alonso1 carlos feliu2 marilena d. cabral3 basilio valladares1 affiliations: 1university institute of tropical diseases and public health of the canary islands, university of la laguna, spain 2laboratory of parasitology, university of barcelona, spain 3department of science and technology, university of cape verde, cape verde correspondence to: pilar foronda email: pforonda@ull.edu.es postal address: faculty of pharmacy, university of la laguna, avda. astrofísico fco. sánchez, 38203 la laguna, canary islands, spain dates: received: 03 nov. 2014 accepted: 02 feb. 2015 published: 29 may 2015 how to cite this article: foronda, p., plata-luis, j., del castillo-figueruelo, b., fernández-álvarez, á., martín-alonso, a., feliu, c. et al., 2015, ‘serological survey of antibodies to toxoplasma gondii and coxiella burnetii in rodents in north-western african islands (canary islands and cape verde)’, onderstepoort journal of veterinary research 82(1), art. #899, 4 pages. http://dx.doi.org/10.4102/ojvr.v82i1.899 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. serological survey of antibodies to toxoplasma gondii and coxiella burnetii in rodents in north-western african islands (canary islands and cape verde) in this research communication... open access • abstract • introduction • materials and methods    • biological samples and study area    • enzyme-linked immunosorbent assay    • indirect immunofluorescence    • statistical analysis • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ coxiella burnetii and toxoplasma gondii are intracellular parasites that cause important reproductive disorders in animals and humans worldwide, resulting in high economic losses. the aim of the present study was to analyse the possible role of peridomestic small mammals in the maintenance and transmission of c. burnetii and t. gondii in the north-western african archipelagos of the canary islands and cape verde, where these species are commonly found affecting humans and farm animals. between 2009 and 2013, 108 black rats (rattus rattus) and 77 mice (mus musculus) were analysed for the presence of coxiella and toxoplasma antibodies by enzyme-linked immunosorbent assay (elisa) and indirect immunofluorescence (ifa), respectively. our results showed a wide distribution of c. burnetii and t. gondii, except for t. gondii in cape verde, in both rodent species. the overall seroprevalence of c. burnetii antibodies was 12.4%; 21.1% for cape verde and 10.2% for the canary islands. with respect to t. gondii, seropositive rodents were only observed in the canary islands, with an overall seroprevalence of 15%. considering the fact that both pathogens can infect a large range of hosts, including livestock and humans, the results are of public health and veterinary importance and could be used by governmental entities to manage risk factors and to prevent future cases of q fever and toxoplasmosis. introduction top ↑ coxiella burnetii and toxoplasma gondii are intracellular pathogens with worldwide distribution (angelakis & raoult 2010; dubey 2010). q fever is caused by c. burnetii and has been found in a large range of domestic and wild animals including mammals, birds, reptiles and arthropods (angelakis & raoult 2010). toxoplasmosis is caused by t. gondii and can be found in all warm-blooded animals, including terrestrial and marine mammals and birds (jenkins et al. 2013; pan et al. 2012; wendte, gibson & grigg 2011). both pathogens cause high economic losses in livestock as a result of reproductive disorders (arricau-bouvery & rodolakis 2005; guatteo et al. 2011; hill, chirukandoth & dubey 2005). in humans, most infections remain asymptomatic; however, q fever and toxoplasmosis can be dangerous to pregnant women and immunosuppressed patients (angelakis & raoult 2010; desmonts & couvreur 1974; dubey & beattie 1988). the main source of human infection with q fever is livestock. pets can also transmit this disease to humans (arricau-bouvery & rodolakis 2005) and oral transmission by ingesting dairy products has been also reported (fishbein & raoult 1992). in the case of toxoplasmosis, the main source of infection is the ingestion of raw or insufficiently cooked meat with tissue cysts, oocysts in water or raw food and via vertical transmission (cook et al. 2000). rodents have been found to be a source of livestock infection with several pathogens, including t. gondii (kijlstra et al. 2008) and c. burnetii (reusken et al. 2011). therefore, and considering the fact that both t. gondii and c. burnetii are endemic in the canary islands (bolaños et al. 2003; rodríguez et al. 2010; rodríguez-ponce, molina & hernández 1995; solá-graffigna 1997; velasco 2010) and the lack of information on these species for cape verde, the aim of the present work was to contribute to the understanding of the epidemiology of the two pathogens in these north-western african archipelagos. to this end, a serological study to detect t. gondii and c. burnetii antibodies in peridomestic rodents from the canary islands and cape verde was carried out. materials and methods top ↑ biological samples and study area the study was carried out in five of the seven canary islands (spain), namely tenerife, el hierro, gran canaria, lanzarote and fuerteventura, and one island belonging to the cape verde republic, namely santiago. both archipelagos are located near the north-west coast of africa. the canary islands are located 100 km off the coast of morocco, between 27°37′n and 29°24′n and 13°23′w and 18°8′w. cape verde is located 550 km off the coast of senegal, between 17°16′n and 14°44′n and 22°37′w and 25°24′w. between 2009 and 2013, 185 rodents from the canaries and cape verde were captured randomly, comprising 108 black rats (rattus rattus) (linnaeus, 1758) and 77 mice (mus musculus) (linnaeus, 1758). the sample size was determined according to the variables host species and island. the sampled areas were determined at random: orgaos, praia and säo domingos in santiago, san cristóbal de la laguna in tenerife, frontera in el hierro, telde, santa brígida and san mateo in gran canaria and the whole islands of fuerteventura and lanzarote. the animals were captured alive using sherman traps in suburban-rural areas. the traps were set in the afternoon and the evening and checked soon after sunrise. animals were taken to the university institute of tropical diseases and public health of the canary islands or to laboratories belonging to cape verde university. the animals were euthanised by cervical dislocation or by carbon dioxide inhalation and bled by cardiac puncture. blood samples were centrifuged and serum was removed and stored in a 1:1 glycerol solution at -20 °c until analysed. enzyme-linked immunosorbent assay an elisa kit was used (coxiella burnetii elisa kit, bio-x diagnostics, belgium); however, anti-rat antibody peroxidase conjugate and the anti-mouse antibody peroxidase conjugate (sigma-aldrich, usa) were used with the serum samples instead of the kit’s anti-goat antibody, which was used in controls. the sera and controls were used at 1:100 dilutions. the plates were read spectrophotometrically at 450 nm with a microplate reader (model 680, bio-rad laboratories, usa). the cut-off value was determined in accordance with the manufacturer’s instructions. indirect immunofluorescence an ifa kit was used (toxo-spot ifi, biomerieux, france) in order to detect antibodies against t. gondii. the serum samples were diluted at 1:40 and 1:80 in phosphate buffered saline (pbs) and the conjugate was made as follows: 400 µl pbs, 10 µl evans blue and 1 µl goat anti-rat or goat anti-mouse antibodies (thermo scientific, usa), both labelled with fluorescein. for each slide, one negative and one positive control were included and the results were determined in accordance with these controls. a positive sample was used as positive control and pbs was used as negative control. statistical analysis data analysis was performed with statistical software spss 20.0 (ibm, usa). logistic regression could not be carried out because of the limited number of outcome events. instead, chi-square contingency tables were used to examine the relationships between rodent species and island studied and the proportion of rodents harbouring antibodies against c. burnetii or t. gondii. fisher’s exact test was used if expected cell counts were < 5. a probability value < 0.05 was considered statistically significant. ethical considerations top ↑ all the animal procedures were performed according to the principles of animal welfare in experimental science (spanish government 2007, 2013). animal trapping and its use were approved by the environmental agencies of the relevant governmental entities, in accordance with act 151/2001, 33/2003, 42/2007 and 4/2010. results top ↑ coxiella burnetii and t. gondii antibodies were found in both species of rodents analysed, with overall prevalences of 12.4% (ci 95% 7.7–17.2) and 11.9% (ci 95% 7.2–16.6), respectively. coxiella burnetii was present in the two archipelagos studied; t. gondii was not found in cape verde (table 1). table 1: seroprevalence of antibodies against coxiella burnetii and toxoplasma gondii in rodents in the canary islands and cape verde. rodents carrying antibodies against c. burnetii were found in all the islands analysed except for tenerife, where antibodies were not found in either host. c. burnetii showed a higher seroprevalence in cape verde (21.1%, ci 95% 8.1–34.0) than in the canary islands (10.2%, ci 95% 5.3–15.1) but without significant differences. significant differences in the proportion of rodents harbouring antibodies against both c. burnetii and t. gondii were found amongst the different islands included in the study (p < 0.01, chi-square contingency table). when data from different hosts were compared, significant differences were observed for c. burnetii between m. musculus (16.9%, ci 95% 7.81–26.0) and r. rattus (4.9%, ci 95% 0.2–9.5) (p < 0.05) collected from the canary islands. however, differences between hosts were not observed when these islands were compared pairwise. the overall prevalence for t. gondii in the canary islands was 15% (ci 95% 9.2–20.7); 17.1% (ci 95% 8.9–25.2) for r. rattus and 12.3% (ci 95% 4.3–20.3) for m. musculus. no significant differences between hosts were found, even when the islands were analysed separately. comparing the seroprevalence of t. gondii between different islands, there was a significant difference for m. musculus between fuerteventura (54.5%, ci 95% 25.1–84.0) and el hierro (4%, ci 95% 0–11.7) islands (p < 0.001, fisher’s exact test). no positive results were found in rodents captured in cape verde. discussion top ↑ this study reveals the wide distribution of t. gondii and c. burnetii in rodents in the canary islands, as antibodies against both pathogens were found in all the islands analysed, except for c. burnetii in tenerife. furthermore, this study also indicates the role of black rats (r. rattus) as reservoirs of t. gondii in santiago, as antibodies against this parasite were found in almost one-third of the individuals analysed. to the authors’ knowledge, there are no previous studies of c. burnetii or t. gondii in rodents from oceanic islands. when comparing the results with previous studies carried out in continental areas, the overall prevalence of t. gondii obtained in the present study (12.4%) is lower than that observed in iran (24.41%) (mosallanejad et al. 2012) but higher than that found in france (5%) (gotteland et al. 2013) or brazil (5.7%) (siqueira et al. 2013). the overall prevalence of c. burnetii was 11.9%, similar to that obtained in the netherlands (7.1%) (reusken et al. 2011). in the canary islands, previous studies involving small ruminant livestock (rodríguez et al. 2010; velasco 2010) and humans (bolaños et al. 2003) have indicated that c. burnetii is endemic and t. gondii is widely distributed in the archipelago (rodríguez-ponce et al. 1995; solá-graffigna 1997). in the case of cape verde, to the authors’ knowledge, there are neither available data for livestock census nor data about c. burnetii and t. gondii. however, based on personal observations during this study, the main livestock animals in cape verde seem to be goats, as in the canary islands (gobierno de canarias 2012). the same occurs in several african continental countries, such as uganda, where goats are not only important to the local economy but also serve as a major source of protein (bisson et al. 2000). considering the fact that c. burnetii and t. gondii can be transmitted from rodents to livestock (kijlstra et al. 2008; reusken et al. 2011), the presence of these pathogens in rodents from the canary islands and cape verde may have influenced the productivity of small ruminant flocks, which depends greatly on their reproductive efficiency (arricau-bouvery & rodolakis 2005; guatteo et al. 2011; hill et al. 2005). however, the results of this study are not only important from an economic point of view; these pathogens are also relevant to public health. both toxoplasmosis and q fever are occupational diseases for people who work with infected animals and/or their products. in this regard, it must be remembered that the rodents captured in this study came from suburban and rural areas where people and their pets live in close contact with rodents and can acquire these pathogens (arricau-bouvery & rodolakis 2005). consequently, this study suggests that the incidence of both q fever and toxoplasmosis, mainly in their mild forms, may have been underestimated in the canary islands and cape verde, especially in immunosuppressed patients such as pregnant women and hiv-positive people, as both pathogens can produce severe disease. for this reason, t. gondii and c. burnetii are important in sub-saharan africa, where hiv is a public health problem (unaids 2013). conclusion top ↑ the present study reveals the presence and wide distribution of t. gondii and c. burnetii in peridomestic rodents from the canary islands and c. burnetii in cape verde, where there were no available data about their presence in these mammals. the two species examined, r. rattus and m. musculus, presented antibodies against both pathogens with similar prevalences. considering the potential health impact and the economic importance of t. gondii and c. burnetii in the archipelagos studied, the results of the study could be useful for governments to improve the management of q fever and toxoplasmosis. however, more studies are required in order to analyse the possible role of these rodent species in the transmission of both pathogens to humans and livestock in this area. acknowledgements top ↑ we thank the islands councils of tenerife, el hierro, gran canaria, lanzarote and fuerteventura. we want to thank leonildo varela and all members of the pci project for their help in taking samples. this study was supported by rede de investigación de centros de enfermedades tropicales – ricet (rd06 0021/0005, fis), ministry of health, madrid, spain; agencia española para la cooperación internacional y el desarrollo under programa de cooperación interuniversitaria (a1/035356/11); the spanish ministry of foreign affairs and cooperation; real federación española de fútbol; semtsi; project semaca (mac73/c169) mac 2007-2013; programa nacional de biodiversidad, ciencias de la tierra y cambio global: subprograma de biología de organismos y sistemas (cgl 2006-04937 and cgl2009-07759bos) of the spanish ministry of science and education; and the canary government (a.m.a., grant number tesis20100083). competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions b.v. 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infections in domestic ruminants in canary islands (spain)’, transboundary and emerging diseases 57(6), 464–464. http://dx.doi.org/10.1111/j.1865-1682.2010.01159.x wendte, j.m., gibson, a.k. & grigg, m.e., 2011, ‘population genetics of toxoplasma gondii: new perspectives from parasite genotypes in wildlife’, veterinary parasitology 182(1), 96–111. http://dx.doi.org/10.1016/j.vetpar.2011.07.018 article information authors: joseph m. genchwere1 christopher j. kasanga2 affiliations: 1tanzania veterinary laboratory agency, mwanza, tanzania2southern african centre for infectious disease surveillance, sokoine university of agriculture, chuo kikuu, tanzania correspondence to: joseph genchwere postal address: po box 129, mwanza, tanzania how to cite this article: genchwere, j.m. & kasanga, c.j., 2014, ‘spatial and temporal distribution of foot-and-mouth disease virus in the lake zone of tanzania’, onderstepoort journal of veterinary research 81(2), art. #724, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.724 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. spatial and temporal distribution of foot-and-mouth disease virus in the lake zone of tanzania in this proceedings... open access • abstract • introduction • materials and methods    • sample collection and methodology    • data analysis • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ this study was conducted to determine the spatiotemporal distribution of foot-and-mouth disease (fmd) virus (fmdv) serotypes and evaluate the awareness of livestock keepers about fmd in tanzania. an observational prospective study involving serological analysis, fmdv antigen detection and questionnaire survey was carried out in the lake zone of tanzania. seroprevalence of antibodies to the nonstructural protein 3abc of fmdv and serotype-specific antigen detection were investigated by using svanovir® fmdv 3abc-ab elisa and indirect-sandwich elisa (selisa), respectively, whilst a structured questionnaire was used to evaluate the awareness of livestock keepers about fmd. during the period of 2010–2011, both serum and tissue (foot-and-mouth epithelia) samples were collected from cattle suspected of fmd in 13 districts of the four regions of the lake zone. a total of 107 (80.5%) out of 133 tested serum samples were seropositive to nonstructural protein 3abc, with at least one sample being positive from all 10 districts screened. fifteen (53.6%) out of 28 tissue epithelial samples collected from fmd cases in eight districts during the course of this study were positive to serotype o fmdv antigen. of these eight districts, serotype o fmdv antigens were detected from seven districts and no other serotypes were recovered from animal samples screened. questionnaire surveys in six districts indicated that livestock keepers in the lake zone were aware of the clinical manifestations (26/29 = 90.0%) and economic impact (23/29 = 79.0%) of fmd in the region. the questionnaire data showed that fmd outbreaks often occurred after rainy seasons (22/29 = 75.9%), with the highest peaks predominantly occurring just after the long rains in may and june, and at the end of the short rains in november and december of each year. the spatial distribution of the fmd cases suggested that serotype o virus exposure was the only widespread cause of the 2010–2011 outbreaks in the lake zone. introduction top ↑ foot-and-mouth disease (fmd) is a highly contagious, vesicular disease of cloven-hoofed animal species (habiela et al. 2010). it is caused by the foot-and-mouth disease virus (fmdv) of the genus aphthovirus and the family picornaviridae (carrillo et al. 2005; oie 2009). there are seven distinct serotypes, namely: o, a, c, south african territories (sat) 1, sat 2, sat 3, and asia 1 (aphis 2007; oie 2009; paton et al. 2009). since fmd was first reported in tanzania in 1927 (tanganyika department of veterinary services 1927; chibunda et al. 2006), efforts to eradicate this economically important disease have not been fruitful, as there are still several fmd outbreaks (long-standing endemic or periodic epidemics) being encountered. the occurrence and geographical distribution of fmdv serotypes is not clearly known and this remains to be fully explored. this study was conducted in order to determine the current spatial and temporal distribution of the fmdv serotypes, to evaluate the awareness of livestock keepers about fmd and its socio-economic impact in the lake zone of tanzania. materials and methods top ↑ sample collection and methodology an observational, prospective study involving serological analysis, fmdv antigen detection and questionnaire survey was carried out. serum and epithelial tissue samples were collected from cattle suspected of fmd in 13 districts during the period of 2010–2011.seroprevalence of antibodies to the nonstructural protein 3abc of fmdv were investigated by using svanovir® fmdv 3abc-ab elisa (svanova biotech ab, sweden) and developed in collaboration with cevan (centro virologia animal), (corepro buenos aires, argentina). serotype-specific antigen detection was investigated by indirect-sandwich elisa (selisa) supplied by the institute for animal health (pirbright laboratory, uk); the procedure was as described by roeder and le blanc smith (1987) and ferris and dawson (1988). structured questionnaires were used to evaluate the awareness of livestock keepers about fmd and its socio-economic impact. data analysis descriptive statistics for laboratory data of both selisa and non-structural protein 3abc elisa were used and frequency distributions were calculated (thrusfield & bertola 2005). prevalence of positive animals was determined by dividing the number of positive serum samples by the total number of samples tested. for questionnaires, data were coded in such a way that variables (species affected, clinical signs, season and economic impacts) represented 1 and 0 for positive and negative awareness, respectively. all data were analysed with the microsoft excel® software (microsoft, usa). results top ↑ a total of 107 (80.5%) out of 133 tested serum samples from cattle were seropositive to nonstructural protein 3abc, with at least one sample being positive from all 10 districts screened (table 1).fifteen (53.6%) out of 28 tissue epithelial samples collected from fmd cases in eight districts during the course of this study were positive to serotype o fmdv antigen. of these eight districts, serotype o fmdv antigens were detected in seven districts and no other serotypes were recovered from animal samples screened (table 2). the questionnaire surveys in six districts indicated that livestock keepers in the lake zone were aware of the clinical manifestations 26/29 (90%) and economic impact 23/29 (79%) of fmd (table 3). the questionnaire data showed that fmd outbreaks often occurred after rainy seasons 22/29 (75.9%), with the highest peaks predominantly occurring just after the long rains (masika) in may and june, and at the end of the short rains (vuli) in november and december (figure 1). figure 1: temporal trends of foot-and-mouth disease outbreaks and its respective frequencies as scored by the respondents. table 1: overall positive sera for 3abc-fmdv antibodies per district of lake zone. table 2: foot-and-mouth disease virus antigen detection using selisa on 28 clinical samples. table 3: farmers’ awareness about foot-and-mouth disease and its economic impacts. discussion top ↑ the study shows that fmd is endemic and widely spread throughout all four regions of the lake zone of tanzania (table 1 and figure 2). the disease is of high economic importance, especially to countries that have an intensive animal industry (mwiine et al. 2010). the questionnaire survey indicates that the group of livestock keepers was aware of the disease. they also described most of the clinical presentations of the disease very well; most of the signs listed for fmd 26/29 (90%), such as mouth and foot lesions, hypersalivation, anorexia and lameness, were consistent with what has been described by other researchers (radostits et al. 1994; jullu 2004; tesfaye 2006; cfsph 2007; swai et al. 2009). the findings of this study indicate a high correlation between livestock keepers’ awareness of fmd and the serological diagnosis of fmdv infection by nsp 3abc-ab elisa. these results also conform to the positive results obtained by selisa test, which indicates 53.6% to be positive for fmdv serotype o in seven districts (table 2). this observation indicates a correlation between the laboratory results and the findings of the questionnaire survey about the awareness of livestock keepers on the clinical manifestation of fmd under the field conditions. seasonal incidence of fmd was found to be high at the end of the long rainy season (may and june) and also at the end of short rainy season (vuli) (november and december), as shown in figure 1. the lowest incidence of fmd outbreak was reported during the long rainy season (masika) (march and april) and during august and september (figure 1). the reasons for this observation could be ascribed to movement of animals searching for pastures and water. many herds of cattle meet at such places, thereby hastening the spread of infection from one herd to another. the fmd study on nsp 3abc antibody detection revealed evidence of infection, and that spatial distribution of the disease in the lake zone was 80.5% (table 1). the study using selisa revealed that the spatial distributions of fmd outbreaks in the year 2010–2011 in the lake zone were caused mainly by serotype o virus (figure 2). these findings are in agreement with rweyemamu et al. (2008), who reported that serotype o was the most widely prevalent serotype in most parts of the world, including tanzania. in this study, the detection of serotype o virus antigens in all four regions of the lake zone, which was reported to be negative by swai et al. (2009) and jullu (2004) in this zone, depicts the possibility of re-introduction of serotype o. this may be the cause of the acute epidemic (sporadic) outbreaks of fmd in almost all the districts of the lake zone. the reintroduction of type o virus could be due to loose and free border movement of animals (livestock and wildlife) to and from neighbouring countries, such as kenya and uganda in eastern africa. furthermore, the spread of serotype o virus within the districts of the lake zone could be associated with movement of animals from one district or region to another during trade and marketing. it could also be associated with the long-distance grazing movements practiced by agro-pastoralists with large herds of cattle searching of pastures and water. this study provides evidence that the presence of fmd is an endemic situation in almost all the districts and regions of the lake zone. serotype o has been detected as the most, and probably the only, prevailing serotype in the lake zone of tanzania during the 2010–2011 outbreaks. serotype o virus has not been detected in the lake zone for years; the research conducted by jullu (2004), by using both selisa and real time polymerase chain reaction (rt-pcr), reported only serotype sat 1 in musoma urban and sat2 in musoma urban and tarime from samples collected in 1999. samples collected in 2003 in ukerewe and mwanza city detected serotypes sat 2. jullu (2004) used the same protocol and kit for selisa as was used in the current study. however, serotype o was detected in the northern zone (arusha), eastern zone (dar es salaam) and southern highland (iringa and mbeya) from samples collected for the period 1997–2003 (jullu 2004). swai et al. (2009) detected only serotypes sat 1 and sat 2 in the lake zone from samples collected between 1997 and 2004. recently, findings by kasanga et al. (2011) could not detect the presence of serotype o in the lake zone from the samples collected from 1967–2009. the detection of only serotype o virus in samples collected in the lake zone during 2010 and 2011, which was not detected by other researchers (jullu 2004; swai et al. 2009; kasanga et al. 2011), provides a great challenge to the routine disease surveillance. the recent occurrence of epidemic (sporadic) outbreaks with the classical clinical signs signifies the naive immunity status of the animals. this situation can be probably due to introduction of either the new strain of serotype o virus or reintroduction of serotype o virus with variant pathogenicity, which was not present some years back. with regard to the location of the lake zone and the widespread finding of the fmdv serotype o in all four regions, the probability of the infection is either from the vaccines or from the neighbouring countries. this is because some farmers, especially from the kagera region, do vaccinate their animals by using fmd polyvalent vaccine from kenya; the rest of the zone has no recent history of fmd vaccination. the current study highlights the fmdv serotype (serotype o) prevalence in the lake zone and also the extent of spatial distribution of fmd in this zone. this is an important step, as the information detailed in this study is useful in vaccine matching and selection of vaccine candidate strains for the region. this will help in the recommendation for an appropriate approach and proper control programme of fmd in tanzania. figure 2: spatial distribution of foot-and-mouth disease and its serotypes in the lake zone of tanzania. conclusion top ↑ the spatial distribution of the fmd cases suggests that serotype o virus exposure was the only widespread cause of the 2010–2011 outbreaks in the lake zone of tanzania. the temporal occurrence of fmd has been noted after rainy seasons, with the highest peaks predominantly encountered just after long rains and at the end of the short rains.the observed agreement between the livestock keepers in the current study and previous veterinary literature regarding most of the exhibited signs of important cattle diseases has proven that in the lake zone, livestock keepers have great knowledge about fmd. this is especially regarding disease outbreak, period of occurrence, diagnosis of the disease and its economic impacts. the research indicates that the disease is endemic in the lake zone. it suggests that extensive and regular sero-surveillance, virus isolation and characterisations of the field fmdv isolates need to be conducted for the possible development of a relevant vaccine from the local circulating field serotype or strains. acknowledgements top ↑ the authors would like to express their thanks to the ministry of livestock and fisheries development for sponsoring this research. special thanks go to dr win mleche (former director of veterinary services, ministry of livestock and fisheries development, tanzania) and dr ezekia ranga (sadc-tads coordinator in tanzania) for their encouragement and unreserved help to provide us with fmdv kits meant for laboratory analysis of samples. they are very grateful to all staff at vic-mwanza for their support during field sample collection and storage and are highly indebted to the livestock keepers: for their utmost patience in responding to our questions during the questionnaire survey and targeted discussions, and for allowing us to collect samples from their animals. this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.m.g. (tanzania veterinary laboratory agency–mwanza) was the research project leader. c.j.k. (sokoine university of agriculture) was the research project supervisor. references top ↑ aphis (animal and plant health inspection service), foot and mouth disease, u.s. department of agriculture, 2007, viewed 10 july 2011, from http://www.aphis.usda.gov carrillo, c., tulman, e.r., delhon, g., lu, z., carreno, a., vagnozzi, et al., 2005, ‘comparative genomics of foot-and-mouth disease virus’, journal of virology 79, 6487–6504. http://dx.doi.org/10.1128/jvi.79.10.6487-6504.2005 cfsph (the center for food security and public health), foot and mouth disease, iowa state university, 2007, viewed 14 july 2011, from http://www.cfsph.iastate.edu chibunda, r.t., makene, v.w., pereka, a.e. & magayane, f.t, 2006, ‘heat intolerance syndrome with foot and mouth disease in cattle in tanzania: occurrence and its effect on animal behaviour and physiological parameters’, tanzania veterinary journal 23(2). ferris, n.p. & dawson, m., 1988, ‘routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot and mouth and swine vesicular diseases’, veterinary microbiology 16, 201–209. http://dx.doi.org/10.1016/0378-1135(88)90024-7 habiela, m., alamin, m.a.g., raouf, y.a. & ali, y.h., 2010, ‘epizootiological study of foot and mouth disease in sudan. the situation after two decades’, veterinarski arhiv 80(1), 11–26. jullu, b., 2004, ‘epidemiological status of fmdv serotypes in tanzania’, dissertation for award of master of veterinary medicine degree at sokoine university of agriculture, morogoro, tanzania. kasanga, c.j., sallu, r., ngeleja, c.m, wadsworth, j., ferris, n., hutchings, g., et al., 2011, ‘molecular biological characteristics of foot-and-mouth disease viruses collected in tanzania from 1967 to 2009’, one health conference, johannesburg, rsa, 15 july 2011. mwiine, f.n., ayebazibwe, c., olaho-mukani, w., alexandersen, s. & tjornehoj, k., 2010, ‘prevalence of antibodies against foot-and-mouth disease virus in cattle in kasese and bushenyi districts in uganda’, international journal of animal and veterinary advance 2(3), 89–96. oie, 2009, ‘foot and mouth disease: terrestrial manual: version adopted by the world assembly of delegates of the oie’, viewed 05 june 2012, from http://www.oie.int paton, d.j., sumption, k.j. & charleston, b., 2009, ‘option for control of foot and mouth disease: knowledge, capability and policy’, philosophical transactions of the royal society b 364, 2657–2667. http://dx.doi.org/10.1098/rstb.2009.0100 radostits, o.m., blood, d.c. & gay, c.c., 1994, veterinary medicine, 8th edn., bailliere tindall, london, 345–372. roeder, p.l. & le blanc smith, p.m., 1987, ‘detection and typing of foot and mouth disease virus byenzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis’, research in veterinary science 43, 225–232. rweyemamu, m., roeder, p. & mackay, d., 2008, ‘epidemiological patterns of foot-and-mouth disease worldwide’, transboundary and emerging diseases 55(1), 57–72. http://dx.doi.org/10.1111/j.1865-1682.2007.01013.x swai, e.s., mrosso, a. & masambu, j.i.g., 2009, ‘occurrence of foot and mouth disease serotypes in tanzania: a retrospective study of tongue epithelial tissue samples’, tanzania veterinary journal 26(1). tanganyika department of veterinary services, 1927, annual report of the tanganyika department of veterinary services, 1927, dar es salaam, tanzania. tesfaye, r.c., 2006, ‘participatory appraisal and seroprevalence study of foot and mouth disease in borana pastoral system, south ethiopia’, faculty of veterinary medicine, addis ababa university, debre zeit, ethiopia, msc. thesis. thrusfield, m.v. & bertola, g., 2005, veterinary epidemiology, 3rd edn., p. 584, blackwell scientific publishers, london. article information authors: isihaka j. haji1,2 imna malele3 boniface namangala2 affiliations: 1department of economic and productive sectors, regional commissioner’s office, tanzania2department of paraclinical studies, university of zambia, zambia 3tsetse and trypanosomosis research institute, tanga, tanzania correspondence to: boniface namangala postal address: po box 32379, lusaka 10101, zambia dates: received: 06 july 2014 accepted: 22 aug. 2014 published: 13 nov. 2014 how to cite this article: haji, i.j., malele, i. & namangala, b., 2014, ‘occurrence of haemoparasites in cattle in monduli district, northern tanzania’, onderstepoort journal of veterinary research 81(1), art. #843, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.843 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. occurrence of haemoparasites in cattle in monduli district, northern tanzania in this original research... open access • abstract • communication • ethical considerations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ haemoparasite infections are among the most economically important cattle diseases in sub-saharan africa. the present study investigated the occurrence of haemoparasites in 295 indigenous cattle from five villages (mswakini, lake manyara, naitolia, makuyuni and nanja) of the monduli district, a wildlife-domestic animal-human interface area in northern tanzania. the data showed that the overall occurrence of haemoparasites in the sampled cattle was 12.5% (95% ci: 8.7% – 16.3%), involving single and mixed infections with theileria parva, anaplasma marginale, babesia bovis, trypanosoma vivax and trypanosoma brucei. the highest haemoparasite occurrence was recorded in lake manyara (18.3%; 95% ci: 8.5% – 28.1%), and the lowest was recorded in nanja (6.5%; 95% ci: 0.4% – 12.6%). this preliminary study, furthermore, provided evidence of the possible arthropod vectors (ticks and tsetse flies) that may be involved in the transmission of haemoparasites to cattle in the monduli district. it is envisaged that this survey will stimulate more studies to determine the prevalence of haemoparasites in livestock by using more sensitive molecular techniques. communication top ↑ in sub-saharan africa, ticks are the most important ectoparasites involved in the transmission of numerous haemoparasite infections such as theileriosis, babesiosis, anaplasmosis and rickettsiosis (gitau et al. 1997; makala et al. 2003; swai et al. 2007). in addition, the haematophagous ticks that contribute to cattle anaemia cause physical damage to the hide and the resultant wounds make the animals susceptible to myiasis (snelson 1995). tsetse flies are the second most important vectors; they are mainly involved in the transmission of bovine trypanosomosis (laohasinnarong et al. 2011). such haemoparasite infections are also an impediment to the introduction of more productive exotic cattle breeds (makala et al. 2003). the zebu cattle (bos indicus) are more resistant than the exotic breeds (bos taurus) against both the haemoparasites and the arthropod vector (jongejan et al. 1988). although haemoparasites may be more accurately diagnosed by the more sensitive molecular techniques such as polymerase chain reaction (pcr) and loop-mediated isothermal amplification (lamp) or by serology, microscopy remains the most reliable, available and affordable test in the resource-imited endemic areas such as tanzania (mitashi et al. 2012).a cross-sectional survey was conducted from november to december 2013 in the monduli district (3°20’s, 36°15’e), which is an important wildlife-domestic animal-human interface area located in northern tanzania, to investigate the occurrence of haemoparasites in the indigenous tanzanian short horn zebu (tshz) and boran crosses. a total of 295 cattle, comprising 255 tshz and 40 boran crosses, were sampled from five villages (mswakini, lake manyara, naitolia, makuyuni and nanja) of the monduli district, of which 203 (68.8%) were female and 92 (31.2%) were male. with regard to age: 41 (13.9%) were under 12 months, 231 (78.3%) were between 12 and 36 months, whilst 23 (7.8%) were older than 36 months. after clinical examination and body condition scoring, as described by munang’andu et al. (2010), blood was collected from the jugular vein into labelled, heparinised tubes. during the sampling exercise, data for animal identification and clinical history of individual cattle were captured on a form. the packed cell volume (pcv) values were determined and giemsa-stained thin blood smears from each animal were examined by microscopy; the blood parasites were identified as previously described (almeria et al. 2001; soulsby 1982). data (± standard error [s.e.]) captured on record sheets were entered, stored and statistically analysed using spss version 16.0. mean pcv in infected and non-infected cattle were compared using the one-way analysis of variance. all statistical tests were considered significant at p < 0.05. clinical examination revealed that 29 (9.8%) cattle were infested with various species of ticks, including amblyomma, hyalomma, rhipicephalus and boophilus species (horak 2009) and that 35 (11.9%) animals were emaciated. a few tsetse flies (glossina pallidipes and glossina swinnertoni) (krinsky 2009) were also seen at some sampling sites. of the 295 cattle sampled, 37 (12.5%; 95% ci: 8.7% – 16.3%) had haemoparasite infections. table 1 summarises the occurrence of single and/or mixed infections with theileria parva, babesia bovis, anaplasma marginale, trypanosoma vivax and trypanosoma brucei in the sampled monduli cattle. the highest haemoparasite infection was recorded from lake manyara village (18.3%; 95% ci: 8.5% – 28.1%), whilst the lowest was from nanja village (6.5%; 95% ci: 0.4% – 12.6%) (table 1); however, there was no statistically significant difference (p = 0.12) in the occurrence of bovine haemoparasite infection observed among villages. the average ages for infected and non-infected cattle were 20.3 months and 18.3 months, respectively. although a tendency towards lower pcv values was observed in infected animals from some villages, in general, there was no significant difference (p = 0.08) in the mean pcvs of cattle that were detected with haemoparasites (29.2% ± 1.7) and the animals in which they were not detected (31.2% ± 0.3). table 1: prevalence of haemoprotozoa in cattle from monduli district, northern tanzania. in the present survey, the occurrence of haemoparasite infection in the indigenous tshz and boran crosses reared communally in monduli district in northern tanzania was determined by microscopy. considering the fact that microscopy may fail to detect haemoparasites in cattle with low parasitaemia, it is possible that the rate of bovine haemoparasite infection would be higher if more sensitive molecular techniques were used (namangala et al. 2012; thekisoe et al. 2007). however, despite its limitations in terms of sensitivity and specificity, microscopy remains the most commonly used definitive diagnostic test of haemoparasite infections in resource-poor countries such as tanzania (laohasinnarong et al. 2011) as it is simple, reliable and inexpensive (mitashi et al. 2012). it is still one of the most widely used diagnostic tests in the field and can simultaneously demonstrate several haemoparasite species, as was the case in the present study. most of the haemoparasite-infected cattle did not manifest clinical disease, apart from poor body condition and anaemia, which could have been due to multiple causes including worm and tick infestation as well as poor pasture during the sampling period. although approximately 12.0% of the cattle were emaciated, only a few emaciated animals tested positive for haemoparasites. moreover, there was no significant difference in the pcv values of infected and non-infected cattle. these data support the notion that the indigenous monduli tshz and boran crosses may be relatively tolerant to haemoparasite infections (taylor 1998). normally, such haemoparasite-infected cattle would be expected to show more severe clinical signs, considering the fact that (1) they generally had a poor body condition score and (2) most of them rarely received veterinary services, hence the observed tick burden, which could have contributed to anaemia. nonetheless, the majority of those cattle were either asymptomatic or only exhibited mild signs, with no case of acute infection. therefore, in addition to possible indirect effects on productivity, such cattle could act as a source of theileriosis, trypanosomosis, anaplasmosis and babesiosis (laohasinnarong et al. 2011; makala et al. 2003; swai et al. 2007) for other susceptible livestock, particularly the more productive exotic animals. it is also noteworthy that such haemoparasite-infected, asymptomatic cattle may serve as reservoirs of the zoonotic trypanosoma brucei rhodesiense and trypanosoma brucei gambiense, which may cause sleeping sickness (welburn et al. 2001). although trypanosoma congolense is one of the common causes of trypanosomosis in cattle, it was not detected in the present study. in agreement with swai and kaaya (2012), a relatively higher occurrence of t. vivax in cattle blood sampled can be reported. this may be as a result of reduced presence of the biological tsetse vectors for t. congolense and increased presence of other haematophagous arthropods, including insect vectors such as tabanus, stomoxy and haematopota species that could easily transmit t. vivax mechanically. according to data obtained in the present study, the occurrence of bovine haemoparasites seems to be associated with proximity to national parks. although not statistically significant, a tendency was observed towards higher occurrences of haemoparasites in cattle from lake manyara (~18%) and naitolia (~14%) villages, which are close to lake manyara national park with abundant arthropod vectors and wild animal reservoirs of haemoparasites (munag’andu et al. 2010), compared to nanja village (6%), which is located much further from lake manyara national park and close to monduli town, with better access to veterinary services. furthermore, the occurrence of bovine haemoparasite infection appears to be linked with age, with calves tending to have a lower infection rate than adults. this may be partially explained by the fact that calves usually graze close to home, as compared to older animals that graze far into the bush, with increased frequency of being bitten by infected arthropod vectors (bett et al. 2004; lisulo et al. 2014). ethical considerations top ↑ blood samples were obtained from cattle whose owners agreed to the sampling after giving their informed consent to participate in the survey. conclusion top ↑ the present study has provided evidence of both the haemoparasites and, potentially, their arthropod vectors (ticks and tsetse flies) responsible for disease transmission to the indigenous tanzanian cattle in monduli district. it is, therefore, envisaged that this survey will stimulate more studies to determine the prevalence of haemoparasites in tanzanian livestock using more sensitive molecular techniques such as pcr and/or lamp. the regular use of insecticides to control the arthropod vectors of haemoparasites in monduli district is highly recommended. acknowledgements top ↑ this study received financial support from the southern africa centre for infectious disease surveillance (sacids). the authors would also like to thank the monduli farmers, the assistant administrative secretary arusha regional secretariat, the department of livestock and fisheries in monduli district council and the technical staff at tanzania veterinary laboratory agency (tvla) arusha centre for their assistance and support. the authors declare that there is no conflict of interest. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article authors’ contributions i.j.h. 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http://dx.doi.org/10.1016/s0140-6736(01)07096-9 masgoret_209-225.indd 209 onderstepoort journal of veterinary research, 76:209–225 (2009) molasses as a possible cause of an “endocrine disruptive syndrome” in calves m.s. masgoret1, c.j. botha1*, j.g. myburgh1, t.w. naudé1, l. prozesky1, v. naidoo1, j.h. van wyk2, e.j. pool3 and g.e. swan1 abstract masgoret, m.s., botha, c.j., myburgh, j.g., naudé, t.w., prozesky, l., naidoo, v., van wyk, j.h., pool, e.j. & swan, g.e. 2009. molasses as a possible cause of an “endocrine disruptive syndrome” in calves. onderstepoort journal of veterinary research, 76:209–225 during the mid 1990s a potentially serious, chronic syndrome was reported in well-managed beef and dairy herds from unrelated parts of south africa. farmers reported that it manifested as various combinations of decreased production, decreased weaning masses, apparent immune breakdown in previously immunocompetent animals, increased reproductive disorders, various mineral imbalances in non-deficient areas and goitre, noticeable as enlarged thyroid glands. the farmers associated this syndrome with certain batches of sugar cane molasses and molasses-based products. the syndrome was reminiscent of an “endocrine disruptive syndrome”. the objective of this study was to evaluate the suspected endocrine disruptive effect of molasses included in cattle feed. using existing in vitro assays, four batches of molasses syrup were screened for possible inclusion in a calf feeding trial. two batches were selected for the trial. thirty-two, 4to 6-week-old, weaned holstein bull calves were included in the single phase, three treatment, parallel design experiment. in two of the groups of calves, two different batches of molasses were included in their rations respectively. the control group was fed a ration to which no molasses was added, but which was balanced for energy and mineral content. the mass gain of the calves was recorded over the 6-month study period. the calves were clinically examined every week and clinical pathology parameters, immune responses and endocrine effects were regularly evaluated. even though endocrine disrupting effects were detected with the in vitro screening assays, these could not be reproduced in the calves in the experiment. the two batches of molasses utilized in the calf feeding trial did not induce major differences in any of the parameters measured, with the exception of a lower mass gain in one of the molasses-fed groups (group 1), which tended towards significance. the results of the study indicate that the two batches of molasses had no endocrine disruptive or immunosuppressive effects in calves. keywords: endocrine disruptive compound, endocrine disruptive syndrome, holstein calves, sugar cane molasses * author to whom correspondence is to be directed. e-mail: christo.botha@up.ac.za 1 department of paraclinical sciences, faculty of veterinary science, university of pretoria, private bag x04, onder stepoort, 0110 south africa accepted for publication 28 october 2008—editor 2 ecophysiology laboratory, department of botany and zoology, university of stellenbosch, stellenbosch, 7600 south africa 3 department of medical bioscience, university of the western cape, bellville, 7535 south africa 210 molasses as cause of “endocrine disruptive syndrome” in calves introduction during the mid 1990s a chronic, potentially serious syndrome was reported in well-managed beef and dairy herds in various unrelated parts of south africa, which manifested as combinations of: • decreased weaning mass; • apparent immune deficiency/incompetence/ breakdown resulting in calf diarrhoea, mastitis, pneumonia, footrot, verminosis in adult stock and relapses of protozoal diseases in animals considered to be immunocompetent; • decreased milk yield and loss in condition, particularly of dairy cows; • increased reproductive disorders with poor conception, resorptions of foetuses and abortions; • various mineral imbalances, such as copper deficiency in non-deficient areas, selenium deficiency in spite of a kidney selenium excess and high kidney lead concentrations; and • goitre manifesting as enlarged and/or cystic thyroids in both adults and aborted foetuses. although the cause was unknown, farmers related this syndrome to the feeding of certain batches of sugar cane molasses and molasses-based products. this was based on a causal relationship, as all the affected animals were feeding on molasses or molasses-based products. further support came from the dramatic improvements observed in health and productivity that often followed the withdrawal of the molasses component from the diet. the reported syndrome shares many similarities with an “endocrine disruptive syndrome” (eds), a relatively new concept in toxicology that has been well described in humans and in a variety of wildlife species (colborn, vom saal & soto 1993; lamb 1996; guillette 2000a & b), but never before in cattle. although this syndrome is yet to be identified in cattle, it has been predicted that, should it ever occur in cattle, it would be characterized by infertility and immunosuppression (rhind 2002). endocrine disruptive compounds (edcs) are exogenous substances or mixtures that alter the functions of the endocrine system and consequently cause adverse health effects in an intact organism or its progeny (ipcs—international programme on chemical safety steering group 1998). most edcs have the ability to act as either hormone agonists or antagonists and/or disrupt production, secretion, transportation, metabolism, receptor binding and excretion of natural hormones which regulate developmental processes and support endocrine homeostasis in the organism (kavlock, daston, derosa, fenner-crisp, gray, kaattari, lucier, luster, mac, maczka, miller, moore, rolland, scott, sheehan, sinks & tilson 1996). this ability of edcs may, amongst others, result in oestrogenic, anti-oestrogenic, androgenic and anti-androgenic effects, which will affect the normal reproductive functions of animals (li & hansen 1996). endocrine disrupting effects are not confined to effects on steroidal hormones; it has been demonstrated that a number of these environmental agents can alter thyroid hormone levels in humans and animals (langer, tajtakova, fodor, kocan, bohov, michalek & kreze 1998; ishihara, sawatsubashi & yamauchi 2003). some of the most important compounds included in this classification are: organohalogen compounds, phthalates, bisphenol a, alkylphenols and other com pounds such as phyto-oestrogens (naturally occurring plant oestrogens) (colborn, dumanoski & myers 1996). a similar group of compounds, referred to as “immunotoxicants”, are pollutants often classified with edcs and are thought to affect the immune system, mainly by disrupting band t-cell homeostasis (sweeny 2002). for a more extensive discussion a full review is available by colborn et al. (1996) and norris & carr (2006). even though there is a paucity of scientific literature on possible reproductive effects of edcs in cattle, concerns have been expressed about the potential effects on reproductive health and immune status of farm animals following exposure to a range of natural and synthetic environmental compounds that disrupt normal hormonal homeostasis (sweeney 2002; brevini, cillo, antonini & gandolfi 2005). in the past few years, more evidence regarding the effects of exposure to multiple pollutants on animal production has become available (meijer, de bree, wagenaar & spoelstra 1999). to date there have not been many controlled studies that have confirmed adverse effects of edcs in cattle, although there is sufficient evidence that there are causal relationships between edcs and production and reproductive functions of farm animals (rhind 2005). furthermore, it is also important to consider the age of the animals at the time of exposure to edcs. their impact might be more apparent in the developing foetus and post-natal offspring as many of the normal homeostatic endocrine feedback mechanisms and the immune system are not yet fully developed and there is a greater potential for low dose edc exposures to exert adverse effects on those systems (crisp, clegg, 211 m.s. masgoret et al. cooper, wood, anderson, baetcke, hoffman, morrow, rodier, schaeffer, tovart, zeeman & patel 1998; sweeny 2002). molasses is concentrated syrup of sugar cane consisting of structural sugars, hemicelluloses and minerals, and is usually obtained as a by-product of sugar refining operations. it is an important component of stock feed in south africa and the rest of the world, serving as a highly fermentable carbohydrate source of energy for growth and maintenance. moreover, molasses is often included in a ration to improve its palatability, improve rumen microbial activity, increase microbial protein synthesis, reduce dustiness of the ration or act as a binder for pelleting (perry, cullison & lowrey 1999). apart from molasses’ ability to deplete copper reserves, attributed to its high sulphur content (beames 1959; arthington & pate 2002) and a condition known as “molasses toxicity” (mcdonald, edwards, greenhalg & morgan 1995), no other deleterious effects caused by it have been reported in the past. due to its widespread use in cattle feed, it was considered necessary to ascertain if there was indeed a problem with molasses. thus, a scientifically controlled study to investigate the suspected toxicity of molasses was essential in order to improve our understanding of the syndrome. the objectives of this project were threefold: • to perform an initial screening of four batches of molasses in order to select two potential harmful batches for inclusion in a calf feeding trial; • to ascertain if specific batches of molasses could reduce growth and production in calves by comparing their mass gain over a 6-month study period; and • to evaluate the safety of molasses by evaluating clinical and clinico-pathological parameters, immune responses and endocrine effects between the groups of calves. materials and methods molasses screening tests molasses syrup samples from four different sugar mills were collected. two of the sugar mills (mills 1 and 2) were situated in the southern part of south africa, where sugar cane is harvested after 2 years of growth—these samples were collected directly from the bulk tanks. the other two sugar mills (mills 3 and 4) were situated in the northern part of south africa, where sugar cane is harvested after 12 months—these samples were collected directly from the production line. the four batches of molasses were analysed by the institute for soil, climate and water (iscw), pretoria to determine their chemical and nutrient composition. as no single in vitro test is able to detect all properties of hormonally active substances a battery of tests (including in vivo tests) were performed to evaluate results (eertmans, dhooge, stuyvaert & comhaire 2003). ethyl acetate and acetone extracts (obtained using analytical grade reagents) of the four batches of molasses were prepared and sent to different laboratories for oestrogenicity, immunotoxicity and thyroid activity assays as described below. oestrogenicity assays a cell proliferation assay, using a human cancer cell line (mcf-7) to measure oestrogenic activity (lippman & huff 1976), was performed by the medical faculty of the texas a & m university, usa. the assay was repeated three times and the average calculated. a “yeast screen” was conducted by the eco physiology laboratory, department of botany and zo ology, university of stellenbosch, south africa. this test utilizes yeast cells, transfected with the human oestrogen receptor (gray, kelce, wiese, tyl, gaido, cook, klinefelter, desaulniers, wilson, zacharewski, waller, foster, laskey, reel, giesy, laws, mclachlan, breslin, cooper, di giulio, johnson, purdy, mihaich, safe & colborn 1997). in addition, a frog vitellogenin (vtg) assay was done. in this study, male frog (xenopus laevis) liver slices were exposed to molasses extracts and the vtg synthesized by the liver slices, as an indication of oestrogenic activity, was measured by means of radio-immunoassay (van wyk, pool & leslie 2003). this assay was performed by the ecophysiology laboratory, department of botany and zoology, university of stellenbosch. immunotoxicity screening tests gamma-interferon (-ifn) was assayed (using a bovigam elisa kit), to evaluate the production of -ifn in response to mitogenic stimulation of bovine peripheral blood mononuclear cells, as an indication of immunosuppressive activity (maue, waters, palmer, whipple, minion, brown & estes 2004). this assay was performed by the medical faculty of the texas a & m university, usa. 212 molasses as cause of “endocrine disruptive syndrome” in calves the production of interleukin-6 (il-6), another indicator of immunosuppressive activity, was also measured by the ecophysiology laboratory, depart ment of botany and zoology, university of stellenbosch. after incubation of molasses samples with human blood, the supernatants were assayed for il-6 concentrations. the difference in il-6 response was then calculated and expressed as a percentage recovery using a control sample containing no extract as 100 % recovery. samples giving recoveries of less than 90 % are considered to indicate inflammatory response suppression (pool, johaar, james, petersen & bouic 1998; pool, robson, smith, van wyk & myburgh 2002). thyroid hormone (th) activity test a widely used screening test, the frog metamorphosis assay based on the assessment of tail resorption by x. laevis tadpoles, was performed to assess thyroid activity (eertmans et al. 2003; turque, palmier, le mevel, alliot & demeneix 2005). in this study the tadpoles were exposed to triiodothyronine (t3) and thyroxine (t4) as well as molasses and combinations of t4 and molasses. tail biopsies from metamorphic tadpoles were collected at 48 and/or 96 h post-treatment. the level of thyroid hormone receptor β (trβ) mrna was evaluated by means of a reverse transcription-polymerase chain reaction (rt-pcr). this assay was performed by the ecophysiology laboratory, department of botany and zoology, university of stellenbosch. calf feeding trial experimental animals, housing and nutrition thirty-two, 4to 6-week-old, weaned holstein bull calves of approximately the same mass, supplied by the same rearing facility, were adapted to their new environment over a 4 week period. during the adaptation period they received the same starter ration on which they were reared. the calves were subsequently included in a single phase, three treatment, parallel design experiment. before inclusion into the study all the animals were clinically examined (normal general appearance and habitus, temperature, respiratory and heart rates, rumen movements, absence of noticeable clinical signs of disease) and clinical pathology parameters (haematology, serum proteins, serum enzyme activities and serum urea nitrogen (sun) and creatinine) were determined. the calves were weighed before commencement of the trial, ranked from the heaviest to the lightest and sorted into replicates of three animals each. within each replicate the calves were randomly allocated, by means of a table of random numbers, to the treatment groups. the calves (n = 32) were allocated to three groups and each group was fed one of the following rations at 3 % body mass: group 1: a ration containing 6 % of molasses (“as is” basis) from mill 1 (n = 11) group 2: a ration containing 6 % of molasses (“as is” basis) from mill 4 (n = 10) control group: a ration to which no molasses was added, but approximately 3 % fermentable sugar (sucrose, glucose and fructose) and minerals (mainly potassium, magnesium and sulphate) equivalent to that of the national average for molasses (as supplied by the sugar milling research institute (smri), university of kwazulu-natal, durban) were added to it to compensate for the mineral and energy content of molasses (n = 11). eragrostis hay (both e. curvula and e. tef) was also available ad libitum and the calves had free access to potable municipal water. the calves were weighed weekly and the amount of the ration to be fed was adjusted accordingly. the rations were balanced by an independent nutritionist to ensure that they were equivalent in all aspects. the three different rations were mixed separately in batches of one ton each. the mixer used for this procedure was cleaned of all loose material before mixing and the control ration without molasses was always mixed first followed by the two molassescontaining rations. the calves were individually housed, in pens, at the university of pretoria´s biomedical research centre, onderstepoort so that each calf only had access to its own feed. animals were allowed access to an adjacent communal camp, for approximately 6 h a day, during weekdays for exercise and socialization. feed and water analysis samples of each batch of feed (eight in total) were collected and submitted for complete feed analyses to up nutrilab, department of animal and wildlife sciences, faculty of natural and agricultural sciences, university of pretoria. the analyses were 213 m.s. masgoret et al. performed according to the laboratory’s official standard operating procedures (giron 1973; association of official analytical chemists 2000). water samples were collected at commencement of the trial and submitted to the iscw for icp-ms analysis. clinical examination the animals were observed daily for any abnormalities. diarrhoea was included as part of the clinical signs, when: • calves exhibited mild diarrhoea (semi-formed to soft) on 3 or more days during the specific week; • calves exhibited moderate diarrhoea (soft to watery) on 2 or more days during the specific week; and • calves exhibited severe diarrhoea (watery) on one or more occasions during the specific week. the calves were clinically examined every week. however, daily clinical examinations were performed whenever an animal’s physical condition deteriorated. due to persistent diarrhoea in a number of calves, faecal samples were collected from six animals on day 90 of the trial and screened for coccidial oocysts and helminth eggs and submitted to the department of veterinary tropical diseases, faculty of veterinary science, university of pretoria for bacterial culture. faecal samples were also sent for virus particle identification by electron microscopy (em) to the department of anatomy and physiology, faculty of veterinary science, university of pretoria body mass the animals were weighed weekly on a calibrated digital built-in floor scale and the weekly mass gain was calculated. clinical pathology clinical pathology parameters were assessed before the commencement of the trial and on days 120 and 148 after the experiment commenced. the following parameters were determined by the clinical pathology laboratory, faculty of veterinary sci ence, university of pretoria: • haematology (red cell count, white cell count [including differential count], thrombocyte count, haematocrit); • serum proteins (total serum proteins, albumin and globulins); • serum enzyme activities (alkaline phosphatase [alp], γ-glutamyltransferase [ggt], lactate dehydrogenase [ldh] and aspartate aminotransferase [ast]); and • serum nitrogenous compounds (urea & creatinine). thyroid hormone analyses the animals were bled once before commencement of the trial (day 0) and three times during the trial to measure triiodothyronine (t3) and thyroxine (t4) concentrations. the latter were at 1 month (day 29), at 3 months (day 86) and at 5 months (day 148). the analyses were performed by the staff of the endocrinology laboratory, faculty of veterinary science using a coat-a-count total t3/t4 method (diagnostic products corporation, usa). testosterone analysis serum of the 32 animals, bled on day 154 of the feeding trial, was submitted to the reproduction laboratory, faculty of veterinary science, university of pretoria, for determination of total testosterone concentrations. the analysis was performed by means of a coat-a-count total testosterone radioimmunoassay (diagnostic products corporation, usa). tests to assess immune status of the calves all the animals, which had been vaccinated at 4 weeks of age against mannheimia haemolytica, infectious bovine rhinotracheitis (ibr) and bovine viral diarrhoea (bvd) with bovi-tect pi® (intervet sa (pty), ltd) at the rearing facility, were bled on day 120 of the feeding trial to assess their immune competence. serum samples were submitted to the department of veterinary tropical diseases, faculty of veterinary science for an indirect fluorescent antibody (fa) test for igg antibodies against ibr. serum samples from day 120 were also sent to the arc-onderstepoort veterinary institute (ovi) to determine brucella antibody titres using the complement fixation test (cft). following blood collection, the calves were immunized (on day 120) with brucella abortus strain 19 vaccine®, (onderstepoort biological products). four weeks later blood was again collected and the cft repeated to measure antibody titres in all the experimental animals (coetzer, thomson & tustin 1994). a “brucella protein allergen skin test” was performed in all the calves on day 170 of the trial. an 214 molasses as cause of “endocrine disruptive syndrome” in calves area of the skin of the neck was selected in each animal and its thickness was measured using a calliper. animals were then injected intradermally with brucellergene ocb® (synbiotics corporation), a brucella protein allergen. the skin thickness was again measured 72 h later (day 173) to evaluate the inflammatory reaction against the allergen. a “lymphocyte transformation test” was done immediately before commencement of the feeding trial and after 4 months (day 120 of the trial), to ascertain the immune competence of the animals. the lymphocytes were challenged with the mitogen, concanavilin a (con-a) (van kleef, macmillan, gunter, allsop, shkap & brown 2000). an unstimulated animal was used as control. this test was performed by the division of molecular biology, ovi. immunoglobulin g (igg) and immunoglobulin m (igm) were first measured at the beginning of the trial to determine a basal level. the animals were bled again at 4 months (day 120) and at 5 months (day 154), before and after the immunization with brucella abortus strain 19 vaccine, to determine their immunoglobulin titres and therefore their immune competence. the titres were determined by the clinical pathology laboratory, faculty of veterinary science, university of pretoria. post-mortem analyses the animals were slaughtered during week 26 of the trial at an approved abattoir. the carcass mass of each animal was recorded at slaughter. samples of various organs or tissues were collected at necropsy. these included: cerebrum, cerebellum, midbrain, brainstem, heart, lung, rumen wall, small and large intestine, several lymph nodes, spleen, liver, kidney, testis, thyroid gland, pancreas, thymus and adrenal gland. samples for histopathology (10 mm thickness) were collected and fixed in 10 % buffered formalin. the tissues were routinely processed, embedded in paraffin wax, cut and mounted on glass slides, and stained with haematoxylin and eosin. the organs were evaluated macroscopically and the histopathology slides were viewed under a light microscope by two independent pathologists. various tissue samples were submitted to the bacteriology division, ovi, for brucella culture. the following tissues were collected: retropharyngeal-, superficial inguinal and prescapular lymph nodes, tonsils, spleen, testis and epididymus. bacterial culture was done using standard techniques (alton, jones, angus & verger 1988). liver samples (100–200 g) were also collected and submitted to the up nutrilab for copper (cu), iron (fe), manganese (mn) and zinc (zn) determination by means of atomic absorption spectrophotometry (giron 1973). statistical analyses the data were captured and arranged in animals per replicate. various statistical analyses were performed by statistical consultation service, pot chefstroom campus of the university of north-west. all statistical calculations were performed using a statistical programme (statsoft. inc. 2005). the significance level was set at p < 0.05. clinical significance was based on the partial eta-square. an eta-square value of 0.14 can be viewed as a large effect (cohen 1988). one-way analysis of variance (anova) on experimental groups was utilized to test for differences in carcass masses, the brucella complement fixation test and testosterone concentration. repeated measures analyses were done on weekly mass gains, t3, t4, igg and igm levels. these were followed by two-way anova on groups within block-replicates. tukey multiple comparisons were performed to compare groups or bonferroni multiple comparisons to compare different times over all groups. a two-way anova on groups within block-replicates was used for monthly mass gain, the brucella skin test and the lymphocyte transformation test. results molasses screening tests the selection of the molasses to be used in the calf feeding trial was based on the laboratory screening tests (table 1) since no major differences were noticeable in the chemical and nutritional composition of the four batches (table 2). molasses from mill 1 showed the highest oestrogenic activity based on the mcf-7 assay as well as in the yeast screen and a marked immunosuppressive activity with the ifn assay. molasses from mill 4 showed the highest immunosuppressive effect with the il-6 assay and a high th activity. based on the results obtained with these laboratory screening tests, molasses from 215 m.s. masgoret et al. from mills 1 and 4 was selected to be used in the feeding trial. calf feeding trial feed and water analysis the mean values of the feed analyses of the eight samples collected during the trial were calculated for each group. the results are presented in table 3. there were no major differences in the composition of the rations fed to the three groups. water analysis results confirmed that all values were within normal ranges. clinical examination bouts of diarrhoea were observed in all the calves before and during the first half of the feeding experiment (fig. 1). however, group 2 had a higher table 1 screening tests results test* mcf-7 yeast screen vtg -ifn il-6 thyroid toxicity laboratory texas stellenbosch stellenbosch texas stellenbosch stellenbosch mill 1 blank mill 1 sample 2 729 ppt 0.5 od 3.0 od 7 μg/mℓ 2–12 μg/mℓ 0.3 od 92 % 0.23 units mill 2 blank mill 2 sample 1 090 ppt 0.5 od 0.6 od negative negative 1.1 od > 90 % 89 % 0.18 units mill 3 blank mill 3 sample 1 090 ppt 0.6 od 1.5 od 2 μg/mℓ 40 μg/mℓ 1.3 od 73 % 0.21 units mill 4 blank mill 4 sample 467 ppt 0.6 od 1.3 od 2 μg/mℓ 5–25 μg/mℓ 1.35 od 68 % 0.32 units * maximum results in bold text table 2 chemical and nutritional analyses of molasses nutritional data mill 1 mill 2 mill 3 mill 4 national average* dry solids (%) fructose (%) glucose (%) sucrose (%) starch (ppm) 77.05 6.7 4.3 27.7 1 342 74.89 7.7 5.2 27.6 1 586 81.45 9.5 7.4 28.1 922 79.03 8.9 6.5 27.6 542 7.4 5.2 31.3 1 900 elements (mg/kg) on an “as is” basis arsenic beryllium boron cadmium chromium cobalt copper lead lithium mercury molybdenum** nickel selenium tin uranium vanadium zinc 0 0.0166 8.5466 0.3039 0 0.614 2.5636 1.1576 0.1509 1.1796 < 0.01 1.8036 3.8844 0.0937 0 0 13.9484 0 0.0188 12.1501 0.3229 0 1.0301 2.6086 1.4927 0.1034 0.9608 – 3.1614 3.6116 0.2538 0 0 16.4702 1.1029 0.027 21.615 0.3215 0.1351 1.6929 3.179 0.9294 0.1587 0.9263 – 4.2833 3.6971 0.1771 0 0 14.3986 2.3084 0.022 9.8574 0.324 0.2778 1.7604 4.8766 1.24 0.1302 1.3735 0.17 13.3637 3.8244 0.0816 0 0 17.598 * information supplied by the sugar milling research institute ** analysed by central analytical laboratories, pretoria 216 molasses as cause of “endocrine disruptive syndrome” in calves number of animals with diarrhoea, but this did not affect their mass gain. no viral particles were noticed with the em scan and no bacterial pathogens were cultured in any of the samples. the diarrhoeal fluid was also negative for coccidial oocysts and helminth eggs. calf 8 from group 1 was hospitalized on day 93 of the trial due to severe persistent diarrhoea; it made an uneventful recovery and was returned to the group. four months after the commencement of molasses feeding, one of the calves from the control group (calf 5) developed an abomasal displacement which was corrected surgically. when the displacement re-occurred on day 162 of the trial, the calf was slaughtered in the abattoir. another animal from group 1 (calf 28) developed an upper respiratory tract infection on day 157, it was treated with an antibacterial agent [danofloxacin, advocin®, pfiz er laboratories (pty) ltd] and a non-steroidal anti-inflammatory drug [flunixin meglumine, cron yxin®, kyron laboratories (pty) ltd.] and subsequently recovered. body mass the mean live body mass of the three groups are depicted in fig. 2. no significant differences between the groups were noticed throughout the trial. the weekly mass gain of each animal was calculated against its initial mass at the beginning of the trial (fig. 3). average weekly mass gains of the two molasses-fed groups were compared with those of the control group (fig. 4). a notable lower mean mass gain, although not statistically significant, occurred in group 1 compared with group 2 and the control group. however, when the experimental error was corrected (due to the large differences in body mass between different replicates) the monthly mass gain was only statistically significantly (p = 0.025) lower in group 1 at 4 months following molasses feeding and at no other time. clinical pathology the mean of the clinical pathology parameters was calculated for each group (tables 4 and 5). all the parameters fluctuated within or near normal ranges. thyroid hormone analyses the results of the t3 and t4 analyses were plotted (fig. 5 and 6). the mean t3 concentrations of the three groups showed similar curves. group 1 showed a slightly lower mean t4 concentration compared to the other two groups at 3 months after commencement of molasses feeding. however, no statistical significant difference could be demonstrated. in the control group an appreciable decrease in mean t4 concentrations occurred from the initial bleeding before the molasses feeding commenced, up to 3 months of molasses feeding. testosterone analysis there were no significant differences amongst the groups with regard to their mean testosterone concentrations (table 6). table 3 feed and mineral analyses on an “as is” basis group 1 group 2 control group mean ± sd dm (g/100 g) moist (g/100 g) ash (g/100 g) cp (g/100 g) c fat (g/100 g) me (mj/kg) 87.2 ± 0.40 12.8 ± 0.40 5.5 ± 0.32 12.7 ± 0.54 3.87 ± 1.37 15.93 ± 0.34 87.1 ± 0.44 12.8 ± 0.44 5.5 ± 0.42 12.8 ± 0.70 3.8 ± 1.23 15.9 ± 0.26 88.0 ± 0.43 11.9 ± 0.43 6.6 ± 0.52 12.5 ± 0.56 3.7 ± 1.26 15.7 ± 0.40 ca (g/100 g) p (g/100 g) mg (g/100 g) cu (mg/kg) fe (mg/kg) zn (mg/kg) mn (mg/kg) k (g/100 g) na (g/100 g) 0.77 ± 0.05 0.38 ± 0.06 0.19 ± 0.03 31.01 ± 6.09 356.11 ± 99.08 121.90 ± 8.55 71.29 ± 14.98 0.99 ± 0.11 0.17 ± 0.05 0.78 ± 0.08 0.37 ± 0.06 0.19 ± 0.03 30.76 ± 4.34 357.37 ± 93.47 125.11 ± 9.81 62.92 ± 13.11 1.04 ± 0.06 0.22 ± 0.07 0.82 ± 0.07 0.46 ± 0.06 0.22 ± 0.03 34.86 ± 9.58 340.86 ± 62.27 140.72 ± 22.01 113.78 ± 59.52 1.32 ± 0.18 0.19 ± 0.07 sd = standard deviation 217 m.s. masgoret et al. t a b l e 4 h a e m a to lo g y p a ra m e te rs n o rm a l ra n g e b e fo re t ri a l d a y 1 2 0 d a y 1 5 4 g ro u p 1 g ro u p 2 c o n tr o l g ro u p g ro u p 1 g ro u p 2 c o n tr o l g ro u p g ro u p 1 g ro u p 2 c o n tr o l g ro u p m e a n ± s d h b ( g /d ℓ) r c c ( x1 0 1 2 /ℓ ) h t (ℓ /ℓ l) m c v ( fℓ ) m c h c ( g /d ℓ ce lls ) r d w ( % ) w c c ( x1 0 9 /ℓ ) a b n m a t (x 1 0 9 /ℓ ) a b n im m ( x1 0 9 /ℓ ) a b l ym p ( x1 0 9 /ℓ ) a b m o n o ( x1 0 9 /ℓ ) a b e o s (x 1 0 9 /ℓ ) a b b a so ( x1 0 9 /ℓ ) t h r c ( x1 0 9 /ℓ ) 5 .0 – 9 .0 0 .2 4 – 4 4 0 – 6 0 3 0 – 3 6 4 – 1 0 .0 0 .6 – 4 .0 0 .0 – 0 .0 1 2 2 .5 – 7 .5 0 .0 3 – 0 .8 4 0 .0 – 2 .4 0 .0 – 0 .2 2 0 0 – 6 0 0 1 0 0 ± 1 4 .5 1 8 .6 ± 1 .2 9 0 .3 0 ± 0 .0 4 3 4 .8 ± 1 .9 5 3 3 .2 5 ± 0 .5 6 2 5 .1 2 ± 2 .0 3 8 .6 8 ± 3 .4 2 2 .9 8 ± 1 .6 2 0 .0 1 ± 0 .0 5 4 .2 8 ± 1 .7 9 0 .8 6 ± 0 .5 8 0 .0 5 ± 0 .1 1 0 .0 3 ± 0 .0 7 1 0 2 2 ± 4 2 7 .3 9 4 .1 ± 9 .8 1 8 .0 ± 0 .6 7 0 .2 8 ± 0 .0 3 3 5 .3 3 ± 1 .1 3 3 .2 3 ± 0 .6 3 2 6 .3 6 ± 2 .3 4 8 .6 6 ± 1 .4 7 3 .3 6 ± 1 .3 0 .0 8 ± 0 .1 4 4 .5 0 ± 2 .0 1 0 .6 4 ± 0 .4 4 0 .0 2 ± 0 .0 5 0 .0 7 ± 0 .0 9 8 0 2 ± 2 8 5 .9 8 8 .3 6 ± 1 1 .3 4 7 .7 5 ± 0 .8 6 0 .2 7 ± 0 .0 3 3 4 .3 5 ± 1 .6 9 3 3 .1 5 ± 0 .6 5 2 5 .9 2 ± 3 .2 6 7 .9 4 ± 3 .1 2 2 .5 8 ± 1 .2 3 0 .0 1 ± 0 .0 4 4 .4 8 ± 2 .3 9 0 .7 6 ± 0 .3 9 0 .0 4 ± 0 .0 9 0 .0 7 3 ± 0 .1 1 9 5 0 ± 2 4 0 .5 1 1 0 ± 4 .9 6 8 .4 4 ± 0 .5 3 0 .3 2 ± 0 .0 1 3 7 .7 8 ± 2 .0 7 3 4 .5 4 ± 0 .2 1 2 4 .4 9 ± 1 .4 8 1 0 .3 0 ± 2 .3 3 4 .0 3 ± 1 .1 6 0 .0 0 ± 0 5 .5 9 ± 1 .4 0 .5 8 ± 0 .3 4 0 .0 7 ± 0 .1 0 .0 4 ± 0 .0 6 6 3 8 .6 ± 7 8 .2 1 1 4 ± 8 .6 8 .5 5 ± 0 .3 4 0 .3 3 ± 0 .0 2 3 8 .5 2 ± 2 .1 2 3 4 .4 5 ± 0 .5 5 2 4 .6 0 ± 1 .2 5 1 0 .4 7 ± 1 .3 3 3 .8 5 ± 0 .7 9 0 .0 0 ± 0 5 .7 3 ± 1 .2 6 0 .8 1 ± 0 .3 6 0 .0 3 ± 0 .0 4 0 .0 6 ± 0 .0 9 5 4 6 .5 ± 8 2 .9 6 1 1 0 .1 ± 6 .4 1 8 .3 8 ± 0 .3 7 0 .3 2 ± 0 .0 2 3 8 .0 5 ± 0 .0 2 3 4 .5 6 ± 1 .7 7 2 5 .0 6 ± 0 .6 4 1 0 .8 1 ± 1 .8 9 4 .2 4 ± 2 .2 1 0 .0 2 ± 0 .9 3 5 .7 4 ± 0 .0 7 0 .7 5 ± 2 .1 8 0 .0 6 ± 0 .3 1 0 .0 0 ± 0 6 4 0 ± 1 1 7 .7 6 1 1 4 ± 6 .9 5 8 .8 2 ± 0 .7 2 0 .3 3 ± 0 .0 2 3 8 .0 4 ± 2 .1 3 4 .0 7 ± 0 .4 3 2 3 .9 3 ± 1 .7 1 9 .5 3 ± 2 .2 4 3 .6 7 ± 1 .5 2 0 .0 0 ± 0 4 .9 6 ± 1 .5 7 0 .8 1 ± 0 .3 4 0 .0 4 ± 0 .0 5 0 .0 3 ± 0 .0 3 6 8 3 ± 9 9 .7 4 1 1 7 .4 ± 7 .8 9 8 .8 3 ± 0 .4 3 0 .3 4 ± 0 .0 2 3 8 .8 ± 2 .1 8 3 4 .2 1 ± 0 .5 7 2 4 .6 4 ± 1 .3 7 1 0 .8 1 ± 1 .7 7 3 .9 8 ± 1 .2 1 0 .0 0 ± 0 5 .6 5 ± 1 .6 1 0 .8 4 ± 0 .3 6 0 .2 5 ± 0 .3 9 0 .0 9 ± 0 .1 2 6 0 4 .4 ± 1 1 .8 6 1 1 3 ± 6 .2 7 8 .5 9 ± 0 .4 8 0 .3 3 ± 0 .0 2 3 8 .4 3 ± 1 .5 8 3 4 .2 0 ± 0 .3 1 2 4 .6 9 ± 1 .7 4 1 0 .2 8 ± 2 .5 3 3 .8 7 ± 1 .4 1 0 .0 0 ± 0 5 .4 6 ± 1 .5 4 0 .8 9 ± 0 .4 4 0 .0 4 ± 0 .1 0 0 .0 3 ± 0 .0 4 6 5 6 ± 1 4 0 .3 6 s d = s ta n d a rd d e vi a tio n t a b l e 5 s e ru m p ro te in s, n itr o g e n o u s co m p o u n d s a n d e n zy m e a ct iv iti e s n o rm a l ra n g e b e fo re t ri a l d a y 1 2 0 d a y 1 5 4 g ro u p 1 g ro u p 2 c o n tr o l g ro u p g ro u p 1 g ro u p 2 c o n tr o l g ro u p g ro u p 1 g ro u p 2 c o n tr o l g ro u p m e a n ± s d t s p ( g /ℓ ) a l b ( g /ℓ ) g l o b ( g /ℓ ) a /g a l p ( u /ℓ ) a s t ( u /ℓ ) g g t ( u /ℓ ) l d ( u /ℓ ) u re a ( m m o l/ℓ ) c re a t (μ m o l/ℓ ) 6 5 – 7 8 2 8 – 3 7 2 8 – 4 2 0 .9 – 1 .4 3 3 – 3 2 8 2 1 – 1 6 7 0 – 4 5 3 8 – 2 8 8 5 3 .6 – 1 0 .7 1 0 – 1 3 3 6 2 .1 ± 4 .0 7 3 3 .3 ± 0 .9 2 2 8 .7 ± 4 .2 4 1 .2 ± 0 .1 9 2 5 2 .8 ± 7 0 .2 6 8 .9 ± 2 5 .5 1 2 0 .2 ± 6 .2 3 1 8 5 7 ± 4 8 6 8 .6 ± 1 .2 6 8 .5 ± 9 .8 8 6 2 .7 3 ± 3 .8 2 3 3 .2 1 ± 2 .1 5 2 9 .5 2 ± 4 .8 5 1 .2 ± 0 .2 3 2 9 6 .8 ± 1 0 2 .1 4 5 1 .0 ± 6 .2 7 1 9 .1 ± 3 .9 8 1 7 0 1 .5 ± 2 3 7 .4 7 .4 ± 0 .7 8 6 5 .6 ± 7 .2 4 6 2 .1 ± 5 .0 9 3 2 .5 ± 2 .2 5 2 9 .5 ± 5 .0 5 1 .1 ± 0 2 0 2 9 7 .4 ± 1 5 4 .6 5 7 .5 ± 1 9 .7 5 2 3 .1 ± 5 .2 0 1 8 1 3 ± 3 9 9 7 .9 ± 1 .5 1 7 0 .4 ± 1 .5 1 6 4 .7 ± 3 .0 2 3 3 .0 ± 0 .9 3 1 .7 ± 2 .6 8 1 .1 ± 0 .0 8 2 7 7 .6 ± 1 0 3 .8 8 0 .7 ± 1 6 .2 8 1 6 .5 ± 3 .7 5 1 8 8 3 .7 ± 2 4 3 .7 3 .9 ± 1 .0 7 9 3 .4 ± 9 .3 9 6 4 .8 6 ± 3 .7 8 3 3 .0 6 ± 1 .6 8 3 1 .8 ± 4 .0 7 1 .1 ± 0 .1 6 2 6 3 .3 ± 6 6 .7 7 1 .0 ± 1 2 .9 1 6 .9 ± 3 .7 8 1 7 9 0 .2 ± 1 7 7 .5 2 .6 ± 0 .9 2 9 1 .6 ± 4 .8 1 6 4 .7 ± 4 .1 8 3 3 .7 ± 1 .7 1 3 1 .0 ± 4 .0 2 1 .1 ± 0 .1 5 2 7 8 .7 ± 7 8 .1 6 8 .5 ± 8 .2 0 1 6 .4 ± 2 .5 0 1 8 9 9 ± 2 3 1 3 .6 ± 1 .3 3 9 2 .0 ± 9 .8 4 6 6 .9 ± 3 .9 9 3 2 .8 ± 1 .4 9 3 4 .2 ± 4 .4 2 1 .0 ± 0 .1 6 2 5 6 .3 ± 6 6 .4 6 8 .7 ± 1 4 .0 0 2 3 .9 ± 5 .6 3 1 9 0 9 ± 2 8 4 3 .1 ± 0 .6 3 8 4 .9 ± 8 .0 0 6 8 .8 5 ± 2 .2 3 3 3 .2 4 ± 2 .8 5 3 5 .6 1 ± 3 .6 8 0 .9 4 ± 0 .1 6 2 3 9 .3 ± 1 1 2 .4 6 9 .3 ± 7 .7 3 2 1 .9 ± 4 .7 2 1 9 1 6 .4 ± 1 9 8 .7 3 .0 4 ± 1 .3 8 5 .8 ± 6 .0 9 6 6 .6 ± 4 .9 4 3 4 .0 ± 1 .9 9 3 2 .7 ± 4 .9 9 1 .1 ± 0 .1 7 2 6 8 .0 ± 8 7 .7 9 6 9 .5 ± 9 .0 5 2 1 .5 ± 4 .3 2 1 8 5 9 .2 ± 2 4 2 .2 3 .4 ± 1 .0 8 8 2 .7 ± 1 0 .1 4 s d = s ta n d a rd d e vi a tio n 218 molasses as cause of “endocrine disruptive syndrome” in calves m a s s ( k g ) 0 14 28 42 56 70 84 98 112 126 140 154 168 days 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 fig. 2 mean live body mass of the three experimental groups through out the trial group 1 group 2 control group n u m b e r o f c a lv e s w it h d ia rr h o e a 7 6 5 4 3 2 1 0 0 14 28 42 56 70 84 98 112 126 140 154 168 182 days fig. 1 diarrhoea in calves from the three experimental groups group 1 group 2 control group m a s s g a in ( k g ) 0 14 28 42 56 70 84 98 112 126 140 154 168 days 200 180 160 140 120 100 80 60 40 20 0 fig. 3 mean weekly mass gains of the three experimental groups group 1 group 2 control group 219 m.s. masgoret et al. m a s s d if fe re n c e ( k g ) 5 0 –5 –10 –15 –20 –25 0 14 28 42 56 70 84 98 112 126 140 154 168 days fig. 4 average weekly mass gains of the two molasses-fed groups compared to those of the control group (green triangle indicates a new batch of feed) group 1 group 2 new feed n m o l/ ℓ 0 29 58 87 116 145 days fig. 5 mean triiodothyronine (t3) concentrations of the three experimental groups at days 0, 29, 86 and 148 of the feeding trial group 1 group 2 control group 2.8 2.5 2.2 1.9 1.6 1.3 1 n m o l/ ℓ days fig. 6 mean thyroxine (t4) concentrations of the three experimental groups at days 0, 29, 86 and 148 of the feeding trial group 1 group 2 control group 0 29 58 87 116 145 80 75 70 65 60 55 50 220 molasses as cause of “endocrine disruptive syndrome” in calves s i* ( s ti m u la ti o n i n d e x ) fig. 7 results of the lymphocyte transformation test of the three experimental groups before and after 4 months of molasses feeding 1 800 1 500 1 200 900 600 300 0 group 1: before group 1: after group 2: before group 2: after control group: before control group: after m g /d ℓ 0 22 44 66 88 110 132 154 days fig. 8 igg levels of the three experimental groups at days 0, 120 and 154 of the trial [normal range of igg: 1700– 2700 mg/dℓ (coetzer et al. 1994)] group 1 group 2 control group 2 200 2 000 1 800 1 600 1 400 1 200 1 000 m g /d ℓ 0 22 44 66 88 110 132 154 days fig. 9 igm levels of the three experimental groups at days 0, 120 and 154 of the trial [normal range of igm: 250– 400 mg/dℓ (coetzer et al. 1994)] group 1 group 2 control group 400 300 200 100 221 m.s. masgoret et al. immune status of the calves using a serum screening dilution of 1:20, all animals in the three groups determined were positive for antibodies against ibr. before the immunization with brucella abortus strain 19 all the animals tested negative for b. abortus antibodies. the antibody titres against b. abortus were similar in the three groups one month after the immunization of the calves (table 7). no statistical significant difference was found between the three experimental groups. the results of the brucella protein allergen skin test showed no statistical significant differences between the groups when skin thickness was measured 72 h after the intradermal administration of the antigen brucellergene ocb® (table 8). with the “lymphocyte transformation test”, the control group showed more transformation at day 120 after the challenge with the mitogen, concanavilin a, but due to a very large standard deviation the difference was not statistically significant. results are presented as a stimulation index (si), where si = mean counts per min of test sample/mean counts per min of unstimulated control. a si higher than 2 was considered to be an indication of concanavilin a-induced proliferation (fig. 7). mean igg titres in all three groups increased consistently during the molasses feeding trial (fig. 8). mean igm concentrations increased after the start of molasses feeding at 4 months, but decreased slightly when determined again at 5 months into the trial (fig. 9). only slight variations occurred between the groups. no statistical significant differences were detected between the three experimental groups. post mortem analyses mean carcass mass of each group is shown in table 9. no significant differences were found between groups. no noteworthy macroor microscopic lesions were detected in the organs/tissues. all calves in the control group tested negative for b. abortus organisms on culture. brucella abortus was cultured in the left pre-scapular lymph node of one animal in group 2 and two animals in group 1. table 6 mean testosterone concentration of the three experimental groups at day 154 of the trial group 1 group 2 control mean ± sd testosterone (nmol/ℓ) 12.09 ± 9.39 14.4 ± 9.27 13.83 ± 8.53 sd = standard deviation table 7 mean brucella abortus antibody titres (cft) of the three experimental groups 4 weeks after immunization with brucella abortus strain 19 vaccine® group 1 group 2 control mean ± sd brucella antibodies (iu) 269.64 ± 198.87 249.00 ± 203.37 278.18 ± 205.87 sd = standard deviation table 8 mean skin thickness of the three experimental groups before and after intradermal injection with brucellergene ocb® group 1 group 2 control before after before after before after mean ± sd skin thickness (mm) 10.68 ± 1.98 13.73 ± 2.28 10.40 ± 2.22 13.68 ± 1.49 11.64 ± 2.18 14.69 ± 2.41 sd = standard deviation 222 molasses as cause of “endocrine disruptive syndrome” in calves the mean liver cu, fe, zn and mn concentrations for each group were calculated (table 10). no relevant differences were found in the levels of these trace elements between the three groups. discussion even though endocrine disrupting activity was detected in laboratory tests, these could not be reproduced in calves under experimental conditions. this illustrates that in vitro/in vivo comparisons can be misleading. in vitro assays do not always reliably predict the in vivo outcome, due to differences in metabolic capabilities of the test systems (and lack of in vivo pharmacokinetics and pharmacodynamics) and the diverse range of mechanisms by which endocrine disrupting chemicals may act (clode 2006). the feeding of molasses to calves has been reported by farmers to decrease production, but no significant differences between the study groups were detected in this study. during this feeding trial, calves from the three groups were kept in similar facilities, under the same conditions and fed diets with comparable composition regarding energy, protein and minerals (tables 2 and 3). drinking water parameters were also within normal limits. although it would seem that group 1 had a lower mean mass gain (compared to the other two groups) (fig. 4), a significant difference only occurred at 4 months of molasses feeding. considering that this was the only significant difference during the whole feeding trial, it is unlikely that this could be attributed to molasses in the feed. although the calves from group 2 exhibited a higher incidence of diarrhoea when compared with the other two groups (fig. 1), it did not appear to affect their mass gain over the whole feeding period. as the results of faecal samples, submitted for bacterial culture and virus particle identification as well as screening for coccidial oocysts and helminth eggs, were negative, the diarrhoea was considered to be of dietary or metabolic origin. the mean mass of the three experimental groups at week 24 of the trial (when the calves were between 30 and 32 weeks of age) ranged from 243–261 kg (fig. 2). although these holstein calves were weaned of milk within days of birth, their masses were comparable with the national average weaning mass of bonsmara calves, a beef breed known for high weaning mass (220 kg at 205 days of age) (agricultural research council, livestock business unit 2006). the farmers also reported increased reproductive disorders with poor conception, and high rates of resorption of foetuses and abortions, which were attributed to an “endocrine disruptive syndrome”. since the animals used in the feeding trial were bull calves, only certain parameters could be evaluated to detect reproductive disorders, viz., testosterone levels and the morphology and histology of the male reproductive organs. no significant difference was detected between the three groups with respect to testosterone concentrations (table 6) and reprotable 9 average carcass mass of the three experimental groups group 1 group 2 control mean ± sd carcass mass (kg) 134.64 ± 23.99 145.3 ± 25.88 141.91 ± 21.04 sd = standard deviation table 10 mean concentrations of cu, fe, zn and mn in liver samples group 1 group 2 control group normal range in cattle*mean ± sd cu (mg/kg wm) fe (mg/kg wm) zn (mg/kg wm) mn (mg/kg wm) 223.3 ± 57.1 40.4 ± 9.4 31.9 ± 8.0 2.9 ± 0.3 218.2 ± 39.3 40.8 ± 3.8 33.8 ± 8.1 2.9 ± 0.5 189.6 ± 40.3 40.5 ± 9.8 35.8 ± 11.5 2.8 ± 0.4 25–100 45–300 25–100 2.5–6 * mineral levels in animal health (puls 1994) wm = wet mass sd = standard deviation 223 m.s. masgoret et al. ductive organs of the bull calves from the three groups showed no visible macroor microscopical abnormalities. the farmers also highlighted apparent immune incompetence/breakdown in their herds resulting in calf diarrhoea, verminosis, relapses of protozoal diseases in previously immunocompetent animals and pneumonia caused by various pathogens. in the current study, calves from the three experimental groups were considered to be immunocompetent. when tested for ibr antibodies at 4 months into the feeding trial, after being vaccinated at a very young age at the rearing facility, all the animals showed positive titres. when challenged with b. abortus vaccine, all the animals seroconverted and the three experimental groups showed similar antibody titres against b. abortus one month after the immunization (table 7). in south africa, titres of 30 iu/mℓ or higher are considered positive in those animals that have been vaccinated between 4 and 8 months of age (coetzer et al. 1994). while all calves in the control group tested negative for b. abortus organisms on culture, only one animal from group 2 and two animals from group 1 tested positive. however, these results cannot be regarded as indicative of immune incompetence. in addition, immunoglobulin concentrations in the three groups showed no significant differences. whereas igg titres increased consistently during the molasses feeding trial (fig. 8), most probably due to natural exposure to different pathogens in the environment, mean igm concentrations increased at 4 months of molasses feeding, but decreased slightly when determined again at 5 months (fig. 9). the lymphocyte transformation test performed at 4 months also corroborated the immune competency of the animals in the three experimental groups (fig. 7). only three calves became sick during the trial, one calf from the control group developed a left sided abomasal displacement, which is not an infectious disease. one calf, from group 1, was hospitalized due to severe persistent diarrhoea and another animal from group 1 was treated for an upper respiratory tract infection, but both recovered. thus, all the parameters measured, indicate that the calves’ immune response was not affected. immunosuppression could, therefore, not be confirmed in the molasses fed calves. various mineral imbalances observed in the field were also attributed to the “endocrine disruptive syn drome”. although mean liver cu concentrations in the three groups were higher than the normal range for cattle (table 10), the concentrations did not reach toxic levels (250-800 mg/kg) (puls 1994) and is ascribed to the high dietary cu levels fed during the trial (table 3). these results not only contradict reports of cu deficiencies on the affected farms, but also molasses feeding experiments conducted in the past. beames (1959) fed different rations to three groups of hereford heifers and concluded that the two groups of animals receiving molasses as a supplement showed significantly depleted liver cu concentrations when compared to a group of heifers receiving no molasses supplement. he concluded that the reduction of cu reserves in the molasses-fed groups could be explained on the basis of high sulphur concentrations and a subsequent copper-molybdenum-inorganic sulphate interaction. arthington & pate (2002), performed experiments on brahman heifers and compared liver cu concentrations between animals given corn vs. molasses supplements. their results indicated that components in molasses-based supplements decreased the accumulation of cu in the liver of beef heifers and they suggested that selenium and molybdenum components in molasses could be partly responsible for this decrease. although dietary levels of fe were considered adequate (100-500 mg/kg)(puls 1994), liver fe concentrations in the three experimental groups were slightly lower than the normal range (table 3), but did not reach deficient levels for cattle (< 30 mg/kg) (puls 1994). iron deficiency is associated with anaemia, reduced growth, poor immune function and weakness (graham 1991; puls 1994). during the trial, no abnormalities in the haematology parameters were noticed and growth rate and immune function were also as expected. despite goitre being reported by the farmers manifesting as enlarged and/or cystic thyroids, no abnormalities were detected during macroand microscopical examination of the thyroid glands of the animals from the three experimental groups. an assessment of thyroid hormone status (t3 and t4) was also performed in the calves at four different times during the feeding trial (fig. 6 and 7). the mean t3 concentrations of the three groups showed similar curves and even though animals in group 1 showed a slightly lower mean t4 concentration compared to the other two groups at 3 months after commencement of molasses feeding, no statistical significant difference could be demonstrated. an appreciable decrease in mean t4 concentration, although not 224 molasses as cause of “endocrine disruptive syndrome” in calves statistically significant, occurred in the control group from the initial bleeding before molasses feeding commenced up to 3 months of molasses feeding. under the conditions of the present study, the two batches of molasses investigated induced no endocrine disruptive or immunosuppressive effects in the holstein bull calves. it can therefore be concluded that these two batches of molasses per se were not deleterious when fed to calves. acknowledgments we thank the south african sugar association and the national research foundation for funding the trial. we also express our gratitude to the following people and institutions who performed various analyses during the trial and who also gave valuable input throughout the project: prof. c. cruywagen, university of stellenbosch; prof. l. guillette jr, university of florida, usa; dr g. rottinghaus, columbia university, usa; dr m. estes, texas a & m university, usa; mr c. von brandis; proff. j. lawrence, h. bertschinger and j. godfroid, and dr j. crafford, university of pretoria; mario smuts, el-maré kilian, roland auer and the staff of the university of pretoria’s biomedical research centre; elsbé myburgh and staff of the section of clinical path ol ogy, university of pretoria; elise ferreira, up nutrilab; drs a. michell and m. van kleef, ovi; prof. f. steyn jr, 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: christopher j. kasanga2 mmeta yongolo1 chanasa mpelumbe-ngeleja1 raphael sallu1 mathias mkama1 joseph masambu1 affiliations: 1tanzania veterinary laboratory agency, tanzania2faculty of veterinary medicine, sokoine university of agriculture, tanzania correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: joseph, j., kasanga, c.j., yongolo, m., mpelumbe-ngeleja, c., sallu, r., mkama, m. et al., 2014, ‘spatial and temporal distribution of foot-and-mouth disease virus in the eastern zone of tanzania’, onderstepoort journal of veterinary research 81(2), art. #738, 1 page. http://dx.doi.org/10.4102/ ojvr.v81i2.738 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. spatial and temporal distribution of foot-and-mouth disease virus in the eastern zone of tanzania in this abstract... open access • abstract • acknowledgements abstract top ↑ this study was conducted to establish the spatial and temporal distribution of footand-mouth disease (fmd) virus (fmdv) serotypes in the ea stern zone of tanzania. observational prospective studies involvi ng serological analysis, and fmdv antigen detection, and retrospective study on fmdv antigen detection were used in this research. seroprevalence of antibodies to the nonstruc tural protein 3abc of fmdv and serotype-specific antigen detection were investigated by using svanovir® fmdv 3abc-ab elisa and indirect-sandwich elisa (selisa), respectively. serum and tissue samples were collected from cattle suspected of fmd in six districts of two regions in the eastern zone of tanzania during the period of 2010 to 2011. a total of 41 (43.6%) out of 94 tested sera in six district were seropositive to non-structural 3abc protein, with the highest seroprevalence of 81.0% in bagamoyo district, followed by kibaha (56.2%), kinondoni (41.7%), ilala (34.8%), kisarawe (16.7%) and temeke (15.4%) districts. three fmdv serotypes, namely o, a and sat 2, were detected in the eastern zone between 2001 and 2011, with type o being the most frequently detected serotype (n = 9; 60.0%) followed by type sat 2 (n = 5; 33.3%) and type a n = 1; 6.7%). these findings indicate that the eastern zone of tanzania is predominantly infected with fmdv serotypes o, a, and sat 2 with different spatial and temporal distribution, and that fmd outbreaks in the zone could be incriminated to at least these three serotypes. these observations imply that a rational control of fmd by vaccination in the eastern zone of tanzania should consider incorporation of serotypes o, a and sat 2 serotypes in the relevant vaccine(s). further studies are required to elucidate the genetic and antigenic characteristics of circulating fmdv strains in the eastern zone of tanzania so that an appropriate fmd control strategy can be recommended in this region. acknowledgements top ↑ this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). article information authors: emmanuel s. swai1 abdu a. hayghaimo1 ayubu a. hassan2 bartholomeo s. mhina2 affiliations: 1ministry of livestock development and fisheries (moldf), dar-es-salaam, tanzania 2tanga municipal livestock office, tanga, tanzania correspondence to: emmanuel swai email: esswai@gmail.com postal address: po box 9152, dar-es-salaam, tanzania dates: received: 23 feb. 2015 accepted: 06 may 2015 published: 12 aug. 2015 how to cite this article: swai, e.s., hayghaimo, a.a., hassan, a.a. & mhina, b.s., 2015, ‘the slaughter of increased numbers of pregnant cows in tanga abattoir, tanzania: a cause for concern?’, onderstepoort journal of veterinary research 82(1), art. #947, 5 pages. http://dx.doi.org/10.4102/ojvr.v82i1.947 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the slaughter of increased numbers of pregnant cows in tanga abattoir, tanzania: a cause for concern? in this original research... open access • abstract • introduction • materials and methods    • study location    • study animals and design    • meat inspection protocol and data collection    • data analysis • ethics statement • results    • slaughter data    • pregnant cows slaughter data • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ information on the level of foetal wastage in slaughtered cattle in tanzania is limited. a three-month observational study (april – june 2014) of animals slaughtered at the tanga abattoir in tanga region, tanzania was carried out to determine the number of pregnant cows slaughtered. the total number of cattle slaughtered during the study period was 3643, representing a monthly kill average of 1214 and a daily kill average of 40. over 98% of the cattle presented to the abattoir for slaughter were local breed (tanzania shorthorn zebu) and most were above 3 years of age. improved breeds of cattle represented only 1.3% of all slaughters. of the cattle slaughtered, 2256 (61.9%) were female and 1387 (38.1%) were male. a total of 655 slaughtered cows were pregnant, representing a foetal wastage of 29.1%. of the 655 recovered foetuses, 333 (50.8%) were male and 322 (49.2%) were female. of the recovered foetuses, 25.8% were recovered in the first, 42.7% in the second and 31.6% in the third trimester. this study indicates cases of significant foetal losses, negatively impacting future replacement stock as a result of the slaughter of pregnant animals. the indiscriminate slaughter of pregnant cows suggests that existing animal welfare legislation is not sufficiently enforced and routine veterinary ante-mortem inspection of trade animals is failing to prevent the high level of foetal wastage. introduction top ↑ the human population in tanzania is projected to increase from the current 43 million to about 63.6 million by 2025 (unpd 2008). rapid population growth will present an important challenge to achieving food security in developing countries such as tanzania. statistics from the world bank and the food and agriculture organization of the united nations (fao) show that globally, livestock production is currently growing faster than any other agricultural sector (robinson et al. 2014). this production growth is driven by the rapidly increasing demand for livestock products. the demand for meat and milk is predicted to at least double over the next two decades (robinson & pozzi 2011). the demand for quantity and quality of livestock products in developing countries is propelled by population growth, rising incomes, urbanisation and socio-economic factors such as human health concerns and changing socio-cultural values (i.e. change in diets) (delgado 2005; thornton 2010). elasticity of demand for livestock products is four to five times higher than for cereals; if real income rises, the demand for livestock products increases faster than the demand for cereals (tarver 1994). livestock farming will continue to play a crucial role as a source of food, livelihood and income generation over the coming decades. tanzania's animal wealth at the end of 2014/2015 included 23.5 million cattle, 15.6 million goats, 7 million sheep, 2.1 million pigs, 36 million village poultry, 0.3 million donkeys and 43.2 million poultry as well as very small numbers of domestic buffalo and one-humped camels (urt 2014). more than 99% of these livestock are kept in low input–low output systems, owned and managed by resource-poor mixed and pastoral producers who operate under a traditional husbandry system, often with little or no access to good and reliable animal husbandry practices, markets and reliable veterinary services. livestock contributes about 30% of agricultural gross domestic product (gdp), derived from an estimated 23.5 million heads of cattle, held by 1.27 million small-scale households and mostly comprised of indigenous east african shorthorn zebu. despite its great leverage potential, the sector is seriously constrained by animal diseases, poor-quality veterinary inputs and service as a result of ineffective regulatory capacity, inadequate and low-quality feeds as a result of seasonal fluctuation, poor production or husbandry technology innovations and inadequate investments to enhance its contribution to the development of the country. however, realisation and expansion of livestock production is constrained by other challenges, including the indiscriminate slaughter of future replacement animal stock. the trend of animal slaughter in abattoirs has shown that not only non-breeding livestock are being slaughtered for meat but also productive pregnant and lactating female animals (adama, shiawoya & michael 2011; gregory & grandin 2007; whitlock & maxwell 2008). these animals are either killed for home consumption, rituals, religious festivals, ceremonies, disease control or to meet immediate financial needs (gregory & grandin 2007). the wastage of conceptus through the slaughter of pregnant female animals is an unethical and uneconomic practice affecting cattle production in tanzania. wastage of a calf at any stage between conception and birth negatively affects herd fertility and growth. the scale and impact of slaughtering pregnant female animals is not fully recorded. there are concerns that the problem is exacerbated by the lack of proper production or market records and the failure to carry out pregnancy checks before slaughter. in order to have a clear understanding of the practices impacting the slaughter of pregnant cows, stakeholder awareness (livestock keepers and traders) and records of traded female stock for slaughter is imperative. abattoir surveys as a tool for disease surveillance and investigation can provide essential information that can be utilised for research and disease control purposes (cadmus & adesokan 2009). the objective of this work was, therefore, to assess the magnitude of slaughter of pregnant cows in tanga region, tanzania by using an abattoir survey as a study tool. materials and methods top ↑ study location the study was conducted at tanga city abattoir, located 330 km northeast of dar-es-salaam, the major city of tanzania. the abattoir, constructed in 1982, provides in the daily meat requirements of the inhabitants of tanga and neighbouring areas. geographically, the city is located between 4°21′ and 6°14′ s and 36°11′ and 38°26′ e. tanga city experiences tropical climate conditions, typified by hot and humid weather throughout the year. annual rainfall is approximately 1100 mm per year, with two distinct rainy seasons: the long rain season between april and may and the short rain season between october and november. the mean annual temperature ranges from 23 °c to 33 °c on average and humidity ranges between 60% and 70% (also see swai & schoonman 2012). at full operation, the abattoir has a daily maximum handling capacity of 120 heads of cattle and 150 small ruminants (sheep and goats). however, as a result of the lack of essential facilities, it presently slaughters around 40 heads of cattle daily. study animals and design the study animals were cattle brought for slaughter from all districts of tanga region and nearby districts of kilimanjaro, arusha and morogoro. some animals were transported to the abattoir using vehicles and others were trekked in. the study design employed was an active abattoir survey, carried out from april to june 2014 (also see swai & schoonman 2012). the main study subject of interest in this survey was traded female cattle stock. after arrival at the abattoir, age, breed, number and origin of the animals were recorded in a purposively designed record form. the age was determined based on dentition and owner's information (forse 1999; turton 1999). for purposes of quality control of the data, duly filled forms were collected regularly and discussed with the meat inspector in charge. determining the precise location of the source of each animal was not possible for various reasons, including a poor recording system and lack of reliable identification methods at farm and marketing points. no pregnancy diagnosis was conducted owing to limited facility and competency. meat inspection protocol and data collection routine meat inspections or examinations were carried out by a para-veterinarian (the resident abattoir meat inspector with a basic animal health background) using recommended standard procedures (fao/unep/who 1994; gracey, collins & huey 1999; urt 2003). inspection or examination procedure employed visual inspection, palpation, and incision of each visceral organ. the uteri of slaughtered cows were retrieved and the uterine horns were opened and inspected for the presence or absence of foetuses. recovered foetuses were examined to ascertain their sex and age, estimated as described by citek et al. (2011). recovered foetuses were stratified into the categories < 3 months (first trimester), 3 to 6 months (second trimester) and > 6 months old (third trimester); these data were posted in the data collection form designed for the purpose. data analysis data were entered, stored and analysed using microsoft excel and epi-info statistical software version 6.04b (cdc 1996). descriptive statistics such as the proportion of all slaughters, frequency of pregnant slaughtered cows and the extent of foetal wastage were generated. the percentage of foetal wastage was calculated as the total number of foetuses recovered divided by the total number of cows slaughtered. ethics statement top ↑ permission to carry out this study was granted by the executive director of tanga city. the director of veterinary services, tanzania issued a research permit letter to conduct this active abattoir surveillance work in tanga city. verbal consent was obtained from each of the traded stock owners after explaining the purpose and importance of the study prior to data collection. results top ↑ slaughter data a total of 3643 cattle were slaughtered between april and june 2014, representing a monthly kill average of 1214 and a daily kill average of 40. over 98% (n = 3596) of the cattle presented to the abattoir for slaughter were local breed (tanzania shorthorn zebu) and above 3 years of age. improved breeds represented only 1.3% of all slaughters. results showed that more (61.9%, n = 2256) female cattle were slaughtered than male cattle (38.1%, n = 1387) during the period under study. the proportions of cattle (female and male) and pregnant cows slaughtered are shown in table 1. table 1: slaughtered cattle and foetal wastage in tanga city abattoir from april to june 2014. pregnant cows slaughter data of the 2256 cows slaughtered, 655 were pregnant, at varied gestation periods. monthly foetal recovery rates (%) are shown in table 1. the monthly foetal wastage ranged from 23.1% to 37.04%, with a mean percentage of 29.1% over the study period. this indicates that at least one out of every four cows brought to the abattoir for slaughter is likely to be pregnant. of the total number of foetuses encountered during the survey period, 333 (50.8%) were male and 322 (49.2%) were female. two foetuses (0.3% [2/655]) were classified as mummified. no twins or multiple foetuses were retrieved during the study. the highest recorded number of foetuses was 273 (april), of which 52.4% were female. of the recovered foetuses, 25.8% were recovered in the first, 42.7% in the second and 31.6% in the third trimester. the highest number of wasted foetuses was recovered during the months of may and june, at the second trimester of pregnancy (figure 1). figure 1: percentage (%) of foetuses recovered by trimester and month of the survey period (n = 655). discussion top ↑ this survey revealed that 29.1% of cows slaughtered over the period april – june 2014 were pregnant. the implication of this finding is that 29.1% of the future productive herd is lost because of this practice. reproduction losses are widely recognised as one of the important constraints to increased cattle production, and foetal wastage as a result of the slaughter of pregnant cows is definitely one of several factors contributing to low livestock production and expansion (willingham, shelton & thompson 1986). given the current livestock and marketing setting, early pregnancy diagnosis may not be possible. lack of staff and competency in pregnancy diagnosis are possible reasons. on the other hand, there are motives for the slaughter of pregnant cows in their various stages of gestation other than lack of competency or gross ignorance. it is possible that livestock keepers or traders sell pregnant cows because phenotypically they appear heavier and consequently sell at better prices than non-pregnant ones. limited financial resources in times of crisis, such as the dry season, may motivate indiscriminate sales of female cattle for slaughter. however, the current study was conducted during the wet season, characterised by an abundant supply of green fodder from most areas likely to be the source of slaughter stock. furthermore, indiscriminate sales of pregnant animals could be a result of culling unwanted or non-productive cows, old or injured animals (muhammad et al. 2009). in addition, some livestock keeping communities believe that foetal meat is more nutritious than meat obtained from mature cows (muhammad, ashiru & abdullahi 2007) – another possible reason for the increased slaughter of pregnant cows. in some areas and communities, particularly where this survey was conducted, evidence of pregnant dairy stock theft for slaughter is often reported. phenotypically, pregnant cows have a better body condition score than non-pregnant cows, which may be the reason for theft preferences and slaughter of these animals. a review of abattoir surveys conducted elsewhere showed variation in the proportion of slaughtered pregnant cows (fayemi & muchenje 2013). a survey conducted in nigeria reported that 5% of the 321 448 cows slaughtered over a period of 3 years were pregnant (cadmus & adesokan 2010). on the other hand, results from ghana revealed 18% (28 410/154 179) of the slaughtered cows over a period of 4 years were pregnant (atawalna et al. 2013). variations between surveys and reports could be as a result of the volume of female animals slaughtered, period and duration of the study, consumer volume, abattoir location, country-specific animal slaughter regulations and laws pertaining to slaughtering female stock. observations gathered from this study and elsewhere (fayemi & muchenje 2013) strongly suggest that the proportion of slaughtered pregnant cows is higher in those more than 3 months pregnant. most of the foetuses recovered in this study were recovered in the second and third trimesters (74%), a finding that is consistent with reports from other studies. wosu (1988), ndi, tambi and agharih (1993) and fayemi et al. (2008) found that 74%, 64.1%, and 75.7% of the foetuses recovered respectively were in the second and third trimesters. the slaughter of pregnant cows, whether intentional or out of ignorance, has negative consequences on the reproductive potential of livestock keepers’ herds. importantly, the high numbers of female foetuses recorded mean that an opportunity for increasing future female breeding stock is compromised. moreover, the slaughter of pregnant cows is likely to frustrate the efforts of breeders, geneticists and nutritionists, as it poses the risk of widening the gap between the amount of meat required to provide sufficient animal protein to meet the needs of meat consumers and the amount of meat actually produced (fayemi & muchenje 2013). this study was limited to visual uterus inspection, therefore we were unable to detect early embryonic foetal development (defined as the age between fertilisation and day 45) by flushing the uterus for detailed collection of embryo and eventual laboratory examination. moreover, most of the cows presented for slaughter do not have any record reflecting their age, last calving and last service dates. this might have contributed to low early embryonic foetal recovery and detection rate in this survey. further studies are needed to elucidate why pregnant cows are sold and slaughtered. such studies should focus on determining possible ways of preventing or minimising these losses. moreover, pregnancy diagnosis should form part of standard operating procedures before any slaughter of female stock brought to the abattoir takes place. training for extension agents, meat inspectors and farmers on simple methods of pregnancy diagnosis is recommended. conclusion top ↑ this survey revealed that the equivalent of one foetus was lost for every four cows slaughtered and most of the foetuses recovered were in the second and third trimesters. the reasons for slaughtering pregnant cows ranged from sheer ignorance, cash constraint, lack of capacity to undertake pregnancy diagnosis and inability to enforce animal welfare legislation, partly as a result of the poor infrastructure available at the holding ground and slaughtering points. policy and regulation efforts should concentrate on instituting routine veterinary checks at holding grounds and abattoirs. livestock keepers, traders and butchers need to be regularly informed about proper animal husbandry practices, including breeding, as well as the implications of slaughtering pregnant cows. acknowledgements top ↑ the authors are grateful to tanga city authority for permission to undertake this study. abattoir attendants are thanked for their cooperation and help. this work was supported by the moldf through surveillance funds. we would also like to thank the director of veterinary services, for the kind permission to publish this work. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions e.s.s. (ministry of livestock development and fisheries) made conceptual contributions, analysed data and prepared the manuscript. a.a.h. (tanga municipal livestock office) and b.s.m. (tanga municipal livestock office) were responsible for data collection and assisted in preparation of the manuscript. a.a.h. (ministry of livestock development and fisheries) was responsible for logistic support and assisted in preparation of the manuscript. references top ↑ adama, j.y., shiawoya, e.l. & michael, n., 2011, ‘incidence of foetal wastages of cows slaughtered in minna abattoir, niger state, nigeria’, journal of applied biosciences 42, 2876–2881. atawalna, j., emikpe, b.o., shaibu, e., mensah, a., eyarefe, o.d. & folitse, r.d., 2013, ‘incidence of foetal wastage in cattle slaughtered at the kumasi abattoir, kumasi, ghana’, global veterinaria 11(4), 399–402. cadmus, s.i. & adesokan, h.k., 2009, ‘causes and implications of bovine organs/offals condemnations in some abattoirs in western nigeria’, tropical animal health and production 41(7), 1455–1463. http://dx.doi.org/10.1007/s11250-009-9334-7 cadmus, s.i.b. & adesokan, h.k., 2010, ‘bovine foetal wastage in south western nigeria: a survey of some abattoirs’, tropical animal health and production 42, 617–621. http://dx.doi.org/10.1007/s11250-009-9465-x centers for disease control (cdc), 1996, epi-info, version 6.04d, atlanta. citek, j., hradecka, e., rehout, v. & hanusova, l., 2011, ‘obstetrical problems and stillbirth in beef cattle’, animal science papers and reports 29(2), 109–118. delgado, c., 2005, ‘rising demand for meat and milk in developing countries: implications for grasslands-based livestock production’, in d.a. mcgilloway (ed.), grassland: a global resource, pp. 29–39, wageningen academic, wageningen. fao/unep/who, 1994, guidelines for echinococcosis/hydatidosis surveillance, prevention and control, food and agriculture organization of the united nations, rome. fayemi, a.o., taiwo, b.b.a., okubanjo, a.o. & adekunmisi, a.a., 2008, ‘frequency of slaughtering gravid cows in some selected parts of ogun state’, proceedings of the 33rd annual conference of the nigeria society of animal production, ayetoro, september 2008, pp. 234–237. fayemi, p.o. & muchenje, v., 2013, ‘maternal slaughter at abattoirs: history, causes, cases and the meat industry’, springer plus 2, 125. http://dx.doi.org/10.1186/2193-1801-2-125 forse, b., 1999, where there is no vet, macmillan, london. gracey, j.f., collins, d.s. & huey, r.j., 1999, meat hygiene, 10th edn, w.b. saunders, london. gregory, n.g. & grandin, t., 2007, animal welfare and meat production, 2nd edn., cabi, wallingford. http://dx.doi.org/10.1079/9781845932152.0000 muhammad, b.f., haruna, i.y., abdulsamad, a.m. & bichi, j.m., 2009, ‘foetal wastage in northern nigeria: the case of gombe abattoir, gombe state’, proceedings of the 13th annual conference of animal science of nigeria, abu zaria, september 15–19, 2008, pp. 124–127. muhammad, i.r., ashiru, r. & abdullahi, a.y., 2007, ‘implications of the slaughter of pregnant ewes and does to the future stock in the semi-arid urban abattoirs’, journal of animal and veterinary advances 6(6), 819–822. ndi, c., tambi, n.e. & agharih, n., 1993, ‘reducing calf wastage from the slaughtering of pregnant cows in cameroon’, world animal review, viewed 29 november 2014 from http://www.fao.org/docrep/v1650t/v1650t0g.htm. robinson, t.p. & pozzi, f., 2011, mapping supply and demand for animal-source foods to 2030, animal production and health working paper no. 2, food and agriculture organization of the united nations, rome. robinson, t.p., wint, g.r.w., conchedda, g., van boeckel, t.p., ercoli, v., palamara, e. et al., 2014, ‘mapping the global distribution of livestock’, plos one 9(5), e96084. http://dx.doi.org/10.1371/journal.pone.0096084 swai, e.s. & schoonman, l., 2012, ‘a survey of zoonotic diseases in trade cattle slaughtered at tanga city abattoir: a cause of public health concern‘, asian pacific journal of tropical biomedicine (2012), 55–60. http://dx.doi.org/10.1016/s2221​-1691(11)60190-1 tarver, d., 1994, ‘urbanisation in africa since 1994’, africa insight (24), 75–78. thornton, p.k., 2010, ‘livestock production: recent trends, future prospects’, philosophical transactions of the royal society b 365, 2853–2867. http://dx.doi.org/10.1098/rstb.2010.0134 turton, j., 1999, cattle teeth: how to estimate the age of cattle, infopak, national department of agriculture, pretoria. united nations population division (unpd), 2008, the 2006 revision and world urbanization prospects: the 2005 revision, population division of the department of economic and social affairs of the united nations secretariat, viewed 25 november 2014, from http://esa.un.org/unpp. united republic of tanzania (urt), 2003, veterinary act no. 16 of 2003, government printer, dar-es-salaam. united republic of tanzania (urt), 2014, budget speech 2014/15, viewed 15 february 2015, from http://www.mifugouvuvi.go.tz/budget-speech-2014/15 whitlock, b.k. & maxwell, h.s., 2008, ‘pregnancy-associated glycoproteins and pregnancy wastage in cattle’, theriogenology 70(3), 550–559. http://dx.doi.org/10.1016/j.theriogenology.2008.05.003 willingham, t., shelton, m. & thompson, p., 1986, ‘an assessment of reproductive wastage in sheep’, theriogenology 26(2), 179–188. http://dx.doi.org/10.1016​/0093-691x(86)90022-1 wosu, l.o. 1988, ‘calf wastage through slaughtering of pregnant cows in enugu abattoir (nigeria)’, revue d’elevage et de médecine vétérinaire des pays tropicaux 41(1), 97–98. article information authors: jeyanthi b.p. gopalraj1 francoise c. clarke1 edward f. donkin2 affiliations: 1department of biology, university of limpopo, medunsa campus, south africa2department of animal and wildlife sciences, university of pretoria, south africa correspondence to: jeyanthi gopalraj postal address: po box 139, medunsa 0204, south africa dates: received: 23 apr. 2013 accepted: 03 july 2013 published: 02 sept. 2013 how to cite this article: gopalraj, j.b.p., clarke, f.c. & donkin, e.f., 2013, ‘assessment of acquired immune response to rhipicephalus appendiculatus tick infestation in different goat breeds’, onderstepoort journal of veterinary research 80(1), art. #614, 5 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.614 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. assessment of acquired immune response to rhipicephalus appendiculatus tick infestation in different goat breeds in this research communication... open access • abstract • introduction • material and methods    • experimental animals    • acquired immune response assessments in goats       • tick infestation       • blood samples       • electrophoresis and serum proteins       • statistical analyses • results    • gamma globulin levels    • comparison between breeds    • engorged female tick mass    • numbers of females completing the blood meal • ethical considerations • discussion    • gamma globulin levels    • engorged female tick mass    • numbers of females completing the blood meal • conclusion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ changes in serum gamma globulin levels, numbers of replete female ticks and engorged tick mass were used as parameters to monitor the acquired immune response (antibody mediated immune response) elicited by rhipicephalus appendiculatus adult tick infestations. three consecutive rhipicephalus appendiculatus adult tick infestations were applied to south african indigenous goats (nguni), saanen goats and cross-bred goats (saanen goats crossed with south african indigenous goats [nguni]) under laboratory conditions. during the three consecutive rhipicephalus appendiculatus adult tick infestations the serum gamma globulin levels increased in all three breeds, whilst the mean replete female tick numbers and engorged tick mass decreased. even though all three goat breeds exhibited an acquired immune response, the south african indigenous goats (nguni) response was significantly higher than that of the saanen and cross-bred goats. however, the acquired immune response elicited by saanen goats was significantly lower when compared with cross-bred goats. introduction top ↑ small-scale south african farmers, especially those in communal areas, often depend on goats for meat and milk. development programmes may include the introduction of milk goats for household milk production (donkin & boyazoglu 2000). it would therefore be preferable if these goats were less susceptible to tick infestation. it has been shown that there are breed differences in goats towards resistance against heartwater, which is a major tick-borne disease (donkin et al. 1992). it is therefore important to determine the potential of different goat breeds to acquire a natural resistance against tick infestation. the present understanding of host-tick relationships and acquired resistance is based on work done with laboratory animals and different cattle breeds (opdebeeck et al. 1988; dipleou 1990; rechav, clarke, els & dauth 1991). many studies have been carried out to verify the acquisition of resistance by hosts when they are immunized by successive tick infestations (jittapalapong et al. 2000a, 2000b; monteiro & bechara 2008, monteiro et al. 2011; caperucci, bechara & camargo-mathias 2009, 2010; veronez et al. 2010; nunes, bechara & camargo-mathias 2011). it is generally accepted that the calves of all breeds are equally susceptible to tick infestation at birth (hewetson 1972) and the ability to acquire a natural resistance varies from breed to breed (dipeolu 1989; spickett et al. 1989). it is characteristic of asian zebu calves (bos indicus) to acquire a higher degree of tick resistance in a shorter period of time than the british breeds (bos taurus) (wagland 1978; latif et al. 1991). however, resistance also fluctuates with season, photoperiod and rainfall (sutherst & utech 1981). studies of serum proteins have shown that the gamma globulin fraction is the only blood serum component that is affected by the development of resistance through tick infestation (willadsen 1980; rechav & dauth 1987). little is known about the tick-host relationship of different goat breeds. resistance to tick feeding has been shown to be an acquired, immunologically mediated phenomenon in goats, associated with cutaneous basophilia to nymphs of the ixodid tick (amblyomma cajennense) after repeated infestations (monteiro & bechara 2008). ixodid ticks remain attached to their hosts for extended periods of time, allowing ample opportunity for the development of immune mediated rejection and resistance mechanisms in the host (champagne 1994). macivor and horak (1987) reported that angora goats were less resistant to tick infestation than boer goats. schwalbach, bechara and camargo-mathias (2003) indicated that the small east african indigenous goat breed possesses a high resistance to tick infestation and higher tolerance to infestation effects than the exotic toggenburg breed. nyangiwe and horak (2007) found that goats are good alternative hosts for most of the economically important ticks that infest cattle in south africa. since rhipicephalus appendiculatus (r. appendiculatus) parasitises wild ungulates and livestock, and is common in goats (bryson et al. 2002a, 2002b), it is important to assess the acquired immune responses of the same goat breeds, such as saanen, south african indigenous goats (nguni) and cross-bred goats (saanen x south african indigenous goats) to infestations with this tick. the brown ear tick, r. appendiculatus, is a three-host tick of great economic importance as it acts as a vector for east coast fever (cattle adaped theileria parva), corridor disease (buffalo associated theileria parva), benign bovine theileriosis (theileria mutans) and tick bite fever in man (rickettsia conori). material and methods top ↑ unfed adults of r. appendiculatus were used from a colony cycled on rabbits and maintained in the laboratory of the department of biology, medunsa campus, university of limpopo at 25 ˚c ± 1˚c, at 75% ± 5% relative humidity (rh) and a photoperiod of approximately 12 hours of light and 12 hours of dark (12l:12d). experimental animals the protocol of this study was approved by the animal ethics committee at the medunsa campus of the university of limpopo. sixteen month-old naïve (not exposed to any pathogens), healthy saanen, south african indigenous (nguni) and cross-bred (saanen x south african indigenous) goats were either kept in pens or in individual metabolic crates and supplied with adequate food (total mixed ration) and water. acquired immune response assessments in goats tick infestation eight goats from each of the above mentioned goat breeds were infested three times with equal numbers of unfed r. appendiculatus males and females. so as not to over-infest the goats, three pairs of ticks were used per one kg host body mass. the unfed adult ticks were released into a linen bag glued to the shaven backs of the experimental animals, allowed to attach and feed and the engorged females were collected. histamine release during feeding can increase vaso-permeability and local discomfort in hosts, but can also lead to pruritus and intensified reflex grooming. since grooming can dislodge ticks, grooming by the goats was limited through the application of leather collars and chaining to either side of the experimental cage, which allowed sufficient space for limited movement necessary for the goats to eat, drink and lie down. the goats were re-infested as soon as the engorged females of the previous infestation had all been collected. replete females were collected on a daily basis and the individual mass of each was determined and recorded. blood samples blood samples of 5 ml from each goat for quantitative serum analysis were collected from the jugular vein in sterile heparin vacutainer tubes (lasec, south africa) at the time of each infestation and again towards the end of each detachment period. blood samples from a group of eight naïve animals from the same breed were taken and analysed as controls. the samples were allowed to clot at room temperature before the serum was retrieved for serological analysis. electrophoresis and serum proteins blood serum protein levels were determined through electrophoresis. the serological analysis was performed on the day of collection by the department of chemical pathology, university of limpopo (medunsa campus). serum protein-electrophoresis was done on an agarose gel using the paragon high resolution electrophoresis kit (beckman instruments, fullerton, california u.s.a) with a barbital buffer, ph 8.8 (0.15 ionic strength). the membranes were scanned with an appraise densitometer (beckman instruments, usa) and separated into albumin α1, α2, β and γ globulin fractions. statistical analyses comparing three or more treatment groups: the significance of differences in the means of engorged female mass and blood parameters were determined by subjecting the data to a one way analysis of variance test (anova) followed by tukey hsd. tests were used to indicate significance. results top ↑ gamma globulin levels the mean gamma globulin levels increased progressively after each tick infestation when compared to the uninfested control animals, as shown for south african indigenous goats (nguni) (table 1). table 1: gamma globulin levels (g/l) in the serum of the test and control south african indigenous goats (nguni) before and after three consecutive infestations of rhipicephalus appendiculatus adult ticks. the mean gamma globulin levels increased progressively after each tick infestation when compared to the uninfested control animals as shown for cross-bred (table 2) goats. table 2: gamma globulin levels (g/l) in the serum of the test and control cross-bred goats (south african indigenous goats x saanen) before and after three consecutive infestations of rhipicephalus appendiculatus adult ticks. the mean gamma globulin levels increased progressively after each tick infestation when compared to the uninfested control animals as shown for saanen (table 3) goats. table 3: gamma globulin levels (g/l) in the serum of the test and control saanen goats before and after three consecutive infestations of rhipicephalus appendiculatus adult ticks. comparison between breeds comparison between breeds in terms of increase in gamma globulin levels in the serum of south african indigenous goats (nguni), cross-bred goats and saanen goats before and after each of three consecutive infestations with r. appendiculatus adult ticks is shown in figure 1. figure 1: increase in gamma globulin levels (g/l) in south african indigenous goats (nguni), cross-bred goats and saanen goats before and after three consecutive infestations of rhipicephalus appendiculatus adult ticks. engorged female tick mass figure 2 shows the mean engorged mass (± sd) of female r. appendiculatus collected from south african indigenous, cross-bred and saanen goats after each of three successive infestations. figure 2: mean engorged mass of female ticks (mg) recovered during three consecutive infestations of rhipicephalus appendiculatus from south african indigenous goats (nguni), cross-bred and saanen goats. numbers of females completing the blood meal the decline in the numbers of r. appendiculatus females that completed their blood meal on the experimental south african indigenous goats, cross-bred goats and saanen goats are shown in figure 3. figure 3: replete females recovered during three consecutive infestations of rhipicephalus appendiculatus from south african indigenous goats (nguni), cross-bred and saanen goats. ethical considerations top ↑ these experiments were conducted in agreement with the provisions enforced by the research ethics committee (bp 06/98) and the animal ethics committee of the medunsa campus of the university of limpopo. goats were kept under controlled laboratory conditions in animal production unit at the experimental farm of medunsa campus of the university of limpopo with sufficient food, water, space and ventilation according to acceptable standards. as per university policy research data and records were maintained. discussion top ↑ gamma globulin levels an increase in the level of the protein globulins in the serum of the hosts indicates the development of acquired humoral immune responses (rechav 1987; rechav & dauth 1987). the higher levels of gamma globulin levels seen in the south african indigenous goats (nguni) were highly significant (p < 0.0001), indicating that three successive infestations of r. appendiculatus elicited a very strong acquired immune response. the cross-bred goats also exhibited significant increases in gamma globulin levels (p < 0.05) from pre-infestation values and after the third infestation (p < 0.05), but not between the first and second infestations. although significant, the acquired resistance elicited was less than the strong immune response elicited in the south african indigenous goats (nguni). the saanen goats also showed a concomitant increase in gamma globulin levels. the increases, although significant, indicated a relatively weak acquired response when compared to the response elicited in both the south african indigenous and the crossbred goats mainly between the pre-infestation value and the values after the first and third infestations (p < 0.05). even though all three breeds exhibited increases in gamma globulin levels to some degree when compared to their pre-infestation values, they were relatively high for the saanen goats compared to the south african indigenous goats and the cross-bred goats (p < 0.0001). this gamma globulin level was statistically significantly higher in the saanen goats compared to the crossbred goats (p < 0.01) as well as in the saanen compared to the south african indigenous goats (p < 0.05); this implys that a saanen goat has a strong immune system compared to the south african indigenous goats and cross-bred goats. engorged female tick mass the mean engorgement mass was found to be a consistently reliable indicator of the effect of an immune response on tick feeding success in laboratory animals (heller-haupt, varma & langi 1981). three repeated infestation of south african indigenous goats with adult r. appendiculatus ticks resulted in a highly significant decline of the mean engorgement mass of female ticks (p < 0.01). this was also true for the cross-bred goats (p < 0.01) as well as for the saanen goats (p < 0.01). the difference between the mean engorged mass of the females from the first and second infestation was not significant, but the difference between the second and third infestation was significant, as was the difference after the first infestation when compared to the third infestation (p < 0.01). there were no significant breed differences in engorged female r. appendiculatus tick mass. numbers of females completing the blood meal as only a few females complete their blood meal successfully on an immune host (brown 1985), the number of ticks recovered from goats after each infestation were also used as an indication of immune response. the numbers of engorged female ticks collected from all three goat breeds decreased with every infestation. repeated infestation of south african indigenous goats with adult r. appendiculatus ticks resulted in a significant decline in replete female tick numbers (p < 0.02) from the first and third infestation tick yield (p < 0.05). numbers of replete female ticks recovered from crossbred goats during three consecutive infestations also decreased significantly from first to the third and last infestation (p < 0.01). the numbers of engorged r. appendiculatus females recovered from the saanen goats after three subsequent infestations also showed a significant decrease in tick yield between the first and last infestation (p <0.01). although the engorged r. appendiculatus female tick numbers declined between infestations, the difference was not significant between breeds (p < 0.97). conclusion top ↑ during the three consecutive infestations the serum gamma globulin levels increased significantly (p < 0.0001) for all of the goat breeds, whilst the mean replete tick mass and the number of replete females collected decreased progressively. even though all the breeds elicited an acquired immune response, the south african indigenous goats (nguni) developed a significantly higher level of acquired immunity (p < 0.01) compared to the saanen goats and cross-bred goats, as measured by serum gamma globulin levels. the difference in mean gamma globulin levels between saanen goats and the cross-breeds implies that the acquired immune response of the saanen goats was lower when compared with cross-bred goats (p < 0.0001). acknowledgement top ↑ this project was funded by the medunsa campus of the university of limpopo. financial assistance by medunsa for this research is highly acknowledged. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions f.c.c. (university of limpopo, medunsa campus) was my supervisor and e.f.d. (university of pretoria) was my co-supervisor. j.b.p.g. (university of limpopo, medunsa campus) was involved in the design of study and in the sequence alignment. f.c.c. was involved in the project design and co-ordination. f.c.c. and e.f.d. participated in the experimental design. j.b.p.g. conducted all of the experiments and collected the data. statistical data analysis and interpretation of the graphs were done by j.b.p.g. during the interpretation of results both f.c.c. and e.f.d. provided conceptual and intellectual contributions. j.b.p.g. drafted the manuscript. e.f.d. was involved with the integration of the breed difference assessment into the manuscript. references top ↑ brown, s.j., 1985, ‘immunology of acquired resistance to ticks’, parasitology today 1, 166–171. http://dx.doi.org/10.1016/0169-4758(85)90175-9 bryson, n.r., tice, g.a., horak, i.g., stewart, c.g. & du plessis, b.j.a., 2002a, ‘ixodid ticks on cattle belonging to small-scale farmers at four communal grazing areas in south africa’, journal of the south african 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tick (boophilus microplus) in brahman (bos indicus) cattle ii, the dynamics of resistance in previously unexposed and exposed cattle’, australian journal of agricultural research 29, 395–400. http://dx.doi.org/10.1071/ar9780395 willadsen, p., 1980, ‘immunity to ticks’, advanced parasitology 18, 293–313. http://dx.doi.org/10.1016/s0065-308x(08)60402-9 article information authors: helena greter1,2 vreni jean-richard1,2 lisa crump1,2 mahamat béchir3 idriss o. alfaroukh4 esther schelling1,2 bassirou bonfoh5 jakob zinsstag1,2 affiliations: 1swiss tropical and public health institute, switzerland2university of basel, switzerland 3centre de support en santé internationale, chad 4institut de recherche en elevage pour le développement, chad 5centre suisse de recherches scientifiques, côte d’ivoire correspondence to: jakob zinsstag postal address: po box, ch-4002 basel, switzerland how to cite this article: greter, h., jean-richard, v., crump, l., béchir, m., alfaroukh, i.o., schelling, e. et al., 2014, ‘the benefits of “one health” for pastoralists in africa’, onderstepoort journal of veterinary research 81(2), art. #726, 3 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.726 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www. sacids.org/ kms/frontend/ index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the benefits of ‘one health’ for pastoralists in africa in this proceedings... open access • abstract • introduction • the sahel ecosystem and its inhabitants • the evolution of ‘one health’ • past research, results and consequences    • demography and health surveillance of mobile pastoralists    • continuing ‘one health’ projects • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ ‘one health’ is particularly suited to serve mobile pastoralists. dinka pastoralists in sudan inspired calvin schwabe to coin the term ‘one medicine’, indicating that there is no difference in paradigm between human and veterinary medicine. our contemporary definition of ‘one health’ is any added value in terms of improved health of humans and animals or financial savings or environmental services resulting from a closer cooperation of human and animal health sectors. here we present a summary of ‘one health’ studies with mobile pastoralists in africa which were done in research partnership, demonstrating such an added value. initial joint human and animal health studies revealed higher livestock vaccination coverage than in the pastoralist community, leading to joint animal and human vaccination intervention studies which demonstrated a better access to primary health care services for pastoralists in chad. further simultaneous animal and human serological studies showed that camel breeding was associated with human q-fever seropositivity. in borana communities in ethiopia, human cases of mycobacterium bovis infection could be related to strains isolated from cattle. a challenge remained with regard to how to assess vaccination coverage in mobile populations. with the advent of mobile phones, health and demographic surveillance could be established for mobile pastoralists and their animals. this presents vast possibilities for surveillance and control of human and animal diseases. pastoralists prefer a ‘one health’ approach and therefore contribute toward the validation of this concept by showing real added value of the cooperation between human and animal health services. introduction top ↑ the human and animal health unit at the swiss tropical and public health institute, basel, switzerland, is a multidisciplinary research unit working with partner institutions in nine countries in africa (ethiopia, kenya, chad, mali, mauritania, côte d’ivoire) and asia (kyrgyzstan, mongolia, vietnam). we focus on health research for mobile pastoralists and the control of zoonotic diseases in developing countries. several of these diseases are emerging and many zoonotic diseases are also categorised as neglected diseases. pastoralist populations in these countries are often marginalised by government healthcare systems because of the remote location of their camps and villages or high mobility (schelling et al. 2010). several of our studies have shown that the application of the ‘one health’ concept is especially valuable in the settings of mobile pastoralism (schwabe 1964). this review summarises the importance of ‘one health’ for pastoralists in africa. the sahel ecosystem and its inhabitants top ↑ the sahelian belt is a semi-arid zone which borders the southern aspect of the sahara desert, spanning africa east to west from ethiopia to mauritania. it forms a corridor through the continent where highly mobile populations have lived for a long time. the ecological zone in the central regions includes a rainy season from june to september and a dry season from october to may. this unique ecosystem led to the development of specific agricultural inventions. one important form is mobile pastoralism which allows for the breeding of large herds of cattle, camels, goats and sheep by following the cycle of pasture growth during the seasons (wiese 2004). many different ethnic groups practise this way of life in the sahelian belt. in chad, for example, the fulani, gorane and arab groups prevail in the lake chad basin (cerezo et al. 2011; jean-richard et al. in press[a]). social science and anthropological studies have investigated the requirements for adequate healthcare in different mobile pastoralist ethnic groups. these studies show, amongst other things, that it is crucial to plan health interventions for populations with respect to their cultural definition of the human body and the forces influencing their health (münch 2012). the evolution of ‘one health’ top ↑ in the early 1960s, calvin w. schwabe, a veterinarian with a background in biology working in southern sudan at the time, observed that dinka pastoralists maintained an integrated attitude toward humans and animals. inspired by this observation, schwabe developed the concept of ‘one medicine’, stating that ‘human and veterinary medicine share a common body of knowledge in anatomy, physiology, pathology and the origins of diseases in all species’ (schwabe 1964) and thereby recognising the mutual benefits available through the connection of veterinary medicine and human health. today, this concept is expanded to ‘one health’: further recognising the inextricable linkage of human, livestock, companion animal and wildlife health and implying an added value to the health and wellbeing of humans and animals (zinsstag et al. 2011). closer cooperation of human and animal healthcare provision can also lead to financial savings in different sectors (zinsstag et al. 2005). the concept of ‘one health’ has important potential, especially in developing countries such as many of the sahelian nations, where our unit works in close collaboration with local partners, communities and ministries. past research, results and consequences top ↑ a 1999–2000 study on the health of mobile pastoralists in chad, where approximately 1000 people and animals were examined, found hardly anyone who did not report a health problem (daugla et al. 2004). more cattle had been vaccinated than children and women; and no children had been vaccinated completely according to the standards of the expanded programme on immunization of the united nations (epi). nevertheless, the people rarely sought care in health centres, coming only at late stages of illness, with the result that many diseases remained untreated (schelling et al. 2005). similar studies in mali showed higher child mortality in mobile pastoralist populations than in the sedentary population (münch 2012). in addition, mobile pastoralists face a higher risk for zoonotic diseases than do sedentary populations because of their deep dependence on and close contact with their livestock (dean et al. 2013). practices such as consumption of raw milk and meat favour the transmission of brucellosis, bovine tuberculosis and anthrax. it has also been possible to show the direct relationship of camel breeding and human q-fever seroprevalence (schelling et al. 2003). on the basis of these findings, a first collaborative intervention project applying ‘one health’ was initiated, planning and implementing a joint vaccination campaign for both livestock and children in mobile pastoralists in chad (béchir et al. 2004; schelling et al. 2007; zinsstag et al. 2005). this campaign showed not only a health benefit but also the economic benefit in terms of savings made by the chadian public health and animal health ministries from closer cooperation between the public health and veterinary sectors. it laid the foundation for further joint health interventions in the country.combined investigations of zoonotic disease surveillance, performed simultaneously in both humans and animals, have also been shown to be highly effective in terms of identifying epidemiological linkages. studies in ethiopia on bovine and human tuberculosis demonstrated that bovine tuberculosis can be transmitted to humans and vice versa (gumi et al. 2012). human nutrition includes a wide variety of animal-source foods. for mobile pastoralists, their entire diet depends on their livestock, as meat and milk are consumed directly or sold in order to gain money to buy cereals (münch 2012). this strong dependence of the people on their livestock shows clearly the importance of healthy herds. herd losses as a result of prolonged dry seasons or disease have a direct effect on the mobile pastoralists, resulting in malnutrition and vitamin deficiencies. because fruits and vegetables are not part of the mobile pastoralist diet, milk is the primary source of vitamin a. this interrelationship was shown by studies on vitamin a in chadian mobile pastoralists which demonstrated a direct relationship between the retinol levels in the women’s blood and in the livestock’s milk (béchir et al. 2012b; zinsstag et al. 2002). such findings serve as indicators for the health status not only of humans but also the ecosystem, as vitamin a levels in milk depend on the pasture quality which is, in turn, dependent on climate (zinsstag et al. 2002). the hostile environment where pastoralists live exposes them to wind, dust, humidity, heat and lack of access to safe drinking water. these conditions also favour the prevalence of parasitic infection which is very high at 63% amongst women (95% ci: 55–72) and 60% amongst children (95% ci: 53–77) (béchir et al. 2012a). demography and health surveillance of mobile pastoralists an estimated 20 million–30 million mobile pastoralists live in the sahelian belt. in most countries, little demographic data is available and, in particular, the assessment of mobile populations was originally very difficult with previously-existing technologies (weibel et al. 2011). today, communication tools such as mobile phones and the expanding mobile communication network open up completely new possibilities. a study aiming to exploit these new capabilities and their usefulness for the development of demographic and health surveillance systems amongst mobile pastoralists was carried out from 2010 to 2012 in chad (jean-richard et al. 2014). groups of mobile pastoralists were enrolled in the study and contacted regularly by mobile phone in order to gather information on the birth, death and health status of the community. about half of the groups already owned mobile phones at the time of enrolment. the study showed that the use of mobile phones for obtaining plausible demographic and health surveillance data is both feasible and well accepted, indicating future promise with regard to expanding the project in chad and adapting the system to mobile populations in other sahelian countries. during this study, demographic data on livestock herds was also collected, revealing the potential to combine both human and animal health surveillance using mobile phones (jean-richard et al. in press[a]). continuing ‘one health’ projects building upon the long-standing relationship with the mobile pastoralist community and the partner institutions in chad, new projects were developed which aimed to meet the demands that arose from the mobile pastoralists. an early perception of the pastoralists was that although access to veterinary care was limited, human health needs were addressed even less often. this led to further work to assess vitamin a status more broadly in the human population and cattle, as well as to explore the link between pasture and cattle. subsequently, their priority was the important economic losses as a result of infection of the livestock with fasciola gigantica. this infection weakens the animals, leading to reduced milk and meat production and an increased number of abortions. fasciola gigantica is a parasite which has a freshwater snail as its intermediate host. this is also the case for schistosomia haematobium and schistosoma mansoni, two parasite species which infect humans. in chad, currently, more data is available on livestock fascioliasis than on human schistosomiasis (jean-richard et al. in press[b]; massenet, abakar & karifene et al. 1995; massenet et al. 2012). an ongoing project will further deepen the understanding of the epidemiology of human schistosomiasis and livestock fascioliasis in mobile pastoralists and their livestock in chad. our work expands the ‘one health’ approach to ‘health in social-ecological systems’, addressing the dynamics of both human and animal populations and their ecosystem determinants (zinsstag et al. 2011; zinsstag 2012). conclusion top ↑ the shared objective of all research in the unit is to improve human and animal health in a sustainable way. the research ideally contributes to the development and implementation of locally adapted and accepted health interventions which are supported by the communities as well as the ministries. applying the ‘one health’ approach in challenging settings such as the sahelian belt has proven to be successful in many of these projects. involving representatives of the mobile pastoralists, the research community and the ministries in the planning of projects and interventions is crucial and leads to strong engagement for common goals and solutions. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.z. (swiss tropical and public health institute; university of basel) was the project leader. e.s. (swiss tropical and public health institute; university of basel), j-r.v. (swiss tropical and public health institute; university of basel), m.b. (centre de support en santé internationale), l.c. (swiss tropical and public health institute; university of basel) and h.g. (swiss tropical and public health institute; university of basel) were responsible for experimental and project design and performed most of the experiments. e.s., i.o.a. (institut de recherche en elevage pour le développement) and b.b. 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animal health to strengthen health systems’, lancet 366(9503), 2142–2145. http://dx.doi.org/10.1016/s0140-6736(05)67731-8 salih_47-53.indd introduction southern sudan is situated approximately between latitudes 4 °n to 10 °n and longitudes 24 °e to 36 °e and has an annual rainfall ranging between 900 and 1 500 mm. livestock are mainly comprised of cattle, goats and sheep. the local people depend primarily on cattle to satisfy their needs for meat, milk and milk by-products, but sheep and goats are also a source of meat. the cattle population of central equatoria state is estimated to be 7.5 million and consists mainly of the local nilotic zebu breed. hoogstraal (1956) reported 49 species of ticks that infest both domestic and wildlife in southern sudan. the most important among these ticks is rhipicephalus appendiculatus (the vector of east coast fever caused by the protozoan parasite theileria parva), which is mainly distributed in areas of high rainfall and moderate temperature, such as the district of kajo kaji, yei, ngangala, torit and katire. in addition, this tick was identified in chukudum, aswa river, palotaka, nimule and juba (morzaria, tat47 onderstepoort journal of veterinary research, 75:47–53 (2008) preliminary survey of ticks (acari: ixodidae) on cattle in central equatoria state, southern sudan d.a. salih1*, i.i. julla2, s.m. hassan3, a.m. el hussein1 and f. jongejan4,5 abstract salih, d.a., julla, i.i., hassan, s.m., el hussein, a.m. & jongejan, f. 2008. preliminary survey of ticks (acari: ixodidae) on cattle in central equatoria state, southern sudan. onderstepoort journal of veterinary research, 75:47–53 in a preliminary survey conducted in 2005, the species composition and seasonality of ticks infesting cattle in central equatoria state, southern sudan was determined. three locations were selected (gumbo, khor rumla and nyaing) and surveyed every 3 months. two cattle herds in each of the three locations were visited four times during the study period. total body collections of ticks were made from each of five cattle (nilotic zebu breed) kept in six different herds. four tick genera and ten species were identified. the tick species identified were amblyomma lepidum, amblyomma variegatum, boophilus annulatus, boophilus decoloratus, hyalomma marginatum rufipes, hyalomma truncatum, rhipicephalus appendiculatus, rhipicephalus evertsi evertsi, rhipicephalus praetextatus and rhipicephalus sanguineus group. the highest number of ticks was collected in october during the rainy season. a finding of great significance was that r. appendiculatus, vector of east coast fever, has now firmly established itself throughout the year with possible implications for cattle production in central equatoria state. keywords: cattle, preliminary survey, southern sudan, ticks * author to whom correspondence is to be directed: e-mail: diaeldin2000@hotmail.com 1 central veterinary research laboratories, al amarat, p.o. box 8067, khartoum, sudan 2 college of veterinary science, university of bahr el ghazal, p.o. box 10739, khartoum, sudan 3 department of parasitology, faculty of veterinary medicine, university of khartoum, p.o. box 32, khartoum north, sudan 4 utrecht centre for tick-borne diseases, department of infectious diseases and immunology, faculty of veterinary medicine, utrecht university, yalelaan 1, 3584 cl utrecht, the netherlands 5 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, onderstepoort, 0110 south africa accepted for publication 28 september 2007—editor 48 ticks (acari: ixodidae) on cattle in southern sudan chell, minor, pederson, julla, rahim, dyson & aarle 1981; julla 1985, 1994). however, information regarding the prevalence of r. appendiculatus and other tick species infesting livestock in southern sudan and their economic impact is lacking. this is mainly due to the lack of adequate laboratory facilities and trained personnel to conduct research on ticks, in addition to long standing civil unrest. this study was initiated to update the current distribution of ticks infesting cattle in central equatoria state, southern sudan. materials and methods study area the study was conducted over 12 months from january to december 2005 around the city of juba (4°50′ n; 31°35′ e), the capital of central equatoria state, southern sudan (fig. 1). the climate around juba is (semi)equatorial, with minimal variations in seasonal temperatures. the highest mean maximum ambient temperature usually occurs during january in the dry season with a daily maximum ranging between 30° and 32 °c. the mean minimum temperature occurs during the rainy season which is from march to november, with temperatures ranging between 19° and 27 °c and a rainfall between 800 and 1 200 mm. collection of ticks a longitudinal survey, with a visit every 3 months, to three locations was conducted. the locations surveyed were khor rumla (15 km south of juba), ny aing (10 km north of juba) and gumbo (10 km south east of juba) (fig. 1). in each locality two cattle herds were regularly inspected at each visit. the visits were conducted in january (dry season), april (after the rain had commenced), july (peak of rainy season) and october (towards the end of the rainy season). from each herd, five animals (long-horned nilotic zebu cattle), three of them being calves (less than 1 year old) and two adult cattle (more than 4 years old) were sampled. at each visit the same herd was sampled, but not the same animals. a total body collection of ticks was made using a pair of blunt forceps. prior to collection, each animal was cast and restrained. the ticks from each animal were collected separately and placed in labelled universal bottles containing 70 % ethanol. on each label, location, animal identification number and date of collection were recorded. in the laboratory, the ticks were identified according to hoogstraal (1956), okello-onen, hassan & essuman (1999) and walker, bouattour, camicas, estrada-peňa, horak, latif, pegram & preston (2003). statistical analysis the data on ticks were subjected to a general linear model (glm) procedure using the statistical anal ysis system (sas) package. sas was used to perform analysis of variance (anova) while mean separations were performed using the ryan-einotgabriel-welsch (regw) multiple range test (day & quinn 1989). correlation analysis was carried out to relate the means of the total body collections with monthly meteorological data. results a total of 2 576 ticks was collected from 120 animals during the study period. four genera and ten species of ticks were identified. the tick species identified were (in order of abundance): amblyomma var ie gatum (38.2 %), hyalomma marginatum rufipes (18.4 %), hyalomma truncatum (15.3 %), rhipi cephalus appendiculatus (11.9 %), rhipicephalus evertsi evertsi (6.3 %), boophilus decoloratus (5.6 %), rhipicephalus praetextatus (2.2 %), boophilus annulatus fig. 1 locations where samples were collected from central equatoria state, southern sudan. top: map of the sudan depicting its state boundaries. black area: central equatoria state. bottom: juba area in central equatoria state depicting the locations where the samples were collected 31°30’ 31°35’ 31°40’ 31°45’ 4°50’ 4°45’ 4°55’ gumbo khor rumla juba nyaing r iv e r n ile n 1:203 888 49 d.a. salih et al. table 1 anova mean squares (me), r-squares (r), coefficient of variance (cv) and f values (f) for total body collections of ticks from cattle in central equatoria state, southern sudan during the year 2005 source of variation me r cv f a. variegatum b. annulatus$ b. decoloratus h. m. rufipes h. truncatum r. appendiculatus r. e. evertsi r. praetextatus 117.30 1.75 35.57 665.02 574.20 23.20 31.07 1.17 0.17 0.20 0.52 0.81 0.73 0.24 0.51 0.16 75.58 214.5 128.18 81.17 117.45 87.73 106.90 141.04 2.12* 2.63*** 10.59*** 41.53*** 26.68*** 3.16*** 10.36*** 1.87ns total 3 555.9 0.75 42.0 30.30*** $ females only ns not significant degrees of freedom = 9 * = p < 0.05, *** = p < 0.001 table 2 means (± se) of total body collection of ticks from cattle at different locations in central equatoria state, southern sudan during the year 2005* tick species locations gumbo nyaing khor rumla a. variegatum b. annulatus$ b. decoloratus h. m. rufipes h. truncatum r. appendiculatus r. e. evertsi r. praetextatus 10.9 ±1.3a 0.2 ± 0.06b 0.8 ± 0.2b 6.0 ± 1.6a 5.8 ± 1.7a 3.0 ± 0.5a 1.1 ± 0.3b 0.6 ± 0.1b 9.6 ± 1.5a 1.0 ± 0.6a 3.8 ± 1.0a 1.0 ± 0.2b 0.0 ± 0.0b 2.7 ± 0.6a 0.9 ± 0.3b 1.0 ± 0.2a 9.0 ±1.2a 0.3 ± 0.08b 0.9 ± 0.2b 5.9 ± 1.4a 4.1 ± 1.2a 3.4 ± 0.5a 2.5 ± 0.5a 0.4 ± 0.1b total 28.2 ± 4.2a 19.9 ± 2.0b 26.4 ± 2.8a $ females only number of observations = 40 in each location means (± se) followed by the same letter in each column are not significantly different at 5 % level based on ryan’s q test (regwq) * collection was carried out four times in each location table 3 means (±se) of total body collection of ticks from cattle in different months in central equatoria state, southern sudan during the year 2005* tick species months january april july october a. variegatum b. annulatus$ b. decoloratus h. m. rufipes h. truncatum r. appendiculatus r. e. evertsi r. praetextatus 6.7 ± 0.8a 0.2 ± 0.07b 0.4 ± 0.1b 0.7 ± 0.1b 0.2 ± 0.1b 3.0 ± 0.5b 1.4 ± 0.3b 0.5 ± 0.1a 11.7 ± 1.4a 0.8 ± 0.3a 3.5 ± 0.7a 0.8 ± 0.2b 0.4 ± 0.1b 2.5 ± 0.5b 0.2 ± 0.1c 0.7 ± 0.2a 12.0 ± 2.1a 0.0 ± 0.0b 0.1 ± 0.07b 2.2 ± 0.5b 0.7 ± 0.2b 1.6 ± 0.2b 1.1 ± 0.2b, c 0.3 ± 0.1a 9.8 ± 2.1a 0.5 ± 0.1a 1.2 ± 0.2b 20.3 ± 1.9a 18.2 ± 2.3a 5.7 ± 0.8a 4.6 ± 0.8a 0.7 ± 0.2a total 13.1 ± 0.9c 20.6 ± 1.6b 17.9 ± 2.2b, c 60.7 ± 4.2a $ females only number of observations = 30 in each month means (± se) followed by the same letter in each column are not significantly different at 5 % level based on ryan’s q test (regwq) * collection was carried out from five animals in six herds in each month 50 ticks (acari: ixodidae) on cattle in southern sudan (1.5 %), amblyomma lepidum (0.4 %) and rhipi cephalus sanguineus group (0.2 %). male ticks usually outnumbered females except for boophilus spp. anova, mean squares, r-squares, coefficient of variance and f values are shown in table 1. the mean tick load per animal was found to be lowest in january (16.2) and the highest in october (53.8). as far as locations are concerned, at nyaing the lowest tick infestation was found (19.9 ± 2.0), which was significantly different (p < 0.05) from the other two locations (gumbo, 28.2 ± 4.2 and khor rumla 26.4 ± 2.8) (table 2). with respect to the season of collection, it was found that tick infestation increased significantly (p < 0.05) from 13.1 ± 0.9 in january (dry season) to 60.7 ± 4.2 in october (towards the end of the rainy season) (table 3). male hosts were found to carry more ticks (29.9 ± 3.3) than female hosts (22.3 ± 2.6). significant differences (p < 0.05) were observed in the male/female tick ratio in the october collection (table 5). data on distribution, abundance, seasonality and correlation between mean tick count and climatic factors are presented separately below for each species. amblyomma lepidum dönitz, 1909 only four males and six females of the east african bont tick were collected during this study. three males were collected from gumbo in april, whereas the fourth one was collected from khor rumla in october, associated with two females, while the other four females were collected from the same location in the same season, but without males. this table 4 correlation analysis between means of total body tick collection and climatic factors in central equatoria state, southern sudan during the year 2005 tick species climatic factors max min rh rain a. variegatum b. annulatus$ b. decoloratus h. m. rufipes h. truncatum r. appendiculatus r. e. evertsi r. praetextatus 0.078 0.235** 0.381*** –0.352*** –0.292*** –0.008 –0.288*** 0.136 0.210* 0.289*** 0.543*** –0.109 –0.093 –0.068 –0.278** 0.105 0.213* –0.065 –0.071 0.391*** 0.333*** 0.008 0.193* –0.074 0.261*** –0.018 0.061 0.083 0.040 0.148 –0.069 0.070 climatic factors (previous month) max min rh rain a. variegatum b. annulatus$ b. decoloratus h. m. rufipes h. truncatum r. appendiculatus r. e. evertsi r. praetextatus 0.001 0.255* 0.457*** –0.465*** –0.408*** –0.106 –0.416*** 0.124 0.233* 0.246* 0.501*** –0.231* –0.219* –0.160 –0.364*** 0.071 0.105 –0.189 –0.313** 0.463*** 0.401** 0.063 0.341*** –0.112 0.177 –0.153 –0.202 0.250* 0.192* 0.072 0.138 –0.119 $ females only * = p < 0.05, ** = p < 0.01, *** = p < 0.001 max = mean maximum temperature min = mean minimum temperature rh = relative humidity rain = rainfall table 5 means (±se) of total body collection of ratio of males to females (m/f) in central equatoria state, southern sudan during the year 2005 ratio location season m/f gumbo nyaing k. rumla january april july october 2.8/1 ± 0.4a 3.0/1 ± 0 .7a 2.1/1 ± 0.4a 4.0/1 ± 0.7a 2.6/1 ± 0.4a, b 2.1/1 ± 0.6b 1.0/1 ± 0.1b means (± se) followed by the same letter in each row are not significantly different at 5 % level based on ryan’s q test (regwq) 51 d.a. salih et al. tick species represent only 0.4 % (10/2576) of all ticks collected. due to the very small number, this species was not included in the statistical analysis. amblyomma variegatum fabricius, 1794 the tropical bont tick was the most predominant tick species, representing 38.2 % (984/2576) of the total number of ticks collected. there was no significant difference in the mean collection among the three locations surveyed, ranging from 9.0 ± 1.2 in khor rumla to 10.9 ± 1.3 in gumbo (table 2). the highest mean (12.0 ± 2.1) was recorded in the middle of the rainy season (table 3). female hosts were found to carry more of this species than male hosts. a positive correlation (r = 0.261, p ≤ 0.001) was found between rainfall and the mean body collection (table 4). boophilus annulatus say, 1821 only one b. annulatus male was collected in january in khor rumla, therefore only females of this species were subjected to statistical analysis. the mean total body collection of this tick species in nyaing (1.0 ± 0.6) was found to be significantly higher (p < 0.05) compared with other locations (table 2). no ticks of this species were collected during july, whereas the highest mean (0.8 ± 0.3) was collected during april (table 3). female abundance was found to be positively correlated with ambient temperatures (maximum and minimum) and negatively correlated with rainfall and relative humidity (r = –0.018, p > 0.05 and r = –0.065, p > 0.05, respectively) (table 4). boophilus decoloratus koch, 1844 the blue tick was found together with b. annulatus in nyaing with the highest mean total per animal (3.8 ± 1.0), which was significantly higher (p < 0.05) as compared with the other locations (table 2). this tick species was found to be abundant in april (3.5 ± 0.7) which was significantly different (p < 0.05) compared with other seasons (table 3). the correlation analysis indicated that this tick species is also affected by climatic factors as for b. annulatus, with the exception of rainfall, which was positively but not significantly correlated (r = 0.061) (table 4). hyalomma marginatum rufipes koch, 1844 the highest mean number of the hairy hyalomma was collected from gumbo (6.0 ± 1.6), but was not significantly different (p > 0.05) from the infestation at khor rumla, while the infestation in these two locations were significantly higher (p < 0.05) compared with the infestation at nyaing (table 2). this tick was most abundant during the rainy season (october) when the highest mean infestation per animal was recorded (20.3 ± 1.9) (table 3). a signif i cant negative correlation was found with mean max imum temperature (r = –0.352, p < 0.001) and mean minimum temperature (r = –0.109, p > 0.05) (table 4). hyalomma truncatum koch, 1844 the african hyalomma was not found at nyaing, whereas the mean tick burdens at the other two locations were not significantly different (p > 0.05) (table 2). this tick species was found to be abundant during october with mean number of 18.2 ± 2.3 (table 3). the effect of climatic factors on this species was found to be the same as for hyalomma m. rufipes (table 4). rhipicephalus appendiculatus neumann, 1901 the brown ear tick was recorded in all locations and seasons with the collection in october (5.7 ± 0.8) being significantly larger (p < 0.05) compared with other collections (table 3). the presence of this tick was found to be negatively but not significantly correlated with mean temperature (maximum, r = –0.008, minimum, r = –0.068) and positively but not significantly correlated with relative humidity (r = 0.008) and rainfall (r = 0.148) (table 4). rhipicephalus evertsi evertsi neumann, 1897 the mean infestation of the red legged tick in khor rumla was 2.5 ± 0.5 which was significantly higher (p < 0.05) than in the other locations (table 2). the infestation in october was the highest (4.6 ± 0.8) and significantly different (p < 0.05) from the other seasons (table 3). there was a significant positive correlation of the mean numbers of this tick species with relative humidity r = 0.193 (p < 0.05) (table 4). rhipicephalus praetextatus gerstäcker, 1873 this tick species was recorded at nyaing (1.0 ± 0.2) with the highest mean infestation and was significantly different (p < 0.05) from the other locations (table 2). the infestation with this tick was not signif icantly different between different seasons (p > 0.05) (table 3). a negative correlation (r = –0.074) was found with relative humidity (table 4). rhipicephalus sanguineus group latreille, 1806 only six ticks belonging to the rhipicephalus sanguineus group were collected during the period of 52 ticks (acari: ixodidae) on cattle in southern sudan study. two females were collected from gumbo in january, while four males were collected from nyaing in the same month. this tick was not included in the statistical analysis. discussion since the previous surveys on ticks in the southern sudan (hoogstraal 1956; anonymous 1983; jongejan, zivkovic, pegram, tatchell, fison, latif & paine 1987; julla 1994), no work has been carried out on ticks in southern sudan due to civil unrest. in the present study, four genera and ten species of ticks were identified (table 1), compared to eight genera and 46 species and subspecies of ticks identified by hoogstraal (1956), five genera and eight species by anonymous (1983) and five genera and 12 species by julla (1994). these differences may be attributed to the fact that the present survey was limited to cattle around juba town only, whereas hoogstraal (1956) included ticks from all livestock and wildlife in equatoria region, as did julla in his survey conducted in 1994. the mean number of ticks per head of cattle in central equatoria state was relatively low throughout the study period with the exception of the october collection (table 3). this may be attributed to the fact that indigenous cattle are generally highly successful in developing resistance to ticks (latif 1984). males constituted the majority of all species with the exception of boophilus spp., which males are difficult to find on the host (tatchell & easton 1986). the highest collection of ticks combined with an equal male/female ratio obtained during october (tables 3 and 5), indicates that the rainy season is favorable for the breeding of ticks in central equatoria state. the small number of a. lepidum ticks (ten specimens) found in this survey confirms its patchy distribution (osman & hassan 2003). hoogstraal (1956) and also anonymous (1983), for instance, did not find this tick species in central equatorial state but julla (1994) did, which may indicate the continuous shifting of its microhabitat. the distribution limits of ticks are not fixed and constant. they are determined by a complicated interplay of factors such as climate, vegetation, host density, host resistance and animal husbandry practices (tatchell & easton 1986). more important is the finding that this tick spe cies is spreading south (w. lako, unpublished data 2006). rhipicephalus appendiculatus is the most important rhipicephalid tick in east and southern africa where it infests a wide variety of domestic and wild ruminants (jongejan & uilenberg 2004). the year-round presence of r. appendiculatus (table 3), confirms that it has firmly established itself in central equatoria state. this finding provides an answer to the query by jongejan et al. (1987) as to “whether this tick is repeatedly introduced by seasonal migration of livestock or is able to maintain itself around ju ba?” and confirms the previous finding of julla (1994). moreover, the latter author indicated that east coast fever and its vector r. appendiculatus has not only become established in juba but has also extended to terekaka area (~ 60 km north of juba). the distribution and abundance of r. appendiculatus are key determinants in the epidemiology of east coast fever (lessard, l’eplattenier, norval, kundert, dolan, croze, walker, irvin & perry 1990). this distribution can be influenced by the climate as well as many other factors such as the presence, abundance, movement of cattle, the resistance of the host to ticks and tick-borne diseases and the vegetation cover. in this study, there was a pronounced effect of climatic factors on the numbers of r. appendiculatus ticks as well as many other tick species (table 4). the presence of r. appendiculatus was found to be negatively correlated with mean maximum temperature (r = –0.008) as well as mean minimum temperature (r = –0.068). this finding is in accord with suggestions of tatchell & easton (1986), who reported that the life cycle of this tick species is very precisely controlled by the combined effect of low temperatures, slowing developmental rates and lack of rain in the dry season preventing early resumption of feeding by adults produced from the first fed nymphs. the most abundant tick species in juba area was a. variegatum (38.2 %) compared with r. appendiculatus (11.9 %). it is known that a. variegatum has a broader distribution than r. appendiculatus, being able to survive in hotter drier environments (tatchell & easton 1986). in those african countries which have one rainy season per year, a. variegatum and r. appendiculatus usually have one generation per year (dipeolu 1989). this fact is clearly evident from the results of the present investigation, i.e. that there is only one generation of r. appendiculatus per year in southern sudan. as far as tick control is concerned, acaricides are only applied when tick numbers are very high. the type of strategic tick control to be implemented in a certain area requires a sound knowledge of vector ecology and disease epidemiology. moreover, the nature of the farming system and socio-economic 53 d.a. salih et al. considerations will also have an important influence in the success of this approach (de castro 1997). here, it is concluded that indigenous zebu cattle are successful in developing resistance to ticks in central equatoria state, as inferred from the low tick burdens detected in the present study. it remains to be shown whether the current situation is the best one possible with respect to keeping losses due to ticks and tick-borne diseases low without much use of acaricides. however, a cross-sectional and longitudinal survey in the same region would be required to answer the fore-going questions. acknowledgements this research was supported by the international foundation for science, stockholm, sweden and the organisation of islamic conference standing committee on scientific and technological cooperation (comstech), islamabad, pakistan through a grant to mr diaeldin ahmed salih (ifs grant 3765-1). this work is published by kind permission of the directorgeneral of research, ministry of animal resources and fisheries, government of south sudan and the director-general, animal resources research corporation, khartoum. references anonymous 1983. tick and tick-borne disease control, the sudan. tick ecology and tick control. ag: gcp/sud/024/den. technical report 1. rome: food and agricultural organ ization. day, r.w. & quinn, g.p. 1989. comparison of treatment after an analysis of variance in ecology. ecological monographs, 59:433–463. de castro, j.j. 1997. sustainable tick and tick-borne disease control in livestock improvement in developing countries. veterinary parasitology, 71:77–97. dipeolu, o.o. 1989. research on ticks of livestock in africa: review of the trends, advances and milestones in tick biology and ecology in the decade 1980–1989. insect science and its application, 10:723–740. hoogstraal, h. 1956. african ixodoidea. i. ticks of the sudan (with special reference to equatoria province and with preliminary reviews of the genera boophilus, margaropus and hyalomma). department of the navy, bureau of medicine and surgery, washington, d.c., usa. jongejan, f., zivkovic, d., pegram, r.g., tatchell, r.j., fison, t., latif, a.a. & paine, g. 1987. ticks (acari: ixodidae) of the blue and white nile ecosystems in the sudan with particular reference to the rhipicephalus sanguin eus group. experimental and applied acarology, 3:331–346. jongejan, f. & uilenberg, g. 2004. the global importance of ticks. parasitology, 129:s1–s12. julla, i.i. 1985. theileriosis in the south sudan, in immunization against theileriosis in africa, edited by a.d. irvin. proceedings of a workshop held at ilrad, nairobi, kenya, 1–5 october 1984: 27–30. julla, i.i. 1994. studies on the epidemiology of theileriosis in equatorial region of the sudan with emphasis on east coast fever. ph.d. thesis, university of khartoum. latif, a.a. 1984. resistance to hyalomma anatolicum anatolicum koch (1844) and rhipicephalus evertsi neumann (1897) (ixodoidea: ixodidae) by cattle in the sudan. insect science and its application, 5:509–511. lessard, p., l’eplattenier, r., norval, r.a.i., kundert, k., dolan, t.t., croze, h., walker, j.b., irvin, a.d. & perry, b.d. 1990. geographical information systems for studying the epidemiology of cattle diseases caused by theileria parva. veterinary record, 17:255–262. morzaria, s.p., tatchell, r.j., minor, r., pederson, v., julla, i., rahim, a., dyson, d. & aarle, p.a.m. 1981. preliminary studies on the epidemiology of theileriosis in eastern equatoria province of the sudan, in advances in the control of theileriosis, edited by a.d. irvin, m.p. cunningham & a.s. young. the hague: martinus njihoff publishers. okello-onen, j., hassan, s.m. & essuman, s. 1999. taxonomy of african ticks: an identification manual. nairobi: icipe science press. osman, a.m. & hassan, s.m. 2003. the ecology and distribution of the east africa tick amblyomma lepidum: a review. the sudan journal of veterinary science & animal hus bandry, 42(1 & 2):45–53. tatchell, r.j. & easton, e. 1986. tick (acari: ixodidae) ecological studies in tanzania. bulletin of entomological research, 76:229–246. walker, a.r., bouattour, a., camicas, j.l., estradapeňa, a., horak, i.g., latif, a.a., pegram, r.g. & preston, p.m. 2003. ticks of domestic animals in africa: a guide to identification of species. edinburgh, scotland: bioscience reports. article information authors: jakob zinsstag1 andrea meisser1 esther schelling1 bassirou bonfoh2 marcel tanner1 affiliations: 1swiss tropical and public health institute, basel, switzerland 2centre suisse de recherches scientifiques en côte d’ivoire, côte d’ivoire correspondence to: jakob zinsstag postal address: po box 4002, basel switzerland and university of basel, switzerland how to cite this proceeding: zinsstag, j., meisser,, a., schelling, e., bonfoh, b. & tanner, m., 2012, ‘from “two medicines” to “one health” and beyond’, onderstepoort journal of veterinary research 79(2), art. #492, 5 pages. http://dx.doi.org/10.4102/ ojvr.v79i2.492 note: proceedings of the conference of the southern african centre for infectious disease surveillance ‘one health’ held at the national institute for communicable diseases, johannesburg, july 2011. copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. from ‘two medicines’ to ‘one health’ and beyond in this proceeding... open access • abstract • introduction • brief history of integrative thinking in medicine and health • operational definition of ‘one health’ and its leverage • examples of the leverage of ‘one health’ • the unfinished ‘one health’ agenda • conceptual extensions of ‘one health’ towards systemic approaches • operationalisation of ‘one health’ • acknowledgment • references abstract top ↑ we first review historic and conceptual background to integrative thinking in medicine. lacking a general theory of ‘one health’, we provide an operational definition of ‘one health’ and its leverage as: any added value in terms of human and animal health, financial savings or environmental benefit from closer cooperation of human and animal health sectors at all levels of organisation. examples of such added value of ‘one health’ are given from the fields of health systems, nutrition and zoonoses control in africa and asia. ‘one health’ must become main-stream rather than a new discipline or new association; it should just become normal that practitioners and professionals in the health, animal and environment sectors work together as closely as possible. current and future challenges in financing clean energy, migration flows, food security and global trade further warrant rethinking of human and animal health services. a conceptual outlook relates health as an outcome of human-environment systems called ‘health in social-ecological systems’. the paper ends with an outlook on the operationalisation of ‘one health’ and its future potential, specifically also in industrialised countries. introduction top ↑ the present paper is summarising and extending an invited key note, given as a video presentation to the first african ‘one health’ conference, held in johannesburg on july 14 and 15, 2011 by the first author (http://www.sacids.org/kms/frontend/?m=103). the human and animal health research unit in the department of epidemiology and public health at the swiss tropical and public health institute (http://www.swisstph.ch) entertains partnerships with countries in east–africa, central–africa and west–africa, central asia and switzerland. here we account for conceptual and practical research work on collaborative efforts between human and animal health, between developing and industrialised countries involving disciplines as diverse as epidemiology, anthropology, cultural sciences, sociology, geography, molecular biology, statistics and mathematics. initially the focus of the research group was on the provision of health care to mobile pastoralists, who are nearly devoid of health services. however, soon questions on the control of zoonoses, diseases transmissible between animals and humans, like bovine tuberculosis, rabies, brucellosis, anthrax and avian influenza, came in the focus of attention. the research group is part of several larger international research networks. it is the health partner in the national centre of competence in research north-south (http://www.north-south.unibe.ch), together with seven swiss research institutions and their partners on all continents except australia. the network covers research partnerships on natural resource management, conflict transformation, governance, water and sanitation, livelihood, urban planning and health and is jointly funded by the swiss national science foundation (snsf) and the swiss agency for development and cooperation (sdc). in this network, health research is a component of a larger development research approach for which we currently work on integrated methods involving interand transdisciplinary approaches. the unit is also part of the european union funded network on integrated control of neglected zoonoses in africa (www.iconzafrica.org). it involves twenty european and african partner institutes. further, the unit collaborates in wellcome trust funded projects on bovine tuberculosis in africa which evolved into an african capacity building program ‘ecosystem and population health: bridging the frontiers in health’ (http://www.afriqueone.net). the last network is the consortium one health next generation oh-nextgen with a strong focus on training young fellows to change the mindset in health system considering health problem at the human, animal and environment interface in different eco-zones. networks have proven essential for reassembling critical mass, interand transdisciplinary collaboration, north-south, south-south and east-west exchanges and comparisons, yielding typically a higher output of research outcomes as could be achieved if each partner worked alone. we provide first a brief historic and conceptual background to integrative thinking in medicine. we will then provide an operational definition of ‘one health’ and its leverage. examples of the leverage of ‘one health’ are given from the fields of health systems, nutrition and zoonoses control in africa and asia. prior to further conceptual extensions the unfinished ‘one health’ agenda will be described. the paper ends with an outlook on how to make ‘one health’ operational and its future potential, specifically also in industrialised countries. brief history of integrative thinking in medicine and health top ↑ we are often asked what is new about ‘one health’. actually nothing, close interactions between human and animals are longstanding but vary by their historical intensity and cultural background. we provide here examples from a more detailed account (zinsstag et al. 2011). in the ancient egyptian culture humans and their animals were seen as belonging to one ‘flock of god’. transformation from humans to animals (metempsychosis) are known from india and africa. fulbe pastoralists in africa, in their myths of creation, see cattle as being an integral part of their society (louanges à la femme 1966). the zhou dynasty in china (11th century − 13th century) maintained the first integrated public health system including medical doctors and veterinarians. the chinese scholar xu dachun stated already in the 18th century, 100 years earlier than rudolf virchow that, ‘the foundations of veterinary medicine are as comprehensive and subtle as those of human medicine and it is not possible to place one above the other’ (driesch & peters 2003). whilest human medicine became a faculty in the medieval european universities (rüegg 2004), veterinary medicine remained in the hand of equerries, the persons in charge of the horses for warfare, until claude bourgelat, founded the first veterinary school in lyon (1761). the end of 19th century with the advent of cellular pathology and microbiology was a period of very close interaction of human and animal health as comparative medicine. one of its protagonists, rudolf virchow, stated in an address to the prussian government on bovine tuberculosis, ‘between animal and human medicine there is no dividing line – nor should there be’. the object is different, but the experience obtained constitutes the basis of all medicine. ‘in the twentieth century veterinary and human medicine evolved in a way as to specialise into more and more sub-disciplines and the influence of comparative medicine decreased. the american epidemiologist calvin schwabe, influenced by his work with dinka pastoralists in sudan coined the term ‘one medicine’ in the 1960s. it means that, there is no difference of paradigm between human and veterinary medicine. both sciences share a common body of knowledge in anatomy, physiology, pathology, on the origines of diseases in all species. we can thus conclude that the modern formulation of ‘one medicine’ has african roots. in the past decades, ‘one medicine’, addressing more and more public and environmental health issues became ‘one health’(zinsstag et al. 2005) and has seen unprecedented revival at the level of international organisations, national governments and academia (zinsstag et al. 2009b) after the outbreaks of major diseases (sars, avian flu, swine fever …). operational definition of ‘one health’ and its leverage top ↑ the scholarly statements on ‘one medicine’ mentioned above have been replaced by an ongoing debate on contemporary definitions and delimitation of what has become ‘one health’. in the past conferences, in particular at the first ‘one health’ conference in melbourne in february 2011, many presenters limited themselves to recognising the interdependence of humans and animals and their environment. in our view, this is a necessary component of ‘one health’, but only part of it. we lack a modern and internationally acknowledged theory of ‘one health’, which may require an in-depth epistemological assessment of all involved disciplines. we propose here a pragmatic operational definition of ‘one health’ as any added value in terms of human and animal health, financial savings or social and environmental benefits from closer cooperation of professionals in the health, animal and environment sectors at all levels of organisation. claiming a ‘one health’, in our view, requires the demonstration of added value to what human and animal health working alone can achieve. specifically a ‘one health’ approach is capable of identifying points of leverage of health of humans and animals from a systemic analysis. examples of the leverage of ‘one health’ top ↑ the presented examples are published as case studies on zoonoses epidemiology, nutrition and public health services. most often zoonoses are investigated either in humans or animals. in the case of zoonotic diseases that are transmissible between humans and animals, integrated study designs investigating health status in humans and animals simultaneously allow an instantaneous identification of the source of a zoonotic disease. for example, in chadian pastoralist human q-fever to camels (schelling et al. 2003). human brucellosis can be eliminated by interventions in animals. from a public health point of view, mass vaccination of livestock to prevent human brucellosis is not profitable in mongolia. but if societal benefits are summed up, including benefits for private households and the livestock sector, the intervention is largely profitable. if costs of brucellosis mass vaccination are shared between the health and livestock sector proportional to their benefits, brucellosis control becomes highly cost-effective (roth et al. 2003). similarly, the cumulative cost of dog rabies mass vaccination and human post-exposure treatment (pet) in n’djaména, chad reaches break-even with the cumulative cost of pet alone after six years (zinsstag et al. 2009a). such comparative assessments can only be made if human and animal health is investigated as a single social-ecological disease system. a shared veterinary laboratory to diagnose brucellosis in febrile patients has brought the collaborating physician in mali to include brucellosis testing as a differential diagnosis to malaria and typhoid fever in an area where raw milk consumption is still prevalent (steinmann et al. 2005). pastoralists in africa depend highly on milk from their animals for their nutrition and the vitamin a status of mobile pastoralist women and children in chad depends directly on the vitamin a levels in the milk of their cows. in the same way a study on the vaccination status of mobile pastoralist children, women and their animals showed that the vaccination coverage of livestock was much higher than that of children and women. joining the vaccination campaigns between the veterinary and public health services reduced the logistic cost by 15% and improved vaccination coverage of children and women, who have otherwise no access to health care (bechir et al. 2004; schelling et al. 2005). work with pastoral communities heavily relayed on collaboration with cultural scientists, who lived for example with kel tamacheq communities in north mali. fluency in local languages and coranic literacy were critical for creating a trustful relationship. informations and data on mother and child health seem to be more accurate from participant observation than from clinical surveys by a medical doctor (münch et al. 2007). in this way a ‘one health’ approach recognises the need for collaboration between medical and cultural sciences. the unfinished ‘one health’ agenda top ↑ the above examples clearly show an added value of closer cooperation between human an animal health for the understanding of the human animal linkage by taking more a societal perspective rather than a public health point of view only. it shows how interventions become profitable or public and animal health status can be improved. much of this dynamic has been taken up but there remain still a huge unfinished agenda (zinsstag et al. 2009b). a recent outbreak of q-fever in the netherlands (enserink 2010a & 2010b) has shown the current limitations of communication between the animal and public health surveillance system. there are obvious reasons why surveillance systems of communicable diseases for humans and animals should be coupled in a single cooperative surveillance system, which informs on outbreaks in all different species simultaneously to the whole system. this would, as the dutch q-fever example shows, reduce time to detection and time to intervention significantly. the control of rift valley fever, another epidemically occurring zoonosis, would largely benefit from joint contingency planning where roles of each sector, how information flows and cost-sharing schemes are jointly decided on based on evidence before an outbreak. similarly there are great public health opportunities in merging human and animal cancer registries (o’brien et al. 2000). geo-referenced detection of cancer incidence in one species could reveal environmental exposure for the other species. canada is spearheading such approaches by its joint surveillance of antimicrobial resistance (cipars, http://www.phacaspcgc.ca/cipars-picra/index-eng.php) or the integrated surveillance of enterobacteriacea (c-enternet). the human-animal bond has far reaching consequences in the case of non-communicable disease like depression or obesity (o’haire 2010). systemic approaches, well known from pastoral counseling (van katwyk 2005) or family therapy could be extended to health care for humans and their pets or pets and their holders (the human-animal bond as an entity). respiratory problems of a dog may be associated to smoking behavior of the dog holder (reif et al. 1992). obesity of a pet may be associated to a health problem of its owner, and hence the owner’s care determines the pet animal’s health. new ways of communication between clinical veterinarians and family doctors require a dialogue and negotiation as to when an interaction makes sense and may lead to improved health of animals and their humans. to prevent fears of institutes being absorbed by larger ones, public and animal health systems should cooperate as equal rights partners respecting each others technical field of competence. ‘one health’ as a mindset, must become mains-stream, rather than a new discipline or institution, it must become normal that professionals throughout all relevant disciplines (e.g. physicians, veterinarians, social scientists and ecologists) work together as closely as possible. current and future challenges in financing, clean energy, increasing migration flows, food insecurity and global trade further warrant rethinking of human and animal health services (see below). the above ‘unfinished’ agenda is also incomplete and warrants each actor’s imagination as to how interactions between the two medicines can yield better health for all. conceptual extensions of ‘one health’ towards systemic approaches top ↑ the closer cooperation between human and animal health has been extended since over a decade by including ecological and eco-systemic aspects, known as eco-system health (www.ecohealth.net), recognising inter-dependency of health of humans and animals and the integrity of eco-systems (forget & lebel 2001; lebel 2002). systems biology, previously concerned mainly with complex processes at cellular and sub-cellular level recognise extensions at higher scales up to populations, for example for explaining the development of persistent infections and phylo-geographic lineages in tuberculosis (gagneux & small 2007; young, stark & kirschner 2008). environmental sciences and work on natural resource management use conceptual approaches called social-ecological systems (ostrom 2007) or human-environment systems, which can easily be applied to a systemic approach to health e.g. in the management of bovine spongiform encephalitis (bse) in switzerland (scholz 2011). studying health in social-ecological systems (hses) opens new ways addressing complex, multivariable, nonlinear, cross-scale and dynamic factors determining the health of humans and animals (zinsstag et al. 2011). hses formally include social sciences and humanities in health research but require further work on epistemological bridges between humanities, economics and natural sciences. as an example on health in a social-ecological system, we can mention the interactions of the socio-political and ecological changes from a planned economy to a market economy in mongolia in 1990, causing the breakdown of public health and veterinary disease control systems. in the same time the privatised livestock production led to a sharp increase of livestock numbers, pasture degradation and animal disease like foot-and-mouth disease (fmd) and brucellosis, which is transmissible to humans. effective reduction of brucellosis incidence in humans requires interventions in livestock. understanding livestock demographic dynamics becomes a key for the planning of sustainable pasture management and the planning of animal health interventions. seeking a stabilisation of the mongolian livestock population in mongolia will be an important element to preserving pastures. potential freedom of important zoonotic and transboundary diseases will provide market access and help stabilising livestock population by increasing offtake. all these complex social-ecological processes determine the health of humans and animals but depend on political and societal forces engaging their respective interests. further work aims at demonstrating added value of a systemic approach on overall societal burden and cost of disease and its control, while preserving ecosystem services and social stability using transdisciplinary approaches. operationalisation of ‘one health’ top ↑ we have shown that ‘one health’ is well integrated into broader conceptual thinking like ‘ecosystem health’ or ‘health in social-ecological systems’. whilst research can disentangle the complex interactions between health, society and ecosystems, demonstrating central points of leverage for future interventions, governments and international agencies aim primarily at making ‘one health’ work in practice. there is not a blueprint for making ‘one health’ operational and there are multiple actors involved and require stakeholders’ involvement, long term partnerships, capacity-building, but also local champions. most of the activities in industrialised countries are mirrored by the ‘one health’ initiative website (http://www.onehealthinitiative.org). the world bank for example engages in the study of structural savings from institutional planning by joining surveillance or laboratory capacity. academic curricula teaching ‘one health’ are developed by several universities, for example the university of calgary in canada and imply the development of methods for ‘one health’ research (zinsstag et al. 2009b). research for development shows that operational models of ‘one health’ require transdisciplinary processes (schelling et al. 2007). academic research extends processes to improved understanding of the interactions by involving stakeholders like communities, authorities and experts for the identification of locally acceptable and adapted health interventions. all processes need to be negotiated between actors since each context is different (meisser, schelling & zinsstag 2011; schelling et al. 2005; schelling et al. 2007). contextual solutions will address the importance of cultural determinants of the human animal relationship. as an example we can mention the dog, which in some cultures has mainly an emotional value with strong human-dog bonds and in others a commodity with commercial value as food supply. researchers and planners aiming at making ‘one health’ operational require high level self reflexive capacity in recognising inter-cultural aspects of the human-animal relationship. key outcomes of a closer cooperation of human and animal health will remain economic savings, health benefits for humans and animals and ecological benefits. not all outcomes such as improved communication and information flows between sectors with subsequent e.g. earlier detection of a disease or appropriate measure can be easily quantified, but can still be captured with qualitative attributes. the potential is endless and potential savings go in the billions, if doctors and veterinarians communicate and interact in a closer way. but we are also very much aware that those in charge of national planning would want to be re-assured by examples of cost saving potential, if not from their own country, at least from a country in their region. acknowledgment top ↑ we acknowledge the collaboration of all members of our research unit and the collaborators at swiss tph. we gratefully acknowledge our partners in chad, ethiopia, mali, mauritania, côte d’ivoire, kyrgyzstan and mongolia. lisa crump thanked for language editing. this paper has been supported by nccr north-south, eu fp7 iconz africa and the consortium afrique one funded by the wellcome trust. references top ↑ bechir, m., schelling, e., wyss, k., daugla, d.m., daoud, s., tanner, m. & zinsstag, j., 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university of pretoria, private bag x04, onderstepoort 0110, south africa dates: received: 14 nov. 2012 accepted: 30 mar. 2013 published: 24 apr. 2013 how to cite this article: petzer, i.m., karzis, j., meyer, i.a. & van der schans, t.j., 2013, ‘a cost-benefit model comparing the california milk cell test and milk electrical resistance test’, onderstepoort journal of veterinary research 80(1), art. #538, 6 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.538 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a cost-benefit model comparing the california milk cell test and milk electrical resistance test in this original research... open access • abstract • introduction • methods    • sample collection    • milk electrical resistance (mer)    • california milk cell test (cmct)    • laboratory procedures    • analysis of data: sensitivity, specificity and determination of cut-off points    • modelling approach    • prices used for model inputs    • cost-benefit model inputs    • true positive cases (sensitivity)    • false negative cases    • true negative cases (specificity)    • false positive cases    • benefit-cost ratios • results and discussion • conclusions • acknowledgements    • competing interests    • authors contributions • references abstract top ↑ the indirect effects of mastitis treatment are often overlooked in cost-benefit analyses, but it may be beneficial for the dairy industry to consider them. the cost of mastitis treatment may increase when the duration of intra-mammary infections are prolonged due to misdiagnosis of host-adapted mastitis. laboratory diagnosis of mastitis can be costly and time consuming, therefore cow-side tests such as the california milk cell test (cmct) and milk electrical resistance (mer) need to be utilised to their full potential. the aim of this study was to determine the relative benefit of using these two tests separately and in parallel. this was done using a partial-budget analysis and a cost-benefit model to estimate the benefits and costs of each respective test and the parallel combination thereof. quarter milk samples (n = 1860) were taken from eight different dairy herds in south africa. milk samples were evaluated by means of the cmct, hand-held mer meter and cyto-microbiological laboratory analysis. after determining the most appropriate cut-off points for the two cow-side tests, the sensitivity and specificity of the cmct (se = 1.00, sp = 0.66), mer (se = 0.92, sp = 0.62) and the tests done in parallel (se = 1.00, sp = 0.87) were calculated. the input data that were used for partial-budget analysis and in the cost-benefit model were based on south african figures at the time of the study, and on literature. the total estimated financial benefit of correct diagnosis of host-adapted mastitis per cow for the cmct, mer and the tests done in parallel was r898.73, r518.70 and r1064.67 respectively. this involved taking the expected benefit of a correct test result per cow, the expected cost of an error per cow and the cost of the test into account. the cmct was shown to be 11% more beneficial than the mer test, whilst using the tests in parallel was shown to be the most beneficial method for evaluating the mastitis-control programme. therefore, it is recommended that the combined tests should be used strategically in practice to monitor udder health and promote a pro-active udder health approach when dealing with host-adapted pathogens. introduction top ↑ when mastitis, due to host-adapted pathogens staphylococcus aureus (s. aureus) and streptococcus agalactiae, (s. agalactiae) is misdiagnosed, the risk of more severe udder parenchymal damage increases. longer periods of bacterial infection and shedding can create a prolonged window of opportunity for clinical mastitis to develop and for contagious transmission to occur (lam 1996; zadocks et al. 2002). in addition to dry-cow treatment, treatment of clinical mastitis forms part of most mastitis-control programmes (swinkels, hogeveen & zadocks 2005). staphylococcus aureus mastitis is highly prevalent in south african dairy herds and is a costly disease (petzer et al. 2009). according to goodger and ferguson 1987 and zepeda et al. 1998, control of s. aureus mastitis through preventive measures, early detection, dry-cow treatment and culling of chronically infected animals can be economically profitable. determining the costs specific to a single disease-control programme requires partial budgeting (smith 1995). in this study, partial-budget analysis was used to determine the relative effects of diagnosing and misdiagnosing true positive and true negative animals. these results were then used to perform a cost-benefit analysis. in this study, the gold standard used for mastitis diagnosis was defined as a somatic cell count (scc) of ≥ 400 000 cells/ml milk and the presence of mastitogenic bacteria (petzer et al. 2009). chronic mastitis, which is associated with a low probability of cure, may be diagnosed by clinical signs. these include palpable tissue changes, a history of repeated clinical mastitis, the presence of bacteria and infection of multiple quarters within a cow (sol et al. 1997; sol et al. 2000; deluyker, van oye & boucher 2005). cow-side tests, such as the california milk cell test (cmct) and milk electrical resistance (mer) are relatively cost effective and are used in the milking parlour for timeous identification of possible udder health problems as part of a mastitis-control programme. the international dairy federation (idf) requirement for a cow-side test is for it to have a sensitivity of at least 70% and a specificity of 99% (mein 2010). the purpose of this study was to determine the most beneficial cut-off points for the diagnosis of mastitis using either of the cow-side tests, when compared to the gold standard. this was done for tests used both separately and in a parallel combination. these cut-off points were then used to calculate sensitivity and specificity. earlier identification of mastitis using cow-side tests is more beneficial to producers in a mastitis-control programme and could provide the producer with valuable information regarding the early treatment of true mastitis cases. this approach supports proactive udder-health management and may assist in the reduction of the mammary infection rate in herds. indirect effects of mastitis treatment, such as prevention of clinical mastitis and transmission of infections to other cows are often overlooked in cost-benefit analysis, which may indicate that treatment is more favourable (st rose et al. 2003; swinkels et al. 2005). methods top ↑ sample collection the order in which the tests were performed in the parlour was as follows: a strip cup was used to identify possible clinical mastitis cases, after which aseptic quarter milk samples were taken, followed by quarter mer readings and the cmct. quarter milk samples (n = 1860) were collected aseptically according to standard procedure (giesecke, du preez & petzer 1994) from eight dairies in south africa. these samples were placed on ice and transported to the milk laboratory, production animal studies, onderstepoort and were analysed within 12 hours of collection. following aseptic sampling, approximately 5 ml milk from each quarter was milked into the handheld mer meter. the reading was recorded and this milk was poured into the corresponding cup of the cmct spatula, in order for the cmct to be performed. this process was repeated for each quarter that was sampled. milk electrical resistance (mer) electrical resistance, the reciprocal of electrical conductivity, was measured in this study. hand-held mer meters were used to evaluate udder health on a quarter basis. this is in contrast to in-line conductivity that is used in commercial dairies, which is measured at cow level. the hand-held mer meters (mast-o-test, durotec, po box 12540, centralhill, port elizabeth 6006, south africa) read electrical resistance in milli-ohms per centimeter (mω/cm). measures of resistance were categorised into three ordinal levels for data analysis, < 24 mω/cm (red light), 24 – 31 mω/cm (orange light) and > 31 mω/cm (green light), based on the manufacturer’s suggestion. an inter-quarter variation in resistance of 15% or more indicated possible udder health problems (mast-o-test, durotec, po box 12540, centralhill, port elizabeth 6006, south africa). california milk cell test (cmct) the cmct spatulas were pre-calibrated to help ensure that equal volumes of milk and reagent were used in each test (schalm, carroll & jain 1971). excess milk was carefully discarded up to the calibration line, ensuring that the volume of milk left in each cup was equal to the volume of reagent to be added. the milk and reagent were thoroughly mixed whilst carefully studying the viscosity of the mixtures. in this study, the same operator performed the reading according to the description of schalm et al. 1971. a weak positive (+) was recorded if during swirling, the mixture remained liquid (as with a negative score) and on tilting the paddle, the mixture flowed over the bottom of the cup in a slimy, streaky layer that was clearly visible. a distinct positive (++) was recorded if after the first one to two swirls of the mixture there was already distinct gel formation. some portions of the distinct positive mixture tended to collect at the centre of the cup, whereas others continued to move around its periphery. upon stopping the swirling motion, the gel-like mixture levelled out to cover the bottom evenly and on tilting the paddle, the mixture flowed over the bottom of the cup in a distinctly slimy non-uniform mass, possibly of streaky appearance. a strong positive (+++) was recorded if after the first one to two swirls the mixture had already formed a jelly that tended to adhere to the centre of the cup, where it formed a peak and left the periphery of the bottom of the cup exposed. after stopping the swirling motion, the mixture levelled out somewhat on the bottom of the cup, but its surface remained uneven and continued to show a distinct peak in its central region (schalm et al. 1971).acid milk is indicated by the mixture becoming distinctly yellow, with a ph 5.2. this is very rare and usually indicates fermentation of lactose by bacteria. alkaline milk is indicated when the reaction causes the mixture to become a deep purple colour, it may be as a result of udder inflammation or indicating that the udder is drying off (schalm et al. 1971). laboratory procedures samples were plated out onto columbia agar base plates with 5% defibrinated bovine blood (quantum biotechnologies (pty) ltd, ferndale, south africa). test plates were incubated for 24 to 48 hours at 37°c ± 1°c. isolated bacteria were identified in accordance with standard laboratory milk culture methodology based on colony morphology, haemolysis, catalase, koh test and gram staining. additional tests for bacterial identification included a strepkit (latex agglutination test from quantum biotechnologies (pty) ltd, ferndale, south africa), staphylase test (quantum biotechnologies (pty) ltd, ferndale, south africa) and the api 20e kit (biomerieux, po box 4328, honeydew 2040) (sandholm et al. 1995; karzis 2005). scc was determined using the fossomatic 5000 (rhine rhur, po box 76167, wendywood 2144). analysis of data: sensitivity, specificity and determination of cut-off points data was collected from 1860 aseptic quarter milk samples from eight different dairy herds in south africa. this data was analysed using a gold standard (criteria used for mastitis positive quarters were: scc ≥ 400 000 cells/ml and bacteria present). then the most appropriate cut-off point for each test was determined using the gold standard in the statistical programme genstat® (payne et al. 2011). using these cut-off points, the sensitivity and specificity of each of the tests was calculated from the data using microsoft excel® 2003. the criteria of the gold standard used for disease positive quarters (scc ≥ 400 000 cells/ml) with bacteria present was taken into account for each test done separately and for both tests done in parallel (dohoo, martin & stryhm 2003; petzer et al. 2009).these findings were used as criteria to determine whether each quarter had an increased scc using the cmct, with (1+, 2+ and 3+) indicating a positive test result and (0) indicating a negative test result. increased scc, as well as udder inflammation, are an indication of a possible mastitis case in a mastitis-control programme. similarly, the criteria used to evaluate udder inflammation of quarters using mer were as follows: an orange light (24 – 31 mω/cm) and red light (< 24 mω/cm) indicated a positive test result, whilst a green light (> 31 mω/cm) indicated a negative test result; the latter being a healthy quarter. both sets of criteria were calculated in genstat® using the gold standard (as described above). the sensitivity and specificity of each of the tests and of the tests in parallel were then inserted into the model as inputs. modelling approach partial-budget analysis was used as part of a cost-benefit model. in a cost-benefit model, economic effects for any given scenario are calculated as total revenues weighed against total costs. in this model, the expected cost or benefit of a specific test was calculated. this was done by determining the likelihood that a specific test would predict a false positive, false negative, true positive or true negative result. an expected cost or benefit was associated with each of these outcomes. for example, correctly identifying a true positive result and acting on it would have certain benefits, such as a reduction in the infection rate and a reduction in the decline of milk yield and milk quality. it also has costs associated, such as the cost of treatment and the cost of additional labour. similarly, incorrectly identifying a true negative result as positive would have costs associated, such as an increase in the loss of milk, an increase in antibiotic residues present in milk and in the bulk tank due to unnecessary treatment, and the cost of treatment. partial-budget analysis was used to calculate the relative costs and benefits. in partial-budget analysis, alternatives are compared without calculating a complete budget for each scenario. instead, only the revenues and costs that are affected by the specific scenario are considered. total returns were calculated as extra returns plus reduced costs. total costs were calculated as reduced returns plus extra costs. both direct and indirect effects of diagnosing and misdiagnosing true positive and true negative animals were taken into account. biological parameters included the clinical outcome of infection with host adapted pathogens for the affected animal, effects at herd level and contagious transmission for true positives (detected and undetected). the sensitivity and specificity for each test were used in the cost-benefit model as a basis for determining the expected costs and benefits of each test. the expected value of each cost or benefit was calculated by determining, from the sensitivity and specificity of each test, the positive and negative predictive values of each test. the expected value of a true positive outcome was calculated as the probability of a test identifying a true positive case multiplied by the cost or benefit of that outcome, and similarly for other outcomes. costs and benefits of correct or incorrect diagnoses were calculated at the cow level (swinkels et al. 2005). this model was applied separately for each of the two cow-side tests, as well as for tests used in parallel in order to determine which of the methods were more beneficial, compared to the gold standard criteria used. a comparison of the value of benefits that could be expected from a given test, based on the cost-benefit model, was used to estimate the value of the mastitis-control programme, using the cmct test, the mer test or a combination thereof (smith 1995). model input variables were based on current south african economic conditions and on literature (keefe 1997; lactodata 2011). the accuracy of the mastitiscontrol programme in correct diagnosis of mastitis using the cmct, mer and the tests in parallel was compared to the gold standard used. statistical analysis was performed using microsoft excel® 2003, by taking the model inputs and prices calculated (see below) into account (dohoo et al. 2003). prices used for model inputs cost of treatment (r45.45) was calculated using the cost of curaclox lc (norbrook (ark ah) po box 10698, centurion 0046), which is commonly used (r15.15 per treatment x three treatments). milk loss as a result of treatment (r360.86) was calculated using the current milk price at the time of the investigation (r2.97/l) x the average milk production per cow per day (27 l) (lactodata 2011) x the number of days for which milk was discarded (4.5 days). this was calculated as the sum of the three treatments of curaclox lc (norbrook (ark ah) po box 10698, centurion 0046) at 12 hourly intervals (1.5 days) and the (72h or three day) withdrawal period of the product after final infusion, for which milk was discarded. the cost of cmct test was calculated by calculating the costs of the following: mastest reagent per test = price of mastest per 5 l (r490)/number of tests per 5 l (888.33) = 0.55 number of 6ml cmct tests done per 5 l = (1000 ml/6 ml per test = 166.66 tests) x 5 l = 888.33 the cost of the cmct paddle = r60/number of tests per 5 l (r888.33) = r0.068 pump bottle = r30/number of tests per 5 l (r888.33) = r0.034 marking pen = r20/number of tests per 5 l (r888.33) = r0.023 the cost of the cmct per cow (r2.70) was calculated using the sum of the cost of mastest reagent per cmct (r0.55), the cost of the cmct paddle (r0.068) per test, the cost of the pump bottle (r0.034) per test and the cost of the marking pen (r0.023) used for calibration per test x four quarters per cow. the cost of the mer test per cow (r5.36) was calculated by the sum of the cost of the handheld mast-o-test meter (r2500/1860 tests = r1.34 per test) (csi-africa 2011, product specifications) x four quarters. cost-benefit model inputs for each test, the cost and benefit of each possible test outcome was calculated. for each possible outcome, positive effects were calculated as benefits and negative effects as costs. for all calculations, the cost of performing the test was included. this model was calculated over the 305 day lactation period for cows infected with host-adapted organisms (keefe 1997). the following four scenarios were analysed for cmct, mer and using the tests in parallel. true positive cases (sensitivity) the benefit of detecting true positive cases of mastitis with cow-side tests was calculated as follows:(culling cost (r6000) x 13% reduced infection rate [19]) + reduced loss of milk (r360.86) – treatment cost (r45.45 x 87%) [19] = r1101.32 (table 1) false negative cases the cost of not detecting true positive cases of mastitis by the cow-side tests was calculated as follows:estimated loss of milk yield due to sub-clinical mastitis (r360.86) x 13% increased infection rate [19] = r46.91 true negative cases (specificity) the detection of a quarter with normal udder health by the test is beneficial and has no cost implications. false positive cases the cost of misdiagnosing a healthy quarter with the test was calculated as follows:the cost of unnecessary treatment (r45.45) + the consequent loss of milk (r360.86) = r406.31 for a given outcome, the expected benefit of the outcome was calculated by multiplying the net benefit with the likelihood of attaining the outcome (i.e. false positive, false negative, true positive, true negative) through the use of this test. the overall cost of the test per cow was subtracted from the expected benefit for all scenarios, since a test is always conducted. benefit-cost ratios benefit-cost ratios were calculated as follows (for each test done separately and for both tests combined):benefit-cost ratios = expected benefit or (expected cost of error + cost of test). results and discussion top ↑ long periods of infection and bacterial shedding due to misdiagnosis create a prolonged window of opportunity for clinical mastitis to develop and for contagious transmission to occur. in addition to dry-cow treatment, treatment of clinical mastitis is part of most mastitis-control programmes. indirect effects of mastitis treatment, such as prevention of repeated clinical mastitis and transmission to other cows, are often overlooked in cost-benefit analysis, but render a specific treatment as being more favourable (waage & aursjoe 1993; lam 1996; hamman & zecconi 1998; zadoks et al. 2002; st rose et al. 2003; swinkels et al. 2005). table 1: a partial-budget analysis using a cost-benefit model to estimate benefits and costs of the cmct, mer test done separately and in parallel. table 2: summary of the cost-benefit model showing expected costs and benefits and the cost-benefit ratio of the cmct and mer test done separately and in parallel, taking cost of test per cow into account. the sensitivity of cmct and the parallel combination of cmct and mer tests are both equal to 1, proving that these methods of diagnosis are equally able to identify true positive animals in the mastitis-control programme, with the expected benefit of test per cow being r1101.32 (table 1). this is a surprising result, as it was expected that the combination of the two tests (measuring two mastitis indicators) should have had an advantage over one test (measuring only one mastitis indicator). the sensitivity of the mer test of 0.92 (table 1), show that it is slightly less able to detect true positive animals in the mastitis-control programme than the cmct test or the tests done in parallel. the specificity of cmct, mer test and the tests done in parallel were 0.66, 0.62 and 0.87 respectively (table 1), indicating that the tests done in parallel were the most beneficial for detecting true negative animals. it also indicated that the cmct was more beneficial than the mer test for the same purpose. the probability of diagnosing a false negative case using the cmct and the tests done in parallel were 0, indicating that these methods were more beneficial in mastitis diagnosis than the mer test (p = 0.08) (table 1), with the cost of error per cow being estimated at r46.91 (table 1). the probability of a false positive case using the mer test was higher than both that of cmct on its own and the tests done in parallel, indicating that the mer test was more likely to misdiagnose true negative animals, with the cost of error per cow being estimated at r406.31 (table 1). partial-budget analysis was used in order to estimate benefits and costs to producers of a mastitis-control programme. the part of the enterprise budget affected by mastitis was separated out so that the effects of mastitis were not overshadowed by some other factor or disease (smith 1995). a cost-benefit model was used as a method for calculating the cost-benefit ratio, which was an index of the rand value of benefits that could be expected from an investment with a given cost associated (smith 1995). the cost-benefit model was also used to calculate the expected costs and benefits of a specific scenario. the sum of the total expected cost of errors per cow (table 1) and the cost to conduct the test per cow (table 2) was subtracted from the expected benefits of test per cow for cmct, mer test. the tests were performed in parallel in order to calculate the total expected benefit of the tests (table 1). the cmct was shown to be more beneficial than the mer test, with a total expected benefit of test per cow of r1228.64 as opposed to that of r1100.29 (table 2). the most beneficial method for diagnosing mastitis in the mastitis-control programme was using both cow-side tests in parallel, with a total expected benefit of test per cow of r1393.25 (table 1). the conclusions made in this study were consistently made on the evaluation of an expected benefit. however, benefit-cost ratios were calculated as being 9.72 for cmct, 7.74 for mer and 24.02 for both tests in parallel (see table 2). conclusions top ↑ the gold standard used for mastitis diagnosis in this study was a scc ≥ 400 000 cells/ml milk and the presence of pathogens (petzer et al. 2009), which can be both costly and time consuming. a cost-benefit model was used to estimate the accuracy of the mastitis-control programme, by comparing the costs and benefits that arise from using the cmct, mer test, or both tests in parallel. misdiagnosis of true mastitis cases by cow-side tests increases the duration of infection when treatment of mastitis is postponed. when used separately, the expected benefit of cmct is 11% more beneficial than the mer test in diagnosing mastitis. the cost-benefit model indicates that, by using the cmct and mer tests in parallel, the total expected benefit of test per cow is the highest and the expected cost of error per cow the lowest. the total expected benefit of both tests in parallel is 13% more than that of the mer alone, and 11% more than that of the cmct alone. the sensitivity and specificity for the cmct test is higher than that of the mer test and similar to that of the two cow-side tests used in parallel. the cost-benefit ratio of using both tests in parallel is 2.5 times that of the cmct test alone, and 3.1 times that of the mer test alone. with all parameters considered, and taking all costs and benefits into account it is clearly beneficial to use both test in parallel. further research is recommended to analyse the data collected from in-line ec meters based on cow samples, comparing the accuracy of the results to cmct results and a combination thereof against the gold standard. future work with respect to modelling and sensitivity analysis is required in order to assess the extent of reduction in infection rates that should be maintained by control programmes in order to ensure that the process of testing and control of yield has a financially viable outcome. acknowledgements top ↑ the authors thank the dairy producers used for their participation and the staff of the milk laboratory, department of production animal studies, university of pretoria, onderstepoort for the analysis of the samples. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors contributions j.k. drafted the manuscript and critically revised it, formatted and edited the manuscript, was involved in the data analysis, calculating the sensitivity and specificity, as well as assisting in calculating the cost-benefit model and determining the model inputs for the partial-budget analysis. i.m.p. assisted in collection of aseptic milk samples and performing of cow-side tests, assisted with model inputs, reviewing the technical aspects of the manuscript. i.a.m. was involved in calculating the cost-benefit model, doing all the excel calculations, calculating the model inputs and technical editing of the article. t.j.v.d.s. was mainly involved in collecting of aseptic samples and performing of all the cow-side tests in the dairy, as well as in editing the manuscript. references top ↑ deluyker, h.a., van oye, s.n. & boucher, j.f., 2005, ‘factors affecting cure and somatic cell count after pirlimycin treatment of subclinical mastitis in lactating cows’, journal of dairy science 88, 604–614. http://dx.doi.org/10.3168/jds.s0022-0302(05)72724-7 dohoo, i., martin, w. 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the profit from strategies to reduce the prevalence of staphylococcus aureus mastitis’, preventive veterinary medicine 33, 183–193. http://dx.doi.org/10.1016/s0167-5877(97)00054-8 article information authors: carmen t. jacobs1 clarke h. scholtz1 affiliation: 1department of zoology and entomology, university of pretoria, south africa correspondence to: carmen jacobs email: ctjacobs@zoology.up.ac.za postal address: private bag x20, hatfield 0028, south africa dates: received: 07 aug. 2014 accepted: 09 dec. 2014 published: 16 apr. 2015 how to cite this article: jacobs, c.t. & scholtz, c.h., 2015, ‘a review on the effect of macrocyclic lactones on dung-dwelling insects: toxicity of macrocyclic lactones to dung beetles’, onderstepoort journal of veterinary research 82(1), art. #858, 8 pages. http://dx.doi.org/10.4102/ojvr.v82i1.858 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a review on the effect of macrocyclic lactones on dung-dwelling insects: toxicity of macrocyclic lactones to dung beetles in this review article... open access • abstract • introduction • published studies    • ivermectin       • lethal and sublethal effect studies       • survival and reproduction studies       • dung decomposition studies       • community structure studies       • dung attractiveness studies    • eprinomectin and doramectin    • moxidectin    • comparative studies: comparison of two products    • comparative studies: comparison of all four products • effect of routes of administration on faecal concentration • the way forward • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ avermectins and milbemycins are commonly used in agro-ecosystems for the control of parasites in domestic livestock. as integral members of agro-ecosystems with importance in maintaining pasture health through dung burial behaviour, dung beetles are an excellent non-target bio-indicator taxon for examining potential detrimental effects of pesticide application. the current review focuses on the relative toxicity of four different anthelmintics (ivermectin, eprinomectin, doramectin and moxidectin) in dung residues using dung beetles as a bio-indicator species. one of the implications of this review is that there could be an effect that extends to the entire natural assemblage of insects inhabiting and feeding on the dung of cattle treated with avermectin or milbemycin products. over time, reduced reproductive rate would result in decreased dung beetle populations and ultimately, a decrease in the rate of dung degradation and dung burial. introduction top ↑ the importance placed on anthelmintics to bring parasite populations under control has resulted in a challenging arms race to develop a product that exhibits the perfect balance between host and non-target organism toxicity and pest resistance. the need for more effective products is becoming increasingly important because pest resistance appears to be keeping pace with the development of new products. pest resistance is arguably one of the top challenges as far as protecting livestock is concerned and probably the main driving force behind parasite control research in the livestock industries (sangster 1999; wolstenholme et al. 2004) as it has been reported in many countries, in a variety of nematodes and against all currently available anthelmintics (sutherland & leathwick 2011). anthelmintics, which control helminth pests by removing them, are grouped according to their common chemistry and mode of action (sangster & dobson 2002; vercruysse & rew 2002). currently, the avermectins (ivermectin, eprinomectin and doramectin) and the milbemycins (moxidectin), collectively known as macrocyclic lactones, are amongst the most effective anthelmintics on the market. the avermectins are naturally produced by strains of a soil-dwelling actinomycete, streptomyces (burg et al. 1979; shoop & soll 2002). all the avermectins have a unique pharmacophore that consists of a 16-membered macrocyclic lactone backbone (shoop & soll 2002) with a disaccharide chain at c-13 (steel 1993; vercruysse & rew 2002). although the avermectins are a glycosidic derivative of the pentacyclic 16-membered lactone (albers-schoenberg et al. 1981; chabala et al. 1980), they do not possess the antifungal and antibacterial properties associated with the macrolide antibiotics (albers-schoenberg et al. 1981; burg et al. 1979; chabala et al. 1980). they act by interfering with invertebrate neurotransmission rather than inhibiting protein synthesis (albers-schoenberg et al. 1981; chabala et al. 1980). ivermectin was the first avermectin to be introduced in 1981 (steel 1993; vercruysse & rew 2002). ivermectin (22, 23-dihydroavermectin) is a disaccharide derivative of the pentacyclic 16-membered lactone (burg et al. 1979; campbell 1985; chabala et al. 1980; römbke et al. 2010). the antiparasitic effect of ivermectin is extremely potent against insects, nematodes and acarines (campbell 1985; putter et al. 1981). although potent, ivermectin is not equally active against all species and is often highly stage specific (campbell 1985), so that a genus known to be susceptible to ivermectin may not be susceptible at all life stages (campbell & benz 1984). abamectin, a combination of 80% avermectin b1a and 20% avermectin b1b, is the starting material for ivermectin. it is effective against nematodes as well as acarines and to date remains the only avermectin or milbemycin to be used in both the animal health and crop industries (shoop, mrozik & fisher 1995). eprinomectin was introduced to the animal health industry in 1997 as an alternative to ivermectin as it was considered to be the only topical endectocide safe for use in lactating dairy animals (shoop et al. 1996b; vercruysse & rew 2002). although ivermectin has no side-effects on the host and has a very broad spectrum of activity, with few exceptions it cannot be used in lactating dairy animals because of the levels of residue that remain in the milk (shoop et al. 1996a, 1996b; vercruysse & rew 2002). doramectin was commercialised in 1993 (vercruysse et al. 1993) and is the easiest avermectin to administer. in a study by grandin, maxwell and lanier (1998), it was found that doramectin caused significantly less discomfort during administration than ivermectin. the milbemycins, although structurally similar and with a similar range of biological activity to the avermectins, differ in substituents in a few of the side chains at the c-13 position and can basically be considered to be deglycosylated avermectins (sangster & dobson 2002; steel 1993; vercruysse & rew 2002). although they were discovered in 1973, before the discovery of ivermectin, they were originally developed for use in crop protection and have been used in veterinary practice from about 1986 only (mckellar & benchaoui 1996; takiguchi et al. 1980). moxidectin, the only milbemycin available on the market as an endectocide, was introduced in 1989 and commercialised worldwide by the early 1990s (mckellar & benchaoui 1996; steel 1993). the milbemycins are highly lipophilic (moxidectin is about 100 times more lipophilic than the avermectins), soluble in organic solvents and insoluble in water, and after an initial increase in its plasma concentration post-administration, it is redistributed throughout the body fat reserves, from which it is slowly released (mckellar & benchaoui 1996). various studies have shown that a characteristic of the avermectins, regardless of the animal or method of administration, is that most of the dose is excreted largely unaltered in the dung, where it retains its insecticidal activity (campbell 1985; steel 1993; strong 1993; wardhaugh & rodriguez-menendez 1988). this is the focus of the present review. published studies top ↑ numerous laboratory and field studies have been undertaken on the effects of avermectins and milbemycin in cattle dung on non-target organisms and on their effects on different aspects of dung beetle biology. countries with large cattle populations were chosen based on food and agriculture organization of the united nations, statistic division (faostat)'s live animal production database (food and agriculture organization of the united nations [fao] 2013). although the methods used were different in each country and changed somewhat over the years, the results have remained more or less consistent. ivermectin ivermectin is the most extensively studied of all the avermectins. the first study that set the scene for interest in the field was that of wall and strong (1987), who conducted an experiment in the uk to investigate the environmental consequences of treating cattle with ivermectin. in contrast to the control dung pats, the experimental pats contained few to no coleoptera or diptera. the results also indicated that there was no visible dung degradation in the ivermectin-treated dung when compared to the controls. this field trial showed that treatment with a ruminal bolus that delivers 40 µg/kg ivermectin per day was enough to disrupt the entire dung-inhabiting insect community. various subsequent studies have simulated or repeated this experiment with variable results. lethal and sublethal effect studies lumaret et al. (1993) studied the effects of ivermectin residues on dung beetles by running a field trial on a farm in spain in spring. dung toxicity was assessed by recording the mortality of the dung beetles feeding on the dung. in addition, the numbers of larvae and pupae were recorded after 29 days. no adult mortality was recorded for the duration of the study but 100% larval and pupal mortality was observed in dung collected on the day after treatment. no differences in offspring numbers between treated and untreated dung were observed from day 6 onwards. a delay in development was observed for beetles bred in treated dung when compared to the control offspring. pitfall traps baited with dung collected 10 and 17 days after treatment were similarly attractive with treated and untreated dung for the first 3 days, and then a peak of attraction occurred between days 4 and 6, when the dung was most attractive and still relatively fresh. from day 6 onwards, the attraction to the treated dung persisted for 30 days whilst the untreated dung became unattractive after day 7. lumaret et al. (1993) proposed that increased attractiveness is a result of biochemical modifications in the dung composition, most likely as a result of protein degradation released by ivermectin therapy. krüger and scholtz (1997) ran a laboratory trial to determine the lethal and sublethal effects of ivermectin residues in dung from animals treated with a single standard injection of ivermectin at 200 µg/kg. laboratory colonies of euoniticellus intermedius were provided with 250 ml of dung twice a week for 2 weeks and monitored for adult mortality as well as for brood ball numbers. brood balls were counted, removed and incubated to monitor for emergence. no results regarding adult survival were reported. there was no significant difference between treated and control populations in the number of brood balls formed; however, on average, the number of adults emerging from treated brood balls was significantly lower than in the controls (similar findings were obtained by fincher [1992]). ivermectin caused 100% mortality in offspring 2–7 days after treatment and significantly fewer emergences from day 14 after treatment when compared to the controls. prolonged development in treated broods (similar to the findings of lumaret et al. [1993]) was also recorded, roughly 2.5 times longer for dung collected 1, 7 and 14 days after treatment and a larval developmental time of 5 weeks compared to the control of 3.5 weeks for dung collected 28 days after treatment. survival and reproduction studies ridsdill-smith (1988) studied the effect of ivermectin on the survival and reproduction of the dung beetle onthophagus binodis in australia. ivermectin had no influence on adult dung beetle survival. immature survival, however, was zero for week 1 after treatment but steadily rose to equal that of the other anthelmintic by week 8 after treatment. there was no untreated control. fincher (1992) compared the effect of 20 µg/kg and 200 µg/kg ivermectin on some dung-inhabiting insects, including the introduced african dung beetle e. intermedius in texas, usa. the results revealed that neither dosage had any significant effect on adult survival, as described by ridsdill-smith (1988) and wardhaugh and rodriguez-menendez (1988), or brood ball production when compared to the controls; however, emergence of adult e. intermedius from brood balls made with dung from cattle that received 200 µg/kg ivermectin was reduced for no more than 2 weeks after treatment (fincher 1992). cruz rosales et al. (2012) evaluated the effect of ivermectin on the survival and fecundity of e. intermedius adults as well as on the survival and development of e. intermedius from egg to adult in mexico. they found that at low concentrations (10 µg/kg) the ivermectin had no effect on the survival or fertility of the adults or on the survival of the larvae, but they did record an increase in the larval development time. at the medium concentration (1 mg/kg) the survival of adults was reduced to almost half and no larvae emerged. at the highest concentration (100 mg/kg) 100% mortality was observed and no oviposition was performed. they concluded that the prolonged development time may cause a phase lag in the field activity cycle, which may reduce the number of e. intermedius individuals and the efficiency of the environmental services that they provide, and that more analyses with higher concentrations between 0.01 ppm and 0.1 ppm of ivermectin are needed to establish lethal concentrations for larvae and adults of e. intermedius. dung decomposition studies wardhaugh and rodriguez-menendez (1988) studied the effect of ivermectin on the development and survival of the dung beetles copris hispanus, bubas bubalus and onitis belial in southern spain. the results showed no adult mortality, reduced egg-laying and reduced juvenile survival as described by ridsdill-smith (1988). a marked reduction in adult feeding activity was observed in treatments suffering the highest mortalities, namely day 1–8 dung, and the inference was made that mortality was a result of the accumulating toxic effects, which suppressed feeding (wardhaugh & rodriguez-menendez 1988). whilst this study was aimed at the development and survival of the dung beetles, a decrease in the rate of dung decomposition as a result of reduction in adult feeding activity was observed. madsen et al. (1990) conducted field as well as laboratory experiments in denmark to show how treating cattle with a single therapeutic ivermectin injection affected the fauna and decomposition of dung pats. the results from the field trial showed that ivermectin had an effect on beetle larvae 1–10 days after treatment but that the number of larvae was not affected by ivermectin applied 20–30 days before collection. the decomposition rate was significantly delayed when compared to control dung but also depended on variables such as climate, season, soil type, faunal inhabitants and microclimate. the results from the laboratory bioassays showed a 95% – 100% mortality rate in musca domestica as well as musca autumnalis for dung collected one day after treatment. there was no clear reduction in excreted ivermectin placed in the field for 7–62 days and the 62-day assay was obscured by natural mortality. most of the variance found in this experiment was attributed to seasonal conditions. sommer et al. (1992) ran a field trial in denmark to assess the impact of ivermectin residues on dung fauna and the resulting effect on dung degradation. according to the arthropods found in the treated dung, there was no significant difference between the residues found in the pour-on and injectable formulations even though the pour-on formulation was 2.5 times the dose of the injectable formulation; however, dung collected from cattle 1–2 days after treatment with the injectable formulation showed delayed dung degradation for up to 45 days but no effect was observed on dung collected 13–14 days after treatment. dung collected from cattle 1–2 days after treatment with the pour-on formulation led to delayed dung degradation for up to 13–14 days after treatment, which was a similar result to that of wardhaugh and rodriguez-menendez (1988) and madsen et al. (1990). iglesias et al. (2011) evaluated the local effects of ivermectin on dung fauna and degradation under different meteorological and biological conditions in the same area in argentina in 2011. the results showed that fewer arthropods were found in the dung of the calves treated with ivermectin, but the difference was not statistically significant. community structure studies krüger and scholtz (1998a, 1998b) conducted a large-scale field study to determine the ecotoxicological effect of ivermectin on the dung beetle community structure under drought and high rainfall conditions. the results showed a large effect on the dung beetle community in the form of significantly lower species richness and evenness as well as increased species dominance in treated dung during drought conditions (krüger & scholtz 1998a). during high rainfall conditions, however, fewer beetle and fly larvae were found in the pats after 7 days, but no effect of ivermectin was detected after a year (krüger & scholtz 1998b). this suggests that these ecotoxicological effects are likely to be more severe in times of drought than under more favourable conditions. kryger, deschodt and scholtz (2005) carried out a long-term, large-scale field study in south africa to assess the effect of ivermectin on the structure of dung beetle communities. no observable effects of ivermectin on the dung beetle communities were found, as the disparities between treated and untreated dung were insignificant and most probably a result of differences in microclimate. species richness and diversity were also unaffected and ecologically similar to the control communities. this study showed that treatment with ivermectin under extensive farming conditions in the south african highveld can be considered safe with regard to the dung beetle communities under high rainfall conditions. strong et al. (1996) carried out a comparative field trial to examine the effects of ivermectin and fenbendazole boluses on dung-colonising diptera and coleoptera in the uk. although there were no significant differences in adult beetle numbers between the treated and untreated dung, not only was there a significant difference in larval and pupal numbers between the ivermectin and fenbendazole treated and untreated dung, but the larvae found in the ivermectin-treated dung were inhibited in their development. pitfall trapping showed no significant difference in adult beetle numbers between treated and untreated dung, although a trend towards higher numbers of beetles attracted to the treated dung was noted. römbke et al. (2010) carried out a field study in spain to determine the effects of ivermectin on the structure and function of dung and soil invertebrate communities. they observed a significantly lower abundance of adult dung beetles on the dung from cattle treated with ivermectin compared to the control group. they also noted that although adult dung beetles were attracted to the ivermectin-spiked dung, the rate of degradation was slower than for the control dung. dung attractiveness studies errouissi and lumaret (2010) studied the effects on the attractiveness to dung beetles of dung treated with ivermectin. they found that the ivermectin-contaminated dung showed a significant attractive effect, which highlighted the danger of wide-spread ivermectin use as this potentially puts the dung beetles’ offspring and, indirectly, future beetle generations’ survival at risk. eprinomectin and doramectin only comparative studies involving the effect of these products on dung beetles were available and are discussed in the next section, but two studies involving effects on other taxa are briefly described. lumaret et al. (2005) examined the larvicidal activity of eprinomectin residues on the dung-inhabiting fly neomyia cornicina in france and found that eprinomectin residues in dung had a significant effect on n. cornicina as no emergences were observed on the dung from days 1–11 but after day 12 the first flies emerged. floate et al. (2008) addressed concerns raised about the use of endectocides affecting birds that feed on dung-breeding insects by testing the toxicity of faecal residues after doramectin treatment. a significant reduction in insect emergence was noted for dung from cattle treated ≤ 4 weeks prior, which was attributed to higher concentrations of the residues. moxidectin fincher and wang (1992) tested the effects of moxidectin on two introduced african species of dung beetle, namely e. intermedius and onthophagus gazella. they found no significant differences between the mean number of brood balls produced by either species or on the emergence of progeny between treated and untreated dung. there also seemed to be no effect on the sex ratio for either species. they concluded that moxidectin seemed to be compatible with beneficial dung-burying beetles when used at the recommended dose. iwasa, suzuki and maruyama (2008) examined the effects of moxidectin on non-target coprophilous insects, more specifically the dung beetle caccobius jessoensis, in cattle dung in field as well as laboratory trials in japan. the results showed that concentrations were at maximum levels 3 days after treatment, showed a marked decline by day 7 and were not detectable by day 21. no significant differences were found between the control and the treated cattle dung with regard to numbers and weight of brood balls as well as emergence rates. results of the field study, again, showed no significant differences between the control and the treated cattle dung. they concluded that moxidectin has no, or at most, the least effect compared to other avermectins on non-target coprophagous insects. comparative studies: comparison of two products comparative studies have been undertaken between ivermectin and doramectin (dadour 2000; suárez et al. 2003; webb et al. 2010); ivermectin and moxidectin (doherty et al. 1994; strong & wall 1994); moxidectin and doramectin (suárez et al. 2009) and moxidectin and eprinomectin (wardhaugh, longstaff & morton 2001). dadour (2000) examined the impact that abamectin and doramectin have on the survival and reproduction of the dung beetle o. binodis. this study was carried out in australia and abamectin, rather than ivermectin, was chosen because it was the first avermectin sold commercially for the treatment of endoparasites in australia. significant adult mortality was observed in abamectin-treated dung 3–6 days after treatment and in doramectin-treated dung 9 days after treatment. whereas abamectin residues had no effect on adult mortality in sexually mature beetles, sexually immature (newly emerged) beetles, which went through a period of intense feeding during which they were exposed to maximum abamectin residues, were found to be much more affected by the residues. in contrast to other studies (fincher 1992; krüger & scholtz 1997), brood ball production was also significantly lower in beetles fed on dung from cattle treated with abamectin for up to 42 days after treatment. brood ball production was also significantly lower in beetles fed on dung from cattle treated with doramectin, but only for 3–6 days after treatment. the enhanced brood mass in beetles fed on dung from doramectin-treated cattle at 24–34 days after treatment could not be explained. according to the high-performance liquid chromatography (hplc) results, doramectin reached maximum concentration on day 3 after treatment, following a linear decline, with an elimination half-life of 15 days (dadour 2000). suárez et al. (2003) compared the effects of ivermectin and doramectin on the invertebrate colonisation of cattle dung in argentina. no significant differences were found in the numbers of adult beetles, regardless of the treatment. faecal residue concentrations for both ivermectin and doramectin were highest in the first few days and remained relatively high throughout the experimental period. doramectin concentrations were higher than ivermectin concentrations, as the results showed that after 180 days of exposure to environmental conditions, dung collected 27 days after ivermectin treatment still contained 56% residue compared to dung collected from doramectin treatment, which contained 75% residue. webb et al. (2010) assessed the abundance and dispersal of dung beetles in response to ivermectin and doramectin treatment on pastured cattle in scotland by running a 2-year field trial. in the field-scale study, significantly more beetles were trapped in fields grazed by cattle treated with an avermectin than in fields where cattle remained untreated. the colonising trials, however, indicated that aphodius beetles preferred colonising dung from untreated cattle rather than dung from cattle treated with doramectin and could discriminate between dung from untreated cattle and dung from cattle treated with doramectin at a spatial scale of at least 70 m. doherty et al. (1994) compared the larvicidal activities of different concentrations of moxidectin and abamectin on o. gazella to assess the level of threat they pose to dung fauna, and consequently dung degradation, in australia. although oviposition was not affected by either treatment, larval survival was affected by all concentrations of abamectin and by all concentrations of moxidectin over 128 µg/kg. in fact, moxidectin at 256 µg/kg and 512 µg/kg produced survival comparable to 4 µg/kg and 8 µg/kg abamectin. strong and wall (1994) compared the relative effects of ivermectin and moxidectin on the colonisation of dung by dung-inhabiting insects in england. there was no significant difference between the three treatments in adult scarabaeidae numbers showing that neither ivermectin nor moxidectin residues repel colonising adult beetles. however, dung collected from ivermectin-treated cattle up to 7 days after treatment showed high larval mortality, unlike moxidectin-treated dung and the control. suárez et al. (2009) demonstrated the effects of moxidectin and doramectin faecal residues on the activity of dung-colonising insects by depositing dung from cattle treated with moxidectin, dung from cattle treated with doramectin and control dung from untreated cattle on a field. comparisons of dung degradation were inconclusive; however, total numbers of insects recovered from control pats were significantly higher than in treated pats. furthermore, a lower adverse effect was observed for moxidectin compared to doramectin with no significant degradation of moxidectin or doramectin observed. wardhaugh et al. (2001) compared eprinomectin to moxidectin by examining the survival and development of onthophagus taurus when fed on dung from treated cattle in australia. the results showed that moxidectin had no effect on the survival or development of the beetles but the opposite was found to be true for eprinomectin. high juvenile mortality and suppressed brood ball production amongst those that survived were recorded. they concluded by designing a model that simulated the effects of eprinomectin residues and suggested that a single treatment of eprinomectin is capable of reducing the next generation by 25% – 35%. comparative studies: comparison of all four products two laboratory studies provided comparative results amongst ivermectin, moxidectin, doramectin and eprinomectin but they were performed under different laboratory conditions (floate 2007; floate, colwell & fox 2002). floate (2006) also wrote a review about the global environmental effects of faecal residues left by treatment of cattle with ivermectin, doramectin, moxidectin and eprinomectin on non-target dung-inhabiting species. pour-on formulations of ivermectin, doramectin, eprinomectin and moxidectin were applied to four groups of heifers in canada at the recommended dose of 500 µg/kg and dung was collected 1, 2, 4 and 6 weeks after treatment. artificial dung pats were then randomly deposited in a block design in a pasture adjacent to grazing cattle and collected again after 8 days to analyse insect populations. to monitor dung beetle activity, dung-baited pitfall traps were placed in the centre and at either end of the study site. based on the number of species affected and duration of suppression, the results showed that treatment of cattle with doramectin, ivermectin, eprinomectin or moxidectin, in descending order of adverse effect and reduced levels of insect activity in the dung but moxidectin was the least likely to affect the natural insect assemblage associated with cattle dung (floate et al. 2002). floate (2006) raised concerns that the use of endectocides in cattle may reduce the insect diversity in canada and lead to the accumulation of undegraded dung on pastures as a result of reduced insect activity required for dung pat degradation. floate (2007) also compared the field effects of ivermectin, doramectin, eprinomectin and moxidectin residues on the attractiveness of dung to dung-colonising insects over 3 years in canada. pitfall traps were set in spring and autumn and re-baited weekly for a month in each season. insect captures were compared between pitfall traps baited with dung from untreated cattle and dung from cattle treated with doramectin, eprinomectin, moxidectin or ivermectin at the recommended dose of 500 µg/kg. twofold and up to sixfold differences in captures between control and treated dung were observed. more specifically, 11 out of 29 cases of attraction and 11 out of 29 cases of repellence were recorded for doramectin, eprinomectin tended to repel insects, with 19 out of 29 cases of repellence, whilst ivermectin (17 out of 25 cases) and moxidectin (17 out of 18 cases) showed a strong attractive effect. floate (2007) concluded that emergence of offspring from field-colonised dung should not be used as a measure of residue toxicity; standardised laboratory tests should still be the preferred method, but rather as a measure of ‘insect activity’, which is a composite measure of residual toxicity, the number and species composition and the mortality factors such as predation, competition and parasitism. effect of routes of administration on faecal concentration top ↑ there are a variety of ways to administer avermectins to cattle, namely subcutaneously by injection, topically in the form of a pour-on and orally in various forms. lumaret et al. (2005) determined the faecal concentrations of pour-on eprinomectin in cattle following treatment at the recommended dose of 500 µg/kg by using hplc. the maximum faecal concentrations were recorded 3 days after treatment. eprinomectin remained detectable in the faeces until 29 days after treatment. lumaret et al. (1993) measured ivermectin concentrations in dung from cattle treated with a single dose of injectable ivermectin at the recommended dose rate of 200 µg/kg by using hplc. chemical analysis of the ivermectin concentration in fresh dung indicated that it increased daily on days 1–4 after treatment, reaching a peak of elimination on day 5 followed by a rapid decrease until day 12, after which the concentration was under the detection limit. one would expect that the injectable formulations would be more effective than the pour-on formulation but this is not always the case. in the denmark field trial by sommer et al. (1992), the concentration of subcutaneously administered ivermectin was compared to the pour-on formulation of ivermectin using hplc. although there was no significant difference between the residue concentrations of the pour-on and injectable formulations, even though the pour-on formulation was 2.5 times the dose of the injectable formulation, the injectable formulation led to a longer period of delayed dung degradation than the pour-on formulation. herd, sams and ashcraft (1996) examined the persistence of ivermectin in faeces by comparing the faecal residues following different modes of administration, namely sustained-release (sr) bolus, pour-on and injectable formulations, in ohio, usa. they emphasised the importance of formulation and route of administration in drug concentration determination, persistence and ecotoxic potential. all faecal concentrations recorded, regardless of mode of administration, were well above concentrations that are lethal or sublethal to beneficial dung-breeding invertebrates. they concluded by stating that the sr bolus and pour-on formulations are likely to be more ecotoxic to non-target organisms than the injectable formulation judging from their higher faecal concentrations, and that the sr bolus formulation is of particular concern because of the persistent excretion of toxic concentrations for prolonged periods of time. the way forward top ↑ most recently, wall and beynon (2012) wrote a review on the impact of macrocyclic lactone parasiticides. they reported that macrocyclic lactone residues from parasiticide treatments may play an important role in the loss of coprophilous insects, which may in turn delay pat decomposition. they added that field studies have provided contradicting results that reflect confounding factors such as weather conditions, pat moisture content, pat location, time of year, dung insect species phenologies, timing and method of application. these factors are important in determining whether the results obtained from experimental and laboratory studies reflect the real impact on the economically important process of dung decomposition. the timely removal of dung from pastures by insects and weathering is both functionally and economically important; if appropriate decomposition does not occur, cattle farmers may suffer considerable economic losses as a result of pasture fouling, increases in dung-breeding pest fly populations and a higher transmission of livestock endoparasites. the benefits of rapid dung removal are therefore rather substantial; not only does it reduce such losses, but it helps to return nutrients to the soil, particularly nitrogen, a large proportion of which would otherwise be lost as ammonia. conclusion top ↑ although it is difficult to recommend a control programme that will suit all forms and styles of livestock farming, a standardised procedure for the testing of antiparasitic remedies needs to be developed in order to accurately compare the toxicity of various products. the best scenario would be to farm holistically, minimising the need for pesticides. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions c.t.j. 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place. the problem of trypanosomosis, affecting both human and animal health, “lies at the heart of africa’s struggle against poverty” and dealing with this disease has the potential to impact on all eight millennium development goals—of the 37 countries with tsetse infestations, 21 are among the world’s 25 poorest (paat 2008). historical overview underpinning today’s analyses of the economics of dealing with tsetse and trypanosomosis are over five decades of research and experience. at this historic moment, celebrating the centenary of the onderstepoort veterinary institute, it seems appropriate to review this work and how it informs the present day dilemmas and decisions. all the more so, since the first large-scale successful tsetse eradication took place here, with the elimination of glossina pallidipes over 11 000 km2 from what was then zululand (du toit 1954). thus, from the late 1940s onwards into the 1950s and 1960s, the economic component of tsetse control work consisted of meticulous recording of the costs of operations. by this time, a number of approaches had been tried and tested for controlling or eliminating tsetse populations. describing bush clearing wilson (1953) records: “the total cost there fore for the clearing done in both 1949 and 1950 over most of the blocks was £200 per mile. the cost 27 onderstepoort journal of veterinary research, 76:27–32 (2009) assessing the economics of animal trypanosomosis in africa—history and current perspectives a.p.m. shaw a p consultants, upper cottage, abbotts ann, andover, sp11 7ba, united kingdom abstract shaw, a.p.m. 2009. assessing the economics of animal trypanosomosis in africa—history and current perspectives. onderstepoort journal of veterinary research, 76:27–32 finding appropriate ways of dealing with the problem of tsetse and trypanosomosis will be an important component of efforts to alleviate poverty in africa. this article reviews the history of economic analyses of the problem, starting with the use of cost to guide choice of technique for tsetse control in the 1950s, followed by work in the 1970s and 1980s linking these to the impact of the disease on livestock productivity, and in the 1990s to its wider impact. in the current situation, with limited resources and a range of techniques for controlling or eliminating tsetse, the cost implications of choosing one technique or another are important and a recent study reviewed these costs. a novel approach to assessing the potential benefits from removing trypanosomosis by creating ‘money maps’ showed that high losses from animal trypanosomosis currently occur in areas with high cattle population densities on the margins of the tsetse distribution and where animal traction is an important component of farming systems. given the importance of the decisions to be made in the next decade, when prioritising and choosing techniques for dealing with tsetse and trypanosomosis, more work needs to be done underpinning such mapping exercises and estimating the true cost and likely impact of planned interventions. 28 assessing the economics of animal trypanosomosis in africa of continuing this method of eradicating glossina palpalis was obviously prohibitive.” they then turned to “the use of 5 per cent ddt sprays on the fringing vegetation along 20 miles of the mbogo river” which “gave dramatic results and g. palpalis was eradicated at a cost of £42 per mile”. thus, by 1950, costs were being analysed in order both to inform choice of technique and to improve tsetse control methods. more systematic cost analyses began in the 1970s with consideration of full overheads and comparison of techniques. thankfully, the tsetse control methods used have changed: jahnke (1974) cites a “grand total of animals shot” between 1944 and 1970 in uganda of 161 867, a figure which is only marginally mitigated by the fact that the number shot per km2 ranged from 2–16 animals. furthermore, seen strictly in terms of government expenditure, game clearance came out better, at roughly half the cost of ground-spraying. the most detailed and com prehensive cost study was undertaken during the late 1980s and early 1990s by barrett (1997) in zim babwe, where so much ground-breaking research work into tsetse control methods was being undertaken. during this period a uniquely wide range of techniques was being used there: ground-spraying, aerial spraying, targets and insecticide-treated cattle (itc). barrett’s study emphasized the fact that for each technique costs varied greatly depending on terrain, fly species and the presence of cattle and quantified how these variables affected cost. the cost studies all highlight the difficulties of calculating tsetse control costs in a manner which enables comparisons to be made across ecosystems, tsetse species, project duration, currencies, tsetse control unit administrative structures and therefore countries and locations. in the early 1970s, benefit-cost studies became a mandatory component of all donor-funded and government projects. meanwhile, these difficulties in comparing costs across localities and projects with different objectives, led to people becoming increasingly aware that decision-making in the tsetse and trypanosomosis field would need to include a benefit component. the first published benefit-cost study on the subject was jahnke (1974), which looked at ground spraying and game clearance in relation to benefits from ranching in uganda, and concluded that given the costs of tsetse control by these methods plus land clearance for development, these schemes barely broke even. since then, numerous benefit-cost studies were undertaken throughout af rica, both in advance of work being undertaken and retrospectively following successful control or elimination. some of these are summarised in shaw (2004). they demonstrate that most interventions to deal with tsetse yield respectable benefit-cost ratios of around 3:1, provided that they are undertaken in areas where trypanosomosis seriously constrains cattle production and that they are sustained. by the 1980s it had become clear that the demands for benefit-cost studies needed to be underpinned by serious field work looking at the direct impacts of the disease on livestock productivity. a large number of studies were undertaken, comparing key parameters (herd structure, mortality, birth rates, mass gain and milk yield) in infected and non-infected individuals, herds and in locations with different levels of tsetse challenge, mostly studying cattle. these consistently showed that the disease has an impact on productivity, but the impacts vary hugely, even across similar locations. the results were summarised in swallow (2000) and updated in shaw (2004), with the following broad conclusions. calf mortality rates were 6–10 % higher in infected populations, but the difference could be up to 20 %. death rates in older animals were 2–8 % higher, but the difference can be negligible if try pan ocides are used effectively (doran 2000). annual calving rates were typically 7 % lower in infected pop ulations. the effect on milk yields was particularly difficult to measure, with impact ranging from a reduction of 2–26 %. in terms of its impact on agriculture, the disease’s impact on the use and effectiveness of draught power is crucial, but there is, in fact, very little numerical data on this. swallow (2000) estimated oxen in a high-risk area in ethiopia to be 38 % less efficient. throughout africa, farmers probably treat draught oxen more than any other category of livestock. for example, in the eastern province of zambia, doran (2000) reports them as being given nearly ten times as many curative trypanocide treatments as bulls and over 50 % more than cows. during the 1990s more work was done on the indirect effects of the disease on land use. in contrast to the ‘direct’ effects of the disease on livestock health, ‘indirect’ effects occur where the presence of tsetse affects people’s production methods by limiting their use of draught power, making it difficult to upgrade livestock breeds, avoiding grazing or settling in certain areas. again, the studies undertaken showed very variable responses to the challenges posed by tsetse infestation (reid & swallow 1998; doran 2000). for example, in some cases tsetse removal accelerated in-migration by farmers and livestock keepers; in others in-migration preceded tsetse control or took place despite the presence of tsetse. thus, following the sahel droughts of the early 1970s 29 a.p.m. shaw and 1980s zebu cattle keepers migrated southwards in large numbers, for example increasing cattle numbers six-fold in northern ivory coast and five-fold in the central african republic (shaw 2004). despite the presence of tsetse, draught power is extensively used in west africa, southern africa, ethiopia and uganda, but at a high cost in terms of trypanocides, lowered productivity and increased mortality. current perspectives faced with the closing of tsetse control departments, a gradual reduction in funding and interest, not just in tsetse control and animal trypanosomosis, but overall in livestock, african scientists set up the pan-african tsetse and trypanosomiasis eradication campaign (pattec). this was endorsed by the african union heads of state in their historic declaration in 2000 in lomé, which committed their countries to finding ways of dealing with the problem of tsetse and trypanosomosis. thus, the new millennium began with this pan-african initiative and with it a renewed need for economic inputs in order to analyse where, when and how best to intervene. refining the costs in order to inform choice of technique in this context, a study was undertaken based on the options for uganda (shaw, torr, waiswa & robinson 2007). this updated the approach used by barrett (1997), siting the estimates in uganda and using as a basis the control approaches being examined by pattec for tsetse elimination, including field studies and an administrative structure. the four methods were trap ping at various intensities, insecticide-treated cattle (itc) with insecticide applied by pour-on or by spraying, aerial spraying using the sequential aerosol technique (sat) and the use of the sterile insect technique (sit) following suppression using sat or sit. the gradual elimination of tsetse with the use of each technique was modelled using a tsetse population dynamics model (vale & torr 2005; http:// www.tsetse.org/). the results for a selection of scenarios are given in fig. 1. this shows that costs vary widely, both within techniques and across techniques. where sufficient cattle are present for itc to be used and the insecticide is applied by spraying, this can be the cheapest technique. aerial spraying is more expensive but can be effective within the shortest time period. where sit is considered a necessary component of a programme, following suppression of the tsetse population by another method, the costs increase substantially. the cost of traps is relatively high, because of the organisation and manpower required to deploy them and is dependent on the density required, itself largely a function the glossina species being targeted. in general terms, costs for elimination, in a straightforward situation of an isolated tsetse population, would thus range from 200–1,500 us$/km2. once non-isolated populations are considered, barriers against reinvasion are needed and costs increase. for example, maintaining barriers for 5 years with reinvasion pressure on one of four sides of a block would add between 20 % and 60 % to costs. if barriers are needed over a larger area, for a longer period, or if reinvasions occur, costs would increase further. these tsetse elimination scenarios need to be compared to the ongoing costs of controlling either the disease or the vector. fig. 2 shows a selection of these costs, estimated over a 20-year period and using the standard practice of discounting (cf. shaw et al. 2007) whereby monetary amounts received or spent in the future are weighted relatively lower than those spent or received in the present. these control approaches would obviously not eliminate the disease, so that some losses would still persist. for the use of trypanocides, the emergence of drug resistance remains a concern. the number of doses per km2 per year would be a function of the cattle population density and the number of doses being given—for example, 1.5 per head per year on average in eastern zambia (doran 2000). for this costing of control options, the costs of itc are all using sprays with the additional option of using spraying in restricted application (ra) to the legs and belly of cattle (bourn, grant, shaw & torr 2005). this method retains the efficacy of the other itc application methods while being substantially cheaper. like other itc methods it can also have an added beneficial effect in reducing tick burdens and the incidence of tick-borne diseases. therefore, in terms of decision-making it can be seen that in many situations effective levels of tsetse control, even if maintained over a long period, can be cheaper, or as cheap as, attempting to eliminate the fly. mapping the benefits the numerous benefit-cost studies had, over the years, demonstrated that for all the techniques developed, sustained tsetse and trypanosomosis control was profitable in specific localities. but, with the more global approach fostered by pattec, it has become necessary, firstly, to be very clear about where the priority areas for intervention are and, 30 assessing the economics of animal trypanosomosis in africa secondly, what levels of expenditure are justified by the losses due to trypanosomosis in different locations. the development of geographic information systems (gis) alongside remote sensing has dramatically increased the extent to which spatial variables can be analysed, linked and mapped. these techniques have much to offer epidemiology and animal health and more specifically in decision-support in the fields of tsetse and trypanosomosis (gilbert, jenner, pender, rogers, slingenbergh & wint 2001; hendrickx, biesemans & de deken 2004). the provision of maps of cattle populations and updated tsetse distributions already highlighted priority areas for intervention. accordingly a study was undertaken (shaw, hendrickx, gilbert, mattioli, codjia, dao, diall, mahama, sidibé & wint 2006) to try and bridge this gap, by producing a ‘money map’ of the impact of animal trypanosomosis for west africa. this study had three components. firstly, a new map of the main cattle breeds and production systems was pro duced. secondly, for each of the four main systems identified, the herd dynamics were modelled for ‘with’ and ‘without’ trypanosomosis scenarios and the differences in monetary output were calculated and projected over a 20-year period. these differences in output represent the losses imposed by the disease, or the potential benefits from its removal. they were then expressed as dollars per bovine currently present in the area. thirdly, a spatial expansion model was used to project where ‘with’ and ‘without’ trypanosomosis cattle populations would migrate over the 20-year period, in relation to the carrying capacity of the land. combining production systems, benefits per bovine and the expansion of cattle populations made it possible to produce a map showing the potential monetary benefits per km2, over 20 years, if trypanosomosis were removed. a section of this map is reproduced in fig. 3. this shows the potential benefits in us$/km2 ranging from under $500 to over $7,000 over 20 years (as with costs, benefits have been discounted at 10 %). this big range reflects, firstly, the cattle distribution and, secondly, the work oxen distribution. the highest levels of benefits, shown in dark green, are in the areas on the northern boundary of the tsetse distribution and there is a strong association with the cotton growing areas where there is intensive use of draught cattle, often partly trypanotolerant crossbred stock. further to the south, in the ‘red’ zones on the map, cattle population densities are much lower, many of the breeds are trypanotolerant so that although trypanosomosis does affect them, the disease usually presents in a less acute form. cattle are kept mainly as a store of wealth and not used as intensively for draught or milk production as they are further north. closer to the west african coast, around the major conurbations, meat prices are higher and there is a substantial demand for milk from urban populations, so that cattle, including some trypano-sensitive zebu breeds, are kept on a more commercial basis, and thus the losses imfig. 2 cost comparisons for ongoing control of tsetse and trypanosomosis at varying intensities source: based on estimates in shaw et al. 2007 and bourn et al. 2004 note: itc: insecticide-treated cattle; ra: restricted application; drugs refer to the total number of doses of trypanocide given to cattle per year per km2. future costs are discounted at 10 % per annum 1 400 1 200 1 000 800 600 400 200 0 u s $ /k m 2 f re e d o f ts e ts e traps 4/km2 traps 8/km2 aerial spray – sat itc pour-on 8/km2 itc spray 8/km2 sterile insect + itc sterile insect + sat field cost field studies administration itc 4/km2 itc 8/km2 itc ra 8/km2 drugs 40/km2 drugs 90/km2 1 400 1 200 1 000 800 600 400 200 0 u s $ /k m 2 c o n tr o ll e d f o r 2 0 y e a rs field cost field studies administration fig. 1 cost comparisons for tsetse elimination source: adapted from shaw et al. 2007 and based on prices in uganda in 2006 note: sat: sequential aerosol technique; itc: insecticide-treated cattle. future costs are discounted at 10 % per annum 31 a.p.m. shaw posed by disease are, therefore, again likely to be higher. implications for decision-making these two recent pieces of work therefore provide basic figures for the key economic variables. while it is tempting simply to juxtapose the money maps and the costs of dealing with tsetse, this would be too simplistic. as in any economic analysis of this type, a number of assumptions had to be made along side extrapolations from existing data, so that the figures give orders of magnitude rather than absolutely accu rate amounts. furthermore, the nature of the tsetse distributions—whether they are isolated, how many fly species are present—need to be considered, alongside other variables determining which techniques are feasible and sustainable (human and cattle population density, vegetation, terrain, etc.). with these caveats, it is possible to generalise along the following lines. looking at the west african money map, the red areas are primarily those with very low cattle population densities (five or fewer per km2). in these areas, 20 years is far too short a time horizon for cattle populations to become established (and there may be constraints other than tsetse) so that investment to deal with tsetse on a large scale would currently be unprofitable. the green areas, where much higher cattle population densities prevail and there is high use of draught oxen, are those areas where the disease is causing the highest losses and should be priority areas. here intervention to control tsetse is likely to be highly cost effective. the focus of decision-making will need to be on identifying any areas where tsetse populations are sufficiently isolated to consider long-term elimination which could be sustained without the need to maintain long-term barriers and the attendant risk of reinvasion and increases in costs. in such areas, choice of technique will need to be guided by cost and appropriateness to the fly species, cattle population density and terrain. where tsetse pop ulations are not isolated and elimination is not currently considered feas ible the presence of high num bers of cattle makes cheaper options for control, such as itc, look very attractive. the areas in the middle range between the green and red range still offer high levels of potential benefits per km2. these tend to be neither isolated nor on the fringes of the tsetse distribution, so that ongoing tsetse control using low cost sustainable methods alongside the judicious use of trypanocides would appear to be the best option. looking to the future with the very welcome renewal of interest in and funding for tsetse and trypanosomosis control speartotal benefit ($/km2) 6 000–7 000 0–500 500–1 000 2 000–3 000 3 000–4 000 5 000–6 000 4 000–5 000 1 000–2 000 7 000+ no tsetse unsuitable/protected water fig. 3 potential benefits from the removal of trypanosomosis in west africa: us$ gained over 20 years source: adapted from shaw et al. 2006 note: future benefits are discounted at 10 % per annum 32 assessing the economics of animal trypanosomosis in africa headed by pattec comes a grave responsibility to ensure that funds are directed to those activities which are most likely to be successful, sustained, appropriate to the locality and most cost effective. economic arguments will therefore continue to be important in guiding decision-making. much information has been gathered over the past five decades, but today’s decisions still rely on up-to-date field information appropriate to each country, socioeconomic environment and tsetse/trypanosomosis interface. new tools, especially gis, offer new insights and help to decision-making. the ‘money map ping’ approach has been welcomed by planners in the field of tsetse and trypanosomosis control and could be adapted to other diseases and pro duction constraints. currently, work is underway with the fao pro-poor livestock policy initiative (pplpi) to apply the ‘mapping the benefits’ concept to the tsetseinfested countries within the inter-gov ernmental author ity on development (igad) countries: ethiopia, kenya, somalia, sudan and uganda. however, new data on livestock productivity and how it is affected by disease is urgently needed to underpin such modelling exercises. similarly, on the cost side of the equation it is vitally important that accurate calculations are made of the true costs of interventions, not just to projects and governments, but also to those who actually have to deal with this disease on a day to day basis and invest their time and money in mitigating its effects—africa’s livestock keepers. acknowledgements the author would like to thank the organising committee for inviting this paper and for funding its presentation at the onderstepoort centenary cel ebrations. her gratitude also goes to the many colleagues and friends with whom she collaborated in undertaking some of the work described here, in particular guy hendrickx, raffaele mattioli, tim robinson, steve torr and william wint, with whom the bulk of this work was done. she would also like to thank dao balabadi, victorin codjia, oumar diall, marius gilbert, charles mahama, ian maudlin, issa sidibé, glyn vale, charles waiswa and sue welburn. references barrett, j. 1997. economic issues in trypanosomiasis control. chatham: natural resources institute. bourn, d., grant, i., shaw, a. & torr, s. 2005. cheap and safe tsetse control for livestock production and mixed farming in africa. aspects of applied biology, 75:81–92. du toit, r. 1954. trypanosomiasis in zululand and the control of tsetse flies by chemical means. onderstepoort journal of veterinary research, 26:317–87. doran, m. 2000. socio-economics of trypanosomosis. impli cations for control strategies within the common fly-belt of malawi, mozambique, zambia and zimbabwe. harare: regional tsetse and trypanosomosis control programme for southern africa (bovine trypanosomosis in southern africa, vol. 3.) gilbert, m., jenner, c., pender, j., rogers, d., slingen bergh, j. & wint, w. 2001. the programme against african trypanosomiasis information system (paatis) in world class parasites. the african trypanosomes, vol. 1, edited by s.n. black & j.r. seed. dordrecht: kluwer academic publishers. hendrickx, g., biesemans, j. & de deken, r. 2004. the use of gis in veterinary parasitology, in gis and spatial analysis in veterinary science, edited by p. durr & a. gatrell. wallingford: cabi publishing. jahnke, h.e. 1974.the economics of controlling tsetse flies and cattle trypanosomiasis examined for the case of uganda. munich: institut für wirtschaftsforschung, weltforum verlag (forschungsbericht der afrika-studienstelle, no. 48). programme against african trypanosomiasis (paat) 2008. paat—on target against poverty 1997–2007. rome: food and agriculture organization of the united nations. reid, r.s. & swallow, b.m. (eds) 1998. an integrated approach to the assessment of trypanosomosis control technologies and their impacts on agricultural production, human welfare and natural resources in tsetse-affected areas of africa, phase 1. nairobi: international livestock research institute: final technical report for ifad tag 284-ilri. shaw, a.p.m. 2004. the economics of african trypanosomiasis, in the trypanosomiases, edited by i. maudlin, p. holmes & m. miles. wallingford: cabi publishing. shaw, a., hendrickx, g., gilbert, m., mattioli, r., codjia, v., dao, b., diall, o., mahama, c., sidibé, i. & wint, w. 2006. mapping the benefits: a new decision tool for tsetse and trypanosomiasis interventions. edinburgh: dfid animal health programme and fao programme against african trypanosomiasis. shaw, a., torr, s., waiswa, c. & robinson, t. 2007. comparative costings of alternatives for dealing with tsetse: es timates for uganda. rome: fao (pro-poor livestock pol icy initiative working paper, no. 40). http://www.fao.org/ag/ againfo/projects/en/pplpi/workingpapers.html swallow, b.m. 2000. impacts of trypanosomiasis on african agriculture. rome: fao (paat technical and scientific series, no. 2). wilson, s.g. 1953. the control of glossina palpalis fuscipes newstead in kenya colony. bulletin of entomological research, 44:711–728 vale, g. & torr, s. 2005. user-friendly models of the costs and efficacy of tsetse control: application to sterilisation and insecticidal techniques. medical and veterinary entomology, 19:293–305. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: ernest ngoepe1 christine fehlner-gardiner2 alexander wandeler2 claude sabeta1 affiliations: 1agriculture research council-onderstepoort veterinary research, university of pretoria, south africa2canadian food inspection agency, canada correspondence to: ernest ngoepe postal address: private bag x04, onderstepoort 0110, south africa dates: received: 13 nov. 2014 accepted: 06 may 2014 published: 11 sept. 2014 how to cite this article: ngoepe, e., fehlner-gardiner, c., wandeler, a. & sabeta, c., 2014, ‘antigenic characterisation of lyssaviruses in south africa’, onderstepoort journal of veterinary research 81(1), art. #711, 9 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.711 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. antigenic characterisation of lyssaviruses in south africa in this original research... open access • abstract • introduction • materials and methods    • brain specimens    • antigenic analysis by immunofluorescence assay    • viruses included in the analysis    • viral rna extraction and reverse transcription polymerase chain reaction    • dna purification, nucleotide sequencing and phylogenetic analysis • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ there are at least six lyssavirus species that have been isolated in africa, which include classical rabies virus, lagos bat virus, mokola virus, duvenhage virus, shimoni bat virus and ikoma lyssavirus. in this retrospective study, an analysis of the antigenic reactivity patterns of lyssaviruses in south africa against a panel of 15 anti-nucleoprotein monoclonal antibodies was undertaken. a total of 624 brain specimens, collected between 2005 and 2009, confirmed as containing lyssavirus antigen by direct fluorescent antibody test, were subjected to antigenic differentiation. the lyssaviruses were differentiated into two species, namely rabies virus (99.5%) and mokola virus (0.5%). furthermore, rabies virus was further delineated into two common rabies biotypes in south africa: canid and mongoose. initially, it was found that the canid rabies biotype had two reactivity patterns; differential staining was observed with just one monoclonal antibody. this difference was likely to have been an artefact related to sample quality, as passage in cell culture restored staining. mongoose rabies viruses were more heterogeneous, with seven antigenic reactivity patterns detected. although mokola viruses were identified in this study, prevalence and reservoir host species are yet to be established. these data demonstrate the usefulness of monoclonal antibody typing panels in lyssavirus surveillance with reference to emergence of new species or spread of rabies biotypes to new geographic zones. introduction top ↑ rabies is a viral zoonotic disease caused by lyssaviruses that are capable of infecting all mammalian species. these viruses belong to the lyssavirus genus of the rhabdoviridae family. the lyssavirus genus consists of 14 recognised viral species based on serological assays, genetic distances of the nucleoprotein gene, topology and consistency of the phylogenetic trees, antigenic patterns in reaction with anti-nucleocapsid monoclonal antibodies and/or other additional characters such as ecological properties, host and geographic range and pathological features (dietzgen et al. 2011; ictv 2014). these includes classical rabies virus (rabv) and the other viral species are referred to as rabies-related viruses. of the 14 species, only six have been identified and isolated in africa to date, namely: rabv, lagos bat virus (lbv), mokola virus (mokv), duvenhage virus (duvv), shimoni bat virus (shibv) and ikoma lyssavirus (ikov). in addition, lleida bat lyssavirus was recently proposed as a putative species of the lyssavirus genus (freuling et al. 2011; marston et al. 2012). within the classical rabies virus two common variants have been identified in southern africa: the canid rabies and mongoose rabies biotypes (king, meredith & thomson 1993; nel et al. 2005; von teichman et al. 1995). rabies in mongooses was first identified in southern africa in the 1800s (snyman 1940; swanepoel 2004). in contrast, rabies in dogs appears to have spread into southern africa via the north-west from angola in the 1940s, and by the 1950s dog rabies had entered zimbabwe and south africa, where it became established in various canid host species in subsequent years (nadin-davis & bingham 2004). in south africa, rabies still remains a significant public and veterinary health threat, as the disease is well maintained by various host species. genetic typing by nucleotide sequence analysis and antigenic typing using discriminatory monoclonal antibody (mab) panels in an indirect immunofluorescence assay (ifa) can be used to differentiate lyssavirus species, as well as variants within the species (favoretto et al. 2002; favoretto et al. 2006; smith 2002). the discriminatory capability of ifa is generally determined by the number of mabs used in a panel (nadin-davis et al. 2010; smith 2002). antigenic typing panels generally depend on mabs specific for the nucleoprotein (n) and phosphoprotein (p); glycoprotein (g) mabs can also be used. in addition, specific mab panels have also been generated and used to map antigenic sites on these proteins to characterise lyssaviruses, and also to distinguish rabv variants persisting in geographically restricted host reservoirs (nadin-davis et al. 2010; rupprecht et al. 1991; smith & king 1996). lyssavirus typing with mabs provides a rapid and inexpensive screening method for large-scale surveillance of both new lyssaviruses (gould et al. 1998) and common or emerging variants of the rabies virus (nadin-davis et al. 2000). this technology was used to differentiate between street virus and the rabies vaccine strain during the oral vaccination campaigns against fox rabies in western europe and in ontario, canada (fehlner-gardiner et al. 2008; schneider et al. 1988). in previous studies, a panel of 80 anti-nucleoprotein mabs was used to differentiate rabies viruses from namibia and south africa (king et al. 1993). it was shown that canid rabies viruses conformed to a single reactivity pattern, whereas mongoose rabies viruses had several variations in their reactivity patterns (king et al. 1993). this retrospective study describes the mab typing of lyssavirus isolates collected in south africa from different animal species between 2005 and 2009 is reported on. materials and methods top ↑ brain specimens the specimens were routinely submitted to the agricultural research council-ondestepoort veterinary institute (arc-ovi) for rabies diagnosis from various provinces of south africa except the western cape and kwazulu-natal (kzn), which submit samples to allerton veterinary laboratory, kzn and western cape provincial veterinary laboratory (figure 1). all the samples were confirmed to be positive for lyssavirus antigen by direct fluorescent antibody test (dfat) using fluorescently labelled goat antiserum raised against purified rabv and mokv ribonucleoproteins (dean, abelseth & atanasiu 1996). a total of 624 brain specimens, collected between 2005 and 2009, from different animal species were analysed by mab-ifa. figure 1: the geographical distribution of lyssavirus variants identified and received in south africa between 2005 and 2009 at the agricultural research councilonderstepoort veterinary institute. antigenic analysis by immunofluorescence assay a panel of 16 murine mabs discriminating between different southern african lyssavirus species and antigenic variants were chosen from the mab collection of the centre of expertise for rabies of the canadian food inspection agency (ottawa, canada). the panel included an anti-human adenovirus type-5 mab (1c5) as negative control and 15 anti-rabies virus nucleoprotein mabs, which included a positive control (38hf2) that had reacted with all tested lyssaviruses. the reactivity patterns for the canid and mongoose rabies virus biotypes, mokv, lbv and duvv established during the selection of mabs for this panel are shown in table 1. table 1: typical reactivity patterns of african lyssaviruses against a panel of 16 monoclonal antibodies. brain smears were prepared on teflon-coated 5 mm well slides and fixed in cold 80% acetone, acs grade (merck chemicals, germany) for at least 20 min. to each well on the slide, 80 μl of mab (in hybridoma supernatant) was added and incubated at 37 °c for 50 min – 60 min. the slides were removed from the incubator and rinsed twice with tris buffer (7.2 ph – 7.4 ph) for 5 min – 10 min, followed by three rinses with phosphate buffered saline (pbs) (7.2 ph – 7.4 ph) for 8 min – 10 min each. after air-drying, 80 μl of fluorescein isothiocyanate-conjugated goat anti-mouse igg antibody (sigma-aldrich, usa) was added to each well and the slides were incubated at 37 °c for 40 min – 60 min. the slides were rinsed with tris buffer for 5 min – 10 min and counterstained with evans blue (0.5%) for 40 s. the slides were dried by blotting them on a paper towel, then they were cover-slipped using aqueous mounting medium (50% pbs, 7.2 ph – 7.4 ph and 50% glycerol) and examined using ultraviolet fluorescence microscopy (carl zeiss, germany). the ifa reactivity for each mab was recorded as positive (+) or negative (-) to generate the overall staining pattern for each isolate. the test was repeated only when non-specific results with either negative or positive controls (1c5 and 38hf2) were obtained. viruses included in the analysis further phylogenetic analyses and comparison with phylogroups determined from previous studies (nel et al. 2005; van zyl, markotter & nel 2010) were carried out on a panel of 21 representative isolates selected from the seven mongoose biotype groups detected with antigenic typing and from six isolates selected from the canid biotype groups. the year of isolation, host species and geographical origin of the mongoose biotype isolates are shown in table 2. table 2: representative mongoose rabies variants from each antigenic group that were included in phylogenetic analyses. viral rna extraction and reverse transcription polymerase chain reaction total viral rna was extracted from original brain material using trizol® reagent (sigma-aldrich, usa) according to the manufacturer’s instructions. for the cdna synthesis of the g-l intergenic region and nucleoprotein genes, 5 µl of total rna (approximately 2 µg) and 20 pmol of the positive sense primers (2 µl), 001lys (n-gene) and g (+) (g-l intergenic region) (table 3) were denatured at 65 °c for 5 min. the reaction mixtures were immediately cooled on ice and rna was reverse transcribed at 42 °c for 60 min in a 20 µl reaction mixture containing 200 u superscript iii reverse transcriptase (invitrogen), 4 µl of reaction buffer, 10 mm deoxynucleotide triphosphates (dntps) and 40 u of rnase inhibitor (promega, usa). the polymerase chain reactions (pcrs) were performed in a total volume of 50 µl containing 1x pcr reaction buffer, 1.5 mm mgcl2, 250 µm of each dntp, 5 µl of cdna, 1.25 u of taq dna polymerase (applied biosystem, usa) and 40 pmol of each of the primers (g [+] and l [-], 001lys and 304) (table 3). the amplification reactions were performed in a geneamp 2400 thermocycler with an initial denaturation of 94 °c for 2 min, followed by 40 cycles (25 cycles) of denaturation at 94 °c, primer annealing at 37 °c for 30 s (42 °c for 90 s) and primer extension at 72 °c for 90 s followed by a final extension at 72 °c for 7 min (values in parenthesis indicate conditions for g-l intergenic region). the amplicons were subsequently analysed by ethidium bromide-stained agarose gel (1%) electrophoresis. table 3: oligonucleotide primers used in the study showing annealing positions and nucleotide sequences. dna purification, nucleotide sequencing and phylogenetic analysis the pcr products were purified using the dna purification kit according to the manufacturer’s instructions (promega, usa). the purified products were sequenced in both directions using both primer sets used in the reverse transcription polymerase chain reaction (rt-pcr) step together with the sequencing kit according to the manufacturer’s instructions (applied biosystems, usa). the sequencing products were electrophoresed on an abi 377 automated sequencer (applied biosystems) and the consensus sequences of 1353 nucleotides (nt) of the n gene and 592 nt (g-l intergenic region) were obtained by using mega 3.1 sequence editor (kumar, tamura & nei 2004). the phylogenetic trees were constructed using the neighbour joining (nj) method in clustal x (saitou & nei 1987). two sets of 18 full-length nucleoprotein and partial g-l intergenic region nucleotide sequences representing previous mongoose rabv phylogroups from southern africa were retrieved from genbank and included for phylogenetic analysis. the branching order of the trees was statistically evaluated by 1000 bootstrap replicates; values of more than 700 (70%) were considered to be statistically significant (hills & bull 1993). results top ↑ of the 624 samples included for analysis, 99.5% (621) had staining patterns consistent with those established for rabv, and 0.5% (3) had patterns consistent with that of mokv. the rabv isolates were identified from various animal species in all provinces of south africa included in the present study. mokola virus isolates were identified in the provinces of mpumalanga (n = 1) from a domestic dog, and eastern cape (n = 2) from domestic cats (figure 1). the rabv isolates were further differentiated into the canid (438/621, 70.5%) and mongoose (183/621, 29.5%) rabies biotypes. the canid rabv biotype was primarily confirmed in rabies cases obtained from domestic dogs (216/438, 49%), black-backed jackals (canis mesomelas) (29/438, 7%) and bat-eared foxes (otocyon megalotis) (29/438, 7%), whereas the mongoose rabv biotype was recovered mainly in herpestidae species, especially the yellow mongoose (82/183, 45%). moreover, there were two mongoose rabies biotype cases detected in limpopo province from a black-backed jackal and a domestic dog (figure 1). the canid rabv biotype isolates fell into two categories, with differences observed only with mab 32gd12 (table 4). the majority of the canid viruses (403/438, 92%) were stained by this mab (reactivity pattern i). although viruses that conformed to canid biotype reactivity pattern ii were less common, they exhibited similar geographic distribution to viruses that conformed to canid biotype reactivity pattern i (figure 1). among the mongoose rabv biotypes, seven antigenic reactivity patterns (a-g) were detected (table 5) and these viruses were recovered from a wide range of different animal species (table 6). mongoose rabies biotype viruses that conformed to group b and d staining patterns were most common (table 5). however, the antigenic reactivity patterns do not appear to have any species-specific association with the different hosts (table 6). table 4: indirect immunofluorescence assay reactivity patterns of canid rabies viruses obtained from south africa. table 5: indirect immunofluorescence assay reactivity patterns obtained for mongoose rabies viruses. table 6: the distribution of mongoose rabies virus variant antigenic reactivity patterns by host species. the pcr amplicons generated from the selected isolates were of the expected size of approximately 850 bp (g-l region) and 1680 bp (n-gene). sequencing yielded about 740 bases (g-l region) and 1400 bases (n-gene) of sequence on average, which were trimmed to a consensus of 592 and 1353 nucleotides (nt) for phylogenetic analysis (data not shown). representative canid rabv isolates conforming to both reactivity patterns (n = 3 for each group) had 98% nucleotide sequence identity in the nucleoprotein-encoding region (data not shown). for the mongoose biotype, phylogenetic analysis of representative isolates from each group (n = 21) revealed trees with similar topologies when analysing either the partial g-l intergenic region or the full-length nucleoprotein nucleotide sequences (figure 2a and figure 2b). isolates corresponding to staining pattern a (n = 3) all clustered within the previously described phylogroup iv, whereas those with staining pattern e (n = 3) and f (n = 3) clustered within phylogroup iii. these isolates also clustered geographically with other isolates that fell within phylogroups iii and iv (figure 2). in contrast, the isolates corresponding to the other staining patterns (b, c, d, f and g) did not group together under a single phylogroup, and did not exhibit geographical clustering, as observed for isolates with staining patterns a, e and f. thus, two of the three isolates with staining patterns b, c and g clustered with phylogroup iii, whilst the third isolate examined fell within phylogroup ii and iv. the three viruses with staining pattern d fell within phylogroups ii, iii and iv. whilst the isolates from staining pattern groups b, c, d, and g did not cluster geographically, they did map to similar geographic areas with other isolates belonging to the same phylogroups (figure 2). figure 2: the neighbour-joining tree based on 1353 nucleotide sequences of nucleoprotein (a) and 592 nucleotide sequences of the g-l intergenic region (b) of the 21 mongoose rabies viruses selected from antigenic groups. discussion top ↑ the geographical distribution of the rabies cases detected between 2005 and 2009 reflects the distribution of the susceptible host species in south africa. for example, bat-eared foxes maintain and transmit rabies in the western regions of the country (sabeta et al. 2007a), whereas black-backed jackals maintain the disease cycles in the northern region of south africa (swanepoel 2004; zulu, sabeta & nel 2009). canid rabies biotype cases were detected in all provinces included in the present study, and although the provinces of kzn and western cape were not represented, the canid rabv biotype is known to be endemic in these regions, with principal reservoirs in domestic dogs and bat-eared foxes, respectively (swanepoel 2004). the majority of mongoose rabv biotype cases were found in north-west, free state and mpumalanga provinces, which is consistent with the distribution of herpestidae species (nel et al. 2005). two mongoose rabv biotype variants were detected in a domestic dog and black-backed jackal in limpopo province, whereas all other cases from this region were of the canid rabv biotype. these detections may have been the result of sporadic introduction from neighbouring zimbabwe and botswana, where mongoose biotype rabv is endemic (bingham et al. 2001; foggin 1988; sabeta et al. 2008; tremlett , wibberley & king 1994). however, as the sampling method was only passive, relying on the state authorities to submit animal rabies suspected cases to the laboratory, it is possible that the mongoose rabv biotype is also established in this region of south africa, but its prevalence underestimated. spill-over of the mongoose rabies biotype from the host species is common; for example, mongoose rabvs were recovered from domestic dogs, sheep and cattle in the present study. in contrast, spillover of the canid rabv biotype into non-canid wildlife species is rarely observed (von teichman et al. 1995). understanding the movement of rabv biotype and particularly spillover of wildlife variants into domestic animals and vice versa is a key aspect in the rabies control in the region. the mab panel used in this study was originally selected to differentiate amongst canid and mongoose biotypes of the rabies virus, as well as the non-rabies lyssavirus species of mokv, lbv and duvv circulating in the region (bingham et al. 2001). several different staining patterns were observed for the rabies viruses in the present study, notably two for the canid biotype and seven for the mongoose biotype. a similar variation in mab reactivity patterns was observed amongst the mongoose viruses in previous studies (king et al. 1993; tremlett et al. 1994). in contrast, a single antigenic reactivity pattern of the canid rabv biotype was previously observed in isolates from south africa and namibia (king et al. 1993). in previous phylogenetic studies, whilst seven canid rabv biotype variants or clusters were observed in the northern provinces of south africa, they conformed to a single antigenic typing pattern (agricultural research council-onderstepoort veterinary institute 2007; zulu 2007). similar observations were made in a study from botswana, where antigenic typing could not differentiate smaller clades or sub-groupings established by phylogenetic analyses (johnson et al. 2004). it is unlikely that the detection of two reactivity patterns for the canid rabv biotype in the present study is due to a superior performance of the current mab panel distinguishing between two variants that have long co-existed in the region. however, if this scenario were the case, observation of some geographic segregation of the variants would be expected, and it was not. given that the observed canid rabv reactivity patterns differed by only one mab (32gd12), it is more likely that the epitope recognised by mab 32gd12 is unstable and the observed staining variability was an artefact of the quality of the tissue that was sampled. when the five canid rabies viruses that had antigenic reactivity pattern ii were grown in cell culture, reactivity with mab 32gd12 was restored, suggesting that the epitope recognised by this mab is indeed unstable (data not shown). this is further supported by the observation that comparison of the n-gene nucleotide sequence of multiple isolates conforming to each reactivity pattern indicated that they were 98% identical, and predicted amino acid sequences of n protein revealed no sequence motif that could explain the specific binding of mab 32gd12 to reactivity pattern indicated that they i isolates alone (data not shown). a phylogenetic study of canid biotype viruses from free state province and surrounding areas, collected between 1995 and 2007, showed similar high nucleotide sequence identity (ngoepe, sabeta & nel 2009). molecular phylogeny studies using both g-l intergenic region and full-length nucleoprotein nucleotide sequences showed that the mongoose rabv biotype from southern africa (zimbabwe and south africa) could be differentiated into five main clades, which are consistent with their geographic localities (nel et. al., 2005; van zyl et. al. 2010). in contrast, in the present study, mab typing revealed seven antigenic reactivity patterns within the mongoose rabv biotype. however, the selected isolates from each antigenic group clustered within just three of the five clades previously determined (nel et al. 2005; van zyl et al. 2010). concordant results were obtained whether the g-l intergenic region or the nucleoprotein nucleotide sequence was used for phylogenetic analysis. a high degree of diversity in mongoose rabies isolates examined with mabs using a different mab panel was also observed in studies in botswana (tremlett et al. 1994). the heterogeneous reactivity patterns observed within the mongoose rabv variant have been interpreted as supporting the hypothesis that the mongoose rabv variant has existed in this region for a longer period than the canid rabv variant and has therefore adapted well into the host species of this specific geographic locality (nadin-davis & bingham 2004). molecular clock analyses have supported this hypothesis and estimate the age of the mongoose rabv to be in the range of 200 years, consistent with anecdotal evidence from the historical record (van zyl et al. 2010). the heterogeneity in mab reactivity patterns observed in the present study is somewhat puzzling; however, not all of the different reaction patterns are associated with a given host species or geographic origin. in addition, only the antigenic types a, e and f are found on distinct branches of the sequence-based phylogenetic tree. antigenic types b, c, d, and g are scattered over three of the five branches of the tree, with the three group d isolates falling into three different clades. it might well be that these are artefacts related to sample quality, as observed with the canid biotypes. these observations highlight the limitation of using the current mab panel for higher resolution studies of rabv sub-variants. the retrospective analysis of the present study confirms not only the presence of the two rabv biotypes but also the rare mokv in south africa. the true prevalence and the host species of mokv are yet to be established. in southern africa, mokv has only been isolated from domestic cats, with one exception from a dog (sabeta et al. 2007b). since domestic cats are unknown to be a reservoir species for any lyssavirus, it is likely that they exist in close association with the true mokv reservoir and are incidental hosts for this lyssavirus species. the ability to identify mokv quickly using mabs is of great importance since the current vaccines do not protect against mokv infections (badrane et al. 2001; von teichman et al. 1998). conclusion top ↑ rabies case surveillance using only pan-lyssavirus reactive antisera in dfat does not provide sufficient information to fully understand the epidemiology of rabies in a region where multiple lyssaviruses are circulating. whilst virus characterisation by genetic sequencing provides definitive information with regard to virus type, mab typing is a useful screening tool that can be done quickly and at comparatively lower cost. this study has illustrated the value of mab typing to identify species of the lyssavirus genus and has contributed to an understanding of the epidemiology of rabies and rabies-related viruses in south africa; however, evaluation of detailed relationships amongst isolates requires the use of more sophisticated molecular techniques that are not impacted to the same degree by sample quality. routine lyssavirus differentiation using mabs should be continued in synergy with nucleotide sequencing in this region. such studies can inform the design of rabies control strategies, may assist in the identification of reservoir species (as in the case of mokv), and may also lead to the identification of new lyssavirus species (kuzmin et al. 2010). acknowledgements top ↑ the authors would like to thank drs livio heath and susan nadin-davis for critically reviewing the manuscript and also ms debra mohale and dr wonderful shumba for their technical support. this work was partly funded by the rabies diagnostic project, onderstepoort veterinary research institute (ovi 15/4/p001) and the european virus archive (eva) (04/17/c215). competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions e.n. 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mesomelas)’, msc thesis, university of pretoria. article information authors: sarah cleaveland1 hawthorne beyer2 katie hampson1,3 daniel haydon1 felix lankester1,4,5 tiziana lembo1 francois-xavier meslin6 michelle morters7 zacharia mtema1,3 maganga sambo3 sunny townsend1 affiliations: 1boyd orr centre for population and ecosystem health, institute of biodiversity, animal health and comparative medicine, college of medical, veterinary and life sciences, university of glasgow, scotland 2arc centre of excellence for environmental decisions, centre for biodiversity and conservation science, university of queensland, australia 3ifakara health institute, ifakara, tanzania 4paul g. allen school of global animal health, washington state university, united states 5lincoln park zoo, chicago, united states 6world health organization, geneva, switzerland 7department of veterinary medicine, university of cambridge, united kingdom correspondence to: sarah cleaveland postal address: graham kerr building, university of glasgow, glasgow g12 8qq, united kingdom how to cite this article: cleaveland, s., beyer, h., hampson, k., haydon, d., lankester, f., lembo, t. et al., 2014, ’the changing landscape of rabies epidemiology and control’, onderstepoort journal of veterinary research 81(2), art. #731, 8 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.731 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the changing landscape of rabies epidemiology and control in this proceedings... open access • abstracts • introduction    • rabies burden of disease studies    • epidemiological modelling    • surveillance approaches    • economic analyses    • dog ecology studies    • international partnerships • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ over the past 20 years, major progress has been made in our understanding of critical aspects of rabies epidemiology and control. this paper presents results of recent research, highlighting methodological advances that have been applied to burden of disease studies, rabies epidemiological modelling and rabies surveillance. these results contribute new insights and understanding with regard to the epidemiology of rabies and help to counteract misperceptions that currently hamper rabies control efforts in africa. the conclusion of these analyses is that the elimination of canine rabies in africa is feasible, even in wildlife-rich areas, through mass vaccination of domestic dogs and without the need for indiscriminate culling to reduce dog population density. furthermore, the research provides valuable practical insights that support the operational planning and design of dog vaccination campaigns and rabies surveillance measures. introduction top ↑ over the past 20 years, much progress has been made in understanding the epidemiology of rabies in africa, supporting the view that canine rabies elimination is both feasible and cost-effective through mass vaccination of domestic dogs (lembo et al. 2010; rupprecht et al. 2008; world health organization [who] 2004; who 2013). this research has resulted in a groundswell of momentum amongst rabies scientists and international human and animal health agencies to drive forward ambitious plans for canine rabies elimination. however, despite huge progress in some parts of the world, most notably latin america where canine rabies elimination is targeted for 2015 (who 2012), substantial challenges remain in africa where few national programmes are in existence. figure 1: scheme showing how misperceptions about dog ecology and rabies epidemiology have negative consequences for rabies control; and identifying the types of research studies that can generate the evidence-base needed for effective control. an enduring problem relates to several misperceptions that currently hamper rabies control efforts in africa (lembo et al. 2010) (figure 1). for example, there is a perception that rabies is relatively insignificant as a disease of public health concern; that rabies is a problem of ‘stray’ dogs that are not accessible for parenteral vaccination; that rabies can only be controlled through culling or reduction in the dog population density; and that wildlife play a major role in sustaining rabies cycles in africa. these misperceptions drive a cycle of neglect, where the implementation of ineffective control measures results in demotivation of policy-makers and veterinary field staff and fuels the erroneous impression that rabies control is futile. here we review the most recent evidence from rabies epidemiology studies that address these misperceptions, highlighting how new approaches and analytical techniques are being applied and illustrating how the results are constantly reinforcing the evidence base as to the feasibility of canine rabies elimination in africa. rabies burden of disease studies top ↑ in common with many ‘neglected’ diseases, a principal factor contributing to the low prioritisation of rabies control has been the lack of information about the burden and impact of rabies, particularly in low-income countries that are most affected by the disease. the development of a probability-tree model for estimating human rabies deaths from the incidence of suspect rabid animal bite injuries and human post-exposure prophylaxis (pep) (cleaveland et al. 2002) has paved the way for several estimates of human rabies incidence in africa and asia (coleman, fèvre & cleaveland 2004; fèvre et al. 2005; hossain et al. 2012; knobel et al. 2005; ly et al. 2009; tenzin et al. 2011). these studies have indicated that rabies incidence in africa, estimated to be ~24 000 deaths per year (knobel et al. 2005), is at least 100 times higher than officially reported.the most recent estimates of rabies burden incorporate more detailed country-level data, including updated information on human pep use. these analyses, part of the global burden of disease studies coordinated by the partners for rabies prevention, have also included consideration of economic losses as a result of premature deaths. overall, using the probability model, global estimates of human rabies deaths are similar to previous studies, with an estimated 61 000 human rabies deaths per year occurring globally (95% ci, 52 200–70 700) and 23 800 in africa (95% ci, 21 000–28 000) (who 2013). however, the apparent consistency in estimates masks substantial local variation, most notably the decline in human rabies deaths in several countries in asia because of increased availability of rabies pep and, in some areas, dog rabies control. whilst any decline in human rabies deaths is to be celebrated, this progress has come at a high cost, with pep costs in asia estimated at around us$1.5 billion. these contribute substantially to the estimated global annual cost of rabies of us$6bn (95% ci, 4.6–7.3), which also includes productivity losses of us$2bn as a result of premature deaths (who 2013). empirical data to both parameterise and validate estimates of human rabies deaths have been generated from community surveys (hossain et al. 2012), large-scale verbal autopsy surveys (suraweera et al. 2012) and active surveillance and contact tracing (hampson et al. 2008). nonetheless, it is clear that these data are still very scarce and that any figures based on models that incorporate incomplete and imprecise data are likely to have a high degree of uncertainty. despite these uncertainties, clinical studies in malawi have demonstrated that rabies is a more common cause of childhood encephalitis than recognised previously and can be misdiagnosed easily as malaria, even by experienced clinicians (mallewa et al. 2007). this may seem surprising, given that rabies exhibits several distinct clinical features in humans, such as aerophobia and hydrophobia, but serves to highlight the importance of improving awareness amongst clinicians and submitting samples for laboratory diagnosis to ensure that the rabies disease burden is not masked by high levels of misdiagnosis, particularly in malaria-endemic areas of africa. although rabies does not have the pandemic potential that characterises the emerging zoonoses of greatest concern to high-income countries (figure 2), its burden and impact cannot be considered negligible. the physical, psychological and economic consequences of rabies are borne by the poorest communities in the world and the continued suffering caused by the disease is entirely preventable and remains a conspicuous failure of the veterinary and medical professions to deliver on existing solutions. figure 2: scheme showing how misperceptions about dog ecology and rabies epidemiology have negative consequences for rabies control; and identifying the types of research studies that can generate the evidence-base needed for effective control. epidemiological modelling the advent of powerful analytical and modelling tools now allows valuable insights regarding the epidemiology and control of rabies to be generated from a wide variety of epidemiological, genetic and geographic data. patch occupancy models, developed from metapopulation theory to explore persistence of species in fragmented habitats (hanski & ovaskainen 2000), have recently been applied to the study of rabies persistence in structured populations in the serengeti ecosystem. in this study, hospital-derived animal-bite injury cases were used as a measure of disease occurrence in villages (‘patches’), exploiting an easily accessible source of data to parameterise spatially-explicit models of disease on a regional scale (beyer et al. 2011). bite-injury case records typify the type of coarse field data that is often the only information available to epidemiologists in africa. these data contain many uncertainties that have previously limited their utility for epidemiological inference. however, the use of state-space models provides a powerful framework for modelling the effects of uncertainty and have allowed us to exploit accessible but ‘noisy’ data in order to generate new insights into regional-scale transmission dynamics. for example, the serengeti model highlighted the importance of spatial structure, with village-to-village transmission of dog rabies being more important in driving regional-scale dynamics than transmission from wildlife-protected areas; and being consistent with results of earlier studies that identified dogs, not wildlife, as being rabies reservoirs in the serengeti (lembo et al. 2008). these models also have potential for the design of spatially-structured vaccination campaigns by identifying communities that contribute most to the persistence of rabies; they could, therefore, be prioritised for vaccination in situations where resources are limited (beyer et al. 2012). whilst much effort has focused on understanding the epidemiology and control of rabies in endemic settings, attention is now turning toward prospects for the elimination of canine rabies. the feasibility of canine rabies elimination has been supported by a strong body of evidence from epidemiological field studies, phylogenetic analyses and epidemiological modelling (summarised in lembo et al. 2010). a key finding has been the low value of r0, which consistently falls between 1.0 and 2.0 for canine rabies in dog populations across the world, despite wide variation in dog densities and demographic characteristics in urban and rural communities in different countries (hampson et al. 2009). the lack of density-dependence in rabies transmission was evidenced again with an r0 estimate of 1.2 for the 2008–2010 rabies outbreak in bali, indonesia in communities with very high dog densities (townsend et al. 2013). these findings have two major implications for rabies control and elimination: firstly, that elimination of canine rabies through vaccination of ~70% dogs is epidemiologically feasible in most settings, including high-density populations; and secondly, that tackling rabies through dog density reduction (e.g. mass culling) is likely to be ineffective (morters et al. 2013). these conclusions are also supported by previous experience. for example, despite substantial reductions in the dog population of flores, indonesia as part of a culling program to control a rabies outbreak in 1996, the disease remains endemic (windiyaningsih et al. 2004). similarly, culling failed to control canine rabies in korea, israel, and bali, whereas subsequent mass dog vaccination programmes have resulted in control of the disease (knobel et al. 2013). in addition to having no demonstrable beneficial impact on the control of rabies, indiscriminate culling of dogs also has substantial ethical and welfare implications and no evidence exists to support its use in rabies control strategies. however, misperceptions remain widespread (presumably because conclusions from research studies run counter to many intuitive assumptions) and culling is still considered a feasible policy option by many veterinary services in africa. engaging with policy-makers therefore remains a high priority to ensure uptake of research findings and to instill confidence as to the feasibility of rabies control and elimination through mass dog vaccination. epidemiological models also provide critical support for the development and design of elimination strategies in terms of understanding elimination dynamics; designing the most cost-effective strategies for disease control and surveillance at national, regional and global levels; and providing guidance for policymakers about expected times to elimination. a spatially-explicit stochastic simulation model of rabies in bali, indonesia, for example, has indicated that time to elimination of rabies is critically dependent on high levels of contiguous vaccination coverage and that even small pockets of low coverage (e.g. involving < 0.5% of the dog population) can cause a significant delay in progress (townsend et al. 2013). in contrast, the probability of rabies elimination is only slightly affected by the timing of campaigns (e.g. intensive synchronised versus more prolonged dog vaccination campaigns), so campaigns can be tailored to optimise logistic efficiency and resource availability in order to achieve the high vaccination coverage required. surveillance approaches surveillance is a critical element of the control and elimination of infectious diseases. as control programmes progress toward elimination, surveillance efforts need to be intensified in order to detect new incursions and to declare freedom from disease with confidence. surveillance remains arguably the weakest element of many national and regional control and elimination strategies, particularly in africa and asia, with well-recognised difficulties associated with detection and confirmation of both animal and human cases. a further problem relates to the potentially long incubation period of rabies (fekadu 1993; hampson et al. 2009), making it difficult to determine whether rabies has truly been eliminated during periods with no detected cases. several questions therefore remain as to the optimum control strategies that should be adopted in the face of low case-detection probabilities, as well as regarding the duration and level of surveillance efforts that are needed in order to provide confidence that rabies has truly been eliminated. although definitions for freedom from canine rabies have been proposed (who 2013), current international guidelines lack quantitative definitions about the level of surveillance that would be required by an effective surveillance system for the purpose of declaring freedom from rabies (oie 2011). recent modelling work provides useful insights, for example, in the use of outbreak simulation techniques to explore the effectiveness of control and elimination strategies. in relatively isolated areas that are not subject to frequent reintroductions (e.g. islands), but which have realistic levels of rabies surveillance (i.e. case-detection probabilities < 10%), mass dog vaccination is more effective at controlling rabies (and is no more costly) than vaccinating only in response to detected outbreaks. these models further indicate that surveillance measures need to be able to detect at least 5% of rabies cases in order to be confident that rabies has been eliminated under the current guidelines for declaration of freedom (townsend et al. 2012). these models were developed initially for populations without repeat introductions but, in reality, high levels of legal and illegal dog movements remain a concern for maintaining rabies-free status in areas where the disease has been eliminated. informal movement of dogs through road and water-based transport can represent a considerable challenge in many areas and, whilst cross-boundary coordination and effective rabies control over large geographic regions should mitigate the risk of new introductions, effective surveillance will still be needed in order to detect and respond to new incursions. maintaining surveillance levels is also important so as to detect outbreaks that may be triggered by rare spill-over transmission from other reservoir hosts, such as bats, and to contain outbreaks that might otherwise have the potential to establish new epidemic cycles. with recognition of the critical importance of strengthening rabies surveillance, much effort is therefore being directed at improvements in detection, reporting and laboratory diagnosis of rabies. examples include the application of mobile phone technologies to enhance reporting of human rabies exposures at bite treatment centres in tanzania (mtema 2013). mobile phone systems also allow for rapid communication between human and animal health sectors to ensure follow up of animal cases; reminder texts to be sent to patients to complete the full course of pep; and improved distribution of vaccine stocks to avoid the vaccine shortages that occur frequently in many of the more remote parts of africa (mtema 2013). the advent of new diagnostic techniques is also helping to overcome some of the logistic, technical and cultural barriers associated with submitting human and animal samples for centralised laboratory diagnosis using conventional fluorescence techniques. these include (1) new diagnostic tests that use light microscopy rather than fluorescence microscopy and can therefore be carried out at local facilities with very high specificity and sensitivity (dürr et al. 2008; lembo et al. 2006); (2) field-based lateral flow devices, that may provide a useful tool for generating surveillance data (markotter et al. 2009) and for empowering and incentivising field workers to engage with rabies surveillance (halliday et al. 2012); (3) new techniques for intra vitam diagnosis of human rabies cases (dacheux et al. 2008; fooks et al. 2009); and (4) culturally-acceptable methods for post-mortem sampling of brain material for human rabies diagnosis (mallewa et al. 2007). although these techniques are improving capacity for laboratory-confirmed diagnosis in africa, attention also needs to be given to improving the reporting of clinical cases, which provides the entry point for building an effective rabies surveillance system. whilst laboratory diagnosis provides robust confirmation of rabies and is important for effective administration of pep, clinical case reporting also needs strengthening as the central pillar of rabies surveillance. for example, surveillance in the smallpox eradication programme was underpinned by clinical case reporting from health centres (henderson & klepac 2013) and during the rinderpest eradication campaigns in africa, participatory surveillance based on the engagement of local communities was the primary surveillance tool in the final stages of eradication (roeder, mariner & kock 2012). given the distinctive nature of dog rabies, recognition amongst local communities in africa can be high (e.g. 74% of cases reported as suspect rabid animals in rural tanzania were confirmed positive on laboratory diagnosis; lembo et al. 2008). whilst clinical surveillance is clearly neither 100% specific nor sensitive, these cases can contribute valuable epidemiological data for the comparative analysis of trends, initiating follow-up investigations and, most critically, providing a stimulus for disease control measures. indeed, the lack of feedback or beneficial response to mitigate disease problems is arguably the greatest barrier to the reporting of zoonoses, particularly within resource-poor systems, and this chronic lack of response (or capacity to respond) is disempowering and demotivating at the grassroots level for healthcare and veterinary workers alike (halliday et al. 2012). therefore, whilst there is no doubt that laboratory diagnostic capacity needs strengthening throughout most of africa, the lack of capacity should not act as a deterrent to initiating rabies control measures. rather, effective rabies control measures should be integrated within responsive surveillance systems and should provide motivation to field staff to improve case reporting and submission of samples. economic analyses whilst confidence is growing as to the epidemiological feasibility of canine rabies control and elimination through mass dog vaccination, a key question relates to the cost and sustainability of control measures, particularly in low-income countries. in tanzania, mass vaccination campaigns have led to dramatic declines in demand for pep (cleaveland et al. 2003) and the concomitant savings to the public health sector generate a potential mechanism for sustaining dog vaccination campaigns. cost-effectiveness analysis, incorporating deterministic models of dog-to-human transmission, indicate that strategies involving dog vaccination and pep are likely, over the longer term, to be more cost effective for preventing human rabies deaths than human pep alone (bögel & meslin 1990; zinsstag et al. 2009). however, the relationship between dog rabies incidence and human pep demand, which is a critical determinant of cost-effectiveness, is likely to vary considerably across different socioeconomic settings. in northern tanzania, for example, the incidence of bite injuries reported at health clinics declines rapidly as the incidence of rabies falls (cleaveland et al. 2003) and can decline to zero in areas where canine rabies has been eliminated (lembo et al. 2010). this suggests that people in these communities seek pep only when they recognise the risk of rabies – they would not seek treatment for bite injuries from non-rabies suspect animals, a situation which presumably reflects both a high level of rabies recognition and the high private costs associated with pep in comparison with household incomes in africa (knobel et al. 2005). in contrast, in many higher-income settings or where rabies occurs more sporadically, the scale of pep use may be driven both by health-seeking behaviour of more affluent and knowledgeable members of the community as well as clinician decision making. in these situations, pressures and uncertainties faced by clinicians regarding the genuine need for pep means that pep use can remain very high even when the incidence of animal rabies cases and risk of exposure to rabies is extremely low (lardon et al. 2010), with important implications for the cost-effectiveness of human rabies prevention through rabies control in the animal reservoir. research findings have also contributed important information for practical field operations, including fundamental questions relating to dog population ecology and ownership patterns, which are central to the design of rabies control measures. dog ecology studies an enduring misperception relating to dog ecology in africa is the widely-held, but erroneous, impression that a large proportion of dogs are ownerless or ‘stray’ dogs that are not accessible for vaccination. this has had major implications for rabies control across africa, with policy makers being reluctant to invest in dog vaccination campaigns and resources rather being directed toward ineffective strategies, such as culling (figure 1). a key finding from dog ecology studies is that, although most dogs in africa are free-roaming, the number of ownerless dogs or those inaccessible for vaccination remains very low. for example, only 1%, 8% and 11% of dogs respectively were unowned in three study sites in n’djamena (kayali et al. 2003). in zimbabwe, all dogs on communal lands were owned (butler & bingham 2000) and in tanzania, < 1% of dogs were unowned in an urban site that was specifically targeted for dog ecology studies on the basis of reports of a large population of ‘stray’ dogs (gsell et al. 2012). although precise estimates of ownerless dogs are difficult to obtain, a robust conclusion remains that the level of dog ownership and dog accessibility is sufficiently high in all these communities to be able to control dog rabies through mass dog vaccination of owned dogs. estimating the size of dog populations has been considered a key operational requirement for mass dog vaccination campaigns. however, although several approaches to determining the dog population size have been described (summarised in the canine rabies blueprint [global alliance for rabies control 2013]), difficulties still remain. for owned dogs, knowledge of the human:dog ratio (hdr) combined with human population data can provide useful preliminary estimates. dog ecology studies from across africa indicate relatively consistent hdr values in rural settings (davlin & vonville 2012; knobel et al. 2008), but the hdr can vary widely, particularly in urban settings. furthermore, human population figures are generally obtained from census data, which may only be collected every 10 years, and projections from these data are usually generated from average population growth rates. however, with rapid changes in human demographics, including high rates of urbanisation in africa (un habitat 2008), population growth rates at a local level can vary widely, leading to uncertainties in projected population sizes and, hence, dog population estimates. therefore, whilst an initial crude estimate of the dog population size can be generated using the projected human population sizes and ‘average’ or ‘typical’ hdrs for planning of first campaigns, these figures should be refined progressively as campaigns are implemented. an important point in designing household dog population surveys is that estimates of hdr need to explicitly include pups, because pups are not always considered as ‘dogs’ in answer to the question about the number of dogs in a household. it is also important to note that young pups (less than 3 months of age) need to be included for rabies vaccination during annual campaigns (a point that is often not well recognised by either the owners of puppies or veterinary officers). determining vaccination coverage levels for puppies separately from adults can be useful in order to identify whether low coverage in pups is a problem that needs to be addressed. despite uncertainties in most approaches to estimating dog population sizes, it is clear that extremely precise data may not be critical at the initial stages of implementing national strategies and more precise data can be generated as campaigns are rolled out. for procurement purposes, estimates should over-estimate rather than underestimate dog population sizes; provision of excess vaccines at the start of a rabies control programme is generally not problematic, provided the shelf life allows for vaccine to be used in successive campaigns. in addition to planning campaigns, knowledge of the dog population size is also important in order to determine vaccination coverage, a critical parameter for monitoring vaccination campaigns. during campaigns, vaccination coverage is often determined from the number of dogs vaccinated during a campaign (or vaccine doses used) divided by the estimated dog population size, but given the uncertainties in dog population sizes, these figures are also likely to be unreliable. it should also be noted that, during recurrent campaigns, using vaccine doses to estimate coverage would only provide a conservative estimate, as dogs vaccinated in the previous year(s) and not during the current campaign would not be included in coverage estimates. however, if using vaccines with 2–3 years’ duration of immunity (as is the case for most commercial vaccines), some of these dogs may still be protected against rabies and contributing to population immunity. because of the critical importance of monitoring vaccination coverage during national control programmes, alternative methods should be adopted to generate more precise values at the community level. household surveys generate accurate and useful data and it is feasible to include them in vaccination campaigns that adopt ‘house-to-house’ strategies. however, logistic and resource constraints may preclude sampling other communities and, as the ‘even-ness’ of coverage is likely to be an important measure (townsend et al. 2013), greater emphasis may need to be given to developing relatively cheap and simple populationand community-based survey tools that could be applied more broadly in all vaccinated communities. other forms of post-vaccination survey include mark-resight surveys, which capture coverage in free-roaming dogs (e.g. kaare et al. 2009; kayali et al. 2003; matter et al. 2000) and could be encouraged as standard methodology for campaigns which are conducted using centralised vaccination stations and in areas with a high proportion of free-roaming dogs. these methods have, in addition, been used to estimate the size of the ownerless dog population (e.g. gsell et al. 2012). international partnerships with strong interdisciplinary partnerships now established, involving the partners for rabies prevention, the tripartite partnership of the world health organization (who), the food and agriculture organization of the united nations (fao) and the world organisation for animal health (oie), as well as other international health and animal welfare agencies, there is a growing international momentum toward canine rabies elimination (lembo et al. 2011). rabies has been included within the who roadmap for neglected tropical diseases (who 2012), with targets set for regional elimination of canine rabies in latin america by 2015 and south-east asia by 2020. to support the development of national rabies control plans, a step-wise strategy has been developed (fao 2013), identifying the key activities and capacities that need to be established at different stages of disease control and elimination. this framework is further supported by practical guidelines (the ‘canine rabies blueprint’) in order to support field operations (lembo 2012). conclusion top ↑ in summary, there are clearly strong grounds for optimism that canine rabies can be controlled and eliminated in africa. multiple strands of evidence from empirical and theoretical research studies generate confidence as to the epidemiological feasibility of canine rabies elimination, with the principal focus of activities directed toward mass dog vaccination. in addition, findings from operational research studies, in tandem with strong international partnerships, provide practical guidance and support needed for the design and implementation of effective national and regional elimination programmes. acknowledgements top ↑ support for research on the epidemiology and surveillance of rabies in tanzania, including support for m.s. (ifakara health institute, tanzania) and k.h. (university of glasgow and ifakara health institute, tanzania) was provided by grants from the wellcome trust (082715, 084615ma and 087393/b/08/z), the ubs optimus foundation and the leverhulme trust (support for h.b. [university of queensland]). s.t. (university of glasgow) was supported by a grant from the uk medical research council (g0901135) and t.l. (university of glasgow) by a grant from the bbsrc dfid cidlid initiative (bb/h009302/1). the authors would like to thank sacids and afrique one for organisation of the one health conference at which this paper was presented. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions the study was conceived by s.c. (university of glasgow), t.l. (university of glasgow) and k.h. (university of glasgow and ifakara health institute, tanzania) and all authors contributed to writing the article. figure 1 was conceived by s.c. and d.h. (university of glasgow) and figure 2 was conceived by s.c., t.l. and s.t. (all university of glasgow) and f-x.m. 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health organization (who), 2012, ‘accelerating work to overcome the global impact of neglected tropical diseases: a roadmap for implementation’, world health organization, geneva, switzerland. who/htm/ntd/2012.1. world health organization (who), 2013, ‘who expert consultation on rabies. 2nd report’, who technical report series, 982, world health organization, geneva, switzerland. zinsstag, j., dürr, s., penny, m.a, mindekem, r., roth, f., gonzalez, s.m. et al., 2009, ‘transmission dynamics and economics of rabies control in dogs and humans in an african city’, proceedings of the national academy of sciences of the united states of america 106(35), 14996–15001. http://dx.doi.org/10.1073/pnas.0904740106 article information authors: chimuka musako1,2 celia abolnik3 affiliations: 1department of veterinary and tsetse control services, ministry of agriculture and livestock, zambia 2department of veterinary tropical diseases, university of pretoria, south africa 3department of production animal studies, university of pretoria, south africa correspondence to: chimuka musako postal address: po box 710070, mansa 10101, zambia dates: received: 15 july 2012 accepted: 10 oct. 2012 published: 07 dec. 2012 how to cite this article: musako, c. & abolnik, c., 2012, ‘determination of the seroprevalence of newcastle disease virus (avian paramyxovirus type 1) in zambian backyard chicken flocks’, onderstepoort journal of veterinary research 79(1), art. #502, 4 pages. http://dx.doi.org/10.4102/ ojvr.v79i1.502 copyright notice: © 2012. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. determination of the seroprevalence of newcastle disease virus (avian paramyxovirus type 1) in zambian backyard chicken flocks in this research communication... open access • abstract • introduction • materials and methods    • sampling    • enzyme-linked immunosorbent assay    • statistical analysis    • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a cross-sectional study was conducted in five provinces and 11 districts of zambia to determine the seroprevalence of newcastle disease in zambian backyard chicken flocks. of the chickens sampled, 73.9% tested positive for avian paramyxovirus type 1 antibodies by means of an enzyme-linked immunosorbent assay. seroprevalence varied amongst the five provinces sampled, ranging from 82.6% in the eastern province to 48.3% in luapula province. seroprevalence also varied amongst the 11 districts sampled, ranging from 91.3% in monze district of southern province to 22.8% in mufulira district of the copperbelt province. overall, the seroprevalence of newcastle disease in zambian backyard chicken flocks has increased since the previous study conducted in 1994. introduction top ↑ newcastle disease (nd), notifiable to the world organisation of animal health (oie 2010), is caused by virulent avian paramyxovirus type 1 (apmv-1) strains. it is a contagious disease of birds that is widely distributed throughout the world, affecting many domestic and wild avian species and causes severe economic losses in the poultry sector (cattoli et al. 2009). although two classes (i and ii) and multiple genotypes are described, only a single serotype of apmv-1 exists (czegledi et al. 2006).the poultry industry in zambia is based on two distinct systems. the first is the commercial system, where broilers or layers are obtained from hatcheries and reared on commercial feed and in properly designed chicken houses. strict vaccination schedules for important avian diseases such as nd are followed, and the lentogenic lasota/46 vaccine strain is widely used. most commercial systems are situated along the railway line in close proximity to major towns. the second system is the village or backyard production system, where chickens scavenge for food and subsist with little input from their owners. vaccination schedules are not followed for these chickens (songolo & katongo 2000). in a previous study (alders, inoue & katongo 1994) the seroprevalence of nd in chickens was determined to be 36.9%, based on the haemagglutination inhibition (hi) titres of 2000 blood samples. seroprevalence varied between provinces, ranging from 29.2% in the northern province to 51.3% in the copperbelt province. recognising that small-scale farming has the potential to make an important developmental contribution to the national economy, the government is now considering improving support to the agricultural sector, thus spurring sectoral growth and productivity. it is anticipated that the right agricultural policy environment will improve sectoral income and savings, and contribute to poverty reduction. the purpose of the current study was to determine updated information on the seroprevalence of nd virus in zambian village chickens, as a starting point to justify an official policy for nd control. materials and methods top ↑ sampling serum samples were collected from various local chicken breeds in various districts of zambia between june and december in both 2009 and 2010. border areas, busy market places and sites near water bodies where wild birds congregate were selected. only chickens that had not been vaccinated against nd and were apparently healthy at the time of sample collection were included in the survey. the formula of cannon and roe (1982) was used to determine the sample size, based on the assumption that the prevalence of nd in the country was 25%, with a 95% probability of detecting at least one infected chicken. eleven districts from five provinces were sampled and 29 households from each district were included in the study. the resulting sample size was 1595. however, owing to logistical problems, only 1012 samples were obtained. a blood sample of 2 ml was collected from the wing vein of each bird. the blood was allowed to clot at room temperature, after which the serum was separated and stored at –20 °c until testing. enzyme-linked immunosorbent assay samples were tested using a commercial enzyme-linked immunosorbent assay (elisa) kit for nd (flockchek®, idexx laboratories, maine) according to the recommended procedure. the laboratory work was performed at the central veterinary research institute in lusaka, zambia. results were read using a microplate reader (multiskan,labsystems). absorbance values were measured at 650 nm. sample/positive ratios (s/p) were calculated according to the recommended procedure. serum samples with s/p ≤ 0.2 were considered to be negative, whereas those with s/p ≥ 0.2 were considered to be positive. statistical analysis the prevalence of nd was calculated for each province and district using spss software (ibm corporation, new york). the prevalence within the provinces and between the districts was compared using fisher’s exact test. analysis of variance was used to determine whether the log-transformed titres from the various provinces were significantly different. values were considered significant at p ≤ 0.05. ethical considerations a blood sample of 2 ml was collected from the wing vein of each bird sampled. the procedure was done as humanely as possible. the birds did not show any undesirable reaction after sample collection. results top ↑ the results regarding nd seroprevalence in each of the five provinces are shown in descending order in table 1. the highest seroprevalence was recorded for the eastern province (82.6%; ci = 74.9% – 90.4%), followed by the southern province (80.0%; ci = 76.7% – 83.3%), the northern province (77.3%; ci = 70.4% – 84.2%), the copperbelt province (51.6%; ci = 44.4% – 58.8%) and finally luapula province (48.3%; ci = 30.1% – 66.5%). the seroprevalence of nd was significantly different (p < 0.001) amongst the provinces. the seroprevalence of nd was also determined at district level. in southern province, the highest seroprevalence was recorded in monze district (91.3%; ci = 85.5% – 97.1%), whilst the lowest was recorded for kazungula district (71.7%; ci = 65.2% – 78.2%). of the two districts that were sampled in the northern province, seroprevalence was highest in mpulungu (87.8%; ci= 78.6% – 97.0%) and lowest in nakonde (71.7%; ci = 62.5% – 80.9%). for the two districts that were sampled in the copperbelt province, the highest seroprevalence was recorded in chililabombwe (80.4%; ci = 72.3% – 88.5%) and the lowest in mufulira (22.8%; ci = 14.2% – 31.4%). only one district in both the eastern and luapula provinces was sampled and therefore seroprevalences could not be compared. the seroprevalence of nd was significantly different (p < 0.001) amongst the districts (see figure 1 for the location of the districts). discussion top ↑ when nd was first reported in zambia in 1952, outbreaks were concentrated along the railway line, where the largest population of poultry occurred (songolo & katongo 2000). in a study conducted by alders et al. (1994), seroprevalence levels varied amongst the provinces, ranging from 29.2% in the northern province to 51.3% in the copperbelt province. in the current study, we demonstrated not only that nd seroprevalence in flocks in the informal sector had increased to 77.3% in the northern province (copperbelt province remained roughly the same at 51.6%) but also that seroprevalence was 82.6% in the eastern province, 80% in the southern province and 48.3% in luapula province. it is likely that the increasing population of chickens provided a sustainable reservoir for the maintenance of nd strains, which could have allowed the infection to persist or facilitated the introduction of viruses more frequently. therefore, although the eastern and northern provinces are not situated along the railway line, the increase in chicken numbers could have contributed to the infection shifting to these areas. table 1: seroprevalence of newcastle disease in each province and district sampled, in descending order. figure 1: map of zambian districts. the samples were collected from apparently healthy, unvaccinated birds, suggesting that the infections were due to circulating avirulent strains. whether these originated from vaccine strains or another source, for example class i or class ii lineage 1 nd viruses, which are common in wild birds (czegledi et al. 2006), is unknown. since only a single serotype of apmv-1 exists, we were unable to distinguish between vaccine-related and field-strain antibodies. the live lentogenic lasota/46 vaccine strain is widely used in the commercial sector and it is possible that some spillover of vaccine into backyard chickens occurred, especially where spent layers end up in villages. it has been shown that vaccines alter the epidemiology of nd to some extent, since they prevent disease but not infection. vaccinated birds exposed to virulent virus strains develop no clinical signs; however, some replication of the infecting virus occurs and birds excrete virulent nd virus. this would probably not be excreted in similar quantities as by susceptible birds, but there would be sufficient virus to infect other birds (mavale 2001; miller et al. 2007). in the zambian situation, active vaccination of village and backyard chickens against nd is rarely practiced owing to cost and problems associated with maintaining the cold chain for the heat-labile live vaccine. there was a slight peak in nd seroprevalence between january and march, and from september to november. a similar trend was reported in mozambique (harun & massango 2001; songolo & katongo 2000). this may be attributed to either a seasonal or a social reason. the period from january to march is cool and humid, with heavy rains, whereas the period from september to november is hot, windy and dry. another important factor associated with the transmission of nd is the keeping of flocks of various ages. this is common in village chicken husbandry and although mortality is higher in young chickens, 100% mortality may occur in adult flocks, depending on the strain (alexander 1998; songolo & katongo 2000; spradbrow 2001). social aspects that affect transmission of nd in zambia include the transport of live chickens during extensive travelling (both locally and across borders) to visit friends and family over the festive season, and increased trading at markets in january to generate income for school fees. conclusion top ↑ although unidentified nd strains are apparently widespread in zambia, little official attention has been given to the control of the disease in chickens since the government ceased subsidised nd vaccinations in the 1980s. the increasing seroprevalence of nd, albeit due to unknown strains, highlights the ease with which the infection can spread. incursion of a highly virulent nd strain to which the poultry population has insufficient immunity could have devastating consequences. however, these could be mitigated by a good vaccination strategy. improved small-scale poultry farming would make an important contribution to the national economy and this could motivate the re-establishment of an official, subsidised nd vaccination programme in zambia. similarly, a vaccination programme amongst village and backyard chickens would limit mortalities, improve flock numbers and ultimately improve food security and generate much-needed income for many poor households in the country. since the last study to isolate and characterise nd strains responsible for losses in the zambian poultry sector was published almost 30 years ago (hussein et al. 1984), there is a need to isolate and genetically characterise currently circulating strains affecting poultry in zambia to allow appropriate control decisions to be taken. acknowledgements top ↑ we wish to acknowledge the following people who contributed to the study: martin simuunza (university of zambia) performed the statistical analysis, oliver chibomba (central veterinary research institute) collected some of the samples and performed some of the experiments and cheelo mweene, gregory bwalya, tingiya sikombe, linford moya (central veterinary research institute) collected some of the samples. the district veterinary officers (ministry of livestock and fisheries) in the various districts from where the samples were collected provided additional information. this work was financially supported by the institute of tropical medicine, belgium. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this paper. authors’ contributions c.m. (ministry livestock and fisheries) was responsible for conceptual design, data analysis and contributed to writing the manuscript. c.a. (university of pretoria) was the study leader, responsible for experimental design, and contributed to writing the manuscript. references top ↑ alders, r.g., inoue, s. & katongo, j.c., 1994, ‘prevalence and evaluation of hitchner b1 and v4 vaccines for the control of newcastle disease in village chickens in zambia’, preventive veterinary medicine 21, 125–132. http://dx.doi.org/10.1016/0167-5877(94)90001-9alexander, d.j., 1998, ‘newcastle disease’, in s.e aiello & a. mays (eds.), the merck veterinary manual, pp. 1941–1942, whitehouse station, nj. cannon, r.m. & roe, r.t., 1982, livestock disease surveys: a field manual for veterinarians, australian bureau of animal health, canberra. cattoli, g., fusaro, a., monne, i., molia, s., lemenach, a., maregeya, b. et al., 2009, ‘emergence of a new genetic lineage of newcastle disease virus in west and central africa – implications for diagnosis and control’, veterinary microbiology 19(142), 3–4. czegledi, a., ujvari, d., somogyi, e., wehmann, e., werner, o. & lomniczi, b., 2006, ‘third genome size category of avian paramyxovirus serotype 1 (newcastle disease virus) and evolutionary implications’, virus research 120, 36–48. http://dx.doi.org/10.1016/j.virusres.2005.11.009, pmid:16766077 harun, m. & massango, f.a., 2001, ‘village poultry production in mozambique: farming systems and ethnoveterinary knowledge in angonia and tsangano districts, tete province’, in r.g. alders & p.b. spradbrow (eds.), sadc planning workshop on newcastle disease control in village chickens: proceedings of an international workshop, aciar proceedings 103, maputo, mozambique, march 06–09, 2000, pp. 76–79. hussein, n.a., sharma, r.n., ando, r. & chizyuka, h.g.b., 1984, ‘a study on newcastle disease virus pathotypes in zambia’, revue scientifique et technique, office international des epizooties, 3, 383–389. mavale, a.p., 2001, ‘country report: mozambique’, in r.g. alders & p.b. spradbrow (eds.), sadc planning workshop on newcastle disease control in village chickens: proceedings of an international workshop, aciar proceedings 103, maputo, mozambique, march 06–09, 2000, pp. 20–25. miller, p.j., king, d.j., afonso, c.l. & suarez, d.l., 2007, ‘antigenic differences among newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge’, vaccine 25, 7238–7246. http://dx.doi.org/10.1016/j.vaccine.2007.07.017, pmid:17719150 national malaria control centre, 2009, enumeration reports, viewed 20 november 2012, from http://www.nmcc.org.zm/report-map.htm oie. see world organisation for animal health. oie, 2010, ‘newcastle disease’, in manual of diagnostic tests and vaccines for terrestrial animals, viewed 21 august 2012, from http://www.oie.int/eng/normes/mmanual/a_summry.htm songolo, a. & katongo, j.c., 2000, ‘country report: zambia’, in r.g. alders & p.b. spradbrow (eds.), sadc planning workshop on newcastle disease control in village chickens: proceedings of an international workshop, aciar proceedings 103, maputo, mozambique, march 06–09, 2000, pp. 43–45. spradbrow, p.b., 2001, ‘the epidemiology of newcastle disease in village chickens’, in r.g. alders & p.b. spradbrow (eds.), sadc planning workshop on newcastle disease control in village chickens: proceedings of an international workshop, aciar proceedings 103, maputo, mozambique, march 06–09, 2000, pp. 53–55. world organisation for animal health, 2010, newcastle disease, viewed 21 august 2012, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.03.14_newcastle_dis.pdf kellerman_19-23.indd introduction at the time of the founding of onderstepoort, theiler, as the director, either controlled or had a hand, in most of the research done at the institute. he was a man of wide interests and included in these interests was botany. he rightly suspected that some of the then unexplained conditions of stock, such as geeldikkop and gousiekte, were in fact plant poisonings. his subsequent research on plant poisonings drew the attention of noted botanists such as j. burtt-davy, i.b. pole evans and a.o.d. mogg, initiating a fruitful collaboration between botanists and veterinarians that has persisted to this day. theiler can thus be said to have laid the foundation for the future sections of toxicology at the institute and faculty (gutsche 1979; kellerman, personal observations 2008). to the best of our knowledge, the unit now known as the section of toxicology came into being in about 1928 upon the appointment of d.g. (douw) steyn as pharmacologist cum toxicologist at the institute. he was succeeded by t.f. adelaar (1948– 1974), t.w. naudé (1974–1976), t.s. kellerman (1976–1998), j.p.j. joubert (1998–2004) and finally dharma naicker (2004 to date), who is currently the acting head of the section. perhaps it is worthwhile to point out that over 90 years, from 1908 to 1998, only five people had headed the section. what is more, save for theiler, all these incumbents actually knew each other, a state of affairs that held considerable benefits for the section, as inter alia continuity of purpose was promoted and new appointees never lacked for mentors to guide them in their research. research on plant poisonings south africa has one of the richest floras in the world, so it comes as no surprise that, included in this wide variety, are some 600 poisonous plants (vahrmeyer 1981). moreover, since many of these 19 onderstepoort journal of veterinary research, 76:19–23 (2009) poisonous plants t.s. kellerman section pharmacology and toxicology, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract kellerman, t.s. 2009. poisonous plants. onderstepoort journal of veterinary research, 76:19–23 south africa is blessed with one of the richest floras in the world, which—not surprisingly—includes many poisonous plants. theiler in the founding years believed that plants could be involved in the aetiologies of many of the then unexplained conditions of stock, such as gousiekte and geeldikkop. his subsequent investigations of plant poisonings largely laid the foundation for the future sections of toxicology at the institute and the faculty of veterinary science (up). the history of research into plant poisonings over the last 100 years is briefly outlined. some examples of sustained research on important plant poisonings, such as cardiac glycoside poisoning and gousiekte, are given to illustrate our approach to the subject and the progress that has been made. the collation and transfer of information and the impact of plant poisonings on the livestock industry is discussed and possible avenues of future research are investigated. 20 poisonous plants plants are unique to the region much of the research on poisonous plants done abroad had little application here. as a result, south african investigators could look only to themselves for answers to the problems caused by many of these plants. in the 1990s, a study of the distribution, diagnosis and estimated economic impact of plant poisonings and mycotoxicoses in south africa was carried out (kellerman, naudé & fourie 1996). the study revealed that the most important poisonings (poisonings by cardiac glycoside-containing plants, geeldikkop, vermeersiekte, gifblaar poisoning, diplodiosis and lantana poisoning) were the same as those with which theiler and the early workers long ago had battled. this finding was expected, as the first plant poisonings brought to their notice were the most damaging and, by inference, those for which farmers most urgently needed answers. to review the history of research on poisonous plants at the institute over the last hundred years in the limited space allowed would be an impossible task. in view of this, only the research on some of the major problems listed in the 1996 study will be briefly outlined, principally to illustrate our approach to the subject and the progress that has been made. cardiac glycoside poisoning according to that study, poisoning with cardiac glycoside-containing (cg) plants, collectively, is the single most important plant poisoning in south africa, annually accounting for about 33 % of all cattle deaths from plant poisonings. two types of cardiac glycosides are contained by south african plants, namely cardenolides and bufadienolides. cardenolides have a single unsaturated five-member lactone ring on c17 of the steroid molecule while bufadienolides have a doubly unsaturated six member (pentadienolide) lactone ring in that position (naudé 1977). all the important cardiac glycoside-containing plants in south africa have bufadienolides as their active principles (kel ler man, coetzer, naudé & botha 2005). poisoning by bufadienolides may be either acute or chronic, depending on whether the bufadienolides contained by them have a cumulative effect or not. acute poisoning affects the respiratory, cardiovascular, gastrointestinal and nervous systems, usually leading either to death or recovery within days. cumulative or krimpsiekte-causing bufadienolides on the other hand involve mainly the nervous system sometimes resulting in sudden death or more commonly, protracted paresis or paralysis. affected animals typically assume a characteristic pose (with the feet together and back arched), lie down frequently and develop protracted paresis /paralysis. a few display persistent torticolis (kellerman et al. 2005). the first report of research on cardiac glycoside poisoning that we could trace was by soga, who in 1891 succeeded in reproducing krimpsiekte by dosing a ‘plakkie’, tylecodon ventricosis, to goats (soga 1891). his finding was confirmed by tomlinson & dixon cited by henning (1926), who also incriminated tylecodon wallichii in the aetiology of the disease. the next notable advance in krimpsiekte and plant-induced cardiac glycoside poisoning was the isolation by naudé & potgieter (1966) of the toxic principle, a bufadienolide, from moraea pallida, followed closely by yet another from t. wallichii by van rooyen & pieterse (1968). consequent to this pioneering work many plants, including several plakkies (tylecodon, cotyledon and kalanchoe spp.), tulp (moraea spp.), slangkop (drimia spp.) and kruidjie-roer-my-nie (melianthus spp.) have been shown similarly to contain bufadienolides (kellerman et al. 2005). knowing the nature of the active principles was very important as this opened up the overseas literature on extensively researched cardiac glycosides such as digitalis. information on the mode of action was particularly helpful, since amongst others, it had bearing on treatment. attention could then be paid to the treatment and control of the poisoning. several therapeutic agents were tested without notable success until joubert & schultz (1982) showed that drenching poisoned animals with activated charcoal gave excellent results. this simple, practical and economical treatment has saved the lives of innumerable animals. the current methods of controlling plant poisonings, namely by the mechanical or chemical eradication of plants, fencing off infested areas, grazing management, etc., all involve changing the environment in some way. however, changing the environment is not always feasible, particularly in communal or extensive commercial farming areas where capital is lacking, cash turnover from livestock production is slow and labour is scarce. since the environment could not easily be changed, means had to be found of manipulating the animals to resist or avoid poisoning. firstly, the feasibility of immunising stock against plant poisonings was investigated. joubert accord21 t.s. kellerman ingly attempted to prepare a vaccine against cardiac glycoside poisoning by conjugating cardiac glycosides with bovine serum albumin. although sheep developed strong immunity to a particular cardiac glycoside in the vaccine, little cross-immunity could be demonstrated against challenges with non-homologous cardiac glycosides or cardiac glycosidecontaining plants (j.p.j. joubert, unpublished data 1982). the issue of developing a vaccine against poisoning by cardiac glycoside-plants is currently being re-examined by c.j. botha (personal communication 2007), with initial promising results. south african stockmen traditionally know that animals that grow up on infested veld learn to avoid certain poisonous plants. this explains why poisoning by these plants (e.g. moraea spp.) occur mostly in naïve stock, newly introduced from non-infested areas. a project was immediately launched to extract and characterise the factor(s) responsible for this conditioned feed aversion (cfa) in yellow tulp, moraea pallida. according to kellerman et al. (2005), ‘sniffer sheep’, previously averted to yellow tulp, were used to sense the presence of the aversive substance in fractions of the plant mixed in maize meal. refusal of the fractions consumed by naïve control sheep indicated the presence of an aversive substance, provided the ‘sniffer sheep’ were still willing to eat pure maize meal. by following the refused fractions—obtained by solvent extraction and chromatographic separation—epoxyscillirosidin, the main toxic principle of yellow tulp, was identified as the ‘aversive substance’ (snyman, kellerman, schultz, joubert, basson & labuschagne 2004). field trials revealed that averted cattle had difficulty in recognizing epoxyscillirisodin in yellow tulp growing naturally on kikuyu pastures. to circumvent this problem, an epoxyscillirosidin-free hexane tulp extract, serving as an ‘identification factor’ for tulp, was dosed in conjunction with the aversion factor, during induction of aversion. some evidence was also found that the epoxyscillirosidin used to induce aversion, could contribute to the poisoning of animals during the aversion process on tulp-infested pastures. partially replacing the epoxyscillirosidin with lithium chloride, a non lethal aversive agent that does not affect the heart, successfully addressed the problem (snyman et al. 2004; snyman, schultz, joubert, basson & labuschagne 2003). the investigation is continuing. gousiekte the fifth most important plant poisoning in the 1996 study (kellerman et al. 1996), gousiekte, is certainly the most unusual. it is typically characterized by apparently healthy ruminant animals suddenly dropping dead from heart failure some 6–8 weeks after ingesting certain members of the rubiaceae (gardenia) family. walker (1908), the government veterinary officer at ermelo, first reported the appearance of gousiekte, a serious new disease which caused the sudden death of sheep. he was convinced that the causative agent was a plant. there the matter rested until 1915, when more than 1 000 sheep died of gousiekte after trekking across a ‘toxic’ farm near kaalfontein in the vicinity of pretoria. in a series of experiments conducted on the farm by theiler and co-workers, they established beyond doubt that pachystigma pygmaeum was responsible for the outbreak (theiler, du toit & mitchell 1923). subsequently, several other members of the rubiaceae have been incriminated by various workers as the aetiology of gousiekte, namely pachystigma thamnus, pachystigma latifolium, fadogia homblei, pavetta harborii and pavetta schumanniana (kellerman et al. 2005). studies on the clinical signs and cardiodynamics of gousiekte (pretorius & terblanche 1967; pretorius, terblanche, van der walt & van ryssen 1973) and the subcellular effects on energy production and the contractile mechanism of the myocardium (snyman, van der walt & pretorius 1982a, b) suggested that the primary lesion in gousiekte was inhibition of the contractile mechanism of the myocardium. in the 1990s, the matter was taken further by fourie and co-workers, who launched an ambitious project to isolate the cardiotoxin and to explain the pathogenesis of the disease. gousiekte is a particularly challenging disease to investigate, inter alia because initially only ruminants were known to be affected, large amounts of plant material of variable toxicity are required to induce the disease, and there is a latent period of approximately six weeks to contend with. in a most accomplished piece of research, a pure toxin was isolated from p. harborii (fourie 1994; fourie, erasmus, schultz & prozesky 1995) and identified by vleggaar as a polyamine, pavetamine (r. vleggaar, personal communication 1997). polyamines are highly biologically active compounds affecting many functions in the body including cell growth. studies in rats by schultz and her co-workers confirmed that pavetamine in common with other polyamines inhibits the synthesis of proteins. ongoing investigations by these workers indicate that pavetamine acts by inhibiting the synthesis of the contractile protein, myosin, in myocardial cells. when the depletion of myosin through natural degradation reaches a level where the contractility 22 poisonous plants of the myocardium is compromised, sudden heart failure ensues. prospects for future research owing to the diligent efforts of veterinarians and other workers over the last hundred years, most of the plant poisonings affecting stock in south africa have been identified and described. amongst others, their botanical, toxicological and pathological aspects have been studied and in many the active principles have been isolated and characterized. although new poisonings, such as the nephrotoxic condition of cattle in the northern cape province caused by nolletia gariepina (du plessis 2004), will still sporadically surface, the era of ‘descriptive’ toxicology is slowly coming to an end. one of the many new priorities would be in depth studies of the pathogenesis of our plant poisonings, preferably at subcellular level (see gousiekte), partly in the expectation that a better understanding of their modes of action might lead to the development of effective therapies. the question of therapy in general has been briefly discussed under the heading of cardiac glycoside poisoning. the most immediate challenge facing researchers today is the control and prevention of plant poisonings in the field. according to the discussions under the heading of cardiac glycoside poisoning, it appeared that manipulating the animal to avoid or resist poisoning had a better chance of success than trying to change the environment. the problems experienced in the development of vaccines against certain plant poisonings were touched on, but this aspect has not yet been fully exploited and should be further investigated. the very promising research on cfa as a means of teaching stock to avoid plant poisoning has been fully discussed elsewhere in this publication. one of the chief attractions of cfa is that, being one of the nature’s own ways of protecting stock, it is completely environmentally friendly. averted animals are able to graze with impunity on infested pastures without the ecology of the system first having been disturbed by human intervention; for instance, by the use of herbicides or the mechanical eradication plants. more fanciful perhaps, is the prospect of introducing genes involved in resistance to plant poisonings from the rumen flora of wild animals into that of cattle and sheep. genetic manipulation of rumen flora could perhaps be considered as one of the avenues of future research on the control of plant poisonings in south africa. transmission of technology steyn was one of the most dominant figures on the toxicological scene in south africa during the first half of the previous century, having published prolifically in both scientific and popular journals. to him must also go the credit for writing the first reference book, the toxicology of plants in south africa, thereby cementing his place in the annals of toxicology at onderstepoort (steyn 1934). a second volume, vergiftiging van mens en dier, followed some 14 years later, but this was aimed mainly at the farming community (steyn 1949). by the early 1980s a pressing need for a modern work arose as the one by steyn, published more than 50 years before, was dated and long out of print. as a result, academics, veterinarians, students and researchers in related fields, amongst others, did not have a south african reference book to consult on plant poisonings. plant poisonings and mycotoxicoses of livestock in southern africa, written in conjunction with coetzer (pathologist) and naudé (toxicologist) duly appeared in 1988 (keller man, coetzer & naudé 1988). having a pathologist as a co-author was particularly useful as pathology plays a pivotal role in the diagnosis of many of our plant poisonings. in the 1990s the manuscript was updated to include information on developments in the field of plant poisoning since 1988. the need for a second edition was made more urgent by the fact that by the late 1990s all the senior researchers in toxicology, save for c.j. botha at the faculty, had either retired or were about to retire, leaving a serious vacuum in this field of expertise at onderstepoort. this gave rise to a worrying lack of mentors to guide the next generation of young researchers responsible for taking toxicology into the future. it was hoped that the second edition, published in 2005 and in which botha was co-opted as a co-author, would help fill this gap (kellerman et al. 2005). conclusion looking back over the past century, onderstepoort (both the institute and faculty), can be seen to have played a leading role in the investigation of the plant poisonings in southern africa. this achievement would not have been possible, however, without the century-long dedication of many south african veterinarians from all over the country and 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(kandelaarbos). 11. die isolering van ‘n bufadienolied. journal of the south african chemical institute, 21:89–90. vahrmeyer, j. 1981. poisonous plants of southern africa that cause stock losses. cape town: tafelberg publishers. walker, j. 1908. gouw-ziekte. a disease of sheep. report of the government veterinary bacteriologist of the transvaal, 1908–1909: 74–99. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice hasle_167-175.indd introduction ticks are important vectors of pathogens affecting humans and livestock, and may themselves cause anaemia, hide damage, and wounds resulting in secondary bacterial infections (fletcher 2007). an understanding of their dispersal mechanisms is crucial towards their effective control. ticks have very limited locomotor ability and rely on their hosts for dispersal. migratory birds can serve as hosts for several tick species (hoogstraal, kaiser, traylor, gaber & guindy 1961; hoogstraal, kaiser, traylor, guindy & gaber 1963; hoogstraal 1972; mehl, michaelsen & lid 1984; olsén, jaenson & bergström 1995), and may transport these with their associated tick-borne pathogens across geographical barriers such as deserts and oceans. in addition, larger, ground-living birds, such as francolin, spurfowl and guineafowl, are important hosts for the immature stages of certain tick species, and are often heavily infested (horak & williams 1986; horak, fourie, novellie & williams 1991a; horak, spickett, braack & williams 1991b; horak & boomker 1998; uys & horak 2005). although passerines and other small birds usually harbour only small numbers of ticks, they often occur in flocks, and thus, because of their large numbers and extraordinary mobility, have the potential of significantly contributing to pathogen dispersal as well as tick gene flow within a region. it 167 onderstepoort journal of veterinary research, 76:167–175 (2009) ticks collected from birds in the northern provinces of south africa, 2004–2006 g. hasle1*, i.g. horak2, g. grieve3, h.p. leinaas4 and f. clarke5 abstract hasle, g., horak, i.g., grieve, g., leinaas, h.p. & clarke, f. 2009. ticks collected from birds in the northern provinces of south africa, 2004–2006. onderstepoort journal of veterinary research, 76:167–175 approximately 3 000 birds, mainly passerines, caught in mist nets in the northern provinces of south africa, were examined for ticks. a total of 178 ticks, belonging to 14 species, were recovered from 83 birds of 43 different species. hyalomma rufipes was the most numerous tick, with 26 larvae and 109 nymphs collected, followed by amblyomma marmoreum, with 13 larvae and two nymphs. despite the study being conducted within the distribution range of amblyomma hebraeum, it was not seen on any passerines, whereas three larger species were infested. the potential for small birds to spread ticks with their associated tick-borne pathogens is discussed. keywords: amblyomma marmoreum, birds, hyalomma rufipes, migration, northern south africa, passerines, ticks * author to whom correspondence is to be directed. e-mail: hasle@reiseklinikken.no 1 oslo travel clinic, st olavs plass 3, no-0165 oslo, norway 2 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa, and department of zoology and entomology, university of the free state, p.o. box 339, bloemfontein, 9300 south africa 3 birdlife northern gauteng ringing group, 344 delphinus street, waterkloof ridge, pretoria, 0181 south africa 4 department of biology, university of oslo, p.o. box 1066 blindern, no-0316 oslo, norway 5 department of biology, university of limpopo (medunsa campus), p.o. box 139, medunsa, 0204 south africa accepted for publication 22 august 2008—editor 168 ticks collected from birds in northern provinces of south africa, 2004–2006 is especially birds that may introduce certain tick species with their associated tick-borne pathogens to new areas, once climate change or human impact has made these habitable for them. birds readily cross fences between wildlife reserves and pastures used by domestic livestock, and may thus transfer both ticks and tick-borne pathogens from their potential wildlife reservoirs to domestic animals. moreover acaricide resistance is a considerable problem in some regions of africa (fletcher 2007), and it is not entirely unlikely that birds play some role in the spread of acaricide-resistant strains of ticks. few data sets exist on the ticks that infest small birds in sub-saharan africa. however, horak et al. (1991a) have recorded the immature stages of two tick species on four species of small birds in the eastern cape province, south africa and in a more recent study van niekerk, fourie & horak (2006) recorded ticks on 39 species of birds in the free state province, south africa. amongst the latter there were 28 different passerine species. our objective in the present study was to provide additional data on ticks and the species of birds that they might infest, as well as on the bird species that could be important in spreading ticks. materials and methods the bird-ringing excursions of amateur ornithologists provide a valuable opportunity for the collection of ectoparasites from the birds they have captured. this study was carried out in collaboration with the members of the pretoria bird ringing club during their activities at various localities in the provinces of gauteng, limpopo, mpumalanga and north west, south africa (table 1). the birds were caught in mist nets and examined for ticks using headmounted magnifying glasses. the examination concentrated on the head of the birds, especially the eyelids, around the beak and in the ears, where the vast majority of ticks usually occur on passerines (mehl et al. 1984; smith 2001). the bare skin under the wings, the brood patch and the region around the cloaca were also frequently examined, but these sites yielded no ticks. the examination for ticks increased the handling time of the birds by about one minute. ticks and other ectoparasites were collected by means of forceps and placed in separate vials containing 70 % ethanol, together with a label recording date, ring number, locality and bird species. we have followed the nomenclature proposed by hockey, dean & ryan (2005) for the birds we examined, and their migration and feeding habits are summarized in appendix 1. approximately 3 000 birds were examined, but unfortunately the documents recording the exact number were lost. results the method of trapping birds in mist nets is suitable only for small birds, and most of the birds caught were passerines. out of a total of approximately 3 000 birds examined, belonging to 43 species, we recovered 178 ticks from 83 of them. the ticks comprised 48 larvae, 124 nymphs and six adults belonging to 14 species (table 2). among these were one argas, one hyalomma and an ixodes species that we could not identify. the birds that harboured most ticks were olive thrush and cape robin-chat. the most numerous tick collected was hyalomma rufipes, comprising 26 larvae and 109 nymphs. the second most common species was amblyomma table 1 localities at which ticks were collected from birds in the northern provinces of south africa no. locality coordinates 1 2 3 4 5 6 7 8 9 10 11 12 13 buffelsdrift, pretoria colbyn, pretoria groenkloof, pretoria jaleoda ntsinini, sagewood cottage nylsvley olifantskop rietvlei samrand, midrand suikerbosrand nr, kareekloof gate sunbird hill, kameelfontein wakkerstroom retirement forest, wakkerstroom 25°35’ s 25°44’ s 25°47’ s 25°57’ s 25°36’ s 24°39’ s 23°58’ s 25°55’ s 25°55’ s 26°31’ s 25°38’ s 27°21’ s 27°18’ s 28°20’ e 28°15’ e 28°12’ e 28°35’ e 30°23’ e 28°42’ e 27°28’ e 28°18’ e 28°08’ e 28°10’ e 28°24’ e 30°06’ e 30°19’ e 169 g. hasle et al. table 2 ticks collected from 83 infested birds in the northern provinces of south africa (immature stages when not otherwise indicated) tick and bird species bird species names no. localities* birds ticks argas species cardinal woodpecker dendropicos fuscescens 1 1 7 amblyomma hebraeum swainson’s spurfowl southern yellow-billed hornbill double-banded sandgrouse pternistis swainsonii tockus leucomelas pterocles bicinctus 1 2 1 1 2 1 1 7 7 amblyomma marmoreum double-banded sandgrouse cattle egret brown-crowned tchagra red-headed weaver red-billed quelea cinnamon-breasted bunting pterocles bicinctus bubulcus ibis tchagra australis anaplectes melanotis quelea quelea emberiza tahapisi 1 1 2 1 1 6 2 2 2 1 1 7 7 1 7, u 7 7 7 haemaphysalis elliptica rattling cisticola levailant’s cisticola long-tailed widowbird cisticola chiniana cisticola tinniens euplectes progne 1 2 1 1 3 1 1 3, 12 8 haemaphysalis hoodi striped pipit1 anthus lineiventris 1 1 5 hyalomma glabrum red-billed quelea cinnamon-breasted bunting quelea quelea emberiza tahapisi 1 1 1 1 7 7 hyalomma rufipes striped kingfisher red-faced mousebird laughing dove brown-crowned tchagra southern boubou crimson-breasted shrike common fiscal magpie shrike southern black tit ashy tit dark-capped bulbul black-chested prinia sabota lark kurrichane thrush olive thrush marico flycatcher southern black flycatcher cape robin-chat wattled starling halcyon chelicuti urocolius indicus streptopelia senegalensis tchagra australis laniarius ferrugineus laniarius atrococcineus lanius collaris corvinella melanoleuca parus niger parus cinerascens pycnonotus tricolor prinia flavicans calendulauda sabota turdus libonyanus turdus olivaceus bradornis mariquensis melaenornis pammelaina cossypha caffra creatophora cinerea 1 1 1 3 2 1 1 1 2 3 2 1 1 2 2 2 2 7 1 3 1 1 13 2 1 1 1 4 7 2 1 2 2 26 5 3 25 1 11 7 7 1, 7, 10 1, 5 7 1 1 7 7 3 10 7 3, 5 10 7 6, u 1, 10, u 10 170 ticks collected from birds in northern provinces of south africa, 2004–2006 marmoreum, with 13 larvae and two nymphs. five specimens of haemaphysa lis elliptica were also recovered, one of these from a rattling cisticola and three from levailant’s cisticola. no other ticks were found on cisticola. between one and four specimens of the remaining tick species were collected. amblyomma hebraeum was not found on any of the smaller birds, but was present on swainson’s spurfowl, southern yellow-billed hornbill and doublebanded sandgrouse. a single rhipicephalus (boophilus) decoloratus larva was collected from a three-banded plover. the only adult ticks recovered were ixodes spinae, ixodes theilerae and rhipicephalus turanicus. the sole argas specimen was a larva collected from a cardinal woodpecker. tick and bird species bird species names no. localities* birds ticks lesser masked-weaver southern masked-weaver red-billed quelea blue waxbill pin-tailed whydah cape longclaw ploceus intermedius ploceus velatus quelea quelea uraeginthus angolensis vidua macroura macronyx capensis 1 4 3 1 1 1 1 19 3 1 1 9 7 7, 11 4, 7 1 7 10 hyalomma species cinnamon-breasted bunting emberiza tahapisi 1 1 7 ixodes pilosus group levaillant’s cisticola yellow-crowned bishop cisticola tinniens euplectes afer 1 1 1 1 2 4 ixodes species bar-throated apalis cape robin-chat blue waxbill apalis thoracica cossypha caffra uraeginthus angolensis 2 1 1 2 1 1 5 5 11 ixodes spinae southern red bishop1, 2 euplectes orix 1 1 9 ixodes theilerae cape batis2 olive thrush3 cape weaver1, 2 batis capensis turdus olivaceus ploceus capensis 1 1 1 1 1 1 13 10 10 rhipicephalus (boophilus) decoloratus three-banded plover charadrius tricollaris 1 1 1 rhipicephalus turanicus marsh owl4 asio capensis 1 3 6 * numbers in this column refer to localities in table 1 1 tick species identity uncertain 2 adult tick: female 3 the same bird also carried two nymphs of h. rufipes 4 adult ticks: one female, two males u = unknown 171 g. hasle et al. discussion most ticks were recovered from birds that feed mainly on the ground, in particular members of the family turdidae. this agrees with the results of an earlier study conducted elsewhere (olsén et al. 1995). only a few of the mainly arboreal bird species that we examined harboured ticks, e.g. the dark-capped bulbul and the lesser maskedweaver (see appendix 1). because these species are often caught in nets, it is sometimes possible to detect even a low rate of tick infestation on them. after an exhaustive study of numerous specimens of all stages of development of the subspecies of the hyalomma marginatum group, apanaskevich & horak (2008) concluded that these ticks should be treated as independent species, namely h. margiappendix 1 species characteristics of the tick-infested birds in the study (hockey et al. 2005) bird species migration habits feeding habitats swainson’s spurfowl* resident and sedentary on ground cardinal woodpecker* resident and sedentary in trees southern yellow-billed hornbill* resident and sedentary. rarely forms small flocks during dry season and drought mainly on the ground striped kingfisher* resident, with some local movement mainly on ground in arid areas red-faced mousebird* generally resident, locally nomadic in response to phenology of fruiting trees. altitudinal migrant flowers and fruits marsh owl* resident where habitat is stable, otherwise nomadic lives on ground, eats rodents laughing dove* largely sedentary with some local nomadic movements open ground double-banded sandgrouse* sedentary. may move in search of water on ground three-banded plover* sedentary. partial intra-african migrant in response to seasonal rainfall on open shores cattle egret* moves over large distances on ground brown-crowned tchagra resident and sedentary on ground southern boubou resident and sedentary on ground crimson-breasted shrike resident and sedentary, but may move locally to riverine woodland during non-breeding season on ground and in trees cape batis resident. altitudinal migration in trees common fiscal mostly resident and sedentary, possibly nomadic small prey usually eaten on ground magpie shrike resident and generally sedentary, but may move locally in response to drought and fires mostly on ground southern black tit resident mainly in trees, eats earthworms after rain ashy tit resident and locally nomadic in bushes, less frequently on ground dark-capped bulbul sedentary. some local dispersal linked to food availability in trees, occasionally on ground rattling cisticola resident low in grass or bushes, or on ground levailant’s cisticola mostly resident, may undertake local movements low down in vegetation 172 ticks collected from birds in northern provinces of south africa, 2004–2006 natum, h. rufipes, h. isaaci and h. turanicum, and we have followed their recommendation. hyalomma rufipes was the most prevalent tick species on small birds in our survey as well as in other studies (hoogstraal et al. 1961; van niekerk et al. 2006). adult h. rufipes feed on large ungulates (norval 1982), and the immature stages feed on birds (horak et al. 1991b; uys & horak 2005; van niekerk et al. 2006), and hares (horak & fourie 1991). it is patchily distributed in africa, europe and western and central asia (walker, bouattour, camicas, estrada-peña, horak, latif, pegram & preston 2003), and is the most important vector of c rimean-congo haemorrhagic fever (cchf) virus to humans in south africa bird species migration habits feeding habitats black-chested prinia resident. probably locally nomadic in bushes, less frequently on ground bar-throated apalis resident and sedentary. some winter movements to lower altitudes partly on ground sabota lark resident and sedentary, locally nomadic in drier part of range on ground kurrichane thrush mainly resident. some altitudinal migration on ground olive thrush mostly resident, altitudinal migrant, and in response to drought mostly on ground marico flycatcher resident mostly on ground southern black flycatcher resident partly on ground cape robin-chat altitudinal migrant spends much time on ground wattled starling nomadic mostly on ground lesser masked-weaver resident, sedentary and local nomad in tree canopies cape weaver mostly resident and sedentary, with some local movements. 5% move >100km on ground southern masked-weaver resident, sedentary and partial migrant on ground, grass stems and trees red-headed weaver in botswana, moves out of deciduous woodland in dry season; in zimbabwe, range contracts in non-breeding season mainly in trees, bushes and creepers red-billed quelea large scale movements throughout range on ground yellow-crowned bishop resident and locally nomadic. migratory in west africa on ground or directly from plants southern red bishop resident and sedentary, some local movement in non-breeding season both on ground and perched in vegetation long-tailed widowbird resident and sedentary, local movement in nonbreeding season. largely on ground blue waxbill mostly resident. may move nomadically in winter on ground and in vegetation pin-tailed whydah resident and sedentary. nomadic in non-breeding season eat seeds on ground cape longclaw resident. form groups on burnt ground in winter on ground striped pipit resident and sedentary, possibly with some local movement on ground cinnamon-breasted bunting resident, but migrant from nov-dec to april-may on ground * = non-passerine species 173 g. hasle et al. (horak, swanepoel & gummow 2002). in africa it may also transmit anaplasma marginale, rickettsia conorii and babesia occultans (walker et al. 2003). unlike other tick species, of which the immature stages tend to infest mainly larger birds, h. rufipes is found on passerines (cumming 1998) as well as on larger species such as crested francolin and helmeted guineafowl (horak et al. 1991b; uys and horak 2005). it is a two-host tick, which, like its close relative h. marginatum, probably remains attached to the host for 12 to 26 days from the start of feeding of the larva to detachment of the engorged nymph (hueli 1979). this prolonged period of attachment plays an important role in the long-distance transportation of ticks with their associated tick-borne pathogens. the immature stages of hyalomma glabrum, which we collected from two birds, infest hares and birds (apanaskevich & horak 2006). this tick, which was previously thought to be hyalomma turanicum, a known vector of cchf, has recently been re-established as a valid species (apanaskevich & horak 2006). adult a. marmoreum, the second most common species recovered in this study, feed nearly exclusively on tortoises (horak, mckay, heyne & spickett 2006). its immature stages feed on a wide range of hosts, including tortoises and birds (horak et al. 2006; van niekerk et al. 2006). this tick may play a role in the transmission of ehrlichia ruminantium to domestic ruminants (norval & horak 2004). amblyomma hebraeum, which in the present study was collected from three of the larger bird species, is the major vector of e. ruminatium in south africa (norval & horak 2004). it also transmits theileria mutans to cattle, and rickettsia africae to humans. its distribution is confined to south-eastern africa (walker et al. 2003), and it is the tick species of which the immature stages have most often been recorded biting humans in south africa (horak, fourie, heyne, walker & needham 2002). the adults prefer large ungulates, while the immature stages parasitize large and small ungulates as well as large ground living birds (walker et al. 2003). within its distribution range the immature stages of a. hebraeum are the most common ticks found on helmeted guineafowl (horak & williams 1986; horak et al. 1991b), and they are also common on crested francolin (uys & horak 2005). despite large numbers of birds being examined in earlier studies, adult ticks were not encountered on them (horak & williams 1986; horak et al. 1991b; uys & horak 2005). amblyomma hebraeum is apparently not spread by small birds, as no ticks of this species were collected from passerines and other small birds in our study, even though it was conducted within the distribution range of the tick. haemaphysalis elliptica (formerly h. leachi) (apanas kevich, horak & camicas 2007), the third most common tick collected (table 2), is a major vector of babesia canis to dogs, and can transmit r. conorii to humans. it is a parasite of large domestic and wild carnivores, and is widespread in southern africa (apanaskevich et al. 2007). true h. leachi is found in the nile delta and north-east africa (walker et al. 2003). the immature stages of h. elliptica are not common parasites of birds. van niekerk et al. (2006) recovered a single nymph from birds in free state province, while horak et al. (1991b) collected only three larvae and two nymphs from 118 helmeted guineafowl, as opposed to 23 778 a. hebraeum and 2 387 a. marmoreum larvae and nymphs from the same birds in a habitat in which all three tick species were abundant. it is not known whether haemaphysalis hoodi and i. theilerae, which parasitize birds, and i. spinae which infests birds, hyraxes and rodents (cumming 1998), or ixodes pilosus, which parasitizes wild and domestic ungulates and dogs, transmit any pathogens (walker et al. 2003). rhipicephalus (boophilus) decoloratus transmits babesia bigemina, a. marginale and borrelia theileri, the first two of which are causes of cattle diseases of immense veterinary importance in south africa. this is a one-host tick (walker et al. 2003), and should be considered an accidental parasite of birds, on which it is unlikely to complete its life cycle. rhipicephalus turanicus belongs to the rhipicephalus sanguineus complex, and could be a vector of rickettsiae of the spotted fever group (matsumoto, ogawa, brouqui, raoult & parola 2005). large carnivores and large ground-living birds are hosts of the adults of this species (walker, keirans & horak 2000), which we collected from a marsh owl. argasids are typically endophilic (burrow or nestdwelling) (hillyard 1996), and this may explain why an argas species was found on a woodpecker. we found no tick species in this study that had not previously been recorded in the same stage of development on birds (cumming 1998; walker et al. 2003). our results agree with those of previous surveys in that the immature stages of h. rufipes are the most common tick species found on passerines and other small land birds, followed by a. mar174 ticks collected from birds in northern provinces of south africa, 2004–2006 moreum, while other ticks seem to be rare or occasional parasites (horak et al. 1991a; van niekerk et al. 2006). strangely, none of the ticks recovered in this study were from migratory birds that breed in temperate regions, and which travel large distances every day during the migration seasons. few south african birds migrate regularly. red-billed quelea and cinnamon-breasted bunting are nomadic when not breeding (sinclair, hockey & tarboton 2002), and may therefore be important in long distance dispersal of ticks. sedentary birds like cape robin-chat may move within their distributional range during winter, or to kwazulu natal, where they are seen as winter visitors (sinclair et al. 2002). some species are altitudinal migrants, e.g. bar-throated apalis and cape robin-chat (hockey et al. 2005). almost all bird species move about in search of food and water, particularly during harsh environmental conditions, and may thereby also transport ticks. conclusion birds that feed on the ground are predisposed to tick infestation, but there are also considerable differences among tick species in their predisposition and ability to infest birds. the immature stages of h. rufipes and a. marmoreum infest small birds, like passerines, but they may also infest larger birds. conversely the immature instars of a. hebraeum infest larger birds, but apparently not passerines. the overall widespread distribution of h. rufipes may in part be ascribed to its tendency to infest passerines. ticks (including acaricide-resistant ticks) with their associated tick-borne pathogens may be dispersed over large distances via bird migration. smaller birds, through their huge numbers, may play a role as hosts for ticks, but no ticks of medical or veterinary importance seem to use small birds as maintenance hosts. acknowledgements this survey is a part of an ectoparasite study approved by the ethical and scientific review committee of the university of limpopo (medunsa campus). g. hasle’s participation in the study was supported by a travel grant from abbott, norway. references apanaskevich, d.a., & horak, i.g. 2006. the genus hy alomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum delpy, 1949 (acari, ixodidae) as a valid species with a redescription of the adults, the first description of its immature stages and notes on its biology. onderstepoort journal of veterinary research, 73:1–12. apanaskevich, d.a., horak, i.g. & camicas, j-l. 2007. redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haemaphysalis (rhipistoma) leachi (audouin, 1826) (ixodida, ixodidae). onderstepoort journal of veterinary research, 74: 181–207. apanaskevich, d.a. & horak, i.g. 2008. the genus hyalomma koch, 1844. v. re-evaluation of the taxonomic rank of taxa comprising the h. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice verwoerd_99-102.indd introduction arnold theiler had already proved the filterability of the etiological agent, and therefore its viral nature, in 1905. he had serially passaged the virus in sheep, believed that it had become attenuated and started using the blood of infected sheep as a primitive vaccine in 1906. as a result bluetongue vaccine became the third viral vaccine produced at onderstepoort, following theiler’s famous rinderpest and human smallpox vaccines. later it was realized that theiler was in fact using a relatively avirulent strain of the virus. although the vaccine gave variable results it must have been reasonably effective as more than one million doses were already sold in 1916 and it was used with only minor improvements until the 1940s. a full description of the disease bluetongue can be found elsewhere (coetzer & tustin 2004) as well as brief historical reviews of research on bluetongue at onderstepoort (bigalke & verwoerd 2008; verwoerd & bigalke 2008). key discoveries and milestones adaptation to cultivation in embryonated eggs following the successful cultivation and attenuation of african horsesickness virus (ahsv) in mouse brains, several attempts during the 1930s to repeat it with bluetongue virus (btv) failed. eventually, in 1940, mason, coles & alexander managed to adapt the virus to growth in embryonated hen’s eggs and also demonstrated attenuation in this system. however, serial passage was difficult and growth erratic. nevertheless, they managed to obtain evidence for the existence of different strains, as was the case for ahsv, and by 1942 six strains have been identified, three of which had been adapted to cultivation in eggs. discovery of the insect vector of bluetongue in 1944, the seminal discovery that members of the orbivirus genus, including both btv and ahsv, is transmitted by culicoides midges was made by r.m. du toit, a veterinary entomologist, at onderste99 onderstepoort journal of veterinary research, 76:99–102 (2009) history of bluetongue research at onderstepoort d.w. verwoerd faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa abstract verwoerd, d.w. 2009. history of bluetongue research at onderstepoort. onderstepoort journal of veterinary research, 76:99–102 research on this economically important disease of ruminants, especially sheep, which had been named bluetongue by farmers in the 19th century, has been part and parcel of the activities at onderstepoort ever since its establishment in 1908 and therefore covers a full century of the ovi’s existence. in view of onderstepoort’s centenary celebration a brief overview of this research is given in terms of the historic milestones which influenced and guided global research on this and other viral diseases of animals. 100 history of bluetongue research at onderstepoort poort. farmers had known for many years that stock losses caused by these viruses could be limited by keeping animals indoors after sundown and by avoiding wetland pastures, which suggested the involvement of an insect vector. the development of a light trap which facilitated the catching of large numbers of these tiny insects finally enabled du toit to prove the biological transmission of both viruses by c. variipennis (= imicola). development of an avianized vaccine the discovery by alexander in 1947 that the optimal temperature for the cultivation of btv in embryonated eggs is 33.5 °c and not 37 °c solved the problem of viral propagation. soon a number of btv strains were attenuated and in the same year a trivalent (soon followed by a quadrivalent) live attenuated vaccine was launched, replacing the old theiler vaccine. initially, however, the vaccine was still produced in sheep. the product was safer and more effective, leading to an increased demand which could not be met using sheep for its production. the vaccine was therefore produced in embryonated eggs but still suspended in sheep’s blood to extend its shelf life. immunity and cross-protection immunological studies carried out by neitz in 1948 on the available btv strains clearly demonstrated for the first time that immunization with a particular strain resulted in lifelong solid immunity to the specific strain but little or no immunity to other strains. it explained the vaccine failures encountered with the monovalent theiler vaccine and also formed the basis for later studies on the serotyping of isolates and for the development of multivalent vaccines giving maximum protection under conditions of varying pre ponderance of different strains. lyophilization to the rescue in 1950, when alexander took over as director of the ovi, he was confronted by a crisis in terms of a short-supply of bluetongue vaccine. following particularly heavy rainfall and a dramatic increase in the price of wool and wool-producing sheep, the demand for vaccine suddenly jumped from 2.5 million doses the previous year to over 6 million. there was a shortage of eggs and low fertility in those available and to make things worse the vaccine produced in embryonated eggs had a short shelf life. to solve the latter problem lyophilisation was introduced, facilitated by the discovery that buffered lactose-peptone is an excellent stabilizer of the virus. vaccine could subsequently be stockpiled for periods of greater demand and production increased to more than 24 million doses by 1958. internationalization of the problem the outbreak of bluetongue in cyprus in 1943 was the first report of the disease outside africa. in 1952 onderstepoort also confirmed the presence of bt in israel. in the same year, a condition named ‘sore muzzle’ was described in california and its similarity to bluetongue noticed. in 1953 alexander was invited to the usa for expert advice and he was able to confirm the diagnosis based on preserved material. the virus was subsequently isolated at onderstepoort and later identified as serotype 10. in 1956 bt outbreaks also occurred in portugal and spain and the virus identified as btv serotype 10. cultivation in cell cultures the next major achievement was the cultivation of the egg-adapted btv in primary lamb kidney cells by haig and co-workers in 1956. it first led to the development of a group-specific complement-fixation test and later to a practical and relatively inexpensive neutralization test used by howell for serotyping isolates. by 1960 he had described 12 and by 1970, 16 distinct serotypes. onderstepoort was in a unique position to assist other countries in identifying and typing the virus responsible for their outbreaks and was appointed in 1963 by the oie as a world reference centre for bluetongue, a responsibility it still carries today. vaccine production in cell cultures passaging the various serotypes in cell cultures and testing for attenuation and immunogenicity was the logical next step in order to convert vaccine production from eggs to cell cultures. the number of serotypes in the polyvalent vaccine, first released in the early 1960s, increased rapidly and by 1968 numbered 14. in the same year production was switched to bhk21 cells in roller cultures, another major improvement. however, protection obtained with the polyvalent vaccine was unsatisfactory due to interference between strains and variation in immunogenicity. for the next decade or so erasmus and weiss concentrated on solving the problem by plaque-purifying each serotype and selecting suitable vaccine strains. by 1982, 15 such strains had been isolated and was incorporated into three pentavalent vaccines which gave a good immunity when correctly administered. 101 d.w. verwoerd molecular biology of bluetongue virus btv was selected for molecular studies by verwoerd in 1964 because so little was known about the structure and replication of this economically important virus. mass production and purification of the virus was the first step before morphological studies and biochemical analysis could start. surprisingly electron microscopy revealed two differently sized particles first thought to be two viruses. at the time all known viruses consisted of a protein capsid containing a genome which was either double-stranded or single-stranded dna or single-stranded rna. our chemical analysis clearly indicated rna but physico-chemical characteristics were those of dna. the conclusion that the virus has a double-stranded rna genome was rather revolutionary and not easily accepted at first. in addition, it was found that the genome consists of ten segments and that the virus possesses a double-layered protein capsid. it was, therefore, clear that we were dealing with a uniquely structured virus, which later gave rise to its classification in a new genus orbivirus (verwoerd, els, de villiers & huismans 1972). molecular genetics during the 1970s, molecular research concentrated on genetic aspects such as the relationship between the ten genome segments and the viral proteins. it was shown that seven of the segments act as genes coding for viral proteins, the other three for non-viral proteins found in the infected cell. much effort was spent on elucidating the role of the various components in the replication of the virus in an attempt to better understand the pathogenesis of the disease. it was also the decade during which many new techniques such as cloning and sequencing of nucleic acids were developed which collectively became known as biotechnology. these techniques were also applied to btv and led inter alia to the development of new diagnostic tests using cloned genome segments as diagnostic probes. first recombinant subunit vaccines the 1980s were characterized by various attempts to develop recombinant subunit vaccines and the further development of improved diagnostic techniques. considerable excitement was created when huismans, van der walt, cloete & erasmus (1987) demonstrated the immunogenicity of a single outer capsid protein (vp2), supporting the feasibility of developing recombinant vaccines. genome libraries were constructed and the first recombinant vaccines developed in collaboration with researchers at oxford, using the baculovirus of insects as vector. various combinations were shown to be effective as monovalent vaccines but unfortunately problems encountered in upscaling production has so far precluded its commercial application. advanced diagnostics during the 1990s, the cloning and sequencing of the ten genome segments of all 21 south african serotypes continued. cross-hybridization of these dna clones identified segment five as the most suitable group-specific probe as it is highly conserved in all serotypes, whereas segment two, coding for an outer protein, is best for serotype-specific probes. in 1991 a series of sequencing studies on btv isolates from various countries led to the discovery of ‘topotypes’, or geographical variants within serotypes which can be used to determine the origin of an outbreak. improving diagnostic tests started to dominate the scene during this period, following the disappointing progress with the development of new generation vaccines. globalization of the disease between 1956 and 1999 limited outbreaks of bt occurred in a number of countries, mostly in the middle east, south america and southern europe. the new millennium with its climate change and increased mobility of man and animals brought about an explosion of new outbreaks and the involvement of northern europe for the first time. since 1999 there have been outbreaks in greece, italy, corsica, the balearic islands and various balkan countries, involving serotypes 2, 4, 9 and 16. in 2002 bt 4 was also reported in spain and portugal. the live attenuated bt vaccine from obp was used in different countries: bt 2, 4, 9 and 16 in italy, bt 2 and 4 in corsica, and bt 2 and 4 in spain and portugal. since 2006 bt 8 has emerged in northern europe, presumably due to global warming, also infecting cat tle in the nether lands, belgium, germany, luxem bourg and france. in the summer of 2007, bt 8 also spread to the uk, switzerland, denmark, spain, poland and italy. it is clear that in spite of all our accumulated knowledge further research is needed to finally conquer the disease. references alexander, r.a. 1947. the propagation of bluetongue virus in the developing chick embryo with particular reference to the temperature of incubation. onderstepoort journal of vet erinary science and animal industry, 22:7–26. 102 history of bluetongue research at onderstepoort bigalke, r.d. & verwoerd, d.w. 2008. a century of research achievements by the onderstepoort veterinary insti tute. transactions of the royal society of south africa, 63:31–51. coetzer, j.a.w. & tustin, r.c. 2004. (eds). infectious diseases of livestock, 2nd ed. cape town: oxford university press. du toit, r.m. 1944. the transmission of bluetongue and horsesickness by culicoides. onderstepoort journal of veterinary science and animal industry, 19:7–16. haig, d.a., mckercher, d.g. & alexander, r.a. 1965. the cytopathogenic action of bluetongue virus on tissue cultures and its application to the detection of antibodies in the serum of sheep. onderstepoort journal of veterinary research, 27:171–177. howell, p.g. 1960. a preliminary antigenic classification of strains of bluetongue virus. onderstepoort journal of veterinary research, 28:357–363. huismans, h., van der walt, n.t., cloete, m. & erasmus, b.j. 1987. isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep. virology, 157:172–179. mason, j.h., coles, j.d.w.a. & alexander, r.a. 1940. cultivation of bluetongue virus in fertile eggs produced on a vitamin deficient diet. nature, 145:1022–1023. neitz, w.o. 1948. immunological studies on bluetongue in sheep. onderstepoort journal of veterinary science and ani mal industry, 23:93–36. verwoerd, d.w., els, h.j., de villiers, e.m. & huismans, h. 1972. structure of the bluetongue virus capsid. journal of virology, 10:783–794. verwoerd, d.w. & bigalke, r.d. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: chikera s. ibe1 barth i. onyeanusi1 joseph o. hambolu1 affiliations: 1department of veterinary anatomy, ahmadu bello university, nigeria correspondence to: chikera ibe postal address: department of veterinary anatomy, ahmadu bello university, zaria 2222, nigeria dates: received: 12 july 2013 accepted: 02 sep. 2013 published: 28 mar. 2014 how to cite this article: ibe, c.s., onyeanusi, b.i. & hambolu, j.o., 2014, ‘functional morphology of the brain of the african giant pouched rat (cricetomys gambianus waterhouse, 1840)’, onderstepoort journal of veterinary research 81(1), art. #644, 7 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.644 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. functional morphology of the brain of the african giant pouched rat (cricetomys gambianus waterhouse, 1840) in this original research... open access • abstract • introduction • materials and methods    • experimental animals and management    • brain extraction and gross morphological assessment • results    • telencephalon    • diencephalon    • mesencephalon    • metencephalon    • myelencephalon • ethical considerations • discussion • conclusion • acknowledgement    • competing interests    • authors' contributions • references abstract top ↑ a gross morphological study of the brain of the african giant pouched rat (cricetomys gambianus waterhouse, 1840) was undertaken in order to document its normal features and assess the structure-function paradigm. the study was conducted by direct observation of 29 adult african giant pouched rats’ brains. in the telencephalon, the cerebral cortex was devoid of prominent gyri and sulci, but the large olfactory bulb and tract relaying impulses to the olfactory cortex were very prominent. the large size of the olfactory bulb correlated with the established sharp olfactory acuity of the rodent. in the mesencephalic tectum, the caudal colliculi were bigger than the rostral colliculi, indicating a more acute sense of hearing than sight. in the metencephalon, the cerebellar vermis, the flocculus and the paraflocculus were highly coiled and, thus, well developed. the myelencephalon revealed a better organised ventral surface than dorsal surface; the cuneate fascicle, the intermediate sulcus and the lateral sulcus were not evident on the dorsal surface, but there were clearly visible pyramids and olivary prominence on the ventral surface. in conclusion, the highly coiled cerebellar vermis, flocculus and paraflocculus, as well as the conspicuous pyramids and olivary prominence are indicative of a good motor coordination and balance in the african giant pouched rat. introduction top ↑ the brain is the control centre of the vital activities that are necessary for survival. it receives sensory impulses from the sensory organs through the spinal cord and cranial nerves, processes these impulses and initiates motor outputs to effector organs. thus, the brain is the organ that is specialised in coordinating activities in relation to changes in the internal and external environments.the adult brain is divided into the forebrain (telencephalon and diencephalon), midbrain (mesencephalon) and hindbrain (metencephalon and myelencephalon). the brain can also be divided into the cerebrum, cerebellum and brainstem. the cerebrum attaches to the brainstem by the cerebral peduncles, while the cerebellum attaches to the brainstem by the cerebellar peduncles. the cerebrum is the main constituent of the telencephalon, while the thalamus, epithalamus, metathalamus and hypothalamus make up the diencephalon (delahunta & glass 2009). the metencephalon is made up of the cerebellum and the pons, also called pons varolii after costanzo varolio (1543–1575), the italian anatomist who discovered it (tubbs et al. 2008). the myelencephalon is composed of the medulla oblongata and caudal portion of the fourth ventricle (darly 1965; wingerd 1988). the cerebrospinal fluid, which provides mechanical protection and nourishment to the brain, circulates through the two lateral ventricles in the telencephalon, the third ventricle in the diencephalon and the fourth ventricle in the hindbrain. the macroscopic appearance of the brain differs in mammals, depending on their different adaptations and lifestyles. for example, animals that lack brachiation as a mode of locomotion, such as members of the order artiodactyla, lack a visible olivary body (adogwa 1985; ghaji 1983; marsden & rowland 1965). the african giant pouched rat (cricetomys gambianus waterhouse, 1840) has been successfully used to detect land mines in mozambique (mott 2004) and diagnose tuberculosis in europe (maggie 2003). it is able to do this because of its high acuity of olfaction, which is a function of the rhinencephalon. also, the poor sense of sight of the rodent, which is a reason for its nocturnal behaviour, has been attributed to the small rostral colliculi, relative to the caudal colliculi (ibe et al. 2010). there is lack of information on the functional morphology of the brain of the african giant pouched rat. therefore, the aim of this research was to study the gross morphology of the brain of the african giant pouched rat and propose some of its morpho-functional paradigms. materials and methods top ↑ experimental animals and management twenty-nine captive and clinically healthy adult african giant pouched rats were used for this study. the animals were captured live from the wild in kaduna state, nigeria, using locally made traps. the traps were made of galvanised metal and were 1.3 m x 0.3 m x 0.1 m in dimension. the rats were transported by road in laboratory cages to the animal pen of the department of veterinary anatomy, ahmadu bello university, zaria; they were acclimatised for one month before the commencement of the experiment. during this period, they were physically examined in the cage under careful restraint. only healthy rats were utilised for this study. the animals were given access to food and drinking water ad libitum throughout the experimental period. brain extraction and gross morphological assessment the body weight of each rat was obtained using a digital electronic balance (citizen scales [1] pvt ltd., switzerland), with a sensitivity of 0.01 g. immediately after chloroform euthanasia in a closed container, physiologic saline solution was used to perfuse the brain through the common carotid artery, in order to flush the brain free of blood. this procedure was immediately followed by perfusion of the brain with 10% phosphate-buffered formalin. each skull was exposed after skinning and stripping off all the facial muscles within 30 minutes of euthanasia. craniotomy, through the calvaria, exposed the dura mater, which was cut with a pair of curved pointed scissors. the falx cerebri and tentorium cerebelli were both pulled from the longitudinal and transverse fissures of the brain, respectively, by gentle traction. the cerebral vein was cut with a pair of sharp scissors. at this stage, the brain (still within the cranium) was fixed in 10% phosphate-buffered formalin for three days; this was done to facilitate its easy extraction according to the method of ramaswamy (1978) (using delicate rongeurs, spatulas, forceps and scissors). the extracted brain was then examined and found to be devoid of any macroscopic pathology. the structural characteristics of the different components of the brain were examined with the naked eye. these included their shape, size, surfaces, borders and angles. the presence or absence of sulci, fissures or prominences was also evaluated. finally, the projections of the different cranial nerves from the myelencephalon were observed with both the naked eye and with the aid of a hand lens. definitions of gross anatomical structures were based on standard information on rodent anatomy (parker & haswell 1974; olds & olds 1979; suckow, weisbroth & franklin 2006). nomina anatomica veterinaria (2005) was used for the nomenclature. results top ↑ the gross anatomical features observed in the present study were consistent in all brain samples that were dissected. telencephalon on a dorsal view of the intact brain (figure 1), the two cerebral hemispheres or neocortex were distinctly divided by a longitudinal fissure and separated from the cerebellum by a transverse fissure. each neocortex was large, diamond-shaped, narrow rostrally and wider caudally. it was devoid of prominent gyri and sulci; however, the bulging of the frontal lobe was evident. the olfactory bulbs were also very prominent and visible from the dorsal view. they were roughly pyramidal in shape. figure 1: dorsal view of the brain of the adult african giant pouched rat. on a ventral view (figure 2), the large olfactory bulbs and tracts relaying impulses to the olfactory cortex were evident. the olfactory nerve fibres, which were rostral to the bulbs, could not be accessed due to their position in the cribriform plate of the ethmoid bones. the large size of the olfactory bulbs was of interest, owing to their importance in the sense of smell of the rodent. the olfactory bulbs gave off the medial, lateral and middle olfactory tracts that conveyed sensory impulses to the olfactory cortex (figure 2: b, c and d). the lateral olfactory tracts continued into their ipsilateral pyriform lobes (figure 2: i). figure 2: ventral view of the brain of the african giant pouched rat. diencephalon on a dorsal view of the brainstem, the medial surface of the thalamus formed the lateral boundary of the third ventricle (figure 3: b and c). caudal to the thalamus and lateral to the rostral colliculus was a small protuberance, the lateral geniculate body. the caudal border of the thalamus was wider than the rostral border, thus overlapping the rostral colliculus and occluding the lateral geniculate body from the dorsal view of the brainstem (figure 3). the rostral colliculus was continuous with the lateral geniculate body through its brachium. below the lateral geniculate body and lateral to the caudal colliculus was the medial geniculate body (figure 3: f). the caudal colliculus was also continuous bilaterally with the medial geniculate body through its brachium. however, the brachia were not clearly visible. the lateral geniculate body was apparently more distinct than the medial geniculate body. the geniculate bodies made up the metathalamus of the diencephalon. the pineal gland (part of the epithalamus) was very small in the african giant pouched rat. figure 3: dorsal view of the brainstem of the african giant pouched rat. ventrally (figure 2), the paired optic nerves (cranial nerve [cn] ii) united at the optic chiasma and gave off optic tracts that ran on the rostral boarder of the tuber cinerium. they were the fourth largest diameter cranial nerve after the trigeminal nerve (cn v), facial nerve (cn vii) and olfactory nerve (cn i) in all of the dissected animals. the oculomotor nerve (cn iii) and trochlear nerve (cn iv) were lost during dissection due to their minute size. the mamillary bodies (figure 2: l) were relatively large, interposed between the pyriform lobes laterally, related to the optic chiasma rostrally, and separated from the pons by the cerebral peduncle and the mesencephalon in its caudal portion. mesencephalon dorsally, the mesencephalon separated the third ventricle from the fourth ventricle (figure 3), while ventrally it extended from the caudal portion of the mamillary body to the most rostral part of the pons (figure 2). the mesencephalic aqueduct passed through the midbrain and connected the third ventricle cranially to the fourth ventricle caudally. the mesencephalic tectum formed the roof of the mesencephalon. it was composed of the corpora quadrigemina. rostral to the corpora quadrigemina was the third ventricle and caudal to the corpora quadrigemina was the fourth ventricle (figure 3). the eminence of the corpora quadrigemina was composed of a pair of rostral colliculi and a pair of caudal colliculi (figure 3: d and e); a transverse fissure separated both. a longitudinal fissure separated each pair of colliculi. the caudal colliculi were oval in shape, while the rostral colliculi were conical. the caudal colliculi were visibly bigger than the rostral colliculi. on the dorsal view of the intact brain, the caudal colliculi were visible through the transverse fissure (figure 1: d), which was suggestive of their large size relative to the rostral colliculi.on sagittal view of the brain (figure 4) the corpus callosum was well developed, broad and sickle-shaped; it was well delineated into the rostrum, genu, truncus and splenium (figure 4: d, e, f and g). also, on this view the rostral colliculi and the rostral part of the caudal colliculi were covered by the cerebral cortex, while the transverse cerebral fissure exposed the caudal half of the caudal colliculi. the commissural fibres of the colliculi were not prominent. this view also showed that the rostral colliculi were smaller than the caudal colliculi (figure 4: j and k). the mesencephalic tegmentum formed the floor of the mesencephalon (figure 4: l), while the substantial nigra separated the tectum from the tegmentum. figure 4: mid-sagittal view of the brain of the african giant pouched rat. metencephalon on dorsal view of the intact brain (figure 1), the cerebellum was very distinct and secondary to the cerebrum in size. the cerebellar hemisphere was highly coiled with a distinct unpaired vermis; the paired flocculus, with its lateral accessory part, the paraflocculus, was also paired (figure 1: h, g and f). this depicted a well-developed cerebellum. the arbor vitae, which constitutes the white matter component of the cerebellar folia, was distinct (figure 4: a). the vermis was the largest cerebellar lobe, while the flocculus was larger than the paraflocculus. on this view, the cerebellum completely covered the fourth ventricle, forming the roof of the ventricle, thus rendering it invisible. on a sagittal view of the vermis (figure 4: t), the rostral lobe was divided by fissures into the following lobules: the lingula (figure 4: c), the central lobule (figure 4: d) and the culmen (figure 4: e). the caudal lobe was divided into the following lobules: declive (figure 4: g); fused folium and tuber (figure 4: h); fused pyramis and uvula (figure 4: j); and the nodulus (figure 4: b), which was separated from the uvula by the uvulonodularis fissure (figure 4: r). the pons of the african giant pouched rat was almost inconspicuous. however, it was observed as a transverse band of nerve fibre caudal to the cerebral crus and rostral to the trapezoid body on the ventral surface of the brain (figure 2: o). caudally, it formed part of the rostral part of the rhomboid fossa. it was pre-trigeminal, as the large trigeminal nerve emerged from the brainstem caudolateral to the pons and rostral to the trapezoid body (figure 2: n1, o and p) myelencephalon the myelencephalon extended from the point of the nuchal flexure, which is caudal to the pyramidal decussation, to the pontomedullary junction. these were the convenient landmarks for isolating the myelencephalon. the central canal of the spinal cord continued rostrally to the medulla oblongata. in the upper half of the myelencephalon, it continued dorsally as the cavity of the fourth ventricle. the myelencephalon was conical in shape, its broad extremity being directed rostrally; it was compressed dorsoventrally. its ventral surface rested on the basilar portion of the occipital bone, while the dorsal surface was partly covered by the cerebellum. it created an impression on the occipital bone. on dorsal view, the most medial part of the myelencephalon was the dorsal median sulcus, which was faintly visible (figure 1: j). the dorsal median sulcus continued on the floor of the fourth ventricle, where it was more visible than in the closed part of the myelencephalon. it divided the myelencephalon into symmetrical halves. in the medulla oblongata, on either side of the dorsal median sulcus the gracile fasciculus was faintly visible. the cuneate fasciculus, the dorsal intermediate sulcus, the lateral funiculus and the dorsal lateral sulcus were not macroscopically visible. rostral to the gracile fasciculus and directly caudal to the obex was the tubercle of gracile nucleus. this tubercle contributed to the caudal border of the floor of the fourth ventricle. the tubercle of the cuneate nucleus was not prominent. the obex (figure 3: k) marked the end of the fourth ventricle and the beginning of the central canal. rostral to the obex was the rhomboid fossa, which was separated into two equal halves by the dorsal median sulcus. the rhomboid fossa, which formed the floor of the fourth ventricle, tapered into the calamus scriptorius rostrally and closed into the central canal at the obex caudally. it was bounded laterally by an inconspicuous sulcus limitans. however, at its greatest width, it extended laterally as the canalis comminicans into the lateral recess. a convex prominence, corresponding to the underlying vestibular nucleus, was evident between the sulcus limitans medially and the medial cerebellar peduncles laterally. the hypoglossal trigone and vagal trigone were not visible. on ventral view, the most rostral part of the myelencephalon was the prominent trapezoid body (figure 2: p). the most medial part was the ventral median fissure (figure 2: v). on either side of the ventral median fissure was the prominent pyramid (figure 2: w). the olivary prominence was situated lateral to each of the pyramids, from which it was separated by the ventral lateral sulcus. the deep ventral lateral sulcus was indicative of the prominent pyramids. the olivary prominence was also distinct (figure 2: r). between the rostral end of this prominence and the trapezoid body, there was a slight depression, corresponding to the pre-olivary sulcus. caudal to the olivary prominence was the post-olivary sulcus. the rootlets of nerves that emerged from these sulci were lost during dissection, thus, could not be evaluated. rostrally, the pyramids ended at the pontomedullary junction. the basal pons served as the landmark for identifying the pontomedullary junction. the transverse bundle of fibres that made up the trapezoid body was parallel to the pons and was immediately caudal to it. caudally, the medulla oblongata could be differentiated from the spinal cord by the prominent transverse depression caudal to the pyramidal decussation at the level of the foramen magnum. ethical considerations top ↑ the procedure for the research project was approved by the ethical committee of ahmadu bello university, zaria, nigeria. the housing and management of the animals were in accordance with that stated in the guide for the care and use of laboratory animals, 8th edition, national research council, usa, downloaded from the national academic press, washington d.c. (http://www.nap.edu/). discussion top ↑ in the present study, apart from the frontal lobe, the cerebral hemisphere in the african giant pouched rat lacked prominent gyri and sulci. the absence of prominent neocortical sulci placed the african giant pouched rat brain in the lissencephalic group. a similar lissencephalic brain has been reported in some sciuromorphs and myomorphs by pilleri, gihr and kraus (1984) and in hystricomorphs by dozo, vucetich and candela (2004). the african giant pouched rat has a large diamond-shaped neocortex, as recorded in the present study. nzalak et al. (2008) recorded a neocortex ratio (relative neocortex weight) of 66% in this rodent. this value is within the range of neocortex ratios for primates, as stipulated by dunbar (1996) who also stated that the neocortex ratio might serve as a measure of cognitive function in animals. this implies that the sense of reasoning and intelligence may be high in the african giant pouched rat, as is found in some primates.well-developed olfactory bulbs were observed in the present study; they were visible from the dorsal view of the intact brain, although they are structures located on the ventral surface of the brain. the conspicuous olfactory bulbs observed in the present study agreed with the findings of nzalak et al. (2008). in some mammals, such as elephants (shoshani, kupsky & marchant 2006), the olfactory bulbs are small and therefore not visible from the dorsal view of intact brain; while in other mammals, such as whales (marino et al. 2003), olfactory structures are absent. according to the morphometric analysis reported by nzalak et al. (2008), the olfactory bulbs of the adult african giant pouched rat are longer than those of the adult african grasscutter (thryonomys swinderianus). rombaux et al. (2006) emphasised that the size of the olfactory bulb is a good indication of the acuity of olfaction in an individual. this may be one of the reasons why the african giant pouched rat is used to detect landmines and diagnose tuberculosis by odour perception. the rodent may also rely heavily on its olfaction, since the sense of sight is poorly developed (morris 1963; ajayi 1975; smithers 1985; ibe et al. 2010). the present study shows that the caudal colliculi are macroscopically bigger than the rostral colliculi in the african giant pouched rat. ibe et al. (2010) showed that the difference in height as well as in diameter of the colliculi in the african giant pouched rat was very highly significant (p < 0.001); the caudal colliculi is wider and higher than the rostral colliculi. the rostral colliculi are bigger than the caudal colliculi in the rabbit (bensley 2009), dog (meyer 1964), calf (schmidt et al. 2009), donkey (oto & haziroğlu 2009) and camel (adogwa 1985; mensah-brown & garey 2006), while the caudal colliculi are bigger than the rostral colliculi in the pig (getty 1975) and cetaceans (marino et al. 2003). mcferland, morgane and jacobs (1969) reported that the caudal colliculi are enlarged in species using ecolocation. this implies that the acoustic system of this nocturnal rodent may be more efficient than the visual system. the present study shows a well-developed vermis in the cerebellum of the african giant pouched rat, which is indicative of good motor coordination and balance. despite the greater brain weight of the african grasscutter relative to that of the african giant pouched rat, as reported by nzalak et al. (2008), the cerebellum weighs relatively more in the african giant pouched rat than in the african grasscutter. the authors recorded a relative weight of 14.0% and 12.5% in the cerebellum of the african giant pouched rat and african grasscutter, respectively. the authors also reported a relatively higher cerebellar length in the african giant pouched rat (23.0%) than in the african grasscutter (20.3%). thus, the cerebellum of the african giant pouched rat is more developed than that of the african grasscutter, which may confer more-efficient motor coordination and balance in the african giant pouched rat than in the african grasscutter. the pons in the african giant pouched rat, as observed in the present study, is small. according to masdern and rowland (1965), the relative sizes of the pons indicate the functional significance of the cortico-pontine and striato-pontine pathways. they also reported that the pons increases in size and complexity, reaching maximum development in primates. this progressive phylogenetic increase explains the inconspicuous pons observed in the study, as rodents are among the first orders of the eutherian mammals (parker & heswell 1974). masdern and rowland (1965) reported that the pons is post-trigeminal in monotremes and pre-trigeminal in marsupials and most species of lower eutherian orders. this is in agreement with the present study in which the pons of the african giant pouched rat, a lower eutherian order, is pretrigeminal. the macroscopic appearance of the myelencephalon of the african giant pouched rat in the present study is similar to that of other rodents, but with slight variations. potter and brueck (1958) reported that the medulla oblongata in the guinea pig is almost completely covered by the cerebellum. this differs from the result obtained in the present study for the african giant pouched rat in which the cerebellum covered only the rostral part of the myelencephalon. the most striking observation of the myelencephalon in the present study was the absence of distinct cuneate fascicle, dorsal lateral sulcus and dorsal intermediate sulcus on the dorsal surface and the presence of macroscopically visible structures on the ventral surface. thus, the ventral surface of the myelencephalon may be more developed than the dorsal surface. in the present study, olivary prominence was observed on the ventral surface of the medulla oblongata in the african giant pouched rat. marsden and rowland (1965) reported that olivary prominence is absent in artiodactyla; but highly developed in primates, especially man. ghaji (1983) and adogwa (1985) reported the absence of this prominence in the camel (an artiodactyl). the presence of the olivary prominence in the present study contributes to the good motor coordination and balance in the african giant pouched rat. the present study showed that pyramids are macroscopically visible on the ventral medulla oblongata of the african giant pouched rat. voogd (1998) recorded inconspicuous pyramids in ungulates. the macroscopically visible pyramids are important in fine motor control of the distal limbs (jennings 2004). therefore, the ability of the african giant pouched rat to maintain balance only on the hind limbs for a reasonable time, especially when fighting, may be a consequence of the developed pyramids that control motor impulses to the hind limbs. conclusion top ↑ the present study has provided some useful information on the macroscopic appearance of the brain of the adult african giant pouched rat in relation to its functions. the study will be of benefit in understanding some of the behaviours of the rodent, which are necessary for its domestication. acknowledgement top ↑ the authors wish to acknowledge the leading contributions of professors s.a. ojo and j.o. ayo of the departments of veterinary anatomy and veterinary physiology, respectively, of the ahmadu bello university, zaria, nigeria. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions c.s.i. (ahmadu bello university) obtained the animals and conducted the research in the departmental laboratory. b.i.o. (ahmadu bello university) supervised the research and edited the manuscript. j.o.h. 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the brain a new procedure’, italian journal of anatomy and embryology 82, 105–110. rombaux, p., mouraux, a., bertrand, b., nicolas, g., duprez, t. & hummel, t., 2006, ‘olfactory function and olfactory bulb volume in patients with postinfectious olfactory loss’, laryngoscope 116, 436–439. http://dx.doi.org/10.1097/01.mlg.0000195291.36641.1e schmidt, m.j., pilatus, u., wigger, a., kramer, m. & oelschlager, h.a., 2009, ‘neuroanatomy of the calf brain as revealed by high-resolution magnetic resonance imaging’, journal of morphology 270, 745–758. http://dx.doi.org/10.1002/jmor.10717 shoshani, j., kupsky, w.j. & marchant, g.h., 2006, ‘elephant brain: part i: gross morphology, functions, comparative anatomy, and evolution’, brain research bulletin 70, 124–157. http://dx.doi.org/10.1016/j.brainresbull.2006.03.016 smithers, r.h.n., 1985, ‘the mammals of the southern african subregion’, quarterly review of biology 60, 362. http://dx.doi.org/10.1086/414487 suckow, m.a., weisbroth, s.h. & franklin, c.l., 2006, the laboratory rat, pp. 230–439, elsevier academic press, oxford. tubbs, r.s., loukas, m., shoja, m.m., apaydin, n., ardalan, m.r., shokouhi, g. et al., 2008, ‘costanzo varolio (constantius varolius 1543-1575) and the pons varolli’, neurosurgery 62, 734–737. http://dx.doi.org/10.1227/01.neu.0000317323.63859.2a voogd, j., 1998, ‘mammals’, in r. nieuwenhuys, p.a.m. van dongen, h.j.n. donkelaar & c. nicholson (eds.), the central nervouse system of vertebrates, springer science online textbook, viewed 30 june 2009, from http://www.springer.com wingerd, b.d., 1988, rat dissection manual, johns hopkins university press, baltimore. article information authors: mark m. rweyemamu1 esron d. karimuribo1 leonard e.g. mboera2 affiliations: 1southern african centre for infectious disease surveillance, sokoine university of agriculture, tanzania2national institute for medical research, dar es salaam, tanzania correspondence to: mark rweyemamu postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: rweyemamu, m.m., karimuribo, e.d. & mboera, l.e.g., 2014, ‘the changing landscape for health research in africa: the focus of the southern african centre for infectious diseases and surveillance’, onderstepoort journal of veterinary research 81(2), art. #799, 2 pages. http://doi:10.4102/ ojvr.v81i2.799 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the changing landscape for health research in africa: the focus of the southern african centre for infectious diseases and surveillance in this preface... open access • introduction • acknowledgements    • competing interests    • authors’ contributions • references introduction top ↑ in april 2013, the southern african centre for infectious diseases and surveillance (sacids) (see http://www.sacids.org) joined forces with the tanzania national institute for medical research to convene the second one health conference in africa, held in arusha, tanzania, with the overarching theme of ‘the changing landscape for health research in africa’.whilst this reflection covered both communicable and con-communicable diseases, the greater emphasis was on infectious or communicable diseases. this reflects the impact of these diseases on human health, well-being and economic development in africa (mboera et al. 2014; rweyemamu, otim-nape & serwadda 2006; rweyemamu et al. 2012). the heavy burden of infectious diseases of humans and animals in africa falls into two categories, namely, (1) exposure to internally or externally generated emerging or re-emerging diseases and (2) the continuing occurrence of major epidemic diseases in an endemic state in africa. it has been reported that about 60% of all infectious pathogens in humans originate from animals, although many of these have completely adapted to their new host and no longer require a non-human animal host for persistence. it is also known that between 60% and 75% of new or emerging infectious diseases of humans in the last half century have originated from animals, of which perhaps 71% of these were of wildlife origin. the drivers of many emerging diseases are mostly related to human behaviour and actions, socio-economic (e.g. globalisation of travel and trade), environmental and ecological factors (jones et al. 2008; lightfoot, rweyemamu & heymann 2013; rweyemamu et al. 2006; taylor et al. 2001). the continuing endemic settings of major infectious diseases in africa constitute a high risk for health and livelihoods and future marginalisation of africa through trade restrictions and socio-political impacts (rweyemamu et al. 2012; waage et al. 2010). in africa, 72% of the disease burden is attributable to poverty, interactions between socio-economic opportunities and the health of animals, people and ecosystems, compared to 27% in the rest of the world. another important aspect of health in africa is climate change and variability, which is reported to have affected africa more heavily than most other parts of the world. generally, vulnerability of individuals and communities to infectious diseases is influenced by multiple factors (environmental, economic and socio-ecological) in addition to host and causative agent factors (food and agriculture organization of the united nations [fao] 2013; mcmichael & woodruff 2004; ndiyoi et al. 2006; world health organization 2011). accordingly, there is an increasing consensus that because of their holistic nature, one health (oh) and/or ecohealth approaches are particularly appropriate for the risk management of infectious diseases in africa (charron 2012; mboera et al. 2014; rweyemamu et al. 2013; zingstagg et al. 2011). furthermore, the world bank and several independent authors have shown that these approaches are cost-effective (grace 2014; rushton 2012; world bank 2012). in pursuance of the same goal in africa, sacids has developed a broad-based, oh-driven approach to infectious diseases in sub-saharan africa through its vision of: a sub-saharan african society protected from devastating infectious diseases affecting the health of humans, animals, i.e. both terrestrial and aquatic, and ecosystems, thereby promoting livelihoods, socio-economic development including market access and the environment. (sacids n.d.) the modus operandi of sacids is that of a virtual centre which links african academic and research institutions that deal with infectious diseases of humans and animals in smart partnership with centres of research excellence in industrialised countries and international research centres. sacids has adopted the community of practice (cop) approach for both its research capacity development programme and its collaboratively themed research programme (rweyemamu et al. 2012, 2013). following an internal review and development of a new business plan to 2020, the programme of sacids now revolves around the following six themes or cops, (1) emerging and vector-borne diseases, (2) bacterial zoonoses including food-borne diseases and anti-microbial resistance, (3) viral diseases of food security importance, (4) cross-cutting oh sciences, (5) oh training (short-courses, annual oh summer schools and oh-based msc courses) and (6) research management and an oh forum, including conferences. whilst the first three themes are disease-category based, themes four and five help sacids to develop a broad-based approach, which includes socio-economics, socio-anthropology, environment, ecosystems, ecohealth, oh-based and information communication and technology driven approaches to disease surveillance, health systems and policy, poverty focus and an examination of such issues as the interaction between agriculture and health (karimuribo et al. 2012; kayunze et al. 2014; mboera et al. 2014; mwabukusi et al. 2014; rweyemamu et al. 2013). that is the sacids approach to the changing landscape for health research in africa, as it strives to evolve into a regional one health forum and research platform in africa (theme six). acknowledgements top ↑ we acknowledge, with thanks, the funding agencies for sacids, especially the wellcome trust through grant wt087546ma, the rockefeller foundation through grants 2008-dsn310, 2009-dsn305 and 2011-dsn307, the google foundation through grant gf-02-2009 and the international development research centre through grant 107030-001. we also acknowledge the specific action financial support received from fao and the foresight programme of the uk government. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions m.m.r. (sokoine university of agriculture) produced the first draft of the manuscript, building on shared vision by the three authors, who have all been involved in the organisation of the one health conference. m.m.r. also undertook the primary literature review. e.d.k. (sokoine university of agriculture) and l.e.g.m. (national institute for medical research) reviewed the text and improved it with additional references and all the three authors agreed upon the final version. references top ↑ charron, d.f., 2012, ecohealth research in practice: innovative applications of an ecosystem approach to health, springer, ottawa, viewed 03 april 2014, from http://www.idrc.ca/en/resources/publications/pages/idrcbookdetails.aspx?publicationid=1051 food and agriculture organization of the united nations, 2013, world livestock 2013 – changing disease landscapes, fao, rome. grace, d., 2014, ‘the business case for one health’, onderstepoort journal of veterinary research 81(2), art. #725, 6 pages. http://dx.doi.org/10.4102/ojvr.v81i2.725 jones k.e., patel, n., levy, m.a., storeygard, a., balk, d., gittleman, j.l. et al.,2008, ‘global trends in emerging infectious diseases’, nature 451, 990–993. http://dx.doi.org/10.1038/nature06536 karimuribo, e.d., sayalel, k., beda, e., short, n., wambura, p., mboera, l.g. et al., 2012, ‘towards one health disease surveillance: the southern african centre for infectious disease surveillance approach’, onderstepoort journal of veterinary research 79(2), art. #454, 7 pages. http://dx.doi.org/10.4102/ojvr.v79i2.454 kayunze, k.a., kiwara, a., lyamuya, e., kambarage, d.m., rushton, j., coker, r. et al., 2014, ‘practice of one health approaches: bridges and barriers in tanzania’, onderstepoort journal of veterinary research 81(2) art. #733, 8 pages. http://dx.doi.org/10.4102/ojvr.v81i2.733 lightfoot, n., rweyemamu, m. & heymann, d.l., 2013. ‘preparing for the next pandemic. greater cross sector collaboration between health, veterinary, wildlife and environmental experts is needed’, british medical journal 346, f364. http://dx.doi.org/10.1136/bmj.f364 mboera, l.e.g., mfinanga, s.g., karimuribo, e.d., rumisha, s.f. & sindato, c., 2014, ‘the changing landscape of public health in sub-saharan africa: control and prevention of communicable diseases needs rethinking’, onderstepoort journal of veterinary research 81(2) art. #734, 6 pages. http://dx.doi.org/10.4102/ojvr. v81i2.734 mcmichael, a. & woodruff, r., 2004, ‘climate change and risk to health’, british medical journal 29(7480), 1416–1417. http://dx.doi.org/10.1136/bmj.329.7480.1416 mwabukusi, m., karimuribo, e.d., rweyemamu, m.m. & beda, e., 2014, ‘mobile technologies for disease surveillance in humans and animals’, onderstepoort journal of veterinary research 81(2), art. #737, 5 pages. http://doi:10.4102/ojvr.v81i2.737 ndiyoi, m., rweyemamu, m. & meadows, k., 2006, ‘strengthening livelihoods through food and nutrition security in vulnerable sadc countries’, mid-term review of osro/raf/510-511/saf, fao, rome, viewed 03 april 2014, from http://typo3.fao.org/fileadmin/user_upload/oed/docs/osroraf510_511saf_2006_er.pdf rushton, j., häsler, b., de haan, n. & rushton, r., 2012. ‘economic benefits or drivers of a ‘one health’ approach: why should anyone invest?’, onderstepoort journal of veterinary research 79(2), art. #461, 5 pages. http://dx.doi.org/10.4102/ojvr. v79i2.461 rweyemamu, m., otim-nape, w. & serwadda, d., 2006, foresight. infectious diseases: preparing for the future: africa, office of science and innovation, london, p. 1–120, viewed 03 april 2014, from http://www.bis.gov.uk/assets/bispartners/foresight/docs/infectious-diseases/a1_id_africa.pdf rweyemamu, m.m., paweska, j., kambarage, d. & namuba, f, 2012, ‘towards one africa, one health: the sacids one health focus on infectious diseases’, onderstepoort journal of veterinary research 79(2), art. #449, 2 pages. http://dx.doi.org/10.4102/ojvr.v79i2.449 rweyemamu, m., kambarage, d., karimuribo, e., wambura, p., matee, m., kayembe, j.m. et al., 2013, ‘development of a one health national capacity in africa: the southern african centre for infectious disease surveillance (sacids) one health virtual centre model’, currents topics in microbiology and immunology 366, 73–91. http://dx.doi.org/10.1007/82_2012_244 southern african centre for infectious diseases and surveillance, n.d., vision and mission, viewed 03 april 2014, from http://www.sacids.org/oweb/sacids/home/about~us/19-35/vision__mission.html taylor, l.h., latham, s.m. & woolhouse, m.e., 2001, ‘risk factors for human disease emergence’, philosophical transactions of the royal society of london. series b: biological sciences 356(1411), 983–989. http://dx.doi.org/10.1098/rstb.2001.0888 waage, j., banerji, r., campbell, o., chirwa, c., collender, g., dieltiens, v. et al., 2010, ‘the millennium development goals: a cross-sectoral analysis and principles for goal setting after 2015’, the lancet 376, 991–1023. http://dx.doi.org/10.1016/s0140-6736(10)61196-8 world bank, 2012, ‘people, pathogens and our planet: the economics of one health’, world bank, washington, dc, viewed 09 december 2013, from http://documents.worldbank.org/curated/en/2012/06/16360943/people-pathogens-planet-economics-one-health world health organization, 2011, facts about health in the african sub-region – factsheet no. 314 who, geneva. zinsstag, j., schelling, e., waltner-toews, d. & tanner, m., 2011, ‘from “one medicine” to “one health” and systemic approaches to health and well-being’, preventive veterinary medicine 101(3–4), 148–156. http://dx.doi.org/10.1016/j. prevetmed.2010.07.003 article information authors: christopher f.l. saarnak1 maria v. johansen1 samson mukaratirwa2 affiliations: 1department of veterinary disease biology, university of copenhagen, denmark2school of life sciences, university of kwazulu-natal, south africa correspondence to: christopher saarnak postal address: dyrlægevej 100, 1870 frederiksberg c, denmark how to cite this article: saarnak, c.f.l., johansen, m.v. & mukaratirwa, s., 2014, ‘advanz: establishing a pan-african platform for neglected zoonotic disease control through a one health approach’, onderstepoort journal of veterinary research 81(2), art. #740, 3 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.740 note: dyrlægevej 100, 1870 frederiksberg c, denmark copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. advanz: establishing a pan-african platform for neglected zoonotic disease control through a one health approach in this proceedings... open access • abstract • introduction • a pan-african platform • future perspectives • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ advocacy for neglected zoonotic diseases (advanz) is a one health neglected zoonotic diseases (nzds) project, funded by the european commission through its 7th framework programme. the initiative aims at persuading decision makers and empowering stakeholders at local, regional, and international levels towards a coordinated fight against nzds. advanz is establishing an african platform to share experiences in the prevention and control of nzds. the platform will compile and package existing knowledge or data on nzds and generate evidence-based algorithms for improving surveillance and control with the ultimate aim of eliminating and eradicating these diseases. the platform will serve as a forum for african and international stakeholders, as well as existing one health and nzd networks and harness and consolidate their efforts in the control and prevention of nzds. the platform had its first meeting in johannesburg, south africa in march 2013. introduction top ↑ endemic and enzootic zoonoses cause about a billion cases of illness and millions of deaths in people every year (karesh et al. 2012). of these zoonoses, diseases such as anthrax, bovine tuberculosis, brucellosis, cysticercosis, echinococcosis (hydatid disease) and rabies are considered ‘neglected’ because they are not adequately addressed at either a national or an international level. the world health organization (who) uses the term neglected zoonotic diseases (nzds) to refer to the six aforementioned zoonoses, but also include leishmaniasis and human african trypanosomiasis (who 2009). the who recognised the need for increasing the global awareness of these diseases as they cause significant morbidity and mortality in the already impoverished rural communities of the world. in 2009, the food and agriculture organization of the united nations (fao) and the world organisation for animal health (oie) joined the who to formulate a concept note on sharing responsibilities and coordinating global activities to address health risks at the animal–human–ecosystems interfaces (the fao-oie-who collaboration 2010). the so-called fao–oie–who tripartite recognised the need to establish an environment in which ministers representing the various sectors within countries can voice their expectations and come to a consensus on future activities, with particular reference to collaboration. whilst the aetiology of the eight nzds may be varied, the interventions to prevent and control them are quite similar, especially at the community level. many of the nzds are closely linked to poverty and to the lack of basic infrastructure such as water and sanitation (molyneux et al. 2011). interventions that include engaging communities, creating public awareness and enhancing basic conditions such as hygiene and biosecurity can have an important effect on reducing the risks of transmission (who 2010). in addition, some of the nzds can be controlled by interventions such as mass vaccination (e.g. rabies [who expert consultation on rabies 2005]) or treatment (e.g. echinococcosis [world health organization 2011]). most importantly, several of the diseases can be controlled by creating a simple but effective change of the day-to-day practices amongst the people at risk, such as proper hand washing and preventing open defecation (who 2010). political will and resources are further requirements for the effective control this group of diseases. nzds are not negligible, as evidenced by their considerable impact on the lives and livelihoods of poor and marginalised populations in particular (grace et al. 2013). although the ‘one health’ approach initially concentrated on addressing emerging zoonotic diseases and pandemic threats in particular, the approach is gaining recognition for addressing nzds as they arise from infections transmitted from domesticated (livestock or companion) or wild animals (who 2009). a pan-african platform top ↑ a new pan-african one health platform on nzds (oh nzd) is currently being initiated. the platform is called advanz (advocacy for neglected zoonotic diseases). at a workshop held in johannesburg, south africa in march 2013, representatives of the various key stakeholders in existing networks met to set up a framework for a pan-african network for oh nzd practitioners. the framework that was agreed upon at the meeting has an interdisciplinary approach, which takes into account the linkages between social, economic and environmental elements affecting human health. the rationale behind the platform is to harness strengths from current disease-specific networks to provide a platform for intersectoral collaboration. the cysticercosis working group in eastern and southern africa, a network that consists of ten member countries, represents a successful south–south–north network that follows a ‘one health’ approach. it is now internationally well recognised, used as a model for other networks and has managed to represent the region in the advocacy for the emerging problem of taenia solium cysticercosis. the proposed advanz platform, which will be based on a similar background, is unique in that multidisciplinary and interdisciplinary networks, groups, private, public and non-governmental organisations and individuals interested in nzds are eligible to be members of the platform, which will allow experiences and best practices in the prevention and control of nzds to be shared (figure 1). members of the platform will likely be representatives of mainly south–south networks working on nzds, private and public organisations, ngos and working groups from individual countries consisting of key stakeholders involved in the fight against nzds. the national groups will both address the burden of nzds in the specific country and serve as the local channel to utilise the resources availed through the platform. the platform is expected to strengthen the interdisciplinary and multidisciplinary cooperation and coordination amongst scientists and control authorities concerned with nzds in africa. the specific objectives of the platform will be to: • coordinate and secure continuous advocacy for the intersectoral approach for surveillance and control of nzds in humans and animals • disseminate information regarding nzds • maintain a forum for continuous communication amongst stakeholders regarding development of evidence-based algorithms for surveillance and control of nzds • inform members about nzd-related research and training activities • inculcate an ‘one health’ approach for control and prevention of nzds. the proceedings of the advanz meeting with stakeholders can be found at http://www.advanz.org. the platform is open for all interested organisations and institutions involved in nzd activities. the corresponding author can be contacted for further information. figure 1: proposed structure of the pan-african one health platform (advanz) for neglected zoonotic diseases. future perspectives top ↑ in november 2014, an international conference entitled ‘neglected zoonoses 4 (nz4)’ will showcase successful nzd approaches, including the advanz platform model. the conference will be organised in association with the aforementioned tripartite, and will be held at the who headquarters in geneva, switzerland. if the model is successful, it can be applied to other regions of the world suffering from nzds. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. this work was supported by the eu grant advanz–advocacy for the fight against neglected zoonotic diseases (fp7 kbbe contract no: 0312030, http://www.advanz.org). authors’ contributions c.f.l.s. (university of copenhagen) wrote the original draft of the article. s.m. (university of kwazulu-natal) and m.v.j. (university of copenhagen) contributed equally to the manuscript. references top ↑ grace, d., holley, c., jones, k., leach, m., marks, n., scoones, i. et al., 2013, ‘zoonoses – from panic to planning’, institute of development studies, brighton.karesh, w.b., dobson, a., lloyd-smith, j.o., lubroth, j., dixon, m.a., bennett, m. et al., 2012, ‘ecology of zoonoses: natural and unnatural histories’, lancet 380(9857), 1936–1945. http://dx.doi.org/10.1016/s0140-6736(12)61678-x molyneux, d., hallaj, z., keusch, g., mcmanus, d., ngowi, h., cleaveland, s. et al., 2011, ‘zoonoses and marginalised infectious diseases of poverty: where do we stand?’, parasites and vectors 4(1), 106. http://dx.doi.org/10.1186/1756-3305-4-106 the fao-oie-who collaboration, 2010, ‘sharing responsibilities and coordinating global activities to address health risks at the animal-human-ecosystems interfaces: a tripartite concept note’, viewed 05 august 2013, from http://www.who.int/influenza/resources/documents/tripartite_concept_note_hanoi_042011_en.pdf who, 2009, integrated control of neglected zoonotic diseases in africa: applying the ‘one health’ concept, who, geneva who, 2010, ‘statement: 3rd international conference on neglected zoonotic diseases: community-based interventions for prevention and control of neglected tropical diseases’, who, 23–24 november 2010, geneva. who expert consultation on rabies, 2005, first report, trs 931, who, geneva, viewed 07 august 2013, from http://www.who.int/rabies/animal/excerpttrs931.pdf world health organization, 2011, ‘report of the who informal working group on cystic and alveolar echinococcosis surveillance, prevention and control, with the participation of the food and agriculture organization of the united nations and the world organisation for animal health’, viewed 07 august 2013, from http://whqlibdoc.who.int/publications/2011/9789241502924_eng.pdf article information authors: beda j. mwang’onde1,2 gamba nkwengulila1 mwita chacha1,3 affiliations: 1department of zoology and wildlife conservation, university of dar es salaam, tanzania 2division of livestock and human diseases vector control, tropical pesticides research institute, tanzania 3wellcome trust fellow, department of zoology and wildlife conservation, tanzania correspondence to: beda mwang’onde postal address: po box 35064, dar es salaam, tanzania how to cite this article: mwang’onde, b.j., nkwengulila, g. & chacha, m., 2014, ‘the risk factors for human cysticercosis in mbulu district, tanzania’, onderstepoort journal of veterinary research 81(2), art. #719, 5 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.719 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www. sacids.org/ kms/frontend/ index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the risk factors for human cysticercosis in mbulu district, tanzania in this proceedings... open access • abstract • introduction • materials and methods    • study area and ethical clearance    • data collection    • statistical analysis • results    • demographic characteristics    • characterisation of risk factors for human cysticercosis • discussion    • indiscriminate defaecation and improper use of toilets    • free-range system of raising pigs    • consumption of undercooked infected pork    • improper and lack of meat hygiene and inspection    • social structure and gender • conclusion    • recommendations • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ the objective of this study was to explore the reasons for the persistence of human cysticercosis (hcc) transmission in mbulu district, northern tanzania. the study was carried out in 25 villages, whereby five major risks were identified. the risks were indiscriminate defaecation and improper use of toilets; a free-range system of keeping pigs; indiscriminate or unregulated slaughtering and inadequate meat hygiene and inspection; consumption of undercooked and porcine cysticerci infected pork; and social structure and roles. all of the identified risks were backed up by the immanent lifestyles of the community involved. these findings are important for the development of intervention strategies in the study area introduction top ↑ human cysticercosis (hcc) is a tissue invasion by the pork tapeworm larvae, taenia solium. humans are definitive hosts of t. solium and are infected by eating undercooked pork that contains viable cysticerci. the cysticercus develops into an adult tapeworm in the gut which produces large numbers of eggs which pass out in the faeces. the disease is among the neglected tropical diseases (ntds) with a negative impact on public health and the piggery industry. it is endemic in most resource poor countries where sanitation is inadequate and free-range pig farming is practiced (garcia et al. 2003). however, as a result of migrations, tourism and globalisation, hcc is also reported in industrialised countries (canas et al. 2005; schantz et al. 1998) where sanitation is adequate. also, there have been case reports of hcc among communities of muslims (shandera & kassy 2006) and orthodox jews (schantz et al. 1998; moore et al. 1995). the features for a global epidemiology of hcc include frequent travel to endemic areas, residence and/or household contact (del brutto 2012). therefore, with globalisation and increased tourism, the disease poses a threat worldwide. the prevalence of hcc in mbulu district is 16.3% (mwang’onde et al. 2012) and that of porcine cysticercosis (pcc) is 17.4% (ngowi et al. 2004a). a computed tomography (ct) scan of hcc sero-positive patients based on cysticercosis wb igg (immunoblot assay test) from the 25 villages of study showed that over 76.0% had lesions suggestive of human neurocysticercosis (ncc). furthermore, hospital-based studies on people with epilepsy (pwe) reported a prevalence of ncc of over 14.0% (winkler et al. 2008; willingham et al. 2008). nevertheless, most studies in tanzania have focused on the prevalence of pcc (phiri et al. 2003; ngowi et al. 2004b; boa et al. 2006; mkupasi et al. 2010); risk factors for pcc (ngowi et al. 2004a); and the control of pcc and its financial implications (ngowi et al. 2007). according to mwang’onde et al. (2012), winkler et al. (2008) and willingham et al. (2008), the prevalence of hcc in mbulu district is worrying. the risks associated with the high prevalence of hcc in the district have never been documented. the present study, therefore, documents the risk factors associated with the persistence of hcc in mbulu district, northern tanzania. materials and methods top ↑ study area and ethical clearance the study was carried out in mbulu district (3°48′–4°30′s, 35–36°e) from september 2011 to april 2013. mbulu district has a population of 320 279 (nbs 2012). the area lies at an estimated altitude of 1000 m – 2400 m above sea level. the climate ranges from semi-arid to sub-humid, with an annual rainfall of < 400 mm and > 1200 mm, respectively. there are two rainy seasons with two peaks of maximum rains. the long rainy season extends from about march to mid-may and the short rainy period from november to december. in between (january through february) is a dry spell and the dry season is between june and october. the relative humidity ranges from 55% to 75% and the mean annual temperature ranges from 15 °c to 24 °c. ethical clearance for the study was granted by the national institute for medical research (nimr), nimr/hq/r.8a/vol. ix/1297. data collection information on sanitation, hygiene, systems of pig raising, knowledge of pcc and hcc, meat eating behaviour, age and gender roles, animal-human interactions and, community’s attitude towards pcc was sought from 80 hcc sero-positives and the general community. an administered questionnaire, face-to-face interviews via field-farmer meetings, radio session dialogues, archives reviews and field observations were the techniques used to obtain information (figure 1). traditional customs regarding the above parameters were also teased out. the study was cross-sectional, thus there were no demarcations or classifications between cases and controls among interviewees which would reduce bias in the identification and estimation of exposure to risk factors. figure 1: techniques used to collect information on hcc risks, (a) field meetings; (b) interview session and (c) dialogue with community members through their local radio. statistical analysis data entry and validation was carried out using ms-excel 2010 version (ms corp., redmond, wa, usa). risk factors were analysed using the statistical package for social sciences version 19.0 (spss statistic 19, 2010 ibm). a logistic regression (odds ratio [or]) (szumilas 2010) analysis was used to measure the association between various identified exposure risks for hcc (confidence interval [ci] = 95%). results top ↑ demographic characteristics mbulu district has a population of 320 279 (0.98:1 female/male ratio) (nbs 2012). the population is skewed to the left as a large proportion (76.3%) of the population is aged 0–29 years, with 4–14-year-olds counting the highest number (50.6%) followed by the 15–29-year-olds (25.7%). males predominate in most age groups except in the 30–44 and 60–79 age groups (table 1). table 1: population structure of mbulu district. characterisation of risk factors for human cysticercosis the mbulu community was historically pastoralist but currently practice mixed farming. members of this community, particularly men, have a high preference for eating bloody or undercooked meat (p = 0.00049). the population of mbulu community is dominated by males (nbs 2012). the age groups from 13–60 are among the highly active who commonly attend local clubs (p = 0.0019) where meat consumption goes together with alcohol intake. in this community, pork is the most consumed meat in local brew clubs (p = 0.0475), followed by beef (p = 0.1675), mutton (p = 0.284) and poultry (p = 0.921). although every household owns a toilet, these either lack frequent visits by household members or are very small for an adult to comfortably utilise. these small and unappealing toilets are located in front of almost every household purposefully to escape penalties from health authorities for not having them. nevertheless, about 12.9% (n = 31) of heads of households amicably expressed their preference for not sharing a latrine with their daughters. this belief provides room for people to relieve themselves in bushes and elsewhere where roaming pigs can easily access human faeces. furthermore, the toilets in some households are located very far from the house and have a very small hole which entertains defaecation around the brims which attracts the attention of roaming pigs. it is also acceptable by an appreciable proportion of the population that indiscriminately disposed children’s stool serves a purpose as a source of food for farmyard animals like pigs, dogs and chickens. in addition, over 2/3 of the latrines in these communities are easily accessed by scavenging pigs. there were no official slaughter slabs in the study area, consequently, and to avoid inconvenience the seller’s house (homestead) becomes the slaughter place at that particular time of business (figure 2). the official meat inspector, if available, inspects pork carcasses at homesteads. water wells down the hill are the reliable source of domestic water (p = 0.00048) with a minority of the population (p = 0.0192) having access to running water. figure 2: the mbulu district, (a) an individually owned slaughtering pen at a pork seller’s house and (b) a slaughtering place near a local brew club. indiscriminate defaecation and improper use of toilets (or = 3.0; 95% ci, 1.028–8.755); a free-range system of keeping pigs (or = 5.0; 95% ci, 1.195–20.922); indiscriminate or unregulated slaughtering, inadequate meat hygiene and inspection (or = 6.5; 95% ci, 2.009–21.033); consumption of undercooked and infected pork (or = 1.25; 95% ci, 0.681–2.293); and social structures and roles (or = 0.75; 95% ci, 0.382–1.471) were found to be the five main risk factors for hcc epidemiology in mbulu. discussion top ↑ the present study documents for the first time the reasons for the persistence of hcc in mbulu district. various risk factors were identified; however, the discussion is mainly on the five risks identified as most potential for the epidemiology of taeniosis-cysticercosis or hcc in mbulu. indiscriminate defaecation and improper use of toilets this is a practice where people in a community do not utilise the available toilet facilities but rather go to relieve themselves in the open, among bushes. the practice pollutes ground waters, contaminates agricultural produce and helps the spread of viral, bacterial and parasitic diseases. the practice is detrimental to human and animal health and to social economic development, as among others it poses a high risk of cysticercosis in areas where pigs are kept as it facilitates the lifecycle of t. solium.the reasons for indiscriminate defaecation and improper use of toilets in mbulu district include the belief that human faeces are a good and readily available source of food for domestic animals, therefore children should relieve themselves in play grounds which roaming pigs, scavenging chickens and pet dogs can easily access. some would like to examine their stool soon after excretion searching for any abnormalities of the gastrointestinal tract (git); others would not like to share a toilet hole with their daughters; and others believe that the process of constructing pit latrines or toilets brings misfortunes among members of the clan; hence defaecation in the bushes is a comfortable and safe practice. considering all these reasons advanced by different households and/or clans, it is reasonable to suggest that these communities, in general, prefer relieving themselves in bushes rather than in toilets. free-range system of raising pigs about 96% of the pigs in mbulu district are kept under a free-range system (ngowi et al. 2004a). this system of keeping pigs in mbulu goes back to ancestral times when pigs were grazed with large and small ruminants in grazing lands; in some communities it is still practiced. they believe the system to be economical in terms of feeds, pens construction and time management. however, elsewhere, vaga (2009) reports the system to be less productive in terms of weight gain per unit of food consumed. raising and allowing pigs to feed on human faeces risks human and porcine cysticercosis (cao et al., 1997; allan et al. 2005). the 25 villages involved in the present study permit pigs to scavenge around the bush and pigsties. such feeding methods not only raises the risk of pcc, but also increases the public health risk of hcc in the community. consumption of undercooked infected pork the manyara region’s ethnic communities include the iraqw, mbugwe, hadzabe, fyomi, maasai, barabaig, nyisanzu and alawa in which, with the exception of the hadzabe who are mostly hunter-gatherers, these ethnic groups share some common values as they are pastoralists by nature. people from these communities prefer grilled or smoked meat to other cooking methods. heavy drinking, particularly by the men in the community, inevitably leads to the consumption of undercooked meat at local social clubs, and the practice perpetuates the taenia spp. life cycle, among other parasitic life, which is a potential health risk for hcc. improper and lack of meat hygiene and inspection in mbulu district, authorised personnel (i.e. para-vets) have often been directing slaughter or butcher men to make cuts in pork carcasses in their absence based on what they used to see from experts or professionals during routine meat inspection. furthermore, the whole district of mbulu has no single legal collective slaughter slab for pigs other than open slaughter places. most pigs are slaughtered without ante mortem inspection, which is a prerequisite procedure, and then a para-vet is consulted for the inspection process. in the case where a carcass is infected with cysticerci, a para-vet would never be consulted and the carcass is set for a barter trade or sold at over 50% less of normal value. this situation allows cysticerci infected pork to enter the markets, thus perpetuating taeniosis in the area. proper meat hygiene and inspection is essential for public health because zoonosis account for 60% of all infectious disease pathogens and 75% of all emerging pathogens (komba et al. 2012). therefore, all animals intended for slaughter should be inspected while live, and post slaughter by authorised personnel to make sure meat for human consumption is not only fit and wholesome but also free from zoonosis (tfda 2003). the tanzania food, drugs and cosmetics authority (tfda) further stipulates that premises for slaughtering animals intended for supply, sell, offer or expose for sale of meat for human consumption shall be registered by the authority, which is not the case in mbulu. among others, the tfda act no. 1, 2003 prescribes the methods, instruments or appliances which may be used to carry out slaughtering; monitor and manage the slaughter and butchery facilities for suitability; marking or labeling of carcasses and viscera; and treatment and disposal of any animal, carcass or viscera, or any part of them for the intended purpose. yet, in mbulu whenever an authorized person rules for the total condemnation of a carcass or part of it, if not bunt to ash or disposed to a level that cannot be easily accessed, it is scavenged and consumed by the community. social structure and gender gender is the relationship between men and women, both perceptual and material and it is a central organising principle of societies, and often governs the processes of production and reproduction, consumption and distribution (fao 1997). based on a general community study on the sero-prevalence of human cysticercosis in mbulu district by mwang’onde et al. (2012), the frequency was higher in males than females. furthermore, the sero-positivity ratio of hcc between females and males was 1:3 (n = 171) from the participation ratio of 1:1.7 (n = 1051). this signifies that regardless of the high population of males (nbs 2012) and the recruitment ratio, males were more frequently infected with cysticerci than females. in this community, women are not allowed to participate in an economic or social activity unless they get the permission of their husbands and this might be the main reason for the low proportion of women participating in this study. furthermore, men’s freedom to attend social functions exposes them to a greater risk of infection with t. solium cysticercosis.according to flintan (2011), men and women have different abilities to access different opportunities in most developing countries, particularly in agro-pastoral communities. these differences also limit women’s integration in the market economy, allowing men to dominate the marketing of farm products. agro-pastoral societies are patrilineal and male-dominated with strongly marked gender roles, a pattern which is extremely similar across the world, hence men are responsible for managing and selling meat animals (fao n.d.). this situation also exists in mbulu district, where males dominate livestock production and marketing. at their livestock markets, famously known as ’mnada’, there is massive consumption of alcoholic drinks and freshly slaughtered meat, including pork, barbecued over open fires. conclusion top ↑ populations accustomed to open or indiscriminate defaecation may require a substantial change in cultural values and behaviour through education towards using fixed-point facilities. without such change, people may not use latrines at all or may use them in a way that undermines the potential health benefits. it is important to understand what stimulates people’s interest in hygiene and sanitation. health is one factor, but convenience, dignity, and social status may provide a stronger motivation. once behaviours are recognised by a community, there is a pressure to conform, and social structures and leaders begin to contribute to the adoption of new behaviour. therefore, to change attitudes towards hygiene and eating behaviour, education programmes should be the central focus, not only in mbulu but also in areas with similar settings. recommendations the principal advantage of the findings of this study on cysticercosis persistency in mbulu paves the way for planning community-based control interventions. thus, the information obtained serves in developing an appropriate strategy for breaking the cycle and therefore control of t. solium cysticercosis in mbulu. since the current study focused on the hcc prevalence based on the general community’s sero-screening, further investigation is needed to examine outpatients and inpatients and environmental contamination by t. solium eggs to evaluate how big the risk of hcc is, in order to enhance the development of the best approach to controlling the disease in mbulu district. acknowledgements top ↑ the authors wish to thank mr. amoniche mtweve from mbulu district council for his invaluable organisational support during field work; we further thank the mbulu district officials for their amicable acceptance of this study in their district. the research for this article was carried out within the framework of the consortium afrique one ‘ecosystem and population health: expanding frontiers in health’. afrique one is funded by the wellcome trust (wt087535ma) and the university of dar es salaam hosted and facilitated the project. competing interests the authors declare that they have no competing interests in the writing of this article. authors’ contributions m.c. (university of dar es salaam and wellcome trust) designed the study. b.j.m. (university of dar es salaam and tropical pesticides research institute), m.c. and g.n. 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mukaratirwa, s., githigia, s., saimo, m., sikasunge, c., maingi, n., lubega, g.w., kassuku, a., michael, l., siziya , s., krecek, r.c., noormahomed, e., vilhena, m., nsengiyum va, g., andriantsimahavandy, a., dorny, p., johansen, m.v. & willingham, a.l., 2004b, ‘taenia solium cystic ercosis in eastern and southern africa: an emerging problem in agricul ture and public health’, southeast asian journal of tropical medicine and public health 35 (1), 266–270. ngowi, h.a., mlangwa, j.e.d., carabin, h., mlozi, m.r.s., kassuku, a. a., kimera, s.i. & willingham, a.l, 2007, ‘financial efficie ncy of health and pig management education intervention in controlling porcine cysticercosis in mbulu district, northern tanzania’, l ivestock research for rural development 19(62), viewed 17 august 2011, from http://www.lrrd.org/ phiri, i.k., ngowi, h., afonso, s., matenga, e., boa, m., mukaratir wa, s., githigia, s., saimo, m., sikasunge, c., maingi, n., lubega, g.w., kassuku, a., michael, l., siziya, s., krecek, r.c., noormahome d, e., vilhena, m., dorny, p. & willingham, a.l., 2003, ‘t he emergence of taenia solium cysticercosis in eastern and sou thern africa as a serious agricultural problem and public hea lth risk’, acta tropica 87, 13–23. http://dx.doi.org/10.1016/s0001-706x(03)00051-2 schantz, p.m., wilkins, p.p. & tsang, v.c.w., 1998. ‘i mmigrants, imaging, and immunoblots: the emergence of neurocystice rcosis as a significant public health problem’, in w.m. shie ld, w.a. craig, j.m hughes (eds.), emerging infections, vol . 2, pp. 213–242, asm press, washington, dc. shandera, w.x. & kassy, j.s., 2006, ‘neurocysticer cosis: current knowledge and advances’, current neur ology and neuroscience reports 6(6), 453–459. http://dx.doi.org/10.1007/s11910-006-0046-3 szumilas, m., 2010, ‘explaining odds ratios’, journal of the canadian academy of child and adolescent psychiatry 19(3), 227–229. pmc2938757 tfda, 2003, the tanzania food, drugs and cosmetics act no. 1, 2003. the government printer, dar es salaam,tanzania, viewed 1 1 februery 2013, from http://www.tfda.or.tz/index/view/acts/ vaga, m., 2009, keeping pigs in a free range system, estonia n university of life sciences, viewed 21 december 2012, from http://oe.confolio.org/ willingham, a.l., harrison, l.j., fèvre, e.m. & p arkhouse, m.e., 2008, ‘inaugural meeting of the cystice rcosis working group in europe’, emerging infectious diseases 14(12), e2. http://dx.doi.org/10.3201/1412.080889 winkler, a.s., blocher, j., auerd, h., gotwalde, t., matuj a, w. & schmutzhard, e., 2008, ‘anticysticercal an d antitoxocaral antibodies in people with epilepsy in rural t anzania’, transactions of the royal society of tropi cal medicine and hygiene 102, 1032–1038. http://dx.doi.org/10.1016/j.trstmh.2008.05.004 modisane_115-121.indd introduction global trade in animals and animal products has grown significantly in recent years. the improvement of global transport systems makes it easier for animal products to be transported much faster and so can animal disease causing agents. the impact caused by these diseases may be localized or global. some diseases are not apparent but cause severe economic losses because of their nature. some that affect man are notorious for causing lower productivity. for these reasons, it is important to prevent, control and eradicate animal diseases using a science-based approach. disease control measures are undertaken to minimize the various impacts they may have. when disease control measures are undertaken, often some unintended impacts are experienced. these undesired results need to be monitored, measured and the cost thereof be determined so as to justify the choice of the measure employed. in addition, it is necessary to take into consideration factors contributing to animal disease occurrence which include climate, vegetation, animal population, interaction of the wild and domesticated animals, farming systems and management of animals, presence of vectors, and type of animal breed (e.g. indigenous sanga and zebu cattle’s resistance to tick-borne diseases). it is also important to ensure that the interaction between wild animals and domesticated animals is limited as this interaction may have undesired effects on both. this interaction can be minimised in a number of ways including fences and movement control. climate and its changing nature has recently become an important consideration since it is believed to influence the epidemiology and spread of some diseases, particularly arthropod-borne ones. understanding of the epidemiology of animal diseases is a very significant consideration in animal disease outbreak control and prevention. 115 onderstepoort journal of veterinary research, 76:115–121 (2009) field services: eradication and control of animal diseases b.m. modisane deputy director-general, disaster management and veterinary services national department of agriculture, pretoria, south africa abstract modisane, b.m. 2009. field services: eradication and control of animal diseases. onderstepoort journal of veterinary research, 76:115–121 prevention, eradication and control of animal diseases, as well as public health assurance are major functions of veterinary authorities. the strategies to control animal diseases differ from disease to disease but are often similar on a disease basis from country to country depending on the main objective of the measure employed. these measures include among others movement control and quarantine, vaccination, treatment and mass slaughtering. however, not every country uses all these control measures at the same time. a combination of measures may be employed to avoid spreading of the disease from infected to clean animals and success is dependent on a variety of factors, including the strength and capacity of the veterinary services, cross border efforts for disease surveillance, political will, diagnostic facilities and financial support. 116 field services the world organisation for animal health (oie) provides adequate guidance on disease prevention and outbreak management and serves as an international standard-setting body and a world reference centre for activities involving animal disease outbreak management and trade involving animals and animal products. however, there are still some animal disease management approaches that are not yet harmonised. as a result, research on animal diseases and outbreak management carried out in different parts of the world at research institutes and universities is necessary and will continue being necessary for many years to come. it is for this reason that approaches to animal disease and outbreak management is constantly changing as new knowledge is gathered and new discoveries are made available to veterinarians. traditional approaches to disease control for purposes of trade are receiving a strong challenge from new concepts like compartmentalisation, commodity-based trade and containment zones. although some of these concepts are not really new disease control approaches, they tend to contribute to new thoughts in disease control. management of disease outbreaks prevention, eradication and control of animal diseases, as well as public health assurance, are major functions of veterinary authorities in most countries. strategies to control animal diseases differ from disease to disease but are often similar for the same diseases from country to country depending on the main objective of the measure employed. the success of these measures is dependent on a variety of factors amongst which the strength and capacity of the veterinary services, cross-border efforts for disease surveillance, political will, diagnostic laboratory and financial support are important (perry & sones 2007). those countries that are successful in effectively managing animal diseases have strong animal disease policies and legislation, normally based on principles and guidelines of the oie’s terrestrial and aquatic animal health codes (2007), and a strong laboratory network guided by the biological standards of the oie (2007). furthermore, these countries have statutory bodies that also monitor the conduct of veterinarians, including those in the private sector. privately practising veterinarians, although independent, are expected to report certain animal diseases to the state once they have been diagnosed. this emphasizes the importance of the existence of strong links between the state veterinary service and private practitioners. prevention of disease outbreaks is more cost effective than eradication and control after outbreaks have taken place. effective and efficient organization of preventative measures following a risk assessment approach is essentially the first step in controlling the spread of disease. in addition, effective movement control and quarantine, vaccination, treatment and mass slaughtering can be used for disease confinement and eradication purposes (perry & sones 2007). each control measure acts by reducing the effective reproductive index of the infective agent in the population. it is not necessary to use all these control measures simultaneously. however, a combination of these measures may be required to avoid spreading of the infective agent from infected to clean animals. movement control and quarantine the prevention of the interaction of wild and domesticated animals is often a necessary step in the prevention of disease outbreaks as some diseases can be easily exchanged between the two. the classical examples where diseases can be exchanged in this manner are foot-and-mouth disease (fmd), classical swine fever, avian influenza and even bovine tuberculosis. separation of wild and domestic animals could be achieved by fencing or other physical or natural barriers like rivers, forests, mountains, thus effectively controlling the movement of animals and confining them to their respective territories. electrified fences are sometimes used for this purpose. such separation can however be resource intensive and has recently been questioned as the cost and benefits have not been assessed, particularly for african countries (perry & sones 2007). in addition to separating wild and domestic animals, movement controls are also essential in separating animals of different disease status, such as vaccinated from unvaccinated animals. it is often necessary that these animals be identifiable in order to detect an animal that is wandering out of its rightful territory on time. such fences need to be checked for intactness, and should be immediately repaired as soon as any damage is detected. the south african approach for the control of foot and mouth disease, as explained in the unpublished fmd protocol of the department of agriculture (2005), is to have the infected area enclosed by an electrified game proof fence, and immediately surrounded by a zone in which all ruminants are vaccinated twice a year and inspected regularly for fmd lesions. this zone is commonly referred to as the buffer zone. all animals in this zone are identi117 b.m. modisane fied as animals that have been vaccinated. the buffer zone is in turn surrounded by a zone in which all ruminants are inspected at less regular intervals but also separated from the vaccinated animals with a fence. this zone is often referred to as the surveillance zone. the surveillance zone is traditionally a disease free area. the same zoning concepts are applied during disease outbreaks, when certain zones like infected zone, buffer zone and surveillance zones are declared. movement control also aims at controlling the translocation of possibly infective animal products either by people or mechanically. it is often necessary that only products that have been rendered safe be allowed out of the infected area into areas that are free from disease. it may be necessary that vehicles and other mechanical means be disinfected with an effective disinfectant. the need for roadblocks is brought about by these procedures. quar antine is an extreme form of movement control where animals, their products and sometimes even handlers are confined to an area with limited movement in order to avoid contact with susceptible animals or their handlers. all the principles explained above are applicable. vaccination without doubt vaccination, when available and applicable, is still the most cost effective means of preventing and controlling and even eradicating infectious animal diseases (pastoret, lombard & schudel 2007). vaccines are normally easily available, also to farmers, unless their application needs special attention. vaccination can, however, interfere with future diagnoses of disease if an inappropriate vaccine has been used. the dawn of marker vaccines has to a large extent contributed to reducing these interferences significantly and led to the ability to differentiate between vaccinated and diseased animals (vannier, capua, le potier, mackay, muylkens, parida, paton & thiry 2007). scudamore (2007) reported that the fmd outbreak in the united kingdom was unprecedented with the need to develop a vaccination policy at the height of an epidemic. he also indicated that during a survey conducted in that country it was very clear that a high percentage of consumers have a propensity not to consume animals that have been vaccinated. however, this new technology, being able to differentiate between diseased and vaccinated animals commonly known as diva, also contributed to continued disapproval by the public of the culling of large numbers of animals when there are other alternatives like vaccination. in an unpublished report of the south african department of agriculture on the outbreak and control of classical swine fever in the eastern cape (2005) it is stated that vaccination in most cases requires the possibility to handle animals individually, often repeatedly for boosters, which can be a challenge in more extensive, free-ranging farming systems without intensive handling facilities. in some instances it is possible to use bait vaccines and still be able to sufficiently and effectively control animal diseases as was the case in the control of rabies in the czech republic (matouch, vitasek, semerad & malena 2007). treatment treatment can be aimed at treating the affected animal against the particular agent of concern or treating the animal to prevent it from being attacked by disease-carrying arthropods like ticks and insects. dipping the animals, together with vaccination where appropriate, is still considered to be one of the most effective strategies against some arthropod borne diseases. dipping, however, has to be properly applied as resistance to some acaricides is developing at an alarming rate (ntondini, van dalen & horak 2008). mass slaughtering the availability of the technology enhancing the differentiation of diseased from vaccinated animals has contributed to an increase in public disapproval of some veterinary prophylactic measures such as mass slaughtering of livestock to control outbreaks of epizootic diseases (scudamore 2007). although this is the case, slaughtering of infected animals will still be necessary in many circumstances. it is emphasized therefore that decision-making in the control of animal disease outbreaks should be a dynamic and flexible process. (ge, mourits & huirne 2007). when a disease is diagnosed at the beginning of an outbreak there are many uncertainties about the consequences and success of control options. in the south african context and probably everywhere else in the world, there is always a possibility of a strong public outcry at the methods employed to control outbreaks of animal diseases. industries and the public should therefore be involved for successful animal disease prevention and control. at the same time, flexible decisionmaking can avoid unnecessary control costs through learning and adjusting. 118 field services new concepts in animal disease control and trade although the above guidelines have been applied for many years and are still applicable, the concept of trading with animals and their products on the basis of geographical freedom from disease is currently being challenged. in the concept, any incursion of the disease in the so called disease free area automatically resulted in loss of trade from the entire country or region. new concepts such as compartmentalization, containment zones and commodity based trade have recently been introduced. these concepts, as well as the dawn of marker vaccines, have changed disease control approaches significantly. compartmentalization trade ‘regions’ and ‘zones’ are based on principles of epidemiological science and risk analysis that assess and manage animal disease risks so that the safety of trade can be ensured by establishing geographical boundaries (scott, zapeda, garber, smith, swayne, rhorer, keller, shimshony, batho, caporale & giovannini 2006). these scientists argue that the boundaries of geographical regions and zones can be breached through numerous epidemiological pathways. the concept of a ‘compartment’ extends the application of a ‘risk boundary’ beyond that of a geographical interface and considers all epidemiological factors that can contribute to the creation of an effective boundary. containment zone the terrestrial animal health code (oie 2008) describes a containment zone as a defined zone around and including suspected or infected establishments, taking into account the epidemiological factors and results of investigations, where control measures to prevent the spread of the infection are applied. the concept has helped to a large extent to spur authorities in a country to move with speed in the control of animal diseases and the necessary establishment of measures to prevent the spread of disease so as to allow trade on safe commodities even if the freedom status of the country by the oie has not been established yet. commodity-based trade in the context of trade, commodities could be anything from live animals to their products or even genetic and pathological material (oie 2008). the concept of trading in commodities only from countries that are free from the disease in question is believed to be trade limiting and unnecessarily wasteful by the british department for international development (2008). it is argued that the ability to export should be determined by the product or animal’s disease status and not by the country’s disease status. the advantage is that this approach allows countries with endemic diseases that cannot be eradicated, to participate. it is also believed that this approach will help many developing countries to participate in international trade even if they have not eradicated certain animal diseases. it is however also argued that this may not necessarily be the case, as once this approach is made a standard, every country, including the developed ones, will be allowed to use the same standard. it is also believed that this will be a disincentive for countries to control animal diseases that can be eradicated. some of these diseases, especially erosion diseases, could become endemic and lead to general low production of food worldwide. the situation in south africa since 2000 since 1994, south africa has joined the global village with regards to tourism and trade, contributing to the occurrence of some diseases. it is now understood that the 2000 fmd outbreak in the kwazulu-natal province occurred as a result of the illegal feeding of swill or galley waste obtained from ships in the durban harbour ((department of agriculture, unpublished 2001). it is also clear that the avian influenza outbreak was probably facilitated by insufficient attempts by ostrich farmers to keep ostriches apart from wild birds, especially water fowl (department of agriculture, unpublished 2005). it is not known how classical swine fever (csf) was introduced into the country. foot-and-mouth disease: camperdown outbreak since 2000, outbreaks in south africa mainly affected the limpopo province, but also occurred in the mpumalanga and kwazulu-natal provinces. these fmd outbreaks led to the suspension of international trade in a number of commodities, including beef and cloven-hoofed animal products. the main outbreak occurred in 2000 when a type o fmd virus was introduced into south africa for the first time. this outbreak was dealt with decisively by veterinary services. the occurrence of the disease affected international agricultural trade negatively, but also affected local trade in diverse agricultural 119 b.m. modisane products such as milk and strawberries. the major concern was the fact that type o fmd virus was diagnosed for the first time in south africa. the natural response was to go ahead and cull all affected and at risk animals. the culling process was slower than desired, however, and the disease was spreading fast. the strategy was consequently changed to that of vaccination after due consideration of a number of factors including the fact that the disease was moving into territories which were difficult to access. foot-and-mouth disease: middelburg outbreak the mpumalanga outbreak of fmd occurred shortly after the one in kwazulu-natal. this outbreak, however, occurred as a result of the contact between buffaloes that escaped from the kruger national park and domestic cattle. cattle from this outbreak area were inadvertently taken to a feedlot in mid delburg from where animals were sold to a slaughterhouse in swaziland. a clinical diagnosis of the infection was made at the abattoir and as a result swa ziland lost its export status and the much needed revenue. the outbreak was approached from a significantly different angle because the animals were confined to a feedlot and only a few pens were affected. the affected animals were culled and buried, whereas those that were still free from the disease were vaccinated and later slaughtered under supervision at designated abattoirs. this again demonstrates that there are no hard and fast rules in disease eradication, especially if we consider that the same disease, although due to a different strain of the virus, was involved at camperdown and middelburg but were controlled differently, depending on the prevailing conditions. equine influenza: cape town equine influenza occurred in horses in the western and eastern cape provinces. it is believed that the disease occurred when infected horses that were certified to have been vaccinated against the disease, entered south africa via cape town from europe. these horses could not be quarantined properly to prevent transmission of the infection. in this case, vaccination and supportive treatment of affected animals was employed. porcine reproductive and respiratory syndrome outbreaks of a few animal diseases unknown to south africa before 2004 raised some concern amongst veterinarians. one of these diseases is caused by salmonella cholerasuis, which was diagnosed for the first time in april 2004 in pigs in the western cape province (department of agriculture, unpublished 2004). not long thereafter, another disease, which had not been diagnosed before in this country, i.e. porcine reproductive and respiratory syndrome (prrs), was discovered and again in pigs in the western cape province. for prrs, isolating infected farms and slaughtering affected herds were used without the need for vaccination. notifiable avian influenza in ostriches notifiable avian influenza (nai) occurred in ostriches in the eastern and western cape provinces in 2004. this disease had been diagnosed before in this country, but had never been reported in its highly pathogenic form. this time it caused high morbidity and mortalities in ostriches and led to suspension of trade in all poultry and poultry products with trading partners. the ostrich industry lost close to r700 million and had to lay off 4 000 workers due to this loss of income during the period (department of agriculture, unpublished 2005). the poultry industry, including ornamental birds, also lost several million rand as they could not export their produce for months causing a severe shortage of poultry meat in this region. south africa had to prove to trading partners that the poultry industry itself was not affected by the disease before they could be allowed to export meat to the region. in this instance, the approach utilized was testing, isolating infected flocks, culling and burying the culled birds. it ended up as an exercise which adversely affected the emotional wellbeing of veterinarians and technicians alike, requiring psychological counselling in some cases. this is an example of an unintended impact that needs to be taken into consideration when disease eradication campaigns are planned. classical swine fever classical swine fever (csf) devastated the pig industry and affected both the eastern and western cape provinces. control measures for this disease continued for more than two years, at a great cost to the state, and included the establishment of road blocks in and around the province and a surveillance programme. animals that tested positive for the disease were put down humanely and buried. food security within the province and in other provinces was affected adversely. brucella canis brucella canis was never before reported in south africa and was introduced into the country in 2005 120 field services in the western cape province. so far it is not known how the disease was introduced, but it is suspected that an infected dog escaped from a ship/sea vessel and found its way inland where it may have infected other dogs. the canine was put down and no further cases were seen. general considerations it is not easy to predict when and how a disease will be introduced into a country. governments therefore have to be vigilant at all times and ensure that animal diseases are kept out of its territory to the best of their abilities. when these diseases are introduced, pre-conceived mitigation strategies should be implemented immediately. if an exotic disease is introduced, the speed of detection and prompt reaction of the veterinary services are of paramount importance in limiting the impact of such an outbreak. this requires surveillance strategies that include all parts of the livestock farming sector, a particular challenge for countries with a large informal and emerging farming component. a strong veterinary service component is essential for executing effective surveillance strategies and effective response to outbreaks. south africa is grateful for the support of international organizations, like the oie, for their assistance in promoting on a political level the need for independent and strong veterinary services systems to safeguard animal and human health, food safety and security as well as international trade in animals and animal products. poor implementation of disease control measures may contribute to localized negative impacts. improper use of chemicals and drugs, in particular, can expose animals, humans and the immediate environment to possible toxic effects, either directly, and/or indirectly, or through residues in livestock products (perry & sones 2007). participatory epidemiology the use of ‘existing veterinary knowledge’ or indigenous knowledge for epidemiological purposes and early detection and the use of local communication routes, e.g. tribal heads and chiefs, often improve the success rate in disease control and eradication. these traditional routes of communication are often an effective means to inform the public of the existence of serious animal diseases, but also of vaccination and inspection days and points. disease control programmes are also more acceptable to the affected community when their traditions are taken into account (mcleod & rushton 2007). geographic information systems a strong veterinary service component is essential for executing effective surveillance strategies and effective response to outbreaks. geographical information systems (gis) are able to provide information from a database and display it on a map. it can, for instance, depict the percentage of animals vaccinated at each dip tank in a particular year. it gives a clear picture of the disease situation in a particular country or area and can provide vast amounts of information during an outbreak such as: • exact location of index cases/infected farms • farms involved • farms located within a specified radius therefore, gis is valuable during the planning of control efforts. conclusion many factors have to be considered when deciding which disease control measure to employ. the approach to disease control differs from situation to situation and depends on available resources. it is advised that decision makers in animal disease control should constantly evaluate the effectiveness of control measures employed and adjust them where necessary. at the same time, disease control approach is moving with the times—as new technologies are discovered. a better understanding of the epidemiology of diseases is also leading to new approaches, such as compartmentalization and containment zones, to facilitate trade in commodities. these new concepts emphasize the necessity for flexibility in disease control decision-making. references ge, l., mourits, m.c.m. & huirne, r.b.m. 2007. towards flexible decision support in the control of animal epidemics. oie scientific and technical review, 26:551–563. matouch, o., vitasek, j., semerad, z. & malena, m. 2007. rabies-free status of the czech republic after 15 years of oral vaccination. oie scientific and technical review, 26: 577–584. mcleod, a. & rushton, j. 2007. participatory epidemiology in disease surveillance and research economics of animal vac cination. oie scientific and technical review, 26:313– 326. ntondini, z., van dalen, e.m.s. & horak, i.g. 2008. the extent of acaricide resistance in 1-, 2and 3-host ticks on communally grazed cattle in the eastern region of the eastern cape province, south africa. journal of the south african veterinary association 70:130–135. 121 b.m. modisane pastoret, p., lombard, m. & schudel, a. 2007. animal vac cination, parts 1 and 2. oie scientific and technical review, 26, april & august 2007. perry, b.d. & sones, k.r. 2007. epidemiological-economic support tools to control fmd in south american endemic settings, in global roadmap for improving the tools to control foot and mouth disease in endemic settings. nairobi: ilri. scott, a., zepeda, c., garber, l., smith, j., swayne, d., rhorer, a., kellar, j., shimshony, a., batho, h., caporale, v. & giovannini, a. 2006. the concept of compartmentalisation. oie scientific and technical review, 25:873–879. scudamore, j.m. 2007. consumer attitudes to vaccination of food-producing animals. oie scientific and technical review, 26:451–459. vannier, p., capua, i., le potier, m.f., mackay d.k.f., muylkens, b., parida, s., paton, d.j. & thiry, e. 2007. marker vaccines and the impact of their use on diagnosis and prophylactic measures. oie scientific and technical review, 26:351–372. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: mohie haridy1,2 madeh sadan3 mosab omar4 hiroki sakai2 tokuma yanai2 affiliations: 1department of pathology and clinical pathology, south valley university, egypt2department of pathogenetic veterinary sciences, gifu university, japan 3department of surgery, anesthesiology and radiology, south valley university, egypt 4department of parasitology, south valley university, egypt correspondence to: mohie haridy postal address: po box 83523, qena 83611, egypt dates: received: 06 nov. 2013 accepted: 11 apr. 2014 published: 28 aug. 2014 how to cite this article: haridy, m., sadan, m., omar, m., sakai, h. & yanai, t., 2014, ‘coenurus cerebralis cyst in the orbit of a ewe’, onderstepoort journal of veterinary research 81(1), art. #707, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.707 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. coenurus cerebralis cyst in the orbit of a ewe in this research communication... open access • abstract • introduction • materials and methods • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a 4-year-old rahmani breed ewe was presented for surgery to the veterinary teaching hospital, south valley university, egypt with enlargement and protrusion of the eye ball, blepharitis and congestion of the conjunctiva. on examination, a cyst 2.5 cm x 3.5 cm in diameter containing sandy fluid was detected in the perioptic nerve fat. histopathological examination revealed that the epithelial lining of the conjunctiva was necrotic and severely infiltrated by neutrophils. the underlying connective tissue was oedematous, hyperaemic and severely infiltrated by neutrophils. desquamation of the corneal epithelium was seen, together with oedema of the stroma. the tissue surrounding the cyst was compressed and the lacrimal glands revealed pressure atrophy. the muscular tissue was atrophied and infiltrated by fat cells. the cyst wall was lined with white scolices protruding from the inner wall. based on the gross and histopathological characteristics of the cyst observed, the cyst was diagnosed as coenurus cerebralis. this is the first report of orbital coenurosis in a sheep. introduction top ↑ coenurus cerebralis is the larval stage of taenia multiceps, an intestinal cestode of wild and domestic canids (the definitive host) (sharma & chauhan 2006). coenurus cerebralis is commonly located in the cerebrum in sheep, goats, cattle and humans. it constitutes a major health problem in sheep and goats worldwide, with significant economic repercussions (haridy et al. 2013; sharma & chauhan 2006). sheep are the usual intermediate hosts of t. multiceps. other herbivorous animals, non-human primates and humans can also be infected. taenia multiceps coenuri are usually found in the brain and spinal cord of animals (jones & pybus 2008). lagomorphs, including hares and rabbits, are the usual intermediate hosts of taenia serialis. infections have also been reported in squirrels, other rodents, cats and humans (jones & pybus 2008). taenia serialis coenuri are usually found in the subcutaneous tissues, muscles and retroperitoneum in animals (ing et al. 1998). causes of exophthalmoses include: orbital abscess, orbital cellulitis, orbital cyst, eosinophilic myositis, lacrimal gland disease, foreign body granuloma, haematoma and trauma (boydell 1991). reports on orbital parasitic cysts are uncommon in veterinary literature and have been attributed to hydatidosis, cysticercosis and t. serialis in a horse, hogs and pet rabbits, respectively (barnett et al. 1988; cardenas-ramirez et al. 1984; holmberg et al. 2007; o’reilly et al. 2002; wills 2001). however, human ocular coenurosis is common and has been reported in uganda and nigeria (ibechukwu & onwukemr 1991; williams & templeton 1971). the present study describes a case of orbital coenurosis in a sheep with a complete pathological picture following enucleation of the left eye. materials and methods top ↑ a 4 year-old rahmani ewe was admitted for surgery to the clinic of surgery, veterinary teaching hospital, south valley university, egypt for enucleation of the left eye. clinical examination revealed blepharitis, congestion of the conjunctiva, and enlargement and protrusion of the eye ball. enucleation of the affected eye ball was performed using the tranquilliser rompun (xylazine hcl) (bayer, uk) at a dose rate of 0.2 mg/kg weight intramuscular (i/m) in combination with local infiltration anaesthesia (xylocaine 2%). after aseptic preparation of the affected eye, incisions were made 3 mm − 5 mm from the eyelid margins, and were connected at the level of the medial and lateral canthi. blunt dissection was applied until the eye ball came free from the orbital cavity and was totally excised. the orbital cavity was packed with sterile gauze, and cephalexin (25 mg/kg weight) was administered intramuscularly for five consecutive days post operatively. the enucleated eye ball was fixed in 10% neutral buffered formalin and embedded in paraffin by routine methods. sections measuring 4 µm in thickness were stained with haematoxylin and eosin (he) (bancroft et al. 1996). results top ↑ clinical signs were mainly: proptosis, blepharitis, congestion of the conjunctiva, chemosis, peri-ocular swelling, especially of the superior fornix (fossa), and enlargement and protrusion of the left eye ball with no vision (figure 1a). the cornea of the eye was opaque, necrosed and covered with faecal matter, with complete loss of vision. during enucleation of the eye, a cyst filled with a translucent sandy fluid was observed in the periorbital fat, after cutting the retractor bulbar muscles. the cyst was loosely located between the retractor bulbar muscles and the orbital fat. the cyst measured 2.5 cm x 3.5 cm and contained a large number of white clusters of scolices (figure 1b). white scolices were protruding from the inner wall of the cyst and the majority of the scolices were located in a longitudinal line at one side of the cyst (figure 1c).microscopically, the epithelium of the conjunctiva was necrotic and severely infiltrated by neutrophils. the underlying connective tissue was oedematous, hyperaemic and severely infiltrated by neutrophils (figure 2a and figure 2b). the corneal epithelium was necrotic, with neutrophil infiltration. the stroma was oedematous and infiltrated by neutrophils. the tissue surrounding the cyst was compressed and lacrimal glands revealed pressure atrophy. the muscular tissue was atrophied and infiltrated by fat cells (figure 2c). there was a minimal cellular reaction in the muscles. fresh unstained samples of the internal fluid of the cyst revealed typical protoscolices of t. multiceps with rose-thorn hooks. the scolices had four suckers and a rostellum armed with a double crown that ranged between 28–30 hooks (figure 3a and figure 3b). the rostellar hooks ranged from 100 µm – 116 µm and 155 µm – 170 µm in length for small and big hooks, respectively, and the suckers ranged from 300 µm – 320 µm each in diameter. figure 1: (a) photograph of a ewe suffering from orbital coenurosis showing blepharitis, congestion of the conjunctiva and enlargement and protrusion of the eye ball; (b) coenurus cyst observed during enucleation of the eye (dotted line) located retrobulbarly in the periocular fat as a transparent cyst with white scolices and (c) scolices located in a longitudinal line at one side of the cyst. figure 2: photomicrograph of the eyeball of the ewe; (a and b) necrosis of the lining epithelium with suppurative conjunctivitis and (c) pressure atrophy of the tissues surrounding the cyst including lacrimal glands and muscular tissue: haematoxylin and eosin. figure 3: fresh unstained sample showing typical taenia multiceps protoscolices (a) whole mounted protoscolex (b) rosellar hooks. discussion top ↑ coenurus cerebralis cysts are often localised in the nervous system, including the brain and spinal cord (haridy et al. 2013). however, their occurrence in the subcutaneous and muscular tissues and other organs has also been recorded, especially in goats (oryan et al. 2010). the extra-cerebral cysts in goats have been erroneously referred to be due to coenurus gaigeri (sharma et al. 1998). the only orbital coenurosis that has been recorded in animals is t. serialis cysts in the orbit of a pet rabbit and chinchilla (holmberg et al. 2007; o’reilly et al. 2002), but it has been frequently recorded in humans (ibechukwu & onwukeme 1991; williams & templeton 1971). the two most common taenia species forming coenuri are t. seralis and t. multiceps. differentiation can be achieved by measuring the lengths of the rostellar hooks (edwards & herbert 1981) and verifying host specificity. in the present study, the scolices had four suckers and a rostellum, as described in the results section. the ranges of values of the dimensions in the present study are similar to those of t. multiceps (edwards & herbert 1981). moreover, the sheep is a common host for c. cerebralis, and coenuri of t. serials have never been reported in sheep. although there have been no reports of c. gaigeri in the central nervous system or eye, it has not been possible to differentiate morphologically between c. gaigeri and c. cerebralis (oryan et al. 2010). recently, c. cerebralis and c. gaigeri were considered to be non-analogous metacestodes for the same tapeworm (kheirandish et al. 2012). moreover, it was possible to differentiate the characteristic features of the coenurus cyst in the present study from hydatid cysts (naghashyan & harutunyan 2001). the cyst wall in the present study was thin, translucent, easily separated from the surrounding tissues, had no vascularity and lacked the laminated layer usually found in hydatid cysts. moreover, the cyst was filled with a transparent watery fluid and the daughter cysts were tightly attached to the germinal layer (naghashyan & harutunyan 2001). human ocular coenurosis involves the vitreous anterior chamber or subconjunctival tissues (ibechukwu & onwukeme 1991; manschot 1976; williams & templeton 1971). it is suggested that the larvae of t. multiceps enter the eye by way of the ciliary arteries or direct inoculation of the larva to the conjunctiva (ibechukwu & onwukeme 1991). however, orbital coenurosis due to t. serialis larvae has been observed in a pet rabbit and chinchilla (holmberg et al. 2007; o’reilly et al. 2002). these species are infected after ingesting eggs (oncospheres) that have been shed in faeces by the definitive host. the oncospheres migrate through the intestinal epithelium and travel through the circulatory system to the predilection sites, including the orbit (heath 1971). in the present study, the cyst was found in the orbital fat, causing exophthalmia of the left eye. proptosis led to exposure keratitis and conjunctival congestion, as well as ulceration of the cornea (barnett et al. 1988). exophthalmoses are caused by space-occupying lesions; usually a retro-bulbar abscess, tumour or cyst (wilcock 2007). in sheep, exophthalmos has been observed in cases of enzootic nasal adenocarcinoma (de las heras et al. 2003) and conidiobolomycosis (silva et al. 2007). there is no knowledge of either parasitic or dermoid cysts being recorded in sheep. coenurus cysts have been reported to cause softening and pressure atrophy of the overlying skull, to the extent of perforation (nooruddin et al. 1996; ozkan et al. 2011). coenurus cysts might creep into the orbital cavity through the optic canal or through softening and perforation of the sphenoid bone. conclusion top ↑ the present study described a unique case of orbital coenurosis in a sheep. taenia multiceps coenurus most likely occasionally lodges in the eyes of domestic ruminants and must be considered as a differential diagnosis for ocular pathology in regions where the parasite is endemic. effective control measures in endemic regions include: public awareness of the epidemiology of the disease, hygienic disposal of offal, prophylactic anti-parasitic drugs on sheep farms, and deworming of dogs. acknowledgements top ↑ the authors would like to thank miss cate swift for her proofreading of this manuscript. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions m.h. (south valley university) was the pathologist and wrote the manuscript draft. m.s. (south valley university) was the surgeon and reported the clinical signs. m.o. (south valley university) was the parasitologist and reported the parasite description. h.s. (gifu university) reviewed the technical and scientific basis of the manuscript and t.y. (gifu university) reviewed the manuscript. references top ↑ bancroft, t.d., stevens, a. & turner, d.r., 1996, theory and practice of histological technique, 4th edn., churchill livingston, new york, london, san francisco, tokyo. barnett, k.c., cottrell, b.d. & rest, j.r., 1988, ‘retrobulbar hydatid cyst in the horse’, equine veterinary journal 20, 136–138. http://dx.doi.org/10.1111/j.2042-3306.1988.tb01478.x boydell, p., 1991, ‘fine needle aspiration biopsy in the diagnosis of exophthalmos’, journal of small animal practice 32, 542–546. http://dx.doi.org/10.1111/j.1748-5827.1991.tb00882.x cardenas-ramirez, l., celis-salgado, p. & hernandez-jauregui, p., 1984, ‘ocular and orbital cysticercosis in hogs’, veterinary pathology 21, 164–167. de las heras, m., ortín, a., cousens, c., minguijón, e. & sharp, j.m., 2003, ‘enzootic nasal adenocarcinoma of sheep and goats’, current topics in microbiology and immunology 275, 201–223. http://dx.doi.org/10.1007/978-3-642-55638-8_8 edwards, g.t. & 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guthrie5 eric denison6 philip s. mellor6 christopher hamblin6 affiliations: 1institute of veterinary, animal and biomedical sciences, massey university, new zealand 2polio communication section, unicef nigeria country office, nigeria 3ross university school of veterinary medicine, st. kitts, west indies 4arc-onderstepoort veterinary institute: parasites, vectors and vector-borne diseases, onderstepoort, south africa 5equine research centre, university of pretoria, south africa 6the pirbright institute, pirbright, united kingdom correspondence to: stuart gordon email: s.j.g.gordon@massey.ac.nz postal address: private bag 11 222, palmerston north 4442, new zealand dates: received: 04 nov. 2014 accepted: 27 jan. 2015 published: 29 may 2015 how to cite this article: gordon, s.j.g., bolwell, c., rogers, c., musuka, g., kelly, p., labuschagne k. et al., ‘the occurrence of culicoides species, the vectors of arboviruses, at selected trap sites in zimbabwe’, onderstepoort journal of veterinary research 82(1), art. #900, 8 pages. http://dx.doi.org/10.4102/ojvr.v82i1.900 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the occurrence of culicoides species, the vectors of arboviruses, at selected trap sites in zimbabwe in this original research... open access • abstract • introduction • materials and methods    • selection of culicoides trap sites in zimbabwe    • trapping, sorting and processing of culicoides samples for identification    • trapping and sorting of culicoides midges in harare for virus isolation    • preparation of culicoides specimens for virus isolation and identification • results    • collection of culicoides across zimbabwe for species identification    • collection of culicoides in harare for species identification and virus isolation • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references • appendix 1 abstract top ↑ a study of the distribution of culicoides species was conducted by establishing 12 light trap sites over five rainy seasons between 1998 and 2003 covering all the geo-climatic natural regions of zimbabwe. in total, 279 919 specimens of culicoides were trapped over a total of 163 trapping nights. the highest median counts of culicoides per trapping night were recorded in natural region iii, which has climatic conditions conducive to the successful development of the larvae. culicoides imicola, the major vector of bluetongue and african horse sickness viruses in africa, was found to be the most abundant species (80.4%), followed by culicoides enderleini (5.9%) and culicoides milnei (5.2%). this study identified 10 species of culicoides that had not been previously described in zimbabwe, including culicoides loxodontis and culicoides miombo, which are members of the c. imicola complex. a total of 23 994 culicoides midges were collected from five trap sites in harare, zimbabwe, with the dominant species, c. imicola, representing 91.6% of the total collection. seventeen arboviruses were isolated from these midges, 15 of which were bluetongue virus. the predominant bluetongue virus serotype was serotype 11, followed by serotypes 1, 8, 12 and 15. bluetongue virus serotypes 1, 2, 8, 10, 12, 15, 16 and 18, detected in this study, had not been previously reported in zimbabwe. introduction top ↑ african horse sickness virus (ahsv), which infects all equine species, and bluetongue virus (btv), which infects sheep and other ruminants, are endemic in zimbabwe. high seroprevalences of ahsv in horses and donkeys and btv in cattle and sheep were reported throughout zimbabwe (gordon 2010), with mortality rates because of african horse sickness and bluetongue reaching 95% (coetzer & guthrie 2004) and 33% (musuka 1999) respectively. based on their economic importance and capacity to spread rapidly between countries, the world organisation for animal health (oie) has designated both these diseases as notifiable animal diseases. equine encephalosis virus (eev), which infects all equine species, and epizootic haemorrhagic disease virus (ehdv), which infects domestic and wild ruminants, have also been detected in zimbabwe (gordon 2010; musuka 1999; paweska et al. 1999). many of these viruses are also enzootic in neighbouring countries, namely botswana (mushi et al. 1998), zambia (mweene et al. 1996), namibia and south africa (meiswinkel, venter & nevill 2004; scacchia et al. 2009; world organisation for animal health 2011). oura et al. (2012) recently documented eev in ethiopia, ghana, gambia and israel. bluetongue today has a nearly worldwide distribution. all of these diseases are caused by double-stranded ribonucleic acid (dsrna) viruses of the family reoviridae and genus orbivirus, which are transmitted almost exclusively by culicoides (diptera: ceratopogonidae) biting midges (meiswinkel et al. 2004; mellor, boorman & baylis 2000). culicoides imicola and culicoides bolitinos have been identified as vectors of ahsv and are widely distributed in sub-saharan africa (meiswinkel & paweska 2003; mellor 1993). culicoides bolitinos has also been implicated as an important potential vector of eev and btv in south africa (meiswinkel & paweska 1998, 2003; venter et al. 2002). previous studies in zimbabwe found c. imicola to be the most abundant livestock-associated culicoides species, followed by culicoides zuluensis (blackburn, searle & phelps 1985; braverman & phelps 1981; braverman et al. 1985; gordon 2010; musuka 1999; musuka, chihota & kelly 1998; phelps, blackburn & searle 1982). the vector competence of most culicoides species in zimbabwe has not been studied in detail and other species may, in future, be identified as vectors of these viruses. agro-ecological classification, known as natural region (nr) classification in zimbabwe, divides the country into five regions based on mean annual rainfall (vincent & thomas 1960). the aim of this study was to identify the species of culicoides distributed across these regions during the period 1998–2003. it is known that climate has a profound influence on the viability and fecundity of the culicoides midges (mellor 1993). to date no differences in disease abundance have been reported between these regions. the monthly abundance and distribution of culicoides species in these five regions was, therefore, determined. in addition, this study aimed to isolate and identify arboviruses isolated from culicoides midges collected in harare over 7 days within the study period. such studies will expand our knowledge of the biology of zimbabwean culicoides species and the potential for transmission of viral pathogens. this information will help assess the risks to both ruminant and equine livestock in zimbabwe attributable to the presence of the culicoides vector and hence the implementation of appropriate integrated control strategies (rawlings et al. 1998). materials and methods top ↑ selection of culicoides trap sites in zimbabwe culicoides midges were collected from a convenience sample of 12 trap sites in zimbabwe. collections were made over five consecutive rainy seasons (november–april) from 1998 to 2003. collection site selection was based on several criteria, including: a minimum number of either horses or ruminants (> 8); historical presence of clinical disease or deaths in animals; presence of suitable culicoides vector breeding conditions (e.g. low-lying areas, such as river banks or marshy fields, with clay soils); security of the equipment; and the availability of an alternating 220v current power supply. trap sites were distributed to include each of the five nrs of zimbabwe (figure 1). two trapping sites were established in nr i, six in nr ii, one in nr iii, two in nr iv and one in nr v. twenty-four individual collections (trapping nights) were made from nr i, 109 from nr ii, 18 from nr iii and 9 from nr iv across the five rainy seasons. trapping was to be conducted for one night every 2 weeks during each rainy season, although this protocol was not followed consistently at each trapping site. only three individual collections were made in nr v during the entire period under study. figure 1: the natural regions of zimbabwe and the locations of the 12 culicoides trapping sites. rainfall is highest in nr i (> 1050 mm per annum) whilst nr ii and iii contain the most arable land and receive 500 mm – 1050 mm of rain per annum. nr i is a specialised and diversified farming region with plantation forestry, fruit and intensive livestock production, whilst nrs ii and iii are suitable for intensive farming based on crops and livestock production. severe mid-season dry spells make nr iii marginal for maize, tobacco and cotton, or for enterprises based on crop production alone. the farming systems, therefore, are based on both livestock (assisted by the production of fodder crops) and cash crops. nr iv and v have a lower annual rainfall (< 600 mm per annum) and experience periodic seasonal droughts and/or severe dry spells during the rainy season. the farming, therefore, is based on extensive livestock ranching and drought-resistant fodder crops (vincent & thomas 1960). trapping, sorting and processing of culicoides samples for identification dusk (18:00) till dawn (06:00) trapping was conducted at each trap site using onderstepoort 220 v down draught suction light traps each fitted with an 8 w black fluorescent (ultraviolet) light tube. the traps were hung on the branches of trees or on the eaves of buildings approximately 1.4 m above ground level and within close proximity to the animals. culicoides midges were collected into phosphate-buffered saline (pbs) and 0.5% chlorhexidine (savlon®, novartis, u.k.). after collection specimens were re-suspended and preserved in 80% ethyl alcohol and stored in the dark at room temperature until sorted, identified and counted. species were identified and counted under a binocular dissecting microscope using a wing pattern atlas of southern african culicoides species (r. meiswinkel, unpublished atlas 1996). for large collections in excess of 1000 midges, the total numbers were estimated by measurement of a minimum of five 1 ml subsamples according to the protocol of van ark and meiswinkel (1992). trapping and sorting of culicoides midges in harare for virus isolation culicoides midges were collected for virus isolation at five sites around harare (17°51′50′s, 31°1′47′e, located in nr ii) using the method described above. these five harare sites formed part of the 12 trappings mentioned previously. these culicoides species were age graded according to the method described by dyce (1969). harare is 1508 m above sea level and situated in the northeast highveld of zimbabwe. it is characterised by moderate subtropical conditions with an average monthly rainfall of 120 mm and air temperature between 15 ºc and 27 ºc. all trap sites were on smallholdings with vertisol soil types and mixed forest vegetation where urban-intensive farming was practised. the main species of livestock were cattle, goats, sheep and horses. the traps were hung from branches or eaves of buildings within close proximity to animal premises. all sites’ traps were operated from dusk (18:00) to dawn (06:00) for seven consecutive days in march 2003. culicoides were collected into a phosphate-buffered saline (pbs) solution containing 100 mg/ml of gentamicin and 0.1% chlorhexidine (savlon®, novartis, u.k.) and stored at 4 ºc until processed. individual species of culicoides were stored at 4 ºc in 2 ml eppendorf tubes (a maximum of 50 flies per eppendorf tube) with 1 ml of pbs containing 100 mg/ml of gentamicin and 0.1% chlorhexidine (savlon®, novartis, u.k.). for each species or species group, the culicoides specimens were sorted into the following categories: males, blood engorged females, non-engorged nulliparous females and non-engorged parous females, according to dyce (1969). preparation of culicoides specimens for virus isolation and identification midges were grouped into individual pools of up to 100 non-engorged females of each species. each pool was homogenised in a mortar using a pestle with 1 ml of eagles maintenance medium containing 1% foetal calf serum and antibiotics, using a modification of the technique proposed by el hussein et al. (1989). each homogenate was subsequently titrated for infectious virus in bhk-21 cells, brains from suckling mice and 11-day-old embryonated hens’ eggs. virus isolates were characterised using serogroup-specific, indirect, sandwich enzyme-linked immunosorbent assay (elisa) for btv (thevasagayam et al. 1996), ahsv (hamblin, mellor & boned 1991), eev (crafford et al. 2003) and ehdv (thevasagayam et al. 1996) antigens. the btv isolates were subsequently serotyped using virus neutralisation index assays (hazrati & ozawa 1965). results top ↑ collection of culicoides across zimbabwe for species identification a total of 279 919 specimens of culicoides, representing 51 different species, were trapped across zimbabwe over five rainy seasons. table 1 shows the numbers of culicoides trapped per night in each nr of zimbabwe and are reported as median values with interquartile ranges (iqrs). the distribution of each of the 10 most abundant culicoides within the nr are presented as percentage values in table 2. the trap sites located in nr iii recorded the highest median count of culicoides species (1376 [iqr 286–1980]), followed by those trap sites located in nr iv (821 [iqr 175–918]). a full list of the different culicoides trapped (in order of abundance) across the five nrs of zimbabwe between 1998 and 2003 is shown as supplementary data in appendix 1 (table 1-a1). table 1: median (and interquartile ranges) counts of culicoides midges per trapping night caught in the five natural regions of zimbabwe across five rainy seasons: 1998–2003 (november–april). table 2: the mean percentage of the 10 most abundant culicoides spp. trapped in each natural region in zimbabwe over the five rainy seasons (1998–2003). the predominant culicoides species trapped was c. imicola (80.4%), followed by culicoides enderleini (5.9%), culicoides milnei (5.2%) and c. zuluensis (4.8%). culicoides imicola was the most abundant species trapped in nr i–iv, with c. enderleini being the most abundant species trapped in nr v. the second most abundant species trapped in each nr was culicoides miombo (nr i), c. milnei (nr ii), c. zuluensis (nr iii), c. miombo (nr iv) and c. bolitinos (nr v). species of culicoides found in every nr were c. imicola, c. enderleini, c. bolitinos and culicoides subschultzei. ten additional species, which have not previously been recorded in zimbabwe, were collected (table 3), bringing the total number of culicoides species recognised in zimbabwe to 60. table 3: the distribution of previously unreported species of culicoides across the five natural regions of zimbabwe (1998–2003). collection of culicoides in harare for species identification and virus isolation a total of 23 994 culicoides specimens were collected from the harare sites. culicoides imicola (21 986/23 994; 91.6%) was the most prevalent of the 26 species captured, followed by c. enderleini (1556/23 994; 6.5%). other species of culicoides regularly captured at harare were c. zuluensis (248/23 994; 1.0%), c. bolitinos (70/23 994; 0.3%) and culicoides brucei (39/23 994; 0.2%). three species novel to zimbabwe, namely c. miombo, culicoides glabripennis and a specimen from the nigripennis group, were identified in the harare collections. significant differences were found in the numbers of culicoides midges collected at the five harare trapping sites. nearly 90% of all the specimens collected were from eight collections made at one of the sites at a horse stud. however, the greatest species diversity was observed at a different horse stud (17 different species). the lowest numbers of culicoides and lowest species diversity were found at a dairy farm on the outskirts of harare that had cattle only and no horses (196 midges belonging to eight species of culicoides). in the harare study, 222 samples of non-engorged parous female culicoides were assayed for btv, ahsv, eev and ehdv. a total of 17 orbiviruses were isolated: 15 btv and 2 eev. fourteen isolates of btv were made from c. imicola and one from culicoides magnus. both the eev isolates were from c. imicola. no ahsv or ehdv were isolated. nine different btvs were identified. three isolates of serotype 11 (3/15; 20%) were made and two isolates each of serotypes 1, 8, 12 and 15 (2/15; 13.3% each) were made. one isolate each of serotypes 2, 10, 16 and 18 were also identified (1/15; 6.7% each). the eev isolates were not serotyped. discussion top ↑ trap sites chosen were believed to be representative of the climatic and geo-physical conditions found within each nr. however, the significance of the distribution of culicoides across the five nrs of zimbabwe must be interpreted with caution. it is important to realise that these five agro-ecological regions were characterised more than 50 years ago (vincent & thomas 1960). mugandani et al. (2012) have argued that this is misleading as it implies that the climatic conditions have remained stable over this time. research, however, points to the contrary; makarau (1999) and low (2005) noted increased variability of rainfall, rain days and temperature in zimbabwe. most meteorological stations in zimbabwe have shown a decline in rainfall over the past 100 years (mugandani et al. 2012). furthermore, corbett and carter (1996) noted that continued use of these agro-ecological regions was at odds with the political, social and agrarian reform that has taken place in zimbabwe since vincent and thomas (1960) established these regions. caution should also be observed when interpreting absolute counts of culicoides as there may be some variation dependent on the trap sites within each nr. the number of host animals and the distance of these animals from the trap site may also influence the numbers of culicoides collected. trap sites in nr ii recorded the greatest diversity of culicoides species, with 43 species identified. the highest median count of culicoides was recorded in trap sites located within nr iii. natural region iii includes the central highveld of zimbabwe, averaging a total annual rainfall of 500 mm – 700 mm, with predominantly greyish brown clay soils and sandy loams derived from granitic rocks (vincent & thomas 1960). high median counts of culicoides midges were recorded in march and april, at the end of the rainy season. numbers of adult flies steadily increase after the start of the rainy season as the availability of breeding sites increases (musuka 1999). similar findings have been reported previously in zimbabwe and south africa, with large populations of culicoides vectors found in areas with high average annual rainfall and summer temperatures (musuka 1999; musuka et al. 2001; venter, nevill & van der linde 1997). temperature influences the abundance of culicoides vectors through its effect on the availability of suitable breeding sites, larval development and adult mortality (mellor & hamblin 2004). high environmental temperatures lead to a shorter time span between blood-feeding events and an increase in the rate of virogenesis within the adult vector, leading to earlier virus transmission, although at high temperatures the rate of adult mortality also increases (mellor & hamblin 2004; wellby et al. 1996). previous studies have demonstrated that soil type appears to be very important in determining the distribution and abundance of c. imicola (meiswinkel 1998; musuka et al. 2001). in concurrence with these previous findings, the largest numbers of c. imicola were found in areas with a high, moisture-retentive clay soil, whilst the lowest numbers were encountered in rapidly draining sandy soils. the biggest collections of culicoides were obtained from a large equine stud on the outskirts of harare. this farm was situated in a low-lying area on the banks of a major river and had clayey soils and open grassland vegetation. this study showed that large numbers of c. imicola were collected in light traps located at sites where horses were stabled indoors at night even though some stables were fitted with protective fly screens. this suggests that c. imicola were still feeding successfully as they depend upon blood meals for the development of their eggs. successful feeding may have been facilitated by the common practice of leaving the upper half of the stable door open for ventilation (meiswinkel 1998). horses kept out in the open at night appear to be more susceptible to midge bites than stabled horses and these outside horses may act as decoys deflecting c. imicola away from the stabled animals. if these decoys are removed or stabled, then the midges may attempt to feed on stabled animals (meiswinkel 1998). this study has identified 10 species of culicoides that previously have not been recorded in zimbabwe (blackburn et al. 1985; musuka 1999; musuka & kelly 2000; musuka et al. 1998, 2001; phelps et al. 1982), bringing the total number of recognised species of culicoides in zimbabwe to 60. of particular note was the identification of two additional c. imicola complex species, culicoides loxodontis and c. miombo, which were distributed across four of the nrs of zimbabwe. the epidemiological significance of these species as vectors of arboviruses in zimbabwe needs further investigation, although the findings of this study significantly add to the available knowledge on the distribution of these species in southern africa. musuka (1999) has shown that c. bolitinos has a widespread distribution in southern africa. furthermore, c. bolitinos, which was found at all five trap sites in harare in the present survey, has been implicated in ahsv, eev and btv transmission in south africa (meiswinkel & paweska 1998, 2003; venter et al. 2002) and, therefore, could play a role in the spread of the disease in zimbabwe. in our study, the highest prevalence (5.7%) of c. bolitinos was recorded in nr v (table 2). this region is characterised by low rainfall and high environmental temperatures, although in south africa c. bolitinos has been reported to be more abundant in the cooler mountainous regions (meiswinkel & paweska 2003; venter et al. 2002). this species breeds in the dung of cattle and buffaloes and the traps in the nr v trap site were located near a cattle pen containing 30 cattle. the trap sites also included a wildlife sanctuary in north zimbabwe that hosts large populations of wild buffaloes (syncerus caffer). the distribution of culicoides species observed during this study closely matched the sero-incidence and prevalence of ahsv, eev, btv and ehdv reported by gordon (2010). in order to fully understand the role of the various culicoides species in the transmission of arboviruses in zimbabwe, further studies are needed to determine the vector competence of the sibling species of the c. imicola group and other culicoides species that are potential vectors in southern africa (musuka et al. 1998). it has recently been demonstrated, for example, that c. magnus could become infected with both btv and ahsv under laboratory conditions (paweska, prinsloo & venter 2003; paweska, venter & mellor 2002; venter et al. 2004, 2009). in this study, virus isolations of eev and btv were made only from c. imicola and c. magnus. according to meiswinkel et al. (2004), c. miombo is suspected to be a vector of btv. in the harare study, however, no viral isolates were obtained from this species (although only nine individual specimens were collected and assayed). no ahsv or ehdv were isolated from any of the trapped culicoides species despite the traps being located at horse studs and cattle farms. low ahsv isolations from culicoides midges were also reported by rawlings et al. (1998) in a study conducted in gambia, which was attributed to an extremely low infection prevalence of ahsv in the vector species of culicoides. these observations were further authenticated by mellor, osborne and jennings (1984), in sudan, who could only make two ahsv isolations out of 7000 culicoides specimens. many viral, genetic and environmental factors can influence the susceptibility of culicoides to infection and some species may be refractory to infection (mellor et al. 1984). it is recommended, however, that large numbers of flies from many different collection sites should be collected and assayed before defining a species as refractory to infection. the predominant serotype of btv isolated from the culicoides vectors was serotype 11, followed by serotypes 1, 8, 12 and 15. previously only btv serotype 11 has been isolated from the culicoides vector in zimbabwe (blackburn et al. 1985), so this is the first time that btv serotypes 1, 2, 8, 10, 12, 15, 16 and 18 have been recorded in the country. the possibility that some of these additional btv serotypes isolated may be vaccine viruses should be considered, however. the vaccine currently used in zimbabwe is an onderstepoort biological product® attenuated cell culture adapted vaccine. serotypes 15, 16 and 18 are not included in this vaccine and thus vaccinated cattle and sheep may not be adequately protected against all btv serotypes present in zimbabwe. there is, therefore, a need to consider the possibility of developing a local polyvalent vaccine that protects against all the btv serotypes circulating in zimbabwe. conclusion top ↑ the greatest diversity and highest median counts of culicoides were found in the central highveld region of zimbabwe, which enjoys climatic and geophysical conditions favouring optimal vector breeding and larval development. the discovery of new culicoides species in zimbabwe adds to the existing knowledge on the distribution of culicoides species in southern africa. these findings, however, highlight the need for further studies to measure the vector competence of existing culicoides species and to fully understand the role of these species in the transmission and epidemiology of arboviruses in zimbabwe. furthermore, the discovery of novel btv serotypes in this study highlights the need to review bluetongue vaccines currently in use and vaccination and control strategies being practised in zimbabwe. acknowledgements top ↑ the authors would like to acknowledge the support and cooperation provided by the horse and livestock establishments in zimbabwe that allowed culicoides trapping to be conducted on their premises during the period under study. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions s.j.g.g. (massey university) wrote the manuscript and acted as project leader. g.n.m. (biomedical research and training institute), p.j.k. (biomedical research and training institute), p.s.m. (the pirbright institute) and c.h. (the pirbright institute) helped to design the project, establish and monitor the trapping sites, sort and process the vectors and assist with the virus isolation and identification. k.l. (arc-onderstepoort veterinary institute: parasites, vectors and vector-borne diseases) and e.d. (the pirbright institute) helped to sort, count and identify the vectors. c.b. (massey university), c.r. (massey university) and a.j.g. (university of pretoria) made conceptual contributions and assisted with the data analysis, statistical work and the editing of the final manuscript. references top ↑ blackburn, n.k., searle, l. & phelps, r.j., 1985, ‘viruses isolated from culicoides (diptera: ceratopogonidae) caught at the veterinary research farm, mazowe, zimbabwe’, journal of the entomological society of southern africa 48, 331–336. braverman, y. & phelps, r.j., 1981, ‘species composition and blood-meal identification in samples of culicoides (diptera: ceratopogonidae) collected near salisbury, zimbabwe in 1976–77’, journal of the entomological society of southern africa 44, 315–323. braverman, y., linley, j.r., marcus, r. & frish, k., 1985, ‘seasonal survival and expectation of infective life of culicoides spp. (diptera: ceratopogonidae) in israel, with implications for bluetongue virus transmission and a comparison of the parous rate in c. imicola from israel and zimbabwe’, journal of medical entomology 22(5), 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onderstepoort journal of veterinary research 59, 183–189. venter, g.j., gerdes, g.h., mellor, p.s. & paweska, j.t., 2004, ‘transmission potential of south african culicoides species for live-attenuated bluetongue virus’, veterinaria italiana 40, 198–203. venter, g.j., groenewald, d., venter, e., hermanides, k.g. & howell, p.g., 2002, ‘a comparison of the vector competence of the biting midges, culicoides (avarita) bolitinos and c. (a.) imicola, for the bryanston serotype of equine encephalosis virus’, medical and veterinary entomology 16(4), 372–377. http://dx.doi.org/10.1046/j.1365-2915.2002.00385.x venter, g.j., nevill, e.m. & van der linde, t.c., 1997, ‘seasonal abundance and parity of stock-associated culicoides species (diptera: ceratopogonidae) in different climatic regions in southern africa in relation to their viral vector potential’, onderstepoort journal of veterinary research 64, 259–271. venter, g.j., wright, i.m., van der linde, t.c. & paweska, j.t., 2009, ‘the oral susceptibility of south african field populations of culicoides to african horse sickness virus’, medical and veterinary entomology 23, 367–378. http://dx.doi.org/10.1111/j.1365-2915.2009.00829.x vincent, v. & thomas, r.g., 1960, an agricultural survey of southern rhodesia: part i: agro-ecological survey, government printer, harare. wellby, m.p., baylis, m., rawlings, p. & mellor, p.s., 1996, ‘effect of temperature on survival and rate of virogenesis of african horse sickness virus in culicoides variipennis sonorensis (diptera: ceratopogonidae) and its significance in relation to the epidemiology of the disease’, bulletin of entomological research 86, 715–720. http://dx.doi.org/10.1017/s0007485300039237 world organisation for animal health (oie), 2011, ‘sub-regional representation for southern africa’, gaborone, botswana, viewed 14 october 2014, from http://www.oie.int/doc/ged/d11781.pdf appendix 1 top ↑ table 1-a1: the distribution of all species of culicoides collected across the five natural regions of zimbabwe (1998–2003). article information authors: christo j. botha1 heleen coetser2 rowena a. schultz2 leonie labuschagne2 deon van der merwe3 affiliations: 1department of paraclinical sciences, university of pretoria, south africa2division of toxicology, agricultural research council-onderstepoort veterinary institute, south africa 3department of diagnostic medicine/pathobiology, kansas state veterinary diagnostic laboratory, kansas state university, united states correspondence to: christo botha postal address: private bag x04, onderstepoort 0110, south africa dates: received: 05 dec. 2012 accepted: 26 apr. 2013 published: 31 may 2013 how to cite this article: botha, c.j., coetser, h., schultz, r.a., labuschagne, l. & van der merwe, d., 2013, ‘spatial variation of epoxyscillirosidine concentrations in moraea pallida (yellow tulp) in south africa’, onderstepoort journal of veterinary research 80(1), art. #543, 6 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.543 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. spatial variation of epoxyscillirosidine concentrations in moraea pallida (yellow tulp) in south africa in this original research... open access • abstract • introduction • materials and methods    • plant material    • epoxyscillirosidine concentration    • soil    • gis spatial analysis    • comparative statistical analysis • results • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ moraea pallida (yellow tulp) poisoning is economically the most important intoxication of livestock in south africa. poisoning varies according to locality, climatic conditions and growth stage of the plant. the primary objective of this study was to determine the concentration of the toxic principle, epoxyscillirosidine, in yellow tulp leaves and to ascertain the variability of epoxyscillirosidine concentrations within and between different locations. a secondary objective was to utilise geographic information systems in an attempt to explain the variability in toxicity. flowering yellow tulp plants were collected at 26 sampling points across 20 districts of south africa. the leaves of five plants per sampling point were extracted and submitted for liquid chromatography/mass spectrometry analysis. a large variation in mean epoxyscillirosidine concentrations, ranging from 3.32 μg/g – 238.27 μg/g, occurred between different geographical regions. the epoxyscillirosidine concentrations also varied tremendously between individual plants (n = 5) collected at the same sampling point, with up to a 24 times difference between the lowest and highest concentration detected. no generalised correlation between epoxyscillirosidine concentrations and soil elemental concentrations could be established. however, samples obtained from the north-eastern part of the sampling region tended to have higher epoxyscillirosidine concentrations compared to samples obtained from the south-western part of the sampling region. higher toxin concentrations in the north-east were associated with statistically significant higher soil concentrations of iron, bismuth, bromide, cadmium, chromium, rubidium, tellurium, thallium, titanium and zinc, whilst soil concentrations of strontium and soil ph, were significantly lower. this study corroborated the contention that epoxyscillirosidine concentration in yellow tulp fluctuates and may explain the variability in toxicity. introduction top ↑ poisoning of livestock by cardiac glycoside-containing plants has the greatest economic impact of all plant-associated poisonings in the republic of south africa. collectively, they are held responsible for 33% of all mortalities from plant poisonings of cattle and 10% of those in small stock (kellerman, naudé & fourie 1996). chemically, two major groups of cardiac glycosides, namely the cardenolides and bufadienolides, are recognised (steyn & van heerden 1998). poisoning of livestock by bufadienolide-containing plants, which surpasses cardenolide-induced poisonings in importance, may either be acute or chronic. tulp poisoning (induced by various moraea species) and slangkop poisoning (caused by various drimia species) induce only acute intoxication, as these species contain non-cumulative bufadienolides (kellerman et al. 2005). on the other hand, members of three genera of the crassulaceae (cotyledon, tylecodon and kalanchoe), generally referred to as plakkies, may cause either acute or chronic poisoning as they contain cumulative, neurotoxic bufadienolides. the chronic form of the poisoning is colloquially referred to as krimpsiekte (botha 2003). tulp poisoning is the most important plant-associated poisoning in south africa and occurs throughout the country. it is predominantly found in newly-introduced cattle or young, naive animals (kellerman, naudé & fourie 1996; kellerman et al. 2005). moraea pallida (yellow tulp) contains a bufadienolide, namely 1α, 2α-epoxyscillirosidine and is predominantly responsible for the intoxication of livestock (enslin et al. 1966; kellerman et al. 2005). however, yellow tulp poisoning is reported to vary according to locality, climatic conditions and growth stage (kellerman et al. 2005). thus, it is assumed that the epoxyscillirosidine concentration in yellow tulp varies between different geographic regions of the country. in a limited study, snyman, schultz & van den berg (2011) linked cardiac glycoside content to values obtained with an immunoassay (digoxin equivalents) and reported variability in yellow tulp toxicity. the first objective of this study was to determine epoxyscillirosidine concentration in yellow tulp leaves collected at different geographical regions in south africa. the second objective of this study was to utilise a geographic information system (gis) to establish if the differences over geographic regions (assuming that the difference is not genetically driven), were due to variations in edaphic parameters that could influence the synthesis and accumulation of epoxyscillirosidine in the plant. materials and methods top ↑ plant material moraea pallida was collected in different geographical regions of south africa during september 2010 – october 2010. ten to 15 plants, growing in close proximity to each other (within approximately 1 m2) in grazing camps, were collected per sampling point by digging up the plants. at each site, global positioning system (gps) sampling point locations were recorded in terms of longitude and latitude using handheld gps sensors and the wgs 84 map datum. a voucher specimen from each site was retained, pressed and mounted on cardboard. the hgw schweickertd herbarium, at the university of pretoria, verified the botanical identification. the voucher specimens were kept at the toxicology biolab, agricultural research council-onderstepoort veterinary institute (arc-ovi). the remaining plants were dried in an oven (protea laboratory equipment) at 60 °c until the mass stabilised. from the batch of plants collected per sampling point, the five plants with the best leaves (longest and broadest) were selected for extraction. epoxyscillirosidine concentration the leaves of the five plants per sampling point were extracted separately. the extraction procedures followed the standardised method used at the toxicology laboratory, arc-ovi (schultz, kellerman & van den berg 2005). briefly, the dried leaves were finely milled; 0.5 g dry plant material was homogenised for five minutes in dichloromethane (80 ml, british drug house [bdh], england) and deionised water (40 ml) to which ten drops of concentrated hydrochloric acid (merck, south africa) were added. the sample was shaken for 30 minutes and then centrifuged (2400 x g) for 30 minutes. the precipitate was removed with a pipette and placed in a clean flask; 40 ml of deionised water with 20 drops ammonia (merck, south africa) was added, shaken for 30 minutes and then centrifuged for 30 minutes. the bottom layer was filtered through sodium sulphate (approximately 20 g) on a whatman 1 filter paper to remove moisture. the filtrate was evaporated to dryness (< 50 °c), after which 2 ml analytical grade methanol (merck, south africa) was added (dissolved in an ultrasonic bath) and filtered (0.45 μ nylon filter). the extracts were submitted to the laboratory of the national horse racing authority, johannesburg to determine the epoxyscillirosidine concentrations. the samples were dried at room temperature, reconstituted in 2 ml methanol (merck, south africa) and vortex mixed for 30 seconds. the samples were then diluted by transferring 100 µl of each sample to a high performance liquid chromatography (hplc) vial and methanol 900 µl and 100 µl of internal standard (proscillaridin a, sigma-aldrich, germany) in methanol (10 µg/ml) were added. samples were submitted for liquid chromatography/mass spectrometry (lc-ms). an agilent 1260 series liquid chromatograph (agilent technologies, south africa) was employed with a waters xselect csh c18 150 mm x 2.1 mm column (5 µm particle size). the mobile phase was 5 mm ammonium acetate and 0.1% formic acid in water:acetonitrile (98/2, v/v) (solvent a) and 5 mm ammonium acetate and 0.1% formic acid in water: acetonitrile, (2/98, v/v) (solvent b). the liquid chromatograph was interfaced to a thermo scientific exactive high-resolution mass spectrometer (thermo scientific, south africa) equipped with a heated-electrospray lonisation probe (hesi-ii), utilising xcalibur software version 2.1 with toxid 2.1.2. soil sub-soil samples (one 500 g per sampling point) were collected from the root zone of the plants following removal of the top 3 cm – 5 cm of soil. the soil samples were submitted to the arc-institute for soil, climate and water (arc-iscw), pretoria for analyses of k, ca, mg, na, ph (h2o), phosphate and resistance. an inductively coupled plasma/mass spectrometry (icp-ms) scan on an ammonium edta extract of the samples, including analyses of li, be, b, ti, v, cr, mn, co, ni, cu, zn, as, br, se, rb, sr, mo, cd, sn, sb, te, i, cs, ba, la, w, pt, hg, tl, pb, and u was performed. correlations between soil elements and median epoxyscillirosidine concentrations were investigated using the pearson product moment correlation method (sigmaplot for windows version 11.0; build 11.2.0.5). statistical significance was assumed at a confidence level of 95% and above. gis spatial analysis spatial distributions of yellow tulp epoxyscillirosidine concentrations were studied in a gis environment (esri 2008: arcmap 10.0; build 2414). the median epoxyscillirosidine concentration for each site was compiled and the data were projected into the africa-albers equal area conic coordinate system. to determine whether the data distribution was spatially autocorrelated or not, a spatial autocorrelation analysis was performed by calculating the global moran’s i-statistic using inverse distance conceptualisation and euclidian distance. to determine whether the data displayed spatial high-low trends, a high-low clustering analysis was performed by calculating a general g-statistic using inverse distance conceptualisation and euclidian distance. comparative statistical analysis visual inspection of the data revealed an apparent, strong difference in epoxyscillirosidine concentrations along a south-west (sw) to north-east (ne) distribution axis. to describe the difference statistically, the data was divided into two groups, ne and sw, based on a dividing line stretching from -26°n, 24°e to -30°n, 29°e. the two groups were compared using a t-test when the data were normal, and had equal variance; otherwise the two groups were compared using the mann-whitney rank sum test. a significant difference between groups was assumed when the difference reached a 95% confidence level (p < 0.050). results top ↑ flowering m. pallida were collected at 26 sampling points across 20 districts of south africa (figure 1). the mean and median epoxyscillirosidine concentrations, as well as the lowest and highest concentrations in the yellow tulp leaves (n = 5) collected at each sampling point, are tabled (table 1). the data is not normally distributed. figure 1: mean epoxyscillirosidine concentrations of flowering moraea pallida leaves (n = 5) collected at 26 locations in south africa to the north-east and south-west of a diagonal divisional line drawn between -26°n, 24°e to -30°n, 29°e. table 1: epoxyscillirosidine concentrations in flowering moraea pallida leaves (n = 5) and variability per sampling point of plants collected during september – october 2010 at various geographical regions of south africa. a large variation in mean epoxyscillirosidine concentrations was noticed between geographical regions, ranging from 3.32 (± 0.56) μg/g at excelsior to 238.27 (± 232.21) μg/g at frankfort. huge differences in epoxyscillirosidine concentrations between individual plants within a plant community were also detected at certain sampling points. the largest variations in individual epoxyscillirosidine concentrations in plants growing in close proximity to each other were recorded at bloemhof, rouxville and bethlehem, with a 24, 20 and 18 times difference between the lowest and highest concentrations respectively. the spatial autocorrelation analysis resulted in a moran’s index i-statistic of 0.649, a z-score of 0.878, with a p-value of 0.380. the spatial distribution of epoxyscillirosidine concentration therefore appeared not to be significantly different from random, but the power of the analysis was poor due to a low sample number. the overall high-low clustering analysis resulted in a general g-statistic of 0.000024, a z-score of -0.287, and a p-value of 0.774, indicating that the overall spatial distribution of high and low values was not significantly different from random. however, there was a statistically significant difference (p < 0.001) in median epoxyscillirosidine concentrations of plants to the sw and ne of a diagonal divisional line drawn from nw to se, based on a dividing line stretching from -26°n, 24°e to -30°n, 29°e, with median concentrations of 5 μg/g and 54 μg/g respectively (figure 1). this sample division also resulted in significant soil differences. significantly higher toxin concentration in the ne was associated with higher soil concentrations of fe, bi, br, cd, cr, rb, te, tl, ti and zn, whilst soil concentrations of sr and soil ph were significantly lower (table 2). table 2: median epoxyscillirosidine concentrations in plants and median soil parameter values, to the north-east and south-west of a diagonal divisional line drawn between -26°n, 24°e and -30°n, 29°e. discussion top ↑ this study corroborates the notion that the toxicity of yellow tulp is highly variable and confirms a previous report of relatively low cardiac glycoside content (reflected as digoxin equivalents) in yellow tulp collected at griekwastad and bloemfontein (snyman, schultz & van den berg 2011). in the current study, yellow tulp leaves collected at belfast, mpumalanga highveld had a high mean epoxyscillirosidine concentration of 136.95 (± 104.94) μg/g. snyman et al. (2011) also reported relatively high cardiac glycoside content (18 438 nmoles/l) in a plant (only one sample) collected at middelburg on the mpumalanga highveld. however, it is difficult to compare as snyman et al. (2011) utilised an indirect analysis (digoxin assay) to estimate the cardiac glycoside content. the differences in epoxyscillirosidine concentration in yellow tulp can explain the variability in toxicity. however, poisoning will depend on the amount of plant material ingested and even though the epoxyscillirosidine concentration in the plant material may be low, if sufficient plant material is ingested it can induce intoxication. this is well illustrated as plant material collected during incidences of tulp poisoning near newcastle and glencoe (annema, private practitioner, personal communication) was found to be quite diverse. the mean epoxyscillirosidine concentration in yellow tulp leaves (n = 5) at newcastle was high at 227.61 (± 114.07) μg/g and relatively low at glencoe at 23.05 (± 15.49) μg/g. in conditioned feed aversion trials, snyman et al. (2004) reported that the lowest oral dose of epoxyscillirosidine that induces clinical signs of poisoning in cattle is 0.10 mg/kg body weight. based on the mean epoxyscillirosidine concentrations found at newcastle and glencoe, it is estimated that a young steer of 300 kg has to ingest 130 g – 1300 g of dry plant material or 0.43 g – 4.30 g of dry plant material per kilogram body weight. these estimated toxic doses for cattle are comparable to lethal oral doses of 1.25 g/kg of dried, milled moraea pallida plant material administered to sheep during experimental studies (swan et al. 1995). in the current study, the epoxyscillirosidine concentrations were only determined in the leaves and not in other aerial parts such as the inflorescence, or in the corms. it is highly probable that the epoxyscillirosidine concentrations in yellow tulp may vary during a growing season. differences in the concentrations of toxic principles in various plant parts and during specific growth stages have been reported for a number of poisonous plants in south africa such as fadogia homblei and lantana camara (kellerman et al. 2005). soil type probably also influences toxicity of yellow tulp. steyn (1934) reported that another bufadienolide-containing plant, cotyledon orbiculata, which grows on sandy soils, is more toxic than plants growing on black clay soils. grosskopf (1964) relayed the opinions of farmers who reported that geigeria ornativa growing in limestone-rich areas is more toxic. many of the earlier research investigations involving gis and toxicology have mainly dealt with pesticide use and their environmental implications, especially to ground water and/or stream water quality (corwin, werle & rhoades 1988; howell, gelinas & slaats 1988; davis & flores 1992; heidtke & auer 1993). numerous scientific reports also exist where gis have been used to analyse heavy metal pollution in the environment (herpin, berlekamp & markert 1996; markert, herpin & berlekamp 1996; frangi & richard 1997; mielke, gonzales & smith 2000; facchinelli, sacchi & mallen 2001; pesch & schroeder 2006). most or all of these studies have centered on risks to human health; nevertheless, they demonstrate the potential of spatial analysis or geoprocessing tools, which are components that are central to a gis in various toxicological problem-solving scenarios. despite the popularity of gis amongst many medical and environmental toxicologists, there are relatively few veterinary toxicological studies that have used gis for problem solving thus far. durr & gatrell (2004) wrote extensively about spatial analysis in veterinary science in general, wherein a majority of their topics discussed epidemiological modelling and management of wildlife diseases. kistemann et al. (2004) studied spatial and temporal distribution of verotoxin-producing escherichia coli amongst humans and cattle in sweden. they compared spatial patterns of the distribution of human infections with spatial patterns of occurrence in cattle using gis. whilst it is true that the use of gis is limited to situations that are spatial in nature, it is also a fact that the immense potential of gis to solve regional and local veterinary toxicological issues is underutilised at present. the moran’s index indicates a degree of spatial autocorrelation in the data. although the power of the analysis is not at a level needed for a reliable result, a degree of autocorrelation in this case may support a conclusion that local effects influence toxin levels, which causes plants that grow close together to have more similar toxin concentrations compared to plants growing in more distant locations. this conclusion is also supported by the regional differences between the ne and sw. one of the advantages of visually evaluating data in a spatial context is that it enables the qualitative detection of data patterns, which can then form the basis for forming testable hypotheses. the elliptical distribution pattern of the sample locations along the ne to sw axis allows for evaluation along that axis (figure 1). although several statistically significant associations between soil parameters and plant toxin concentration were found (table 2), inferences cannot be made regarding the causal relationship of these associations. however, these results do suggest that further experiments are needed to investigate the potential influence of soil nutrients and soil ph on toxin production. due to the distribution pattern of the sample sites, the presence or absence of toxin concentration differences along other distribution axes could not be supported or disproved. potential factors that could influence toxin concentrations, but were not investigated in the current study include: regional climatic differences; genetic differences between populations; and local stressors such as variations in insect or other herbivore pressure, pathogens and intraand interspecific competition. the variation of epoxyscillirosidine concentrations within populations over time also needs to be investigated, including variations in relation to plant growth stages. these data and an increased understanding of the factors that determine poisoning risk will allow an assessment of change in risk that might be expected when environmental changes occur, such as climate change and/or land use change. the most important environmental parameters that determine epoxyscillirosidine poisoning can then be identified and broader geographic areas can be delineated in a gis environment as potential areas of yellow tulp poisoning. acknowledgements top ↑ the authors would like to extend their gratitude to the veterinarians, students and farmers who collected and submitted plant material and soil samples. we would also like to thank mrs nina van vliet, arc-institute for soil, climate and water, pretoria for the soil analysis and dr magda rösemann at the laboratory of the national horse racing authority, johannesburg for the lc-ms analysis. this research is based upon work supported financially by the national research foundation, south africa. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.j.b. (university of pretoria) was the principal investigator. h.c. (agricultural research council-onderstepoort veterinary institute), r.a.s. (agricultural research council-onderstepoort veterinary institute) and l.l. (agricultural research council-onderstepoort veterinary institute) prepared the plant samples and performed the extraction procedures. d.v.d.m. (kansas state university) was responsible for the gis analysis. c.j.b., d.v.d.m. and r.a.s. compiled and edited the manuscript. references top ↑ botha, c.j., 2003, ‘krimpsiekte, a paretic/paralytic syndrome of small stock in south africa: studies on the aetiology of krimpsiekte, the toxicokinetics and dynamic effects of cotyledoside and seasonal fluctuation of cotyledoside concentration in plant material’, phd thesis, the norwegian school of veterinary science.corwin, d.l., werle, j.w. & rhoades, j.d., 1988, ‘the use of computer-assisted mapping techniques to delineate potential areas of salinity development in soils. 1. a conceptual introduction 2. field verification of the threshold-model approach’, hilgardia 56, 1–32.  davis, r.m. & flores, p.i., 1992, ‘identifying potential pollutant point sources in an area of high ground-water consumption’, ground water monitoring and remediation 12, 116–119. http://dx.doi.org/10.1111/j.1745-6592.1992.tb00041.x durr, p.a. & gatrell, a.c., 2004, gis and spatial analysis in veterinary science, cabi publishing, wallingford. http://dx.doi.org/10.1079/9780851996349.0000 enslin, p.r., naudé, t.w., potgieter, d.j.j. & van wyk, a.j., 1966, ‘1α, 2α-epoxyscillirosidine, the main toxic principle of homeria glauca (wood and evans) n.e. br’, tetrahedron 22, 3213–3220. http://dx.doi.org/10.1016/s0040-4020(01)82303-6 esri, 2008, what is gis? gis.com, a guide to geographic information systems, viewed 04 december 2012, from http://www.gis.com/whatisgis/ facchinelli, a., sacchi, e., & mallen, l., 2001, ‘multivariate statistical and gis-based approach to identify heavy metal sources in soils’, environmental pollution 114, 313–324. http://dx.doi.org/10.1016/s0269-7491(00)00243-8 frangi, j.p. & richard, d., 1997, ‘heavy metal soil pollution cartography in northern france’, science of the total environment 205, 71–79. http://dx.doi.org/10.1016/s0048-9697(97)00184-8 grosskopf, j.f.w., 1964, our present knowledge of ‘vermeersiekte’ (geigeria poisoning). technical communication no. 21. department of agricultural technical services, republic of south africa. heidtke, t.m. & auer, m.t., 1993, ‘application of a gis-based nonpoint source nutrient loading model for assessment of land development scenarios and water quality in owasco lake, new york’, water science and technology 28, 595–604. herpin, u., berlekamp, j. & markert, b., 1996, ‘the distribution of heavy metals in a transect of the three states the netherlands, germany and poland, determined with the aid of moss monitoring’, science of the total environment 187, 185–198. http://dx.doi.org/10.1016/0048-9697(96)05141-8 howell, g., gelinas, r. & slaats, j., 1988, ‘identification of surface water acidification sources in nova scotia canada’, water pollution research journal of canada 23, 520–531. kellerman, t.s., coetzer, j.a.w., naudé, t.w. & botha, c.j., 2005, plant poisonings and mycotoxicoses of livestock in southern africa, 2nd edn., oxford university press, cape town. kellerman, t.s., naudé, t.w. & fourie, n., 1996, ‘the distribution, diagnosis and estimated economic impact of plant poisonings and mycotoxicoses in south africa’, onderstepoort journal of veterinary research 63, 65–90. pmid:8856758 kistemann, t., zimmer, s., vagsholm, i. & andersson, y., 2004, ‘gis-supported investigation of human ehec and cattle vtec o157 infections in sweden: geographical distribution, spatial variation and possible risk factors’, epidemiology and infection 132, 495–505. http://dx.doi.org/10.1017/s0950268803001729, pmid:15188718, pmcid:2870128 markert, b., herpin, u. & berlekamp, j., 1996, ‘a comparison of heavy metal deposition in selected eastern european countries using the moss monitoring method, with special emphasis on the “black triangle”’, science of the total environment 193, 85–100. http://dx.doi.org/10.1016/s0048-9697(96)05333-8 mielke, h.w., gonzales, c.r. & smith, m.k., 2000, ‘quantities and associations of lead, zinc, cadmium, manganese, chromium, nickel, vanadium, and copper in fresh mississippi delta alluvium and new orleans alluvial soils’, science of the total environment 246, 249–259. http://dx.doi.org/10.1016/s0048-9697(99)00462-3 pesch, r. & schroeder, w., 2006, ‘spatiotemporal variability of metal accumulation in mosses. analysis of measurement data and metadata by statistics and gis’, nova hedwigia 82, 447–466. http://dx.doi.org/10.1127/0029-5035/2006/0082-0447 schultz, r.a., kellerman, t.s. & van den berg, h., 2005, ‘the role of fluorescence polarization immuno-assay in the diagnosis of plant-induced cardiac glycoside poisoning of livestock in south africa’, onderstepoort journal of veterinary research 72, 189–201. http://dx.doi.org/10.4102/ojvr.v72i3.195, pmid:16300187 snyman, l.d., kellerman, t.s., schultz, r.a., joubert, j.p.j., basson, k.m. & labuschagne, l., 2004, ‘conditioned feed aversion as a means of preventing intake of yellow-tulp (homeria pallida) by livestock’, in t. acamovic, c.s. stewart & t.w. pennycott (eds.), poisonous plants and related toxins: 6th international symposium on poisonous plants proceedings, glasgow, scotland, 2001, pp. 531–539, cabi publishing, wallingford. snyman, l.d., schultz, r.a. & van den berg, h., 2011, ‘variability of yellow tulp (moraea pallida bak.) toxicity’, journal of the south african veterinary association 82(2), 131–132. http://dx.doi.org/10.4102/jsava.v82i2.48, pmid:22135929 steyn, d.g., 1934, the toxicology of plants in south africa. central news agency ltd, south africa. steyn, p.s. & van heerden, f.r., 1998, ‘bufadienolides of plant and animal origin’, natural product reports 15(4), 397–398. http://dx.doi.org/10.1039/a815397y, pmid:9736996 swan, g.e., schultz, r.a., kellerman, t.s., mülders, m.s.g., maartens, b.p. & van der walt, j.j., 1995, ‘in vivo effects of a novel calcium antagonist (r56865) against induced epoxyscillirosidin and tulp poisoning in sheep’, onderstepoort journal of veterinary research 62, 163–166. pmid:8628568 ngeranwa_199-205.indd introduction anaplasmosis is an infectious rickettsial disease caused by anaplasma marginale and anaplasma centrale in cattle and anaplasma ovis in sheep and goats (theiler 1910, 1911; lestoquard 1924). the disease is acute or subacute in cattle, although subclinical infections are not uncommon. the severity of the disease in cattle is directly related to age: in animals less than 1 year it is usually subclinical; in yearlings and 2-year-olds it is moderately severe, and in older cattle it is severe and often fatal (potgieter & stoltsz 2004). sheep and goats often suffer only mild anaplasmosis, but occasionally goats suffer severe clinical disease (splitter, antony & twiehause 1956; kimberling 1988; shompole, waghela, rurangirwa & mcguire 1989; stoltsz 2004). anaplasma phagocytophilum, previously known as ehrlichia phagocytophila (dumler, barbet, bekker, dasch, palmer, ray, rikihisa & rurangira 2001), occurs widespread in humans and domestic animals. anaplasmosis is widely distributed throughout tropical and subtropical areas of the world, as well as in some temperate areas (soulsby 1982). the disease 199 onderstepoort journal of veterinary research, 75:199–205 (2008) detection of anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay j.j.n. ngeranwa1, 2, s.p. shompole1, e.h. venter2, a. wambugu1, j.e. crafford2 and b.l. penzhorn2* abstract ngeranwa, j.j.n., shompole, s.p., venter, e.h., wambugu, a., crafford, j.e. & penzhorn, b.l. 2008. detection of anaplasma antibodies in wildlife and domestic species in wildlifelivestock interface areas of kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay. onderstepoort journal of veterinary research, 75:199–205 the seroprevalence of anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, thomson’s gazelle, grant’s gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife/livestock interface areas of kenya. serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (ci-elisa), using a recombinant antigen (msp-5) from anaplasma marginale surface membrane. a monoclonal antibody, fc-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. the results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from kenya, which indicated a low seroprevalence. the differences are attributed to the accurate analytical method used (ci-elisa), as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers. keywords: anaplasma, ci-elisa, kenya, seroprevalence, wildlife-livestock interface * author to whom correspondence is to be directed. e-mail: banie.penzhorn@up.ac.za 1 kenya agricultural research institute, p.o. box 29231, nairobi, kenya 2 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa current address: department of biochemistry & biotechnology, kenyatta university, p.o. box 43844, nairobi, kenya accepted for publication 24 april 2008—editor 200 anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of kenya is common in africa, the middle east, southern europe, the far east, central and south america and the united states of america (soulsby 1982). anaplasma infections in wildlife, both natural and experimental, as well as occurrence of anaplasma antibodies in wildlife have been reported world-wide (kuttler 1984). among african wildlife, subclinical occurrence of anaplasma marginale, either natural or after artificial infection, has been confirmed in the african buffalo, syncerus caffer (potgieter 1979), eland, taurotragus oryx (peirce 1972; ngeranwa, venter, penzhorn, soi, mwanzia & nyongesa 1998), black wildebeest, connochaetes gnou (neitz 1935), blue wildebeest, connochaetes taurinus (smith, brocklesby, bland, purnell, brown & payne 1974), grey duiker, sylvicapra grimmia (neitz & du toit 1932) and blesbok, damaliscus dorcas phillipsi (neitz & du toit 1932). anaplasma marginale was successfully transmitted from a naturally infected giant african rat, cricetomys gambianus, to a bovine (dipeolu, akinboade & adetunji 1981). subclinical occurrence of anaplasma ovis, either natural or after artificial infection, has been confirmed in eland (enigk 1942; ngeranwa et al. 1998) and blesbok (neitz 1939), and anaplasma centrale can be artificially established in blesbok (neitz & du toit 1932). the occurrence of unidentified anaplasma spp., based on positive serological assays or presence of organisms visible on blood smear examination, has been reported in african buffalo (brocklesby & vidler 1966), blue wildebeest (brocklesby & vidler 1965; kuttler 1965; löhr & meyer 1973; burridge 1975), coke’s hartebeest, alcelaphus buselaphus cokei (löhr & meyer 1973), thomson’s gazelle, gazella thomsonii (löhr & meyer 1973), grant’s gazelle, gazella granti (löhr, ross & meyer 1974), gerenuk, litocranius walleri (brocklesby & vidler 1965), impala, aepyceros melampus (kuttler 1965; löhr et al. 1974), sable antelope, hippotragus niger (grobler 1981; thomas, wilson & mason 1982), waterbuck, kobus ellipsiprymnus (kuttler 1965; löhr et al. 1974) and giraffe, giraffa camelopardalis (brocklesby & vidler 1966; löhr & meyer 1973; augustyn & bigalke 1974; löhr et al. 1974). giraffe would appear to be the only african wildlife species in which clinical anaplasmosis has been described in free-ranging animals. severe clinical signs were reported in two cases (löhr & meyer 1973; augustyn & bigalke 1974). in both instances, death occurred in association with anaplasma parasitaemia and severe anaemia. anaplasmosis has been described in captive addax, addax nasomaculatus (ebedes & reyers 1984). in the coast range area of california, anaplasma spp. infections are maintained in black-tailed deer, odocoileus hemionus, populations in the absence of cattle (christensen, osebold & douglas 1962). these deer also serve as reservoirs for infection of cattle. anaplasma phagocytophilum can infect whitetailed deer, odocoileus virginianus, and other cervids (dugan, yabsley, tate, mead, munderloh, herron, stallknecht, little & davidson 2006). the role of free-ranging african wildlife as reservoirs for infection of livestock has not been elucidated, however. the major surface protein 5 (msp-5) is conserved and regarded as a group-specific antigen among anaplasma species. a msp-5 recombinant protein together with a specific monoclonal antibody (mab) (anaf16c1) has been well characterized and when used in a competitive inhibition enzyme-linked immunosorbent assay (ci-elisa) they could detect group-specific antibody to all recognized anaplasma species in cattle and goats (de echaide, knowles, mcguire, palmer & suarez mcelwain 1988; visser, mcguire, palmer, davis, shkap, pipano & knowles 1992; ndung’u, aguirre, rurangirwa, mcelwain, mc guire, knowles & palmer 1995; knowles, de echaide, palmer, mcguire, stil ler & mcelwain 1996; rey na-bello, cloeckart, vizcaino, gonzatti, aso, dubray & zygmunt 1998; molloy, bowles, knowles, mcel wain, bock, kingston, blight & dalgleish 1999). this ci-elisa was used to detect group-specific antibodies to anaplasma in wildlife-livestock interface areas in kenya. materials and methods study area this study was primarily carried out in the machakos area of kenya, where wildlife populations share the grazing with cattle, sheep and goats. a few specimens from other areas in kenya were also included. blood specimens were collected from eland (n = 12), blue wildebeest (n = 58), kongoni, damaliscus korrigum (n = 120), impala (n = 7), thomson’s gazelle (n = 8), grant’s gazelle (n = 5), giraffe (n = 3) and plains zebra, equus quagga (n = 11) at the athi river slaughter house, machakos (60 km south of nairobi) and its surroundings during routine game cropping. giraffe specimens (n = 13) were collected from nakuru national park (165 km northwest of nairobi) and eland specimens from the field in the nakuru region (n = 41) and baobab ranch, mom201 j.j.n. ngeranwa et al. basa (n = 2). sera from livestock were collected from the machakos area, as well as from the laikipia and thika districts. the negative controls (n = 10) were from captive-born buffalo calves raised in tickfree surroundings (wildlife disease research project, kabete, nairobi, kenya). in live animals, blood was collected by venipuncture from the jugular vein into 5 mℓ tubes. blood from shot or slaughtered animals was collected into tubes when the animal was exsanguinated. the blood was allowed to clot; the serum was decanted into stoppered tubes and frozen until processed in the laboratory. ci-elisa the ci-elisa with recombinant msp-5 was modified from a previously described assay used to detect antibody against msp-5 in a. ovis infected goats (ndung’u et al. 1995). the recombinant anaplasma antigen msp-5 and a mab anaf16c1, were supplied by washington state university, usa. antimouse antibody was produced and conjugated to horseradish peroxidase (hrpo) at the biotech nology section, kenya agricultural research institute, kabete, kenya. briefly, pam104a-transformed e. coli xl-1 blue was grown overnight in 50 mℓ of luria-bertani broth containing 150 mg of ampicillin per mℓ. the e. coli was harvested by centrifugation at 1 000 g for 10 min at 4 °c. the pellet was washed with 10 mℓ of a modified proteinase inhibition buffer (pi buffer) [50 mm tris-hc1 (ph 8.0) containing 5 mm edta and 1 mm phenylmethylsulfonyl fluoride]. the pellet was dissolved in 5 mℓ pi buffer containing 1 mg lysozyme per mℓ and incubated on ice for 20 min. nonidet p-40 was added to a concentration of 1 %, the mixture was vortexed briefly and incubated on ice for 10 min. the solution was sonicated twice on ice at 100 watts for 1 min pausing for 15 s. after sonication the mixture was centrifuged at 12 000 g for 20 min at 4 °c. the supernatant (the antigen) was recovered and was stored at 4 °c. before use, the msp-5 antigen and anaf16c1 mab were titrated in a checkerboard titration to determine the optimum working dilutions. immulon 2 plates (dynatec-usa) were coated overnight at room temperature with 40 μℓ of antigen diluted with coating buffer (0.03 m nahco3, 0.015 m na2co3, ph 9.6). the following day the contents of the wells were discarded and the plates washed three times with pbs (ph 7.4) containing 0.05 % tween 20 (pbs-t). after washing, the coated plates were blocked by adding 200 μℓ of pbs-t containing 5 % skimmed milk powder per well and incubated for 1 h at room temperature. after blocking and emptying the wells, 40 μℓ per well of neat test serum were added in duplicates. all the subsequent incubations were done at room temperature for 45 min with mechanical shaking. the following controls were included: the first row was left as the blank and only pbs was added to keep the wells from drying out; in the second row, 40 μℓ of freshly diluted anaf16c1 mab were added; in the third and fourth rows, 10 different known negative sera were added, each well with a different serum sample; in the fifth row, known positive serum was added. after incubation, the contents of the wells were discarded, 50 μℓ of the anaf16c1 mab were added per well except for the blank row in which pbs was again added. the plates were incubated and then washed three times with pbs-t before 50 μℓ of the hrpo-conjugated anti-mouse antibody were added to each well, including the blank wells. incubation was done as above. after this incubation, the plates were washed three times with pbs-t with 5 min soaking between each wash. the substrate [2,2’-azino-di(3-ethylbenzothiazoline sulfonate)] (abts) containing 0.05 % h2o2 (30 % v/v) was added, 100 μℓ per well, incubated at room temperature for 30 min and the optical density was determined in a spectrophotometer at 410 nm wave length. the cut-off values were computed by calculating the mean and standard deviations of the negative controls. any value which was less than the product of the mean of the negative controls and three standard deviations was considered positive while any reading above this product, was considered negative. results the results are given in table 1. discussion a high seroprevalence of anaplasma antibodies was found in all species investigated (table 1). these results are in contrast to those of kuttler (1965), where only 7/117 wildlife sera were positive for anaplasma spp. on the complement-fixation test (cft). in a more recent survey also using the cft, only 1/10 buffaloes from a ranch in laikipia was seropositive to anaplasma spp. (kimber, lubroth, dubovi, berninger & demaar 2002). prevalence rates in wildlife approaching 75 % were reported by löhr et al. (1974), based on the card agglutination test (cat) and the indirect fluorescent antibody test 202 anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of kenya (ifat). the differences may arise from the different methods used, as well as species-specific differences. the ifat was significantly more sensitive for detection of cattle infected with anaplasma spp. (97 %); the cat and cft were less so (84 % and 79 %, respectively) (gonzalez, long & todorovic 1978). in an experimental study in the usa, the cft gave false positive and suspicious reactions when applied to serum samples of known anaplasma-negative pronghorn, antilocapra americana, bighorn sheep, ovis canadensis, and elk, cervus canadensis (howe, hepworth, blunt & thomas 1964). false negative reactions also occurred with known positive deer sera. with the capillary tube-agglutination test, 96 % accuracy was obtained with known negative wildlife sera, but 49 % false-negative reactions occurred on known positive sera (howe et al. 1964). it has also been shown that cf titres in anaplasma-carrier deer fall to levels below the sensitivity of the diagnostic test (christensen, osebold & rosen 1958). it is interesting to note that 27 black-faced impalas, a. m. petersi, in northern namibia were seronegative to anaplasma on the cat (karesh, rothstein, green, reuter, braselton, torres & cook 1997). whether this was due to insensitivity of the test or dearth of vectors in an arid environment is a moot point. the high seroprevalence (72.7 %) in plains zebras is of interest, as occurrence of anaplasma spp. in zebras has apparently not been reported previously. anaplasmosis has been reported in domestic horses; the causative organism in that case was named anaplasma equi (brion 1943). anaplasma phagocytophilum should also be borne in mind. although it has not been reported from african wildlife, a. phago cytophilum can infect horses (madigan, richter, kimsey, barlough & bakken 1995) and other livestock (hoffman-lehman, meli, dreher, gönczi, deplazes, braun, engels, schüpbach, jörger, thoma, griot, stärk, willi, schmidt, kocan & lutz 2004). infected domestic animals could have been imported into kenya, and a. phagocytophilum may have spread to wildlife. serological cross-reactivity between anaplasma marginale and anaplasma phagocytophilum has been demonstrated (dreher, de la fuente, hoffmann-lehmann, meli, pustera, kocan, woldehiwet, braun, regula, stärk & lutz 2005; stirk, alleman, barbet, sorenson, wamsley, gaschen, luckschander, wong, chu, foley, bjoersdorff, stuen & knowles 2007). high seroprevalence was also found in bovines, sheep and goats, with overall prevalences of 97 %, 90 % and 85 %, respectively (table 1). earlier work done in kenya using the cft on bovine sera found a prevalence of 26 % of positive cases and 26 % of suspicious ones (kuttler 1965). in more recent work, where diagnosis was based only on clinical signs, the incidence of bovine anaplasmosis over a oneyear period was found to be 15–57 % (mulei & rege 1989). these findings could not have been accurate, as diagnosis based on clinical signs is not sensitive enough and subclinical cases and carrier animals would have been missed. the authors also used blood smears to confirm their diagnosis, a method that may not help as the organisms are known to disappear from the blood more than 16–26 days into the disease (henning 1956; ristic 1962). other than the methods used, differences may also occur based on the area where the studies were carried out. it is noteworthy that the study area covered by mulei & rege (1989) is a high-potential one where zero-grazing management is practised by most farmers. with this management, the number of vectors and carrier animals is minimal. the present study, on the other hand, focused on areas of wildlife-livestock interaction, which are generally more arid rangelands where tick control is practised to reduce tick burdens rather than to eradicate ticks. table 1 seroprevalence of antibodies to anaplasma spp. in various species at the wildlife/livestock interface in kenya species district no. positive eland machakos nakuru mombasa 12/12 (100 %) 3/4 (75 %) 2/2 (100 %) blue wildebeest machakos 56/58 (96.5 %) kongoni machakos 112/120 (93.3 %) impala machakos 7/7 (100 %) thomson’s gazelle machakos 6/8 (75 %) grant’s gazelle machakos 4/5 (80 %) giraffe machakos nakuru 3/3 (100 %) 11/13 (84.6 %) plains zebra machakos 8/11 (72.7 %) cattle thika machakos laikipia 29/29 (100 %) 31/31 (100 %) 82/88 (93.2 %) sheep thika machakos 24/30 (80 %) 20/20 (100 %) goats machakos 17/20 (85 %) 203 j.j.n. ngeranwa et al. the high seroprevalence in kenyan wildlife is not unique. seroprevalence of up to 100 % has been reported in wildlife at some localities in the usa, while at other localities it was zero, suggesting either a lack of vectors and/or carriers, or that sample sizes may have been too small (jessup, goff, stiller, oliver, bleich & boyce 1993). in the machakos area, where most of our samples came from, wildlife and livestock grazed together. the cattle reared on these farms are of indigenous breeds, hence relatively resistant to anaplasmosis (soulsby 1982), which allows relaxed tick control. in most of these areas, theileriosis is also not common as the principal tick vector, rhipicephalus appendiculatus, does not occur, providing yet another reason for not applying strict tick control measures. these reasons can explain the high prevalence rates obtained in this study. the findings of this study confirm that wildlife carry anaplasma organisms in kenya and could serve as reservoirs of infection for domestic animals. acknowledgements this study was financed by the netherlands government, through the wildlife disease research project, to whom we express our deep appreciation. we also thank the director, kenya agricultural research institute (karl), for availing time, money and facilities without which this work 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice article information authors: lundi ntantiso1 chantel de beer2 tanguy marcotty3 abdalla a. latif2,4 affiliations: 1makhathini research station, jozini, south africa2parasites, vectors and vector-borne diseases programme, agricultural research council – onderstepoort veterinary institute, south africa 3institute of tropical medicine, antwerp, belgium 4department of tropical veterinary diseases, university of pretoria, south africa correspondence to: abdalla latif postal address: private bag x05, onderstepoort 0110, south africa dates: received: 19 jan. 2014 accepted: 12 may 2014 published: 12 sept. 2014 how to cite this article: ntantiso, l., de beer, c., marcotty, t. & latif, a.a., 2014, ‘bovine trypanosomosis prevalence at the edge of hluhluwe-imfolozi park, kwazulu-natal, south africa’, onderstepoort journal of veterinary research 81(1), art. #762, 8 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.762 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. bovine trypanosomosis prevalence at the edge of hluhluwe-imfolozi park, kwazulu-natal, south africa in this original research... open access • abstract • introduction • research method and design    • study area    • trypanosomosis survey    • sampling and analysis    • tsetse population monitoring at the study sites    • statistical analyses • results    • tsetse abundance at the three diptanks    • trypanosome surveys at the three diptanks    • trypanosome surveys at the seven diptanks • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the northern kwazulu-natal (nkzn) region of south africa is the southern limit of the african tsetse belt. entomological information on glossina brevipalpis and glossina austeni was generated following the outbreak of trypanosomosis in cattle in 1990. however, these data have not been supported by parallel studies on epidemiology of the disease and therefore there has been no control policy in place. this study presented the first intensive investigations to address the epidemiology of trypanosomosis in nkzn. tsetse abundance, trypanosome herd average prevalence (hap), herd average anaemia (haa) and herd average packed cell volume (ha-pcv) were investigated at three communal diptanks located at the edge of hluhluwe-imfolozi park by monthly sampling from june 2006 – november 2007. seasonal trypanosome surveys were conducted at seven other communal diptanks. glossina brevipalpis prevalence was high at two of the diptanks, mvutshini and ekuphindisweni, but low at ocilwane, whilst g. austeni was only collected from mvutshini. this high and low tsetse challenge presented different disease scenarios. cattle at mvutshini and ekuphindisweni had the highest hap of 12.3% and 8.9% respectively, both significantly different (p = 0.001) from the hap obtained from cattle at ocilwane (2.9%). these two cattle herds also had the highest haa, 27.7% and 33.4% respectively, whilst cattle at ocilwane had the lowest, 11.1% (p = 0.001). conversely, cattle at ocilwane had the highest ha-pcv, ranging between 29.0% and 32.0%, whilst cattle at mvutshini and ekuphindisweni had the lowest ha-pcv (24.0% – 29.0%). by combining the data from the three diptanks (1318 observations), 62.0% of the infected cattle were found anaemic, compared to 20.0% in the uninfected group. trypanosome seasonal surveys showed that cattle at all the seven diptanks were infected with trypanosomes; mean hap, haa and ha-pcv of 10.2%, 46.6% and 23.7%, respectively. this study generated information on the epidemiological factors related to the wide spread of trypanosome-infected cattle and tsetse flies. trypanosomosis is a disease of economic importance impacting the livelihood of resource-poor farmers in nkzn. introduction top ↑ in south africa, animal trypanosomosis (also known as nagana) is restricted to parts of northern kwazulu-natal (nkzn) province, covering an area of 16 000 km2 extending from north of the umfolozi river to the mozambique border (sigauque et al. 2000). the disease coincides with the presence of two species of tsetse, glossina austeni and glossina brevipalpis (diptera: glossinidae) (kappmeier 2000). south africa historically had four species of tsetse flies, namely glossina m. morsitans, glossina pallidipes, g. brevipalpis and g. austeni. glossina m. morsitans disappeared shortly after the devastating rinderpest epidemic of 1896 that resulted in the removal of most bovid animals. glossina pallidipes was eradicated by 1953, primarily using aerial application of dichlorodiphenyltrichloroethane (ddt). the two so-called minor vectors species, g. brevipalpis and g. austeni, continue to exist in the thickest coastal bush forest areas that were not suitable for g. pallidipes and considered unsuitable for cattle grazing (du toit 1954). the introduction of eucalyptus plantations for commercial purposes has been associated with artificial changes in land cover and the plantations, with their surroundings, became protected areas. thicket expanded considerably after cattle farmers shifted to game ranching, thus increasing the conserved and protected tsetse areas. this man-made habitat resulted in the extension of the distribution and multiplication of both tsetse species, compared to the 1954 distribution maps (esterhuizen et al. 2006).in 1952, isolated cases of nagana were reported from zululand, with most cases occurring around lake st. lucia, well known for high infestations with g. brevipalpis and g. austeni (du toit 1954). kappmeier, nevill and bagnall (1998) reviewed the nagana situation up to 1990. the authors showed a rise in infections with trypanosomes on a number of farms in the lower mkuze area. in 1987, an outbreak in the nibela area was associated with clinical disease and mortalities. in 1990, during a severe drought, about 10 000 cattle died of nagana and 116 000 were treated with ethidium bromide in the low-lying areas of zululand (kappmeier et al. 1998). other control measures included the use of pyrethroid formulations in diptanks to protect cattle against ticks and to reduce the challenge by the vector tsetse fly. these were indications that nagana was not eliminated and the two remaining tsetse species, g. brevipalpis and g. austeni, continue to play a major role after the eradication of g. pallidipes. sixteen years after the nagana outbreak in 1990, a once-off sampling of 76 cattle at one diptank at the edge of hluhluwe-imfolozi park showed that the incidence of nagana caused mainly by trypanosoma congolenese had increased since 1990 (van den bossche et al. 2006). these authors concluded that the disease contributes significantly to the overall disease situation in nkzn and recommended that further research was needed to develop appropriate control methods. in a more recent study, shaw et al. (2014) worked out the economic benefits obtained from intervening against tsetse and trypanosomosis in six countries in east africa. the results of the study indicated the potential benefits in the region to be around $2.5 billion, with an average benefit per square kilometre varying from $500 – $3300. the current incidence of trypanosomosis in nkzn is unknown; it is thought to be relatively high and is a cause of concern to veterinarians and livestock farmers as there has been no national control policy in place for the last 20 years. the aim of this present study was to unravel the epidemiology of nagana in cattle where tsetse flies are known to occur, and to obtain baseline data against which the success of future control and the benefits of operations can be measured. research method and design top ↑ study area the study was conducted in hlabisa municipality, around the edges of hluhluwe-imfolozi park and along the isimangaliso (st. lucia) wetlands park, which are the two major conservation and protected areas in the district. this area falls within the 16 000 km2 tsetse belt in nkzn where both g. brevipalpis and g. austeni occur (kappmeier-green 2002). the vegetation consists of natural bush and sand forest plantations. average annual temperature ranges from 22 °c to 28 °c, rainfall is around 950 mm in summer and 260 mm in winter. this district has 200 000 head of cattle and 57 585 small stock. the human population in the area is about 572 340, of which 15 719 are stock owners (emslie 2005). trypanosomosis survey a total of 10 communal diptanks (cattle move freely) were selected for trypanosomosis surveillance (figure 1). three diptanks located at the edge of hluhluwe-imfolozi park were selected for regular monthly surveys: ekuphindisweni, mvutshini and ocilwane. seasonal trypanosome surveys were conducted in cattle at seven other communal diptanks located at various tsetse-infested areas, of which four are located at the edge of isimangaliso (st. lucia) wetland park. cattle at any of the diptanks constituted one herd as they graze together and get the same animal husbandry management; for example, they were rounded up for dipping on the same day. each herd consists of about 1000 cattle owned by more than 100 small-scale farmers (communal farming). cattle were sampled on the day when presented for dipping from june 2006 to november 2007. each herd was sampled randomly; two to three animals were sampled in a crush of 30–40. the data generated over the study period at one diptank were considered as repeated observations on a herd over time. figure 1: study area in northern kwazulu-natal province, south africa with diptank locations, (1) ekuphindisweni, (2) mvutshini, (3) ocilwane, (4) bukhipha, (5) nhlwathi, (6) mzineni, (7) nibela, (8) gwenyambili, (9) qakwini and (10) mahlambanyathi. circled diptanks indicate surveillance sites. the total number of cattle sampled at ekuphindisweni, mvutshini and ocilwane were 398, 371 and 315, respectively. cattle at five diptanks (gwenyambili, mahlambanyathi, nhlwathi, qakwini and nibela) were surveyed 2–5 times per year to cover two seasons, mainly winter (may–september) and summer (october–april), whilst one sampling was carried out at two diptanks (mzineni and bukhipha). a total of 726 cattle were sampled. treatment against trypanosomosis by the local communities in nkzn has not been a regular practice and in most cases is non-existent. for ethical reasons, parasitologically positive as well as negative animals with a packed cell volume (pcv) equal to or lower than 24% were treated with diminazene aceturate (berenil® r.t.u; intervet s.a., johannesburg, south africa) at a dose of 3.5 mg/kg body weight. sampling and analysis blood was collected from the tail or jugular veins using 10 ml vacutainer tubes coated with edta (bd vacutainer®; bd, plymouth, united kingdom) as anticoagulant. blood from each sample was decanted into plain microhaematocrit centrifuge capillary tubes (marienfeld-superior, lauda-königshofen, germany), sealed with cristseal and centrifuged for 5 min at 9000 rpm. after centrifugation, the pcv was determined. animals with a pcv of 24% or less were considered anaemic (murray & dexter 1988; van den bossche, shumba & makhambera 2000). the buffy coat of each sample was extruded onto a microscope slide, covered with a cover slip and examined for motile trypanosomes under a compound microscope using 40× magnifications.trypanosoma prevalence in the present study refers to infections with t. congolense, the dominant species infecting cattle in the area (mamabolo et al. 2009; motloang et al. 2012; van den bossche et al. 2006) and identification of the parasites in the buffy coat during examination, based on its characteristic motility, difference from other species and stained thin smear examination. the level of bovine trypanosomosis of a herd at a specific site was calculated as the proportion of cattle with trypanosome infection and referred to as the ‘herd average prevalence of trypanosome infection’ (hap) (van den bossche & rowlands 2001). the percentage anaemia (i.e. percentage of cattle in a herd with pcv of 24% or less) was calculated. the pcv of cattle at each sampling was averaged and referred to as ‘herd average pcv’ (ha-pcv) (van den bossche & rowlands 2001). furthermore, the percentage of anaemic cattle in a herd was referred to as ‘herd average anaemia’ (haa). hap, ha-pcv and haa values are considered as indicators of the health status of a herd (trail et al. 1991; trail et al. 1985) and were obtained from herds at each diptank. tsetse population monitoring at the study sites four odour-baited h-traps (kappmeier 2000) were deployed and remained in place permanently for the duration of the study at ekuphindisweni (may 2006 – december 2008), mvutshini (may 2005 – december 2008) and ocilwane (november 2007 – december 2008) diptanks. flies were collected once every 2 weeks, identified to species and counted. the apparent density (flies per trap per day) was calculated for the three diptank sites (esterhuizen et al. 2006). statistical analyses haa and hap data were analysed using logistic regressions. categorical explanatory variables were the diptanks and the month of sampling. in addition, the buffy coat result (positive or negative) was used as explanatory variable of haa. pcv data were analysed in a linear regression using the buffy coat result, the location (one of the three diptanks) and the sampling month as discrete explanatory variables. p-values < 0.05 were considered statistically significant. surveillance data were averaged per sampling site and date. confidence intervals (95%) were calculated assuming a binomial distribution of haa and hap and a normal distribution of pcv. results top ↑ tsetse abundance at the three diptanks both fly species were collected from mvutshini diptank (figure 2). in the areas around ekuphindisweni (lying at the northernmost side of hluhluwe-imfolozi park) and ocilwane (lying at the southern side of the park) diptanks, only g. brevipalpis was present (figure 2). the apparent density of g. brevipalpis at ocilwane diptank was lower than at ekuphindisweni and mvutshini diptanks. the apparent densities of both species were higher in the summer months (october–april) and lower in the winter months (may–september). figure 2: tsetse populations of, (a) glossina brevipalpis and (b) glossina austeni at ekuphindisweni, mvutshini and ocilwane diptanks. trypanosome surveys at the three diptanks comparisons of hap, haa and ha-pcv are shown in figure 3. cattle at mvutshini and ekuphindisweni diptanks had the highest hap, both significantly different (p ≤ 0.002) from the hap obtained from cattle at ocilwane. the herds at mvutshini and ekuphindisweni diptanks also had significantly higher haa than at ocilwane, even when using the buffy coat result as explanatory variable (p < 0.001). conversely, cattle at ocilwane diptank had the highest ha-pcv even when using the buffy coat result as explanatory variable (about 3.0% more; p < 0.001). infected animals had a pcv 4.2% lower (95.0% ci: 3.2–5.2) than uninfected animals (p < 0.01). figure 3: comparing herd average packed cell volume, trypanosomes herd average prevalence and herd average anaemia in positive and negative cattle at the three diptanks, (a, d, g, j, m) ekuphindisweni, (b, e, h, k, n) mvutshini and (c, f, i, l, o) ocilwane (estimates and 95% confidence intervals without interaction between explanatory variables). trypanosome surveys at the seven diptanks table 1 shows the results of trypanosome surveys conducted at seven communal diptanks. generally, t. congolense infection in cattle was found at all of the diptanks surveyed; hap 10.2% ± 9.1%, haa 46.6% ± 21.0% and ha-pcv 23.7% ± 1.6%. the standard deviation values were high for hap and haa parameters because of the different levels of trypanosome challenge at each location. the highest hap (range 15.4% – 34.4%, n = 4) was recorded at gwenyambili diptank. this high infection in the cattle herds produced high values of haa (range 35.0% – 75.0%). this diptank is located inside a dense indigenous forest, a suitable tsetse habitat next to isimangaliso wetland park. the rainy seasons did not result in improvement of the health condition of trypanosome-infected cattle as demonstrated by results obtained from ha-pcv, which approached or were below 25.0%. table 1: trypanosome survey at the seven communal diptanks showing means ± standard deviation and 95% confidence interval results of the herd average prevalence, herd average anaemia and herd average packed cell volume. discussion top ↑ in 1990, during a severe drought in the tsetse-infested low lying areas of zululand, about 10 000 cattle died of nagana and 116 000 were treated during this outbreak using ethidium bromide (kappmeier et al. 1998). further control measures included the use of pyrethroid dip after 4 years to reduce the challenge by the vector tsetse fly. sixteen years after the 1990 outbreak, van den bossche et al. (2006) found the hap of cattle sampled at one communal diptank at the edge of hluhluwe-imfolozi park to be 34% and the haa 83%. this survey demonstrated that nagana was still prevalent and control was required. the present results also demonstrated that the haa at the seven surveyed diptanks was high (up to 60%). nagana is considered a neglected disease in south africa because no control policy has been implemented for over 20 years. the present study presents the first intensive epidemiological investigations to address the problem of animal trypanosomosis in south africa since the 1990 outbreak and this information could be used in future control operations.the relationship between trypanosome hap, haa and ha-pcv was investigated in cattle at three communal diptanks for 15 months. these three epidemiological parameters were previously shown to reflect the health condition in cattle in relation to tsetse challenge (van den bossche & rowlands 2001). although the percentage of haa in infected cattle is high, more ‘uninfected’ animals could also have been anaemic, but trypanosome infections may have gone undetected by the conventional buffy coat examination employed in this study. the results obtained by marcotty et al. (2008) confirmed the low sensitivity of the buffy coat test, which, despite parasite concentrations, failed to detect 66.0% of infected cattle in their study. this is the result of low parasitaemia levels in field cattle. again, van den bossche et al. (2006) found the infection rate in cattle at hluhluwe-imfolozi park (our study sites) to be 34.0% using the buffy coat and 60.5% by using the polymerase chain reaction (pcr) molecular tool. moreover, mamabolo et al. (2009) found 91.0% positive for t. congolense using pcr analysis of samples collected from cattle at mvutshini. other sensitive molecular tools, when compared with conventional diagnostics, revealed higher levels of trypanosome infections in field cattle (geysen, delespaux & geerts 2003). these studies confirm the low sensitivity of the buffy coat technique and support our results of a high percentage of aparasitaemic cattle with anaemia. cattle at ocilwane diptank are subjected to irregular low tsetse challenge along the southernmost edges of the reserve. it is known that this is the most southerly distribution limit of tsetse in nkzn (hendrickx 2002). infections in cattle at ocilwane were detected at only 5 out of 15 monthly samplings. only a few animals were infected (2.2%) but all of the infected ones became anaemic (100%). there are some speculations about the infected cattle at ocilwane: the natural population of g. austeni is very low at the study sites and emigration from the southernmost limit of this species may allow little fly movement to the diptank site; alternatively, the h-trap is not optimum for attracting and catching the species, although it was demonstrated to be the best available (kappmeier 2000). all of the seven infected cattle became anaemic, which strongly suggests that these flies previously fed on buffaloes, thus transmitting highly virulent strains (motloang et al. 2012). cattle at the diptank could not have moved to graze inside the park as there was no report of fatal corridor disease in the herd for the duration of the study. buffaloes in the parks in kzn are known carriers of theileria parva, the cause of corridor disease (t. parva infections in cattle associated with carrier buffalo) (mbizeni et al. 2013). this is a unique situation where the interface with the irregular tsetse challenge and the presence of trypanosome reservoirs in game produced an epidemic character of the disease. such interfaces are found along the kasungu national park and nkhotakota game reserve in malawi, along the malawi national park and along the kwando river in the zambezi (formerly caprivi) region of namibia (van den bossche et al. 2000). anaemia caused by trypanosome infections is the result of the parasite damaging red blood cells by releasing biochemical molecules and is non-regenerative (murray & dexter 1988; murray et al. 1979; nok & balogun 2003; suliman & feldman 1989). the pcv of individual animals and the ha-pcv are useful indicators of anaemia and in trypanosome endemic areas are the most typical signs of nagana in domestic animals (marcotty et al. 2008; murray & dexter 1988; trail et al. 1991). in endemic tsetse areas, parasitologically negative animals that have a low pcv are regarded as having trypanosome infections. in the present study, 62% of the infected animals, using direct parasitological methods, were found to be anaemic and therefore the 20% of anaemic cattle that were aparasitaemic should be considered trypanosome-infected and receive treatment. there are some cattle breeds in western and eastern africa that have been shown to be tolerant to trypanosome infection and able to limit development of anaemia by a process of erythropoiesis balancing the depletion of red blood cells (naessens, teale & sileghem 2002). the cattle types in this study have no history of trypanotolerence, thus anaemia in an infected animal becomes chronic and progressive in absence of treatment. the survey conducted at the seven diptanks under tsetse challenge highlighted the magnitude of nagana as a major risk to animal health. the hap was very high (up to 31%), and consequently haa was also high (up to 70%). anaemia in cattle is clearly related to trypanosome infections as cattle continued to be anaemic during the rainy season and received no drug treatment. this is also demonstrated by comparing the ha-pcv and haa conditions in cattle under high and low tsetse challenge; that is, mvutshini and ekuphindisweni versus ocilwane, and the other seven diptanks versus ocilwane. several epidemiological factors and their associations with the vector tsetse fly, livestock, presence of game and the climate have been identified in trypanosomosis. the infection rate in tsetse flies is of prime importance (connor & van den bossche 2004). motloang et al. (2012) investigated the vector competence of g. brevipalpis and g. austeni collected from the same communal diptanks in the present study around hluhluwe-imfolozi park and from other farms and game parks. they found the infection with mature parasites in g. austeni, which is considered the main vector, to be 8%. moreover, g. austeni collected from the same sites and which fed on susceptible cattle under controlled conditions subsequently transmitted t. congolense and the animals had to be treated. low fly population density can cause a serious disease problem (jenni et al. 1980; molyneux & jefferies 1986; roberts 1981). this observation supports the hap in cattle at all diptanks surveyed with low or no apparent g. austeni presence. the apparent abundance of g. brevipalpis was substantially higher than that of g. austeni in three main vegetation types in zululand (esterhuizen et al. 2005). it was not possible to attribute these findings to the real population density of the two species or to sampling bias favouring g. brevipalpis catches, although the h-trap was developed to target the two species and was more efficient than any other tested traps for g. austeni (kappmeier 2000; kappmeier & nevill 1999). a total of 59 diptanks (herds) were sampled in 1994 (bagnall, in kappmeier et al. 1998) to determine the prevalence of anaemia in cattle. the highest prevalence of trypanosomosis was recorded in ubombo district (our study sites) at 10% – 15%. van den bossche et al. (2006) found 34% of cattle at mvushini diptank were infected with trypanosomes. these results obtained over an extended period of time indicate that the prevalence of nagana has in fact increased and our present results have confirmed this trend. on the other hand, the tsetse distribution and abundance has also increased. our results have, for the first time, reported the presence of g. brevipalpis at ocilwane diptank, previously thought to be the southernmost limit of the tsetse distribution. commercial cattle farmers have shifted to game ranching and resorts, thus increasing the conserved and protected tsetse areas in national and provincial game parks and nature reserves. these man-made habitats have resulted in the extension of the distribution and multiplication of two tsetse species compared to the 1954 distribution maps (esterhuizen et al. 2006). the limited grazing areas and degraded communal land have therefore forced cattle to move to such tsetse habitats, exposing them to nagana challenge. conclusion top ↑ the present study showed that nagana is prevalent at all 10 diptanks surveyed and affects mainly the resource-poor farmers. in south africa, there is no control policy for tsetse and nagana and treatment using trypanocidal drugs is still not accessible to resource-poor farmers. the only response is to ‘manage the crisis’ when the problem is out-of-hand by the introduction of pyrethroid chemical acaricides in dips, which eventually kills the flies and ticks. however, the dipping policy is not enforced by law and has proved to be unsustainable over the years, as demonstrated in this study by the high level of trypanosome infections in cattle following removal of pyrethroid dips since 1992. what is required is a politically supported decision to eradicate tsetse flies in order to increase cattle productivity and alleviate poverty. a strategy for area-wide control to establish a tsetse-free zone in south africa has been advocated (kappmeier-green, potgieter & vreysen 2007). integrated control including treatment is an option but is also not sustainable because of migration and emigration of tsetse flies from the conservation and protected game parks, resulting in a high risk game–livestock–tsetse interface. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions a.a.l. 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edge of hluhluwe-imfolozi park, kwazulu-natal province, south africa’, onderstepoort journal of veterinary research 73, 77–79. http://dx.doi.org/10.4102/ojvr.v73i1.172 van den bossche, p. & rowlands, g.j., 2001, ‘the relationship between the parasitological prevalence of trypanosomal infections in cattle and herd average packed cell volume’, acta tropica 78, 163–170. http://dx.doi.org/10.1016/s0001-706x(00)00182-0 van den bossche, p., shumba, p. & makhambera, p., 2000, ‘the distribution and epidemiology of bovine trypanosomosis in malawi’, veterinary parasitology 88, 163–176. http://dx.doi.org/10.1016/s0304-4017(99)00222-8 article information authors: esmey b.e. moema1 pieter h. king1 johnny n. rakgole2 chantélle baker3 affiliations: 1department of biology, university of limpopo, medunsa campus, south africa2department of virology, university of limpopo, medunsa campus, south africa 3electron microscope unit, university of limpopo, medunsa campus, south africa correspondence to: esmey moema postal address: po box 139, medunsa 0204, south africa dates: received: 22 apr. 2013 accepted: 24 june 2013 published: 25 sept. 2013 how to cite this article: moema, e.b.e., king, p.h., rakgole, j.n. & baker, c., 2013, ‘descriptions of diplostomid metacercariae (digenea: diplostomidae) from freshwater fishes in the tshwane area’, onderstepoort journal of veterinary research 80(1), art. #611, 7 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.611 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. descriptions of diplostomid metacercariae (digenea: diplostomidae) from freshwater fishes in the tshwane area in this research communication... open access • abstract • introduction • materials and methods    • light microscopy    • scanning electron microscopy    • polymerase chain reaction • results    • polymerase chain reaction • discussion • recommendations • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the metacercarial (larval) stages of diplostomid digeneans are known to inhabit freshwater fish, causing tissue damage in the process. due to their widespread diversity, little is known about their life cycle. the classification of these parasitic stages to the species level using only the morphology is very challenging due to the lack of genitalia; they are regarded to be the most important structures in the identification of these organisms. in this study, additional morphological information through light and scanning electron microscopy is given for two different diplostomids found in the cranial cavity of clarias gariepinus and the vitreous chambers of tilapia sparrmanii and pseudocrenilabrus philander. the diplostomid metacercaria inhabiting the cranial cavity of clarias gariepinus was morphologically identified as diplostomulum (tylodelphys) mashonense and an unknown metacercaria of the genus diplostomum was found in the vitreous chambers of pseudocrenilabrus philander and tilapia sparrmanii. both parasitic species’ 28s recombinant deoxyribonucleic acid genomic regions were successfully amplified using dig 125/1500r primer pairs. the assay yielded a product of approximately 1300 base pairs as seen on the gel images. there were 14 nucleotide differences over the entire analysed sequences resulting in a 1.1% (14/1273) nucleotide difference. in line with the morphological characteristics of these parasites, there seemed to be a slight difference in their genetic make-up. the application of molecular techniques on digenetic trematodes seems very promising and may yield great potential in future descriptions of morphologically similar parasitic species. introduction top ↑ according to gibson, jones and bray (2001), adult parasites of the family diplostomidae are parasitic in many birds and mammals. larval forms of this family use a variety of freshwater snails as first intermediate hosts and various fish species as second intermediate hosts. beverley-burton (1963) from zimbabwe and niewiadomska (1984, 1986) from poland pioneered early studies on the adults of the genus diplostomum. larval forms belonging to the genus diplostomulum were also previously studied from the eyes and brain of freshwater fish from various parts of the world. these included the work done by: pandey (1973) from india; prudhoe and hussey (1977), chibwana et al. (2013) from tanzania and nigeria; mashego and saayman (1989) and madanire-moyo, luus-powell and olivier (2010) from south africa; hendrickson (1978a, 1978b) and höglund and thulin (1992) from the baltic sea. the aim of this research was to study the morphology and phylogeny of two diplostomid metacercariae; one from the eyes and the other from the cranial cavity of different freshwater fish species from two localities in the proximity of tshwane, gauteng province, south africa. materials and methods top ↑ diplostomid metacercariae were collected through dissections of three freshwater fish species, namely pseudocrenilabrus philander weber, 1897, tilapia sparrmanii a. smith, 1840 and clarias gariepinus burchell, 1822 (c. gariepinus). they were collected from the supersand dam (25º35´02.42´´s, 28º10´38.98´´e, altitude 1198 m above sea level [a.s.l.]) situated ± 2 km north of the bon accord dam in the onderstepoort area, gauteng and the boekenhoutskloof farm dam (25º32´45.18´´s, 28º26´12.47´´e, altitude 1214 m, a.s.l.), approximately 15 km north of the roodeplaat dam nature reserve in the proximity of tshwane, gauteng province, south africa. live forms of the metacercariae were studied using a compound microscope with the aid of vital stains. specimens were fixed in 2.5% phosphate buffered glutaraldehyde (ph = 7.2–7.4), 5% saline buffered solution and 70% ethanol respectively and stored until required. standard techniques for light and scanning electron microscopy (sem) were employed to study the internal and external ultrastructures of these parasites. polymerase chain reaction (pcr) techniques were also performed pertaining to molecular structures on the same species. light microscopy specimens fixed in 70% ethanol were stained in haematoxylin or eosin and van cleave’s haematoxylin staining solutions. stained specimens were mounted on slides using dpx mounting medium. projection drawings of mounted specimens were made by means of a drawing tube attached to a nikon compound microscope (nikon, tokyo, japan). for each description, a maximum of 20 specimens were drawn and measured, with all measurements given in micrometres (μm). morphological measurements are presented in the text as follows: minimum to maximum values, followed by the average value and standard deviation in parentheses. all light micrographs were taken using a nikon coolpix®990 digital camera fitted to the compound microscope. scanning electron microscopy specimens fixed in 2.5% gluteraldehyde were washed in millonig’s phosphate buffer (ph = 7.2) and dehydrated through an graded series of ethanol (from 70% ethanol to 100% ethanol) for 30 seconds to one minute in each concentration. they were processed as follows: overnight critical point dried (polaron, watford, uk), mounted on aluminium stubs, sputter coated with gold (emscope; quorum technologies, ashford, uk) and examined using a leica stereoscan 420 sem (leica electron optics, uk). polymerase chain reaction specimens for pcr were suspended in 5% saline buffered solution (200 µl) and frozen in a -70 ˚c freezer until required. during the extraction process, specimens were thawed and excess buffered saline solution (bss: 800 ml distilled water, 8 g nacl, 0.2 g kcl, 1.44 g na2hpo4 and 0.24 g kh2po4; ph = 7.4) was removed. deoxyribonucleic acids (dna) were extracted using a qiaamp dna mini kit (qiagen, valencia, ca, usa) following the manufacturer’s instructions. two parasite samples obtained from different locations, the brain and eyes of two fishes, were extracted in duplicates. in brief, specimens were transferred to 220 µl lysis solution (200 µl atl buffer and 20 µl proteinase k) in microcentrifuge tubes. samples were incubated at 56 ˚c for four hours followed by the addition of 200 µl of absolute ethanol. the dna in the lysate was allowed to bind to the spin columns and washed twice with the supplied buffers before being eluted in 200 µl of elution buffer. the extracted dna of parasites was stored at -20 ˚c until analysis. the 28s rdna was amplified using the primers dig12 (5–aagcatatcactaagcgg–3) and 1500r (5–gctatcctgagggaaacttcg–3) (tkach et al. 1999; tkach, pawloski & mariaux 2000). the pcr reaction was performed in a final volume of 25 µl. the reagents at a final concentration of 0.2 mm for dntps, 1.5 mm mgcl2, 0.2 µm each of primers and 1 u per reaction of taq polymerase (bioline, biotaq, london, uk) were used. the pcr products were sequenced on the abi 3100xl sequencing platform (life technologies, foster city, ca, usa). the sequences were edited using chromaspro v1.5 (technelysium pty, ltd, queensland, australia). additional sequences were downloaded from genbank (accession numbers are shown on the phylogenetic tree in the results section) and aligned using clustalw within bioedit (hall 1999). the phylogenetic trees were constructed using mega v5.22 (tamura et al. 2011).this research project met the requirements of the medunsa research ethics committee (bp 05/2005) and the animal ethics committee (aec 02/05) of the university of limpopo, south africa. fish species that were collected were kept under controlled laboratory conditions with sufficient light, air and food according to acceptable standards. they were anaesthetised using clove oil prior to dissection. results top ↑ metacercaria: diplostomid metacercaria a [diplostomulum (tylodelphys) mashonense] (figure 1a). figure 1: light microscope projection drawings, (a) diplostomid metacercaria a [diplostomulum (tylodelphys) mashonense] from the cranial cavity of clarias gariepinus collected from boekenhoutskloof farm dam and (b) diplostomid metacercaria b from the vitreous chamber of tilapia sparrmanii and pseudocrenilabrus philander collected from supersand dam, gauteng province. the host is clarias gariepinus sampled from the boekenhouts-kloof farm dam in the gauteng province and its preferred site in the host is the brain cavity.the metacercariae were found unencysted in the cranial cavity of the sharptooth catfish, c. gariepinus. a few were collected around the frontal lobe, however, the majority of these larval stages had migrated posteriorly into the cavity. the numbers varied from a minimum of eight to over a hundred specimens per fish host. the metacercaria appeared to be off-white and elongated (figure 1a; figures 2a and 2b). the foliate body measured 946 μm–1917 μm (1409 μm ± 302 μm) long x 240 μm–485 μm (375 μm ± 75 μm) wide. the body was comprised of a long and distinct forebody, followed by a small hindbody. it had an oral sucker (figure 2c), which was situated at the anterior end of the body. it measured 36 μm–90 μm (70 μm ± 17 μm) x 42 μm–96 μm (72 μm ± 14 μm). the tegument around the oral sucker was surrounded by non-ciliated serrated receptors (figure 2c). bordering the oral sucker were two pseudosuckers (figure 1a), 54 μm–120 μm (73 μm ± 22 μm) x 24 μm–84 μm (48 μm ± 16 μm), which were situated bilaterally at the anterior end of the body. the oral sucker (figure 2c) continued into a muscular pharynx measuring 24 μm–60 μm (46 μm ± 13 μm) x 30 μm–60 μm (47 μm ± 10 μm). a short oesophagus measuring 30 μm–108 μm (40 μm ± 27 μm) x 12 μm–24 μm (19 μm ± 4 μm) bifurcated into two long intestinal caeca, 772 μm–1371 μm (1111 μm ± 247 μm) x 18 μm–36 μm (28 μm ± 7 μm), within the hindbody at the posterior end of the body. figure 2: light and scanning electron micrograph, (a) light micrograph, (b) whole mount, (c) oral sucker, (d) acetabulum, (e) holdfast organ, (f) anterior body tegument, (g) body tegument at the acetabular region and (h) non-ciliated receptors on body tegument. a spherical acetabulum that was similar in size to the oral sucker (figure 2d), was situated midventrally on the body and measured 36 μm–90 μm (71 μm ± 17 μm) x 30 μm–96 μm (67 μm ± 21 μm). the tegument around the acetabulum wall (figure 2d) had no sensory receptors, but had ridges that were equally distributed around it. an oval-shaped holdfast organ (figure 2e) with a deep fissure measured 156 μm–198 μm (180 μm ± 15 μm) x 24 μm–150 μm (101 μm ± 48 μm) and was situated between the acetabulum and the posterior end of the body. genital primodia, consisting of two testes, occurred anteriorly in the hindbody. the anterior testis was asymmetrical, measuring 102 μm–467 μm (199 μm ± 120 μm) x 114 μm–263 μm (179 μm ± 50 μm), and lay in line with the junction of the forebody and the hindbody. it was also placed more to the right in the hindbody. the posterior testis on the other hand was bilobed and measured 96 μm–155 μm (126 μm ± 27 μm) x 138 μm–221 μm (176 μm ± 24 μm) (figure 1a).the excretory system was not observed due to the copious amount of calcareous bodies found in the body. the anterior body tegument was relatively smooth in the area around the oral sucker (figure 2f), but became unevenly ridged posteriorly (figure 2g) with non-ciliated papillae all over the body surface (figure 2b–h). there were no spines on the body tegument. metacercaria: diplostomid metacercaria b (figure 1b). the hosts are tilapia sparrmanii and pseudocrenilabrus philander sampled from the supersand dam in the gauteng province and its preferred site in the host is the vitreous chamber. these metacercariae were found unencysted in the vitreous chamber of small southern mouth-brooders (p. philander) and banded tilapian (t. sparrmanii) species. the number of specimens varied from a minimum of two to a maximum of 12 per fish host. the metacercaria (figure 1b; figures 3a and 3b) was whitish in colour with an elongated body. the spatulate body measured 832 μm–1263 μm (1019 μm ± 138 μm) long x 257 μm–407 μm (340 μm ± 50 μm) wide. the body was comprised of a well-developed oral sucker (figure 3c), which measured 42 μm–72 μm (55 μm ± 9 μm) x 30 μm–60 μm (51 μm ± 10 μm). this sucker was located subterminally at the anterior end of the body and the tegument around it was not armed with receptors. well-developed pseudosuckers (figure 1b and figure 3a) measuring 24 μm–42 μm (34 μm ± 6 μm) x 36 μm–84 μm (59 μm ± 15 μm) flanked the oral sucker. figure 3: light and scanning electron micrographs, (a) light micrograph, (b) whole mount, (c) oral sucker, (d) acetabulum, (e) holdfast organ, (f) non-ciliated receptors dorso-anteriorly on the body, (g) body tegument with many non-ciliated receptors and (h) excretory pore. the mouth that was located in the oral sucker connected to a muscular pharynx measuring 30 μm–54 μm (43 μm ± 8 μm) x 30 μm–54 μm (43 μm ± 6 μm). the pharynx lead to a very short oesophagus that measured 24 μm–84 μm (39 μm ± 18 μm) x 18 μm–24 μm (19 μm ± 2 μm). the oesophagus then bifurcated into two long intestinal caeca, 557 μm–988 μm (717 μm ± 141 μm) x 18 μm–42 μm (29 μm ± 7 μm), ending at the posterior end of the holdfast organ. a spherical acetabulum (figure 3d) measuring 30 μm–66 μm (51 μm ± 12 μm) in diameter was situated along two-thirds of the body length. it was somewhat smaller than the oral sucker.an elliptical holdfast organ (figure 3e) with hymen measured 96 μm–180 μm (131 μm ± 29 μm) x 42 μm–96 μm (68 μm ± 18 μm) and was situated at the posterior end of the forebody. a conspicuous v-shaped excretory bladder (figure 1b and figure 3a) measuring 138 μm–240 μm (172 μm ± 32 μm) x 72 μm–132 μm (98 μm ± 21 μm) was situated at the posterior end of the hindbody. no flame cells were observed due to the rod-shaped calcareous bodies in the body. the genital primodia were also not observed, most probably due to the developmental stage of the parasites that were examined. an excretory pore was present at the posterior end of the hindbody (figure 3h). the non-ciliated receptors were observed posterior to the oral sucker (figure 3f) surrounding the acetabulum (figure 3d) and in most of the body tegument (figures 3g and 3h). polymerase chain reaction parasite samples were successfully amplified using the dig 125/1500r primer pairs. three of the extracts tested positive: two from the brain and one from the eye. the assay yielded a product of about 1300 base pairs (bp) as seen on the gel images. brain sample sequences were 100% similar. the sequence of eye sample differed from the one obtained from the brain by having one base pair longer in the sequenced region. in addition, there were 14 nucleotide differences over the entire analysed sequences resulting in a 1.1% (14/1273) nucleotide difference. table 1 shows the specific locations of the base differences. the sequences were submitted to genbank under the following accession numbers: kf189071 (brain) and kf189072 (eye). table 1: differences in the nucleotides across the genomes of two diplostomid metacercariae from the eyes of tilapia sparrmanii and pseudocrenilabrus philander, and the brain cavity of clarias gariepinus. nucleotide positions were only based on the analysed regions of the two sequences. a phylogenetic tree (figure 4) was constructed using the two sample sequences along with the similar sequences downloaded during a basic local alignment search tool (blast) analysis from the genbank database. figure 4: a phylogenetic tree showing the brain and eye diplostomids in relation to other parasites from the genbank database. discussion top ↑ in this study, two dissimilar diplostomids were found to infect three fish species. diplostomid metacercaria a was only found in the cranial cavity of c. gariepinus, whereas diplostomid metacercaria b was only found in the vitreous chamber of t. sparrmanii and p. philander. from this study it is evident that the two diplostomids are host specific and site specific due to the niches where they were sampled.both the cranial cavity and the eye diplostomid metacercariae were found unencysted at their respective sites. they were found to be moving freely in the cerebrospinal fluid in the region of the frontal lobes and vitreous humour of the eye, respectively. eight catfish that were dissected and examined in the present study were infected with the larvae in their cranial cavity. however, they did not show any signs of distress caused by these parasites. khalil (1963) described diplostomum tregenna, nazmi gohar, 1932 from the cranial nerves of clarias lazera from the river nile. he observed three stages of this parasite; the third stage was well-developed showing genitalia. beverley-burton (1963) described an adult diplostomula of diplostomulum (tylodelphys) mashonense (d. [t.] mashonense); the metacercariae were discovered in the cranial cavity of clarias mossambicus, which was synonymous to c. gariepinus in southern rhodesia (now known as zimbabwe). similarly, mashego and saayman (1989) described d. (t.) mashonense collected from c. gariepinus from dams around the limpopo province (formerly known as lebowa). the latter authors’ observations were that the metacercariae concentrated more on the frontal lobes of the brain in young hosts and thereafter they were found to have moved posteriorly as the hosts aged. this phenomenon was also observed during the present research study. these larvae were found to be very similar to the one described by the abovementioned authors and further, it was also very difficult to observe the excretory system including the flame cells. the metacercariae found in the cranial cavity of c. gariepinus during our investigation can therefore be identified as the larval stage of d. (t.) mashonense. tilapia sparrmanii specimens were found to be more frequently infected by eye diplostomid metacercariae than p. philander. it is still unclear as to why this should be the case, but immunity and the daily activities of the fish species may possibly have an influence. hendrickson (1978a, 1978b) made observations about the eye diplostomids diplostomum scheuringi, hughes, 1929 and diplostomum spathaceum respectively that were sampled from wyoming fish. however, in his study observations were that some fish species were less infected than others and that these parasites had little, if any, effect on the sight of the fish. pandey (1973) described another type of eye diplostomid, diplostomulum opthalmi (pandey 1970), from freshwater fish in india. the eye diplostomid metacercaria observed in the present study is very similar to the one described by hendrickson (1978a, 1978b) and pandey (1973). however, it differs in the body tegument and size, has suckers that are spined and there is an absence of pseudosuckers. in order to classify this diplostomid species as a new species, the adult stage of this larval parasite will have to be found or experimentally raised. only then would the taxonomic status of this species be able to be commented on. although the diplostomid metacercariae in the present study seem morphologically similar to the metacercariae described by other researchers, morphological studies are simply not sufficient. the morphology of organisms may undergo micro-evolutionary adaptation over time as the environment changes due to many external factors. factors like ph, temperature, nutrients, light, health, age, as well as angles of observation may introduce variation of the same organism and are therefore very subjective even with standardised guidelines (huyse, poulin & théron 2005). phylogenetic studies were thus conducted on these metacercarial stages. in line with the morphological characteristics of the two parasites, there seemed to be a slight difference in their genetic make-up. from the 1273 bp of a 28s region, a 1.03% difference exists at dna level. this is thought to be a highly conserved region for most organisms and should therefore be common to organisms of the same species. lower homology values will be expected for non-conserved regions of these organisms, such that their full length sequences could differ by as much as 15%. the power of phylogenetic analysis is undermined by the lack of parasite sequences in the genbank database and therefore renders a limitation into complete characterisation of these organisms. the phylogenetic tree thus constructed, clusters the parasites in the present study with those that belong to the superfamily diplostomoidea and can unfortunately only resolve the samples studied to the order strigeidida. there are mixed families within the same node when analysing this phylogenetic tree to the family level. this is the first study to report on the larger fragment of the 28s rdna of diplostomids from the cranial cavity of c. gariepinus and the eyes of p. philander and t. sparrmanii. presently there are no related sequences in the genbank database. however, there is a recent report on phylogenetic studies of diplostomids collected from the brain of c. gariepinus sampled from nigeria and tanzania, of which the sequences covered up to the its-2 region (chibwana et al. 2013). unfortunately the sequences of the present study cannot be compared to those generated in the previous work due to the fact that they both target exclusive dna regions. the only sequence that links to the sequences from our study (and that of chibwana et al. 2013) is alaria alata (accession number jf820609). since there are no related sequences in the database, we can only base our nomenclature on the morphology. this was a pilot study from the department of biology, which aimed to assess the possibility of applying molecular biology techniques to the investigation of larval digenetic parasites. the application of molecular techniques on digenetic trematodes seems very promising and may yield great potential in future descriptions of these parasitic species. recommendations top ↑ we recommend a full length genome analysis of these two organisms in order to establish more definitive evidence of whether they belong to the same species. further than that, archived specimens that have been described using biological and morphological characteristics should be genetically analysed in order to increase the information in the database of these organisms in genbank. acknowledgements top ↑ sem equipment utilised during this investigation was supported by the department of science and technology in partnership with the national research foundation of south africa. the department of virology, medunsa campus allowed us to utilise their pcr laboratories for molecular work. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions e.b.e.m. (university of limpopo) was the principal investigator and this study forms part of her doctoral thesis. p.h.k. (university of limpopo) was the postgraduate supervisor of this study. c.b. (university of limpopo) was a co-worker involved with all aspects of micrography and technical finishing of photographic plates. j.n.r. (university of limpopo) was involved in the phylogenetic studies of the present study. references top ↑ beverley-burton, m.m., 1963, ‘a new strigeid, diplostomum (tylodelphys) mashonense n. sp., (trematoda: diplostomidae), from the grey heron, ardea cinerea l. in southern rhodesia, with an experimental demonstration of part of the life cycle’, revue de zoologie et de botanique africaines lxvii, 291–308.chibwana, f.d., blasco-costa, i., georgiva, s., hosea, k.m., nkwengulila, g., scholz, t. & kostadinova, a., 2013, ‘a first insight into the barcodes for african diplostomids (digenea: diplostomatidae): brain parasites in clarias gariepinus (siluriformes: clariidae)’, infection, genetics and evolution 17, 62–70. http://dx.doi.org/10.1016/j.meegid.2013.03.037, pmid:23542455 gibson, d.i., jones, a. & bray, r.a., 2001, ‘keys to the trematoda, vol. 1’, cabi publishing, london. hall, t.a., 1999, ‘bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 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diplostomulum stage of diplostomum tregenna nazmi gohar, 1932 with an experimental demonstration of part of the life cycle’, journal of helminthology 37(3), 199–206. http://dx.doi.org/10.1017/s0022149x00003783 madanire-moyo, g.n., luus-powell, w.j. & olivier, p.a.s., 2010, ‘ecology of metazoan parasites of clarias gariepinus (osteicthyes: clariidae) from the nwanedi-luphephe dams of the limpopo river system, south africa’ african zoology 45(2), 233–243. http://dx.doi.org/10.3377/004.045.0202 mashego, s.n. & saayman, j.e., 1989, ‘digenean trematodes and cestodes of clarias gariepinus (burchell, 1822) in lebowa, south africa, with taxonomic notes’, south african journal of wildlife research 19, 17–20. niewiadomska, k., 1984, ‘present status of diplostomum spathaceum (rudolphi, 1819) and differentiation of diplostomum pseudospathaceum nom. nov (trematoda: diplostomidae)’, systematic parasitology 6, 81–86. http://dx.doi.org/10.1007/bf02185515 niewiadomska, k., 1986, ‘verification of the life-cycles of diplostomum spathaceum (rudolphi, 1819) and d. pseudospathaceum niewiadomska, 1984 (trematoda: diplostomidae)’, systematic parasitology 8, 23–31. pandey, k.c., 1973, ‘studies on metacercariae of freshwater fishes of india. ii. description of a known and five unknown strigeid metacercariae from lucknow’, journal of indian zootomy 14(3), 155–166. prudhoe, s. & hussey, c.g., 1977, ‘some parasitic worms in fresh-water fishes and fish predators from the transvaal, south africa’, african zoology 12, 113–148. tamura, k., peterson, d., peterson, n., stecher, g., nei, m. & kumar, s., 2011, ‘mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods’, molecular biology and evolution 28(10), 2731–2739. http://dx.doi.org/10.1093/molbev/msr121, pmid:2736382, pmcid:pmc3203626 tkach, v., grabda-kazubska, b., pawlowski, j. & swiderski, z., 1999, ‘molecular and morphological evidences for close phylogenetic affinities of the genera macrodera, leptophallus, metaleptophallus and paralepodema (digenea: plagiorchidea)’, acta parasitologica 44, 170–179. tkach, v., pawlowski, j. & mariaux, j., 2000, ‘phylogenetic analysis of the suborder plagiorchiata (platyhelminthes: digenea) based on partial isrdna sequences’, international journal of parasitology 30, 83–93. http://dx.doi.org/10.1016/s0020-7519(99)00163-0 article information authors: kenneth hammond-aryee1 lesley s. van helden2 paul d. van helden1 affiliations: 1division of molecular biology and human genetics, sa mrc centre for tuberculosis research, dst/nrf centre of excellence for biomedical and tuberculosis research, stellenbosch university, south africa 2veterinary services, western cape government agriculture, south africa correspondence to: kenneth hammond-aryee email: kenhammond@sun.ac.za postal address: po box 241, tygerberg 8000, south africa dates: received: 01 june 2015 accepted: 26 aug. 2015 published: 03 nov. 2015 how to cite this article: hammond-aryee, k., van helden, l.s. & van helden, p.d., 2015, ‘the prevalence of antibodies to toxoplasma gondii in sheep in the western cape, south africa’, onderstepoort journal of veterinary research 82(1), art. #993, 5 pages. http://dx.doi.org/10.4102/ojvr.v82i1.993 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the prevalence of antibodies to toxoplasma gondii in sheep in the western cape, south africa in this research communication... open access • abstract • introduction • materials and methods    • study area and climate    • cat presence on the farm    • blood collection    • serological investigations    • statistical analysis • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the seroprevalence of toxoplasma gondii antibodies in a sample of 292 merino sheep farmed in a semi-intensive manner in the overberg region of the western cape, south africa, was investigated. antibody seroprevalence was determined by enzyme-linked immunosorbent assay. of the total sample, 23 sheep tested positive for t. gondii antibodies (8%; 95% ci: 4.7688–10.9846). there was no statistically significant relationship between seroprevalence and age of the sheep. the highest seroprevalence was found in sheep between 28 and 40 months old; a total of 19 sheep were seropositive by 40 months. no seropositive sheep were found in the age group between 16 and 28 months. the seroprevalence reported in this study is higher than what has previously been reported for the western cape (6%) and across south africa on average (4.7%). as sheep farming is economically significant in south africa, the presence of t. gondii amongst sheep may pose a production threat to the small-stock industry as well as to public health and food security. we therefore recommend further surveillance to identify high-risk animal populations so that local control measures can be put in place. introduction top ↑ toxoplasma gondii is an apicomplexan, obligate intracellular protozoan parasite of global importance. t. gondii infection causes the disease toxoplasmosis in humans and animals and its antibodies are known to be present in about a third of the global human population, although local and regional prevalences vary widely. toxoplasma gondii is very successful as a pathogen owing to its ability to infect almost all mammals and birds (dubey 2002). toxoplasmosis is found worldwide, but is more common at lower altitudes and in warm and humid climates. members of the family felidae are the only known definitive hosts for t. gondii. cats can become infected by feeding on prey already infected with dormant t. gondii cysts or tachyzoites and also by drinking oocyst-contaminated water. infected cats are known to shed infective oocysts in their faeces 5–12 days post ingestion of oocysts (al kappany et al. 2010; dubey 1995; elmore et al. 2010), thereby contaminating the environment and posing a risk of transmission to other species. toxoplasmosis causes substantial economic losses in sheep farming globally and was first described in ovines in 1954 (buxton et al. 2007; jones & dubey 2012). primary t. gondii infections in livestock, in particular sheep and goats, pose a health risk to these animals, as infection is known to cause abortions, stillbirths and neonatal mortalities. in the united kingdom, for example, ovine toxoplasmosis causes up to 2% of foetal losses per annum (buxton et al. 2007; innes et al. 2009). in humans and animals, t. gondii infections are acquired post natally by the ingestion of tissue cysts in partially cooked meat, infective oocysts in food or water contaminated with infected felid faeces, or handling of tissues of animals infected with tissue cysts. infection can also occur by vertical transmission from mother to foetus in humans, sheep, goats and small rodents (hill & dubey 2002; jones & dubey 2012; smith 1993). in humans, infection can also occur via blood transfusions and organ transplantation, although this is rare. infection has been known to occur via inhalation of aerosols containing infective oocysts or from contact with contaminated soils in both humans and animals. in sheep, t. gondii infection is mainly acquired post natally, as congenital infections usually lead to abortions. rarely, congenitally affected lambs are born, which can be a possible route for infection in humans (dubey & welcome 1988; williams et al. 2005). there are various risk factors reported to be associated with t. gondii infection in sheep. seroprevalence in sheep is known to increase with age and is therefore higher in ewes or rams than in lambs (dubey 2009). other risk factors for t. gondii infection in sheep include the presence of cats on farms, the nature of farming and management practices (commercial vs non-commercial; intensive, semi–intensive, free range or open), climatic conditions and geographic location, presence of surface drinking water sources and size of the farm (abu samra et al. 2007; andrade et al. 2013; mainar et al. 1996). although infected ewes do not always show symptoms of clinical toxoplasmosis, t. gondii infection has been noted as an important cause of ovine abortions in the united states and europe (dubey 2009), with seroprevalence in sheep ranging from 20.8% (huffman et al. 1981) to 73.8% (dubey & welcome 1988) in the united states. it has also been suggested that some breeds of sheep may be more susceptible to t. gondii infection than others (dubey & welcome 1988; williams et al. 2005). in south africa, seroprevalence studies have been reported in human populations (both asymptomatic and hiv and/or aids cohorts) as well as in animal populations, as reviewed by hammond-aryee, esser and van helden (2014a). however, these studies are limited and few of them are from the post-hiv era. there have been even fewer studies in animal populations and only one focused on sheep, reporting seroprevalence as 4.3% via enzyme-linked immunosorbent assay (elisa) and 5.6% via indirect fluorescent antibody (ifa) tests (abu samra et al. 2007). in that study, the western cape was also mentioned as the province with the highest consumption of mutton in south africa. the ingestion of partially cooked, undercooked or raw meat has been documented as a significant mode of t. gondii infection worldwide. studies in europe have shown that ingestion of undercooked lamb was a risk factor in the acquisition of t. gondii infection in a cohort of pregnant women (cook et al. 2000). in a us study, 50% of a cohort of 131 women who had vertically transmitted t. gondii to their infants recalled having eaten raw or uncooked mutton sometime during their pregnancies (boyer et al. 2005). as t. gondii can be transmitted to humans by the ingestion of mutton or lamb, sheep may have an important role in the epidemiology of toxoplasmosis. investigation into the presence or absence of t. gondii antibodies in a population of sheep will provide significant insight into the risk of toxoplasmosis in a particular ecosystem. the current study focused on the seroprevalence of t. gondii antibodies in a flock of sheep in south africa. it contributes to the knowledge about this important pathogen and the role of animals in the epidemiology of toxoplasmosis. materials and methods top ↑ study area and climate sheep were sampled from a farming area in bredasdorp in the overberg region of the western cape, south africa. the area has a mediterranean climate and receives about 350 mm of rain per year, most of which is in winter. december is usually associated with the lowest rainfall (< 20 mm), whereas august is associated with the highest rainfall (40 mm – 50 mm). the average midday temperatures are between 17.5 °c in winter and 26.2 °c in summer. livestock farmed in the region include mostly sheep and cattle. the sample consisted of 292 merino sheep (4 rams and 288 ewes) selected randomly from a flock of approximately 1000–1500. the sheep were farmed in a semi-intensive manner, grazing planted pastures and crop stubble. the rams were between 16 and 64 months old, whilst the ewes were between 4 and 76 months old, with the modal age group being between 4 and 16 months (figure 1). figure 1: age distribution of study sample. cat presence on the farm at the time of sampling, there were 10 domestic cats living on the farm. blood collection in may 2014, blood samples were collected from the 292 sheep via venipuncture. blood was collected into anticoagulant-containing tubes and immediately put on ice, after which it was transported to our laboratories and centrifuged at 3500 g for 5 min to isolate the plasma. the plasma was then stored at −80 °c for further analysis. serological investigations an elisa was used for the detection of igg antibodies to t. gondii in sheep plasma samples. the elisa test was performed via a commercially available enzyme immunoassay kit (idexx toxotest ab, idexx laboratories, switzerland) according to the manufacturer's instructions. briefly, thawed sheep plasma samples and controls were diluted at a ratio of 1:400 in wash buffer to prevent non-specific reactions. the diluted plasma samples were then dispensed into microtitre plates precoated with inactivated t. gondii antigen and mixed by gentle shaking. the microplate was covered with an adhesive plate cover and incubated at 37 °c for 60 min. each well was then washed three times with approximately 300 ml buffer in each wash. all residual wash solution was removed by tapping the microplate gently onto an absorbent material. bound antigen–antibody complexes were then conjugated with 100 μl peroxidase-labelled anti-ruminant igg conjugate. the plate was covered and incubated at 37 °c for 60 min. the described wash step was repeated to eliminate any residual unbound complexes. enzyme-bound complexes were visualised by adding 100 μl enzyme substrate to the wells and incubating the plate at 26 °c for 15 min. the enzyme–substrate reaction was stopped by adding 100 μl stop solution to each well of the microplate. the absorbance was read on a photometer at a wavelength of 450 nm. positive and negative controls provided in the kit were included on each microplate per batch of test samples. the optical densities of the positive control (pcx) and the samples (samplea450) were corrected by subtracting the optical density of the negative control (ncx). the samples were analysed relative to the positive and negative controls: s/p is a ratio of the cosrrected optical density of the sample to the corrected optical density of the positive control for each sample. statistical analysis a pearson chi square test was performed using sas/stat statistical analysis software (version 9.3). correlations between the serological status of sheep and their age were investigated. differences were deemed statistically significant if p ≤ 0.05. ethical considerations top ↑ ethical clearance for this study was obtained from the animal health research ethics committee of the western cape department of agriculture (ethical clearance certificate g13/89). results top ↑ of the total sample, 23 sheep (8%; 95% ci: 4.7688–10.9846) tested positive for t. gondii antibodies. figure 2 shows that distinct t. gondii antibody seroprevalence patterns were evident in the different age categories shown in figure 1. figure 2: seroprevalence of toxoplasma gondii antibodies in a sample of sheep, organised according to age. the highest seroprevalence (34%) was observed in animals between 28 and 40 months old, followed by a seroprevalence of 16.7% in the 64–76-month-old group. the seroprevalence in the 52–64-month-old group was 4.2%, whereas a seroprevalence of 2.1% was seen in both the 4–16-month-old group and the 40–52-month-old group. no seropositive sheep were observed in the 16–28-month-old group. discussion top ↑ infectious diseases, such as toxoplasmosis, have a substantial impact on animal productivity in sheep-farming regions of the world and some may remain undetected in flocks for prolonged periods, leading to unforeseen and unexplained abortions, foetal or newborn deaths and infertility. in some cases, these diseases lead to persistent or recurring infection in herds, resulting in poor reproductive output in the long term. reproductive losses resulting from these diseases are a threat to the long-term economic viability of such flocks. toxoplasma gondii antibodies have been detected in naturally exposed sheep flocks worldwide. reported seroprevalence varies from as low as 3% in pakistan (zaki 1995) to as high as 68% (deconinck et al. 1996) in the ivory coast and 95.7% in turkey (mor & arslan 2007). toxoplasma gondii seroprevalence in small ruminants has been reported from some parts of africa. hove, lind and mukaratirwa (2005) reported a seroprevalence of 67.9% in a population of sheep and goats from different parts of zimbabwe. they also reported an eightfold difference in seroprevalence between sheep from a large commercial farm (10%) and sheep reared under a communal grazing system (80%), reporting the presence of many domestic cats and a high household density as potential risk factors for t. gondii infection. in botswana, seroprevalences of between 10% (binta et al. 1998) and 30% (sharma et al. 2003) have been reported in a population of goats. in ghana, van der puije et al. (2000) reported a seroprevalence of 33.2% in sheep and 26.8% in goats, with a higher seroprevalence reported in female animals (35.8%) than in male animals (21.1%). significant differences in seroprevalence were found between breeds, age groups and ecological zones from which the animals were sampled. in uganda, bisson et al. (2000) reported a seroprevalence of 31% in a population of goats, with significantly higher seroprevalence in goats from urban areas than from rural areas. a strong positive correlation was demonstrated between age and seroprevalence. in south africa, abu samra et al. (2007) reported a seroprevalence of 5.6% using an ifa test and 4.3% according to an elisa. the mean seroprevalence in the western cape, as determined by elisa, was 6%. the authors also reported a significantly higher seroprevalence in sheep from commercial farms than in sheep from rural or informal-sector farms. a significant correlation was found between seroprevalence and the average minimum temperature. sheep that were managed in an extensive manner had a significantly lower seroprevalence (1.8%) than those managed in a semi-intensive or fully intensive system (5.3%). the seroprevalence reported from the current study (8%) is higher than the mean provincial seroprevalence reported by abu samra et al. (2007). this may be a result of the farm from which the sample was selected being a commercial farm with semi-intensive management, as both of these factors have been shown to contribute to increased risk for seroprevalence. most seropositive sheep had acquired the infection by 4 years of age. seroprevalence is known to increase with age, with 95% of susceptible ewes seroconverting by 6 years of age (dubey 2009). in our study, the highest number of seropositive sheep was between 28 and 40 months old; there was no significant relationship between seroprevalence and age of the sheep. these observations may be due to the sample being a convenience sample over which the investigator had no control. conclusion top ↑ transmission of t. gondii to humans and non-food animals is likely to be proportional to the t. gondii seroprevalence within food animal populations. there is a need to actively survey at-risk populations such as feral cats, wildlife and food animals within the ecosystem to design appropriate interventions for managing and preventing disease transmission. in africa, where poverty, poor hygiene and a high burden of hiv infection exist, t. gondii infection is likely to have substantial implications for the health and economic well-being of the people of this continent (hammond-aryee, esser & van helden 2014b). the seroprevalence reported in this study is higher than the previously reported figures for the western cape (6%) and overall in south africa (4.7%) (abu samra et al. 2007). our study was more limited than the comparative study, but our results do suggest ongoing transmission of t. gondii in livestock. cats on sheep farms may be a major risk factor for transmission of t. gondii to livestock, as suggested by a recent study in which t. gondii anti-igg antibody seroprevalence amongst feral cats was found to be 37.1% (hammond-aryee et al. in press). in south africa sheep are farmed not only for meat production but also for the production of wool. an infectious disease such as toxoplasmosis can therefore have both health and economic implications in the country. we recommend ongoing surveillance to identify high-risk animal populations so that local control measures can be put in place to prevent interspecies spread of the disease. acknowledgements top ↑ the authors acknowledge financial support from the south african medical research council: centre for tuberculosis research, department of science and technology/national research foundation centre of excellence for biomedical tuberculosis research, division of molecular biology and human genetics at the faculty of medicine and health sciences, stellenbosch university. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions p.d.v.h. (stellenbosch university), l.s.v.h. (western cape government agriculture) and k.h-a. 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pakistan’, journal of the pakistan medical association 45(1), 4–5. pmid: 7731085. article information authors: colin musara1 camille vaillant2 affiliations: 1department of preclinical veterinary studies, university of zimbabwe, zimbabwe2department of veterinary preclinical studies, university of liverpool, united kingdom correspondence to: colin musara postal address: po box mp 167, mount pleasant, zimbabwe dates: received: 04 sept. 2012 accepted: 21 dec. 2012 published: 05 mar. 2013 how to cite this article: musara, c. & vaillant, c., 2013, ‘immunohistochemical studies of the enteric nervous system and interstitial cells of cajal in the canine stomach’, onderstepoort journal of veterinary research 80(1), art. #518, 4 pages. http://dx.doi.org/10.4102/ ojvr.v80i1.518 copyright notice: © 2013. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. immunohistochemical studies of the enteric nervous system and interstitial cells of cajal in the canine stomach in this research communication... open access • abstract • introduction • materials and methods    • ethical considerations • results • discussion • conclusion • acknowledgements    • competing interests    • authors' contributions • references abstract top ↑ the distribution of interstitial cells of cajal (icc), the probable pacemakers in gastrointestinal motility, was investigated using an antigenic marker of gastric icc known as c-kit. antiserum raised against the general neuronal marker protein gene peptide 9.5 (pgp) as well as the nitrergic neuronal marker neuronal nitric oxide synthase (nnos) were used to investigate the distribution of gastric nerves. polyclonal goat anti-human c-kit was reliable in labelling icc in the stomach. two classes of icc were identified according to their distribution: icc-my distributed around the periphery of myenteric ganglia and icc-im in the circular and longitudinal muscle layers. the neuronal marker pgp was reliably consistent in revealing the density and distribution of the enteric nervous system. density of nerve fibres was higher in circular smooth muscle than in longitudinal smooth muscle. from nnos immunohistochemistry, it is evident that inhibitory (nitrergic) nerves constitute a substantial fraction of the enteric nervous system. introduction top ↑ the canine stomach is divided into three parts, namely, the fundus, corpus and antrum. the fundus, by adaptive relaxation, is responsible for storage of ingesta whereas the corpus acts in unision with the antrum to mix and grind food particles before propelling them aborally. these functions rely on an efficient gastric motor system composed of pacemaker cells, neurons and smooth muscle cells. the gastric pacemaker located in the greater curvature of the corpus sets the basic electrical rhythm upon which contraction is superimposed. the origin of electrical activity in gastric smooth muscle is not known for certain but available evidence indicates involvement of interstitial cells of cajal (icc) (hall et al. 1993; huizinga et al. 1995; sanders, don koh & ward 2006; ward et al. 1995). these are small fusiform or stellate cells with prominent nuclei and arborisations that intertwine to form a network within the gastrointestinal musculature (sanders 1996) and myenteric plexus (burns et al. 1996).regulation and integration of gastric motility is mainly by the enteric nervous system with extrinsic sympathetic and parasympathetic nerves playing a role in modulation only. motor nerves may be either excitatory or inhibitory. the interplay between these opposing actions determines gastric motility. excitatory neurons for gastric smooth muscle include those secreting acetylcholine, substance p, enkephalin and galanin whereas those releasing vasoactive intestinal polypeptide, cholecystokinin, somatostatin and neurotensin are inhibitory. recently a subpopulation of neurons producing nitric oxide has been identified in the gastrointestinal tract (moncada, palmer & higgs 1991). nitric oxide is an important mediator of relaxation of stomach smooth muscle (li & rand 1990). it is synthesised from l-arginine in a reaction catalysed by neuronal nitric oxide synthase (nnos). after synthesis, nitric oxide diffuses out to exert its biological effects via the second messenger molecule cyclic guanosine monophosphate. disturbances of motor function are common in mammalian gastrointestinal disorders (lyford et al. 2002). in the dog, for example, gastric dilatation-volvulus is an acute disease characterised by gastric retention, accumulation of gas in the stomach, gastric dilatation and twisting of the stomach about its longitudinal axis (burrows & ignazewski 1990). the objective of the present study was to investigate the normal morphology of the motor-regulating system in the canine stomach, namely icc and the enteric neurons. this would contribute towards establishing a morphological basis for diagnosis of congenital as well as degenerative motor deficits of the stomach and possibly tumours of icc. materials and methods top ↑ icc express proteins which are useful markers in immunohistochemical studies of non-living tissues. one such protein is c-kit which is involved in the growth, differentiation and migration of icc (rottapel et al. 1995). the general marker for the enteric nervous system is protein gene peptide 9.5 (krammer et al. 1993; thompson et al. 1983), which occurs in large amounts in the neurons of the vertebrate nervous system. expression of neuronal nitric oxide synthase in nitric oxide-producing neurons allows the enzyme to be used as a marker in immunohistochemical studies (bredt & snyder 1990).tissue samples from the corpus and antrum of the stomach were collected from nine dogs during surgery. the breeds of dogs included a bull mastiff, an irish setter, a jack russell, a collie and six crossbreeds. samples were collected in phosphate-buffered saline (pbs) maintained at 4 °c. each sample was divided into two parts for preparation of fixed and unfixed sections. for unfixed tissue, portions were cut from each sample under a dissecting microscope, embedded in optimal cutting temperature compound (oct, miles company) and snap-frozen in isopentone cooled with liquid nitrogen. the frozen tissue was stored in a freezer at −40 °c until further use. fixed tissue was prepared by immersing the samples for 24 h in freshly prepared 4% formaldehyde in 0.1 m sodium cacodylate buffer at ph 7.4 followed by cryopreservation in 20% sucrose-sodium cacodylate buffer for another 24 h after which samples were dissected, snap-frozen and stored as for unfixed tissue. all sections for immunohistochemistry were pre-treated with 0.3% hydrogen peroxide in pure ethanol for 20 min to block endogenous peroxidase activity (vanderwiden et al. 1996). primary and secondary antisera were diluted in pbs with 2% bovine serum albumin and 0.25% sodium azide. the horseradish peroxidase used in this study was diluted in pbs only. each experiment involved four incubations; a 24-hour incubation of samples at 4 °c with primary anti serum, followed by a one-hour incubation with biotinylated secondary antiserum at room temperature to trace sites of bound antibody. sites of bound antibody were detected by incubation with biotinylated streptavidin horseradish peroxidase for one hour at room temperature, and a final incubation in substrate (table 1) (shu & fan 1988) for 20 min at room temperature. the reaction was stopped by rinsing the sections in 0.1 m acetate buffer. table 1: composition of substrate solution for immunoperoxidase reaction. in all experiments, a negative control was run by substituting primary antibody with pbs. labelling for the neuronal marker pgp gave consistent results and was therefore used as the positive control. the procedure employed for immunohistochemistry is summarised in table 2. table 2: markers and antibodies for immunohistochemical studies of the gastric motor-regulating system. for examination, sections were counterstained by immersion in eosin for clearer morphological definition. coverslips were mounted using the resin distren xylene plasticizer, dpx (agar scientific company) after sections had been dehydrated by passing them through serial ethanol (50%, 70%, 100% and 100%) and immersion in the resin solvent xylene. all sections were examined under a standard light microscope at magnifications of 100–400. whole mounts were evaluated and photomicrographs processed at final magnifications 125 and 500. ethical considerations the study was conducted using non-living tissue obtained from surgical procedures carried out in the university of liverpool small animal hospital during routine clinical work. results of the investigation were treated in confidentiality until submission of the manuscript. results top ↑ polyclonal antiserum against c-kit gave no staining in any of the samples fixed in formaldehyde, most likely because of sensitivity of c-kit to the fixation process. however, application of goat anti-human c-kit to unfixed sections successfully labelled icc (figures 1 and 2). two classes of icc were identified according to their distribution. one class of cells, known as icc-my, was distributed around the periphery of myenteric ganglia and at the level of the myenteric plexus. these cells were fusiform in shape and had relatively few processes. the other class of icc comprised a population of stellate cells diffusely distributed in the circular and longitudinal muscle layers. figure 1: overview of the density and distribution of icc in the canine corpus. figure 2: distribution of icc-my at the periphery of myenteric ganglia in the corpus. neurons in the corpus and antrum of the stomach were consistently labelled by antibodies raised against the general neuronal marker pgp (figures 3 and 4). in all samples, coarse bundles of fibres were observed in the serosa, myenteric plexus and submucosa. nerve cell bodies were scattered between nerve bundles within the myenteric ganglia. individual nerve fibres rather than bundles were more common within the mucosa and circular as well as longitudinal muscle layers. fibre density was highest in the circular muscle layer as compared to the longitudinal muscle layer. several cell bodies were observed in the myenteric ganglia interspersed between bundles of nnos-positive fibres. nerve fibres with nnos-like immunoreactivity were also diffusely distributed throughout the circular and longitudinal muscle layers, either singly or in small groups (figure 5). figure 3: relative density of nerve fibres in the circular and longitudinal muscle layers, as well as nerve cell bodies in the myenteric ganglia. figure 4: a typical nerve bundle in the circular smooth muscle of the corpus. figure 5: normal density and distribution of nitrergic nerve fibres in the circular smooth muscle of the antrum. discussion top ↑ in the past, a number of techniques have been used in the morphological study of icc. the earliest of these procedures employed methylene blue and zinc iodide-osmic acid stains. however, these were of limited use, largely because of lack of specificity and the requirement for fresh tissue (christensen 1992). later on, electron microscopy became the method of choice for visualisation of icc (sanders 1996). recently, immunohistochemical methods that demonstrate the presence of specific proteins have become useful in the morphological identification of icc (bernadini et al. 2012). prior to the present study, no single histochemical or immunohistochemical method labelled icc in the canine stomach with sufficient accuracy and consistency. the receptor tyrosine kinase (c-kit) has been detected in icc in man (vanderwiden et al. 1996) and mice (huizinga et al. 1995), amongst other species. c-kit is a large protein of molecular weight about 160 kilodalton. in this study, icc was labelled by polyclonal goat anti-human c-kit (santa cruz biotechnology). staining for c-kit in unfixed sections of the stomach revealed an overview of the density and distribution of icc in the circular smooth muscle, myenteric ganglia and longitudinal smooth muscle at ×125 magnification. at higher magnification (×500), cell morphology and size could be clearly defined. assessment of the density, distribution and architecture of nerve fibres and cell bodies of the enteric nervous system has been used in diagnosis of neurological motor deficits such as hirschsprung’s disease in humans (cotran, kumar & robbins 1994). in the present investigation, all three morphological features were well-defined. reduction of non-adrenergic inhibitory nerves has been identified as a cause of impaired motility observed in some cases of diminished nerve fibre density (tomita, munakata & korusu 1994). since nitrergic nerves form a major component of inhibitory nerves of the gastrointestinal tract, the investigation was extended to ascertain the morphological features of nitric oxide-producing neurons in the stomach. staining for nnos revealed a large component of nitrergic nerves in the circular smooth muscle. this finding supports the growing concept that inhibitory motor control makes an important contribution to normal motor function in the gastrointestinal tract. in conclusion, observations made in the present study lend weight to the paradigm that in considering the gastro-intestinal motor system, attention should be drawn to all three (instead of just two) components, namely, smooth muscle, neurons and icc. conclusion top ↑ owing to their specificity, immunohistochemical methods cannot be readily extrapolated from one species to another. in the present study, gastric icc and enteric neurons in the dog were successfully labelled using antibodies raised against goat anti-human c-kit, pgp 9.5 and nnos. thus the density and distribution of enteric neurons and icc in the canine stomach were revealed. immunohistochemical methods are a valuable diagnostic tool in that they allow comparison of the morphology of the motor-regulating system between normal animals and those with gastric motor dysfunction. in future, application of electrophysiological techniques would be necessary to complement diagnostic effort from immunohistochemistry. acknowledgements top ↑ we would like to thank the european union for funding this research work through the uz-university of liverpool link programme. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions c.m. (university of zimbabwe) conducted all experiments and c.v. 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1southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania2central veterinary research institute, zambia 3tanzania veterinary laboratory agency, tanzania 4university of zambia, school of veterinary medicine, zambia 5southern african development community secretariat, botswana correspondence to: frank banda postal address: po box 33980, lusaka, zambia how to cite this article: banda, f., kasanga, c.j., sallu, r., sinkala, y., sinkombe, t.w., mulumba, m. et al., 2014, ‘investigation of foot-and-mouth disease outbreaks in the mbala and kazungula districts of zambia’, onderstepoort journal of veterinary research 81(2), art. #721, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.721 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. investigation of foot-and-mouth disease outbreaks in the mbala and kazungula districts of zambia in this proceedings... open access • abstract • introduction • materials and methods    • study area    • sample collection    • virus isolation and typing • results • discussion • conclusion and recommendations • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ foot-and-mouth disease (fmd) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. it is known to be endemic in zambia, with periodic outbreaks occurring in different geographical areas of the country. this study was conducted to investigate the presence of fmd virus (fmdv) in reported fmd-suspected cases in cattle from the kazungula and mbala districts of zambia. sixty epithelial tissues or oesophageal-pharyngeal (op) scrapings (probang samples) were collected from mbala (n = 51) and kazungula (n = 9) and examined for fmdv. the fmdv viral rna and serotypes were examined by realtime reverse transcription polymerase chain reaction (qrt-pcr) and antigen enzymelinked immunosorbent assay (elisa), respectively. twenty-two samples (36.7%) were positive for the fmdv genome by qrt-pcr with cycle threshold (ct) values ranging from 13 to 31. the fmdv-positive samples from epithelial tissues showed relatively higher ct values compared to those obtained from op scrapings, irrespective of geographical location. forty percent (40%; n = 4) of epithelial tissues from mbala were serotyped into sat 2 serotype by antigen elisa. kazungula samples were serotyped into sat 1. these findings indicated that mbala and kazungula districts had fmd outbreaks in 2012 that were ascribed to at least fmdv serotype sat 2 and sat 1 field strains. furthermore, regular interaction between buffalos from the mosi-o tunya park and domestic animals from surrounding areas could contribute to the occurrence of regular fmd outbreaks in kazungula, whilst the uncontrolled animal movements across borders between mbala and nsumbawanga could be responsible for disease outbreaks in mbala. in-depth molecular biological studies, including sequencing and phylogeny of the viruses, should be conducted to elucidate the complex epidemiology of fmd in zambia, thereby providing valuable information needed for the rational control strategy of fmd in zambia and neighbouring countries. introduction top ↑ foot-and-mouth disease (fmd) is the most contagious disease of mammals and has a great potential for causing severe economic loss in susceptible cloven-hoofed animals (oie terrestrial manual 2010). it is one of the most economically important infectious diseases of livestock as it can severely constrain international trade of animals and animal products (li-na et al. 2011). the agent of the disease, fmd virus (fmdv) is a single-stranded, positive-sense rna virus in the genus aphthovirus, family picornaviridae. there are seven immunologically-distinct serotypes of the virus, namely: types o, a, c, asia 1 and the south african serotypes sat 1, sat 2 and sat 3. infection with one serotype does not confer immunity against another (ferris et al. 2006). this needs to be taken into consideration when developing intervention strategies in endemic settings; it is important to take into account the characteristics of the different serotypes in different ecological systems (loth et al. 2011). further collection and analysis of samples, together with improved local epidemiological investigation into fmd outbreaks in countries in sub-saharan africa, is required to improve our understanding of the complex epidemiology of fmd in the region.foot-and-mouth disease is endemic in africa and the epidemiology of the disease is more complicated than in other parts of the world (vosloo et al. 2006), as six of the seven serotypes (o, a, c, sat 1, sat 2 and sat 3) have occurred in africa (rweyemamu et al. 2008; vosloo et al. 2002). in addition to having endemic infection by six of the seven serotypes of fmdv, serotype distribution differs between regions and intratypic variants within serotypes occur (vosloo et al. 2002). in southern africa, sat 1, sat 2 and sat 3 serotypes of fmdv are maintained by large numbers of african buffaloes (syncerus caffer), which provide a potential source of infection to domestic livestock and wild animals (vosloo & thomson 2004; thomson et al. 2003). in zambia, fmd is endemic and the first outbreak was reported in 1933 (overby & zyambo 1983; perry & hedger 1984), but its epidemiology is still unknown. data available indicate that there are three high-risk areas identified where fmd has occurred repeatedly (figure 1). rampant outbreaks of fmd are still observed in zambia (ministry of livestock and fisheries development (mlfd) 2010; knowles 2010) with the recent ones occurring in january and may 2012 in the mbala and kazungula districts, respectively. the genotypes of circulating fmdv serotypes in zambia have not been studied. furthermore, the occurrence and distribution pattern of the known serotypes in the country are not fully exploited and hence, poorly understood (mulenga, pers. comm., 2012). in this study, a panel of virological-detection assays, including antigen enzyme-linked immunosorbent assay (elisa) and rna extraction followed by one-step real time reverse transcription-polymerase chain reaction (qrt-pcr), were used. this was done in order to determine the presence of the fmd viral genome and typing for the fmdv serotype responsible for the fmd outbreaks in mbala and kazungula districts of zambia. figure 1: map of zambia showing the provinces and the areas at risk of footandmouth disease. materials and methods top ↑ study area the fmd outbreaks in cattle were suspected in january 2012 in the mbala district in the northern province and in may 2012 in the kazungula district in the southern province of zambia (figure 2 and figure 3). the mbala district is bordered with the nsumbawanga region of tanzania, whilst the kazungula district is in the zambezi basin. in both areas, there is subsistence-type of farming practice. the mbala district has a livestock population of 21 560, whilst the kazungula district has 65 125. in the kazungula distict there is greater possibility of interaction between livestock and wildlife, as most of it is in a game management area (gma). in the mbala district there is less chance of interaction between the two because the nearest game park, katavi national park, is approximately 600 km away on the tanzanian side; there are, however, reports of interaction of livestock in the border areas. figure 2: map showing the study area and the sampling sites in mbala district of the northern province of zambia. figure 3: map showing the study area and the sampling sites in kazungula district of the southern province of zambia. sample collection epithelial tissues and probang samples were collected from fmd-suspected animals. all of the 60 (100%) samples examined were from bovine species. of these, 47 (78.33%) were probang tissue samples, whilst 13 (21.66%) were epithelial tissues (three mouth tissues and 10 foot tissues). samples were collected in duplicate from mbala (n = 51) and kazungula (n = 9). areas where the samples were collected are shown in (figure 2 and figure 3). seventeen (17) of the 51 samples collected from mbala came from itokoto, which is an area with no active cases, but is in close proximity to the outbreak area. the tissue specimens were collected as described by kitching and donaldson (1987) and the probang samples were taken as described in the oie terrestrial manual (2010). the epithelial tissues were transported from the collection site to the diagnostic laboratory in 0.04 m phosphate buffer (ph 7.2 – ph 7.6) with 50% glycerol at 4 °c. the probang samples were placed in transport fluid (composed of 0.08 m phosphate buffer containing 0.010% bovine serum albumin, 0.002% phenol red and antibiotics) and adjusted to ph 7.2 (oie terrestrial manual 2010). all specimens were packaged as described elsewhere (kitching & donaldson 1987) and shipped to the tanzania veterinary laboratory agency (tvla); copies were also submitted to the regional reference laboratory of the botswana vaccine institute (bvi). virus isolation and typing epithelium samples were ground in phosphate buffered saline (pbs) using sand and a pestle and mortar to give a 10% suspension. these suspensions were tested in an antigen-detection elisa (roeder & le blanc smith 1987) both at tvla and bvi. total rna was extracted from the ground tissue suspension and the oesophageal-pharyngeal (op) samples using qiaamp viral rna kit (qiagen, germany) following manufacturer’s instructions. to determine the presence of fmd viral rna, samples were also tested by one-step qrt-pcrs targeting the 3d regions of the fmdv genome (callahan et al. 2002) using an agpath-id one-step qrt-pcr kit (applied biosystems, usa) (p/n am1005, 4387424, 4388519) according to manufactures instructions. a 7500 fast real-time pcr system machine (applied biosystems, usa) was used. at bvi, virus isolation was performed on two samples submitted; there was no virus isolated on the first and second passage. pcr, followed by sequencing, was performed later. results top ↑ out of a total of 257 cattle that were physically examined in both areas, 33 (12.8%) animals showed clinical signs and lesions suggestive of fmd. the principal clinical signs were salivation and lameness. mouth lesions consisted of erosions and ulcers, which were mainly on the tongue and dental pad. foot lesions comprised of erosions on the interdigital spaces and the coronary bands. on the latter, the lesions were so severe that the hoof tended to separate from the coronary band. the most-affected cattle showed foot lesions and, in most cases, healing mouth lesions where observed. no deaths were recorded during the outbreak in both districts.using a positive cut-off cycle threshold (ct) value of 32.0 (shaw et al. 2007), 22 (36.7%) samples were positive for fmdv genome by qrt-pcr (figure 4), with ct values ranging from 13 to 31. after repeat testing, 38 (63.3%) samples still showed ct values of ‘no ct’; hence, they were considered to be no virus detected (nvd) and where excluded from further study. all the 13 (100%) of the epithelial tissues examined came out positive. the lowest ct value of 13 was recorded from a sample from the kazungula district, whilst the highest was a sample examined from the mbala district, with a ct value of 30. no virus was detected in all 17 samples collected from the itokoto village, an area with no active cases of fmd. in general, samples collected from the kazungula district showed lower ct values than those collected from the mbala district. the ct values ranged between 13 and 18, indicating higher levels of viral rna in the samples collected from the kazungula district than those collected from the mbala district (table 1). lower ct values were observed for most epithelial tissues than those from the probang tissues, indicating that there were higher levels of viral rna in the epithelial tissues than in the op samples. at tvla, four of the 13 epithelial samples from mbala demonstrated fmdv sat 2 antigens by elisa. at bvi, fmdv sat 1 was demonstrated from the kazungula samples and fmdv sat 2 from the mbala samples. history of previous vaccinations with serotype o in some areas was recorded in the mbala district, whilst in the kazungula district there was no history of vaccinations against fmdv. table 1: description of samples that were used in this study detailing real time reverse transcriptance polymerase chain reaction and antigen elisa results. figure 4: graph showing amplification plot for one step real-time polymerase chain reaction. discussion top ↑ longjam et al. (2011) stated that for effective control of fmd, outbreaks should be detected at an early stage and persistent infections should be recognised to prevent further transmittance. the observation of high ct values from samples in both outbreaks and clinical signs of fmd, coupled with the confirmation of sat 2 from the mbala samples and sat 1 from the kazungula samples at both tvla and bvi, confirmed that the majority of the animals in these herds had been infected by fmdv. due to the foot lesions characteristic of fmd, some animals were observed as having difficulties in moving; hunter (1997) also observed this. using a positive cut-off ct value of 32.0 (shaw et al. 2007), it was noted that only eight of the 47 (17.02%) probang samples examined had ct values below 32.0. this is contrary to the epithelial tissues, which showed a 100% positivity rate. it must be noted that the fmdv genome-positive samples from the epithelial tissues showed relatively higher ct values compared to those obtained from the op scraping; this was irrespective of geographical location. this could be attributed to the fact that there was much higher replication of the virus in the epithelial tissues compared to the op samples; hence, more viral rna was recovered from the epithelial cells than the op cells. this could also be because epithelial tissues were from active clinical cases, whilst op samples were from those recovering or from non-clinical cases. the one-step qrt-pcr generally showed much higher ct values for the kazungula samples than for the mbala samples (table 1). one of the contributing factors to this difference could be that different serotypes of fmdv could be responsible for the outbreaks in the two regions. furthermore, samples from mbala were collected in january 2012 and were subjected to longer storage time before being finally tested, as compared to the ones from kazungula, which were only stored for two weeks before being examined. the storage facilities at central veterinary research institute (cvri) were also questionable in terms of maintaining the cold chain due to frequent power cuts. since samples were transported from zambia to tanzania by road, the maintenance of cold chain was also questionable, despite the fact that dry ice was used, as it took almost 36 h for samples to arrive at the laboratory. these facts were also found to be true by mwiine et al. (2009), who reported that the low rate of virus, despite healing lesions suggestive of fmdv, was probably due to long transportation times for samples and difficulties in maintaining the cold chain during both transportation and storage. it is worthwhile to note also that all the op samples collected in the areas with no active cases of fmd were deemed to be no virus detected (nvd) by one-step qrt-pcr. this was a good negative control for the study and also showed the extent of the outbreaks. during the investigation of the outbreak in the kazungula district, a discussion with local people and wildlife officials in the area revealed that each year during the rainy season a herd of buffalo moves up into areas where there is human habitation and, at times, mingles with local herds of cattle. these buffalo, however, return to the game park after the rainy season. the fence that was in the kazungula district and was meant to separate wildlife and domestic animals has since been vandalised and is no longer standing. this leads to free interaction of cattle and wildlife from the nearby mosi-o tunya national park. in this outbreak, however, no buffalo were observed interacting with cattle. furthermore, vaccinations where not conducted in some camps within the district. it is known that buffalo are important for their role in maintaining fmd infection and their ability of infecting other susceptible species in sub-saharan africa (thomson & bastos 2004; thomson et al. 2003). they have been shown to be the source of infection for impala and domestic animals in proximity of the kruger national park (knp) and other game parks in southern africa (thomson & bastos 2004). in the mbala district, previous outbreaks of fmd have been believed to be extensions of outbreaks in the sumbawanga district of tanzania, which borders the mbala district of zambia (overby & zyambo 1983; j. banda, pers. comm., 2012). despite the fact that there is no game park with herds of buffalo in the mbala district, katavi national park in the sumbawanga region of tanzania is known to house a number of buffalo and these could be speculated to be the sources of the fmd outbreaks in this region. furthermore, during the outbreak investigation, a discussion with the local people revealed that, fortnightly, there are organised local markets (commonly known as munanda) in the sumbawanga district and at these markets cattle fetch a much lower price compared to the price of cattle in zambia. villagers also revealed that cattle traders usually pass by the villages on their way home from these markets with their animals and at times ask for shelter to rest for a night or two. during this time, the traders’ animals interact with the local animals. intermarriages are also common on the borders and cattle are usually used for payment of the bride. this entails movement of animals from one family to another. in addition, pockets of farmers deliberately do not vaccinate their animals. the sat 2 fmdv outbreaks in mbala could be extensions from tanzania, which has previously reported high incidences of fmd outbreaks along the borders (picado et al. 2010). however, it should also be noted that there is a possibility of having more fmdv serotypes circulating in the area and that they could not be detected during this study. previous reports (knowles 2010) have indicated outbreaks of fmdv sat 1 in 2009 and fmdv serotype o in 2010 in the same areas. conclusion and recommendations top ↑ this study has detected the fmdv genome in the samples collected from both the mbala and kazungula districts. furthermore, four (40%) out of the total of 10 epithelial samples examined from the mbala district using antigen elisa at tvla, showed high prevalence of antigens to fmdv serotypes sat 2; this was also confirmed by results obtained from bvi. the study also detected fmdv sat1 antigens from the kazungula samples. it can thus be concluded that zambia had fmd outbreaks in both the kazungula and mbala districts in 2012. vosloo et al. (2003) stated that current outbreaks of fmd should be researched in order to ensure that vaccine strains could be appropriately matched against the strains currently in the field. it was with this background that this study was conducted. this study could not conclude the sources of these outbreaks, although information on the ground indicated that they could be from buffalo in the kazungula district. this would be due to the regular interaction between buffalo from the mosi-o tunya national park and the domestic animals from the surrounding areas. cross-border movements of animals between the mbala district and the sumbawanga region also play a major role in the fmd outbreaks in these areas. thus, there is a need to conduct a phylogenetic analysis of the sat2 outbreak in the mbala district in order to infer the genetic relationship of viruses circulating in the two regions. it is also recommended that cattle owners who take their livestock to graze in national parks should be sensitised on the potential risk of transmission of fmdv from cattle to wildlife. restriction of animal movement must be enforced to minimise further trans-boundary transmission of the disease. this could be done by erecting cordon lines, setting up more veterinary camps and increasing the number of veterinary assistants (va) in fmd high-risk areas, as the areas are too vast for the number of vas currently manning them. because of the porous borders, a regional fmd control strategy should be developed. furthermore, animal identification and the monitoring of animal movements are necessary to identify the cross-border movements and market chain interactions of ruminants; this would lead to improved border and movement controls. stricter adherence to protocols for the issuance of movement permits should also be followed. it also recommended that in order to characterise the circulating fmdv serotypes, more systematic epidemiological and molecular studies be carried out. these studies should be done not only in cattle but also in small ruminants and buffalo to ascertain their roles in the regular fmd outbreaks in zambia. therefore, for zambia to properly control fmd there is a need to invest in scientifically valid epidemiological investigations in order to help devise a national strategic plan. this would include re-evaluation of the vaccines used and the role of wildlife animals in the transmission and maintenance of fmd. acknowledgements top ↑ this study was supported by the southern africa centre for infectious disease surveillance (sacids) and the sadc–tads project in zambia. the authors would like to thank george thobokwe for virus isolation and typing and milner mukubwali for digitising maps of the kazungula and mbala districts. this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions f.b. (sokoine university of agriculture and central veterinary research institute, zambia) prepared the manuscript, collected samples, performed most of the experiments and edited the manuscript. c.j.k. (sokoine university of agriculture) undertook the experimental design and edited the manuscript. r.s. (tanzania veterinary laboratory agency) performed some laboratory work. y.s. (university of zambia) collected samples and proofread the manuscript. t.w.s. (central veterinary research institute, zambia) prepared the samples. m.m. (southern african development community secretariat, botswana) proofread the manuscript and designed the project. m.m.r. (sokoine university of agriculture) designed the project. p.n.w. (sokoine university of agriculture) offered mentorship and helped to prepare the manuscript. references top ↑ callahan, j.d., brown, f. & osorio, f.a., 2002, ‘use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus’, journal of the american veterinary medical association 220(11),1636–1642.  http://dx.doi.org/10.2460/javma.2002.220.1636 ferris, n.p., king, d.p., reid, s.m., hutchings, g.h., shawa, a.e., paton, d.j et al. 2006, ‘foot-and-mouth disease virus: a first inter-laboratory comparison trial to evaluate virus isolation and rt-pcr detection methods’, veterinary microbiology 117, 130–140. http://dx.doi.org/10.1016/j.vetmic.2006.06.001 hunter, p., 1997, ‘vaccination as a means of control of foot and mouth disease in sub-saharan africa’, vaccine 16(2/3), 261–264. knowles, n., 2010, ‘fao world reference laboratory for fmd’, molecular epidemiology reports, viewed on 22 june 2011, from http://www.wrlfmd.org/fmd_genotyping/africa/zam.htm kitching, r.p. & donaldson a.i., 1987, ‘collection and transportation of specimens for vesicular virus investigation’, revise science techniques office international epizotics 6, 263–272. li-na, m., jie, z., hao-tai, c., jian-hua, z., yao-zhong, d. & yong-sheng, l., 2011, ‘an overview on elisa techniques for fmd’, virology journal 8, 419. http://dx.doi.org/10.1186/1743-422x-8-419 longjam, n., deb, r., sarmah, a.k., tayo, t., awachat, v.b. & saxena, v.k., 2011, ‘a brief review on diagnosis of foot-and-mouth disease of livestock: conventional to molecular tools’, veterinary medicine international 2011, 905768. http://dx.doi.org/10.4061/2011/905768 loth, l., osmani, m.g., kalam, m.a., chakraborty, r.k., wadsworth, j., knowles, n.j., hammond, j.m. & benigno, c., 2011, ‘molecular characterization of foot-and-mouth disease virus: implications for disease control in bangladesh’, transboundary and emerging diseases 58, 240–246. http://dx.doi.org/10.1111/j.1865-1682.2011.01206.x mweene, a.s., pandey, g.s., sinyangwe, p., nambota, a., samui, k. & kida, h., 1996, ‘viral diseases of livestock in zambia’, japanese journal of veterinary research 44(2), 89–105. mwiine, f.n., ayebazibwe, c., olaho-mukani, w., alexandersen, s., balinda, s.n., masembe, c., et al., 2009, ‘serotype specificity of antibodies against foot-and-mouth disease virus in cattle in selected districts in uganda’, transboundary and emerging diseases 57(5), 365–74. http://dx.doi.org/10.1111/j.1865-1682.2010.01157.x office international des epizooties (oie), 2010, ‘foot-and-mouth disease’, in manual of standards for diagnostic tests and vaccines for terrestrial animals, viewed on 10 july 2011, from www.oie.int/manual-of-diagnostic-tests-and-vaccines-for-terrestrial-animals. overby, e. & zyambo, g.g.n., 1983, ‘foot and mouth disease outbreaks in zambia’, review science technical office of international epizootics 2(1), 189–197. perry, b.d. & hedger, r.s., 1984, ‘history and epidemiology of foot and mouth disease in zambia: a review’, tropical animal health production 16, 107–114. http://dx.doi.org/10.1007/bf02239854 picado, a., speybroeck, n., kivaria, f., mosha, r.m., sumaye, r.d., casal, j. & berkvens, d., 2010, ‘foot-and-mouth disease in tanzania from 2001 to 2006’, transboundary and emerging diseases, 865–1682. roeder, p.l., le blanc, r. & smith, p.m., 1987, ‘the detection and typing of foot-and-mouth disease virus by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis’, research in veterinary science 43, 225–232. rweyemamu, m., roeder, p., mackay, d., sumption, k., brownlie, j. & leforban, y., 2008, ‘planning for the progressive control of foot-and-mouth disease worldwide’, transboundary and emerging diseases 55, 1 73–87. shaw, a. e., reid, s.m., ebert, k., hutchings, g.h., ferris, n.p. & king, d.p., 2007, ‘implementation of one step real-time rt-pcr protocol for diagnosis of foot-and-mouth disease’, journal of virological methods 143, 81–85. http://dx.doi.org/10.1016/j.jviromet.2007.02.009 thomson, g.r. & bastos, a.d.s., 2004, in coetzer, j.a.w., tustin, r.c. (eds.), ‘foot-and-mouth disease’, infectious diseases of livestock, 2nd edn., oxford university press, south africa, pp. 1324–1365. thomson, g.r., vosloo, w. & bastos, a.d., 2003, ‘foot and mouth disease in wildlife’, virus research 91, 145/161. vosloo, w., bastos, a.d.s. & boshoff, c.i., 2006, ‘retrospective genetic analysis of sat-1 type foot-and-mouth disease outbreaks in southern africa’, archives of virology 151, 285–298. vosloo, w., bastos, a.d.s., sahle, m., sangare, o. & dwarka, r.m., 2003, ‘virus topotypes and the role of wildlife in foot and mouth disease in africa’, in proceedings of the southern and east african experts panel on designing successful conservation and development interventions at the wildlife/livestock interface: implications for wildlife, livestock and human health, ahead (animal health for the environment and development) forum, iucn vth world parks congress (steven a. osofsky) 14th and 15th september 2003, durban, south africa, p. 24. vosloo, w., bastos, a.d., sangare, o., hargreaves, s.k. & thomson, g.r., 2002, ‘review of the status and control of foot and mouth disease in sub-saharan africa’, review science technical 21, 437–449. http://dx.doi.org/10.1007/s00705-005-0629-3 vosloo, w. & thomson, g.r., 2004, ‘natural habitats in which foot-and-mouth disease viruses are maintained’, in e. domingo & f. sobrino (eds.), foot-and-mouth disease: current perspectives, horizon bioscience, great britain. ministry of livestock and fisheries, 2010, ministry of livestock and fisheries annual report, p. 22, government printer, lusaka, zambia. thomson_129-134.indd introduction it is well known that sub-saharan africa (s-sa) is one of the least developed regions of the world and it therefore follows that more effective development is the most urgent requirement of the subcontinent today. the fundamental question facing the veterinary fraternity is: what can we contribute to addres sing the development issue of our region? it needs to be remembered that about 150 million of the world’s 800 million rural poor live in s-sa. that defines the extent of the problem facing us and it is undeniable that not enough is being done to address the problem. on the other hand, what needs to be done to correct this situation and how to achieve it are much more difficult to establish. the reasons for that being so in the context of animal disease, together with alternative and more appropriate approaches, will be proposed here. our subcontinent has many natural and abundant natural resources, one of the most fundamentally valuable being our wildlife heritage. unfortunately, in many regions of s-sa this heritage is a pitiful remnant of what it once was but, in parts of eastern and southern africa, achievements in the field of conser129 onderstepoort journal of veterinary research, 76:129–134 (2009) currently important animal disease management issues in sub-saharan africa g.r. thomson sadc foot and mouth disease project, private bag 0095, gaborone, botswana abstract thomson, g.r. 2009. currently important animal disease management issues in sub-saharan africa. onderstepoort journal of veterinary research, 76:129–134 the present international approach to management of transboundary animal diseases (tads) is based on the assumption that most can be eradicated; consequently, that is the usual objective adopted by international organizations concerned with animal health. however, for sub-saharan africa and southern africa more particularly, eradication of most tads is impossible for the foreseeable future for a variety of technical, financial and logistical reasons. compounding this, the present basis for access to international markets for products derived from animals requires that the area of origin (country or zone) is free from trade-influencing tads. the ongoing development of transfrontier conservation areas (tfcas), extending across huge areas of southern africa, therefore presents a development conundrum because it makes creation of geographic areas free from tads more difficult and brings development based on wildlife conservation on the one hand and that based on livestock production on the other into sharp conflict. sub-saharan africa is consequently confronted by a complex problem that contributes significantly to retarded rural development which, in turn, impedes poverty alleviation. in southern africa specifically, foot-and-mouth disease (fmd) presents the greatest problem in relation to access to international markets for animal products. however, it is argued that this problem could be overcome by a combination between (1) implementation of a commodity-based approach to trade in products derived from animals and (2) amendment of the international standards for fmd specifically (i.e. the fmd chapter in the terrestrial animal health code of the world organisation for animal health [oie]) so that occurrence of sat serotype viruses in free-living african buffalo need not necessarily mean exclusion of areas where buffalo occur from international markets for animal products. this would overcome a presently intractable constraint to market access for southern african countries and enable conservation and livestock production to be more effectively integrated, to the benefit of both. 130 animal disease management issues in sub-saharan africa vation have been remarkable. the transfrontier conservation area (tfca)—defined as “the area or com ponent of a large ecological region that straddles the boundaries of two or more countries encompassing one or more protected areas as well as multiple land-use areas”—initiative is an example of new ambitious ideas to conserve this resource, the objective in this case being to develop a large number of interconnected conservation areas that straddle the borders of many countries (because wilderness areas are largely located at the borders of countries in the region). ultimately, it is hoped that in southern africa alone about 120 million hectares will be covered by 13 tfcas (bengis 2005) although up to 20 have been proposed. there is a problem in relation to tfcas, however, that few conservationists acknowl edge, i.e. how to achieve economic sustainability of these many large tfcas? it is frequently glibly stated that tourism will provide the economic base for tfcas without credible argument being supplied to support the viability of that idea. it is intuitively difficult to imagine that tourism alone will provide economic sustenance to millions of people—an estimated 1.2 million live within the borders of the kavango-zambesi (kaza) tfca alone—covering such an extensive area. furthermore, the needs and livelihood traditions of people living within or close to tfcas have often been overlooked, creating an inherently unstable political environment (wol mer 2007). it seems obvious therefore that only a diversity of livelihood opportunities could hope to be adequate in relation to the future economic sustainability of tfcas. an unfortunate accompaniment of our wildlife heritage is a number of diseases of livestock—maintained and sometimes spread by wildlife—that have been a major trial for livestock farmers’ for centuries, although indigenous livestock have managed to adapt to some of them to a variable degree (bengis, kock, thomson & bigalke 2004). such diseases, e.g. those caused by the sat serotypes of foot-andmouth disease (fmd) virus, african horsesickness, african swine fever, lumpy skin disease, rift valley fever (there is a long list), can be devastating to livestock (some are also zoonotic) and are therefore greatly feared in developed parts of the world. this has simply resulted in prohibition of imports of animals or products derived from animals from s-sa to developed parts of the world. trade bans and resulting limitation of economic activity within the livestock sector are among the reasons why livestock farming in most of s-sa has not come close to levels of production that potentially exist. as a consequence, countries in s-sa generally and southern africa more specifically, where livestock production is generally more commercialized, are faced with a conundrum: conser va tion of our unique wildlife heritage will increasingly be penalized by limited access to international markets for commodities derived from livestock. this, paradoxically, also includes commodities that could be derived from wildlife through consumptive utilization. problems associated with present approaches to the management of infectious animal diseases in sub-saharan africa the fear of transboundary animal diseases (tads— diseases that are able to spread quickly and affect a large number of animals over a wide geographic area in a short period of time), together with insistence that imports of animal products are derived from locations where tads do not occur, have resulted in distorted policies in respect of disease man agement and trade in animal products. inter national organisations, supported by aid programmes have, for example, focused on attempts aimed at eventual eradication of animal diseases (sometimes euphemistically referred to as ‘progressive control’). however, with few exceptions, eradication of these diseases is currently impossible because they are maintained by free-living mammals and/or arthropods. technologies to achieve eradication in such situations simply do not exist. the international veterinary fraternity, furthermore, seems to have shortsightedly espoused the medical approach to highly contagious human diseases such as smallpox, polio and measles, i.e. initiatives aimed at eradication based on mass vaccination and requiring enormous financial resources. it is true that in the case of rinderpest eradication has probably been achieved after many years of effort and at great cost but there are technical reasons why this approach was successful in this instance but unlikely to be similarly successful in most other cases. quite simply, the vaccine used against rinderpest in the last several decades is unparalleled in terms of efficacy and affordability. it is furthermore clear in retrospect that wildlife were not able to sustain the infection once it disappeared from cattle populations, despite predictions to the contrary (rossiter 2004). diseases such as fmd and contagious bovine pleuropneumonia (cbpp) have been touted as the next appropriate candidates for eradication. in the case of cbpp (where wildlife are not directly involved) there 131 g.r. thomson are reasons why, with presently available vaccine and strategies, this is doomed to failure (thomson 2005). for the sat serotypes of fmd the prospect for eradication in the foreseeable future approaches zero because sat serotypes of fmd have evolved in a unique relationship with african buffalo (syncerus caffer) populations (thomson & bastos 2004) and, therefore, eradication could only be achieved by extermination of buffalo populations or development of a mechanism that would free buffalo from infection with sat serotypes. neither option is presently possible on the scale required for ethical, logistical and financial reasons. the fact is that the epidemiologies of most tads, not to mention performance characteristics of vaccines and other control mechanisms, preclude any realistic prospect of success for eradication. consequently, to continue down this well-trodden path makes little sense. rather, while we await technologies that will make it possible to for us to eliminate all or some tads, it makes more sense to focus efforts on managing the impact of tads because we will have to continue to live with most of them for years to come. the reflex ‘stamp out’ reaction to outbreaks of tads is another consequence of the fixation with freedom from tads. the net result is that ‘stamping out’ (slaughter and disposal of all animals involved in tads outbreaks, including in-contact animals) often results in large numbers of animals being destroyed with the consequence that the effects of human intervention frequently far outweigh the effects of the disease. in the process the livelihoods of many poor people may be destroyed with little overall benefit to the disease situation (penrith & thomson 2004; scoones & foster 2007). it is argued here that the ingrained—but essentially irrational—idea that access to international markets for animal products can only be achieved through geographic freedom from tads is in urgent need of correction. if that could be achieved prospects for future rural development in s-sa would improve remarkably. managing the impacts of tads it needs to be acknowledged that, certainly in southern africa, tads have been managed pragmatically and largely successfully in the past. the remarkable technical achievements of various components of ‘onderstepoort’ over the last 100 years attest to this. however, a different type of problem confronts us today—the need to apply available technologies to manage the impacts of tads in circumstances where the diseases themselves cannot be eliminated or effectively controlled. in the past, management of animal disease was seen as an end in itself and the veterinary fraternity concerned itself very little with issues such as socio-economic development of rural areas or the environmental impact of animal disease management practices. these, how ever, are the modern imperatives which we need to help address, failing which our branch of science will become increasingly irrelevant or, more likely, usurped by others. the animal disease which, in southern africa, has had the greatest impact on trade in livestock commodities (animals and products derived from animals) is fmd; other tads may occasionally be problematic but are not really significant at present. the underlying problem is that most healthy pop ulations of african buffalo harbour sat serotypes of fmd virus on a continuous basis (thomson & bas tos 2004). this means that only localities where buffalo do not occur (or alternatively those few where the buffalo are free from infection with fmd virus) qualify for recognition as being free from fmd. this situation will obviously be worsened in future by the expansion of tfcas because the inevitable consequence of their establishment will be that numbers and distribution of buffalo will expand. international trade in commodities derived from livestock when it comes to access to international markets for food products there are two sets of barriers that may preclude access for imported products; tariff and non-tariff barriers. the world trade organisation (wto) was created by the un to ensure that trade should be as free as possible without endangering the people, animals or plant life (or the environment generally) of the importing country. international guidelines and standards for ensuring this are the responsibility of the wto’s various standard-setting bodies. non-tariff barriers are addressed by two agreements: the agreement on sanitary and phytosanitary measures (sps agreement) and the agreement on technical barriers to trade (tbt agreement), the latter mainly concerning product specification, packaging and labelling. the sps agreement deals with standards for human food products and animal and plant diseases, i.e. measures to prevent their spread through trade. standards for these issues are set by three separate international stand132 animal disease management issues in sub-saharan africa ard-setting bodies, viz. the codex alimentarius commission, the world organisation for animal health (oie) and the international plant protection convention (ippc), respectively. for so-called terrestrial animals (essentially mammals, birds and insects such as bees), the international standards are set by the oie and contained in the terrestrial animal health code (tahc). in effect, standards for trade in products that contain animal tissues or secretions are governed by the codex alimentarius (i.e. standards set by the commission) and the oie’s tahc. however, the ground rules for these two sets of standards differ; those of the codex alimentarius define standards for the products themselves whereas the tahc is based on the fundamental presumption that safe products can basically only be derived from countries, zones (regions of a country) or compartments where important tads do not occur. thus compliance with tahc requirements requires demonstration that the locality of production (country, zone or compartment) is free from tads that influence trade. this creates an enormous problem for many developing countries, especially those in s-sa, for reasons explained above. fundamentally, the question is, is this fair and reasonable or might there be an alternative? commodity-based trade to overcome this problem a different basis for approaching trade in animal commodities was proposed. this is based on the fact that some commodities do not pose a significant risk of transmitting tads because the causative agent is not present in those commodities or at least in amounts which enable transmission. so, for example, bovine milk does not contain the agent that causes bovine spongiform encephalopathy (bse), even in cows suffering from the diseases (oie 2008). furthermore, it has been known for millennia that processing of products produced from healthy animals—a fundamental departure point of human food safety is that only products from healthy animals may enter the human food chain—can decisively reduce the potential of products to transmit both human and animal pathogens which may inadvertently be present. on this basis, it was argued that a commodity-based approach could safely overcome the need to prove geographic freedom from tads as the basis for access to regulated, high-value markets for products derived from animals (thomson, tambi, hargreaves, leyland, catley, van’t klooster & penrith 2004). the oie, after initial opposition, has accepted the principle of commodity-based trade (cbt, www.oie. int) but so far has not developed standards which would facilitate widespread application. never theless, an ad hoc group has been constituted by the oie which is presumably working on this issue. the african union (kurwijila 2007) and the southern african development community (b. hulman, personal communication 2008) have also endorsed the principles behind the cbt approach. in the meantime, ways in which cbt could be applied to particular commodities have been under investigation outside the oie. a publication soon to be published argues that beef derived from healthy cattle and from which the bones and major lymph nodes have been removed (so-called de-boned beef) holds low risk for transmission of fmd and a range of other tads-causing agents (thomson, ley land & donaldson, in press). further processing of de-boned beef would obviously lower the risk of beef containing such pathogens. the issue of how compliance with standards that will hopefully soon be forthcoming can be enforced has also been addressed. the problem is ensuring credible certification of commodities, especially those that have been processed (thomson, perry, leyland, catley, penrith & donaldson 2006). the argument advanced by that publication is that independent, third-party certification—as is increasingly being applied to international agri-business—holds consid erable advantage although the issue remains controversial. present international standards required for recognition of a country or zone (region) as being free from fmd and their effects on southern africa it is presently possible for countries that are members of the oie to apply for recognition of its territory or part thereof (zone or region) as being free from fmd (officially, the fmd status of member countries is provided in the form of a ‘list’ on the oie website (www.oie.int). in order to achieve this status, applicant countries need to prove that no outbreak of fmd has occurred in the country or zone for the last 1–2 years (depending on whether vaccination is employed as a control strategy or not) and also that both the domestic livestock and wildlife populations (i.e. all susceptible species) show no evidence of ‘circulating fmd virus’ for at least a year. 133 g.r. thomson it has been demonstrated over many years that it is possible to prevent transmission of sat viruses from buffalo to cattle by separation of buffalo and cattle populations, primarily through the use of fencing (such fencing also needs to prevent antelope crossing because they can act as intermediaries in the transmission of fmd from buffalo to cattle) (sutmoller, thomson, hargreaves, foggin & ander son 2000; vosloo, thompson, botha, bengis & thomson, in press). vaccination of cattle kept adjacent to infected buffalo populations provides further protection. it has even proven possible, in the kasane area of botswana for example, to protect cattle populations which mix freely with infected buffalo from sat infection over a 20 year period by vaccinating the cattle every 4 months (m. letshwenyo, personal communication 2008). admittedly, this system has broken down recently, probably due to the inability of the current vaccine to protect against one or more of the sat2 topotypes circulating in buffalo popu lations. it is therefore clear that the growing numbers and distribution of buffalo implicit in the development of tfcas in southern africa will increasingly constrain livestock production by limiting access for animal products manufactured in the region to international markets. this will constitute a significant obstacle to rural development in an area where it is desperately needed. could a more appropriate system be devised without increasing the risk of fmd spread through trade commodities? it is accepted by the oie that, in some circumstances, the status of domestic livestock in countries or zones in respect of tads need not necessarily be influenced by the status of wildlife populations. so, for example, the status of a country’s poultry industry need not be negatively influenced by the presence of highly pathogenic avian influenza (hpai) and newcastle disease viruses in wild birds (tahc— www.oie.int). it is argued here that this principle is equally applicable in the context of sat serotype infections in southern africa. such provision would enable countries to establish domestic animal populations demonstrably free from circulating fmd virus without necessarily requiring infected buffalo to be excluded from the country or zone. combining the above principle (dissociation of fmd presence in buffalo from the fmd status of domestic livestock) with a commodity-based approach for specific commonly traded products would facilitate trade in defined commodities produced in southern africa that present an acceptably low risk for transmission of fmd (and most other tads-causing infections) than the present system based exclusively on geographic freedom from fmd in both domestic and wild animal populations. for example and as explained above, there is good evidence that deboned beef produced from healthy cattle by conventional methods presents an acceptably low risk of spreading fmd and other tads. if such beef were produced from cattle populations proven free from fmd virus, a safer trading system than that presently in operation would prevail but, at the same time, enable wider access to international markets. as a further measure, processing of the beef (i.e. beneficiation) would provide additional safe-guards. in this way a matrix of risk-reduction measures could be applied to ensure safe trade in products derived from animals. what needs to be done to implement necessary changes implicit in this proposal for fmd? 1. the oie (perhaps through the regional commission for africa) be requested to amend chapter 8.5 of the tahc to accept the possibility that domestic livestock populations (cattle specifically) can be maintained free of infection with sat viruses even when infected buffalo are present in the country or zone, i.e. the two species can be effectively sequestered from each other using physical (e.g. fencing) and/or immunological means (vaccination). 2. development by the oie of specific standards for commonly traded products derived from animals (such a de-boned beef and its derivatives) would ensure that products prepared from healthy animals according to processes that render the risk of fmd virus (or other tads-causing agents) being inadvertently present, acceptably low (i.e. provide an appropriate level of protection as required by the wto). conclusion the basis on which animal health standards for trade in commodities derived from animals rests differs fundamentally from all other international standards applied to trade in goods and services. it is argued that there is no rational basis for the present approach to animal health standards based on the concept of geographic freedom of the area of production from important transboundary animal diseases. adoption of a commodity-based approach by the relevant international standard-setting body 134 animal disease management issues in sub-saharan africa concerned (the oie) would abrogate this dichotomy. it would also broaden access to international markets for all countries, especially those in southern africa, without necessarily increasing the risk of spreading tads. to become a reality this will require that the oie develops product-specific standards for commonly traded commodities (both primary products and those that have been processed) and also develops appropriate ways through which attainment of those standards can be reliably certified. more specifically, the current requirement for the area of production of animal products to be free from fmd creates a difficulty because transfrontier conservation areas are being created over vast areas of sub-saharan africa. this means that numbers and distribution of buffalo—the reservoirs of sat serotype infections in southern africa—will increase significantly in future. that in turn will make access to international markets for beef and other animal commodities more difficult and impede integrated rural development. proposals are therefore made for amendments to the current fmd chapter of the oie’s terrestrial animal health code that will resolve this problem. references bengis, r.g., kock, r.a., thomson, g.r. & bigalke, r.d. 2004. infectious diseases of animals in sub-saharan africa: the wildlife/livestock interface, in infectious diseases of livestock, 2nd ed., edited by j.a.w. coetzer & r.c.tustin. cape town: oxford university press. bengis, r.g. 2005. transfrontier conservation area initiatives in sub-saharan africa: some animal health challenges, in conservation and development interventions at the wildlife/ livestock interface, edited by s.a. osofsky, iucn gland: switzerland & cambridge. kurwijila, r. 2007. african union statement on livestock commodity-based trade, in a commodity-based trade approach for livestock products. london: department for international development. oie (world organisation for animal health), 2008. terrestrial animal health code, chapter 11.6. paris: office international des epizooties. www.oie.int. penrith, m-l. & thomson, g.r. 2004. special factors affecting the control of livestock diseases in sub-saharan africa, in infectious diseases of livestock, 2nd ed., edited by j.a.w. coetzer & r.c. tustin. cape town: oxford university press. rossiter, p.b. 2004. rinderpest, in infectious diseases of livestock, 2nd ed., edited by j.a.w coetzer & r.c.tustin. cape town: oxford university press. scoones, i. & foster, p. 2007. hpai and international policy processes—a scoping study. http://www.steps-centre.org/ img/leftnav_bg_2.gif. sutmoller, p., thomson, g.r., hargreaves, s.k., foggin, c.m. & anderson, e.c. 2000. the foot-and-mouth disease risk posed by african buffalo within wildlife conservancies to the cattle industry of zimbabwe. preventive veterinary medicine, 44:43–60. thomson, g.r. 2005. contagious bovine pleuropneumonia and poverty: a strategy for addressing the effects of the disease in sub-saharan africa. dfid animal health programme, centre for tropical veterinary medicine, university of edinburgh. thomson, g.r. & bastos, a.d.s. 2004. foot and mouth disease, in infectious diseases of livestock, 2nd ed., edited by j.a.w. coetzer & r.c.tustin. cape town: oxford university press. thomson, g.r., tambi, e.n., hargreaves, s.k., leyland, t.j., catley, a.p., van ‘t klooster, g.g.m. & penrith, m-l. 2004. international trade in livestock and livestock products: the need for a commodity-based approach. veterinary record, 155:429–433. thomson, g.r., perry b., leyland, t.j., catley, a.p., penrith, m-l. & donaldson, a.i. 2006. certification for regional and international trade in livestock commodities: the need to balance credibility and enterprise. veterinary record, 159:53–57. thomson, g.r., leyland, t.j. & donaldson, a.i. in press. de-boned beef—an example of a commodity for which specific standards could be developed to ensure an appropriate level of protection for international trade. transboundary and emerging diseases. vosloo, w., thompson, p.n., botha, b., bengis, r.g. & thomson, g.r. in press. longitudinal study to investigate the role of impala (aepyceros melampus) in foot and mouth disease maintenance in the kruger national park, south africa. transboundary and emerging diseases. wolmer, w. 2007. from wilderness vision to farm invasions: conservation & development in zimbabwe’s southeast lowveld. james currey: oxford/weaver: harare/african academic press: hollywood ca. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: alemnesh jufare1 nesibu awol2 fanos tadesse3 yisehak tsegaye2 birhanu hadush2 affiliations: 1alage agricultural technical vocational education training college, ethiopia 2college of veterinary medicine, mekelle university, ethiopia 3college of agricultural and veterinary medicine, addis ababa university, ethiopia correspondence to: nesibu awol email: nesibuawol@yahoo.com postal address: po box 2084, mekelle, ethiopia dates: received: 26 june 2014 accepted: 27 jan. 2015 published: 30 apr. 2015 how to cite this article: jufare, a., awol, n., tadesse, f., tsegaye, y. & hadush, b., 2015, ‘parasites of pigs in two farms with poor husbandry practices in bishoftu, ethiopia’, onderstepoort journal of veterinary research 82(1), art. #839, 5 pages. http://dx.doi.org/10.4102/ojvr.v82i1.839 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. parasites of pigs in two farms with poor husbandry practices in bishoftu, ethiopia in this original research... open access • abstract • introduction • material and methods    • study area       • study design and study animals       • sample collection and analysis    • data analysis • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ a cross-sectional study was conducted from november 2011 to april 2012 on a total of 384 pigs from two privately owned intensive farms in bishoftu, ethiopia. the objectives of the study were to identify and determine the prevalence of common parasites of pigs. for the determination of gastrointestinal (git) parasites, faecal samples were collected from the study animals and subjected to standard parasitological examination techniques. physical examination was conducted for the presence of skin parasitic lesions and skin scrapings were collected to determine prevalence of ectoparasites. the overall prevalence of git parasites in the pigs was 25% (96/384). examination of faecal samples revealed the ova or oocysts of four different gastrointestinal parasites, namely coccidia (12%), strongyles (5.2%), ascaris suum (4.9%) and trichuris suis (2.9%). mixed infection by at least two parasite species was observed in 3.65% (14/384) of the pigs. the only ectoparasite species identified was sarcoptes scabiei var. suis, with a prevalence of 2.6%. this study indicates that pig parasites are a major problem in the study area, hence implementation of strategic control measures and appropriate hygienic management systems are recommended to reduce the prevalence of parasites. introduction top ↑ pig production is the fastest growing livestock sector worldwide and this trend is expected to continue over the coming years. it is believed to mitigate the deficiency of animal protein and considered a tool to fight poverty in the tropics (food and agriculture organization of the united nations [fao] 2012). over the past decade, rearing of domestic pigs has become an increasingly popular livestock strategy in east africa (food and agriculture organization statistics division [faostat] 2005; phiri et al. 2003). the lack of grazing land for ruminants and the recognition by farmers of quicker and higher returns on their investment have contributed to an increased interest in pig production (phiri et al. 2003; serres 2001). pigs have lower social prestige than cattle, but they are inexpensive to raise and are therefore a popular option for resource-poor farmers (dewey et al. 2011). moreover, the high fecundity and feed conversion efficiency, early maturity, short generation interval, relatively small space requirement and ability to produce maximally under varied management conditions are some of the advantages of pig production compared to other livestock rearing (lekule & kyvsgaard 2003). the growth in pig production plays an important role in contributing to national gross domestic product and general economic growth by providing an additional animal protein source for human consumption, generating employment and reducing poverty (oluwafemi 2008). pig production in ethiopia is believed to be in its infant stage (hailu et al. 2014). the pig population in the country is estimated to be 29 000 heads, representing 0.1% of the african pig population (faostat 2005). so far there are no large intensive swine farms in ethiopia. small-scale pig production as an agricultural activity was only recently introduced in all parts of ethiopia and is mainly concentrated around the bishoftu area. in many rural parts of ethiopia, pig production is characterised by extensive production systems in which animals are allowed to scavenge at backyard and municipal garbage dumping sites (abdu & gashaw 2010). this extensive husbandry system coupled with poor environmental hygiene and non-selective feeding behaviour of pigs has been indicated as a major risk factor for infection of pigs with helminths and other gastrointestinal (git) parasites; therefore pigs may act as potential reservoir hosts of human git parasites (zewdneh et al. 2013). git parasitic diseases constitute an important constraint to the pig industry in most parts of the world (bakut, shinggu & nwosu 1997; onah & chiejina 1995). in the tropical and sub-tropical areas, as a result of minimum management attention given to pigs, parasitic infections in pigs are estimated to be second to african swine fever in importance and are major constraints to efficient pig production of all age groups (hale, stewart & marti 1986; permin et al. 1999; sangeeta, prasad & singh 2002). the importance of parasitic diseases in pigs is chiefly economical as subclinical infection delays the achievement of market weight because of poor feed conversion rates, growth rate and general health status (hale & stewart 1987; stewart & hale 1988). parasitic diseases also predispose pigs to concurrent infections by depressing the immunity of infected animals, whilst some can result in condemnation of organs or carcasses, causing additional losses in the pig industry (hale & stewart 1998). several studies have been conducted to determine the occurrence and economic importance of parasites in pigs and various parasite species have been identified worldwide. these studies have identified ascaris spp., trichuris spp., oesophagostomum spp., trichinella spp., strongylus spp. (caballero-hernández et al. 2004; kagira et al. 2008; nganga, karanja & mutune 2008), eimeria spp., isospora spp. and cryptosporidium spp. (nosal & eckert 2005) as the most common git parasites of pigs. the most common external parasite of pigs is sarcoptes scabiei var. suis, although in some cases they may be infested by demodex phylloides and haematopinus suis (damriyasa et al. 2004; davis & moon 1990). information on epidemiological data and economic significance of git parasites in pigs in ethiopia is scant. available information is limited to a survey covering smallholder farmers in and around holeta (abdu & gashaw 2010), burayu district (kumsa & kifle 2014) and tigray (zewdneh et al. 2013), where ascaris suum, oesophagostomum spp., s. scabiei var. suis and eimeria spp. were reported to be prevalent. information on prevalence, types of parasite and management practices helps to formulate pig husbandry and extension programmes. in addition, knowledge about parasite species can be used as baseline information to design effective parasite control measures (kumsa & kifle 2014). therefore, this study was conducted in bishoftu, ethiopia, with the objective of determining the prevalence of common parasites and risk factors for their occurrence in intensively managed small-scale private pig farms. material and methods top ↑ study area the study was conducted from november 2011 to april 2012 in bishoftu. this town is located at 9°n and 40°e in east showa zone, ada‘aliban district, oromia region, 47 km south-east of addis ababa. the altitude is about 1850 m above sea level. bishoftu experiences an average annual rainfall of 800 mm, having a bimodal pattern, with the main rainy season extending from june to september (during which 84% of the rain is expected) and the short rainy season from march to may. the mean annual minimum and maximum temperatures are 12 °c and 27 °c respectively, with an overall average of 18.7 °c. the mean relative humidity is 61.3%. it has three agro-ecological zones, namely midland (94%), highland (3%) and lowland (3%) (national metrological surveillance agency [nmsa] 2003). study design and study animals a cross-sectional study was designed to address the objective of this investigation. although two large and five small pig farms were available, only two farms’ owners were willing to participate, so these two farms were selected for inclusion. a sample size of 384 pigs was recruited for the study following the formula described by thrusfield (2005). study subjects were randomly selected regardless of their health status, age, sex and body condition. for each recruited study subject, a general physical examination was conducted and data on age, sex and body condition score were recorded on a structured data sheet. data on age of individual study subjects was retrieved from farm record books whilst animals were categorised as piglets (< 10 weeks), growers (10–16 weeks) or adult pigs (> 16 weeks), according to keshaw et al. (2009). the body condition score was determined based on assessment of fat cover on their spine and transverse spinal process (holness 1991). generally, as found by teshale (2005), the husbandry practice of the farms was not of a high standard; they were poorly managed with limited husbandry and health care practices. in addition, both farms had similar management practices and owned 1338 pigs (farm 1) and 652 pigs (farm 2) at the time of study. sample collection and analysis fresh faecal samples were collected directly from the rectum of the 384 pigs (289 from farm 1 and 95 from farm 2). the samples were placed in separate plastic containers and transported in an ice box to the parasitology laboratory of the college of agricultural and veterinary medicine, addis ababa university for immediate processing or stored in the refrigerator at 4 °c for a day before processing. the faeces were examined by the centrifugal faecal floatation technique for eggs of nematodes, cestodes and coccidian oocysts, and the sedimentation technique for the presence of trematode eggs (soulsby 1982; urquhart et al. 1996). during physical examination from pigs that had clinical skin lesions, skin scrapings were collected by scraping the edges of the lesions using scalpel blades until capillary bleeding was seen and were preserved in labelled bottles containing 10% formalin and transported to the laboratory. thereafter, they were placed for 30 min in petri dishes containing 10% potassium hydroxide. the samples were examined for the presence of mites and identification was carried out according to the standard techniques recommended by soulsby (1982) and urquhart et al. (1996). data analysis the data were entered into a microsoft excel spreadsheet and coded appropriately. for data analysis, spss version 16 was used. descriptive statistics were used to determine the prevalence of parasites in pigs. the chi-square test was used to determine the association between the infection and the risk factors such as age, body condition score and sex. in all cases, 95% confidence intervals and p < 0.05 were set for significance. results top ↑ of the total 384 pigs examined, 25% (96/384) were found to harbour one or more parasite species. mixed infection was observed in 3.65% (n = 14) of the pigs. in this study, four git parasites and one species of ectoparasite were identified. the git parasites identified were coccidia (12%), a. suum (4.9%), strongyles (5.2%) and trichuris suis (2.9%). the prevalence of mange mite infestation was 2.6% (n = 10) and only s. scabiei var suis was identified from all 10 skin scraping samples collected from the suspected lesions. the frequency of occurrence and prevalence of parasites identified in this study is summarised in table 1. table 1: prevalence of parasites identified by faecal and skin scraping examination in 384 pigs in bishoftu. the occurrence of parasitic infestation was higher in grower pigs (29.7%) than in piglets (19.9%) and adult pigs (23.1%). there was no significant difference (p > 0.05) in infection rate of git parasites amongst the different risk factors. however, the occurrence of parasites was significantly higher in farm 1 than in farm 2 (p < 0.05), with a prevalence of 31.1% in farm 1 and 15.1% in farm 2. in addition, the occurrence of s. scabiei var. suis was also significantly higher (p < 0.05) in piglets than in growers and adults. the distribution of parasites according to sex, age and body condition score of swine is listed in table 2. table 2: distribution of parasites according to farm, sex, age and body condition score of pigs in bishoftu. discussion top ↑ of the 384 pigs examined, 23.96% (n = 92) were found to harbour one or more parasite species. however, this is lower than the findings of abdu and gashaw (2010) in holeta, ethiopia, keshaw et al. (2009) in the west indies, and nganga et al. (2008) and kagira et al. (2008) in kenya, who recorded prevalences of 30.4%, 68.78%, 67.8% and 84.2%, respectively. these variations in the prevalence of pig parasitic infections could be a result of the difference in management systems, breed of pig, nutrition, climatic factors and animal health extension services in countries. the statistically significant difference (p = 0.007) in the prevalence of parasites between the two farms (31.1% for farm 1 and 15.1% for farm 2) could be ascribed to the difference in the number of samples taken from each farm. the overall prevalence of coccidia in this study was 12%, which is higher than that reported by abdu and gashaw (2010), who recorded 5.6% prevalence in and around holeta, ethiopia from semi-intensive and extensive farming systems. studies conducted by keshaw et al. (2009) in the west indies, weka and ikeh (2009) in jos metropolis, nigeria and weng et al. (2005) in china also indicated prevalences of 88%, 15.6% and 47.2%, respectively. this variation could be because of the difference in pig husbandry practices in the various study areas. several species of eimeria and isospora suis have been identified from pigs (cañon-franco, henão-agudelo & pérez-bedoya 2012; eysker et al. 1994; mundt et al. 2005). in piglets, coccidiosis causes poor performance during the fattening period as well as diarrhoea, and also predisposes the animal to secondary bacterial and viral infections (koudela & vítovec 1998; lindsay, blagburn & powe 1992; stuart et al. 1982). the prevalence of strongyle ova in this study was 5.2%. this figure is lower than those reported by kagira et al. (2008) in kenya, marufu et al. (2008) in zimbabwe and keshaw et al. (2009) in the west indies, who reported a prevalence of 37%, 14% and 44%, respectively. the most common strongyles identified were oesophagostomum spp., hyostrongylus rubidus and trichostrongylus axei (keshaw et al. 2009; marufu et al. 2008; nganga et al. 2008). these parasites are responsible for loss of appetite, poor growth rate and poor feed conversion efficiency, and also predisposes animals to other pathogens and death in pigs (stewart & hoyt 2006). ascaris suum was recovered at a prevalence of 4.9%. this is lower than those reported by abdu and gashaw (2010) in holeta and zewdneh et al. (2013) in tigrai, ethiopia, nganga et al. (2008) and kagira et al. (2008) in kenya and permin et al. (1999) in ghana. larval migration of a. suum is one of the causes of milk spot hepatic lesions in growing pigs. it also depresses weight gain by up to 40% and feed conversion efficiencies by up to 25% (polley & mostert 1980). the thick-shelled eggs of a. suum are resistant to adverse environmental factors as well as chemicals and can maintain infectivity for long periods of time (roepstorff & nansen 1998). the prevalence of t. suis in this study was 2.9%. studies conducted by zewdneh et al. (2013) in tigrai (ethiopia), kagira et al. (2008) in kenya, marufu et al. (2008) in zimbabwe, permin et al. (1999) in ghana, weng et al. (2005) in china, keshaw et al. (2009) in the west indies and nissen et al. (2011) in uganda reported a prevalence of 0.3%, 7%, 4.7%, 4.6%, 5.2%, 38% and 17%, respectively. sporadic disease caused by heavy infestation by t. suis is more common in pigs and is associated with watery diarrhoea that usually contains blood. the most important feature of trichuris spp. is the longevity of the eggs in the environment, which can be up to 3 or 4 years (urquhart et al. 1996). the overall prevalence of s. scabiei var. suis in this study was 2.6%, which was lower than that reported by abdu and gashaw (2010) in extensive and semi-intensive swine farms in and around holeta, ethiopia, where a prevalence of 16.2% was reported. there was a significant difference (p = 0.048) in the prevalence of s. scabiei var. suis amongst the age groups, with a prevalence of 5.4%, 0.8% and 1.5% in adults, growers and piglets, respectively. both sarcoptic and demodectic mange mites have been reported as the causal agent for swine mange mite infestation (soulsby 1982; urquhart et al. 1996). however, sarcoptic mange caused by s. scabiei var. suis is the most common and serious ectoparasitic problem in swine (das et al. 2010; galuppi et al. 2007). conclusion top ↑ this study clearly shows that parasitic infections are prevalent in pigs in the study area. however, the attention given to pig diseases in general and parasitic diseases in particular so far has not been sufficient. in the absence of detailed studies on parasitic diseases of pigs in ethiopia, their negative impact on pig production will continue unabated. therefore, comprehensive studies are necessary to obtain a clear epidemiological picture of pig parasitic diseases, their burden and their impact on production. in addition, appropriate control measures such as strategic application of acaricides, deworming with appropriate drugs and good sanitation should be undertaken to reduce the impact of parasitic diseases on pig health and production. acknowledgements top ↑ the authors thank the owners of swine farms for their cooperation during sample collection and the staff of the parasitology laboratory of the college of agricultural and veterinary medicine, addis ababa university for their support during sample processing. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions a.j. (alage agricultural technical vocational education training college) designed the study, conducted laboratory work and prepared the manuscript. n.a. (mekelle university), f.t. (addis ababa university), y.t. (mekelle university) and b.h. 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royal veterinary college, hawkshead lane, north mymms, hatfield, hertfordshire, al9 7ta, united kingdom how to cite this article: kock, r., 2014, ‘drivers of disease emergence and spread: is wildlife to blame?’, onderstepoort journal of veterinary research 81(2), art. #739, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.739 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. drivers of disease emergence and spread: is wildlife to blame? in this proceedings... open access • abstract • introduction • disease emergence factors and wildlife • conclusion • acknowledgements    • competing interests • references abstract top ↑ the global focus on wildlife as a major contributor to emerging pathogens and infectious diseases (eids) in humans and domestic animals is not based on field, experimental or dedicated research, but mostly on limited surveys of literature, opinion and the assumption that biodiversity harbours pathogens. the perceived and direct impacts of wildlife, from being a reservoir of certain human and livestock pathogens and as a risk to health, are frequently overstated when compared to the global burden of disease statistics available from who, oie and fao. however organisms that evolve in wildlife species can and do spill-over into human landscapes and humans and domestic animal population and, where these organisms adapt to surviving and spreading amongst livestock and humans, these emerging infections can have significant consequences. drivers for the spill-over of pathogens or evolution of organisms from wildlife reservoirs to become pathogens of humans and domestic animals are varied but almost without exception poorly researched. the changing demographics, spatial distribution and movements, associated landscape modifications (especially agricultural) and behavioural changes involving human and domestic animal populations are probably the core drivers of the apparent increasing trend in emergence of new pathogens and infectious diseases over recent decades. introduction top ↑ the global focus on wildlife as a major contributor to emerging pathogens and infectious diseases (eids) in humans and domestic animals is not based on field, experimental or dedicated research, but mostly on surveys of literature that report new diseases corrected for reporting bias (e.g. jones et al. 2008; jones et al. 2013). in addition to hotspot mapping, such publications link diseases to possible pathogen origins from various non-domestic animal species. the assessment of these reported trends in eids and their impacts is significantly biased by research regarding human immunodeficiency virus (hiv) and aids, severe acute respiratory syndrome (sars) and highly pathogenic avian influenza (hpai), all of which have an indirect wildlife link. except for aids, the aforementioned diseases have taken a relatively small toll on human life but have incurred huge costs, mostly related to fear induced by perceived pandemic threats and high case fatality rates and resulting from the syndrome’s importance being amplified by global media. political pressure has resulted in strong government responses and international investment in pathogen research and addressing the spread of these agents. however, it is surprising that little has been done so far to deal with the main drivers and amplifiers of the diseases, for example, through a shift in animal production systems or closure of key epidemiological nodes such as live or wet markets (fournié et al. 2013). the potential for similar events remains or is even more likely than before, given the ever-expanding markets and associated domestic animal production systems, which are conducive to the evolution and amplification of new pathogens. this is further enhanced by the increasing movement and contact between humans, domestic animals and wildlife (wallace & kock 2012). disease emergence factors and wildlife top ↑ reported causes for and drivers of the apparent trends in disease emergence lack, in most cases, strong evidence-based research (jones et al. 2013). however, there is circumstantial evidence to suggest that novel epidemics and disease syndromes are the result of changing agroecology and human behaviour and movements, intensifying interfaces with certain wildlife species and climate change (roche & guégan 2011). the inclusion of wildlife in the epidemiology and evolution of eids is justified (keesing et al. 2010), but the role of wildlife is often misrepresented. the idea that biodiversity is the origin of many infectious agents is stating the obvious: where else would new infectious agents come from? it is not that the ‘pathways’ for emergence are in any way unnatural; rather, it is the opportunities for emergence that are changing and probably on the increase. the most dramatic example of this is the emergence of aids from the spillover of simian immunodeficiency virus from sooty mangabey and chimpanzees into humans and the subsequent genetic changes (through mutation, recombination or reassortment) enabling the evolution of human pathogens hiv-1 and hiv-2 (gao et al. 1999). bush meat, the fragmentation of forest and the development of road systems facilitated the virus’s spread to urban centres and eventually to the global human population, causing the most significant epidemic disease in modern times. wildlife species are a reservoir of some emerging pathogens, but zoonotic transmission is uncommon and often involves a domestic animal bridge (shown in brackets), for example crimean–congo haemorrhagic fever (livestock [sang 2011]), japanese encephalitis (pigs [impoinvil et al. 2011]), nipah virus infection (pigs), hendra virus infection (horses [field 2010]), plague, trypanosomosis (livestock [funk et al. 2013]). some of these examples of emerging zoonoses are a direct result of human interference, for example, the establishment of the pig industry and fruit farms in areas with high-density bat populations in the case of nipah virus (pulliam et al. 2012). an additional driver of zoonotic emergence from wildlife is, the creation of wildlife habitats in high-density human settlements or recreational areas for example lyme disease (levi et al. 2012) in the united states of america and the slaughter and consumption of primates and other bush meat in african causes the spillover of ebola virus to humans (leroy, gonzalez & baize 2011). it is suggested that such bridges have, over time, resulted in the establishment of a range of human diseases, for example diphtheria, measles, campylobacteriosis small pox, pertussis and mumps (wolfe, dunavan & diamond 2007). the advent of modern livestock systems is also associated with diseases such as leptospirosis, cysticercosis and echinococcosis and an array of zoonotic infections by pathogens such as the japanese encephalitis virus, highly pathogenic avian influenza viruses, norovirus, hepatitis e virus, campylobacter spp., escherichia coli o157:h7 (toxin code) and the epidemic strain of salmonella enterica serotype typhimurium definitive type 104 (altekruse, cohen & swerdlow 1997; jones et al. 2013). figure 1 provides an estimate of global wildlife-related mortalities in humans as a proportion of total incidences in an average year. a log scale was used as the wildlife contribution would otherwise not appear in most cases, which is an indication of how rare they are. figure 1: global mortality estimates of infections from a wildlife source in humans as a proportion of total incidence. many wildlife species, from a wide range of taxa, act as temporary or intermediate disease hosts. they can transmit and amplify infections over space and time but are not long-term reservoirs (table 1). occasionally, wildlife species are victims of or spillover hosts from domestic animals and even humans (table 2). table 1: examples of wildlife taxa that are important hosts, carriers or vectors of human or domestic animal pathogens but not the maintenance hosts table 2: examples of wildlife taxa that are impacted by spillover infection from domestic animal or human reservoirs of disease. it is interesting that some infections are well tolerated by wild reservoir hosts, as illustrated by the association between wild birds and avian influenza viruses (alexander 2000) and bats and lyssa viruses (calisher et al. 2006), and the role of communities with respect to infective agents and hosts, transmission and their pathogenicity has recently become a prominent research topic (johnson & thieltges 2010; randolph & dobson 2012). however, when the contact between wild species and domestic animals or humans is intensified, spillover occurs with direct pathogenic impact or opportunity for evolution of these viruses in new hosts, as evidenced by the emergence of hpai and sars (tang et al. 2009). figure 2 provides a simplified model of this process. figure 2: model of pathogen evolution from the biosphere through contact between wildlife and domestic animals or humans. conclusion top ↑ in conclusion, evidence suggests that we should not see wildlife as a direct source of pathogen threats, but rather as an indirect source of candidate pathogens. a smarter solution than trying to identify cryptic potential infection from wildlife would be to address human behaviour and actions, which are driving most of the emerging disease syndromes. if science provides the evidence and critical control points, and health sectors and society engage within the developing human landscape, pathogen emergence need not be catastrophic in its impact, and humans and domestic animals can continue to co-exist with wildlife in a rich, resilient natural world. acknowledgements top ↑ competing interests the author declares that he has no financial or personal relationship(s) that may have inappropriately influenced him in writing this article. references top ↑ alexander, d.j., 2000, ‘a review of avian influenza in different bird species’, veterinary microbiology 74, 3–13.altekruse, s.f., cohen, m.l. & swerdlow, d.l., 1997, ‘emerging foodborne diseases’, emerging infectious diseases 3(3), 285–293. http://dx.doi.org/10.3201/eid0303.970304 bengis, r.g., kock, r.a. & fischer, j., 2002, ‘infectious animal diseases: the wildlife/livestock interface’, review scientifique technique office internationale des epizooties 21(1), 53–65. bermejo, m., rodríguez-teijeiro, j.d., illera, g., 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400 species of freshwater snails (including limpets) occur in africa, some of which form an important part of freshwater ecosystems for inland fisheries where they provide a valuable source of food. lymnaeid snails are the first intermediate hosts for several fasciola spp. the main snail host for the liver fluke, fasciola gigantica, is lymnaea natalensis which is widely distributed in africa (brown 1994). another study by van eeden, brown & oberholzer (1965) indicated that this species also has a wide distribution range in south africa that extends into the western cape (as far as the knysna district), kwa-zulu natal and the eastern mpumalanga provinces. surveys executed over the past 2 years in farm dams and other waterbodies around pretoria showed l. natalensis to be the most abundant freshwater snail in this area. the aim of this study was to examine different cercariae shed by l. natalensis in dams surrounding pretoria, gauteng province, as well as to gather information on other life stages within the life cycles of these parasites. materials and methods lymnaea natalensis specimens were collected over a period of 2 years using metal scoops as described 215 onderstepoort journal of veterinary research, 75:215–223 (2008) cercariae developing in lymnaea natalensis krauss, 1848 collected in the vicinity of pretoria, gauteng province, south africa e.b.e. moema1, p.h. king1* and c. baker2 abstract moema, e.b.e, king, p.h. & baker, c. 2008. cercariae developing in lymnaea natalensis krauss, 1848 collected in the vicinity of pretoria, gauteng province, south africa. onderstepoort journal of veterinary research, 75:215–223 freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis and fasciolosis. snails were collected on several occasions in the proximity of pretoria, south africa and their cercarial sheddings were studied. this article describes three different types of cercariae shed by the freshwater snail, lymnaea natalensis, viz. a fork-tailed cercaria of a trichobilharzia sp., an avian parasite belonging to the family schistosomatidae, an echinostomatid cercaria of the family echinostomatidae, also avian parasites and a xiphidiocercaria of the family plagiorchiidae which parasitise avians and amphibians. the morphology of these cercariae was studied by light and scanning electron microscopy. keywords: cercariae, lymnaea natalensis, parasites, schistosoma, snails, trichobilharzia, sp. ∗ author to whom correspondence is to be directed: e-mail: pking@ul.ac.za 1 department of biology, university of limpopo, medunsa campus, p.o. box 139, medunsa, 0204 south africa 2 electron microscope unit, university of limpopo, medunsa campus, p.o. box 84, medunsa, 0204 south africa accepted for publication 21 may 2008—editor 216 cercariae in lymnaea natalensis krauss, 1848 collected in gauteng province, south africa by van eeden (1960). snails found under water lilly leaves were also collected manually. in the laboratory they were kept in plastic containers and fed fish flakes. they were exposed individually to daylight (but not direct sunlight) to stimulate the natural shedding of cercariae. the cercariae were stained with nile blue sulphate or neutral red vital stains and studied by light microscopy while alive. drawings were made with a drawing tube and measurements were derived from these drawings. in all cases 20 specimens were measured and all measurements given in the descriptions are in micrometres (μm). minimum and maximum values are indicated with the mean value and standard deviation in brackets. no other intra-molluscan stages were collected. for scanning electron microscopy (sem), specimens were fixed overnight in 2.5 % glutaraldehyde, washed in millonig’s phosphate buffer (ph = 7.2) and dehydrated through a graded ethanol series for 30 s to 1 min in each concentration. thereafter, they were critical point dried (polaron, uk) overnight, mounted on stubs, sputter coated with gold (emscope, uk) and examined using a leica stereoscan 420 scanning electron microscope (leica electron optics, uk) at the electron microscope unit of the university of limpopo (medunsa campus). in a preliminary life cycle study, two mallard ducks and two wisstar rats were exposed to the fork-tailed cercariae. a small number of cercariae were placed with the experimental animals and left for 1 h. dissection of the animals took place 3 weeks later. results three types of cercariae were identified, namely the fork-tailed type, and two single-tailed types, one of which is an echinostome and the other a xiphidiocercaria. fork-tailed cercaria (fig. 1a–b and 2a–h) the cercaria (fig. 1a–b and 2a) comprises an oval body and long tail stem terminating in two furcal rami. the body measures 233–257 (243 + 9) long x 59–79 (63 + 7) wide. the oral sucker is protrusible, measures 61–73 (68 + 4) x 40–46 (44 + 1), and a few ciliated sensory receptors surround it (fig. 2c). prominent openings of the penetration gland ducts (fig. 2d) are also visible by sem. the oesophagus, 12–28 (21 + 4) x 2.9–4.5 (3.7 + 1), bifurcates anteriorly to the acetabulum into two intestinal caeca, 12–21 (15 + 2) x 3–6 (5 + 1), that end blindly at the mid-level of the first penetration glands. one of the most characteristic features of this cercaria is the two dark pigmented eyespots, measuring 5–6 (5.5 + 1) x 5–6 (5.5 + 1) (fig. 2b), that are situated in the mid-body bilateral to the blind-ending caeca. the acetabulum (fig. 2e), 13–18 (16 + 1) x 12–19 (16 + 2) is very protrusible and is situated 78–97 (86 + 8) from the oral sucker and 67–92 (73 + 8) from the posterior end of the body. five pairs of penetration glands (fig.1a) were observed between the eyespots and excretory bladder. the first pair is coarsely granulated, situated anteriorly to the acetabulum and measures 43–45 (44 + 1) x 22–24 (23 + 1). the remaining pairs are finely granulated and situated posteriorly to the acetabulum, measuring 19–25 (22 + 4) x 4–16 (10 + 8). the penetration ducts extend anteriorly to the oral sucker where they open exteriorly (fig. 1a). the excretory bladder is y-shaped measuring 6–13 (9 + 3) x 7–16 (10 + 3) (fig. 1a). from the bladder an excretory duct extends posteriorly into the tail that terminates in an opening on the tip of each caudal ramus (fig. 2h). the main excretory duct on either side of the bladder extends anteriorly and divides opposite to the acetabulum to form anterior and posterior collecting ducts. six pairs of flame cells were observed in the body, three occurring posteriorly and three anteriorly to the acetabulum. the flame cell formula is 2 (3 + 3) = 12. the body tegument displays many backwardly directed spines (fig. 2f and g), which decrease in number towards the posterior end of the body. the tail stem (fig. 1a) is narrower than the body and measures 341–371 (353 + 13) x 30–50 (39 + 7). the furcal rami, measuring 178–199 (154 + 7) x 15– 36 (25 + 7) (fig. 2h), is shorter than the tail stem. both the tail stem and furcal rami are covered by less and shorter backwardly directed spines (fig. 2g and h). no sensory receptors were observed on the tail tegument. remarks the cercaria is classified as a brevifurcate-apharyngeate distome cercaria according to the taxonomic key of frandsen & christensen (1984). it is placed in the family schistosomatidae and is commonly known as an avian cercaria as the adult parasites are mostly found in ducks and geese (appleton 1986). this cercaria with its characteristic schistosome morphology has one unique feature, i.e. a pair of dark pigmented eyespots, which differentiates it from human schistosome cercariae. 217 e.b.e. moema, p.h. king & c. baker 80 μm 30 μm 40 μm 10 μm 40 μm 10 μm 20 μm 20 μm a b c d e f g h fig. 1 light microscope projection drawings of three types of cercariae shed by lymnaea natalensis: an apharyngeal-brevifurcate distiome cercaria (a) head and (b) forked tail; an echinostomatid cercaria (c) head, (d) single tail and (e) collar spines; and a xiphidio cercaria (f) head, (g) stylet and (h) single tail 218 cercariae in lymnaea natalensis krauss, 1848 collected in gauteng province, south africa three weeks after infection only two spindle-shaped eggs were recovered from the faeces of one of the mallard ducks. the other duck and the rats remained uninfected. the eggs were identified as those belonging to the genus trichobilharzia according to the descriptions of avian schistosome eggs (type 3) by appleton (1982, 1986). this avian cercaria was thus identified as a trichobilharzia sp. it has been recorded that this type of cercaria is able to penetrate human skin, and in so doing causes cercarial dermatitis, as a result of an allergic response (picard & jousson 2001). since humans are not suitable hosts the cercariae do not enter the blood stream to mature but perish after penetrating the skin (cheng 1986). echinostome cercaria (fig. 1c–e and 3a–h) the cercaria (fig. 1c–e and 3a) has a large oval head and a long single tail. the body (fig. 1c and 3b) measures 232–292 (262 + 21) x 52–99 (82 + 15). the most distinctive feature of this cercaria is the horseshoe-shaped collar (fig. 1e and 3f), consisting of 27 individual spines. they are grouped into four corner spines measuring 10.5–14.9 (12.1 + 1.6) x 2.9–4.5 (3.6 + 0.7) (fig. 3e), five lateral spines, 8.5–14.9 (12.4 + 1.9) x 2.9–4.5 (3.9 + 0.7) and nine dorsal spines arranged into two alternating rows of four oral spines measuring 10.5–11.9 (11.2 + 0.8) x 2.9–4.5 (3.6 + 0.8) alternating with five aboral spines, 11.9–14.9 (13.6 + 1.3) x 2.9–4.5 (3.3 + 0.6) (fig. 3f). an oval-shaped and very protrusible oral sucker measures 36–60 (47 + 10) x 26–67 (45 + 12) at the anterior end of the body. this sucker is surrounded by many long-ciliated receptors (fig. 3c, e and g) as well as multi-ciliated (cluster) receptors (fig. 3e and g) consisting of groups with 6 to 12 short cilia. these receptors are situated mostly dorsal and dorso-laterally to the oral sucker (fig. 3g). the oral sucker extends into a small pre-pharyngeal sac measuring 16–24 (19 + 2.5) x 14–25 (21 + 2.8), filled with two large granules of 8.9–14.9 (10.8 + 1.9) in diameter (fig. 1c). it continues into a prepharynx measuring 9–15 (12 + 2) x 4–7 (6 + 1) that extends into a prominent pharynx, 11–37 (18 + 7) x 10–15 (13 + 2). this leads to an oesophagus measuring 45–82 (74 + 11) x 7–16 (10 + 3) that bifurcates to form two intestinal caeca measuring 75–135 (106 + 23) x 9–16 (12 + 3) that terminate at the posterior end of the body. the acetabulum (fig. 3d) is oval, well-developed and measures 37–63 (50 + 9) x 40– 70 (55 + 9), and is further also situated at a position 49–127 (82 + 23) posterior to the oral sucker and 45–93 (62 + 13) from the end of the body. the excretory bladder is bipartite with the most posterior part larger and oval, measuring 18–45 (32 + 9) x 15–33 (26 + 6), and preceded by a smaller oval part measuring 7–10 (9 + 1) x 19–22 (21 + 1) (fig. 1c). the main excretory ducts on either side of the bladder extend anteriorly towards the pharyngeal region where they fold back posteriorly thus forming anterior and posterior collecting ducts in the midbody. these collecting ducts branch to form capillaries ending in flame cells. eight flame cells were observed anteriorly and six posterior to the acetabulum. the flame cell formula is as follows: 2 [(2 + 1+ 2 + 2) + (1 + 1 +2 + 1 + 2)] = 28. a caudal excretory duct was also observed to extend into the anterior part of the tail stem where it branches at a quarter of the tail length into two ducts that open to the outside through two excretory openings (fig. 1c). locomotion is provided by a single muscular tail (fig. 1d) that is slightly longer than the head, measuring 297–306 (300 + 4) x 30–59 (37 + 9). it displays various long ciliated papillae arranged bilaterally on the tail stem (fig. 3h). finfolds are absent. the tegument of both the body and tail is aspinous and unevenly ridged (fig. 3f and h). remarks the cercaria is classified as an echinostome cercaria according to the keys provided by frandsen & christensen (1984). porter (1938) described an echinostomatid cercaria with a collar of 26 spines, cercaria middelburgensis that was shed by l. natalensis collected from a lake in middleburg, mpu malanga province. this cercaria differs from the description of the present cercaria in respect of size, most body structures and the number and arrangement of the collar spines. fain (1953) described cercaria bruynoghei and cercaria decora with 27 spines in the collar. these cercariae differ from the present specimen in the species of the snail host, general sizes of all structures, position of the acetabulum and flame cell formula. similarly, jansen van rensburg (2001) described a 27-spined echinostomatid cercaria from l. natalensis from the okavango delta, botswana. most body structures are similar to those of the present material but the prepharyngeal sac is absent. in contrast to porter (1938), king & van as (1997) described a 27-spined echinostomatid cercaria that was shed by bulinus tropicus from the free state province. most of the cercarial features also appear similar to 219 e.b.e. moema, p.h. king & c. baker ba dc fe hg 100 μm 40 μm 300 μm 1 μm 1 μm 300 μm 1 μm 1 μm fig. 2 apharyngeal-brevifurcate distome cercaria. light micrographs of (a) whole mount; (b) head of cercaria; (c–h) scanning electron micrographs of cercaria: (c) oral sucker (os) surrounded by sensory receptors (scr); (d) sensory receptors (scr) and penetration gland ducts (pgd); (e) acetabulum (ac); (f) tegumental features of body; (g) tegument features of body posteriorly and tail; (h) tail with finfold (ff) and excretory opening (eo) 220 cercariae in lymnaea natalensis krauss, 1848 collected in gauteng province, south africa fig. 3 echinostomatid cercaria. light micrographs of (a) whole mount; (b) head of cercaria; (c–h) scanning electron micrographs of cercaria: (c) oral sucker (os) surrounded by long ciliated sensory receptors; (d) acetabulum (ac); (e) sensory receptors along the oral sucker and collar showing corner (cs) and lateral spines (lsp); (f) tegumental features and collar spines, lateral spines (ls) and dorsal spines (ds); (g) long ciliated (lcr) sensory and cluster receptors (cr); (h) tail features with long ciliated receptors (lcr) ba dc fe hg 150 μm 80 μm 1 μm 2 μm 2 μm 1 μm 1 μm 3 μm 221 e.b.e. moema, p.h. king & c. baker those of this study, with the exception of the content of the prepharyngeal sac and the species of the first intermediate host. xiphidiocercaria (fig. 1f–h and 4a–h) the cercaria has a stout oval body and a very short single tail (fig. 1f, h and 4a). the body is flattened dorso-ventrally, measuring 172–187 (176 + 5) x 43– 55 (49 + 3). a well-developed oral sucker (fig. 4c), measuring 36–43 (38 + 2) x 30–37 (33 + 3) is situated at the anterior end of the body. the most characteristic feature of this cercaria, however, is a simple stylet (fig. 1g and 4b), measuring 18–21 (19 + 1) x 3–5 (3 + 1), that is situated dorsally to the oral sucker. the oral sucker leads to a pre-pharynx that is longer than it is wide, measuring 22–27 (25 + 2) x 7–12 (8 + 2). it extends into a well-developed pharynx, 12– 18 (15 + 2) x 10–15 (12 + 1). the pharynx extends into an oesophagus, measuring 21–31 (23 + 3) x 7– 12 (9 + 1), and bifurcates into two intestinal caeca anterior to the acetabulum and which extend to reach the posterior margin of the acetabulum. the caeca measure 120–123 (121 + 1) long x 9–11 (10 + 1) wide. the acetabulum is covered by spines of similar length to those on the rest of the body (fig. 4d). mid-body, the acetabulum measures 30–36 (32 + 2) x 28–37 (31 + 3) and is situated 45–48 (47 + 1) from the oral sucker. three pairs of coarsely granulated penetration glands with distinct nuclei were observed at the sides of the acetabulum (fig. 1f). the first pair of penetration glands measures 9–12 (10 + 1) x 9–18 (14 + 3), the second pair, 10–13 (11 + 2) x 9–21 (13 + 4) and the third pair, 6–12 (9 + 2) x 9–16 (11 + 2). penetration gland ducts extend to the oral sucker where they open to the exterior. the excretory bladder consists of two parts: a larger posterior part, measuring 16–30 (20 + 4) x 16–37 (24 + 8) and a smaller anterior part, 21–31 (23 + 3) x 21–45 (28 + 8). the main excretory ducts extend bilaterally from the anterior bladder to the level of the acetabulum where they fold back posteriorly giving rise to anterior and posterior collecting ducts that eventually form capillaries that terminate in flame cells. three flame cells were observed anteriorly and two posteriorly. the flame cell formula is 2 [(2 + 1) + (2)] = 10. locomotion is provided by a short single tail (fig. 1h and 4h) measuring 148–164 (152 + 6) x 22–24 (23 + 1), that is aspinous and lacks dorso-ventral finfolds. the body tegument is evenly granulated and covered by numerous minute backwardly pointing spines (fig. 4g). sensory receptors are few, but receptors with long cilia were observed on the sides of the body (fig. 4f). short ciliated receptors were also observed on the ventral surface of the body (fig. 4e). most receptors with short cilia were observed surrounding the oral sucker (fig. 4c), whereas nonciliated receptors were observed alongside the acetabulum (fig. 4d). remarks this cercaria is classified as an armatae xiphidio cercaria according to the classification keys proposed by frandsen & christensen (1984) and haseeb (1984). frandsen & christensen (1984) are of the opinion that xiphidio cercariae belonging to the armatae group develop into intestinal parasites in all groups of vertebrates. in an earlier work, porter (1938) described the following nine xiphidio cercariae with three pairs of penetration glands and five to six flame cells on each side of the body: cercaria elizabethae (porter, 1938) from port elizabeth, eastern cape province; c. grobelaaria from oudtshoorn, western cape prov ince; c. hartebeestia from the hartbeestpoort dam, north west province; c. nymphaea from sydenham, kwazulu natal province; c. spruitensis from the hartbeestpoort dam; c. stonei from komatipoort, mpumalanga province; c. umhlotunzana from clair wood, kwa-zulu natal province; c. veta from hoopstad, free state province; and c. vuurensis from middleburg, mpumalanga province. of the nine xiphidio cercariae originally described six were shed by l. nata lensis, i.e. c. elizabethae, c. hartebeestia, c. nymphaea, c. stonei, c. umhlotunzana and c. vuurensis. the cercaria in this study differs from these six cercariae in the morphology of the xiphidio spine, intestinal caecal length, position and arrangement of the penetration glands and morphology of the tail. fain (1953) and vercammen-grandjean (1960) described five and two xiphidio cercariae with three pairs of penetration glands, respectively. all of these cercariae differ from the present cercarial type by the size and orientation of the penetration glands, flame cell formula, intestinal caeca length and form of the stylet. in contrast, king & van as (2001) described three xiphidio cercariae shed by b. tropicus in the free state that all differ in morphology when compared to the present cercaria, e.g. the position and number of penetration glands in the body. 222 cercariae in lymnaea natalensis krauss, 1848 collected in gauteng province, south africa fig. 4 xiphidio cercaria. light micrographs of (a) whole mount; (b) anterior stylet (s); (c–h) scanning electron micrographs of cercaria: (c) oral sucker (os) and stylet opening (so); (d) acetabulum (ac); (e) short ciliated receptors (scr); (f) long ciliated receptors (lcr); (g) tegumental features of the body; (h) tail (t) features ba dc fe hg 60 μm 10 μm 1 μm 1 μm 300 μm 100 μm 300 μm 1 μm 223 e.b.e. moema, p.h. king & c. baker discussion the apharyngeal-brevifurcate distome fork-tailed cer caria was found to be an avian schistosome cercaria. the life cycle studies in the mallard ducks and wisstar rats unfortunately produced only two trichobilharzia eggs in the faeces of one of the mallard ducks. according to appleton (2003) 16 adults and 12 cercariae of avian schistosomes have been described from the afrotropical region. nine trichobilharzia species have been described from birds in central africa and madagascar, and appleton (1982, 1986) recorded five different types of avian schistosome eggs from faecal samples collected at various localities in south africa. three trichobilharzia cercariae were described from l. natalensis in africa, but since the cercaria in the present study is the smallest and the resulting eggs clearly fall within trichobilharzia type 3 as was classified by appleton (1982, 1986 and 2003), this cercaria may well be different from those described earlier. the life cycle therefore needs to be repeated using a larger number of ducks of different species in order to find the adult form and to verify the characteristic eggs. the echinostomatid cercaria with its collar with 27 spines shed by l. natalensis was recovered from most localities. although the life cycle of this parasite is unknown, l. natalensis was found to host echinostomatid metacercarial cysts in the mantle. this snail therefore also serves as second intermediate host for this parasite. the xiphidiocercaria described in this study was found to be the most abundant single-tailed cercaria in the habitats examined in the proximity of pretoria. the life cycle of this parasite is also unknown, but other xiphidio stages were also found during this study. xiphidio metacercariae were found encysted in various second intermediate hosts, i.e. in the gill chambers of freshwater shrimps, in the mantle of l. natalensis and occasionally also in the intestine of barbus paludinosus. the cysts found in freshwater shrimps were bigger in size and the cyst wall was thinner compared to the cysts in l. natalensis and b. paludinosus. although it appears as if freshwater shrimps are the most suitable second intermediate host, surveys have shown that they were not always present, especially during winter months. studies have also shown that the highest infection with cysts occurs in l. natalensis. references appleton, c.c. 1982. the eggs of some blood-flukes (trematoda: schistosomatidae) from south african birds. south african journal of zoology, 17:147–150. appleton, c.c. 1986. occurrence of avian schistosomatidae (trematoda) in south african birds as determined by a faecal survey. south african journal of zoology, 21:60–67. appleton, c.c. 1996. freshwater molluscs of southern africa. pietermaritzburg, south africa: university of natal press. appleton, c.c. 2003. the avian schistosomatidae of sub-saharan africa with particular reference to cercaria herini, a cause of dermatitis in people. proceedings of workshop on african freshwater malacology, kampala, uganda. brown, d.s. 1994. freshwater snails of africa and their medical importance, 2nd ed. london: taylor & francis ltd. cheng, t.c. 1986. general parasitology, 2nd ed. orlando, florida: academic press inc. fain, a. 1953. contribution à l’étude des formes larvaires des trematodes au congo belge et spécialment de la larvae de schistosoma mansoni. mémoires institut royal colonial belge section de sciences naturalles et médicales, 22:1– 312. frandsen, f. & christensen, n.o. 1984. an introductory guide to the identification of cercariae from african freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance. acta tropica, 41:181–202. haseeb, m.a. 1984. studies on larval trematodes infecting freshwater snails in pakistan. x. non-virgulate xiphidio cercariae. zeitschrift für parasitenkunde, 70:637–654. jansen van rensburg, c.j. 2001. snail borne larval trematodes of the okavango delta, botswana. m.sc. dissertation, university of the free state. king, p.h. & van as, j.g. 1997. description of the adult and larval stages of tylodelphys xenopi (trematoda: diplosto matidae) from southern africa. journal of african zoology, 83: 287–295. king, p.h. & van as, j.g. 2001. cercariae shed by bulinus tropicus (krauss, 1848) in the free state, south africa. journal of african zoology, 36:95–105. picard, d. & jousson, o. 2001. genetic variability among cercariae of the schistosomatidae (trematoda: digenea) causing swimmer’s itch in europe. parasite, 8:237–242. porter, a. 1938. the larval trematoda found in certain south african mollusca with special reference to schistosomiasis (bilharziasis). publications of the south african institute of medical research, 8:1–492. van eeden, j.a. 1960. key to the genera of south african freshwater and estuarine gastropods (mollusca). annals of transvaal museum, 24:1–17. van eeden, j.a., brown, d.s. & oberholzer, g. 1965. the distribution of freshwater molluscs of medical and veterinary importance in southeastern africa. annals of tropical medicine and parasitolology, 59:413–424. vercammen-grandjean, p.h. 1960. les trematodes du lac kivi sud. musée royal de l’afrique centrale, tervuren. belgique annales novelle serie in 4˚ sciences zoologiques, 5:1–171. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice madekurozwa_59-65.indd introduction extensive research has been conducted on the innervation of the ovary in both birds (gilbert 1965; 1968; 1969; unsicker, seidel, hofmann, muller, schmidt & wilson 1983) and mammals (lakomy, do boszynska & szteyn 1983; dees, hiney, mcar thur, johnson, dissen & ojeda 2006). early reports described the ovary as being supplied by predominantly sympathetic nerves, which form a component of the ovarian nerve plexus (gilbert 1965; 1968; 1969). these sympathetic nerves form part of a neuronal pathway through which the central nervous system exerts a regulatory influence on the ovary (gerendai, kocsis & halasz 2002). the sympathetic nerves supplying the ovary are generally adrenergic in nature. these extrinsic adrenergic nerves have a myriad of functions, which include the regulation of blood flow (lakomy et al. 1983), folliculogenesis (curry, lawrence & burden 1984; kannisto, owman, rosengren & walles 1984), and ovarian steroidogenesis (weiss, dail & ratner 1982; sporrong, kannisto, owman, sjoberg & walles 1985; aguado 2002; delgado, sosa, dominguez, casais, aguado & rastrilla 2004). in addition to the extrinsic sympathetic nerves supplying the ovary, an intrinsic neuronal system has also been identified. the intrinsic innervation of the ovary is composed of neuronal cell bodies, which are generally catecholiminergic (d’albora & barcia 1996; d’albora, lombide & ojeda 2000; anesetti, lombide, d’albora & ojeda 2001). although the exact role of ovarian neuronal cell bodies is unclear, they are thought to be involved in the inhibition of steroidogenesis and in the regulation of ovarian blood flow (d’albora, anesetti, lombide, dees & ojeda 2002). it is clear from the literature that both the extrinsic and intrinsic ovarian innervation is vital to the normal functioning of the ovary. indeed it has been 59 onderstepoort journal of veterinary research, 75:59–65 (2008) an immunohistochemical study of ovarian innervation in the emu (dromaius novaehollandiae) m-c. madekurozwa* department of anatomy and physiology, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa abstract madekurozwa, m-c. 2008. an immunohistochemical study of ovarian innervation in the emu (dromaius novaehollandiae). onderstepoort journal of veterinary research, 75:59–65 the present study investigated the distribution of nerves in the ovary of the emu. the neuronal markers, protein gene product 9.5, neurofilament protein and neuron specific enolase demonstrated the constituents of the extrinsic and intrinsic ovarian neural systems. the extrinsic neural system was composed of ganglia in the ovarian stalk, as well as nerve bundles, which were distributed throughout the ovary. isolated neuronal cell bodies, in the medulla and cortex, formed the intrinsic neural system. an interesting finding of the study was the presence of nerve bundles, circumscribed by lymphocytes, in the ovarian stalk. the findings of the study indicate that the distribution of nerve fibres and neuronal cell bodies in the emu ovary is similar, but not identical to that of the domestic fowl and ostrich. keywords: emu, immunohistochemistry, innervation, ovary * e-mail: madex@op.up.ac.za accepted for publication 10 october 2007—editor 60 ovarian innervation in the emu (dromaius novaehollandiae) suggested that ovarian pathologies may result from a dysfunction in innervation (anesetti et al. 2001; lara, dorfman, venegas, luza, luna, mayerhofer, guimaraes, rosa e silva & ramirez 2002). to date much of the research conducted on the distribution and function of ovarian nerves has focused on mammals (weiss et al. 1982; lakomy et al. 1983; curry et al. 1984; dees, hiney, schultea, mayerhofer, danil chik, dissen & ojeda 1995; dees et al. 2006). furthermore, apart from a recent study conducted on the ovary of the immature ostrich (kimaro & madekurozwa 2006), most of the research carried out on birds has been limited to the domestic fowl (gilbert 1965; 1968; 1969; unsicker et al. 1983). although the emu is an economically important ratite there is currently a lack of information on the ovarian innervation of this avian species. the present study describes the distribution of nerve fibres in the ovary of the emu using antibodies against neurofilament protein (np), protein gene product 9.5 (pgp 9.5) and neuron specific enolase (nse). neurofilament protein is expressed specifically in neurons (ohara, gahara, miyake, teraoka & kitamura 1993), while pgp 9.5 and nse are cytoplasmic markers of both neuronal and neuroendocrine cells (martin, fraile, peina do, arenas, elices, alonso, paniagua, martin & san ta maria 2000; theodoropoulos, tsigka, mihalopoulou, tsoukala, lazaris, patsouris & ghikonti 2005). materials and methods a total of eleven female emus aged 18 months and weighing 35–40 kg were used in the present study. the birds originated from a commercial farm in the north west province of south africa. all the birds had active ovaries, which contained approximately 30 predominantly yellow-yolk follicles, with the diameters of the largest follicles ranging from 20 to 30 mm. the birds were sampled in march. the emu being a short day breeder typically begins to lay in april in the southern hemisphere (minnaar & minnaar 1997). the birds were slaughtered at a commercial abattoir, employing a standard slaughter protocol. ovarian tissue samples were obtained from the birds 10–15 min after slaughter. the tissue samples were then immersion-fixed in either 4 % paraformaldehyde (ph 7.2) or bouin’s fluid for 12 h. some samples were fixed in bouin’s fluid because the antibody against pgp 9.5 reacts better with antigens in paraffin sections. the samples fixed in paraformaldehyde were then placed for 24 h at 4 °c, in a 30 % sucrose solution made up in 0.01 m phosphate buffered saline solution (pbs, ph 7.4). thereafter, they were snap-frozen in oct compound (sakura, ca, usa) in isopentane slurry and then stored at –80 °c. tissue samples fixed in bouin’s fluid were processed routinely for histology and embedded in paraffin wax. the immunostaining technique was performed on 10 μm-thick cryostat sections and 5 μm-thick paraffin sections, using a lsab-plus kit (dakocytomation, denmark). the cryostat sections were air-dried for 60 min at room temperature after which they were rinsed in pbs. endogenous peroxidase activity in both cryostat and paraffin sections was blocked, using a 3 % (v/v) hydrogen peroxide solution in water for 5 min. the slides were then rinsed in pbs for 5 min. thereafter, the paraffin sections were microwaved at 750 w for two cycles of 7 min each, and after being allowed to cool for 20 min, they were rinsed with pbs and then incubated for 60 min at room temperature in a solution containing a polyclonal antibody against pgp 9.5, at a dilution of 1:50. the cryostat sections were incubated for 30 min in a solution containing monoclonal antibodies against np, at a dilution of 1:25, and a ready-to-use solution of antibodies against nse, for 60 min. after this incubation all tissue sections were rinsed with pbs and then incubated for 15 min in a solution containing a biotinylated secondary antibody (lsab-plus kit, dakocytomation, denmark). thereafter, the slides were rinsed in pbs and subsequently incubated for 15 min with the streptavidin peroxidase component of the lsab-plus staining kit. tissue sections were then rinsed in pbs and bound antibody was visualized after the addition of a 3,3’-diaminobenzidine tetrachloride solution (lsab-plus kit, dakocyto mation, denmark). tissue sections were counterstained with mayer’s haematoxylin before being dehydrated in graded concentrations of ethanol. in the negative controls the primary antibodies were replaced with either normal mouse or rabbit serum. a histological section of brain tissue was used as a positive control. results the ovary was composed of an ovarian stalk, medulla and cortex. numerous nerve bundles immunopositive for nse, np and pgp 9.5 coursed through the ovarian stalk and medulla (fig. 1). some of the nerve bundles were associated with blood vessels. in addition, a few nerve bundles in the ovarian stalk were closely associated with lymphoid follicles (fig. 61 m-c. madekurozwa fig. 1 np immunoreactive nerve bundles (arrows) in the medulla. some of the nerve bundles are associated with blood vessels (v). arrowheads: melanocytes fig. 2 np immunoreactive nerve bundle (arrows) surrounded by lymphocytes (l) fig. 3 np immunonegative neuronal cell bodies (arrows) form a ganglion in the ovarian stalk. closely associated with the neuronal cell bodies is a np immunopositive nerve bundle (n) fig. 4 nse immunoreactive neuronal cell bodies (arrows) in the medulla. n: nerve bundle fig. 5 pgp 9.5 immunopositive nerve fibres (arrows) in the upper limits of the cortex. arrowheads: melanocytes. g: germinal epithelium fig. 6 infiltration of an atretic previtellogenic follicle by connective tissue stroma (s) and np immunoreactive nerve fibres (arrows) 62 ovarian innervation in the emu (dromaius novaehollandiae) 2). ganglia, composed of clusters of neuronal cell bodies were present in the ovarian stalk (fig. 3). the neuronal cell bodies were characterized by the presence of a large round nucleus with a prominent nucleolus. the cytoplasm of these cell bodies was immunopositive for nse and pgp 9.5, but immunonegative for np. two to three layers of fibrocytes circumscribed the ganglia. isolated neuronal cell bodies were demonstrated in the medulla. in some instances the neuronal cell bodies were associated with nerve bundles (fig. 4). the cortex contained primordial, pre-vitellogenic and vitellogenic follicles. nerve bundles in the cortex were not closely associated with developing primordial and previtellogenic follicles, but rather occurred in the connective tissue stroma between the follicles. these cortical nerve bundles branched to form a neural network in the superficial regions of the cortex (fig. 5). although nerve fibres were not seen around developing previtellogenic follicles, they were observed in atretic previtellogenic follicles during the infiltration of the follicle by connective tissue (fig. 6). in addition to nerve fibres occasional neuronal cell bodies were observed in the cortex. vitellogenic follicles were suspended by follicular stalks, which were extensions of the cortex. several nerve bundles coursed through the follicular stalks and branched to form perifollicular nerve fibres in the superficial tunic and theca externa of the developing vitellogenic follicles (fig. 7). most of the perifollicular nerve fibres were associated with blood vessels. the early stages of atresia in vitellogenic follicles were characterized by the proliferation of theca interna and granulosa cells. a neural network was pres ent in the region between the theca interna and granulosa cell layer. the theca interna and granulosa cells subsequently differentiated into interstitial gland cells, which infiltrated both the theca externa and oocyte. eventually the interstitial gland cells abutted intramural veins, which were located in the outer limits of the theca externa. at this stage of atresia a dense network of nerve fibres was observed between the interstitial gland cells and intramural veins (fig. 8). the interstitial gland cells eventually dispersed to form stromal interstitial gland cells, which were located in the superficial regions of the cortex. nerve fibres were observed coursing between the stromal interstitial gland cells. discussion the neuronal markers used in the study have clearly demonstrated the components of both the extrinsic and intrinsic neural systems of the emu ovary. the extrinsic system was composed of ganglia in the ovarian stalk and nerve bundles in the ovarian stalk, medulla, cortex and follicular stalks. isolated neuronal cell bodies in the medulla and cortex formed the intrinsic neural system. as in the domestic fowl (gilbert 1965; 1968; 1969), extrinsic nerve bundles entered the ovary through the ovarian stalk where they subsequently formed a nerve plexus. the ovarian nerve plexus in both the emu and domestic fowl (gilbert 1965; 1968; 1969; unsicker et al. 1983) contained several encapsulated ganglia. in the present study the nerves forming the plexus were immunopositive for nse, np fig. 7 pgp 9.5 immunopositive nerves (arrows) in the theca externa (te) of a developing vitellogenic follicle. ti: theca interna. g: granulosa cell layer fig. 8 a network of nse immunoreactive nerve fibres (arrows) is present at the interface between intramural veins (m) and interstitial gland cells (ig) in an atretic vitellogenic follicle 63 m-c. madekurozwa and pgp 9.5, while the ganglia were immunopositive for nse and pgp 9.5, but immunonegative for np. in the emu, ostrich (kimaro & madekurozwa 2006) and domestic fowl (gilbert 1965; 1968) nerves in the ovarian stalk coursed into the medulla, cortex and follicular stalks. the role of nerves in blood regulation is well established (lakomy et al. 1983). indeed, the close association between nerves and blood vessels has been reported in the ovaries of the rat (lawrence & burden 1980), domestic fowl (unsicker et al. 1983), and pig (lakomy et al. 1983). in the present study, perivascular nerve fibres were demonstrated throughout the ovary. admittedly, the antibodies used precluded the identification of adrenergic and cholinergic nerves. both cholinergic and adrenergic nerves have been demonstrated in the domestic fowl (gilbert 1969). however, it was conceded that most of the nerves were adrenergic. this is supported by research conducted in the rat in which most perivascular nerves are adrenergic (lawrence & burden 1980). although the association between nerves and blood vessels has been extensively reported, the relationship between nerves and lymphoid follicles has received far less attention. an interesting finding of the present study was the close association between nerves and lymphoid follicles in the ovarian stalk. research conducted by felten, ackerman, wiegand & felten (1987) on the spleen suggests that neurotransmitters released from nerve endings regulate the migration, maturation, proliferation and activation of lymphocytes. the relevance of the close association of nerve bundles and lymphoid follicles in the emu ovary is unclear. the distribution of nerve fibres in the medulla, cortex, follicular stalks and perifollicular regions of birds appears to be fairly uniform (gilbert 1968; 1969; kimaro & madekurozwa 2006). indeed the distribution pattern of perifollicular nerve fibres in the developing follicles of the emu varied little from that of the ostrich (kimaro & madekurozwa 2006). in both ratites nerves formed a network in the thecal layer. no nerve fibres were seen within the granulosa cell layer. the presence of a thecal neural network and the lack of nerve fibres within the granulosa are consistent with observations made in the domestic fowl (gilbert 1965; 1969). it is thought that in mammals perifollicular nerve fibres play a role in folliculogenesis (curry et al. 1984; kannisto et al. 1984; malamed, gibney & ojeda 1992) and ovulation (bahr, kao & nalbandov 1974; luna, cortes, flores, hernandez, trujillo & dominguez 2003). in addition, based on the findings of the present study, it is probable that perifollicular nerves are involved in the differentiation of thecal and granulosa cells into interstitial gland cells during atresia. this is evidenced by the presence of numerous nerve fibres in regions of interstitial gland formation in atretic vitellogenic follicles. the presence of a neural network in contact with differentiating interstitial gland cells is not surprising, as it has been shown that nerves have a profound effect on the development, maintenance and function of corpora lutea in the bovine (kotwica, bogacki & rekawiecki 2002), pig (jana, dzienis, panczyszyn, rogozinska, wojtkiewicz, skobowiat & majewski 2005) and rat (casais, delgado, sosa & rastrilla 2006). in addition to the extrinsic neural system described above, the ovaries of birds and mammals contain an intrinsic neural system. the exact function of the intrinsic neural system is unclear. however, it is thought that this neural system is concerned not only with follicular maturation and blood regulation, but also with the inhibition of steroidogenesis (d’albora et al. 2002). the intrinsic neural system is composed of isolated neuronal cell bodies. as in the domestic fowl (gilbert 1965; 1968), the neuronal cell bodies in the emu were characterized by the presence of a round nucleus with a prominent nucleolus. significantly the results of the present study not only confirm the findings of gilbert (1965; 1968), with regard to the morphology of neuronal cell bodies, but also further characterize the physiological nature of these cells. the fact that the neuronal cell bodies were immunopositive for both nse and pgp suggests that they possess a neuroendocrine function. this is supported by research on the rat ovary in which neuronal cell bodies have been shown to produce catecholamines as evidenced by the presence of tyrosine hydroxylase, an enzyme involved in the synthesis of norepinephrine. the distribution and number of neuronal cell bodies appear to be species specific. in the emu a few neuronal cell bodies were demonstrated in the medulla and occasionally in the cortex. likewise, in the ostrich a few neuronal cell bodies were observed in the medulla (kimaro & madekurozwa 2006). in contrast, numerous neuronal cell bodies were observed in the follicular stalks supporting mature follicles in the domestic fowl (gilbert 1965). in this species very few neuronal cell bodies were observed in the cortex or in association with developing follicles (gilbert 1969). furthermore no neuronal cell bodies were observed in the medulla. in both the immature ostrich (kimaro & madekurozwa 2006) and the emu neuronal cell bodies were occasionally associated 64 ovarian innervation in the emu (dromaius novaehollandiae) with nerve bundles. similar observations have been made in the human ovary where it is thought that the association of neuronal cell bodies with nerve bundles implies a regulatory association between the intrinsic and extrinsic neural systems of the ovary (anesetti et al. 2001). in conclusion, this study has described the salient features of the extrinsic and intrinsic neural systems of the emu ovary. the distribution of nerve fibres in the emu ovary is similar to that of the domestic fowl (gilbert 1965; 1968; 1969) and ostrich (kimaro & madekurozwa 2006). a 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livestock, ministry of agriculture, livestock and fisheries, kenya 3emergency centre for transboundary animal diseases, food and agriculture organization of the united nations, kenya 4international centre for insect physiology and ecology, nairobi, kenya 5college of humanities and social sciences, university of nairobi, kenya correspondence to: nelson owange postal address: po box 36001, nairobi 00200, kenya dates: received: 27 feb. 2014 accepted: 21 aug. 2014 published: 20 nov. 2014 how to cite this article: owange, n.o., ogara, w.o., kasiiti, j., gathura, p.b., okuthe, s., sang, r. et al., 2014, ‘perceived risk factors and risk pathways of rift valley fever in cattle in ijara district, kenya’, onderstepoort journal of veterinary research 81(1), art. #780, 7 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.780 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. perceived risk factors and risk pathways of rift valley fever in cattle in ijara district, kenya in this original research... open access • abstract • introduction • materials and methods    • study area    • sampling method    • key informant interviews    • data analysis • results    • qualitative ranking of perceived rift valley fever risk factors    • qualitative ranking of perceived rift valley fever entry pathways    • qualitative ranking of perceived rift valley fever exposure or spread pathways    • qualitative ranking of perceived rift valley fever outbreak consequences • discussion • conclusion    • recommendations • acknowledgements    • competing interests    • authors’ contributions • references • footnotes abstract top ↑ ijara district in kenya was one of the hotspots of rift valley fever (rvf) during the 2006/2007 outbreak, which led to human and animal deaths causing major economic losses. the main constraint for the control and prevention of rvf is inadequate knowledge of the risk factors for its occurrence and maintenance. this study was aimed at understanding the perceived risk factors and risk pathways of rvf in cattle in ijara to enable the development of improved community-based disease surveillance, prediction, control and prevention. a cross-sectional study was carried out from september 2012 to june 2013. thirty-one key informant interviews were conducted with relevant stakeholders to determine the local pastoralists’ understanding of risk factors and risk pathways of rvf in cattle in ijara district. all the key informants perceived the presence of high numbers of mosquitoes and large numbers of cattle to be the most important risk factors contributing to the occurrence of rvf in cattle in ijara. key informants classified high rainfall as the most important (12/31) to an important (19/31) risk factor. the main risk pathways were infected mosquitoes that bite cattle whilst grazing and at watering points as well as close contact between domestic animals and wildlife. the likelihood of contamination of the environment as a result of poor handling of carcasses and aborted foetuses during rvf outbreaks was not considered an important pathway. there is therefore a need to conduct regular participatory community awareness sessions on handling of animal carcasses in terms of preparedness, prevention and control of any possible rvf epizootics. additionally, monitoring of environmental conditions to detect enhanced rainfall and flooding should be prioritised for preparedness. introduction top ↑ rift valley fever (rvf) is a viral disease caused by rift valley fever virus (rvfv), a member of the genus phlebovirus, family bunyaviridae. it is a mosquito-borne viral zoonosis that periodically causes disease outbreaks in humans and livestock (davies, linthicum & james 1985). the virus is transmitted to humans through direct contact with tissues and blood of infected animals as well as bites from infected mosquitoes. the disease has been endemic in sub-saharan africa since 1912 (peters & linthicum 1994). periodic outbreaks have been reported in many african countries, saudi arabia and yemen (al-afaleg et al. 2003; centers for disease control and prevention [cdc] 2007; gerdes 2004; woods & karpati 2002). outbreaks of rvf in ijara district have been associated with unusually heavy rainfall that causes extensive flooding of basins and low-lying grassland depressions called dambos, triggering mass emergence of aedes mosquitoes (anyangu et al. 2010). in 1997/1998 and 2006/2007, massive outbreaks of rvf occurred in east africa, both associated with el niño events (cdc 2007; woods & karpati 2002), with an estimated 27 500 human cases and more than 600 deaths reported in 1997/1998 in kenya alone. historical outbreaks of rvf ‘since the early 1950s have been associated with cyclical patterns of the el niño/southern oscillation (enso) phenomenon, which results in elevated and widespread rainfall over the rvf-endemic areas of africa’ (anyamba et al. 2010:43). the two outbreaks (in 1997/1998 and 2006/2007) were the most notable in terms of public health and socio-economic impact (murithi et al. 2010). rift valley fever virus has distinct endemic and epidemic cycles. during the endemic cycle, the virus persists in inter-epidemic periods through vertical transmission in aedes mosquito eggs (davies et al. 1985). flooding of mosquito habitats can introduce rvfv into domestic animal populations by the emergence of vertically infected aedes mosquitoes. epidemic cycles are driven by the subsequent expansion of various culex mosquito populations, which serve as excellent secondary vectors if immature mosquito habitats remain flooded long enough (anyamba et al. 2010). apart from the mosquito transmission to domestic animals and humans during the epidemic cycle, aerosols and contact between infected animals and humans can transmit the virus to humans (anyamba et al. 2010). the virus is amplified in people and animals. flat topography, presence of water-retaining soil types and dense bush cover are important factors for flooding and/or mosquitoes breeding (anyangu et al. 2010). risk pathway analysis involves investigation of the possibility of entry, release, exposure and eventual consequences of the disease. it helps establish the routes the disease follows for possible entry, establishment and spread (breiman et al. 2010). factors such as vectors, hosts, animal movement patterns and the role of wildlife are used to estimate the probability of occurrence of the disease. in the usa, risk pathway analysis for emerging and re-emerging diseases has been adopted in order to establish exact areas to target for better control and prevention (kasari et al. 2008). breiman et al. (2010) documented that rvfv can enter a new area through infected mosquitoes and their eggs, infected livestock, infected wildlife, infected humans and smuggling (terrorist activity). the virus can then spread through mosquito bites, environmental contamination by infected carcasses or aborted foetuses and movement of infected animals and humans, leading to massive losses of lives, livelihoods and trade. about 90% of the population of ijara is directly dependent on livestock for food and as a source of income. during the last outbreak, a ban on livestock trade and imposition of quarantine resulted in severe economic losses of more than $9.3 million (murithi et al. 2010). in ijara district, cattle are driven over long distances towards the tana river delta or into boni forest, passing through various ecosystems. ijara district1 was one of the hotspots during the last rvf outbreaks in the arid and semi-arid north-eastern province of kenya (nguku et al. 2010). the nomadic and semi-nomadic pastoralist communities maintain large livestock herds even in circumstances of limited pasture and water. outbreaks of rvf have caused major disruptions to public health and the economic mainstay of this population. the movement of these viruses amongst animals and vectors with occasional involvement of human populations under the influence of environmental factors required further study. the study used key informant interviews to investigate the perceived risk factors and risk pathways associated with rvf in cattle along the livestock movement corridors in ijara. it was part of a bigger project whose overall objective was to bring about a better understanding of the environmental, biotic and socio-economic drivers of the emergence of rvf and other arboviruses and the viable control options in the arid and semi-arid north-eastern province of kenya, with a focus on ijara district, a major hotspot of the disease. the information from this study can be used to create awareness as well as to formulate prevention and mitigation measures for rvf. materials and methods top ↑ study area the study was carried out in the arid and semi-arid region of north-eastern kenya, with ijara district at the centre of the study. ijara district is bordered by garissa and fafi districts to the north, somalia to the east, lamu district to the south and tana river district to the west (figure 1). it lies at approximately 33°e, 6°n and 43°e, 5°s and is devoid of mountains. it is characterised by low, undulating plains at an altitude ranging between 0 m.a.s.l. and 90 m.a.s.l. figure 1: map of study area showing the relationship between the study area, the surrounding districts and somalia. figure 2 is a map of kenya showing the areas where rvf outbreaks occurred and their relationship with the study area. figure 2: map of kenya showing rift valley fever endemicity and the relationship with the study area. the study area (figure 1) falls in ecological zone v-iv with a total forest cover of 2484 km². boni forest, which is an indigenous open canopy forest that forms part of the northern zanzibar-inhamdare coastal forest mosaic, covers a major part of the study area. a section of the forest, the boni national reserve, is under the management of the kenya wildlife service as a protected conservation area. the soil types are black cotton and alluvial types, temperatures range between 15 ºc and 38 ºc, bimodal rainfall ranges between 700 mm and 1000 mm per annum, and average relative humidity is 68 mm. migration in the district is occasioned by search for pasture during the dry seasons and involves movement of people and livestock to the tana river delta and the boni forest area, where water and pasture are abundant long after the rainy season. other movements in search of pasture and water are towards fafi and garissa districts and somalia. sampling method a cross-sectional study of the perceived risk factors and risk pathways of rvf for cattle in ijara was carried out between august 2012 and june 2013. purposive sampling was used to select 31 individuals who were later interviewed in order to describe the perceived risk factors and risk pathways for rvf in cattle as understood by the locals (chambers 1981, 1994; world bank 2004). targeted stakeholders for 31 key informant interviews included local leaders, veterinary officers, animal health assistants, community-based animal health workers, kenya wildlife service personnel and local administrative officers. key informant interviews the key informant interviews were used to identify and rank the rvf-associated risk factors and risk pathways as perceived by the local pastoralists in the study area. it is important to note that some key informants were technical experts who may have given their opinion rather than the local communities’ perception. the key informants listed the risk factors and used informal ranking, which was later cross-checked by a more formal pair-wise and matrix ranking and scoring. the identified pathways were also qualitatively ranked as high or high importance (4), medium or important (3), low or less important (2) or negligible or not important (1) or high (3), moderate (2) and low (1) in the study area by the respondents. these qualitative rankings had no set criteria and depended on the key informants’ responses. data analysis at the end of the data collection process, all information gathered was ranked and qualitative measures of perceived importance of risk factors and pathways summarised to address the objectives of the study. results top ↑ qualitative ranking of perceived rift valley fever risk factors according to the respondents, availability of vectors (31/31 = 4), large number of cattle (29/31 = 4; 2/31 = 3) and high rainfall (12/31 = 4; 19/31 = 3) are rated most important and/or important risk factors associated with rvf in ijara (table 1). there was varied perception on soil types, dambos, bushy vegetation, wildlife and flat topography as risk factors, ranging from 3 (important) to 1 (not important). all the respondents rated drought as not important as a risk factor associated with rvf. high temperature was also rated as a less important to not important risk factor. table 1: summarised results for qualitative ranking of rift valley fever risk factors in cattle as perceived by the pastoralists’ key informants in ijara, kenya. qualitative ranking of perceived rift valley fever entry pathways as shown in table 2, the perceived entry risk pathways for rvf in ijara district according to the key informants were infected mosquitoes, infected domestic animals, infected aborted foetuses and fluids and infected wild animals. all the respondents were in agreement that infected mosquitoes constituted the most likely entry pathway for rvf (31/31). most of the respondents (30/31) were in agreement that infected domestic animals could be classified as high means of rvf entry into ijara district, whilst only one respondent classified it as medium means of rvf entry (1/31). the majority of the respondents (30/31) perceived virus smuggling from neighbouring somalia to be a negligible (unlikely) means of rvf virus entry. table 2: summarised results for qualitative ranking of rift valley fever entry risk pathway in cattle as perceived by the pastoralists’ key informants in ijara, kenya. qualitative ranking of perceived rift valley fever exposure or spread pathways the perceived exposure or spread risk pathway were bites from infected mosquitoes at livestock watering points, around cattle bomas, in bushy environments coming in contact with cattle as well as the exposure to contaminated pasture and environment by infected aborted foetuses and fluids. most of the respondents perceived spread of rvfv through mosquitoes’ bite to be one of the possible forms of spread, whilst environmental contamination by infected aborted foetuses and fluids was categorised as a medium to low-risk pathway (table 3 and figure 3). figure 3: endemic (on left) and epidemic (on right) life cycles of rift valley fever involving close association between heavy rainfall conditions, vector aedes and culex mosquitoes, domestic animals and humans. table 3: summarised results for qualitative ranking of rift valley fever exposure risk pathway in cattle as perceived by the pastoralists’ key informants in ijara, kenya. qualitative ranking of perceived rift valley fever outbreak consequences the most highly rated consequences are morbidity (30/31), quarantine (31/31) and ban on trade (31/31). ratings for abortion, mortality, loss of appetite, reduced production and vaccination consequences ranged between high, medium and low for each consequence (table 4). table 4: summarised results for qualitative ranking of rift valley fever outbreak consequences in cattle as perceived by the pastoralists’ key informants in ijara, kenya. discussion top ↑ the informants rated availability of vectors, large number of cattle and high rainfall highly as perceived risk factors associated with rvf in ijara. these findings were supported by those of anyangu et al. (2010), who showed a strong association between severe infections of rvf and handling of a large number of animals, closeness to water sources and mosquitoes in the 2006/2007 outbreak. respondents’ low rating on the role of wildlife in rvf transmission does not correspond with the findings of evans et al. (2008), who detected rvf antibodies in warthogs, gerenuk, waterbuck and buffalo. the risk pathway analysis was based on three possible stages: assessment (entry, exposure and consequence), communication and management. there were basically three pathways for consideration for the above analysis in relation to the possible entry of the rvf virus into the study area, transmission and spread of the virus and release of the virus to neighbouring areas. the entry risk pathways for rvf in ijara district mentioned by the pastoralists were through infected mosquitoes, infected domestic animals, infected aborted foetuses and fluids and infected wild animals, which were corroborated by the study carried out by breiman et al. (2010). the perceived exposure or spread risk pathways of infected mosquitoes at livestock watering points, around cattle bomas and in bushy environments coming into contact with cattle as well as the exposure to pasture and environment contaminated by infected aborted foetuses and fluids were indications of the communities’ understanding of the risk factors associated with rvf. the likelihood of virus smuggling across the border with somalia was rated as negligible. it is also important to note that the respondents considered aborted foetuses as a less important pathway in the entry and spread of rvf, a factor which does not correspond with the findings of anyangu et al. (2010) that aborted foetuses were the single most important factors directly associated with severe rvf infections in humans during the 2006/2007 outbreak, compared to the presence of mosquitoes, water bodies and contact with livestock, which were jointly associated. table 5 provides a summary of the risk pathway analysis. table 5: a summary discussion table of risk pathway analysis for rift valley fever in cattle in ijara, kenya. conclusion top ↑ availability of the rvfv vectors (mosquitoes) and hosts (cattle) as well as high rainfall were the main risk factors understood by the locals to enable epidemics of rvf in ijara. the main perceived routes of entry, exposure and spread of rvf in ijara were infected mosquitoes, infected domestic and wild animals and environmental contamination from poor disposal of infected carcasses. however, the locals did not consider transmission from contaminated environment important. the close association between cattle and wildlife was not perceived to be amongst the pathways of rvf transmission in the study area. the areas of contact with wildlife were bushy grazing areas and watering points. it is important to note that rvfv is endemic in ijara district. as a result, the likelihood of its entry into ijara may not be applicable since it is already present. this explains why the communities’ perception about the source and/or spread of rvf is the same, namely the role played by mosquitoes. recommendations although an rvf decision support tool has been developed for the horn of africa (international livestock research institute [ilri]/food and agriculture organization of the united nations [fao] 2009), it seems it is not actually implemented in ijara. furthermore, management of cattle and wildlife carcasses would cause a big loss to the pastoralists in ijara in cases of rvf outbreak given their current perception of their being a low-risk factor. there is therefore a need to implement the rvf decision support tool, especially education and creation of awareness on proper ways of handling both domestic and wild animal carcasses to mitigate or prevent the possibility of rvfv transmission. awareness should also include the likelihood of rvfv transmission between domestic and wild animals by limiting wild-domestic animal interaction to a minimum. on the other hand, strategic vaccination, if carried out, can help protect the animals and reduce losses in case of an outbreak. continuous surveillance of rvf in ijara in domestic animals, wildlife, humans and vectors as well as environmental monitoring of rainfall pattern should be carried out together with all the stakeholders to provide early warning signs that can help in preparedness, control and prevention of the occurrence of rvf. acknowledgements top ↑ we wish to express our thanks to the international development research centre (idrc) for financial support that facilitated this research. we also appreciate the overwhelming support and assistance from members of the idrc rvf ecohealth project management committee. we recognise the invaluable support from the field key informant interview facilitators (james wauna [international centre for insect physiology and ecology {icipe}], denis njeru gitonga [district livestock production officer, ijara], omar chatsi [deputy district veterinary officer, ijara]), the herd owners, herdsmen, kenya wildlife service (kws) personnel and local leaders who offered overwhelming support during interviews. lastly, we recognise the administrative support from the icipe. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions the study forms part of a bigger project funded by the idrc, implemented by the icipe’s lead agency and two implementing partners, the state department of veterinary services of the government of kenya and the university of nairobi. r.s. (icipe), h.a. (icipe), m.m. (ministry of agriculture, livestock and fisheries), j.k. (ministry of agriculture, livestock and fisheries), w.o-o. (university of nairobi) were the project initiators and guided the holistic project design and implementation. t.t.o.l. (icipe) was engaged in the design of the study, producing and helping in the interpretation of gis maps. n.o.o. (university of nairobi) carried out the study, led the design, actual fieldwork, analysis and presentation of the findings under the supervision of w.o.o. (university of nairobi), m.m., s.o. (emergency centre for transboundary animal diseases) and p.b.g. (university of nairobi), who guided and supervised every stage of the study. references top ↑ al-afaleg a.i., abu elzein, e.m., mousa, s.m. & abbas, a.m., 2003, ‘a retrospective study on rift valley fever in saudi arabia’, revue scientifique et technique, office international des épizooties 22, 867–871. anyangu, a.s., gould, l.h., sharif, s.k., nguku, p.m., omolo, j.o., mutonga, d. et al., 2010, ‘risk factors for severe rift valley fever infection in kenya, 2007’, american journal of tropical medicine and hygiene 83(suppl. 2), 14–21. http://dx.doi.org/10.4269/ajtmh.2010.09-0293 anyamba, a., linthicum, j.k., small, j., britch, s.c., pak, e., de la rocque, s. et al., 2010, ‘prediction, assessment of the rift valley fever activity in east and southern africa 2006–2008 and possible vector control strategies’, american journal of tropical medicine and hygiene 83(suppl. 2), 43–51. http://dx.doi.org/10.4269/ajtmh.2010.09-0289 breiman, f.r., minjauw, b., sharif, s.k., ithondeka, p. & njenga, m.k., 2010, ‘rift valley fever: scientific pathways toward public health prevention and response’, american journal of tropical medicine and hygiene 83(suppl. 2), 1–4. http://dx.doi.org/10.4269/ajtmh.2010.83s2a01 centers for disease control and prevention (cdc), 2007, ‘rift valley fever outbreak – kenya, november 2006-january 2007’, morbidity and mortality weekly report 56, 73–76. chambers, r., 1981, ‘rapid rural appraisal: rationale and repertoire’, ids discussion paper no. 155, institute for development studies, sussex. chambers, r., 1994, ‘participatory rural appraisal: analysis of experience’, world development 22(9), 1253. http://dx.doi.org/10.1016/0305-750x(94)90003-5 davies, f.g., linthicum, k.j. & james, a.d., 1985, ‘rainfall and epizootic rift valley fever’, bulletin of the world health organization 63, 941–943. evans, a., gakuya, j.t., paweska, m., rostal, l., akoolo, l., van vuren, p.j. et al., 2008, ‘prevalence of antibodies against rift valley fever in kenyan wildlife’, 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macharia, j.m., hightower, a., luman, e.t. et al., 2010, ‘rift valley fever in kenya: history of epizootics and identification of vulnerable districts’, epidemiology and infection 139(3), 372–380. http://dx.doi.org/10.1017/s0950268810001020 nguku, m.p., sharif, s.k., mutonga, d., amwayi, s., omolo, j., mohammed, o. et al., 2010, ‘an investigation of a major outbreak in kenya: 2006–2007’, american journal of tropical medicine and hygiene 83(suppl. 2), 5–13. http://dx.doi.org/10.4269/ajtmh.2010.09-0288 peters, c.j. & linthicum, k.j., 1994, ‘rift valley fever’, in g.w. beran (ed.), handbook of zoonoses, 2nd edn., section b: viral zoonoses, pp. 125–138, crc press, boca raton. world bank, 2004, ‘monitoring and evaluations: some tools, methods and approaches’, viewed 20 august 2014, from http://www.worldbank.org/ieg/ecd/tools/ woods, c.w. & karpati, a.m., 2002, ‘an outbreak of rift valley fever in north eastern kenya, 1997–1998’, emerging infectious diseases 8, 138–144. http://dx.doi.org/10.3201/eid0802.010023 footnotes top ↑ 1.ijara district is amongst the four districts (lujis, lagdera, fafi and ijara) of garissa county after the promulgation of the new constitution in kenya in 2010. ijara district was carved out of garissa district (the region of current garissa county was originally called garissa district) even before the new constitution. many publications still use garissa district to mean garissa county including ijara district. however, some publications differentiate between ijara and garissa district. in this publication, garissa district refers to the district bordering ijara district to the north (see figure 1). schoeman_59-66.indd epidemiology babesiosis is a disease of worldwide significance and was first recognised in 1888 as a cause of fever, haemolytic anaemia, haemoglobinuria and death of cattle. members of the genus babesia readily parasitize the red blood cells of dogs, causing progressive anaemia. canine babesia are morphologically classified into large and small forms, both exhibiting a worldwide distribution (table 1). babesia canis and another novel, as yet unnamed babesia sp. detected in the usa (large babesia) and babesia gibsoni and babesia annae (small ba besia) have been documented to infect dogs (bir kenheuer, neel, ruslander, levy & breidschwerdt 2004). three main spe cies of large babesia infect dogs, namely babesia vogeli, b. canis and babesia rossi. these three species are antigenically distinct, transmitted by different vectors and differ widely in pathogenicity and geographic distribution (uilenberg, franssen, perie & spanjer 1989). babesia vogeli is the least pathogenic. it occurs in france, australia, japan, brazil, south africa and the usa and usually causes mild disease in adult dogs, but severe disease in some puppies (matjila, penzhorn, bekker, nijhof & jongejan 2004). babesia canis is widespread in europe (it affects more than 400 000 dogs per year in france alone) and asia and is of intermediate pathogenicity. babesia rossi occurs predominantly in southern af rica and is ostensibly the most virulent of the subspecies. improved pcr techniques have lately allowed for better definition of these parasites (matjila, leisewitz, jongejan & penzhorn 2008). the smaller parasite, b. gibsoni occurs principally in the middle east, southern asia, japan, north africa, south america and is an emerging infectious disease in the usa, as well as having been detected lately in italy, hungary and australia (muhlnickel et al. 2002). a more virulent subspecies of b. gibsoni has recently been identified in california (kjemtrup, wainwright, miller, penzhorn & carreno 2006) a babesia microti-like piroplasma, b. annae (also known as theileria annae) has been found to be 59 onderstepoort journal of veterinary research, 76:59–66 (2009) canine babesiosis j.p. schoeman department of companion animal clinical studies, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract schoeman, j.p. 2009. canine babesiosis. onderstepoort journal of veterinary research, 76:59– 66 babesiosis is a tick-borne disease affecting humans and many domestic and wild animals. domestic animals showing appreciable morbidity and mortality include dogs, cats, cattle and horses. both canine and feline babesiosis are diseases characterised by haemolytic anaemia, icterus and haemoglobinuria. canine babesiosis can range from chronic or subclinical to peracute and fatal, depending on the virulence of the species and the susceptibility of the host. this paper deals with canine babesiosis with specific reference to pathogenesis, clinical findings, complications, diagnosis and treatment, as well as newly identified prognostic factors in babesia rossi babesiosis. 60 canine babesiosis endemic in dogs in northwest spain (camacho, pallas, gestal, guitan, olmeda, telford & spielman 2003). most recently another small piroplasm causing a disease dubbed ‘bloody ears’ has been identified in brazil (loretti & barros 2005). various species of ticks such as rhipicephalus sanguineus, dermacentor spp. and haemaphysalis ellip ticum can transmit the large babesia of dogs, whereas b. gibsoni is transmitted by haemaphysalis bispinosa and haemaphysalis longicornis. babesia annae is thought to be transmitted by ixodes hexagonus (lobetti 2006). both trans-stadial and transovarial transmission can occur and ticks are believed to remain infective for several generations. babesia spp. can also be transmitted by blood transfusion. strong circumstantial evidence exist that b. gibsoni is transmitted by dog bites (birkenheuer, correa, levy & breitschwerdt 2005), whilst transplacental transmission from dam to offspring has recently been proven as an additional mode of transmission (fukumoto, suzuki, igarashi & xuan 2005). pathogenesis and clinical signs babesia spp. cause disease mostly in young dogs, although dogs of all ages can be affected. the incubation period of canine babesiosis varies from 10– 21 days for b. canis and 14–28 days for b. gibsoni. the female ticks feed on their host for about one week only and have left the host by the time disease develops. the severity of the disease depends on the species of babesia, the presence of concurrent infections and the age and immune status of the host. the disease presentation varies widely from peracute to chronic or even subclinical. babesia rossi, the domtable 1 geographical spread of canine babesiosis species of babesia tick vector country endemicity large babesia b. rossi h. elliptica south africa endemic b. canis dermacentor reticularis france endemic spain endemic hungary endemic switzerland small outbreaks germany small outbreak belgium three cases netherlands small outbreak b. vogeli r. sanguineus usa endemic in the south, especially in kennels, but spreading okinawa, japan endemic france sample from one dog australia endemic south africa endemic brazil endemic small babesia b. gibsoni – californian isolate h. bispinosa and h. longicornis usa endemic in the west b. gibsoni – asian isolate usa endemic, spreading northwards (detected in 29 states) north and east africa endemic asia endemic australia three pitbull terriers middle east endemic hungary two dogs (no pcr confirmation) italy one case (no pcr confirmation) b. annae spain endemic 61 j.p. schoeman inant species found in south africa, is very virulent and causes peracute and acute disease. clinical signs include pale mucous membranes, depression, tachy cardia, tachypnoea, anorexia, weak ness, splenomegaly and fever. it is thought that the clinical signs are the result of tissue hypoxia following the anaemia and a concomitant systemic inflammatory response syndrome caused by marked cytokine release (lobetti 2006). the pathogenesis of the anaemia is incompletely understood; intravascular and extravascular haemolysis take place, but other mechanisms such as poor bone marrow response are thought to play a role as well. the anaemia is (perhaps counter-intuitively) not correlated to the degree of parasitaemia and dogs start to improve after parasiticidal treatment, even though their haematocrits generally drop further, before starting to rise (jacobson, reyers, berry & viljoen 1996). some cases show additional immune-mediated breakdown of red blood cells and dogs that show in-saline-positive red blood cell agglutination have to be carefully monitored for rapid decreases in haematocrit. mortality for babesia spp. infections range from around 12 % for b. rossi to approxi mately 1 % for b. vogeli (lobetti 2006). the severe form of the disease is characterized by marked haemolytic anaemia, severe acid-base abnormalities (leisewitz, jacobson, de morais & reyers 2001) with frequent secondary multiple organ failure and complications such as acute renal failure (arf), hepatopathy with marked icterus, hypoglycaemia (keller, jacobson, nel, de clerq, thompson & schoeman 2004), acute respiratory distress syndrome (ards), cerebral pathology and additional immune-mediated red blood cell destruction (imha) (jacobson 2006). a small subset of dogs presents with high haematocrits (relative haemoconcentration), despite vigorous haemolysis, due to presumed shifting of fluid from the intravascular to the extravascular component. these dogs are at increased risk of developing arf or cerebral complications, as well as other organ failures (welzl, leisewitz, jacobson, vaughanscott & myburgh 2001). pancreatitis is frequently associated with other complications, and has a mortality rate of 20 %. icterus, vomition, melaena, abdominal pain and diarrhoea are common findings in dogs with suspected pancreatitis. in addition, 65 % of the dogs with pancreatitis also had icterus, 30 % had ards, 30 % had imha, 15 % had arf, while 10 % had haemoconcentration and another 10 % had cerebral syndrome concomitant with the pancreatitis. it is postulated that pancreatitis is the formerly described ‘gut’ form of babesiosis (mohr, lobetti & van der lugt 2000). babesia vogeli causes a moderate, often clinically unapparent infection in mature dogs. the parasitaemia in b. vogeli also seems to be very low and as such the infection may frequently be missed during the routine examination of a blood smear. subclinical infections are common in adult dogs infected with b. vogeli, but puppies tend to present with marked anaemia (irwin & hutchinson 1991). this is mostly a disease that is endemic in greyhound kennels in the usa and particular care should be taken when relocating greyhounds that can harbour subclinical infections. babesia canis infections result in a more variable pathogenicity, intermediate between b. rossi and b. vogeli. a recent study from italy, presumably describing b. canis infection, reported anaemia in the majority of dogs and thrombocytopaenia in all cases (furlanello, fiorio, caldin, lubas & solano-gallego 2005). babesia gibsoni infection may follow a hyper-acute, acute or chronic course. the acute course is the most common, and is characterized by fever, lethargy, haemolytic anaemia, thrombocytopenia, lymphadenopathy and splenomegaly (conrad, thomford, yamane, whiting, bosma uno, holshuh & shelly 1991). the hyper-acute state is rare and is characterized by shock and extensive tissue damage. this is mostly a disease of american pitbull and staf fordshire bull terriers that is putatively transmitted via dog bites (birkenheuer et al. 2005). dogs with subclinical b. gibsoni infections have been reported in australia and the usa, where they are pcr positive, but neither show microscopic parasitaemia, nor clinical illness (macintire, boudreaux, west bourne, wright & conrad 2002). such cases can have dire consequences if imported into non-endemic areas. much of the apparent discrepancy in the clinical picture of b. gibsoni infections in the usa has lately been explained when distinct genotypical differences have been detected between the californian and the asian isolate occurring in the mid-western usa. the californian isolate seems to be a more virulent species and is found in breeds other than pitbull terriers (kjemtrup et al. 2006). diagnosis diagnosis of acute cases infected with b. canis is based on the classic clinical presentation and the 62 canine babesiosis demonstration of the parasites within red blood cells on diff-quick stained, thin capillary blood smears. the large babesias are typically seen as paired, 2.4 x 5 μm-sized bodies, although some red blood cells can contain up to 6–8 pyriform to round bodies. babesia gibsoni, the small babesia of dogs, is typically found as single, annular bodies measuring 1 x 3.2 μm. the recognition of these small parasites require good staining technique and considerable practice, as many red blood cells in anaemic dogs are vacuolated and pitted. blood smears are usually taken from the ear margin. the degree of parasitaemia can differ from 0.05–10 % of counted red blood cells, depending on the virulence of the species and has been found to be higher when patients are co-infected with ehrlichia spp. (van heerden, reyers & stewart 1983). due to the virulence of b. rossi, the mere presence of parasites in one red blood cell is enough to confirm the diagnosis. in this species, as well as in b. vogeli, parasitaemia has been shown to be higher in capillary than in central blood at presentation (irwin & hutchinson 1991; böhm, leisewitz, thompson & schoeman 2006). ironically, the converse has been shown when a higher parasitaemia was found in central blood rather than capillary blood in dogs experimentally infected with b. vogeli (ewing 1966). in more chronic cases, due to less virulent species such as b. canis and b. vogeli, where parasitaemia may be below the microscopic detection limit, diagnosis is more problematic and a presumptive diagnosis is often based on suggestive historical findings, physical examination findings and positive indirect fluorescent antibody titres or pcr. thick smears (not alcohol fixed) may be helpful in detecting the parasite in cases of very low parasitaemia. another way in which to increase the likelihood of finding parasites is to search along the periphery of the blood smear, as parasitized red blood cells tend to marginate during the making of the smear. recent or active infection can be confirmed by the demonstration of increasing antibody titres over 2–3 weeks. an improved elisa has recently been developed that is able to distinguish between infection with b. gibsoni and the b. canis species on serology alone (verdida, hara et al. 2004). some sub-clinical infections require bone marrow aspirate cytology or smears prepared from red cells just below the buffy coat to demonstrate the parasite. a diagnosis should not be based solely on seropositivity, because clinically normal dogs in or from endemic areas can be seropositive. the high sensitivity and specificity of a newly developed pcr probe should allow the detection of low parasitaemias in subclinically infected cases and may be the most useful test in screening dogs newly imported into babesia-free countries. the rest of the blood smear findings are classically those reflecting the underlying regenerative, haemolytic anaemia and include marked anisocytosis, poly chromasia, reticulocytosis and normoblastaemia. it takes the bone marrow approximately 3–5 days to respond to an acute red blood cell breakdown and, therefore, the blood smear findings in acute cases may appear to reflect those of a nonregenerative anaemia. thrombocytopenia is a hallmark of the disease, regardless of the babesia spp. involved and is often marked, but yet petechiation or epistaxis is very rarely seen, except in cases with concomitant ehrlichia infections (furlanello et al. 2005; kettner, reyers & miller 2003). the pathophysiology of the thrombocytopenia remains undetermined, but sequestration or consumption is postulated. other haematological findings may include spherocytosis, in cases with secondary immune-mediated haemolytic anaemia and a left shift neutrophilia due to the marked systemic inflammatory response. acute babesiosis can easily be misdiagnosed as prim ary immune-mediated haemolytic anaemia, espe cially if parasitaemias are low, only central venous blood is submitted or when laboratory personnel and clinicians are unfamiliar with the appearance of the parasites. in this regard a positive antiglobulin (coombs’ test) may add to the confusion, since most b. canis and b. gibsoni infected dogs have been found to be positive with this test. the same diagnostic conundrum has been found in b. gibsoni-infected cases with thrombocytopenia, in the absence of detectable parasitaemia. it is important to remember that thrombocytopenia in these infections precedes the onset of anaemia and parasitaemia. further laboratory findings include elevation of liver enzymes such as alp, alt and ast—more so in the patients with marked icterus, reflecting the concomitant hepatopathy in these cases. serum potassium is often low, especially in icteric cases. serum bilirubin concentrations are elevated, commensurate with the degree and rapidity of onset of the anaemia and the severity of the accompanying he pat opathy. azotaemia is present in many dehydrated cases and in those with acute renal failure (lo betti & jacob son 2001). urea is frequently disproportionately raised to creatinine and is elevated for reasons other than renal disease (de scally, leisewitz, lobetti & thompson 2006). urinalysis may show bili63 j.p. schoeman rubinuria, haemoglobinuria, proteinuria, renal tubular epithelial cells and granular casts. acid base abnormalities are common with metabolic acidosis (due to raised lactate and raised chloride ion concentration) and respiratory alkalosis being most common. severe disease almost always causes mixed acid base imbalances (leisewitz et al. 2001). treatment the primary goals are to eliminate the parasite and reverse the life-threatening anaemia. diminazene aceturate, trypan blue and imidocarb dipropionate are effective against b. canis (table 2). mildly to moderately affected dogs normally make an uneventful recovery following antibabesial therapy alone. the epidemiology of the disease seems to suggest that dogs do develop a state of premunition after repeated infections. however, in non-endemic areas, it would be ideal to sterilise the infection and therefore render the dogs non-infective for ticks, in order to prevent the organism from becoming established in that country. imidocarb dipropionate at a dose of either 7.5 mg/kg once off or 7 mg/kg given twice with a 14-day interval have been shown to sterilise the infection. diminazene dose calculation should be meticulous due to its low therapeutic index, especially in puppies and the drug should not be repeated within an interval of shorter than a 3 week period (miller, swan, lobetti & jacobson 2005). the absolute minimum interval might be longer in certain breeds and individuals, which appear to show a propensity to develop severe cerebral toxicity with classic cerebellar sulci haemorrhages. in the author’s experience this complication is fortunately quite rare. trypan blue is one of the oldest drugs, but is still used in some parts of the world. neither diminazene, nor trypan blue is capable of sterilising the infection and their use in non-endemic areas is therefore discouraged. the first treatment that has been shown to be effective against b. gibsoni is a combination of atovaquone and azithromycin (birkenheuer, levy & breitschwerdt 2004). these parasites are very difficult to clear with conventional therapy and dogs usually become chronic carriers or present with recurrent episodes of acute babesiosis. markedly anaemic animals or those with any of the abovementioned complications require a variety of supportive treatments, depending on the severity of the case and range from single to multiple blood transfusions, corticosteroid drug administration for the concomitant immune-mediated red blood cell destruction, aggressive colloidal and crystalloid fluid support with added electrolytes such as potassium chloride and diuretics in cases with acute renal failure. assisted respiration is frequently necessary for cases with pulmonary oedema due to acute respiratory distress syndrome. cross matching is not necessary for the first transfusion in dogs, because dogs do not have naturally occurring allo-antibodies. to further minimize delayed transfusion reactions or the risk of sensitising patients that might invariably require future transfusions in endemic areas, only dea 1.1 and 1.2 negative donors are used. whole blood can safely be used in cases of canine babesiosis. the decision to administer a blood transfusion is a multi-facetted one and is not determined solely by the patient’s haematocrit, but rather by the clinical condition of the animal and the rapidity of onset of the anaemia. table 2 drugs used in the treatment of babesiosis babesia spp. drug dose route of administration duration of therapy b. canis diminazene aceturate (berenil®) 3.5 mg/kg im/sc one dose b. canis trypan blue (trypan blue®) 10 mg/kg strictly iv one dose followed by imidocarb or diminazene 1 week later b. canis imidocarb dipropionate (imizol®) 7 mg/kg im two doses, 14 days apart b. gibsoni atovaquone and azithromycin 13.3 mg/kg q 8 h 10 mg/kg q 24 h po po 10 consecutive days b. felis primaquin phosphate 0.5 mg/kg im daily for 1–3 days 64 canine babesiosis as such, dogs with haematocrits as low as 0.07 ℓ/ℓ can survive without transfusion, whereas some dogs with haematocrits as high as 0.20 ℓ/ℓ have to be transfused. given the stringent criteria for donor selection and the significant owner and veterinarian effort needed to procure safe blood, it should be con sidered a precious resource. due to this and other equally cogent arguments, the use of component therapy (i.e. red blood cells or plasma) has gained favour, allowing a single unit of blood to meet the needs of several patients. acute canine babesiosis cases are not necessarily volume depleted and mostly require oxygen-carrying capacity only (in some cases with arf or ards volume expansion may actually be harmful), and thus benefit most from the use of packed red blood cells. blood is initially transfused slowly (2 mℓ/kg/h) for the first 30–60 min, while observing the patients for transfusion reactions, such as a sudden rise in body temperature and/or respiratory rate and lip and ear pinna swelling. prognosis the prognosis is generally quite good, with approximately 85–90 % of cases surviving the illness, depending on the level of care and the babesia spp. involved (keller et al. 2004; schoeman, rees & herrtage 2007). the use of blood transfusions has a major impact on survival in severely anaemia animals. cases with haemoconcentrated babesiosis and cases developing acute renal failure, acute respiratory distress syndrome or cerebral babesiosis have the worst prognosis and mortality can be greater than 50 %—in some cases approaching 100 %, despite intensive, technically advanced interventions (welzl et al. 2001). lactate concentrations of > 5 mmol/ℓ on admission are associated with increased mortality, but lactate > 2.5 mmol/ℓ and/or increase after admission and/or failure to decrease to < 50 % of admission value, is strongly predictive of death. lactate persistently > 4.4 mmol/ℓ strongly predicts death and lactate > 4.4 mmol/ℓ at 24 h after admission correctly predicts death in every case. hyperlactataemia is present in approximately half of hospitalised cases. extremely high values (> 10 mmol/ℓ) are not uncommon. lac tate correlates negatively with glucose. hyperlactataemia is also positively associated with clinical collapse (nel, lobetti, keller & thompson 2004). acute renal failure (arf) is rare. renal involvement in babesiosis is diagnosed on the basis of persistently elevated creatinine despite appropriate fluid therapy. renal involvement in babesiosis resembles the functional renal failure of sepsis. oliguria is an ominous sign (lobetti & jacobson 2001). hypotension is common in babesiosis and is associated with clinical collapse (jacobson, lobetti & vaughan-scott 2000). clinical collapse is also associated with hypoglycaemia (keller et al. 2004). this appears to be a very simple and useful prognostic factor (böhm et al. 2006). a small study, comparing 3 dogs that died naturally with 31 other dogs, showed higher cardiac troponin i values in the non-survivors. this suggested that high cardiac troponin i values are associated with a poor outcome. from these results, ctni also correlated with disease severity (lobetti, dvir & pearson 2002). hypoglycaemia (glucose < 3.3 mmol/ℓ) is present in c. 20 % of cases. hypoglycaemia is associated with stupor and collapse. miosis disappears after dextrose infusion. dogs that are collapsed (non-ambulatory) at presentation have an 18-fold increased risk for hypoglycaemia. pups less than 6 months of age are at risk of developing hypoglycaemia. the risk is slightly increased in dogs with severe anaemia, icterus or vomition. response to treatment with intravenous dextrose is sometimes dramatic. hypoglycaemia is an important differential diagnosis for cerebral babesiosis. when glycaemic status is taken into account, the incidence of true cerebral babesiosis is very low (keller et al. 2004). timing, source of blood (venous versus capillary) and counting method cause large variability in parasite counts. a significant positive correlation between high parasitemia and mortality has recently been demonstrated. there is also a significant positive correlation between capillary and venous parasitaemia, with capillary parasitaemias being consistently higher than venous ones, making the capillary the preferential site for obtaining diagnostic smears (böhm et al. 2006). at presentation to the hospital, dogs that died had a significantly higher median cortisol concentration (482 nmol/ℓ) than other admitted dogs that survived (150 nmol/ℓ). conversely, the total and free thyroxine concentrations in all but one of the dogs that died were below the limit of detection and significantly lower (2.7 nmol/ℓ and 0.12 pmol/ℓ, respectively) than in other admitted dogs that survived (7.4 nmol/ℓ and 0.4 pmol/ℓ, respectively). six of 11 dogs (55 %) with a serum cortisol above 400 nmol/ℓ died, whereas all dogs with admission serum cortisol concentrations less than 260 nmol/ℓ survived (schoe65 j.p. schoeman man et al. 2007). in addition, dogs that died also had increased acth-stimulated cortisol concentrations, indicating that acute up-regulation of adrenal function is associated with poor outcome (schoeman & herrtage 2008). outcome is not affected by systemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome positive status, nor the number of organs involved, but rather by the organ system affected, such as the lungs, kidneys or brain (welzl et al. 2001). prevention regular control of the tick vectors by routinely dipping or spraying pets or using tick collars or spot-on preparations is the only effective way of preventing this disease in most parts of the world. ticks must feed on the host for at least 3 days in order to transmit b. rossi and owners should therefore be encouraged to examine their dogs daily for the presence of ticks. a vaccine against b. rossi has recently become commercially available in south africa (nobivac piro®) and research into developing vaccines for all species of canine babesiosis is ongoing. blood donors in endemic areas should be regularly tested to prevent the spread of the disease via blood transfusion. acknowledgements acknowledgement is due to all the lecturers and res idents, past and present, at the department of companion animal clinical studies at onderstepoort and to all tireless babesiosis researchers worldwide, who have over many 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journal of veterinary medical science, 66:1517–1521. welzl, c., leisewitz, a.l., jacobson, l.s., vaughanscott, t. & myburgh, e. 2001. systemic inflammatory response syndrome and multiple-organ damage/dysfunction in complicated canine babesiosis. journal of the south african veterinary association, 72:158–162. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: carin i. boshoff1,2,3 armanda d.s. bastos2 mzwandi m. dube4 livio heath1 affiliations: 1agricultural research council-onderstepoort veterinary institute, transboundary animal diseases programme, south africa 2mammal research institute, department of zoology & entomology, university of pretoria, south africa 3department of biomedical sciences, tshwane university of technology, south africa 4central veterinary laboratory, ministry of agriculture, swaziland correspondence to: carin boshoff email: boshoffci@tut.ac.za postal address: private bag x68, pretoria 0001, south africa dates: received: 09 sept. 2014 accepted: 03 oct. 2014 published: 03 dec. 2014 how to cite this article: boshoff, c.i., bastos, a.d.s., dube, m.m. & heath, l., 2014, ‘first molecular assessment of the african swine fever virus status of ornithodoros ticks from swaziland’, onderstepoort journal of veterinary research 81(1), art. #846, 5 pages. http://dx.doi.org/10.4102/ojvr.v81i1.846 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. first molecular assessment of the african swine fever virus status of ornithodoros ticks from swaziland in this research communication... open access • abstract • introduction • materials and methods    • swaziland as the study area    • study design    • tick collection    • molecular analysis • results • discussion • conclusion and future research • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ african swine fever (asf) is an economically significant haemorrhagic disease of domestic pigs. it is caused by the african swine fever virus (asfv), a deoxyribonucleic acid (dna) arbovirus. argasid ticks of the genus ornithodoros, which are widely distributed throughout southern africa, play a primary role in virus maintenance and spread within the endemic sylvatic cycle. the asf status of swaziland is unknown, but this land-locked country is surrounded by asf-positive countries, has a burgeoning pig industry and sylvatic cycle hosts present within its borders. in this first assessment of asf status, warthog burrows in seven nature reserves and game management areas in swaziland were investigated for tick and virus presence. tick infestation rates of between 33.3% – 88.8% were recovered for the four ornithodoros-infested reserves. a total of 562 ticks were screened for virus genome presence using a duplex polymerase chain reaction (pcr) that targets the c-terminal end of the p72 gene of the asfv and confirms dna integrity through amplification of the 16s rrna tick host gene. all samples were negative for virus genome presence and positive for the tick genome target. nucleotide sequencing of the latter confirmed that ornithodoros ticks from swaziland are identical to those from the kruger national park in south africa across the gene region characterised. whilst this first evaluation of asf presence in swaziland indicates that the virus does not appear to be present in the key virus vector, the presence of sylvatic cycle hosts, together with the country's proximity to asf-affected countries calls for expanded investigations and regular monitoring of the asf status of swaziland. introduction top ↑ african swine fever (asf) is an infectious viral haemorrhagic disease of domestic pigs. it is caused by the african swine fever virus (asfv), the the only known dna arbovirus and sole member of the asfarviridae family (king et al. 2012). warthog-associated argasid ticks of the genus ornithodoros, which are widely distributed throughout southern africa, play a primary role in maintenance and spread of the virus within the endemic sylvatic cycle, and virtually all regions in which the sylvatic hosts co-occur are asf-positive (jori & bastos 2009). the sylvatic cycle of asfv established between wild suids and ticks allows for the circulation and perpetual maintenance of the virus. currently, no vaccines are available to control the disease and prevention relies on regulated control zones such as the one in south africa (figure 1a), and on strict bio-security measures that prevent contact between domestic pigs and ornithodoros ticks (penrith, thomson & bastos 2004). figure 1: map indicating (a) swaziland surrounded by the african swine fever control zone (indicated in red) and (b) the topography of swaziland, the game reserves sampled in this study and ornithodoros tick presence or absence. swaziland is situated between the kwazulu-natal and mpumalanga provinces of south africa, to the south and north, respectively; it shares its eastern border with mozambique (figure 1a). an important objective of livestock development in swaziland is to increase animal production to meet the growing demand for animal protein. recent increases in small-scale pig farming, particularly in communities in close proximity to game reserves containing sylvatic cycle hosts, has signalled the need for assessment of asf status, as it is an important but unevaluated constraint for pig production. no prior surveys have been conducted; the asf status of swaziland is currently unknown and assumed negative, despite the presence of both invertebrate and wild suid species associated with the sylvatic cycle and the encirclement of this land-locked country by asf-positive regions. as such, swaziland is currently considered to be an area that is under constant threat of incursion, but if found to be asf-positive, can potentially also serve as an uncontrolled reservoir of infection. this present survey was therefore undertaken to determine the distribution of sylvatic cycle ornithodoros ticks and possible presence of the virus in this invertebrate reservoir. the results of the study are crucial for formulation of appropriate disease-control strategies for this and neighbouring countries, and complement ongoing efforts by the swaziland government and the food and agriculture organization (fao), with support of the european union (eu), to reverse the country's declining agricultural productivity and to increase pig production (fao 2011). materials and methods top ↑ swaziland as the study area swaziland is the smallest country in africa, and is bordered in the north, west and south by south africa and by mozambique in the east. it has a land area of just over 17 000 km2 and contains four separate geographical regions that run from north to south. these regions vary in altitude from 1800 m.a.s.l. – 200 m.a.s.l. along the eastern border with mozambique, the lubombo mountain ridge has an altitude of around 600 m. along the north-western border, the rainforest highveld region, with an average altitude of 1200 m, lies on the edge of the escarpment; the middleveld has an average altitude of 700 m.a.s.l., with the lowveld of swaziland being around 250 m.a.s.l. swaziland's climate ranges from temperate to subtropical. the weather depends on the region of the country, with the lowland areas being subtropical, with temperatures reaching up to 42 °c during peak summer, whereas at higher altitudes in the highveld regions, temperatures in summer average 20 °c and the weather is cloudy and cool. rain falls mostly during the summer months, with the winter months of june–august being dry. annual rainfall is highest on the highveld in the west, with between 1000 mm – 2000 mm being recorded, with the lowveld in the east recording between 500 mm – 900 mm per annum (atlapedia 2014). study design in swaziland, there are national, private and community-owned protected areas including national parks, nature reserves, wildlife sanctuaries and game reserves. for the present study, access was granted for sampling in seven nature reserves and game parks (table 1), and included parks located near the borders with south africa and mozambique. these parks form part of the songimvelo-malolotja transfrontier conservation areas (tfca) and the lubombo conservancy, which incorporate south africa and mozambique, respectively. as warthog censuses have not been performed, exact numbers of these sylvatic vertebrate host species are unknown for the seven parks included in the present study. table 1: game parks and infestation rates of warthog burrows with ornithodoros ticks in swaziland. in order to ensure consistency between sampling opportunities, a single team consisting of a sampling team leader and field assistant performed the collection under the field guidance of a knowledgeable ranger at each specific park. estimated collection days per park were determined based on the size of a park, with a maximum of 5 days and a minimum of 2 days being allocated for hlane (22 000 ha) and mbuluzi (3000 ha), respectively. storm water pipes on dirt and tarred roads, and anthills near roads, which can both serve as overnight warthog shelters, as well as warthog burrows in the field, were inspected for tick presence. tick collection ticks were collected according to a modified manual collection method (jori et al. 2013). briefly, sand was collected from each burrow and spread in a thin layer across a large black plastic sheet, which was placed in the sun. as soft ticks are photophobic and do not tolerate warm temperatures, this encourages ornithodoros tick movement and facilitates tick detection and collection. a minimum time span of 30 min per burrow was allowed, to ensure that tick movement was elicited and that all visible ticks were collected. ticks were hand-picked, labelled and transported to the onderstepoort veterinary institute, transboundary animal disease programme (ovi-tadp) laboratory for analysis, in sealed containers, under the necessary permits. individual ticks were temporarily incapacitated by refrigeration, photographed and sorted microscopically into the various nymphal stages. adult ticks were sorted on the basis of gender, and each tick was placed in an individual, labelled tube. molecular analysis individual ticks were weighed and placed into 1.5 ml eppendorf tubes and frozen for 10 min at -86 °c prior to the addition of phosphate-buffered saline (pbs). each tick was ground with a pestle and dna was extracted from 100 µl of the 10% weight in volume (w/v) homogenate using a modified silica-guanidium thiocyanate method (boom et al. 1990). a duplex polymerase chain reaction (pcr), incorporating a tick mitochondrial 16s gene target as internal control, was performed when screening for asfv genome presence (bastos et al. 2009). by incorporating primers that target the p72 gene of the asf virus genome and those that target a conserved region of the mitochondrial 16s rrna gene of ornithodoros ticks, presence of virus genome and nucleic acid integrity could, respectively, be determined. genomic amplification was performed in a final volume of 50 µl in the presence of 0.2 mm dntp, 0.5 µm of each primer, 1u thermostable go taq g2 dna polymerase (promega corp., madison, wi, usa) and 3 µl of each dna extract. thermal cycling conditions prescribed for c-terminal p72 gene amplification (bastos et al. 2003) were used. the pcr products were evaluated by 1.5% agarose gel electrophoresis against a 100-bp ladder (promega corp., madison, wi, usa). in this assay, amplification of a 478 bp band confirms virus presence and amplification of the 313 bp host gene target verifies dna integrity. the dna fragments of the expected size were excised from the gel and purified. nucleotide sequences were generated for the tick mitochondrial gene target (inqaba biotechnologies, pretoria, south africa) of a geographically representative subset of tick samples. the resulting partial 16s rrna sequences, submitted to genbank under accession numbers kj940548–kj940550, were complemented with homologous data prior to phylogenetic inference in mega 5 (tamura et al. 2011). results top ↑ ornithodoros ticks were found to be present in four of the seven game reserves that were sampled (table 1 and figure 1b). tick infestation rates varied from 33.3% – 88.8% between the four tick-infested parks (table 1). a total of 562 ticks were collected, of which 105 were female, 113 were male and the remaining 344 were nymphs. all ticks tested by duplex pcr were positive for the host 16s rrna gene, but negative for asfv, suggesting that despite the presence of sylvatic cycle hosts, the virus appears to be absent from ornithodoros ticks in the game reserves that were evaluated. discussion top ↑ ticks were only observed in parks situated in the eastern region of the country and were absent in the parks in the mountainous western region. swaziland has a bowl-like terrain, being predominantly mountainous with forest escarpments in the north-western side, flattened throughout the centre of the country and becoming mountainous in the north-east along the mozambique border. the game reserves that were positive for ornithodoros tick presence all fell within the eastern savannah area and were associated with lower altitude localities (figure 1b). despite intensive searching and re-visiting of some of the parks, warthog burrows proved difficult to locate, particularly in the western half of the country, which is mountainous and densely vegetated. these sampling difficulties and the high altitudes likely underlie the comparatively lower number of burrows sampled in mlilwane and nisela, and the relatively low overall tick numbers from western swaziland. similar difficulties were encountered in malawi, where large differences in occurrence of ornithodoros ticks were observed, leading the authors of that study to speculate that altitude may be an important factor influencing tick distribution (haresnape & mamu 1986). the slight male bias in adult ticks collected in swaziland is consistent with previous studies (arnot, du toit & bastos 2009; thomson et al. 1983), but the adult to nymph ratio of 39%:61% was nearly three-fold higher than the average adult to nymph ratio of 13%:87% from a previous tick survey conducted in south africa (thomson et al. 1983). this adult bias is important, as there is an on average nine-fold higher infection rate in adults versus nymphs (calculated based on results of the thomson et al. 1983 study). this, together with the 1.4 fold higher sensitivity of pcr versus isolation (basto et al. 2006), mitigates concerns regarding the adequacy of the swaziland sample size and underscores the advantages of performing adult-tick-biased surveys (arnot et al. 2009). sequencing of the host mitochondrial gene of ticks sampled from three infested reserves was used to confirm the ornithodoros porcinus species designation and to evaluate phylogeographical variation through comparison to reference data from southern and east africa. bastos et al. (2009) identified three geographically distinct o. porcinus lineages, with south african samples clustering within the south-east lineage. the gene phylogeny (figure 2) confirmed that the sampled soft ticks from hlane, mlawula and mbuluzi game reserves in swaziland are identical to each other and all fall within the geographically distinct o. porcinus south-east lineage (bastos et al. 2009). the ticks from swaziland had 100% and 99.63% pairwise sequence identities to ticks from two south african localities, namely the kruger national park (knp) and mkuze game reserve (mgr), respectively, across the gene region sequenced. figure 2: neighbour joining tree depicting 16s rrna gene relationships of ornithodoros ticks sampled from three swaziland wildlife reserves. swaziland is surrounded by countries and regions where asf has previously been reported. in mozambique, bordering swaziland to the east, disease control is challenging and the disease is known to be endemic in the country (penrith et al. 2007). in south africa, the disease is endemic in mpumalanga province, which borders swaziland to the north, and measures that prevent contact between pigs and sylvatic hosts of asfv are implemented with success to prevent spread of the disease. in a study performed in mgr, in kwazulu-natal province, which borders swaziland to the south, it was shown that the virus was not present in any of the ticks sampled, signalling a possible change in the asf status of this game reserve since the last survey in the late 1970s (arnot et al. 2009). together, these negative results from swaziland and mkuze, once substantiated with additional, more extensive studies, will assist in delineating, with greater confidence, the southernmost distributional range of asf-positive ticks in south africa. swaziland has an estimated population of 42 000 domestic pigs, but as the country is presumed to be asf negative, there are no bio-security measures in place to prevent the spread of the disease. as asf is an economically important disease with implications for all burgeoning pig industries across sub-saharan africa (penrith et al. 2013), the apparent absence of the virus indicated by this preliminary pcr-based tick assessment suggests that swaziland should continue to focus efforts on minimising incursions in order to retain this status. conclusion and future research top ↑ fifty-eight burrows were examined for the presence of ornithodoros ticks from seven nature reserves in swaziland. a total of 562 ticks were tested for the presence of asfv. the relatively low number of warthog burrows and ticks was likely due to topography and to the relatively low numbers of warthogs in the parks, as no warthogs were observed by the sampling team. the hypothesis that asfv may be absent from sylvatic cycle hosts in swaziland needs to be more fully investigated. it is proposed that parks in adjacent countries forming part of the trans-frontier parks be sampled to confirm the presence or absence of the ticks and the virus. the preliminary results highlight the need for additional research, including serological surveys of warthogs in all the parks and of domestic pigs across the country, and testing of these wild suids and domestic pigs for prior exposure to ornithodoros ticks with an enzyme-linked immunosorbent assay (elisa) capable of detecting optsgp1 argasid salivary protein antibodies (díaz-martín et al. 2011) in order to clarify all epidemiological aspects that impact asf status in swaziland. acknowledgments top ↑ the authors wish to thank the ministry of agriculture, swaziland for permission to perform this study, jason kilian and sean nord for providing field assistance, and lara nicholson and eduard roos for laboratory assistance. this research was supported by european community's seventh framework programme (fp7/2007-2013) under grant agreement kbbe211691asfrisk. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.i.b. (agricultural research council-onderstepoort veterinary institute) performed all of the experiments and prepared the manuscript. a.d.s.b. (university of pretoria) assisted with the data analysis and preparation of the manuscript. m.m.d. (central veterinary laboratory) was responsible for sample collection approval and coordinating the fieldwork in swaziland. l.h. 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s., 2011, ‘mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods’, molecular biology and evolution 28, 2731–2739. http://dx.doi.org/10.1093/molbev/msr121 thomson, g., gainaru, m., lewis, a., biggs, h., nevill, e., van der pypekamp, h. et al., 1983, ‘african swine fever. the relationship between african swine fever virus, the warthog and ornithodoros species in southern africa’, in p.j. wilkinson (ed.), commission of the european communities, report eur 2466 en, pp. 85–100. allsopp_81-88.indd introduction heartwater is a lethal bacterial disease of domestic and some wild ruminants. it is caused by the intracellular rickettsia ehrlichia ruminantium which is transmitted by ticks of the genus amblyomma, and the disease is prevalent wherever the tick vectors occur (provost & bezuidenhout 1987). the endemic area includes the whole of sub-saharan africa, and also the french antillean islands of guadeloupe, antigua and marie galante, to which infected amblyomma variegatum ticks were introduced probably in the eighteenth century (maillard & maillard 1998). in the sub-saharan region the disease is one of the major causes of stock losses, and it has been estimated that more than 150 million animals are at risk in the area (minjauw & mcleod 2003). typically the infection causes a high fever, nervous signs, hydropericardium, hydrothorax and oedema of the lungs and brain, and death. the first known reference to what was subsequently thought to have been heartwater was made in 1838 by louis trichardt (neitz 1947). while trekking through the northern province of south africa many of his sheep died 3 weeks after they had suffered a severe tick infestation. sixty years later dixon (1898) and edington (1898) showed that the disease could be passaged by the transfer of blood from infected to susceptible animals, and lounsbury (1900) confirmed that the vector in south africa was amblyomma hebraeum. heartwater at onderstepoort when the onderstepoort veterinary research institute was opened in 1908 the director, sir arnold theiler, had been working on the disease since at least 1904 (theiler 1904). theiler continued to work on heartwater, and in 1925 he invited the canadian 81 onderstepoort journal of veterinary research, 76:81–88 (2009) trends in the control of heartwater b.a. allsopp department of veterinary tropical diseases, faculty of veterinary science, university of pretoria private bag x04, onderstepoort, 0110 south africa abstract allsopp, b.a. 2009. trends in the control of heartwater. onderstepoort journal of veterinary research, 76:81–88 heartwater is an economically serious tick-borne disease of ruminants caused by the intracellular bacterium ehrlichia ruminantium. the disease has traditionally been controlled by four different approaches: controlling the tick vector by dipping, establishing endemic stability, performing immunisation by infection and treatment, and preventing the disease by regular administration of prophylactic antibiotics. the first three of these methods are subject to failure for various epidemiological reasons, and serious disease outbreaks can occur. prophylaxis is effective, but very expensive, and the logistics are daunting when large herds of animals are involved. the development of a safe, cheap and effective vaccine is the only likely way in which heartwater can be economically controlled, and over the past 15 years three new types of experimental vaccine have been developed: inactivated, attenuated, and recombinant vaccines. these new vaccines have shown varying degrees of promise, but none is as yet sufficiently successful to be marketable. we describe the experimental products, and the various technical and biological difficulties which are being encountered, and report on ways in which new technologies are being used to improve vaccine effectiveness. 82 control of heartwater rickettsiologist edmund cowdry to onderstepoort to investigate the disease. cowdry demonstrated the causative organism in the tissues of infected ruminants and ticks (cowdry 1925a, b) and initially he named it rickettsia ruminantium. the name was later changed to cowdria ruminantium (moshkovski 1947) and much more recently molecular phylogenetic evidence has shown that the heartwater agent is in fact a species of ehrlichia which is now known as e. ruminantium (dumler, barbet, bekker, dasch, palmer, ray, rikihisa & rurangirwa 2001). work on heartwater has continued at onderstepoort up until the present day, and some notable advances have been made. in 1945 the introduction of antibiotics led to the development of a method of control by infection and treatment which is still in use (neitz & alexander 1945). in 1985 a technique for the continuous in vitro culture of the organism was introduced after at least 50 years of effort (bezuidenhout, paterson & barnard 1985). most recently the sequencing and annotation of the e. ruminantium genome has been completed (collins et al. 2005), the first whole genome to be determined in africa. two new experimental vaccines have also been developed during recent years, a recombinant vaccine in 2003 (collins, pretorius, van kleef, brayton, allsopp, zweygarth & allsopp 2003) and an attenuated vaccine in 2005 (zweygarth, josemans, van strijp & steyn 2005). control methods for heartwater the current methods used for heartwater control include the use of acaricides to control the tick vector, antibiotic prophylaxis (peregrine 1994), immunization by infection and treatment (van der merwe 1987), farming with animal breeds resistant to the dis ease (camus, maillard, ruff, pepin, naves & matheron 1996), and establishment of endemic stability (tice, bryson, stewart, du plessis & de waal 1998). all of these methods have serious drawbacks. prophylactic antibiotic treatment is widely used by commercial angora goat farmers in the eastern cape province of south africa because these animals are highly susceptible to heartwater. it is, however, a very expensive option, the logistics are daunting and there is always the likelihood that antibiotic resistance may develop. tick control with acaricides, although also expensive, has been in use for more than a century (dixon 1899) and it was in the past very effective. today, however, acaricide resistance is widespread and the environmental pollution caused by the chemicals is generally deplored. also of serious concern is the fact that animals undergoing an intensive dipping regimen lose any immunity to the disease and subsequent tick control failures can lead to catastrophic heartwater losses, as occurred in zimbabwe in the 1970s (norval 1979). the establishment of endemic stability, using limited dipping to control ticks to manageable levels, can be highly successful, but if herd immunity breaks down then serious disease outbreaks can occur. this can happen if new immunotypes enter an area where immunity to the previously prevalent strains offers no or limited protection, and such a situation is very likely to occur as widespread genetic exchange is known to take place in the field between different strains of e. ruminantium (allsopp & allsopp 2007). ultimately the only long term control method which will be cost effective is immunization, and an infection and treatment regimen has been widely used in southern africa since its development at onderstepoort in 1945 (neitz & alexander 1945). this ‘vaccine’ is commercially available and it consists of a cryopreserved preparation of blood taken from a sheep infected with virulent e. ruminantium organisms of the ball 3 genotype (haig 1952). the blood is injected intravenously into the animals to be immunized, their temperatures are monitored daily, and antibiotic treatment is administered when the infection becomes established, but before serious disease occurs. there are several practical disadvantages to this procedure. firstly, e. ruminantium organisms rapidly lose infectivity even at 4° c (brayton, collins, van strijp & allsopp 2003), so the infective blood must be preserved on dry ice and thawed immediately before inoculation which makes the infection and treatment method inappropriate for use in rural areas. secondly, the procedure must be supervised by trained staff because of the need for intravenous injection and subsequent careful monitoring of the animals’ condition. thirdly, because live organisms are involved the procedure cannot be used in areas where heartwater is not endemic. the most serious disadvantage, however, is that the duration and effectiveness of immunity is uncertain. the ball 3 genotype used in this procedure is known to offer only limited protection against some common virulent genotypes (collins et al. 2003). however, because ball 3 produces a temperature rise two or three days before serious disease is established it is unlikely to lead to stock deaths during the immunization procedure, whereas other common virulent genotypes like welgevonden (du plessis, van gas, olivier & bezuidenhout 1989) can cause death very shortly after a rapid tempera83 b.a. allsopp ture rise and are therefore not suitable for use in infection and treatment. development of improved vaccines there are three possible types of improved vaccine each of which has received extensive attention from researchers: inactivated, live attenuated, and recombinant. we will survey recent developments and future prospects for all three types. inactivated vaccines the first inactivated heartwater vaccine, developed in guadeloupe using the gardel strain of e. ruminantium, was prepared from elementary bodies partially purified from tissue culture material and chemically inactivated using sodium azide (martinez, maillard, coisne, sheikboudou & bensaid 1994). the vaccine was formulated with freund’s adjuvant and used to immunize goats, each of which received an initial and a booster inoculation. when subsequently challenged with homologous tissue culture material 50–80 % of the animals were found to be protected while 100 % of untreated controls died. the same methodology was later used in zimbabwe, where the vaccine was prepared using the crystal springs strain, inactivated with β-propiolactone, and also formulated with freund’s adjuvant. this vaccine was used to immunize sheep, each of which received five inoculations over a period of months, and in this case 50–100 % of the animals were protected against a homologous needle challenge which killed 60 % of untreated controls (mahan, andrew, tebele, burridge & barbet 1995). similar inactivated vaccines have been used over the succeeding 12 years, using different strains of e. ruminantium and different commercially acceptable adjuvants. when a needle challenge with a heterologous strain, or a tick challenge, is given the inactivated vaccines are less protective than in the original experiments (mahan, smith, kumbula burridge & barbet 2001; faburay, geysen, ceesay, marcelino, alves, taoufik, postigo, bell-sakyi & jongejan 2007). protection levels are even lower when the vaccines are tested in the field against a natural tick challenge with organisms of differing immunogenicities. a summary of all the inactivated vaccine field trials conducted by one research group over a period of 8 years showed that overall mortality levels could be reduced, in sheep from 78 % (unvaccinated) to 54 % (vaccinated) and in cattle from 67 % (unvaccinated) to 19 % (vaccinated) (mahan, barbet & burridge 2003). one early difficulty with these inactivated vaccines was that large amounts of material had to be used, in multiple inoculations, and the cost of production from tissue culture was unacceptably high. there have, however, been marked improvements in culture technology for e. ruminantium in recent years, which have put commercial vaccine preparation within reach (marcelino, sousa, verissimo, cunha, carrondo & alves 2006; marcelino, vachiery, amaral, roldao 2007). the problem that remains to be addressed is the disappointing levels of protection achieved against natural tick challenge in the field, especially in africa. the high mortalities of vaccinated animals in this situation are likely to be the result of infection with non-cross protecting immunotypes. it is therefore of great significance for vaccine development to note that there is extensive polymorphism between e. ruminantium strains, particularly in southern africa (allsopp, van heerden, steyn & allsopp 2003; allsopp & allsopp 2007), and moreover there is large-scale recombination between the different genotypes (allsopp, van strijp, faber, josemans & allsopp 2007). so while inactivated vaccines could be useful in the caribbean, where there is limited genetic variability within the e. ruminantium population, it may be difficult to achieve the improvements in protectivity in the african situation which would be required to make these vaccines commercially viable. recombinant vaccines the observation that inactivated heartwater vaccines can stimulate protective immunity indicates that the development of a successful recombinant vaccine, using selected e. ruminantium genes, is theoretically possible. a recombinant vaccine should be cheaper to manufacture, and easier to store than an inactivated vaccine, and recombinant heartwater vaccine development is a very active area of research. the first attempts to develop such a vaccine used the map1 gene of e. ruminantium, cloned into an expression vector together with a viral enhancerpromoter, which was tested for its ability to protect mice against a lethal homologous challenge (nyika, mahan, burridge, mcguire, rurangirwa & bar bet 1998). protection levels ranging from 23–88 % were obtained in several different experiments using this naked dna vector alone, and subsequently attempts were made to improve the protection levels by boosting with recombinant map1 protein (nyika, barbet, burridge & mahan 2002). this procedure enhanced protection levels in mice from a range of 13–27 % without boosting to a range of 53–67 % with boosting. 84 control of heartwater there are some experimental indications that the map1 gene might not be the best choice for recombinant vaccine development. for example, purified map1 protein inoculated into goats and a sheep induced high specific antibody titres but conferred no protection against homologous challenge (van kleef, neitz & de waal 1993). then it must be remembered that e. ruminantium map1 genes are highly polymorphic (allsopp, dorfling, maillard, bensaid, haydon, van heerden & allsopp 2001) so any vaccine using this gene would have to include variants of all important virulent strains. the complete e. ruminantium genome contains 888 annotated genes (collins et al. 2005), any of which are potentially available for use in experimental vaccines, how ever identifying those which code for antigens which stimulate the required protective t-cell response is not a trivial exercise (esteves et al. 2004). one research group has attempted to identify suitable genes by screening e. ruminantium expression libraries (barbet, whitmire, kamper, simbi, ganta, moreland, mwangi, mcguire & mahan 2001). clones were selected whose expression products were both recognized by anti-e. ruminantium antibodies and also stimulated proliferation of peripheral blood mononuclear cells (pbmc) from cattle immunised against e. ruminantium by infection and treatment. the selected clones were expressed in recombinant bacteria and pools of bacterial lysates containing the e. ruminantium gene products were used to immunize mice. some pools of recombinants stimulated protective immunity against homologous challenge in 58–89 % of the immunized mice, levels of protection which were no improvement on those obtained using the map1 gene. a striking feature of all the e. ruminantium immunization trials in mice discussed above is the highly variable levels of immunity which were reported. even more problematic is the observation that some genes confer immunity in the mouse model but this protection is not reproducible in ruminants (louw, brayton, collins, pretorius, van strijp & allsopp 2002; collins et al. 2003). these observations led one laboratory to begin conducting all e. ruminantium vaccination trials in sheep, and the results have been much more reproducible. in some of the most successful experiments a cocktail of four e. ruminantium genes, cloned in a dna vaccine vector and used to immunize sheep, was found to stimulate complete protection against a virulent needle challenge with both homologous, and five different heterologous, e. ruminantium strains (collins et al. 2003). this experimental vaccination was, however, not very effective when used in the field against a natural heartwater-infected tick challenge, when levels of protection only reached 20 %. the four genes in the protective cocktail are thought to be components of an abc transporter system but it was not known whether the protection which they offered was specific to their function. the map1 gene had been shown to provide some protection when presented as a dna vaccine, even though there were reasons to believe that it might not be a good choice for recombinant vaccine development, so further work was carried out to determine how the protection stimulated by the putative abc transporter genes compared with that stimulated by other cocktails of e. ruminantium genes (pretorius, collins, steyn, van strijp, van kleef & allsopp 2007). the protection was shown to be specific to the abc transporter genes, since cocktails of other e. ruminantium orfs presented in the same vaccine system failed to provide any protection. furthermore, the levels of protection stimulated by each of the putative abc transporter genes presented individually was similar to that stimulated by the four-gene cocktail. the protection against field challenge remained poor, however, and this appeared to be a result of markedly enhanced virulence of e. ruminantium organisms injected by the tick, as compared to those which are present in infected mammalian blood (pretorius et al. 2007). heterologous prime-boosting is a well tried strategy for improving dna vaccine protection levels, it involves boosting the initial immunity with recombinant protein or a recombinant viral vaccine vector (vorder meier, lowrie & hewinson 2003; gilbert et al. 2006) enhancing the protection levels of the experimental e. ruminantium recombinant vaccine has been attempted by boosting with either recombinant protein or recombinant lumpy skin disease virus (lsdv) (pretorius, van kleef, collins, tshikudo, louw, faber, van strijp & allsopp 2008). re combinant protein boosting appeared to be more efficient than boosting with rlsdv, since only lymphocytes isolated from animals which received the protein boost showed specific proliferation and increased interferon gamma expression when exposed to the recombinant e. ruminantium proteins. complete protection against an experimental needle challenge was retained, but the levels of protection against an infected tick challenge in the field were not improved at all. in summary, the development of a recombinant vaccine shows great promise, especially when using a prime-boost configuration, but there are problems to be resolved. the algorithms used for selecting 85 b.a. allsopp vaccine candidate genes in silico are currently unreliable, and in vivo stimulation of cytokine secretion or t-cell responses does not correlate with the protective ability of genes. it is also evident that protection against a needle challenge is not a reliable predictor of immunity against a natural tick challenge, so a way must be found to implement a laboratorybased tick challenge to simulate the natural situation in a controlled environment. it is also important to note that candidate vaccines must be tested in ruminants, since tests in the mouse model do not give reproducible results nor can the findings be reliably extrapolated to predict how the vaccine will behave in ruminants. attenuated vaccines attenuated vaccines are the oldest form of vaccine, and as they are based on pathogens which cause no clinical disease, but which retain their immunogenic properties, they can stimulate solid long-lasting immunity. the main difficulty is that traditional methods for attenuating virulent organisms are imprecise processes which take indeterminate lengths of time. the first strain of e. ruminantium to be attenuated was the senegal strain, which attenuated after culture in endothelial cells over 11 passages and 239 days (jongejan 1991). when inoculated into sheep and goats the attenuated organism caused no clinical reaction and the animals were completely protected against a subsequent homologous needle challenge. when field trials were conducted, however, the protection levels were lower, with reductions in mortality, as compared to unvaccinated controls, from 70–43 % in one study (gueye, jongejan, mbengue, diouf & uilenberg 1994), and from 100–25 % in another (faburay et al. 2007). immunity to the senegal strain provides limited protection against other e. ruminantium strains (jongejan, thielemans, briere & uilenberg 1991) while the welgevonden strain, in contrast, provides good cross-protection against a range of other virulent strains (collins et al. 2003). in principle, therefore, the welgevonden strain would be a much better candidate from which to develop an attenuated vaccine, but unfortunately it failed to attenuate after culture through hundreds of passages over several years (gueye et al. 1994; zweygarth, vogel, josemans & horn 1997). a common technique to induce attenuation of a pathogen is to culture it in an abnormal host cell, and the welgevonden strain was induced to infect a canine macrophage-monocyte cell line (dh82) by adding cycloheximide to the culture medium in order to slow the metabolism of the host cells. after continuous propagation in dh82 cells the organism was found to have lost all virulence for mice between passages 25 and 50. since dh82 cells are cancerous and not suitable for vaccine use the attenuated strain was re-adapted to bovine endo thelial cells after 61 passages in dh82 cells (zwey garth et al. 2005). when the re-adapted attenuated welgevonden strain was used to infect sheep or goats there was a brief rise in body temperature, without any other adverse symptoms, and the animals were subsequently found to be fully protected against a lethal needle challenge with both the homologous, and four different heterologous, e. ruminantium strains (zweygarth et al. 2005). further testing of this promising attenuated vaccine has been undertaken (zweygarth, josemans, van strijp & steyn 2008) and a summary of the results in different ruminant species, administered by different routes, is shown in table 1. the intravenous route was the most effective, providing 100 % protection in merino sheep and boer goats, and while the protection in angora goats was only 90 % these animals are known to be exceptionally sensitive to e. ruminantium (du plessis, jansen & prozesky 1983). the protection rate in cattle was also less than ideal, at 83 %, but it should be noted that the table 1 immunising ability of attenuated e. ruminantium welgevonden (after zweygarth et al. 2008) species dose of elementary bodies route challenge protection (%) merino sheep merino sheep merino sheep merino sheep boer goats angora goats friesian cattle 1.1 x 105 various 6.7 x 105 2.6 x 105 various 0.9 x 104–1.3 x 105 1.7 x 105–2.2 x 106 iv iv sc im iv iv iv homologous heterologous (five different strains) homologous homologous homologous homologous heterologous (gardel) 100 100 83 80 100 90 83 note: all animals were needle challenged with blood stabilates of virulent e. ruminantium 86 control of heartwater cattle were challenged with the gardel strain, to which they are very sensitive, because they are not particularly sensitive to the welgevonden strain. attempts to use an intramuscular or subcutaneous route of inoculation led to less effective protection than when using the intravenous route. ideally a vaccine would provide lifelong immunity, and some experiments have been conducted to determine the duration of the immunity which this attenuated vaccine provides. so far these have only been carried out in merino sheep, and the immunity remains at 100 % for at least 6 months, but then falls to 80 % after one year (zweygarth et al. 2008). it is likely, however, that under normal field conditions the natural heartwater challenge would prevent early loss of immunity. estimates made of the possible production costs of this attenuated welgevonden vaccine suggest that the culture costs for 60 sheep vaccine doses would be around us $1. this compares very favourably with the current cost of $27 for 60 doses of ball 3 infected blood for infection and treatment. further development of this promising attenuated vaccine is needed, and the first priority is to carry out tick challenge experiments on immunized animals. several commercial vaccine development criteria must also be addressed, such as possible formulation of the vaccine for intramuscular or subcutaneous inoculation, which would be much more convenient that the intravenous route, and testing of retention of effectiveness after lyophilization, since a lyophilized vaccine would not need a cold chain for distribution. the perennial concern with attenuated vaccines is the possibility that they may revert to virulence, perhaps as the result of recombination in the field with wild types of the pathogen. this, however, is not of major consequence in the endemic area, since new genes would not be added to the existing environment and widespread recombination between strains is already known to occur. obviously this attenuated vaccine would not be useable in non-endemic areas, but it would be an excellent stopgap vaccine for use in africa pending the development of a recombinant vaccine. summary the last ten years have seen the most significant developments in e. ruminantium vaccination since the introduction of the infection and treatment procedure in 1945, with exciting advances in both recombinant and attenuated vaccines. a recombinant vaccine would be the ideal solution to heartwater control, especially for export, since it would be useable anywhere in the world. this would be essential in the event of any spread of the disease outside the current endemic area, such as the long feared possibility of a cattle egret mediated spread of heartwater infected ticks from the lesser antilles to the american mainland (deem 1998). another advantage of a recombinant vaccine would be the relative ease with which the evolution of vaccine genes in the wild could be followed, allowing the vaccine formulation to be changed to keep up with pathogen changes. the research needed to develop a recombinant vaccine is, however, likely to be relatively time-consuming and expensive, and in the interim the new attenuated welgenvonden vaccine shows great promise. the development of this vaccine to the level of commercialization should be undertaken as a matter of urgency. acknowledgements i would like to thank all my colleagues and collaborators with whom i have worked on heartwater during the last 17 years. my thanks go, in alphabetical order, to the following: dr maria allsopp, dr kelly brayton, dr nicola collins, dr etienne de villiers, ms erika faber, prof. frans jongejan, mrs antoinette josemans, prof. fourie joubert, ms junita liebenberg, ms elmarié louw, dr alri pretorius, ms helena steyn, dr henriette van heer den, dr mirinda van kleef, ms fransie van strijp and dr erich zweygarth. references allsopp, m.t., dorfling, c.m., maillard, j.c., bensaid, a., haydon, d.t., van heerden, h. & allsopp, b.a. 2001. ehrlichia ruminantium major antigenic protein gene (map1) variants are not 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: gerald zirintunda1,2 justine ekou1 affiliations: 1department of animal production and management, busitema university, uganda 2department of livestock health and entomology, ministry of agriculture, animal industry and fisheries, uganda correspondence to: justine ekou email: jekou@covab.mak.ac.ug postal address: po box 236, tororo, uganda dates: received: 23 sep. 2014 accepted: 19 jan. 2015 published: 11 june 2015 how to cite this article: zirintunda, g. & ekou, j., 2015, ‘occurrence of porcine cysticercosis in free-ranging pigs delivered to slaughter points in arapai, soroti district, uganda’, onderstepoort journal of veterinary research 82(1), art. #888, 5 pages. http://dx.doi.org/10.4102/ojvr.v82i1.888 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. occurrence of porcine cysticercosis in free-ranging pigs delivered to slaughter points in arapai, soroti district, uganda in this original research... open access • abstract • introduction • materials and methods • results • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ poverty, hunger and the need for production of pigs with meagre or zero inputs have made most farmers release their pigs to range freely, thus creating a pig-human cycle that maintains taenia solium, the pig tapeworm and cause of porcine cysticercosis, in the ecosystem. a preliminary study was designed to establish the prevalence of porcine cysticercosis by postmortem examination of the tongue and carcass of free-range pigs from february to april 2014 in arapai subcounty, soroti district, eastern uganda. the tongue of each pig was extended and examined before deep incisions were made and the cut surfaces were examined. the rest of the carcasses were examined for cysts. out of 178 pigs examined, 32 were qualitatively positive for porcine cysticercosis, representing a prevalence of 18.0%. this high prevalence represents a marked risk to the communities in the study area of neurocysticercosis, a debilitating parasitic zoonosis. proper human waste disposal by use of pit latrines, confinement of free-range pigs and treatment with albendazole and oxfendazole are recommended. introduction top ↑ pig production has increasingly become an important activity in uganda, with the pig population increasing in the last three decades from 0.19 million to 3.6 million (ministry of agriculture, animal industry and fisheries [maaif] & uganda bureau of statistics 2009; uganda bureau of statistics 2013). in comparison to other animal rearing enterprises, pig production requires minimal inputs and relatively smaller space (eusebio 1980), which makes pig farming popular. it is thus not surprising that more than 1.1 million families, about 18% of the total households in uganda, own pigs (maaif & uganda bureau of statistics 2009). this rapid increase in production has been matched by a rapid increase in consumption of pork within the country, driven not only by population growth but also by a combination of rising income and changing preferences associated with urbanisation. uganda has the highest per capita consumption of pork in sub-saharan africa, with a 2011 estimate of 3.4 kg/person/year representing a ten-fold increase in the last 30 years (ballantyne 2012). however, programmes promoting pig production have not emphasised proper management and public health concerns. poverty and lack of resources have driven farmers or communities to rear pigs extensively with very minimal inputs, hence exposing them to the risk of porcine cysticercosis. porcine cysticercosis is caused by the metacestodes (cysticerci) of the cestode taenia solium, and it is endemic in uganda (world organization for animal health 2014; waiswa et al. 2009). taenia solium is a zonootic tapeworm that is maintained by a pig-human cycle in the ecosystem. the infection is contracted by pigs when they either ingest human faeces containing infective eggs or when feeding on pasture contaminated with t. solium eggs (carrique-mas et al. 2001). if people consume raw or inadequately cooked pork from infected animals, the larval cysts can develop into the adult stage tapeworm in their intestines, where gravid proglottids containing infective eggs detach from the adult tapeworm and are excreted in the faeces (garcia et al. 2003). in places where open defaecation is common the faeces containing these infective eggs are consumed directly or from contaminated pasture by pigs, and the lifecycle is perpetuated (ito et al. 2006; lescano et al. 2007). humans can also act as an aberrant intermediate host for t. solium if there is faecal-oral contamination with the infective eggs. in such cases the larval stage can be found in human muscle, heart, eyes, skin or central nervous system, causing human cysticercosis (flisser, rodríguez-canul & willingham 2006). the most serious form of human cysticercosis is when the larval form develops in the brain, a condition called neurocysticercosis (ncc). adult t. solium infestation in humans is associated with subclinical conditions of malnutrition and anasarca due to larval migration through the tissues (delgado-azanero et al. 2007). human ncc may manifest with headaches, blindness, hydrocephalus, chronic meningitis and dementia (carabin et al. 2005). ncc contributes to epilepsy in regions where pigs are free-ranging and hygiene is poor (blocher et al. 2011; rottbeck et al. 2013). the prevalence of cysticercosis was determined to be 11.7% amongst patients with epilepsy and 2.8% amongst controls who were normal individuals in families of burundi (newell et al. 1997), indicating that cysticercosis causes epilepsy. there is evidence of a high prevalence of ncc infecting people in villages where pigs are raised (phiri et al. 2003). age increases the risk of being positive for cysticercosis in pigs where open-air defaecation and free-range pig raising are practised (jayashi et al. 2012). approximately 50 000 individuals die globally every year of ncc caused by the parasitic intermediate stages of t. solium (ito et al. 2006). the purpose of the present study was therefore to estimate the prevalence of porcine cysticercosis amongst pigs delivered for slaughter in arapai in the soroti district of eastern uganda. materials and methods top ↑ the study was done in arapai subcounty which is located in the northern part of soroti district in eastern uganda. figures 1 and 2 show the location of soroti district in uganda and the location of arapai subcounty in soroti. figure 1: map of uganda showing location of soroti district (shaded red). figure 2: map soroti district showing location of arapai (shaded). the 16 most popular slaughter points for pig trade in the subcounty were selected for inclusion in the study, namely five slaughter places in aloet parish, eight in arapai market, two in temele trading centre and one in apida trading centre. a cross-sectional qualitative study in which the tongue, cardiac muscles and thigh muscles were examined for the presence of cysts was performed. the head of a slaughtered pig was set aside; the tongue was fully extended, dried with a smooth towel and examined for cysts on both the dorsum and ventrum before it was incised and the cut surfaces were examined. visual inspection of the carcass, its cut surfaces and the organs within it was done. the external and internal masseters and the pterygoid muscles were examined and two incisions made into each, the cuts being parallel to the bone and right through the muscle. the pericardium was examined visually. the heart was incised once lengthwise through the left ventricle and interventricular septum to expose the interior and cut surfaces for examination. in addition two deep incisions were made into the left ventricle. after removal of the peritoneum the muscles of the diaphragm were examined visually and incised once. the oesophagus was examined visually. the gracilis muscle was incised once parallel to the pubic symphisis. all pigs were examined consistently. cysts were identified as oval, about 10 mm × 5 mm or larger, with a delicate, fairly translucent, white parasite membrane and host capsule. within the cyst a pale fluid and the scolex, visible as a white dot within the cyst, usually invaginating midway along the long axis of the cyst, was considered diagnostic (world organization for animal health 2014). results top ↑ as shown on table 1, a total of 178 pigs were examined and 32 were found to be positive for cysticercosis, indicating an overall prevalence of 18.0% in the study area. aloet and arapai market recorded similar results of 14.9% and 14.6% respectively, whilst temele and apida also had similar results of 28.6% each. table 1: occurrence of porcine cysticercosis amongst free-range pigs delivered to various slaughter places in arapai subcounty, soroti district, eastern uganda. discussion top ↑ the overall observed porcine cysticercosis prevalence in this study was 18.0%. this was higher than the prevalence determined by tongue and necropsy examination previously reported in eastern and southern provinces of zambia (mwape et al. 2012; sikasunge et al. 2007), the teso district of western kenya (mutua et al. 2007), north west cameroon (shey-njila et al. 2003), angónia district of mozambique (pondja et al. 2010), homa bay district of kenya (eshitera et al. 2012) and the eastern cape province, south africa (krecek et al. 2008). a prevalence as high as 18.0% based on tests of low sensitivity should cause concern. the tongue test is 70% sensitive and 100% specific; the enzyme-linked immunosorbent assay (elisa) has a sensitivity and specificity of 75%, whilst enzyme-linked immune-electro transfer blot (eitb) has a sensitivity and specificity of 100% (gonzalez et al. 1990). the prevalence may be projected to be about 40% had more sensitive tests such as eitb been used. shey-njila et al. (2003) found 4.44% by tongue examination and 27.7% prevalence by elisa in the same population. sikasunge et al. (2007) found 12.7% by tongue examination and 32.1% by elisa, pondja et al. (2010) 12.7% by tongue examination and 34.9% by elisa, eshitera et al. (2012) 5.6% by tongue examination and 32.6% by elisa, krecek et al. (2008) 11.9% by tongue examination and 33.3% by eitb, whilst gonzalez et al. (1990) found 23.4% by tongue examination, 37.7% by elisa and 51.9% by eitb. thus the observed prevalence was very high given the low sensitivity of the test methods employed, which is seriously concerning since the true prevalence is probably much higher. the higher prevalence in arapai subcounty was thought to be due to the very low latrine coverage and the free-ranging rearing system, which enables pigs to scavenge in the environment and to consume human faeces. in soroti pit latrine coverage is 71% and 94% of pigs are reared either free-ranging or tethered in bushes where they are at risk of acquiring porcine cysticercosis (uganda bureau of statistics 2009; zirintunda 2011). most farmers are poor and food insecure, and equally their pigs lack sufficient food, as reported in other studies (adesehinwa, makinde & oladele 2003; chimonyo et al. 2005; halimani et al. 2012). the free-ranging pigs are also able to move long distances away from their owners’ premises where they access eggs of t. solium, even if their owners are free from infection and have access to latrines. the pigs are not fed commercial rations since most owners also have very little to eat, and therefore pigs are exposed to human faeces whilst scavenging. the farmers in soroti have no regular strategies to control worms amongst themselves or amongst their pigs. this could lead to a higher prevalence of cysticercosis when compared to other places where deworming programmes have been implemented. the prevalence of cysticercosis was similar for aloet and arapai sub-counties and for temele and apida slaughter points; this was possibly because of almost the same magnitude of those factors that affect porcine cysticercosis, like latrine coverage and use. aloet and arapai slaughter points had a slightly lower prevalence compared to temele and apida. aloet is a township with moderate pit latrine coverage whilst arapai is a cattle market with some sanitary facilities. in addition, both places receive pigs for slaughter from distant places and through middlemen who probably carry out pre-transit lingual examination of the pigs before delivery (nsadha et al. 2014). conclusion top ↑ in conclusion, the prevalence of porcine cysticercosis in arapai, soroti district is very high. since it is a zonoosis, the human population in arapai is at a high risk of the maladies associated with porcine cysticercosis, like ncc and epilepsy. it is also possible for cysticercosis to occur in people without brain involvement, and for clinical symptoms to appear to be absent (somers et al. 2006). confinement of pigs should be adopted to prevent continuous transmission of porcine cysticercosis (pouedet et al. 2002). vaccination of pigs with crude extracts of t. solium metacestodes and oncosphere antigens (flisser et al. 2004; molinari et al. 1997) may also be helpful. treatment with drugs such as albendazole and oxfendazole is of value, as the cysts may lose their fluid and collapse. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions g.z. 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organization for animal health, 2014, cysticercosis, oie terrestrial manual, viewed 10 august 2014, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.09.05_cysticercosis.pdf zirintunda, g., 2011, ‘assessment of human faecal environmental contamination and the prevalence of porcine cysticercosis in soroti district, uganda’, dissertation submitted in part fulfilment for the degree of master of science in international animal health, royal (dick) school of veterinary studies, edinburgh. cloete_17–31.indd introduction foot-and-mouth disease (fmd) virus serotypes sat 1, 2 and 3 are endemic in african buffalo (syncerus caffer) populations in the kruger national park (knp) and adjoining game farms in the extreme north-eastern corner of south africa (vosloo, bastos, sangare, hargreaves & thomson 2002a; vosloo, boshoff, dwarka & bastos 2002b). the control zone consists of the endemic game parks and the buffer zone that is divided into a zone with and one without vaccination of cattle (directorate of animal health, department of agriculture 2006). the rest of the country has been declared free of fmd without vaccination by the office international des epizooties (oie) in 1996. a total of ± 55 000 cattle are vaccinated against the disease twice annually in the buffer zone where vaccination is practised. in addition, the erection and maintenance of cattle and game-proof fences along all the international borders and western knp border and adjoining game parks are used as control measures to prevent introduction of fmd through contact between livestock and infected game. the movement of livestock within, into and out of the infected, buffer and 17 onderstepoort journal of veterinary research, 75:17–31 (2008) evaluation of different adjuvants for foot-and-mouth disease vaccine containing all the sat serotypes m. cloete1*, b. dungu1, 2, l.i. van staden1, n. ismail-cassim1 and w. vosloo1, 3 abstract cloete, m., dungu, b., van staden, l.i., ismail-cassim, n. & vosloo, w. 2008. evaluation of different adjuvants for foot-and-mouth disease vaccine containing all the sat serotypes. onderstepoort journal of veterinary research, 75:17–31 foot-and-mouth disease (fmd) is an economically important disease of cloven-hoofed animals that is primarily controlled by vaccination of susceptible animals and movement restrictions for animals and animal-derived products in south africa. vaccination using aluminium hydroxide gel-saponin (as) adjuvanted vaccines containing the south african territories (sat) serotypes has been shown to be effective both in ensuring that disease does not spread from the endemic to the free zone and in controlling outbreaks in the free zone. various vaccine formulations containing antigens derived from the sat serotypes were tested in cattle that were challenged 1 year later. both the as and isa 206b vaccines adjuvanted with saponin protected cattle against virulent virus challenge. the oilbased isa 206b-adjuvanted vaccine with and without stimulators was evaluated in a field trial and both elicited antibody responses that lasted for 1 year. furthermore, the isa 206 adjuvanted fmd vaccine protected groups of cattle against homologous virus challenge at very low payloads, while pigs vaccinated with an emergency isa 206b-based fmd vaccine containing the sat 1 vaccine strains were protected against the heterologous sat 1 outbreak strain. keywords: adjuvant, cattle, control strategies, foot-and-mouth disease, pigs, south africa, vaccines * author to whom correspondence is to be directed. e-mail: cloetemi@arc.agric.za 1 transboundary animal diseases programme, onderstepoort veterinary institute, private bag x05, onderstepoort, 0110 south africa 2 onderstepoort biological products, private bag x07, onderstepoort, 0110 south africa 3 department of veterinary tropical diseases, university of pretoria, pretoria, 0110 south africa accepted for publication 22 october 2007—editor 18 adjuvants for foot-and-mouth disease vaccine surveillance zones is controlled by an officially administered permit and identification system. movement of livestock from the infected and buffer zones to the free zone is not permitted, except for direct slaughter for local consumption at dedicated abattoirs situated in the perimeter of the free zone (brückner, vosloo, cloete, dungu & du plessis 2004). the current vaccine used for cattle contains strains of all three sat serotypes of fmd virus in an aqueous-based vaccine with aluminium hydroxide gelsaponin (as) as adjuvant. this vaccine has several deficiencies that include the induction of short-lived antibody responses and the consequent need for frequent application of the vaccine (hunter 1998), and it does not elicit good humoral immunity in pigs (van bekkum, bool & vermeulen 1967). the oil-adjuvanted fmd vaccines are suitable for all susceptible species, including pigs. they can also possibly act as slow release vaccines, thus providing longer lasting protection than the aqueous vaccines (hunter 1998), which means that annual vaccination can be implemented in the fmd control zone of south africa instead of the current double initial vaccination and 6-monthly boosters required with the aqueous vaccines. oil-adjuvanted vaccines have been used successfully in the fmd eradication campaigns in south america (dora, coelho nunes, goular de silveira, jorgens, rosenberg & astudillo 1984; bahnemann & mesquita 1987; sutmoller, barteling, olascoaga & sumption 2003). commercially available single water-in-oil (w/o) adjuvants, montanide isa 25 and isa 50, and double water-in-oil-in-water (w/o/w) adjuvants, isa 206, without the addition of saponin, elicited protective humoral immune responses against european fmd serotypes in cattle (barnett, pullen, williams & doel 1996; iyer, ghosh, singh & deshmuhk 2001), sheep (barnett, keel, reid, armstrong, statham, voyce, aggarwal & cox 2004; cox, barnett, dani & salt 1999; patil, bayra, ramakrishna, hugar, misra, prabhudas & natarajan 2002b), goats (patil, bayra, ramakrishna, hugar, misra & natarajan 2002a) and pigs (barnett et al. 1996; barnett, cox, aggarwal, gerber & mccullough 2002; barnett & carabin 2002). the w/o adjuvants induced faster and better protective immune responses than the w/o/w adjuvants (barnett et al. 1996; iyer et al. 2001), while the addition of saponin to the w/o fmd vaccine enhanced the antibody titres especially at lower payloads (smitsaart, mattion, filippi, robiolo, periolo, la torre & bellinzoni 2000). the addition of saponin to isa 206 adjuvanted fmd vaccines containing o1 campos enhanced the specific antibody responses in cattle and pigs (smitsaart, esponoza, sanguinetti, fillipi, ham & bellinzoni 2004). oilbased fmd vaccines containing antigens from the international vaccine bank, pirbright were shown to be more stable at + 4 °c than the as vaccine formulations (barnett & doel 1992; barnett et al. 1996). barnett & statham (2002) developed a novel approach for the formulation and storage of oil vaccines that involves the layering of the individual components of fmd vaccine in the same primary container and then storing the product at ultra-low temperatures. the advantage is that the shelf life can be substantially extended. another advantage of oil adjuvanted fmd vaccines is that they can provide early protection against infection. isa 206 adjuvanted fmd vaccines containing the o, a, c and asia 1 fmd serotypes without saponin and given as a once-off vaccination protected cattle (barnett et al. 1996; iyer et al. 2001; cox, voyce, parida, reid, hamblin, paton & barnett 2005; cox, voyce, parida, reid, hamblin, hutchings, paton & barnett 2006), sheep (cox et al. 1999), goats (barnett et al. 2002; patil et al. 2002a) and pigs (barnett et al. 1996; barnett et al. 2002; barnett & carabin 2002) within 4–7 days post vaccination (pv). cattle were protected against direct contact challenge following emergency vaccination (cox et al. 2005), and an increase in payload reduced subclinical infection, leading to fewer persistently infected animals (cox et al. 2006). cattle vaccinated with a commercial isa 206 adjuvanted monovalent o1 manisa vaccine (merial, pirbright uk, 3pd50) were partially protected 4 days pv, and fully protected 7 days pv after virulent virus challenge (golde, pacheco, duque, doel, penfold, ferman, gregg & rodriquez 2005). the performance of selected oil adjuvants containing the sat serotypes of fmd virus has been investigated previously (hunter 1996). preliminary trials with oil-adjuvanted vaccines without saponin containing sat strains showed that w/o/wand w/oadjuvanted vaccines elicited higher antibody responses than the as vaccines (hunter 1996), and should therefore be able to protect for longer time periods than the as vaccines. the aims of this investigation were to evaluate different oil-adjuvanted fmd vaccine formulations to determine which formulation would protect cattle from homologous virulent virus challenge, to perform a field evaluation of the oil-adjuvanted fmd vaccine intended to replace the current aqueous vaccine used in areas surrounding the knp where fmd vaccination is practised, to determine whether 19 m. cloete et al. the addition of saponin to the w/o/w emulsion will positively affect the vaccine as previously demonstrated (smitsaart et al. 2004), and finally, to determine the level of protection induced by oil-based vaccines using lower payloads as well as their efficacy in pigs using the sat strains. materials and methods preparation of different vaccine formulations table 1 summarises the composition of fmd vaccine formulations and their intended use in this study. vaccines were formulated according to the manufacturers’ instructions. animal trials to determine the efficacy of the various vaccines evaluation of different oil-adjuvanted vaccine formulations four groups of six fmd-free cattle were vaccinated with fmd vaccine formulations 1–4 (table 1). animals vaccinated with vaccine formulation 1 were given booster vaccinations after 4 weeks, while animals receiving vaccine formulations 2–4 were boosted after 8 weeks. samples were collected at 4-week intervals for 40 weeks after vaccination for serological testing. the two groups vaccinated with the vaccine formulations 1 and 2 were challenged 50 weeks pv and those vaccinated with vaccine formulations 3 and 4 at 56 weeks pv with sat 2b. one control animal was included in each of the challenge tests. they were brought into the containment facilities at the transboundary animal diseases programme (tadp), and challenged with 104 tissue culture infectious doses (tcid50) tissue culture adapted sat 2b strain administered intradermally into the tongue (kitching, barnett & donaldson 2000; stellmann, terr, favre, brun & fontaine 1977). the animals were monitored for clinical signs of fmd and their temperatures recorded daily for 10 days. sera were collected before challenge and 10 and 18 days after challenge for antibody titre determination. animals were considered protected from live virus challenge when they did not show any fmd-like lesions on any of their four feet. safety testing of isa 206b-based fmd vaccine three fmd-free cattle were inoculated intradermally into the tongue at ten points using 0.1 mℓ of the fmd vaccine formulation 5 containing monovalent sat 2b antigen and saponin. on day 4 the cattle were injected with 3 mℓ vaccine subcutaneously in the neck. the animals were monitored for a further 6 days. the vaccine was considered safe if no fmd lesions were observed on the tongue and feet, and if there was no swelling at the site of injection. potency test of isa 206b-based fmd vaccines with decreased payload two groups of five fmd-free cattle were vaccinated with a 0.25 dose (0.5 mℓ instead of 2.0 mℓ) of either monovalent formulation 5 or pentavalent fmd vaccine formulation 6 (table 1). sera were collected on days 0, 7, 14, 21 and 31 for testing for antibodies. the cattle vaccinated with the monovalent sat 2 vaccine 5 and two unvaccinated controls, free from fmd, were challenged 21 days pv with 105 tcid50 of live sat 2b. the cattle vaccinated with pentavalent vaccine 6, and two unvaccinated controls were challenged with 105 cattle infectious doses of sat 3 strain. the animals were monitored for clinical signs of fmd and their temperatures recorded daily for 10 days. they were considered protected from live virus challenge when they did not show any fmd-like lesions on any of their feet, while the vaccine was deemed protective if three or more animals were protected from clinical fmd. potency test of isa 206b-based fmd vaccine following emergency vaccination in november 2000 an fmd outbreak caused by sat 1 occurred in a cattle feedlot in south africa (brückner, vosloo, du plessis, kloeck, connoway, ekron, weaver, dickason, schreuder, marais & mogajane 2002). a pig breeding facility was also located on the premises and approximately 50 000 pigs were vaccinated with emergency fmd vaccine formulation 7 (table 1) (brückner et al. 2002). five vaccinated pigs were brought to the containment facilities at tapd 36 days pv. the five pigs as well as two unvaccinated controls, free from fmd, were challenged with 105.3 tcid50 per 0.1 mℓ (pig kidney titrated) sat 1 outbreak strain (swl 3/00/1) using 0.1 mℓ per site, intradermally into each of the two digits of the heel bulb of one foot (de leeuw, tiessink & van bekkum 1979). the animals were monitored for clinical signs of fmd and their temperatures recorded daily for 10 days. animals were considered protected from live virus challenge when they did not show any fmd-like lesions on any of their feet or in their mouths (de leeuw et al. 1979), excluding the infected and adjacent claw. the vac20 adjuvants for foot-and-mouth disease vaccine t a b l e 1 s u m m a ry o f f m d v a cc in e f o rm u la tio n s u se d in t h is in ve st ig a tio n v a c c in e n o . a d ju v a n t c o m p o n e n ts im m u n o s ti m u la n t v a c c in e a n ti g e n s a p p li c a ti o n 1 a lu m in iu m h yd ro xi d e ( 2 5 % a lh yd ro g e l) 0 .5 % c h lo ro fo rm 0 .1 2 5 % b a yt ri l 1 % f o e ta l c a lf se ru m ( f c s ) 0 .5 % a n tifo a m 2 x p h o sp h a te b u ff e re d s a lin e ( p b s ) q u il a s a p o n in (1 m g p e r d o se ) 3 μ g p e r d o se o f e a ch : s a t 1 k n p 1 9 6 /9 1 /1 ( s a t 1 a ) s a r 9 /8 1 /1 ( s a t 1 b ) s a t 2 k n p 1 9 /8 9 /2 ( s a t 2 a ) z im 7 /8 3 /2 ( s a t 2 b ) s a t 3 k n p 1 0 /9 0 /3 ( s a t 3 ) c o m p a ri so n o f d iff e re n t f m d va cc in e a d ju va n ts f o r p ro te ct io n o f ca tt le 2 5 0 % m o n ta n id e i s a 2 0 6 b ( w /o /w ) 0 .5 % c h lo ro fo rm 0 .1 2 5 % b a yt ri l, p b s 3 5 0 % m o n ta n id e i s a 5 0 ( w /o ) 4 m o n ta n id e i m s 1 3 1 3 co m p ri si n g w a te rd is p e rs e d li q u id n a n o p a rt ic le s co m b in e d w ith im m u n o st im u la tin g c o m p o u n d (a u co u tu ri e r, d u p u is & g a n n e 2 0 0 1 ) 5 5 0 % m o n ta n id e i s a 2 0 6 b 0 .5 % c h lo ro fo rm 0 .1 2 5 % b a yt ri l, p b s q -v a c s a p o n in (5 m g p e r d o se ) 3 μ g p e r d o se s a t 2 b s a fe ty a n d p o te n cy t e st s 6 5 0 % m o n ta n id e i s a 2 0 6 b s a m e a s 5 q -v a c s a p o n in 3 μ g p e r d o se o f e a ch : s a t 1 a a n d s a t 1 b s a t 2 a a n d s a t 2 b s a t 3 p o te n cy t e st 7 5 0 % m o n ta n id e i s a 2 0 6 b s a m e a s 5 n o n e 1 μ g p e r d o se o f s a t 1 a a n d s a t 1 b e m e rg e n cy s a t 1 v a cc in e fo r va cc in a tio n o f 5 0 0 0 0 p ig s in m id d e lb u rg , m p u m a la n g a 8 5 0 % m o n ta n id e i s a 2 0 6 b s a m e a s 5 n o n e 3 μ g p e r d o se o f e a ch : s a t 1 a a n d s a t 1 b s a t 2 a s a t 3 f ie ld e va lu a tio n o f f m d v a cc in e a d ju va n ts in m p u m a la n g a 9 5 0 % m o n ta n id e i s a 2 0 6 b s a m e a s 5 q u il a s a p o n in 1 0 a lh yd ro g e l s a m e a s 1 21 m. cloete et al. cine was deemed protective if three or more animals were protected from clinical fmd. field evaluation of different oil-based vaccine formulation vaccine formulations 8–10 (table 1) were evaluated in cattle at two dip tanks named daantjie and msogwaba, respectively, in the white river district, located in the buffer zone where cattle are vaccinated. unvaccinated calves were used in the study and were bled, tagged and information logged. at both dip tanks, two groups of 50 calves each were vaccinated with vaccine formulations 8 and 9. the third group vaccinated with vaccine formulation 10 consisted of 47 calves at daantjie and 33 at mso gwaba. sera were collected 1, 2, 4, 12, 25 and 55 weeks pv at daantjie and 1, 3, 22, 55 and 82 weeks pv at msogwaba. the cattle vaccinated with the vac cine formulation 10 were boosted 4 and 24 weeks pv, while cattle vaccinated with vaccine formulations 8 and 9 were boosted at 12 weeks pv. all the groups of cattle at msogwaba were re-vaccinated at week 55. serological testing all sera were tested for antibodies to sat 1, sat 2 and sat 3 using the liquid phase blocking elisa (lpbe) (hamblin, barnett & hedger 1986) with virus homologous to the vaccine strains (sat 1: sar9/81/1, sat 2: zim7/83/2 and sat 3: knp10/90/3). virus neutralization tests (vnt) were performed as described by rweyemamu, booth, head & pay (1978), using the homologous virus to each of the five vaccine strains. the neutralizing antibody titre of the serum was expressed as the reciprocal of the dilution that neutralizes 50 % of the virus. the values were represented as geometric mean titre expressed in log10 (patil et al. 2002a). for both tests a cut-off of > 1.6log10 was taken as seroconversion to a serotype of fmd virus. statistical analyses were performed using genstat® for windows® (7th edition) at a 1 % confidence level (payne 2003). results evaluation of the ability of different oiladjuvanted fmd vaccines to protect cattle from virulent virus challenge fmd vaccine formulations 1–4 (table 1) were tested in four groups of six cattle. they were vaccinated and boosted, sera were collected at 4-weekly intervals and the neutralizing antibody titres determined. the average vnt titres for the six animals vaccinated with the different vaccine formulations at each time point is shown for sat 1 (fig. 1a and b), sat 2 (fig. 1c and d) and sat 3 (fig.1 e). for all the five sat antigens included in formulations 1–4, the isa 206b (formulation 2) and isa 50 (formulation 3) vaccines induced higher average antibody titres in animals that received a booster vaccination at week 8, as well as longer lasting immune responses, than for formulations 1 (as) and 4 (ims 313) (fig. 1a–e). the antibody titres elicited by the isa 206b (formulation 2) and isa 50 (3) formulations showed a steady decline over time from 12 weeks pv and, in general, were still positive at weeks 32–40 (fig. 1a–e), while the antibody titres for the sat 2b antigen remained positive for the duration of the trial (fig. 1d). there was no statistical difference between the average antibody titres of formulations 2 and 3 (fig. 1a–e). the average immune response elicited by the antigens present in the as vaccine (formulation 1) had largely disappeared by week 12 (fig. 1a–c), except for the sat 2b (fig. 1d) and sat 3 antigens (fig. 1e), where neutralizing antibodies could be demonstrated until week 24. only sat 3 (fig. 1e) elicited an increase in average antibody titres following the booster vaccination at week 4 with the as vaccine. the initial antibody responses to the ims1313 vaccine (formulation 4) were low for all the antigens and were of short duration (most average figures were below the cut-off level at weeks 20–28) (fig. 1a–e), with animals only seroconverting to the sat 2a antigen after receiving the booster vaccination at week 8 (fig. 1c). in contrast, the sat 1b antigen induced an average titre of 2.2 log10 at week 3 (fig. 1b). challenge with virulent fmd sat 2b strain vaccine formulations 1 and 2 protected four of six cattle each. the control animal succumbed to clinical disease. of the animals that were vaccinated with vaccine formulation 3, one was protected after challenge, while none of the cattle vaccinated with vaccine formulation 4 were protected. the control animal did not develop clinical disease, indicating a technical problem with the infection of that particular animal, but the fact that most of the vaccinated animals were diseased indicated that the virus was viable and that the challenge was successful. all the groups of cattle developed high levels of neutralizing antibodies against sat 2a (fig. 1c) and sat 2b (fig. 1d) following challenge with this serotype, while cross reaction with the other serotypes was also observed (fig. 1 a–e). such cross-reactions have been observed during active infection (cottral 1972a, b). 22 adjuvants for foot-and-mouth disease vaccine a 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 weeks post vaccination 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 l o g 1 0 v ir u s n e u tr a li za ti o n t it re a c a a a b b b a b a a a a a a a a b b b b b bb b a a a a aa a a a a a a a a a a a a a a a a a a a a b 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 weeks post vaccination 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 l o g 1 0 v ir u s n e u tr a li za ti o n t it re a a a aa a a b c b b a a a a b a b a b a b c b b a a a b b b b b b b b a a a a a a a a a a a a a a a a a a a 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 weeks post vaccination 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 l o g 1 0 v ir u s n e u tr a li za ti o n t it re c a b a b a a a b b b b b a b a a a b b b b b b bb b a b a a a a b a ba b a a a a a a bb b aa a a a a a a a 23 m. cloete et al. fig. 1 evaluation of fmd vaccine formulations 1–4 cattle vaccinated with formulation 1 received a booster at 4 weeks, while all other groups received boosters at 8 weeks. neutralizing titres were determined using vnt and titres ≥ 1/45 or ≥ 1.6log10 are deemed positive (the cut-off is shown). the neutralizing titres elicited by the different sat antigens present in the different vaccine formulations for weeks 4–40 were analysed using a one-sided anova to test for differences in immune responses between the different vaccine formulations. the data were acceptably normal and variances were homogenous at the 1 % confidence level. the neutralizing titres determined at weeks 50–59 were analysed differently via analysis of an unbalanced design using genstat regression to test for differences in immune responses between the different vaccine formulations. the data were acceptably normal with heterogeneous treatment variances. the treatment means were separated using fishers’ protected t-test least significant difference (lsd) at the 1 % level of significance (snedecor & cochran 1980). the points on the graph represent the average titres of the six cattle in each group per collection date. titres to sat 1a (a), sat 1b (b), sat 2a (c) and sat 2b (d) and sat 3 (e) are indicated in separate graphs. if the probability for difference was < 0.01, the statistically significant differences between the groups vaccinated with different vaccine formulations at each time point are denoted by different letters, and when the probability was > 0.01, it indicated no differences in immune response and are denoted by the same letter. formulation 1 formulation 2 formulation 3 formulation 4 d 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 weeks post vaccination 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 l o g 1 0 v ir u s n e u tr a li za ti o n t it re a a a a a a a a b b b b b b b b c c a a b a a a a a a b b b b a a a b b b b a a a b a b a a a a a a e 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 weeks post vaccination 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 l o g 1 0 v ir u s n e u tr a li za ti o n t it re a a a a a a a bb b b b b b b b a b a b b a a a a a b b a a b b b a a b a b a a a a aa a a a a a a a a 24 adjuvants for foot-and-mouth disease vaccine fig. 2 evaluation of fmd vaccine formulations 8, 9 and 10 at two dip tanks the animals vaccinated with vaccine formulation 10 were revaccinated at 4 and 24 weeks while all those that received vaccine formulations 8 and 9 were revaccinated at 12 weeks. all three groups received booster vaccinations at 55 weeks at msogwaba dip tank. the graphs represent the average titres against the different vaccine formulations as determined using a lpb-elisa, and ≥ 1.6log10 was taken as positive. the data were analyzed using an unbalanced design as described in the legend of fig 1. average titres to sat 1 (a), sat 2 (b), and sat 3 (c) fmd vaccines strains at daantjie and sat 1 (d), sat 2 (e) and sat 3 (f) at msogwaba dip tanks are indicated in separate graphs. the numbers of sera collected and analysed at each time point are indicated in brackets. if the probability for difference was < 0.01, the statistically significant differences between the groups vaccinated with different vaccine formulations at each time point are denoted by different letters, and when the probability was > 0.01, there were no differences in immune response and are denoted by the same letter. the dotted line at the bottom of the graphs from week 4 represents extended intervals between sampling periods. formulation 8 formulation 9 formulation 10 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 0 1 2 4 12 25 55 weeks post vaccination e l is a t it re a (5 0 ) (5 0 ) (4 7 ) a (3 7 ) a (4 7 ) a (4 5 ) a (4 4 ) a (3 6 ) a (4 1 ) a (4 7■) a (3 2 ) a (3 1▲) a (4 3■) a (4 6♦) a (2 9▲) b (3 2 ) a (4 5 ) b (3 8 ) a b (3 2 ) a (2 1 ) a (4 2▲) 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 0 1 2 4 12 25 55 weeks post vaccination e l is a t it re b a (3 8 ) a (3 2 ) a (2 1 ) b (3 2 ) a (4 3 ) a (4 6 ) a (2 9 ) a (4 7■) a (3 2♦) a (3 1 )a (4 4 ) a (3 6♦) a (4 1 ▲) (4 7 ) (5 0 ) a (3 7 ) a (4 7 ) a (4 5 ) a (4 5♦; 4 2■) (5 0 ) 25 m. cloete et al. fig. 2 (cont.) 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 0 1 3 22 55 82 weeks post vaccination e l is a t it re d (5 0 ) (3 3 ) a (4 1■) a (4 0♦) b (3 0 ) a (3 3■) a (4 3♦) b (2 6 ) b (2 8 ) a (3 6♦) a (4 2■) b (3 3 ) a (2 5 ) b (1 0 ) (5 0 ) a (2 3 ) a (2 4 ) a (1 4 ) 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 0 1 2 4 12 25 55 weeks post vaccination e l is a t it re c b (3 8 ) b (3 2 ) a (2 1 ) a (3 2▲) a (4 5♦) a (4 2 ) a (4 3♦) a (4 6 ) a (2 9▲) a (4 7♦) a (3 2■) a (3 1 ) a (4 4 ) a (3 6 ) a (4 1▲) (4 7 ) (5 0 ) a (3 7 ) a (4 7 ) a (4 5 ) (5 0 ) safety testing and potency testing of isa 206badjuvanted fmd vaccines with reduced payloads formulation 5 did not cause any adverse reactions at the sites of injection, indicating that the adjuvant/ antigen combination is safe for use. similarly, no adverse reactions were reported in the study using formulations 1–4 or in the large scale field evaluation for vaccines 8–10 in the white river area. the protective efficacy of the isa 206b-adjuvanted fmd vaccines was also tested using decreased payloads of antigen. in the first potency test five cattle were vaccinated with a 0.25 dose of vaccine formulation 5. after they and two control cattle were challenged with 105 tcid50 of sat 2a virus, four of the five vaccinated cattle were protected. both control animals developed lesions on all four feet. cattle vaccinated with a 0.25 dose of vaccine formulation 6 developed neutralizing antibodies against the sat 1 and 2 antigens in the vaccine by day 14, but not against sat 3 (results not shown). after they and two unvaccinated controls were challenged with sat 3 strain, four of the five vaccinated cattle were protected, while the fifth animal developed fmd lesions on only one of its feet. both the control animals developed lesions on all four feet. formulation 8 formulation 9 formulation 10 26 adjuvants for foot-and-mouth disease vaccine 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 0 1 3 22 55 82 weeks post vaccination e l is a t it re e (5 0 ) (3 3 ) a (4 1■) a (4 0♦) a (3 0▲) a (3 3■) a (2 6 ) a (4 3♦) a (2 8▲) a (3 6■) a (4 2 ) a (3 3 ) b (2 5 ) a b (1 0 ) (5 0 ) a (2 3 ) a (2 4 ) a (1 4 ) challenge of pigs with heterologous virus following emergency vaccination with an isa 206b-adjuvanted sat 1 fmd vaccine all the vaccinated pigs were protected while both control animals developed lesions in their mouths and on all four feet. field evaluation of isa 206-based trivalent fmd vaccine the different fmd vaccine formulations were subsequently tested in the field and compared to the standard as vaccine to allow for larger numbers of animals and to investigate the efficacy of all the formulations. groups of animals were vaccinated with formulations 8–10 at the two different dip tanks, sera collected on a regular basis and antibodies to all three serotypes determined using the lpbe. the numbers varied per collection date as some animals were sold, died or the farmers did not arrive at the dip tank with the animals. the results for the daantjie (fig. 2a–c) and msogwaba (fig. 2d–f) dip tanks are presented separately. at daantjie vaccine 9 (isa 206b with saponin) induced a marginally better response to all the antigens at week 1, indicating that the addition of 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 0 1 3 22 55 82 weeks post vaccination e l is a t it re f (5 0 ) (3 3 ) b (4 1 ) b (4 0 ) a (3 0 ) a (3 3 ) a (4 3 ) a (2 6 ) b (2 8 ) a (3 6 ) b (4 2 ) b (3 3 ) a (2 5 ) b (1 0 ) (5 0 ) a (2 3 ) a (2 4 ) a (1 4 ) fig. 2 (cont.) formulation 8 formulation 9 formulation 10 27 m. cloete et al. an immunostimulant to the oil-based vaccine could quicken the immune response, but by week 2 formulation 8, containing isa 206b without saponin, induced somewhat higher titres, although none of these values were statistically different (fig. 2a–c). the titres elicited by all the antigens in all the vaccine formulations declined from week 3 and by week 25 the average titre for formulation 10, the as vaccine, was negative (fig. 2a–c). there were no positive titres to the sat 3 antigen present in the oil formulations from week 25 (fig. 2c). the titres induced by the sat 1 and 2 antigens in the oil formulations remained positive until week 55 (fig. 2a and b). the group of cattle vaccinated with formulation 10, the as vaccine, and given a booster vaccination at week 24, had positive titres against all three antigens at week 55 (fig. 2a–c). the immune responses at the msogwaba dip tank (fig. 2d–f) were more variable, with little difference between the formulations when measuring antibody titres to sat 2 (fig. 2e), while the two isa 206b formulations (8 and 9) induced similar responses to sat 1, which were significantly higher than the response induced by the as vaccine (formulation 10) (fig. 2d). only vaccine 10 induced high levels of antibodies to sat 3 (± 2.2 log10) at week 1, but these declined to a similar level to the antibodies elicited by formulations 8 and 9 (1.7 log10–1.9 log10) at week 3 (fig. 2f). at week 22 only antibodies to sat 1 (fig. 2d) could be detected in all 3 formulations, while no titres to sat 2 (fig. 2e) and sat 3 (fig. 2f) could be demonstrated. the cattle vaccinated with formulation 10 received a booster vaccination at week 24, resulting in a positive titre against the sat 2 antigen at week 55 (fig. 2e), but not against the sat 1 antigen (fig. 2d). at week 55 the antibody titres against the sat 1 and 2 antigens in formulation 8 were positive (fig. 2d and e) while no antibodies were induced by the sat 3 antigen in all three vaccine formulations (fig. 2f). the booster vaccinations at week 55 in all the groups of cattle resulted in positive titres to all three antigens in all the formulations at week 82 (fig. 2d–f). discussion it is imperative that research should be intensified to develop more effective vaccines containing the sat serotypes and to investigate the use of different adjuvants and other boosters that could elicit longer lasting immunity, as gathering animals at frequent intervals for vaccination in extensive farming systems can create logistical problems. the fmd isa 206b (formulation 2) and isa 50 (formulation 3) containing antigens from the three sat serotypes and containing saponin as immunostimulant elicited higher and longer lasting vnt antibody titres in cattle than the as (formulation 1) and ims 1313 (formulation 4) vaccines. in the group of cattle vaccinated with fmd vaccine adjuvanted with isa 206b and containing saponin, a booster vaccination after 8 weeks did not elicit initial high antibody responses, but titres above the 1.6 log10 cut-off titre were maintained for 28–50 weeks. the cattle vaccinated with the as fmd vaccine that received a booster vaccination at 4 weeks only maintained antibody titres above the cut-off titre for 8–12 weeks. hunter (1996) reported similar findings in a preliminary evaluation of oil-adjuvanted fmd vaccines containing the sat serotypes. cattle vaccinated with a monovalent sat 1 vaccine containing different oil adjuvants (w/o, w/o/w and incomplete freund’s) without saponin as immunostimulant, maintained vnt titres higher than 2.2 log10 for at least 6 months, whereas the cattle vaccinated with the as vaccine only maintained the vnt titres above the cut-off of 1.6 log10 for 2–3 months (hunter 1996), which corresponded with our findings. groups of cattle vaccinated with w/o/w-adjuvanted fmd vaccine containing the same sat 1–3 vaccine strains described in this investigation developed neutralizing antibody responses of at least 2.2 log10 within 7 days and maintained antibody levels of above the cut-off of 1.6 log10 for the duration of the trial, which was up to 330 days (hunter 1996). the difference in initial immune responses between the two studies could possibly be ascribed to the payload of antigen present in the vaccine formulations, since hunter (1996) used a payload of 12 μg of each antigen per dose, whereas the cattle in this trial were vaccinated with 3 μg of each antigen per dose. in this investigation four out of six of the cattle vaccinated with the as and isa 206b-adjuvanted fmd vaccines were protected against live fmd virus challenge 50 weeks pv despite the fact that no neutralizing antibodies could be detected in the first group. in a follow-up potency test with an isa 206badjuvanted fmd vaccine (formulation 5) containing sat 1–3 with added saponin as immunostimulant, cattle were protected against sat 3 challenge using a payload of 0.75 μg of fmd sat antigen per dose, even though the cattle did not seroconvert to sat 3 before challenge. similarly, 0.75 μg per dose of sat 2b in an isa 206b adjuvant containing saponin (formulation 6) was sufficient to protect cattle from homologous live virus challenge. only one of the six cattle vaccinated with the isa 50 (formula28 adjuvants for foot-and-mouth disease vaccine tion 3) vaccine was protected from live virus challenge, despite the presence of neutralizing antibodies to the sat 2b antigen at the time of challenge. these results show that a high neutralizing antibody response does not necessarily lead to protection and that cattle with a negative antibody titre can still be protected against homologous virus challenge. the cattle might succumb to heterologous challenge, but this has not been tested. in a previous challenge experiment only 3/4 and 2/5 cattle vaccinated but not boosted with a w/o/w-adjuvanted vaccine without saponin containing sat 1–3 antigens were protected when challenged 7 and 11 months after vaccination, respectively, although they received a much higher payload of antigen, and still had detectable antibody titres (hunter 1996). therefore, it seems that the booster vaccination and the addition of saponin may have had an effect on the protection levels of the vaccinated animals in our study. the cattle vaccinated with the as fmd vaccine (formulation 1) were probably protected by cellular immunity caused by the presence of saponin, since it induces strong th1 and th2 responses and moderate ctl responses to some proteins, probably as a result of forming mixed protein-saponin micelles (cox & coulter 1997). barnett and co-workers (2004) demonstrated that sheep vaccinated with as vaccine containing o1 lausanne elicited both neutralizing antibody and gamma interferon responses. cd8+ response in cattle following infection with fmd similarly showed that cellular responses might be involved during fmd infection (childerstone, cedillobaron, foster-cuevas & parkhouse 1999). pigs vaccinated with a 1 μg per dose of emergency sat 1 fmd vaccine formulation 6 without saponin were fully protected from heterologous virus challenge. phylogenetic comparisons indicated that the outbreak virus clustered in the same topotype as the vaccine strains (brückner et al. 2002), while cross neutralization tests indicated a close antigenic relationship to the sat 1 vaccine strains (r-value of 0.6) (j.j. esterhuysen, personal communication 2001). these results demonstrate that isa 206b-adjuvanted fmd vaccine without saponin can protect pigs against live heterologous virus challenge 36 days pv if the challenge strain is antigenically and genetically closely related to the vaccine strains. in a recent study, pigs vaccinated with a w/o/w fmd vaccine at a payload of 3 μg per dose all developed fmd signs when challenged with homologous live virus 7 days pv, whereas pigs vaccinated with an increased payload of 12 μg per dose were partially protected when challenged at 7 days pv, and completely protected when challenged 14 days pv (eblé, bouma, de bruin, van hemert-kluitenberg, van oirschot & dekker 2004) these pigs developed weak iga responses following vaccination that increased significantly after challenge on day 7 pv. how ever, protection was correlated with the neutralizing antibody response and not the iga responses ((eblé, bouma, weerdmeester, stegeman & dekker 2007). an fmd vaccine containing o1 manisa (3 μg/mℓ) and adjuvanted with w/o/w emulsion protected pigs from heterologous virus challenge (o taiwan), although the challenge strain was not genetically or antigenically (r-value of 0.4) closely related (eblé, de bruin, bouma, van hemert-klui tenberg & dekker 2006). the montanide® ims 1313 adjuvant (fmd vaccine formulation 4) consisting of nanoparticles containing an immunostimulant in a water-based formulation was also evaluated in this investigation. cattle vaccinated with vaccine formulation 4 containing saponin as immunostimulant that received a booster vaccination at 8 weeks were not protected from live virus challenge 1 year after vaccination. the ims 1313 adjuvant containing saponin did not show any auto-boost effects, even though nanoparticles can act as slow-release vaccines (cox & coulter 1997). preliminary results of the evaluation of microsphere-encapsulated fmd vaccine containing the sat 1–3 serotypes as slow-release vaccines also showed disappointing results (unpublished data 2005). ims-adjuvanted fmd vaccines presented con trasting results in mice in which they elicited simi lar or higher antibody titres than the isa 206-adjuvanted fmd vaccine (quattrocchi, fon de vila, pap palardo, sadira & zamorano 2006), while also inducing interleukin 2 (il-2), il-4 and gamma interferon responses and protecting mice at 7 days pv (quattrocchi, bianco, fondevila, pappalardo, sa dira & zamorano 2004). the field evaluation of the isa 206b-adjuvanted fmd vaccine in the presence or absence of saponin as immunostimulant yielded different results at daantjie and msogwaba dip tanks even though the same vaccine formulations were used at both dip tanks. this demonstrates clearly the influence of the vaccination campaign at different localities where it is possible that vaccines were not applied in an optimal manner. the supervision of the experiment at each location was reliant on different people and, in addition, there were staff changes during the course of the experiment that could possibly have had an influence on the accuracy of the sera and data collection. furthermore, the data evaluation at msogwaba was influenced by the low numbers of animals 29 m. cloete et al. bled on certain dates, for example at week 55 only 28 % of the animals initially recruited for the study were presented at the dip tank. it is also possible that cattle could accidentally have been vaccinated during the routine bi-annual vaccination campaign, explaining why the titres against the sat 1 and 2 antigens in isa 206b vaccine formulation without saponin (formulation 8) at msogwaba dip tank were positive at week 55 (fig. 2d and e). smitsaart and co-workers (2000) evaluated w/o fmd vaccine formulations containing saponin as immunostimulant in different cloven-hoofed domestic animal species. cattle vaccinated with w/o-adjuvanted fmd vaccine with saponin at a payload of 9.7 μg per dose developed significantly higher initial antibody titres than cattle vaccinated with a w/o vaccine at a payload of 38.8 μg per dose without saponin (smitsaart et al. 2000). the cattle vaccinated with w/o vaccine with added saponin developed higher igg1 and igg2 responses than the vaccine without added saponin. in a follow-up trial isa 206-based fmd vaccines consisting of monovalent o1 campos (payloads of 5 μg and 20 μg per dose) were evaluated in the presence or absence of saponin. the enhancing effect of saponin was only apparent in cattle at low antigen payloads when compared to isa 206-based vaccines lacking saponin (smitsaart et al. 2004). although high antigen payloads were not tested in our investigation, no obvious advantageous effect of saponin in the isa 206b-adjuvanted vaccine was observed when compared to the isa 206b vaccine without saponin when using low payloads. in this investigation 67 % of cattle were protected for 1 year after receiving a primary vaccination and boosted 8 weeks later with isa 206b-adjuvanted fmd vaccine containing saponin. this is in contrast to a previous study in which only 40 % of cattle were protected 1 year after receiving a single vaccination with a w/o/w-adjuvanted fmd vaccine containing a higher sat antigen payload but no saponin (hunter 1996). it is therefore necessary to investigate further the potential benefits of adding saponin to the oil-based vaccines using sat antigens. the results emanating from this study show that isa 206band as-adjuvanted vaccines may be equally suitable for use in south africa, since both provided equal levels of protection against challenge. however, this study was based on a small number of animals, and it is possible that the isa-based vaccines would provide better protection, as high antibody levels have been shown to correlate with protection (barnett, statham, vosloo & haydon 2003). owing to the extensive farming practices prevalent in the buffer zone in sa it is not always possible to access animals on a regular basis for vaccination and the longer-lasting isa 206b-adjuvanted vaccine would therefore be preferable. additionally, the antibody levels are more readily detectable, which is advantageous when serosurveillance is performed to monitor the efficacy of the vaccination campaign. however, further research is required to optimize the vaccines having an auto boost effect, which would eliminate the need for boosters. the effect of antigen payload must be investigated to determine whether an increased payload can lead to an increase in the duration of neutralizing antibody body levels in cattle following a single vaccination. acknowledgements we thank the department of agriculture and the red meat research and development trust in sa for financial assistance. dr stephane ascarateil, seppic, airliquide, paris, france, supplied the sample montanide isa 206b, isa 50v and ims 1313 emulsions used in this study. dr ben du plessis and personnel of the mpumalanga veterinary services, nelspruit, mpumalanga assisted with communication to the local farmers of the msogwaba and daantjie dip tanks to participate in this project, and to allow their cattle to form part of this investigation. the late mr billy phologane, mr jan esterhuysen and ms karin boshoff performed the virus neutralization tests and elisas. drr comfort phiri and alison lubisi performed the animal work for the cattle potency tests. mr chris van vuuren and personnel assisted with the cattle trials at kaalplaas, onderstepoort, and ms kerstin malan formulated the vaccines used in the study. ms marie smith and nicolene thiebaut of the arc biometry unit carried out the statistical analysis used in this study. dr mary-louise penrith critically reviewed the manuscript. without all their assistance, this paper would not be possible. references aucouturier, j., dupuis, l. & ganne, v. 2001. adjuvants designed for veterinary and human vaccines. vaccine, 19: 2666–2672. bahnemann, h.g. & mesquita, j.a. 1987. oil-adjuvanted vaccine against foot-and-mouth disease. bulletin centro panamericano fiebre altosa, 53:25–50. barnett, 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veterinary medicine and public health, sokoine university of agriculture, tanzania correspondence to: tebogo kgotlele postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: kgotlele, t., kasanga, c.j., kusiluka, l.j.m. & misinzo, g., 2014, ‘preliminary investigation on presence of peste des petits ruminants in dakawa, mvomero district, morogoro region, tanzania’, onderstepoort journal of veterinary research 81(2), art. #732, 3 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.732 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. preliminary investigation on presence of peste des petits ruminants in dakawa, mvomero district, morogoro region, tanzania in this proceedings... open access • abstracts • introduction • material and methods    • study area    • samples    • sample preparation and laboratory analysis • results and discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstracts top ↑ peste des petits ruminants (ppr) is an acute viral disease of small ruminants characterised by the sudden onset of depression, fever, oculonasal discharges, sores in the mouth, foul-smelling diarrhoea and death. for many years, in africa, the disease was mainly confined to west and central africa but it has now spread southwards to previously ppr-free countries including tanzania, democratic republic of congo and angola. the disease was first reported in tanzania in 2008 when it was confined to the northern zone districts bordering kenya. presence of the disease has also been confirmed in southern tanzania especially mtwara region. recently, a suspected outbreak of ppr in dakawa area, mvomero district, morogoro region was reported. clinical samples (lungs, intestines, lymph nodes, whole blood and sera) from suspected goats (n = 8) and sheep (n = 1) were submitted to sokoine university of agriculture for analysis. molecular diagnosis by amplification of the nucleoprotein gene and the fusion gene of ppr virus (pprv) using pprv specific primers was done. five goats and the sheep were positive for pprv after performing rt-pcr. to our knowledge, this is the first report confirming the presence of ppr in the mvomero district of the morogoro region, tanzania. hence, more efforts should be put in place to prevent the spread of ppr in tanzania. introduction top ↑ peste des petits ruminants (ppr) is a highly infectious disease of small domestic ruminants and small wild ruminants, such as antelopes, impala and gazelles (muse et al. 2012a). the disease is currently considered one of the main transboundary animal diseases that constitute a threat to small ruminant production in many developing countries (banyard et al. 2010). the disease is caused by the peste des petits virus (pprv), a member of the genus morbillivirus of the paramyxoviridae family (kwiatek et al. 2010). the ppr virus can be transmitted when animals sneeze or cough, therefore the virus is found in discharge from the eyes, nose and mouth of infected animals and affected animals that do not show signs of the disease (roeder et al. 1999). contact and movement of animals from affected to unaffected areas play an important role in transmitting the disease, especially where a communal grazing system is practiced. as a result, ppr is on the list of economically important animal diseases whose occurrence has to be reported to the world organisation for animal health (oie) (fao-empres 2009).peste des petits ruminants was first described in west africa in the 1940s (khan et al. 2008). but with the advent of specific diagnostic tests, the geographical distribution of the disease was found to have expanded in the late 1980s and mid-1990s (fao-empres 2009). currently, ppr is present in central, east and west africa, asia, and the near and middle east (geerts 2009). it is believed that the development of trade relations, transport, tourism and migration of wild animals susceptible to ppr have contributed to the spread of the disease beyond the boundaries of west africa (kaukarbayevich 2009). in tanzania, ppr was first reported in 2008 in the northern zone districts bordering kenya (karimuribo et al. 2011). the disease was later introduced into southern tanzania in 2009 through newly purchased goats from a livestock market located about 700 km away on the outskirts of dar es salaam city (muse et al. 2012a). there is no official confirmation of one of the possible routes of ppr transmission from the northern parts to the southern parts of tanzania. the objective of this study was to confirm whether suspected cases seen in goats and sheep in mvomero district, morogoro region were caused by pprv and establish one of the possible routes of transmission of ppr from northern to southern tanzania. because of the geographical position of morogoro, it is the centre of the regional roads network to the coast, dar es salaam, dodoma and iringa. material and methods top ↑ study area the study was carried out in dakawa, mvomero district in morogoro region. morogoro region is located on the eastern side of tanzania mainland. the area was purposively targeted following reports of suspected ppr outbreak. samples clinical samples (lungs, intestines, lymph nodes, whole blood and sera) from eight goats and one sheep were submitted to sokoine university of agriculture for analysis. sample preparation and laboratory analysis buffy coat was prepared from whole blood using histopaque®-1077 (sigma-alderich, st. louis), transferred into a sterile microfuge tube and stored at -80 ºc until rna extraction. tissues from the same animal were pooled together and homogenised in f-12 nutrient mixture (ham) 1x (invitrogen, new york) to make a 10% tissue suspension. the tissue suspension was then centrifuged at room temperature and stored at -80 ºc until rna extraction. viral rna was recovered from buffy coat, sera and homogenised tissue samples using qiaamp viral rna mini extraction kit (qiagen, hilden, germany) according to the manufacturer’s instructions. extracted rna was amplified in a 25 µl reaction using agpath-id one-step rt-pcr kit (applied biosystems, courtaboeuf, france) using pprv-specific primers: np3 (5’-tctcggaaatcgcctcacagactg-3’) and np4 (5’-cctcctcctggtcctccagaatct-3’) from couacy-hymann et al. (2002), pprv-f-f(5’gagactgagtttgtgacctacaagc-3’) and pprv-f-r (5’-atcacagtgttaaagcctgtagagg-3’) from forsyth and barrett (1995). amplification was carried out in geneamp pcr system 9700 (applied biosystems, foster city, usa) followed by electrophoresis on 1.5% agarose gel. the dna was visualised by ultraviolet fluorescence with positive samples showing 351 bp bands for np3/np4 and 322 bp bands for pprv-f-f/pprv-f-r primers. results and discussion top ↑ clinical signs observed were nasal discharges, diarrhoea and oral ulcers, as shown in figure 1. these observations are consistent with previous reports from muse et al. (2012a, 2012b) done on ppr in tanzania. figure 1: ppr-suspected animals showing (a) diarrhoea and (b) erosions on the palate and gums (see arrow heads). post mortem showed enlarged and congested lymph nodes associated with the gastrointestinal and/or respiratory tract, as shown in figure 2. these findings have been reported by roeder et al. (1999), centre for food security and public health (cfsph) (2008), oie (2008) and chauhan et al. (2009). figure 2: congested lymph nodes in the (a) respiratory and (b) gastrointestinal systems of ppr-suspected animals (see arrow heads). virus presence was confirmed in tissue samples using reverse transcription polymerase chain reaction using pprv-specific primers targeting nucleoprotein and fusion protein genes. six out of the nine animals were positive for fusion protein while only four animals were positive for nucleoprotein. nucleoprotein gene transcripts are more abundant in infected tissues compared to transcripts of the fusion gene required for viral attachment and transmission on epithelial tissues, thus the ability of primers to detect the virus is affected by the stage of infection and time of sample collection (luka et al. 2012). conclusion top ↑ this study confirms for the first time the presence of ppr in the morogoro region in tanzania. since vaccination against ppr has never been carried out, this is a result of natural transmission of the virus under field conditions. given the position of the morogoro region, more studies need to be carried out in order to put in place effective control and prevention measures to contain the possible spread of the disease to other regions of tanzania. acknowledgements top ↑ the authors would like to thank mr kochuchu and farmers in dakawa for their cooperation, contributions and time. this study was supported by a grant from the wellcome trust to the southern african centre for infectious disease surveillance (sacids), sokoine university of agriculture, morogoro, tanzania. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions t.k. (sokoine university of agriculture) was responsible for project design, prepared the samples and performed most of the experiments. c.j.k. (sokoine university of agriculture), l.j.m.k. (sokoine university of agriculture) and g.m. 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veterinary research 79(2), 49–54. http://dx.doi.org/10.4102/ojvr.v79i2.457 muse, e.a., matondo, r.b., karimuribo, e.d., misinzo, g., albano, m.o. & gitao, g.c., 2012b, ‘clinico-pathological findings of the 2011 outbreak of peste des petits ruminants (ppr) in tandahimba district, southern tanzania’, research opinions in animal and veterinary sciences 2(4), 256–262. oie, 2008, manual of diagnostic tests and vaccines for terrestrial animals, viewed 22 may 2012, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.07.11_ppr.pdf , pp. 1036–1046, world organization for animal health, paris. roeder, p.l., obi, t.u., taylor, w. & diallo, a., 1999, recognizing peste des petits ruminants: a field manual, food and agriculture organization emergency prevention system (fao-empres) (livestock) animal health service, rome. article information authors: christopher j. kasanga1 wataru yamazaki2 valerie mioulet3 donald p. king3 misheck mulumba4 ezekia ranga6 jimis deve7 cornelius mundia8 patrick chikungwa9 laureta joao10 philemon n. wambura5 mark m. rweyemamu1 affiliations: 1southern african centre for infectious disease surveillance, sokoine university of agriculture, tanzania2department of microbiology, university of miyazaki, japan 3the pirbright institute, united kingdom 4southern african development community secretariat, botswana 5faculty of veterinary medicine, sokoine university of agriculture, tanzania 6ministry of livestock development and fisheries, tanzania 7southern african development community, transboundary animal diseases section, mozambique 8southern african development community, transboundary animal diseases section, zambia 9southern african development community, transboundary animal diseases section, malawi 10southern african development community, transboundary animal diseases section, angola correspondence to: christopher kasanga postal address: po box 3019, chuo kikuu, morogoro, tanzania how to cite this article: kasanga, c.j., yamazaki, w., mioulet, v., king, d.p., mulumba, m., ranga, e. et al., 2014, ‘rapid, sensitive and effective diagnostic tools for foot-and-mouth disease virus in africa’, onderstepoort journal of veterinary research 81(2) art. #727, 5 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.727 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www. sacids.org/ kms/frontend/ index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. rapid, sensitive and effective diagnostic tools for foot-and-mouth disease virus in africa in this proceedings... open access • abstract • introduction • research method and design    • samples    • rna extraction    • real-time rt-pcr assay    • rt-lamp assay    • specificity of rt-lamp assay • results • ethical considerations • trustworthiness • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ speed is paramount in the diagnosis of highly infectious diseases, such as foot-and-mouth disease (fmd), as well as for emerging diseases; however, simplicity is required if a test is to be deployed in the field. recent developments in molecular biology have enabled the specific detection of fmd virus (fmdv) by reverse-transcription loop-mediated isothermal amplification (rt-lamp), real-time reverse-transcription polymerase chain reaction (rt-qpcr) and sequencing. rt-lamp enables amplification of the fmdv rna-dependent rna polymerase 3d(pol) gene at 63 °c (in the presence of a primer mixture and both reverse transcriptase and bst dna polymerase) for 1 h, whilst rt-qpcr amplifies the same gene in approximately 2 h 30 min. in this study, we compared the sensitivity and effectiveness of rt-lamp against rt-qpcr for the detection of the fmdv 3d(pol) gene in 179 oesophageal-pharyngeal scraping samples (collected by probang) obtained from clinically healthy cattle and buffalo in malawi, mozambique and tanzania in 2010. the fmdv detection rate was higher with rt-lamp (30.2%; n = 54) than with rt-qpcr (17.3%; n = 31). all samples positive by rt-qpcr (cq ≤ 32.0) were also positive for the rt-lamp assay; and both assays proved to be highly specific for the fmdv target sequence. in addition, the vp1 sequences of 10 viruses isolated from positive samples corresponded to the respective fmdv serotypes and genotypes. our findings indicate that the performance of rt-lamp is superior to rt-qpcr. accordingly, we consider this test to have great potential with regard to the specific detection and surveillance of infectious diseases of humans and animals in resource-compromised developing countries. introduction top ↑ foot-and-mouth disease (fmd) is a highly contagious disease that affects cloven-hooved animals such as cattle, sheep, goats and pigs. fmd outbreaks have occured worldwide, resulting in significant economic losses (knowles & samuel 2003). early identification of fmd virus (fmdv) is, therefore, critical for the control of disease and to minimise losses that could occur in livestock. rapid and accurate diagnosis of fmdv is required for effective disease control. fmd cannot be distinguished clinically from other vesicular diseases, such as swine vesicular disease (svd), vesicular exanthema of swine (ves) and vesicular stomatitis (vs); similarities that can pose challenges for early confirmation of field outbreaks. routine laboratory diagnosis of fmd can be performed by a combination of antigen-capture enzyme-linked immunosorbent assay (elisa) and virus isolation (oie 2012). antigen-capture elisa only takes 4 h to perform; however, this test is only suitable for epithelium samples and does not have the analytical sensitivity required to confirm the negative status of premises (oie 2012; reid et al. 2001). in contrast, viral isolation is highly sensitive, but can take long time: up to two weeks to deliver a definitive answer. several reverse-transcription polymerase chain reaction (rt-pcr) and real-time rt-pcr (rt-qpcr) assays have been developed for the detection of fmdv (callahan et al. 2002; reid et al. 2000, 2001). rt-qpcr assays, which are highly sensitive and rapid and do not require electrophoresis, are now used widely for routine diagnosis of fmdv (reid et al. 2009; shaw et al. 2007). however, because these assays require a precision thermal cycler with a fluorescence detector and commercially-available kit-form reagents, these assays can be expensive to perform. given these limitations, a rapid, simple, sensitive, and more cost-effective assay for diagnosis of fmd in clinical samples is required. a recently-developed novel nucleic acid amplification method, termed loop-mediated isothermal amplification (lamp) (nagamine, hase & notomi 2002), is a promising candidate for rapid and easy detection of fmdv cdna. lamp is based on the principle of autocycling strand displacement dna synthesis performed by the large fragment of bst dna polymerase. a combined mix of amv reverse transcriptase and bst dna polymerase enables simultaneous reverse transcription and dna amplification to be undertaken in the same tube. reverse-transcription loop-mediated isothermal amplification (rt-lamp) assays are known to be more sensitive than conventional gel-based reverse-transcription polymerase chain reaction (rt-pcr) assays. in addition, they are fast and easy to perform since they require only a simple incubator, such as a heating block or a water bath, to provide a constant temperature for the reaction (parida et al. 2006). the aim of this study was to assess the performance of rt-qpcr and rt-lamp assays for detection of the rna-dependent rna polymerase 3d(pol) gene of the fmdv genome from oesophageal-pharyngeal (op) scraping samples (collected by probang) obtained from cattle and buffalo for the purposes of active surveillance. research method and design top ↑ samples oesophageal-pharyngeal scrapings were collected from clinically healthy cattle and buffalo in malawi (lengwe national park [np]), mozambique (marromeu np) and tanzania (katavi np) in 2010. the samples were collected in separate vials containing transport media, then were transported to the food and agriculture organization (fao) world reference laboratory for fmd (wrlfmd) at pirbright and stored at -20 °c until use. rna extraction total rna was extracted from 600 µl elution buffer–sample mixture (140 µl original op sample mixed with 460 µl elution buffer) using the rneasy 96 biorobot 9604 kit (catalogue number 967142) on an automated robotic platform (magna pure lc, roche). the resultant total rna was eluted in 50 µl nuclease-free h2o and stored in aliquots of 10 µl at -80 °c until required. real-time rt-pcr assay an established one-step rt-qpcr specific for the 3d(pol) region of fmdv (reid et al. 2009, adapted from callahan et al. 2002) was used in this study. this one-step real-time rt-pcr assay was performed (in duplicate) using an mx3005p qpcr system (stratagene, la jolla, ca, usa). briefly, each 25 μl pcr reaction contained 0.8 µm each of the 3df/3dr primers (100 μm stock concentration, applied biosystems), 0.3 µm 3dp taqman probe (labelled with fam and tamra as reporter and quencher, respectively) and 0.5 µl superscript iii/platinum taq enzyme mix (supplied with the superscript® iii platinum® one-step qrt-pcr kit; catalogue number 11732-088, life technologies, paisley uk) to amplify 5 μl of template rna. the amplification involved incubation of reverse transcription reactions at 48 °c for 45 min, then a 10 min denaturation at 95 °c, followed by 50 cycles of 15 s for denaturation at 95 °c and 60 s combined annealing/extension at 60 °c. following amplification, quantification cycle (cq) values were assigned and the average cq value of the results from duplicate samples was used for analysis. the primers and probe used are shown in table 1. table 1: primers and probes used for one-step rt-qpcr and rt-lamp for the detection of fmdv. rt-lamp assay multiplex rt-lamp assays were performed in a pcr reaction tube (pcr snapstrip 0.2 ml natural, catalogue number 3240-00, anachem, luton, uk) as described previously (yamazaki et al. 2012). briefly, a 25 μl volume reaction mix that contained 2 μl template rna, 0.15 u amv reverse transcriptase (invitrogen, carlsbad, ca, usa), 8 u bst dna polymerase (large fragment; new england biolabs, ipswich, ma, usa), 20 mm tris-hcl (ph 8.8, cambridge bioscience, cambridge, uk), 10 mm kcl (sigma-aldrich), 8 mm mgso4 (new england biolabs), 10 mm (nh4)2so4 (sigma-aldrich), 0.1% tween20 (sigma-aldrich), 0.8 m betaine (sigma-aldrich), 1.4 μm each dntp (ge healthcare, little chalfont, uk), 1.6 μm each inner primer (8-fip and 8-bip), 0.2 μm each outer primer (81-f3 and 81-b3) and 0.8 μm each loop primer (81-lf and 81-lb). each rt-lamp reaction was incubated at 63 °c for 1 h, followed by 80 °c for 5 min. amplified products were detected at 650 nm using a loopamp exia turbidimeter (teramecs, kyoto, japan). a reaction was considered positive when the differential value of the turbidity reached 0.1 ftu within 1 h. a tp value (time of positivity, in minutes) was designated as the time at which this differential value reached this threshold. the evaluation of the multiplex rt-lamp assays was performed using blind coded samples. specificity of rt-lamp assay the rt-lamp assay specificity was assessed using samples positive for swine vesicular disease virus (svdv), vesicular stomatitis virus (vsv) and vesicular exanthema of swine virus (vesv) that cause infections that are clinically indistinguishable from fmd. results top ↑ a total number of 179 op samples were tested for the 3d(pol) region of the fmdv genome by both rt-qpcr and rt-lamp assays. thirty-one samples (17.3%) tested positive for fmdv by rt-qpcr whilst 54 samples (30.2%) were positive for fmdv by rt-lamp. the detection rate of fmdv was, on average, three to four times higher in samples collected from buffalo compared with those obtained from cattle in the livestock-wildlife interface areas of katavi, lengwe and marromeu national parks in tanzania, malawi and mozambique respectively. the summary of results is shown in table 2. table 2: summary of results for oesophageal-pharyngeal samples tested during this study. the multiplex rt-lamp assay demonstrated higher sensitivity (54/179) in detection of fmdv rna from op probang scraping samples than rt-qpcr (31/179). all samples that produced a positive result by rt-qpcr (with cq values ≤32.0) were positive by rt-lamp assay. the 23 samples that tested positive by rt-lamp but negative by rt-qpcr had cq values ranging from 32.5 to 38.5. the rt-lamp assay was shown to be specific for fmdv as all the rna samples positive for svdv, vsv and vesv were negative for fmdv. in addition, 10 samples that were positive for fmdv by both rt-lamp and rt-qpcr were sequenced for the vp1 coding region and all sequences corresponded to the respective serotypes and/or genotypes of the virus. ethical considerations top ↑ this study was conducted in accordance with relevant national and international guidelines on handling animals and the arrive guidelines (kilkenny et al. 2010) for reporting in vivo animal experiments were adhered to. trustworthiness top ↑ the experiment was considered to be both reliable and valid. reliability of the study was tested by repeating the same procedure for rt-lamp and rt-qpcr on positive control rna obtained from the reference fmdv isolate o1 manisa (tur/8/69), which yielded the same results on repeated trials. the experimental procedures were performed with care and interpretation of results was done according to the established standards. discussion top ↑ rapid, sensitive and specific detection methods for fmdv are necessary for deploying rational control methods for fmd in a specific geographic region. rt-qpcr and rt-lamp are two sensitive and rapid diagnostic methods for fmdv. in contrast to rt-pcr (either real time or gel-based), the design of rt-lamp assays is particularly challenging because a larger number of oligonucleotides is required in order to perform the test. in this study, the performance of rt-qpcr and rt-lamp for fmdv detection on op samples was evaluated. the performance of rt-lamp was higher (30.2%; n = 54) than rt-qpcr (17.3%; n = 31) (table 2). several studies have shown that the diagnostic sensitivity of rt-lamp assays for fmdv is high, ranging from 82.7% – 98.5% when tested against material comprising several fmdv serotypes (chen et al. 2011; dukes, king & alexandersen 2006; shao et al. 2010). the findings of this study indicate that rt-lamp is relatively more sensitive than rt-qpcr in the detection of the fmdv 3d(pol) gene.the rt-lamp assay used in this study amplified the 3d(pol) region for 1 h at 63 °c whereas real-time rt-pcr took almost 2 h and 30 min in a thermocycler. the fact that rt-lamp takes less time and does not necessarily require a thermocycler implies that there is high potential for its application in the field and in areas where specialised laboratory equipment is not available. the op samples analysed during this study were collected from clinically health cattle and african buffalo in livestock–wildlife interface areas. it has been reported previously that fmdv detection rates in op samples are usually low in persistently-infected animals (vosloo et al. 1996). the sensitivity of rt-lamp with regard to detecting cases of fmdv that were negative by rt-qpcr (cq values 32.5–38.5) raises the possibility that the rt-lamp could be suitable for the identification of animals with persistent fmdv infections and could thus be applied to the surveillance of infectious diseases in africa in the regions where they are known to be endemic. as most countries in africa are planning to adopt a strategic control programme for fmd through vaccination and controlled animal movements, a wider knowledge and understanding of the dynamics and epidemiology of fmdv infection should be taken into consideration. this will require the rapid identification of high-risk ‘hotspots’ as well as potentially-infected and fmd-free zones. this task will, however, need the development of simple and cost-effective diagnostic tools such as rt-lamp that can be deployed under field conditions in order to identify fmdv infection and to define the spatiotemporal distribution of virus serotypes in the region. previous studies have shown the heterogeneity of fmdv serotypes in southern africa (kasanga et al. 2012; vosloo et al. 2002). this heterogeneity highlights the need for continuous surveillance of fmd using rapid, simple and cost-effective methods that are able to discriminate serotypes and/or subtypes. the rt-qpcr and rt-lamp assays evaluated during this study could not discriminate serotypes and/or subtypes, indicating that further research is required in order to develop rt-qpcr and rt-lamp assays for this purpose, which could then be used in areas of high fmdv heterogeneity. limitations of the study top ↑ possible limitations that could affect the results of the current study include the storage condition(s) of the op samples and the time taken to transport the samples to the fao world reference laboratory for fmd where the analysis was performed. recommendations further in-depth studies are required in order to evaluate the detection ability of rt-lamp and rt-qpcr on field samples and to compare the performance of the two assays under different conditions such as temperature and moisture. conclusion top ↑ in conclusion, we have evaluated the performance of rt-qpcr and rt-lamp assays in the detection of the 3d(pol) region of the fmdv genome from op probang scrapings. we found that rt-lamp was superior to rt-qpcr in the detection of fmdv from op probang samples and could be used for surveillance of fmd in cattle and buffalo in developing african countries. acknowledgements top ↑ we thank participating farmers around katavi, lengwe and marromeu national parks, as well as gaothlele thobokwe, for the op sample collection. this study was supported jointly by the african development bank (sadc tads), the southern african centre for infectious disease surveillance (sacids) through the wellcome trust grant (wt087546ma), the uk department of the environment, food and rural affairs (defra) who provided support to the fmd reference laboratory through a research project (se2940) at the pirbright institute and the japan society for the promotion of science by young researcher overseas visits program for vitalizing brain circulation. the authors also wish to acknowledge the support provided by the biotechnology and biological sciences research council, the department for international development and the scottish government for their support through the combating infectious diseases of livestock for international development initiative (project bb/h009302/1) and european union fp7-kbbe-2011-5 under grant agreement number 289364 (rapidia-field) to the pirbright laboratory where analysis of samples was performed. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.j.k. (sokoine university of agriculture) participated in the study design, the experimental work, the analysis and interpretation of the data and drafted the manuscript. w.y. (university of miyazaki), v.m (the pirbright laboratory) and d.p.k. (the pirbright laboratory) participated in the study design, the experimental work and drafting of the manuscript. m.m., j.d., c.m., p.c., l.j. (all southern african development community) and e.r. (ministry of livestock development and fisheries) participated in sample collection and the writing of the manuscript. p.n.w and m.m.r (both sokoine university of agriculture) participated in the study design and the writing of the manuscript. all authors read and approved the final manuscript. references top ↑ callahan, j.d., brown, f., osorio, f.a., sur, j.h., kramer, e., long, g.w. et al., 2002, ‘use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus’, journal of the american veterinary medical association 220(11), 1636–1642. http://dx.doi.org/10.2460/javma.2002.220.1636chen, h.t., zhang, j., liu, y.s. & liu, x.t., 2011, ‘rapid typing of foot-and-mouth disease serotype asia 1 by reverse transcription loop-mediated isothermal amplification’, virology journal 8, 489. http://dx.doi.org/10.1186/1743-422x-8-489 dukes, j.p., king, d.p. & alexandersen, s., 2006, ‘novel reverse transcription loop-mediated 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ferris, n.p. & king, d.p., 2007, ‘ implementation of a one-step real-time rt-pcr protocol for diagnosis of foot-and-mouth disease’, journal of virological methods, 143(1), 81–85. http://dx.doi.org/10.1016/j.jviromet.2007.02.009 vosloo, w., bastos, a.d., kirkbride, e., esterhuysen, j.j., janse van rensburg d., bengis., r.g. et al., 1996, ‘persistent infection of african buffalo (syncerus caffer) with sat-type foot-and-mouth disease viruses: rate of fixation of mutations, antigenic change and interspecies transmission’, journal of general virology 77(pt 7), 1457–1467. http://dx.doi.org/10.1099/0022-1317-77-7-1457 vosloo, w., bastos, a.d., sangare, o., hargreaves, s.k. & thomson, g.r., 2002, ‘ review of the status and control of foot and mouth disease in sub-saharan africa’, revue scientifique et technique (international office of epizootics) 21(3), 437–449. yamazaki, w., mioulet, v., murray, l., madi, m., haga, t., misawa, n. et al., 2012, ‘development and evaluation of multiplex rt-lamp assays for rapid and sensitive detection of foot-and-mouth disease virus’, journal of v irological methods 192(1–2), 18–24. mitema_155-159.indd introduction drugs, hormones and pesticides are chemical compounds used for alleviation of various diseases in animals. they can be used either for curative, specific disease control or prophylaxis. these compounds can also be used as production aids in food producing animals to enhance mass gain for early market (mitema 2004). hormones, in particular the steroids, have been used as production aid com155 onderstepoort journal of veterinary research, 76:155–159 (2009) improved management of drugs, hormones and pesticides in africa e.s. mitema department of public health, pharmacology and toxicology, faculty of veterinary medicine university of nairobi, p.o. box 29053, nairobi, kenya abstract mitema, e.s. 2009. improved management of drugs, hormones and pesticides in africa. onderstepoort journal of veterinary research, 76:155–159 drugs, hormones and pesticides are chemical compounds used for alleviation of various diseases in animals. there are many classes of drugs which have been used and in the case of natural steroid hormones these have been used to increase mass gain by stimulating protein anabolism. pesticides have been used for many years in the control of ectoparasites which transmit important human and livestock diseases. the purpose of the present article is to review procedures for management of veterinary products to facilitate national and international trade. these compounds and/or their metabolites have the potential to cause undesirable health effects to either target animals or consumers. most african countries do not have competent authorities to conduct risk analysis for veterinary drug and pesticide residues in edible tissues. because of the possible undesirable health effects from residues of veterinary compounds, the fao/who established expert groups to establish acceptable daily intake and maximum residue levels (mrls) for each drug or pesticide. in the case of natural steroids like oestradiol, progesterone and testosterone implants, no withdrawal period is required since there is no risk to the consumer. bulls can have levels of testosterone ranging from 535–10 950 pg/g, heifers 92–250 and treated steers 100 pg/g, respectively. data to enable approval of drugs and pesticides is to a large extent similar and include toxicity studies, reproductive studies, stability studies, safety, efficacy, tissue residue depletion studies and environmental impact. good practice in the use of acaricides as indicated on the label is inevitable so that residue levels of these compounds remain below the specified mrl. enactment and enforcement of legislations by various countries for the control of registration, sale, distribution and usage of ethical products should be enforced including use of prescriptions by veterinarians. good practice in the use of veterinary drugs is the recommended or authorized usage of drugs. it should be enforced to ensure safe animal products for human consumption and to facilitate regional or international trade. in conclusion, for efficient production of animal protein from food producing animals all veterinary products should be approved prior to use, residue monitoring programs should be implemented; veterinarians and producers must use these compounds prudently using recommended good practices. 156 management of drugs, hormones and pesticides in africa pounds for increased mass gain by stimulating protein anabolism to attain early market maturity. pesti cides have been used for many years as either sprays, pour-ons, in dips or in baths to control ectoparasites like ticks, flees, mite, lice, etc. exam ples of acaridices which have been used include inorganic arsenicals, organochlorine compounds (bhc, lindane and toxaphene), organophosphates (dichlorvos and chlorphentyphos), carbamates (sevin), natural pyrethrins, formamidines (amitraz) synthetic pyrethins (cypermethrin, alpha cypermethrin, deltamethrin, permethrin, etc). tick resistance to acaricides is quite rampant and hence prudent use and monitoring of acaricides to minimize resistance is important. ticks transmit important livestock diseases like anaplasmosis, babesiosis and theileriosis. theileriosis (east coast fever) is a very fatal disease to exotic cattle in eastern africa and unless proper tick control is practiced, there can be serious economic losses. the purpose of this presentation is to review procedures for the management of veterinary products to facilitate national and international trade. drugs drugs are phamacotherapeutic armaments used regularly for therapy and disease control in animals. there are many classes of drugs used which include, among others, antimicrobial agents, antihelminics, antiprotozoal agents, hormones and anti-inflammatory compounds. chemical compounds are xenobiotic in the mammalian body and have to be metabolized to safe and water soluble metabolites. drug compounds and/or their metabolites have the potential to cause undesirable health effects to either target animals or consumers. undesirable health effects may include among others acute or chronic toxic effects and long term effects like carcinogenic, teratogenic, genotoxic, reproductive or developmental disorders. in the case of antimicrobial agents, antimicrobial resistance development can occur due to non-prudent use of these compounds. international trade implications most african countries do not have competent authorities to conduct risk analysis for veterinary drug and pesticide residues in edible tissues. risk analysis is a science-based decision to protect consumers from possible hazards from residues. risk analysis comprises risk assessment, risk communication and risk management. because of the possible undesirable health effects from residues of veterinary compounds, the fao/who established expert groups to establish maximum residue levels (mrls) for each drug or pesticide. the role of the expert committee on food additives and the joint meeting on pesticide residues is to establish health-based guid ance values for residues of veterinary drugs and pesticides, respectively, by establishing an acceptable daily intake (adi). adi refers to the total amount of drug residues and metabolites in edible tissues which human beings can consume throughout life without causing undesirable effects and is expressed as mg/person/day. an adi is an output of a risk assessment of the compound, following application of the first two steps of risk assessment paradigm: hazard identification and hazard characterization. an adi is derived from the noel or lowest observed level (loel) from either appropriate toxicological, pharmacological, microbiological or epidemiological end point applying appropriate safety factor. once an adi has been established, maximum residue limit (mrl) can be derived taking cognisance of other factors. an adi derived from toxicological end point utilizes appropriate laboratory animals. established mrl is apportioned to various edible tissues like fat, kidney, liver, meat, eggs, milk and eggs according to dietary patterns. mrl refers to the maximum amount of chemical residue legally permitted to be in edible tissues or recognized as safe and is expressed as mg/kg. hormones steroid hormones are produced mainly in the gonads and major compounds used for anabolic purposes are oestradiol, progesterone and testosterone. endogenous steroid tissue hormone levels are high in most of normal untreated bulls, pregnant heifers and cows and thus proper use of natural steroids as implants do not significantly increase tissue levels above those found in animals’ physiological state (who 1982). bulls can have testosterone levels ranging from 535–10,950 pg/g, heifers 92–250 and a treated steer 100 pg/g, respectively (roche 1991). in the case of natural steroids like oestradiol, proges terone and testosterone implants, no withdrawal period is required. anabolic steroids (nadrolone, norethandrolone, ethylestrenol) are derivatives of tes tosterone that enhances anabolic effects with reduced androgenic effects when synthetic non-steroidal oestrogens include stilbene or zeranol. anabolic steroids stimulate haematopoiesis, appetite 157 e.s. mitema and mass gain. adverse effects of anabolic steroids include hepatotoxicity, masculinization and early closure of bone epiphyses in young animals. because of their undesirable health effects, their use by producers as implants should follow strict label instructions. in most countries, especially the european union, their use as growth promoters has been banned. the proper use of steroids is defined as implantation of steroids in a site of the body that does not enter the human food chain for instance ear implantation and proper adherence to designated withholding period prior to slaughter. acaricides ectoparasiticides should be regulated for use in most african countries to facilitate use of quality products. in kenya, acaricides are regulated by pest control products board (cap 355), which is a different agency from the one controlling drugs and hormones. data to enable approval of pesticides to a large extent is similar to those for drugs and include toxicity studies, reproductive studies, stability studies, safety, efficacy, tissue residue depletion studies and environmental impact among others (blagburn & lindsay 2001). good practice in the use of acaricides as indicated on the label is inevitable so that residue levels of these compounds are below the specified mrl for each compound. the fao/who’s joint meeting on pesticide residue is the expert committee that establishes the mrls of the acaricides for use in food producing animals. african countries should adopt the codex standards of the fao/who on pesticide residues to facilitate improved national and international trade. interventions in proper management of veterinary compounds adoption of the codex standard in order to promote regional and international trade among member states in africa, prudent procedures in the management of veterinary products is inevitable. edible tissues of animal origin should contain residue levels of veterinary drugs and pesticides below the recommended mrl to facilitate trade. one of the world trade organization treaties for instance sanitary and phytosanitary measures requires that all edible animal products should use the codex standard. the codex standard is the basis for international trade for edible animal products. mrls of the various drugs and pesticides established become adopted as the codex standard for the respective compound. african governments through their respective veterinary regulatory authorities should adopt the codex standards to facilitate trade. the risk management for chemical residues is through adherence to the withdrawal period for each particular veterinary compound administered to food animals. legislative framework enactment and enforcement of legislations by various countries for the control of registration, importation/sale, distribution and usage of ethical products should be encouraged. although most african countries have regulatory legislation, these are, however, not properly enforced. lack of enforcement may be due to either political interference, lack of awareness on risks associated with these compounds, lack of trained personnel or lack of financial resources. in the case of veterinary products’ regulation, it is important that all drugs should be registered before market authorization in respective countries. prescriptions by veterinarians for ethical products is important before products are used. registration of all products registration of veterinary products ensures that these compounds are safe, efficacious and of good quality to target animals. one of the registration requirements is that the residues of these products are safe to consumers in case of food producing animals. environmental safety of metabolites of veterinary compounds is gaining importance especially for products eliminated via faeces and would otherwise contaminate the environment and alter biodiversity. review of veterinary compounds before market authorization is a tedious process that requires the presence of legally established competent authority. the review process requires that drug companies/sponsors supply relevant data to support pharmaceutical quality, residue study, tox i cological study, environmental impact assessment, safety to target animals and consumer or clinical efficacy. most african countries do not have well trained scientists to conduct and review processes before drug approval and use of experts from other countries with competent regulatory authorities may be necessary. review and approval of pesticides for the control of ectoparasites in most countries in africa is governed by different legislation. the mission of regulation of pesticides is basically to ensure safe, efficacious and quality products. environmental safety of pesticides is given a lot of consideration since these compounds find their way in the envi158 management of drugs, hormones and pesticides in africa ronment. tissue residue depletion data for specific pesticides is also assessed to ensure that mrl levels are observed to facilitate international trade. establishment of surveillance programmes for residue monitoring of various drugs, hormones and acaricides should be implemented by all countries. good agricultural practices drugs the use of veterinary products, especially in food producing animals, should be closely regulated by the concerned regulatory authority, the professionals, producers and the industry. good practice in the use of veterinary drugs is the recommended or authorized usage of drugs. it envisages that veterinarians take control of the usage of drugs and advise on pesticide application. the veterinarians should give adequate information to producers concerning withdrawal periods as recommended by the manufacturers before animal products are sold or slaughtered for human consumption. in order to achieve proper usage of drugs to ensure safe animal products for human consumption and facilitation of regional or international trade, the following guidelines should be observed and where possible enforced by the concerned parties: • accurate diagnosis should be obtained where possible before treatment, which should be guided by principles of maximum effectiveness combined with minimum risk. specific treatments should employ few products and avoid combination therapy. • veterinarians and producers should avoid overuse or misuse of drugs to minimize development of resistance, for instance in the use of antibacterial agents or anthelmintics. antimicrobial or anthelmintic resistance tends to cause delays in clinical recovery and escalates treatment costs. • the use of prescriptions for ethical drugs should be enforced to ensure correct dosage, site and route of administration. • whenever products are used off-label or extralabel, due to lack of authorized product or certain disease conditions, veterinarians in using these products in food producing animals should exercise a lot of considerations and ensure that extended withdrawal period time is assigned for the drug prior to marketing of either milk, meat or eggs. extra-label use should only be used by a qualified veterinarian or somebody under his/her supervision. • records of all veterinary products administered to food animals should be kept including quantities, date of administration and the identity of animal. records should be kept for at least 2 years and be available when required by competent authority. • all medicinal products should be stored properly according to the label instructions and in compliance with national laws. all expired drugs medicines/products should be disposed of safely according to the label instruction. • all antimicrobial treatments should follow recommended guidelines on prudent use to minimize antimicrobial resistance among food borne patho gens like salmonella spp., campylobacter spp., escherichia coli and enterococcus faecium (who 2000). veterinarians should use antibiotics judiciously following a clear clinical indication and after antimicrobial susceptibility test has been conducted. pesticides good agricultural practice in the management of acaricides is quite important to minimize environmental contamination and development of tick resistances. • the right formulation (dip, spray, collars and pour-on) should be applied to food animals. a spray formulation product should not be used in the dip or as pour-on. • pesticides must be stored in a dry place and protected from extremes of temperature. most pesticides can retain their activity for at least two years. they must be kept out of reach of children. • pesticides containers should be thoroughly washed and washings including either the spray or dip material properly disposed. the washed containers must not be used to contain food materials. • regular acaricide efficacy surveillance on ticks should be conducted by regulatory authorities to protect the animals from tick infestation. in conclusion, for efficient production of animal protein from food producing animals, it is almost a must to use drugs, acaricides and to some extent natural steroids. organic production of edible animal products in africa is still possible in some parts of the continent especially among pastoralists, but this is becoming less common since most local people use 159 e.s. mitema dewormers. good clinical veterinary practices and good agricultural practices in the use of veterinary products on food producing animals are crucial to enable producers to market their products without trade hindrances. all veterinary products should be approved prior to use and veterinarians and producers must use these compounds prudently. references blagburn, b.l. & lindsay, d.s. 2001. ectoparasiticides, in pharmacology and therapeutics, 8th ed., edited by h. richard adams. ames: iowa state university. mitema, e.s. 2004. prudent use of veterinary drugs. impact on safe animal products for increased productivity. kenya veterinarian, 27:1–2. roche, j.f. 1991. growth promoters, in veterinary applied pharmacology and therapeutics, 5th ed., edited by g.c. bran der, d.m. pugh, r.j. bywater & w.l. jenkins. london: bailliére tindall. world health organisation 1982. health aspects of residues of anabolics in meat. report on who working group, bilthoven, 10–13 nov. 1981. copenhagen: who regional office for europe. world health organisation 2000. global principles for the containment of antimicrobial resistance due to antimicrobial use in animals intended for food. journal of veterinary pharmacology and therapeutics, 24:385–390. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: mohammad t. rahimi1,2 ahmad daryani1,2 shahabeddin sarvi1,2 azar shokri1,2 ehsan ahmadpour1,3 saeed h. teshnizi4 azade mizani1,2 mahdi sharif1,2 affiliations: 1toxoplasmosis research centre, mazandaran university of medical sciences, sari, iran 2department of parasitology and mycology, sari medical school, mazandaran university of medical sciences, sari, iran 3infectious and tropical diseases research centre, tabriz university of medical sciences, tabriz, iran 4paramedical school, hormozgan university of medical science, bandar abbas, iran correspondence to: mahdi sharif email: msharifmahdi@yahoo.com postal address: department of parasitology and mycology, sari medical school, mazandaran university of medical sciences, pc 48168-95475, sari, iran dates: received: 19 june 2014 accepted: 13 nov. 2014 published: 30 apr. 2015 how to cite this article: rahimi, m.t., daryani, a., sarvi, s., shokri, a., ahmadpour, e., mizani, a. et al., 2015, ‘cats and toxoplasma gondii: a systematic review and meta-analysis in iran’, onderstepoort journal of veterinary research 82(1), art. #823, 10 pages. http://dx.doi.org/10.4102/ojvr.v82i1.823 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. cats and toxoplasma gondii: a systematic review and meta-analysis in iran in this review article... open access • abstract • introduction • research method and design    • database search    • data collection    • statistical methods • results • discussion • limitations of the study    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ toxoplasma gondii is a cosmopolitan zoonotic intracellular coccidian of the phylum apicomplexa infecting warm-blooded animals and human beings. this protozoan causes a significant public health problem in humans and imposes considerable economic losses and damages to husbandry industries. the final host, cats, accounts for all of these significant burdens. hence the present study was designed to analyse and review the overall prevalence rate of t. gondii infection in cats in iran for the first time. in the present study data collection (published and unpublished papers, abstracts of proceedings of national parasitology congresses and dissertations) was systematically undertaken on electronic databases including pubmed, google scholar, ebsco, science direct, scopus, magiran, irandoc, iranmedex and scientific information database. a total of 21 studies from 1975 to 2013 reporting prevalence of toxoplasma infection in cats from different areas in iran met the eligibility criteria. the pooled proportion of toxoplasmosis using the random-effect model amongst cats was estimated at 33.6% (95% confidence interval [ci] 22.05–46.41). the prevalence rate of cat toxoplasmosis in various regions of iran ranged from 1.2% to 89.2%. firstly, this study establishes a crude prevalence rate of t. gondii infection in cats. secondly, it discusses the role of significant risk factors including sex, age and being either household or stray cats, in the epidemiology of the disease. furthermore, the current study determines gaps and drawbacks in the prior studies that are useful to keep in mind to assist in designing more accurate investigations in future. introduction top ↑ toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite which belongs to the phylum apicomplexa that infects all species of warm-blooded animals (flegr et al. 2003). in humans it is considered to be one of the most common parasites, based on serological investigations that estimate that up to a third of the world's population has been exposed to this widespread zoonotic agent. the overall seroprevalence rate of toxoplasmosis amongst the general population in iran is 39.3% (95% confidence interval [ci] 33.0% – 45.7%) (bahrami et al. 2011; daryani et al. 2014; dubey & jones 2008; sharif et al. 2007). even though the majority of toxoplasmosis cases in immune-competent individuals are either asymptomatic or mild, first exposure to t. gondii during pregnancy can lead to transplacental transmission to the embryo, with serious pathological signs including hydrocephalus, microcephaly, blindness, abortion and death of the foetus (dunn et al. 1999; havelaar, kemmeren & kortbeek 2007). in addition, t. gondii is considered to be an opportunistic and life-threatening parasite in immune-compromised groups, encompassing those with hiv and aids, cancer and organ transplant recipients who receive immunosuppressive drugs (tenter, heckeroth & weiss 2000). felids play a pivotal role for t. gondii as definitive hosts, and interestingly are known as the only final hosts that produce oocysts in their faeces, contaminating soil, food and water (cenci-goga et al. 2011; dubey 2004). even though the final host excretes oocysts for a short period of time (only 1–2 weeks), millions of oocysts may be excreted. oocysts can survive in the environment for several months and are noticeably resistant to freezing, drying and disinfectants, whereas they are not heat-resistant and are destroyed at 70 °c for 10 min (dubey & beattie 1988; dubey & jones 2008). infection both in the definitive and the intermediate host usually occurs either through ingestion of infected tissue cysts or oocysts. felids are more likely to shed oocysts followed by ingestion of tissue cysts rather than oocysts. surprisingly, a cat must ingest at least 1000 oocysts in order to develop an infection, although ingestion of just one bradyzoite is enough for a cat to acquire t. gondii infection (dubey 2008). clinical manifestations of cats infected with t. gondii include depression, anorexia and fever, followed by peritoneal effusion, hypothermia, icterus and dyspnoea. moreover, some other symptoms of toxoplasmosis are diarrhoea, weight loss, muscle hyperaesthesia, fever, anorexia, seizures, ataxia, pancreatitis and anterior or posterior uveitis (dubey & lappin 2006). furthermore, the coccidian phase of the entero-epithelial cycle is seen solely in the definitive feline host. the extra-intestinal development of t. gondii is the same for all hosts, including all warm-blooded vertebrates (dubey 2005). the disease is important both in the medical and veterinary fields. toxoplasmosis causes significant economic losses and damages to animal husbandry due to stillbirths and neonatal mortality in sheep and goats (dubey & beattie 1988; hartley & marshall 1957). in spite of the need, currently there is no effective vaccine, even though many efforts have been conducted to develop a vaccine and are ongoing. furthermore, no approved treatment exists for clinical toxoplasmosis in cats. drugs including pyrimethamine, sulphonamides, trimethoprim and clindamycin, either alone or in combination, that have been prescribed to treat cats with clinical toxoplasmosis have shown varied results (dabritz et al. 2007). cats have a key and crucial role in the epidemiology of toxoplasmosis, so expanding the basic knowledge about t. gondii infection in cats is a matter of importance. it is worth mentioning that epidemiological investigations are still the most useful method for evaluating the status of t. gondii infection. despite the multitude of publications on toxoplasmosis in cats from iran, there is no systematic review and meta-analysis that can describe the status of toxoplasmosis in the final host in this country. therefore the objective of the current systematic review and meta-analysis was to determine the weighted prevalence of t. gondii infection and describe the epidemiological features of infection in cats in iran. research method and design top ↑ database search to gather information a precise and comprehensive search was performed on all scientific publications (full texts and abstracts) from february to april in 2013 (figure 1). the following nine databases were included: five english databases (pubmed, google scholar, ebsco, science direct and scopus) and four persian databases (magiran, irandoc, iranmedex and the scientific information database [sid]). in addition to published articles, dissertations and all proceedings of national parasitology congresses held in iran from 1975 to 2013 were carefully evaluated. in order to avoid missing any articles, whole references of papers were also meticulously checked. figure 1: flowchart describing the study design process. the search terms used alone or in combination were ‘toxoplasma gondii’, ‘toxoplasmosis’, ‘toxoplasma infection’, ‘animal toxoplasmosis’, ‘cat’, ‘feline’, ‘epidemiology’, ‘prevalence’, ‘iran’ and ‘anti-toxoplasma antibodies’. data collection was limited to items in english and persian. data collection all cross-sectional studies carried out to estimate the prevalence of toxoplasmosis, diagnosed by different methods using serological, molecular and parasitological tests on cats, were included. repetitive papers were excluded. the data that were collected for the current study were as follows: year of publication, first author, study areas, sample size, number of males and females, prevalence rate, age of samples, diagnostic tests, time of the study, and involving either domestic or stray cats. for this purpose a data extraction form was used. the quality of the meta-analysis was evaluated using the strengthening the reporting of observational studies in epidemiology (strobe) checklist, which included 22 items that we considered essential for good reporting of observational studies. these items were related to the article's title, abstract, introduction, methods, results and discussion sections. scores under 7.75 were considered to indicate bad quality, of 7.76–15.5 low quality, 15.6–23.5 moderate and more than 23.6 high quality (von elm et al. 2007). statistical methods the crude and the weighted prevalence estimates as well as the 95% ci for each study that was included were calculated. a forest plot was used to visualise the heterogeneity amongst studies. the heterogeneity was expected in advance, and statistical methods such as i2 and cochrane's q test (with a significance level of p < 0.1) were used to quantify variations. for the purpose of meta-analysis we assumed that the included studies were random samples from the populations under study, and a random-effect model was employed. meta-regression and subgroup analyses were employed to assess the cause of heterogeneity amongst the selected studies, and egger's regression test and funnel plotting were used to evaluate publication bias. proportions of individual studies and overall prevalence were presented using forest plots. the meta-analysis was performed using the trial version of statsdirect statistical software (http://www.statsdirect.com). results top ↑ from the nine databases, 21 studies met the eligibility criteria and were included in the current systematic review and meta-analysis. a mean score of 16.8 using the strobe checklist (von elm et al. 2007) was obtained for the 21 studies that were analysed. a flowchart depicts the study design process (figure 1). a total number of 2145 cats was examined for toxoplasmosis from 1975 to 2013 in different areas of iran, and 662 cases were diagnosed as positive using different diagnostic methods (tables 1 and 2). table 1: publications on cat toxoplasmosis included for meta-analysis. table 2: studies of cat toxoplasmosis included for meta-analysis based on stool examination. during a period of 39 years, nine different types of diagnostic methods were employed to evaluate t. gondii infection in cats, as follows: the modified agglutination test (mat), direct agglutination test (dat), indirect immunofluorescent assay (ifa), latex agglutination test (lat), immunochromatography test (ict) and sabin and feldman test (sft), wet smear, flotation and polymerase chain reaction (pcr). the most frequently used diagnostic method for t. gondii assessment in cats in iran was the ifa (5 studies), followed by flotation (4 studies), mat (3 studies), lat (2 studies), dat (2 studies), ict (2 studies), sft, pcr and wet smear (1 study for each). the pooled proportion of toxoplasmosis, using the random-effect model, amongst cats in iran over the 39-year period was estimated at 33.6% (95% ci 22.05–46.41) and a forest plot diagram of the current study was drawn (figure 2). a wide variation was observed in the prevalence estimates of different studies (q statistic = 742.3, df = 20, p < 0.0001 and i² = 99%). figure 2: forest plot diagram of the current systematic review and meta-analysis. the prevalence rate of cat toxoplasmosis in various regions of iran was between 1.2% and 89.2% in khorasan and tehran respectively. the prevalence rate of toxoplasmosis in cats in different parts of iran is shown in figure 3. amongst the studies included, only three studies compared stray cats with domestic cats for toxoplasmosis (akhtardanesh et al. 2010; haddadzadeh et al. 2006; raeghi & sedeghi 2011). they found a statistically significant difference in the number of positive stray cats and domestic cats. the results based on age distribution were mentioned in 6 out of 21 surveys. a significantly higher prevalence rate of t. gondii was observed in older animals as compared with younger ones. regarding sex, just two studies reported a significant difference, with male cats showing a higher prevalence rate of toxoplasmosis than females (farhang 2010; raeghi & sedeghi 2011). the source of faeces in all the studies was the rectum of the examined cats. figure 3: prevalence of toxoplasmosis in cats in different provinces. results of heterogeneity of meta-analysis for two groups (stray and domestic cats) showed that they were not homogeneous (p < 0.0001). overall seroprevalence rates for stray and domestic cats were 0.38 (95% ci 0.22–0.55) and 0.33 (95% ci 0.19–0.47) respectively, and the pooled estimate was 0.36 (95% ci 0.25–0.47) (figure 4). figure 4: prevalence of seropositivity in terms of type of cats (stray or domestic). there was a significant difference between sub-groups of sexes (p < 0.0001), and random meta-analysis showed that seroprevalence rates of toxoplasmosis in males and females were 0.21 (95% ci 0.23–0.59) and 0.31 (95% ci 0.19–0.43), respectively, and the pooled estimate was 0.36 (95% ci 0.25–0.47) (figure 5). figure 5: prevalence of seropositivity by sex of cats. differences between sub-groups of ages (juvenile and adult) were significant (p < 0.0001) in only two studies, and random meta-analysis showed seroprevalence rates of toxoplasmosis in juveniles and adults to be 0.545 (95% ci 0.540–0.550) and 0.392 (95% ci 0.286–0.562) respectively. the pooled estimate was 0.481 (95% ci 0.386–0.788) (table 3). table 3: prevalence of seropositivity by age of cats. egger's regression test and a funnel plot were carried out for assessment of publication bias, and results showed that publication bias was significant (figure 6). figure 6: funnel plots from egger. five studies employed stool examination on 582 cats and 110 cases (18.9%) were reported as positive. discussion top ↑ the current study is the first systematic review and meta-analysis of cat toxoplasmosis in iran. it provides valuable data about the prevalence of toxoplasmosis in cats from 1975 to 2013. the overall prevalence rate of toxoplasmosis amongst cats in iran was estimated to be 33.6%. the worldwide seroprevalence of toxoplasmosis in domestic cats (felis catus) was estimated to be 30% – 40% (dubey & beattie 1988), and our findings roughly correspond to this range. besides, previous studies have shown that the prevalence rates of t. gondii antibodies in cat populations vary greatly, from 0% to 100%, and depend on particular criteria including age, method of survey, number of animals studied and geographical area (dubey 2005; dubey & beattie 1988). several surveys have been performed on cat toxoplasmosis in iran's neighbouring countries. an investigation on anti-t. gondii antibodies in 99 cats in ankara, turkey, using sft and ifa showed prevalences of 40.3% and 34.3% respectively (özkan et al. 2008), which is in close alignment with the present study. in another survey in nigde, turkey, the prevalence of antibodies to t. gondii employing sft in stray cats was reported to be 76.4% (karatepe et al. 2009). in saudi arabia the prevalence rate of t. gondii antibodies in cats was determined to be 15.2% using the indirect haemagglutination (iha) test (hossain et al. 1986). a study on stray and domestic cats using lat in lahore, pakistan, showed that 56% of cases were seropositive for t. gondii (domestic cats 48% and stray cats 64%) (shahzad et al. 2006). in addition, the seroprevalence rate of t. gondii infection in cats was reported to be 60% in faisalabad, pakistan using lat (ahmad et al. 2001). the infection rates in cats in lebanon and iraq were reported to be 78.1% and 100%, respectively (deeb, sufan & digiacomo 1985; khairy, alaa & ahmad 2010). the findings of the current study are in agreement with those of dubey (1998), who reported a 30% – 80% seroprevalence rate in cats in the united states of america (usa). a high prevalence of toxoplasmosis of cats in some areas may be due to the following factors: humid and temperate climate; absence of routine treatment for feline toxoplasmosis; and a considerable abundance of cats. it was confirmed that there is conformity between climate and the prevalence rate of toxoplasmosis, and regional prevalence varied in conformity with different climates. it is usually more prevalent in warm, humid climates and at lower altitudes compared to cold or dry districts. this fact is associated with longer viability of t. gondii oocysts in a warm and humid environment (tutuncu et al. 2003). sporulated oocysts of t. gondii can persist in the environment (particularly in moist shaded soil or sand) for several months owing to their resistance to variable environ­mental conditions (dubey & beattie 1988; webster 2001). in the usa the prevalence rate in the drier southwest, including new mexico, utah and arizona, was lower (16.1%) than in humid climates such as hawaii (59.2%), in accordance with the abovementioned climate template for t. gondii infection (dubey & jones 2008). in contrast, in some areas of iran, such as mazandaran province, the findings are not in accordance with that climate template. iran's climate ranges from arid or semi-arid to subtropical along the caspian coast and in the northern forests. in the north of the country temperatures seldom decrease below freezing in winter, and the area usually remains humid for the rest of the year. in summer the temperature rarely exceeds 29 °c (84.2 °f) (modarres & de paulo rodrigues da silva 2007). in the past different diagnostic tests were used in t. gondii surveys in cats. ifa was the most frequently employed test for the diagnosis of cat toxoplasmosis. this test was introduced in 1992 and considered a more reliable test than other serological diagnostic methods due to its significant sensitivity and specificity. this method is a relatively simple assay for evaluating the infection of animals, and also is particularly useful for screening a large number of specimens, which may explain why most studies used this method (chejfec 1999; de la luz galvan-ramirez et al. 2012; silva et al. 2002). in this systematic review and meta-analysis sex, age and type of cat (stray or domestic) can be probable causes of homogeneity, as well as publication bias. the role of risk factors including sex, age and being stray or domestic in the prevalence of toxoplasmosis is undeniable. in the current review there was a statistically significant difference between sexes (p < 0.05), and a higher infection rate of toxoplasmosis was seen in male cats compared to females. this may be related to lifestyle, as male cats have more of a tendency to wander and thus more access to contaminated sources; spending more time outdoors may increase their exposure to infection. the age of animals is another major factor in prevalence of toxoplasmosis. since young animals were less infected than older ones, it is expected that with increasing age exposure to t. gondii infection also increases (miró et al. 2004). in iran a significant relationship was observed between age at sampling and prevalence rate of toxoplasmosis amongst cats. these findings were in accordance with the results of some studies in other countries, which reported an increased prevalence of toxoplasmosis in older compared to younger cats (frenkel et al. 1995; maruyama et al. 2003; vollaire, radecki & lappin 2005). it has been mentioned that newborn kittens are more dangerous than adult cats for transmission of this infectious disease, since they excrete oocysts for 1–2 weeks. on the other hand, kittens infected transplacentally or via milk exhibit more severe symptoms and frequently die due to pulmonary or hepatic disease (buxton & rodger 2008). in the present study, haddadzadeh et al. (2006), akhtardanesh et al. (2010) and raeghi and sedeghi (2011) showed that the prevalence of toxoplasmosis in stray cats was noticeably higher than in domestic cats. in general, stray cats are more prone to t. gondii infection compared to household cats. the findings confirmed that stray cats have a tendency to have a higher prevalence rate than cats kept indoors. an acceptable justification for this fact might be that stray cats could acquire the infection through catching wild rodents, birds and reptiles, raw food scraps and so on. cat owners can decrease their cats’ exposure risk of acquiring t. gondii infection by keeping their cats indoors and also not feeding them uncooked meat and milk. in addition, the food source of the animal is significant in transmission and also for completion of the lifecycle of the parasite (dubey et al. 2006). integrated control strategies and measures should be considered to prevent and control toxoplasmosis in both stray and household cats, which will have important implications for the control of toxoplasmosis in humans and other important intermediates such as sheep, goats and cattle. limitations of the study top ↑ the collected data are limited just to 10 out of 31 provinces, and there is a paucity of data for cat toxoplasmosis in the majority of provinces of iran. although many efforts have been made to determine the prevalence of toxoplasmosis in cats in different parts of iran, some gaps in prior studies were evident. recommendations not enough attention was paid during sampling to the role of major factors including sex, age, and being stray or companion cats, despite their key role in the epidemiology of the disease. therefore, considering all of the abovementioned parameters is necessary in order to overcome these shortcomings in future. the present study will form the basis of further studies that will enable us to deepen our knowledge of the epidemiology of t. gondii. conclusion top ↑ based on the current results, stray cats are probably the major source of t. gondii infection in iran. this study accentuates some valuable and interesting points: the prevalence rate of toxoplasmosis in cats in iran is high (33.6%), and this considerable infection rate of final hosts can be considered a potential danger to public health and animals due to high contamination of the environment by oocysts. acknowledgements top ↑ we are very grateful to drs hamid badali and mohsen aarabi for their helpful consultations and comments on the manuscript. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions m.s. 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epidemiology (strobe) statement: guidelines for reporting observational studies’, preventive medicine 45, 247–251. http://dx.doi.org/10.1016/j.ypmed.2007.08.012 webster, j.p., 2001, ‘rats, cats, people and parasites: the impact of latent toxoplasmosis on behaviour’, microbes and infection 3, 1037–1045. http://dx.doi.org/10.1016/s1286-4579(01)01459-9 vale_41-45.indd introduction a century ago, when onderstepoort veterinary institute began, bruce (1895) had already shown that tsetse transmit the trypanosomes that cause the usually fatal diseases of nagana in livestock and sleeping sickness in humans. his work, in zululand, raised the prospect of overcoming these diseases that severely limit african heath, agriculture and transport. since then we have found several trypanocidal drugs but their use alone is problematic (barrett, coombs & mottramm 2004): there is widespread resistance to the veterinary prophylactics, for humans there is no prophylactic and the curatives are dangerously toxic. these difficulties and the apparent impossibility of developing a vaccine and the logistical problems of restocking with the few trypanotolerant breeds of cattle, mean that trypanosomosis is best tackled by controlling tsetse, with drugs as temporary expedients. much has been done to implement various methods of tsetse control, but the flies continue to occupy + 10 million km2 (rogers & robinson 2004), much as in bruce’s time. the present essay addresses the prospects for better progress by discussing the available techniques, the scale and organization of their implementation, and ways of improving these matters. techniques the widespread application of residual insecticide is now unacceptable. host destruction and bush clearing are also outmoded as deliberate tactics, but will continue de facto due to wider and denser settlement (jordan 1986). the main options, and their pros and cons, are as follows: sequential aerial spraying this technique involves 4–5 applications of non-residual insecticide, sprayed in the cool season to fos41 onderstepoort journal of veterinary research, 76:41–45 (2009) prospects for controlling trypanosomosis g.a. vale southern african centre for epidemiological modelling and analysis university of stellenbosch, stellenbosch abstract vale, g.a. 2009. prospects for controlling trypanosomosis. onderstepoort journal of veterinary research, 76:41–45 the best technical package for the future comprises trypanocidal drugs for temporary relief and the use of insecticide-treated cattle, artificial baits and aerial spraying to attack the vector, to so give more lasting security. whether this can speed the previously slow progress will depend on overcoming past hindrances to tsetse control: sporadic support, disputes over its desirability, difficulties of sustaining international operations, and poor planning in some instances. the pan-african tsetse and trypanosomiasis campaign intends to speed the progress but will fail unless it improves its image by breaking its association with the sterile insect technique and quickly executing some cheap and effective operations in large areas. even then, there could be severe brakes due to africa’s political and financial instability. overall, the pace of control is likely to increase, but perhaps only a little. 42 prospects for controlling trypanosomosis ter descent of droplets (allsopp & hursey 2004). excellent results are possible, especially with the newly improved methods of aircraft guidance, and large areas can be tackled quickly (kgori, modo & torr 2006), but the aircraft must fly low at night, precluding application in mountainous terrain. moreover, the technique gives no protection against invasion, and establishment costs are substantial, so that operations in less than about 1 000 km2 are uneconomic. baits stationary artificial baits (vale & torr 2004) consist of either traps or pyrethroid-coated screens, i.e. targets. traps are costlier than targets and less efficient, but since they retain the flies they are useful for surveys. with the savannah species of tsetse the performance of traps and targets is enhanced many times by adding the odour attractants identified from cattle, but the riverine species respond poorly to odours. pyrethroid can also be applied to cattle, whose wanderings enhance fly-bait contact. the cost-effectiveness of cattle treatment is improved by restricting application of insecticide to where most tsetse feed, i.e. the legs and belly of larger animals (torr, maudlin & vale 2007). baits can form barriers to invasion (hargrove 2003) but cattle are useable only where they occur, and targets can be stolen. sterile insect technique (sit) this technique releases sterile males to swamp wild males and so reduce breeding (feldmann 2004). however, sit demands the costly and lengthy establishment of large and complex rearing facilities, and requires insecticidal campaigns to suppress the wild tsetse population prior to sterile male release. moreover, control by reducing the breeding of the long-lived tsetse is inherently much slower than killing the pest (vale & torr 2005). field costs tables of costs should be viewed cautiously since they can misleadingly compare techniques used for distinctive purposes under different conditions, and with various costing procedures. standardizing on the basic field costs of government-run clearance of an isolated population of savannah tsetse, the costs are 761 us$/km2 for sit, $380 for aerial spraying, $283 for artificial baits, $56 for whole body spraying of 8 cattle per km2 and $12 if application is restricted to the legs and belly (shaw, torr, waiswa & robinson 2006). the high costs of sit are greater if the suppression expenses are added, and would rise further if mixed infestations were tackled, requiring the release of sterile males of each species present. choice of techniques given that insecticidal methods are relatively cheap and simple, and that their efficacy is proven by their clearance of huge areas (e.g. torr, hargrove & vale 2005; kgori et al. 2006), it is best to employ only those techniques. they should be used according to the following rules: • if the operational area is not isolated, make it so by an invasion barrier of baits. then, to eliminate the isolated population, put insecticide on cattle if these are present. if not, then: – if the area is fairly flat and greater than about 1 000–2 000 km2, use aerial spraying, especially if there is a risk of target theft. – if not, use targets. • if a campaign employs a mosaic of control methods to suit varying terrain and land use, run the techniques to produce a synchronized effect, so that tsetse movement does not allow some flies to avoid treatment by any method. natural and artificial baits are the most compatible since they can be applied at any season to work at similar speeds. combining baits with aerial spraying may require the partial overlapping of treated areas. • have targets ready to deploy promptly in any residual pocket left after aerial spraying, before the pocket expands. planning whatever techniques are used, their timing, location and intensity must be planned with a ‘feel’ for the pop ulation dynamics of tsetse. problems resulting from the absence of such a feel are exemplified by the aerial spraying of the 25 000 km2 of the okavango delta, botswana, in 18 years from 1973–1991 (hargrove 2003). annual campaigns were piecemeal, with inadequate attention to the routes and rates of reinvasion. hence, there was a poor effect until the better planning in 2001/02, with targets placed for an effective invasion barrier (kgori et al. 2006). to strengthen the prospects for good planning elsewhere, we might encourage the use of user-friendly models of tsetse populations (www.tsetse.org). for example, one such model has compared the cost43 g.a. vale effectiveness of sit and insecticidal methods (vale & torr 2005), drawn lessons from the results of the recent spraying and target campaigns in botswana (kgori et al. 2006) and helped to predict the costs of all control methods (shaw et al. 2006). implementation tsetse control has been implemented on a range of scales, by a variety of agencies, but it consists of just two basic set-ups. firstly, there are large campaigns run by governments, often with donor funds, to clear many thousands of square kilometers. secondly, the development of cheap and relatively simple bait techniques has allowed smaller self-help schemes (dransfield & brightwell 2004). the relative merits of each set-up depend first on the appropriate scale of operations. scale for any single technique applied in one area, the fixed overheads mean that the costs per unit area decline as the operation becomes larger. however, when operational areas are small, they usually cover settlements where insecticide-treated cattle are appropriate for control. expansion from such areas will embrace unsettled places where the more costly aerial spraying or targets are needed. then there are the establishment costs for more than just one technique. moreover, ground access must be developed and maintained for any technique in unsettled places—for the associated surveys and monitoring if not necessarily for the control measures themselves. hence, the average costs per unit area tend to increase with the scale of control. conversely, the more the operations embrace unsettled areas, or places where livestock farming is inherently unproductive, the less the average benefit per unit area. the upshot is that the sustainable use of the cheaper control methods is beneficial in highly productive settlements (shaw 2004). operations nearby can be justified to counter invasion, but wider control is uneconomical—and downright damaging when it encourages unregulated settlement where wildlife enterprises are the appropriate land users (cumming & lynam 1997). against these arguments for limited control, larger campaigns have the benefit of clearing more of the invasion sources, thereby offering a more permanent solution. indeed, the technical ideal is the clearance of whole fly-belts, removing forever the invasion threat. hence, lively debate arises about the required scale of control (dfid 2003). for now a compromise is needed: the only large unsettled areas that should be tackled are those that will not be damaged by land use change and which can be conveniently held against invasion, as exemplified by the recent work in botswana (kgori et al. 2006). elsewhere, less ambitious schemes would be appropriate. the balance within this compromise will be determined by the support given to the distinctive types of implementing agency involved. agencies progress towards fly-belt clearance requires international coordination and donor help. the possibilities were explored as one aim of the regional tsetse and trypanosomosis control programme (rttcp) for malawi, mozambique, zambia and zimbabwe, started in 1986 with ec support. the technical option to clear the 380 000 km2 of the fly-belt common to all four countries was proved, but it was confirmed as neither practicable nor worth it in the foreseeable future. hence, the rttcp wound down in the late 1990s (rttcp 1999). another chance for international co-operation originated in the technical success of sit on unguja island (vreysen, saleh, ali, abdulla, zhu, juma, dyck, masangi, mkonyi & feldmann 2000) and the publicity given to it by the international atomic energy agency (iaea). sub sequently, the sit forum was created with iaea encouragement, and lobbied successfully for the establishment of the pan-african tsetse and trypanosomosis eradication campaign (pattec) in 2000, supported by the african union and aimed at rapid continental clearance. a sit laboratory is already built in ethiopia and more are discussed (enserink 2007). however, many of the traditional donors for tsetse control believe that the aims of pattec are not achievable, and that even large national operations should not be supported (dfid 2003). even if marked progress towards continental clearance were made by pattec, or via an accumulation of separate government campaigns, it would take many decades to achieve. hence, we must learn now to cope better with tsetse locally, in ways sustainable with little or no external funding. much of the focus is therefore on the cheapest and simplest options, i.e. baits, implemented by ranches and local communities (dransfield & brightwell 2004). the restricted application of insecticide to cattle is the most promising bait method, not simply because it is much cheaper but also because cattle usually occur in the very districts where trypanosomosis is an immediate problem. 44 prospects for controlling trypanosomosis however, self-help is hindered by beliefs, from precedent, that tsetse control is a government responsibility. specialist help is required with planning, monitoring and procurement. moreover, where invasion is a problem there is a limit to how small an area can be tackled effectively. this explains why in tanzania the national ranching company could use insecticide-treated cattle successfully in 2 500 km2 of the kagera region, whereas the technique was less successful on mkwaja ranch that was a tenth of the size, with cattle in only part of the area (hargrove 2003). therefore, to minimize any invasion problem, the area treated by one local community should adjoin those of other communities, governments or pattec. cost reduction with any implementing agency, and at any scale, the prospects for support depend largely on reducing the costs—not just the field costs quoted above, but also the overheads. shaw et al. (2006) estimated the overheads to be 213–240 us$/km2 for a full complement of administration, surveys, monitoring, ecological impact assessments and socio-economic studies. however, while the administrative and survey costs are unavoidable for sound planning and management, the other cost components are usually deemed necessary only with schemes run largely through governments and donor funding. hence, opportunities for cost reduction occur not only via the coordination of public and private attacks on adjacent infestations but also by partnerships in which the private sector implements control in its particular area and governments offer there the minimum of specialist help with planning, surveys and public health matters. such a system is now established in uganda (kabasa 2007). this involves the restricted application of insecticide to 37 000 head of cattle in five districts covering a total of 10 000 km2. it is particularly exciting since treatment expenses have been reduced to just two us cents per animal per month, even when private vets are paid to assist, so that field costs are around $1 per km2 per year. however, in areas where invasion pressure is more serious, it would be necessary to employ baits as invasion barriers, the basic costs of which in government-run schemes are around 120 us$/km2 protected per year if artificial baits are used, or about $22 if insecticide-treated cattle are employed (shaw et al. 2006). while treated cattle are by far the cheaper, they are at permanent risk of trypanosomosis near the invasion front—tsetse alighting on treated cattle usually feed before dying and so can transmit disease. thus, targets are technically better as barriers. how, then, could target costs be lowered towards those for cattle treatment? the performance of odours to use with targets for savannah tsetse might be doubled by identifying the complete range of attractants in cattle odour (torr, mangwiro & hall 2006). we need to re-check the apparently poor response of riverine tsetse to odours—it being expected a priori that odours would be important in habitats where visibility is poor. moreover, it was recently shown that for riverine tsetse in kenya the normal target size, i.e. 1 m2, can be reduced to 1/16 with only a halving of effectiveness (jenny lindh, personal communication). this raises the possibility of deploying handkerchief-sized baits that are cheap, disposable and bio-degradable. therefore, in overview, while we cannot guarantee continental clearance of tsetse in the next 100 years, we now have the prospects of effective compromise and cooperation, of being freer of trypanosomosis where immediately needed, and of improving the technologies and implementation arrangements that can help most. acknowledgements the author thanks dr alexandra shaw and proff. d. cumming, j. hargrove, i. maudlin and s. torr for comments on the draft. references allsopp, r. & hursey, b.h. 2004. insecticidal control of tsetse, in the 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factory. science, 317:310–313. 45 g.a. vale feldmann, u. 2004. the sterile insect technique as a component of area-wide integrated pest management of tsetse, in the trypanosomiases, edited by i. maudlin, p.h. holmes & m.a. miles. wallingford: cabi. hargrove, j.w. 2003. tsetse eradication: sufficiency, necessity, and desirability. research report, dfid animal health programme. centre for tropical veterinary medicine, university of edinburgh, uk. jordan, a.m. 1986. trypanosomiasis control and african rural development. london: longman. kabasa, j.d. 2007. public-private partnership works to stamp out sleeping sickness in uganda. trends in parasitology, 23: 191–192. kgori, p.m., modo, s. & torr, s.j. 2006. the use of aerial spraying to eliminate tsetse from the okavango delta of botswana. acta tropica, 99:184–199. rogers, d.j. & robinson, t.p. 2004. tsetse distribution, in the trypanosomiases, edited by i. maudlin, p.h. holmes & m.a. miles. wallingford: cabi. shaw, a.p.m. 2004. economics 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: leonard e.g. mboera1 sayoki g. mfinanga2 esron d. karimuribo3 susan f. rumisha1 calvin sindato4 affiliations: 1national institute for medical research, tanzania2muhimbili medical research centre, tanzania 3sokoine university of agriculture, tanzania 4tabora medical research centre, tanzania correspondence to: esron karimuribo postal address: po box 3021, chuo kikuu, morogoro, tanzania how to cite this article: mboera, l.e.g., mfinanga, s.g., karimuribo, e.d., rumisha, s.f. & sindato, c., 2014, ‘the changing landscape of public health in sub-saharan africa: control and prevention of communicable diseases needs rethinking’, onderstepoort journal of veterinary research 81(2), art. #734, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i2.734 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the changing landscape of public health in sub-saharan africa: control and prevention of communicable diseases needs rethinking in this proceedings... open access • abstract • introduction • communicable diseases, ecosystems and livelihoods • vulnerability to communicable diseases • climate change or variability and communicable diseases • challenges in the control of communicable diseases • integrated disease control employing an ecosystem approach • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ in sub-saharan africa, communicable diseases (cds) are the leading public health problems and major causes of morbidity and mortality. cds result in significant individual suffering, disrupting daily life, threatening livelihoods and causing one-third of the years lost to illness or death worldwide. this paper aims to analyse the current strategies in the control and prevention of cds in sub-saharan africa and proposes an ecohealth approach in relation to current changing epidemiological profiles. whilst in recent years the burden of hiv and aids, tuberculosis and malaria have helped to mobilise large amounts of funding and expertise to help address them, many cds, particularly those affecting the poor, have been neglected. people living in rural areas are also likely to be politically marginalised and living in degraded environments. they often lack assets, knowledge and opportunities to gain access to health care or protect themselves from infections. new diseases are also emerging at unprecedented rates and require attention. many cds are rooted in environmental and livelihood conditions and mediated by social and individual determinants. it is now increasingly recognised that a much broader, coordinated and multi-sectoral ecohealth approach is required to address cds in sub-saharan africa. an ecohealth approach has been shown to be more robust in public health interventions than the traditional medical approach. the approach helps to generate an understanding of ecosystem factors that influence the emergence and spread of both old and new diseases, considers temporal and spatial dimensions of disease infection and allows systems thinking. in conclusion, establishing intersectoral and multisectoral linkages is important to facilitate joint efforts to address cds at the national, district and community levels. introduction top ↑ a communicable disease (cd) is an illness due to a specific infectious agent or its toxic product that arises through transmission of that agent or its products from an infected person, animal or inanimate reservoir (e.g. from a food source or contaminated water) to a susceptible host. cds comprise clinically evident illness resulting from the infection, presence and growth of pathogenic biological agents in an individual host organism. they can be spread by direct contact with an infected person or animal, through ingestion of contaminated food or water, by disease vectors or by contact with contaminated surroundings like animal droppings or contaminated air. demographic and environmental factors such as population growth, increased urbanisation and alteration of habitats of disease vectors may promote the spread of infectious diseases (saker et al. 2004). whilst health systems face an increasing burden from chronic non-cds, cds continue to be the single most important contributor to the burden of disease in developing countries. cds have remained leading causes of morbidity and mortality in sub-saharan africa for various reasons, including human sociocultural factors, inequity, weak health systems, limited national budgets and poor governance and accountability. others include antimicrobial drug and insecticide resistance, environmental changes that favour vector population increase and demographic factors such as land use patterns, significant human migration and displacements. population growth and migration, environmental change, transformation of landscape and globalisation of trade and economies have changed the kinds of health challenges faced by populations around the world. despite substantial gains in socio-economic development around the world, and enormous advances in sanitation and medical knowledge and technologies, many cds continue to pose an unacceptable burden to people in developing countries. a further threat is posed to people all around the world by the emergence of new infectious diseases, most of them zoonotic in nature, which appear to be on the rise (jones et al. 2008). about 60.0% of all infectious pathogens of humans originate from animals, although many of these are totally adapted to their new host and no longer require a non-human animal host for persistence. various publications suggest between 60.0% and 75.0% of new or emerging infectious diseases of humans in the last half century originated from animals, of which about 71.0% of these were of wildlife origin (jones et al. 2008). important zoonoses that have posed health threats in africa include: avian influenza, ebola, marburg, lassa fever, rift valley fever, new arenavirus (lujo) outbreak, anthrax, plague, human african trypanosomiasis, bovine brucellosis, bovine tuberculosis, porcine cysticercosis and rabies. it has been estimated that over half (54.3%) of emerging infectious disease events are caused by bacteria or rickettsia, reflecting a large number of drug-resistant microbes (jones et al. 2008). the drivers of many emerging disease are mostly related to human behaviour, livelihoods, environmental and ecological factors. the main risk factors for human and animal epidemic diseases revolve around systems of governance; movement of humans, animals and commodities; and human behaviour (rweyemamu, otim-nape & serwadda 2006). communicable diseases, ecosystems and livelihoods top ↑ the persistency of most cds and the emergence of zoonoses are complex and multifactorial, driven by factors which include evolving ecology, microbial adaptation, human demographics and behaviour, international travel and trade, agricultural practices, technology and industry. the persistence or re-emergence of infectious diseases, and the emergence of some new ones, may be indicative of wider problems affecting the dynamics of socio-ecological systems. ecosystem health examines changes in the biological, physical, social and economic environments and relates these changes to impacts upon human health. major cds that are endemic in africa constitute a high risk for health and livelihoods and future marginalisation of africa through trade restrictions and sociopolitical impacts. in general in africa cds constitute a disproportionately higher burden level than in the rest of the world, with 72% of the disease burden attributable to poverty, interactions between socio-economic opportunities and the health of animals, people and ecosystems. it is estimated that about 40% of the burden of disease in sub-saharan africa is environmentally determined (smith, corvalan & kjellstrom 1999). another important aspect of health is climate change and variability which is reported to have impacted more heavily in africa than most other parts of the world. generally, vulnerability of individuals and communities to cds is influenced by multiple factors (environmental, economic and socio-ecological) in addition to host and causative agent factors (weiss & mcmichael 2004; wilson 1995). vulnerability is also tightly linked to inequity with respect to access to resources, decision-making power and capacity to cope. the continued prevalence of illness and vulnerability itself stems in part from the continued inequality within and between households, communities and countries. inequality affects vulnerability directly by constraining the options available to communities, households and individuals when faced with external shocks, and indirectly through its various links to poverty. complex health problems such as cds are difficult to solve without understanding socio-economic and environmental contexts. it has already been recognised that socio-economic and environmental factors affect health, exposure to illness, risk for illness-producing behaviours, and the household or community response to the respective health problem. the modelling of human health from the perspective of its interaction with the environment was initially highlighted by the experience of the biomedical world and the spread of infectious diseases. yet the world is subject to the influences of many complex factors that can undermine the health of human beings and that cannot be controlled by a biomedical approach alone. a new approach to human health constitutes a bridge between public health, a strategy for integrated management of the environment and an ecosystem approach to promoting human health. the ecosystem approach to human health offers an unequalled opportunity to promote human health through an enlightened approach to management of the ecosystem. ecosystem management relates to natural and environmental resources, but also should take into account the many human components as well as the socio-economic and cultural factors relevant to the living environment. the ecosystem approach to human health is also dependent on a participatory and transdisciplinary research methodology that remains sensitive to the needs and aspirations of different social groups, including the differences between genders. this means that the ecosystem approach to human health promotes a holistic view of human health, livelihoods and environmental sustainability (charron 2012). in the context of ecosystems, health is a positive characteristic of human communities. it implies both the availability of and accessibility to resources. specific positive outcomes include food security, good nutrition, low levels of disease, reproductive capacity, a sense of well-being and access to knowledge (waltner-toews & kay 2002). this means that health is a social construct negotiated in the context of a better understanding of the constraints and opportunities provided by the ecosystems of which people forms an integral part. despite this understanding, for a number of years, conventional cds research and control has tended to choose specific outcomes and view them as the result of a linear chain of events. it is envisaged that a more complex and realistic view requires identification of certain kinds of livelihood factors, which, although they are possible determinants of communicable disease transmission, also generate money to improve the well-being of the population. considering this view, resolution of health-related issues (including cds, education, nutrition and livelihoods) requires going beyond traditional health sector concerns and paying attention to the ecological and socio-economic context. thus, it is not surprising to find out that programmes that are entirely unrelated to the conventional health sector, such as agriculture, water and infrastructure development projects, have a major, usually contradictory, effect on human health. it has been argued that conventional programmes aimed at creating environments that promote health are simplistic and if they achieve their goals they do so by accident rather than by design. most cds represent complex, multidimensional health problems with a host of interacting variables ranging from the parasite, vector, human host and local health-delivery systems to land use and climate change. a sound understanding of the nature and dynamics of certain ecosystem variables and their relationship to cds transmission is a necessary step in identifying and addressing interventions that may reduce diseases whilst increasing household productivity. vulnerability to communicable diseases top ↑ large groups of people continue to be vulnerable to cds due to poverty, absence of adequate infrastructure, lack of access to health services, and degraded living environments. similar to adaptations that people make to their changing environment, infectious disease agents also adjust to their changing surroundings. the pathogens that cause disease are in a state of perpetual adaptation, which can lead to the emergence of ‘new’ diseases or the spread of known diseases to previously unaffected areas. diseases that experience a marked change in distribution, incidence and/or behaviour are often referred to as ‘emerging or re-emerging diseases’. factors that lead to the adaptation of infectious agents are complex and dynamic, ranging from deforestation, irrigation, species competition, and human and animal migration patterns to drug resistance and changing vector lifecycle due to climate variability (mcmichael et al. 1996; patz et al. 2005) understanding the determinants of health, beyond exposure to parasites and viruses, is fundamental to understanding how communities and individuals adapt to avoid risk and illness. factors such as the ability to mitigate increased vulnerability associated with illness, access to timely diagnosis and care, and access to adequate nutrition all play a role in managing vulnerability and disease progression. these factors are in turn affected by behavioural and societal norms, such as the ability to control resources needed for health-seeking and protection behaviour and the perception that specific symptoms merit an immediate investment of time and resources (jones & williams 2004). climate change or variability and communicable diseases top ↑ there is a close link between local climate and the occurrence or severity of cds. the effects of climate change and variability on ecosystem health are multiple and include interrelated environmental factors, food security factors, health factors, socio-economic factors and governance factors. the impacts of climate change are global, affecting mostly people from developing countries who depend most directly on their ecosystems for survival and, because of poverty, have the least capacity to adapt to the rapid changes that are affecting their environment. the major impacts of climate change include severe floods, frequent and prolonged droughts, reduced water supply, decline in both crop and livestock yields, culminating in food shortages and health disorders. the impact of climate change and variability on health and livelihoods varies between different ecological patterns. populations in the drylands are likely to face decreased water availability, with implications for the consumption of safe food and disease profiles. climate changes have been implicated in contributing to the spread of cds, especially vector-borne diseases. the risk of cds may increase due to changes and survival of pathogens, population movements, vector dynamics and changes in the natural ecosystems. such changes include deforestation, agriculture and animal husbandry, water development projects, urbanisation, loss of biodiversity, introduction of alien species, and climate change. there are many factors that affect vulnerability and how households manage their health and well-being in relation to climate change. the resilience of many ecosystems is likely to be exceeded by an unprecedented combination of disturbances associated with climate change and other drivers such as land use change and overexploitation of resources. understanding how different factors influence access to resources and the social structures that govern behaviour is essential in appreciating health problems and their prevention, especially in view of a changing climate. drylands in sub-saharan africa are under constant threat from multiple stresses and challenges, which occur as a result of a complex interplay of natural and human-induced processes. people in the semiarid and arid areas of tanzania suffer from poverty, food insecurity, ecological fragility and social vulnerability as well as cds. increasingly, scarce water sources concentrate people and animals in specific locations which can be hotspots for cd transmission. over many years, communities in sub-saharan africa have lived with variability in rainfall and frequent droughts using a range of coping strategies. adaptation to climate variability has been a survival strategy that has been well refined over time. however, increasing variability and frequency of unusual weather events place the livelihoods of those most vulnerable to climate change at risk. challenges in the control of communicable diseases top ↑ appropriate technical solutions exist to detect and treat most of the high impact cds, whether through chemotherapy, chemoprophylaxis or applying better management practices. poor execution or inability to apply management practices and known solutions for causes of the underlying poverty, capacity, infrastructure, knowledge and policy has prevented health services in countries in sub-saharan africa to control cds. the absence of effective health systems and weak disease surveillance in the region means that at best there is syndromic information on disease with little specific local knowledge. this means that some diseases are probably over-reported and many others under-reported. pharmaceutical costs are likely to remain high given the reluctance of commercial pharmaceutical companies to invest in accessible, affordable products for use in animals to prevent or reduce the risk of zoonotic infection, since these are of little commercial value. this is in addition to weak community infrastructure (housing, animal facilities, waste systems, water supplies), poor nutrition and poor hygienic practices mostly due to lack of basic facilities and weak or remote health services. education and awareness can also play a part – although there is no evidence that this in itself is a basic constraint, it will help in specific interventions and improved practices. given the basic living conditions, exposure to the environment and reliance on animals for survival and daily food, poor communities are at risk from zoonosis, with a disproportionate number of women and children exposed through their particular domestic roles in animal management. there is a general consensus that sub-saharan africa is lagging behind in the implementation of the un millennium development goals (mdgs). considering that four of the eight mdgs are linked directly to cds whilst the other four are linked indirectly, the continuing burden of cds in africa contributes to the slow progress with regard to the mdgs and in general result in undernutrition (waage et al. 2010). underlying trends to this disease status are increasing natural and man-made shocks and emergencies which further increase vulnerability of african communities (ndiyoi, rweyemamu & meadows 2006). studies on future risk of cd have led to the realisation that such diseases are and will continue to be a formidable challenge to human welfare and economic development in sub-saharan africa over the next two decades, thereby impeding the region’s ability to meet the targets of the mdgs (brownlie et al. 2005, 2006; fao/oie/who 2008; jones et al. 2008; king et al. 2006; kock et al. 2012; rweyemamu et al. 2006; taylor, latham & woolhouse 2001; woolhouse 2008; woolhouse, gowtage-sequeria & evans 2006; world bank 2010; zinsstag et al. 2011). it is therefore evident that environmental, economic and socio-ecological factors are inextricably linked with burden of disease. the latter is generally measured in terms of commonest diseases; direct and indirect costs incurred on disease treatment, control and prevention; years lived with disability; and years of life lost due to disease. rarely captured in the burdens are the linkages with animals and the environment. this is particularly relevant to the livestock livelihood communities. a considerable body of research pinpoints weak health systems as critical impediment to improved health across africa (anyangwe & mtonga 2007). likewise, sub-saharan african countries are faced with a number of problems due to a limited financial and human resource base. weak and dysfunctional health systems are the major reasons why many effective low-cost health interventions remain underutilised whilst people continue to suffer and die in the midst of existing opportunities. in many cases, the failure to deliver affordable and effective interventions to predictable, preventable and treatable cds is a major weakness in the health systems of many developing countries. health systems research can help identify best practices and prioritise areas that need strengthening (de savigny & adam 2009). whilst health systems in general have received worldwide attention, little has been done to strengthen facility, district and national institutions responsible for stewardship of health systems in the region. ministries of health in sub-saharan africa maintain two major systems for collecting and analysing information regarding cds, namely the health management information system and the integrated disease surveillance and response strategy. accurate epidemiological surveillance data are essential for adequate disease control. surveillance requires impact indicators, based on standardised case definitions on morbidity and mortality. definitions will vary, depending on diagnostic capabilities at different levels of the health care system, and most importantly, whether the case definition is based on confirmatory tests or not. adequate case definition serves to provide indicators of disease epidemiology, which can be used for needs assessment, situation analysis and to evaluate the effectiveness of the disease control programmes. they also serve to guide and adapt control activities. the effectiveness of a health information system at the national, district and facility levels depends on the ability of staff to utilise the information properly. in many sub-saharan african countries, these systems are not functioning properly. integrated disease control employing an ecosystem approach top ↑ in sub-saharan africa there is a bidirectional link between livelihoods, ecosystem and health. environmental changes, especially those brought about by agricultural and water development as well as construction projects, often lead to changes in the ecology of some diseases. for instance, agriculture, which accounts for the occupation of the majority of rural people in sub-saharan africa and serves as food source for its urban population, is closely tied to human health (hawkes & ruel 2006). good health affects agriculture by boosting people’s capacity for work and thus increasing agricultural productivity. on the other hand, unintended side-effects associated with certain agricultural production systems may increase human exposure to vectors of diseases (ijumba & lindsay 2001; mboera et al. 2007). in agricultural communities, poor health directly reduces income and productivity, further decreasing people’s ability to address poor health and inhibiting economic development more severely, whilst in the population at large, malnutrition and disease patterns influence market demand for agricultural products. examining health in an ecosystemic context is therefore important because environment present not only opportunities for improving health but also risks to ill health. most cds are linked to the environment (prüss-üstün & corvalán 2006; weiss & mcmichael 2004). understanding the determinants of health is fundamental to understanding how communities and individuals adapt to avoid risk and illness. the prevailing approach to cd control, a highly successful one, has focused on mass immunisation or rapid diagnosis, isolation and treatment for non-vaccine preventable diseases. the emergence late in the 20th century of antimicrobial resistance, the difficulties in finding effective vaccines for diseases such as hiv and malaria, the experiences from sars and pandemic influenza have highlighted the need for alternative or complementary approaches that emphasise disease prevention, in addition to control. for vector-borne disease like malaria, vector-eradication campaigns relying primarily on insecticides (either using insecticide-treated nets or indoor residual spraying) have remained the most widely used approach. meanwhile, changes in the ecology of vectors have accelerated because of environmental and climate changes, changes in host ecology, and selection pressures from insecticides. because microbes and vectors are living organisms, they will continue to adapt, change and pose threats to human health. an ecosystem approach to preventing cds considers drivers of risk in terms of the underlying ecological, social, cultural, political and economic factors of transmission dynamics. like other health problems, the ecology and transmission of most cds can be linked to interactions among several factors, such as demographic changes, poverty, urbanisation, deforestation, changes in agriculture modes of production, changed relationships between people and animals, natural resource management, gender differences and cultural patterns. an understanding of the complex interactions between these factors, and their local manifestations and risk factors for disease, require research across current boundaries of scientific disciplines and sectors (boischio et al. 2009; spiegel et al. 2005). the prevention of disease and the prevention of serious harm to livelihoods from such diseases depend on sound and resilient environments in ecological, social and economic terms. it has been realised that there is interplay between livestock, wildlife and humans, and therefore between animal and human diseases. thus, collaborative efforts between different sectors are required to realise effective control and/or eradication of emerging cds in sub-saharan africa (karimuribo et al. 2010). there is a need to promote collaborations between human and animal health sectors if meaningful prevention and control of emerging and re-emerging diseases is to be realised in sub-saharan africa. it is therefore essential to build intersectoral policy options that target disease prevention (bazzani & wiese 2012). bringing different people and their contributions together in pursuit of a shared goal is a widely recognised strategy to harness capacity to address complex societal challenges, including cds. working together across sectors and disciplines to tackle complex interactions between health, environment and equity is now widely recognised to be beneficial in development research (charron 2012). despite major progress in understanding cds and substantial investments into control, the diseases remain major public health problems and pose serious challenges to development. the burden of cds in sub-saharan africa is influenced by poverty, rapid and transformative economic development, and chronically weak health systems. under these conditions, innovative approaches are needed to develop and deliver cds interventions in more context-adapted, effective and sustainable ways. cd control needs to be better integrated within highly variable local realities of community livelihoods, environments and health services. integrated disease control can be defined as applied research that explores and creates synergies among environmental, health systems and community-based approaches to disease control. it has the potential to tackle root causes of cds beyond the medical and public health aspects of the disease. a number of studies have indicated that an ecohealth approach is the most appropriate way to address cds. the approach strives for improved human health and well-being, based on sustainable ecosystems, with more equitable development and less poverty. the ecohealth approach supports systems-based, policy-relevant research on the relationships between ecosystems, human livelihoods and health to define, assess and mitigate priority problems that affect the well-being of people and the services provided by ecosystems they depend upon. in seeking to improve human health and well-being whilst simultaneously maintaining ecosystems and their services, the ecohealth approach puts forward an approach that requires a holistic framing of health-environment issues. in doing so, ecosystem and human dimensions along with gender and social equity issues are considered through different disciplines and non-academic knowledge, working together in a trans-disciplinary fashion and at the research-policy interface. multistakeholder participation is encouraged, particularly those from the community and research end-users, including policy makers. conclusion top ↑ delivering public health services requires functional and effective country-level health systems, including capable innovative health leadership, adequate financial resources, qualified healthcare providers, effective human resource systems, effective health information systems and adequate physical infrastructure. to be effective, intervention tools need to be usable within the available health system framework and implemented appropriately so that the end-user is able to benefit from them. the new approach to disease control should take into consideration the following: (1) socio-ecological and socio-economic approach as key elements for the risk management of disease; (2) understanding ecological processes (social, cultural, environmental and economic factors) contributing to disease occurrence and persistence at a time of rapid population growth, urbanisation, food system and environmental change; (3) improving specific determination of disease aetiologies and their drivers in poor and vulnerable communities; and (4) appropriate policies and health delivery systems.further progress in controlling cds almost certainly requires some combination of biomedical technologies and social and environmental strategies coordinated among relevant sectors. addressing cds requires intersectoral cooperation and strong partnerships. many cds are rooted in environmental and livelihood conditions and mediated by social and individual determinants. it is now increasingly recognised that a much broader, coordinated and intersectoral ecohealth approach is required to address them. an ecohealth approach has been shown to be more robust in public health interventions than the traditional medical approach. the approach helps to generate an understanding of ecosystem factors that influence the emergence and spread of both emerging and re-emerging diseases, considers temporal and spatial dimensions of disease infection and allows systems thinking. in conclusion, the establishment of intersectoral linkages is important to facilitate joint efforts to address cds at the national, district and community levels. acknowledgements top ↑ c.s. is a phd student supported by wellcome trust grant wt087546ma to sacids and the contribution of e.d.k. was supported by rockefeller foundations grants 2008-dsn310 and 2009-dsn305. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions l.e.g.m. 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to health and well-being’, preventive veterinary medicine 101, 148–156. http://dx.doi.org/10.1016/j.prevetmed.2010.07.003 reviewer acknowledgement open accesshttp://www.ojvr.org page 1 of 1 the onderstepoort journal of veterinary research recognises the value and importance of the peer reviewer in the overall publication process – not only in shaping the individual manuscript, but also in shaping the credibility and reputation of our journal. we are committed to the timely publication of all original, innovative contributions submitted for publication. as such, the identification and selection of reviewers who have expertise and interest in the topics appropriate to each manuscript are essential elements in ensuring a timely, productive peer review process. we would like to take this opportunity to thank all reviewers who participated in shaping this volume of the onderstepoort journal of veterinary research: akinbowale jenkins ali halajian anita michel anna-mari bosman annemarie 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sunelle strydom tanguy marcotty tom aire tony shakespeare tshepo matjila vanessa steenkamp vinny naidoo weicheng bei wilmien luus-powell we appreciate the time taken to perform your review successfully. horak_69-74.indd context the most recently compiled world list of valid tick names contains the names of 692 ixodid and 186 argasid tick species as well as that of nuttalliella namaqua, the only species in this genus (nava, guglielmone & mangold 2008). of these 879 species, 80 ixodid and 25 argasid species and n. namaqua occur in south africa. twenty-one of the 80 ixodid species and two of the argasid species have distributions confined to this country (theiler 1962; walker 1991; walker, keirans & horak 2000), and n. namaqua has only been recorded in south africa and namibia. forty-six of the 80 ixodid and 16 of the 25 argasid species and n. namaqua have been described since 1908, and this review is confined to the latter 63 ticks. twenty-four of these descriptions were written, or contributed to by people in south africa, and the remainder by researchers in england, france, germany, israel, russia, mozambique and the usa. the south african authors and the south african ticks that they have described as being new to science since 1908 are summarized in tabular format. a similar format has been followed for tick species with south african distributions, but described by foreign researchers during the same period. the earliest description of a tick that has a strictly south african distribution is that by de geer in 1778 for amblyomma sylvaticum, a tick of tortoises and more particularly the angulate tortoise, chersina angulata (horak, mckay, henen, heyne, hofmeyr & de villiers 2006). the most recent description is that of haemaphysalis colesbergensis, a tick of domestic and wild felids (apanaskevich & horak 2008c). south africans have in recent times also been involved in some of the more controversial name changes of well-known ticks of veterinary importance. the name changes of the common blue tick, boophilus decoloratus and the asiatic blue tick boophilus microplus to rhipicephalus (boophilus) decoloratus and rhipicephalus (boophilus) microplus were first published by horak, camicas & keirans (2002) in their world list of valid tick names. 69 onderstepoort journal of veterinary research, 76:69–74 (2009) a century of tick taxonomy in south africa i.g. horak department of veterinary tropical diseases, faculty of veterinary science, university of pretoria onderstepoort, 0110 south africa abstract horak, i.g. 2009. a century of tick taxonomy in south africa. onderstepoort journal of veterinary re search, 76:69–74 eighty ixodid tick species, 25 argasid tick species and nuttalliella namaqua occur in south africa. twenty-one of the 80 ixodid species and two of the argasid species occur only in this country, while n. namaqua is present only in south africa and namibia. forty-six of the 80 ixodid species and 16 of the 25 argasid species as well as n. namaqua have been described as new species since 1908. people working in south africa have written or contributed to the descriptions of 24 of these 63 new species, while foreign researchers have described the remainder. new species indigenous to south africa are still being discovered, while the names of some species, well known because of their veterinary importance, have been altered. 70 a century of tick taxonomy in south africa table 1 authors who have worked in south africa and the south african tick species that they have described as new to science since 1908 south african author and species south african author and species bedford, g.a.h. (1912–1938) walker, jane (1966–2000) argas peringueyi (bedford & hewitt 1925) rhipicephalus theileri bedford & hewitt, 1925 haemaphysalis cooleyi bedford, 1929 ixodes elongatus bedford, 1929 nuttalliella namaqua bedford, 1931 argas striatus bedford, 1932 rhipicephalus distinctus bedford, 1932 haemaphysalis hyracophila hoogstraal, walker & neitz, 1971 argas africolumbae hoogstraal, kaiser, walker, ledger, converse & rice, 1975 ixodes neitzi clifford, walker & keirans, 1977 rhipicephalus zambeziensis walker, norval & corwin, 1981 ixodes catherinei keirans, clifford & walker, 1982 ixodes corwini keirans, clifford & walker, 1982 rhipicephalus lounsburyi walker, 1990 rhipicephalus neumanni walker, 1990 rhipicephalus exophthalmos keirans & walker, 1993 rhipicephalus oreotragi walker & horak, 2000 rhipicephalus warburtoni walker & horak, 2000 du toit, r. (1929–1964) rhipicephalus glabroscutatum du toit, 1941 theiler, gertrud (1940–1983) argas eboris (theiler, 1959) ixodes drakensbergensis clifford, theiler & baker, 1975 rhipicephalus arnoldi theiler & zumpt, 1949 horak i.g. (1974–present) haemaphysalis colesbergensis apanaskevich & horak, 2008 zumpt, f. (sa institute of medical research) (1948–1980) rhipicephalus muehlensi zumpt, 1943* * zumpt was working at the bernard nocht-institut in hamburg at the time he described this species table 2 tick species with south african distributions described as new to science by foreign researchers since 1908 tick species and foreign authors* tick species and foreign authors* ixodid ticks amblyomma nuttalli dönitz, 1909 haemaphysalis aciculifer warburton, 1913 haemaphysalis hoodi warburton & nuttall, 1909 haemaphysalis pedetes hoogstraal, 1972 haemaphysalis silacea robinson, 1912 haemaphysalis zumpti hoogstraal & el kammah, 1974 ixodes alluaudi neumann, 1913 ixodes aulacodi arthur, 1956 ixodes bakeri arthur & clifford, 1961 ixodes bedfordi arthur, 1959 ixodes cavipalpus nuttall & warburton, 1908 ixodes myotomys clifford & hoogstraal, 1970 ixodes procaviae arthur & burrow, 1957 ixodes rhabdomysae arthur, 1959 ixodes spiculae arthur, 1956 ixodes spinae arthur, 1958 ixodes theilerae arthur, 1953 ixodes transvaalensis clifford & hoogstraal, 1966 rhipicentor bicornis nuttall & warburton, 1908 rhipicentor nuttalli cooper & robinson, 1908 rhipicephalus evertsi mimeticus dönitz, 1910 rhipicephalus follis dönitz, 1910 rhipicephalus gertrudae feldman-muhsam, 1960 rhipicephalus simpsoni nuttall, 1910 rhipicephalus sulcatus neumann, 1908 rhipicephalus turanicus pomerantsev, 1936 rhipicephalus zumpti santos dias, 1950 argasid ticks argas arboreus kaiser, hoogstraal & kohls, 1964 argas streptopelia kaiser, hoogstraal & horner, 1970 argas theilerae hoogstraal & kaiser, 1970 argas walkerae kaiser & hoogstraal, 1969 argas zumpti hoogstraal, kaiser & kohls, 1968 carios boueti (roubad, & colas-belcour, 1933) carios confusus (hoogstraal, 1955) carios cordiformis (hoogstraal & kohls, 1967) carios faini (hoogstraal, 1960) ornithodoros compactus walton, 1962 ornithodoros porcinus walton, 1962 ornithodoros zumpti heisch & guggisberg, 1953 * not listed in references 71 i.g. horak table 3 south african ticks, other than rhipicephalus species, described by foreign researchers, for which additional descriptions have been produced by researchers in south africa since 1908 species and authors* descriptions by researchers working in south africa** amblyomma hebraeum koch, 1844 amblyomma marmoreum koch, 1844 amblyomma nuttalli dönitz, 1909 amblyomma sylvaticum (de geer, 1778) amblyomma tholloni neumann, 1899 aponomma exornatum (koch, 1844) aponomma latum (koch, 1844) aponomma transversale (lucas, 1844) dermacentor rhinocerinus (denny, 1843) haemaphysalis aciculifer warburton, 1913 haemaphysalis elliptica (koch, 1844) haemaphysalis parmata neumann, 1905 haemaphysalis silacea robinson, 1912 hyalomma glabrum delpy, 1949 hyalomma rufipes koch, 1844 hyalomma truncatum koch, 1844 ixodes alluaudi neumann, 1913 ixodes bakeri arthur & clifford, 1961 margaropus winthemi karsch, 1879 rhipicentor bicornis nuttall & warburton, 1908 rhipicentor nuttalli cooper & robinson, 1908 walker & olwage (1987) theiler & salisbury (1959); walker & olwage (1987) theiler & salisbury (1959) theiler (1943a) walker & olwage (1987) theiler (1945a) theiler (1945b) theiler (1945b) keirans (1993) theiler (1945c) apanaskevich, horak & camicas (2007) theiler (1945c) theiler (1945c) apanaskevich & horak (2006) apanaskevich & horak (2008a) apanaskevich & horak (2008b) theiler (1941) clifford, walker & keirans (1976) walker & laurence (1973); gothe (1967) theiler (1961) theiler (1961) * not listed in references ** listed in references table 4 south african rhipicephalus species described by foreign researchers, for which additional descriptions have been produced since 1908 by researchers in south africa rhipicephalus species and original authors* descriptions by researchers in south africa** rhipicephalus (boophilus) decoloratus (koch, 1844) rhipicephalus (boophilus) microplus (canestrini, 1887) rhipicephalus appendiculatus neumann, 1901 rhipicephalus capensis koch, 1844 rhipicephalus evertsi evertsi neumann, 1897 rhipicephalus follis dönitz, 1910 rhipicephalus gertrudae feldman-muhsam, 1960 rhipicephalus kochi dönitz, 1905 rhipicephalus lunulatus neumann, 1907 rhipicephalus maculatus neumann, 1901 rhipicephalus muehlensi zumpt, 1943 rhipicephalus nitens neumann, 1904 rhipicephalus oculatus neumann, 1901 rhipicephalus sanguineus (latreille, 1806) rhipicephalus simpsoni nuttall, 1910 rhipicephalus simus koch, 1844 rhipicephalus sulcatus neumann, 1908 rhipicephalus tricuspis dönitz, 1906 rhipicephalus turanicus pomerantsev, 1936 rhipicephalus zumpti santos dias, 1950 gothe (1967); arthur & londt (1973); heyne (1986) gothe (1967); londt & arthur (1975); heyne (1986) walker, norval & corwin (1981) walker, keirans & horak (2000) walker et al. (2000) walker et al. (2000) walker et al. (2000) walker et al. (2000) walker, keirans, pegram & clifford (1988); walker et al. (2000) theiler & robinson (1953b); walker et al. (2000) salisbury (1959); walker et al. (2000) walker et al. (2000) keirans, walker, horak & heyne (1993); walker et al. (2000) pegram, keirans, clifford & walker (1987b); walker et al. (2000) theiler 1949; walker et al. 2000 pegram, walker, clifford & keirans (1987c); walker et al. (2000) pegram, clifford, walker & keirans (1987a); walker et al. (2000) walker et al. (1988; 2000) pegram et al. (1987a); walker et al. (2000) walker et al. (2000) * not listed in references ** listed in references 72 a century of tick taxonomy in south africa this was done after molecular and morphological studies by murrell, campbell & barker (2000) and beati & keirans (2001) had indicated that the genus rhipicephalus was paraphyletic in respect of the genus boophilus. murrell & barker (2003) later confirmed these name changes. the yellow dog tick of south africa, previously lumped with haemaphysalis leachi of north and east africa, has been reinstated as haemaphysalis elliptica, an old taxon originally described from the cape of good hope by koch in 1844 (apanaskevich, horak & camicas 2007). while the bont-legged ticks, to which sub-specific status within the hyalomma marginatum species group had previously been assigned, have all been raised to specific status (apanaskevich & horak 2008a). furthermore the tick previously known as hyalomma marginatum turanicum in south africa has been reinstated as hyalomma glabrum, the only hyalomma with a strictly southern hemisphere, and with that a strictly south african distribution (apanaskevich & horak 2006). three of the abovementioned ticks are of considerable veterinary importance in that r. (b.) decoloratus and r. (b.) microplus are the vectors of babesia species, the cause of babesiosis or redwater in domestic cattle (de vos, de waal & jackson 2004), while h. elliptica, and not h. leachi, is now the only proven vector of babesia canis rossi the cause of the virulent form of babesiosis encountered in domestic dogs in south africa (lewis, penzhorn, lopez-rebollar & de waal 1996; apanaskevich et al. 2007). a fourth member of this group, hyalomma rufipes, is the principal vector in south africa of the virus causing crimean-congo haemorrhagic fever in humans (horak, swanepoel & gummow 2002). the name changes are thus not only of academic importance, but of scientific concern as they are oft used in the literature concerning the diseases that they transmit and in which the correct use of taxonomic epithets is essential. acknowledgements i have been fortunate in having two monographs to consult, namely those by theiler (1962) and walker (1991), and i pay tribute to both authors and to their attention to detail. references all authors mentioned in the descriptive text have been listed, but only south african authors or foreign authors that worked in south africa at the time at which they described particular tick species have been included in the list of references. apanaskevich, d.a. & horak, i.g. 2006. the genus hyalomma koch, 1844. i. reinstatement of hyalomma (euhyalomma) glabrum delpy, 1949 (acari, ixodidae) as a valid species with a redescription of the adults, the first description of its immature stages and notes on its biology. onderstepoort journal of veterinary research, 73:1–12. apanaskevich, d.a., horak, i.g. & camicas, j-l. 2007. redescription of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haem aphy salis (rhipistoma) leachi (audouin, 1826) (ixodida, ixodidae). onderstepoort journal of veterinary research, 74: 181–207. apanaskevich, d.a. & horak, i.g. 2008a. the genus hy alomma koch, 1844. v. re-evaluation of the taxonomic rank of taxa comprising the h. (euhyalomma) marginatum group of species (acari: ixodidae) with redescription of all parasitic stages and notes on biology. international journal of acarology, 34:13–42. apanaskevich, d.a. & horak, i.g. 2008b. the genus hyalomma. vi. systematics of h. (euhyalomma) truncatum and the closely related species, h. (e.) albiparmatum and h. (e.) nitidum (acari, ixodidae). experimental and applied acarology, 44:115–136. apanaskevich, d.a. & horak, i.g. 2008c. two new species of african haemaphysalis ticks (acari: ixodidae), carnivore parasites of the h. (rhipistoma) leachi group. journal of para sitology, 94:594–607. arthur, d.r & londt, j.g.h. 1973. the parasitic life cycle of boophilus decoloratus (koch, 1844). journal of the ento mological society of southern africa, 36:87–116. beati, l. & keirans, j.e. 2001. analysis of the systematic relationships among ticks of the genera rhipicephalus and boophilus (acari: ixodidae) based on mitochondrial 12s ribosomal dna gene sequences and morphological characters. journal of parasitology, 87:32–48. bedford, g.a.h. & hewitt, j. 1925. descriptions of two new species of ticks, with notes on rare and hitherto unrecorded species from south africa. south african journal of natural history, 5:259–266. bedford, g.a.h. 1929. notes on some south african ticks, with descriptions of three new species. annual report of the director of veterinary services, union of south africa, 15: 493–499. bedford, g.a.h. 1931. nuttalliella namaqua, a new genus and species of tick. parasitology, 23:230–232. bedford, g.a.h. 1932. description of argas striatus, a new species of tick. 18th report of the director of veterinary services and animal industry, union of south africa, 18:221–222. bedford, g.a.h. 1932. a synoptic check-list and host-list of the ectoparasites found on south african mammalia, aves and reptilia (2nd ed.). 18th report of the director of veterinary services and animal industry, union of south africa, 18:223– 523. clifford, c.m., theiler, gertrud & baker, maureen, 1975. ixodes (afrixodes) drakensbergensis n. sp. from domestic and wild animals in natal, republic of south africa. onderstepoort journal of veterinary research, 42:33–44. clifford, c.m., walker, jane b. & keirans, j.e. 1976. ixodes (afrixodes) bakeri arthur & clifford, 1961 (acarina: ixodidae): descriptions of the male and immature stages from rodents and insectivores and notes on its biology in south africa. onderstepoort journal of veterinary research, 43:105–112. 73 i.g. horak clifford, c.m., walker, jane b. & keirans, j.e. 1977. ixodes (afrixodes) neitzi, n. sp. (acarina: ixodidae) from the mountain reedbuck in south africa. onderstepoort journal of veterinary research, 44:143–150. de vos, a.j., de waal, d.t. & jackson, l.a. 2004. bovine babesiosis, in infectious diseases of livestock, edited by j.a.w. coetzer & r.c. tustin. cape town: oxford university press. du toit, r. 1941. description of a tick rhipicephalus glabroscutatum, sp. nov. (ixodidae), from the karroo areas of the union of south africa. onderstepoort journal of veterinary science and animal industry, 16:115–118. gothe, r. 1967. ticks in the south african zoological survey collection. part xii. the genera boophilus curtice, 1891 and margaropus karsch, 1879. onderstepoort journal of veterinary research, 34:81–107. heyne, heloise 1986. differentiation of boophilus decoloratus and boophilus microplus. journal of the south african veterinary association, 57:251–252. hoogstraal, h., walker, jane b. & neitz, w.o. 1971. notes on african haemaphysalis ticks. vii. h. (rhipistoma) hyracophila sp. nov. (ixodoidea: ixodidae), a parasite of hyraxes in south africa. journal of parasitology, 57:417–425. hoogstraal, h., kaiser, m.n., walker, jane b., ledger, j.a., converse, j.d. & rice, r.c.a. 1975. obser vations on the subgenus argas (ixodoidea: argasidae: argas). 10. a. (a.) africolumbae, n. sp., a pretoria virus-infected parasite of birds in southern and eastern africa. journal of medical entomology, 12:194–201. horak, i.g., camicas, j.-l. & keirans, j.e. 2002. the ixodidae, amblyommidae and nuttalliellidae (acari: ixodida): a world list of valid tick names. experimental and applied acarology, 28:7–54. horak i.g., swanepoel r. & gummow, b. 2002. the distribution of hyalomma spp. and human cases of crimeancongo haemorrhagic fever in south africa, in proceedings of the 10th conference of the association of institutions for tropical veterinary medicine, copenhagen, denmark 20–23 august 2001: 501–509. horak, i.g., mckay, i.j., henen, b.t., heyne, heloise, hofmeyr, margaretha d. & de villiers, a.l. 2006. parasites of domestic and wild animals in south africa. xlvii. ticks of tortoises and other reptiles. onderstepoort journal of veterinary research, 73:215–227. keirans, j.e., clifford, c.m. & walker, jane b. 1982. the ixodes (afrixodes) oldi group (acari: ixodidae) from subsaharan africa with descriptions of five new species. journal of medical entomology, 19:309–329. keirans, j.e. 1993. dermacentor rhinocerinus (denny, 1843) (acari: ixodida: ixodidae): rediscription of the male, female and nymph and first description of the larva. onderstepoort journal of veterinary research, 60:59–68. keirans, j.e. walker, jane b., horak, i.g. & heyne, heloise 1993. rhipicephalus exophthalmos sp. nov., a new tick species from southern africa, and redescription of rhipicephalus oculatus neumann, 1901, with which it has hitherto been confused (acari: ixodida: ixodidae). onderstepoort journal of veterinary research, 60:229–246. keirans, j.e. & walker, jane b. 1993. rhipicephalus exophthalmos sp. nov., in keirans, j.e., walk er, jane b. & horak, i.g. 1993. rhipicephalus exophthalmos sp. nov., a new tick species from southern africa, and redescription of rhipicephalus oculatus neumann, 1901, with which it has hitherto been confused (acari: ixodida: ixodidae). onder stepoort journal of veterinary research, 60:229–246. lewis, b.d., penzhorn, b.l., lopez-rebollar, l.m. & de waal, d.t. 1996. isolation of a south african vector-specific strain of babesia canis. veterinary parasitology, 63:9–16. londt, j.g.h. & arthur, d.r. 1975. the structure and parasitic life cycle of boophilus microplus (canestrini, 1888) in south africa (acarina: ixodidae). journal of the entomological society of southern africa, 38:321–340. murrell, a.n., campbell, j.h. & barker, s.c. 2000. phylogenetic analysis of the rhipicephaline ticks indicates that the genus rhipicephalus is paraphyletic. molecular phylogenetics and evolution, 16:1–7. murrell, a. & barker, s.c. 2003. synonomy of boophilus curtice, 1981 with rhipicephalus koch, 1844 (acari: ixo didae). systematic parasitology, 56:169–172. nava, s., guglielmone, a.a. & mangold, a.j. 2008. an overview of the systematics and evolution of ticks. frontiers in bioscience, in press. pegram, r.g., clifford, c.m., walker, jane b. & keirans, j.e. 1987a. clarification of the rhipicephalus sanguineus group (acari, ixodoidea, ixodidae). i. r. sulcatus neu mann 1908 and r. turanicus pomarantsev, 1936. systematic parasitology, 10:3–26. pegram, r.g., keirans, j.e., clifford, c.m. & walker, jane b. 1987b. clarification of the rhipicephalus sanguineus group (acari, ixodoidea, ixodidae). ii. r. sanguineus (latreille, 1806) and related species. systematic parasitology, 10:27–44. pegram, r.g., walker, jane b., clifford, c.m. & keirans, j.e. 1987c. comparisons of populations of the rhipicephalus simus group: r. simus, r. praetextatus and r. muhsamae (acari: ixodidae). journal of medical entomology, 24:666–682. salisbury, lois e. 1959. ticks in the south african zoological survey collection. part x. rhipicephalus mühlensi zumpt. onderstepoort journal of veterinary research, 28:125–132. theiler, gertrud 1941. ticks in the south african zoological survey collection. part i. ixodes alluaudi neumann, 1913, a primitive tick parasitic on shrews. onderstepoort journal of veterinary science and animal industry, 17:51–60. theiler, gertrud 1943. ticks in the south african zoological survey collection. part ii. onderstepoort journal of veterinary science and animal industry, 18:85–89. theiler, gertrud 1945a. ticks in the south african zo ological survey collection. part iii. the ornate aponommas. onderstepoort journal of veterinary science and animal industry, 20:165–178. theiler, gertrud 1945b. ticks in the south african zoological survey collection. part iv. the inornate aponommas. onderstepoort journal of veterinary science and animal industry, 20:179–190. theiler, gertrud 1945c. ticks in the south african zoo logical survey collection. part v. three african haemaphysalids parasitic on domestic stock. onderstepoort journal of veterinary science and animal industry, 20:191–207. theiler, gertrud 1949. ticks in the south african zoological survey collection. part vi. little known african 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: leendert d. snyman1 bradley c. flett2,3 rowina a. schultz1 affiliations: 1department of toxicology, agricultural research council, onderstepoort veterinary institute, south africa2crop protection (pathology), agricultural research council, grain crop institute, potchefstroom, south africa 3unit of environmental sciences and development, north-west university, potchefstroom campus, south africa correspondence to: leendert snyman postal address: private bag x05, onderstepoort 0110, south africa dates: received: 30 jan. 2014 accepted: 07 apr. 2014 published: 12 nov. 2014 how to cite this article: snyman, l.d., flett, b.c. & schultz, r.a., 2014, ‘studies towards optimising the isolation of diplonine, a neurotoxin isolated from cultures of stenocarpella maydis (berk.) sacc.’, onderstepoort journal of veterinary research 81(1), art. #765, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.765 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. studies towards optimising the isolation of diplonine, a neurotoxin isolated from cultures of stenocarpella maydis (berk.) sacc. in this research communication... open access • abstract • communication • extraction with water • fractionation by cation exchange chromatography • neurotoxic clinical signs in a guinea pig • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ diplonine, a mycotoxin that induces neurotoxic clinical signs in the guinea pig, resembling those occurring in cattle and sheep with diplodiosis, was isolated previously from a stenocarpella maydis culture. knowledge of the chemical properties of the toxin, which was characterised as a substituted ß-cyclopropylamino acid, enabled amendments in the present study to the initial steps of the isolation procedure. extraction with water and fractionation by cation exchange chromatography improved the efficiency of isolation, potentially allowing the preparation of larger amounts of the toxin. communication top ↑ as laboratory observations indicated that diplonine might be more soluble in water than in methanol, extraction of the neurotoxin with water was investigated. the same s. maydis culture used by schultz et al. (2011) (experiment 1) was used in this study. this culture was isolated from a s. maydis-infected maize kernel, onto potato dextrose agar (pda) (flett & mclaren 1994) and maintained as a culture on a pda slant in the agricultural research council – grain crops institute (arc–gci) collection. schultz et al. (2011) found a methanolic extract of this culture to be severely to extremely toxic using a guinea pig bio-assay, with typical paretic signs as a guide after being dosed via a stomach tube (table 1). bio-assays in this study were similarly performed. in the present study, culture material (100 g) was extracted with 250 ml water (saturated with chloroform) for 24 h on a mechanical shaker (labotec sp4; labotec, midrand, south africa). the extract was centrifuged in an allegro x-22r centrifuge (beckmann coulter inc., johannesburg, south africa) at 4500 rpm for 40 min. the supernatant was decanted and the pellet stirred up with a volume of water equal to the decanted supernatant. after being centrifuged under the same conditions the supernatants were combined and defatted twice by extraction with equal volumes of hexane (merck ar; merck [pty] ltd, johannesburg, south africa). bio-assaying of the dried extract at a dose equivalent to 101 g culture/kg body weight (bw) resulted in moderate neurotoxic signs in a guinea pig, whilst a dose equivalent to 174 g culture/kg bw proved to be fatally toxic as it resulted in mortality of the guinea pig 6 h after being dosed. a dose equivalent to 62 g culture/kg bw did not elicit neurological signs in a guinea pig. a comparison of these dosing rates with that of the methanol extracts (table 1) indicated a much higher yield of the neurotoxin when extracted with water. table 1: toxicity of various fractions as reflected by the severity of neurotoxic signs in a guinea pig. extraction with water top ↑ as laboratory observations indicated that diplonine might be more soluble in water than in methanol, extraction of the neurotoxin with water was investigated. the same s. maydis culture used by schultz et al. (2011) (experiment 1) was used in this study. this culture was isolated from a s. maydis-infected maize kernel, onto potato dextrose agar (pda) (flett & mclaren 1994) and maintained as a culture on a pda slant in the agricultural research council – grain crops institute (arc–gci) collection. schultz et al. (2011) found a methanolic extract of this culture to be severely to extremely toxic using a guinea pig bio-assay, with typical paretic signs as a guide after being dosed via a stomach tube (table 1). bio-assays in this study were similarly performed. in the present study, culture material (100 g) was extracted with 250 ml water (saturated with chloroform) for 24 h on a mechanical shaker (labotec sp4; labotec, midrand, south africa). the extract was centrifuged in an allegro x-22r centrifuge (beckmann coulter inc., johannesburg, south africa) at 4500 rpm for 40 min. the supernatant was decanted and the pellet stirred up with a volume of water equal to the decanted supernatant. after being centrifuged under the same conditions the supernatants were combined and defatted twice by extraction with equal volumes of hexane (merck ar; merck [pty] ltd, johannesburg, south africa). bio-assaying of the dried extract at a dose equivalent to 101 g culture/kg body weight (bw) resulted in moderate neurotoxic signs in a guinea pig, whilst a dose equivalent to 174 g culture/kg bw proved to be fatally toxic as it resulted in mortality of the guinea pig 6 h after being dosed. a dose equivalent to 62 g culture/kg bw did not elicit neurological signs in a guinea pig. a comparison of these dosing rates with that of the methanol extracts (table 1) indicated a much higher yield of the neurotoxin when extracted with water. fractionation by cation exchange chromatography top ↑ as diplonine is an amino acid derivative, purification of the water extract by ion exchange chromatography seemed to be a logical first step in the chromatographic purification of diplonine. by using cation exchange chromatography, all non-ionic and negatively charged substances at a ph of 3.5 could be eliminated in one step, including arabitol, which severely hampered the purification of diplonine (unpublished observations) in the previous study (snyman et al. 2011). fractionation of the water extract by means of cation exchange chromatography was performed with pre-cooled liquids (± 5 °c) as follows: the ph of an amount of the water extract (84 ml) equivalent to 40 g s. maydis culture was adjusted to ph 3.5 by addition of glacial acetic acid (merck ar; merck [pty] ltd, johannesburg, south africa). the extract was passed through 70 g amberlite ir-120 h cation exchange resin (bdh; mcl [pty] ltd, johannesburg, south africa), packed in a 55 mm diameter glass column, at a rate of 1 drop/s. the resin was then washed with 300 ml water followed by elution with 200 ml 1 m ammonium hydroxide (merck ar; merck [pty] ltd, johannesburg, south africa).the dried eluate (325 mg) elicited moderate signs of neurotoxicity when dosed to a 155 g guinea pig (equivalent to 258 g culture/kg bw). a replicate yielded 298 mg of the dried eluate and induced mild neurological signs when dosed to a 145 g guinea pig (equivalent to 275 g culture/kg bw). comparison of these dosing rates (culture equivalents) with that of the water extracts (table 1) indicated a marked loss of toxicity during cation exchange chromatography. when comparing the dosing rates (mg dried residue/kg bw) of the eluates from the cation exchange column with that of pure diplonine (table 1), it was evident that although considerable purification had been achieved by cation exchange chromatography, impurities were still present. in contrast to the water extract, only 30% of pure diplonine (20 mg) was lost when passing it through the column, which might indicate suboptimal chromatographic conditions when fractionating the extract. the dosing rate (mg dried residue/kg bw) of the ammonia eluate nevertheless compares favourably with that of the toxic fraction obtained after two fractionations on silica gel with the chloroform : methanol : ammonia mobile phase (3353 mg/kg bw) in the original isolation of diplonine (snyman et al. 2011). the published method was more expensive, took longer and was more laborious to perform. the amended procedure shows potential for large-scale preparation of diplonine, especially if the loss of toxicity during cation exchange chromatography can be reduced by further optimisation of the chromatographic conditions. neurotoxic clinical signs in a guinea pig top ↑ the neurological signs induced in the guinea pigs by the various fractions largely corresponded with those of the pure toxin, diplonine. the clinical signs elicited by diplonine had previously only been described (snyman et al. 2011) but not photographically documented. for this reason, a series of photographs (figures 3–7) showing progression of intoxication in the guinea pig dosed with diplonine have been included. the photographs corroborate the resemblance to clinical signs in cattle and sheep, and thereby the suggestion that diplonine might be the neurotoxin responsible for diplodiosis in cattle and sheep (snyman et al. 2011). additionally, this photographic record may serve as an important reference for further studies on diplodiosis when guinea pigs are used as a model.in conclusion, the procedures investigated seem to be promising in producing the higher quantities of diplonine required to prove that diplonine is the neurotoxin that causes diplodiosis in cattle and sheep. this has, to some extent, already been demonstrated by the neurotoxic clinical signs that were induced in a guinea pig, as photographically illustrated in this article. figure 1: the chemical structures of diplonine (1 or 2). figure 2: schematic representation of the original isolation of diplonine. figure 3: guinea pig (in front) immediately after dosing with diplonine, showing no clinical signs (normal). figure 4: guinea pig 12 h after dosing with diplonine, showing paresis in the hindquarters (mildly affected). figure 5: guinea pig 24 h after dosing with diplonine, experiencing frequent falling (moderately affected). figure 6: guinea pig 36 h after dosing with diplonine, exhibiting lateral recumbency (severely affected). figure 7: guinea pig 48 h after dosing with diplonine, showing no clinical signs apart from righting reflexes (recovered). acknowledgements top ↑ the maize trust is gratefully acknowledged for funding this research. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions b.c.f. (arc–gci) was responsible for the s. maydis cultures. l.d.s. (arc–ovi) performed the extraction and compiled the manuscript. r.a.s. (arc–ovi) was responsible for the clinical trials. references top ↑ flett, b.c. & mclaren, n.w., 1994, ‘optimum disease potential for evaluating resistance to stenocarpella maydis ear rot in corn hybrids’, plant diseases 78, 587–589. http://dx.doi.org/10.1094/pd-78-0587kellerman, t.s., coetzer, j.a.w., naudé, t.w. & botha, c.j., 2005, plant poisonings and mycotoxicoses of livestock in southern africa, 2nd edn., oxford university press, cape town. schultz, r.a., snyman l.d., basson, k.m. & labuschagne, l., 2011, ‘the use of a guinea pig model in detecting diplodiosis, a neuromycotoxicosis of ruminants’, in f. riet-correa, j. pfister, a.l. schild & t. wierenga (eds.), poisoning by plants, mycotoxins and related toxins: 8th international symposium on poisonous plants, uk proceedings, joão pessoa-paraiba, brazil, 04–08 may 2009, pp. 520–523, cabi, wallingford. snyman, l.d., kellerman, t.s., vleggaar, r., flett, b.c., basson, k.m. & schultz, r.a., 2011, ‘diplonine, a neurotoxin isolated from cultures of the fungus stenocarpella maydis (berk.) sacc. that induces diplodiosis’, journal of agricultural and food chemistry 59, 9039–9044. http://dx.doi.org/10.1021/jf202735e fasina_39-46.indd introduction fifty-nine countries, including nigeria, have been affected by highly pathogenic notifiable avian influenza (hpnai) h5n1 strains since 2004 (oie 2006; who 2006). the livestock sector, especially poultry, plays a very important economic role within the resource poor populations of the developing nations of the world. it provides food (animal protein), income, employment and foreign exchange for countries that trade their animals and animal products (sonaiya, branckaert & gueye 1999; mcdermott, coleman & randolph 2000; fao 2002). the poultry sector represents a major source of income in nigeria. it contributed approximately 4.45 % of the total animal contribution to the agricultural gross domestic product (gdp) in 2004 (central bank of nigeria 2004). the over 140 million birds are composed of about 60 % backyard poultry stock and about 40 % commercial or semi-commercial birds (adene & oguntade 2007). about 75 % of the commercial birds are layer stock and they are responsible for the mass production of eggs and poultry meat in nigeria. poultry is vitally important to the rural poor since it is the most widespread form of livestock in nigeria that the poor rural individuals can afford to keep as a source of income and assets. however, estimating the economics and other financial parameters in the nigerian poultry industry is extremely 39 onderstepoort journal of veterinary research, 75:39–46 (2008) the financial cost implications of the highly pathogenic notifiable avian influenza h5n1 in nigeria f.o. fasina1, 2*, m.m. sirdar3 and s.p.r. bisschop2 abstract fasina, f.o., sirdar, m.m. & bisschop, s.p.r. 2008. the financial cost implications of the highly pathogenic notifiable avian influenza h5n1 in nigeria. onderstepoort journal of veterinary research, 75:39–46 nigeria and several other nations have recently been affected by outbreaks of the asian h5n1 strain of highly pathogenic notifiable avian influenza (hpnai) virus, which affects the poultry sector most heavily. this study analysed previous methods of assessing losses due to avian influenza, and used a revised economic model to calculate costs associated with the current avian influenza outbreaks. the evaluation used epidemiological data, production figures and other input parameters to determine the final costs. an infection involving 10 % of the commercial bird population will cost nigeria about $245 million and a worse scenario may lead to a loss of around $700 million. the results urge governments to invest more in measures aimed at the effective prevention of hpnai and to consider the huge economic losses associated with the disease. finally, an inter-disciplinary approach to managing and controlling hpnai outbreaks is encouraged. keywords: avian influenza, economics, hpnai, h5n1, nigeria, poultry * author to whom correspondence is to be directed. e-mail: daydupe2003@yahoo.co.uk viral research department, national veterinary research institute, vom, nigeria 2 poultry reference centre, department of production animal studies, faculty of veterinary science, university of pretoria, onderstepoort, 0110 south africa 3 military cooperative corporation, khartoum, sudan accepted for publication 21 august 2007—editor 40 financial cost implications of avian infl uenza h5n1 in nigeria difficult since data collection is often incomplete or fragmented. hpnai has caused high levels of mortality, restriction in international trade, infection of various animal species, endangered food security and carries potential for a human pandemic (fao 2002; european commission 2004; otte, nugent & mcleod 2004; holtz-eakin 2005; rushton, viscarra, bleich & mcleod 2005; ducatez, olinger, owoade, de landt sheer, ameerlaan, niesters, osterhaus, fouchier & muller 2006; oie 2006). increasingly, cases of emerging and re-emerging livestock diseases as well as diseases with emergency potential like hpnai, are becoming more connected with higher densities of livestock, increasing trade resulting in the movement of people and products and breaches in biosecurity at various levels (national, regional and farm), despite improving technologies and advances in information dissemination and management practices. although diverse aspects of the avian influenza virus are being investigated worldwide, studies on the socio-economic aspects of avian influenza are limited (otte et al. 2004; holtz-eakin 2005). losses associated with livestock diseases may be easy to identify, but it is extremely difficult to attach a cost implication, and these cost implications are often incomplete (howe 1985; otte et al. 2004; holtz-eakin 2005). a review of socio-economic evaluation models of previous workers indicated that the cost of any illness is the sum total of direct, indirect, intangible and control costs (oluokun & david-west 1981; lasley 1986; rushton, thornton & otte 1999; szucs 1999; otte et al. 2004; verbiest & castillo 2004; holtz-eakin 2005; world bank 2006a). such a model was used earlier by oluokun et al. (1981) in evaluating costs associated with a rinderpest outbreak in nigeria. hanson & hanson (1983) also suggested that loss of any kind must be interpreted in terms of population at risk, and that knowledge of morbidity and mortality rates are important in estimating the cost implication. the costs due to hpnai outbreaks is less significant than those associated with the post outbreak effects on market, trade, enzootic potential, productivity, dwindling interest in poultry enterprises and the attendant zoonotic and food security risks. in this study, each of these factors was considered in estimating the cost implications of hpnai on the commercial layer industry, based on the population affected, the population at risk and economics of alternative control measures. these evaluations mathematically estimate the cost implications of hpnai h5n1 in nigeria, and the implications for disease control are discussed. materials and methods for the financial cost evaluation of hpnai in nigeria, the actual situation and scenarios of mild (10 %) and severe (70 %) generalised outbreaks in the commercial flocks were selected. in nigeria, the commercial layer is very important, accounting for almost 90 % of all egg production (adene & oguntade 2007). similarly, ~99 % of all infected poultry populations are commercial layers and layer breeders (data retrieved from national veterinary research institute, nigeria, december 2006). our estimates deal only with this segment which often operates with little to no biosecurity. a number of assumptions were made: 1. hpnai caused 100 % mortality in affected flocks, either through pathologic death or control measures by destruction. 2. one hundred percent cessation in egg production was assumed, based on published reports (capua & marangon 2000). 3. hpnai caused a loss of 6 months in layer/layer breeder systems (downtime and raising new stock to point of lay). 4. laying birds were in full production and would lay 284 eggs (80 % production) for one laying cycle, and layer breeders would lay 265 eggs (75 % production). fifty percent of the breeders’ offspring would have market value (pullet) and 50 % would be cockerels with zero value. two hundred chicks per breeder hen are expected and approximately 100 of these chicks will be valued stock (average production standards). 5. all deaths in the poultry population in nigeria occurring during the study period (january to august 2006) arose from hpnai or factors associated with it. other baseline data were obtained from resource inventory management, nigeria national livestock resource survey and faostat-glipha (fao 2006a, b and c). it is difficult to place an economic value on human beings affected by hpnai. the affected human population was not economically assessed. prevention of the spread of the disease in livestock would prevent its introduction in the human population. 41 f.o. fasina, m.m. sirdar & s.p.r. bisschop mathematical models ci = ps {ʊ + β + δ + γ} or ci = psʊ + psβ + psδ + psγ where ci = cost implications p = population of poultry s = susceptibility rate of population ʊ = direct losses: losses from mortalities (cost due to mortality of poultry and values of chicks lost from breeders) β = indirect losses: egg and meat loss (value of direct loss of eggs due to yield reduction, cost of rejection of poultry meat and eggs, and cost associated with glut) δ = intangible losses: opportunity cost (cost of rearing replacement stock to production or sale point, cost of feeding to point of production, cost of retaining facilities and staff during downtime and rearing stage, and cost of destroying remaining population of animals) γ = miscellaneous costs (cost of intense campaign to win back consumer confidence, cost of control and administrative/governmental policies, and external inputs) * all calculations were done in naira (n) (nigerian currency) and converted to us dollars ($) at an exchange rate of $1 = n128.50. details of final data used for the calculations are found in tables 1 and 2. total chicken population in nigeria = 140 000 000 commercial chickens = 40 % of 140 000 000 = 56 000 000 commercial layers and layer breeders = 75 % of 56 000 000 = 42 000 000 commercial layers = 90 % of 42 000 000 = 37 800 000 layers in production = 75 % of 37 800 000 = 28 350 000 at 80 % hen-day production: number of eggs per day = 22 680 000 number of eggs per annum [eggs in 12 months (365 days)] = 8 278 200 000 total number of trays = 275 940 000 (30 eggs per tray) trays per annum at $2.18 per tray, the total annual value of eggs from all commer cial layers will be = $601,549,200 layer breeders = 10 % of 42 000 000 = 4 200 000 layer breeders in production = 75 % of 4 200 000 = 3 150 000 at 75 % production: total expected chicks per breeder per annum (100 are saleable pullets) = ~200 chicks total expected number of valued chicks (pullets) per annum = 100 x 3 150 000 if chicks price range between $0.70 and $1.13 with an average of $0.93: total value of chicks expected would be = 100 x 3 150 000 x $0.93 = $294,163,424 total value of chicks and eggs expected from layer breeders and commercial layers = $294,163,424 + $601,549,200 = $895,712,624 calculating for ʊ (direct costs) psʊ1 = actual determined direct value based on the outbreak situation (january to august) psʊ2 = estimated direct value based on mild scenario of hpnai outbreak (10 % losses in commercial poultry population). psʊ3 = estimated direct value based on severe scenario of hpnai outbreak (70 % losses in commercial poultry population). psʊ = market value of birds + value of chicks lost psʊ1 = $5,732,460 + $1,074,023 = $6,806,483 psʊ2 = $37,926,000 + $29,416,342 = $67,342,342 psʊ3 = $265,482,000 + $205,914,397 = $471,396,397 calculating for β (indirect costs) psβ = cost (glut) costs associated with glut: reduction in price observed x (total annual national production [trays per annum] – trays lost to mortality in hpnai) psβ1 cost (glut) 1 = ($2.28 – $2.02) x (275 940 000 – 5 650 704) = $42,068,373 psβ2 cost (glut) 2 = ($2.28 – $1.56) x (275 940 000 – 27 594 000) = $154,612,296 psβ3 cost (glut) 3 = ($2.28 – $1.16) x (275 940 000 – 193 158 000) = $83,748,327 calculating for δ (intangible costs) since intangible costs are costs of rearing replacement stock, facilities retention, staff retention, downtime cost and destruction/ disposal of remaining of affected flocks, therefore psδ = replacement cost + downtime cost + destruction/disposal cost replacement cost = (99.985 % cost for raising pullets to pol* + 0.015 % cost for layer breeders pullets to pol) x total number lost * pol: point of lay bird downtime cost for facilities = facility cost per bird per annum x downtime period per annum x number of birds n100 per bird per annum† x 3/12 months‡ x number of birds † $778.21 per 1 000 birds per annum for retaining poultry pen (field investigations and data from poultry producers, 2006) ‡ average downtime period is 2–4 months (~3 months) destruction/disposal costs are borne by government as well as part of the cost of control. psδ1 = {(0.99985 x $4.28 + 0.015 x $13.23) x 785 570) + ($0.78 x 3/12 x 785 570)} = $3,516,156 42 financial cost implications of avian infl uenza h5n1 in nigeria psδ2 = {(0.99985 x $4.28 + 0.015 x $13.23) x 4 200 000) + ($0.78 x 3/12 x 4 200 000)} = $18,798,906 psδ3 = {(0.99985 x $4.28 + 0.015 x $13.23) x 29 400 000) + ($0.78 x 3/12 x 29 400 000)} = $131,592,341 calculating for γ (miscellaneous costs) nigerian government budget allocation for 2005 used as a guide for 2006. compensation reported till date = $182,640 (www.nigeria.gov. ng/avian%20flu%20center) table 1 types and number of birds affected between 10 january and 31 august 2006 species affected number percentage chicken: layer/pullet§ chicken: broiler/cockerel chicken: layer breeder guinea fowl/quail duck/goose ostrich* turkey wild bird (multi species) 770 826 2 755 11 501 19 148 218 101 2 98.12 0.004 0.015 0.000024 0.000188 0.000278 0.000129 0.0000025 total 785 570 ~100 § include local , backyard and free range laying hens * ostriches numbers were estimated based on field investigation table 2 parameters used in assessing the economic impacts s/no. description symbol basic data actual scenario mild scenario severe scenario 1 population size at risk (layers and breeders) p 42 000 000 0.0056 % (758 570) 10 % (4 200 000) 70 % (29 400 000) 2 susceptible population s 100 % 100 % 100 % 100 % 3 mortality/disposal 100 % 100 % 100 % 100 % 4 commercial layer population affected 37 800 000 774 069 3 780 000 26 460 000 5 layer breeder population affected 4 200 000 11 501 420 000 2 940 000 6 total market value of adult birds (commercial layer at ~$7 and layer breeders at ~$27.30) layer breeder $264,600,000 $114,660,000 $5,418,483 $313,977 $26,460,000 $11,466,000 $185,220,000 $80,262,000 total $379,260,000 $5,732,460 $37,926,000 $265,482,000 7 value of eggs at ~$2.18 (layers only) and meat (old lay value at ~$4.36/bird) per annum eggs meat $601,549,200 $164,808,000 $15,974,720 $3,374,941 $60,154,920 $16,480,800 $421,084,440 $115,365,600 total $944,899,200 $19,349,661 $76,635,720 $536,450,040 8 value of chicks expected $294,163,424 $1,074,023 $29,416,342 $205,914,397 9 proportion in production 75 % 75 % 75 % 75 % 10 mean egg price per tray* n280 ($2.18) n260 ($2.02) n200 ($1.56) < n150 ($1.16) 11 delay in next production pre-outbreak period 6 months 6 months 6 months * average egg price derived from field data collected before, during and after the crises period of outbreak. note that egg price per tray of 30 eggs was progressively dropping as outbreak situation worsened. layer represents commercial layers, bbeeders represents layer breeders. other data were derived from undp 2006 43 f.o. fasina, m.m. sirdar & s.p.r. bisschop other funds and materials acknowledged by the government include monetary and non-monetary income. monetary income 1. world bank special emergency fund = $50,000,000 (of which $7,000,000 has been released (who 2006) 2. three banks = $171,206 non-monetary income 3. dfid = 15 000 protective personnel equipment (ppe) 4. fao = 7 500 protective personnel equipment and 750 ℓ (diskol) 5. who = 10 000 doses of tamiflu 6. usaid = 1 425 protective personnel equipment 7. israel government = 1.5 tonnes of medical equipment expenditure items 3–7 were assessed in monetary value as below: 1. dfid = 15 000 protective personnel equipment = $1,781,250 (at $118.75/ppe*) 2. fao = 7 500 protective personnel equipment and 750 ℓ (diskol) (~$20/ℓ†) = $905,625 3. who = 10 000 doses of tamiflu = $800,000 (at $80 per dose of ten tablets§) 4. usaid = 1 425 protective personnel equipment = $169,219 5. israel government = 1.5 tonnes of medical equipment = $?? (details not available to do actual costing) total = $2,856,094 * http://www.gallawaysafety.com/disposableprotectiveclothing-c-76.html § http://www.coreynahman.com/tamiflu.html † price of comparable virucidal (disinfectant) (onderste poort vet erinary animal hospital) other organisations, including eu and unicef, were also acknowledged by the government. assuming that all other donations is included in the government spending, psγ = 50 % (expenditure items a, b, c, d) + 100 % (expenditure item e) + reported compensation + non-monetary expenditure * note that items a–e are listed in table 3 psγ = $108,655 + $155,642 + $182,640 + $2,856,094 psγ = $3,303,031 psγ1 = psγ2 = psγ3 it is impossible to correlate government spending to the scale of the outbreak; this amount was left unchanged for all scenarios. it seems reasonable to assume that this spending would in fact increase in the event of more severe outbreak. psγ = psγ1 = psγ2 = psγ3 = $3,303,031 results using the above values, the total cost implication was calculated as follows: ci = psʊ + psβ + psδ + psγ actual cost implication ci = {$6,806,483 + $42,068,373 + $3,516,156 + $3,303,031} ci = $55,694,043 scenario a (mild generalised outbreaks 10 % commercial flock) ci = {$67,342,342 + $154,612,296 + $18,798,906 + $3,303,031} ci a = $244,056,575 scenario b (severe generalised outbreaks 70 % commercial flock) ci = {$471,396,397 + $83,748,327 + $131,592,341 + $3,303,031} ci b = $690,040,096 table 3 budgets and allocations for 2005 fiscal year department classification no. expenditure items 2005 allocation % estimated to be spent on hpnai fma&rd 06200002501004 publicity and advertisement (a) $22,757 50 fma&rd 02500002000240 animal disease control (b) $77,821 50 fma&rd 02500002000241 national veterinary quarantine services (c) $77,821 50 nvri 02500002000202 strengthening of central and outstation laboratories (d) $38,911 50 nvri 02500002000205 research and studies (avian influenza) (e) $155,642 100 total $372,952 $264,297 fma&rd federal ministry of agriculture and rural development nvri national veterinary research institute source: nigerian government (2006a and b) 44 financial cost implications of avian infl uenza h5n1 in nigeria these costs do not include the price of medical supplies donated. although our analyses did not consider the broiler industry, we are aware that there was a reported monthly regional export market losses of 12 000 tonnes of poultry meat (personal communication, poultry association of nigeria 2006). these losses translated to 144,000 tonnes/annum and at an average cost of n350/kg of meat, the broiler industry in nigeria will have recorded annual direct losses of $392,217,899. discussion in 2004, nigeria (west africa’s regional centre for trade and commerce) had approximately 140 million poultry of which 119 million were estimated to be laying birds (fao 2006a). about 40 % of the total poultry flock is commercial or semi-commercial and this accounts for the majority of an annual producfig. 1 importance of cost of animal disease outbreak peopleresources disease production goods and services consumption cost valuedisease loss * input of resources is expected to yield goods and services for the benefit of individuals. animal disease situations prevent quality goods and services from reaching human and come at a huge cost and great loss of values * adapted from mcinerney 1988 fig. 2 graphs a and b showing evaluation of cost of animal diseases note: area between standard and disease graph is the cost. several other avian diseases cause partial loss of poultry production. however, few other diseases attract culling of the affected flock p e rc e n ta g e p ro d u c ti o n 100 50 0 weeks in production standard disease 521 the impact of other diseases on egg production b note: area between standard and disease graph is the cost. hpnai cause complete loss in poultry production either through cessation of production, death or culling. most of the nigerian farmers reported above 90 % reduction in production and the entire flock was finally culled (100 % loss) [analysis of the nigerian situation] weeks in production p e rc e n ta g e p ro d u c ti o n 100 50 0 standard hpnai 521 the impact of hpnai on egg output a 45 f.o. fasina, m.m. sirdar & s.p.r. bisschop tion of over 476 000 metric tonnes of eggs (0.8 % of the world total) and 211 000 metric tonnes of meat (0.3 % of the world total) (fao 2006c). laying hens contribute huge resources to the national poultry flock and this emphasizes the importance of commercial layer flocks for nigeria’s economy. laying hens not only have production value, but old birds (spent hens) also serve as a major source of eggs and meat for resource poor families. the nigerian poultry industry ranks second in importance to petroleum, the country’s major source of income (ducatez et al. 2006). currently, the layer industry has lost about $60 million as a result of hpnai h5n1 between january and august, 2006. nigeria’s gross national income was $55.9 billion and the gross domestic product was $72.1 billion as at 2004 (world bank 2006b). this figure, therefore, is a huge economic loss by any assessment. these losses are independent of losses associated with the downstream sectors and broiler industry. economists suggest that the price paid for livestock disease should not be assigned monetary values alone (hanson & hanson 1983; howe 1985; mcinerney 1988). the cost implication of the disease is, however, important as a starting point to assessing the true effect of the outbreak (fig. 1). previous estimates of the cost of avian influenza outbreaks using direct costs grossly undervalue costs associated with hpnai. a mild scenario of infection affecting 10 % of the commercial laying bird population will cost the country in the region of $245 million and a worsening situation may lead to losses of around $700 million in the layer industry alone. from the results, any severe outbreaks of hpnai in a country like nigeria will mean an extremely huge economic loss and will negatively affect the agricultural industry in the subregion. graphical representation of the effect of highly pathogenic avian influenza (fig. 2) proved that these previous methods may not be sufficient to estimate the cost of this disease. this study considered that the productive lifespan and the potential value of the animals involved should be taken into consideration if a comprehensive evaluation of the cost of animal disease is to be done. a point-of-lay commercial bird or a breeder chicken, although may cost less than a rooster/broiler or a turkey respectively at any point, is more valuable than the latter in term of economic benefit since the laying hen or the breeder will bring economic benefit for at least a year. “economic value is not simply prices” (mcinerney 1988). apart from financial losses, the hpnai h5n1 outbreak also had severe impacts on trade and tourism, created scarcity/unavailability of animal protein due to public health misconceptions, led to higher prices for alternative and often lesser quality products, and increased the costs of livestock farming. there are concerns that hpnai may become enzootic in the sub-region or in the african continent, which may then become a source of infection or reinfection to other parts of the world. efforts to step up controls at the borders, surveillance and effective analysis systems are considered justified by the huge resources that will be lost due to such outbreak, if calculated over the productive life. there is a need for restructuring of the poultry industry which aims for higher levels of biosecurity. a scientifically based contingency plan and fair compensation schemes also needs to be developed by all governments. this must be established and tested periodically before the outbreak of any disease, since time lost to decision-making during disease outbreaks has huge economic impacts. conclusion a separate analysis of the socio-economic changes forced on the affected farmers and the costs of different control efforts is still necessary to assist the decision makers in prioritizing all efforts aimed at controlling hpnai in nigeria. an assessment of the effect of the hpnai on other categories of service providers, including day-old-chick suppliers, feed millers and other input suppliers, the hospitality industry, and sole traders in poultry products as well as animal pharmaceutical industries will also be essential to comprehensively assess the overall effect of hpnai. acknowledgements we thank dr celia abolnik of the agricultural research council at onderstepoort and dr peter smith from the faculty of veterinary science for reviewing the manuscript, and prof. bruce gummow for his guidance. data collection was supported in part by funds provided by the poultry reference centre in the department of production animal studies, faculty of veterinary science, university of pretoria. references adene, d.f. & 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http://www.who.int/csr/disease/avian_influenza/country/cases_table yahoo uk 2006. currency converter. http://uk.finance.yahoo.com/currency-converter/convert article information authors: yona sinkala1,2 martin simuunza1 john b. muma1 dirk u. pfeiffer3 christopher j. kasanga4 aaron mweene1 affiliations: 1department of disease control, university of zambia, zambia 2ministry of agriculture and livestock, zambia 3department of veterinary clinical sciences, royal veterinary college, united kingdom 4department of veterinary microbiology and parasitology, sokoine university of agriculture, tanzania correspondence to: yona sinkala postal address: po box 32379, lusaka, zambia note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. postal address: po box 32379, lusaka, zambia copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. foot and mouth disease in zambia: spatial and temporal distributions of outbreaks, assessment of clusters and implications for control in this proceedings... open access • abstract • introduction • materials and method    • study area    • data collection       • data management and geocoding       • statistical analysis • results • discussion    • implications for control measures • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ zambia has been experiencing low livestock productivity as well as trade restrictions owing to the occurrence of foot and mouth disease (fmd), but little is known about the epidemiology of the disease in these endemic settings. the fundamental questions relate to the spatio-temporal distribution of fmd cases and what determines their occurrence. a retrospective review of fmd cases in zambia from 1981 to 2012 was conducted using geographical information systems and the satscan software package. information was collected from peer-reviewed journal articles, conference proceedings, laboratory reports, unpublished scientific reports and grey literature. a space–time permutation probability model using a varying time window of one year was used to scan for areas with high infection rates. the spatial scan statistic detected a significant purely spatial cluster around the mbala–isoka area between 2009 and 2012, with secondary clusters in sesheke–kazungula in 2007 and 2008, the kafue flats in 2004 and 2005 and livingstone in 2012. this study provides evidence of the existence of statistically significant fmd clusters and an increase in occurrence in zambia between 2004 and 2012. the identified clusters agree with areas known to be at high risk of fmd. the fmd virus transmission dynamics and the heterogeneous variability in risk within these locations may need further investigation. introduction top ↑ foot and mouth disease (fmd) is endemic in zambia and several other african countries and impacts negatively on the livelihoods of the people (perry & hedger 1984; perry & stones 2007). fmd is the single most important economic animal disease affecting regional trade in livestock, wildlife and other agricultural products within the southern african development community and common market for east and southern africa. zambia, like many other african countries, is looking for ways to derive maximum benefit from trade in livestock and wildlife resources through prescribed international standards such as disease-free zones and the progressive control pathway. many economists consider regional trade in the subregion as a vehicle for economic growth, poverty reduction and achievement of the much desired millennium development goals.since the first case in 1933, several outbreaks have occurred in zambia and areas with a high fmd risk have been described (chilonda et al. 1999; mweene et al. 1996; overby & zyambo 1983; perry & hedger 1984; zyambo 1975). these studies were all conducted more than two decades ago. however, the epidemiological pattern of fmd may not remain static in space and time owing to both its highly contagious nature (radostits, blood & gay 1994) and the behaviour of human populations. the most recent study (chilonda et al. 1999) concluded that the epidemiology of fmd transmission in zambia is not adequately understood. since then several outbreaks have occurred within zambia and the surrounding region, with an increased frequency observed since 2004 (thobokwe et al. 2010; thomson, penrith & fosgate 2012). the reasons for the recent outbreaks in zambia are not well known, but elsewhere in the region it is suspected to be as a result of the breakdown in the once effective control measures of the 1980s and 1990s. therefore, the spatial aggregation of fmd cases needs to be assessed and updated to help design effective control measures. the epidemiology of fmd in sub-saharan africa is further complicated by: • the presence of reservoir african buffalo (syncerus caffer) and several other host species • the presence of six of the seven known fmd virus (fmdv) serotypes, of which the most notable are the southern african territory (sat) serotypes • mutations of the fmdv • rampant wildlife and livestock movement (vosloo et al. 1995) • the knowledge, attitude and perception of smallholder livestock farmers and traders about fmd (perry & stones 2007). although the transmission of fmd from carriers (buffalo or cattle) to cattle is not fully understood, evidence exist that such transmission occurs only rarely (condy & hedger 1974; rweyemamu 1984). despite several cohort studies regarding the trigger of this transmission, there have been no conclusive findings (rweyemamu et al. 2008; thomson et al. 1992; vosloo, bastos & boshoff 2005; vosloo, bastos & boshoff 2006). this knowledge gap creates challenges in understanding predictors of future outbreaks necessary for effective preventive control in endemic settings where buffalo and cattle co-exist. these cohort studies are expensive and it may be difficult to mimic what happens in nature in an experimental setup. in addition, findings regarding the epidemiology of fmd in a wildlife species in one ecological region may not be able to be extrapolated to other regions because of ecological and viral variability (vosloo et al. 2009). therefore, research is recommended to understand what factors could pertain to the epidemiology of the disease in potential high-risk areas materials and method top ↑ study area zambia extends over 752 000 km2 and has a population of 13.4 million, of which the majority (61%) are concentrated in the rural areas (cso 2010). the country has more grazing land than arable land. most rural zambian populations depend on livestock for their livelihood. traditional farmers own 84% of the estimated 3.4 m cattle, 68% of the 450 000 sheep, 97% of the 1 m goats and 90% of the 1.5 m pigs (ministry of livestock and fisheries development 2011). fmd is a notifiable disease of national economic importance in zambia, which compels the government to assume the cost of control during an outbreak. the disease is often under-reported and not all outbreaks that are sampled are analysed owing to poor sampling, storage and despatch techniques. fmd in zambia has always been mild in indigenous cattle, often causing only transient paralysis (occult form of the disease) (mweene et al. 1996; zyambo 1975). most traditional animals are not closely monitored except during ploughing periods. data collection the study was a retrospective fmd case series. a case is defined as a clinical presentation of fmd-like lesions and confirmed by laboratory analysis. because of the social economic status of cattle, fmd is reported only for this species in zambia. there is a notable absence of records of fmd in pigs, sheep and goats, except for a passing reference to natural cases of fmd in pigs in 1935 and an outbreak in the southern province in 1979 (chilonda et al. 1999). data on fmd cases recorded in zambia between 1981 and 2012 were obtained from peer-reviewed journal articles, conference proceedings, reference laboratory reports, unpublished scientific reports and grey literature. the grey literature included records from the national epidemiology unit, the virology database of the central veterinary research laboratory, annual reports from the ministry of agriculture, fmd reports from the animal health institute (pirbright), the onderstepoort veterinary institute and the botswana vaccine institute. the case data included date of occurrence, location, number of cases, population at risk (where available), serotype involved, vaccination and vaccine used. data management and geocoding the disease data consisted of 178 fmd-positive cases in 86 locations over the 31-year period. the cases were from villages and veterinary camps. a spatial coordinate system was used for geo-referencing the cases using the available smallest administrative units of village or veterinary camp (figure 2). two files were developed for use in satscan (free software developed by kulldorff [2012]): a case file in spreadsheet format detailing location identification, number of cases and the date the cases were reported, and a geocoded file that contained location identification and the latitude and longitudes of the locations of cases. both file types were saved as a space-delimited format. the coordinates for the identified clusters and the fmd cases were projected to the universal transverse mercator (utm) zone 35s world geodetic system (wgs) 1984 and represented using arcgis version 10.1 (environmental systems research institute, redlands, california). a topographic map of zambia was used as a reference for relating cases and clusters to geographical features such as roads, national parks, rivers and international boundaries. statistical analysis spatial analysis can be used on disease outbreak data to describe geographical patterns of disease (e.g. clusters). disease clusters can help in identifying common environmental factors or sources of exposure (chhetri, perez & thurmond 2010; premashthira et al. 2011). the null hypothesis was that the number of infected villages or veterinary camps, their location and the time frame of infection were similar. therefore, the disease likelihood ratio was expected to be constant over the whole territory and time frame under analysis. the analysis included the hypothesis that the number of cases reported within a certain geographical area was higher than that observed outside that area during the epidemic and was assessed by a purely spatial test. clustering was also assessed across different time intervals by the space–time test. a hypothesis could then be generated to understand disease epicentres – how the disease is sustained over time in endemic situations and the variation in risk within a perceived high-risk area. spatial scan statistics (kulldorff & nagarwalla 1995) are frequently used for geographical disease cluster detection and cluster evaluation in disease surveillance. the advantages of using the satscan program are that it can adjust for confounding variables, resolves pre-selection bias as it searches for clusters without specifying their size or location, gives a single p-value as the likelihood ratio-based test takes account of multiple testing, and can be applied to a whole region to detect clusters (kulldorff et al. 2005). the satscan software scans for temporal, spatial and spatial–temporal clusters. briefly, it selects scanning windows of continuously varying sizes over a spatial study area. for each location and size of the scanning window, the alternative hypothesis is that there is an elevated rate within the window compared with outside. the most likely cluster was determined by maximising a likelihood function over all the zones. once the window with the greatest likelihood ratio statistic has been identified, the sampling distribution of the likelihood ratio is evaluated using a monte carlo test (9999 repetitions). a likelihood ratio was created by dividing the maximum likelihood value by another likelihood value based on the null hypothesis. we used space–time retrospective analysis using the space–time permutation probability model, scanning for areas with high rates. a one-year unit was used as case data are available only per year. results top ↑ the space–time test identified four significant clusters in different time periods (years) and the null hypothesis was subsequently rejected. the most likely cluster was located in the kasama district (n = 10), with a radius of 167 km, between 2009 and 2012. the likely secondary clusters were located in senanga (n = 13), with a radius of 279 km, between 2007 and 2008, itezhitezhi (n = 48), with a radius of 137 km, between 2004 and 2005, and katapazi (livingstone) (n = 3), with a radius of 39 km in 2012 (figure 1, figure 2, figure 3 and table 1). figure 1: graph of fmd cases from 1981–2012, showing an increase in the number of cases per five year period. figure 2: spatial distribution of cases of foot and mouth disease in zambia (according to serotype and topotype) within the three high-risk areas. figure 3: clusters of foot and mouth disease in zambia as identified by satscan. table 1: space–time foot and mouth disease clusters in zambia as identified using the space–time permutation probability model. the study did not control for covariates or confounders but used molecular epidemiology reference laboratory reports on the likely source of the outbreaks to discuss potential factors. discussion top ↑ the results demonstrate the spatial distribution of fmd in zambia. four significant fmd clusters were indentified, including two distinct clusters within the lower zambezi basin (figure 3). although based on geo-referenced and case report data, this report should not be construed as an evaluation of the fmd epidemics that occurred between 1981 and 2012; rather, it is intended as a hypothetical (yet realistic) scenario that facilitates the retrospective exploration and generation of hypotheses on epidemic spread. the findings are limited to the data set investigated and do not factor in the level of under-reporting, unconfirmed samples, livestock or wildlife densities and serotype variations. therefore, the findings should best be regarded as evidence of associations that may support specific hypotheses. the data are compatible with the assertion that fmd in zambia shows two epidemiological patterns. one is related to serotypes o, sat 1 and sat 2 being maintained and spread by livestock movement along the border with tanzania (perry & hedger 1984). this is where the primary cluster was located (around kasama) between 2009 and 2012, with most index cases being in the mbala district. the second is the sat 1 and sat 2 serotypes in the kafue flats and zambezi basin being maintained and spread by both african buffalo and domestic livestock movement. the sat viruses may also become established in cattle, leading to cyclic incidences as naïve populations increase if the basic reproduction number is not reduced to less than 1 (rweyemamu et al. 2008). in the kafue flats, itezhitezhi was identified as the centre of the 2004–2005 cluster, with the index case in katengwa, namwala district. these two areas are within the kafue flats, where more than 600 000 cattle compete with approximately 9000 buffalo (1000 from the lochnivar national park; 8000 from the kafue national park) for grazing and water annually (zambia wildlife authority 2007; chilonda et al. 1999). the high contact rates between cattle and wildlife (buffalo) in the dry season is believed to be the source of most of the fmd outbreaks. historically, outbreak index cases in this area have generally been identified on the fringes of the lochnivar national park in namwala, mazabuka or monze (overby & zyambo 1983; perry & hedger 1984; zyambo 1975). this primary source of exposure, together with secondary movement of infected cattle, may be the major reason for the spatial clustering observed (zyambo 1975). in the zambezi basin, two distinct clusters around senanga (2007–2008) and livingstone (2012) have been identified. whilst the centre of the cluster was located around the senanga district, the index case was in mwandi (sesheke) from where infections spread to kazungula, senanga, mongu and kalabo. this outbreak was associated with serotype sat 2 and two plausible sources of origin have emerged. one source is attributed to buffalo, as the area lies on a buffalo migratory route between the chobe national park in botswana and the sichifulo game management area in zambia. this is supported by molecular evidence that associated the virus to the buffalo strain that was isolated from chobe national park earlier (fao_wrlfmd 2012a). the second source is attributed to the movement of cattle from neighbouring namibia (bastos et al. 2003; mweene et al. 1996) and molecular evidence shows an association between the outbreak virus and the one isolated from a namibian outbreak in cattle earlier (fao_wrlfmd 2012a). the livingstone katapazi cluster of 2012 was associated with serotype sat 1 and showed no relationship with any other virus isolated in the area (fao_wrlfmd 2012b), which supports the suspicion that it may have come from contact with buffalo that were spotted in the area. in our view, the two lower zambezi clusters, although close to each other, should be considered separately as the sources of risk are most likely different and considerations may have to be made in disease control approaches. data are available only for certain localities and under-reporting is likely as diseases such as fmd are known to spread spatially unless adjacent areas are devoid of susceptible animals. under-reporting and the aggregation of cases to veterinary camps or districts may negatively impact the ability to determine the exact spatial location of the outbreak accurately, particularly so in small clusters (< 800 km2) (jones & kulldorff 2012). jones and kulldorff (2012) also report that the likelihood of spatially overestimating outbreaks by including geographical areas outside the actual disease cluster increases with aggregated data. the creation of windows that may include areas that have not reported fmd (e.g. west and south of the primary cluster) is one of the inherent limitations of satscan (jones & kulldorff 2012; rivas et al. 2006). our study supports the alternative hypothesis that there is an elevated risk for those areas located within the identified clusters compared with those beyond. the reasons for the difference in risk for the areas located within and beyond the clusters require further investigation. the factor associated with fmd clustering may be either within or beyond the location where the disease is observed (rivas et al. 2006). for example, in the case of market-driven spread, the cluster location may be different from the market. fmd outbreaks in zambia have long been associated with livestock movement along trade routes (bastos et al. 2003; perry & hedger 1984; zyambo 1975). these movements are largely driven by incentives related to differences in price of livestock and therefore the epidemiology of fmd cannot be separated from the economics of livestock marketing (rushton 2008). understanding the behaviour of small-scale farmers and livestock traders alike may provide clues to disentangling the endemicity (perry & rich 2007) of the disease. therefore, the role of people networks in the epidemiology of fmd may need to be investigated further. implications for control measures the identified clusters may be taken into consideration when formulating new disease control strategies, especially in the context of the planned disease-free zones (republic of zambia 2011). as the risk is not static and may change with the changing disease dynamics, more targeted surveillance within and beyond the identified clusters is recommended. the kafue flats and the two zambezi basin clusters lie within the zambezi kavango transfrontier conservation area (kaza-tfca 2013), where free roaming of wildlife (buffalo) is being promoted without regard for the fmd risks. it is therefore necessary to profile fmd clusters with a view to influence control measures if benefits are to be realised from livestock trade. conclusion top ↑ the fmd clusters may not be stated depending on exposing factors and therefore evaluation is periodically relevant in these endemic settings. the exposure to fmdv in the identified clusters may be a result of epidemiological, ecological and sociological processes. understanding these processes may lead to innovative solutions that should be considered in the fmd control pathway for zambia and southern africa. our study therefore provides a basis for risk-based surveillance and further investigations into risk determinants within the identified clusters. acknowledgements top ↑ this work was supported by a grant (wt087546ma) from the wellcome trust to the southern africa centre for infectious disease surveillance (sacids). the authors are grateful to the veterinary department of the zambian ministry of agriculture and livestock for permission to use the fmd outbreak data and the relevant documents referenced in this study. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions y.s. (university of zambia and ministry of agriculture and livestock, zambia) collected the data, conducted the analysis and prepared the manuscript. m.s. (university of zambia), j.b.m. (university of zambia) and c.j.k. (sokoine university of agriculture) edited the manuscript. a.m. (university of zambia) and d.u.p. 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to: achenef meleku postal address: po box 196, gondar, ethiopia dates: received: 24 nov. 2013 accepted: 21 may 2014 published: 12 nov. 2014 how to cite this article: kebede, h., melaku, a. & kebede, e., 2014, ‘constraints in animal health service delivery and sustainable improvement alternatives in north gondar, ethiopia’, onderstepoort journal of veterinary research 81(1), art. #713, 10 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.713 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. constraints in animal health service delivery and sustainable improvement alternatives in north gondar, ethiopia in this original research... open access • abstract • introduction • research method and design    • study area    • design       • questionnaire survey       • in situ observation of infrastructural setup       • retrospective data collection       • discussions with district agricultural officers       • discussions with concerned regional bureaus • results    • reported livestock diseases       • cattle       • small ruminants       • poultry       • equine       • small animals    • current status of service delivery       • existing practices by farmers       • expenditure on veterinary services       • existing practices in government service delivery       • existing premises in government service delivery       • existing practices and facilities in private veterinary service       • reason for engagement in private service       • farmers’ preference • level of satisfaction • expectations for privatisation • constraints for veterinary service delivery    • government veterinary service    • private veterinary service • retrospective assessment    • annual budget allocation in government service    • annual physical plan execution report • group discussion    • district officials    • regional bureaus • discussion    • disease epidemiology    • status of existing service    • options for service delivery improvement • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ poor livestock health services remain one of the main constraints to livestock production in many developing countries, including ethiopia. a study was carried out in 11 districts of north gondar, from december 2011 to september 2012, with the objective of identifying the existing status and constraints of animal health service delivery, and thus recommending possible alternatives for its sustainable improvement. data were collected by using pre-tested questionnaires and focus group discussion. findings revealed that 46.34% of the responding farmers had taken their animals to government veterinary clinics after initially trying treatments with local medication. more than 90.00% of the clinical cases were diagnosed solely on clinical signs or even history alone. the antibacterial drugs found in veterinary clinics were procaine penicillin (with or without streptomycin), oxytetracycline and sulphonamides, whilst albendazole, tetramisole and ivermectin were the only anthelmintics. a thermometer was the only clinical aid available in all clinics, whilst only nine (45.00%) clinics had a refrigerator. in the private sector, almost 95.00% were retail veterinary pharmacies and only 41.20% fulfilled the requirement criteria set. professionals working in the government indicated the following problems: lack of incentives (70.00%), poor management and lack of awareness (60.00%) and inadequate budget (40.00%). for farmers, the most frequent problems were failure of private practitioners to adhere to ethical procedures (74.00%) and lack of knowledge of animal diseases and physical distance from the service centre (50.00%). of all responding farmers, 58.54% preferred the government service, 21.14% liked both services equally and 20.33% preferred the private service. farmers’ indiscriminate use of drugs from the black market (23.00%) was also mentioned as a problem by private practitioners. sustainable improvement of animal health service delivery needs increased awareness for all stakeholders and a well-regulated private service in order to mitigate the constraints apparent in the government service. introduction top ↑ globally, agriculture provides a livelihood for more people than any other industry. growth in agricultural production and productivity is necessary in order to raise rural incomes, to support the increasing numbers of people dependent on this industry (upton 2004) and to meet the food and raw material needs of fast growing urban populations. enhancing agricultural productivity contributes to industrial growth by providing cheap labour, capital investment, foreign currency and markets for manufactured consumer goods. agriculture plays a key role in reducing poverty because most of the world’s poor live in rural areas that are largely dependent on agriculture; food prices also determine the cost of living for the urban poor. livestock provide over half of the value of global agricultural output and one third in developing countries (upton 2004). increased livestock production and higher self-sufficiency would reduce the need for foreign aid and increase gross domestic product as a result of the export of livestock and their products. livestock production contributes to rural livelihoods, employment and poverty relief, integrating with and complementing crop production, acting as a savings bank and providing a buffer against risks (sen & chander 2003; upton 2004).poor livestock health services remain one of the main constraints to the development of livestock production in many developing countries. in sub-saharan africa, losses resulting from disease are estimated at $2 billion per year, of which half is attributable to direct losses from mortality and the other half to indirect losses as a consequence of reduced growth, fertility and ability to work (de haan & bekure 1991). these losses caused by animal disease are not simply restricted to lower outputs; in addition, disease prevents the introduction of livestock into certain areas (e.g. in africa large tracts of land with high yielding potential are lost because of african trypanosomosis) and skin diseases, such as dermatophilosis, preclude the use of more productive animals, including crossbred dairy cattle, improved pigs and poultry breeds. trade embargos are enforced by importing countries as a result of the presence of highly contagious diseases, creating another important bottleneck for the sector, reducing trade and the inflow of foreign currency. furthermore, certain livestock diseases are zoonotic, which means that the control of animal disease is not only of economic importance, but also of social and political significance. consequently, disease control services will continue to be an important factor in the development of the livestock sector (cheneau, el idrissi & ward 2004; chilonda & van huylenbroeck 2001). as in many developing countries, most veterinary services in ethiopia are currently provided by the government. veterinary services, like many publicly provided livestock services to smallholder farmers in ethiopia, are economically inefficient and, in many cases, may actually cost the government more than their value to the farmer. the pattern of budgetary allocations has resulted in the animal health public sector provision meeting less than 30% of demand (cheneau et al. 2004; de haan & bekure 1991). the current global economic and political climate is forcing sharp reductions in government expenditure on state-funded services and a need for increased transparency and public accountability. the government of ethiopia is making ongoing efforts to privatise many businesses in the service sector, but it seems that no priority is being given to veterinary services. it is now time to explore opportunities for a more decentralised, client-oriented, cost-effective approach to the delivery of animal health services (silkin & kasirye 2002). with the changing global economic situation, public services are being challenged to perform more effectively. thus the concept of privatisation is finding wide acceptance as an alternative. veterinary services are no exception to this trend. privatisation means relying on society’s private institutions rather than on those provided by the government. it is the act of reducing the role of government, or of increasing the role of private sector, in an activity or in the ownership of assets. privatisation of veterinary services has become a necessity for several reasons. these include the reduction in financial resources, the small size of the budget allocated to the livestock sector, the difficulty experienced by the public sector in meeting the demands placed upon them, as well as the increasing inability of the government structure to absorb all the veterinarians and animal health assistants being trained (tber 1995). privatisation in the context of the delivery of animal services relates to the transfer of activities, functions, responsibilities and property from the public to the private sector. it should be seen as a process of refocusing public sector veterinary services by devolving responsibilities, not merely as a means of reducing government expenditure. in planning for such changes, it is important to consider current and future projected consumer demand, disease epidemiology, changing livestock systems, socioeconomic growth, the availability of suitably qualified professionals, current and future infrastructure needs and the views of the existing clinical veterinary service. this will enable preparation of a sustainable programme that will help to facilitate effective privatisation of the animal health services (mlangwa & kisauzi 1994; sparagano 1999). the objectives of this study were therefore to identify existing constraints to animal health service delivery and to recommend possible alternatives for sustainable improvement of animal health service delivery in the north gondar administrative zone. research method and design top ↑ study area north gondar is located in amhara national regional state, bordering sudan in the west, south gondar and west gojam in the south, waghemra in the east and tigray in the north. it encompasses different agro-climatic zones, including kola (lowland), woina dega (midland) and dega (highland). north gondar zone harbours the north-western lowlands, which have huge agricultural potential, especially for livestock and cash crops such as sesame and cotton. mixed farming systems (both livestock and crop production) predominate in the area.the study was conducted in 11 districts (out of 22), namely metema, gendawuha ketema zuria, chilga, dembia, gondar zuria, gondar ketema, lay armachiho, wegera, dabat, debark ketema and debark. design questionnaire survey three administrative units (locally called kebele) were randomly selected from total list of administrative units in each district using a simple random sampling method. a list of the farmers living in each administrative unit was taken from local development workers and systematic random sampling was used to select a total of five individuals. the list of farmers to be included in the study was dispatched to respective local agents before beginning the survey so that they could arrange time to come to centres during the schedule.using thrusfield’s (2005) derivation for sample size, the following assumptions were made and the number of individuals to be included was estimated. as the objective of the survey was more of a qualitative study, 10% error was inferred and, because no published work was encountered, 50% was used for prevalence of problems. the following formula was used: [eqn 1] where n = number of individuals to be included, zα = confidence level (90% [z90%] confidence level was used), pexp = expected prevalence of farmers experiencing constraints in the service and d = level of absolute precision. furthermore, because of the seasonality of farm activities, a 50% response rate was expected. therefore, to obtain 68 (50%) responses, 136 individuals were needed: 136/11 districts = 13 farmers from each district. from each district, 3 kebeles were selected (13/3 = 4.3 [i.e. 5] farmers per kebele). recall papers were distributed for 15 farmers from each district, where a minimum of 10 and maximum of 15 farmers responded to calls for the questionnaire. a structured and pre-tested questionnaire format was used to collect information from each district (total = 123) and all veterinary practitioners involved in both the public (n = 20) and private sector (n = 17) available during the study period. the questionnaire was designed to assess the occurrence of common diseases in the area, the treatment regimens adopted when disease develops and the level of satisfaction with the existing animal health service delivery, including reasons for any dissatisfaction. during the questionnaire survey, it was attempted to correlate farmers’ local perception of disease signs and symptoms with scientific contexts. for individuals in veterinary services (private and public), the methods used for routine diagnosis and commonly used therapeutic agents were also assessed. an assessment was also made of the costs, including professional services and therapeutic agents provided by private practitioners and the public service, together with any charges made. in situ observation of infrastructural setup a checklist (based on standards set by the regional bureau of agriculture) was prepared and both private (n = 17) and government (n = 20) animal health service delivery centres operating during the survey period were included, inspected and observations recorded for all available premises and facilities. retrospective data collection retrospective data over 5 years were collected from the agricultural district offices in order to assess budgetary and administrative constraints relating to planned activities and the ability to implement them with the available resources. the 5-year regional drug price record was assessed for the most common and routinely used drugs in public service delivery to characterise the trend. average collective cost per year for each total unit of drugs considered was used to describe the trend. discussions with district agricultural officers after the individuals in the public and private animal health service delivery sectors and farmers had been interviewed, the problems raised were summarised and forwarded to individuals in leading positions in the ministry of agriculture (moa) at district, zonal and regional levels, whose views were obtained with respect to the existing constraints summarised from the survey and future prospects for the service, including options to improve it. discussions with concerned regional bureaus after completion of the assessment survey, a checklist was also prepared for the regional bureau of agriculture, regional livestock agency, bureau of finance and economic development and the regional veterinary diagnostic laboratory. these checklists were targeted to assess the administrative and policy issues related to improvement strategies for the sustainable development of animal health delivery at the regional level. in the discussions, the regional laboratory and livestock agency, senior experts and middle-level professionals were involved. the existing constraints in terms of service delivery, options for improvement, opportunities and threats presented by privatisation, as well as what type of service to privatise were all raised for discussion.discussions with the regional bureau of finance and economic development and the regional bureau of agriculture, including directors, took place in order to evaluate the necessary policies for privatisation and the technical feasibility in terms of existing personnel and resource management. results top ↑ reported livestock diseases cattle infectious and non-infectious diseases were described. farmers included lumpy skin disease more frequently (46.34%), whilst anthrax and gastrointestinal parasites were more frequently mentioned by professionals in the public and private services. foot and mouth disease was frequently mentioned by both professionals and farmers. small ruminants small ruminants (sheep and goats) in the zone were also affected by several diseases. sheep and goat pox was the disease most frequently mentioned by farmers and professionals working in the public sector. gastrointestinal parasitism was mentioned most frequently by professionals working in the private sector. poultry farmers and professionals were also asked to list diseases that affect poultry. newcastle disease was the most frequently mentioned, followed by coccidiosis, fowl pox and ectoparasites. equine of the diseases that affect equines, anthrax was most frequently mentioned, followed by mange, colic, african horse sickness and back sores. small animals the most frequently mentioned disease of pets (dogs and cats) in the zone was rabies. ectoparasites, internal parasites and flea allergic dermatitis were the most common problems indicated by farmers, government workers and private practitioners, respectively. current status of service delivery existing practices by farmers assessment of animal disease management practices indicated that 46.34% of the respondents had taken their animals to veterinary clinics (government) after initially trying local medicaments. of the respondents, 19.50% habitually administered modern medicines obtained from private or illegal sources themselves when their animals became sick (table 1). table 1: accustomed practices undertaken by farmers to combat animal health problems. expenditure on veterinary services the estimated average costs incurred by livestock owners for animal disease related problems in different species per year are summarised in table 2. a greater amount of money was spent on cattle than any other species. table 2: average cost (in us dollars) incurred for animal disease, as estimated by respondents. existing practices in government service delivery of the total number of public veterinary clinics visited (n = 20), only 10% were known to use laboratory diagnostic aids for faecal egg screening. in none of the government clinics were either local or general anaesthetic agents evident, which indicates surgical procedures were not performed, or if they were, they were unethical, without pain management. the only antibiotics found in all of the veterinary clinics were penicillin (+) streptomycin combination, oxytetracycline and sulphonamides. the anthelmentics found were albendazole, tetramisole and ivermectin and there were no antifungal agents. the disinfectants found in nearly 20% of government clinics were potassium permanganate and gentian violet. more than 90% of the clinical cases were diagnosed solely on the clinical signs or the history alone. in almost all sites, anthelmentics were given based on the history taken from the owner. existing premises in government service delivery from the results of the visits, it was evident that all of the government clinics (n = 20) lacked appropriate facilities to restrain and handle clinical cases and they were not well equipped. a thermometer was the only instrument available in all the clinics. only nine (45%) of the clinics had a refrigerator, without any reserve power backups, implying less than 50% of clinics were able to handle vaccines or drugs that need cold chain storage. none of the clinics had a stereomicroscope for diagnosis of ectoparasites. existing practices and facilities in private veterinary service almost 95.0% of private service facilities were private drug retail shops owned by veterinarians and assistant veterinarians and, of those visited, only 41.2% were shown to fulfil the criteria set by the regional bureau of agriculture. almost all drug shops handled pesticides (for control of crop pests) and antibiotics (for veterinary use) in the same dispensing area. in 2.0% of the drug retail shops, there were complaints about the sale of expired drugs and, in 30.0%, clinical services for which they were not licensed were seen to be given during the survey. reason for engagement in private service a question posed to private practitioners asked why they were engaged in private business. nearly 35.29% of them answered that they did so in order to acquire money to live; 23.53% answered ‘for greater profitability’. farmers’ preference of all responding farmers, 58.54% preferred the government service, 21.14% liked both services equally and 20.33% preferred the private service. the reasons mentioned by farmers for their preference for either the public or private sector are listed in table 3 and table 4. about 47.22% of farmers preferred government service because of cost. the farmers who preferred the private service attributed their preference to the availability of service whenever it was needed. the existing service delivery under the current animal health service was seen to be unsatisfactory, both in the private and public service systems, in terms of both quality and range. many respondents were seen to use veterinary drugs without any veterinary supervision because of the distance from clinics or their lack of availability in the existing clinics. table 3: reasons indicated by farmers for preference of government service. table 4: reasons indicated by farmers for preference of private service. level of satisfaction top ↑ farmers were asked to indicate their level of satisfaction when they used the service, either in the government or private sector. about 37.7% of farmers were satisfied with the private service and fairly satisfied with the public service (figure 1). figure 1: level of satisfaction of farmers with private and government veterinary services. expectations for privatisation top ↑ possible impacts of privatisation for the future were assessed. most (75%) of the respondents said that privatisation would have a positive effect on veterinary service delivery in the zone. reductions in the quality of service and job losses were also mentioned by respondents (table 5). table 5: government service staff expectations of outcomes of privatisation schemes. constraints for veterinary service delivery top ↑ government veterinary service constraints for veterinary service delivery in the public sector were related to management, clients (livestock owners) and professionals working in government. some of the problems related to management were lack of awareness, shortage of budget and also the minimal attention given to the sector. amongst client-related problems, distance from the service centre was most frequently (50%) mentioned by respondents. lack of motivation either because of inadequate salary or financial resource availability was the most important problem conveyed by the professionals (70%) (table 6). table 6: problems in public veterinary service. private veterinary service the constraints in private veterinary service delivery are listed in table 7. problems related to cost were most frequently mentioned by respondents (40%). failure to adhere to accepted norms and some unethical practices were also mentioned by some respondents (30%). table 7: constraints in private veterinary service. retrospective assessment top ↑ annual budget allocation in government service comparison was made between the planned and allocated budget in the public veterinary service. no district had been satisfied during the last 5 years because the budget allocation was lower in all districts than the planned budget (figure 2). there was also no indication of any increment in the annual government budget allocation to match the increase in drug prices; rather, there was a relative decline in the budget (figure 3). thus, an inverse relationship was observed between the annual budget allocation and the price of drugs (correlation ‘r’ = -0.67). figure 2: average planned and allocated budget in ethiopian birr, 2007–2012. figure 3: the average price of drugs (in ethiopian birr) and the allocated budget, 2007–2012. annual physical plan execution report the average annual vaccination and execution of the treatment plan are shown in figure 4 and figure 5, respectively. the vaccination performance ranged from 50% to 75%, whilst the therapeutic plan execution performace was greater than 70% during the last 5 years. however, the vaccination performance should also be evaluated based on the epidemiology of diseases and the number of animals in need of vaccination. figure 4: average vaccination plan versus actual execution, 2008–2011. figure 5: average annual animal health service plan and execution record, 2008–2011. group discussion top ↑ district officials representatives from district offices of agriculture as well as members of the animal health service team were involved in the group discussions undertaken at all assessment study sites. problems experienced by individual farmers and animal health service personnel were presented and discussed and possible solutions were proposed. problems raised by farmers regarding the government service included shortage of different drug types and absence of an accessible regular daily service from animal health assistants. strengths included reliability of drugs when available and their value for money. people in the private animal health sector highlighted unfair price competition with government clinics, lack of implementation of laws on illegal drug circulation and lack of ethical practice by individuals working in adjacent services. the public sector raised issues around adequate staffing to support a dual mobile and ambulatory-based health service (during the mobile service delivery, a single animal health assistant is expected to cover three neighbouring peasant associations in rural service centres). lack of incentives was also cited at almost all of the study sites. engagement of public service delivery staff in non-animal health service activities was also raised in 60% of the group discussions; exceptions were metema, dembia, debark and chilga districts.district representatives agreed partly on the questions raised by farmers and individuals engaged in service delivery. solutions proposed included reinforcing and refreshing the illegal trade (drug) regulatory team, implementing the new regional livestock agency and sequential implementation of a regional plan for service cost recovery. from group discussions, the issue of privatisation was also encouraged as a solution. regional bureaus from discussions with individuals from regional bureaus of economic development, it was evident that the government remained committed to its responsibilities for the delivery of public services for the benefit of the community. in this respect, the provision of an animal health service available to the vast majority of the community, especially farmers, remained an important commitment, with the aim of providing a reliable service to address the deficits. the failure to privatise agricultural support services in other african countries was highlighted, suggesting that privatisation may be difficult to implement in ethiopia. however, for the long term, privatising at least parts of the veterinary service cannot be overlooked. so far, policies to either support or impede privatisation have not been prepared at a regional level with specific reference to agricultural services. the absence of an agency to manage privatisation at a regional level was also raised.during the discussion, the following constraints for animal health service delivery were listed: • deficiency in budget allocation • gaps in control of illegal drug circulation and implementation of ethical practices • absence of adequate training and capacity building for existing government staff • poor routine practice in procedures relating to animal health service delivery • the need for existing professionals in public service to engage in non-professional duties and responsibilities in the office of agriculture • problems related to paraprofessional practice (paravets) • the low level of incentives to encourage individuals into the veterinary service – basic pay and related allowances • poor support services for animal health • shortage of laboratory facilities, including reagents and materials • poor networking with other stakeholders – university faculties and multiplication centres • free service delivered by the government service and constraints upon the necessary facilities, diagnostic tools, prophylaxis and treatment. the option of privatisation was raised in all of the group discussions. pre-requisites included the implementation of an appropriate code of practice in the public and private sectors, a change in attitude of both farmers and professionals towards a quality service and the establishment of cost recovery for government services. concerns regarding privatisation of the service were also raised with respect to business drivers becoming predominant before the proper implementation of a code of practice to ensure that strong ethical values are maintained, in order to change attitudes, so that delivery of a quality animal health service to farmers could be achieved. the issue of what to privatise was also discussed. there was strong debate about whether to recommend that the vaccination service should be handled by private service or not. the issue of covering the costs of transboundary disease vaccination by the moa and difficulties with the requirement for cold chain maintenance were also discussed. it was then concluded that, although it may be difficult at the initial stage, providing a quality private service could help solve the problems and that the moa can play a role in its facilitation. discussion top ↑ disease epidemiology the north gondar administrative zone comprises highland, upland and lowland agro-ecologies. these diverse agro-ecological zones create an ideal environment for the prevalence of a variety of animal diseases. government and private sector professionals and farmers described several livestock diseases. these included major viral, bacterial, parasitic and metabolic diseases affecting cattle, sheep, goats, horses, cats and dogs. the presence of various animal diseases in the zone was also reported by tegegne et al. (2009). these diseases result in important economic losses by either directly killing animals or indirectly by reducing their productivity. status of existing service the existing service delivery under the current animal health system was considered to be unsatisfactory in both private and public sectors in terms of quality and effective widespread delivery. from personal observation of respondents, a considerable number of veterinary drugs were being administered without veterinary supervision because of remoteness of clinics or non-availability of adequate services in existing clinics. willingness to pay for a quality service was also assessed for general clinical examination, castration, caesarean section, normal delivery, both open and closed castration as well as wound management. nearly 52.2% of responding farmers were willing to pay suggested acceptable prices. the remaining respondents would not volunteer acceptable prices because they felt that services should be provided by the government or that the service deliverer should determine prices. respondents in areas of greater economic potential emphasised the value of a quality service rather than being influenced by price. the indicated acceptable prices were comparable to regional prices studied for the implementation of cost recovery under the moa in selected milk shed areas of the region (swedish international development authority 1994). options for service delivery improvement privatisation of veterinary services was preferred as a possible solution for improving animal health service delivery by respondents in the present survey. similar ideas put forward by sen and chander (2003) suggested privatisation of veterinary services as one strategy for providing an efficient veterinary service. cheneau et al. (2004) also reported on the improvement in veterinary services in developing countries as a result of privatisation. consequently, many countries in the world have started to implement or have already implemented privatisation. the availability of drugs and their use in animals are significantly higher in countries that have privatised veterinary services and drug supplies (cameroon, central african republic, côte d’ivoire, ghana, kenya, mali and senegal) (sen & chander 2003). as a consequence, many african governments are moving to privatise their veterinary services. the government of zambia is also encouraging free competition amongst veterinarians (mlangwa, chilonda & pandey 1997).despite successes in different parts of the world, privatisation is not necessarily free from problems. some of the respondents in this research suggested that the quality of the veterinary service might actually be reduced by privatisation. private practitioners may fail to adhere to norms and professional ethics. they may also concentrate in and around urban areas, leaving remote rural communities isolated (cheneau et al. 2004; sen & chander 2003). they may also focus solely on profit rather than providing effective quality services. in ethiopia, there is a free market ideology and there are policies that promote the private sector. there has been some progress in veterinary services. almost all importation and distribution of veterinary drugs is handled by private companies. any veterinary professional can obtain a license and set up in private practice. in any district, there are one or more private veterinary pharmacies and, in big towns such as gondar, there are private veterinary clinics. despite this, the process of transferring decades of a state-monopolised service into the private sector is still slow. accordingly, no progress has been made in delineating public and private responsibilities with respect to animal health service delivery and in promoting private practice. private veterinary practice has been promoted under the european union-funded parc programme. in 1993, a condition of continued funding was that this programme should show greater progress and the most recent report from the veterinary privatisation promotion office showed that compared with other countries considerable advances have indeed been made. the report also showed that between 1995 and 2001, the percentage of private clinics rose from 6.0% to 14.7% of the total and the percentage of private health posts from 1.0% to 6.5% (silkin & kasirye 2002). livestock owners do spend some money on veterinary services. the value presented in table 3 is a rough estimate, as most farmers do not keep records. however, it does indicate the willingness of farmers to pay for some veterinary services. the sustainability of a private service depends on the profitability of livestock owners. the types of production systems, livestock densities and the market price of the animals all impact upon profitability for the private practitioner (umali, feder & de haan 1992). there is resistance in the public sector to charging real costs (full cost recovery) for the services it provides. it is, however, essential to do so if the private practitioner is to be assured of an adequate income and if the economy is to grow. the government service is confronted by many problems. respondents mentioned inadequate availability of instruments, lack of awareness, shortage of budget and insufficient government attention to the sector, resulting in an inability to maintain full service provision. the government budgets have not kept pace with the increased livestock population and price of drugs. each year, no satisfactory budget has been allocated to the districts. the presence of significant constraints in the government gave rise to concerns about the efficiency and efficacy of the public sector veterinary service and the diverse demands of farmers have led to a search for alternative ways of providing the service. privatisation could be the solution to these problems (sen & chander 2003). recommendations top ↑ based on the conclusions from this study, the existing constraints for veterinary service delivery could be ameliorated using the following recommendations:• increased privatisation, with a continuing reduced but properly resourced government service. the benefits need to be demonstrated to farmers in livestock production so that there is an obvious working partnership, which can be seen to be beneficial in terms of good disease control and improved productivity through ready access to a robust service. this needs to include an outreach service for ongoing education of the farming community. • the government service delivery should cease to provide a free service and implement cost recovery programmes, so that private veterinary service can be encouraged to play its role in animal health service delivery with appropriate ethical standards. • strategies developed for privatisation of veterinary services should underline the possible pitfalls for their sustainable implementation. • policies, rules and regulations regarding privatisation of veterinary services need to be clearly established in advance. • implementation of privatisation programmes needs to be phased, monitored and demonstrated to work. • if the government prefers to continue with its commitment to the existing level of public service, then appropriate resources must be made available, including recruitment and outreach education, to ensure the expected quality of service delivery and the understanding of disease control measures by the farming community. • the privatisation process requires initial seed money for those would be private practitioners. to address this case, the saving and credit associations and the government structure must make a deal and devise a strategy to make credit available and accessible. • the non-governmental organisations working in livestock productivity should also consider supporting the privatisation procedure and associated seed money issues as one area of focus to enable privatisation to be a working platform in improving animal health and productivity. • inclusion of entrepreneurship and business management courses in the veterinary medicine curriculum needs to be considered, or else arrangements should be made to offer such courses after graduation to let the new graduates consider providing private veterinary service as an alternative job market and to ultimately survive and thrive in it. conclusion top ↑ according to this study, the range of endemic diseases and the severity of their impact on livestock production were perceived to be critical by professionals in government service and the private sector, as well as the farming community. the most important diseases that were included in the world organisation for animal health’s former list a of notifiable diseases were highlighted because of their wide distribution and frequency of occurrence: foot and mouth disease, lumpy skin disease, peste des petits ruminants, newcastle disease, african horse sickness and contagious caprine pleuropneumonia. the most frustrating disease of zoonotic importance, rabies, was also highlighted because of its occurrence in all study areas.the status of the veterinary service is in its infancy in terms of both widespread coverage and standardised service provision in both the government and private sectors. there was a view that the budget allocation was insufficient to provide quality service delivery that is accessible to the whole community. the yearly budget allocation does not match the requirements for resources to spend on trained personnel, equipment, facilities and drug price inflation, thus precluding implementation of the 5-year action plan. the attitude of officers in the government sector towards a privatised veterinary service seemed to be favourable in terms of encouraging the private sector to be involved, rather than a total service transfer from the public to private sector. a considerable proportion of professional respondents from study districts were somewhat pessimistic about the current trends in the public sector if they continue and made suggestions for service improvement. in addition, respondents also attributed constraints for the quality of service delivery from the government veterinary service to management-related constraints in resource allocation, shortage of the required tools and facilities and a lack of awareness and perception of the needs of livestock owners. private service was judged to be constrained by problems related to the subsidised government service, gaps in implementation of legal and ethical practices, absence of institutions for securing the finance required to start businesses, lack of trust by farmers in a private service, as well as an unwillingness or inability to pay for services delivered. deficiencies in access to services by farmers have also led farmers to use modern veterinary drugs as well as traditional remedies without veterinary supervision. the regional bureaus of agriculture as well as the bureau of finance and economic development have studied the issues relating to privatisation of the veterinary service. some officials maintained that the service provision should be a responsibility of the government. others have raised the point that there is no particular policy directive that would either preclude or facilitate the implementation of a private veterinary service. the only regulations related to private veterinary practice were encountered in the regional livestock agency. consequently, from the observations made to date, a slow step by step engagement with a privatised service could be a solution to the future sustainable management of the animal health service. acknowledgements top ↑ competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions h.k. (university of gondar) was the project leader who formulated the primary proposal, was involved in data collection and writing of the manuscript. a.m. (university of gondar) was instrumental in data collection and analysis. e.k. (university of gondar) was involved in data collection and editing of the final draft. references top ↑ cheneau, y., el idrissi, a.h. & ward, d., 2004, ‘an assessment of the strengths and weaknesses of current veterinary systems in the developing world’, revue scientifique et technique, office international des épizooties 23(1), 351–359.chilonda, p. & van huylenbroeck, g., 2001, ‘a conceptual framework for the economic analysis of factors influencing decision-making of small-scale farmers in animal health management’, revue scientifique et technique, office international des épizooties 20(3), 687–700. de haan, c. & bekure s., 1991, animal health services in sub-saharan africa: initial experiences with new approaches, alpan network paper 29, international livestock centre for africa, addis ababa. mlangwa, j.e.d., chilonda, p. & pandey, g.s., 1997, ‘veterinary ethics in the liberalized market: the zambian environment’, revue scientifique et technique, office international des épizooties 16, 739–745. mlangwa, j.e.d. & kisauzi, d.n., 1994, ‘systems approach to animal health services delivery in sub-saharan africa the case of privatisation’, revue scientifique et technique, office international des épizooties 13, 673–685. sen, a. & chander, m., 2003, ‘privatization of veterinary services in developing countries: a review’, tropical animal health and production 35, 223–236. http://dx.doi.org/10.1023/a:1023343429498 swedish international development authority, 1994, ‘study on cost recovery of veterinary services provided by ministry of agriculture in selected districts of amhara region’, sida, bahir dar. silkin, t. & kasirye, f., 2002, veterinary services in the horn of africa: where are we now? a review of animal health policies and institutions focusing in pastoral areas , community-based animal health and participatory epidemiology unit, pan african programme for the control of epizootics, viewed 15 april 2013, from http://sites.tufts.edu/capeipst/files/2011/03/silkin-kasirye-pdf-version.pdf sparagano, o., 1999, ‘privatisation of animal health services in the tropics’, tropical animal health and production 31, 191–192. http://dx.doi.org/10.1023/a:1005173731087 tber, a., 1995, privatization of veterinary practice: evaluation of the experience of african countries in this field, mimeo, memphis. tegegne, a., mengistie, t., desalew, t., teka, w. & dejen, e., 2009, ‘transhumance cattle production system in north gondar, amhara region, ethiopia: is it sustainable?’, working paper 14, improving productivity and market success of ethiopian farmers project, international livestock research institute, addis ababa. thrusfield, m., 2005, veterinary epidemiology, 3rd edn., blackwell, edinburgh. umali, d.l., feder, g. & de haan, c., 1992, the balance between public and private sector activities in the delivery of livestock services, world bank discussion paper 163, world bank, washington, dc. upton, m., 2004, the role of livestock in economic development and poverty reduction, pplpi working paper 10, food and agriculture organization, rome. article information authors: john d. grewar1,2 peter n. thompson2 carina w. lourens3 alan j. guthrie3 affiliations: 1western cape department of agriculture: veterinary services, elsenburg, south africa 2department of production animal studies, university of pretoria, south africa 3equine research centre, university of pretoria, south africa correspondence to: alan guthrie email: alan.guthrie@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 10 apr. 2015 accepted: 18 aug. 2015 published: 30 sept. 2015 how to cite this article: grewar, j.d., thompson, p.n., lourens, c.w. & guthrie, a.j., 2015, ‘equine encephalosis in thoroughbred foals on a south african stud farm’, onderstepoort journal of veterinary research 82(1), art. #966, 4 pages. http://dx.doi.org/10.4102/ojvr.v82i1.966 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. equine encephalosis in thoroughbred foals on a south african stud farm in this research communication... open access • abstract • introduction • research method and design    • body temperature data collection    • blood and serum collection    • virological and serological methods    • case definition    • statistical methods • results    • outbreak identification    • pre-outbreak and post-outbreak antibody levels    • test results • ethical considerations • discussion • conclusion • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ thoroughbred foal body temperature data were collected from shortly after birth until shortly after weaning during the 2007/2008 season on a stud farm in the western cape province of south africa. equine encephalosis (ee) caused by ee virus (eev) serotype 4 (eev-4) occurred in the foal group during the first autumn after their birth (march and april 2008). a descriptive study was undertaken to provide data on the eev maternal antibody status, the association between pyrexia and eev infection, and the incidence of infection amongst the foals prior to and during the episode. this included the frequent capturing of foal body temperature data and regular collection of serum and whole blood during pyretic episodes. infection by eev was determined using both virological and serological methods. a high ee incidence of at least 94% occurred amongst the foal cohort, despite the fact that 37% of foals had previously shown maternal antibody to eev-4. pyrexia in foals was not directly associated with ee infection and 41% of infected foals showed no detectable pyretic episode. information obtained from this ee episode showed the high incidence of eev infection in foals during the first autumn after their birth. monitoring foal body temperature can alert farmers to outbreaks of infectious disease, such as ee. these results are relevant to the epidemiology of ee and facilitate greater understanding of it as a differential diagnosis of african horse sickness (ahs), given that ee and ahs have similar epidemiologic profiles. introduction top ↑ equine encephalosis (ee) is an arboviral disease of equids that is transmitted by culicoides midges (paweska & venter 2004). the causative agent of ee is ee virus (eev), a member of the orbivirus genus in the reoviridae family. eev infections of horses are typically unapparent or manifest as only a mild or subclinical disease (crafford et al. 2011). clinical signs of ee can include inappetance, pyrexia, mucous membrane congestion and icterus. in some cases, however, the clinical signs may be similar to those seen in cases of african horse sickness (ahs), which is caused by the closely related ahs virus (ahsv) that is also transmitted by haematophagous culicoides midges (howell, guthrie & coetzer 2004). the viraemic period in eev-infected horses is generally brief, and horses do not act as long-term carriers of the virus (erasmus et al. 1970). the mortality rate of ee is less than 5% (howell et al. 2004). although the first definitive description of ee in south africa occurred in 1967 (erasmus et al. 1970), it is speculated that in 1910, when theiler described what he called ‘ephemeral fever’ (theiler 1910), he was describing ee (guthrie, pardini & howell 2009). seven different serotypes of eev have been identified in southern africa, and are assigned as numerical isolates, namely serotype one (bryanston), serotype two (cascara), serotype three (gamil), serotype four (kaalplaas), serotype five (kyalami), serotype six (potchefstroom) and serotype seven (e21/20) (howell et al. 2002). eev is transmitted by a number of different species of culicoides midges and there are eev-competent vector populations of these midges circulating in the western cape province (paweska & venter 2004; theodoridis et al. 1979; venter, koekemoer & paweska 2006). horse populations in south africa have a high eev seroprevalence, and all seven serotypes of eev have been prevalent within the western cape province (howell et al. 2002, 2008; paweska & venter 2004). whilst there have been prior studies on the seroprevalence of eev infection of horses in south africa, this study provides data on the eev maternal antibody status, the association between pyrexia and eev infection, and the incidence of infection amongst thoroughbred foals prior to and during a natural infection with eev. the major importance of eev infection of horses in a regional context is its similar epidemiology to that of ahsv infections; however, it is to be stressed that morbidity and mortality of ee is minimal, in marked contrast to those associated with outbreaks of ahs. research method and design top ↑ body temperature data collection temperature data from 127 thoroughbred foals in the 2007/2008 season were obtained by scanning temperature-sensitive microchips that were inserted into the nuchal ligament of each foal shortly after birth. the microchips used were destron fearing™ lifechip® with bio-thermo® technology (destron fearing, usa). after being scanned, the microchip returned a string including both the unique identifier of the microchip as well as the body temperature of that foal according to international organisation for standardisation (iso) standard 11785. microchips were inserted according to manufacturer specifications by a registered veterinarian. foals were born and resident on a thoroughbred stud in the western cape province of the republic of south africa. a single body temperature measurement was collected from each foal on each weekday from august 2007 until end of july 2008, with recordings beginning at approximately 7 am each day. recording of each foal’s temperature began on the first weekday morning after that foal was microchipped; 83% of foals had their first body temperature recorded less than 2 days after birth. a total of 34 foals were removed from the study (all for reasons unrelated to eev infection) prior to the outbreak that began in mid-march 2008; thus the final study cohort consisted of 93 foals. data collection included a total of 9556 temperature scans from the initial scan on 06 august 2007 up to and including scans on 30 april 2008. the total number of temperature scans during the estimated outbreak period of 15 march 2008–30 april 2008 was 2526 scans. blood and serum collection serum samples were collected from each foal by jugular venipuncture at monthly intervals using serum vacuum tubes (becton dickenson vacutainer™, becton dickenson, usa) from birth until after the outbreak. at least one serum sample from every foal was selected from each of the pre-outbreak and post-outbreak periods for testing. whole blood samples for virus isolation (vi) were collected by jugular venipuncture from pyretic foals into heparinised (becton dickenson vacutainer™ 102 iu lh) vacuum tubes, and nine haemolysed samples were excluded from testing. virological and serological methods virus isolation was performed as described (quan et al. 2008) with cytopathic agents identified as eev by a group-specific antigen capture enzyme linked immunosorbent assay (c-elisa) (crafford et al. 2003). eev isolates were serotyped using a serotype-specific plaque inhibition neutralisation test after the technique described (porterfield 1960) and modified (quan et al. 2008), in which electrical insulating fish-spine beads filled with type-specific antiserum were used to indicate homologous virus-antibody neutralisation of virus inoculated vero cells. serotype-specific neutralising antibodies to eev were detected by using a serum neutralisation test (snt) performed in microtitre plates using eev-serotype four (eev-4) as the challenge virus as previously described (howell et al. 2002). case definition pyrexia was determined through statistical process control and was defined as any foal body temperature greater than 39.9 °c or any foal body temperature greater than 1.96 standard deviations above the running mean of the six previous body temperature readings for that individual foal. cases of ee that occurred during march and april of 2008 were identified by either or both of the following criteria: eev isolation from whole blood samples collected from a pyretic foal during the outbreak seroconversion of foals to eev, defined as a fourfold or greater increase in antibody titre between serum samples collected prior to 15 march 2008 and after 30 april 2008. statistical methods data analysis was performed using chi-squared and fisher’s exact tests of association using ncss software (ncss [2004], kaysville, utah, usa). a p-value < 0.05 was considered significant. box plots were produced using r (r [2013], r foundation for statistical computing, vienna, austria). results top ↑ outbreak identification analysis of temperatures of foals obtained via the implanted microchips identified a cluster of pyretic episodes between the second half of march and the end of april 2008, with a total of 53/93 (57%) foals having at least one pyretic episode during this period. prior to this period there was an average of seven pyretic episodes per month from the foal cohort, and after april 2008 this dropped to an average of three episodes per month. pre-outbreak and post-outbreak antibody levels between january and march 2008, prior to the outbreak of ee, 34/93 (37%) foals had detectable levels of snt antibodies to eev-4, the titres of which were waning as foals aged (figure 1). based on the titres waning with age and the time of year during which samples were collected, it is assumed that these pre-outbreak titres were maternally derived. figure 1: boxplot of foal age-grouped equine encephalosis virus – serotype 4 antibody titres determined by a serum neutralisation test from the same cohort of foals (n = 90) sampled between january and march 2008 (before ee episode – blue box fill) and after 30 april 2008 (after ee episode – orange box fill). test results virus isolation was undertaken on blood collected from 44 of the 53 foals that experienced pyretic episodes, and eev was isolated from 37 (84%) of these foals. the 37 virus isolates were all confirmed to be eev-4 using the eev group-specific c-elisa and serotype-specific plaque inhibition tests. serum neutralisation test assays confirmed that most foals (90%; n = 84) seroconverted to eev over the outbreak period. of the nine foals that did not seroconvert to eev, three foals were not evaluated as they were removed from the study cohort during the outbreak period; six others had equivocal serological responses that did not meet the criteria for seroconversion but were still consistent with possible exposure to eev. eev was not isolated from six foals that experienced a pyretic episode and seroconverted to the virus. in total, 87/93 foals (94%) were considered to have become infected with eev-4 during the outbreak between mid-march and the end of april 2008. in contrast, only 51 eev-infected foals (59%) had a pyretic episode during this period. there was no significant association between pyrexia and eev infection (p = 0.3). re-evaluation of the body temperature data using pyrexia cut-offs at 39 °c and 39.5 °c still showed no significant association between pyrexia and ee infection (p > 0.3). ethical considerations top ↑ ethics approval of research was granted by the university of pretoria’s animal use and care committee (protocol v075/07). owner/manager consent was obtained prior to the research and was a criterion of the ethics approval. discussion top ↑ we describe the occurrence of ee amongst thoroughbred foals at a stud in the western cape province of south africa during march and april 2008. post-outbreak snt titre levels were markedly higher than the waning maternal antibody titres prior to the outbreak (figure 1), illustrating the high incidence of ee outbreak infection and subsequent seroconversion. the high incidence of infection is consistent with the results of other sero-epidemiological studies performed in south africa, where seroprevalence was found to be 77% (paweska & venter 2004) and 84% (howell et al. 2008). the high incidence amongst the foal cohort precluded determination of any specific risk factors associated with eev infection of horses on this farm. ee is described as a generally mild to subclinical infection that in some cases can manifest signs similar to ahs (howell et al. 2004). pyrexia monitoring identified the outbreak of ee but only 59% of infected foals exhibited this clinical sign of disease. there were no deaths attributed to ee during the outbreak, which is consistent with the low mortality rate described previously (howell et al. 2004). whilst this study concluded that pyrexia in foals is not directly associated with ee, it is evident that if farmers monitor foal body temperature regularly and frequently, an outbreak of ee (and potentially other infectious diseases) can be identified based on an increase in the number of foals presenting with pyrexia within a short period of time. pyrexia as a result of eev infection prompting a disease outbreak response has been described during the 2008/2009 eev outbreak amongst horses in israel (aharonson-raz et al. 2011). conclusion top ↑ this study has confirmed that eev maternal antibody may not prevent eev infection of foals, most likely because of the serotype-specific nature of maternal antibody (howell et al. 2008) as well as waning maternal antibody levels prior to the high-risk outbreak season. the fact that at least 34 of 93 foals (37%) had maternal antibodies indicates that their dams were previously exposed to eev-4. the risk period for arbovirus infection in thoroughbred foals, particularly those diseases transmitted by culicoides spp. like ahs and ee, is in the first autumn following their birth. this susceptibility reflects not only the necessary environmental conditions that provide for peak populations of the insect vector, but also the set management principle in the thoroughbred breeding industry in the southern hemisphere of having foals born from august through november. acknowledgments top ↑ the authors wish to thank craig carey, dr jim antrobus and the rest of the management and labour team of arc-en-ciel stud farm for their invaluable help. thanks also to the equine research centre and veterinary genetics laboratory staff, in particular dr marc jukel and dr melvyn quan, for their help with the project. the authors also are grateful to dr jim maclachlan for his review of and contributions to this manuscript. funding for this study was generously supplied by the equine research centre, faculty of veterinary science, university of pretoria, south africa. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions j.d.g. (western cape department of agriculture) performed the research under the supervision of and wrote the manuscript with input from a.j.g. (university of pretoria) and p.n.t. (university of pretoria). c.w.l. (university of pretoria) performed the serological laboratory analysis and provided technical information on the serum neutralisation assay methods and interpretation. a.j.g. planned the overarching project on which this research was based. p.n.t. and j.d.g. provided statistical analysis and interpretation of applicable results. references top ↑ aharonson-raz, k., steinman, a., bumbarov, v., maan, s., maan, n.s., nomikou, k. et al., 2011, ‘isolation and phylogenetic grouping of equine encephalosis virus in israel’, emerging infectious diseases 17(10), 1883–1886. pmid: 22000361, http://dx.doi.org/10.3201/eid1710.110350 crafford, j.e., guthrie, a.j., van vuuren, m., mertens, p.p.c., burroughs, j.n., howell, p.g. et al., 2003, ‘a group-specific, indirect sandwich elisa for the detection of equine encephalosis virus antigen’, journal of virological methods 112, 129–135. pmid: 12951221, http://dx.doi.org/10.1016/s0166-0934(03)00202-7 crafford, j.e., guthrie, a.j., van vuuren, m., mertens, p.p.c., burroughs, j.n., howell, p.g. et al., 2011, ‘a competitive elisa for the detection of group-specific antibody to equine encephalosis virus’, journal of virological methods 174, 60–64. pmid: 21458496, http://dx.doi.org/10.1016/j.jviromet.2011.03.024 erasmus, b.j., adelaar, t.f., smit, j.d., lecatsas, g. & toms, t., 1970, ‘the isolation and characterization of equine encephalosis virus’, bulletin de l’office international des épizooties 74, 781–789. guthrie, a.j., pardini, a.d. & howell, p.g., 2009, ‘equine encephalosis’, in t.s. mair & r.e. hutchinson (eds.), infectious diseases of the horse, pp. 132–137, equine veterinary journal ltd, cambridgeshire. howell, p.g., groenewald, d., visagie, c.w., bosman, a.m., coetzer, j.a.w. & guthrie, a.j., 2002, ‘the classification of seven serotypes of equine encephalosis virus and the prevalence of homologous antibody in horses in south africa’, onderstepoort journal of veterinary research 69, 79–93. pmid: 12092781. howell, p.g., guthrie, a.j. & coetzer, j.a.w., 2004, ‘equine encephalosis’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, 2nd edn., vol. 2, pp. 1247–1251, oxford university press, oxford. howell, p.g., nurton, j.p., nel, d., lourens, c.w. & guthrie, a.j., 2008, ‘prevalence of serotype specific antibody to equine encephalosis virus in thoroughbred yearlings in south africa (1999–2004)’, onderstepoort journal of veterinary research 75, 153–161. pmid: 18788209, http://dx.doi.org/10.4102/ojvr.v75i2.14 paweska, j.t. & venter, g.j., 2004, ‘vector competence of culicoides species and the seroprevalence of homologous neutralizing antibody in horses for six serotypes of equine encephalosis virus (eev) in south africa’, medical and veterinary entomology 18, 398–407. porterfield, j., 1960, ‘a simple plaque-inhibition test for the study of arthropod-borne viruses’, bulletin of the world health organisation 22, 373–380. pmid: 14434290 quan, m., van vuuren, m., howell, p.g., groenewald, d. & guthrie, a.j., 2008, ‘molecular epidemiology of the african horse sickness virus s10 gene’, journal of general virology 89, 1159–1168. pmid: 18420793, http://dx.doi.org/10.1099/vir.0.83502-0 theiler, a., 1910, ‘notes on a fever in horses simulating horse-sickness’, transvaal agricultural journal 8(32), 581–586. theodoridis, a., nevill, e.m., els, h.j. & boshoff, s.t., 1979, ‘viruses isolated from culicoides midges in south africa during unsuccessful attempts to isolate bovine ephemeral fever virus’, onderstepoort journal of veterinary research 46(4), 191–198. pmid: 551368 venter, g.j., koekemoer, j.j.o. & paweska, j.t., 2006, ‘investigations on outbreaks of african horse sickness in the surveillance zone in south africa’, revue scientifique et technique, office international des épizooties 25, 1097–1109. pmid: 17361773 hulman_147-153.indd introduction the southern african development community (sadc) was established in 1980 as a regional economic community now comprising 15 member states (including the republic of seychelles) in the southern african region with headquarters in ga borone, botswana. the sadc vision is one of a common future, a future in a regional community that will ensure economic well-being, improvement of standards of living and quality of life, freedom and social justice, peace and security for the peoples of southern africa. this shared vision is anchored on the common values, principles and historical and cultural affinities that exist between the peoples of southern africa. as from 2001, sadc has embarked on a course to deepen regional integration through restructuring, a process which involves mainly reforms of institutions and management systems. under this new structure, sadc has centralised the coordination of 147 onderstepoort journal of veterinary research, 76:147–153 (2009) livestock policy and trade issues in sadc b. hulman senior programme manager, sadc secretariat, p. box 0095, gaborone, botswana abstract hulman, b. 2009. livestock policy and trade issues in sadc. onderstepoort journal of veterinary research, 76:147–153 as from 2001, the southern african development community (sadc) has embarked on a course to deepen regional integration through restructuring. under the new structure sadc has centralised the coordination of its activities to the secretariat in gaborone. the former sector coordinating units have been merged into four directorates, one of which is the food, agriculture and natural resources (fanr) directorate, which comprises, amongst others, the livestock development unit (ldu). the ldu, under the aegis of the fanr, formulates policies for regional livestock development in order to respond to the objectives of the regional indicative strategic development plan (risdp), and which are mainly to: • contribute to improved food security. • promote wealth creation. • enhance rural livelihood. • enhance livestock as a tradable and consumable commodity. following the launch of the sadc economic partnership agreement (epa) negotiations, the eight sadc epa member states identified sanitary and phytosanitary and technical barriers to trade to be major trade barriers for access to international markets, especially the eu market where standards are normally set beyond international standards. sadc has already brought some of the issues related to beef exports to the oie regional commission for africa as sadc member states feel that a few of the present requirements do not have a scientific basis. the paper discusses the process that the ldu follows in the formulation of policies and strategies in regional livestock development with the objective of bolstering intra and extra regional trade in livestock and livestock products. 148 livestock policy and trade issues in sadc its activities to the secretariat in gaborone. cen tralisation has meant merging the former 21 sectors into four directorates, one of which is the directorate of food, agriculture and natural resources (fanr), which includes the livestock development unit (ldu). the role of the ldu is to contribute to the promotion of regional integration and sustainable livestock production through the coordination of animal disease control strategies, human resource development, marketing and utilisation of livestock products in order to create employment, improve the standard of living of the people and to meet regional food security objectives. the ldu works with member states through the live stock technical committee and its four subcommittees, which are task forces meeting regularly to consult member states on issues related to: • epidemiology of animal diseases • diagnostics and testing facilities including development of quality assurance and harmonization of standard operating procedures • productivity of farm animals and natural resources (rangelands) and production and marketing of processed products of animal origin • ensuring that products of livestock origin conform to food safety standards the recommendations of the sub-committees form the basis of policy formulations for regional livestock development which are enshrined in the objectives of the regional indicative strategic develop ment plan (risdp). the risdp has the following priorities under the intervention area of sustainable food security: • ensuring food security • ensuring access to food • improving safety and nutritional value of food • ensuring disaster preparedness for food security • ensuring equitable and sustainable use of the environment and natural resources • strengthening institutional frameworks and capacity building. the risdp will provide sadc member states, including sadc institutions, with a coherent and comprehensive development agenda on social and economic policies for the next 15 years. policy issues livestock plays an important role in the livelihoods of the farmers of the region as it contributes to more than 30 % of the agricultural gdp. seventy percent of the livestock farmers are smallholders and it is be fitting that during policy formulations special atten tion be devoted to the problems faced by the smallholder farming community. a workshop organised by sadc in july 2000 provided an opportunity for key stakeholders to deliberate and reach consensus on priority issues for regional integration and protocol development in the sadc sectors of crops, livestock and agricultural research and training. the expected output was to clearly identify issues where integration is needed, articulate why this was an area for integration, i.e. what are the objectives to be achieved through integration; and formulate strategies, mechanisms and structures that could be put in place to implement integration. the priority issues for regional integration in the livestock sector were, and still are, deemed to be: • the control of transboundary animal diseases and disease vectors • quality assurance of veterinary drugs, vaccines and animal feed ingredients, imported from outside the sadc region as well as those produced within the region • sanitary standards and regulations for animals and their products, including risk assessment • optimal use of animal genetic resources in the region • information exchange on aspects relating to the above with special reference to “best practice” technology, and to early warning systems • capacity development to implement policies and strategies in the livestock sector • research that is specifically oriented towards problem solving. the workshop came up with a set of guidelines for protocol development and issues for policy formulations in livestock with the overall objective to achieve regional integration, and sustainable use of animal resources. member states undertook to assist in the development of the protocols and strategies for the regional development of the livestock sector. the overall objective would be achieved by addressing the following issues. farm animal genetic resources (fangr) ensure regional commitment to good management practices, conservation of and sustainable use of 149 b. hulman fangr to ensure their long-term availability and accessibility in order to optimise benefits for all users of fangr in sadc member states: • develop and harmonise common policies. • share information on the availability, use and characteristics of fangr in the region and beyond. • create a user-friendly and accessible regional database. • characterise fangr. livestock census the objective is to develop sustainable management and utilisation of natural resources and to generate an essential planning tool. as a strategy it would be useful to undertake annual censuses by using the most cost-effective method. the data will assist to establish a regional database. livestock identification it is expected that livestock identification will help to achieve better security against cross-border stock theft and will ensure better disease surveillance monitoring. with the need nowadays to comply with food safety requirements it has become essential to develop and promote national legislations for animal identification, especially for trade. animal feeds introduce and maintain regional statutory controls for the production and use of animal feeds and develop regional standards for quality assurance. in order to achieve this, it is essential to establish: • minimum standards for premises, operational procedures, hygiene, storage and transport • independent statutory quality control bodies at national level • registration and licensing protocols. transboundary disease control the objective is to facilitate the co-ordination and control of transboundary animal diseases (tads) through the development of the following strategies: • harmonise disease control strategies in accordance with the oie recommended guidelines. • share information and establish an early warning information system. • strengthen collaboration between the sadc livestock sector and international organisations such as au-ibar, fao, oie, iaea, afdb, undp, etc. • facilitate the establishment of national epidemiological units, databases and information exchange. registration of veterinary biologicals and medicinal products there is a need to regulate the importation, distribution and use of veterinary biologicals and medicinal products and for this it is essential to: • create or strengthen national databases on biologicals and medicinal products. • establish national registration and licensing bodies. • identify regional institutions with the capacity to produce veterinary biologicals and drugs. • identify a regional body to assist with the registration of veterinary biologicals and drugs. information management system the development of a standardised and harmonised livestock and animal health information management system for the region was also felt to be a priority so as to facilitate timeous risk assessment, especially for trading purposes. for this, it is necessary to identify suitable software and hardware for regional use and undertake regional training on information management systems and risk assessment. quality assurance and standardisation standardise and improve diagnostic capabilities of veterinary laboratories in the region by the following strategies: • harmonise the quality assurance procedures of member states. • identify independent quality assurance auditors. • establish a regional accreditation system for diagnostic laboratories. abattoir hygiene standards and meat inspection establishment of minimum standards for abattoirs and meat processing facilities for the region through the development of the following strategies: • set and harmonise minimum training standards for meat inspection personnel. 150 livestock policy and trade issues in sadc • set and harmonise minimum standards for abattoirs and meat inspection facilities. • establish and harmonise operational procedures for abattoirs and processing plants for trade in the region. hygiene standards for milk and dairy processing and manufacturing establish, set and harmonise minimum standards for milk processing and dairy manufacturing plants bearing in mind the existing international standards. private sector engagement create an enabling policy environment for the development of a vibrant private sector in the livestock industry by: • promoting the commissioning of core function analysis studies by member states • promoting the commercialisation of government services, where possible, as a first step towards privatisation such as artificial insemination, feed production and vaccine production • encouraging smart partnerships between the government, private sector and the farming community • providing specific support to enhance competition and accountability in the private sector. import and export of animals and animal products the overall objective is to promote intra and extra regional trade in animals and animal products in the sadc countries and for this to be achievable there is need to: • establish port health authorities at national level. • harmonise import requirements into the sadc region. • harmonise import/export certification system to facilitate trade in the region. • build national and regional capacity to ensure compliance. • harmonise import/export certification for movement of companion animals and racehorses. animal welfare with the aim to promote humane handling, use and transportation of animals, it is essential to develop a code of conduct for the use, handling and slaughter of animals and to promote civic education on animal welfare. addressing the policy issues the importance of agriculture for the region cannot be overemphasised. regardless of their economic base, there is overwhelming evidence that all economies in sadc tend to be driven by agricultural conditions when it comes to growth rate, stability, control of inflation, particularly food prices, and overall reduction of poverty. the key principles underlying regional integration are: • that each country takes the regional dimension into account, and should therefore be prepared to accept compromises and trade-offs. • the principle of balance equity and mutual benefit agreed upon continues. since it recognises the significant physical, economic and social disparities that exist among the sadc countries. • the free movement of factors of production (capital, labour, goods and services) is central to economic integration. • the facilitation and promotion of investments and trade in goods and services are at the core of the integration process. • the twin pillars of real political commitment by governments and the effective participation by stakeholders are fundamental to the process. the above have served as guidelines for the formulation of certain policy issues in the livestock sector. the fanr directorate has recently embarked on the formulation of a regional agricultural policy, which will include a livestock development component, to further assist in the consolidation of regional integration. the ldu of the fanr directorate of the sadc secretariat has implemented projects and programmes which have assisted member states to develop strategies which can result in enhancing production and productivity in the livestock sector. the sadc farm animal genetic resources project has enabled sadc countries to characterise and evaluate the production potential of indigenous livestock breeds as well as the need to conserve and utilise these invaluable animal resources which are well adapted to the environment prevailing in the region. the outcome of this first intervention should now be extended to the farming community so that there can be a community-based approach to the conservation and utilisation of farm animal genetic 151 b. hulman resources, with special reference to indigenous breeds. with the restructuring of sadc, this issue which was kept in abeyance has come to the forefront and will be taken on board through the regional agricultural policy. the promotion of integration in the livestock sector is another project which has addressed the issues of information management in the livestock sector. the core objective of this project is to develop a livestock information management system (lims) for livestock in the region. lims has developed modules to receive data on animal health, animal production, marketing and trade, and to analyse these data so as to generate reports for dissemination in member states in order to enable decision makers to take informed decision on livestock development in the region. the project has also provided opportunities to foster public/private partnerships which are essential to further develop the livestock sector. the project has an important component of capacity building in the fields of animal health, animal production and marketing with the collaboration of regional training institutions. sadc secretariat is implementing another project which involves strengthening of institutions, such as veterinary services and veterinary laboratories, for the risk management of tads in the sadc region. the overall objective of this project is to make livestock a tradable commodity and to enhance the credibility of the veterinary services of the participating member states. the sadc foot-and-mouth disease (fmd) project is addressing issues related to the control of fmd in three sadc member states, but the results will be shared with all the member states. the sadc fmd and sadc tads projects are expected to establish the southern african commission for the control of tads (sacct) with inputs from international organisations such as fao and oie so that the region can develop long term solutions for the control of tads. another sadc project, viz. food safety—capacity building on residue control, is expected to have the following outcomes: • food safety regulatory frameworks at national and sadc regional level strengthened • regulatory framework for harmonized registration and quality control of livestock protection products and veterinary drugs at national and sadc regional level strengthened • analytical capability of national and regional laboratories strengthened and assistance towards accreditation provided • coordination and communication between national and regional authorities involved in the regulatory frameworks on sanitary and phytosanitary (sps) relevant issues enhanced • a network of sps subject matter specialists established and public awareness on, and knowledge of sps issues in the sadc region, improved. sadc fully endorses the recommendations of the oie conference on veterinary medicinal products in africa held in dakar and recommends that the above project implements the activities as per its work plan. sadc will endeavour to establish or identify a centre of excellence for the registration, quality control and safe disposal of veterinary drugs, biologicals and chemicals, with a view to harmonisation and regional recognition. livestock trade issues in the sadc sadc is working in close collaboration with the codex alimentarius commission and the oie in the development of guidelines to help member states to formulate national and regional norms and standards with regards to food of animal origin. the print livestock project has recently launched two regional studies; a first one on “value added information management systems (vaims)” and the second one on “zoning, animal identification and traceability study (zaits)”, which will be able to focus on commodity-based approaches and improve the qualitative and quantitative assessment of value chains in order to foster trade. the commodity and value chain focus to regional and international trade in livestock and livestock products is recognised by sadc as an option for livestock development. sadc has recommended that member states promote debate on this approach to foster its adoption by relevant standard setting bodies, such as the oie. sadc welcomes the oie debate on compartmentalisation in relation to fmd control and trade in beef and is confident that the recommendations will bolster trade. sadc has finished the revision of the sps annexes to the sadc trade protocol and reaffirms the oie standards in the trading of livestock products. sadc advocates the promotion of improved marketing of live stock products through a commodity approach which includes attainment of international food safety standards. in this respect 152 livestock policy and trade issues in sadc particular emphasis should be devoted to facilitating better access to international markets for de-boned and processed beef. the objectives of the sadc trade protocol signed in august 1996 lay down the foundation for regional trade development and access to regional as well as to international markets. the objectives are to: • further liberalise intra-regional trade in goods and services on the basis of fair, mutually equitable and beneficial trade arrangements, complemented by protocols in other areas. • ensure efficient production within sadc reflecting the current and dynamic comparative advantages of its members. • contribute towards the improvement of the climate for domestic, cross-border and foreign investments. • enhance the economic development, diversification and industrialisation of the region • establish a free trade area in the sadc re gion. the free trade area (fta) was established during the recent summit of heads of sadc member states held in august, 2008. twelve member states are establishing the fta under the protocol on trade. in the near future, angola, the democratic republic of congo and the seychelles are set to join. sadc is also negotiating an economic partnership agreement (epa) with the european com mis sion. the main objective of the epa is to consolidate the regional integration process and to foster growth and development in the region, as well as to establish a new and wto-compliant legal basis for governing bi-regional trade relations. it is expected that the full epa will be concluded by 31 december 2008 in order to consolidate even further the regional and development dimension of the epa. there is no secret, the ‘globalisation’ which implies that from now onwards to the year 2020, most (if not all) the economies will have to converge towards total market liberalisation. this ‘globalisation’ is an irreversible process which is already confirmed at the level of the relations between the european union and the acp countries, with epa that are being discussed. the acp economies must use the epa as an opportunity to enhance business competitiveness that will contribute to economic growth. the acp countries must regard the epa as another opportunity to modernise their economies to face the irreversible globalisation process. finally, sadc in collaboration and synergy with member states is gradually improving the establishment of its regional agricultural information management system (aims) to provide quality analysis of data collected in member states on the major agricultural and the livestock sector’ trends, using recognized methods. sadc has submitted the following for consideration by the oie regional commission for africa and the oie scientific commission so as to allow increased trade in beef between beef exporting sadc member states and the european union: • the 30 months age limit of bovines specified for the safety of de-boned muscle meat for freedom from the bse agent be reconsidered by the oie scientific commission and be removed as this age limit is not based on science. • the necessity for extremely costly and logistically difficult surveillance to be conducted when a satisfactory risk assessment has shown that the risk of bse being present in a country is negligible (e.g. through the use of the geographical bse risk [gbr] assessment method), to be reconsidered by the oie. • the oie scientific commission reconsiders the freedom from fmd of de-boned beef derived from animals that have undergone ante and post mortem inspection, the carcass matured at +2 and 4 °c for a minimum of 24 h to ensure that the ph of the meat is 6 and below, and the removal of the major lymph nodes. • sadc recognises the threat to transboundary animal disease control posed by the creation of transfrontier conservation areas (tfca). however, the tfca movement is a reality and ways need to be found to accommodate the conflicting objectives of more effective tads control on the one hand and the tfca movement on the other. references comesa 2006. report on the state of integration in comesa. 11th summit of the comesa heads of state and government, djibouti, 16 november 2006. meite, v. 2008. competitiveness enhancement of acp business in the epa context. sunday standard, botswana. eaf-info@ cde.int sadc 2000. proceedings of a community building workshop on crops, livestock production and animal disease control, food security, and research and training, gaborone, 3–5 july 2000. gaborone, botswana: sadc secretariat. sadc 2004. protocol on trade. gaborone, botswana: sadc sec retariat. sadc policy briefing. 2008. transboundary animal diseases and market access: future options for the beef industry in southern africa, 7 and 8 april 2008, pretoria, south africa. brighton: institute of development studies. 153 b. hulman sadc 2008. sadc handbook on free trade area, growth development and wealth creation. gaborone, botswana: sadc secretariat thomson, g.r. et. al. 2004. international trade in livestock and livestock products: the need for a commodity-based approached. the veterinary record, 2 october 2004. thomson, g.r. 2008. a short overview of regional positions on foot and mouth disease control in southern africa in transboundary animal diseases and market access: future options for the beef industry in southern africa. working paper 2, brighton: institute of development studies. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: zuhura i. kimera1 robinson h. mdegela2 consolatha j.n. mhaiki3 esron d. karimuribo2 faith mabiki4 hezron e. nonga2 james mwesongo4 affiliations: 1department of fisheries development, ministry of livestock and fisheries development, tanzania 2department of veterinary medicine and public health, sokoine university of agriculture, tanzania 3department of soil science, sokoine university of agriculture, tanzania 4department of physical science, sokoine university of agriculture, tanzania correspondence to: hezron nonga email: nongahezron@yahoo.co.uk postal address: po box 3021, morogoro 67125, tanzania dates: received: 24 nov. 2014 accepted: 27 aug. 2015 published: 27 nov. 2015 how to cite this article: kimera, z.i., mdegela, r.h., mhaiki, c.j.n., karimuribo, e.d., mabiki, f., nonga, h.e. et al., 2015, ‘determination of oxytetracycline residues in cattle meat marketed in the kilosa district, tanzania’, onderstepoort journal of veterinary research 82(1), art. #911, 5 pages. http://dx.doi.org/10.4102/ojvr.v82i1.911 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. determination of oxytetracycline residues in cattle meat marketed in the kilosa district, tanzania in this research communication... open access • abstract • introduction • materials and methods    • sample collection    • sample analysis    • control samples    • data analysis • ethical considerations • results    • control samples • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ oxytetracycline is used to treat various diseases in cattle. however, its use may be associated with unacceptable residue levels in food. oxytetracycline residues in tissues from indigenous cattle were determined in a cross-sectional study conducted in the kilosa district, tanzania, between november 2012 and april 2013. a total of 60 tissue samples, including muscle, liver and kidney, were collected from slaughterhouses and butchers and analysed for oxytetracycline using high-performance liquid chromatography. oxytetracycline residues were found in 71.1% of the samples, of which 68.3% were above acceptable regulatory levels. the mean concentration of oxytetracycline across tissues was 3401.1 µg/kg ± 879.3 µg/kg; concentrations in muscle, liver and kidney were 2604.1 µg/kg ± 703.7 µg/kg, 3434.4 µg/kg ± 606.4 µg/kg and 3533.1 µg/kg ± 803.6 µg/kg, respectively. high levels of oxytetracycline residue in meat from indigenous cattle may pose a health threat to consumers in kilosa. the findings possibly reflect a general lack of implementation of recommended withdrawal periods, ignorance about drug use and lack of extension services. strict regulation of the use of antimicrobial drugs in the livestock industry and associated testing of animal-derived food sources prior to marketing are required. introduction top ↑ in tanzania, livestock farming generally follows traditional practices whereby pastoralists and agro-pastoralists rear mostly local breeds for meat and milk production (ministry of livestock and fisheries development 2010). livestock farming faces several constraints, including diseases, poor genetic potential of animals, poor management and nutrition, and drought (mellau, nonga & karimuribo 2010). owing to limited extension services and poor animal health delivery systems, farmers buy veterinary drugs from veterinary shops and treat their livestock themselves. however, when drugs are administered by non-professionals, correct dosages and withdrawal periods are unlikely to be observed, which poses a potential hazard to human health (barton 2000). the possible effects include toxic or allergic reactions, development of bacterial resistance and disturbance of normal intestinal microflora composition (abbasi et al. 2011; pena et al. 2005; uekane, neto & gomes 2011). the use of antimicrobial agents in food-producing animals has become a notable public health concern, especially in developing countries where such drugs are administered indiscriminately (bedada & zewde 2012; muriuki et al. 2001; olatoye & basiru 2013). the increased use of antimicrobials in animal production is due to their being applied both therapeutically and prophylactically; some are also routinely added to animal feeds at sub-therapeutic levels for growth promotion (bedada & zewde 2012; nonga et al. 2010). oxytetracycline is one of the most commonly used antibiotics in livestock production in tanzania and other african countries (katakweba et al. 2012; olufemi & agboola 2009). apart from being a broad-spectrum antibiotic, oxytetracycline is also cheap, readily available from veterinary shops and accessed easily, without restrictions, by farmers (nonga et al. 2009; olatoye & basiru 2013). katakweba et al. (2012) reported that a number of drugs, such as oxytetracycline, are used abusively to treat and protect cattle against various diseases. moreover, informal vendors are often seen selling oxytetracycline and other tetracycline-based drugs at informal markets and along the road, without any prescription being required or restrictions imposed (bedada & zewde 2012; karimuribo et al. 2013). to protect humans from harmful effects of veterinary drug residues in animal-derived food sources, the united nations food and agriculture organization (fao) and the world health organization (who) have set standards for maximum residue limits in foods. these limits apply to the parent drug or chemical and its metabolites that may accumulate and be deposited or stored within the cells, tissues or organs following administration of the compound. the acceptable maximum residue limits for tetracycline-based compounds, including chlortetracycline and oxytetracycline, are set at 200 µg/kg, 600 µg/kg and 1200 µg/kg for cattle-derived muscle, liver and kidney, respectively. withdrawal periods of 5–20 days are recommended before slaughter, depending on the species and the nature of the food products (blanchflower et al. 1997). however, regulatory bodies in tanzania have not yet set withdrawal periods for veterinary drugs and farmers rely only on the directions given in the package insert. this information is always written in english, a language the majority of tanzanian farmers do not understand. therefore, farmers rarely comply with the recommendations and usage is also not monitored by the responsible regulatory authorities. consequently, veterinary drug residues are likely to be present in food of animal origin. this study sought to investigate the presence of excessive concentrations of oxytetracycline residues in cattle meat marketed in the kilosa district, tanzania. materials and methods top ↑ sample collection this study was conducted in the kilosa district (5°55’–7°53's, 36°30’–37°30'e), which is located approximately 300 km west of dar es salaam in east central tanzania. the district is divided into three zones, namely kilosa, gairo and mikumi, and spans a land area of 19 056 km2. the human population of the district is documented as 438 175 (national bureau of statistics 2012). samples were obtained from cattle slaughtered at the kilosa, gairo and mikumi slaughter slabs and at the parakuyo and chakwale livestock markets. records obtained during sampling indicated that the animals originated from ten different villages across the district. animals were selected for sampling using simple random sampling techniques. information such as the name of the owner or supplier, the village of origin, any pathological lesions at the time of sampling and inspection status prior to slaughter was collected before sampling. three samples (muscle, liver and kidney) of 100 g – 200 g each were obtained from each of 20 animals, yielding 60 tissue samples in total. all samples were collected in separate polythene bags and transported on ice to the analytical laboratory at the sokoine university of agriculture (faculty of veterinary medicine). the samples were stored in a freezer at −20 °c for approximately 1 week and thawed at room temperature for eight hours before analysis. sample analysis high-performance liquid chromatography (hplc) was used to analyse the samples. all the reagents and chemicals were of hplc or analytical grade. reagents included oxytetracycline standard (sigma, st louis), oxalic acid dihydrate, citric acid monohydrate and disodium ethylenediaminetetraacetate (na2edta) (techno pharmachem, india), anhydrous disodium hydrogen phosphate (carlo ebra, milan), methanol, acetonitrile and hplc water (carlo ebra, milan). whatman membranes, microsyringe membrane filters (chromafil ca 20/25s), nylon membranes (p/n 0235-0301) and cronus c-18 solid-phase extraction cartridges (200 mg/3 ml, labhut) were used for the chromatography steps. each tissue sample (5 g) was homogenised three times in a mcllvaine buffer–edta solution (20 ml, 20 ml and 10 ml) and collected in a 50-ml polypropylene centrifuge tube. the mixture was then centrifuged at 4000 g for 10 min and the supernatant was filtered through a single whatman filter, pre-moistened with 2 ml mcllvaine buffer–edta solution, into a 250-ml sidearm flask. the solid-phase extraction was conducted by conditioning the extraction cartridge with 20 ml methanol followed by 20 ml hplc-grade water. the final sample extract was applied to an 18-carbon cartridge, which was subsequently washed with 20 ml hplc-grade water. oxytetracycline was eluted with 6 ml methanolic oxalic acid solution into a 10-ml volumetric flask, which was then filled with water to volume. muscle, liver and kidney sample extracts were analysed for oxytetracycline residues according to aoac official method 995.09 (aoac international 2000), with some modifications. the hplc instrument (shimadzu 20ad) was fitted with an autosampler (sil-20 aht) and a uv detector at 350 nm was used for analysis. a reversed-phase 18-carbon column (150 mm × 4.60 mm; particle size, 5 µm; supelco) was used at 25 °c for separation. the sample injection volume was 1 µl at a flow rate of 0.8 ml/min. a low-pressure gradient system, consisting of water and methanol, acetonitrile and aqueous oxalic acid (10:30:60) as the mobile phase, was applied for a retention time of 15 min. to determine residues in the samples, they were analysed concurrently with the oxtetracycline standard solutions (0.05 µg/ml, 0.1 µg/ml, 0.25 µg/ml, 0.5 µg/ml and 1.0 µg/ml). the extract from each sample was injected in duplicate to obtain an average peak height of positive samples. samples were considered positive for oxytetracycline residue if their retention time and peak corresponded to that of the reference standards. the retention time of the reference standard was 4.3 min. oxytetracycline residues in tissue sample extracts were quantified against the aforementioned concentrations of the oxytetracycline reference standards. the standards were analysed in duplicate and the peak areas appropriate to specific standard concentrations were measured. these were used to calculate the residue concentrations in sample extracts. control samples as it was not possible to obtain cattle that had not been treated with oxytetracycline or other veterinary drugs prior to slaughter, three oxytetracycline-free guinea pigs (all of the same age, sex and weight) raised at the faculty of veterinary medicine, sokoine university of agriculture, were used as controls. one guinea pig served as negative control, whereas the other two were injected with 20% oxytetracycline (laprovet, indre-et-loire) at 10 mg/kg body weight and 20 mg/kg body weight, respectively. after 24 h the three control animals were placed in a gas chamber (61.4% co2, 20.3% o2 and 18.29% n2) for 5 min before being humanely killed. muscle, liver and kidney tissue samples (5 g each) were subjected separately to the extraction, clean-up and elution procedures as described for the test samples derived from cattle. two samples of each of the three tissue types were taken from each of the control animals. the control samples were run through the hplc column under similar conditions to the oxytetracycline standard solutions. a blank sample eluted from the solid-phase extraction cartridge was included to check for the analytical column efficiency during extraction. operational conditions of the hplc instrument were tested to ensure the robustness of the method. these checks included varying the percentage of solvents in the mobile phase (methanol: 10% – 30%; acetonitrile: 10% – 30%; oxalic acid: 50% – 70%), the column temperature (from 25 °c to 40 °c)and the flow rate (from 0.6 ml/min to 1 ml/min). the ph of the buffer was changed from 2.0 to 7.0. stability of the sample and standard solutions at room temperature was also tested. data analysis the data were analysed using epi info (version 7) (centre for disease control, atlanta, usa). the chi-square statistic and confidence intervals were used to compare proportions; a probability of p < 0.5 was considered statistically significant. descriptive statistics were used to compute means, standard deviations and range. analysis of variance (anova) was used to compare differences in means of continuous variables. ethical considerations top ↑ permission for this study was granted by the executive directors of the kilosa district council and ethical approval for the study was obtained from the ethical committee of the sokoine university of agriculture. the university issued a research permit letter on behalf of the tanzanian commission for science and technology. results top ↑ control samples the non-spiked samples from the control animals peaked at a different time from that of the analytical standards, whereas both oxytetracycline-spiked samples peaked at 4.3 min as expected. the higher concentration of oxytetracycline was associated with a higher peak. the peak for extracted oxytetracycline was also detected at 4.3 min and was of similar height to the spiked control sample. between 79% and 83% oxytetracycline was recovered from the samples. the limit of detection and the limit of quantification were 1.936 mg/kg and 6.7 mg/kg, respectively. the correlation coefficients associated with the linear regression for the analytical oxytetracycline standard (figure 1) and test samples (figure 2) were r2 = 0.92 and r2 = 0.94, respectively. (the linear regression equation is shown in figure 2). for quantification purposes, the best-fit line was expected to be 99%, but based on the local environment, nature of the equipment and the laboratory used, the obtained fits were considered adequate. figure 1: combined chromatogram of the negative control sample (without oxytetracycline) and the sample spiked with 10 mg/kg 20% oxytetracycline. figure 2: calibration curve depicting the best-fit line from analytical oxytetracycline standards. of the 20 animals sampled, 17 (85%) tested positive for oxytetracycline residues. moreover, 68.3% of the positive tissue samples contained oxytetracycline residues above the acceptable levels for muscle, liver and kidney (food and agriculture organization/world health organization 2014). the mean concentration of oxytetracycline residues across all tissues was 3401.1 µg/kg ± 879.3 µg/kg. for the respective tissue types, the mean concentrations were 2604.1 µg/kg ± 703.7 µg/kg for muscle, 3434.4 µg/kg ± 606.4 µg/kg for liver and 3533.1 µg/kg ± 803.6 µg/kg for kidney tissue. oxytetracycline concentrations were higher than the acceptable levels in all tissue types sampled from animals from the kilosa and mikumi zones (table 1). lower concentrations of oxytetracycline were found in samples collected from cattle slaughtered in the gairo zone (table 1). table 1: oxytetracycline concentration in cattle tissue from the kilosa district, tanzania. discussion top ↑ the purpose of this study was to determine the presence of oxytetracycline residues in beef from indigenous cattle in the kilosa district, tanzania. the results showed a high residual presence of oxytetracycline (71.1%), with a notable number of positive samples being above the acceptable maximum residue levels recommended for meat by the who and fao. the finding can probably be attributed to widespread use of oxytetracycline for treatment and prevention of cattle diseases and is possibly exacerbated by failure to observe withdrawal periods (karimuribo et al. 2013). higher levels of oxytetracycline residue were found in liver and kidney tissue than in muscle, which can be attributed to their being organs of metabolism and excretion and therefore they are at greater risk of exposure to residues (olatoye & ehinmowo 2010). after administration, oxytetracycline enters all tissues and body fluids, but higher concentrations are found in the kidney, liver, bile, lungs and bones (aiello & moses 2010). oxytetracycline is excreted mainly via urine and bile, which explains the high concentrations of residue observed in kidney and liver tissues in this study. as liver and kidney are considered luscious offal from cattle and are popular amongst most meat consumers in tanzania, the detection of high levels of oxytetracycline residues in these tissues is of importance to public health. the proportions of oxytetracycline-positive samples found in this study were higher than reported in other studies (olufemi & agboola 2009), although bedada and zewde (2012) reported a comparable proportion of oxytetracycline-positive muscle tissue samples (71.3%; n = 384) from cattle in ethiopia. however, in a study analysing muscle tissue from cattle in the morogoro and dodoma municipalities, tanzania, only 41.2% of samples tested positive for oxytetracycline residues (mmbando 2004). oxytetracycline residues in muscle tissue were reported in 45.6% and 54.4% of samples in studies from kenya (muriuki et al. 2001) and nigeria (olufemi & agboola 2009), respectively, which are both relatively low compared to levels seen in the current study. this difference may be due to different sample types, laboratory methods and possible variation in oxytetracycline use depending on the animal management system of the locality. nisha (2008) reported that indiscriminate use of antibiotics to treat pyrexia, inflammation, wounds and viral diseases is associated with high levels of residues in edible tissues of food-producing animals. the high incidence of oxytetracycline residues observed in the current study probably reflects cattle being sold for slaughter whilst under a therapeutic or prophylactic regimen of oxytetracycline or animals being slaughtered before the end of the withdrawal period (5–7 days when the antibiotic has been administered at a dose of 10 mg/kg for 7 days [aiello & moses 2010]). it is also of concern that these levels of oxytetracycline were found in tissue from indigenous cattle, because more than 98% of the cattle population in tanzania (approximately 21 million) are indigenous breeds and the main source of meat consumed in tanzania (national bureau of statistics 2012). in addition, there is no official monitoring programme and consumer response towards the dangers posed by drug residues is passive. thus, there is a risk of sustained consumer exposure to antibiotic residues and the associated effects on human health. the high levels of antibiotic residues found may be due to insufficient knowledge about drug use and the lack of extension services. livestock keepers in kilosa are mostly maasai, mang'ati or sukuma, who are known pastoralists in tanzania, and livestock extension officers’ access to these farmers is problematic. there are few livestock field officers available and the majority are found in the vicinity of town centres such as gairo. in the kilosa and mikumi zones, no veterinary services were offered because of the areas’ remoteness and poor infrastructure. the higher oxytetracycline residue levels found in samples from villages in the latter zones may be due to the lack of veterinary services, including extension services. the role of livestock extension officers is to advise farmers on proper animal management systems and disease control programmes, including vaccination. our findings support the conclusion of muriuki et al. (2001) that variation in residue levels – even from the same district – reflects the variation in animal husbandry practices as used by different livestock keepers and in different areas. easy access to antibiotics such as oxytetracycline, together with a lack of awareness, insufficient extension activities and inadequate usage guidelines from manufacturers, may lead to misuse and overuse of the drug and possibly failure to observe withdrawal periods. these actions may contribute to the presence of high levels of antibiotic residues in meat (nisha 2008). the lack of farmers’ awareness of the possible side-effects of antimicrobials and other drugs in humans also has to be considered (karimuribo et al. 2013). administration of drugs to food-producing animals requires consideration not only of effects on the animal but also of effects in humans who consume food from these animals. the high levels of antibiotic residues found in this study suggest that the public consuming animal products originating from the kilosa district may have been exposed to antimicrobial residues. our results, together with those of mmbando (2004) and nonga et al. (2009) about antibiotic residues in broiler chickens, suggest that some communities are exposed to small doses of antimicrobials from various animal food sources. this practice may contribute to the development of microbial resistance. conclusion top ↑ the findings of our study may be indicative of the inappropriate use and management of veterinary drugs by livestock keepers in the kilosa district specifically, but also more generally in tanzania. we therefore recommend stricter regulation of the use of veterinary drugs in the livestock industry as well as the inspection of livestock products prior to marketing. furthermore, livestock keepers need to be educated on the importance of adhering to the recommended drug withdrawal periods and possible human health effects associated with presence of veterinary drug residues in food of animal origin. veterinarians and livestock officers should also promote alternative management options aimed at good animal husbandry and disease control measures. acknowledgements top ↑ the authors acknowledge financial support from the project enhancing pro-poor innovations in natural resources and agriculture value chains (epinav). we appreciate the support and cooperation from the livestock keepers and field officers in the study area. technical support rendered by m. mugusi and j. fitwangile is acknowledged. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions z.i.k. (ministry of livestock and fisheries development) developed the research proposal, collected data, performed the laboratory analyses, analysed the data and drafted the manuscript. r.h.m. (sokoine university of agriculture) and f.m. (sokoine university of agriculture) planned the study, supervised data collection and analysis and interpretation of results, and proofread the manuscript before submission. c.j.n.m. (sokoine university of agriculture) and j.m. (sokoine university of agriculture) contributed to standardising the methods and performed laboratory analyses. e.d.k. (sokoine university of agriculture) and h.e.n. (sokoine university of agriculture) supervised all aspects of the study. references top ↑ abbasi, m.m., babaei, h., ansarin, m., nourdadgar, a. & nemati, m., 2011, ‘simultaneous determination of tetracyclines residues in bovine milk samples by solid phase extraction and hplc-fl method’, advanced pharmaceutical bulletin 1(1), 34–39. http://dx.doi.org/10.5681/apb.2011.005 aiello, s.e. & moses, m.a. 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(clarias gariepinus) in ibadan, nigeria’, world journal of fish and marine sciences 5(3), 302–309. olufemi, o.i. & agboola, e.a., 2009, ‘oxytetracycline residues in edible tissues of cattle slaughtered in akure, nigeria’, internet journal of food safety 11, 62–66. pena, a., pelantova, n., lino, m.c., silveira, m.i.n. & solich, p., 2005, ‘validation of an analytical methodology for determination of oxytetracycline and tetracycline residues in honey by hplc with fluorescence detection’, journal of agriculture and food chemistry 53, 3784–3788. pmid: 15884797, http://dx.doi.org/10.1021/jf050065r uekane, t.m., neto, f.r.a. & gomes, l.n.f., 2011, ‘development and validation of a method for the analysis of tetracyclines in chicken-muscle by liquid chromatography-electrospray-mass spectrometry in tandem (lc-esi-ms/ms)’, química nova 34(1), 43–48. http://dx.doi.org/10.1590/s0100-40422011000100009 article information authors: seraphine n. esemu1 roland n. ndip2,3 lucy m. ndip1,4,5 affiliations: 1laboratory for emerging infectious diseases, university of buea, cameroon 2department of microbiology and parasitology, university of buea, cameroon 3department of biochemistry and microbiology, university of fort hare, south africa 4department of biomedical sciences, university of buea, cameroon 5center for tropical diseases, university of texas medical branch, united states correspondence to: lucy ndip email: lndip@yahoo.com postal address: po box 63, buea, cameroon dates: received: 27 june 2014 accepted: 16 sept. 2014 published: 25 nov. 2014 how to cite this article: esemu, s.n., ndip, r.n. & ndip, l.m., 2014, ‘genetic diversity of ehrlichia ruminantium strains in cameroon’, onderstepoort journal of veterinary research 81(1), art. #840, 5 pages. http://dx.doi.org/10.4102/ojvr.v81i1.840 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. genetic diversity of ehrlichia ruminantium strains in cameroon in this original research... open access • abstract • introduction • materials and methods    • study sites and samples    • nested map1 polymerase chain reaction    • sequencing and sequence analysis of map1 pcr products • results    • polymerase chain reaction amplification of ehrlichia ruminantium dna from amblyomma variegatum ticks and cattle    • genetic diversity of ehrlichia ruminantium in cameroon • discussion • acknowledgments    • competing interests    • authors’ contributions • references abstract top ↑ in order to investigate the extent of genetic diversity among ehrlichia ruminantium strains in cameroon, a partial fragment (800 bp) of the e. ruminantium map1 gene was amplified by nested polymerase chain reaction in 121 of 156 e. ruminantium pcs20-positive dna samples extracted from ticks and cattle collected from two ranches. deoxyribonucleic acid sequencing of the map1 gene products indicated the presence of at least 21 genotypes at the nucleotide level and 16 genotypes at the amino acid level circulating within the study sites. some of the genotypes were identical to antigua (u50830), blaaukrans (af368000) or umbanein (u50835), whilst the others were new genotypes. twenty-four representative sequences were deposited in genbank and given accession numbers jx477663 – jx477674 (for sequences of tick origin) and jx486788 – jx486799 (for sequences of cattle origin). knowledge of e. ruminantium strain diversity could be important in understanding the epidemiology of heartwater. introduction top ↑ ehrlichia ruminantium, the causative agent of heartwater in domestic and wild ruminants, is an obligate intracellular gram-negative and pleomorphic bacterium. it is transmitted by ticks of the genus amblyomma (dumler et al. 2001; uilenberg 1983), with amblyomma variegatum being reported as the most efficient vector of this pathogen (waghela et al. 1991). heartwater has been reported to be a substantial obstacle to the improvement of livestock production throughout sub-saharan africa (dumler et al. 2001; faburay et al. 2008; uilenberg 1983). reports of the presence of heartwater in cameroon have been based on: molecular testing (esemu et al. 2013); serology (awa 1997); microscopic demonstration of morulae in giemsa-stained brain impression smears of grey matter from deceased cattle (ndi et al. 1998); clinical diagnosis (merlin, tsangueu & ronsvoal 1986; ndi et al. 1998); and the presence of the heartwater vector, a. variegatum (awa 1997; merlin et al. 1986; merlin, tsangueu & ronsvoal 1987; ndi et al. 1998; ndip et al. 2004; stachurski 2000). in addition to tick control, immunisation is an important prevention strategy; however, a successful immunisation programme should be informed by appropriate epidemiological data, which include the genetic characteristics of the circulating strains. in cameroon, the economic impact of heartwater in domestic ruminants is recognised, but not yet well-documented (ndi et al. 1998). the control of heartwater is mainly by tick control, the main tick control strategy being hand-picking and the application of acaricides through spray races, dips or using knapsack sprayers (ndi et al. 1998). so far, there has not been any report of the control of heartwater by immunisation. several strains of e. ruminantium have been reported in sub-saharan africa (esemu, ndip & ndip 2011) and they exhibit different levels of genetic diversity. the genetic diversity within the e. ruminantium map1 gene has not shown any evidence of geographic clustering (allsopp et al. 1997; allsopp et al. 2001; faburay et al. 2008). the map1 gene, one of the 16 paralogous genes that make up the map1 multigene family, has been used to understand genetic diversity among e. ruminantium strains (allsopp et al. 2001; martinez et al. 2004) and it remains the best tool for characterising genetic diversity among african, caribbean and madagascan strains of e. ruminantium (raliniaina et al. 2010). previous studies have been aimed at detecting e. ruminantium in a. variegatum ticks, based on the pcs20 polymerase chain reaction (pcr) assay, and have revealed a high degree of similarity within the pcs20 region of the e. ruminantium strains that were identified (esemu et al. 2013). in the present study, genetic diversity among the e. ruminantium strains circulating between a. variegatum tick vector and the cattle host by analysis of the map1 gene was investigated. materials and methods top ↑ study sites and samples one hundred and fifty-six e. ruminantium-pcs20-positive dna samples were extracted from a. variegatum ticks (144) and cattle (12) originating from two ranches (société de développement et d'exploitation des productions animales [sodepa] dumbo ranch [sdr] and upper farms ranch [ufr]), as previously described by esemu et al. (2013), were included in this study. of these 156 dna samples, 59 were from sdr (49 extracted from a. variegatum ticks and 10 from cattle blood) and 97 from ufr (95 extracted from a. variegatum ticks and 2 from cattle blood). nested map1 polymerase chain reaction nested map1 pcr was performed for the 156 dna samples using the following primers: map1nt (5'-ctcg taag aagt gcgt taat-3') as the external forward primer; map1ct1 (5’-ttaa aata caaa cctt cctc c-3’) as the external reverse primer; map1lp (5’-cttg gtgt gtcc tttt ctga-3’) as the internal forward primer and map1ct2 (5’-cctt cctc caat ttct atac c-3’) as the internal reverse primer (martinez et al. 2004). the individual reaction mixture for the first round of pcr was made up of template deoxyribonucleic acid (dna) (5 µl), 12.5 µl pcr master mix (2x) (toptaq™ master mix, qiagen, hilden, usa) and 0.5 µl of each primer from a working solution of 20 µm (final concentration of 0.4 µm), and nuclease-free pcr water to make up 25 µl total individual reaction volume. deoxyribonucleic acid amplification was carried out in a geneamp pcr system 2700 thermal cycler (applied biosystems, usa) under the following cycling conditions: for the first round of pcr reaction, initial denaturation was performed at 94 °c for 3 min, followed by 35 cycles of denaturation (94 °c for 45 s), primer annealing (53 °c for 45 s) and primer extension (72 °c for 45 s). the final extension was at 72 °c for 10 min and the reaction was stopped by cooling to 4 °c until the samples were collected. for the second round of pcr amplification, aliquots of 1 µl of pcr product from the first round of pcr amplification were used as the dna template. the pcr amplification conditions for the second round were optimised as follows: initial denaturation at 94 °c for 3 min, followed by 40 cycles of denaturation (94 °c for 1 min), primer annealing (57 °c for 1 min) and primer extension (72 °c for 1 min). the final extension was at 72 °c for 10 min and holding at 4 °c until the samples were collected. the expected pcr product size after the second round of pcr amplification was 800 bp. each batch of pcr run had one negative control, in which sterile distilled water was used as the template. the pcr products were analysed on a 1% agarose gel (agtc bioproducts, florida, usa). sequencing and sequence analysis of map1 pcr products to determine the extent of genetic diversity of e. ruminantium strains from a. variegatum ticks and cattle in cameroon, amplified map1 pcr products were sequenced from both ends with the nested map1 pcr primers (inqaba biotec, south africa). twenty-four high-quality sequences of the positive map1 pcr products, 12 (six from sdr and six from ufr) of tick origin and 12 (ten from sdr and two from ufr) of cattle origin were selected for analysis and aligned with clustalw (hall 1999). the blast program (national center for biotechnology information, bethesda, md) (morgulis et al. 2008) was used for similarity matches between the e. ruminantium strains reported in the present study and reference strains deposited in genbank. similarity matches between e. ruminantium strains reported in the present study were determined in a pair-wise sequence alignment using bioedit version 7.0.9 (hall 1999). phylogenetic relationships were determined with mega6 (tamura et al. 2013). all sequences were deposited in genbank. results top ↑ polymerase chain reaction amplification of ehrlichia ruminantium dna from amblyomma variegatum ticks and cattle amplified nested pcr products of the expected size (800 bp) were detected in 109 (75.7%) of the 144 e. ruminantium pcs20-positive tick dna samples that were tested. the map1 gene was amplified from all of the 12 (100%) e. ruminantium pcs20-positive cattle dna samples. all pcr products were separated in a 1% agarose gel, stained with ethidium bromide, visualised and photographed in a molecular imager gel doc xr system (bio-rad, hercules, ca, usa). genetic diversity of ehrlichia ruminantium in cameroon sequence analysis of map1 pcr products revealed several genotypes of e. ruminantium circulating in a. variegatum ticks and the cattle host in the study sites. alignment of the map1 sequences of all the strains revealed deletions and insertions concentrated within three variable regions. single nucleotide polymorphisms (snps) were dispersed throughout the dna sequences. similarly, alignment of the map1 amino acid sequences of all the strains revealed deletions and insertions concentrated within three variable regions (figure 1). figure 1: comparison of the amino acid sequences at the three variable regions of the mapi coding sequences of ehrlichia ruminantium strains from cameroon and six reference strains (accession number put in parentheses). based on nucleotide sequence identity, the e. ruminantium strains sequenced in this study were placed into different groups (table 1) with some groups having more than one genotype. identical nucleotide sequences were obtained for only three pairs among the 24 strains sequenced: cmr buea 34 and cmr dumbo 48 (group 1), cmr buea 89 and cmr dumbo 52 (group 3) and cmr buea 20 and cmr dumbo 7 (group 5). table 1: classification of the cameroonian ehrlichia ruminantium strains based on the percentage identities† at the nucleotide level and the number of genotypes in each group. these results indicated the presence of more e. ruminanium genotypes in sdr than in ufr. of the 21 genotypes at the nucleotide level, 13 were present only in sdr, whilst five were present only in ufr. three genotypes were present in both sdr and ufr. ten genotypes were detected in cattle, whilst eight were detected in a. variegatum ticks. three identical genotypes were detected in both a. variegatum ticks and cattle. some of the e. ruminantium map1 genotypes were identical to different strains reported elsewhere in african and caribbean countries, such as antigua (u50830) from the caribbean island, blaaukrans (af368000) from south africa and umbanein (u50835) from sudan. the phylogenetic tree of cameroonian e. ruminantium strains with some reference strains at the amino acid level revealed clustering of some strains. all strains in group 5 clustered with antigua, strains in group 2 clustered with blaaukrans, group 4 clustered with umbanein and gardel and group 8 clustered with kiswani (figure 2). the partial sequences of the map1 gene of the e. ruminantium strains analysed in the present study were deposited in genbank and given accession numbers jx477663 – jx477674 (for sequences of tick origin) and jx486788 – jx486799 (for sequences of cattle origin). figure 2: phylogenetic tree based on the partial amino acid sequences of map1 of 24 ehrlichia ruminantium strains reported in this study, and seven reference strains (bold and accession numbers put in parentheses). discussion top ↑ several strains of e. ruminantium, the causative agent of heartwater, have been reported throughout the endemic regions of africa and the caribbean (esemu et al. 2011). the control of heartwater by immunisation will potentially be hampered by genetic diversity among these strains (allsopp et al. 1999; allsopp et al. 2001; faburay et al. 2008; martinez et al. 2004; reddy et al. 1996). consequently, the effectiveness of any future heartwater vaccine will probably be dependent on the strains incorporated into the vaccine cocktail. recently, a high prevalence of e. ruminantium pcs20-positives in dna samples extracted from a. variegatum ticks (esemu et al. 2013) was reported in cameroon; however, information on the extent of genetic diversity among the e. ruminantium strains in cameroon remains to be established. the present study, therefore, aimed to bridge this knowledge gap by genotyping the e. ruminantium strains detected in cameroon in order to add to the epidemiological information currently available and provide data that may be useful for any future heartwater control programme. analysis of the map1 gene was therefore the method of choice because other authors have reported its use in demonstrating genetic diversity within strains from africa and the caribbean (allsopp et al. 1999; allsopp et al. 2001; faburay et al. 2008; martinez et al. 2004). the present study is the first report of genetic diversity among e. ruminantium strains in cameroon. out of the 156 e. ruminantium strains analysed in the present study, 121 (77.6%) gave specific positive amplicons of the map1 gene. the nested map1 pcr assay failed to amplify 35 (22.4%) of the pcs20 positive e. ruminantium strains. all of the e. ruminantium strains that had map1-negative results were associated with weak positive pcs20 signals. within the limited geographical area considered in the present study, there were at least 21 genotypes at the nucleotide level and 16 genotypes at the amino acid level, indicating the presence of silent mutations within the map1 gene. elsewhere, several strains of e. ruminantium have been reported in limited geographical areas (faburay et al. 2008; martinez et al. 2004). notable deletions and insertions were observed at three variable regions corresponding to the three variable regions delineated by reddy et al. (1996). also, numerous snps resulting in both synonymous and non-synonymous mutations were observed throughout the nucleotide sequence of all strains. the results of the present study are therefore in line with results reported elsewhere (reddy et al. 1996). the genotypes were compared with previously published map1 sequences deposited in genbank. interestingly, the cameroonian strains had affinity with both african and caribbean strains, forming several clusters with strains from africa and the caribbean. even though the map1 protein is immunodominant, it does not stimulate a protective response in sheep and goats (van kleef, neitz & de waal 1993). in addition, if the protein did stimulate protection, it would be expected to be under selection pressure and this has been shown not to be the case (allsopp et al. 2001). the reason for the observed genetic diversity of map1 is unknown, but it is most unlikely to be directly related to the ability of different strains to stimulate cross-protective immunity. the genetic diversity reported within the map1 gene can, nevertheless, be used as a marker for e. ruminantium strain differences. this great diversity may be as a result of more than one introduction of e. ruminantium in cameroon from e. ruminantium-infected ticks and ruminants. at the amino acid level, six genotypes showed 100% identity with antigua, two with blaaukrans and two with umbanein. this affinity may be due to multiple introductions of e. ruminantium-infected ticks and/or ruminants from other parts of the country, and even beyond, to the study sites. the amino acid phylogram (figure 2) indicated that there was no clustering in the e. ruminantium strains from cameroon and the results from the present study support the report of allsopp et al. (2001), which indicated clearly that there is no geographical distribution clustering among map1 variants. future studies with more strains from more locations in cameroon may provide additional insight into the diversity of e. ruminantium. understanding the diversity of e. ruminantium strains in a. variegatum ticks and cattle is essential for epidemiological and control purposes. acknowledgments top ↑ we are grateful to the laboratory for emerging infectious diseases, university of buea, for the facilities used to carry out this study. competing interests the authors declare that they have no competing interests. authors’ contributions s.n.e. (university of buea) performed the experiments, participated in the sequence alignment and analysis, and drafted the manuscript. r.n.n. (university of buea) conceived the study, provided materials for the molecular genetic studies, participated in the sequence alignment and analysis, and revised the manuscript. l.m.n. 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infection): current status’, advances in veterinary science and comparative medicine 27, 427–480. van kleef, m., neitz, a.w. & de waal, d.t., 1993, ‘isolation and characterization of antigenic proteins of cowdria ruminantium’, revue d’élevage et de médecine vétérinaire des pays tropicaux 46, 157–164. waghela, s.d., rurangirwa, f.r., mahan, s.m., yunker, c.e., crawford, t.g., barbet, a.f., et al., 1991, ‘a cloned dna probe indentifies cowdria ruminantium in amblyomma variegatum ticks’, journal of clinical microbiology 29, 2571–2577. penrith_91-95.indd introduction african swine fever (asf) is a devastating haemorrhagic fever of pigs that causes up to 100 % mortality (penrith, thomson & bastos 2004b). it is arguably the most serious constraint for pig production wherever it occurs. it is endemic in most sub-saharan african countries where pigs are produced, and where pigs, because of their high reproductive potential and ability to convert low quality feed into high quality protein, could play a crucial role in poverty alleviation (penrith et al. 2004b). it is also of great importance because of its remarkable potential for transboundary spread, which was amply demonstrated in the second half of the last century when it escaped from africa to affect several countries in western europe, with trans-atlantic spread to the caribbean and brazil, and again in 2007 when it appeared in the caucasus, with subsequent involvement of all the countries in that region (wil kinson 1989; penrith et al. 2004b; world organisation for animal health 2007, 2008). in spite of ongoing efforts, to date there is no vaccine against asf. new generation vaccines may possibly offer some hope for the future (lewis, zsak, burrage, lu, kutish, neilan & rock 2000), but there are still many challenges (neilan, zsak, lu, burrage, kutish & rock 2004; argilaguet, pérez-martin, gallardo, nofrarias, pujols, pérez-filgueira, blanco, salguero, escribano & rodriguez 2008). distribution and epidemiology of asf asf is presumed to have evolved in southern and eastern africa, where a sylvatic cycle of maintenance and transmission involving the natural hosts and vectors, namely warthogs (phacochoerus spp.) and argasid ticks (ornithodoros moubata complex), occurs (penrith et al. 2004b). the ticks inhabit the burrows of warthogs and feed on their blood, transmitting the virus in the process. warthogs are impervious to the pathogenic effects of the virus, do not develop viraemia and exhibit no clinical signs, 91 onderstepoort journal of veterinary research, 76:91–95 (2009) african swine fever mary-louise penrith1, 2 1 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria 2 tadscientific, 40 thomson street, colbyn, 0083 pretoria abstract penrith, m-l. 2009. african swine fever. onderstepoort journal of veterinary research, 76:91–95 african swine fever (asf) is a devastating haemorrhagic fever of pigs that causes up to 100 % mortality, for which there is no vaccine. it is caused by a unique dna virus that is maintained in an ancient cycle between warthogs and argasid ticks, making it the only known dna arbovirus. asf has a high potential for transboundary spread, and has twice been transported from africa to other continents— europe and subsequently the caribbean and brazil (1957, 1959) and the caucasus (2007). it is also a devastating constraint for pig production in africa. research at onderstepoort veterinary institute has made and is making important contributions to knowledge of this disease, focusing on the cycle in warthogs and tampans and transmission from that cycle to domestic pigs, resistance to its effects in domestic pigs, and the molecular genetic characterisation and epidemiology of the virus. 92 african swine fever although the virus may be extracted from their lymphoid tissues. however, neonatal warthogs that spend the first few weeks of their lives in the burrows develop sufficiently high viraemia to infect new ticks, although they do not develop clinical disease (thomson 1985). the ticks are able to maintain and transmit the virus to pigs for several years (oleagapérez, pérez-sanchez & encinas-grandes 1990; astigarraga, oleaga-pérez, pérez-sanchez & en cinas-grandes 1995) and they also transmit the virus transovarially, transstadially and sexually (plowright, parker & pierce 1969; plowright, perry & peirce 1970; plowright, perry & greig 1974; rennie, wilkinson & mellor 2001). like warthogs, the other wild african pigs (potamochoerus spp., hylochoerus meinertzhageni) are susceptible to infection with asf virus but not to its pathogenic effects (plowright, thomson & neser 1994; anderson, hutchings, mu karati & wilkinson 1998). the introduction of domestic pigs into the region, probably first in angola and mozambique, resulted in a susceptible host that almost invariably died of the disease. the occurrence of the disease in areas where o. moubata complex ticks inhabited pig sties allowed the establishment of a domestic cycle of viral circulation, and over time in some areas pigs developed a higher degree of resistance, resulting in lower mortality, although the virulence of the viruses infecting them remained unchanged (haresnape, lungu & mamu 1985, 1987; penrith, thomson, bastos, phiri, lubisi, botha, esterhuysen, du plessis, macome & pinto 2004c). how and when the disease spread to west africa is uncertain, but in spite of the absence of ornithodoros spp. in association with either pigs or warthogs, asf became endemic in senegal, probably guinea bissau, and cameroon (penrith et al. 2004b). since no long term carrier state has been demonstrated in recovered pigs, and there is considerable evidence that such a state does not occur (valadão 1969; wilkinson, wardley & williams 1983; pujols-romeu, badiolasaiz, pérez de rozas, se g ura-cardona, sacogalvany 1991; penrith et al. 2004c). viral circulation in these countries depends upon an endless supply of naïve pigs to infect, a circumstance that is strongly favoured by traditional free-ranging pig husbandry systems (penrith, lopes pereira, lopes da silva, quembo, nhamusso & banze 2007). in 1957 and again in 1959, asf spread from angola to portugal, probably in infected pork subsequently fed as swill (wilkinson 1989). the disease became established in the iberian peninsula, from which it was only eradicated in 1994 (davies 1994). the presence in pig sties of a suitable tick host, ornithodoros erraticus, helped to prolong the time taken for eradication (pérez-sánchez, astigarraga, oleagasánchez & encinas grandes 1994), and a small outbreak related to ticks in abandoned pig sties in portugal occurred in 1999 (basto, nix, boinas, mendes, silva, cartaxeiro, portugal, leitão, dixon & martins 2006). during the 1970s and 1980s several countries in western europe experienced outbreaks that were quickly eradicated, but asf persists in sardinia, which became infected in 1987, owing to circulation in dense populations of free-ranging domestic pigs with occasional involvement of wild boar as well (manelli, sotgia, patta, oggiano, carboni, cossu & laddomada 1998). during the same period outbreaks occurred in cuba, hispaniola (haiti and dominican republic), and brazil, and were only eradicated at considerable cost (butler & gibbs 1984; lyra 2006). from 1994 africa experienced an upsurge of asf that resulted in a pandemic that swept through several west african countries never previously affected (el-hicheri, gómez-tejedor, penrith, davies, dou ati, edoukou & wojciechowski 1998; world or ganisation for animal health 1996–2002; otesile, ajuwa pe, odemuyiwa, akpavie, olaifa, odaibo, olaleye & adetosoye 2005). additionally, kenya experienced outbreaks for the first time in 30 years, the previously asf-free southern provinces of mo zambique suffered major outbreaks, and the island of madagascar became infected for the first time in 1997 (roger, rantovonjato, vola & uilenberg 2001; penrith et al. 2004b). most of the affected countries had difficulty in controlling the disease and even those in which asf has not become endemic experience sporadic incursions. mauritius was infected, probably from madagascar, in 2007 (world organ isation for animal health 2007). in 2007 a dramatic change in distribution occurred when asf was confirmed in the republic of georgia, with subsequent spread to armenia, azerbaijan and russia. the source of the virus, a genotype ii virus previously described from mozambique and madagascar (bastos, penrith, macome, pinto & thomson 2004) that is not related to the genotype i virus that previously invaded europe, is likely to be galley waste that was available to free-ranging pigs around the port of poti on the georgian black sea coast (unpublished reports to fao 2007). the fact that asf was confirmed in dead wild boars in chechnya, on the border with georgia (world organisation for animal health 2007), is a matter for concern, since it complicates control and eradication. 93 m-l. penrith milestones in research on asf asf was first recognised as a disease entity distinct from classical swine fever and related to contact between domestic pigs and warthogs in kenya (montgomery 1921). the ability of ornithodoros ticks to maintain and transmit the virus was first described in spain by sanchez-botija in 1963, and this led to the confirmation of the pivotal role played by o. moubata in the sylvatic cycle in africa (plowright et al. 1969). studies by haresnape and her colleagues (haresnape et al. 1985, 1987) in malawi revealed the existence of a domestic cycle involving or nithodoros in a population of pigs of which a fairly high proportion had antibodies to asf yet were clearly healthy and productive. all asf viruses are regarded as belonging to a single serotype, since attempts to distinguish them by serotyping proved unreliable (vigário, terrinha & moura nunes 1974). the use of restriction fragment length polymorphism (rflp) made it possible to study the genetic relationships between asf viruses (wesley & tuthill 1984; blasco, aguero, amendral & viñuela 1989). nucleotide sequencing using the p72 structural protein has permitted further advances in the genetic characterization of asf viruses, which has proven extremely useful in indicating the possible or probable origin of outbreak viruses and the relationships between outbreaks (bastos, penrith, cruciere, edrich, hutchings, roger, couacyhyman & thomson 2003; bastos et al. 2004). prospects for control in the future, it may be possible to improve control of asf by the use of effective new generation vaccines, and/or by exploiting the natural resistance that has been demonstrated to occur in some populations of domestic pigs. in the meantime, we are faced with the difficulty that the conventional control methods of ‘stamping out’ and destroying large numbers of pigs is increasingly unacceptable for ethical and environmental reasons and is in fact impossible to implement successfully in countries where resources in terms of both finance and veterinary services are limited (penrith & thomson 2004a). the only viable alternative is farmer-based control, where the focus is on prevention. a directly transmitted disease like asf can be adequately controlled by biosecurity measures, but the application of these in the areas most affected by asf implies a change from traditional extensive low input husbandry to more intensive systems that place a higher demand on the producer (penrith et al., 2007). it is likely that such a change would need to be market driven rather than depending entirely on considerations of disease prevention. the onderstepoort contribution to asf research and control asf was first reported in south africa in 1928 (steyn 1928, 1932; de kock, robinson & keppel 1940). onderstepoort has made valuable contributions in terms of research and has also provided international support for control of asf. studies carried out at onderstepoort contributed to knowledge of the pathogenic, immunogenic and haemadsorbing properties of asf viruses and in particular provided considerable information about the sylvatic cycle and the respective roles of warthogs and ticks in maintenance and transmission of the virus (thomson, gainaru & van dellen 1979; thomson, gainaru, lewis, biggs, nevill, van der pypekamp, gerber, esterhuysen, bengis, bezuidenhout & condy 1980; 1981; thomson 1985). when asf emerged as a serious and widespread problem for traditional pig producers throughout sub-saharan africa, research was initiated into the nature of natural resistance evident in the domestic pig population in north-western mozambique, adjacent to the district in malawi where a similar situation was described (haresnape et al. 1985, 1987). it was hoped that a genetic basis for resistance could be established, but the results indicated that resistance is apparently not inherited (penrith et al. 2004c). the project provided impetus for molecular genetic studies on asf virus to be initiated at onderstepoort (bastos et al. 2003, 2004). these studies have been continued and expanded (lubisi, bastos, dwarka & vosloo 2005, 2007), and there are currently several internationally funded projects under way at ovi that are making contributions to the molecular epidemiology of asf, especially in eastern and southern africa, to improved diagnosis of asf and, perhaps, eventually to the development of an effective vaccine against this devastating disease. in response to the asf crisis in sub-saharan africa that occurred in the 1990s, the food and agriculture organization of united nations provided technical support for diagnosis and control to affected countries that applied for it. the onderstepoort veterinary institute provided expert consultants for technical cooperation projects in mozambique, kenya, côte d’ivoire, benin, cabo verde, ghana, gambia and mauritius during the period 1995–2007. 94 african swine fever references anderson, 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: folorunso o. fasina1 dauda g. bwala1 evelyn madoroba2 affiliations: 1department of production animal studies, university of pretoria, south africa 2bacteriology section, agricultural research council–onderstepoort veterinary institute, south africa correspondence to: folorunso fasina email: dayo.fasina@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 08 may 2015 accepted: 27 aug. 2015 published: 27 nov. 2015 how to cite this article: fasina, f.o., bwala, d.g. & madoroba, e., 2015, ‘investigation of multidrug-resistant fatal colisepticaemia in weanling pigs’, onderstepoort journal of veterinary research 82(1), art. #986, 6 pages. http://dx.doi.org/10.4102/ojvr.v82i1.986 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. investigation of multidrug-resistant fatal colisepticaemia in weanling pigs in this research communication... open access • abstract • communication • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ escherichia coli is usually a benign commensal of the gut microflora. however, when e. coli acquires virulence genes it can multiply rapidly and cause disease through colonisation of the intestinal mucosa. escherichia coli can become a significant pathogen in young pigs. we report an investigation of fatal colisepticaemia in weanling pigs from emerging farms where piglets and weaners were diarrhoeic and the mortality rate ranged between 15% and 70% in each litter. faecal and tissue samples were processed for histopathology, bacteriology and molecular biology (multiplex and monoplex polymerase chain reaction) and we recovered enteroaggregative multidrug-resistant e. coli producing east-1 enterotoxin. an association between poor housing conditions and the observed cases was established and future management programmes were recommended to reduce the impact of such pathogens. enteroaggregative e. coli is becoming a major problem in the pig industry. it therefore becomes necessary to establish the full impact of e. coli on the south african pig industry and to determine the geographic extent of the problem. communication top ↑ rotavirus, escherichia coli, clostridium perfringens, isospora suis, transmissible gastro-enteritis virus and enterococcus durans are amongst the most common diarrhoea-causing pathogens in piglets and are sometimes associated with neonatal and weanling deaths (johnson et al. 1992; martins et al. 2000; vu-khac, holoda & pilipcinec 2004). escherichia coli is generally a benign commensal of the gut microflora. however, when the bacterium acquires virulence genes it can multiply rapidly and colonise the intestinal mucosa by using surface proteins (fimbriae). the subsequent production of heat-stable or heat-labile toxins causes disease (parma et al. 2000). diarrhoea caused by e. coli affects all categories of young pigs (piglets, weaners and growers) to different degrees (henton & engelbrecht 1997; nagy & fekete 1999; vu-khac et al. 2006). recent reports have indicated that prevalence and isolation of antimicrobial-resistant e. coli are on the increase (enne et al. 2008; luppi et al. 2015; toledo et al. 2012). post-weaning diarrhoea (pwd) is a recurrent problem in weaned pigs (3–4 weeks of age) and previous studies have associated this condition with the f4 (mainly k88ac) and f18 fimbriae virulence factors (nagy & fekete 1999; vu-khac et al. 2006). in south africa specifically, henton and engelbrecht (1997) have serotyped 674 isolates from pigs and found that types o149, o141, o9, o20, o8 and a few others are prevalent in south africa (arranged here in descending order of prevalence). f4 was associated with 46.9% of the isolates. similar reports have been obtained from other smallholder farms and emerging pig farms despite slight variations in the pattern of presentation. in december 2013, carcasses of two five-week-old large white–landrace cross weanlings were presented to the pathology laboratory of the faculty of veterinary science, university of pretoria (accession number s04444-13). earlier, a report based on routine visits of the porcine herd health team to the farm of origin (a start-up farm with 60 sow units) indicated that certain design and management errors had predisposed piglets in the farrowing unit to wet floors, cold draughts and hypothermia and the associated consequences. suggestions for correction were offered. by the time the carcasses were presented for post-mortem examination, 14 sows had produced an average of 12 piglets per litter, of which 11 per litter were weaned at 4 weeks (weaning weight ≈ 6.7 kg; n = 151). pwd and mortality started 3–7 days post weaning and continued for the next 2 weeks, ultimately becoming self-limiting. approximately 80% of the piglets experienced pwd to some extent. the mortality rate varied from 15% to 70% in affected litters. necropsy revealed severe generalised congestion, severe segmental catarrhal enteritis, moderate nephrosis and mild acute hepatosis. generalised lymphadenomegaly due to cortical hyperplasia and diffuse moderate interstitial pneumonia with atelectasis were also observed. multifocal villus crypts filled with a monopopulation of small bacterial rods histologically typical of e. coli were observed in tissue samples collected for histopathology (figure 1a–c). the rod-like organisms adhered to enterocyte brush borders and were scattered within the lamina propria, extending to the lamina muscularis of the small intestine. the intestinal mucosa was not well differentiated and goblet cells were sparse, with a high mitotic rate (figure 1d), indicative of regeneration and repair. figure 1: histopathology of the intestinal and respiratory system tissue from the studied animals, stained with haematoxylin–eosin. (a) overview of a transverse section of intestine; (b) intestinal lumen, with normal lymphocytes and plasma cells in the lamina, mild congestion of enterocytes and lymphoid cells (arrow) and normal mucosal cells; (c) lumen filled with bacteria and debris (arrow), normal enterocytes and goblet cells; (d) section of the intestine, showing normal enterocytes, congestion, mild haemorrhage and autolytic cells (arrow); (e) transverse section of the pig trachea showing the respiratory epithelium, blood vessels and hyaline cartilage; (f) interstitial alveolar wall thickening (arrow); (g) interstitial pneumonia and congestion; (h) consolidated lung tissue with numerous bronchioles and congestion. multifocal atelectasis was observed in the lungs, interspersed by areas of marked interstitial pneumonia. large numbers of mononuclear cells, mainly macrophages and lymphocytes, had infiltrated and distended the alveolar walls (figure 1e–h). multifocal areas of protein-rich alveolar oedema were observed. the lymph nodes showed numerous lymphoblasts within follicles, with prominent medullary congestion. based on the presumptive aetiological diagnosis, diarrhoeic faeces were collected from sick piglets during a follow-up visit to the farm. culture on blood agar revealed both rough and smooth non-haemolytic e. coli organisms (pathology laboratory accession number b04025-14). during an in-vitro antimicrobial sensitivity evaluation, these organisms were found to be resistant to ampicillin, kanamycin, trimethoprim–sulphadimidine, oxytetracycline and tylosin, but susceptible to cetriofur, enrofloxacin and florfenicol. subsequent faecal samples collected between january 2014 and june 2015 from this farm and others with matching production statuses revealed similar patterns of clinical signs, pathology and bacterial cultures. however, variable patterns of antibiotic resistance were observed, based primarily on the predominant antibiotics used on the farms. all isolates were subcultured on blood agar and molecular characterisation was conducted. for this purpose, we conducted an investigation into the virulence factors associated with pwd using standardised protocols for polymerase chain reaction (pcr) and previously determined primers (table 1). table 1: primer sequences of escherichia coli virulence factors tested in the study. escherichia coli dna was extracted using cell lysis. bacterial cells were boiled at 99 °c for 15 min, followed by centrifugation. the supernatant containing crude dna extracts was used in multiplex and monoplex pcr reactions targeting the following virulence factors: heat-labile toxin (lt), heat-stable toxin a (sta), heat-stable toxin b (stb), shiga toxins stx1, stx2 and stx2e, enteroaggregative heat-stable enterotoxin (east-1), adhesin involved in diffuse adherence 1 (aida-1), porcine attachingand effacing-associated factor (paa) and fimbriae f4, f5, f6, f41 and f18. primers used in the multiplex and monoplex pcr reactions were combined as described by mohlatlole et al. (2013). the primers that were used for dna amplification in the pcr reactions are listed in table 1. the composition of the 25-µl pcr reactions was as follows: 2.5 µm of each primers, 5 µl crude dna extract, 12.5 µl pcr master mix (fermentas) and sterile dnase-free water (fermentas). the conditions for amplification were as follows: 10 min initial denaturation at 94 °c, followed by 30 cycles of denaturation at 94 °c for 30 s, annealing at 56 °c for 30 s and extension at 72 °c for 1 min. to ensure complete amplification of the pcr products, the thermocycling conditions included 7 min of extension at 72 °c. monoplex pcr reactions were set up to confirm east toxins in the samples. similar pcr amplification conditions were used, except for the volume of water being adjusted to a final volume of 25 µl using sterile dnase-free water. reference and control samples were included for all pcr reactions. for this purpose, reference samples that are known to be positive for enterotoxins and fimbriae were obtained from the culture collection of the bacteriology section of the agricultural research council – onderstepoort veterinary institute (arc–ovi). the strains included b41 (f5:f41:sta), 1883–1 (f18:sta), 1883–2 (f41:f5:sta), 1883–3 (f41:f5:f6:sta), 1883–4 (f4:f5:lt:stb), 1474 k12-k99 pienk (f5:sta), 1474 k12-k99 geel (f5:sta) and k99 (f5:sta). in addition, isolates that were known to be positive for east-1, paa, aida-1 and e. coli attaching and effacing (eae) factor reference samples were included. e. coli atcc 25922 was used as the negative control for virulence factors. the pcr results indicated that of the 15 virulence factors tested, only east-1 yielded a positive band. although most outbreaks of e. coli-derived diarrhoea in animals have been associated with strains with more than one virulence factor, in this case only east-1 toxin was observed (both e. coli isolates were positive for the 125-base-pair toxin; see figure 2). east toxins, produced by enteroaggregative e. coli, are antigenically related to the heat-stable toxins produced by enterotoxigenic e. coli and are known to induce fluid secretion (veilleux & dubreuil 2006). nevertheless, the products encoded by east have also been found to be both diarrhoeagenic and non-pathogenic (ruan et al. 2012). although our results confirmed that e. coli was involved in causing disease and the associated mortalities in this case, involvement of other organisms was not explored. the east-1 toxins have been found in south african pigs in a previous study (mohlatlole et al. 2013), which reported that 22.5% of the e. coli isolates carried the east-1 gene. similar results have been obtained in studies from korea and the czech republic, although higher prevalences were obtained in sucklers compared with weaner pigs (lee et al. 2008; zajacova, konstantinova & alexa 2012). as the east-1 toxin could exacerbate the pathogenesis of e. coli diarrhoea in weaned pigs (choi et al. 2001), it will be necessary to determine the spatio-temporal extent of these organisms in the south african pig population to understand the epidemiology of the infection in the country. furthermore, no e. coli vaccine available in south africa confers protection against the east-1 toxin, which may partially explain the high mortality observed in some of the cases. figure 2: gel electrophoresis showing east-1 positive amplicons. lanes: 1, 100-bp plus dna ladder; 2, stb negative; 3, lt negative; 4, e. coli atcc 25922 negative control strain; 5, sta negative; 6 and 7, east positive. based on the outcome of the pathology and pcr results, a conclusive diagnosis of colisepticaemia was made. at weaning, piglets are subjected to a variety of stress factors, including the withdrawal of the dam and sow's milk, a dip in the concentration of cd4+ and cd8+ cells, neutrophils and lymphocytes, and a post-weaning syndrome that consists of pwd, oedema disease and endotoxin shock (almond & kirk 2010; van beers-schreurs et al. 1992). the change in gut microflora associated with the changing diet around the time of weaning may lead to e. coli overgrowth and subsequent enteritis and endotoxaemia. on this farm, several cases of diarrhoea were observed in the farrowing and weaner pens and respiratory distress was observed in young weaners. although the e. coli-associated enteritis could have compromised the integrity of the intestinal villi and reduced the efficiency of feed utilisation, the cause of death in these weaner pigs was e. coli-associated endotoxaemia. no cases of piglet anaemia were observed in the herd and the oxygen-carrying capacity of haemoglobin did not appear compromised. the consolidation and interstitial alveolar wall thickening observed in the lungs notably reduced the capacity of the lungs in complying with their primary role of oxygen–carbon dioxide exchange at the alveolar level. fast-growing weaner pigs need an increasing level of such gaseous exchange to meet their physiologic needs at this stage. it becomes vitally important to reduce other associated complications (e.g. digestive or respiratory) arising from e. coli infection at the time of weaning. we recommended changes in management protocol at this farm to ensure dry floors and hygiene in the farrowing and weaner units. establishment of a vaccination protocol that includes routine e. coli vaccination of sows 2 weeks before parturition is also recommended to boost colostral immunity and decrease the magnitude of antibiotic treatment in subsequent outbreaks. acknowledgements top ↑ we thank the staff of the pathology section of the department of paraclinical sciences, the microbiology laboratory of the department of veterinary tropical diseases and the porcine herd health students (php 650) of the faculty of veterinary science, university of pretoria, who all participated in the investigation. we also appreciate the cooperation of the farmer. the molecular biology work was carried out at the arc–ovi, onderstepoort, south africa. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions f.o.f. (university of pretoria) and d.g.b. (university of pretoria) drafted and revised the manuscript. f.o.f. attended to the case and performed clinical sampling and follow-up. e.m. 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from diarrhoeic piglets in the slovak republic’, journal of veterinary medicine series b 51, 343–347. pmid: 15525362. vu-khac, h., holoda, e., pilipcinec, e., blanco, m., blanco, j.e., mora, a. et al., 2006, ‘serotypes, virulence genes, and pfge profiles of escherichia coli isolated from pigs with post weaning diarrhoea in slovakia’, bmc veterinary research 2, 10. pmid: 16549022, http://dx.doi.org/10.1186/1746-6148-2-10 zajacova, z.s., konstantinova, l. & alexa, p., 2012, ‘detection of virulence factors of escherichia coli focused on prevalence of east-1 toxin in stool of diarrheic and non-diarrheic piglets and presence of adhesion involving virulence factors in asta positive strains’, veterinary microbiology 154, 369–375. pmid: 21864997, http://dx.doi.org/10.1016/j.vetmic.2011.07.029 article information authors: gabriel m. shirima1 seleman. n. masola2 obeid. n. malangu3 brant a. schumaker4 affiliations: 1tanzania veterinary laboratory agency, dar es salaam, tanzania 2department of animal health, national livestock research institute, tanzania 3veterinary investigation centre, mpwapwa, tanzania 4department of veterinary science, university of wyoming, united states correspondence to: gabriel shirima postal address: po box 9254, dar es salaam 15487, tanzania dates: received: 19 may 2014 accepted: 25 aug. 2014 published: 25 nov. 2014 how to cite this article: shirima, g.m., masola, s.n., malangu, o.n. & schumaker, b.a., 2014, ‘outbreak investigation and control case report of brucellosis: experience from livestock research centre, mpwapwa, tanzania’, onderstepoort journal of veterinary research 81(1), art. #818, 4 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.818 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. outbreak investigation and control case report of brucellosis: experience from livestock research centre, mpwapwa, tanzania in this original research... open access • abstract • introduction • materials and methods    • study area and animals    • outbreak investigation       • case definition       • serological screening of livestock       • serological screening of humans    • data storage and analysis • results    • epidemiologic investigation       • magnitude of the problem and herd history    • intensive follow-up       • serology and control measures • discussion • acknowledgements    • competing interests    • authors’ contribution • references abstract top ↑ brucellosis screening was conducted between 2005 and 2010 at the national livestock research institute headquarters, mpwapwa, tanzania, following an abortion storm in cattle. the initial screening targeted breeding herds; 483 cattle were screened using the rose bengal plate test (rbpt) followed by the competitive enzyme-linked immunosorbent assay (c-elisa) as a confirmatory test. the seropositivity on c-elisa was 28.95% in 2005; it subsequently declined to 6.72%, 1.17%, 0.16% and 0.00% in 2006, 2007, 2009 and 2010, respectively. brucella seropositivity was not detected in goats. seropositivity declined following institution of stringent control measures that included: gradual culling of seropositive animals through slaughter; isolation and confinement of pregnant cows close to calving; proper disposal of placentas and aborted foetuses; the use of the s19 vaccine; and restricted introduction of new animals. it was thought that the source of this outbreak was likely to have been from the introduction of infected animals from another farm. furthermore, humans were found with brucellosis antibodies. out of 120 people screened, 12 (10%) were confirmed seropositive to brucella antigen exposure by c-elisa analysis. the majority of the seropositive individuals (80%) were milkers and animal handlers from the farm. nine individuals had clinical signs suggestive of brucellosis. all cases received medical attention from the district hospital. this achievement in livestock and human health showed that it is possible to control brucellosis in dairy farms, compared to pastoral and agro-pastoral farms, thus providing evidence to adopt these strategies in dairy farms thought to be at risk. introduction top ↑ brucellosis is an infectious and contagious bacterial disease. it primarily affects domestic and wild animals and has both economic and public health implications. it is of economic importance as it causes financial losses from abortions, sterility, decreased milk production, veterinary fees and costs of replacement animals (radostits et al. 2000). in humans, it is characterised by headaches, joint pain, undulating fever and general body malaise (bouley et al. 2012). brucellosis has a worldwide distribution and is also an important disease in tanzania. the history of brucellosis in tanzania (historically tanganyika) dates back to 1928, when an outbreak of abortions was reported in exotic dairy animals introduced into the country. the affected animals were kept in engare nanyuki, arusha and were confirmed brucellosis positive (kitalyi 1984). from the time that the disease was introduced into the country, it has never been controlled. brucellosis continued to spread in pastoral, agro-pastoral and dairy farming systems (shirima et al. 2007). mahlau and hammond (1962) reported three outbreaks in indigenous cattle in the maswa district, lake zone, where in all cases the seropositivity was greater than 20% in the affected herds. this was followed by an abattoir survey in the same zone where seropositivity in cattle and goats was 15.0% and 1.3%, respectively (mahlau & hammond 1962). nevertheless, extensive surveillance covering different farming systems was conducted by jiwa et al. (1996); it revealed a prevalence of 10.8% in the same zone, with variable seropositivity noted in government ranches (15.8%), dairies (6.3%) and traditional herds (4.3%). similar surveys conducted in other zones revealed brucellosis seropositivity at varying levels (maiseli 1992; shirima et al. 2007; swai & schoonman 2009; weinhaupl et al. 2000). lack of regular brucellosis screening in indigenous herds, ranches and dairy farms has resulted in the disease spreading countrywide (jiwa et al. 1996; minga & balemba 1990; shirima et al. 2007). despite this spread, it is not certain which brucella species are in circulation; knowledge is necessary for implementing the appropriate control strategies. the present paper describes investigation of a disease outbreak following an abortion storm in 2005 in a cattle herd belonging to a research institution, and devises various integrated control strategies. materials and methods top ↑ study area and animals the present study was conducted at the national livestock research institute (nlri) (currently known as the tanzania livestock research institute [taliri]), mpwapwa, which is located in the dodoma region in the central zone of tanzania. since 1907, the institute has been mandated to conduct livestock production research. the taliri also has a farm that keeps cattle, goats, sheep and pigs for research purposes. the centre is known for developing a cattle breed known as the mpwapwa breed (dual-purpose breed). outbreak investigation brucellosis outbreaks were first reported at the taliri in 1937 following the introduction of animals (department of veterinary services 1937); however, the disease has been controlled until this latest event, when a similar disease presenting with an abortion storm was suspected. a team of five veterinarians, one from the tanzania veterinary laboratory agency (tvla), dar es salaam, three from the veterinary investigation centre (vic), mpwapwa, and one from taliri, mpwapwa, were assigned to carry out the disease outbreak investigation. the epidemiologic investigation procedures employed to carry out the assignment included: case description, diagnosis verification, magnitude determination, and intensive follow-up (world organisation for animal health [oie] 2014). case definition and other epidemiological information were obtained through person-to-person interviews. case definition history provided by the farm veterinarian indicated that pregnant animals expected to calve within 2–3 months were aborting. clinically aborted animals had no significant clinical signs. based on herd records, aborted cows were in the third trimester. for the purpose of the present investigation, the definition for a positive brucellosis abortion case was based on three categories: • suspected cases were those animals characterised by abortion at the third trimester with or without retained placenta and no fever noted. • probable cases fulfilled the suspect criteria and epidemiological information, and also tested positive on rose bengal plate test (rbpt). • confirmed cases fulfilled probable case criteria and also tested positive on competitive enzyme-linked immunosorbent assay (c-elisa). serological screening of livestock as part of intensive follow-up, blood samples were collected for screening. plain vacutainers were used to collect whole blood from each animal and labelled using individual animal ear-tag numbers. blood samples were left at the ambient temperature for at least 30 min after collection, to avoid problems of albumin coagulation that prevents sera formation during centrifugation. samples were centrifuged at 3022 g for 5 min, using a mobile spin centrifuge (vulcon technologies, usa) at vic-mpwapwa. tubes were removed and sera were decanted into eppendorf tubes (eppendorf-netheler-hinz gmbh, hamburg, germany) in duplicate. all sera were labelled and kept in the freezer at approximately -20 °c. the rbpt and c-elisa kits, with control sera, were used for screening and confirmation, respectively. the antigen used for screening was brucella abortus antigen, which was kindly donated by the veterinary laboratory agency (vla) weybridge, uk (batch numbers sg269 and sg276). the rbpt and c-elisa tests were performed as described by vla protocol (perret et al. 2001). interpretations of results were based on agglutination for rbpt and visual observation for any colour development and optic density readings for c-elisa using the elisa reader at 450 nm. the samples were considered to be positive when the binding ratio was greater than 10 and at a cut-off point of 0.83 (optic density). the first screening was performed in june 2005, when 483 out of 490 pregnant and lactating cows were sampled. seven animals were not in the herd during bleeding and were thus not screened. the follow-up activities were conducted between may and june on an annual basis until 2010 (table 1). screening was skipped in 2008 due to logistic reasons; in 2006, the number was almost doubled due to inclusion of experimental animals. the farm had four groups of animals that were kept separate (production herd, experimental herd, yearlings with both males and females, and goat flock). the experimental herd, yearlings and goat flock were tested once and not followed because they were all negative. table 1: number of cattle screened annually at tanzania livestock research institute, mpwapwa. serological screening of humans people working in the institute were approached after receiving permission from the institute director and in consultation with the district medical officer. individual consent was also sought before participation in the present study. the people enrolled were taliri residents who either drank milk from the farm or worked with taliri animals. the taliri residents who neither drank milk from the farm nor worked with animals were excluded. based on their willingness to participate, in 2006 120 residents were screened using rbpt and confirmed by c-elisa. clinical signs suggestive of brucellosis were collected from each individual after sampling. data storage and analysis data generated were entered in a microsoft excel 2007 spread sheet. some of the variables collected from interviews were summarised using narrative text, whereas serological data were analysed using descriptive statistics. results top ↑ epidemiologic investigation magnitude of the problem and herd history based on the previous records, the incidences of abortion storms were not reported at taliri for decades. during the first visit, 30 out of the 126 heavily pregnant cows were found to have aborted within a period of 2 weeks. the herd structure consisted of 40 non-pregnant heifers, 180 yearlings, 80 breeding bulls, and 490 pregnant and lactating cows. there were also 200 goats present on the farm, but they were kept separate from the cattle. the geographical boundaries protected the farm animals from co-mingling with other neighbouring herds. two years before the incidences of abortion storm in cattle, 100 cattle were introduced into the herd from a different farm belonging to another institution. these animals were screened for brucellosis on the farm of origin and found to be seronegative by the rbpt before being introduced into the taliri farm. intensive follow-up serology and control measures the first screening targeted mature female cows in the production herd (pregnant and lactating cows) where 483 out of 490 cows were tested using the rbpt, followed by the c-elisa as a confirmatory test. in 2005, seropositivity was 28.95%, which subsequently declined to 6.72%, 1.17%, 0.16%, and 0.00% in 2006, 2007, 2009 and 2010, respectively (table 1). the experimental herd and goat flock was tested once, and found to be seronegative. out of the 120 human sera that were tested, seropositivity was 10% (table 2). seropositivity was higher in men (12%) compared to females (7%), with no statistically significant difference (p = 0.728); however, out of the 10 infected men, 8 (80%) were engaged in milking, herding the cattle and assisting during calving. nine seropositive individuals had joint pains, body weakness and irregular fevers, whereas three showed no symptoms. table 1: human serum samples tested using competitive enzyme immunosorbent assay at tanzania livestock research institute, mpwapwa. control and eradication of brucellosis through test and slaughter, coupled with vaccination and restriction of animal movements, was adopted with some modification, based on the resources and transmission pathways to control the disease. gradual culling of marked seropositive cattle, isolation of pregnant cows into designated pens 2 weeks before calving, proper disposal of placentas and aborted foetuses, vaccination of eligible calves (6–8 months) using the s19 vaccine, and restricted introduction of new animals were integrated during the entire period; however, vaccinated animals were not screened for brucellosis to avoid cross-reaction with the test resulting in false positives. marked seropositive cattle were isolated and slaughtered under veterinarian supervision within the institute at certain intervals (gradual culling) for salvage purposes. residents were advised to boil milk destined for human consumption. discussion top ↑ in the present study, the confirmatory diagnosis of bovine brucellosis was attained by using serological tests. the c-elisa seropositivity in cattle was associated with a history of abortion storm and was in agreement with several other studies (schelling et al. 2003; shirima et al. 2007; swai 1997). the results of the present study indicate that brucellosis infection could be present on dairy farms that have had no history of the disease, in the absence of routine surveillance. introduction of animals originating from other herds has probably been the source of infection on the farm. although these animals were tested before purchasing, false negative cases could have been the source of infection. false negative cases resulting from screening with serological tests such as rbpt were probably due to the failure to detect chronic infections, recently aborted cases, or an inherent weakness of the assay whose sensitivity ranges from 63% – 99% (bishop, bosman & herr 1994; jeff 2013). in several countries, control and eradication of brucellosis has been achieved through test and slaughter, vaccination, and restriction of animal movements (mcdermott, grace & zinsstag 2013). in other countries, similar methods have been adopted with some modification, based on the available resources and transmission pathways. in the present study, gradual culling of marked seropositive cattle, isolation of pregnant cows into designated pens 2 weeks before calving, proper disposal of placentas and aborted foetuses, vaccination of eligible calves (6–8 months) using the s19 vaccine, and restricting introduction of new animals were integrated during the entire period. although the risk factors for human transmission were beyond the scope of the present investigation, the majority of seropositive humans were animal handlers and/or milk drinkers. the human results indicated that > 80% of seropositive cases were mainly due to occupation; this was consistent with other observations (niwael 2001; minja 2002; schelling et al. 2003; shirima et al. 2007, 2010). the remaining proportion could probably be due to drinking unboiled milk originating from infected cows. these findings may necessitate public education to promote boiling of milk before drinking, and to properly handle animals during calving and aborted foetuses by using protective materials such as latex gloves. although seropositive animals that were destined for gradual culling were isolated, extra care was required by attendants to reduce the risk of exposure to humans. the integration of different approaches employed for brucellosis control at the taliri-mpwapwa farm has shown significant success in reducing disease incidence. it can therefore be concluded that brucellosis can be controlled and prevented with the appropriate human resources, good collaboration with the existing diagnostic institutions, and awareness of brucellosis-control strategies. owner awareness enhances the compliance to biosecurity measures, as well as the agreement to test and slaughter seropositive animals. control strategies used at the taliri-mpwapwa farm are strongly recommended for other organised farms, with or without modifications, depending on farm husbandry and management. acknowledgements top ↑ the authors appreciate the financial support provided by the director of the taliri-mpwapwa farm during the entire course of this work. thanks to the director of central veterinary laboratory (cvl), dar es salaam, for granting permission and the premises to analyse the samples. all residents who participated in this study are also recognised. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contribution g.m.s. (tanzania veterinary laboratory agency) was the team leader and ran the c-elisa and interpreted the data. s.n.m. (national livestock research institute) and o.n.m. (veterinary investigation centre) were responsible for designing, serum separation and implementing control measures. b.a.s. (university of wyoming) performed the critical revision of the manuscript. references top ↑ bishop, g.c., bosman, p.p. & herr, s., 1994, ‘bovine brucellosis’, in j.a.w. coetzer, g.r. thomson & r.c. tustin (eds.), infectious diseases of livestock with special reference to southern africa, vol. 2, pp. 1053–1066, oxford university press, uk. bouley, a.j., biggs, h.m., stoddard, r.a., morrissey, a.b., bartlett, j.a., afwamba, i.a. et al., 2012, ‘brucellosis among hospitalized febrile patients in northern tanzania’, american journal of tropical medicine and hygiene 87(6), 1105–1111. http://dx.doi.org/10.4269/ajtmh.2012.12-0327 department of veterinary services, 1937, annual report, tanganyika. jeff, a., 2013, brucellosis diagnostics, wyoming state university, veterinary laboratory. jiwa, s.f.h., kazwala, r.r., tungaraza, r., kimera, s.i. & kalaye, w.j., 1996, ‘bovine brucellosis serum agglutination test prevalence and breed predisposition according to prevalent management systems in the lake zone of tanzania’, preventive veterinary medicine 26, 341–346. http://dx.doi.org/10.1016/0167-5877(95)00543-9 kitalyi, j., 1984, ‘bovine brucellosis in government parastatal and ujamaa village dairy farms in the central zone of tanzania: assessment of control measures in some of farms’, in proceedings of 2nd tanzania veterinary association scientific conference, arusha, tanzania, december, 1984. mahlau, e.a. & hammond, j.a., 1962, ‘a brucellosis survey of the western areas of tanganyika’, bulletin of epizootic disease in africa 10, 511–516. maiseli, n.g., 1992, livestock diseases and diagnosis in the southern highlands regions of tanzania, ministry report, ministry of agriculture. mcdermott, j., grace, d. & zinsstag, j., 2013, ‘economics of brucellosis impact and control in low-income countries’, revue scientifique et technique, office international des epizooties 32(1), 249–261. minga, u.m. & balemba, o.b., 1990, ‘elisa: a new serodiagnosis test for brucellosis in tanzania’, in proceedings of the 8th tanzania veterinary association scientific conference, arusha, tanzania, december, 1990. minja, k.s.g., 2002, ‘prevalence of brucellosis in indigenous cattle: implication for human occupational groups in hanang and babati districts of tanzania’, master of veterinary medicine thesis, sokoine university of agriculture, tanzania. niwael, m.m., 2001, ‘epidemiological study of brucellosis in humans and animals in babati and hanang districts of tanzania’, master of veterinary medicine thesis, sokoine university of agriculture, tanzania. perret, l., brew, s., stack, j., tucker, j. & macmillan, a.p., 2001, guide to the eia techniques used in the diagnosis of brucellosis, veterinary laboratory agency, new haw, addlestone surrey, kt15 3nb, uk. radostits, o.m., gay, c.c., blood, d.c. & hinchcliff, k.w., 2000, veterinary medicine. a text book of the diseases of cattle, sheep, pigs, goats and horses, 9th edn., p. 1963, harcourt publishers ltd., london. schelling, e., digumbaye, c., daoud, s., nicollete, j., boerlin, p., tanner, m. et al., 2003, ‘brucellosis and q fever seroprevalences of nomadic pastoralists and their livestock in chad’, preventive veterinary medicine 61, 279–293. http://dx.doi.org/10.1016/j.prevetmed.2003.08.004 shirima, g., fitzpatrick, j.m., kunda, j.s., mfinanga, g.s., kazwala, r.r., kambarage, d.m. et al., 2010, ‘the role of livestock keeping in human brucellosis trends in livestock keeping communities in tanzania’, tanzania journal of health research 12(3), n.p. http://dx.doi.org/10.4314/thrb.v12i3.51261 shirima, g.m., cleaveland, s., kazwala, r.r., kambarage, d.m., nigel, f., mcmillan, a. et al., 2007, ‘sero-prevalence of brucellosis in smallholder dairy, agropastoral, pastoral, beef ranch and wildlife animals in tanzania’, bulletin of animal health and production in africa 55, 13–21. swai, e.m., 1997, ‘studies on the prevalence of bovine brucellosis and reproductive performance in small scale dairy cattle herds in dar-es-salaam and morogoro regions’, master of veterinary medicine thesis, sokoine university of agriculture, tanzania. swai, e.s. & schoonman, l., 2009, ‘human brucellosis: seroprevalence and risk factors related to high risk occupational groups in tanga municipality, tanzania’, zoonoses and public health 56(4), 183–187. http://dx.doi.org/10.1111/j.1863-2378.2008.01175.x weinhaupl, i., schopf, k.c., khaschabi, d., kapaga, a.m. & msami, h.m., 2000, ‘investigation on the prevalence of bovine brucellosis in dairy cattle in dar-es-salaam region and in zebu cattle in lugoba area tanzanaia’, tropical animal health and production 32, 147–154. http://dx.doi.org/10.1023/a:1005231514467 world organisation for animal health (oie), 2014, guidelines for animal disease control, viewed 23 may 2014, from http:// www.oie.int article information authors: louise j. biggs1 chris a.p. carrington1 vinny naidoo2 affiliations: 1department of production animal clinical studies, university of pretoria, south africa2biomedical research centre, university of pretoria, south africa correspondence to: vinny naidoo postal address: private bag x04, onderstepoort 0110, south africa dates: received: 31 oct. 2013 accepted: 15 mar. 2014 published: 22 aug. 2014 how to cite this article: biggs, l.j., carrington, c.a.p. & naidoo, v., 2014, ‘evaluation of medicinal turpentine used for the prevention of bovine babesiosis in southern kwazulu-natal and the eastern free state’, onderstepoort journal of veterinary research 81(1), art. #705, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.705 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. evaluation of medicinal turpentine used for the prevention of bovine babesiosis in southern kwazulu-natal and the eastern free state in this original research... open access • abstract • introduction • material and methods    • in vitro anti-babesial effect    • toxicity and clinical pathology study    • data analysis • ethical approval • results    • babesia cultures    • animal phase study • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ medicinal turpentine has been used extensively in the eastern free state and kwazulu-natal provinces of south africa with reportedly excellent results. it is believed that it is able to prevent and treat babesiosis (redwater) in cattle. redwater is an often-fatal disease in cattle and results in losses of large numbers every year in south africa. this study was initiated in an attempt to investigate the validity of the use of the turpentine as a medicinal agent. using a semi in vitro screening assay, babesia caballi grown in primary equine erythrocytes was exposed to various concentrations of turpentine in comparison to diminazene and imidocarb. the turpentine had no parasiticidal effect following direct exposure. during the recovery phase, the previously exposed parasites appeared to grow more slowly than the controls. in comparison, diminazene and imidocarb were 100% effective in killing the parasites. in a subsequent tolerance study in adult cattle (n = 6) at 1x (2 ml), 3x and 5x the recommended dose, the product was non-toxic. irritation was noted at the injection site with the higher dose. the only major finding on clinical pathology was a general increase in globulins, without a concurrent change in native babesia antibody titres. it was concluded that it is unlikely that medicinal turpentine is an effective treatment against babesiosis. introduction top ↑ the genus babesia belongs to the phylum apicomplexa, class sporozoasida, order eucoccidiorida, suborder piroplasmorina and family babesiidae (de vos & potgieter 2004). the two species of economic importance in southern africa are babesia bigemina (african redwater) and babesia bovis (asiatic or european redwater). babesiosis, the disease caused by the parasite, is principally a vector-borne disease transmitted by rhipicephalid ticks (du plessis, de waal & stoltsz 1994). once the host is infected, the parasite has an incubation period of approximately 14 days for b. bigemina, and shorter for b. bovis, at 9 days. clinical signs that develop include: pyrexia, listlessness, anorexia, a decrease in or cessation of rumen movements, decreased milk production, and for b. bovis especially, neurological signs. due to the erythrolysis induced by parasites exiting the erythrocytes, animals are also anaemic. after being infected and surviving, animals develop what is known as premunity. in this state, the animals appear to be resistant to further infection due to some parasites remaining latent in the blood (de vos & potgieter 2004). if the disease remains untreated, the mortality rate may reach 90%.treatment of babesiosis in cattle usually involves the use of diminazene and imidocarb, both of which are diamidines (de vos & potgieter 2004). whilst both drugs are effective, they have limited prophylactic effects. another method commonly used is vaccination, as cattle develop a durable immunity after a single infection with b. bigemina and b. bovis (carrington, du plessis & naidoo 2011). whilst many of the farmers have relied on vaccination, as recommended by the manufacturer (onderstepoort biological products, south africa), losses still continue to occur. this has resulted in the local farmers using medicinal turpentine, both as a therapeutic and preventative agent. from informal discussions with members of this farming community, ten farmers were able to confirm that they used the product, based on recommendations from a neighbour; therefore, the origin of treatment was unknown. from these discussions, the standard dose used was seen to be 2 ml, administered subcutaneously every three months. the farmers were also of the belief that treatment resulted in an overall reduction in the total incidence of babesiosis on their farms, even though they were unable to confirm conclusively that their cattle were even infected with babesia.  turpentine oil is naturally derived and is mainly composed of the monoterpenes alpha-pinene and beta-pinene (mercier, prost & prost 2009). the oil is obtained as a by-product from the distillation of resin obtained from pine trees (pinus spp.) during the kraft process and is widely used as a complementary medicine, with a history of use extending from ancient times (burt 2004; kleppe 1970; mercier et al. 2009). whilst the predominant use of the medicine is in chest rubs and for the management of respiratory conditions, it has also been reported to be effective in the treatment of both external and internal parasites (daubney 1930; sheather 1923). turpentine oil has been evaluated in controlled research studies for its effect against internal parasites (krause et al. 2007; vreden et al. 1992). in a rodent model of malaria, subcutaneous administration of turpentine oil 24 h or 5 min before inoculation of sporozites resulted in 80% and 35% reduction of schizont development, respectively (vreden et al. 1992). to date, the effects of turpentine on in-vivo or in vitro babesia infections have not been evaluated. however, with the similarity in pathogenesis between malaria and babesiosis, the use of the product may be valid (krause et al. 2007). in the opinion of the authors, if effective against babesia, turpentine could control babesia either by inhibiting parasite growth or by somehow acting as an adjuvant to pre-existing premunity. as a first step in validating the use of turpentine, the in vitro effect of turpentine on babesia was evaluated and the safety in a bovine model of overdose was established. as a secondary objective, changes in humoral immunity and lymphocyte counts were also evaluated. the treatment response of infected cattle was not evaluated in the present study, as it was believed to be unethical to proceed with an in-vivo study prior to obtaining any information on in vitro efficacy. material and methods top ↑ in vitro anti-babesial effect babesia caballi was grown in primary equine red blood cells, using a cell free medium, as previously described (naidoo et al. 2005). this species was used for the study, because, whilst attempts were also made to culture b. bigemina in primary bovine red blood cells, the parasite repeatedly failed to grow. following a period of 48 h to allow for parasite establishment (day 0), the cultures were exposed to turpentine (20.0 µg/ml, 10.0 µg/ml, 5.0 µg/ml, 2.5 µg/ml) (n = 6) in the media for 48 h (day 3). the turpentine was bought in south africa and was supplied with a certificate of analysis by the company (0.880 g/ml). thereafter, the media with turpentine were removed and the cultures were allowed an additional 48 h to re-establish (day 5). the positive controls were diminazene (n = 6, 10 µg/well) and imidocarb dipropionate (n = 3, 10 µg/well), whilst untreated wells served as the negative control. a thin blood smear using a slide-on-slide technique was made from the cultures on days 0, 3 and 5, and stained with giemsa (sigma aldrich, south africa). parasitaemia was determined by the number of infected cells in an area of 5000 cells under 100x magnification. results are presented as the percentage parasitaemia. toxicity and clinical pathology study twenty-four cattle (18–24-month-old replacement heifers) without prior vaccination against babesia were used in the study. the animals (n = 6) were treated subcutaneously at the recommended dose (2 ml or 4.25 mg/kg), 3x the recommended dose (6 ml or 12.75 mg/kg), 5x the recommended dose (10 ml or 21.25 mg/kg) or saline (2 ml). the doses were selected to obtain the best representation of toxicity and changes in clinical pathology. the animals were housed under natural field conditions, in order to simulate natural exposure to parasites, as per normal farm practice (see ethical clearance).blood samples (edta and serum) were collected prior to treatment and at 18 h, 48 h and 120 h post injection. as part of the evaluation, the clinical pathology laboratory also evaluated thin blood smears of all samples. the blood was analysed immediately using an automated counter (dr bouwer and partners inc, south africa) for changes in haematology before and 48 h after treatment. frozen serum samples from all time points were analysed 2 weeks later for specific clinical chemistry parameters (ast, ggt, urea, creatinine, creatine kinase, tsp, albumin and globulin) (onderstepoort clinical pathology laboratory, south africa). serum samples were also analysed by indirect fluorescent antibody test for changes in b. bovis or b. bigemina antibodies pre-treatment and post-treatment (day 5) (onderstepoort veterinary institute, south africa) (tønnesen et al. 2006). clinical monitoring took place from 0–120 h post turpentine or saline injection, and 37 days later. parameters monitored included presence of salivation, restlessness, head shaking, licking of the injection site, looking at the injection site and swelling at the injection site. swellings were measured with callipers. changes in body temperature were also monitored. on day 37, the animals’ additional evaluation included measuring skin thickness and pregnancy evaluation by rectal palpation. data analysis all results were analysed using the spss 20 (ibm, south africa). normality was tested with a shapiro-wilk’s test. a paired t-test was used to assess for changes to pre-treatment values per treatment group, whilst an anova was used to test for differences between groups at selected time points. when significance was evident on anova, a dunnett post-hoc test was used to ascertain if any of the groups were significantly different to the control. biological significance was evaluated against the reference range provided by the evaluating laboratories. the serology results for b. bovis and b. bigemina before and after treatment were evaluated by means of a pearson’s chi-square test. ethical approval top ↑ this study was approved by the animal use and care committee of the university of pretoria. results top ↑ babesia cultures results are reported only for b. caballi, as b. bigemina failed to grow. no reasons could be found for the latter, as both parasites were placed under the same conditions, except for the source of the red cells. no change was evident in b. caballi growth in the turpentine-treated and negative control wells (figure 1), whilst those with diminazene and imidocarb showed negligible growth (0.01% vs 0%). following subculture of the treated well, the parasite numbers of the turpentine treatment cultures further increased, albeit at a lower percentage than the control group. imidocarb-treated and diminazene-treated wells showed no recovery of the parasites on subculture. no significant difference was present between the treated and control wells for any of the evaluated time points. figure 1: percentage parasitaemia following direct exposure of babesia caballi cultures to turpentine and percentage parasitaemia following subculture in the absence of turpentine. animal phase study the animals remained healthy for the duration of the study, despite the turpentine-treated animals all showing a mild febrile reaction (39.08 ± 0.19, 39.78 ± 0.70, 39.78 ± 0.33 and 39.58 ± 0.23 for the control and three turpentine groups in ascending order, respectively). the mild febrile reaction occurred concurrently with an injection site reaction characterised as palpable masses under the skin. reactions were most severe 2 days and 3 days post treatment, with 0, 1, 2, and 2 animals in the saline, 2 ml, 6 ml and 10 ml turpentine groups, respectively showing severe reactions. despite the febrile reactions, most of the animals showed an increase in mass by day 37 post treatment. for no apparent reason, three animals in the 10 ml turpentine group developed corneal opacity 2 days after administration. the opaque corneas did not, however, interfere with the animals’ mass, feed intake or behaviour. the corneal opacity in two of these animals had resolved by day 5 of the study, whilst the third had resolved by day 37. whilst not specifically evaluated in the study, at least two animals per group were confirmed pregnant at dosing. no abortions were recorded for the period of monitoring. subsequent telephonic discussion with the farm owner confirmed that all animals calved successfully. all of the evaluated clinical pathology parameters measured prior to the start of the study (0 h) were not statistically different (table 1), with the exception of eosinophil counts (p-value = 0.02), with the 2 ml turpentine dose being higher than the control group on post hoc analysis (p-value = 0.009). this difference was not considered to be of biological significance, as it was within the reference interval (0.00 × 109/l – 2.4 × 109/l) specified by the laboratory. at 18 hours, only the neutrophil counts were found to be significantly different on anova (p = 0.015), with the 2 ml (normal dose) (p-value = 0.009) and the 10 ml (5x normal dose) (p-value = 0.035) dosing groups being significantly different from the control group. however, with the evident change being within the reference interval, this change is not considered to be of biological significance. the laboratory reported no babesia parasites for any of the blood smears evaluated. no differences in the clinical chemistry were present in the 18 h and 48 h samples (table 2). for the 120 h time point, a significant difference was present for globulins (p = 0.46) (table 3), with the 6 ml (p-value = 0.035) and the 10 ml (p-value = 0.049) groups being significantly different from the control group. for the 6 ml group, all six animals, and five animals for the 10 ml group, were outside the globulin reference interval (28 g/l – 42 g/l). total serum proteins (tsp) demonstrated significant difference in the 6 ml group (p = 0.038). all animals tested serologically positive for exposure to either b. bigemina or b. bovis, despite having no prior history of vaccination. when the animals were evaluated for the presence or absence of positive titres (> 1/80) to b. bovis (22 positive before and 18 after treatment) or b. bigemina (18 before and 7 after), no changes in titres were evident before or after treatment. due to the unexpected decrease in the number of positive animals for the latter, the contract laboratory reanalysed all samples and was able to reproduce this peculiar result.  table 1: haematological parameters obtained for the treatment groups before (0 h) and after (18 h) treatment with turpentine in comparison to the control groups. table 2: clinical chemistry parameters obtained for the treatment groups before (0 h) and after (18 h) treatment with turpentine in comparison to the control groups. table 3: clinical chemistry parameters obtained for the treatment groups after treatment (48 h and 120 h) with turpentine in comparison to the control groups. discussion top ↑ in the management of babesiosis, the methods available to induce immunity are to decrease the parasite burden through the use of drugs such as diminazene or imidocarb (vial & gorenflot 2006). the alternative method would be to vaccinate the animal, either with the live parasite in combination with treatment or to use a sub-unit protein vaccine that stimulates an immune response directly (timms et al. 1984). for the present study, both of these effects were evaluated indirectly. for the former, the direct effect of turpentine on the parasite was evaluated in blood cultures, at various concentrations, for death or altered capacity to divide. the immune stimulatory effect was evaluated through in vivo changes in babesia-specific antibody titres and circulating leucocyte counts. in both cases, higher concentrations than clinically recommended were used.for the cell culture experiment, the cultures were exposed to four different concentrations of turpentine oil. these concentrations were calculated based on the dose of 2 ml used by the famers, which are believed to be unlikely to achieve a plasma concentration above 20 µg/ml. the primary culture revealed no primary parasiticidal activity on the cultured organism. the subculture did show reduced growth of 30% in comparison to the controls, which may indicate a potential ‘post-antibiotic’ effect. in a study using the same parasite, naidoo et al. (2005) speculated that this effect was due to the product’s innate ability to interfere with cell division, with the result that the parasites could not divide at the same rate as the control group (static effect). nonetheless, the effect seen is considered to be minor and is probably insufficient to offer protection, as both imidocarb and diminazene resulted in 100% parasite clearance. the failure of turpentine to produce a visible effect in vitro was also not unexpected, despite farmers reporting the successful management of treated animals with clinical signs of babesiosis. a similar negative result was reported in 1918 when turpentine was tried as a means of treating horses with theileria equi (formerly nutella equi, later b. equi and more recently t. equi) (de kock 1918). according to this historic report, the treated horses showed no signs of improvement and died from classical equine anaemia. whilst it may be argued that the absence of a positive result was due to this study making use of b. caballi and not b. bovis or b. bigemina, in reality both these species are sensitive to imidocarb dipropionate and diminazene under clinical conditions, making it likely that the turpentine would allow for the sufficient clearing of both these parasites. with regard to the direct effect on the immune system, healthy animals under field conditions were exposed to a single intramuscular administration of turpentine. all treatments were well tolerated with a general increase in mass in all the treated animals on day 37. the administered turpentine did, however, induce a mild to moderate inflammatory reaction at the injection site that was reversible with time. this reaction occurred in conjunction with a mild increase in body temperature and globulins, the latter most likely being a non-specific change resulting from the acute inflammatory response. the only other major clinical change evident was an increase in the occurrence of corneal opacity in the animals receiving the higher turpentine dose and a high incidence of injection site reactions. for the former, no explanation could be found and it was of minimal importance to the animals as they continued to feed without any discomfort. whilst the turpentine was not expected to increase the antibody response directly, it was considered to be theoretically possible, albeit very unlikely, that the turpentine could act as an adjuvant to a naturally acquired premunity, as these animals grazed on infected fields. to evaluate for this effect, animals were evaluated for changes in the anti-babesia antibody titres as well as changes in circulating leucocyte counts. whilst changes were evident in serum globulin concentration, this change was not associated with changes in the globulin titres against both b. bovis and b. bigemina. therefore, this increase would be an indication of an inflammatory response, especially when considered in conjunction with the injection site reaction, as opposed to a specific humoral immunity response. the absence of change in peripheral lymphocyte and monocyte counts was an unexpected finding. previous studies in the mouse showed that turpentine oil increased plasma concentrations of interleukin-6 (il-6) (tsujinaka et al. 1997). with il6 known to be an important stimulator of lymphocyte differentiation (horn, henze & heidrich 2000), an increase in lymphocyte counts was expected. in addition, in vitro bovine lymphocyte cultures exposed to babesia antigens showed a direct proliferative response after 6 days of exposure (tetzlaff et al. 1992). in the absence of any meaningful clinical pathological changes, it is believed that the maximum of 10 ml (± 200 µl/kg, 5x utilised dose) was insufficient to stimulate any meaningful response in cattle. conclusion top ↑ the lack of an in vitro parasiticidal effect and the poor static effect of medicated turpentine, in combination with the lack of a specific humoral response and a non-specific lymphocytic cellular immune response, suggest that medicinal turpentine is not a direct anti-babesial compound. however, to conclusively demonstrate this effect, an infectious model for post-turpentine exposure may be required to determine whether the product has an indirect effect. based on the somewhat slower growth of babesia in the culture systems it may be possible, albeit doubtful, that the product slows down babesia growth, thereby allowing for the generation of protective immunity in vivo under field conditions. acknowledgements top ↑ many thanks to mrs antoinette josemans of the onderstepoort veterinary institute, for assistance with the babesia cultures, the staff of both the clinical pathology laboratories for their assistance and the owners of the farms for the use of their cattle, staff and facilities for the tolerance study. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions l.j.b. (university of pretoria) participated in the study design, carried out the laboratory and clinical research and drafted the manuscript as a post-graduate student; c.a.p.c. (university of pretoria) acted as student supervisor, participated in the study design and edited the manuscript; v.n. 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sauerwein, r.w., 1992, ‘cytokines inhibit the development of liver schizonts of the malaria parasite plasmodium berghei in vitro’, european journal of immunology 22, 2271–2275. http://dx.doi.org/10.1002/eji.1830220914 article information authors: osama m. ishag1 intisar k. saeed2 yahia h. ali2 affiliations: 1rabak veterinary research laboratory, white nile state, sudan 2department of virology, veterinary research institute, soba, sudan correspondence to: osama ishag email: samsovet@yahoo.com postal address: po box 293, white nile state tx 79371, sudan dates: received: 31 oct. 2014 accepted: 01 june 2015 published: 21 aug. 2015 how to cite this article: ishag, o.m., saeed, i.k. & ali, y.h., 2015, ‘peste des petits ruminants outbreaks in white nile state, sudan’, onderstepoort journal of veterinary research 82(1), art. #897, 4 pages. http://dx.doi.org/10.4102/ojvr.v82i1.897 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. peste des petits ruminants outbreaks in white nile state, sudan in this research communication... open access • abstract • introduction • outbreak investigation • discussion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ eight outbreaks of peste des petits ruminants in sheep and goats were reported in white nile state, sudan, between 2008 and 2009. a mortality rate of 4.2% was reported across the different outbreaks. clinically the disease was characterised by high fever, ocular and nasal discharge, pneumonia, ulceration of the mucous membranes, diarrhoea and death. the post-mortem findings included necrotic lesions in the mouth and gastrointestinal tract, and swollen, oedematous lymph nodes associated with the lungs and intestine. of the 209 serum samples tested by competitive enzyme-linked immunosorbent assay, 113 (54%) were found positive. peste des petits ruminants virus was confirmed in tissues, nasal swabs and blood samples by immunocapture enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction and isolation of the virus in culture of lamb testicle cells. introduction top ↑ peste des petits ruminants (ppr) is an acute, highly contagious viral disease of sheep and goats (diallo et al. 2007; kwiatek et al. 2007). the ppr virus (pprv) is classified as a member of the genus morbillivirus in the family paramyxoviridae. the genome, which consists of single-stranded negative-sense rna of approximately 16 kb, encodes eight proteins, namely a nucleocapsid protein (n), a phosphoprotein (p), a matrix protein (m), a fusion protein (f), a haemagglutinin protein (h), a large polymerase protein (l) and two non-structural proteins (c and v) (bailey et al. 2005; singh et al. 2004). genetic classification of pprv has identified four lineages by partial sequencing of the f and n genes (rossiter 2004). the economic impacts of the disease are due to high morbidity and mortality. morbidity of between 50% and 100% has been reported, and mortality of 20% – 100% (roeder & obi 1999; singh & prasad 2008). lower mortality is common in endemic areas (roeder & obi 1999), whereas higher mortality is generally observed when pprv infection is associated with other diseases, such as sheep and goat pox (dhar et al. 2002). the mortality rate may vary between species and it has been reported that the case-fatality rate can reach 55% – 85% in goats, 10% in sheep and 50% in camels (khalafalla et al. 2010; radostits et al. 2007). high abortion rates have been reported in goats (abubakar, ali & khan 2008). ppr is considered to be the main constraint to increasing sheep and goat production in countries where it is endemic and the most economically important viral disease of small ruminants where it occurs (nanda et al. 1996; rossiter 2004). diagnosis of ppr is based on clinical examination, post-mortem lesions and laboratory tests. the main symptoms of the disease are fever, pneumonia with nasal and ocular discharges, and diarrhoea. in addition to viral isolation, laboratory tests to detect the antigen, genome and antibodies are available. the competitive enzyme-linked immunosorbent assay (celisa) test used for detection of ppr antibodies is very sensitive and specific (libeau et al. 1995). libeau et al. (1994) also developed an immunocapture elisa (icelisa) for detection of pprv antigen, which is reliable and can be used to differentiate ppr from rinderpest. ppr historically occurred in west africa, the middle east and south asia, and more recently has also been reported from northern sub-saharan africa. the distribution of ppr has expanded during the recent past, with the disease detected for the first time in uganda and kenya in 2007, in tanzania in 2009, in morocco in 2008 (kwiatek et al. 2011), in tunisia in 2007 (ayari-fakhfakh et al. 2011) and in angola in 2012 (world organisation for animal health [oie] 2012). in sudan, ppr was first reported by el hag (1973) and since then regular outbreaks have been reported, mainly in sheep and goats (kwiatek et al. 2011). however, it has not previously been confirmed in white nile state. this report describes outbreaks that occurred amongst sheep and goats there in 2008/2009. outbreak investigation top ↑ white nile state is situated in the south of northern sudan, (13 16’27”n; 33 26’59”e) and has a total area of 39 701 km2 (figure 1). the population of the state is estimated at 1.73 million inhabitants, about two-thirds of whom live in rural areas (central bureau of statistics 2009). the small ruminant population is estimated at about 1.8 million sheep and close to 1.7 million goats, with a rural household having, on average, eight goats and one sheep. the main production systems are nomadic pastoralism and agro-pastoralism. small ruminants often feed in open pastures on natural grazing, where they are herded by children or women. livestock and its production is considered an important source of protein, livestock provide saving and insurance for rural households and 32% of farm income to rural households in white nile state. figure 1: map of the sudan, with the area of study indicated. over a period of 2 years (2008–2009), outbreaks of a fatal disease in sheep and goats were reported to veterinary authorities in white nile state. the first case of the disease was reported to the rabak veterinary clinic. the animals presented with signs of pyrexia with inappetence, discharge from the eyes, nose and mouth, diarrhoea and death. symptomatic treatment was given to sick animals without significant response. the disease then also occurred in different parts of the state. in all areas where the disease was reported that were visited, animals were examined, clinical signs and mortality rates were recorded, and epidemiological data were collected. samples collected included 209 serum samples (38 from goats and 171 from sheep), 20 whole-blood samples and 80 swabs taken from sheep and goats with and without signs of ppr. twenty tissue samples from lung and lymph nodes were also collected, homogenised as a 20% suspension in phosphate buffered saline and stored at -20 °c until use. antibody detection using a celisa kit (cirad, montpellier) and antigen detection using an icelisa kit (cirad, montpellier) were performed according to the manual provided with the kits. virus isolates were prepared from lamb testicle cells in a disposable tissue culture flask. for molecular diagnosis, rna was extracted from tissue samples using an rna extraction kit (rneasy mini kit, qiagen, germany) according to the manufacturer’s recommended protocol. the onestep rt-pcr kit (qiagen, germany) was used for amplification of the n protein, following the manufacturer’s recommended protocol. reverse-transcription polymerase chain reaction (rt-pcr) amplified the n protein gene using primers as described by couacy-hymann et al. (2002). clinical signs observed varied between individual animals, but the most commonly observed signs were sudden death of apparently healthy animals, especially in the younger age groups, serous discharge from the eyes, nose and mouth, which later became thick and yellowish, epithelial necrosis of the lips, inner cheeks and upper surface of the tongue, watery foul-smelling diarrhoea, disturbed breathing, dyspnoea and sneezing in an attempt to clear the nose, and death (table 1). the overall mortality rate due to ppr was 4.2%, as shown in table 2. table 1: clinical signs and date of reported cases in white nile state, sudan. table 2: number of animals affected and mortality rate during the 2008–2009 outbreak of peste des petits ruminants in white nile state, sudan. at post-mortem examination, the main findings were necrotic and erosive mucosa inside the lips and on the dorsal surface of the tongue, and congestion of the lung and small intestine; the associated lymph nodes were congested, enlarged and soft. as shown in table 3, 113 serum samples were found positive for pprv by celisa, with more adult animals affected than young animals. table 3: analysis of serum samples as tested by competitive enzyme-linked immunosorbent assay. using icelisa, pprv antigen was detected in 11% of swab samples, 35% of tissue samples and 30% of whole-blood samples (table 4). table 4: analysis of swab, tissue and whole-blood samples as tested by immunocapture enzyme-linked immunosorbent assay. pprv was isolated successfully in lamb testicle cells. typical pprv cytopathic effects (cpe) appeared on day 14 post inoculation and the final harvest of the infected cells was on day 25 post inoculation. two further passages were made on lamb testicle cells. of the seven tissue samples (lungs and lymph nodes) tested by rt-pcr, all were found positive. discussion top ↑ during 2008 and 2009 eight outbreaks of an unknown fatal disease, with symptoms of pneumonia and diarrhoea, were reported in sheep and goats in white nile state, sudan. based on the epidemiological, clinicopathological and virological findings, the disease was confirmed to be ppr. data collected from the field indicated that farmers had no knowledge about ppr before the outbreaks, although the disease had been reported in all states bordering white nile state. many of the rural population cross the state with their animals in search of green pastures and water and therefore contagious diseases such as ppr can be transmitted from sick to healthy animals when they mix whilst grazing and drinking. field and laboratory investigations indicated that the disease manifested as acute to peracute in the younger age groups. the features of the disease were discharge from the eyes, nose and mouth, necrotic stomatitis, diarrhoea and pneumonia, in line with symptoms that have been reported previously (aiello & mays 1998; khan et al. 2008; roeder & obi 1999). abubakar et al. (2009) stated that the seroprevalence of ppr is high in sheep and goats older than 2 years, but al-majali et al. (2008), ozkul et al. (2002) and singh et al. (2004) reported that sheep and goats between 4 months and 2 years are more likely to be seropositive for ppr. the study showed that there was no significant difference in the number of seropositive sheep and goats older than 2 years than younger ones and more sheep were seropositive than goats; however, megersa et al. (2011), reported a higher seroprevalence in goats than in sheep and linked it to higher fecundity in goats than in sheep. it was noted in this study that climatic factors had a marked influence on the spread of ppr, with most outbreaks occurring in the colder months, similar to reports by durojaiye, obi and ojo (1983). pprv antigen was confirmed by icelisa in samples that were taken from sick animals and at post-mortem examinations of animals that had recently died of ppr. all tissue samples tested for the pprv genome were found positive by means of rt-pcr. this result agrees with previous reports that described the high sensitivity of rt-pcr using np3/np4 primers for detection of ppr nucleic acids (couacy-hymann et al. 2009; kumar et al. 2007). isolation of pprv in lamb testicle cell culture produced a cpe consisting of rounding of cells and syncytia formation at 14 days post inoculation. this finding is similar to that described by hamdy et al. (1976) and intisar (2002), although intisar (2002) reported that growth of pprv in lamb testicle cells was slower, with a cpe observed only 17–20 days post innoculation. after pprv had been diagnosed and confirmed to be the cause of this outbreak, a mass vaccination campaign was undertaken by the veterinary authority in white nile state and quarantine and movement control were instituted to prevent spread of the disease. the possibility that the severity of the disease observed in the outbreaks was due to the appearance of a new lineage (lineage iv) in sudan was considered. however, an investigation of samples from various regions in sudan, including a sample from these outbreaks, indicated a gradual replacement of lineage iii by lineage iv from about mid 2000 (kwiatek et al. 2011). the low mortality observed (see table 2) suggests possible previous exposure, which may have rendered a relatively high proportion of the older animals immune, whereas young animals developed severe symptoms. acknowledgements top ↑ the authors would like to thank the international atomic energy agency (iaea) for providing celisa, icelisa and onestep rt-pcr kits. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions o.m.i. (rabak veterinary research laboratory) was the project leader and was responsible for the study design, sample collection and data analysis. i.k.s. (veterinary research institute) performed some experiments and y.h.a. 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http://dx.doi.org/10.1023/b:verc.0000042875.30624.67 singh, b. & prasad, s., 2008, ‘modelling of economic losses due to some important disease in goats in india’, agriculture economics research review 21, 297–302. world organisation for animal health (oie), 2012, peste des petits ruminants, angola, viewed on 15 october 2014, from http://www.oie.int/wahis_2/public/wahid.php/reviewreport/review?page_refer=mapfulleventreport&reportid=12408 article information authors: christo j. botha1 matthew j. legg2 mariëtte truter3 michael sulyok4 affiliations: 1department of paraclinical sciences, university of pretoria, south africa2private practitioner, benoni, south africa 3biosystematics division, agricultural research council-plant protection research institute, south africa 4department of agrobiotechnology (ifa-tulln), university of natural resources and life sciences, austria correspondence to: christo botha postal address: private bag x04, onderstepoort 0110, south africa dates: received: 14 july 2014 accepted: 25 aug. 2014 published: 12 nov. 2014 how to cite this article: botha, c.j., legg, m.j., truter, m. & sulyok, m., 2014, ‘multitoxin analysis of aspergillus clavatus-infected feed samples implicated in two outbreaks of neuromycotoxicosis in cattle in south africa’, onderstepoort journal of veterinary research 81(1), art. #848, 6 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.848 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. multitoxin analysis of aspergillus clavatus-infected feed samples implicated in two outbreaks of neuromycotoxicosis in cattle in south africa in this research communication... open access • abstract • introduction • case histories    • case 1    • case 2 • materials and methods    • fungal isolation    • mycotoxin analysis • results    • fungal identification    • mycotoxin analysis • discussion • acknowledgement    • competing interests    • authors’ contributions • references abstract top ↑ aspergillus clavatus intoxication is a highly fatal neuromycotoxicosis of ruminants, especially cattle. it is caused by the ingestion of infected sprouting grain and sorghum beer residue. locomotor disturbances, tremors and paralysis are observed. histologically, degeneration and necrosis of larger neurons in the medulla oblongata, the midbrain, the thalamus and the ventral horns of the spinal cord are observed. although a range of mycotoxins such as patulin, cytochalasin e and pseurotin a have been isolated, there is limited information on which specific mycotoxin or group of mycotoxins are involved during outbreaks of intoxication in livestock. in the present study, two outbreaks of a. clavatus poisoning in cattle are briefly described. feed samples were collected for fungal identification, and culture and multitoxin analysis. a range of fungal metabolites were detected, and the estimated concentrations (μg/kg) are provided. both the sprouting barley and brewer’s grain were predominantly infected with a. clavatus and, to a lesser extent, rhizopus arrhizus. the only common aspergillus secondary metabolite present in all the samples was pseurotin a. patulin and cytochalasin e were present in the sprouting barley samples, as well as the a. clavatus isolates cultured on malt extract agar for 2 weeks; however, neither of these mycotoxins could be detected in the brewer’s grain sample. introduction top ↑ ingestion of aspergillus clavatus-infected feed may induce a highly fatal neuromycotoxicosis in ruminants, especially in cattle; however, sheep have also been poisoned (gilmour et al. 1989; kellerman et al. 2005; riet-correa et al. 2013; schlosberg et al. 1991). this intoxication occurs sporadically in south africa (kellerman et al. 2005), but has also been reported in other countries (riet-correa et al. 2013; sabater-vilar et al. 2004; schlosberg et al. 1991). infected sprouting grains and malt are usually the cause, although, in south africa, sorghum beer residue (maroek) has also been implicated. aspergillus clavatus is a saprophytic fungus that often overgrows other fungal contaminants in the feed and is then visible as a greenish-blue mould (el-hage & lancaster 2004; kellerman et al. 1976; kellerman et al. 1984).the clinical signs of a. clavatus poisoning in cattle include: hypersensitivity, tremors (which are not consistently present), ataxia, progressive paresis and paralysis. affected animals walk with a stiff-legged gait, take short steps and sometimes knuckle over at the fetlocks. they may stagger and fall before paralysis and death set in. neurological signs that are not evident at rest can be precipitated following exercise. in addition, drooling of saliva and constipation have also been reported (el-hage & lancaster 2004; kellerman et al. 1976; kellerman et al. 1984). chronically affected cattle can survive for several weeks. there appears to be an extended latent period or a cumulative effect. during the experimental reproduction of this neurotoxicity, kellerman and co-workers (1976) noted that cattle only became sick 7–10 days after commencement of feeding the poisonous ration or sorghum beer residue. el-hage and lancaster (2004) reported that new cases appeared up to 18 days after removal of infected sprouted barley. gross lesions are visible in the skeletal muscles of severely affected animals that exhibit clinical signs for longer periods (kellerman et al. 2005). a greyish-white appearance of the larger muscles of the hindlimbs and forelimbs, especially near their origins and insertions, has been reported (kellerman et al. 1976; loretti et al. 2003; riet-correa et al. 2013). the most important microscopic lesions, which are also useful for diagnostic confirmation, occur in the brain and spinal cord. degeneration and necrosis of larger neurons (chromatolysis and cytoplasmic vacuolation) in nuclei of the medulla oblongata, the midbrain, the thalamus and the ventral horns of the spinal cord have been described (kellerman et al. 1976; loretti et al. 2003). furthermore, hyaline degeneration and necrosis of skeletal muscles and myocardial degeneration and necrosis have also been noted (kellerman et al. 1976; loretti et al. 2003). aspergillus clavatus synthesises mycotoxins such as: patulin (lopez-diaz & flannigan 1977); cytochalasin e (büchi et al. 1973; lopez-diaz & flannigan 1977); tremorgenic metabolites such as tryptoquivalone (clardy et al. 1975) and tryptoquivaline and its related toxic metabolites (büchi et al. 1977; clardy et al. 1975). case histories top ↑ case 1 in november 2013, a group of young heifers and bulls exhibited neurological signs such as: knuckling over at the fetlocks (figure 1), muscle fasciculation, hyperaesthesia, weakness of the hind quarters, ataxia, stumbling and recumbency. when handled, some collapsed and displayed mild seizure-like activity with paddling, but when left undisturbed were able to rise again. the farmer produced sprouting barley to feed the animals. the sprouting barley was heavily contaminated with fungi (figure 2); two samples were collected and submitted for fungal identification and mycotoxicological analysis. figure 1: an affected heifer exhibiting flexion (over-knuckling) of the fetlocks. figure 2: aspergillus clavatus-infected sprouting barley. case 2 in march 2014, 12 friesland dairy cows died acutely after exhibiting moderate muscle tremor, aggressive behaviour and paralysis. the cows were fed a total mixed ration containing silage, brewer’s grain, maize stover and a commercial concentrate. a set of tissue samples was collected and submitted for histopathology. distinctive microscopic lesions in the central nervous system confirmed a. clavatus poisoning. later, three animals became recumbent with the hindlimbs extended in an abnormal position behind them. eventually, a total of 32 highly prized dairy cows succumbed. the brewer’s grain and commercial pelleted ration were submitted for mycological identification and mycotoxicological screening.there is limited information on which specific toxin or group of toxins are involved during outbreaks of intoxication (lopez-diaz & flannigan 1977; sabater-vilar et al. 2004). although a range of mycotoxins have been isolated from toxic a. clavatus strains, none of the metabolites have been administered to ruminants in order to reproduce the intoxication (mckenzie et al. 2004). the objective of the present study was to submit feed samples implicated in the two recent outbreaks and a. clavatus isolates cultured from the barley for comprehensive screening for fungal metabolites. materials and methods top ↑ fungal isolation two sprouting barley samples from case 1 and one brewer’s grain sample and one commercial pelleted ration from case 2, were visually inspected and isolations made directly from surface fungal growth by plating small clumps of fungal spores and/or mycelia onto potato dextrose agar (pda) (merck, germany) amended with penicillin (250 mg/l). isolations were also made from the plant material. seeds (n = 25) and root segments (n = 25, 10    mm long) from the sprouting barley and malt residue (n = 50) from brewer’s grain were surface disinfected with 1% sodium hypochlorite (3 min), rinsed twice with sterile water, blotted dry and plated onto pda amended with penicillin. plates were incubated for 7–14 days, at 25 °c under cool-white fluorescent light. the plates were checked regularly and selected colonies were transferred to half-strength pda. isolates retrieved were identified based on culture and morphological characteristics. aspergillus isolates were plated to czapak yeast agar (cya), czapek yeast agar with 20% sucrose (cy20s) and malt extract agar (mea) (klich 2002) and incubated in the dark at 25 °c for 7 days. one plate of cya was incubated at 37 °c for 7 days. rhizopus isolates were plated to pda and mea and incubated in the dark at 25 °c and 37 °c for 7 days (schipper 1984). selected single-spore isolates were deposited in the culture collection of the national collections of fungi, biosystematics division, plant protection research institute, agricultural research council (arc-ppri), pretoria, south africa. mycotoxin analysis to comply with international phytosanitary regulations the feed samples collected from both outbreaks (barley 1, barley 2 and brewers grain; table 1) were air dried and finely milled before being shipped to austria for multitoxin screening. in addition, two a. clavatus isolates (ppri 13831 and ppri 13832; table 1) were also submitted for mycotoxin screening. a standardised extraction procedure was followed (sulyok et al. 2006). briefly: 5 g of each sample were extracted for 90 min with 20 ml of acetonitrile/water/acetic acid (79:20:1, v/v/v) on a rotary shaker (gfl 3017, gfl, burgwedel, germany). the crude extracts were diluted 1+1 (v+v) with acetonitrile/water/acetic acid (20:79:1, v/v/v) and 5 µl of the diluted extract were injected. table 1: fungal metabolites and estimated concentrations (µg/kg) in feed samples and aspergillus clavatus isolates. table 1 (continues...): fungal metabolites and estimated concentrations (µg/kg) in feed samples and aspergillus clavatus isolates. detection and quantification were performed with a liquid chromatography/tandem mass spectrometry (lc-ms/ms) system (qtrap5500, applied biosystems, foster city, california, united states of america) equipped with an electrospray ionisation (esi) source (turboionspray, agilent technologies, waldbronn, germany) and an ultra-high performance liquid chromatography system (1290 series, agilent technologies, waldbronn, germany) (malachova et al. 2014; vishwanath et al. 2009). chromatographic separation was performed at 25 °c on a gemini c18-column, 150 mm × 4.6 mm inner diameter (i.d.), 5 μm particle size, equipped with a c18 security guard cartridge, 4 mm × 3 mm i.d. (all from phenomenex, torrance, california, usa). elution was carried out in binary gradient mode. both mobile phases contained 5 mm ammonium acetate and were composed of methanol/water/acetic acid 10:89:1 (v/v/v; eluent a) and 97:2:1 (v/v/v; eluent b), respectively. after an initial time of 2 min at 100% a, the proportion of b was increased linearly to 50% within 3 min. further linear increase of b to 100% within 9 min was followed by a hold-time of 4 min at 100% b and 2.5 min column re-equilibration at 100% a. the flow rate was 1000 μl/min. the esi-ms/ms was performed in the scheduled multiple reaction monitoring (smrm) mode both in positive and negative polarities in two separate chromatographic runs (malachova et al. 2014; vishwanath et al. 2009). the smrm detection window of each analyte was set to the respective retention time ± 27 s and ± 42 s in positive and negative modes, respectively. the target scan time was set to 1s. confirmation of positive analyte identification was obtained by the acquisition of two smrms per analyte (with the exception of moniliformin and 3-nitropropionic acid that each exhibit only one fragment ion), which yields 4.0 identification points according to commission decision 2002/657/ec (european union 2002). analyst® software version 1.5.1 (ab sciex, foster city, california, usa) was used to control the lc-ms/ms instrument, as well as for automatic and manual integration of the peak (malachova et al. 2014; vishwanath et al. 2009). quantification of the > 300 metabolites included in the method was done based on linear, 1/x weighed calibration curves derived from the analysis of serial dilutions of a multi-analyte stock solution. results were not corrected for apparent recoveries. results top ↑ fungal identification both the sprouting barley and brewer’s grain were predominantly infected with a. clavatus and, to a lesser extent, rhizopus arrhizus. in contrast, the commercial pelleted ration yielded only rhizopus and no aspergillus colonies. plated plant material from the sprouting barley resulted in 70% a. clavatus and 50% r. arrhizus colonies, whereas the brewer’s grain resulted in 100% a. clavatus and 20% r. arrhizus colonies when plated on the respective agars. several plant material segments were colonised by both fungi. aspergillus clavatus isolates (ppri 13831, 13832, 14650 and 14651) had colony diameters of 38 mm – 42 mm on cya, 40 mm – 45 mm on mea, 43 mm – 48 mm on cy20s and 20 mm on cya at 37    °c. stipes were 500 µm – 1500 µm x 20 µm – 30 µm, smooth-walled, colourless, and expanding gradually into clavate vesicles, 50 µm – 75 µm wide. conidial zone extended from 50 µm – 150 µm down from the top of the vesicles; heads were uniseriate with phialides 7 µm – 9 µm x 3.5 µm. conidia were smooth-walled, ellipsoidal, 4 µm – 6 µm x 3 µm – 4 µm, and dull turquoise in mass. colonies of r. arrhizus (ppri 14572, only one isolate selected for preservation) were greyish brown. rhizoids were brownish. sporangiophores on stolons were up to 1500 µm in length, up to 18 µm in width, brown, single or aggregated in small groups. sporangia were greyish black, powdery in appearance, up to 175 µm in diameter. sporangiospores were subglobose to ellipsoidal, with ridges on the surface, up to 8 µm in length. mycotoxin analysis the multitoxin screening results are tabulated (table 1). discussion top ↑ patulin is considered to be a major contributor to the neurotoxicity induced by a. clavatus (sabater-vilar et al. 2004). riet-correa et al. (2013) suggested that patulin analysis can aid in confirming a diagnosis of a. clavatus poisoning. in the current investigations, patulin was detected in the sprouting barley samples collected during the first outbreak and both isolates also synthesised this metabolite. the concentrations were considered to be high, as maximum tolerable levels ranging from 10 µg/kg – 50 µg/kg have been set in the european union for a range of apple-based and fruit-based commodities, including baby food (commission regulation 1881/2006). on the other hand, patulin could not be detected (< limit of detection [lod]) in the brewer’s grain sample collected during the second outbreak, despite histological confirmation of a. clavatus poisoning. kellerman et al. (1976) could also not demonstrate patulin in toxic fractions isolated with column chromatography from an extract prepared from sorghum beer residue (maroek) collected during an outbreak in south africa. however, despite mycological verification of a.  clavatus contamination of the brewer’s grain collected during the second outbreak, not all fungal strains are toxic, which might explain the absence of patulin in the sample (loretti et al. 2003). another common metabolite, cytochalasin e, was present in the barley samples as well as the isolates, but was also below the limit of detection in the brewer’s grain sample.the only common aspergillus secondary metabolite present in all the samples was pseurotin a, although at a comparatively lower concentration in the brewer’s grain. at relatively high concentrations, pseurotin a is reported to be cytotoxic to cell lines (a2780 human ovarian carcinoma cells and rat pheochromocytoma cells [pc12 cells]). on the other hand, at lower concentrations it induces neurite outgrowth in pc12 cells. the authors concluded that pseurotin a may possess neuroprotective properties that may prevent or be useful to treat human diseases related to dysfunction of the nervous system such as senile dementia and alzheimer’s disease (komagata et al. 1996). it is thus impossible to conclude that pseurotin a is involved in the neurotoxicity seen in cattle. the tremorgenic compound, tryptoquivaline f was also present in the barley samples, but the concentration could not be determined, as no standard is available. no consistent pattern with respect to the presence of mycotoxins could be discerned when the incriminated feed samples collected during the two outbreaks were compared (table 1). although mycological identification alone is usually insufficient to confirm a diagnosis and analytical tests are advisable, the question still remains as to which mycotoxin or combination of mycotoxins are involved. the specific mycotoxin or group of mycotoxins responsible for the neurotoxicity induced during these outbreaks could not be identified. furthermore, when sampling feed for mycotoxin analysis, it should be remembered that the toxic portion of the feed could have all been consumed and mycotoxins are not homogeneously distributed in a feed sample (sabater-vilar et al. 2004). in both cases investigated, the feed was overgrown by a. clavatus, possibly because this fungus grows and sporulates more aggressively. flannigan (1986) reported that the optimum temperature and relative humidity for growth of a. clavatus is 20 °c – 35 °c and 93% – 98%, respectively. higher temperatures in the hydroponically cultivated barley sprouts will favour growth and a. clavatus will dominate the fungal population (lopez-diaz & flannigan 1977; mckenzie et al. 2004). at 25 °c, the synthesis of specifically cytochalasin e is favoured when compared to patulin production (lopez-diaz & flannigan 1977). in the first outbreak, the stock owner changed the process of producing sprouting barley. where the hydroponically spouted barley was usually produced in trays in a refrigerated container, the batch implicated as the cause of the neurological signs was not produced in a refrigerated environment where the temperature and humidity could be controlled. the nutritional advantage of feeding sprouting grain produced hydroponically is doubtful (mckenzie et al. 2004). this practice is fraught with danger and livestock veterinarians should warn farmers about the risks of fungal contamination. acknowledgement top ↑ the authors would like to thank dr martin ferreira, potchefstroom, for information pertaining to the second outbreak. this work is based upon research supported by the national research foundation of south africa. the lc–ms/ms system was funded by the federal country lower austria and co-financed by the european regional development fund of the european union. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions c.j.b. (university of pretoria) was the principal investigator, m.j.l. (private practitioner) investigated the first outbreak, m.t. (agricultural research council-plant protection research institute) performed the mycological culturing and identification and m.s. (university of natural resources and life sciences) conducted the multitoxin analysis. all authors read and approved the final manuscript. references top ↑ büchi, g., luk, k.c., kobbe, b. & townsend, j.m., 1977, ‘four new mycotoxins of aspergillus clavatus related to tryptoquivaline’, journal of organic chemistry 42, 244–246. http://dx.doi.org/10.1021/jo00422a015büchi, g., kitaura, y., yuan, s.s., wright, h.e., clardy, j., demain, a.l. et al., 1973, ‘structure of cytochalasin e, a toxic metabolite of aspergillus clavatus’, journal of the american chemical society 95, 5423–5425. http://dx.doi.org/10.1021/ja00797a060 clardy, j., springer, j.p., koenig, t., büchi, g., matsuo, k. & wightman, r., 1975, ‘tryptoquivaline and tryptoquivalone, two tremorgenic metabolites of aspergillus clavatus’, journal of the american chemical society 97, 663–665. http://dx.doi.org/10.1021/ja00836a045 commission decision of 12 august 2002 implementing council directive 96/23/ec concerning the performance of 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cytochalasin e by aspergillus clavatus during malting of barley and wheat’, international journal of food microbiology 35, 129–136. http://dx.doi.org/10.1016/s0168-1605(96)01211-1 loretti, a.p., colodel, e.m., driemeier, d., corrêa, a.m., bangel, j.j. & ferreiro, l., 2003, ‘neurological disorder in dairy cattle associated with consumption of beer residues contaminated with aspergillus clavatus’, journal of veterinary diagnostic investigation 15, 123–132. http://dx.doi.org/10.1177/104063870301500206 malachova, a., sulyok, m., beltran, e., berthiller, f. & krska, r., 2014, ‘optimization and validation of a quantitative liquid chromatography-tandem mass spectrometric method covering 295 bacterial and fungal metabolites including all regulated mycotoxins in four model food matrices’, journal of chromatography a 1362, 145–156. http://dx.doi.org/10.1016/j.chroma.2014.08.037 mckenzie, r.a., kelly, m.a., shivas, rg., gibson, j.a., cook, p.j., widderick, k. et al., 2004, ‘aspergillus clavatus tremorgenic neurotoxicosis in cattle fed sprouted grains’, australian veterinary journal 82, 635–638. http://dx.doi.org/10.1111/j.1751-0813.2004.tb12614.x riet-correa, f., rivero, r., odriozola, e., de lourdes adrien, m., medeiros, r.m.t. & schild, a.l., 2013, ‘mycoxicoses of ruminants and horses’, journal of veterinary diagnostic investigation 25, 692–708. http://dx.doi.org/10.1177/1040638713504572 sabater-vilar, m., maas, r.f.m., de bosschere, h., ducatelle, r. & fink-gremmels, j., 2004, ‘patulin produced by an aspergillus clavatus isolated from feed containing malting residues associated with a lethal neurotoxicosis in calttle’, mycopathologia 158, 419–426. http://dx.doi.org/10.1007/s11046-005-2877-x schipper, m.a., 1984, ‘a revision of the genus rhizopus’, studies in mycology 25, 1–34. schlosberg, a., zadikov, i., perl, s., yakobson, b., varod, y., elad, d. et al., 1991, ‘aspergillus clavatus as the probable cause of a lethal mass neurotoxicosis in sheep’, mycopathologia 114, 35–39. http://dx.doi.org/10.1007/bf00436689 sulyok, m., berthiller, f., krska, r. & schuhmacher, r., 2006, ‘development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize’, rapid communications in mass spectrometry 20, 2649–2659. http://dx.doi.org/10.1002/rcm.2640 vishwanath, v., sulyok, m., labuda, r., bicker, w. & krska, r., 2009, ‘simultaneous determination of 186 fungal and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry’, analytical and bioanalytical chemistry 395, 1355–1372. http://dx.doi.org/10.1007/s00216-009-2995-2 article information authors: japhta m. mokoele1,2 b. tom spencer1 leo a.m.g. van leengoed1,3 folorunso o. fasina1 affiliations: 1department of production animal studies, university of pretoria, south africa2limpopo department of agriculture, limpopo, south africa 3department of farm animal health, utrecht university, the netherlands correspondence to: folorunso fasina postal address: private bag x04, onderstepoort post office, south africa dates: received: 31 mar. 2014 accepted: 05 june 2014 published: 12 nov. 2014 how to cite this article: mokoele, j.m., spencer, b.t., van leengoed, l.a.m.g. & fasina, f.o., 2014, ‘efficiency indices and indicators of poor performance among emerging small-scale pig farmers in the limpopo province, south africa’, onderstepoort journal of veterinary research 81(1), art. #774, 12 pages. http://dx.doi.org/10.4102/ ojvr.v81i1.774 copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. efficiency indices and indicators of poor performance among emerging small-scale pig farmers in the limpopo province, south africa in this original research... open access • abstract • introduction • materials and methods    • study area and data collection    • data collection and management • results • market • discussion • conclusions • acknowledgements    • competing interests    • authors’ contributions • references • appendix 1 abstract top ↑ limpopo is a very important area for pig production in terms of animal populations and contributions to transboundary animal disease spread. emerging small-scale pig farmers (esspf) are being encouraged to establish operations and spread in south africa; however, for these farmers to perform optimally, they need to understand the basics of animal agriculture and contribute to enhancing biosecurity and efficient production systems. in the present study, the limitations to efficient production amongst esspf were evaluated and some improvements were suggested. it was found that the esspf are dominated by males and include a large percentage of older persons. a total of 26.54% of these farmers have post-matriculation qualifications. undefined and indigenous breeds still dominate their animal genetics. the animal health technicians are the preferred channels by which farmers report diseases to the authorities (52.47%) and only one out of five (20.37%) will preferably report a disease situation direct to a veterinarian. these farmers do not vaccinate their stock, and knowledge of biosecurity is poor. antimicrobials, especially tetracyclines, are abused. animals that are slaughtered within the community or sold at local sale points, pension pay stations and auction markets are likely candidates for disease spread. it is recommended that the younger generations are retained and incentivised in animal agriculture. improved training on management, health, biosecurity and better market access must be provided for the esspf, whilst efforts should made to consolidate these farmers into small cooperatives. the current government agricultural support system will need to be reworked to benefit the resource-poor farmers. collaborative efforts in disease reporting and management among veterinarians, animal health technicians and extension officers will become necessary. finally, the creation of a progressive quality grading system for esspf should be planned by the industry and this should be attached to a reward system that will encourage these farmers to target good farming practice. introduction top ↑ pigs are of high economic importance, especially among the poor. they contribute to human nutrition, food security, poverty alleviation, enhanced livelihood and creation of employment for the rural community (antwi & seahlodi 2011; dietze 2011; mergenthaler, weinberger & qaim 2009). in addition, they provide a less-expensive source of animal protein for urban diets compared with cattle, sheep and goats (ironkwe & amefule 2008). whilst pig farming, as part of animal agriculture, is central to the development of rural farmers, the real contribution of emerging small-scale pig farmers (esspf) to the rural economy is not well assessed and somewhat doubtful. these economic contributions by esspf are constrained by management, health, housing, feeding and marketing constraints (antwi & seahlodi 2011; chikazunga et al. 2007).in south africa, it is suggested that there were approximately 125 000 production sows in 2010/2011, with approximately 100 000 sows being held commercially and the remaining 25 000 being kept by small-scale farmers. the south african pork industry contributes around 2.15% to the primary agricultural sector, and whilst 2 616 000 pigs were slaughtered in 2011, the total pork production was put at 203 375 tonnes in cold dressed mass (food and agriculture organization corporate statistical database [faostat] 2014). limpopo is a major pig-producing province in south africa and, in 2011, contributed approximately 24.00% of the total recorded pig production (department of agriculture, forestry and fisheries [daff] 2012). only about 11 700 of all the sows in the province are registered as commercial sows and there is a widespread distribution of non-registered and disorganised medium-scale and small-scale pig farms. in view of its location and poorly controlled boundaries with other countries, previous studies have indicated that limpopo has played a key role in some transboundary animal disease disseminations and is responsible almost exclusively for new introductions of african swine fever into south africa (boshoff et al. 2007). since all the index cases, to date, of outbreaks of african swine fever in south africa have originated from limpopo and the majority of these outbreaks were linked with small-scale pig farms, it is critical to understand the management and production systems of this category of farms in order to effectively carry out proactive disease prevention and management. the present study was conducted in order to collect basic management and health parameters from esspf in limpopo, so that their production efficiency and contributions in terms of biosecurity and health could be evaluated. materials and methods top ↑ study area and data collection five districts in limpopo province were studied, namely: sekhukhune, capricorn, waterberg, vhembe and mopani (figure 1). these districts have partially documented records (n = 85) of esspf; limpopo’s department of agriculture is in the process of fully documenting the records of these pig farmers. this will enable targeted interventions in terms of infrastructure development, veterinary extension services, and production and marketing inputs to be carefully planned to assist these farmers. a purposive sampling method was used to select esspf (n = 185) from the study sites, including the 85 small-scale pig farmers enlisted on limpopo’s department of agriculture list and an additional 100 farms that fall within this category but were not listed. the expansion of the list beyond the department list became necessary because the preliminary data from the field suggested that there were many unlisted esspf in the province (steyn et al. 1994). the inclusion criterion for the present study was pig farms with ≤ 50 sows, located within the five districts of limpopo, that have been active in pig production for at least one year. a participatory research model approach was used (raman, sanghi & chambers 1989; thrusfield 1995). figure 1: map of limpopo, showing study sites 2012–2013. data collection and management based on available literature, expert opinions and primary objectives of the present study, a semi-structured questionnaire (see appendix 1 for content) was designed at the department of production animal studies, university of pretoria. this was pilot tested amongst veterinary students (n = 12) and a few extension veterinarians (n = 2). the questionnaire was later reviewed and validated during organised consultative forums by selected pig veterinarians in the field, the research committees of limpopo department of agriculture and the faculty of veterinary science, university of pretoria, as well as state veterinarians and animal health technicians of the limpopo department of agriculture. the instrument was used to collect data from the selected participants using a participatory method, as described by thrusfield (1995). a total of 185 questionnaires were collected from the field, but 20 were filtered out due to missing values and inconsistent data. another respondent was removed because he had increased his herd size to a 150 sow unit. a total of 164 respondents (88.65%) were included in the analysis. the data from completed questionnaires were coded, captured and filtered using a microsoft excel® (microsoft, usa) spreadsheet, and descriptive statistics were performed. the analyses performed included: proportional percentages; measures of central tendencies; percentiles and graphs of farm characteristics; production parameters; management and health parameters; and operational efficiencies using stata v9.0 (stata corporation, lakeway drive, college station, texas, usa). efficiency indices for piglets weaned per sow and for average days to oestrus were calculated by dividing the category mean by mean total number of sows for the category. correlation coefficients were calculated for farm parameters that were thought to influence one another in the analysed data. results top ↑ male farmers represented the majority of the participating respondents (76.83%), which is a reflection of the population structure amongst the esspf (table 1a). similarly, the farmers who were ≥ 46 years old were 78.53% of the total respondents; 5.52% were ≤ 35 years and 15.95% were 36–45 years old. in addition, a total of 77.78% of the respondents were married (table 1a). the majority of the farmers interviewed (63.80%) were engaged full time in their pig farming activities, whilst only 26.90% were involved part time. table 1a: characteristics of emerging small-scale pig farmers, limpopo, south africa. table 1b: characteristics of emerging and small-scale pig farms, limpopo, south africa. it was noted that 26.54% of the total respondents had post-matriculation qualifications. although the definition of breeds was based on phenotypic characteristics of the pigs observed on the farms, 61.18% of the breeds used were indigenous or not well described, 28.29% were large white, 10.53% were landrace and 7.24% were duroc breeds (table 1a). a total of 2.47% esspf farmers had previously benefited from the comprehensive agricultural support programme (casp) of the limpopo department of agriculture. the average number of sows per farm was 7.4 and the average number of days for a sow to return to oestrus was 42.9 days after weaning the litter, whilst the number of piglets weaned per sow per year was 4.85 pigs (table 1b). at this rate there would need to be some 50 000 sows (unregistered) in limpopo to make up half of the 24.00% of slaughter pigs that are not accounted for by the 11 700 registered sows mentioned in the introduction above. it is questionable whether this really is the case. the majority of the respondents (92.36%) did not provide a heat source for their piglets, especially in the cold winter, and this greatly impacted on increased levels of mortalities. in addition, 44.00% lost piglets primarily due to overlay and hypothermia, whilst 62.67% claimed that piglets were lost principally to multiple causes (table 2). the interviewed farmers complained about skin conditions in their herds and approximately 46.84% of the respondents identified skin diseases (primarily mange) as being the most important disease complex on their farms (table 2). other disease complexes that were rated lower by the farmers included: reproductive (12.66%), enteric (8.86%), respiratory (6.33%), musculoskeletal (5.05%) and mixed infections (39.87%) (table 2). table 2: management and health parameters of emerging and small-scale pig farms, limpopo, south africa. a total of 52.47% of the esspf preferred to report to and use the service of animal health technicians. state veterinarians were the first point of contact for one out of five farmers (20.37%), and 17.28% preferred to contact the extension officers first. knowledge and implementation of biosecurity was poor amongst the surveyed farmers, as only 8.07% washed their hands before or after farming activities, and only 1.24% utilised footbaths in their farms. although 65.84% indicated that they had built fences, it was noted that these fences were not purpose built for biosecurity and were only extensions of the human accommodations. most importantly, 98 (77.00%) of the farmers did not vaccinate their pigs against major pig diseases, including parvovirus infection, leptospirosis and erysipelas. terramycin was the most abused drug used by 30.38% of the responding esspf, as no diagnosis was reached before treatment was implemented in most cases. market top ↑ the majority of the respondents (82.61%) preferred or were obliged to sell their pigs at local points and within communities, 9.32% would sell at the auctions and 14.09% at formal abattoirs or supermarkets. a total of 40.67% of the farmers transported their pigs to the slaughter facilities and only 1.33% of these farmers transported live pigs from different sources in the same vehicle (table 3). these transportations vary widely between < 50 km and 400 km, depending on the distance of the major abattoir or auction points (figure 2). figure 2: distance analyses of movement from farms to major slaughter and marketing points, (a) belfast auction, (b) phalaborwa abattoir, (c) bronkhorstspruit abattoir and (d) thabazimbi abattoir. table 3: market-related characteristics of emerging and small-scale pig farmers, limpopo, south africa. an evaluation of the price that the farmers received per pig sold revealed that there is a great lack of coordination in pricing and no template exists to standardise sales. for example, a 32-week-old pig (50 kg – 70 kg) sells for approximately zar 500 compared to zar 1200 for a 20-week-old pig (≈ 70 kg) in a formal market. it should, however, be emphasised that these pigs often have a high bone-to-meat ratio when compared with those originating from a commercial operation. although a good proportion (41.25%) of the farmers claimed to use concentrates in feeding their pigs, evidence based on the drawn-up checklist contradicted this assertion. it appeared that most of the farmers used kitchen remnants and mixed portions, and only supplemented with vegetables and concentrate feeds (figure 3). a total of 43.83% used boreholes as sources of water for their piggery but a proportion (32.10%) used village streams and other water sources like rivers (9.26%) and municipal water (6.78%) (figure 3). figure 3: main source of feed and water for the pigs. the majority of the farmers (81.76%) had less than 10 sows (figure 4, table 4), and this category of farmers appeared to have the best efficiency index compared to other categories in terms of number of piglets weaned per sow per year (n = 5.19). it appeared that the higher the number of sows per esspf, the lower the efficiency of weaning per individual sow on the farm (table 4). figure 4: evaluation of farm sow population against average number of piglets weaned per year. table 4: efficiency index of piglets weaned per sow. the average number of days after weaning for sows to return to oestrus in the essp farms was 42.9 days (table 5), but there was significant difference amongst the different categories (1–10, 11–20, 21–30, 31–40, 41–50) (p < 0.0001). sows of those farmers with between 1–10 sows took approximately 49.9 days to return to oestrus, whilst those with between 21 and 30 sows returned to oestrus within 16 days (table 5, figure 5); however, some farms with less than 10 sows took up to 210 days for their sows to return to oestrus (figure 5). table 5: efficiency index of return to oestrus per sow. figure 5: evaluation of farm sow population against average number of days to return to oestrus. overall, the level of education was negatively correlated with all of the farm parameters assessed, except the piglets per sow per year, where it had a very poor correlation. the total piglets weaned was positively correlated with the total number of piglets born per sow per year (39.86%) and the number of sows on the farm was positively correlated with the total piglets weaned (30.97%) (table 6). table 6: correlation coefficient of sows with certain farm and farmer parameters. discussion top ↑ the present study’s findings and analyses revealed some deficiencies. it is a difficult task to collect critical production parameters where no records exist to validate the collected information, and the farmers’ perceptions and recall are the only forms of validation. in the present study, efforts were made to use check questions and interviewees’ observational analyses to validate some of the data collected from the farmers. despite this challenge, the present study has revealed that most of the esspf in limpopo province are males (77.00%), a fact that has raised questions about the issue of women in agriculture and economic empowerment in this sector. it appears that the pig industry has a large imbalance in terms of ownership and gender, and there may be a need to transform the industry and provide more opportunities with a critical focus on women from rural areas. it should be understood, however, that pig farming is labour-intensive in terms of input; few women may be willing to get involved in such activities. whilst gender equity is one of the major standpoints of the land reform policies, to date, female ownership of land and other means of agricultural production, especially in the rural areas, is still viewed with some degree of abhorrence in the region (anon 2014; cross & hornby 2002; kalabamu 2006).the majority of the farmers were older than 45 years of age, which is an indication that the younger generation prefer not to get involved in agriculture but would rather migrate to urban areas in search of salaried jobs (brooks et al. 2013). this observation may also be an indication of late entry into animal agriculture. a similar trend has been observed in other studies (oladele, kolawole & antwi 2013; schembri et al. 2013). currently, it is widely believed that only the poor become involved in rural farming and that it is not a financially rewarding activity. it is recommended that policies be implimented that encourage younger individuals to be retained in the rural areas at all levels in order to reduce gross emigration to the city, whilst boosting agricultural productivity in the rural areas. because pig farming in the rural areas is labour intensive, larger families and married people are at an advantage, as two or more people are involved. the outcome of the present study’s analysis indicates that 78.00% of all respondents were married. the majority of the farmers were also educated up to the end of high school (58.64%). previous workers have confirmed that the level of education has a positive relationship on market access (lubungu, chapoto & tembo 2012). since farmers with post-secondary education constituted just over a quarter of the total respondents, it is expected that these educated farmers positively influence the market and open access for the less educated ones. it is important to emphasise that the majority of the respondents still kept indigenous and crossbred pigs; these results are comparable with other data from india (70.00%) (nath et al. 2013). these indigenous and undefined pigs have been known to under-perform compared with the exotic breeds in terms of litter size, litter weight, birth weight, weaning weight and average daily weight gain, and they often have poor access to veterinary services (halimani et al. 2010). extension services must be targeted towards encouraging farmers to adopt the improved breeds of pigs in rural farm operations. although the department of agriculture, forestry and fisheries has made certain provisions available for funding of animal infrastructure in terms of the casp, only 2.47% of the respondents have benefited. it will be necessary for government departments to fine-tune agricultural policies and remove unnecessary bottlenecks that impede the development of esspf so that planned programmes and policies can reach the targeted beneficiaries. in the current programme, the farmers are supported in terms of provision of 10 sows or a housing unit for 10–50 sows. a realistic economic model has indicated that farmers will need between 150 and 250 sows to be commercially viable. whilst the government may not be able to provide this level of support for all the esspf, the options of forming them into smaller cooperatives to benefit from economies of scale and become more competitive in terms of input supplies and marketing should be explored (mashala 2012). in addition, financial institutions should be encouraged to provide low-interest agricultural financing to farmers with viable projects. with regard to productivity, analysis indicated that the esspf are poorly productive, compared with the commercial operations. they weaned an average of 4.85 piglets per sow per year, which is 19.00% of the standard for the south african pig industry (26 piglets per sow per year) and it took much longer for the sows to return to oestrus (42.9 days compared with 4–6 days) (fasina et al. 2012). the statistics generated in the present study are incompatible with economically feasible and viable pig production; there will be a need for a major shift if the essp farms are to be commercially viable and sustainable in south africa. at the same time as consideration is given to these statistics, they should also be carefully examined, since large variations exist between the farmers’ operational efficiencies in each category. hypothermia is a major source of neonatal and piglet mortalities; it also predisposes piglets to other causes of mortalities including diseases, crushing and starvation (kammersgaard, pedersen & jørgensen 2011; pedersen et al. 2012). hypothermia will significantly reduce the ability of piglets to access the sow and get colostrum within the first 6 h – 12 h after birth to meet their nutritional requirements, to benefit from maternal immunity and protect against diseases. regular supervision in the farrowing house would help to reduce the levels of mortalities in terms of prevention of hypothermia, regular feeding, reducing illnesses and maintaining sow’s udder health (shankar, madhusudhan & harish 2009); this was lacking in the surveyed farms. the great majority of the respondents (92.36%) did not provide any heat source and the high level of pre-weaning mortality observed in the farms of esspf in limpopo can be attributed to this situation. pedersen et al. (2012) confirmed that mortality associated with hypothermia can be seven times more severe in piglets subjected to suboptimal environmental and floor temperatures in the first week of birth (kammersgaard et al. 2011). building of simple, but cosy, creeps using local materials can be a cheap but good alternative to using electricity to heat up creep areas. this should potentially reduce hypothermia and encourage piglets to suckle more, with consequent reduction in piglet pre-weaning mortality, and boost performance. pigs are exposed to a variety of predisposing factors and pathogens causing diseases. primary diseases and conditions of concern in the industry include production-limiting diseases, respiratory complexes and lameness-associated problems, amongst others. in the present study’s analyses, for ease of recognition by the esspf, disease complexes were grouped as syndromes. the disease conditions were grouped as respiratory, enteric, skin or integumentary, musculoskeletal and reproductive syndromes. a total of 46.84% of the respondents indicated that skin conditions were a major challenge on their farms. further enquiries from respondents confirmed that the conditions in pigs were observed as scratching, with discoloured or thickened skin, scabs and hair loss, among others, which are indications of sarcoptic mange (arends, stanislaw & gerdon 1990). mange significantly depresses growth rate and feed efficiency, and it is expected that a considerable loss in days-to-market prevails amongst the pigs from these farms for that reason. many of the farmers sell their pigs at an average of 8 months, a loss of about 90 days compared to the commercial operations. a total of 22.15% of the respondents mentioned ivermectin as a frequently used medicine on the farms. it is particularly concerning that terramycin is used routinely by about 30.00% of the farmers, without proper diagnosis and consideration of its consequences. it is recommended that a community-specific farm-health plan be constructed to target this group, using state veterinarians and animal health technicians. such health plans must include messages on the importance of vaccination, antibiotic abuse and biosecurity, areas, where serious deficiencies were observed amongst the respondents (food and agriculture organization [fao] 2010; fasina et al. 2012). vaccination against very important production-limiting diseases of pigs in south africa (parvovirus, leptospirosis and erysepelas, as well as escherichia coli) must be included in such protocols. similarly, it would be beneficial to use vaccines on a cost-sharing basis between farmers. since the farmers use the services of animal health technicians, extension officers and veterinarians, there is a need for coordination among these professionals to maximise the impact of state veterinary extension services and block loopholes that may exist with individual efforts. data, which are comparable to the present study’s statistics, about increased reliance on technicians rather than the veterinarians by esspf, has been reported elsewhere (alawneh et al. 2014). it should be emphasised that disease complexes among the essp farms pose major risks to the commercial operations. therefore the inclusion of private veterinarians and large commercial farms in sponsoring veterinary extension services amongst these individuals whilst serving the role of mentors and patrons must be critically evaluated. the extension messages must also target how market access can be created for these farmers and a suitable grading system that will encourage them to maximise productivity and move towards good farming practices. the role of veterinary extension and the use of multiple professionals to meet the challenges of animal health services have been emphasised previously (hernández-jover et al. 2008; mockshell, ilukor & birner 2014). the determinants of cooperation and referrals between paraprofessionals and veterinarians have been previously identified to include mobile phone ownership, training, cumulative annual assessment, and membership of paraprofessional bodies and association (ilukor, nielsen & birner 2014). it is necessary to facilitate inter-professional relationships amongst animal health service providers in rural south africa by provision of the identified facilities. nutrition and feeding are very important components of animal production and health; in the present study, it was concluded that the esspf are more likely to swill-feed the pigs, with potential consequences of spreading diseases, including exotic pig diseases like classical swine fever, foot-and-mouth disease and porcine respiratory and reproductive syndrome. similarly, since the swill is not evaluated for its nutritional qualities, it becomes difficult to assess whether this feed meets the nutritional requirement of the different classes of pigs on the farms. it is probable that the weaning and growing pigs are underfed and this is responsible for their taking longer to reach appropriate market weights (manchidi 2009; viljoen 1993). water is a critical resource in any pig production unit, as it is required for cleaning the pens, drinking purposes and cooling the pigs. in many of the farm units included in the present study, pigs get water only twice a day, whilst certain piggeries only supply water every other day. limited access to water has negative effects on pigs, as it retards their growth potential and affects many other biochemical processes, resulting in conditions such as salt toxicity (manchidi 2009). based on estimation, the greater the distance between the household or production site and water source, the higher the probability of not providing water regularly. since about 45.06% of the farmers only depended on village streams or other distant sources for water, pigs reared under these conditions are likely to suffer degrees of water deprivation. however if a farmer wants to proceed with a regular supply of feed and water, despite the distance between the farm and sources of supplies, a higher capital cost will be incurred and realistic profitable operation will become more difficult (mabuza & ngubane 2010). finally, the south african pig retail industry is broadly classified into a dual market structure: the high-value markets (processors and supermarkets) for commercial pig farmers and the low-value markets (local auctions, pension pay points for the esspf). whilst the high-value markets pay premium prices for quality products, the emerging small-scale pig farmers get paid below the market values for their pigs (antwi & seahlodi 2011). the present study’s findings confirmed this assertion, since no coordinated pricing and standardised sale template exists for this category of farmers. the use of extension services to improve sectoral marketing and possible formation into cooperatives is critical. conclusions top ↑ the authors’ acknowledge: the limpopo department of agriculture for providing funds for this survey as part of a postgraduate programme; the director of veterinary services in limpopo province, dr mampane and his team of managers in the districts, and all the veterinary officials for their help with collection of data in their respective districts. the department of production animal studies and the faculty of veterinary science, onderstepoort partially supported this work with funds and permitted the use of their resources for postgraduate training. mrs ingrid booysen of the department of geography, geoinformatics and meteorology kindly produced the maps. acknowledgements top ↑ the authors’ acknowledge: the limpopo department of agriculture for providing funds for this survey as part of a postgraduate programme; the director of veterinary services in limpopo province, dr mampane and his team of managers in the districts, and all the veterinary officials for their help with collection of data in their respective districts. the department of production animal studies and the faculty of veterinary science, onderstepoort partially supported this work with funds and permitted the use of their resources for postgraduate training. mrs ingrid booysen of the department of geography, geoinformatics and meteorology kindly produced the maps. competing interests the authors declare that they have no financial or personal relationship(s) that may have inappropriately influenced them in writing this article. authors’ contributions j.m.m. (university of pretoria), b.t.s. (university of pretoria) and f.o.f. (university of pretoria) formulated the hypothesis and questionnaire design and validation; l.a.m.g.v.l. 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n., hernandez-jover, m., toribio, j.a.l.m.l. & holyoake, p.k., 2013, ‘demographic and production practices of pig producers trading at saleyards in eastern australia’, australian veterinary journal 91, 507–516. http://dx.doi.org/10.1111/avj.12131 shankar, b.p., madhusudhan, h.s. & harish, d.b., 2009, ‘pre-weaning mortality in pig – causes and management’, veterinary world 2(6), 236–239. steyn, a.g.w., smit, c.f., du toit s.h.c. & strasheim, c., 1994, modern statistics in practice, van schaik publishers, pretoria. thrusfield, m., 1995, veterinary epidemiology, 2nd edn., blackwell science ltd., london. viljoen, j., 1993, ‘nutrient requirements of growing pigs – birth to marketing’, in e.h. kemm (ed.), pig production in south africa, bulletin 427, pp. 66–75, arc-irene animal production institute, pretoria. appendix 1 top ↑ appendix 1 appendix 1 continues appendix 1 continues bruckner_141-146.indd introduction the creation of the office international des epi zo oties (oie) as an independent inter-governmental international organisation in 1924, offered the international community well before the creation of the united nations (oie 2008a), a totally new and much needed international decision-making forum to guard against the regional and global spread of infectious and trade sensitive animal diseases and zoonosis. from the 28 founding countries, membership has since grown to 172 countries and territories. known today as the world organisation for animal health, the initial objectives of the oie remain just as relevant as when it was founded. these objectives have been expanded through the years to encompass the overall objective of promoting global animal health. in 1994, the world trade organization (wto) mandated the oie as the international reference organisation for international standards, guidelines and recommendations related to global animal health with the main purpose of facilitating international trade in terrestrial and aquatic animals and their products and to avoid the introduction of pathogens via international trade in animals and animal products, while at the same time preventing countries from setting up unjustified sanitary barriers to inhibit trade (wto 1995). the oie in pursuance of this mandate has also initiated several other measures to facilitate the international trade in terrestrial and aquatic animals and their products, the most important being to assist members to identify their needs for veterinary serv141 onderstepoort journal of veterinary research, 76:141–146 (2009) the role of the world organisation for animal health (oie) to facilitate the international trade in animals and animal products g.k. brückner world organisation for animal health (oie), 12 rue de prony 75017, paris, france abstract brückner, g.k. 2009. the role of the world organisation for animal health (oie) to facilitate the international trade in animals and animal products. onderstepoort journal of veterinary research, 76:141–146 the international trade in animals and animal products has become a sensitive issue for both developed and developing countries by posing an important risk for the international spread of animal and human pathogens whilst at the same time being an essential activity to ensure world-wide food security and food safety. the oie has since its founding in 1924, applied a democratic and transparent decision-making process to continuously develop and review international standards for animal health and zoonoses to facilitate trade in animals and animal products. the role of the oie is also mandated by the world trade organization (wto) as international reference point for standards related to animal health. in support of its overall objective of promoting animal health world-wide, the oie has also launched several other initiatives such as the improvement of the governance of veterinary services within its member countries and territories and to enhance the availability of diagnostic and scientific expertise on a more even global geographical distribution. several trade facilitating concepts such as country, zonal and compartment freedom from disease as well the trade in disease free commodities has been introduced to enhance the trade in animals and animal products for all its members including those from developing and transitional countries who are still in the process of enhancing to full compliance with international sanitary standards. 142 oie to facilitate international trade in animals and animal products ice delivery to progress towards compliance with oie international standards. the oie internal standards, guidelines and recommendations for animal and aquatic health the negotiation process between importing and exporting countries to initiate the trade in animals and animal products is usually underpinned by the mutual understanding between trade partners that trade would be accepted or allowed by the importing country if the exporting country can give acceptable sanitary guarantees to protect animal and human health in accordance with the appropriate level of sanitary protection determined and required by the importing country for importing that specific animal or animal product. to facilitate the negotiation process and to discourage importing countries to insist on sanitary guarantees from exporting countries that would inhibit or prevent trade or to require sanitary requirements that are not scientifically justifiable, the wto has for this very reason mandated the oie within the san itary and phytosanitary agreement (sps agreement) of the wto, as the international reference organization for setting standards for the international trade in animals and animal products. a similar man date was given in terms of the sps agreement to the codex alimentarius commission (codex) for food safety and the international plant protection con vention (ippc) for matters related to plant health. some of the key principles within the sps agreement are that standards set by the oie should be science based and must be such that the main aim would be to protect animal and human health without imposing unattainable conditions for trade. countries that are signatories to the sps agreement and who are also members of the oie are obliged to honor this important requirement in trade negotiations (brück ner 2004). countries are therefore encouraged to apply the minimum standards, guidelines and recom mendations set by the oie and wherever possible incorporate it into their national legislation. should a standard required by an importing country be higher than that required in the oie standards recommended for trade purposes, it must be scientifically justified and based on a risk assessment on the importing commodity (wto 1995). oie standards are by default already the outcome of a risk assessment and need in general not again be subjected to a risk assessment for trade purposes. the terrestrial animal health code and the aquatic animal health code of the oie contain the standards to be applied in the international trade in terrestrial and aquatic animals and their products. these standards are democratically adopted by the members of the oie after been debated by specialist ad hoc groups under guidance of the oie specialist com missions: the scientific commission for animal dis eases, the biological standards com mission, the aqua tic animal health standards com mission and the terrestrial animal health standards commis sion. standards are revised and updated on a continuous basis. the companion complementary volumes, the oie manual of diag nostic tests and vac cines for terrestrial animals (oie 2008b) and the similar manual for aquatic animals, specify reference techniques for diagnosing animal diseases, tests that are prescribed and recommended for trade purposes, and export certification and quality requirements for vaccines for specific animal diseases. while the focus of this paper will mainly be on animal health issues related to terrestrial animals, the same principles apply to aquatic animals. oie standards reflect a horizontal and a vertical dimension. the horizontal standards are described in volume i and vertical standards in volume ii of the 2008 edition of the code. horizontal standards are those describing generic aspects, such as ethics in international trade or the quality of national veter inary services, a sine qua non condition for importing countries to trust the reliability of health certificates accompanying consignments of animals and products in cross-border trade. these certificates must be issued exclusively by the veterinary services under the full responsibility of the government of the exporting country. other guidelines and recommendations to facilitate trade are also described in the code such as procedures for pathogen inactivation; disposal of carcasses and dead animals; recommendations for disinfection, transport of animals by land, air or sea; identification and traceability of animals and products; requirements for border control and quarantine station; import risk analysis; equivalence; obligations and ethics in international trade; zoning and compartmentalisation; transfer of biological material and other animal welfare considerations such as slaughter of animals. vertical standards are those describing specific recommendations for each of the oie listed animal diseases and zoonoses. the focus is on the animal health situation in the exporting country and assumes that either the disease is not present in the importing country or is the subject of a control or 143 g.k. brückner eradication programme. these standards specify for each disease an incubation period based on a safe risk mitigation waiting period for trade purposes; the commodities that can be traded irrespective of the disease status of a country for that specific disease; the requirements to certify a country, zone or compartment free from that disease and the risk mitigation measures than can be applied by the exporting country or required by an importing country before an animal or product is imported. general surveillance guidelines are described that could be applied for most diseases but for some diseases such as foot-and-mouth disease (fmd), highly pathogenic avian influenza and bluetongue, specific surveillance guidelines have been developed to prove absence of the circulating pathogen or to confirm claims for freedom from disease. the oie offers a voluntary dispute settlement mechanism for mediating trade conflicts between oie members. this is a science-based approach for find ing alternative solutions and resolving differences, as distinct from the legalistic approach used in the formal wto system. the role of the oie is to assist the parties to arrive at a scientifically sound conclusion. oie standards are developed on the basis of a highly meticulous risk analysis taking into account the fact there is no such thing as zero risk. the wealth and range of the standards published in this way enable importing countries to reduce the use of sometimes arbitrary risk analysis methods, and to favour a systematic use of oie standards, as recommended in the sps agreement (wto 1995). trade facilitating initiatives by the oie other than the provision of international standards, recommendations and guidelines more than 70 % of the 172 current members of the oie are from developing and transitional countries. it would therefore be naïve to accept that all members of the oie would have equal opportunities, capacity, expertise, financial means, infrastructure and level of veterinary service delivery to also equally apply these standards. the oie also fully realizes that the disease situation varies from country to country and that for some it would be much easier to give assurances of disease freedom or absence of disease than for others. more than 70 % of the current oie listed diseases still occur for example on the african continent—either sporadically or in an endemic form while in some countries in the northern hemisphere, only five to ten of these diseases might still be present (erlacher-vindel, brückner & vallat 2007). evaluation of veterinary service delivery to assist those members who have indicated the wish to know their current level of performance in applying international standards for veterinary service delivery, the oie has initiated a process to assess the performance of the veterinary services of countries to identify their needs in eventually moving towards compliance with oie standards. this process known as the pvs evaluation process, applies a specific methodology where trained oie experts visit countries wishing to be evaluated. the focus of the evaluation is on assessing performance of the country within the context of specific critical components for veterinary service delivery: human, physical and financial resources; technical authority and capability; interaction with stakeholders and access to markets. the level of advancement of a country is assessed in detail in respect of the critical competencies within each of these four components. an initial pvs evaluation is ideally followed by either a second evaluation or a more detailed gap analysis to assess the financial and other resources needed to advance within the critical competencies of each component. more than a diagnostic instrument, the oie pvs tool promotes a culture of raising awareness and continual improvement, which can be used either passively or actively depending on the level of interest, priorities and commitment of the veterinary services and its stakeholders. in the passive mode, the oie pvs tool helps to raise awareness and improve the understanding of all sectors including other administrations regarding the fundamental components and critical competencies the veterinary services must have in order to function effectively (oie 2008a). the active mode is where the maximum outcomes are realised but this mode requires a sustained commitment by both the public and private sectors. in this mode, performance is assessed, differences are explored and priorities are established, strategic actions are outlined, investments are evaluated and agreed to, and commitments made and implemented. continuity of this process requires a true partnership between the public and the private sectors. leadership on the part of the public sector is a fundamental and critical determinant of success. according to g. funes (personal communication 2008) of the current 172 members of the oie, 80 countries have to date requested to be subjected to 144 oie to facilitate international trade in animals and animal products a pvs evaluation with 36 (21 %) from africa representing 70 % of the current 52 countries in africa that are members of the oie. improvement of veterinary diagnostic capacity integral to the pvs assessment process, is the identification of the need to establish scientific and technological expertise within these countries to enable them to become self-sufficient in the early detection and diagnosis of diseases and to be able to provide the scientific justification for certification of animals and animal products for trade. the oie, realising that the majority of its 177 reference laboratories and 29 collaborating centres and expertise are located mostly in developed countries in the northern hemisphere, accepted that this ideal can only be achieved by actively moving towards scientific capacity building with the eventual aim of establishing focal points of expertise and more reference laboratories within developing countries. to enable the realisation of this ideal, the concept of twinning between laboratories was born, the aim of which is to ensure eventually an even global geographical distribution of expertise and reference laboratories allowing easier access to experts and for the rapid detection and diagnosis of disease. the principle of the twinning initiative is that an agreement is reached between a candidate laboratory in a developing country and a parent oie reference laboratory or oie collaborating centre for which the parent laboratory over a specified period helps the candidate laboratory to acquire the necessary expertise to eventually be able to qualify to become a designated oie reference laboratory for a particular disease. the oie has obtained substantial donor support to facilitate and fund this process (erlacher-vindel et al. 2007). in some instances, countries, due to the lack of resources and scientific expertise, are prone to exploitation in trade related issues such as the imposing of a ban on all imports from a country that has notified an outbreak of an oie listed disease. such a decision taken by some members to impose a total ban on imports of animals and products without referring to the relevant provisions of the code for these commodities is unacceptable. though this approach is acknowledged for the few days it might take while awaiting more precise information on the animal health situation in the affected country, it should rapidly be replaced by protection measures based on oie standards and recommendations for each of the commodities concerned. it is therefore important to acknowledge that developing and intransition countries need a veterinary scientific community that is able to participate in the preparation of standards and to obtain the necessary expertise to challenge non-justifiable trade restrictions. to this end, the twinning concept will create the much needed opportunities to enable developing and intransition countries to become scientifically competent to debate on an equal footing the scientific justification and application of standards. the concepts of disease-free countries, zones, compartments and commodities as trade facilitating measures since the early 1990s, the oie has been given by the international committee, composed of the delegates of the oie member countries, the responsibility of compiling a list of member countries or zones that are officially recognised as being free from certain diseases (oie 2008a). for this purpose, a clearly defined and impartial procedure for declaring a member country free from a disease was identified. in may 1995 a new procedure was adopted by the international committee giving a mandate to the oie scientific commission for animal diseases to examine in detail dossiers submitted by the delegates of member countries in support of a claim that their countries or zones within their countries could be considered free of fmd in accordance with the provisions of the terrestrial animal health code. in 1996 the first official list of oie member countries that were fmd free without using vaccination was published after adoption by the international committee. this process has been expanded to also include rinderpest, contagious bovine pleuropneumonia (cbpp) and bovine spongiform encephalopathy (bse) (oie 2008a). however, it also became evident that country freedom due to factors such as high maintenance costs was not attainable and sustainable by all oie members. this realisation resulted in the introduction of the concept where zones within an infected country could be officially recognised free from disease— with or without vaccination (brückner 2004). this process was again later refined to further facilitate trade from countries for which the achievement of neither country freedom nor zonal freedom was possible by introducing the concept of compartmentalisation. while the maintenance of either country or zonal freedom is based on maintaining separation of populations of different health status through mainly geographic entities, the concept of compartmentalisation is based on ensuring the disease free status of an establishment through the application 145 g.k. brückner of strict bio-security management practices. the concept of compartmentalisation is not a totally new concept as in certain countries in africa and also elsewhere in the world, the principle of separating animal populations of different health status through bio-security management practices, was already applied for many years for diseases such as african swine fever and bovine tuberculosis. the oie fully recognises and acknowledges that advancements in science, new knowledge on the epidemiology of diseases and risk mitigation measures that can be applied to render products of animal origin safe from disease, allows for acceptance of the concept that for certain diseases and certain animal products, a specific animal product or commodity could under certain provisions be certified safe for trade purposes. this could even be applied in the event where a certain disease is present in a country but where a specific animal product does not pose a risk for transmitting that disease. the concept of safe commodity trade has therefore already been incorporated into the code where for example milk and milk products, semen, hides and skins, gelatin and collagen from hides and skins and deboned skeletal muscle from cattle under 30 months can be traded without restriction from a country infected with bse (oie 2008a). the same principle is also applicable for deboned bovine meat that has undergone maturation and allowing for a lowering in ph below 6 to render it safe from fmd virus. further research is needed to expand this concept as a trade facilitating measure for other oie listed diseases. the application of this concept either on its own or in combination with either compartmentalisation or zonal freedom from disease would especially facilitate trade from those countries for which country or zonal freedom is not a sustainable option. to promote the development of this concept, the oie will continue to encourage agrifood industries and research institutions to further the development of research programmes on unresolved issues of major importance to the development of world trade. a better knowledge for example of the conditions under which the fmd virus survives or is killed during the meat maturing process might well have a major impact on the normative provisions and the way in which oie members apply the standards contained in the code chapter on fmd. this is an important issue, given that more than 100 oie members are still infected with this disease. it would however, be naïve and irresponsible to adopt an approach based solely on the systematic inactivation of pathogens in products, which could result in members relaxing surveillance activities for animal diseases and policies on the prevention and control of potential biological disasters. the positive impact of animal health policies on poverty reduction and public health is in itself ample justification for financing and maintaining surveillance networks and rapid response mechanisms to deal with animal health threats and risks. to guarantee the effectiveness of surveillance at the national, regional and worldwide level, a nonnegotiable pre-requisite is for all members to comply with oie standards on the quality and evaluation of veterinary services. in addition to their surveillance mission, the veterinary services are also responsible for the reliability of the veterinary certificates they issue. these certificates accompany every consignment of animals or animal products transported in international trade globally. com pliance with oie standards for quality of the veterinary services of a country ensures that these certificates are issued under conditions that guarantee their reliability, so that granting access to regional and global markets for all will not pose a threat to the safety of international trade. conclusion developing countries are increasingly coming under pressure to improve their delivery of veterinary services as a prerequisite for entering the competitive arena of international trade in animals and animal products. the demands placed on developing countries by predominantly developed countries to comply with international disease prevention standards have also resulted in increasing demands on the financial, human and technological resources of these developing countries. the oie in pursuance of its mandate offered in the agreement on the application of sanitary and phytosanitary measures of the wto and the mandate given to the oie by its international committee is committed to facilitate and promote as far as possible within its resources, the international trade in animals and animal products for all its members. achieving this goal does not allow for shortcuts, will take time and demands a rational and scientific approach for future decision-making. realising that compliance with oie standards, guidelines and recommendations are not immediately achievable by all, the exploitation of and research into still more alternatives to facilitate trade without sacrificing the importance of good veterinary governance and the need for eventual compliance with standards, offers 146 oie to facilitate international trade in animals and animal products a major challenge to not only the oie but to all international organisations seeking a more liberalized trading environment and market access for all. references brückner, g.k. 2004. working towards compliance with international standards. revue scientifique et technique, oie, 23:95–107. brückner, g.k., linnane, s. & diaz, f. 2007. responses to a questionnaire on networking between oie reference labora tories and oie collaborating centres, in first international conference of oie reference laboratories and collaborating centres, edited by m. lombard & b. dodet. developmental biology, 128:77–82. oie 2008a. terrestrial animal health code, 17th ed. paris: office international des epizooties. oie 2008b. manual of diagnostic tests and vaccines for terrestrial animals, 6th ed. paris: office international des epi zooties. erlacher-vindel, e., brückner, g.k. & vallat, b. 2007. the oie concept of laboratory twinning, in first international conference of oie reference laboratories and collaborating centres, edited by m. lombard & b. dodet. developmental biology, 128:119–123. wto 1995.the results of the uruguay round of multilateral trade negotiations: the legal texts: agreement on the application of disease control and phytosanitary measures. geneva: world trade organisation. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (coated fogra27 \050iso 12647-2:2004\051) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [850.394 1133.858] >> setpagedevice article information authors: joshua mbanga1 yvonne o. nyararai1 affiliations: 1department of applied biology and biochemistry, national university of science and technology, zimbabwe correspondence to: joshua mbanga email: joshmbanga@gmail.com postal address: po box ac 939, ascot, bulawayo 00263, zimbabwe dates: received: 15 july 2014 accepted: 15 oct. 2014 published: 07 apr. 2015 how to cite this article: mbanga, j. & nyararai, y.o., 2015, ‘virulence gene profiles of avian pathogenic escherichia coli isolated from chickens with colibacillosis in bulawayo, zimbabwe’, onderstepoort journal of veterinary research 82(1), art. #850, 8 pages. http://dx.doi.org/10.4102/ojvr.v82i1.850 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. virulence gene profiles of avian pathogenic escherichia coli isolated from chickens with colibacillosis in bulawayo, zimbabwe in this original research... open access • abstract • introduction • materials and methods    • sample collection    • dna extraction    • virulence genotyping    • sequencing of polymerase chain reaction products to confirm amplification of the regions of interest • results    • escherichia coli virulence gene screening    • single polymerase chain reactions and sequencing • discussion • limitations    • recommendations • conclusion • acknowledgements    • competing interests    • authors’ contributions • references • appendix 1 abstract top ↑ colibacillosis, a disease caused by avian pathogenic escherichia coli (apec), is one of the main causes of economic losses in the poultry industry worldwide. this study was carried out in order to determine the apec-associated virulence genes contained by e. coli isolates causing colibacillosis in chickens. a total of 45 e. coli isolates were obtained from the diagnostics and research branch of the central veterinary laboratories, bulawayo, zimbabwe. these isolates were obtained from chickens with confirmed cases of colibacillosis after postmortem examination. the presence of the iuta, hlyf, ompt, frz, sitd, fimh, kpsm, sita, sopb, uvry, pstb and vat genes were investigated by multiplex polymerase chain reaction (pcr) assay. of the 45 isolates, 93% were positive for the presence of at least one virulence gene. the three most prevalent virulence genes were iuta (80%), fimh (33.3%) and hlyf (24.4%). the kpsm, pstb and ompt genes had the lowest prevalence, having been detected in only 2.2% of the isolates. all 12 virulence genes studied were detected in the 45 apec isolates. virulence gene profiles were constructed for each apec isolate from the multiplex data. the apec isolates were profiled as 62.2% fitting profile a, 31.1% profile b and 6.7% profile c. none of the isolates had more than seven virulence genes. virulence profiles of zimbabwean apec isolates are different from those previously reported. zimbabwean apec isolates appear to be less pathogenic and may rely on environmental factors and stress in hosts to establish infection. introduction top ↑ in chickens, colibacillosis refers to any local or systemic infection caused entirely or partially by escherichia coli (omer et al. 2010). colibacillosis is economically important worldwide as it results in high morbidity and mortality of affected birds (dho-moulin & fairbrother 1999; dziva & stephens 2008). the infection is generally initiated or enhanced by predisposing agents, such as mycoplasmal or viral infections and environmental factors (dho-moulin & fairbrother 1999). however, management approaches based only on protecting poultry against predisposing conditions have proved largely ineffective for controlling outbreaks of avian colibacillosis (barnes, nolan & vaillancourt 2008). antimicrobial drugs remain important in reducing both incidence and mortality associated with this disease (zakeri & kashefi 2012) but there is increasing evidence that avian pathogenic e. coli (apec) is becoming more resistant to antimicrobial agents (saidi, marifakureva & mbanga 2013; sharada, ruban & thiyageeswaran 2009; skyberg et al. 2006). a vaccine based approach for the control of outbreaks of avian colibacillosis is therefore highly desirable (olsen, christensen & bisgaard 2012). currently available vaccines are not totally effective (schouler et al. 2012). this is mainly because of the diverse characteristics of apec strains, which prevent the identification of common properties that could be used as a basis for vaccination (schouler et al. 2012). several virulence factors have been associated with the virulence of apec, including those encoding for adhesins, toxins, iron acquisition systems, autotransporters, sugar metabolism, serum resistance proteins, and capsule as well as lipopolysaccharide complexes (ewers, janssen & wieler 2003; li et al. 2005; schouler et al. 2012). however, numerous studies have demonstrated that these virulence factors are rarely all present in the same isolate and that they can occur either individually or polygenically with varying frequencies in clinical isolates (delicato et al. 2003; vandekerchove et al. 2005). this all indicates that apec strains constitute a heterogeneous group. the accurate identification of virulent strains of e. coli and the virulence genes they possess is essential if genes that can serve as vaccine targets are to be identified. virulence gene studies are therefore important as they not only aid in the characterisation of pathogenic strains of e. coli but may eventually lead to the development of effective vaccines. work on virulence genes in the western world and most of asia is on the rise, but in africa very little information seems to be available (randall et al. 2012; van der westhuizen & bragg 2012). in this study, 12 different apec virulence genes were used to characterise zimbabwean apec isolates. the 12 genes used in this study included the iuta, hlyf, ompt, frz, sitd, fimh, kpsm, sita, sopb, uvry, pstb and vat genes (full names provided in table 2). these have all been previously reported in literature, and some have been shown to occur in high frequencies in apec isolates from other countries (johnson et al. 2008; kafshdouzan et al. 2013; schouler et al. 2012). a number of studies have reported the successful use of multiplex polymerase chain reaction (pcr) in the detection of virulence genes in e. coli isolated from different organisms like dogs (pass, odedra & batt 2000), pigs (schierack et al. 2006) and poultry (ewers et al. 2005). this study used multiplex pcr developed by van der westhuizen and bragg (2012) to detect virulence genes in apec isolates. virulence gene profiles were then constructed for each apec isolate from the multiplex data. this study was carried out in order to provide information on the virulence factors of e. coli isolated from chickens with confirmed cases of colibacillosis in zimbabwe. materials and methods top ↑ sample collection all of the 45 e. coli isolates used in this study were obtained from the diagnostics and research branch of the central veterinary laboratories, bulawayo. these isolates were obtained from chickens with confirmed cases of colibacillosis after postmortem examination. confirmatory tests were carried out on the isolates and the identification of e. coli was performed according to methods described by barrow and feltham (1993). biochemical tests included the gram stain and the catalase, oxidase, indole and citrate tests. dna extraction bacterial strains were subcultured overnight at 37 °c in luria-bertani (lb) broth (oxoid, basingstoke, hampshire, uk) and genomic deoxyribonucleic acid (dna) was extracted using a standard phenol-chloroform method (sambrook & russell 2001). to check for purity, dna was run along a 1% ethidium bromide–stained agarose gel (sigma-aldrich, st louis, usa) with a 1 kb dna ladder (thermo scientific, waltman, massachusetts, usa) in tris-borate-edta (tbe) buffer for 1 hr at 100 v and then viewed using the uvipro silver gel documentation system (uvitec, cambridge, uk). the concentration of dna was estimated by comparing the band light intensity to the band intensity on the 1 kb ladder on the uvipro silver gel documentation system. dna concentration of samples ranged from 75 ng/0.5 µg to 100 ng/0.5 µg. virulence genotyping the presence of genes encoding virulence factors was detected using multiplex pcr amplification. four multiplex pcr assays were used to detect 12 virulence genes (table 1). the multiplex design was according to that reported by van der westhuizen and bragg (2012), with slight changes in the primer and final magnesium chloride concentrations. the effected changes were using primer concentrations of 0.5 µm for the frz, sitd, fimh, ompt, iuta, pstb and sopb genes and adjusting the final magnesium chloride (mgcl2) concentration to 3 mm for all multiplex reactions. the primers used in this study are listed in table a1 in appendix 1. all primers used were obtained from inqaba biotech, pretoria, south africa. three microlitres of each of the dna samples were mixed with all necessary components for amplification in a 0.2 ml pcr tube (perkin-elmer, waltman, massachusetts, usa) in a 25 µl reaction. the reaction mixture included 2.5 µl of ×10 pcr dream taq buffer (thermo scientific, waltman, massachusetts, usa), 2 µl of deoxynucleotide triphosphates (dntps) 10 mm; 0.25 µl of dream taq polymerase (thermo scientific, waltman, massachusetts, usa), 5u/µl nuclease-free water to maintain a total volume of 25 µl. the appropriate primers ranging from 0.5 µm to 2 µm were added and the mgcl2 concentration was adjusted to a final concentration of 3 mm, as shown in table 1. negative controls comprised a water control. an applied biosystems geneamp® pcr system 9700 was used for the pcr thermal cycling conditions with an initial denaturation step at 94 °c for 5 min, 35 cycles (denaturation at 94 °c for 30 sec, annealing at 63 °c for 45 sec, extension at 72 °c for 1 min and 45 sec) and a final elongation step at 72 °c for 10 min. the amplified products were then run along a 1% ethidium bromide–stained agarose gel with a 100 bp dna ladder (thermo scientific, waltman, massachusetts, usa) in tbe buffer for 1 hr at 100 v and then viewed using the uvipro silver gel documentation system (uvitec, cambridge, uk). table 1: final primer concentrations used in the different multiplex polymerase chain reactions. the multiplex pcrs described were used to screen for the presence of 12 virulence genes in the apec isolates in duplicate. prevalence of each virulence gene was calculated (table 2) and virulence gene profiles were then assigned to each apec isolate (table 3). table 2: frequency of the 12 virulence genes in 45 avian pathogenic escherichia coli isolates. table 3: presence or absence of expected amplicons and virulence profiles of avian pathogenic escherichia coli isolates. sequencing of polymerase chain reaction products to confirm amplification of the regions of interest nine of the most prevalent genes (table 2) were amplified using single pcr and 10 µl of each sample was sent for sequencing at inqaba biotech, pretoria, south africa. pcr products were purified and concentrated by excising them from a 1% agarose gel using a sterile scalpel and then using a zymo research dna clean and concentrator-5 kit (epigenetics company, irvine, california, usa). purification was carried out at the national university of science and technology (nust). sequencing was performed using an automated abi-prism 3100 genetic analyser (applied biosystems, foster city, usa) according to the manufacturer's instructions. dna sequence data (chromatographs and sequences) were sent back by email for analysis. sequence results were analysed using basic local alignment search tool (blast) in the national centre for biotechnology information (ncbi) databases to confirm the identity of the amplified regions. results top ↑ escherichia coli virulence gene screening all 45 isolates obtained from the central veterinary laboratories (cvl), bulawayo were positively identified and confirmed to be e. coli through culturing and biochemical tests. after successful dna isolation and quantification, the dna of each of the 45 apec isolates was subjected to four different multiplex pcrs. each multiplex reaction amplified three apec virulence gene regions. this was done in order to screen the apec isolates for 12 virulence associated genes. figure 1 shows results for multiplex 1, which targeted the frz, sitd and fimh virulence genes. some of the apec isolates (14, 15 and 16) had all three genes present (figure 1). multiplex 2 targeted the kpsm, sita and vat virulence genes. only one isolate (isolate 16) had all three genes, whilst isolates 12, 28 and 30 only had the vat gene (figure 2). figure 3 shows results for multiplex 3, which assayed for the presence of the ompt, iuta and pstb genes. most of the apec isolates had a 302 bp amplicon consistent with the iuta gene. multiplex 4 assayed for the presence of the sopb, uvry and hlyf virulence genes. figure 4 shows the amplification of two bands 450 bp (hlyf) and 797 bp (sopb) for isolates 2, 3, 4, 5 and 7 and only one band (450 bp) for isolate 6. the most prevalent virulence associated genes in the apec isolates tested were the iuta gene (80%), fimh (33.3%), hlyf (24.4%) and sopb (20%) (table 2). the data obtained from electrophoresis agarose gels were used to assign virulence gene profiles to each apec isolate (table 3). figure 1: multiplex 1. lane m indicates the lane containing the marker, generuler 100 bp plus dna ladder (thermo scientific, waltham, massachusetts, usa), whilst lane n indicates the negative control. figure 2: multiplex 2. lane m indicates the lane containing the marker, generuler 100 bp plus dna ladder (thermo scientific, waltham, massachusetts, usa) whilst lane n indicates the negative control. figure 3: multiplex 3. lane m indicates the lane containing the marker, generuler 100 bp plus dna ladder (thermo scientific, waltham, massachusetts, usa). isolates 24–44 all had the iuta (302 bp) gene. figure 4: multiplex 4. lane m indicates the lane containing the marker, generuler 100 bp plus dna ladder (thermo scientific, waltham, massachusetts, usa) whilst lane n indicates the negative control. single polymerase chain reactions and sequencing nine of the most prevalent genes were successfully amplified using single pcr and produced the predicted sized bands during electrophoresis. to further confirm the identity of the amplicons, the bands obtained through electrophoresis were purified, concentrated and sent for sequencing. the sequences obtained (data not shown) were analysed using blastn and blastx and the amplicons were confirmed to be part of the genes of interest (data not shown). discussion top ↑ in this study, 45 e. coli isolates were obtained from chickens with confirmed cases of colibacillosis and screened for 12 virulence genes commonly associated with pathogenicity in apec. some of the virulence factors investigated have only been discovered recently. ninety-three percent of the e. coli isolates in this study had at least one virulence gene, suggesting that the isolates used could have been apec. the iuta gene had the highest prevalence at 80% (table 2). this gene has been well characterised and is one of the five genes of the aerobactin operon. it encodes an outer membrane protein involved in the high-affinity binding of fe3-aerobactin and can be plasmid located (johnson et al. 2006) or chromosomally encoded in some apec strains (schouler et al. 2012). the aerobactin system plays a role in the persistence and generation of lesions in apec-infected chickens. the present findings are in agreement with other studies that found an equally high prevalence of the iuta gene in apec isolates. these include the studies of rodriguez-siek et al. (2005), johnson et al. (2008), and schouler et al. (2012), who found prevalence rates of 80.2%, 80.8% and 82.7% respectively. the type 1 fimbrial adhesion gene (fimh) had a prevalence of 33.3% in this study. fimh is thought to contribute to the protection of e. coli from host heterophils (mellata et al. 2003). its role in the virulence of apec strains remains controversial, with conflicting reports (li et al. 2005). other studies have found higher occurrences of the fimh gene, notably that of rodriguez-siek et al. (2005), who found a prevalence of 98.1% for this gene in 524 apec isolates. interestingly, in a study by van der westhuizen and bragg (2012) using 10 zimbabwean apec isolates, all 10 had the fimh gene present. the hlyf gene has been shown to contribute to iron uptake (morales et al. 2004; williams & warner 1980). hlyf has been well documented in chickens suffering from colibacillosis (van der westhuizen & bragg 2012). the hlyf gene had a prevalence rate of 24.4% in the current study (table 2). this disagrees with several studies that have found higher prevalence rates (johnson et al. 2008; kafshdouzan et al. 2013). another study, however, found a lower prevalence rate of a related gene, hlye (jin et al. 2008). the plasmid partitioning protein encoded by sopb is common in various plasmids associated with virulence characteristics in apec (van der westhuizen & bragg 2012). the sopb gene had a prevalence rate of 20%, which agrees with results from the study carried out on 10 zimbabwean apec isolates by van der westhuizen and bragg (2012), as 30% of their isolates had the sopb gene. the vacuolating autotransporter toxin (vat) gene, which has been shown to induce cytotoxic effects in host cells (parreira & gyles 2003), was present in 17.8% of the isolates. this low prevalence rate agrees with other studies, in which detection rates of 39.8% (ewers et al. 2007), 25.5% (jin et al. 2008) and 33.4% (johnson et al. 2008) were found in apec. the sita and sitd genes are part of the sitabcd system. gene sita encodes a periplasmic binding protein of the sitabcd transport system, which is involved in iron and manganese transport and can be both chromosomally and plasmid located (mellata, touchman & curtiss 2009; sabri, léveillé & dozois 2006; schouler et al. 2004). sitabcd has been shown to play a role in virulence (sabri et al. 2008). the prevalence of the sita (11.1%) and sitd (13.3%) genes (table 2) is comparable to that found by van der westhuizen and bragg (2012) but differs from those reported by schouler et al. (2012) and johnson et al. (2008) for the sita gene and by ewers et al. (2007) for the sitb gene. the presence of these two genes agrees with the results of other scholars who believe that in e. coli sitabcd-encoding genes are associated with clinical strains isolated from extra-intestinal infections from poultry and human urinary tract infections (rodriguez-siek et al. 2005; schouler et al. 2004). the frz operon was present in 8.9% of the e.coli isolates (table 2). work by rouquet et al. (2009) suggested that the gene products from the frz operon are used by e.coli to promote growth in serum during oxygen-restricted conditions. a link between the expression of this locus and e. coli pathogenic abilities was confirmed by experiments showing its role in promoting bacterial fitness under stressful conditions, including oxygen restriction or the late stationary phase of growth, and in promoting growth in chicken serum or the intestinal tract during in vivo competition assays (schouler et al. 2012). the results of this study agree with those of van der westhuizen and bragg (2012) with regard to the zimbabwean isolates they worked on; however, they differ from work carried out by schouler et al. (2012) in which they found a prevalence of 53.4% for the frz operon in 352 apec isolates. a transcriptional regulator of iron uptake genes in apec, uvry (li et al. 2005) has only recently been used to screen for apec using multiplex pcr (van der westhuizen & bragg 2012). in this study, this gene had a low prevalence rate of 4.4% (table 2). this differs from the 100% reported by van der westhuizen and bragg (2012) for the 10 zimbabwean isolates they studied. the ompt, pstb and kpsm genes all had a low prevalence rate of 2.2%. the ompt gene encodes the episomal outer membrane protease that cleaves colicins (cavard & lazdunski 1990). other studies on apec have shown higher detection rates of this gene (johnson et al. 2008; kafshdouzan et al. 2013; rodriguez-siek et al. 2005). the kpsm gene encodes proteins required for translocation of e. coli group ii capsular polysaccharide across the inner membrane (clarke, pearce & roberts 1999). prevalence rates of the kpsm gene reported for apec isolates have tended to be low: 15.7% (johnson et al. 2008), 15.8% (rodriguez-siek et al. 2005) and 0% for zimbabwean isolates (van der westhuizen & bragg 2012). the pstb gene, which is part of the pstscab operon, has been shown to increase resistance to polymyxin, rabbit serum and acid shock (lamarche et al. 2005). the pstb gene contributes to virulence but is still relatively new in the diagnostic context (lamarche et al. 2005). as a result very little has been published on this virulence gene in apec. virulence profiles were generated for each e. coli isolate used in this study (table 3). the apec isolates were profiled as 62.2% fitting profile a, 31.1% profile b and 6.7% profile c. none of the isolates had more than seven virulence genes. these findings agree with those of van der westhuizen and bragg (2012). they found the zimbabwean apec isolates to have fewer virulence genes (with most having less than 11/18 virulence genes studied) than the south african apec isolates, which all had between 12 and 18 virulence genes. the low number of virulence genes in the isolates assayed in this study could be a result of the fact that these isolates possess other virulence genes that were not screened for. numerous studies have demonstrated that virulence genes are rarely all present in the same isolate. different isolates may harbour different associations of virulence genes and belong to specific subpathotypes, with each subpathotype characterised by the type of lesions it produces in poultry with avian colibacillosis (maturana et al. 2011; olsen et al. 2012). another possibility is that the isolates used in this study belong to strains that are not highly pathogenic and that environmental or other disease factors caused stress in the hosts, which allowed them to be infected and led to colibacillosis (van der westhuizen & bragg 2012). the risk for colibacillosis is known to increase with increasing infection pressure in the environment. good housing, hygiene and avoiding overcrowding are very important in reducing infection rates (saidi et al. 2013). another reason, although less likely, could be that the strains used in this study were not the causative agents in the confirmed colibacillosis cases. this is because isolation of an e. coli strain from a pathological lesion is not a sufficient criterion to classify it as a pathogen (schouler et al. 2012). limitations top ↑ potential biases in this study are related to the relatively small sample size of e. coli isolates used. in addition, in vivo tests of the isolates to determine their pathogenicity could have improved the study. in retrospect, more virulence genes could have been screened and positive controls for all 12 genes studied could have been used. recommendations screening for more virulence factors using a larger sample size could provide more definite conclusions. also, a pathogenicity test of isolates using the 1-day-old chick lethality test to correlate virulence profiles with pathogenicity is recommended. it might be interesting to study apec from other poultry sources like ducks, geese and turkeys to determine relationships with apec-causing colibacillosis in chickens. conclusion top ↑ the study revealed that the virulence profiles of zimbabwean apec isolates may be different from those reported in other studies, which generally show a high prevalence of the virulence genes we investigated in apec isolates causing colibacillosis in poultry (ewers et al. 2007; johnson et al. 2008; kafshdouzan et al. 2013). this suggests that other virulence genes not investigated in the present study may be important in virulence of zimbabwean apec isolates or, more likely, that these isolates are less pathogenic and rely on environmental factors and stress in hosts to establish infection. acknowledgements top ↑ the authors thank the cvl, bulawayo for provision of isolates, especially zwelabo sibanda. special thanks go to the international network for the availability of scientific publications (inasp) for their assistance through authoraid. the study was supported by the nust research board. competing interests the authors declare that they have no financial or personal relationship(s) which may have inappropriately influenced them in writing this article. authors’ contributions j.m. (nust) was responsible for the project design, was the principle supervisor and carried out experimental work on the project. y.o.n. 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adriaensen, c., zaleska, m., hernalsteens, j.p., de baets, l. et al., 2005, ‘virulence-associated traits in avian escherichia coli: comparison between isolates from colibacillosis-affected and clinically healthy layer flocks’, veterinary microbiology 108, 75–87. http://dx.doi.org/10.1016/j.vetmic.2005.02.009 williams, p.h. & warner, p.j., 1980, ‘colv plasmid-mediated, colicin v independent iron uptake system of invasive strains of escherichia coli’, infection and immunity 29, 411–416. zakeri, a. & kashefi, p., 2012, ‘antimicrobial susceptibilities of avian escherichia coli isolates in tabriz, iran’, african journal of biotechnology 11(19), 4467–4470. appendix 1 top ↑ table a1: primers used for amplifying regions in avian pathogenic escherichia coli virulence genes. article information authors: raphael s. sallu1,2 christopher j. kasanga2 mkama mathias1,2 mmeta yongolo1 chanasa mpelumbe-ngeleja1 misheck mulumba3 ezekia ranga4 philemon wambura2 mark rweyemamu2 nick knowles5 donald king5 affiliations: 1tanzania veterinary laboratory agency, dar es salaam, tanzania2southern african centre for infectious diseases surveillance, sokoine university of agriculture, tanzania 3southern african development community secretariat, gaborone, botswana 4ministry of livestock development and fisheries, dar es salaam, tanzania 5world reference laboratory for foot-and-mouth disease, institute for animal health, united kingdom correspondence to: raphael sallu postal address: po box 9254, dar es salaam, tanzania how to cite this article: sallu, r.s., kasanga, c.j., mathias, m., yongolo, m., mpelumbe-ngeleja, c., mulumba, m. et al., 2014, ‘molecular survey for foot-and-mouth disease virus in livestock in tanzania, 2008–2013’, onderstepoort journal of veterinary research 81(2), art. #736, 6 pages. http://doi:10.4102/ ojvr.v81i2.736 note: proceedings of the 2nd one health conference in africa. jointly organised by the southern african centre for infectious disease surveillance and the tanzania national institute for medical research, held at the snow crest hotel in arusha, tanzania from 16th to 19th april 2013: http://www.sacids.org/ kms/frontend/index.php?m=119. copyright notice: © 2014. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. molecular survey for foot-and-mouth disease virus in livestock in tanzania, 2008–2013 in this proceedings... open access • abstract • introduction • research method and design    • viruses    • rna extraction    • real-time pcr and vp1 sequencing    • sequences and phylogenetic analyses • results    • real-time pcr and typing    • phylogenetic analysis of serotype o, a and sat1 viruses • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ phylogeography data are of paramount importance in studying the molecular epidemiology dynamics of foot-and-mouth disease virus (fmdv). in this study, epithelial samples and oesophageal-pharyngeal fluids were collected from 361 convalescent animals (cattle and buffaloes) in the field throughout tanzania between 2009 and 2013. the single plex real-time rt-pcr (qrt-pcr) assay for rapid and accurate diagnosis of fmdv employing the callahan 3df-2, 3df-r primers and callahan 3dp-1 probe were used. preparation of the samples was performed according to the oie manual, with a kenya o serotype obtained from the attenuated vaccine serving as a positive control and samples collected from healthy animals serving as true negatives. the results indicated that 53.49% of samples (n = 176) were positive for fmdv genome by qrt-pcr, with ct values ranging from 14 to 32. in addition, molecular typing of the fmdv genome positive samples using serotype specific primers revealed the existence of several serotypes: serotype south africa territory 1 (sat1) (34.25%, n = 60), serotype a (68.92%, n = 98), serotype o (59.20%, n = 98) and sat2 (54.54%, n = 96). the virus protein 1 sequences analysis for 35 samples was performed and the collective results indicated: 54.28% serotype o, 25.71% serotype a, 14.28% serotype sat1 and 2.85% serotype sat2. therefore in this study, both the phylogenetic trees and spatial distribution of serotypes elucidated the phylodynamics of multiple fmdv field strains in tanzania and neighbouring countries. introduction top ↑ foot-and-mouth disease virus (fmdv) belongs to the genus aphthovirus, family picornaviridae which principally infects cloven-hoofed animals including wildlife animals. the virus is a positive sense, single-stranded rna virus and is categorised into seven serotypes: a, o, c, asia 1, south africa territory 1, 2 and 3 (sat1, sat2 and sat3) (domingo et al. 2003). fmdv can be genetically classified based on its geographic origin (topotypes); for example, the serotype sat1 can be grouped into eight topotypes (i–viii) based on nucleotide differences (within virus protein 1 [vp1] coding sequence) of up to 15% (samuel & knowles 2001). the serotype sat1 topotype iii is found in tanzania, zambia, malawi, kenya and zimbabwe according to the study conducted by vosloo et al. (2002).foot-and-mouth disease (fmd) is highly contagious and, combined with its high antigenic diversity, this makes the disease difficult to control. the disease has caused significant economic losses as a result of the deaths of young animals, decreased productivity and trade sanctions against livestock and animal products from infected regions. this disease is endemic in most countries of sub-saharan africa (vosloo et al. 2002) and is commonly reported in southern african development community (sadc) countries. to date, efforts to control this disease have not been very successful. this is contributed mainly to the lack of knowledge on the actual fmdv serotypes and subtypes (topotypes) circulating in these countries. the knowledge of the serotypes and subtypes is a prerequisite for effective regional disease control strategies. the epidemiology of fmd in tanzania is influenced by two different patterns, viz. a cycle in which wildlife plays a role in maintaining and spreading the disease to other susceptible domestic animals and wild ungulates and a cycle that is maintained within domestic animals and that is independent of wildlife (kivaria 2003). in southern africa, the former cycle predominates as a result of the presence of the african buffalo (syncerus caffer), the only wildlife species for which long-term maintenance of fmdv has been described (hedger, forman & woodford 1973). in the sadc region, both cycles probably occur, whilst in west africa, because of the absence of significant numbers of wildlife hosts, the virus is believed to be maintained primarily within the domestic animal cycle. in southern africa, a number of countries have been able to control fmdv by separating infected buffalo from livestock and by limited use of vaccination. from the epidemiological surveillance conducted by rweyemamu et al. (2008), it has been observed that tanzania lies in the great lakes cluster, which comprises the countries of the east african community (i.e. tanzania, kenya, uganda, rwanda and burundi), as well as the eastern part of the democratic republic of congo (drc). this region not only has large livestock populations but also the highest concentration of wildlife in the world. farming is dominated by agro-pastoral communities and is characterised by communal grazing and migrations. the eastern drc is heavily dependent on trade livestock from uganda, tanzania, rwanda and burundi. this great lakes cluster probably contains the most complicated fmd situation in the world, with several fmd primary endemic foci. five serotypes (o, a, c, sat1 and sat2) are endemic in this cluster and the sixth serotype (sat3) was isolated in wildlife (african buffalo) in uganda in 1970 (hedger et al. 1973); although, it has never been isolated from the livestock in the great lakes cluster. in tanzania, the common bovine fmdv serotypes observed are sat1, sat2 and o. these serotypes were identified by c-elisa antigen during a period of seven years, 2002–2009. the serotypes were observed from the following regions, namely: kagera (type o), kilimanjaro (sat2), morogoro (sat2), pwani (sat1 and sat2), rukwa (sat2), singida (sat2) and tabora (sat1) (central veterinary laboratory 2009). the previous effort to control this disease showed futile results, as there are reported cases where there was a re-emergence of the disease after the animals have been vaccinated. also, the use of quarantine and culling strategies are very expensive to execute and difficult because of the geography of the country. research method and design top ↑ viruses the viruses used in this study were obtained from all endemic areas of tanzania from the outbreak cases from 2008 to 2013. collection of the samples followed the world organisation of animal health (oie) guidelines (oie 2012) and included all bio-data such as the location where the animal was found, name of the area and farmer, type of animal, age and sex. besides this, also the type of the farming system practiced by farmer was noted, that is zero grazing or range. the epithelium, probang and serum samples were collected from the clinical animals, especially cattle and pigs, which normally show disease symptoms. the epithelium samples were taken from the affected muzzle, gums, tongue and hooves. the oro-pharyngeal samples were stored in liquid nitrogen, whilst the epithelium samples were stored in a 50:50 mixture of pbs and glycerol at ph of 7.2 and stored in cold chain as serum samples. before the epithelium tissues were ground, drying was performed using the blotted dry absorbent to reduce glycerol content and weighed. an epithelial suspension was prepared by grinding the sample in sterile pestle and mortal with sterile sand. the suspension was centrifuged at 2000 rpm for 10 min and the supernatant taken for the molecular work. rna extraction the rna extraction was conducted directly from the 361 samples from epithelium tissues suspension, sera and oro-pharyngeal sample using qiaamp® viral rna kit (qiagen, hilden, germany). besides the 361 samples, extraction was also conducted to additional samples to form a positive extracted control (serotype o kenya vaccine strain) and a negative extracted control. the 361 samples were tested using real rt-pcr as described by shaw et al. (2007). samples were screened using the callahan 3d probe (6-fam 5’ – tcc ttt gca cgc cgt ggg ac – 3’ tamra), 3d forward (5’ – act ggg ttt tac aaa cct gtg a – 3’) and 3d reverse (5’ – gcg agt cct gcc acg ga – 3’) primers (eurogentec, liege, belgium). the master mix kit used in this assay was invitrogen superscript iii platinum one step qrt-pcr system (cat. no. 11732-020), with the final volume per reaction as 20.0 µl, plus 5.0 µl from each template rna. the reaction mix per one reaction was as follows: 2× reaction mix at 12.5 µl, nuclease free water 1.0 µl, primer 3df 2.0 µl (10 µm), primer 3dr 2.0 µl (10 µm), probe 3dp 1.5 µl (10 µm), rox 1:10 pre-diluted superscript iii rt 0.5 µl and platinum taqmix 0.5 µl. the pcr regime was as follows: 30 min for 60 °c (reverse transcriptase step), 10 min at 95 °c (inactivation reverse transcriptase or activation dna polymerase), 15 s for 95 °c (denaturation) and 1 min at 60 °c (annealing and elongation), with the two last steps performed for 50 cycles. measurement fluorescence was taken at the end of second step at stage 3. the results interpretation was as follows: samples with ct < 32 were classified as positive, samples with 32 < ct < 50 were ambiguous and marked for retesting and samples with no ct were classified as negative. the cycle threshold value (ct value) was fixed automatically from the pre-loaded machine software (7500 software v. 2.0.5). fmdv positive samples were further analysed for rt-pcr and vp1 sequencing. real-time pcr and vp1 sequencing one primer combination was used for the rt–pcr of fmdv serotypes, as follows: serotype o: o-1c244f/eur-2b52r, serotype a: a-1c612f/eur-2b52r, serotype c: c-1c536f/eur-2b52r, serotype asia1: as1-1c530f/eur-2b52r, serotype sat1: sat1-1c559f/sat-2b208r, serotype sat2: sat2-p1-1223f/sat-2b208r and serotype sat3: sat3-p1-1222f/sat-2b208r (table 1). one-step rt-pcr was carried out using the qiagen one-step rt-pcr kit (cat. no. 210210). the thermal profiles used for amplification of the vp1 sequence of various serotypes were as follows: fmdv o and asia1: 42 °c for 30 min, 94 °c for 5 min, 35 cycles at 94 °c for 60 s, 60 °c for 60 s and 72 °c for 90 s, followed by a final extension of 72 °c for 5 min. conditions were the same for the other serotypes, except that extension temperatures were 55 °c for a and c and 50 °c for sat1, sat2 and sat3. temperature cycling was carried out using the geneamp® pcr systems 9700 (applied biosystems, foster city, usa). standard 2% (w/v) agarose gel (seakem® le agarose, rockland, usa) was prepared by dissolving 3 g agarose in 150 ml 1× tbe electrophoresis buffer (0.040 m tris-borate, 0.001 m edta, ph 8.0) in an erlenmeyer flask. the mixture was heated in a hot plate or microwave to allow the agarose to dissolve and form a gel. the gel was allowed to cool to about 20 °c before adding 3 µl of ethidium bromide (10 mg/ml). the gel was then poured into a horizontal gel tray fitted with appropriate combs. after about 40 min of gel polymerisation, the combs were carefully removed and the tray immersed in an electrophoresis tank containing electrophoresis buffer (1× tbe). then 3 µl of dna products of each individual sample was mixed with 2 µl of bromophenol blue dye diluted 6× (3:1 water: 6× dye, 50 mm edta, 50 mm nacl, 50% glycerol) and then loaded into separate lanes (slots) of the submersed agarose gel. the samples were run at 120 volts for 45 min. after the run, the gel was removed and photographed under uv light using a video capture system (molecular imager® gel dox xr system 170-8170 with flowgen is 1000; bio-rad, seoul, korea). sequences and phylogenetic analyses the pcr products were directly sequenced on both strands to obtain the complete vp1 sequences, which were compared with the other relevant fmdv vp1 sequences within the same serotypes. computer-assisted comparisons of the nucleotide sequences were made to find the similarities of nucleotides sequences in the national centre for biotechnology information (http://www.ncbi.nlm.nih.gov/), using the blastn search program. also, for further sequence comparisons, another sequence resource used was from the european bioinformatics institute (http://www.ebi.ac.uk/service/) maintained by the european molecular biology laboratories. vp1 sequences data were assembled and analysed using seqman ii (dnastar lasergene 8.0, madison, usa). this led to final sequence of 636–639 nucleotides of the vp1 coding region. nucleotide sequences that encoded amino acid from the vp1 gene were translated to the (deduced) amino acid sequences by the editseq. to compare with ingroup sequences, additional sequences from the genbank were used for the vp1 region. alignment of vp1 sequences was performed using clustal w algorithm method (megalign; dnastar, madison, usa) and bio-edit 7.0. these alignments were used to construct distance matrices by using the kimura 2-parameter nucleotide substitution model in the program mega 5.1 (tamura et al. 2007). midpoint-rooted neighbour-joining trees were then constructed with mega 5.1 software. the robustness of the tree topology was assessed with 1000 bootstrap replicates by using the model in mega 5.1. results top ↑ real-time pcr and typing the results from different primer combinations (table 1) indicated that 53.49% of samples (n = 176) were positive for fmdv genome by qrt-pcr, with ct values ranging from 14 to 32. in addition, molecular typing of the fmdv genome positive samples using serotype specific primers revealed the existence of several serotypes: serotype sat 1 (34.25%, n = 60), serotype a (68.92%, n = 98), serotype o (59.20%, n = 98) and sat 2 (54.54%, n = 96). the vp1 sequences analysis collectively across all 35 samples was performed and the results indicated: 54.28% serotype o, 25.71% serotype a, 14.28% serotype sat1 and 2.85% serotype sat2. table 1: detailed information of the virus protein 1 primers used in the study. phylogenetic analysis of serotype o, a and sat1 viruses complete vp1 sequence analysis data from the analysed samples, together with the archived data from across africa, were used. serotype o predominated the fmdvs examined, with all 20 of the fmdv serotype o tanzania isolates genotyped in this study clustered into a single topotype: east africa (ea)-2 (figure 1). the results from different parts of tanzania revealed that the same topotype is circulating and is responsible for causing the majority of outbreaks. isolate o/tan-cvl-2010-0037 is 100% similar to o/tan-cvl-2011-0046, which is from the tabora region. the phylogenetic analysis from these isolates concurs with the geographical distribution of the virus with respect to outbreaks (kasanga et al. 2012). the serotype o viruses were found in tabora, serengeti, musoma, kahama, mwanza, rorya, iringa, mbeya, rukwa and the pwani region, which amounts to the majority of the country. figure 1: midpoint-rooted neighbour-joining tree (based on the complete virus protein 1 coding sequence) showing the relationships between the foot-andmouth disease virus serotype o isolates from tanzania (coloured red) and other contemporary and reference viruses. all the isolates in this serotype were grouped in the africa topotype (genotype 1) of the serotype viruses (figure 2) as it was previously identified in the country (kasanga et al. 2012). the isolates a/tan/11/2008 and a/tan/12/2008, which were reported to be 100% similar, are also similar to isolate a/tan-cvl-2008-0155 from the rukwa region, with high bootstrap support. isolate a/tan/11/2009 was 100% similar to isolate a/tan-cvl-2009-52 from tabora. for the serotype a topotype africa (g1), the recorded nucleotide difference in vp1 coding region of 0.0% – 15.6%, with any other topotypes defined for fmd type a viruses reported in africa. these serotype a viruses were found in regions such as mtwara, tabora, kigoma, rukwa and dar es salaam and this shows the virus is expanding its territory, as previously reported (kasanga et al. 2012). figure 2: midpoint-rooted neighbour-joining tree (based on the complete virus protein 1 coding sequence) showing the relationships between the foot-andmouth disease virus serotype a isolates from tanzania (coloured blue) and other contemporary and reference viruses. for the phylogenetic analysis of sat1 (figure 3), it can be concluded that all fmd type sat1 viruses isolated in tanzania and analysed to date were placed within the topotype 1nwz and the nucleotide differences in vp1 coding region of 15% – 60%. the nucleotide number of composition was 663 and the average nucleotide match was from 645–660 with the unknown strain. all bootstrap of 70% and above are shown (figure 3). the isolates numbered sat1/tan-cvl-2012-0359 and sat1/tan-cvl-2012-0361 are 100% similar and were obtained in the same area in the morogoro region. the five sat1 isolates used in this study, sat1/tan-cvl-2012-0354, sat1/tan-cvl-2012-0355, sat1/tan-cvl-2012-0359, sat1/tan-cvl-2012-0360 and sat1/tan-cvl-2012-0361 are clustered together in a group with isolates sat1/tan/11/2012, sat1/tan/23/2012, sat1/tan/25/2012 and sat1/tan/27/2012 obtained from the northern part of tanzania (figure 3). since the detection of sat1 in the country in 1971 (rweyemamu & loretu 1972, 1973), this serotype has been responsible for many outbreaks, especially in the wildlife and livestock interface. figure 3: midpoint-rooted neighbour-joining tree (based on the complete virus protein 1 coding sequence) showing the relationships between the foot-andmouth disease virus serotype south africa territory 1 isolates from tanzania (coloured yellow) and other contemporary and reference viruses. discussion top ↑ this study on the molecular survey of fmdv was focused on the viruses circulating in tanzania from 2008 to 2013. the phylogenetic analysis of vp1 sequences has been widely used to identify and characterise fmdv isolates (klein et al. 2006; knowles et al. 2009; konig et al. 2007). the final study included 18 type o, 9 type a and 6 sat1 field isolates collected from different endemic areas of country. the results showed that the o type is the one responsible for most of the outbreaks in the country. the serotype o is endemic and has spread widely in the country since the late 1950s (rweyemamu 1972; vosloo et al. 2002). this serotype seems to be widespread in the northern, southern, western and eastern regions of tanzania, but not in the central parts of the country (kasanga et al. 2012). serotype o, together with the a and sat1 serotypes, are the primary causes of sporadic emergencies in some parts of tanzania (kasanga et al. 2012). the previous studies indicated some enigma relating to the dynamics of serotype a in tanzania. but from 2009 to 2012, the incidence dynamics of this serotype started to change; in 2009 there was one outbreak in nzega (tabora) caused by isolate a/tan-cvl-2009-0052. this isolate is 100% similar to the one reported by kasanga et al. (2012), which caused the outbreak in the eastern coastal zone. but, in this current study, it was revealed that the serotype a is spreading fast in tanzania, as it was found in nzega, mtwara, tabora, kibondo (kigoma), dar es salaam and sumbawanga (rukwa). in a previous study (kasanga et al. 2012), this serotype was only reported in regions such as iringa, morogoro, pwani, dar es salaam and dodoma. according to rweyemamu and loretu (1972), serotype a was regularly reported in the northern, northern-lake and central zones of tanzania up to 1971. the samples collected and analysed from 1997 to 2004 revealed no presence of the serotype a (swai, mrosso & masambu 2009). with this current study, it seems that this serotype’s reappearance is contrary to the previous reports. the spread and reappearance of serotype a could possibly be caused by the movement of animals across the border or within the country by nomadic livestock keepers. therefore, a detailed study should be conducted regarding to the disappearance and reappearance of serotype a, which makes a genetic comparison between the old and the new strains which are appearing. conclusion top ↑ the present study provides a comprehensive picture of the phylogenetic relationships of fmdv isolates, focusing on the circulating viruses in tanzania, thus filling a gap of knowledge in the country regarding the molecular epidemiology of this economically important virus. as fmdv is rapidly spreading in the country, further collection and analysis of samples, together with the improvement of the local epidemiological investigation of fmd outbreaks is recommended. intensive phylogeography studies should be carried out that will locate particular serotype or topotype that is responsible for the disease outbreak. this is a very important step in controlling the spread of the disease and also for the production of a specific vaccine which will cater for a specific topotype. in conclusion, this study provides an overview of the molecular epidemiology of fmdv in tanzania. although only a limited number of ad hoc samples were available for the sequence analysis, this study highlights the spread of fmdv in tanzania. acknowledgements top ↑ the authors thank the researchers and staff from the veterinary investigation centres for sample collection during fmd outbreaks. we also appreciate the assistance provided by farmers during the surveillance exercises. we recognise the contribution of staff at the centre for infectious disease and biotechnology – temeke during the laboratory analysis of samples. we are grateful to the world reference laboratory, pirbright, uk which assisted us during the data analysis. the ministry of livestock development and fisheries, united republic of tanzania funded the technical advice. the authors also acknowledge the welcome trust foundation through southern african centre for infectious disease surveillance (sacids) for providing financial support for phd work. last, but not least, the first author wishes to thank all his supervisors at sokoine university of agriculture, world reference laboratory for foot and mouth disease-pirbright, tanzania veterinary laboratory agency and the royal veterinary college. this work was supported by the wellcome trust grant wt087546ma to the southern african centre for infectious diseases & surveillance (sacids). competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions r.s.s. (tanzania veterinary laboratory agency) was involved in the laboratory work, surveillance and data analysis. p.w. (sokoine university of agriculture), m.r. (sokoine university of agriculture) and c.j.k. (sokoine university of agriculture) were the first authors’ supervisors in laboratory work and academic advisors. m.y. (tanzania veterinary laboratory agency), m.m. (tanzania veterinary laboratory agency) and c.m.-n. (tanzania veterinary laboratory agency) where the first authors’ technical advisors during surveillance work and sample preparation work. d.k. (world reference laboratory for the foot and mouth disease-pirbright) and n.k. (world reference laboratory for the foot and mouth disease-pirbright) were involved in data analysis. references top ↑ central veterinary laboratory, 2009, virology annual report, ministry of livestock and fisheries development, centre for infectious diseases, temeke, dar es salaam-tanzania.domingo, e., escarmis c., baronowski, e., ruiz-jarabo, c.m., carrillo e., neun˜z. j.i. et al., 2003, ‘ evolution of foot-and-mouth disease virus’, virus research 91, 47–63. http://dx.doi.org/10.1016/s0168-1702(02)00259-9 hedger, r.s., forman a.j. & woodford m.h., 1973, ‘foot-and-mouth disease in east african buffalo’, bulletin of epizootic diseases of africa 21, 99–101. kasanga, c.j., sallu, r., kivaria, f., mkama, m., masambu, j., yongolo, m. et al., 2012, ‘foot-and-mouth disease virus serotypes detected in tanzania from 2003 to 2010: conjectured status and future prospects’, onderstepoort journal of veterinary research 79(2), art. #462. http://dx.doi.org/10.4102/ojvr.v79i2.462 kivaria, f.m., 2003, ‘foot-and-mouth disease in tanzania: an overview of its national status’, veterinary quarterly 25, 72–78. http://dx.doi.org/10.1080/01652176.2003.9695147 klein, j., parlak, u., ozyoruk, f. & christensen, l.s., 2006, ‘the molecular epidemiology of foot-and-mouth disease virus serotype a and o from 1998 to 2004 in turkey’, bmc veterinary research 2(35), 1–13. knowles, n.j., nazem shirazi, m.h., wadsworth, j., swabey, k.g., stirling, j.m., statham, r.j. et al., 2009, ‘ recent spread of a new strain (a-irn-05) of foot-and-mouth disease virus type a in the middle east’, transboundary emerging diseases 56, 157–169. http://dx.doi.org/10.1111/j.1865-1682.2009.01074.x konig, g.a., palma, e.l., maradei, e. & piccone, m.e., 2007, ‘molecular epidemiology of foot-and-mouth disease virus types a and o isolated in argentina during the 2000–2002 epizootics’, veterinary microbiology 124, 1–15. http://dx.doi.org/10.1016/j.vetmic.2007.03.015 oie world organisation for animal health, 2012, terrestrial animal health code. chapter 8.5: foot-and-mouth disease, oie, paris. rweyemamu, m.m. & loretu, k., 1972, ‘observations on foot-and-mouth disease in tanzania’, bulletin of epizootic diseases of africa 20, 101–104. http://dx.doi.org/10.1111/j.1865-1682.2007.01013.x rweyemamu, m.m. & loretu, k., 1973, ‘observations on the first epidemic of foot-and-mouth disease type sat1 in tanzania’, bulletin of epizootic diseases of africa 21, 393–400. rweyemamu, m.m., roeder p., mackay d., sumption k., brownlie j., leforban y. et al., 2008, ‘epidemiological patterns of foot-and-mouth disease worldwide’, transboundary and emerging diseases 55(1), 57–72. samuel, a.r. & knowles n.j., 2001, ‘foot-and-mouth disease type o viruses exhibit genetically and geographically distinct evolutionary lineages topotypes’, journal of general virology 82, 609–621. tamura, k., dudley, j., nei, m. & kumar, s., 2007. ‘mega4: molecular evolution genetics analysis (mega) software version 5.1’, molecular biology evolution 24, 1596–1599. http://dx.doi.org/10.1093/molbev/msm092 shaw, a.e., reid, s.m., ebert, k., hutchings, g.h., ferris, n.p. & king, d.p., 2007, ‘implementation of one step real-time rt-pcr protocol for diagnosis of foot-and-mouth disease’, journal of virology methods 143, 81–85. http://dx.doi.org/10.1016/j.jviromet.2007.02.009 swai, e.s., mrosso, a. & masambu, j.i.g., 2009, ‘occurrence of foot-and-mouth disease serotypes in tanzania: a retrospective study of tongue epithelial tissue samples’, tanzania veterinary journal 26, 7–12. http://dx.doi.org/10.4314/tvj.v26i1.49232 vosloo, w., bastos, a.d., sangare, o., hargreaves, s.k. & thomson, g.r., 2002, ‘review of the status and control of foot-and-mouth disease in sub-saharan africa’, revue scientifique et technique (oie) 21, 437–444. dematos_103-108.indd introduction a single survey on the ticks that infest dogs has been conducted in zimbabwe, another in mozambique, while seven such surveys have been conducted in south africa, and a total of 24 ixodid and one argasid tick species recovered (goldsmid 1963; horak, jacot guillarmod, moolman & de vos 1987b; horak 1995; bryson, horak, höhn & louw 2000; horak, emslie & spickett 2001; jacobs, fourie, kok & horak 2001; horak & matthee 2003; neves, afonso & horak 2004; nyangiwe, horak & bryson 2006). in addition to the above 24 species, dias (1991) has recorded amblyomma tholloni, a tick of african elephants, and rhipicephalus meuhlensi, a tick of nyalas, on dogs in mozambique. subsequent to the above-mentioned surveys, one of the most commonly encountered ticks on dogs in southern africa, namely haemaphysalis leachi, has been reinstated as haemaphysalis elliptica, an old taxon from the cape of good hope (apanaskevich, horak & camicas 2007). with the exception of the ticks in the surveys conducted by goldsmid (1963) and jacobs et al. (2001), the majority of ticks identified as h. leachi in the afore-mentioned surveys have been re-examined and re-identified as h. elliptica (apanaskevich et al. 2007). 103 onderstepoort journal of veterinary research, 75:103–108 (2008) ixodid ticks on dogs belonging to people in rural communities and villages in maputo province, mozambique c. de matos1, c. sitoe2, l. neves3, n.r. bryson4 and i.g. horak5* abstract de matos, c., sitoe, c., neves, l., bryson, n.r. & horak, i.g. 2008. ixodid ticks on dogs belonging to people in rural communities and villages in maputo province, mozambique. onderstepoort journal of veterinary research, 75:103–108 the species composition and geographic distribution of ixodid ticks infesting domestic dogs owned by people in rural communities and villages in maputo province was established by collecting ticks from dogs at each of 27 localities spread throughout the province. ticks were collected from a total of 132 dogs, and nine species belonging to four genera were identified. one dog was infested with six species, three with five and 13 with four species. haemaphysalis elliptica followed by rhipicephalus simus were present on dogs at most localities, and their geographic distribution in maputo province has been mapped for the first time. keywords: dogs, geographic distribution, haemaphysalis elliptica, ixodid ticks, maputo province, mozambique, rhipicephalus simus * author to whom correspondence is to be directed: e-mail: ivan.horak@up.ac.za 1 direcção de ciências animais, instituto de investigação agrária de mozambique, c.p. 1922, maputo, mozambique, and department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 direcção de ciências animais, instituto de investigação agrária de mozambique, c.p. 1922, maputo, mozambique 3 faculdade de veterinária, universidade eduardo mondlane, c.p. 257, maputo, mozambique 4 department of veterinary tropical diseases, faculty of vet erinary science, university of pretoria, private bag x04, onder stepoort, 0110 south africa 5 department of veterinary tropical diseases, faculty of veterinary science, university of pretoria, private bag x04, onder stepoort, 0110 south africa, and arc-onderstepoort veter inary institute, onderstepoort, 0110 south africa, and department of zoology and entomology, university of the free state, p.o. box 339, bloemfontein, 9300 south africa accepted for publication 14 january 2008—editor 104 ixodid ticks on dogs in maputo province, mozambique during the course of surveys conducted in south africa, the seasonal abundances of haemaphysalis elliptica, rhipicephalus gertrudae, rhipicephalus san guineus and rhipicephalus simus have been determined on dogs (horak 1982; horak et al. 1987b, 2001; jacobs et al. 2001; horak & matthee 2003). with the exception of r. sanguineus, the geographic distributions of these ticks in south africa have been mapped (howell, walker & nevill 1978; walker, keirans & horak 2000). the adults of the abovementioned five ticks, and those of the ixodes pilosus group and of ixodes rubicundus, may use dogs as hosts (horak et al. 1987b; jacobs et al. 2001; horak & matthee 2003). however, with the exception of the adults of r. sanguineus, they may also infest other animals (walker 1991). as is the case with the adults, the immature stages of r. sanguineus infest only dogs (walker et al. 2000), while those of the i. pilosus group infest dogs and a variety of small and large mammals (walker 1991). the immature stages of i. rubicundus infest elephant shrews, elephantulus spp., and smith’s red rock rabbits, pronolagus rupestris (stampa 1959; fourie, horak & woodall 2005), whereas those of the other species nearly exclusively infest rodents (norval & mason 1981; norval 1984; matthee, horak, beaucornu, durden, ueckermann & mcgeogh 2007). most of the dogs in the survey conducted in mo zambique by neves et al. (2004) were examined in the province of maputo. this is the southernmost province and has a seaboard of approximately 320 km, and the landscape rises from the indian ocean in the east to about 800 m above sea level in the west. the vegetation along the coast is described as shrubland with patches of wetland, mangrove and deciduous trees along river shores and on dunes. inland it is dominated by woodland, and in the north-west district of magude by associations of savannah and mopane (colophospermum mopane). the inland vegetation of the south was characterized by forest that has been extensively denuded for the production of charcoal. the southern border of the province abuts on north-eastern kwazulu-natal province, south africa, where a survey of the ticks infesting dogs belonging to persons in rural communities has been conducted (horak et al. 2001). the western border of maputo province adjoins the eastern border of the kruger national park, south africa, in which a survey on the ticks infesting wild carnivores has been conducted (horak, braack, fourie & walker 2000). the present communication records the results of a tick survey conducted on domestic dogs owned by people in 27 rural communities and villages spread throughout the province of maputo. materials and methods thirty rural communal cattle dip-tanks or villages, evenly spread throughout maputo province were selected for the survey. ticks were to be collected, whenever possible, from five dogs at each of these localities. in addition, ticks were collected from 24 dogs at two villages in south-western maputo province during a rabies inoculation campaign, as well as from two dogs at two villages in the south-east of the province. to minimize stress to the dogs as well as injuries to the researchers during the collections, the animals were usually muzzled and thereafter restrained by their owners. the ticks were stored in 70 % ethyl alcohol in internally labelled vials for later identification and counting. the geographic coordinates of each locality at which ticks were collected were recorded and used to plot the distributions of the two most commonly collected species, namely h. elliptica and r. simus. results and discussion no dogs were available at seven of the chosen localities, and less than five dogs were examined at five. a total of 132 dogs was examined and 1 768 ticks were collected. of the nine species that could be identified, h. elliptica and r. simus were the most numerous and the most widely distributed (table 1, fig. 1). one of the dogs was infested with six tick species, three with five and 13 with four species, and the largest number of ticks collected from a single dog was 41. combining the results of the present survey with those of the survey conducted by neves et al. (2004) and the ticks listed by dias (1991), a total of 16 tick species has now been collected from dogs in mozambique. amblyomma hebraeum adult ticks of this species prefer large herbivorous animals as hosts (norval 1983; horak, macivor, petney & de vos 1987a; horak, golezardy & uys 2007). these large ticks are also occasionally found on dogs and other medium-sized animals, particularly when the former are in poor health, as some of the dogs in the present survey were. the larvae infest a large variety of hosts, including large and small herbivores, carnivores and ground-frequenting birds, while some of these animals are also infested by nymphs (norval 1983; horak et al. 1987a, 2000). 105 c. de matos et al. the immature stages are almost invariably present on free-running domestic dogs within the tick’s distribution range in rural areas in southern africa (horak et al. 1987b, 2001; neves et al. 2004; nyan giwe et al. 2006). although the present survey was designed to collect adult ticks, some larvae and a fairly large number of a. hebraeum nymphs were recovered, undoubtedly because of the latter’s large size, particularly when they are engorged. haemaphysalis elliptica this tick has been collected from dogs in all the surveys conducted in south africa as well as in the one previously conducted in mozambique. not only was it present on dogs at all except two of the 27 sampling localities in the current survey, but it was recovered from 100 of the 132 dogs examined (fig. 1a, table 1). free-ranging domestic dogs and the larger wild felids are particularly susceptible to infestation with the adults of h. elliptica (horak et al. 1987b, 2000, 2001; neves et al. 2004). its immature stages infest murid rodents and have been collected from these animals in many regions of south africa (fourie, horak & van den heever 1992; braack, horak, jordaan, segerman & louw 1996; petney, horak, howell & meyer 2004; horak, fourie & braack 2005; matthee et al. 2007). however, no systematic collections of ticks from these small mammals appear to have been done in mozambique. haemaphysalis elliptica is widely distributed in south africa (howell et al. 1978) and its distribution extends into southern mozambique and zimbabwe (apanaskevich et al. 2007). its presence in the southern part of the country is confirmed by the present survey (fig. 1a). in central and northern mozambique its distribution overlaps that of the southern distribution of haemaphysalis leachi sensu strictu (apanaskevich et al. 2007). in the past it was presumed that h. leachi was responsible for the transmission of babesia canis rossi, the cause of virulent babesiosis in domestic dogs in south africa (lewis, penzhorn, lopez-reboltable 1 ticks collected from 132 dogs at 27 rural localities in maputo province, mozambique tick species no. of positive localities no. of dogs infested number of ticks collected larvae nymphs males females total amblyomma hebraeum haemaphysalis elliptica haemaphysalis spp. ixodes cavipalpus rhipicephalus appendiculatus rhipicephalus evertsi evertsi rhipicephalus pravus group rhipicephalus sanguineus rhipicephalus simus rhipicephalus turanicus 16 26 4 1 7 2 1 8 24 9 57 100 6 2 13 2 1 21 98 22 48 0 0 0 0 0 0 1 0 0 195 9 0 0 37 0 0 8 0 0 24 252 3 1 2 2 0 120 334 39 4 331 4 2 0 1 2 67 259 23 271 592 7 3 39 3 2 196 593 62 fig. 1 localities at which (a) haemaphysalis elliptica and (b) rhipicephalus simus were collected from dogs in maputo province, mozambique haemaphysalis elliptica mozambique south africa swaziland maputumaputu matolamatola marracuene sedemarracuene sede moamba sede magude sedemagude sede bella vistabella vista n naamacha sedenaamacha sede a rhipicephalus simus mozambique south africa swaziland maputumaputu matolamatola marracuene sedemarracuene sede moamba sede magude sedemagude sede bella vistabella vista n naamacha sedenaamacha sede b 106 ixodid ticks on dogs in maputo province, mozambique lar & de waal 1996). it is now apparent that lewis et al. (1996) conducted their experiments with h. elliptica and that it is this tick that is the vector of b. canis rossi (apanaskevich et al. 2007). the role of h. leachi sensu strictu as a vector of b. canis rossi or other pathogenic organisms that may affect domestic dogs still needs clarification. ixodes cavipalpus according to walker (1991) i. cavipalpus is probably the most widely distributed ixodes sp. in the afrotropical region. amongst a variety of other hosts, it has been collected from both small and large domestic and wild carnivores (theiler 1962; walker 1991), and two males and six females have been collected from domestic dogs in harare, zimbabwe (goldsmid 1963). in the present survey, the only collections consisted of a single male and two females taken from dogs at the mahubo dip-tank between boane and bela vista in the south of the province. not only dogs, but also cattle and goats examined at the same locality were infested. rhipicephalus appendiculatus adult r. appendiculatus infests cattle, goats and large wild bovids (walker et al. 2000; nyangiwe & horak 2007), and its immature stages are found on the same hosts as the adults as well as on smaller bovids and scrub hares (horak & fourie 1991; walker et al. 2000). all stages of development may, however, infest domestic dogs and wild carnivores (goldsmid 1963; horak et al. 2000, 2001; nyangiwe et al. 2006). in the present study, nymphs and males were collected from 13 dogs at seven localities. infestation of dogs with this tick species has, in isolated instances, epidemiological implications for the transmission of theileria parva, the cause of east coast fever and corridor disease in domestic cattle (lawrence, perry & williamson 2004). many resource-limited rural hunters use dogs to run down their prey, often cutting, or lifting fences between farms to gain access to wildlife. the dogs could either introduce r. appendiculatus onto a previously tick-free property, or acquire infestation during the hunt and bring the ticks back to their owner’s property. rhipicephalus sanguineus all stages of development of r. sanguineus infest domestic dogs (horak 1982), which walker et al. (2000) believe are its only true hosts. although our collections focused on adult ticks, one larva and eight nymphs were collected, confirming the host preference of these stages. the tick’s life cycle is adapted to the resting places of dogs in man-made structures such as sheds, dog kennels and human dwellings (howell et al. 1978; horak 1982). its prevalence and intensity of infestation are significantly higher on city or urban dogs, which are usually confined to the properties of their owners, than on rural dogs (neves et al. 2004). its presence on 21 dogs at eight of the rural sampling localities in the present survey suggests that these animals were at least for some part of the day, chained, or caged at the homes of their owners. rhipicephalus simus adult r. simus has a preference for domestic and wild carnivores, zebras, equus spp., warthogs, phaco choerus africanus, and domestic cattle (walker et al. 2000). in a recent survey conducted in the eastern cape province, south africa, 29 dogs at 14 sampling localities were infested with this tick (nyangiwe et al. 2006), whereas no r. simus were collected from 360 cattle at 72 localities, including the 14 at which dogs were infested (nyangiwe & horak, unpublished data 2007). in the present study there also appeared to be a slight preference for dogs rather than for cattle, in that r. simus was collected from 98 dogs (74.2 %) of the 132 examined compared to 102 cattle (70.3 %) of 145 examined. its immature stages infest murid rodents (norval & mason 1981; braack et al. 1996; horak et al. 2005), and it is thus more common on dogs in rural environments, where rodents are likely to be plentiful, than on city or urban dogs (horak et al. 1987b, 2001; neves et al. 2004). with the exception of the sabié dip-tank in the central western region of maputo province, the chiango dip-tank in the east, and one of the villages in the south-east, r. simus was present on dogs throughout the province (fig. 1b). this distribution lies within that mapped for this tick in southern africa (walker et al. 2000). rhipicephalus turanicus like r. sanguineus, r. turanicus has a very widespread distribution outside africa, and these two ticks are often mistaken for each other, particularly as the adults of both infest domestic dogs (walker et al. 2000). unlike r. sanguineus, however, r. turanicus does not make use of man-made structures as an integral part of its life cycle. furthermore, whereas r. sanguineus infests only domestic dogs during all stages of development, adult r. turanicus 107 c. de matos et al. infests a variety of hosts in addition to domestic dogs, amongst these are cats, sheep, goats, horses, wild carnivores, hares and birds, and its immature stages probably infest rodents and other small mammals, but not dogs (horak, spickett, braack, penzhorn, bagnall & uys 1995; horak et al. 2000; walker et al. 2000). because r. turanicus is a parasite of wildlife as well as domestic animals, it is encountered on animals in suburban, rural and wildlife environments (walker et al. 2000). moreover, it would seem from the restricted late summer abundance of its adults (horak et al. 2001), that it is only likely to complete one life cycle annually in south africa, whereas r. sanguineus often completes more than one (horak 1982; horak & matthee 2003). acknowledgements we express our sincere appreciation to the directorate of animal science, mozambique for permitting c. de matos and c. sitoe to conduct this survey as part of their official duties. we are most grateful to the dog owners who assisted with the collection of ticks from their animals, and to dr roy williams of the arc-onderstepoort veterinary institute for mapping the distribution of the ticks. the project was financed by funds provided to c. de matos by the sida/sarec-joint inive-veternary faculty research project # 27, and by funds provided to i.g. horak by the national research foundation. references apanaskevich, d.a., horak, i.g. & camicas, j.-l. 2007. re description of haemaphysalis (rhipistoma) elliptica (koch, 1844), an old taxon of the haemaphysalis (rhipistoma) leachi group from east and southern africa, and of haemaphysalis (rhipistoma) leachi (audouin, 1826) (ixodida, ixodidae). onderstepoort journal of veterinary research, 74:181–208. braack, l.e.o., horak, i.g., jordaan, leonora c., seger man, joyce & louw, j.p. 1996. the comparative host status of red veld rats (aethomys chrysophilus) and bushveld gerbils (tatera leucogaster) for epifaunal arthropods in the southern kruger national park, south africa. onderstepoort journal of veterinary research, 63:149–158. bryson, n.r., horak, i.g., höhn, e.w. & louw, j.p. 2000. ectoparasites of dogs belonging to people in resource-poor communities in north west province, south africa. journal of the south african veterinary association, 71:175–179. dias, j.a. travassos santos. 1991 (1993). some data concerning the ticks (acarina, ixodoidea) presently known in mozambique. 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onderstepoort journal of veterinary research, 58:81–105. walker, jane b., keirans, j.e. & horak, i.g. 2000. the genus rhipicephalus (acari, ixodidae): a guide to the brown ticks of the world. cambridge: cambridge university press. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (europe iso coated fogra27) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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/untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [600 600] /pagesize [595.276 841.890] >> setpagedevice article information authors: joanna klećkowska-nawrot1 karolina goździewska-harłajczuk1 renata nowaczyk1 krzysztof krasucki2 affiliations: 1department of animal physiology and biostructure, university of environmental and life sciences, poland 2department of descriptive and clinical anatomy, centre of biostructure research, medical university of warsaw, poland correspondence to: joanna klećkowska-nawrot email: lestat_v@poczta.onet.pl postal address: kozuchowska 1/3, 51-631 wroclaw, poland dates: received: 21 aug. 2014 accepted: 21 nov. 2014 published: 25 mar. 2015 how to cite this article: klećkowska-nawrot, j., goździewska-harłajczuk, k., nowaczyk, r. & krasucki, k., 2015, ‘functional anatomy of the lacrimal gland in african black ostrich struthio camelus domesticus in the embryonic and postnatal period’, onderstepoort journal of veterinary research 82(1), art. #872, 12 pages. http://dx.doi.org/10.4102/ojvr.v82i1.872 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. functional anatomy of the lacrimal gland in african black ostrich struthio camelus domesticus in the embryonic and postnatal period in this original research... open access • abstract • introduction • research methods and design    • animals    • macroscopic evaluation    • histological analysis    • histochemical analysis • results    • gross anatomy    • group 1       • morphometry       • histology and histometry       • histochemical analysis    • group 2       • morphometry       • histology and histometry       • histochemical analysis    • groups 3 and 4       • morphometry       • histology and histometry       • histochemical analysis • ethical considerations • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the aim of the present study was morphological and histochemical analysis of the lacrimal gland (lg) in african black ostrich struthio camelus domesticus in the embryonic and postnatal period. studies were conducted on 50 ostriches aged between the 28th day of incubation until 7 months old. tissue sections were stained with haematoxylin and eosin, azan trichrome, periodic acid-schiff, alcian blue ph 2.5, aldehyde fuchsin and hale's dialysed iron. the lg in ostrich was classified as a tubulo-acinar type. the primordia of the lobes were determined in the lg structure on the 28th day of incubation, whilst the weakly visible lobes with acini and tubules were observed on the 40th day of incubation. morphometric studies of the lg showed steady growth, characterised by an increase in both length and width. histometric measurements of lobe size showed little difference between the first, second and third age groups, whilst in the fourth age group a marked increase in size of lobes was observed. the study showed that, apart from morphological changes, during the growth of the lg the character of acid mucopolysaccharides changed. sulphated acid mucopolysaccharides were indicated, particularly with aldehyde fuchsin (af) staining in the fourth age group. the hale's dialysed iron (hdi) staining showed a low concentration of carboxylated acid mucopolysaccharides in the first and second age groups and a higher concentration in the third and fourth age groups. periodic acid-schiff staining (pas)-positive cells were observed in each age group, but only a small number of cells with a weakly pas-positive reaction were demonstrated in the first age group. introduction top ↑ the lacrimal and harderian glands are the main orbital glands belonging to the lacrimal apparatus (international committee on veterinary gross anatomical nomenclature 2012; jordan 1990; paynter 1993). both glands have been described in amphibians, reptiles, birds and mammals (sakai 1988; shirama & hokano 1992). the anatomical structure of these glands is characterised by great variability (chieffi baccari, di matteo & minucci 1992; dimitrov 2011a, 2011b; rehorek et al. 2005; schechter, warren & mircheff 2010). in birds the lacrimal gland (lg) is less developed and of smaller size than the harderian gland (dimitrov 2011a; dimitrov & genchev 2011). the lg is located within the peri-orbita in the dorsotemporal part of the orbit and secretes via multiple excretory ducts into the conjunctival space beneath the lower eyelid (harris et al. 2008; jones, pierce & ward 2007; kern 2007). the harderian gland is located medioventrally to the eyeball, near the inter-orbital septum (boydak & aydin 2009; burns 1992; dimitrov & genchev 2011; kozlu & altunay 2011; mobini 2012). in birds the lacrimal apparatus has been implicated as a part of the head-associated lymphatic tissue system (rehorek et al. 2005, citing burns 1992) in which the harderian gland is the lympho-epithelial organ (dimitrov 2011a; ohshima & hiramatsu 2002) and a site of immune response (baba et al. 1990; boydak & aydin 2009; kozlu & altunay 2011; payne 1994). the lg is responsible for production of tear fluid which, together with the harderian gland and conjunctiva-associated lymphoid tissue, helps to maintain corneal health (lavach 1990; mohammadpour 2009). the lg produces serous, mucous or seromucous fluid (lavach 1990). the lg excretory products are part of the tear film that moistens and lubricates the anterior eye surface and also provides nutrients (dartt 2009; funaki, hodges & dartt 2010; klećkowska-nawrot & dzięgiel 2008; klećkowska-nawrot et al. 2013; payne 1994; zagon et al. 2012). the tear film protects the anterior surface of the cornea and both superior and inferior conjunctival sacs (jordan 1990; payne 1994), and allows gas exchange between the air and the epithelium (lavach 1990; mohammadpour 2009). the tear film consists of three layers: a surface lipid layer, a middle aqueous layer, and an inner mucous layer (walcott 1998) covering the exposed surface of the eye, namely the cornea and conjunctiva (hodges & dartt 2003). the lg produces the constituents of the aqueous layer (hodges & dartt 2003). the tears contain several soluble antimicrobial factors that protect the ocular surface (davidson & kuonen 2004). the lg-specific proteins found at highest concentrations in tears are lactoferrin, tear-specific pre-albumin and lysozyme (kijlstra & kuizenga 1994). a clear cornea with a smooth, well-lubricated facade is a prerequisite for clear vision. this is particularly important for animals that inhabit dry, hot and sandy areas (mohammadpour 2009). struthio camelus domesticus, also known as african black ostrich, is usually referred to by breeders as ‘black necks’. the african black ostrich is the result of many years’ selection. this breed was created by crossing two subspecies: the north african ostrich, struthio camelus camelus, and the southern african ostrich, struthio camelus australis (busse 1990; horbańczuk 2003). african black ostrich is characterised by a milder temperament, better quality of feathers, as well as better growth than the others (horbańczuk 1997). ocular diseases can have a significant impact on the condition and productivity of breeding animals. the majority of dry eye symptoms are the result of chronic inflammation of the lg, which decreases the ability of the eye to respond to environmental factors (sorgolu, yucel & aktas 2003; stern et al. 2004). the lg secretory function and tear film composition play an important role in eye physiology and pathology (flanagan & willcox 2009; kawashima et al. 2012). the aim of the present study was morphological and histochemical analysis of the lg during the embryonic and postnatal period in ostriches. detailed knowledge of macroscopic and microscopic lg morphology can be a basis for diagnosis and treatment of ocular diseases in ostriches. research methods and design top ↑ animals the studies were performed on 50 african black ostriches aged between the 28th day of incubation and 7 months. the study included 12 pre-hatching birds (28, 33 and 40 days of incubation) and 38 post-hatching birds (24, 48, 72 hours after hatching; 2, 3, 5 weeks of age; 5, 6, 7 months of age). the animals were divided into four age groups on the basis of histological differences. the embryos were obtained from incubated eggs provided by an ostrich farm in namyslow, poland. extracted embryos were classified into different developmental stages based on the days of incubation. the post-hatching birds from the ostrich farm in namyslow were collected in 2010–2011. all the birds died as a result of natural causes. mummified or underdeveloped embryos and nestlings were excluded from this study. the lg was dissected from embryos and birds immediately after death and fixed in 4% buffered formaldehyde for 48 hours. macroscopic evaluation macroscopic evaluation of the lg was performed using a stereoscopic zeiss stemi 2000-c microscope (carl zeiss, jena, germany). solutions of 0.5% – 4% acetic acid (ch3cooh), and 70% ethyl alcohol (c2h5oh) were used for the clear presentation of lg anatomical structure. lg morphology was described using topographic anatomical methods, namely holotypy and syntypy. morphometric measurements (length and width) of glands were obtained using an electronic slide caliper with an accuracy of 0.1 mm. data were statistically processed by statistical software (microsoft office professional plus 2010, microsoft corporation, redmond, wa, usa). histological analysis the dissected lgs were immediately fixed in 4% buffered formaldehyde for 48 hours, rinsed in running water for 24 hours and then processed in a vacuum tissue processor (etp) (rvg3, intelsint, italy), embedded in paraffin and cut on sliding microtome slide 2003 (pfma.g., germany) into 3 µm – 4 µm sections. all samples were stained with h&e and azan trichrometo demonstrate the general structure. the histological slides were examined with a zeiss axio scope a1 light microscope (carl zeiss, jena, germany) for the histological description (zawistowski 1965). histochemical analysis histochemical analysis was conducted as follows: periodic acid-schiff staining (pas) to identify the presence of muco-substances (neutral or acidic) and glycoproteins; alcian blue (ab) ph 2.5 to show sialylated glycoproteins; hale's dialysed iron (hdi) and aldehyde fuchsin (af) to demonstrate sulphated acid mucopolysaccharides (sam) and carboxylated acid mucopolysaccharides (cam) and elastic tissue was demonstrated by af staining. slides were examined with a zeiss axio scope a1 light microscope (carl zeiss, jena, germany) with an axio vision system (axio scope a1, carl zeiss, jena, germany). pas, ab ph 2.5, af and hdi staining scoring systems were based on standard protocols that have been described previously (spicer & henson 1967). anatomical terminology used was according to paynter (1993). results top ↑ gross anatomy the lg of the ostriches became macroscopically visible by the 28th day of incubation, and appeared as a uniform, undivided and flattened gland. in all age groups this gland was bright red and oval, and positioned in the dorsolateral angle of the orbit, between the lateral and dorsal rectus muscles, close to the pyramidal third eyelid muscle and tendon of the pyramidal muscle. the lg secretes via multiple ducts that open into the conjunctival space beneath the lower eyelid. the nasolacrimal duct penetrated the lacrimal bone and rostrum maxillae premaxillae bone and opened into the nasal cavity. group 1 morphometry the mean size of the lg in the first age group of pre-hatching birds (length × width with secondary duct [sd]) was 4.92 mm (± 0.9) × 2.65 mm (± 0.5) (figure 1). figure 1: morphometric parameters of the lacrimal gland in african black ostrich in the embryonic and postnatal period. histology and histometry the lg was covered by a thin connective tissue capsule with septa extending into the gland and dividing it into primordia or lobes (figure 3a). the loose connective tissue was composed of adipocytes, a large amount of blood vessels, fibroblasts, rare lymphocytes, and collagenous and reticular fibres (figure 3f). the numerous fine elastic fibres in the capsule and gland septa were demonstrated with af staining (figure 4d). the average thickness of connective tissue interlobular septa was 34.26 µm (± 6.5) (figure 2). amongst primordia of the lobes, lobules with cubic cells within the epithelial layer and a wide lumen were found on the 28th and 33rd days of incubation (figures 3b and 3d). there was no division into acini and tubules (figures 3c and 3f). the average size of lobes was 205.07 µm (± 49.6) (figure 2). on the 40th day of incubation a clear division into lobes with interlobular connective tissue was observed, and the acini and ducts were present (figure 3e). in the central part of the lg the lobes had 3–4 acini and tubules, whilst closer to the periphery of the gland the number of acini and tubules was higher (7–12). the mean outer diameter of the glandular acini on the 40th day of incubation was 40.7 µm (± 7.0) and the mean outer diameter of the secondary ducts was 130.61 µm (± 24.5) (figure 2). the lg primary ducts were absent in the first group of birds. figure 2: histometric parameters of the lacrimal gland in african black ostrich in the embryonic and postnatal period. figure 3: light micrograph of the lacrimal gland in african black ostrich in group 1. (a) 28th day of incubation, (b) 28th day of incubation, (c) 40th day of incubation, (d) 33rd day of incubation, (e) 40th day of incubation and (f) 28th day of incubation. histochemical analysis examination of the lg with pas staining showed numerous cells with a weakly positive reaction (−/+) that contained pas-positive granules. pas staining detected the presence of neutral and acid mucopolysaccharides and glycoproteins (figure 4a and table 1). ab ph 2.5 staining showed the presence of slightly positive granules (−/+) (light blue colour) in cells or single cells with blue granules that were considered positive (+) cells with ab ph 2.5 (figure 4b and table 1). this reaction indicated the presence of sialylated glycoproteins. hdi staining demonstrated a slightly positive reaction (−/+) in only a few cells, which were weakly blue in colour (figure 4c and table 1). hdi staining detects the presence of cam. af staining gave a positive reaction (+/++), with a purple colour that indicated the presence of sam (figure 4d and table 1). figure 4: light micrograph of the lacrimal gland in african black ostrich in group 1. (a) 33rd day of incubation, (b) 33rd day of incubation, (c) 40th day of incubation and (d) 28th day of incubation. table 1: histochemical analysis of the lacrimal gland in african black ostrich in the embryonic and postnatal period. group 2 morphometry the mean size of the lg in the second age group (length × width × with sd) was 7.22 mm (± 1.3) × 3.02 mm (± 0.4) (figure 1). histology and histometry in this group of birds the slightly marked lobes observed were comparable to those in the first age group. the average thickness of connective tissue interlobular septa was 27.57 µm (± 3.2) (figure 2). the average size of lobes in this group was 216.91 µm (± 38.9) (figure 2). each lobe contained from 2 to 7 acini and ducts (figure 5a). the primary and secondary ducts were well defined and branched (figures 5b and 5f). the acini were composed of tall conical cells with a small lumen and were surrounded by myoepithelial cells (figure 5c). the mean outer diameter of the glandular acini was 42.52 µm (± 3.4) (figure 2). the ducts had one layer of cubic epithelium and a large and irregular lumen (figures 5b and 5c). the mean outer diameter of the primary ducts in this group was 142.31 µm (± 13.7) and of the secondary ducts was 119.27 µm (± 8.6) (figure 2). figure 5: light micrograph of the lacrimal gland in african black ostrich in group 2. (a) 24th hour after hatching, (b) 24th hour after hatching, (c) 72th hour after hatching, (d) 72th hour after hatching, (e) 24th hour after hatching, and (f) 48th hour after hatching. histochemical analysis in this group of animals pas staining of lg demonstrated the presence of secretory cells containing pas-positive (+/++) granules irregularly located in glandular acini and ducts (figure 5d and table 1). staining with ab ph 2.5 indicated the presence of positive granules (+/++) in glandular acini and duct epithelial cells (figure 5e and table 1). hdi staining showed a weakly positive reaction ( /+) in the peripheral part of lobes and a positive reaction (++) in the central part of lobes amongst the ducts, indicating the presence of cam (figure 5f and table 1). in addition, the af staining showed weakly positive granules (−/+) in acini and ducts, indicating the presence of sam (table 1). groups 3 and 4 morphometry the mean size of lg in group 3 (length × width with sd) was 9.73 mm (± 1.9) × 4.54 mm (± 1.3) (figure 1) and in group 4 was 14.52 mm (± 2.4) × 5.68 mm (± 1.2) (figure 1). histology and histometry significant changes in the acini and secondary and primary tubules were observed in the group 3 and 4 birds. in these age groups less interlobular connective tissue and a clear division into lobes were observed in comparison to the first and second age groups (figures 6a, 6c and 8a). the average thickness of interlobular septa in the third age group was 17.41 µm (± 4.9) and in the fourth age group 25.23 µm (± 6.7) (figure 2). the average size of lobes in the third age group was 215.7 µm (± 25.8) and in the fourth age group 402.49 µm (± 30.8) (figure 2). the acini were composed of tall conical cells that formed a small lumen and were surrounded by myoepithelial cells. the myoepithelial cells lay around the basal membrane of the acini (figures 6b, 6d and 8c). the nuclei of the acini cells were ovoid and located in the basal compartment of the cytoplasm. these cells had granular, basophilic, vacuolated cytoplasm (figure 6b, 8c and 8d). the mean outer diameter of the glandular acini in the third age group was 52.67 µm (± 11.4) and in the fourth age group 73.63 µm (± 12.7) (figure 2). the secondary tubules were lined with cuboidal cells and had a large and irregular lumen. the nuclei of these cells had an oval shape and were located in the basal part of cells (figures 6b, 6d and 8c). the mean outer diameter of the secondary ducts in the third age group was 109.28 µm (± 7.1) and in the fourth age group was 130.08 µm (± 13.1) (figure 2). in the central part of each lobe from 2 to 4 primary ducts were present. the mean outer diameter of the primary ducts in the third age group was 165.75 µm (± 19.2) and in fourth age group 208.51 µm (± 20.6) (figure 2). figure 6: light micrograph of the lacrimal gland in african black ostrich in group 3. (a) 2-weeks-old, (b) 2-weeks-old, (c) 3-weeks-old and (d) 3-weeks-old. histochemical analysis the differences in histochemical findings between the third and fourth age groups of birds are indicated in table 1. group 3: pas staining demonstrated the presence of secretory cells containing pas-positive granules evaluated as (+) and not many cells with a stronger reaction, evaluated as (+/++), irregularly located in glandular acini and primary and secondary ducts (figure 7a and table 1). staining with ab ph 2.5 in the lg of 2-week-old ostriches demonstrated the presence of weakly positive granules (−/+) in the glandular units and ducts (table 1), whilst in the lg of 3-and 5-week-old birds this staining showed the presence of positive granules (++) in all acini and ducts (figure 7b). hdi staining showed the presence of positive granules evaluated as (++) both in acini and in secondary ducts, and indicated the presence of cam (figure 7c and table 1). af staining showed weakly positive granules (−/+) in acini and secondary ducts that also indicated the presence of cam (figure 7d and table 1). figure 7: light micrograph of the lacrimal gland in african black ostrich in group 3. (a) 3-weeks-old, (b) 3-weeks-old, (c) 5-weeks-old and (d) 2-weeks-old. figure 8: light micrograph of the lacrimal gland in african black ostrich in group 4. (a) 6-months-old, (b) 6-months-old, (c) 5-months-old and (d) 7-months-old. group 4: in 6-month-old birds a weakly pas-positive (−/+) reaction was observed in a small number of glandular cells (figure 9b and table 1), whereas in 7-month-old birds a pas-positive reaction, evaluated as (+++), was evident in all acini and ducts (figure 9a). staining with ab ph 2.5 indicated the presence of strongly positive granules (+++) located in glandular acini and primary and secondary ducts (figures 9c and 9d, table 1). hdi staining indicated a medium-positive reaction (++), similar to that in the third age group of ostriches, indicating the presence of cam (figures 10a and 10b, table 1). in addition, the af staining showed the presence of positive granules evaluated at (++/+++) in acini and ducts, indicating the presence of sam (figures 10c and 10d, table 1). figure 9: light micrograph of the lacrimal gland in african black ostrich in group 4. (a) 7-months-old, (b) 6-months-old, (c) 6-months-old and (d) 5-months-old. figure 10: light micrograph of the lacrimal gland in african black ostrich in group 4. (a) 7-months-old, (b) 5-months-old, (c) 6-months-old and (d) 5-months-old. ethical considerations top ↑ this study was approved by the ii local ethical committee in wroclaw, poland (no. 19/2010). discussion top ↑ the lg is poorly described in birds compared to mammals (burns 1976; henker et al. 2013; mohammadpour 2009). the lg gland in each age group of ostriches examined was located in the region of the posterior commissure of the eyelid, in the dorsolateral angle of the orbit between the lateral and dorsal rectus muscles, close to the pyramidal third eyelid muscle and tendon of the pyramidal muscle. similar localisation of this gland has been demonstrated in other avian species, e.g. turkey, chickens and ducks (burns 1979; dimitrov 2011b). the lg in pigs is also located in the dorsolateral angle of the orbit, but between the dorsal oblique muscle and the lateral rectus muscle, and appears as a soft and pale structure (henker et al. 2013), whereas the lg was bright red in the ostriches examined. in the present study the ostrich lg appeared at around the 28th day of incubation as a uniform, undivided, oblate gland. development of the lg is an example of an epithelial-mesenchymal interaction (johnston et al. 1979). the tubular invagination, extension and branches give rise to the lobular structure of the mature gland (kammandel et al. 1999). according to chieffi baccari, di matteo and minucci (1995) the lg in the lizard podarcis s. sicula appears on about the 22nd day of development. during development the acini fill up the preformed mesenchymal sac (chieffi baccari et al. 1995). the lg in pigs becomes macroscopically visible at the 42nd day of gestation (klećkowska-nawrot & dzięgiel 2008). studies of the histological structure of the lg have been reported in various birds, but not in ostriches (burns 1976; dimitrov & genchev 2011). in ostriches the lg was characterised by an increase in both length and width. the largest gland was observed in the fourth age group of ostriches, in which the largest increase in length was demonstrated compared to the other age groups. the entire surface of the lg was covered by a connective tissue capsule. the interstitium consisted of some collagen fibres separating the glandular lobe, as previously reported by others (harris et al. 2008; schechter et al. 2010). in ostriches the lg consists of tubules lined with secretory epithelium and organised into lobes that drain into ducts; these anastomose into larger ducts that finally drain onto the ocular surface. the lg of the ostrich has been classified as a compound tubular-acinar gland, as in japanese quails (dimitrov & genchev 2011). according to other authors the cells of the secretory epithelium in tubules and the acini are columnar with basally located nuclei. the duct cells are similarly organised, although more cuboidal in shape (walcott 1998). studies of the lg in turkey and chicken broilers demonstrated differences in the number of lobules between these birds (dimitrov 2011b). in the present study the size of the glandular lobes was evaluated. histometric measurements of glandular lobe size showed little difference amongst the first, second and third age groups; however, in group 4 a marked increase in lobe size was observed. different results were obtained in measurement of interlobular septa thickness, which decreased from the first to the third age group, with an increase of thickness in the fourth age group. this may be related to the significant increase in lobe size in birds of the fourth age group. on the other hand, the outer diameter of acini showed an increase from the first to the fourth age group of ostriches. this increase in size was small in the first and second age groups and larger in the third and fourth age groups. varied results were obtained in measurement of the outer diameter of the primary and secondary glandular ducts. there were no primary glandular ducts in the first age group, whilst from the second to the fourth age groups a steady increase was demonstrated in the outer diameter of the primary ducts. secondary glandular ducts were observed in each age group, but only from the 40th day of incubation in the first age group. the outer diameter of these ducts was high in the first age group. the outer diameter of the secondary ducts underwent a slight decrease in groups 2 and 3, increasing again in the fourth age group and reaching a size similar to the first age group. the number of secretory ducts in turkey and chicken broilers decreased from the periphery towards the central part of glandular lobules (dimitrov 2011b). there were no plasma cells in any of the groups of birds studied, and only a few lymphocytes were observed, confirming other reports that the harderian gland is responsible for the production of immunoglobulin in birds (khan et al. 2007; kozlu & altunay 2011; nasrin et al. 2013). different observations were made by burns (1979). surgical removal of the harderian gland in the domestic fowl resulted in increased secretory activity of the lg and also in an increase in goblet cell numbers along the length of the lg duct. plasma cells were more numerous in the lg in birds that were operated on (burns 1979). histochemical analysis of ostriches’ lg showed differences in the proportion of secreted sam to cam in acini and duct epithelial cells. in each age group this proportion was evaluated on the basis of hdi and af staining. sam were indicated particularly by af staining of the lg in the fourth age group of ostriches; however, sam were not observed with either hdi or af staining in the third age group. furthermore, hdi staining did not demonstrate sam in any age group of ostriches. instead hdi staining showed low concentrations of cam in the first and second age groups, and a higher concentration in the third and fourth age groups. very different results were obtained with pas staining. in the lg of examined birds, pas-positive cells were observed in each age group; however, only a small number of cells with a weakly pas-positive reaction was demonstrated in the first age group. in the second age group a medium pas-positive reaction was observed, but in the third age group the pas-positive reaction was weaker. similar results were obtained by millar et al. (1996), who demonstrated a small number of pas-positive acini cells in the superior and inferior lg of the rabbit. results of research on morphological changes in rat lg indicate that ageing is associated with alteration in the ability of acinar cells to synthesise and secrete proteins (draper et al. 2003). according to sakai (1989) the lobules of the lg comprise a branched duct system and terminal acini with two types of secretory cells: acidic cells positive both for pas and ab ph 2.5 and neutral cells positive for pas and negative for ab ph 2.5. the ab ph 2.5 staining of the lg in examined ostriches indicated the strongest positive reaction in the fourth age group, both in acinar epithelium and in primary and secondary duct epithelium. in the same group a strong pas-positive reaction was demonstrated in the 7-month-old birds. in the other age groups pas and ab ph 2.5 staining showed a slight difference. chieffi baccari et al. (1992) also proved that the lg is composed of two cell types characterised by histochemical staining with ab ph 2.5 and pas. rare pas-positive cells were found scattered in the acinar epithelium, as in the first age group of ostriches examined in this study. conclusion top ↑ in the ostriches that were examined in this study, steady growth of the lg was observed with an increase in both length and width. together with the growth of the gland, a marked increase in size of the lobes and glandular acini was observed, with apparent increase in dimensions of the gland structures. within the lobes a linear increase in lobular size was observed. the development of primary ducts was present from the second to the fourth age groups. the study showed that apart from morphological changes, during the growth of lg the character of acid mucopolysaccharides varied. histological study showed no plasma cells in any group of birds, and confirmed other reports that not the lg but the harderian gland is responsible for the production of immunoglobulin in birds. acknowledgements top ↑ this research was supported by statutory research and development activity funds assigned to the faculty of veterinary medicine, wroclaw university of environmental and life sciences. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions j.k-n. (university of environmental and life science) obtained the research material, performed the study in the departmental laboratory and edited manuscript. k.g-h. (university of environmental and life science) supervised the research and edited the manuscript. r.n. (university of environmental and life science) supervised the research and edited manuscript. k.k. 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rats is related to opioidergic signaling pathways’, investigative ophthalmology & visual science 53, 3234–3240. http://dx.doi.org/10.1167/iovs.11-9051 zawistowski, s., 1965, technika histologiczna oraz podstawy histopatologii, wydawnictwo lekarskie pzwl, warsaw. article information authors: geoff brown1 estelle h. venter2 paul morley3 henry annandale4 affiliations: 1department of production animal studies, university of pretoria, south africa 2department of veterinary tropical diseases, university of pretoria, south africa 3diagnostic medicine center, colorado state university, united states 4onderstepoort veterinary academic hospital, university of pretoria, south africa correspondence to: geoff brown email: geoff.brown@up.ac.za postal address: private bag x04, onderstepoort 0110, south africa dates: received: 17 dec. 2014 accepted: 09 mar. 2015 published: 15 june 2015 how to cite this article: brown, g., venter, e.h., morley, p. & annandale, h., 2015, ‘the effect of rift valley fever virus clone 13 vaccine on semen quality in rams’, onderstepoort journal of veterinary research 82(1), art. #919, 8 pages. http://dx.doi.org/10.4102/ojvr.v82i1.919 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the effect of rift valley fever virus clone 13 vaccine on semen quality in rams in this original research... open access • abstract • introduction • materials and methods    • experimental animals • ethical consideration    • vaccination procedure    • blood collection    • antibody testing    • semen collection    • semen evaluation    • exclusion of animals from statistical analysis    • statistical procedures • results    • clinical findings    • temperature variation    • antibody testing    • semen collection    • exclusions from statistical analysis    • descriptive statistics: semen evaluation data    • logistic regression analysis • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ rift valley fever (rvf) is an arthropod-borne viral disease of importance in livestock and humans. epidemics occur periodically in domestic ruminants. people in contact with infected livestock may develop disease that varies from mild flu-like symptoms to fatal viraemia. livestock vaccination may assist in disease control. rift valley fever virus (rvfv) clone 13 is a relatively new vaccine against rvf, derived from an avirulent natural mutant strain of rvfv, and has been shown to confer protective immunity against experimental infection with rvfv. the hypothesis tested in the current trial was that rams vaccinated with rvfv clone 13 vaccine would not experience a reduction in semen quality (measured by evaluating the percentage progressively motile and percentage morphologically normal spermatozoa in successive ejaculates) relative to unvaccinated control animals. ram lambs were screened for antibodies to rvfv using a serum neutralisation test. animals without detectable antibodies (n = 23) were randomly allocated to either a test group (n = 12) or a control group (n = 11). animals in the test group were vaccinated with rvfv clone 13 vaccine. daily rectal temperature measurements and weekly semen and blood samples were taken from all animals. seven animals were eliminated from the statistical analysis because of potential confounding factors. logistic regression analysis was performed on data gathered from the remaining animals to determine whether an association existed between animal group, rectal temperature and semen quality parameters. no correlation existed between the treatment group and values obtained for the semen quality parameters measured. there was no statistically significant post-vaccination decline in the percentage of live morphologically normal spermatozoa, or the percentage of progressively motile spermatozoa, either when assessed amongst all animals or when assessed within individual groups. a repeat study with a larger sample size and a more comprehensive pre-screening process may be indicated to avoid the inclusion of unsuitable animals. introduction top ↑ rift valley fever (rvf) (phlebovirus, bunyaviridae) is a mosquito-borne viral disease of ruminants with significant economic and public health implications. the virus may cause fatal disease in juvenile and adult animals and may induce abortion in pregnant animals (flick & bouloy 2005). it is also a zoonosis that may have debilitating or life-threatening effects (gerdes 2004). people who live and work with livestock are at greatest risk of contracting the disease (swanepoel & coetzer 2004). vaccination of livestock helps prevent the spread of disease by reducing the population of viraemic animals that may infect vectors. vaccination of dams confers colostral immunity to offspring, which reduces juvenile mortality (gerdes 2004). in south africa, three vaccines are available: a modified-live vaccine (mlv), a killed vaccine, and a vaccine derived from an avirulent natural mutant strain of rift valley fever virus (rvfv clone 13). rvfv clone 13 vaccine (onderstepoort biological products, onderstepoort, south africa) is produced from the avirulent strain 74hb59 of rvfv, derived originally by passage of a non-fatal human case of rvf in the central african republic through mice and vero cells. this strain lacks approximately 70% of the s-segment of the ribonucleic acid (rna) genome of the virus coding for the non-structural protein nss, which was found to be the determinant of virulence (ikegami & makino 2009; von teichman et al. 2010). this deletion renders the virus unable to revert to virulence in vivo (pepin et al. 2010). several bacterial, viral and protozoal diseases have been shown to cause a reduction in semen quality in mammals. this may be mediated by a local effect on scrotal thermoregulation, as in bovine scrotal cutaneous dermatophilus congolensis infection (sekoni 1993) or the hypothesised effect of scrotal oedema caused by eperythrozoon wenyonii infection (montes et al. 1994). ovine orchitis caused by arcanobacterium pyogenes was shown to cause a reduction in semen quality, either directly through bacterial invasion of spermatogenic tissue or by general inflammation caused by epididymo-orchitis (gouletsou et al. 2004). arteriviruses, such as equine viral arteritis or porcine respiratory and reproductive syndrome, have a tropism for testicular tissue that may directly affect spermatogenesis (prieto & castro 2005). bluetongue disease has been associated with infertility in male ruminants. this may occur either as a result of systemic pyrexia or microvascular lesions in the testis caused by orbivirus infection (kirschvink, raes & saegermann 2009; osburn 1994). vaccination with mlv vaccines has been cited by anecdotal accounts and proven by controlled trials to cause alterations in the spermiogram. vaccination with a mlv strain of bluetongue virus was shown to cause a significant transient reduction in semen quality (bréard et al. 2007). by contrast, the use of an inactivated bluetongue vaccine was reported to have no detrimental effect on semen quality (leemans et al. 2012). interestingly, vaccination of boars with a live, recombinant pseudorabies virus vaccine resulted in no significant difference in semen quality pre-vaccination and post-vaccination (castro et al. 1992). the use of inactivated porcine circovirus vaccines was found by researchers to have no statistically significant effect on semen quality (caspari et al. 2014). the purpose of the current study was to determine the effects (if any) of rvfv clone 13 vaccine on semen quality in rams, and to quantify such effects by measuring differences in semen motility, sperm morphology and rectal temperature over a period of approximately one ovine spermatogenic cycle of 42 days. materials and methods top ↑ experimental animals merino ram lambs aged between 9 and 14 months from a reserve flock were tested for antibodies to rvfv using a serum neutralisation test (snt). twenty-eight animals were tested once using the snt, and seropositive animals were excluded from participation in the study. animals were not screened for antibodies to any other diseases prior to inclusion in the trial. the resultant 23 seronegative rams were randomly divided into two groups of approximately equal size: group 1: animals vaccinated with rvfv clone 13 vaccine (n = 12). group 2: unvaccinated control animals (n = 11). semen was collected from all animals prior to vaccination of test animals. semen was then collected at weekly intervals for 42 days after vaccination. ethical consideration top ↑ ethical approval for the study was obtained from the university of pretoria research ethics committee (project v020-12). all procedures were carried out by a veterinarian. the study was performed at knoffelfontein farm, philipstown, northern cape, south africa. this facility is a department of agriculture, forestry and fisheries approved centre for assisted animal reproduction. all animals were housed together in an outdoor pen with sufficient shelter and ad libitum access to food and water. vaccination procedure on day 0 of the study, animals in the test group (n = 12) were vaccinated by subcutaneous injection with rvfv clone 13 vaccine (onderstepoort biological products) (obp) (batch number not specified). vaccine administration was performed as directed by the manufacturer, using a 3 ml syringe (braun omnifix®, melsungen, germany) and a three quarters inch 21 gauge needle (terumo, louvain, belgium). animals in the control group (n = 11) were unvaccinated. no inert placebo was used. blood collection blood was collected weekly by jugular venipuncture. approximately 3 ml of blood per ram was aspirated into a plain serum tube (vacutainer, becton-dickinson, woodmead, south africa). tubes were refrigerated at 4 °c for 48 hours until separation of serum and cellular components occurred. serum was placed in a fresh tube and frozen at -18 °c until delivery to the laboratory. antibody testing antibody testing was performed using a snt at the virology laboratory of the department of veterinary tropical diseases (dvtd), university of pretoria, according to the oie terrestrial manual (world organisation for animal health [oie] 2013) and slightly modified to the standard operating procedures (sop) of the laboratory. semen collection semen was collected by means of an artificial vagina (av) or by electrostimulation. where an av was used, one ram at a time was introduced into a pen containing a restrained ewe in oestrus. some rams showed normal sexual behaviour and attempted to mount the ewe, whereupon the penis was diverted into the av and semen was collected in a warmed collection vial. in animals where av semen collection failed, a lubricated electroejaculation probe (ruakara ram probe®, shoof international, cambridge, new zealand) was introduced into the rectum to the level of the prostate. electrical stimulation was intermittently applied until ejaculation occurred. semen was collected from an exteriorised penis into a warmed collection vial. semen evaluation semen evaluation comprised assessment of individual progressive motility and sperm morphology. all evaluations were performed by the same operator in order to ensure repeatability and consistency. all equipment used in semen handling was warmed to 37 °c. findings were recorded on a semen evaluation form according to the format set out by nöthling and irons (2008) and entered into a computer spreadsheet (excel® 2010, microsoft corporation, redmond, washington). individual sperm motility was evaluated by examining a droplet of extended semen (triladyl®, minitüb, tiefenbach, germany). a single droplet of extended semen was placed between a coverslip and microscope slide. ten fields at 200× magnification phase-contrast microscopy were evaluated according to a method previously described (nöthling & dos santos 2012). the percentages of individual progressive, aberrant and immotile sperm per field were estimated and recorded. the mean values for 10 fields described the subjective net motility of the ejaculate. sperm morphology was evaluated using 1000× oil-immersion bright-field light microscopy of an eosin-nigrosin stained smear, fixed under a coverslip using a mounting medium (entellan® merck millipore international, billerica, massachusetts). two hundred spermatozoa were evaluated per ejaculate. percentages of morphologically normal sperm, live morphologically normal sperm, sperm with nuclear defects, and sperm with tail or acrosomal defects were calculated. exclusion of animals from statistical analysis animals with progressive motility of less than 50% on day 0 were excluded from statistical analysis. such animals would not pass a breeding soundness examination under real-world conditions, and motility was deemed unlikely to improve significantly with repeated collections. animals that showed antibodies to rvfv prior to the onset of the trial (but after the initial screening process) were excluded from statistical analysis. animals that displayed a fever response for five or more consecutive daily measurements, starting at day 0, were excluded in order to remove persistent fever as a confounding factor. a reference of 39.1 °c ± 0.5 °c was used for normal ovine rectal temperature; any temperature greater than or equal to 39.6 °c was considered a fever response (kahn & line 2005). statistical procedures data were analysed using statistical analysis software (sas) (sas institute, cary, north carolina). descriptive statistics were generated from the data using sas plugin for ms excel. the effect of vaccination on percentage progressively motile spermatozoa was assessed using repeated-measures logistic regression analysis (sas genmod function). the primary outcome assessed was whether vaccination affected breeding soundness. this could not be directly assessed, as the intervening variable of temperature existed, and therefore it was assessed whether animals with higher body temperatures also had poorer progressive semen motility. a secondary outcome assessed was whether vaccination had any effect on temperature. from this, a conclusion could be drawn on whether vaccinated animals had poor semen quality relative to unvaccinated animals. results top ↑ clinical findings animals used in the study were young, peripubertal ram lambs. only three out of the 23 animals that passed the initial antibody screening process were recorded as having two permanent teeth; the remainder had only deciduous teeth. the modal body condition score for the entire group of rams was 2.5 out of 5. the mean scrotal circumference of all animals was 29.1 cm. no swelling, pain or redness was noted at vaccination sites in any vaccinated animals at any time during the study period. no animal became sufficiently ill at any time during the trial to warrant treatment or exclusion from the trial. temperature variation the mean rectal temperature of all animals (in both the vaccinated and the control group) throughout the study period was 39.34 °c ± 0.41 °c (mean ± s.d.). a maximum of 41.4 °c was measured in one animal on day 2 of the trial period, and a minimum of 37.4 °c measured in one animal on day 24 of the trial period. the mean daily rectal temperature of all animals in the control group throughout the study period was 39.27 °c ± 0.36 °c (mean ± s.d.). the mean daily rectal temperature of all animals in the vaccinated group throughout the study period was 39.41 °c ± 0.43 °c (mean ± s.d.). the highest daily average rectal temperature recorded in all animals (in both the vaccinated and the control group) throughout the study was a temperature of 39.73 °c ± 0.35 °c (mean ± s.d.), recorded on day 29. antibody testing one animal from the control group tested positive using snt for rvfv antibodies, without exposure to vaccine antigen. of the vaccinated animals, all except two exhibited an antibody response. most vaccinated animals displayed a measurable antibody titre on the third test (day 21 after vaccination) (table 1). table 1: summary of serum neutralisation test results. semen collection electroejaculation (ee) was the predominant means of semen collection throughout the study. of the 161 semen collections performed during the study, 115 were by ee and 46 were by av. exclusions from statistical analysis one ram from the control group was excluded from the trial because it seroconverted between the screening process and the first blood sampling. five animals were excluded because they had poor semen quality (< 50% progressively motile) on day 0 of the trial. one animal was excluded because of a persistent febrile response noted from day 0 of the trial. descriptive statistics: semen evaluation data descriptive statistics were generated for both vaccinated and control groups, grouped together. the descriptive statistics for progressive motility and live morphologically normal sperm are displayed in table 2. there was no significant temporal decline in the mean number of live, morphologically normal spermatozoa and the mean number of progressively motile spermatozoa throughout the trial (tables 3 and 4). table 2: percentage progressive motility and live, morphologically normal sperm of vaccinated and control groups assessed together. table 3: percentage progressively motile spermatozoa. table 4: percentage morphologically normal sperm throughout trial. logistic regression analysis when animals (both vaccinated and control) were grouped together and evaluated as a single group, progressive sperm motility on day 0 of all animals was found to correlate strongly with progressive motility after day 0 throughout the trial (p = 0.0062). when progressive motility throughout the trial was evaluated according to group (vaccinated vs control), no significant difference was found between groups (p = 0.0499), therefore animal group was not shown to correlate with a difference in percentage progressively motile spermatozoa. when compared within groups, values for progressively motile on day 0 were shown to correspond significantly with subsequent values through the trial (p = 0.0321). daily temperature values were not found to have a significant association with group (p = 0.8606). the occurrence of fever (rectal temperature ≥ 39.6 °c) on any day throughout the trial was not found to have a significant association with animal groups (vaccinated or control) (p = 0.6665). when treatment group as well as progressive motility on day 0 were evaluated in the same multivariable regression model for their effects on progressive motility throughout the trial, it was found that treatment group was not significantly associated with a reduction in progressive motility (p = 0.3325), but that progressive motility on day 0 correlated significantly with subsequent progressive motility (p = 0.0321) (table 5). table 5: summary of correlations between measured variables: single variable models. body temperature (measured once a week, on the day of semen collection) and the values for progressively motile sperm on day 0 were evaluated in a two-variable regression model to assess their combined effect on the percentage of progressively motile sperm through the study. in this model it was found that the weekly measured temperature was not significantly correlated with progressive motility throughout the trial (p = 0.3711), but that the values for progressively motile sperm on day 0 from a given animal correlated strongly with subsequent pro­gressive motility from that animal evaluated during the trial (p = 0.0002). similarly, body temperature (measured once a week, on the day of semen collection) and the values for progressively motile sperm on day 0 were evaluated in a two-variable regression model for their combined effect on percentage live, morphologically normal sperm throughout the trial. it was found that the weekly temperature value was not significantly correlated with the value for live, morphologically normal sperm through the study (p = 0.8785) but that progressive motility on day 0 correlated significantly with the value for live, morphologically normal sperm throughout the study (p = 0.0190). discussion top ↑ very few research trials have been performed involving rvfv clone 13 vaccine (dungu et al. 2010; von teichman et al. 2011). the current trial did not evaluate the protective effect of rvfv clone 13 vaccine by exposing animals to a challenge trial. obtaining enough animals proved difficult, as they needed to be unvaccinated, serologically naïve rams. some rams were therefore too young to reliably pass a breeding soundness examination. this complicated the data analysis, as a significant number of animals needed to be excluded from statistical calculations on the grounds of poor semen quality at the start of the trial. a large proportion of younger animals were randomly allocated to the vaccinated group. this resulted in more exclusions for poor semen quality occurring in this group. an improvement on the implemented randomisation model may have been to allocate animals to two groups based on the results of their initial semen quality analysis, to ensure an even distribution between groups prior to the administration of the vaccine to one of the groups. various factors may have caused poor semen quality in a large proportion of trial animals. male animals store sperm in the epididymides prior to ejaculation. prolonged sperm storage (for example lack of ejaculation as a result of absent or reduced sexual activity) may result in ‘aged’ spermatozoa that exhibit a lower than normal progressive motility and a higher proportion of epididymal defects and loose heads. this phenomenon was reviewed by barth and oko (1989), and an evolutionary biology review characterised such defects as ‘post-meiotic senescence’ (pizzari et al. 2007). hence, repeated semen collections may have resulted in an improvement in semen quality as aged spermatozoa were ejaculated and replaced with newly produced spermatozoa. importantly, younger animals with small scrotal circum­ferences and no permanent teeth were over-represented amongst those excluded for poor semen quality. this might suggest that these animals were not adequately mature prior to first semen collection and evaluation. in addition to the physiological reasons for poor semen quality discussed above, events during collection and handling of the semen sample may affect its quality. animals that have their semen collected by ee are more likely to contaminate the sample with urine or produce an oligospermic ejaculate that consists mainly of accessory gland fluid. an av is well known to provide a more consistent semen sample than ee, which is more reflective of true semen quality (hulet, foote & blackwell 1964). had a larger pool of animals been available, a better option may have been to exclude animals that failed to mount an av, and then to randomise animals by semen quality as discussed above. a single animal from the control group was excluded from the statistical analysis because of seroconversion to rvfv. this suggests that the animal was exposed to rvfv between the initial pre-enrolment screening test and the first pre-vaccination test. assuming high sensitivity and specificity of the snt, the fact that no other animals exhibited antibody titres at this stage of the trial might indicate a low-intensity occurrence of rvfv in the region where the trial was conducted. alternatively, an imperfect sensitivity of the snt test used may have resulted in a false-negative result at the initial screening, which could have been avoided by multiple testing or the parallel use of another diagnostic test. when temperatures were averaged amongst groups and all animals were included (including those excluded from subsequent statistical analysis), it was found that throughout the trial, vaccinated animals had a slightly higher average temperature (39.41 °c ± 0.43 °c) than control animals (39.27 °c ± 0.36 °c). the significance and underlying causes of this finding are open to debate. the authors speculate that this is purely as a result of natural variation and would normalise with increased sample size; however, some vaccine effect cannot be ruled out. importantly, when logistic regression analysis was performed, it was found that rectal temperature had no correlation with either progressive motility or percentage live, morphologically normal sperm. this suggests that a febrile response to vaccination (if any) was minimal and was insufficient to induce abnormalities in the spermiogram. interestingly, in prior work by dungu et al. (2010), no clone 13 vaccinated animals exhibited temperature reactions above 40 °c at any stage of the trial. there were several instances in the present study in which rectal temperatures in both control and vaccinated animals were elevated above 40 °c. this may be as a result of significant differences in environmental conditions under which animals were kept. in the trial by dungu et al. (2010), animals were kept in an indoor, temperature-controlled confinement facility as a virulent infective virus was used. in the present trial, animals were kept outdoors during the early karoo summer, and had to contend with significant variation in environmental temperature. as discussed previously, leemans et al. (2012) found that inactivated bluetongue virus had no adverse effect on semen quality in rams. the authors attributed this outcome to an absence of febrile response to vaccination, compared to the findings of a previous study using live vaccine by bréard et al. (2007) and the effect of a natural infection (kirschvink et al. 2009). this result contrasts with the findings of dungu et al. (2010) and von teichman et al. (2011) that sheep and calves vaccinated with the live (smithburn) rvfv vaccine failed to develop a post-vaccination temperature response. no previous research has been performed that evaluates the effect of a live rvfv vaccine on semen quality, but it is known that the live rvfv vaccine may induce teratogenesis in pregnant animals, and its use should be avoided in these animals if at all possible (coetzer & barnard 1977). it may therefore be hypothesised that, as the live rvfv vaccine was not found to induce a febrile response in sheep or calves, it may be safe for use in male animals used for breeding. this line of reasoning may require further investigation. the day 0 value for percentage progressive motility in this study was found to be highly predictive of subsequent values for progressive motility throughout the trial (p = 0.0062) when both groups (vaccinated and control) were assessed together. similarly, the day 0 value for percentage progressively motile sperm was found to be predictive of subsequent values for this parameter (p = 0.0321) when assessed within the group. these findings suggested that an animal with high-quality semen on day 0 was likely to continue to produce high-quality semen throughout the trial, and an animal with poor-quality semen on day 0 was likely to continue to produce poor-quality semen. interestingly, there was a right-shift in distribution of percentage progressive motility when comparing values on day 0 and values after day 0 amongst both control and vaccinated groups, indicating a temporal improvement in semen quality amongst all animals as the trial progressed. as discussed previously, this apparent improvement may have occurred as a result of repeated ejaculations, thereby eliminating aged epididymal sperm that would have shown poor progressive motility. two animals from the vaccinated group failed to display measurable antibody titres to rvfv at any stage during the trial. these animals were subsequently excluded from the trial on the grounds of poor semen quality. however, the fact that these animals failed to seroconvert after vaccination is worthy of special mention. in previously reported work (dungu et al. 2010), two out of 17 vaccinated animals exhibited a weak antibody response to a 106 pfu dose of rvfv clone 13 vaccine, which was nonetheless able to protect them against challenge with live virus. in an earlier study by barnard (1979), it was noted that after two inoculations with the smithburn live virus vaccine, two out of five cattle failed to develop an antibody response detectable by the snt. these animals were nonetheless immune when challenged with live virus. one reason for this might be that the snt is not sensitive enough in detecting neutralising antibodies. alternatively, antibody-mediated humoral immunity may not be the most important immune response that protects animals against rvfv infection. conclusion top ↑ clone 13 vaccine was found to be capable of inducing seroconversion in vaccinated rams. these animals did not experience significant deterioration in semen quality post-vaccination. therefore, according to these findings, rvfv clone 13 is a vaccine that can be used safely in breeding rams. conclusions drawn from this trial must be interpreted in the context of its small sample size. the fact that two animals out of 12 failed to seroconvert within 42 days after vaccination warrants further investigation. a challenge trial may assist in confirming whether or not the vaccine is protective. a repeat of the trial with a larger sample size may confirm, with greater statistical certainty, that the vaccine has no ill effect on semen quality parameters while remaining effective in protecting animals against clinical disease. acknowledgements top ↑ financial support was provided by onderstepoort biological products. the authors would like to express their gratitude to the staff of knoffelfontein farm, sr willemien jooste and dr cizelle naudé for assistance with animal handling and semen collection. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced the outcomes and conclusions they may have drawn. authors’ contributions the research was conducted as part of a master's degree in veterinary medicine (gynaecology) dissertation by g.b. (university of pretoria). the practical work was carried out by g.b. and supervised by e.h.v. (university of pretoria) and h.a. (university of pretoria). p.m. (colorado state university) assisted with the statistical analysis. g.b. wrote and submitted the manuscript. e.h.v. and h.a. assisted in the writing of the manuscript. references top ↑ barnard, b.j., 1979, ‘rift valley fever vaccine – antibody and immune response in cattle to a live and an inactivated vaccine’, journal of the south african veterinary association 50(3), 155–157. barth, a.d. & oko, r.j., 1989, abnormal morphology of bovine spermatozoa, iowa state university press, ames. bréard, e., pozzi, n., sailleau, c., durand, b., catinot, v., sellem, e. et al., 2007, ‘transient adverse effects on the quality of an attenuated bluetongue virus vaccine on the quality of ram semen’, veterinary record 160(13), 431–435. http://dx.doi.org/10.1136/vr.160.13.431 caspari, k., henning, h., schreiber, f., maass, p., gössl, r., schaller, c. et al., 2014, ‘impact of porcine circovirus type 2 (pcv2) vaccination on boar semen quality and quantity using two different vaccines’, theriogenology 82, 574–579. http://dx.doi.org/10.1016/j.theriogenology.2014.05.017 castro, j.m., del pozo, m., gil, j., imaz, m.a., simarro, i. & rillo, s.m., 1992, ‘effect of a bioengineered pseudorabies (aujeszky's disease) vaccine on the semen quality of boars’, theriogenology 38, 165–173. http://dx.doi.org/10.1016/0093-691x(92)90228-j coetzer, j.a. & barnard, b.j., 1977, ‘hydrops amnii with hydranencephaly and arthrogryposis with wesselsbron disease and rift valley fever viruses as aetiological agents’, onderstepoort journal of veterinary research 44, 119–126. dungu, b., louw, i., lubisi, a., hunter, p., von teichman, b.f. & bouloy, m., 2010, ‘evaluation of the efficacy and safety of the rift valley clone 13 vaccine in sheep’, vaccine 4581–4587. http://dx.doi.org/10.1016/j.vaccine.2010.04.085 flick, r. & bouloy, m., 2005, ‘rift valley fever virus’, current molecular medicine 5, 827–834. http://dx.doi.org/10.2174/156652405774962263 gerdes, g.h., 2004, ‘rift valley fever’, revue scientifique et technique, office international des épizooties 23(2), 613–624. gouletsou, p.g., fthenakis, g.c., cripps, p.j., pappaioannou, n., lainas, t., psalla, d. et al., 2004, ‘experimentally induced orchitis associated with arcanobacterium pyogenes: clinical, ultrasonographic, seminological and pathological features’, theriogenology 62, 1307–1328. http://dx.doi.org/10.1016/j.theriogenology.2004.01.008 hulet, c.v., foote, w.c. & blackwell, r.l., 1964, ‘effects of natural and electrical ejaculation on predicting fertility in the ram’, journal of animal science 23, 418–424. ikegami, t. & makino, s., 2009, ‘rift valley fever vaccines’, vaccine 27(suppl. 4), d69–d72. http://dx.doi.org/10.1016/j.vaccine.2009.07.046 kahn, c.m. & line, s. (eds.), 2005, ‘table of normal body temperature values’, the merck veterinary manual, 9th edn., p. 2582, merck, whitehouse station. kirschvink, n., raes, m. & saegermann, c., 2009, ‘impact of a natural bluetongue serotype 8 infection on semen quality of belgian rams in 2007’, veterinary journal 182, 244–251. http://dx.doi.org/10.1016/j.tvjl.2008.06.008 leemans, j., raes, m., saegerman, c., sustronck, b., makoschey, b. & kirschvink, n., 2012, ‘effect of an inactivated bluetongue serotype 8 vaccine on semen quality in rams’, vete rinary journal 193, 567–569. http://dx.doi.org/10.1016/j.tvjl.2012.01.020 montes, a.j., wolfe, d.f., welles, e.g., tyler, j.w. & tepe, e., 1994, ‘infertility associated with eperythrozoon wenyonii in a bull (abstract)’, journal of the american veterinary medical association 204(2), 261–263. nöthling, j.o. & dos santos, i.p., 2012, ‘which fields under a coverslip should one assess to estimate sperm motility?’, theriogenology 77, 1686–1697. http://dx.doi.org/10.1016/j.theriogenology.2011.12.014 nöthling, j.o. & irons, p.c., 2008, ‘a simple multidimensional system for the recording and interpretation of sperm morphology in bulls’, theriogenology 69, 603–611. http://dx.doi.org/10.1016/j.theriogenology.2007.11.007 osburn, b.i., 1994, ‘the impact of bluetongue virus on reproduction’, comparative immunology, microbiology and infectious diseases 17(3), 189–196. http://dx.doi.org/10.1016/0147-9571(94)90042-6 pepin, m., bouloy, m., bird, b.h., kemp, a. & paweska, j., 2010, ‘rift valley fever virus (bunyaviridae: phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention’, veterinary research 41(6), 61. http://dx.doi.org/10.1051/vetres/2010033 pizzari, t., dean, r., pacey, a., moore, h. & bonsall, m.b., 2007, ‘the evolutionary ecology of preand post-meiotic sperm senescence’, trends in ecology and evolution 23(3), 131–140. http://dx.doi.org/10.1016/j.tree.2007.12.003 prieto, c. & castro, j.m., 2005, ‘porcine reproductive and respiratory syndrome virus infection in the boar: a review’, theriogenology 63, 1–16. http://dx.doi.org/10.1016/j.theriogenology.2004.03.018 sekoni, v.o., 1993, ‘effects of severe chronic scrotal dermatophilus congolensis (kirchi) infection on semen characteristics in zebu/friesian crossbred bulls and effect of long-term terramycin chemotherapy’, theriogenology 40, 211–223. http://dx.doi.org/10.1016/0093-691x(93)90355-9 swanepoel, r. & coetzer, j.w., 2004, ‘rift valley fever’, in j.a.w. coetzer & r.c. tustin (eds.), infectious diseases of livestock, 2nd edn., vol. 2, pp. 1037–1070, oxford university press, cape town. von teichman, b., engelbrecht, a., zulu, g., dungu, b., pardini, a. & bouloy, m., 2011, ‘safety and efficacy of rift valley fever smithburn and clone 13 vaccines in calves’, vaccine 29, 5771–5777. http://dx.doi.org/10.1016/j.vaccine.2011.05.055 von teichman, b.f., louw, i., engelbrecht, a., heath, j.a. & smit, t.k., 2010, ‘onderstepoort rift valley fever virus vaccines’, proceedings of the 9th annual congress of the southern african society for veterinary epidemiology and preventive medicine, pretoria, august 18–20, 2010, pp. 25–30. world organisation for animal health (oie), 2013, manual of diagnostic tests and vaccines for terrestrial animals 2012, chapter 2.1.14., viewed 04 march 2014, from http://www.oie.int/fileadmin/home/eng/health_standards/tahm/2.01.14_rvf.pdf bredin_1-9.indd introduction giraffes, giraffa camelopardalis, (linnaeus, 1758) require an estimated 20 g calcium (ca) and 10 g phos phorus (p) per day from birth to 5 years of age for the growth of their unique skeletons, and if they are to maintain a ca:p ratio in their bones of 2:1 (mitchell & skinner 2003; mitchell, van schalkwyk & skinner 2005). thereafter, daily requirements in males and females are lower but in females are increased by specific stresses such as lactation (mitchell et al. 2005). recent analyses of giraffe skeletal biology (van schalkwyk, skinner & mitchell 2004; mitchell et al. 2005) suggested that by selective browsing giraffes are likely to be able to obtain sufficient ca for skeletal growth. sources of sufficient p are more obscure. one possibility is that they obtain p by eating bones. osteophagia is an often observed behaviour in giraffes (pattern 1940; nesbitt-evans 1970; western 1971; wyatt 1971; leuthold & leuthold 1972; hall-martin 1974; kok & opperman 1980; hampton 2002), and it seems to be reported more frequently in giraffes than almost any other ruminant except for domestic animals on p deficient pasture. its occurrence is highest in the winter months when the nutrient quality of browse declines (langman 1978). osteophagia has many causes. boredom, habit and taste contribute, but theiler, green & du toit (1924) established that osteophagia in cattle (bos taurus/ indicus), could be eliminated by supplementary feed1 onderstepoort journal of veterinary research, 75:1–9 (2008) can osteophagia provide giraffes with phosphorus and calcium? i.p. bredin1, j.d. skinner1 and g. mitchell1, 2* abstract bredin, i.p., skinner, j.d. & mitchell, g. 2008. can osteophagia provide giraffes with phosphorus and calcium? onderstepoort journal of veterinary research, 75:1–9 the daily requirement for calcium and phosphorus by giraffes to sustain the growth and maintenance of their skeletons is large. the source of sufficient calcium is browse. the source of necessary phosphorus is obscure, but it could be osteophagia, a frequently observed behaviour in giraffes. we have assessed whether bone ingested as a result of osteophagia can be digested in the rumen. bone samples from cancellous (cervical vertebrae) and dense bones (metacarpal shaft) were immersed in the rumens of five sheep, for a period of up to 30 days, and the effect compared to immersion in distilled water and in artificial saliva for 30 days. distilled water had no effect on the bones. dense bone samples were softened by exposure to the saliva and rumen fluid, but did not lose either calcium or phosphorus. in saliva and rumen fluid the cancellous bone samples also softened, and their mass and volume decreased as a result of exposure to saliva, but in neither fluid did they lose significant amounts of calcium and phosphorus. we conclude that although saliva and rumen fluid can soften ingested bones, there is an insignificant digestion of bones in the rumen. keywords: calcium, giraffe, osteophagia, phosphorus * author to whom correspondence is to be directed. email: mitchg@uwyo.edu 1 veterinary wildlife unit, faculty of veterinary science, private bag x04, university of pretoria, onderstepoort, 0110 south africa 2 department of zoology and physiology, university of wyoming, 1000 e university avenue, laramie, wy 82071, usa accepted for publication 2 august 2007—editor 2 can osteophagia provide giraffes with phosphorus and calcium? ing with p in the form of bone meal. the appetite for bones observed in p deficient cattle is innate, specific, and cued mainly by the smell of bones. it is associated with a decline in the inorganic phosphate fraction of blood plasma and a withdrawal of ca and p from the reserves in bones (denton, blair-west, mckinley & nelson 1986; blair-west, denton, nelson, mckinley, radden & ramshaw 1989; underwood & suttle 1999). a giraffe’s diet on average has a ca:p ratio of 7.7:1 (pellew 1984; mitchell & skinner 2003), and if fed to cattle a diet with this ca:p ratio would result in clinical signs of phosphate deficiency and osteophagia (mcdowell 1992; underwood & suttle 1999). for osteophagia to be an effective adaptive behaviour to supply p (and/or ca), and assuming that ingested bones or bone fragments enter the rumen, then the ingested bones must be small enough to pass through the rumen into the abomasum, or must be able to be digested in the rumen, and p and ca released in a soluble, absorbable form. phosphorus can be absorbed from the rumen (wadhwa & care 2002). however, removal of ca and p from bone and their absorption occurs best in a low ph environment. in carnivores, for example, gastric ph is approximately 2 and bones and bone fragments can readily reach the stomach where they are dissolved. in ruminants abomasal ph is somewhat higher. in giraffes it is 3.6 ± 0.1 and is identical to that of five other wild ruminant species (3.6 ± 0.4) (maloiy, clemens & kamau 1982), but nevertheless is sufficiently acidic to dissolve bone and release p from it. however, in ruminants, unlike in monogastric animals, bones cannot directly enter the abomasum. the break down of bone is also likely to depend on chewing and possibly rumination, but the time of exposure to these processes is brief and intermittent. prolonged exposure in the rumen itself is a more likely site of digestion. we report here, therefore, an investigation into whether ca and p ingested as bone can be released from bone in the rumen in significant quantities and/ or if bone can be reduced to a form that can reach the abomasum and small intestine. as far as could be determined no study on the digestion of bones in the rumen has been done previously. our study suggests that such digestion is poor. materials and methods bone samples calcium and p extraction from two types of bone was determined: cancellous (porous) and compact (dense) bone, which replicate the range of bone types ingested by giraffes. bone samples were obtained from giraffe bones used in our previous studies to establish the mineral composition of both types (van schalkwyk et al. 2004; mitchell et al. 2005). for this study cancellous bone was obtained from third, fourth and fifth cervical vertebrae and compact bone from a single metacarpus shaft. to standardize surface areas, the bone samples were cut into cubes, the side lengths of which were approximately 1.7 cm. the surface area of each cube was thus about 17.5 cm3 and their volumes about 5 cm3 (fig. 1; tables 3 and 4). measurement of digestion digestion was assessed from changes in bone mass, volume and density, and from changes in the ca and p composition. bone mass, volume and density initial mass of the bone samples was recorded using a mettler toledo bloc pb 153-s scale (mettler, microsep, rsa) to an accuracy of 0.1 g. volumes of the bone samples were determined by the displacement of water in volumetric flasks, which measured changes in volume to 0.1 mℓ. density was calculated by dividing dry mass by volume of water displaced, assuming that at 20 °c the density of water is 1 g/mℓ. (khan, khan, khan & khanam 1997), and was recorded as g/cm3. volume, mass, and density measurements were repeated both preand post-treatment to determine any significant changes. post-treatment, all samples were oven dried following harris (1970) after volume measurements were taken and before mass was determined. ca and p analysis of the bones after initial mass and volume were measured, all the samples of bone were defatted using petroleum ether as described by the association of official analytical chemists (aoac), official method 945.16 (horwitz 2000) at nutrilab, department of animal and wildlife science, faculty of natural and agri cultural sciences, university of pretoria. the lipid free samples were weighed (± 0.001 g) and then oven dried after the method of harris (1970). cancellous bone samples were ground to a powder using a custom-made iron pestle and mortar. for compact bone samples this method produced a coarse powder, which was further ground using a motor-driven mill (mikro-feinmühle-culatti mfc, 3 i.p. bredin, j.d. skinner & g. mitchell janke ika®—labortechnik, 50/60 hz, 200 w) to particles of approximately 1 mm3 in size. duplicate samples of powdered bone samples, weigh ing 0.5 ± 0.003 g were oven dried and ashed in a muffle furnace at 550 °c for 4.0 h. the dry ashing technique followed the aoac official method 999.11 (horwitz 2000). samples were left to cool overnight and placed in a desiccator for 30 min prior to determining the ash mass. the ash residue was dissolved in an acid solution, filtered and diluted to a volume of 100 mℓ. dissolved ash solutions for ca analysis were diluted 50 times with distilled water and a further ten times with lanthanum chloride (lacl3, 0.5 %). solutions for p analysis were diluted 50 times with distilled water. phosphorus concentrations were measured using an auto analyser ii (techicon™, bran & lübbe, germany) according to the aoac official method 965.17 (horwitz 2000). calcium concentrations were measured in an atomic absorption spectrophotometer (perkin-elmer 5100pc, usa) using the aoac official method 935.13 (horwitz 2000). in both cases a difference of less than 10 % between duplicates was accepted. for larger differences analysis of those particular samples was repeated. calcium and p concentrations measured were converted to mg/g of ash ([volume x dilution x reading] ÷ sample mass), and expressed as a percentage. the mean percentage multiplied by the original mass of the bone sample was used to calculate total ca (g), total p (g), and total non-ca + p (g) minerals in each bone sample. experimental animals five mature, rumen-fistulated merino sheep wethers were used for the trial (animal use and care committee, faculty of veterinary science, university of pretoria approval number v068/04). sheep were used as experimental animals because their rumen fluid composition is identical to that of wild ruminants including giraffes (giesecke & van gylswyk 1975) (table 5) and because they exhibit osteophagia (brothwell 1976; bazely 1989). the animals were housed at the experimental farm of the faculty of biological and agricultural science, university of pretoria, under the supervision of the department of animal and wildlife science, faculty of natural and agricultural sciences, university of pretoria. the sheep were housed individually in cement-floor pens (3 x 2 m), which were covered by a roof, and were fed a ration of good quality teff (eragrostis tef) hay (table 1). water was available ad libitum. the hay was milled to 1 cm length. the sheep were fed the ration for 16 days prior to the start of the trial. feed consumption rate was estimated at approximately 2 kg per wether per day, which is above normal consumption rates for ewes on a maintenance diet (perry, cullison & lowery 1999). the sheep were weighed at the start of the trial and three times afterwards (10, 20 and 30 days). their body masses ranged from 38.1 kg to 72.0 kg at the start of the trial and they maintained body mass for the duration of the experiment. rumen fluid ph (as a marker of rumen health) was measured at each time interval using the model iq 150 handheld ph/mv/temperature meter or model iq 120 ph meter with a silicon chip sensor (i.q. scientific instruments, inc., san diego, usa). blood samples from all five sheep were taken at the start of the trial and at the respective time intervals to determine plasma ca and p concentrations. after collection, the blood sam ples were stored on ice and centrifuged within 1 h. preparation of blood plasma for inorganic p analysis was done following the procedures described by lit tle, robison, playne & haydock (1971). after precipitation the solutions were filtered through glass microfibre paper (9.0 cm gf/a whatman ltd., eng land) into acid-cleaned 30 mℓ. mccartney bottles. the remaining plasma in the centrifuged tubes was pipetted into individual, sealable tubes for analysis of inorganic ca levels. all samples were refrigerated at 5 °c. calcium and p concentrations in plasma were analysed using the same methods described above for bone sample analysis, except that to minimize interference by p on the spectrophotometer, the plasma samples for ca analysis were diluted 50 times with lanthanum chloride (lacl3, 0.1%). table 1 eragrostis tef hay fed to the sheep throughout the trial. all values are on a dm basis dm (g/100 g) ge (mj/kg)1 moist (g/100 g) ash (g/100 g) cp (g/100 g)2 cf (g/100 g) ca (g/100 g) p (g/100 g) ca:p 100 18.5 0 4.72 7.16 34.91 0.28 0.32 0.9:1 1 according to j. van ryssen (personal communication 2006) 2 “dumas” method was used for crude protein analysis (official method 990.03, horwitz 2000) 4 can osteophagia provide giraffes with phosphorus and calcium? experimental design the assumption was made that there would be significant evidence of digestion in the three fluids used, viz. rumen fluid, artificial saliva and distilled water, if the cubed bone samples were immersed in them for 30 days, the artificial saliva and distilled water being used to control for the effects of rumen digestion. ingested bone is exposed to saliva at the time of ingestion and possibly at intervals during rumination. saliva has a similar ph and osmolarity to rumen fluid, and contains chemicals similar to those found in rumen fluid, although at different concentrations (wadhwa & care 2002). all the samples used were placed in individual nylon bags (pore size 53 μm; nutrilab) and kept in distilled water, artificial saliva and rumen fluid via the rumen fistula’s for 10, 20 and 30 days. at the appropriate time intervals a number of the bone samples were removed from the respective fluid they were suspended in. these were washed under tap water and oven dried according to harris’ (1970) method for analysis. fifteen samples of each bone type were randomly assigned to the three treatments. five of each type were analysed at each time interval as follows: fifteen samples of each type were placed in distilled water at ph 5.86–6.38 and 39 °c. another 15 samples of each type were placed in an artificial saliva solution and incubated at 39 °c. this solution lacked saliva enzymes but contained 9.8 g/ℓ na hco3, 0.57 g/ℓ kcl, 0.47 g/ℓ nacl, 0.12 g/ℓ mgso4.7h2o, 0.04 g/ℓ anhydrous cacl2 and 3.17 g/ℓ anhydrous na2hpo4, to which 4 mℓ/ℓ hydrochloric acid was added to reduce the ph to rumen ph of 6.5 (mcdougall 1948). the last 15 samples of each bone type were suspended in the rumens of the sheep. the nylon bags containing these samples were attached to a 120 g mass so that they would remain submerged in the rumen fluid. statistical analysis a two-tailed student’s t-test was used to compare differences between control samples and test samples. p-values < 0.05 were regarded as significant. results rumen fluid, saliva, distilled water and blood chemistry distilled water and rumen ph remained constant over the 30 days of the experiment (table 2). the ph of saliva increased over time from 6.8 to 7.3. this table 2 rumen, saliva, distilled water (dh2o) and blood chemistry fluid type n before treatment 10 days 20 days 30 days rumen ph (mean ± sd) saliva ph (mean ± sd) dh2o ph (mean ± sd plasma ca (mmol/ℓ) plasma p (mmol/ℓ) 5 7 7 5 5 6.4 ± 0.2 6.8 ± 0.2 6.5 ± 0.1 2.4 ± 0.2 1.4 ± 0.4 6.5 ± 0.2 7.0 ± 0.2 6.1 ± 0.4 2.4 ± 0.1 1.4 ± 0.3 6.5 ± 0.3 7.4 ± 0.2 6.5 ± 0.7 2.4 ± 0.1 1.7 ± 0.7 6.5 ± 0.3 7.3# 6.4 ± 0.4 2.5 ± 0.3 1.5 ± 0.5 # 2 measurements only table 3 effects of distilled water (dh2o), artificial saliva, and rumen fluid on metacarpus shaft bone samples bone variable before treatment1 dh2o after 30 days artificial saliva after 30 days rumen fluid after 30 days mass (g) volume (mℓ) density (g/cm3) total ash (g per sample) % ca % p total ca (g per sample) total p (g per sample) non-ca + p (g per sample) 9.8 ± 0.9 5.2 ± 0.5 1.9 ± 0.1 7.0 ± 1.0 25.6 ± 0.9 11.6 ± 0.2 2.5 ± 0.4 1.1 ± 0.2 3.3 ± 0.5 9.5 ± 1.3 5.0 ± 0.6 1.9 ± 0.1 6.9 ± 0.9 25.4 ± 0.5 11.5 ± 0.2 2.5 ± 0.3 1.1 ± 0.2 3.4 ± 0.4 10.7 ± 0.4 5.5 ± 0.4 1.9 ± 0.1 7.6 ± 0.3 24.7 ± 1.0 11.7 ± 0.2 2.7 ± 0.1 1.3 ± 0.1 3.7 ± 0.1 10.0 ± 1.4 5.3 ± 0.5 1.9 ± 0.1 7.2 ± 1.1 25.6 ± 1.4 11.4 ± 0.5 2.6 ± 0.5 1.1 ± 0.2 3.4 ± 0.5 1 forty five bone samples were used to determine pre-treatment means for mass, volume and density, and five for assessment of fluid effects on mass, volume and density at 30 days. fifteen samples were used to determine means for pre-treatment mineral content and five for the effect of fluids on mineral content at 30 days bold = significant (p < 0.05) using the t-test, compared to pre-treatment samples 5 i.p. bredin, j.d. skinner & g. mitchell fig. 1a and b physical appearance of blocks prepared from cervical vertebrae (a) and metacarpus shaft (b) after 30 days a = bone sample prior to immersion in a fluid, b = bone sample immersed in distilled water for 30 days, c = bone sample immersed in rumen fluid for 30 days, and d = bone sample immersed in artificial saliva for 30 days increase was not a result of the presence of bones as the ph of the saliva solution was similar in bonefree and bone-containing saliva. plasma ca concentra tion remained constant at 2.4 ± 0.1 mmol/ℓ (table 2). plasma p concentration was more variable, ranging from 1.4 ± 0.3 to 1.7 ± 0.7 mmol/ℓ (table 2). these ca and p values are within the normal range for sheep (hurwitz 1996; underwood & suttle 1999). bone samples no significant effects of immersion were measurable after exposure for 10 and 20 days. some effects were seen after 30 days (fig. 1; tables 3 and 4). physical appearance the physical appearance of the two types of bones after 30 days of exposure to the various treatments is shown in fig. 1a and b. distilled water had no obvious macroscopic effects. artificial saliva produced visible erosion of the cervical vertebrae but had no eroding effect on samples derived from the metacarpus. rumen fluid did not produce visible erosion of bones but did discolour them. both rumen fluid and artificial saliva caused softening of the cervical vertebrae samples, and cracking of the metacarpus shaft samples. chemical and physical analysis as might have been predicted, the composition of metacarpal bone samples after 30 days of immersion, was unaffected by any of the three fluids (table 3), except in so far as they softened after immersion in rumen fluid suggesting that their structure was altered. table 3 shows, however, an apparent significant increase in total ash and non-ca + p content table 4 the effects of distilled water (dh2o), artificial saliva, and rumen fluid on cervical vertebrae bone samples bone variable before treatment1 dh2o after 30 days artificial saliva after 30 days rumen fluid after 30 days mass (g) volume (mℓ) density (g/cm3) total ash (g per sample) % ca % p total ca (g per sample) total p (g per sample) non-ca + p (g per sample) 5.1 ± 0.7 4.9 ± 0.8 1.1 ± 0.1 2.6 ± 0.4 21.1 ± 0.7 9.5 ± 0.6 1.0 ± 0.1 0.4 ± 0.1 1.2 ± 0.2 5.2 ± 0.6 5.4 ± 0.9 1.0 ± 0.1 3.4 ± 0.5 22.0 ± 0.8 10.2 ± 0.8 1.2 ± 0.2 0.6 ± 0.1 1.6 ± 0.2 3.5 ± 0.5 3.5 ± 0.5 1.0 ± 0.1 3.0 ± 0.4 25.5 ± 1.9 11.6 ± 1.1 1.1 ± 0.2 0.5 ± 0.1 1.4 ± 0.1 5.6 ± 0.4 5.7 ± 0.4 1.0 ± 0.1 3.2 ± 0.2 21.8 ± 0.8 9.7 ± 0.5 1.2 ± 0.1 0.5 ± 0.1 1.6 ± 0.1 1 forty five bone samples were used to determine pre-treatment means for mass, volume and density, and five for assessment of fluid effects on mass, volume and density at 30 days. fifteen samples were used to determine means for pre-treatment mineral content and five for the effect of fluids on mineral content at 30 days bold = significant (p < 0.05) using the t-test, compared to pre-treatment samples a c b d a c b d a b 6 can osteophagia provide giraffes with phosphorus and calcium? of metacarpal samples after exposure to artificial saliva. this result can be related to the sig nificantly higher mass of the bones that were analysed. it is not a biological effect. cancellous bone samples, on the other hand, were affected by exposure to the fluids, albeit minimally. distilled water had no effect apart from an anomalous increase in total ash and the non-ca + p minerals. artificial saliva solution had several significant effects. the mass and volume of the samples decreased significantly over the 30-day period, confirming the visible effects of saliva shown in fig. 1a. the percentage of ca and p in the samples increased significantly over the period, either because the bone samples absorbed ca and p from the saliva solution or because of a loss of some other component such as protein. this percentage increase in ca and p did not, however, translate into increased absolute amounts of ca and p because the mass of the bones decreased. the amount of ca and p lost from the bones as a result of the change in mass was calculated to be 0.5 g ca and 0.1 g p over the 30-day period, which are trivial amounts compared to daily physiological requirements. in rumen fluid the total ash content of cancellous bones increased probably because of absorption of minerals other than ca and p from the rumen fluid: it is the non-ca plus p fraction of the ash that appeared to increase. another possibility for this increase is that it represented the consequences of colonization of the bone samples by microbes. we did not analyse this possibility. discussion the giraffe skeleton is unique as it constitutes a greater proportion of its body mass than is the case in other similar sized mammals, and it elongates faster than any other mammalian skeleton. the absolute amounts of ca and p required by giraffes to support this growth are two to three fold more than the amount required by similar sized mammals such as buffaloes (van schalkwyk et al. 2004; mitchell et al. 2005). the origin of the ca needed almost certainly is browse. the origin of sufficient p is less obvious, although pellew (1984) showed significant selection by giraffe cows for p-rich browse in the wet season of east africa, and by bulls throughout the year. a possible alternative source of p is osteophagia. osteophagia is a well documented phenomenon in african ungulates and occurs in all types of ruminants both domestic and wild, and especially in giraffes (theiler et al. 1924; nesbitt-evans 1970; western 1971; wyatt 1971; leuthold & leuthold 1972; sutcliffe 1973; hall-martin 1974; sekulic & estes 1977; langman 1978; kok & opperman 1980; hampton 2002). it has a distinct geographical distribution that depends largely on the p content of the parent rock on which food plants are growing (sutcliffe 1973), and factors such as excessive ca, aluminium or iron, which can reduce the availability of p to plants (sutcliffe 1973). the craving for bones can be attributed primarily to a p deficiency (theiler et al. 1924; denton 1982; denton et al. 1986), but its function may be related more to maintaining a proper ca to p ratio than simply increasing the intake of one of the two (barrette1985). it also has a distinct seasonal occurrence being more common in winter than summer (langman 1978). the fact that it is more frequently reported in giraffes than other wild ruminants, suggests that the unique p demands of the giraffe skeleton is the cause. if osteophagia has evolved as an adaptation for providing minerals and specifically p, it could be extable 5 the composition of rumen fluid in wild ruminants and sheep variable east africa* south africa# giraffe◊ sheep‡ rumen ph rumen dm (%) rumen nh3-n mg.100 mℓ –1 fermentation rate (mℓ gas.gdm–1.h–1 total vfa mmol.ℓ–1 % acetic acid % propionic acid % butyric acid 6.5 ± 0.2 17.6 ± 3.2 18.8 ± 3.7 4.7 ± 1.0 156.3 ± 5.3 75.0 ± 1.6 15.0 ± 1.7 9.2 ± 0.8 6.5 ± 0.1 17.0 ± 1.2 10.0 ± 6.6 4.2 ± 0.5 137.9 ± 11.8 73.8 ± 3.8 15.3 ± 2.3 9.8 ± 3.6 6.5 ± 0.2 13.8 13.6 ± 2.9–24.6 ± 2.1 3.8 ± 0.1 158.3 ± 3.5 75.8 14.2 9.0 6.5 ± 0.3 10.1–10.5 12.4 ± 5.8–18.2 ± 8.3 3.6–6.0 113–126 77–81 13–19 2–4 * species in east africa were buffalo, eland, waterbuck, oryx, gerenuk, goats, and giraffe (data from maloiy et al. 1982) # species in south africa were buffalo, wildebeest, oryx, impala, springbok, and kudu (data from giesecke & van gylswyk 1975) ◊ data from maloiy et al. 1982; odenyo et al. 1999 ‡ data from this study, from hungate (1966), church (1979), and odenyo et al. 1999 7 i.p. bredin, j.d. skinner & g. mitchell pected to be both directed and selective. moreover, ingested bones or bone fragments too large to enter the lower gastrointestinal tract (abomasum and duodenum) directly should be able to be digested in the upper digestive tract (reticulo-rumen). however there is no evidence that giraffes select, for example, cancellous bone, which is easier to crush, over dense bone, which even with sophisticated machinery is difficult to reduce to powder or small particles. the many observations made of osteophagia in giraffes show that the bones selected range from fresh to weathered, and from dense nonporous bones to bones that are highly porous and often brittle. furthermore giraffes, and ruminants in general, do not have mouth parts designed for crushing and grinding bone. their molar teeth are adapted for grinding herbage, although sutcliffe (1973) has reported that deer chew bones in a “cigar-like manner”. bones are a potentially large source of minerals and consist of approximately 460 g mineral per kg, 360 g protein per kg, and 180 g fat per kg (mcdonald, edwards, greenhalg & morgan 2002). calcium and p are the two most abundant mineral elements constituting about 36 % and 17 %, respectively, of the mineral component of the bones we used and of other mature bone (underwood & suttle 1999). thus, if bones were digested in the rumen and were reduced to a size that allows them to pass through the reticulo-omasal orifice to enter the abomasum and small intestine where further digestion and absorption is more certain, then osteophagia would be a highly advantageous behaviour, especially if the rumen is itself adapted to digest bone. our results show, however, that there was little digestion of bone in either artificial saliva or the rumen at least in the first 30 days of immersion. insignificant amounts of ca and p were removed. both types of bone softened in the rumen but this digestion was not associated with significant loss of ca or p. immersion in artificial saliva resulted in some digestion. in saliva cancellous bone not only softened, its mass and volume decreased, and ca and p were also removed in proportion to the decline in mass (bone density remained constant), although the absolute amounts removed are miniscule compared to daily requirements. these effects were produced despite the absence of the digestive enzymes normally found in saliva. the elution of minerals by saliva could simply be an effect of ph, but this is unlikely. at the start of our trials the ph of distilled water, saliva and rumen fluid was acidic and similar, but only saliva had the effects reported here. moreover the ph of the saliva solution became more alkaline with time (table 2). the softening of cancellous bone and dense bone, taken together with our observation that the samples became softer the longer they were in the fluids, suggests that immersion could facilitate mechanical digestion during rumination. the possibility exists therefore, that if giraffe saliva contains digestive enzymes and if it is of similar chemical composition to the artificial saliva solution used in this investigation, then through the mechanical effect of chewing, and the chemical action of saliva, minerals may be released from bones, especially cancellous bones. potentially more important consequences of this effect are that re-chewed, softened, bones could be more susceptible to rumen digestion and that bones could be reduced to the size that could pass through the rumen without further digestion to enter the lower gastrointestinal tract. they also may be more susceptible to acid digestion in the abomasum. we are doubtful though if these are likely scenarios. prolonged and continuous exposure to saliva (10 or more days) obviously does not occur. the most plausible reasons for the lack of effect found in this study are that the bones were not exposed for a sufficiently long time for digestion to occur, or that proteolytic activity in the microbial population in the rumens of the sheep was low, and/ or that the rumen fluid of sheep differs from that of giraffes and other ruminants. the possibility that a longer time is required for digestion seems unlikely but cannot be excluded by the data obtained. minerals are required daily, after 30 days the amount of mineral released was trivial, and the degree of bone digestion was small. we are not convinced that longer exposure would have altered any of these findings significantly. reduced proteolysis is a more likely reason. proteolysis would release ca and p from bone. the diet provided was relatively low in protein (7 % of dm) and so proteolysis might have been lower than it could have been: low protein hay is associated with low rumen ammonia-n production which is a marker of proteolytic activity (van gylswyk 1970). however, this diet accurately mimics the composition of ingested feed during the winter by giraffes. thus even if this is the reason for the lack of digestion that was found, it is likely that a similar low rate of digestion will occur in the giraffe rumen in winter. the composition of rumen fluid in sheep has been studied in detail by church (1979) and hungate (1966). giesecke & van gylswyk (1975), maloiy et 8 can osteophagia provide giraffes with phosphorus and calcium? al. (1982) and odenyo, mcsweeney, palmer, negassa & osuji (1999) have analysed the rumen contents of ten wild ruminant species including giraffes, and in free-ranging sheep and goats in east africa (maloiy et al. 1982; odenyo et al. 1999) and south africa (giesecke & van gylswyk 1975). a summary of these data is shown in table 5. these species include selective foragers (giraffe and gerenuk), bulk or roughage feeders (buffalo, waterbuck, wildebeest, oryx, sheep) and intermediate feeders (eland, kudu, impala and springbok). as table 5 shows, the composition of rumen fluid varies little between species. the only report that is not consistent with these data is that of jones, meyer, bechaz, stoltz, palmer & van der merwe (2001) who found that the rumen ph of browsers, including giraffes was lower (5.8 ± 0.1) than that of grazers (6.7 ± 0.1). they could find no consequences of the lower ph, however, and our data show that ph itself has little effect on the digestion of bones. indeed, jones et al. (2001) showed that at the higher ph nitrogen and dry matter digestion were greater than they were at the lower ph. the general conclusion is that there is little difference in rumen fluids between african ruminants with different morphological adaptations of the gut, or between wild and domestic ruminants, or between those of different size, or because of locality (giesecke & van gylswyk 1975; gordon & illius 1994; robbins, spalinger & van hoven 1995). we conclude that differences between rumen fluids are insignificant, do not account for the lack of bone digestion in our study, and that differences in rumen digestion between species, if it exists, is unlikely to be large. the data presented here shows, therefore, that although osteophagia is a potentially large source of ca and p for ruminants, there was little digestion of bones in the rumen, at least in the model and for the time period used here. it is possible that if large quantities of very weathered bones are incubated in the rumen for a long time after repeated exposure to saliva by rumination, that digestion may occur. our data suggests, however, that even this scenario is unlikely to provide sufficient p for skeletal growth. if p (and ca) is to be obtained from bone or bone fragments then they must reach the abomasum. acknowledgements we acknowledge the assistance of at dekker of the state veterinary services, skukuza; louis van schalk wyk of the peace parks foundation, hoedspruit; prof. w. van niekerk, prof. j. van ryssen, roelf coertze and jacqueline els of the department of animal and wildlife science, faculty of natural and agricultural sciences, university of pretoria; elise ferreira, truida smit, carol semenya, tilla basson and alpheus phosa of the nutrilab, uni versity of pretoria; and proff. h. groeneveld and m. van der linde for statistical help. i.p. bredin was the recipient of the blundell memorial scholarship, eriksen trust and university of pretoria postgraduate bursaries. funds to support 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and buffalo (syncerus caffer) skeletons. journal of zoology (london), 264:307–315 wadhwa, d.r. & care, a.d. 2002. the absorption of phosphate ions from the ovine reticulorumen. the veterinary journal, 163:182–186. western, d. 1971. giraffe chewing a grant’s gazelle carcass. east african wildlife journal, 9:156–157. wyatt, j.r. 1971. osteophagia in masai giraffe. east african wildlife journal, 9:157. abstract introduction materials and methods results discussion conclusion acknowledgements references about the author(s) dikeledi p. malatji biotechnology platform, agricultural research council, south africa department of wildlife and animal science, university of pretoria, south africa anna m. tsotetsi parasites, vectors and vector-borne diseases program, agricultural research council, south africa department of zoology and entomology, university of free state, qwaqwa campus, south africa este van marle-koster department of wildlife and animal science, university of pretoria, south africa farai c. muchadeyi biotechnology platform, agricultural research council, south africa citation malatji, d.p., tsotetsi, a.m., van marle-koster, e. & muchadeyi, f.c., 2016, ‘a description of village chicken production systems and prevalence of gastrointestinal parasites: case studies in limpopo and kwazulu-natal provinces of south africa’, onderstepoort journal of veterinary research 83(1), a968. http://dx.doi.org/10.4102/ojvr.v83i1.968 original research a description of village chicken production systems and prevalence of gastrointestinal parasites: case studies in limpopo and kwazulu-natal provinces of south africa dikeledi p. malatji, anna m. tsotetsi, este van marle-koster, farai c. muchadeyi received: 13 apr. 2015; accepted: 07 nov. 2015; published: 12 may 2016 copyright: © 2016. the author(s). licensee: aosis. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the majority of rural households in developing countries own village chickens that are reared under traditional scavenging systems with few inputs and exposure to various parasitic infestations. understanding of the village chicken farming system and its influence on helminth infestation is a prerequisite for optimal prevention and control strategies. this study investigated the village chicken production system and associated gastrointestinal parasites in 87 households from limpopo (n = 39) and kwazulu-natal (n = 48) provinces of south africa. a total of 191 village chicken faecal samples and 145 intestines were collected to determine the prevalence of gastrointestinal parasites in villages of limpopo and kwazulu-natal provinces, respectively. the faecal floatation analysis of samples from limpopo and kwazulu-natal provinces indicated infestations by ascaridia galli (18.77%), heterakis gallinarum (15.56%) and capillaria spp. (4.00%); tapeworms choanotaenia infundibulum (2.10%) and raillietina cesticillus (6.00%) and eimeria spp. (29.46%). mixed infestations were observed in five (4.90%) samples from limpopo province and in only four (4.49%) from kwazulu-natal province, of which 1.12% were a mixture of c. infundibulum and eimeria spp. and 3.37% a combination of h. gallinarum and eimeria spp. in limpopo, 2.94% of the chickens were positive for h. gallinarum and eimeria spp., whilst 0.98% had a. galli and capillaria spp. infestations. further investigation is needed to understand the impact of gastrointestinal parasites on village chicken health and production and develop appropriate intervention and control strategies feasible for small-holder farmers. introduction village chickens are poultry mostly owned by village communities in rural areas of africa (thekisoe, mbati & bisschop 2004) and other developing countries (muchadeyi et al. 2004; muhiye 2007). they play a vital role through their contribution to the socio-economic and cultural lives of small-holder farmers (nyoni & masika 2012; van marle-köster et al. 2008). village chickens can be used as tokens of appreciation for services rendered and are often given to visitors as gifts (kusina & kusina 1999). their role in national economies is through improved nutritional status and income of many small-holder farmers as well as landless and marginalised communities (muchadeyi et al. 2004; tarwireyi & fanadzo 2013). a majority of chicken populations in africa are kept under traditional scavenging systems (mcainsh et al. 2004; mtileni et al. 2009) that are often characterised by low productivity and high mortality. village chickens are left to scavenge to meet their nutritional needs (muchadeyi et al. 2004; mwale & masika 2009), which predisposes them to predators (kusina & kusina 1999; pedersen 2002), diseases and parasites (swatson et al. 2003) that coexist in the scavenging environment. parasite infestation contributes to poor production and can cause mortality in severe cases (soulsby 1982). gastrointestinal parasites are the most prevalent parasites affecting the productivity of village chickens (mwale & masika 2011). their prevalence in village chickens has been studied in different countries, and to a lesser extent in south africa (mukaratirwa & khumalo 2010; mwale & masika 2011). different species of endo-parasites have been identified (muhairwa et al. 2007; permin et al. 2002), including endo-parasites such as eimeria spp. and helminths (norton & ruff 2003). parasitic infestation rates have been shown to differ amongst different production systems (permin et al. 1999) because of variations in environmental and management factors. improved poultry management practices are responsible for reduction in the incidence of parasitic infestations (puttalakshmamma et al. 2008). kaufmann (2011) indicated that village chickens do not only harbour a wide spectrum of helminths but are also associated with relatively high intensity of infestations compared to commercial chickens with more frequent incidences in free range than intensive systems. the small-holder farming sector of south africa is similar to those in other african and developing countries that are characterised by low production inputs, exposure of chickens to diseases and parasites and compromised biosecurity and veterinary interventions (acavomic et al. 2005). therefore, the aim of the study was to describe typical village chicken production systems in selected village farms in limpopo and kwazulu-natal provinces of south africa and to determine the prevalence of gastrointestinal parasites in these communal low-input farming systems. such information is considered important for the development and implementation of effective control programmes. materials and methods study sites and animal populations this study was conducted in 18 villages in two agro-ecological zones of the limpopo and kwazulu-natal provinces of south africa (figure 1). these provinces were targeted based on the existence of free-range village chicken production and the contrasting environmental conditions between them. mopani district in limpopo province is situated within the subtropical zone. it can be very hot in summer, reaching maximum temperatures of 38 °c. winters are mild during the day and cold during the nights. vhembe district in limpopo province experiences a hot semi-arid climate with hot temperatures most of the year. average annual precipitation amounts to 372 mm with extremely dry winters. uthukela district municipality in kwazulu-natal province covers an area of approximately 11 500 km². it experiences heavy snow on the mountains in winter. it is located in the western boundary of kwazulu-natal province. umzinyathi district has a temperate climate. frost occurs only in parts of umzinyathi in winter. rainfall varies from more than 800 mm in endumeni and umvoti to less than 400 mm in parts of msinga. eighty-seven households were randomly selected from villages of the two provinces from november 2012 to february 2013. information was gathered from veterinary extension officers on chicken ownership in the two provinces. households in each village were then selected on the basis of the availability of free-ranging chickens and the willingness of the chicken owners to participate in the study. figure 1: map of south africa showing sampled villages (red dots) in limpopo and kwazulu-natal provinces. questionnaire survey one-on-one interviews with farmers were conducted with assistance from the agricultural extension officers from the department of agriculture in vhembe district, limpopo province, and from community extension personnel of the mdantsane non-governmental organization (ngo) in tugela ferry in kwazulu-natal province between november 2012 and february 2013. standardised questionnaires were administered to the 87 randomly selected households from villages of limpopo (n = 39) and kwazulu-natal (n = 48) provinces. the questionnaires were semi-structured with both closed and open-ended questions that were designed to capture information on the village chicken production systems with emphasis on the different livestock species kept by farmers, roles of village chickens, chicken nutrition, housing and health management and access of farmers to agricultural extension services. information was also captured during these interviews on any chicken disease or clinical signs observed by farmers in their flocks. farmers were then asked to rank the prevalent diseases or clinical signs in order of importance. chicken production parameters that included number of eggs/clutch/hen, number of egg clutches per hen per annum and number of eggs that hatched per clutch were recorded. farmers in both provinces did not keep farm records, and therefore, data collected were based on farmer recall. sample collection and parasite identification a total of 191 free-ranging village chicken faecal samples were collected from individual village chickens from the same households interviewed in limpopo (102 faecal samples from 34 households) and kwazulu-natal provinces (89 faecal samples from 47 households). freshly voided faecal samples were collected by following chickens and monitoring them within the household, chicken pens and the surroundings. after collection, faecal samples were stored at 4 °c until further analysis to prevent the eggs of parasites from hatching. the modified quantitative mcmaster floatation technique was used to examine faecal samples (maff 1986). in addition to the faecal samples, 145 live mature chickens were purchased from the same villages in limpopo (n = 99) and kwazulu-natal (n = 46). these chickens were slaughtered and the gastrointestinal tracts were removed from the proventriculus to the cloaca after which each region was cut open by dissection following the world association for the advancement of veterinary parasitology guidelines for evaluating the effectiveness of anthelmintics in chickens and turkeys (yazwinski et al., 2003). all gastrointestinal parasites visible to the naked eye were recovered from the git of the chickens using thumb forceps, washed and stored in 70% ethanol at ambient temperature awaiting parasite identification. identification of each parasite was carried out based on the morphological parameters using the helminthological keys (norton & ruff 2003; soulsby 1982). data analysis statistical analysis system was used to analyse questionnaire-derived variables such as flock size, flock composition, diseases, disease clinical signs, internal parasites, external parasites, vaccination, treatment and access to veterinary services. descriptive statistics using generalized linear model procedures, surveymeans and surveyfreq procedures were computed and presented as tables and graphs. the level of significance was considered at p ≤ 0.05. for each province, the number of eggs/clutch/hen, number of egg clutches per hen per annum and number of eggs that hatched per clutch were averaged into mean clutch size, mean number of clutches and average hatchability, respectively. the prevalence of each recovered and identified git parasite was calculated as the number of chickens infested with that particular parasite species, divided by the total number of chickens sampled (thrusfield 1990). the prevalence of the gastrointestinal parasites was calculated per province using statistical analysis system (sas 2003). the mean intensity was determined by dividing the total number of recovered parasites of a particular species by the number of chickens infested with that parasite (bush et al. 1997). abundance was calculated by dividing the number of parasites of a particular species by the total number of chickens examined (bush et al. 1997). results village chicken flock sizes and composition a total of 858 and 1351 village chickens were reported by the village farmers in limpopo and kwazulu-natal provinces, respectively (table 1). the least square means ± standard error of flock size per household was 22.03 ± 2.85 for limpopo province and 28.40 ± 2.57 for kwazulu-natal province. flock sizes varied between farms within provinces and about 44.68% of the farmers from both provinces had between 10 and 30 chickens. twenty-seven percent of the farms owned 30–50 chickens and 8.5% owned 50–100 chickens. only 6.3% of the interviewed farmers had fewer than 10 chickens. cock:hen ratios of 1:3 and 1:4 were observed in limpopo and kwazulu-natal provinces, respectively. table 1: least squares means ± standard error of the flock sizes and composition in limpopo and kwazulu-natal provinces of south africa. role of village chickens village chickens were predominantly kept for providing meat for household consumption (51% and 37%), sale (15% and 2%) and a combination of meat and sale (25.6% and 29.2%) and for meat and eggs (5.1% and 4.2%) in limpopo and kwazulu-natal provinces, respectively, as well as other functions including investment and rituals (table 2). most farmers indicated that they preferred village chicken meat as a supplement to their nutritional diets because it is tastier compared to intensively raised commercial breeds. families also benefited indirectly from rearing village chickens through the use of manure for vegetable gardens. farmers also indicated that chickens control weeds and insect pests by foraging. table 2: the percentage of farmers reporting the different roles of village chickens in the selected villages of limpopo and kwazulu-natal province of south africa. village chicken production systems in addition to rearing village chickens, farmers in both provinces kept goats and cattle, as well as sheep, pigs and donkeys (figure 2). chickens were the predominant species in both provinces, followed by cattle and goats. in limpopo, 94.9% farmers provided chicken housing at night. chicken structures in this province were made from locally available materials such paper boxes, scrap wood from discarded household furniture and wooden poles. farmers in kwazulu-natal province did not provide housing for their chickens, which find shelter in trees and homestead kitchens. village chickens in both provinces were left to scavenge around the homestead and surroundings for food and water during daytime. figure 2: livestock owned by farmers in the study areas of limpopo and kwazulu-natal provinces. the typical diet for the village chickens consisted of locusts and other insects, earthworms, grass, discarded food and vegetables. scavenging was supplemented with kitchen leftovers (16.72% and 4.59%), maize grain (28.73% and 52.26%), commercial feed (16.68% and 1.15%) and a combination of maize grain and husks and other leftovers (30.64% and 1.15%) in limpopo and kwazulu-natal provinces, respectively. because of the nature of the production system practised, farmers did not have a well-organised and controlled chicken-breeding programme. broody hens would naturally incubate their own eggs, and none of the farmers practised artificial incubation. hens laid an average of three clutches per year with an average of 12.08 and 13.64 eggs per clutch in limpopo and kwazulu-natal provinces, respectively. the average hatchability rate was 76.32% and 78.31% for limpopo and kwazulu-natal provinces, respectively. farmers increased their flocks by sharing chickens (6.89% and 22.98%) amongst friends and relatives in limpopo and kwazulu-natal provinces, respectively. over 71.80% of the farmers reported that newcastle disease was the most important constraint that caused chicken mortality in both the provinces. of the farms in limpopo and kwazulu-natal provinces, 23% and 49%, respectively, were affected by newcastle disease. other diseases of importance were fowlpox and infectious bursal disease. the most predominant chicken disease clinical signs observed by farmers in descending order of importance in both provinces were generalised weakness (3.44% and 11.49), swollen eyes (6.89% and 6.10%), diarrhoea (8.05% and 0.00%) and coughing (5.75% and 1.15%). other clinical signs included chickens being unable to walk (3.44% and 2.29%) and salivation (1.15% and 0.00%) in limpopo and kwazulu-natal provinces, respectively. of all farmers interviewed during this study, only 6.89% in limpopo and 29.32% in kwazulu-natal had observed gastrointestinal parasites in chicken droppings. a proportion of 11.49% and 66.23% of the farmers observed the presence of external parasites on their chickens in limpopo and kwazulu-natal provinces, respectively. vaccination, treatment and access to veterinary interventions ninety-four percent of the farmers interviewed in kwazulu-natal province used nobilis® nd clone 30 vaccine that was provided by mdantsane ngo to vaccinate their chickens against newcastle disease. other farmers in kwazulu-natal province used this vaccine to treat their chickens against diseases as they only administered it when chickens had already developed clinical signs. the frequency of vaccination varied amongst farms depending on the knowledge they had and also on the clinical signs observed in the chicken flocks. most of the farmers (52.10%) administered nd clone vaccine once every 2 months, whereas 14.58% used it every month. only 12.50% of the farmers used it once per annum. a total of 10.42% of the farmers used vaccination randomly or when the need arose. farmers in limpopo province did not use any vaccination to manage the health of their animals. in limpopo province, 17 (43.59%) of the farmers interviewed used ethno-veterinary medicine such as aloe, garlic and hot chilli pepper for treating chicken diseases and parasites. farmers indicated that they had more faith in the use of piperazine, jeyes fluid and laundry powders in that order. none of the farmers interviewed in kwazulu-natal province used ethno-veterinary medicine, and none of the farmers from either province used antibiotics and/or anthelmintic to treat diseases and parasites. it was also observed that all farmers (100.00%) in the surveyed villages of limpopo province had never received extension support from the department of agriculture. in kwazulu-natal, a total of 47 farmers (97.92%) received veterinary interventions provided by the mdantsane ngo. prevalence of gastrointestinal parasites fourteen (15.73%) and 43 (42.16%) chicken faecal samples were positive for gastrointestinal parasites in kwazulu-natal and limpopo provinces, respectively. six different parasite species that included nematodes ascaridia galli, heterakis gallinarum and capillaria spp., tapeworms choanotaenia infundibulum and raillietina cesticillus and protozoa (coccidia) eimeria spp. were identified in both kwazulu-natal and limpopo provinces (table 3). five (4.90%) samples from limpopo and four (4.49%) from kwazulu-natal had mixed infestations in which the animal was infested by more than one parasitic species. ascaridia galli was the most prevalent parasite at 17.65% in limpopo followed by eimeria spp. (13.73%), h. gallinarum (8.82%), r. cesticillus (4.90%), capillaria spp. (2.94%) and c. infundibulum at 1.00%. the results from kwazulu-natal province indicated a lower prevalence of gastrointestinal parasites except for eimeria spp. at 15.6% (table 3). the prevalence of h. gallinarum was 6.74% and that of a. galli, capillaria spp. and r. cesticillus was 1.10% in kwazulu-natal province. no trematodes were observed in either province. in this study, variation in the prevalence of these parasites was observed between the two different provinces, although they were not statistically significant. table 3: prevalence (%), least squares means (lsm ± se) and range of gastrointestinal parasite species from faecal samples from village chickens of limpopo and kwazulu-natal provinces of south africa. twenty-nine (64.44%) of the 45 chickens slaughtered in kwazulu-natal were positive for either one or two adult parasite species. mixed infestations of a. galli and tapeworm were observed in 27.59% of the infested animals, whilst the remaining 72.41% were positive for only one parasite species. in limpopo province, 36 (36.36%) of the 99 slaughtered chickens were positive for at least one parasite species. tapeworms were the most prevalent parasites at 75.00%, followed by a. galli at 52.78% and h. gallinarum at 8.33%. mixed infestations were observed in 15 (41.67%) of the intestines, 6.67% of which were with a. galli and h. gallinarum and 93.33% were with a. galli and tapeworms. the tapeworms were not identified to species. a total of 201 parasites from 36 positive chickens and 228 from 29 positive chickens were observed in limpopo and kwazulu-natal provinces, respectively (table 4). the average number of worms recovered per animal was 5.9 ± 5.43 in limpopo and 7 ± 12.39 in kwazulu-natal province. the chickens that harboured more worms were observed in kwazulu-natal province, where a total of 150 a. galli parasites were recovered from a single chicken. however, the chicken that carried these 150 parasites was not included in the statistical analysis as it was considered an outlier that was going to inflate the average number of worms per animal. in kwazulu-natal province, the average intensity of infestation was highest for a. galli, with an average worm count of 7.2 ± 13.76 per chicken. the highest mean abundance of infestation was seen in a. galli (3.61 ± 10.28 worms per chicken) followed by tapeworm (1.35 ± 2.13 worms per chicken) (table 5). in≈limpopo, the intensity was high for h. gallinarum at 8.67 ± 2.32 worms per chicken. mean abundance of infestation was high at 1.11 ± 3.27 worms per chicken for tapeworms. table 4: total worm count and mean worm intensity for slaughtered free-range chickens from the selected villages in limpopo and kwazulu-natal provinces of south africa. table 5: mean worm abundance ± standard deviation from the intestines of slaughtered chickens from limpopo and kwazulu-natal provinces of south africa. discussion this study described the village chicken production systems and associated parasite infestations in two provinces of south africa. village chicken production in south africa is similar to most small-holder farming systems where the chickens are exposed to the harsh environmental and production challenges coupled with farmers having limited resources to manage their flocks (acavomic et al. 2005). therefore, an understanding of the dynamics and challenges of helminth infestation in the context of the production systems is crucial to the development and implementation of effective parasite control strategies. this study sampled from villages of limpopo and kwazulu-natal provinces of south africa, which are similar to villages in most african and other developing countries. the proportion of hens was high in both provinces, which is in contrast to other studies, which observed village chicken flocks being dominated by chicks (maphosa et al. 2004; muchadeyi et al. 2004). it was expected that high rates of helminth infestations would be observed with increased flock sizes because of the increased number of animals per unit area that leads to larger amounts of faeces deposited on the ground and possibly increased infectivity per unit area (permin & nansen 1998). the relatively low levels of helminth infestations observed regardless of flock numbers in kwazulu-natal could be attributed to the extensive management of these chickens and the hot and dry conditions during sampling, which negatively affect the development of parasite eggs into infective stages and their survival in the environment (permin & nansen 1998). farmers in both provinces practised mixed livestock farming and owned goats, cattle, sheep and pigs in addition to chickens. however, the presence of other animal species in mixed livestock farming systems can expose and increase the risk of parasite infestation in village chickens as other species could act as carriers of certain parasites (permin & nansen 1998). the majority of chickens in this study scavenged for their feed, an observation that was consistent with other studies (muchadeyi et al. 2007; mwale & masika 2009). whilst this is a viable option for resource-limited farmers, scavenging for feed results in poor quality nutrition and also exposes the chickens to predation, diseases and parasites (acamovic et al. 2005). scavenging chickens are also exposed to the open air and environment and have greater contact with host organisms such as insects and the earthworm where they can be infested. insects and earthworms are intermediate hosts that may indirectly transmit the parasite eggs and infective stage of nematodes to chickens on consumption (butcher & miles 2009). the prevalence of parasites in limpopo province would probably be lower if the chicken housing was well managed with cleaning, removal of the droppings and regular application of disinfectants (kusina & kusina 1999; pedersen 2002). farmers in the surveyed villages were also observed to exchange animals with their neighbours and nearby community members. according to the conventional commercial chicken management practices (kitalyi 1998), new and introduced stock needs to be isolated and monitored for a certain period of time so that the farmer does not introduce diseases and parasites to the farms. such practices, although challenging for small-holder communal farmers, can help in reducing the introduction and spread of new pathogens between neighbouring farms and/or communities in village chicken and other livestock production systems. the reported diseases and clinical signs were merely based on farmer’s perceptions and their limited knowledge of clinical signs. the majority of the farmers did not have access to veterinary extension services, whilst those in kwazulu-natal province received extension services from a ngo working in that area. the administration of vaccines randomly without clear indications for the need for vaccinations and proper procedures being followed could result in poor response to vaccines. the prevalence of a. galli in both provinces was higher than the 10.0% – 14.5% reported in kenya (irungu, kimani & kisia 2004). however, the prevalence of nematode infestations (combination of a. galli and h. gallinarum prevalence) was low compared with those observed in zambian villages, which revealed that 28.8% and 32.8% of the chickens were infested with a. galli and h. gallinarum, respectively (phiri et al. 2007) and those in palestinian chickens, where the prevalence of a. galli and h. gallinarum was 75.6% and 68.9%, respectively (rayyan & al-hindi 2010). in this study, variation in the prevalence of these parasites was observed between the two different provinces, although they were not statistically significant. mukaratirwa and khumalo (2010) observed more parasite species and relatively higher prevalence of a. galli, h. gallinarum and capillaria spp. in coastal kwazulu-natal than was observed in this study. the significantly lower prevalence of a. galli and r. cesticillus parasites in kwazulu-natal and the relatively lower prevalence of the other nematodes and tapeworms were probably because of the generally dry summer months in the sampled localities of msinga, in contrast to limpopo province where it rained during the days of sampling. in both provinces, all parasitised chickens harboured 1–6 helminth species, which was comparable to a study by mukaratirwa and khumalo (2010) but less than that from other studies that observed up to 7, 10 and 13 species of gastrointestinal helminths (bersabeth 1999; permin et al. 2002; phiri et al. 2007). the level of mixed infestation observed in this study was expected and is common in village chickens (phiri et al. 2007). no trematodes were observed in the faecal samples and git of the village chickens in this study, which is in agreement with findings from previous studies in different populations (abdelqader et al. 2008; mukaratirwa & khumalo 2010). however, this was in contrast with the study by mwale and masika (2011) who identified the trematodes postharmostomum gallium and postharmostomum commutatum. according to those authors, trematodes require a wide range of intermediate hosts such as dragonflies and freshwater snails to complete their life cycle, which may not be available in most production systems; therefore, they are rare. conclusion the study described the chicken production systems typical for small-holder village chicken farming in the limpopo and kwazulu-natal provinces of south africa. as observed in similar systems, village chickens contribute to the livelihood of the many families in marginalised communities of south africa. the scavenging production system coupled with minimal management inputs and lack of knowledge on chicken health exposes the village chickens to different diseases and various internal and external parasites. economically important parasites such as a. galli and eimeria spp. were prevalent in both provinces, with variations in worm burdens and infection intensity. overall, this study presents gastrointestinal parasites as a problem affecting the village chickens of limpopo and kwazulu-natal provinces. prevention and control of parasites in these farming systems are compromised by the mixed farming systems, the limited resources at the farmers’ disposal for chicken management and the absence of biosecurity measures to avoid disease and parasite transmission amongst chickens and interspecies. acknowledgements this study was funded jointly by the agricultural research council-biotechnology platform and the national research foundation under the zambia/south africa bilateral research program. ms malatji received an nrf-department of science and technology professional development program research fellowship and university of pretoria phd support bursary. 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(ed.), 1990, epidemiología veterinaria, acribia, zaragoza. van marle-köster, e., hefer, c.a., nel, l.h. & groenen, m.a.m., 2008, ‘genetic diversity and population structure of locally adapted south african chicken lines: implications for conservation’, south african journal of animal science 38, 271–281. yazwinski, t.a., chapman, h.d., davis, r.b., letonja, t., pote, l., maes, l. et al., 2003, ‘world association for the advancement of veterinary parasitology (w.a.a.v.p.) guidelines for evaluating the effectiveness of anthelmintics in chickens and turkeys’, veterinary parasitology 116, 159–173. vaughan_189-198.indd introduction the south african association for marine biological research, incorporating sea world, oceanographic research institute and the dolphinarium relocated from their old premises of 46 years on durban’s beach front in march and april 2004 to the new point development’s marine theme park, ushaka marine world in durban, south africa. netting of fishes from the large exhibits could cause physical damage to struggling fishes, increasing the risk of opportunistic secondary bacterial infections (inoue, dos santos neto & morales 2004) which could not be treated in time for the opening of ushaka marine world on 30 april 2004. an ideal method to facilitate the removal of fishes from exhibits would be the use of a general anaesthetic. characteristics of a suitable anaesthetic for this purpose would include ease of administration and stability, rapid recovery of anaesthetised fishes once re-introduced into untreated water and the dependency of anaesthetic duration upon concentration (dunn & koester 1985). “2-phenoxyeth a nol” (merck laboratories, johannesburg) was selected to facilitate the removal of fishes from the two main aquarium exhibits, the open ocean exhibit, housing the highest diversity of fishes (table 1), and the shark tank housing two large adult zambezi sharks, carcharhinus leucas valenciennes, 1839, eight adult spotted ragged-tooth sharks, carcharias taurus rafinesque, 1810, and several sympatric teleost species (table 2). 2-phenoxyethanol is relatively inexpensive and remains viable in long-term exposure (kaiser & vine 1998) and is also available in bulk in south africa. deacon, white & hecht (1997) recommend 2-phenoxyethanol as a highly suitable anaesthetic for repeatedly exposed fishes. 2-phenoxyethanol also showed a more notable sedation than ms-222 in an experiment on gilthead sea bream, sparus aurata (molinero & gonzalez 1995) and is considered a desirable anaesthetic for the transportation of fishes by yanar & kumlu (2001). 189 onderstepoort journal of veterinary research, 75:189–198 (2008) 2-phenoxyethanol as anaesthetic in removing and relocating 102 species of fishes representing 30 families from sea world to ushaka marine world, south africa d.b. vaughan¹*, m.r. penning² and k.w. christison³ abstract vaughan, d.b., penning, m.r. & christison, k.w. 2008. 2-phenoxyethanol as anaesthetic in removing and relocating 102 species of fishes representing 30 families from sea world to ushaka marine world, south africa. onderstepoort journal of veterinary research, 75:189–198 2-phenoxyethanol was used as an anaesthetic to translocate 102 species of fishes representing 30 families from the sea world aquarium on durban’s beachfront to ushaka marine world. most fishes responded well to a final anaesthetic concentration of 0,150 mℓ/ℓ and there were no mortalities. keywords: anaesthetic, fishes, 2-phenoxyethanol, public aquarium, translocation * author to whom correspondence is to be directed. email: dvaughan@aquarium.co.za 1 two oceans aquarium, dock road, victoria and alfred water front, cape town, 8000 south africa 2 south african association for marine biological research, ushaka marine world, 1 king shaka avenue, point precinct, durban, 4000 south africa 3 department of biodiversity and conservation biology, university of the western cape, private bag x17, bellville, 7535 south africa accepted for publication 21 april 2008—editor 190 2-phenoxyethanol as anaesthetic in relocating fi shes pole syringe anaesthetic technique using ketamine hydrochloride and xylazine hydrochloride was not feasible for facilitating translocation of the c. leucas, and c. taurus, weighing in excess of 170 kg each because of the restriction in dart-syringe size and therefore efficient dosage administration. repeated darting could not guarantee successful induction or the safety of the operator. each exhibit of fishes was moved separately on different days. material and methods the shark tank was first to have its animals relocated on 13 april 2004. the fishes in the open ocean exhibit were relocated 2 days later. incoming seawater supplies and filtration systems were shut down. aeration of both exhibits was maintained for circulation and sustaining desirable dissolved oxygen levels during the procedure. the shark tank and the open ocean exhibit volumes were calculated at 386 400 ℓ and 850 500 ℓ, respectively. temperature, dissolved oxygen and ph levels were monitored using a ysi 85 hand-held temperature, dissolved oxygen, conductivity and salinity meter, and a cyberscan ph and orp meter. 2-phenoxyethanol was thoroughly mixed with tap water in 450 ℓ plastic drums alongside the exhibits and poured directly into the water. concentration increments of 0.050 mℓ/ℓ, equating to 19.32 ℓ of 2-phenoxyethanol, were added every 30 min to the shark tank to a final concentration of 0.150 mℓ/ℓ. the initial concentration for the open ocean exhibit was 0.030 mℓ/ℓ equating to 25 ℓ of 2-phenoxyethanol. after 30 min, increments of 0.006 mℓ/ℓ (5 ℓ of 2-phenoxyethanol) were added every 15 min until the concentration of 0.150 mℓ/ℓ was obtained. the concentration was increased to 0.250 mℓ/ℓ only to anaesthetise the last fish species, cobia, rachy centron canadum linnaeus, 1776, once all other fishes had been removed. small concentration increments spread over a longer period of time in the open ocean exhibit enabled specific species of fishes of a higher diversity than the shark tank to be removed upon their potential differences in reaction to concentrations and exposure times to 2-phenoxyeth anol. excitation events were recorded and the fishes were removed according to their effective induction to anaesthesia upon loss of equilibrium. the zambezi sharks were guided into pvc stretchers upon the first signs of anaesthesia, after which the spotted ragged-tooth sharks were similarly removed, to prevent physical injury to themselves through collision with tank walls and viewing panels. all other anaesthetised fishes were collected in transparent pvc bags by a team of divers. they were removed to awaiting transport tanks containing 2 000 ℓ, 4 000 ℓ or 8 000 ℓ volumes of seawater on the back of two flatbed trucks to be delivered in relay to the new aquarium exhibits. all the transport tanks except one of 2 000 ℓ capacity, contained a 2-phenoxy ethanol concentration of 0.100 mℓ/ℓ. the untreated transport tank was reserved for potentially sensitive species to the affects of the 2-phenoxethanol, so that they could be revived quickly to prevent possible mortalities. all transport tanks were separately filtered using diatom cartridge filters on pump-recirculation, and oxygenated using medical-grade oxygen diffused directly into the water via airstones and flexible gas tubing. after transporting and delivery of two loads of fishes to the new aquarium, all the seawater in the transport tanks was drained and replaced and re-dosed with 2-phenoxyethanol at the same concentration. results/observations water quality parameters throughout the exercise for both exhibits were similar and relatively stable. temperatures for both exhibits remained unchanged at 22.6 °c, and 22.9 °c for the open ocean exhibit and shark tank, respectively, with a stable ph for each exhibit at 7.77 and 7.86 throughout the exercise. dissolved oxygen levels were measured at the anaesthetic induction time per species. one hundred and two species of fishes representing 30 families were successfully anaesthetised using 2-phenoxy ethanol (tables 1 and 2). both zambezi sharks displayed excitation at 0.100 mℓ/ℓ at 50 min, characterised by a marked increase in swimming speed, and became disorientated at 0.150 mℓ/ℓ at 72.9 % dissolved oxygen and 70 min, when they were removed promptly to avoid injury. none of the spotted ragged-tooth sharks showed any changes in behaviour throughout the procedure and were removed from the shark tank after both c. leucas were removed. the remaining fishes were removed by the team of divers from the shark tank after all the sharks had been removed. all species of the family carangidae (kingfishes) from the shark tank displayed excitation (table 2), yet this was completely absent in all the carangid species in the open ocean exhibit anaesthetised at the same final concentration of 0.150 mℓ/ℓ (table 1). the crocodile needlefish, tylosurus crocodilus croco dilus peron & lesueur, 1821, from the open ocean 191 d.b. vaughan, m.r. penning & k.w. christison t a b l e 1 f is h e s a n a e st h e tis e d a n d r e m o ve d f ro m t h e o p e n o ce a n e xh ib it a t s e a w o rl d s c ie n ti fi c n a m e s e n g li s h n a m e s n u m b e rs e x c it a ti o n l e v e l a n a e s th e ti c l e v e l/ in d u c ti o n t im e f a m il y : a lb u li d a e ( b o n e fi s h e s ) a lb u la v u lp e s l in n a e u s, 1 7 5 8 b o n e fis h 5 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : b e lo n id a e ( n e e d le fi s h e s ) t yl o su ru s cr o co d ilu s cr o co d ilu s p e ro n & l e s u e u r, 1 8 2 1 c ro co d ile n e e d le fis h 1 0 .0 4 8 m ℓ/ ℓ; 7 5 m in 0 .0 4 8 m ℓ/ ℓ; 7 5 m in f a m il y : s e rr a n id a e ( r o c k c o d s ) c e p h a lo p h o lis s o n n e ra ti v a le n ci e n n e s, 1 8 2 8 t o m a to r o ck co d 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in e p in e p h e lu s ca e ru le o p u n ct a tu s b lo ch , 1 7 9 0 w h ite sp o tt e d r o ck co d 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : h a e m u li d a e ( r u b b e rl ip s a n d g ru n te rs ) p le ct o rh in ch u s ch u b b i r e g a n , 1 9 1 9 d u sk y ru b b e rl ip s 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p le ct o rh in ch u s g ib b o su s l a ce p e d e , 1 8 0 2 h a rr y h o tli p s 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p le ct o rh in ch u s p la yf a ir i p e lle g ri n , 1 9 1 4 w h ite b a rr e d r u b b e rl ip s 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o m a d a sy s co m m e rs o n n ii l a ce p e d e , 1 8 0 1 s p o tt e d g ru n te r 5 5 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o m a d a sy s ka a ka n c u vi e r, 1 8 3 0 ja ve lin g ru n te r 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o m a d a sy s m a cu la tu m b lo ch , 1 7 9 7 s a d d le g ru n te r 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o m a d a sy s m u lti m a cu la tu m p la yf a ir , 1 8 6 6 c o ck g ru n te r 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o m a d a sy s st ri a tu m g ilc h ri st & t h o m p so n , 1 9 0 8 s tr ip e d g ru n te r 5 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : l u tj a n id a e ( s n a p p e rs ) l u tja n u s b o h a r f o rs sk a l, 1 7 7 5 t w in sp o t sn a p p e r 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l u tja n u s g ib b u s f o rs sk a l, 1 7 7 5 h u m p b a ck s n a p p e r 8 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l u tja n u s ka sm ir a f o rs sk a l, 1 7 7 5 b lu e b a n d e d s n a p p e r 7 7 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l u tja n u s ru ss e lli i b le e ke r, 1 8 4 9 r u ss e ll’ s sn a p p e r 7 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : s p a ri d a e ( s e a b re a m s ) a ca n th o p a g ru s b e rd a f o rs sk a l, 1 7 7 5 r iv e rb re a m 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h ry so b le p h u s p u n ic e u s g ilc h ri st & t h o m p so n , 1 9 0 8 s lin g e r 1 9 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in d ip lo d u s ce rv in u s h o tt e n to tu s s m ith , 1 8 4 4 z e b ra 1 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in d ip lo d u s sa rg u s ca p e n si s s m ith , 1 8 4 4 b la ck t a il 1 7 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p a ch ym e to p o n g ra n d e g u n th e r, 1 8 5 9 b ro n ze b re a m 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p a g e llu s b e llo tt ii n a ta le n si s s te in d a ch n e r, 1 9 0 2 r e d t jo rtjo r 7 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o ly st e g a n u s p ra e o rb ita lis g u n th e r, 1 8 5 9 s co ts m a n 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in r h a b d o sa rg u s h a lu b i s te in d a ch n e r, 1 8 8 1 c a p e s tu m p n o se 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in r h a b d o sa rg u s sa rb a f o rs sk a l, 1 7 7 5 n a ta l s tu m p n o se 6 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in 192 2-phenoxyethanol as anaesthetic in relocating fi shes s c ie n ti fi c n a m e s e n g li s h n a m e s n u m b e rs e x c it a ti o n l e v e l a n a e s th e ti c l e v e l/ in d u c ti o n t im e f a m il y : l e th ri n id a e ( e m p e ro rs ) g n a th o d e n te x a u re o lin e a tu s l a ce p e d e , 1 8 0 2 g lo w fis h 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l e th ri n u s cr o ci n e u s s m ith , 1 9 5 9 y e llo w fin e m p e ro r 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l e th ri n u s h yp se lo p te ru s b le e ke r, 1 8 7 3 m o za m b iq u e e m p e ro r 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l e th ri n u s n e b u lo su s f o rs sk a l, 1 7 7 5 b lu e e m p e ro r 1 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in l e th ri n u s sa n g u in e u s s m ith , 1 9 5 5 c u tt h ro a t e m p e ro r 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : c o ra c in id a e ( g a lj o e n s ) c o ra ci n u s m u lti fa sc ia tu s p e lle g ri n , 1 9 1 4 b a n d e d g a ljo e n 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : k y p h o s id a e ( s e a c h u b s ) k yp h o su s b ig ib b u s l a ce p e d e , 1 8 0 1 g re y ch u b 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : e p h ip p id a e ( b a tf is h e s ) p la ta x p in n a tu s l in n a e u s, 1 7 5 8 d u sk y b a tf is h 3 4 n o n e o b se rv e d 0 .0 6 0 m ℓ/ ℓ; 1 0 5 m in f a m il y : m o n o d a c ty li d a e ( m o o n ie s ) m o n o d a ct yl u s fa lc ifo rm is l a ce p e d e , 1 8 0 0 c a p e m o o n y 1 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : m u ll id a e ( g o a tf is h e s ) p a ru p e n e u s m a cr o n e m a l a ce p e d e , 1 8 0 1 b a n d -d o t g o a tf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : p o m a c a n th id a e ( a n g e lf is h e s ) a p o le m ic h th ys t ri m a cu la tu s l a ce p e d e , 1 8 3 1 t h re e sp o t a n g e lfi sh 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p o m a ca n th u s im p e ra to r b lo ch , 1 7 8 7 e m p e ro r a n g e lfi sh 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : c h a e to d o n ti d a e ( b u tt e rf ly fi s h e s ) c h a e to d o n a u ri g a f o rs sk a l, 1 7 7 5 t h re a d fin b u tt e rf ly fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n g u tt a tis si m u s b e n n e tt , 1 8 2 3 g o rg e o u s g u ss y 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n lu n u la l a ce p e d e , 1 8 0 3 h a lfm o o n b u tt e rf ly fis h 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n u n im a cu la tu s b lo ch , 1 7 8 7 l im e sp o t b u tt e rf ly fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n k le in ii b lo ch , 1 7 9 0 w h ite sp o tt e d b u tt e rf ly fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n m a d a g a sk a ri e n si s a h l, 1 9 2 3 p e a rl y b u tt e rf ly fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n m a rl e yi r e g a n , 1 9 2 1 d o u b le sa sh b u tt e rf ly fis h 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h a e to d o n v a g a b u n d u s l in n a e u s, 1 7 5 8 v a g a b o n d b u tt e rf ly fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f o rc ip ig e r fla vi ss im u s jo rd a n & m cg re g o r, 1 8 9 8 l o n g n o se b u tt e rf ly fis h 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in h e n io ch u s m o n o ce ro s c u vi e r, 1 8 3 1 m a sk e d c o a ch m a n 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in t a b l e 1 co n tin u e d 193 d.b. vaughan, m.r. penning & k.w. christison s c ie n ti fi c n a m e s e n g li s h n a m e s n u m b e rs e x c it a ti o n l e v e l a n a e s th e ti c l e v e l/ in d u c ti o n t im e f a m il y : o p le g n a th id a e ( k n if e ja w s ) o p le g n a th u s ro b in so n i r e g a n , 1 9 1 6 n a ta l k n ife ja w 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : c a ra n g id a e ( k in g fi s h e s ) c a ra n g o id e s ca e ru le o p in n a tu s r u p p e ll, 1 8 3 0 c o a st a l k in g fis h 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c a ra n g o id e s fu lv o g u tt a tu s f o rs sk a l, 1 7 7 5 y e llo w sp o tt e d k in g fis h 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c a ra n x se xf a sc ia tu s q u o y & g a im a rd , 1 8 2 5 b ig -e ye k in g fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in m e g a la sp is c o rd yl a l in n a e u s, 1 7 5 8 t o rp e d o s ca d 1 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p se u d o ca ra n x d e n te x b lo ch & s ch n e id e r, 1 8 0 1 w h ite k in g fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in t ra ch in o tu s a fr ic a n u s s m ith , 1 9 6 7 s o u th e rn p o m p a n o 3 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in t ra ch in o tu s b o tla s h a w , 1 8 0 3 l a rg e s p o t p o m p a n o 1 0 n o n e o b se rv e d 0 .0 7 2 m ℓ/ ℓ; 1 3 5 m in f a m il y : r a c h y c e n tr id a e ( c o b ia ) r a ch yc e n tr o n c a n a d u m l in n a e u s, 1 7 7 6 p ro d ig a l s o n 1 n o n e o b se rv e d 0 .2 5 0 m ℓ/ ℓ; 4 0 0 m in f a m il y : c ir rh it id a e ( h a w k fi s h e s ) c ir rh iti ch th ys o xy ce p h a lu s b le e ke r, 1 8 5 5 s p o tt e d h a w kf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : p o m a c e n tr id a e ( d a m s e lf is h e s ) a b u d e fd u f n o ta tu s d a y, 1 8 6 9 d u sk y d a m se l 5 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in a b u d e fd u f va ig ie n si s q u o y & g a im a rd , 1 8 2 5 s e rg e a n t m a jo r 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : l a b ri d a e ( w ra s s e s ) b o d ia n u s b ilu n u la tu s l a ce p e d e , 1 8 0 1 s a d d le b a ck h o g fis h 9 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in b o d ia n u s d ia n a l a ce p e d e , 1 8 0 1 d ia n a ’s h o g fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in b o d ia n u s p e rd iti o q u o y & g a im a rd , 1 8 3 4 g o ld sa d d le h o g fis h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c h e ili n u s tr ilo b a tu s l a ce p e d e , 1 8 0 1 t ri p le ta il w ra ss e 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c o ri s g a im a rd a fr ic a n a s m ith , 1 9 5 7 a fr ic a n c o ri s 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in h a lic h o e re s ir id is r a n d a ll & s m ith , 1 9 8 2 r a in b o w w ra ss e 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in h e m ig ym n u s fa sc ia tu s b lo ch , 1 7 9 2 b a rr e d t h ic kl ip 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in h o lo g ym n o su s d o lia tu s l a ce p e d e , 1 8 0 1 r in g e d w ra ss e 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : s c a ri d a e ( p a rr o tf is h e s ) s ca ru s g h o b b a n f o rs sk a l, 1 7 7 5 b lu e b a rr e d p a rr o tf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in s ca ru s ru b ro vi o la ce u s b le e ke r, 1 8 4 7 e m b e r p a rr o tf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in t a b l e 1 co n tin u e d 194 2-phenoxyethanol as anaesthetic in relocating fi shes s c ie n ti fi c n a m e s e n g li s h n a m e s n u m b e rs e x c it a ti o n l e v e l a n a e s th e ti c l e v e l/ in d u c ti o n t im e f a m il y : m u g il id a e ( m u ll e ts ) m u g il ce p h a lu s l in n a e u s, 1 7 5 8 f la th e a d m u lle t 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : a c a n th u ri d a e ( s u rg e o n fi s h e s ) a ca n th u ru s tr io st e g u s l in n a e u s, 1 7 5 8 c o n vi ct s u rg e o n 9 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in a ca n th u ru s d u ss u m ie ri v a le n ci e n n e s, 1 8 3 5 p e n ci lle d s u rg e o n 6 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in a ca n th u ru s n ig ro fu sc u s f o rs sk a l, 1 7 7 5 b ro w n s u rg e o n 1 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in a ca n th u ru s te n n e n ti g u n th e r, 1 8 6 1 l ie u te n a n t su rg e o n 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in a ca n th u ru s th o m p so n i f o w le r, 1 9 2 3 c h o co la te s u rg e o n 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in a ca n th u ru s xa n th o p te ru s v a le n ci e n n e s, 1 8 3 5 y e llo w fin s u rg e o n 6 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c te n o ch a e tu s st ri a tu s q u o y & g a im a rd , 1 8 2 5 s tr ip e d b ri st le to o th 5 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in c te n o ch a e tu s st ri g o su s b e n n e tt , 1 8 2 8 s p o tt e d b ri st le to o th 6 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in n a so b re vi ro st ri s v a le n ci e n n e s, 1 8 3 5 s p o tt e d u n ic o rn 4 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in n a so li tu ra tu s s ch n e id e r, 1 8 0 1 o ra n g e -s p in e u n ic o rn 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in n a so u n ic o rn is f o rs sk a l, 1 7 7 5 b lu e sp in e u n ic o rn 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p a ra ca n th u ru s h e p a tu s l in n a e u s, 1 7 7 6 p a le tt e s u rg e o n 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in z e b ra so m a s co p a s c u vi e r, 1 8 2 9 t w o to n e t a n g 2 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : s ig a n id a e ( r a b b it fi s h e s ) s ig a n u s su to r v a le n ci e n n e s, 1 8 3 5 w h ite sp o tt e d r a b b itf is h 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : s c o m b ri d a e ( t u n a s ) s a rd a o ri e n ta lis t e m m in ck & s ch le g e l, 1 8 4 4 s tr ip e d b o n ito 1 3 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in e u th yn n u s a ff in is c a n to r, 1 8 4 9 e a st e rn li tt le t u n a 2 4 0 .1 0 2 m ℓ/ ℓ; 2 1 0 m in 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in f a m il y : b a li s ti d a e ( t ri g g e rf is h e s ) b a lis to id e s co n sp ic ill u m b lo ch & s ch n e id e r, 1 8 0 1 c lo w n t ri g g e rf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in b a lis to id e s vi ri d e sc e n ce b lo ch & s ch n e id e r, 1 8 0 1 d o tt y tr ig g e rf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in p se u d o b a lis te s fu sc u s b lo ch & s ch n e id e r, 1 8 0 1 r ip p le d t ri g g e rf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in s u ff la m e n b u rs a b lo ch & s ch n e id e r, 1 8 0 1 b o o m e ra n g t ri g g e rf is h 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 3 3 0 m in t a b l e 1 co n tin u e d 195 d.b. vaughan, m.r. penning & k.w. christison t a b l e 2 f is h e s a n a e st h e tis e d a n d r e m o ve d f ro m t h e s h a rk t a n k a t s e a w o rl d s c ie n ti fi c n a m e e n g li s h n a m e n u m b e rs e x c it a ti o n l e v e l a n a e s th e ti c l e v e l/ in d u c ti o n t im e f a m il y : c a rc h a rh in id a e ( r e q u ie m s h a rk s ) c a rc h a rh in u s le u ca s v a le n ci e n n e s, 1 8 3 9 z a m b e zi s h a rk 2 0 .1 0 0 m ℓ/ ℓ; 5 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : o d o n ta s p id id a e ( r a g g e d -t o o th s h a rk s ) c a rc h a ri u s ta u ru s r a fin e sq u e , 1 8 1 0 s p o tt e d r a g g e d -t o o th 8 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : c a e s io n id a e ( f u s il ie rs ) c a e si o t e re s s e a le , 1 9 0 6 b e a u tif u l f u si lie r 2 0 .0 5 0 m ℓ/ ℓ; 3 0 m in 0 .0 5 0 m ℓ/ ℓ; 3 0 m in f a m il y : s p a ri d a e ( s e a b re a m s ) d ip lo d u s sa rg u s ca p e n si s s m ith , 1 8 4 4 b la ck t a il < 2 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in l ith o g n a th u s m o rm yr u s l in n a e u s, 1 7 5 8 s a n d s te e n b ra s < 1 5 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in r h a b d o sa rg u s th o rp e i s m ith , 1 9 7 9 b ig e ye s tu m p n o se 6 n o n e o b se rv e d 0 .1 0 0 m ℓ/ ℓ; 4 7 m in f a m il y : m o n o d a c ty li d a e ( m o o n ie s ) m o n o d a ct yl u s fa lc ifo rm is l a ce p e d e , 1 8 0 0 c a p e m o o n y < 1 5 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : c h a e to d o n ti d a e ( b u tt e rf ly fi s h e s ) c h a e to d o n b la ck b u rn ii d e sj a rd in s, 1 8 3 6 b ro w n b u rn ie 1 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : c a ra n g id a e ( k in g fi s h e s ) a le p e s d je d a b a f o rs sk a l, 1 7 7 5 s h ri m p s ca d < 2 0 0 0 .1 5 0 m ℓ/ ℓ; 7 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in c a ra n g o id e s ca e ru le o p in n a tu s r u p p e ll, 1 8 3 0 c o a st a l k in g fis h < 2 0 0 .1 5 0 m ℓ/ ℓ; 7 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in c a ra n x se xf a sc ia tu s q u o y & g a im a rd , 1 8 2 5 b ig e ye k in g fis h < 1 0 0 .1 5 0 m ℓ/ ℓ; 7 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in d e ca p te ru s m a cr o so m a b le e ke r, 1 8 5 1 s le n d e r sc a d < 1 0 0 0 .1 0 0 m ℓ/ ℓ; 3 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in s e ri o la r iv o lia n a v a le n ci e n n e s, 1 8 3 3 l o n g fin y e llo w ta il 1 0 .1 5 0 m ℓ/ ℓ; 7 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in t ra ch in o tu s a fr ic a n u s s m ith , 1 9 6 7 s o u th e rn p o m p a n o < 2 0 0 0 .1 0 0 m ℓ/ ℓ; 4 5 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in t ra ch in o tu s b o tla s h a w , 1 8 0 3 l a rg e sp o tt e d p o m p a n o < 1 4 0 .1 5 0 m ℓ/ ℓ; 7 0 m in 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : e c h e n e id e a ( r e m o ra s ) r e m o ra r e m o ra l in n a e u s, 1 7 5 8 r e m o ra 6 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : p o m a c e n tr id a e ( d a m s e lf is h e s ) a b u d e fd u f va ig ie n si s q u o y & g a im a rd , 1 8 2 5 s e rg e a n t m a jo r < 4 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in p le ct ro g ly p h id o d o n le u co zo n u s b le e ke r, 1 8 5 9 s a sh d a m se l < 1 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in t o ta l n u m b e rs g iv e n a s e st im a tio n s a re a p p ro xi m a te n u m b e rs o f in d iv id u a ls p e r sh o a l r e m o ve d 196 2-phenoxyethanol as anaesthetic in relocating fi shes s c ie n ti fi c n a m e e n g li s h n a m e n u m b e rs e x c it a ti o n l e v e l a n a e s th e ti c l e v e l/ in d u c ti o n t im e f a m il y : l a b ri d a e ( w ra s s e s ) t h a la ss o m a h e b ra ic u m l a ce p e d e , 1 8 0 1 g o ld b a r w ra ss e < 1 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in t h a la ss o m a lu n a re l in n a e u s, 1 7 5 8 c re sc e n tta il w ra ss e < 1 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : m u g il id a e ( m u ll e ts ) l iz a r ic h a rd so n ii s m ith , 1 8 4 6 s o u th e rn m u lle t < 1 0 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in f a m il y : a c a n th u ri d a e ( s u rg e o n fi s h e s , u n ic o rn fi s h e s ) a ca n th u ru s tr io st e g u s l in n a e u s, 1 7 5 8 c o n vi ct s u rg e o n < 4 0 n o n e o b se rv e d 0 .1 5 0 m ℓ/ ℓ; 7 0 m in t o ta l n u m b e rs g iv e n a s e st im a tio n s a re a p p ro xi m a te n u m b e rs o f in d iv id u a ls p e r sh o a l r e m o ve d t a b l e 2 co n tin u e d 197 d.b. vaughan, m.r. penning & k.w. christison exhibit was the most sensitive of all fishes to 2-phenoxyethanol. excitation was brief yet violent, followed by a sudden and complete loss of equilibrium at 0.048 mℓ/ℓ and 83.2 % dissolved oxygen after 75 min after the introduction of the initial increment, and was removed to the untreated transport tank where it made a full recovery. beautiful fusilier, caesio teres seale, 1906 displayed a short period of excitation at 0.050 mℓ/ℓ before losing equilibrium at 81.6 % dissolved oxygen and 30 min. dusky batfish, platax pin natus linnaeus, 1758 became anaesthetised at 0.060 mℓ/ℓ and 78 % dissolved oxygen and 105 min without excitation. largespotted pompano, trachino tus botla shaw, 1803, from the open ocean exhibit became anaesthetised at 0.072 mℓ/ℓ and 71.9 % dissolved oxygen and 135 min, and at 0.150 mℓ/ℓ in the shark tank at 72.9 % dissolved oxygen and 70 min. bigeye stumpnose, rhabdo sargus thorpei smith, 1979, became anaesthetised at 0.100 mℓ/ℓ at 79.8 % dissolved oxygen and 47 min without displaying excitation. excitation was evident in the little eastern tuna, euthynnus affinis cantor, 1849, at 0.102 mℓ/ℓ and 210 min, but they only became anaesthetised at 0.150 mℓ/ℓ at 71.9 % dissolved oxygen and 330 min. all other fishes except the cobia, r. canadum, became anaesthetised at 0.150 mℓ/ℓ and 71.9 % dissolved oxygen and 330 min without excitation, and were easily removed from the exhibits. the cobia was the last fish to be removed from the open ocean exhibit after increasing the dosage to 0.250 mℓ/ℓ, at 89.6 % dissolved oxygen and 430 min. no mortalities resulted from the use of 2-phenoxyethanol on any of the fishes during anaesthesia or transport to ushaka marine world. discussion hseu, yeh, chu & ting (1998) outlined a drawback in the use of 2-phenoxyethanol, in that it requires high anaesthetic concentrations in comparison to ms-222 (argent chemical laboratories) and quinal dine (sigma-aldrich) in fishes, and that the effective concentration for most fishes is 0.200 mℓ/ℓ to 0.600 mℓ/ℓ. recently, velíšec, wlasow, gomulka, svobodova & novotny (2007) indicated that 2-phenoxyethanol also has a relatively low therapeutic index, reported as 1:2.6. the majority of fishes anaesthetised throughout their translocation of fishes from sea world to ushaka marine world showed optimal anaesthesia at the concentration of 0.150 mℓ/ℓ, with some species effec tively anaesthetised at lower dosages, and one species (r. canadum) requiring additional anaesthetic to facilitate successful removal. yanar & kum lu (2001) and velíšec et al. (2007) indicated that the effective concentration of an anaesthetic leading to induction in fishes varied according to their sex, age, water quality parameters, overall biomass and physiological state, and the duration of the exposure. all t. botla from the shark tank were juveniles of less than 20 cm in length. those from the open ocean exhibit were adults of approximately 50 cm in length. velíšec & svobodova (2004a) indicated that juvenile rainbow trout, oncorhynchus mykiss walbaum, 1792, are more sensitive to 2 phen oxyethanol than adults. this could explain the difference in effective induction of t. botla from the shark tank and open ocean exhibit, where more than double the dosage of 2-phenoxyethanol for the adult fish from the open ocean exhibit was required for the same anaesthetic effect. once the desired level of anaesthesia had been reached in both exhibits, several hours passed before all fishes were eventually removed. their anaesthetic state remained stable and did not progress with time. velíšec & svobodova (2004a); velíšec et al. (2007) also consider that sensitivity to anaesthetics is influenced by the enhancement of decreasing or low dissolved oxygen concentration, but the most important factor affecting the anaesthetic efficiency of 2-phenoxyethnol is temperature, where higher temperatures produce better anaesthetic efficiency (velí šec & svobodova 2004b). dissolved oxygen levels dropped slightly in each exhibit from the beginning of the exercise to first induction, but remained stable at 71.9 % and 72.9 % for the open ocean exhibit and shark tank, respectively. this initial drop is thought to be the result of shutting down the filtration processes and incoming make-up water just prior to the introduction of the first anaesthetic increment. the last dissolved oxygen reading for the open ocean exhibit was taken at induction of the last remaining individual fish, r. canadum, and was notably higher at 89.6 % after the removal of all other fishes, which indicates that the stable dissolved oxygen levels observed reflect the equilibrium between biomass and maintained artificial aeration. aeration is therefore recommended for similar future exercises. yanar & kumlu (2001) stated, following an experiment with european seabass, dicentrarchus labrax linnaeus, 1758, that an increase in anaesthetic concentration decreased the induction time. this is evident in comparing the time to induction at 0.150 mℓ/ℓ, 198 2-phenoxyethanol as anaesthetic in relocating fi shes obtained at 330 min and 70 min for the open ocean exhibit and shark tank respectively, but excitation was displayed in all carangids in the shark tank but not in those from the open ocean exhibit. it is therefore possible that time to anaesthetic concentration, and not necessarily the concentration alone could have played an important role in controlling the onset of excitation in this family of fishes. molinero & gonzalez (1995) consider that reactions of fishes to 2-phenoxyethanol and ms-222 include a change in respiratory rate and pigmentation. tytler & hawkins (1981) suggested that retaining fishes in an anaesthetic bath for too long leads to the fading of respiration and finally complete respiratory and cardiac failure, but hajec, kłyszejko & dziaman (2006) confirmed that lower effective concentrations of the anaesthetic clove oil provided longer periods of general anaesthesia with respiration being maintained. although fishes in both exhibits became increasingly less responsive to external stimuli, such as sudden movements and the presence of divers in the water through the progression of concentration increments and time, no obvious pigmentation changes or notable changes in respiration rates for the entire duration of the exercise were observed. shoaling species such as southern pompano, trachi notus africanus smith, 1967, shrimp scad, alepes djedaba forsskal, 1775, and coastal kingfish, carangoides caeruleopinnatus ruppell, 1830, assumed tight shoals until just prior to anaesthesia when they became disorientated, and dispersed throughout the water column before losing equilibrium. no information is available in which the successful use of 2-phenoxyethanol to anaesthetise adult c. leucas, and c. taurus, is outlined. both these species reacted favourably to this anaesthetic, allowing safe anaesthesia without risk of injury to the sharks or handlers, at the concentration of 0.150 mℓ/ℓ and 72.9 % dissolved oxygen, and 70 min. the translocation of all fishes from one public aquarium to another is a rare occurrence, and one which requires careful planning. the use of 2-phenoxyethanol as a reliable and stable anaesthetic was paramount to the successful and relatively stressfree procedure which included transportation by road. references deacon, n., white, h., & hecht, t. 1997. isolation of the effective concentration of 2-phenoxyethanol for anaesthesia in the spotted grunter (pomadays commersonnii), and its effect on growth. aquarium sciences and conservation, 1: 19–27. dunn, r.f. & koester, d.m. 1985. anesthetics in elasmobranchs: a review with emphasis on halothane-oxygen-nitrous oxide. journal of aquariculture and aquatic sciences, 5:44–52. hajek, g.j., klyszejko, b. & dziaman, b. 2006. the anaesthetic effect of clove oil on common carp (cyprinus carpio l.). acta ichthyologica et piscatoria, 36:93–97. hseu, j., yeh, s., chu, y. & ting, y. 1998. comparison of efficacy of five anesthetics in goldlined sea bream (sparus sarba). acta zoologica taiwanica, 9:35–41. inoue, l.a.k.a., dos santos neto, c. & morales, g. 2004. standardization of 2-phenoxyethanol as anaesthetic for juvenile brycon cephalus (gunther, 1867): the use in field procedures. ciência rural (santa maria), 34:563–565. kaiser, h. & vine, n. 1998. the effect of 2-phenoxyethanol and transport packing density on the post-transport survival rate and metabolic activity in the goldfish (carassius auratus). aquarium sciences and conservation, 2:1–7. molinero, a. & gonzalez, j. 1995. comparative effects of ms 222 and 2-phenoxyethanol on gilthead sea bream (sparus aurata l.) during confinement. comparative bio chemistry and physiology, 111:405–414. tytler, p. & hawkins, a.d. 1981. vivisection, anesthetics and minor surgery, in aquarium systems, edited by a.d. hawkins. new york: academic press. velíšek, j. & svobodova, z. 2004a. anaesthesia of rainbow trout (oncorhynchus mykiss) with 2-phenoxyethanol: acute toxicity and biochemical blood profile. acta veterinaria brno, 73:379–384. velíšek, j. & svobodova, z. 2004b. anaesthesia of common carp (cyprinus carpio l.) with 2-phenoxyethanol: acute toxicity and effects on biochemical blood profile. acta veterinaria brno, 73:247–252. velíšek, j., wlasow, t., gomulka, p., svobodova, z. & novonty, l. 2007. effects of 2-phenoxyethanol anaesthesia on sheatfish (silurus glanis l.). veterinarni medicina, 52: 103–110. yanar, m. & kumlu, m. 2001. the anaesthetic effects of quinaldine sulphate and/or diazepam on sea bass (dicentrarchus labrax) juveniles. turk 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice article information authors: wilfred tembo1,2 hezron e. nonga1 affiliations: 1department of veterinary medicine and public health, sokoine university of agriculture, tanzania 2department of veterinary services, ministry of agriculture and livestock, zambia correspondence to: hezron nonga email: nongahezron@yahoo.co.uk postal address: po box 3021, morogoro 67125, tanzania dates: received: 22 july 2014 accepted: 17 dec. 2014 published: 16 apr. 2015 how to cite this article: tembo, w. & nonga, h.e., 2015, ‘a survey of the causes of cattle organs and/or carcass condemnation, financial losses and magnitude of foetal wastage at an abattoir in dodoma, tanzania’, onderstepoort journal of veterinary research 82(1), art. #855, 7 pages. http://dx.doi.org/10.4102/ojvr.v82i1.855 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a survey of the causes of cattle organs and/or carcass condemnation, financial losses and magnitude of foetal wastage at an abattoir in dodoma, tanzania in this original research... open access • abstract • introduction • materials and methods    • study area and animals    • study design and data collection    • estimation of financial loss attributed to organ and/or carcass condemnations    • data analysis • results    • slaughtered cattle and conditions in retrospective study    • slaughtered cattle and conditions in december 2013    • financial losses as a result of condemnations    • numbers of pregnant cows slaughtered • ethics statement • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ slaughterhouses provide a safeguard that prevents the public from consuming meat of poor quality or meat which may be infected with zoonotic diseases. this work reviews a 3-year database of cattle that were slaughtered and inspected between 2010 and 2012 at dodoma abattoir, tanzania. in addition, meat inspection was undertaken for 1 month (december 2013). the aim of this study was to establish causes of organ and carcass condemnations and their financial implications as well as the magnitude of slaughter of pregnant cows at dodoma abattoir. during retrospective study, it was found that a total of 9015 (10.5%) lungs, 6276 (7.3%) intestines, 5402 (6.3%) livers, 3291 (3.8%) kidneys and 41 (0.05%) carcasses were condemned. pulmonary emphysema (3.4%), fasciolosis (4.5%), pimply gut (5.7%), kidney congenital cysts (1.9%) and hydatidosis (3.1%) were major causes of organ condemnations. this large number of condemned edible organs and/or carcasses implies that public health considerations result in deprivation of valuable protein. occurrence of hydatidosis, cysticercosis, fasciolosis and tuberculosis illustrates the possible public health problem and presence of environmental infections. of the 794 cows slaughtered in december 2013, 46% were pregnant. financial loss as a result of organ and/or carcass condemnations was estimated at $9892. condemnation of organs and/or carcasses and indiscriminate slaughter of pregnant cows represent a significant loss of meat and revenue and a reduction in growth of future herds, which has a negative effect on the livestock industry. this justifies appropriate surveillance and disease control programmes coupled with strict enforcement of legislation governing animal welfare to curb the slaughter of pregnant animals. introduction top ↑ tanzania mainland has about 50 million ha of land suitable for grazing and has the third largest livestock population in africa, after sudan and ethiopia (ministry of livestock development and fisheries [mldf] 2012). tanzania has a total of 21 400 889 cattle, 99.4% of which are reared by smallholder farmers (mldf 2012). this places beef production amongst the most important economic and rural livelihood activities in tanzania. indeed, indigenous cattle provide more than 70% of meat as well as 67% of milk consumed in tanzania (mellau, nonga & karimuribo 2010a). the contribution of livestock is not limited to its share in the total gross domestic product (gdp); it also contributes to the national food supply (meat, eggs, milk) and to crop production through the provision of manure and draught power (mldf 2012). however, the potential contribution of livestock cannot be fully exploited because of losses that occur as a result of morbidity and mortality as a result of livestock diseases. some of these losses can only be observed at slaughterhouses during antemortem and postmortem examinations. the number of organs and carcasses condemned for various reasons implies serious economic losses to farmers and the livestock industry in the country (mellau, nonga & karimuribo 2011). in addition, condemnations also reduce the availability of meat required by the human population to meet their protein and mineral requirements. apart from the losses resulting from condemnations, others occur as a result of slaughter of pregnant cows, a situation that obviously adds to the bulk of losses to the farmer and the livestock industry as a whole. in view of the increasing human population, tanzania requires a corresponding increase in its cattle population and improved herd health management. however, the increase in cattle population cannot be fully attained because of foetal losses that occur as a result of slaughter of pregnant cows. previous studies have found that up to 8.6% of cows slaughtered in arusha were pregnant (mellau, nonga & karimuribo 2011) whilst 14.4% cows slaughtered at yola modern abattoir in adamawa state, nigeria were pregnant (ardo, lawal & aliyara 2013). slaughter of pregnant cows at slaughterhouses threatens the growth of the cattle population with serious repercussions for any country's livestock industry. dodoma is one of the regions with a high number of livestock in tanzania. it is a capital city where all parliamentary sessions and many important government and political party activities are undertaken. dodoma abattoir is the main source of beef for all the delegates to these functions, as well as for the general population of the municipality. dodoma region is amongst the drier areas of tanzania with variable climatic conditions and unpredictable rainfall, which may have a direct influence on the patterns of livestock diseases. however, there has been no study on diseases of cattle raised in such drier areas of tanzania. the quality and safety of beef consumed in dodoma is unknown, as there has been no study on common pathological lesions encountered in slaughtered cattle. causes of organ and carcass condemnations and the associated financial implications have not been documented. although similar surveys have been conducted in other regions, it is important to carry out the study because the prevalence and incidence of diseases differ according to geographical areas. furthermore, such data could be a convenient and inexpensive source of information that could be used to monitor disease trends and possible emergence and re-emergence of pathogens. moreover, the extent of slaughter of pregnant cows in the region is not known although there have been limited reports in some areas of tanzania (maro 2014; mellau, nonga & karimuribo 2011). the aim of this study was to provide baseline data on the common causes of slaughterhouse condemnations of organs and/or carcasses and to estimate the financial losses that occur at dodoma abattoir. materials and methods top ↑ study area and animals the survey was conducted at an abattoir in dodoma municipality, which lies between latitude 6.1731°s and longitude 35.749°e. the municipality has a human population of 410 956 (population and housing census of tanzania [phct] 2013). dodoma region covers an area of 2669 km2 and has 2 083 588 people (phct 2013) and about 1 185 501 cattle (mldf 2012). it receives a mean annual rainfall of less than 570 mm and the wet season starts in november and ends in may. the mean minimum and maximum temperatures vary according to altitude but average at 16 ° c and 29 ° c, respectively. the bulk of the cattle slaughtered come from dodoma itself and the adjoining areas of arusha and singida regions. dodoma abattoir is owned by tanzania meat company and it slaughters an average of 81 cattle per day. study design and data collection this study involved the use of both retrospective (secondary) and prospective (primary) data. the retrospective study involved retrieval of slaughter records of a 3-year period from 2010 to 2012. as a means of quality control of data, all records with no proper diagnosis of organ and/or carcass lesions and ambiguous information on species and slaughter dates were excluded from the study. routine meat inspection is carried out by qualified meat inspectors with a diploma in animal health and production who have undertaken special training in meat inspection, meat processing and pathology of farm animals. the meat inspectors perform their work under the supervision of qualified veterinarians. during routine meat inspection, the disease conditions were grossly diagnosed based on pathological changes such as colour, size, morphology, consistency and presence of lesions or parasites as described by gracey (1986). judgement on whether organs and/or carcasses were fit or unfit for human consumption was based on guidelines provided by tanzania's meat inspection guidelines (united republic of tanzania [urt] 1962) and as described by gracey (1986) and the food and agriculture organization of the united nations (fao) (1994). at the end of meat inspection every day, all partially and totally condemned carcasses and organs were taken to the abattoir laboratory for further examination and identification of the lesions and parasites, if any. in case of doubt, if lesions would need further investigation and as a means of external validation, the abattoir submitted samples to dodoma veterinary investigation centre (vic) for diagnostic confirmation. collection of prospective primary data was carried out through active meat inspection conducted in december 2013 using the same approach as for the retrospective study. estimation of foetal loss was arrived at by observation and palpation of uteri of all slaughtered cows. estimation of financial loss attributed to organ and/or carcass condemnations estimation of financial loss attributed to organ and/or carcass condemnations was established based on the data recorded in december 2013. the parameters used for assessing the financial loss were the average weight of organs and/or carcasses in kilograms and average prices per kilogram of organs and/or carcasses in us dollars ($). financial loss for the 1-month period was established by direct computation of the product (by multiplication) of the average weight of one organ or carcass, the average price (per kg of organ or carcass) and the total number of condemned organs and/or carcasses, as described by mwabonimana et al. (2009). the annual financial loss was obtained by assuming that the parameters (organ and carcass condemnation rates per month and average meat prices) remained relatively constant during the year. data analysis the data were entered into a microsoft excel spread-sheet and the proportions (%) of lesions were calculated considering the number affected against the total number of animals slaughtered and inspected. a chi squared test was used to compare the proportions (%) of lesions obtained in the wet and dry seasons and at a critical probability of p < 0.05 using the stat calc function on epi info™ (version 7, centers for disease control, atlanta, usa). furthermore, foetal losses were quantified as proportion (%) of the cows’ uteri that were found with foetuses over the total number of cows that were slaughtered during the prospective study. results top ↑ slaughtered cattle and conditions in retrospective study based on meat inspection records, a total of 85 980 cattle were slaughtered and inspected at dodoma abattoir during the period 2010–2012. of the slaughtered cattle, 29.4% (n = 25 298) had pathological conditions that lead to condemnations of organs and/or carcasses (table 1). a total of 667 (0.8%) carcasses had lesions or pathological conditions and of these, 41 (0.05%) were totally condemned because of jaundice (0.03%; n = 27), cysticercosis (0.01%; n = 12) and tuberculosis (0.002%; n = 2). the rest were provisionally passed for human consumption after being chilled for 10 days at -10 ° c for cases of cysticercosis, chilled for 24 h for cases of mild jaundice and mild haemorrhages, and trimmed for bruises. table 1: number and percentage of organs and/or carcasses condemned according to condition at dodoma abattoir, tanzania for the period 2010–2012. of all the cattle that were slaughtered during the 3-year period, a total of 25 924 (30.2%) organs and/or carcasses were condemned. organ-specific condemnations were: 9015 lungs (10.5%) – the highest number of condemnations – followed by 6276 (24.8%) intestines, 5402 (21.4%) livers and 3291 (13%) kidneys, 580 (0.7%) hearts and 410 (0.5%) aortas (table 1). the least frequently condemned organs were the spleen (0.8%), plucks (0.3%) and head (0.004%). of the lungs that were condemned, emphysema (32.8%) was the leading cause of condemnations, followed by hydatidosis (27.3%) and haemorrhages (21.9%). lumps in the gut as a result of oesophagostomosis, i.e. pimply gut (78%), was the main cause of condemnations of intestines. fasciolosis accounted for 72.3% of condemned liver followed by calcified cysts (16.3%). of 3291 kidneys that were condemned, congenital cysts (48.7%) and hydronephrosis (31.2%) were the main reasons for condemnation. it was further found that a total of 49 571 (57.7%) and 36 409 (42.4%) cattle were slaughtered in the wet and dry seasons, respectively. significant differences (p < 0.05) were observed in the occurrence of some conditions between the wet and dry seasons. calcified cysts, peritonitis, abscesses, pimply gut and splenomegaly were more prevalent during the wet seasons. on the other hand, jaundice, bruises, emphysema, lung haemorrhages, hydronephrosis, renal calculi, onchocercosis and pericarditis were more prominent in the dry seasons. no significant differences (p > 0.05) were observed in other conditions between the wet and dry seasons. slaughtered cattle and conditions in december 2013 in the primary data, 2438 cattle were slaughtered and inspected, of which 794 (32.6%) were cows, 641 (26.3%) were bulls and 1003 (41.1%) were castrates. a total of 2329 (95.5%) cattle were the common local shorthorn zebu cattle whilst the rest (4.5%) were crossbreeds of exotic dairy and beef cattle. the stock movement permits at the abattoir indicated that most of the cattle (98%) came from kizota livestock market in dodoma whilst the rest (2%) came from individual suppliers who delivered cattle directly to the abattoir. even though 47 (1.9%) of cattle were emaciated and 446 (18.3%) were soiled with dung and mud, no animal was condemned at antemortem inspection during the prospective study. of all the cattle that were slaughtered in december 2013, 1247 (51.1%) had diseases or conditions that led to condemnations. one carcass was totally condemned because of generalised tuberculosis whilst another carcass was condemned as a result of cysticercosis. organ assessment showed that 325 (13.3%) lungs, 186 (7.6%) livers, 279 (11.4%) intestines, 84 (3.4%) kidneys, 32 (1.3%) hearts and 334 (13.7%) aortas were condemned. onchocercosis (13.7%) was responsible for all aortas that were condemned. the rest were pimply gut (10.2%), fasciolosis (4.1%), pulmonary emphysema (5.2%), calcified cysts (3.7%), congenital cysts (1.7%), hydatidosis (2.7%), enteritis (1.2%), pericarditis (0.5%) and cysticercosis (0.1%). financial losses as a result of condemnations the prices that were used in the estimation of financial losses were those that prevailed in dodoma municipality during the prospective study. the average weight of organs and/or carcasses, total number of organs and/or carcasses condemned, average unit cost per kg of organs and/or carcasses and the total financial losses attributable to organ and carcass condemnations are indicated in table 2. the total cost of all organs and carcasses during 1 month was $9892. assuming a relatively constant rate of condemnations and prices of meat, the annual financial loss was estimated at $118 702. table 2: financial loss in prospective study carried out in december 2013 at dodoma abattoir, tanzania. numbers of pregnant cows slaughtered a total of 794 cows were slaughtered and their uteri examined for pregnancy. of all cows slaughtered, 365 (46.0%) were found to be pregnant. ethics statement top ↑ permission to carry out this study was granted by the dodoma municipal director and ethics approval for the study was given by the ethical committee of sokoine university of agriculture (sua), morogoro, tanzania. the vice chancellor of sua issued a research permit letter on behalf of the tanzanian commission for science and technology (costech) that permitted the researcher from the university to conduct research at dodoma municipal abattoir. the management of the abattoir also issued a permission letter for the abattoir records to be used for research and to conduct a 31-day survey of causes of organ and/or carcass condemnations, financial losses associated with those condemnations and the magnitude of slaughter of pregnant animals at the abattoir. verbal consent was obtained from each of the cattle owners who had sent animals for slaughter at the abattoir after explaining the purpose and importance of the study prior to data collection. discussion top ↑ the present study has revealed that a number of conditions result in the condemnation of organs and carcasses and thus have financial implications. furthermore, the apparently prevalent slaughter of pregnant cows is of animal welfare concern. the condemnation of organs and carcasses reduces the availability of necessary protein, vitamins and minerals and deprives the farmers and cattle traders of valuable income. the existence of tuberculosis, hydatidosis, fasciolosis and cysticercosis, despite the low prevalence of some of these conditions, is an indication of a clear public health threat to meat handlers, livestock keepers and meat consumers. therefore, the overall economic loss, the public health risk and the negative impact on the national livestock industry cannot be overemphasised. because of their anatomical and histological characteristics, lungs are perhaps the organs most exposed to physical, chemical and biological injuries. this is supported by the findings of the current study, which revealed that 35.6% of all condemned organs were lungs. this was higher than the results obtained in arusha by mellau, nonga and karimuribo (2011). elsewhere, in ismailia, egypt, ahmed, ismail and dessouki (2013) reported that lungs contributed up to 44.6% of all condemned organs. this is in agreement with findings by mellau, nonga and karimuribo (2010b), who reported that emphysema had contributed 13.1% to organ condemnations at arusha abattoir in tanzania. ruminants, particularly cattle, have well-developed interlobular septa and lack of collateral ventilation, making them more susceptible to interstitial emphysema (mellau, nonga & karimuribo 2010b). pulmonary emphysema is associated with diseases such as east coast fever; it may also be caused by obstruction of airflow or by extensive gasping respiration during the slaughter process (fao 1994). improper stunning, delayed slaughter after stunning and delayed hoisting after slaughter may also have contributed to the high number of lungs with emphysema and haemorrhages (personal observation). exposure of animals to stress factors like dust, overcrowding and exhaustion from long treks in search of pasture and water during the dry season may also contribute to respiratory conditions (kusiluka & kambarage 1996). liver condemnation because of fasciolosis occurred in 4.5% of all cattle that were slaughtered. this prevalence is comparable to 6.7% found in arusha (mwabonimana et al. 2009). however, mellau, nonga and karimuribo (2010a) found prevalence to be almost twice as high (8.6%) in the same area. a very high liver condemnation percentage as a result of fasciolosis (up to 30%) has been reported by kamwela, kassuku and nonga (2013) in sumbawanga, and nzalawahe and komba (2013) in kigoma, tanzania. studies conducted in ethiopia and nigeria (mohammed, hailemariam & mindaye 2012; mulugeta, begna & tsegaye 2011; njoku-tony 2011) have also revealed higher occurrence of fasciolosis. this shows that fasciolosis is a large burden in cattle in most african countries. although fasciolosis rarely cause mortalities in cattle, its effects result in reduced production and condemnation of livers during meat inspection in abattoirs (kambarage et al. 1995). kidneys accounted for 13% of all organs and/or carcasses condemned. the main causes for kidney condemnation were congenital cysts and hydronephrosis. the results for hydronephrosis were similar to those reported by mellau, nonga and karimuribo (2011) in arusha, who found a prevalence of 1.9%. renal calculi, nephritis, necrosis and melanosis were other causes of kidney condemnation although at very low rates. both hydronephrosis and renal calculi were observed more often in the dry seasons. this was probably as a result of scarcity of water for animals, which predisposes animals to these renal conditions. pimply gut (oesophagostomosis) was the leading cause of condemnations of cattle intestines in the current study. incidence was lower than reported by mellau, nonga and karimuribo (2011) in arusha and by cadmus and adesokan (2009) in nigeria. in addition, the results indicated that pimply gut was observed more during the wet than the dry seasons, presumably as a result of favourable conditions for increased helminth activity in endemic areas. it is therefore imperative that control of helminths is stepped up during the wet season in order to reduce wastage of valuable meat from condemnations. the results of this study have also shown the presence of hydatidosis in cattle, which is of public health importance and has an economic impact, especially in the rural poor communities where extensive grazing is practised (berhe 2009; ernest et al. 2009). these results are in agreement with nonga and karimuribo (2009), who found an infection rate of 4.2% in arusha, tanzania. however, higher prevalence was reported in morocco, ethiopia and kenya (azlaf & dakkak 2006; berhe 2009; njoroge et al. 2002). variations in prevalence of hydatidosis in cattle may be as a result of differences in the ecosystems, grazing patterns and status of echinococcosis in stray dogs, which are the definitive hosts. because of its zoonotic importance, livestock extension and public health education need to be strengthened in order to reduce the disease burden. the low prevalence of cysticercosis observed in the present study was in agreement with mellau, nonga and karimuribo (2011), who found 0.3% in arusha. however, these results were lower than those reported by kebede (2008) in ethiopia (18.5%). in addition, tuberculosis was found in 0.01% of slaughter cattle, which is within the range of 0.1% found by mellau, nonga and karimuribo (2011) in arusha and 0.3% in tanga by swai and schoonman (2012). in spite of low prevalence recorded for the two zoonotic diseases, data suggest that they are endemic to tanzania and therefore more concerted efforts to reduce their potential public health impact are needed. results from the present study revealed that the estimated financial loss resulting from organ and carcass condemnations was around $118 702 per annum. liver condemnations alone accounted for a financial loss of $2706 per month, which is comparable with results obtained in rukwa by kamwela et al. (2013). similarly, mwabonimana et al. (2009) found financial loss as a result of fasciolosis to be $1169 in arusha. the annual financial loss in the present study was comparable with results obtained from a study conducted in nigeria (cadmus & adesokan 2009), which indicated an annual financial loss of $110 968. this loss of revenue by farmers, traders and the livestock industry has serious financial implications and a negative impact on the socio-economic wellbeing of those involved in the livestock value chain. therefore disease control strategies should be implemented strictly. the present study revealed that a high number of slaughtered cows were pregnant. the results were comparable to the findings by maro (2014) in tanga, tanzania, who found that 40.5% of slaughtered cows were pregnant. elsewhere, in the western province of zambia, zulu et al. (2013) reported 35.7% foetal wastage. however, our results indicated a much higher foetal wastage than the 8.6% reported in arusha (mellau, nonga and karimuribo 2011), as well as the 5% (cadmus & adesokan 2010) and 14.4% (ardo et al. 2013) reported in nigeria. the huge foetal wastage threatens growth of the livestock industry and therefore undermines government efforts to increase food production. moreover, the slaughter of pregnant cows disregards animal welfare legislation (urt 2008). in addition, the quality of meat from pregnant cows may be reduced by the presence of increased hormonal levels in tissues. conclusion top ↑ generally, meat inspection in abattoirs provides useful information regarding the quality of meat and does not require many resources. on the other hand, abattoir surveys have limitations because diagnosis of diseases and conditions based on gross pathology has low sensitivity, so that some conditions are likely to be missed at routine meat inspection. the prevalence may also have been underestimated because of generally poor record keeping. based on high organ condemnation rates observed during this study, effective extension, implementation and periodic review of routine livestock disease surveillance systems, including an effective trace-back system, are recommended. this will help to reduce the burden of diseases in animals. furthermore, extension services to farmers should accompany pregnancy diagnosis in animals intended for slaughter at farm level in order to withdraw pregnant animals from slaughter. moreover, strict enforcement of animal welfare legislation is advocated in order to curb the problem of slaughtering pregnant animals. acknowledgements top ↑ the authors extend their gratitude to the african development bank (adb), through the sadc-tads project for kindly financing this research. the cooperation shown by the abattoir management, meat inspectors and entire staff of the tanzania meat company (tmc), dodoma abattoir is highly appreciated. competing interests the authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this article. authors’ contributions w.t. (sokoine university of agriculture) was responsible for proposal development, data collection, data analysis and drafting the manuscript. h.e.n. (sokoine university of agriculture) was responsible for the study plan, supervision of data collection, data analysis and interpretation, and structuring and proofreading the manuscript. references top ↑ ahmed, a.m., ismail, s.a.s. & dessouki, a.a., 2013, ‘pathological lesions survey and economic loss for male cattle slaughtered at ismailia abattoir’, international food research journal 20(2), 857–863. ardo, m.b., lawal, h. & aliyara, y.h., 2013, ‘economic implication of bovine foetal wastage in yola modern abattoir, adamawa state, nigeria’, international journal for agro veterinary and medical sciences 7(2), 1–10. http://dx.doi.org/10.5455/ijavms.202 azlaf, r. & dakkak, a., 2006, ‘epidemiological study of the cystic echinococcosis in morocco’, veterinary parasitology 137, 83–93. http://dx.doi.org/10.1016/j.vetpar.2006.01.003 berhe, g., 2009, ‘abattoir survey on cattle hydatidosis in tigray region of ethiopia’, tropical animal health and production 41, 1347–1352. 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hailemariam, z. & mindaye, s., 2012, ‘major causes of liver condemnation and associated financial loss at kombolcha elfora abattoir, south wollo, ethiopia’, european journal of applied science 4(4), 140–145. mulugeta, s., begna, f. & tsegaye, e., 2011, ‘prevalence of bovine fasciolosis and its economic significance in and around assela, ethiopia’, global journal of medical research 11(3), 1–7. mwabonimana, m.f., kassuku, a.a., ngowi, h.a., mellau, l.s.b., nonga, h.e. & karimuribo, e.d., 2009, ‘prevalence and economic significance of bovine fasciolosis in slaughtered cattle at arusha abattoir, tanzania’, tanzania veterinary journal 26(2), 68–74. njoku-tony, r.f., 2011, ‘bovine fasciolosis among slaughtered cattle in selected abattoirs in imo state, nigeria’, world rural observations 3(1), 59–63. njoroge, e.m., mbithi, p.m.f., gathuma, j.m., wachira, t.m., gathura, p.b., magambo, j.k. et al., 2002, ‘study of cystic echinococcosis in slaughter animals in three selected areas of northern turkana, kenya’, veterinary parasitology 104, 85–91. http://dx.doi.org/10.1016/s0304-4017(01)00614-8 nonga, h.e. & karimuribo, e.d., 2009, ‘a retrospective survey of hydatidosis in livestock in arusha, tanzania, based on abattoir data during 2005–2007’, tropical animal health and production 41(7), 1253–1257. http://dx.doi.org/10.1007/s11250-009-9308-9 nzalawahe, j. & komba, e.v.g., 2013, ‘occurrence and seasonal predisposition of fasciolosis in cattle and goats slaughtered in kasulu district abattoir, western tanzania’, research opinions in animal and veterinary sciences 3(11), 395–400. population and housing census of tanzania (phct), 2013, ‘the 2012 phct general report’, national bureau of statistics, dar es salaam. swai, e.s. & schoonman, l., 2012, ‘a survey of zoonotic diseases in trade cattle slaughtered at tanga city abattoir: a cause of public health concern’, asian pacific journal of tropical biomedicine 2(1), 55–60. http://dx.doi.org/10.1016/s2221-1691(11)60190-1 united republic of tanzania (urt), 1962, meat inspection guidelines, government printer, dar es salaam. united republic of tanzania (urt), 2008, the animal welfare act, government printer, dar es salaam. zulu, v., mwanza, a.m., banda, f.c., yasuda, j. & yoshida, m., 2013, ‘cattle reproductive wastage in zambia: a case of mongu abattoir’, bulletin of the faculty of agriculture, kagoshima university 63, 49–54. junker_225-235.indd introduction despite the remarkable diversity of south african birdlife, knowledge concerning their helminth parasites is scant (ortlepp 1937, 1938a, b, 1963; versterpatsinska-kloryga 1987) and even sparser on the structure of their helminth communities. a first step was taken by crowe (1977), who compared the influence of sex, age and habitat on the in testinal helminths of helmeted guineafowls, numi da meleagris (linnaeus, 1758), at kimberley, northern cape province, south africa. thereafter, alexander & mclaughlin (1997) provided a comprehensive analysis of the helminth communities of four species of ducks at barberspan, south africa. it is also apparent from bush’s (1990) chapter on helminth communities in avian hosts, that considerably more information on helminth community dynamics in birds from aquatic environments than those from terrestrial habitats is available. this paper analyses the composition and structure of the helminth community of 15 helmeted guineafowls in the limpopo province, even though small numbers of hosts were available and a larger sample might have a different outcome. data on the various 225 onderstepoort journal of veterinary research, 75:225–235 (2008) the helminth community of helmeted guineafowls, numida meleagris (linnaeus, 1758), in the north of limpopo province, south africa k. junker1, l. debusho2 and j. boomker1* abstract junker, k., debusho, l. & boomker, j. 2008. the helminth community of helmeted guineafowls, numida meleagris (linnaeus, 1758), in the north of limpopo province, south africa. onderstepoort journal of veterinary research, 75:225–235 the helminths of 15 helmeted guineafowls were collected in the north of limpopo province, south africa. a total of 11 cestode, ten nematode and a single acanthocephalan species were present. species richness ranged from 8 to 16 species per host, and nine core and nine secondary species accounted for 40.9 % of the component parasite community. the remaining 18.2 % comprised satellite species. core species represented 91 % of all the worms present. individual intensities ranged from 66 to 2 724 per host and overdispersion was pronounced. there were no significant differences regarding the abundance and species richness between male and female hosts. the number of component species and overall abundance did not differ significantly between juvenile and adult hosts, but cyrnea parroti was significantly more abundant, and the prevalence of hadjelia truncata was higher in young birds than in adults. in contrast, gongylonema congolense and porogynia paronai were absent in juveniles, but had a prevalence of 60 % and 70 %, respectively, in adults. pairwise spearman’s rank correlation yielded one positive and 10 negative significant species correlations. a single trematode, dicrocoelium macrostomum, was collected from five of nine guineafowls, but was not included in the helminth community study. keywords: acanthocephala, cestoda, helmeted guineafowls, nematoda, numida meleagris * author to whom correspondence is to be directed. e-mail: joop.boomker@up.ac.za 1 department of veterinary tropical diseases, university of pretoria, private bag x04, onderstepoort, 0110 south africa 2 department of statistics, university of pretoria, private bag, south africa accepted for publication 28 may 2008—editor 226 helminth community of helmeted guineafowls in limpopo province, south africa helminth species collected have been presented in a companion publication (junker & boomker 2007a). material and methods during july 2005 to november 2006 the gastro intestinal helminths of 15 helmeted guineafowls on a farm about 60 km west of musina (messina), limpopo province (22°22’ s, 29°30’ e), were examined as detailed in junker & boomker (2007a). three of the birds shot in may 2006 and two collected in july 2006 were young birds, between 6 and 10 months old (siegfried 1966), the remainder were adults. three of the juveniles were females and two males, and the adults comprised four females and six males. the terms prevalence, intensity and abundance are used in accordance with the proposals of margolis, esch, holmes, kuris & schad (1982) and bush, lafferty, lotz & shostak (1997). infrapopulation and infracommunity follow bush & holmes (1986a, b), metapopulation follows riggs & esch (1987) and component parasite community is used as defined by holmes & price (1986). as suggested by bush & holmes (1986a) and alexander & mclaughlin (1997), species with a prevalence of 70 % and higher, were categorized as core species, those with prevalences of < 40 % as satellite species and those with prevalences > 40 % but < 70 % as secondary spe cies. a summary of these definitions is to be found in esch, shostak, marcogliese & goater (1990). a wilcoxon rank sum test was performed to determine differences in species richness, as well as in the abundance of the various species between male and female hosts, juveniles and adults and between birds shot in winter and spring. a variance ratio test of schluter (1984) and mcculloch (1985) was used to detect species association with presence-absence data for all parasites, parasites in the small intestine (si) and parasites in the caeca. pairwise spearman’s rank correlation for every possible species combination in the small intestine, gizzard and caeca respectively were calculated. to avoid possible distortions inherent to this form of analysis, double zero matches, i.e. absence of both species from a host, were eliminated. of the 14 helminth species present in the small intestine only the single acanthocephalan and the 12 cestodes were included in the analysis, because the occurrence of two nematodes, ascaridia numidae and an unidentified subulurid, was restricted to one and two hosts respectively, while a third nematode, sicarius caudatus, utilized the si as well as the gizzard. we tested for a correlation between subulura dentigera and subulura suctoria from the caeca only, as subulura sp. most probably represents either of the former two nematodes. significance was set at the 95 % level throughout. in the absence of scoleces, counts were not always possible for all the cestodes of a particular host. while these hosts were included in analyses based on presence/absence data, they were excluded from the sample pool in the wilcoxon rank sum tests pertaining to the abundance of helminths at species level. results a total of 11 951 helminths representing ten nematode, 11 cestode and a single acanthocephalan species were recovered from the alimentary canal of the 15 guineafowls. data on their prevalence, intensity of infection and abundance, as well as their feed ing guild classification and status as core, secondary or satellite species are summarized in table 1. in all likelihood, raillietina sp. and subulura sp. are representatives of the remaining species of these two genera listed in table 1. a single trematode species, dicrocoelium macrostomum, was present in five of nine guineafowls examined for this parasite. although included in the general results and discussion, these trematodes do not form part of the community study outlined below. following the classification of bush (1990) four feeding guilds, i.e. organisms using the same feedingmode, without regard to their taxonomic affinity, were present in the helminth community. the trematode guild, feeding actively on semi-solid food materials such as blood, bile, mucus and intestinal debris as well as directly absorbing nutrients through their tegumental surface, was restricted to the liver and represented by a single species. the nematode guild, being mucosal and engulfing tissue and/or lumen contents, occupied the largest number of sites along the alimentary canal, namely the crop, proventriculus, gizzard as well as the small and large intestine. the females of tetrameres numida are an exception in so far as they inhabit the glands of the proventriculus, where they suck blood. the cestodes and acanthocephalans occurred in the small intestine only. mediorhynchus gallinarum has a short neck and its attachment to the mucosa remains superficial. the larger part of its abdomen is suspended freely in the intestinal lumen, absorbing nutrients via the body surface (junker & boomker 227 k. junker, l. debusho & j. boomker t a b l e 1 h e lm in th s re co ve re d f ro m 1 5 h e lm e te d g u in e a fo w ls in m u si n a , l im p o p o p ro vi n ce , s o u th a fr ic a p a ra s it e g u il d s it e s ta tu s n o . o f in f. h o s ts p re v a le n c e (% ) in te n s it y a b u n d a n c e m e d ia n m e a n s d r a n g e m e d ia n m e a n s d a c a n th o c e p h a la m e d io rh yn ch u s g a lli n a ru m l s i c 1 5 1 0 0 2 3 5 5 .7 7 8 .3 2 – 2 3 1 2 3 5 5 .7 7 8 .3 c e s to d a a b u la d zu g n ia g u tt e ra e d a va in e a n a n a h ym e n o le p is c a n ta n ia n a n u m id e lla n u m id a o ct o p e ta lu m n u m id a o rt le p p o le p is m u lti u n ci n a ta p o ro g yn ia p a ro n a i r a ill ie tin a a n g u st a r a ill ie tin a p in tn e ri r a ill ie tin a s te in h a rd ti r a ill ie tin a s p . l m m l l m l l l l l s i s i s i s i s i s i s i s i s i s i s i c s a t s e c c c c s e c s e c c s e c c 1 2 5 a 6 1 0 1 0 1 3 7 8 1 2 8 1 1 8 0 3 6 4 0 6 7 6 7 8 7 4 7 5 3 8 0 5 3 7 3 1 3 6 3 1 4 7 9 1 1 6 .5 1 0 4 2 7 .5 1 3 1 1 .7 5 .8 4 2 .7 5 5 .9 9 1 .9 9 .3 1 2 .3 1 0 .3 5 .3 4 9 .0 1 5 .8 8 .2 4 .4 7 0 .4 7 2 .7 1 1 0 .7 5 .2 1 3 .3 7 .9 3 .9 6 0 .2 8 .8 1 – 2 8 1 – 1 0 1 – 1 2 4 1 – 2 0 3 1 – 3 6 0 2 – 1 4 5 – 3 9 1 – 2 5 2 – 1 2 4 – 1 3 7 6 – 2 8 7 .5 0 0 2 .5 6 7 0 0 .5 2 0 8 9 .2 1 .7 1 0 .7 3 5 .9 6 1 .2 7 5 .3 5 .1 3 .8 1 7 .8 1 0 .9 8 .8 3 .5 3 5 .7 6 3 .4 9 9 .4 6 .1 1 0 .4 7 .6 4 .1 4 1 .2 1 0 .5 n e m a to d a a sc a ri d ia n u m id a e c yr n e a p a rr o ti g o n g yl o n e m a c o n g o le n se h a d je lia t ru n ca ta s ic a ri u s ca u d a tu s s u b u lu ra d e n tig e ra s u b u lu ra s u ct o ri a s u b u lu ra s p . u n id e n tif ie d s u b u lu ri d t e tr a m e re s n u m id a n n n n n n n n n n s i g iz c ro p g iz g iz , s i c a e ca c a e ca c a e ca s i p ro v s a t c s e c s e c s e c s e c c s e c s a t s a t 1 1 5 6 8 8 8 1 5 6 2 5 6 1 0 0 4 0 5 3 5 3 5 3 1 0 0 4 0 1 3 3 3 4 7 1 9 2 1 .5 1 5 3 4 5 1 5 2 .5 2 4 .0 1 3 .8 2 3 .0 1 .6 2 .1 1 5 .9 5 3 6 .3 4 4 .0 2 .5 2 .4 0 .0 1 8 .2 2 2 .0 0 .5 1 .7 1 3 .4 5 8 9 .2 6 5 .4 0 .7 1 .7 4 2 – 7 5 2 – 6 1 1 – 2 1 – 6 1 – 3 1 9 – 2 2 1 4 1 – 1 7 0 2 – 3 1 – 5 0 7 0 1 1 1 3 7 0 0 0 0 0 .3 1 3 .8 9 .2 0 .9 1 .1 8 .5 5 3 6 .3 1 7 .6 0 .3 0 .8 1 1 8 .2 1 7 .6 0 .9 1 .6 1 2 .5 5 8 9 .2 4 5 1 .5 1 .4 a d a ta f ro m 1 4 h o st s o n ly l = lu m e n a l a b so rb e r; m = m u co sa l a b so rb e r; n = n e m a to d e s i = s m a ll in te st in e , g iz = g iz za rd , p ro v = p ro ve n tr ic u lu s c = c o re s p e ci e s; s a t = s a tll ite s p e ci e s; s e c = s e co n d a ry s p e ci e s 228 helminth community of helmeted guineafowls in limpopo province, south africa 2006). it is therefore included in bush’s (1990) category of lumenal absorbers, together with the majority of the larger cestodes. small and delicate cestodes such as davainea nana, hymenolepis cantani ana and ortleppolepis multiuncinata, whose entire body is virtually buried amongst the mucosal villi, constitute the fourth guild, namely that of mucosal absorbers. except for the monoxenous nematode ascaridia nu midae, all members of the component community have indirect life-cycles. the component community comprised nine core spe cies as well as nine secondary species, each representing 40.9 % of the total number of species, and four satellite species accounting for 18.2 % of the species present (table 1). despite their prevalence of 67 % being slightly below the 70 % threshold, we have arbitrarily included numidella numida and octopetalum numida with the core species, as they were two of the most numerous helminths recovered in this study. the core species accounted for 91 % of all individuals, the secondary species for table 2 number of helminth species recovered from 15 helmeted guineafowls in musina, limpopo province, south africa host number of species total number of species acanthocephalans cestodes nematodes gfm1 gfm2 gfm3 gfm4 gfm5 gfm6 gfm7 gfm8 gfm9 gfm10 gfm11 gfm12 gfm13 gfm14 gfm15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 9 7 7 7 4 5 5 5 5 5 8 6 7 5 6 6 4 5 4 4 6 3 6 5 2 5 5 3 5 5 16 12 13 12 9 12 9 12 11 8 14 12 11 11 12 average 1 6.1 4.5 11.6 sd 0 1.4 1.2 2.0 range 1 5–9 2–6 8–16 0 1 2 3 4 5 6 7 8 n u m b e r o f g u in e a fo w ls 400 800 1 200 1 600 2 000 number of helminths 0 1 2 3 4 5 6 7 8 9 11 12 13 14 16 number of component species n u m b e r o f g u in e a fo w ls i n fe c te d fig. 1 frequency distribution of the total number of helminth species found in helmeted guineafowls in musina, lim popo province, south africa. the number of individual hosts infected by a certain number of helminth species is indicated by the vertical bars fig. 2 the frequency distribution of the total number of helminths in individual helmeted guineafowls in musina. helminth burdens were grouped into size classes (0– 400, 401–800, etc. to 2 000+ helminths per guinea fowl) represented on the x-axis. the y-axis displays the number of guineafowls infected with a certain size class of helminth burdens 229 k. junker, l. debusho & j. boomker t a b l e 3 p a ir w is e s p e a rm a n ’s r a n k co rr e la tio n s fo r h e lm in th s in t h e s m a ll in te st in e o f h e lm e te d g u in e a fo w ls . o n ly s p e ci e s si g n ifi ca n tly c o rr e la te d w ith a t le a st o n e o th e r sp e ci e s h a ve b e e n in cl u d e d . t h e c o rr e la tio n c o e ff ic ie n ts a re d is p la ye d in t h e u p p e r ri g h t co rn e r o f th e m a tr ix . t h e lo w e r le ft h a lf in cl u d e s th e r e sp e ct iv e p -v a lu e s fo r e a ch p a ir o f sp e ci e s c o re s p e c ie s s e c o n d a ry s p e c ie s s a te ll it e s p e c ie s 1 2 3 4 5 6 7 8 9 1 0 1 . m e d io rh yn ch u s g a lli n a ru m 0 .5 7 6 * – 0 .4 6 4 0 .0 9 6 0 .0 6 0 – 0 .2 0 2 0 .1 9 8 – 0 .1 6 5 – 0 .0 4 2 – 0 .4 2 7 2 . n u m id e lla n u m id a 0 .0 3 1 * – 0 .6 6 2 * – 0 .5 7 0 – 0 .4 1 9 0 .3 3 5 0 .0 6 0 – 0 .2 8 2 – 0 .5 3 2 – 0 .4 5 7 3 . o ct o p e ta lu m n u m id a 0 .0 8 2 0 .0 1 0 * 0 .3 0 3 0 .3 5 6 – 0 .8 3 5 * – 0 .8 9 5 * 0 .0 3 5 0 .2 7 5 – 0 .5 8 7 4 . o rt le p p o le p is m u lti u n ci n a ta 0 .8 2 0 0 .1 4 0 0 .4 6 7 0 .1 1 2 – 0 .6 6 4 – 0 .7 1 1 0 .2 7 5 0 .5 4 4 – 0 .7 9 4 * 5 . r a ill ie tin a p in tn e ri 0 .8 6 0 0 .2 2 8 0 .3 4 7 0 .8 1 1 – 0 .4 4 7 – 0 .6 9 6 * 0 .1 5 6 0 .1 7 0 – 0 .4 3 9 6 . h ym e n o le p is c a n ta n ia n a 0 .5 2 8 0 .4 6 3 0 .0 0 5 * 0 .1 5 0 0 .2 6 7 0 .1 0 5 – 0 .7 8 2 * – 0 .8 0 1 0 .8 0 0 7 . p o ro g yn ia p a ro n a i 0 .4 9 8 0 .8 7 9 < 0 .0 0 0 1 * 0 .0 7 4 0 .0 2 5 * 0 .8 6 6 – 0 .4 4 6 – 0 .8 1 7 * – 0 .1 1 2 8 . r a ill ie tin a a n g u st a 0 .5 7 4 0 .3 7 5 0 .9 1 5 0 .5 5 0 0 .6 8 9 0 .0 2 2 * 0 .1 9 6 – 0 .3 8 3 – 0 .8 1 0 * 9 . r a ill ie tin a s te in h a rd ti 0 .0 9 2 0 .1 1 3 0 .4 7 4 0 .3 4 3 0 .7 1 7 0 .0 5 6 0 .0 1 3 * 0 .3 4 9 – 0 .8 4 6 * 1 0 . d a va in e a n a n a 0 .1 4 6 0 .2 5 5 0 .0 1 0 0 .0 3 3 * 0 .2 7 6 0 .2 0 0 0 .8 1 1 0 .0 0 8 * 0 .0 1 6 * * d a ta p e rt a in in g t o s ig n ifi ca n tly c o rr e la te d p a ir s (p < 0 .0 5 ) a re in b o ld a n d m a rk e d w ith a n a st e ri sk 230 helminth community of helmeted guineafowls in limpopo province, south africa 8.6 % and satellite species made up 0.4 % of the total worm count. infracommunities in the helmeted guineafowls from musina were moderately species rich, ranging from 8 to 16 species, with a mean number of 11.6 ± 2. sixty percent of the hosts were infected with 12 or more species (table 2, fig. 1). the total number of helminths in individual guineafowls was highly aggregated and ranged from 66 to 2 724. in ten of the 15 hosts the intensity of infection was below 800 (fig. 2), but three guineafowls had worm burdens of 1 457, 1 496 and 2 724 worms and when combined, these accounted for 48 % of the component parasite community. subulura suctoria, which was by far the most common of all the helminths recovered, constituted 86, 74 and 81 % of the total worm load of the above three hosts. excluding s. suctoria, the acanthocephalans and cestodes, especially o. numida and n. numida, occurred in higher numbers than nematodes. cyrnea parroti, gongylonema congolense, s. dentigera and subulura sp. were moderately abundant with occasional high numbers in individual hosts. the abundance of the remaining nematodes was low, ranging from one to six in single hosts. the wilcoxon rank sum test yielded no significant evidence of differences between male and female hosts or between the winter and spring season in respect of species richness. despite the group of five juvenile helmeted guineafowls including the two birds with the lowest number of helminth species, and three of the five birds harboured a lower than average number of helminths, no significant differences were found between the number of component species and overall abundance seen in juvenile versus adult hosts. however, when the wilcoxon rank sum test was performed at species level, some differences related to host age became apparent. of the gizzard nematodes, the abundance of c. parroti was significantly higher in young guineafowls, averaging 26.4 ± 27.7, than in adults, in which the mean abundance was 7.5 ± 6.6 (p = 0.0312), and, although not statistically significant, the prevalence of hadjelia truncata was twice as high in young birds than in older ones (80 % vs 40 %). conversely, gongylonema congolense from the crop was absent in young birds, but had a prevalence of 60 % in adult guineafowls. the abundance of this parasite was thus significantly higher in adults (p = 0.0451). similarly, the cestode porogynia paronai occurred in 70 % of the adult birds, but was not found in the juveniles. therefore, its abundance was significantly lower in the latter hosts (p = 0.032). the abundance of subulura sp. was significantly higher in juvenile birds than in adults (p = 0.0156). the pairwise spearman’s rank correlation test yielded 11 significantly correlated species pairs in the small intestine, of which one was positive and 10 negative. the results are presented in table 3. the gizzard nematodes, c. parroti and h. truncata, were positively correlated, whereas s. dentigera and s. suc toria from the caeca were negatively correlated. both results were, however, not significant. discussion helmeted guineafowls are non-selective omnivores feeding on a large variety of dietary items that, among others, include arthropods. saayman (1966) re covered a wide variety of prey taxa, namely orthop tera (four families), coleoptera (five families), isop tera, hemiptera, lepidoptera, hymenoptera, dip tera, myriapoda and araneida, from the crops of 36 helmeted guineafowls in the eastern cape province. notwithstanding their being a sedentary species, the birds can cover a considerable distance during their daily forays (del hoyo, elliot & sargatal 1994). these characteristics and a well structured, complex alimentary canal are among the major host factors contributing to parasite community richness (kennedy, bush & aho 1986). this might explain why, despite the harsh climatic conditions and the largely undiversified mopani (colophospermum mopane) veld habitat of the study area (acocks 1988), the helminth community of helmeted guineafowls from musina is diverse. the inclusion of live food in their diet, up to 12 % of the annual total, but higher during the summer months when insects are abundant (mentis, poggenpoel & maguire 1975), may also account for the dominance of helminths with an indirect life cycle in the guineafowls. we attribute the low prevalence and intensity of d. nana and especially of a. numidae to the arid environment characteristic of the study area. ascaridia spp. were also the only nematodes with a direct life cycle recovered from guineafowls by verster & ptasinska-kloryga (1987). their eggs are resistant and can survive for several months in suitable moist soil conditions (anderson 1992), but these were certainly not met in the present study area. furthermore, earth worms can harbour eggs and larvae, thus serv231 k. junker, l. debusho & j. boomker ing as paratenic hosts (anderson 1992), but environmental conditions were not conducive for this route of transmission either. no information is available as to the intermediate hosts of d. nana, but we assume that they are similar to those used by the congeneric davainea proglottina, namely snails and slugs (anderson 1992). little evidence of these invertebrates was seen in or around water troughs and one would not expect them to occur in large number under the prevailing conditions. thus, despite the high numbers of available final hosts, the scarcity of intermediate hosts seems to limit these parasites. similarly, the absence of the trematode guild from the small intestine of the guineafowls appears to be related to the availability of intermediate hosts. most digeneans are dependant on molluscs or very rarely an annelid intermediate host for completion of their life cycles (gibbons, jones & khalil 1996). hence, they are more frequently associated with an aquatic habitat. hodasi (1969, 1976) concluded that trematodes were rare parasites in gallinaceous birds. it is difficult to determine the host specificity of helminths, and whether a certain parasite is regarded as a specialist or a generalist is often subjective, especially as helminths which are specialists in a certain host can nevertheless occur in other, often related hosts (bush 1990). based on the host-parasite check list of guineafowls of junker & boomker (2007b), we consider the following helminths as generalists: s. suctoria, g. congolense, h. truncata and possibly a. numidae as well as the cestode h. cantaniana and the acanthocephalan m. gallinarum. each of these has been reported from a variety of hosts. many of the remaining helminths collected during this study are currently recorded from the guineafowl genera numida and/or guttera only, such as the nematodes s. dentigera and t. numida or the cestodes o. numida, raillietina angusta, raillietina pintneri and raillietina steinhardti. numidella numida which is equally common in guineafowls in the usa was also found in turkeys and domestic chickens in that country. however, failure to experimentally infect the latter hosts with the parasite (jones 1933) led reid (1962) to believe that chickens and turkeys were not natural hosts. while s. dentigera, t. numida, n. numida, o. numida, r. angusta, r. pintneri and r. steinhardti would therefore seem to be specialists in guineafowls, this, at best tentative, classification might simply reflect a general lack of data and could well change as more information on other gamebirds, such as korhaans, bustards, francolins, spurfowls and quails, becomes available. in an environment where high temperatures combined with low rainfall jeopardize successful completion of helminth life cycles, spreading the risk of transmission between various final hosts would appear a more reliable way to assure high parasite survival rates than a specialist approach. we would therefore expect the generalists to outweigh the specialists. similarly, helminths collected from the guineafowls in this study use a wide range of intermediate hosts and are often not limited to a specific host or even host taxon. numidella numida, for example, is reported to use ground and dung beetles as well as grasshoppers (reid 1962), and the common nematode, s. suctoria makes use of coleopterans, dermapterans and orthopterans (anderson 1992). this strategy of spreading the risk of transmission between several intermediate hosts, all serving as prey to the final host, might well explain the aforementioned helminths’ success in colonizing their final hosts, resulting in a prevalence of 100 % in s. suctoria, even under adverse environmental conditions. however, intermediate host data are usually very generalized in respect of the taxonomic status of the hosts. hence, as more life cycle data become available especially elucidating parasite-intermediate host associations at species level, this picture of lack of specificity might change. the aggregated pattern of dispersion seen in our data is common in parasite communities (pielou 1974; bush & holmes 1986a, b; alexander & mclaughlin 1997) and is a result of a number of factors, such as differences in the individual host’s immune competence, feeding preferences and species specific host behaviour (petney, van ark & spickett 1990; horak & boomker 2000). saayman (1966) demonstrated a pronounced difference in feeding-preferences between different members of the same guineafowl flock both in the amount of food consumed as well as the composition of crop contents. the higher the food intake and the higher the percentage of insect matter in the individual’s diet, the higher the probability of ingesting an infected intermediate host and becoming infected. a further reason for the aggregation of helminths in certain host individuals is the fact that a single infected guineafowl can excrete hundreds of nematode eggs in its faeces and a single tapeworm proglottid can contain hundreds of hexacanth larvae. consequently, dung beetles, or other insects, feed232 helminth community of helmeted guineafowls in limpopo province, south africa ing on contaminated faeces or around contaminated patches can be exposed to large numbers of parasite eggs during a single meal. reid (1962) records up to 50 cysticercoids of n. numida in infected intermediate hosts, up to 930 cysticercoids of skrjabinia cesticillus were present in a single beetle, while dung beetles have been found to contain 100 or more cysticercoids of h. cantaniana. thus the ingestion of a single infected intermediate host can lead to the presence of a large number of helminths in individual final hosts. nine core species were identified within the helminth community of helmeted guineafowls at musina. the helminth infracommunity of a single crested guinea fowl, guttera edouardi, from a nearby locality examined by junker & boomker (2007a) suggests a considerable overlap between the two parasite com munities. nine helminth species were present in the crested guineafowl, of which seven are core species and two are secondary species in helmeted guineafowls. this can probably be attributed to much the same feeding habits, exposing them to a similar pool of intermediate arthropod hosts. core species are usually the first to appear in juvenile birds (hair 1975) and our data reflect the high colonization ability of these species, in that their proportional density in juvenile birds was distinctly higher than that seen in the overall host population (60.6 % vs 40.9 %). in contrast, the percentage of secondary species in juvenile birds was 34.8 % compared to 40.9 % in the overall population, and satellite species averaged 4.6 % in comparison with an overall average of 18.2 %. pairwise spearman’s rank correlation detected 11 significant correlations between helminth species in the small intestine. of these, the only significant positive correlation occurred between the acantho cephalan m. gallinarum and the cestode n. numida, in that their intensities increased or decreased in unison. positive associations between species can be due to several factors, amongst others the use of a common intermediate host. in this case a positive association in the source community would merely be transferred to the target community and would not necessarily reflect an interaction of the two species in the final host (lotz & font 1994). as is the case with many of the other parasite species collected in our study, there is no data on the intermediate hosts used by m. gallinarum in south africa. its north american counterpart, mediorhynchus grandis, however, has been reported to use several species of grasshoppers as intermediate hosts (moore 1962), and grasshoppers also form part of the life cycle of n. numida (mohler 1936; reid 1962). whether a source community is the origin of the positive correlation between these two species, or if one parasite indeed changes the habitat in the final host in such a way as to facilitate the colonization by the other, would necessitate experimental studies. conversely, n. numida had a significant negative correlation with o. numida, which also uses orthopterans as intermediate hosts (gwyun & hamilton 1935), and o. numida was negatively, albeit not significantly so, correlated with m. gallinarum. another positive correlation, although not significant (p = 0.0819), was found between c. parroti and h. truncata in the gizzard. the few data available on their intermediate hosts suggest that these do not overlap. cyrnea parroti has been reported from orthopteran intermediate hosts and h. truncata from beetles (anderson 1992). their positive correlation might be a result of the fact that both seem to make use of a window period during the development of their host in which the latter is more susceptible to infection (see below). negative correlations between species, where an increased intensity of the one leads to a decreased intensity of the other, may result from competition for resources such as carbohydrates or attachment sites (smyth & mcmanus 1989). or it could indicate that the presence of one species alters the habitat to such an extent that it is less suitable for the other. smyth & mcmanus (1989) report a number of substances that are produced by hymenolepis diminuta and which might act as inhibitory factors, producing a crowding effect. moreover, the host’s immune response triggered by a certain species could well make this host less susceptible to subsequent colonization by other parasites. some of the factors influencing parasite community patterns in other hosts seem to be of little importance in structuring the helminth communities of helmeted guineafowls. one of these is age. moore, freehling, horton & simberloff (1987) concluded that age can occasionally have an important influence on the prevalence and intensity of helminth infections of bobwhite quail, colinus virginianus (lin naeus, 1758), and pence (1990) reported changes in host age over seasons to be one of the factors most frequently cited when discussing prevalence and intensity. however, in the present study neither overall abundance nor species richness in juvenile guineafowls differed significantly from those in adults. in contrast, crowe (1977) reported that juvenile helmeted guineafowls, i.e. birds younger than 10 233 k. junker, l. debusho & j. boomker months, from the kimberley district, south africa had significantly higher burdens of cestodes and acanthocephalans than adults, and davies, junker, jansen, crowe & boomker (in preparation) found higher burdens of s. suctoria, o. numida and m. gallinarum in juveniles during a study on helmeted guineafowls in the free state province. forrester, conti, bush, campbell & frohlich (1984) found no significance in the differences between the prevalence of helminth species in chick and adult bobwhites, but the intensity of infection of a single helminth species was higher in chicks than in adults. when studying the helminth communities in willets, tringa semipalmatus (gmelin, 1789) (= catop tro phorus semipalmatus), both on their breeding grounds (freshwater) and in their wintering habitat (saltwater), bush (1990) found young birds to be depauperate, but within the course of 2 weeks the diversity of their helminth communities increased considerably and, in the case of helminths with freshwater life cycles, at 3 months of age no longer differed from those of adult birds. several factors could influence the prevalence and intensity of helminths in guineafowls of different ages. young birds might well be more susceptible to helminth infections when compared to adults, as has been suggested by a number of authors (ackert & reid 1937; biester & schwarte 1959; soulsby, 1969). however, this would be counterbalanced by a time-dependant higher probability of previous exposure to infected intermediate hosts, and thus to the various parasites, in older birds, therefore evening out differences between different ages on component community level. on the other hand, it is well documented that the diet of juvenile helmeted guineafowls and other gamebirds consists of a larger percentage of arthropods than that of adults (del hoyo et al. 1994; crowe 2000), increasing their exposure to possible intermediate hosts. some age-related differences on metapopulation level, i.e. when singling out certain parasite species from the musina hosts, were observed. cyrnea parroti, whose predilection site is under the lining of the gizzard, was significantly more abundant in juvenile birds. we observed a distinct hardening of the gizzard lining in adult guineafowls which was not nearly as pronounced in the younger birds and which could easily impede establishment of this parasite in older hosts. this phenomenon might also explain why the prevalence of h. truncata, using the same site, decreased from 80 % in juveniles to only 40 % in older guineafowls. dogiel (1964) suggested that the normal development of a host, such as a thickening of skin, could result in a habitat being no longer suitable for the parasite, hence leading to resistance against the latter. the same mechanism is obviously not in play with g. congolense, which lives in tunnels under the crop mucosa. while the observed thickening of the mucosa should make colonization with g. congolense more difficult with increasing host age, this parasite was not found in any of the younger birds, but was present in 60 % of the older hosts. a possible explanation might be that g. congolense is only a secondary species indicating that its prevalence in the entire ecosystem is lower than that of a core species such as c. parroti. consequently, age, if seen as an increase of the probability of prior exposure to a certain parasite with time, might have a more pronounced influence on the distribution pattern of this particular parasite. using the same reasoning, one could expect the prevalence of h. truncata, also a secondary species, to increase in adult birds. as has been discussed this is not the case. however, the hardening of the crop mucosa never seems as pronounced as that of the gizzard’s and, while the latter would seem likely to form a suitable barrier against the establishment of parasites, this is not necessarily so in the former. similarly to c. congolense, the cestode p. paronai had a significantly higher abundance in adult guineafowls, being absent in young birds. little is known about the life cycle of this parasite except that it is one of the cestodes making use of sites other than the small intestine, in this case the bile ducts of guineafowls (smyth & mcmanus 1989). junker & boomker (2007a) have reported immature stages of this parasite from the liver/bile ducts and adults from the small intestine of helmeted guineafowls. whether morphological changes, such as the size of the bile ducts, or biochemical changes, such as the bile composition, during the ontogenesis of the guineafowl hosts in some way facilitate the migration and establishment of developing p. paronai has to remain speculation. on the other hand, a change in the prey preference in growing birds, possibly taking larger prey items not formerly included in the diet, may expose older guineafowls to a wider range of parasites. the significantly higher abundance of subulura sp. in juvenile birds can be attributed to the fact that the population of subulura spp. in these hosts was main ly represented by infective larvae that do not yet display sufficient diagnostic characters to distinguish between the two species s. dentigera and s. 234 helminth community of helmeted guineafowls in limpopo province, south africa suctoria. we consider this a result of the fact that infections in the juvenile hosts had been recently acquired, thus comprising a high number of immatures, as opposed to the more mature infections found in older guineafowls. host gender was another determinant that had no significant influence on the distribution of worm burdens and species richness within the guineafowl pop ulation from musina. similar results were obtained by crowe (1977), who attributed the absence of sexual variation in helminth infections to the fact that there is little behavioural or dietary difference between sexes outside the breeding season. all hosts in our and also crowe’s (1977) study were collected during the non-breeding season, extending from march to october in south africa (del hoyo et al. 1994). helmeted guineafowls collected in the free state province during august 2007, however, showed sex related differences regarding the intensities of some helminths (davies et al. in preparation). possible reasons for this given by the latter authors are a difference in the length of the small intestine and caeca between males and females, as demonstrated by prinsloo (2003), resulting in a larger habitat in the females. moreover, females have a relatively higher intake of insects prior to breeding, which is often aided by the male’s foraging for its mate (hockey, dean & ryan 2005). when discussing helminth communities in avian hosts, bush (1990) concluded that host age and sex played a minor role, whereas the overall environment and habitat diversity therein exercised a major influence on the patterns of helminth communities. he argued that the latter would directly influence the “supply” of helminths available in the system. keeping in mind that the current set of data was based on a limited number of hosts, and that a larger sample size might change the emerging picture, it nevertheless suggests that helmeted guineafowls are no exception to this general pattern. acknowledgements we thank dr w.j. luus-powell and mr h.e. hattingh, university of limpopo, for providing logistic support to collect the guineafowls, and mr k. meyer and mr m. storm for making the latter available to us. mr r. watermeyer, university of pretoria, is thanked for his assistance with the processing of the cestodes. ms r. owen, university of pretoria, has greatly assisted with the statistical analyses. this study 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [595.276 841.890] >> setpagedevice article information authors: modesta makungu1,2 hermanus b. groenewald1 wencke m. du plessis3 michelle barrows4 katja n. koeppel5 affiliations: 1department of anatomy and physiology, university of pretoria, south africa 2department of veterinary surgery and theriogenology, sokoine university of agriculture, tanzania 3ross university school of veterinary medicine, st. kitts, west indies 4veterinary services and conservation medicine, bristol zoo gardens, united kingdom 5johannesburg zoo, johannesburg, south africa correspondence to: modesta makungu email: modesta_makungu@yahoo.com postal address: private bag x04, onderstepoort 0110, south africa dates: received: 12 mar. 2015 accepted: 13 may 2015 published: 15 july 2015 how to cite this article: makungu, m., groenewald, h.b., du plessis, w.m., barrows, m. & koeppel, k.n., 2015, ‘thoracic limb morphology of the red panda (ailurus fulgens) evidenced by osteology and radiography’, onderstepoort journal of veterinary research 82(1), art. #953, 10 pages. http://dx.doi.org/10.4102/ojvr.v82i1.953 copyright notice: © 2015. the authors. licensee: aosis openjournals. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. thoracic limb morphology of the red panda (ailurus fulgens) evidenced by osteology and radiography in this original research... open access • abstract • introduction • materials and methods    • radiography    • osteology    • bone measurements       • statistical analysis • ethical considerations • results    • clavicle    • scapula    • humerus    • radius and ulna    • carpus    • metacarpus and digits    • sesamoid bones    • other findings • discussion • conclusion • acknowledgements    • competing interests    • authors’ contributions • references abstract top ↑ the red panda (ailurus fulgens) is distributed primarily in the himalayas and southern china. it is classified as a vulnerable species by the international union for conservation of nature. the aim of this study was to describe the normal osteology and radiographic anatomy of the thoracic limb of the red panda. radiography of the right thoracic limb was performed in seven captive adult red pandas. radiographic findings were correlated with bone specimens from three adult animals. the scapula was wide craniocaudally and presented with a large area for the origin of the teres major muscle. the square-shaped major tubercle did not extend proximal to the head of the humerus. the medial epicondyle was prominent. a supracondylar foramen was present. the radial tuberosity and sesamoid bone for the abductor digiti i longus were prominent. the accessory carpal bone was directed palmarolaterally. metacarpal bones were widely spread. the thoracic limb morphology of the red panda evidenced by osteology and radiography indicated flexibility of the thoracic limb joints and well-developed flexor and supinator muscles, which are important in arboreal quadrupedal locomotion. knowledge gained during this study may prove useful in identifying skeletal material or remains and diagnosing musculoskeletal diseases and injuries of the thoracic limb. introduction top ↑ the red panda (ailurus fulgens) is classified as a vulnerable species by the international union for conservation of nature (iucn 2014). it is an arboreal animal commonly kept in zoological gardens. the red panda is affected by musculoskeletal diseases and injuries similar to those reported in domestic cats and dogs and radiography is commonly used as the first diagnostic imaging modality. diseases and injuries involving the musculoskeletal system of the thoracic limb, such as osteoarthritis, hyperostotic bone disease and fractures, have been reported in the red panda (lynch, mccracken & slocombe 2002; philippa & ramsay 2011; preece 2011). knowledge of the normal osteology and radiographic anatomy is important for accurate interpretation and diagnosis of musculoskeletal diseases involving the thoracic limb. the normal osteology and radiographic anatomy of the thoracic limb in companion animals is well documented (nickel et al. 1986; smallwood & spaulding 2013; thrall & robertson 2011), which serves as a reference for diagnosis of musculoskeletal diseases. little information is available on the osteology of the thoracic limb of the red panda (carlsson 1925) and to our knowledge, the normal radiographic anatomy of the thoracic limb has not been described in this species. the aim of this study was to describe the morphology of the thoracic limb of the red panda evidenced by osteology and radiography as a reference for clinical use and identification of skeletal material and skeletal remains. materials and methods top ↑ radiography radiography of the right thoracic limb was performed in seven intact, skeletally mature, adult captive red pandas belonging to the johannesburg zoo. of the seven animals, five were male and two were female. the age of the animals ranged from 1.4 years to 14.3 years (mean 7.3 ± 5.4 years).the mean weight of the animals was 4.5 kg ± 0.7 kg (range: 3.7 kg − 5.6 kg). radiography was performed under general anaesthesia during annual health examinations using a table top technique. in six animals, an eva-hf525 x-ray machine (comed medical systems, korea) was used at a source-to-image distance (sid) of 95 cm. an automatic x-ray film processor (model: cp-345; elk corporation, japan) was used. mammography films (um-ma, fujifilm, japan) were used in combination with um-ma screens (fujifilm europe, germany). all radiographs were obtained using a peak kilovoltage (kvp) range of 46−50 and a charge of 5 mas. radiographic images were digitised using a digital camera (canon 5dmark2, canon, japan). in one animal, radiography was performed using a siemens polymat 50 x-ray machine (siemens, germany) and images were obtained using a computed radiography system (fuji axim fcr capsula xl, fujifilm, japan) at an sid of 100 cm. a kvp range of 48–60 and charge range of 4 mas – 5 mas were used. previous radiographs of the right thoracic limb of two red pandas, which were taken at the bristol zoo gardens for clinical evaluation before skeletal maturity (10 and 7 months old, respectively), were retrieved and evaluated for the location of physes. osteology bone specimens of skeletally mature adult animals (one from the national museum of scotland and two euthanised animals from the johannesburg zoo) were used for the osteology study. photographs of bone specimens were obtained using a digital camera (canon 5dmark2, canon, japan). a report on the myology of the thoracic limb of the red panda (fisher et al. 2009) was used to locate the sites for the origins, insertions and functions of the various muscles of the thoracic limb. bone measurements bone measurements of the right thoracic limb were performed on radiographic images of skeletally mature captive red pandas. this excludes measurements for the scapula, which were performed on bone specimens. the maximum lengths of bones were measured from the proximal to the distal extremities. the maximum lengths of the humerus, radius and ulna were measured on the mediolateral (ml) view, whereas those of the metacarpals, phalanges and sesamoid bones were measured on the dorsopalmar (dpa) view of the manus. the craniocaudal (crcd) diameters of the humerus, radius and ulna were measured on the ml view at mid diaphysis. the ml diameter of the sesamoid bone for the abductor digiti i longus was measured on the dpa view of the manus as the maximum diameter perpendicular to the maximum length. radiographic measurements were not compensated for magnification. statistical analysis statistical analysis was performed using the statview statistical package (sas institute, usa). mean, range and standard deviation (s.d.) were calculated. data were expressed as mean ± s.d. ethical considerations top ↑ this study was approved by the animal use and care committee of the university of pretoria. results top ↑ measurements of bones described in the figures are shown in tables 1–3. table 1: measurements of the scapula, humerus, radius and ulna in captive red pandas. table 2: radiographic measurements of metacarpal bones and phalanges in captive red pandas. table 3: radiographic measurements of the sesamoid bones in captive red pandas. clavicle a rudimentary clavicle was seen in two animals (aged 14.3 and 1.4 years) as an area of mineral opacity in the soft tissues just cranial to the supraglenoid tubercle, as shown on the ml view of the shoulder joint of the older animal (figure 1a). on the caudocranial (cdcr) view of the shoulder joint, the rudimentary clavicle was seen distomedial to the minor tubercle (figure 1b) in this animal. figure 1: radiographs of the right shoulder joints of (a, b) a 14.3-year-old male and (c) a skeletally immature red panda. mediolateral views are shown in (a) and (c) and a caudocranial view in (b). scapula the scapula was wide craniocaudally with a convex cranial margin (figure 2a). the proximal half of the caudal margin was almost convex, whereas the distal half was fairly straight with a rough surface (figure 2a). the mediodistally directed coracoid process was stout (figure 2b, c) and the infraglenoid tubercle was well developed (figure 2b). on the ml view of the shoulder joint (figure 1a), the coracoid process was seen as a curvilinear area of increased mineral opacity caudal to the supraglenoid tubercle. the articular surface of the glenoid cavity was concave and extended proximal to the lateral area of the supraglenoid tubercle (figure 2c). figure 2: (a) lateral, (b) caudal and (c) cranial views of a specimen of the right scapula of an adult red panda. black arrow shows the proximal extension of the articular surface of the glenoid cavity. the infraspinous fossa was deep and concave, whereas the supraspinous fossa was convex (figure 2a). caudal to the proximal half of the infraspinous fossa, the lateral surface presented a large area for the origin of the teres major muscle (figure 2a). the spine of the scapula was slanted slightly towards the infraspinous fossa (figure 2a). the craniodistally directed hamate process was prominently large and flattened mediolaterally (figure 2a−c). on the ml view of the shoulder joint (figure 1a), the hamate process projected cranially beyond the cranial margin of the supraglenoid tubercle. on the cdcr view, it projected distally beyond the level of the glenoid cavity (figure 1b). the medial surface of the scapula presented with a triangular facies serrata. humerus the humerus was the longest bone of the thoracic limb (table 1). the major and minor tubercles did not extend higher than the head of the humerus (figure 3a and figure 4a−c). the major tubercle was almost square, with a flat proximal margin (figure 4a). when viewed medially, the proximal and cranial margins of the minor tubercle formed an approximately right angle (figure 4c). on the ml view of the shoulder joint, the major and minor tubercles were further distal than the head of the humerus (figure 1a). the cranial and proximal margins of the minor tubercle appeared as a square area of increased mineral opacity caudal to the intertubercular groove (figure 1a). on the cdcr view of the shoulder joint, the major and minor tubercles were fairly flattened (figure 1b). figure 3: mediolateral radiographs of (a) the right humerus and (b) the radius and ulna of two male red pandas. bones of a 1.4-year-old animal are shown in (a) and those of a 2.5-year-old animal are shown in (b). figure 4: bone specimen of (a–c) the right proximal and (d–f) the distal humerus of an adult red panda. cranial views are shown in (a) and (d). lateral views are shown in (b) and (e). medial and caudal views are shown in (c) and (f), respectively. the area for the insertion of the infraspinatus muscle (facies m. infraspinati) was prominent, ovoid, elongated in a proximodistal direction and presented as a shallow fossa (figure 4b). it was at the same level as the head of the humerus (figure 4b). the wide and prominent area for the insertion of the subscapularis muscle faced caudomedially (figure 4c). the tuberosity for the teres minor muscle was not seen. the medial epicondyle was well developed and larger than the lateral epicondyle (figure 4d, f). the medial and lateral epicondyles presented with shallow depressions for the origin of the flexors and extensors of the carpus and digits, respectively. on the ml view of the elbow joint, the medial epicondyle was square, whereas the lateral epicondyle formed an obtuse angle (figure 5a). on the crcd view of the elbow joint, the medial and lateral margins of the medial and lateral epicondyles, respectively, appeared undulating (figure 5b). figure 5: radiographs of the right elbow joint of (a) a 1.4-year-old, (b) a 10.3-year-old and (c) a skeletally immature male red panda. mediolateral views are shown in (a) and (c) and a craniocaudal view is shown in (b). the lateral supracondylar crest was prominent (figure 4d−f) and projected caudally beyond the cortical bone on the ml view of the elbow joint (figure 5a). the medially located supracondylar foramen sloped from caudomedial to craniodistal (figure 4d, f). on the crcd view of the elbow joint, the supracondylar foramen was represented by two ovoid radiolucent areas just proximal to the medial epicondyle (figure 5b). the ovoid olecranon fossa sloped from proximolateral to distomedial (figure 4f and figure 5b). it was moderately deep and elongated mediolaterally (figure 4f). the cranial surface of the condyle of the humerus had well-defined radial and coronoid fossae (figure 4d). the articular surface of the condyle of the humerus presented with a larger grooved and medially located trochlea humeri and a small laterally located capitulum humeri (figure 4d). the cranial surface of the trochlea humeri had a prominent medial trochlea lip (figure 4d), whereas the caudal surface was concave (figure 4f). the proximal and distal physes of the humerus (figure 1c and figure 5c) were similar to those of the domestic cat and dog. radius and ulna the ulna was longer and relatively larger than the radius (figure 3b; table 1). the head of the radius sloped from lateral to medial and was elliptical in outline (figure 6a, b). it had a concave articular fovea (figure 6a, b) and was demarcated from the body by a distinct neck (figure 5 and figure 6). the medial half of the cranial surface of the head of the radius was flat, whereas its lateral counterpart was convex (figure 6a). the caudal surface of the head of the radius presented with a smooth band, the articular circumference, for articulation with the radial notch of the ulna (figure 6b). figure 6: bone specimens of (a, b) the right proximal and (c, d) the distal radius of adult red pandas. cranial views are shown in (a) and (c). caudal views are shown in (b) and (d). white arrows in (c) and (d) indicate a prominent crest for the insertion of the brachioradialis muscle. the body of the radius curved cranially along the longitudinal axis of the bone (figure 3b). the proximal half of the cranial surface of the body presented with a flat surface (figure 6a). the radial tuberosity was prominent and located relatively further distally (figure 5a and figure 6b). it had a longitudinal groove (figure 6b). the trochlea of the radius had three cranial grooves for the passage of tendons (figure 6c). medially, it presented with a prominent crest (figure 6c and figure 6d). the ulnar notch was concave and elongated craniocaudally. the ulna curved laterally along the longitudinal axis of the bone. the tuber olecrani was square (figure 5a and figure 7a). on the ml view of the elbow joint, a thin, straight radiopaque line was seen to run almost parallel with the proximal margin of the tuber olecrani, representing the physeal scar of the tuber olecrani (figure 5a). the medial coronoid process was prominent and rounded (figure 5a and figure 7b). the anconeal process sloped from lateral to medial (figure 7b), with its lateral margin wrapped proximally against the lateral surface (figure 7a). the craniolaterally located radial notch was at the same level as the body of the ulna and consisted of a single articular facet (figure 7a). just distal to the medial coronoid process, the body of the ulna presented with a deep narrow groove for the insertion of the brachialis muscle (figure 7a). figure 7: (a) lateral, (b) cranial and (c) medial views of bone specimens of (a, b) the right proximal and (c) the distal ulna of adult red pandas. the head of the ulna was well developed (figure 7c). the articular circumference was markedly elevated from the body of the ulna and formed almost a right angle with the styloid process (figure 7c). the styloid process was large and bulbous (figure 7c). on the ml view of the elbow joint, the physes of the tuber olecrani and proximal part of the radius (figure 5c) were similar to those of the domestic cat and dog. the physes of the distal part of the radius and ulna appeared transverse (figure 8c), similar to those of the domestic cat. figure 8: (a) dorsopalmar and (b, c) mediolateral radiographs of the right carpus of red pandas. the carpus of a 1.4-year-old female animal is shown in (a) and (b), whereas that of a skeletally immature animal is shown in (c). carpus the carpus consisted of seven carpal bones: the inter­medioradial, ulnar, accessory, and the first, second, third and fourth carpal bones (figure 8a). the intermedioradial carpal bone was the largest carpal bone. palmaromedially it presented with a tuberosity (figure 8a). medially the bone presented with an ovoid articular facet for the sesamoid bone of the abductor digiti i longus. the palmarolaterally directed accessory carpal bone was prominent and almost the same size as the intermedioradial carpal bone (figure 8a). it had two articular surfaces for the ulnar carpal bone and styloid process of the ulna (figure 8). on the ml view of the carpus, the physis of the accessory carpal bone appeared sagittal (figure 8c), similar to that of the domestic cat and dog. the fourth carpal bone was the largest bone in the distal row (figure 8a). the third carpal bone was flattened mediolaterally and comma shaped on the dpa view of the carpus (figure 8a). the second carpal bone was the smallest bone in the distal row and was almost triangular (figure 8a). the first carpal bone was well developed and articulated distally with the first metacarpal bone (figure 8a). metacarpus and digits five metacarpal (mc) bones and digits were seen (figure 9). all mc bones were almost equally developed with regard to width (figure 9). the mc bones were widely spread and relatively short in relation to the length of the digits (figure 9). the first digit had only two phalanges, namely the proximal and distal phalanges. the other digits (ii−v) had three phalanges: proximal, middle and distal phalanges (figure 9). the distal articular surface of the middle phalanx was symmetrical and convex (figure 10a). the proximal articular surface of the distal phalanx was concave and parallel to the horizontal plane. figure 9: dorsopalmar radiograph of the right manus of a 1.4-year-old female red panda. figure 10: (a) dorsal and (b) lateral views of bone specimens of the middle phalanx and sesamoid bone for the abductor digiti i longus, respectively, of an adult red panda. (c) mediolateral radiograph of the right carpus of an 11.3-year-old male red panda. sesamoid bones a large sesamoid bone of the abductor digiti i longus was seen in all animals (figure 8a and figure 10b). on the dpa view of the carpus, it was seen medial to the intermedioradial carpal bone (figure 8a). on the ml view of the carpus, it was not seen owing to its superimposition on the carpal bones (figure 8b). each metacarpophalangeal joint had paired proximal sesamoid bones on the palmar aspect (figure 9). other findings on the ml view of the carpus, four animals had one bone and two animals had two bones on the palmar aspect of the fifth mc bone, which are likely sesamoid bones (figure 8b and figure 10c). (one animal did not have any bones on the palmar aspect of the fifth mc bone.) on the dpa view of the carpus, the bones were superimposed on the abaxial palmar aspect of the fifth mc bone and therefore poorly visible (figure 8a). osteoarthrosis was seen in the elbow of two animals and in the carpus of three animals, all older than nine years. the two animals with elbow osteoarthrosis had concurrent osteoarthrosis of the carpus. of the two joints, the elbow was more severely affected. elbow osteoarthrosis was characterised by sclerosis of the trochlea notch, poor visualisation of the medial coronoid process, thickening of the cranial cortex of the distal part of the humerus and the presence of osteophytes dorsally on the anconeal process, the cranial aspect of the humeroradial joint, around the trochlea notch and the lateral epicondylar crest. osteoarthrosis of the carpus was characterised by poor delineation of the carpal bones, osteophytes on the distal part of the radius and styloid process of the radius and enthesophytes on the lateral and medial margins of the bases of the metacarpal bones, distal part of the sesamoid bone for the abductor digiti i longus, lateral aspect of the fourth carpal bone and ulnar carpal bone. discussion top ↑ less cursorial animals inhabit high-structured habitats such as dense forests, whereas cursorial animals inhabit more open terrain (gonyea 1978). the thoracic limb of less cursorial animals is capable of a greater range and variety of movements compared with cursorial animals (hopwood 1947). the difference in movements of the thoracic limb between these two groups of animals is a result of variation in their feeding habits and the environments in which they live (gonyea 1978; hopwood 1947). in the red panda, movement on and between the small terminal branches of trees is facilitated by a high degree of flexibility of the pectoral and pelvic girdles and limb joints (roberts & gittleman 1984). the wide scapula observed in the red panda accommodates well-developed muscles, which stabilise the shoulder joint and prevent luxation. fisher et al. (2009) reported stout tendons of insertion of the supraspinatus and infraspinatus muscles, which stabilise the shoulder joint in the red panda. in arboreal quadrupedal animals, the thoracic limb is frequently used in an abducted position and therefore the muscles that stabilise the shoulder joint are well developed (roberts & davidson 1975). the presence of a prominent, large hamate process, which provides the origin of the deltoideus pars acromialis (fisher et al. 2009), and the location of the insertion of the infraspinatus muscle at the same level as the head of the humerus, enhance the abduction function of the shoulder joint in the red panda. similar findings have also been reported in some african mammals (hopwood 1947; taylor 1974). the presence of the large area for the origin of the teres major muscle, well-developed medial epicondyle, deep groove for the insertion of the brachialis muscle and prominent further distally located radial tuberosity observed in the red panda indicate the presence of strong flexor muscles of the thoracic limb. strong flexor muscles of the thoracic limb are important in arboreal quadrupeds as the thoracic limb is frequently held in a flexed position during locomotion (hopwood 1947). the teres major and brachialis muscles flex the shoulder and elbow joints, respectively (fisher et al. 2009). the medial epicondyle provides the origin for the flexor muscles of the carpus and digits (fisher et al. 2009). the radial tuberosity provides the insertion for the biceps brachii, which flexes the elbow joint (fisher et al. 2009). the radial tuberosity was also found to be larger and located further distally in the african palm civet (nandinia binotata), a climbing species, which points to the strength of the biceps brachii in flexing the elbow joint (taylor 1974). the concave caudal surface of the trochlea humeri in the red panda increases the stability of the elbow joint when the joint is in a flexed position (fleagle & simons 1995). the prominent medial trochlea lip counteracts the adducting forces at the elbow joint produced by the carpal and digital flexors when the antebrachium and manus are in a pronated position (fleagle & simons 1995). the oblique orientation of the olecranon fossa observed in the red panda, sloping from proximolateral to distomedial, indicates that the thoracic limb moves through an arch during locomotion, away from the parasagittal plane of the body, as in most feline species (gonyea 1978). this phenomenon most likely reflects an adaptation to the arboreal environment in which it lives. in felids, it was found that species that are exclusive forest dwellers have a greater angle of inclination of the olecranon fossa compared with those that inhabit more open terrain (gonyea 1978). the power to rotate the antebrachium, which is of great importance to a climbing species (hopwood 1947), is also important because the manus is used as a prehensile organ for food manipulation tasks such as grasping and bending bamboo to bring the leaves within reach of the mouth (antón et al. 2006; roberts & gittleman 1984). in the red panda, the power to rotate the antebrachium was indicated by the presence of the following features: the elliptically shaped head of the radius, stout and well-developed body and head of the ulna, well-developed muscles of supination and a craniolaterally located radial notch with a single articular facet at the same level as the body of the ulna (gonyea 1978; taylor 1974). during pronation and supination, the elliptically shaped head of the radius acts as a cam, which imparts the eccentric motion of the radius (gonyea 1978). the stout and well-developed body and head of the ulna act as a pivot, which supports the radius during pronation and supination. the prominent lateral supracondylar crest provides the origin for the brachioradialis muscle, which supinates the antebrachium (fisher et al. 2009). a distinct flattening on the proximal half of the cranial surface of the radius accommodates the supinator muscle, which supinates the antebrachium (fisher et al. 2009). in addition, the abductor digiti i longus assists in supination (antón et al. 2006). the deep-sited, cranially located radial notch, with two articular facets, restricts rotation of the radius (gonyea 1978; taylor 1974). the palmarolaterally directed accessory carpal bone, large sesamoid bone for the abductor digiti i longus and widely spread mc bones indicate flexibility of the manus in the red panda, which is important for arboreal locomotion and food manipulation (antón et al. 2006; taylor 1974). the palmarolaterally directed accessory carpal bone enables greater leverage to be exerted on the manus during ulnar deviation (taylor 1974). a large sesamoid bone for the abductor digiti i longus provides the insertion of the muscle, which abducts the carpal joint and carpometacarpal joint of digit i (fisher et al. 2009) and also supinates the manus (antón et al. 2006). in addition, the sesamoid bone of the abductor digiti i longus acts as a ‘false-thumb’ for the grasping actions of the manus (antón et al. 2006). widely spread and relatively short mc bones provide a wide space and allow greater effectiveness of the muscles responsible for small movements of the digits (taylor 1974). the paired proximal sesamoid bones provide insertions for a lateral and medial belly of flexores breves profundi muscles (fisher et al. 2009). these muscles flex the metacarpophalangeal joints and may abduct or adduct the digits (fisher et al. 2009). the presence of osteoarthrosis in animals older than nine years in this study, without medical history of trauma, suggests that the likely cause is primary osteoarthrosis. it is likely that these two joints are subjected to more stress as a result of arboreal locomotion; however, this warrants further investigation. a review of feline osteoarthrosis indicated that for a large proportion of animals presenting with osteoarthrosis, there was no obvious cause, suggesting a primary osteoarthrosis (lascelles 2010). the small bones located on the palmar aspect of mc v are likely sesamoid bones. however, in this study it was difficult to ascertain which muscle they are associated with. the symmetrical shape of the distal articular surface of the middle phalanx and the parallel orientation to the horizontal plane of the proximal articular surface of the distal phalanx observed in this study are different from felids’ (gonyea & ashworth 1975) and indicate that the claws of the red panda are slightly retractile (taylor 1974) or not able to retract at all (gonyea & ashworth 1975). conclusion top ↑ morphology of the thoracic limb of the red panda evidenced by osteology and radiography indicated flexibility of the thoracic limb joints and well-developed flexor and supinator muscles, which are important in arboreal quadrupedal locomotion. knowledge of the normal osteology and radiographic anatomy of the thoracic limb of the red panda may prove useful in identifying skeletal material or remains and diagnosis of musculoskeletal diseases and injuries of the thoracic limb. acknowledgements top ↑ the authors would like to thank the organization for women in science for the developing world (owsd), the swedish international development cooperation agency (sida), the university of pretoria, the johannesburg zoo and the bristol zoo gardens for supporting this study. the national museum of scotland is acknowledged for supplying bone specimens. we are also grateful to dr georgina cole, ms fania mohlala, and the sisters and animal handlers of the johannesburg zoo and the onderstepoort veterinary academic hospital for their assistance during radiographic examination. mrs charmaine vermeulen and mrs wilma olivier of the university of pretoria assisted with photography and administrative work, respectively. competing interests the authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. authors’ contributions h.b.g. (university of pretoria), w.m.d.p. (ross university school of veterinary medicine) and m.b. (bristol zoo gardens) designed the project and supervised the research. k.n.k. (johannesburg zoo) performed general anaesthesia and supervised clinical procedures. m.m. 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